Design and synthesis of an androgen receptor pure antagonist (CH5137291) for the treatment of castration-resistant prostate cancer

Article abstract of DOI:10.1016/j.bmc.2010.10.023

A series of 5,5-dimethylthiohydantoin derivatives were synthesized and evaluated for androgen receptor pure antagonistic activities for the treatment of castration-resistant prostate cancer. Since CH4933468, which we reported previously, had a problem wit

Full text of DOI:10.1016/j.bmc.2010.10.023

Bioorganic & Medicinal Chemistry 18 (2010) 8150–8157
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design and synthesis of an androgen receptor pure antagonist (CH5137291)
for the treatment of castration-resistant prostate cancer
⇑
⇑
Hitoshi Yoshino , Haruhiko Sato , Takuya Shiraishi, Kazutaka Tachibana, Takashi Emura, Akie Honma,
Nobuyuki Ishikura, Toshiaki Tsunenari, Miho Watanabe, Ayako Nishimoto, Ryo Nakamura,
Toshito Nakagawa, Masateru Ohta, Noriyuki Takata, Kentaro Furumoto, Kazuya Kimura, Hiromitsu Kawata
Research Division, Chugai Pharmaceutical Co., Ltd, 1-135, Komakado, Gotemba, Shizuoka 412-8513, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 5,5-dimethylthiohydantoin derivatives were synthesized and evaluated for androgen receptor
pure antagonistic activities for the treatment of castration-resistant prostate cancer. Since CH4933468,
which we reported previously, had a problem with agonist metabolites, novel thiohydantoin derivatives
were identified by applying two strategies. One was the replacement of the alkylsulfonamide moiety by a
phenylsulfonamide to avoid the production of agonist metabolites. The other was the replacement of the
phenyl ring with a pyridine ring to improve in vivo potency and reduce hERG affinity. Pharmacological
assays indicated that CH5137291 (17b) was a potent AR pure antagonist which did not produce the
agonist metabolite. Moreover, CH5137291 completely inhibited in vivo tumor growth of LNCaP-BC2, a
castration-resistant prostate cancer model.
Received 4 August 2010
Revised 8 October 2010
Accepted 9 October 2010
Available online 15 October 2010
Keywords:
Androgen receptor pure antagonist
Prostate cancer
Metabolite
hERG inhibition
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
also inhibited tumor growth of the LNCaP xenograft dose-depen-
dently in mice.
Prostate cancer is the most common cancer amongst men in the
USA and the second most common malignant cause of male death
worldwide.1 Since the growth of prostate cancer is dependent on
androgen, androgen receptor (AR) antagonists such as flutamide
(1) (a precursor of the active form hydroxyflutamide (2)) and bica-
lutamide (3) are clinically used to treat prostate cancer2 (Chart 1).
These AR antagonists exhibit good efficacy in many cases and com-
prise an important part of effective therapeutics.3–6 However, after
a short period of response, the tumor progresses through treat-
ment to a castration-resistant prostate cancer (CRPC) stage.7 There-
fore, treatment of CRPC would benefit from novel AR antagonists.
We previously reported that hydroxyflutamide and bicalutamide
have partial agonistic activities at high concentrations in vitro.8
Therefore, we hypothesized that the AR antagonists exhibiting no
agonist activities, so-called AR pure antagonists9,10 would be effi-
cacious against CRPC.
However, CH4933468 did not show antitumor activity against a
mouse LNCaP-BC2 xenograft model,13 an AR hypersensitive CRPC
model, because of the existence of a small amount of 3-dealkylated
thiohydantoin metabolite (5) with strong AR agonistic activity.
Therefore, the amount of 5 was evaluated in vitro and in vivo to
predict the production of 5 in human (Table 1). In in vitro liver
microsome experiments with various animal species, the concen-
tration of the metabolite was determined to be less than 0.1%.
However, from in vivo pharmacokinetic studies after oral adminis-
tration of CH4933468, the AUC of metabolite 5 was found to be in
the range of 0.005–8.6% in various animals. Especially, a high con-
centration of 5 was detected in rats. In parallel, we evaluated the
HO
O
R
H
N
H
N
O
S
O
O2N
F3C
NC
F3C
F
We recently reported that
a
thiohydantoin derivative,
O
CH4933468 (4),11 with a sulfonamide terminal side chain, not only
showed AR pure antagonistic activities in the reporter gene assay
(RGA) but also inhibited the growth of LNCaP-BC2,12 a bicaluta-
mide-resistant cell line modeling CRPC. Furthermore, CH4933468
R=H flutamide (1)
R=OH hydroxyflutamide (2)
bicalutamide (3)
O
NC
Cl
N
N
SO2NH2
S
CH4933468 (4)
⇑
Corresponding authors. Tel.: +81 550 87 8638; fax: +81 550 87 5326.
E-mail address: yoshinohts@chugai-pharm.co.jp (H. Yoshino).
Chart 1. Structure of AR antagonists.
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.10.023

H. Yoshino et al. / Bioorg. Med. Chem. 18 (2010) 8150–8157
8151
Table 1
OEt
CN
O
Agonist metabolite (5) of CH4933468 and its amount in vitro and in vivo in several
animal species
H2N
O
a
NC
NCS
NCS
NC
N
NH
O
O
Cl
Cl
S
5
6a
6b
NC
N
NC
N
NH
N
SO2NH2
O
Cl
Cl
S
N
S
H
CH4933468 (4)
agonist metabolite (5)
NC
NC
F3C
N
b
c
N
agonist EC5 = 0.2 nM
agonist EC5 > 10000 nM
antagonist sIC50 = 200 nM
F3C
S
7
O
H2N
SO2NBn2
In vitro liver microsome concd @ 30 mina
(% of intact CH4933468)
In vivo PKb AUC%
of intact
Br
Br
O
e
d
NC
NH2
Human
Mouse
Rat
Dog
Monkey
0.022%
0.038%
0.075%
<0.001%
0.027%
NC
N
H
Br
0.7%c
R
8.6%c
R
R=Cl
R=CF3
8a
9a
9b
R=Cl
R=CF3
0.005%d
0.15%c
8b
O
O
a
Lower limit of quantification: 0.1 nM, substrate concn: 10 mM, enzyme concn:
NC
N
f
NC
N
N
SO2NBn2
1 mg/protein/mL. The values were determined by
duplicate.
a single experiment run in
N
SO2NH2
R
S
R
S
b
Lower limit of quantification: 0.1 nM, n = 3.
Dose: 100 mg/kg multiple.
Dose: 100 mg/kg single.
11a
11b
10a
10b
R=Cl
R=CF3
R=Cl
R=CF3
c
d
Scheme 1. Reagents and conditions: (a) Et3N, toluene, 100 °C; (b) Et3N, THF, reflux;
(c) 6 N HCl, 1,4-dioxane, 80 °C; (d) iPr2NEt, CH2Cl2, rt; (e) (i) NaH, THF, 45 °C; (ii)
NaH, ClCSOPh, THF, rt; (f) concd H2SO4, rt.
pharmacological effects of the agonist metabolite in vitro. The
addition of metabolite 5 at a concentration of only 0.1% to
CH4933468 affected the pure AR antagonistic activities of
CH4933468 (data not shown). These results showed that it was dif-
ficult to develop CH4933468 as an AR pure antagonist.
Based on the above results, we applied a novel modification
strategy to obtain AR pure antagonists without the propensity to
produce agonist metabolites. Our strategy was to mimic the struc-
ture of the alkyl sulfonamide by incorporating an aryl sulfonamide
(Chart 2).
by esterification gave 15a and 15b, respectively. Cyclization of
the thiohydantoin ring with corresponding isothiocyanates in the
presence of DMAP followed by deprotection of the benzyl group
gave target molecules 17a, 17b, 17c, and 17d.
Compound 22 was prepared by a different route due to the dif-
ficulty of the synthesis of 4-aminopyridine-2-sulfonamide. Specif-
ically, 2-chloropyridin-4-ylamine (18) was treated with PMBSH in
the presence of tBuOK to give 19. Alkylation of 19 with 2-bromo-2-
methylpropionic acid, followed by esterification gave 20. After
cyclization of the thiohydantoin ring, deprotection of PMB group
with TFA gave thiol 21. Treatment of 21 with KNO3 and SO2Cl2, fol-
lowed by ammonia, afforded target compound 22.
2. Chemistry
The agonist metabolite 5 and 3-phenylthiohydantoin analogs
were prepared according to the synthetic sequence outlined in
Scheme 1. Isothiocyanate 6a and aminoisobutyric acid ester were
treated with Et3N to give compound 5. Isothiocyanate 6b was trea-
ted with 2-methyl-2-phenylaminopropanenitrile and Et3N,
followed by hydrolysis with 6 N HCl to give 3-phenyl thiohydan-
toin 7. For the synthesis of phenyl sulfonamides 11a and 11b,
bromide derivatives 9a and 9b were used as intermediates. Alkyl-
ation of 3-amino-N,N-bis(phenylmethyl)benzenesulfonamide14
with 9a and 9b and NaH, followed by thiohydantoin ring formation
with ClCSOPh and NaH, gave compounds 10a and 10b, respec-
tively. Deprotection of the benzyl group on sulfonamide by
3. Results and discussion
The synthesized compounds were evaluated for their in vitro
agonist/antagonist activities to AR using the same procedures as
previously reported.11 In RGA, a ‘pure antagonist’ was defined to
have a EC5 value greater than 10,000 nM. Cell growth was also
determined in LNCaP-BC2 cells as previously reported.11 Agonistic
activity was expressed as + or ꢀ according to the dose–response
curve of growth. A ‘pure antagonist’ was expressed as ꢀ.
Phenyl sulfonamide compounds 11a and 11b showed almost
the same activity as CH4933468 in the in vitro RGA and cell growth
inhibition assays. Since 3-phenyl thiohydantoin (7) showed partial
agonist activities, these results were attributed to the sulfonamide
moiety. In addition, as expected, compound 11a did not produce
the agonist metabolite 5 either in vitro or in vivo (data not shown).
So, we succeeded in converting an alkyl linker to an aryl linker with
the activities maintained without the production of the agonist
metabolite.
To confirm the in vivo activities of the compounds, antiandro-
genic activities were evaluated on the basis of seminal vesicle
(SV) wet weight in castrated mice.17 However, phenyl sulfonamide
derivatives 11a and 11b tended to show weaker activities in vivo
than CH4933468. Since these compounds were metabolically
stable in human liver microsome and showed acceptable perme-
ability in a parallel artificial membrane permeation assay (PAMPA),
weak in vivo antiandrogenic activities might be mainly attributed
to low solubility.
15
concentrated H2SO4 afforded target compounds 11a and 11b.
Pyridine sulfonamide derivatives were synthesized according to
the synthetic sequence shown in Scheme 2. Dibromopyridines 12a
and 12b were treated with iPrMgCl and SO2Cl2,16 followed by ami-
dation with dibenzylamine to give corresponding bromo sulfona-
mides 13a and 13b. An amino group on the pyridine ring was
installed by treatment with p-methoxybenzylamine (PMBNH2),
followed by deprotection of the PMB group with TFA. Alkylation
of 14a and 14b with 2-bromo-2-methylpropionic acid, followed
O
O
NC
N
NC
N
N
N
SO2NH2
SO2NH2
R
R
S
S
Chart 2. Modification strategy to avoid agonist metabolite formation.

8152
H. Yoshino et al. / Bioorg. Med. Chem. 18 (2010) 8150–8157
ity. Especially, 4-pyridine compound 22 drastically reduced hERG
binding. On the other hand, 5-pyridine compounds 17c and 17d re-
sulted in only slightly reduced hERG binding. The results indicated
that the position of the nitrogen on the pyridine ring significantly
affects hERG channel binding. The replacement of the phenyl ring
by a pyridine ring contributed to solve both issues at the same
time.
Among the compounds, 17b (CH5137291) attracted the most
attention as an AR pure antagonist. First, the existence of the
3-dearylated thiohydantoin metabolite (23)19 was examined (Table
3). Since 23 also showed strong agonistic activity in RGA like com-
pound 5, the amount of 23 produced after oral administration of
CH5137291 was a significant issue. In the in vitro liver microsome
stabilityexperimentswith CH5137291, the productionof 23 was be-
low the lower limit of quantification (0.001%) in any animal species,
including human. After oral administration of CH5137291, less than
0.1% of metabolite 23 was determined in rat and was not determined
in other species. In addition, other metabolites which showed AR
agonist activity were not detected in any species. These results
encouraged us to believe that the amount of the agonist metabolite
23 produced from CH5137291 would be acceptable in clinical
development.
Since CH5137291 showed weak hERG inhibition in vitro, poten-
tial cardiovascular effects of CH5137291 were evaluated using
anesthetized dogs. Fortunately, no significant adverse changes of
electrocardiographic parameters including QT prolongation were
observed (data not shown).
To confirm the in vivo bicalutamide-resistant antitumor effect
of CH5137291, we examined the LNCaP-BC2 mouse xenograft
model. Treatment with CH5137291 (10 and 100 mg/kg) inhibited
tumor growth in this model (Fig. 1a). On the other hand, bicaluta-
mide (10 and 100 mg/kg) did not inhibit tumor growth. This ten-
dency was clarified by the relative PSA (prostate specific antigen;
the transcriptional product of AR) concentration results. In the case
of CH4933468, the relative PSA increased significantly.13 Treat-
ment with CH5137291 completely inhibited production of PSA
(Fig. 1b). In contrast, bicalutamide did not completely inhibit pro-
duction of PSA. These results were corroborated by the in vitro ago-
nist activities of bicalutamide.
a, b
c, d
Br
X
Y
Br
X
Y
H2N
X
Y
SO2NBn2
Br
SO2NBn2
12a X=N, Y=CH
12b X=CH, Y=N
13a X=N, Y=CH
13b X=CH, Y=N
14a X=N, Y=CH
14b X=CH, Y=N
O
O
6a or 6b
HO
e
MeO
g
f
Br
HN
X
Y
SO2NBn2
15a X=N, Y=CH
15b X=CH, Y=N
O
O
N
NC
N
NC
h
N
N
X
SO2NBn2
X
Y
SO2NH2
R
R
S
S
Y
R=Cl, X=N, Y=CH
R=CF3, X=N, Y=CH
R=Cl, X=CH, Y=N
R=CF3, X=CH, Y=N
16a
16b
16c
16d
17a R=Cl, X=N, Y=CH
17b R=CF3, X=N, Y=CH
17c
17d
R=Cl, X=CH, Y=N
R=CF3, X=CH, Y=N
O
HO
H2N
SPMB
H2N
Cl
f
i
Br
e
N
N
18
19
O
O
N
6b
MeO
g
j
NC
F3C
HN
SPMB
N
SH
N
S
N
20
k, l
O
21
NC
F3C
N
N
SO2NH2
S
N
22
Scheme 2. Reagents and conditions: (a) iPrMgCl, hexane, rt, then SO2Cl2, 0 °C;
(b) Bn2NH, Et3N, CH2Cl2, rt; (c) PMBNH2, iPr2NEt, 1,3-dimethly-2-imidazolidinone
(DMI), microwave, 100 °C; (d) TFA, microwave, 100 °C; (e) iPr2NEt, 1,4-dioxane,
60 °C; (f) trimethylsilyl diazomethane, MeOH, toluene, rt; (g) DMAP, toluene,
100 °C; (h) concd H2SO4, rt; (i) PMBSH, tBuOK, DMI, microwave, 120 °C; (j) TFA,
microwave, 120 °C; (k) KNO3, SO2Cl2, 0 °C; (l) NH3 aq, rt.
In addition, 11a and 11b strongly inhibited the human ether-
a-go-go related gene (hERG) potassium channel. Therefore, further
investigation was necessary to address issues of weak in vivo po-
tency and potent hERG inhibitory activity. According to Zhao et al.,
both solubility and hERG affinity are highly related to the lipophilic-
ity of the molecule.18 On the other hand, our previous research
showed that the thiohydantoin ring and left side phenyl ring were
important for AR binding.11 Therefore, we focused our efforts on
the conversion of the right side phenyl ring. Specifically, we replaced
the phenyl ring with pyridine rings to reduce lipophilicity.
All pyridine derivatives showed AR pure antagonistic activity in
both RGA and LNCaP-BC2 (Table 2). Although there were some dif-
ferences in potency between RGA and LNCaP-BC2, all compounds
in which the phenyl ring had been replaced by a pyridine ring
showed acceptable biological activity.
Concerning the in vitro ADME profile, some improvements were
observed after replacing the phenyl ring with a pyridine ring.
Although the metabolic stability of the pyridine derivatives was
slightly lower than that of the phenyl derivatives, the solubility
of the pyridine derivatives was improved. In the mouse PK screen-
ing, all of the pyridine derivatives had higher AUC values than 11a
and 11b. Among them, the AUCs for 2-pyridine derivatives 17a and
17b were tenfold higher than that of 11b. On the other hand,
4-pyridine compound 22 showed only a slight increase in AUC
value. The in vivo SV wet weights in mice for 17a and 17b showed
potent antiandrogenic activities, indicating that increased oral AUC
values might lead to improved in vivo activity. As expected, the
pyridine derivatives showed a reduction in hERG inhibitory activ-
4. Conclusion
Starting from AR pure antagonist CH4933468, which had the
problem of forming an agonist metabolite, novel thiohydantoin
derivatives were identified through two strategies: the replace-
ment of the alkylsulfonamide by a phenylsulfonamide to reduce
the production of the agonist metabolites and the replacement of
the phenyl ring by a pyridine ring to improve in vivo potency
and reduce hERG affinity. Pharmacological assays indicated that
CH5137291 (17b) was a potent AR pure antagonist which would
not produce the agonist metabolite 23. Moreover, CH5137291
completely inhibited in vivo tumor growth and PSA production
in the LNCaP-BC2 xenograft model, a bicalutamide-resistant mod-
el. In addition to that, the results of early safety studies including a
2-week mouse safety study indicated that CH5137291 was well-
tolerated. These results indicate that CH5137291 is expected to
contribute to novel therapeutic approaches against CRPC.
5. Experimental
5.1. Chemistry: instruments
Column chromatography was carried out on Merck Silicagel 60
(230–400 mesh) if not otherwise specified. Reverse phase column
chromatography was carried out on Fuji Silysia Chromatorex Pro.
No. DM2035MT. NH silica gel column chromatography was carried

H. Yoshino et al. / Bioorg. Med. Chem. 18 (2010) 8150–8157
8153
Table 2
Activities of thiohydantoin derivatives
O
NC
N
N
R2
R1
S
No.
R1
R2
RGAa
LNCaP-BC2b
Human liver MS
stabilityc
Residue (%)
PBSd
g/mL)
PAMPA
(cm/s)
In vivo
SVe
ED50
In vivo PKf
hERGg
Inhibition% at
(
l
EC5
(nM)
sIC50
(nM)
Agonist
Antagonist
IC50 (nM)
AUC
(mg/mL h)
+/ꢀ
(mg/kg)
10 lM
3
4
30
>10,000
200
200
+
ꢀ
600
10
99
100
<3
13
2.5Eꢀ05
2.5Eꢀ05
3
10
NT
7.4
17
Cl
(CH2)3SO2NH2
21h
7
CF3
3000
200
+
100
80
NT
6.9Eꢀ07
NT
NT
NT
75h
39h
58
41
SO2NH2
11a
11b
Cl
CF3
>10,000
>10,000
400
300
ꢀ
ꢀ
100
100
100
100
<3
11
1.6Eꢀ06
8.1Eꢀ06
84
63
669i
26
10
N
17a
17b
Cl
CF3
>10,000
>10,000
600
700
ꢀ
ꢀ
100
100
100
94
5
38
1.3Eꢀ06
1.4Eꢀ05
6
7
SO2NH2
684h
SO2NH2
22
CF3
>10,000
1000
ꢀ
80
97
29
1.2Eꢀ05
NT
101i
0
N
163i
52
33
SO2NH2
17c
17d
Cl
CF3
>10,000
>10,000
2000
1000
ꢀ
ꢀ
50
30
95
95
76
60
1.3Eꢀ05
1.4Eꢀ05
NT
6
167h
N
NT: not tested.
a
EC5 and IC50 values were determined by a single experiment run in duplicate.
b
c
d
e
f
IC50 values were determined by a single experiment run in triplicate, A ‘pure antagonist’ was expressed as ꢀ.
NADPH added and residue (%) were determined after 15 min..
Phosphate buffer (pH 7.4).
In vivo antiandrogenic activities on seminal vesicle (SV) wet weight in mice (n = 4 or 5).
Oral administration in mice (n = 2), dose 10 mg/kg.
Using whole cell patch-clamp technique, the values were determined by a single experiment run in duplicated.
Vehicle 5% arabic gum.
Vehicle 5% DMSO and 20% Cremophor EL.
g
h
i
Table 3
GCMS-QP5050A (EI). High resonance mass spectra (HRMS) were
recorded by a Micromass Q-Tof Ultima API mass spectrometer.
Agonist metabolite (23) of CH5137291 and its amount in vitro and in vivo in several
animal species
O
O
5.1.1. 2-Chloro-4-(4,4-dimethyl-5-oxo-2-thioxoimidazolidin-1-
yl)benzonitrile (5)
NC
F3C
N
NC
F3C
N
NH
N
N
SO2NH2
To a solution of 2-chloro-4-isothiocyanatobenzonitrile (6a)
(15.0 g, 77.1 mmol) and 2-aminoisobutyric acid ethyl ester hydro-
chloride (19.4 g, 116 mmol) in toluene (500 mL) was added Et3N
(26.9 mL, 193 mmol) at 0 °C. The mixture was then stirred at
100 °C for 30 min. After cooling to room temperature, water was
added to the reaction mixture and extracted with AcOEt. The or-
ganic layer was washed with 2 N HCl, saturated aq NaHCO3 and
brine and then dried with MgSO4. After filtering, the solvent was
distilled off at reduced pressure. The residue was recrystallized
from AcOEt/hexane (1:1) to give 5 (18.6 g, 86%) as a colorless solid.
Rf 0.34 (AcOEt/hexane = 1:1); mp: 228–229 °C, 1H NMR (400 MHz,
CDCl3) d: 7.47 (1H, dd, J = 1.8, 8.3 Hz), 7.48 (1H, br s), 7.62 (1H, d,
J = 1.8 Hz), 7.80 (1H, d, J = 8.3 Hz); HRMS calcd for C12H11ClN3OS
280.0306. Found 280.0305.
S
S
CH5137291 (17b)
agonist EC5 > 10000 nM
antagonist sIC50 = 700 nM
agonist metabolite (23)
agonist EC 5 = 1.0 nM
In vitro liver microsome concn @ 30 mina
(% of intact CH5137291)
In vivo SDPKb
AUC% of intact
Human
Mouse
Rat
Dog
Monkey
<0.001%
<0.001%
<0.001%
<0.001%
<0.001%
ND
0.023%
NT
ND
ND: not detected, NT: not tested.
Lower limit of quantification: 0.1 nM, substrate conc: 10 mM, enzyme concn:
1 mg/protein/mL. The values were determined by
a
5.1.2. 4-(3-Phenyl-4,4-dimethyl-5-oxo-2-thioxoimidazolidin-1-
yl)-2-trifluoromethylbenzonitrile (7)
a single experiment run in
duplicate.
To a solution of 4-isothiocyanato-2-trifluoromethylbenzonitrile
(6b) (678 mg, 2.97 mmol) and 2-methyl-2-phenylaminopropane-
nitrile (356 mg, 2.22 mmol) in THF (7 mL) was added Et3N
(1.07 mL, 7.77 mmol), the mixture was then refluxed for 5 h. After
cooling to room temperature, solvent was distilled off at reduced
pressure. The residue was dissolved in 1,4-dioxane (5 mL) and
6 N HCl (5 mL) and the mixture was refluxed for 1 h. After cooling
to room temperature, water was added to the reaction mixture and
extracted with CH2Cl2. The organic layer was washed with brine,
b
Lower limit of quantification: 1 nM, dose: 500 mg/kg single (mouse, rat), 10 mg/
kg single (monkey). Administration vehicle: 0.5% CMC suspension (mouse, rat), 5%
DMSO/20% Cremophore (monkey), n = 3.
out on Fuji Silysia NH-DM1020. Rf was determined with Merck Sil-
icagel 60 F254 plates. 1H NMR spectra were recorded on Bruker ARX
300, Varian Mercury300 or JEOL ECP-400. Mass spectra (MS) were
measured by a Thermo Electron LCQ Classic (ESI) or a Shimadzu

8154
H. Yoshino et al. / Bioorg. Med. Chem. 18 (2010) 8150–8157
(400 MHz, CDCl3) d: 7.51 (1H, dd, J = 1.8, 8.8 Hz), 7.48 (1H, br s),
7.64 (1H, d, J = 8.8 Hz), 7.92 (1H, d, J = 1.8 Hz), 8.60 (1H, br s).
Tumor volume
a
5.1.5. N,N-Dibenzyl-3-[3-(4-cyano-3-trifluoromethylphenyl)-
5,5-dimethyl-4-oxo-2-thioxoimidazolidin-1-
yl]benzenesulfonamide (10b)
To a solution of 9b (11.4 g, 34.0 mmol) and 3-amino-N,N-
bis(phenylmethyl) benzenesulfonamide (12.0 g, 34.0 mmol) in
THF (100 mL) was added NaH (1.63 g, 40.8 mmol) at room temper-
ature, the mixture was heated at 45 °C for 1 h. After cooling to 0 °C,
NaH (8.16 g, 204 mmol) and phenyl chlorothionoformate (17.6 g,
102 mmol) were added then stirred for 5 min. After further stirring
at room temperature for 1 h, the mixture was diluted with
tBuOMe. To saturated aq NH4Cl was slowly added the reaction
mixture at 0 °C and extracted with tBuOMe. The organic layer
was washed with brine and dried with MgSO4. After filtering, the
solvent was distilled off at reduced pressure. Purification by silica
gel column chromatography (AcOEt/hexane = 2:3) and NH silica
gel chromatography (AcOEt/hexane = 2:3) gave 10b (12.4 g, 56%)
as a brown amorphous solid. Rf 0.32 (AcOEt/hexane = 1:2); 1H
NMR (400 MHz, CDCl3) d: 1.60 (6H, s), 4.37 (4H, s) 7.00–7.10 (4H,
m), 7.18–7.30 (6H, m), 7.51–7,55 (1H, m), 7.67 (1H, t, J = 8.0 Hz),
7.82–7.86 (2H, m), 7.92–8.01 (3H, m); MS (ESI) m/z 649 ([M+H]+).
Relative PSA concentration*
b
5.1.6. N,N-Dibenzyl-3-[3-(3-chloro-4-cyanophenyl)-5,5-
dimethyl-4-oxo-2-thioxoimidazolidin-1-
yl]benzenesulfonamide (10a)
This compound was prepared from 9a using a procedure similar
to that described for 10b. Rf 0.30 (AcOEt/hexane = 1:2); 1H NMR
(400 MHz, CDCl3) d: 1.58 (6H, s), 4.37 (4H, s) 7.02–7.10 (4H, m),
7.20–7.30 (6H, m), 7.51–7,55 (1H, m), 7.64–7.70 (2H, m), 7.81–
7.86 (2H, m), 7.93 (1H, d, J = 8.1 Hz); MS (EI) m/z 615 ([M]+).
*: Relative PSA concentration (day 17 of administration) / PSA
concentration (day 0 of administration)
Figure 1. Antitumor activity in LNCaP-BC2 mouse xenografts. LNCaP-BC2 was
subcutaneously inoculated into castrated 6-week-old male SCID mice. When the
tumor size reached 90–400 mm3, the mice were randomized into groups and orally
administered either a test compound or a vehicle (5% gum arabic) in seven cycles of
5 days on, 2 days off. (n = 5 animals per group). After 17 days, the tumor size and
plasma PSA level were measured. *Relative PSA concentration (day 17 of admin-
istration)/PSA concentration (day 0 of administration).
5.1.7. 3-[3-(4-Cyano-3-trifluoromethylphenyl)-5,5-dimethyl-4-
oxo-2-thioxoimidazolidin-1-yl]benzenesulfonamide (11b)
Compound 10b (12.4 g, 19.1 mmol) was dissolved in concd
H2SO4 at 0 °C, the mixture was stirred at room temperature for
15 min. After cooling to 0 °C, iced water was added and extracted
with AcOEt. The organic layer was washed with saturated aq NaH-
CO3 and brine, and then dried with MgSO4. After filtering, the sol-
vent was distilled off at reduced pressure. Purification by silica gel
column chromatography (AcOEt/hexane = 2:1) gave 11a (7.53 g,
84%) as a colorless solid. Rf 0.34 (AcOEt/hexane = 2:1); 1H NMR
(400 MHz, CDCl3) d: 1.60 (6H, s), 4.94 (2H, s), 7.53–7.57 (1H, m),
7.74 (1H, t, J = 8.0 Hz), 7.83 (1H, dd, J = 1.8, 8.0 Hz), 7.90–7.93
(1H, m), 7.96 (1H, d, J = 1.8 Hz), 8.00 (1H, d, J = 8.0 Hz), 8.06–8.10
then dried with MgSO4. After filtering, the solvent was distilled off
at reduced pressure. Purification by silica gel column chromatogra-
phy (AcOEt/hexane = 1:2) gave 7 (56 mg, 6.5%). 1H NMR (400 MHz,
CDCl3) d: 1.60 (6H, s), 7.29–7.33 (2H, m), 7.52–7.59 (3H, m), 7.83–
7.88 (1H, m), 7.96–8.00 (2H, m); HRMS calcd for C19H15F3N3OS
390.0882. Found 390.0882.
(1H, m); HRMS calcd for
469.0608.
C19H16F3N4O3S2 469.0610. Found
5.1.3. 2-Bromo-N-(4-cyano-3-trifluoromethylphenyl)-2-
methylpropionamide (9b)
To a solution of 4-amino-2-trifluoromethylbenzonitrile (8b)
(10.0 g, 53.7 mmol) in CH2Cl2 (110 mL) were added iPr2NEt
(10.3 mL, 59.1 mmol) and 2-bromobutyryl bromide (7.24 mL,
59.1 mmol)at 0 °C and the mixturewas stirredat roomtemperature.
After stirring for 2 h, 2 N HCl was added at 0 °C and the reaction mix-
ture was extracted with CH2Cl2. The organic layer was washed with
brine and dried with MgSO4. After filtering, the solvent was distilled
off at reduced pressure. The residue was recrystallized from AcOEt/
hexane (1:3) to give 9b (18.0 g, quant). Rf 0.55 (AcOEt/hexane = 1:2);
mp: 126–127 °C, 1H NMR (400 MHz, CDCl3) d: 7.82 (1H, d, J = 8.4 Hz),
7.92 (1H, d, J = 2.4 Hz), 8.06 (1H, s), 8.76 (1H, br s).
5.1.8. 3-[3-(3-Chloro-4-cyanophenyl)-5,5-dimethyl-4-oxo-2-
thioxoimidazolidin-1-yl]benzenesulfonamide (11a)
This compound was prepared from 10a using a procedure sim-
ilar to that described for 11b. Yield 79%. Rf 0.44 (AcOEt/hex-
ane = 2:1); 1H NMR (400 MHz, CDCl3) d: 1.60 (6H, s), 4.95 (2H, s),
7.49–7.56 (2H, m), 7.68 (1H, d, J = 1.8 Hz), 7.73 (1H, t, J = 8.0 Hz),
7.82 (1H, d, J = 8.4 Hz), 7.89–7.92 (1H, m), 8.07 (1H, d, 8.0 Hz);
HRMS calcd for C18H16ClN4O3S2 435.0347. Found 435.0348.
5.1.9. 6-Bromopyridine-2-sulfonic acid dibenzylamide (13a)
1 M iPrMgCl in THF (15 mL, 15.0 mmol) was added dropwise to
2,6-dibromopyridine (12a) (2.36 g, 9.96 mmol) and the mixture
was stirred at room temperature for 90 min, To a solution of SO2Cl2
(1.60 mL, 19.9 mmol) in hexane (75 mL) was slowly added the
above mixture at 0 °C. After stirring for 10 min, the reaction mix-
ture was distilled off at reduced pressure and the residue was di-
5.1.4. 2-Bromo-N-(3-chloro-4-cyanophenyl)-2-
methylpropionamide (9a)
This compound was prepared from 4-amino-2-chlorobenzoni-
trile (8a) using a procedure similar to that described for 9b. Yield
73%. Rf 0.43 (AcOEt/hexane = 1:3); mp: 135–136 °C, 1H NMR

H. Yoshino et al. / Bioorg. Med. Chem. 18 (2010) 8150–8157
8155
luted with heptane. The mixture was distilled off at reduced pres-
sure and the residue was dissolved in CH2Cl2 (35 mL). To the above
solution were added dibenzylamine (1.90 mL, 9.92 mmol) and Et3N
(2.5 mL, 17.9 mmol) at 0 °C. After stirring at room temperature for
1 h, water was added and extracted with CH2Cl2. The organic layer
was washed with brine and dried with MgSO4. After filtering, the
solvent was distilled off at reduced pressure. Purification by silica
gel column chromatography (AcOEt/hexane = 1:4) gave 13a
(2.17 g, 52%) as a brown oil. Rf 0.31 (AcOEt/hexane = 1:4); 1H
NMR (400 MHz, CDCl3) d: 4.50 (4H, s), 7.18–7.26 (10H, m), 7.58
(1H, d, J = 8.1 Hz), 7.65 (1H, t, J = 7.3 Hz), 7.85 (1H, d, J = 7.3 Hz);
MS (ESI) m/z 417, 419 ([M+H]+).
ane = 1:2); 1H NMR (400 MHz, CDCl3) d: 1.51 (6H, s), 3.64 (3H, s),
4.45 (4H, s), 5.00 (1H, s), 6.54 (1H, d, J = 8.4 Hz), 7.07–7.10 (4H,
m), 7.18–7.21 (6H, m), 7.33 (1H, d, J = 7.0 Hz), 7.52 (1H, t,
J = 7.9 Hz); MS (ESI) m/z 476 ([M+Na]+).
5.1.14. 2-(5-Dibenzylsulfamoylpyridin-3-ylamino)-2-
methylpropionic acid methyl ester (15b)
This compound was prepared from 14b using a procedure sim-
ilar to that described for 15a. Yield 14%. 1H NMR (400 MHz, CDCl3)
d: 1.58 (6H, s), 3.69 (3H, s), 4.33 (4H, s), 7.00–7.30 (11H, m), 8.11
(1H, d, J = 1.8 Hz), 8.39 (1H, d, J = 1.8 Hz); MS (ESI) m/z 454
([M+H]+).
5.1.10. 5-Bromopyridine-3-sulfonic acid dibenzylamide (13b)
This compound was prepared from 3,5-dibromopyridine (12b)
using a procedure similar to that described for 13a. Yield 43%. 1H
NMR (400 MHz, CDCl3) d: 4.41 (4H, s), 7.10–7.36 (10H, m), 7.97
(1H, s), 8.78 (1H, d, J = 1.8 Hz), 8.86 (1H, d, J = 1.8 Hz); MS (ESI)
m/z 417, 419 ([M+H]+).
5.1.15. 6-[3-(3-Chloro-4-cyanophenyl)-5,5-dimethyl-4-oxo-2-
thioxoimidazolidin-1-yl]pyridine-2-sulfonic acid
dibenzylamide (16a)
To a solution of 15a (63.0 mg, 0.139 mmol) and 6a (135 mg,
0.691 mmol) in toluene (0.3 mL) was added 4-dimethylaminopyr-
idine (DMAP) (43.0 mg, 0.352 mmol) and the mixture was stirred
at 100 °C for 16 h. After cooling to room temperature, the solvent
was distilled off at reduced pressure. Purification by silica gel col-
umn chromatography (AcOEt/hexane = 1:2) gave 16a (22.5 mg,
26%). 1H NMR (400 MHz, CDCl3) d: 1.70 (6H, s), 4.51 (4H, s),
7.06–7.14 (4H, m), 7.22–7.30 (6H, m), 7.49 (1H, dd, J = 1.8,
8.1 Hz), 7.66 (1H, d, J = 1.8 Hz), 7.85 (1H, d, J = 8.1 Hz), 7.91–8.00
(2H, m), 8.09 (1H, d, J = 6.6 Hz); MS (ESI) m/z: 616 ([M+H]+).
5.1.11. 6-Aminopyridine-2-sulfonic acid dibenzylamide (14a)
To a solution of 13a (1.05 g, 2.53 mmol) in DMI (5 mL) were
added p-methoxybenzylamine (0.490 mL, 3.79 mmol) and iPr2NEt
(0.878 mL, 5.04 mmol) and the mixture was rapidly heated by
microwave irradiation at 100 °C for 30 min. After cooling to room
temperature, 0.5 N HCl was added and extracted with AcOEt. The
organic layer was washed with brine and dried with MgSO4. After
filtering, the solvent was distilled off at reduced pressure. The res-
idue was dissolved in TFA (5 mL) and the mixture was heated by
microwave irradiation at 100 °C for 10 min. After cooling to room
temperature, the solvent was distilled off at reduced pressure. To
the residue was added saturated aq NaHCO3 and extracted with
CH2Cl2. The organic layer was washed with brine and dried with
MgSO4. After filtering, the solvent was distilled off at reduced pres-
sure. Purification by silica gel column chromatography (AcOEt/
hexane = 1:2) gave 14a (551 mg, 62%). Rf 0.26 (AcOEt/hex-
ane = 1:2); 1H NMR (400 MHz, CDCl3) d: 4.44 (4H, s), 4.65 (2H, br
s), 6.62 (1H, d, J = 8.1 Hz), 7.13–7.15 (4H, m), 7.20–7.24 (6H, m),
7.33 (1H, d, J = 7.3 Hz), 7.56 (1H, t, J = 7.9 Hz); MS (ESI) m/z 354
([M+H]+).
5.1.16. 6-[3-(4-Cyano-3-trifluoromethylphenyl)-5,5-dimethyl-4-
oxo-2-thioxoimidazolidin-1-yl]pyridine-2-sulfonic acid
dibenzylamide (16b)
This compound was prepared from 15a and 6b using a proce-
dure similar to that described for 16a. Yield 21%. 1H NMR
(400 MHz, CDCl3) d: 1.71 (6H, s), 4.51 (4H, s), 7.07–7.13 (4H, m),
7.20–7.30 (6H, m), 7.81 (1H, dd, J = 1.8, 8.1 Hz), 7.93–8.07 (4H,
m), 8.08 (1H, d, J = 7.6 Hz); MS (ESI) m/z: 650 ([M+H]+).
5.1.17. 5-[3-(3-Chloro-4-cyanophenyl)-5,5-dimethyl-4-oxo-2-
thioxoimidazolidin-1-yl]pyridine-3-sulfonic acid
dibenzylamide (16c)
This compound was prepared from 15b and 6a using a proce-
dure similar to that described for 16a. Yield 40%. 1H NMR
(400 MHz, CDCl3) d: 1.58 (6H, s), 4.41 (4H, s), 7.07–7.13 (4H, m),
7.20–7.30 (6H, m), 7.51 (1H, dd, J = 1.8, 8.3 Hz), 7.68 (1H, d,
J = 1.8 Hz), 7.84 (1H, d, J = 8.3 Hz), 8.05 (1H, t, J = 2.2 Hz), 8.72
(1H, d, J = 2.2 Hz), 9.03 (1H, d, J = 2.2 Hz); MS (ESI) m/z: 616
([M+H]+).
5.1.12. 5-Aminopyridine-3-sulfonic acid dibenzylamide (14b)
This compound was prepared from 13b using a procedure sim-
ilar to that described for 14a. Yield 34%. 1H NMR (400 MHz, CDCl3)
d: 4.35 (4H, s), 7.08–7.30 (11H, m), 8.21 (1H, d, J = 1.8 Hz), 8.40 (1H,
d, J = 1.8 Hz); MS (ESI) m/z 354 ([M+H]+).
5.1.13. 2-(6-Dibenzylsulfamoylpyridin-2-ylamino)-2-
methylpropionic acid methyl ester (15a)
5.1.18. 5-[3-(4-Cyano-3-trifluoromethylphenyl)-5,5-dimethyl-4-
oxo-2-thioxoimidazolidin-1-yl]pyridine-3-sulfonic acid
dibenzylamide (16d)
This compound was prepared from 15b and 6b using a proce-
dure similar to that described for 16a. Yield 73%. 1H NMR
(400 MHz, CDCl3) d: 1.60 (6H, s), 4.42 (4H, s), 7.07–7.13 (4H, m),
7.20–7.30 (6H, m), 7.83 (1H, dd, J = 1.8, 8.4 Hz), 7.96 (1H, d,
J = 1.8 Hz), 8.01 (1H, d, J = 8.4 Hz), 8.06 (1H, t, J = 2.2 Hz), 8.73
(1H, d, J = 2.2 Hz), 9.04 (1H, d, J = 2.2 Hz); MS (ESI) m/z: 650
([M+H]+).
To a crude solution of 14a (550 mg, 1.56 mmol) and iPr2NEt
(5.40 mL, 31.0 mmol) in 1,4-dioxane (5 mL) was added a solution
of 2-bromo-2-methylpropionic acid (2.60 g, 15.6 mmol) in 1,4-
dioxane (1 mL) and the mixture was stirred at 90 °C for 13 h. After
cooling to 0 °C, 2 N NaOH (10 mL) and MeOH (20 mL) were added
and the mixture was heated at 70 °C for 30 min. After cooling to
room temperature, the solvent was distilled off at reduced pres-
sure. The residue was acidified (pH 5) with 5 N HCl and the mixture
was extracted with AcOEt. The organic layer was washed with
brine and dried with MgSO4. After filtering, the solvent was dis-
tilled off at reduced pressure and the residue was dissolved in tol-
uene (2 mL) and MeOH (2 mL). To the above solution was added a
2 M solution of (trimethylsilyl)diazomethane in hexane (1.0 mL,
2.0 mmol) and the mixture was stirred at room temperature for
30 min. After distilling off the solvent, the residue was purified
by silica gel column chromatography (AcOEt/hexane = 1:2) to give
15a (328 mg, 46%) as a colorless solid. Rf 0.26 (AcOEt/hex-
5.1.19. 6-[3-(3-Chloro-4-cyanophenyl)-5,5-dimethyl-4-oxo-2-
thioxoimidazolidin-1-yl]pyridine-2-sulfonic acid amide (17a)
This compound was prepared from 16a using a procedure sim-
ilar to that described for 11a. Yield 65%. 1H NMR (400 MHz, DMSO-
d6) d: 1.69 (6H, s), 7.62 (2H, br s), 7.77 (1H, dd, J = 1.5, 8.4 Hz), 7.97
(1H, d, J = 7.7 Hz), 8.08 (1H, d, J = 1.5 Hz), 8.11 (1H, d, J = 8.0 Hz),
8.21 (1H, d, J = 8.4 Hz), 8.26 (1H, dd, J = 7.7, 8.0 Hz); HRMS calcd
for C17H15ClN5O3S2 436.0299. Found 436.0300.

8156
H. Yoshino et al. / Bioorg. Med. Chem. 18 (2010) 8150–8157
5.1.20. 6-[3-(4-Cyano-3-trifluoromethylphenyl)-5,5-dimethyl-4-
oxo-2-thioxoimidazolidin-1-yl]pyridine-2-sulfonic acid amide
(17b)
This compound was prepared from 16b using a procedure sim-
ilar to that described for 11a. Yield 36%. 1H NMR (400 MHz, DMSO-
d6) d: 1.72 (6H, s), 7.64 (2H, br s), 7.99 (1H, d, J = 7.7 Hz), 8.10–8.18
(2H, m), 8.27 (1H, dd, J = 7.7, 8.0 Hz), 8.37 (1H, s), 8.43 (1H, d,
(1 mL) was added DMAP (34.0 mg, 0.279 mmol) and the mixture
stirred at 80 °C for 1 day. After further addition of 4-isothiocyanat-
o-2-trifluoromethylbenzonitrile (127 mg, 0.558 mmol), the mix-
ture was stirred at 80 °C for 1 day. After cooling to room
temperature, purification by silica gel column chromatography
(AcOEt/hexane = 3:2)
gave
4-{3-[2-(4-methoxybenzylsulfa-
nyl)pyridin-4-yl]-4,4-dimethyl-5-oxo-2-thioxoimidazolidin-1-yl}-
2-trifluoromethylbenzonitrile (127 mg, 50%). The above obtained
compound (127 mg, 0.235 mmol) was dissolved in TFA (2.0 mL)
and the mixture heated by microwave irradiation at 120 °C for
30 min. After cooling to room temperature, the solvent was dis-
tilled off at reduced pressure. Purification by reverse phase chro-
matography (H2O/MeCN/0.1% TFA = 90:10:1–50:50:1) gave 21
(48.1 mg, 49%). 1H NMR (300 MHz, CDCl3) d: 1.67 (6H, s), 5.30
(1H, s), 6.80 (1H, dd, J = 2.0, 6.9 Hz), 7.49 (1H, d, J = 2.0 Hz), 7.65
(1H, d, J = 6.9 Hz), 7.80 (1H, dd, J = 2.1, 8.4 Hz), 7.92 (1H, d,
J = 2.1 Hz), 8.00 (1H, d, J = 8.4 Hz); MS (ESI) m/z: 423 ([M+H]+).
J = 8.4 Hz); HRMS calcd for
C18H15F3N5O3S2 470.0563. Found
470.0562.
5.1.21. 5-[3-(3-Chloro-4-cyanophenyl)-5,5-dimethyl-4-oxo-2-
thioxoimidazolidin-1-yl]pyridine-3-sulfonic acid amide (17c)
This compound was prepared from 16c using a procedure sim-
ilar to that described for 11a. Yield 24%. 1H NMR (400 MHz, CDCl3)
d: 1.62 (6H, s), 5.15 (2H, br s), 7.52 (1H, d, J = 8.4 Hz), 7.67 (1H, d,
J = 1.8 Hz), 7.83 (1H, d, J = 8.4 Hz), 8.19 (1H, d, J = 1.8 Hz), 8.76
(1H, d, J = 2.2 Hz), 9.24 (1H, d, J = 1.8 Hz); HRMS calcd for
C17H15ClN5O3S2 436.0299. Found 436.0298.
5.1.26. 4-[3-(4-Cyano-3-trifluoromethylphenyl)-5,5-dimethyl-4-
oxo-2-thioxoimidazolidin-1-yl]pyridine-2-sulfonic acid amide
(22)
5.1.22. 5-[3-(4-Cyano-3-trifluoromethylphenyl)-5,5-dimethyl-4-
oxo-2-thioxoimidazolidin-1-yl]pyridine-3-sulfonic acid amide
(17d)
This compound was prepared from 16d using a procedure sim-
ilar to that described for 11a. Yield 52%. 1H NMR (400 MHz, CDCl3)
d: 1.64 (6H, s), 5.19 (2H, br s), 7.82 (1H, d, J = 8.4 Hz), 7.95 (1H, br
s), 8.01 (1H, d, J = 8.4 Hz), 8.20 (1H, t, J = 1.8 Hz), 8.78 (1H, d,
J = 1.8 Hz), 9.25 (1H, d, J = 1.8 Hz); HRMS calcd for C18H15F3N5O3S2
470.0563. Found 470.0556.
To a solution of 21 (31.0 mg, 0.0734 mmol) and KNO3 (14.8 mg,
0.147 mmol) in MeCN (1.0 mL) was added SO2Cl2 (0.0118 mL,
0.147 mmol) at 0 °C, After stirring at 0 °C for 30 min, concd NH3
was added. After stirring at room temperature for 30 min, water
was added and extracted with AcOEt. The organic layer was
washed with brine and dried with MgSO4. After filtering, the sol-
vent was distilled off at reduced pressure. Purification by reverse
phase chromatography (H2O/MeCN = 80:20–55:45) gave 22
(6.1 mg, 18%). 1H NMR (300 MHz, CDCl3) d: 1.64 (6H, s), 5.30 (2H,
br s), 7.52 (1H, dd, J = 2.0, 5.2 Hz), 7.80 (1H, dd, J = 2.1, 8.2 Hz),
7.92 (1H, d, J = 2.1 Hz), 7.99 (1H, d, J = 8.2 Hz), 8.03 (1H, d,
J = 2.0 Hz), 8.88 (1H, d, J = 5.2 Hz); HRMS calcd for C18H15F3N5O3S2
470.0563. Found 470.0558.
5.1.23. 2-(4-Methoxybenzylsulfanyl)pyridin-4-ylamine (19)
To
a
solution of 2-chloropyridin-4-ylamine (18) (1.28 g,
-toluenethiol
9.96 mmol) in DMI (8 mL) were added 4-methoxy-
a
(1.67 mL, 12.0 mmol) and tBuOK (2.46 g, 21.9 mmol) and the mixture
washeated bymicrowaveirradiationat120 °Cfor2 h. Aftercoolingto
room temperature, water was added and extracted with tBuOMe. The
organic layer was washed with brine and dried with MgSO4. After fil-
tering, the solvent was distilled off at reduced pressure. Purification
by reverse phase chromatography (H2O/MeCN/0.1% TFA = 90:10:1–
50:50:1) gave 19 (583 mg, 24%). MS (ESI) m/z 247 ([M+H]+).
5.2. Biology
5.2.1. Reporter gene assay
Hela cells were co-transfected with MMTV-Luc-Hyg and pSG5-
hAR-neo using a FuGENE™ 6 transfection reagent. The transfected
5.1.24. 2-[2-(4-Methoxybenzylsulfanyl)pyridin-4-ylamino]-2-
methylpropionic acid methyl ester (20)
cells were selected in DMEM containing 500
300 g/mL hygromycin and 10% FBS and the cloned 11A11B2 cells
were maintained in DMEM containing 400 g/mL neomycin,
200 g/mL hygromycin and 10% FBS. Pre-starved 11A11B2 cells
lg/mL neomycin,
l
To a solution of 19 (103 mg, 0.415 mmol) and 2-bromo-2-meth-
ylpropionic acid (76.3 mg, 0.457 mmol) in 1,4-dioxane (2 mL) was
added iPr2NEt (0.0796 mL, 0.457 mmol), the mixture was stirred at
60 °C for 5 h. After further addition of iPr2NEt (0.159 mL,
0.914 mmol) and 2-bromo-2-methylpropionic acid (153 mg,
0.914 mmol), the mixture was stirred at 80 °C for 2 h. To the reac-
tion mixture was added 2 N NaOH in MeOH (3 mL) and the mixture
was heated at 80 °C for 15 min. After cooling to room temperature,
the solvent was distilled off at reduced pressure and the residue
was dissolved in THF (2 mL). To the above solution was added
diazomethane in Et2O solution (prepared from N-methyl-N0-ni-
tro-N-nitrosoguanidine and 6 N NaOH) until the yellow color did
not disappear. To the mixture was added acetic acid until the yel-
low solution disappeared. The solvent was distilled off at reduced
pressure. Purification by silica gel column chromatography
(AcOEt/hexane = 2:3) gave 20 (96.6 mg, 67%). 1H NMR (300 MHz,
CDCl3) d: 1.55 (6H, s), 3.71 (3H, s), 3.78 (3H, s), 4.32 (2H, s), 4.53
(1H, s), 6.14 (1H, dd, J = 2.4, 5.8 Hz), 6.24 (1H, d, J = 2.4 Hz), 6.82
(2H, d, J = 8.7 Hz), 7.30 (2H, d, J = 8.7 Hz), 8.05 (1H, d, J = 5.8 Hz).
l
l
were plated onto 96-well plates at 1 ꢁ 104 cells/well in phenol
red-free DMEM containing 3% DCC-FCS. Following overnight
attachment, the cells were treated with 1, 10, 100, 1000 or
10,000 nmol/L of test compound in the absence or presence of
DHT (0.1 nmol/L) for 48 h. The luciferase activity of each sample
was measured using a Bright-Glo™ luciferase assay system.
5.2.2. In vitro LNCaP-BC2 cell growth assay
Pre-starved LNCaP-BC2 cells were plated onto 96-well plates in
phenol red-free RPMI 1640 containing 5% DCC-FBS. Following
overnight attachment, the cells were treated with the test com-
pound in the absence or presence of 0.01 nM R1881 for 6 days. Cell
proliferation was determined using a CellTiter 96Ò AQueous One
Solution Cell Proliferation Assay kit (Promega).
5.2.3. In vivo antitumor activities on LNCaP-BC2 xenograft in
mice
LNCaP-BC2 (2 ꢁ 106 cells) was subcutaneously inoculated into
castrated 6-week-old male SCID mice (CLEA Japan). When the tu-
mor size reached 90–400 mm3, the animals were randomized
and orally administered an agent or the agent vehicle (5% gum ara-
bic) at 10 mL/kg body weight. The agents were administered once a
5.1.25. 4-[3-(2-Mercaptopyridin-4-yl)-4,4-dimethyl-5-oxo-2-
thioxoimidazolidin-1-yl]-2-trifluoromethylbenzonitrile (21)
To a solution of 20 (96.6 mg, 0.279 mmol) and 4-isothiocyanat-
o-2-trifluoromethylbenzonitrile (127 mg, 0.558 mmol) in toluene

H. Yoshino et al. / Bioorg. Med. Chem. 18 (2010) 8150–8157
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Acknowledgments
The authors thank Ms. Hitomi Suda of Chugai Pharmaceutical
Co., Ltd for HRMS measurements of the compounds, and Editing
Services of Chugai Pharmaceutical Co., Ltd for assistance with
English usage.
13. To be published.
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