Structure-activity relationships of bioisosteric replacement of the carboxylic acid in novel androgen receptor pure antagonists

Article abstract of DOI:10.1016/j.bmc.2010.03.036

A series of 5,5-dimethylthiohydantoin derivatives were synthesized and evaluated for androgen receptor pure antagonistic activities for the treatment of hormone refractory prostate cancer. CH4933468 (32d) with a sulfonamide side chain not only exhibited antagonistic activity with no agonistic activity in the reporter gene assay but also inhibited the growth of bicalutamide-resistant cell lines. This compound also inhibited tumor growth of the LNCaP xenograft in mice dose-dependently.

Full text of DOI:10.1016/j.bmc.2010.03.036

Bioorganic & Medicinal Chemistry 18 (2010) 3159–3168
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Structure–activity relationships of bioisosteric replacement of the
carboxylic acid in novel androgen receptor pure antagonists
a,
a
a
a
Hitoshi Yoshino a, , Haruhiko Sato , Kazutaka Tachibana , Takuya Shiraishi , Mitsuaki Nakamura ,
Masateru Ohta a, Nobuyuki Ishikura a, Masahiro Nagamuta a, Etsuro Onuma a, Toshito Nakagawa a,
Shinichi Arai a, Koo-Hyeon Ahn b, Kyung-Yun Jung b, Hiromitsu Kawata a
*
*
a Research Division, Chugai Pharmaceutical Co., Ltd, 1-135, Komakado, Gotemba, Shizuoka 412-8513, Japan
b C&C Research Laboratories, 146-141, Annyung-dong, Hwasung-city, Kyunggi-do, 445-976, Republic of Korea
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 hormone refractory prostate cancer. CH4933468 (32d)
with a sulfonamide side chain not only exhibited antagonistic activity with no agonistic activity in the
reporter gene assay but also inhibited the growth of bicalutamide-resistant cell lines. This compound also
inhibited tumor growth of the LNCaP xenograft in mice dose-dependently.
Received 8 February 2010
Revised 13 March 2010
Accepted 16 March 2010
Available online 19 March 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Androgen receptor
Pure antagonist
Prostate cancer
Bioisostere
Sulfonamide
1. Introduction
chains to a nonsteroidal known androgen ligand, RU56187 (4),
which have been reported to have binding affinity for AR. As ex-
Prostate cancer is the most common cancer amongst men in
the USA and the second most common malignant cause of male
death worldwide after lung cancer.1 Since the growth of prostate
cancer is dependent on androgen, androgen receptor (AR)
antagonists such as flutamide (1) (a precursor of its active form
hydroxyflutamide (2)) and bicalutamide (3) (Chart 1) are
clinically used to treat prostate cancer.2 These antiandrogens
exhibit good efficacy in many cases and comprise an important
part of effective therapeutics.3–6 However, a considerable prob-
lem with these antiandrogens is that recurrence occurs after a
short period of response.7 Hydroxyflutamide and bicalutamide
have partial agonistic activities at high concentrations in vitro,8
which may contribute to recurrence. Therefore, research into
new antiandrogenic agents that exhibit no agonistic activities,
so-called ‘AR pure antagonists’, has been conducted.9–12
pected, we recently reported a thiohydantoin derivative (5) with
a carboxyl terminal side chain that showed pure antagonistic activ-
ities in vitro and oral AR antagonistic activity in vivo.15
In further experiments, however, the carboxylic acid derivatives
exhibited weak cell growth inhibition in T877A mutation AR cell
line LNCaP,
cancer cell line. Moreover, compound 5 also showed weak antago-
nistic activity in LNCaP-BC2, bicalutamide-resistant and
androgen-hypersensitive prostate cell line established in our
a common androgen-dependent human prostate
a
OH H
H
R
H
O
S
O2N
F3C
flutamide (1) R=H
N
NC
F3C
N
F
O
O
O
Estrogen pure antagonists have been reported to have a side
chain which inhibits the folding of helix 12 of the estrogen receptor
(ER) ligand binding domain (LBD).13,14 Since AR and ER belong to
the nuclear receptor superfamily and the tertiary structure of the
AR LBD is similar to that of ER LBD, we thought that the same
strategy would be effective.9,10 Therefore, we introduced side
bicalutamide (3)
hydroxyflutamide (2) R=OH
O
O
NC
F3C
N
NC
F3C
N
N
N
CO2H
S
S
5
RU56187 (4)
Chart 1. Structure of AR antagonists.
* Corresponding authors. Tel.: +81 550 87 8638; fax: +81 550 87 5326 (H.Y.).
E-mail address: yoshinohts@chugai-pharm.co.jp (H. Yoshino).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.03.036

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H. Yoshino et al. / Bioorg. Med. Chem. 18 (2010) 3159–3168
group.16 Because these cell lines reflect clinical hormone refractory
prostate cancer, it is necessary to identify potent and orally active
AR pure antagonists not only in wild type RGA but also in AR
mutant cell lines. In this paper, we describe two strategies to in-
crease antiandrogenic activities. One is bioisosteric replacement
of the carboxylic acid side chain to find functional groups that
would effectively inhibit the folding of helix 12 of the AR LBD.
The other is optimization of the substituent groups on the phenyl
ring to increase AR affinity.
introduction of the methyl group of compound 28 by treatment
with LDA and MeI gave 29a and 29b at a ratio of 2 to 1. Further
replacement of the chloride group by nitrile followed by treatment
with 6 N HCl afforded 30a and 30b, respectively. Isothiocyanates
were synthesized by treatment of the corresponding anilines with
thiophosgene. Target compounds 32a–f were prepared by coupling
the isothiocyanates with amine 17a followed by acidic hydrolysis
of the N,N-dimethylimino group.
3. Biological assays
2. Chemistry
The synthesized compounds were evaluated for their in vitro
binding affinities and agonist/antagonist activities to AR using
the same procedures as previously reported.15 The binding affinity
to AR was determined by displacement of [3H]-mibolerone with
the test compound utilizing CHO-K1/hAR cells. The reporter gene
assay initially used a transient assay system until a stable assay
system using hAR-transfected HeLa cells was utilized. The 0.1 nM
DHT-induced transcriptional activity was set at 100%. A ‘pure
antagonist’ was defined as having a 5% effective concentration
(EC5) value greater than 10,000 nM. To estimate the potential
antagonistic activity, the transcriptional activity value determined
by the agonistic assay was subtracted from that of the antagonistic
activity. The resulting value was termed the subtracted antagonis-
tic activity (sIC50). The sIC50 value was determined from the
dose–response curve of the subtracted antagonistic activity. Cell
growth was determined in LNCaP cells and bicalutamide-resistant
LNCaP-BC2 cells. Agonistic activity was expressed as + or ꢀ accord-
ing to the dose–response curve of growth. A ‘pure antagonist’ was
expressed as ꢀ. Bicalutamide showed pure antagonistic activity in
LNCaP cells but clearly exhibited agonistic activity in LNCaP-BC2
cells.
Some of the requisite analogs were prepared according to the
synthetic sequence outlined in Scheme 1. Carboxylic acid 5 was
treated with ClCO2Et in the presence of Et3N followed by ammonia
to give the corresponding amide 6. Multistep reactions of
compound 7 afforded amine 8. Compound 8 was treated with
TMS isocyanate to afford urea 9. Compound 8 was also treated with
chlorosulfonyl isocyanate and tert-BuOH followed by TFA to afford
sulfamide 10. Tetrazole 13 was synthesized through multistep
reactions starting from material 11. Protection of tetrazole with
p-methoxybenzyl (PMB) group gave regioisomers of 12a and 12b
as a mixture. This mixture was treated with 4-CN-3-CF3-PhNCS
followed by deprotection of PMB group to give tetrazole 13.
Sulfonic acid derivative 15 was synthesized from compound 14.
Sulfonamide derivatives were synthesized according to the
synthetic sequence shown in Scheme 2. In the case of compounds
16a and 16b, the sulfonamide group of compound 17 was pro-
tected with N,N-(dimethylimino)methylene group. After cycliza-
tion with 4-CN-3-CF3-PhNCS, deprotection of sulfonamide group
with 6 N HCl afforded corresponding sulfonamide 18. In the case
of compound 21, PMB group was used to protect the sulfonamide
group. Monomethyl sulfonamide 24 was protected with the Boc
group, but dimethyl sulfonamide 27 was directly given from
treatment with isothiocyanate.
4. Results and discussion
Further optimization of the substituent groups on the phenyl
rings of sulfonamide derivatives is shown in Scheme 3. The
In vitro activities of the thiohydantoin derivatives with various
terminal side chains are shown in Table 1. Although compound 5
O
O
a
NC
F3C
N
NC
F3C
N
N
CO2H
N
CONH2
S
O
S
5
6
NC
F3C
N
e
O
N
N
NHCONH2
NHSO2NH2
O
b, c, d
S
NC
F3C
N
N
NH2
9
Cl
N
f, g
O
S
8
7
O
NC
F3C
N
S
PMB
10
N
O
O
N
N
NC
N
H
H
c, k, l
h, i, d, j,
N
N
12a
N
O
CN
N
N
NC
F3C
N
N
N
N
N
S
11
NC
N
13
H
N
N
PMB
12b
O
m, c, n
O
Cl
S
NC
F3C
N
N
SO3H
O
O
S
15
14
Scheme 1. Reagents and conditions: (a) ClCO2Et, Et3N, THF, 0 °C then NH4OH, rt; (b) aminoisobutyric acid methyl ester, K2CO3, NaI, DMF, 80 °C; (c) 4-CN-3-CF3-PhNCS, Et3N,
THF, rt; (d) NH2NH2/H2O, CH2Cl2, EtOH, rt; (e) trimethylsilyl isocyanate, CH2Cl2, rt; (f) chlorosulphonyl isocyanate, tert-BuOH, CH2Cl2; (g) TFA, CH2Cl2, rt; (h) NaN3, AlCl3, 1,4-
dioxane, reflux; (i) p-methoxybenzylalcohol, DEAD, PPh3, CH2Cl2, rt; (j) acetone cyanohydrin, Et3N, MeOH, 50 °C; (k) 2 N HCl, MeOH, 60 °C; (l) TFA, 80 °C; (m) aminoisobutyric
acid methyl ester, K2CO3, n-Bu4NI, DMF, MeCN, reflux; (n) Me4NCl, DMF, reflux.

H. Yoshino et al. / Bioorg. Med. Chem. 18 (2010) 3159–3168
3161
O
c, d
(CH )nSO2N=CHNMe
a, b
2
2
NC
F3C
N
RO2C
N
SO2NH2
Cl(CH )n
2
N
H
(CH )nSO2NH2
2
S
17a (n=3, R=Et)
17b (n=4, R=Me)
16a (n=3)
16b (n=4)
18a (n=3)
18b(n=4)
O
O
N
O
c, d, h
SO2Cl e, f, g
NC
F3C
N
SO2N(PMB)2
N
O
NC
N
SO2NH2
H
S
19
20
21
i, j
c, k
NC
F3C
N
N
Cl
SO2NHMe
SO2NHMe
EtO2C
N
SO2NMe(Boc)
H
S
22
23
24
O
b
c
NC
F3C
N
Cl
SO2NMe2
N
SO2NMe2
MeO2C
N
SO2NMe2
H
25
S
26
27
Scheme 2. Reagents and conditions: (a) DMF dimethylacetal, DMF, rt; (b) 2-aminoisobutyric acid methyl or ethyl ester, K2CO3, NaI, DMF, 80 °C; (c) 4-CN-3-CF3-PhNCS, Et3N,
THF, rt; (d) 6 N HCl, 1,4-dioxane, reflux; (e) PMB2NH, Et3N, CH2Cl2, rt; (f) NH2NH2/H2O, EtOH, rt; (g) acetone cyanohydrin, Et3N, MeOH, 50 °C; (h) TFA, anisole, reflux; (i) Boc2O,
DMAP, CH3CN, rt; (j) 2-aminoisobutyric acid ethyl ester, K2CO3, NaI, DMF, MeCN, 80 °C; (k) TFA, CH2Cl2, rt.
though all of the compounds which had between two to four
H
N
b, c
Cl
methylene groups in the sulfonamide side chain showed pure
antagonistic activities, those with three methylene groups showed
the most potent activities. The results indicate the same tendencies
as the carboxylic acid side chains,15 but the sulfonamide
derivatives bound more strongly to AR than the carboxylic acid
derivative. The difference between the binding affinities of
compound 18a and 5 might come from the different charge states
of the compound. Based on the calculated pKa values, compound
18a is neutrally charged and 5 is negatively charged. The binding
of neutral compound 18a is stronger than that of the negatively
charged compound. Therefore, the sulfonamide side chain has
the desired lipophilicity and pKa value for AR antagonistic activity.
For the next step, we turned our attention to the optimization of
the phenyl group with a sulfonamide terminal side chain (Table
2). According to Refs. 17,18 an electron-withdrawing group such
as a nitrile group is essential for AR binding, so we mainly opti-
mized the trifluoromethyl group to other substituent groups. With
compound 32a, the replacement of the trifluoromethyl group with
hydrogen resulted in a drastic loss of activity. Methyl-substituted
compound 32b had less potency than trifluoromethyl in the RGA
assay but was as active as 18a in the cell growth assays. Although
that result indicates the growth inhibition of 32b had some causes
other than AR signal, the reason cannot be clearly explained.
Although, methoxy-substituted compound 32c showed almost
the same potency in the RGA assay, agonistic activities were
observed in the cell growth inhibition assays. Among them,
chloro-substituted derivative 32d showed the most potent pure
antagonistic activities not only in RGA but also in all cell growth
inhibition. With respect to the substituent R1, compounds with
an electron-withdrawing group at R1 showed stronger affinity
than those with an electron-donating group. Our docking model
suggests that substituent R1 interacts with the main chain carbonyl
group Met745 of AR (Fig. 1). In the case of compounds 32d (R1 = Cl)
and 18a (R1 = CF3), dꢀ of R and d+ of the carbon atom of Met745
makes contact at an appropriate distance, 3.4 and 3.0 Å, respec-
tively. In the case of compound 32c (R1 = OMe), the distance be-
tween OMe and the carbon atom of Met745 was slightly longer
than that of 32d and 18a (3.8 Å). This difference in distance might
be the reason for the weaker binding of compound 32c. In the case
of compound 32b (R1 = Me), there is no dꢀ in the substituent R1.
We introduced a methyl group to compound 18a to improve AR
binding. However, compound 32e with a methyl group introduced
at position R2 showed enhanced agonistic activity, but the
NC
F3C
NH2
F3C
O
H
N
a
Cl
F3C
29a
30a
30b
O
28
b, c
H
N
Cl
NC
F3C
NH2
F3C
O
29b
R3
R2
R3
R2
O
d, e, f
NC
NH2
NC
N
N
SO2NH2
R1
R1
S
31a R1=R2=R3=H
32a R1=R2=R3=H
R1=Me,R2=R3=H
R1=OMe,R2=R3=H
R1=Cl,R2=R3=H
R1=Me,R2=R3=H
31b
31c
31d
30a
30b
32b
32c
32d
32e
32f
R1=OMe,R2=R3=H
R1=Cl,R2=R3=H
R1=CF3,R2=Me,R3=H
R1=CF3,R2=H,R3=Me
R1=CF3,R2=Me,R3=H
R1=CF3,R2=H,R3=Me
Scheme 3. Reagents and conditions: (a) MeI, n-BuLi, THF, ꢀ30 °C; (b) CuCN, NMP,
170 °C; (c) concd HCl, EtOH, reflux; (d) thiophosgene, THF, 0 °C; (e) 17a, DMAP, THF,
reflux; (f) 6 N HCl, 1,4-dioxane, 80 °C.
with a carboxylic acid showed strong pure antagonistic activity in
transient RGA, it showed not only weak binding affinity to AR but
also weak antagonistic activity in stable RGA and cell growth
inhibition assays. Overall, antagonistic activities in stable RGA
tended to be weaker than those in transient RGA. Although amide
6 and urea 9 exhibited improved binding affinity, they showed
agonistic activities in transient RGA. On the other hand, tetrazole
13 and sulfonic acid 15, which are generally known as carboxylic
acid bioisosteres, did not show AR binding activity. Sulfamide 10
and sulfonamide 18a showed the desired androgen pure antago-
nistic activities in all the cell lines assayed. Particularly, the cell
growth inhibition of 18a was more than 10 times more potent than
that of 5. Because we found that sulfonamide had the desired po-
tency, we expanded the research to further derivatizations of the
sulfonamide side chains. Although N-methyl sulfonamide 24
showed potent antagonistic activities in RGA, agonistic activity
was observed in LNCaP-BC2 cell growth. In the case of N,N-di-
methyl sulfonamide 27, more potent agonistic activities were
observed in transient RGA and LNCaP. These results indicated that
an increase in lipophilicity leads to increased agonistic activities.
Next, we evaluated the effect of the length of the alkyl chain. Even

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H. Yoshino et al. / Bioorg. Med. Chem. 18 (2010) 3159–3168
Table 1
In vitro activities of thiohydantoin derivatives
O
NC
N
N
R
F3C
S
No.
R
Binding
RGA transient
sIC50
RGA stable
sIC50
LNCaP
Antagonist IC50
LNCaP-BC2
Antagonist IC50
clog P
IC50 (nM)
EC5
EC5
Agonist
Agonist
(nM)
(nM)
(nM)
(nM)
+/ꢀ
(nM)
+/ꢀ
(nM)
3
4
5
6
200
15
3900
480
800
>1000
>1000
>1000
400
170
80
200
<1
>10,000
3600
650
>10,000
>10,000
>10,000
>10,000
>10,000
110
100
ND
25
0.08
190
1
+
550
NT
400
55
200
89
NT
NT
18
Me
(CH2)3CO2H
+
ND
NT
ꢀ
130
290
490
260
>10,000
>10,000
64
64
21
160
230
>10,000
>10,000
350
>10,000
NT
2000
560
490
1000
NT
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
+
ꢀ
ꢀ
+
2000
400
1600
780
ND
3.27
2.49
2.33
2.33
2.91
1.37
2.16
2.81
3.14
1.93
2.14
(CH2)3CONH2
(CH2)3NHCONH2
(CH2)3NHSO2NH2
(CH2)3-5-tetrazolyl
(CH2)3SO3H
(CH2)3SO2NH2
(CH2)3SO2NHMe
(CH2)3SO2NMe2
(CH2)2SO2NH2
(CH2)4SO2NH2
ꢀ
9
ꢀ
10
13
15
18a
24
27
21
18b
ꢀ
NT
NT
ꢀ
NT
NT
ND
>10,000
1400
NT
7200
>10,000
320
350
NT
550
1300
160
190
3700
750
1400
+
30
NT
79
NT
ꢀ
770
>1000
>10,000
>10,000
ꢀ
ꢀ
ꢀ
110
NT: Not tested, ND: Not determined.
Table 2
In vitro activities of sulfonamide derivatives
R3
O
NC
N
N
SO2NH2
R1
R2
S
No.
R1
R2
R3
Binding
RGA stable
LNCaP
Agonist Antagonist IC50
LNCaP-BC2
Metabolic stability (human liver MS)
Residue (%) after 15 min
IC50
(nM)
EC5
(nM)
sIC50
(nM)
Agonist Antagonist IC50
+/ꢀ
(nM)
+/ꢀ
(nM)
5
3900
400
>5000
>1000
1900
440
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
2000
2000
320
>10,000
1900
330
190
110
2300
ꢀ
2000
160
NT
300
1900
410
1100
NT
ꢀ
400
18
NT
51
ND
12
ND
NT
97
95
92
18a CF3
32a
32b Me
32c OMe
32d Cl
H
H
H
H
H
Me
H
H
H
H
H
H
H
ꢀ
ꢀ
H
NT
ꢀ
NT
ꢀ
94
+
+
100
100
89
ꢀ
ꢀ
32e CF3
32f
260
+
+
CF3
Me >5000
>10,000
NT
NT
91
NT: Not tested, ND: Not determined.
introduction of a methyl group at R3 (32f) decreased the AR bind-
ing affinity. Change in the location of the side chain sulfonamide
group would result in a slight change in the phenyl ring.
Metabolic stability of the sulfonamide derivatives was exam-
ined in human liver microsomes. All of the compounds tested
were metabolically stable. Among them, compound 32d showed
significantly high metabolic stability as well as potent pure antag-
onistic activities. To confirm the in vivo activities of 32d, the
antiandrogenic activities were evaluated by seminal vesicle (SV)
wet weights in castrated mice (data not shown).15 Compound
32d inhibited dose-dependent SV wet weight gain by 10 mg/body
from sc administration of testosterone propionate (ED50 = 10 mg/
kg). In addition, we confirmed the in vivo efficacy of 32d against
prostate cancer. We used LNCaP xenograft models of castrated
SCID mice (Fig. 2). As shown in the growth inhibition of LNCaP cells
in vitro, 32d inhibited tumor growth of LNCaP xenograft in mice
dose-dependently at the same level as bicalutamide. Hence, we be-
lieve 32d (CH4933468) is a promising candidate for development
as an orally available antiandrogen for the treatment of bicaluta-
mide-resistant prostate cancer.
Met745
Met745
δ+
O
3.4 Å
1
R
S
O
S
N
H2N
N
CN
O
O
18a R1=CF3
32d R1=Cl
3.0 Å
3.4 Å
3.8 Å
5. Conclusion
32c R1=OMe
We have discovered novel thiohydantoin derivatives which
have a sulfonamide side chain. The compounds showed pure
Figure 1. Docking model of compound 32d to AR.

H. Yoshino et al. / Bioorg. Med. Chem. 18 (2010) 3159–3168
3163
6.1.1. 4-[3-(4-Cyano-3-trifluoromethylphenyl)-5,5-dimethyl-4-
oxo-2-thioxoimidazolidin-1-yl]butyramide (6)
900
800
700
600
500
400
300
200
100
0
Vehicle
Bicalutamide 3mg/kg
Bicalutamide 30mg/kg
Bicalutamide 300mg/kg
Castration
To a solution of 4-[3-(4-cyano-3-trifluoromethylphenyl)-5,5-di-
methyl-4-oxo-2-thioxoimidazolidin-1-yl]butyric acid15 (5) (2.30 g,
6.80 mmol) in THF (50 mL) were added Et3N (2.8 mL, 20.4 mmol)
and ClCO2Et (1.00 mL, 10.2 mmol) at 0 °C. After stirring for 3 min,
ammonia hydroxide (10 mL) was added to the reaction mixture.
After stirring for 15 min at room temperature, water was added
to the reaction mixture 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. Purification by sil-
ica gel column chromatography (AcOEt/hexane = 10:1) gave 6
(868 mg, 55%) as a colorless amorphous solid. Rf 0.22 (AcOEt); 1H
NMR (300 MHz, CDCl3) d: 1.62 (6H, s), 2.15–2.24 (2H, m), 2.41
(2H, t, J = 6.7 Hz), 3.78–3.84 (2H, m), 5.38 (1H, br s), 5.59 (1H, br
s), 7.74 (1H, dd, J = 1.8, 8.3 Hz), 7.92 (1H, d, J = 1.8 Hz), 7.97 (1H,
d, J = 8.3 Hz); MS (EI) m/z 398 ([M]+); HRMS Calcd for
C17H18F3N4O2S 399.1097. Found 399.1090.
Days
6.1.2. 4-[3-(3-Aminopropyl)-4,4-dimethyl-5-oxo-2-
thioxoimidazolidin-1-yl]-2-trifluoromethylbenzonitrile (8)
900
800
700
600
500
400
300
200
100
0
Vehicle
32d (CH4933468) 3mg/kg
32d (CH4933468) 30mg/kg
32d (CH4933468) 300mg/kg
Castration
To a solution of 2-(3-chloropropyl)isoindole-1,3-dione (7)
(2.00 g, 8.94 mmol) in DMF (40 mL) were added 2-aminoisobutyric
acid methyl ester HCl salt (2.75 g, 17.9 mmol), K2CO3 (4.94 g,
35.8 mmol) and NaI (1.34 g, 8.94 mmol); the mixture was then
stirred at 80 °C for 12 h. After cooling to room temperature, water
was added to the reaction mixture 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.
Purification by silica gel column chromatography (AcOEt/
hexane = 1:1) gave 2-[3-(1,3-dioxo-1,3-dihydro-isoindol-2-yl)pro-
pylamino]-2-methylpropionic acid methyl ester (2.21 g, 81%).
To a solution of the above obtained compound (400 mg,
1 mmol) in THF (7 mL) were added 3-trifluoromethyl-4-cyanois-
othiocyanate15 (330 mg, 1.45 mmol) and Et3N (0.036 mL,
0.26 mmol); the mixture was stirred at room temperature for
15 h; water was then added to the reaction mixture 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. Purification by silica gel column chromatography (AcOEt/
hexane = 1:2) gave 4-{3-[3-(1,3-dioxo-1,3-dihydroisoindol-2-
yl)propyl]-4,4-dimethyl-5-oxo-2-thioxoimidazolidin-1-yl}-2-trif-
luoromethylbenzonitrile (630 mg, 96%).
Days
Figure 2. Antitumor activities on LNCaP xenograft in mice. LNCaP was subcutane-
ously inoculated into 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 = 6 animals per group).
To
a solution of the above obtained compound (5.30 g,
antagonistic activities not only in RGA assays but also in cell
growth assays in LNCaP and LNCaP-BC2, a bicalutamide-resistant
prostate cancer cell line. Among the compounds, CH4933468
exhibited antiandrogenic activities. Furthermore, CH4933468
inhibited tumor growth of the LNCaP xenograft in mice dose-
dependently at a level similar to bicalutamide. This compound is
promising as a candidate for the development of novel agents for
bicalutamide refractory prostate cancer.
10.6 mmol) in CH2Cl2 (40 mL) and EtOH (100 mL) was added
NH2NH2/H2O (1.65 mL, 53.0 mmol), the mixture was stirred at
room temperature. After stirring for 2 days, the solvent was dis-
tilled off at reduced pressure. To the residue was added CH2Cl2,
insoluble material was filtered. Distillation of the filtrate at re-
duced pressure gave a crude product of 8 (3.80 g, 62%). This com-
pound was used in the next step without purification. Rf 0.38
(NH silica gel, CH2Cl2/MeOH = 20:1); 1H NMR (300 MHz, CDCl3)
d: 1.60 (6H, s), 1.92–2.03 (2H, m), 2.84 (2H, t, J = 6.7 Hz), 3.79–
3.85 (2H, m), 7.77 (1H, dd, J = 1.5, 8.7 Hz), 7.90 (1H, d, J = 1.5 Hz),
7.95 (1H, d, J = 8.7 Hz).
6. Experimental
6.1. Chemistry: instruments
6.1.3. 3-[3-(4-Cyano-3-trifluoromethylphenyl)-5,5-dimethyl-4-
oxo-2-thioxoimidazolidin-1-yl]propylurea (9)
Column chromatography was carried out on Merck Silica Gel 60
(230–400 mesh) if not otherwise specified. NH silica gel column
chromatography was carried out on Fuji Silysia NH-DM1020. Rf
was determined with Merck Silica Gel 60 F254 plates. 1H NMR spec-
tra were recorded on JEOL EX-270, Bruker ARX 300, Varian Mer-
cury300 or JEOL ECP-400. Mass spectra (MS) were measured by a
Thermo Electron LCQ Classic (ESI) or a Shimadzu GCMS-QP5050A
(EI). High resonance mass spectra (HRMS) were recorded by a
Micromass Q-Tof Ultima API mass spectrometer.
To a solution of 8 (100 mg, 0.270 mmol) in CH2Cl2 (3 mL) was
added trimethylsilyl isocyanate (0.073 mL, 0.540 mmol) and the
mixture was stirred at room temperature for 12 h, water was then
added to the reaction mixture and extracted with CH2Cl2. The or-
ganic layer was washed with brine and dried with MgSO4. After
filtering, the solvent was distilled off at reduced pressure. Purifica-
tion by silica gel column chromatography (CH2Cl2/MeOH = 20:1)
gave 9 (58 mg, 52%) as a colorless amorphous solid. Rf 0.26 (NH

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H. Yoshino et al. / Bioorg. Med. Chem. 18 (2010) 3159–3168
silica gel, CH2Cl2/MeOH = 20:1); 1H NMR (300 MHz, CDCl3) d: 1.60
(6H, s), 2.00–2.06 (2H, m), 3.28–3.35 (2H, m), 3.80–3.86 (2H, m),
4.41 (2H, br s), 5.09 (1H, t, J = 5.7 Hz), 7.76 (1H, dd, J = 1.9,
8.0 Hz), 7.89 (1H, d, J = 1.9 Hz), 7.96 (1H, d, J = 8.0 Hz); MS (ESI)
m/z 414 ([M+H]+) HRMS Calcd for C17H19F3N5O2S 414.1206. Found
414.1233.
12b (1.20 g, 96%) as a mixture. Rf 0.50 (AcOEt/hexane = 2:1); 1H
NMR (300 MHz, CD3OD) d: 1.41 and 1.42 (6H, s), 1.86–2.02 (2H,
m), 2.71–2.85 (2H, m), 2.83 and 2.97 (2H, t, J = 7.5 Hz), 5.45 and
5.64 (2H, s), 6.89 (2H, d, J = 8.8 Hz), 7.16 and 7.32 (2H, d, J = 8.8 Hz).
6.1.6. 4-{4,4-Dimethyl-5-oxo-3-[3-(1H-tetrazol-5-yl)propyl]-2-
thioxoimidazolidin-1-yl}-2-trifluoromethylbenzonitrile (13)
To a crude solution of 12a and 12b (620 mg, 2.00 mmol) in THF
(7 mL) were added 3-trifluoromethyl-4-cyanoisothiocyanate15
(450 mg, 2.00 mmol) and Et3N (0.450 mL, 3.20 mmol). The mixture
was stirred at room temperature for 15 h, and water was then
added to the reaction mixture 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.
Purification by silica gel column chromatography (AcOEt/hex-
6.1.4. 3-[3-(4-Cyano-3-trifluoromethylphenyl)-5,5-dimethyl-4-
oxo-2-thioxoimidazolidin-1-yl]propylsulfamide (10)
N-Chlorosulfonyl-tert-butylcarbamate was prepared by the
addition of tert-BuOH (0.031 mL, 0.32 mmol) to a solution of chlo-
rosulfonyl isocyanate (0.028 mL, 0.32 mmol) in CH2Cl2 (1 mL) at
0 °C.19 To
a solution of 8 (120 mg, 0.324 mmol) and Et3N
(0.050 mL, 0.36 mmol) in CH2Cl2 (1.1 mL) was slowly added pre-
pared N-chlorosulfonyl-tert-butylcarbamate at 0 °C, the mixture
was stirred for 1 h. After further stirring for 2 h at room tempera-
ture, water was added to the reaction mixture 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 to give a crude product of N-tert-butyloxycarbonyl-N0-(3-(4-
cyano-3-trifluoromethylphenyl)-5,5-dimethyl-4-oxo-2-thioxoimi-
dazolidin-1-yl]-propyl)sulfamide (111 mg, 62%).
To a solution of the above obtained compound (105 mg,
0.19 mmol) in CH2Cl2 (2 mL) was added TFA (0.147 mL) and the
mixture was stirred at room temperature for 12 h. To the reaction
mixture was added water and then extracted with CH2Cl2. The
organic layer was washed with brine and dried with MgSO4. After
filtering, the solvent was distilled off at reduced pressure. Purifica-
tion by silica gel column chromatography (AcOEt/hexane = 3:2)
gave 10 (65 mg, 82%) as a colorless amorphous solid. Rf 0.22
(AcOEt/hexane = 1:1); 1H NMR (300 MHz, CDCl3) d: 1.61 (6H, s),
2.07–2.17 (2H, m), 3.25–3.32 (2H, m), 3.85–3.90 (2H, m), 4.58
(2H, br s), 4.91 (1H, t, J = 6.8 Hz), 7.76 (1H, dd, J = 1.8, 8.3 Hz),
7.89 (1H, d, J = 1.8 Hz), 7.96 (1H, d, J = 8.3 Hz); HRMS Calcd for
C16H19F3N5O3S2 450.0875. Found 450.0896.
ane = 2:1) gave
a crude product of 4-(5-imino-3-{3-[2-(4-
methoxybenzyl)-2H-tetrazol-5-yl]propyl}-4,4-dimethyl-2-thioxo-
imidazolidin-1-yl)-2-trifluoromethylbenzonitrile (280 mg, 26%).
To a solution of the above obtained compound (280 mg,
0.516 mmol) in MeOH (15 mL) was added 2 N HCl (6 mL), the mix-
ture was stirred at 60 °C for 2 h. After cooling to room temperature,
water was added to the reaction mixture 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.
Purification by silica gel column chromatography (AcOEt/hex-
ane = 2:1) gave 4-(3-{3-[2-(4-methoxybenzyl)-1H-tetrazol-5-yl]-
propyl}-4,4-dimethyl-5-oxo-2-thioxoimidazolidin-1-yl)-2-trif-
luoromethylbenzonitrile (275 mg, 98%).
The above obtained compound (150 mg, 0.277 mmol) was dis-
solved in TFA (10 mL) and the mixture was stirred at 80 °C for
2 h. After cooling to room temperature, 2 N HCl (6 mL) was added
and the mixture was stirred at 60 °C for 2 h. After cooling to room
temperature, the solvent was distilled off at reduced pressure. The
residue was purified by silica gel column chromatography (CHCl3/
MeOH = 9:1) to give 13 (92.0 mg, 78%) as a colorless amorphous
solid. Rf 0.28 (AcOEt/hexane = 2:1); 1H NMR (300 MHz, CD3OD) d:
1.51 (6H, s), 2.19–2.30 (2H, m), 2.98 (2H, t, J = 7.6 Hz), 3.75–3.81
(2H, m), 3.80 (3H, s), 5.66 (2H, s), 6.89 (2H, d, J = 8.5 Hz), 7.34
(1H, d, J = 8.5 Hz), 7.76 (1H, dd, J = 1.5, 8.3 Hz), 7.87 (1H, d,
J = 1.5 Hz), 7.94 (1H, d, J = 8.3 Hz); HRMS Calcd for C17H17F3N7OS
424.1161. Found 424.1134.
6.1.5. 2-{3-[1-(4-Methoxybenzyl)-1H-tetrazol-5-yl]
propylamino}-2-methylpropionitrile (12a) and 2-{3-[2-(4-
methoxybenzyl)-2H-tetrazol-5-yl]propylamino}-2-
methylpropionitrile (12b)
To a solution of 4-(1,3-dioxo-1,3-dihydroisoindol-2-yl)butyro-
nitrile (11) (2.30 g, 10.8 mmol) in 1,4-dioxane (10 mL) were added
NaN3 (2.11 g, 32.4 mmol) and AlCl3 (1.31 g, 9.80 mmol), the mix-
ture was heated at 130 °C for 36 h. After cooling to room tempera-
ture, AcOEt and 3 N HCl were added. After stirring for 30 min,
water was added to the reaction mixture and then extracted with
AcOEt. The organic layer was washed with brine and dried with
Na2SO4. After filtering, the solvent was distilled off at reduced pres-
sure. Purification by trituration (AcOEt/hexane = 2:1) gave 2-[3-
(1H-tetrazol-5-yl)propyl]isoindole-1,3-dione (1.72 g, 62%).
6.1.7. 3-[3-(4-Cyano-3-trifluoromethylphenyl)-5,5-dimethyl-4-
oxo-2-thioxoimidazolidin-1-yl]propane-1-sulfonic acid (15)
To a solution of 3-chloropropane-1-sulfonic acid 2,2-dimethyl-
propyl ester (14)20 (405 mg, 1.77 mmol) in MeCN (10 mL) and
DMF (2 mL) were added 2-amino-isobutyric acid methyl ester
HCl salt (816 mg, 5.31 mmol), K2CO3 (1.54 g, 11.2 mmol) and n-tet-
rabutylammonium iodide (654 mg, 1.77 mmol), the mixture was
stirred at 80 °C for 2 days. After cooling to room temperature,
water was added to the reaction mixture 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.
Purification by silica gel column chromatography (AcOEt/hex-
ane = 1:1) gave 2-[3-(2,2-dimethylpropoxysulfonylpropylamino]-
2-methylpropionic acid methyl ester (126 mg, 23%).
The above compound was treated with a procedure similar to
that described for 8 to obtain 3-[3-(4-cyano-3-trifluoromethyl-
phenyl)-5,5-dimethyl-4-oxo-2-thioxoimidazolidin-1-yl]propane-
1-sulfonic acid 2,2-dimethylpropyl ester (yield 78%).
To a solution of the above obtained compound (118 mg,
0.233 mmol) in DMF (6 mL) was added tetramethylammonium
chloride (128 mg, 1.17 mmol) and the mixture was heated under
reflux for 6 h. After cooling, water was added and the reaction mix-
ture was extracted with CH2Cl2. The organic layer was washed
with water and brine, and then dried over MgSO4. After filtration
To
a solution of the above obtained compound (1.70 g,
6.60 mmol) in CH2Cl2 (85 mL) were added p-methoxybenzyl alco-
hol (855 mg, 6.67 mmol), PPh3 (1.75 g, 6.67 mmol) and diethyl azo-
dicarboxylate (DEAD) (1.15 g, 6.60 mmol) and the mixture was
stirred at room temperature. After stirring for 12 h, the solvent
was distilled away at reduced pressure. To the residue were added
CH2Cl2 (50 mL), EtOH (10 mL) and NH2NH2/H2O (11 g), the mixture
was stirred at room temperature for 12 h. After filtering, purifica-
tion by silica gel column chromatography (CH2Cl2/MeOH = 5:1)
gave a mixture of 3-[1-(4-methoxybenzyl)-1H-tetrazol-5-yl]pro-
pylamine and 3-[2-(4-methoxybenzyl)-2H-tetrazol-5-yl]propyl-
amine (980 mg, 60%).
To
a solution of the above obtained mixture (980 mg,
3.96 mmol) in MeOH (20 mL) was added acetone cyanohydrin
(1.00 mL), the mixture was stirred at 50 °C for 4 h. Distillation of
the solvent at reduced pressure gave crude products of 12a and

H. Yoshino et al. / Bioorg. Med. Chem. 18 (2010) 3159–3168
3165
and evaporation of the solvent under reduced pressure, the result-
ing residue was purified by silica gel column chromatography
(AcOEt/MeOH = 10:1) to give 15 (85 mg, 84%) as a colorless amor-
phous solid. 1H NMR (300 MHz, CD3OD) d: 1.80 (6H, s), 2.48–2.54
(2H, m), 3.12 (2H, t, J = 3.1 Hz), 4.10–4.16 (2H, m), 8.11 (1H, dd,
J = 1.5, 8.4 Hz), 8.28 (1H, d, J = 1.5 Hz), 8.31 (1H, d, J = 8.4 Hz); MS
(ESI) m/z: 436 [M+H]+; HRMS Calcd for C16H15F3N3O4S2 ([MꢀH]ꢀ)
434.0461. Found 434.0476.
J = 8.4 Hz); MS (ESI) m/z 433 ([MꢀH]ꢀ); HRMS Calcd for
C16H18F3N4O3S2 435.0766. Found 435.0797.
6.1.11. 4-[3-(4-Cyano-3-trifluoromethylphenyl)-5,5-dimethyl-4-
oxo-2-thioxoimidazolidin-1-yl]butane-1-sulfonamide (18b)
This compound was prepared from 17b using a procedure sim-
ilar to that described for 18a. Colorless amorphous solid. Rf 0.18
(AcOEt/hexane = 2:1); 1H NMR (300 MHz, CDCl3) d: 1.60 (6H, s),
1.97–2.04 (4H, m), 3.21–3.26 (2H, m), 3.72–3.77 (2H, m), 4.66
(2H, s), 7.77 (1H, dd, J = 1.9, 8.5 Hz), 7.89 (1H, d, J = 1.9 Hz), 7.96
(1H, d, J = 8.5 Hz). MS (ESI) m/z 447 ([MꢀH]ꢀ); HRMS Calcd for
C17H20F3N4O3S2 449.0923. Found 449.0901.
6.1.8. 2-(3-{[1-Dimethylaminomethylidene]sulfamoyl}propyl-
amino)-2-methylpropionic acid ethyl ester (17a)
To a solution of compound 3-chloropropane-1-sulfonic acid
amide (16a) (4.00 g, 25.4 mmol) in DMF (20 mL) was added N,N-
dimethylformamide dimethyl acetal (3.70 mL, 43.3 mmol), stirred
at room temperature for 1 h. After the addition of AcOEt, the
organic layer was washed with water and dried over MgSO4. After
filtration, the solvent was distilled off under reduced pressure to
give 3-chloropropane-1-sulfonic acid 1-dimethylaminomethylide-
neamide (3.05 g, 57%).
2-Aminoisobutyric acid ethyl ester hydrochloride (4.33 g,
28.2 mmol) and K2CO3 (7.80 g, 56.4 mmol) were dissolved in DMF
(30 mL) and stirred at room temperature for 30 min. To this solution,
a solution of the above obtained compound (3.00 g, 10.2 mmol) in
DMF (20 mL) and NaI (2.11 g, 14.1 mmol) were added and stirred
at 80 °C for 15 h. After cooling to room temperature, water was
added and the reaction mixture was extracted with AcOEt. The
organic layer was washed with water and dried over MgSO4. After
filtration, the solvent was distilled off under reduced pressure. The
resulting residue was purified by NH silica gel column chromatogra-
phy (AcOEt/hexane = 4:1) to give 17a (1.81 g, 44%) as a colorless oil.
Rf 0.37 (AcOEt/MeOH = 4:1); 1H NMR (300 MHz, CDCl3) d: 1.29 (6H,
s), 1.89–1.94 (2H, m), 2.57 (2H, t, J = 6.8 Hz), 3.04 (3H, s), 3.07–3.11
(2H, m), 3.13 (3H, s), 3.70 (3H, s), 8.03 (1H, s); MS (ESI) m/z 330
([M+Na]+).
6.1.12. 2-[(Cyanodimethylmethyl)amino]ethanesulfonic acid
bis(4-methoxybenzyl)amide (20)
Bis(4-methoxybenzyl) amine (900 mg, 3.50 mmol) was dis-
solved in CH2Cl2 (20 mL) and cooled to 0 °C. To this solution,
Et3N (1.02 mL) was added and then 2-(1,3-dioxo-1,3-dihydroisoin-
dol-2-yl)ethanesulfonyl chloride (19) (1.05 g, 3.84 mmol) was
added in small portions, followed by stirring at room temperature
for 3 h. After the addition of water, the reaction mixture was ex-
tracted with CH2Cl2. The organic layer was washed with brine
and dried over MgSO4. After filtration and evaporation of the sol-
vent under reduced pressure, the resulting residue was purified
by silica gel column chromatography (AcOEt) to give 2-phthalimi-
doethanesulfonic acid bis(4-methoxybenzyl) amide (1.40 g, 81%).
To the suspension of the above obtained compound (1.40 g,
2.83 mmol) in EtOH (15 mL) was added NH2NH2/H2O (0.151 mL),
the mixture was stirred at room temperature for 12 h. The reaction
solution was filtered and the filtrate was concentrated under
reduced pressure. The resulting residue was purified by silica gel
column chromatography (CH2Cl2/MeOH = 20:1) to give 2-aminoe-
thanesulfonic acid bis(4-methoxybenzyl)amide (460 mg, 45%).
To a solution of the above obtained compound (450 mg,
1.23 mmol) in MeOH (5 mL) was added acetone cyanohydrin
(0.136 mL) and stirred at room temperature for 12 h. After further
addition of acetone cyanohydrin (0.226 mL), the mixture was
heated and maintained at 40–50 °C for 3 h. The reaction solution
was concentrated under reduced pressure and purified by silica
gel chromatography (CH2Cl2/MeOH = 50:1) to give 20 (330 mg,
62%). Rf 0.67 (CH2Cl2/MeOH = 20:1); 1H NMR (300 MHz, CDCl3) d:
1.44 (6H, s), 1.95 (1H, br s), 3.00–3.16 (4H, m), 3.82 (6H, s), 4.30
(4H, s), 6.89 (4H, d, J = 8.7 Hz), 7.23 (4H, d, J = 8.7 Hz).
6.1.9. 2-(4-{[1-Dimethylaminomethylidene]sulfamoyl}-
butylamino)-2-methylpropionic acid methyl ester (17b)
This compound was prepared from 16b using a procedure sim-
ilar to that described for 17a. Colorless oil. Rf 0.08 (AcOEt/
MeOH = 3:1); 1H NMR (300 MHz, CDCl3) d: 1.29 (6H, s), 1.56–
1.62 (2H, m), 1.78–1.92 (2H, m), 2.47 (2H, t, J = 7.2 Hz), 2.98–3.03
(2H, m), 3.04 (3H, s), 3.13 (3H, s), 3.70 (3H, s), 8.03 (1H, s).
6.1.10. 3-[3-(4-Cyano-3-trifluoromethylphenyl)-5,5-dimethyl-4-
oxo-2-thioxoimidazolidin-1-yl]propane-1-sulfonamide (18a)
To a solution of 17a (2.20 g, 7.50 mmol) in THF (34 mL) were
6.1.13. 2-[3-(4-Cyano-3-trifluoromethylphenyl)-5,5-dimethyl-4-
oxo-2-thioxoimidazolidin-1-yl]ethanesulfonamide (21)
To a solution of 20 (220 mg, 0.510 mmol) in THF (4.5 mL) were
added Et3N (0.014 mL, 0.100 mmol)) and 4-cyano-3-trifluorometh-
ylphenyl isothiocyanate15 (116 mg, 0.508 mmol); the mixture was
stirred at room temperature for 3 h. The reaction solution was con-
centrated under reduced pressure and purified by silica gel column
chromatography (CH2Cl2/MeOH = 40:1) to give 2-[3-(4-cyano-3-tri-
fluoromethylphenyl)-4-imino-5,5-dimethyl-2-thioxoimidazolidin-
1-yl]ethanesulfonic acid bis(4-methoxybenzyl)amide (259 mg,
77%).
added
Et3N
(0.21 mL,
1.51 mmol)
and
4-cyano-3-trif-
luoromethylphenylisothiocyanate15 (1.71 g, 7.49 mmol), the mix-
ture was stirred at room temperature for 2 h. The reaction
solution was concentrated and the resulting residue was purified
by NH silica gel column chromatography (AcOEt/hexane = 4:1) to
give 3-[3-(4-cyano-3-trifluoromethylphenyl)-5,5-dimethyl-4-oxo-
2-thioxoimidazolidin-1-yl]propane-1-sulfonic acid 1-dimethylam-
inomethylideneamide (2.60 g, 71%)
To
a solution of the above obtained compound (2.60 g,
5.31 mmol) in 1,4-dioxane (25 mL) was added 6 N HCl (25 mL),
the resulting mixture was heated under reflux for 1 h. After cooling
to room temperature, water was added and the reaction mixture
was extracted with CH2Cl2. The organic layer was washed with
water and dried over MgSO4. After filtration and concentration un-
der reduced pressure, the resulting residue was purified by silica
gel column chromatography (AcOEt/hexane = 4:1) to give 18a
(1.62 g, 70%) as a colorless amorphous solid. Rf 0.18 (AcOEt/hex-
ane = 2:1); 1H NMR (300 MHz, CDCl3) d: 1.62 (6H, s), 2.36–2.46
(2H, m), 3.28 (2H, t, J = 7.1 Hz), 3.90–3.95 (2H, m), 4.85 (2H, s),
7.77 (1H, dd, J = 2.3, 8.4 Hz), 7.90 (1H, d, J = 2.3 Hz), 7.97 (1H, d,
To a solution of the above obtained compound (259 mg,
0.392 mmol) in 1,4-dioxane (2.5 mL) was added 6 N HCl (2.5 mL),
the mixture was heated under reflux for 1 h. After cooling to room
temperature, water was added and the reaction mixture was
extracted with CH2Cl2. The organic layer was washed with brine
and dried over MgSO4. After filtration and concentration under
reduced pressure, the resulting residue was purified by silica
gel column chromatography (AcOEt/hexane = 1:1) to give
2-[3-(4-cyano-3-trifluoromethylphenyl)-5,5-dimethyl-4-oxo-2-thi
oxoimidazolidin-1-yl]ethanesulfonic acid bis-(4-methoxybenzyl)
amide (144 mg, 56%).

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H. Yoshino et al. / Bioorg. Med. Chem. 18 (2010) 3159–3168
The above obtained compound (140 mg, 0.211 mmol), TFA
(300 MHz, CDCl3) d: 1.23 (6H, s), 1.82–1.92 (2H, m), 2.59 (2H, t,
J = 6.7 Hz), 2.81 (6H, s), 2.95–3.00 (2H, m), 3.64 (3H, s).
(1 mL) and anisole (0.02 mL) were mixed and stirred at room tem-
perature for 2 h, followed by heating under reflux for 1 h. After
cooling to room temperature, water was added and the reaction
mixture was extracted with AcOEt. The organic layer was washed
with brine and dried over MgSO4. After filtration and concentration
under reduced pressure, the resulting residue was purified by silica
gel column chromatography (AcOEt/hexane = 4:1) to give 21
(64 mg, 72%) as a colorless amorphous solid. Rf 0.21 (AcOEt/hex-
ane = 3:1); 1H NMR (300 MHz, CDCl3) d: 1.64 (6H, s), 3.67–3.72
(2H, m), 4.17–4.22 (2H, m), 4.88 (2H, br s), 7.76 (1H, dd, J = 1.8,
8.5 Hz), 7.88 (1H, d, J = 1.8 Hz), 7.97 (1H, d, J = 8.5 Hz); HRMS Calcd
for C15H16F3N4O3S2 421.061. Found 421.0642.
6.1.17. 3-[3-(4-Cyano-3-trifluoromethylphenyl)-5,5-dimethyl-4-
oxo-2-thioxoimidazolidin-1-yl]propane-1-sulfonic acid
dimethylamide (27)
This compound was prepared from 26 using a procedure similar
to that described for 18a. Yield 56%; Rf 0.77 (CH2Cl2/ace-
tone = 20:1); 1H NMR (300 MHz, CDCl3) d: 1.62 (6H, s), 2.32–2.43
(2H, m), 2.91 (6H, s), 3.04 (2H, t, J = 6.9 Hz), 3.89–3.95 (2H, m),
7.78 (1H, dd, J = 1.8, 8.3 Hz), 7.90 (1H, d, J = 1.8 Hz), 7.96 (1H, d,
J = 8.3 Hz); MS (ESI) m/z 485 ([M+Na]+); HRMS Calcd for
C18H22F3N4O3S2 463.1079. Found 463.1092.
6.1.14. 2-(4-{N-methyl-N-(tert-butoxycarbonyl)sulfamoyl}-
butylamino)-2-methylpropionic acid ethyl ester (23)
6.1.18. N-(4-Chloro-2-methyl-3-trifluoromethylphenyl)-2,2-
dimethylpropionamide (29a) and N-(4-chloro-2-methyl-5-
trifluoromethylphenyl)-2,2-dimethylpropionamide (29b)
To a solution of 3-chloropropane-1-sulfonic acid methylamide
(22)21 (1.08 g, 6.29 mmol) were added Boc2O (2.06 g, 9.43 mmol)
and 4-dimethylaminopyridine (DMAP) (77 mg, 0.630 mmol) in
CH3CN (12.6 mL); the mixture was stirred at room temperature
for 17 h. After the addition of water, the reaction mixture was ex-
tracted with CH2Cl2 and the organic layer was dried over MgSO4.
After filtration, the solvent was distilled off under reduced pressure
to give N-methyl-N-(tert-butyloxycarbonyl)-3-chloropropanesulf-
onamide (1.65 g, 96%).
To a solution of 2-aminoisobutyric acid ethyl ester hydrochlo-
ride (592 mg, 3.53 mmol) in CH3CN (5 mL) and DMF (1 mL) was
added K2CO3 (1.02 g, 7.38 mmol), the mixture was stirred at room
temperature for 1 h. To a solution of the mixture were added the
above obtained compound (800 mg, 2.94 mmol) and NaI (441 mg,
2.94 mmol), the reaction mixture was maintained at 80 °C for
22 h. After cooling to room temperature, water was added and
the reaction mixture was extracted with AcOEt. The organic layer
was washed with brine and dried over MgSO4. After filtration
and evaporation of the solvent under reduced pressure, the result-
ing residue was purified by silica gel column chromatography
(AcOEt/hexane = 1:1) to give 23 (813 mg, 75%). Rf 0.32 (AcOEt/hex-
ane = 1:1); 1H NMR (300 MHz, CDCl3) d: 1.27 (3H, t, J = 7.1 Hz), 1.28
(6H, s), 1.54 (9H, s), 1.87–1.92 (2H, m), 2.59 (2H, t, J = 6.5 Hz), 3.19
(3H, s), 3.54–3.59 (2H, m), 4.16 (2H, q, J = 7.1 Hz).
To
a solution of N-(4-chloro-3-trifluoromethylphenyl)-2,2-
dimethylpropionamide (28) (11.2 g, 40.0 mmol) in THF was slowly
added n-BuLi (1.6 M in hexane, 60 mL) at ꢀ30 °C, the mixture was
stirred at the same temperature for 45 min. To the reaction mix-
ture was added MeI (5.1 mL, 55.0 mmol) at ꢀ30 °C, the mixture
was stirred at the same temperature for 30 min. After the addition
of water, the reaction mixture was extracted with AcOEt. The or-
ganic layer was washed with water and dried over MgSO4. After fil-
tration, the reaction solution was concentrated under reduced
pressure and purified by silica gel column chromatography
(CH2Cl2) to give 29a (8.63 g, 74%) and 29b (2.12 g, 18%).
Compound 29a: Rf 0.79 (CH2Cl2); 1H NMR (400 MHz, CDCl3) d:
1.35 (9H, s), 2.35–2.37 (3H, m), 7.21 (1H, br s), 7.34 (1H, d,
J = 8.7 Hz), 7.77 (1H, d, J = 8.7 Hz); MS (ESI) m/z 294 ([M+H]+).
Compound 29b: Rf 0.88 (CH2Cl2); 1H NMR (400 MHz, CDCl3) d:
1.35 (9H, s), 2.28 (3H, s), 7.32 (1H, s), 8.31 (1H, s); MS (ESI) m/z
294 ([M+H]+).
6.1.19. 4-Amino-3-methyl-2-trifluoromethylbenzonitrile (30a)
Under nitrogen atmosphere, CuCN (177 mg, 1.98 mmol) was
added to a solution of compound 29a (340 mg, 1.16 mmol) in
NMP (3.5 mL). The mixture was heated and maintained at 200 °C
for 4 days. After cooling to room temperature, water was added.
The precipitate was filtered, washed with water and dried. To a
solution of the resulting solid in EtOH (2.7 mL) was added concd
HCl (2.7 mL) and the mixture was refluxed for 2 h. After cooling
to 0 °C, 2 N NaOH was added to neutralize the reaction mixture
followed by extraction with CH2Cl2. The organic layer was washed
with brine and dried over MgSO4. After filtration and evaporation
of the solvent under reduced pressure, the resulting residue was
purified by NH silica gel column chromatography (CH2Cl2) to give
30a (200 mg, 86%). Rf: 0.43 (CH2Cl2); 1H NMR (400 MHz, CDCl3) d:
1.56 (9H, s), 4.38 (2H, br s), 6.82 (1H, d, J = 8.4 Hz), 7.46 (1H, d,
J = 8.4 Hz); MS (EI) m/z 200 ([M]+).
6.1.15. 3-[3-(4-Cyano-3-trifluoromethylphenyl)-5,5-dimethyl-4-
oxo-2-thioxoimidazolidin-1-yl]propane-1-sulfonic acid
methylamide (24)
Compound 23 was treated with a procedure similar to that de-
scribed for 18a to obtain tert-butyl 3-[3-(4-cyano-3-trifluorometh-
ylphenyl)-5,5-dimethyl-4-oxo-2-thioxoimidazolidin-1-yl]pro-
pane-N-methylsulfonylcarbamate (yield 100%).
To a solution of the above obtained compound (300 mg,
0.547 mmol) in CH2Cl2 (2.7 mL) was slowly added TFA (0.421 mL)
at 0 °C, and the mixture stirred at room temperature for 5.5 h.
The reaction solution was concentrated under reduced pressure
and purified by silica gel column chromatography (AcOEt/hex-
6.1.20. 4-Amino-5-methyl-2-trifluoromethylbenzonitrile (30b)
This compound was prepared from 29b using a procedure sim-
ilar to that described for 30a. Yield 20%; Rf: 0.48 (CH2Cl2); 1H NMR
(400 MHz, CDCl3) d: 2.20 (9H, s), 4.30 (2H, br s), 6.94 (1H, s), 7.45
(1H, s); MS (EI) m/z 200 ([M]+).
ane/CH2Cl2 = 1:1:1) to give 24 (235 mg, 96%) as
a colorless
amorphous solid. Rf 0.18 (AcOEt/hexane = 1:1); 1H NMR
(300 MHz, CDCl3) d: 1.62 (6H, s), 2.33–2.39 (2H, m), 2.84 (3H, d,
J = 5.2 Hz), 3.16 (2H, t, J = 7.1 Hz), 3.89–3.94 (2H, m), 4.35 (1H, q,
J = 5.2 Hz), 7.77 (1H, dd, J = 1.7, 8.4 Hz), 7.90 (1H, d, J = 1.7 Hz),
7.96 (1H, d, J = 8.4 Hz); MS (ESI) m/z 447 ([MꢀH]ꢀ); HRMS Calcd
for C17H20F3N4O3S2 449.0923. Found 449.0941.
6.1.21. 3-[3-(3-Chloro-4-cyanophenyl)-5,5-dimethyl-4-oxo-2-
thioxoimidazolidin-1-yl]propane-1-sulfonamide (32d)
Under nitrogen atmosphere, thiophosgene (27.5 mL, 361 mmol)
was added to a solution of 4-amino-2-chloro-benzonitrile (31d)
(50.0 g, 327 mmol) in THF (1500 mL) at 0 °C. After stirring at 5 °C
for 1 h, water was added and the reaction mixture was extracted
with Et2O. The organic layer was washed with brine and dried over
MgSO4. After filtration, the solvent was distilled off under reduced
6.1.16. 2-(3-Dimethylsulfamoylpropylamino)-2-
methylpropionic acid methyl ester (26)
This compound was prepared from 3-chloropropane-1-sulfonic
acid dimethylamide (25) using a procedure similar to that de-
scribed for 17a. Yield 29%; Rf 0.43 (AcOEt/MeOH = 10:1); 1H NMR

H. Yoshino et al. / Bioorg. Med. Chem. 18 (2010) 3159–3168
3167
pressure. The resulting residue was recrystallized from acetone/
hexane to give 2-chloro-4-isothiocyanatobenzonitrile (44.5 g,
70%). 1H NMR (300 MHz, CDCl3) d: 7.19 (1H, dd, J = 2.1, 8.7 Hz),
7.35 (1H, d, J = 2.1 Hz), 7.65 (1H, d, J = 8.7 Hz).
To a solution of 17a (100 mg, 0.330 mmol) in THF (0.7 mL) were
added the above obtained compound (63.3 mg, 0.330 mmol) and
DMAP (60.4 mg, 0.494 mmol) and the mixture was heated at
60 °C under nitrogen atmosphere. After stirring for 45 min, water
was added to the reaction mixture 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. Purifica-
tion by silica gel column chromatography (CH2Cl2/MeOH = 20:1)
8.18 (1H, d, J = 8.1 Hz); MS (ESI) m/z 449 ([M+H]+); HRMS Calcd
for C17H20F3N4O3S2 449.0923. Found 449.0911.
6.1.26. 3-[3-(4-Cyano-2-methyl-5-trifluoromethylphenyl)-5,5-
dimethyl-4-oxo-2-thioxoimidazolidin-1-yl]propane-1-
sulfonamide (32f)
Rf 0.33 (CH2Cl2/MeOH = 20:1); 1H NMR (400 MHz, DMSO-d6) d:
1.54 (3H, s), 1.58 (3H, s), 2.12–2.25 (2H, m), 2.22 (3H, s), 3.05–3.15
(2H, m), 3.75–3.90 (2H, m), 6.87 (2H, s), 8.22 (1H, s), 8.30 (1H, s);
HRMS Calcd for C17H20F3N4O3S2 449.0923. Found 449.0937.
6.2. Competitive androgen receptor binding assay
gave
3-[3-(3-chloro-4-cyanophenyl)-5,5-dimethyl-4-oxo-2-thi-
oxoimidazolidin-1-yl]propane-1-sulfonic acid 1-dimethylami-
nomethylideneamide (136 mg, 91%). 1H NMR (400 MHz, CDCl3) d:
1.60 (6H, s), 2.32–2.38 (2H, m), 3.06 (3H, s), 3.13 (2H, t,
J = 6.6 Hz), 3.16 (3H, s), 3.93–3.95 (2H, m), 7.44 (1H, dd, J = 1.5,
8.4 Hz), 7.60 (1H, d, J = 1.5 Hz), 7.78 (1H, d, J = 8.4 Hz), 8.07 (1H,
s); MS (ESI) m/z 460 ([M+H]+).
To a solution of the above obtained compound (136 mg,
0.299 mmol) in 1,4-dioxane (2.4 mL) was added 6 N HCl (1.2 mL);
the resulting mixture was heated under reflux for 1 h. After cooling
to room temperature, water was added and the reaction mixture
was extracted with CH2Cl2. The organic layer was washed with
water and dried over MgSO4. After filtration and concentration un-
der reduced pressure, the resulting residue was purified by silica
gel column chromatography (CH2Cl2/MeOH = 30:1) to give 32d
(104 mg, 86%) as a colorless amorphous solid. Rf: 0.23 (CH2Cl2/
MeOH = 20:1); 1H NMR (400 MHz, DMSO-d6) d: 1.53 (6H, s),
2.12–2.22 (2H, m), 3.10 (2H, t, J = 7.3 Hz), 3.81 (2H, t, J = 7.3 Hz),
6.86 (2H, s), 7.65 (1H, d, J = 8.1 Hz), 7.96 (1H, s), 8.15 (1H, d,
J = 8.1 Hz); MS (ESI) m/z 401 ([M+H]+; HRMS Calcd for
C15H18ClN4O3S2 401.0503. Found 401.0517.
CHO-K1/hAR cells (5 ꢁ 104/well) were plated onto 24-well
plates and cultured for 2 days. Adhered cells were washed with
PBS(-) and replaced with phenol red-free DMEM containing
0.34 nmol/L [3H]-mibolerone in the presence or absence of test
compound. Nonspecific binding of [3H]-mibolerone was
determined separately by adding 200-fold excess of cold miboler-
one. Following a 2 h incubation at 37 °C, cells were washed with
PBS(-) and solubilized in 10 mmol/L Tris–HCl, pH 6.8 containing
2% SDS and 10% glycerol. Radioactivity was counted using a
scintillation counter.
6.3. Reporter gene assay
6.3.1. Transient reporter gene assay
Twenty four hours before transfection, the Hela cells were pla-
ted at 1 ꢁ 104 cells/well in phenol red-free DMEM containing 3%
DCC-FCS onto 96-well plates. The cells were co-transfected with
GM-LUC (50 ng/well) and pSG5-hAR (10 ng/well) using
a
FuGENE™ 6 Transfection Reagent (Roche). six hours after the
transfection, 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
(Promega).
Compounds 32a–c, e and f were prepared using a procedure
similar to that described for 32d.
6.1.22. 3-[3-(4-Cyanophenyl)-5,5-dimethyl-4-oxo-2-
thioxoimidazolidin-1-yl]propane-1-sulfonamide (32a)
Rf 0.30 (AcOEt/hexane = 1:1); 1H NMR (400 MHz, CDCl3) d: 1.60
(6H, s), 2.35–2.48 (2H, m), 3.28 (2H, t, J = 7.0 Hz), 3.88–3.98 (2H,
m), 4.70 (2H, br s), 7.51 (2H, d, J = 8.4 Hz), 7.79 (2H, d, J = 8.4 Hz);
MS (ESI) m/z 367 ([M+H]+; HRMS Calcd for C15H19N4O3S2
367.0893. Found 367.0899.
6.3.2. Stable reporter gene assay
Hela cells were co-transfected with MMTV-Luc-Hyg and pSG5-
hAR-neo using a FuGENE™ 6 Transfection Reagent. The transfected
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
l
6.1.23. 3-[3-(4-Cyano-3-methylphenyl)-5,5-dimethyl-4-oxo-2-
thioxoimidazolidin-1-yl]propane-1-sulfonamide (32b)
1H NMR (300 MHz, CDCl3) d: 1.58 (6H, s), 2.37–2.42 (2H, m),
2.59 (3H, s), 3.25 (2H, t, J = 6.9 Hz), 3.15 (3H, s), 3.88–3.94 (2H,
m), 5.00 (2H, br s), 7.26–7.32 (2H, m), 7.72 (1H, d, J = 8.2 Hz),
8.07 (1H, s); MS (ESI) m/z 379 ([MꢀH]ꢀ; HRMS Calcd for
C16H21N4O3S2 381.1049. Found 381.1049.
l
were plated at 1 ꢁ 104 cells/well in phenol red-free DMEM con-
taining 3% DCC-FCS onto 96-well plates. 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.
6.1.24. 3-[3-(4-Cyano-3-methoxyphenyl)-5,5-dimethyl-4-oxo-2-
thioxoimidazolidin-1-yl]propane-1-sulfonamide (32c)
6.4. In vitro cell growth assay LNCaP and LNCaP-BC2
Rf 0.20 (CH2Cl2/MeOH = 20:1); 1H NMR (400 MHz, DMSO-d6) d:
1.54 (6H, s), 2.12–2.25 (2H, m), 3.08–3.17 (2H, m), 3.82 (2H, t,
J = 7.7 Hz), 3.91 (3H, s), 6.87 (2H, s), 7.14 (1H, d, J = 8.1 Hz), 7.36
(1H, s), 7.87 (1H, d, J = 8.1 Hz); MS (ESI) m/z 397 ([M+H]+; HRMS
Calcd for C16H21N4O4S2 397.0998. Found 397.1031.
Pre-starved LNCaP or LNCaP-BC2 cells were plated onto 96-well
plates in phenol red-free RPMI 1640 containing 5% DCC-FBS. Fol-
lowing overnight attachment, the cells were treated with the test
compound in the absence or presence of R1881 (LNCaP: 0.1 nM,
LNCaP-BC2: 0.01 nM) for 6 days. Cell proliferation was determined
by CellTiter 96Ò AQueous One Solution Cell Proliferation Assay
(Promega).
6.1.25. 3-[3-(4-Cyano-2-methyl-3-trifluoromethylphenyl)-5,5-
dimethyl-4-oxo-2-thioxoimidazolidin-1-yl]propane-1-
sulfonamide (32e)
6.5. In vitro metabolic stability in mouse liver microsome
Rf 0.20 (CH2Cl2/MeOH = 20:1); 1H NMR (400 MHz, DMSO-d6) d:
1.56 (3H, s), 1.58 (3H, s), 2.15–2.20 (2H, m), 2.23 (3H, s), 3.10 (2H, t,
J = 8.1 Hz), 3.78–3.90 (2H, m), 6.87 (2H, s), 8.00 (1H, d, J = 8.1 Hz),
To a 0.1 M potassium phosphate buffer (pH 7.4) containing 1 mM
NADPH and 0.5 mg/mL mouse liver microsome, test compound

3168
H. Yoshino et al. / Bioorg. Med. Chem. 18 (2010) 3159–3168
(final concn 1 mM) was added to start the reaction. After incubation
for 15 min at 37 °C, CH3CN was added to stop the reaction. The con-
centration of the test compound was measured by LC/MS/MS
(QTRAP, Applied Biosystems).
ces Ford of Chugai Pharmaceutical Co., Ltd for assistance with Eng-
lish usage.
References and notes
1. Xia, Y. Q.; Jemal, M. Rapid Commun. Mass Spectrom. 2009, 23, 2125.
2. Stewart, A. B.; Lwaleed, B. A.; Douglas, D. A.; Birch, B. R. Curr. Med. Chem.
Anticancer Agents 2005, 5, 603.
6.6. Molecular modeling. Docking model of compound 32d to
AR
3. Kennealey, G. T.; Furr, B. J. Urol. Clin. North Am. 1991, 18, 99.
4. Blackledge, G. R. Eur. Urol. 1996, 29, 96.
This model was built based on the X-ray crystal structure of hu-
man AR in complex with the ligand R1881 (PDB ID: 1e3g).22 3D
structures of compound 32d were modeled using software SYBYL23
with a Tripos force field.24 Compound 32d was manually docked
into AR such that (i) the binding mode of the cyanophenyl moiety
of compound 32d was similar to that of bicalutamide (PDB ID:
1z95)17 and (ii) the thiohydantoin ring was modeled so that the
side chain attached to the thiohydantoin ring of 32d was directed
to helix 12 of AR. After checking the bumps between the compound
and AR, energy minimization of the compound/AR complex was
performed using a molecular mechanics method with the Tripos
force field on the condition that the coordinates of AR are fixed.
Conformations obtained for 32d are local minimum energy
conformations.
5. Tyrrell, C. J.; Altwein, J. E.; Klippel, F.; Varenhorst, E.; Lunglmayr, G.; Boccardo,
F.; Holdaway, I. M.; Haefliger, J. M.; Jordaan, J. P. J. Urol. 1991, 146, 1321.
6. Eisenberger, M. A.; Blumenstein, B. A.; Crawford, E. D.; Miller, G.; McLeod, D. G.;
Loehrer, P. J.; Wilding, G.; Sears, K.; Culkin, D. J.; Thompson, I. M., Jr.; Bueschen,
A. J.; Lowe, B. A. N. Eng. J. Med. 1998, 339, 1036.
7. Wirth, M. P.; Froschermaier, S. E. Urol. Res. 1997, 25, S67.
8. Kemppainen, J. A.; Wilson, E. M. Urology 1996, 48, 157.
9. Ishioka, T.; Tanatani, A.; Nagasawa, K.; Hashimoto, Y. Bioorg. Med. Chem. Lett.
2003, 13, 2655.
10. Hashimoto, Y.; Miyachi, H. Bioorg. Med. Chem. 2005, 13, 5080.
11. Tachibana, K.; Imaoka, I.; Yoshino, H.; Emura, T.; Kodama, H.; Furuta, Y.; Kato,
N.; Nakamura, M.; Ohta, M.; Taniguchi, K.; Ishikura, N.; Nagamuta, M.; Onuma,
E.; Sato, H. Bioorg. Med. Chem. 2007, 15, 174.
12. Tachibana, K.; Imaoka, I.; Yoshino, H.; Kato, N.; Nakamura, M.; Ohta, M.;
Kawata, H.; Taniguchi, K.; Ishikura, N.; Nagamuta, M.; Onuma, E.; Sato, H.
Bioorg. Med. Chem. Lett. 2007, 17, 5573.
13. Brunner, N.; Frandsen, T. L.; Holst-Hansen, C.; Bei, M.; Thompson, E. W.;
Wakeling, A. E.; Lippman, M. E.; Clarke, R. Cancer Res. 1993, 53, 3229.
14. Bowler, J.; Lilley, T. J.; Pittam, J. D.; Wakeling, A. E. Steroids 1989, 54, 71.
15. Tachibana, K.; Imaoka, I.; Shiraishi, T.; Yoshino, H.; Nakamura, M.; Ohta, M.;
Kawata, H.; Taniguchi, K.; Ishikura, N.; Tsunenari, T.; Saito, H.; Nagamuta, M.;
Nakagawa, T.; Takanashi, K.; Onuma, E.; Sato, H. Chem. Pharm. Bull. (Tokyo)
2008, 56, 1555.
6.7. In vivo antitumor activities on LNCaP Xenograft in mice
LNCaP (2 ꢁ 106 cells) was subcutaneously inoculated into non-
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
day in seven cycles of 5 days on, 2 days off; the tumor size was
measured at time points designated to be appropriate.
16. Kawata, H.; Ishkura, N.; Watanabe, M.; Nishimoto, A.; Tsunenari, T.; Aoki, Y. The
Prostate, in press.
17. Bohl, C. E.; Gao, W.; Miller, D. D.; Bell, C. E.; Dalton, J. T. Proc. Natl. Acad. Sci.
U.S.A. 2005, 102, 6201.
18. Soderholm, A. A.; Lehtovuori, P. T.; Nyronen, T. H. J. Med. Chem. 2005, 48, 917.
19. Abdaoui, M.; Dewynter, G.; Aouf, N.; Favre, G.; Morere, A.; Montero, J. L. Bioorg.
Med. Chem. 1996, 4, 1227.
20. Adamczyk, M.; Chen, Y. Y.; Mattingly, P. G. Tetrahedron Lett. 2001, 42, 4285.
21. White, Emil H.; Lim, Hyung M. J. Org. Chem. 1987, 52, 2162.
22. Matias, P. M.; Donner, P.; Coelho, R.; Thomaz, M.; Peixoto, C.; Macedo, S.; Otto,
N.; Joschko, S.; Scholz, P.; Wegg, A.; Basler, S.; Schafer, M.; Egner, U.; Carrondo,
M. A. J. Biol. Chem. 2000, 275, 26164.
Acknowledgments
23.
‘SYBYL 7.3,’ Tripos International, St. Louis.
The authors thank Ms. Hitomi Suda of Chugai Pharmaceutical
Co., Ltd for HRMS measurements of the compounds and Ms. Fran-
24. Clark, M.; Cramer, R. D., III; Van, O. N. J. Comput. Chem. 1989, 10, 982.