Synthesis and pharmacological evaluation of novel arylpiperazine derivatives as nonsteroidal androgen receptor antagonists

Article abstract of DOI:10.1248/cpb.52.1330

The search for novel antiandrogens by high-throughput screening (HTS) of the Yamanouchi chemical library led to the discovery of the lead compound (5), which possesses an arylmorpholine moiety. Through the optimization of the lead compound (5), we have found a series of novel arylpiperazine derivatives. Among them, 4-[4-cyano-(3-trifluoromethyl)phenyl]-N-(4-fluorophenyl)piperazine-1- carboxamide (22; YM-92088) exhibited a potent AR antagonistic activity with an IC₅₀ value of 0.47 μM in the reporter assay, which is more potent than bicalutamide (4) which has an IC₅₀ value of 0.89 μM.

Full text of DOI:10.1248/cpb.52.1330

1330
Chem. Pharm. Bull. 52(11) 1330—1333 (2004)
Vol. 52, No. 11
Synthesis and Pharmacological Evaluation of Novel Arylpiperazine
Derivatives as Nonsteroidal Androgen Receptor Antagonists
Isao KINOYAMA,*,a Nobuaki TANIGUCHI,a Toru YODEN,b Hiroshi KOUTOKU,a Takashi FURUTANI,a
a
Masafumi KUDOH,a and Minoru OKADA
a Institute for Drug Discovery Research, Yamanouchi Pharmaceutical Co., Ltd.; 21 Miyukigaoka, Tsukuba, Ibaraki
305–8585, Japan: and b Institute for Technology Development, Yamanouchi Pharmaceutical Co., Ltd.; 3–17–1 Hasune,
Itabashi-ku, Tokyo 174–8612, Japan.
Received July 30, 2004; accepted August 30, 2004; published online August 30, 2004
The search for novel antiandrogens by high-throughput screening (HTS) of the Yamanouchi chemical
library led to the discovery of the lead compound (5), which possesses an arylmorpholine moiety. Through the
optimization of the lead compound (5), we have found a series of novel arylpiperazine derivatives. Among them,
4-[4-cyano-(3-trifluoromethyl)phenyl]-N-(4-fluorophenyl)piperazine-1-carboxamide (22; YM-92088) exhibited a
potent AR antagonistic activity with an IC50 value of 0.47 mM in the reporter assay, which is more potent than
bicalutamide (4) which has an IC50 value of 0.89 mM.
Key words androgen receptor; antagonist; antiandrogen; prostate cancer
The androgen receptor (AR) is a member of the nuclear nonsteroidal AR antagonists exhibit some adverse effects
receptor superfamily and acts as a ligand-dependent intracel- such as mastodynia, gynaecomastia and hepatotoxicity15—22)
;
lular transcription factor.1—3) The AR is also responsible for therefore potent AR antagonists with less adverse effects are
mediating the physiological actions of the androgens, namely highly desirable.
testosterone and 5a-dihydrotestosterone (DHT). Androgens
To identify novel AR antagonists, we conducted an HTS of
play a critical role in normal male development and the the Yamanouchi chemical library using a reporter assay,
subsequent maintenance of secondary male characteristics which measured the inhibitory activity of test compounds on
such as muscle mass, bone mass, strength, fat distribution, androgen-induced activation of AR. Compound 5 was
and spermatogenesis.4—6) The male sexual accessory organs, discovered as an HTS hit with an associated IC50 value of
such as the prostate, are stimulated to grow and are maintai- 3.1 mM. Through optimization of the lead compound (5), we
ned in size and secretory function by the continued actions of have found a series of novel arylpiperazine derivatives.
androgens. It is well established that androgens play a major Herein, we describe the results of our studies on the synthe-
role in human prostate disorders.7) Thus compounds that ses and structure–activity relationships of these arylpiper-
block the action or synthesis of androgens have been proven azine derivatives as novel nonsteroidal AR antagonists.
useful in the treatment of diseases such as prostate cancer,
benign prostatic hypertrophy, hirsutism, and acne.8—11)
Chemistry
AR antagonists,12,13) including cyproterone acetate (1),14)
flutamide (2),15—17) nilutamide (3),18) and bicalutamide
(4),19—22) have been used clinically for the treatment of
prostate cancer (Fig. 1). Cyproterone acetate (1), which is
classified as a steroidal AR antagonist, possesses progesta-
tional activity and suppresses the secretion of gonadotrop-
hins, both of which are unwanted side effects. A number of
nonsteroidal AR antagonists have been reported in the litera-
ture23—30) and three of these, flutamide (2), nilutamide (3),
and bicalutamide (4), are used clinically in conjunction with
gonadotropin-releasing hormone (GnRH) agonists.31) These
Compounds selected for biological evaluation were
Reagents: (a) Cyclic amines, DMF; (b) (4-F)PhCHO, NaBH(OAc)3, AcOH, CH2Cl2;
(c) (4-F)PhSO2Cl, Et3N, THF; (d) (4-F)PhCOCl, Et3N, THF; (e) (4-F)PhCOCH2Br,
K2CO3, DMF; (f) (4-F)PhCO(CH2)2Cl, K2CO3, NaI, DMF; (g) (4-F)PhNCO, CH2Cl2;
(h) NaBH4, THF.
Fig. 1. Structures of Androgen Antagonists Used in Therapeutics
Chart 1
∗ To whom correspondence should be addressed. e-mail: kinoyama@yamanouchi.co.jp
© 2004 Pharmaceutical Society of Japan

November 2004
1331
prepared as described in Chart 1. Compounds 11—16 were cyano analogue 9 showed weak activity, the introduction of
prepared by the ipso substitution of 4-fluoro-2-trifluorome- an additional trifluoromethyl group (11), having a similar
thylbenzonitrile with the corresponding cyclic amines; good moiety as compared to bicalutamide (4), led to a 6-fold
yields were observed. Synthesis of the benzyl derivative (17) increase in the AR antagonistic activity relative to compound
was achieved by the reductive amination of the intermediate 5. These results suggested that the electron-withdrawing
piperazine (14) with 4-fluorobenzaldehyde. The piperazine effects and/or the hydrophobicity of the trifluoromethyl
(14) was treated with 4-fluorophenylsulfonyl chloride or group may be very important for AR antagonistic activity.
4-fluorobenzoyl chloride to afford 18 and 19 respectively, in Subsequently, we carried out the replacement of the morpho-
excellent yields. Alkylation of the piperazine (14) with 4-flu- line ring with other cyclic amines. Among these cyclic amine
orophenacyl bromide or 3-chloro-4ꢀ-fluoropropiophenone analogues (12—15), only the N-methyl-piperazine derivative
with sodium iodide afforded 20 and 21, respectively. Treat- (15) exhibited a potent inhibitory activity. Therefore we
ment of 14 with 4-fluorophenyl isocyanate provided the urea carried out further modifications of the substituents on the
derivative (22). Reduction of the carbonyl group of 20 with piperazine framework (Table 2).
sodium borohydride gave the corresponding secondary alco-
hol analogue (23).
The 4-fluorophenyl derivative (16) exhibited a 2-fold
enhancement in inhibitory activity over compound 15
(IC50ꢁ0.54, 1.0 mM, respectively). Insertion of a methylene
linkage between the piperazine and the 4-fluorophenyl ring,
Results and Discussion
All the analogues prepared were evaluated for their AR such as 17, resulted in a 3-fold decrease in its inhibitory
antagonistic activity using a reporter assay; the resulting IC50 activity relative to 16. Compound 18, which contained a
values are listed in Tables 1 and 2.
4-fluorophenylsulfonyl moiety like bicalutamide, was less
We selected compound 5, which was discovered from HTS active than 17. On the other hand, the corresponding amide
with an IC50 value of 3.1 mM, as the lead compound and derivative (19) exhibited more potent AR antagonistic activ-
started to make modifications to compound 5. Firstly, we ity than compounds 17 and 18. These results suggested that
investigated the effect of the substituents on the phenyl ring. the conformation of the piperazine framework may be impor-
The removal of the amino group of 5 resulted in a slight tant. Insertion of a methylene group into compound 19, such
decrease in the AR antagonistic activity (compound 6). as 20 and 21, led to a decrease in potency indicating that the
Furthermore we try to replace the toxicity concerned nitro long distance between the 4-fluorophenyl ring and piperazine
group by other groups. As shown in compounds 7 and 8, framework was unfavorable. Reduction of the carbonyl group
introduction of an electron-donating group afforded very (23) resulted in a slight increase in inhibitory activity relative
deleterious effects on the inhibitory activity. Although the to 20. Interestingly, the urea derivative (22) exhibited the
most potent AR antagonistic activity in this series with an
Table 1. AR Antagonistic Activities of Arylmorpholine and Arylpiper-
IC50 value of 0.47 mM, which is more potent than bicalu-
azine Derivatives
Compound
Structure
IC50 (mM)a)
Table 2. AR Antagonistic Activities of Arylpiperazine Derivatives
5
6
7
8
9
3.1
4.3
Compound
Structure
Me
IC50 (mM)a)
1.0
NEb)
10%b)
43%b)
15
16
0.54
17
18
19
1.8
2.6
11
12
13
14
0.51
48%b)
NEb)
0.81
20
21
2.3
51%b)
18%b)
22
0.47
(YM-92088)
15
4
1.0
23
1.5
0.89
4
0.89
a) Compounds were tested for their ability to inhibit AR mediated transcriptional
activation using a reporter assay. b) Percent inhibition at 10 mM. NE: No effect.
a, b) Refer to Table 1.

1332
Vol. 52, No. 11
(DMSO-d6) d: 2.78 (3H, s), 3.10 (2H, br), 3.35 (4H, br), 4.22 (2H, br), 7.34
(1H, dd, Jꢁ8.8, 2.4 Hz), 7.43 (1H, d, Jꢁ2.4 Hz), 7.93 (1H, d, Jꢁ8.8 Hz),
11.29 (1H, br). FAB-MS m/z: 270 (MꢃHꢃ). Anal. Calcd for C13H14N3F3·
HCl·0.1H2O: C, 50.77; H, 4.98; N, 13.66; Cl, 11.53; F, 18.53. Found: C,
50.61; H, 4.95; N, 13.74; Cl, 11.48; F, 18.62.
tamide (4) with an IC50 value of 0.89 mM.
Conclusion
We have succeeded in the identification of a novel lead
compound as an AR antagonist through an HTS. The origi-
nal lead 5 inhibited the AR mediated transcriptional activity
with an IC50 value of 3.1 mM. Modification of the lead 5
resulted in the identification of the arylpiperazine derivatives
with improved AR antagonistic activity.
4-[4-(4-Fluorophenyl)piperazin-1-yl]-2-(trifluoromethyl)benzonitrile
(16) Compound 16 was prepared from 10 and 1-fluorophenylpiperazine in
1
78% yield, pale yellow solid. mp 104—105 °C (EtOH). H-NMR (DMSO-
d6) d: 3.19—3.26 (4H, m), 3.57—3.67 (4H, m), 6.97—7.12 (4H, m), 7.31
(1H, dd, Jꢁ8.8, 2.4 Hz), 7.38 (1H, d, Jꢁ2.4 Hz), 7.87 (1H, d, Jꢁ8.8 Hz).
FAB-MS m/z: 350 (MꢃHꢃ). Anal. Calcd for C18H15N3F4: C, 61.89; H, 4.33;
N, 12.03; F, 21.75. Found: C, 61.69; H, 4.22; N, 12.03; F, 21.96.
Compound 22 exhibited the most potent AR antagonistic
activity with an IC50 value of 0.47 mM in the reporter assay,
which is more potent than bicalutamide (4) which has an IC50
value of 0.89 mM. Compound 22 was also found to possess
potent AR binding affinity (Kiꢁ68 nM for rat prostate AR
4-[4-(4-Fluorobenzyl)piperazin-1-yl]-2-(trifluoromethyl)benzonitrile
Hydrochloride (17) To a solution of 14 (1.5 g, 5.88 mmol) in AcOH
(0.34 ml, 5.88 mmol) and CH2Cl2 (20 ml) was added benzaldehyde (0.63 ml,
5.88 mmol) and NaBH(OAc)3 (1.87 g, 8.82 mmol). After stirring at ambient
temperature for 4 h, the solution was concentrated in vacuo. The residue was
diluted with saturated aqueous NaHCO3 and extracted with AcOEt. The
organic layer was concentrated in vacuo. The residue was purified by silica
gel column chromatography (CHCl3/MeOHꢁ50/1) to give the free base of
the title compound (2.08 g, 97%) as a colorless oil. The free base of 17
(470 mg, 1.29 mmol) was dissolved in AcOEt and the solution was treated
with 4 M solution of HCl in AcOEt. The precipitate was filtered off and
washed with AcOEt to give the title compound (408 mg, 79%) as a colorless
solid. mp 221—223 °C. 1H-NMR (DMSO-d6) d: 3.02—3.20 (2H, m),
3.25—3.40 (2H, m), 3.45—3.60 (2H, m), 4.14—4.30 (2H, m), 4.32—4.45
(2H, m), 7.26—7.35 (3H, m), 7.41 (1H, d, Jꢁ2.0 Hz), 7.67—7.78 (2H, m),
7.92 (1H, d, Jꢁ8.8 Hz), 11.99 (1H, br). FAB-MS m/z: 364 (MꢃHꢃ). Anal.
Calcd for C19H17N3F4·HCl: C, 57.08; H, 4.54; N, 10.51; Cl, 8.87; F, 19.01.
Found: C, 57.14; H, 4.32; N, 10.54; Cl, 8.77; F, 19.29.
4-{4-[(4-Fluorophenyl)sulfonyl]piperazin-1-yl}-2-(trifluoromethyl)-
benzonitrile (18) To a mixture of 14 (300 mg, 1.18 mmol) and Et3N
(232 mg, 2.29 mmol) in THF (5 ml) was added 4-fluorobenzenesulfonyl
chloride (274 mg, 1.41 mmol) and stirred at room temperature for 2 h. The
reaction mixture was added saturated aqueous NaHCO3, stirred for 5 h,
extracted with AcOEt, and washed with H2O. The organic layer was dried
over Na2SO4, concentrated in vacuo and crystallized from AcOEt/
n-hexane to give the title compound (400 mg, 82%) as a colorless solid. mp
162—163 °C. 1H-NMR (DMSO-d6) d: 2.98—3.10 (4H, m), 3.49—3.64
(4H, m), 7.16—7.26 (1H, m), 7.30 (1H, d, Jꢁ2.0 Hz), 7.45—7.56 (2H, m),
7.79—7.91 (3H, m). FAB-MS m/z: 414 (MꢃHꢃ). Anal. Calcd for
C18H15N3O2F4S: C, 52.30; H, 3.66; N, 10.16; F, 18.38; S, 7.76. Found:
C, 52.31; H, 3.61; N, 10.26; F, 18.77; S, 7.74.
3
against H-mibolerone)32) and no intrinsic AR agonistic ac-
tivity at concentrations up to 10 mM. Furthermore, compound
22 significantly inhibited the growth of prostate weights in
rats (data not shown). These results suggest that compound
22 (YM-92088) has potential as a novel nonsteroidal AR
antagonist. We will report the details in due course.
Experimental
In general, all reagents and solvents were of commercial quality and were
used without further purification unless otherwise noted. Melting points
were determined on a Yanaco MP-500D micro melting point apparatus
without correction. 1H-NMR spectra were measured with a JMN-EX400
spectrometer; chemical shifts are expressed in d units using tetramethylsi-
lane as the standard (in NMR description, sꢁsinglet, dꢁdoublet, tꢁtriplet,
mꢁmultiplet and brꢁbroad peak). MS spectra were determined with a
JEOL JMS-LX2000 spectrometer. Elemental analysis was performed with a
Yanaco MT-5 microanalyzer (C, H, N) and Yokogawa IC-7000S ion
chromatographic analyzer (halogens) and were within ꢂ0.4% of theoretical
values.
Compounds 5—9 were prepared according to literatures.33—37)
4-Morpholino-2-trifluoromethylbenzonitrile (11) To a solution of
4-fluoro-2-(trifluoromethyl)benzonitrile (10, 600 mg, 3.17 mmol) in N,N-di-
methylformamide (DMF, 10 ml) was added morpholine (1.1 g, 12.7 mmol)
at ambient temperature and stirred at 80 °C for 21 h. The reaction mixture
was diluted with H2O. The precipitate was filtered off and washed with H2O
to give the title compound (775 g, 95%) as a colorless solid. mp 200 °C.
1H-NMR (DMSO-d6) d: 3.43 (4H, t, Jꢁ4.9 Hz), 3.73 (4H, t, Jꢁ4.9 Hz), 7.25
(1H, dd, Jꢁ8.8, 2.5 Hz), 7.32 (1H, d, Jꢁ2.5 Hz), 7.86 (1H, d, Jꢁ
8.8 Hz). FAB-MS m/z: 257 (MꢃHꢃ). Anal. Calcd for C12H11N2OF3: C, 56.25;
H, 4.33; N, 10.93; F, 22.24. Found: C, 56.23; H, 4.26; N, 11.01; F, 22.39.
The following compounds 12—16 were prepared using a procedure simi-
lar to described for 11 from the fluoride 10 and the corresponding amines.
4-Piperidino-2-trifluoromethylbenzonitrile (12) Compound 12 was
prepared from 10 and piperidine in 70% yield, colorless solid. mp
68—69 °C. 1H-NMR (DMSO-d6) d: 1.48—1.68 (6H, m), 3.40—3.52
(4H, m), 7.20 (1H, dd, Jꢁ9.3, 2.4 Hz), 7.26 (1H, d, Jꢁ2.4 Hz), 7.78 (1H, d,
Jꢁ9.3 Hz). FAB-MS m/z: 255 (MꢃHꢃ). Anal. Calcd for C13H13N2F3: C,
61.41; H, 5.15; N, 11.02; F, 22.42. Found: C, 61.34; H, 5.10; N, 11.04; F,
22.60.
4-(3-Oxopiperazin-1-yl)-2-(trifluoromethyl)benzonitrile (13) Com-
pound 13 was prepared from 10 and 3-oxopiperazine in 33% yield, pale
yellow solid. mp 205—207 °C (AcOEt). 1H-NMR (DMSO-d6) d: 3.29—
3.37 (2H, m), 3.61—3.68 (2H, m), 3.98 (2H, s), 7.17 (1H, dd, Jꢁ9.1,
2.7 Hz), 7.24 (1H, d, Jꢁ2.7 Hz), 7.85 (1H, d, Jꢁ9.1 Hz), 8.26 (1H, br). FAB-
MS m/z: 270 (MꢃHꢃ). Anal. Calcd for C12H10N3OF3: C, 53.54; H, 3.74; N,
15.61; F, 21.17. Found: C, 53.42; H, 3.51; N, 15.65; F, 21.12.
4-Piperazin-1-yl-2-(trifluoromethyl)benzonitrile Hydrochloride (14)
Compound 14 was prepared from 10 and piperazine in 73% yield, colorless
solid. mp 269—271 °C (MeOH/AcOEt). 1H-NMR (DMSO-d6) d: 3.16—
3.24 (4H, m), 3.67—3.77 (4H, m), 7.31 (1H, dd, Jꢁ8.8, 2.4 Hz), 7.40 (1H,
d, Jꢁ2.4 Hz), 7.91 (1H, d, Jꢁ8.8 Hz), 9.45 (1H, br). FAB-MS m/z: 256
(MꢃHꢃ). Anal. Calcd for C12H12N3F3·HCl: C, 49.41; H, 4.49; N, 14.41; Cl,
12.15; F, 19.54. Found: C, 49.21; H, 4.47; N, 14.46; Cl, 12.04; F, 19.63.
4-(4-Methylpiperazin-1-yl)-2-(trifluoromethyl)benzonitrile Hydrochl-
oride (15) Compound 15 was prepared from 10 and 1-methylpiperazine in
76% yield, colorless solid. mp 226—228 °C (EtOH/AcOEt). 1H-NMR
4-[4-(4-Fluorobenzoyl)piperazin-1-yl]-2-(trifluoromethyl)benzonitrile
(19) To a mixture of 14 (300 mg, 1.18 mmol) and Et3N (178 mg,
1.96 mmol) in THF (5 ml) was added 4-fluorobenzoyl chloride (223 mg,
1.41 mmol) and stirred at room temperature for 1 h. The reaction mixture
was added saturated aqueous NaHCO3, stirred for 30 min, extracted with
AcOEt, and washed with H2O. The organic layer was dried over Na2SO4,
concentrated in vacuo and crystallized from AcOEt/n-hexane to give the title
1
compound (308 mg, 69%) as a colorless solid. mp 171—173 °C. H-NMR
(DMSO-d6) d: 3.40—3.84 (8H, m), 7.23 (1H, dd, Jꢁ8.8, 2.4 Hz), 7.27—
7.34 (3H, m), 7.50—7.57 (2H, m), 7.87 (1H, d, Jꢁ8.8 Hz). FAB-MS m/z:
378 (MꢃHꢃ). Anal. Calcd for C19H15N3OF4: C, 60.48; H, 4.01; N, 11.14;
F, 20.14. Found: C, 60.49; H, 4.02; N, 11.19; F, 20.20.
4-{4-[2-(4-Fluorophenyl)-2-oxoethyl]piperazin-1-yl}-2-(trifluoro-
methyl)benzonitrile (20) To a mixture of 14 (500 mg, 1.96 mmol) and
K2CO3 (300 mg, 2.16 mmol) in DMF (10 ml) was added 4-fluorophenacyl
bromide (425 mg, 1.96 mmol) and stirred at room temperature for 1 h. The
reaction mixture was diluted with H2O, extracted with AcOEt, and washed
with H2O. The organic layer was dried over Na2SO4, and concentrated
in vacuo. The residue was purified by silica gel column chromatography
(CHCl3/MeOHꢁ50/1) and crystallized from AcOEt/i-Pr2O to give the title
compound (709 mg, 92%) as a colorless solid. mp 162 °C. 1H-NMR
(DMSO-d6) d: 2.65 (4H, d, Jꢁ4.8 Hz), 3.47 (4H, d, Jꢁ4.8 Hz), 3.93 (2H, s),
7.24 (1H, dd, Jꢁ8.8, 2.4 Hz), 7.31 (1H, d, Jꢁ2.4 Hz), 7.33—7.39 (2H, m),
7.83 (1H, d, Jꢁ8.8 Hz), 8.05—8.13 (2H, m). FAB-MS m/z: 392 (MꢃHꢃ).
Anal. Calcd for C20H17N3OF4: C, 61.38; H, 4.38; N, 10.74; F, 19.42. Found:
C, 61.49; H, 4.15; N, 10.64; F, 19.54.
4-{4-[3-(4-Fluorophenyl)-3-oxopropyl]piperazin-1-yl}-2-(trifluoro-
methyl)benzonitrile (21) To a mixture of 14 (280 mg, 1.10 mmol), NaI
(1.65 g, 11.0 mmol) and K2CO3 (230 mg, 1.65 mmol) in DMF (10 ml) was
added 3-chloro-4ꢀ-fluoropropiophenone (230 mg, 1.21 mmol) and stirred at
80 °C for 14 h. The reaction mixture was diluted with H2O. The precipitate

November 2004
1333
was filtered off and washed with H2O, and recrystallized from AcOEt/i-Pr2O
X: (luciferase activity of AR/CHO#3)/(luciferase activity of SV/CHO#10)
in the presence of 0.3 nM of DHT and the tested compound
The concentration of compounds showing 50% of AR antagonistic activ-
ity, IC50 values, were obtained by nonlinear analysis using statistical analysis
to give the title compound (334 mg, 75%) as a colorless solid. mp 144—
1
145 °C. H-NMR (DMSO-d6) d: 2.45—2.85 (6H, m), 3.15—3.55 (6H, m),
7.18—7.42 (4H, m), 7.83 (1H, d, Jꢁ8.3 Hz), 8.02—8.15 (2H, m). FAB-MS
m/z: 406 (MꢃHꢃ). Anal. Calcd for C21H19N3OF4: C, 62.22; H, 4.72; system (SAS).
N, 10.37; F, 18.75. Found: C, 61.94; H, 4.75; N, 10.34; F, 19.13.
Acknowledgements We thank the staff of the Division of Analytical
Science Laboratories for the elemental analysis and spectral measurements.
4-[4-Cyano-3-(trifluoromethyl)phenyl]-N-(4-fluorophenyl)piperazine-
1-carboxamide (22) To a solution of 14 (780 mg, 2.64 mmol) in CH2Cl2
(10 ml) was added 4-fluorophenyl isocyanate (0.36 ml, 3.17 mmol) and
stirred at room temperature for 4 h. The reaction mixture was concentrated
in vacuo. The residue was purified by silica gel column chromatography
(n-hexane/AcOEtꢁ1/3) and crystallized from MeOH/Et2O to give the title
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column chromatography (CHCl3/MeOHꢁ30/1) to give the free base of the
title compound (170 mg, 51%) as a colorless oil. The free base of 23 was
dissolved in AcOEt and the solution was treated with 4 M solution of HCl in
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Evaluation of Transcriptional Activity for Human Androgen Recep-
tor (a) Establishment of CHO cells stably transfected with human andro-
gen receptor gene and MMTV-luciferase reporter gene or SV40-luciferase
gene;
Chinese hamster ovary (CHO) cells were maintained in Alpha-modified
Eagle’s medium supplemented with 10% fetal bovine serum (FBS). The cul-
ture medium of neomycin-resistant clone cells was supplemented with 10%
dextran-coated charcoal-stripped FBS (DCC-FBS) and 500 mg/ml of
neomycin. The CHO cells were transfected at 40—70% confluence in 10-cm
petri dishes with a total of 20 mg DNA (pMAMneoLUC; MMTV-luciferase
reporter plasmid and pSG5-hAR; human androgen receptor expression
plasmid, or SV40-LUC; SV40-luciferase reporter plasmid containing
neomycin resistant gene) by calcium phosphate mediated transfection. The
stable transfected cells were selected in the culture medium supplemented
with neomycin. The selected clone was designated as AR/CHO#3 (human
AR gene and MMTV-luciferase reporter gene integrated CHO cell) or
SV/CHO#10 (SV-40-luciferase reporter gene integrated CHO cell), respec-
tively;
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(b) Activities of the tested compounds to inhibit androgen receptor medi-
ated transcription induced by DHT (AR antagonistic activity).
The stable transfected AR/CHO#3 or SV/CHO#10 cells were plated onto
96 well luminoplates (Packard) at a density of 2ꢄ104 cells/well, respectively.
Four to 8 h later, the medium was changed to the medium containing DMSO,
0.3 nM of DHT, or 0.3 nM of DHT and the tested compound. At the end of
incubation, the medium was removed and then cells were lysed with 20 ml of
lysis buffer [25 mM Tris–HCl (pH 7.8), 2 mM dithiothreitol, 2 mM 1,2-cyclo-
hexanediamine-tetraacetic acid, 10% glycerol and 1% TritonX-100].
Luciferase substrate [20 mM Tris–HCl (pH 7.8), 1.07 mM (MgCO3)4
Mg(OH)2·5H2O, 2.67 mM MgSO4·7H2O, 0.1 mM EDTA, 33.3 mM dithiothre-
itol, 0.27 mM coenzyme A, 0.47 mM luciferin, 0.53 mM ATP] was added and
luciferase activity was measured with a ML3000 luminometer (Dynatech
Laboratories). AR antagonistic activities were calculated by formula below.
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AR antagonistic activity (%)ꢁ100(IꢅX)/(IꢅB)
I: (luciferase activity of AR/CHO#3)/(luciferase activity of SV/CHO#10)
in the presence of 0.3 nM of DHT
B: (luciferase activity of AR/CHO#3)/(luciferase activity of SV/CHO#10)
in the presence of DMSO
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Synthesis, 12, 1147—1148 (1990).