4-(Anilino)pyrrole-2-carboxamides: Novel non-steroidal/non-anilide type androgen antagonists effective upon human prostate tumor LNCaP cells with mutated nuclear androgen receptor

Article abstract of DOI:10.1016/j.bmc.2008.05.063

Various 4-(anilino)pyrrole-2-carboxamides were designed and synthesized as novel androgen receptor (AR) antagonists without steroidal or anilide structure, based on our strategy for developing full antagonists of nuclear receptors. Introduction of a bulky

Full text of DOI:10.1016/j.bmc.2008.05.063

Bioorganic & Medicinal Chemistry 16 (2008) 6799–6812
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
4-(Anilino)pyrrole-2-carboxamides: Novel non-steroidal/non-anilide type
androgen antagonists effective upon human prostate tumor LNCaP cells
with mutated nuclear androgen receptor
Ken-ichi Wakabayashi a, Keisuke Imai b, Hiroyuki Miyachi a, Yuichi Hashimoto a, Aya Tanatani c,
*
a Institute of Molecular & Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan
b Drug Discovery Research, Astellas Pharma Inc., Miyukigaoka 21, Tsukuba, Ibaraki 305-8585, Japan
c Department of Chemistry, Faculty of Science, Ochanomizu University, 2-1-1 Otsuka, Bunkyo-ku, Tokyo 112-8610, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Various 4-(anilino)pyrrole-2-carboxamides were designed and synthesized as novel androgen receptor
(AR) antagonists without steroidal or anilide structure, based on our strategy for developing full antago-
nists of nuclear receptors. Introduction of a bulky N-alkyl group, such as a cyclohexylmethyl or benzyl
group, increased the binding affinity for wild-type AR and the potency for growth inhibition of andro-
gen-dependent SC-3 cells. Among the compounds obtained, N-[4-[(benzyl)(4-nitrophenyl)amino]-1-
methylpyrrole-2-carbonyl]pyrrolidine (22) is as potent an AR antagonist as the typical anilide-type AR
antagonists hydroxyflutamide and bicalutamide. Further, compound 22 had potent binding affinity for
T877A mutated AR, and dose-dependently inhibited the testosterone-induced production of prostate-
specific antigen in LNCaP cells bearing T877A AR.
Received 28 February 2008
Revised 13 May 2008
Accepted 28 May 2008
Available online 18 June 2008
Keywords:
Androgen
Antagonist
Pyrrole-2-carboxamide
Mutated receptor
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
and its AR-antagonistic activity was found as an undesired side
effect.
Androgen receptor (AR) is a member of the nuclear receptor
superfamily,1 and is functionally regulated by the binding of andro-
gens, such as testosterone (1, Fig. 1) and its more potent metabolite,
dihydrotestosterone (DHT, 2). These ligands induce sequential con-
formational changes of the receptor that result in receptor–protein
and receptor–DNA interactions. Typical AR antagonists antagonize
the biological responses elicited by endogenous and/or exogenous
androgens by competitively inhibiting the binding of the latter to
AR, and are expected to be effective for the treatment of andro-
gen-dependent prostate cancer.
The AR antagonists so far known can be classified into two struc-
tural types, that is, steroidal and non-steroidal compounds.2–4 Mod-
ification of the structures of endogenous androgens, such as 1 and 2,
afforded steroid-type derivatives, including cyproterone acetate (3),
but these compounds often possess side effects that arise from
cross-reactivity with other steroid hormone nuclear receptors, such
as progesterone and estrogen receptors, and it is generally difficult
to separate effects on androgenic activities from those on anabolic
activities. For example, cyproterone acetate (3) was originally
developed as a progestin that suppresses gonadotropin release,5
The non-steroidal AR antagonists not only overcome these
difficulties, but also achieve tissue selectivity. Moreover, non-ste-
roidal skeletons represent robust scaffolds whose structural
modification/development affords wide structural diversity as
compared with the steroidal derivatives. For these reasons, vari-
ous non-steroidal AR antagonists have been synthesized, of
which some, such as flutamide (4) and bicalutamide (Casodex,
6), are clinically used to treat prostate cancer (Fig. 1).6–9 These
AR antagonists have a common anilide structure with electron-
withdrawing substituents. Although these anilide-type AR antag-
onists are useful for the treatment of prostate cancer in the early
stage, prostate cancer often advances to a ‘hormone-refractory’
state in which the disease progresses even in the presence of
continued androgen ablation or antagonist therapy, suggesting
the development of androgen-independent prostate cancer
cells.10 In some cases, these antagonists may act as AR agonists
toward the androgen-independent prostate cancer cells, and
aggravate the disease. One possible reason for the hormone-
refractory state is considered to be the mutation of AR.11–13
For example, hydroxyflutamide (5), an active form of 4, and 6
act as AR agonists toward T877A and W741L/W741C AR
mutants, respectively.14,15 Thus, new types of non-steroidal AR
antagonists are needed to treat prostate cancers resistant to
the known AR antagonists.16–21
* Corresponding author. Tel./fax: +81 3 5978 2716.
E-mail address: tanatani.aya@ocha.ac.jp (A. Tanatani).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.05.063

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K. Wakabayashi et al. / Bioorg. Med. Chem. 16 (2008) 6799–6812
O
OH
OH
OAc
O
O
O
Cl
testosterone (1)
5α-dihydrotestosterone (2)
cyproterone acetate (CPA, 3)
H
N
H
N
R
OH
O O2S
F
O
O2N
NC
CF3
CF3
4 R = H, flutamide
bicalutamide (Casodex, 6)
5 R = OH, hydroxyflutamide
Figure 1. Structures of steroidal and non-steroidal androgen ligands.
Recently, X-ray crystallographic investigations of nuclear recep-
tor–ligand interactions have uncovered possible mechanisms of the
agonistic/antagonistic actions.22–24 Thus, the ligand binding domain
2. Results and discussion
2.1. Design and synthesis of novel AR antagonists
(LBD) of nuclear receptors contains 12 a-helices and two short b-
turns, and the C-terminal helix (H12) in the LBD plays a critical role
in the ligand-dependent activation of the receptor. When the ago-
nist binds to the receptor, H12 is repositioned and serves as a ‘lid’
on the ligand-binding pocket to stabilize the ligand–receptor com-
plex.25 This agonist-induced conformational change of the receptor
results in the dissociation of a corepressor and the recruitment of a
coactivator. On the other hand, the binding of the antagonist to nu-
clear receptor does not induce the proper folding of H12, and conse-
quently H12 is displaced from the active conformation of the
nuclear receptor, and folds over the activation function 2 (AF2) re-
gion to disrupt coactivator recruitment or to stabilize the recep-
tor–corepressor complex.26
Our previous studies on the SARs of 8 and its derivatives as AR
antagonists indicated that a general structural feature for AR-
Taking into account these molecular findings, it is one of effective
molecular designs of nuclear receptor antagonists to disturb the
proper conformation of H12.27 Based on this idea, we have recently
developed novel vitamin D antagonists.28,29 Computer-assisted
docking studies suggested that the bulky substituent on the side
chain of vitamin D skeleton inhibits the folding of H12 of vitamin
D nuclear receptor to elicit the antagonistic activity. We performed
similar structural analyses for AR antagonists, and developed iso-
xazolone derivatives as novel non-steroidal AR antagonists.27 That
is, an isoxazolone derivative 7 (Fig. 2) was obtained as an AR ligand
candidate by virtual screening of a database of commercially avail-
able molecules. The structural development of 7 afforded the potent
AR antagonist 8 (ISOP-4), bearing a bulky chlorophenyl group that
may disturb the H12 folding. Compound 8 possessed high binding
affinity for AR and potently suppressed the growth of an androgen-
dependent Shionogi carcinoma cell line, SC-3 (IC50 = 0.4 lM). In par-
ticular, 8 acted as an antagonist toward the mutantAR (T877A) in the
LNCaPcellline, althoughits potencyis nothigh. Theresultsindicated
our strategy that H12-folding inhibitor-type AR antagonists might
be effective in both wild-type and mutated ARs, and also suggested
that the development of non-steroidal/non-anilide type AR antago-
nists would be feasible. Recently, we reported another type of AR
antagonists, 4-(anilino)pyrrole-2-carboxamide derivatives (9, and
10), based on 8 as a lead compound.30 These compounds showed
moderate AR binding affinity and inhibited the growth of SC-3 cells
at concentrations of the order of 10 lM, although these compounds
are weaker AR antagonists than 5 or 6. In this paper, we describe the
development of novel AR antagonists possessing higher affinity than
5 and6 for both wild-type and mutant Ars, by using 9 and 10 as lead
compounds.
Figure 2. Design of novel non-steroidal/non-anilide type AR antagonists. The IC50
values in SC-3 growth-inhibitory assay are shown in parentheses.

K. Wakabayashi et al. / Bioorg. Med. Chem. 16 (2008) 6799–6812
6801
antagonistic activity consists of two aromatic rings connected by a
suitable linking group, with one benzene ring bearing a dialkylami-
no group, that is, such structures meet the requirements for bind-
ing to the receptor (Fig. 2).27 Comparison of the partial agonistic
activity of 7 and the antagonistic activity of 8 showed that the
chlorophenyl group of 8 is thought to disturb the folding of H12
of AR. Similarly, the pyrrolecarboxamides, 9 and 10, have two aro-
matic rings linked by a nitrogen atom.30 In these compounds, the
terminal pyrrolidine ring is attached to the aromatic ring through
the carbonyl group, affording a more stable amide structure. When
we compare the molecular structures of 8 and 9/10, the pyrrole-
carboxamides are smaller in size than the corresponding moiety
of 8, and this might reduce the AR binding affinity. In other words,
the N-(pyrrolyl)aniline skeleton of 9 and 10 may correspond to the
phenylmethanoisoxazolone structure of 8. Therefore, introduction
of a bulky substituent corresponding to the chlorophenyl group
of 8 into 9/10 should be effective for the development of novel
AR antagonists. We thus designed and synthesized novel (N, N-
disubstituted amino)pyrrolecarboxamide derivatives as shown in
Schemes 1–3.
1). In the case of an alkyl halide bearing a hydroxyl group, direct
N-alkylation proceeded in low yield (31% for compound 17). There-
fore, the hydroxyl group was protected with an acetyl (for
compound 16) or tert-butyldimethylsilyl group (for compounds
25 and 26), and then deprotected after the N-alkylation. In order
to evaluate substituent effects on the phenyl ring of 22, various
N-aryl-N-benzylaminopyrrolecarboxamides, 35–40, were synthe-
sized (Scheme 2). Ethyl 4-nitropyrrole-2-carboxylate (27) was
converted to the aminopyrrolecarboxamide 28 in four steps.30
Compound 28 was treated with substituted bromobenzene in the
presence of Pd2(dba)3, BINAP, and Cs2CO3 to afford compounds
29–34,31 which were N-benzylated to give compounds 35–40.
The pyrrolidine ring of 8 is important for the AR-antagonistic
activity. Therefore, several modifications of the pyrrolidine ring
of 22 were performed, that is, introduction of ester (44), hydroxy-
methyl (45), carboxylate (46), and various carboxamides (47–52)
(Scheme 3). N-Methylation followed by catalytic hydrogenation
of ethyl 4-nitropyrrole-2-carboxylate (27) afforded 42. Condensa-
tion of 42 and 1-bromo-4-nitrobenzene in the presence of
Pd2(dba)3, BINAP, and Cs2CO3 afforded 43 in 56% yield. In this reac-
tion, when we used t-BuONa instead of Cs2CO3,43 was obtained in
lower yield. Benzylation of 43 gave 44 in 94% yield, and 44 was re-
duced with LiAlH4 to afford 45 in 68% yield, or saponified with
The N,N-disubstituted 4-(anilino)pyrrole-2-carboxamides 11–
17 and 18–24 were synthesized from 9 and 10, respectively, by
treatment with NaH, followed by addition of alkyl halide (Scheme
11 R1 = CH3
R1
12 R1 = CH2CH3
13 R1 = CH(CH3)2
14 R1 = CH2-cyclohexyl
15 R1 = benzyl
H
N
N
a)
N
N
N
N
a, b) for 16
O
O
16 R1 = 4-hydroxybenzyl
17 R1 = 4-(hydroxymethyl)benzyl
9
18 R1 = CH3
19 R1 = CH2CH3
20 R1 = CH(CH3)2
21 R1 = CH2-cyclohexyl
22 R1 = benzyl
R1
N
H
N
a)
N
N
N
O2N
N
a, c) for 26
O
O2N
O
10
23 R1 = 4-fluorobenzyl
24 R1 = 4-methylbenzyl
25 R1 = 4-hydroxybenzyl
26 R1 = 4-(hydroxymethyl)benzyl
Scheme 1. Syntheses of (N, N-disubstituted amino)pyrrolecarboxamides. Reagents: (a) Alkyl halide, NaH, DMF; (b) K2CO3, MeOH; (c) TBAF, DMF.
R2
H
O2N
H2N
N
e)
a - d)
OEt
N
N
N
H
N
N
O
O
O
27
28
29-34
29, 35 R2 = 2-NO2
30, 36 R2 = 4 NO2, 3-CF3
31, 37 R2 = 4-CN
R2
-
N
e)
N
32, 38 R2 = 4-CF3
33, 39 R2 = 4-acetyl
34, 40 R2 = 4-COOCH3
N
O
35-40
Scheme 2. Syntheses of N-aryl-N-benzylaminopyrrolecarboxamides. Reagents and conditions: (a) 15% KOH, reflux; (b) (COCl2)2, CH2Cl2, DMF then pyrrolidine, CH3CN;
(c) NaH, CH3I, DMF; (d) 10% Pd/C, H2, AcOEt; (e) R3-PhBr, BINAP, Pd2(dba)3, Cs2CO3, toluene, 80 °C; (f) BnBr, NaH, DMF.

6802
K. Wakabayashi et al. / Bioorg. Med. Chem. 16 (2008) 6799–6812
H
N
O2N
X
OEt
OEt
a, b)
c)
OEt
N
H
N
O
N
O
O2N
O
41 X = NO2
42 X = NH2
43
27
d)
e)
N
N
OEt
OH
N
N
O2N
O2N
O
44
45
f)
R3
N
g) for 48
N
N
OH
R4
h) for 47, 49 - 51
h, i) for 52
N
O2N
N
O2N
O
O
47 - 52
46
Scheme 3. Syntheses of N-aryl-N-benzylaminopyrrole derivatives. Reagents and conditions: (a) NaH, CH3I, DMF; (b) 10% Pd/C, H2, AcOEt; (c) 1-bromo-4-nitrobenzene, BINAP,
Pd2(dba)3, Cs2CO3, toluene, 80 °C; (d) BnCl, NaH, DMF; (e) LiAlH4, THF, 0 °C; (f) 30% KOH, MeOH, THF; (g) diethylamine, HOBt, EDCI, CH2Cl2; (h) MsCl, Et3N, THF, 0 °C then
R1R2NH; (i) concd HCl, MeOH, 65 °C.
potassium hydroxide to produce 46 in 96% yield. The key interme-
diate 46 was converted to various amides 47–52 by using HOBt/
EDCI or MsCl/Et3N as coupling reagents.
(IC50 = 0.44
say conditions, and showed similar inhibitory activity to
(IC50 = 0.35 M) and 6 (IC50 = 0.27 M). In this case, also, the intro-
lM) did not show apparent cytotoxicity under the as-
5
l
l
duction of a further substituent, such as a fluoro, methyl, or hydro-
xyl group, on the benzyl group of 22 decreased the potency in
either of the assays.
2.2. Biological activities
The test compounds were first evaluated by using a combina-
tion of two in vitro assay systems, that is, a competitive binding as-
say of wild-type AR (wtAR) using [3H]testosterone and a growth
inhibition assay using androgen-dependent SC-3 cells.32 Activity
of the selected compounds toward mutated AR was analyzed by
the use of LNCaP cells, which express T877A point-mutated
AR.33–35 Quantitative assessment of androgenic and anti-andro-
genic activities of the compounds was performed by measurement
of the amount of prostate-specific antigen (PSA) produced by
LNCaP cells. PSA is a marker molecule of prostate tumor malig-
nancy and its production is known to be induced by androgens.36,37
None of the pyrrolecarboxamides exhibited AR-agonistic activ-
ity in our assay systems. The effect of the introduction of N-substit-
uents into compound 9 on the AR-antagonistic activities is shown
in Table 1. Introduction of a small N-substituent (compounds 11–
13) did not affect the activity, while introduction of a bulky substi-
tuent such as a cyclohexylmethyl or a benzyl group caused an
increase of the activity: the wtAR binding affinities of 14
Since some compounds bearing the xylidine moiety, such as 14
and 15, exhibited significant cytotoxicity, we examined further
structural modification, using 22 as the lead compound. For this
purpose, we synthesized several analogs of 22 having an elec-
tron-withdrawing group other than a nitro group on the benzene
ring, and determined their activities (Table 3). Among the synthe-
sized compounds, 22 was the most potent. Interestingly compound
36, having the same substituents (3-CF3, 4-NO2) as flutamide (4),
showed weaker activity than 22. Compound 37, having a 4-cyano
group, showed similar potency to 22, while replacement of the ni-
tro group of 22 with a trifluoromethyl, an acetyl, or an ester group
resulted in relatively strong binding affinity, but weaker antagonis-
tic activity as compared with 22 in SC-3 cell assay.
Next, we examined the modification of the terminal carboxam-
ide group. Except for compounds 49,51, and 52, all the derivatives
shown in Table 4 exhibited potent wtAR binding affinity, but the
potency in SC-3 cell growth inhibition assay did not correlate with
the binding affinity. The ester derivative 44 (Ki = 0.18
IC50 = 0.95 M) was nearly as active as compound 22 in both as-
says, while a carboxylic acid derivative 46 showed higher wtAR
binding affinity (Ki = 0.075 M) and exhibited partial agonistic
lM,
(Ki = 0.49
9 by one order of magnitude, and these compounds are as active
as 5 (Ki = 0.43 M). However, they showed high cytotoxicity in
SC-3 cell assay at concentrations above 1 M. Compounds 16 and
l
M) and 15 (Ki = 0.35
lM) are more potent than that of
l
l
l
l
activity in SC-3 growth inhibition assay (data not shown). Based
on these results, the amide derivatives seem to be more effective
as AR antagonists than the corresponding ester or carboxylic acid
derivatives, and compound 22 was the most potent AR antagonist
among the synthesized compounds, at least in our two assay
systems.
In the structural development of 8 into pyrrolecarboxamides,
we introduced a pyrrole ring instead of one of the two benzene
rings of 8. In order to confirm the significance of the pyrrolecarbox-
amide skeleton, the biological activities of the benzenecarboxa-
17, bearing a polar hydroxyl group, showed weaker wtAR affinity
than the corresponding derivative 15, but these compounds are
less toxic, and they inhibited SC-3 cell growth with similar potency
to 4. Similar SARs were observed among the derivatives of com-
pound 10 bearing a polar nitro group on the anilino moiety (Table
2). Among the synthesized compounds, the N-cyclohexylmethyl
(21, Ki = 0.52
much greater wtAR affinity and more potent SC-3 cell growth inhi-
bition. Unlike 14 and 15, compounds 21 (IC50 = 0.88 M) and 22
lM) and N-benzyl analogs (22, Ki = 0.11 lM) showed
l

K. Wakabayashi et al. / Bioorg. Med. Chem. 16 (2008) 6799–6812
6803
Table 1
wtAR binding affinity and antagonistic activity of compounds 9 and 11–17
R1
N
N
N
O
Compound
R1
wtAR binding affinitya (Ki,
l
M)
SC-3 growth-inhibitory activityb (IC50
, lM)
4
5
6
Flutamide
Hydroxyflutamide
(R)-Bicalutamide
H
CH3
CH2CH3
3.7 0.9
4.2 0.5
0.35 0.02
0.27 0.05
15.0 0.2
26
0.43 0.03
0.049 0.012
3.1 1.0
6.1 0.3
2.7 0.8
9
11
12
13
22.5 2.1
10
CH(CH3)2
2.7 1.6
c
14
15
16
0.49 0.54
0.35 0.13
0.69 0.05
2.2 1.8
—
c
—
OH
6.6 4.8
5.8 0.2
OH
17
a
Competitive binding assay using 10 nM [3H]testosterone and wtAR with test compounds. The conditions were the same as for other tables and Figure 3.
b
The antagonistic activity was assessed in terms of IC50 for the growth of SC-3 cells induced by 10 nM testosterone. The conditions were the same as for other tables and
Figure 3.
c
Toxicity was observed at 1 lM test compound.
Table 2
wtAR binding affinity and antagonistic activity of compounds 10 and 18–26
R1
N
N
N
O2N
O
Compound
R1
wtAR binding affinity (Ki,
l
M)
SC-3 growth-inhibitory activity (IC50, lM)
10
18
19
20
H
CH3
CH2CH3
CH(CH3)2
4.9 0.7
3.6 0.6
2.0 0.5
3.4 0.9
>30
>30
15.5 3.5
14.5 2.1
21
22
23
0.52 0.42
0.11 0.02
0.80 0.08
0.88 0.18
0.44 0.08
4.0 0.5
F
CH3
OH
24
0.81 0.22
4.7 0.4
25
26
0.73 0.02
1.8 0.5
4.8 0.7
5.5 2.6
OH

6804
K. Wakabayashi et al. / Bioorg. Med. Chem. 16 (2008) 6799–6812
Table 3
wtAR binding affinity and antagonistic activity of compounds 35–40
N
N
R2
N
O
Compound
R2
wtAR binding affinity (Ki,
lM)
SC-3 growth-inhibitory activity (IC50
0.44 0.08
, lM)
22
35
36
37
38
39
40
4-NO2
2-NO2
3-CF3, 4-NO2
4-CN
4-CF3
0.11 0.03
1.3 0.1
b
—
a
—
2.5 0.7
0.61 0.23
1.7 0.6
2.0 0.8
6.1 3.1
0.13 0.06
0.27 0.03
0.14 0.05
0.18 0.04
4-Acetyl
4-COOCH3
a
52% inhibition at 30
l
M test compound.
b
27% inhibition at 1
lM test compound.
Table 4
wtAR binding affinity and antagonistic activity of compounds 44–52
Compound
X (-CO-NR3R4)
wtAR binding affinity (Kj,
l
M)
SC-3 growth-inhibitory activity (IC50
0.44 0.08
, lM)
22
0.11 0.03
44
45
46
47
–COOEt
–CH2OH
–COOH
–CONH2
0.18 0.15
0.17 0.06
0.075 0.042
0.22
0.95 0.28
2.1 0.1
—
d
1.95
48
0.10 0.01
0.99 0.59
a
49
—
6.1 3.8
1.7 0.1
1.0 0.8
13.7 6.1
50
51
0.11 0.01
b
—
c
52
—
a
25% inhibition at 1
25% inhibition at 3
42% inhibition at 1
l
l
l
M test compound.
M test compound.
M test compound.
b
c
d
Compound 46 showed partial agonistic activity alone, and weak antagonistic activity toward 10 nM testosterone (IC50: 6.2
l
M).
mides 53 and 54 were examined. The activities of 53 and 54 were
considerably decreased, especially in the SC-3 cell growth inhibi-
tion assay (Fig. 3), which confirms that the pyrrole ring is impor-
tant for potent AR-antagonistic activity.
AR gene mutations in the LBD, which alter the ligand specificity
and/or functional activity, are thought to contribute to the ability
of some antagonists of wtAR to exhibit agonistic activity toward
mutated ARs. LNCaP prostate cancer cells express AR with a
T877A point mutation on helix 11, and wtAR antagonists such as
5 promoted the growth of these cells,33–35 while 6 inhibited their
proliferation. Therefore, we examined the binding affinity of se-
lected compounds for T877A AR from homogenized LNCaP cells
(Table 5). Under our experimental conditions, both
(Ki = 0.003 M) and 6 (Ki = 0.010 M) had potent affinities for
T877A AR, and compound 22 (Ki = 0.005 M) showed higher affin-
5
l
l
l
ity for LNCaP than did 6 (Table 5). It should be noted that direct
comparison of Ki values themselves determined for T877A AR in
this paper with those for wtAR determined in this paper is not

K. Wakabayashi et al. / Bioorg. Med. Chem. 16 (2008) 6799–6812
6805
O
N
N
N
N
O2N
O2N
O
53
54
Ki, 0.34 μMa; IC50, 3.7 μMb
Ki, 0.37 μMa; IC50, 4.9 μMb
awtAR binding affinity, and bSC-3 growth inhibition activity
Figure 3. Structures and activities of benzenecarboxamides 53 and 54.
appropriate, because the former and the latter were assessed by
using full-size intact T877A AR extracted from human LNCaP cells
and recombinant human AR ligand binding domain, respectively.
Concerning the cell assay, species difference between murine SC-
3 and human LNCaP cells might not be excluded. Even though, it
is interesting that compound 15, which has a wtAR affinity compa-
rable with that of 22, exhibited only very weak binding affinity for
T877A AR. Other compounds bearing electron-withdrawing sub-
stituents (compounds 37–40) showed similar binding affinity to-
ward both wt and T877A ARs. On the other hand, compound 51,
which lacks affinity for wtAR, showed a T877A AR affinity as potent
as those of 5 and 6. This result suggests that the binding domain of
T877A AR may have a larger pocket near the residues that interact
with the carboxamide moiety of 22 as a result of the mutation. It
has been reported that other steroid hormones also can bind the
mutated T877A receptor, promoting the growth of mutated pros-
tate cancer cells.33–35
Then, we determined the AR-agonistic and -antagonistic activi-
ties of selected compounds by measuring with an ELISA the
amount of prostate-specific antigen (PSA) produced by LNCaP cells
(Fig. 4). Agonistic activity can be assessed in terms of enhanced
production of PSA and antagonistic activity can be detected in
terms of inhibition of PSA production induced by 1. Compound 5
promoted PSA production as well as 1. A carboxyl derivative 46
with wtAR partial agonistic activity also increased the PSA produc-
tion, while 6 and all the carboxamides examined showed no ago-
nistic activity, except for 48 at high concentration. Among them,
Figure 4. The effect on PSA production of test compounds (a) alone and (b) in the
presence of 10 nM testosterone (1) in LNCaP cells. The amount of PSA produced in
the presence of test compounds was normalized to that produced in the presence of
10 nM testosterone (1) alone, taken as 100%.
bicalutamide (6) and the carboxamides 22, 36–38 showed AR-
antagonistic activity in LNCaP cells (Fig. 4b). Compounds 39 and
40 have relatively weak binding ability for T877A AR (25–35 times
weaker binding than compound 22), and consistently with this,
they did not show AR-antagonistic activity. Compound 48, which
has the most potent binding ability to T877A AR (Ki = 0.003 lM),
did not inhibit PSA production. Compound 22, as well as 6, tended
to suppress the proliferation of LNCaP cells, while 5 increased
LNCaP cell growth (data not shown). Thus, compound 22 acted as
a potent AR antagonist toward both wtAR and T877A AR.
Table 5
Binding affinity for T877A mutated AR
The SARs of this series of AR ligands indicate that our strategy is
useful for the design of novel AR antagonists. The introduction of a
bulky N-substituent into the weak AR antagonists 9 and 10 in-
creased both the AR affinity and the antagonistic activity. A xyli-
dine derivative 15 did not bind to T877A AR, which means that
the xylene moiety of compound 15 seems to be important for
selective binding to wtAR. The aromatic ring with an electron-
withdrawing group is favorable for enhancing the binding affinity
of the compound to the mutated AR. Another significant structural
feature is the carboxamide group. The carboxyl derivative 46 has a
high binding affinity for wtAR and acted as a partial agonist toward
wtAR. The diethylamide 48 is a potent antagonist for wtAR, but not
for T877A AR. Introduction of the bulky substituent on the polar
functional group (compounds 49, 51, and 52) decreased the wt
AR binding affinity, while compound 51 showed potent binding
affinity for T877A AR. Thus, the effect of the polar substituent on
the 2-position of the pyrrole ring is also significant for both binding
affinity and activation/inactivation of wild-type and mutated ARs.
We examined computer-assisted docking calculations of our novel
compounds using the reported crystal structures of AR LBD. How-
ever, only the crystal structures of AR LBD-agonist complexes
Compound
Ki, l
Ma
Hydroxyflutamide (5)
(R)-Bicalutamide (6)
0.003 0.001 (0.43)
0.010 0.004 (0.051)
(0.35)b
0.005 0.001 (0.11)
0.14 ((0.80)
0.13 (0.81)
0.33 (0.73)
1.4 (1.8)
0.049 0.007 (—)
0.006 0.003 (0.13)
0.036 0.009 (0.27)
0.13 0.06 (0.14)
0.18 0.01 (0.18)
0.007 0.001 (0.18)
0.10 0.07 (0.075)
0.003 0.001 (0.10)
0.027 0.011 (0.11)
0.009 0.007 (—)
15
22
23
24
25
26
36
37
38
39
40
44
46
48
50
51
a
Competitive binding assay using 1 nM [3H]testosterone and soluble fraction of
homogenized LNCaP cells with test compounds. Ki values toward wtAR are shown
in parentheses.
b
20% inhibition at 3 lM test compound.

6806
K. Wakabayashi et al. / Bioorg. Med. Chem. 16 (2008) 6799–6812
(active conformations) have been reported so far, including T877A
AR-bicyclic isoindoledione derivative38 and W741L AR-615 (agonist
for this mutant) complexes. In our computational study using the
active form of wtAR, several structures were picked up as possible
stable receptor–ligand complexes. More detailed analyses on the
interaction of novel AR antagonists with AR LBDs, both wild-type
and mutants, including theoretical and X-ray crystallographical
analyses, are ongoing.
121.0, 119.6, 112.2, 109.9, 49.3, 46.2, 41.0, 36.5, 26.5, 24.0, 21.5.
MS m/z 311 (M+), 312 (MH+); HRMS calcd for C17H21N3O (M)
311.1988, found 311.2017.
4.2.3. N-[4-[(3,5-Dimethylphenyl)(ethyl)amino]-1-methylpyr-
role-2-carbonyl]pyrrolidine (12)
The title compound was prepared from compound 9 and ethyl
iodide by procedure A (27%). Colorless crystals: mp 86–87 °C (n-
hexane–ethyl acetate): 1H NMR (CDCl3) d 6.57 (s, 1H), 6.48 (s,
2H), 6.41 (s, 1H), 6.36 (s, 1H), 3.86 (s, 3H), 3.62 (br, 4H), 3.58 (q,
2H, J = 7.2 Hz), 2.22 (s, 6H), 1.92–1.90 (m, 4H), 1.19 (t, 3H,
J = 7.2 Hz).13C NMR (125 MHz, CDCl3) d 161.6, 148.5, 138.6,
129.1, 124.9, 122.4, 119.5, 112.2, 111.2, 49.4, 47.3, 46.2, 36.6,
26.6, 24.1, 21.6, 12.4. MS m/z 325 (M+), 326 (MH+). Anal. Calcd
for C20H27N3O: C, 73.81; H, 8.36; N, 12.91. Found: C, 73.93; H,
8.27; N, 12.96.
3. Conclusion
In line with our strategy for developing full antagonists of nu-
clear receptors, we have designed and synthesized a series of AR
antagonists with a pyrrolecarboxamide scaffold. Among them,
compound 22 displayed high binding affinity for wtAR and potent
growth-inhibitory activity against SC-3 cells. Further, compound
22 exhibited antagonistic activity against T877A AR, having a po-
tency comparable with that of (R)-bicalutamide (6). Although it
is unknown whether compound 22 antagonizes other mutated
ARs, including W741L/W741C AR mutants, for which bicalutamide
(6) acts as an agonist, compound 22 is considered to be promising
as a scaffold for the development of novel agents for anti-AR ther-
apy of prostate cancer.
4.2.4. N-[4-[(3,5-Dimethylphenyl)(iso-propyl)amino]-1-methyl-
pyrrole-2-carbonyl]pyrrolidine (13)
The title compound was prepared from compound 9 and isopro-
pyl iodide by procedure A at 100 °C for 17 h (13%). Yellow oil: 1H
NMR (CDCl3) d 6.49 (s, 1H), 6.34 (s, 3H), 6.28 (d, 1H, J = 1.7 Hz),
4.19 (sept, 1H, J = 6.4 Hz), 3.89 (s, 3H), 3.62 (br, 4H), 2.21 (s, 6H),
1.92 (m, 4H), 1.10 (d, 6H, J = 6.4 Hz); 13C NMR (125 MHz, CDCl3)
d 161.7, 149.9, 138.3, 138.3, 125.9, 124.9, 123.6, 118.5, 115.1,
111.4, 49.5, 47.0, 46.3, 36.7, 26.6, 24.1, 21.7, 20.7; MS m/z 339
(M+), 340 (MH+); HRMS calcd for C21H29N3O (M) 339.2311, found
339.2310.
4. Experimental
4.1. General information
Routine thin layer chromatography (TLC) was performed on sil-
ica gel 60 F254 plates (Merck, Germany). Column chromatography
was done using silica gel 60 spherical or silica gel 60 N spherical
(Kanto Chemical Co., Inc., Japan). Melting points were determined
on a MP-J3 melting point apparatus (Yanaco, Japan) and are uncor-
rected. Elemental analyses were carried out in the Microanalytical
Laboratory, Faculty of Pharmaceutical Sciences, University of To-
kyo, and were within 0.3% of the theoretical values. Concerning
oily/non-crystalline compounds, their purity was confirmed by
thin layer chromatography and proton nuclear magnetic resonance
(NMR) spectroscopy. Fast atom bombardment mass spectra (FAB-
MS) were measured with a MS-JEOL JMS-HX110 mass spectrome-
ter using a nitrobenzyl alcohol matrix as appropriate. NMR spectra
data were obtained on a JEOL ALPHA500 spectrometer (500 MHz).
Chemical shifts are given in parts per million (ppm) downfield
from internal reference TMS in d units, and coupling constants (J
values) are given in hertz (Hz). Compounds 9, 10, and 28 were pre-
pared by the methods previously reported.30
4.2.5. N-[4-[(Cyclohexylmethyl)(3,5-dimethylphenyl)amino]-1-
methylpyrrole-2-carbonyl]pyrrolidine (14)
The title compound was prepared from compound 9 and cyclo-
hexylmethyl bromide by procedure A at 100 °C for 17 h (13%). Yel-
low oil; 1H NMR (CDCl3) d 6.51 (s, 1H), 6.42 (s, 2H), 6.36 (s, 1H),
6.33 (s, 1H), 3.86 (s, 3H), 3.62 (br, 4H), 3.26 (d, 2H, J = 6.8 Hz),
2.22 (s, 6H), 1.93–1.90 (m, 4H), 1.81 (d, 2H, J = 6.8 Hz), 1.74–1.68
(m, 4H), 1.22–1.12 (m, 3H), 0.97–0.88 (m, 2H); 13C NMR
(125 MHz, CDCl3) d 161.6, 150.0, 138.3, 130.8, 124.7, 122.3,
119.0, 112.1, 111.2, 59.9, 49.4, 46.2, 36.6, 31.2, 26.6, 25.9, 24.1,
21.6, 21.6; MS m/z 393 (M+); HRMS calcd for C25H35N3O (M)
393.2624, found 393.2626.
4.2.6. N-[4-[(Benzyl)(3,5-dimethylphenyl)amino]-1-methylpy-
rrole-2-carbonyl]pyrrolidine (15)
The title compound was prepared from compound 9 and benzyl
bromide by procedure A (58%). White crystals: mp 130 °C (n-hex-
ane–ethyl acetate); 1H NMR (CDCl3) d 7.34–7.27 (m, 4H), 7.22 (t,
1H, J = 6.8 Hz), 6.54 (s, 3H), 6.43 (s, 1H), 6.37 (s, 1H), 4.80 (s, 2H),
3.80 (s, 3H), 3.59–3.57 (m, 4H), 2.19 (s, 6H), 1.91–1.88 (m,
4H);13C NMR (125 MHz, CDCl3) d 161.5, 148.8, 138.8, 129.0,
126.7, 125.8, 121.6, 120.4, 112.7, 110.4, 57.4, 48.0, 47.3, 36.5,
25.2, 21.6; MS m/z 387 (M+), 388 (MH+); Anal. Calcd for
4.2. Synthesis
4.2.1. General synthetic procedure A for compounds 11–26
Alkyl halide (1.1–1.5 equiv) was added to a suspension of 9 or
10 (1 equiv) and sodium hydride (1.5–2 equiv) in DMF at 0 °C.
The reaction mixture was stirred until 9 or 10 was consumed, then
poured into water, and extracted with ethyl acetate. The organic
layer was washed with brine, dried over MgSO4, and evaporated.
The residue was purified by silica gel column chromatography or
preparative TLC.
C25H29N3O: C, 77.48; H, 7.54; N, 10.84. Found: C, 77.42; H, 7.59;
N, 10.82.
4.2.7. N-[4-[(3,5-Dimethylphenyl)(4-hydroxyphenyl)amino]-1-
methylpyrrole-2-carbonyl]pyrrolidine (16)
4-(Chloromethyl)phenyl acetate (104
lL, 0.67 mmol) was added
to a suspension of 9 (100 mg, 0.34 mmol), triethylamine (94
l
L,
4.2.2. N-[4-[(3,5-Dimethylphenyl)(methyl)amino]-1-methylpyr-
role-2-carbonyl]pyrrolidine (11)
0.67 mmol), and KI (112 mg, 0.67 mmol) in DMF (2 mL) at 0 °C.
The reaction mixture was stirred at room temperature for 16.5 h,
then poured into water, and extracted with ethyl acetate. The or-
ganic layer was washed with brine, dried over MgSO4, and evapo-
rated. The residue was purified by silica gel column
chromatography (n-hexane–ethyl acetate = 3:2; 1:1) and prepara-
tive TLC (n-hexane–ethyl acetate = 1:4) to give N-[4-[(4-acetoxyl-
The title compound was prepared from compound 9 and methyl
iodide by procedure A (73%). Yellow oil: 1H NMR (CDCl3) d 6.54 (d,
1H, J = 1.7 Hz), 6.49 (s, 2H), 6.42 (s, 1H), 6.36 (d, 1H, J = 1.7 Hz), 3.86
(s, 3H), 3.63 (br, 4H), 3.18 (s, 3H), 2.24 (s, 6H), 1.93–1.90 (m, 4 H).
13C NMR (125 MHz, CDCl3) d 161.6, 149.8, 138.3, 131.7, 124.6,

K. Wakabayashi et al. / Bioorg. Med. Chem. 16 (2008) 6799–6812
6807
phenyl)(3,5-dimethylphenyl)amino]-1-methylpyrrole-2-carbonyl]
pyrrolidine as a colorless oil (110 mg, 76%); 1H NMR (CDCl3) d 7.31
(d, 2H, J = 8.5 Hz), 7.01 (d, 2H, J = 8.5 Hz), 6.51 (d, 1H, J = 2.1 Hz),
6.42 (s, 2H), 6.41 (s, 1H), 6.35 (d, 1H, J = 2.1 Hz), 4.78 (s, 2H), 3.81
(s, 3H), 3.62–3.56 (m, 4H), 2.29 (s, 3H), 2.20 (s, 6H), 1.94–1.88
(br, 4H); MS m/z 445 (M+), 446 (MH+), HRMS calcd for
343 (MH+), HRMS calcd for C18H22N4O3 (M) 342.1692, found
342.1683.
4.2.11. N-[4-[(4-Nitrophenyl)(iso-propyl)amino]-1-methylpy-
rrole-2-carbonyl]pyrrolidine (20)
The title compound was prepared from compound 10 and iso-
propyl bromide by procedure A at 50 °C for 2 d (83%). Yellow oil;
1H NMR (CDCl3) d 7.96 (d, 2H, J = 9.4 Hz), 6.59 (d, 2H, J = 9.4 Hz),
6.51 (d, 1H, J = 1.7 Hz), 6.23 (d, 1H, J = 1.7 Hz), 4.25 (sept, 1H,
J = 6.4 Hz), 3.88 (s, 3H), 3.61 (br, 4H), 1.92–1.89 (m, 4H), 1.13 (d,
6H, J = 6.4 Hz); 13C NMR (125 MHz, CDCl3) d 161.2, 154.8, 137.1,
125.9, 125.7, 125.1, 121.1, 113.8, 111.8, 49.4, 48.3, 46.3, 36.7,
26.5, 24.0, 20.6; MS m/z 356 (M+), 357 (MH+), HRMS calcd for
C19H24N4O3 (M) 356.1848, found 356.1851.
C27H31N3O3(M) 445.2365, found 445.2364. K2CO3 (18 mg,
0.13 mmol) was added to a solution of N-[4-[(4-acetoxylphe-
nyl)(3,5-dimethylphenyl)amino]-1-methylpyrrole-2-carbonyl]pyr-
rolidine (58 mg, 0.13 mmol) in methanol (500
lL) and water
(50 L) at room temperature, and the mixture was stirred for
l
4.5 h. The solution was acidified to about pH 1 with 2 M HCl aq
and extracted with ether. The organic layer was washed with brine,
dried over MgSO4, and evaporated. The residue was purified by
preparative TLC (n-hexane–ethyl acetate = 1:4) to give 16 (35 mg,
67%). White powder: mp 208.5–210 °C (acetone):1H NMR
(DMSO-d6) d 9.22 (s, 1H), 7.06 (d, 2H, J = 8.5 Hz), 6.76 (d, 1H,
J = 1.7 Hz), 6.66 (d, 2H, J = 8.5 Hz), 6.39 (s, 2H), 6.34 (d, 1H,
J = 1.7 Hz), 6.25 (s, 1H), 4.63 (s, 2H), 3.67 (s, 3H), 3.55–3.38 (br,
4H), 2.08 (s, 6H), 1.82–1.79 (m, 4H);13C NMR (125 MHz, DMSO-
d6) d 160.6, 155.9, 148.9, 137.4, 129.6, 129.2, 127.7, 124.4, 121.8,
118.8, 115.0, 111.8, 109.8, 55.5, 48.7, 45.9, 35.7, 26.0, 23.6, 21.3;
MS m/z 403 (M+), 404 (MH+), HRMS calcd for C25H29N3O2 (M)
403.2260, found 403.2282.
4.2.12. N-[4-[(Cyclohexylmethyl)(4-nitrophenyl)amino]-1-methyl-
pyrrole-2-carbonyl]pyrrolidine (21)
The title compound was prepared from compound 10 and
cyclohexylmethyl bromide by procedure A at 70 °C for 2 d (84%).
Orange foam; 1H NMR (CDCl3) d 8.00 (d, 2H, J = 9.4 Hz), 6.67 (d,
2H, J = 9.4 Hz), 6.59 (d, 1H, J = 1.3 Hz), 6.32 (d, 1H, J = 1.3 Hz),
3.88 (s, 3 H), 3.63 (br, 4H), 3.45 (d, 2H, J = 6.8 Hz), 1.94–1.92 (m,
4H), 1.77–1.65 (m, 6H), 1.25–1.17 (m, 3H), 0.99–0.95 (m, 2H);13
C
NMR (125 MHz, CDCl3) d 161.2, 154.8, 137.2, 128.0, 125.8, 125.7,
122.7, 111.9, 110.9, 59.8, 49.4, 46.3, 36.7, 36.6, 31.0, 29.5, 26.5,
26.3, 25.8, 24.0; MS m/z 410 (M+), 411 (MH+), HRMS calcd for
4.2.8. N-[4-[(3,5-Dimethylphenyl)(4-hydroxymethylbenzyl)
amino]-1-methylpyrrole-2-carbonyl]pyrrolidine (17)
C23H30N4O3 (M) 410.2318, found 410.2301.
4-Hydroxymethylbenzyl bromide (34 mg, 0.17 mmol) was
added to a suspension of 9 (25 mg, 0.084 mmol), triethylamine
(23 lL, 0.17 mmol), and KI (28 mg, 0.17 mmol) in DMF (1 mL) at
4.2.13. N-[4-[(Benzyl)(4-nitrophenyl)amino]-1-methylpyrrole-
2-carbonyl]pyrrolidine (22)
0 °C. The reaction mixture was stirred at 80 °C for 2.5 h, then was
poured into water, and extracted with ethyl acetate. The organic
layer was washed with brine, dried over MgSO4, and evaporated.
The residue was purified by preparative TLC (n-hexane–ethyl ace-
tate = 1:1) to give 17 (11 mg, 31%). Colorless oil; 1H NMR (CD3OD)
d 7.25 (s, 4H), 6.64 (s, 1H), 6.45 (s, 2H), 6.37 (s, 1H), 6.32 (s, 1H),
4.74 (s, 2H), 4.54 (s, 2H), 3.70 (s, 3H), 3.58–3.48 (m, 4H), 2.10 (s,
6H), 1.91–1.86 (m, 4H);13C NMR (125 MHz, CD3OD) d 163.7, 150.6,
141.0, 140.0, 139.2, 132.3, 128.1, 128.0, 125.6, 122.9, 120.7, 113.7,
111.8, 65.0, 57.5, 50.8, 47.4, 36.3, 27.3, 25.0, 21.7; MS m/z 417 (M+),
418 (MH+), HRMS calcd for C26H31N3O2(M) 417.2416, found
417.2414.
The title compound was prepared from compound 10 and ben-
zyl bromide by procedure A (84%). Orange crystals: mp 116–
116.5 °C (acetone–methanol); 1H NMR (CDCl3) d 8.01 (d, 2H,
J = 9.4 Hz), 7.34 (t, 2H, J = 7.2 Hz), 7.28 (t, 1H, J = 7.2 Hz), 7.26 (d,
2H, J = 7.2 Hz), 6.74 (d, 2H, J = 9.4 Hz), 6.63 (d, 1H, J = 2.1 Hz),
6.39 (d, 1H, J = 2.1 Hz), 4.90 (s, 2H), 3.84 (s, 3H), 3.62–3.59 (m,
4H), 1.94–1.86 (m, 4H); 13C NMR (125 MHz, CDCl3) d 161.1,
154.1, 137.9, 137.0, 128.8, 127.9, 127.3, 126.3, 125.8, 125.7,
122.3, 112.2, 110.5, 57.3, 49.4, 46.3, 36.7, 26.5, 24.1; MS m/z 404
(M+), 405 (MH+); HRMS calcd for C23H24N4O3 (M) 404.1848, found
404.1875; Anal. Calcd for C23H24N4O3: C, 68.30; H, 5.98; N, 13.85.
Found: C, 68.35; H, 6.05; N, 13.86.
4.2.14. N-[4-[(4-Fluorobenzyl)(4-nitrophenyl)amino]-1-methyl-
pyrrole-2-carbonyl]pyrrolidine (23)
4.2.9. N-[4-[(Methyl)(4-nitrophenyl)amino]-1-methylpyrrole-
2-carbonyl]pyrrolidine (18)
The title compound was prepared from compound 10 and 4-flu-
orobenzyl chloride by procedure A at 70 °C for 1 h (46%). Orange
oil; 1H NMR (CDCl3) d 7.98 (d, 2H, J = 9.4 Hz), 7.21 (dd, 2H, J = 8.5,
5.5 Hz), 7.00 (t, 2H, J = 8.5 Hz), 6.71 (d, 2H, J = 9.4 Hz), 6.60 (d,
1H, J = 2.1 Hz), 6.34 (d, 1H, J = 2.1 Hz), 4.85 (s, 2H), 3.84 (s, 3H),
3.61–3.57 (m, 4H), 1.94–1.90 (m, 4H);13C NMR (125 MHz, CDCl3)
d 160.2 (d, J = 246 Hz), 154.0, 138.1, 132.7, 128.1 (d, J = 7 Hz),
127.7, 126.0, 125.8, 122.3, 115.6 (d, J = 20 Hz), 112.2, 110.5, 56.6,
49.4, 46.3, 36.7, 26.5, 24.1; MS m/z 422 (M+), 423 (MH+), HRMS
calcd for C23H23N4O3 (M) 422.1754, found 422.1761.
The title compound was prepared from compound 10 and
methyl iodide by procedure A (90%). Yellow powder; mp 158–
159 °C (n-hexane–ethyl acetate); 1H NMR (CDCl3) d 8.02 (d, 2H,
J = 9.4 Hz), 6.69 (d, 1H, J = 9.4 Hz), 6.63 (d, 1H, J = 2.1 Hz), 6.35 (d,
1H, J = 2.1 Hz), 3.86 (s, 3H), 3.62 (br, 4H), 3.30 (s, 3H), 1.94–1.91
(m, 4H);13C NMR (125 MHz, CDCl3) d 161.2, 154.5, 137.6, 128.8,
125.8, 125.7, 122.1, 111.7, 110.2, 49.4, 46.3, 41.3, 36.7, 26.6, 24.1;
MS m/z 328 (M+), 329 (MH+), HRMS calcd for C17H20N4O3 (M)
328.1535, found 328.1537; Anal. Calcd for C17H20N4O3: C, 62.18;
H, 6.14; N, 17.06. Found: C, 62.26; H, 6.10; N, 17.20.
4.2.10. N-[4-[(Ethyl)(4-nitrophenyl)amino]-1-methylpyrrole-2-
carbonyl]pyrrolidine (19)
4.2.15. N-[4-[(4-Methylbenzyl)(4-nitrophenyl)amino]-1-methyl-
pyrrole-2-carbonyl]pyrrolidine (24)
The title compound was prepared from compound 10 and ethyl
iodide by procedure A (92%). Orange oil: 1H NMR (CDCl3) d 7.98 (d,
2H, J = 9.4 Hz), 6.63 (d, 2H, J = 9.4 Hz), 6.61 (d, 1H, J = 1.7 Hz), 6.33
(d, 1H, J = 1.7 Hz), 3.87 (s, 3H), 3.65 (q, 2H, J = 6.8 Hz), 3.62 (br, 4H),
1.93–1.90 (m, 4H), 1.22 (t, 3H, J = 6.8 Hz);13C NMR (125 MHz,
CDCl3) d 161.1, 153.9, 137.2, 126.7, 125.9, 125.8, 122.8, 111.5,
111.1, 49.4, 47.6, 46.3, 36.7, 26.5, 24.0, 12.2; MS m/z 342 (M+),
The title compound was prepared from compound 10 and 4-
methylbenzyl bromide by procedure A (79%). Orange oil; 1H NMR
(CDCl3) d 7.99 (d, 2H, J = 9.4 Hz), 7.13 (s, 4H), 6.73 (d, 2H,
J = 9.4 Hz), 6.62 (d, 1H, J = 2.1 Hz), 6.37 (d, 1H, J = 2.1 Hz), 4.85 (s,
2H), 3.84 (s, 3H), 3.61–3.58 (m, 4H), 2.33 (s, 3H), 1.91–1.89 (m,
4H);13C NMR (125 MHz, CDCl3) d 161.2, 154.1, 137.9, 137.0,
133.9, 129.4, 128.0, 126.3, 125.8, 125.7, 122.4, 112.2, 110.6, 49.4,

6808
K. Wakabayashi et al. / Bioorg. Med. Chem. 16 (2008) 6799–6812
46.3, 36.7, 26.6, 24.1, 21.0; MS m/z 418 (M+), 419 (MH+), HRMS
calcd for C24H26N4O3 (M) 418.2005, found 418.2005.
ture was heated at 80 °C until the aryl bromide was consumed.
The mixture was diluted with ethyl acetate, washed with water
and brine, dried over MgSO4, and evaporated. Otherwise, the mix-
ture was filtered through Celite and eluted with dichloromethane,
and the filtrate was evaporated. The residue was purified by silica
gel column chromatography.
4.2.16. N-[4-[(4-Hydroxybenzyl)(4-nitrophenyl)amino]-1-methyl-
pyrrole-2-carbonyl]pyrrolidine (25)
A
solution of 4-tert-butyl-dimethylsilyloxybenzyl bromide
(74 mg, 0.25 mmol) in DMF (1 mL) was added to a suspension of
10 (70 mg, 0.23 mmol) and sodium hydride (14 mg, 0.35 mmol)
in DMF (1 mL) at 0 °C. The reaction mixture was stirred at room
temperature for 20 min, then poured into water, and extracted
with ethyl acetate. The organic layer was washed with brine, dried
over MgSO4, and evaporated. The residue was purified by silica gel
column chromatography (chloroform–methanol = 20:1) to give 25
(79 mg, 66%) as orange powder: mp 223–224 °C (n-hexane–ethyl
4.2.19. N-[4-(2-Nitrophenylamino)-1-methylpyrrole-2-carbo-
nyl]pyrrolidine (29)
The title compound was prepared from 28 and 2-bromonitro-
benzene by procedure B (39%). Vermillion oil; 1H NMR (CDCl3) d
9.12 (s, 1H), 8.16 (dd, 1H, J = 8.5, 1.2 Hz), 7.33 (t, 1H, J = 7.2 Hz),
7.09 (dd, 1H, J = 8.5, 1.2 Hz), 6.69–6.67 (m, 2H), 6.66 (d, 1 H,
J = 1.2 Hz), 6.42 (d, 1H, J = 1.7 Hz), 3.88 (s, 3H), 3.64 (br, 4H), 1.94
(m, 4H); MS m/z 314 (M+), 315 (MH+); HRMS calcd for
acetate); 1H NMR (DMSO-d6)
d 9.29 (s, 1H), 7.97 (d, 2H,
J = 9.4 Hz), 7.07 (d, 2H, J = 8.5 Hz), 6.69 (d, 1H, J = 2.1 Hz), 6.80 (d,
2H, J = 9.4 Hz), 6.69 (d, 2H, J = 8.5 Hz), 6.46 (d, 1H, J = 2.1), 4.81 (s,
2H), 3.71 (s, 3H), 3.54 (br, 2H), 3.41 (br, 2H), 1.82–1.79 (m,
4H);13C NMR (125 MHz, DMSO-d6) d 160.3, 156.4, 154.1, 136.6,
127.8, 127.1, 127.0, 125.7, 125.4, 122.5, 115.3, 112.3, 110.1, 79.1,
56.0, 48.7, 45.9, 36.0, 26.0, 23.6; MS m/z 420 (M+), 421 (MH+),
HRMS calcd for C23H24N4O3 (M) 420.1798, found 420.1800.
C16H18N4O3 (M) 314.1379, found 314.1343.
4.2.20. N-[4-(4-Nitro-3-trifluoromethylphenylamino)-1-methyl
pyrrole-2-carbonyl]pyrrolidine (30)
The title compound was prepared from 28 and 1-bromo-4-ni-
tro-3-trifluoromethylbenzene by procedure B (70%). Pale yellow
powder: mp 202–203 °C (n-hexane–ethyl acetate); 1H NMR
(CDCl3) d 7.95 (d, 1H, J = 9.0 Hz), 7.08 (d, 1H, J = 2.1 Hz), 6.83 (dd,
1H, J = 9.0, 2.6 Hz), 6.62 (d, 1H, J = 1.7 Hz), 6.36 (d, 1H, J = 1.7 Hz),
6.23 (s, 1H), 3.85 (s, 3H), 3.63 (br, 4H), 1.96–1.93 (m, 4H); 13C
4.2.17. N-[4-[(4-Hydroxymethylbenzyl)(4-nitrophenyl)amino]-
1-methylpyrrole-2-carbonyl]pyrrolidine (26)
A solution of 4-(tert-butyl-dimethylsilyloxymethyl)benzyl bro-
mide (71 mg, 0.23 mmol) in DMF (1 mL) was added to a suspension
of 10 (64 mg, 0.20 mmol) and sodium hydride (14 mg, 0.35 mmol)
in DMF (1 mL) at 0 °C. The reaction mixture was stirred at 80 °C for
1 h, then poured into water, and extracted with ethyl acetate. The
organic layer was washed with brine, dried over MgSO4, and evap-
orated. The residue was purified by preparative TLC (n-hexane–
ethyl acetate = 1:2) to give N-[4-[[(4-tert-butyl-dimethylsilyl-
oxymethyl)benzyl](4-nitrophenyl)amino]-1-methylpyrrole-2-car-
bonyl]pyrrolidine as an orange oil (88 mg, 79%). 1H NMR (CDCl3) d
8.00 (d, 2H, J = 9.4 Hz), 7.29 (d, 2H, J = 8.1 Hz), 7.21 (d, 2H,
J = 8.1 Hz), 6.73 (d, 2H, J = 9.4 Hz), 6.62 (d, 1H, J = 1.7 Hz), 6.38 (d,
1H, J = 1.7 Hz), 4.88 (s, 2H), 4.72 (s, 2H), 3.84 (s, 3H), 3.62–3.59
(m, 4H), 1.94–1.91 (m, 4H), 0.94 (s, 9H), 0.09 (s, 6H);13C NMR
(125 MHz, CDCl3) d 161.1, 154.1, 140.6, 137.8, 135.5, 127.9,
126.4, 126.3, 125.8, 125.7, 122.4, 112.2, 110.6, 64.5, 57.1, 49.4,
46.3, 36.7, 26.5, 25.9, 24.0, 18.3, -5.3; MS m/z 548 (M+), 549
(MH+), HRMS calcd for C30H40N4O4Si (M) 548.2819, found
548.2793. A solution of TBAF in THF was added to a solution
of N-[4-[[(4-tert-butyl-dimethylsilyloxymethyl)benzyl](4-nitro-
phenyl)amino]-1-methylpyrrole-2-carbonyl]pyrrolidine (78 mg,
0.14 mmol) in THF (3 ml) at room temperature. The reaction mix-
ture was stirred for 3 h, then diluted with ethyl acetate, and
washed with water and saturated NH4Cl aq The organic layer
was dried over MgSO4 and evaporated. The residue was purified
by preparative TLC (n-hexane–ethyl acetate = 1:2) to give 26
(60 mg, 97%). Orange foam:1H NMR (CDCl3) d 7.97 (d, 2H,
J = 9.4 Hz), 7.30 (d, 2H, J = 8.1 Hz), 7.20 (d, 2H, J = 8.1 Hz), 6.69
(d, 2H, J = 9.4 Hz), 6.61 (d, 1H, J = 2.1 Hz), 6.37 (d, 1H, J = 2.1 Hz),
4.87 (s, 2H), 4.63 (s, 2H), 3.79 (s, 3H), 3.57 (m, 4H), 1.91–1.89
(m, 4H);13C NMR (125 MHz, CDCl3) d 161.1, 154.0, 140.3, 137.8,
136.1, 127.9, 127.3, 126.4, 125.7, 125.6, 122.3, 112.1, 110.5,
64.5, 57.0, 49.4, 46.3, 36.6, 26.4, 24.0; MS m/z 434 (M+), 435
(MH+), HRMS calcd for C24H26N4O4 (M) 434.1954, found 434.1984.
NMR (125 MHz, CDCl3)
d 161.3, 151.7, 137.0, 126.6 (q,
J = 33.3 Hz), 125.9, 122.4 (q, J = 273 Hz), 121.5, 120.9, 113.8, 111.8
(q, J = 7.4 Hz), 109.8, 49.5, 46.4, 36.6, 26.5, 24.1; MS m/z 382
(M+), 383 (MH+); HRMS calcd for C17H17F3N4O3 (M) 382.1253,
found 382.1263.
4.2.21. N-[4-(4-Cyanophenylamino)-1-methylpyrrole-2-carbo-
nyl]pyrrolidine (31)
The title compound was prepared from 28 and 4-bromobenzo-
nitrile by procedure B (32%). Pale yellow crystals: mp 294 °C (n-
hexane–ethyl acetate); 1H NMR (CDCl3) d 7.40 (d, 2H, J = 8.9 Hz),
6.72 (d, 2H, J = 8.5 Hz), 6.62 (d, 1H, J = 1.7 Hz), 6.37 (d, 1H,
J = 1.7 Hz), 5.50 (s, 1H), 3.86 (s, 3H), 3.63 (br, 4H), 2.40 (s, 3H),
1.95–1.92 (m, 4H); MS m/z 294 (M+); Anal. Calcd for C17H18N4O:
C, 69.37; H, 6.16; N, 19.03. Found: C, 69.33; H, 6.19; N, 19.06.
4.2.22. N-[4-(4-Trifluoromethylphenylamino)-1-methylpyrrole-
2-carbonyl]pyrrolidine (32)
The title compound was prepared from 28 and 4-bromotrifluo-
romethylbenzene by procedure B (51%). Pale yellow foam; 1H NMR
(CDCl3) d 7.37 (d, 2H, J = 8.5 Hz), 6.76 (d, 2H, J = 8.5 Hz), 6.60 (d, 1H,
J = 1.7 Hz), 6.37 (d, 1H, J = 1.7 Hz), 5.50 (s, 1H), 3.84 (s, 3H), 3.63 (br,
4H), 1.93–1.91 (m, 4H); MS m/z 382 (M+), 383 (MH+); HRMS calcd
for C17H17F3N4O3 (M) 382.1253, found 382.1263.
4.2.23. N-[4-(4-Acetylphenylamino)-1-methylpyrrole-2-carbonyl]
pyrrolidine (33)
The title compound was prepared from 28 and 4-bromoaceto-
phenone by procedure B (56%). Pale yellow powder: mp 181–
182 °C (n-hexane–dichloromethane); 1H NMR (CDCl3) d 7.79 (d,
2H, J = 9.0 Hz), 6.72 (d, 2H, J = 9.0 Hz), 6.61 (d, 1H, J = 1.7 Hz), 6.38
(d, 1H, J = 1.7 Hz), 5.71 (s, 1H), 3.84 (s, 3H), 3.63 (br, 4H), 2.48 (s,
3H), 1.93–1.90 (m, 4H); 13C NMR (125 MHz, CDCl3) d 196.3, 161.4,
151.9, 130.7, 127.4, 125.3, 122.6, 121.3, 112.2, 110.0, 60.3, 49.5,
46.4, 36.6, 26.6, 26.0, 24.1, 14.1; MS m/z 311 (M+), 312 (MH+); HRMS
calcd for C18H21N3O2 (M) 311.1634, found 311.1605.
4.2.18. General procedure B for compounds 29–34
A well-dried flask was evacuated and backfilled with argon. The
flask was charged with Pd2(dba)3 (1–10 mol%Pd), NaO-t-Bu or
Cs2CO3 (1.4 equiv), BINAP or 2-(di-tert-butylphosphino)biphenyl
(1–10 mol%), aryl bromide (0.9 equiv), and 2830 (1.0 equiv) in tolu-
ene, then evacuated and backfilled with argon. The reaction mix-
4.2.24. N-[4-(4-Methoxycarbonylphenylamino)-1-methylpyr-
role-2-carbonyl]pyrrolidine (34)
The title compound was prepared from 28 and methyl 4-bro-
mobenzoate by procedure B (66%). White powder, mp 186–

K. Wakabayashi et al. / Bioorg. Med. Chem. 16 (2008) 6799–6812
6809
187 °C (n-hexane–dichloromethane); 1H NMR (CDCl3) d 7.83 (d,
4.2.30. N-{4-[(4-Acetylphenyl)(benzyl)amino]-1-methylpyrrole-
2-carbonyl}pyrrolidine (39)
2H, J = 8.5 Hz), 6.71 (d, 2H, J = 8.5 Hz), 6.59 (d, 1H, J = 1.7 Hz),
6.37 (d, 1H, J = 1.7 Hz), 5.73 (s, 1H), 3.83 (s, 6H) 3.61 (br, 4H),
1.92–1.89 (m, 4H); 13C NMR (125 MHz, CDCl3) d 167.2, 161.4,
151.4, 131.4, 125.1, 122.8, 121.1, 119.0, 112.2, 110.0, 51.5, 49.4,
46.3, 36.5, 26.5, 24.0; MS m/z 327 (M+), 328 (MH+); HRMS calcd
for C18H21N3O3 (M) 327.1583, found 327.1601.
The title compound was prepared from 33 and benzyl bromide
by procedure C (55%). Colorless oil; 1H NMR (CDCl3) d 7.76 (d, 2H,
J = 9.2 Hz), 7.33–7.23 (m, 5H), 6.77 (d, 2H, J = 9.2 Hz), 6.60 (d, 1H,
J = 1.8 Hz), 6.39 (d, 1H, J = 1.8 Hz), 4.88 (s, 2H), 3.83 (s, 3H), 3.61–
3.58 (m, 4H), 2.47 (s, 3H), 1.93–1.90 (m, 4H); 13C NMR (125 MHz,
CDCl3) d 196.3, 161.4, 152.9, 137.9, 130.3, 128.7, 128.6, 127.1,
126.6, 126.5, 125.5, 122.4, 112.5, 110.9, 57.0, 49.5, 46.3, 36.7,
26.6, 26.0, 24.1; MS m/z 401 (M+), 402 (MH+); HRMS calcd for
4.2.25. General procedure C for compounds 35–40
Benzyl bromide (1.1–1.5 equiv) was added to a suspension of
29–34 (1 equiv) and sodium hydride (1.5–2 equiv) in DMF at
0 °C. The reaction mixture was stirred at 0 °C until the starting
material was consumed, then poured into water, and extracted
with ethyl acetate. The organic layer was washed with brine, dried
over MgSO4 and evaporated. The residue was purified by silica gel
column chromatography and preparative TLC.
C25H27N3O2 (M) 401.2103, found 401.2147.
4.2.31. N-{4-[(Benzyl)(4-methoxycarbonylphenyl)amino]-1-
methylpyrrole-2-carbonyl}pyrrolidine (40)
The title compound was prepared from 34 and benzyl bromide
by procedure C (35%). Colorless oil; 1H NMR (CDCl3) d 7.78 (d, 2H,
J = 9.0 Hz), 7.30 (t, 2H, J = 7.7 Hz), 7.25 (d, 2H, J = 7.7 Hz), 7.22 (t,
1H, J = 7.7 Hz), 6.75 (d, 2H, J = 9.0 Hz), 6.58 (d, 1H, J = 1.7 Hz),
6.38 (d, 1H, J = 1.7 Hz), 4.85 (s, 2H), 3.81 (s, 3H), 3.81 (s, 3H),
3.60–3.57 (m, 4H), 1.91–1.88 (m, 4H); 13C NMR (125 MHz, CDCl3)
d 167.2, 161.4, 152.8, 138.1, 131.0, 128.8, 128.6, 127.0, 126.5,
125.4, 122.4, 118.3, 112.5, 110.9, 57.0, 51.5, 49.4, 46.3, 36.7, 26.6,
24.1; MS m/z 417 (M+), 418 (MH+); HRMS calcd for C25H27N3O3
(M) 417.2052, found 417.2070.
4.2.26. N-[4-[(Benzyl)(2-nitrophenyl)amino]-1-methylpyrrole-
2-carbonyl]pyrrolidine (35)
The title compound was prepared from 29 and benzyl bromide
by procedure C (17%). Vermilion oil; 1H NMR (CDCl3) d 7.66 (dd,
1H, J = 1.7, 8.1 Hz), 7.38–7.34 (m, 3H), 7.30 (t, 2H, J = 7.7 Hz),
7.24–7.20 (m, 2H), 6.95 (dt, 1H, J = 1.3, 8.1 Hz), 6.26 (d, 1H,
J = 2.1 Hz), 6.01 (d, 1H, J = 2.1 Hz), 4.80 (s, 2H), 3.69 (s, 3H), 3.51
(br, 4H), 1.88–1.85 (m, 4H); 13C NMR (125 MHz, CDCl3) d 161.6,
142.6, 138.0, 133.0, 131.6, 128.6, 127.1, 127.1, 126.0, 124.6,
123.1, 120.9, 117.4, 106.0, 58.4, 49.3, 46.2, 36.3, 26.6, 24.1; MS
m/z 404 (M+), 405 (MH+); HRMS calcd for C25H27N3O3 (M)
404.1848, found 404.1845.
4.2.32. Ethyl 1-methyl-4-nitropyrrole-2-carboxylate (41)
Methyl iodide (2 mL, 32 mmol) was added to a suspension of
ethyl 4-nitropyrrole-2-carboxylate (5.3 g, 29 mmol) and sodium
hydride (1 g, 43 mmol) in DMF (50 mL) at 0 °C. The reaction mix-
ture was stirred at room temperature for 30 min, then poured into
water at 0 °C, and extracted with ethyl acetate. The organic layer
was washed with brine, dried over MgSO4, and evaporated. The
residue was recrystallized from n-hexane–ethyl acetate to give
41 (5.5 g, 28 mmol, 97%) as white crystals. 1H NMR (CDCl3) d
7.58 (d, 1H, J = 1.7 Hz), 7.41 (t, 1H, J = 1.7 Hz), 4.31 (q, 2H,
4.2.27. N-[4-[(Benzyl)(4-nitro-3-trifluoromethylphenyl)
amino]-1-methylpyrrole-2-carbonyl]pyrrolidine (36)
The title compound was prepared from 30 and benzyl bromide
by procedure C (71%). Yellow needles. mp 158–159 °C (n-hexane–
ethyl acetate); 1H NMR (CDCl3) d 7.92 (d, 1H, J = 9.4 Hz), 7.35 (t, 2H,
J = 7.7 Hz), 7.29 (t, 1H, J = 7.7 Hz), 7.24 (d, 2H, J = 7.7 Hz), 7.18 (d,
1H, J = 3.0 Hz), 6.82 (dd, 1H, J = 9.4, 3.0 Hz), 6.63 (d, 1H,
J = 2.1 Hz), 6.37 (d, 1H, J = 2.1 Hz), 4.90 (s, 2 H), 3.85 (s, 3H), 3.60
(br, 4H), 1.95–1.92 (m, 4 H); 13C NMR (125 MHz, CDCl3) d 161.0,
152.5, 136.5, 136.3, 128.9, 128.5, 127.6, 127.4, 126.4, 126.2 (q,
J = 31.4 Hz), 126.1, 122.3 (q, J = 274 Hz), 122.0, 114.0, 111.9 (q,
J = 7.4 Hz), 110.1, 57.5, 49.4, 46.3, 36.7, 26.5, 24.1; MS m/z 472
(M+), 473 (MH+); HRMS calcd for C24H23F3N4O3 (M) 472.1722,
found 472.1712; Anal. Calcd for C24H23F3N4O3: C, 61.01; H, 4.91;
N, 11.86. Found: C, 60.93; H, 5.00; N, 11.70.
J = 6.8 Hz), 3.98 (s,
3 C NMR
H), 1.37 (t, 1H, J = 6.8 Hz);13
(125 MHz, CDCl3) d 160.2, 135.2, 127.5, 123.2, 112.6, 60.9, 37.9,
14.2; MS m/z 199 (MH+).
4.2.33. Ethyl 4-amino-1-methylpyrrole-2-carboxylate (42)
10% Pd/C was added to a solution of 41 (1.0 g, 5.1 mmol) in
ethyl acetate (20 mL) at room temperature. The reaction mixture
was vigorously stirred under positive pressure of hydrogen for
1 h at room temperature and then filtered through Celite. The Cel-
ite was rinsed with ethyl acetate, and the combined filtrates were
evaporated to give 42 as colorless oil (0.85 g, 99%). 1H NMR (CDCl3)
d 6.43 (d, 1H, J = 1.7 Hz), 6.31 (d, 1H, J = 1.7 Hz), 4.21 (q, 2H,
J = 7.3 Hz), 3.77 (s, 3H), 2.94 (s, 2H), 1.29 (t, 3H,J = 7.3 Hz); 13C
NMR (125 MHz, CDCl3) d 160.0, 130.7, 120.3, 117.4, 108.0, 59.5,
36.2, 14.3; MS m/z 199 (MH+).
4.2.28. N-{4-[(Benzyl)(4-cyanophenyl)amino]-1-methylpyrrole-
2-carbonyl}pyrrolidine (37)
The title compound was prepared from 31 and benzyl bromide
by procedure C (36%). Colorless oil; 1H NMR (CDCl3) d 7.36 (d, 2H,
J = 9.4 Hz), 7.33 (t, 2H, J = 7.2 Hz), 7.28–7.24 (m, 3H), 6.75 (d, 2H,
J = 9.4 Hz), 6.37 (d, 1H, J = 2.1 Hz), 4.85 (s, 2H), 3.83 (s, 3H), 3.61–
3.59 (m, 4H), 1.94–1.91 (m, 4H); 13C NMR (125 MHz, CDCl3) d
161.2, 152.1, 137.4, 133.2, 128.7, 128.1, 127.1, 126.3, 125.6,
122.4, 120.4, 113.1, 110.7, 98.6, 56.9, 49.4, 46.3, 36.7, 26.5, 24.0;
MS m/z 384 (M+), 385 (MH+); HRMS calcd for C24H24N4O (M)
384.1950, found 384.1975.
4.2.34. Ethyl 1-methyl-4-(4-nitrophenyl)aminopyrrole-2-carbo-
xylate (43)
A well-dried flask was evacuated and backfilled with argon. The
flask was charged with 42 (0.85 g, 5.1 mmol), toluene (20 mL), BIN-
AP (0.31 g, 10 mol%), Cs2CO3 (2.3 g, 7.1 mmol), Pd2(dba)3 (230 mg,
10 mol%Pd), and 1-bromo-4-nitrobenzene (1.0 g, 5.0 mmol), then
evacuated and backfilled with argon. The reaction mixture was
stirred at 80 °C for 6 h, then diluted with ethyl acetate, washed
with water and brine, dried over MgSO4, and evaporated. The res-
idue was purified by silica gel column chromatography (n-hexane–
ethyl acetate = 5:1; 4:1; 3:1; 2:1; 1:1) to give 43 as a yellow
powder (0.81 g, 56%). 1H NMR (CDCl3) d 8.05 (d, 2 H, J = 9.4 Hz),
6.82 (d, 1H, J = 1.7 Hz), 6.75 (d, 1H, J = 1.7 Hz), 6.72 (d, 2H,
J = 9.4 Hz), 5.93 (s, 1H), 4.28 (q, 2H, J = 7.3 Hz), 3.93 (s, 3 H), 1.35
(t, 3H, J = 7.3 Hz);13C NMR (125 MHz, CDCl3) d 160.8, 152.5,
4.2.29. N-{4-[(Benzyl)(4-trifluoromethylphenyl)amino]-1-methyl-
pyrrole-2-carbonyl}pyrrolidine (38)
The title compound was prepared from 32 and benzyl bromide
by procedure C (30%). Colorless oil; 1H NMR (CDCl3) d 7.35–7.23
(m, 7H), 6.80 (d, 2H, J = 9.0 Hz), 6.60 (d, 1H, J = 1.7 Hz), 6.39 (d,
1H, J = 1.7 Hz), 4.85 (s, 2H), 3.83 (s, 3H), 3.62–3.59 (m, 4H), 1.93–
1.90 (m, 4H); MS m/z 427 (M+), 428 (MH+); HRMS calcd for
C24H24F3N3O (M) 427.1871, found 427.1880.

6810
K. Wakabayashi et al. / Bioorg. Med. Chem. 16 (2008) 6799–6812
138.8, 126.3, 124.3, 122.4, 121.9, 113.7, 112.1, 60.2, 37.0, 14.4; MS
4.2.39. 4-[(Benzyl)(4-nitrophenyl)amino]-1-methylpyrrole-2-
carboxamide (47)
m/z 289 (M+), 290 (MH+).
The title compound was prepared from 46 and ammonia by
procedure D (78%). Yellow powder. mp 208–209 °C (n-hexane–
ethyl acetate); 1H NMR (CDCl3) d 8.02 (d, 2H, J = 9.4 Hz), 7.35 (t,
2H, J = 7.3 Hz), 7.29 (t, 1H, J = 7.3 Hz), 7.25 (d, 2H, J = 7.3 Hz), 6.74
(d, 2H, J = 9.4 Hz), 6.71 (d, 1H, J = 1.7 Hz), 6.51 (d, 1H, J = 1.7 Hz),
5.45 (br, 2H), 4.91 (s, 2H), 3.94 (s, 3H);13C NMR (125 MHz, CDCl3)
d 162.6, 153.9, 138.3, 136.9, 128.9, 128.4, 127.5, 126.3, 125.8,
124.6, 124.0, 112.3, 110.1, 57.3, 37.1; MS m/z 350 (M+), 351
(MH+), HRMS calcd for C19H18N4O3 (M) 350.1379, found 350.1334.
4.2.35. Ethyl 4-[(benzyl)(4-nitrophenyl)amino]-1-methylpyrrole-
2-carboxylate (44)
Benzyl bromide (0.39 mL, 3.4 mmol) and potassium iodide
(0.47 g, 2.8 mmol) were added to a suspension of 43 (0.81 g,
2.8 mmol) and sodium hydride (0.10 g, 4.2 mmol) in DMF
(25 mL) at 0 °C, under argon. The reaction mixture was stirred at
room temperature for 4.5 h, then poured into water, and extracted
with ethyl acetate. The organic layer was washed with brine, dried
over MgSO4, and evaporated. The residue was purified by silica gel
column chromatography (n-hexane–ethyl acetate = 5:1; 4:1; 3:1)
to give 44 as a yellow foam (1.0 g, 94%). 1H NMR (CDCl3) d 7.98
(d, 2H, J = 9.4 Hz), 7.31 (t, 2H, J = 7.3 Hz), 7.24–7.21 (m, 3H), 6.84
(d, 1H, J = 2.1 Hz), 6.71 (d, 1H, J = 2.1 Hz), 6.70 (d, 2H, J = 9.4 Hz),
4.88 (s, 2H), 4.26 (q, 2H, J = 7.2 Hz), 3.88 (s, 3 H), 1.31 (t, 3H,
4.2.40. N,N-Diethyl 4-[(4-nitrophenyl)(benzyl)amino]-1-methyl-
pyrrole-2-carboxamide (48)
Diethylamine (58 lL, 0.57 mmol) was added to a solution of 46
(50 mg, 0.14 mmol), HOBt (19 mg, 0.14 mmol), and EDCI (27 mg,
0.14 mmol) in dichloromethane (1.0 mL) at room temperature, un-
der argon. The reaction mixture was stirred at room temperature
for 16 h, then poured into water. The mixture was extracted with
ethyl acetate. The organic layer was washed with brine, dried over
MgSO4, and evaporated. The residue was purified by preparative
TLC (n-hexane–ethyl acetate = 2:1; two times) to give 48 as an or-
ange oil (52 mg, 92%). 1H NMR (CD3OD) d 7.91 (d, 2H, J = 9.4 Hz),
7.29–7.25 (m, 4H), 7.20 (t, 1H, J = 6.4 Hz), 6.83 (d, 1H, J = 2.1 Hz),
6.77 (d, 2H, J = 9.4 Hz), 6.22 (d, 1H, J = 2.1 Hz), 4.90 (s, 2H), 3.65
(s, 3 H), 3.47 (q, 4H, J = 6.8 Hz), 1.16 (t, 6H, J = 6.8 Hz);13C NMR
(125 MHz, CD3OD) d 165.2, 155.8, 138.9, 138.8, 129.7, 129.4,
128.3, 127.9, 127.0, 126.6, 123.0, 113.4, 109.3, 58.0 44.7, 41.4,
35.7, 13.8; MS m/z 406 (M+), 407 (MH+); HRMS calcd for
J = 7.2 Hz);13
C NMR (125 MHz, CDCl3) d 160.7, 153.8, 138.1,
136.8, 128.8, 128.7, 127.3, 126.1, 125.7, 125.2, 122.1, 114.4,
112.3, 60.1, 57.2, 37.0, 14.3; MS m/z 379 (M+), 380 (MH+); HRMS
calcd for C21H21N3O4 (M) 379.1532, found 379.1520.
4.2.36. 4-[(Benzyl)(4-nitrophenyl)amino]-2-hydroxymethyl-1-
methylpyrrole (45)
A solution of 44 (53 lL, 0.14 mmol) in THF (1.2 mL) was added
to a suspension of lithium aluminum hydride (8.0 mg, 0.21 mmol)
in THF (0.3 mL) at 0 °C. The reaction mixture was stirred at 0 °C for
1.5 h, and then quenched with water (10
lL) and 2 M NaOH aq
(10 L) at 0 °C. MgSO4 was added to the solution, and the whole
l
solution was filtered. The filtrate was evaporated, and the residue
was purified by preparative TLC (n-hexane–ethyl acetate = 1:1) to
give 45 as an orange oil (32 mg, 68%). 1H NMR (CDCl3) d 7.97 (d,
2H, J = 9.4 Hz), 7.33–7.23 (m 5H), 6.71 (d, 2H, J = 9.4 Hz), 6.56 (d,
1H, J = 1.7 Hz), 6.03 (d, 1H, J = 1.7 Hz), 4.89 (s, 2H), 4.57 (s, 2H),
3.65 (s, 3H);13C NMR (125 MHz, CDCl3) d 154.2, 137.6, 137.1,
131.4, 128.7, 128.3, 127.2, 126.1, 125.7, 119.0, 112.1, 106.5, 57.4,
56.6, 33.9; MS m/z 337 (M+), 338 (MH+); HRMS calcd for
C23H26N4O3 (M) 406.2005, found 406.2015.
4.2.41. N,N-Di-n-butyl-4-[(benzyl)(4-nitrophenyl)amino]-1-
methylpyrrole-2-carboxamide (49)
The title compound was prepared from 46 and di-n-butylamine
by procedure D (77%). Orange oil; 1H NMR (CDCl3) d 7.98 (d, 2H,
J = 9.4 Hz), 7.32 (t, 2H, J = 7.3 Hz), 7.26 (t, 1H, J = 7.3 Hz), 7.24 (d,
2H, J = 7.3 Hz), 6.73 (d, 2H, J = 9.4 Hz), 6.62 (d, 1H, J = 2.1 Hz),
6.18 (d, 1H, J = 2.1 Hz), 4.90 (s, 2H), 3.70 (s, 3H), 3.44 (t, 4H,
J = 7.7 Hz), 1.56 (br, 4H), 1.28 (br, 4H), 0.90 (br, 6H);13C NMR
(125 MHz, CDCl3) d 163.3, 154.1, 137.8, 137.0, 128.7, 128.0,
127.3, 126.3, 126.1, 125.7, 120.9, 112.1, 108.0, 57.3, 48.9, 45.2,
35.6, 30.3, 20.0, 13.7; MS m/z 462 (M+), 463 (MH+), HRMS calcd
for C27H34N4O3 (M) 462.2631, found 462.2587.
C19H19N3O3 (M) 337.1426, found 337.1440.
4.2.37. 4-[(Benzyl)(4-nitrophenyl)amino]-1-methylpyrrole-2-
carboxylic acid (46)
A suspension of 44 (1.0 g, 2.6 mmol) in 30% KOH aq (15 mL),
methanol (15 mL), and tetrahydrofuran (7 mL) was heated at
110 °C for 2 h. The solution was cooled and acidified to about pH
1 with 2 M HCl aq and then extracted with ether. The organic layer
was washed with brine, dried over MgSO4, and evaporated to give
46 (0.89 g, 96%). Yellow powder. mp 185.5–186.5 °C (n-hexane–
ethyl acetate); 1H NMR (CDCl3) d 8.02 (d, 2 H, J = 9.4 Hz), 7.35 (t,
2 H, J = 7.3 Hz), 7.28 (t, 1H, J = 7.3 Hz), 7.25 (d, 2H, J = 7.3 Hz),
7.00 (d, 1H, J = 2.1 Hz), 6.80 (d, 1H, J = 2.1 Hz), 6.75 (d, 2H,
J = 9.4 Hz), 4.92 (s, 2H), 3.92 (s, 3 H);13C NMR (125 MHz, CDCl3) d
164.1, 153.7, 138.4, 136.8, 129.2, 128.9, 127.5, 126.7, 126.3,
125.8, 120.9, 116.5, 112.4, 57.1, 37.3; MS m/z 351 (M+), 352
(MH+). Anal. Calcd for C19H17N3O4: C, 64.95; H, 4.88; N, 11.96.
Found: C, 64.84; H, 4.90; N, 12.01.
4.2.42. N-[4-[(Benzyl)(4-nitrophenyl)amino]-1-methylpyrrole-
2-carbonyl]piperidine (50)
The title compound was prepared from 46 and piperidine by
procedure D (91%). Yellow powder. Mp 111–112 °C (n-hexane–
ethyl acetate); 1H NMR (CDCl3) d 8.01 (d, 2H, J = 9.4 Hz), 7.33 (t,
2H, J = 7.3 Hz), 7.27 (t, 1H, J = 7.3 Hz), 7.25 (d, 2H, J = 7.3 Hz), 6.75
(d, 2H, J = 9.4 Hz), 6.62 (d, 1H, J = 1.7 Hz), 6.17 (d, 1H, J = 1.7 Hz),
4.90 (s, 2H), 3.73 (s, 3 H), 3.64–3.61 (m, 4H), 1.71–1.68 (m, 2H),
1.61–1.56 (m, 4H);13C NMR (125 MHz, CDCl3) d 162.0, 154.1,
137.9, 137.0, 128.7, 128.0, 127.3, 126.4, 125.7, 125.4, 121.5,
112.2, 109.1, 57.2, 46.2, 35.7, 26.1, 24.6; MS m/z 418 (M+), 419
(MH+), HRMS calcd for C24H26N4O3 (M) 418.2005, found 406.2009.
4.2.38. General procedure D for compounds 47, 49–52
Methanesulfonyl chloride (1.1 equiv) was added to a solution of
46 (1 equiv) and triethylamine (3 equiv) in THF at 0 °C, under ar-
gon. The reaction mixture was stirred at 0 °C for 30 min. Amine
(1.5 equiv) was then added, and the reaction mixture was stirred
at 0 °C until 46 was consumed. The mixture was poured into satu-
rated NH4Cl, and extracted with ethyl ester. The organic layer was
washed with brine, dried over MgSO4, and evaporated. The residue
was purified by silica gel column chromatography or preparative
TLC.
4.2.43. N-[4-[(Benzyl)(4-nitrophenyl)amino]-1-methylpyrrole-
2-carbonyl]indoline (51)
The title compound was prepared from 46 and indoline by pro-
cedure D (53%). Yellow powder. mp 203–208 °C (chloroform–ethyl
acetate); 1H NMR (DMSO-d6) d 8.00 (d, 2H, J = 9.4 Hz), 7.99 (br, 1H),
7.35–7.29 (m, 4H), 7.24 (t, 2H, J = 6.8 Hz), 7.15 (d, 1H, J = 2.1 Hz),
7.14 (d, 1H, J = 7.3 Hz), 6.99 (t, 1H, J = 7.3 Hz), 6.84 (d, 2H,
J = 9.4 Hz), 6.70 (d, 1H, J = 2.1 Hz), 4.99 (s, 2H), 4.23 (t, 2H,
J = 8.1 Hz), 3.73 (s, 3H), 3.07 (t, 2H, J = 8.1 Hz);13
C NMR

K. Wakabayashi et al. / Bioorg. Med. Chem. 16 (2008) 6799–6812
6811
(125 MHz, DMSO-d6) d 160.1, 154.0, 142.9, 137.2, 136.9, 132.6,
128.6, 127.4, 127.0, 126.7, 126.5, 125.7, 125.3, 124.9, 123.6,
123.3, 116.6, 112.4, 110.6, 56.5, 50.5, 35.9, 28.0; MS m/z 452
(M+), 453 (MH+), HRMS calcd for C27H24N4O3 (M) 452.1848, found
452.1845; Anal. Calcd for C27H24N4O3: C, 71.67; H, 5.35; N, 12.38.
Found: C, 71.39; H, 5.45; N, 12.27.
and incubated at 27 °C until the OD600 reached 0.5–0.6. Follow-
ing the addition of IPTG to a concentration of 1 mM, incubation
was continued for an additional 4.5 h. Cells were harvested by
centrifugation at 4000g at 4 °C for 15 min and stored at °80 °C
until use. All subsequent operations were performed at 4 °C.
The bacterial pellet obtained from 40 mL of culture was resus-
pended in 1 mL of ice-cold TEGDM buffer (10 mM Tris, 1 mM
EDTA, 10% glycerol, 10 mM DTT, 10 mM sodium molybdate). This
suspension was subjected to sonication using 7 s  12 bursts on
ice (USP-600A sonicator, Shimadzu, Japan), and crude GST-hAR-
LBD fraction was prepared by centrifugation of the suspension
at 12,000g for 30 min at 4 °C. This supernatant protein, crude
4.2.44. (3S,4S)-N-[4-[(Benzyl)(4-nitrophenyl)amino]-1-methyl-
pyrrole-2-carbonyl]-3,4-dihydroxypyrrolidine (52)
(3S,4S)-N-[4-[(Benzyl)(4-nitrophenyl)amino]-1-methylpyrrole-
2-carbonyl]-3,4-di(methoxymethoxy)pyrrolidine was prepared
from 46 and (3S,4S)-di(methoxymethoxy)pyrrolidine by procedure
D, and hydrolyzed (46 mg, 0.088 mmol) in methanol (1.5 mL) and
2 M HCl aq at 65 °C. After 1 h, the mixture was poured into water
and extracted with ethyl acetate. The organic layer was washed
with brine, dried over MgSO4, and evaporated. The residue was
purified by silica gel column chromatography (chloroform–metha-
nol = 40:1; 20:1) to give 52 (28 mg, 73%) as a yellow powder. mp
212.5–213 °C (n-hexane–ethanol); 1H NMR (CD3OD) d 8.00 (d,
2H, J = 9.4 Hz), 7.33–7.29 (m, 4H), 7.25–7.24 (m, 1H), 6.89 (d, 1H,
J = 1.7 Hz), 6.84 (d, 2H, J = 9.4 Hz), 6.49 (d, 1H, J = 1.7 Hz), 4.99 (d,
1H, J = 17.7 Hz), 4.93 (d, 1H, J = 17.7 Hz), 4.10 (d, 2H, J = 14.1 Hz),
3.86–3.81 (m, 2H), 3.78 (s, 3H), 3.56 (d, 1H, J = 11.5 Hz), 3.47 (d,
1H, J = 11.5 Hz);13C NMR (125 MHz, CD3OD) d 164.1, 155.9, 139.1,
138.9, 129.8, 129.5, 128.3, 127.9, 126.6, 126.6, 124.6, 113.6,
112.5, 76.3, 74.6, 58.1, 56.1, 53.4, 36.6; MS m/z 436 (M+), 437
(MH+); HRMS calcd for C23H24N4O5(M) 436.1747, found 436.1761.
receptor fraction, was diluted to
a protein concentration of
0.3–0.5 mg/mL and used in binding assays as GST-hAR-LBD frac-
tion. Total protein was determined by using a Coomassie Protein
Assay Reagent and Albumin Standard (Pierce). Binding studies
were performed by incubating increasing concentrations
(100 nM to 100 lM) of test compound (dissolved in DMSO) with
the GST-hAR-LBD fraction in the presence of a saturating concen-
tration of [3H]testosterone (10 nM) at 4 °C for 12–18 h. Non-spe-
cific binding was assessed by addition of a 1000-fold excess of
non-radioactive testosterone. Separation of bound and free radio-
activity was achieved by the charcoal method. After incubation
was completed, 500 lL of 1.5% (w/v) dextran-coated charcoal
(Sigma) was added to each reaction solution. After 20 min, the
solution was centrifuged at 550g for 10 min (high-speed refriger-
ated micro centrifuge, TOMY MX-15). The protein fraction
(350 lL) was collected and the radioactivity was determined
4.2.45. N-[4-[(Benzyl)(4-nitrophenyl)amino]phenylcarbonyl]
pyrrolidine (53)
with a liquid scintillation counter. All experiments were per-
formed in duplicate.
The title compound was prepared from ethyl 4-(4-nitro-
phenyl)aminobenzoate by the procedure shown in Scheme 3. Yel-
low powder. Mp 81–82 °C (n-hexane–dichloromethane); 1H NMR
(CDCl3) d 8.04 (d, 2H, J = 9.4 Hz), 7.60 (t, 2H, J = 8.6 Hz), 7.31 (m,
5H), 7.33 (d, 2H, J = 8.5 Hz), 6.81 (d, 2H, J = 9.4 Hz), 5.13 (s, 2H),
3.66 (t, 2H, J = 6.8 Hz), 3.49 (t, 2H, J = 6. 8 Hz), 1.98 (quint, 2H,
J = 6.8 Hz), 1.91 (quint, 2H, J = 6.8 Hz);13C NMR (125 MHz, CDCl3)
d 168.7, 152.7, 147.2, 139.3, 136.7, 134.9, 129.3, 129.0, 127.6,
125.7, 125.6, 114.4, 56.5, 53.4, 49.7, 46.3, 26.5, 24.4; MS m/z 401
(M+), 402 (MH+), HRMS calcd for C24H24N3O3(MH+) 402.1818,
found 402.1858.
4.4. SC-3 growth inhibition assay
Shionogi carcinoma-3 (SC-3) cells were cloned from Shionogi
carcinoma 115 cells, which were established from a mouse breast
cancer. SC-3 shows androgen-dependent growth.32 In this assay,
androgenic and anti-androgenic activities of test compounds were
determined in terms of SC-3 growth promotion by the test com-
pound alone and inhibition of testosterone-induced cell growth
by the test compound, respectively. SC-3 cells were cultured in
the presence of MEM supplemented with 2% FBS and 10 nM testos-
terone at 37 °C 5% CO2. All experiments were performed in tripli-
cate or more. For SC-3 cell growth inhibition assay, the cells
were trypsinized, and seeded into 96-well plates at 2.0 Â 104 cell/
mL in MEM containing 2% DCC-FBS. Various concentrations of test
4.2.46. N-[3-[(Benzyl)(4-nitrophenyl)amino]phenylcarbonyl]
pyrrolidine (54)
The title compound was prepared from ethyl 3-(4-nitro-
phenyl)aminobenzoate by the procedure shown in Scheme 3. Or-
ange oil; 1H NMR (CDCl3) d 8.01 (d, 2H, J = 9.4 Hz), 7.38 (m, 9H),
6.74 (d, 2H, J = 9.4 Hz), 5.05 (s, 2H), 3.61 (t, 2H, J = 6.8 Hz), 3.33
(t, 2H, J = 6.8 Hz), 1.95 (quint, 2H, J = 6.8 Hz), 1.87 (quint, 2H,
compound (from 0.1 lM to 100 lM DMSO solution) and/or testos-
terone in DMSO solution (final concentration 10 nM) were added
on the next day. Then the plates were incubated at 37 °C 5% CO2
for 3 days, and the cell number was determined using the WST-1
method with a Cell Counting Kit (Dojindo) and an MPR-A4i2 micro
plate reader (Tosoh, Japan). The number of cells in wells with tes-
tosterone alone was defined as 100%. The concentration of the test
compound that inhibited the increase of the cell number induced
by 10 nM testosterone by 50% was quantified (IC50) after log-logit
transformation.
J = 6.8 Hz);13
C NMR (125 MHz, CDCl3) d 168.4, 152.9, 145.7,
139.7, 136.7, 130.0, 128.9, 127.9, 127.5, 126.4, 125.7, 125.4,
125.2, 113.7, 56.6, 49.5, 46.2, 26.4, 24.3; MS m/z 401 (M+), 402
(MH+), HRMS calcd for C24H24N3O3 (MH+) 402.1818, found
402.1810.
4.3. Receptor binding assay
4.5. Receptor binding assay with LNCaP cells
Binding affinities of test compounds for hAR (human andro-
gen receptor) were measured in competition experiments using
[3H]testosterone and cytosolic fraction of hAR-LBD (hAR ligand-
binding domain)-transformed Escherichia coli, as described previ-
ously.27 A hAR-LBD expression plasmid vector, which encodes
GST-hAR-LBD (627–919 aa, EF domain) fusion protein under
the lac promoter (provided by Prof. S. Kato, Univ. of Tokyo),
was transfected into E. coli strain HB-101. The bacteria (10 mL)
were cultured for two nights, then added to 1 L of LB medium,
LNCaP cells were cultured in the presence of RPMI1640 supple-
mented with 10% FBS at 37 °C under an atmosphere of 5% CO2 in
air. Cells were harvested with a cell scraper and by centrifugation
at 800g at 4 °C for 5 min, and stored at À80 °C until use. The col-
lected cells (ca. 1.2 g) were suspended in 21 mL of the cell extrac-
tion buffer (10 mM Tris, 1.5 mM EDTA, 0.25 M sucrose, 10 mM
sodium molybdate, 1 mM PMSF). This suspension was subjected
to homogenization (0 °C, 1000 rpm) and a crude fraction was

6812
K. Wakabayashi et al. / Bioorg. Med. Chem. 16 (2008) 6799–6812
11. Veldscholte, J.; Berrevoets, C. A.; Ris-Stalpers, C.; Kuiper, G. G.; Jenster, G.;
Trapman, J.; Brinkmann, A. O.; Mulder, E. J. Steroid Biochem. Mol. Biol. 1992, 41,
665–669.
12. Suzuki, H.; Akakura, K.; Komiya, A.; Aida, S.; Akimoto, S.; Shimazaki, J. Prostate
1996, 29, 153–158.
13. Hara, T.; Miyazaki, J.; Araki, H.; Yamaoka, M.; Kanzaki, N.; Kusaka, M.;
Miyamoto, M. Cancer Res. 2003, 63, 149–153.
14. Poujol, N.; Wurtz, J. M.; Tahiri, B.; Lumbroso, S.; Nicolas, J. C.; Moras, D.; Sultan,
C. J. Biol. Chem. 2000, 275, 24022–24031.
15. Bohl, C. E.; Gao, W.; Miller, D. D.; Bell, C. E.; Dalton, J. T. Proc. Natl. Acad. Sci.
U.S.A. 2005, 102, 6201–6206.
16. Bohl, C. E.; Chang, C.; Mohler, M. L.; Chen, J.; Miller, D. D.; Swaan, P. W.; Dalton,
J. T. J. Med. Chem. 2004, 47, 3765–3776.
17. Fujii, S.; Goto, T.; Ohta, K.; Hashimoto, Y.; Suzuki, T.; Ohta, S.; Endo, Y. J. Med.
Chem. 2005, 48, 4654–4662.
18. Kinoyama, I.; Taniguchi, N.; Toyoshima, A.; Nozawa, E.; Kamikubo, T.;
Imamura, M.; Matsuhisa, A.; Samizu, K.; Kawanimani, E.; Niimi, T.; Hamada,
N.; Koutoku, H.; Furutani, T.; Kudoh, M.; Okada, M.; Ohta, M.; Tsukamoto, S.
J. Med. Chem. 2006, 49, 716–726.
19. Sun, C.; Robl, J. A.; Wang, T. C.; Huang, Y.; Kuhns, J. E.; Lupisella, J. A.; Beehler, B.
C.; Golla, R.; Sleph, P. G.; Seethala, R.; Fura, A.; Krystek, S. R., Jr.; An, Y.; Malley,
M. F.; Sack, J. S.; Salvati, M. E.; Grover, G. J.; Ostrowski, J.; Hamann, L. G. J. Med.
Chem. 2006, 49, 7596–7599.
20. Higuchi, R. I.; Arienti, K. L.; Lopez, F. J.; Mani, N. S.; Mais, D. E.; Caferro, T. R.;
Long, Y. O.; Jones, T. K.; Edwards, J. P.; Zhi, L.; Schrader, W. T.; Negro-Vilar, A.;
Marschke, K. B. J. Med. Chem. 2007, 50, 2486–2496.
21. McGinley, P. L.; Koh, J. T. J. Am. Chem. Soc. 2007, 129, 3822–3823.
22. Brzozowski, A. M.; Pike, C. W.; Dauter, Z.; Hubbard, R. E.; Bonn, T.; Engström,
O.; Öhman, L.; Greene, G. L.; Gustafsson, J.-A.; Carlquist, M. Nature 1997, 389,
753–758.
prepared by centrifugation of the suspension at 105,000g for 1 h
for 4 °C. This supernatant protein, crude receptor fraction, was
diluted to a protein concentration of 0.25 mg/mL with the cell
extraction buffer. Binding studies were performed by incubating
increasing concentrations of test compound (dissolved in DMSO)
with receptor fraction in the presence of a saturating concentra-
tion of [3H]testosterone (1 nM) at 4 °C for 12–18 h. Non-specific
binding was assessed by addition of a 1000-fold excess of non-
radioactive testosterone. Separation of bound and free radioac-
tivity was achieved by the charcoal method. After incubation
was completed, 500 lL of 1.5% (w/v) dextran-coated charcoal
(Sigma) was added to each reaction solution. After 20 min, the
solution was centrifuged at 500g for 10 min. The protein fraction
(350 lL) was collected and the radioactivity was determined
with a liquid scintillation counter. All experiments were per-
formed in duplicate.
4.6. PSA production assay of LNCaP by ELISA
LNCaP cells maintained in RPMI1640 medium supplemented
with 10% FBS were plated at the concentration of 3 Â 105 cell/mL
with an appropriate concentration (0–10 lM) of a test compound
and incubated in the same medium for 48 h at 37 °C under an
atmosphere containing 5% CO2 in the presence or absence of
10 nM testosterone. After the incubation, the supernatant was col-
lected and the amount of PSA contained in the supernatant was
quantified by the use of ELISA PSA Assay Kit (Cayman Co. Ltd)
according to the protocol recommended by the supplier. The
amount of PSA produced in the presence of 10 nM testosterone
alone was defined as 100% (Fig. 4).
23. Carlberg, C. J. Cell. Biochem. 2003, 88, 274–281.
24. 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, 26164–26171.
25. Gonzalez, M. M.; Samenfeld, P.; Peräkylä, M.; Carlberg, C. Mol. Endocrinol. 2003,
17, 2028–2038.
26. Wurtz, J.; Bourguet, W.; Renaud, J. P.; Vivat, V.; Chambon, P.; Moras, D.;
Gronemeyer, H. Nat. Struct. Biol. 1996, 3, 87–94.
27. Ishioka, T.; Tanatani, A.; Nagasawa, K.; Hashimoto, Y. Bioorg. Med. Chem. Lett.
2003, 13, 2655–2658.
28. Kato, Y.; Nakano, Y.; Sano, H.; Tanatani, A.; Kobayashi, H.; Shimazawa, R.;
Koshino, H.; Hashimoto, Y.; Nagasawa, K. Bioorg. Med. Chem. Lett. 2004, 14,
2579–2583.
Acknowledgments
29. Nakano, Y.; Kato, Y.; Imai, K.; Ochiai, E.; Namekawa, J.-i.; Ishizuka, S.;
Takenouchi, K.; Tanatani, A.; Hashimoto, Y.; Nagasawa, K. J. Med. Chem. 2006,
49, 2398–2406.
30. Wakabayashi, K.; Miyachi, H.; Hashimoto, Y.; Tanatani, A. Bioorg. Med. Chem.
2005, 13, 2837–2846.
31. Wolfe, J. P.; Tomori, H.; Sadighi, J. P.; Yin, J.; Buchwald, S. L. J. Org. Chem. 2000,
65, 1158–1174.
32. Kasayama, S.; Saito, H.; Kouhara, H.; Sumitani, S.; Sato, B. J. Cell Physiol. 1993,
154, 254–261.
This study was supported by Kato Memorial Bioscience Founda-
tion, and Grants-in Aid for Scientific Research from the Ministry of
Education, Culture, Sports, Science and Technology, Japan, and the
Japan Society for the Promotion of Science. K.W. acknowledges the
support of the Japan Society for the Promotion of Science in the
form of a Fellowship for Japanese Junior Scientists.
33. Fenton, M. A.; Shuster, T. D.; Fertig, A. M.; Taplin, M. –E.; KolvenBag, G.; Bubley,
G. J.; Balk, S. P. Clin. Cancer Res. 1997, 3, 1383–1388.
References and notes
34. Veldscholte, J.; Ris-Stalpers, C.; Kuiper, G. G. J. K.; Jenster, G.; Berrevoets, C.;
Claassen, E.; van Rooij, H. C. J.; Trapman, J.; Brinkmann, A. O.; Mulder, E.
Biochem. Biophys. Res. Commun. 1990, 173, 534–540.
35. Taplin, M.-E.; Bubley, G. J.; Ko, Y.-J.; Small, E. J.; Upton, M.; Rajeshkumar, B.;
Balk, S. P. Cancer Res. 1999, 59, 2511–2515.
1. Freedman, L. P. Molecular Biology of Steroid and Nuclear Hormone Receptors;
Birkhauser: Boston, MA, 1998.
2. Negro-Vilar, A. J. Clin. Endocrinol. Metab. 1999, 84, 3459–3462.
3. Gao, W.; Bohl, C. E.; Dalton, J. T. Chem. Rev. 2005, 105, 3352–3370.
4. Gao, W. Q.; Dalton, J. T. Drug Discov. Today 2007, 12, 241–248.
5. Neri, R. O. Adv. Sex Horm. Res. 1976, 2, 233–262.
36. Riegman, P. H.; Vlietstra, R. J.; van der Korput, J. A.; Brinkmann, A. O.; Trapman,
J. Mol. Endocrinol. 1991, 5, 1921–1930.
6. Blackledge, G. Cancer 1993, 1, 3830–3833.
37. Lee, C.; Sutkowski, D. M.; Sensibar, J. A.; Zelner, D.; Kim, I.; Amsel, I.; Shaw, N.;
Prins, G. S.; Kozlowski, J. M. Endocrinology 1995, 136, 796–803.
38. Salvati, M. E.; Balog, A.; Shan, W.; Wei, D. D.; Pickering, D.; Attar, R. M.; Geng, J.;
Rizzo, C. A.; Gottardis, M. M.; Weinmann, R.; Krystek, S. R.; Sack, J.; An, Y.; Kish,
K. Bioorg. Med. Chem. Lett. 2005, 15, 271–276.
7. Maucher, A.; von Angerer, E. J. Cancer Res. Clin. Oncol. 1993, 119, 669–674.
8. Sufrin, G.; Coffey, D. S. Invest. Urol. 1976, 13, 429–434.
9. Narayana, A. S.; Loening, S. A.; Culp, D. A. Br. J. Urol. 1981, 53, 152–153.
10. Scher, H. I.; Steineck, G.; Kelly, W. K. Urology 1995, 46, 142–148.