Design, synthesis, and biological evaluation of 4-arylmethyl-1- phenylpyrazole and 4-aryloxy-1-phenylpyrazole derivatives as novel androgen receptor antagonists

Article abstract of DOI:10.1016/j.bmc.2012.02.005

A series of 4-arylmethyl-1-phenylpyrazole and 4-aryloxy-1-phenylpyrazole compounds B were designed, synthesized, and evaluated for their potential as new-generation androgen receptor (AR) antagonists therapeutically effective against castration-resistant

Full text of DOI:10.1016/j.bmc.2012.02.005

Bioorganic & Medicinal Chemistry 20 (2012) 2338–2352
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis, and biological evaluation of 4-arylmethyl-1-phenylpyrazole
and 4-aryloxy-1-phenylpyrazole derivatives as novel androgen receptor
antagonists
Satoshi Yamamoto a, , Naoki Tomita a, Yuri Suzuki b, Tomohiko Suzaki a, Tomohiro Kaku a, Takahito Hara a,
⇑
Masuo Yamaoka a, Naoyuki Kanzaki a, Atsushi Hasuoka a, Atsuo Baba a, Mitsuhiro Ito a
a Pharmaceutical Research Division, Takeda Pharmaceutical Company Ltd, 26-1, Muraokahigashi 2-chome, Fujisawa, Kanagawa 251-8555, Japan
b CMC Center, Takeda Pharmaceutical Company Ltd, 17-85, Jusohommachi 2-chome, Yodogawa-ku, Osaka 532-8686, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 4-arylmethyl-1-phenylpyrazole and 4-aryloxy-1-phenylpyrazole compounds B were designed,
synthesized, and evaluated for their potential as new-generation androgen receptor (AR) antagonists
therapeutically effective against castration-resistant prostate cancer (CRPC). Introduction of a bulky
amide substituent (R2) to the terminal aryl ring of the 4-arylmethyl group favored the reduction of ago-
nistic activity and improved the pharmacokinetic (PK) properties. Similarly, introduction of a bulky sub-
stituent in the 4-aryloxy derivatives also resulted in improved PK properties. Compounds 28h and 44b
exhibited potent antitumor effects against a CRPC model of LNCaP-hr cell line in a mouse xenograft
model. On the contrary, bicalutamide showed only partial suppression of tumor growth. These results
suggest that the novel pyrazole derivatives are new-generation AR antagonists, different from the
‘first-generation’ antagonists such as bicalutamide in a CRPC treatment model.
Received 9 January 2012
Revised 2 February 2012
Accepted 2 February 2012
Available online 10 February 2012
Keywords:
Androgen receptor antagonists
Castration-resistant prostate cancer
4-Arylmethyl-1-phenylpyrazole derivatives
4-Aryloxy-1-phenylpyrazole derivatives
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
such as bicalutamide show significant synergistic effects by blocking
adrenal androgen signals as well as by suppressing transient in-
Prostate cancer (PC) is the most common cancer in American
males and the second-leading cause of cancer deaths after lung can-
cer.1 Treatment of PCs can be of various types such as surgery, radi-
ation therapy, and endocrine therapy. These therapies are provided
alone or in combination depending on the stage of the PCs, age of the
patients, and so on. In endocrine therapy, progression of PCs is sup-
pressed by inhibiting the action and/or secretion of androgens,
which play a pivotal role in the proliferation of PC cells.2 Secretion
of testosterone (95% from the testis) is prevented by surgical or
chemical castration using gonadotropin-releasing hormone (GnRH)
analogs. Androgen receptor (AR) antagonists are used in the treat-
ment of PCs; AR antagonists suppress the action of androgens by
inhibiting their binding to the ARs. Currently, AR antagonists such
as cyproterone acetate,3 flutamide,4,5 nilutamide,6,7 and bicaluta-
mide8–17 are used for clinical purposes (Fig. 1). When a combination
of AR antagonists and chemical castration is used, AR antagonists
crease in testosterone levels induced by GnRH analogs. This ‘com-
bined androgen blockade (CAB)’ therapy is at least initially
effective for PC treatment.15 However, in spite of the initial success
of the PC treatment, there is a considerable population of patients
who develop castration-resistant prostate cancer (CRPC) after the
prolonged use of an AR antagonist.18–20 In such cases, discontinua-
tion of CAB therapy or change to another AR antagonist should be
considered.21 Despite its risk of severe adverse effects, docetaxel, a
microtubule-stabilizing agent, is the only approved chemothera-
peutic agent against CRPC.22 Thus, it is necessary to develop effective
new-generation AR antagonists against CRPC. In recent years, inten-
siveclinicalandnonclinicalstudieshavebeencarriedout.23–28 AnAR
antagonist MDV-3100 is reported to be effective against CRPC, and it
is currently under phase 3 clinical trials.29,30 Because of its unique
pharmaceutical properties, this agent is categorized as a ‘second-
generation’ AR antagonist.
We discovered novel 1-arylmethyl-4-(4-cyanophenyl)pyrrole
derivatives 1 as orally available AR antagonists (Fig. 2).31 Among
these antagonists, compound 2 showed antitumor effects against
bicalutamide-resistant LNCaP-cxD2 cell line as well as androgen
dependent JDCaP cell line in a mouse xenograft model upon oral
administration.31 In optimizing pyrroles 1, we found that cyano-
phenyl moiety a, which might correspond to the A ring of dihydro-
testosterone (DHT), was favorable for the antagonistic activity.
Abbreviations: AR, androgen receptor; CRPC, castration-resistant prostate can-
cer; CAB, combined androgen blockade; DHT, dihydrotestosterone; SAR, structure–
activity relationship; EDC, N-[3-(dimethylamino)propyl]-N 0-ethylcarbodiimide
hydrochloride; DMT-MM, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpho-
lin-4-ium chloride.
⇑
Corresponding author. Tel.: +81 466 32 1219; fax: +81 466 29 4455.
E-mail address: yamamoto_satoshi@takeda.co.jp (S. Yamamoto).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2012.02.005

S. Yamamoto et al. / Bioorg. Med. Chem. 20 (2012) 2338–2352
2339
Figure 1. Known androgen receptor antagonists.
Figure 2. Design of 1-(4-cyanophenyl)-4-substitutedpyrazole AR antagonists.
Our structure–activity relationship (SAR) also suggested that the
arylmethyl moiety b, which did not mimic any moiety of DHT,
was an important contributor to the strong antagonistic activity.
To date some studies on other AR antagonists possessing a cyano-
phenyl moiety and a second aryl ring have been reported.27,28,32–34
In addition to optimizing pyrroles 1, we investigated a new ser-
ies of compounds for novel AR antagonists by modifying the cen-
tral pyrrole ring. A basic scaffold A was designed based on the
importance of a and b for antagonistic activity (Fig. 2). This scaffold
contains a cyanophenyl group, a central ring Y, a linker Q, and a
terminal ring M. In contrast to our previous pyrrole studies
(Y1 = N), the Y1 atom of ring Y was fixed at carbon so that a hetero
atom or carbon could be introduced at Q1 or Q2. We set out to ex-
plore novel AR antagonists based on scaffold A, and found that pyr-
azole derivative 3 showed AR antagonistic activity. It is noteworthy
that compound 3 possessed an activity profile that was deemed
appropriate for efficacy against CRPC: strong AR antagonistic activ-
ity without any significant agonistic activity. Thus we selected 3 as
the lead for a new series of AR antagonists, and studied substituent
modifications at R1 on the cyanophenyl group, linker X, and termi-
nal ring T (compound B) to obtain compounds with good efficacy
and pharmacokinetic (PK) properties. In this paper, we describe
the design, synthesis, and biological evaluation of pyrazole com-
pounds as novel AR antagonists.
2. Chemistry
4-Benzyl-1-phenylpyrazoles 3, 16a–c, and 18a–c were prepared
as shown in Scheme 1. Reaction of benzyl halides 4–7 with 2,4-pen-
tanedione gave diketones 8–11. Cyclization of 8–11 with hydrazine
hydrate gave 4-benzylpyrazoles 12–15, which were arylated with
4-fluorobenzonitriles to afford 4-benzyl-1-phenylpyrazoles 3,
16a–c, and 17a–c. Basic hydrolysis of esters 17a–c gave carboxylic
acids 18a–c. Scheme 2 illustrates the synthesis of 1-phenyl-4-phen-
yloxypyrazoles 25 and 27. The reaction of phenols 19 and 20 with 3-
chloropentan-2,4-dione afforded phenyl ether 21 and 22, which
were treated with hydrazine hydrate to give 4-phenoxypyrazoles
23 and 24. Coupling of 23 and 24 with 2-chloro-4-fluorobenzonitrile
afforded 25 and 26, respectively. Ester 26 was subjected to basic
hydrolysis to afford carboxylic acid 27. 4-Benzyl-1-phenylpyrazoles
28a–i and 1-phenyl-4-phenyloxypyrazoles 29a,b, which possessed
an amide moiety on the terminal benzene ring, were synthesized as

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S. Yamamoto et al. / Bioorg. Med. Chem. 20 (2012) 2338–2352
Scheme 1. Synthesis of compounds 3, 16a–c, and 18a–c. Reagents and conditions: (a) 2,4-Pentanedione, EtONa, EtOH, reflux; (b) hydrazine hydrate, EtOH, reflux, 17–72% for
two steps; (c) 4-fluorobenzonitrile (3), 2-chloro-4-fluorobenzonitrile (16a and 17a–c), 4-fluoro-2-(trifluoromethyl)benzonitrile (16b) or 3-chloro-4-fluorobenzonitrile (16c),
NaH, DMF, 0 °C to room temp, 44%–quant.; (d) 1 N NaOH, MeOH, THF, 50 °C, 66%–quant.
Scheme 2. Synthesis of compounds 25 and 27. Reagents and conditions: (a) 3-Chloropentan-2,4-dione, Cs2CO3, acetone, reflux; (b) 3-chloropentan-2,4-dione, K2CO3, DMF,
80 °C; (c) hydrazine hydrate, AcOH, room temp, 29% (23) and 52% (24) for two steps; (d) NaH, 2-chloro-4-fluorobenzonitrile, DMF, 0 °C, 53% (25); (e) 1 N NaOH, MeOH, THF,
40 °C, 72% from 24 (two steps).
Scheme 3. Synthesis of compounds 28a–i and 29a,b. Reagents and conditions: (a) EDC, HOBt, amines (RaH), DMF, room temp, 20–93%; (b) DMT-MM, amines (RaH), THF,
isopropanol, room temp, 60–89%.
shown in Scheme 3. Condensation of acids 18a–c and 27 with
amines was carried out using N-[3-(dimethylamino)propyl]-N0-eth-
ylcarbodiimide hydrochloride (EDC) or 4-(4,6-dimethoxy-1,3,-
5-triazin-2-yl)-4-methylmorpholin-4-ium chloride (DMT-MM).
DMT-MM was used for the condensation of amines possessing an
alcoholic hydroxyl group, so that acylation of the amino group
proceeded selectively over esterification of the hydroxyl
group.

S. Yamamoto et al. / Bioorg. Med. Chem. 20 (2012) 2338–2352
2341
Scheme 4. Synthesis of compounds 33, 36, and 38. Reagents and conditions: (a) NaH, 2-chloro-4-fluorobenzonitrile, DMF, 0 °C, 58%; (b) N-bromosuccinimide, AcOH, room
temp, 57%; (c) 4-fluorophenylboronic acid, PS-Ph3P-Pd, Na2CO3, DMF, H2O, 150–170 °C (microwave), 20%; (d) N-iodosuccinimide, MeCN, room temp, 77%; (e) 4-
fluorophenylstyrene, Pd(OAc)2, NaHCO3, tetrabutylammonium chloride, DMF, 120–130 °C (microwave), 67%; (f) H2, Pd-C, EtOAc, room temp, 34%; (g) POCl3, DMF, 0–80 °C,
56%; (h) 4-fluorophenylmagnesium bromide, THF, 0 °C, 80%.
1-Phenyl-4-phenylpyrazole compound 33, 1-phenyl-4-(2-phen-
ylethyl)pyrazole derivative 36, and 4-( -hydroxybenzyl)-1-
dines 42 and 43. Acylation of 42 and 43 afforded compounds
44a,b and 45.
a
phenylpyrazole derivative 38 were prepared as shown in
Scheme 4. Compounds 33 and 36 were synthesized by a palla-
dium-catalyzed coupling reaction. 3,5-Dimethylpyrazole 30 was
deprotonated with sodium hydride and then treated with
2-chloro-4-fluorobenzonitrile to give 1-phenylpyrazole 31. Pyra-
zole 31 was halogenated at the 4-position by N-halogenosuccini-
mide to give 4-halogenopyrazoles 32 and 34. Suzuki coupling of
4-bromopyrazole 32 with 4-fluorophenylboronic acid afforded 4-
phenylpyrazole 33. 4-Iodopyrazole 34 was allowed to react with
4-fluorostyrene under modified Heck reaction conditions35 to give
olefin 35. 4-(2-Phenylethyl)pyrazole 36 was obtained by the cata-
3. Results and discussion
The synthesized compounds were evaluated using in vitro and
in vivo biological tests as follows. The binding affinities of the com-
pounds to the ARs were measured by the AR binding inhibitory test
(Tables 1–4). The antagonistic and agonistic activities of the com-
pounds toward the ARs were determined by using the AR reporter
assay system (Tables 1–4). In these assays, we used the wild-type
AR and the T877A mutant-type AR, and estimated their efficacy
against PCs, including flutamide-resistant PC. To evaluate their po-
tential against CRPC, we established LNCaP-hr cell line as a model
of CRPC.36 This cell line expresses high levels of AR with a single
mutation (T877A, inherited from the parent LNCaP-FGC cells).
The cells are highly responsive to androgens and exhibit continu-
ous growth in androgen-depleted medium. LNCaP-hr cells produce
high levels of prostate specific antigen (PSA) in vitro in the absence
of any androgens, indicating the constitutive activity of AR. We
performed an in vitro assay using LNCaP-hr cells; the amount of
PSA secreted by LNCaP-hr cells was measured after the addition
of the test compound (Tables 2–4). The PK profile of the com-
pounds was analyzed by a mouse cassette dosing test (Tables 2–
4). The in vivo therapeutic efficacy of the compounds against CRPC
lytic hydrogenation of 35. 4-(a-Hydroxybenzyl)-1-phenylpyrazole
38 was also prepared from 31. Formylation of the pyrazole skele-
ton of 31 by Vilsmeier reaction afforded 4-formyl-1-phenylpyra-
zole 37. Reaction of 37 with 4-fluorophenylmagnesium bromide
yielded compound 38.
1-Phenyl-4-pyridyloxypyrazole derivatives 44a,b and 45 were
synthesized from 37 as described in Scheme 5. Baeyer-Villiger oxi-
dation of 37 and subsequent solvolysis afforded 4-hydroxy-1-
phenylpyrazole 39. Coupling of 39 with 2-bromo-5-nitropyridine
and 5-bromo-2-nitropyridine gave 1-phenyl-4-pyridyloxypyraz-
oles 40 and 41, respectively. Reduction of the nitro group of 40
and 41 using zinc powder afforded the corresponding aminopyri-

2342
S. Yamamoto et al. / Bioorg. Med. Chem. 20 (2012) 2338–2352
Scheme 5. Synthesis of compounds 44a,b and 45. Reagents and conditions: (a) (1) mCPBA, MeCN, EtOAc, room temp, (2) Et3N, MeOH, room temp, 69% for two steps;
(b) 2-bromo-5-nitropyridine (40) or 5-bromo-2-nitropyridine (41), Cs2CO3, DMF, 100 °C, 56% (40) and 80% (41); (c) zinc powder, AcOH, THF, room temp, 72% (42) and 83%
(43); (d) acetic anhydride, pyridine, 0 °C to room temp, 70% (44a) and 63% (45); (e) 2,2-dimethylpropanoyl chloride, Et3N, THF, 0 °C, 92%.
Table 1
In vitro activities of compounds 3, 16a–c, 25, 33, 36, and 38
F
X
N
N
3
R1
2
NC
a
Compd
X
R1
Binding IC50
(
lM)
Reporter
a
a
Antagonist IC50
Wild
0.70
0.075
0.11
>10
0.67
6.2
(
l
M)
Agonist EC50
Wild
>10
1.1
>10
>10
>10
>10
>10
>10
>10
(lM)
Wild
T877A
T877A
T877A
3
16a
16b
16c
25
33
36
38
CH2
CH2
CH2
CH2
H
0.091
0.00047
0.0014
1.5
0.11
0.18
0.053
0.026
0.054
0.060
0.17
>10
>10
>10
2-Cl
2-CF3
3-Cl
2-Cl
2-Cl
2-Cl
2-Cl
0.00070
0.00095
0.35
0.0063
0.033
0.018
0.0083
0.12
0.062
0.062
>10
>10
>10
>10
0.82
0.11
0.27
0.14
0.18
0.47
O
Bond
(CH2)2
CH(OH)
1.1
0.048
0.33
1.6
>10
Bicalutamide
a
IC50 and EC50 values shown are the mean values of duplicate measurements.
was evaluated by performing animal experiments in which LNCaP-
hr cells were subcutaneously injected into a mouse xenograft mod-
el (Figs. 4 and 5). In these studies, we sought to discover novel AR
antagonists that primarily possess strong binding affinity and
antagonistic activity without significant agonistic activity, which
was deemed important for efficacy against CRPC.
X. The AR binding affinity was found to be highly dependent on
the linker length. Removal (33, X = bond) or elongation (36,
X = (CH2)2) of the linker diminished the binding affinities, indicat-
ing that an atomic length of 1 unit was favorable. Interestingly,
conversion of the methylene linker (16a, X = CH2) to an ether linker
(25, X = O) resulted in reduction of the agonistic activity in addition
to enhancement of the antagonistic activity against T877A mutant-
type AR. In contrast, when a hydroxyl group was introduced in the
methylene linker (38, X = CH(OH)), weak agonistic activity was
observed. Thus we find that the linker X is very sensitive to deter-
mining the balance between agonistic and/or antagonistic activity.
On the basis of these results, we focused on the strong binder 16a,
and studied reduction of the observed agonistic activity via an
The SAR data obtained from the initial synthetic study of
compounds B are shown in Table 1. In the substituents on the
cyanophenyl moiety, introduction of
a chloro group or a
trifluoromethyl group at the 2-position greatly increased the bind-
ing affinity, although agonistic activity was also observed (16a,
16b). Shifting of the chloro group to the 3-position decreased the
binding affinity (16c). For compound 16a, we analyzed the linker

S. Yamamoto et al. / Bioorg. Med. Chem. 20 (2012) 2338–2352
2343
Table 2
In vitro activities and PK profiles (mouse cassette dosing) of compounds 28a–i
3
O
4
2
Ra
N
N
Cl
NC
a
Cmpd
Posi-tion
Ra
Binding IC50
(lM)
Reporterb antagonist
LNCaP-hr PSA secretion
% of controlc
Mouse cassette dosingd
10 mg/kg po
a
IC50
(l
M)
Wild
T877A
Wild
>10
0.70
0.42
1.3
T877A
1
l
M
10
l
Me
AUC (
lg h/mL)
MRT (h)
28a
28b
28c
28d
28e
28f
2
3
4
4
4
4
NH2
NH2
NH2
NHMe
NMe2
NHtBu
4.2
1.5
>10
1.9
0.14
0.11
6.3
NTf
21
76
NT
21
33
NT
35
NT
NT
NT
57
12.3
0.6
5.0
NT
NT
2.2
2.0
3.2
NT
NT
4.0
0.26
0.016
0.027
0.92
0.11
0.20
0.010
0.013
0.46
0.10
4.0
0.97
0.77
14.1
OH
OH
28g
28h
4
4
0.16
0.34
0.099
0.16
2.7
2.1
1.1
33
27
NT
31
30.5
38.9
3.3
3.9
HN
HN
0.59
28i
4
0.51
0.70
2.4
>10
35
NT
6.2
2.4
N
O
a
b
c
d
e
f
IC50 and EC50 values shown are the mean values of duplicate measurements.
Agonistic activities of 28a–i were EC50 >10 lM for wild-type or T877A mutant-type AR.
n = 3.
n = 3.
Bicalutamide exhibited more than 100% secretion of control at 10
Not tested.
lM.
Figure 3. Introduction of a Bulky Amide Moiety in Arylmethyl Group for Inhibition of Folding of Helix 12.
Table 3
In vitro activities and PK profiles (mouse cassette dosing) of compounds 29a and 29b
O
Ra
O
N
N
Cl
NC
a
a
Compd
Ra
Binding IC50
(
l
M)
Reporterb antagonist IC50
(lM)
LNCaP-hr PSA secretion % of controlc
10
Mouse cassette dosingd 10 mg/kg, po
Wild
1.1
T877A
0.055
0.18
Wild
1.8
T877A
0.18
1
l
M
lM
AUC (
lg h/mL)
MRT (h)
4.8
OH
29a
29b
33
22
>100
22
24.0
HN
N
O
1.3
3.0
0.72
13.9
3.0
a
b
c
IC50 and EC50 values shown are the mean values of duplicate measurements.
Agonistic activities of 29a and 29b were EC50 >10 lM for wild-type or T877A mutant-type AR.
n = 3.
n = 3.
d

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S. Yamamoto et al. / Bioorg. Med. Chem. 20 (2012) 2338–2352
Table 4
In vitro activities and PK profiles (mouse cassette dosing) of compounds 44a,b and 45
H
Rb
N
O
Y2
Y1
O
N
N
Cl
NC
Y1
Y2
Rb
Binding IC50
(
lM)
Reporterb antagonist IC50
(l
M)
LNCaP-hr PSA secretion % of controlc
Mouse cassette dosingd 10 mg/kg, po
a
a
Compd
Wild
T877A
Wild
T877A
1
l
M
10
l
M
AUC (
l
g h/mL)
MRT (h)
44a
44b
45
N
N
CH
CH
CH
N
Me
tBu
Me
5.4
4.3
1.2
0.27
0.34
0.083
>10
4.0
7.1
1.6
0.19
0.35
8
8
11
14
5
39
18.2
29.5
0.13
2.8
4.2
1.9
a
b
c
IC50 and EC50 values shown are the mean values of duplicate measurements.
Agonistic activities of 44a,b and 45 were EC50 >10 lM for wild-type or T877A mutant-type AR.
n = 3.
n = 3.
d
alternative approach to linker X modification. Apart from 16a, we
selected compound 25, which showed strong antagonistic activity
free from agonistic activity, for further investigation.
similar to the 4-benzylpyrazole compounds, 2-hydroxy-2-methyl-
propylamide derivative 29a exhibited moderate PK profile and
activities in binding and reporter gene assays. However, unlike 4-
benzylpyrazole compounds, the 29a exhibited reduced inhibitory
activity against LNCaP-hr PSA secretion at high concentrations.
On the other hand, morpholine derivative 29b exhibited increased
inhibitory activity in an LNCaP-hr PSA assay. The PK profile of 29b
was not as good as that of 29a. Considering the good PK profile of
the compounds, we also studied replacement of the phenyloxy
moiety with a heteroaryloxy group.31 The results for compounds
44a, 44b, and 45 are shown in Table 4. None of these compounds
To analyze the reduction of the agonistic activity of 16a, we fo-
cused on the folding of helix 12 of the AR ligand binding domain
(LBD).37–40 Folding of the helix is an important factor in the expres-
sion of agonistic activity. Therefore, inhibition of folding is an effec-
tive method for reducing agonistic activity. We hypothesized that
the arylmethyl group (X–T–R2 in B) inhibited folding, as shown
in Figure 3, and that the size of the 4-fluorobenzyl group of 16a,
which possessed AR agonistic activity as well as antagonistic activ-
ity, was not sufficient for the inhibition. On the basis of this
hypothesis, we investigated whether introduction of a bulky group
into the benzyl moiety could result in steric repulsion to reduce the
agonistic activity. With the goal of obtaining compounds with good
PK properties, we set out to prepare compounds with relatively
polar bulky groups, such as amide compounds (C in Figure 3).
The results are shown in Table 2. Notably, none of the evaluated
showed agonistic activity (EC50 >10 lM) in a reporter assay of
wild-type or mutated AR. Pyridin-2-yloxy compounds 44a and
44b exhibited moderate PK profiles. Compound 44b had moderate
binding affinity and antagonistic activity. It was noteworthy that
44a and 44b showed strong inhibitory activity against LNCaP-hr
PSA secretion. Pyridin-3-yloxy compound 45 showed higher bind-
ing affinities and antagonistic activities than did 44a. On the other
hand, the PK profile of pyridin-2-yloxy compounds was better than
that of pyridin-3-yloxy compounds. The AUC value of 44a in mouse
cassette dosing was approximately 100-fold as large as that of
45. On the basis of these results, 44b was selected for further
evaluations.
We evaluated the antitumor effects of 28h, 44b, and bicaluta-
mide in mouse xenograft model using LNCaP-hr cells. Bicalutamide
is reported to show long duration of plasma concentration in men
(mean t1/2 of R-isomer and S-isomer is 4.2 days and 19 h, respec-
tively41). Long duration was also observed in mice (data not
shown). On the basis of this data for bicalutamide and the PK data
for 28h and 44b (Tables 2 and 4), these compounds were orally
administered twice (28h, 44b) or once (bicalutamide) a day for
4 weeks. Tumor volume and plasma PSA concentration were mea-
sured after the treatment period. As shown in Figs. 4 and 5, com-
pounds 28h and 44b showed extremely potent tumor growth
inhibition by T/C values (volume change in a test compound/vol-
ume change in control) of 3% and 2% at doses of 40 and 50 mg/
kg, bid, respectively. The plasma PSA levels in these treatment
groups were also markedly reduced to 23% and 17% of those in
the vehicle treatment groups, respectively. In addition, these com-
pounds resulted in almost no loss of body weight. On the other
hand, bicalutamide showed only partial suppression of tumor
growth (T/C value of 37%) and had almost no effect on plasma
PSA level even at a dose of 100 mg/kg, qd. These results, as well
compounds exhibited agonistic activity (EC50 >10 lM) in a reporter
assay of wild-type or mutated AR. Among the 3 regioisomers of
carboxamides (28a–c) used in the reporter antagonist assay, the
4-carboxamide compound 28c was the most potent. The secondary
amide exhibited higher antagonistic activity than that of the ter-
tiary amide (28d vs 28e). Carboxamides of polar amines had better
PK properties than that of simple alkyl amides (28f vs 28g or 28h),
presumably because of their increased hydrophilicity. In particular,
2-hydroxy-2-methylpropylamide derivative 28h showed moderate
PK profile and inhibited LNCaP-hr PSA secretion, for which bicalu-
tamide exhibited no inhibition (more than 100% secretion of con-
trol at 10 lM). It was noteworthy that 28h showed significantly
different response in CRPC in vitro model from bicalutamide. We
thus selected 28h for further studies.
Next, we studied the modification of 4-phenyloxypyrazole
compound 25, which showed AR antagonistic activity without
significant agonistic activity. The data for the PK profile of this
compound was insufficient for further evaluation (mouse cassette
dosing, 10 mg/kg, po AUC = 1.00
l
gꢀh/mL, MRTpo = 2.2 h). As
described above, in the case of the 4-benzylpyrazole derivatives,
better PK profiles and biological activities were obtained by the
introduction of a bulky amide substituent at the 4-position of the
benzyl moiety. To identify 4-aryloxypyrazole compounds which
had improved PK profiles and activities, we applied this SAR infor-
mation to 4-aryloxylpyrazole derivatives. As shown in Table 3,

S. Yamamoto et al. / Bioorg. Med. Chem. 20 (2012) 2338–2352
2345
(A)
(B)
control (castration + vehicle)
28h 20mg/kg, p.o., bid
28h 40mg/kg, p.o., bid
Bicalutamide 100mg/kg, p.o., qd
control (castration + vehicle)
28h 20mg/kg, p.o., bid
28h 40mg/kg, p.o., bid
1000
900
800
700
600
500
400
300
200
100
0
T/C (%)
100
24
22
20
18
16
14
Bicalutamide 100mg/kg, p.o., qd
##
#
37
*
15
3
**
**
0
7
14
21
28
0
7
14
Days
21
28
Days
(C)
Compound
Dose
(mg/kg, p.o.)
–
Mean SE (ng / mL)
Day 28
T / C
(%)
100
88
Control (castration + vehicle)
31.4 8.2
28h
28h
Bicalutamide
20, bid
40, bid
100, qd
27.6 11.1
7.2 3.9
23
44.0 12.4
142
Figure 4. Antitumor effects of 28h and bicalutamide against LNCaP-hr cell line in mouse xenograft model (n = 6 animals per group). (A) Tumor volume (mm3). (B) Body
weight (g). (C) Plasma PSA (ng / mL). T/C indicates increase in a test compound group during the treatment period/increase in a control group during the treatment
period ꢁ 100. ⁄P <0.05, Steel test versus control. ⁄⁄P <0.025, Shirley–Williams test versus control. #P <0.05 Student’s t-test versus control. ##P <0.025 Williams test versus
control.
(A)
(B)
control (castration + vehicle)
44b 50mg/kg, p.o., bid
1000
control (castration +
vehicle)
24
22
20
18
16
14
900
800
700
600
500
400
300
200
100
0
44b 50mg/kg, p.o., bid
T/C (%)
100
##
*
2
0
7
14
21
28
0
7
14
21
28
Days
Days
(C)
Compound
Dose
Mean SE (ng / mL)
Day 28
T / C
(mg/kg, p.o.)
(%)
100
17
Control (castration + vehicle)
–
17.8 3.1
3.0 1.2#
44b
50, bid
Figure 5. Antitumor effects of 44b against LNCaP-hr cell line in mouse xenograft model (n = 6 animals per group). (A) Tumor volume (mm3). (B) Body weight (g). (C) Plasma
PSA (ng/mL). T/C indicates increase in a test compound group during the treatment period/increase in a control group during the treatment period ꢁ 100. ⁄P <0.01, Steel test
versus control. #P <0.01, Student’s t-test versus control. ##P <0.001, Student’s t-test versus control.
as the significantly different activity observed in CRPC in vitro
model between compounds 28h and 44b in contrast to bicaluta-
mide, suggested that 28h and 44b represent a class of new-gener-
ation AR antagonists effective against CRPC model and that their
properties are different from those of ‘first-generation’ antagonists
such as bicalutamide.

2346
S. Yamamoto et al. / Bioorg. Med. Chem. 20 (2012) 2338–2352
in vacuo. The residue was purified by silica gel column chromatog-
4. Conclusion
raphy (hexane–EtOAc) to give methyl 4-(2-acetyl-3-oxobutyl)ben-
zoate 11 (4.56 g) as a colorless gum. This product 11 (4.56 g,
18.4 mmol) was mixed with EtOH (90 mL) and hydrazine hydrate
(0.983 mL, 20.2 mmol), and the mixture was refluxed for 2 h. After
cooling, the mixture was concentrated in vacuo, and the residue
was recrystallized from toluene to give 15 (3.86 g, 72% from 7) as
colorless tabular crystals. 1H NMR (300 MHz, CDCl3) d: 2.14 (6H,
s), 3.79 (2H, s), 3.90 (3H, s), 7.17 (2H, d, J = 8.7 Hz), 7.93 (2H, d,
J = 8.7 Hz), 9.47 (1 H, br s).
In the course of investigating new-generation AR antagonists
therapeutically effective against CRPC, we designed, synthesized,
and evaluated the activities of 4-arylmethyl-1-phenylpyrazole
and 4-aryloxy-1-phenylpyrazole compounds B. In 4-arylmethyl
derivatives, the introduction of a bulky amide group at the 4-posi-
tion of the benzyl group afforded 28h with significantly reduced
agonistic activity and improved pharmacokinetics. In 4-aryloxy
derivatives, the introduction of a bulky substituent at the 4-posi-
tion of the aryloxy group and replacement of the phenyloxy moiety
with a pyridyloxy group improved the pharmacokinetics and gave
44b. Oral administration of 28h and 44b induced extremely potent
antitumor effects against LNCaP-hr cell line, a CRPC model, in a
mouse xenograft. On the other hand, administration of bicaluta-
mide caused only partial suppression of the growth of LNCaP-hr.
It was indicated that the 4-arylmethyl-1-phenylpyrazole and 4-
aryloxy-1-phenylpyrazole compounds represent a class of new-
generation AR antagonists effective against CRPC model and that
their properties are different from those of ‘first-generation’ AR
antagonists such as bicalutamide.
5.1.2. 4-(4-Fluorobenzyl)-3,5-dimethyl-1H-pyrazole (12)
Compound 12 was prepared in a manner similar to that de-
scribed for 15 in 55% yield as a white solid. 1H NMR (300 MHz,
DMSO-d6) d: 1.98 (3H, s), 2.08 (3H, s), 3.63 (2H, s), 6.74–7.29
(4H, m), 11.98 (1H, br s).
5.1.3. 4-[4-(4-Fluorobenzyl)-3,5-dimethyl-1H-pyrazol-1-yl]-
benzonitrile (3)
To an ice-cooled solution of 12 (0.200 g, 0.979 mmol) in DMF
(2.0 mL) was added NaH (60% in mineral oil, 0.047 g, 1.18 mmol),
and the mixture was stirred at 0 °C for 30 min. To the mixture
was added 4-fluorobenzonitrile (0.237 g, 1.96 mmol), and the mix-
ture was stirred at 0 °C for 2 h and room temperature for 18 h. The
mixture was acidified with saturated aqueous solution of NH4Cl
and extracted twice with EtOAc. The organic layers were com-
bined, washed with brine, dried over anhydrous MgSO4, and con-
centrated in vacuo. The residue was purified by silica gel column
chromatography (hexane–EtOAc). The product was recrystallized
from hexane–EtOAc to give 3 (0.131 g, 44%) as white crystals, mp
100–101 °C. 1H NMR (300 MHz, CDCl3) d: 2.19 (3H, s), 2.30 (3H,
s), 3.76 (2H, s), 6.91–7.03 (2H, m), 7.05–7.16 (2H, m), 7.55–7.64
(2H, m), 7.70–7.78 (2H, m). Anal. Calcd for C19H16FN3: C, 74.74;
H, 5.28; N, 13.76. Found: C, 74.68; H, 5.23; N, 13.74.
5. Experimental section
5.1. Chemistry
Melting points were determined with a Yanagimoto melting
point apparatus or a Büchi melting point apparatus B-545 and
are uncorrected. 1H NMR spectra were obtained at 300 MHz on a
Bruker DPX-300 spectrometer. Chemical shifts are given in d values
(ppm) using tetramethylsilane as the internal standard. Peak mul-
tiplicities are expressed as follows: s, singlet; d, doublet; t, triplet;
q, quartet; dd, doublet of doublet; br, broad; br s, broad singlet; m,
multiplet. The HPLC analyses were performed using a Shimadzu
UFLC instrument. Elution was done with a gradient of 5–90% sol-
vent B in solvent A (solvent A was 0.1% TFA in water, and solvent
B was 0.1% TFA in acetonitrile) through a L-column 2 ODS
5.1.4. Methyl 2-[(3,5-dimethyl-1H-pyrazol-4-yl)methyl]-
benzoate (13)
Compound 13 was prepared in a manner similar to that de-
scribed for 15 in 26% yield as a white solid. 1H NMR (300 MHz,
CDCl3) d: 2.08 (6H, s), 3.90 (3H, s), 4.12 (2H, s), 7.21–7.28 (2H,
m), 7.32–7.40 (1H, m), 7.88 (1H, dd, J = 7.9 and 1.5 Hz).
(3.0 ꢁ 50 mm, 2
l
m) column at 1.2 mL minꢂ1. Area% purity was
measured at 254 nm. Elemental analyses were carried out by
Takeda Analytical Laboratories Ltd. Reactions were followed by
TLC on Silica Gel 60 F 254 precoated TLC plates (E. Merck) or NH
TLC plates (Fuji Silysia Chemical Ltd). Chromatographic separations
were carried out on Silica Gel 60 (0.063–0.200 mm, Merck KGaA),
basic silica gel (ChromatorexÒ NH, 100–200 mesh, Fuji Silysia
Chemical Ltd) or Purif-Pack (Si or NH, Moritex Corporation) using
the indicated eluents. Yields are unoptimized. Preparative HPLC
separations were performed by Unipoint system (Gilson, Inc.)
outfitted with YMC ODS column (30 ꢁ 75 mm or 50 ꢁ 20 mm,
YMC Co., Ltd). Samples were eluted with a gradient of 5–100%
water in acetonitrile containing 0.1% TFA, and detected by the UV
absorbance at 220 nm and 254 nm. Microwave reactions were
performed using Optimizer system (Personal Chemistry, Inc.) or
Initiator Sixty system (Biotage, Inc.). The polymer-bound reagents
were pre-conditioned prior to use.
5.1.5. Methyl 3-[(3,5-dimethyl-1H-pyrazol-4-yl)methyl]-
benzoate (14)
Compound 14 was prepared in a manner similar to that de-
scribed for 15 in 17% yield as a white solid. 1H NMR (300 MHz,
CDCl3) d: 2.15 (6H, s), 3.78 (2H, s), 3.90 (3H, s), 7.27–7.38 (2H,
m), 7.79–7.93 (2H, m).
5.1.6. 2-Chloro-4-[4-(4-fluorobenzyl)-3,5-dimethyl-1H-pyrazol-
1-yl]benzonitrile (16a)
Compound 16a was prepared in a manner similar to that de-
scribed for 3 in quantitative yield as colorless gum. 1H NMR
(300 MHz, CDCl3) d: 2.18 (3H, s), 2.32 (3H, s), 3.76 (2H, s), 6.97
(2H, t, J = 8.7 Hz), 7.05–7.13 (2H, m), 7.51 (1H, dd, J = 8.5 and
2.1 Hz), 7.70–7.77 (2H, m). Anal. Calcd for C19H15ClFN3: C, 67.16;
H, 4.45; N, 12.37. Found: C, 67.02; H, 4.57; N, 12.20.
5.1.1. Methyl 4-[(3,5-dimethyl-1H-pyrazol-4-yl)methyl]-
benzoate (15)
To a mixture of 2,4-pentanedione (6.72 mL, 65.4 mmol), sodium
ethoxide (20%w/w in EtOH, 8.55 mL, 21.8 mmol), and EtOH
(46.0 mL) warmed at 50 °C was added a solution of methyl 4-(bro-
momethyl)benzoate 7 (5.00 g, 21.8 mmol) in EtOH (40.0 mL) drop-
wise over 30 min. The mixture was refluxed for 2 h, cooled to room
temperature, and concentrated in vacuo. The residue was mixed
with water and extracted with EtOAc. The organic layer was
washed with brine, dried over anhydrous MgSO4, and concentrated
5.1.7. 4-[4-(4-Fluorobenzyl)-3,5-dimethyl-1H-pyrazol-1-yl]-2-
(trifluoromethyl)benzonitrile (16b)
Compound 16b was prepared in a manner similar to that de-
scribed for 3 in 87% yield as a white solid, mp 111–112 °C. 1H
NMR (300 MHz, CDCl3) d: 2.19 (3H, s), 2.35 (3H, s), 3.77 (2H, s),
6.91–7.03 (2H, m), 7.04–7.13 (2H, m), 7.75–7.82 (1H, m), 7.88–
7.96 (1H, m), 8.02 (1H, d, J = 1.9 Hz). Anal. Calcd for C20H15F4N3:
C, 64.34; H, 4.05; N, 11.25. Found: C, 64.22; H, 4.02; N, 11.36.

S. Yamamoto et al. / Bioorg. Med. Chem. 20 (2012) 2338–2352
2347
5.1.8. 3-Chloro-4-[4-(4-fluorobenzyl)-3,5-dimethyl-1H-pyrazol-
1-yl]benzonitrile (16c)
5.1.15. 4-(4-Fluorophenoxy)-3,5-dimethyl-1H-pyrazole (23)
A mixture of 4-fluorophenol 19 (1.00 g, 8.92 mmol), 3-chlorop-
entan-2,4-dione (1.17 mL, 9.81 mmol), Cs2CO3 (3.20 g, 9.81 mmol),
and acetone (20 mL) was refluxed for 5 h. After cooling, white solid
was removed by filtration and the filtrate was concentrated in va-
cuo. The residue was purified by silica gel column chromatography
(hexane–EtOAc) to give 3-(4-fluorophenoxy)pentane-2,4-dione 21
as a pale yellow gum (1.30 g). This product 21 (1.20 g, 5.71 mmol)
was dissolved in acetic acid (12 mL), and hydrazine hydrate
(0.333 mL, 6.85 mmol) was added to the solution. The mixture
was stirred at room temperature for 1 h, concentrated in vacuo,
and neutralized with saturated aqueous solution of NaHCO3. The
mixture was extracted twice with EtOAc. The organic layers were
combined, washed with brine, dried over anhydrous MgSO4, and
concentrated in vacuo. The residue was purified by silica gel col-
umn chromatography (hexane–EtOAc) to give 23 (0.499 g, 29%
from 19) as a yellow solid. 1H NMR (300 MHz, CDCl3) d: 2.11 (6H,
s), 6.78–6.87 (2H, m), 6.91–7.00 (2H, m).
Compound 16c was prepared in a manner similar to that de-
scribed for 3 in 89% yield as a white solid, mp 109–110 °C. 1H
NMR (300 MHz, CDCl3) d: 2.04 (3H, s), 2.17 (3H, s), 3.77 (2H, s),
6.98 (2H, t, J = 8.7 Hz), 7.05–7.14 (2H, m), 7.55 (1H, d,
J = 8.3 Hz), 7.66–7.73 (1H, m), 7.84 (1H, s). Anal. Calcd for
C19H15ClFN3: C, 67.16; H, 4.45; N, 12.37. Found: C, 67.20; H,
4.43; N, 12.40.
5.1.9. Methyl 2-{[1-(3-chloro-4-cyanophenyl)-3,5-dimethyl-1H-
pyrazol-4-yl]methyl}benzoate (17a)
Compound 17a was prepared in a manner similar to that de-
scribed for 3 in 60% yield as a white solid. 1H NMR (300 MHz,
CDCl3) d: 2.12 (3H, s), 2.26 (3H, s), 3.91 (3H, s), 4.20 (2H, s),
7.01 (1H, d, J = 7.9 Hz), 7.30 (1H, d, J = 6.8 Hz), 7.36–7.44 (1H,
m), 7.53 (1H, dd, J = 8.5 and 2.1 Hz), 7.70–7.80 (2H, m), 7.90–
7.96 (1H, m).
5.1.10. Methyl 3-{[1-(3-chloro-4-cyanophenyl)-3,5-dimethyl-
1H-pyrazol-4-yl]methyl}benzoate (17b)
5.1.16. Methyl 4-[(3,5-dimethyl-1H-pyrazol-4-yl)oxy]benzoate
(24)
Compound 17b was prepared in a manner similar to that de-
scribed for 3 in 53% yield as a white solid. 1H NMR (300 MHz,
CDCl3) d: 2.18 (3H, s), 2.34 (3H, s), 3.84 (2H, s), 3.91 (3H, s),
7.28–7.41 (2H, m), 7.52 (1H, dd, J = 8.5 and 2.1 Hz), 7.70–7.77
(2H, m), 7.84 (1H, s), 7.89 (1H, d, J = 7.5 Hz).
To a solution of methyl 4-hydroxybenzoate 20 (5.00 g,
32.9 mmol) in DMF (125 mL) were added K2CO3 (9.10 g,
65.8 mmol) and 3-chloropentane-2,4-dione (7.84 mL, 65.8 mmol)
at 0 °C. The mixture was stirred at 80 °C for 30 min, cooled to room
temperature, and filtrated to remove an insoluble solid. The filtrate
was diluted with EtOAc, washed with saturated aqueous solution
of NaHCO3 and brine, dried over anhydrous Na2SO4, and concen-
trated in vacuo to give methyl 4-(1-acetyl-2-oxopropoxy)benzoate
22 (11.8 g). A mixture of 22 (11.8 g), hydrazine hydrate (1.94 mL,
39.5 mmol), and acetic acid (40 mL) was stirred at room tempera-
ture for 1 h. The reaction mixture was concentrated in vacuo, neu-
tralized with saturated aqueous solution of NaHCO3 at 0 °C, and
extracted twice with EtOAc. The organic layers were combined,
washed with brine, dried over anhydrous MgSO4, and concentrated
in vacuo to give a yellow solid. The residue was purified by silica
gel column chromatography (hexane–EtOAc) to afford 24
(4.20 g, 52% from 20) as a white solid. 1H NMR (300 MHz, CDCl3)
d: 2.11 (6H, s), 3.89 (3H, s), 6.91 (2H, d, J = 7.7 Hz), 7.98 (2H, d,
J = 7.7 Hz).
5.1.11. Methyl 4-{[1-(3-chloro-4-cyanophenyl)-3,5-dimethyl-
1H-pyrazol-4-yl]methyl}benzoate (17c)
Compound 17c was prepared in a manner similar to that de-
scribed for 3 in 70% yield as a white solid. 1H NMR (300 MHz,
DMSO-d6) d: 2.09 (3H, s), 2.39 (3H, s), 3.83 (3H, s), 3.87 (2H, s),
7.32 (2H, d, J = 7.9 Hz), 7.71–7.78 (1H, m), 7.85–7.97 (3H, m),
8.08 (1H, d, J = 8.3 Hz).
5.1.12. 4-{[1-(3-Chloro-4-cyanophenyl)-3,5-dimethyl-1H-
pyrazol-4-yl]methyl}benzoic acid (18c)
To a suspension of 17c (0.500 g, 1.32 mmol) in THF (5.0 mL) and
MeOH (5.0 mL) at 50 °C was added 1 N NaOH (5.0 mL, 5.0 mmol)
dropwise. The mixture was stirred at 50 °C for 1 h to give a color-
less solution. The solution was cooled to room temperature, neu-
tralized with 2 N HCl (2.5 mL), and extracted twice with EtOAc.
The organic extracts were combined, washed with brine, dried over
anhydrous MgSO4, and concentrated in vacuo to give 18c (0.455 g,
94%) as a white solid. 1H NMR (300 MHz, DMSO-d6) d: 2.10 (3H, s),
2.40 (3H, s), 3.86 (2H, s), 7.29 (2H, d, J = 8.3 Hz), 7.75 (1H, dd, J = 8.5
and 2.1 Hz), 7.86 (2H, d, J = 8.7 Hz), 7.95 (1H, d, J = 1.9 Hz), 8.08
(1H, d, J = 8.7 Hz), 12.83 (1H, br s).
5.1.17. 2-Chloro-4-[4-(4-fluorophenoxy)-3,5-dimethyl-1H-
pyrazol-1-yl]benzonitrile (25)
To an ice-cooled mixture of 23 (0.100 g, 0.485 mmol) and DMF
(1.0 mL) was added NaH (60% in mineral oil, 0.023 g, 0.582 mmol),
and the mixture was stirred at 0 °C for 30 min. To the mixture was
added 2-chloro-4-fluorobenzonitrile (0.151 g, 0.970 mmol), and
the mixture was stirred at 0 °C for 1 h. The mixture was mixed with
saturated aqueous solution of NaHCO3 and extracted twice with
EtOAc. The organic layers were combined, washed with brine,
dried over anhydrous MgSO4, and concentrated in vacuo. The
residue was purified by silica gel column chromatography (hex-
ane–EtOAc) to afford 25 (0.088 g, 53%) as a white solid, mp 118–
119 °C. 1H NMR (300 MHz, CDCl3) d: 2.13 (3H, s), 2.32 (3H, s),
6.82–6.91 (2H, m), 6.96–7.05 (2H, m), 7.55 (1 H, dd, J = 8.7 and
1.9 Hz), 7.72–7.81 (2H, m). Anal. Calcd for C18H13ClFN3O: C,
63.26; H, 3.83; N, 12.29. Found: C, 63.22; H, 3.78; N, 12.35.
5.1.13. 2-{[1-(3-Chloro-4-cyanophenyl)-3,5-dimethyl-1H-
pyrazol-4-yl]methyl}benzoic acid (18a)
Compound 18a was prepared in a manner similar to that de-
scribed for 18c in 66% yield as a white solid. 1H NMR (300 MHz,
DMSO-d6) d: 2.00 (3H, s), 2.30 (3H, s), 4.15 (2H, s), 7.06 (1H, d,
J = 7.5 Hz), 7.31 (1H, t, J = 7.5 Hz), 7.40–7.48 (1H, m), 7.74 (1H,
dd, J = 8.5 and 2.1 Hz), 7.78–7.85 (1H, m), 7.94 (1H, d, J = 2.3 Hz),
8.08 (1H, d, J = 8.3 Hz), 13.01 (1H, br s).
5.1.14. 3-{[1-(3-Chloro-4-cyanophenyl)-3,5-dimethyl-1H-
pyrazol-4-yl]methyl}benzoic acid (18b)
5.1.18. 4-{[1-(3-Chloro-4-cyanophenyl)-3,5-dimethyl-1H-
pyrazol-4-yl]oxy}benzoic acid (27)
Compound 18b was prepared in a manner similar to that de-
scribed for 18c in quantitative yield as a white solid. 1H NMR
(300 MHz, DMSO-d6) d: 2.10 (3H, s), 2.40 (3H, s), 3.87 (2H, s),
7.39–7.46 (2H, m), 7.72–7.82 (3H, m), 7.96 (1H, d, J = 2.3 Hz),
8.08 (1H, d, J = 8.7 Hz), 12.94 (1H, br s).
To a solution of 24 (7.18 g, 29.2 mmol) in DMF (75 mL) was
added NaH (60% in mineral oil, 1.40 g, 35.0 mmol) at 0 °C. After
stirring at 0 °C for 30 min, 2-chloro-4-fluorobenzonitrile (9.07 g,
58.3 mmol) was added. The mixture was stirred at 0 °C for 2 h,
neutralized with saturated aqueous solution of NH4Cl, and

2348
S. Yamamoto et al. / Bioorg. Med. Chem. 20 (2012) 2338–2352
extracted twice with EtOAc. The organic layers were combined,
washed with brine, dried over anhydrous MgSO4, and concentrated
in vacuo. The residue was purified by silica gel column chromatog-
raphy (hexane–EtOAc) to give methyl 4-{[1-(3-chloro-4-cyano-
phenyl)-3,5-dimethyl-1H-pyrazol-4-yl]oxy}benzoate 26 (14.5 g)
as a white solid. To a warmed solution of 26 (14.5 g) in THF
(220 mL) and methanol (220 mL) was added 1 N NaOH (110 mL)
at 40 °C. The resulting colorless solution was stirred at the same
temperature for 2 h, cooled on ice, and acidified with 1 N HCl.
The mixture was concentrated in vacuo, and the residue was ex-
tracted twice with EtOAc. The organic layers were combined,
washed with brine, dried over anhydrous MgSO4, and concentrated
in vacuo. The residue was purified by silica gel column chromatog-
raphy (hexane–EtOAc) to afford 27 (7.68 g, 72% from 24) as a white
solid. 1H NMR (300 MHz, DMSO-d6) d: 2.06 (3H, s), 2.30 (3H, s),
7.06 (2H, d, J = 8.3 Hz), 7.81 (1H, d, J = 8.5 Hz), 7.94 (2H, d,
J = 8.1 Hz), 8.01 (1H, s), 8.13 (1H, d, J = 8.7 Hz), 12.77 (1 H, br s).
5.1.23. 4-{[1-(3-Chloro-4-cyanophenyl)-3,5-dimethyl-1H-
pyrazol-4-yl]methyl}-N-methylbenzamide (28d)
Compound 28d was prepared in a manner similar to that de-
scribed for 28f in 22% yield as a white solid, mp 173–174 °C. 1H
NMR (300 MHz, CDCl3) d: 2.17 (3H, s), 2.32 (3H, s), 3.01 (3H, d,
J = 4.9 Hz), 3.83 (2H, s), 6.12 (1H, br s), 7.19 (2H, d, J = 8.3 Hz),
7.52 (1H, d, J = 8.3 Hz), 7.65–7.82 (4H, m). Anal. Calcd for
C
21H19ClN4Oꢀ0.5H2O: C, 65.03; H, 5.20; N, 14.45. Found: C, 65.37;
H, 5.01; N, 14.48.
5.1.24. 4-{[1-(3-Chloro-4-cyanophenyl)-3,5-dimethyl-1H-
pyrazol-4-yl]methyl}-N,N-dimethylbenzamide (28e)
Compound 28e was prepared in a manner similar to that de-
scribed for 28f in 20% yield as a white solid, mp 131–132 °C. 1H
NMR (300 MHz, CDCl3) d: 2.21 (3H, s), 2.34 (3H, s), 3.00 (3H, s),
3.12 (3H, s), 3.83 (2H, s), 7.17 (2H, d, J = 8.3 Hz), 7.37 (2H, d,
J = 8.3 Hz), 7.53 (1H, dd, J = 8.5 and 2.1 Hz), 7.72–7.80 (2H, m).
Anal. Calcd for C22H21ClN4O: C, 66.49; H, 5.45; N, 14.10. Found:
C, 66.46; H, 5.36; N, 14.15.
5.1.19. N-tert-Butyl-4-{[1-(3-chloro-4-cyanophenyl)-3,5-
dimethyl-1H-pyrazol-4-yl]methyl}benzamide (28f)
To a solution of 18c (2.00 g, 5.47 mmol), N-[3-(dimethyl-
5.1.25. 4-{[1-(3-Chloro-4-cyanophenyl)-3,5-dimethyl-1H-pyra-
zol-4-yl]methyl}-N-(2-hydroxy-2-methylpropyl)benzamide (28h)
A suspension of 26c (2.00 g, 5.47 mmol), 4-(4,6-dimethoxy-
1,3,5-triazin-2-yl)-4-methylmorpholin-4-ium chloride (2.27 g, 8.21
mmol) and 1-amino-2-methylpropan-2-ol (0.585 g, 6.56 mmol) in
THF (25 mL) and 2-propanol (25 mL) was stirred at room temper-
ature for 19 h. The mixture was concentrated in vacuo, acidified
with 1 N HCl, and extracted twice with EtOAc. The organic layers
were combined, washed with saturated aqueous solution of NH4Cl
and brine, dried over anhydrous Na2SO4, and concentrated in
vacuo. The residue was purified by silica gel column chroma-
tography (hexane–EtOAc). The product was recrystallized from
hexane–EtOAc to give 28h (2.06 g, 89%) as a white solid, mp
171–172 °C. 1H NMR (300 MHz, DMSO-d6) d: 1.09 (6H, s), 2.11
(3H, s), 2.40 (3H, s), 3.24 (2H, d, J = 6.0 Hz), 3.84 (2H, s), 4.54 (1H,
s), 7.26 (2H, d, J = 8.1 Hz), 7.73–7.80 (3H, m), 7.94 (1H, d,
J = 1.8 Hz), 8.08 (1H, d, J = 8.7 Hz), 8.15 (1H, t, J = 6.0 Hz). Anal.
Calcd for C24H25ClN4O2: C, 65.97; H, 5.77; N, 12.82. Found: C,
66.12; H, 5.71; N, 12.87.
amino)propyl]-N0-ethylcarbodiimide
hydrochloride
(1.57 g,
8.21 mmol), and 1-hydroxybenzotriazole (1.11 g, 8.21 mmol) in
DMF (20 mL) was added tertbutylamine (0.862 mL, 8.21 mmol).
After stirring at room temperature for 13 h, the mixture was mixed
with 10% aqueous solution of NaHSO4 and extracted twice with
EtOAc. The organic layers were combined, washed with 1 N HCl,
saturated aqueous solution of NaHCO3 and brine, dried over anhy-
drous MgSO4, and concentrated in vacuo. The residue was purified
by silica gel column chromatography (hexane–EtOAc). The product
was recrystallized from hexane–EtOAc to give 28f (1.81 g, 79%) as a
white solid, mp 135–136 °C. 1H NMR (300 MHz, CDCl3) d: 1.46 (9H,
s), 2.17 (3H, s), 2.32 (3H, s), 3.82 (2H, s), 5.89 (1H, br s), 7.17 (2H, d,
J = 8.3 Hz), 7.51 (1 H, dd, J = 8.6 and 2.0 Hz), 7.62–7.67 (2H, m), 7.74
(2H, dd, J = 5.2 and 3.1 Hz). Anal. Calcd for C24H25ClN4O: C, 68.48;
H, 5.99; N, 13.31. Found: C, 68.49; H, 5.97; N, 13.34.
5.1.20. 2-{[1-(3-Chloro-4-cyanophenyl)-3,5-dimethyl-1H-
pyrazol-4-yl]methyl}benzamide (28a)
Compound 28a was prepared in a manner similar to that de-
scribed for 28f in 88% yield as a white solid, mp 208–209 °C. 1H
NMR (300 MHz, CDCl3) d: 2.03 (3H, s), 2.32 (3H, s), 3.94 (2H, s),
7.02 (1H, d, J = 6.8 Hz), 7.17–7.46 (4H, m), 7.73 (1H, dd, J = 8.5
and 2.1 Hz), 7.80 (1H, s), 7.93 (1H, d, J = 1.9 Hz), 8.08 (1H, d,
J = 8.7 Hz). Anal. Calcd for C20H17ClN4Oꢀ0.2AcOEt: C, 65.32; H,
4.90; N, 14.65. Found: C, 65.36; H, 4.82; N, 14.50.
5.1.26. 4-{[1-(3-Chloro-4-cyanophenyl)-3,5-dimethyl-1H-
pyrazol-4-yl]methyl}-N-(2-hydroxyethyl)benzamide (28g)
Compound 28g was prepared in a manner similar to that de-
scribed for 28h in 60% yield as a white solid, mp 146–147 °C. 1H
NMR (300 MHz, CDCl3) d: 2.17 (3H, s), 2.32 (3H, s), 2.56 (1H, br
s), 3.58–3.67 (2H, m), 3.84 (2H, s), 3.95 (2H, d, J = 9.4 Hz), 6.58
(1H, br s), 7.20 (2H, d, J = 7.9 Hz), 7.51 (1H, d, J = 7.9 Hz), 7.67–
7.78 (4H, m). Analytical HPLC showed 100% purity.
5.1.21. 3-{[1-(3-Chloro-4-cyanophenyl)-3,5-dimethyl-1H-
pyrazol-4-yl]methyl}benzamide (28b)
5.1.27. 2-Chloro-4-{3,5-dimethyl-4-[4-(morpholin-4-ylcarbon-
yl)benzyl]-1H-pyrazol-1-yl}benzonitrile (28i)
Compound 28b was prepared in a manner similar to that de-
scribed for 28f in 73% yield as a white solid, mp 200–201 °C. 1H
NMR (300 MHz, DMSO-d6) d: 2.10 (3H, s), 2.40 (3H, s), 3.83 (2H,
s), 7.26–7.42 (3H, m), 7.65–7.80 (3H, m), 7.95 (2H, d, J = 2.3 Hz),
8.08 (1H, d, J = 8.7 Hz). Anal. Calcd for C20H17ClN4O: C, 65.84; H,
4.70; N, 15.36. Found: C, 65.60; H, 4.77; N, 15.03.
Compound 28i was prepared in a manner similar to that de-
scribed for 28f in 82% yield as a white solid, mp 141–142 °C. 1H
NMR (300 MHz, CDCl3) d: 2.19 (3H, s), 2.34 (3H, s), 3.35–3.94
(8H, m), 3.82 (2H, s), 7.18 (2H, d, J = 7.9 Hz), 7.34 (2H, d,
J = 8.3 Hz), 7.52 (1H, dd, J = 8.5 and 2.1 Hz), 7.71–7.78 (2H, m).
Anal. Calcd for C24H23ClN4O2ꢀ0.2H2O: C, 65.73; H, 5.38; N, 12.78.
Found: C, 65.72; H, 5.36; N, 12.86.
5.1.22. 4-{[1-(3-Chloro-4-cyanophenyl)-3,5-dimethyl-1H-
pyrazol-4-yl]methyl}benzamide (28c)
Compound 28c was prepared in a manner similar to that de-
scribed for 28f in 64% yield as a white solid, mp 200–201 °C. 1H
NMR (300 MHz, DMSO-d6) d: 2.10 (3H, s), 2.40 (3H, s), 3.83 (2H,
s), 7.24 (2H, d, J = 8.3 Hz), 7.29 (1H, br s), 7.71–7.82 (3H, m), 7.89
(1H, br s), 7.95 (1H, d, J = 2.3 Hz), 8.08 (1H, d, J = 8.7 Hz). Anal. Calcd
for C20H17ClN4O: C, 65.84; H, 4.70; N, 15.36. Found: C, 65.70; H,
4.69; N, 15.29.
5.1.28. 4-{[1-(3-Chloro-4-cyanophenyl)-3,5-dimethyl-1H-pyra-
zol-4-yl]oxy}-N-(2-hydroxy-2-methylpropyl)benzamide (29a)
Compound 29a was prepared in a manner similar to that de-
scribed for 28h in 78% yield as a white solid, mp 194–195 °C. 1H
NMR (300 MHz, CDCl3) d: 1.30 (6H, s), 2.14 (3H, s), 2.19 (1H, s),
2.32 (3H, s), 3.48 (2H, d, J = 6.0 Hz), 6.47–6.54 (1H, m), 6.94–7.00
(2H, m), 7.56 (1H, dd, J = 8.6 and 2.2 Hz), 7.74–7.82 (4H, m). Anal.

S. Yamamoto et al. / Bioorg. Med. Chem. 20 (2012) 2338–2352
2349
Calcd for C23H23ClN4O3: C, 62.94; H, 5.28; N, 12.77. Found: C,
62.91; H, 5.31; N, 12.80.
NMR (300 MHz, CDCl3) d: 2.31 (3H, s), 2.45 (3H, s), 7.48 (1H,
dd, J = 8.5 and 2.1 Hz), 7.70 (1H, d, J = 1.9 Hz), 7.76 (1H, d,
J = 8.3 Hz).
5.1.29. 2-Chloro-4-{3,5-dimethyl-4-[4-(morpholin-4-ylcarbon-
yl)phenoxy]-1H-pyrazol-1-yl}benzonitrile (29b)
5.1.34. 2-Chloro-4-{4-[(E)-2-(4-fluorophenyl)ethenyl]-3,5-
dimethyl-1H-pyrazol-1-yl}benzonitrile (35)
Compound 29b was prepared in a manner similar to that de-
scribed for 28f in 93% yield as a white solid, mp 156–157 °C. 1H
NMR (300 MHz, CDCl3) d: 2.15 (3H, s), 2.31 (3H, s), 3.70 (8H, br
s), 6.92–6.98 (2H, m), 7.37–7.43 (2H, m), 7.56 (1H, dd, J = 8.5 and
2.1 Hz), 7.76 (1H, d, J = 8.5 Hz), 7.79 (1H, d, J = 1.9 Hz). Anal. Calcd
for C23H21ClN4O3: C, 63.23; H, 4.84; N, 12.82. Found: C, 63.28; H,
4.76; N, 12.78.
A
mixture of 34 (0.100 g, 0.280 mmol), 4-fluorostyrene
(0.05 mL, 0.42 mmol), Pd(OAc)2 (0.0031 g, 0.014 mmol), NaHCO3
(0.059 g, 0.700 mmol), Bu4NCl (0.078 g, 0.280 mmol), and DMF
(1.0 mL) was heated at 120 °C for 15 min and 130 °C for 15 min un-
der microwave irradiation. The mixture was cooled to room tem-
perature and extracted twice with EtOAc. The organic layers
were combined, washed with brine, dried over anhydrous MgSO4,
and concentrated in vacuo. The residue was purified by silica gel
column chromatography (hexane–EtOAc) followed by preparative
HPLC separation to afford 35 (0.066 g, 67%) as a white solid. 1H
NMR (300 MHz, CDCl3) d: 2.45 (3H, s), 2.50 (3H, s), 6.74 (1H, d,
J = 16.2 Hz), 6.84 (1H, d, J = 16.2 Hz), 7.06 (2H, t, J = 8.7 Hz), 7.40–
7.55 (3H, m), 7.72–7.79 (2H, m).
5.1.30. 2-Chloro-4-(3,5-dimethyl-1H-pyrazol-1-yl)benzonitrile
(31)
To a cooled suspension of NaH (60% in mineral oil, 6.24 g,
156 mmol) in DMF (100 mL) at 5 °C was added 3,5-dimethylpyra-
zole 30 (15.0 g, 156 mmol) dropwise. After stirring at 5 °C for 30
min, 2-chloro-4-fluorobenzonitrile (24.3 g, 156 mmol) was added
dropwise, and the whole was stirred at 0 °C for 1 h. The mixture
was diluted with water. The resulting precipitates were collected
by filtration, washed with water and hexane, and dried in air.
The resulting solid was purified by silica gel column chromatogra-
phy (NH silica gel, hexane–EtOAc). The product was recrystallized
from hexane–EtOAc to afford 31 (21.0 g, 58%) as a colorless pow-
der. 1H NMR (300 MHz, CDCl3) d: 2.29 (3H, s), 2.42 (3H, s), 6.07
(1H, s), 7.51 (1H, dd, J = 8.5 and 2.1 Hz), 7.70–7.76 (2H, m).
5.1.35. 2-Chloro-4-{4-[2-(4-fluorophenyl)ethyl]-3,5-dimethyl-
1H-pyrazol-1-yl}benzonitrile (36)
A mixture of 35 (0.291 g, 0.827 mmol), 5% Pd-C (0.352 g,
0.165 mmol), and EtOAc (7.0 mL) was stirred at room temperature
under H2 atmosphere. After 3 h, Pd-C was removed by filtration
and the filtrate was concentrated in vacuo. The residue was purified
bypreparative HPLCto give36(0.101 g, 34%)asawhitesolid, mp96–
97 °C. 1H NMR (300 MHz, CDCl3) d: 2.06 (3H, s), 2.17 (3H, s), 2.61–
2.81 (4H, m), 6.91–7.00 (2H, m), 7.00–7.09 (2H, m), 7.43 (1H, dd,
J = 8.5 and 2.1 Hz), 7.65–7.74 (2H, m). Anal. Calcd for C20H17ClFN3:
C, 67.89; H, 4.84; N, 11.88. Found: C, 67.73; H, 4.80; N, 11.74.
5.1.31. 4-(4-Bromo-3,5-dimethyl-1H-pyrazol-1-yl)-2-chloroben-
zonitrile (32)
To a solution of 31 (7.30 g, 31.5 mmol) in AcOH (50 mL) was
added bromine (5.54 g, 34.7 mmol) dropwise. After stirring at
room temperature for 30 min, the mixture was diluted with water.
The resulting precipitates were collected by filtration, washed with
water, and dried in air. Recrystallization from EtOAc–hexane gave
32 (5.60 g, 57%) as a white powder. 1H NMR (300 MHz, CDCl3) d:
2.30 (3H, s), 2.42 (3H, s), 7.49 (1H, dd, J = 8.5 and 2.0 Hz), 7.71
(1H, d, J = 2.0 Hz), 7.76 (1H, d, J = 8.5 Hz).
5.1.36. 2-Chloro-4-(4-formyl-3,5-dimethyl-1H-pyrazol-1-yl)-
benzonitrile (37)
The Vilsmeier reagent was prepared by adding phosphoryl chlo-
ride (14.4 mL, 155 mmol) dropwise to ice cold DMF (65 mL) under
stirring. The mixture was then stirred for 15 min at 0 °C. To the Vils-
meier reagent was added a solution of 31 (15.0 g, 64.7 mmol) in DMF
(90.0 mL). The mixture was heated to 80 °C and stirred for 21 h. The
reaction was quenched with water at 0 °C. Insoluble solid was col-
lected by filtration, washed with MeOH and diisopropyl ether, and
dried to give 37 as a yellow solid (4.58 g, 27%). The filtrate was ex-
tracted twice with EtOAc. The organic layers were combined,
washed with saturated aqueous solution of NaHCO3 and brine, dried
over anhydrous Na2SO4, and evaporated to give a yellow solid. This
solid was diluted with EtOAc and the insoluble solid was separated.
This solid was dried to afford 37 (2.64 g, 16%) as a yellow solid. The
filtrate was purified by silica gel column chromatography (hex-
ane–EtOAc) to give 37 (2.15 g, 13%) as a yellow solid. 1H NMR
(300 MHz, CDCl3) d: 2.53 (3H, s), 2.67 (3H, s), 7.51 (1H, d,
J = 8.3 Hz), 7.73 (1H, s), 7.81 (1H, d, J = 8.5 Hz), 10.05 (1H, s).
5.1.32. 2-Chloro-4-[4-(4-fluorophenyl)-3,5-dimethyl-1H-
pyrazol-1-yl]benzonitrile (33)
A mixture of polystyrene-bound triphenylphosphine palladium
(0) (PS-PPh3-Pd, 0.1 mmol/g, 0.025 g, 0.0025 mmol), 32 (0.100 M in
1,1-dimethoxyethane, 2.0 mL, 0.200 mmol), 4-fluorophenylboronic
acid (0.111 M in 1,1-dimethoxyethane, 2.0 mL, 0.222 mmol), and
0.5 M aqueous solution of Na2CO3 (0.60 mL, 0.300 mmol) was
heated at 150 °C for 4 min and at 170 °C for 6 min under micro-
wave irradiation. The resin reagent was removed by filtration
and the filtrate was concentrated in vacuo. The residue was puri-
fied by preparative HPLC to give 33 (0.013 g, 20%) as a solid, mp
114–115 °C. 1H NMR (300 MHz, DMSO-d6) d: 2.23 (3H, s), 2.38
(3H, s), 7.28–7.42 (4H, m), 7.79 (1H, dd, J = 8.7 and 2.1 Hz), 7.99
(1H, d, J = 1.9 Hz), 8.13 (1H, d, J = 8.5 Hz). Analytical HPLC showed
100% purity.
5.1.37. ( )-2-Chloro-4-{4-[(4-fluorophenyl)(hydroxy)methyl]-
3,5-dimethyl-1H-pyrazol-1-yl}benzonitrile (38)
To an ice-cooled suspension of 37 (0.100 g, 0.385 mmol) in THF
(2.0 mL) was added a THF solution of 4-fluorophenylmagnesium
bromide (1.0 M, 0.77 mL, 0.77 mmol). After stirring at 0 °C for
2 h, the reaction was quenched with saturated aqueous solution
of NH4Cl, and the mixture was extracted twice with EtOAc. The or-
ganic layers were combined, washed with brine, dried over anhy-
drous MgSO4 and concentrated in vacuo. The residue was
purified by silica gel column chromatography (hexane–EtOAc).
The product was recrystallized from hexane–EtOAc to afford 38
5.1.33. 2-Chloro-4-(4-iodo-3,5-dimethyl-1H-pyrazol-1-
yl)benzonitrile (34)
A mixture of 31 (5.00 g, 21.6 mmol), N-iodosuccinimide (4.86 g,
710 21.6 mmol), and acetonitrile (100 mL) was stirred at room
temperature for 6 days. The mixture was concentrated in vacuo,
and the residue was purified by silica gel column chromatography
(hexane–EtOAc) to give a white solid. This solid was washed with
diisopropyl ether to give 34 as a white solid (5.05 g, 65%). The
diisopropyl ether extract was concentrated and recrystallized
from hexane–EtOAc to give 34 (0.95 g, 12%) as a white solid. 1H
(0.109 g, 80%) as
a
white solid, mp 145–146 °C. 1H NMR
(300 MHz, CDCl3) d: 2.03 (1H, d, J = 3.6 Hz), 2.18 (3H, s), 2.34 (3H,
s), 5.92 (1H, d, J = 2.6 Hz), 7.05 (2H, dd, J = 8.6 and 4.6 Hz), 7.36

2350
S. Yamamoto et al. / Bioorg. Med. Chem. 20 (2012) 2338–2352
(2H, dd, J = 8.2 and 5.4 Hz), 7.48 (1H, dd, J = 8.5 and 2.1 Hz), 7.70
(1H, d, J = 2.1 Hz), 7.74 (1H, d, J = 8.3 Hz). Anal. Calcd for
5.1.42. 4-{4-[(6-Aminopyridin-3-yl)oxy]-3,5-dimethyl-1H-
pyrazol-1-yl}-2-chlorobenzonitrile (43)
C
19H15ClFN3O: C, 64.14; H, 4.25; N, 11.81. Found: C, 64.24; H,
Compound 43 was prepared in a manner similar to that de-
scribed for 42 in 83% yield as a yellow solid. 1H NMR (300 MHz,
CDCl3) d: 2.15 (3H, s), 2.33 (3H, s), 4.40 (2 H, br s), 6.50 (1H, dd,
J = 9.0 and 0.7 Hz), 7.13 (1H, dd, J = 9.0 and 2.9 Hz), 7.54 (1H, dd,
J = 8.5 and 2.1 Hz), 7.72–7.79 (3H, m).
4.29; N, 11.85.
5.1.38. 2-Chloro-4-(4-hydroxy-3,5-dimethyl-1H-pyrazol-1-yl)-
benzonitrile (39)
To a solution of 37 (2.00 g, 7.70 mmol) in MeCN (42 mL) and
EtOAc (26 mL) was added m-chloroperbenzoic acid (3.99 g,
23.1 mmol) at room temperature. After stirring at room tempera-
ture for 16 h, the mixture was diluted with EtOAc, washed with
aqueous solution of Na2S2O3, saturated aqueous solution of NaH-
CO3 and brine, dried over anhydrous Na2SO4, and concentrated in
vacuo. The residue was dissolved in methanol (30 mL), and trieth-
ylamine (3.22 mL, 23.1 mmol) was added. The mixture was stirred
at room temperature for 1 h and concentrated in vacuo to give a
yellow solid. The solid was diluted with toluene/acetone and insol-
uble solid was collected by filtration to afford 39 (1.10 g, 58%) as a
yellow solid. The filtrate was purified by silica gel column
chromatography (hexane–EtOAc) to give 39 (0.21 g, 11%) as a yel-
low solid. 1H NMR (300 MHz, DMSO-d6) d: 2.14 (3H, s), 2.34 (3H, s),
7.69 (1H, d, J = 8.5 Hz), 7.87 (1H, s), 8.02 (1H, d, J = 8.7 Hz), 8.35
(1H, br s).
5.1.43. N-(6-{[1-(3-Chloro-4-cyanophenyl)-3,5-dimethyl-1H-
pyrazol-4-yl]oxy}pyridin-3-yl)acetamide (44a)
To a solution of 42 (0.087 g, 0.256 mmol) in pyridine (1 mL) was
added acetic anhydride (0.036 mL, 0.384 mmol) at 0 °C. The mix-
ture was stirred at 0 °C for 0.5 h and at room temperature for
17 h. The mixture was diluted with EtOAc, neutralized with 1 N
HCl, and extracted twice with EtOAc. The organic layers were com-
bined, washed with 1 N HCl, saturated aqueous solution of NaHCO3
and brine, dried over Na2SO4, and concentrated in vacuo. The res-
idue was purified by silica gel column chromatography (NH silica
gel, hexane–EtOAc). The product was recrystallized from hep-
tane–EtOAc to afford 44a (0.068 g, 70%) as a yellow solid, mp
195–196 °C. 1H NMR (300 MHz, CDCl3) d: 2.14 (3H, s), 2.20 (3H,
s), 2.32 (3H, s), 6.97 (1H, dd, J = 8.0 and 1.2 Hz), 7.20 (1H, br s),
7.57 (1H, dd, J = 8.5 and 2.1 Hz), 7.74 (1H, d, J = 8.5 Hz), 7.80 (1H,
d, J = 1.9 Hz), 8.06–8.16 (2H, m). Anal. Calcd for C19H16ClN5O2: C,
59.77; H, 4.22; N, 18.34. Found: C, 59.77; H, 4.21; N, 18.19.
5.1.39. 2-Chloro-4-{3,5-dimethyl-4-[(5-nitropyridin-2-yl)oxy]-
1H-pyrazol-1-yl}benzonitrile (40)
5.1.44. N-(6-{[1-(3-Chloro-4-cyanophenyl)-3,5-dimethyl-1H-
pyrazol-4-yl]oxy}pyridin-3-yl)-2,2-dimethylpropanamide (44b)
To a solution of 42 (1.39 g, 4.09 mmol) in THF (100 mL) were
added 2,2-dimethylpropanoyl chloride (0.55 mL, 4.50 mmol) and
triethylamine (1.25 mL, 9.00 mmol) at 0 °C. After stirring at 0 °C
for 2 h, the mixture was diluted with EtOAc, neutralized with 1 N
HCl, and extracted twice with EtOAc. The organic layers were
combined, washed with saturated aqueous solution of NH4Cl and
brine, dried over anhydrous Na2SO4, and concentrated in vacuo.
The residue was purified by silica gel column chromatography
(hexane–EtOAc). The product was recrystallized from heptane–
EtOAc to afford 44b (1.59 g, 92%) as a yellow solid, mp 191–
192 °C. 1H NMR (300 MHz, CDCl3) d: 1.33 (9H, s), 2.14 (3H, s),
2.32 (3H, s), 6.95 (1H, d, J = 9.3 Hz), 7.27 (1H, br s), 7.57 (1H, dd,
J = 8.5 and 2.1 Hz), 7.74 (1H, d, J = 8.7 Hz), 7.80 (1H, d, J = 2.1 Hz),
8.07–8.17 (2H, m). Anal. Calcd for C22H22ClN5O2: C, 62.34; H,
5.23; N, 16.52. Found: C, 62.60; H, 5.21; N, 16.63.
To a solution of 39 (0.356 g, 1.44 mmol) in DMF (18 mL) were
added 2-bromo-5-nitropyridine (0.438 g, 2.16 mmol) and Cs2CO3
(0.703 g, 2.16 mmol). After stirring at 100 °C for 3 h and cooled to
room temperature, the mixture was diluted with EtOAc, acidified
with saturated aqueous solution of NH4Cl, and extracted twice
with EtOAc. The organic layers were combined, washed with satu-
rated aqueous solution of NH4Cl and brine, dried over anhydrous
Na2SO4, and concentrated in vacuo. The residue was purified by sil-
ica gel column chromatography (hexane–EtOAc). The product was
recrystallized from heptane–EtOAc to afford 40 (0.295 g, 56%) as a
yellow solid. 1H NMR (300 MHz, CDCl3) d: 2.16 (3H, s), 2.32 (3H, s),
7.15 (1H, d, J = 9.1 Hz), 7.57 (1H, dd, J = 8.5 and 2.3 Hz), 7.76 (1H, d,
J = 8.5 Hz), 7.80 (1H, d, J = 2.1 Hz), 8.53 (1H, dd, J = 9.1 and 2.6 Hz),
9.05 (1H, d, J = 2.3 Hz).
5.1.40. 2-Chloro-4-{3,5-dimethyl-4-[(6-nitropyridin-3-yl)oxy]-
1H-pyrazol-1-yl}benzonitrile (41)
Compound 41 was prepared in a manner similar to that de-
scribed for 40 in 80% yield as a yellow solid. 1H NMR (300 MHz,
CDCl3) d: 2.17 (3H, s), 2.34 (3H, s), 7.43 (1H, dd, J = 9.0 and
2.9 Hz), 7.56 (1H, dd, J = 8.5 and 2.1 Hz), 7.77–7.82 (2H, m), 8.28
(1H, dd, J = 8.9 and 0.4 Hz), 8.37 (1H, dd, J = 2.9 and 0.5 Hz).
5.1.45. N-(5-{[1-(3-Chloro-4-cyanophenyl)-3,5-dimethyl-1H-
pyrazol-4-yl]oxy}pyridin-2-yl)acetamide (45)
Compound 45 was prepared in a manner similar to that de-
scribed for 44a in 63% yield as a yellow solid, mp 186–187 °C. 1H
NMR (300 MHz, CDCl3) d: 2.15 (3H, s), 2.20 (3H, s), 2.33 (3H, s),
7.23 (1H, d, J = 2.8 Hz), 7.55 (1H, dd, J = 8.5 and 2.1 Hz), 7.72–7.80
(2H, m), 7.83 (1H, br s), 8.00 (1H, d, J = 3.0 Hz), 8.15 (1H, d,
J = 9.1 Hz). Anal. Calcd for C19H16ClN5O2: C, 59.77; H, 4.22; N,
18.34. Found: C, 59.54; H, 4.23; N, 18.16.
5.1.41. 4-{4-[(5-Aminopyridin-2-yl)oxy]-3,5-dimethyl-1H-
pyrazol-1-yl}-2-chlorobenzonitrile (42)
To a solution of 40 (0.050 g, 0.135 mmol) in acetic acid (0.5 mL)
and THF (1 mL) were added zinc powder (0.089 g, 1.35 mmol) and
hydrochloric acid (0.023 mL). After stirring at room temperature
for 4 h, the mixture was diluted with EtOAc, basified with 25%
NH4OH, and extracted twice with EtOAc. The organic layers were
combined, washed with brine, dried over anhydrous Na2SO4, and
concentrated in vacuo. The residue was suspended in acetone/tol-
uene and the insoluble solid was filtered off. The filtrate was puri-
fied by silica gel column chromatography (hexane–EtOAc) to afford
42 (0.033 g, 72%) as a yellow solid. 1H NMR (300 MHz, CDCl3) d:
2.13 (3H, s), 2.33 (3H, s), 3.49 (2H, br s), 6.79 (1H, dd, J = 8.7 and
0.6 Hz), 7.11 (1H, dd, J = 8.7 and 3.0 Hz), 7.56 (1H, dd, J = 8.5 and
2.1 Hz), 7.64 (1H, dd, J = 2.9 and 0.5 Hz), 7.72 (1H, d, J = 8.5 Hz),
7.79 (1H, d, J = 2.1 Hz).
5.2. Cassette dosing in mice
All experiments were conducted in accordance with the regula-
tions of Animal Care and Use Committee of the Takeda Pharmaceu-
tical Company Ltd. Test compounds were administered as a
cassette dosing to mice. After oral administration, blood samples
were collected. The blood samples were centrifuged to obtain the
plasma fraction. The plasma samples were deproteinized with ace-
tonitrile containing an internal standard. After centrifugation, the
supernatant was diluted with LC mobile phase and centrifuged
again. The compound concentrations in the supernatant were mea-
sured by LC/MS/MS.

S. Yamamoto et al. / Bioorg. Med. Chem. 20 (2012) 2338–2352
2351
5.3. Biology
caliper and expressed in mm3 using the formula 0.5 ꢁ a ꢁ b2,
where a is the largest diameter and b is largest diameter perpen-
dicular to a. Body weight was measured weekly. At the end of
experiments, blood samples were collected to measure the serum
PSA levels by ELISA (Markit M PA; Dainippon Sumitomo Pharma,
Japan). Antitumor activity was expressed as T/C% (increase in
tumor volume in a test compound group during the treatment
period/increase in tumor volume in a vehicle group during the
treatment period ꢁ 100).
5.3.1. AR binding inhibitory assay (wild-type and T877A
mutant-type AR)
After FreeStyle293F (Invitrogen) cells were transfected with
pcDNA3.1 containing an androgen receptor (AR) gene (wild-type
AR or T877A mutant-type AR) by using 293 fectin transfection re-
agent (Invitrogen), these cells were seeded into an Erlenmeyer
flask (Corning, 1L, 430518) at 1.1 ꢁ 106 cells/mL in FreeStyle293
Expression Medium (Invitrogen). After 48 h shaking incubation
(125 rpm) at 37 °C in a 8% CO2 atmosphere, these cells were
washed with TEG Buffer (10 mM Tris-HCl (pH 7.2), 50 mM EDTA,
10% Glycerol), and suspended with TEGM Buffer (10 mM Tris–
HCl (pH 7.2), 1 mM EDTA, 10% glycerol, 10 mM Na2MoO4, 1 mM
DTT, 1 mM 2-ME, 1 ꢁ Complete protease inhibitor tablet (Roche)).
After freezing and thawing to lyse cells, lysate was centrifuged at
228,000ꢁg at 4 °C for 20 min. The supernatant was stored at
ꢂ80 °C as AR cell lysate. To cell lysate solution containing an AR
Acknowledgements
We thank Dr. Osamu Uchikawa, Dr. Keiji Kamiyama, Dr. Toshi-
masa Tanaka, and Dr. Yumi N. Imai for helpful discussions; Mr.
Masaharu Nakayama, Mr. Takashi Santou, Mr. Masahiko Hattori,
Mr. Hideo Araki, Ms. Mayumi Imai, and Ms. Kazuyo Nakamura
for the excellent technical assistance for conducting biological
assays.
or a T877A mutant-type AR were added [17-a
-methyl-3H] mibol-
erone (final 3 nM, PerkinElmer NET-919) and a compound, and
the mixture was incubated at 4 °C for 3 h. B (Bound)/F (Free) were
separated by the dextran/charcoal method.42 The label count of B
was measured, and the inhibitory rate of the compound was
calculated.
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