17,20-Lyase inhibitors. Part 3: Design, synthesis, and structure-activity relationships of biphenylylmethylimidazole derivatives as novel 17,20-lyase inhibitors

Article abstract of DOI:10.1016/j.bmc.2011.02.009

A novel series of biphenylylmethylimidazole derivatives and related compounds were synthesized as inhibitors of 17,20-lyase, a key enzyme in the production of steroid hormones, and their biological activities were evaluated. In an attempt to identify potent and selective inhibitors of 17,20-lyase over the related CYP3A4 enzyme, a homology model for human 17,20-lyase was developed using the X-ray crystallographic structure of the mammalian CYP2C5 enzyme. With the aid of molecular modeling, optimization of the biphenyl moiety was performed to give an acetamide derivative, which was resolved by HPLC to give the active (-)-enantiomer. The obtained active enantiomer showed not only potent inhibition of both rat and human 17,20-lyase,with IC₅₀ values of 14 and 26 nM, respectively, but also excellent selectivity (>300-fold) for inhibition of 17,20-lyase over CYP3A4. Moreover, the active enantiomer significantly reduced both serum testosterone and DHEA concentrations in a monkey model after single oral administration. Asymmetric synthesis of the active enantiomer was also developed via a chiral intermediate using a diastereoselective Grignard reaction.

Full text of DOI:10.1016/j.bmc.2011.02.009

Bioorganic & Medicinal Chemistry 19 (2011) 2428–2442
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
17,20-Lyase inhibitors. Part 3: Design, synthesis, and structure–activity
relationships of biphenylylmethylimidazole derivatives as novel
17,20-lyase inhibitors
Tomohiro Kaku a, , Saori Tsujimoto a, Nobuyuki Matsunaga a, Toshimasa Tanaka b, Takahito Hara c,
⇑
Masuo Yamaoka c, Masami Kusaka c, Akihiro Tasaka d
a Medicinal Chemistry Research Laboratories, Takeda Pharmaceutical Company, Ltd, 17-85, Jusohonmachi 2-chome, Yodogawa-ku, Osaka 532-8686, Japan
b Discovery Research Center, Takeda Pharmaceutical Company, Ltd, 17-85, Jusohonmachi 2-chome, Yodogawa-ku, Osaka 532-8686, Japan
c Pharmacology Research Laboratories, Takeda Pharmaceutical Company, Ltd, 17-85, Jusohonmachi 2-chome, Yodogawa-ku, Osaka 532-8686, Japan
d Environment and Safety Department, Takeda Pharmaceutical Company, Ltd, 17-85, Jusohonmachi 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 novel series of biphenylylmethylimidazole derivatives and related compounds were synthesized as
inhibitors of 17,20-lyase, a key enzyme in the production of steroid hormones, and their biological activ-
ities were evaluated. In an attempt to identify potent and selective inhibitors of 17,20-lyase over the
related CYP3A4 enzyme, a homology model for human 17,20-lyase was developed using the X-ray crys-
tallographic structure of the mammalian CYP2C5 enzyme. With the aid of molecular modeling, optimiza-
tion of the biphenyl moiety was performed to give an acetamide derivative, which was resolved by HPLC
to give the active (ꢀ)-enantiomer. The obtained active enantiomer showed not only potent inhibition of
both rat and human 17,20-lyase,with IC50 values of 14 and 26 nM, respectively, but also excellent selec-
tivity (>300-fold) for inhibition of 17,20-lyase over CYP3A4. Moreover, the active enantiomer significantly
reduced both serum testosterone and DHEA concentrations in a monkey model after single oral admin-
istration. Asymmetric synthesis of the active enantiomer was also developed via a chiral intermediate
using a diastereoselective Grignard reaction.
Received 27 November 2010
Revised 4 February 2011
Accepted 5 February 2011
Available online 12 February 2011
Keywords:
17,20-Lyase
Testosterone
DHEA
Prostate cancer
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
therapeutic target for prostate cancer to address some of these
issues.6–8 To date, several steroidal and nonsteroidal inhibitors of
The prostate is an androgen-dependent organ and androgens
are essential for growth and maintenance of prostate function. As
around 80% of patients with prostate cancer respond to hormonal
ablation,1 the current standard treatment for prostate cancer is
surgical castration or medical castration, which involves the
administration of luteinizing hormone–releasing hormone (LH–
RH) agonists2 such as leuprorelin or goserelin. In the past decade,
combination therapy of an LH–RH agonist with an anti-androgen,
referred to as maximum androgen blockade (MAB), has also been
used in the medical treatment of advanced prostate cancer.3 How-
ever, unfavorable effects of anti-androgens such as anti-androgen
withdrawal syndrome have been observed in some clinical cases.4
Additionally, recent studies have suggested that residual adrenal
androgens remaining after castration could be responsible for the
development of castration-resistant prostate cancer.5
17,20-lyase9–43 have been reported, of which a few, for example,
YM-11626 and abiraterone acetate10 (Fig. 1), have been evaluated
in clinical studies. The structural features of these inhibitors in-
clude a lipophilic moiety such as a steroidal skeleton or a steroid
mimetic fused ring with a heteroaromatic ring, which may bind
heme iron in the targeted enzyme.
Our previous studies found that some steroid mimetic struc-
tures such as stilbene, biphenyl, naphthalene and benzothiophene
rings were suitable for lipophilic moieties of 17,20-lyase inhibitors,
and several inhibitors obtained using a steroid A, C-ring mimetic
approach have already been published.44 During the same period,
two studies by Hartmann et al. also reported a steroid mimetic ap-
proach using a biphenyl ring,29,30 but our studies were continued
independently.45
The objective of this study was to identify potent 17,20-lyase
inhibitors with selectivity over the cytochrome P450 enzymes, par-
ticularly CYP3A4, a major drug-metabolizing enzyme. This is be-
cause inhibitors of the P450 family of enzymes have the
potential to cause drug–drug interactions or significant systemic
side effects due to poor selectivity among the P450 enzymes. Here,
17,20-Lyase, also known as CYP17A1, is a key enzyme in
androgen biosynthesis that has been proposed as an alternative
⇑
Corresponding author. Tel.: +81 6 6300 6546; fax: +81 6 6300 6306.
E-mail address: Kaku_Tomohiro@takeda.co.jp (T. Kaku).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.02.009

T. Kaku et al. / Bioorg. Med. Chem. 19 (2011) 2428–2442
2429
the suitable boronic acid 19 was investigated as an alternative ap-
proach to give biphenylylmethylimidazole derivatives and related
compounds (I) (method C).
The synthesis of compounds 5–8 is shown in Scheme 1. Addi-
tion of lithium species generated from n-BuLi with commercially
available 2a–3b to ketone 4 gave good-to-moderate yields of 5–
8. Scheme 2 depicts the preparation of compounds 13–18. Treating
1,3- or 1,4-dibromobenzene with a substoichiometric amount of n-
BuLi in tetrahydrofuran (THF), followed by addition to ketone 9
gave bromide 10a or 10b in high yield. Compounds 13–18 were
synthesized via a palladium-catalyzed Suzuki coupling reaction
of 10a or 10b with the corresponding arylboronic acids 11a–d, fol-
lowed by detritylation using pyridine hydrochloride in methanol
(MeOH). As shown in Scheme 3, in all cases in which suitable aryl-
boronic acids were unavailable, 10b was successfully converted to
the boronic acid 19, which was then subjected to Suzuki coupling
reaction without purification to give 21a–e. Deprotection of 21a–e
was performed by the same procedure used for 13 to give low-to-
moderate yields of compounds 22–26.
Figure 1. Structures of previously developed 17,20-lyase inhibitors.
we describe the synthesis and biological evaluations of
biphenylylmethylimidazole derivatives and related compounds
(I), focusing on the improvement of selectivity for 17,20 lyase over
CYP3A4. In addition, asymmetric synthesis of the selected com-
pound (ꢀ)-17 using a diastereoselective Grignard reaction is de-
scribed in detail.
The synthesis of analogs of 17, in which the isopropyl group was
replaced by hydrogen or other alkyl groups, is shown in Scheme 4.
Coupling of lithium species, generated from 1,4-dibromobenzene
and n-BuLi, with N-tritylated aldehyde 2749 was performed in
the same manner as for 10a, followed by oxidation of 28 with man-
ganese dioxide (MnO2) to give ketone 29. The ketone 29 was then
subjected to Suzuki coupling reaction to give the acetamide
derivative 30, which was treated with Grignard reagent to give
good-to-moderate yields of 31a–c. As deprotection of 31a or 31c
using pyridine hydrochloride in MeOH gave an unsatisfactory yield
(39–59%), an alternative method using Pd/C (10%) under a hydro-
gen atmosphere was investigated for the deprotection of 31b and
found to give a good yield of the desired compound 33. Otherwise,
deprotection of the acetamide derivative 30 with pyridinium
hydrochloride provided 35, which was then subjected to reduction
with sodium borohydride to give a good yield of the alcohol 36.
2. Results and discussion
2.1. Molecular design
As we have previously reported, naphthylmethylimidazole
derivative 1 (Fig. 2) and its related compounds, which together
form a new class of 17,20-lyase inhibitor with a hydroxymethylim-
idazole ring that acts as a ligand for heme iron in the active site of
17,20-lyase, have shown promising in vitro activity for further
development.46 Unfortunately, compound 1 was found to be a po-
tent inhibitor of CYP3A4, with an IC50 value of less than 1000 nM
(Kaku and co-workers, manuscript in press). Therefore, aiming to
improve the selectivity for 17,20-lyase over CYP3A4, scaffold hop-
ping was achieved in this series of inhibitors by using a steroid A,
C-ring mimetic approach to develop biphenylylmethylimidazole
derivatives and related compounds (I) with distinct structural fea-
tures in the hydroxymethylimidazole ring, as shown in Figure 2.
2.3. Asymmetric synthesis of compound (ꢀ)-17
2.2. Chemistry
The synthetic route toward chiral (ꢀ)-17 is shown in Scheme 5.
Strategies for the synthesis of biphenylylmethylimidazole
derivatives and related compounds (I) are summarized in Figure 3.
Lithiation of biphenyl derivatives 2a–3b followed by addition to
ketone 447 gave biphenylylmethylimidazole derivatives and re-
lated compounds (I) (method A). Otherwise, coupling of lithium
species generated from 1,3- or 1,4-dibromobenzene and n-butyl-
lithium (n-BuLi) with tritylated ketone 9 provided the key interme-
diates 10a and 10b. The biphenylylmethylimidazole derivatives
and related compounds (I) can be synthesized via a palladium-
catalyzed Suzuki coupling reaction of bromide 10a or 10b with
boronic acid derivatives (11a–d), followed by deprotection (meth-
od B). It was difficult to convert all the arylbromides 20a–e48 to the
corresponding aryl boronic acids; hence, the conversion of 10b to
The preparation of a-hydroxyketone 41 began from chiral silyle-
ther 38, which was easily prepared from the known morpholine
amide 3750 or achiral 40. Chiral silylether 38 was treated with lith-
ium species generated from 1,4-dibromobenzene and n-BuLi to
yield 39, followed by removal of the tert-butyldimethylsilyl (TBS)
group with tetrabutylammonium fluoride (TBAF) to give a-hydrox-
yketone 41 in moderate chemical yield with 96% enantiomeric ex-
cess (ee) (in two steps). The enantiomeric purity of the obtained
chiral compounds was determined by high-performance liquid
chromatography (HPLC) on a Chiralpak AD column. Alternatively,
osmium-catalyzed asymmetric dihydroxylation of the correspond-
ing enol ethers derived from 40 gave the desired
a-hydroxyketone
41 in good chemical yield with excellent ee (98% ee).
Figure 2. Molecular design of novel 17,20-lyase inhibitors.

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T. Kaku et al. / Bioorg. Med. Chem. 19 (2011) 2428–2442
Figure 3. Retrosynthesis of novel biphenyl derivatives and related compounds as 17,20-lyase inhibitors.
To construct the chiral center of compound (ꢀ)-17, a chelation-
controlled Grignard reaction was used for the synthesis of
(2R,3S)-diol 42; compound 41 with 96% ee was treated with
isopropylmagnesium bromide (i-PrMgBr) to give (2R,3S)-diol 42
together with some by-products, including reduction product 420.
Isolation of 42 from the other by-products proved troublesome;
therefore, optical purity of (2R,3S)-diol 42 was not determined.
Swern oxidation of (2R,3S)-diol 42 gave 43, which was then
converted to bromide 44 at moderate yield. After protection of
44 with a trimethylsilyl group, imidazole ring closure was per-
formed with formamidine acetate in saturated NH3 solution in
Scheme 1. Reagents and conditions: (a) (1) 2a–3b, n-BuLi, THF, ꢀ78 °C, then (2) 4,
ꢀ78 °C to rt, 39–82%.
Scheme 2. Reagents and conditions: (a) (1) n-BuLi, Et2O, ꢀ78 °C, then (2) 9, ꢀ78 to ꢀ50 °C, 82–90%; (b) substituted phenylboronic acid 11a–d, Pd(PPh3)4, 2 M Na2CO3, DME,
reflux, 32–96%; (c) pyridine hydrochloride, MeOH, 60 °C, 21–79%.

T. Kaku et al. / Bioorg. Med. Chem. 19 (2011) 2428–2442
2431
Scheme 3. Reagents and conditions: (a) (1) n-BuLi; (2) trimethoxyborane (B(OMe)3), ꢀ78 °C to rt; (3) 1 N HCl, compound 19 was not isolated; (b) substituted arylbromide
20a–e, Pd(PPh3)4, 2 M Na2CO3, DME, reflux, 23–72%; (c) pyridine hydrochloride, MeOH, 60 °C, 26–61%.
Scheme 4. Reagents and conditions: (a) (1) n-BuLi, THF, ꢀ78 to ꢀ50 °C, then (2) 27, 56%; (b) MnO2, CH2Cl2, rt, 78%; (c) 11c, Pd(PPh3)4, 2 M Na2CO3, toluene, EtOH, reflux, 76%;
(d) RMgBr, THF, 0 °C, 61% to quant.; (e) For 31a, 31c, pyridine hydrochloride, MeOH, 60 °C, 39–59%, for 31b, H2, 10%Pd/C, 1 N HCl, EtOH, rt, 84%; (f) pyridine hydrochloride,
MeOH, 60 °C, 88%; (g) NaBH4, MeOH, 0 °C to rt, 76%.
MeOH and THF to give 45 at good chemical yield with 90% ee.
Unfortunately, the conversion of -hydroxyketone 41 into 45 pro-
2.4. Structure–activity relationships (SARs) for 17,20-lyase and
CYP3A4 inhibitors
a
ceeded with decreased optical purity, whereas recrystallization of
the N-tritylated imidazole 46 with 90% ee led to improved optical
purity of 46, up to 98.8% ee. Compound 46 was then subjected to
Suzuki coupling reaction to give a good yield of the acetamide
derivative 47 with 99.1% ee. Deprotection of compound 47 using
90% formic acid resulted in decreased enantiomeric excess; how-
ever, an alternative approach using catalytic hydrogenation of 47
was performed to give (ꢀ)-17 without any loss of optical purity.
Finally, the absolute configuration of compound (ꢀ)-17 was con-
firmed to be S by X-ray crystallographic analysis of the precursor
46 (Fig. 4).
All compounds synthesized as racemates were tested in vitro
for inhibition of rat 17,20-lyase, recombinant human 17,20-lyase
and CYP3A4. Selected compounds were resolved by HPLC and the
resulting optical isomers were tested using the same in vitro assay.
Results of the initial SAR study of m- and p-biphenyl com-
pounds 5–8 and 13–16 are shown in Table 1. The unsubstituted
compounds 5 and 7 demonstrated promising potency against
17,20-lyase, with IC50 values of 15 and 33 nM, respectively, for
rat enzymes, and 18 and 33 nM, respectively, for human enzymes.
However, these compounds also showed potent inhibitory activity

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T. Kaku et al. / Bioorg. Med. Chem. 19 (2011) 2428–2442
Scheme 5. Reagents and conditions: (a) TBSCl, imidazole, DMF, 0 °C to rt, 98%; (b) (1) n-BuLi, THF, ꢀ78 °C, then (2) 38, 92%; (c) TBAF, THF, 0 °C, 82% (96%ee); (d) (1) NaHMDS,
TBSCl, THF, ꢀ70 °C to rt, then; (2) AD-mix-b, CH3SO2NH2, tert-BuOH, H2O, 5 °C to rt, 87% (98%ee); (e) i-PrMgBr, THF, 0 °C, 59%; (f) oxalylchloride (COCl)2, DMSO, triethylamine
(Et3N), CH2Cl2, ꢀ70 °C to rt, 84%; (g) pyridinium bromide perbromide, THF, rt, 65%; (h) (1) TMSOTf, 2,6-lutidine, THF, 0 °C to rt, then (2) formamidine acetate, satd NH3, MeOH,
THF, rt, 74% (90% ee); (i) (1) trityl chloride, Et3N, DMF, 0 °C to rt; (2) recrystallization from hexane–AcOEt, 63% (98.8% ee); (j) 3-acetamidophenylboronic acid 11c, Pd(PPh3)4,
2 M Na2CO3, toluene, EtOH, reflux, 87% (99.1% ee); (k) 90% formic acid, THF, reflux, 83% (91.7% ee); (l) H2 (4 atm), 10% Pd/C, EtOH, 70 °C, 61% (99.0% ee).
against CYP3A4. Regarding the m- biphenyl derivatives, all 40-
substituted compounds (6, 13 and 14) showed potent inhibition
of rat 17,20-lyase, but insufficient selectivity between 17,20-lyase
and CYP3A4 inhibition. Furthermore, the introduction of a large
substituent, such as a methoxy group, on to the phenyl ring led
to decreased activity of human 17,20-lyase.
On the other hand, p-biphenyl derivatives, other than 8, exhib-
ited potent inhibitory activities against both rat and human 17,20-
lyase and the introduction of an appropriate substituent into the
benzene ring was tolerated. Additionally, as a general trend, p-
biphenyl derivatives showed improved selectivity of 17,20-lyase
over CYP3A4 inhibition compared with the corresponding m-
biphenyl derivatives. These results encouraged us to focus on fur-
ther modification of the p-biphenyl derivatives.
To increase the inhibitory activities of p-biphenyl derivatives, a
homology model of human 17,20-lyase was developed using the X-
ray crystallographic structure of the mammalian CYP2C5 enzyme.
Among several inhibitors, we selected (S)-15 as one of the repre-
sentative compound that was used in the docking study in the
homology model of human 17,20-lyase. This was because the race-
mate 15 showed potent inhibition against human 17,20-lyase with
Figure 4. ORTEP drawing of 46.
an IC50 value of 27 nM and both of the enantiomers of 15 are

T. Kaku et al. / Bioorg. Med. Chem. 19 (2011) 2428–2442
2433
Table 1
Effects of meta or para substitution and R substituents on inhibition of rat and human 17,20-lyase and human CYP3A4 enzymes
HO
N
R
p-
N
H
m-
Compound
Meta or para substitution
R
Enzyme inhibition IC50 (nM)
17,20-Lyase
Ratioa
CYP3A4
Rat
Human
5
6
13
14
7
8
15
16
m-
m-
m-
m-
p-
p-
p-
p-
H
OMe
F
Cl
H
OMe
F
Cl
15
10
8.3
19
33
120
28
18
130
19
49
33
54
27
28
1200
2000
2100
2400
3700
4900
5300
7600
67
15
111
49
112
91
196
271
29
a
Ratio is given by IC50 value of CYP3A4 inhibition/IC50 value of human 17,20-lyase inhibition.
expected to have inhibitory activity against human 17,20-lyase,
based on the previously reported results on compound 1 and its
enantiomers.46 The docking experiment was designed on the
assumption that (S)-15 should be coordinated to the heme iron
via the nitrogen of the imidazole ring, and the proposed binding
mode of (S)-15 is shown in Figure 5. Results of docking studies
indicated that the biphenyl ring of (S)-15 occupied a hydrophobic
region in the active site of the enzyme and there was still enough
space between the I-helix and the F-helix to introduce an appropri-
ate substituent at the C-30 or C-40 position of the benzene ring of
the inhibitor.
Table 2
Effects of R1 and R2 substituents and X on inhibition of rat and human 17,20-lyase and
human CYP3A4 enzymes
HO
N
N
H
X
R2
R1
Compound
X
R1
R2
Enzyme inhibition IC50 (nM)
Additionally, it was postulated that reducing the lipophilicity of
inhibitors would improve the selectivity for 17,20-lyase over
CYP3A4. That is because, in general, the CYP family of enzymes
are known to favor lipophilic molecules as substrates or inhibitors.
Based on the observations, the introduction of a polar substitu-
ent at the C-30 or C-40 position on the benzene ring or replacement
of the benzene ring with pyridine ring in this series of inhibitors
seems to be a promising strategy to enhance inhibitory activity
against 17,20-lyase and improve the selectivity for 17,20-lyase
over CYP3A4. Therefore, the effects of polar substituents on the
benzene ring and replacement of the benzene ring with pyridine
ring in p-biphenyl derivatives were investigated (Table 2). As a re-
sult, compound 17 with an acetamide group at C-30 position,
showed potent activity against both rat and human 17,20-lyase
and the selectivity for inhibition of 17,20 lyase over CYP3A4 was
17,20-Lyase
CYP3A4
Rat
Human
17
18
22
23
24
25
26
C
C
C
C
C
N
N
NHAc
H
CONHMe
SO2NHMe
NHCONHMe
H
H
NHAc
H
H
H
17
24
49
24
120
44
7100
1000
8100
3300
5500
71
24
160
21
H
H
210
53
150
36
5200
>10000
NHAc
similar to that observed with 16. On the other hand, 18 with an
acetamide group at the C-40 position, exhibited less potent activity
against human 17,20-lyase and decreased selectivity for 17,20-
lyase versus CYP3A4. Among the various substituent groups at
the C30-position, the acetamide group provides optimal inhibitory
activity against 17,20-lyase and selectivity for the inhibition of
17,20-lyase over CYP3A4. Furthermore, 25 exhibited less potent
activity against human 17,20-lyase compared with 7. In contrast,
26 bearing an acetamide group on the pyridine ring showed potent
inhibition of human 17,20-lyase and reduced CYP3A4 inhibition. In
summary, the introduction of an appropriate polar substituent
such as an acetamide group on the benzene or pyridine rings of
the inhibitor was tolerated, as predicted by results of the docking
study of (S)-15.
The effects of substituents at the linker position between the
biphenyl and imidazole rings are summarized in Table 3. When
the isopropyl (i-Pr) group of 17 was replaced with other alkyl
groups or hydrogen at the linker position, small alkyl substituents
such as methyl, ethyl, and cyclopropyl (cyclo-Pr) groups were
shown to maintain inhibitory activity against human 17,20-lyase.
On the other hand, replacement of the i-Pr group of 17 with hydro-
gen decreased the inhibitory activity against human 17,20-lyase.
Figure 5. Docking analysis of (S)-15 in the homology model of 17,20-lyase.

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T. Kaku et al. / Bioorg. Med. Chem. 19 (2011) 2428–2442
Table 3
Table 5
Effects of R substituents on inhibition of rat and human 17,20-lyase and human
CYP3A4 enzymes
In vivo effects of S-(ꢀ)-17 on serum testosterone and DHEA levels after single oral
dosing (3 mg/kg) in monkeys
HO
R
Compound
% of average 0 h values, mean SD
Serum testosterone Serum DHEA
2 h 5 h
110 29 98 33
52 8a
23
N
N
H
2 h
5 h
Vehicle
S-(ꢀ)-17 (3 mg/kg)
116 95
23
148 53
13 6b
5
9
NHAc
n = 3.
a
P <0.05 versus vehicle (t-test).
P <0.05 versus vehicle (Welch test).
Compound
R
Enzyme inhibition IC50 (nM)
17,20-Lyase CYP3A4
Human
b
Rat
serum testosterone and DHEA concentrations in male cynomolgus
monkeys. These biological results indicate that (ꢀ)-17 might be
useful for the treatment of both hormone-dependent and castra-
tion-resistant prostate cancer.
17
32
33
34
36
i-Pr
Me
Et
cyclo-Pr
H
17
62
25
81
320
24
38
40
45
77
7100
>10,000
>10,000
5500
>10,000
3. Conclusion
We have reported the synthesis and SAR of a new class of 17,20-
lyase inhibitors. On the basis of molecular modeling, compounds
17, 26 and 33, bearing an acetamide group on the benzene or pyr-
idine rings, were synthesized and their biological activities were
evaluated. Optical resolution of these inhibitors by HPLC gave the
biologically active enantiomers (ꢀ)-17, (ꢀ)-26 and (ꢀ)-33, respec-
tively, which showed potent activities against both rat and human
17,20-lyase, with excellent selectivity (>300-fold) for the inhibition
of 17,20-lyase over CYP3A4. Among these enantiomers, (ꢀ)-17 sig-
nificantly reduced both serum testosterone and DHEA concentra-
tions in a monkey model. From the data presented here, we
concluded that the p-biphenyl derivatives and related compounds,
as well as the naphthalene derivative 1, showed promising profiles
for further development.46 Further investigation of this series
should provide further insights to the selectivity for 17,20-lyase
over the cytochrome P450 enzymes.
These results suggest that the introduction of a small alkyl group
into the linker position was necessary to increase the inhibitory
activity against human 17,20-lyase, and that the i-Pr group gives
optimal potency.
Finally, we selected compounds 17, 26, and 33 as candidates for
chiral resolution by HPLC, to evaluate whether the interaction with
enzymes such as 17,20-lyase and CYP3A4 by these inhibitors is ste-
reospecific. Table 4 shows the biological activities of the obtained
optical isomers. Interestingly, each enantiomer had different inhib-
itory activity against 17,20-lyase and all of the (ꢀ)-enantiomers
had potent activity against both rat and human 17,20 lyase, with
greater than 300-fold selectivity for the inhibition of 17,20-lyase
over CYP3A4. These findings suggest that the chiral configuration
at the linker position between the biphenyl and imidazole rings
plays an important role in inhibition of 17,20-lyase and the selec-
tivity of 17,20-lyase over CYP3A4 inhibition.
2.5. In vivo efficacy
4. Experimental
Among synthesized inhibitors, the active enantiomer (ꢀ)-17,
which showed potent inhibitory activity against human 17,20-
lyase in vitro, was selected as a representative for the evaluation
of in vivo activity. In a monkey model, both testicular androgen
testosterone and adrenal androgen dehydroepiandrosterone
(DHEA) concentrations were investigated. As shown in Table 5, sin-
gle oral doses of (ꢀ)-17 3 mg/kg significantly decreased both
Melting points were determined using a BUCHI Melting Point B-
545 apparatus and are uncorrected. Infrared (IR) spectra were re-
corded using a SHIMADZU FT-IR-8200PC spectrometer. 1H NMR
spectra were recorded using a Varian Gemini-200 or Varian Mer-
cury-300 spectrometer; chemical shifts are given in ppm with tet-
ramethylsilane as an internal standard, and coupling constants (J)
are measured in hertz (Hz). The following abbreviations are used:
s = singlet, d = doublet, t = triplet, m = multiplet, br s = broad sin-
glet. Reactions were followed by thin-layer chromatography
(TLC) on Silica Gel 60 F254 precoated TLC plates (Merck, Darmstadt,
Germany). Column chromatography was performed using Silica
Gel 60 (Merck, Darmstadt, Germany). Compounds 2a–b, 3a–b,
11a–d, 20a–b, 20d–e, 40 were commercially available, compounds
4,47 20c,4827,49 and 3750 were prepared in the same manner as de-
scribed previously, and compound 9 was easily prepared from
compound 4 and trityl chloride in the usual manner.
Table 4
Inhibitory effects of ( )-17, ( )-26 and ( )-33 enantiomers on rat and human 17,20-
lyase and human CYP3A4 enzymes
HO
*
R
N
N
H
X
NHAc
4.1. General procedure for method A and 1-[1,10-biphenyl]-3-yl-
1-(1H-imidazol-4-yl)-2-methyl-1-propanol (5)
Compound
X
R
Enzyme inhibition IC50 (nM)
17,20-Lyase CYP3A4
Human
n-BuLi in hexane (1.6 M; 6.4 mL, 10.2 mmol) was added to a
cooled (ꢀ78 °C) solution of 2a (2.11 g, 9.05 mmol) in THF (30 mL)
and the mixture was stirred at ꢀ78 °C for 1 h. A solution of 4
(400 mg, 2.89 mmol) in THF (10 mL) was added to the mixture
and the solution was allowed to warm to rt. The reaction was
quenched with aqueous NH4Cl solution and the aqueous phase
was extracted with AcOEt. The extract was dried over MgSO4 and
Rat
(ꢀ)-17
(+)-17
(ꢀ)-26
(+)-26
(ꢀ)-33
(+)-33
C
C
N
N
C
C
i-Pr
i-Pr
i-Pr
i-Pr
Et
14
770
33
26
340
26
92
22
>10,000
6700
>10,000
>10,000
>10,000
>10,000
>1000
19
Et
450
240

T. Kaku et al. / Bioorg. Med. Chem. 19 (2011) 2428–2442
2435
concentrated under reduced pressure. The residue was chromato-
graphed on silica gel (CH2Cl2/MeOH = 20:1) and recrystallized from
AcOEt–hexane to give 5 (450 mg, 1.54 mmol, 53%) as a colorless
powder. Melting point (mp) 180–183 °C (AcOEt–hexane). 1H
NMR (CDCl3+CD3OD) d: 0.83 (3H, d, J = 6.6 Hz), 0.99 (3H, d,
J = 6.6 Hz), 2.55–2.73 (1H, m), 6.97 (1H, d, J = 1.0 Hz), 7.28–7.50
(7H, m), 7.54–7.62 (2H, m), 7.72–7.77 (1H, m). IR (KBr): 3200,
1005, 799 cmꢀ1. Anal. Calcd for C19H20N2O: C, 78.05; H, 6.89; N,
9.58. Found: C, 77.84; H, 6.86; N, 9.36. Compounds 6–8 were pre-
pared in the same manner as described for the preparation of 5.
3.50 (1H, s), 6.72 (1H, d, J = 1.2 Hz), 7.09–7.16 (6H, m), 7.30–7.38
(14H, m). IR (KBr): 1489, 1445, 1159, 1009, 909, 812, 747, 735,
702, 660 cmꢀ1. Anal. Calcd for C32H29N2OBr: C, 71.51; H, 5.44; N,
5.21. Found: C, 71.54; H, 5.48; N, 5.20.
4.7. 1-(40-Fluoro-[1,10-biphenyl]-3-yl)-2-methyl-1-(1-trityl-1H-
imidazol-4-yl)-1-propanol (12a)
Under N2 atmosphere, a mixture of 10a (1.01 g, 1.88 mmol), 4-
fluorophenylboronic acid (413 mg, 2.95 mmol) and Pd(PPh3)4 (0)
(190 mg, 0.16 mmol) in dimethyloxyethane (DME)/2 M Na2CO3
(20 mL/8 mL) was refluxed for 18 h. The resulting mixture was ex-
tracted with AcOEt and the organic layer was dried over MgSO4
and concentrated under reduced pressure. The residue was chro-
matographed on silica gel (hexane/AcOEt = 4:1) and recrystallized
from AcOEt–hexane to give 12a (705 mg, 1.28 mmol, 68%) as a pale
yellow powder. Mp 141–143 °C (AcOEt–hexane). 1H NMR (CDCl3)
d: 0.75 (3H, d, J = 6.8 Hz), 0.93 (3H, d, J = 6.8 Hz), 2.35–2.52 (1H,
m), 3.70 (1H, s), 6.78 (1H, d, J = 1.0 Hz), 7.04–7.18 (8H, m), 7.22–
7.38 (12H, m), 7.44–7.55 (3H, m), 7.63 (1H, s). IR (KBr): 1512,
1480, 1233, 1159 cmꢀ1. Anal. Calcd for C38H33N2OF: C, 82.58; H,
6.02; N, 5.07. Found: C, 82.43; H, 5.83; N, 4.91. Compounds 12b–
f were prepared in the same manner as described for the prepara-
tion of 12a.
4.2. 1-(1H-Imidazol-4-yl)-1-(40-methoxy-[1,10-biphenyl]-3-yl)-2-
methyl-1-propanol (6)
Yield 74%. Mp 74–77 °C (AcOEt–hexane). 1H NMR (CDCl3) d:
0.82 (3H, d, J = 6.9 Hz), 0.98 (3H, d, J = 6.9 Hz), 2.50–2.74 (1H, m),
3.83 (3H, s), 6.89–6.99 (3H, m), 7.26–7.55 (6H, m), 7.71–7.76
(1H, m). IR (KBr): 2969, 1516, 1480, 1248, 1181 cmꢀ1. Anal. Calcd
for C20H22N2O2: C, 74.51; H, 6.88; N, 8.69. Found: C, 74.45; H,
7.17; N, 8.40.
4.3. 1-[1,10-Biphenyl]-4-yl-1-(1H-imidazol-4-yl)-2-methyl-1-
propanol (7)
Yield 83%. Mp 182–183 °C (AcOEt–hexane). 1H NMR
(CDCl3+CD3OD) d: 0.83 (3H, d, J = 6.8 Hz), 0.99 (3H, d, J = 6.8 Hz),
2.57–2.74 (1H, m), 6.98 (1H, d, J = 1.2 Hz), 7.30–7.47 (3H, m),
7.50–7.65 (7H, m). IR (KBr): 3142, 2965, 1487, 826, 762 cmꢀ1. Anal.
Calcd for C19H20N2Oꢁ0.1H2O: C, 77.57; H, 6.92; N, 9.52. Found: C,
77.57; H, 6.95; N, 9.61.
4.8. 1-(40-Chloro[1,10-biphenyl]-3-yl)-2-methyl-1-(1-trityl-1H-
imidazol-4-yl)-1-propanol (12b)
Yield 70%. Mp 157 °C (AcOEt–hexane). 1H NMR (CDCl3) d: 0.75
(3H, d, J = 6.7 Hz), 0.94 (3H, d, J = 6.7 Hz), 2.35–2.55 (1H, m), 3.70
(1H, s), 6.78 (1H, d, J = 1.4 Hz), 7.05–7.60 (23H, m), 7.65 (1H, s).
4.4. 1-(1H-Imidazol-4-yl)-1-(40-methoxy[1,10-biphenyl]-4-yl)-2-
methyl-1-propanol (8)
IR (KBr): 1493, 1476, 1445, 909 cmꢀ1
.
Anal. Calcd for
C38H33N2OClꢁH2O: C, 79.94; H, 5.86; N, 4.91. Found: C, 79.85; H,
Yield 39%. Mp 200–201 °C (AcOEt). 1H NMR (CDCl3+CD3OD) d:
0.83 (3H, d, J = 6.6 Hz), 0.98 (3H, d, J = 6.6 Hz), 2.52–2.76 (1H, m),
3.85 (3H, s), 6.92–7.02 (3H, m), 7.38–7.62 (7H, m). IR (KBr): 3218,
1497, 1252, 1038, 1007, 816 cmꢀ1. Anal. Calcd for C20H22N2O2: C,
74.51; H, 6.88; N, 8.69. Found: C, 74.64; H, 6.89; N, 8.53.
5.85; N, 4.80.
4.9. 1-(40-Fluoro-[1,10-biphenyl]-4-yl)-1-(1-trityl-1H-imidazol-
4-yl)-2-methyl-1-propanol (12c)
Yield 80%. Mp 213–214 °C (AcOEt–hexane). 1H NMR (CDCl3) d:
0.77 (3H, d, J = 6.6 Hz), 0.92 (3H, d, J = 6.6 Hz), 2.42–2.49 (1H, m),
3.53 (1H, s), 6.78 (1H, s), 7.06–7.15 (7H, m), 7.337.57 (17H, m).
IR (KBr): 1493, 1445, 1223, 1159, 818, 748, 733, 702 cmꢀ1. Anal.
Calcd for C38H33N2OF: C, 81.52; H, 6.08; N, 5.00. Found: C, 81.58;
H, 6.08; N, 4.92.
4.5. General procedure for method B and 1-(3-bromophenyl)-2-
methyl-1-(1-trityl-1H-imidazol-4-yl)-1-propanol (10a)
n-BuLi (1.6 M; 50 mL, 80 mmol) was added to a cooled (ꢀ78 °C)
solution of 1,3-dibromobenzene (29.02 g, 123 mmol) in diethyl-
ether (250 mL) and the mixture was stirred at ꢀ78 °C for 30 min.
A solution of 9 (20.0 g, 53 mmol) in THF (100 mL) was added and
the solution was allowed to warm to ꢀ50 °C. After stirring at
ꢀ50 °C for a further 30 min, the reaction was quenched with aq
NH4Cl solution. The mixture was extracted with AcOEt and dried
over MgSO4. After removal of the solvent, the residue was recrys-
tallized from AcOEt–hexane to give 10a (25.30 g, 47 mmol, 90%)
as a colorless powder. Mp 164–165 °C (AcOEt–hexane). 1H NMR
(CDCl3) d: 0.72 (3H, d, J = 6.8 Hz), 0.90 (3H, d, J = 6.8 Hz), 2.22–
2.44 (1H, m), 3.65 (1H, s), 6.73 (1H, d, J = 1.6 Hz), 7.06–7.19 (7H,
m), 7.26–7.39 (11H, m), 7.46 (1H, dt, J = 7.8, 1.3 Hz), 7.59 (1H, t,
J = 1.8 Hz). IR (KBr): 1493, 1472, 1445, 702 cmꢀ1. Anal. Calcd for
4.10. 1-(40-Chloro-[1,10-biphenyl]-4-yl)-1-(1-trityl-1H-imidazol-
4-yl)-2-methyl-1-propanol (12d)
Yield 85%. Mp 211–212 °C (AcOEt–hexane). 1H NMR (CDCl3) d:
0.76 (3H, d, J = 7.0 Hz), 0.92 (3H, d, J = 7.0 Hz), 2.42–2.49 (1H, m),
3.53 (1H, s), 6.78 (1H, s), 7.13–7.15 (6H, m), 7.32–7.59 (18H, m).
IR (KBr): 1485, 1445, 1094, 1005, 909, 812, 747, 733, 700 cmꢀ1
.
Anal. Calcd for C38H33N2OCl: C, 79.69; H, 5.88; N, 4.89. Found: C,
79.48; H, 6.14; N, 4.72.
C
32H29N2OBr: C, 71.51; H, 5.44; N, 5.21. Found: C, 71.43; H, 5.60;
4.11. N-{40-[1-Hydroxy-2-methyl1-(1-trityl-1H-imidazol-4-yl)-
N, 5.38. Compound 10b was prepared in the same manner as de-
scribed for the preparation of 10a.
propyl][1,10-biphenyl]-3-yl}acetamide (12e)
Yield 96%. 1H NMR (CDCl3) d: 0.76 (3H, d, J = 6.6 Hz), 0.92 (3H, d,
J = 6.6 Hz), 2.20 (3H, s), 2.38–2.56 (1H, m), 3.55 (1H, s), 6.77 (1H, d,
J = 1.2 Hz), 7.06–7.20 (6H, m), 7.24–7.76 (18H, m). IR (KBr): 3063,
1674, 1557, 1483, 1445 cmꢀ1. FAB-MS m/z: 592.2935 (calcd for
4.6. 1-(4-Bromophenyl)-2-methyl-1-(1-trityl-1H-imidazol-4-yl)-
1-propanol (10b)
Yield 82%. Mp 145–146 °C (AcOEt–hexane). 1H NMR (CDCl3) d:
0.71 (3H, d, J = 6.6 Hz), 0.89 (3H, d, J = 6.6 Hz), 2.30–2.44 (1H, m),
C40H38N3O2: 592.2964).

2436
T. Kaku et al. / Bioorg. Med. Chem. 19 (2011) 2428–2442
4.12. N-{40-[1-Hydroxy-2-methyl-1-(1-trityl-1H-imidazol-4-yl)-
C
21H23N3O2 ꢁ 0.75 H2O: C, 69.50; H, 6.80; N, 11.58. Found: C,
69.12; H, 6.91; N, 11.45. FAB-MS m/z: 350.1825 (calcd for
21H24N3O2: 350.1869).
propyl][1,10-biphenyl]-4-yl}acetamide (12f)
C
Yield 32%. Mp 244–248 °C (AcOEt). 1H NMR (CDCl3) d: 0.77 (3H,
d, J = 6.6 Hz), 0.93 (3H, d, J = 6.6 Hz), 2.20 (3H, s), 2.30–2.56 (1H, m),
3.53 (1H, s), 6.77 (1H, d, J = 1.4 Hz), 7.08–7.14 (6H, m), 7.27–7.38
(10H, m), 7.43–7.58 (8H, m). IR (KBr): 2971, 1671, 1535,
1493 cmꢀ1. Anal. Calcd for C40H37N3O2: C, 81.19; H, 6.30; N, 7.10.
Found: C, 81.01; H, 6.37; N, 6.99.
4.18. N-{40-[1-Hydroxy-1-(1H-imidazol-4-yl)-2-methylpropyl]
[1,10-biphenyl]-4-yl}acetamide (18)
Yield 21%. Mp 199–201 °C (AcOEt). 1H NMR (CDCl3+CD3OD) d:
0.82 (3H, d, J = 6.8 Hz), 0.98 (3H, d, J = 6.8 Hz), 2.17 (3H, s), 2.49–
2.70 (1H, m), 6.96 (1H, d, J = 0.8 Hz), 7.44–7.60 (9H, m). IR (KBr):
4.13. 1-(40-Fluoro[1,10-biphenyl]-3-yl)-1-(1H-imidazol-4-yl)-2-
methyl-1-propanol (13)
3173,
21H23N3O2ꢁ0.1AcOEt: C, 71.75; H, 6.70; N, 11.73. Found: C,
71.70; H, 6.65; N, 11.90.
1667,
1534,
1499 cmꢀ1
.
Anal.
Calcd
for
C
A mixture of 12a (620 mg, 1.12 mmol) and pyridine hydrochlo-
ride (270 mg, 2.34 mmol) in MeOH (20 mL) was stirred at 60 °C for
4 h. The mixture was made alkaline with aq NaHCO3 solution and
concentrated under reduced pressure. The resulting mixture was
extracted with AcOEt and the combined organic layers were dried
over MgSO4. After removal of the solvent, the residue was chro-
matographed on silica gel (CH2Cl2/MeOH = 40:1) and recrystallized
from AcOEt–hexane to give 13 (274 mg, 0.88 mmol, 79%) as a col-
orless powder. Mp 154–156 °C (AcOEt–hexane). 1H NMR
(CDCl3+CD3OD) d: 0.82 (3H, d, J = 6.8 Hz), 0.98 (3H, d, J = 6.8 Hz),
2.40–2.80 (1H, m), 6.70–7.16 (3H, m), 7.30–7.59 (6H, m), 7.72
(1H, s). IR (KBr): 3187, 1514, 1236, 1005, 795 cmꢀ1. Anal. Calcd
for C19H19N2OF: C, 73.53; H, 6.17; N, 9.03. Found: C, 73.48; H,
5.94; N, 9.02. Compounds 14–18 were prepared in the same man-
ner as described for the preparation of 13.
4.19. General procedure for method C and 4-[1-hydroxy-2-
methyl-1-(1-trityl-1H-imidazol-4-yl)propyl] phenylboronic acid
(19)
n-BuLi (1.6 M; 14 mL, 23 mmol) was added to a cooled (ꢀ78 °C)
solution of 1,4-dibromobenzene (5.10 g, 22 mmol) in diethylether
(30 mL) and THF (40 mL) and the mixture was stirred at ꢀ78 °C
for 15 min. A solution of 10b (6.50 g, 17 mmol) in THF (30 mL)
was added dropwise to the mixture. After stirring at ꢀ78 °C for
15 min, n-BuLi (1.6 M; 20 mL, 33 mmol) was added dropwise.
The resulting mixture was further stirred at ꢀ78 °C for 15 min
and trimethoxyborane (15 mL, 134 mmol) was added. The mixture
was allowed to warm to rt and further stirred for 1 h. HCl (1 N) was
added to the reaction mixture and the solution was extracted with
AcOEt. The combined organic layers were concentrated under re-
duced pressure. The residue was redissolved in AcOEt and the solu-
tion was washed with 1 N NaOH and brine, before being dried over
MgSO4. Concentration of the solvent under reduced pressure gave
crude 19 (4.00 g) as a colorless amorphous solid. This compound
was used for the next step without further purification.
4.14. 1-(40-Chloro[1,10-biphenyl]-3-yl)-1-(1H-imidazol-4-yl)-2-
methyl-1-propanol (14)
Yield 76%. Mp 169 °C (AcOEt–hexane). 1H NMR (CDCl3) d: 0.82
(3H, d, J = 6.8 Hz), 0.98 (3H, d, J = 6.8 Hz), 2.50–2.78 (1H, m), 6.99
(1H, d, J = 1.0 Hz), 7.33–7.44 (4H, m), 7.45–7.56 (4H, m), 7.78
(1H, s). IR (KBr): 2969, 1476, 1092, 1013 cmꢀ1. Anal. Calcd for
4.20. 40-[1-Hydroxy-2-methyl-1-(1-trityl-1H-imidazol-4-
yl)propyl]-N-methyl[1,10-biphenyl]-3-carboxamide (21a)
C19H19N2OCl: C, 69.83; H, 5.86; N, 8.57. Found: C, 69.98; H, 5.98;
N, 8.50.
4.15. 1-(40-Fluoro[1,10-biphenyl]-4-yl)-1-(1H-imidazol-4-yl)-2-
methyl-1-propanol (15)
Under N2 atmosphere a mixture of crude 19 (3.44 g), 3-bromo-
phenyl-N-methylcarboxamide 20a (1.10 g, 5.14 mmol) and
Pd(PPh3)4 (0) (210 mg, 0.18 mmol) in DME (20 mL) and 2 M
Na2CO3 solution (20 mL) was refluxed for 16 h. The resulting mix-
ture was extracted with AcOEt and the combined organic layers
were dried over MgSO4 and concentrated under reduced pressure.
The residue was chromatographed on silica gel (hexane/
AcOEt = 1:2) to give 21a (1.00 g, 1.69 mmol, 33%) as a pale yellow
amorphous solid. 1H NMR (CDCl3) d: 0.76 (3H, d, J = 6.7 Hz), 0.93
(3H, d, J = 6.7 Hz), 2.30–2.54 (1H, m), 3.02 (3H, d, J = 4.8 Hz), 3.58
(1H, s), 6.33 (1H, br s), 6.78 (1H, d, J = 1.4 Hz), 7.04–7.20 (6H, m),
7.22–7.38 (9H, m), 7.39–7.76 (8H, m), 7.98 (1H, t, J = 1.4 Hz). IR
Yield 69%. Mp 198–199 °C (AcOEt–hexane). 1H NMR (CDCl3) d:
0.85 (3H, d, J = 7.0 Hz), 1.00 (3H, d, J = 7.0 Hz), 2.60–2.74 (1H, m),
3.42 (1H, br s), 7.02–7.16 (3H, m), 7.48–7.66 (7H, m), 9.16 (1H,
br s). IR (KBr): 3241, 1493, 1397, 1242, 1009, 814, 781, 762, 623,
511 cmꢀ1 Anal. Calcd for C19H19N2OF: C, 75.53; H, 6.17; N, 9.03.
.
Found: C, 73.43; H, 6.09; N, 9.03.
4.16. 1-(40-Chloro[1,10-biphenyl]-4-yl)-1-(1H-imidazol-4-yl)-2-
methyl-1-propanol (16)
(KBr): 3295, 2969, 1644, 1549, 1121 cmꢀ1
. FAB-MS m/z:
Yield 76%. Mp 197–198 °C (AcOEt–hexane). 1H NMR (CDCl3) d:
0.83 (3H, d, J = 7.0 Hz), 0.98 (3H, d, J = 7.0 Hz), 2.58–2.75 (1H, m),
3.38 (1H, br s), 7.00 (1H, s), 7.37 (2H, d, J = 8.4 Hz), 7.48–7.64
(7H, m), 9.24 (1H, br s). IR (KBr): 3200, 1485, 1364, 1190, 1094,
1028, 1005, 808, 781 cmꢀ1. Anal. Calcd for C19H19N2OCl: C, 69.83;
H, 5.86; N, 8.57. Found: C, 69.80; H, 5.85; N, 8.65.
592.2999 (calcd for C40H38N3O2:592.2964). Compounds 21b–e
were prepared in the same manner as described for the prepara-
tion of 21a.
4.21. 40-[1-Hydroxy-2-methyl-1-(1-trityl-1H-imidazol-4-yl)-
propyl]-N-methyl[1,10-biphenyl]-3-sulfonamide (21b)
Yield 24%. Mp 148–149 °C (AcOEt). 1H NMR (CDCl3) d: 0.76 (3H,
d, J = 6.8 Hz), 0.93 (3H, d, J = 6.8 Hz), 2.36–2.58 (1H, m), 2.69 (3H, d,
J = 5.6 Hz), 3.57 (1H, s), 4.38–4.50 (1H, m), 6.79 (1H, s), 7.08–7.20
(6H, m), 7.28–7.40 (10H, m), 7.48–7.65 (5H, m), 7.80 (2H, d,
J = 8.0 Hz), 8.07 (1H, s). IR (KBr): 2969, 1495, 1472, 1445, 1327,
1161 cmꢀ1. Anal. Calcd for C39H37N3O3S: C, 74.61; H, 5.94; N,
6.69. Found: C, 74.71; H, 6.00; N, 6.69.
4.17. N-{40-[1-Hydroxy-1-(1H-imidazol-4-yl)-2-methylpropyl]
[1,10-biphenyl]-3-yl}acetamide (17)
Yield 48%. 1H NMR (CDCl3+CD3OD) d: 0.82 (3H, d, J = 6.8 Hz),
0.98 (3H, d, J = 6.8 Hz), 2.17 (3H, s), 2.51–2.74 (1H, m), 6.96 (1H,
d, J = 1.0 Hz), 7.25–7.39 (3H, m), 7.42–7.56 (5H, m), 7.68 (1H, s).
IR (KBr): 3210, 2971, 1672, 1557, 1483 cmꢀ1. Anal. Calcd for

T. Kaku et al. / Bioorg. Med. Chem. 19 (2011) 2428–2442
2437
4.22. N-{40-[1-Hydroxy-2-methyl-1-(1-trityl-1H-imidazol-4-yl)-
propyl][1,10-biphenyl]-3-yl}-N0-methylurea (21c)
4.28. 1-(1H-Imidazol-4-yl)-2-methyl-1-[4-(2-pyridinyl)phenyl]-
1-propanol (25)
Yield 34%. 1H NMR (CDCl3) d: 0.75 (3H, d, J = 6.7 Hz), 0.92 (3H, d,
J = 6.7 Hz), 2.38–2.56 (1H, m), 2.78 (3H, d, J = 4.6 Hz), 3.52 (1H, s),
5.00–5.20 (1H, m), 6.79 (1H, d, J = 1.0 Hz), 6.90 (1H, br s), 7.06–
7.18 (6H, m), 7.22–7.39 (14H, m), 7.42 (2H, d, J = 8.6 Hz), 7.51
(2H, d, J = 8.6 Hz). IR (KBr): 2967, 1669, 1557 cmꢀ1. Anal. Calcd
for C40H38N4O2ꢁ0.2AcOEt: C, 78.48; H, 6.39; N, 8.97. Found: C,
78.45; H, 6.53; N, 8.99.
Yield 26%. 1H NMR (CDCl3) d: 0.82 (3H, d, J = 6.8 Hz), 0.98 (3H, d,
J = 6.6 Hz), 2.57–2.70 (1H, m), 6.95 (1H, d, J = 1.2 Hz), 7.18–7.24
(1H, m), 7.49 (1H, d, J = 1.2 Hz), 7.62–7.79 (4H, m), 7.91 (2H, d,
J = 8.4 Hz), 8.66 (1H, d, J = 4.8 Hz). IR (KBr): 1590, 1468, 1435,
829, 781, 733 cmꢀ1. Anal. Calcd for C18H19N3Oꢁ0.4H2O: C, 71.93;
H, 6.64; N, 13.98. Found: C, 71.69; H, 6.55; N, 13.98.
4.29. N-{6-(4-[1-Hydroxy-1-(1H-imidazol-4-yl)-2-methylpropyl]
phenyl)-2-pyridyl}acetamide (26)
4.23. 2-Methyl-1-[4-(2-pyridinyl)phenyl]-1-(1-trityl-1H-
Yield 42%. Mp 206–208 °C (AcOEt–MeOH). 1H NMR
(CDCl3+CD3OD) d: 0.81 (3H, d, J = 6.7 Hz), 0.98 (3H, d, J = 6.7 Hz),
2.22 (3H, s), 2.52–2.74 (1H, m), 6.97 (1H, d, J = 1.0 Hz), 7.44 (1H,
d, J = 7.8 Hz), 7.52 (1H, d, J = 1.0 Hz), 7.59 (2H, d, J = 8.4 Hz), 7.76
(1H, t, J = 7.8 Hz), 7.83 (2H, d, J = 8.4 Hz), 8.09 (1H, d, J = 7.8 Hz).
IR (KBr): 3177, 2967, 1651, 1559, 1451 cmꢀ1. Anal. Calcd for
imidazol-4-yl)-1-propanol (21d)
Yield 72%. Mp 226–227 °C (chloroform–MeOH–hexane). 1H
NMR (CDCl3) d: 0.75 (3H, d, J = 6.8 Hz), 0.93 (3H, d, J = 6.4 Hz),
2.39–2.53 (1H, m), 3.60 (1H, s), 6.78 (1H, d, J = 1.4 Hz), 7.10–7.35
(17H, m), 7.58–7.63 (2H, m), 7.71–7.78 (2H, m), 7.89–7.93 (2H,
m), 8.66–8.69 (1H, m). IR (KBr): 1588, 1491, 1466, 1447, 1435,
C20H22N4O2ꢁ0.3AcOEt: C, 67.57; H, 6.53; N, 14.87. Found: C,
1003, 781, 747, 702 cmꢀ1
.
67.39; H, 6.63; N, 14.89.
4.30. 4-Bromophenyl(1-trityl-1H-imidazol-4-yl)methanol (28)
4.24. N-{6-(4-[1-Hydroxy-2-methyl-1-(1-trityl-1H-imidazol-4-
yl)propyl]phenyl)-2-pyridinyl}acetamide (21e)
Compound 28 was prepared in the same manner as described
for the preparation of 10a. Yield 56%. Mp 214–215 °C (AcOEt–
MeOH–THF–hexane). 1H NMR (CDCl3) d: 3.56 (1H, br s), 5.71 (2H,
d, J = 4.4 Hz), 6.58 (1H, s), 7.07–7.13 (7H, m), 7.25–7.44 (12H, m).
IR (KBr): 1493, 1445, 1128, 1011, 909, 747, 733, 702 cmꢀ1. Anal.
Calcd for C29H23N2OBr: C, 70.31; H, 4.68; N, 5.65. Found: C,
69.93; H, 4.86; N, 5.31.
Yield 23%. Mp 137 °C (AcOEt–hexane). 1H NMR (CDCl3) d: 0.74
(3H, d, J = 6.8 Hz), 0.92 (3H, d, J = 6.8 Hz), 2.20 (3H, s), 2.34–2.58
(1H, m), 3.60 (1H, s), 6.77 (1H, d, J = 1.4 Hz), 7.06–7.20 (6H, m),
7.28–7.42 (10H, m), 7.44 (1H, dd, J = 0.8, 7.9 Hz), 7.59 (2H, d,
J = 8.4 Hz), 7.75 (1H, t, J = 7.9 Hz), 7.83 (2H, d, J = 8.4 Hz), 8.05–
8.16 (2H, m). IR (KBr): 2969, 1732, 1690, 1447 cmꢀ1. Anal. Calcd
for C39H36N4O2ꢁ1.1AcOEt: C, 75.58; H, 6.55; N, 8.12. Found: C,
75.52; H, 6.46; N, 8.17. Compounds 22–26 were prepared in the
same manner as described for the preparation of 13.
4.31. 4-Bromophenyl(1-trityl-1H-imidazol-4-yl)methanone (29)
A
mixture of 28 (30.0 g, 60.6 mmol) and MnO2 (52.6 g,
606 mmol) in CH2Cl2 (1000 mL) was stirred at rt for 26 h. The mix-
ture was filtered through Celite, and the filtrate was concentrated
in vacuo. The residue was recrystallized from AcOEt–Et2O–hexane
to give 29 (23.3 g, 47.2 mmol, 78%) as a colorless powder. Mp 145–
146 °C (AcOEt–Et2O–hexane). 1H NMR (CDCl3) d: 7.10–7.19 (6H,
m), 7.31–7.41 (9H, m), 7.52 (1H, d, J = 1.4 Hz), 7.68 (2H, d,
J = 8.4 Hz), 7.77 (1H, d, J = 1.4 Hz), 8.21 (2H, d, J = 8.4 Hz). IR
4.25. 40-[1-Hydroxy1-(1H-imidazol-4-yl)-2-methylpropyl]-N-
methyl[1,10-biphenyl]-3-carboxamide (22)
Yield 42%. 1H NMR (CDCl3+CD3OD) d: 0.81 (3H, d, J = 6.9 Hz),
0.98 (3H, d, J = 6.9 Hz), 2.40–2.80 (1H, m), 3.00 (3H, s), 6.96 (1H,
s), 7.30 (1H, d, J = 1.4 Hz), 7.38–7.60 (5H, m), 7.60–7.74 (2H, m),
7.93 (1H, s). IR (KBr): 3277, 2969, 1645, 1547 cmꢀ1. Anal. Calcd
for C21H23N3O2ꢁ0.75H2O: C, 69.50; H, 6.80; N, 11.58. Found: C,
69.53; H, 7.16; N, 11.28.
(KBr): 1644, 1520, 1213, 887, 756, 747, 702 cmꢀ1
.
4.32. N-[40-[(1-Trityl-1H-imidazol-4-yl)carbonyl][1,10-biphenyl]-
3-yl]acetamide (30)
Compound 30 was prepared in the same manner as described
for the preparation of 12a. Yield 76%. Mp 198–199 °C (AcOEt–hex-
ane). 1H NMR (CDCl3) d: 2.20 (3H, s), 7.14–7.21 (6H, m), 7.36–7.44
(12H, m), 7.54–7.77 (6H, m), 8.33 (2H, d, J = 8.4 Hz). IR (KBr): 1671,
1645, 1603, 1553, 1524, 756, 702 cmꢀ1. Anal. Calcd for C37H29N3O2:
C, 81.15; H, 5.34; N, 7.67. Found: C, 80.89; H, 5.58; N, 7.45.
4.26. 40-[1-Hydroxy-1-(1H-imidazol-4-yl)-2-methylpropyl]-N-
methyl[1,10-biphenyl]-3-sulfonamide (23)
Yield 52%. 1H NMR (CDCl3+CD3OD) d: 0.81 (3H, d, J = 6.6 Hz),
0.98 (3H, d, J = 6.6 Hz), 2.20–2.70 (4H, m), 6.98 (1H, d, J = 1.2 Hz),
7.48–7.65 (6H, m), 7.74–7.83 (2H, m), 8.04 (1H, t, J = 1.9 Hz). IR
4.33. N-[40-[1-Hydroxy-1-(1-trityl-1H-imidazol-4-yl)ethyl][1,10-
biphenyl]-3-yl]acetamide (31a)
(KBr): 3287, 2971, 1474, 1308, 1161 cmꢀ1
C
.
Anal. Calcd for
20H23N3O3Sꢁ0.5H2O: C, 60.89; H, 6.13; N, 10.65. Found: C, 61.10;
H, 6.31; N, 10.78.
Methylmagnesium bromide (1.0 M in THF, 4.4 mL, 4.38 mmol)
was added dropwise to a cooled (0 °C) solution of 30 (800 mg,
1.46 mmol) in THF (14 mL). After being stirred for 20 min at 0 °C,
the reaction was quenched with saturated NH4Cl solution. The
resulting mixture was extracted with AcOEt and the combined or-
ganic layers were washed with brine and dried over MgSO4. After
removal of the solvent in vacuo, the residue was recrystallized
from AcOEt–MeOH–hexane to give 31a (823 mg, 1.46 mmol,
quant.) as a colorless powder. Mp 157–158 °C (AcOEt–MeOH–hex-
ane). 1H NMR (CDCl3) d: 1.81 (3H, s), 2.20 (3H, s), 3.37 (1H, s), 6.79
4.27. N-{40-[1-Hydroxy-1-(1H-imidazol-4-yl)-2-
methylpropyl][1,10-biphenyl]-3-yl}-N0-methylurea (24)
Yield 61%. 1H NMR (CDCl3+CD3OD) d: 0.77 (3H, d, J = 6.6 Hz),
0.94 (3H, d, J = 6.6 Hz), 2.40–2.70 (1H, m), 2.71 (3H, s), 6.89 (1H,
s), 7.02–7.50 (9H, m). IR (KBr): 3260, 1665, 1557 cmꢀ1. Anal. Calcd
for C20H24N4O2ꢁ0.5AcOEt: C, 66.65; H, 7.12; N, 14.13. Found: C,
66.89; H, 7.00; N, 14.28.

2438
T. Kaku et al. / Bioorg. Med. Chem. 19 (2011) 2428–2442
(1H, d, J = 1.4 Hz), 7.12–7.20 (8H, m), 7.31–7.52 (16H, m), 7.65 (1H,
br s). IR (KBr): 1672, 1553, 1483, 1445, 909, 747, 733, 700 cmꢀ1
1485, 831, 791 cmꢀ1
70.11; H, 6.41; N, 12.26. Found: C, 70.43; H, 6.48; N, 12.01.
.
Anal. Calcd for C20H21N3O2ꢁ0.4H2O: C,
.
Anal. Calcd for C38H33N3O2 ꢁ 0.5AcOEt: C, 79.05; H, 6.14; N, 6.91.
Found: C, 79.13; H, 6.33; N, 6.71. Compounds 31b,c were prepared
in the same manner as described for the preparation of 31a.
4.40. N-[40-[1-Hydroxy(1H-imidazol-4-yl)methyl][1,10-
biphenyl]-3-yl]acetamide (36)
4.34. N-[40-[1-Hydroxy-1-(1-trityl-1H-imidazol-4-
Sodium borohydride (NaBH4) (172 mg, 4.55 mmol) was added
to a cooled (0 °C) solution of 35 (500 mg, 1.97 mmol) in MeOH
(40 mL) and the mixture was stirred at rt for 3 h. The mixture
was diluted with H2O and the aqueous phase was extracted with
AcOEt. The combined organic layers were dried over MgSO4 and
concentrated under reduced pressure. The residue was chromato-
graphed on silica gel (CH2Cl2/MeOH = 10:1 to 4:1) and recrystal-
lized from AcOEt–diethylether–hexane to give 36 (461 mg,
1.50 mmol, 76%) as a colorless powder. Mp 227–228 °C (AcOEt–
diethylether–hexane). 1H NMR (CDCl3+CD3OD) d: 2.17 (3H, s),
5.85 (1H, s), 6.74 (1H, s), 7.38–7.59 (8H, m), 7.74 (1H, s). IR
(KBr): 1667, 1651, 1607, 1561, 1485, 1325, 793, 775 cmꢀ1. Anal.
Calcd for C18H17N3O2ꢁ0.3H2O: C, 69.13; H, 5.67; N, 13.44. Found:
C, 69.21; H, 5.81; N, 13.21.
yl)propyl][1,10-biphenyl]-3-yl]acetamide (31b)
Yield 86%. 1H NMR (CDCl3) d: 0.86 (3H, t, J = 7.6 Hz), 2.12–2.20
(5H, m), 3.35 (1H, s), 6.77 (1H, s), 7.13–7.22 (6H, m), 7.26–7.48
(18H, m), 7.66 (1H, s). IR (KBr): 1674, 1609, 1557, 1485, 1445,
747, 733, 702 cmꢀ1. Anal. Calcd for C39H35N3O2 ꢁ 0.5AcOEt: C,
79.20; H, 6.32; N, 6.76. Found: C, 79.23; H, 6.40; N, 6.93.
4.35. N-[40-[Cyclopropyl(hydroxy)(1-trityl-1H-imidazol-4-
yl)methyl][1,10-biphenyl]-3-yl]acetamide (31c)
Yield 61%. Mp 230–231 °C (AcOEt–MeOH–hexane). 1H NMR
(CDCl3) d: 0.41–0.49 (4H, m), 1.47–1.55 (1H, m), 2.20 (3H, s), 3.26
(1H, s), 6.82 (1H, d, J = 1.4 Hz), 7.11–7.41 (19H, m), 7.50–7.53 (5H,
m), 7.65 (1H, s). IR (KBr): 1671, 1591, 1559, 1483, 1445, 731,
702 cmꢀ1. Anal. Calcd for C40H35N3O2: C, 81.47; H, 5.98; N, 7.13.
Found: C, 81.25; H, 6.01; N, 6.92. Compounds 32, 34, and 35 were
prepared in the same manner as described for the preparation of 13.
4.41. Optical resolution of 17
Optical resolution of 17 (2.00 g) was performed using preparative
HPLC on a Chiralpak AD column (Daicel Chemical Industries, 50
mmID ꢂ 500 mmL, eluent: hexane/EtOH = 50:50, flow rate: 70 mL/
min, temperature: 25 °C) to yield (ꢀ)-17 (970 mg, >99.9% ee) and
(+)-17 (920 mg, 99.9% ee), respectively. The HPLC retention time of
(ꢀ)-17 was 10.8 min and that of (+)-17 was 26.3 min under the ana-
lytical condition (Chiralpak AD column, 4.6 mmID ꢂ 250 mmL, elu-
ent: hexane/EtOH = 50:50, flow rate: 0.5 mL/min, temperature: rt).
[(ꢀ)-17: Anal. Calcd for C21H23N3O2 ꢁ 0.5H2O ꢁ 0.1AcOEt: C, 69.99;
H, 6.81; N, 11.44. Found: C, 70.22; H, 7.13; N, 11.27. (+)-17: Anal.
Calcd for C21H23N3O2 ꢁ 0.75H2O: C, 69.50; H, 6.80; N, 11.58. Found:
4.36. N-[40-[1-Hydroxy-1-(1H-imidazol-4-yl)ethyl][1,10-
biphenyl]-3-yl]acetamide (32)
Yield 59%. 1H NMR (CDCl3+CD3OD) d: 1.89 (3H, s), 2.16 (3H, s),
6.89 (1H, s), 7.27–7.52 (8H, m), 7.69 (1H, s). IR (KBr): 3031, 1672,
1609, 1591, 1559, 1483, 1397, 1312, 791 cmꢀ1. Anal. Calcd for
C
19H19N3O2ꢁ0.3H2O: C, 69.83; H, 6.05; N, 12.86. Found: C, 69.98;
H, 6.05; N, 12.61.
C, 69.68; H, 7.00; N, 11.43. ½a D20
ꢃ
= +16.5 (c 0.912, MeOH).
4.37. N-[40-[Cyclopropyl(hydroxy)-1H-imidazol-4-ylmethyl]
4.42. Optical resolution of 26
[1,10-biphenyl]-3-yl]acetamide (34)
Optical resolution of 26 (3.00 g) was performed using prepara-
tive HPLC on a Chiralpak AD column (50 mmID ꢂ 500 mmL, eluent:
hexane/EtOH = 30:70, flow rate: 70 mL/min, temperature: 25 °C) to
yield (ꢀ)-26 (1410 mg, 99.8% ee) and (+)-26 (1180 mg, >99.9% ee),
respectively. The HPLC retention time of (ꢀ)-26 was 8.6 min and
that of (+)-26 was 23.9 min under the analytical condition (Chir-
alpak AD column, 4.6 mmID ꢂ 250 mmL, eluent: hexane/
EtOH = 30:70, flow rate: 0.5 mL/min, temperature: rt). (ꢀ)-26:
Yield 39%. 1H NMR (CDCl3+CD3OD) d: 0.47–0.60 (4H, m), 1.57–
1.64 (1H, m), 2.17 (3H, s), 7.00 (1H, s), 7.36–7.58 (8H, m), 7.71
(1H, s). IR (KBr): 3148, 1667, 1591, 1555, 1485, 831, 791 cmꢀ1
.
Anal. Calcd for C21H21N3O2ꢁ0.5CHCl3ꢁ0.7H2O: C, 61.53; H, 5.50; N,
10.01. Found: C, 61.46; H, 5.67; N, 9.81.
4.38. N-[40-(1H-Imidazol-4-ylcarbonyl)[1,10-biphenyl]-3-yl]-
acetamide (35)
½
a 2D0
ꢃ
= ꢀ8.3 (c 0.362, MeOH). (+)-26: ½a D20
ꢃ
= +6.9 (c 0.376, MeOH).
Yield 88%. Mp 253–254 °C (AcOEt–MeOH–hexane). 1H NMR
(CDCl3+CD3OD) d: 2.19 (3H, s), 7.34–7.47 (2H, m), 7.47–7.59 (1H,
m), 7.72–7.84 (5H, m), 8.04 (2H, d, J = 8.4 Hz). IR (KBr): 1671, 1638,
4.43. Optical resolution of 33
Optical resolution of 33 (280 mg) was performed using prepara-
tive HPLC on a Chiralpak AD column (50 mmID ꢂ 500 mmL, eluent:
hexane/EtOH/diethylamine = 85:15:0.1, flow rate: 60 mL/min, tem-
perature: 25 °C) to yield (ꢀ)-33 (53 mg, >99.9% ee) and (+)-33
(67 mg, 99.6% ee), respectively. The HPLC retention time of (ꢀ)-33
was 43.0 min and that of (+)-33 was 59.2 min under the analytical
condition (Chiralpak AD column, 4.6 mmID ꢂ 250 mmL, eluent:
hexane/EtOH = 85:15, flow rate: 1.0 mL/min, temperature: rt). (ꢀ)-
1599, 1559, 1437, 1399, 1345, 768 cmꢀ1
.
Anal. Calcd for
18H15N3O2: C, 70.81; H, 4.95; N, 13.76. Found: C, 70.72; H, 4.70;
N, 13.58.
C
4.39. N-[40-[1-Hydroxy-1-(1H-imidazol-4-yl)propyl][1,10-
biphenyl]-3-yl]acetamide (33)
33: ½a 2D0
ꢃ
= ꢀ2.24 (c 1.03, EtOH). (+)-33: ½a D20
ꢃ
= +1.69 (c 1.06, EtOH).
Under H2 atmosphere, a mixture of 31b (1.31 g, 2.27 mmol) and
10% Pd/C (1.31 g) in 1 N HCl (2.3 mL) and EtOH (22 mL) was vigor-
ously stirred at rt for 8.5 h. The mixture was made alkaline with
NaHCO3 (210 mg) and filtered through Celite. After removal of
the solvent in vacuo, the residue was chromatographed on silica
gel (CHCl3/MeOH = 10:1 to 4:1) to give 33 (640 mg, 1.91 mmol,
84%) as a colorless amorphous solid. 1H NMR (CDCl3+CD3OD) d:
0.89 (3H, t, J = 7.6 Hz), 2.17–2.32 (5H, m), 6.91 (1H, s), 7.30–7.53
(8H, m), 7.69 (1H, s). IR (KBr): 3148, 1667, 1609, 1591, 1557,
4.44. Asymmetric synthesis of N-[40-[(1S)-1-Hydroxy-1-(1H-
imidazol-4-yl)-2-methylpropyl]-1,10-biphenyl-3-yl]acetamide
((ꢀ)-17): synthesis of 4-[(2R)-2-(tert-butyldimethylsilyloxy)-
propanoyl]morpholine (38)
Imidazole (12.26 g, 180.1 mmol) was added dropwise to a
cooled (0 °C) solution of (2R)-2-hydroxy-1-(morpholin-4-yl)

T. Kaku et al. / Bioorg. Med. Chem. 19 (2011) 2428–2442
2439
propan-1-one 37 (24.09 g, 151.3 mmol)47 in N,N-dimethylformam-
ide (DMF) (150 mL), followed by tert-butyldimethylsilyl chloride
(TBSCl) (23.06 g, 153.0 mmol) and the mixture was stirred at 0 °C
for 30 min, allowed to warm to rt and stirred for a further 16 h.
The reaction mixture was poured into water and the aqueous
phase was extracted with AcOEt. The combined organic phase
was washed with water and brine, dried over MgSO4 and evapo-
rated to give 38 (40.70 g, 98%) as a pale yellow oil. This compound
was used without purification in the next step. IR (KBr): 1651,
AcOEt and the extract was washed with brine and dried over MgSO4.
After removal of the solvent, the residue was chromatographed on
silica gel (hexane/AcOEt = 10:1 to 4:1) to give 41 (468 mg,
2.04 mmol, 87%) as a pale yellow oil. The optical purity of the ob-
tained 41 was 98.0% ee as determined by HPLC (Chiralpak AD).
4.48. (2R,3S)-3-(4-Bromophenyl)-4-methylpentane-2,3-diol (42)
Solution 41 (100 mg, 0.44 mmol; 96.3% ee) in THF (3 mL) was
added dropwise to a cooled (0 °C) solution of i-PrMgBr (0.75 M in
THF; 2.9 mL, 2.2 mmol) and the mixture was stirred at 0 °C for
4.5 h. The reaction was quenched with satd aq NH4Cl and the aque-
ous phase was extracted with AcOEt. The combined organic layers
were washed with brine, dried over MgSO4 and concentrated in va-
cuo. The residue was chromatographed on silica gel (hexane/
AcOEt = 3:1) to give 42 (70.8 mg, 0.6 mmol, 59%) with some by-
products. This compound was used without further purification
in the next step. 1H NMR (CDCl3) d: 0.82 (3H, d, J = 7.0 Hz), 0.88
(3H, d, J = 6.6 Hz), 1.01 (3H, d, J = 6.2 Hz), 1.57 (1H, s), 2.23–2.36
(1H, m), 2.41 (1H, s), 4.25 (1H, dt, J = 6.2, 6.2 Hz), 7.22–7.27 (2H,
m), 7.44–7.51 (2H, m). IR (KBr): 2975, 1489, 1393, 1076, 1009,
1119, 831 cmꢀ1 1H NMR (CDCl3) d: 0.11 (6H, s), 0.91 (9H, s), 1.41
.
(3H, d, J = 6.6 Hz), 3.60–4.00 (8H, m), 4.57 (1H, q, J = 6.6 Hz).
4.45. (2R)-1-(4-Bromophenyl)-2-[[tert-butyl(dimethyl)silyl]oxy]-
propan-1-one (39)
n-BuLi (1.6 M; 62.9 mL, 101 mmol) was added to a cooled
(ꢀ78 °C) solution of 1,4-dibromobenzene (25.6 g, 109 mmol) in
THF (350 mL) and the mixture was stirred at ꢀ78 °C for 20 min. A
solution of 38 (22.0 g, 80.5 mmol) in THF (50 mL) was added to the
mixture and the solution was stirred at ꢀ78 °C for 45 min. The reac-
tion was quenched with aq NH4Cl and the mixture was extracted
with AcOEt. The combined organic layers were washed with brine
and dried over MgSO4. After removal of the solvent in vacuo, the res-
idue was chromatographed on silica gel (hexane/AcOEt = 40:1) to
give 39 (25.3 g, 73.7 mmol, 92%) as a pale yellow oil. 1H NMR (CDCl3)
d: 0.02 (3H, s), 0.07 (3H, s), 0.87 (9H, s), 1.50 (3H, d, J = 7.0 Hz), 4.84
(1H, q, J = 7.0 Hz), 7.56–7.61 (2H, m), 7.94–8.00 (2H, m). IR (KBr):
986, 882, 818 cmꢀ1
.
4.49. (3S)-3-(4-Bromophenyl)-3-hydroxy-4-methylpentane-2-
one (43)
Dimethyl sulfoxide (DMSO) (10.9 mL, 154 mmol) was added
dropwise to a cooled (–70 °C) solution of oxalyl chloride
1684, 1586, 1256, 1146, 1071, 920, 835, 777 cmꢀ1
.
(6.71 mL, 38.4 mmol) in CH2Cl2 (150 mL). After stirring for
10 min, a solution of 41 (10.5 g, 38.4 mmol) in CH2Cl2 (100 mL)
was added dropwise, and the reaction mixture was stirred at a
temperature of ꢀ50 to ꢀ40 °C for 30 min. The mixture was then
cooled to ꢀ70 °C, and triethylamine (53.5 mL, 384 mmol) was
added dropwise, and the solution was allowed to warm to rt. The
resulting mixture was diluted with satd aq NH4Cl solution and ex-
tracted with CH2Cl2. The combined organic layers were washed
with brine and dried over MgSO4. After removal of the solvent in
vacuo, the residue was chromatographed on silica gel (hexane/
AcOEt = 10:1) to give 43 (8.72 g, 32.2 mmol, 84%) as a pale yellow
amorphous solid. 1H NMR (CDCl3) d: 0.88 (6H, d, J = 6.6 Hz), 2.14
(3H, s), 2.69–2.82 (1H, m), 4.44 (1H, s), 7.40–7.45 (2H, m), 7.47–
7.53 (2H, m). IR (KBr): 2971, 1709, 1487, 1358, 1169, 1142, 1009,
816 cmꢀ1. Anal. Calcd for C12H15O2Br: C, 53.15; H, 5.58; N, 0.00;
Br, 29.47. Found: C, 52.94; H, 5.59; N, 0.07.
4.46. (2R)-1-(4-Bromophenyl)-2-hydroxypropan-1-one (41)
Tetrabutylammonium fluoride (21.5 g, 21.5 mmol) was added
to a cooled (0 °C) solution of 39 (25.7 g, 74.9 mmol) in THF
(250 mL) and the mixture was stirred at 0 °C for 15 min. The mix-
ture was diluted with satd aq NH4Cl solution and the aqueous
phase was extracted with diisopropylether. The combined organic
layers were washed with brine and dried over MgSO4. After re-
moval of the solvent in vacuo, the residue was chromatographed
on silica gel (hexane/AcOEt = 4:1) to give 41 (14.1 g, 61.6 mmol,
82%) as a pale yellow oil. The optical purity of the obtained 41
was 96.3% ee as determined by HPLC using a Chiralpak AD column
with hexane/EtOH = 85:15 as an eluent. 1H NMR (CDCl3) d: 1.44
(3H, d, J = 7.0 Hz), 3.70 (1H, d, J = 6.6 Hz), 5.05–5.18 (1H, m),
7.64–7.69 (2H, m), 7.77–7.82 (2H, m). IR (KBr): 1717, 1485, 1402,
1358, 1175, 1073, 1011, 824 cmꢀ1
.
4.50. (3S)-1-Bromo-3-(4-bromophenyl)-3-hydroxy-4-
4.47. Alternative method for (2R)-1-(4-bromophenyl)-2-
methylpentane-2-one (44)
hydroxypropan-1-one (41); asymmetric oxidation with AD-mix-
b™
Pyridinium bromide perbromide (11.2 g, 34.9 mmol) was added
to a stirred solution of 43 (7.89 g, 29.1 mmol) in THF (150 mL) and
the mixture was stirred at rt for 12 h. The reaction mixture was di-
luted with aqueous sodium thiosulfate solution and the aqueous
phase was extracted with AcOEt. The combined organic layers were
washed with brine and dried over MgSO4. After removal of the sol-
vent in vacuo, the residue was chromatographed on silica gel (hex-
ane/AcOEt = 10:1) to give 44 (6.58 g, 18.8 mmol, 65%) as a pale
yellow powder. 1H NMR (CDCl3) d: 0.78 (3H, d, J = 7.0 Hz), 0.95
(3H, d, J = 7.0 Hz), 2.72–2.85 (1H, m), 4.11 (1H, d, J = 14.2 Hz), 4.19
(1H, d, J = 14.2 Hz), 7.37–7.44 (2H, m), 7.48–7.55 (2H, m). IR (KBr):
40%sodiumbis(trimethylsilyl)amidesolution(NaHMDS)inTHF–
cumene (2.46 g, 4.70 mmol) at ꢀ70 °C was added to a solution of 1-
(4-bromophenyl)propan-1-one 40 (500 mg, 2.35 mmol) in THF
(10 mL) and stirred for 30 min. A solution of TBSCl (429 mg,
2.84 mmol) in hexane (2 mL) was added to the mixture and stirred
at ꢀ70 °C for 30 min, allowed to warm to rt, and stirred for an addi-
tional 100 min. The mixture was diluted with water and the aqueous
phase was extracted with AcOEt. The organic layers were washed
with brine, dried over MgSO4, and concentrated under reduced pres-
sure. The remaining residue was dissolved in tert-butanol (tert-
BuOH) (15 mL) and H2O (12 mL) and the mixture was cooled to
5 °C, followed by the addition of methanesulfonamide (226 mg,
2.37 mmol) and AD-mix-b (3.32 g, 2.37 mmol). After being stirred
at 5 °C for 22 h and at rt for a further 15 h, sodium sulfite (2.35 g,
18.6 mmol) was added. The mixture was stirred for an additional
2 h and diluted with water. The aqueous phase was extracted with
2969, 1726, 1487, 1076, 1026, 1009, 814 cmꢀ1
.
4.51. (1S)-1-(4-Bromophenyl)-1-(1H-imidazol-4-yl)-2-
methylpropan-1-ol (45)
Trimethylsilyl trifluoromethanesulfonate (TMSOTf) (8.01 mL,
44.3 mmol) was added dropwise to a cooled (0 °C) solution of 44

2440
T. Kaku et al. / Bioorg. Med. Chem. 19 (2011) 2428–2442
(6.20 g, 17.7 mmol) and 2,6-lutidine (6.18 mL, 53.1 mmol) in anhy-
drous THF (50 mL), and the reaction mixture was stirred at 0 °C for
30 min and allowed to warm to rt. After stirring for a further
90 min, the mixture was diluted with 1 N HCl followed by extrac-
tion with AcOEt. The combined organic layers were washed with
satd NaHCO3 solution followed by brine, before drying over MgSO4.
The solution was concentrated in vacuo to give crude (3S)-1-bro-
mo-3-(4-bromophenyl)-4-methyl-3-[(trimethylsilyl)oxy]pentane-
2-one as a colorless amorphous solid. This material, without
further purification, was mixed with THF (6 mL) and formamidine
acetate (2.95 g, 28.3 mmol) and added to a saturated ammonia
solution in MeOH (30 mL); the resulting mixture was stirred at rt
for 22 h. After removing excess NH3 and MeOH, the residue was
chromatographed on silica gel (CH2Cl2/MeOH = 50:1 to 10:1) to
give 45 (3.89 g, 13.2 mmol, 74%) as a pale yellow amorphous solid.
The optical purity of the obtained 45 was 90.0% ee as determined
by HPLC (Chiralpak AD). 1H NMR (CDCl3) d: 0.78 (3H, d,
J = 6.6 Hz), 0.95 (3H, d, J = 7.0 Hz), 2.50–7.64 (1H, m), 6.95 (1H, d,
J = 1.2 Hz), 7.43 (4H, s), 7.55 (1H, d, J = 1.2 Hz). IR (KBr): 2971,
(c 1.10, MeOH). FAB-MS m/z: 350.1834 (calcd for
21H24N3O2:350.1869).
C
4.55. Inhibition of rat 17,20-lyase activity in vitro
Inhibition of rat enzymes was determined according to a previ-
ously described method,51 with some modifications. Testes excised
from 13-week-old male Sprague–Dawley (SD) rats were homoge-
nized, and testicular microsomes were prepared by a series of cen-
trifugation. The reaction mixture contained 75 mM phosphate
buffer (pH 7.4), 7
hydroxyprogesterone (NEN), 5 mM NADPH (Oriental Yeast), and
1–1000 nM test compound in a total volume of 20 L. The concen-
l a-
g microsome protein, 10 nM [1,2-3H]-17
l
tration of reagents was expressed as the final concentration in the
reaction mixture. The test compounds were serially diluted with
dimethylformamide, and then diluted fivefold with distilled water
before 5
terminated by the addition of 40
incubation at 37 °C, then vortexing for 30 s and brief centrifuga-
tion; 30 L of the organic phases were applied to silica gel TLC
lL were added to the reaction mixture. The reaction was
lL of ethyl acetate after 15 min
1485, 1395, 1009, 814, 733 cmꢀ1
.
l
plates (Whatman, LHPK). The substrate and the products
androstenedione and testosterone were separated using the
toluene–acetone (7:2) solvent system. Detection of the spots and
measurement of the radioactivity as PSL were performed using a
BAS2000 Bio-image analyzer (FUJIX). The concentrations of the test
compounds necessary to reduce the concentration of the products
by 50% (IC50) were calculated.
4.52. (1S)-1-(4-Bromophenyl)-2-methyl-1-(1-trityl-1H-
imidazol-4-yl)propan-1-ol (46)
Trityl chloride (1.00 g, 3.59 mmol) was added to a cooled (0 °C)
mixture of 45 (1.01 g, 3.42 mmol, 90.0% ee) and triethylamine
(0.715 mL, 5.13 mmol) in DMF (10 mL), and the reaction mixture
was stirred at rt for 2.5 h. After being diluted with H2O, the mixture
was extracted with AcOEt. The combined organic layers were
washed with H2O and brine successively, followed by drying over
MgSO4. The solution was concentrated in vacuo and the obtained
residue was recrystallized from hexane–AcOEt to give 46 (1.16 g,
2.16 mmol, 63%) as a colorless powder. The optical purity of the ob-
tained 46 was 98.8% ee as determined by HPLC (Chiralpak AD).
Recrystallization of 46 with 98.8% ee from hexane–AcOEt provided
needle crystals with 99.5% ee for X-ray crystallographic analysis.
The spectral data of 46 were identical to those of the racemate
10b except for elemental analysis and optical rotation. Anal. Calcd
for C32H29N2OBr: C, 71.51; H, 5.44; N, 5.21; Br, 14.87. Found: C,
4.56. Inhibition of human 17,20-lyase activity in vitro
Inhibition of human enzymes was determined as described
above for rat enzymes. The reaction mixture contained 75 mM
phosphate buffer (pH 7.4), 1 mM magnesium chloride, 0.5 pmol
of recombinant P450c17 (Biotechnology Laboratories, Takeda
Pharmaceutical Company Limited), 0.5 pmol recombinant cyto-
chrome b5 (Pan Vera), 20.8 ng recombinant NADPH-cytochrome
P450 reductase (Pan Vera), 10 nM [1,2-3H]-17
lone (Amersham), 5 mM NADPH (Oriental Yeast), and 1–1000 nM
test compounds in a total volume of 20 L. Dilution of test com-
pounds and termination of the reaction were performed as de-
scribed above for rat 17,20-lyase assays. Organic phases (30 L)
a-hydroxypregneno-
l
71.45; H, 5.66; N, 5.02. ½a D20
ꢃ
= +34.6 (c 1.00, MeOH).
l
were applied to silica gel thin-layer chromatography plates (What-
man, LHPK). The substrate and the product, DHEA, were separated
using the cyclohexane–ethyl acetate (3:2) solvent system.
4.53. N-[40-[(1S)-1-Hydroxy-2-methyl-1-(1-trityl-1H-imidazol-4-
yl)-propyl]-1,10-biphenyl-3-yl]acetamide (47)
Compound 47 was prepared from 46 (300 mg, 0.56 mmol, 98.8%
ee) and 3-acetamidephenylboronic acid (130 mg) and Pd(PPh3)4
(0) (32 mg, 0.028 mmol) as a colorless amorphous solid (288 mg,
0.49 mmol, 87%) using the same method described for the prepara-
tion of 12a. The optical purity of 47 was 99.1% ee as determined by
HPLC (Chiralpak AD). The spectral data of 47 were identical to
those of the racemate 12e except for optical rotation.
4.57. Inhibition of CYP3A4 activity in vitro
The reaction mixture contained 50 mM phosphate buffer (pH
7.4), 10 pmol/mL recombinant CYP3A4 (Gentest), 100 lM testos-
terone, NADPH regenerating system (0.5 mM NADP (Oriental
Yeast)), 5 mM glucose-6-phosphate (Oriental Yeast), 1 mM MgCl2,
1.5 unit/mL G-6-P dehydrogenase (Oriental Yeast)), and 1 or
½
a 2D0
= +46.6 (c 0.49, MeOH).
ꢃ
10 lM test compound in a total volume of 200 lL. The total micro-
some protein content was adjusted by control microsome protein
(Gentest). The reaction mixture was incubated for 30 min at
4.54. N-[40-[(1S)-1-Hydroxy-1-(1H-imidazol-4-yl)-2-
methylpropyl]-1,10-biphenyl-3-yl]acetamide (S-(ꢀ)-17)
37 °C and terminated by adding 200
lL of acetonitrile. After addi-
tion of 400 L water, the reaction mixture was centrifuged at
l
A solution of 47 (86.3 mg, 0.143 mmol) and 10% Pd/C (85 mg) in
EtOH (10 mL) was vigorously stirred at 70 °C for 9 h under H2
(4 atm) atmosphere. The mixture was filtered, and the filtrate was
concentrated under reduced pressure. The residue was chromato-
graphed on silica gel (CH2Cl2/MeOH = 10:1) to give (ꢀ)-17
(30.7 mg, 0.088 mmol, 61%) as a colorless amorphous solid. The opti-
cal purity of the obtained (ꢀ)-17 was 99.0% ee as determined by
HPLC (Chiralpak AD). The spectral data of (ꢀ)-17 were identical to
14,000 rpm for 10 min. The 6-hydroxytestosterone contents in
the supernatants were determined using a HPLC system (Shimadzu
LC-10, column: Inertsil ODS-3 [4.6 ꢂ 150 mm] GL Sciences).
4.58. Homology modeling
Multiple alignment using the amino acid sequences of human
CYP enzymes was performed using ClustalW and revised by hand.
According to the alignment, side chains of the CYP2C5 crystal
those of the racemate 17 except for optical rotation. ½a D20
= ꢀ15.7
ꢃ

T. Kaku et al. / Bioorg. Med. Chem. 19 (2011) 2428–2442
2441
structure obtained from the Protein Data Bank (PDB code 1DT6)
were replaced by the corresponding residues of CYP17 using the
homology module of Insight II (v2000, Accelrys Inc.). Using the
Search-Loop function of Insight II, conformations of the insertions
and deletions in the alignment were created. After some manual
adjustments to remove large steric hindrances, the whole structure
was subjected to energy minimization for 1000 steps with steepest
descent minimization and, subsequently, 5000 steps with conju-
gate gradient minimization, using the Discover-ESFF force field
(v98.0, Accelrys Inc.). During the minimization procedure, the
dielectric constant was set to 4ꢄr, where r is the distance between
two interacting atoms, and the force constant of tethering con-
straints for the backbone of structurally conserved regions and
heme was set to 40 kcal/Å2 to prevent large movements from the
initial positions.
crystal X-ray analysis, Ms H. Shinohara and Mr T. Masaki for tech-
nical support, and Dr F. W. Dahlquist (University of Oregon) for
kindly providing vector pCWori. This work was funded by
Millennium Pharmaceuticals, Inc. The authors would also like to
acknowledge FireKite for editorial assistance in the development
of this manuscript, which was supported by funding from
Millennium Pharmaceuticals, Inc.
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A single crystal (0.50 ꢂ 0.20 ꢂ 0.16 mm) of 46 was obtained by
recrystallization from AcOEt. The reflection data were collected
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C32H29BrN2O;
M = 537.50; orthorhombic; space group P212121 (#19); cell con-
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experiments. All procedures were performed according to proto-
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