Design, synthesis, and biological evaluation of 3-aryl-3-hydroxy-1- phenylpyrrolidine derivatives as novel androgen receptor antagonists

Article abstract of DOI:10.1016/j.bmc.2012.11.001

We designed and synthesized a series of 3-aryl-3-hydroxy-1- phenylpyrrolidine derivatives D and evaluated their potential as novel androgen receptor (AR) antagonists therapeutically effective against castration-resistant prostate cancer (CRPC). Introducti

Full text of DOI:10.1016/j.bmc.2012.11.001

Bioorganic & Medicinal Chemistry 21 (2013) 70–83
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis, and biological evaluation of
3-aryl-3-hydroxy-1-phenylpyrrolidine derivatives as novel
androgen receptor antagonists
⇑
Satoshi Yamamoto , Hiromi Kobayashi, Tomohiro Kaku, Katsuji Aikawa, Takahito Hara, Masuo Yamaoka,
Naoyuki Kanzaki, Atsushi Hasuoka, Atsuo Baba, Mitsuhiro Ito
Pharmaceutical Research Division, Takeda Pharmaceutical Company Ltd, 26-1, Muraokahigashi 2-chome, Fujisawa, Kanagawa 251-8555, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
We designed and synthesized a series of 3-aryl-3-hydroxy-1-phenylpyrrolidine derivatives D and evalu-
ated their potential as novel androgen receptor (AR) antagonists therapeutically effective against castra-
tion-resistant prostate cancer (CRPC). Introduction of a methyl group at the 2-position (R2) of the
pyrrolidine ring increased the AR binding affinity. The (2S,3R) configuration of the pyrrolidine ring was
favorable for the AR antagonistic activity. It was found that introduction of an amide substituent (R1)
and a pyridin-3-yl group (Q) was effective for reducing the AR agonistic activity which appeared during
the optimization of lead compound 6. Compound 54 showed potent antitumor effects against a CRPC
model of LNCaP-hr cell line in a mouse xenograft, in which bicalutamide exhibited only partial suppres-
sion of tumor growth. Thus, the pyrrolidine derivatives such as 54 are novel AR antagonists, and their
properties having efficacy against CRPC are distinct from those of a representative first-generation antag-
onist, bicalutamide.
Received 26 September 2012
Revised 30 October 2012
Accepted 1 November 2012
Available online 12 November 2012
Keywords:
Androgen receptor antagonists
Castration-resistant prostate cancers
3-Aryl-3-hydroxy-1-phenylpyrrolidine
derivatives
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
demonstrated the clinical evidence of AR signaling to be an impor-
tant target in CRPC.21 Taking into account the numerous unmet
Growth of prostate cancer (PC) is primarily stimulated by
androgen. Androgen receptor (AR) antagonists, which block andro-
gen signal, have been used for the treatment of PC. Cyproterone
acetate,1 flutamide,2,3 nilutamide,4,5 and bicalutamide6–15 are
known as AR antagonists used for clinical purposes (Fig. 1). A com-
bination use of AR antagonists such as bicalutamide and surgical or
chemical castration using gonadotropin-releasing hormone ana-
logues, referred to as ‘combined androgen blockade (CAB) therapy,’
has significant effects against PC. However, prolonged use of an AR
antagonist results in development of castration-resistant prostate
cancer (CRPC).16–18 In the cases, the AR antagonists such as bicalu-
tamide become partial agonists,17 and discontinuation of CAB ther-
medical needs of CRPC, development of novel AR antagonists is
an important approach for the treatment of CRPC. Recently, an
AR antagonist enzalutamide (MDV-3100) that has been reported
to be effective against CRPC21,23,24 has been approved in the US
(Fig. 1). Because of its unique pharmaceutical properties, this agent
is categorized as a ‘second-generation’ AR antagonist in contrast to
‘first-generation’ antagonists such as bicalutamide or flutamide.
We reported novel 1-arylmethyl-4-phenylpyrrole compounds A
(represented by compound 1) and 1-arylmethyl-4-phenylpyrazole
and 1-aryloxy-4-phenylpyrazole compounds B (represented by
compounds 2 and 3) as orally available AR antagonists effective
against PCs, including CRPC (Fig. 2).25,26 Our structure–activity
relationship (SAR) studies for these compounds show that the cya-
nophenyl group a and the arylmethyl/aryloxy moiety b are impor-
tant contributors to the strong AR antagonistic activity.25,26 Some
studies on other AR antagonists having a cyanophenyl group and
a second aryl ring were also reported.27–32
apy or change to another AR antagonist should be considered.19
A
microtubule-stabilizing agent, docetaxel,20 and an androgen bio-
synthesis inhibitor, abiraterone acetate,21,22 are currently approved
for the treatment of CRPC. A phase 3 study of abiraterone acetate
Taking into account the importance of regions a and b, we
investigated a new series of compounds as novel AR antagonists
represented as scaffold C by modifying the pyrrole/pyrazole central
ring of compounds A and B (Fig. 2). Scaffold C consists of a p-cya-
nophenyl group, a central ring Z, a linker L, and a terminal ring M.
Compound 4, which possessed 3-hydroxypyrrolidine moiety, was
Abbreviations: PC, prostate cancer; AR, androgen receptor; CRPC, castration-
resistant prostate cancer; CAB, combined androgen blockade; PSA, prostate-specific
antigen; DHT, dihydrotestosterone; SAR, structure–activity relationship; EDC, N-[3-
(dimethylamino)propyl]-N0-ethylcarbodiimide hydrochloride.
⇑
Corresponding author. Tel: +81 466 32 1219; fax: +81 466 29 4455.
E-mail address: satoshi.yamamoto@takeda.com (S. Yamamoto).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.11.001

S. Yamamoto et al. / Bioorg. Med. Chem. 21 (2013) 70–83
71
Figure 1. Chemical structure of known androgen receptor (AR) antagonists.
also found to show high AR binding affinity. Thus, an aliphatic ring
system possessing a hydroxyl group, such as 3-hydroxypyrrolidine,
was employed for the central ring Z. It was anticipated that the Z
ring with a hydroxyl moiety would contribute to strong binding
affinity to the AR. We set out to explore novel AR antagonists with
scaffold C and found that 3-benzyl-3-hydroxypyrrolidine deriva-
tive 5 and 3-hydroxy-3-phenylpyrrolidine 6 possessed AR antago-
nistic activity. In addition to the antagonistic activity, compound 5
exhibited AR agonistic activity. On the other hand, compound 6
showed AR antagonistic activity without any significant agonistic
activity. This AR activity profile was deemed appropriate for effi-
cacy against CRPC. Thus, we selected compound 6 as a lead for
new series of AR antagonists and studied terminal ring Q and sub-
stituents R1–R3 on Q, central pyrrolidine, and p-cyanophenyl group
(compound D) to obtain compounds with good efficacy and phar-
macokinetic (PK) properties.
Figure 2. Design of 3-aryl-1-(4-cyanophenyl)-3-hydroxypyrrolidine AR antagonists.

72
S. Yamamoto et al. / Bioorg. Med. Chem. 21 (2013) 70–83
Scheme 1. Synthesis of Compounds 4–6, 25–32, and 37. Reagents and conditions: (a) (1) sodium bis(2-methoxyethoxy)aluminium dihydride, THF, 70 °C, (2) 2-chloro-4-
fluorobenzonitrile (10 and 13), 4-fluoro-2-methoxybenzonitrile (11), or 4-fluorobenzonitrile (12), Li2CO3, DMSO, 100 °C, 61–74% for two steps; (b) 2-chloro-4-
fluorobenzonitrile, Li2CO3, DMSO, 100 °C, 67%; (c) pyridine SO3 complex, Et3N, DMSO, room temp, 16–97%; (d) (COCl)2, DMSO, Et3N, CH2Cl2, ꢀ78 °C to 0 °C, 73%; (e) 1,4-
diiodobenzene (19) or 2,5-dibromopyridine (21–24 and 35), nBuLi, Et2O, THF, ꢀ78 °C, 36–59%; (f) Grignard reagent, THF, 5 °C to room temp, 9–29%; (g) 2,5-dibromopyridine,
nBuLi, toluene, ꢀ78 °C, 57%; (h) CO, Pd(OAc)2, 1,10-bis(diphenylphosphino)ferrocene, Et3N, DMF, EtOH, 70 °C, 53–98% (29–32) and 6% (27, for two steps); (i) (1) sodium bis(2-
methoxyethoxy)aluminium dihydride, THF, 70 °C, (2) BnOCOCl, Et3N, THF, room temp, 79% for two steps; (j) 4-methylmorpholine N-oxide, tetrapropylammonium
perruthenate, MS-4A, MeCN, room temp, 32%; (k) CO, [1,10-bis(diphenylphosphino)ferrocene]palladium(II) dichloride dichloromethane adduct, Et3N, MeOH, 60 °C, 86% (28)
and 66% (36); (l) (1) H2, Pd/C, MeOH, room temp, (2) 2,4-difluorobenzonitrile, Li2CO3, DMSO, 100 °C, 8% for two steps.
with 4-fluorobenzonitriles or benzyloxycarbonyl chloride resulted
in 1-substituted pyrrolidines 10–13 or 33. Oxidation of 4, 10–13
and 33 gave pyrrolidin-3-ones 14–18 and 34. Regioselective lithia-
tion of 2,5-dibromopyridine at the 5-position in diethyl ether34 and
subsequent coupling with 15–18 and 34 afforded 3-hydroxy-3-
(pyridin-5-yl)pyrrolidines 21–24 and 35, respectively. On the other
hand, lithiation of 2,5-dibromopyridine in toluene generated 5-
bromo-2-lithiopyridine,34 which was coupled with 15 to yield
2. Chemistry
1-(4-Cyanophenyl)-3-hydroxypyrrolidine
4 and 1-(4-cyano-
phenyl)-3-aryl (or benzyl) pyrrolidines 5, 6, 25–32, and 37 were
prepared as shown in Scheme 1. Coupling of 2-chloro-4-fluoroben-
zonitrile and pyrrolidine 9 afforded the 3-hydroxypyrrolidine 4.
Reduction of chiral pyrrolidones 7 and 833 by sodium bis(2-
methoxyethoxy)aluminium dihydride followed by the reaction

S. Yamamoto et al. / Bioorg. Med. Chem. 21 (2013) 70–83
73
Scheme 2. Synthesis of Compounds 45–54. Reagents and conditions: (a) LiOH, EtOH, THF, H2O, room temp to 50 °C, 45%; (b) NaOH, EtOH, THF, H2O, room temp, 60%–quant.;
(c) EDC, HOBt ammonium salt, DMF, room temp, 61%; (d) amines (R1aR1bH), EDC, HOBt, DMF, THF, room temp, 21–98%; (e) pyrrolidin-2-one, N,N0-dimethylethylenediamine,
K3PO4, CuI, toluene, 80 °C, 75%.
3-hydroxy-3-(pyridin-2-yl)pyrrolidine 20. Compounds 14 and 15
were reacted with Grignard reagents or lithiobenzene to afford
3-hydroxypyrrolidines 5, 6, 19, 25, and 26. The reactions to obtain
19–24, 26, and 35 from chiral 2-methylpyrrolidin-3-ones 15–18
and 34 were stereoselective (the detail of the stereochemistry is
discussed below). Esters 27–32 and 36 were obtained by palla-
dium-catalyzed carbonization reactions of aryl bromide/iodide
19–24 and 35 under CO atmosphere in corresponding alcohol,
respectively. Compound 37 was synthesized by deprotection of
the Cbz group of 36 by hydrogenation reaction in the presence of
Pd/C followed by reaction with 2,4-difluorobenzonitrile under ba-
sic condition.
Figure 4. Plausible mechanism of reaction of 15 with 2-bromo-5-lithiopyridine.
Scheme 2 illustrates the synthesis of carboxamides 45–50 and
52–54 as well as pyrrolidone derivative 51. Basic hydrolysis of es-
ters 27–32 and 37 gave corresponding carboxylic acids 38–44.
Condensation of carboxylic acids 38–44 with amines was per-
formed using N-[3-(dimethylamino)propyl]-N0-ethylcarbodiimide
hydrochloride (EDC) to give amides 45–50 and 52–54. Copper-
catalyzed coupling reaction of bromopyridine 21 with pyrrolidin-
2-one in the presence of N,N0-dimethylethylenediamine and
35
K3PO4 afforded pyrrolidone derivative 51.
We determined the absolute stereochemical configuration of 51
by X-ray crystallographic analysis (Fig. 3). The configuration of the
substituents on the 2- and 3-positions of the pyrrolidine ring of 51
proved to be (2S,3R). This result suggested that bromopyridine 21
has (2S,3R) configuration, which is the same as the stereochemical
Figure 3. ORTEP drawing of 51. Ellipsoids are drawn at the 20% probability level.

74
S. Yamamoto et al. / Bioorg. Med. Chem. 21 (2013) 70–83
against normal and flutamide-resistant PCs. In addition, to evaluate
the potential of the compounds against CRPC, we used LNCaP-hr
cell line as a model of CRPC.26,36 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 continuous growth in an androgen-depleted medium.
LNCaP-hr cells produce high levels of prostate specific antigen
(PSA) in vitro in the absence of any androgens, indicating the con-
stitutive activity of AR. We measured the amount of PSA secreted
by LNCaP-hr cells after the addition of the test compound (Table 2).
The solubility of the compounds was analyzed in pH 6.8 solution.
In these studies, we sought to discover novel AR antagonists that
primarily possess strong binding affinity and antagonistic activity
without significant agonistic activity.
The SAR trends from the initial synthetic study of compounds D
are shown in Table 1. Introduction of a methyl group with S-config-
uration at the 2-position (R2) of the pyrrolidine ring (3-hydroxyl
group was cis to the 2-methyl) increased the AR binding affinity,
although AR agonistic activity was also observed against T877A
mutant-type AR (25 vs 26). Next, reduction of the AR agonistic
activity of 26 was investigated with a focus on folding of helix 12
in the AR ligand-binding domain (LBD)37–40 on the basis of the
SAR information on pyrazole compounds.26 Because helix 12 fold-
ing is an important factor for the expression of AR agonistic activ-
ity, inhibition of the folding was supposed to be one of the effective
approaches for reduction of the agonistic activity. We hypothe-
sized that terminal moiety Q–R1 in compound D favors inhibition
of helix 12 folding, and anticipated that increasing bulkiness of
the moiety by replacing the fluoro group in compound 26 with lar-
ger R1 substituents (compound E) could result in steric repulsion to
reduce the AR agonistic activity (Fig. 6). The SAR study on pyrazole
compounds revealed that introduction of amide substituents cor-
responding to R1 removed the AR agonistic activity.26 Thus, we
set out to prepare compounds possessing amide substituents at
R1 (F in Fig. 6). Although the agonistic activity was notably dimin-
ished by replacement with amide substituents, unlike that in
pyrazole compounds, residual agonistic activity was still observed
Figure 5. NOE correlation of 54.
configuration of 51. The plausible mechanism for the stereoselec-
tive formation of 21 is shown in Figure 4: 2-bromo-5-lithiopyri-
dine can attack pyrrolidin-3-one 15 only from the opposite site
of 2S-methyl group of 15 because of the steric repulsion with the
methyl group.
The relative stereochemical configuration of the substituents on
the 2- and 3-positions of the pyrrolidine ring of 54 was determined
by nuclear magnetic resonance (NMR) analysis (data not shown).
Significant nuclear Overhauser effect (NOE) correlations were ob-
served between H-14 and H-2, H-4b as well as H-19 and H-4
a
(Fig. 5). These observations showed that the methyl group on the
2-position and the pyridine ring on the 3-position of 54 are in trans
configuration. On the basis of the results of X-ray crystallographic
analysis of 51, the reaction of pyrrolidin-3-one 34 with 2-bromo-5-
lithiopyridine was also expected to give (2S,3R) isomer 35. Thus,
compound 54 becomes (2S,3R) isomer having 2-methyl and 3-pyr-
idyl groups with trans configuration.
3. Results and discussion
The binding affinity and antagonistic/agonistic activity of the
synthesized compounds to the ARs were measured by the AR bind-
ing inhibition test and the AR reporter gene assay, respectively
(Tables 1 and 2). In these assays, we used the wild-type AR and a
T877A mutant-type AR to estimate the efficacy of our compounds
(45 and 46, EC50 = 10
lM). Interestingly, replacement of the
Table 1
Binding inhibitory and antagonistic activities of 3-aryl-3-hydroxy-1-phenylpyrrolidine derivatives
R1
HO
X1
X2
R2
3
N
Cl
NC
Binding
Reporter
Antagonist
Agonist
a
b
b
b
Compd.
R1
X1
X2
R2
C-3
IC50
(l
M)
IC50
(l
M)
EC50
Wild
(l
M)
Wild
T877A
Wild
0.38
0.068
0.017
0.017
0.0086
0.12
T877A
T877A
6
25
26
45
46
47
48
49
H
F
F
CH
CH
CH
CH
CH
CH
N
CH
CH
CH
CH
CH
N
CH
CH
CH
H
H
RS
RS
R
R
R
R
R
R
S
0.37
0.18
0.13
0.25
0.065
>10
0.009
0.01
0.10
0.099
10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
0.0062
>10
>10
0.02
10
10
5.1
>10
>10
>10
>10
0.015
0.036
0.0081
0.042
0.018
0.016
0.10
3.0
0.097
0.12
S-Me
S-Me
S-Me
S-Me
S-Me
S-Me
R-Me
0.008
0.027
0.032
0.068
0.044
1.1
CONH2
CONHMe
CONHMe
CONHMe
CONMe2
CONHMe
0.025
1.7
N
N
50
1.6
6.4
0.33
>10
1.6
0.47
>10
Bicalutamide
Dihydrotestosterone (DHT)
0.054
0.0021
0.0026
a
Absolute configuration at 3-position of pyrrolidine.
IC50 and EC50 values shown are the mean values of duplicate measurements.
b

S. Yamamoto et al. / Bioorg. Med. Chem. 21 (2013) 70–83
75
Table 2
In vitro activities of compounds 48 and 51–54
R1
HO
S
R
N
N
R3
NC
Binding
Reportera
LNCaP-hr
Antagonist
PSA secretion
% Of controlc
R1
R3
IC50
(l
M)
IC50
(l
M)
b
b
Compd
Wild
T877A
0.10
Wild
T877A
0.099
1
l
M
10 l
Md
48
51
CONHMe
O
Cl
Cl
0.044
0.025
39
101
0.013
0.087
0.011
0.16
17
35
N
52
53
54
CONHMe
CONHMe
CONHMe
OMe
H
F
0.12
7.6
0.83
0.24
5.3
1.7
0.062
0.40
0.29
0.73
1.4
1.7
87
95
41
NTe
42
35
a
b
c
Agonistic activities of 48 and 51–54 were EC50 >10 lM for wild-type or T877A mutant-type AR.
IC50 and EC50 values shown are the mean values of duplicate measurements.
n = 3.
d
e
Bicalutamide exhibited more than 100% secretion of control at 10
Not tested.
lM.
Figure 6. Introduction of an amide group to inhibit folding of helix 12.
benzene ring of 46 (X1 = X2 = CH) with a pyridine (X1 = N, X2 = CH)
reduced the observed agonistic activity (48, EC50 >10 M). On the
other hand, nicotinamide derivative 47 (X1 = CH, X2 = N) showed
(EC50 >10 lM). On the other hand, methoxy group diminished inhi-
l
bition of PSA secretion (52). Removal of the chloro group (53,
R3 = H) also decreased PSA secretion inhibition. Conversion of the
amide group at R1 with pyrrolidin-2-one moiety resulted in com-
pound 51, which failed to show concentration-dependent inhibi-
tory activity. Because bicalutamide exhibited no inhibition
against LNCaP-hr PSA secretion (more than 100% secretion of con-
weak agonistic activity (EC50 = 5.1 lM). Thus, we found that the
nitrogen atom at X1 in compounds F was sensitive to determine
the balance between agonistic and/or antagonistic activity. It was
also found that N-methylamide group at R1 could improve the sol-
ubility (1.7, 7.0, 42, and 45
l
g/mL in pH 6.8 solution for 25, 45, 46,
trol at 10
as 54 showed significantly different response in CRPC in vitro mod-
el. Compound 54 was orally available in mice (AUC = 231.7 g h/
mL, MRT = 4.07 h at a dose of 30 mg/kg, p.o. (n = 3)). On the basis
of these biological and physicochemical profiles, we selected 54
for further evaluation.
Compound 54 was evaluated for its in vivo therapeutic efficacy
against CRPC in comparison with bicalutamide, using a mouse
xenograft model in which LNCaP-hr cells were subcutaneously in-
jected. It was reported that oral administration of bicalutamide
showed long duration of plasma concentration in men (mean t1/2
of R-isomer and S-isomer, 4.2 days and 19 h, respectively41). Long
duration was also observed in mice (data not shown). On the basis
of these data for bicalutamide and the PK data for 54 described
above, these compounds were orally administered once (bicaluta-
mide) or twice (54) daily for 4 weeks. Tumor volume and plasma
PSA concentration were measured after the treatment period.
Compound 54 showed potent inhibition of tumor growth with
T/C values (volume change in a test compound/volume change
lM), it should be noted that pyrrolidine compound such
and 48, respectively). The secondary amide exhibited higher AR
antagonistic activity than the tertiary amide (48 vs 49). Further,
we studied the effect of configuration of the substituents at the
2- and 3-position in the pyrrolidine ring on the antagonistic activ-
ity. Compound 50, an antipode of 48, showed less potent antago-
nistic activity than that of 48. This result suggested that the
(2S,3R) configuration of the methyl and hydroxyl groups in the pyr-
rolidine compounds was favorable for the antagonistic activity.
Next, we evaluated compound 48, which possessed strong AR
antagonistic activity without any significant agonistic activity, for
the inhibitory activity against PSA secretion by LNCaP-hr cells, to
estimate the potential activity against CRPC. The compound, how-
ever, did not inhibit the PSA secretion at high concentrations. Thus,
we sought to enhance the inhibitory activity of compound 48. In
assessing the substituent at R3 on the cyanophenyl moiety, we
found that replacement of the chloro group of compound 48 with
a fluoro group increased inhibitory activity against PSA secretion
(54) (Table 2). Compound 54 did not possess any agonistic activity
l

76
S. Yamamoto et al. / Bioorg. Med. Chem. 21 (2013) 70–83
in control) of 11% at a dose of 30 mg/kg, bid (Fig. 7). The plasma
PSA level in the 30 mg/kg, bid treatment group was also markedly
reduced to 18% of that in the vehicle treatment group. In addition,
compound 54 demonstrated inhibition of body weight loss caused
by the tumor burden. On the other hand, bicalutamide showed
only partial suppression of tumor growth (T/C value of 48%) even
at a dose of 100 mg/kg, qd. Almost no reduction of plasma PSA
level was observed, as anticipated from in vitro studies in an
LNCaP-hr PSA secretion system (more than 100% of control at
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; dt, doublet of triplet; br s, broad
singlet; m, multiplet. Specific rotations were measured with a JAS-
CO P-1030 digital polarimeter. The HPLC analyses were performed
using a Shimadzu UFLC instrument. Elution was done with a gradi-
ent of 5–90% solvent B in solvent A (solvent A was 0.1% TFA in
water, and solvent B was 0.1% TFA in acetonitrile) through a L-col-
10 lM). These results suggested that compound 54 was effective
against CRPC model and that the property of this compound
is different from that of the first-generation antagonist,
bicalutamide.
4. Conclusion
umn 2 ODS (3.0 ꢁ 50 mm, 2
l
m) column at 1.2 mL minꢀ1. Area %
To explore novel AR antagonists therapeutically effective
against CRPC, we designed, synthesized, and evaluated the effica-
cies of 3-aryl-3-hydroxy-1-phenylpyrrolidine compounds D. The
chiral pyrrolidine compounds were synthesized using stereoselec-
tive arylation of chiral 2-methylpyrrolidin-3-one derivatives. Intro-
duction of an amide group to the 3-aryl moiety and replacement of
the benzene ring with a pyridine notably reduced AR agonistic
activity to generate a potent AR antagonist 48. The (2S,3R) config-
uration of the pyrrolidine ring of 48 was favorable for the AR antag-
onistic activity. Oral administration of compound 54, discovered by
further modification of 48, showed antitumor effects against
LNCaP-hr cell line, a CRPC model, in a mouse xenograft. On the
other hand, bicalutamide only showed partial suppression of the
growth of LNCaP-hr. Our data suggested that the pyrrolidine com-
pounds D such as 54 are novel AR antagonists effective against
CRPC model, and their properties are different from those of the
first-generation AR antagonist, bicalutamide.
purity was measured at 254 nm. Mass spectra (MS) were deter-
mined using Waters micromass ZQ or Shimadzu LCMS-2020. High
resolution mass spectra (HRMS) were recorded by Shimadzu
LCMS-IT-TOF. Elemental analyses were carried out by Takeda Ana-
lytical 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.
5.1.1. 2-Chloro-4-(3-hydroxypyrrolidin-1-yl)benzonitrile (4)
A
suspension of 2-chloro-4-fluorobenzonitrile (10.0 g,
64.3 mmol), pyrrolidin-3-ol 9 (5.60 g, 64.3 mmol), and lithium car-
bonate (7.12 g, 96.4 mmol) in DMSO (100 mL) was stirred at 100 °C
(A) 900
T/C(%) (B)27
control (castration + vehicle)
54 10 mg/kg, p.o., bid
54 30 mg/kg, p.o., bid
control (castration + vehicle)
54 10 mg/kg, p.o., bid
54 30 mg/kg, p.o., bid
800
700
600
500
400
300
200
100
0
25
100
Bicalutamide
23
Bicalutamide
100 mg/kg, p.o., qd
100 mg/kg, p.o., qd
58
48
21
19
17
15
13
11
0
7
14
21
28
0
7
14
21
28
Days
Days
(C)
Dose
Plasma PSA
T / C
Compound
Mean SE (ng / mL)
Day 28
(mg/kg, p.o.)
–
(%)
100
139
18
Control (castration + vehicle)
23.8 6.1
54
54
10, bid
30, bid
100, qd
33.0 14.8
4.3 3.9*
Bicalutamide
45.6 9.7
191
Figure 7. Antitumor effects of 54 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
#
<0.025, Shirley-Williams test vs. control. P <0.025 Williams test versus control.

S. Yamamoto et al. / Bioorg. Med. Chem. 21 (2013) 70–83
77
for 3 h. The mixture was diluted with H2O. The precipitated com-
pound was collected by filtration, air-dried, and recrystallized from
hexane–EtOAc to give 4 (9.56 g, 67%) as light brown crystals, mp
130.5–131 °C. 1H NMR (300 MHz, CDCl3) d: 1.68 (1H, d,
J = 4.0 Hz), 2.00–2.30 (2H, m), 3.25–3.36 (1H, m), 3.37–3.49 (1H,
m), 3.49–3.64 (2H, m), 4.55–4.80 (1H, m), 6.41 (1H, dd, J = 8.8
and 2.4 Hz), 6.56 (1H, d, J = 2.4 Hz), 7.42 (1H, d, J = 8.8 Hz). MS
(ESI) m/z 223 [(M+H)+]. Anal. Calcd for C11H11ClN2O: C, 59.33; H,
4.98; N, 12.58; Cl, 15.92. Found: C, 59.38; H, 4.93; N, 12.53; Cl,
15.78.
47.2 mmol) and dichloromethane (20 mL) slowly at ꢀ78 °C. After
stirring at ꢀ78 °C for 0.5 h, a solution of 4 (3.00 g, 13.5 mmol) in
dichloromethane (100 mL) was added, and the mixture was stirred
at ꢀ40 °C for 0.5 h. To the mixture was added triethylamine
(13.1 mL, 94.0 mmol), and the resulting mixture was stirred at
0 °C for 20 min. Aqueous solution of NH4Cl was added, and the
mixture was diluted with H2O and EtOAc. The organic layer was
washed with H2O and brine, dried over anhydrous Na2SO4, and
concentrated in vacuo. The residue was recrystallized from hex-
ane–EtOAc to give 14 (2.18 g, 73%) as light brown crystals, mp
162–163 °C. 1H NMR (300 MHz, CDCl3) d: 2.82 (2H, t, J = 7.8 Hz),
3.74–3.85 (4H, m), 6.52 (1H, dd, J = 8.8 and 2.4 Hz), 6.67 (1H, d,
J = 2.4 Hz), 7.52 (1H, d, J = 8.8 Hz). MS (ESI) m/z 219 [(MꢀH)ꢀ].
5.1.2. 2-Chloro-4-[(2S,3S)-3-hydroxy-2-methylpyrrolidin-1-
yl]benzonitrile (10)
To a solution of (4S,5S)-4-hydroxy-5-methylpyrrolidin-2-one
733 (7.00 g, 60.8 mmol) in THF (120 mL) was added sodium bis(2-
methoxyethoxy)aluminium dihydride (70% in toluene, 59.7 mL,
215 mmol) slowly at 5 °C. After stirring at 70 °C for 3 h, the reac-
tion mixture was cooled to 5 °C followed by addition of sodium
carbonate decahydrate (26.1 g, 91.2 mmol). The mixture was stir-
red at room temperature for 16 h, diluted with THF, and the precip-
itate was filtered off. The filtrate was concentrated in vacuo, and
the residue was dissolved in DMSO (80 mL). To the solution was
added lithium carbonate (8.99 g, 122 mmol) and 2-chloro-4-fluo-
robenzonitrile (9.46 g, 60.9 mmol), and the mixture was stirred
at 100 °C for 1 h. The mixture was diluted with EtOAc and H2O,
and the organic layer was dried over anhydrous MgSO4 and con-
centrated in vacuo. The residue was purified by silica gel column
chromatography (hexane–EtOAc) to give 10 (10.7 g, 74%) as a col-
orless solid. 1H NMR (300 MHz, CDCl3) d: 1.18 (3H, d, J = 6.6 Hz),
1.82 (1H, d, J = 5.5 Hz), 2.01–2.14 (1H, m), 2.25–2.35 (1H, m),
3.19–3.28 (1H, m), 3.43–3.51 (1H, m), 3.89–3.98 (1H, m), 4.43–
4.52 (1H, m), 6.43 (1H, dd, J = 8.9 and 2.4 Hz), 6.56 (1H, d,
J = 2.4 Hz), 7.42 (1H, d, J = 8.9 Hz). MS (ESI) m/z 237 [(M+H)+].
5.1.7. 2-Chloro-4-[(2S)-2-methyl-3-oxopyrrolidin-1-
yl]benzonitrile (15)
To a solution of 10 (7.00 g, 29.6 mmol) and triethylamine
(33.0 mL, 23.7 mmol) in DMSO (100 mL) was added sulfur trioxide
pyridine complex (23.5 g, 148 mmol), and the resulting mixture
was stirred at room temperature for 1 h. The mixture was diluted
with EtOAc and H2O, and the organic layer was 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 15 (2.58 g, 37%) as
colorless crystals, mp 97.5–98 °C. 1H NMR (300 MHz, CDCl3) d:
1.34 (3H, d, J = 6.8 Hz), 2.68–2.89 (2H, m), 3.63–3.86 (2H, m),
4.03 (1H, q, J = 6.8 Hz), 6.56 (1H, dd, J = 8.8 and 2.4 Hz), 6.70 (1H,
d, J = 2.4 Hz), 7.50 (1H, d, J = 8.8 Hz). MS (ESI) m/z 233 [(MꢀH)ꢀ].
5.1.8. 2-Methoxy-4-[(2S)-2-methyl-3-oxopyrrolidin-1-
yl]benzonitrile (16)
Compound 16 was prepared in a manner similar to that de-
scribed for 15 in 16% yield as
a
yellow powder. 1H NMR
(300 MHz, CDCl3) d: 1.35 (3H, d, J = 6.6 Hz), 2.73–2.82 (2H, m),
3.68–3.82 (2H, m), 3.92 (3H, s), 4.05 (1H, q, J = 6.9 Hz), 6.11 (1H,
d, J = 1.8 Hz), 6.25 (1H, dd, J = 8.7 and 2.4 Hz), 7.40 (1H, d,
J = 8.7 Hz). MS (ESI) m/z 231 [(M+H)+].
5.1.3. 4-[(2S,3S)-3-Hydroxy-2-methylpyrrolidin-1-yl]-2-
methoxybenzonitrile (11)
Compound 11 was prepared in a manner similar to that de-
scribed for 10 in 61% yield as a light yellow powder. 1H NMR
(300 MHz, CDCl3) d: 1.19 (3H, d, J = 6.6 Hz), 1.91 (1H, d,
J = 5.7 Hz), 1.99–2.17 (1H, m), 2.24–2.34 (1H, m), 3.19–3.32 (1H,
m), 3.41–3.53 (1H, m), 3.88 (3H, s), 3.91–4.01 (1H, m), 4.40–4.54
(1H, m), 5.98 (1H, d, J = 2.1 Hz), 6.14 (1H, dd, J = 8.7 and 2.3 Hz),
7.32 (1H, d, J = 8.7 Hz). MS (ESI) m/z 233 [(M+H)+].
5.1.9. 4-[(2S)-2-Methyl-3-oxopyrrolidin-1-yl]benzonitrile (17)
Compound 17 was prepared in a manner similar to that de-
scribed for 15 in 97% yield as a yellow oil. 1H NMR (300 MHz,
CDCl3) d: 1.34 (3H, d, J = 6.6 Hz), 2.75–2.80 (2H, m), 3.71–3.77
(2H, m), 4.05 (1H, q, J = 6.6 Hz), 6.67 (2H, d, J = 9.0 Hz), 7.54 (2H,
d, J = 9.0 Hz). MS (ESI) m/z 201 [(M+H)+].
5.1.4. 4-[(2S,3S)-3-Hydroxy-2-methylpyrrolidin-1-
yl]benzonitrile (12)
5.1.10. 2-Chloro-4-[(2R)-2-methyl-3-oxopyrrolidin-1-
yl]benzonitrile (18)
Compound 12 was prepared in a manner similar to that de-
scribed for 10 in 65% yield as
a
yellow powder. 1H NMR
Compound 18 was prepared in a manner similar to that de-
scribed for 15 in 63% yield as a light brown solid. 1H NMR
(300 MHz, CDCl3) d: 1.34 (3H, d, J = 6.8 Hz), 2.68–2.89 (2H, m),
3.63–3.86 (2H, m), 4.03 (1H, q, J = 6.8 Hz), 6.56 (1H, dd, J = 8.8
and 2.4 Hz), 6.70 (1H, d, J = 2.4 Hz), 7.50 (1H, d, J = 8.8 Hz).
(300 MHz, CDCl3) d: 1.18 (3H, d, J = 6.3 Hz), 1.96–2.17 (2H, m),
2.22–2.37 (1H, m), 3.16–3.31 (1H, m), 3.40–3.54 (1H, m), 3.88–
4.02 (1H, m), 4.39–4.54 (1H, m), 6.50–6.56 (2H, m), 7.42–7.48
(2H, m). MS (ESI) m/z 203 [(M+H)+].
5.1.5. 2-Chloro-4-[(2R,3R)-3-hydroxy-2-methylpyrrolidin-1-
yl]benzonitrile (13)
5.1.11. 4-(3-Benzyl-3-hydroxypyrrolidin-1-yl)-2-
chlorobenzonitrile (5)
Compound 13 was prepared in a manner similar to that de-
scribed for 10 using (4R,5R)-4-hydroxy-5-methylpyrrolidin-2-one
833 in 67% yield as a colorless solid. 1H NMR (300 MHz, CDCl3) d:
1.18 (3H, d, J = 6.4 Hz), 1.83 (1H, d, J = 5.5 Hz), 2.02–2.16 (1H, m),
2.32–2.39 (1H, m), 3.16–3.33 (1H, m), 3.39–3.55 (1H, m), 3.87–
4.01 (1H, m), 4.39–4.56 (1H, m), 6.43 (1H, dd, J = 8.9 and 2.4 Hz),
6.56 (1H, d, J = 2.4 Hz), 7.41 (1H, d, J = 8.9 Hz).
To a solution of 14 (200 mg, 0.906 mmol) in THF (10 mL) was
added 1.03 M benzylmagnesium bromide in THF (3.52 mL,
3.63 mmol) at 5 °C, and the mixture was stirred at room tempera-
ture for 0.5 h. The mixture was diluted with aqueous solution of
NH4Cl and EtOAc. The organic layer was dried over anhydrous
MgSO4, and concentrated in vacuo. The residue was purified by sil-
ica gel column chromatography (hexane–EtOAc). The product was
recrystallized from hexane–EtOAc to give 5 (26 mg, 9%) as colorless
crystals, mp 149.5–151 °C. 1H NMR (300 MHz, CDCl3) d: 1.65 (1H,
s), 1.93–2.07 (1H, m), 2.07–2.33 (1H, m), 2.94–3.10 (2H, m), 3.22
(1H, dd, J = 10.6 and 1.2 Hz), 3.36–3.86 (3H, m), 6.37 (1H, dd,
5.1.6. 2-Chloro-4-(3-oxopyrrolidin-1-yl)benzonitrile (14)
To a solution of oxalyl chloride (2.94 mL, 34.3 mmol) in dichlo-
romethane (80 mL) was added a mixture of DMSO (3.35 mL,

78
S. Yamamoto et al. / Bioorg. Med. Chem. 21 (2013) 70–83
J = 8.9 and 2.4 Hz), 6.51 (1H, d, J = 2.4 Hz), 7.22–7.46 (6H, m). MS
(ESI) m/z 313 [(M+H)+]. HRMS Calcd for C18H17ClN2O 313.1102.
Found 313.1088. Analytical HPLC showed 100% purity.
(300 MHz, CDCl3) d: 1.33 (3H, d, J = 6.4 Hz), 2.24 (1H, s), 2.27–
2.37 (1H, m), 2.41–2.54 (1H, m), 3.24–3.33 (1H, m), 3.63–3.73
(1H, m), 3.89 (3H, s), 4.02 (1H, q, J = 6.5 Hz), 6.02 (1H, d,
J = 2.3 Hz), 6.19 (1H, dd, J = 8.7 and 2.3 Hz), 7.37 (1H, d,
J = 8.7 Hz), 7.43–7.49 (1H, m), 7.54–7.61 (1H, m), 8.41 (1H, dd,
J = 2.6 and 0.8 Hz). MS (ESI) m/z 388 [(M+H)+].
5.1.12. 2-Chloro-4-(3-hydroxy-3-phenylpyrrolidin-1-
yl)benzonitrile (6)
To a solution of 14 (208 mg, 0.943 mmol) in THF (10 mL) was
added 1.0 M phenylmagnesium bromide in THF (3.52 mL,
3.52 mmol) at 5 °C, and the mixture was stirred at room tempera-
ture for 0.5 h. The reaction mixture was diluted with H2O and
EtOAc. The organic layer was dried over anhydrous Na2SO4, and
concentrated in vacuo. The residue was purified by silica gel col-
umn chromatography (hexane–EtOAc). The product was recrystal-
lized from hexane–EtOAc to give 6 (76 mg, 27%) as colorless
crystals, mp 122–126 °C. 1H NMR (300 MHz, CDCl3) d: 1.99 (1H,
s), 2.33–2.45 (1H, m), 2.46–2.61 (1H, m), 3.50–3.83 (4H, m), 6.44
(1H, dd, J = 8.8 and 2.4 Hz), 6.59 (1H, d, J = 2.3 Hz), 7.29–7.47 (4H,
m), 7.48–7.56 (2H, m). MS (ESI) m/z 299 [(M+H)+]. Anal. Calcd for
C17H15ClN2O: C, 68.34; H, 5.06; N, 9.38; Cl, 11.87. Found: C,
68.35; H, 5.20; N, 9.28; Cl, 11.78.
5.1.16. 4-[(2S,3R)-3-(6-Bromopyridin-3-yl)-3-hydroxy-2-
methylpyrrolidin-1-yl]benzonitrile (23)
Compound 23 was prepared in a manner similar to that de-
scribed for 21 in 24% yield as a light yellow solid. 1H NMR
(300 MHz, CDCl3) d: 1.32 (3H, d, J = 6.3 Hz), 2.25 (1H, s), 2.27–2.37
(1H, m), 2.42–2.53 (1H, m), 3.22–3.32 (1H, m), 3.62–3.72 (1H, m),
4.02 (1H, q, J = 6.3 Hz), 6.59 (2H, d, J = 9.0 Hz), 7.46 (1H, dd, J = 8.4
and 0.6 Hz), 7.50 (2H, d, J = 9.0 Hz), 7.57 (1H, dd, J = 8.4 and
2.7 Hz), 8.39 (1H, dd, J = 2.7 and 0.6 Hz). MS (ESI) m/z 358 [(M+H)+].
5.1.17. 4-[(2R,3S)-3-(6-Bromopyridin-3-yl)-3-hydroxy-2-
methylpyrrolidin-1-yl]-2-chlorobenzonitrile (24)
Compound 24 was prepared in a manner similar to that de-
scribed for 21 in 18% yield as a colorless powder. 1H NMR
(300 MHz, CDCl3) d: 1.33 (3H, d, J = 6.4 Hz), 2.19 (1H, s), 2.29–
2.40 (1H, m), 2.41–2.54 (1H, m), 3.21–3.35 (1H, m), 3.62–3.73
(1H, m), 4.01 (1H, q, J = 6.4 Hz), 6.49 (1H, dd, J = 8.9 and 2.4 Hz),
6.62 (1H, d, J = 2.4 Hz), 7.44–7.51 (2H, m), 7.54–7.60 (1H, m),
8.38–8.41 (1H, m). MS (ESI) m/z 392 [(M+H)+].
5.1.13. 4-[(2S,3S)-3-(5-Bromopyridin-2-yl)-3-hydroxy-2-
methylpyrrolidin-1-yl]-2-chlorobenzonitrile (20)
To a solution 2,5-dibromopyridine (1.21 g, 5.11 mmol) in tolu-
ene (8.5 mL) was added 1.6 M butyllithium in hexane (3.84 mL,
6.14 mmol) dropwise at ꢀ78 °C under argon atmosphere. After
stirring at ꢀ78 °C for 1 h, was added a solution of 15 (1.00 g,
4.26 mmol) in toluene (10 mL). The mixture was stirred at
ꢀ78 °C for 0.5 h, and allowed to warm to room temperature. The
mixture was diluted with H2O, and extracted with EtOAc. The or-
ganic layer was washed with brine, dried over anhydrous MgSO4,
and concentrated in vacuo. The residue was purified by silica gel
column chromatography (hexane–EtOAc) to give 20 (0.949 g,
57%) as a colorless solid. 1H NMR (300 MHz, CDCl3) d: 1.29 (3H,
d, J = 6.4 Hz), 2.22–2.36 (1H, m), 2.42–2.53 (1H, m), 3.34–3.46
(1H, m), 3.65–3.78 (1H, m), 3.91 (1H, q, J = 6.3 Hz), 4.99 (1H, s),
6.49 (1H, dd, J = 8.9 and 2.4 Hz), 6.62 (1H, d, J = 2.4 Hz), 7.02 (1H,
dd, J = 8.5 and 0.8 Hz), 7.45 (1H, d, J = 8.9 Hz), 7.83 (1H, dd, J = 8.5
and 2.3 Hz), 8.63 (1H, dd, J = 2.3 and 0.8 Hz). MS (ESI) m/z 392
[(M+H)+].
5.1.18. 2-Chloro-4-[3-(4-fluorophenyl)-3-hydroxypyrrolidin-1-
yl)benzonitrile (25)
Compound 25 was prepared in a manner similar to that de-
scribed for 6 in 28% yield as a colorless solid, mp 163–164.5 °C.
1H NMR (300 MHz, CDCl3) d: 2.04 (1H, s), 2.29–2.60 (2H, m),
3.53–3.87 (4H, m), 6.44 (1H, dd, J = 8.7 and 2.3 Hz), 6.58 (1H, d,
J = 2.3 Hz), 7.05–7.17 (2H, m), 7.43 (1H, d, J = 8.7 Hz), 7.46–7.55
(2H, m). MS (ESI) m/z 317 [(M+H)+]. Anal. Calcd for C17H14ClFN2O:
C, 64.46; H, 4.45; N, 8.84; Cl, 11.19; F, 6.00. Found: C, 64.61; H,
4.61; N, 8.76; Cl, 11.08; F, 5.78.
5.1.19. 2-Chloro-4-[(2S,3R)-3-hydroxy-3-(4-fluorophenyl)-2-
methylpyrrolidin-1-yl]benzonitrile (26)
Compound 26 was prepared in a manner similar to that de-
scribed for 6 in 29% yield as a colorless solid, mp 95.5–97 °C. 1H
NMR (300 MHz, CDCl3) d: 1.32 (3H, d, J = 6.4 Hz), 1.99 (1H, s),
2.26–2.51 (2H, m), 3.15–3.30 (1H, m), 3.56–3.69 (1H, m), 4.04
(1H, q, J = 6.4 Hz), 6.49 (1H, dd, J = 8.8 and 2.4 Hz), 6.62 (1H, d,
J = 2.4 Hz), 6.97–7.10 (2H, m), 7.31–7.41 (2H, m), 7.45 (1H, d,
J = 8.8 Hz). MS (ESI) m/z 331 [(M+H)+]. Anal. Calcd for
5.1.14. 4-[(2S,3R)-3-(6-Bromopyridin-3-yl)-3-hydroxy-2-
methylpyrrolidin-1-yl]-2-chlorobenzonitrile (21)
To a solution 2,5-dibromopyridine (656 mg, 2.77 mmol) in Et2O
(40 mL) was added 1.6 M butyllithium in hexane (1.73 mL,
2.77 mmol) dropwise at ꢀ78 °C under argon atmosphere. After
stirring at ꢀ78 °C for 0.5 h, was added a solution of 15 (500 mg,
2.13 mmol) in THF (5 mL). The mixture was stirred at ꢀ78 °C for
0.5 h, and allowed to warm to room temperature. The mixture
was diluted with aqueous solution of NH4Cl and EtOAc. The organic
layer was dried over anhydrous 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 21 (415 mg, 50%) as light yellow crystals, mp
201 °C. 1H NMR (300 MHz, CDCl3) d: 1.33 (3H, d, J = 6.4 Hz), 2.19
(1H, s), 2.29–2.40 (1H, m), 2.41–2.54 (1H, m), 3.21–3.35 (1H, m),
3.62–3.73 (1H, m), 4.01 (1H, q, J = 6.4 Hz), 6.49 (1H, dd, J = 8.9
and 2.4 Hz), 6.62 (1H, d, J = 2.4 Hz), 7.44–7.51 (2H, m), 7.54–7.60
(1H, m), 8.38–8.41 (1H, m). MS (ESI) m/z 392 [(M+H)+].
C
18H16ClFN2O: C, 65.36; H, 4.88; N, 8.47; Cl, 10.72; F, 5.74. Found:
C, 65.45; H, 5.07; N, 8.33; Cl, 10.64; F, 5.55. ½a D25
ꢀ118.9 (c 0.17,
ꢂ
MeOH).
5.1.20. Ethyl 4-[(2S,3R)-1-(3-chloro-4-cyanophenyl)-3-hydroxy-
2-methylpyrrolidin-3-yl]benzoate (27)
To a suspension of 1,4-diiodobenzene (330 mg, 1.00 mmol) in
Et2O (5.0 mL) was added 1.6 M butyllithium in hexane (0.75 mL,
1.20 mmol) at -78 °C under argon atmosphere. After stirring at
ꢀ78 °C for 0.5 h, was added a solution of 15 (235 mg, 1.00 mmol)
in THF (2.0 mL). The mixture was stirred at ꢀ78 °C for 0.5 h and then
at room temperature for 16 h. The mixture was diluted with aque-
ous solution of NH4Cl and EtOAc. The organic layer was washed with
brine, dried over anhydrous MgSO4, and concentrated in vacuo. The
residue was purified by silica gel column chromatography (hexane–
EtOAc) to give 2-chloro-4-[(2S,3R)-3-hydroxy-3-(4-iodophenyl)-2-
methylpyrrolidin-1-yl]benzonitrile 19 (120 mg) as a yellow solid.
5.1.15. 4-[(2S,3R)-3-(6-Bromopyridin-3-yl)-3-hydroxy-2-
methylpyrrolidin-1-yl]-2-methoxybenzonitrile (22)
Compound 22 was prepared in a manner similar to that de-
scribed for 21 in 59% yield as a light yellow powder. 1H NMR

S. Yamamoto et al. / Bioorg. Med. Chem. 21 (2013) 70–83
79
A
mixture of the compound (120 mg), palladium(II) acetate
5.1.24. Ethyl 5-[(2S,3R)-1-(4-cyanophenyl)-3-hydroxy-2-
(12.0 mg, 0.053 mmol), 1,10-bis(diphenylphosphino)ferrocene
(30.0 mg, 0.054 mmol), triethylamine (0.075 mL, 0.54 mmol), DMF
(2.0 mL), and EtOH (2.0 mL) was stirred at 70 °C for 4 h under carbon
monoxide atmosphere. The mixture was diluted with H2O, and ex-
tracted with EtOAc. The organic layer was washed with saturated
aqueous solution of NaHCO3 and brine, dried over anhydrous
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 27 (23 mg, 6%) as colorless
crystals, mp 129–130 °C. 1H NMR (300 MHz, CDCl3) d: 1.32 (3H, d,
J = 6.3 Hz), 1.38 (3H, t, J = 7.2 Hz), 2.05 (1H, s), 2.32–2.50 (2H, m),
3.22–3.32 (1H, m), 3.62–3.70 (1H, m), 4.07 (1H, q, J = 6.3 Hz), 4.37
(2H, q, J = 7.2 Hz), 6.49 (1H, dd, J = 8.7 and 2.4 Hz), 6.63 (1H, d,
J = 2.4 Hz), 7.42–7.50 (3H, m), 8.03 (2H, d, J = 8.7 Hz). MS (ESI) m/z
385 [(M+H)+].
methylpyrrolidin-3-yl]pyridine-2-carboxylate (31)
Compound 31 was prepared in a manner similar to that de-
scribed for 29 in 97% yield as a brown oil. 1H NMR (300 MHz,
CDCl3) d: 1.34 (3H, d, J = 6.6 Hz), 1.43 (3H, t, J = 7.2 Hz), 2.21 (1H,
s), 2.30–2.40 (1H, m), 2.45–2.57 (1H, m), 3.22–3.33 (1H, m),
3.62–3.73 (1H, m), 4.05–4.15 (1H, m), 4.47 (2H, q, J = 7.2 Hz),
6.60 (2H, d, J = 9.0 Hz), 7.51 (2H, d, J = 9.0 Hz), 7.89 (1H, dd,
J = 8.4 and 2.4 Hz), 8.12 (1H, dd, J = 8.4 and 0.6 Hz), 8.72 (1H, dd,
J = 2.4 and 0.6 Hz). MS (ESI) m/z 352 [(M+H)+].
5.1.25. Ethyl 5-[(2R,3S)-1-(3-chloro-4-cyanophenyl)-3-hydroxy-
2-methylpyrrolidin-3-yl]pyridine-2-carboxylate (32)
Compound 32 was prepared in a manner similar to that de-
scribed for 29 in 98% yield as a brown oil. 1H NMR (300 MHz,
CDCl3) d: 1.35 (3H, d, J = 6.4 Hz), 1.44 (3H, t, J = 7.1 Hz), 2.24 (1H,
s), 2.31–2.44 (1H, m), 2.46–2.59 (1H, m), 3.22–3.36 (1H, m),
3.61–3.74 (1H, m), 4.07 (1H, q, J = 6.4 Hz), 4.47 (2H, q, J = 7.1 Hz),
6.50 (1H, dd, J = 8.8 and 2.4 Hz), 6.63 (1H, d, J = 2.4 Hz), 7.47 (1H,
d, J = 8.8 Hz), 7.88 (1H, dd, J = 8.3 and 2.4 Hz), 8.13 (1H, d,
J = 8.3 Hz), 8.72 (1H, d, J = 2.4 Hz). MS (ESI) m/z 386 [(M+H)+].
5.1.21. Methyl 6-[(2S,3S)-1-(3-chloro-4-cyanophenyl)-3-
hydroxy-2-methylpyrrolidin-3-yl]pyridine-3-carboxylate (28)
To a solution of 20 (800 mg, 2.04 mmol) and triethylamine
(0.34 mL, 2.44 mmol) in MeOH (20 mL) was added [1,10-bis(diphen-
ylphosphino)ferrocene]palladium(II) dichloride dichloromethane
adduct (166 mg, 0.203 mmol), and the resulting mixture was
stirred at 60 °C overnight under carbon monoxide atmosphere.
The mixture was diluted with H2O, and extracted with EtOAc. The
organic layer was washed with brine, dried over anhydrous MgSO4,
and concentrated in vacuo. The residue was purified by silica gel
column chromatography (hexane–EtOAc) to give 28 (653 mg,
86%) as a brown amorphous. 1H NMR (300 MHz, CDCl3) d: 1.30
(3H, d, J = 6.4 Hz), 2.26–2.38 (1H, m), 2.45–2.57 (1H, m),
3.38–3.50 (1H, m), 3.68–3.80 (1H, m), 3.89–4.00 (4H, m), 5.28
(1H, s), 6.50 (1H, dd, J = 8.7 and 2.3 Hz), 6.63 (1H, d, J = 2.7 Hz),
7.19 (1H, dd, J = 8.1 and 0.9 Hz), 7.45 (1H, d, J = 9.1 Hz), 8.31 (1H,
dd, J = 8.3 and 2.3 Hz), 9.16 (1H, d, J = 2.3 Hz). MS (ESI) m/z 372
[(M+H)+].
5.1.26. Benzyl (2S,3S)-3-hydroxy-2-methylpyrrolidine-1-
carboxylate (33)
To a suspension of 7 (7.00 g, 60.8 mmol) in THF (120 mL) was
added sodium bis(2-methoxyethoxy)aluminium dihydride (70%
in toluene, 60 mL, 216 mmol) at 0 °C. After stirring at 70 °C for
4 h, the reaction mixture was cooled to 0 °C followed by addition
of sodium carbonate decahydrate (52.2 g, 182 mmol). The mixture
was stirred at room temperature overnight, and the precipitate
was filtered off. The filtrate was concentrated in vacuo, and the res-
idue was dissolved in THF (120 mL). To the solution was added tri-
ethylamine (12.7 mL, 91.1 mmol) and benzyl chloroformate
(11.0 mL, 78.0 mmol) at 0 °C. After stirring at room temperature
for 18 h, H2O was added and extracted with EtOAc. The organic
layer was washed with diluted hydrochloric acid and brine, dried
over anhydrous MgSO4, and concentrated in vacuo. The residue
was purified by silica gel column chromatography (hexane–EtOAc)
to give 33 (11.3 g, 79%) as a light brown oil. 1H NMR (300 MHz,
CDCl3) d: 1.15–1.30 (3H, m), 1.66 (1H, d, J = 5.1 Hz), 1.82–1.97
(1H, m), 2.00–2.15 (1H, m), 3.40–3.55 (2H, m), 3.90–4.00 (1H, m),
4.29–4.39 (1H, m), 5.05–5.22 (2H, m), 7.30–7.40 (5H, m). MS
(ESI) m/z 236 [(M+H)+].
5.1.22. Ethyl 5-[(2S,3R)-1-(3-chloro-4-cyanophenyl)-3-hydroxy-
2-methylpyrrolidin-3-yl]pyridine-2-carboxylate (29)
A mixture of 21 (250 mg, 0.637 mmol), palladium(II) acetate
(29.0 mg, 0.129 mmol), 1,10-bis(diphenylphosphino)ferrocene
(71.0 mg, 0.128 mmol), triethylamine (0.18 mL, 1.29 mmol), DMF
(5.0 mL), and EtOH (2.0 mL) was stirred at 70 °C for 16 h under car-
bon monoxide atmosphere. The mixture was diluted with aqueous
solution of NaHCO3 and EtOAc. The organic layer was washed with
H2O and brine, dried over anhydrous MgSO4, and concentrated in
vacuo. The residue was purified by silica gel column chromatogra-
phy (hexane–EtOAc) to give 29 (130 mg, 53%) as a colorless solid.
1H NMR (300 MHz, CDCl3) d: 1.35 (3H, d, J = 6.4 Hz), 1.44 (3H, t,
J = 7.1 Hz), 2.24 (1H, s), 2.31–2.44 (1H, m), 2.46–2.59 (1H, m),
3.22–3.36 (1H, m), 3.61–3.74 (1H, m), 4.07 (1H, q, J = 6.4 Hz),
4.47 (2H, q, J = 7.1 Hz), 6.50 (1H, dd, J = 8.8 and 2.4 Hz), 6.63 (1H,
d, J = 2.4 Hz), 7.47 (1H, d, J = 8.8 Hz), 7.88 (1H, dd, J = 8.3 and
2.4 Hz), 8.13 (1H, d, J = 8.3 Hz), 8.72 (1H, d, J = 2.4 Hz). MS (ESI)
m/z 386 [(M+H)+].
5.1.27. Benzyl (2S)-2-methyl-3-oxopyrrolidine-1-carboxylate
(34)
To a solution of 33 (13.5 g, 117 mmol) in MeCN (100 mL) was
added molecular sieves 4A (13.3 g), 4-methylmorpholine N-oxide
(10.0 g, 85.4 mmol), and tetrapropylammonium perruthenate
(0.40 g, 1.14 mmol). After stirring at room temperature for 20 h,
the mixture was concentrated in vacuo. To the residue was added
EtOAc, and the precipitate was filtered off. The filtrate was concen-
trated in vacuo, and the residue was purified by silica gel column
chromatography (hexane–EtOAc) to give 34 (8.74 g, 32%) as a yel-
low oil. 1H NMR (300 MHz, CDCl3) d: 1.34 (3H, d, J = 6.6 Hz), 2.50–
2.70 (2H, m), 3.60–3.75 (1H, m), 3.90–4.10 (2H, m), 5.15 (1H, d,
J = 12.3 Hz), 5.20 (1H, d, J = 12.3 Hz), 7.30–7.40 (5H, m). MS (ESI)
m/z 234 [(M+H)+].
5.1.23. Ethyl 5-[(2S,3R)-1-(4-cyano-3-methoxyphenyl)-3-
hydroxy-2-methylpyrrolidin-3-yl]pyridine-2-carboxylate (30)
Compound 30 was prepared in a manner similar to that de-
scribed for 29 in 66% yield as a colorless powder. 1H NMR
(300 MHz, CDCl3) d: 1.35 (3H, d, J = 6.4 Hz), 1.43 (3H, t,
J = 7.2 Hz), 2.31–2.43 (2H, m), 2.48–2.60 (1H, m), 3.24–3.38 (5H,
m), 3.62–3.75 (1H, m), 4.08 (1H, q, J = 6.2 Hz), 4.47 (2H, q,
J = 7.1 Hz), 6.03 (1H, d, J = 2.3 Hz), 6.20 (1H, dd, J = 8.7 and
2.1 Hz), 7.37 (1H, d, J = 8.5 Hz), 7.89 (1H, dd, J = 8.2 and 2.4 Hz),
8.12 (1H, dd, J = 8.3 and 0.8 Hz), 8.73–8.76 (1H, m). MS (ESI) m/z
382 [(M+H)+].
5.1.28. Benzyl (2S,3R)-3-(6-bromopyridin-3-yl)-3-hydroxy-2-
methylpyrrolidine-1-carboxylate (35)
To a solution of 2,5-dibromopyridine (2.64 g, 11.1 mmol) in
Et2O (165 mL) was added 1.6 M butyllithium in hexane (7.0 mL,
11.2 mmol) at -78 °C under argon atmosphere. After stirring at -
78 °C for 0.5 h, was added a solution of 34 (2.00 g, 8.57 mmol) in
THF (30 mL) dropwise. The mixture was stirred at ꢀ78 °C for

80
S. Yamamoto et al. / Bioorg. Med. Chem. 21 (2013) 70–83
0.5 h, and allowed to warm to room temperature. The mixture was
diluted with aqueous solution of NH4Cl and EtOAc. The organic
layer was dried over anhydrous MgSO4, and concentrated in vacuo.
The residue was purified by silica gel column chromatography
(hexane–EtOAc) to give 35 (1.57 g, 36%) as a yellow oil. 1H NMR
(300 MHz, CDCl3) d: 1.27–1.33 (3H, m), 2.13–2.26 (3H, m), 3.60–
3.76 (2H, m), 3.91–4.06 (1H, m), 5.08–5.22 (2H, m), 7.29–7.39
(5H, m), 7.44–7.49 (1H, m), 7.63 (1H, dd, J = 8.3 and 2.6 Hz), 8.50
(1H, d, J = 2.1 Hz). MS (ESI) m/z 391 [(M+H)+].
brine, dried over anhydrous MgSO4, and concentrated in vacuo.
The residue was washed with hexane–diisopropyl ether to give
39 (464 mg, 88%) as a light brown powder. 1H NMR (300 MHz,
DMSO-d6) d: 1.16 (3H, d, J = 6.2 Hz), 2.21–2.34 (1H, m), 2.44–2.57
(1H, m), 3.25–3.43 (1H, m), 3.56–3.69 (1H, m), 4.24 (1H, q,
J = 6.2 Hz), 5.98 (1H, s), 6.63 (1H, dd, J = 8.9 and 2.4 Hz), 6.75 (1H,
d, J = 2.3 Hz), 7.57 (2H, d, J = 8.9 Hz), 7.84 (1H, dd, J = 8.3 and
0.8 Hz), 8.29 (1H, dd, J = 8.3 and 2.3 Hz), 8.93 (1H, dd, J = 2.1 and
0.8 Hz), 13.33 (1H, br s). MS (ESI) m/z 358 [(M+H)+].
5.1.29. Methyl 5-{(2S,3R)-1-[(benzyloxy)carbonyl]-3-hydroxy-2-
methylpyrrolidin-3-yl}pyridine-2-carboxylate (36)
5.1.33. 5-[(2S,3R)-1-(3-Chloro-4-cyanophenyl)-3-hydroxy-2-
methylpyrrolidin-3-yl]pyridine-2-carboxylic acid (40)
Compound 36 was prepared in a manner similar to that de-
scribed for 28 in 66% yield as a yellow oil. 1H NMR (300 MHz,
CDCl3) d: 1.30 (3H, d, J = 5.7 Hz), 2.17–2.31 (2H, m), 2.62 (1H, s),
3.63–3.76 (1H, m), 4.00 (3H, s), 4.01–4.10 (1H, m), 5.09–5.21 (2H,
m), 7.30–7.39 (5H, m), 7.92 (1H, dd, J = 8.2 and 2.4 Hz), 8.10 (2H,
d, J = 8.3 Hz), 8.85 (1H, dd, J = 2.5 and 0.8 Hz). MS (ESI) m/z 371
[(M+H)+].
To a solution of 29 (95 mg, 0.246 mmol) in THF (3.0 mL) and
EtOH (3.0 mL) was added 1 N NaOH solution (0.99 mL, 0.99 mmol),
and the resulting mixture was stirred at room temperature for 2 h.
The mixture was neutralized with 1 N HCl and concentrated in va-
cuo (not to driness). The precipitated compound was collected by
filtration and washed with H2O to give 40 (78 mg, 89%) as a color-
less powder. 1H NMR (300 MHz, DMSO-d6) d: 1.18 (3H, d,
J = 6.4 Hz), 2.24–2.47 (2H, m), 3.15–3.34 (1H, m), 3.56–3.72 (1H,
m), 4.15 (1H, q, J = 6.2 Hz), 6.03 (1H, br s), 6.70 (1H, dd, J = 8.9
and 2.3 Hz), 6.84 (1H, d, J = 2.3 Hz), 7.63 (1H, d, J = 8.9 Hz), 7.91–
7.96 (1H, m), 8.01 (1H, d, J = 8.1 Hz), 8.70 (1H, d, J = 1.7 Hz). MS
(ESI) m/z 358 [(M+H)+].
5.1.30. Methyl 5-[(2S,3R)-1-(4-cyano-3-fluorophenyl)-3-
hydroxy-2-methylpyrrolidin-3-yl]pyridine-2-carboxylate (37)
A mixture of 36 (13.86 g, 35.4 mmol), 10% Pd/C (50% wet,
0.50 g), and MeOH (60 mL) was stirred at room temperature under
H2 atmosphere for 3 h. After filtration through a pad of Celite, the
filtrate was concentrated in vacuo, and the residue was dissolved
in DMSO (120 mL). To the solution was added lithium carbonate
(6.63 g, 89.7 mmol) and 2,4-difluorobenzonitrile (7.29 g,
52.4 mmol), and the resulting mixture was stirred at 100 °C for
2 h. The mixture was diluted with H2O and EtOAc. The organic
layer was washed with brine, dried over anhydrous Na2SO4, and
concentrated in vacuo. The residue was purified by silica gel col-
umn chromatography (hexane–EtOAc) to give 37 (1.04 g, 8%) as a
light yellow oil. 1H NMR (300 MHz, CDCl3) d: 1.35 (3H, d,
J = 6.4 Hz), 2.33–2.43 (1H, m), 2.47 (1H, s), 2.49–2.58 (1H, m),
3.28 (1H, dt, J = 10.1 and 7.7 Hz), 3.61–3.72 (1H, m), 4.00 (3H, s),
4.01–4.10 (1H, m), 6.30–6.43 (2H, m), 7.41 (1H, dd, J = 8.7 and
7.6 Hz), 7.88 (1H, dd, J = 8.2 and 2.4 Hz), 8.10–8.15 (1H, m), 8.71–
8.75 (1H, m). MS (ESI) m/z 356 [(M+H)+].
5.1.34. 5-[(2S,3R)-1-(4-Cyano-3-methoxyphenyl)-3-hydroxy-2-
methylpyrrolidin-3-yl]pyridine-2-carboxylic acid (41)
Compound 41 was prepared in a manner similar to that de-
scribed for 40 in 95% yield as a colorless powder. 1H NMR
(300 MHz, DMSO-d6) d: 1.21 (3H, d, J = 6.5 Hz), 2.30–2.45 (2H,
m), 3.23–3.32 (1H, m), 3.55–3.70 (1H, m), 3.87 (3H, s), 4.14 (1H,
q, J = 6.5 Hz), 6.00 (1H, br s), 6.22 (1H, d, J = 1.7 Hz), 6.30 (1H, dd,
J = 8.7 and 1.8 Hz), 7.40 (1H, d, J = 8.7 Hz), 7.93–8.05 (2H, m),
8.68 (1H, d, J = 1.5 Hz). MS (ESI) m/z 354 [(M+H)+].
5.1.35. 5-[(2S,3R)-1-(4-Cyanophenyl)-3-hydroxy-2-
methylpyrrolidin-3-yl]pyridine-2-carboxylic acid (42)
Compound 42 was prepared in a manner similar to that de-
scribed for 40 in 60% yield as a light brown solid. 1H NMR
(300 MHz, DMSO-d6) d: 1.18 (3H, d, J = 6.0 Hz), 2.28–2.48 (2H,
m), 3.18–3.40 (1H, m), 3.55–3.65 (1H, m), 4.08 (1H, q, J = 6.0 Hz),
5.97 (1H, br s), 6.71 (2H, d, J = 9.0 Hz), 7.55 (2H, d, J = 9.0 Hz),
7.90 (1H, dd, J = 8.1 and 2.1 Hz), 7.98 (1H, d, J = 8.1 Hz), 8.64 (1H,
d, J = 2.1 Hz). MS (ESI) m/z 324 [(M+H)+].
5.1.31. 4-[(2S,3R)-1-(3-Chloro-4-cyanophenyl)-3-hydroxy-2-
methylpyrrolidin-3-yl]benzoic acid (38)
A mixture of 27 (740 mg, 1.92 mmol), lithium hydroxide mono-
hydrate (120 mg, 2.85 mmol), EtOH (5.0 mL), THF (5.0 mL), and
H2O (10 mL) was stirred at room temperature for 1.5 h and then
at 50 °C for 2 h. The mixture was diluted with aqueous NaOH solu-
tion, washed with EtOAc, acidified with hydrochloric acid, and ex-
tracted with a mixture of EtOAc and MeOH. The organic layer was
washed with brine, dried over anhydrous MgSO4, and concentrated
in vacuo. The residue was recrystallized from MeOH–Et2O to give
38 (308 mg, 45%) as colorless crystals, mp 293–295 °C. 1H NMR
(300 MHz, DMSO-d6) d: 1.16 (3H, d, J = 6.0 Hz), 2.22–2.40 (2H,
m), 3.15–3.35 (1H, m), 3.55–3.70 (1H, m), 4.07 (1H, q, J = 6.0 Hz),
5.79 (1H, s), 6.67 (1H, dd, J = 9.0 and 2.4 Hz), 6.81 (1H, d,
J = 2.4 Hz), 7.53 (2H, d, J = 8.4 Hz), 7.61 (1H, d, J = 9.0 Hz), 7.89
(2H, d, J = 8.4 Hz), 12.88 (1H, br s). MS (ESI) m/z 357 [(M+H)+].
5.1.36. 5-[(2R, 3S)-1-(3-Chloro-4-cyanophenyl)-3-hydroxy-2-
methylpyrrolidin-3-yl]pyridine-2-carboxylic acid (43)
Compound 43 was prepared in a manner similar to that de-
scribed for 40 in quantitative yield as a yellow solid. 1H NMR
(300 MHz, CDCl3) d: 1.18 (3H, d, J = 6.4 Hz), 2.24–2.47 (2H, m),
3.15–3.34 (1H, m), 3.56–3.72 (1H, m), 4.15 (1H, q, J = 6.2 Hz),
6.03 (1H, br s), 6.70 (1H, dd, J = 8.9 and 2.3 Hz), 6.84 (1H, d,
J = 2.3 Hz), 7.63 (1H, d, J = 8.9 Hz), 7.91–7.96 (1H, m), 8.01 (1H, d,
J = 8.1 Hz), 8.70 (1H, d, J = 1.7 Hz). MS (ESI) m/z 358 [(M+H)+].
5.1.37. 5-[(2S,3R)-1-(4-Cyano-3-fluorophenyl)-3-hydroxy-2-
methylpyrrolidin-3-yl]pyridine-2-carboxylic acid (44)
Compound 44 was prepared in a manner similar to that de-
5.1.32. 6-[(2S,3S)-1-(3-Chloro-4-cyanophenyl)-3-hydroxy-2-
methylpyrrolidin-3-yl]pyridine-3-carboxylic acid (39)
scribed for 40 in 90% yield as
a
colorless solid. 1H NMR
To a solution of 28 (550 mg, 1.48 mmol) in THF (13 mL) and
EtOH (13 mL) was added 1 N NaOH solution (7.4 mL, 7.40 mmol),
and the resulting mixture was stirred at room temperature for
5 h. The mixture was diluted with H2O, neutralized with 1 N HCl,
and extracted with EtOAc. The organic layer was washed with
(300 MHz, DMSO-d6) d: 1.19 (3H, d, J = 6.2 Hz), 2.31–2.45 (2H,
m), 3.19–3.32 (1H, m), 3.57–3.67 (1H, m), 4.08–4.19 (1H, m),
6.03 (1H, s), 6.55–6.68 (2H, m), 7.54–7.61 (1H, m), 7.93–8.04
(2H, m), 8.69 (1H, dd, J = 2.3 and 0.8 Hz). MS (ESI) m/z 342
[(M+H)+].

S. Yamamoto et al. / Bioorg. Med. Chem. 21 (2013) 70–83
81
5.1.38. 4-[(2S,3R)-1-(3-Chloro-4-cyanophenyl)-3-hydroxy-2-
J = 8.3 and 2.3 Hz), 8.91 (1H, s). MS (ESI) m/z 371 [(M+H)+]. HRMS
methylpyrrolidin-3-yl]benzamide (45)
Calcd for C19H19ClN4O2 371.1269. Found 371.1247. Analytical HPLC
A
solution of 38 (120 mg, 0.336 mmol), N-[3-(dimethyl-
showed 99.0% purity. ½a D25
ꢀ109.5 (c 0.20, MeOH).
ꢂ
amino)propyl]-N0-ethylcarbodiimide
hydrochloride (97.0 mg,
0.504 mmol), and 1-hydroxy-1H-benzotriazole ammonium salt
(77.0 mg, 0.504 mmol) in DMF (5.0 mL) was stirred at room tem-
perature over night. The reaction mixture was diluted with H2O,
and extracted with EtOAc. The organic layer was washed with sat-
urated aqueous solution of NaHCO3 and brine, dried over anhy-
drous MgSO4, and concentrated in vacuo. The residue was
purified by silica gel column chromatography (MeOH–EtOAc).
The product was recrystallized from hexane–EtOAc to give 45
(73 mg, 61%) as colorless crystals, mp 265–266 °C. 1H NMR
(300 MHz, DMSO-d6) d: 1.16 (3H, d, J = 6.3 Hz), 2.25–2.40 (2H,
m), 3.15–3.35 (1H, m), 3.55–3.65 (1H, m), 4.08 (1H, q, J = 6.3 Hz),
5.73 (1H, s), 6.68 (1H, d, J = 8.7 Hz), 6.81 (1H, d, J = 2.4 Hz), 7.31
(1H, br s), 7.47 (2H, d, J = 2.4 Hz), 7.61 (1H, d, J = 8.4 Hz), 7.82
(2H, d, J = 8.7 Hz), 7.89 (1H, br s). MS (ESI) m/z 356 [(M+H)+]. Anal.
Calcd for C19H18ClN3O2: C, 64.13; H, 5.10; N, 11.81; Cl, 9.96. Found:
5.1.42. 5-[(2S,3R)-1-(3-Chloro-4-cyanophenyl)-3-hydroxy-2-
methylpyrrolidin-3-yl]-N-methylpyridine-2-carboxamide (48)
Compound 48 was prepared in a manner similar to that de-
scribed for 54 in 21% yield as colorless crystals, mp 205–205.5 °C.
1H NMR (300 MHz, CDCl3) d: 1.36 (3H, d, J = 6.4 Hz), 2.30–2.43
(1H, m), 2.45–2.60 (1H, m), 2.78 (1H, s), 3.02 (3H, d, J = 5.1 Hz),
3.20–3.34 (1H, m), 3.59–3.75 (1H, m), 4.06 (1H, q, J = 6.4 Hz),
6.50 (1H, dd, J = 8.8 and 2.4 Hz), 6.63 (1H, d, J = 2.4 Hz), 7.47 (1H,
d, J = 8.8 Hz), 7.83 (1H, dd, J = 8.2 and 2.4 Hz), 7.94 (1H, br s),
8.01–8.06 (1H, m), 8.50 (1H, d, J = 1.5 Hz). MS (ESI) m/z 371
[(M+H)+]. Anal. Calcd for C19H19ClN4O2: C, 61.54; H, 5.16; N,
15.11; Cl, 9.56. Found: C, 61.50; H, 5.19; N, 14.97; Cl, 9.50. ½a D25
ꢂ
ꢀ158.0 (c 0.15, MeOH).
5.1.43. 5-[(2S,3R)-1-(3-Chloro-4-cyanophenyl)-3-hydroxy-2-
methylpyrrolidin-3-yl]-N,N-dimethylpyridine-2-carboxamide
(49)
C, 63.91; H, 5.21; N, 11.70; Cl, 9.86. ½a D25
ꢀ157.1 (c 0.19, MeOH).
ꢂ
5.1.39. 5-[(2S,3R)-1-(4-Cyano-3-fluorophenyl)-3-hydroxy-2-
methylpyrrolidin-3-yl]-N-methylpyridine-2-carboxamide (54)
A mixture of 44 (2.82 g, 8.26 mmol), 2.0 M methylamine in THF
(12.4 mL, 24.8 mmol), N-[3-(dimethylamino)propyl]-N0-ethylcar-
bodiimide hydrochloride (1.90 g, 9.91 mmol), 1-hydroxybenzotria-
zole (1.34 g, 9.92 mmol), and DMF (40 mL) was stirred at room
temperature for 14 h. The reaction mixture was diluted with H2O
and EtOAc. The organic layer was washed with brine, dried over
anhydrous Na2SO4, and concentrated in vacuo. The residue was
purified by silica gel column chromatography (hexane–EtOAc).
The product (2.57 g) was combined with another lot (1.00 g), and
the whole was recrystallized from hexane–EtOAc to give 54
(2.67 g, 66%, 100% ee) as colorless crystals, mp 196–197 °C. 1H
NMR (300 MHz, CDCl3) d: 1.37 (3H, d, J = 6.4 Hz), 2.29–2.42 (1H,
m), 2.47–2.62 (1H, m), 3.01 (3H, d, J = 5.1 Hz), 3.10 (1H, s), 3.18–
3.31 (1H, m), 3.60–3.72 (1H, m), 4.05 (1H, q, J = 6.3 Hz), 6.32 (1H,
dd, J = 12.4 and 2.3 Hz), 6.39 (1H, dd, J = 8.9 and 2.3 Hz), 7.40
(1H, dd, J = 8.7 and 7.5 Hz), 7.79 (1H, dd, J = 8.2 and 2.4 Hz),
7.88–7.99 (2H, m), 8.45–8.49 (1H, m). MS (ESI) m/z 355 [(M+H)+].
Anal. Calcd for C19H19FN4O2: C, 64.40; H, 5.40; N, 15.81; F, 5.36.
Compound 49 was prepared in a manner similar to that de-
scribed for 54 in 51% yield as colorless crystals, mp 150–151 °C.
1H NMR (300 MHz, CDCl3) d: 1.33 (3H, d, J = 6.3 Hz), 2.25–2.35
(1H, m), 2.45–2.55 (1H, m), 3.04 (3H, s), 3.13 (3H, s), 3.19–3.29
(1H, m), 3.60 (1H, s), 3.60–3.70 (1H, m), 4.03 (1H, q, J = 6.3 Hz),
6.49 (1H, dd, J = 8.3 and 2.4 Hz), 6.62 (1H, d, J = 2.4 Hz), 7.40–7.50
(2H, m), 7.72 (1H, dd, J = 8.1 and 2.4 Hz), 8.45 (1H, d, J = 2.4 Hz).
MS (ESI) m/z 385 [(M+H)+]. HRMS Calcd for C20H21ClN4O2
385.1426. Found 385.1401. Anal. Calcd for C20H21ClN4O2: C,
62.42; H, 5.50; N, 14.56. Found: C, 62.28; H, 5.48; N, 14.56. ½a D25
ꢂ
ꢀ168.8 (c 0.16, MeOH).
5.1.44. 5-[(2R,3S)-1-(3-Chloro-4-cyanophenyl)-3-hydroxy-2-
methylpyrrolidin-3-yl]-N-methylpyridine-2-carboxamide (50)
Compound 50 was prepared in a manner similar to that de-
scribed for 54 in 32% yield as colorless crystals, mp 202–203 °C.
1H NMR (300 MHz, DMSO-d6) d: 1.18 (3H, d, J = 6.3 Hz), 2.25–
2.45 (2H, m), 2.79 (3H, d, J = 4.8 Hz), 3.18–3.28 (1H, m), 3.55–
3.70 (1H, m), 4.16 (1H, q, J = 6.3 Hz), 6.01 (1H, s), 6.70 (1H, dd,
J = 9.0 and 2.1 Hz), 6.85 (1H, d, J = 2.1 Hz), 7.63 (1H, d, J = 9.0 Hz),
7.90–8.00 (2H, m), 8.56 (1H, s), 8.75 (1H, d, J = 4.8 Hz). MS (ESI)
m/z 371 [(M+H)+]. Anal. Calcd for C19H19ClN4O2: C, 61.54; H,
5.16; N, 15.11; Cl, 9.56. Found: C, 61.40; H, 5.22; N, 14.79, Cl,
Found: C, 64.39; H, 5.45; N, 15.84; F, 5.31. ½a D25
ꢀ172.0 (c 0.84,
ꢂ
MeOH).
5.1.40. 4-[(2S,3R)-1-(3-Chloro-4-cyanophenyl)-3-hydroxy-2-
methylpyrrolidin-3-yl]-N-methylbenzamide (46)
9.40. ½a 2D5
ꢂ
+142.8 (c 0.16, MeOH).
Compound 46 was prepared in a manner similar to that de-
scribed for 54 in 69% yield as colorless crystals, mp 215–216 °C.
1H NMR (300 MHz, DMSO-d6) d: 1.16 (3H, d, J = 6.3 Hz), 2.25–
2.35 (2H, m), 2.76 (3H, d, J = 4.5 Hz), 3.28–3.38 (1H, m), 3.55–
3.65 (1H, m), 4.07 (1H, q, J = 6.3 Hz), 5.73 (1H, s), 6.67 (1H, dd,
J = 9.0 and 2.1 Hz), 6.81 (1H, d, J = 2.1 Hz), 7.48 (2H, d, J = 8.4 Hz),
7.60 (1H, d, J = 9.0 Hz), 7.77 (2H, d, J = 8.4 Hz), 8.35 (1H, q,
J = 4.5 Hz). MS (ESI) m/z 370 [(M+H)+]. Anal. Calcd for
5.1.45. 5-[(2S,3R)-1-(4-Cyano-3-methoxyphenyl)-3-hydroxy-2-
methylpyrrolidin-3-yl]-N-methylpyridine-2-carboxamide (52)
Compound 52 was prepared in a manner similar to that de-
scribed for 54 in 46% yield as colorless crystals, mp 221–223 °C.
1H NMR (300 MHz, CDCl3) d: 1.36 (3H, d, J = 6.2 Hz), 2.30–2.41
(1H, m), 2.46–2.56 (2H, m), 3.02 (3H, d, J = 5.1 Hz), 3.25–3.34
(1H, m), 3.63–3.76 (1H, m), 3.89 (3H, s), 4.05–4.13 (1H, m), 6.04
(1H, d, J = 2.1 Hz), 6.21 (1H, dd, J = 8.7 and 2.1 Hz), 7.38 (1H, d,
J = 8.7 Hz), 7.86 (1H, dd, J = 8.2 and 2.4 Hz), 7.96 (1H, s), 8.07–
8.13 (1H, m), 8.53 (1H, d, J = 1.5 Hz). MS (ESI) m/z 367 [(M+H)+].
Anal. Calcd for C20H22N4O3: C, 65.56; H, 6.05; N, 15.29. Found: C,
C
20H20ClN3O2: C, 64.95; H, 5.45; N, 11.36; Cl, 9.59. Found: C,
64.90; H, 5.53; N, 11.24, Cl, 9.54. ½a D25
ꢀ168.8 (c 0.16, MeOH).
ꢂ
5.1.41. 6-[(2S,3S)-1-(3-Chloro-4-cyanophenyl)-3-hydroxy-2-
methylpyrrolidin-3-yl]-N-methylnicotinamide (47)
65.28; H, 6.17; N, 15.17. ½a D25
ꢀ151.3 (c 0.18, MeOH).
ꢂ
Compound 47 was prepared in a manner similar to that de-
scribed for 54 in 98% yield as an orange amorphous. 1H NMR
(300 MHz, CDCl3) d: 1.29 (3H, d, J = 6.1 Hz), 2.26–2.37 (1H, m),
2.44–2.56 (1H, m), 3.05 (3H, d, J = 4.9 Hz), 3.37–3.50 (1H, m),
3.67–3.78 (1H, m), 3.93 (1H, q, J = 6.7 Hz), 5.22 (1H, s), 6.11 (1H,
br s), 6.49 (1H, dd, J = 8.7 and 2.3 Hz), 6.63 (1H, d, J = 2.7 Hz),
7.19 (1H, d, J = 8.3 Hz), 7.45 (1H, d, J = 8.7 Hz), 8.10 (1H, dd,
5.1.46. 5-[(2S,3R)-1-(4-Cyanophenyl)-3-hydroxy-2-
methylpyrrolidin-3-yl]-N-methylpyridine-2-carboxamide (53)
Compound 53 was prepared in a manner similar to that de-
scribed for 54 in 54% yield as colorless crystals, mp 207–208 °C.
1H NMR (300 MHz, DMSO-d6) d: 1.19 (3H, d, J = 6.0 Hz), 2.22–
2.48 (2H, m), 2.78 (3H, d, J = 4.8 Hz), 3.10–3.25 (1H, m), 3.53–
3.63 (1H, m), 4.09 (1H, q, J = 6.0 Hz), 5.98 (1H, s), 6.72 (2H, d,

82
S. Yamamoto et al. / Bioorg. Med. Chem. 21 (2013) 70–83
J = 8.7 Hz), 7.56 (2H, d, J = 8.7 Hz), 7.95–8.00 (2H, m), 8.50 (1H, s),
8.75 (1H, q, J = 4.8 Hz). MS (ESI) m/z 337 [(M+H)+]. Anal. Calcd for
thawing to lyse cells, lysate was centrifuged at 228,000ꢁg at 4 °C
for 20 min. The supernatant was stored at ꢀ80 °C as AR cell lysate.
To cell lysate solution containing an AR or a T877A mutant-type AR
C
19H22N4O2: C, 67.84; H, 5.99; N, 16.66. Found: C, 67.79; H, 5.89;
N, 16.67. ½a 2D5
ꢂ
ꢀ184.4 (c 0.20, MeOH).
were added [17-a
-methyl-3H] mibolerone (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.43 The label count of B was measured, and the inhibitory
rate of the compound was calculated.
5.1.47. 2-Chloro-4-{(2S,3R)-3-hydroxy-2-methyl-3-[6-(2-
oxopyrrolidin-1-yl)pyridin-3-yl]pyrrolidin-1-yl}benzonitrile
(51)
A suspension of 21 (204 mg, 0.520 mmol), pyrrolidin-2-one
(0.047 mL,
0.619 mmol),
N,N0-dimethylethylenediamine
5.4.2. AR reporter gene assay (wild-type, T877A mutant-type)
Cos-7 (5 ꢁ 106 cells) were sown in a 150 cm2 flask (Corning),
and cultured in culture medium (DMEM medium containing 10%
Dextran Charcoal (DCC)-Fetal Bovine Serum (FBS), 2 mM gluta-
mine) for 24 h. pcDNA3.1 (Invitrogen) containing AR genes (wild-
type, T877A mutant-type), and pGL3-MMTV-luc vector containing
luciferase gene bound at the downstream of an AR promoter de-
rived from Mouse Mammary Tumor Virus (MMTV) were co-
transfected by using SuperFect transfect ion reagent (QIAGEN).
After culturing at 37 °C in a 5% CO2 atmosphere for 4 h, these cells
were harvested and plated in a 96 well plate (10,000 cells/well)
(0.011 mL, 0.102 mmol), tripotassium phosphate (220 mg,
1.04 mmol), and copper(I) iodide (10.0 mg, 0.0525 mmol) in tolu-
ene (10 mL) was stirred at 80 °C for 3 h. The reaction mixture
was diluted with H2O and EtOAc. The organic layer was washed
with brine, dried over anhydrous Na2SO4, and concentrated in va-
cuo. The residue was purified by silica gel column chromatography
(hexane–EtOAc). The product was recrystallized from hexane–
EtOAc to give 51 (155 mg, 75%) as colorless crystals, mp 171–
174 °C. 1H NMR (300 MHz, CDCl3) d: 1.33 (3H, d, J = 6.4 Hz),
2.03–2.20 (2H, m), 2.26 (1H, s), 2.30–2.39 (1H, m), 2.42–2.53 (1H,
m), 2.66 (2H, t, J = 8.0 Hz), 3.20–3.29 (1H, m), 3.63 (1H, ddd,
J = 10.2, 8.3 and 4.4 Hz), 4.02–4.11 (3H, m), 6.48 (1H, dd, J = 8.8
and 2.4 Hz), 6.62 (1H, d, J = 2.3 Hz), 7.45 (1H, d, J = 8.7 Hz), 7.76
(1H, dd, J = 8.8 and 2.6 Hz), 8.31 (1H, d, J = 2.6 Hz), 8.37 (1H, d,
J = 8.9 Hz). MS (ESI) m/z 397 [(M+H)+]. Anal. Calcd for
and cultured for 2 h. Dihydrotestosterone (DHT, final 0.1 lM) and
a compound were added, and the cells were further cultured for
24 h, after which the luciferase activity was measured. The inhibi-
tory rate by the compound was calculated with the luciferase
activity induced by the addition of 0.1 lM DHT.
C
21H21ClN4O2: C, 63.55; H, 5.33; N, 14.12; Cl, 8.93. Found: C,
63.46; H, 5.30; N, 13.96; Cl, 8.84. ½a D25
ꢂ
ꢀ152.5 (c 0.79, MeOH).
5.4.3. PSA secretion assay in LNCaP-hr cells
LNCaP-hr cells were seeded into 24-well plates at 40,000 cells/
well in phenol red-free RPMI1640 containing 10% DCC-FBS, and on
5.2. Determination of the absolute configuration of 51
the next day, test compounds or bicalutamide of 1–10 lM were
A colorless platelet (0.25 ꢁ 0.20 ꢁ 0.10 mm3) of 51 was ana-
added. The PSA levels in the conditioned media were determined
with an enzyme-immunoassay kit (Dainippon Pharmaceutical
Co., Ltd) 3 days after the treatment with the compounds.
lyzed with a Rigaku RAXIS RAPID diffractometer using graphite
monochromated Cu K
a radiation to obtain the following crystal
data: C21H21ClN4O2, crystal system orthorhombic, space group
P212121 (#19), lattice parameters a = 9.476(5) Å, b = 10.592(6) Å,
c = 18.483(9) Å,
5.4.4. Antitumor effects against LNCaP-hr cell line in mouse
xenograft model
a = 90°, b = 90°, c
= 90°, V = 1855(6) Å3, Z = 4,
T = 100 K. Of the 16,594 reflections collected, 3304 were unique
(Rint = 0.166). The refinement converged with R1 = 0.072 and
wR2 = 0.243 for 642 reflections with I >2r(I). The absolute configu-
ration of 51 was established as (2S,3R) based on the Flack param-
eter,42 0.11(5). Further details of the X-ray structure data are
available on request from the Cambridge Crystallographic Data
Centre (deposition number CCDC 902808).
Five-week-old male BALB/c athymic nude mice were purchased
from Charles River Japan (Kanagawa, Japan) and maintained on a
12/12 h light/dark cycle (light on at 8 am) with constant tempera-
ture (25 °C) and given free access to food and water. One hundred
microliters of LNCaP-hr cell suspension in PBS/Matrigel (BD Biosci-
ences; 1:1) at a cell density of 5 ꢁ 107 cells/mL was inoculated into
the flank region of each mouse after castration on the same day.
When the average tumor volume reached to approximately 100–
300 mm3, grouping was made to give the similar average tumor
volume (n = 6 for each group). From the next day of the grouping,
54 at a dose of 10, 30 mg/kg, twice daily (bid), bicalutamide at a
dose of 100 mg/kg, once daily or vehicle (0.5% methylcellulose),
twice daily was orally administered to the mice for 28 days. The tu-
mor size was measured with a caliper and expressed in mm3 using
the formula 0.5 ꢁ a ꢁ b2, where a is the largest diameter and b is
largest diameter perpendicular 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 dur-
ing the treatment period ꢁ 100).
5.3. Solubility determination
Small volumes of the compound DMSO solutions were added to
the aqueous buffer solution (pH 6.8). After incubation, precipitates
were separated by filtration. The solubility was determined by
HPLC analysis of each filtrate.
5.4. Biology
5.4.1. 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 293fectin transfection re-
agent (Invitrogen), these cells were seeded into an Erlenmeyer flask
(Corning, 1L, 430518) at 1.1 ꢁ 106 cells/mL in FreeStyle293 Expres-
sion 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, 1ꢁ
Complete protease inhibitor tablet (Roche)). After freezing and
Acknowledgments
We thank Dr. Keiji Kamiyama, Dr. Tomoyasu Ishikawa, and Dr.
Yumi N. Imai for helpful discussions; Ms. Keiko Higashikawa for
the help in X-ray crystallographic analysis; Mr. Motoo Iida for
the help in NMR analysis; Mr. Masaharu Nakayama, Mr. Takashi
Santou, Mr. Masahiko Hattori, Mr. Hideo Araki, Ms. Mayumi Imai,

S. Yamamoto et al. / Bioorg. Med. Chem. 21 (2013) 70–83
83
24. Tran, C.; Ouk, S.; Clegg, N. J.; Chen, Y.; Watson, P. A.; Arora, V.; Wongvipat, J.;
Smith-Jones, P. M.; Yoo, D.; Kwon, A.; Wasielewska, T.; Welsbie, D.; Chen, C. D.;
Higano, C. S.; Beer, T. M.; Hung, D. T.; Scher, H. I.; Jung, M. E.; Sawyers, C. L.
Science 2009, 324, 787.
and Ms. Kazuyo Nakamura for the technical assistance for conduct-
ing biological assays.
25. Yamamoto, S.; Matsunaga, N.; Hitaka, T.; Yamada, M.; Hara, T.; Miyazaki, J.;
Santou, T.; Kusaka, M.; Yamaoka, M.; Kanzaki, N.; Furuya, S.; Tasaka, A.;
Hamamura, K.; Ito, M. Bioorg. Med. Chem. 2012, 20, 422.
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