Synthesis and structure-activity relationships of novel, potent, orally active hypoxia-inducible factor-1 inhibitors

Article abstract of DOI:10.1016/j.bmc.2014.07.020

Hypoxia-inducible factor-1 (HIF-1) is the chief transcription factor regulating hypoxia-driven gene expression. HIF-1 overexpression is associated with poor prognosis in several cancers and therefore represents an attractive target for novel antitumor agents. We explored small molecule inhibitors of the HIF-1 pathway. Using high-throughput-screening, we identified benzanilide compound 1 (IC₅₀= 560 nM) as a seed. Subsequent extensive derivatization led to the discovery of compounds 43a and 51d, with anti-HIF-1 activities in vitro (IC₅₀= 21 and 0.47 nM, respectively), and in vivo. Additionally, 43a (12.5-100 mg/kg) also displayed in vivo anti-tumor efficacy, without influencing body weight.

Full text of DOI:10.1016/j.bmc.2014.07.020

Bioorganic & Medicinal Chemistry 22 (2014) 5513–5529
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and structure–activity relationships of novel, potent,
orally active hypoxia-inducible factor-1 inhibitors
a
a
a
a
Satoshi Nagao a, , Yoshinobu Yamane , Setsuo Funasaka , Keigo Tanaka , Kazuki Miyazaki ,
Yoshihiko Kotake a, Jun-ichi Kamata a, Saori Watanabe-Miyano a, Osamu Toyama a, Yoichi Ozawa a,
Yoshiharu Mizui a,b, Kiyoshi Okamoto a, Daisuke Ito a
⇑
a Eisai Co., Ltd, 5-1-3 Tokodai, Tsukuba-shi, Ibaraki 300-2635, Japan
b H3 Biomedicine Inc., 300 Technology Square, Cambridge, MA 02139, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Hypoxia-inducible factor-1 (HIF-1) is the chief transcription factor regulating hypoxia-driven gene
expression. HIF-1 overexpression is associated with poor prognosis in several cancers and therefore
represents an attractive target for novel antitumor agents. We explored small molecule inhibitors of
the HIF-1 pathway. Using high-throughput-screening, we identified benzanilide compound 1 (IC50 = 560
nM) as a seed. Subsequent extensive derivatization led to the discovery of compounds 43a and 51d, with
anti-HIF-1 activities in vitro (IC50 = 21 and 0.47 nM, respectively), and in vivo. Additionally, 43a
(12.5–100 mg/kg) also displayed in vivo anti-tumor efficacy, without influencing body weight.
Ó 2014 Elsevier Ltd. All rights reserved.
Received 2 June 2014
Revised 14 July 2014
Accepted 14 July 2014
Available online 30 July 2014
Keywords:
Hypoxia-inducible factor-1
Hypoxia
Structure–activity relationships
Cancer
Inhibitor
Small molecule
1. Introduction
proteins (PHD) 1–3 on proline residues 402 and/or 564. Subse-
quently, HIF-1
a
is polyubiquitinated and rapidly degraded by the
is
It is generally accepted that cancer cells in hypoxic regions resist
existing chemotherapies and radiation therapy,1–3 and are associ-
ated with an increased risk of invasion, metastasis, treatment
failure, and patient mortality.3–5 Hypoxia in itself produces various
disadvantages for both normal and cancer cells, such as decreased
energy production and induction of apoptosis. However, cancer
cells can adapt to the hypoxic environment by activating the
hypoxia-inducible factor (HIF) pathway to induce the expression
of a wide variety of genes involved in glycolysis, angiogenesis,
hematopoiesis, survival pathways, and invasion.6–10 HIF-1 is a tran-
26S proteasome. Under hypoxic conditions, however, HIF-1
a
highly stabilized, dimerizes with HIF-1b, and translocates to the
nucleus. The HIF-1 complex then becomes transcriptionally acti-
vated and contributes to cancer malignancy.6–12
Overexpression of HIF-1a has been reported in many types of
cancers, including colon, breast, lung, gastric, skin, ovarian, pancre-
atic, prostate and renal carcinomas.13 It is significantly associated
with patient mortality in several different types of cancers affect-
ing the brain, breast, cervix, and lung.5 These findings indicated
that HIF-1a represented an attractive therapeutic target for a wide
scription factor, which consists of an
a
/b heterodimer. HIF-1
a
is an
range of cancers and considerable effort has therefore been
devoted to the identification of HIF-1 inhibitors. Various types of
small molecules have been reported to inhibit the HIF-1 path-
way.14–16 However, no HIF-1 pathway inhibitors have yet been
approved for clinical use and there is still a need for a novel com-
pound with potent HIF-1 inhibitory activity.
oxygen-regulated protein and HIF-1b is a constitutively expressed
protein, irrespective of oxygen concentration. Under normoxic con-
ditions, the von Hippel–Lindau (VHL) protein recognizes and binds
to HIF-1a, which is hydroxylated by prolyl hydroxylase-domain
The present study describes our identification of novel HIF-1
inhibitors. High-throughput screening (HTS) using a cell-based
reporter assay17 identified N-(4-iodophenyl)-4-(morpholinometh-
yl)-benzamide (compound 1), which had submicromolar activity
(IC50 = 560 nM) under hypoxic conditions. This compound reduced
Abbreviations: HIF-1, hypoxia-inducible factor-1; VHL, von Hippel–Lindau; PHD,
prolyl hydroxylase; HTS, high-throughput screening; NBS, N-bromosuccinimide;
MNBA, 2-methyl-6-nitrobenzoic anhydride; DIBAL, diisobutylaluminum hydride;
VEGF-PLAP, vascular endothelial growth factor-placental alkaline phosphatase.
⇑
Corresponding author. Tel.: +81 (0)29 847 5882; fax: +81 (0)29 847 5677.
E-mail address: s-nagao@hhc.eisai.co.jp (S. Nagao).
http://dx.doi.org/10.1016/j.bmc.2014.07.020
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

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S. Nagao et al. / Bioorg. Med. Chem. 22 (2014) 5513–5529
a heteroaromatic core. Condensation was carried out either using
N
H
N
O
2-methyl-6-nitrobenzoic anhydride (MNBA) or via acid chloride
from 12, followed by reduction using lithium borohydride.
Subsequent tosylation and amination with morpholine gave 6,
15a–b with pyridine, and 18 with thiophene. These benzanilide
compounds had some sub-optimal physicochemical properties, as
they were highly crystalline, and/or raised concerns regarding their
chemical/metabolic amide stability. With the aim of improving
these physicochemical properties, we explored isosteres of the
amide moiety or alternatives to morpholine. E-Olefine 21 was
synthesized from phosphonate 1920 and aldehyde 20 using a
conventional Horner–Wadsworth–Emmons reaction.21 The subse-
quent reduction of methyl ester, oxidation of alcohol and reduc-
tive amination afforded the desired E-olefine compound 24
(Scheme 3).22–24 Compounds 26, 27, and 30 were synthesized from
the already known compounds 25, 1, and 28, respectively. Com-
pounds 32a–d and 35a–d were synthesized via nucleophilic sub-
stitution to benzylchloride 31 using the amine derivative
(Scheme 4).25 Compound 38 without benzylamine was formed
using 2d and 36. Sonogashira cross-coupling reactions with aro-
matic or cyclic aliphatic acetylene using Pd and CuI as catalysts
produced the corresponding compounds 41a–e and 43a–b, respec-
tively (Scheme 5).26–28 Compounds 51a,c,d were prepared from
O
I
Figure 1. Benzanilide compound 1.
the HIF-1
a
protein level without affecting its mRNA level.18 Based
on these data, compound 1 was derivatized to produce compounds
that showed in vitro activity at subnanomolar concentrations and
in vivo efficacy in the U251 mouse xenograft model, without
changes in body weight.
2. Chemistry
First, we explored the optimal substitution of the para-iodo
moiety of benzanilide compound 1 (Fig. 1) to create more potent
compounds. Derivatization of benzanilide was conducted as
shown in Scheme 1. Benzanilide derivatives, 1 and 4a–c,e,f, were
synthesized by condensation followed amidation. To investigate
the optimal position of a substituent, ortho-, meta-, and para-
benzylmorpholine were prepared for the amide structure using
similar synthetic routes (Scheme 2).19 To explore an alternative
to the benzanilide structure, we synthesized compounds bearing
Cl
N
Cl
a
H
N
b
H
N
H2N
O
R1
Cl
+
R1
R1
O
O
O
2a: R1 = H
4a:
4b:
4c:
R
1 = H
R1 = o-I
1 = m-I
3
R1 = o-I
2b:
2c:
R
1 = m-I
R
2d: R1 = p-I
1: R1 = p-I
R1 = p-CF3
R1 = p-CF3
R1 = p-OMe
2e:
2f:
4e:
4f:
R1 = p-OMe
Scheme 1. Reagents and conditions: (a) saturated NaHCO3 aq, THF, room temperature (RT) or Et3N, THF, RT; (b) morpholine, RT or morpholine, THF, RT.
Cl
Cl
N
N
Cl
Cl
a
c
H
N
b
b
H
N
H2N
N
N
O
O
N
Cl
+
CF3
O
O
O
O
O
O
CF3
CF3
CF3
CF3
5
3
6
H
N
H
N
H2N
H2N
OH
+
+
CF3
CF3
2e
2e
7
8
H
N
H
N
a
d
H
N
b
Cl
O
O
Me O
Me O
N
CF3
Br
CF3
Y
CF3
O
9
10
11
O
O
HO
H
MeO
g
f
e
H
H2N
N
+
Y
N
MeO
X
X
Y
OH
O
CF3
X
O
CF3
CF3
O
2e
12a: X = N, Y = CH
12b: X = CH, Y = N
13a: X = N, Y = CH
13b: X = CH, Y = N
14a: X = N, Y = CH
14b: X = CH, Y = N
N
H
b
TsO
H
Y
N
O
Y
N
X
X
O
O
CF3
CF3
15a: X = N, Y = CH
15b: X = CH, Y = N
H
N
O
O
H
N
h
i
b
HO
OH
O
Cl
O
N
OH
O
S
S
S
S
O
CF3
CF3
16
17
18
Scheme 2. Reagents and conditions: (a) saturated NaHCO3 aq, THF, room temperature (RT) or Et3N, THF, 0 °C; (b) then morpholine, RT, or morpholine, Et3N, DMF, RT; (c)
SOCl2, reflux then saturated NaHCO3 aq, THF, RT; (d) NBS, benzoyl peroxide, CCl4, reflux; (e) SOCl2, reflux then Et3N, THF, RT or MNBA, Et3N, DMAP, DCM, RT; (f) LiBH4, THF,
RT; (g) TsCl, DMAP, pyridine, Et3N, THF–DCM, RT; (h) NaBH4, MeOH, 0 °C to RT; (i) SOCl2, reflux then H2N–C6H4–CF3, Et3N, THF, RT.

S. Nagao et al. / Bioorg. Med. Chem. 22 (2014) 5513–5529
5515
O
O
O
HO
a
b
c
O
P
MeO
MeO
+
OEt
CF3
CF3
OEt
CF3
19
20
21
22
H
O
N
d
O
CF3
CF3
23
24
N
H
N
N
e
H2N
O
O
+
I
O
I
25
2d
26
Me
N
N
N
H
f
O
N
O
O
O
I
I
HCl
1
27
O
N
O
g
N
O
+
Cl
N
H
O
NH2
I
I
28
29
30
Scheme 3. Reagents and conditions: (a) NaH, THF, 0 °C to room temperature (RT); (b) DIBAL, toluene, ꢀ78 °C to RT; (c) MnO2, DCM, RT; (d) morpholine, NaBH(OAc)3, AcOH,
1,2-dichloroethane, RT; (e) NaBH(OAc)3, THF, RT; (f) MeI, NaH, DMF, RT then 4 N HCl in EtOAc; (g) pyridine, THF, RT.
R2
Cl
H
N
b
H
N
Cl
H2N
a
Cl
+
CF3
O
O
O
CF3
CF3
2d
3
31
32a-d
N
N
N
2
2
a
c
: R
=
=
: R
=
N
N
N
N
c
b
N
b : R2
d : R2
=
N
N
N
Me
Me
N
N
N
H
H
H
N
d
e
N
N
HN
N
N
N
R3
R3
R3
Boc
R4
O
R4
O
R4
O
CF3
CF3
CF3
33a-b
34a-d
35a-d
Me
a : R3
b : R3
=
=
, R4 = H
, R4 = H
a : R3
b : R3
=
=
, R4 = H
3
, R4 = H
, R4 = H
Me
a
: R
=
Me
Me
, R4 = H
3
b
Me
: R
=
Me
: R3 = H, R4
=
: R3 = H, R5
=
c
c
Me
Me
Me
Me
d : R3 = H, R4
=
d : R3 = H, R5
=
H
H
N
d
H2N
f
N
N
HN
N
OH
+
Boc
Boc
CF3
O
O
O
CF3
CF3
36
2d
37
N
H
N
g
N
O
CF3
38
Scheme 4. Reagents and conditions: (a) Et3N, THF, 0 °C to room temperature (RT); (b) amine, Et3N, DMF, RT; (c) 2-methylpiperazine Et3N, DMF, 60 °C; (d) TFA, RT or TFA,
DCM, 0 °C to RT; (e) 3-(chloromethyl)pyridine hydrochloride, Et3N, DMF, 60 °C; (f) MNBA, Et3N, DMAP, dichloromethane, RT; (g) 4-pyridinecarboxaldehyde, NaBH(OAc)3,
AcOH, 1,2-dichloroethane, THF, RT.
benzylchloride 50 and chiral piperazine 47a,c,d, which were syn-
para-substituent was indicated from the lower activity obtained
using 4a, 4b, and 4c. Replacement of the para-iodophenyl moiety
with the hydrophilic para-methoxy group in compound 4f resulted
in a 25-fold decrease in potency compared to compound 1,
whereas para-trifluoromethyl moiety 4e was slightly more potent
than compound 1. This result indicated that a hydrophobic moiety
on the para-position of aniline was required to elicit potent HIF-1
inhibitory activity.
thesized as shown in Scheme 6.29,30
3. Results and discussion
The in vitro activities of the benzanilide derivatives on HIF-1
transcription, quantified using a human glioma cell line (U251)
expressing a PLAP reporter gene under the control of a VEGF
promoter containing the active HIF-1 binding site (VEGF-PLAP),
are summarized in Tables 1–6. Table 1 highlights the effect of mod-
ification of the para-iodophenyl moiety. The importance of the
To investigate whether the phenyl moiety of the benzanilide
compounds could be replaced by another cyclic moiety, we syn-
thesized pyridine derivatives 6, 15a–b and a thiophene derivative

5516
S. Nagao et al. / Bioorg. Med. Chem. 22 (2014) 5513–5529
N
N
H
H
N
b
c
c
N
N
N
N
N
O
O
I
R5
d: R5 = 1-hydroxycyclohexyl
40
41a-e
a: R5 = phenyl
Cl
H
N
Cl
b: R5 = 3-pyridyl e: R5 = 1-adamantyl
c: R5 = cyclohexyl
H2N
a
Cl
+
I
O
O
I
3
2d
39
N
H
N
N
H
N
d
N
H2N
N
N
H2N
N
O
O
O
O
I
R5
42
43a-b
5 = phenyl
a:
R
b: R5 = 1-adamantyl
Scheme 5. Reagents and conditions: (a) saturated NaHCO3 aq, THF, room temperature (RT) or Et3N, THF, RT; (b) 3-pyridylmethylpiperazine, Et3N, DMF, RT; (c) HC„C-R5,
PdCl2(PPh3)2 or Pd(PPh3)4, CuI, Et3N, DMF, RT or 100 °C; (d) (2-aminopyridin-3-yl)(piperazin-1-yl)methanone, Et3N, DMF, 50 °C.
d
NO2
NO2
NO2
O
O
N
O
O
N
O
O
N
R6
a, b
c
e
d
NH
Me
S
R6
S
R6
S
NH
Me
N
N
N
Boc
HN
N
N
N
N
Me
Me
Me
44
45
46a-b
47a,c-d
R6 = CH2OMe
R
a :R6 = H
a : R6 = H
6 = CH2O(CH2)2OMe
:R6 = CH2OMe
c
: R6 = Cl
b
d :R6 = CH2O(CH2)2OMe
R6
Cl
H
N
N
H2N
H
N
N
N
H2N
Me
f
g
h
O
+
O
I
2d
48
49
50
51a,c-d
a :R6 = H
:R6 = CH2OMe
d :R6 = CH2O(CH2)2OMe
c
Scheme 6. Reagents and conditions: (a) 2-nitrobenzene-1-sulfonyl chloride, Et3N, THF, 0 °C to room temperature (RT); (b) TFA, DCM, 0 °C to RT; (c) 3-(chloromethyl)pyridine,
Et3N, DMF, 60 °C; or 2-chloro-5-(chloromethyl)pyridine, Et3N, DMF, 60 °C; (d) mercaptoacetic acid, LiOH, DMF, RT; (e) MeOCH2SnBu3 or MeO(CH2)2OCH2SnBu3, Pd(PPh3)4,
DMF, 120 °C; (f) PdCl2(PPh3)2, CuI, Et3N, DMF, RT; (g) 4-chloromethylbenzoyl chloride, Et3N, THF, 0 °C; (h) piperazine derivatives, Et3N, DMF, 70 °C.
Table 1
Table 2
Effect of substitution of R1 on in vitro biological activity
Effect of ring A and B on in vitro biological activity
N
H
N
O
N
H
N
R1
B
O
O
A
O
No.
R1
VEGF/PLAP IC50 (lM)
No.
A
B
VEGF/PLAP IC50
0.28
(lM)
4a
4b
4c
1
4e
4f
H
o-I
m-I
p-I
p-CF3
p-OMe
5.4
>20
2.2
0.56
0.28
13.9
4e
CF3
CF3
N
6
8
4.0
>10
>10
CF3
18. As shown in Table 2, a heteroaromatic ring, including pyridine
and thiophene, resulted in a >5-fold decrease in potency relative
to compound 4e, except for picolinamide 15a. One possible expla-
nation for the comparable inhibitory activity of 15a and 4e is that
the basicity/polarity of the pyridine nitrogen was compromised
by the adjacent carboxamide functionality. However, the picolina-
mide 15a showed unacceptable toxicity compared with the
benzanilide 4e in the mouse model (data not shown) and we
therefore selected benzanilide. Replacement of the six membered
11
15a
0.23
N
N
15b
18
4.5
1.6
S

S. Nagao et al. / Bioorg. Med. Chem. 22 (2014) 5513–5529
5517
Table 3
Table 5
Effect of ring C on in vitro biological activity
Effects of substitution of R5 and R7 on in vitro biological activity
No.
C
VEGF/PLAP IC50 (lM)
N
H
N
N
N
R7
O
O
C
R5
CF3
No.
R5
R7
VEGF/PLAP IC50 (lM)
H
N
4e
24
0.28
0.88
41a
41b
Ph
3-Pyridyl
0.0079
0.034
O
N
41c
41d
0.0031
>0.2
N
O
C
HO
I
41e
43a
43b
0.00097
0.021
H
N
1
0.56
2.5
O
O
Ph
N
H
N
26
NH
0.0058
2 O
Me
N
HCl
27
30
2.2
2.5
O
The structure–activity relationships (SAR) of R2 are highlighted
in Table 4. Replacement of the morpholine group with a benzylpi-
perazine group in compound 32a resulted in a >10-fold increase in
potency, relative to compound 4e. The pyridylmethylpiperazine
groups, such as 4-pyridyl and 3-pyridyl analogues, also had equi-
potent inhibitory activity to benzylpiperazine 32a. The importance
of an amine moiety at the benzylic position was demonstrated by
compound 38 which lacked a nitrogen atom at this position and
exhibited >10-fold decrease in potency, relative to compound
32b. Compound 32d possessed an open piperazine ring and
showed an approximate 9-fold decrease in potency relative to
compound 32c. Compound 35a, where a chiral methyl moiety
was introduced in the piperazine ring, exhibited more potent
HIF-1 inhibition compared to 32c.
N
H
benzene/pyridine rings with five membered heterocyclic rings,
such as thiophene, led to decreased activity. Additionally, replace-
ment with meta- and ortho-substitutions 8 and 11 resulted in
>35-fold decrease in potency, relative to compound 4e. These
results suggested that para-substitution in ring B was required
for potent HIF-1 inhibitory activity. The E-olefin compound 24
showed moderate inhibitory activity (Table 3). In contrast, the
aniline 26 and methylated amide hydrochloride 27 showed less
HIF-1 inhibition than did amide 1. In addition, retro amide 30
resulted in an approximately 5-fold decrease in potency, com-
pared to the seed compound 1.
An increase in potency was observed with the para-phenylacet-
ylene 41a, compared to compound 32c (Table 5). The potency of
Table 4
Effect of substitution of R2 on in vitro biological activity
R2
H
N
O
CF3
No.
R2
VEGF/PLAP IC50
0.28
(lM)
No.
R2
VEGF/PLAP IC50
0.0033
(lM)
N
N
4e
35a
O
N
N
N
N
Me
Me
N
N
N
32a
32b
0.013
35b
35c
0.013
0.030
N
N
N
N
N
N
N
0.0079
Me
N
N
N
N
38
0.11
35d
0.035
Me
N
32c
32d
0.014
0.12
N
N
N
N
N
Me
Me

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S. Nagao et al. / Bioorg. Med. Chem. 22 (2014) 5513–5529
Table 6
41a led us to explore hydrophobic substituents further. The
Effects of substitution of R6 on in vitro biological activity
cyclohexylacetylene 41c and adamantylacetylene 41e showed at
least 2-fold improvement in potency, relative to phenylacetylene
41a. In particular, adamantylacetylene 41e exhibited the highest
potency (IC50 = 0.97 nM). In contrast, 3-pyridyl acetylene 41b and
hydroxycyclohexylacetylene 41d resulted in 4-fold and over
25-fold reductions in potency relative to phenylacetylene 41a,
respectively. These data clearly indicated that hydrophobic
substituents on the para-position of the benzanilide moiety were
essential for potent HIF-1 inhibitory activity. Compounds 43a
and 43b with 2-aminonicotinic amide also displayed good activity
(IC50 = 21 nM and 5.8 nM, respectively), however they were
slightly less effective than compounds 41a and 41e, bearing the
3-pyridylmethyl moiety.
R6
N
H
N
N
N
Me
O
No.
R6
H
VEGF/PLAP IC50 (lM)
51a
51c
51d
0.0026
0.0011
0.00047
CH2OMe
CH2O(CH2)2OMe
Finally, benzanilide 51a, synthesized to incorporate the positive
SARs described above, exhibited potent HIF-1 inhibition, as
expected (Table 6). Furthermore, we explored the effect of substi-
tution on pyridine in 51a and discovered that substitution of the
6-position on pyridine effectively increased inhibitory activity. In
particular, analogue 51d with a methoxyethoxymethyl moiety
showed a sub-nM IC50. This compound was >1000 times more
potent than seed compound 1.
The benzanilide derivatives 41a, 43a, and 51d with effective
HIF-1 inhibitory activity in vitro were evaluated for suppression
of HIF-1
a protein level in vivo. Tumor samples from the mouse
xenograft model where 41a and 43a had been administered orally
at a dosage of 50 and 100 mg/kg displayed significantly less HIF-
1
a
protein, but HIF-1
treatment (Fig. 2). Treatment with 51d, which had shown the
most potent in vitro activity, strongly reduced HIF-1 levels at
a
mRNA was not reduced at all,18 24 h after
a
doses of 6.25 and 12.5 mg/kg. However body weight decreased
to 92% compared with control at the dose of 12.5 mg/kg of 51d
(data not shown). Next, we evaluated the antitumor efficacy of
43a in the U251 mouse xenograft model (Fig. 3). Daily oral
administration of 12.5–100 mg/kg 43a for 11 days significantly
delayed tumor growth, without significant body weight loss.
Compound 43a also suppressed HIF-1a protein levels 3 h after
oral administration of 12.5 mg/kg (Fig. 4), and could be adminis-
tered daily for approximately 50 days at 50 mg/kg in survival
model using a U251 intracranial xenograft (data not shown).
The detailed mechanism underlying HIF-1
benzanilide compounds was unclear. To investigate whether
HIF-1 synthetic or degradation pathways were affected by these
compounds, we experimented with 41a in a present of an iron
chelator and proteasome inhibitor. The degradation of HIF-1
a inhibition by these
a
a
needs to activate prolyl hydroxylase, which is activated in the
presence of iron (II) and oxygen. Iron chelators, such as deferox-
amine and cobalt, have been reported to interfere with the degra-
dation of HIF-1
these induce HIF-1
Interestingly, compound 41a inhibited the induction of HIF-1
protein accumulation under hypoxic conditions, but it did not
inhibit HIF-1 accumulation induced by deferoxamine (Fig. 5).
a
by suppressing prolyl hydroxylase. As a result,
a
accumulation under normoxic conditions.
a
a
On the other hand, proteasome inhibitors, such as MG-132, pre-
vented HIF-1 complex degradation by inhibiting the ubiquitin-
proteasome system. Compound 41a did not suppress hypoxia
induced HIF-1
some inhibitor MG-132 (Fig. 6). Additionally, in vitro and
in vivo experiments showed no change of HIF-1 mRNA in the
presence of compound 41a.18 These results suggested that benza-
nilide compounds may enhance the degradation of HIF-1 under
a protein accumulation in the presence of protea-
a
Figure 2. Pharmacodynamic profiles of 41a, 43a, and 51d. Mice bearing U251
tumor xenografts were treated with a single dose of 41a, 43a, or 51d at the
a
indicated dosage. Tumors were harvested 24 h after dosing. The amounts of HIF-1
a
hypoxic conditions through accelerating the proline hydroxyl-
ation dependent ubiquitin–proteasome pathway but not synthetic
in the tumors were measured by an ELISA and expressed as a percentage of the
vehicle control. Data points represent the mean of three animals.

S. Nagao et al. / Bioorg. Med. Chem. 22 (2014) 5513–5529
5519
Figure 3. Compound 43a suppressed U251 tumor xenograft growth without changes in body weight. Mice bearing U251 xenografts received oral administration of 43a or
vehicle daily for 11 days. The y-axis indicates tumor volume (mm3). Data points represent the mean of five animals, ⁄P < 0.05 compared with the vehicle control; bars
represent the standard deviation.
120%
100%
80%
60%
40%
20%
0%
12.5 mg/kg
25 mg/kg
50 mg/kg
100 mg/kg
0
8
16
24
time (hr)
Figure 6. Compound 41a failed to suppress hypoxia-induced HIF-1
a protein
Figure 4. The time-course of changes in the amount of HIF-1
xenografts following 43a treatment. U251 tumor-bearing mice were orally treated
with indicated dosage of 43a. Tumors were collected at 3, 12, 16 and 24 h after
a
protein in U251
accumulation in the presence of proteasome inhibitor MG132. Untransfected
U251 cells (4 ꢁ 104 cells/well in 96-well plates) were incubated with different
concentrations of 41a for 6 h under either normoxia (21% O2), hypoxia (2% O2), or
treatment. The amount of HIF-1
a
protein in total tumor extracts were determined
hypoxia in the presence of 25 lM MG132. The amounts of HIF-1a protein were then
with ELISA. Means SD are shown.
measured by ELISA. Data points represent the mean of two wells.
4. Conclusions
We explored the development of potent and orally active HIF-1
pathway inhibitors using benzanilide analogues, starting from the
HTS hit compound 1. SAR analyses indicated that: (1) a hydropho-
bic moiety was preferred at the 4-position of aniline in compound
1; (2) the piperazinomethyl and carboxyanilide needed to be in a
para-configuration; and (3) benzyl- or pyridylmethyl-piperazine,
instead of morpholine, improved HIF-1 pathway inhibition. In par-
ticular, adamantyl-acetylene and (S)-4-((6-substituted-4-pyridin-
3-yl)methyl)-2-methylpiperazine moieties dramatically improved
the compounds’ anti-reporter expression activity in vitro and 51d
achieved a sub-nM IC50. Furthermore, we confirmed that represen-
tative benzanilide compounds decreased HIF-1a protein levels
in vivo.
We discovered a HIF-1 inhibitor with fairly potent in vitro and
in vivo activity. As the detailed mechanism underlying these
effects is still unclear, additional studies are required for further
development of this compound series.
Figure 5. Compound 41a failed to suppress the deferoxamine induced HIF-1
a
protein accumulation. Untransfected U251 cells (4 ꢁ 104 cells/well in 96-well
plates) were incubated for 6 h with different concentrations of 41a under either
5. Experimental sections
5.1. Chemistry
normoxia (21% O2), hypoxia (2% O2), or normoxia in the presence of 100
deferoxamine. The amounts of HIF-1 protein were then measured by ELISA. Data
points represent the mean of two wells.
lM
a
5.1.1. General methods
pathway. Further studies are needed to clarify the precise mech-
anism of benzanilide action on the HIF-1 pathway.
1H and 13C nuclear magnetic resonance (NMR) spectra were
recorded on
a Bruker Avance spectrometer (operating at

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S. Nagao et al. / Bioorg. Med. Chem. 22 (2014) 5513–5529
600 MHz for 1H and 150 MHz for 13C). Chemical shifts were
expressed in ppm (d) from the residual CHCl3 signal at dH
7.26 ppm and dC 77.0 ppm in CDCl3 or the residual CHD2SOCD3
signal at dH 2.50 ppm and dC 39.5 ppm in CD3SOCD3 (s = singlet,
d = doublet, t = triplet, q = quadruplet, m = multiplet, and
br = broad). Coupling constants (J) are given in Hertz (Hz). High-
resolution mass spectra (HRMS) were recorded on a Thermo Fisher
LTQ Orbitrap XL spectrometer using electrospray ionization (ESI)
and a Waters GCT Premier using electron ionization (EI). Column
chromatography was carried out using silica gel 60 (spherical)
5.1.6. 4-(Morpholinomethyl)-N-(4-(trifluoromethyl)phenyl)
benzamide (4e)
The title compound was prepared from 2e using a method anal-
ogous to that described for 4a in 86.5% yield as a white solid.
1H NMR (600 MHz, CDCl3): d = 2.47 (br s, 4H), 3.58 (s, 2H), 3.73
(t, J = 4.5 Hz, 4H), 7.49 (d, J = 8.3 Hz, 2H), 7.63 (d, J = 8.7 Hz, 2H),
7.78 (d, J = 8.7 Hz, 2H), 7.84 (d, J = 8.3 Hz, 2H), 7.98 (br s, 1H); 13C
NMR (150 MHz, CDCl3): d = 53.6, 62.9, 66.9, 119.7, 124.1, 126.3,
126.4, 127.1, 129.5, 133.3, 141.0, 142.7, 165.6; HRMS–ESI m/z
[M+H]+ calcd for C19H20F3N2O+2: 365.1471, found: 365.1472.
(40–50 lm, Kanto Chemical Co., Inc.), Chromotorex (200–350
mesh, Fuji Silysia Chemical Ltd) and a Hi-Flash™ column (silica
gel and NH-silica gel, Yamazen Corporation). Chemicals and sol-
vents used in the study were commercially available.
5.1.7. N-(4-Methoxyphenyl)-4-(morpholinomethyl)benzamide
(4f)
The title compound was prepared from 2f using a method anal-
ogous to that described for 4a in 83.4% yield as a white solid.
1H NMR (600 MHz, CDCl3): d = 2.45 (br s, 4H), 3.55 (s, 2H), 3.72
(t, J = 4.7 Hz, 4H), 3.81 (s, 3H), 6.91 (d, J = 8.7 Hz, 2H), 7.45 (d,
J = 8.1 Hz, 2H), 7.53 (d, J = 8.7 Hz, 2H), 7.73 (br s, 1H), 7.81 (d,
J = 8.1 Hz, 2H); 13C NMR (150 MHz, CDCl3): d = 53.6, 55.5, 63.0,
67.0, 114.3, 122.0, 127.0, 129.3, 131.0, 133.9, 142.1, 156.6, 165.4;
HRMS–ESI m/z [M+H]+ calcd for C19H23N2O+3: 327.1703, found:
327.1707.
5.1.2. 4-(Morpholinomethyl)-N-phenylbenzamide (4a)
To a stirred solution of 2a (100 mg, 0.529 mmol) in THF (2 mL)
and saturated NaHCO3 aqueous solution (0.4 mL) was added drop-
wise 4-(chloromethyl)benzoyl chloride (100 mg, 0.529 mmol) in
tetrahydrofuran (THF) (0.5 mL) at 0 °C and stirred at 0 °C for
30 min. Morpholine (231 lL, 2.65 mmol) was added and the reac-
tion mixture was stirred at room temperature overnight. The reac-
tion mixture was then poured into saturated NaHCO3 and
extracted with ethyl acetate (EtOAc). The organic extract was
washed with brine, dried over Na2SO4, filtered and then concen-
trated. The crude solid was washed with n-hexane/diethyl ether
and filtered to produce compound 4a as a white solid (141 mg,
0.477 mmol, 90.2%).
5.1.8. 4-(Morpholinomethyl)-N-(5-(trifluoromethyl)pyridin-2-
yl)benzamide (6)
To a stirred solution of 5 (85.6 mg, 0.528 mmol) in THF (2 mL)
were added triethylamine (147 lL, 1.06 mmol) and 4-(chloro-
methyl)benzoyl chloride 3 (100 mg, 0.528 mmol) at 0 °C. At the
same temperature, the mixture was stirred for 200 min. To the stir-
red reaction mixture was added morpholine (460 lL, 5.28 mmol)
at room temperature. The reaction mixture was stirred at room
temperature for 38 h. The mixture was diluted with water and
EtOAc. The organic extract was washed with brine, dried over
Na2SO4, filtrated and then concentrated. The residue was purified
with column chromatography on NH silica gel (n-heptane/
EtOAc = 3:1–1:2). The resulting solid was washed with n-hexane/
Et2O and filtrated to afford the title compound 6 as a white solid
(56.7 mg, 0.155 mmol, 29.4%).
1H NMR (600 MHz, CDCl3): d = 2.46 (br s, 4H), 3.56 (s, 2H), 3.72
(t, J = 4.5 Hz, 4H), 7.13–7.18 (m, 1H), 7.37 (t, J = 7.8 Hz, 2H), 7.46 (d,
J = 7.9 Hz, 2H), 7.64 (d, J = 7.8 Hz, 2H), 7.78–7.85 (m, 3H); 13C NMR
(150 MHz, CDCl3): d = 53.6, 62.9, 67.0, 120.1, 124.5, 127.0, 129.1,
129.4, 133.9, 137.9, 142.2, 165.5; HRMS–ESI m/z [M+H]+ calcd for
C
18H21N2O+2: 297.1598, found: 297.1603.
5.1.3. N-(2-Iodophenyl)-4-(morpholinomethyl)benzamide (4b)
The title compound was prepared from 2b using a method anal-
ogous to that described for 4a in 33.9% yield as a white solid.
1H NMR (600 MHz, CDCl3): d = 2.47 (br s, 4H), 3.58 (s, 2H), 3.73
(t, J = 4.5 Hz, 4H), 6.88 (dd, J = 7.8, 7.8 Hz, 1H), 7.41 (t, J = 7.8 Hz,
1H), 7.50 (d, J = 7.9 Hz, 2H), 7.82 (d, J = 7.8 Hz, 1H), 7.93 (d,
J = 7.9 Hz, 2H), 8.28 (br s, 1H), 8.46 (d, J = 7.8 Hz, 1H); 13C NMR
(150 MHz, CDCl3): d = 53.7, 62.9, 67.0, 90.1, 121.7, 126.0, 127.2,
129.4, 129.5, 133.4, 138.3, 138.8, 142.7, 165.1; HRMS–ESI m/z
[M+H]+ calcd for C18H20IN2O+2: 423.0564, found: 423.0567.
1H NMR (600 MHz, CDCl3): d = 2.46 (br s, 4H), 3.58 (s, 2H), 3.73
(t, J = 4.7 Hz, 4H), 7.51 (d, J = 8.1 Hz, 2H), 7.89 (d, J = 8.1 Hz, 2H),
7.98 (dd, J = 8.9, 2.1 Hz, 1H), 8.51–8.58 (m, 2H), 8.74 (br s, 1H);
13C NMR (150 MHz, CDCl3): d = 53.7, 62.9, 67.0, 113.4, 122.7,
123.5, 127.3, 129.5, 132.4, 135.8, 143.4, 145.4, 154.1, 165.6;
HRMS–ESI m/z [M+H]+ calcd for C18H19F3N3O+2: 366.1424, found:
366.1423.
5.1.4. N-(3-Iodophenyl)-4-(morpholinomethyl)benzamide (4c)
The title compound was prepared from 2c using a method anal-
ogous to that described for 4a in 32.3% yield as a white solid.
1H NMR (600 MHz, CDCl3): d = 2.45 (br s, 4H), 3.56 (s, 2H),
3.72 (t, J = 4.5 Hz, 4H), 7.09 (dd, J = 8.2, 8.2 Hz, 1H), 7.44–7.50
(m, 3H), 7.62 (dd, J = 8.2, 1.1 Hz, 1H), 7.74–7.85 (m, 3H), 8.05
(s, 1H); 13C NMR (150 MHz, CDCl3): d = 53.7, 62.9, 67.0, 94.2,
119.3, 127.0, 128.8, 129.4, 130.5, 133.4, 133.5, 139.1, 142.6,
165.4; HRMS–ESI m/z [M+H]+ calcd for C18H20IN2O+2: 423.0564,
found: 423.0566.
5.1.9. 3-(Morpholinomethyl)-N-(4-(trifluoromethyl)phenyl)
benzamide (8)
A stirred solution of 3-(chloromethyl)benzoic acid 7 (100 mg,
0.586 mmol) in SOCl2 (5 mL) was refluxed for 85 min. The reaction
mixture was evaporated. To a stirred solution of 2e (94.4 mg,
0.586 mmol) in THF (4 mL) and saturated NaHCO3 aqueous solu-
tion (1 mL) was added prepared acid chloride in THF at room tem-
perature. The reaction mixture was stirred at room temperature for
100 min. To the reaction mixture was added morpholine (511 lL,
5.86 mmol). The reaction mixture was stirred at room temperature
for 20 h. The mixture was diluted with water and EtOAc. The
organic extract was washed with brine, dried over Na2SO4, filtrated
and then concentrated. The crude solid was washed with n-hex-
ane/Et2O and filtrated to afford the title compound 8 as a white
solid (172 mg, 0.472 mmol, 80.6%).
5.1.5. N-(4-Iodophenyl)-4-(morpholinomethyl)benzamide (1)
The title compound was prepared from 2d using a method anal-
ogous to that described for 4a in 94.2% yield as a white solid.
1H NMR (600 MHz, CDCl3): d = 2.41–2.50 (m, 4H), 3.56 (s, 2H),
3.69–3.75 (m, 4H), 7.43 (d, J = 8.0 Hz, 2H), 7.46 (d, J = 8.0 Hz, 2H),
7.67 (d, J = 8.0 Hz, 2H), 7.76–7.84 (m, 3H); 13C NMR (150 MHz,
CDCl3): d = 53.6, 62.9, 67.0, 87.7, 121.9, 127.0, 129.4, 133.5, 137.7,
138.0, 142.6, 165.4; HRMS–ESI m/z [M+H]+ calcd for C18H20IN2O2+:
423.0564, found: 423.0565.
1H NMR (600 MHz, CDCl3): d = 2.47 (br s, 4H), 3.57 (s, 2H), 3.72
(t, J = 4.7 Hz, 4H), 7.46 (dd, J = 7.7 Hz, 1H), 7.55 (d, J = 7.7 Hz, 1H),
7.63 (d, J = 8.3 Hz, 2H), 7.76–7.82 (m, 3H), 7.86 (s, 1H), 8.02 (br s,
1H); 13C NMR (150 MHz, CDCl3): d = 53.7, 63.0, 66.9, 119.7, 124.1,
125.9, 126.3, 126.4, 127.7, 128.9, 133.0, 134.6, 139.0, 141.0,

S. Nagao et al. / Bioorg. Med. Chem. 22 (2014) 5513–5529
5521
165.8; HRMS–ESI– m/z [M+H]+ calcd for C19H20F3N2O+2:365.1471,
mmol), triethylamine (694
lL, 4.98 mmol) and 4-dimethylamino-
found: 365.1471.
pyridine (203 mg, 1.66 mmol) in DCM (15 mL) was stirred at room
temperature for 10 min. To the stirred mixture was added 4-ami-
5.1.10. 2-Methyl-N-(4-(trifluoromethyl)phenyl)benzamide (10)
To a stirred solution of 2e (300 mg, 1.86 mmol) in THF (6.0 mL)
nobenzotrifluoride (309 lL, 2.49 mmol). The mixture was stirred
at room temperature for 20.5 h. The mixture was diluted with
water and EtOAc. The organic extract was washed with brine, dried
over Na2SO4, filtrated and then concentrated. The crude solid was
washed with n-heptane/Et2O and filtrated to afford the title com-
pound 13b as a light brown solid (400 mg, 1.23 mmol, 74.3%).
1H NMR (600 MHz, CDCl3): d = 4.06 (s, 3H), 7.67 (d, J = 8.5 Hz,
2H), 7.81 (d, J = 8.5 Hz, 2H), 8.09 (br s, 1H), 8.28 (d, J = 8.2 Hz,
1H), 8.37 (dd, J = 8.2, 1.8 Hz, 1H), 9.20 (d, J = 1.8 Hz, 1H); 13C
NMR (150 MHz, CDCl3): d = 53.3, 120.1, 123.9, 125.1, 126.5,
127.2, 132.9, 136.6, 140.2, 147.9, 150.5, 163.0, 164.7; HRMS–ESI
m/z [M+H]+ calcd for C15H12F3N2O+3: 325.0795, found: 325.0793.
were added triethylamine (519
lL, 3.72 mmol) and o-toluoyl chlo-
ride 9 (291 L, 2.23 mmol) at 0 °C. The mixture was stirred at room
l
temperature for 3 h. The mixture was diluted with water and
EtOAc. The organic extract was washed with brine, dried over
Na2SO4, filtrated through a NH silica gel pad and then concentrated
to afford the title compound 10 as a colorless solid (quant.).
1H NMR (600 MHz, CDCl3): d = 2.51 (s, 3H), 7.25–7.31 (m, 2H),
7.39 (dd, J = 7.6, 7.6 Hz, 1H), 7.48 (d, J = 7.6 Hz, 1H), 7.58–7.66
(m, 3H), 7.74 (d, J = 7.9 Hz, 2H); 13C NMR (150 MHz, CDCl3):
d = 19.8, 119.4, 124.1, 126.0, 126.3, 126.4, 126.5, 130.7, 131.5,
135.8, 136.7, 141.0, 168.1; HRMS–ESI m/z [M+H]+ calcd for
C15H13F3NO+: 280.0944, found: 280.0945.
5.1.14. 5-(Hydroxymethyl)-N-(4-(trifluoromethyl)phenyl)
picolinamide (14a)
5.1.11. 2-(Morpholinomethyl)-N-(4-(trifluoromethyl)phenyl)
benzamide (11)
To a stirred solution of 13a (500 mg, 1.54 mmol) in THF (30 mL)
was added 2 M LiBH4 in THF solution (3.08 mL, 6.16 mmol) at room
temperature. The reaction mixture was stirred at room tempera-
ture for 80 min. The mixture was diluted with water and EtOAc.
The aqueous layer was extracted with EtOAc. The combined
organic extracts were washed with water and brine, dried over
Na2SO4, filtrated and then concentrated. The residue was purified
with column chromatography on NH silica gel (n-heptane/
EtOAc = 1:1). The target fractions were collected and evaporated
to afford the title compound 14a as a white solid (269 mg,
0.908 mmol, 58.9%).
To a stirred solution of 10 (100 mg, 0.358 mmol) in CCl4 (4 mL)
were added N-bromosuccinimide (70.1 mg, 0.394 mmol) and ben-
zoyl peroxide (4.34 mg, 0.0179 mmol) at room temperature. The
stirred reaction mixture was refluxed for 110 min. The reaction
mixture was cooled to room temperature and filtrated. The filtrate
was evaporated. To the residue was added THF (1 mL) and mor-
pholine (311 lL, 3.57 mmol) at room temperature. The reaction
mixture was stirred at room temperature for overnight. The mix-
ture was diluted with water and EtOAc. The organic extract was
washed with brine, dried over Na2SO4, filtrated and then concen-
trated. The residue was purified with column chromatography on
NH silica gel (n-heptane/EtOAc = 5:1). The resulting solid was
washed with n-hexane/Et2O and filtrated to afford the title com-
pound 11 as a white solid (15.4 mg, 0.0423 mmol, 11.8%).
1H NMR (600 MHz, CDCl3): d = 2.59 (br s, 4H), 3.68 (s, 2H), 3.79
(t, J = 4.3 Hz, 4H), 7.24 (d, J = 7.4 Hz, 1H), 7.43–7.50 (m, 2H), 7.63 (d,
J = 8.3 Hz, 2H), 7.82 (d, J = 8.3 Hz, 2H), 8.03 (dd, J = 7.4, 1.5 Hz, 1H),
11.87 (br s, 1H); 13C NMR (150 MHz, CDCl3): d = 52.8, 62.8, 66.7,
120.1, 124.1, 126.0, 126.3, 128.9, 130.8, 131.6, 132.1, 132.6,
137.0, 142.0, 166.9; HRMS–ESI m/z [M+H]+ calcd for C19H20F3N2O2+:
365.1471, found: 365.1468.
1H NMR (600 MHz, CDCl3): d = 1.97–2.10 (m, 1H), 4.86 (br s,
2H), 7.64 (d, J = 8.3 Hz, 2H), 7.88–7.95 (m, 3H), 8.28 (d, J = 7.9 Hz,
1H), 8.62 (d, J = 1.1 Hz, 1H), 10.16 (br s, 1H); 13C NMR (150 MHz,
CDCl3): d = 62.4, 119.3, 122.4, 124.1, 126.1, 126.4, 136.1, 139.7,
140.7, 146.6, 148.6, 162.2; HRMS–ESI m/z [M+H]+ calcd for
C
14H12F3N2O+2: 297.0845, found: 297.0845.
5.1.15. 6-(Hydroxymethyl)-N-(4-(trifluoromethyl)phenyl)
nicotinamide (14b)
The title compound was prepared from 13b using a method
analogous to that described for 14a in 42.3% yield as a white solid.
1H NMR (600 MHz, CDCl3): d = 3.46 (br s, 1H), 4.87 (s, 2H), 7.45
(d, J = 7.9 Hz, 1H), 7.66 (d, J = 8.3 Hz, 2H), 7.79 (d, J = 8.3 Hz, 2H),
7.96 (br s, 1H), 8.17–8.26 (m, 1H), 9.06 (s, 1H); 13C NMR
(150 MHz, CDCl3): d = 64.2, 120.0, 120.5, 124.0, 126.5, 126.9,
129.0, 136.0, 140.5, 146.9, 163.1, 163.7; HRMS–ESI m/z [M+H]+
calcd for C14H12F3N2O+2: 297.0845, found: 297.0844.
5.1.12. Methyl 6-((4-(trifluoromethyl)phenyl)carbamoyl)
nicotinate (13a)
A
stirred mixture of 6-(methoxycarbonyl)nicotinic acid
(300 mg, 1.66 mmol) in SOCl2 (3 mL) was refluxed for 40 min.
The reaction mixture was evaporated. To a stirred solution of 4-
aminobenzotrifluoride (250
l
L, 1.99 mmol) in THF (6 mL) were
5.1.16. 5-(Morpholinomethyl)-N-(4-(trifluoromethyl)phenyl)
picolinamide (15a)
To a stirred solution of 14a (269 mg, 0.908 mmol) in DCM
(6 mL) and THF (1 mL) were added 4-dimethylaminopyridine
added triethylamine (1.16 mL, 8.3 mmol) and prepared acid chlo-
ride in THF at room temperature. The reaction mixture was stirred
at room temperature for 50 min. The mixture was diluted with
water and EtOAc. The organic extract was washed with brine, dried
over Na2SO4, filtrated and then concentrated. The crude solid was
washed with n-heptane/Et2O and filtrated to afford the title com-
pound 19a as a white solid (360 mg, 1.11 mmol, 66.9%).
(20 mg,
6.29 mmol) and pyridine (110
ture. The reaction mixture was stirred at room temperature for
25 min. To the stirred mixture was added triethylamine (253 L,
0.164 mmol),
p-toluenesulfonyl
chloride
(1.2 g,
lL, 1.36 mmol) at room tempera-
l
1H NMR (600 MHz, CDCl3): d = 4.01 (s, 3H), 7.66 (d, J = 8.3 Hz,
2H), 7.92 (d, J = 8.3 Hz, 2H), 8.39 (d, J = 7.9 Hz, 1H), 8.53 (dd,
J = 7.9, 1.5 Hz, 1H), 9.22 (d, J = 1.5 Hz, 1H), 10.16 (br s, 1H); 13C
NMR (150 MHz, CDCl3): d = 52.8, 119.5, 122.2, 124.1, 126.4,
126.4, 128.7, 139.1, 140.4, 149.3, 152.2, 161.3, 164.9; HRMS–ESI
m/z [M+H]+ calcd for C15H12F3N2O+3: 325.0795, found: 325.0792.
1.83 mmol). The mixture was stirred at room temperature for
70 min. The mixture was diluted with water and EtOAc. The
organic extract was washed with brine, dried over Na2SO4, filtrated
and then concentrated. To the residue in DMF (5 mL) were added
morpholine (791 lL, 9.07 mmol) and triethylamine (1.26 mL,
9.07 mmol) at room temperature. The mixture was stirred at room
temperature for 10 min. The mixture was diluted with water and
EtOAc. The organic extract was washed with brine, dried over
Na2SO4, filtrated and then concentrated. The residue was purified
with column chromatography on NH silica gel (n-heptane/
EtOAc = 12:1–4:1). The target fractions were collected and
5.1.13. Methyl 5-((4-(trifluoromethyl)phenyl)carbamoyl)
picolinate (13b)
A mixture of 6-(methoxycarbonyl)nicotinic acid 12b (300 mg,
1.66 mmol), 2-methyl-6-nitrobenzoic anhydride (572 mg, 1.66

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S. Nagao et al. / Bioorg. Med. Chem. 22 (2014) 5513–5529
evaporated. The resulting solid was washed with n-hexane/Et2O
and filtrated to afford the title compound 15a as a white solid
(196 mg, 0.535 mmol, 59.0%).
added NaH (757 mg, 18.9 mmol, 60% w/w) at 0 °C under nitrogen.
To the mixture was added 4-(trifluoromethyl)benzaldehyde 20 at
0 °C. At the same temperature, the mixture was stirred for
70 min. The reaction mixture was stirred at room temperature
for 35 min. The mixture was diluted with saturated NH4Cl and
EtOAc. The organic extract was washed with water and brine, dried
over MgSO4, filtrated and then concentrated. The crude solid was
washed with n-hexane and filtrated to afford the title compound
21 as a white solid (3.92 g, 12.8 mmol, 74.4%).
1H NMR (600 MHz, CDCl3): d = 2.43–2.53 (m, 4H), 3.61 (s, 2H),
3.70–3.76 (m, 4H), 7.64 (d, J = 8.3 Hz, 2H), 7.88–7.95 (m, 3H),
8.26 (d, J = 7.9 Hz, 1H), 8.58 (d, J = 1.1 Hz, 1H), 10.16 (br s, 1H);
13C NMR (150 MHz, CDCl3): d = 53.6, 60.3, 66.9, 119.3, 122.3,
124.1, 126.0, 126.4, 137.4, 138.3, 140.8, 148.4, 148.6, 162.3;
HRMS–ESI m/z [M+H]+ calcd for C18H19F3N3O+2: 366.1424, found:
366.1422.
1H NMR (600 MHz, CDCl3): d = 3.93 (s, 3H), 7.21 (d, J = 1.9 Hz,
2H), 7.58 (d, J = 8.3 Hz, 2H), 7.62 (s, 4H), 8.05 (d, J = 8.3 Hz, 2H);
13C NMR (150 MHz, CDCl3): d = 52.1, 124.1, 125.7, 126.6, 126.9,
129.6, 129.8, 130.1, 130.1, 140.2, 141.0, 166.7; HRMS–ESI m/z
[M+H]+ calcd for C17H14F3O+2: 307.0940, found: 307.0937.
5.1.17. 6-(Morpholinomethyl)-N-(4-(trifluoromethyl)phenyl)-
nicotinamide (15b)
The title compound was prepared from 14b using a method
analogous to that described for 15a in 29.7% yield as a light yellow
solid.
5.1.21. (E)-(4-(4-(Trifluoromethyl)styryl)phenyl)methanol (22)
To a stirred suspension of 21 (1.0 g, 3.27 mmol) in toluene
(60 mL) was added dropwise DIBAL in toluene (3.24 mL,
3.27 mmol, 1.01 M) at ꢀ78 °C under nitrogen. At the same temper-
ature, the mixture was stirred for 70 min. The stirred mixture was
allowed to warm to room temperature. The mixture was stirred for
30 min. The reaction mixture was recooled to ꢀ78 °C. At the same
temperature, DIBAL in toluene (4.86 mL, 4.91 mmol, 1.01 M) was
added to the reaction mixture. The stirred mixture was warmed
to room temperature. The mixture was stirred for 30 min. To the
stirred mixture was added 5 N HCl. The mixture was stirred for
90 min. The mixture was diluted with EtOAc. The organic extract
was washed with water and brine, dried over MgSO4, filtrated
and then concentrated. The crude solid was washed with n-hexane
and filtrated to afford the title compound 22 as a white solid
(884 mg, 3.18 mmol, 97.1%).
1H NMR (600 MHz, CDCl3): d = 2.49–2.57 (m, 4H), 3.72–3.77 (m,
6H), 7.61 (d, J = 7.9 Hz, 1H), 7.65 (d, J = 8.7 Hz, 2H), 7.78 (d,
J = 8.7 Hz, 2H), 8.02 (s, 1H), 8.19 (dd, J = 7.9, 2.1 Hz, 1H), 9.04 (d,
J = 2.1 Hz, 1H); 13C NMR (150 MHz, CDCl3): d = 53.8, 64.6, 66.9,
119.9, 123.2, 123.9, 126.4, 126.8, 128.7, 135.8, 140.5, 147.4,
162.5, 163.9; HRMS–ESI m/z [M+H]+ calcd for C18H19F3N3O2+:
366.1424, found: 366.1418.
5.1.18. 5-(Hydroxymethyl)thiophene-2-carboxylic acid (17)
To a stirred solution of 5-formyl-2-thiophenecarboxylic acid 16
(5.0 g, 32.0 mmol) in MeOH (160 mL) was added NaBH4 (1.82 g,
48.0 mmol) at 0 °C under nitrogen. The mixture was stirred at
room temperature for 8 h. To the reaction mixture was added ace-
tone and then evaporated. The residue was poured into 2 N HCl
diluted with EtOAc. The aqueous layer was extracted with EtOAc.
The combined organic extracts were washed with brine, dried over
MgSO4, filtrated and then concentrated. The crude solid was
washed with n-hexane/Et2O and filtrated to afford the title com-
pound 17 as a light brown solid (quant.).
1H NMR (600 MHz, CDCl3): d = 1.68 (t, J = 4.8 Hz, 1H), 4.72 (d,
J = 4.8 Hz, 2H), 7.12 (d, J = 16.1 Hz, 1H), 7.19 (d, J = 16.1 Hz, 1H),
7.38 (d, J = 7.9 Hz, 2H), 7.53 (d, J = 7.9 Hz, 2H), 7.57–7.63 (m, 4H);
13C NMR (150 MHz, CDCl3): d = 65.1, 124.2, 125.6, 126.6, 127.0,
127.2, 127.4, 129.3, 130.8, 136.1, 140.8, 141.0; HRMS–ESI m/z
[MꢀH]+ calcd for C16H12F3Oꢀ: 277.0846, found: 277.0838.
1H NMR (600 MHz, DMSO-d6): d = 4.66 (s, 2H), 5.66 (br s, 1H),
6.99 (d, J = 3.8 Hz, 1H), 7.58 (d, J = 3.8 Hz, 1H), 12.90 (br s, 1H);
13C NMR (150 MHz, DMSO-d6): d = 58.5, 124.3, 132.6, 133.0,
154.4, 163.0; HRMS–ESI m/z [M+H]+ calcd for C6H7O3S+:
159.0110, found: 159.0112.
5.1.22. (E)-4-(4-(Trifluoromethyl)styryl)benzaldehyde (23)
To a stirred mixture of 22 (884 mg, 3.18 mmol) in DCM
(100 mL) was added manganese (IV) oxide (8.8 g, 101 mmol) at
room temperature. The mixture was stirred at room temperature
for 75 min. The reaction mixture was filtrated through a pad of Cel-
iteÒ. The filtrate was evaporated to afford the title compound 23 as
a white solid (quant.).
5.1.19. 5-(Morpholinomethyl)-N-(4-(trifluoromethyl)phenyl)-
thiophene-2-carboxamide (18)
A stirred solution of 17 (100 mg, 0.631 mmol) in SOCl2 (2 mL)
was refluxed for 1 h. The reaction mixture was evaporated. To a
stirred solution of 2e (102 mg, 0.631 mmol) in THF (3 mL) were
added triethylamine (440 lL, 3.16 mmol) and prepared acid
chloride in THF at room temperature. The reaction mixture was
stirred at room temperature for 35 min. To the reaction mixture
was added morpholine (550 lL, 6.31 mmol). The reaction mixture
1H NMR (600 MHz, CDCl3): d = 7.21–7.29 (m, 2H), 7.64 (s, 4H),
7.68 (d, J = 7.9 Hz, 2H), 7.90 (d, J = 7.9 Hz, 2H), 10.02 (s, 1H); 13C
NMR (150 MHz, CDCl3): d = 124.1, 125.8, 127.0, 127.2, 129.8,
130.1, 130.3, 130.5, 135.8, 140.0, 142.6, 191.5; HRMS–ESI m/z
[M+H]+ calcd for C16H12F3O+: 277.0835, found: 277.0832.
was stirred at room temperature for 22 h. The mixture was
diluted with water and EtOAc. The organic extract was washed
with brine, dried over Na2SO4, filtrated and then concentrated.
The crude solid was washed with n-hexane/Et2O and filtrated
5.1.23. (E)-4-(4-(4-(Trifluoromethyl)styryl)benzyl)morpholine
(24)
To a stirred mixture of 23 (879 mg, 3.18 mmol) in 1,2-dichloro-
to afford the title compound 18 as
a
brown solid (96 mg,
ethane (30 mL) were added morpholine (416
lL, 4.77 mmol), AcOH
0.259 mmol, 41.1%).
(179 L, 3.18 mmol) and NaBH(OAc)3 (1.01 g, 4.77 mmol) at room
l
1H NMR (600 MHz, CDCl3): d = 2.53 (br s, 4H), 3.70–3.76 (m,
6H), 6.95 (d, J = 3.8 Hz, 1H), 7.53 (d, J = 3.8 Hz, 1H), 7.61 (d,
J = 8.7 Hz, 2H), 7.73 (d, J = 8.7 Hz, 2H), 7.77 (br s, 1H); 13C NMR
(150 MHz, CDCl3): d = 53.5, 57.7, 66.9, 119.6, 124.0, 126.2, 126.3,
126.4, 129.1, 137.3, 140.8, 149.1, 160.0; HRMS–ESI m/z [M+H]+
calcd for C17H18F3N2O2S+: 371.1036, found: 371.1033.
temperature. The mixture was stirred at room temperature for 2 h.
The reaction mixture was poured into saturated NaHCO3 aqueous
solution and diluted with EtOAc. The organic extract was washed
with water and brine, dried over Na2SO4, filtrated and then concen-
trated. The crude solid was washed with n-hexane and filtrated to
afford the title compound 24 as a white solid (834 mg, 2.40 mmol,
75.5%).
5.1.20. (E)-Methyl 4-(4-(trifluoromethyl)styryl)benzoate (21)
1H NMR (600 MHz, CDCl3): d = 2.46 (br s, 4H), 3.51 (s, 2H), 3.72
(t, J = 4.5 Hz, 4H), 7.10 (d, J = 16.2 Hz, 1H), 7.18 (d, J = 16.2 Hz, 1H),
7.34 (d, J = 8.1 Hz, 2H), 7.49 (d, J = 8.1 Hz, 2H), 7.60 (s, 4H); 13C NMR
To
a
stirred solution of methyl 4-((diethoxyphospho-
ryl)methyl)benzoate 1920 (5.42 g, 18.9 mmol) in THF (60 mL) was

S. Nagao et al. / Bioorg. Med. Chem. 22 (2014) 5513–5529
5523
(150 MHz, CDCl3): d = 53.7, 63.1, 67.0, 124.2, 125.6, 126.5, 126.7,
5.1.27. 4-(Chloromethyl)-N-(4-(trifluoromethyl)phenyl)-
benzamide (31)
The title compound was prepared from 2d and 3 using a
method analogous to that described for 39 in 95.9% yield as a white
solid.
126.9, 129.2, 129.6, 130.9, 135.6, 138.2, 140.8; HRMS–ESI m/z
[M+H]+ calcd for C20H21F3NO+: 348.1570, found: 348.1570.
5.1.24. 4-Iodo-N-(4-(morpholinomethyl)benzyl)aniline (26)
To a stirred mixture of 4-(morpholinomethyl)benzaldehyde
2522 (100 mg, 0.487 mmol) in THF (2 mL) were added 4-iodoani-
line (160 mg, 0.731 mmol) and NaBH(OAc)3 (206 mg, 0.974 mmol)
at room temperature. The mixture was stirred at room tempera-
ture for 16 h. The reaction mixture was diluted with saturated
NaHCO3 aqueous solution and EtOAc. The organic extract was
washed with water and brine, dried over Na2SO4, filtrated and
then concentrated. The residue was purified with column chro-
matography on silica gel (n-heptane/EtOAc = 3:2–1:1). The result-
ing solid was washed with n-hexane and filtrated to afford the
title compound 26 as colorless crystals (67.6 mg, 0.166 mmol,
34.0%).
1H NMR (600 MHz, DMSO-d6): d = 4.85 (s, 2H), 7.61 (d,
J = 7.9 Hz, 2H), 7.73 (d, J = 8.7 Hz, 2H), 7.98 (d, J = 7.9 Hz, 2H),
8.01 (d, J = 8.7 Hz, 2H), 10.59 (s, 1H); 13C NMR (150 MHz, DMSO-
d6): d = 45.3, 120.1, 123.6, 124.3, 125.9, 128.1, 128.8, 134.3,
141.4, 142.7, 165.5; HRMS–ESI m/z [M+H]+ calcd for C15H12ClF3
NO+: 314.0554, found: 314.0550.
5.1.28. 4-((4-Benzylpiperazin-1-yl)methyl)-N-(4-
(trifluoromethyl)phenyl)benzamide (32a)
The title compound was prepared from 31 using a method anal-
ogous to that described for 40 in 84.3% yield as a white solid.
1H NMR (600 MHz, CDCl3): d = 2.49 (br s, 8H), 3.52 (s, 2H), 3.57
(s, 2H), 7.22–7.34 (m, 5H), 7.46 (d, J = 8.3 Hz, 2H), 7.62 (d,
J = 8.3 Hz, 2H), 7.77 (d, J = 8.3 Hz, 2H), 7.81 (d, J = 8.3 Hz, 2H),
7.94 (br s, 1H); 13C NMR (150 MHz, CDCl3): d = 53.0, 53.2, 62.5,
63.0, 119.7, 124.1, 126.3, 126.4, 127.0, 127.0, 128.2, 129.2, 129.5,
133.1, 138.0, 141.0, 143.3, 165.7; HRMS–ESI m/z [M+H]+ calcd for
C26H27F3N3O+: 454.2101, found: 454.2092.
1H NMR (600 MHz, CDCl3): d = 2.43 (br s, 4H), 3.48 (s, 2H), 3.70
(t, J = 4.5 Hz, 4H), 4.07 (br s, 1H), 4.28 (d, J = 4.9 Hz, 2H), 6.41 (d,
J = 8.7 Hz, 2H), 7.27–7.32 (m, 4H), 7.40 (d, J = 8.7 Hz, 2H); 13C
NMR (150 MHz, CDCl3): d = 47.8, 53.6, 63.1, 67.0, 78.1, 115.1,
127.3, 129.5, 137.0, 137.7, 137.8, 147.6; HRMS–ESI m/z [M+H]+
calcd for C18H22IN2O+: 409.0771, found: 409.0766.
5.1.25. N-(4-Iodophenyl)-N-methyl-4-(morpholinomethyl)-
benzamide hydrochloride (27)
5.1.29. 4-((4-(Pyridin-4-ylmethyl)piperazin-1-yl)methyl)-N-(4-
(trifluoromethyl)phenyl)benzamide (32b)
To a stirred solution of N-(4-Iodophenyl)-4-(morpholinometh-
yl)benzamide 4d (100 mg, 0.237 mmol) in DMF (2 mL) was added
NaH (8.53 mg, 0.356 mmol, 60% w/w) washed with pentane at
room temperature under nitrogen. The mixture was stirred for
The title compound was prepared from 31 using a method anal-
ogous to that described for 40 in 91.9% yield as a white solid.
1H NMR (600 MHz, CDCl3): d = 2.50 (br s, 8H), 3.51 (s, 2H), 3.59
(s, 2H), 7.25–7.28 (m, 2H), 7.46 (d, J = 8.3 Hz, 2H), 7.62 (d,
J = 8.7 Hz, 2H), 7.78 (d, J = 8.7 Hz, 2H), 7.83 (d, J = 8.3 Hz, 2H),
8.02 (br s, 1H), 8.53 (d, J = 5.7 Hz, 2H); 13C NMR (150 MHz, CDCl3):
d = 53.1, 53.1, 61.7, 62.5, 119.7, 123.9, 124.1, 126.2, 126.4, 127.1,
129.4, 133.2, 141.1, 143.1, 147.6, 149.7, 165.7; HRMS–ESI m/z
[M+H]+ calcd for C25H26F3N4O+: 455.2053, found: 455.2045.
25 min. To the reaction mixture was added MeI (14.8 lL,
0.237 mmol). The mixture was stirred at room temperature for
50 min. The reaction mixture was poured into water and extracted
with EtOAc. The organic extract was washed with water and brine,
dried over Na2SO4, filtrated and then concentrated. The residue
was purified with column chromatography on NH silica gel (n-hep-
tane/EtOAc = 2:1). The resulting oil was treated with 4 N HCl in
EtOAc to afford the title compound 27 as a white solid (101 mg,
0.214 mmol, 90.1%).
5.1.30. 4-((4-(Pyridin-3-ylmethyl)piperazin-1-yl)methyl)-N-(4-
(trifluoromethyl)phenyl)benzamide (32c)
The title compound was prepared from 31 using a method anal-
ogous to that described for 40 in 92.3% yield as a white solid.
1H NMR (600 MHz, CDCl3): d = 2.49 (br s, 8H), 3.52 (s, 2H), 3.58
(s, 2H), 7.23–7.28 (m, 1H), 7.46 (d, J = 8.3 Hz, 2H), 7.62 (d,
J = 8.5 Hz, 2H), 7.66 (d, J = 7.6 Hz, 1H), 7.78 (d, J = 8.5 Hz, 2H),
7.83 (d, J = 8.3 Hz, 2H), 8.00 (s, 1H), 8.50 (dd, J = 4.7, 1.4 Hz, 1H),
8.54 (d, J = 1.4 Hz, 1H); 13C NMR (150 MHz, CDCl3): d = 53.0, 53.1,
60.2, 62.4, 119.7, 123.3, 124.1, 126.2, 126.3, 127.1, 129.4, 133.2,
133.6, 136.7, 141.1, 143.2, 148.6, 150.5, 165.7; HRMS–ESI m/z
[M+H]+ calcd for C25H26F3N4O+: 455.2053, found: 455.2042.
1H NMR (600 MHz, DMSO-d6): d = 2.95–3.05 (m, 2H), 3.11 (d,
J = 12.1 Hz, 2H), 3.35 (s, 3H), 3.77 (t, J = 11.7 Hz, 2H), 3.91
(d, J = 12.1 Hz, 2H), 4.26 (br s, 2H), 7.00 (d, J = 8.7 Hz, 2H), 7.33
(d, J = 7.6 Hz, 2H), 7.50 (d, J = 7.6 Hz, 2H), 7.60 (d, J = 8.7 Hz, 2H),
11.27 (br s, 1H); 13C NMR (150 MHz, DMSO-d6): d = 37.6, 50.6,
58.4, 63.0, 92.0, 128.3, 129.3, 130.4, 130.9, 137.2, 137.8, 144.0,
168.8; HRMS–ESI m/z [M+H]+ calcd for C19H22IN2O+2: 437.0720,
found: 437.0715.
5.1.26. 4-Iodo-N-(4-(morpholinomethyl)phenyl)benzamide (30)
To a stirred mixture of 4-(morpholinomethyl)aniline 2823,24
(50 mg, 0.260 mmol) in THF (3 mL) were added pyridine
(30.8 mg, 0.390 mmol) and 4-iodobenzoyl chloride (76.2 mg,
0.286 mmol) at room temperature. The mixture was stirred at
room temperature for 2 h. The reaction mixture was diluted with
1 N NaOH aqueous solution, THF and EtOAc. The organic extract
was washed with water and brine, dried over MgSO4, filtrated
and then concentrated. The crude solid was recrystallized from
EtOAc/Et2O/n-hexane. The suspension was filtrated and washed
with Et2O to afford the title compound 30 as light brown solid
(84.0 mg, 0.199 mmol, 76.5%).
5.1.31. N1,N2-Dimethyl-N1-(pyridin-3-ylmethyl)ethane-1,2-
diamine
To a stirred solution of N,N0-dimethylethylenediamine (1.55 mL,
11.9 mmol) in DMF (8.0 mL) was added triethylamine (3.3 mL,
23.8 mmol) at room temperature under nitrogen. To the mixture
was added dropwise 3-bromomethylpyridine hydrobromide
(1.0 g, 3.95 mmol) at room temperature. The mixture was stirred
at room temperature for 3.5 h. The reaction mixture was diluted
with water and CHCl3. The aqueous layer was extracted with
CHCl3. The combined organic extracts were dried over MgSO4, fil-
trated and then concentrated. The residue was purified with col-
umn chromatography on NH silica gel (EtOAc/MeOH = 1:0–9:1)
to afford the title compound as colorless liquid (562 mg,
3.14 mmol, 79.4%).
1H NMR (600 MHz, CDCl3): d = 2.44 (br s, 4H), 3.49 (s, 2H), 3.71
(t, J = 4.7 Hz, 4H), 7.34 (d, J = 8.3 Hz, 2H), 7.53–7.64 (m, 4H), 7.75
(br s, 1H), 7.84 (d, J = 8.3 Hz, 2H); 13C NMR (150 MHz, CDCl3):
d = 53.6, 62.9, 67.0, 98.9, 120.1, 128.6, 129.9, 134.4, 134.4, 136.6,
138.0, 164.9; HRMS–ESI m/z [M+H]+ calcd for C18H20IN2O2+:
423.0564, found: 423.0561.
1H NMR (600 MHz, CDCl3): d = 2.19 (s, 3H), 2.42 (s, 3H), 2.55 (t,
J = 5.9 Hz, 2H), 2.68 (t, J = 5.9 Hz, 2H), 3.52 (s, 2H), 7.22–7.30 (m,
1H), 7.66 (d, J = 7.9 Hz, 1H), 8.46–8.55 (m, 2H); 13C NMR

5524
S. Nagao et al. / Bioorg. Med. Chem. 22 (2014) 5513–5529
(150 MHz, CDCl3): d = 36.4, 41.9, 49.2, 56.7, 59.9, 123.3, 134.5,
136.5, 148.5, 150.3; HRMS–ESI m/z [M+H]+ calcd for C10H18N3+:
180.1495, found: 180.1493.
5.1.36. (R)-4-((2-Methylpiperazin-1-yl)methyl)-N-(4-
(trifluoromethyl)phenyl)benzamide (34b)
The title compound was prepared from 33b using a method
analogous to that described for 34a in 78.0% as a colorless solid.
1H NMR (600 MHz, CDCl3): d = 1.12 (d, J = 6.4 Hz, 3H), 2.08 (td,
J = 10.9, 3.2 Hz, 1H), 2.33–2.44 (m, 1H), 2.56–2.66 (m, 2H), 2.76–
2.88 (m, 2H), 2.92 (dd, J = 12.1, 1.9 Hz, 1H), 3.23 (d, J = 13.8 Hz,
1H), 4.09 (d, J = 13.8 Hz, 1H), 7.48 (d, J = 7.9 Hz, 2H), 7.62 (d,
J = 8.7 Hz, 2H), 7.78 (d, J = 8.7 Hz, 2H), 7.82 (d, J = 7.9 Hz, 2H),
8.00 (br s, 1H); 13C NMR (150 MHz, CDCl3): d = 16.7, 46.4, 52.7,
53.6, 56.7, 58.2, 119.7, 124.1, 126.2, 126.3, 127.0, 129.3, 132.9,
141.1, 144.3, 165.7; HRMS–ESI m/z [M+H]+ calcd for C20H23F3N3O+:
378.1788, found: 378.1780.
5.1.32. 4-((Methyl(2-(methyl(pyridin-3-ylmethyl)amino)
ethyl)amino)methyl)-N-(4-(trifluoromethyl)phenyl)
benzamide (32d)
The title compound was prepared from 31 using a method anal-
ogous to that described for 40 in 72.4% yield as a white solid.
1H NMR (600 MHz, CDCl3): d = 2.26 (s, 3H), 2.31 (s, 3H), 2.39–
2.47 (m, 4H), 3.46 (s, 2H), 3.54 (s, 2H), 7.24–7.29 (m, 1H), 7.37
(d, J = 7.9 Hz, 2H), 7.60–7.65 (m, 3H), 7.76 (d, J = 7.9 Hz, 2H), 7.86
(d, J = 8.3 Hz, 2H), 8.11 (s, 1H), 8.50 (dd, J = 4.7, 1.3 Hz, 1H), 9.27
(br s, 1H); 13C NMR (150 MHz, CDCl3): d = 43.2, 43.7, 54.4, 54.6,
59.3, 62.6, 119.7, 123.4, 124.2, 125.9, 126.2, 127.4, 129.1, 133.9,
134.5, 136.9, 141.8, 143.8, 148.0, 150.1, 166.9; HRMS–ESI m/z
[M+H]+ calcd for C25H28F3N4O+: 457.2210, found: 457.2199.
5.1.37. (R)-4-((3-Methylpiperazin-1-yl)methyl)-N-(4-
(trifluoromethyl)phenyl)benzamide (34c)
The title compound was prepared from 31 using a method anal-
ogous to that described for 40 in 64.4% as a colorless solid.
1H NMR (600 MHz, CDCl3): d = 1.02 (d, J = 6.0 Hz, 3H), 1.70 (t,
J = 10.6 Hz, 1H), 2.05 (td, J = 11.0, 3.4 Hz, 1H), 2.74 (d, J = 10.6 Hz,
2H), 2.84–2.98 (m, 3H), 3.51–3.60 (m, 2H), 7.48 (d, J = 7.9 Hz,
2H), 7.63 (d, J = 8.5 Hz, 2H), 7.78 (d, J = 8.5 Hz, 2H), 7.83 (d,
J = 7.9 Hz, 2H), 7.94 (br s, 1H); 13C NMR (150 MHz, CDCl3):
d = 20.0, 45.9, 50.5, 53.8, 61.4, 62.9, 119.7, 124.1, 126.3, 126.4,
127.0, 129.5, 133.1, 141.0, 143.3, 165.7; HRMS–ESI m/z [M+H]+
calcd for C20H23F3N3O+: 378.1788, found: 378.1778.
5.1.33. (S)-tert-Butyl 3-methyl-4-(4-((4-(trifluoromethyl)
phenyl)carbamoyl)benzyl)piperazine-1-carboxylate (33a)
The title compound was prepared from 31 using a method anal-
ogous to that described for 40 in quant as a colorless solid.
1H NMR (600 MHz, CDCl3): d = 1.12 (d, J = 6.0 Hz, 3H), 1.46 (s,
9H), 2.04–2.17 (m, 1H), 2.43–2.51 (m, 1H), 2.61 (d, J = 11.3 Hz,
1H), 2.81–3.00 (m, 1H), 3.04–3.16 (m, 1H), 3.28 (d, J = 14.0 Hz,
1H), 3.59–3.85 (m, 2H), 4.03 (d, J = 13.2 Hz, 1H), 7.48 (d,
J = 7.9 Hz, 2H), 7.63 (d, J = 8.3 Hz, 2H), 7.78 (d, J = 8.3 Hz, 2H),
7.83 (d, J = 7.9 Hz, 2H), 7.97 (br s, 1H); 13C NMR (150 MHz, CDCl3):
d = 15.3, 28.4, 43.3, 44.2, 50.6, 55.0, 57.7, 79.6, 119.7, 124.1, 126.3,
126.4, 127.1, 129.2, 133.1, 141.0, 144.0, 154.7, 165.6; HRMS–ESI
m/z [M+H]+ calcd for C25H31F3N3O+3: 478.2312, found: 478.2301.
5.1.38. (S)-4-((3-Methylpiperazin-1-yl)methyl)-N-(4-
(trifluoromethyl)phenyl)benzamide (34d)
The title compound was prepared from 31 using a method anal-
ogous to that described for 40 in 70.8% as a colorless solid.
1H NMR (600 MHz, CDCl3): d = 1.01 (d, J = 6.0 Hz, 3H), 1.70 (t,
J = 10.5 Hz, 1H), 2.02–2.08 (m, 1H), 2.74 (d, J = 10.5 Hz, 2H), 2.84–
2.98 (m, 3H), 3.51–3.60 (m, 2H), 7.48 (d, J = 7.9 Hz, 2H), 7.63 (d,
J = 8.5 Hz, 2H), 7.78 (d, J = 8.5 Hz, 2H), 7.83 (d, J = 7.9 Hz, 2H),
7.95 (br s, 1H); 13C NMR (150 MHz, CDCl3): d = 20.0, 45.9, 50.5,
53.8, 61.4, 62.9, 119.7, 124.1, 126.2, 126.4, 127.0, 129.5, 133.1,
141.0, 143.3, 165.7; HRMS–ESI m/z [M+H]+ calcd for C20H23F3N3O+:
378.1788, found: 378.1778.
5.1.34. (R)-tert-Butyl 3-methyl-4-(4-((4-(trifluoromethyl)
phenyl)carbamoyl)benzyl)piperazine-1-carboxylate (33b)
The title compound was prepared from 31 using a method anal-
ogous to that described for 40 in quant as a colorless solid.
1H NMR (600 MHz, CDCl3): d = 1.12 (d, J = 6.4 Hz, 3H), 1.46 (s,
9H), 2.05–2.17 (m, 1H), 2.42–2.51 (m, 1H), 2.61 (d, J = 11.7 Hz,
1H), 2.81–3.00 (m, 1H), 3.04–3.16 (m, 1H), 3.28 (d, J = 13.6 Hz,
1H), 3.59–3.86 (m, 2H), 4.03 (d, J = 13.6 Hz, 1H), 7.47 (d,
J = 8.1 Hz, 2H), 7.63 (d, J = 8.3 Hz, 2H), 7.78 (d, J = 8.3 Hz, 2H),
7.83 (d, J = 8.1 Hz, 2H), 7.98 (br s, 1H); 13C NMR (150 MHz,
CDCl3): d = 15.4, 28.4, 43.3, 44.4, 50.5, 55.1, 57.7, 79.7, 119.7,
124.1, 126.2, 126.4, 127.1, 129.2, 133.1, 141.1, 144.0, 154.7,
165.6; HRMS–ESI m/z [M+H]+ calcd for C25H31F3N3O+3: 478.2312,
found: 478.2301.
5.1.39. (S)-4-((2-Methyl-4-(pyridin-3-ylmethyl)piperazin-1-
yl)methyl)-N-(4-(trifluoromethyl)phenyl)benzamide (35a)
To a solution of 34a (200 mg, 0.529 mmol) in DMF (3 mL) were
added triethylamine (295 lL, 2.12 mmol) and 3-chloromethylpyri-
dine hydrochloride (95.5 mg, 0.582 mmol) at room temperature.
The mixture was stirred at 60 °C for overnight. The residue was
diluted with EtOAc and water. The organic extract was dried over
Na2SO4, filtrated and then concentrated. The residue was purified
with column chromatography on NH silica gel (EtOAc/
MeOH = 1:0–9:1) to afford the title compound 35a as a white solid
(216 mg, 0.461 mmol, 87.1%).
5.1.35. (S)-4-((2-Methylpiperazin-1-yl)methyl)-N-(4-
(trifluoromethyl)phenyl)benzamide (34a)
A solution of 33a (530 mg, 1.11 mmol) in trifluoroacetic acid
(3 mL) was stirred at room temperature for 55 min. The mixture
was evaporated. The residue was diluted with water and 5 N NaOH
aqueous solution. The mixture was extracted with EtOAc. The
organic extract was washed with brine, dried over Na2SO4, filtrated
and then concentrated. The crude solid was washed with n-hep-
tane/Et2O to afford the title compound 34a as a white solid
(361 mg, 0.957 mmol, 86.2%).
1H NMR (600 MHz, CDCl3): d = 1.12 (d, J = 6.4 Hz, 3H), 2.00–2.10
(m, 1H), 2.17–2.28 (m, 2H), 2.53 (br s, 1H), 2.58–2.69 (m, 3H), 3.25
(d, J = 13.6 Hz, 1H), 3.46–3.51 (m, 2H), 4.07 (d, J = 13.6 Hz, 1H),
7.23–7.28 (m, 1H), 7.46 (d, J = 8.1 Hz, 2H), 7.60–7.68 (m, 3H),
7.78 (d, J = 8.3 Hz, 2H), 7.82 (d, J = 8.1 Hz, 2H), 8.00 (br s, 1H),
8.50 (dd, J = 4.7, 1.4 Hz, 1H), 8.53 (d, J = 1.4 Hz, 1H); 13C NMR
(150 MHz, CDCl3): d = 17.0, 51.3, 53.3, 55.3, 57.7, 60.1, 60.6,
119.7, 123.3, 124.1, 126.2, 126.3, 127.0, 129.3, 133.0, 133.6,
136.7, 141.1, 144.2, 148.6, 150.4, 165.7; HRMS–ESI m/z [M+H]+
calcd for C26H28F3N4O+: 469.2210, found: 469.2197.
1H NMR (600 MHz, CDCl3): d = 1.12 (d, J = 6.0 Hz, 3H), 2.08 (td,
J = 10.9, 3.2 Hz, 1H), 2.34–2.43 (m, 1H), 2.56–2.66 (m, 2H), 2.76–
2.88 (m, 2H), 2.92 (dd, J = 12.3, 2.1 Hz, 1H), 3.23 (d, J = 14.0 Hz,
1H), 4.09 (d, J = 14.0 Hz, 1H), 7.48 (d, J = 8.1 Hz, 2H), 7.62 (d,
J = 8.5 Hz, 2H), 7.78 (d, J = 8.5 Hz, 2H), 7.82 (d, J = 8.1 Hz, 2H),
8.00 (br s, 1H); 13C NMR (150 MHz, CDCl3): d = 16.7, 46.4, 52.7,
53.6, 56.7, 58.2, 119.7, 124.1, 126.2, 126.3, 127.0, 129.3, 132.9,
141.1, 144.4, 165.7; HRMS–ESI m/z [M+H]+ calcd for C20H23F3N3O+:
378.1788, found: 378.1780.
5.1.40. (R)-4-((2-Methyl-4-(pyridin-3-ylmethyl)piperazin-1-
yl)methyl)-N-(4-(trifluoromethyl)phenyl)benzamide (35b)
The title compound was prepared from 34b using a method
analogous to that described for 35a in 54.8% as a light brown solid.

S. Nagao et al. / Bioorg. Med. Chem. 22 (2014) 5513–5529
5525
1H NMR (600 MHz, CDCl3): d = 1.12 (d, J = 6.0 Hz, 3H), 2.00–2.11
5.1.44. 4-((1-(Pyridin-4-ylmethyl)piperidin-4-yl)methyl)-N-(4-
(m, 1H), 2.17–2.28 (m, 2H), 2.53 (br s, 1H), 2.57–2.70 (m, 3H), 3.25
(d, J = 14.0 Hz, 1H), 3.45–3.52 (m, 2H), 4.07 (d, J = 14.0 Hz, 1H),
7.23–7.28 (m, 1H), 7.46 (d, J = 8.1 Hz, 2H), 7.59–7.68 (m, 3H),
7.78 (d, J = 8.3 Hz, 2H), 7.82 (d, J = 8.1 Hz, 2H), 8.01 (br s, 1H),
8.50 (dd, J = 4.9, 1.5 Hz, 1H), 8.53 (d, J = 1.5 Hz, 1H); 13C NMR
(150 MHz, CDCl3): d = 17.0, 51.3, 53.3, 55.3, 57.7, 60.1, 60.6,
119.7, 123.3, 124.1, 126.2, 126.3, 127.0, 129.3, 133.0, 133.6,
136.7, 141.1, 144.2, 148.6, 150.4, 165.7; HRMS–ESI m/z [M+H]+
calcd for C26H28F3N4O+: 469.2210, found: 469.2197.
(trifluoromethyl)phenyl)benzamide (38)
A solution of 37 (191 mg, 0.413 mmol) in trifluoroacetic acid
(3 mL) was stirred at 0 °C for 15 min. The mixture was evaporated.
The residue was diluted with EtOAc and saturated NaHCO3 aque-
ous solution. The organic extract was washed with water and
brine, dried over Na2SO4, filtrated and then concentrated. The res-
idue was suspended in 1,2-dichloroethane (3 mL) and THF (6 mL).
To the suspension were added 4-pyridinecarboxaldehyde (78.9
0.826 mmol), NaBH(OAc)3 (131 mg, 0.62 mmol) and acetic acid
(23.6 L, 0.413 mmol) at room temperature. The mixture was stir-
lL,
l
5.1.41. (R)-4-((3-Methyl-4-(pyridin-3-ylmethyl)piperazin-1-
yl)methyl)-N-(4-(trifluoromethyl)phenyl)benzamide (35c)
The title compound was prepared from 34c using a method
analogous to that described for 35a in 60.7% as a white solid.
1H NMR (600 MHz, CDCl3): d = 1.13 (d, J = 6.0 Hz, 3H), 1.99–2.10
(m, 1H), 2.17–2.29 (m, 2H), 2.54 (br s, 1H), 2.58–2.70 (m, 3H), 3.22
(d, J = 13.6 Hz, 1H), 3.50–3.57 (m, 2H), 4.02 (d, J = 13.6 Hz, 1H),
7.22–7.28 (m, 1H), 7.46 (d, J = 7.9 Hz, 2H), 7.60–7.67 (m, 3H),
7.78 (d, J = 8.3 Hz, 2H), 7.82 (d, J = 7.9 Hz, 2H), 8.02 (br s, 1H),
8.46–8.51 (m, 1H), 8.54 (d, J = 1.1 Hz, 1H); 13C NMR (150 MHz,
CDCl3): d = 17.0, 51.1, 53.4, 55.2, 55.3, 60.6, 62.4, 119.7, 123.3,
124.1, 126.2, 126.3, 127.1, 129.4, 133.1, 134.4, 136.6, 141.1,
143.2, 148.4, 150.4, 165.7; HRMS–ESI m/z [M+H]+ calcd for C26H28-
F3N4O+: 469.2210, found: 469.2196.
red at room temperature for 110 min. The residue was diluted with
EtOAc and water. The organic extract was washed with brine, dried
over Na2SO4, filtrated and then concentrated. The crude solid was
washed with n-hexane/Et2O to afford the title compound 38 as a
white solid (138 mg, 0.304 mmol, 73.7%).
1H NMR (600 MHz, CDCl3): d = 1.29–1.41 (m, 2H), 1.53–1.65 (m,
3H), 1.96 (t, J = 11.2 Hz, 2H), 2.62 (d, J = 6.8 Hz, 2H), 2.83 (d,
J = 11.2 Hz, 2H), 3.48 (s, 2H), 7.23–7.30 (m, 4H), 7.62
(d, J = 8.3 Hz, 2H), 7.74–7.83 (m, 4H), 8.02 (br s, 1H), 8.52
(d, J = 5.3 Hz, 2H); 13C NMR (150 MHz, CDCl3): d = 32.1, 37.6, 43.0,
53.8, 62.0, 119.7, 123.9, 124.1, 126.2, 126.3, 127.1, 129.6, 132.1,
141.1, 145.5, 147.9, 149.6, 165.7; HRMS–ESI m/z [M+H]+ calcd for
C
26H27F3N3O+: 454.2101, found: 454.2090.
5.1.45. 4-(Chloromethyl)-N-(4-iodophenyl)benzamide (39)
To a stirred solution of 2d (20.0 g, 91.3 mmol) in THF (300 mL)
were added triethylamine (25.0 mL, 183 mmol) and 4-(chloro-
methyl)benzoyl chloride (17.3 g, 91.3 mmol) at 0 °C. The mixture
was stirred at room temperature for 3.5 h. The reaction mixture
was diluted with water, EtOAc and THF. The organic extract was
washed with brine, dried over MgSO4, filtrated and then concen-
trated. The crude solid was washed with Et2O and filtrated to
afford the title compound 39 as a white solid (32.2 g, 86.7 mmol,
94.9%).
5.1.42. (S)-4-((3-Methyl-4-(pyridin-3-ylmethyl)piperazin-1-
yl)methyl)-N-(4-(trifluoromethyl)phenyl)benzamide (35d)
The title compound was prepared from 34d using a method
analogous to that described for 35a in 60.9% as a white solid.
1H NMR (600 MHz, CDCl3): d = 1.13 (d, J = 6.4 Hz, 3H), 1.98–
2.10 (m, 1H), 2.17–2.29 (m, 2H), 2.53 (br s, 1H), 2.59–2.69 (m,
3H), 3.22 (d, J = 13.8 Hz, 1H), 3.51–3.55 (m, 2H), 4.01 (d,
J = 13.8 Hz, 1H), 7.22–7.28 (m, 1H), 7.45 (d, J = 7.9 Hz, 2H),
7.60–7.67 (m, 3H), 7.78 (d, J = 8.3 Hz, 2H), 7.82 (d, J = 7.9 Hz,
2H), 8.01 (br s, 1H), 8.48 (dd, J = 4.7, 1.2 Hz, 1H), 8.54 (d,
J = 1.2 Hz, 1H); 13C NMR (150 MHz, CDCl3): d = 16.9, 51.1, 53.4,
55.2, 55.3, 60.6, 62.4, 119.7, 123.3, 124.1, 126.2, 126.3, 127.1,
129.4, 133.1, 134.4, 136.6, 141.1, 143.2, 148.4, 150.4, 165.7;
HRMS–ESI m/z [M+H]+ calcd for C26H28F3N4O+: 469.2210, found:
469.2196.
1H NMR (600 MHz, DMSO-d6): d = 4.84 (s, 2H), 7.59 (d,
J = 8.3 Hz, 2H), 7.61–7.64 (m, 2H), 7.68–7.71 (m, 2H), 7.95 (d,
J = 8.3 Hz, 2H), 10.34 (s, 1H); 13C NMR (150 MHz, DMSO-d6):
d = 45.4, 87.4, 122.4, 128.0, 128.8, 134.5, 137.3, 138.9, 141.2,
165.2; HRMS–ESI m/z [M+H]+ calcd for C14H12ClNO+: 371.9647,
found: 371.9644.
5.1.43. tert-Butyl 4-(4-((4-
(trifluoromethyl)phenyl)carbamoyl)benzyl)piperidine-1-
carboxylate (37)
5.1.46. N-(4-Iodophenyl)-4-((4-(pyridin-3-ylmethyl)piperazin-
1-yl)methyl)benzamide (40)
To a stirred solution of 39 (5.0 g, 13.5 mmol) in DMF (42 mL)
were added triethylamine (3.8 mL) and 1-(pyridin-3-ylmethyl)-
piperazine (3.2 g, 18.1 mmol) at room temperature under nitrogen.
The stirred mixture was heated at 50 °C for 3 h. The reaction mix-
ture was cooled to room temperature and diluted with water, THF
and EtOAc. The organic extract was washed with water, dried over
Na2SO4, filtrated and then concentrated. The crude solid was
washed with Et2O/EtOAc and filtrated to afford the title compound
40 as a white solid (6.33 g, 12.4 mmol, 91.5%).
To a stirred solution of 4-((1-(tert-butoxycarbonyl)piperidin-
4-yl)methyl)benzoic acid 3625 (722 mg, 2.26 mmol) in dichloro-
methane (36.1 mL) were added triethylamine (848 lL,
6.10 mmol), 4-dimethylaminopyridine (276 mg, 2.26 mmol) and
2-methyl-6-nitrobenzoic anhydride (780 mg, 2.26 mmol) at room
temperature. The mixture was stirred at room temperature for
25 min. To the mixture was added 4-aminobenzotrifluoride
(284 lL, 2.26 mmol). The mixture was stirred at room tempera-
ture for overnight. The reaction mixture was diluted with water
and EtOAc. The organic extract was dried over MgSO4, filtrated
and then concentrated. The residue was purified with column
chromatography on silica gel (n-heptane/EtOAc = 4:1–2:1) to
afford the title compound 37 as a light yellow solid (760 mg,
1.64 mmol, 72.7%).
1H NMR (600 MHz, CDCl3): d = 2.48 (br s, 8H), 3.52 (s, 2H), 3.56
(s, 2H), 7.23–7.27 (m, 1H), 7.40–7.47 (m, 4H), 7.62–7.70 (m, 3H),
7.80 (d, J = 7.9 Hz, 2H), 7.85 (s, 1H), 8.50 (dd, J = 4.9, 1.3 Hz, 1H),
8.53 (d, J = 1.3 Hz, 1H); 13C NMR (150 MHz, CDCl3): d = 53.0, 53.1,
60.2, 62.5, 87.7, 121.9, 123.3, 127.0, 129.4, 133.4, 133.6, 136.7,
137.8, 138.0, 142.9, 148.6, 150.5, 165.5; HRMS–ESI m/z [M+H]+
calcd for C24H26IN4O+: 513.1146, found: 513.1148.
1H NMR (600 MHz, CDCl3): d = 1.10–1.21 (m, 2H), 1.45 (s, 9H),
1.55–1.65 (m, 2H), 1.65–1.76 (m, 1H), 2.57–2.69 (m, 4H), 4.08 (br
s, 2H), 7.25–7.29 (m, 2H), 7.62 (d, J = 8.7 Hz, 2H), 7.75–7.84 (m,
4H), 7.98 (br s, 1H); 13C NMR (150 MHz, CDCl3): d = 28.5, 31.9,
38.0, 43.0, 44.0, 79.4, 119.7, 124.1, 126.2, 126.4, 127.1, 129.6,
132.2, 141.1, 145.1, 154.8, 165.7; HRMS–ESI m/z [M+H]+ calcd for
5.1.47. N-(4-(Phenylethynyl)phenyl)-4-((4-(pyridin-3-
ylmethyl)piperazin-1-yl)methyl)benzamide (41a)
To a stirred solution of 40 (3.0 g, 5.85 mmol) in DMF (50 mL)
were added triethylamine (2.4 mL, 17.3 mmol), CuI (113 mg,
0.593 mmol) and PdCl2(PPh3)2 (207 mg, 0.295 mmol) at room
C
25H30F3N2O+3: 463.2203, found: 463.2192.

5526
S. Nagao et al. / Bioorg. Med. Chem. 22 (2014) 5513–5529
temperature under nitrogen. To the mixture was dropwise added
phenylacetylene (1.9 mL, 17.3 mmol) at room temperature. The
mixture was stirred at room temperature for 20 h. The reaction
mixture was diluted with EtOAc, THF and water. The organic
extract was washed with water, dried over MgSO4, filtrated and
then concentrated. The residue was purified with column chroma-
tography on NH silica gel (EtOAc). The resulting solid was washed
with EtOAc/Et2O and filtrated to afford the title compound 41a as a
light brown solid (2.61 g, 5.36 mmol, 91.7%).
1H NMR (600 MHz, CDCl3): d = 1.72 (br s, 6H), 1.94–2.02 (m,
9H), 2.48 (br s, 8H), 3.52 (s, 2H), 3.56 (s, 2H), 7.23–7.28 (m, 1H),
7.38 (d, J = 8.7 Hz, 2H), 7.43 (d, J = 7.9 Hz, 2H), 7.56 (d, J = 8.7 Hz,
2H), 7.65 (d, J = 7.9 Hz, 1H), 7.77–7.83 (m, 3H), 8.50 (dd, J = 4.7,
1.4 Hz, 1H), 8.54 (d, J = 1.4 Hz, 1H); 13C NMR (150 MHz, CDCl3):
d = 28.0, 30.1, 36.4, 42.9, 53.0, 53.1, 60.2, 62.5, 79.0, 98.2, 119.5,
120.1, 123.3, 127.0, 129.4, 132.4, 133.6, 133.6, 136.7, 137.2,
142.7, 148.6, 150.5, 165.3; HRMS–ESI m/z [M+H]+ calcd for
C
36H41N4O+: 545.3275, found: 545.3273.
1H NMR (600 MHz, CDCl3): d = 2.49 (br s, 8H), 3.52 (s, 2H), 3.57 (s,
2H), 7.23–7.27 (m, 1H), 7.31–7.37 (m, 3H), 7.45 (d, J = 8.1 Hz, 2H),
7.50–7.56 (m, 4H), 7.65 (d, J = 8.3 Hz, 3H), 7.82 (d, J = 8.1 Hz, 2H),
7.89 (s, 1H), 8.50 (dd, J = 4.5, 1.3 Hz, 1H), 8.54 (d, J = 1.3 Hz, 1H);
13C NMR (150 MHz, CDCl3): d = 53.0, 53.1, 60.2, 62.5, 89.1, 89.2,
119.1, 119.7, 123.3, 123.3, 127.0, 128.2, 128.3, 129.4, 131.5, 132.5,
133.5, 133.6, 136.7, 138.0, 142.9, 148.6, 150.5, 165.4; HRMS–ESI
m/z [M+H]+ calcd for C32H31N4O+: 487.2492, found: 487.2495.
5.1.52. tert-Butyl 4-(2-aminonicotinoyl)piperazine-1-
carboxylate
To a solution of tert-butyl 1-piperazinecarboxylate (5.18 g,
27.8 mmol) in DMF (40 mL) were added triethylamine (7.4 mL,
53.4 mmol), 2-aminonicotinic acid (3.7 g, 26.8 mmol) and ben-
zotriazol-1-yloxytris(dimethylamino)phosphonium
hexafluoro-
phosphate (14.2 g, 32.1 mmol) at room temperature under
nitrogen. The stirred mixture was heated at 60 °C for 2 h. The reac-
tion mixture was cooled to room temperature and diluted with
water and EtOAc. The aqueous layer was extracted with EtOAc. The
combined organic extracts were washed with water and brine, dried
over Na2SO4, filtrated and then concentrated. The crude solid was
purified with column chromatography on NH silica gel (n-heptane/
EtOAc 1:1–1:2) to afford the title compound as a light orange solid
(7.67 g, 25.0 mmol, 93.4%).
5.1.48. N-(4-(Pyridin-3-ylethynyl)phenyl)-4-((4-(pyridin-3-
ylmethyl)piperazin-1-yl)methyl)benzamide (41b)
The title compound was prepared from 40 using a method anal-
ogous to that described for 41a in 85.5% yield as a light yellow
solid.
1H NMR (600 MHz, CDCl3): d = 2.49 (br s, 8H), 3.52 (s, 2H), 3.57
(s, 2H), 7.23–7.30 (m, 2H), 7.45 (d, J = 8.3 Hz, 2H), 7.55 (d, J = 7.9 Hz,
2H), 7.64–7.71 (m, 3H), 7.78–7.85 (m, 3H), 8.00 (s, 1H), 8.48–8.57
(m, 3H), 8.76 (d, J = 1.5 Hz, 1H); 13C NMR (150 MHz, CDCl3):
d = 53.0, 53.1, 60.2, 62.5, 85.7, 92.4, 118.3, 119.8, 120.5, 123.0,
123.3, 127.0, 129.4, 132.6, 133.4, 133.6, 136.7, 138.3, 138.5,
143.0, 148.4, 148.6, 150.5, 152.2, 165.5; HRMS–ESI m/z [M+H]+
calcd for C31H30N5O+: 488.2445, found: 488.2445.
1H NMR (600 MHz, CDCl3): d = 1.47 (s, 9H), 3.44–3.51 (m, 4H),
3.58 (br s, 4H), 5.18 (s, 2H), 6.67 (dd, J = 7.5, 4.9 Hz, 1H), 7.35
(dd, J = 7.5, 1.6 Hz, 1H), 8.13 (dd, J = 4.9, 1.6 Hz, 1H); 13C NMR
(150 MHz, CDCl3): d = 28.4, 44.2, 80.4, 113.0, 113.1, 136.3, 150.1,
154.5, 157.1, 169.0; HRMS–ESI m/z [M+H]+ calcd for C15H23N4O3+:
307.1765, found: 307.1745.
5.1.49. N-(4-(Cyclohexylethynyl)phenyl)-4-((4-(pyridin-3-
ylmethyl)piperazin-1-yl)methyl)benzamide (41c)
5.1.53. 4-((4-(2-Aminonicotinoyl)piperazin-1-yl)methyl)-N-(4-
iodophenyl)benzamide (42)
The title compound was prepared from 40 using a method anal-
ogous to that described for 41a in 76.3% yield as a white solid.
1H NMR (600 MHz, CDCl3): d = 1.29–1.42 (m, 3H), 1.49–1.59 (m,
3H), 1.72–1.80 (m, 2H), 1.84–1.93 (m, 2H), 2.27–2.70 (m, 9H), 3.52
(s, 2H), 3.56 (s, 2H), 7.22–7.28 (m, 1H), 7.39 (d, J = 8.3 Hz, 2H), 7.43
(d, J = 8.1 Hz, 2H), 7.57 (d, J = 8.1 Hz, 2H), 7.65 (d, J = 7.9 Hz, 1H),
7.80 (d, J = 8.3 Hz, 2H), 7.83 (s, 1H), 8.49 (dd, J = 4.7, 1.4 Hz, 1H),
8.53 (d, J = 1.4 Hz, 1H); 13C NMR (150 MHz, CDCl3): d = 24.9, 25.9,
29.7, 32.7, 53.0, 53.1, 60.2, 62.5, 80.1, 94.2, 119.6, 120.1, 123.3,
127.0, 129.4, 132.4, 133.6, 136.7, 137.2, 142.7, 148.6, 150.5,
165.4; HRMS–ESI m/z [M+H]+ calcd for C32H37N4O+: 493.2962,
found: 493.2962.
To a tert-butyl 4-(2-aminonicotinoyl)piperazine-1-carboxylate
(1.94 g, 6.33 mmol) was cooled to 0 °C. At the same temperature,
a cooled trifluoroacetic acid (36 mL, 485 mmol) was added. The
mixture was stirred at 0 °C for 50 min. The mixture was diluted
with toluene and then evaporated. To the mixture were added
DMF (24 mL), triethylamine (6.8 mL, 49.1 mmol) and 4-(chloro-
methyl)-N-(4-iodophenyl)benzamide (1.8 g, 4.84 mmol) at room
temperature under nitrogen. The stirred mixture was heated at
50 °C for 5 h, cooled to room temperature. The reaction mixture
was stirred at room temperature for 12 h. The reaction mixture
was diluted with water, THF and EtOAc. The organic extract was
washed with water, dried over Na2SO4, filtrated and then concen-
trated. The crude solid was washed with Et2O/EtOAc and filtrated
to afford the title compound 42 as a light brown solid (2.22 g,
4.10 mmol, 84.7%).
5.1.50. N-(4-((1-Hydroxycyclohexyl)ethynyl)phenyl)-4-((4-
(pyridin-3-ylmethyl)piperazin-1-yl)methyl)benzamide (41d)
The title compound was prepared from 40 using a method anal-
ogous to that described for 41a in 89.8% yield as a light yellow
solid.
1H NMR (600 MHz, DMSO-d6): d = 2.41 (br s, 4H), 3.28–3.64 (m,
6H), 5.91 (s, 2H), 6.58 (dd, J = 7.5, 4.9 Hz, 1H), 7.35 (dd, J = 7.5,
1.8 Hz, 1H), 7.46 (d, J = 8.3 Hz, 2H), 7.62 (d, J = 9.0 Hz, 2H), 7.68
(d, J = 9.0 Hz, 2H), 7.90 (d, J = 8.3 Hz, 2H), 7.99 (dd, J = 4.9, 1.8 Hz,
1H), 10.29 (s, 1H); 13C NMR (150 MHz, DMSO-d6): d = 52.5, 61.3,
67.0, 87.2, 111.7, 114.0, 122.4, 127.7, 128.7, 133.5, 136.0, 137.2,
139.0, 141.9, 149.1, 156.0, 165.4, 167.2; HRMS–ESI m/z [M+H]+
calcd for C24H25IN5O+2: 542.1047, found: 542.1046.
1H NMR (600 MHz, CDCl3): d = 1.22–1.36 (m, 1H), 1.52–1.81 (m,
7H), 1.93–2.08 (m, 2H), 2.20–2.84 (m, 9H), 3.52 (s, 2H), 3.56 (s, 2H),
7.23–7.28 (m, 1H), 7.40–7.46 (m, 4H), 7.61 (d, J = 7.9 Hz, 2H), 7.66
(d, J = 7.9 Hz, 2H), 7.80 (d, J = 8.3 Hz, 2H), 7.89 (s, 1H), 8.50 (dd,
J = 4.9, 1.5 Hz, 1H), 8.54 (d, J = 1.5 Hz, 1H); 13C NMR (150 MHz,
CDCl3): d = 23.4, 25.2, 40.1, 53.0, 53.1, 60.2, 62.5, 69.1, 84.0, 92.6,
118.8, 119.6, 123.3, 127.0, 129.4, 132.5, 133.5, 133.6, 136.7,
137.9, 142.8, 148.6, 150.4, 165.4; HRMS–ESI m/z [M+H]+ calcd for
5.1.54. 4-((4-(2-Aminonicotinoyl)piperazin-1-yl)methyl)-N-(4-
(phenylethynyl)phenyl)benzamide (43a)
C
32H37N4O+2: 509.2911, found: 509.2913.
To a stirred solution of 42 (100 mg, 0.185 mmol) in DMF
(1.8 mL) were added triethylamine (39
lL, 0.277 mmol), phenyl-
5.1.51. N-(4-(Adamantan-1-ylethynyl)phenyl)-4-((4-(pyridin-3-
ylmethyl)piperazin-1-yl)methyl)benzamide (41e)
The title compound was prepared from 40 using a method anal-
ogous to that described for 41a in 46.3% yield as a white solid.
acetylene (41 L, 0.369 mmol), CuI (3.8 mg, 0.02 mmol) and
l
PdCl2(PPh3)2 (7.3 mg, 0.01 mmol) at room temperature under
nitrogen. The mixture was stirred at room temperature for 6 h.
The reaction mixture was diluted with EtOAc and water. The

S. Nagao et al. / Bioorg. Med. Chem. 22 (2014) 5513–5529
5527
organic extract was washed with brine, dried over Na2SO4, filtrated
and then concentrated. The residue was purified with column
chromatography on NH silica gel (n-heptane/EtOAc = 1:3–EtOAc/
MeOH = 19:1) to afford the title compound 10a as a light yellow
solid (91.2 mg, 0.177 mmol, 96.0%).
afford the title compound 46a as light yellow oil (545 mg,
1.45 mmol, 85.0%).
1H NMR (600 MHz, CDCl3): d = 1.30 (d, J = 6.8 Hz, 3H), 2.13 (td,
J = 11.5, 3.6 Hz, 1H), 2.28 (dd, J = 11.3, 3.6 Hz, 1H), 2.57 (d,
J = 11.3 Hz, 1H), 2.76 (d, J = 11.3 Hz, 1H), 3.34–3.46 (m, 2H), 3.47–
3.61 (m, 2H), 4.06–4.14 (m, 1H), 7.22–7.29 (m, 1H), 7.62–7.73
(m, 4H), 8.06 (dd, J = 7.4, 1.7 Hz, 1H), 8.47–8.56 (m, 2H); 13C NMR
(150 MHz, CDCl3): d = 16.2, 41.1, 50.0, 53.0, 57.5, 59.8, 123.4,
124.3, 130.9, 131.7, 133.4, 133.8, 136.3, 147.8, 148.8, 150.2;
HRMS–ESI m/z [M+H]+ calcd for C17H21N4O4S+: 377.1278, found:
377.1265.
1H NMR (600 MHz, CDCl3): d = 2.48 (br s, 4H), 3.55–3.77 (m,
6H), 5.15 (s, 2H), 6.65 (dd, J = 7.6, 5.0 Hz, 1H), 7.31–7.38 (m, 4H),
7.46 (d, J = 8.3 Hz, 2H), 7.50–7.57 (m, 4H), 7.66 (d, J = 8.6 Hz, 2H),
7.84 (d, J = 8.3 Hz, 2H), 7.91 (s, 1H), 8.07–8.16 (m, 1H); 13C NMR
(150 MHz, CDCl3): d = 53.2, 62.3, 89.1, 89.2, 113.0, 113.4, 119.2,
119.7, 123.3, 127.2, 128.2, 128.3, 129.3, 131.6, 132.5, 133.8,
136.3, 137.9, 142.1, 149.9, 157.0, 165.3, 168.6; HRMS–ESI m/z
[M+H]+ calcd for C32H30N5O+2: 516.2394, found: 516.2393.
5.1.58. (S)-4-((6-Chloropyridin-3-yl)methyl)-2-methyl-1-((2-
nitrophenyl)sulfonyl)piperazine (46b)
5.1.55. N-(4-(Adamantan-1-ylethynyl)phenyl)-4-((4-(2-
aminonicotinoyl)piperazin-1-yl)methyl)benzamide (43b)
The title compound was prepared from 42 using a method anal-
ogous to that described for 43a in 59.8% yield as a light yellow
solid.
To a stirred solution of 45 (1.98 g, 6.59 mmol) in DMF (15 mL)
were added triethylamine (2.0 mL, 14.3 mmol) and 2-chloro-5-
chloromethylpyridine (1.28 g, 7.91 mmol) at room temperature
under nitrogen. The stirred mixture was heated at 60 °C for 5.5 h.
The mixture was diluted with EtOAc and water. The organic extract
was washed with water and brine, dried over Na2SO4, filtrated and
then concentrated. The residue was purified with column chroma-
tography on NH silica gel (n-heptane/EtOAc = 4:1–1:1) to afford
the title compound 46b as yellow oil (quant.).
1H NMR (600 MHz, CDCl3): d = 1.72 (br s, 6H), 1.93–2.01 (m,
9H), 2.48 (br s, 4H), 3.52–3.75 (m, 6H), 5.15 (s, 2H), 6.65 (dd,
J = 7.6, 4.9 Hz, 1H), 7.35 (dd, J = 7.6, 1.5 Hz, 1H), 7.39 (d,
J = 8.7 Hz, 2H), 7.45 (d, J = 8.1 Hz, 2H), 7.56 (d, J = 8.1 Hz, 2H),
7.78–7.85 (m, 3H), 8.11 (dd, J = 4.9, 1.5 Hz, 1H); 13C NMR
(150 MHz, CDCl3): d = 28.0, 30.1, 36.4, 42.9, 53.2, 62.3, 79.0, 98.2,
113.0, 113.4, 119.5, 120.2, 127.1, 129.3, 132.4, 133.9, 136.3,
137.1, 142.0, 149.9, 157.0, 165.2, 168.6; HRMS–ESI m/z [M+H]+
calcd for C36H40N5O+2: 574.3177, found: 574.3175.
1H NMR (600 MHz, CDCl3): d = 1.29 (d, J = 6.4 Hz, 3H), 2.15 (td,
J = 11.6, 3.5 Hz, 1H), 2.28 (dd, J = 11.3, 3.5 Hz, 1H), 2.54 (d,
J = 11.0 Hz, 1H), 2.74 (d, J = 11.3 Hz, 1H), 3.35–3.43 (m, 2H), 3.47–
3.53 (m, 1H), 3.57 (d, J = 13.2 Hz, 1H), 4.06–4.14 (m, 1H), 7.26–
7.31 (m, 1H), 7.61–7.72 (m, 4H), 8.07 (dd, J = 7.4, 1.8 Hz, 1H),
8.29 (d, J = 1.8 Hz, 1H); 13C NMR (150 MHz, CDCl3): d = 16.1, 41.0,
49.9, 53.0, 57.4, 58.9, 124.1, 124.4, 130.9, 131.8, 132.5, 133.5,
133,7, 139.1, 147.8, 149.8, 150.5; HRMS–ESI m/z [M+H]+ calcd for
5.1.56. (S)-2-Methyl-1-((2-nitrophenyl)sulfonyl)piperazine (45)
To a stirred solution of (S)-tert-butyl 3-methylpiperazine-1-car-
boxylate (2.01 g, 10 mmol) in THF (20 mL) were added triethyl-
amine (2.8 mL, 20.2 mmol) and 2-nitrobenzenesulfonyl chloride
(2.72 g, 12.3 mmol) at 0 °C under nitrogen. The mixture was stirred
at room temperature for 70 min. The mixture was diluted with
EtOAc and water. The organic extract was washed with brine, dried
over MgSO4, filtrated and then concentrated. The residue was puri-
fied with column chromatography on NH silica gel (EtOAc only) to
afford the product A.
To a stirred solution of the product A in dichloromethane
(20 mL) was added trifluoroacetic acid (7.5 mL, 101 mmol) at room
temperature. The mixture was stirred at room temperature for
85 min. The mixture was evaporated. The residue was diluted with
EtOAc and saturated NaHCO3 aqueous solution. The aqueous layer
was extracted with EtOAc. The combined organic extracts were
dried over MgSO4, filtrated and then concentrated. The residue
was purified with column chromatography on NH silica gel
(EtOAc/MeOH = 1:0–9:1) to afford the title compound 45 as a yel-
low solid (quant.).
C
17H20ClN4O4S+: 411.0888, found: 411.0878.
5.1.59. (S)-3-Methyl-1-(pyridin-3-ylmethyl)piperazine (47a)
To a stirred solution of 46a (314 mg, 0.834 mmol) in DMF
(8.0 mL) were added lithium hydroxide (200 mg, 8.34 mmol) and
mercaptoacetic acid (290 lL, 4.17 mmol) at room temperature
under nitrogen. The mixture was stirred at room temperature for
24 h. The mixture was diluted with THF and EtOAc. The resulting
suspension was filtrated through a pad of CeliteÒ. The filtrate
was evaporated. The residue was purified with column chromatog-
raphy on NH silica gel (EtOAc/MeOH = 1:0–4:1) to afford the title
compound 47a as yellow oil (80.6 mg, 0.421 mmol, 50.5%).
1H NMR (600 MHz, CDCl3): d = 1.01 (d, J = 6.4 Hz, 3H), 1.70 (t,
J = 10.4 Hz, 1H), 2.04 (td, J = 11.2, 3.0 Hz, 1H), 2.73 (d, J = 11.2 Hz,
2H), 2.83–2.98 (m, 3H), 3.45–3.54 (m, 2H), 7.23–7.29 (m, 1H),
7.67 (d, J = 7.6 Hz, 1H), 8.50 (d, J = 4.9 Hz, 1H), 8.54 (s, 1H); 13C
NMR (150 MHz, CDCl3): d = 19.9, 45.8, 50.5, 53.6, 60.5, 61.2,
123.3, 133.6, 136.7, 148.5, 150.5; HRMS–ESI m/z [M+H]+ calcd for
C11H18N+3: 192.1495, found: 192.1493.
1H NMR (600 MHz, CDCl3): d = 1.29 (d, J = 6.8 Hz, 3H), 2.70–2.80
(m, 2H), 2.91–3.00 (m, 2H), 3.28 (td, J = 12.7, 3.4 Hz, 1H), 3.49 (d,
J = 13.2 Hz, 1H), 3.99–4.08 (m, 1H), 7.62–7.73 (m, 3H), 8.04–8.10
(m, 1H); 13C NMR (150 MHz, CDCl3): d = 15.2, 41.5, 45.9, 49.6,
50.6, 124.3, 130.9, 131.7, 133.4, 133.9, 147.8; HRMS–ESI m/z
[M+H]+ calcd for C11H16N3O4S+: 286.0856, found: 286.0850.
5.1.60. (S)-1-((6-(Methoxymethyl)pyridin-3-yl)methyl)-3-
methylpiperazine (47c)
To a stirred solution of 46b (465 mg, 1.13 mmol) in DMF
(5.0 mL) were added tributyl(methoxymethyl)stannane30 (550
mg, 1.64 mmol) and tetrakis(triphenylphosphine)palladium(0)
(131 mg, 0.113 mmol) at room temperature under nitrogen. The
stirred mixture was heated at 120 °C for 6 h. The mixture was
diluted with water and EtOAc. The organic extract was washed
with brine, dried over Na2SO4, filtrated and then concentrated.
The residue was purified with column chromatography on NH sil-
ica gel (n-heptane/EtOAc = 3:1–2:3) to afford the product A.
To a stirred solution of the product A in DMF (3.0 mL) were
added lithium hydroxide (134 mg, 5.61 mmol) and mercaptoacetic
5.1.57. (S)-2-Methyl-1-((2-nitrophenyl)sulfonyl)-4-(pyridin-3-
ylmethyl)piperazine (46a)
To a stirred solution of 45 (485 mg, 1.70 mmol) in DMF (5.0 mL)
were added triethylamine (711 lL, 5.10 mmol) and 3-chlorometh-
ylpyridine hydrochloride (423 mg, 2.58 mmol) at room tempera-
ture under nitrogen. The stirred mixture was heated at 80 °C for
5 h. The mixture was diluted with EtOAc and water. The organic
extract was washed with water and brine, dried over Na2SO4, fil-
trated and then concentrated. The residue was purified with column
chromatography on NH silica gel (n-heptane/EtOAc = 3:1–1:3) to
acid (195
lL, 2.81 mmol) at room temperature under nitrogen. The
mixture was stirred at room temperature for 24 h. The mixture was

5528
S. Nagao et al. / Bioorg. Med. Chem. 22 (2014) 5513–5529
diluted with THF and EtOAc. The resulting suspension was filtrated
through a pad of CeliteÒ. The filtrate was evaporated. The residue
was purified with column chromatography on NH silica gel
(EtOAc/MeOH = 1:0–4:1) to afford the title compound 47c as yel-
low oil (57.0 mg, 0.242 mmol, 21.4%).
NMR (150 MHz, CDCl3): d = 28.0, 30.1, 36.4, 42.9, 45.3, 78.9, 98.3,
119.6, 120.3, 127.4, 128.9, 132.4, 134.8, 136.9, 141.3, 164.9;
HRMS–ESI m/z [M+H]+ calcd for C26H27ClNO+: 404.1776, found:
404.1761.
1H NMR (600 MHz, CDCl3): d = 1.01 (d, J = 6.4 Hz, 3H), 1.68 (t,
J = 10.4 Hz, 1H), 2.03 (td, J = 11.2, 3.2 Hz, 1H), 2.73 (d, J = 11.0 Hz,
2H), 2.82–2.97 (m, 3H), 3.45–3.52 (m, 5H), 4.58 (s, 2H), 7.38 (d,
J = 7.9 Hz, 1H), 7.68 (dd, J = 7.9, 1.7 Hz, 1H), 8.48 (d, J = 1.7 Hz,
1H); 13C NMR (150 MHz, CDCl3): d = 19.9, 45.8, 50.5, 53.6, 58.8,
60.3, 61.1, 75.4, 121.0, 132.3, 137.5, 149.8, 157.1; HRMS–ESI m/z
[M+H]+ calcd for C13H22N3O+: 236.1757, found: 236.1750.
5.1.65. N-(4-((Adamantan-1-ylethynyl)phenyl)-4-(((S)-2-
methyl-4-(pyridin-3-ylmethyl)piperazin-1-
yl)methyl)benzamide (51a)
The title compound was prepared from 50 and 47a using a
method analogous to that described for 40 in 21.3% yield as a yel-
low solid.
1H NMR (600 MHz, CDCl3): d = 1.11 (d, J = 6.4 Hz, 3H), 1.72 (br s,
6H), 1.94–2.08 (m, 10H), 2.16–2.27 (m, 2H), 2.52 (br s, 1H), 2.56–
2.70 (m, 3H), 3.24 (d, J = 13.6 Hz, 1H), 3.48 (s, 2H), 4.07 (d,
J = 13.6 Hz, 1H), 7.21–7.28 (m, 1H), 7.38 (d, J = 8.5 Hz, 2H), 7.44
(d, J = 7.9 Hz, 2H), 7.57 (d, J = 8.5 Hz, 2H), 7.65 (d, J = 7.6 Hz, 1H),
7.77–7.84 (m, 3H), 8.49 (dd, J = 4.7, 1.4 Hz, 1H), 8.53 (d,
J = 1.4 Hz, 1H); 13C NMR (150 MHz, CDCl3): d = 16.9, 28.0, 30.1,
36.4, 42.9, 51.3, 53.3, 55.3, 57.7, 60.1, 60.6, 79.0, 98.1, 119.5,
120.1, 123.3, 126.9, 129.3, 132.4, 133.4, 133.6, 136.7, 137.2,
143.7, 148.6, 150.4, 165.4; HRMS–ESI m/z [M+H]+ calcd for
5.1.61. Tributyl((2-methoxyethoxy)methyl)stannane
To a stirred solution of diisopropylamine (5.0 mL, 35.7 mmol) in
THF (80 mL) was added n-BuLi (11.0 mg, 28.6 mmol, 2.59 M) at
ꢀ78 °C under nitrogen. At the same temperature, the mixture
was stirred for 1 h. To the mixture was added tri-N-butyltin
hydride (7.0 mL, 26.0 mmol) at ꢀ78 °C. The mixture was stirred
at 0 °C for 30 min. The mixture was recooled to ꢀ78 °C. To the mix-
ture was 2-methoxyethoxymethyl chloride (3.5 mL, 30.9 mmol),
followed by stirred at the same temperature for 30 min. The stirred
mixture was warmed to room temperature over 4 h. The mixture
was diluted with saturated NH4Cl aqueous solution and Et2O. The
aqueous layer was extracted with Et2O. The combined organic
extracts were washed with brine, dried over Na2SO4, filtrated and
then concentrated. The residue was purified with column chroma-
tography on silica gel (n-heptane/EtOAc = 1:0–4:1) to afford the
title compound as colorless oil (5.72 g, 15.1 mmol, 58.0%).
1H NMR (600 MHz, CDCl3): d = 0.85–0.92 (m, 15H), 1.23–1.34
(m, 6H), 1.45–1.55 (m, 6H), 3.37 (s, 3H), 3.46–3.53 (m, 4H), 3.77–
3.81 (m, 2H); 13C NMR (150 MHz, CDCl3): d = 9.1, 13.7, 27.3, 29.1,
59.1, 62.6, 71.9, 74.5; HRMS–EI m/z [M+H]+ calcd for C12H27O2SnÅ+:
323.1028, found: 323.0999.
C
37H43N4O+: 559.3431, found: 559.3411.
5.1.66. N-(4-(Adamantan-1-ylethynyl)phenyl)-4-(((S)-4-((6-
(methoxymethyl)pyridin-3-yl)methyl)-2-methylpiperazin-1-
yl)methyl)benzamide (51c)
The title compound was prepared from 50 and 47c using a
method analogous to that described for 40 in 79.0% yield as light
yellow amorphous.
1H NMR (600 MHz, CDCl3): d = 1.11 (d, J = 6.0 Hz, 3H), 1.72 (br s,
6H), 1.93–2.08 (m, 10H), 2.19–2.26 (m, 2H), 2.51 (br s, 1H), 2.57–
2.69 (m, 3H), 3.19–3.27 (m, 1H), 3.48 (s, 5H), 4.06 (d, J = 13.6 Hz,
1H), 4.57 (s, 2H), 7.35–7.40 (m, 3H), 7.44 (d, J = 8.3 Hz, 2H), 7.56
(d, J = 8.0 Hz, 2H), 7.66 (dd, J = 7.9, 1.7 Hz, 1H), 7.76–7.83 (m, 3H),
8.47 (d, J = 1.7 Hz, 1H); 13C NMR (150 MHz, CDCl3): d = 17.0, 28.0,
30.1, 36.4, 42.9, 51.3, 53.3, 55.3, 57.7, 58.8, 59.8, 60.5, 75.4, 79.0,
98.1, 119.5, 120.1, 121.0, 126.9, 129.3, 132.3, 132.4, 133.4, 137.2,
137.4, 143.8, 149.8, 157.1, 165.4; HRMS–ESI m/z [M+H]+ calcd for
5.1.62. (S)-1-((6-((2-methoxyethoxy)methyl)pyridin-3-
yl)methyl)-3-methylpiperazine (47d)
The title compound was prepared from 46b using a method
analogous to that described for 47c in 35.1% as yellow oil.
1H NMR (600 MHz, CDCl3): d = 1.01 (d, J = 6.4 Hz, 3H), 1.68 (t,
J = 10.4 Hz, 1H), 2.03 (td, J = 11.0, 3.0 Hz, 1H), 2.72 (d, J = 11.0 Hz,
2H), 2.82–2.97 (m, 3H), 3.39–3.42 (m, 3H), 3.44–3.52 (m, 2H),
3.62 (t, J = 4.7 Hz, 2H), 3.73 (t, J = 4.7 Hz, 2H), 4.68 (s, 2H), 7.44
(d, J = 7.9 Hz, 1H), 7.67 (dd, J = 7.9, 1.5 Hz, 1H), 8.48 (d, J = 1.5 Hz,
1H); 13C NMR (150 MHz, CDCl3): d = 19.9, 45.8, 50.5, 53.6, 59.1,
60.3, 61.1, 70.1, 71.9, 74.0, 121.2, 132.3, 137.5, 149.7, 157.2;
HRMS–ESI m/z [M+H]+ calcd for C15H26N3O+2: 280.2020, found:
280.2012.
C
39H47N4O+2: 603.3694, found: 603.3669.
5.1.67. N-(4-(Adamantan-1-ylethynyl)phenyl)-4-(((S)-4-((6-((2-
methoxyethoxy)methyl)pyridin-3-yl)methyl)-2-
methylpiperazin-1-yl)methyl)benzamide (51d)
The title compound was prepared from 50 and 47d using a
method analogous to that described for 40 in 52.3% yield as light
yellow amorphous.
1H NMR (600 MHz, CDCl3): d = 1.11 (d, J = 6.0 Hz, 3H), 1.72 (br s,
6H), 1.94–2.06 (m, 10H), 2.18–2.25 (m, 2H), 2.51 (br s, 1H), 2.57–
2.69 (m, 3H), 3.23 (d, J = 14.0 Hz, 1H), 3.40 (s, 3H), 3.47 (s, 2H),
3.59–3.64 (m, 2H), 3.70–3.74 (m, 2H), 4.06 (d, J = 14.0 Hz, 1H),
4.68 (s, 2H), 7.38 (d, J = 8.7 Hz, 2H), 7.41–7.46 (m, 3H), 7.56 (d,
J = 8.0 Hz, 2H), 7.66 (dd, J = 7.9, 1.7 Hz, 1H), 7.76–7.83 (m, 3H),
8.45 (d, J = 1.7 Hz, 1H); 13C NMR (150 MHz, CDCl3): d = 17.0, 28.1,
30.1, 36.4, 42.9, 51.3, 53.3, 55.3, 57.7, 59.1, 59.8, 60.5, 70.2, 71.9,
74.0, 79.0, 98.1, 119.5, 120.1, 121.2, 126.9, 129.2, 132.3, 132.4,
133.4, 137.2, 137.5, 143.8, 149.7, 157.2, 165.4; HRMS–ESI m/z
[M+H]+ calcd for C41H51N4O+3: 647.3956, found: 647.3929.
5.1.63. 4-(Adamantan-1-ylethynyl)aniline (49)
The title compound was prepared from 2d and 48 using a
method analogous to that described for 41a in 76.1% yield as a
brown solid.
1H NMR (600 MHz, CDCl3): d = 1.70 (br s, 6H), 1.92–2.00 (m,
9H), 3.69 (br s, 2H), 6.56 (d, J = 8.7 Hz, 2H), 7.18 (d, J = 8.7 Hz,
2H); 13C NMR (150 MHz, CDCl3): d = 28.1, 30.0, 36.5, 43.1, 79.5,
96.0, 113.7, 114.7, 132.8, 145.7; HRMS–ESI m/z [M+H]+ calcd for
C
18H22N+: 252.1747, found: 252.1737.
5.2. Biology
5.1.64. N-(4-(Adamantan-1-ylethynyl)phenyl)-4-
(chloromethyl)benzamide (50)
5.2.1. Cells and compounds
The title compound was prepared from 49 using a method anal-
ogous to that described for 31 in 85.3% yield as a white solid.
1H NMR (600 MHz, CDCl3): d = 1.72 (br s, 6H), 1.93–2.01 (m,
9H), 4.63 (s, 2H), 7.39 (d, J = 8.7 Hz, 2H), 7.50 (d, J = 8.1 Hz, 2H),
7.56 (d, J = 8.7 Hz, 2H), 7.80 (s, 1H), 7.85 (d, J = 8.1 Hz, 2H); 13C
Human U251 glioma cells were purchased from the Riken Cell
Bank (Ibaraki, Japan). U251/vascular endothelial growth factor
(VEGF)-placental alkaline phosphatase (PLAP) cells harbored a
plasmid in which the PLAP reporter gene was under the control
of a VEGF promoter, as described previously.17 The cells were

S. Nagao et al. / Bioorg. Med. Chem. 22 (2014) 5513–5529
5529
cultured in Dulbecco’s modified Eagle’s medium, supplemented
with 10% fetal bovine serum, 100 U/mL penicillin, and 100 g/mL
measured twice weekly, and the relative tumor volumes (the ratio
of tumor volume to initial size before treatment) were calculated.
l
streptomycin. The cells were incubated at 37 °C in a humidified
atmosphere containing 5% CO2 and either 21% O2 (normoxic condi-
tions) or 2% O2 (hypoxic conditions).
Acknowledgments
For the cellular assays, benzanilide compounds were prepared
in dimethyl sulfoxide (DMSO) and then diluted in the culture med-
ium. The final concentration of DMSO in any incubation mixture
did not exceed 0.2% (v/v). For the animal studies, 41a, 43a, and
51d were formulated in DMSO/Tween-80 (35:65), which was
diluted 5-fold with saline immediately before administration.
Following exposure to the compounds indicated, untransfected
The authors would like to thank the colleagues who helped to
generate all of the data reported in this manuscript. In particular,
special thanks are owed to Makoto Asano for a commitment to
the Project, Eri Ena, Satoko Sasaki, and Yumi Asai for NMR and
HRMS analyses.
Supplementary data
U251 cells in 96-well plates were solubilized in 100 lL lysis buffer
(50 mM Tris [pH 7.5], 300 mM NaCl, 10% glycerol, 3 mM EDTA,
1 mM MgCl2, 20 mM beta-glycerophosphate, 25 mM NaF, 1% Triton
X-100, and 1 mM Na3VO4). The lysates were diluted fivefold with
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2014.07.020.
PBS containing 1% bovine serum albumin, and 100
lL was assayed
References and notes
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a
a
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HIF-1 reporter activity was determined as previously
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Untransfected U251 cells were injected subcutaneously into the
flank or hind leg of female BALB/cA (nu/nu) athymic mice, and
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and diameter was the shortest dimension. All experiments were
approved by the Animal Care and Use Committee at the Eisai Tsu-
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Shocker (Yasui Kikai, Osaka, Japan). To determine the amount of
HIF-1a present, the fragmented tumors were lysed in lysis buffer
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[50 mM Tris (pH 7.5), 300 mM NaCl, 10% glycerol, 3 mM EDTA,
1 mM MgCl2, 20 mM b-glycerophosphate, 25 mM NaF, 1% Triton
X-100, and 1 mM Na3VO4) and centrifuged at 1500ꢁg for 5 min.
The total protein concentration of the sample supernatant was
determined using a BCA protein assay kit (Thermo Fisher Scientific,
Waltham, MA). The HIF-1
a concentration in the supernatant was
determined by ELISA, as described above (Section 5.2.1),18 and nor-
malized to the total protein concentration.
5.2.5. Antitumor studies
Mice bearing untransfected U251 xenografts were stratified
into groups of 5 animals with approximately equal mean tumor
volumes. The animals received a 10 mL/kg oral administration of
43a or vehicle once daily for 11 days. The tumor volumes were