Angiogenesis inhibitors identified by cell-based high-throughput screening: Synthesis, structure-activity relationships and biological evaluation of 3-[(E)-styryl]benzamides that specifically inhibit endothelial cell proliferation

Article abstract of DOI:10.1016/j.bmc.2011.12.058

Proliferation of endothelial cells is critical for angiogenesis. We report orally available, in vivo active antiangiogenic agents which specifically inhibit endothelial cell proliferation. After identifying human umbilical vein endothelial cell (HUVEC) pr

Full text of DOI:10.1016/j.bmc.2011.12.058

Bioorganic & Medicinal Chemistry 20 (2012) 1442–1460
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Angiogenesis inhibitors identified by cell-based high-throughput screening:
Synthesis, structure–activity relationships and biological evaluation of 3-[(E)-
styryl]benzamides that specifically inhibit endothelial cell proliferation
Kihito Hada a,b, , Atsushi Suda a, Kohsuke Asoh a, Takuo Tsukuda a, Masami Hasegawa a, Yasuko Sato a,
⇑
Kotaro Ogawa a, Shino Kuramoto a, Yuko Aoki a, Nobuo Shimma a, Tsutomu Ishikawa b, Hiroshi Koyano a
a Kamakura Research Laboratories, Chugai Pharmaceutical Co., Ltd., 200 Kajiwara, Kamakura, Kanagawa 247-8530, Japan
b Graduate School of Pharmaceutical Sciences, Chiba University, 1-33 Yayoi, Inage Chiba 263-8522, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Proliferation of endothelial cells is critical for angiogenesis. We report orally available, in vivo active anti-
angiogenic agents which specifically inhibit endothelial cell proliferation. After identifying human
umbilical vein endothelial cell (HUVEC) proliferation inhibitors from a cell-based high-throughput
screening (HTS), we eliminated those compounds which showed cytotoxicity against HCT116 and vascu-
lar endothelial growth factor receptor 2 (VEGFR-2) inhibitory activity. Evaluations in human Calu-6 xeno-
graft model delivered lead compound 1. Following extensive lead optimization and alteration of the
scaffold we discovered 32f and 32g, which both inhibited the proliferation and tube formation of HUVEC
without showing inhibitory activity against any of 25 kinases or cytotoxicity against either normal fibro-
blasts or 40 cancer cell lines. Upon oral administration, 32f and 32g had good pharmacokinetic profiles
and potent antitumor activity and decreased microvessel density (MVD) in Calu-6 xenograft model. Com-
bination therapy with a VEGFR inhibitor enhanced the in vivo efficacy. These results suggest that 32f and
32g may have potential for use in cancer treatment.
Received 7 December 2011
Revised 27 December 2011
Accepted 28 December 2011
Available online 5 January 2012
Keywords:
Angiogenesis
Endothelial cell
HUVEC
3-[(E)-styryl]benzamide
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
inhibitors targeting VEGFRs, primarily VEGFR-2 (also known as
KDR) have been the most studied and three multi-kinase inhibitors
Angiogenesis, the formation of new blood vessels from existing
vasculature, plays an important role in tumor growth and metasta-
sis.1 The growth of new blood vessels involves the proliferation of
endothelial cells in response to specific growth stimuli such as vas-
cular endothelial growth factor (VEGF), one of the most potent
tumor angiogenic factors, and the migration of these endothelial
cells to the tumor site to form new capillaries supplying oxygen
and nutrition to the growing tumor.2 Evidence shows that inhibi-
tion of angiogenesis can suppress the progression of tumor growth.
Indeed, the clinical benefit of angiogenesis inhibitors has been
demonstrated by bevacizumab, a recombinant humanized mono-
clonal antibody to VEGF, which was approved for the treatment
of colorectal cancer in combination with 5-FU/CPT-11 in 2004.3
By binding to VEGF, bevacizumab prevents it from binding to the
receptor (VEGFR), thus inhibiting endothelial cell proliferation
and tube formation.4 In other words, inhibiting endothelial cell
proliferation can lead to antiangiogenesis.5
with potent VEGFR-2 inhibition, sunitinib,6 sorafenib,7 and pazopa-
nib8 have been approved for the treatment of advanced cancers.
Despite their clinical benefits, drug resistance and on-target
adverse events such as hypertension, proteinuria and hemorrhage
are observed during treatment with VEGFR inhibitors.9–13 Thus,
there is still a need for angiogenesis inhibitors which could over-
come these drawbacks through a different mode of action from that
of VEGFR inhibitors. This premise prompted us to search for new
small-molecule angiogenesis inhibitors.
Cell-based high-throughput screening (HTS) of our chemical
library by applying human umbilical vein endothelial cell
(HUVEC) antiproliferative assays followed by counter assays
identified lead compound 1 (RO0123743), which inhibits angio-
genesis both in vitro and in vivo and does not show cytotoxicity
or VEGFR-2 inhibition. As a result of extensive chemical modifi-
cations, compounds 32f and 32g were identified as potent and
specific endothelial proliferation inhibitors with good physico-
chemical properties, metabolic stability, and significant oral
efficacy in a human xenograft model. Herein, we describe iden-
tifying lead compound 1 and optimizing it efficiently into 32f
and 32g. The results of their biological evaluations are also
described.
To date, a large number of small-molecule angiogenesis inhibi-
tors have been reported. Among them, receptor tyrosine kinase
⇑
Corresponding author. Tel.: +81 467 47 6069; fax: +81 467 2248.
E-mail address: hadakht@chugai-pharm.co.jp (K. Hada).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.12.058

K. Hada et al. / Bioorg. Med. Chem. 20 (2012) 1442–1460
1443
R3
R1
R2
OH
R3
R1
O
a
R4
+
Cl
R4
R2
2a, R1 = Me, R2 = Cl
2b, R1 = H, R2 = Cl
2c, R1 = Me, R2 = H
3a, R3 = OMe, R4 = Ac
4a, R1 = Me, R2 = H, R3 = OMe, R4 = Ac
4b, R1 = H, R2 = Cl, R3 = OMe, R4 = Ac
4c, R1 = Me, R2 = Cl, R3 = H, R4 = CN
3b, R3 = H, R4 = CN
O
O
O
b
O
O
Cl
Cl
1
5
O
O
O
c
d
R1
Cl
O
O
O
Cl
O
O
O
O
8a, R1 = Me
8b, R1 = H
6
7
O
O
e
f
R1
Cl
O
R1
Cl
O
OH
NH2
O
O
9a, R1 = Me
9b, R1 = H
10a, R1 = Me
10b, R1 = H
O
O
O
g
h
O
O
9a
10a
N
O
Cl
Cl
12
11
Scheme 1. Reagents and conditions: (a) K2CO3, DMF, 80 °C; (b) Et3SiH, TFA, rt, 6 h, 36%; (c) MOMCl, SnCl4, CH2Cl2, 0 °C, 5 h, 60%; (d) 2a–b, K2CO3, DMF, 80 °C; (e) 20% KOH,
MeOH, reflux, 1 h; (f) 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), 1-hydroxybenzotriazole (HOBT), NH4Cl, N,N-diisopropylethylamine (DIPEA), DMF, rt; (g)
TMSCHN2, toluene, MeOH, rt, 2.5 h, 95%; (h) (HCHO)n, HCO2H, CH3CN, reflux, 14 h, 77%.
HO
HO
O
O
O
O
a
b
c
O
H
O
H
O
HO
O
O
O
O
O
O
13
14
15
16
O
O
O
O
O
d, e
g
f
H
N
H
H
N
OH
N
NH2
O
O
O
O
O
Cl
Cl
Cl
17
18
19
Scheme 2. Reagents and conditions: (a) MgCl2, Et3N, 1,2-dichloroethane, 40 °C, 1 h, then (HCHO)n, 70 °C, 3 h, 76%; (b) Me2SO4, K2CO3, acetone, reflux, 1 h, 86%; (c) NaClO2,
NaH2PO4, 2-methyl-2-butene, tert-BuOH-H2O, rt, 50 min, 100%; (d) (COCl)2, cat.DMF, CH2Cl2, rt, 2.5 h. (e) 4-chloroaniline, DIPEA, CH2Cl2, rt, 1 h, 76% (2 steps); (f) 20% KOH,
MeOH, reflux, 30 min, 94%; (g) EDC, HOBT, NH4Cl, DIPEA, DMF, rt, 17 h, 93%.
was conducted as outlined in Scheme 1. Compounds 4a–c were
prepared by coupling phenols 2a–c with the corresponding benzyl
chlorides 3a–b under basic conditions. Reduction of the carbonyl
group of 1 with Et3SiH in trifluoroacetic acid (TFA) provided 5.
2. Chemistry
The synthesis of analogues 4–12 bearing different functional
groups from those of lead compound 1 on benzyl phenyl ether

1444
K. Hada et al. / Bioorg. Med. Chem. 20 (2012) 1442–1460
O
O
O
O
O
a
O
P
a
O
O
O
Cl
O
O
H
O
O
O
O
O
Cl
7
24
15
(E/Z)-20
O
O
b
c
O
P
c
b
O
O
OH
OH
O
O
Cl
O
25
(E/Z)-21
O
O
d
O
P
O
O
O
NH2
NH2
NH2
R
O
O
+
O
Cl
26
27a-u
O
NH2
Cl
Scheme 4. Reagents and conditions: (a) triethyl phosphite, 160 °C, 2.5 h, 95%; (b)
5 M NaOH, MeOH, 60 °C, 1.5 h, 96%; (c) EDC, HOBT, NH4Cl, DIPEA, DMF, rt, 20 h,
62%; (d) substituted benzaldehydes, sodium tert-pentoxide, DMF, rt.
22
23
Scheme 3. Reagents and conditions: (a) (4-chlorobenzyl)triphenylphosphonium
chloride, NaOEt, EtOH, rt, 1 h, 92%; (b) 20% KOH, MeOH, reflux, 2 h, 95%; (c) EDC,
HOBT, NH4Cl, DIPEA, DMF, rt, 16 h, 33% for 22 and 62% for 23, respectively.
28 gave 29a–e. Hydrolysis of 29a–e followed by condensation fur-
nished the target compounds 31a–e.
Compounds 32a–h were prepared by the synthetic route out-
lined in Scheme 6. Carboxylic acid (E)-21 was adopted as a com-
mon intermediate to synthesize amides with various solubilizing
groups. Horner-Wadsworth-Emmons reaction of 15 with diethyl
(4-chlorobenzyl)phosphonates gave stilbene (E)-20 as a sole iso-
mer. Hydrolysis of the ester afforded carboxylic acid (E)-21. Com-
pounds 32a–h were prepared by condensation of (E)-21 via acid
chlorides with various amines.
Compounds 9–12 were prepared from commercially available
ethyl 4-methoxybenzoate (6) via 3–5 synthetic steps. Thus, reac-
tion of 6 with methoxymethyl (MOM) chloride in the presence of
SnCl4 provided 7.14 Coupling of 7 with phenols 2a–b in the pres-
ence of K2CO3 gave the corresponding benzyl phenyl ethers 8a–b.
Compounds 8a–b were hydrolyzed under basic conditions to give
9a–b. Esterification of the carboxylic acid 9a with trimethylsilyl-
diazomethane afforded methyl ester 11. Carboxylic acids 9a–b
were condensed with NH4Cl to give the corresponding amides
10a–b. Nitrile 12 was obtained from 10a by direct conversion of
the amide group by aldehyde-catalyzed water transfer.15
3. Results and discussion
3.1. Lead generation from cell-based HTS
Amide derivative 19 was prepared as shown in Scheme 2.
Formylation of 13 was performed in a similar way to the procedure
of Skattebøl and co-workers.16 Methylation of 14 using methyl
iodide afforded 15. Pinnick oxidation17 of 15 afforded carboxylic
acid 16. Reaction of 16 with 4-chloroaniline via acid chloride pro-
vided 17. Amide 19 was prepared by hydrolysis of ethyl ester in 17
followed by condensation of 18. To obtain stilbene analogues, we
adopted the synthetic methods shown in Scheme 3–6. Stilbenes
22 and 23 were synthesized as shown in Scheme 3. Wittig reaction
of 15 with (4-chlorobenzyl)triphenylphosphonium chloride gave
(E)-20 and (Z)-20 as a 1:2 mixture. Ester hydrolysis followed by
condensation gave amides, which were separated into 22 [(E)-iso-
mer] and 23 [(Z)-isomer].
4-Methoxy-3-[(E)-styryl]benzamide analogues 27a–u described
here were synthesized as outlined in Scheme 4. We selected 26 as a
key intermediate to synthesize 27a–u because Horner-Wadsworth-
Emmons reaction with commercially available aldehydes gives
derivatives with various substituents on the A phenyl ring. Arbuzov
reaction of 7 with triethyl phosphite afforded 24. Hydrolysis of the
ethyl ester group in 24 under basic conditions provided acid 25,
which was converted to amide 26. Horner-Wadsworth-Emmons
reaction of 26 with different aldehydes gave compound 27a–u.
3-[(E)-2-(4-chlorophenyl)vinyl]benzamides 31a–e were syn-
thesized by the method shown in Scheme 5. We chose 28 as an
intermediate to facilitate derivatization of the methoxy moiety of
22. Horner-Wadsworth-Emmons reaction of 14 with diethyl (4-
chlorobenzyl)phosphonate provided a stilbene 28. Alkylation of
The evaluation cascade used to obtain our lead compounds is
shown in Figure 1. As a primary screening, high-throughput
VEGF-stimulated HUVEC proliferation assays at 4 lM were per-
formed on 280,000 compounds. The compounds which showed
over 50% inhibition against HUVEC growth were further evaluated
with a cell growth inhibition assay using a human colorectal cancer
cell line, HCT116, and a VEGFR-2 inhibition assay to eliminate non-
selective cytotoxics and VEGFR-2 inhibitors. We identified 14 lead
candidates which have more than 5-fold selectivity (defined as the
IC50 ratio of HCT116 to HUVEC) and no VEGFR-2 inhibition. Those
candidates which showed tumor growth inhibition (TGI) in a hu-
man lung cancer (Calu-6) xenograft model (chosen for its sensitiv-
ity to antiangiogenic agents18) and microvessel density (MVD)
reduction in the xenograft tissues were nominated as the lead
compounds. We believe that this proof-of-concept confirmation
in animal models is essential when generating leads from
cell-based screening. Among the lead candidates, 1 (RO0123743)
was the most promising lead compound showing antiproliferative
activity against HUVEC (IC50 = 1.3
activity against HCT116 (IC50 = 34 M) and no VEGFR-2 inhibition
(IC50 >50 M) in vitro. In vivo, moderate activity (TGI = 34%) in
lM), weak antiproliferative
l
l
the Calu-6 xenograft model was observed when 1 was orally
administered once a day for 11 consecutive days (600 mg/kg),
and antiangiogenic activity was confirmed by MVD reduction
(15%) in the xenograft tissue (Fig. 2).

K. Hada et al. / Bioorg. Med. Chem. 20 (2012) 1442–1460
1445
R
O
29a-c for b
29d-e for c
HO
HO
a
O
O
H
O
O
O
O
O
Cl
Cl
14
28
29a, R = Et
29b, R = n-Pr
29c, R = HO(CH ) O(CH )
2 2
2 2
R
O
R
O
N
N
d
e
29d, R =
29e, R =
*
OH
NH2
O
O
N
Cl
Cl
*
30a-e
31a-e
Scheme 5. Reagents and conditions: (a) diethyl (4-chlorobenzyl)phosphonate, sodium tert-pentoxide, DMF, 0 °C, 2 h, 71%; (b) alkyl halides, K2CO3, CH3CN, 60 °C; (c)
Br(CH2)2Br, K2CO3, DMF, 80 °C, then amines; (d) 20% KOH, MeOH, reflux; (e) EDC, HOBT, NH4Cl, DIPEA, DMF, rt.
O
O
O
O
O
R2
N
a
b
c
H
O
O
OH
R3
O
O
O
O
Cl
Cl
Cl
15
(E)-20
(E)-21
32a-h
Scheme 6. Reagents and conditions: (a) diethyl (4-chlorobenzyl)phosphonates, sodium tert-pentoxide, toluene-THF, 0 °C; (b) 20% KOH, MeOH, 65 °C, 80% (2 steps); (c)
(COCl)2, cat.DMF, CH2Cl2, rt, then amines, DIPEA, CH2Cl2, rt.
Cellular Antiproliferation of
VEGF-stimulated HUVEC and HCT116
O
O
IC50 (HCT116) / IC50 (HUVEC) > 5
O
Cl
VEGFR-2 Phosphorylation Inhibition
1 (RO0123743)
IC50 > 50 μM
in vitro
in vivo
HUVEC IC 50 = 1.3 μM
HCT116 IC50 = 34 μM
VEGFR-2 IC 50 > 50 μM
TGI = 34% (600 mg/kg)
MVD reduction = 15%
Tumor Growth Inhibition and MVD Reduction
in Animal Model
Figure 1. Evaluation cascade.
Figure 2. Structure and biological profiles of lead compound 1 (RO0123743).
3.2. Lead optimization
We started structural optimization of lead compound 1 by opti-
mizing functional groups around the benzyl phenyl ether moiety,
using the same evaluation cascade as that of for lead identification
(Table 1). The terminal acetyl group on the B phenyl ring was mod-
ified first. Replacement of the acetyl group (1) with a cyano group
(12) or a methyl ester group (11) maintained the antiproliferative
adjacent to the chloro group of the A phenyl ring did not affect
antiproliferative activity on either HUVEC (IC50 = 1.2 and 0.66
for 4b and 10b, respectively) or HCT116 (IC50 = 39 and >50
l
l
M
M
for 4b and 10b, respectively) compared to those of 1 and 10a.
However, removal of the chloro group (4a) at R2-position or
methoxy group (4c) at R3-position resulted in the reduction of
activity against HUVEC (IC50 = 1.5 and 2.3
respectively) while an ethyl group (5) showed a reduction in activ-
ity (IC50 = 40 M). Higher activity was obtained with an analogue
lM for 12 and 11,
antiproliferative activity on HUVEC (IC50 = 2.8 and 11 lM for 4a
and 4c, respectively), indicating that substituents at both R2- and
R3-positions were necessary for a potent inhibition of HUVEC
proliferation.
l
carrying an amide (10a). These results suggest that a hydrogen
acceptor at R4-position is essential for the antiproliferative activity
against HUVEC and a hydrogen donor enhances the activity. Com-
Analogues 10a–b were selected for a VEGFR-2 inhibition assay
and were found to show no VEGFR inhibition (IC50 >50 lM), so
pound 10a also had no activity against HCT116 (IC50 >50 lM),
we further evaluated their in vivo efficacy (Table 2). Compound
10b showed improved antitumor and antiangiogenic activity
(62% TGI and 27% MVD reduction, respectively) after once-daily
oral administration for 11 consecutive days at 600 mg/kg. In con-
trast, compound 10a displayed weak TGI (14%) and no MVD reduc-
tion. Mouse liver microsomal clearances of 10a and 10b (414 and
resulting in high selectivity (IC50 ratio of HCT116 to HUVEC >78).
Carboxylic acid (9a) did not inhibit the proliferation of HUVEC or
HCT116, presumably due to low cell penetration.
The necessity for substituents at R1-, R2- and R3-positions was
examined by deletion studies. Deletion of the methyl group

1446
K. Hada et al. / Bioorg. Med. Chem. 20 (2012) 1442–1460
Table 1
Cell growth inhibition of benzyl phenyl ethers against HUVEC and HCT116
R3
B
R1
R2
O
R4
A
Compd
Substituents
R3
Cell growth inhibition/IC50
(
l
M)
Selectivitya
R1
R2
Cl
R4
HUVEC
HCT116
HCT116/HUVEC
*
1
Me
MeO
MeO
MeO
1.3
34
26
8.6
33
O
O
O
*
*
4a
Me
H
2.8
1.2
24
39
4b
H
Cl
4c
5
Me
Me
Cl
Cl
H
MeO
CN
Et
11
40
45
43
4.1
1.1
OH
*
9a
Me
Me
H
Cl
Cl
Cl
MeO
MeO
MeO
>50
>50
>50
>50
NDb
>78
>75
O
NH2
NH2
O
*
*
*
10a
10b
0.64
0.66
O
O
O
11
12
Me
Me
Cl
Cl
MeO
MeO
2.3
1.5
23
10
CN
>50
>33
a
IC50 ratio of HCT116 to HUVEC.
Not determined.
b
97
l
L/min/mg, respectively) might explain the weak in vivo
potent inhibition of HUVEC proliferation. Compound 22 showed no
VEGFR-2 inhibition (IC50 >50 M) and improved in vivo antitumor
efficacy of 10a.
l
With the preferred amide, methoxy, and chloro groups kept in
place on the A and B phenyl rings, our next effort focused on alter-
ing the benzyl phenyl ether bond. Compound 10b still had weak
and antiangiogenic activity (71% TGI and 60% MVD reduction,
respectively) after once-daily oral administration for 11 consecu-
tive days at 300 mg/kg.
antiproliferative activity against HUVEC (IC50 = 0.66
l
M), presum-
To further improve the antiproliferative activity against HUVEC,
intensive derivatization on A phenyl ring of 22 was performed
(Table 4). Replacement of the chlorine atom at 4-position of 22 with
bromine (27c), or fluorine (27f) resulted in a significant reduction of
antiproliferative activity against HUVEC. Replacement of the chlo-
rine atom with electron-withdrawing groups (27d, 27g and 27h)
or electron-donating groups (27b, 27e and 27i) at 4-position also
decreased inhibition of HUVEC proliferation while antiproliferative
activity against HCT116 was less affected. As we expected from the
result of the deletion studies, compounds carrying a substituent at
2- or 3-position (27j–r) decreased the antiproliferative activity on
ably due to a high degree of conformational flexibility of the ether
bond. Since the conformational restriction is one of the common
practices for improvement of activity, we reduced the flexibility
of 10b. As shown in Table 3, replacement of the ether bond with
a trans-double bond (22) significantly enhanced the antiprolifera-
tive activity against HUVEC (IC50 = 0.087 lM) while maintaining
high selectivity (IC50 ratio of HCT116 to HUVEC = 82). A cis-double
bond (23) and an amide bond (19) decreased inhibition of HUVEC
proliferation. These results suggest that fixing position between A
and B phenyl rings by hydrophobic trans-olefin is favorable for the
Table 2
VEGFR-2 inhibition and in vivo antitumor efficacy of 10a and 10b
O
O
NH2
O
R
Cl
Compd
R
VEGFR-2 inhibition/IC50
(lM)
Microsomal clearancea
(
lL/min/mg)
In vivo antitumor efficacy/TGIb (%)
MVD reductionc (%)
10a
10b
Me
H
>50
>50
414
97
14
62
0
27
a
b
c
Clearance of test compound when incubated with mouse liver microsomes.
Compound 10a and 10b were orally administered at 600 mg/kg in the Calu-6 xenograft model.
Microvessel density reduction in the Calu-6 xenograft tissue.

K. Hada et al. / Bioorg. Med. Chem. 20 (2012) 1442–1460
1447
Table 3
Optimization of benzyl phenyl ether bond
O
NH2
L
O
Cl
Compd
L
Cell growth inhibition/IC50
(lM)
Selectivitya
Solubility in
Microsomal
clearanceb
(lL/mim/mg)
In vivo antitumor
efficacy/TGI (%)
MVD reductionc
(%)
FaSSIF (
l
g/mL)
HUVEC
HCT116
HCT116/HUVEC
H
N
*
*
19
5.5
>50
>9
5
16
NDd
NDd
*
*
*
O
*
22
23
0.087
2.9
7.1
29
82
10
16
110
544
71e
60e
137
NDd
NDd
a
b
c
IC50 ratio of HCT116 to HUVEC.
Clearance of test compound when incubated with mouse liver microsomes.
Microvessel density reduction in the Calu-6 xenograft tissue.
Not determined.
d
e
Compound 22 was orally administered at 300 mg/kg in the Calu-6 xenograft model.
Table 4
27u were less potent than 22. Overall, 4-monochloro substituent
(22) was the most favorable for A phenyl ring.
In vitro structure–activity relationships on A phenyl ring of 3-[(E)-styryl]benzamides
Despite its potent inhibition of HUVEC proliferation and good
selectivity to HCT116, compound 22 had low solubility in fasted
O
state simulated intestinal fluid (FaSSIF)19 (16
lg/mL) and moderate
2
B
mouse liver microsomal clearance (110
lL/min/mg), presumably
NH2
3
due to high lipophilicity20 (logD = 4.0 at pH 7.4) (Table 3). Further
optimization of 22 to improve the solubility and the metabolic sta-
bility by introducing solubilizing groups led us eventually to iden-
tify 32f and 32g. We first tried to improve them by modifying the
methoxy group on B phenyl ring (Table 5). An abrupt loss of activ-
ity was, however, observed with solubilizing groups (31c–e) as
well as with substituents such as ethoxy (31a) or n-propoxy
(31b) groups, suggesting that substituents at the position of the
methoxy motif fit in a space limited in size. We explored the idea
of introducing a solubilizing group at amide nitrogen. Amides 32a–
c were first prepared to test whether the modification of the amide
moiety is tolerable. Mono-substituted amides 32a and 32c main-
tained antiproliferative activity against and selectivity to HUVEC,
while N,N-dimethyl amide 32b had diminished activity suggesting
that a hydrogen donor is necessary for a potent inhibition of HU-
VEC proliferation. This observation is consistent with that of the
R4 on benzyl phenyl ether. Introduction of hydroxylated alkyl
groups at amide nitrogen, as illustrated by ethanol 32d, 1,2-prop-
anediols 32e–g, and 1,3-propanediol 32h had moderate to good
levels of antiproliferative activity against HUVEC (IC50 = 0.25–
R
4
A
O
Compd
R
Cell growth inhibition/IC50
(lM)
Selectivitya
HUVEC
HCT116
HCT116/HUVEC
27a
27b
27c
27d
27e
27f
27g
27h
27i
H
4-t-Bu
4-Br
2.4
0.37
0.56
0.88
1.6
2.1
3.1
4.7
8.8
3.9
8.3
24
36
20
22
35
48
24
16
22
19
23
>50
15
19
26
11
>50
36
>50
>50
11
15
54
39
40
30
11
5.2
4.7
2.2
5.9
>6.0
0.6
0.4
13
4.8
>8.8
4
2.2
>19
20
4-CF3
4-Me
4-F
4-CN
4-NO2
4-OMe
3-Me
3-F
3-Cl
3-CN
2-F
2-Me
2-OMe
2-OCF3
2-Cl
2,4-diCl
3,4-diCl
2-F-4-Cl
27j
27k
27l
27m
27n
27o
27p
27q
27r
27s
27t
27u
49
2.0
2.3
5.7
8.9
23
2.6
0.56
1.7
1.5
bility (317, 225, and 263
showed good stability in mouse liver microsomes (10, 7, and
12 L/min/mg, respectively) while keeping antiproliferative activ-
l
M). Among them, 1,2-propanediols 32e–g improved the solu-
l
g/mL in FaSSIF, respectively) and
l
>10
>5.9
ity against HUVEC and high selectivity (IC50 ratio of HCT116 to
HUVEC = 30, 27, and 84, respectively). Chirality of 1,2-propanediols
(32f and 32g) did not affect antiproliferative activity against either
HUVEC or HCT116. From these results, chiral 32f and 32g were se-
lected for intensive in vitro and in vivo profiling.
a
IC50 ratio of HCT116 to HUVEC.
HUVEC. Larger substituents (27l–m, 27p–r) at 2- or 3-position had a
tendency to inhibit HUVEC proliferation less potently compared to
the unsubstituted compound 27a (IC50 = 2.4
l
M), indicating limited
3.3. Biological evaluation
spaces at 2- and 3-positions. The same tendency was observed in
2,4- and 3,4-disubstituted compounds 27s–u. 3,4-Dichloro-substi-
tuted compound 27t and 2,4-disubstituted compounds 27s and
As an indicator of in vitro antiangiogenic activity, the effect of 32f
and 32g on tube formation was evaluated using an Angiogenesis Kit

1448
K. Hada et al. / Bioorg. Med. Chem. 20 (2012) 1442–1460
Table 5
In vitro structure–activity relationships on B phenyl ring of 3-[(E)-styryl]benzamides
R1
O
R3
N
B
R2
A
O
Cl
Compd
Substituents
R2
Cell growth inhibition/IC50
(l
M)
Selectivitya
Solubility in FaSSIFb
g/mL)
Microsomal clearancec
L/min/mg)
(l
(l
R1
R3
HUVEC
HCT116
HCT116/HUVEC
31a
31b
31c
Et
n-Pr
H
H
H
H
H
H
0.19
1.3
11
1.7
>50
38
8.9
38
1.5
10
4
74
123
83
247
HO(CH2)2O(CH2)2–
N
31d
31e
H
H
H
8.4
9.5
4.9
7.5
0.6
0.8
109
164
288
251
*
N
H
N
*
32a
32b
32c
32d
Me
Me
Me
Me
Me
Me
Et
H
Me
H
0.19
2.5
0.16
0.91
16
35
15
17
84
14
94
19
24
NDd
9
331
NDd
187
73
(CH2)2OH
H
16
OH
32e
32f
32g
Me
Me
Me
H
H
H
0.40
0.48
0.25
12
13
21
30
27
84
317
225
263
10
7
OH
OH
OH
*
OH
*
OH
12
*
*
OH
OH
32h
Me
H
1.5
14
9.3
251
8
a
b
c
IC50 ratio of HCT116 to HUVEC.
FaSSIF: fasted state simulated intestinal fluid.
Clearance of test compound when incubated with mouse liver microsomes.
Not determined.
d
(a)
(b)
4 μΜ
20 μΜ
32f
32g
100
90
80
70
60
50
40
30
20
10
0
Vehicle
4 μM
20 μM
32f
32g
Compound
Figure 3. Inhibition of tube formation by 32f and 32g in a HUVEC and fibroblast co-culture system. (a) Images of 32f and 32g at concentrations of 0, 4, and 20
Percentage inhibition by 32f and 32g in a tube formation assay.
lM. (b)
(Kurabo Industries Ltd.) which is composed of a co-culture of HU-
VEC and fibroblasts.21 As shown in Figure 3, the tube formation
was strongly inhibited by 32f and 32g at the concentrations of 4
and 20 lM (4 lM: 38% and 41%; 20 lM: 86% and 93% for 32f and
32g, respectively). No morphological damage of normal fibroblast
cells was observed at either concentration. Both compounds also

K. Hada et al. / Bioorg. Med. Chem. 20 (2012) 1442–1460
1449
Table 6
Cell growth inhibition of 32f and 32g against various cell lines
Cell line
Tumor type
IC50
/
l
M (selectivitya)
Cell line
Tumor type
IC50/l
M (Selectivitya)
32f
32g
32f
32g
HUVEC
HCT116
WiDr
COLO205
COLO320DM
DLD1
HT29
HCT15
QG56
Colon
Colon
Colon
Colon
Colon
Colon
Colon
Colon
Lung
Lung
Lung
Lung
Lung
0.48 (1)
13 (27)
7 (15)
0.25 (1)
27 (108)
15 (60)
18 (72)
61 (244)
28 (112)
16 (64)
29 (116)
21 (84)
24 (96)
30 (120)
18 (72)
54 (216)
34(136)
38 (152)
15 (60)
7.2 (29)
69 (276)
63 (252)
1.4 (6)
DU145
PC3
Prostate
Prostate
Prostate
Pancreas
Pancreas
Pancreas
Pancreas
Gastric
Gastric
Gastric
Liver
Liver
5.3 (11)
24 (50)
8.6 (18)
55 (115)
47 (98)
27 (56)
39 (81)
5.7 (12)
15 (31)
52 (108)
29 (60)
16 (33)
30 (63)
38 (79)
38 (79)
20 (42)
30 (63)
29 (60)
31 (65)
17 (35)
23 (92)
17 (68)
21 (84)
63 (252)
67 (268)
51 (204)
49 (196)
26 (104)
23 (92)
52 (208)
27 (108)
28 (112)
30 (120)
46 (184)
50 (200)
30 (120)
30 (120)
36 (144)
38 (152)
20 (80)
22Rv1
AsPC-1
Capan-1
BxPC-3
Panc-1
MKN45
MKN-28
NCI-N87
Huh-7
Hep G2
HuH-1
C32
18 (38)
46 (96)
19 (40)
10 (21)
20 (42)
23 (48)
15 (31)
19 (40)
13 (27)
40 (83)
25 (52)
39 (81)
4.9 (10)
4.5 (9)
NCI-H460
NCI-H460-PTX250
A549
Calu-6
Liver
NCI-H441
MDA-MB-231
KPL-4
T47D
MDA-MB-435s
MCF7
Lung
Melanoma
Leukemia
Leukemia
Leukemia
Leukemia
Leukemia
Leukemia
Breast
Breast
Breast
Breast
Breast
Ovarian
Ovarian
10C9
CCRF-CEM
CEM/C2
K562
JOK-1
KG-1a
74 (154)
65 (135)
0.92 (2)
9.5 (20)
IGROV1
IGROV1/T8
12 (48)
a
IC50 ratio of cancer cell line to HUVEC.
Table 7
Oral pharmacokinetic parameters in nude micea
Compd
CL (mL/min/Kg)b
tmax (h)
Cmax (ng/mL)
t1/2 (h)
AUC0–24 h (ngꢀh/mL)
F (%)
32f
32g
5.8
5.6
1.0
1.0
8270
9370
7.8
8.5
86400
90200
100
90c
a
b
c
Compounds 32f and 32g were dosed at 30 mg/kg.
CL/F (mL/min/kg).
Oral bioavailability when 32g was dosed at 10 mg/kg.
exhibit less antiproliferative activity against 40 cancer cell lines
than against HUVEC (Table 6). These results indicate that 32f and
32g suppressed angiogenesis in vitro without showing cytotoxicity.
Additional in vitro profiling revealed that 32f and 32g showed no
As expected from the fact that 32f has no VEGFR-2 inhibitory
activity, 32f in combination with sunitinib, a multi-kinase inhibitor
with potent VEGFR-2 inhibition, enhanced the antitumor activity
that either compound was capable of alone (Fig. 5). Add-on was
possible to maximum tolerated dose (MTD) (80 mg/kg) of sunitinib
without body weight loss. This result suggests that 32f may be
used for combination therapy with other antitumor agents includ-
ing VEGFR inhibitors.
inhibitory activity against 25 kinases (IC50 >50 lM) including VEG-
FR-2, platelet-derived growth factor receptor (PDGFR) and fibro-
blast growth factor receptor 2 (FGFR2), whose activities are
related to angiogenesis (see Supplementary data). This result im-
plies a different mode of action from that of kinase inhibitors.
Pharmacokinetic parameters in nude mice of 32f and 32g are
shown in Table 7. Both compounds had low clearance
(5.6–5.8 mL/min/kg) and high bioavailability (F = 90–100%), result-
ing in sustained exposure (AUC = 86400 and 90200 ngꢀh/mL for 32f
and 32g, respectively) after single 30 mg/kg oral dosing.
The in vivo antitumor activity of 32f and 32g was evaluated in
the Calu-6 xenograft model (Fig. 4). Once-daily oral administration
of the compounds for 11 consecutive days inhibited the growth of
tumor in a dose-dependent manner in the range of 1 to 10 mg/kg
without apparent body weight loss (Fig. 4a and b). From the results
shown in Figure 4a, the ED50 values of 32f and 32g for tumor
growth inhibition were determined as 3.2 and 2.3 mg/kg, respec-
tively. MVD in Calu-6 xenograft tissue was determined by immu-
nohistochemistry 24 hours after the final administration. The
result demonstrated a significant decrease of MVD in the tissue
as compared to control (Fig. 4c). As shown in Table 6, 32f and
32g showed weak antiproliferative activity against the Calu-6 can-
4. Conclusion
The orally available 3-[(E)-styryl]benzamides 32f and 32g have
been shown to be potent HUVEC proliferation inhibitors both
in vitro and in vivo. These compounds exemplify a unique type
of angiogenesis inhibitor that specifically inhibits endothelial cell
proliferation and does not show cytotoxicity or inhibition of angi-
ogenesis-related kinases. In the lead generation and optimization
stages, proof-of-concept confirmation of antiangiogenic activity
in the animal model via oral administration prompted the identifi-
cation of these unique compounds. By reason of their potent
in vivo efficacy and their safety, 32f and 32g appear to have major
potential for use in cancer treatment. The studies for clarifying the
molecular targets are currently underway in our laboratories.
5. Experimental
5.1. Chemistry: instruments
cer cell line (IC50 = 40 and 54 lM for 32f and 32g, respectively).
Taken together, these results demonstrate that 32f and 32g
showed growth inhibition of the xenografted tumor through anti-
angiogenic activity.
Commercially available reagents and solvents were used with-
out further purification. Reactions were monitored by TLC on silica

1450
K. Hada et al. / Bioorg. Med. Chem. 20 (2012) 1442–1460
32f
32g
(a)
(c)
600
500
400
300
200
100
0
32f
32g
control
control
1 mg/kg
1 mg/kg
3 mg/kg
10 mg/kg
1 mg/kg
3 mg/kg
10 mg/kg
3 mg/kg
10 mg/kg
0
4
8
12
0
4
8
12
Days after 1st treatment
Days after 1st treatment
Control
(b)
120
110
100
90
120
110
100
90
60
50
40
30
20
10
0
1 mg/kg 3 mg/kg 10 mg/kg
32f
32g
80
80
0
4
8
12
0
4
8
12
32f
32g
-10
Days after 1st treatment
Days after 1st treatment
Compound
Figure 4. (a) In vivo antitumor activity in the Calu-6 xenograft model. Compounds 32f and 32g were orally administered once a day for 11 days at 1, 3, and 10 mg/kg to tumor
bearing Balb/c nude mice. (b) Body weight change (%). (c) MVD reduction in the Calu-6 xenograft tissue. Photographs are immunohistochemical staining of tumor tissues by
anti-mouse CD31 antibody (ꢂ10 magnification). Endothelial cells are stained brown.
gel 60 F254 precoated TLC plates (E. Merck) or NH TLC plates
(E. Merck). Melting points were determined with a Yanaco MP-S3
apparatus and are uncorrected. Proton nuclear magnetic resonance
(1H NMR) spectra were obtained at 400 or 500 MHz on a JEOL JNM-
ECA400 or a JEOL JNM-A500 spectrometer. Carbon nuclear mag-
netic resonance (13C NMR) spectra were obtained at 100 or
125 MHz on a JEOL JNM-ECA400 or a JEOL JNM-A500 spectrometer.
All NMR chemical shifts are reported as d values in parts per mil-
lion (ppm), and coupling constants (J) are given in hertz (Hz).
The splitting pattern abbreviations are as follows: s, singlet; d, dou-
blet; t, triplet; q, quartet; br s, broad singlet; br t, broad triplet; m,
unresolved multiplet due to the field strength of the instrument;
dd, doublet of doublet; dt, doublet of triplet; ddd, doublet of dou-
blet of doublet; dddt, doublet of doublet of doublet of triplet, dtt;
doublet of triplet of triplet. Chromatographic separations were car-
ried out on prepacked silica gel columns (KP-Sil) or prepacked ba-
sic silica gel columns (KP-NH), supplied by Biotage. Yields are
unoptimized. Purity of all final products was determined by LC–
MS to be >95%. The LC–MS analyses were performed using a
Waters LC–MS instrument equipped with a Waters ACQUITY SQD
(ESI mode) and a Waters ACQUITY UPLC instrument. Elution was
done with a gradient of 5–100% solvent B in solvent A (solvent A:
0.1% formic acid in water, solvent B: 0.1% of formic acid in acetoni-
trile) in 1 min followed by 0.4 min at 100% B through a AscentisÒ
5.1.1. General procedure for the synthesis of compounds 4a–c
A mixture of 2a–c (1.1 equiv), 3a–b (1 equiv) and K2CO3
(1.1 equiv) in DMF was stirred at 80 °C for 3 h. After cooling to
room temperature, the mixture was diluted with EtOAc. The organ-
ic layer was washed with water and brine, dried over anhydrous
Na2SO4 and concentrated in vacuo. The residue was purified by
flash chromatography (SiO2, n-hexane/EtOAc) to give the desired
products 4a–c.
5.1.2. 1-[4-Methoxy-3-[(3-methylphenoxy)methyl]phenyl]etha
none (4a)
Compound 4a was obtained from 2c and 3a as a colorless oil in
93% yield. 1H NMR (400 MHz, CDCl3) d: 2.34 (3H, s), 2.57 (3H, s),
3.93 (3H, s), 5.09 (2H, s), 6.76–6.89 (3H, m), 6.95 (1H, d,
J = 8.7 Hz), 7.18 (1H, t, J = 7.7 Hz), 7.96 (1H, dd, J = 8.7, 2.3 Hz),
8.11 (1H, d, J = 2.3 Hz). MS (ESI+) 271 [M+H]+. HRMS (ESI+) calcd
for C17H19O3 [M+H]+ 271.1329, found 271.1329.
5.1.3. 1-[3-[(4-Chlorophenoxy)methyl]-4-methoxyphenyl]etha
none (4b)
Compound 4b was obtained from 2b and 3a as a colorless oil in
98% yield. 1H NMR (400 MHz, CDCl3) d: 2.56 (3H, s), 3.94 (3H, s),
5.08 (2H, s), 6.93 (2H, d, J = 9.3 Hz), 6.95 (1H, d, J = 8.8H), 7.23
(2H, d, J = 9.3 Hz), 7.96 (1H, dd, J = 8.8, 2.2 Hz), 8.07 (1H, d,
J = 2.2 Hz). MS (ESI+) 291, 293 (Cl isotope) [M+H]+. HRMS (ESI+)
calcd for C16H16ClO3 [M+H]+ 291.0782, found 291.0783.
Express C18 column (50 mm 2.1 mm, 2.7 lm particles) at
1 mLꢀminꢁ1. Area % purity was measured at 254 nm. High-resolu-
tion mass spectra (HRMS) were measured with a ThermoFisherSci-
entific LTQ Orbitrap XL spectrometer. Optical rotations were
measured at 25 °C on a JASCO DIP-1000 digital polarimeter using
a 1.0-cm, 1-mL cell.
5.1.4. 3-[(4-Chloro-3-methylphenoxy)methyl]benzonitrile (4c)
Compound 4c was obtained from 2a and 3b as a colorless pow-
der in 93% yield. 1H NMR (400 MHz, CDCl3) d: 2.35 (3H, s), 5.05 (2H,

K. Hada et al. / Bioorg. Med. Chem. 20 (2012) 1442–1460
1451
brine, dried over anhydrous Na2SO4, and concentrated in vacuo.
The residue was recrystallized from n-hexane/EtOAc (30:1) to give
7 (23.8 g, 60%) as colorless plates. 1H NMR (400 MHz, CDCl3) d:
1.39 (3H, t, J = 7.0 Hz), 3.95 (3H, s), 4.36 (2H, q, J = 7.0 Hz), 4.66
(2H, s), 6.92 (1H, d, J = 8.3 Hz), 8.03 (1H, dd, J = 8.3, 2.1 Hz), 8.05
(1H, d, J = 2.1 Hz). MS (ESI+) 229, 231 (Cl isotope) [M+H]+. HRMS
(ESI+) calcd for C11H14ClO3 [M+H]+ 229.0626, found 229.0628.
1,200
1,000
800
600
400
200
0
(a)
Control
32f (150 mg/kg)
sunitinib (80 mg/kg)
32f (150 mg/kg) + sunitinib (80 mg/kg)
5.1.7. Ethyl 3-[(4-Chloro-3-methylphenoxymethyl)]-
4-methoxybenzoate (8a)
A mixture of 7 (4.00 g, 17.5 mmol), 2a (2.74 g, 19.2 mmol) and
K2CO3 (2.66 g, 19.3 mmol) in DMF (40 mL) was stirred at 80 °C
for 13 h. After cooling to room temperature, the solvent was re-
moved in vacuo. The residue was diluted with EtOAc and the insol-
uble solid was filtered and the cake was washed with EtOAc. The
filtrate was concentrated in vacuo and the residue was purified
by flash chromatography (SiO2, n-hexane/EtOAc = 10:1 to 5:1) to
give 8a (5.91 g, quant.) as a colorless powder. 1H NMR (400 MHz,
CDCl3) d: 1.38 (3H, t, J = 7.1 Hz), 2.35 (3H, s), 3.92 (3H, s), 4.35
(2H, q, J = 7.1 Hz), 5.05 (2H, s), 6.78 (1H, dd, J = 8.7, 2.9 Hz), 6.90
(1H, d, J = 2.9 Hz), 6.93 (1H, d, J = 8.7 Hz), 7.23 (1H, d, J = 8.7 Hz),
8.03 (1H, dd, J = 8.7, 2.2 Hz), 8.14 (1H, d, J = 2.2 Hz). MS (ESI+)
335, 337 (Cl isotope) [M+H]+. HRMS (ESI+) calcd for C18H20ClO4
[M+H]+ 335.1045, found 335.1042.
treatment
0
5
10
15
Days after 1st treatment
120
(b)
110
100
90
5.1.8. Ethyl 3-[(4-chlorophenoxy)methyl]-4-methoxybenzoate
(8b)
Compound 8b was prepared in a manner similar to that de-
scribed for 8a in quantitative yield as a colorless powder. 1H
NMR (400 MHz, CDCl3) d: 1.38 (3H, t, J = 7.1 Hz), 3.92 (3H, s),
4.35 (2H, q, J = 7.1 Hz), 5.06 (2H, s), 6.93 (1H, d, J = 8.5 Hz), 6.94
(2H, d, J = 9.3 Hz), 7.24 (2H, d, J = 9.3 Hz), 8.03 (1H, dd, J = 8.5,
2.2 Hz), 8.14 (1H, d, J = 2.2 Hz). MS (ESI+) 321, 323 (Cl isotope)
[M+H]+. HRMS (ESIꢁ) calcd for C17H16ClO4 [MꢁH]ꢁ 319.0732, found
319.0734.
80
0
5
10
15
Days after 1st treatment
Figure 5. (a) In vivo antitumor activity of 32f in the Calu-6 xenograft model in
combination with sunitinib. Compound 32f (150 mg/kg) and sunitinib (80 mg/kg)
were orally administered once a day for 11 days to tumor bearing Balb/c nude mice.
(b) Body weight change (%).
5.1.9. 3-[(4-Chloro-3-methylphenoxymethyl)]-4-methoxyben-
zoic acid (9a)
To a solution of 8a (5.86 g, 17.5 mmol) in MeOH (50 mL) was
added 20% aqueous solution of KOH (20 mL) and the mixture
was stirred for 1 h under reflux. The reaction mixture was diluted
in water (100 mL) and then cooled to 0 °C. The reaction mixture
was adjusted to pH of 4 by addition of 37% aqueous solution of
HCl. The resulting precipitate was filtered, washed with water to
give 9a (5.17 g, 96%) as a colorless powder. 1H NMR (400 MHz,
DMSO-d6) d: 2.29 (3H, s), 3.90 (3H, s), 5.07 (2H, s), 6.85 (1H, dd,
J = 8.8, 3.1 Hz), 7.04 (1H, d, J = 3.1 Hz), 7.15 (1H, d, J = 8.8 Hz),
7.29 (1H, d, J = 8.8 Hz), 7.93 (1H, dd, J = 8.8, 2.2 Hz), 7.96 (1H, d,
J = 2.2 Hz), 12.69 (1H, br s). MS (ESI+) 307, 309 (Cl isotope)
[M+H]+. HRMS (ESI+) calcd for C16H16ClO4 [M+H]+ 307.0732, found
307.0731.
s), 6.72 (1H, dd, J = 8.7, 3.0 Hz), 6.85 (1H, d, J = 3.0 Hz), 7.24 (1H, d,
J = 8.7 Hz), 7.50 (1H, dd, J = 7.7, 7.6 Hz), 7.61–7.66 (2H, m), 7.73
(1H, s). MS (ESIꢁ) 256, 258 (Cl isotope) [MꢁH]ꢁ. HRMS (ESIꢁ) calcd
for C15H11ClNO [MꢁH]ꢁ 256.0524, found 256.0530.
5.1.5. 1-Chloro-4-[(5-ethyl-2-methoxyphenyl)methoxy]-2-meth-
ylbenzene (5)
To a solution of 1 (50 mg, 0.16 mmol) in TFA (1 mL) was added
Et3SiH (320 lL, 2.00 mmol) and the mixture was stirred for 6 h.
The reaction mixture was concentrated in vacuo and the residue
was purified by flash chromatography (SiO2, n-hexane/
EtOAc = 10:1) to give 5 (17 mg, 36%) as a colorless oil. 1H NMR
(400 MHz, CDCl3) d: 1.17 (3H, t, J = 7.6 Hz), 2.48 (3H, s), 2.53 (2H, q,
J = 7.6 Hz), 3.90 (3H, s), 3.97 (2H, s), 6.71 (1H, d, J = 8.7 Hz), 6.74
(1H, s), 6.83 (1H, d, J = 8.3 Hz), 6.99 (1H, d, J = 2.2 Hz), 7.03 (1H, dd,
J = 8.3, 2.2 Hz), 7.13 (1H, d, J = 8.7 Hz). MS (ESIꢁ) 289, 291 (Cl isotope)
[MꢁH]ꢁ. HRMS (ESIꢁ) calcd for C17H16ClO2 [MꢁH]ꢁ 289.0990, found
289.0989.
5.1.10. 3-[(4-Chlorophenoxy)methyl]-4-methoxybenzoic acid
(9b)
Compound 9b was prepared in a manner similar to that de-
scribed for 9a in 99% yield as a colorless powder. 1H NMR
(400 MHz, DMSO-d6) d: 3.90 (3H, s), 5.09 (2H, s), 7.03 (2H, d,
J = 9.1 Hz), 7.16 (1H, d, J = 8.5 Hz), 7.34 (2H, d, J = 9.1 Hz), 7.95
(1H, dd, J = 8.5, 2.2 Hz), 7.96 (1H, d, J = 2.2 Hz), 12.67 (1H, s). MS
(ESI+) 293, 295 (Cl isotope) [M+H]+. HRMS (ESIꢁ) calcd for
5.1.6. Ethyl 3-(chloromethyl)-4-methoxybenzoate (7)
15H12ClO4 [MꢁH]ꢁ 291.0419, found 291.0420.
To a solution of 6 (28.0 mL, 172 mmol) and methoxymethyl chlo-
ride (26.0 mL, 342 mmol) in CH2Cl2 (500 mL) at 0 °C was added SnCl4
(10 mL, 85.5 mmol)dropwiseover 15 min. Thereactionmixture was
stirred for 5 h at 0 °C and then poured into water (1 L). The organic
layer was separated and the aqueous layer was extracted twice with
CH2Cl2 (200 mL). The organic layers were combined, washed with
C
5.1.11. 3-[(4-Chloro-3-methylphenoxy)methyl]-4-methoxy-
benzamide (10a)
To
a solution of 9a (5.12 g, 16.7 mmol), NH4Cl (1.34 g,
25.1 mmol), and 1-hydroxybenzotriazole (HOBT) (3.07 g,

1452
K. Hada et al. / Bioorg. Med. Chem. 20 (2012) 1442–1460
20.0 mmol) in DMF (50 mL) were added 1-ethyl-3-(3-dimethyl-
aminopropyl)carbodiimide (EDC) (3.84 g, 20.0 mmol) and N,N-
diisopropylethylamine (DIPEA) (8.7 mL, 49.9 mmol). The mixture
was stirred at room temperature for 23 h and concentrated in va-
cuo. The residue was diluted with EtOAc. The organic layer was
washed with a saturated aqueous solution of NaHCO3, dried over
anhydrous Na2SO4 and concentrated in vacuo. The residue was
purified by flash chromatography (SiO2, n-hexane/EtOAc = 1:1 to
EtOAc) to give 10a (4.48 g, 88%) as a colorless powder. 1H NMR
(400 MHz, DMSO-d6) d: 2.30 (3H, s), 3.88 (3H, s), 5.04 (2H, s),
6.85 (1H, dd, J = 8.8, 3.0 Hz), 7.04 (1H, d, J = 3.0 Hz), 7.10 (1H, d,
J = 8.8 Hz), 7.21 (1H, br s), 7.30 (1H, d, J = 8.8 Hz), 7.87 (1H, br s),
7.89 (1H, dd, J = 8.8, 2.2 Hz), 7.95 (1H, d, J = 2.2 Hz). MS (ESI+)
306, 308 (Cl isotope) [M+H]+. HRMS (ESI+) calcd for C16H17ClNO3
[M+H]+ 306.0891, found 306.0889.
Na2SO4, and concentrated in vacuo. The crude residue was recrys-
tallized from MeOH-water (3:4) to give 14 (27.0 g, 76%) as colorless
prisms. Melting point 67–68 °C (lit.22 69–70 °C). 1H NMR
(400 MHz, CDCl3) d: 1.41 (3H, t, J = 7.1 Hz), 4.39 (2H, q,
J = 7.1 Hz), 7.03 (1H, d, J = 8.8 Hz), 8.19 (1H, dd, J = 8.8, 2,0 Hz),
8.31 (1H, d, J = 2.0 Hz), 9.95 (1H, s), 11.38 (1H, s). MS (ESI+) 195
[M+H]+. HRMS (ESIꢁ) calcd for C10H9O4 [MꢁH]ꢁ 193.0495, found
193.0496.
5.1.16. Ethyl 3-formyl-4-methoxybenzoate (15)
To a solution of 14 (72.0 g, 371 mmol) in acetone were added
K2CO3 (61.5 g, 445 mmol) and Me2SO4 (58.5 g, 463 mmol). The
mixture was stirred for 1 h under reflux. After cooling to room tem-
perature, the insoluble solid was filtered and the cake was washed
with EtOAc (500 mL). The filtrate was diluted with EtOAc (500 mL)
and saturated aqueous solution of NaHCO3 (400 mL). The organic
layer was separated, and the aqueous layer was extracted with
EtOAc (1000 mL ꢂ 2). The organic layers were combined, washed
with brine (300 mL), dried over anhydrous Na2SO4, filtered, and
the solvent was removed under reduced pressure. The residue
was recrystallized from acetonitrile–water (5:4) to give 15
(66.7 g, 86%) as colorless needles. Melting point 77–79 °C. 1H
NMR (400 MHz, CDCl3) d: 1.39 (3H, t, J = 7.1 Hz), 4.00 (3H, s),
4.37 (2H, q, J = 7.1 Hz), 7.04 (1H, d, J = 8.8 Hz), 8.25 (1H, dd,
J = 8.8, 2.5 Hz), 8.49 (1H, d, J = 2.5 Hz), 10.45 (1H, s). MS (ESI+)
209 [M+H]+. HRMS (ESI+) calcd for C11H13O4 [M+H]+ 209.0808,
found 209.0809.
5.1.12. 3-[(4-Chlorophenoxy)methyl]-4-methoxybenzamide
(10b)
Compound 10b was prepared in a manner similar to that de-
scribed for 10a in 96% yield as a colorless powder. 1H NMR
(400 MHz, DMSO-d6) d: 3.88 (3H, s), 5.05 (2H, s), 7.04 (2H, d,
J = 9.1 Hz), 7.11 (1H, d, J = 8.8 Hz), 7.22 (1H, br s), 7.34 (2H, d,
J = 9.1 Hz), 7.88 (1H, br s), 7.90 (1H, dd, J = 8.8, 2.2 Hz), 7.94 (1H,
d, J = 2.2 Hz). MS (ESI+) 292, 294 (Cl isotope) [M+H]+. HRMS (ESI+)
calcd for C15H15ClNO3 [M+H]+ 292.0735, found 292.0732.
5.1.13. Methyl 3-[(4-chloro-3-methylphenoxy)methyl]-4-
methoxybenzoate (11)
To a solution of 9a (20 mg, 0.065 mmol) in MeOH (1 mL) and
toluene (3.5 mL) was added 10% TMSCHN2 in hexane solution
(0.34 mL, 0.21 mmol) and the mixture was stirred for 2.5 h at room
temperature. The reaction mixture was concentrated in vacuo and
the residue was purified by flash chromatography (SiO2, n-hexane/
EtOAc = 20:1) to give 11 (20 mg, 95%) as a colorless powder. 1H
NMR (400 MHz, CDCl3) d: 2.35 (3H, s), 3.89 (3H, s), 3.93 (3H, s),
5.05 (2H, s), 6.78 (1H, dd, J = 8.8, 2.8 Hz), 6.90 (1H, d, J = 2.8 Hz),
6.94 (1H, d, J = 8.8 Hz), 7.23 (1H, d, J = 8.8 H z), 8.03 (1H, dd,
J = 8.8, 2.0 Hz), 8.14 (1H, d, J = 2.0 Hz). MS (ESI+) 321, 323 (Cl iso-
tope) [M+H]+. HRMS (ESI+) calcd for C17H18ClO4 [M+H]+ 321.0888,
found 321.0887.
5.1.17. 5-Ethoxycarbonyl-2-methoxybenzoic acid (16)
To a solution of 15 (500 mg, 2.40 mmol), NaH2PO4 (288 mg,
2.40 mmol), 2-methyl-2-butene (1.12 mL, 10.6 mmol) in tert-BuOH
(15 mL) and water (4 mL) was added NaClO2 (739 mg, 8.17 mmol)
and the mixture was stirred for 50 min at room temperature. The
reaction mixture was adjusted to pH of 4 by addition of 1 M HCl.
The aqueous layer was extracted with CH2Cl2. The organic layers
were combined, washed with brine, dried over anhydrous Na2SO4,
and concentrated in vacuo to give 16 as a colorless powder
(536 mg, quant.). 1H NMR (400 MHz, DMSO-d6) d: 1.32 (3H, t,
J = 7.1 Hz), 3.91 (3H, s), 4.30 (2H, q, J = 7.1 Hz), 7.26 (1H, d,
J = 8.8 Hz), 8.09 (1H, dd, J = 8.8, 2.2 Hz), 8.23 (1H, d, J = 2.2 Hz),
12.96 (1H, br s). MS (ESI+) 225 [M+H]+. HRMS (ESIꢁ) calcd for
5.1.14. 3-[(4-Chloro-3-methylphenoxy)methyl]-4-methoxy-
benzonitrile (12)
C
11H11O5 [MꢁH]ꢁ 223.0601, found 223.0604.
To a solution of 10a (20 mg, 0.066 mmol) in CH3CN (1 mL) at
room temperature were successively added formic acid (0.25 mL)
and paraformaldehyde (10 mg, 0.34 mmol). The reaction mixture
was then refluxed for 12 h, and the resulting solution was cooled
to room temperature. The crude mixture was concentrated in va-
cuo and the residue was purified by flash chromatography (SiO2,
n-hexane/EtOAc = 15:1 to 1:1) to give 12 (15 mg, 77%) as a color-
less powder. 1H NMR (400 MHz, CDCl3) d: 2.36 (3H, s), 3.94 (3H,
s), 5.04 (2H, s), 6.75 (1H, dd, J = 8.8, 3.0 Hz), 6.87 (1H, d,
J = 3.0 Hz), 6.96 (1H, d, J = 8.6 Hz), 7.24 (1H, d, J = 8.8 Hz), 7.62
(1H, dd, J = 8.6, 2.1 Hz), 7.77 (1H, d, J = 2.1 Hz). MS (ESIꢁ) 286,
288 (Cl isotope) [MꢁH]ꢁ. HRMS (ESI+) calcd for C16H15ClNO2
[M+H]+ 288.0786, found 288.0786.
5.1.18. Ethyl 3-[(4-chlorophenyl)carbamoyl]-4-methoxybenzoate
(17)
To a mixture of 16 (101 mg, 0.50 mmol) in CH2Cl2 (5 mL) and
DMF (2
lL) at room temperature was added oxalyl chloride
(59 L, 0.68 mmol) and the mixture was stirred for 2.5 h at room
l
temperature. The reaction mixture was concentrated in vacuo to
give ethyl 3-chlorocarbonyl-4-methoxybenzoate (109 mg, quant.)
as a colorless powder. 1H NMR (400 MHz, CDCl3) d: 1.41 (3H, t,
J = 7.1 Hz), 4.00 (3H, s), 4.40 (2H, q, J = 7.1 Hz), 7.05 (1H, d,
J = 8.8 Hz), 8.26 (1H, dd, J = 8.8, 2.2 Hz), 8.75 (1H, d, J = 2.2 Hz). To
a
solution of 4-chloroaniline (63 mg, 0.50 mmol) and DIPEA
(235 L, 1.35 mmol) in CH2Cl2 (2 mL) at room temperature was
l
added a solution of ethyl 3-chlorocarbonyl-4-methoxybenzoate
(109 mg, 0.50 mmol) in CH2Cl2 (3 mL) and the mixture was stirred
for 1 h at room temperature. The reaction mixture was washed
with saturated aqueous solution of NH4Cl and brine, dried over
anhydrous Na2SO4, filtered, and concentrated in vacuo. The residue
was purified by flash chromatography (SiO2, n-hexane/CH2Cl2 = 1:1
to CH2Cl2) to give 17 (114 mg, 76%) as a colorless powder. 1H NMR
(400 MHz, CDCl3) d: 1.40 (3H, t, J = 7.1 Hz), 4.13 (3H, s), 4.38 (2H, q,
J = 7.1 Hz), 7.10 (1H, d, J = 8.8 Hz), 7.33 (2H, dd, J = 8.8 Hz), 7.64
(2H, d, J = 8.8 Hz), 8.21 (1H, dd, J = 8.8, 2.5 Hz), 8.94 (1H, d,
J = 2.5 Hz), 9.63 (1H, br s). MS (ESI+) 334, 336 (Cl isotope) [M+H]+.
5.1.15. Ethyl 3-formyl-4-hydroxybenzoate (14)
To a solution of 13 (30.0 g, 197 mmol) and Et3N (165 mL,
1.18 mol) in 1,2-dichloroethane was added MgCl2 (93.9 g,
986 mmol) and the mixture was stirred for 1 h at 40 °C. Then para-
formaldehyde (59.2 g, 1.97 mol) was added and the reaction mix-
ture was stirred for 3 h at 70 °C. After cooling to 0 °C, 1 M HCl
(1000 mL) was added. The mixture was filtered, and washed with
CH2Cl2 (500 mL). The organic layer was separated, washed with
1 M HCl (500 mL) and brine (500 mL), dried over anhydrous

K. Hada et al. / Bioorg. Med. Chem. 20 (2012) 1442–1460
1453
HRMS (ESI+) calcd for C17H17ClNO4 [M+H]+ 334.0841, found
334.0836.
(1H, dd, J = 8.8, 2.2 Hz), 7.88 (0.5H, dd, J = 8.8, 2.2 Hz), 8.21 (0.5H, d,
J = 2.2 Hz), 12.7 (1.5H, br s). MS (ESI+) 289, 291 (Cl isotope) [M+H]+.
HRMS(ESIꢁ) calcd for C16H12ClO3 [M+H]+ 287.0469, found287.0469.
5.1.19. 3-[(4-Chlorophenyl)carbamoyl]-4-methoxybenzoic acid
(18)
5.1.23. 3-[(E)-2-(4-Chlorophenyl)vinyl]-4-methoxybenzamide
(22) and 3-[(Z)-2-(4-Chlorophenyl)vinyl]-4-methoxybenzamide
(23)
To a solution of (E/Z)-21 (96 mg, 0.33 mmol), NH4Cl (27 mg,
0.51 mmol), and HOBT (51 mg, 0.33 mmol) in DMF (1.3 mL) were
To a suspension of 17 (50 mg, 0.15 mmol) in MeOH (1 mL) was
added 20% aqueous solution of KOH (0.4 mL) and the mixture was
stirred for 30 min under reflux. After cooling to room temperature,
water (2 mL) and 1 M HCl (1.5 mL) was added. The resulting pre-
cipitate was filtered, washed with water to give 18 (43 mg, 94%)
as a colorless powder. 1H NMR (400 MHz, DMSO-d6) d: 3.95 (3H,
s), 7.28 (1H, d, J = 8.8 Hz), 7.41 (2H, d, J = 8.8 Hz), 7.76 (2H, d,
J = 8.8 Hz), 8.07 (1H, dd, J = 8.8, 2.2 Hz), 8.12 (1H, d, J = 2.2 Hz),
10.32 (1H, s), 12.91 (1H, s). MS (ESI+) 306, 308 (Cl isotope)
[M+H]+. HRMS (ESI+) calcd for C15H13ClNO4 [M+H]+ 306.0528,
found 306.0528.
added EDC (96 mg, 0.50 mmol) and DIPEA (173 lL, 1.00 mmol).
The mixture was stirred at room temperature for 16 h. The reaction
mixture was diluted with EtOAc. The organic layer was washed
with saturated aqueous solution of NaHCO3 and brine, dried over
anhydrous Na2SO4 and concentrated in vacuo. The residue was
purified by flash chromatography (SiO2, n-hexane/EtOAc = 7:1 to
EtOAc) to give 22 (31 mg, 33%) as a colorless powder and 23
(59 mg, 62%) as a colorless powder. Compound 22: 1H NMR
(400 MHz, DMSO-d6) d: 3.91 (3H, s), 7.10 (1H, d, J = 8.8 Hz), 7.26
(1H, br s), 7.29 (1H, d, J = 16.5 Hz), 7.42 (1H, d, J = 16.5 Hz), 7.43
(2H, d, J = 8.5 Hz), 7.60 (2H, d, J = 8.5 Hz), 7.82 (1H, dd, J = 8.8,
2.2 Hz), 7.91 (1H, br s), 8.20 (1H, d, J = 2.2 Hz). 13C NMR
(100 MHz, DMSO-d6) d: 55.8, 110.8, 123.1, 124.5, 125.7, 126.4,
127.9, 128.0, 128.6, 128.7, 131.8, 136.0, 158.5, 167.2. MS (ESI+)
288, 290 (Cl isotope) [M+H]+. HRMS calcd for C16H15ClNO2
[M+H]+ 288.0786, found 288.0787. Compound 23: 1H NMR
(400 MHz, CDCl3) d: 3.86 (3H, s), 5.48 (2H, br s), 6.62 (1H, d,
J = 12.1 Hz), 6.66 (1H, d, J = 12.1 Hz), 6.94 (1H, d, J = 8.8 Hz), 7.13
(2H, d, J = 8.8 Hz), 7.17 (2H, d, J = 8.8 Hz), 7.51 (1H, d, J = 2.2 Hz),
7.80 (1H, dd, J = 8.8, 2.2 Hz). MS (ESI+) 288, 290 (Cl isotope)
[M+H]+. HRMS (ESI+) calcd for C16H15ClNO2 [M+H]+ 288.0786,
found 288.0780.
5.1.20. N3-(4-Chlorophenyl)-4-methoxybenzene-1, 3-
dicarboxamide (19)
To
0.23 mmol), and HOBT (28 mg, 0.18 mmol) in DMF (2 mL) were
added EDC (35 mg, 0.18 mmol) and DIPEA (79 L, 10.5 mmol)
a solution of 18 (46 mg, 0.15 mmol), NH4Cl (12 mg,
l
and the mixture was stirred at room temperature for 17 h. The sol-
vent was removed and the resulting residue was purified by flash
chromatography (SiO2, CH2Cl2/MeOH = 50:1 to 20:1) to give 19
(43 mg, 93%) as a colorless powder. 1H NMR (400 MHz, DMSO-d6)
d: 3.92 (3H, s), 7.23 (1H, d, J = 8.8 Hz), 7.29 (1H, br s), 7.40 (2H, d,
J=8.8 Hz), 7.76 (2H, d, J = 8.8 Hz), 7.96 (1H, br s), 8.02 (1H, dd,
J = 8.8, 2.2 Hz), 8.10 (1H, d, J = 2.2 Hz), 10.32 (1H, s). MS (ESI+)
305, 307 (Cl isotope) (M+H). HRMS (ESI+) calcd for C15H14ClN2O2
[M+H]+ 305.0687, found 305.0686.
5.1.21. Ethyl 3-[2-(4-chlorophenyl)vinyl]-4-methoxybenzoate
((E/Z)-20)
5.1.24. Ethyl 3-(diethoxyphosphorylmethyl)-4-methoxyben-
zoate (24)
To a solution of 15 (101 mg, 0.48 mmol) and (4-chlorobenzyl)tri-
phenylphosphonium chloride (215 mg, 0.51 mmol) in EtOH (5 mL)
was added 20% ethanol solution of NaOEt (283 lL, 0.72 mmol). The
A mixture of 7 (25.0 g, 109 mmol) and triethyl phosphite
(26.2 mL, 153 mmol) was stirred at 160 °C for 2.5 h. The reaction
mixture was cooled to room temperature, and excess triethyl phos-
phite was removed by distillation to give 24 (34.2 g, 95%) as a col-
orless oil. 1H NMR (400 MHz, CDCl3) d: 1.26 (6H, t, J = 7.1 Hz), 1.37
(3H, t, J = 7.1 Hz), 3.26 (2H, d, J = 21.4 Hz), 3.90 (3H, s), 4.04 (4H,
quintet, J = 7.1 Hz), 4.33 (2H, q, J = 7.1 Hz), 6.88 (1H, d, J = 8.5 Hz),
7.94 (1H, ddd, J = 8.5, 2.2, 2.2 Hz), 7.98 (1H, dd, J = 2.2 Hz). MS
(ESI+) 331 [M+H]+. HRMS (ESI+) calcd for C15H24O6P [M+H]+
331.1305, found 331.1297.
mixture was stirred for 1 h at room temperature. 1 M HCl (0.8 mL)
was added, and the mixture was extracted with EtOAc. The organic
layer was separated, washed with brine, dried over anhydrous
Na2SO4, filtered, and concentratedin vacuo. The residue was purified
by flash chromatography (SiO2, n-hexane/EtOAc = 50:1 to 5:1) to
give a colorless oil (E/Z)-20 (141 mg, 92%) as a mixture of E/Z isomers
in 1:2 ratio. 1H NMR (400 MHz, CDCl3) d: 1.29 (3H, t, J = 7.1 Hz), 1.41
(1.5H, t, J = 7.1 Hz), 3.84 (3H, s), 3.95 (1.5H, s), 4.25 (2H, q, J = 7.1 Hz),
4.38 (1H, q, J = 7.1 Hz), 6.62 (1H, d, J = 12.4 Hz), 6.66 (1H, d,
J = 12.4 Hz), 6.89–6.93 (1.5H, m), 7.11–7.17 (4.5H, m), 7.32 (1H, d,
J = 8.5 Hz), 7.41 (0.5H, d, J = 16.5 Hz), 7.47 (1H, d, J = 8.5 Hz), 7.83
(1H, d, J = 2.2 Hz), 7.94 (1H, dd, J = 8.5, 2.2 Hz), 7.96 (0.5H, d, J = 8.8,
2.2 Hz), 8.26 (0.5H, d, J = 2.2 Hz). MS (ESI+) 317, 319 (Cl isotope)
[M+H]+. HRMS (ESI+) calcd for C18H18ClO3 [M+H]+ 317.939, found
317.936.
5.1.25. 3-(Diethoxyphosphorylmethyl)-4-methoxybenzoic acid
(25)
To a solution of 24 (2.01 g, 6.08 mmol) in MeOH (40 mL) was
added 5 M NaOH aqueous solution (7.2 mL, 36.0 mmol), and the
mixture was stirred at 60 °C for 1.5 h. After cooling to room tem-
perature, the solvent was removed in vacauo. The residue was di-
luted with water and washed with diethyl ether. The aqueous
solution was separated, acidified with 1 M HCl solution and ex-
tracted with EtOAc. The extract was washed with brine, dried over
anhydrous Na2SO4, filtered and concentrated in vacuo to give 25
(1.76 g, 96%) as a colorless powder. 1H NMR (400 MHz, DMSO-d6)
d: 1.16 (6H, t, J = 7.1 Hz), 3.22 (2H, d, J = 21.4 Hz), 3.86 (3H, s),
3.93 (4H, quintet, J = 7.1 Hz), 7.07 (1H, d, J = 8.8 Hz), 7.83 (1H,
ddd, J = 8.8, 2.2, 2.2 Hz), 7.85 (1H, dd, J = 2.2, 2.2 Hz), 12.61 (1H,
s). MS (ESI+) 303 [M+H]+. HRMS (ESI+) calcd for C13H20O6P
[M+H]+ 303.0992, found 303.0985.
5.1.22. 3-[2-(4-chlorophenyl)vinyl]-4-methoxybenzoic acid ((E/
Z)-21)
To a solution of (E/Z)-20 (131 mg, 0.41 mmol) in MeOH (1.3 mL)
was added 20% aqueous solution of KOH (0.26 mL) and the mixture
was stirred for 2 h under reflux. After cooling to room temperature,
1 M HCl (1.5 mL) was added. The resulting precipitate was filtered,
washed with water to give a colorless powder (E/Z)-21 (113 mg,
95%) as a mixture of E/Z isomers in 1:2 ratio. 1H NMR (400 MHz,
DMSO-d6) d: 3.84 (3H, s), 3.94 (1.5H, s), 6.63 (1H, d, J = 12.6 Hz),
6.66 (1H, d, J = 12.6 Hz), 7.13 (1H, d, J = 8.8 Hz), 7.14 (0.5H, d,
J = 8.8 Hz), 7.17 (2H, d, J = 8.8 Hz), 7.29 (2H, d, J = 8.5 Hz), 7.30
(0.5H, d, J = 16.5 Hz), 7.43 (1H, d, J = 8.5 Hz), 7.43 (0.5H, d,
J = 16.5 Hz), 7.64 (1H, d, J = 2.2 Hz), 7.64 (1H, d, J = 8.5 Hz), 7.85
5.1.26. 3-(Diethoxyphosphorylmethyl)-4-methoxybenzamide
(26)
To
a solution of 25 (1.65 g, 5.45 mmol), NH4Cl (439 mg,
8.21 mmol), and HOBT (838 mg, 5.45 mmol) in DMF (27 mL) were

1454
K. Hada et al. / Bioorg. Med. Chem. 20 (2012) 1442–1460
added EDC (1.57 g, 8.18 mmol) and DIPEA (2.85 mL, 16.4 mmol).
The mixture was stirred at room temperature for 20 h and concen-
trated in vacuo. The residue was diluted with an aqueous solution
of NH4Cl and extracted with EtOAc. The extract was dried over
anhydrous Na2SO4 and concentrated in vacuo. The residue was
purified by flash chromatography (basic-SiO2, CH2Cl2/MeOH = 50:1
to 5:1) to give 26 (1.02 g, 62%) as a colorless powder. 1H NMR
(400 MHz, DMSO-d6) d: 1.15 (6H, t, J = 7.1 Hz), 3.17 (2H, d,
J = 22.0 Hz), 3.84 (3H, s), 3.92 (4H, quintet, J = 7.1 Hz), 7.02 (1H, d,
J = 8.8 Hz), 7.15 (1H, br s), 7.76–7.82 (3H, m). MS (ESI+) 302
[M+H]+. HRMS (ESI+) calcd for C13H21NO5P [M+H]+ 302.1152, found
302.1146.
7.39 (1H, d, J = 16.4 Hz), 7.56 (1H, d, J = 16.4 Hz), 7.72 (2H, d,
J = 8.2 Hz), 7.79 (2H, d, J = 8.2 Hz), 7.85 (1H, dd, J = 8.8, 2.2 Hz),
7.93 (1H, br s), 8.24 (1H, d, J = 2.2 Hz). MS (ESI+) 322 [M+H]+. HRMS
(ESI+) calcd for C17H15F3NO2 [M+H]+ 322.1049, found 322.1049.
5.1.32. 4-Methoxy-3-[(E)-2-(4-tolyl)vinyl]benzamide (27e)
The compound 27e was obtained as a colorless powder accord-
ing to the general procedure in 89% yield. 1H NMR (400 MHz,
DMSO-d6) d: 2.31 (3H, s), 3.91 (3H, s), 7.08 (1H, d, J = 8.8 Hz),
7.19 (2H, d, J = 8.2 Hz), 7.25 (1H, d, J = 16.8 Hz), 7.25 (1H, br s),
7.36 (1H, d, J = 16.8 Hz), 7.47 (2H, d, J = 8.2 Hz), 7.80 (1H, dd,
J = 8.8, 2.2 Hz), 7.92 (1H, br s), 8.19 (1H, d, J = 2.2 Hz). MS (ESI+)
268 [M+H]+. HRMS (ESI+) calcd for C17H18NO2 [M+H]+ 268.1332,
found 268.1332.
5.1.27. General procedure for the synthesis of compounds 27a–
u
To a solution of benzaldehydes (1.2 equiv) and 26 (1.0 equiv) in
DMF was added sodium tert-pentoxide (2.0 equiv). The mixture
was stirred at room temperature for 1 h. Two workup procedures
were used. Procedure A: Saturated aqueous solution of NH4Cl
was added to the reaction mixture, and the resulting precipitate
was filtered, washed with water, and dried in vacuo to give the de-
sired products 27a–c, 27e–f, 27i–l, 27n–p, 27r. Procedure B: The
reaction mixture was diluted with EtOAc and washed with water
and brine. The organic layer was dried over Na2SO4, filtered and
concentrated in vacuo. The crude reaction mixture was purified
by flash chromatography (SiO2, n-hexane/EtOAc) to give the de-
sired products 27d, 27g–h, 27m, 27q, 27s–u.
5.1.33. 3-[(E)-2-(4-Fluorophenyl)vinyl]-4-methoxybenzamide
(27f)
The compound 27f was obtained as a colorless powder accord-
ing to the general procedure in 57% yield. 1H NMR (400 MHz,
DMSO-d6) d: 3.91 (3H, s), 7.09 (1H, d, J = 8.2 Hz), 7.20 (2H, dd,
J = 8.8, 8.8 Hz), 7.25 (1H, br s), 7.28 (1H, d, J = 17.0 Hz), 7.35 (1H,
d, J = 17.0 Hz), 7.62 (2H, dd, J = 8.8, 5.5 Hz), 7.81 (1H, dd, J = 8.2,
2.2 Hz), 7.91 (1H, br s), 8.18 (1H, d, J = 2.2 Hz). MS (ESI+) 272
[M+H]+. HRMS (ESI+) calcd for C16H15FNO2 [M+H]+ 272.1081, found
272.1083.
5.1.34. 3-[(E)-2-(4-Cyanophenyl)vinyl]-4-methoxybenzamide
(27g)
5.1.28. 4-Methoxy-3-[(E)-styryl]benzamide (27a)
The compound 27g was obtained as a colorless powder accord-
ing to the general procedure in 51% yield. 1H NMR (400 MHz,
DMSO-d6) d: 3.92 (3H, s), 7.12 (1H, d, J = 8.8 Hz), 7.25 (1H, br s),
7.37 (1H, d, J = 16.5 Hz), 7.58 (1H, d, J = 16.5 Hz), 7.76 (2H, d,
J = 8.2 Hz), 7.81 (2H, d, J = 8.2 Hz), 7.86 (1H, dd, J = 8.8, 2.2 Hz),
7.90 (1H, br s), 8.23 (1H, d, J = 2.2 Hz). MS (ESI+) 279 [M+H]+. HRMS
(ESI+) calcd for C17H15N2O2 [M+H]+ 279.1128, found 279.1130.
The compound 27a was obtained as a colorless powder accord-
ing to the general procedure in 94% yield. 1H NMR (400 MHz,
DMSO-d6) d: 3.91 (3H, s), 7.09 (1H, d, J = 8.5 Hz), 7.25 (1H, br s),
7.27–7.31 (2H, m), 7.36–7.43 (3H, m), 7.57 (2H, d, J = 7.7 Hz),
7.82 (1H, dd, J = 8.5, 2.2 Hz), 7.93 (1H, br s), 8.21 (1H, d,
J = 2.2 Hz). 13C NMR (125 MHz, DMSO-d6) d: 55.7, 110.8, 122.7,
124.8, 125.5, 126.2, 126.3, 127.6, 128.5, 128.6, 129.3, 137.1,
158.4, 167.2. MS (ESI+) 254 [M+H]+. HRMS calcd for C16H16NO2
[M+H]+ 254.1176, found 254.1178.
5.1.35. 4-Methoxy-3-[(E)-2-(4-nitrophenyl)vinyl]benzamide
(27h)
The compound 27h was obtained as a yellow powder according
to the general procedure in 35% yield. 1H NMR (400 MHz, DMSO-
d6) d: 3.94 (3H, s), 7.13 (1H, d, J = 8.8 Hz), 7.27 (1H, br s), 7.46
(1H, d, J = 16.5 Hz), 7.65 (1H, d, J = 16.5 Hz), 7.85 (2H, d,
J = 8.5 Hz), 7.87 (1H, dd, J = 8.8, 2.2 Hz), 7.91 (1H, br s), 8.23 (2H,
d, J = 8.5 Hz), 8.26 (1H, d, J = 2.2 Hz). MS (ESI+) 299 [M+H]+. HRMS
(ESI+) calcd for C16H15N2O4 [M+H]+ 299.1026, found 299.1027.
5.1.29. 3-[(E)-2-(4-tert-Butylphenyl)vinyl]-4-
methoxybenzamide (27b)
The compound 27b was obtained as a colorless powder accord-
ing to the general procedure in 99% yield. 1H NMR (400 MHz,
DMSO-d6) d: 1.29 (9H, s), 3.91 (3H, s), 7.08 (1H, d, J = 8.8 Hz),
7.24 (1H, br s), 7.27 (1H, d, J = 16.8 Hz), 7.36 (1H, d, J = 16.8 Hz),
7.40 (2H, d, J = 8.2 Hz), 7.49 (2H, d, J = 8.2 Hz), 7.80 (1H, dd,
J = 8.8, 2.2 Hz), 7.93 (1H, br s), 8.19 (1H, d, J = 2.2 Hz). MS (ESI+)
310 [M+H]+. HRMS (ESI+) calcd for C20H24NO2 [M+H]+ 310.1802,
found 310.1799.
5.1.36. 4-Methoxy-3-[(E)-2-(4-methoxyphenyl)vinyl]benzamide
(27i)
The compound 27i was obtained as a colorless powder accord-
ing to the general procedure in quantitative yield. 1H NMR
(400 MHz, DMSO-d6) d: 3.77 (3H, s), 3.90 (3H, s), 6.94 (2H, d,
J = 8.5 Hz), 7.07 (1H, d, J = 8.2 Hz), 7.21 (1H, br s), 7.21 (1H, d,
J = 17.0 Hz), 7.26 (1H, d, J = 17.0 Hz), 7.51 (2H, d, J = 8.5 Hz), 7.78
(1H, dd, J=8.2, 1.9 Hz), 7.89 (1H, br s), 8.16 (1H, d, J = 1.9 Hz). MS
(ESI+) 284 [M+H]+. HRMS (ESI+) calcd for C17H18NO3 [M+H]+
284.1281, found 284.1279.
5.1.30. 3-[(E)-2-(4-Bromophenyl)vinyl]-4-methoxybenzamide
(27c)
The compound 27c was obtained as a colorless powder accord-
ing to the general procedure in 92% yield. 1H NMR (400 MHz,
DMSO-d6) d: 3.95 (3H, s), 7.09 (1H, d, J = 8.8 Hz), 7.26 (1H, br s),
7.27 (1H, d, J = 16.5 Hz), 7.43 (1H, d, J = 16.5 Hz), 7.53 (2H, d,
J = 8.8 Hz), 7.56 (2H, d, J = 8.8 Hz), 7.82 (1H, dd, J = 8.8, 2.2 Hz),
7.91 (1H, br s), 8.20 (1H, d, J = 2.2 Hz). MS (ESI+) 332, 334 (Br iso-
tope) [M+H]+. HRMS (ESI+) calcd for C16H15BrNO2 [M+H]+
332.0281, found 332.0279.
5.1.37. 4-Methoxy-3-[(E)-2-(3-tolyl)vinyl]benzamide (27j)
The compound 27j was obtained as a colorless powder accord-
ing to the general procedure in 55% yield. 1H NMR (400 MHz,
DMSO-d6) d: 2.34 (3H, s), 3.91 (3H, s), 7.09 (1H, d, J = 8.2 Hz),
7.09 (1H, d, J = 7.7 Hz), 7.22 (1H, br s), 7.25 (1H, d, J = 16.5 Hz),
7.26 (1H, t, J = 7.7 Hz), 7.35 (1H, d, J = 7.7 Hz), 7.39 (1H, d,
J = 16.5 Hz), 7.39 (1H, s), 7.81 (1H, dd, J = 8.2, 2.2 Hz), 7.90 (1H, br
s), 8.19 (1H, d, J = 2.2 Hz). MS (ESI+) 268 [M+H]+. HRMS (ESI+) calcd
for C17H18NO2 [M+H]+ 268.1332, found 268.1333.
5.1.31. 4-Methoxy-3-[(E)-2-[4-
(trifluoromethyl)phenyl]vinyl]benzamide (27d)
The compound 27d was obtained as a colorless powder accord-
ing to the general procedure in 63% yield. 1H NMR (400 MHz,
DMSO-d6) d: 3.93 (3H, s), 7.12 (1H, d, J = 8.8 Hz), 7.28 (1H, br s),

K. Hada et al. / Bioorg. Med. Chem. 20 (2012) 1442–1460
1455
5.1.38. 3-[(E)-2-(3-Fluorophenyl)vinyl]-4-methoxybenzamide
(27k)
5.1.44. 4-Methoxy-3-[(E)-2-[2-
(trifluoromethoxy)phenyl]vinyl]benzamide (27q)
The compound 27k was obtained as a colorless powder accord-
The compound 27q was obtained as a colorless powder accord-
ing to the general procedure in 63% yield. 1H NMR (400 MHz,
DMSO-d6) d: 3.92 (3H, s), 7.13 (1H, d, J = 8.8 Hz), 7.27 (1H, br s),
7.40–7.46 (4H, m), 7.50 (1H, d, J = 16.8 Hz), 7.87 (1H, dd, J = 8.8,
2.2 Hz), 7.88–7.91 (1H, m), 7.98 (1H, br s), 8.16 (1H, d, J = 2.2 Hz).
MS (ESI+) 338 [M+H]+. HRMS (ESI+) calcd for C17H15F3NO3 [M+H]+
338.0999, found 338.0998.
ing to the general procedure in 79% yield. 1H NMR (400 MHz,
DMSO-d6) d: 3.92 (3H, s), 7.07–7.12 (2H, m), 7.25 (1H, br s), 7.30
(1H, d, J = 16.5 Hz), 7.38–7.43 (3H, m), 7.46 (1H, d, J = 16.5 Hz),
7.83 (1H, dd, J = 8.8, 2.2 Hz), 7.91 (1H, br s), 8.20 (1H, d,
J = 2.2 Hz). MS (ESI+) 272 [M+H]+. HRMS (ESI+) calcd for
C
16H15FNO2 [M+H]+ 272.1081, found 272.1082.
5.1.39. 3-[(E)-2-(3-Chlorophenyl)vinyl]-4-methoxybenzamide
(27l)
5.1.45. 3-[(E)-2-(2-Chlorophenyl)vinyl]-4-methoxybenzamide
(27r)
The compound 27l was obtained as
a
colorless powder
The compound 27r was obtained as a colorless powder accord-
ing to the general procedure in 63% yield. 1H NMR (400 MHz,
DMSO-d6) d: 3.92 (3H, s), 7.12 (1H, d, J = 8.8 Hz), 7.27 (1H, br s),
7.30 (1H, ddd, J = 7.7, 7.7, 1.6 Hz), 7.37 (1H, dd, J = 7.7, 7.7 Hz),
7.41 (1H, d, J = 16.5 Hz), 7.49 (1H, d, J = 7.7 Hz), 7.55 (1H, d,
J = 16.5 Hz), 7.81 (1H, dd, J = 7.7, 1.6 Hz), 7.86 (1H, dd, J = 8.8,
2.2 Hz), 7.99 (1H, br s), 8.18 (1H, d, J = 2.2 Hz). MS (ESI+) 288, 290
(Cl isotope) [M+H]+. HRMS (ESI+) calcd for C16H15ClNO2 [M+H]+
288.0786, found 288.0788.
according to the general procedure in 57% yield. 1H NMR
(400 MHz, DMSO-d6) d: 3.92 (3H, s), 7.10 (1H, d, J = 8.8 Hz),
7.24 (1H, br s), 7.28 (1H, d, J = 17.0 Hz), 7.31 (1H, d, J = 8.5 Hz),
7.40 (1H, dd, J = 8.5, 7.7 Hz), 7.46 (1H, d, J = 17.0 Hz), 7.54 (1H,
d, J = 7.7 Hz), 7.63 (1H, s), 7.83 (1H, dd, J = 8.8, 2,2 Hz), 7.89
(1H, br s), 8.19 (1H, d, J = 2.2 Hz). MS (ESI+) 288, 290 (Cl isotope)
[M+H]+. HRMS (ESI+) calcd for C16H15ClNO2 [M+H]+ 288.0786,
found 288.0788.
5.1.40. 3-[(E)-2-(3-Cyanophenyl)vinyl]-4-methoxybenzamide
(27m)
5.1.46. 3-[(E)-2-(2,4-Dichlorophenyl)vinyl]-4-methoxybenz-
amide (27s)
The compound 27m was obtained as a colorless powder accord-
ing to the general procedure in 53% yield. 1H NMR (400 MHz,
DMSO-d6) d: 3.92 (3H, s), 7.11 (1H, d, J = 8.8 Hz), 7.26 (1H, br s),
7.33 (1H, d, J = 16.5 Hz), 7.54 (1H, d, J = 16.5 Hz), 7.58 (1H, dd,
J = 7.7, 7.7 Hz), 7.72 (1H, d, J = 7.7 Hz), 7.85 (1H, dd, J = 8.8,
2.2 Hz), 7.91 (1H, br s), 7.92 (1H, d, J = 7.7 Hz), 8.06 (1H, s), 8.21
(1H, d, J = 2.2 Hz). MS (ESI+) 279 [M+H]+. HRMS (ESI+) calcd for
The compound 27s was obtained as a colorless powder accord-
ing to the general procedure in 51% yield. 1H NMR (400 MHz,
DMSO-d6) d: 3.91 (3H, s), 7.12 (1H, d, J = 8.5 Hz), 7.26 (1H, br s),
7.43 (1H, d, J = 16.5 Hz), 7.44 (1H, dd, J = 8.5, 1.6 Hz), 7.50 (1H, d,
J = 16.5 Hz), 7.65 (1H, d, J = 1.6 Hz), 7.84 (1H, d, J = 8.5 Hz), 7.86
(1H, dd, J = 8.5, 2.2 Hz), 7.98 (1H, br s), 8.17 (1H, d, J = 2.2 Hz).
MS (ESI+) 322, 324 (Cl isotope) [M+H]+. HRMS (ESI+) calcd for
C
17H15N2O2 [M+H]+ 279.1128, found 279.1129.
C
16H14Cl2NO2 [M+H]+ 322.0396, found 322.0399.
5.1.41. 3-[(E)-2-(2-Fluorophenyl)vinyl]-4-methoxybenzamide
(27n)
5.1.47. 3-[(E)-2-(3,4-Dichlorophenyl)vinyl]-4-methoxybenz-
amide (27t)
The compound 27n was obtained as a colorless powder accord-
ing to the general procedure in 83% yield. 1H NMR (400 MHz,
DMSO-d6) d: 3.92 (3H, s), 7.12 (1H, d, J = 8.8 Hz), 7.22–7.27 (3H,
m), 7.33 (1H, ddd, J = 7.7, 6.6, 1.6 Hz), 7.38 (1H, d, J = 16.5 Hz),
7.51 (1H, d, J = 16.5 Hz), 7.75 (1H, ddd, J = 7.1, 6.6, 1.6 Hz), 7.85
(1H, dd, J = 8.8, 2.2 Hz), 7.99 (1H, br s), 8.22, (1H, d, J = 2.2 Hz).
MS (ESI+) 272 [M+H]+. HRMS (ESI+) calcd for C16H15FNO2 [M+H]+
272.1081, found 272.1083.
The compound 27t was obtained as a colorless powder accord-
ing to the general procedure in 65% yield. 1H NMR (400 MHz,
DMSO-d6) d: 3.91 (3H, s), 7.10 (1H, d, J = 8.8 Hz), 7.26 (1H, br s),
7.28 (1H, d, J = 16.5 Hz), 7.47 (1H, d, J = 16.5 Hz), 7.58 (1H, dd,
J = 8.2, 2.2 Hz), 7.61 (1H, d, J = 8.2 Hz), 7.83 (1H, dd, J = 8.8,
2.2 Hz), 7.84 (1H, d, J = 1.6 Hz), 7.89 (1H, br s), 8.18 (1H, d,
J = 2.2 Hz). MS (ESI+) 322, 324 (Cl isotope) [M+H]+. HRMS (ESI+)
calcd for C16H14Cl2NO2 [M+H]+ 322.0396, found 322.0395.
5.1.42. 4-Methoxy-3-[(E)-2-(2-tolyl)vinyl]benzamide (27o)
The compound 27o was obtained as a colorless powder accord-
ing to the general procedure in 91% yield. 1H NMR (400 MHz,
DMSO-d6) d: 2.42 (3H, s), 3.91 (3H, s), 7.10 (1H, d, J = 8.8 Hz),
7.16–7.24 (3H, m), 7.27 (1H, d, J = 16.5 Hz), 7.27 (1H, br s), 7.46
(1H, d, J = 16.5 Hz), 7.60 (1H, d, J = 7.1 Hz), 7.83 (1H, dd, J = 8.8,
1.9 Hz), 7.96 (1H, br s), 8.19 (1H, d, J = 1.9 Hz). MS (ESI+) 268
[M+H]+. HRMS (ESI+) calcd for C17H18NO2 [M+H]+ 268.1332, found
268.1331.
5.1.48. 3-[(E)-2-(4-Chloro-2-fluorophenyl)vinyl]-4-methoxy-
benzamide (27u)
The compound 27u was obtained as a colorless powder accord-
ing to the general procedure in 35% yield. 1H NMR (400 MHz,
DMSO-d6) d: 3.91 (3H, s), 7.11 (1H, d, J = 8.8 Hz), 7.26 (1H, br s),
7.30 (1H, dd, J = 8.2, 2.2 Hz), 7.32 (1H, d, J = 16.5 Hz), 7.46 (1H,
dd, J = 11.0, 2.2 Hz), 7.50 (1H, d, J = 16.5 Hz), 7.78 (1H, dd, J = 8.2,
8.2 Hz), 7.85 (1H, dd, J = 8.8, 2.2 Hz), 7.96 (1H, br s), 8.20 (1H, d,
J = 2.2 Hz). MS (ESI+) 306, 308 (Cl isotope) [M+H]+. HRMS (ESI+)
calcd for C16H14ClFNO2 [M+H]+ 306.0692, found 306.0692.
5.1.43. 4-Methoxy-3-[(E)-2-(2-methoxyphenyl)vinyl]benzamide
(27p)
5.1.49. Ethyl 3-[(E)-2-(4-chlorophenyl)vinyl]-4-hydroxy-
benzoate (28)
The compound 27p was obtained as a colorless powder accord-
ing to the general procedure in 97% yield. 1H NMR (400 MHz,
DMSO-d6) d: 3.86 (3H, s), 3.90 (3H, s), 6.97 (1H, dd, J = 7.7,
7.1 Hz), 7.04 (1H, d, J = 8.2 Hz), 7.08 (1H, d, J = 8.8 Hz), 7.24 (1H,
br s), 7.27 (1H, ddd, J = 8.2, 7.1, 1.6 Hz), 7.38 (1H, d, J = 16.8 Hz),
7.48 (1H, d, J = 16.8 Hz), 7.60 (1H, dd, J = 7.7, 1.6 Hz), 7.81 (1H,
dd, J = 8.8, 2.2 Hz), 7.98 (1H, br s), 8.15 (1H, d, J = 2.2 Hz). MS
(ESI+) 284 [M+H]+. HRMS (ESI+) calcd for C17H18NO3 [M+H]+
284.1281, found 284.1280.
To a solution of diethyl 4-chlorobenzylphosphonate (1.19 g,
4.54 mmol) in DMF (10 mL) at 0 °C was added sodium tert-pentox-
ide (1.37 g, 11.8 mmol) and the mixture was stirred for 20 min at
0 °C. Then a solution of 14 (842 mg, 4.33 mmol) in DMF (5 mL)
was added and the reaction mixture was stirred for 2 h at 0 °C.
The reaction mixture was poured into saturated aqueous solution
of NH4Cl (30 mL) and extracted with EtOAc (100 mL). The organic
layer was separated, washed with water and brine, dried over

1456
K. Hada et al. / Bioorg. Med. Chem. 20 (2012) 1442–1460
MgSO4, filtered and concentrated in vacuo. The residue was puri-
(2H, d, J = 8.2 Hz), 7.39 (1H, d, J = 16.5 Hz), 7.44 (2H, d, J = 8.2 Hz),
7.93 (1H, dd, J = 8.2, 1.8 Hz), 8.25 (1H, d, J = 1.8 Hz). MS (ESI+)
374, 376 (Cl isotope) [M+H]+. HRMS (ESI+) calcd for C21H25ClNO3
[M+H]+ 374.1517, found 374.1507.
fied by flash chromatography (SiO2, n-hexane/EtOAc = 3:1 to 2:1)
to give 28 (925 mg, 71%) as
a
colorless powder. 1H NMR
(400 MHz, CDCl3) d: 1.41 (3H, t, J = 7.1 Hz), 4.38 (2H, q,
J = 7.1 Hz), 5.74 (1H, br s), 6.84 (1H, d, J = 8.8 Hz), 7.14 (1H, d,
J = 16.5 Hz), 7.31 (2H, d, J = 8.8 Hz), 7.32 (1H, d, J = 16.5 Hz), 7.45
(2H, d, J = 8.8 Hz), 7.85 (1H, dd, J = 8.8, 2.1 Hz), 8.23 (1H, d,
J = 2.1 Hz). MS (ESI+) 303, 305 (Cl isotope) [M+H]+. HRMS (ESI+)
calcd for C17H16ClO3 [M+H]+ 303.0782, found 303.0778.
5.1.55. Ethyl 3-[(E)-2-(4-chlorophenyl)vinyl]-4-[2-(4-
methylpiperazin-1-yl)ethoxy]benzoate (29e)
Compound 29e was prepared in a manner similar to that de-
scribed for 29d in 66% yield as a colorless oil. 1H NMR (400 MHz,
CDCl3) d: 1.41 (3H, t, J = 7.1 Hz), 2.30 (3H, s), 2.49 (4H, br s), 2.68
(4H, br s), 2.92 (2H, t, J = 5.8 Hz), 4.23 (2H, t, J = 5.8 Hz), 4.38 (2H,
q, J = 7.1 Hz), 6.90 (1H, d, J = 8.5 Hz), 7.17 (1H, d, J = 16.5 Hz), 7.32
(2H, d, J = 8.2 Hz), 7.38 (1H, d, J = 16.5 Hz), 7.44 (2H, d, J = 8.2 Hz),
7.93 (1H, dd, J = 8.5, 1.9 Hz), 8.25 (1H, d, J = 1.9 Hz). MS (ESI+)
429, 431 (Cl isotope) [M+H]+. HRMS (ESI+) calcd for C24H30ClN2O3
[M+H]+ 429.1939, found 429.1930.
5.1.50. General procedure for the synthesis of compounds 29a–c
A mixture of 28 (1 equiv), alkyl halides (1.5 equiv), K2CO3
(2 equiv) in CH3CN was stirred at 60 °C for 24 h. The insoluble solid
was filtered and the cake was washed with CH3CN. The filtrate was
concentrated in vacuo and the residue was purified by flash chroma-
tography (SiO2, n-hexane/EtOAc) to give the desired products 29a–c.
5.1.51. Ethyl 3-[(E)-2-(4-chlorophenyl)vinyl]-4-ethoxybenzoate
(29a)
5.1.56. General procedure for the synthesis of compounds
30a–d
The compound 29a was obtained as a colorless powder according
to the general procedure in 99% yield. 1H NMR (400 MHz, CDCl3) d:
1.41 (3H, t, J = 7.1 Hz), 1.52 (3H, t, J = 6.9 Hz), 4.18 (2H, q,
J = 6.9 Hz), 4.38 (2H, q, J = 7.1 Hz), 6.90 (1H, d, J = 8.7 Hz), 7.17 (1H,
d, J = 16.5 Hz), 7.33 (2H, d, J = 8.6 Hz), 7.42 (1H, d, J = 16.5 Hz), 7.47
(2H, d, J = 8.6 Hz), 7.93 (1H, dd, J = 8.7, 2.1 Hz), 8.26 (1H, d,
J = 2.1 Hz). MS (ESI+) 331, 333 (Cl isotope) [M+H]+. HRMS (ESI+) calcd
for C19H20ClO3 [M+H]+ 331.1095, found 331.1096.
To a solution of 29a–d (0.14 mmol) in MeOH (3 mL) was added
20% aqueous solution of KOH (0.6 mL) and the mixture was stirred
for 1 h under reflux. After removal of MeOH, 1 M HCl (2.4 mL) was
added. The resulting precipitate was filtered, washed with water to
give the desired products 30a–d.
5.1.57. 3-[(E)-2-(4-Chlorophenyl)vinyl]-4-ethoxybenzoic acid
(30a)
The compound 30a was obtained as a colorless powder accord-
ing to the general procedure in 87% yield. 1H NMR (400 MHz,
DMSO-d6) d: 1.44 (3H, t, J = 7.1 Hz), 4.20 (2H, q, J = 7.1 Hz), 7.13
(1H, d, J = 8.8 Hz), 7.33 (1H, d, J = 16.6 Hz), 7.42 (1H, d,
J = 16.6 Hz), 7.43 (2H, d, J = 8.8 Hz), 7.63 (2H, d, J = 8.8 Hz), 7.86
(1H, dd, J = 8.8, 2.0 Hz), 8.20 (1H, d, J = 2.0 Hz), 12.73 (1H, br s).
MS (ESI+) 303, 305 (Cl isotope) [M+H]+. HRMS (ESIꢁ) calcd for
5.1.52. Ethyl 3-[(E)-2-(4-chlorophenyl)vinyl]-4-
propoxybenzoate (29b)
The compound 29b was obtained as a colorless powder accord-
ing to the general procedure in 99% yield. 1H NMR (400 MHz,
CDCl3) d: 1.11 (3H, t, J = 7.4 Hz), 1.41 (3H, t, J = 7.1 Hz), 1.92 (2H,
tq, J = 7.4, 6.6 Hz), 4.06 (2H, t, J = 6.6 Hz), 4.38 (2H, q, J = 7.1 Hz),
6.91 (1H, d, J = 8.7 Hz), 7.18 (1H, d, J = 16.7 Hz), 7.33 (2H, d,
J = 8.4 Hz), 7.42 (1H, d, J = 16.7 Hz), 7.46 (2H, d, J = 8.4 Hz), 7.93
(1H, dd, J = 8.7, 2.1 Hz), 8.26 (1H, d, J = 2.1 Hz). MS (ESI+) 345, 347
(Cl isotope) [M+H]+. HRMS (ESI+) calcd for C20H22ClO3 [M+H]+
345.1252, found 345.1253.
C
17H14ClO3 [MꢁH]ꢁ 301.0626, found 301.0620.
5.1.58. 3-[(E)-2-(4-Chlorophenyl)vinyl]-4-propoxybenzoic acid
(30b)
The compound 30b was obtained as a colorless powder accord-
ing to the general procedure in 97% yield. 1H NMR (400 MHz,
DMSO-d6) d: 1.05 (3H, t, J = 7.3 Hz), 1.84 (2H, tq, J = 7.3, 6.4 Hz),
4.11 (2H, t, J = 6.4 Hz), 7.14 (1H, d, J = 8.8 Hz), 7.34 (1H, d,
J = 16.6 Hz), 7.43 (1H, d, J = 16.6 Hz), 7.44 (2H, d, J = 8.8 Hz), 7.62
(2H, d, J = 8.8 Hz), 7.85 (1H, dd, J = 8.8, 2.0 Hz), 8.20 (1H, d,
J = 2.0 Hz). MS (ESI+) 317, 319 (Cl isotope) [M+H]+. HRMS (ESIꢁ)
calcd for C18H16ClO3 [MꢁH]ꢁ 315.0782, found 315.0775.
5.1.53. Ethyl 3-[(E)-2-(4-chlorophenyl)vinyl]-4-[2-(2-
hydroxyethoxy)ethoxy]benzoate (29c)
The compound 29c was obtained as a colorless powder accord-
ing to the general procedure in 95% yield. 1H NMR (400 MHz,
CD3OD) d: 1.40 (3H, t, J = 7.1 Hz), 3.64–3.75 (4H, m), 3.95 (2H, t,
J = 4.6 Hz), 4.31 (2H, t, J = 4.6 Hz), 4.36 (2H, q, J = 7.1 Hz), 7.10
(1H, d, J = 8.8 Hz), 7.25 (1H, d, J = 16.5 Hz), 7.34 (2H, d, J = 8.5 Hz),
7.48 (1H, d, J = 16.5 Hz), 7.54 (2H, d, J = 8.5 Hz), 7.91 (1H, dd,
J = 8.8, 1.9 Hz), 8.24 (1H, d, J = 1.9 Hz). MS (ESI+) 391, 393 (Cl iso-
tope) [M+H]+. HRMS (ESI+) calcd for C21H24ClO5 [M+H]+ 391.1306,
found 391.1309.
5.1.59. 3-[(E)-2-(4-Chlorophenyl)vinyl]-4-[2-(2-
hydroxyethoxy)ethoxy]benzoic acid (30c)
The compound 30c was obtained as a colorless powder accord-
ing to the general procedure in 87% yield. 1H NMR (400 MHz,
DMSO-d6) d: 3.52–3.58 (4H, m), 3.86 (2H, t, J = 4.7 Hz), 4.27 (2H,
t, J = 4.7 Hz), 4.64 (1H, t, J = 4.7 Hz), 7.16 (1H, d, J = 8.8 Hz), 7.37
(1H, d, J = 16.6 Hz), 7.43 (2H, d, J = 8.8 Hz), 7.44 (1H, d,
J = 16.6 Hz), 7.63 (2H, d, J = 8.8 Hz), 7.85 (1H, dd, J = 8.8, 2.4 Hz),
8.20 (1H, d, J = 2.4 Hz), 12.75 (1H, br s). MS (ESI+) 363, 365 (Cl iso-
tope) [M+H]+. HRMS (ESIꢁ) calcd for C19H18ClO5 [MꢁH]ꢁ 361.0837,
found 361.0832.
5.1.54. Ethyl 3-[(E)-2-(4-chlorophenyl)vinyl]-4-(2-
dimethylaminoethyloxy)benzoate (29d)
A
mixture of 28 (151 mg, 0.50 mmol), 1,2-dibromoethane
(0.43 mL, 4.97 mmol), K2CO3 (344 mg, 2.49 mmol) in DMF (3 mL)
was stirred at 80 °C for 1 h. To the mixture was added dimethyl-
amine solution (2.0 M in THF, 2.5 mL, 5.0 mmol), and the mixture
was stirred at 80 °C for 1 h. The reaction mixture was diluted with
EtOAc (30 mL). The organic layer was washed with water and
brine, dried over Na2SO4, filtered and concentrated in vacuo. The
residue was purified by flash chromatography (SiO2, CH2Cl2/
MeOH = 50:1 to 10:1) to give 29d (109 mg, 58%) as a colorless
oil. 1H NMR (400 MHz, CDCl3) d: 1.41 (3H, t, J = 7.1 Hz), 2.39 (6H,
s), 2.84 (2H, t, J = 5.8 Hz), 4.20 (2H, t, J = 5.8 Hz), 4.38 (2H, q,
J = 7.1 Hz), 6.91 (1H, d, J = 8.2 Hz), 7.17 (1H, d, J = 16.5 Hz), 7.32
5.1.60. 3-[(E)-2-(4-Chlorophenyl)vinyl]-4-(2-
dimethylaminoethoxy)benzoic acid (30d)
The compound 30c was obtained as a colorless powder accord-
ing to the general procedure in 92% yield. 1H NMR (400 MHz,
DMSO-d6) d: 2.28 (6H, s), 2.76 (2H, t, J = 5.8 Hz), 4.22 (2H, t,
J = 5.8 Hz), 7.15 (1H, d, J = 8.5 Hz), 7.37 (1H, d, J = 17.0 Hz), 7.42
(1H, d, J = 17.0 Hz), 7.44 (2H, d, J = 8.5 Hz), 7.60 (2H, d, J = 8.5 Hz),

K. Hada et al. / Bioorg. Med. Chem. 20 (2012) 1442–1460
1457
7.84 (1H, dd, J = 8.5, 2.2 Hz), 8.18 (1H, d, J = 2.2 Hz). MS (ESI+) 346,
348 (Cl isotope) [M+H]+. HRMS (ESI+) calcd for C19H21ClNO3 [M+H]+
346.1204, found 346.1194.
DMSO-d6) d: 2.27 (6H, s), 2.73 (2H, t, J = 5.9 Hz), 4.19 (2H, t,
J = 5.9 Hz), 7.11 (1H, d, J = 8.8 Hz), 7.24 (1H, br s), 7.35 (1H, d,
J = 16.4 Hz), 7.42 (1H, d, J = 16.4 Hz), 7.45 (2H, d, J = 8.8 Hz), 7.57
(2H, d, J = 8.8 Hz), 7.80 (1H, dd, J = 8.8, 2.2 Hz), 7.90 (1H, br s),
8.18 (1H, d, J = 2.2 Hz). MS (ESI+) 345, 347 (Cl isotope) [M+H]+.
HRMS (ESI+) calcd for C19H22ClN2O2 [M+H]+ 345.1364, found
345.1355.
5.1.61. 3-[(E)-2-(4-Chlorophenyl)vinyl]-4-[2-(4-
methylpiperazin-1-yl)ethoxy]benzoic acid (30e)
To a solution of 29e (59 mg, 0.14 mmol) in MeOH (3 mL) was
added 20% aqueous solution of KOH (0.6 mL) and the mixture was
stirred for 2.5 h under reflux. After removal of MeOH, 1 M HCl
(2.4 mL) was added and the aqueouslayer was extractedwith EtOAc.
The organic layers were combined, washed with brine (100 mL),
dried over anhydrous Na2SO4, filtered, and concentrated in vacuo
to give 30e (50 mg, 90%) as a colorless powder. 1H NMR (400 MHz,
DMSO-d6) d: 2.14 (3H, s), 2.33 (4H, br s), 2.54 (4H, br s), 2.80 (2H, t,
J = 5.5 Hz), 4.22 (2H, t, J = 5.5 Hz), 7.13 (1H, d, J = 8.8 Hz), 7.34 (1H,
d, J = 17.0 Hz), 7.42 (1H, d, J = 17.0 Hz), 7.43 (2H, d, J = 8.5 Hz), 7.60
(2H, d, J = 8.5 Hz), 7.83 (1H, dd, J = 8.8, 2.2 Hz), 8.18 (1H, d,
J = 2.2 Hz). MS (ESI+) 401, 403 (Cl isotope) [M+H]+. HRMS (ESI+) calcd
for C22H26ClN2O3 [M+H]+ 401.1626, found 401.1616.
5.1.67. 3-[(E)-2-(4-Chlorophenyl)vinyl]-4-[2-(4-
methylpiperazin-1-yl)ethoxy]benzamide (31e)
The compound 31e was obtained as a colorless powder accord-
ing to the general procedure in 82% yield. 1H NMR (400 MHz,
DMSO-d6) d: 2.13 (3H, s), 2.31 (4H, br s), 2.53 (4H, br s), 2.79
(2H, t, J = 5.7 Hz), 4.21 (2H, t, J = 5.7 Hz), 7.12 (1H, d, J = 8.8 Hz),
7.24 (1H, br s), 7.34 (1H, d, J = 16.6 Hz), 7.42 (1H, d, J = 16.6 Hz),
7.45 (2H, d, J = 8.3 Hz), 7.58 (2H, d, J = 8.3 Hz), 7.80 (1H, dd,
J = 8.8, 2.0 Hz), 7.89 (1H, br s), 8.19 (1H, d, J = 2.0 Hz). MS (ESI+)
400, 402 (Cl isotope) [M+H]+. HRMS (ESI+) calcd for C22H27ClN3O2
[M+H]+ 400.1786, found 400.1778.
5.1.62. General procedure for the synthesis of compounds 31a–e
To a solution of 30a–e (1 equiv), NH4Cl (2 equiv), and HOBT
(1 equiv) in DMF were added EDC (1.5 equiv) and DIPEA (4 equiv)
and the mixture was stirred at room temperature for 8 h. Water
was added, and the resulting precipitate was filtered and washed
with water to give the desired products 31a–e.
5.1.68. 3-[(E)-2-(4-Chlorophenyl)vinyl]-4-methoxybenzoic acid
((E)-21)
To a solution of diethyl 4-chlorobenzylphosphonate (13.3 g,
50.4 mmol) in toluene (150 mL) at 0 °C was added sodium tert-
pentoxide (8.35 g, 72.1 mmol) and the mixture was stirred for
20 min at 0 °C. Then a solution of 15 (10.0 g, 48.0 mmol) in THF
(40 mL) was added dropwise over 20 min and the reaction mixture
was stirred for 1.5 h at 0 °C. The reaction mixture was poured into
saturated aqueous solution of NH4Cl (300 mL) and extracted with
EtOAc (350 mL ꢂ 2). The organic layers were combined, washed
with brine (100 mL), dried over anhydrous Na2SO4, filtered, and
concentrated in vacuo to give crude (E)-20 (16.4 g). 1H NMR
(400 MHz, CDCl3) d: 1.41 (3H, t, J = 7.2 Hz), 3.98 (3H, s), 4.38 (2H,
q, J = 7.2 Hz), 6.92 (1H, d, J = 8.8 Hz), 7.15 (1H, d, J = 16.4 Hz), 7.32
(2H, d, J = 8.6 Hz), 7.41 (1H, d, J = 16.4 Hz), 7.47 (2H, d, J = 8.6 Hz),
7.95 (1H, dd, J = 8.8, 2.4 Hz), 8.26 (1H, d, J = 2.4 Hz). To a solution
of crude (E)-20 (16.0 g) in MeOH (100 mL) was added 20% aqueous
solution of KOH (30 mL). The mixture was stirred for 2 h at 65 °C,
and then cooled to 0 °C. The reaction mixture was adjusted to pH
of 3 by addition of 1 M HCl (170 mL). The resulting precipitate
was filtered, washed with water and n-hexane/EtOAc (10:1). The
residue was recrystallized from EtOH (700 mL) to give (E)-21
(11.2 g, 80%) as colorless needles. 1H NMR (400 MHz, DMSO-d6)
d: 3.94 (3H, s), 7.15 (1H, d, J = 8.8 Hz), 7.30 (1H, d, J = 16.6 Hz),
7.43 (2H, d, J = 8.4 Hz), 7.43 (1H, d, J = 16.6 Hz), 7.64 (2H, d,
J = 8.4 Hz), 7.88 (1H, dd, J = 8.8, 2.1 Hz), 8.20 (1H, d, J = 2.1 Hz),
12.8 (1H, br s). MS (ESI+) 289, 291 (Cl isotope) [M+H]+. HRMS
(ESIꢁ) calcd for C16H12ClO3 [MꢁH]ꢁ 287.0469, found 287.0470.
5.1.63. 3-[(E)-2-(4-Chlorophenyl)vinyl]-4-ethoxybenzamide
(31a)
The compound 31a was obtained as a colorless powder accord-
ing to the general procedure in 96% yield. 1H NMR (400 MHz,
DMSO-d6) d: 1.42 (3H, t, J = 6.9 Hz), 4.17 (2H, q, J = 6.9 Hz), 7.09
(1H, d, J = 8.6 Hz), 7.27 (1H, br s), 7.31 (1H, d, J = 16.7 Hz), 7.44
(1H, d, J = 16.7 Hz), 7.45 (2H, d, J = 8.6 Hz), 7.60 (2H, d, J = 8.6 Hz),
7.81 (1H, dd, J = 8.6, 2.2 Hz), 7.92 (1H, br s), 8.21 (1H, d,
J = 2.2 Hz). MS (ESI+) 302, 304 (Cl isotope) [M+H]+. HRMS (ESI+)
calcd for C17H17ClNO2 [M+H]+ 302.0942, found 302.0938.
5.1.64. 3-[(E)-2-(4-Chlorophenyl)vinyl]-4-propoxybenzamide
(31b)
The compound 31b was obtained as a colorless powder accord-
ing to the general procedure in 85% yield. 1H NMR (400 MHz,
DMSO-d6) d: 1.04 (3H, t, J = 7.4 Hz), 1.83 (2H, tq, J = 7.4, 6.5 Hz),
4.08 (2H, t, J = 6.5 Hz), 7.09 (1H, d, J = 8.6 Hz), 7.26 (1H, br s),
7.32 (1H, d, J = 16.5 Hz), 7.44 (1H, d, J = 16.5 Hz), 7.45 (2H, d,
J = 8.6 Hz), 7.59 (2H, d, J = 8.6 Hz), 7.81 (1H, dd, J = 8.6, 2.2 Hz),
7.92 (1H, br s), 8.20 (1H, d, J = 2.2 Hz). MS (ESI+) 316, 318 (Cl iso-
tope) [M+H]+. HRMS (ESI+) calcd for C18H19ClNO2 [M+H]+
316.1099, found 316.1092.
5.1.69. General procedure for the synthesis of compounds 32a–
5.1.65. 3-[(E)-2-(4-Chlorophenyl)vinyl]-4-[2-(2-
h
hydroxyethoxy)ethoxy]benzamide (31c)
To a mixture of (E)-21 (501 mg, 1.74 mmol) in CH2Cl2 (15 mL)
The compound 31c was obtained as a colorless powder accord-
ing to the general procedure in 83% yield. 1H NMR (400 MHz,
DMSO-d6) d: 3.51–3.58 (4H, m), 3.85 (2H, t, J = 4.7 Hz), 4.24 (2H,
t, J = 4.7 Hz), 4.63 (1H, t, J = 4.7 Hz), 7.11 (1H, d, J = 8.8 Hz), 7.25
(1H, br s), 7.35 (1H, d, J = 16.6 Hz), 7.43 (1H, d, J = 16.6 Hz), 7.44
(2H, d, J = 8.8 Hz), 7.59 (2H, d, J = 8.8 Hz), 7.80 (1H, dd, J = 8.8,
2.2 Hz), 7.90 (1H, br s), 8.19 (1H, d, J = 2.2 Hz). MS (ESI+) 362, 364
(Cl isotope) [M+H]+. HRMS (ESI+) calcd for C19H21ClNO4 [M+H]+
362.1154, found 362.1147.
and DMF (7 lL) at 0 °C was added oxalyl chloride (331 mg,
2.60 mmol) and the mixture was stirred for 13 h at room temper-
ature. The reaction mixture was concentrated in vacuo to give 3-
[(E)-2-(4-chlorophenyl)vinyl]-4-methoxy-benzoyl
chloride
(535 mg, quant.) as a colorless powder. 1H NMR (400 MHz, CDCl3)
d: 3.99 (3H, s), 6.97 (1H, d, J = 8.8 Hz), 7.14 (1H, d, J = 16.5 Hz), 7.33
(2H, d, J = 8.8 Hz), 7.36 (1H, d, J = 16.5 Hz), 7.47 (2H, d, J = 8.8 Hz),
8.05 (1H, dd, J = 8.8, 2.2 Hz), 8.30 (1H, d, J = 2.2 Hz). To a solution
of amines (2.5 equiv) and DIPEA (5 equiv) in CH2Cl2 at room tem-
perature was added a solution of 3-[(E)-2-(4-chlorophenyl)vinyl]-
4-methoxy-benzoyl chloride (1 equiv) in CH2Cl2 and the mixture
was stirred for 1 h at room temperature. The reaction mixture
was washed with saturated aqueous solution of NH4Cl and brine,
dried over anhydrous Na2SO4, filtered, and concentrated in vacuo.
5.1.66. 3-[(E)-2-(4-Chlorophenyl)vinyl]-4-(2-
dimethylaminoethoxy)benzamide (31d)
The compound 31d was obtained as a colorless powder accord-
ing to the general procedure in 76% yield. 1H NMR (400 MHz,

1458
K. Hada et al. / Bioorg. Med. Chem. 20 (2012) 1442–1460
The residue was purified by flash chromatography (SiO2, n-hexane/
EtOAc) to give the desired products 32a–h.
(2H, dd, J = 5.5, 5.5 Hz), 3.41 (1H, ddd, J = 13.6, 5.5, 5.5 Hz), 3.65
(1H, dddt, J = 6.0, 5.5, 5.5, 5.5 Hz), 3.92 (3H, s), 4.59 (1H, t,
J = 5.5 Hz), 4.83 (1H, d, J = 6.0 Hz), 7.12 (1H, d, J = 8.7 Hz), 7.30 (1H,
d, J = 16.5 Hz), 7.44 (1H, d, J = 16.5 Hz), 7.44 (2H, d, J = 8.5 Hz), 7.62
(2H, d, J = 8.5 Hz), 7.83 (1H, dd, J = 8.7, 2.3 Hz), 8.19 (1H, d,
J = 2.3 Hz), 8.38 (1H, t, J = 5.5 Hz). 13C NMR (125 MHz, DMSO-d6) d:
42.9, 55.8, 63.8, 70.4, 110.9, 123.3, 124.6, 125.4, 126.6, 128.0,
128.1, 128.5, 128.7, 131.9, 136.1, 158.5, 166.0. MS (ESI+) 362, 364
(Cl isotope) [M+H]+. HRMS (ESI+) calcd for C19H21ClNO4 [M+H]+
5.1.70. 3-[(E)-2-(4-Chlorophenyl)vinyl]-4-methoxy-N-
methylbenzamide (32a)
Compound 32a was obtained from (E)-21 and methylamine
hydrochloride as
a
colorless powder in 84% yield. 1H NMR
(400 MHz, DMSO-d6) d: 2.79 (3H, d, J = 4.4 Hz), 3.91 (3H, s), 7.10
(1H, d, J = 8.8 Hz), 7.27 (1H, d, J = 16.5 Hz), 7.42 (1H, d,
J = 16.5 Hz), 7.43 (2H, d, J = 8.5 Hz), 7.61 (2H, d, J = 8.5 Hz), 7.78
(1H, dd, J = 8.8, 2.2 Hz), 8.14 (1H, d, J = 2.2 Hz), 8.35 (1H, br q,
J = 4.4 Hz). MS (ESI+) 302, 304 (Cl isotope) [M+H]+. HRMS (ESI+)
calcd for C17H17ClNO2 [M+H]+ 302.0942, found 302.0943.
362.1154, found 362.1152. ½a D25
ꢃ
+14.3° (c 0.2, MeOH).
5.1.76. 3-[(E)-2-(4-Chlorophenyl)vinyl]-N-[(2S)-2,3-dihydroxy-
propyl]-4-methoxybenzamide (32g)
Compound 32g was obtained from (E)-21 and (S)-3-aminopro-
5.1.71. 3-[(E)-2-(4-Chlorophenyl)vinyl]-4-methoxy-N,N-
dimethylbenzamide (32b)
pane-1,2-diol as a
colorless powder in 92% yield. 1H NMR
(500 MHz, DMSO-d6) d: 3.21 (1H, ddd, J = 13.6, 5.5, 5.5 Hz), 3.35
(2H, dd, J = 5.5, 5.5 Hz), 3.41 (1H, ddd, J = 13.6, 5.5, 5.5 Hz), 3.65
(1H, dddt, J = 6.0, 5.5, 5.5, 5.5 Hz), 3.92 (3H, s), 4.59 (1H, t,
J = 5.5 Hz), 4.84 (1H, d, J = 6.0 Hz), 7.12 (1H, d, J = 8.7 Hz), 7.29
(1H, d, J = 16.5 Hz), 7.44 (1H, d, J = 16.5 Hz), 7.44 (2H, d,
J = 8.5 Hz), 7.62 (2H, d, J = 8.5 Hz), 7.82 (1H, dd, J = 8.7, 2.3 Hz),
8.19 (1H, d, J = 2.3 Hz), 8.39 (1H, t, J = 5.5 Hz). 13C NMR
(125 MHz, DMSO-d6) d: 42.9, 55.8, 63.8, 70.4, 110.9, 123.3, 124.6,
125.4, 126.6, 128.0, 128.1, 128.5, 128.7, 131.9, 136.1, 158.5,
166.0. MS (ESI+) 362, 364 (Cl isotope) [M+H]+. HRMS (ESI+) calcd
Compound 32b was obtained from (E)-21 and dimethylamine
hydrochloride as a yellow oil in quantitative yield. 1H NMR
(400 MHz, CDCl3) d: 3.08 (6H, br s), 3.91 (3H, s), 6.89 (1H, d,
J = 8.5 Hz), 7.06 (1H, d, J = 16.5 Hz), 7.30 (2H, d, J = 8.8 Hz), 7.33
(1H, dd, J = 8.5, 2.2 Hz), 7.40 (1H, d, J = 16.5 Hz), 7.44 (2H, d,
J = 8.8 Hz), 7.67 (1H, d, J = 2.2 Hz). MS (ESI+) 316, 318 (Cl isotope)
[M+H]+. HRMS (ESI+) calcd for C18H19ClNO2 [M+H]+ 316.1099,
found 316.1097.
5.1.72. 3-[(E)-2-(4-Chlorophenyl)vinyl]-N-ethyl-4-
methoxybenzamide (32c)
for C19H21ClNO4 [M+H]+ 362.1154, found 362.1154. ½a 2D5
ꢁ14.5°
ꢃ
(c 0.2, MeOH).
Compound 32c was obtained from (E)-21 and ethylamine
hydrochloride as
a
colorless powder in 86% yield. 1H NMR
5.1.77. 3-[(E)-2-(4-Chlorophenyl)vinyl]-N-[2-hydroxy-1-(hydroxy-
methyl)ethyl]-4-methoxybenzamide (32h)
(400 MHz, DMSO-d6) d: 1.13 (3H, t, J = 7.1 Hz), 3.25–3.31 (2H, m),
3.90 (3H, s), 7.09 (1H, d, J = 8.8 Hz), 7.26 (1H, d, J = 16.5 Hz), 7.41
(1H, d, J = 16.5 Hz), 7.42 (2H, d, J = 8.5 Hz), 7.60 (2H, d, J = 8.5 Hz),
7.78 (1H, dd, J = 8.8, 2.2 Hz), 8.13 (1H, d, J = 2.2 Hz), 8.37 (1H, brt,
J = 5.5 Hz). MS (ESI+) 316, 318 (Cl isotope) [M+H]+. HRMS (ESI+)
calcd for C18H19ClNO2 [M+H]+ 316.1099, found 316.1098.
Compound 32h was obtained from (E)-21 and 2-aminopro-
pane-1,3-diol as
a
colorless powder in 95% yield. 1H NMR
(500 MHz, DMSO-d6) d: 3.54 (4H, dd, J = 6.0, 6.0 Hz), 3.92 (3H, s),
3.98 (1H, dtt, J = 8.2, 6.0, 6.0 Hz), 4.68 (2H, t, J = 6.0 Hz), 7.11 (1H,
d, J = 8.7 Hz), 7.30 (1H, d, J = 16.5 Hz), 7.44 (1H, d, J = 16.5 Hz),
7.44 (2H, d, J = 8.5 Hz), 7.62 (2H, d, J = 8.5 Hz), 7.83 (1H, dd,
J = 8.7, 2.3 Hz), 7.93 (1H, d, J = 8.2 Hz), 8.18 (1H, d, J = 2.3 Hz). 13C
NMR (125 MHz, DMSO-d6) d: 53.8, 55.8, 60.3, 110.7, 123.4, 124.5,
125.6, 126.9, 128.0, 128.1, 128.7, 128.7, 131.9, 136.1, 158.4,
166.0. MS (ESI+) 362, 364 (Cl isotope) [M+H]+. HRMS calcd for
C19H21ClNO4 [M+H] 362.1154, found 362.1152.
5.1.73. 3-[(E)-2-(4-Chlorophenyl)vinyl]-N-(2-hydroxyethyl)-4-
methoxybenzamide (32d)
Compound 32d was obtained from (E)-21 and 2-aminoethanol
as a pale yellow powder in 81% yield. 1H NMR (400 MHz, DMSO-
d6) d: 3.30 (2H, dt, J = 5.8, 5.5 Hz), 3.52 (2H, dt, J = 5.8, 5.8 Hz),
3.91 (3H, s), 4.74 (1H, t, J = 5.8 Hz), 7.10 (1H, d, J = 8.5 Hz), 7.28
(1H, d, J = 16.5 Hz), 7.43 (1H, d, J = 16.5 Hz), 7.43 (2H, d,
J = 8.5 Hz), 7.61 (2H, d, J = 8.5 Hz), 7.81 (1H, dd, J = 8.5, 2.2 Hz),
8.17 (1H, d, J = 2.2 Hz), 8.39 (1H, t, J = 5.5 Hz). MS (ESI+) 332, 334
(Cl isotope) [M+H]+. HRMS calcd for C18H19ClNO3 [M+H]+
332.1048, found 332.1048.
5.2. HUVEC proliferation assay
For the serial dilution of the compound, 5 lL of a solution was
repeatedly diluted with the equal volume of DMSO onto 96-well
plates. Human umbilical vein endothelial cells (HUVECs) were cul-
tured in RPMI1640 supplemented with 10% FCS, containing 50
lg/
5.1.74. 3-[(E)-2-(4-Chlorophenyl)vinyl]-N-(2,3-dihydroxypropyl)-
4-methoxybenzamide (32e)
mL heparin and 30 g/mL endothelial cell growth supplement
l
(ECGS). Cells treated with a range of concentrations of the com-
pounds and seeded at 3000 cells/well in RPMI1640 supplemented
with 5% FCS, containing 20 ng/mL vascular endothelial growth fac-
tor (VEGF) and culture medium as a reference. The plates were
incubated for 4 days at 37 °C in a humidified incubator containing
Compound 32e was obtained from (E)-21 and 3-aminopropane-
1,2-diol as a colorless powder in 93% yield. 1H NMR (400 MHz,
DMSO-d6) d: 3.21 (1H, ddd, J = 13.6, 5.5, 5.5 Hz), 3.35 (2H, dd,
J = 5.5, 5.5 Hz), 3.41 (1H, ddd, J = 13.6, 5.5, 5.5 Hz), 3.63 (1H, dddt,
J = 6.0, 5.5, 5.5, 5.5 Hz), 3.91 (3H, s), 4.58 (1H, t, J = 5.5 Hz), 4.83
(1H, d, J = 6.0 Hz), 7.13 (1H, d, J = 8.8 Hz), 7.29 (1H, d, J = 16.5 Hz),
7.43 (1H, d, J = 16.5 Hz), 7.43 (2H, d, J = 8.5 Hz), 7.61 (2H, d,
J = 8.5 Hz), 7.82 (1H, dd, J = 8.8, 2.0 Hz), 8.19 (1H, d, J = 2.0 Hz),
8.38 (1H, t, J = 5.5 Hz). MS (ESI+) 362, 364 (Cl isotope) [M+H]+.
HRMS calcd for C19H21ClNO4 [M+H]+ 362.1154, found 362.1152.
5% CO2. On the last day, 10–20 lL of Cell Counting Kit-8 (Dojindo
Laboratories, Tokyo, Japan) was added to the culture in each well
and the plates were further incubated for a few hours at 37 °C in
a humidified incubator containing 5% CO2. After incubation, absor-
bance in each well was measured at 450 nm using a micro-plate
reader (BIO-RAD Model 3550). The antiproliferative activity of each
compound for VEGF-induced condition was calculated by the for-
mula (1-T/C) ꢂ 100 (%), where T and C represent mean difference
absorbance at 450 nm of the cells treated with compounds (T)
and that of the untreated control cells (C). The IC50 values were
calculated using an Excel function entitled mod100LinearIC50
developed by Roche Palo Alto Research Center (CA, USA).
5.1.75. 3-[(E)-2-(4-Chlorophenyl)vinyl]-N-[(2R)-2,3-dihydroxy-
propyl]-4-methoxybenzamide (32f)
Compound 32f was obtained from (E)-21 and (R)-3-aminopro-
pane-1,2-diol as
(500 MHz, DMSO-d6) d: 3.21 (1H, ddd, J = 13.6, 5.5, 5.5 Hz), 3.36
a
colorless powder in 90% yield. 1H NMR

K. Hada et al. / Bioorg. Med. Chem. 20 (2012) 1442–1460
1459
5.3. Tube formation assay
volumes of CH3CN/MeOH (1:1 v/v) including an internal standard,
then thoroughly mixed to precipitate plasma protein. The suspen-
sions were transferred to 96-well MultiScreen filtration plates
The Angiogenesis Kit (Kurabo Industries Ltd.) was charged with
the test compound at final concentration of 4
lM and 20
l
M, and
(0.45
lm) and filtrated by centrifugation at 1000ꢂg for 5 min at
incubated in a CO2 incubator (37 °C, 5%). After 11 days of incuba-
tion, capillary-like tubes formed were fixed with 70% ethanol and
visualized with a CD31 Staining Kit (also produced by Kurabo
Industries Ltd.). Under a microscope, stain images of the wells were
photographed, stored as an image file, and measured quantita-
tively for the area of capillary-like tube formation with Kurabo
angiogenesis image analysis software. The inhibition rates (%) of
the test compound-charged wells were calculated, with the control
taken as 100%.
4 °C (LX-130, Tomy Seiko). The filtrate was applied to a liquid chro-
matography-tandem mass spectrometry (LC-MS/MS) system
(API3000, Applied Biosystems/MDS SCIEX) to measure concentra-
tions of the compounds. The lower limit of quantification (LLOQ)
was 20 ng/mL for each compound. The pharmacokinetic parameters
were calculated by non-compartmental analysis using Watson ver.
6.3 (ThermoFisher Scientific).
5.7. Antitumor test
5.4. Solubility assay (LYSA)
Cell suspension of human lung carcinoma cell line Calu-6 was
prepared using Hanks’ balanced salt solution implanted subcuta-
neously in the flank region of female Balb/c nude mice, respectively
at 5.0 ꢂ 106 cells per mouse. When the volume of the tumor
reached 150 mm3, the test compound was orally administered
once daily for 11 days. The tumor volume was calculated from
the equation 1/2 ꢂ (long diameter ꢂ short diameter2). The tumor
growth inhibition rate was calculated from changes in the tumor
volume in the test compound treatment group relative to changes
in that of the control group.
Samples were prepared in triplicate from 10 mM dimethylsulf-
oxide stock solutions. After evaporation (1 h) of dimethylsulfoxide
with a centrifugal vacuum evaporator, the compounds were
dissolved in FaSSIF, and shaken for 2 h. The solutions were filtered
using a microtiter filter plate (Whatman UNIFILTER) and the fil-
trate was analyzed by UV measurement or HPLC-UV. In addition,
a four point calibration curve is prepared from the 10 mM stock
solutions and used to determine the solubility of the compounds.
The results are expressed in
lg/mL. Starting from a 10 mM stock
solution, the measurement range for MW 500 was 0–666
lg/mL.
5.8. Measurement of the number of blood vessels in the tumor
5.5. Liver microsomal stability assay
5.0 ꢂ 106 Cells of human lung carcinoma cell line Calu-6 were
implanted subcutaneously in the flank region of female Balb/c
nude mice. When the tumor volume reached 150 mm3, the test
compound was orally administered once daily for 11 days.
Twenty-four hours after the final administration, xenograft tissue
was removed from the mice, and a middle portion of the long
diameter of the tumor was embedded, as a block 2 to 3 mm thick,
in O.C.T. Compound, and preserved as a frozen tissue specimen.
Frozen sections were prepared, and blood vessels in the tumor tis-
sue were stained by an immunohistological method using anti-
mouse CD31 antibody. The stained tissue was photographed under
a microscope, and the images were stored as an image file. The
number of the stained blood vessels was measured by Image-Pro
Plus (Nippon Roper K.K.). The rate of blood vessel density decrease
was calculated as a rate compared to the blood vessel density de-
crease in the control group.
Mouse or human microsome incubations were conducted by an
automated procedure implemented on a Biomek FX (Beckman
Coulter). Compounds (5 lM) were incubated in microsomes at
1.0 mg protein/mL in a 50 mM potassium phosphate buffer, pH
7.4, at 37 °C. Cofactor (NADPH) was produced by a generating sys-
tem (glucose 6-phosphate, 3.2 mM; b-NADP, 2.6 mM; MgCl2,
6.5 mM). Addition of the NADPH generating system to the pre-
warmed microsomes containing the test compound started the
reaction. Aliquots (50
lL) were taken at four defined time points
within 60 min and transferred into 100
l
L of MeOH containing
an internal standard. Concentration of each compound was ana-
lyzed by LC–MS/MS using an ODS-3 RP 18 column (GL Sciences).
Quantitative detection was achieved on a API 365 instrument (AB
Sciex) using electron spray ionization. Concentrations were deter-
mined by ratio of test compound and internal standard peaks and
given as a percentage of the concentration measured at the first
5.9. Antitumor test of 32f in combination with sunitinib
time point (substrate depletion). Intrinsic clearance (CL, in lL/
min/mg microsomal protein) is the rate constant of the first-order
decay of the test compound, normalized for the protein concentra-
tion in the incubation.
Cell suspension of human lung carcinoma cell line Calu-6 was
prepared using Hanks’ balanced salt solution and implanted subcu-
taneously in the flank region of female and Balb/c nude mice at
5.0 ꢂ 106 cells per mouse. When the volume of the tumor reached
200 mm3, the mice were randomized into 4 groups (n = 5), and the
following treatment was implemented orally once daily for 11 days:
(1) vehicle, (2) 32f (150 mg/kg), (3) sunitinib (80 mg/kg), (4) combi-
nation of both test compounds. The tumor volume was calculated
from the equation 1/2 ꢂ (long diameter ꢂ short diameter2). The
tumor growth inhibition rate was calculated from changes in the
tumor volume in the test compounds treatment group relative to
changes in that of the control group.
5.6. Pharmacokinetic studies
Pharmacokinetic studies of 32f and 32g were performed in fe-
male nude mice following an oral dose of 30 mg/kg. The compounds
were dissolved in DMSO/Cremophor EL/1% Tween 80 (1:1:8 v/v/v)
and orally administered to animals through a stomach sonde. Blood
of mice (n = 2/time point) was collected from the retro-orbital vein
using a heparinized capillary or by heart puncture using a dispos-
able syringe with a 25G needle under light isoflurane anesthesia
at the scheduled time points of 30 min, 1, 2, 4, 7 and 24 h after
administration. Blood samples were immediately centrifuged by a
hematocrit centrifuge (Model 3220, Kubota) at 12,000 rpm for
3 min or by a centrifuge at 3000 rpm for 10 min at 4 °C (LX-130,
Tomy Seiko) to separate the plasma. Plasma samples were immedi-
ately frozen on dry ice and stored in a deep freezer set at ꢁ80 °C un-
Acknowledgments
The authors thank Dr. Nobuya Ishii for helpful discussions;
Kiyoaki Sakata and Toshihiko Fujii for technical assistance with cel-
lular assays; Yukako Tachibana for kinase assays; Kiyoshi Yoshinari
for physicochemical property assays and liver microsomal stability
assays; Hitomi Suda and Yuichiro Ishiguro for HRMS measurement.
til analysis. The plasma sample (25 lL) was added with three

1460
K. Hada et al. / Bioorg. Med. Chem. 20 (2012) 1442–1460
Kondo, S. I.; Kato, K.; Toi, M.; Umezawa, K. Int. J. Oncol. 2001, 18, 1009; (h)
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