Design, synthesis and biological evaluation of arylcinnamide hybrid derivatives as novel anticancer agents

Article abstract of DOI:10.1016/j.ejmech.2014.05.028

The combination of two pharmacophores into a single molecule represents one of the methods that can be adopted for the synthesis of new anticancer molecules. A series of novel antiproliferative agents designed by a pharmacophore hybridization approach, combining the arylcinnamide skeleton and an α-bromoacryloyl moiety, was synthesized and evaluated for its antiproliferative activity against a panel of seven human cancer cell lines. In addition, the new derivatives were also active on multidrug-resistant cell lines over-expressing P-glycoprotein. The biological effects of various substituents on the N-phenyl ring of the benzamide portion were also described. In order to study the possible mechanism of action, we observed that 4p slightly increased the Reactive Oxygen Species (ROS) production in HeLa cells, but, more importantly, a remarkable decrease of intracellular reduced glutathione content was detected in treated cells compared with controls. These results were confirmed by the observation that only thiol-containing antioxidants were able to significantly protect the cells from induced cell death. Altogether our results indicate that the new derivatives are endowed with good anticancer activity in vitro, and their properties may result in the development of new cancer therapeutic strategies.

Full text of DOI:10.1016/j.ejmech.2014.05.028

European Journal of Medicinal Chemistry 81 (2014) 394e407
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design, synthesis and biological evaluation of arylcinnamide hybrid
derivatives as novel anticancer agents
,
a
,
a
Romeo Romagnoli a , Pier Giovanni Baraldi , Maria Kimatrai Salvador ,
*
*
Mariem Chayah a, M. Encarnacion Camacho a, Filippo Prencipe a, Ernest Hamel b,
Francesca Consolaro c, Giuseppe Basso c, Giampietro Viola c
,
*
a Dipartimento di Scienze Chimiche e Farmaceutiche, Via Fossato di Mortara 17, Università di Ferrara, 44121 Ferrara, Italy
b Screening Technologies Branch, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, Frederick National Laboratory for
Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, MD 21702, USA
c Dipartimento di Salute della Donna e del Bambino, Laboratorio di Oncoematologia, Università di Padova, 35131 Padova, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
The combination of two pharmacophores into a single molecule represents one of the methods that can
be adopted for the synthesis of new anticancer molecules. A series of novel antiproliferative agents
designed by a pharmacophore hybridization approach, combining the arylcinnamide skeleton and an a-
Received 13 February 2014
Received in revised form
6 May 2014
bromoacryloyl moiety, was synthesized and evaluated for its antiproliferative activity against a panel of
seven human cancer cell lines. In addition, the new derivatives were also active on multidrug-resistant
cell lines over-expressing P-glycoprotein. The biological effects of various substituents on the N-phenyl
ring of the benzamide portion were also described. In order to study the possible mechanism of action,
we observed that 4p slightly increased the Reactive Oxygen Species (ROS) production in HeLa cells, but,
more importantly, a remarkable decrease of intracellular reduced glutathione content was detected in
treated cells compared with controls. These results were confirmed by the observation that only thiol-
containing antioxidants were able to significantly protect the cells from induced cell death. Altogether
our results indicate that the new derivatives are endowed with good anticancer activity in vitro, and their
properties may result in the development of new cancer therapeutic strategies.
Accepted 9 May 2014
Available online 10 May 2014
Keywords:
Apoptosis
Phenylcinnamides
Michael acceptor
In vitro antiproliferative activity
GSH depletion
Oxidative stress
Ó 2014 Elsevier Masson SAS. All rights reserved.
1. Introduction
By the screening of a 100-member library of amides, compound 1a
was identified as one of the most active derivatives, with moderate
Molecular hybridization, which covalently combines into a sin-
gle molecule two different bioactive molecules with complemen-
tary pharmacophoric functions or with different mechanisms of
action, is an effective tool to design highly active novel anticancer
agents [1e9]. Antitumor activity of cinnamic acid derivatives was
explored by many research groups [10e17]. Several laboratories
reported that the phenylcinnamide scaffold showed anticancer
activity against various human cancer cell lines. Phenylcinnamide
derivatives with general structure 1 (Chart 1) are a class of com-
pounds initially identified by Hergenrother et al. as potential
anticancer agents [18,19], with a moderate cytotoxic activity (IC50
activity against U-937 (lymphoma) and HeLa (cervical) cancer cells
(IC50 values of 3.0 and 11.3 M, respectively). This derivative
m
arrested the cell cycle at the G2/M phase and induced apoptotic cell
death in the HeLa cancer cell line. Since the 3,4,5-trimethoxyphenyl
substituent was demonstrated to be an essential structural
requirement for optimal biological activity in numerous tubulin
inhibitors, the antiproliferative activity of 1a was associated with
inhibition of tubulin polymerization. Increasing the bulk of the
meta-substituent on the phenyl ring of the styryl portion, from
methoxy to benzyloxy, to furnish derivative 1b, led to an increase in
antiproliferative activity relative to 1a, with IC50 values of 1.8 and
ranging from 1 to 10
mM). These compounds interact with micro-
2.1 mM against U-937 and HeLa cells, respectively. Raffa et al.
tubules by interfering with the dynamics of tubulin polymerization.
described a series of cinnamoyl anthranilates with general struc-
ture 2 that inhibited the proliferation of human leukemia K562 cells
with IC50 values ranging from 0.57 to 5 mM [20]. COMPARE analysis,
* Corresponding authors.
E-mail addresses: rmr@unife.it (R. Romagnoli), pgb@unife.it (P.G. Baraldi),
giampietro.viola1@unipd.it (G. Viola).
effects on tubulin polymerization and on cell cycle distribution
indicated that these compounds act as antitubulin agents.
http://dx.doi.org/10.1016/j.ejmech.2014.05.028
0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.

R. Romagnoli et al. / European Journal of Medicinal Chemistry 81 (2014) 394e407
395
OCH3
OCH3
para-position on the phenyl of the benzylidene fragment, corre-
sponding to compounds 4aep and 4qer, respectively. The struc-
tureeactivity relationship (SAR) was investigated by the insertion of
different substituents [both electron-releasing (CH3, alkoxy, OCF3,
SCF3, morpholin-4-yl) and electron-withdrawing (F and Cl) groups]
at different positions on the phenyl ring of the benzamide portion.
R
CONH2
NH
O
RO
N
OCH3
H
O
2
1a, R=CH3
1b, R=CH2C6H5
R=Cl, Br, I or CH3.
2. Chemistry
Br
H
The synthetic route for the synthesis of compounds 4aer is
outlined in Scheme 1. The two isomeric nitrocinnamic acids 5a and
5b [28] were converted to acyl chlorides in CH2Cl2 by POCl3 and
then reacted in situ with the appropriate commercially available
aniline (or 4-methylbenzylamine for the preparation of 4h) to
furnish the corresponding nitro arylcinnamides 6aep and 6qer,
respectively. The subsequent reduction of 6aep and 6qer using
tin(II) chloride in refluxing ethanol yielded the related amino de-
rivatives 7aep and 7qer, respectively, which were converted to the
N
NH
H
HCl
NH2
N
O
N
N
H
O
4
PNU-166196 or Brostallicin (3)
O
Br
O
H
hybrid compounds 4aep and 4qer by condensation with a-bro-
moacrylic acid using 1-ethyl-3-[3-(dimethylamino)propyl]carbo-
diimide hydrochloride (EDCl) in dimethylformamide.
Ar
N
Ar
O
N
H
N
H
4q-r
O
N
4a-p
H
Br
4a, Ar=C6H5
3. Biological results and discussion
4b, Ar=napht-1-yl
4c, Ar=4-F-C6H5
4d, Ar=3-F-C6H5
4e, Ar=3,4-F2-C6H4
4f, Ar=4-Cl-C6H5
4q, Ar=4-OCH3-C6H5
4r, Ar=3,4,5-(OCH3)3-C6H3
3.1. In vitro antiproliferative activities
In Table 1, we report the antiproliferative effects of a-bromoa-
cryloylamido arylcinnamides 4aer against the growth of human
4g, Ar=4-CH3-C6H5
4h, Ar=4-CH3-C6H4CH2
4i, Ar=4-OCH3-C6H5
4j, Ar=4-OC2H5-C6H5
4k, Ar=4-OCF3-C6H5
CO2H
O2N
CO2H
or
4l, Ar=4-SCF3-C6H5
5b
4m, Ar=3,4-(OCH3)2-C6H4
4n, Ar=3,5-(OCH3)2-C6H4
4o, Ar=3,4,5-(OCH3)3-C6H3
4p, Ar=4-(morpholin-4'-yl)-C6H5
O2N
5a
a
O
O
O2N
Ar
N
Chart 1. Structure of phenylcinnamides 1a, 1b and 2, brostallicin (3) and
cryloylamido arylcinnamides 4aer.
a-bromoa-
Ar
or
N
H
H
6a-p
O2N
6q-r
The compound
low chemical reactivity and devoid of cytotoxic effects
(IC50 > 120 M on murine leukemia L1210 cells) [21]. The -bro-
moacryloyl moiety is present in a series of potent anticancer
distamycin-like minor groove binders, including PNU-166196
(brostallicin, 3), which was evaluated as a first-line single agent
chemotherapy in patients with advanced or metastatic soft tissue
a-bromoacrylic acid is an alkylating agent with
b
m
a
O
O
H2N
Ar
N
Ar
or
N
H
H
7a-p
H2N
7q-r
sarcoma [22,23]. It has been hypothesized that the reactivity of the a-
c
bromoacryoyl moiety results from a first-step Michael-type nucle-
ophilic attack, followed by a further reaction of the former vinylic
bromo substituent alpha to the carbonyl, leading successively either
to a second nucleophilic substitution or to a beta elimination [24].
O
Br
O
H
Ar
N
Ar
O
N
N
H
H
4q-r
O
N
4a-p
6-7a, 4a, Ar=C6H5
6-7b, 4b, Ar=napht-1-yl
6-7c, 4c, Ar=4-F-C6H5
6-7d, 4d, Ar=3-F-C6H5
6-7e, 4e, Ar=3,4-F2-C6H4
6-7f, 4f, Ar=4-Cl-C6H5
6-7g, 4g, Ar=4-CH3-C6H5
6-7h, 4h, Ar=4-CH3-C6H4CH2
6-7i, 4i, Ar=4-OCH3-C6H5
6-7j, 4j, Ar=4-OC2H5-C6H5
6-7k, 4k, Ar=4-OCF3-C6H5
6-7l, 4l, Ar=4-SCF3-C6H5
6-7m, 4m, Ar=3,4-(OCH3)2-C6H4
6-7n, 4n, Ar=3,5-(OCH3)2-C6H4
6-7o, 4o, Ar=3,4,5-(OCH3)3-C6H3
H
Phenylcinnamides are characterized by the presence of an a,b-
Br
unsaturated carbonyl moiety, which can be considered as an Michael
acceptor, an active pharmacophore often employed in the design of
anticancer drugs [10,25]. Because of their ability to interact with
cellular nucleophiles, Michael acceptors are often employed as a
powerful tool in the design of anticancer agents [26,27]. Combining
two bioactive pharmacophores, corresponding to the phenyl-
cinnamide skeleton and an a-bromoacryoyl moiety, within a unique
structure might result in compounds that exhibit synergistic anti-
cancer effects. In a continuing study of hybrid compounds containing
6-7q, 4q, Ar=4-OCH3-C6H5
6-7r, 4r, Ar=3,4,5-(OCH3)3-C6H3
the a-bromoacryloyl moiety as potential anticancer drugs, we syn-
thesized a novel series of hybrid compounds with general structure
4, in which this moiety was linked to the arylcinnamide scaffold.
In particular, we synthesized two different series of compounds,
6-7p, 4p, Ar=4-(morpholin-4'-yl)-C6H5
Scheme 1. Reagents: a. ArNH2, POCl3, TEA, CH2Cl2; b: SnCl2 H2O, EtOH, reflux; c: a-
characterized by the a-bromoacryloyl moiety located at the meta- or
bromoacrylic acid, EDCI, DMF, rt, 18 h.

396
R. Romagnoli et al. / European Journal of Medicinal Chemistry 81 (2014) 394e407
Table 1
In vitro cell growth inhibitory effects of compounds 1aeb, 4aer and 7o.
a
Compd
IC50
(
mM)
HeLa
HT-29
LoVo
CEM
Jurkat
SEM
MCF-7
4a
4b
4c
4d
4e
4f
4g
4h
4i
7.3 ꢀ 2.1
1.4 ꢀ 0.1
16.6 ꢀ 5.9
6.5 ꢀ 0.6
36.6 ꢀ 2.6
4.6 ꢀ 0.4
8.7 ꢀ 3.9
7.9 ꢀ 1.0
1.2 ꢀ 0.3
2.7 ꢀ 0.3
2.3 ꢀ 0.2
3.4 ꢀ 0.3
61.2 ꢀ 11.1
39.8 ꢀ 2.8
1.9 ꢀ 0.4
3.6 ꢀ 0.2
2.3 ꢀ 0.5
1.6 ꢀ 0.4
3.1 ꢀ 0.3
3.7 ꢀ 0.9
63.1 ꢀ 6.5
44.1 ꢀ 0.9
3.6 ꢀ 0.2
3.9 ꢀ 0.18
0.19 ꢀ 0.09
5.0 ꢀ 0.7
0.68 ꢀ 0.22
6.2 ꢀ 1.8
1.1 ꢀ 0.26
27.3 ꢀ 3.9
2.8 ꢀ 0.5
13.6 ꢀ 3.8
3.3 ꢀ 0.5
2.69 ꢀ 0.2
11.0 ꢀ 1.3
2.5 ꢀ 0.5
2.2 ꢀ 0.8
2.5 ꢀ 0.3
5.5 ꢀ 1.0
43.3 ꢀ 1.8
30.1 ꢀ 1.4
2.8 ꢀ 0.4
3.0 ꢀ 0.4
1.8 ꢀ 0.3
2.6 ꢀ 0.2
3.0 ꢀ 0.2
3.0 ꢀ 0.6
55.4 ꢀ 11.1
27.0 ꢀ 4.5
2.4 ꢀ 0.8
1.16 ꢀ 0.11
4.8 ꢀ 0.8
1.8 ꢀ 0.44
16.6 ꢀ 4.5
0.83 ꢀ 0.25
8.4 ꢀ 3.7
16.8 ꢀ 4.8
1.2 ꢀ 0.4
1.0 ꢀ 0.2
26.0 ꢀ 4.1
1.0 ꢀ 0.3
0.62 ꢀ 0.31
0.09 ꢀ 0.03
1.19 ꢀ 0.45
0.19 ꢀ 0.04
1.3 ꢀ 0.2
1.9 ꢀ 0.1
0.54 ꢀ 0.07
5.9 ꢀ 1.0
0.013 ꢀ 0.006
0.46 ꢀ 0.02
0.27 ꢀ 0.05
0.55 ꢀ 0.13
0.026 ꢀ 0.01
0.098 ꢀ 0.033
13.0 ꢀ 4.0
0.55 ꢀ 0.19
2.6 ꢀ 0.16
0.24 ꢀ 0.04
0.74 ꢀ 0.17
0.47 ꢀ 0.15
0.93 ꢀ 0.12
30.3 ꢀ 4.5
30.1 ꢀ 2.1
0.19 ꢀ 0.05
0.98 ꢀ 0.33
0.29 ꢀ 0.03
0.04 ꢀ 0.01
1.4 ꢀ 0.47
0.41 ꢀ 0.05
43.3 ꢀ 4.8
12.5 ꢀ 0.9
3.5 ꢀ 0.5
2.8 ꢀ 0.3
1.5 ꢀ 0.12
1.7 ꢀ 0.4
1.5 ꢀ 0.3
1.9 ꢀ 0.3
0.79 ꢀ 0.25
2.0 ꢀ 0.13
29.0 ꢀ 3.4
19.0 ꢀ 3.2
0.56 ꢀ 0.18
2.2 ꢀ 0.18
0.88 ꢀ 0.19
0.43 ꢀ 0.1
1.6 ꢀ 0.3
0.55 ꢀ 0.08
0.85 ꢀ 0.03
30.3 ꢀ 0.9
29.3 ꢀ 2.2
0.15 ꢀ 0.02
1.21 ꢀ 0.32
0.14 ꢀ 0.04
0.51 ꢀ 0.11
0.06 ꢀ 0.01
2.1 ꢀ 0.9
0.07 ꢀ 0.03
0.18 ꢀ 0.04
19.3 ꢀ 2.3
4j
4k
4l
15.3 ꢀ 2.5
12.7 ꢀ 2.6
4m
4n
4o
4p
4q
4r
7o
1a
1b
0.021 ꢀ 0.005
0.011 ꢀ 0.004
0.059 ꢀ 0.025
0.61 ꢀ 0.08
0.022 ꢀ 0.005
0.025 ꢀ 0.013
40.9 ꢀ 13.6
23.4 ꢀ 4.1
0.003 ꢀ 0.0004
0.04 ꢀ 0.017
0.018 ꢀ 0.003
0.057 ꢀ 0.013
0.08 ꢀ 0.03
0.11 ꢀ 0.09
2.81 ꢀ 2.8
0.49 ꢀ 0.17
53.1 ꢀ 4.6
16.5 ꢀ 2.6
2.2 ꢀ 0.39
45.2 ꢀ 5.6
43.0 ꢀ 3.0
21.7 ꢀ 5.0
18.2 ꢀ 1.9
17.3 ꢀ 6.0
8.5 ꢀ 2.3
a
IC50 ¼ compound concentration required to inhibit tumor cell proliferation by 50%. Data are expressed as the means ꢀ SE from the doseeresponse curves of at least three
independent experiments.
cervix carcinoma (HeLa), colorectal carcinoma (HT-29 and LoVo),
lymphoblastic leukemia (CEM, Jurkat and SEM) and breast carci-
noma (MCF-7) cells, using the phenylcinnamides 1a and 1b as
positive controls. Compound 1b, with IC50’s ranging from 2.2 to
Relative to the activity of derivative 4c, insertion of an additional
fluorine atom at the meta-position to yield 4e increased anti-
proliferative activity (4e1300 fold) against all cancer cell lines.
By comparing the effect of substituents with opposite electronic
properties at the para-position of phenyl ring, a 2e10 fold increase
in potency was observed by replacing the electron withdrawing
chlorine atom (4f) with an electron-donating methyl group (4g).
The cell growth inhibitory activities of N-40-tolyl and N-40-
methylbenzyl derivatives 4g and 4h, respectively, were very similar
against the HeLa, LoVo and MCF-7 cells, while 4h had significantly
reduced activity (from 2 to 7 fold) as compared with 4g against the
remaining cell lines.
The antiproliferative activity of hybrid compounds can be
further characterized in terms of the substitution pattern and the
number of methoxy groups on the phenyl ring. The results show
that mono-, di- or trimethoxy substitution on the 30, 40, and/or 50-
positions of the phenyl ring (compounds 4i, 4meo and 4qer) were
well tolerated and increased activity, usually substantially, as
compared with the unsubstituted benzamide derivative 4a.
A single methoxy substituent on the para-position of the phenyl
ring (compound 4i) led to a 7e15 fold increase in antiproliferative
activity relative to 4a. Comparing 4i with 4m, an additional
methoxy group at the meta-position had little effect on activity
against HeLa, HT-29, CEM and MCF-7, but there were 2-, 3- and 20-
fold increases in activity against the Jurkat, LoVo and SEM cells,
respectively. The 30,40-dimethoxy derivative 4m was generally (6
out of 7 cell lines) more active than the isomeric 30,50-dimethoxy
analogue 4n. Finally, adding a third methoxy group to produce the
30, 40, 50-trimethoxyphenyl derivative 4o caused a reduction in ac-
tivity against five of the seven cancer cell lines.
21.7
terpart 1a. With only two exceptions (4k and 4l), all the molecules
that were generated by the hybridization of the -bromoacryloyl
mM, was 1.5- to 12-fold more potent than its methoxy coun-
a
moiety with the arylcinnamide system were more active than 1a.
For the benzyloxy derivative 1b, its activity was lower than that of
derivatives 4b, 4eej, 4mer against the LoVo, CEM, Jurkat and SEM
cells. Among the hybrid compounds, six of them (4e, 4i, 4meo and
4q) exhibited potent activity, with double-digit nanomolar IC50
values against both the CEM and SEM cell lines. The validity of the
hybridization approach was confirmed comparing the potency of
compound 4o with that of the amino phenylcinnamide derivative
7o. This latter compound was 30e700 fold less active than the
corresponding
strating that the presence of a
a
-bromoacryloylamido derivative 4o, demon-
-bromoacryloyl moiety significantly
a
enhanced antiproliferative activity.
With the exception of the CEM cells, compound 4b bearing the
more lipophilic 1-naphthyl moiety exerted a more pronounced
antiproliferative activity toward cell lines tested in comparison
with the unsubstituted phenyl derivative 4a.
The antiproliferative activities of the hybrid molecules were
influenced by the substituents on the phenyl ring of the aniline/
benzylamino moiety. A comparison of the para-position substitu-
tion on the phenyl ring demonstrated that ERGs such as methyl (4g
and 4h), methoxy (4i) or ethoxy (4j) and morpholin-4-yl (4p)
increased antiproliferative activity compared with the unsub-
stituted benzene derivative 4a, whereas trifluoromethoxy (OCF3,
4k) or trifluoromethylsulfanyl (SCF3, 4l) substituents produced a
dramatic loss of potency.
Introduction of a weak electron-withdrawing group, a para-
fluorine atom (4c), decreased the activity relative to 4a on six
cancer cell lines, the exception being the MCF-7 cells. Moving the
fluorine from the para- (4c) to the meta-position (4d) enhanced
antiproliferative activity 4e15 fold in six of the seven cell lines, the
exception being the CEM cells. Antiproliferative activity increased
2e30 fold by increasing halogen size from fluorine to chlorine (4f).
Turning specifically to the para-substituted phenyl derivatives,
replacing 40-methoxy (4i) with the 40-ethoxy homologue (com-
pound 4j) produced a 2e4 fold reduction in activity. This loss of
activity became much more pronounced if the methoxy group was
replaced with
a
bulkier trifluromethoxy (4k) or tri-
fluoromethoxysulfanyl (4l) moiety.
This activity loss cannot be explained simply as being caused by
a steric effect of the substitution at the para-position of the phenyl
ring because insertion of an electron poor heterocycle such as

R. Romagnoli et al. / European Journal of Medicinal Chemistry 81 (2014) 394e407
397
Table 3
morpholin-4-yl (compound 4p) enhanced antiproliferative activity
8e100 fold relative to 4a with six of the seven cancer cell lines (the
exception was the CEM cells). Changing the location of a-bromoa-
cryloylamido moiety from the meta- in 4i to the para-position in 4q
had little effect on activity, except in the LoVo cells. Similarly, there
was little difference in activity between the two isomeric trime-
thoxyphenyl derivatives 4o and 4r, except in two cell lines (LoVo
and SEM cells).
In vitro cell growth inhibitory effects of compounds 4p and 4r on drug-resistant cell
lines.
a
Compd
IC50
(
mM)
LoVo
LoVoDoxo
Resistance ratiob
4p
4r
0.51 ꢀ 0.11
2.1 ꢀ 0.9
1.05 ꢀ 0.44
1.37 ꢀ 0.24
11.3 ꢀ 0.35
2.0
0.6
118
Doxorubicin
0.09 ꢀ 0.043
CEM
CEMVble100
Resistance ratiob
3.2. Evaluation of cytotoxicity in human peripheral mononuclear
cells (PBMC)
4p
4r
0.61 ꢀ 0.08
0.41 ꢀ 0.09
0.7
0.6
105
0.025 ꢀ 0.013
0.002 ꢀ 0.0005
0.015 ꢀ 0.005
0.021 ꢀ 0.008
Vinblastine
a
To obtain more insights into the cytotoxic potential of these new
compounds for normal human cells, two of the most active com-
pounds (4p and 4r) were assayed in vitro against human peripheral
mononuclear cells (PBMC) obtained from healthy donors. As
showed in Table 2, compounds 4r and 4p showed, in resting PBMC,
IC50 ¼ compound concentration required to inhibit tumor cell proliferation by
50%. Data are presented as the means ꢀ SE from the doseeresponse curves of at least
three independent experiments.
b
The values express the ratio between the IC50’s determined in resistant and non-
resistant cell lines.
a lesser degree of toxicity having an IC50 of about 3.5e4.0 mM that is
roughly 10e100 fold lower respect to the lymphoblastic cell lines
Jurkat and CEM. On the contrary they proved cytotoxic only for
PHA-stimulated PBMC, suggesting that these compounds acts only
in proliferating cells.
To further evaluate if the new derivatives interfered with the
microtubule network, we examined the effects of 4r and 4p on
HeLa cells by immunofluorescence microscopy. Shown in Fig. 1
(Panel A) is the normal microtubule network of untreated cells.
Following 24 h of treatment with 4p or 4r at 2.5 or 5.0 mM (Fig. 1,
panels BeD), the microtubule network was not substantially
modified in comparison with the untreated cells. Altogether these
results were consistent with the conclusion that the anti-
proliferative activity of these hybrid compounds was not derived
from a direct interaction with tubulin and that it is unlikely that
they act as microtubule binding agents.
3.3. Effects of phenylcinnamides on drug-resistant cell lines
Drug resistance is an important therapeutic problem caused by
the appearance of tumor cells endowed with differing mechanisms
that confer resistance against a variety of anticancer drugs [29,30].
Among the most common mechanisms of resistance are those
related to the over-expression of a cellular membrane protein
called P-gp that mediates the efflux of various structurally unre-
lated drugs [29,30]. In this context, we evaluated sensitivity of two
of the most active compounds 4p and 4r in two multidrug-resistant
cell lines, one derived from a colon carcinoma (LoVoDoxo) [31], the
other derived from a T-cell leukemia (CEMVblꢁ100) [32], both
expressing high levels of P-gp. As shown in Table 3, compounds 4p
and 4r were equally potent toward parental cells and cells resistant
to vinblastine or doxorubicin, suggesting that these compounds
might be useful in the treatment of drug refractory tumors.
3.5. Analysis of cell cycle effects
The effects of a 24 h treatment with different concentrations of
4r and 4p on cell cycle progression in HeLa cells were determined
by flow cytometry (Fig. 2, Panels A and B). The compounds caused a
significant G2/M arrest in a concentration-dependent manner in
the cell line tested, with a rise in G2/M cells occurring at a con-
centration as low as 2 mM. Importantly, the increase in G2/M phase
cells was accompanied by a remarkable reduction in S phase cells
for both compounds.
3.4. In vitro inhibition of tubulin polymerization
3.6. Phenylcinnamides induce apoptosis in Hela cells
Previous studies have shown that compound 1a most likely
derived its antiproliferative activity from an interaction at the
colchicine site of tubulin, causing inhibition of tubulin polymeri-
zation [19]. To investigate whether the antiproliferative activities of
the most active hybrid compounds (4e, 4i, 4m, 4oep, 4r) were
related to an interaction with the microtubule system, these mol-
ecules were evaluated for their in vitro inhibition of the polymeri-
zation of purified tubulin [33]. All tested compounds did not inhibit
To characterize the mode of cell death induced by 4r and 4p, a
biparametric cytofluorimetric analysis was performed using PI,
which stains DNA and enters only dead cells, and fluorescent
immunolabeling of the protein annexin-V, which binds to phos-
phatidylserine in a highly selective manner [34]. Dual staining for
annexin-V and with PI permits discrimination between live cells
(annexin-Vꢁ/PIꢁ), early apoptotic cells (annexin-Vþ/PIꢁ), late
apoptotic cells (annexin-Vþ/PIþ) and necrotic cells (annexin-Vꢁ/
PIþ). As shown in Fig. 3 (Panels A and B), Hela cells treated with the
two compounds for 24 h showed an accumulation of annexin-V
positive cells that further increased after 48 h in comparison with
the untreated cells.
tubulin assembly at concentrations as high as 20 mM, indicating
that they were inactive as inhibitors of tubulin polymerization.
Table 2
Cytotoxicity of 4p and 4r for human peripheral blood mononuclear cells (PBMC).
Cell line
IC50 (m
M)a
3.7. Phenylcinnamides induce caspase-dependent cell death
4p
4r
3.5 ꢀ 1.8
To evaluate if the apoptotic cell death induced by 4p and 4r is
caspase-dependent, Hela cells were treated with the two com-
pounds in the absence or presence of the pan-caspase inhibitor z-
VAD.fmk. As shown in Fig. 4 (panel A), the inhibition of caspases
b
PBMCresting
PBMCPHA
4.0 ꢀ 1.9
c
0.50 ꢀ 0.27
0.24 ꢀ 0.12
Values are the mean ꢀ SEM for three separate experiments.
a
Compound concentration required to reduce cell growth inhibition by 50%.
PBMC not stimulated with PHA.
b
c
significantly increased cell viability suggesting
a caspase-
PBMC stimulated with PHA.
dependent process of cell death.

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R. Romagnoli et al. / European Journal of Medicinal Chemistry 81 (2014) 394e407
Fig. 1. Effects of compounds 4p (panels B and C) and 4r (panels D and E) on microtubule networks in Hela cells. Panel A, untreated cells. Cells were incubated with 2.5
and D) or 5.0 M (panels C and E) of the compounds for 24 h and then stained with anti- -tubulin primary antibody and secondary Alexa-conjugated antibody and then observed by
confocal microscopy (magnification 20ꢂ). Cells were also counterstained with DAPI to visualize the nuclei.
mM (panels B
m
b
To determine which caspases were involved in phenyl-
cinnamide-induced cell death, the expression of caspases was
evaluated by immunoblot analysis. We observed an activation, in a
time- and concentration-dependent manner, of the effector
caspase-3 and cleavage of its substrate PARP (Fig. 4, panel B).
Interestingly, the two compounds did not induce activation of
caspase-9, the major initiator caspase of the mitochondrial
apoptotic pathway (Fig. 4, panel B). Indeed a clear activation of
caspase-8 was observed for both compounds, suggesting that the
induced apoptosis followed the extrinsic pathway.
3.8. Phenylcinnamides induce downregulation of Bcl-2 family
proteins and DNA damage
To further study the mechanism of apoptosis induction by
phenylcinnamides, we evaluated the expression of proteins of the
Bcl-2 family. As shown in Fig. 5, the anti-apoptotic protein Bcl-2
was decreased by treatment with 4r in a time dependent manner.
Also 4p produced similar effects, but they were more evident after
48 h of treatment. The expression of another anti-apoptotic protein,
Mcl-1, was decreased by both compounds at 2.5 mM, especially at
48 h. In contrast, two pro-apoptotic proteins of the Bcl-2 family, Bak
and Bax, remained practically unchanged after treatment. Of
particular interest was the nearly complete disappearance of Bid
expression after treatment with either compounds an effect that is
clearly evident after only a 24 h treatment at the lowest concen-
tration used. Bid is a pro-apoptotic member of the Bcl-2 family, and
many studies have demonstrated that, following apoptotic stimuli,
activation of caspase-8 induces cleavage of Bid [35,36].
Fig. 2. Percentage of cells in each phase of the cell cycle in HeLa cells treated with the
4r (Panel A) and 4p (Panel B) at the indicated concentrations for 24 h. Cells were fixed
and labeled with PI and analyzed by flow cytometry as described in the experimental
section.
We also investigated whether 4r and 4p induced DNA damage
by examining the expression of phosphorylated histone H2AX at

R. Romagnoli et al. / European Journal of Medicinal Chemistry 81 (2014) 394e407
399
Fig. 3. Flow cytometric analysis of apoptotic cells after treatment of HeLa cells with 4r
(Panel A) or 4p (Panel B) at the indicated concentrations after incubation for 24 or 48 h.
The cells were harvested and labeled with annexin-V-FITC and PI and analyzed by flow
cytometry. Data are represented as means of three independent experiments. For the
sake of clarity, error bars were omitted.
Fig. 4. (A). Percentage of cell viability after a 48 h incubation of HeLa cells with 4r or
4p (2.5 M) in the presence or in the absence of z-VAD.fmk (100
three independent experiments. **P < 0.01 vs. 4p or 4r treated cells. (B). Western blot
analysis of caspase-3, cleaved caspase-9, caspase-8 and PARP after treatment of HeLa
cells with 4r or 4p at the indicated concentrations and for the indicated times. To
m
m
M). Means ꢀ SEM of
confirm equal protein loading, each membrane was stripped and reprobed with anti-b-
actin antibody.
Ser139 (gH2A.X).
gH2A.X phosphorylation occurs shortly after DNA
double strand break (DSB) induction, thus identifying
early sensitive indicator of DSBs, whether induced by ionizing ra-
diation, oxidative stress or chemical agents [37]. As shown in Fig. 5
g
H2A.X as an
which the mitochondrial potential was monitored by flow cytom-
etry with the fluorescent probe 5,50,6,60-tetrachlo-1,10,3,30-tetrae-
thylbenzimidazol-carbocyanine (JC-1) (Fig. 6, panel B). We found
significant mitochondrial depolarization beginning after a 12 h
treatment with 4r. Since the mitochondrial membrane depolari-
zation has been associated with mitochondrial production of ROS
[41,42], these findings suggest that ROS, detected by H2DCFDA prior
to the onset of apoptosis, were not produced as a consequence of
mitochondrial damage.
More importantly, to prove that ROS are involved in the mech-
anism of cell death of compounds 4p and 4r we analyzed cell
viability in the presence of different antioxidants, including
tocopherol acetate (TOC), 2,6-di-tert-butylhydroxyanisole (BHA),
N-acetylcysteine (NAC), reduced glutathione (GSH) and dithio-
threitol (DTT). Surprisingly, as shown in Fig. 7 (panels A and B), only
the thiol-containing scavengers (NAC, GSH and DTT) significantly
increased cell viability. This suggests that ROS do not play a major
role in the antiproliferative effects observed with 4p and 4r.
(panel B), after a 24 h treatment 4r induced gH2A.X expression,
while 4p induced this effect only after 48 h. These findings show
that both compounds induce DNA damage. Bid is also involved in
promoting apoptosis following DNA damage and contributes to
induction of a cell cycle arrest [38]. Thus, our findings that phe-
nylcinnamides induce DNA damage along with strong activation of
Bid could explain the arrest of the cell cycle observed after treat-
ment of cells with these compounds.
3.9. Phenylcinnamides induce ROS production
To better understand the mechanism of action of 4r, we
analyzed ROS production. HeLa cells were treated for different
times and with different concentrations of 4r, and the levels of
intracellular ROS were monitored using two fluorescent probes,
20,70-dichlorodihydrofluorescein diacetate (H2DCFDA) and hydro-
ethidine (HE) [39]. As shown in Fig. 6 (panel A), flow cytometric
analysis showed an early but modest increase (1e6 h) in the
H2DCFDA-positive cells, and this occurred in a concentration-
dependent manner. Using HE, which mainly detects superoxide
anion [40], we did not detect any increase in fluorescence emission
(data not shown), suggesting that superoxide anion does not play a
major role. In addition, a time-course study was performed, in
3.10. Phenylcinnamides induce GSH depletion
Since only thiol containing scavengers protect from cellular
death induced by phenylcinnamides, we determined whether
these compounds caused a decrease in intracellular GSH content.
Therefore, we analyzed HeLa cells for changes in their GSH levels

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R. Romagnoli et al. / European Journal of Medicinal Chemistry 81 (2014) 394e407
Fig. 5. Western blot analysis of Bcl-2, Bax, Bak, Mcl-1and Bid (Panel A), and gH2A.X
(panel B) after treatment of Hela cells with 4p and 4r at the indicated concentrations
and for the indicated times. To confirm equal protein loading, each membrane was
stripped and reprobed with anti-b-actin antibody.
Fig. 6. Production of ROS (Panel A) and assessment of mitochondrial membrane po-
tential (Djmt) (Panel B) following treatment of HeLa cells with compound 4r at the
indicated concentrations. A. Cells were stained with H2DCFDA at the indicated times
and analyzed by flow cytometry to measure production of ROS. B. The change in Djmt
was measured by flow cytometry using JC-1 at the indicated times.
using the fluorescent probe 5-chloromethylfluorescein diacetate
(CMFDA) and flow cytometry. CMFDA is a membrane permeable
dye used for determining intracellular levels of GSH [43]. As shown
in Fig. 8 (Panels A and B), 24 h incubations with either 4p or 4r,
concentration-dependent manners, substantially reduced CMFDA
fluorescence, indicating GSH depletion. Moreover, the effect was
significantly counteracted by addition of NAC to the culture me-
dium GSH is the most abundant thiol in mammalian cells and is
involved in many cellular processes, including antioxidant defense,
drug detoxification, cell signaling, and cell proliferation [43e45].
Intracellular GSH loss is an early feature in the progression of cell
death in response to different apoptotic stimuli and, because of its
action as a primary intracellular antioxidant in the cells, a reduction
in intracellular GSH content is generally believed to reflect gener-
ation of ROS [46]. Nevertheless, our findings suggest that phenyl-
cinnamides induce apoptosis in HeLa cells by directly interacting
with GSH or other intracellular thiols independently of ROS gen-
eration. In this context, we speculate that a possible mechanism of
displayed high antiproliferative activity towards a panel of seven
cancer cell lines, with one-digit micromolar to double-digit nano-
molar IC50 values. The new derivatives had antiproliferative activity
that was significantly greater than that of the two parent com-
pounds 1a and 1b against six of the seven cancer cell lines. The least
active compounds were derivatives with fluorine (4c), tri-
fluoromethoxy (4k) and trifluoromethylsulfanyl (4l) at the para-
position of the phenyl ring. A positive effect was observed by the
replacement of phenyl with 1-naphthyl (compounds 4a and 4b,
respectively), with 4b having IC50 values of 0.68e6.5
mM as
compared with 0.19e27.3 M for 4a. Comparing the activity of
m
hybrid compound 4o with that of the corresponding amino phe-
nylcinnamide 7o, the latter showed weak or no antiproliferative
activity (IC50 ¼ 2.81e63.1
and the insertion of the
molecular change, leading to a significant increase in potency
(IC50 ¼ 0.018e2.3 M for 4o).
mM) against the panel of cancer cell lines,
action of phenylcinnamides results from the reactivity of the
a-
a
-bromoacryloyl moiety was an important
bromoacryoyl moiety and its forming an irreversible adduct with
GSH through a Michael-type nucleophilic attack. This would readily
lead to depletion of intracellular thiols [47].
m
Preliminary mechanism of action studies demonstrated that the
most potent hybrid compounds did not inhibit tubulin polymeri-
zation, but their activity seems to be related to depletion of intra-
cellular GSH. Studies are underway to determine the precise
molecular mechanism that leads to the decrease in intracellular
GSH levels. Whether this occur through a direct chemical reaction
or through interfering with the enzymatic synthesis of GSH re-
mains to be determined. Understanding the precise mechanism
may result in development of new cancer therapeutic strategies
4. Conclusions
The observation that the
phenylcinnamide and the -bromoacryloyl group are capable of
undergoing Michael addition and thus can act as trapping agents of
cellular nucleophiles led us to prepare and evaluate a series of
bromoacryloylamido arylcinnamide derivatives with general
structure 4. These compounds incorporate the two moieties within
their structures. We found that most of the new hybrid compounds
a,b-unsaturated carbonyl system of
a
a
-
and possibly new drugs using phenylcinnamides as
chemotype.
a lead

R. Romagnoli et al. / European Journal of Medicinal Chemistry 81 (2014) 394e407
401
5. Experimental protocols
5.1. Chemistry
5.1.1. Materials and methods
1H and 13C NMR spectra were recorded on a Bruker AC 200 and
Varian 400 Mercury Plus spectrometer, respectively. Chemical
shifts (d) are given in ppm upfield from tetramethylsilane as in-
ternal standard, and the spectra were recorded in appropriate
deuterated solvents, as indicated. Positive-ion electrospray ioniza-
tion (ESI) mass spectra were recorded on a double-focusing Fin-
nigan MAT 95 instrument with BE geometry. Melting points (mp)
were determined on a Buchi-Tottoli apparatus and are uncorrected.
All products reported showed 1H and 13C NMR spectra in agree-
ment with the assigned structures. The purity of tested compounds
was determined by combustion elemental analyses conducted by
the Microanalytical Laboratory of the Chemistry Department of the
University of Ferrara with a Yanagimoto MT-5 CHN recorder
elemental analyzer. All tested compounds yielded data consistent
with a purity of at least 95% as compared with the theoretical
values. All reactions were carried out under an inert atmosphere of
dry N2, unless otherwise indicated. Standard syringe techniques
were used for transferring dry solvents. Reaction courses and
product mixtures were routinely monitored by TLC on silica gel
(precoated F254 Merck plates), and compounds were visualized
with aqueous KMnO4. Flash chromatography was performed using
230e400 mesh silica gel and the indicated solvent system. Organic
solutions were dried over anhydrous Na2SO4.
Fig. 7. Effect of ROS scavengers on cell death induced by compounds 4r and 4p. HeLa
cells were treated with the two compounds at 2.5 M for 48 h in the presence of BHA
(10 M) TOC (100 M) NAC (100 M), GSH (1 mM) or DTT (1 mM). Cell viability was
m
5.1.2. General procedure A for the synthesis of compounds (6aer)
To a solution of nitrocinnamic acid 5a or 5b (386 mg, 2.00 mmol)
in dry chloroform (10 mL) containing triethylamine (0.20 mL,
145 mg, 1.43 mmol) and the appropriate aniline or p-tolylme-
thanamine (2.40 mmol, 1.2 equiv.) was added dropwise POCl3
(0.37 mL, 613 mg, 4.00 mmol, 2 equiv.) at 0 ꢃC with stirring. After
10 min, triethylamine (0.21 mL, 145 mg, 1.43 mol) was slowly
added, and the reaction mixture was stirred for another 30 min at
0 ꢃC. After this time, crushed ice was added to the reaction, and the
product was extracted with CH2Cl2 (3 ꢂ 15 mL). The combined
organic phases were washed successively with 5% HCl (10 mL),
saturated aqueous NaHCO3 (10 mL), water (10 mL), and brine
(10 mL), dried over Na2SO4 and concentrated under reduced pres-
sure. The residue was purified by flash column chromatography on
silica gel or by crystallization.
m
m
m
measured by the MTT assay. Data are represented as means ꢀ S.E.M. for four inde-
pendent experiments. *p < 0.01 vs compound alone.
5.1.2.1. (E)-3-(3-nitrophenyl)-N-phenylacrylamide
(6a).
Following general procedure A, the residue purified by crystalli-
zation from ethyl ether yielded 6a as a yellow solid. Yield 52%, mp
203e205 ꢃC. 1H NMR (200 MHz, d6-DMSO)
d: 7.08 (m, 2H), 7.35 (t,
J ¼ 7.2 Hz, 2H), 7.75 (m, 4H), 8.06 (d, J ¼ 8.0 Hz, 1H), 8.23 (dd, J ¼ 8.0
and 1.6 Hz, 1H), 8.47 (d, J ¼ 1.6 Hz, 1H), 10.3 (s, 1H). MS (ESI):
[Mþ1]þ ¼ 269.2.
5.1.2.2. (E)-N-(naphthalen-1-yl)-3-(3-nitrophenyl)acrylamide (6b).
Following general procedure A, the residue purified by crystalli-
zation from ethyl ether yielded 6b as a yellow solid. Yield 72%, mp
193e195 ꢃC. 1H NMR (200 MHz, d6-DMSO)
d
: 7.06 (d, J ¼ 15.6 Hz,
1H), 7.46 (m, 2H), 7.70 (m, 2H), 7.84 (m, 4H), 8.09 (d, J ¼ 7.8 Hz, 1H),
8.23 (dd, J ¼ 7.8 and 1.6 Hz, 1H), 8.43 (d, J ¼ 1.6 Hz, 1H), 8.49 (d,
J ¼ 15.6 Hz, 1H), 10.5 (s, 1H). MS (ESI): [Mþ1]þ ¼ 319.2.
Fig. 8. Compounds 4p (panel A) and 4r (panel B) induced GSH depletion in HeLa cells.
Cells were treated with the indicated concentration of 4p or 4r and 100 mM NAC, as
indicated. After 24 h incubations, the cells were stained with the fluorescent probe
CMFDA and analyzed by flow cytometry. Data represent means ꢀ S.E.M. of three in-
dependent experiments. *p < 0.01 vs compound alone.
5.1.2.3. (E)-N-(4-fluorophenyl)-3-(3-nitrophenyl)acrylamide
(6c).
Following general procedure A, the residue purified by crystalli-
zation from CH2Cl2 yielded 6c as a pink solid. Yield 48%, mp 213e
215 ꢃC. 1H NMR (200 MHz, d6-DMSO)
d: 6.96 (d, J ¼ 15.8 Hz, 1H),

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R. Romagnoli et al. / European Journal of Medicinal Chemistry 81 (2014) 394e407
7.24 (m, 2H), 7.74 (m, 4H), 8.06 (d, J ¼ 7.8 Hz, 1H), 8.22 (dd, J ¼ 7.8
and 2.0 Hz, 1H), 8.48 (d, J ¼ 2.0 Hz, 1H), 10.4 (s, 1H). MS (ESI):
[Mþ1]þ ¼ 287.2.
crystallization from ethyl ether yielded 6k as a white solid. Yield
83%, mp 208e210 ꢃC. 1H NMR (200 MHz, d6-DMSO)
: 7.00 (d,
J ¼ 15.8 Hz, 1H), 7.09 (d, J ¼ 8.2 Hz, 2H), 7.24 (d, J ¼ 8.2 Hz, 2H), 7.33
(d, J ¼ 15.6 Hz, 1H), 7.84 (t, J ¼ 8.8 Hz, 1H), 8.07 (d, J ¼ 8.6 Hz, 1H),
8.25 (dd, J ¼ 8.6 and 2.0 Hz, 1H), 8.47 (s, J ¼ 2.0 Hz, 1H), 10.6 (s, 1H).
MS (ESI): [Mþ1]þ ¼ 299.1.
d
5.1.2.4. (E)-N-(3-fluorophenyl)-3-(3-nitrophenyl)acrylamide
(6d).
Following general procedure A, the residue purified by crystalli-
zation from CH2Cl2:acetone 10:1 (v/v) yielded 6d as a white solid.
Yield 56%, mp 193e195 ꢃC. 1H NMR (200 MHz, d6-DMSO)
d
: 6.84 (m,
5.1.2.12. (E)-3-(3-nitrophenyl)-N-(4-(trifluoromethylthio)phenyl)
acrylamide (6l). Following general procedure A, the residue puri-
fied by crystallization from ethyl ether yielded 6l as a white solid.
1H), 6.96 (d, J ¼ 15.8 Hz, 1H), 7.41 (m, 2H), 7.72 (m, 3H), 8.07 (d,
J ¼ 7.8 Hz, 1H), 8.27 (dd, J ¼ 7.8 and 1.6 Hz, 1H), 8.48 (d, J ¼ 1.6 Hz,
1H), 10.5 (s, 1H). MS (ESI): [Mþ1]þ ¼ 287.1.
Yield 68%, mp 204e206 ꢃC. 1H NMR (200 MHz, d6-DMSO)
d: 6.66 (d,
J ¼ 8.6 Hz, 2H), 6.90 (d, J ¼ 16.0 Hz, 1H), 7.34 (d, J ¼ 8.6 Hz, 2H), 7.54
(t, J ¼ 8.0 Hz, 1H), 7.81 (d, J ¼ 16.0 Hz, 1H), 8.08 (d, J ¼ 8.0 Hz, 1H),
8.24 (dd, J ¼ 8.0 and 1.4 Hz, 1H), 8.49 (d, J ¼ 1.4 Hz, 1H), 10.7 (s, 1H).
MS (ESI): [Mþ1]þ ¼ 369.1.
5.1.2.5. (E)-N-(3,4-difluorophenyl)-3-(3-nitrophenyl)acrylamide
(6e). Following general procedure A, the residue purified by crys-
tallization from ethyl ether yielded 6e as a gray solid. Yield 53%, mp
103e105 ꢃC. 1H NMR (200 MHz, d6-DMSO)
d
: 6.94 (d, J ¼ 15.8 Hz,
1H), 7.39 (m, 2H), 7.71 (d, J ¼ 7.8 Hz, 1H), 7.79 (d, J ¼ 15.8 Hz, 1H),
7.94 (m, 1H), 8.07 (d, J ¼ 7.8 Hz, 1H), 8.23 (dd, J ¼ 8.2 and 1.4 Hz, 1H),
8.47 (d, J ¼ 1.4 Hz, 1H), 10.6 (s, 1H). MS (ESI): [Mþ1]þ ¼ 305.2.
5.1.2.13. (E)-N-(3,4-dimethoxyphenyl)-3-(3-nitrophenyl)acrylamide
(6m). Following general procedure A, the residue purified by
crystallization from CH2Cl2 yielded 6m as an orange solid. Yield
46%, mp 159e161 ꢃC. 1H NMR (200 MHz, d6-DMSO)
d: 3.63 (s, 3H),
5.1.2.6. (E)-N-(4-chlorophenyl)-3-(3-nitrophenyl)acrylamide
(6f).
3.76 (s, 3H), 6.91 (d, J ¼ 8.8 Hz,1H), 6.95 (d, J ¼ 15.6 Hz,1H), 7.24 (dd,
J ¼ 8.6 and 2.2 Hz, 1H), 7.42 (d, J ¼ 2.2 Hz, 1H), 7.74 (m, 2H), 8.04 (d,
J ¼ 8.2 Hz, 1H), 8.22 (dd, J ¼ 8.2 and 2.0 Hz, 1H), 8.46 (d, J ¼ 2.0 Hz,
1H), 10.2 (s, 1H). MS (ESI): [Mþ1]þ ¼ 329.2.
Following general procedure A, the residue purified by crystalli-
zation from a mixture of ethyl ether:acetone 10:2 (v/v) yielded 6f as
a white solid. Yield 61%, mp 232e234 ꢃC. 1H NMR (200 MHz, d6-
DMSO)
d
: 7.01 (d, J ¼ 15.6 Hz, 1H), 7.38 (d, J ¼ 8.8 Hz, 2H), 7.75 (m,
4H), 8.06 (d, J ¼ 7.8 Hz, 1H), 8.24 (dd, J ¼ 7.8 and 1.4 Hz, 1H), 8.47 (d,
5.1.2.14. (E)-N-(3,5-dimethoxyphenyl)-3-(3-nitrophenyl)acrylamide
(6n). Following general procedure A, the crude residue purified by
flash chromatography, using ethyl acetate:petroleum ether 1:1
(v:v) for elution, furnished 6n as a yellow solid. Yield 54%, mp 172e
J ¼ 1.4 Hz, 1H), 10.6 (s, 1H). MS (ESI): [Mþ1]þ ¼ 303.1.
5.1.2.7. (E)-3-(3-nitrophenyl)-N-p-tolylacrylamide
(6g).
Following general procedure A, the residue purified by crystalli-
zation from acetone yielded 6g as a white solid. Yield 56%, mp 200e
174 ꢃC. 1H NMR (200 MHz, d6-DMSO)
d: 3.82 (s, 6H), 6.24 (s, 2H),
6.97 (s, 1H), 7.01 (d, J ¼ 15.8 Hz, 1H), 7.66 (d, J ¼ 15.8 Hz, 1H), 7.74 (t,
J ¼ 8.2 Hz, 1H), 8.04 (d, J ¼ 8.2 Hz, 1H), 8.27 (dd, J ¼ 8.2 and 2.0 Hz,
1H), 8.45 (d, J ¼ 2.0 Hz, 1H), 10.4 (s, 1H). MS (ESI): [Mþ1]þ ¼ 329.2.
202 ꢃC. 1H NMR (200 MHz, d6-DMSO)
d: 2.27 (s, 3H), 6.97 (d,
J ¼ 15.6 Hz, 1H), 7.13 (d, J ¼ 8.4 Hz, 2H), 7.57 (d, J ¼ 8.4 Hz, 2H), 7.66
(d, J ¼ 15.6 Hz, 1H), 7.74 (m, 1H), 8.05 (d, J ¼ 8.2 Hz, 1H), 8.22 (dd,
J ¼ 8.2 and 2.2 Hz, 1H), 8.46 (d, J ¼ 2.2 Hz, 1H), 10.2 (s, 1H). MS (ESI):
[Mþ1]þ ¼ 283.2.
5.1.2.15. (E)-N-(3,4,5-trimethoxyphenyl)-3-(3-nitrophenyl)acryl-
amide (6o). Following general procedure A, the crude residue pu-
rified by flash chromatography, using EtOAc:petroleum ether 8:2
(v:v) for elution, furnished 6o as a yellow solid. Yield 54%, mp 190e
5.1.2.8. (2E)-N-(4-methylbenzyl)-3-(3-nitrophenyl)acrylamide (6h).
Following general procedure A, the residue purified by crystalli-
zation from acetone yielded 6h as a white solid. Yield 56%, mp 131e
192 ꢃC. 1H NMR (200 MHz, d6-DMSO)
d: 3.63 (s, 3H), 3.77 (s, 6H),
6.94 (d, J ¼ 15.8 Hz, 1H), 7.09 (s, 2H), 7.64 (d, J ¼ 15.8 Hz, 1H), 7.78 (t,
J ¼ 7.8 Hz, 1H), 8.04 (d, J ¼ 7.8 Hz, 1H), 8.23 (dd, J ¼ 7.8 and 2.0 Hz,
1H), 8.44 (d, J ¼ 2.0 Hz, 1H), 10.2 (s, 1H). MS (ESI): [Mþ1]þ ¼ 359.2.
133 ꢃC. 1H NMR (200 MHz, d6-DMSO)
d: 2.31 (s, 3H), 4.35 (d,
J ¼ 5.8 Hz, 2H), 6.84 (d, J ¼ 15.8 Hz, 1H), 7.13 (d, J ¼ 8.4 Hz, 2H), 7.18
(d, J ¼ 8.4 Hz, 2H), 7.57 (d, J ¼ 15.7 Hz, 1H), 7.71 (t, J ¼ 8.0 Hz, 1H),
8.00 (d, J ¼ 8.0 Hz, 1H), 8.20 (dd, J ¼ 8.0 and 2.0 Hz, 1H), 8.40 (d,
J ¼ 2.0 Hz, 1H), 8.62 (t, J ¼ 5.8 Hz, 1H). MS (ESI): [Mþ1]þ ¼ 297.2.
5.1.2.16. (E)-N-(4-morpholinophenyl)-3-(3-nitrophenyl)acrylamide
(6p). Following general procedure A, the crude residue purified by
flash chromatography, using EtOAc for elution, furnished 6p as a
yellow solid. Yield 62%, mp 210e212 ꢃC. 1H NMR (200 MHz, d6-
5.1.2.9. (E)-N-(4-methoxyphenyl)-3-(3-nitrophenyl)acrylamide (6i).
Following general procedure A, the residue purified by crystalli-
zation from CH2Cl2 yielded 6i as a yellow solid. Yield 58%, mp 195e
DMSO)
d
: 3.09 (t, J ¼ 4.6 Hz, 4H), 3.75 (t, J ¼ 4.6 Hz, 4H), 6.99 (m,
2H), 7.64 (m, 3H), 7.77 (m, 2H), 8.04 (d, J ¼ 7.8 Hz, 1H), 8.25 (dd,
J ¼ 7.8 and 1.4 Hz, 1H), 8.46 (d, J ¼ 1.4 Hz, 1H), 10.1 (s, 1H). MS (ESI):
[Mþ1]þ ¼ 354.2.
197 ꢃC. 1H NMR (200 MHz, d6-DMSO)
d: 3.74 (s, 3H), 6.90 (d,
J ¼ 15.8 Hz, 1H), 6.94 (d, J ¼ 9.0 Hz, 2H), 7.60 (d, J ¼ 9.0 Hz, 2H), 7.74
(m, 2H), 8.08 (d, J ¼ 7.8 Hz,1H), 8.22 (dd, J ¼ 7.8 and 2.0 Hz,1H), 8.46
(d, J ¼ 2.0 Hz, 1H), 10.2 (s, 1H). MS (ESI): [Mþ1]þ ¼ 299.2.
5.1.2.17. (E)-N-(4-methoxyphenyl)-3-(4-nitrophenyl)acrylamide
(6q). Following general procedure A, the residue purified by crys-
tallization from petroleum ether yielded 6q as a yellow solid. Yield
5.1.2.10. (E)-N-(4-ethoxyphenyl)-3-(3-nitrophenyl)acrylamide (6j).
Following general procedure A, the crude residue purified by flash
chromatography, using ethyl acetate:petroleum ether 1:1 (v:v) for
elution, furnished 6j as a yellow solid. Yield 56%, mp 180e182 ꢃC. 1H
74%, mp 113e115 ꢃC. 1H NMR (200 MHz, d6-DMSO)
d: 3.81 (s, 3H),
6.94 (d, J ¼ 15.8 Hz, 1H), 7.09 (d, J ¼ 9.0 Hz, 2H), 7.34 (d, J ¼ 9.0 Hz,
2H), 7.64 (d, J ¼ 15.8 Hz, 1H), 7.90 (d, J ¼ 8.8 Hz, 2H), 8.27 (d,
J ¼ 8.6 Hz, 2H), 10.6 (s, 1H). MS (ESI): [Mþ1]þ ¼ 299.1.
NMR (200 MHz, d6-DMSO)
d
: 1.32 (t, J ¼ 6.8 Hz, 3H), 4.02 (q,
J ¼ 6.8 Hz, 2H), 6.93 (m, 3H), 7.64 (m, 3H), 7.74 (m, 1H), 8.05 (d,
J ¼ 7.8 Hz, 1H), 8.21 (dd, J ¼ 7.8 and 2.2 Hz, 1H), 8.46 (d, J ¼ 2.2 Hz,
1H), 10.2 (s, 1H). MS (ESI): [Mþ1]þ ¼ 283.2.
5.1.2.18. (E)-N-(3,4,5-trimethoxyphenyl)-3-(4-nitrophenyl)acryl-
amide (6r). Following general procedure A, the residue purified by
crystallization from ethyl ether yielded 6r as a yellow solid. Yield
5.1.2.11. (E)-N-(4-trifluoromethoxyphenyl)-3-(4-nitrophenyl)acryl-
amide (6k). Following general procedure A, the residue purified by
67%, mp 196e198 ꢃC. 1H NMR (200 MHz, d6-DMSO)
d
: 3.63 (s, 3H),
3.77 (s, 6H), 6.94 (d, J ¼ 15.8 Hz, 1H), 7.10 (s, 2H), 7.63 (d, J ¼ 15.8 Hz,

R. Romagnoli et al. / European Journal of Medicinal Chemistry 81 (2014) 394e407
403
1H), 7.86 (d, J ¼ 8.8 Hz, 2H), 8.28 (d, J ¼ 8.8 Hz, 2H), 10.3 (s, 1H). MS
(ESI): [Mþ1]þ ¼ 359.1.
5.1.3.7. (E)-3-(3-aminophenyl)-N-p-tolylacrylamide
(7g).
Following general procedure B, the crude residue purified by flash
chromatography, using EtOAc:petroleum ether 3:7 (v:v) for elution,
furnished 7g as a yellow solid. Yield, 74%, mp 181e183 ꢃC. 1H NMR
5.1.3. General procedure B for the synthesis of compounds (7aer)
(200 MHz, d6-DMSO) d: 2.26 (s, 3H), 5.22 (bs, 2H), 6.64 (m, 2H), 6.71
To
a suspension of nitroarylcinnamide derivatives 6aer
(d, J ¼ 8.8 Hz, 2H), 7.11 (m, 3H), 7.35 (d, J ¼ 15.6 Hz, 1H), 7.56 (d,
(1.00 mmol) in absolute ethanol (10 mL) was added SnCl2.2H2O
(1.13 g, 5.00 mmol), and the stirring mixture was refluxed for 1.5 h.
After this time, the reaction was cooled to room temperature and
treated with cold water (15 mL), and the suspension was carefully
adjusted to pH 8 with NaHCO3. The mixture was extracted with
EtOAc (3 ꢂ 15 mL), the combined organic phase was washed with
water (10 mL) and brine (10 mL), dried over anhydrous Na2SO4 and
concentrated under reduced pressure. The crude residue was sub-
jected to column chromatography on silica gel or purified by
crystallization with ethyl ether.
J ¼ 8.8 Hz, 2H), 10.1 (s, 1H). MS (ESI): [Mþ1]þ ¼ 253.2.
5.1.3.8. (2E)-3-(3-aminophenyl)-N-(4-methylbenzyl)acrylamide
(7h). Following general procedure B, the crude residue purified by
flash chromatography, using EtOAc:petroleum ether 6:4 (v:v) for
elution, furnished 4h as a yellow solid. Yield 55%; mp 83e85 ꢃC. 1H
NMR (200 MHz, d6-DMSO)
d
: 2.30 (s, 3H), 4.32 (d, J ¼ 5.8 Hz, 2H),
5.18 (bs, 2H), 6.59 (m, 4H), 7.03 (m, 5H), 7.24 (d, J ¼ 15.8 Hz, 1H),
8.54 (t, J ¼ 5.8 Hz, 1H). MS (ESI): [Mþ1]þ ¼ 267.2.
5.1.3.9. (E)-3-(3-aminophenyl)-N-(4-methoxyphenyl)acrylamide
(7i). Following general procedure B, the crude residue purified by
flash chromatography, using EtOAc:petroleum ether 6:4 (v:v) for
elution, furnished 7i as a yellow solid. Yield 93%, mp 173e175 ꢃC. 1H
5.1.3.1. (E)-3-(3-aminophenyl)-N-phenylacrylamide
(7a).
Following general procedure B, the crude residue purified by flash
chromatography, using EtOAc:petroleum ether 1:1 (v:v) for elution,
furnished 7a as a yellow solid. Yield 76%, mp 55e57 ꢃC. 1H NMR
NMR (200 MHz, d6-DMSO) d: 3.73 (s, 3H), 5.22 (bs, 2H), 6.62 (m,
(200 MHz, d6-DMSO)
d: 5.23 (bs, 2H), 6.64 (m, 2H), 6.74 (d,
2H), 6.76 (m, 2H), 6.88 (d, J ¼ 9.0 Hz, 2H), 7.07 (t, J ¼ 7.8 Hz,1H), 7.34
(d, J ¼ 15.6 Hz, 1H), 7.58 (d, J ¼ 9.0 Hz, 2H), 10.0 (s, 1H). MS (ESI):
[Mþ1]þ ¼ 329.1.
J ¼ 6.6 Hz, 2H), 7.08 (m, 2H), 7.33 (m, 3H), 7.71 (d, J ¼ 8.6 Hz, 2H),
10.2 (s, 1H). MS (ESI): [Mþ1]þ ¼ 239.1.
5.1.3.2. (E)-3-(3-aminophenyl)-N-(naphthalen-1-yl)acrylamide (7b).
Following general procedure B, the crude residue purified by flash
chromatography, using EtOAc:petroleum ether 1:1 (v:v) for elution,
furnished 7b as a yellow solid. Yield 72%, mp 132e134 ꢃC. 1H NMR
5.1.3.10. (E)-3-(4-aminophenyl)-N-(4-ethoxyphenyl)acrylamide (7j).
Following general procedure B, the crude residue purified by flash
chromatography, using EtOAc:petroleum ether 1:1 (v:v) for elution,
furnished 7j as a yellow solid. Yield 85%, mp 125e127 ꢃC. 1H NMR
(200 MHz, d6-DMSO)
d
: 5.48 (bs, 2H), 6.66 (d, J ¼ 15.6 Hz, 1H), 6.89
(200 MHz, CDCl3)
d
: 1.31 (t, J ¼ 6.8 Hz, 3H), 4.00 (q, J ¼ 6.8 Hz, 2H),
(m, 2H), 7.10 (m, 2H), 7.49 (m, 4H), 7.69 (d, J ¼ 15.6 Hz, 1H), 7.82 (m,
5.22 (bs, 2H), 6.58 (m, 1H), 6.69 (m, 3H), 6.87 (d, J ¼ 9.2 Hz, 2H), 7.03
(t, J ¼ 7.8 Hz, 1H), 7.41 (d, J ¼ 15.6 Hz, 1H), 7.57 (d, J ¼ 9.2 Hz, 2H),
10.0 (s, 1H). MS (ESI): [Mþ1]þ ¼ 269.2.
2H), 8.42 (d, J ¼ 1.6 Hz, 1H), 10.4 (s, 1H). MS (ESI): [Mþ1]þ ¼ 319.2.
5.1.3.3. (E)-3-(3-aminophenyl)-N-(4-fluorophenyl)acrylamide (7c).
Following general procedure B, the crude residue purified by flash
chromatography, using EtOAc:petroleum ether 1:1 (v:v) for elution,
furnished 7c as a white solid. Yield 52%, mp 174e176 ꢃC. 1H NMR
5.1.3.11. (E)-3-(3-aminophenyl)-N-(4-trifluoromethoxyphenyl)acryl-
amide (7k). Following general procedure B, the crude residue pu-
rified by flash chromatography, using EtOAc for elution, furnished
7k as a yellow solid. Yield 83%, mp > 300 ꢃC. 1H NMR (200 MHz, d6-
(200 MHz, d6-DMSO) d: 5.23 (bs, 2H), 6.62 (m, 1H), 6.73 (m, 3H),
7.04 (d, J ¼ 8.0 Hz,1H), 7.17 (m, 2H), 7.37 (d, J ¼ 15.6 Hz, 1H), 7.72 (m,
DMSO)
d
: 5.24 (bs, 2H), 6.64 (m, 2H), 6.72 (m, 2H), 7.20 (t, J ¼ 7.8 Hz,
2H), 10.2 (s, 1H). MS (ESI): [Mþ1]þ ¼ 257.2.
1H), 7.32 (d, J ¼ 8.2 Hz, 2H), 7.39 (d, J ¼ 15.6 Hz, 1H), 7.82 (d,
J ¼ 9.0 Hz, 2H), 10.4 (s, 1H). MS (ESI): [Mþ1]þ ¼ 323.1.
5.1.3.4. (E)-3-(3-aminophenyl)-N-(3-fluorophenyl)acrylamide (7d).
Following general procedure B, the crude residue purified by flash
chromatography, using EtOAc:petroleum ether 1:1 (v:v) for elution,
furnished 7d as a white solid. Yield 61%, mp 90e92 ꢃC. 1H NMR
5.1.3.12. (E)-3-(3-aminophenyl)-N-(4-(trifluoromethylthio)phenyl)
acrylamide (7l). Following general procedure B, the crude residue
purified by flash chromatography, using EtOAc for elution, fur-
nished 7l as a yellow solid. Yield 55%, mp 173e175 ꢃC. 1H NMR
(200 MHz, d6-DMSO)
d
: 5.34 (bs, 2H), 6.26 (d, J ¼ 15.6 Hz, 1H), 6.71
(m, 1H), 6.81 (m, 2H), 6.89 (m, 1H), 7.06 (m, 1H), 7.34 (m, 2H), 7.39
(d, J ¼ 15.6 Hz, 1H), 7.75 (dd, J ¼ 10.6 and 1.2 Hz, 1H), 10.4 (s, 1H). MS
(ESI): [Mþ1]þ ¼ 257.2.
(200 MHz, d6-DMSO) d: 5.42 (bs, 2H), 6.66 (m, 2H), 6.78 (m, 2H),
7.09 (t, J ¼ 8.0 Hz, 1H), 7.42 (d, J ¼ 15.6 Hz, 1H), 7.66 (d, J ¼ 8.8 Hz,
2H), 7.84 (d, J ¼ 8.4 Hz, 2H), 10.5 (s, 1H). MS (ESI): [Mþ1]þ ¼ 339.2.
5.1.3.5. (E)-3-(3-aminophenyl)-N-(3,4-difluorophenyl)acrylamide
(7e). Following general procedure B, the crude residue purified by
flash chromatography, using EtOAc:petroleum ether 1:1 (v:v) for
elution, furnished 7e as a yellow solid. Yield 78%, mp 126e128 ꢃC.
5.1.3.13. (E)-3-(3-aminophenyl)-N-(3,4-dimethoxyphenyl)acryl-
amide (7m). Following general procedure B, the crude residue
purified by flash chromatography, using EtOAc:petroleum ether 7:3
(v:v) for elution, furnished 7m as a yellow solid. Yield>95%, mp
1H NMR (200 MHz, d6-DMSO)
d: 5.23 (bs, 2H), 6.67 (m, 3H), 6.74 (m,
81e83 ꢃC. 1H NMR (200 MHz, d6-DMSO)
d: 3.72 (s, 3H), 3.74 (s, 3H),
1H), 7.04 (t, J ¼ 8.0 Hz, 1H), 7.40 (m, 2H), 7.93 (m, 2H), 10.4 (s, 1H).
5.43 (bs, 2H), 6.62 (m, 2H), 6.77 (m, 2H), 6.88 (d, J ¼ 8.8 Hz,1H), 7.09
(t, J ¼ 8.2 Hz, 1H), 7.17 (dd, J ¼ 8.2 and 2.0 Hz, 1H), 7.42 (m, 2H), 10.0
(s, 1H). MS (ESI): [Mþ1]þ ¼ 299.1.
MS (ESI): [Mþ1]þ ¼ 275.2.
5.1.3.6. (E)-3-(3-aminophenyl)-N-(4-chlorophenyl)acrylamide (7f).
Following general procedure B, the crude residue purified by
crystallization from Et2O furnished 7f as an orange solid. Yield 70%,
5.1.3.14. (E)-3-(3-aminophenyl)-N-(3,5-dimethoxyphenyl)acryl-
amide (7n). Following general procedure B, the crude residue pu-
rified by crystallization from Et2O furnished 7n as a yellow solid.
mp 170e172 ꢃC. 1H NMR (200 MHz, d6-DMSO)
d: 5.88 (bs, 2H), 6.73
(m, 3H), 6.86 (m, 1H), 7.13 (t, J ¼ 8.0 Hz, 1H), 7.48 (m, 3H), 7.74 (d,
Yield >95%, mp 55e57 ꢃC. 1H NMR (200 MHz, d6-DMSO)
d: 3.86 (s,
J ¼ 9.0 Hz, 2H), 10.3 (s, 1H). MS (ESI): [Mþ1]þ ¼ 273.1.
6H), 5.12 (bs, 2H), 6.63 (m, 2H), 6.70 (m, 2H), 6.95 (m, 3H), 7.12 (t,

404
R. Romagnoli et al. / European Journal of Medicinal Chemistry 81 (2014) 394e407
J ¼ 8.2 Hz, 1H), 7.36 (d, J ¼ 15.6 Hz, 1H), 10.1 (s, 1H). MS (ESI):
[Mþ1]þ ¼ 299.2.
procedure C, the crude residue purified by flash chromatography,
using EtOAc:petroleum ether 1:2 (v:v) for elution, furnished 4b as a
yellow solid. Yield 48%, mp 170e172 ꢃC. 1H NMR (400 MHz, d6-
5.1.3.15. (E)-3-(3-aminophenyl)-N-(3,4,5-trimethoxyphenyl)acryl-
amide (7o). Following general procedure B, the crude residue pu-
rified by flash chromatography, using EtOAc for elution, furnished
7o as a yellow solid. Yield 85%, mp 78e80 ꢃC. 1H NMR (200 MHz, d6-
DMSO)
d
: 6.36 (d, J ¼ 3.2 Hz, 1H), 6.79 (d, J ¼ 3.2 Hz, 1H), 6.95 (d,
J ¼ 15.6 Hz, 1H), 7.48 (m, 4H), 7.64 (m, 4H), 7.72 (d, J ¼ 9.2 Hz, 1H),
7.82 (d, J ¼ 15.6 Hz, 1H), 8.11 (s, 1H), 8.44 (d, J ¼ 1.8 Hz, 1H), 10.4 (s,
1H). 10.5 (s, 1H). 13C NMR (100 MHz, d6-DMSO)
d: 115.2, 118.6, 119.8,
DMSO)
d
: 3.63 (s, 3H), 3.75 (s, 6H), 5.25 (bs, 2H), 6.62 (m, 2H), 6.72
121.8, 122.5, 124.3, 124.8, 125.0, 126.4, 126.6, 127.2, 127.4, 128.4,
129.3, 129.7, 133.3, 135.0, 136.7, 138.8, 139.9, 161.1, 163.5. MS (ESI):
[M]þ ¼ 420.2, [Mþ2]þ ¼ 422.5. Anal. calcd for C22H17BrN2O2: C,
62.72; H, 4.07; N, 6.65; found: C, 62.53; H, 3.92; N, 6.48.
(m, 2H), 7.09 (m, 3H), 7.36 (d, J ¼ 15.6 Hz, 1H), 10.1 (s, 1H). MS (ESI):
[Mþ1]þ ¼ 328.6.
5.1.3.16. (E)-3-(3-aminophenyl)-N-(4-morpholinophenyl)acrylamide
(7p). Following general procedure B, the crude residue purified by
flash chromatography, using EtOAc for elution, furnished 7p as a
yellow solid. Yield 75%, mp 239e241 ꢃC. 1H NMR (200 MHz, d6-
5.1.4.3. 2-Bromo-N-(3-{(1E)-3-oxo-3-[(4-fluorophenyl)amino]prop-
1-en-1-yl}phenyl)-acrylamide (4c). Following general procedure C,
the crude residue purified by flash chromatography, using
EtOAc:petroleum ether 4:6 (v:v) for elution, furnished 4c as a yel-
low solid. Yield 66%, mp 194e196 ꢃC. 1H NMR (400 MHz, d6-DMSO)
DMSO)
d: 3.05 (t. J ¼ 5.0 Hz, 4H), 3.74 (t. J ¼ 5.0 Hz, 4H), 5.22 (bs,
2H), 6.62 (d, J ¼ 9.6 Hz, 2H), 6.75 (m, 3H), 6.89 (d, J ¼ 9.6 Hz, 2H),
7.07 (t, J ¼ 9.0 Hz, 1H), 7.32 (d, J ¼ 15.6 Hz, 1H), 7.54 (d, J ¼ 9.0 Hz,
1H), 9.98 (s, 1H). MS (ESI): [Mþ1]þ ¼ 324.2.
d
: 6.34 (d, J ¼ 3.2 Hz,1H), 6.76 (d, J ¼ 15.8 Hz,1H), 6.78 (d, J ¼ 3.2 Hz,
1H), 7.18 (m, 2H), 7.39 (m, 1H), 7.51 (d, J ¼ 15.6, 1H), 7.59 (m, 2H),
7.73 (m, 2H), 8.08 (s, 1H), 10.3 (s, 1H). 10.4 (s, 1H). 13C NMR
5.1.3.17. (E)-3-(4-aminophenyl)-N-(4-methoxyphenyl)acrylamide
(7q). Following general procedure B, the crude residue purified by
flash chromatography, using EtOAc:petroleum ether 1:1 (v:v) for
elution, furnished 7q as a yellow solid. Yield 68%, mp 190e192 ꢃC.
(100 MHz, d6-DMSO) d: 115.2, 115.6, 118.6, 120.8, 120.9, 121.9, 122.4,
124.4, 124.9, 126.1, 129.4, 135.1, 135.6, 138.9, 139.9, 155.7
(J ¼ 249.5 Hz),161.2, 163.3. MS (ESI): [M]þ ¼ 388.5, [Mþ2]þ ¼ 390.6.
Anal. calcd for C18H14BrFN2O2: C, 55.55; H, 3.63; N, 7.20; found: C,
55.38; H, 3.47; N, 7.01.
1H NMR (200 MHz, CDCl3)
d: 3.72 (s, 3H), 5.62 (bs, 2H), 6.39 (d,
J ¼ 15.6 Hz, 1H), 6.55 (d, J ¼ 8.6 Hz, 2H), 6.87 (d, J ¼ 9.0 Hz, 2H), 7.26
(d, J ¼ 8.6 Hz, 2H), 7.33 (d, J ¼ 15.6 Hz, 1H), 7.56 (d, J ¼ 9.0 Hz, 2H),
9.81 (s, 1H). MS (ESI): [Mþ1]þ ¼ 269.2.
5.1.4.4. 2-Bromo-N-(3-{(1E)-3-oxo-3-[(3-fluorophenyl)amino]prop-
1-en-1-yl}phenyl)-acrylamide (4d). Following general procedure C,
the crude residue purified by flash chromatography, using
EtOAc:petroleum ether 4:6 (v:v) for elution, furnished 4d as a
yellow solid. Yield 52%, mp 144e146 ꢃC. 1H NMR (400 MHz, d6-
5.1.3.18. (E)-3-(4-aminophenyl)-N-(3,4,5-trimethoxyphenyl)acryl-
amide (7r). Following general procedure B, the crude residue pu-
rified by flash chromatography, using EtOAc for elution, furnished
7r as a yellow solid. Yield 65%, mp 86e88 ꢃC. 1H NMR (200 MHz,
DMSO)
J ¼ 15.8 Hz, 1H), 6.94 (m, 1H), 7.39 (m, 4H), 7.54 (d, J ¼ 15.6 Hz, 1H),
7.58 (m, 1H), 7.71 (m, 1H), 8.09 (s, 1H), 10.4 (s, 1H). 10.5 (s, 1H). 13
NMR (100 MHz, d6-DMSO) : 106.4, 106.9, 110.3, 110.7, 115.6, 119.3,
122.7, 122.9,125.2,125.6,126.8,130.1,131.0, 131.2, 135.7, 139.6,141.2,
d: 6.34 (d, J ¼ 3.2 Hz, 1H), 6.78 (d, J ¼ 3.2 Hz, 1H), 6.86 (d,
CDCl3)
d
: 3.80 (s, 3H), 3.86 (s, 6H), 5.98 (s, 2H), 6.27 (d, J ¼ 15.2 Hz,
C
1H), 6.64 (d, J ¼ 8.6 Hz, 2H), 6.93 (bs, 2H), 7.33 (d, J ¼ 8.6 Hz, 2H),
d
7.62 (d, J ¼ 15.2 Hz, 1H), 10.3 (s, 1H). MS (ESI): [Mþ1]þ ¼ 329.2.
141.5, 141.8, 160.4 (J
¼
239.5 Hz), 161.9, 164.4. MS (ESI):
5.1.4. General procedure C for the synthesis of compounds (4aer)
To an ice-cooled solution of amino derivative 7aer (1.00 mmol)
in dry DMF (5.0 mL) were added a mixture of EDCI (383 mg,
[M]þ ¼ 388.6, [Mþ2]þ ¼ 390.6. Anal. calcd for C18H14BrFN2O2: C,
55.55; H, 3.63; N, 7.20; found: C, 55.9; H, 3.48; N, 7.05.
2.00 mmol) and
a
-bromoacrylic acid (2.00 mmol, 306 mg). The
5.1.4.5. 2-Bromo-N-(3-{(1E)-3-oxo-3-[(3,4-difluorophenyl)amino]
prop-1-en-1-yl}phenyl)-acrylamide (4e). Following general proce-
dure C, the crude residue purified by flash chromatography, using
EtOAc:petroleum ether 1:1 (v:v) for elution, furnished 4e as a white
reaction mixture was stirred at room temperature for 18 h and then
concentrated under reduced pressure. The residue was dissolved
with a mixture of CH2Cl2 (15 mL) and water (5 mL), and the organic
phase was washed with brine (5 mL), dried over Na2SO4 and
evaporated to dryness in vacuo. The resulting crude residue was
purified by column chromatography on silica gel.
solid. Yield 52%, mp 152e154 ꢃC. 1H NMR (400 MHz, d6-DMSO)
d:
6.34 (d, J ¼ 3.2 Hz, 1H), 6.78 (m, 2H), 7.40 (m, 4H), 7.55 (d, J ¼ 15.6,
1H), 7.60 (m, 1H), 7.92 (m, 1H), 8.10 (s, 1H), 10.4 (s, 1H). 10.5 (s, 1H).
13C NMR (100 MHz, d6-DMSO)
d: 108.0, 108.2, 115.5, 117.5, 117.6,
5.1.4.1. N-{3-[(1E)-3-anilino-3-oxoprop-1-en-1-yl]phenyl}-2-
bromoacrylamide (4a). Following general procedure C, the crude
residue purified by flash chromatography, using EtOAc:petroleum
ether 1:1 (v:v) for elution, furnished 4a as a white solid. Yield 63%,
118.6, 122.0, 124.5, 124.9, 126.1, 129.4, 133.6 (d, J ¼ 28.4 Hz), 134.9,
136.3 (d, J ¼ 28.4 Hz), 138.9, 140.5, 161.2, 163.5. MS (ESI):
[M]þ ¼ 406.4, [Mþ2]þ ¼ 408.6. Anal. calcd for C18H13BrF2N2O2: C,
53.09; H, 3.22; N, 6.88; found: C, 52.88; H, 3.01; N, 6.73.
mp 132e134 ꢃC. 1H NMR (400 MHz, d6-DMSO)
d
: 6.34 (d, J ¼ 3.2 Hz,
1H), 6.78 (d, J ¼ 3.2 Hz, 1H), 6.83 (d, J ¼ 16.0 Hz, 1H), 7.07 (t,
J ¼ 8.4 Hz, 1H), 7.36 (m, 2H), 7.43 (t, J ¼ 8.0 Hz, 2H), 7.53 (d,
J ¼ 16.0 Hz, 1H), 7.60 (d, J ¼ 8.0 Hz, 1H), 7.69 (dd, J ¼ 8.0 and 1.2 Hz,
2H), 8.08 (d, J ¼ 1.2 Hz, 1H), 10.3 (s, 1H). 10.4 (s, 1H). 13C NMR
5.1.4.6. 2-Bromo-N-(3-{(1E)-3-oxo-3-[(4-chlorophenyl)amino]prop-
1-en-1-yl}phenyl)-acrylamide (4f). Following general procedure C,
the crude residue purified by flash chromatography, using ethyl
acetate:petroleum ether 6:4 (v:v) for elution, furnished 4f as a
yellow solid. Yield 51%, mp 214e216 ꢃC. 1H NMR (400 MHz, d6-
(100 MHz, d6-DMSO) d: 118.6, 119.1 (2C), 121.8, 122.6, 123.3, 124.4,
124.9, 126.1, 128.8 (2C), 129.4, 135.1, 138.9, 139.3, 139.8, 161.2, 163.3.
DMSO)
J ¼ 15.6 Hz, 1H), 7.39 (m, 4H), 7.52 (m, 2H), 7.76 (d, J ¼ 9.0 Hz, 2H),
8.12 (s,1H),10.4 (s,1H).10.5 (s,1H). 13C NMR (100 MHz, d6-DMSO)
d
: 6.34 (d, J ¼ 3.2 Hz, 1H), 6.78 (d, J ¼ 3.2 Hz, 1H), 6.80 (d,
MS (ESI): [M]þ
¼
370.5, [Mþ3]þ
¼
372.7. Anal. calcd for
C
18H15BrN2O2: C, 58.24; H, 4.07; N, 7.55; found: C, 58.01; H, 3.88; N,
d:
7.38.
118.6, 120.7 (2C), 121.9, 122.3, 124.5, 124.9, 126.1, 126.9, 128.7 (2C),
129.4, 135.0, 138.2, 138.9, 140.2, 161.2, 163.5. MS (ESI):
[Mþ1]þ ¼ 404.9, [Mþ3]þ ¼ 406.7. Anal. calcd for C18H14BrClN2O2: C,
53.29; H, 3.48; N, 6.91; found: C, 53.02; H, 3.22; N, 6.74.
5.1.4.2. 2-Bromo-N-(3-{(1E)-3-oxo-3-[(naphthalen-1-yl)amino]
prop-1-en-1-yl}phenyl)-acrylamide (4b). Following general

R. Romagnoli et al. / European Journal of Medicinal Chemistry 81 (2014) 394e407
405
5.1.4.7. 2-Bromo-N-(3-{(1E)-3-oxo-3-[(p-tolyl)amino]prop-1-en-1-
yl}phenyl)-acrylamide (4g). Following general procedure C, the
crude residue purified by flash chromatography, using EtOAc:pe-
troleum ether 6:4 (v:v) for elution, furnished 4g as a white solid.
138.5, 138.9, 140.3, 143.6, 161.2, 183.5. MS (ESI): [M]þ ¼ 454.5,
[Mþ2]þ ¼ 456.7. Anal. calcd for C19H14BrF3N2O3: C, 50.13; H, 3.10; N,
6.15; found: C, 49.94; H, 0.97; N, 6.01.
Yield 66%, mp 198e200 ꢃC. 1H NMR (400 MHz, d6-DMSO)
d: 2.27 (s,
5.1.4.12. 2-Bromo-N-(3-{(1E)-3-oxo-3-[(4-(trifluoromethylthio)
3H), 6.34 (d, J ¼ 3.4 Hz, 1H), 6.78 (d, J ¼ 3.4 Hz, 1H), 6.86 (d,
J ¼ 15.8 Hz, 1H), 7.12 (d, J ¼ 8.4 Hz, 2H), 7.43 (m, 3H), 7.56 (s, 1H),
7.61 (d, J ¼ 8.4 Hz, 2H), 8.07 (s, 1H), 10.2 (s, 1H). 10.4 (s, 1H). 13C NMR
phenyl)amino]prop-1-en-1-yl}phenyl)-acrylamide
(4l).
Following general procedure C, the crude residue purified by flash
chromatography, using EtOAc:petroleum ether 6:4 (v:v) for elution,
furnished 4l as a white solid. Yield 58%, mp 125e127 ꢃC. 1H NMR
(100 MHz, d6-DMSO) d: 20.9, 119.0, 119.6 (2C), 122.2, 123.2, 124.8,
125.4, 126.6, 129.6 (2C), 129.9, 132.8, 135.7, 137.2, 139.3, 140.0, 161.6,
(400 MHz, d6-DMSO)
d
: 6.34 (d, J ¼ 3.0 Hz, 1H), 6.78 (d, J ¼ 3.0 Hz,
163.6. MS (ESI): [M]þ ¼ 384.6, [Mþ2]þ ¼ 386.6. Anal. calcd for
1H), 6.88 (d, J ¼ 15.6 Hz, 1H), 7.44 (m, 2H), 7.56 (m, 2H), 7.67 (d,
J ¼ 8.6 Hz, 2H), 7.88 (d, J ¼ 8.6 Hz, 2H), 8.11 (s, 1H), 10.4 (s, 1H). 10.6
C
19H17BrN2O2: C, 59.23; H, 4.45; N, 7.27; found: C, 59.02; H, 4.31; N,
7.14.
(s, 1H). 13C NMR (100 MHz, d6-DMSO)
d: 115.9, 118.5, 119.9 (2C),
121.9, 124.5, 124.8, 126.0, 128.0, 129.3, 134.8, 137.3 (2C), 138.8, 140.7,
142.1, 161.1, 163.7. MS (ESI): [Mþ1]þ ¼ 470.8, [Mþ3]þ ¼ 472.7. Anal.
calcd for C19H14BrF3N2O2S: C, 48.42; H, 2.99; N, 5.94; found: C,
48.21; H, 2.77; N, 5.78.
5.1.4.8. 2-Bromo-N-(3-{(1E)-3-[(4-methylbenzyl)amino]-3-oxoprop-
1-en-1-yl}phenyl)acrylamide (4h). Following general procedure C,
the crude residue purified by flash chromatography, using
EtOAc:petroleum ether 6:4 (v:v) for elution, furnished 4h as a white
solid. Yield 53%, mp 180e182 ꢃC. 1H NMR (400 MHz, d6-DMSO)
d:
5.1.4.13. 2-Bromo-N-(3-{(1E)-3-oxo-3-[(3,4-dimethoxyphenyl)
amino]prop-1-en-1-yl}phenyl)-acrylamide (4m). Following general
procedure C, the crude residue purified by flash chromatography,
using EtOAc:petroleum ether 7:3 (v:v) for elution, furnished 4m as
a yellow solid. Yield 78%, mp 108e110 ꢃC. 1H NMR (400 MHz, d6-
2.28 (s, 3H), 4.37 (d, J ¼ 5.6 Hz, 2H), 6.32 (d, J ¼ 3.2 Hz, 1H), 6.63 (d,
J ¼ 15.8 Hz, 1H), 6.76 (d, J ¼ 3.2 Hz, 1H), 7.17 (m, 4H), 7.46 (m, 3H),
7.58 (d, J ¼ 8.4 Hz, 1H), 7.95 (s, 1H), 8.63 (t, J ¼ 5.6 Hz, 1H), 10.3 (s,
1H). 13C NMR (100 MHz, d6-DMSO)
d: 20.6, 41.9, 118.6, 121.4, 122.4,
123.9, 124.8, 126.0, 127.2 (2C), 128.8 (2C), 129.2, 135.2, 135.8, 136.2,
138.5, 138.7, 161.0, 164.6. MS (ESI): [M]þ ¼ 398.4, [Mþ2]þ ¼ 400.6.
Anal. calcd for C20H19BrN2O2: C, 60.16; H, 4.80; N, 7.02; found: C,
59.92; H, 4.72; N, 6.88.
DMSO)
d
: 3.73 (s, 3H), 3.75 (s, 3H), 6.34 (d, J ¼ 3.2 Hz, 1H), 6.79 (m,
3H), 6.90 (d, J ¼ 8.6 Hz,1H), 7.20 (dd, J ¼ 8.6 and 2.6 Hz,1H), 7.43 (m,
3H), 7.54 (dd, J ¼ 8.2 and 2.2 Hz, 1H), 8.11 (d, J ¼ 2.2 Hz, 1H), 10.1 (s,
1H). 10.4 (s, 1H). 13C NMR (100 MHz, d6-DMSO)
d: 55.2, 55.6, 104.2,
111.0, 112.0, 118.5, 121.7, 122.7, 124.2, 124.8, 126.0, 129.3, 132.8, 135.1,
138.8, 139.3, 144.9, 148.5, 161.1, 162.9. MS (ESI): [M]þ ¼ 430.6,
[Mþ2]þ ¼ 432.7. Anal. calcd for C20H19BrN2O4: C, 55.70; H, 4.44; N,
6.50; found: C, 55.55; H, 4.21; N, 6.34.
5.1.4.9. 2-Bromo-N-(3-{(1E)-3-oxo-3-[(4-methoxyphenyl)amino]
prop-1-en-1-yl}phenyl)-acrylamide (4i). Following general proce-
dure C, the crude residue purified by flash chromatography, using
EtOAc:petroleum ether 6:4 (v:v) for elution, furnished 4i as a white
solid. Yield 52%, mp 193e195 ꢃC. 1H NMR (400 MHz, d6-DMSO)
d:
5.1.4.14. 2-Bromo-N-(3-{(1E)-3-oxo-3-[(3,5-dimethoxyphenyl)
amino]prop-1-en-1-yl}phenyl)-acrylamide (4n). Following general
procedure C, the crude residue purified by flash chromatography,
using ethyl acetate:petroleum ether 1:1 (v:v) for elution, furnished
4n as a yellow solid. Yield 52%, mp 91e93 ꢃC. 1H NMR (400 MHz, d6-
3.73 (s, 3H), 6.34 (d, J ¼ 3.2 Hz, 1H), 6.78 (m, 2H), 6.89 (d, J ¼ 9.0 Hz,
2H), 7.37 (m, 2H), 7.42 (d, J ¼ 15.8 Hz, 1H), 7.58 (m, 3H), 8.06 (s, 1H),
10.2 (s, 1H). 10.4 (s, 1H). 13C NMR (100 MHz, d6-DMSO)
d: 55.4, 114.2
(2C), 119.7, 121.1, 121.8 (2C), 121.9, 124.3, 127.1, 128.6, 128.7, 134.7,
137.2, 139.0, 140.2, 155.4, 161.4, 163.5. MS (ESI): [M]þ ¼ 400.5,
[Mþ2]þ ¼ 402.7. Anal. calcd for C19H17BrN2O3: C, 56.87; H, 4.27; N,
6.98; found: C, 56.75; H, 4.13; N, 6.78.
DMSO)
d
: 3.73 (s, 6H), 6.24 (t, J ¼ 2.4 Hz, 1H), 6.33 (d, J ¼ 3.2 Hz, 1H),
6.76 (d, J ¼ 15.8 Hz, 1H), 6.78 (d, J ¼ 3.2 Hz, 1H), 6.94 (d, J ¼ 2.4 Hz,
2H), 7.38 (m, 2H), 7.49 (d, J ¼ 15.8 Hz, 1H), 7.57 (m, 1H), 8.08 (s, 1H),
10.2 (s, 1H). 10.4 (s, 1H). 13C NMR (100 MHz, d6-DMSO)
d: 55.0 (2C),
5.1.4.10. 2-Bromo-N-(4-{(1E)-3-oxo-3-[(4-ethoxyphenyl)amino]
prop-1-en-1-yl}phenyl)-acrylamide (4j). Following general proce-
dure C, the crude residue purified by flash chromatography, using
EtOAc:petroleum ether 1:1 (v:v) for elution, furnished 4j as a white
95.4, 97.5 (2C), 118.6, 121.9, 122.6, 124.4, 124.9, 126.1, 129.4, 135.1,
138.9, 140.0, 140.8, 160.5 (2C), 161.2, 163.4. MS (ESI): [M]þ ¼ 430.6,
[Mþ2]þ ¼ 432.7. Anal. calcd for C20H19BrN2O4: C, 55.70; H, 4.44; N,
6.50; found: C, 55.48; H, 4.18; N, 6.30.
solid. Yield 54%, mp 201e203 ꢃC. 1H NMR (400 MHz, d6-DMSO)
d:
1.31 (t, J ¼ 7.2 Hz, 3H), 3.98 (q, J ¼ 7.2 Hz, 2H), 6.33 (d, J ¼ 3.2 Hz,1H),
6.78 (d, J ¼ 3.2 Hz, 1H), 6.82 (d, J ¼ 15.5 Hz, 1H), 6.88 (d, J ¼ 8.8 Hz,
2H), 7.37 (d, J ¼ 8.0 Hz, 1H), 7.44 (t, J ¼ 8.0 Hz, 1H), 7.53 (d,
J ¼ 15.6 Hz, 1H), 7.59 (m, 3H), 8.06 (s, 1H), 10.1 (s, 1H). 10.4 (s, 1H).
5.1.4.15. 2-Bromo-N-(3-{(1E)-3-oxo-3-[(3,4,5-trimethoxyphenyl)
amino]prop-1-en-1-yl}phenyl)-acrylamide (4o). Following general
procedure C, the crude residue purified by flash chromatography,
using EtOAc:petroleum ether 7:3 (v:v) for elution, furnished 4o as a
yellow solid. Yield 70%, mp 93e95 ꢃC. 1H NMR (400 MHz, d6-DMSO)
13C NMR (100 MHz, d6-DMSO)
d: 14.7, 63.1, 114.5 (2C), 118.5, 120.6
(2C), 121.7, 122.8, 124.3, 124.9, 126.1, 129.4, 132.3, 135.2, 138.8, 139.3,
154.6, 161.2, 162.9. MS (ESI): [M]þ ¼ 414.6, [Mþ2]þ ¼ 416.5. Anal.
calcd for C20H19BrN2O3: C, 57.84; H, 4.61; N, 6.75; found: C, 57.74; H,
4.47; N, 6.58.
d
: 3.63 (s, 3H), 3.77 (s, 6H), 6.34 (d, J ¼ 3.2 Hz,1H), 6.79 (m, 2H), 7.09
(s, 2H), 7.37 (m, 2H), 7.49 (d, J ¼ 15.8 Hz, 1H), 7.58 (d, J ¼ 7.8 Hz, 1H),
8.07 (s, 1H), 10.2 (s, 1H). 10.4 (s, 1H). 13C NMR (100 MHz, d6-DMSO)
d: 55.6 (2C), 60.0, 96.8 (2C), 118.6, 121.7, 122.5, 124.2, 124.8, 126.0,
129.3, 133.4, 135.0, 135.3, 138.8, 139.6, 152.6 (2C), 161.1, 163.1. MS
(ESI): [M]þ ¼ 460.7, [Mþ2]þ ¼ 462.9. Anal. calcd for C21H21BrN2O5:
C, 54.68; H, 4.59; N, 6.07; found: C, 54.56; H, 4.42; N, 5.88.
5.1.4.11. 2-Bromo-N-(3-{(1E)-3-oxo-3-[(4-trifluoromethoxyphenyl)
amino]prop-1-en-1-yl}phenyl)-acrylamide (4k). Following general
procedure C, the crude residue purified by flash chromatography,
using EtOAc:petroleum ether 6:4 (v:v) for elution, furnished 4k as a
white solid. Yield 48%, mp 126e128 ꢃC. 1H NMR (400 MHz, d6-
5.1.4.16. 2-Bromo-N-(3-{(1E)-3-oxo-3-[(4-morpholinophenyl)amino]
prop-1-en-1-yl}phenyl)-acrylamide (4p). Following general proce-
dure C, the crude residue purified by flash chromatography, using
ethyl acetate for elution, furnished 4p as a yellow solid. Yield 53%,
DMSO)
2H), 7.39 (m, 2H), 7.54 (m, 2H), 7.84 (d, J ¼ 8.8 Hz, 2H), 8.09 (s, 1H),
10.4 (s, 1H). 10.5 (s, 1H). 13C NMR (100 MHz, d6-DMSO)
: 118.6,
120.5 (2C), 121.7 (2C), 121.9, 122.2, 124.5, 124.9, 126.1, 129.4, 135.0,
d: 6.34 (d, J ¼ 3.2 Hz, 1H), 6.86 (m, 2H), 6.89 (d, J ¼ 9.0 Hz,
d
mp 210e212 ꢃC. 1H NMR (400 MHz, d6-DMSO)
4H), 3.72 (t. J ¼ 4.8 Hz, 4H), 6.34 (d, J ¼ 3.2 Hz, 1H), 6.78 (d,
d
: 3.06 (t. J ¼ 5.2 Hz,

406
R. Romagnoli et al. / European Journal of Medicinal Chemistry 81 (2014) 394e407
J ¼ 3.2 Hz, 1H), 6.82 (d, J ¼ 16.0 Hz, 1H), 6.94 (d, J ¼ 9.6 Hz, 2H), 7.36
(d, J ¼ 7.6 Hz, 1H), 7.42 (t, J ¼ 7.6 Hz, 1H), 7.52 (d, J ¼ 16.0 Hz, 1H),
7.57 (m, 3H), 8.06 (s, 1H), 10.1 (s, 1H). 10.4 (s, 1H). 13C NMR
described [48]. The IC50 was defined as the compound concen-
tration required to inhibit cell proliferation by 50%. Some experi-
ments were performed in the presence of different scavengers, such
as BHA, GSH, TOC, DTT or NAC, all purchased from SigmaeAldrich
(Milano, Italy).
(100 MHz, d6-DMSO) d: 48.7 (2C), 66.0 (2C), 115.4 (2C), 118.5, 120.1
(2C), 121.6, 122.8, 124.2, 124.8, 126.0, 129.3, 131.4, 135.2, 138.7, 139.0,
147.2, 161.0, 162.6. MS (ESI): [Mþ1]þ ¼ 455.8, [Mþ3]þ ¼ 457.9. Anal.
calcd for C22H22BrN3O3: C, 57.90; H, 4.86; N, 9.21; found: C, 57.69; H,
4.73; N, 9.01.
5.2.2. Cell growth inhibitory activity in peripheral blood
mononuclear cells (PBMC)
PBMC from healthy donors were obtained by separation on
Lymphoprep (Fresenius KABI Norge AS) gradient. After extensive
washing, cells were resuspended (1.0 ꢂ 106 cells/mL) in RPMI-1640
with 10% fetal bovine serum and incubated overnight. For cyto-
toxicity evaluations in proliferating PBL cultures, non-adherent
cells were resuspended at 5 ꢂ 105 cells/mL in growth medium,
5.1.4.17. 2-Bromo-N-(4-{(1E)-3-oxo-3-[(4-methoxyphenyl)amino]
prop-1-en-1-yl}phenyl)-acrylamide (4q). Following general proce-
dure C, the crude residue purified by flash chromatography, using
EtOAc:petroleum ether 1:1 (v:v) for elution, furnished 4q as a
cream-colored solid. Yield 53%, mp 219e221 ꢃC. 1H NMR (400 MHz,
d6-DMSO)
d
: 3.73 (s, 3H), 6.33 (d, J ¼ 3.2 Hz,1H), 6.64 (d, J ¼ 15.8 Hz,
containing 2.5 mg/mL PHA (Irvine Scientific). Different concentra-
1H), 6.77 (d, J ¼ 3.2 Hz, 1H), 6.90 (d, J ¼ 8.8 Hz, 2H), 7.49 (d,
tions of the test compounds were added, and viability was deter-
mined 72 h later by the MTT test. For cytotoxicity evaluations in
resting PBMC cultures, non-adherent cells were resuspended
(5 ꢂ 105 cells/mL) and treated for 72 h with the test compounds, as
described above.
J ¼ 15.8 Hz, 1H), 7.61 (m, 4H), 7.72 (d, J ¼ 8.8 Hz, 2H), 10.0 (s, 1H).
10.4 (s, 1H). 13C NMR (100 MHz, d6-DMSO)
d: 55.6, 114.4 (2C), 120.9
(2C), 121.1 (2C), 121.8, 125.3, 126.6, 128.7 (2C), 131.1, 133.0, 139.5,
140.0, 155.7, 161.6, 163.6. MS (ESI): [M]þ ¼ 400.8, [Mþ2]þ ¼ 402.9.
Anal. calcd for C19H17BrN2O3: C, 56.87; H, 4.27; N, 6.98; found: C,
56.67; H, 4.11; N, 6.64.
5.2.3. Effects on tubulin polymerization and on colchicine binding
to tubulin
5.1.4.18. 2-Bromo-N-(4-{(1E)-3-oxo-3-[(3,4,5-trimethoxyphenyl)
amino]prop-1-en-1-yl}phenyl-acrylamide (4r). Following general
procedure C, the crude residue purified by flash chromatography,
using EtOAc:petroleum ether 1:1 (v:v) for elution, furnished 4r as a
yellow solid. Yield 62%, mp 93e95 ꢃC. 1H NMR (400 MHz, d6-DMSO)
Bovine brain tubulin was purified as described previously [33].
To evaluate the effect of the compounds on tubulin assembly
in vitro [49], varying concentrations were preincubated with 10 mM
tubulin in glutamate buffer at 30 ꢃC and then cooled to 0 ꢃC. After
addition of GTP, the mixtures were transferred to 0 ꢃC cuvettes in a
recording spectrophotometer and warmed to 30 ꢃC, and the as-
sembly of tubulin was observed turbidimetrically.
d
: 3.71 (s, 3H), 3.76 (s, 6H), 6.33 (d, J ¼ 3.2 Hz,1H), 6.74 (d, J ¼ 3.2 Hz,
1H), 6.77 (d, J ¼ 15.2 Hz, 1H), 7.09 (s, 2H), 7.43 (d, J ¼ 15.2 Hz, 1H),
7.58 (d, J ¼ 8.8 Hz, 2H), 7.72 (d, J ¼ 8.8 Hz, 2H), 10.1 (s, 1H). 10.4 (s,
1H). 13C NMR (100 MHz, d6-DMSO)
d
: 55.6 (2C), 60.0, 96.9 (2C),
5.2.4. Immunofluorescence analysis
120.4 (2C), 121.0, 124.7, 126.0, 128.2 (2C), 130.4, 133.5, 135.3, 139.4,
Cells were fixed in cold 4% formaldehyde for 15 min, rinsed and
stored prior to analysis. Primary antibody staining was performed
for b-tubulin (mouse, monoclonal 1:1000, SigmaeAldrich, Milano,
Italy). After incubation, cells were washed and incubated with an
Alexa conjugated secondary antibody (1:2000, Life Technologies,
Monza, Italy). Cells were counterstained with 40,6-diamidin-2-
phenylindole (DAPI) (1:10,000, SigmaeAldrich, Milano, Italy). Im-
ages were obtained on a video-confocal microscope (Vico, Eclipse
Ti80, Nikon), equipped with a digital camera.
139.6, 152.6 (2C), 161.0, 163.4. MS (ESI): [M]þ
¼
460.7,
[Mþ2]þ ¼ 462.8. Anal. calcd for C21H21BrN2O5: C, 54.68; H, 4.59; N,
6.07; found: C, 54.5; H, 4.38; N, 5.92.
5.2. Biological assays
5.2.1. Cell growth inhibitory activity
Human T-cell leukemia (Jurkat and CEM) and acute B-cell
lymphoblastic leukemia (SEM) cells were grown in RPMI-1640
medium (Gibco Milano Italy). Human cervix carcinoma (HeLa),
breast adenocarcinoma (MCF-7) and colon adenocarcinoma (HT-29
and LoVo) cells were grown in DMEM medium (Gibco, Milano,
Italy). Both media were supplemented with 115 units/mL of peni-
5.2.5. Annexin-V assay
Surface exposure of phosphatidylserine on apoptotic cells was
measured by flow cytometry with a Coulter Cytomics FC500
(Beckman Coulter) by adding Annexin-V-FITC to cells according to
the manufacturer’s instructions (Annexin-V Fluos, Roche Diag-
nostic). Simultaneously the cells were stained with PI. Excitation
was set at 488 nm, and the emission filters were set at 525 nm and
585 nm for FITC and DAPI, respectively.
cillin G (Gibco, Milano, Italy), 115 mg/mL streptomycin (Invitrogen,
Milano, Italy) and 10% fetal bovine serum (Invitrogen, Milano, Italy).
CEMVblꢁ100 cells are a multidrug-resistant line selected against
vinblastine [32] and were grown in complete RPMI 1640 medium
supplemented with vinblastine 100 ng/mL. LoVoDoxo cells are a
doxorubicin resistant subclone of LoVo cells [33] and were grown in
complete Ham’s F12 medium supplemented with doxorubicin
5.2.6. Flow cytometric analysis of cell cycle distribution
For flow cytometric analysis of DNA content, 2.5 ꢂ 105 Hela cells
in exponential growth were treated with different concentrations
of the test compounds for 24 and 48 h. After an incubation period,
the cells were collected, centrifuged and fixed with ice-cold ethanol
(70%). The cells were then treated with lysis buffer containing
RNAse A and 0.1% Triton X-100, and then stained with PI. Samples
were analyzed on a Cytomic FC500 flow cytometer (Beckman
Coulter). DNA histograms were analyzed using MultiCycle for
Windows (Phoenix Flow Systems).
(0.1
mg/mL). Individual wells of a 96-well tissue culture microtiter
plate were inoculated with 100
m
L of complete medium containing
8 ꢂ 103 cells. The plates were incubated at 37 ꢃC in a humidified 5%
CO2 incubator for 18 h prior to the experiments. Stock solutions
(10 mM) of the different compounds were obtained by dissolving
them in DMSO. After medium removal, 100 mL of the drug solution,
dissolved in complete medium at different concentrations, was
added to each well and incubated at 37 ꢃC for 72 h. The percentage
of DMSO in the medium never exceeded 0.25%. This was also the
maximum DMSO concentration in all cell-based assays described
below. Cell viability was measured by the (3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyl tetrazolium bromide (MTT)) assay as previously
5.2.7. Assessment of mitochondrial changes and ROS production
The mitochondrial membrane potential was measured with the
lipophilic cation JC-1 (Molecular Probes, Eugene, OR, USA), while

R. Romagnoli et al. / European Journal of Medicinal Chemistry 81 (2014) 394e407
407
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fluorescent dyes HE (Molecular Probes) and H2DCFDA (Molecular
Probes), as previously described [50].
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5.2.8. Detection of the intracellular GSH content
Cellular GSH levels were analyzed using CMFDA (Molecular
Probes) [42]. Cells were treated with the test compound for
different times. Cells were harvested, centrifuged and incubated in
the presence of a solution of 5 m
M CMFDA at 37 ꢃC for 30 min.
Cytoplasmic esterases convert non-fluorescent CMFDA to fluores-
cent 6-chloromethylfluorescein, which can then react with GSH.
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