European Journal of Medicinal Chemistry 97 (2015) 32e41

Contents lists available at ScienceDirect

European Journal of Medicinal Chemistry

journal homepage: http://www.elsevier.com/locate/ejmech

Short communication

Design, synthesis and antibacterial activity of cinnamaldehyde

derivatives as inhibitors of the bacterial cell division protein FtsZ
Xin Li a, Juzheng Sheng b, Guihua Huang c, Ruixin Ma d, Fengxin Yin b, Di Song a,
Can Zhao a, Shutao Ma a, *
a
Department of Medicinal Chemistry, Key Laboratory of Chemical Biology (Ministry of Education), School of Pharmaceutical Sciences, Shandong University,
44 West Culture Road, Jinan 250012, China
b
Institute of Biochemical and Biotechnological Drug, Key Laboratory of Chemical Biology of Natural Products (Ministry of Education),
School of Pharmaceutical Sciences, Shandong University, 44 West Culture Road, Jinan 250012, China
c
Department of Pharmaceutics, School of Pharmaceutical Sciences, Shandong University, 44 West Culture Road, Jinan 250012, China
d
Affiliated Hospital of Medical College, Qingdao University, Qingdao 266003, China

a r t i c l e i n f o a b s t r a c t

Article history: In an attempt to discover potential antibacterial agents against the increasing bacterial resistance, novel
Received 10 August 2014 cinnamaldehyde derivatives as FtsZ inhibitors were designed, synthesized and evaluated for their anti-
Received in revised form bacterial activity against nine significant pathogens using broth microdilution method, and their cell
22 April 2015
division inhibitory activity against four representative strains. In the in vitro antibacterial activity, the
Accepted 23 April 2015
Available online 24 April 2015
newly synthesized compounds generally displayed better efficacy against Staphylococcus aureus
ATCC25923 than the others. In particular, compounds 3, 8 and 10 exerted superior or comparable activity
to all the reference drugs. In the cell division inhibitory activity, all the compounds showed the same
Keywords:
Antibacterial activity
trend as their in vitro antibacterial activity, exhibiting better activity against S. aureus ATCC25923 than
Cell division inhibitory activity the other strains. Additionally, compounds 3, 6, 7 and 8 displayed potent cell division inhibitory activity
Cinnamaldehyde derivatives with an MIC value of below 1 mg/mL, over 256-fold better than all the reference drugs.
Design © 2015 Elsevier Masson SAS. All rights reserved.
FtsZ
Synthesis

1. Introduction division proteins. Once the recruitment is accomplished, filaments

The rapidly increasing bacterial resistance to conventional an-
tibiotics has resulted in the enormous difficulty in fighting against
bacterial infections [1e3]. Especially, the recently emerging New
Delhi metallo-b-lactamase 1 (NDM-1) superbugs has made almost
all of the first-line clinical antibiotics ineffective [4]. The weak or no
activity of the originally powerful antibiotics in clinic against the
resistant bacteria [5,6] has necessitated a proactive effort to
discover novel antimicrobial pharmacophores with new mecha-
nisms [7]. The filamentous temperature-sensitive protein Z (FtsZ)
plays an important role in the bacterial cell division and is widely
conserved in the bacterial kingdom as well as absent in the mito-
chondria of higher eukaryotes. In the process of bacterial cell di-
vision, FtsZ forms single-stranded filaments and then the highly
dynamic Z-ring scaffold, followed by the recruitment of other cell
* Corresponding author.
E-mail address: mashutao@sdu.edu.cn (S. Ma).
bends and Z ring contracts, leading to the closure of the septum and
then the completement of the cell division [8,9]. As FtsZ is attractive
and largely unexploited as a new target for the development of
antimicrobials, it has aroused many interests among researchers for
the exploration of novel antimicrobials against resistant bacteria in
recent years [10e12].
The recently intensive investigation of FtsZ inhibitors as anti-
bacterial agents leads to various anti-FtsZ molecules, such as cin-
namaldehyde, totarol and PC190723 (Fig. 1) [8,13,14]. Among these
inhibitors, cinnamaldehyde displays broad-spectrum antibacterial
activity with MIC values of 1000 and 500 mg/mL against Escherichia
coli (E. coli) and Bacillus subtilis (B. subtilis), respectively [13]. In the
target identification, FtsZ has been confirmed to be the target of
cinnamaldehyde with the application of standard molecular
biology experiments. Moreover, the STD-NMR spectroscopy
[15e19] and in silico docking model with AutoDock software have
further indicated that the binding region of cinnamaldehyde is
located in the C-terminal region of FtsZ around the T7 loop [13].
Furthermore, its congener, trans-cinnamic acid with mild

http://dx.doi.org/10.1016/j.ejmech.2015.04.048
0223-5234/© 2015 Elsevier Masson SAS. All rights reserved.
 X. Li et al. / European Journal of Medicinal Chemistry 97 (2015) 32e41
OH
O N
cinnamaldehyde totarol
Fig. 1. Structures of cinnamaldehyde, totarol and PC190723.
antibacterial activity, has also been substantiated to target FtsZ
using light scattering assay. However, cinnamaldehyde is to be
studied as a lead compound of FtsZ inhibitors against the growing
bacterial resistance.
As a continuous work of our previous investigation searching for
novel and potent anti-FtsZ agents [20,21], we have carried out a
novel program to design and synthesize a new library of cinna-
maldehyde derivatives for screening potential FtsZ inhibitors. It is
hoped that the cinnamaldehyde derivatives could exhibit more
potent anti-FtsZ effect with a higher antibacterial efficacy and a
broader antibacterial spectrum.
2. Chemistry
In the present work, the target compounds 3e39 were synthe-
sized from substituted benzaldehyde 1 as outlined in Scheme 1. The
reaction of 1 with malonic acid gave cinnamic acid 2 in the presence
of pyridine through the Knoevenagel condensation. The chlorina-
tion of 2 with oxalyl chloride was followed by the amidation re-
action with different amides in the presence of triethylamine to
provide the target compounds 3e39. The newly synthesized com-
pounds were characterized by MS, 1H NMR and IR, and all the
spectral data were in agreement with the proposed structures.
3. Antibacterial evaluation
All the synthesized compounds (3e39) were determined for
their biological activity, including the in vitro antibacterial activity
and the cell division inhibitory activity. The in vitro antibacterial
activity was performed with the application of the standard broth
microdilution method recommended by NCCLS [22]. Cinnamic acid,
curcumin and ciprofloxacin were selected as the reference agents in
this test. Among these references, cinnamic acid and curcumin
were used as the FtsZ-targeted references. And ciprofloxacin, as a
representative fluoroquinolone antibacterial, was applied as the
reference targeting bacterial DNA gyrase. It binded to the GyrA
subunit of DNA gyrase belonging to the type II topoisomerase
family, and then traped the double strand cleaved DNA
gyrase
complex, leading to bacterial cell death [23]. The tested strains
includes three strains of Staphylococcus aureus (S. aureus)
ATCC25923, S. aureus ATCC29213 (MRSA) and S. aureus PR, two
strains of Streptococcus pyogenes (S. pyogenes) PS and S. pyogenes
PR, one strain of B. subtilis ATCC9372 and one strain of Pseudomonas
aeruginosa (P. aeruginosa) ATCC27853, one clinically isolated strain
of Staphylococcus epidermidis (S. epidermidis), and one strain of
E. coli ATCC25922.
The cell division inhibitory activity of these compounds against
B. subtilis, E. coli, P. aeruginosa and S. aureus was assessed by
analyzing the cell length with the application of phase-contrast
light microscopy [24]. The minimum drug concentration at which
33
O NH2
F F
S
O
N
Cl
PC190723
filamentation of B. subtilis, E. coli and P. aeruginosa or ballooning of
S. aureus appeared was recorded as cell division inhibitory con-
centration indicating their on-target activity. The results of mini-
mum antibacterial activity and cell division inhibitory activity in
the unit of mg/mL are shown in Table 1 and Table 2.
The cinnamaldehyde derivatives were derived from cinna-
maldehyde which displayed a definitely FtsZ-targeted antibacterial
activity and influenced FtsZ polymerization. To determine the ef-
fects of these newly synthetic compounds on the polymerization of
FtsZ, a standard light scattering assay was carried out for three
representatives. The inhibition of selected compounds on FtsZ
polymerization was shown in Fig. 2.
As the polymerization and depolymerization of FtsZ depended
on the hydrolysis of GTP, the production of inorganic phosphate
could reflect the activity of FtsZ. To further validate the FtsZ-
targeted mechanism of cinnamaldehyde derivatives, the light
scattering assay was carried out for three representatives. The in-
hibition of selected compounds on FtsZ GTPase was shown in Fig. 3.
4. Results and discussion
In the in vitro antibacterial activity, all the newly synthesized
compounds displayed superior or comparable efficacy to the pre-
cursor cinnamic acid, indicating a successful modification in this
program. Especially, the subseries of compounds containing 2-
methylbenzimidazolyl moiety generally showed better efficacy
than the others against all the tested strains. It was owing to the
special structure of this fragment that was able to interact with
receptors via hydrogen bonds and/or hydrophobic interactions. As
the previous study revealed that cinnamaldehyde bound around
the T7 loop of the C-terminal domain [13] and PC190723 bound to
FtsZ in a cleft between helix seven (H7) and the C-terminal domain,
it was rational to deduce that cinnamaldehyde derivatives might
partially bound to the binding region of PC190723 [8]. And the 2-
methylbenzimidazolyl moiety was predicted to bind into the hy-
drophobic channel formed by amino acid residues I172, E185, N188,
I228 and I230, which also were the binding sites of the thiazolo-
pyridine portion of PC190723 [8]. Additionally, the nitrogen atoms
of 2-methylbenzimidazolyl moiety might form hydrogen bonds
with amino acid residues R191 and Q192, which formed hydrogen
bonds with the phenoxy ether of PC190723 as well [8]. Among
these outstanding compounds, 3, 8 and 10 exhibited the most
excellent activity with MIC values of 4, 10 and 4 mg/mL against
S. aureus ATCC25923, comparable or superior to all the references.
Furthermore, 4 and 10 exerted antibacterial activity with the same
MIC value of 4 mg/mL against S. epidermidis, over 32-fold better than
all the reference drugs. These outstanding compounds, except 3
having no substituent at the benzene ring, had chlorine atom(s) at
the 4- or/and 2-position of the benzene ring, implying that the
substituents at both positions contributed to the entire
34 X. Li et al. / European Journal of Medicinal Chemistry 97 (2015) 32e41

Scheme 1. Synthetic route for the cinnamaldehyde derivatives (3e39). Regents and Conditions: (a) malonic acid, pyridine, reflux for 12 h; (b) oxalyl chloride, THF, 0  C for 20 min;
(c) DMF, rt for 24 h.

antibacterial activity. Furthermore, 6 with a fluorine atom at the 4-
position has the comparable activity to its 4-chlorine congener 8
against all the tested strains. However, 4-methoxy derivative 9
showed a decreased activity against most pathogens tested. All the
results above indicated that the small group at the 4- or/and 2-
position of the benzene ring might increase the entire activity
against various pathogens. This might be due to the binding region
of cinnamaldehyde derivatives that was volume-limited and had
the existing amino acid residues L209, L200, N208 and G205
around the T7 loop forming hydrophobic interactions with the 2,4-
disubstituted benzene ring [13]. In the other subseries, the amides
with two substituents generally displayed better efficacy than
those with one substituent, revealing hydrophobic interaction
existing between the ligand and the receptor around the amide
group or no hydrogen bond donator at the receptor near the
binding site. In this subseries, 20, 28, 29 and 30 exhibited better
antibacterial activity against S. aureus ATCC25923 and S. aureus PR
than the others. For instance, 20 and 28 exerted the same efficacy
against S. aureus ATCC25923 and S. aureus PR with MIC values of 32
and 64 mg/mL, respectively.
In the cell division inhibitory assay, most of the tested com-
pounds showed more potency against S. aureus ATCC25923 than
the other tested strains. And 2-methylbenzimidazolyl derivatives
displayed more advantages in general than the others in the inhi-
bition of the tested pathogens, exerting similar trends to their
in vitro antibacterial activity. Among these derivatives, 3, 6, 7 and 8
exhibited the most potent activity with MIC values of 0.25, 0.50,
0.50, and 0.50 mg/mL, respectively, similar to the previously
discovered most potent FtsZ inhibitors [8]. In the other subseries,
16, 20, 25, 26, 28, 29 and 30 exerted the apparent inhibition of
 X. Li et al. / European Journal of Medicinal Chemistry 97 (2015) 32e41 35

Table 1
Cinnamaldehyde derivatives with their in vitro antibacterial activity (mg/mL).

Compounds B. subtilis E. coli P. aeruginosa S. aureus S. aureus S. aureus S. epidermidisg S. pyogenes S. pyogenes
ATCC9372a ATCC25922b ATCC27853c ATCC25923d ATCC29213e PRf PSh PRi

3 128 128 128 4 >128 64 32 128 128

4 128 128 128 32 128 64 4 >128 >128
5 128 128 128 64 128 64 16 128 >128
6 128 128 128 16 >128 64 16 128 64
7 128 128 128 16 >128 128 128 64 16
8 128 128 128 8 >128 64 16 32 8
9 128 128 128 128 128 64 32 >128 >128
10 128 128 128 4 >128 16 4 >128 64
11 >128 128 128 >128 >128 >128 >128 64 64
12 >128 128 >128 >128 >128 >128 128 128 128
13 128 128 128 >128 >128 >128 >128 >128 >128
14 >128 >128 >128 >128 >128 >128 ND ND ND
15 >128 >128 >128 >128 >128 >128 ND ND ND
16 128 >128 >128 128 >128 >128 >128 32 128
17 >128 >128 >128 >128 >128 >128 ND ND ND
18 >128 >128 >128 >128 >128 >128 ND ND ND
19 >128 >128 >128 >128 >128 >128 ND ND ND
20 128 128 128 32 >128 64 128 >128 128
21 >128 128 128 >128 >128 >128 >128 128 >128
22 >128 128 128 >128 >128 >128 >128 64 >128
23 >128 >128 128 >128 >128 >128 >128 64 128
24 >128 >128 >128 >128 >128 >128 >128 128 >128
25 >128 >128 >128 >128 >128 >128 >128 128 128
26 >128 >128 >128 >128 >128 >128 ND ND ND
27 >128 >128 >128 >128 >128 >128 ND ND ND
28 128 128 128 >128 >128 >128 >128 >128 >128
29 >128 >128 >128 >128 >128 >128 >128 >128 >128
30 >128 >128 >128 >128 >128 >128 >128 >128 >128
31 >128 128 128 >128 >128 >128 64 >128 128
32 >128 >128 128 >128 128 >128 32 128 128
33 >128 >128 >128 >128 >128 >128 >128 >128 >128
34 128 128 >128 >128 >128 >128 >128 >128 >128
35 128 128 >128 >128 >128 >128 >128 >128 >128
36 128 128 128 32 >128 64 >128 >128 >128
37 128 128 128 64 >128 64 >128 >128 >128
38 128 128 >128 64 128 >128 >128 >128 >128
39 128 128 128 >128 >128 >128 >128 >128 >128
Cinnamic >128 >128 >128 >128 >128 >128 >128 >128 >128
acid
Curcumin 32 >128 >128 >128 >128 >128 ND >128 ND
Ciprofloxacin 8 8 4 8 4 8 >128 8 >128
a
B. subtilis ATCC9372: penicillin-susceptible strain.
b
E. coli ATCC25922: penicillin-susceptible strain.
c
P. aeruginosa ATCC27853: penicillin-susceptible strain.
d
S. aureus ATCC25923: penicillin-susceptible strain.
e
S. aureus ATCC29213: methicillin-resistant strain.
f
S. aureus PR: penicillin-resistant strain isolated clinically, not characterized.
g
S. epidermidis: penicillin-resistant strain isolated clinically, not characterized.
h
S. pyogenes PS: penicillin-susceptible strain.
i
S. pyogenes PR: penicillin-resistant strain; ND: not determine.

bacterial cell division with MIC values of below 128 mg/mL. All the
results described above revealed that the amides with hydrophobic
bulky group might increase their cell division inhibitory activity
and the basic substituents at the amide nitrogen probably
contributed to the entire cell division inhibition.
In the light scattering assay, all the tested compounds displayed
an obvious inhibition on FtsZ polymerization in a dose-dependent
manner (Fig. 2). Among them, compound 10 displayed the best
activity in the various concentration range, and it exhibited sig-
nificant activity even at the concentration of 30 mg/mL, similar to
compound 6 at the concentration of 120 mg/mL. The results indi-
cated that these compounds inhibited the proliferation of bacteria
via influencing the function of FtsZ.
In the GTPase assay, all the tested compounds showed an
apparent effect on the GTPase activity of FtsZ and the effect was
dose-dependent. Especially, compound 10 showed an inhibition of
50% at the concentration of 30 mg/mL, superior to the other
compounds. The inhibition of GTPase activity resulted in the
instability of the FtsZ polymer, leading to the abnormal bacterial
cell division and then the cell death.
In this program, all the active compounds are promising, rep-
resenting a new array of molecules with novel scaffold targeting
FtsZ. The further biological investigation of these compounds is
underway.
5. Conclusions
In conclusion, a novel library of cinnamaldehyde derivatives were
designed, synthesized and tested for their in vitro antibacterial ac-
tivity and cell inhibitory activity against various pathogen strains,
including Gram-positive and -negative bacteria. In addition, the light
scattering assay and GTPase assay were carried out for the further
validation of the FtsZ-targeted mechanism. Generally, the cinna-
maldehyde derivative 3e12, bearing 2-methylbenzimidazolyl
36 X. Li et al. / European Journal of Medicinal Chemistry 97 (2015) 32e41

Table 2
Cinnamaldehyde derivatives with their cell division inhibitory activity (mg/mL).

Compounds B. subtilis ATCC9372a E. coli ATCC25922b P. aeruginosa ATCC27853c S. aureus ATCC25923d

3 >128 >128 >128 0.25

4 >128 128 128 16
5 >128 128 128 >128
6 >128 >128 >128 0.5
7 >128 >128 >128 0.5
8 >128 >128 >128 0.5
9 >128 >128 >128 >128
10 >128 >128 >128 1
11 >128 128 >128 64
12 >128 >128 >128 64
13 >128 128 128 >128
14 >128 >128 >128 >128
15 >128 >128 >128 >128
16 128 >128 128 32
17 >128 >128 >128 >128
18 >128 >128 >128 >128
19 >128 >128 >128 >128
20 >128 128 128 4
21 32 >128 >128 64
22 32 >128 >128 32
23 >128 >128 >128 >128
24 128 >128 >128 >128
25 >128 >128 >128 >128
26 >128 >128 >128 >128
27 >128 >128 >128 >128
28 128 128 128 >128
29 >128 >128 128 >128
30 >128 >128 >128 64
31 >128 >128 >128 >128
32 >128 >128 >128 128
33 64 >128 >128 64
34 128 128 128 64
35 >128 128 >128 >128
36 >128 128 >128 8
37 128 128 128 32
38 128 128 >128 16
39 >128 128 128 >128
Cinnamic acid >128 >128 >128 >128
Curcumin 16 >128 ND >128
Ciprofloxacin >128 >128 >128 >128
a
B. subtilis ATCC9372: penicillin-susceptible strain.
b
E. coli ATCC25922: penicillin-susceptible strain.
c
P. aeruginosa ATCC27853: penicillin-susceptible strain.
d
S. aureus ATCC25923: penicillin-susceptible strain.

fragment, exhibited better activity than the others against all the
strains, and many compounds in this subseries showed superior or
comparable antibacterial activity to the references. Moreover, these
active compounds commonly displayed better activity against
S. aureus ATCC25923 than the other strains. In addition, almost all
the compounds exhibited similar in vitro antibacterial activity
against B. subtilis ATCC 9372, E. coli ATCC 25922 and P. aeruginosa
ATCC 27853, suggesting little or no effects of different amide frag-
ments of these molecules on the antibacterial activity. In cell division
inhibitory activity, the compounds showed preferable activity
against the S. aureus ATCC25923 to the other strains with the min-
imum cell division inhibitory activity of 0.25 mg/mL, over 512-fold
better than all the references. Moreover, three representatives
were selected for the further evaluation using light scattering assay
and GTPase assay, and the results strongly validated the mechanism
of these compounds. These results indicated that the subseries were
promising and worth of further investigation for potent FtsZ-
targeted antibacterial agents especially against S. aureus ATCC25923.
were of analytical grade. Reactions progress was monitored by
thin-layer chromatography (TLC) on 0.25-mm pre-coated silica
GF254 plates (Qingdong Yumingyuan silica gel reagent factory,
Shandong, China, YUYUAN). Flash column chromatography was
carried out with the indicated solvents using silica gel 60 (particle
size 0.040e0.063 mm, Qingdong Yumingyuan silica gel reagent
factory, Shandong, China, YUYUAN). Mass spectra were obtained on
the API 4000 instrument (Applied Biosystems, Connecticut, USA)
1
H nuclear magnetic resonance (1H NMR) spectra were recorded on
the Bruker Avance DRX 400 spectrometer (Bruker, Switzerlands)
using appropriate deuterated solvents and were expressed in parts
per million (d, ppm) downfield from tetramethylsilane (internal
standard). Infrared (IR) spectra were recorded on the Nicolet Nexus
470FT-IR spectrometer (Wisconsin, USA) using KBr pellets. Melting
points were uncorrected and determined with the X-6 melting
point apparatus (Beijing Tianchengwode Biotech Co. Ltd, Beijing,
China). Bacterial morphometric analysis was determined on the
Olympus CKX41 microscope.

6. Experimental protocol 6.1.1. Substituted cinnamic acids (2)

To a solution of the substituted benzaldehyde (50 mmol) and
6.1. Chemistry malonic acid (150 mmol) in DMF (30 mL) was added pyridine
(50 mmol) and stirred for 3e5 h at 90  C. After adding water (60 mL),
All necessary reagents and chemicals used in the present study the reaction solution was acidified (pH 1) with concentrated
 X. Li et al. / European Journal of Medicinal Chemistry 97 (2015) 32e41
Fig. 2. Inhibition of FtsZ polymerization by 6 (A), 8 (B) and 10 (C).
hydrochloric acid and then was cooled to 0  C. Then the residue was
filtered, washed with cold water (2  10 mL) and dried in vacuum for
12 h to afford corresponding crude product 2 in yields ranging from
30.2 to 100.0%, which was used directly without further purification.
6.1.2. General procedure for cinnamaldehyde derivatives (3e31)
To a solution of intermediate 2 (1.35 mmol) in THF (10 mL) at
0  C was slowly added oxalyl chloride (4.05 mmol). The reaction
solution was stirred for 15e25 min at the same temperature and
concentrated in vacuo to give substituted cinnamoyl chloride. The
resulting crude product and TEA (4.05 mmol) was dissolved in DMF
(5 mL) in an ice bath, and the corresponding amine (2.03 mmol) in
DMF (2 mL) was dropwise added. The resulting mixture was slowly
warmed to room temperature and stirred for 18 h at the same
temperature. After adding water (20 mL), the compounds 3e39
were extracted with ethyl acetate (3  20 mL), and then the
37
Fig. 3. Inhibition of GTPase activity of FtsZ by cinnamaldehyde derivatives 6, 8 and 10.
combined organic layers were was washed with saturated NaHCO3
solution (2  10 mL) and brine (2  10 mL), respectively. The
washed organic layer was dried over anhydrous Na2SO4, filtered
and evaporated under reduced pressure to dryness. The residue
was purified by flash chromatography (dichloromethane/methanol,
30:1 or petroleum ether/ethyl acetate, 1:1) to give compounds
3e39 in yields ranging from 42.9 to 91.7%.
6.1.2.1. (E)-1-(2-methyl-1H-benzo[d]imidazol-1-yl)-3-pshenylprop-
2-en-1-one (3). White solid, yield 42.9%, mp 95e98  C, Rf ¼ 0.62
(dichloromethane/methanol, 10:1); 1H NMR (400 MHz, DMSO-d6,
d ppm) 7.94 (d, 1H, J ¼ 15.6 Hz), 7.89e7.87 (m, 2H), 7.79e7.77 (m,
1H), 7.66e7.64 (m, 1H), 7.52e7.47 (m, 4H), 7.36e7.30 (m, 2H), 2.79
(s, 3H); IR (KBr): 3373, 3056, 2973, 2927, 1698, 1619, 1576, 1546,
1495, 1455, 1424, 1384, 1350, 1333, 1310, 1292, 1269, 1238, 1206,
1167, 1112, 1091, 1036 cm
1; HRMS (ESI) m/z calcd. for C17H14N2O
263.1179 [MþH]þ, found: 263.1184.
6.1.2.2. (E)-3-(2-chlorophenyl)-1-(2-methyl-1H-benzo[d]imidazol-1-
yl)prop-2-en-1-one (4). Light yellow solid, yield 63.6%, mp
106e108  C, Rf ¼ 0.61 (dichloromethane/methanol, 10:1); IR (KBr):
3384, 3060, 2972, 2926, 1701, 1621, 1545, 1457, 1379, 1332, 1302,
1286, 1238, 1172, 1130, 1113, 1052, 1035 cm
1; 1H NMR (400 MHz,
DMSO-d6, d ppm) 8.16 (d, 1H, J ¼ 15.6), 8.11 (dd, 1H, J ¼ 1.6, J ¼ 2.0),
7.82e7.80 (m, 1H), 7.66e7.63 (m, 1H), 7.61e7.60 (m, 1H), 7.55e7.50
(m, 2H), 7.48 (dd, 1H, J ¼ 0.8, J ¼ 0.8), 7.36e7.33 (m, 2H), 2.80 (s, 3H);
MS (ESI) m/z calcd for C17H13ClN2O 296.07; found [MþH]þ 297.5.
6.1.2.3. (E)-3-(2-methoxyphenyl)-1-(2-methyl-1H-benzo[d]imidazol-
1-yl)prop-2-en-1-one (5). White solid, yield 75.8%, mp 87e89  C,
Rf ¼ 0.61 (dichloromethane/methanol, 10:1); 1H NMR (400 MHz,
DMSO-d6, d ppm) 8.12 (d, 1H, J ¼ 16 Hz), 7.85e7.83 (m, 1H),
7.81e7.79 (m, 1H), 7.66e7.64 (m, 1H), 7.55e7.48 (m, 2H), 7.36e7.30
(m, 2H), 7.17 (d, 1H, J ¼ 8.4 Hz), 7.07 (t, 1H, J ¼ 7.4 Hz), 3.93 (s, 3H),
2.78 (s, 3H); IR (KBr): 3053, 3019, 2951, 2840, 1685, 1619, 1611, 1572,
1549, 1489, 1455, 1432, 1380, 1348, 1339, 1308, 1279, 1247, 1236,
1167, 1124, 1113, 1051, 1037, 1019 cm
1; HRMS (ESI) m/z calcd. for
C18H16N2O2 293.1285 [MþH]þ, found: 293.1289.
6.1.2.4. (E)-3-(4-fluorophenyl)-1-(2-methyl-1H-benzo[d]imidazol-1-
yl)prop-2-en-1-one (6). White solid, yield 67.6%, mp 138e140  C,
Rf ¼ 0.59 (dichloromethane/methanol, 10:1); 1H NMR (400 MHz,
DMSO-d6, d ppm) 7.99e7.93 (m, 3H), 7.79e7.77 (m, 1H), 7.66e7.64
(m, 1H), 7.45 (d, 1H, J ¼ 15.6 Hz), 7.37e7.32 (m, 4H), 2.79 (s, 3H); IR
(KBr): 3069, 2937, 1698, 1620, 1598, 1546, 1508, 1456, 1418, 1380,
1350, 1339, 1310, 1296, 1271, 1229, 1198, 1161, 1115, 1037, 1001 cm
1;
38 X. Li et al. / European Journal of Medicinal Chemistry 97 (2015) 32e41
HRMS (ESI) m/z calcd. for C17H13FN2O 281.1085 [MþH]þ, found:
281.1088.
6.1.2 .5. (E) -1-(2-m ethyl-1 H-b enzo[d]imidazol-1-yl)-3-(4-
nitrophenyl)prop-2-en-1-one (7). Brown solid, yield 90.6%, mp
178e181  C, Rf ¼ 0.60 (dichloromethane/methanol, 10:1); 1H NMR
(400 MHz, DMSO-d6, d ppm) 8.32 (d, 2H, J ¼ 8.8 Hz), 8.16 (d, 2H,
J ¼ 8.8 Hz), 8.02 (d, 1H, J ¼ 16 Hz), 7.82e7.79 (m, 1H), 7.70 (d, 1H,
J ¼ 16 Hz), 7.67e7.65 (m, 1H), 7.36e7.33 (m, 2H), 2.80 (s, 3H); IR
(KBr): 3380, 3080, 2973, 2926, 1700, 1622, 1600, 1549, 1514, 1455,
1428, 1341, 1309, 1291, 1268, 1237, 1163, 1114, 1034 cm
1; HRMS
(ESI) m/z calcd. for C17H13N3O3 308.1030 [MþH]þ, found: 308.1035.
6.1.2.6. (E)-3-(4-chlorophenyl)-1-(2-methyl-1H-benzo[d]imidazol-1-
yl)prop-2-en-1-one (8). White solid, yield 60.6%, mp 94e97  C,
Rf ¼ 0.55 (dichloromethane/methanol, 10:1); IR (KBr): 3473, 3369,
3081, 2972, 2927, 1695, 1624, 1591, 1568, 1539, 1491, 1454, 1436,
1408, 1382, 1346, 1314, 1286, 1266, 1240, 1208, 1155, 1104, 1092,
1063, 1012 cm
1; 1H NMR (400 MHz, DMSO-d6, d ppm) 7.95e7.91
(m, 3H), 7.79e7.77 (m, 1H), 7.66e7.64 (m, 1H), 7.58 (d, 2H, J ¼ 2.4),
7.50 (d, 1H, J ¼ 15.6), 7.36e7.31 (m, 2H), 2.79 (s, 3H); MS (ESI) m/z
calcd for C17H13ClN2O 296.07; found [MþH]þ 297.5.
6.1.2.7. (E)-3-(4-methoxyphenyl)-1-(2-methyl-1H-benzo[d]imidazol-
1-yl)prop-2-en-1-one (9). White solid, yield 81.8%, mp 83e86  C,
Rf ¼ 0.57 (dichloromethane/methanol, 10:1); 1H NMR (400 MHz,
DMSO-d6, d ppm) 7.92 (d, 1H, J ¼ 15.6 Hz), 7.86 (d, 2H, J ¼ 8.8 Hz),
7.77e7.74 (m, 1H), 7.65e7.63 (m, 1H), 7.35e7.30 (m, 3H), 7.06 (d, 2H,
J ¼ 8.8 Hz) 3.84 (s, 3H), 2.78 (s, 3H); IR (KBr): 3012, 2973, 2931,
2839, 1691, 1600, 1570, 1541, 1514, 1453, 1426, 1378, 1357, 1313,
1298, 1255, 1166, 1109, 1038, 1027, 1008 cm
1; HRMS (ESI) m/z
calcd. for C18H16N2O2 293.1285 [MþH]þ, found: 293.1286.
6.1.2.8. (E)-3-(2,4-dichlorophenyl)-1-(2-methyl-1H-benzo[d]imida-
zol-1-yl)prop-2-en-1-one (10). Light yellow solid, yield 83.3%, mp
108e111  C, Rf ¼ 0.61 (dichloromethane/methanol, 10:1); IR (KBr):
3058, 2925, 1698, 1617, 1609, 1582, 1550, 1469, 1456, 1387, 1352,
1335, 1308, 1294, 1275, 1237, 1168, 1113, 1099, 1052, 1035 cm
1; 1H
NMR (400 MHz, DMSO-d6, d ppm) 8.15e8.06 (m, 2H), 7.81e7.79 (m,
2H), 7.66e7.63 (m, 1H), 7.61e7.56 (m, 2H), 7.35e7.32 (m, 2H), 2.80
(s, 3H); MS (ESI) m/z calcd for C17H12Cl2N2O 330.03; found [MþH]þ
331.3.
6.1.2.9. (E)-3-(3,4-dimethoxyphenyl)-1-(2-methyl-1H-benzo[d]imi-
dazol-1-yl)prop-2-en-1-one (11). Yellow solid, yield 80.6%, mp
135e138  C, Rf ¼ 0.53 (dichloromethane/methanol, 10:1); 1H NMR
(400 MHz, DMSO-d6, d ppm) 7.90 (d, 1H, J ¼ 15.6 Hz), 7.76e7.74 (m,
1H), 7.66e7.64 (m, 1H), 7.51 (d, 1H, J ¼ 1.6 Hz), 7.45 (dd, 1H,
J ¼ 1.6 Hz, J ¼ 1.6 Hz), 7.38 (d, 1H, J ¼ 15.6 Hz), 7.35e7.30 (m, 2H),
7.07 (d, 1H, J ¼ 8.4 Hz), 3.84 (d, 6H, J ¼ 1.2 Hz), 2.78 (s, 3H); IR (KBr):
3073, 3054, 2993, 2937, 2839, 1683, 1609, 1593, 1580, 1515, 1455,
1425, 1382, 1340, 1307, 1274, 1254, 1235, 1174, 1159, 1138, 1114, 1035,
1024 cm
1; HRMS (ESI) m/z calcd. for C19H18N2O3 323.1390
[MþH]þ, found: 323.1391.
6.1.2.10. (E)-1-(2-methyl-1H-benzo[d]imidazol-1-yl)-3-(3,4,5-
trimethoxyphenyl)prop-2-en-1-one (12). White solid, yield 66.7%,
mp 172e175  C, Rf ¼ 0.61 (dichloromethane/methanol, 10:1); 1H
NMR (400 MHz, DMSO-d6, d ppm) 7.88 (d, 1H, J ¼ 15.6 Hz),
7.76e7.74 (m, 1H), 7.66e7.64 (m, 1H), 7.49 (d, 1H, J ¼ 15.6 Hz),
7.34e7.31 (m, 2H), 7.25 (s, 2H), 3.85 (s, 6H), 3.74 (s, 3H), 2.79 (s, 3H);
IR (KBr): 2971, 2937, 2841, 2823, 1700, 1624, 1579, 1542, 1504, 1455,
1421, 1378, 1324, 1298, 1269, 1241, 1169, 1126, 1111, 1035, 1004 cm
1;
HRMS (ESI) m/z calcd. for C20H20N2O4 353.1496 [MþH]þ, found:
353.1498.
6.1.2.11. N-(4-methoxyphenyl)cinnamamide (13). Brown solid, yield
64.7%, mp 150e153  C, Rf ¼ 0.83 (dichloromethane/methanol,
10:1); 1H NMR (400 MHz, DMSO-d6, d ppm) 10.10 (s, 1H), 7.64e7.55
(m, 5H), 7.47e7.40 (m, 3H), 6.93e6.91 (m, 2H), 6.82 (d, 1H,
J ¼ 15.6 Hz), 3.74 (s, 3H); IR (KBr): 3248, 3126, 3060, 2931, 2832,
1654, 1619, 1600, 1540, 1509, 1464, 1449, 1412, 1344, 1297, 1239,
1178, 1108, 1038 cm
1; HRMS (ESI) m/z calcd. for C16H15NO2
254.1176 [MþH]þ, found: 254.1177.
6.1.2.12. (E)-N-butyl-3-(4-cyanophenyl)acrylamide (14). White
solid, yield 80.8%, mp 98e100  C, Rf ¼ 0.60 (dichloromethane/
methanol, 10:1); 1H NMR (400 MHz, DMSO-d6, d ppm) 8.21 (t, 1H,
J ¼ 5.4 Hz), 7.88 (d, 2H, J ¼ 8.4 Hz), 7.75 (d, 2H, J ¼ 8.4 Hz), 7.48 (d,
1H, J ¼ 16 Hz), 6.77 (d, 1H, J ¼ 16 Hz), 3.22e3.17 (m, 2H), 1.49e1.42
(m, 2H), 1.37e1.29 (m, 2H), 0.90 (t, 3H, J ¼ 7.4 Hz); IR (KBr): 3265,
3069, 2971, 2924, 2861, 2228, 1657, 1621, 1552, 1508, 1466, 1431,
1411, 1336, 1308, 1281, 1229, 1158, 1119 cm
1; HRMS (ESI) m/z calcd.
for C14H16N2O 229.1335 [MþH]þ, found: 229.1332.
6.1.2.13. (E)-N-butyl-3-(4-nitrophenyl)acrylamide (15). Yellow
solid, yield 57.7%, mp 121e124  C, Rf ¼ 0.62 (dichloromethane/
methanol, 10:1); 1H NMR (400 MHz, DMSO-d6, d ppm) 8.27e8.22
(m, 3H), 7.83 (d, 2H, J ¼ 8.8 Hz), 7.52 (d, 1H, J ¼ 16 Hz), 6.82 (d, 1H,
J ¼ 16 Hz), 3.22e3.17 (m, 2H), 1.48e1.42 (m, 2H), 1.35e1.30 (m, 2H),
0.92e0.87 (m, 3H); IR (KBr): 3305, 3064, 2956, 2934, 2873, 1654,
1615, 1594, 1549, 1515, 1473, 1455, 1411, 1342, 1221, 1108, 1022 cm
1;
HRMS (ESI) m/z calcd. for C13H16N2O3 249.1234[MþH]þ, found:
249.1231.
6.1.2.14. (E)-N-(3-(2-methyl-1H-imidazol-1-yl)-5-(trifluoromethyl)
phenyl)-3-(4-nitrophenyl)acrylamide (16). Yellow solid, yield 44.2%,
mp 167e170  C, Rf ¼ 0.47 (dichloromethane/methanol, 10:1); 1H
NMR (400 MHz, DMSO-d6, d ppm) 10.87 (s, 1H), 8.29 (d, 1H,
J ¼ 8.8 Hz), 8.21 (d, 1H, J ¼ 1.2 Hz), 8.11e8.08 (m, 1H), 8.02 (s, 1H),
7.92e7.90 (m, 1H), 7.78e7.71 (m, 1H), 7.42 (d, 1H, J ¼ 40.4 Hz),
7.00e6.94 (m, 2H), 6.82 (s, 1H), 5.83 (d, 1H, J ¼ 47.6 Hz), 2.19e2.16
(m, 3H); IR (KBr): 3354, 3099, 2973, 2927, 1687, 1638, 1621, 1577,
1519, 1496, 1449, 1405, 1383, 1339, 1317, 1258, 1239, 1203, 1165,
1127, 1111, 1080, 1049, 1010 cm
1; HRMS (ESI) m/z calcd. for
C20H15F3N4O3 417.1169 [MþH]þ, found: 417.1171.
6.1.2.15. (E)-3-(4-nitrophenyl)-N-propylacrylamide (17). Yellow
solid, yield 91.7%, mp 125e128  C, Rf ¼ 0.56 (dichloromethane/
methanol, 10:1); 1H NMR (400 MHz, DMSO-d6, d ppm) 8.27e8.25
(m, 3H), 7.84e7.82 (d, 2H, J ¼ 8.8 Hz), 7.55e7.51 (d, 1H, J ¼ 16 Hz),
6.83 (d, 1H, J ¼ 16 Hz), 3.19e3.14 (m, 2H), 1.54e1.45 (m, 2H),
0.92e0.86 (m, 3H); IR (KBr): 3299, 3257, 3077, 2964, 2923, 2875,
1655, 1623, 1594, 1555, 1516, 1462, 1428, 1413, 1348, 1337, 1286,
1250, 1226, 1182, 1156, 1109 cm
1; HRMS (ESI) m/z calcd. for
C12H14N2O3 235.1077 [MþH]þ, found: 235.1075.
6.1.2.16. (E)-3-(4-nitrophenyl)-N-(prop-2-ynyl)acrylamide (18).
White solid, yield 61.5%, mp 234e237  C, Rf ¼ 0.67 (dichloro-
methane/methanol, 10:1); 1H NMR (400 MHz, DMSO-d6, d ppm)
8.72 (t, 1H, J ¼ 5.6 Hz), 8.27 (d, 2H, J ¼ 8.8 Hz), 7.85 (d, 2H,
J ¼ 8.8 Hz), 7.58 (d, 1H, J ¼ 16 Hz), 6.82 (d, 1H, J ¼ 16 Hz), 4.03e4.00
(m, 2H), 3.19 (t, 1H, J ¼ 2.4 Hz); IR (KBr): 3258, 3077, 2961, 2927,
2851, 1662, 1625, 1600, 1558, 1513, 1420, 1346, 1331, 1261, 1226,
1183, 1110, 1041 cm
1; HRMS (ESI) m/z calcd. for C12H10N2O3
231.0764 [MþH]þ, found: 231.0766.
6.1.2.17. (E)-N-isopropyl-3-(4-nitrophenyl)acrylamide (19).
White solid, yield 66.7%, mp 165e167  C, Rf ¼ 0.48 (dichloro-
methane/methanol, 10:1); 1H NMR (400 MHz, DMSO-d6, d ppm)
8.26 (d, 2H, J ¼ 8.8 Hz), 8.16 (d, 1H, J ¼ 7.6 Hz), 7.82 (d, 2H,
 X. Li et al. / European Journal of Medicinal Chemistry 97 (2015) 32e41
J ¼ 8.8 Hz), 7.52 (d, 1H, J ¼ 16 Hz), 6.79 (d, 1H, J ¼ 15.6), 4.00e3.93
(m, 1H), 1.13 (d, 6H, J ¼ 6.8 Hz); IR (KBr): 3303, 3078, 2974, 2925,
2850, 1655, 1618, 1598, 1543, 1515, 1467, 1407, 1346, 1277, 1224, 1173,
1130, 1109, 1004 cm
1; HRMS (ESI) m/z calcd. for C12H14N2O3
235.1077 [MþH]þ, found: 235.1076.
6.1.2.18. (E)-3-(4-methoxyphenyl)-N-(pyridin-3-yl)acrylamide (20).
White solid, yield 67.9%, mp 150e154  C, Rf ¼ 0.50 (dichloro-
methane/methanol, 10:1); 1H NMR (400 MHz, DMSO-d6, d ppm)
10.35 (s, 1H), 8.84 (d, 1H, J ¼ 2.4 Hz), 8.29e8.27 (m, 1H), 8.17e8.14
(m, 1H), 7.61e7.57 (m, 3H), 7.37 (dd, 1H, J ¼ 4.8 Hz, J ¼ 4.8 Hz), 7.02
(d, 2H, J ¼ 8.8 Hz), 6.70 (d, 1H, J ¼ 15.6 Hz), 3.81 (s, 3H); IR (KBr):
3242, 3184, 3119, 3059, 3000, 1682, 1622, 1604, 1589, 1553, 1513,
1477, 1427, 1346, 1304, 1287, 1251, 1194, 1169, 1129, 1113, 1025 cm
1;
HRMS (ESI) m/z calcd. for C15H14N2O2 255.1128 [MþH]þ, found:
255.1128.
6.1.2.19. (E)-3-(2,4-dichlorophenyl)-N-propylacrylamide (21).
White solid, 88.1%, mp 144e146  C, Rf ¼ 0.12 (petroleum ether/
ethyl acetate, 2:1); 1H NMR (400 MHz, DMSO-d6, d ppm) 8.23 (s,
1H), 7.72 (s, 2H), 7.66e7.62 (d, 1H, J ¼ 15.6 Hz), 7.51e7.49 (d, 1H,
J ¼ 8 Hz), 6.71e6.67 (d, 1H, J ¼ 11.6 Hz), 3.15e3.14 (d, 2H, J ¼ 5.6 Hz),
1.50e1.45 (q, 2H, J ¼ 7.2 Hz), 0.90e0.86 (t, 3H, J ¼ 7.2 Hz); MS (ESI)
m/z calcd for C12H13Cl2NO 257.04; found [MþH]þ 258.3.
6.1.2.20. (E)-3-(2,4-dichlorophenyl)-N-isopropylacrylamide (22).
White solid, 89.4%, mp 178e180  C, Rf ¼ 0.18 (petroleum ether/
ethyl acetate, 2:1); 1H NMR (400 MHz, DMSO-d6, d ppm) 8.14e8.12
(d, 1H, J ¼ 6.8 Hz), 7.72e7.61 (m, 3H), 7.52e7.50 (d, 1H, J ¼ 8 Hz),
6.68e6.64 (d, 1H, J ¼ 15.6 Hz) 3.98e3.95 (m, 1H), 1.12e1.10 (d, 6H,
J ¼ 6.4 Hz); MS (ESI) m/z calcd for C12H13Cl2NO 257.04; found
[MþH]þ 258.2.
6.1.2.21. (E)-N-butyl-3-(2,4-dichlorophenyl)acrylamide (23).
White solid, 89.2%, mp 146e149  C, Rf ¼ 0.40 (petroleum ether/
ethyl acetate, 2:1); 1H NMR (400 MHz, DMSO-d6, d ppm) 8.21 (s,
1H), 7.72 (s, 2H), 7.66e7.62 (d, 1H, J ¼ 15.6 Hz), 7.51e7.49 (d, 1H,
J ¼ 8.4 Hz), 6.70e6.66 (d, 1H, J ¼ 15.6 Hz), 3.19e3.17 (d, 2H,
J ¼ 5.6 Hz), 1.44e1.43 (d, 2H, J ¼ 6.4 Hz), 1.32e1.30 (d, 2H, J ¼ 7.2 Hz),
0.91e0.87 (t, 3H, J ¼ 7.2 Hz); MS (ESI) m/z calcd for C13H15Cl2NO
271.05; found [MþH]þ 272.5.
6.1.2.22. (E)-3-(2,4-dichlorophenyl)-N-hexylacrylamide (24).
White solid, 87.6%, mp 104e106  C, Rf ¼ 0.63 (petroleum ether/
ethyl acetate, 2:1); 1H NMR (400 MHz, DMSO-d6, d ppm) 8.22 (s,
1H), 7.72e7.71 (m, 2H), 7.66e7.62 (d, 1H, J ¼ 15.6 Hz), 7.51e7.49 (d,
1H, J ¼ 8 Hz), 6.71e6.67 (d, 1H, J ¼ 15.6 Hz), 3.18e3.17 (d, 2H,
J ¼ 5.6 Hz), 1.45 (s, 2H), 1.27 (s, 6H), 0.87 (s, 3H); MS (ESI) m/z calcd
for C15H19Cl2NO 299.08; found [MþH]þ 300.5.
6.1.2.23. (E)-3-(2,4-dichlorophenyl)-N-(4-methoxyphenyl)acryl-
amide (25). White solid, 85.7%, mp 205e208  C, Rf ¼ 0.43 (petro-
leum ether/ethyl acetate, 2:1); 1H NMR (400 MHz, DMSO-d6,
d ppm) 10.21 (s, 1H), 7.80e7.78 (d, 1H, J ¼ 6 Hz), 7.76 (s, 2H),
7.63e7.61 (d, 2H, J ¼ 8.4 Hz), 7.56e7.54 (d, 1H, J ¼ 8 Hz), 6.94e6.91
(d, 2H, J ¼ 8.8 Hz), 6.89e6.85 (d, 1H, J ¼ 16 Hz), 3.74 (s, 3H); MS (ESI)
m/z calcd for C16H13Cl2NO2 321.03; found [MþH]þ 322.4.
6.1.2.24. (E)-3-(3,4-dimethoxyphenyl)-N-(prop-2-ynyl)acrylamide
(26). White solid, yield 83.3%, mp 144e147  C, Rf ¼ 0.88
(dichloromethane/methanol, 10:1); 1H NMR (400 MHz, DMSO-d6,
d ppm) 8.43 (t, 1H, J ¼ 5.6 Hz), 7.40 (d, 1H, J ¼ 15.6 Hz), 7.17e7.11
(m, 2H), 6.99 (d, 1H, J ¼ 8 Hz), 6.51 (d, 1H, J ¼ 15.6 Hz), 3.99e3.97
(m, 2H), 3.79 (d, 6H, J ¼ 4 Hz), 3.14 (t, 1H, J ¼ 2.6 Hz); IR (KBr): 3251,
3014, 2961, 2910, 2852, 1652, 1622, 1595, 1580, 1529, 1515, 1469,
39
1455, 1417, 1435, 1339, 1303, 1260, 1242, 1216, 1167, 1138, 1039,
1017 cm
1; HRMS (ESI) m/z calcd. for C14H15NO3 246.1125 [MþH]þ,
found: 246.1120.
6.1.2.25. (E)-N-butyl-3-(3,4-dimethoxyphenyl)acrylamide (27).
White solid, yield 71.4%, mp 112e115  C, Rf ¼ 0.50 (dichloro-
methane/methanol, 10:1); 1H NMR (400 MHz, DMSO-d6, d ppm)
7.96 (t, 1H, J ¼ 5.4 Hz), 7.34 (d, 1H, J ¼ 16 Hz), 7.14e7.09 (m, 2H), 6.98
(d, 1H, J ¼ 8.4 Hz), 6.50 (d, 1H, J ¼ 15.6 Hz), 3.79 (d, 6H, J ¼ 5.6 Hz),
3.19e3.14 (m, 2H), 1.47e1.40 (m, 2H), 1.36e1.23 (m, 2H), 0.89 (t, 3H,
J ¼ 7.4 Hz); IR (KBr): 3292, 3007, 2961, 2934, 2872, 1652, 1614, 1579,
1549, 1513, 1462, 1437, 1419, 1363, 1320, 1263, 1213, 1192, 1160, 1142,
1037, 1026 cm
1; HRMS (ESI) m/z calcd. for C15H21NO3 264.1594
[MþH]þ, found: 264.1595.
6.1.2.26. (E)-3-(4-fluorophenyl)-1-morpholinoprop-2-en-1-one (28).
White solid, yield 78.6%, mp 134e137  C, Rf ¼ 0.48 (dichloro-
methane/methanol, 10:1); 1H NMR (400 MHz, DMSO-d6, d ppm)
7.81e7.78 (m, 2H), 7.51 (d, 1H, J ¼ 15.2 Hz), 7.27e7.20 (m, 3H),
3.71e3.57 (m, 8H); IR (KBr): 3069, 2972, 2916, 2867, 1648, 1610,
1598, 1511, 1428, 1410, 1364, 1306, 1267, 1213, 1196, 1163, 1113, 1073,
1049 cm
1; HRMS (ESI) m/z calcd. for C13H14FNO2 236.1081
[MþH]þ, found: 236.1084.
6.1.2.27. (E)-3-(2-chlorophenyl)-1-(4-((4-chlorophenyl) (phenyl)
methyl)piperazin-1-yl)prop-2-en-1-one (29). Yellow solid, yield
62.3%, mp 82e84  C, Rf ¼ 0.75 (dichloromethane/methanol, 10:1);
1
H NMR (400 MHz, DMSO-d6, d ppm) 7.97e7.94 (m, 1H), 7.81e7.77
(d, 1H, J ¼ 15.2 Hz), 7.52e7.50 (m, 1H), 7.48e7.46 (d, 2H, J ¼ 8.4 Hz),
7.43e7.41 (m, 2H), 7.40e7.36 (m, 4H), 7.32 (t, 2H, J ¼ 7.6 Hz), 7.27 (d,
1H, J ¼ 15.2 Hz), 7.24 (d, 1H, J ¼ 1.2 Hz), 4.41 (s, 1H), 3.73e3.60 (m,
4H), 2.32 (s, 4H); IR (KBr): 3419, 3060, 3025, 2917, 2809, 1647, 1608,
1488, 1472, 1434, 1369, 1306, 1263, 1227, 1202, 1144, 1089, 1052,
1038 cm
1; HRMS (ESI) m/z calcd. for C26H24Cl2N2O 451.1338
[MþH]þ, found: 451.1340.
6.1.2.28. (E)-1-(4-((4-chlorophenyl) (phenyl)methyl)piperazin-1-yl)-
3-(2-methoxyphenyl)prop-2-en-1-one (30). White solid, yield
73.2%, mp 128e131  C, Rf ¼ 0.59 (dichloromethane/methanol,
10:1); 1H NMR (400 MHz, DMSO-d6, d ppm) 7.78e7.70 (m, 2H),
7.48e7.41 (m, 3H), 7.38e7.30 (m, 4H), 7.23e7.20 (m, 1H), 7.15 (d, 1H,
J ¼ 15.6 Hz), 7.05 (d, 1H, J ¼ 8.4 Hz), 6.98e6.94 (m, 1H), 5.76 (s, 2H),
4.40 (s, 1H), 3.84 (s, 3H), 3.64 (d, 4H, J ¼ 40.4 Hz), 2.42 (d, 4H,
J ¼ 78.4 Hz); IR (KBr): 3427, 3026, 2998, 2959, 2919, 2807, 2760,
1643, 1600, 1488, 1463, 1427, 1370, 1312, 1246, 1226, 1195, 1162,
1144, 1106, 1085, 1044, 1000 cm
1; HRMS (ESI) m/z calcd. for
C27H27ClN2O2 447.1834 [MþH]þ, found: 447.1835.
6.1.2.29. (E)-3-(2,4-dichlorophenyl)-1-morpholinoprop-2-en-1-one
(31). White solid, 82.1%, mp 144e147  C, Rf ¼ 0.13 (petroleum
ether/ethyl acetate, 2:1); 1H NMR (400 MHz, DMSO-d6, d ppm)
8.08e8.06 (d, 1H, J ¼ 8.4 Hz), 7.79e7.75 (d, 1H, J ¼ 15.2 Hz), 7.71 (s,
1H), 7.52e7.50 (d, 1H, J ¼ 8 Hz), 7.38e7.34 (d, 1H, J ¼ 15.2 Hz), 3.73
(s, 2H), 3.61e3.58 (m, 6H); MS (ESI) m/z calcd for C13H13Cl2NO2
285.03; found [MþH]þ 286.3.
6.1.2.30. (E)-3-(2,4-dichlorophenyl)-N,N-diethylacrylamide (32).
White solid, 87.5%, mp 95e98  C, Rf ¼ 0.37 (petroleum ether/ethyl
acetate, 2:1); 1H NMR (400 MHz, DMSO-d6, d ppm) 8.06e8.04 (d,
1H, J ¼ 8.4), 7.77e7.71 (m, 2H), 7.50e7.48 (d, 1H, J ¼ 8.4 Hz),
7.22e7.18 (d, 1H, J ¼ 15.6 Hz), 3.54e3.52 (d, 2H, J ¼ 6.8 Hz),
3.39e3.37 (d, 2H, J ¼ 6.8 Hz), 1.17e1.13 (t, 3H, J ¼ 6.4 Hz), 1.10e1.06
(t, 3H, J ¼ 6.4 Hz); MS (ESI) m/z calcd for C13H15Cl2NO 271.05; found
[MþH]þ 272.5.
40 X. Li et al. / European Journal of Medicinal Chemistry 97 (2015) 32e41
6.1.2.31. (E)-1-(4-((4-chlorophenyl) (phenyl)methyl)piperazin-1-yl)-
3-(2,4-dichlorophenyl)prop-2-en- 1-one (33). Light yellow solid,
89.5%, mp 73e76  C, Rf ¼ 0.24 (petroleum ether/ethyl acetate, 2:1);
1
H NMR (400 MHz, DMSO-d6, d ppm) 8.01e7.99 (d, 1H, J ¼ 8.4 Hz),
7.74 (s, 1H), 7.70 (s, 1H), 7.48e7.46 (m, 3H), 7.43e7.41 (m, 4H),
7.32e7.30 (m, 3H), 7.24e7.22 (m, 1H), 4.42 (s, 1H), 3.72 (s, 2H), 3.60
(s, 2H), 2.32 (s, 4H); MS (ESI) m/z calcd for C26H23Cl3N2O 484.09;
found [MþH]þ 485.5.
6.1.2.32. (E)-3-(3,4-dimethoxyphenyl)-N,N-diethylacrylamide (34).
White solid, yield 56.0%, mp 88e90  C, Rf ¼ 0.59 (dichloromethane/
methanol, 10:1); 1H NMR (400 MHz, DMSO-d6, d ppm) 7.44 (d, 1H,
J ¼ 15.2 Hz), 7.31 (d, 1H, J ¼ 2 Hz), 7.21 (dd, 1H, J ¼ 2 Hz, J ¼ 2 Hz),
6.98 (d, 1H, J ¼ 4.8 Hz), 6.95 (d, 1H, J ¼ 2.4 Hz) 3.80 (d, 6H, J ¼ 12 Hz),
3.53 (d, 2H, J ¼ 6.8 Hz), 3.37 (d, 2H, J ¼ 7.2 Hz), 1.15 (t, 3H, J ¼ 6.8 Hz),
1.07 (t, 3H, J ¼ 6.8 Hz); IR (KBr): 3389, 3075, 2964, 2930, 2839, 1639,
1593, 1512, 1478, 1459, 1423, 1378, 1363, 1305, 1261, 1228, 1161, 1138,
1079, 1037, 1018 cm
1; MS (ESI) m/z calcd. for C15H21NO3 [MþH]þ,
found: 264.3.
6.1.2.33. (E)-3-(3,4-dimethoxyphenyl)-1-morpholinoprop-2-en-1-
one (35). White solid, yield 55.6%, mp 120e123  C, Rf ¼ 0.45
(dichloromethane/methanol, 10:1); 1H NMR (400 MHz, DMSO-d6,
d ppm) 7.47 (d, 1H, J ¼ 15.2 Hz), 7.36 (d, 1H, J ¼ 1.6 Hz), 7.21 (dd,
1H, J ¼ 2 Hz, J ¼ 1.6 Hz), 7.11 (d, 1H, J ¼ 15.2 Hz), 6.96 (d, 1H,
J ¼ 8.4 Hz), 3.80 (d, 6H, J ¼ 13.2 Hz), 3.72 (s, 2H), 3.61e3.57 (m, 6H);
IR (KBr): 3016, 2967, 2928, 2847, 1644, 1596, 1515, 1457, 1431, 1409,
1364, 1331, 1301, 1266, 1253, 1241, 1193, 1145, 1108, 1046,
1020 cm
1; HRMS (ESI) m/z calcd. for C15H19NO4 278.1387 [MþH]þ,
found: 278.1386.
6.1.2.34. (E)-3-(3,4-dimethoxyphenyl)-N,N-dimethylacrylamide (36).
Yellow solid, yield 58.3%, mp 100e113  C, Rf ¼ 0.57 (dichloro-
methane/methanol, 10:1); 1H NMR (400 MHz, DMSO-d6, d ppm)
7.42 (d, 1H, J ¼ 15.2 Hz), 7.34 (d, 1H, J ¼ 2 Hz), 7.10 (dd, 1H, J ¼ 1.6 Hz,
J ¼ 1.6 Hz), 7.08 (d, 1H, J ¼ 15.2 Hz), 6.96 (d, 1H, J ¼ 8.4 Hz),
3.82e3.77 (m, 6H), 3.17 (s, 3H), 2.93 (s, 3H); IR (KBr): 2931, 2838,
1645, 1600, 1512, 1479, 1438, 1424, 1394, 1341, 1314, 1265, 1229,
1164, 1138, 1021 cm
1; HRMS (ESI) m/z calcd. for C13H17NO3
236.1281 [MþH]þ, found: 236.1279.
6.1.2.35. (E)-N,N-diethyl-3-(3,4,5-trimethoxyphenyl)acrylamide (37).
White solid, yield 56.0%, mp 128e130  C, Rf ¼ 0.58 (dichloro-
methane/methanol, 10:1); 1H NMR (400 MHz, DMSO-d6, d ppm)
7.43 (d, 1H, J ¼ 15.6 Hz), 7.03 (d, 3H, J ¼ 15.2 Hz), 3.84 (d, 6H,
J ¼ 8 Hz), 3.68 (s, 3H), 3.54 (d, 2H, J ¼ 6.8 Hz), 3.40e3.35 (m, 2H),
1.15 (t, 3H, J ¼ 6.8 Hz), 1.07 (t, 3H, J ¼ 6.8 Hz); IR (KBr): 2969, 2939,
2839, 1649, 1596, 1582, 1505, 1463, 1421, 1345, 1324, 1283, 1248,
1232, 1185, 1141, 1124, 1099, 1073, 1001 cm
1; HRMS (ESI) m/z calcd.
for C16H23NO4 294.1700 [MþH]þ, found: 294.1704.
6.1.2.36. (E)-1-morpholino-3-(3,4,5-trimethoxyphenyl)prop-2-en-1-
one (38). White solid, yield 57.7%, mp 127e130  C, Rf ¼ 0.71
(dichloromethane/methanol, 10:1); 1H NMR (400 MHz, DMSO-d6,
d ppm) 7.47 (d, 1H, J ¼ 15.2 Hz), 7.18 (d, 1H, J ¼ 15.2 Hz), 7.05 (s,
2H), 3.83 (s, 6H), 3.74 (s, 2H), 3.68 (s, 3H), 3.59 (d, 6H, J ¼ 19.2 Hz);
IR (KBr): 3447, 2952, 2848, 1644, 1596, 1584, 1506, 1455, 1419, 1341,
1323, 1296, 1273, 1251, 1228, 1193, 1157, 1122, 1066, 1046 cm
1;
HRMS (ESI) m/z calcd. for C16H21NO5 308.1492 [MþH]þ, found:
308.1497.
6.1.2.37. (E)-N,N-dimethyl-3-(3,4,5-trimethoxyphenyl)acrylamide
(39). Brown solid, yield 54.2%, mp 120e123  C, Rf ¼ 0.57
(dichloromethane/methanol, 10:1); 1H NMR (400 MHz, DMSO-d6,
d ppm) 7.42 (d, 1H, J ¼ 15.6 Hz), 7.15 (d, 1H, J ¼ 15.2 Hz), 7.04 (s,
2H), 3.83 (s, 6H), 3.68 (s, 3H), 3.18 (s, 3H), 2.93 (s, 3H); IR (KBr):
3063, 2999, 2942, 2849, 1654, 1614, 1580, 1503, 1467, 1454, 1421,
1396, 1336, 1314, 1267, 1247, 1187, 1123, 1001 cm
1; HRMS (ESI) m/z
calcd. for C14H19NO4 266.1387 [MþH]þ, found: 266.1382.
6.2. Antibacterial evaluation
6.2.1. In vitro antibacterial assay
The minimum inhibitory concentration (MIC) of the tested
compounds was determined applying tube dilution method rec-
ommended by NCCLS [25]. Bacterial strains were incubated on
MHA (Mueller Hinton Agar) medium at 37  C for 24 h. The bacteria
solution was prepared by suspension in 10 mL of sterile water for
colonies from culture on MHA medium. MHB (Mueller Hinton
Broth) was used for bacteria in this test. The cell density of each
inoculum was adjusted in sterile water of a 0.5 McFarland standard.
In this method, various concentrations of the cinnamaldehyde de-
rivatives were prepared from 128 to 0.25 mg/mL in sterile tubes No.
1 to 10. 100 mL sterile Mueller Hinton Broth (MHB) was poured in
each sterile tube followed by addition of 200 mL test compound in
tube 1. Two fold serial dilutions were carried out from tube 1 to
tube 10 and excess broth (100 mL) was discarded from the last tube
No. 10. To each tube, 100 mL of standard inoculum (1.5  108 cfu/mL)
was added. Cinnamic acid, curcumin and ciprofloxacin were used as
controls. Turbidity was observed after incubating the inoculated
tubes at 37  C for 24 h. The last tube with no growth of microor-
ganism was recorded to represent the MIC value expressed in mg/
mL.
6.2.2. Cell division inhibitory assay
Cell division inhibitory activity of the tested compounds was
performed as described previously [24]. Overnight cultures were
grown in starvation medium supplemented with 1% hydrolyzed
casein and then diluted in starvation medium supplemented with
3% hydrolyzed casein (B. subtilis) or in Mueller-Hinton medium
(S. aureus, E. coli and P. aeruginosa) and grown at 37  C. The culture
was diluted to A600 of ~0.06, and 10 mL aliquots were added to
transparent 96-well microtiter plates containing dilutions of the
cinnamaldehyde derivatives, cinnamic acid, curcumin and cipro-
floxacin as controls in 100 mL volumes of medium. After incubation
for approximately 5 h (4e5 generations) at 37  C, 20 mL culture
samples were transferred to poly-L-lysine-coated slides for micro-
scopy. Cell morphology was assessed by phase-contrast light mi-
croscopy. Lowest concentration at which filamentation of B. subtilis,
E. coli and P. aeruginosa or ballooning of S. aureus was recorded
represented the cell division inhibitory activity indicating on-target
activity.
6.2.3. Expression and purification of E. coli FtsZ
FtsZ (GeneBank accession number: NC_000913) was recombi-
nant expressed in BL21 (DE3) cells as a soluble N-His6-tagged
fusion protein. The genomic DNA isolated from E. coli ATCC 25922
was used as the template for polymerase chain reactions (PCR). And
the complete open reading frame (ORF) of FtsZ protein was
amplified with the 50 primer FtsZ-F [50 - Gcatgactggtggacagc
aaatgggtcgcGGATCCTTTGAACCAATGAACTTA-30 ] and the 30 primer
FtsZ -R [50 - CggatctcagtggtggtggtggtggtgCTCGAGTTAATCGCT-
TACGCAGG-30 ]. Then, the PCR product was ligated with corre-
sponding predigested pET28a(þ) by Gibson assembly combination
[26]. The ligation products were transformed into E. coli DH5a cells.
Plasmid containing FtsZ was confirmed by DNA sequencing and
then transformed into E. coli BL21 (DE3).
The transformed cells were grown in LB medium (10 g/L tryp-
tone, 5 g/L yeast extract, 10 g/L NaCl) supplemented with 100 mg/ml
kanamycin at 37  C. The temperature was decreased to 22  C when
 X. Li et al. / European Journal of Medicinal Chemistry 97 (2015) 32e41
the A600 reached 0.4e0.8, followed by the addition of isopropyl-1-
thio-b-D-galactopyranoside (0.2 mM) to induce the expression of
FtsZ proteins. After shaking for 18 h, the cells were pelleted and
lysed by sonication. FtsZ proteins were then purified using Ni
Sepharose 6 Fast Flow (GE healthcare life sciences, Pittsburgh, PA,
USA) following the protocol provided by the manufacturer. The
protein product was analyzed by 10% SDS-PAGE stained with Coo-
massie Blue.
6.2.4. Light scattering assay
The inhibition of cinnamaldehyde derivatives for E. coli FtsZ was
measured using a standard light scattering assay on a Hitachi
fluorescence spectrophotometer (model F-7000). The excitation
and emission wavelengths were set at 400 nm and a slit width of
5 nm. The gain was set at 400 V and the cuvette chamber was
maintained at 30  C by a circulating water bath. E. coli FtsZ (12 mM)
was incubated in the polymerization buffer (50 mM MES, 5 mM
MgCl2, 50 mM KCl, pH 6.5) containing various concentrations of a
cinnamaldehyde derivative and was added to a fluorometer cuvette
with a 1-cm path length. The data were collected for 120 s to
establish a baseline. Then polymerization was initiated with the
addition of 2 mM GTP and determined for 2000 s. Compound stock
solutions were prepared in DMSO and the percentage of DMSO was
maintained at 2% for all experiments [27,28].
6.2.5. GTPase assay
A standard Malachite Green/ammonium molybdate assay was
used to determine the production of inorganic phosphate during
the assembly of FtsZ. E. coli FtsZ (10 mM) was incubated in poly-
merization buffer (50 mM MES, 5 mM MgCl2, 50 mM KCl, pH 6.5)
with different concentrations of a cinnamaldehyde derivative at
room temperature for 15 min. Then 50 mM GTP was added to the
reaction mixture and incubated at 37  C for 30 min. Subsequently,
Malachite Green reagent (20% v/v) was added to the reaction
mixtures and the reaction mixtures were centrifuged at 13 000 rpm
for 90 s to remove the protein debris. The samples (100 mL) were
transferred to a 96-well plate, and the absorbance of each well was
measured at 620 nm. The background absorbance was subtracted
from all the readings. A phosphate standard curve was created with
a Malachite Green Phosphate Assay Kit (Bioassay Systems). Com-
pound stock solutions were prepared in DMSO and the percentage
of DMSO was maintained at 2% for all experiments [29].
Acknowledgments
This research was supported financially by the National Natural
Science Foundation of China (20872081 and 21072114), the Nat-
ural Science Foundation of Shandong (ZR2010HM092), and Chi-
naeAustralia Centre for Health Sciences Research (CACHSR,
2014GJ06).
41
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2015.04.048.
References
[1] M.H. Miceli, J.A. Díaz, S.A. Lee, Lancet. Infect. Dis. 11 (2011) 142e151.
[2] B. Spellberg, R. Guidos, D. Gilbert, J. Bradley, H.W. Boucher, W.M. Scheld,
J.G. Bartlett, J. Edwards, Clin. Infect. Dis. 46 (2008) 155e164.
[3] I.M. Gould, M.Z. David, S. Esposito, J. Garau, G. Lina, T. Mazzei, G. Peters, Int. J.
Antimicrob. Agents 39 (2012) 96e104.
[4] K.K. Kumarasamy, M.A. Toleman, T.R. Walsh, J. Bagaria, F. Butt,
R. Balakrishnan, U. Chaudhary, M. Doumith, C.G. Giske, S. Irfan, Lancet. Infect.
Dis. 10 (2010) 597e602.
[5] R.H. Deurenberg, E.E. Stobberingh, Curr. Mol. Med. 9 (2009) 100e115.
[6] M.E. de Kraker, M. Wolkewitz, P.G. Davey, H. Grundmann, Antimicrob. Agents
Chemother. (2011) 1598e1605.
[7] Y.-T. Tan, D.J. Tillett, I.A. McKay, Mol. Med. Today 6 (2000) 309e314.
[8] D.J. Haydon, N.R. Stokes, R. Ure, G. Galbraith, J.M. Bennett, D.R. Brown,
P.J. Baker, V.V. Barynin, D.W. Rice, S.E. Sedelnikova, Science 321 (2008)
1673e1675.
[9] E. Bi, J. Lutkenhaus, Nature (1991) 161e164.
[10] T. Matsui, J. Yamane, N. Mogi, H. Yamaguchi, H. Takemoto, M. Yao, I. Tanaka,
Acta Crystallogr. D. Biol. Crystallogr. 68 (2012) 1175e1188.
[11] N.L. Elsen, J. Lu, G. Parthasarathy, J.C. Reid, S. Sharma, S.M. Soisson, K.J. Lumb,
J. Am. Chem. Soc. 134 (2012) 12342e12345.
[12] M. Kaul, A.K. Parhi, Y. Zhang, E.J. LaVoie, S. Tuske, E. Arnold, J.E. Kerrigan,
D.S. Pilch, J. Med. Chem. 55 (2012) 10160e10176.
[13] P. Domadia, S. Swarup, A. Bhunia, J. Sivaraman, D. Dasgupta, Biochem..
Pharmacol. 74 (2007) 831e840.
[14] R. Jaiswal, T.K. Beuria, R. Mohan, S.K. Mahajan, D. Panda, Biochemistry 46
(2007) 4211e4220.
[15] Y. Chen, K. Bjornson, S.D. Redick, H.P. Erickson, Biophys. J. 88 (2005) 505e514.
[16] M.R. Caplan, H.P. Erickson, J. Biol. Chem. 278 (2003) 13784e13788.
[17] L. Romberg, M. Simon, H.P. Erickson, J. Biol. Chem. 276 (2001) 11743e11753.
[18] C. Lu, M. Reedy, H.P. Erickson, J. Bacteriol. 182 (2000) 164e170.
[19] J. Mingorance, S. Rueda, P. Go mez-Puertas, A. Valencia, M. Vicente, Mol.
Microbiol. 41 (2001) 83e91.
[20] S. Ma, C. Cong, X. Meng, S. Cao, H. Yang, Y. Guo, X. Lu, S. Ma, Bioorg. Med.
Chem. Lett. 23 (2013) 4076e4079.
[21] S. Ma, R. Wang, Y. Wang, J. Cao, S. Ma, Lett. Drug Des. Discov. 10 (2013)
320e326.
[22] J.H. Jorgensen, Methods for Dilution Antimicrobial Susceptibility Tests for
Bacteria that Grow Aerobically: Approved Standard: NCCLS Document
M7eA3, NCCLS, 1993.
[23] P.S. Hameed, V. Patil, S. Solapure, U. Sharma, P. Madhavapeddi, A. Raichurkar,
M. Chinnapattu, P. Manjrekar, G. Shanbhag, J. Puttur, V. Shinde,
S. Menasinakai, S. Rudrapatana, V. Achar, D. Awasthy, R. Nandishaiah,
V. Humnabadkar, A. Ghosh, C. Narayan, V.K. Ramya, P. Kaur, S. Sharma,
J. Werngren, S. Hoffner, V. Panduga, C.N.N. Kumar, J. Reddy, K.N.M. Kumar,
S. Ganguly, S. Bharath, U. Bheemarao, K. Mukherjee, U. Arora, S. Gaonkar,
M. Coulson, D. Waterson, V.K. Sambandamurthy, S.M. de Sousa, J. Med. Chem.
57 (2014) 4889e4905.
[24] N.R. Stokes, J. Sievers, S. Barker, J.M. Bennett, D.R. Brown, I. Collins,
V.M. Errington, D. Foulger, M. Hall, R. Halsey, J. Biol. Chem. 280 (2005)
39709e39715.
[25] J.M. Andrews, J. Antimicrob. Chemother. 48 (2001) 5e16.
[26] D.G. Gibson, L. Young, R.-Y. Chuang, J.C. Venter, C.A. Hutchison, H.O. Smith,
Nat. Meth. 6 (2009) 343e345.
[27] A. Mukherjee, J. Lutkenhaus, J. Bacteriol. 181 (1999) 823e832.
[28] D. Awasthi, K. Kumar, S.E. Knudson, R.A. Slayden, I. Ojima, J. Med. Chem. 56
(2013) 9756e9770.
[29] K. Kumar, D. Awasthi, S.Y. Lee, I. Zanardi, B. Ruzsicska, S. Knudson, P.J. Tonge,
R.A. Slayden, I. Ojima, J. Med. Chem. 54 (2010) 374e381.