European Journal of Medicinal Chemistry 97 (2015) 32e41

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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

bends and Z ring contracts, leading to the closure of the septum and
The rapidly increasing bacterial resistance to conventional an- then the completement of the cell division [8,9]. As FtsZ is attractive
tibiotics has resulted in the enormous difficulty in fighting against and largely unexploited as a new target for the development of
bacterial infections [1e3]. Especially, the recently emerging New antimicrobials, it has aroused many interests among researchers for
Delhi metallo-b-lactamase 1 (NDM-1) superbugs has made almost the exploration of novel antimicrobials against resistant bacteria in
all of the first-line clinical antibiotics ineffective [4]. The weak or no recent years [10e12].
activity of the originally powerful antibiotics in clinic against the The recently intensive investigation of FtsZ inhibitors as anti-
resistant bacteria [5,6] has necessitated a proactive effort to bacterial agents leads to various anti-FtsZ molecules, such as cin-
discover novel antimicrobial pharmacophores with new mecha- namaldehyde, totarol and PC190723 (Fig. 1) [8,13,14]. Among these
nisms [7]. The filamentous temperature-sensitive protein Z (FtsZ) inhibitors, cinnamaldehyde displays broad-spectrum antibacterial
plays an important role in the bacterial cell division and is widely activity with MIC values of 1000 and 500 mg/mL against Escherichia
conserved in the bacterial kingdom as well as absent in the mito- coli (E. coli) and Bacillus subtilis (B. subtilis), respectively [13]. In the
chondria of higher eukaryotes. In the process of bacterial cell di- target identification, FtsZ has been confirmed to be the target of
vision, FtsZ forms single-stranded filaments and then the highly cinnamaldehyde with the application of standard molecular
dynamic Z-ring scaffold, followed by the recruitment of other cell 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].
* Corresponding author.
E-mail address: [email protected] (S. Ma). 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 33

O NH2

F F
OH
S
O
N
O N

Cl
cinnamaldehyde totarol PC190723

Fig. 1. Structures of cinnamaldehyde, totarol and PC190723.

antibacterial activity, has also been substantiated to target FtsZ filamentation of B. subtilis, E. coli and P. aeruginosa or ballooning of
using light scattering assay. However, cinnamaldehyde is to be S. aureus appeared was recorded as cell division inhibitory con-
studied as a lead compound of FtsZ inhibitors against the growing centration indicating their on-target activity. The results of mini-
bacterial resistance. mum antibacterial activity and cell division inhibitory activity in
As a continuous work of our previous investigation searching for the unit of mg/mL are shown in Table 1 and Table 2.
novel and potent anti-FtsZ agents [20,21], we have carried out a The cinnamaldehyde derivatives were derived from cinna-
novel program to design and synthesize a new library of cinna- maldehyde which displayed a definitely FtsZ-targeted antibacterial
maldehyde derivatives for screening potential FtsZ inhibitors. It is activity and influenced FtsZ polymerization. To determine the ef-
hoped that the cinnamaldehyde derivatives could exhibit more fects of these newly synthetic compounds on the polymerization of
potent anti-FtsZ effect with a higher antibacterial efficacy and a FtsZ, a standard light scattering assay was carried out for three
broader antibacterial spectrum. representatives. The inhibition of selected compounds on FtsZ
polymerization was shown in Fig. 2.
2. Chemistry As the polymerization and depolymerization of FtsZ depended
on the hydrolysis of GTP, the production of inorganic phosphate
In the present work, the target compounds 3e39 were synthe- could reflect the activity of FtsZ. To further validate the FtsZ-
sized from substituted benzaldehyde 1 as outlined in Scheme 1. The targeted mechanism of cinnamaldehyde derivatives, the light
reaction of 1 with malonic acid gave cinnamic acid 2 in the presence scattering assay was carried out for three representatives. The in-
of pyridine through the Knoevenagel condensation. The chlorina- hibition of selected compounds on FtsZ GTPase was shown in Fig. 3.
tion of 2 with oxalyl chloride was followed by the amidation re-
action with different amides in the presence of triethylamine to 4. Results and discussion
provide the target compounds 3e39. The newly synthesized com-
pounds were characterized by MS, 1H NMR and IR, and all the In the in vitro antibacterial activity, all the newly synthesized
spectral data were in agreement with the proposed structures. compounds displayed superior or comparable efficacy to the pre-
cursor cinnamic acid, indicating a successful modification in this
3. Antibacterial evaluation program. Especially, the subseries of compounds containing 2-
methylbenzimidazolyl moiety generally showed better efficacy
All the synthesized compounds (3e39) were determined for than the others against all the tested strains. It was owing to the
their biological activity, including the in vitro antibacterial activity special structure of this fragment that was able to interact with
and the cell division inhibitory activity. The in vitro antibacterial receptors via hydrogen bonds and/or hydrophobic interactions. As
activity was performed with the application of the standard broth the previous study revealed that cinnamaldehyde bound around
microdilution method recommended by NCCLS [22]. Cinnamic acid, the T7 loop of the C-terminal domain [13] and PC190723 bound to
curcumin and ciprofloxacin were selected as the reference agents in FtsZ in a cleft between helix seven (H7) and the C-terminal domain,
this test. Among these references, cinnamic acid and curcumin it was rational to deduce that cinnamaldehyde derivatives might
were used as the FtsZ-targeted references. And ciprofloxacin, as a partially bound to the binding region of PC190723 [8]. And the 2-
representative fluoroquinolone antibacterial, was applied as the methylbenzimidazolyl moiety was predicted to bind into the hy-
reference targeting bacterial DNA gyrase. It binded to the GyrA drophobic channel formed by amino acid residues I172, E185, N188,
subunit of DNA gyrase belonging to the type II topoisomerase I228 and I230, which also were the binding sites of the thiazolo-
family, and then traped the double strand cleaved DNA
gyrase pyridine portion of PC190723 [8]. Additionally, the nitrogen atoms
complex, leading to bacterial cell death [23]. The tested strains of 2-methylbenzimidazolyl moiety might form hydrogen bonds
includes three strains of Staphylococcus aureus (S. aureus) with amino acid residues R191 and Q192, which formed hydrogen
ATCC25923, S. aureus ATCC29213 (MRSA) and S. aureus PR, two bonds with the phenoxy ether of PC190723 as well [8]. Among
strains of Streptococcus pyogenes (S. pyogenes) PS and S. pyogenes these outstanding compounds, 3, 8 and 10 exhibited the most
PR, one strain of B. subtilis ATCC9372 and one strain of Pseudomonas excellent activity with MIC values of 4, 10 and 4 mg/mL against
aeruginosa (P. aeruginosa) ATCC27853, one clinically isolated strain S. aureus ATCC25923, comparable or superior to all the references.
of Staphylococcus epidermidis (S. epidermidis), and one strain of Furthermore, 4 and 10 exerted antibacterial activity with the same
E. coli ATCC25922. MIC value of 4 mg/mL against S. epidermidis, over 32-fold better than
The cell division inhibitory activity of these compounds against all the reference drugs. These outstanding compounds, except 3
B. subtilis, E. coli, P. aeruginosa and S. aureus was assessed by having no substituent at the benzene ring, had chlorine atom(s) at
analyzing the cell length with the application of phase-contrast the 4- or/and 2-position of the benzene ring, implying that the
light microscopy [24]. The minimum drug concentration at which 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- binding site. In this subseries, 20, 28, 29 and 30 exhibited better
position has the comparable activity to its 4-chlorine congener 8 antibacterial activity against S. aureus ATCC25923 and S. aureus PR
against all the tested strains. However, 4-methoxy derivative 9 than the others. For instance, 20 and 28 exerted the same efficacy
showed a decreased activity against most pathogens tested. All the against S. aureus ATCC25923 and S. aureus PR with MIC values of 32
results above indicated that the small group at the 4- or/and 2- and 64 mg/mL, respectively.
position of the benzene ring might increase the entire activity In the cell division inhibitory assay, most of the tested com-
against various pathogens. This might be due to the binding region pounds showed more potency against S. aureus ATCC25923 than
of cinnamaldehyde derivatives that was volume-limited and had the other tested strains. And 2-methylbenzimidazolyl derivatives
the existing amino acid residues L209, L200, N208 and G205 displayed more advantages in general than the others in the inhi-
around the T7 loop forming hydrophobic interactions with the 2,4- bition of the tested pathogens, exerting similar trends to their
disubstituted benzene ring [13]. In the other subseries, the amides in vitro antibacterial activity. Among these derivatives, 3, 6, 7 and 8
with two substituents generally displayed better efficacy than exhibited the most potent activity with MIC values of 0.25, 0.50,
those with one substituent, revealing hydrophobic interaction 0.50, and 0.50 mg/mL, respectively, similar to the previously
existing between the ligand and the receptor around the amide discovered most potent FtsZ inhibitors [8]. In the other subseries,
group or no hydrogen bond donator at the receptor near the 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 compounds. The inhibition of GTPase activity resulted in the
results described above revealed that the amides with hydrophobic instability of the FtsZ polymer, leading to the abnormal bacterial
bulky group might increase their cell division inhibitory activity cell division and then the cell death.
and the basic substituents at the amide nitrogen probably In this program, all the active compounds are promising, rep-
contributed to the entire cell division inhibition. resenting a new array of molecules with novel scaffold targeting
In the light scattering assay, all the tested compounds displayed FtsZ. The further biological investigation of these compounds is
an obvious inhibition on FtsZ polymerization in a dose-dependent underway.
manner (Fig. 2). Among them, compound 10 displayed the best
activity in the various concentration range, and it exhibited sig- 5. Conclusions
nificant activity even at the concentration of 30 mg/mL, similar to
compound 6 at the concentration of 120 mg/mL. The results indi- In conclusion, a novel library of cinnamaldehyde derivatives were
cated that these compounds inhibited the proliferation of bacteria designed, synthesized and tested for their in vitro antibacterial ac-
via influencing the function of FtsZ. tivity and cell inhibitory activity against various pathogen strains,
In the GTPase assay, all the tested compounds showed an including Gram-positive and -negative bacteria. In addition, the light
apparent effect on the GTPase activity of FtsZ and the effect was scattering assay and GTPase assay were carried out for the further
dose-dependent. Especially, compound 10 showed an inhibition of validation of the FtsZ-targeted mechanism. Generally, the cinna-
50% at the concentration of 30 mg/mL, superior to the other 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 were of analytical grade. Reactions progress was monitored by
strains, and many compounds in this subseries showed superior or thin-layer chromatography (TLC) on 0.25-mm pre-coated silica
comparable antibacterial activity to the references. Moreover, these GF254 plates (Qingdong Yumingyuan silica gel reagent factory,
active compounds commonly displayed better activity against Shandong, China, YUYUAN). Flash column chromatography was
S. aureus ATCC25923 than the other strains. In addition, almost all carried out with the indicated solvents using silica gel 60 (particle
the compounds exhibited similar in vitro antibacterial activity size 0.040e0.063 mm, Qingdong Yumingyuan silica gel reagent
against B. subtilis ATCC 9372, E. coli ATCC 25922 and P. aeruginosa factory, Shandong, China, YUYUAN). Mass spectra were obtained on
ATCC 27853, suggesting little or no effects of different amide frag- the API 4000 instrument (Applied Biosystems, Connecticut, USA)
1
ments of these molecules on the antibacterial activity. In cell division H nuclear magnetic resonance (1H NMR) spectra were recorded on
inhibitory activity, the compounds showed preferable activity the Bruker Avance DRX 400 spectrometer (Bruker, Switzerlands)
against the S. aureus ATCC25923 to the other strains with the min- using appropriate deuterated solvents and were expressed in parts
imum cell division inhibitory activity of 0.25 mg/mL, over 512-fold per million (d, ppm) downfield from tetramethylsilane (internal
better than all the references. Moreover, three representatives standard). Infrared (IR) spectra were recorded on the Nicolet Nexus
were selected for the further evaluation using light scattering assay 470FT-IR spectrometer (Wisconsin, USA) using KBr pellets. Melting
and GTPase assay, and the results strongly validated the mechanism points were uncorrected and determined with the X-6 melting
of these compounds. These results indicated that the subseries were point apparatus (Beijing Tianchengwode Biotech Co. Ltd, Beijing,
promising and worth of further investigation for potent FtsZ- China). Bacterial morphometric analysis was determined on the
targeted antibacterial agents especially against S. aureus ATCC25923. 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 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.

Fig. 2. Inhibition of FtsZ polymerization by 6 (A), 8 (B) and 10 (C).

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,
hydrochloric acid and then was cooled to 0  C. Then the residue was Rf ¼ 0.61 (dichloromethane/methanol, 10:1); 1H NMR (400 MHz,
filtered, washed with cold water (2  10 mL) and dried in vacuum for DMSO-d6, d ppm) 8.12 (d, 1H, J ¼ 16 Hz), 7.85e7.83 (m, 1H),
12 h to afford corresponding crude product 2 in yields ranging from 7.81e7.79 (m, 1H), 7.66e7.64 (m, 1H), 7.55e7.48 (m, 2H), 7.36e7.30
30.2 to 100.0%, which was used directly without further purification. (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,
6.1.2. General procedure for cinnamaldehyde derivatives (3e31) 1549, 1489, 1455, 1432, 1380, 1348, 1339, 1308, 1279, 1247, 1236,
To a solution of intermediate 2 (1.35 mmol) in THF (10 mL) at 1167, 1124, 1113, 1051, 1037, 1019 cm
1; HRMS (ESI) m/z calcd. for
0  C was slowly added oxalyl chloride (4.05 mmol). The reaction C18H16N2O2 293.1285 [MþH]þ, found: 293.1289.
solution was stirred for 15e25 min at the same temperature and
concentrated in vacuo to give substituted cinnamoyl chloride. The 6.1.2.4. (E)-3-(4-fluorophenyl)-1-(2-methyl-1H-benzo[d]imidazol-1-
resulting crude product and TEA (4.05 mmol) was dissolved in DMF yl)prop-2-en-1-one (6). White solid, yield 67.6%, mp 138e140  C,
(5 mL) in an ice bath, and the corresponding amine (2.03 mmol) in Rf ¼ 0.59 (dichloromethane/methanol, 10:1); 1H NMR (400 MHz,
DMF (2 mL) was dropwise added. The resulting mixture was slowly DMSO-d6, d ppm) 7.99e7.93 (m, 3H), 7.79e7.77 (m, 1H), 7.66e7.64
warmed to room temperature and stirred for 18 h at the same (m, 1H), 7.45 (d, 1H, J ¼ 15.6 Hz), 7.37e7.32 (m, 4H), 2.79 (s, 3H); IR
temperature. After adding water (20 mL), the compounds 3e39 (KBr): 3069, 2937, 1698, 1620, 1598, 1546, 1508, 1456, 1418, 1380,
were extracted with ethyl acetate (3  20 mL), and then the 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: 6.1.2.11. N-(4-methoxyphenyl)cinnamamide (13). Brown solid, yield
281.1088. 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
6.1.2 .5. (E) -1-(2-m ethyl-1 H-b enzo[d]imidazol-1-yl)-3-(4- (m, 5H), 7.47e7.40 (m, 3H), 6.93e6.91 (m, 2H), 6.82 (d, 1H,
nitrophenyl)prop-2-en-1-one (7). Brown solid, yield 90.6%, mp J ¼ 15.6 Hz), 3.74 (s, 3H); IR (KBr): 3248, 3126, 3060, 2931, 2832,
178e181  C, Rf ¼ 0.60 (dichloromethane/methanol, 10:1); 1H NMR 1654, 1619, 1600, 1540, 1509, 1464, 1449, 1412, 1344, 1297, 1239,
(400 MHz, DMSO-d6, d ppm) 8.32 (d, 2H, J ¼ 8.8 Hz), 8.16 (d, 2H, 1178, 1108, 1038 cm
1; HRMS (ESI) m/z calcd. for C16H15NO2
J ¼ 8.8 Hz), 8.02 (d, 1H, J ¼ 16 Hz), 7.82e7.79 (m, 1H), 7.70 (d, 1H, 254.1176 [MþH]þ, found: 254.1177.
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, 6.1.2.12. (E)-N-butyl-3-(4-cyanophenyl)acrylamide (14). White
1428, 1341, 1309, 1291, 1268, 1237, 1163, 1114, 1034 cm
1; HRMS solid, yield 80.8%, mp 98e100  C, Rf ¼ 0.60 (dichloromethane/
(ESI) m/z calcd. for C17H13N3O3 308.1030 [MþH]þ, found: 308.1035. 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,
6.1.2.6. (E)-3-(4-chlorophenyl)-1-(2-methyl-1H-benzo[d]imidazol-1- 1H, J ¼ 16 Hz), 6.77 (d, 1H, J ¼ 16 Hz), 3.22e3.17 (m, 2H), 1.49e1.42
yl)prop-2-en-1-one (8). White solid, yield 60.6%, mp 94e97  C, (m, 2H), 1.37e1.29 (m, 2H), 0.90 (t, 3H, J ¼ 7.4 Hz); IR (KBr): 3265,
Rf ¼ 0.55 (dichloromethane/methanol, 10:1); IR (KBr): 3473, 3369, 3069, 2971, 2924, 2861, 2228, 1657, 1621, 1552, 1508, 1466, 1431,
3081, 2972, 2927, 1695, 1624, 1591, 1568, 1539, 1491, 1454, 1436, 1411, 1336, 1308, 1281, 1229, 1158, 1119 cm
1; HRMS (ESI) m/z calcd.
1408, 1382, 1346, 1314, 1286, 1266, 1240, 1208, 1155, 1104, 1092, for C14H16N2O 229.1335 [MþH]þ, found: 229.1332.
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), 6.1.2.13. (E)-N-butyl-3-(4-nitrophenyl)acrylamide (15). Yellow
7.50 (d, 1H, J ¼ 15.6), 7.36e7.31 (m, 2H), 2.79 (s, 3H); MS (ESI) m/z solid, yield 57.7%, mp 121e124  C, Rf ¼ 0.62 (dichloromethane/
calcd for C17H13ClN2O 296.07; found [MþH]þ 297.5. 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,
6.1.2.7. (E)-3-(4-methoxyphenyl)-1-(2-methyl-1H-benzo[d]imidazol- J ¼ 16 Hz), 3.22e3.17 (m, 2H), 1.48e1.42 (m, 2H), 1.35e1.30 (m, 2H),
1-yl)prop-2-en-1-one (9). White solid, yield 81.8%, mp 83e86  C, 0.92e0.87 (m, 3H); IR (KBr): 3305, 3064, 2956, 2934, 2873, 1654,
Rf ¼ 0.57 (dichloromethane/methanol, 10:1); 1H NMR (400 MHz, 1615, 1594, 1549, 1515, 1473, 1455, 1411, 1342, 1221, 1108, 1022 cm
1;
DMSO-d6, d ppm) 7.92 (d, 1H, J ¼ 15.6 Hz), 7.86 (d, 2H, J ¼ 8.8 Hz), HRMS (ESI) m/z calcd. for C13H16N2O3 249.1234[MþH]þ, found:
7.77e7.74 (m, 1H), 7.65e7.63 (m, 1H), 7.35e7.30 (m, 3H), 7.06 (d, 2H, 249.1231.
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, 6.1.2.14. (E)-N-(3-(2-methyl-1H-imidazol-1-yl)-5-(trifluoromethyl)
1298, 1255, 1166, 1109, 1038, 1027, 1008 cm
1; HRMS (ESI) m/z phenyl)-3-(4-nitrophenyl)acrylamide (16). Yellow solid, yield 44.2%,
calcd. for C18H16N2O2 293.1285 [MþH]þ, found: 293.1286. 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,
6.1.2.8. (E)-3-(2,4-dichlorophenyl)-1-(2-methyl-1H-benzo[d]imida- J ¼ 8.8 Hz), 8.21 (d, 1H, J ¼ 1.2 Hz), 8.11e8.08 (m, 1H), 8.02 (s, 1H),
zol-1-yl)prop-2-en-1-one (10). Light yellow solid, yield 83.3%, mp 7.92e7.90 (m, 1H), 7.78e7.71 (m, 1H), 7.42 (d, 1H, J ¼ 40.4 Hz),
108e111  C, Rf ¼ 0.61 (dichloromethane/methanol, 10:1); IR (KBr): 7.00e6.94 (m, 2H), 6.82 (s, 1H), 5.83 (d, 1H, J ¼ 47.6 Hz), 2.19e2.16
3058, 2925, 1698, 1617, 1609, 1582, 1550, 1469, 1456, 1387, 1352, (m, 3H); IR (KBr): 3354, 3099, 2973, 2927, 1687, 1638, 1621, 1577,
1335, 1308, 1294, 1275, 1237, 1168, 1113, 1099, 1052, 1035 cm
1; 1H 1519, 1496, 1449, 1405, 1383, 1339, 1317, 1258, 1239, 1203, 1165,
NMR (400 MHz, DMSO-d6, d ppm) 8.15e8.06 (m, 2H), 7.81e7.79 (m, 1127, 1111, 1080, 1049, 1010 cm
1; HRMS (ESI) m/z calcd. for
2H), 7.66e7.63 (m, 1H), 7.61e7.56 (m, 2H), 7.35e7.32 (m, 2H), 2.80 C20H15F3N4O3 417.1169 [MþH]þ, found: 417.1171.
(s, 3H); MS (ESI) m/z calcd for C17H12Cl2N2O 330.03; found [MþH]þ
331.3. 6.1.2.15. (E)-3-(4-nitrophenyl)-N-propylacrylamide (17). Yellow
solid, yield 91.7%, mp 125e128  C, Rf ¼ 0.56 (dichloromethane/
6.1.2.9. (E)-3-(3,4-dimethoxyphenyl)-1-(2-methyl-1H-benzo[d]imi- methanol, 10:1); 1H NMR (400 MHz, DMSO-d6, d ppm) 8.27e8.25
dazol-1-yl)prop-2-en-1-one (11). Yellow solid, yield 80.6%, mp (m, 3H), 7.84e7.82 (d, 2H, J ¼ 8.8 Hz), 7.55e7.51 (d, 1H, J ¼ 16 Hz),
135e138  C, Rf ¼ 0.53 (dichloromethane/methanol, 10:1); 1H NMR 6.83 (d, 1H, J ¼ 16 Hz), 3.19e3.14 (m, 2H), 1.54e1.45 (m, 2H),
(400 MHz, DMSO-d6, d ppm) 7.90 (d, 1H, J ¼ 15.6 Hz), 7.76e7.74 (m, 0.92e0.86 (m, 3H); IR (KBr): 3299, 3257, 3077, 2964, 2923, 2875,
1H), 7.66e7.64 (m, 1H), 7.51 (d, 1H, J ¼ 1.6 Hz), 7.45 (dd, 1H, 1655, 1623, 1594, 1555, 1516, 1462, 1428, 1413, 1348, 1337, 1286,
J ¼ 1.6 Hz, J ¼ 1.6 Hz), 7.38 (d, 1H, J ¼ 15.6 Hz), 7.35e7.30 (m, 2H), 1250, 1226, 1182, 1156, 1109 cm
1; HRMS (ESI) m/z calcd. for
7.07 (d, 1H, J ¼ 8.4 Hz), 3.84 (d, 6H, J ¼ 1.2 Hz), 2.78 (s, 3H); IR (KBr): C12H14N2O3 235.1077 [MþH]þ, found: 235.1075.
3073, 3054, 2993, 2937, 2839, 1683, 1609, 1593, 1580, 1515, 1455,
1425, 1382, 1340, 1307, 1274, 1254, 1235, 1174, 1159, 1138, 1114, 1035, 6.1.2.16. (E)-3-(4-nitrophenyl)-N-(prop-2-ynyl)acrylamide (18).
1024 cm
1; HRMS (ESI) m/z calcd. for C19H18N2O3 323.1390 White solid, yield 61.5%, mp 234e237  C, Rf ¼ 0.67 (dichloro-
[MþH]þ, found: 323.1391. 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,
6.1.2.10. (E)-1-(2-methyl-1H-benzo[d]imidazol-1-yl)-3-(3,4,5- J ¼ 8.8 Hz), 7.58 (d, 1H, J ¼ 16 Hz), 6.82 (d, 1H, J ¼ 16 Hz), 4.03e4.00
trimethoxyphenyl)prop-2-en-1-one (12). White solid, yield 66.7%, (m, 2H), 3.19 (t, 1H, J ¼ 2.4 Hz); IR (KBr): 3258, 3077, 2961, 2927,
mp 172e175  C, Rf ¼ 0.61 (dichloromethane/methanol, 10:1); 1H 2851, 1662, 1625, 1600, 1558, 1513, 1420, 1346, 1331, 1261, 1226,
NMR (400 MHz, DMSO-d6, d ppm) 7.88 (d, 1H, J ¼ 15.6 Hz), 1183, 1110, 1041 cm
1; HRMS (ESI) m/z calcd. for C12H10N2O3
7.76e7.74 (m, 1H), 7.66e7.64 (m, 1H), 7.49 (d, 1H, J ¼ 15.6 Hz), 231.0764 [MþH]þ, found: 231.0766.
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, 6.1.2.17. (E)-N-isopropyl-3-(4-nitrophenyl)acrylamide (19).
1421, 1378, 1324, 1298, 1269, 1241, 1169, 1126, 1111, 1035, 1004 cm
1; White solid, yield 66.7%, mp 165e167  C, Rf ¼ 0.48 (dichloro-
HRMS (ESI) m/z calcd. for C20H20N2O4 353.1496 [MþH]þ, found: methane/methanol, 10:1); 1H NMR (400 MHz, DMSO-d6, d ppm)
353.1498. 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 39

J ¼ 8.8 Hz), 7.52 (d, 1H, J ¼ 16 Hz), 6.79 (d, 1H, J ¼ 15.6), 4.00e3.93 1455, 1417, 1435, 1339, 1303, 1260, 1242, 1216, 1167, 1138, 1039,
(m, 1H), 1.13 (d, 6H, J ¼ 6.8 Hz); IR (KBr): 3303, 3078, 2974, 2925, 1017 cm
1; HRMS (ESI) m/z calcd. for C14H15NO3 246.1125 [MþH]þ,
2850, 1655, 1618, 1598, 1543, 1515, 1467, 1407, 1346, 1277, 1224, 1173, found: 246.1120.
1130, 1109, 1004 cm
1; HRMS (ESI) m/z calcd. for C12H14N2O3
235.1077 [MþH]þ, found: 235.1076. 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-
6.1.2.18. (E)-3-(4-methoxyphenyl)-N-(pyridin-3-yl)acrylamide (20). methane/methanol, 10:1); 1H NMR (400 MHz, DMSO-d6, d ppm)
White solid, yield 67.9%, mp 150e154  C, Rf ¼ 0.50 (dichloro- 7.96 (t, 1H, J ¼ 5.4 Hz), 7.34 (d, 1H, J ¼ 16 Hz), 7.14e7.09 (m, 2H), 6.98
methane/methanol, 10:1); 1H NMR (400 MHz, DMSO-d6, d ppm) (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),
10.35 (s, 1H), 8.84 (d, 1H, J ¼ 2.4 Hz), 8.29e8.27 (m, 1H), 8.17e8.14 3.19e3.14 (m, 2H), 1.47e1.40 (m, 2H), 1.36e1.23 (m, 2H), 0.89 (t, 3H,
(m, 1H), 7.61e7.57 (m, 3H), 7.37 (dd, 1H, J ¼ 4.8 Hz, J ¼ 4.8 Hz), 7.02 J ¼ 7.4 Hz); IR (KBr): 3292, 3007, 2961, 2934, 2872, 1652, 1614, 1579,
(d, 2H, J ¼ 8.8 Hz), 6.70 (d, 1H, J ¼ 15.6 Hz), 3.81 (s, 3H); IR (KBr): 1549, 1513, 1462, 1437, 1419, 1363, 1320, 1263, 1213, 1192, 1160, 1142,
3242, 3184, 3119, 3059, 3000, 1682, 1622, 1604, 1589, 1553, 1513, 1037, 1026 cm
1; HRMS (ESI) m/z calcd. for C15H21NO3 264.1594
1477, 1427, 1346, 1304, 1287, 1251, 1194, 1169, 1129, 1113, 1025 cm
1; [MþH]þ, found: 264.1595.
HRMS (ESI) m/z calcd. for C15H14N2O2 255.1128 [MþH]þ, found:
255.1128. 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-
6.1.2.19. (E)-3-(2,4-dichlorophenyl)-N-propylacrylamide (21). methane/methanol, 10:1); 1H NMR (400 MHz, DMSO-d6, d ppm)
White solid, 88.1%, mp 144e146  C, Rf ¼ 0.12 (petroleum ether/ 7.81e7.78 (m, 2H), 7.51 (d, 1H, J ¼ 15.2 Hz), 7.27e7.20 (m, 3H),
ethyl acetate, 2:1); 1H NMR (400 MHz, DMSO-d6, d ppm) 8.23 (s, 3.71e3.57 (m, 8H); IR (KBr): 3069, 2972, 2916, 2867, 1648, 1610,
1H), 7.72 (s, 2H), 7.66e7.62 (d, 1H, J ¼ 15.6 Hz), 7.51e7.49 (d, 1H, 1598, 1511, 1428, 1410, 1364, 1306, 1267, 1213, 1196, 1163, 1113, 1073,
J ¼ 8 Hz), 6.71e6.67 (d, 1H, J ¼ 11.6 Hz), 3.15e3.14 (d, 2H, J ¼ 5.6 Hz), 1049 cm
1; HRMS (ESI) m/z calcd. for C13H14FNO2 236.1081
1.50e1.45 (q, 2H, J ¼ 7.2 Hz), 0.90e0.86 (t, 3H, J ¼ 7.2 Hz); MS (ESI) [MþH]þ, found: 236.1084.
m/z calcd for C12H13Cl2NO 257.04; found [MþH]þ 258.3.
6.1.2.27. (E)-3-(2-chlorophenyl)-1-(4-((4-chlorophenyl) (phenyl)
6.1.2.20. (E)-3-(2,4-dichlorophenyl)-N-isopropylacrylamide (22).
methyl)piperazin-1-yl)prop-2-en-1-one (29). Yellow solid, yield
White solid, 89.4%, mp 178e180  C, Rf ¼ 0.18 (petroleum ether/
62.3%, mp 82e84  C, Rf ¼ 0.75 (dichloromethane/methanol, 10:1);
ethyl acetate, 2:1); 1H NMR (400 MHz, DMSO-d6, d ppm) 8.14e8.12 1
H NMR (400 MHz, DMSO-d6, d ppm) 7.97e7.94 (m, 1H), 7.81e7.77
(d, 1H, J ¼ 6.8 Hz), 7.72e7.61 (m, 3H), 7.52e7.50 (d, 1H, J ¼ 8 Hz),
(d, 1H, J ¼ 15.2 Hz), 7.52e7.50 (m, 1H), 7.48e7.46 (d, 2H, J ¼ 8.4 Hz),
6.68e6.64 (d, 1H, J ¼ 15.6 Hz) 3.98e3.95 (m, 1H), 1.12e1.10 (d, 6H,
7.43e7.41 (m, 2H), 7.40e7.36 (m, 4H), 7.32 (t, 2H, J ¼ 7.6 Hz), 7.27 (d,
J ¼ 6.4 Hz); MS (ESI) m/z calcd for C12H13Cl2NO 257.04; found
1H, J ¼ 15.2 Hz), 7.24 (d, 1H, J ¼ 1.2 Hz), 4.41 (s, 1H), 3.73e3.60 (m,
[MþH]þ 258.2.
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,
6.1.2.21. (E)-N-butyl-3-(2,4-dichlorophenyl)acrylamide (23).
1038 cm
1; HRMS (ESI) m/z calcd. for C26H24Cl2N2O 451.1338
White solid, 89.2%, mp 146e149  C, Rf ¼ 0.40 (petroleum ether/
[MþH]þ, found: 451.1340.
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, 6.1.2.28. (E)-1-(4-((4-chlorophenyl) (phenyl)methyl)piperazin-1-yl)-
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), 3-(2-methoxyphenyl)prop-2-en-1-one (30). White solid, yield
0.91e0.87 (t, 3H, J ¼ 7.2 Hz); MS (ESI) m/z calcd for C13H15Cl2NO 73.2%, mp 128e131  C, Rf ¼ 0.59 (dichloromethane/methanol,
271.05; found [MþH]þ 272.5. 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,
6.1.2.22. (E)-3-(2,4-dichlorophenyl)-N-hexylacrylamide (24). J ¼ 15.6 Hz), 7.05 (d, 1H, J ¼ 8.4 Hz), 6.98e6.94 (m, 1H), 5.76 (s, 2H),
White solid, 87.6%, mp 104e106  C, Rf ¼ 0.63 (petroleum ether/ 4.40 (s, 1H), 3.84 (s, 3H), 3.64 (d, 4H, J ¼ 40.4 Hz), 2.42 (d, 4H,
ethyl acetate, 2:1); 1H NMR (400 MHz, DMSO-d6, d ppm) 8.22 (s, J ¼ 78.4 Hz); IR (KBr): 3427, 3026, 2998, 2959, 2919, 2807, 2760,
1H), 7.72e7.71 (m, 2H), 7.66e7.62 (d, 1H, J ¼ 15.6 Hz), 7.51e7.49 (d, 1643, 1600, 1488, 1463, 1427, 1370, 1312, 1246, 1226, 1195, 1162,
1H, J ¼ 8 Hz), 6.71e6.67 (d, 1H, J ¼ 15.6 Hz), 3.18e3.17 (d, 2H, 1144, 1106, 1085, 1044, 1000 cm
1; HRMS (ESI) m/z calcd. for
J ¼ 5.6 Hz), 1.45 (s, 2H), 1.27 (s, 6H), 0.87 (s, 3H); MS (ESI) m/z calcd C27H27ClN2O2 447.1834 [MþH]þ, found: 447.1835.
for C15H19Cl2NO 299.08; found [MþH]þ 300.5.
6.1.2.29. (E)-3-(2,4-dichlorophenyl)-1-morpholinoprop-2-en-1-one
6.1.2.23. (E)-3-(2,4-dichlorophenyl)-N-(4-methoxyphenyl)acryl- (31). White solid, 82.1%, mp 144e147  C, Rf ¼ 0.13 (petroleum
amide (25). White solid, 85.7%, mp 205e208  C, Rf ¼ 0.43 (petro- ether/ethyl acetate, 2:1); 1H NMR (400 MHz, DMSO-d6, d ppm)
leum ether/ethyl acetate, 2:1); 1H NMR (400 MHz, DMSO-d6, 8.08e8.06 (d, 1H, J ¼ 8.4 Hz), 7.79e7.75 (d, 1H, J ¼ 15.2 Hz), 7.71 (s,
d ppm) 10.21 (s, 1H), 7.80e7.78 (d, 1H, J ¼ 6 Hz), 7.76 (s, 2H), 1H), 7.52e7.50 (d, 1H, J ¼ 8 Hz), 7.38e7.34 (d, 1H, J ¼ 15.2 Hz), 3.73
7.63e7.61 (d, 2H, J ¼ 8.4 Hz), 7.56e7.54 (d, 1H, J ¼ 8 Hz), 6.94e6.91 (s, 2H), 3.61e3.58 (m, 6H); MS (ESI) m/z calcd for C13H13Cl2NO2
(d, 2H, J ¼ 8.8 Hz), 6.89e6.85 (d, 1H, J ¼ 16 Hz), 3.74 (s, 3H); MS (ESI) 285.03; found [MþH]þ 286.3.
m/z calcd for C16H13Cl2NO2 321.03; found [MþH]þ 322.4.
6.1.2.30. (E)-3-(2,4-dichlorophenyl)-N,N-diethylacrylamide (32).
6.1.2.24. (E)-3-(3,4-dimethoxyphenyl)-N-(prop-2-ynyl)acrylamide White solid, 87.5%, mp 95e98  C, Rf ¼ 0.37 (petroleum ether/ethyl
(26). White solid, yield 83.3%, mp 144e147  C, Rf ¼ 0.88 acetate, 2:1); 1H NMR (400 MHz, DMSO-d6, d ppm) 8.06e8.04 (d,
(dichloromethane/methanol, 10:1); 1H NMR (400 MHz, DMSO-d6, 1H, J ¼ 8.4), 7.77e7.71 (m, 2H), 7.50e7.48 (d, 1H, J ¼ 8.4 Hz),
d ppm) 8.43 (t, 1H, J ¼ 5.6 Hz), 7.40 (d, 1H, J ¼ 15.6 Hz), 7.17e7.11 7.22e7.18 (d, 1H, J ¼ 15.6 Hz), 3.54e3.52 (d, 2H, J ¼ 6.8 Hz),
(m, 2H), 6.99 (d, 1H, J ¼ 8 Hz), 6.51 (d, 1H, J ¼ 15.6 Hz), 3.99e3.97 3.39e3.37 (d, 2H, J ¼ 6.8 Hz), 1.17e1.13 (t, 3H, J ¼ 6.4 Hz), 1.10e1.06
(m, 2H), 3.79 (d, 6H, J ¼ 4 Hz), 3.14 (t, 1H, J ¼ 2.6 Hz); IR (KBr): 3251, (t, 3H, J ¼ 6.4 Hz); MS (ESI) m/z calcd for C13H15Cl2NO 271.05; found
3014, 2961, 2910, 2852, 1652, 1622, 1595, 1580, 1529, 1515, 1469, [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)- 2H), 3.83 (s, 6H), 3.68 (s, 3H), 3.18 (s, 3H), 2.93 (s, 3H); IR (KBr):
3-(2,4-dichlorophenyl)prop-2-en- 1-one (33). Light yellow solid, 3063, 2999, 2942, 2849, 1654, 1614, 1580, 1503, 1467, 1454, 1421,
89.5%, mp 73e76  C, Rf ¼ 0.24 (petroleum ether/ethyl acetate, 2:1); 1396, 1336, 1314, 1267, 1247, 1187, 1123, 1001 cm
1; HRMS (ESI) m/z
1
H NMR (400 MHz, DMSO-d6, d ppm) 8.01e7.99 (d, 1H, J ¼ 8.4 Hz), calcd. for C14H19NO4 266.1387 [MþH]þ, found: 266.1382.
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 6.2. Antibacterial evaluation
(s, 2H), 2.32 (s, 4H); MS (ESI) m/z calcd for C26H23Cl3N2O 484.09;
found [MþH]þ 485.5. 6.2.1. In vitro antibacterial assay
The minimum inhibitory concentration (MIC) of the tested
6.1.2.32. (E)-3-(3,4-dimethoxyphenyl)-N,N-diethylacrylamide (34). compounds was determined applying tube dilution method rec-
White solid, yield 56.0%, mp 88e90  C, Rf ¼ 0.59 (dichloromethane/ ommended by NCCLS [25]. Bacterial strains were incubated on
methanol, 10:1); 1H NMR (400 MHz, DMSO-d6, d ppm) 7.44 (d, 1H, MHA (Mueller Hinton Agar) medium at 37  C for 24 h. The bacteria
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), solution was prepared by suspension in 10 mL of sterile water for
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), colonies from culture on MHA medium. MHB (Mueller Hinton
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), Broth) was used for bacteria in this test. The cell density of each
1.07 (t, 3H, J ¼ 6.8 Hz); IR (KBr): 3389, 3075, 2964, 2930, 2839, 1639, inoculum was adjusted in sterile water of a 0.5 McFarland standard.
1593, 1512, 1478, 1459, 1423, 1378, 1363, 1305, 1261, 1228, 1161, 1138, In this method, various concentrations of the cinnamaldehyde de-
1079, 1037, 1018 cm
1; MS (ESI) m/z calcd. for C15H21NO3 [MþH]þ, rivatives were prepared from 128 to 0.25 mg/mL in sterile tubes No.
found: 264.3. 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
6.1.2.33. (E)-3-(3,4-dimethoxyphenyl)-1-morpholinoprop-2-en-1- tube 1. Two fold serial dilutions were carried out from tube 1 to
one (35). White solid, yield 55.6%, mp 120e123  C, Rf ¼ 0.45 tube 10 and excess broth (100 mL) was discarded from the last tube
(dichloromethane/methanol, 10:1); 1H NMR (400 MHz, DMSO-d6, No. 10. To each tube, 100 mL of standard inoculum (1.5  108 cfu/mL)
d ppm) 7.47 (d, 1H, J ¼ 15.2 Hz), 7.36 (d, 1H, J ¼ 1.6 Hz), 7.21 (dd, was added. Cinnamic acid, curcumin and ciprofloxacin were used as
1H, J ¼ 2 Hz, J ¼ 1.6 Hz), 7.11 (d, 1H, J ¼ 15.2 Hz), 6.96 (d, 1H, controls. Turbidity was observed after incubating the inoculated
J ¼ 8.4 Hz), 3.80 (d, 6H, J ¼ 13.2 Hz), 3.72 (s, 2H), 3.61e3.57 (m, 6H); tubes at 37  C for 24 h. The last tube with no growth of microor-
IR (KBr): 3016, 2967, 2928, 2847, 1644, 1596, 1515, 1457, 1431, 1409, ganism was recorded to represent the MIC value expressed in mg/
1364, 1331, 1301, 1266, 1253, 1241, 1193, 1145, 1108, 1046, mL.
1020 cm
1; HRMS (ESI) m/z calcd. for C15H19NO4 278.1387 [MþH]þ,
found: 278.1386. 6.2.2. Cell division inhibitory assay
Cell division inhibitory activity of the tested compounds was
6.1.2.34. (E)-3-(3,4-dimethoxyphenyl)-N,N-dimethylacrylamide (36). performed as described previously [24]. Overnight cultures were
Yellow solid, yield 58.3%, mp 100e113  C, Rf ¼ 0.57 (dichloro- grown in starvation medium supplemented with 1% hydrolyzed
methane/methanol, 10:1); 1H NMR (400 MHz, DMSO-d6, d ppm) casein and then diluted in starvation medium supplemented with
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, 3% hydrolyzed casein (B. subtilis) or in Mueller-Hinton medium
J ¼ 1.6 Hz), 7.08 (d, 1H, J ¼ 15.2 Hz), 6.96 (d, 1H, J ¼ 8.4 Hz), (S. aureus, E. coli and P. aeruginosa) and grown at 37  C. The culture
3.82e3.77 (m, 6H), 3.17 (s, 3H), 2.93 (s, 3H); IR (KBr): 2931, 2838, was diluted to A600 of ~0.06, and 10 mL aliquots were added to
1645, 1600, 1512, 1479, 1438, 1424, 1394, 1341, 1314, 1265, 1229, transparent 96-well microtiter plates containing dilutions of the
1164, 1138, 1021 cm
1; HRMS (ESI) m/z calcd. for C13H17NO3 cinnamaldehyde derivatives, cinnamic acid, curcumin and cipro-
236.1281 [MþH]þ, found: 236.1279. floxacin as controls in 100 mL volumes of medium. After incubation
for approximately 5 h (4e5 generations) at 37  C, 20 mL culture
6.1.2.35. (E)-N,N-diethyl-3-(3,4,5-trimethoxyphenyl)acrylamide (37). samples were transferred to poly-L-lysine-coated slides for micro-
White solid, yield 56.0%, mp 128e130  C, Rf ¼ 0.58 (dichloro- scopy. Cell morphology was assessed by phase-contrast light mi-
methane/methanol, 10:1); 1H NMR (400 MHz, DMSO-d6, d ppm) croscopy. Lowest concentration at which filamentation of B. subtilis,
7.43 (d, 1H, J ¼ 15.6 Hz), 7.03 (d, 3H, J ¼ 15.2 Hz), 3.84 (d, 6H, E. coli and P. aeruginosa or ballooning of S. aureus was recorded
J ¼ 8 Hz), 3.68 (s, 3H), 3.54 (d, 2H, J ¼ 6.8 Hz), 3.40e3.35 (m, 2H), represented the cell division inhibitory activity indicating on-target
1.15 (t, 3H, J ¼ 6.8 Hz), 1.07 (t, 3H, J ¼ 6.8 Hz); IR (KBr): 2969, 2939, activity.
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. 6.2.3. Expression and purification of E. coli FtsZ
for C16H23NO4 294.1700 [MþH]þ, found: 294.1704. FtsZ (GeneBank accession number: NC_000913) was recombi-
nant expressed in BL21 (DE3) cells as a soluble N-His6-tagged
6.1.2.36. (E)-1-morpholino-3-(3,4,5-trimethoxyphenyl)prop-2-en-1- fusion protein. The genomic DNA isolated from E. coli ATCC 25922
one (38). White solid, yield 57.7%, mp 127e130  C, Rf ¼ 0.71 was used as the template for polymerase chain reactions (PCR). And
(dichloromethane/methanol, 10:1); 1H NMR (400 MHz, DMSO-d6, the complete open reading frame (ORF) of FtsZ protein was
d ppm) 7.47 (d, 1H, J ¼ 15.2 Hz), 7.18 (d, 1H, J ¼ 15.2 Hz), 7.05 (s, amplified with the 50 primer FtsZ-F [50 - Gcatgactggtggacagc
2H), 3.83 (s, 6H), 3.74 (s, 2H), 3.68 (s, 3H), 3.59 (d, 6H, J ¼ 19.2 Hz); aaatgggtcgcGGATCCTTTGAACCAATGAACTTA-30 ] and the 30 primer
IR (KBr): 3447, 2952, 2848, 1644, 1596, 1584, 1506, 1455, 1419, 1341, FtsZ -R [50 - CggatctcagtggtggtggtggtggtgCTCGAGTTAATCGCT-
1323, 1296, 1273, 1251, 1228, 1193, 1157, 1122, 1066, 1046 cm
1; TACGCAGG-30 ]. Then, the PCR product was ligated with corre-
HRMS (ESI) m/z calcd. for C16H21NO5 308.1492 [MþH]þ, found: sponding predigested pET28a(þ) by Gibson assembly combination
308.1497. [26]. The ligation products were transformed into E. coli DH5a cells.
Plasmid containing FtsZ was confirmed by DNA sequencing and
6.1.2.37. (E)-N,N-dimethyl-3-(3,4,5-trimethoxyphenyl)acrylamide then transformed into E. coli BL21 (DE3).
(39). Brown solid, yield 54.2%, mp 120e123  C, Rf ¼ 0.57 The transformed cells were grown in LB medium (10 g/L tryp-
(dichloromethane/methanol, 10:1); 1H NMR (400 MHz, DMSO-d6, tone, 5 g/L yeast extract, 10 g/L NaCl) supplemented with 100 mg/ml
d ppm) 7.42 (d, 1H, J ¼ 15.6 Hz), 7.15 (d, 1H, J ¼ 15.2 Hz), 7.04 (s, kanamycin at 37  C. The temperature was decreased to 22  C when
 X. Li et al. / European Journal of Medicinal Chemistry 97 (2015) 32e41 41

the A600 reached 0.4e0.8, followed by the addition of isopropyl-1- Appendix A. Supplementary data
thio-b-D-galactopyranoside (0.2 mM) to induce the expression of
FtsZ proteins. After shaking for 18 h, the cells were pelleted and Supplementary data related to this article can be found at http://
lysed by sonication. FtsZ proteins were then purified using Ni dx.doi.org/10.1016/j.ejmech.2015.04.048.
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- References
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