Synthesis, structure, and biological assay of cinnamic amides as potential EGFR kinase inhibitors

Article abstract of DOI:10.1007/s00044-012-0093-z

A series of derivatives of cinnamic amide (compounds 2a-2v) were synthesized and evaluated for antiproliferative activities against the human breast cancer cell line MCF-7- and EGFR-inhibitory activities. The structures of compounds 2b and 2i were determined by single-crystal X-ray diffraction analysis. Compounds 2f and 2j showed moderate EGFR inhibitory activity with IC₅₀ values of 5.16 and 7.37 μM, respectively. Docking simulation of compound 2f was carried out to illustrate the binding mode of the molecule into the EGFR active site. Structure-activity relationship analysis found that the N-phenyl rings are required for enhancing the activities.

Full text of DOI:10.1007/s00044-012-0093-z

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2013) 22:986–994
DOI 10.1007/s00044-012-0093-z
ORIGINAL RESEARCH
Synthesis, structure, and biological assay of cinnamic amides
as potential EGFR kinase inhibitors
•
Mao Zhang Xiang Lu Hong-Jia Zhang Na Li
•
•
•
•
Yu Xiao Hai-Liang Zhu Yong-Hao Ye
•
Received: 4 November 2011 / Accepted: 14 May 2012 / Published online: 29 May 2012
Ó Springer Science+Business Media, LLC 2012
Abstract A series of derivatives of cinnamic amide
(compounds 2a–2v) were synthesized and evaluated for
antiproliferative activities against the human breast cancer
cell line MCF-7- and EGFR-inhibitory activities. The
structures of compounds 2b and 2i were determined by
single-crystal X-ray diffraction analysis. Compounds 2f
and 2j showed moderate EGFR inhibitory activity with
IC50 values of 5.16 and 7.37 lM, respectively. Docking
simulation of compound 2f was carried out to illustrate the
binding mode of the molecule into the EGFR active site.
Structure–activity relationship analysis found that the
N-phenyl rings are required for enhancing the activities.
designed to modulate or inhibit molecular targets are found
to show high selectivities and low toxicities (Seymour,
1999). One family of the targets is receptor tyrosine
kinases (RTKs), which play a crucial role in signal trans-
duction pathways that regulate various cellular mecha-
nisms (Herbst, 2004). Among the RTKs, epidermal growth
factor receptor tyrosine kinase (known as EGFR, HER-1,
or ErbB-1) is considered to be of great value in cancer
therapy (Traxler, 2003; Zhang et al., 2007). Possibly owing
to overexpressions, mutations, or autocrine expressions of
ligands (EGR or TGFa) (Slamon et al., 1987), hyperacti-
vation of EGFR induces aggressive tumor progression
and reduces sensitivity to antitumor drugs (Yarden and
Sliwkowski, 2001; Oda et al., 2005). EGFR overexpression
is widely found in breast cancer (Lv et al., 2010a, b), ovarian
cancer (Bull-Phelps et al., 2008), lung cancer (Yamamoto
et al., 2009), and hormone-refractory prostate cancer
(Wikstrand et al., 1997). Thus, searching for small mole-
cules to inhibit the EGFR activities shows great signifi-
cance in developing new anticancer agents (Bridges, 1999).
Cinnamic acid, which is ubiquitous in cinnamon oil and
many other balsams, is a naturally occurring aromatic fatty
acid of low toxicity with a long history of human exposure
(Liu et al., 1995). Commercially, it is widely applied in the
perfume industry because of its floral odor. Certain
substituted cinnamic amides containing cyano or fluoro
moieties are of particular value due to their inhibitory
effect in mitochondrial pyruvate transport (Hoskins, 1984).
Cinnamic amides were reported to have many different
biological activities such as anticancer, antimitotic, anti-
oxidant, and seed-germination inhibitory effects (Leslie
et al., 2010; Natella et al., 1999; Cutillo et al., 2003).
However, to our knowledge, few reports have been dedi-
cated to the EGFR inhibitory activity and the structure–
activity relationship (SAR) of cinnamic amide derivatives.
Keywords Cinnamic amides ꢀ MCF-7 ꢀ EGFR ꢀ
Docking simulation ꢀ Structure–activity relationship
Introduction
Conventional cytotoxic drugs for cancer chemotherapy are
associated with serious toxic side effects, while drugs
M. Zhang ꢀ Y. Xiao ꢀ Y.-H. Ye (&)
College of Plant Protection, Jiangsu Key Laboratory of Pesticide
Science, Nanjing Agricultural University, Nanjing 210095,
People’s Republic of China
e-mail: yeyh@njau.edu.cn
X. Lu ꢀ H.-J. Zhang ꢀ H.-L. Zhu (&)
State Key Laboratory of Pharmaceutical Biotechnology, Nanjing
University, Nanjing 210093, People’s Republic of China
e-mail: zhuhl@nju.edu.cn
N. Li
College of Science, Jiangsu Key Laboratory of Pesticide
Science, Nanjing Agricultural University, Nanjing 210095,
People’s Republic of China
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Med Chem Res (2013) 22:986–994
987
Herein, our interest is to seek how simple substituents on
the N-phenyl ring of cinnamic amides affect the EGFR
inhibitory activities. Therefore, we select a series of sub-
stituents for their electronic properties to modify the
N-phenyl ring leaving the other phenyl ring as a constant.
The synthesis route, biological assay and the SARs are
described here. Docking simulations were performed by
the X-ray crystallographic structure of the EGFR in com-
plex with inhibitors to explore the binding modes of these
compounds at the active site.
The crystal data are presented in Table 1, and Fig. 1a, b
give perspective views of 2b and 2i with the atomic
labeling system. Selected bond lengths, bond angles, and
torsion angles are listed in Table 2. All the bond lengths
and angles are in the normal ranges.
Biological assay
The in vitro antiproliferative activities of the cinnamic
amides 2a–2v are exhibited in Table 3. The research was
carried out by the MTT colorimetric assay on the human
tumor cell line MCF-7 which overexpresses EGFR. The
IC50 values, the concentration (lM) at which only 50 %
survival of cells was allowed, were calculated. As shown in
Table 3, compound 2f was found to show the most potent
activity with IC50 value of 2.01 lM.
Furthermore, compounds 2a–2v were also evaluated for
their ability to inhibit the autophosphorylation of EGFR
kinase by a solid-phase ELISA assay. A number of syn-
thesized compounds displayed moderate EGFR kinase
inhibitory activities, which can be seen in Table 3. Owing
to that EGFR is an important factor to breast carcinoma
cancer, the results show the same trend with the antipro-
liferative activities against MCF-7. Compounds 2f and 2j
displayed the most potent inhibitory activities (IC50 = 5.16
and 7.37 lM, respectively).
Results and discussion
Chemistry
In order to investigate how substituents on the N-phenyl
ring influence the activities, 20 substituted anilines and two
alkylamines were treated to react with cinnamoyl chloride.
The substituents introduced here include –Me, –iPr, –OMe,
–OEt, –F, –Cl, and –Br. The synthesis route outlined in
Scheme 1 gave satisfactory yields.
The crystal structures of compounds 2b and 2i
Among these compounds, crystal structures of compounds
2b and 2i were determined by X-ray diffraction analysis.
Scheme 1 Synthesis of cinnamic amides 2a–2v. Reagents and conditions: i SOCl2, 80–90 °C, reflux for 2 h; ii CH2Cl2, DMAP, NaHCO3, 2–3 h
123

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Med Chem Res (2013) 22:986–994
SAR analysis
Table 1 Crystallographic data for compounds 2b and 2i
Compounds
2b
2i
It could be observed in Table 3 that there exists a gap of
activities between 2a–2t and 2u–2v, which demonstrates
that N-phenyl rings are required for enhancing the activities.
For 2a–2t, there is a weak fluctuation in the inhibitory
activities showing that o- [ p- [ m- substituents and
replacements of Me groups by alkoxyl groups (RO) or hal-
ogens groups (X) result in the increase of activities in gen-
eral. But, the IC50 values of 2a–2t cover such narrow ranges
(threefold for MCF-7 and 4.5-fold for EGFR), revealing that
the addition of simple groups to the N-phenyl rings does not
seem to be an essential point for enhancing the inhibitory
activities. Thus, we do not declare it to be a general rule. We
hope this finding could be of help for future exploring.
Empirical formula
Formula weight
Crystal system
Space group
C16H15NO
237.29
C17H17NO2
267.32
Orthorhombic
Pbca
Monoclinic
P21/c
Crystal color
White
Pale gray
18.012(7)
10.205(4)
7.753(3)
90.00
˚
a (A)
14.227(17)
9.504(11)
19.78(2)
90.00
˚
b (A)
˚
c (A)
a (°)
b (°)
c (°)
90.00
93.418(4)
90.00
90.00
˚
V (A)
2674(6)
8
1422(2)
4
Z
Dcalc (g cm-3
h range (°)
hkl range
)
1.179
1.248
Binding model of compound 2f into EGFR
2.51–24.87
2.27–27.63
-9 B h B 17;
-11 B k B 11;
-23 B l B 24
-22 B h B 22;
-11 B k B 12;
-9 B l B 9
In order to help to explain the SARs observed at the EGFR
inhibition, molecular docking of the most potent inhibitor
2f into the ATP binding site of EGFR kinase was per-
formed on the binding model based on the EGFR complex
structure (1M17.pdb), which is depicted in Fig. 2. In the
binding model, compound 2f is nicely bound to the region
with the MeO group projecting toward the HO of THR 830
forming a more optimal hydrogen bond interaction.
Moreover, carbonyl group of compound 2f also forms a
H-bond with the HO of THR 766. Together with the SAR
study, this molecular docking result suggests the MeO to be
favorable for enhancing the activity.
F(0 0 0)
1,008
568.0
No. collected refl.
No. ind refl. (Rint
12,708
10,425
)
2,734 (0.061)
2,734/0/164
0.073
2,887 (0.062)
2,887/0/182
0.082
Data/restraints/parameters
Absorption coefficient
(mm-1
)
R1; wR2 [I [ 2r(I)]
R1; wR2 (all data)
GOOF
0.0580; 0.1417
0.0943; 0.1737
1.038
0.0494; 0.1173
0.0777; 0.1377
1.034
Fig. 1 a ORTEP view showing
the atom-labeling scheme with
thermal ellipsoids drawn at
30 % probability for compound
2b. b ORTEP view of
compound 2i drawn in the same
way as 2b
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Med Chem Res (2013) 22:986–994
989
Table 2 Selected bond lengths, bond angles, and torsion angles of
compounds 2b and 2i
Table 3 The antiproliferative activities and EGFR inhibitory activi-
ties of compounds 2a–2v
Compounds
2b
2i
Compounds
MCF-7 (IC50/lM)
EGFR (IC50/lM)
˚
Bond lengths (A)
2a
2b
2c
5.24 0.94
3.17 0.54
5.83 0.97
3.26 0.59
5.39 1.08
2.01 0.28
2.34 0.35
2.43 0.46
3.30 0.50
2.38 0.33
2.56 0.41
4.14 0.83
4.65 0.90
2.89 0.52
2.37 0.38
4.37 0.61
3.49 0.63
4.00 0.77
4.47 0.92
4.05 0.64
13.40 2.42
11.13 1.78
0.22 0.03
23.16 0.62
12.29 0.37
19.69 0.61
12.69 0.32
14.58 0.41
5.16 0.14
9.05 0.32
9.35 0.27
10.23 0.27
7.37 0.20
10.02 0.25
11.69 0.28
13.23 0.44
12.88 0.39
9.86 0.27
14.59 0.31
13.71 0.37
16.17 0.40
13.93 0.36
15.74 0.49
38.46 0.92
29.85 0.78
0.34 0.01
C1–C2
1.369(4)
1.463(3)
1.316(3)
1.477(3)
1.231(3)
1.339(3)
1.426(3)
1.502(4)
1.375(3)
1.465(3)
1.321(3)
1.477(3)
1.230(2)
1.352(2)
1.419(2)
C4–C7
C7–C8
2d
2e
C8–C9
O1–C9
2f
N1–C9
2g
2h
2i
N1–C10
C
15–C16 (2b)
O2–C13 (2i)
O2–C16 (2i)
1.373(2)
1.422(3)
1.495(3)
2j
2k
2l
C
16–C17 (2i)
Bond angles (°)
H1A–N1–C9
C9–N1–C10
C1–C2–C3
2m
2n
2o
2p
2q
2r
117.0
117.5
125.93(18)
120.1(3)
122.7(2)
114.44(19)
127.5(2)
121.6(3)
109.5
124.94(16)
120.3(2)
N1–C9–O1
122.61(18)
114.84(17)
128.12(19)
N1–C9–C8
C4–C7–C8
2s
C
C
C
10–C15–C16 (2b)
15–C16–H16A (2b)
13–O2–C16 (2i)
2t
2u
2v
Erlotinib
118.26(15)
107.6(2)
110.2
O2–C16–C17 (2i)
16A–C16–C17 (2i)
H
Torsion angles (°)
C3–C4–C7–C8
-17.7(4)
-178.8(2)
174.1(2)
-178.9(2)
-47.8(3)
-3.1(4)
-171.2(2)
-178.69(17)
-166.19(18)
177.74(17)
45.2(3)
C4–C7–C8–C9
C7–C8–C9–N1
N1–C10–C11–C12
C
11–C10–N1–C9
O1–C9–N1–C10
0.8(3)
N1–C10–C15–C16 (2b)
-2.9(4)
C
C
C
11–C10–C15–C16 (2b)
11–C12–C13–O2 (2i)
17–C16–O2–C13 (2i)
178.5(3)
-179.08(16)
177.67(19)
Conclusions
A series of cinnamic amides were synthesized. The crystal
structures of 2b and 2i were determined. All these com-
pounds were evaluated for antiproliferative activities
against the human cancer cell line MCF-7- and EGFR-
inhibitory activities, and the two series of activities show
the same trend. Moreover, docking simulations were per-
formed to give the probable binding modes of compound 2f
into the ATP binding site of EGFR kinase. Biological assay
shows that N-phenyl rings are required to enhance the
activities. Although certain rules are found in the SAR
Fig. 2 Binding model of compound 2f with EGFR kinase. Note Only
interacting residues are displayed for clarity. Ligand and interacting
key residues are represented as stick models, while the rest are
represented as ribbons. The H-bonds are displayed as green dotted
lines (Color figure online)
study, no strong evidences of the data support the beneficial
substituents, revealing that the simple substitution on the
N-phenyl does not bring a leap in activities. The results of
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Med Chem Res (2013) 22:986–994
this study may be helpful for researching new potential
antitumor agents.
mixture of EtOAc and hexane (1:1), final compounds 2a–
2v were obtained.
(E)-N-phenylcinnamamide (2a)
Experimental part
White crystal. yield 82 %. m.p. 148–149 °C, dH (CDCl3):
6.55 (d, J 15.6, 1H, H-8), 7.14 (t, J 7.2, 1H, H-40),
7.34–7.42 (m, 5H, H-3,4,5,30,50), 7.54 (d, J 6.0, 2H, H-2,6),
7.62 (d, J 6.0, 2H, H-20,60), 7.76 (d, J 15.6, 1H, H-7). m/z
(ESI): 224.1 [(M ? H)?]; 222.0 [(M – H)-]. (Anal. Calcd.
for C15H13NO: C, 80.72; H, 5.83; N, 6.78. Found: C, 80.39;
H 6.27; N, 6.41 %.)
Chemistry general
In this study, 22 corresponding cinnamic amides were
prepared. Different amines acted as nucleophilic reagents
to react with cinnamoyl chloride (1). Reactions were
monitored by TLC using silica gel-coated glass slides
(silica gel 60 GF 254, Qingdao Haiyang Chemical, China).
Detections were done in UV (254 nm). Melting points
were measured on a SPSIC WRS-1B digital melting-point
(E)-N-(o-tolyl)cinnamamide (2b)
1
apparatus (Shanghai, China); uncorrected. H-NMR spec-
White crystal. yield 86 %. m.p. 175–177 °C. dH (CDCl3):
2.33 (s, 3H, –CH3); 6.58 (d, J 15.6, 1H, H-8); 7.13 (m, 2H,
H-40,50); 7.22 (m, 2H, H-30,60); 7.38 (m, 3H, H-3,4,5); 7.55
(d, J 7.0, 2H, H-2,6); 7.80 (d, J 15.6, 1H, H-7), 7.99 (br. s,
1H, NH). m/z (ESI): 238.1 [(M ? H)?]; 250.1
[(M ? Na)?]. (Anal. Calcd. for C16H15NO: C, 81.01; H,
6.33; N, 5.91. Found: C, 80.63; H, 6.67; N, 6.24 %.)
tra were recorded on a Bruker Avance III 400 NMR
spectrometer with CDCl3 as solvent. The chemical shifts
(d) are reported in ppm with reference to internal TMS and
coupling constants (J) are given in Hz. ESI–MS spectra
were recorded on a Mariner System 5304 mass spectrom-
eter. All reagents bought from Aldrich (USA), Aladdin
(China) or Sinopharm Chemical Reagent Co., Ltd (China)
were of pure analytical grades and used without further
treatments.
(E)-N-(m-tolyl)cinnamamide (2c)
White crystal. yield 81 %. m.p. 111–112 °C. dH (CDCl3):
2.35 (s, 3H, –CH3); 6.55 (d, J 15.6, 1H, H-8); 6.95 (d, 1H,
H-40); 7.23 (t, J 7.8, 1H, H-50); 7.40–7.43 (m, 4H,
H-3,4,5,20); 7.49–7.54 (m, 3H, H-2,6,60); 7.75 (d, J 15.6,
1H, H-7). m/z (ESI): 238.1 [(M ? H)?]; 250.1
[(M ? Na)?]. (Anal. Calcd. for C16H15NO: C, 81.01; H,
6.33; N, 5.91. Found: C, 81.38; H, 6.07; N, 5.67 %.)
General procedure for the synthesis and purification
of the cinnamic amides
The series of compounds were synthesized according to
literatures with some modifications (Narasimhan et al.,
2004; Zeller et al., 2010). A mixture of cinnamic acid
(0.14 g, 1 mmol) and SOCl2 (15 mL, acted as both reactant
and solvent) was stirred and refluxed at 80–90 °C for 2 h.
Remaining SOCl2 was removed by distillation under
reduced pressure. Thus, the intermediate cinnamoyl chlo-
ride (1) was achieved, which was used directly in the next
step without any further purification.
(E)-N-(p-tolyl)cinnamamide (2d)
White crystal. yield 84 %. m.p. 168–170 °C. dH (CDCl3):
2.34 (s, 3H, –CH3); 6.54 (d, J 14.8, 1H, H-8); 7.16 (d, J 7.2,
2H, H-20,60); 7.38 (m, 3H, H-3,4,5); 7.46–7.53 (m, 4H,
H-2,6,30,50); 7.75 (d, J 14.8, 1H, H-7). m/z (ESI): 238.1
[(M ? H)?]; 250.1 [(M ? Na)?]. (Anal. Calcd. for
C16H15NO: C, 81.01; H, 6.33; N, 5.91. Found: C, 81.40; H,
6.62; N, 5.57 %.)
Intermediate 1 was then dissolved in anhydrous CH2Cl2
(40–50 mL), and NaHCO3 (excessive, about 1.0 g) was
added to the solution. Stirring was continued for 5 min to
destroy residual SOCl2. Then, 4-dimethylaminopyridine
(DMAP, 10 mg) was added to the mixture followed by
another 1–2 min stirring. Afterward, appropriate substi-
tuted aniline or alkylamine (1 mmol) was added slowly
(finished in 1 min) and the mixture was stirred at r.t. for
2–3 h.
(E)-N-(4-isopropylphenyl)cinnamamide (2e)
Yellow crystal. yield 73 %. m.p. 159–160 °C. dH (CDCl3):
1.24 (d, J 7.2, 6H, 2 9 –CH3); 2.89 (m, J 6.8, 1H, –CH–);
6.55 (d, J 15.6, 1H, H-8); 7.21 (d, J 8.4, 2H, H-20,60);
7.36–7.41 (m, 3H, H-3,4,5); 7.53 (m, 4H, H-2,6,30,50); 7.75
(d, J 15.6, 1H, H-7). m/z (ESI): 266.2 [(M ? H)?]; 264.1
[(M – H)-]. (Anal. Calcd. for C18H19NO: C, 81.51; H,
7.17; N, 5.28. Found: C, 81.18; H, 6.78; N, 5.62 %.)
The reaction mixture was washed with HCl solution
(5 %, 50 mL) and NaOH solution (5 %, 50 mL) several
times. The CH2Cl2 solution was collected and dried by
anhydrous Na2SO4. Further purification was carried out by
silica gel column chromatography with petroleum ether/
EtOAc (10:1) as eluent. After recrystallization from a
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Med Chem Res (2013) 22:986–994
991
(E)-N-(2-methoxyphenyl)cinnamamide (2f)
(m, 3H, H-3,4,5); 7.43 (m, 2H, H-2,6); 7.52 (d, J 6.2, 2H,
H-20,60); 7.76 (d, J 15.6, 1H, H-7). m/z (ESI): 242.1
[(M ? H)?]; 240.0 [(M – H)-]. (Anal. Calcd. for
C15H12FNO: C, 74.69; H, 4.98; N, 5.81. Found: C, 74.98;
H, 5.37; N, 5.52 %.)
Yellow crystal. yield 79 %. m.p. 140–141 °C. dH (CDCl3):
3.93 (s, 3H, –OCH3); 6.60 (d, J 15.6, 1H, H-8); 6.91 (d, J 8.0,
1H, H-30); 6.99–7.10 (m, 2H, H-40,50); 7.41 (m, 3H,
H-3,4,5); 7.58 (d, J 6.8, 2H, H-2,6); 7.76 (d, J 15.6, 1H, H-7);
7.98 (d, J 7.2, 1H, H-60); 8.54 (br. s, 1H, NH). m/z (ESI):
254.1 [(M ? H)?]. (Anal. Calcd. for C16H15NO2: C, 75.89;
H, 5.93; N, 5.53. Found: C, 76.22; H, 5.57; N, 5.87 %.)
(E)-N-(2,4-difluorophenyl)cinnamamide (2l)
Pale purple crystal. yield 72 %. m.p. 140–141 °C. dH
(CDCl3): 6.58 (d, J 15.2, 1H, H-8); 6.90 (m, 2H, H-30,50);
7.40 (m, 3H, H-3,4,5); 7.45 (m, 1H, H-60); 7.55 (d, J 6.0,
2H, H-2,6), 7.77 (d, J 15.6, 1H, H-7); 8.42 (br. s, 1H, NH).
m/z (ESI): 260.1 [(M ? H)?]. (Anal. Calcd. for
C15H11F2NO: C, 69.50; H, 4.25; N, 5.41. Found: C, 69.13;
H, 4.61; N, 5.16 %.)
(E)-N-(4-methoxyphenyl)cinnamamide (2g)
White powder. yield 88 %. m.p. 163–164 °C. dH (CDCl3): 3.79
(s, 3H, –OCH3); 6.55 (d, J 15.5, 1H, H-8); 6.88 (d, J 8.3, 2H,
H-30,50); 7.36 (m, 3H, H-3,4,5); 7.47–7.55 (m, 4H, H-2,6,20,60);
7.74 (d, J 15.5, 1H, H-7). m/z (ESI): 254.1 [(M ? H)?]; 276.1
[(M ? Na)?]. (Anal. Calcd. for C16H15NO2: C, 75.89; H, 5.93;
N, 5.53. Found: C, 76.35; H, 5.63; N, 5.30 %.)
(E)-N-(3,5-difluorophenyl)cinnamamide (2m)
White crystal. yield 86 %. m.p. 154–155 °C. dH (CDCl3):
6.51 (d, J 15.6, 1H, H-8); 6.58 (tt, J 7.2, 2.0 1H, H-40); 7.25
(m, 2H, H-20,60); 7.40 (m, 3H, H-3,4,5); 7.54 (m, 2H,
H-2,6); 7.78 (d, J 15.6, 1H, H-7). m/z (ESI): 260.1
[(M ? H)?]. (Anal. Calcd. for C15H11F2NO: C, 69.50; H,
4.25; N, 5.41. Found: C, 69.17; H, 4.64; N, 5.12 %.)
(E)-N-(2-ethoxyphenyl)cinnamamide (2h)
White powder. yield 79 %. m.p. 123–124 °C. dH (CDCl3):
1.53 (t, J 7.2, 3H, –CH3); 4.18 (q, J 7.2, 2H, –OCH2–); 6.62
(d, J 15.6, 1H, H-8); 6.92 (d, J 8.0, 1H, H-30); 7.05 (m, 2H,
H-40,50); 7.42 (m, 3H, H-3,4,5); 7.61 (d, J 6.9, 2H, H-2,6);
7.78 (d, J 15.2, 1H, H-7); 8.00 (br. s, 1H, H-60); 8.56 (br. s,
1H, NH). m/z (ESI): 268.1 [(M ? H)?]; 290.2
[(M ? Na)?]. (Anal. Calcd. for C17H17NO2: C, 76.40; H,
6.37; N, 5.24. Found: C, 76.18; H, 6.66; N, 4.95 %.)
(E)-N-(3,4,5-trifluorophenyl)cinnamamide (2n)
White powder. yield 72 %. m.p. 170–171 °C. dH (CDCl3):
6.49 (d, J 15.6, 1H, H-8); 7.34–7.42 (m, 2H, H-20,60);
7.40–7.42 (m, 3H, H-3,4,5); 7.54–7.57 (m, 2H, H-2,6); 7.78
(d, J 15.6, 1H, H-7). m/z (ESI): 278.1 [(M ? H)?]; 276.1
[(M – H)-]. (Anal. Calcd. for C15H10F3NO: C, 64.98; H,
3.61; N, 5.05. Found: C, 65.33; H, 3.27; N, 5.43 %.)
(E)-N-(4-ethoxyphenyl)cinnamamide (2i)
Pale gray lamellar crystal. yield 83 %. m.p. 150–152 °C. dH
(CDCl3): 1.41 (t, J 7.2, 3H, –CH3); 4.01 (q, J 6.8, 2H, –
OCH2–); 6.54 (d, J 15.6, 1H, H-8); 6.87 (d, J 8.4, 2H,
H-30,50); 7.37(m, 3H, H-3,4,5); 7.52 (m, 4H, H-2,6,20,60);
7.74 (d, J 15.6, 1H, H-7). m/z (ESI): 268.1 [(M ? H)?];
290.2 [(M ? Na)?]. (Anal. Calcd. for C17H17NO2: C, 76.40;
H, 6.37; N, 5.24. Found: C, 76.75; H, 6.59; N, 4.97 %.)
(E)-N-(2-chlorophenyl)cinnamamide (2o)
White crystal. yield 81 %. m.p. 137–139 °C. dH (CDCl3):
6.61 (d, J 15.2, 1H, H-8); 7.07 (t, J 8.8, 1H, H-40); 7,26 (m,
1H, H-30); 7.33 (t, J 7.6, 1H, H-50); 7.40–7.44 (m, 3H,
H-3,4,5); 7.59 (m, 2H, H-2,6); 7.79 (d, J 15.6, 1H, H-7);
7.81 (br. s, 1H, H-60); 8.55 (br. s, 1H, NH). m/z (ESI): 258.2
[(M ? H)?]; 280.1 [(M ? Na)?]. (Anal. Calcd. for
C15H12ClNO: C, 69.90; H, 4.66; N, 5.44. Found: C, 70.26;
H, 4.90; N, 5.02 %.)
(E)-N-(2-fluorophenyl)cinnamamide (2j)
White crystal. yield 76 %. m.p. 116–117 °C. dH (CDCl3):
6.58 (d, J 15.2, 1H, H-8); 7.06–7.19 (m, 3H, H-30,40,50);
7.40 (dd, 3H, H-3,4,5); 7.52 (d J 6.8, 1H, H-60); 7.57 (dd,
2H, H-2,6); 7.78 (d, J 15.6, 1H, H-7); 8.48 (br. s, 1H, NH).
m/z (ESI): 242.1 [(M ? H)?]; 240.0 [(M – H)-]. (Anal.
Calcd. for C15H12FNO: C, 74.69; H, 4.98; N, 5.81. Found:
C, 74.30; H, 5.41; N, 5.50 %.)
(E)-N-(3-chlorophenyl)cinnamamide (2p)
White crystal. yield 84 %. m.p. 124–125 °C. dH (CDCl3):
6.54 (d, J 15.6, 1H, H-8); 7.12 (d, J 7.6, 1H, H-40); 7.28 (t,
J 7.8, 1H, H-50); 7.36 (s, 1H, H-20); 7.40–7.41 (m, 3H,
H-3,4,5); 7.47 (d, J 8.0, 1H, H-60); 7.54 (m, 2H, H-2,6);
7.78 (d, J 15.6, 1H, H-7). m/z (ESI): 258.1 [(M ? H)?];
280.1 [(M ? Na)?]. (Anal. Calcd. for C15H12ClNO: C, C,
(E)-N-(4-fluorophenyl)cinnamamide (2k)
White crystal. yield 79 %. m.p. 155–157 °C. dH (CDCl3):
6.54 (d, J 15.2, 1H, H-8); 7.04 (t, J 8.4, 2H, H-30,50); 7.38
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Med Chem Res (2013) 22:986–994
(E)-N-(tert-butyl)cinnamamide (2v)
69.90; H, 4.66; N, 5.44. Found: C, 70.32; H, 4.94; N,
5.17 %.)
White powder. yield 85 %. m.p. 139–141 °C. dH (CDCl3):
1.44 (s, 9H, 3 9 –CH3); 6.32 (d, J 15.6, 1H, H-8); 7.35 (m,
3H, H-3,4,5); 7.48 (d, J 6.8, 2H, H-2,6); 7.57 (d, J 15.6, 1H,
H-7). m/z (ESI): 204.3 [(M ? H)?]. (Anal. Calcd. for
C13H17NO: C, 76.85; H, 8.37; N, 6.90. Found: C, 76.46; H,
8.74; N, 6.58 %.)
(E)-N-(4-chlorophenyl)cinnamamide (2q)
White crystal. yield 89 %. m.p. 188–189 °C. dH (CDCl3):
6.54 (d, J 15.6, 1H, H-8); 7.32 (d, J 8.8, 2H, H-30,50); 7.40
(m, 2H, H-20,60); 7.53–7.55 (m, 3H, H-3,4,5); 7.59 (d, J 8.0,
2H, H-2,6); 7.77 (d, J 15.6, 1H, H-7). m/z (ESI): 258.1
[(M ? H)?]; 280.1 [(M ? Na)?]. (Anal. Calcd. for
C15H12ClNO: C, 69.90; H, 4.66; N, 5.44. Found: C, 69.56;
H, 4.36; N, 5.75 %.)
Crystallographic studies
X-ray single-crystal diffraction data for compounds 2b and
2i were collected on a Bruker SMART APEX CCD dif-
fractometer at 296 (2) K using Mo Ka radiation
(E)-N-(2,4-dichlorophenyl)cinnamamide (2r)
˚
(k = 0.71073 A) by the x scan mode. The program
SAINT was used for integration of the diffraction profiles.
Structure was solved by direct methods by means of the
SHELXS program of the SHELXTL package and refined
by full-matrix least-squares methods by means of SHELXL
(Sheldrick, 1997). All non-hydrogen atoms of compounds
2b and 2i were refined with anisotropic thermal parameters.
All hydrogen atoms were placed in geometrically idealized
positions and constrained to ride on their parent atoms.
White powder. yield 81 %. m.p. 171–173 °C. dH (CDCl3):
6.58 (d, J 15.5, 1H, H-8), 7.29 (d, J 8.8, 1H, H-50), 7.41 (m,
4H, H-3,4,5,30), 7.58 (m, 2H, H-2,6), 7.74 (br. s, 1H, H-60),
7.78 (d, J 15.5, 1H, H-7), 8.52 (d, J 8.8, 1H, NH). m/z
(ESI): 293.0 [(M ? H)?]. (Anal. Calcd. for C15H11Cl2NO:
C, 61.64; H, 3.77; N, 4.69. Found: C, 61.30; H, 4.03; N,
4.34 %.)
(E)-N-(3,5-dichlorophenyl)cinnamamide (2s)
Cell proliferative activities assay
Pale purple crystal. yield 78 %. m.p. 142–143 °C. dH
(CDCl3): 6.51 (d, J 15.6, 1H, H-8); 7.13 (s, 1H, H-40);
7.27–7.29 (m, 3H, H-3,4,5); 7.42 (m, 2H, H-2,6); 7.57 (m,
2H, H-20,60); 7.78 (d, J 15.6, 1H, H-7). m/z (ESI): 293.0
[(M ? H)?]. (Anal. Calcd. for C15H11Cl2NO: C, 61.64; H,
3.77; N, 4.69. Found: C, 61.26; H, 4.12; N, 4.39 %.)
The antiproliferative activities of compounds 2a–2v were
determined as described (Lv et al., 2010a, b; Li et al.,
2010) by a standard (MTT)-based colorimetric assay
(Sigma). Briefly, cell lines were seeded at a density of
7 9 103 cells/well in 96-well microtiter plates (Costar).
After 24 h, exponentially growing cells were exposed to
the indicated compounds at final concentrations ranging
from 0.1 to 100 lg mL-1. After 48 h, cell survival was
determined by the addition of an MTT solution (10 lL of
5 mg mL-1 MTT in PBS). After 4 h, 100 lL of 10 % SDS
in 0.01 N HCl was added and the plates were incubated at
37 °C for a further 18 h; optical absorbance was measured
at 570 nm on an LX300 Epson Diagnostic microplate
reader. Survival ratios are expressed in percentages with
respect to untreated cells. IC50 values were determined
from replicates of six wells from at least two independent
experiments.
(E)-N-(2-bromophenyl)cinnamamide (2t)
White crystal. yield 89 %. m.p. 148–149 °C. dH (CDCl3):
6.60 (d, J 15.6, 1H, H-8); 7.01 (t, J 8.0, 1H, H-40); 7.37 (t,
J 8.0, 1H, H-50); 7.41–7.45 (m, 3H, H-3,4,5); 7.56–7.61 (m,
3H, H-2,6,30); 7.79 (d, J 15.6, 1H, H-7); 7.81 (br. s, 1H,
H-60); 8.53 (br. s, 1H, NH). m/z (ESI): 302.0 [(M ? H)?].
(Anal. Calcd. for C15H12BrNO: C, 59.62; H, 3.97; N, 4.64.
Found: C, 59.25; H, 4.33; N, 4.40 %.)
(E)-N-dodecylcinnamamide (2u)
EGFR inhibitory assay
White crystal. yield 91 %. m.p. 73–74 °C. dH (CDCl3):
0.88 (t, J 5.8, 3H, –CH3); 1.25–1.32 (m, 18H, –C9H18–);
1.57 (m, 2H, –CH2–); 3.38 (m, 2H, –NCH2–); 6.38 (d,
J 15.5, 1H, H-8); 7.35–7.37 (m, 3H, H-3,4,5); 7.50 (d,
J 6.8, 2H, H-2,6); 7.62 (d, J 15.5, 1H, H-7). m/z (ESI):
316.3 [(M ? H)?]; 338.3 [(M ? Na)?]. (Anal. Calcd. for
C21H33NO: C, 80.00; H, 10.48; N, 4.44. Found: C, 79.78;
H, 10.84; N, 4.09 %.)
The preparation, purification, and the inhibitory assay of
EGFR were conducted as described (Lv et al., 2010a, b; Li
et al., 2010). A 1.6-kb cDNA encoded for the EGFR cyto-
plasmic domain (amino acids 645–1,186) was cloned into
baculoviral expression vector pFASTBacHTc. A sequence
that encodes (His)6 was located at the 50 upstream to the
EGFR sequence. Sf-9 cells were infected for 3 days for
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Med Chem Res (2013) 22:986–994
993
protein expression. Sf-9 cell pellets were solubilized at 0 °C
in a buffer at pH 7.4 containing 50 mM HEPES, 10 mM
NaCl, 1 % Triton, 10 lM ammonium molybdate, 100 lM
sodium vanadate, 10 lg mL-1 aprotinin, 10 lg mL-1 leu-
peptin, 10 lg mL-1 pepstatin, and 16 lg mL-1 benzami-
dine HCl for 20 min followed by 20 min centrifugation.
Crude extract supernatant was passed through an equili-
brated Ni–NTA superflow-packed column and washed with
10 mM and then 100 mM imidazole to remove nonspecif-
ically bound material. Histidine-tagged proteins were eluted
with 250 and 500 mM imidazole and dialyzed against
50 mM NaCl, 20 mM HEPES, 10 % glycerol, and
1 lg mL-1 each of aprotinin, leupeptin, and pepstatin for
2 h. The entire purification procedure was performed at
4 °C or on ice.
Drawing Standard; Cambridge Soft corporation, USA 2009)
then they were energetically minimized by MOPAC with
100 iterations and minimum RMS gradient of 0.10. The
Gasteiger–Hu¨ckel charges of ligands were assigned. The
crystal structures of EGFR (PDB code:1M17) complex were
retrieved from the RCSB Protein Data Bank (http://www.
rcsb.org/pdb/home/home.do). All bound waters and ligands
were eliminated from the protein and the polar hydrogens and
the Kollman-united charges were added to the proteins.
Acknowledgments This research was co-supported by the National
Basic Research Program of China (2010CB126104), National Natural
Science Foundation of China (30901854), Fundamental Research
Funds for the Central Universities (KYZ201107), and Open Research
Fund Program of Jiangsu Key Laboratory of Pesticide Science
(NYXKT201004).
The EGFR kinase assay was set up to assess the level of
autophosphorylation based on DELFIA/Time-Resolved
Fluorometry. Compounds 2a–2v were dissolved in 100 %
DMSO and diluted to the appropriate concentrations with
25 mM HEPES at pH 7.4. In each well, 10 lL of com-
pound was incubated with 10 lL (5 ng for EGFR) of
recombinant enzyme (1:80 dilution in 100 mM HEPES) for
10 min at room temperature. Then, 10 lL of 5 mM buffer
(containing 20 mM HEPES, 2 mM MnCl2, 100 lM
Na3VO4, and 1 mM DTT) and 20 lL of 0.1 mM ATP-
50 mM MgCl2 was added for 1 h. Positive and negative
controls were included in each plate by incubation of
enzyme with or without ATP–MgCl2. At the end of incu-
bation, liquid was aspirated and plates were washed three
times with wash buffer. A 75 lL (400 ng) sample of
europium-labeled antiphosphotyrosine antibody was added
to each well for another 1 h of incubation. After washing,
enhancement solution was added and the signal was
detected by Victor (WallacInc.) with excitation at 340 nm
and emission at 615 nm. The percentage of autophospho-
rylation inhibition by the compounds was calculated by the
following equation: 100 %- [(negative control)/(positive
control - negative control)]. The IC50 values were
obtained from the curves of percentage inhibition with
eight concentrations of the compound. As the contaminants
in the enzyme preparation are fairly low, the majority of
the signal detected by the antiphosphotyrosine antibody is
from EGFR.
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