Synthesis of novel cinnamanilides as potential immunosuppressive agents

Article abstract of DOI:10.1016/j.ejmech.2011.10.027

A series of new cinnamanilides (6-40) were synthesized and their immunosuppressive activity and cytotoxicity were evaluated. Most of the cinnamanilides showed good immunosuppressive activity. Among the synthesized compounds, (Z)-N-(4-bromophenyl)-2-methox

Full text of DOI:10.1016/j.ejmech.2011.10.027

European Journal of Medicinal Chemistry 47 (2012) 585e593
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Synthesis of novel cinnamanilides as potential immunosuppressive agents
,
b
a
b
,
a
Lei Shi a , Lu Wang , Zhi Wang , Hai-Liang Zhu
, Qiao Song
*
**
a Department of Medicinal Chemistry, China Pharmaceutical University, Nanjing 210009, People’s Republic of China
b State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, People’s Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of new cinnamanilides (6e40) were synthesized and their immunosuppressive activity and
cytotoxicity were evaluated. Most of the cinnamanilides showed good immunosuppressive activity.
Among the synthesized compounds, (Z)-N-(4-bromophenyl)-2-methoxy-3-(4-methoxyphenyl)acryl-
amide (37) and (Z)-2-methoxy-3-(4-methoxyphenyl)-N-p-tolylacrylamide (38) exhibited potent immu-
Received 29 July 2011
Received in revised form
12 October 2011
Accepted 12 October 2011
Available online 20 October 2011
nosuppressive activity (IC50 ¼ 1.77 ꢀ 0.33 and 0.94 ꢀ 0.13
mM) without significant cytotoxicity.
Ó 2011 Elsevier Masson SAS. All rights reserved.
Keywords:
Cinnamanilides
Synthesis
Immunosuppressive activity
1. Introduction
In this study, 35 novel cinnamanilides were synthesized and
screened for their immunosuppressive activity. Most of the
synthetic compounds exhibited potent immunosuppressive
activity.
Immunosuppressive drugs are widely used for the treatment of
some autoimmune diseases, such as systemic lupus erythematosus,
rheumatoid arthritis, psoriasis and glomerulonephritis, and in
organ transplantations [1,2]. T-lymphocytes play an integral role in
transplant rejection and autoimmune diseases. Cyclosporine A
(CsA), [3] tacrolimus, [4] and sirolimus (rapamycin) [4,5] are clini-
cally important therapeutic immunosuppressants, which exert
their immunosuppressive effects by inhibiting the proliferation of
T-lymphocytes [6,7]. However, despite their undeniable clinical
advantages, the immunosuppressive drugs in current clinical use
such as glycocorticoids [8], CsA, tacrolimus, and sirolimus, etc., had
rather serious side effects including renal toxicity, liver toxicity,
infection, malignancy, cosmetic consequences, and other unwanted
effects [9e16]. Thus there is a pressing need for novel potential
immunosuppressive agents with high efficacy and low toxicity. A
program to produce synthetic compounds is therefore ongoing in
our laboratory to identify new immunosuppressants [17].
2. Results and discussion
2.1. Chemistry
The route that enabled the synthesis of the 2-methoxy cinna-
manilides is outlined in Scheme 1. The synthesis of these cinna-
manilides started from
a series of commercially available
p-substituted benzaldehydes 1ae1e. Condensation of 1ae1e with
N-acetylglycine in refluxing acetic anhydride and sodium acetate
for 1 h gave the oxazole intermediates 2ae2e in 72e86% yield.
Hydrolysis of these oxazole intermediates 2ae2e with 3 M HCl
under reflux for 3 h provided the pyruvic acid derivatives 3ae3e in
62e76% yield. Methylation of 3ae3e with dimethyl sulfate in 10%
NaOH at room temperature for 4 h and followed by acidification
with 3 M HCl for 0.5 h furnished methylated products 4ae4e in
80e86% yield. Condensation of 4ae4e with a series of p-substituted
anilines 5fe5l with 1-ethyl-3-(3-dimethylaminopropyl) carbodii-
mide hydrochloride (EDC.HCl) in refluxing CH2Cl2 for 6e8 h gave
the 2-methoxy cinnamanilides 6e40 in 68e84% yield. The struc-
tures and chemical features of these newly synthesized cinnama-
nilides were summarized in Table 1.
Cinanserin (20-(3-dimethylaminopropylthio)cinnamanilide) has
been shown to be a potent antagonist of serotonin [18], that also
exhibits analgesic activity [19] and possess immunosuppressive
activity [20]. Some other compounds containing the cinnamide
template also showed potent immunosuppressive activity [21,22].
Cinnamanilides 10, 14 and 32 were successfully crystallized and
their structures were determined by single-crystal X-ray diffraction
analysis. The molecular structures of cinnamanilides 10, 14 and 32
* Corresponding author. Tel./fax: þ86 25 83 27 1445.
** Corresponding author. Tel.: þ86 25 8359 2572; fax: þ86 25 8359 2672.
E-mail addresses: shilei0828@gmail.com (L. Shi), zhuhl@nju.edu.cn (H.-L. Zhu).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.10.027

586
L. Shi et al. / European Journal of Medicinal Chemistry 47 (2012) 585e593
Scheme 1. Synthesis of 2-methoxy cinnamanilides: (a) N-acetylglycine, Ac2O, CH3COONa, reflux, 1 h, 72e86% yield; (b) 3 M HCl, reflux, 3 h, 62e76% yield; (c) 10% NaOH, (CH3)2SO4,
rt, 4 h; (d) 3M HCl, rt, 0.5 h, 80e86%; (e) EDC, CH2Cl2, reflux, 6e8 h, 68e84% yield.
are shown in Fig.1a, b and c, respectively. The collection data details
are condensed in Table 2, and the selected geometrical parameters
are given in Table 3. Hydrogen bonds for cinnamanilides 10, 14 and
32 are listed in Table 4. Crystallographic data for the structural
analysis have been deposited with the Cambridge Crystallographic
Data Centre Nos. 698515 for 10, 698516 for 14 and 698517 for 32.
Copies of this information can be obtained free of charge from The
Director, CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (fax: þ44
1223 336 033; e-mail: deposit@ccdc.cam.ac.uk or www: http://
www.ccdc.cam.ac.uk).
showed moderate immunosuppressive activity with the IC50’s
M.
ranging from 9.48 ꢀ 1.02 to 17.00 ꢀ 1.99
m
As can been seen from the immunosuppressive activity of all the
synthesized compounds (Fig. 2 and Table 5), compounds 13e33
with halogenated R1 substituent generally showed weak to
moderate activity except compound 18 (R1 ¼ F, R2 [ isopropyl),
which exhibited potent immunosuppressive activity with
IC50 ¼ 4.66 ꢀ 0.70
mM. This result indicated that electron with-
drawing R1 substituents such as halogen are not benefit for the
activity. Compounds 34e40 with the same electron donating R1
substituent (R1 ¼ methoxyl) showed distinct activity due to their
different R2 substituents. Among them, compounds 38 with elec-
tron donating R2 substituent (R2 ¼ methyl) exhibited the most
2.2. Biological activity
The spleen cells from 6 to 8 week-old BALB/c mice were co-
potent immunosuppressive activity with IC50 ¼ 0.94 ꢀ 0.13
mM,
stimulated by CD3/CD28 at the presence of 5
mg/mL of different
which suggested that electron donating R2 substituents are favor-
able for the activity. However, Compounds 39 and 40 with electron
donating R2 substituent (isopropyl or methoxyl) showed weak
activity, which suggested that the activity may also be relevant to
the size of molecule. Compounds 35e37 with different halogenated
R2 substituent showed distinct activities in the following order: Br
(37) > Cl (36) > F (35). Compounds 6e12 with the same R1
substituent (-H) also showed distinct activity due to their different
newly synthesized compounds for 48 h. The cell survival rate was
taken by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl trtrazo-
lium bromide) assay (Fig. 2). Some of the synthesized cinnamani-
lides (compounds 6e8, 10, 11, 15, 18, 19, 21, 23, 24, 31 and 36e38)
showed good immunosuppressive activity with the inhibition rate
over 50% at 5
mg/mL. The inhibition rate of the other cinnamanilides
(9, 12e14, 16, 17, 20, 22, 25-30, 32-35, 39 and 40) was below 50% at
5
mg/mL.
R2 substituents in the following order: isopropyl (11)
(7) > methyl (10) and Cl (8) > H (6) > Br (9) > methoxyl (12).
> F
The IC50 values of compounds 6e8, 10, 11, 15, 18, 19, 21, 23, 24, 31
and 36e38 were tested on CD3/CD28 co-stimulated mouse spleen
cells in the presence of different concentrations of compound. The
results are listed in Table 5. Among these compounds, 37 and 38
exhibited the most potent immunosuppressive activity with IC50’s
In conclusion, the immunosuppressive activity of cinnamani-
lides with R1 substituents of methoxy group or hydrogen were
more potent than those with a R1 substituent of a halogen. The data
indicated that an electronic-donating R1 substitute was favorable
for the immunosuppressive activity generally. The electronic-
donating R2 substitute (methyl group and isopropyl) was also
conducive to the activity generally. The activity may also be
1.77 ꢀ 0.33 and 0.94 ꢀ 0.13
18 also showed potent immunosuppressive activity with the IC50’s
ranging from 3.46 ꢀ 0.68 to 4.94 ꢀ 0.66 M. The other compounds
mM, respectively. Compounds 7, 11 and
m

L. Shi et al. / European Journal of Medicinal Chemistry 47 (2012) 585e593
587
Table 1
indicated that these compounds did not exhibit significant cyto-
toxicity to un-activated mouse spleen cells.
Physical properties of 2-methoxy cinnamanilides.
R2
O
4. Experimental section
N
H
4.1. Chemistry
OCH3
R1
All chemicals (reagent grade) used were purchased from Sigma
Aldrich (U.S.A) and Sinopharm Chemical Reagent Co., Ltd (China).
TLC was run on the silica gel coated aluminum sheets (silica gel 60
GF254, E. Merk, Germany) and visualized in UV light (254 nm).
Melting points (uncorrected) were determined on an XT4 MP
apparatus (Taike Corp., Beijing, China). ESI-MS spectra were recor-
ded on a Mariner System 5304 Mass spectrometer, and 1H NMR
spectra were recorded on a Bruker DPX-300, AV-300 or AV-500
spectrometer at 25 ꢁC with TMS and solvent signals allotted as
Compound
R1
R2
Formula
16H15NO2
C16H14FNO2
C16H14ClNO2
C16H14BrNO2
C17H17NO2
mp., ꢁC
Yield, %
6
7
8
9
H
H
H
H
H
H
H
F
F
F
F
F
F
F
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Br
Br
Br
Br
Br
Br
Br
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
H
F
Cl
Br
C
108e110
142e143
118e120
139e141
111e112
145e147
135e137
132e133
149e150
153e154
162e163
139e140
183e185
141e142
95e97
145e146
172e173
177e179
145e146
173e175
178e180
139e141
158e159
172e173
175e177
157e158
165e167
180e182
88e90
80
68
82
75
78
83
76
76
82
80
84
75
80
77
79
80
83
75
72
76
81
77
75
78
80
69
82
72
73
82
78
80
75
82
69
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
CH3
CH(CH3)2
OCH3
H
F
Cl
Br
CH3
CH(CH3)2
OCH3
H
F
Cl
Br
CH3
CH(CH3)2
OCH3
H
F
Cl
Br
CH3
CH(CH3)2
OCH3
H
F
Cl
Br
CH3
CH(CH3)2
OCH3
C19H21NO2
C17H17NO3
C16H14FNO2
C16H13F2NO2
C16H13ClFNO2
C16H13BrFNO2
C17H16FNO2
C19H20FNO2
C17H16FNO3
C16H14ClNO2
C16H13ClFNO2
C16H13Cl2NO2
C16H13BrClNO2
C17H16ClNO2
C19H20ClNO2
C17H16ClNO3
C16H14BrNO2
C16H13BrFNO2
C16H13BrClNO2
C16H13Br2NO2
C17H16BrNO2
C19H20BrNO2
C17H16BrNO3
C17H17NO3
internal standards. Chemical shifts were reported in ppm (d).
Elemental analyses were performed on a CHN-O-Rapid instrument.
4.2. Crystal structure determination
Crystal structure determination of compounds 10, 14 and 32
were carried out on a Nonius CAD4 diffractometer equipped with
graphite-monochromated MoK
a
(
l
¼ 0.71073 Ǻ) radiation. The
structure was solved by direct methods and refined on F2 by full-
matrix least-squares methods using SHELX-97 [23]. All the non-
hydrogen atoms were refined anisotropically. All the hydrogen
atoms were placed in calculated positions and were assigned fixed
isotropic thermal parameters at 1.2 times the equivalent isotropic U
of the atoms to which they are attached and allowed to ride on their
respective parent atoms. The contributions of these hydrogen
atoms were included in the structure-factors calculations.
C17H16FNO3
C17H16ClNO3
C17H16BrNO3
C18H19NO3
C20H23NO3
C18H19NO4
151e153
113e115
118e120
114e116
130e132
162e164
4.3. General procedure for the preparation of the oxazole
derivatives 2ae2e
A mixture of p-substituted benzaldehydes 1ae1e (30 mmol, 1
eq), N-acetylglycine (4.56 g, 39 mmol, 1.3 eq) and sodium acetate
(3.20 g, 39 mmol, 1.3 eq) in acetic anhydride (15 mL) was stirred at
reflux for 1 h. The reaction was quenched with ice water and
vigorously stirred for 1 h in an ice bath to allow precipitation.
Filtration afforded 2ae2e.
relevant to the size of molecule. The bigger molecule is not benefit
for the activity.
In order to examine the cytotoxicity of the synthesized cinna-
manilides, we treated activated or un-activated mouse spleen cells
with different concentrations of the potent immunosuppressive
compounds 11, 18, 37 and 38 for 48 h. The results are shown in
Fig. 3. These compounds did not affect the survival rate of un-
activated cells significantly, which suggested that they should not
be toxic for normal tissues, and the inhibitory effect on activated
mouse spleen cells by these compounds should be specific.
4.3.1. (Z)-4-benzylidene-2-methyloxazol-5(4H)-one (2a)
Yellow powder, yield: 72%. 1H NMR (300 MHz, d6-DMSO): 2.49
(s, 3H, CH3); 7.22 (s, 1H, CH); 7.48e7.52 (m, 3H, ArH); 8.16e8.19 (m,
2H, ArH). MS (ESIþ) m/z 188 (M þ H)þ. Anal. Calcd for C11H9NO2: C,
70.58; H, 4.85; N, 7.48; Found: C, 70.75; H, 4.90; N, 7.46.
4.3.2. (Z)-4-(4-fluorobenzylidene)-2-methyloxazol-5(4H)-one (2b)
Yellow powder, yield: 85%. 1H NMR (300 MHz, d6-DMSO): 2.39
(s, 3H, CH3); 7.25 (s, 1H, CH); 7.35 (t, J ¼ 8.8 Hz, 2H, ArH); 8.23e8.28
(m, 2H, ArH). MS (ESIþ) m/z 206 (M þ H)þ. Anal. Calcd for
C11H8FNO2: C, 64.39; H, 3.93; N, 6.83; Found: C, 64.26; H, 3.96; N,
6.86.
3. Conclusion
A series of new cinnamanilides were synthesized and their
immunosuppressive activity and cytotoxicity were evaluated.
Compounds 37 and 38 exhibited potent immunosuppressive
activity with IC50’s 1.77 ꢀ 0.33 and 0.94 ꢀ 0.13
m
M, respectively. In
4.3.3. (Z)-4-(4-chlorobenzylidene)-2-methyloxazol-5(4H)-one (2c)
Yellow powder, yield: 83%. 1H NMR (300 MHz, d6-DMSO): 2.40
(s, 3H, CH3); 7.24 (s, 1H, CH); 7.58 (d, J ¼ 8.6 Hz, 2H, ArH); 8.20 (d,
J ¼ 8.8 Hz, 2H, ArH). MS (ESIþ) m/z 222 (M þ H)þ. Anal. Calcd for
C11H8ClNO2: C, 59.61; H, 3.64; N, 6.32; Found: C, 59.78; H, 3.62; N,
6.35.
general, electronic-donating substituents at R1 and R2 were favor-
able for the immunosuppressive activity of the synthesized cin-
namanilides. The synthesized cinnamanilides with smaller
molecular size showed more potent activity. Cytotoxicity assaying
of the compounds with potent immunosuppressive activity

588
L. Shi et al. / European Journal of Medicinal Chemistry 47 (2012) 585e593
a
b
c
Fig. 1. Molecular structures of compounds a) 10, b) 14 and c) 32. Displacement ellipsoids are drawn at the 30% probability level.
Table 2
4.3.4. (Z)-4-(4-bromobenzylidene)-2-methyloxazol-5(4H)-one (2d)
Yellow powder, yield: 86%. 1H NMR (300 MHz, d6-DMSO): 2.40
(s, 3H, CH3); 7.24 (s, 1H, CH); 7.58 (d, J ¼ 8.6 Hz, 2H, ArH); 8.20 (d,
J ¼ 8.8 Hz, 2H, ArH). MS (ESIþ) m/z 266 (M þ H)þ. Anal. Calcd for
C11H8BrNO2: C, 49.65; H, 3.03; N, 5.26; Found: C, 49.72; H, 3.02; N,
5.23.
Crystallographical and experimental data for cinnamanilides 10, 14 and 32.
Compound
10
14
32
Formula
C17H17NO2
267.32
C16H13F2NO2
289.27
C19H20BrNO2
374.27
Mr
Crystal size [mm3] 0.30 ꢂ 0.10 ꢂ 0.10 0.30 ꢂ 0.20 ꢂ 0.10 0.40 ꢂ 0.15 ꢂ 0.15
Crystal system
Space group
a [Ǻ]
Monoclinic
P21/c
6.2470(12)
31.964(6)
8.935(3)
90
126.24(2)
90
1439.0(6)
4
Monoclinic
P21/c
11.328(2)
5.4930(11)
22.269(5)
90
98.63(3)
90
1370.0(5)
4
Triclinic
Pꢃ1
4.3.5. (Z)-4-(4-methoxybenzylidene)-2-methyloxazol-5(4H)-one
(2e)
5.5840(11)
9.3800(19)
17.889(4)
103.23(3)
95.01(3)
99.62(3)
891.6(3)
2
B [Ǻ]
C [Ǻ]
Yellow powder, yield: 75%. 1H NMR (300 MHz, d6-DMSO): 2.37
(s, 3H, CH3); 3.83 (s, 3H, OCH3); 7.08 (d, J ¼ 8.6 Hz, 2H, ArH); 7.19 (s,
1H, CH); 8.18 (d, J ¼ 8.8 Hz, 2H, ArH). MS (ESIþ) m/z 218 (M þ H)þ.
Anal. Calcd for C12H11NO3: C, 66.35; H, 5.10; N, 6.45; Found: C,
66.63; H, 5.07; N, 6.49.
a
b
g
[ꢁ]
[ꢁ]
[ꢁ]
V [Å3]
Z
Dc [g cmꢃ3
]
1.234
0.081
1.402
0.111
1.394
2.314
m
[mmꢃ1
]
4.4. General procedure for the preparation of the pyruvic acid
derivatives 3ae3e
F(000)
Max/min trans.
range [ꢁ]
Index ranges
(h, k, l)
Reflections
collected/unique
Data/restraints/
parameters
Rint
568
600
384
0.9920/0.9762
1.27/25.00
ꢃ7/0,ꢃ38/0,
ꢃ8/10
0.9890/0.9675
1.82/25.24
ꢃ13/13,0/6,0/26
0.7228/0.4579
1.18/25.17
0/6, ꢃ11/11,
ꢃ21/21
q
A suspension of oxazole derivatives 2ae2e (20 mmol) in aq. HCl
(3 M, 20 mL) was stirred at reflux for 3 h. The mixture was cooled to
room temperature to allow crystallization. Filtration afforded the
title compound 3ae3e.
2745/2590
2658/2473
2473/0/190
3567/3205
2509/0/181
0.0523
3205/0/208
0.0356
1.172
0.0335
1.112
4.4.1. (Z)-2-hydroxy-3-phenylacrylic acid (3a)
Goodness-of-fit on 1.029
Pale yellow crystal, yield: 68%. 1H NMR (300 MHz, d6-DMSO):
6.39 (s, 1H, CH); 7.23e7.37 (m, 3H, ArH); 7.75 (d, J ¼ 7.2 Hz, 2H,
ArH); 9.24 (s, 1H, OH); 13.21 (s, 1H, COOH). MS (ESIþ) m/z 165
(M þ H)þ. Anal. Calcd for C9H8O3: C, 65.85; H, 4.91; Found: C, 66.01;
H, 4.94.
F2
u
R1,
R1,
R2 [I > 2
R2
s
(I)]a 0.0861/0.1946
0.0742/0.1920
0.1283/0.2796
0.316/-0.308
0.1065/0.2588
0.1645/0.3186
1.496/-1.607
a
u
0.1896/0.2604
0.231/-0.277
Largest diff. peak
and hole (eǺꢃ3
)
a
R1 ¼ SjjFoj-jFcjj/SjFoj, wR2 ¼ [Sw(Fo2-Fc2)2/Sw(Fo2)2]1/2
.

L. Shi et al. / European Journal of Medicinal Chemistry 47 (2012) 585e593
589
9.45 (s, 1H, OH); 13.33 (s, 1H, COOH). MS (ESIþ) m/z 243 (M þ H)þ.
Table 3
Selected bond lengths/Ǻ and angles/ꢁ for cinnamanilides 10, 14 and 32.
Anal. Calcd for C9H7BrO3: C, 44.47; H, 2.90; Found: C, 44.69; H, 2.88.
Compound
10
14
32
4.4.5. (Z)-2-hydroxy-3-(4-methoxyphenyl)acrylic acid (3e)
White powder, yield: 62%. 1H NMR (300 MHz, d6-DMSO): 3.76 (s,
3H, OCH3); 6.37 (s, 1H, CH); 6.92 (d, J ¼ 8.7 Hz, 2H, ArH); 7.71 (d,
J ¼ 8.7 Hz, 2H, ArH); 8.96 (s, 1H, OH); 13.02 (s, 1H, COOH). MS (ESIþ)
m/z 195 (M þ H)þ. Anal. Calcd for C10H10O4: C, 61.85; H, 5.19; Found:
C, 62.04; H, 5.23.
Bond lengths/Ǻ
C(4)eC(7)
C(7)eC(8)
C(8)eO(1)
C(8)eC(10)
C(9)eO(1)
C(10)eO(2)
C(10)eN(1)
C(11)eN(1)
C(14)eC(17)
C(1)eF(1)
1.461(7)
1.319(7)
1.375(5)
1.483(7)
1.431(6)
1.233(5)
1.354(6)
1.418(6)
1.513(8)
e
1.445(6)
1.327(6)
1.390(5)
1.503(6)
1.425(7)
1.218(5)
1.351(6)
1.404(5)
e
1.462(10)
1.316(11)
1.387(8)
1.501(10)
1.425(10)
1.236(9)
1.364(9)
1.394(9)
1.519(11)
e
4.5. General procedure for the preparation of methylation products
4ae4e
1.333(5)
1.350(5)
e
C(14)-F(2)
e
e
Br(1)eC(1)
C(17)eC(18)
C(17)eC(19)
e
1.899(8)
1.479(15)
1.512(14)
Dimethyl sulfate (5 mL) was added dropwise into a suspension
of pyruvic acid derivatives 3ae3e (10 mmol) in 10% NaOH (15 mL)
in ice-water bath and then the mixture was stirred at room
temperature for 4 h. After that, 3 M HCl (15 mL) was added and
stirred for another 0.5 h to precipitate. Filtration afforded the title
compound 4ae4e.
e
e
e
e
Bond angles/ꢁ
C(3)eC(4)eC(7)
C(5)eC(4)eC(7)
C(8)-C(7)-C(4)
117.6(5)
124.9(5)
131.1(5)
123.7(5)
120.3(4)
116.0(4)
122.1(5)
122.3(4)
115.6(4)
121.2(5)
119.2(5)
121.7(7)
120.6(7)
123.8(4)
114.8(4)
e
118.7(4)
124.8(4)
132.2(4)
122.3(4)
121.1(4)
116.3(4)
124.4(4)
121.5(4)
114.1(4)
123.7(4)
117.4(4)
e
118.8(7)
124.1(6)
131.4(7)
123.9(7)
119.9(6)
116.2(6)
123.4(7)
121.8(6)
114.8(6)
123.6(7)
118.0(7)
121.3(8)
122.5(8)
126.6(6)
115.7(6)
e
C(7)eC(8)eO(1)
C(7)eC(8)eC(10)
O(1)eC(8)eC(10)
O(2)eC(10)eN(1)
O(2)eC(10)eC(8)
N(1)eC(10)eC(8)
C(12)eC(11)eN(1)
C(16)eC(11)eN(1)
C(15)eC(14)eC(17)
C(13)eC(14)eC(17)
C(10)eN(1)eC(11)
C(8)eO(1)eC(9)
F(1)eC(1)eC(2)
4.5.1. (Z)-2-methoxy-3-phenylacrylic acid (4a)
Pale yellow powder, yield: 80%. 1H NMR (300 MHz, d6-DMSO):
3.74 (s, 3H, OCH3); 6.93 (s, 1H, CH); 7.34e7.45 (m, 3H, ArH); 7.78 (d,
J ¼ 7.1 Hz, 2H, ArH); 13.00 (s, 1H, COOH). MS (ESIþ) m/z 179
(M þ H)þ. Anal. Calcd for C10H10O3: C, 67.14; H, 5.66; Found: C,
67.01; H, 5.69.
e
128.1(4)
113.0(4)
118.7(4)
119.3(5)
119.6(5)
118.3(4)
e
4.5.2. (Z)-3-(4-fluorophenyl)-2-methoxyacrylic acid (4b)
Pale yellow powder, yield: 83%. 1H NMR (300 MHz, d6-DMSO):
3.73 (s, 3H, OCH3); 6.93 (s, 1H, CH); 7.24 (t, J ¼ 9.0 Hz, 2H, ArH);
7.81e7.86 (m, 2H, ArH); 13.09 (s, 1H, COOH). MS (ESIþ) m/z 197
(M þ H)þ. Anal. Calcd for C10H9FO3: C, 61.22; H, 4.62; Found: C,
61.45; H, 4.65.
F(1)eC(1)eC(6)
e
e
F(2)eC(14)eC(15)
F(2)eC(14)eC(13)
C(2)eC(1)eBr(1)
C(6)eC(1)eBr(1)
C(18)eC(17)eC(19)
C(18)eC(17)eC(14)
C(19)eC(17)eC(14)
e
e
e
e
e
120.5(7)
117.9(6)
113.6(11)
114.1(8)
110.4(8)
e
e
e
e
4.5.3. (Z)-3-(4-chlorophenyl)-2-methoxyacrylic acid (4c)
Pale yellow powder, yield: 86%. 1H NMR (300 MHz, d6-DMSO):
3.73 (s, 3H, OCH3); 6.89 (s,1H, CH); 7.47 (d, J ¼ 8.6 Hz, 2H, ArH); 7.78
(d, J ¼ 8.6 Hz, 2H, ArH); 13.07 (s, 1H, COOH). MS (ESIþ) m/z 213
(M þ H)þ. Anal. Calcd for C10H9ClO3: C, 56.49; H, 4.27; Found: C,
56.38; H, 4.25.
e
e
e
e
4.4.2. (Z)-3-(4-fluorophenyl)-2-hydroxyacrylic acid (3b)
Pale yellow crystal, yield: 72%. 1H NMR (300 MHz, d6-DMSO):
6.41 (s, 1H, CH); 7.17 (t, J ¼ 8.9 Hz, 2H, ArH); 7.78e7.83 (m, 2H, ArH);
9.27 (s, 1H, OH). MS (ESIþ) m/z 183 (M þ H)þ. Anal. Calcd for
C9H7FO3: C, 59.35; H, 3.87; Found: C, 59.46; H, 3.85.
4.5.4. (Z)-3-(4-bromophenyl)-2-methoxyacrylic acid (4d)
Pale yellow powder, yield: 85%. 1H NMR (300 MHz, d6-DMSO):
3.73 (s, 3H, OCH3); 6.87 (s, 1H, CH); 7.59 (d, J ¼ 8.6 Hz, 2H, ArH); 7.71
(d, J ¼ 8.6 Hz, 2H, ArH); 13.09 (s, 1H, COOH). MS (ESIþ) m/z
257(M þ H)þ. Anal. Calcd for C10H9BrO3: C, 46.72; H, 3.53; Found: C,
46.75; H, 3.52.
4.4.3. (Z)-3-(4-chlorophenyl)-2-hydroxyacrylic acid (3c)
White powder, yield: 70%. 1H NMR (300 MHz, d6-DMSO): 6.40
(s, 1H, CH); 7.40 (d, J ¼ 8.6 Hz, 2H, ArH); 7.78 (d, J ¼ 8.6 Hz, 2H, ArH);
9.48 (s, 1H, OH); 13.30 (s, 1H, COOH). MS (ESIþ) m/z 199 (M þ H)þ.
Anal. Calcd for C9H7ClO3: C, 54.43; H, 3.55; Found: C, 54.48; H, 3.52.
4.5.5. (Z)-2-methoxy-3-(4-methoxyphenyl)acrylic acid (4e)
Pale yellow powder, yield: 80%. 1H NMR (300 MHz, d6-DMSO):
3.67 (s, 3H, OCH3); 3.77 (s, 3H, OCH3); 6.87 (s, 1H, CH); 6.95 (d,
J ¼ 8.4 Hz, 2H, ArH); 7.71 (d, J ¼ 8.4 Hz, 2H, ArH); 13.05 (s, 1H,
COOH). MS (ESIþ) m/z 209(M þ H)þ. Anal. Calcd for C11H12O4: C,
63.45; H, 5.81; Found: C, 63.72; H, 5.78.
4.4.4. (Z)-3-(4-bromophenyl)-2-hydroxyacrylic acid (3d)
White powder, yield: 76%. 1H NMR (300 MHz, d6-DMSO): 6.37 (s,
1H, CH); 7.53 (d, J ¼ 8.6 Hz, 2H, ArH); 7.70 (d, J ¼ 8.6 Hz, 2H, ArH);
Table 4
Hydrogen bonds for cinnamanilides 10, 14 and 32 [Å and ꢁ].
4.6. General procedure for the preparation of 2-methoxy
cinnamanilides 6e40
Compound
D-H.A
D-H
H.A
D.A
D-H.A
10a
N(1)eH(1A).O(2)#1
N(1)eH(1A).O(1)
N(1)eH(1A).O(2)#1
N(1)eH(1A).O(2)#1
0.86
0.86
0.86
0.86
2.17
2.25
2.50
2.57
2.944(5)
2.682(5)
3.292(5)
3.356(8)
149.6
111.4
152.9
152.7
14b
A solution of 4ae4e (2 mmol) and EDC.HCl (3 mmol) in CH2Cl2
(10 mL) was added phenylamine 5fe5l (2 mmol), respectively.
Stirred at refluxing for 6e8 h and evaporated of the solvent, the
residue was purified by column chromatography on silica gel,
eluting with petroleum ether/EtOAc (3/1) to furnish title compound
6e40.
32c
a
Symmetry transformations used to generate equivalent atoms: #1 xꢃ1,
ꢃyþ1/2,zꢃ1/2.
b
c
Symmetry transformations used to generate equivalent atoms: #1 x,yþ1,z.
Symmetry transformations used to generate equivalent atoms: #1 xꢃ1,y,z.

590
L. Shi et al. / European Journal of Medicinal Chemistry 47 (2012) 585e593
Fig. 2. The spleen cells from 6 to 8 week-old BALB/c mice were co-stimulated by CD3/CD28 at the present of 5 mg/ml of different compounds for 48 h. The cell survival rate was
taken by MTT assay.
4.6.1. (Z)-2-methoxy-N,3-diphenylacrylamide (6)
4.6.5. (Z)-2-methoxy-3-phenyl-N-p-tolylacrylamide (10)
White powder, 1H NMR (300 MHz, d6-DMSO): 3.71 (s, 3H,
OCH3); 6.80 (s, 1H, CH); 7.08e7.12 (m, 1H, ArH); 7.32e7.45 (m, 5H,
ArH); 7.74e7.79 (m, 4H, ArH); 10.11 (s, 1H, NH). MS (ESIþ) m/z 254
(M þ H)þ. Anal. Calcd for C16H15NO2: C, 75.87; H, 5.97; N, 5.53;
Found: C, 75.78; H, 5.91; N, 5.61.
Colorless crystal, 1H NMR (300 MHz, d6-DMSO): 2.28 (s, 3H,
CH3); 3.70 (s, 3H, OCH3); 6.78 (s, 1H, CH); 7.13 (d, J ¼ 7.4 Hz, 2H,
ArH); 7.31e7.36 (m, 1H, ArH); 7.40e7.45 (m, 2H, ArH); 7.65 (d,
J ¼ 8.4 Hz, 2H, ArH); 7.74 (d, J ¼ 7.3 Hz, 2H, ArH); 10.03(s, 1H, NH).
MS (ESIþ) m/z 268 (M þ H)þ. Anal. Calcd for C17H17NO2: C, 76.38; H,
6.41; N, 5.24; Found: C, 76.51; H, 6.47; N, 5.18.
4.6.2. (Z)-N-(4-fluorophenyl)-2-methoxy-3-phenylacrylamide (7)
White powder, 1H NMR (300 MHz, d6-DMSO): 3.70 (s, 3H,
OCH3); 6.81 (s, 1H, CH); 7.16e7.22 (m, 2H, ArH); 7.32e7.36 (m, 1H,
ArH); 7.40e7.45 (m, 2H, ArH); 7.74e7.82 (m, 4H, ArH); 10.19 (s, 1H,
NH). MS (ESIþ) m/z 272 (M þ H)þ. Anal. Calcd for C16H14FNO2: C,
70.84; H, 5.20; N, 5.16; Found: C, 71.13; H, 5.25; N, 5.14.
4.6.6. (Z)-N-(4-isopropylphenyl)-2-methoxy-3-phenylacrylamide
(11)
White powder, 1H NMR (300 MHz, d6-DMSO): 1.16 (d, J ¼ 6.9 Hz,
6H, CH3); 2.81e2.88 (m,1H, CH); 3.70 (s, 3H, OCH3); 6.78 (s,1H, CH);
7.21 (d, J ¼ 8.6 Hz, 2H, ArH); 7.31e7.36 (m, 1H, ArH); 7.40e7.45 (m,
2H, ArH); 7.67 (d, J ¼ 8.6 Hz, 2H, ArH); 7.73e7.77 (m, 2H, ArH); 10.04
(s, 1H, NH). MS (ESIþ) m/z 296 (M þ H)þ. Anal. Calcd for C19H21NO2:
C, 77.26; H, 7.17; N, 4.74; Found: C, 77.21; H, 7.23; N, 4.72.
4.6.3. (Z)-N-(4-chlorophenyl)-2-methoxy-3-phenylacrylamide (8)
White powder, 1H NMR (300 MHz, d6-DMSO): 3.70 (s, 3H,
OCH3); 6.82 (s, 1H, CH); 7.34e7.43 (m, 5H, ArH); 7.74e7.84 (m, 4H,
ArH); 10.26 (s, 1H, NH). MS (ESIþ) m/z 288 (M þ H)þ. Anal. Calcd for
C16H14ClNO2: C, 66.79; H, 4.90; N, 4.87; Found: C, 66.93; H, 4.95; N,
4.94.
4.6.7. (Z)-2-methoxy-N-(4-methoxyphenyl)-3-phenylacrylamide
(12)
White powder, 1H NMR (300 MHz, d6-DMSO): 3.69 (s, 3H,
OCH3); 3.74 (s, 3H, OCH3); 6.78 (s, 1H, CH); 6.92 (d, J ¼ 8.7 Hz, 2H,
ArH); 7.33e7.45 (m, 3H, ArH); 7.66e7.75 (m, 4H, ArH); 10.00 (s, 1H,
NH). MS (ESIþ) m/z 284 (M þ H)þ. Anal. Calcd for C17H17NO3: C,
72.07; H, 6.05; N, 4.94; Found: C, 71.83; H, 6.08; N, 5.01.
4.6.4. (Z)-N-(4-bromophenyl)-2-methoxy-3-phenylacrylamide (9)
White powder, 1H NMR (300 MHz, d6-DMSO): 3.70 (s, 3H,
OCH3); 6.82 (s, 1H, CH); 7.34e7.45 (m, 3H, ArH); 7.52e7.55 (m, 2H,
ArH); 7.74e7.79 (m, 4H, ArH); 10.25 (s, 1H, NH). MS (ESIþ) m/z 332
(M þ H)þ. Anal. Calcd for C16H14BrNO2: C, 57.85; H, 4.25; N, 4.22;
Found: C, 57.81; H, 4.27; N, 4.25.
4.6.8. (Z)-3-(4-fluorophenyl)-2-methoxy-N-phenylacrylamide (13)
White powder, 1H NMR (300 MHz, d6-DMSO): 3.70 (s, 3H,
OCH3); 6.79 (s, 1H, CH); 7.11 (d, J ¼ 7.3 Hz, 1H, ArH); 7.23e7.37 (m,
4H, ArH); 7.76e7.84 (m, 4H, ArH); 10.11 (s, 1H, NH). MS (ESIþ) m/z
272 (M þ H)þ. Anal. Calcd for C16H14FNO2: C, 70.84; H, 5.20; N, 5.16;
Found: C, 70.92; H, 5.24; N, 5.13.
Table 5
In vitro inhibitory effects of the synthetic cinnamanilides on mouse spleen cells co-
stimulated by CD3/CD28.
Compound
R1
R2
IC50 [mM]
4.6.9. (Z)-N,3-bis(4-fluorophenyl)-2-methoxyacrylamide (14)
Colorless crystal, 1H NMR (300 MHz, d6-DMSO): 3.70 (s, 3H,
OCH3); 6.80 (s, 1H, CH); 7.15e7.28 (m, 4H, ArH); 7.77e7.83 (m, 4H,
ArH); 10.16 (s, 1H, NH). MS (ESIþ) m/z 290 (M þ H)þ. Anal. Calcd for
C16H13F2NO2: C, 66.43; H, 4.53; N, 4.84; Found: C, 66.58; H, 4.54; N,
4.78.
6
7
8
H
H
H
H
H
F
F
F
Cl
Cl
Cl
Br
OCH3
OCH3
OCH3
H
F
Cl
15.22 ꢀ 1.38
4.94 ꢀ 0.66
17.00 ꢀ 1.99
16.59 ꢀ 1.72
3.46 ꢀ 0.68
9.48 ꢀ 1.02
4.66 ꢀ 0.70
13.46 ꢀ 1.20
14.00 ꢀ 1.80
11.01 ꢀ 0.74
12.72 ꢀ 1.20
12.87 ꢀ 1.74
14.73 ꢀ 1.39
1.77 ꢀ 0.33
0.94 ꢀ 0.13
10
11
15
18
19
21
23
24
31
36
37
38
CH3
CH(CH3)2
Cl
CH(CH3)2
OCH3
F
4.6.10. (Z)-N-(4-chlorophenyl)-3-(4-fluorophenyl)-2-
methoxyacrylamide (15)
Br
White powder, 1H NMR (300 MHz, d6-DMSO): 3.70 (s, 3H,
OCH3); 6.82 (s, 1H, CH); 7.26 (t, J ¼ 8.9 Hz, 2H, ArH); 7.39e7.42 (m,
2H, ArH); 7.79e7.84 (m, 4H, ArH); 10.26 (s, 1H, NH). MS (ESIþ) m/z
306 (M þ H)þ. Anal. Calcd for C16H13ClFNO2: C, 62.86; H, 4.29; N,
4.58; Found: C, 62.90; H, 4.32; N, 4.55.
CH3
CH3
Cl
Br
CH3

L. Shi et al. / European Journal of Medicinal Chemistry 47 (2012) 585e593
591
Fig. 3. Stimulated or non-stimulated spleen cells from 6 to 8 week-old BALB/c mice were incubated with different amount of compound 11, 18, 37 and 38 for 48 h, the cell toxicity of
these compounds were measured by MTT assay.
4.6.11. (Z)-N-(4-bromophenyl)-3-(4-fluorophenyl)-2-
methoxyacrylamide (16)
2H, ArH); 7.48 (d, J ¼ 8.4 Hz, 2H, ArH); 7.77 (d, J ¼ 8.6 Hz, 4H, ArH);
10.16 (s, 1H, NH). MS (ESIþ) m/z 288 (M þ H)þ. Anal. Calcd for
C16H14ClNO2: C, 66.79; H, 4.90; N, 4.87; Found: C, 68.07; H, 4.84; N,
4.91.
White powder, 1H NMR (300 MHz, d6-DMSO): 3.67 (s, 3H,
OCH3); 6.79 (s, 1H, ArH); 7.24 (t, J ¼ 8.9 Hz, 2H, ArH); 7.50 (d,
J ¼ 8.8 Hz, 2H, ArH); 7.72e7.81 (m, 4H, ArH); 10.23 (s, 1H, NH). MS
(ESIþ) m/z 350 (M þ H)þ. Anal. Calcd for C16H13BrFNO2: C, 54.88; H,
3.74; N, 4.00; Found: C, 55.02; H, 3.72; N, 4.04.
4.6.16. (Z)-3-(4-chlorophenyl)-N-(4-fluorophenyl)-2-
methoxyacrylamide (21)
White powder, 1H NMR (300 MHz, d6-DMSO): 3.71 (s, 3H,
OCH3); 6.77 (s, 1H, CH); 7.19 (t, J ¼ 9.0 Hz, 2H, ArH); 7.48 (d,
J ¼ 8.6 Hz, 2H, ArH); 7.76e7.81 (m, 4H, ArH); 10.24 (s, 1H, NH). MS
(ESIþ) m/z 306 (M þ H)þ. Anal. Calcd for C16H13ClFNO2: C, 62.85; H,
4.29; N, 4.58; Found: C, 62.80; H, 4.31; N, 4.62.
4.6.12. (Z)-3-(4-fluorophenyl)-2-methoxy-N-p-tolylacrylamide (17)
White powder, 1H NMR (300 MHz, d6-DMSO): 2.27 (s, 3H, CH3);
3.69 (s, 3H, OCH3); 6.77 (s,1H, CH); 7.14 (d, J ¼ 8.2Hz, 2H, ArH); 7.25 (t,
J ¼ 9.0 Hz, 2H, ArH); 7.64 (d, J ¼ 8.4 Hz, 2H, ArH); 7.77e7.82 (m, 2H,
ArH); 10.01(s, 1H, NH). MS (ESIþ) m/z 286 (M þ H)þ. Anal. Calcd for
C17H16FNO2: C, 71.56; H, 5.65; N, 4.91; Found: C, 71.83; H, 5.61; N, 5.02.
4.6.17. (Z)-N,3-bis(4-chlorophenyl)-2-methoxyacrylamide (22)
White powder, 1H NMR (300 MHz, d6-DMSO): 3.71 (s, 3H,
OCH3); 6.78 (s, 1H, CH); 7.41 (d, J ¼ 8.8 Hz, 2H, ArH), 7.48 (d,
J ¼ 8.4 Hz, 2H, ArH); 7.76e7.83 (m, 4H, ArH); 10.30 (s, 1H,
4.6.13. (Z)-3-(4-fluorophenyl)-N-(4-isopropylphenyl)-2-
methoxyacrylamide (18)
White powder, 1H NMR (300 MHz, d6-DMSO): 1.17 (d, J ¼ 7.0 Hz,
6H, CH3); 2.79e2.88 (m, 1H, CH); 3.67 (s, 3H, OCH3); 6.75 (s, 1H,
CH); 7.17e7.26 (m, 4H, ArH); 7.64 (d, J ¼ 8.4 Hz, 2H, ArH); 7.75e7.80
(m, 2H, ArH); 9.99 (s, 1H, NH). MS (ESIþ) m/z 314 (M þ H)þ. Anal.
Calcd for C19H20FNO2: C, 72.82; H, 6.43; N, 4.47; Found: C, 72.95; H,
6.45; N, 4.43.
NH). MS (ESIþ) m/z 322 (M
C16H13Cl2NO2: C, 59.65; H, 4.07; N, 4.35; Found: C, 59.87; H,
4.18; N, 4.32.
þ
H)þ. Anal. Calcd for
4.6.18. (Z)-N-(4-bromophenyl)-3-(4-chlorophenyl)-2-
methoxyacrylamide (23)
White powder, 1H NMR (300 MHz, d6-DMSO): 3.71 (s, 3H,
OCH3); 6.77 (s, 1H, CH); 7.47e7.55 (m, 4H, ArH), 7.74e7.78 (m, 4H,
ArH); 10.29 (s, 1H, NH). MS (ESIþ) m/z 366 (M þ H)þ. Anal. Calcd for
C16H13BrClNO2: C, 52.41; H, 3.57; N, 3.82; Found: C, 52.48; H, 3.54;
N, 3.86.
4.6.14. (Z)-3-(4-fluorophenyl)-2-methoxy-N-(4-methoxyphenyl)
acrylamide (19)
White powder, 1H NMR (300 MHz, d6-DMSO): 3.69 (s, 3H,
OCH3); 3.74 (s, 3H, OCH3); 6.78 (s, 1H, CH); 6.92 (d, J ¼ 9.1 Hz, 2H,
ArH); 7.25 (t, J ¼ 9.0 Hz, 2H, ArH); 7.67 (d, J ¼ 9.1 Hz, 2H, ArH);
7.77e7.82 (m, 2H, ArH); 9.99(s, 1H, NH). MS (ESIþ) m/z 302
(M þ H)þ. Anal. Calcd for C17H16FNO3: C, 67.76; H, 5.35; N, 4.65;
Found: C, 67.63; H, 5.41; N, 4.63.
4.6.19. (Z)-3-(4-chlorophenyl)-2-methoxy-N-p-tolylacrylamide
(24)
White powder, 1H NMR (300 MHz, d6-DMSO): 2.27 (s, 3H, CH3);
3.70 (s, 3H, OCH3); 6.73 (s, 1H, CH); 7.15 (d, J ¼ 8.4 Hz, 2H, ArH);
7.47(d, J ¼ 8.4 Hz, 2H, ArH); 7. 64 (d, J ¼ 8.4 Hz, 2H, ArH); 7.76 (d,
J ¼ 8.6 Hz, 2H, ArH); 10.07 (s, 1H, NH). MS (ESIþ) m/z 302 (M þ H)þ.
Anal. Calcd for C17H16ClNO2: C, 67.66; H, 5.34; N, 4.64; Found: C,
57.59; H, 5.39; N, 4.65.
4.6.15. (Z)-3-(4-chlorophenyl)-2-methoxy-N-phenylacrylamide
(20)
White powder, 1H NMR (300 MHz, d6-DMSO): 3.72 (s, 3H,
OCH3); 6.75 (s, 1H, CH); 7.11 (t, J ¼ 7.3 Hz, 1H, ArH); 7.32e7.37 (m,

592
L. Shi et al. / European Journal of Medicinal Chemistry 47 (2012) 585e593
4.6.20. (Z)-3-(4-chlorophenyl)-N-(4-isopropylphenyl)-2-
methoxyacrylamide (25)
(s, 1H, CH); 7.21 (d, J ¼ 8.6 Hz, 2H, ArH); 7.59e7.71 (m, 6H, ArH);
10.09 (s, 1H, NH). MS (ESIþ) m/z 374 (M þ H)þ. Anal. Calcd for
C19H20BrNO2: C, 60.97; H, 5.39; N, 3.74; Found: C, 60.90; H, 5.43; N,
3.79.
White powder, 1H NMR (300 MHz, d6-DMSO): 1.20 (d, J ¼ 7.0 Hz,
6H, CH3); 2.82e2.90 (m, 1H, CH); 3.71 (s, 3H, OCH3); 6.74 (s, 1H,
CH); 7.21 (d, J ¼ 8.4 Hz, 2H, ArH); 7.48 (d, J ¼ 8.4 Hz, 2H, ArH); 7.67
(d, J ¼ 8.4 Hz, 2H, ArH); 7.76 (d, J ¼ 8.6 Hz, 2H, ArH); 10.08 (s, 1H,
NH). MS (ESIþ) m/z 330 (M þ H)þ. Anal. Calcd for C19H20ClNO2: C,
69.19; H, 6.11; N, 4.25; Found: C, 69.35; H, 6.06; N, 4.29.
4.6.28. (Z)-3-(4-bromophenyl)-2-methoxy-N-(4-methoxyphenyl)
acrylamide (33)
White powder, 1H NMR (300 MHz, d6-DMSO): 3.68 (s, 3H,
OCH3); 3.71 (s, 3H, OCH3); 6.68 (s, 1H, CH); 6.89 (d, J ¼ 9.0 Hz, 2H,
ArH); 7.58 (d, J ¼ 8.6 Hz, 2H, ArH); 7.62e7.67 (m, 4H, ArH); 10.01 (s,
1H, NH). MS (ESIþ) m/z 362 (M þ H)þ. Anal. Calcd for C17H16BrNO3:
C, 56.37; H, 4.45; N, 3.87; Found: C, 56.45; H, 4.44; N, 3.83.
4.6.21. (Z)-3-(4-chlorophenyl)-2-methoxy-N-(4-methoxyphenyl)
acrylamide (26)
White powder, 1H NMR (300 MHz, d6-DMSO): 3.71 (s, 3H,
OCH3); 3.75 (s, 3H, OCH3); 6.73 (s, 1H, CH); 6.92 (d, J ¼ 8.8 Hz, 2H,
ArH); 7.47 (d, J ¼ 8.4 Hz, 2H, ArH); 7.67 (d, J ¼ 8.8 Hz, 2H, ArH); 7.75
(d, J ¼ 8.4 Hz, 2H, ArH); 10.01(s, 1H, NH). MS (ESIþ) m/z 318
(M þ H)þ. Anal. Calcd for C17H16ClNO3: C, 64.24; H, 5.08; N, 4.41;
Found: C, 64.56; H, 5.03; N, 4.35.
4.6.29. (Z)-2-methoxy-3-(4-methoxyphenyl)-N-phenylacrylamide
(34)
White powder, 1H NMR (300 MHz, d6-DMSO): 3.68 (s, 3H,
OCH3); 3.80 (s, 3H, OCH3); 6.82 (s, 1H, CH); 7.0 (d, J ¼ 8.9 Hz, 2H,
ArH); 7.07e7.12 (m, 1H, ArH); 7.30e7.36 (m, 2H, ArH); 7.70e7.79
(m, 4H, ArH); 9.99 (s, 1H, NH). MS (ESIþ) m/z 284 (M þ H)þ. Anal.
Calcd for C17H17NO3: C, 72.07; H, 6.05; N, 4.94; Found: C, 72.29; H,
6.14; N, 4.92.
4.6.22. (Z)-3-(4-bromophenyl)-2-methoxy-N-phenylacrylamide
(27)
White powder, 1H NMR (300 MHz, d6-DMSO): 3.71 (s, 3H,
OCH3); 6.72 (s, 1H, CH); 7.08e7.13 (m, 1H, ArH); 7.32e7.37 (m, 2H,
ArH); 7.61 (d, J ¼ 8.6 Hz, 2H, ArH); 7.70 (d, J ¼ 8.6 Hz, 2H, ArH); 7.76
(d, J ¼ 7.9Hz, 2H, ArH); 10.16 (s, 1H, NH). MS (ESIþ) m/z 332
(M þ H)þ. Anal. Calcd for C16H14BrNO2: C, 57.85; H, 4.25; N, 4.22;
Found: C, 57.92; H, 4.24; N, 4.28.
4.6.30. (Z)-N-(4-fluorophenyl)-2-methoxy-3-(4-methoxyphenyl)
acrylamide (35)
White powder, 1H NMR (300 MHz, d6-DMSO): 3.67 (s, 3H,
OCH3); 3.80 (s, 3H, OCH3); 6.83 (s, 1H, CH); 7.00 (d, J ¼ 8.8 Hz, 2H,
ArH); 7.18 (t, J ¼ 9.0 Hz, 2H, ArH); 7.72 (d, J ¼ 8.8 Hz, 2H, ArH);
7.78e7.82 (m, 2H, ArH); 10.07 (s, 1H, NH). MS (ESIþ) m/z 302
(M þ H)þ. Anal. Calcd for C17H16FNO3: C, 67.76; H, 5.35; N, 4.65;
Found: C, 67.65; H, 5.31; N, 4.62.
4.6.23. (Z)-3-(4-bromophenyl)-N-(4-fluorophenyl)-2-
methoxyacrylamide (28)
White powder, 1H NMR (300 MHz, d6-DMSO): 3.71 (s, 3H,
OCH3); 6.74 (s, 1H, CH); 7.16e7.22 (m, 2H, ArH); 7.61 (d, J ¼ 9.0 Hz,
2H, ArH); 7.69 (d, J ¼ 9.0 Hz, 2H, ArH); 7.76e7.81 (m, 2H, ArH); 10.24
(s, 1H, NH). MS (ESIþ) m/z 350 (M þ H)þ. Anal. Calcd for
C16H13BrFNO2: C, 54.88; H, 3.74; N, 4.00; Found: C, 54.75; H, 3.79; N,
4.06.
4.6.31. (Z)-N-(4-chlorophenyl)-2-methoxy-3-(4-methoxyphenyl)
acrylamide (36)
White powder, 1H NMR (300 MHz, d6-DMSO): 3.67 (s, 3H,
OCH3); 3.80 (s, 3H, OCH3); 6.84 (s, 1H, CH); 7.00 (d, J ¼ 8.8 Hz, 2H,
ArH); 7.38e7.41 (m, 2H, ArH); 7.72 (d, J ¼ 9.0 Hz, 2H, ArH);
7.80e7.84 (m, 2H, ArH); 10.15 (s, 1H, NH). MS (ESIþ) m/z 318
(M þ H)þ. Anal. Calcd for C17H16ClNO3: C, 64.26; H, 5.08; N, 4.41;
Found: C, 64.40; H, 5.09; N, 4.35.
4.6.24. (Z)-3-(4-bromophenyl)-N-(4-chlorophenyl)-2-
methoxyacrylamide (29)
White powder, 1H NMR (300 MHz, d6-DMSO): 3.71 (s, 3H,
OCH3); 6.74 (s, 1H, CH); 7.40 (d, J ¼ 8.8 Hz, 2H, ArH); 7.61 (d,
J ¼ 8.4 Hz, 2H, ArH); 7.69 (d, J ¼ 8.4 Hz, 2H, ArH); 7.80 (d, J ¼ 8.9 Hz,
2H, ArH); 10.27 (s, 1H, NH). MS (ESIþ) m/z 366 (M þ H)þ. Anal. Calcd
for C16H13BrClNO2: C, 52.41; H, 3.57; N, 3.82; Found: C, 52.53; H,
3.62; N, 3.79.
4.6.32. (Z)-N-(4-bromophenyl)-2-methoxy-3-(4-methoxyphenyl)
acrylamide (37)
White powder, 1H NMR (300 MHz, d6-DMSO): 3.67 (s, 3H,
OCH3); 3.80 (s, 3H, OCH3); 6.83 (s, 1H, CH); 7.00 (d, J ¼ 8.7 Hz, 2H,
ArH); 7.52 (d, J ¼ 8.7 Hz, 2H, ArH); 7.70e7.79 (m, 4H, ArH); 10.13 (s,
1H, NH). MS (ESIþ) m/z 362 (M þ H)þ. Anal. Calcd for C17H16BrNO3:
C, 56.37; H, 4.45; N, 3.87; Found: C, 56.43; H, 4.47; N, 3.85.
4.6.25. (Z)-N,3-bis(4-bromophenyl)-2-methoxyacrylamide (30)
White powder, 1H NMR (300 MHz, d6-DMSO): 3.71 (s, 3H,
OCH3); 6.75 (s, 1H, CH); 7.53 (d, J ¼ 8.8 Hz, 2H, ArH); 7.61 (d,
J ¼ 8.6 Hz, 2H, ArH); 7.69 (d, J ¼ 8.6 Hz, 2H, ArH); 7.75 (d, J ¼ 8.8 Hz,
2H, ArH); 10.27 (s, 1H, NH). MS (ESIþ) m/z 410 (M þ H)þ. Anal. Calcd
for C16H13Br2NO2: C, 46.75; H, 3.19; N, 3.41; Found: C, 46.94; H,
3.25; N, 3.33.
4.6.33. (Z)-2-methoxy-3-(4-methoxyphenyl)-N-p-tolylacrylamide
(38)
White powder, 1H NMR (300 MHz, d6-DMSO): 2.27 (s, 3H, CH3);
3.70 (s, 3H, OCH3); 3.79 (s, 3H, OCH3); 6.80 (s, 1H, CH); 6.99 (d,
J ¼ 8.8 Hz, 2H, ArH); 7.13 (d, J ¼ 8.4 Hz, 2H, ArH); 7.65 (d, J ¼ 8.4 Hz,
2H, ArH); 7.70 (d, J ¼ 8.8 Hz, 2H, ArH); 9.88 (s, 1H, NH). 13C NMR (d6-
DMSO): 198.6, 159.9, 158.8, 135.5, 133.9, 131.1, 129.6, 127.4, 120.5,
114.7, 114.3, 56.0, 44.3, 20.9. MS (ESIþ) m/z 298 (M þ H)þ. Anal.
Calcd for C18H19NO3: C, 72.71; H, 6.44; N, 4.71; Found: C, 72.46; H,
6.48; N, 4.80.
4.6.26. (Z)-3-(4-bromophenyl)-2-methoxy-N-p-tolylacrylamide
(31)
White powder, 1H NMR (300 MHz, d6-DMSO): 2.28 (s, 3H, CH3);
3.70 (s, 3H, OCH3); 6.71 (s, 1H, CH); 7.15 (d, J ¼ 8.4 Hz, 2H, ArH);
7.59e7.71 (m, 6H, ArH); 10.09 (s, 1H, NH). MS (ESIþ) m/z 346
(M þ H)þ. Anal. Calcd for C17H16BrNO2: C, 58.97; H, 4.66; N, 4.05;
Found: C, 59.18; H, 4.60; N, 4.03.
4.6.34. (Z)-N-(4-isopropylphenyl)-2-methoxy-3-(4-
methoxyphenyl)acrylamide (39)
4.6.27. (Z)-3-(4-bromophenyl)-N-(4-isopropylphenyl)-2-
methoxyacrylamide (32)
White powder, 1H NMR (300 MHz, d6-DMSO): 1.19 (d, J ¼ 6.9 Hz,
6H, CH3); 2.80e2.89 (m, 1H, CH); 3.67 (s, 3H, OCH3); 3.79 (s, 3H,
OCH3); 6.80 (s, 1H, CH); 6.99 (d, J ¼ 8.8 Hz, 2H, ArH); 7.20 (d,
J ¼ 8.6 Hz, 2H, ArH); 7.67 (d, J ¼ 8.6 Hz, 2H, ArH); 7.71 (d, J ¼ 8.8 Hz,
Colorless crystal, 1H NMR (300 MHz, d6-DMSO): 1.19 (d,
J ¼ 6.9 Hz, 6H, CH3); 2.81e2.88 (m, 1H, CH); 3.70 (s, 3H, OCH3); 6.71

L. Shi et al. / European Journal of Medicinal Chemistry 47 (2012) 585e593
593
2H, ArH); 9.91 (s, 1H, NH). MS (ESIþ) m/z 326 (M þ H)þ. Anal. Calcd
for C20H23NO3: C, 73.82; H, 7.12; N, 4.30; Found: C, 73.98; H, 7.07; N,
4.32.
and 10
the end of culture. Then 90
7.2) was added to each well for 6 h and the absorbance value at
570 nm was collected by microplate reader. The percentage of cell
growth inhibition was determined using the following formula:
m
g/mL). 20
m
L MTT (5 mg/mL) reagent was added 4 h before
L of lysis buffer (10% SDS, 50% DMF, pH
m
4.6.35. (Z)-2-methoxy-N,3-bis(4-methoxyphenyl)acrylamide (40)
White powder, 1H NMR (300 MHz, d6-DMSO): 3.67 (s, 3H,
OCH3); 3.74 (s, 3H, OCH3); 3.79 (s, 3H, OCH3); 6.80 (s, 1H, CH); 6.90
(d, J ¼ 9.0 Hz, 2H, ArH); 6.99 (d, J ¼ 8.8 Hz, 2H, ArH); 7.66e7.72 (m,
4H, ArH); 9.88 (s, 1H, NH). MS (ESIþ) m/z 326 (M þ H)þ. Anal. Calcd
for C18H19NO4: C, 68.99; H, 6.11; N, 4.47; Found: C, 68.85; H, 6.08; N,
4.51.
Inhibitory rate ð%Þ ¼ f1 ꢃ ½Compounds ðOD570Þ
ꢃ Background ðOD570Þꢄ=
ꢂ ½Control ðOD570Þ
ꢃ Background ðOD570Þꢄg ꢂ 100
4.7. Biological assay
4.7.1. Materials
Acknowledgments
Stock solutions of compounds were prepared with dime-
thylsulfoxide (DMSO, Sigma) and diluted with RPMI-1640 medium
containing 10% fetal bovine serum (FBS). MTT was purchased from
Sigma (St Louis, Mo). CD28 and CD3 were purchased from BD
Pharmingen (San Diego, CA).
The work was financed by grants (Project 21102182 and
30772627) from National Natural Science Foundation of China.
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