The synthesis and biological evaluation of some caffeic acid amide derivatives: E-2-Cyano-(3-substituted phenyl)acrylamides

Article abstract of DOI:10.1016/j.bmcl.2009.02.081

A series of caffeic acid amide derivatives 2-cyano-(3-substituted phenyl)acrylamides were synthesized via Knoevenogal condensation of substituted benzaldehydes with cyanoacetamides. The structure of compound 1f was determined as E-isomer by X-ray diffractive analysis. The biological screening tests in vitro showed that compound 1b has obvious inhibitory activities against human gastric carcinoma cell line BGC-823, human nasopharyngeal carcinoma cell line KB and human hepatoma cell line BEL-7402 with IC₅₀ values of 5.6 μg/mL, 13.1 μg/mL and 12.5 μg/mL, respectively. Some preliminary structure-activity relationships (SAR) were also proposed which may provide a direction for further study.

Full text of DOI:10.1016/j.bmcl.2009.02.081

Bioorganic & Medicinal Chemistry Letters 19 (2009) 1861–1865
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
The synthesis and biological evaluation of some caffeic acid amide derivatives:
E-2-Cyano-(3-substituted phenyl) acrylamides
Wei Zhou a, Hai-bo Li b, Chun-nian Xia a, Xian-ming Zheng c, Wei-xiao Hu a,
*
a College of Pharmaceutical Science, Zhejiang University of Technology, Hangzhou, 310014, PR China
b Nantong Institute for the Control of Pharmaceutical and Biological Products, Nantong, Jiangsu 226001, PR China
c Zhejiang Huafang Pharmaceutical Co., Ltd, Taizhou, Zhejiang 318020, PR 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 caffeic acid amide derivatives 2-cyano-(3-substituted phenyl)acrylamides were synthesized
via Knoevenogal condensation of substituted benzaldehydes with cyanoacetamides. The structure of
compound 1f was determined as E-isomer by X-ray diffractive analysis. The biological screening tests
in vitro showed that compound 1b has obvious inhibitory activities against human gastric carcinoma cell
line BGC-823, human nasopharyngeal carcinoma cell line KB and human hepatoma cell line BEL-7402
Received 14 September 2008
Revised 18 February 2009
Accepted 20 February 2009
Available online 25 February 2009
with IC50 values of 5.6 lg/mL, 13.1 lg/mL and 12.5 lg/mL, respectively. Some preliminary structure–
activity relationships (SAR) were also proposed which may provide a direction for further study.
Keywords:
Synthesis
Biological activity
E-2-Cyano-(3-substituted phenyl)
acrylamides
Ó 2009 Elsevier Ltd. All rights reserved.
Cyanoacetamides
To find the synthetic or naturally occurring small molecules
which have significant biological activity or therapeutic use still
deserves current interests. In the last decade, much attention has
been focused on the caffeic acid and its derivatives. Lots of re-
searches have shown that some caffeic acid derivatives possess a
wide range of biological activities such as anti-cancer activity,1–5
anti-bacterial activity,6 anti-oxidation,7,8 and anti-viral activity.9
Typically, the caffeic acid phenylethanolester (CAPE), firstly iso-
lated from beeswax in 1988, was proved to have remarkable
Initially, we try to prepare the title compounds by two steps:
firstly by Knoevenagel condensation of substituted benzaldehydes
with cyanoacetic esters to give 2-cyano-(3-substituted phenyl) ac-
rylic esters, then followed by condensation with various aryl or ali-
cyclic amines to form amides. In the second step, however, the
condensation proceeded in a poor yield for the lack of enough
activity of the ester group caused by its delocalization with vicinal
pi-bond. Then we try to synthesize the 2-cyano-(3-substituted
phenyl)acrylamides by Knoevenagel condensations of substituted
benzaldehydes with cyanoacetamides.
anti-cancer activities.5
A derivative of caffeic acid amide,
Entacapone, chemically named E-2-cyano-N,N-diethyl-3-(3,4-
dihydroxy-5-notrophenyl)acrylamide, was a selective catechol O-
methyltransferase (COMT) inhibitory agent and has been clinically
used as an anti-parkinsonism drug for near ten years.10–12 Besides,
there are researches revealed that some derivatives of Entacapone
possess obvious anti-cancer activity.13,14 Although there have been
lots of reports involving the biological activities of CAPE and its
analogues, study on the relationship between the structure and
its biological activity for this kind of compounds is still in demand.
Due to our interest in this, we have synthesized ten caffeic acid
amides derivatives 2-cyano-(3-substituted phenyl)acrylamides
and further investigated their biological activities. We report here
the synthesis, X-ray structure and inhibitory activities against
BGC-823, KB, BEL-7402 and acetylcholinesterase of these compounds.
Most of N-substituted cyanoacetamides are not available and
need to be prepared. Although there have been several literature
methods available for its preparation, there are some disadvantages
in these methods such as needing high temperature and high pres-
sure,15 needing microwave,16 using poisonous reagent17 or using
lithium amide at very low temperature,18 which are inconvenient
in the laboratory preparation. Here we developed a convenient
method for the preparation of cyanoacetamides, that is, by condens-
ing cyanoacetic acid with amines under the presence of DCC as
coupling agent. By means of this method, the intermediate cyanoac-
etamides (3k–3q) were synthesized in a moderate yield except the
cyanoacetamide (3r), which prepared by reacting ethyl cyanoace-
tate with aqueous ammonia according to the standard procedure.19
The starting materials nitro-substituted benzaldehydes were pre-
pared from vanillin according to the literature methods.20
As a result, the synthesis of 2-cyano-(3-substituted phenyl)-
acrylamides were achieved in moderate to high yield via
* Corresponding author. Tel.: +86 571 88320557; fax: +86 57185133199.
E-mail address: huyang@mail.hz.zj.cn (W. Hu).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.02.081

1862
W. Zhou et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1861–1865
R4
O
R4
O
O
O
DCC
R5 CH2Cl2
+
OH HN
NC CH2
CHO
NC CH2
N
H2O
NC CH2
OEt +
O
NC CH2
3r
NH2
NH3
R4
R5
;
4k-q
3k-q
CH
N
R4
O
R5
,
N
H
CH3COOH
CN
R3
N
+
R3
R5
reflux, toluene
CN
R2
R1
R1
R2
1a-j
3k-r
2
Scheme 1. Synthetic route of 2-cyano-(3-substituted phenyl)acrylamides.
Knoevenagel condensations of substituted benzaldehydes with cya-
noacetamides in the presence of piperidine as catalyst (Scheme 1).25
The whole synthetic route was shown in Scheme 1. The synthe-
sis of all title compounds were summarized in Table 1. The synthe-
sis of cyanoacetamides and their characterizations were
summarized in Table 2.
ally confirmed by elemental analysis. For these compounds, it
seemed that they can exist in either configuration of cis(Z) or
trans(E) for a C–C double bond involving in the molecular struc-
ture. Unfortunately, it is difficult to determine their geometries
based on the direct spectroscopic evidences since the a-cyanocaf-
feamido products contain single vinylic hydrogen that does not al-
low facile NMR assignment based on vicinal proton–proton
coupling. According to the observation of a single vinylic signal
The structures of all title compounds were characterized by IR,
1H NMR and EI-MS, besides, four new compounds were addition-
Table 1
Synthesis and 1H NMR data of 2-yano-(3-substituted phenyl)acrylamides
Entry
2
ÀNR4R5
Yield Mp, °C [lit]
(%)
1H NMR,
r (DMSO-d6, 400 MHz)
CHO
Ph
N
1a
88
89
265–267
217–219
7.29–7.33 (m, 5H), 7.41–7.45 (m, 5H), 7.61 (d, J = 2.0 Hz,1H), 7.83 (d, J = 2.0 Hz, 1H),
7.95 (s, 1H, C@CH)
Ph
HO
HO
NO2
NO2
OH
CHO
H
N
1b*
2.31 (s, 3H), 6.96 (d, 1H), 7.25 (t, 1H), 7.46 (d, 1H), 7.49 (s, 1H), 7.84 (d, J = 2.0 Hz, 1H), 7.99
(d, J = 2.0 Hz, 1H), 8.16 (s, 1H, C@CH), 10.25 (s, 1H, –NH)
CH3
OH
CHO
H
N
1c
95
98
40
42
82
211–214
3.75 (s, 3H), 6.94 (d, 2H), 7.56 (d, 2H), 7.83 (d, J = 1.6 Hz, 1H), 7.99 (d, J = 1.6 Hz, 1H), 8.14 (s,
1H, C@CH), 10.20 (s, 1H, –NH)
OCH3
[210–214]21
HO
HO
HO
NO2
NO2
NO2
OH
CHO
H
N
1d
1e*
1f*
1g
244–247
7.20 (d, 1H), 7.40 (t, 1H), 7.60 (d, 1H), 7.82 (s, 1H), 7.84 (d, J = 2.0 Hz, 1H), 8.00 (d, J = 2.0 Hz,
1H), 8.18 (s, 1H, C@CH), 10.48 (s, 1H, –NH)
[242–248]13
Cl
OH
CHO
H
N
225–226
217–218
7.29 (t, 1H), 7.38 (t, 1H), 7.55 (d, 1H), 7.63 (d, 1H), 7.83 (d, J = 2.0 Hz, 1H), 8.00 (d, J = 2.0 Hz,
1H), 8.21 (s, 1H, C@CH), 9.95 (s, 1H, –NH)
Cl
OH
CHO
H
N
O2N
H3CO
3.90 (s, 3H), 7.21 (d, 1H), 7.29 (d, 1H), 7.40 (t, 1H), 7.57 (d, 1H), 7.79 (s, 1H), 7.88 (s, 1H,
C@CH), 7.90 (d, 1H), 10.52 (s, 1H, –NH)
Cl
Cl
OH
CHO
H
N
231–234
3.95 (s, 3H), 7.20 (d, 1H), 7.41 (t, 1H), 7.60 (d, 1H), 7.83 (s, 1H), 7.94 (d, J = 1.6 Hz, 1H), 8.18
(d, J = 1.6 Hz, 1H), 8.27 (s, 1H, C@CH), 10.52 (s, 1H, –NH)
[229–231]13
H3CO
NO2
OH

W. Zhou et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1861–1865
1863
Table 1 (continued)
Entry
2
ÀNR4R5
Yield Mp, °C [lit]
(%)
1H NMR,
r (DMSO-d6, 400 MHz)
CHO
1h
67
78
53
207–208
1.55–1.63 (m, 6H), 3.51 (t, 4H), 7.63 (s, 1H, C@CH), 7.75 (d, J = 2.4 Hz, 1H), 7.90 (d, J = 2.4 Hz,
1H)
N
[206–207]21
HO
NO2
NO2
NO2
OH
CHO
1i
186–190
3.59 (m, 4H), 3.62 (m, 4H), 7.68 (s, 1H, C@CH), 7.77 (d, J = 2.0 Hz, 1H), 7.93 (d, J = 2.0 Hz, 1H)
N
O
[190–192]21
HO
HO
OH
CHO
1j
–NH2
300–303
7.73 (s, 1H, –NH), 7.77 (d, J = 2.0 Hz, 1H), 7.85 (s, 1H, –NH), 7.93 (d, J = 2.0 Hz, 1H), 8.05 (s,
1H, C@CH)
[296–298]22
OH
*
New compound.
in the 1H NMR spectrum (see Table 1), we believe that only one
geometric isomer exists in each compound. Furthermore, by means
of the X-ray diffractive analysis of single-crystal, the structure of
compound 1f was undoubtedly confirmed as trans-configuration
(Fig. 1).23 Based on the above facts, accordingly, these title com-
pounds prepared in this paper were assumed to be in trans-geom-
etry. The selectivity in configuration may derive from the repulsion
between the substituted phenyl group and amide group, which are
two bulky moieties bonding on C–C double bond.
The screening tests of antitumor activities in vitro for all title
compounds and cis-diamminedichloroplatinum (DDP) as a positive
control were carried out on human gastric carcinoma cell line BGC-
823, human nasopharyngeal carcinoma cell line KB and human
hepatoma cell line BEL-7402 with MTT assay. The IC50 values of
Table 2
Synthesis and characterizations of the intermediate cyanoacetamides
Entry –NR4R5
Reaction Yield (%) Mp (°C) [lit]
Solvent for recryst. IR
r
(cmÀ1
)
1H NMR, d CDCl3
time (h)
Ph
3k
6
4
52
61
154–156 [151–152]18 CH2Cl2
2275, 1676
3.43 (s, 2H), 7.25–7.45 (m, 10H)
N
Ph
H
N
3l
134–137
CH2Cl2
3273, 2256, 1668
2.35 (s, 3H), 3.54 (s, 2H),
7.02–7.33 (m, 4H)
CH3
H
3m
1.5
73
128–131 [131]24a
Petro. ether
3309, 2280, 1657
DMSO-d6, 3.72 (s, 3H),
3.84 (s, 2H), 6.8–7.4 (m, 4H)
N
OCH3
H
N
3n
4
4
73
69
135–137
THF
3273, 2256, 1668
3254, 2261, 1671
DMSO-d6, 3.92 (s, 2H), 7.15–7.74 (m, 4H)
Cl
H
N
3o
116–119 [118]24b
CH2Cl2
3.61 (s, 2H), 7.11–7.42 (m, 4H)
Cl
3p
7
5
2
60
73
70
87–89 [85–86]24c
86–88 [85–86]18
CH2Cl2
EtOH
2256, 1643
2280, 1660
1.57–1.65 (m, 6H), 3.38 (t, 2H),
3.47 (s, 2H), 3.55 (t, 2H)
N
3q
3.90–3.45 (m, 4H), 3.63 (t, 2H),
3.46 (t, 2H), 3.48(s, 2H)
N
O
3r
–NH2
120–121 [119–120]19 EtOH (95%)
3408, 3203, 2271, 1686 3.58 (s, 2H), 7.32 (br d, 1H),
7.64 (br d, 1H)

1864
W. Zhou et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1861–1865
Table 4
Inhibitory rates of all title compounds against acetylcholinesterase
Compound
Concentration (mol/L)
Inhibitory rate (%)
1a
1b
1c
1d
1e
1f
1g
1h
1i
2.5 Â 10À5
2.5 Â 10À5
2.5 Â 10À5
2.5 Â 10À5
2.5 Â 10À5
2.5 Â 10À5
2.5 Â 10À5
2.5 Â 10À5
2.5 Â 10À5
2.5 Â 10À5
7.38
2.73
4.64
4.37
0.95
2.19
1.64
0.27
3.00
1.09
1j
Figure 1. Molecular structure diagram of compound 1f with 30% probability
displacement ellipsoids.
Acknowledgements
Table 3
The authors acknowledge the Natural and Science Foundation
of Zhejiang Province for funding (Project M203027). We also
appreciate the National Center for Drug screening, Shanghai, China,
for the helps in the screening tests of anti-acetylcholinesterase
activity.
Values IC50 of all title compounds against BGC-823, KB and BEL-7402
Compound
BGC-823 (
l
g/mL)
KB (
lg/mL)
BEL-7402 (lg/mL)
1a
1b
1c
1d
1e
1f
1g
1h
1i
>100
5.6
56.2
13.1
>100
40.1
24
65.3
21.1
>100
>100
>100
0.6
104
12.5
29.2
72.7
37.4
>100
56.6
>100
>100
>100
1.77
45.4
>100
10.5
>100
26.1
>100
>100
>100
0.7
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.02.081.
1j
DDP
References and notes
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inhibitory cell growing for these compounds were summarized in
Table 3.
In Table 3, it is clear that those compounds with phenyl or
substituted phenyl in the amide moiety of the molecular struc-
ture(compound 1a, 1b, 1c, 1d, 1e, 1f and 1g) possess more strong
inhibitory activities than that with alicyclic substituents (com-
pound 1h and 1i) or hydrogen (compound 1j). This suggests that
an aryl substituent in the molecular amide moiety maybe more
preferred than an aliphatic or alicyclic substituent. Comparing
their structures and IC50 values of compound 1b–1e, we can con-
clude that the nature and position of substituent in the phenyl of
amide moiety exert an effect on the molecule’s inhibitory activi-
ties, and the m-methyl substituent in phenyl is more preferred
than p-methoxy and m or o-chloro substituent. Besides, compound
1g shows more strong inhibitory activities than compound 1d and
1f, which indicates that the substituents on the other phenyl bond-
ing to the C–C double bond also affect its activities.
In these compounds, compound 1b shows a better results than
others—its IC50 values against BGC-823, KB and BEL-7402 reach to
5.6 lg/mL, 13.1 lg/mL and 12.5 lg/mL, respectively. So, it would
be a valuable lead compound for further study, although its IC50
is larger approximately by an order of magnitude than that of
the general antitumor agent DDP.
Inspired by the fact that Entacapone has selective COMT inhib-
itory activity and has been clinically used as anti-parkinsonism
drug, we have carried out the screening test of inhibitory activities
against acetylcholinesterase for these compounds. Unfortunately,
all compounds showed a very limited activity—the inhibitory rate
being under 7.38% in the concentration of 2.5 Â 10À5 mol/L (Table
4). Maybe, this indicates that the aryl substituent in the amide
moiety of these compounds should be replaced by the aliphatic
substituent, such as the diethyl substituent in the Entacapone.
The further investigation on the SAR is ongoing.
5. Grunberger, D.; Banerjee, R.; Eisinger, K.; Oltz, E. M.; Efros, L.; Caldwell, M.;
Esteves, V.; Nakanishi, K. Experientia 1988, 44, 230.
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7. Son, S.; Lewis, B. A. J. Agric. Food. Chem. 2002, 50, 486.
8. Chen, J. H.; Ho, C. T. J. Agric. Food. Chem. 1997, 45, 2374.
9. Fesen, M. R.; Pommier, Y.; Leteurtre, F.; Hirogushi, S.; Yung, J.; Kohn, K. Biochem.
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Clin. Neurosci. 2007, 14, 424.
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Pharmacoeconomics 2002, 20, 617.
13. Yasunori, K.; Hisao, T.; Koichi, S.; Hiroto, H.; Hideo, N.; Toshiko, O. EP 0,537,742,
1993.
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I. D. J. Med. Chem. 1993, 36, 425.
15. Cossey, A. L.; Harris, R. L. N.; Huppatz, J. L.; Phillips, J. N. Aust. J. Chem. 1976, 29,
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16. Shao, J. G.; Wang, P. Y.; Zheng, M.; Zhong, Q. Chin. J. Yangzhou Univ. 1998, 1, 17.
17. Seeger, Q.; Juergen, H. DE 2,538,254, 1977.
18. Bhawal, B. M.; Khanapure, S. P.; Edward, B. R. Synth. Commun. 1990, 20, 3235.
19. Corson, B. B.; Scott, R. W.; Vose, C. E.. In Organic Syntheses Coll; Blatt, A. H., Ed.;
John Wiley and Sons: New York, 1963; Vol. IV, p 977.
20. Grenier, J. L.; Cotelle, N.; Catteau, J. P.; Cotelle, P. J. Phys. Org. Chem. 2000, 13,
511.
21. Zhao, R. Master.Thesis, China Pharmaceutical University, May 2003.
22. Johannes, B. R.; Evert, H. K.; Juhani, H. E.; Kalevi, K. S.; Juhani, K. P.; Yvonne, L. B;
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1988.
23. Crystals of compound 1f for structural analysis were obtained by slow evaporation
of acetonitrile. Crystal data: C17H12N3ClO5, M = 373.75, triclinic, a = 8.166(3),
b = 10.320(4), c = 10.696(4) Å,
a
= 90.040(5), b = 108.302(5),
c
= 102.979(5)°,
U = 808(5) Å3, T = 293(2) K, space group P1, Z = 2, Dc = 1.536 g/cm3,
l
(Mo-
ꢀ
Ka , 3460 reflections measured, 2860 unique (Rint = 0.065)
) = 0.273 mmÀ1
which were used in all calculations. Fine R1 = 0.091, wR(F2) = 0.256 (all data).
Full crystallographic details of 1f have been deposited at the Cambridge
Crystallographic Data Center and allocated the deposition number CCDC
634551.

W. Zhou et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1861–1865
1865
24. Sadtler Research Laboratories, SADTLER Standard Infrared Grating Spectra,
Philadelphia: Researchers, Editors and Publishers, 1976, (a) 50056K, (b)
50057K, and (c) 45799P.
(10 mmol) N-(3-methylphenyl)-2-cyanoacetamide, 2.0 g (11 mmol) 3,4-
dihydroxy-5-nitrobenzaldehyde and 50 mL toluene were added 0.35 mL
piperidine and 1.3 mL acetic acid. Then the mixture was heated to reflux
until the TLC test shows the reaction is complete. Cooled the mixture to
room temperature, then the precipitate was filtered, washed and then
recrystallized with absolute alcohol to afford 3.0 g crystal compound 1b,
yield 89%, mp 217–219 °C. IR(KBr, cmÀ1): 3368, 3315, 2215, 1666, 1614,
1582, 1546, 1490, 1249, 738, 625. 1H NMR, d (DMSO-d6, 400 MHz): 2.31 (s,
3H), 6.96 (d, J = 7.6 Hz, 1H), 7.25 (t, J = 7.6 Hz, 1H), 7.46 (d, J = 7.6 Hz, 1H),
7.49 (s, 1H), 7.84 (d, J = 2.0 Hz, 1H), 7.99 (d, J = 2.0 Hz, 1H), 8.16 (s, 1H,
C@CH), 10.25 (s, 1H, –NH). EI-MS: 339 (M+), 321, 263, 233, 185, 159, 131,
107, 91, 77, 65, 53. Elemental analysis (calcd data in parentheses): C: 60.25
(60.18), H: 3.78 (3.86), N: 12.29 (12.38). For other experimental details, see
Supplementary data.
25. Representative synthetic procedure for compound 1b: (a) To
5.4 g(50 mmol) 3-methylaniline in 15 mL dichloromethane was added drop
wise solution of 4.2 g (50 mmol) cyanoacetic acid in 30 mL
dichloromethane at room temperature. After the addition, solution of
a mixture of
a
a
10.5 g (50 mmol) DCC in 20 mL dichloromethane was added to the mixture.
Then the mixture was heated to reflux until the TLC test shows the reaction
come to the end. Cool the mixture to room temperature, then the resulted
precipitate was filtered and washed with dichloromethane. The filtrate was
concentrated under vacuum to give crude product, which was recrystallized
with dichloromethane to afford 5.3 g crystal product N-(3-methylphenyl)-2-
cyanoacetamide, yield 61%, mp 134–139 °C. (b) To
a mixture of 1.8 g