Bioorganic & Medicinal Chemistry Letters 19 (2009) 1861–1865

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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
Received 14 September 2008 via Knoevenogal condensation of substituted benzaldehydes with cyanoacetamides. The structure of
Revised 18 February 2009 compound 1f was determined as E-isomer by X-ray diffractive analysis. The biological screening tests
Accepted 20 February 2009
in vitro showed that compound 1b has obvious inhibitory activities against human gastric carcinoma cell
Available online 25 February 2009
line BGC-823, human nasopharyngeal carcinoma cell line KB and human hepatoma cell line BEL-7402
with IC50 values of 5.6 lg/mL, 13.1 lg/mL and 12.5 lg/mL, respectively. Some preliminary structure–
Keywords:
activity relationships (SAR) were also proposed which may provide a direction for further study.
Synthesis
Biological activity
Ó 2009 Elsevier Ltd. All rights reserved.
E-2-Cyano-(3-substituted phenyl)
acrylamides
Cyanoacetamides

To find the synthetic or naturally occurring small molecules Initially, we try to prepare the title compounds by two steps:
which have significant biological activity or therapeutic use still firstly by Knoevenagel condensation of substituted benzaldehydes
deserves current interests. In the last decade, much attention has with cyanoacetic esters to give 2-cyano-(3-substituted phenyl) ac-
been focused on the caffeic acid and its derivatives. Lots of re- rylic esters, then followed by condensation with various aryl or ali-
searches have shown that some caffeic acid derivatives possess a cyclic amines to form amides. In the second step, however, the
wide range of biological activities such as anti-cancer activity,1–5 condensation proceeded in a poor yield for the lack of enough
anti-bacterial activity,6 anti-oxidation,7,8 and anti-viral activity.9 activity of the ester group caused by its delocalization with vicinal
Typically, the caffeic acid phenylethanolester (CAPE), firstly iso- pi-bond. Then we try to synthesize the 2-cyano-(3-substituted
lated from beeswax in 1988, was proved to have remarkable phenyl)acrylamides by Knoevenagel condensations of substituted
anti-cancer activities.5 A derivative of caffeic acid amide, benzaldehydes with cyanoacetamides.
Entacapone, chemically named E-2-cyano-N,N-diethyl-3-(3,4- Most of N-substituted cyanoacetamides are not available and
dihydroxy-5-notrophenyl)acrylamide, was a selective catechol O- need to be prepared. Although there have been several literature
methyltransferase (COMT) inhibitory agent and has been clinically methods available for its preparation, there are some disadvantages
used as an anti-parkinsonism drug for near ten years.10–12 Besides, in these methods such as needing high temperature and high pres-
there are researches revealed that some derivatives of Entacapone sure,15 needing microwave,16 using poisonous reagent17 or using
possess obvious anti-cancer activity.13,14 Although there have been lithium amide at very low temperature,18 which are inconvenient
lots of reports involving the biological activities of CAPE and its in the laboratory preparation. Here we developed a convenient
analogues, study on the relationship between the structure and method for the preparation of cyanoacetamides, that is, by condens-
its biological activity for this kind of compounds is still in demand. ing cyanoacetic acid with amines under the presence of DCC as
Due to our interest in this, we have synthesized ten caffeic acid coupling agent. By means of this method, the intermediate cyanoac-
amides derivatives 2-cyano-(3-substituted phenyl)acrylamides etamides (3k–3q) were synthesized in a moderate yield except the
and further investigated their biological activities. We report here cyanoacetamide (3r), which prepared by reacting ethyl cyanoace-
the synthesis, X-ray structure and inhibitory activities against tate with aqueous ammonia according to the standard procedure.19
BGC-823, KB, BEL-7402 and acetylcholinesterase of these compounds. The starting materials nitro-substituted benzaldehydes were pre-
pared from vanillin according to the literature methods.20
* Corresponding author. Tel.: +86 571 88320557; fax: +86 57185133199. As a result, the synthesis of 2-cyano-(3-substituted phenyl)-
E-mail address: huyang@mail.hz.zj.cn (W. Hu). acrylamides were achieved in moderate to high yield via

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

O R4 O R4 O O
DCC
NC CH2 OH + HN NC CH2 N NC CH2 OEt + NH3 H2O NC CH2 NH2
R5 CH2Cl2 R5 ;
4k-q 3r
3k-q
O R4
CHO
O R4 CH N
N N , CH3COOH CN R5
H
+
R3 R5 R3
CN reflux, toluene
R1 R2
R1 R2
2 3k-r 1a-j

Scheme 1. Synthetic route of 2-cyano-(3-substituted phenyl)acrylamides.

Knoevenagel condensations of substituted benzaldehydes with cya- ally confirmed by elemental analysis. For these compounds, it
noacetamides in the presence of piperidine as catalyst (Scheme 1).25 seemed that they can exist in either configuration of cis(Z) or
The whole synthetic route was shown in Scheme 1. The synthe- trans(E) for a C–C double bond involving in the molecular struc-
sis of all title compounds were summarized in Table 1. The synthe- ture. Unfortunately, it is difficult to determine their geometries
sis of cyanoacetamides and their characterizations were based on the direct spectroscopic evidences since the a-cyanocaf-
summarized in Table 2. feamido products contain single vinylic hydrogen that does not al-
The structures of all title compounds were characterized by IR, low facile NMR assignment based on vicinal proton–proton
1
H NMR and EI-MS, besides, four new compounds were addition- coupling. According to the observation of a single vinylic signal

Table 1
Synthesis and 1H NMR data of 2-yano-(3-substituted phenyl)acrylamides

Entry 2 NR4R5 Yield Mp, °C [lit] 1

H NMR, r (DMSO-d6, 400 MHz)
(%)

CHO
Ph
1a N 88 265–267 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),
HO NO2 Ph 7.95 (s, 1H, C@CH)
OH
CHO
H
N
1b* 89 217–219 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
HO NO2 CH3 (d, J = 2.0 Hz, 1H), 8.16 (s, 1H, C@CH), 10.25 (s, 1H, –NH)
OH

CHO

H
1c N OCH3 95 211–214 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,
HO NO2 [210–214]21 1H, C@CH), 10.20 (s, 1H, –NH)
OH

CHO
H
N
1d 98 244–247 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,
HO NO2 [242–248]13 1H), 8.18 (s, 1H, C@CH), 10.48 (s, 1H, –NH)
Cl
OH

CHO
H
N
1e* 40 225–226 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,
HO NO2 1H), 8.21 (s, 1H, C@CH), 9.95 (s, 1H, –NH)
Cl
OH

CHO
O2N H
N
1f* 42 217–218 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,
H3CO C@CH), 7.90 (d, 1H), 10.52 (s, 1H, –NH)
Cl
OH

CHO
H
N
1g 82 231–234 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
H3CO NO2 [229–231]13 (d, J = 1.6 Hz, 1H), 8.27 (s, 1H, C@CH), 10.52 (s, 1H, –NH)
Cl
OH
 W. Zhou et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1861–1865 1863

Table 1 (continued)
Entry 2 NR4R5 Yield Mp, °C [lit] 1
H NMR, r (DMSO-d6, 400 MHz)
(%)

CHO

1h N 67 207–208 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,
HO NO2 [206–207]21 1H)
OH

CHO

1i N O 78 186–190 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)
HO NO2 [190–192]21
OH

CHO

1j –NH2 53 300–303 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,
HO NO2 [296–298]22 1H, C@CH)
OH
*
New compound.

in the 1H NMR spectrum (see Table 1), we believe that only one between the substituted phenyl group and amide group, which are
geometric isomer exists in each compound. Furthermore, by means two bulky moieties bonding on C–C double bond.
of the X-ray diffractive analysis of single-crystal, the structure of The screening tests of antitumor activities in vitro for all title
compound 1f was undoubtedly confirmed as trans-configuration compounds and cis-diamminedichloroplatinum (DDP) as a positive
(Fig. 1).23 Based on the above facts, accordingly, these title com- control were carried out on human gastric carcinoma cell line BGC-
pounds prepared in this paper were assumed to be in trans-geom- 823, human nasopharyngeal carcinoma cell line KB and human
etry. The selectivity in configuration may derive from the repulsion 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 (cm 1
) 1
H NMR, d CDCl3
time (h)

Ph
3k N 6 52 154–156 [151–152]18 CH2Cl2 2275, 1676 3.43 (s, 2H), 7.25–7.45 (m, 10H)
Ph

H
N
3l 4 61 134–137 CH2Cl2 3273, 2256, 1668 2.35 (s, 3H), 3.54 (s, 2H),
CH3 7.02–7.33 (m, 4H)

H
3m N OCH3 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)

H
N
3n 4 73 135–137 THF 3273, 2256, 1668 DMSO-d6, 3.92 (s, 2H), 7.15–7.74 (m, 4H)
Cl

H
N
3o 4 69 116–119 [118]24b CH2Cl2 3254, 2261, 1671 3.61 (s, 2H), 7.11–7.42 (m, 4H)
Cl

3p N 7 60 87–89 [85–86]24c CH2Cl2 2256, 1643 1.57–1.65 (m, 6H), 3.38 (t, 2H),
3.47 (s, 2H), 3.55 (t, 2H)

3q N O 5 73 86–88 [85–86]18 EtOH 2280, 1660 3.90–3.45 (m, 4H), 3.63 (t, 2H),
3.46 (t, 2H), 3.48(s, 2H)

3r –NH2 2 70 120–121 [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 (%)

5
1a 2.5  10 7.38
5
1b 2.5  10 2.73
5
1c 2.5  10 4.64
5
1d 2.5  10 4.37
5
1e 2.5  10 0.95
5
1f 2.5  10 2.19
5
1g 2.5  10 1.64
5
1h 2.5  10 0.27
5
1i 2.5  10 3.00
5
1j 2.5  10 1.09

Figure 1. Molecular structure diagram of compound 1f with 30% probability

displacement ellipsoids.

Acknowledgements

Table 3 The authors acknowledge the Natural and Science Foundation

Values IC50 of all title compounds against BGC-823, KB and BEL-7402 of Zhejiang Province for funding (Project M203027). We also
Compound BGC-823 (lg/mL) KB (lg/mL) BEL-7402 (lg/mL) appreciate the National Center for Drug screening, Shanghai, China,
1a >100 56.2 104 for the helps in the screening tests of anti-acetylcholinesterase
1b 5.6 13.1 12.5 activity.
1c 45.4 >100 29.2
1d >100 40.1 72.7
1e 10.5 24 37.4 Supplementary data
1f >100 65.3 >100
1g 26.1 21.1 56.6
1h >100 >100 >100
Supplementary data associated with this article can be found, in
1i >100 >100 >100 the online version, at doi:10.1016/j.bmcl.2009.02.081.
1j >100 >100 >100
DDP 0.7 0.6 1.77
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ture(compound 1a, 1b, 1c, 1d, 1e, 1f and 1g) possess more strong Esteves, V.; Nakanishi, K. Experientia 1988, 44, 230.
inhibitory activities than that with alicyclic substituents (com- 6. Grange, J. M.; Davey, R. W. J. R. Soc. Med. 1990, 83, 159.
pound 1h and 1i) or hydrogen (compound 1j). This suggests that 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.
an aryl substituent in the molecular amide moiety maybe more 9. Fesen, M. R.; Pommier, Y.; Leteurtre, F.; Hirogushi, S.; Yung, J.; Kohn, K. Biochem.
preferred than an aliphatic or alicyclic substituent. Comparing Pharmacol. 1994, 48, 595.
their structures and IC50 values of compound 1b–1e, we can con- 10. Gordin, A.; Kaakkola, S.; Teravainen, H. J. Neural Trans. 2004, 111, 1343.
11. Muller, T.; Erdmann, C.; Muhlack, S.; Bremen, D.; Przuntek, H.; Woitalla, D. J.
clude that the nature and position of substituent in the phenyl of Clin. Neurosci. 2007, 14, 424.
amide moiety exert an effect on the molecule’s inhibitory activi- 12. Palmer, C. S.; Nuijten, M. J. C.; Schmier, J. K.; Subedi, P.; Snyder, E. H.
ties, and the m-methyl substituent in phenyl is more preferred Pharmacoeconomics 2002, 20, 617.
13. Yasunori, K.; Hisao, T.; Koichi, S.; Hiroto, H.; Hideo, N.; Toshiko, O. EP 0,537,742,
than p-methoxy and m or o-chloro substituent. Besides, compound 1993.
1g shows more strong inhibitory activities than compound 1d and 14. Bruke, T. R., Jr.; Lim, B.; Marquez, V. E.; Li, Z. H.; Bolen, J. B.; Stefanova, I.; Horak,
1f, which indicates that the substituents on the other phenyl bond- 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,
ing to the C–C double bond also affect its activities. 1039.
In these compounds, compound 1b shows a better results than 16. Shao, J. G.; Wang, P. Y.; Zheng, M.; Zhong, Q. Chin. J. Yangzhou Univ. 1998, 1, 17.
others—its IC50 values against BGC-823, KB and BEL-7402 reach to 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.
5.6 lg/mL, 13.1 lg/mL and 12.5 lg/mL, respectively. So, it would
19. Corson, B. B.; Scott, R. W.; Vose, C. E.. In Organic Syntheses Coll; Blatt, A. H., Ed.;
be a valuable lead compound for further study, although its IC50 John Wiley and Sons: New York, 1963; Vol. IV, p 977.
is larger approximately by an order of magnitude than that of 20. Grenier, J. L.; Cotelle, N.; Catteau, J. P.; Cotelle, P. J. Phys. Org. Chem. 2000, 13,
the general antitumor agent DDP. 511.
21. Zhao, R. Master.Thesis, China Pharmaceutical University, May 2003.
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itory activity and has been clinically used as anti-parkinsonism Topias, M. P.; Olavi, N. E. A.; Pentti, P.; Kyllikki, P. A.; Johan, P. J. DE 3,740,383,
drug, we have carried out the screening test of inhibitory activities 1988.
23. Crystals of compound 1f for structural analysis were obtained by slow evaporation
against acetylcholinesterase for these compounds. Unfortunately, of acetonitrile. Crystal data: C17H12N3ClO5, M = 373.75, triclinic, a = 8.166(3),
all compounds showed a very limited activity—the inhibitory rate 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 P 1,  Z = 2, Dc = 1.536 g/cm3, l(Mo-
being under 7.38% in the concentration of 2.5  10 5 mol/L (Table
Ka) = 0.273 mm 1, 3460 reflections measured, 2860 unique (Rint = 0.065)
4). Maybe, this indicates that the aryl substituent in the amide which were used in all calculations. Fine R1 = 0.091, wR(F2) = 0.256 (all data).
moiety of these compounds should be replaced by the aliphatic Full crystallographic details of 1f have been deposited at the Cambridge
substituent, such as the diethyl substituent in the Entacapone. Crystallographic Data Center and allocated the deposition number CCDC
634551.
The further investigation on the SAR is ongoing.
 W. Zhou et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1861–1865 1865

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