Synthesis and antitumor activity of feruloyl and caffeoyl derivatives This paper is dedicated to Prof. Wei-xiao Hu for his lifelong commitment to mentoring graduate students

Article abstract of DOI:10.1016/j.bmcl.2014.08.024

We developed two efficient protocols for the synthesis of feruloyl and caffeoyl derivatives from commercial vanillin and veratraldehyde. Pharmacological activities were assessed against a panel of human cancer cell lines in vitro. Most synthesized compounds demonstrated attractive cytotoxicity. Several new compounds demonstrated significant antiproliferative and cytotoxic activities against HeLa and Bewo tumor cell lines. In particular, 5-nitro caffeic adamantyl ester showed broad spectrum of tumor inhibition in 10 cell lines, and reduced tumor weight by 36.7% in vivo when administered at a dose of 40 mg kg^-1.

Full text of DOI:10.1016/j.bmcl.2014.08.024

Bioorganic & Medicinal Chemistry Letters xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and antitumor activity of feruloyl and caffeoyl derivatives
Hui-zhen Chen a, You-bao Chen b, Ya-ping Lv b, Fang Zeng b, Juan Zhang b, Yong-lie Zhou c, Han-bing Li b,
Li-fei Chen d, Bin-jie Zhou e, Jian-rong Gao b, Chun-nian Xia b,
⇑
a Sir Run Run Shaw Hospital, Zhejiang University, Hangzhou 310016, People’s Republic of China
b College of Pharmaceutical Science, Zhejiang University of Technology, Hangzhou 310014, People’s Republic of China
c Zhejiang Province People’s Hospital, Hangzhou 310014, People’s Republic of China
d Zhejiang Hisun Pharmaceutical Co. Ltd., 46 Waisha Road, Jiaojiang District, Taizhou City, Zhejiang Province 318000, People’s Republic of China
e Ningbo Green-Health Pharma-ceutical Co., Ltd, Fenghua Xiwu Town Foreign Technological Garden, Ningbo 315505, 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:
We developed two efficient protocols for the synthesis of feruloyl and caffeoyl derivatives from commer-
cial vanillin and veratraldehyde. Pharmacological activities were assessed against a panel of human
cancer cell lines in vitro. Most synthesized compounds demonstrated attractive cytotoxicity. Several
new compounds demonstrated significant antiproliferative and cytotoxic activities against HeLa and
Bewo tumor cell lines. In particular, 5-nitro caffeic adamantyl ester showed broad spectrum of tumor
inhibition in 10 cell lines, and reduced tumor weight by 36.7% in vivo when administered at a dose of
Received 5 May 2014
Revised 6 August 2014
Accepted 8 August 2014
Available online xxxx
This paper is dedicated to Prof. Wei-xiao Hu
for his lifelong commitment to mentoring
graduate students
40 mg kgÀ1
.
Ó 2014 Elsevier Ltd. All rights reserved.
Keywords:
Feruloyl
Caffeoyl
Antitumor
SARs
Naturally occurring feruloyl and caffeoyl derivatives are widely
distributed throughout the plant kingdom.1 Epidemiological
studies demonstrate that a diet rich in fruit and vegetables, which
contain abundant caffeoyl derivatives, can reduce cancer risk in
humans. This suggests that certain dietary constituents may be
effective in preventing cancer.2 There is a large body of evidence
demonstrating that feruloyl or caffeoyl derivatives, such as
curcumin, caffeic acid, caffeic acid phenethyl ester, chlorogenic
observations have led us to focus on caffeic analogues as a poten-
tial scaffold endowed with inhibitory effects on cancer.
Feruloyl and caffeoyl derivatives were prepared using two
convenient methods as shown in Scheme 1. Ferulates and caffeates
were prepared by Knoevenagel condensation of the substituted
benzaldehydes14–17 with mono-malonic esters in a one-pot
manner.18 Acetyl cinnamic acids were prepared by Perkin reaction,
followed by the mixed carbonic anhydride method19 to obtain feru-
loyl amides, and demethylated to obtain caffeoyl amides. Chloro-
genic acid was separated and purified from honeysuckle using the
HSCCC method (High Speed Counter-Current Chromatography).12
The ester condensation protocol can be very useful and applied
to a wide range of cinnamic esters with a structure that is difficult
to synthesize via other documented methods such as: acid-
catalyzed esterification, alkylation of caffeic acid with halohydro-
carbons, esterification via acyl chlorides, coupling reaction with
DCC as coupling agent, transesterification and Wittig reaction.13
We have ever attempted to extended this protocol to prepare feru-
loyl and caffeoyl amides, but it was too difficult to separate pure
amides by column chromatography, for the concentrated reaction
solutions were very dark and gummy. Furthermore, yields of the
desired amides were barely detectable due to excessive amounts
of undetermined by-products. Fortunately, when we attempted
acid, L-cichoric acid, and rosmarinic acid possess a wide range of
biological activities including anti-cancer,3–7 anti-bacterial,8 anti-
oxidative,9,10 and antiviral activities.11 Although there is substan-
tial evidence demonstrating the biological activities of feruloyl
and caffeoyl compounds, little is known about the relationship
between the structure and biological activity of these compounds.
In our previous work, SAR studies showed that four compounds
inhibited against HIV-1integrase and some caffeates exhibited
cytotoxicity with micromolar range. Moreover, compounds with
nitro-substitutions, multiple cycloalkyl groups, or a combination
of the two factors, showed superior inhibitory effects. These
⇑
Corresponding author. Tel./fax: +86 571 88320320.
E-mail address: xcn77@zjut.edu.cn (C.-N. Xia).
http://dx.doi.org/10.1016/j.bmcl.2014.08.024
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Chen, H.-z.; et al. Bioorg. Med. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.bmcl.2014.08.024

2
H.-z. Chen et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
Scheme 1. Synthesis of feruloyl and caffeoyl derivatives.
synthesis of 4-acetylvanillin, it was found that 4-acetylvanillin can
easily form 4-acetyl feruloyl acid when catalyzed by sodium ace-
tate at reflux temperature, followed by the mixed carbonic anhy-
dride method to obtain feruloyl amides, and which can be
demethylated to form caffeoyl amides. All synthesized esters and
amides are summarized in Table 1, which shows the trans (E) con-
figuration confirmed by the 1H NMR spectra, a subset of the com-
pounds were characterized by X-ray analysis.20
The antitumor activities of esters and amides against six human
tumor cell lines including HeLa, Siha, Bewo, HL-60, SGC-7901 and
HepG-2, were evaluated by MTT assay in vitro. Cisplatin and Doxo-
rubicin were used as positive control in the MTT assay. The struc-
tures and anti-tumor activities are summarized in Table 1.
As shown in Table 1, most of feruloyl and caffeoyl compounds
possessed potent anti-cancer activities. I-24 and I-30 showed good
anti-Hela tumor potency. I-30 showed potent Siha inhibition. In
particular, nearly all compounds had significant cytotoxic effects
against Bewo and HL-60 with small IC50 values. For the Bewo cell
line, of the forty compounds screened, eighteen compounds
showed good to excellent inhibitor activities, and IC50 values of I-
and I-30 showed good SCG-7901 inhibitory activity. Compounds
I-15, I-21 and I-30 showed potent HepG-2 inhibition. This observa-
tion suggests that the good to excellent inhibitory activities of the
aforementioned compounds do not simply correlate with the size
of the ester moiety or substitutions with electron-withdrawing
group. The results of Bewo were not repeated with the other five
tumor cell lines, so we can conclude that feruloyl and caffeoyl
compounds possess higher selectivity for the Bewo cell line when
compared to the other five cell lines.
When different electron-withdrawing groups such as NO2, Cl
and Br were present, the compounds showed more potent activity;
it is obvious that a strong electron withdrawing group such as NO2
contributes to increased activity, as can be seen with compounds I-
18, I-20, I-21, I-25, I-26, I-27, I-28, I-29 and I-30. These evidences
would confirm that analogues with resonance election-drawing
group decrease election density to stabilize the corresponding
conformers responsible for the higher activity as well.18 However,
the activities decreased with caffeic adamantyl amides II-1, II-2
and II-3 when Cl or Br groups were introduced. The importance
of the catechol entity was obviously greater for the activities of
caffeoyl amides than for feruloyl amides. In addition, solubility
problems were present with compounds containing adamantyl
derivatives when used in the MTT assay, particularly in the case
of feruloyl adamantyl amides.
14 and I-24 were less than 1
2–56 times more effective than the two positive controls (Cisplatin
IC50 1.07 M, Doxorubicin IC50 20.37 M). Also there are 16 com-
pounds that showed good inhibition on HL-60 cell line. I-20, I-21
lM. Both compounds were at least
l
l
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H.-z. Chen et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
3
Table 1
Inhibitions of caffeoyl derivatives on tumor cells
Entry
Compound
Tumor cells (IC50: lM)
X
Y
R1
ROH/RNH2
HeLa
Siha
Bewo
HL-60
SGC-7901
HepG-2
OH
OH
OH
I-1
I-2
I-3
H
H
H
H
H
H
H
H
H
52.53
23.27
28.61
60.26
34.09
44.19
17.27
6.20
12.32
8.94
>100
>100
>100
81.55
63.95
>100
5.18
11.80
I-4
H
H
H
30.14
48.16
5.90
11.76
97.71
91.63
OH
OH
OH
I-5
I-6
I-7
H
H
H
H
H
H
H
H
H
67.22
19.17
40.80
30.72
39.08
40.97
8.64
8.99
5.42
11.50
12.34
7.54
89.17
>100
>100
>100
>100
>100
OH
I-8
I-9
H
H
H
H
H
H
13.77
16.69
33.77
41.99
3.18
3.47
12.46
7.92
>100
95.80
69.70
OH
80.04
OH
OH
I-10
I-11
H
H
H
H
H
H
23.60
23.86
30.47
51.53
7.26
2.75
9.32
8.32
>100
38.05
45.54
NO2
OH
95.54
OH
I-12
I-13
H
H
H
H
H
H
21.51
34.94
35.75
45.93
2.77
4.46
8.29
33.99
55.96
44.71
54.29
OH
13.12
I-14
I-15
H
H
H
H
H
H
38.22
>100
49.94
>100
0.41
5.10
17.43
17.18
24.08
4.95
OH
OH
>100
10.23
OH
OH
I-16
I-17
H
H
H
H
H
H
29.84
23.48
52.26
74.27
30.70
28.22
10.78
8.00
90.27
70.45
50.82
85.64
OH
I-18
I-19
I-20
I-21
I-22
I-23
I-24
H
H
H
H
H
H
H
H
H
37.98
10.91
ND
66.92
17.17
ND
2.96
4.38
ND
16.65
18.23
7.01
8.49
ND
14.26
ND
40.87
ND
OH
OH
OH
OH
NO2
H
H
Cl
Br
NO2
H
8.18
6.75
ND
ND
H
17.52
ND
28.49
ND
8.24
ND
4.44
ND
H
OH
H
31.57
8.04
88.71
39.51
5.80
0.36
11.25
3.40
40.09
44.80
48.43
14.77
OH
H
H
I-25
I-26
H
H
H
H
H
20.60
13.91
73.74
51.77
35.86
9.31
3.35
12.03
ND
31.37
ND
OH
NO2
52.88
OH
I-27
I-28
H
H
Cl
H
H
38.71
22.69
33.77
29.02
51.94
20.01
18.60
15.15
69.48
ND
ND
ND
OH
Br
OH
OH
OH
OH
I-29
I-30
I-31
H
H
H
H
H
>100
1.28
>100
6.74
>100
>100
5.63
8.15
4.07
29.68
4.60
ND
29.97
3.84
ND
NO2
H
H
CH3
59.94
70.01
92.35
(continued on next page)
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H.-z. Chen et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
Table 1 (continued)
Entry
Compound
ROH/RNH2
HO
Tumor cells (IC50: lM)
X
Y
R1
H
HeLa
ND
Siha
ND
Bewo
HL-60
SGC-7901
>100
HepG-2
>100
OH
OH
I-32
H
H
ND
>100
HO
OH
O
II-1
II-2
II-3
II-4
II-5
II-6
II-7
H
H
H
10.16
50.03
33.23
>100
66.77
91.26
98.42
>100
41.28
18.93
>100
1.09
8.18
29.78
ND
ND
ND
ND
ND
ND
ND
30.06
ND
ND
ND
ND
ND
ND
ND
NH2
H
Cl
Br
H
H
68.36
60.85
>100
68.19
26.69
44.49
41.41
35.65
>100
>100
18.74
73.19
67.87
49.82
NH2
NH2
NH2
NH2
NH2
NH2
NH2
H
H
H
CH3
CH3
CH3
CH3
CH3
67.61
59.54
38.90
34.82
82.37
H
NO2
H
NO2
H
Cl
Br
II-8
H
Control
Doxorubicin
Cisplatin
0.33
0.97
2.04
13.97
1.07
20.37
0.42
10.40
ND
ND
ND
ND
ND—Not detective.
Table 2
Inhibition of I-30 on four additional tumor cell lines (IC50: lM)
O
HO
O
Cell lines
BEL-7404
A549
BCG832
MCF-7
HL-60
HO
NO2
IC50
Doxorubicin
Cisplatin18
21.43
ND
7.1
4.41
ND
9.7
8.22
ND
1.9
4.09
ND
8.8
0.52
0.23
3.12
Control
Figure 1. The influence of I-30 on cell morphology of HL-60 (Â400). (a) Control cells, (b) 2.5
lg/mL, (c) 5 lg/mL, and (d) 10 lg/mL.
As shown in Table 1, the compound I-30, 5-nitro caffeic ada-
mantyl ester, had a broad-range of biological activities. I-30
showed higher activities against all the tested cancer cell lines than
most compounds. To verify the proposed broad-range activity of
I-30, we tested four additional tumor cell lines for cytotoxicity
alongside a repetition of HL-60. (Table 2)
Based on the in vitro anti-tumor data, I-30 was chosen as a lead
compound to test the morphology (Fig. 1) and flow cytometer
analysis (Fig. 2) of the HL-60 cell line. The results showed that
I-30 significantly induced the level of apoptosis in HL-60 cells
in vitro at low concentrations (Fig. 2).
Based on our in vitro anti-tumor and acute toxicity data, we
further evaluated the in vivo anti-tumor activity of I-30 against
S-180 tumor-bearing mice with CTX as positive control. The
results showed that I-30 can inhibit S-180 tumor by 36.7%, but
its efficacy is lower than that of CTX, which inhibited tumors
by 54.8%, although it showed efficiency anti-tumor activity
in vitro. Given that the in vivo studies were performed on murine
tumor cells, which are different from the in vitro tested cells, our
results are still promising considering the possibility of selective
inhibition. Further work needs to be done with human tumor
cells.
I-30 significantly inhibited proliferation of HL-60 cells in
a
dose-dependent and time-dependent manner. Within certain dose
ranges, the inhibition was time-effect relationship. Preliminary
results from flow cytometric analyses revealed that compounds
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H.-z. Chen et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
5
Figure 2. DNA cell cycle analysis in HL-60 cells with increasing doses of I-30 (a) control cells, (b) 2
0.5 g/mL.
lg/mL, (c) 1
l
g/mL, (d) 2
l
g/mL (repeated), (e) 1
lg/mL (repeated), (f)
l
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In conclusion, a series of feruloyl and caffeoyl derivatives were
synthesized and tested for in vitro anti-tumor activity against
several cancer cell lines. The SAR study of these compounds led
to the identification of a new caffeate, I-30, as a highly potent anti-
cancer compound with broad range inhibitory activities. Further
chemo-biological study of I-30 with regards to its anti-tumor path-
way and its enzymatic targets are ongoing in our laboratory, along
with additional in vivo studies.
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Acknowledgments
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We would also like to acknowledge Dr. Sheng-qiang Tong for
assistance with HSCCC and Prof. Ru-song Zhang of Zhejiang
Chinese Medical University for assistance with evaluating the
acute toxicity and anti-tumor activities. This research was sup-
ported by the Science and Technology Development Fund (2013)
of Zhejiang University of Technology.
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Supplementary data
20. (a) Xia, C. N.; Hu, W. X.; Rao, G. W. Acta Crystallogr. 2004, E60, o913; (b) Xia, C.
N.; Hu, W. X.; Zhou, W. Acta Crystallogr. 2007, E63, o2482; (c) Xia, C. N.; Zhou,
W.; Hu, W. X. Acta Crystallogr. 2005, E61, o1783; (d) Xia, C. N.; Hu, W. X.; Zhou,
W. Acta Crystallogr. 2006, E62, o1112; (e) Xia, C. N.; Hu, W. X.; Zhou, W. Acta
Crystallogr. 2006, E62, o4118; (f) Xia, C. N.; Hu, W. X.; Zhou, W. Acta Crystallogr.
2005, E61, o3757; (g) Xia, C. N.; Hu, W. X.; Zhou, W. J. Chem. Crystallogr. 2008,
38, 583; (h) Xia, C. N.; Hu, W. X.; Zhou, W. Acta Crystallogr. 2006, E62, o3900; (i)
Xia, C. N.; Hu, W. X.; Zhou, W. Acta Crystallogr. 2007, E63, o4795. The
aforementioned crystallographic details of I-2, I-3, I-5, I-7, I-9, I-10, I-12, I-13
and I-22, have been deposited at the Cambridge Crystallographic Data Center
and allocated the deposition number CCDC 239302, CCDC 647584, CCDC
272826, CCDC 272824, CCDC 614946, CCDC 290011, CCDC 272827, CCDC
614948 and CCDC 673003, respectively..
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2014.08.
024. These data include MOL files and InChiKeys of the most
important compounds described in this article.
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Please cite this article in press as: Chen, H.-z.; et al. Bioorg. Med. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.bmcl.2014.08.024