Antiproliferative activity and SARs of caffeic acid esters with mono-substituted phenylethanols moiety

Article abstract of DOI:10.1016/j.bmcl.2016.12.007

A series of CAPE derivatives with mono-substituted phenylethanols moiety were synthesized and evaluated by MTT assay on growth of 4 human cancer cell lines (Hela, DU-145, MCF-7 and ECA-109). The substituent effects on the antiproliferative activity were systematically investigated for the first time. It was found that electron-donating and hydrophobic substituents at 2′-position of phenylethanol moiety could significantly enhance CAPE's antiproliferative activity. 2′-Propoxyl derivative, as a novel caffeic acid ester, exhibited exquisite potency (IC₅₀?=?0.4?±?0.02 & 0.6?±?0.03?μM against Hela and DU-145 respectively).

Full text of DOI:10.1016/j.bmcl.2016.12.007

Accepted Manuscript
Antiproliferative Activity and SARs of Caffeic Acid Esters with Mono-substi-
tuted Phenylethanols Moiety
Jin Xie, Fengzhi Yang, Man Zhang, Celine Lam, Yixue Qiao, Jia Xiao,
Dongdong Zhang, Yuxuan Ge, Lei Fu, Dongsheng Xie
PII:
S0960-894X(16)31274-4
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.12.007
BMCL 24496
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
12 September 2016
15 November 2016
2 December 2016
Please cite this article as: Xie, J., Yang, F., Zhang, M., Lam, C., Qiao, Y., Xiao, J., Zhang, D., Ge, Y., Fu, L., Xie,
D., Antiproliferative Activity and SARs of Caffeic Acid Esters with Mono-substituted Phenylethanols Moiety,
Bioorganic & Medicinal Chemistry Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.2016.12.007
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Antiproliferative Activity and SARs of Caffeic Acid Esters with
Mono-substituted Phenylethanols Moiety
Jin Xie, Fengzhi Yang, Man Zhang, Celine Lam, Yixue Qiao, Jia Xiao, Dongdong Zhang, Yuxuan Ge,
Lei Fu*, Dongsheng Xie*
School of Pharmacy, Shanghai Jiao Tong University, 800 Dongchuan Rd., Shanghai 200240, China
* Corresponding authors: Lei Fu, Ph.D., Tel./Fax. 86-21-3420-4791, E-Mail: leifu@sjtu.edu.cn,
Dongsheng Xie, Ph.D., Tel. 86-21-3420-4787, E-Mail: dshxie@sjtu.edu.cn,
Abstract
A series of CAPE derivatives with mono-substituted phenylethanols moiety were synthesized and
evaluated by MTT assay on growth of 4 human cancer cell lines (Hela, DU-145, MCF-7 and ECA-109).
The substituent effects on the antiproliferative activity were systematically investigated for the first
time. It was found that electron-donating and hydrophobic substituents at 2’-position of phenylethanol
moiety could significantly enhance CAPE’s antiproliferative activity. 2’-Propoxyl derivative, as a
novel caffeic acid ester, exhibited exquisite potency (IC50 = 0.4±0.02 & 0.6±0.03 μM aganist Hela and
DU-145 respectively).
Keywords: caffeic acid esters; CAPE derivatives; antiproliferative activity; structure-activity
relationships
As a main active constituent of propolis caffeic acid phenethyl ester (CAPE) has demonstrated
widely and significantly cytotoxic1,2, antiproliferative3-6 and anticancer effects7-10. CAPE could induce
apoptosis by specifically inhibiting nuclear factor kappa B (NF-B) and modulating the redox state of
cancer cells.11,12 Actually, oncogene transformed cells showed increased sensitivity to CAPE-induced
toxicity through apoptosis, suggesting that a direct relationship may exist between the cytotoxicity of
CAPE and the induction of apoptosis.13 CAPE can also suppress 5-lipoxygenase and other tumor
promoter-mediated oxidative processes showing anticancer activity.14-17 In addition, CAPE has
potential to suppress tumor invasion and metastasis by inhibition of vascular endothelial growth factor
(VEGF)-induced tube formation and down-regulation of matrix metalloproteinases (MMPs).18-20 In
view of these, to develop novel caffeic acid derivatives may provide promising
Figure 1. Structural modification of CAPE
Despite the fact that many derivatives of caffeic acid or its analogues are also biologically active,
CAPE is still one of the few compounds with significantly and widely cytotoxic, antiproliferative
activity, and other inhibitory effects on carcinosis. In the structural modifications of CAPE for higher
potency, most of attempts in the change of caffeic acid moiety (Fig. 1, section A), such as alkylation
and positional change of phenolic hydroxyl groups, introduction of other substituents into catechol, and
saturation of the trans-unsaturated bond, were proved inconductive or adverse to the enhancement of
cytotoxic or antiproliferative activity.2, 21-26 On the contrary, those attempts focusing on the replacement
1

of phenethyl alcohol with other fatty alcohols particularly having longer carbon chains (Fig. 1, section
B) provided a number of active caffeic acid esters, which remained or slightly improved CAPE’s
cytotoxic or antiproliferative activity.27-29 Nevertheless, CAPE derivatives with variously substituted
phenethyl alcohol moiety have never been systematically investigated. As a result, there is a lack of
all-sided and in-depth analysis and summary of CAPE derivatives on cytotoxic or antiproliferative
activity, as yet. In order to find new caffeic acid derivatives with potentially more potent anticancer
activity than CAPE, and complete the SARs of caffeic acid esters on antiproliferative activity, we
synthesized a series of CAPE derivatives with mono-substituted phenethyl alcohol moiety, and
systematically investigated their inhibitory property on growth of 4 human cancer cell lines (Hela,
DU-145, MCF-7 and ECA-109). Compared with 3’-position and 4’-position substitution, 2’-position
substitution demonstrated notable substituent effects that 2’-electron-donating and hydrophobic
substituents could significantly improve CAPE’s antiproliferative activity.
Scheme 1. General synthesis of CAPE derivatives
Reagents and conditions: (i). LiAlH4, THF, 0 °C to reflux, 4 h, 50-85%; (ii). SOCl2, MeOH, RT to
reflux, 3 h, 100%; (iii). NaBH4, THF/MeOH, RT to reflux, 16 h, 70-85%; (iv). 1% mol Yb(OTf)3,
CH3NO2, reflux, 40-180 min, 18-61%.
Scheme 2. Synthesis of 2’-alkoxyl CAPE derivatives
Reagents and conditions: (i). RBr, K2CO3, DMF, RT to 50 °C, 6-24 h, 24-92%; (ii). NaBH4,
THF/MeOH, RT to reflux, 16 h, 70-85%; (iii). 1% mol Yb(OTf)3, CH3NO2, reflux, 40-60 min, 9-39%.
Many caffeic acid esters are not readily prepared by general direct esterification methods including
condensation and alkylation in view of numerous by-products, low reactivity and particularly the
restricted scope of application. In this presentation, we prepared variously mono-substituted CAPE
derivatives from caffeic acid and corresponding 2-phenylethanols by means of ytterbium triflate
catalytic esterification, which was recently developed in our laboratory as a convenient and practical
method.30 As shown in Scheme 1, most 2-phenylethanols, due to commercial issue, were prepared
from corresponding phenylacetic acids using LiAlH4 as reducing agent. Nitro and cyano substituted
2

phenylacetic acids were first converted to methyl phenylacetates and then reduced to 2-phenylethanols
by NaBH4 in THF/MeOH. The final CAPE derivatives were rapidly synthesized by refluxing caffeic
acid and various 2-phenylethanols in nitromethane using 1 mol% ytterbium triflate as the only catalyst.
2’-Alkoxyl CAPE derivatives such as 2’-ethoxyl, 2’-propoxyl and 2’-benzyloxy derivatives were
prepared from methyl 2’-hydroxylphenylacetates (Scheme 2). By treating with potassium carbonate
and excess alkyl bromide in DMF, 2’-hydroxylphenylacetates were first converted to
2’-alkoxylphenylacetates, and then reduced by NaBH4 in THF/MeOH to afford
2’-alkoxylphenylethanols as the materials for next esterification.
Table 1. Antiproliferative activity of mono-substituted CAPE derivatives
IC50 (μM)a
Hela
3’
ECA-109
3’
DU-145
3’
MCF-7
3’
R
NO2
CN
2’
4’
2’
4’
2’
4’
2’
4’
55.7±2.8
b
52.2±3.2
51.9±2.7
42.2±2.3
7.5±0.4
20.1±0.7
29.7±1.4
5.4±0.3

106.3±6.4
60.2±3.0
35.8±1.7
55.3±3.3
22.5±0.6
56.8±2.9
25.8±1.1
45.8±2.2
67.7±3.2
74.0±4.5

68.6±3.3
57.1±3.0
48.1±2.3
20.5±0.8
31.5±1.8
32.5±1.4
16.7±0.7

133.3±10.7
88.2±4.6
51.9±2.9
83.2±4.0
28.4±1.3
40.3±2.2
20.9±1.0
49.4±2.1
110.6±5.2
60.5±2.9

35.9±1.7
68.4±3.2
51.8±2.3
15.6±0.7
25.3±1.2
13.8±0.6
21.4±1.0

105.6±4.7
52.7±2.7
12.8±0.9
24.9±1.0
22.1±1.1
35.4±1.6
36.9±1.8
51.9±3.0
67.5±3.5
48.2±1.6

33.1±1.2
30.5±1.3
14.5±0.8
1.6±0.07
24.9±1.3
18.6±0.6
26.3±1.2

153.0 ±9.0
77.6±3.8
29.9±1.5
13.8±0.6
17.6±0.8
28.2±1.5
20.6±1.6
53.2±2.4
78.0±3.8
F
29.4±1.6
15.9±0.7
11.3±0.4
4.9±0.2
2.3±0.1
29.4±1.4
1.6±0.07
39.7±1.4
26.6±1.5
14.5±0.7
9.4±0.3
8.2±0.4
40.2±1.9
5.9±0.3
53.8±2.6
19.2±0.6
16.7±0.8
7.6±0.3
6.6±0.3
24.4±1.1
2.0±0.08
23.8±1.2
10.4±0.5
14.6±0.7
12.2±0.7
2.5±0.1
21.8±0.9
7.1±0.3
CF3
Cl
Br
CH3
OH
OCH3
CAPE
Taxol
1.6±0.07
33.0±1.4
0.036±0.002
32.4±1.2
47.5±2.4
0.94±0.04
10.1±0.3
16.0±0.8
0.011±0.0006
22.0±0.9
28.8±1.2
0.63±0.03
a
Values are means of two independent experiments, each performed in triplicate, P < 0.05 by SPSS
Statistics; b not synthesized and tested
For a deep insight of the SARs of substituted phenethyl alcohol moiety, various substituents with
different electronic property along with different occupying positions were systematically investigated.
As shown in Table 1, most of tested CAPE derivatives showed widely antiproliferative activity;
moreover, a specific derivative usually showed nonsignificant differences of activity against 4 tested
cancer cell lines, e.g. 2’-Cl derivative (IC50 = 11.3±0.4, 14.5±0.7, 16.7±0.8 and 14.6±0.7 μM
respectively), 3’-CN derivative (IC50 = 51.9±2.7, 57.1±3.0, 68.4±3.2 and 30.5±1.3 μM respectively),
and 4’-OCH3 derivative (IC50 = 67.7±3.2, 110.6±5.2, 67.5±3.5 and 78.0±3.8 μM respectively), etc. The
phenomena suggested there may be a common molecular mechanism of antiproliferation in CAPE
derivatives.
Nevertheless, against a specific cancer cell line, there were significant differences of inhibitory
potency among these CAPE derivatives. For instance, 2’-CH3, 2’-OCH3 and 3’-OCH3 derivatives
exhibited excellent inhibitory activity (IC50 = 1 - 2 μM) against Hela cell line, but 4’-OCH3 and 4’-NO2
derivatives showed poor activity (IC50 = 67.7±3.2 and 106.3±6.4 μM). Likewise, 2’-Br, 2’-CH3 and
2’-OCH3 derivatives exhibited good inhibitory activity (IC50 = 5 - 10 μM) aganist ECA-109 cell line,
but 4’-CF3, 4’-OCH3 and 4’-NO2 derivatives showed poor activity (IC50 = 83.2±4.0, 110.6±6.4 and
133.3±10.7 μM respectively). Obviously, the substituents of phenylethanols moiety, with different
3

electronic property, steric hindrance and substitution position, exercised a significant influence over the
antiproliferative activity of CAPE derivatives.
With respect to the electronic property, strong electron-withdrawing substituents such as nitro and
cyano usually decreased CAPE’s antiproliferative activity. On the contrary, electron-donating
substituents particularly at 2’-position of phenethyl alcohol were much favorable to increase molecular
activity, e.g. 2’-CH3 and 2’-OCH3 derivatives, which enhanced the antiproliferative potency of CAPE
for 2-20 times against 4 tested cancer cell lines. However, probably due to its hydrophilic property,
hydroxyl group, despite the electron-donating conjugative effect, seemed not to be able to increase
CAPE’s potency. Toward Hela cell, the IC50 values of 2’-substituted derivatives followed the sequence
of -CH3 ≈ -OCH3 < -Br < -Cl < -CF3 < -F < -NO2, which is well consistent with the
electron-withdrawing ability of corresponding substituents. Similar tendency was also found in the
other three cancer cell lines (Fig. 2).
Figure 2. The antiproliferative potency (IC50s) comparison of variously 2’-substituted CAPE
derivatives
120
100
80
60
40
20
0
120
100
80
60
40
20
0
ECA-109
Hela
2' 3' 4'
2' 3' 4'
CAPE
CN
CF3
Cl
Br CH3
NO2
F
Cl Br CH3 OCH3
CAPE NO2 CN
F
CF3
OCH3
120
100
80
60
40
20
0
120
100
80
60
40
20
0
DU-145
MCF-7
3'
2'
4'
2'
3'
4'
NO
CF3 Cl
CH3 OCH3
CAPENO2
Br
CF3
CH3 OCH3
CAPE
CN
CN
Cl
Br
F
F
2
Figure 3. The antiproliferative potency (IC50s) comparison of position isomeric CAPE derivatives
4

With respect to the substitution position, we found that 2’-isomers of Cl, Br, CH3 and OCH3
substituted derivatives had better inhibitory activity than their 3’ and 4’-isomers against all tested
cancer cell lines and the IC50 values approximately followed the sequence of 2’-position < 3’-position <
4’-position (Fig. 3). Strong electron-withdrawing substituted derivatives such as NO2, F and CF3
derivatives didn’t abide by this rule. 3’-NO2 and 3’-CF3 derivatives seemed more potent than their 2’
and 4’-isomers, but F-substituted derivatives were highly irregular. In addition, compared to
4’-monatomic substitunents, 4’-polyatomic substituents such as 4’-NO2, 4’-CN and 4’-OCH3,
significantly reduced CAPE’s antiproliferative potency, which indicated an adverse steric effect of
these CAPE derivatives when they bind to a specific target.
Based on the above SARs, further investigation was focused on 2’-electron-donating substituents
with higher hydrophobicity. Compared with methyl and methoxyl group, ethyl, ethoxyl, propoxyl and
benzyloxy groups have larger hydrophobic surface while retaining electron-donating capacity,
therefore they may lead to higher potency. As we expected, these novel CAPE derivatives, except
2’-benzoxyl derivative, showed much better antiproliferative activity than CAPE (Table 2).
Particularly, 2’-propoxyl derivative exhibited exquisite potency (IC50 = 0.4±0.02 & 0.6±0.03 μM
against Hela and DU-145, 80 and 25 times more potent than CAPE respectively), which are the best
performance among caffeic acid esters so far. Using 2’-propoxyl derivative as a probe molecule will
help to identify and validate specific targets for anticancer activity of caffeic acid esters in the future.
Although hydrophobic property of 2’-substituents is very advantageous to molecular antiproliferative
activity, bulkily substituted derivative i.e. 2’-benzoxyl derivative didn’t show excellent potency.
Table 2. Antiproliferative activities of electron-donating and hydrophobic CAPE derivatives
IC50 (μM)a
R
Hela
ECA-109
7.3±0.3
9.2±0.4
5.3±0.2
15.1±0.6
26.6±1.3
DU-145
4.7±0.2
MCF-7
8.7 a±0.5*
8.8±0.4
5.4±0.2
5.6±0.3
16.8±0.7
C2H5
4.2±0.2
1.5±0.06
0.4±0.02
3.2±0.2
OC2H5
0.8±0.04
0.6±0.03
9.1±0.4
OC3H7-n
OC3H7-i
OCH2C6H5 11.9±0.5
24.0±1.3
a
Values are means of two independent experiments, each performed in triplicate; P < 0.05 by SPSS
Statistics
In summary, we synthesized a series of variously mono-substituted CAPE derivatives, and
systematically investigated their antiproliferative activity. An important SAR was found that
electron-donating and hydrophobic substituents at 2’-position of phenylethanol moiety could
significantly improve molecular potency. It promoted us to synthesize 2’-propoxyl CAPE derivative,
which afforded the most antiproliferative potency of caffeic acid esters against Hela and DU-145, so
far. This study not only supplements the current SARs of caffeic acid esters for antiproliferative
activity, but also further manifests the existence of a specific target that caffeic acid derivatives may
bind to and act antiproliferative activity.
Acknowledgements
5

This work was supported by the Specialized Research Fund for the Doctoral Program of Higher
Education of China (No. 20110073120081) and National Undergraduate Innovative Test Program
(IPP13135 and IAP9079).
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Graphical abstract
The electronic effect of 2’-substituents on CAPE’s antiproliferative
activity.
8