Synthesis, cytotoxicity of new 4-arylidene curcumin analogues and their multi-functions in inhibition of both NF-κB and Akt signalling

Article abstract of DOI:10.1016/j.ejmech.2012.07.039

A series of new 4-arylidene curcumin analogues (4-arylidene-1,7- bisarylhepta-1,6-diene-3,5-diones) were synthesized and found to be potent antiproliferative agents against a panel of cancer cell lines at submicromolar to low micromolar concentrations by

Full text of DOI:10.1016/j.ejmech.2012.07.039

European Journal of Medicinal Chemistry 55 (2012) 346e357
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, cytotoxicity of new 4-arylidene curcumin analogues and their
multi-functions in inhibition of both NF- B and Akt signalling
k
Yinglin Zuo, Jianing Huang, Binhua Zhou, Shuni Wang, Weiyan Shao, Cuige Zhu, Li Lin, Gesi Wen,
*
Hongyang Wang, Jun Du, Xianzhang Bu
School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510275, 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 new 4-arylidene curcumin analogues (4-arylidene-1,7-bisarylhepta-1,6-diene-3,5-diones)
were synthesized and found to be potent antiproliferative agents against a panel of cancer cell lines at
submicromolar to low micromolar concentrations by SRB assay. Their inhibitory abilities against NF-kB
was evaluated by High Content Analysis (HCA) based immunofluorescence assay; and the Akt signalling
inhibition was determined by fluorescence polarization assay and western blot respectively. The
StructureeActivity Relationship was discussed. Our results revealed that 4-arylidene curcumin analogues
may work in a multi-targets manner in cancer cell.
Received 22 May 2012
Received in revised form
18 July 2012
Accepted 20 July 2012
Available online 31 July 2012
Keywords:
4-Arylidene curcumin analogues
Cytotoxicity
Ó 2012 Elsevier Masson SAS. All rights reserved.
NF-kB inhibition
Akt inhibition
Docking
1. Introduction
proliferation and survival makes Akt kinase as promising target for
therapeutic intervention [13,19e22]. Efforts targeting Akt have
Nuclear factor-
k
B (NF-
k
B) proteins are a family of structurally
increased in recent years, many inhibitors have been described
[13,20e23] and some of them are in clinical development [24].
Curcumin (1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptad-
iene-3,5-dione), a dietary yellow spice and pigment isolated from
the rhizome of Curcuma longa, has been found exhibiting various
bioactivities [25]. Curcumin can inhibit Akt signalling in human
prostate cancer cells [26], and cause cell damage by inactivating the
Akt-related cell survival pathway and release of cytochrome c
[27,28]. On the other hand, one of the predominant targets of cur-
related eukaryotic transcription factors with a conserved retic-
uloendotheliosis (Rel) domain [1]. NF- B is constitutively activated
k
in different types of haematologic cancers and solid tumours [2e5]
and promotes the tumour proliferation, invasion and metastasis
[6,7]; moreover, it allows the malignant cells to escape apoptosis
and therefore contributes to the radiation and chemotherapy
resistance of cancer cells [8e10]. Accumulating evidence suggests
that the inhibition of NF-kB activation can improve the efficacy of
current chemotherapeutic regimens [11,12] during the past
decades.
cumin is the NF-
directly inhibit IKK and proteasomes 26S [31,32] to block NF-
activation. Furthermore, curcumin inhibits NF- B activation and
NF- B-regulated gene expression through inhibition of IKK and Akt
activation [33]. Unfortunately, although curcumin is proved to be
an effective chemo-preventive agent to tumour initiation and
proliferation without significant toxic, genotoxic and teratogenic
properties [34], and has been subjected to many clinical trials in
various human cancer therapies [25,35,36], the clinical potential of
curcumin remains limited due to its relatively low potency and
poor bioavailability [37], and highly active and clinically promising
curcuminoid remain to be developed.
kB cell signalling pathway [29,30], curcumin can
kB
Akt (PKB, protein kinase B) is a serine/threonine kinase func-
tions as critical regulator of cell survival and proliferation, it is also
part of the PI3K-Akt-mTOR pathway that encompasses signalling
with cell growth, proliferation, and survival [13,14]. The deregula-
tion of Akt through inactivation of PTEN [15], point mutation, or
overexpression can result in aberrant signalling [16]. High levels of
Akt activity are observed in a large number of human cancers and
are correlated with chemotherapy resistance and poor prognosis
[17,18]. The importance of Akt mediated signalling in tumour
k
k
In our previous work [38], we found that 4-arylidene curcumin
analogues can strongly decrease the growth of various lung cancer
* Corresponding author. Tel./fax: þ86 20 39943054.
E-mail address: phsbxzh@mail.sysu.edu.cn (X. Bu).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.07.039

Y. Zuo et al. / European Journal of Medicinal Chemistry 55 (2012) 346e357
347
cells with 10e60 fold improved potency over curcumin. Further
mechanism investigations revealed that these analogues can
substituted curcumin analogues may increase the molecular
stability [39]. The synthesis was conducted according to the
previously reported procedure [40] (Scheme 1). Briefly, a boric
acetylacetone anhydride complex was prepared first by refluxing
acetylacetone with boric anhydride in EtOAc to avoid unwanted
Knoevenagel reaction at C-3 of acetylacetone, and then aldol
condensation of protected acetylacetone with different aromatic
aldehydes afforded the products 1e4 in 48.53%e66.70% yields
respectively.
significantly inhibit the activation of NF-
However, although our data indicated an important role of IKK
NF- B in functions of reported 4-arylidene curcumin analogues, the
discordance between their cytotoxicity and the IKK /NF- B inhi-
kB via IKKb blockage.
b/
k
b
k
bition ability could be observed, suggesting possible multi-target
effects of 4-arylidene curcumin analogues in cells.
During the analysis of the interaction between 4-arylidene
curcumin analogues and IKK
shaped 4-arylidene curcumin analogues can fit well with the ATP
binding site of IKK [38]. On the other hand, regardless of the very
b
, we noticed that the propeller-
New 4-arylidene curcumin analogues (Fig. 1, 5e30) were
synthesized according to previously developed method [38] by
coupling 1e4 with various aromatic aldehydes in toluene with
AcOH/piperidine as a catalyst (Scheme 1). The easy chemistry
makes it convenient to synthesis large amount of compounds for
biological evaluations. In present work, four catalogues 5e15,
16e21, 22e26 and 27e30 (were synthesized from 1e4 respec-
tively) were obtained successfully in 46.46%e90.55% yields.
b
detailed analysis of kinase catalytic cleft in literature, simply con-
necting the four major regions (Adenine region; Phosphate-binding
region; Entrance region and a linker region, Sugar region) of ATP
binding pocket in kinase presents a propeller-shape. Reasonably,
a small molecule with propeller-shape may favour its interaction
with the ATP binding pocket of kinase if each branch of the
propeller of both molecule and binding pocket is suitable in volume
and shape. Taking account of the revealed functions of curcumin
analogues on Akt involved pathway as mentioned above, we
assumed that the propeller-shaped 4-arylidene curcumin
analogues would also target Akt well. In this work, we describe the
synthesis and cytotoxicity of new 4-arylidene curcumin analogues
against five cancer cell lines from different tissues, and the
2.2. New 4-arylidene curcumin analogues strongly inhibit
proliferation of various human cancer cell lines
After obtained the proposed compounds, their antiproliferative
activities were evaluated by SRB method [41,42]. Four tumour cell
lines from different tissues (human nasopharyngeal carcinoma cell
line, CNE2; human colon carcinoma cell line, SW480; human breast
adenocarcinoma cell line, MCF-7; human hepatoma cell line,
HepG2) along with human lung carcinoma cell line A549 were
chosen in current work. The antiproliferative results are summa-
rized in Table 1. As shown in Table 1, all the traditional curcumin
analogues (1e4) showed moderate to poor antitumour activity
against the 5 tested cancer cell lines. Remarkably, all the new 4-
arylidene curcumin analogues have significantly improved activi-
ties over their parent 1,3-diketones curcumin analogues 1e4 (5e15,
16e21, 22e26 and 27e30 were synthesized from 1e4 respectively).
Most of the 4-arylidene curcumin analogues showed IC50 in the
submicromolar concentration range against 5 tested cell lines
except 4-arylidene curcumin analogues 22, 23, 24 (synthesized
from 3) and 27e29 (synthesized from 4) with slightly lower
discovery of their inhibitory abilities on both NF-
kB and Akt
signalling.
2. Results and discussion
2.1. Chemistry
To expand the structure diversity, classical 1,3-diketones cur-
cumin analogues (Fig. 1, 1e4) were first synthesized by reactions
of acetylacetone with 2,3-dimethoxybenzaldehyde, 2,5-dimetho
xybenzaldehyde,
2,4-dimethoxybenzaldehyde
and
2,4,6-
trimethoxybenzaldehyde respectively. Instead of hydroxyl, the
methoxyl substitution was chosen in 1e4 because the methoxyl
Fig. 1. Structure of curcumin, compounds 1e4 and 5e30.

348
Y. Zuo et al. / European Journal of Medicinal Chemistry 55 (2012) 346e357
Scheme 1. Synthesis of curcumin, compounds 1e4 and 5e30. Reagents and conditions: (a) EtOAc; (b) B(n-BuO)3, n-BuNH2, HCl; (c) piperidine, AcOH, toluene, 140 ꢀC.
activities. These results are in accordance with our previous work
and further indicate that the 4-arylidene curcumin analogues are
potent anticancer agents.
2.3. New 4-arylidene curcumin analogues inhibit NF-
translocation
kB
Among 1e4, compounds with o- or p-dimethoxy substitutions
on the benzene ring (1 and 2) are generally show higher anti-
proliferation activities than compounds with m-dimethoxyl or tri-
methoxyl substitutions (3 and 4). Interestingly, there is a similar
tendency among the 4-arylidene curcumin analogues, 5e15, 16e21
(synthesized from 1e2 respectively) show potent activities against
the five tested cancer cell lines, when the substitution groups on
the aromatic ring of the 4-arylidene moieties are same, compounds
synthesized from 3e4 respectively showed obviously lower activ-
ities than that from 1e2 (6, 16 vs. 22, 27; 11, 18 vs. 23, 28; 12, 19 vs.
24, 29; 13, 20 vs. 25, 30; 15, 21 vs. 26 respectively). This data
indicated that methoxyl substitution position on the precursor of
4-arylidene curcumin analogues plays a role in their antiprolifera-
tion activity. From the 4-arylidene moieties side of view, however, it
seems that the substitutions have no apparent effect on their
antiproliferation activities.
Normally, NF-
and translocated into the nucleus to induce specific gene expres-
sion. To evaluate the effects of obtained compounds on NF-
signalling, a high content analysis (HCA) based immunofluores-
cence assay was used to visualize the dynamic movement of the
kB is in the cytoplasm and it should be activated
kB
NF-
various conditions. The assay was performed on A549 cells by
using CellomicsÒ NF-
B Activation HCS Reagent Kit (Thermo
kB p65 subunit between the cytoplasm and nucleus under
k
Scientific) in combination with the ArrayScan HCS Reader (Thermo
Scientific), and the enclosed experimental protocol was followed.
A549 cells were plated on 384-well plates and treated with test
compounds or controls at 37 ꢀC for 30 min, after stimulated by
TNF
rabbit anti-p65 NF-
incubated with DyLightÔ 488-conjugated goat anti-rabbit IgG
a, cells were then fixed, permeabilized and incubated with
k
B antibody. After that, cells were washed and
(stain NF-kB) along with Hoechst 33342 (stain nucleus). Images
Table 1
Antiproliferative activity of the target compounds 1e30 against 5 cancer cell lines.a
Compound
Human cancer cell line (IC50
A549
,
m
M)
CNE2
SW480
MCF-7
HepG2
Curcumin
1
2
3
4
5
6
7
8
11.58 ꢁ 0.80
11.46 ꢁ 3.57
16.30 ꢁ 0.79
44.69 ꢁ 5.31
>50
5.76 ꢁ 0.47
10.17 ꢁ 1.92
12.05 ꢁ 4.00
15.55 ꢁ 0.07
>50
15.66 ꢁ 0.39
>50
>50
>50
>50
20.69 ꢁ 5.58
12.86 ꢁ 1.35
5.56 ꢁ 0.61
10.45 ꢁ 0.22
16.54 ꢁ 6.26
0.58 ꢁ 0.05
0.20 ꢁ 0.06
0.20 ꢁ 0.01
0.22 ꢁ 0.01
0.30 ꢁ 0.03
0.23 ꢁ 0.01
0.22 ꢁ 0.06
0.24 ꢁ 0.07
0.21 ꢁ 0.01
0.24 ꢁ 0.01
0.27 ꢁ 0.10
0.34 ꢁ 0.13
0.26 ꢁ 0.01
0.22 ꢁ 0.02
0.28 ꢁ 0.06
0.21 ꢁ 0.01
0.47 ꢁ 0.05
0.51 ꢁ 0.01
1.76 ꢁ 0.27
1.19 ꢁ 0.19
0.32 ꢁ 0.09
0.39 ꢁ 0.10
0.67 ꢁ 0.03
0.81 ꢁ 0.12
0.66 ꢁ 0.04
0.51 ꢁ 0.09
12.78 ꢁ 1.41
14.67 ꢁ 0.12
>50
>50
>50
0.34 ꢁ 0.01
0.13 ꢁ 0.01
0.17 ꢁ 0.01
0.23 ꢁ 0.06
0.63 ꢁ 0.02
0.32 ꢁ 0.01
0.20 ꢁ 0.01
0.20 ꢁ 0.01
0.24 ꢁ 0.03
0.26 ꢁ 0.04
0.27 ꢁ 0.13
0.19 ꢁ 0.01
0.40 ꢁ 0.07
0.22 ꢁ 0.03
0.22 ꢁ 0.04
0.19 ꢁ 0.01
0.39 ꢁ 0.04
0.59 ꢁ 0.07
2.07 ꢁ 0.11
1.09 ꢁ 0.31
0.30 ꢁ 0.01
0.38 ꢁ 0.05
0.58 ꢁ 0.14
1.06 ꢁ 0.04
0.73 ꢁ 0.13
0.34 ꢁ 0.02
0.61 ꢁ 0.01
0.28 ꢁ 0.01
0.28 ꢁ 0.01
0.40 ꢁ 0.01
0.63 ꢁ 0.04
0.40 ꢁ 0.05
0.26 ꢁ 0.05
0.22 ꢁ 0.02
0.30 ꢁ 0.03
0.41 ꢁ 0.03
0.22 ꢁ 0.02
0.34 ꢁ 0.03
0.55 ꢁ 0.04
0.38 ꢁ 0.03
0.42 ꢁ 0.07
0.45 ꢁ 0.09
0.73 ꢁ 0.06
0.97 ꢁ 0.18
2.83 ꢁ 0.02
1.78 ꢁ 0.54
0.85 ꢁ 0.13
0.76 ꢁ 0.09
1.35 ꢁ 0.27
1.58 ꢁ 0.03
1.32 ꢁ 0.26
0.68 ꢁ 0.07
1.04 ꢁ 0.03
0.43 ꢁ 0.03
0.41 ꢁ 0.05
0.65 ꢁ 0.02
0.84 ꢁ 0.01
0.59 ꢁ 0.11
0.33 ꢁ 0.01
0.26 ꢁ 0.05
0.42 ꢁ 0.04
0.56 ꢁ 0.03
0.32 ꢁ 0.03
0.41 ꢁ 0.02
0.56 ꢁ 0.11
0.51 ꢁ 0.01
0.49 ꢁ 0.08
0.50 ꢁ 0.03
1.12 ꢁ 0.04
1.52 ꢁ 0.11
3.35 ꢁ 0.03
1.48 ꢁ 0.17
0.85 ꢁ 0.07
1.37 ꢁ 0.23
1.49 ꢁ 0.12
1.95 ꢁ 0.03
1.45 ꢁ 0.16
0.98 ꢁ 0.02
1.83 ꢁ 0.51
0.48 ꢁ 0.02
0.56 ꢁ 0.08
0.84 ꢁ 0.16
0.72 ꢁ 0.08
0.52 ꢁ 0.03
0.30 ꢁ 0.06
0.36 ꢁ 0.08
0.46 ꢁ 0.02
0.38 ꢁ 0.19
0.31 ꢁ 0.03
0.40 ꢁ 0.07
0.85 ꢁ 0.06
0.46 ꢁ 0.07
0.58 ꢁ 0.02
0.43 ꢁ 0.02
0.99 ꢁ 0.06
2.14 ꢁ 0.56
5.45 ꢁ 1.47
2.60 ꢁ 0.78
0.97 ꢁ 0.31
1.60 ꢁ 0.14
1.88 ꢁ 0.14
3.18 ꢁ 0.92
1.46 ꢁ 0.22
1.03 ꢁ 0.32
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
a
Data shown are the average values from at least two independent experiments with standard error (SE).

Y. Zuo et al. / European Journal of Medicinal Chemistry 55 (2012) 346e357
349
were recorded on ArrayScan HCS Reader, the levels of NF-
k
B
2.4. Selected 4-arylidene curcumin analogues inhibit Akt1 in low
micromolar concentrations
translocation were calculated based on the fluorescence intensity
difference between in the nucleus and in the cytoplasm. The IC50
values of test compounds were summarized in Table 2. Repre-
sentative immunofluorescence images from compound 6 was
shown in Fig. 2, these images clearly showed that the nucleus
To probe the possible effects of 4-arylidene curcumin analogues
on Akt1 kinase, compound 6, 19 and 26 were selected for Akt1
inhibition test. We chose 6, 19 and 26 because they are represen-
tative compounds of the investigated catalogues which covered the
translocation of NF-kB was dose-dependently inhibited by
compound 6.
high to low activities in both antiproliferation and NF-kB inhibition
Data in Table 2 revealed interesting StructureeActivity Rela-
assay (6 from 5e15, 19 from 16e21 and 26 from 22e26). A fluo-
tionship (SAR). Similar with the antiproliferation activity,
rescence polarization based assay was performed by using AKT1/
4-arylidene curcumin analogues 5e26 show much higher NF-
k
B
PKBa KinEASEÔ FP Fluorescein Green Assay kit (Upstate, Millipore
inhibitory activities over their parent curcumin analogues 1, 2 or 3
with several exceptions (9, 17 and 24). Among the 4-arylidene cur-
cumin analogues, compounds with 2,3-dimethoxyl (5e8, 10e15)
and 2,5-dimethoxyl (16, 18e21) substitutions (from 1 and 2
Corporation) to obtain the IC50 values. In this assay, a phosphory-
lated peptide substrate STK has been labelled with a green fluo-
rescent dye and worked as phosphorylated tracer, which could bind
to a phospho-specific STK antibody to form a high molecular
weight complex with high polarization value. In kinase reaction,
non-fluorescently labelled STK substrate 3 is phosphorylated by the
Akt1; the phosphorylated product competes with the tracer for
binding to the phospho-specific STK antibody. The addition in the
binding of the tracer to antibody resulting in an increase in the
fluorescence polarization value which indicating an inhibition of
Akt1 by compound added. As expected, compound 6, 19 and 26
show good activity in Akt1 kinase inhibition, with an IC50 of
respectively) generally show much higher NF-
ties (IC50 < 6.0 M) than that with 2,4-dimethoxyl (22e24, 26) and
2,4,6-trimethoxyl (27e30) substitutions (from 3 and 4 respectively),
22e24, 26 show high IC50 values (above 10.0 M) and no apparent
activity could be observed from analogues 27e30 (IC50 > 50.0 M).
kB inhibitory activi-
m
m
m
These observations are also in accordance with their antiprolifera-
tion activities in general. We noticed that all of methoxyl substitu-
tions mentioned above locate atortho- and/or para- position of the
unsaturated ketone linker in the less active analogues 22e24 and
26e30. Furthermore, compound 9 containing the 2,4,6-trimethoxyl
substitutions also show no apparent activity (IC50 > 50.0 M) in
a,
b
0.81 mM, 5.02 mM and 1.66 mM respectively (Table 3).
m
2.5. Phosphorylation of Akt downstream substrate GSK3b could be
despite of being prepared from 1. All the information indicates that
blocked by Akt1 inhibitor 6
an electron conjugation effect of the substitutions may give key
contribution to the NF-kB inhibitory activities of tested compounds.
In order to further evaluate the effect of the found inhibitor on
cellular Akt signalling, the phosphorylation state of Akt down-
This assumption was further supported by observed phenomena
that the 3-halogen (F or Br) substitution on 4-arylidene moiety of
11e12, 18e19 decreased the activity definitely, by comparing the
activities of analogues which derived from the same parent curcu-
min analogues (e.g. 1 or 2). Though the steric effects may also be
explanation of the lower activity of 27e30, this possibility was
reduced by comparing the activity of 8, 12e14 with that of 9, all of
which have larger triple-substitutions on 4-arylidene moiety but
exhibit different activities. Nevertheless, a few exceptions such as 17
and 25 were observed and the detail mechanism needs further
exploration.
stream substrate GSK3
ence or absence of representative compound 6 which was found to
be the most potent compound against A549 cell growth (IC50
0.13 M) and Akt activity (IC50, 0.81 M) in current work. After
treated with various concentrations of 6 (0.125, 0.25, 0.5, and
1.0 M), the A549 cell was lysed and analyzed by western blot. As
shown in Fig. 3, the phosphorylation of GSK3 was significantly
b in A549 cell were examined in the pres-
,
m
m
m
b
suppressed by compound 6 in a dose dependent manner. This
result indicates 4-arylidene curcumin analogue 6 can block Akt
function in cell level.
Table 2
Inhibition activity of tested compounds on NF-k
B activation/translocation.a,b
R0
R
2,3-Dimethoxyl
2,5-Dimethoxyl
2,4-Dimethoxyl
2,4,6-Trimethoxyl
Cpd.
IC50
,
m
M
Cpd.
IC50
,
mM
Cpd.
IC50
,
m
M
Cpd.
IC50, mM
4-Fluoro
4-Hydroxyl-3-methoxyl
3,4-Dimethoxyl
3,4,5-Trimethoxyl
2,4,6-Trimethoxyl
5
6
7
8
9
1.72 ꢁ 0.02
1.71 ꢁ 0.61
1.67 ꢁ 0.28
1.32 ꢁ 0.07
>50
\
\
\
22
\
\
\
\
\
27
\
\
\
\
16
17
\
2.65 ꢁ 1.07
10.42 ꢁ 1.29
>50
\
\
\
14.36 ꢁ 4.38
\
\
\
\
\
\
2,3-Dimethoxyl
10
11
12
13
14
15
1
2.39 ꢁ 0.45
5.25 ꢁ 1.82
5.53 ꢁ 1.32
2.62 ꢁ 0.19
1.63 ꢁ 0.26
1.26 ꢁ 0.02
>50
\
\
\
\
\
\
3-Fluoro-4-hydroxyl
3-Bromo-4-hydroxyl-5-methoxyl
3,5-Dimethoxyl-4-hydroxyl
2,3,4-Trimethoxyl
3-Hydroxyl -4-methoxyl
No 4-arylidene moiety
18
19
20
\
21
2
3.01 ꢁ 0.33
4.19 ꢁ 0.64
0.95 ꢁ 1.19
\
23
24
25
\
26
3
22.98 ꢁ 2.26
28
29
30
\
\
4
>50
>50
>50
\
>50
3.93 ꢁ 0.15
\
1.78 ꢁ 0.41
12.28 ꢁ 0.25
\
>50
>50
>50
a
Curcumin was used as reference compound and its IC50 on NF-
Data shown are the average values from at least two independent experiments with standard error (SE).
k
B activation inhibition was found to be 21.36 ꢁ 5.23
m
M.
b

350
Y. Zuo et al. / European Journal of Medicinal Chemistry 55 (2012) 346e357
Fig. 3. Effect of compound 6 on phosphorylation of Akt downstream substrate GSK3
b
in cell level. A549 cells were pretreated with compound 6 for 12 h, cell lysates were
prepared and analyzed for the phosphorylation state of the Akt kinase downstream
substrate GSK3b via western blot.
binding site of Akt1 kinase by using Surflex-Dock in Sybyl 7.3.5
(Tripos Inc.) since there is no crystal data of Akt1-ATP complex
available. Several reported crystals which is in complex with an ATP
competitive inhibitor were adopted in our first attempts, we found
that the hinge region residues Glu 228 and Ala 230 in a crystal
structure of Akt1 kinase (PDB code: 3MV5) have hydrogen bonds
respectively with the adenine moiety of ATP (Fig. 4A), which are
conserved in ATP and kinase binding complex. Furthermore, ATP
also showed a binding pose similar with the reported general
manner of kinase-ATP complex (Fig. 4A) [43]. Therefore, 3MV5
were chosen and the docking method was considered to be
validated.
Then, molecular docking study was performed using
compounds 5e26. The results showed that all the 4-arylidene
curcumin analogues adopt
a propeller-shaped conformation
(Supporting information, Table S1) in the propeller-shaped pocket
of the ATP binding site, despite of the direction of each branch of
the “propeller” varied. Furthermore, most of the compounds with
IC50 below 0.5
mM (compounds 6, 11, 15, 16, 20) against all five
tested cancer cell lines exhibit two or more hydrogen bonds in the
hydrophobic adenine binding region except 12 and 13. The repre-
sentative binding modes of 6, 19 and 26 were showed in Fig. 4BeD.
The 4-arylidene moieties of 6 and 26 occupy the hydrophobic
adenine binding region (Fig. 4B and D), and have hydrogen bonds
formed with hinge region residues Glu 228 and Ala 230 respec-
tively. Interestingly, though substitutions on 4-arylidene moiety of
compound 6 (40-OH and 30-OMe) and 26 (30-OH and 40-OMe) are
different, they adopt a similar binding mode. The 4-arylidene
moiety of compound 19 does not occupy the adenine binding
region and no hydrogen bonds formed with residues in this region
(Fig. 4C), it may be partly resulted from the relative larger steric
effects on the 4-arylidene moiety. These observations are in
accordance with the data from kinase inhibition assay, in which the
compound 19 show relative lower Akt1 inhibitory activity than
both 6 and 26. The docking analysis further supported that Akt
kinase would be one target of 4-arylidene curcumin analogues.
Fig. 2. Inhibition of TNFa induced NF-kB activation by compound 6. Example images of
NF-
(DMSO) for 30 min, followed by stimulation with TNF
vehicle (DMSO) treatment, NF- B is located at cytoplasm, upon TNF
is activated and translocated to the nucleus. Preincubation of the cells with increasing
k
B subcellular localization. A549 cells were treated with compounds or vehicle
(10 ng/ml) for 30 min. In the
treatment, NF-
a
k
a
kB
concentrations of compound 6 dose-dependently inhibited the TNFa-induced NF-kB
translocation to the nucleus.
2.7. Discussion
2.6. Molecular docking
In our previous report [38], we have found that 4-arylidene
To further explore the interactions of new 4-arylidene curcumin
analogues with Akt, an ATP molecular was first docked into ATP
curcumin analogues can inhibit the activation of NF-
k
B via IKK
b
blockage. In this work, we further confirmed the ability of NF-
kB
inhibition with SAR information. However, the discordance
between their cytotoxicity and the NF- B inhibition activity sug-
Table 3
Akt1 kinase inhibition activity.a
k
gested possible multi-target effects of 4-arylidene curcumin
analogues in cells. Based on the results above, the Akt inhibition
may be partly responsible for this issue. Interestingly, compound 6
Compound
IC50 (mM)
6
19
26
0.81 ꢁ 0.24
5.02 ꢁ 0.81
1.66 ꢁ 0.38
with most potent ability against A549 cell growth (IC50, 0.13
show strong NF- B (IC50, 1.71 M) and Akt (IC50, 0.81 M) inhibition
among the tested compounds, indicating a close correlation of the
mM)
a
k
m
m
Data shown are the average values from two
independent experiments with standard error (SE).

Y. Zuo et al. / European Journal of Medicinal Chemistry 55 (2012) 346e357
351
Fig. 4. Binding modes of selected compounds. Lipophilic potential surface (using a channel method) of Akt1 kinase (PDB code: 3MV5) ATP binding site with represented molecules.
Small molecular and residues of Akt were represented in stick and coloured by atom type. The colour ramps for lipophilic potential (LP) ranges from brown (highest lipophilic
area of the molecular) to blue (highest hydrophilic area). Z-clipping to remove the N terminal region for visualization facility. (A) ATP. (B) Compound 6. (C) Compound 19.
(D) Compound 26.
anticancer activity with the multi-target effects. Noticeably,
compound 19 (IC50, 0.22e0.58 M) is more potent than compound
26 (IC50, 0.38e1.60 M) in the antiproliferative activity though with
lower Akt1 inhibitory ability, we found it has higher ability (IC50
4.19 M) in NF- B inhibition than 26 (IC50, 12.28 M). Discordances
between cytotoxicity (most IC50 in submicromolar concentration
range) and the NF- B/Akt1 inhibition activity (most IC50 in low
micromolar concentration range) of title compounds could be still
observed, the synergistic effects of NF- B and Akt1 inhibition,
instead of the simply accumulation of both activities, would be
a possible explanation.
Combination of all SAR information from antiproliferation and
Akt1/NF-kB inhibition, it may be concluded that a hydrogen bond
acceptor and a hydrogen bond receptor should be included in one
branch of the “propeller” of the molecule to design next generation
of potent anticancer agent based on 4-arylidene curcumin
analogue, while a 2,4- or 2,4,6 methoxyl substitutions should be
avoided. Though the halogen substitution didn’t affect the anti-
proliferation in current work, it made a negative contribution on
the NF-kB inhibition. Furthermore, it could be seen that one of the
branch of the propeller-sharped 4-arylidene curcumin analogue
occupy the hydrophilic phosphate-binding region in kinase Akt1
further discovered and the results revealed that 4-arylidene cur-
cumin analogues may work in a multi-targets behaviour in cellular
level.
m
m
,
m
k
m
4. Experiments
k
4.1. General
k
All reagents used were commercially available. Solvents were
treated using standard techniques. Reactions were monitored by
TLC on a glass plate coated with silica gel with fluorescent indicator
(GF254). Column chromatography was performed on silica gel
(200e300 mesh). 1H NMR and 13C NMR spectra were recorded
using TMS as an internal standard with a Burker BioSpin Ultrashield
400 NMR system at 400 MHz and 100 MHz, respectively. The Purity
of target compounds (>95%) was determined on a DIONEX Ulti-
mate 3000 HPLC System(Chromeleon SR9 Build 2673); column,
AcclaimÒ120 C18, 5
m
m, 4.6 ꢂ 250 mm; mobile phase, solvent A:
0.1% trifluoroacetic acid (TFA) in water, solvent B: 0.1% TFA in
CH3CN, flow rate ¼ 1 ml/min; UV wavelength, 254 nm; tempera-
ture, ambient; for curcumin and compound 1e4, start 70% B, linear
gradient to 95% B in 30 min; for compound 5e30, start 30% B, linear
gradient to 95% B in 30 min; compound purities were calculated as
the percentage peak area of the analyzed compound, retention
times (tR) were calculated in minutes, a list of all purities is given in
the Supporting Information, Table S2. High resolution mass spectra
(HRMS) were recorded on Shimadzu LCMS-IT-TOF.
(current work) and IKKb [38], introduction of a hydrophilic moiety
in one branch of the molecule may improve the molecular polari-
zation as well as the interactions of molecule with kinase.
3. Conclusions
We designed and synthesized a series of new 4-arylidene cur-
cumin analogues. SRB assay revealed their potent antiproliferative
activities against various cancer cell lines, further confirmed the
potent anticancer activity of 4-arylidene curcumin analogues. The
4.2. (1E,4Z,6E)-5-Hydroxy-1,7-bis(4-hydroxy-3-methoxyphenyl)
hepta-1,4,6-trien-3-one (curcumin)
Curcumin was prepared according to Pedersen method [40]
NF-kB and Akt1 inhibitory activities of title compounds were
with slight modification. To a solution of 2,4-pentanedione

352
Y. Zuo et al. / European Journal of Medicinal Chemistry 55 (2012) 346e357
(30 mmol) in EtOAc (40 ml), boric anhydride (0.7 equiv) was
added and stirred at 70 ꢀC for
h. Then 4-hydroxy-3-
4.3.1. (1E,6E)-1,7-Bis(2,3-dimethoxyphenyl)-4-(4-
fluorobenzylidene)hepta-1,6-diene-3,5-dione (5)
1
methoxybenzaldehyde (2 equiv) and tributyl borate (4 equiv)
were added and the mixture was stirred for further 30 min. After
that, the temperature was raised to 85 ꢀC, and butylamine
(0.2 equiv)/EtOAc solution was added dropwise. After for 20 h
stirring at 85 ꢀC, the mixture was then hydrolyzed by adding 1 N
HCl at 60 ꢀC and strring for 0.5 h at 60 ꢀC. The organic layer was
separated, and the aqueous layer was extracted with EtOAc. The
combined organic layer were washed to neutral and dried over
anhydrous sodium sulphate. After removal of the solvent in
reduced pressure, the crude product was purified by recrystalliza-
tion from EtOH to yield yellow crystals 5.58 g (50.49% yield). HPLC
tR ¼ 4.88 min. The product was confirmed by comparing it with
previously synthesized curcumin [38].
Yellow oil, yield 77.01%; HPLC tR ¼ 22.78 min; 1H NMR
(400 MHz, CDCl3)
d
8.09 (d, J ¼ 15.8 Hz, 1H), 7.86 (d, J ¼ 16.5 Hz, 1H),
7.86 (s, 1H), 7.51e7.45 (m, 2H), 7.19 (d, J ¼ 15.7 Hz, 1H), 7.17 (dd,
J ¼ 8.3, 1.4 Hz, 1H), 7.10 (dd, J ¼ 8.0, 1.4 Hz, 1H), 7.07e6.98 (m, 4H),
6.96e6.88 (m, 2H), 6.91 (d, J ¼ 16.5 Hz,1H), 3.87 (s, 3H), 3.84 (s, 3H),
3.83 (s, 3H), 3.62 (s, 3H); 13C NMR (100 MHz, CDCl3)
d 198.50,
187.33, 165.07, 162.56, 153.26, 153.18, 149.20, 148.92, 142.33, 140.46,
140.44, 140.14, 139.78, 132.55, 132.46, 129.89, 129.86, 128.87, 128.51,
128.34, 124.39, 124.26, 123.74, 120.02, 119.44, 116.22, 116.00, 114.94,
114.58, 61.47, 61.41, 55.98, 55.97; HRMS calcd for C30H27O6F
[M ꢃ H]ꢃ: 501.1713, found 501.1718.
4.3.2. (1E,6E)-1,7-Bis(2,3-dimethoxyphenyl)-4-(4-hydroxy-3-
methoxybenzylidene)hepta-1,6-diene-3,5-dione (6)
The synthesis of compounds 1e4 was carried out by the same
procedure as described for preparation of curcumin, and 1H NMR of
1 [44] and 3 [45] nicely match literature reported data.
Orange powder, yield 60.31%; HPLC tR ¼ 20.95 min; 1H NMR
(400 MHz, CDCl3)
d
8.06 (d, J ¼ 15.7 Hz, 1H), 7.90 (d, J ¼ 16.4 Hz, 1H),
7.84 (s, 1H), 7.22 (d, J ¼ 15.7 Hz, 1H), 7.18 (dd, J ¼ 8.0, 1.1 Hz, 1H), 7.11
(dd, J ¼ 8.0, 1.2 Hz, 1H), 7.08 (dd, J ¼ 8.3, 2.0 Hz, 1H), 7.05 (t,
J ¼ 7.9 Hz, 1H), 7.02 (t, J ¼ 8.0 Hz,1H), 7.01 (d, J ¼ 2.0 Hz,1H), 6.94 (d,
J ¼ 16.4 Hz, 1H), 6.93 (td, J ¼ 8.2, 1.4 Hz, 2H), 6.86 (d, J ¼ 8.3 Hz, 1H),
5.89 (s, 1H), 3.87 (s, 3H), 3.84 (s, 3H), 3.83 (s, 3H), 3.82 (s, 3H), 3.62
4.2.1. (1E,4Z,6E)-1,7-Bis(2,3-dimethoxyphenyl)-5-hydroxyhepta-
1,4,6-trien-3-one (1)
Yellow crystals, yield 51.20%; HPLC tR ¼ 11.80 min; 1H NMR
(400 MHz, CDCl3)
J ¼ 8.0, 1.4 Hz, 2H), 7.07 (t, J ¼ 8.0 Hz, 2H), 6.94 (dd, J ¼ 8.1, 1.4 Hz,
2H), 6.70 (d, J ¼ 16.1 Hz, 2H), 5.89 (s, 1H), 3.89 (s, 12H).
d
15.90 (s, 1H), 7.97 (d, J ¼ 16.1 Hz, 2H), 7.20 (dd,
(s, 3H); 13C NMR (100 MHz, CDCl3)
d 199.22, 187.47, 153.19, 153.11,
149.03, 148.83, 148.38, 146.66, 141.71, 141.66, 139.43, 138.31, 128.96,
128.59, 128.41, 125.83, 125.79, 124.35, 124.21, 123.86, 119.94, 119.38,
114.93, 114.76, 114.36, 112.50, 61.43, 61.33, 55.91; HRMS calcd for
C31H30O8 [M ꢃ H]ꢃ: 529.1862, found 529.1869.
4.2.2. (1E,4Z,6E)-1,7-Bis(2,5-dimethoxyphenyl)-5-hydroxyhepta-
1,4,6-trien-3-one (2)
Yellow crystals, yield 48.53%; HPLC tR ¼ 12.72 min; 1H NMR
(400 MHz, CDCl3)
d
15.95 (s, 1H), 7.95 (d, J ¼ 16.0 Hz, 2H), 7.09 (d,
4.3.3. (1E,6E)-4-(3,4-Dimethoxybenzylidene)-1,7-bis(2,3-
dimethoxyphenyl)hepta-1,6-diene-3,5-dione (7)
J ¼ 2.9 Hz, 2H), 6.91 (dd, J ¼ 9.0, 2.9 Hz, 2H), 6.86 (d, J ¼ 9.0 Hz, 2H),
6.69 (d, J ¼ 16.0 Hz, 2H), 5.89 (s, 1H), 3.86 (s, 6H), 3.81 (s, 6H); 13C
Yellow powder, yield 83.50%; HPLC tR ¼ 23.51 min; 1H NMR
NMR (100 MHz, CDCl3)
d
183.73, 153.62, 153.01, 135.59, 125.02,
(400 MHz, CDCl3)
d
8.06 (d, J ¼ 15.7 Hz, 1H), 7.90 (d, J ¼ 16.4 Hz, 1H),
124.65, 116.94, 113.14, 112.51, 101.64, 56.13, 55.84; HRMS calcd for
7.86 (s, 1H), 7.22 (d, J ¼ 15.7 Hz, 1H), 7.18 (dd, J ¼ 7.9, 1.2 Hz, 1H),
7.14e7.09 (m, 2H), 7.05 (t, J ¼ 8.0 Hz,1H), 7.02 (t, J ¼ 8.0 Hz,1H), 7.02
(d, J ¼ 1.5 Hz, 1H), 6.94 (d, J ¼ 16.4 Hz, 1H), 6.97e6.90 (m, 2H), 6.81
(d, J ¼ 8.4 Hz, 1H), 3.87 (s, 3H), 3.86 (s, 3H), 3.84 (s, 3H), 3.83 (s, 3H),
C23H24O6 [M ꢃ H]ꢃ: 395.1495, found 395.1485.
4.2.3. (1E,4Z,6E)-1,7-Bis(2,4-dimethoxyphenyl)-5-hydroxyhepta-
1,4,6-trien-3-one (3)
3.81 (s, 3H), 3.62 (s, 3H); 13C NMR (100 MHz, CDCl3)
d 198.48,
Yellow crystals, yield 50.12%; HPLC tR ¼ 12.69 min; 1H NMR
187.09, 152.67, 152.59, 150.83, 148.46, 148.39, 148.25, 141.04, 140.80,
138.77, 138.31, 128.27, 128.00, 127.75, 125.74, 124.85, 123.94, 123.81,
123.05, 119.25, 118.74, 114.38, 114.00, 112.35, 110.71, 60.84, 60.74,
55.39, 55.34, 55.33, 55.29; HRMS calcd for C32H32O8 [M þ Na]þ:
567.1995, found 567.1999.
(400 MHz, CDCl3)
d
7.89 (d, J ¼ 16.0 Hz, 2H), 7.48 (d, J ¼ 8.6 Hz, 2H),
6.62 (d, J ¼ 16.0 Hz, 2H), 6.52 (dd, J ¼ 8.5, 2.4 Hz, 2H), 6.46 (d,
J ¼ 2.4 Hz, 2H), 5.80 (s, 1H), 3.88 (s, 6H), 3.84 (s, 6H).
4.2.4. (1E,4Z,6E)-5-Hydroxy-1,7-bis(2,4,6-trimethoxyphenyl)hepta-
1,4,6-trien-3-one (4)
4.3.4. (1E,6E)-1,7-Bis(2,3-dimethoxyphenyl)-4-(3,4,5-
trimethoxybenzylidene)hepta-1,6-diene-3,5-dione(8)
Yellow oil, yield 70.39%; HPLC tR ¼ 24.37 min; 1H NMR
Orange power, 66.70%; HPLC tR ¼ 10.86 min; 1H NMR (400 MHz,
CDCl3)
d
8.05 (d, J ¼ 16.1 Hz, 2H), 6.99 (d, J ¼ 16.1 Hz, 2H), 6.12 (s,
4H), 5.78 (s, 1H), 3.88 (s, 12H), 3.85 (s, 6H); 13C NMR (100 MHz,
(400 MHz, CDCl3)
d
8.07 (d, J ¼ 15.7 Hz, 1H), 7.89 (d, J ¼ 16.4 Hz, 1H),
CDCl3)
d
184.82, 162.66, 161.27, 131.07, 124.36, 106.67, 101.66, 90.57,
7.81 (s, 1H), 7.23 (d, J ¼ 15.7 Hz, 1H), 7.18 (dd, J ¼ 7.9, 1.2 Hz,1H), 7.09
(dd, J ¼ 8.0, 1.4 Hz, 1H), 7.06 (t, J ¼ 8.0 Hz,1H), 7.03 (t, J ¼ 8.0 Hz, 1H),
6.97e6.90 (m, 2H), 6.93 (d, J ¼ 16.4 Hz, 1H), 6.74 (s, 2H), 3.87 (s, 3H),
3.85 (s, 3H), 3.83 (s, 3H), 3.83 (s, 3H), 3.80 (s, 6H), 3.63 (s, 3H); 13C
55.77, 55.42; HRMS calcd for C25H28O8 [M þ Na]þ: 479.1682, found
479.1680.
NMR (100 MHz, CDCl3)
d 198.16, 187.15, 152.77, 152.75, 152.69,
4.3. General procedure for the synthesis of 5e30
148.61, 148.37, 141.21, 140.68, 139.85, 139.73, 139.26, 128.47, 128.29,
128.02, 127.82, 124.05, 123.90, 123.08, 119.41, 118.90, 114.52, 114.17,
107.53, 60.91, 60.85, 60.44, 55.69, 55.45; HRMS calcd for C33H34O9
[M þ Na]þ: 597.2101, found 597.2095.
Compounds 5e30 were synthesized according to a previous
method [38]. In general, 2.0 mmol of 1, 2, 3 or 4 and 4.0 mmol of the
corresponding benzaldehyde as well as 100 ml toluene were added
to a two-neck rounded flask equipped with a water dispenser.
Pyridine (8.0 mg, 0.1 mmol) and acetic acid (9.6 mg, 0.16 mmol)
were added as catalysts. The reaction mixture was stirred at 140 ꢀC
for 16 h, the generated water was removed by water dispenser
during the whole reaction. Then the reaction mixture was evapo-
rated under vacuum, the residue was used for column chroma-
tography to get the product.
4.3.5. (1E,6E)-1,7-Bis(2,3-dimethoxyphenyl)-4-(2,4,6-
trimethoxybenzylidene)hepta-1,6-diene-3,5-dione (9)
Yellow powder, yield 89.36%; HPLC tR ¼ 24.53 min; 1H NMR
(400 MHz, CDCl3)
d
8.02 (d, J ¼ 15.8 Hz, 1H), 8.00 (s, 1H), 7.79 (d,
J ¼ 16.3 Hz, 1H), 7.23 (d, J ¼ 15.8 Hz,1H), 7.19 (dd, J ¼ 8.0, 1.3 Hz, 1H),
7.06 (dd, J ¼ 7.9, 1.4 Hz,1H), 7.03 (t, J ¼ 8.0 Hz, 1H), 7.00 (t, J ¼ 8.0 Hz,
1H), 6.91 (dd, J ¼ 8.2, 1.4 Hz, 1H), 6.89 (dd, J ¼ 8.0, 1.5 Hz, 1H), 6.87

Y. Zuo et al. / European Journal of Medicinal Chemistry 55 (2012) 346e357
353
(d, J ¼ 16.3 Hz, 1H), 6.03 (s, 2H), 3.86 (s, 3H), 3.84 (s, 3H), 3.83 (s,
146.86, 141.51, 141.04, 138.85, 138.17, 137.55, 128.28, 127.96, 127.73,
123.99, 123.84, 123.10, 119.28, 118.78, 114.49, 114.08, 107.67, 60.87,
60.78, 55.74, 55.38; HRMS calcd for C32H32O9 [M ꢃ H]ꢃ: 559.1968,
found 559.1977.
3H), 3.79 (s, 3H), 3.73 (s, 6H), 3.69 (s, 3H); 13C NMR (100 MHz,
CDCl3)
d 195.10, 189.57, 163.73, 159.68, 152.96, 152.92, 148.63,
148.30, 138.40, 137.96, 137.33, 134.92, 129.05, 128.82, 128.30, 125.25,
124.03, 124.00, 119.50, 118.94, 113.84, 113.73, 105.64, 90.27, 61.09,
60.96, 55.68, 55.65, 55.27, 54.82; HRMS calcd for C33H34O9
[M þ Na]þ: 597.2101, found 597.2105.
4.3.10. (1E,6E)-1,7-Bis(2,3-dimethoxyphenyl)-4-(2,3,4-
trimethoxybenzylidene)hepta-1,6-diene-3,5-dione (14)
Yellow powder, yield 75.38%; HPLC tR ¼ 22.76 min; 1H NMR
4.3.6. (1E,6E)-4-(2,3-Dimethoxybenzylidene)-1,7-bis(2,3-
dimethoxyphenyl)hepta-1,6-diene-3,5-dione (10)
(400 MHz, CDCl3)
d
8.15 (s, 1H), 8.06 (d, J ¼ 15.8 Hz, 1H), 7.83 (d,
J ¼ 16.5 Hz, 1H), 7.26 (d, J ¼ 15.7 Hz, 1H), 7.19 (dd, J ¼ 7.9, 1.3 Hz,
1H), 7.14 (d, J ¼ 8.8 Hz, 1H), 7.09 (dd, J ¼ 8.0, 1.3 Hz, 1H), 7.05 (t,
J ¼ 8.0 Hz, 1H), 7.01 (t, J ¼ 8.0 Hz, 1H), 6.94 (dd, J ¼ 8.1, 1.3 Hz, 1H),
6.91 (dd, J ¼ 8.1, 1.4 Hz, 1H), 6.88 (d, J ¼ 16.5 Hz, 1H), 6.56 (d,
J ¼ 8.9 Hz, 1H), 3.96 (s, 3H), 3.87 (s, 3H), 3.85 (s, 3H), 3.82 (s, 9H),
Yellow oil, yield 46.46%; HPLC tR ¼ 23.12 min; 1H NMR
(400 MHz, CDCl3)
d
8.18 (s, 1H), 8.08 (d, J ¼ 15.8 Hz, 1H), 7.81 (d,
J ¼ 16.5 Hz, 1H), 7.25 (d, J ¼ 15.8 Hz, 1H), 7.19 (dd, J ¼ 7.9, 1.3 Hz, 1H),
7.07 (dd, J ¼ 7.9, 1.5 Hz, 1H), 7.05 (t, J ¼ 8.0 Hz, 1H), 7.01 (t, J ¼ 8.0 Hz,
1H), 6.98 (dd, J ¼ 7.7, 1.8 Hz,1H), 6.94 (dd, J ¼ 8.2, 1.6 Hz, 1H), 6.93 (t,
J ¼ 8.8 Hz, 1H), 6.90 (dd, J ¼ 7.9, 1.8 Hz, 1H), 6.89 (dd, J ¼ 8.0, 1.8 Hz,
1H), 6.86 (d, J ¼ 16.4 Hz, 1H), 3.91 (s, 3H), 3.87 (s, 3H), 3.85 (s, 3H),
3.83 (s, 3H), 3.83 (s, 3H), 3.64 (s, 3H); 13C NMR (100 MHz, CDCl3)
3.65 (s, 3H); 13C NMR (100 MHz, CDCl3)
d 197.98, 187.61, 155.66,
153.00, 152.74, 152.61, 148.50, 148.20, 141.62, 140.52, 138.88,
138.84, 135.89, 128.32, 128.16, 127.89, 125.22, 123.92, 123.87,
123.43, 120.08, 119.41, 118.77, 114.28, 114.04, 107.07, 61.25, 60.78,
60.70, 60.37, 55.54, 55.41, 55.37; HRMS calcd for C33H34O9
[M þ Na]þ: 597.2101, found 597.2093.
d
197.97, 188.20, 153.22, 153.08, 152.78, 149.10, 148.78, 148.54,
141.33, 141.22, 139.82, 136.47, 128.92, 128.83, 128.53, 128.13, 124.23,
124.11, 123.95, 122.05, 120.01, 119.45, 114.58, 114.43, 61.45, 61.32,
61.31, 55.94, 55.91, 55.89; HRMS calcd for C32H32O8 [M þ Na]þ:
567.1995, found 567.1988.
4.3.11. (1E,6E)-1,7-Bis(2,3-dimethoxyphenyl)-4-(3-hydroxy-4-
methoxybenzylidene)hepta-1,6-diene-3,5-dione (15)
Yellow powder, yield 79.68%; HPLC tR ¼ 20.92 min; 1H NMR
4.3.7. (1E,6E)-1,7-Bis(2,3-dimethoxyphenyl)-4-(3-fluoro-4-
hydroxybenzylidene)hepta-1,6-diene-3,5-dione (11)
(400 MHz, CDCl3)
d
8.06 (d, J ¼ 15.7 Hz, 1H), 7.87 (d, J ¼ 16.5 Hz, 1H),
7.82 (s, 1H), 7.20 (d, J ¼ 15.7 Hz, 1H), 7.18 (dd, J ¼ 7.9, 1.1 Hz, 1H), 7.12
(dd, J ¼ 8.0, 1.2 Hz, 1H), 7.08e7.00 (m, 4H), 6.93 (d, J ¼ 16.4 Hz, 1H),
6.97e6.89 (m, 2H), 6.77 (d, J ¼ 8.3 Hz, 1H), 5.60 (s, 1H), 3.87 (s, 6H),
3.84 (s, 3H), 3.82 (s, 3H), 3.62 (s, 3H); 13C NMR (100 MHz, CDCl3)
Yellow powder, yield 83.60%; HPLC tR ¼ 21.29 min; 1H NMR
(400 MHz, CDCl3)
d
8.07 (d, J ¼ 15.7 Hz, 1H), 7.88 (d, J ¼ 16.5 Hz,
1H), 7.77 (s, 1H), 7.23 (dd, J ¼ 11.5, 2.0 Hz, 1H), 7.18 (dd, J ¼ 8.4,
2.0 Hz, 1H), 7.17 (d, J ¼ 15.7 Hz, 1H), 7.17 (dd, J ¼ 7.8, 1.2 Hz, 1H),
7.13 (dd, J ¼ 8.0, 1.4 Hz, 1H), 7.05 (t, J ¼ 8.0 Hz, 1H), 7.03 (t,
J ¼ 8.0 Hz, 1H), 6.94 (d, J ¼ 16.5 Hz, 1H), 6.94 (dd, J ¼ 7.7, 1.4 Hz,
1H), 6.93 (dd, J ¼ 8.0, 1.4 Hz, 1H), 6.93 (t, J ¼ 8.6 Hz, 1H), 5.99 (s,
1H), 3.86 (s, 3H), 3.84 (s, 3H), 3.83 (s, 3H), 3.64 (s, 3H); 13C NMR
d
198.99, 187.42, 153.08, 152.99, 148.91, 148.90, 148.68, 145.71,
141.75, 141.37, 139.34, 138.54, 128.82, 128.65, 128.39, 126.65, 124.25,
124.16, 124.09, 123.68, 119.76, 119.32, 116.30, 114.64, 114.32, 110.64,
61.39, 61.25, 55.84, 55.83, 55.81; HRMS calcd for C31H30O8
[M ꢃ H]ꢃ: 529.1862, found 529.1862.
(100 MHz, DMSO)
d 198.22, 188.14, 152.87, 152.77, 151.91, 149.50,
148.36, 148.01, 147.81, 147.69, 140.34, 140.07, 138.65, 137.68, 128.16,
128.13, 128.02, 128.00, 127.57, 124.80, 124.74, 124.66, 124.39,
122.56, 119.23, 119.12, 118.21, 118.17, 118.07, 117.88, 115.55, 115.16,
61.00, 60.95, 55.86; HRMS calcd for C30H27O7F [M ꢃ H]ꢃ: 517.1663,
found 517.1660.
4.3.12. (1E,6E)-1,7-Bis(2,5-dimethoxyphenyl)-4-(4-hydroxy-3-
methoxybenzylidene)hepta-1,6-diene-3,5-dione (16)
Yellow powder, yield 89.20%; HPLC tR ¼ 21.88 min; 1H NMR
(400 MHz, CDCl3)
d
8.06 (d, J ¼ 15.7 Hz, 1H), 7.88 (d, J ¼ 16.4 Hz, 1H),
7.81 (s, 1H), 7.19 (d, J ¼ 15.7 Hz, 1H), 7.08 (dd, J ¼ 8.1, 1.9 Hz, 1H), 7.08
(d, J ¼ 3.1 Hz, 1H), 7.03 (d, J ¼ 2.0 Hz, 1H), 6.98 (d, J ¼ 16.3 Hz, 1H),
6.98 (d, J ¼ 3.0 Hz, 1H), 6.91 (dd, J ¼ 9.0, 3.1 Hz, 1H), 6.90 (dd, J ¼ 9.0,
3.1 Hz, 1H), 6.87 (d, J ¼ 8.2 Hz, 1H), 6.83 (d, J ¼ 9.0 Hz, 1H), 6.79 (d,
J ¼ 9.0 Hz, 1H), 5.89 (s, 1H), 3.82 (s, 6H), 3.78 (s, 3H), 3.75 (s, 3H),
4.3.8. (1E,6E)-4-(3-Bromo-4-hydroxy-5-methoxybenzylidene)-1,7-
bis(2,3-dimethoxyphenyl)hepta-1,6-diene-3,5-dione (12)
Yellow powder, yield 88.65%; HPLC tR ¼ 23.79 min; 1H NMR
(400 MHz, CDCl3)
d
8.07 (d, J ¼ 15.7 Hz, 1H), 7.88 (d, J ¼ 16.4 Hz, 1H),
3.74 (s, 3H); 13C NMR (100 MHz, DMSO)
d 198.59, 188.06, 153.26,
7.75 (s, 1H), 7.30 (d, J ¼ 1.8 Hz, 1H), 7.20 (d, J ¼ 15.7 Hz, 1H), 7.18 (dd,
J ¼ 7.9, 1.3 Hz, 1H), 7.11 (dd, J ¼ 7.9, 1.3 Hz, 1H), 7.04 (q, J ¼ 8.2 Hz,
2H), 6.97 (d, J ¼ 1.9 Hz, 1H), 6.96e6.91 (m, 2H), 6.93 (d, J ¼ 16.5 Hz,
1H), 6.16 (s, 1H), 3.87 (s, 3H), 3.85 (s, 3H), 3.84 (s, 3H), 3.82 (s, 3H),
152.68, 152.47, 149.65, 147.56, 141.10, 139.67, 138.12, 137.41, 127.76,
125.07, 124.55, 123.59, 122.78, 121.91, 118.79, 117.60, 115.86, 114.31,
113.23, 113.13, 112.98, 112.43, 56.16, 56.10, 55.68, 55.59, 55.48;
HRMS calcd for C31H30O8 [M ꢃ H]ꢃ: 529.1862, found 529.1870.
3.66 (s, 3H); 13C NMR (100 MHz, CDCl3)
d 198.64, 187.29, 153.24,
153.17, 149.15, 148.92, 147.16, 145.40, 142.16, 139.96, 139.85, 139.72,
128.88, 128.44, 128.33, 128.32, 126.59, 124.41, 124.26, 123.66,
120.02, 119.47, 114.95, 114.52, 111.23, 108.55, 61.50, 61.39, 56.43,
55.97; HRMS calcd for C31H29O8Br [M ꢃ H]ꢃ: 607.0968, found
607.0975.
4.3.13. (1E,6E)-4-(3,4-Dimethoxybenzylidene)-1,7-bis(2,5-
dimethoxyphenyl)hepta-1,6-diene-3,5-dione (17)
Yellow powder, yield 90.55%; HPLC tR ¼ 24.38 min; 1H NMR
(400 MHz, CDCl3)
d
8.07 (d, J ¼ 15.7 Hz, 1H), 7.88 (d, J ¼ 16.3 Hz, 1H),
7.82 (s,1H), 7.19 (d, J ¼ 15.7 Hz,1H), 7.12 (dd, J ¼ 8.5, 2.1 Hz,1H), 7.08
(d, J ¼ 3.0 Hz, 1H), 7.04 (d, J ¼ 2.0 Hz, 1H), 6.99 (d, J ¼ 16.3 Hz, 1H),
6.98 (d, J ¼ 3.1 Hz, 1H), 6.91 (dd, J ¼ 9.0, 3.1 Hz, 1H), 6.90 (dd, J ¼ 8.9,
3.1 Hz, 1H), 6.83 (d, J ¼ 9.0 Hz, 1H), 6.82 (d, J ¼ 8.4 Hz, 1H), 6.79 (d,
J ¼ 9.0 Hz, 1H), 3.87 (s, 3H), 3.82 (s, 3H), 3.81 (s, 3H), 3.78 (s, 3H),
4.3.9. (1E,6E)-1,7-Bis(2,3-dimethoxyphenyl)-4-(4-hydroxy-3,5-
dimethoxybenzylidene)hepta-1,6-diene-3,5-dione (13)
Yellow powder, yield 83.72%; HPLC tR ¼ 20.53 min; 1H NMR
(400 MHz, CDCl3)
d
8.06 (d, J ¼ 15.7 Hz, 1H), 7.91 (d, J ¼ 16.4 Hz, 1H),
3.75 (s, 3H), 3.74 (s, 3H); 13C NMR (100 MHz, CDCl3)
d 199.05,
7.82 (s, 1H), 7.23 (d, J ¼ 15.7 Hz, 1H), 7.18 (dd, J ¼ 7.9, 1.2 Hz, 1H), 7.10
(dd, J ¼ 8.0,1.3 Hz, 1H), 7.05 (t, J ¼ 8.0 Hz,1H), 7.02 (t, J ¼ 8.0 Hz,1H),
6.94 (d, J ¼ 16.4 Hz, 1H), 6.97e6.91 (m, 2H), 6.77 (s, 2H), 5.78 (s, 1H),
3.87 (s, 3H), 3.85 (s, 3H), 3.83 (s, 9H), 3.64 (s, 3H); 13C NMR
187.42, 153.39, 153.39, 153.35, 153.19, 151.06, 148.75, 141.86, 140.99,
139.70, 138.94, 128.02, 126.32, 125.13, 124.35, 123.54, 123.19, 118.21,
117.20, 113.83, 113.16, 112.74, 112.53, 112.39, 110.97, 56.02, 55.98,
55.85, 55.76, 55.75, 55.70; HRMS calcd for C32H32O8 [M þ Na]þ:
567.1995, found 567.1988.
(100 MHz, CDCl3)
d 198.68, 187.13, 152.68, 152.60, 148.47, 148.26,

354
Y. Zuo et al. / European Journal of Medicinal Chemistry 55 (2012) 346e357
4.3.14. (1E,6E)-1,7-Bis(2,5-dimethoxyphenyl)-4-(3-fluoro-4-
hydroxybenzylidene)hepta-1,6-diene-3,5-dione (18)
7.78 (s, 1H), 7.49 (d, J ¼ 8.6 Hz, 1H), 7.40 (d, J ¼ 8.7 Hz, 1H), 7.10 (d,
J ¼ 15.6 Hz, 1H), 7.08 (dd, J ¼ 8.2, 2.1 Hz, 1H), 7.05 (d, J ¼ 1.9 Hz, 1H),
6.96 (d, J ¼ 16.2 Hz, 1H), 6.85 (d, J ¼ 8.2 Hz, 1H), 6.47 (td, J ¼ 8.2,
2.3 Hz, 2H), 6.42 (d, J ¼ 2.4 Hz, 1H), 6.38 (d, J ¼ 2.3 Hz, 1H), 5.87 (s,
1H), 3.83 (s, 3H), 3.83 (s, 3H), 3.81 (s, 6H), 3.78 (s, 3H); 13C NMR
Yellow powder, yield 86.79%; HPLC tR ¼ 22.18 min; 1H NMR
(400 MHz, CDCl3)
d
8.08 (d, J ¼ 15.7 Hz, 1H), 7.85 (d, J ¼ 16.4 Hz, 1H),
7.74 (s, 1H), 7.23 (dd, J ¼ 11.6, 2.1 Hz, 1H), 7.19 (dd, J ¼ 8.4, 2.0 Hz,
1H), 7.14 (d, J ¼ 15.7 Hz, 1H), 7.06 (d, J ¼ 3.0 Hz, 1H), 7.00 (d,
J ¼ 3.0 Hz, 1H), 6.98 (d, J ¼ 16.3 Hz, 1H), 6.93 (t, J ¼ 8.6 Hz, 1H), 6.91
(dd, J ¼ 9.0, 3.1 Hz, 1H), 6.90 (dd, J ¼ 9.0, 3.1 Hz, 1H), 6.83 (d,
J ¼ 9.0 Hz, 1H), 6.79 (d, J ¼ 9.0 Hz, 1H), 5.80 (s, 1H), 3.82 (s, 3H), 3.78
(s, 3H), 3.76 (s, 3H), 3.75 (s, 3H); 13C NMR (100 MHz, DMSO)
(100 MHz, CDCl3)
d 199.75, 187.45, 163.56, 163.11, 160.38, 160.29,
148.12, 146.64, 142.66, 140.41, 139.99, 138.78, 130.78, 125.95, 125.62,
120.61, 116.93, 116.14, 114.84, 112.56, 105.63, 105.46, 98.22, 98.20,
55.74, 55.41, 55.39; HRMS calcd for C31H30O8 [M ꢃ H]ꢃ: 529.1862,
found 529.1871.
d
198.27, 188.16, 153.30, 152.78, 152.57, 151.95, 149.55, 147.77, 147.65,
140.32, 139.69, 139.13, 137.90, 128.03, 128.01, 127.64, 124.91, 124.85,
123.57, 122.74, 121.76, 118.89, 118.18, 118.15, 118.06, 117.87, 117.67,
113.20, 113.15,112.90, 112.58, 56.10, 56.04, 55.63, 55.56; HRMS calcd
for C30H27O7F [M ꢃ H]ꢃ: 517.1663, found 517.1655.
4.3.19. (1E,6E)-1,7-Bis(2,4-dimethoxyphenyl)-4-(3-fluoro-4-
hydroxybenzylidene)hepta-1,6-diene-3,5-dione (23)
Yellow powder, yield 83.80%; HPLC tR ¼ 21.42 min; 1H NMR
(400 MHz, CDCl3)
d
8.06 (d, J ¼ 15.6 Hz, 1H), 7.80 (d, J ¼ 16.3 Hz, 1H),
7.71 (s, 1H), 7.48 (d, J ¼ 8.6 Hz, 1H), 7.42 (d, J ¼ 8.7 Hz, 1H), 7.23 (dd,
J ¼ 11.7, 2.0 Hz, 1H), 7.18 (dd, J ¼ 8.4, 1.9 Hz, 1H), 7.06 (d, J ¼ 15.5 Hz,
1H), 6.96 (d, J ¼ 16.3 Hz, 1H), 6.91 (t, J ¼ 8.7 Hz,1H), 6.48 (dd, J ¼ 8.6,
2.5 Hz, 1H), 6.47 (dd, J ¼ 8.7, 2.5 Hz, 1H), 6.41 (d, J ¼ 2.3 Hz, 1H), 6.37
(d, J ¼ 2.3 Hz, 1H), 6.06 (s, 1H), 3.83 (s, 6H), 3.81 (s, 3H), 3.78 (s, 3H);
4.3.15. (1E,6E)-4-(3-Bromo-4-hydroxy-5-methoxybenzylidene)-
1,7-bis(2,5-dimethoxyphenyl)hepta-1,6-diene-3,5-dione (19)
Yellow powder, yield 89.81%; HPLC tR ¼ 24.65 min; 1H NMR
(400 MHz, CDCl3)
d
8.07 (d, J ¼ 15.7 Hz, 1H), 7.85 (d, J ¼ 16.4 Hz, 1H),
7.71 (s, 1H), 7.29 (d, J ¼ 1.9 Hz, 1H), 7.17 (d, J ¼ 15.7 Hz, 1H), 7.07 (d,
J ¼ 3.0 Hz,1H), 6.99e6.96 (m, 2H), 6.95 (d, J ¼ 16.4 Hz, 1H), 6.92 (dd,
J ¼ 8.9, 2.8 Hz, 1H), 6.91 (dd, J ¼ 9.0, 3.0 Hz, 1H), 6.84 (d, J ¼ 9.0 Hz,
1H), 6.80 (d, J ¼ 9.0 Hz, 1H), 6.18 (s, 1H), 3.83 (s, 3H), 3.82 (s, 3H),
3.78 (s, 3H), 3.76 (s, 3H), 3.75 (s, 3H); 13C NMR (100 MHz, CDCl3)
13C NMR (100 MHz, CDCl3)
d 199.90, 187.77, 163.82, 163.33, 160.48,
160.40, 152.20, 149.79, 146.81, 146.68,144.02, 140.79, 139.49, 139.07,
131.03, 130.93, 127.99, 127.97, 125.86, 125.80, 125.18, 120.09, 117.93,
117.90, 117.64, 117.46, 116.65, 115.86, 105.72, 105.55, 98.11, 55.38,
55.36, 55.34, 55.33; HRMS calcd for C30H27O7F [M ꢃ H]ꢃ: 517.1663,
found 517.1670.
d
198.75, 187.45, 153.62, 153.59, 153.41, 147.13, 145.25, 142.53,
140.38, 140.10, 139.60, 128.46, 127.99, 126.87, 124.48, 123.66, 123.28,
118.62, 117.60, 114.09, 113.28, 112.73, 112.59, 111.22, 108.48, 56.49,
56.23, 55.99, 55.94; HRMS calcd for C31H29O8Br [M ꢃ H]ꢃ:
607.0968, found 607.0979.
4.3.20. (1E,6E)-4-(3-Bromo-4-hydroxy-5-methoxybenzylidene)-
1,7-bis(2,4-dimethoxyphenyl)hepta-1,6-diene-3,5-dione (24)
Yellow powder, yield 86.48%; HPLC tR ¼ 23.87 min; 1H NMR
(400 MHz, CDCl3)
d
8.05 (d, J ¼ 15.6 Hz, 1H), 7.80 (d, J ¼ 16.3 Hz, 1H),
4.3.16. (1E,6E)-1,7-Bis(2,5-dimethoxyphenyl)-4-(4-hydroxy-3,5-
dimethoxybenzylidene)hepta-1,6-diene-3,5-dione (20)
7.68 (s, 1H), 7.49 (d, J ¼ 8.6 Hz, 1H), 7.41 (d, J ¼ 8.7 Hz, 1H), 7.29 (d,
J ¼ 1.9 Hz, 1H), 7.08 (d, J ¼ 15.6 Hz, 1H), 7.01 (d, J ¼ 1.8 Hz, 1H), 6.94
(d, J ¼ 16.3 Hz, 1H), 6.48 (dd, J ¼ 8.6, 2.3 Hz, 1H), 6.47 (dd, J ¼ 8.6,
2.3 Hz, 1H), 6.42 (d, J ¼ 2.3 Hz, 1H), 6.38 (d, J ¼ 2.3 Hz, 1H), 6.12 (s,
1H), 3.84 (s, 3H), 3.83 (s, 3H), 3.82 (s, 3H), 3.81 (s, 3H), 3.79 (s, 3H);
Yellow powder, yield 88.68%; HPLC tR ¼ 21.55 min; 1H NMR
(400 MHz, CDCl3)
d
8.07 (d, J ¼ 15.7 Hz, 1H), 7.88 (d, J ¼ 16.3 Hz, 1H),
7.79 (s, 1H), 7.18 (d, J ¼ 15.7 Hz, 1H), 7.09 (d, J ¼ 3.0 Hz, 1H), 6.97 (d,
J ¼ 16.3 Hz, 1H), 6.97 (d, J ¼ 3.0 Hz, 1H), 6.91 (dd, J ¼ 9.0, 3.1 Hz, 1H),
6.90 (dd, J ¼ 9.0, 3.1 Hz, 1H), 6.84 (d, J ¼ 9.0 Hz, 1H), 6.79 (d,
J ¼ 9.0 Hz, 1H), 6.78 (s, 1H), 5.78 (s, 1H), 3.84 (s, 6H), 3.82 (s, 3H),
3.78 (s, 3H), 3.75 (s, 3H), 3.75 (s, 3H); 13C NMR (100 MHz, DMSO)
13C NMR (100 MHz, CDCl3)
d 199.17, 187.27, 163.76, 163.30, 160.58,
160.44, 147.09, 145.05, 143.07, 140.52, 140.36, 138.58, 131.08, 130.96,
128.29, 126.96, 125.58, 120.62, 117.04, 116.25, 111.20, 108.37, 105.72,
105.54, 98.38, 98.36, 56.36, 55.57, 55.55, 55.54; HRMS calcd for
C31H29O8Br [M ꢃ H]ꢃ: 607.0968, found 607.0963.
d
199.03, 187.40, 153.47, 153.44, 153.25, 147.04, 141.77, 141.44,
139.79, 139.13, 137.39, 128.03, 124.84, 124.44, 123.61, 123.27, 118.25,
117.28, 113.89, 113.26, 112.59, 112.47, 107.92, 56.28, 56.10, 56.04,
55.84, 55.78; HRMS calcd for C32H32O9 [M ꢃ H]ꢃ: 559.1968, found
559.1978.
4.3.21. (1E,6E)-1,7-Bis(2,4-dimethoxyphenyl)-4-(4-hydroxy-3,5-
dimethoxybenzylidene)hepta-1,6-diene-3,5-dione (25)
Yellow powder, yield 87.20%; HPLC tR ¼ 20.71 min; 1H NMR
(400 MHz, CDCl3)
d
8.06 (d, J ¼ 15.6 Hz, 1H), 7.84 (d, J ¼ 16.2 Hz, 1H),
4.3.17. (1E,6E)-1,7-Bis(2,5-dimethoxyphenyl)-4-(3-hydroxy-4-
methoxybenzylidene)hepta-1,6-diene-3,5-dione (21)
7.76 (s, 1H), 7.49 (d, J ¼ 8.6 Hz, 1H), 7.39 (d, J ¼ 8.7 Hz, 1H), 7.09 (d,
J ¼ 15.6 Hz, 1H), 6.96 (d, J ¼ 16.2 Hz, 1H), 6.80 (s, 2H), 6.47 (td,
J ¼ 8.6, 2.3 Hz, 2H), 6.42 (d, J ¼ 2.3 Hz, 1H), 6.38 (d, J ¼ 2.3 Hz, 1H),
5.73 (s, 1H), 3.84 (s, 3H), 3.83 (s, 9H), 3.81 (s, 3H), 3.79 (s, 3H); 13C
Yellow powder, yield 87.57%; HPLC tR ¼ 21.88 min; 1H NMR
(400 MHz, CDCl3)
d
8.06 (d, J ¼ 15.7 Hz, 1H), 7.85 (d, J ¼ 16.4 Hz, 1H),
7.78 (s, 1H), 7.17 (d, J ¼ 15.7 Hz, 1H), 7.07 (d, J ¼ 2.4 Hz, 2H), 7.05 (dd,
J ¼ 8.4, 2.1 Hz, 1H), 6.99 (d, J ¼ 3.1 Hz, 1H), 6.97 (d, J ¼ 16.5 Hz, 1H),
6.90 (dd, J ¼ 8.9, 3.1 Hz, 1H), 6.89 (dd, J ¼ 8.9, 3.1 Hz, 1H), 6.83 (d,
J ¼ 9.0 Hz, 1H), 6.78 (d, J ¼ 9.0 Hz, 1H), 6.78 (d, J ¼ 8.4 Hz, 1H), 5.60
(s, 1H), 3.87 (s, 3H), 3.82 (s, 3H), 3.78 (s, 3H), 3.75 (s, 3H), 3.74 (s,
NMR (100 MHz, CDCl3) d 199.44, 187.16, 163.52, 163.09, 160.37,
160.25, 146.92, 142.34, 140.33,139.94, 139.40, 137.09, 130.75, 130.73,
125.60, 124.95, 120.62, 116.92, 116.13, 107.81, 105.60, 105.43, 98.22,
98.18, 56.15, 55.41, 55.38; HRMS calcd for C32H32O9 [M ꢃ H]ꢃ:
559.1968, found 559.1969.
3H); 13C NMR (100 MHz, CDCl3)
d 199.05, 187.56, 153.32, 153.31,
153.11, 148.84, 145.66, 141.99, 141.08, 139.70, 138.81, 128.01, 126.71,
124.24, 123.98, 123.49, 123.08, 118.25, 117.24, 116.35, 113.77, 112.97,
112.53, 112.37, 110.62, 55.96, 55.78, 55.71, 55.64; HRMS calcd for
C31H30O8 [M ꢃ H]ꢃ: 529.1862, found 529.1872.
4.3.22. (1E,6E)-1,7-Bis(2,4-dimethoxyphenyl)-4-(3-hydroxy-4-
methoxybenzylidene)hepta-1,6-diene-3,5-dione (26)
Yellow powder, yield 83.19%; HPLC tR ¼ 20.31 min; 1H NMR
(400 MHz, CDCl3)
d
8.06 (d, J ¼ 15.6 Hz, 1H), 7.81 (d, J ¼ 16.3 Hz,
1H), 7.76 (s, 1H), 7.50 (d, J ¼ 8.7 Hz, 1H), 7.43 (d, J ¼ 8.7 Hz, 1H),
7.09 (d, J ¼ 15.5 Hz, 1H), 7.09 (d, J ¼ 2.2 Hz, 1H), 7.07 (dd, J ¼ 8.4,
2.2 Hz, 1H), 6.96 (d, J ¼ 16.3 Hz, 1H), 6.78 (d, J ¼ 8.4 Hz, 1H), 6.49
(dd, J ¼ 8.7, 2.3 Hz, 1H), 6.47 (dd, J ¼ 8.7, 2.3 Hz, 1H), 6.42 (d,
J ¼ 2.4 Hz, 1H), 6.38 (d, J ¼ 2.4 Hz, 1H), 5.61 (s, 1H), 3.88 (s, 3H),
4.3.18. (1E,6E)-1,7-Bis(2,4-dimethoxyphenyl)-4-(4-hydroxy-3-
methoxybenzylidene)hepta-1,6-diene-3,5-dione (22)
Yellow powder, yield 86.52%; HPLC tR ¼ 21.10 min; 1H NMR
(400 MHz, CDCl3)
d
8.05 (d, J ¼ 15.6 Hz, 1H), 7.83 (d, J ¼ 16.3 Hz, 1H),

Y. Zuo et al. / European Journal of Medicinal Chemistry 55 (2012) 346e357
355
3.84 (s, 3H), 3.84 (s, 3H), 3.82 (s, 3H), 3.79 (s, 3H); 13C NMR
(100 MHz, CDCl3) 199.54, 187.40, 163.50, 163.11, 160.38, 160.23,
148.63, 145.62, 142.72, 140.05, 139.96, 139.26, 130.77, 127.02,
125.76, 123.89, 120.60, 116.93, 116.37, 116.19, 110.60, 105.56, 105.46,
98.19, 98.16, 55.78, 55.41; HRMS calcd for C31H30O8 [M ꢃ H]ꢃ:
529.1862, found 529.1866.
7.4, supplemented with 10% foetal bovine serum, 100
m
g/ml strep-
d
tomycin and 100 units/ml penicillin) in a carbon dioxide incubator
(37 ꢀC, 5% CO2, 90% relative humidity).
4.5. In vitro cytotoxicity
Cytotoxicity study of human cancer cell lines of different
tissues was determined using 96-well tissue culture plates. Sul-
forhodamine B (SRB) assay [41,42] was performed to evaluate cell
viability and to obtain the IC50 values. It measures cellular protein
content to determine cell density. Compounds stock solutions
(10 mM) were serially diluted with DMSO, and then various
concentrations of compounds in DMSO were further diluted with
complete growth medium to obtain working test solutions of
required concentrations. The final DMSO concentration was 0.5%.
Cells were plated at 5000 cells/well. The cells were allowed to
grow in carbon dioxide incubator (37 ꢀC, 5% CO2, 90% RH) for
4.3.23. (1E,6E)-4-(4-Hydroxy-3-methoxybenzylidene)-1,7-
bis(2,4,6-trimethoxyphenyl)hepta-1,6-diene-3,5-dione (27)
Yellow powder, 81.83%; HPLC tR
(400 MHz, CDCl3)
7.74 (s, 1H), 7.48 (d, J ¼ 15.7 Hz, 1H), 7.33 (d, J ¼ 16.3 Hz, 1H), 7.10 (d,
J ¼ 2.0 Hz, 1H), 7.07 (dd, J ¼ 8.3, 2.0 Hz, 1H), 6.84 (d, J ¼ 8.2 Hz, 1H),
6.07 (s, 2H), 6.04 (s, 2H), 5.81 (s, 1H), 3.82 (s, 3H), 3.82 (s, 6H), 3.81
(s, 3H), 3.80 (s, 3H), 3.77 (s, 6H); 13C NMR (100 MHz, CDCl3)
¼
20.53 min; 1H NMR
8.20 (d, J ¼ 15.7 Hz,1H), 8.04 (d, J ¼ 16.3 Hz,1H),
d
d
201.38, 188.43, 163.63, 163.14, 161.58, 161.47, 147.86, 146.59,
139.56, 139.23, 138.63, 135.36, 127.37, 126.19, 125.48, 122.72, 114.64,
112.44,106.28,105.52, 90.26, 90.23, 55.58, 55.47, 55.45, 55.19, 55.17;
HRMS calcd for C33H34O10 [M ꢃ H]ꢃ: 589.2074, found 589.2067.
24 h. Then test compounds in complete growth medium (100 mL)
were added to the wells in triplicate. The plates were further
incubated for 48 h. The cell growth was stopped by gently
4.3.24. (1E,6E)-4-(3-Fluoro-4-hydroxybenzylidene)-1,7-bis(2,4,6-
trimethoxyphenyl)hepta-1,6-diene-3,5-dione (28)
layering trichloroacetic acid (50%, 50 mL) on top of the medium in
all the wells. The plates were incubated at 4 ꢀC for 1 h to fix the
cells attached to the bottom of the wells. The liquid of all the
wells was gently pipetted out and discarded. The plates were
washed five times with distilled water to remove trichloroacetic
acid, growth medium, low molecular weight metabolites, serum
proteins etc. and air-dried. The plates were stained with SRB dye
Yellow powder, 82.40%; HPLC tR
(400 MHz, CDCl3)
¼
20.88 min; 1H NMR
8.21 (d, J ¼ 15.7 Hz, 1H), 8.02 (d, J ¼ 16.4 Hz, 1H),
d
7.67 (s, 1H), 7.45 (d, J ¼ 15.7 Hz, 1H), 7.32 (d, J ¼ 16.4 Hz, 1H), 7.26
(dd, J ¼ 11.8, 2.1 Hz, 1H), 7.19 (dd, J ¼ 8.4, 2.0 Hz, 1H), 6.90 (t,
J ¼ 8.7 Hz, 1H), 6.06 (s, 2H), 6.03 (s, 2H), 3.82 (s, 3H), 3.81 (s, 9H),
3.78 (s, 6H); 13C NMR (100 MHz, CDCl3)
d
201.70, 188.80, 164.09,
(0.4% in 1% acetic acid, 100
washed five times with 1% acetic acid and then air-dried. The
adsorbed dye was dissolved in Tris base solution (150 L, 10 mM,
mL) for 30 min. The plates were
163.49, 161.85, 161.77, 152.24, 149.84, 146.63, 146.50, 140.28, 140.05,
138.47, 136.29, 127.91, 126.97, 126.36, 122.48, 117.80, 117.59, 117.41,
106.39,105.57, 90.34, 55.58, 55.30, 55.27; HRMS calcd for C32H31O9F
[M ꢃ H]ꢃ: 577.1874, found 577.1869.
m
pH 10.4) and plates were gently shaken for 1 h on an orbital
shaker. The optical density (OD) was recorded on a TECAN
infiniteÒ M200 pro multimode reader at 515 nm. The cell growth
was determined by subtracting mean OD value of respective
blank from the mean OD value of experimental set. Percent
growth in presence of test compounds was calculated consid-
ering the growth in absence of any test compounds as 100% and
in turn percent growth inhibition in presence of test compounds
was calculated. The data was fitted by using nonlinear regression
in GraphPad Prism 5, the IC50 values was obtained from the
doseeresponse curves. All data are obtained as average values
from triplicate samples, and the experiments were repeated at
least two times.
4.3.25. (1E,6E)-4-(3-Bromo-4-hydroxy-5-methoxybenzylidene)-
1,7-bis(2,4,6-trimethoxyphenyl)hepta-1,6-diene-3,5-dione (29)
Yellow powder, 76.57%; HPLC tR
(400 MHz, CDCl3)
7.65 (s, 1H), 7.46 (d, J ¼ 15.7 Hz, 1H), 7.29 (d, J ¼ 16.4 Hz, 1H), 7.28 (d,
J ¼ 1.9 Hz, 1H), 7.06 (d, J ¼ 1.9 Hz, 1H), 6.07 (s, 2H), 6.04 (s, 2H), 3.82
(s, 9H), 3.82 (s, 3H), 3.80 (s, 3H), 3.78 (s, 6H); 13C NMR (100 MHz,
¼
23.22 min; 1H NMR
8.22 (d, J ¼ 15.7 Hz,1H), 8.00 (d, J ¼ 16.4 Hz,1H),
d
DMSO)
d 199.19, 187.58, 163.89, 163.50, 161.40, 161.25, 147.98,
145.75, 140.65, 137.19, 136.76, 134.67, 126.98, 126.41, 125.83, 121.57,
112.83, 109.40, 105.09, 104.45, 91.14, 91.06, 56.08, 56.05, 55.97,
55.61, 55.58; HRMS calcd for C33H33O10Br [M ꢃ H]ꢃ: 667.1179,
found 667.1175.
4.6. High content screening based NF-
kB translocation assay
4.3.26. (1E,6E)-4-(4-Hydroxy-3,5-dimethoxybenzylidene)-1,7-
bis(2,4,6-trimethoxyphenyl)hepta-1,6-diene-3,5-dione (30)
Yellow powder, 79.28 HPLC tR ¼ 20.17 min; 1H NMR (400 MHz,
CellomicsÒ NF-
kB Activation HCS Reagent Kit (Thermo Scien-
tific) was used and the enclosed experimental protocol was fol-
lowed. A549 cells were plated in 384-well plates (PerkinElmer,
CDCl3)
d
8.21 (d, J ¼ 15.7 Hz, 1H), 8.04 (d, J ¼ 16.3 Hz, 1H), 7.72 (s,
Packard ViewPlateÒ, Product No. 6007460) at 2000 cells/25
mL/well
1H), 7.46 (d, J ¼ 15.7 Hz, 1H), 7.32 (d, J ¼ 16.3 Hz, 1H), 6.82 (s, 2H),
and grown for 24 h. Test compounds in DMSO were diluted with
complete growth medium to obtain working solutions. Then test
6.06 (s, 2H), 6.04 (s, 2H), 5.70 (s, 1H), 3.82 (s, 6H), 3.82 (s, 3H), 3.82
(s, 3H), 3.81 (s, 6H), 3.77 (s, 6H); 13C NMR (100 MHz, CDCl3)
d
201.12,
compounds in complete growth medium (25
wells in duplicate. The final DMSO concentration was 0.5%. The
plates were incubated at 37 ꢀC for 30 min. TNF
(10 L) was added
(10 ng/mL, final, SigmaeAldrich, St.Louis, MO) to cells to stimulate
NF- B translocation for 30 min. Cells were then fixed with pre-
warmed (37 ꢀC) paraformaldehyde (25
L, 4%, Thermo Scientific,
mL) were added to the
188.21, 163.63, 163.17, 161.71, 161.56, 146.82, 140.40, 139.22, 138.29,
136.69, 135.38, 127.63, 125.41, 123.02, 107.78, 106.51, 105.76, 90.39,
90.35, 56.14, 55.60, 55.58, 55.33, 55.29; HRMS calcd for C34H36O11
[M ꢃ H]ꢃ: 619.2179, found 619.2169.
a
m
k
m
4.4. Cell culture
Product No. 28906) for 10 min. Then cells were permeabilized with
permeabilization buffer (Triton X-100) for 10 min, washed twice
Cells (human lung carcinoma cell line, A549; human nasopha-
ryngeal carcinoma cell line, CNE2; human colon cell line, SW480;
human breast adenocarcinoma cell line, MCF-7; human hepatoma
cell line, HepG2.) were grown in tissue culture flasks in complete
growth medium (RPMI-1640 medium with 2 mM glutamine, pH
with PBS. Rabbit anti-p65 NF-kB antibody was added and incubated
at room temperature for 1 h. Cells were washed three times and
incubated with DyLightÔ 488-conjugated goat anti-rabbit IgG
(stain NF-
k
B) along with Hoechst 33342 (stain nucleus) at room
temperature for 1 h. After washing, 50
mL PBS was added and the

356
Y. Zuo et al. / European Journal of Medicinal Chemistry 55 (2012) 346e357
plates were evaluated on ArrayScan HCS Reader (DyLight 488
conjugates: excitation at 494 nm, emission at 532 nm, Hoechst Dye:
excitation at 350 nm, emission at 461 nm). The ArrayScan HCS
Reader and the Cytoplasm to Nucleus Translocation BioApplication
software were used to plate handing, focussing, cell image acqui-
4.9. Molecular docking
Small molecular preparation. The 3D structure of ATP was
extracted from the crystal structure of phosphorylase kinase (PDB
code: 1PHK), considered as energy minimized. The geometry of
compound 6 was energy minimized using density functional
theory. The calculation was performed with the B3LYP hybrid
functional and the 6-31G basis set implemented in Gaussian 03,
revision E.01, software package [46]. The 3D structures of other
4-arylidene curcumin analogues were constructed based on the
energy minimized 3D structure of compound 6, and further energy
minimized using Gaussian 03.
Docking analysis. Molecular docking studies of compounds
with the ATP binding pocket were performed with SurflexeDock in
Sybyl 7.3.5 (Tripos Inc.). The ligand in crystal structure of Akt1-
inhibitor complex (PDB code: 3MV5) was chosen as a reference to
construct the protomol. Threshold is 0.5, bloat is 0, additional
starting conformation per molecular is set to 5, other parameters
are set as default.
sition, analysis, and quantification of NF-
NF- B translocation were calculated and expressed as the differ-
ence between average fluorescence intensity in the nucleus and in
the cytoplasm. After stimulation with TNF , the inhibitory effect of
test compounds on TNF induced NF- translocation was
expressed as a percentage of fluorescence intensity difference (in
nucleus and in cytoplasm) in control wells (TNF only) after sub-
tracting background (no TNF ). The IC50 of test compounds in this
NF- translocation assay stands for the concentration of
kB activation. The levels of
k
a
a
kB
a
a
kB
a compound required to induce 50% inhibition. All data are average
values from duplicate samples, and the experiments were repeated
at least twice.
4.7. Akt kinase inhibition assay
Acknowledgements
AKT1/PKBa KinEASEÔ FP Fluorescein Green Assay kit for fluo-
rescence polarization experiments and Akt1 enzyme were
purchased from Upstate, Millipore Corporation (Charlottesville,
VA). The enclosed experimental protocol of the KinEASEÔ kit was
We are indebted to the National High-tech R&D 863 Program
(No. 2008AA02Z304), National Program on Key Basic Research
Project 973 Program (No.2011CB935800), National Nature Science
Foundation of China (No. 30973619 & 81172931) and the Funda-
mental Research Funds for the Central Universities of China
(09ykpy66) for financial support of this study.
followed. Total reaction volume per well was 25.0 mL. In Corning
Costar 384-well black plates, various concentrations of compounds
in DMSO was diluted with buffer containing 50 mM HEPES (pH 7.2),
0.01% BSA, 5 mM MgCl2, 1 mM DTT. STK Substrate 3 (final
concentration 10
70% activity) were added to each well and incubated for 10 min at
25 ꢀC, ATP (final concentration 100
M) was added to start the
reactions. After 1 h incubation at 25 ꢀC, the reactions were
quenched with 5 L STK stop mix including the phosphorylated STK
tracer, 5
mM) and Akt1(concentration needed to achieve
Appendix A. Supplementary material
m
Supplementary material associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/j.ejmech.
2012.07.039.
m
m
L STK antibody mix were then added and the mixture
were incubated for 6 h at 25 ꢀC before reading. Fluorescence
polarization were recorded by using a TECAN infiniteÒ M1000
multimode reader at 25 ꢀC, excitation: 470 nm, emission: 530 nm,
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