Synthesis and identification of new 4-arylidene curcumin analogues as potential anticancer agents targeting nuclear factor-κB signaling pathway

Article abstract of DOI:10.1021/jm1004545

A series of curcumin analogues including new 4-arylidene curcumin analogues (4-arylidene-1,7-bisarylhepta-1,6-diene-3,5-diones) were synthesized. Cell growth inhibition assays revealed that most 4-arylidene curcumin analogues can effectively decrease the growth of a panel of lung cancer cells at submicromolar and low micromolar concentrations. High content analysis technology coupled with biochemical studies showed that this new class of 4-arylidene curcumin analogues exhibits significantly improved NF-κB inhibition activity over the parent compound curcumin, at least in part by inhibiting IκB phosphorylation and degradation via IKK blockage; selected 4-arylidene curcumin analogues also reduced the tumorigenic potential of cancer cells in a clonogenic assay.

Full text of DOI:10.1021/jm1004545

8260 J. Med. Chem. 2010, 53, 8260–8273
DOI: 10.1021/jm1004545
Synthesis and Identification of New 4-Arylidene Curcumin Analogues as Potential Anticancer Agents
Targeting Nuclear Factor-KB Signaling Pathway
Xu Qiu,†,§ Yuhong Du,‡,§ Bin Lou,‡ Yinglin Zuo,† Weiyan Shao,† Yingpeng Huo,† Jianing Huang,† Yanjun Yu,† Binhua Zhou,†
Jun Du,† Haian Fu,*,‡ and Xianzhang Bu*,†
†School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510275, China, and ‡Department of Pharmacology and
Emory Chemical Biology Discovery Center, Emory University School of Medicine, 1510 Clifton Road, Atlanta, Georgia 30322, United States.
§ These authors contributed equally to this work.
Received April 13, 2010
A series of curcumin analogues including new 4-arylidene curcumin analogues (4-arylidene-1,7-
bisarylhepta-1,6-diene-3,5-diones) were synthesized. Cell growth inhibition assays revealed that most
4-arylidene curcumin analogues can effectively decrease the growth of a panel of lung cancer cells at sub-
micromolar and low micromolar concentrations. High content analysis technology coupled with bio-
chemical studies showed that this new class of 4-arylidene curcumin analogues exhibits significantly
improved NF-κB inhibition activity over the parent compound curcumin, at least in part by inhibiting
IκB phosphorylation and degradation via IKK blockage; selected 4-arylidene curcumin analogues also
reduced the tumorigenic potential of cancer cells in a clonogenic assay.
Introduction
proteasome-mediated processing of p100 to generate p52, re-
sulting in the production of an active p52/RelB dimer and its
translocation to the nucleus.
Nuclear factor-κB (NF-κBa) proteins are a family of struc-
turally related eukaryotic transcription factors with a con-
served reticuloendotheliosis (Rel) domain.1 They influence
gene expression events that impact a large number of physio-
logical processesincludingimmuneresponse, cellsurvival, dif-
ferentiation, and proliferation.2 Before cell stimulation, NF-
κB dimers are inactive in the cytoplasm and mainly regulated
by two distinct signaling pathways.3 The canonical NF-κB acti-
vation pathway has been extensively studied. Prior to activa-
tion, NF-κB dimers composed of RelA (also known as p65),
c-REL, and p50 are retained in the cytoplasm by inhibitoryκB
(IκB) proteins. Various extracellular signals, such as those
from microbial infections, viral infections, and proinflamma-
tory cytokines, activate the IκB kinase (IKK) complex. The
IKK complex targets NF-κB-bound IκBs by phosphorylating
two conserved serine residues, which drives ubiquitin-medi-
ated degradation of IκB and leads to the liberation and subse-
quent nuclear translocation of released NF-κB dimers. Phos-
phorylation of IκB mainly depends on the IKKβ catalytic
subunit of the IKK complex in the canonical pathway. In the
second pathway of NF-κB activation, NF-κB, typically exists
as an RelB/p100 dimer and remains inactivated by p100
instead of IκB. After stimulation by certain signals, such as the
tumor-necrosis factor (TNF) cytokine family member lym-
photoxin B (LTβ), an IKKR homodimer, along with NF-κB
inducing kinase (NIK), induces the phosphorylation and
Recent advances have revealed key roles of NF-κB signal-
ing inpathologicalconditions such asoncogenesis and inflam-
mation. NF-κB is commonly overexpressed and constitutively
activated in different types of hematologic cancers and solid
tumors.4-7 Constitutive activation of NF-κB promotes tumor
proliferation, invasion, and metastasis;8,9 moreover, NF-κB
activation allows malignant cells to escape apoptosis and there-
fore contributes to radiation and chemotherapy resistance in
cancer cells.10-12 Noticeably, many chemotherapeutic agents
have been found to activate NF-κB via various approaches,
leading to chemoresistance and subsequent failure of chemo-
therapy.13,14 Accumulating evidence suggests that inhibition
of NF-κB activation can prevent tumor resistance to chemo-
therapeutic agents, shift the death-survival balance toward
apoptosis, and improve the efficacy of current chemothera-
peutic regimens.15,16
A large number of compounds have been reported to inhibit
NF-κB by interacting with key macromolecules in the signal-
ing pathway. Many natural dietary agents, such as soya iso-
flavone,17 resveratrol,18 and curcumin,19 have been found to
inhibit NF-κB and induce apoptosis in tumor cells. Curcumin
(diferuloylmethane), a yellow spice and pigment isolated from
the rhizome of Curcuma longa, has been used as a spice in
South Asia for centuries. It has attracted much attention
because of its broad range of bioactivities,20 including anti-
oxidative, anti-inflammatory, and anticancer activities, as
well as its pharmacological safety and potency as chemopre-
ventive agent.21 One of the predominant targets of curcumin is
the NF-κB cell signaling pathway.22 Curcumin directly inhib-
its IKK and the 26S proteasome23,24 to block NF-κB acti-
vation. Unfortunately, the clinical potential of curcumin
remains limited because of its relatively low potency and poor
*To whom correspondence should be addressed. For H.F.: phone,
404-727-0365; fax, 404-727-0365; e-mail: hfu@emory.edu. For X.B.:
phone and fax, 020-39942054; e-mail, phsbxzh@mail.sysu.edu.cn.
a Abbreviations: NF-κB, nuclear factor-κB; Rel, reticuloendothelio-
sis; IκB, inhibitor of κB; IKK, inhibitor of κB kinase; NIK, NF-κB
inducing kinase; TNF, tumor-necrosis factor; LTβ, lymphotoxin B;
GST, glutathione S-transferase; TCA, trichloroacetic acid; SRB, sul-
forhodamine B.
r
pubs.acs.org/jmc
Published on Web 11/11/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23 8261
Figure 1. Structures of 1,3-diketones curcumin analogues (1-6), monoketone curcumin analogues (7-10), 4-arylidene crucumin analogues
(13-37), and 4-hydroxymethylene curcumin analogues (38-40).
bioavailability.25 Attempts have been made by others to chem-
ically modify curcumin in order to increase its activity against
cancer and NF-κB.26-31 However, most of these investiga-
tions have focused on design, synthesis, and activity of 1,3-
diketones and monoketone analogues of curcumin. However,
curcuminoids, such as Knoevenagel condensates of curcumin
analogues, contain a novel skeleton and remain quite less in-
vestigated.28,32,33 In 2006, Ajit P. Zambre et al. reported that
copper(II) conjugates of Knoevenagel condensates of curcu-
min showed potential in inhibiting TNFR induced NF-κB
activation.28 Nevertheless, highly active and clinically promis-
ing curcuminoids remain to be developed. A systematic in-
vestigation of current curcumin analogues would greatly
facilitate the development of new curcumin analogues for
therapeutic interventions. In the presentstudy, wedescribe the
synthesis and identification of new 4-arylidene curcumin
analogues (Knoevenagel condensates of 1,3-diketones curcu-
min analogues with aromatic aldehydes) as a new class of
potential anticancer agents. Screening of the synthesized
compounds with high content analysis technology, coupled
with biochemical studies, revealed that 4-arylidene curcumin
analogues have significantly improved IKK/NF-κB inhibi-
tion activity and elevated cytotoxicity over the parent com-
pound curcumin. These compounds effectively decreased
growth and reduced the tumorigenic potential of cancer cells,
as seen in a clonogenic assay.
Results and Discussion
Chemistry. To search for potent analogues with favorable
medicinal properties, we extended the molecular diversity of
curcuminiods by designing three types of curcuminoids: 1,3-
diketones curcumin analogues, monoketone curcumin ana-
logues, and 4-arylidene curcumin analogues (4-arylidene-1,
7-bisarylhepta-1,6-diene-3,5-diones) (Figure 1).
Classical 1,3-diketones (1-6) including curcumin (1) and a
representative 1,3-diketone derivative with known activity
(3)34 were synthesized using a previously reported proce-
dure35 with slight modification (Scheme 1). Briefly, to pro-
tect the C-3 of acetylacetone from an unwanted Knoevenagel
reaction, a boric acetylacetone anhydride complex was pre-
pared first by refluxing acetylacetone with boric anhydride
in EtOAc. The final products 1-6 were synthesized by aldol

8262 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23
Qiu et al.
condensation of protected acetylacetone aromatic aldehydes
as described.
(38-40, Figure 1), which possess absolute diketone moieties,
were designed as references. 38, 39, and 40 were synthesized
by refluxing triethoxymethane and Ac2O with 1, 2, and 3,
respectively (Scheme 3).
Monoketone curcumin analogues have been extensively
investigated because of their potential anticancer activities.
7 has been reported to have high activity in antitumor assays36
and was therefore considered; 9 and 10 are asymmetric an-
alogues of 7. These three compounds were included in our
work to represent monoketone curcumin analogues. 7 and
the intermediate 11 were synthesized by treating 3,4,5-tri-
methoxybenzaldehyde with excess acetone in the presence of
KOH. 9 and 10 were synthesized by condensing 11 with
veratraldehyde and 3-methylthiophene-2-carbaldehyde, re-
spectively. Furthermore, another new compound, 8, was pre-
pared by condensing 12 and 3,4,5-trimethoxybenzaldehyde.
The intermediate 12 was obtained using the same conditions
as in the synthesis of 1-6 except the excess protected acet-
ylacetone (Scheme 2).
Compared to the large number of traditional 1,3-dike-
tones and monoketone curcumin analogues that have been
designed and evaluated, investigation of Knoevenagel con-
densates of curcuminoids remains scarce. In our current
work, 23 new 4-arylidene curcumin analogues (Knoevenagel
condensates 13-17, 19-28, 30-37) were designed and syn-
thesized by coupling 1-4 with different aromatic aldehydes
in toluene with AcOH and piperidine as a catalyst. The known
4-arylidene curcumin analogues 18 and 29 were synthesized
under the same conditions except with acetylacetone and
aldehydes as reactants (Scheme 1).
New 4-Arylidene Crucumin Analogues Exhibit Potent In-
hibitory Activity against Caner Cells. In order to evaluate the
anticancer activities of new curcumin derivatives, we tested
the effects of synthesized compounds on the growth of A549
lung adenocarcinoma cells. For comparison, dose-response
experiments were carried out to obtain GI50 values for each
compound as previously described,37 with curcumin (1) as a
control. As summarized in Table 1, all the traditional 1,3-
diketone cucumin analogues (1-6) showed moderate to
poor activity against A549 growth, while improved antipro-
liferation activities of monoketone analogues 7 and 9 were
observed (GI50 of 0.53 and 1.21 μM respectively).
At the same time, the thiophene ring moiety bearing 10
(GI50 = 9.31 μM) showed decreased activity compared to
7 and 9, indicating that S replacement of the O atom in the
heterocycle of 9 made a negative contribution to its activity.
As a regioisomer of 4, the Knoevenagel condensate 8 ex-
hibited much higher activity (GI50=0.53 μM) than 4. 8 can
also be regarded as a monoketone curumin analogue with an
acetyl substitution. When the activities of 7-9 and 1-6 were
compared, it was revealed that the spacing of the two aro-
matic rings most likely influences the activity of curcumin
analogues against A549 growth. The close activities of 8
and 7 indicated that the acetyl group has no apparent effect
on activity against A549 growth.
The 4-arylidene modification can induce a change of a
partial enolic diketone, which is universal in 1,3-diketones
curcumin analogues, to an absolute diketone moiety, possi-
bly affecting bioactivity. To explore the effect of this transi-
tion, three new 4-hydroxymethylene curcumin analogues
Remarkably, most of the 4-arylidene curcumin analogues
(4-arylidene-1,7-bisarylhepta-1,6-diene-3, 5-diones, 13-37)
showed potent anticancer activities with a GI50 in the sub-
micromolar range (0.23-0.93 μM), except 15, 19, 29, and 37
which exhibited slightly lower activities (GI50 of 1.12-
2.62 μM). The potency of 4-arylidene analogues was improved
by 10- to 60-fold over the parent compound curcumin in this
assay. 4-Hydroxymethylene curcumin analogues (38-40),
however, showed poor activity, suggesting the importance of
Scheme 1. Synthesis of Compounds 1-6 and 13-37a
Scheme 3. Synthesis of Compounds 38-40a
a Reagents and conditions: (a) B2O3, B(n-BuO)3, n-BuNH2; (b) HCl;
(c) piperidine, AcOH, toluene, 140 °C.
a Reagents and conditions: (a) triethoxymethane, Ac2O, 140 °C, 5 h.
Scheme 2. Synthesis of Compounds 7-10a
a Reagents and conditions: (a) excessacetone, KOH/H2O, MeOH;(b) pre-mixed acetylacetone and B(n-BuO)3, n-BuNH2, 80 °C to roomtemperature;
(c) HCl, room temperature; (d) 3,4,5-trimethoxybenzaldehyde, DMF, reflux; (e) veratraldehyde or 3-methylthiophene-2-carbaldehyde, KOH/H2O,
MeOH.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23 8263
Table 1. Effect of Curcumin and Its Analogues on Growth of A549
Cellsa
Growth Inhibition of A549 Cells
compd
GI50 (μM) compd GI50 (μM) compd GI50 (μM)
curcumin
2
15.23
>25
5.80
19.26
>25
>25
0.55
0.53
1.21
9.31
1.64
0.93
1.12
16
17
18
19
20
21
22
23
24
25
26
27
28
0.23
0.53
0.31
2.62
0.58
0.55
0.42
0.67
0.63
0.67
0.37
0.53
0.65
29
30
31
32
33
34
35
36
37
38
39
40
2.00
0.43
0.69
0.36
0.78
0.55
0.42
0.64
1.31
>25
>25
>25
3
4
5
6
7
8
9
10
13
14
15
a Human adenocarcinoma A549 cells were seeded for 12 h, subse-
quently incubated with varying concentrations of curcumin and its ana-
logues for 48 h and then examined for cell viability by the SRB assay.
GI50 is the concentration of compounds that causes 50% inhibition com-
pared to the vehicle control (0.25% DMSO). Samples were run in quin-
tuplicate, and the results are the mean of at least two independent tests.
the third arylvinyl moiety. Importantly, the cytotoxic activ-
ity of these 4-arylidene curcumin analogues was not limited
to A549 cells. In additional to A549 cells, other adenocarci-
noma cells H1944, squamous cell carcinoma cells H157, and
large cell carcinoma cells H460 were tested with selected
4-arylidene curcumin analogues. These compounds effec-
tively decreased viability of all three major types of lung cancer
cells tested. Most selected 4-arylidene analogues potently
inhibited the growth of lung cancer cells with GI50 values at
submicromolar concentrations (low to 0.07 μM) and induced
apoptotic cell death. Data of representative compounds 17,
21, 35 are shown in Figure 2 (see Supporting Information for
additional data from viability and apoptosis assays). These
studies identified a new class of curcumin analogues with
significantly improved activity against cancer cell growth.
Inhibition of TNFr Induced Nuclear Translocation of NF-KB
by New Curcuminoids. One of the major curcumin targets
critical for cancer survival is IκB kinase, IKK.38 IKK is a key
regulator of NF-κB activation. Activated NF-κB is localized
at the nucleus to promote transcription, which can be trig-
gered by TNFR.39 Thus, we utilized nuclear translocation of
NF-κB in response to TNFR as a primary readout to exam-
ine the mechanism of action of new curcuminoids in com-
parison to curcumin. An automated imaging system for high
content analysis was employed to monitor the cytoplasmic/
nuclear localization of NF-κB in response to TNFR.37 A549
cells in a 384-well plate format were treated with various test
compounds before TNFR was added to trigger the nuclear
translocation of the NF-κB p65 subunit. As shown in Figure 3
and Table 2, curcumin (1) attenuated TNFR-induced nuclear
redistribution of NF-κB with an IC50 of 11.6 μM, consistent
with previous reports.37
Figure 2. Inhibition of lung cancer cell viability by curcumin and its
analogues. Cells were grown in 384-well plates and were treated with
curcumin, 17, 21, or 35 as indicated for 72 h. Cell viability was
assessed by the Alamar Blue method and expressed as % control
(DMSO). Results with a panel of lung cancer cells are shown (lung
adenocarcinoma, A549 H1944, H1792; squamous cell carcinoma,
H157; squamous cell carcinoma, H157). Results from one repre-
sentative experiment are shown.
indicating the disadvantages of thiophene replacement with
respect to NF-κB inhibition.
Interestingly, except 19 and 30, the newly designed 4-aryl-
idene crucumin analogues (13-37) exhibited significant
inhibitory activities against NF-κB translocation with IC50
values in the low micromolar range (1.0-4.9 μM). 17 (IC50=
1.0 μM) and 21 (IC50=1.1 μM) showed more than 10-fold
greater potency in blocking the nuclear localization of NF-κB
than curcumin. The low activity of 19 may have resulted
from its dimethylamine moiety, which might be protonized
under the test conditions used, leading to unfavorable target
interactions. 30, however, remained unclear. Nevertheless,
these synthesized 4-arylidene curcumin analogues generally
showed superior activity to the classical 1,3-diketones ana-
logues (1-6) while the monoketone analogues (7-9) dis-
played moderate activity, suggesting a strong correlation
between a compound’s molecular skeleton and its activity.
From a comparison of the chemical structures of all three
types of curcuminoids, the presence of absolute diketone
moieties and 4-arylidene substitutions in 13-37 are unique
over others (1-7, 9, 10). Both factors may contribute to the
increased activities of the tested 4-arylidene curcumin ana-
logues. To explore the nature of these phenomena, we fur-
ther designed and evaluated the NF-κB inhibitory activity
of 4-hydroxymethylene curcumin analogues 38-40. 38-40
showed only poor inhibitory activity against NF-κB activa-
tion (Table 2), indicating the importance of the 4-arylidene
substitution for activity. On the other hand, although the inhib-
itory activities varied, most of the triarylvinyl condensates
The synthesized compounds were tested in the same assay
and showed different degrees of activity against TNFR in-
duced NF-κB activation. IC50 values of the tested curcumi-
noids are summarized in Table 2. As shown in Table 2, most
of the traditional 1,3-diketones curcumin analogues (2-4, 6)
were less active than curcumin. Three monoketone curcumin
analogues (7-9) showed improved activities (IC50 of 4.8-
9.0 μM). Consistent with its cytotoxity profile, 10 was much
less active (IC50 =24.3 μM) than 7-9 in NF-κB inhibition,

8264 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23
Qiu et al.
Table 2. Inhibitory Effect of Compounds on NFκB Activation/Trans-
locationa
IC50 (μM)
IC50 (μM)
IC50 (μM)
compd
curcumin 9.5
mean SD compd mean SD compd mean SD
3.00
2.19
16
17
18
19
20
21
22
23
24
25
26
27
28
1.8
1.0
1.5
>40
1.3
1.1
1.4
1.4
4.9
1.5
1.0
1.5
1.7
1.12
0.55
0.95
29
30
31
32
33
34
35
36
37
38
39
40
1.0
1.05
2
>40
>40
1.0
3
>40
>40
8.5
1.05
1.56
3.32
2.14
1.0
4
1.4
2.7
5
1.01
0.45
0.26
1.48
1.96
0.04
0.05
0.04
0.17
6
>40
7.9
1.8
1.7
7
4.26
1.85
3.44
8
4.8
9.0
1.3
2.8
1.35
1.02
9
10
13
14
15
24.3 7.01
39.3
>40
>40
3.0
3.3
3.8
2.01
2.37
2.13
a A549 cells were seeded in 384-well plates for 15 h, subsequently
incubated with varying concentrations of curcumin and its analogues for
30 min and then stimulated with TNFR. The inhibitory effect of test
compounds on TNFR induced NF-κB translocation was expressed as
percentage of fluorescence intensity difference (in nucleus and in cyto-
plasm) in the control wells (TNFR only) after subtracting background
(no TNFR treatment). The IC50 of NF-κB translocation stands for the
concentration of a compound required to induce 50% inhibitory effect.
Data points from each experiment were obtained as average values from
triplicate samples. Data shown are the average from three independent
experiments with standard deviation (SD).
Noticeably, the data in Tables 1 and 2 suggest an impor-
tant correlation between the antiproliferative activity of test-
ed compounds and their NF-κB inhibitory activity. Just like
1-6 and 10, which showed low activities in both A549 cell
growth and NF-κB activation inhibition, 7-9, 38-40, and
most 4-arylidene curcumin analogues behaved consistently
in both assays. These results strongly support the notion that
the NF-κB pathway is one of the major molecular targets for
curcumin and its analogues.
Enhanced Inhibition of IKB Kinase Activity by 4-Arylidene
Curcumin Analogues. NF-κB is mainly activated by IKKβ in
the well-defined canonical signaling pathway.40 Because of
this, we selected three potent curcumin analogues 17, 21, and
35 to directly test their effects on IKK enzymatic activity,
with the parent compound curcumin as a control. Two assays
were utilized: (i) a cell based IKK activation assay, which
detects phosphorylated IκB and subsequent IκB degrada-
tion, and (ii) a reconstituted in vitro kinase assay which di-
rectly measures the transfer of γ-32P from ATP to recom-
binant IκB. Upon stimulation of A549 cells with TNFR,
activated IKKβ can catalyze the phosphorylation of IκB at
Ser32 and Ser36, followed by degradation of phosphorylated
IκB, leading the release and nuclear translocation of NF-κB.
Antibodies against both IκB and Ser32 phosphorylated IκB
were used to detect the status of IκB as a readout of IKK
activity.
Figure 4 shows the Western blotting results of IκB phos-
phorylation (A) and subsequent degradation (B). Without
the stimuli of TNFR, IκB showed a basal level of phosphor-
ylation and no degradation (the first column). TNFR induced
significant IκB phosphorylation and subsequent degradation
of the protein (the second column). After treatment of cells
with different compounds for 4 h, curcumin could block IκB
phosphorylation and degradation when high concentrations
were used, whereas 17, 21, and 35 potently block the IκB
phosphorylation and degradation in a lower concentration.
Dose-response curves for IκB phosphorylation suggested
Figure 3. Inhibition of the TNFR induced NF-κB activation by
new curcuminoids, 17, 18, 20, and 21. Results from one representa-
tive experiment are shown. (A) Example images of NF-κB subcel-
lular localization. A549 cells were treated with compounds or
vehicle (DMSO) for 30 min, followed by stimulation with TNFR
(10 ng/mL) for 30 min. In the vehicle (DMSO) treatment, NF-κB is
located at cytoplasm. Upon TNFR treatment, NF-κB is activated
and translocated to the nucleus. Preincubation of the cells with
increasing concentrations of compounds, with compound 21 as an
example, dose-dependently inhibited the TNFR-induced NF-κB
translocation to the nucleus. (B) Dose-response curves of the
inhibitory effect of test compounds on TNFR-induced NF-κB
activation. The inhibitory effect of the compound on TNFR induced
NF-κB translocation (activation) was expressed as % of control =
ΔFI(compound)/ΔFI(TNFR only control). ΔFI is the measured NF-κB fluo-
rescence (green) intensity difference between nucleus and cytoplasm.
Data shown are average values from triplicate samples with SD.
with different substituted groups (except 19 and 30) were
active at low micromolar concentrations. It might also be
concluded that the triarylvinyl scaffolds of 4-arylidene cur-
cumin analogues play a key role in their NF-κB inhibition
activities as a pharmacophore, though further investigation
is still needed to confirm that hypothesis.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23 8265
Figure 5. Effect of curcumin analogues on the catalytic activity of
recombinant IKK in vitro. Recombinant IKKβ was incubated with
increasing concentrations of test compounds as indicated. Addition
of Mg/[γ-32P] cocktail with purified GST-I-κB started the reactions,
which were continued for 30 min at 30 °C. Proteins were separated
by SDS-PAGE and processed for radiolabeled GST-I-κB (32P-
I-κB) and stained total GST-I-κB (GST- I-κB). Controls include
reactions without IKKβ (lane 1 from left) or without compound
(lane 2).
Figure 6. Compounds 17, 21, and 35 inhibit the colony formation
of A549 cells. A549 cells were seeded in 12-well plate at a density of
200 cells/well and incubated overnight. The cells were then treated
with compounds or vehicle (DMSO) for 3 days. The medium and
compound were replaced for every 3 days. After a total of 9 days of
incubation, cells were fixed and stained with SRB. The image of the
plate was scanned, and the colonies were counted. (B) The colonies
were counted and normalized to DMSO control. The data shown
are the average of triplicate samples with SD.
Figure 4. Effect of curcumin analogues on in vivo activity of IKK.
(A) In vivo IKK activity as revealed by phosphorylation of IκB.
A549 cells were pretreated with test compounds for 30 min prior to
adding TNFR (10 ng/mL). Whole cell lysates were prepared after
7 min of TNFR treatment and analyzed for the phosphorylation state
of IκB with anti-pS32 antibody via Western blotting. Then anti-
bodies on the membrane were stripped and the membrane was re-
probed for total IκB with antiserum against IκB as indicated. (B) IκB
stability assay. A549 cells were pretreated with test compounds at
indicated dosage prior to the addition of TNFR. Cells were cultured
in the presence of TNFR (10 ng/mL) for an additional 20 min, lysed,
and analyzed for total IκB levels by Western blot.
inhibition assay was performed with recombinant IKKβ and
its substrate GST-IκB.37 The addition of curcumin in the range
of tested concentrations had no obvious effect on IKKβ
catalyzed incorporation of 32P into its substrate GST-IκB
(Figure 5). On the other hand, incubation of compounds 17,
21, and 35 induced a dose-dependent inhibition of IKKβ.
Thus, the structural modifications of these novel curcumin
analogues led to drastically enhanced inhibitory activity over
curcumin in this defined in vitro IKK kinase assay.
Inhibition of Lung Cancer Clonogenic Activity by 4-Aryl-
idene Curcumin Analogues. To compare the anticancer effi-
cacy of curcumin and its new analogues, we investigated the
effect of these compounds on colony formation properties of
lung cancer cells, a readout for tumorigenesis.41 A549 ade-
nocarcinoma cells readily formed colonies in a solid matrix.
The addition of the new triarylvinyl curcumin analogues 17,
21, and 35 drastically inhibited the number of colonies formed
by A549 cells (Figure 6). At concentrations less than 0.2 μM,
17 and 21 reached ∼50% inhibition, while 35 required ap-
proximately 0.4 μM to reach the same level of inhibition. For
comparison, curcumin inhibited colony forming activity at
much higher concentrations, with detectable inhibition at
IC50 values of 2.8 (17), 2.2 (21), and 5.0 μM (35), respectively,
with a similar trend for IkB degradation. Those results sug-
gest that 17, 21, and 35 block TNF-R induced NF-κB acti-
vation at least in part by inhibiting IκB phosphorylation and
degradation. Again, the 4-arylidene curcumin analogues show
much higher activities than curcumin.
The phosphorylation of IκB mainly depends on the IKKβ
catalytic subunit of the IKK complex in the canonical path-
way. It is possible that the inhibitory activities of curcumin
and derivatives may be due to direct inhibition to the IKKβ
kinase. To test this model, an in vitro reconstituted IKK

8266 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23
Qiu et al.
Figure 7. Binding of compound 17 and ATP with the IKKβ catalytic domain homology model. Left: Overlay of compound 17 (colored by
atom type) and ATP (purple) in the ATP binding pocket. The molecules are represented in stick. Typical hydrogen bonds of compound 17
(green dotted line) and ATP (brown dotted line) with the homology model are shown. Right: Surface representation of the IKKβ catalytic
domain homology model with compound 17 (colored by atom type) and ATP (purple) docking into the ATP binding pocket. The kinase surface
was mapped by lipophilic potential (from orange to blue, lipophilic potential decrease) and Z-clipping to remove the N terminal region for
visualization facility. Compound 17 adopts a propeller-shaped conformation in the pocket.
4 μM. These studies demonstrate that these 4-arylidene cur-
cumin analogues have significantly improved anticancer ac-
tivity over the parental curcumin.
Consistent with their enhanced activity against a major
molecular target, the representative 4-arylidene curcumin ana-
logues effectively inhibited the viability of three types of lung
cancer cells and attenuated the clonogenic activity of A549
cells. On the basis of our finding, it can be concluded that the
triarylvinyl skeleton of 4-arylidene curcumin analogues acts
as a phamacophore, and the 4-arylmethylene modification is
an effective strategy to improve the NF-κB inhibitory activ-
ity of curcumin analogues.
Molecular Modeling of the IKKβ/17 Complex. To gain
insights into the potential binding pose of the 4-arylidene cur-
cumin analogues in IKKβ, a molecular docking analysis was
carried out. Since three-dimensional structural data are un-
available for IKKβ, a homology model of the IKKβ catalytic
domain was built based on four templates (PDB codes 2JC6,
1A06, 2QNJ, and 2A2A) from the Protein Data Bank (PDB)
(see Experiments for details). Discovery Studio 2.1 (Accelrys
Inc.) and Sybyl 7.3.5 (Tripos Inc.) were used for the homol-
ogy model building and energy minimization of the IKKβ
catalytic domain, respectively. The docking of the selected
compound, 17, to the IKKβ homology model suggests that
17 could potentially bind to the ATP pocket of IKKβ(Figure7).
Compound 17 adopts a propeller-shaped conformation in
the pocket. The 4-arylidene moiety of 17 occupied the main
hydrophobic adenine binding pockets (Figure 7, region A).
The 40-OH and 30-OMe in 4-arylidene moiety of 17 have
hydrogen bonds respectively with hinge region residues Glu97
and Cys99, which are the same residues targeted by the
adenine moiety during the interaction of ATP with IKKβ
catalytic domain. Furthermore, one of the 1,7-diaryl moieties
of compound 17 lies along the phosphate binding pockets
(Figure 7, region P) with a hydrogen bond between a 30-OMe
and residue Thr180. The other side of 1,7-diaryl moieties
extends from the ribose binding pockets (Figure 7, region R)
to the hydrophilic solvent-exposed entrance (Figure 7, region
E), keeps orthogonal with the 4-arylidene moiety of 17, and
has a hydrogen bond between a 40-OMe and residue Asp103
in the entrance region of the catalytic domain. It should be
cautioned that this computer model provides a starting point,
and details of the structure-activity relationship (SAR) have
to be experimentally tested in future work.
Although presented data support an important role of
IKKβ in the NF-κB pathway in mediating the effect of re-
ported compounds, some observed differences in compound
potency between cell viability assay and the IKKβ/NF-κB
assays could be due to a number of factors. For example,
(i) the status of compounds in cell culture and in vitro con-
ditions could be different. The employed compounds could
be further metabolized in cells to generate multiple active
species, while in vitro kinase assays only measure the activity
of the test compounds. (ii) Although IKKβ could be an
important target of the tested curcumin analogues as pro-
posed, the efficacy of the test compounds on cell survival is
also determined by the cell’s genetic background. Cells har-
boring different oncogene mutations, deletions, or amplifi-
cations could show different sensitivities to test compounds
that target IKKβ. Our data in Figure 2 strongly support this
model. (iii) The suppression of cell growth by these curcumin
analogues could be due to their inhibition of other targets in
addition to IKKβ. These potential targets include the cyto-
protective antioxidant system. Whether and how these
4-arylidene curcumin analogues inhibit other targets in
addition to IKKβ in cancer cells requires further investiga-
tion. Regardless, because of their potent IKKβ inhibitory
activity, these novel triarylvinyl curcuminoids represent new
opportunities for therapeutic development against cancer and
other NF-κB dependent disorders, including various in-
flammatory diseases. Detailed SAR analysis is still required
and is being actively pursued by our laboratory.
In summary, our design and synthesis have led to the gen-
eration of a novel series of curcumin analogues. Biochemical
and cell biology based studies coupled with high content
analysis have led to the identification of a novel class of
4-arylidene curcumin analogues as a new generation of highly
potent curcuminoids. These compounds exhibited signifi-
cantly improved potency in blocking IKK activity and the
NF-κB pathway as revealed by both in vivo and in vitro
kinase assays and pathway analysis.
Experiments
General. All reagents used were commercially available. Sol-
vents 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
1
performed on silica gel (100-200 mesh). H NMR and 13C
NMR spectra were recorded with Varian INOVA 500NB or

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23 8267
Bruker Ultrashield 400 MHz spectrometer using TMS as an in-
ternal standard. Purity of target compounds was determined by
reversed-phase HPLC analyses (Hypersil BDS-C18, 250 μm ꢀ
4.6 μm) with a flow rate of 0.5 mL/min. The samples were eluted
with a gradient of constant 70% A (2-4 and 40), 90% A (38 and
39), or 50% A (others), where solvent A was CH3CN and sol-
vent B was 0.1% TFA in water, using UV monitor at 254 nm for
detection. Retention times (tR) were calculated in minutes, and
purity was calculated as % total area. All compounds were >95%
pureby HPLC. The HR-MS analysis was performed on a Thermo
Finnigan MAT95XP-Trap instrument.
Chemistry. Synthesis of 1-10. 5-Hydroxy-1,7-bis(4-hydroxy
-3-methoxyphenyl)hepta-1,4,6-trien-3-one (1), 5-hydroxy-1,7-
bis(4- methoxyphenyl)hepta-1,4,6-trien-3-one (2), 1,7-bis(3,4-
dimethoxyphenyl)-5-hydroxyhepta-1,4,6-trien-3-one (3), 5-hydroxy-
1,7-bis(3,4,5-trimethoxyphenyl)hepta-1,4,6-trien-3-one (4),
5-hydroxy-1,7-bis(5-methylfuran-2-yl)hepta-1,4,6-trien-3-one (5),
and 1,7-di(furan-2-yl)-5-hydroxyhepta-1,4,6-trien-3-one (6) were
synthesized according to our previous report.35
(E)-4-(3,4,5-Trimethoxyphenyl)but-3-en-2-one (11) and (1E,4E)-
1,5-Bis(3,4,5-trimethoxyphenyl)penta-1,4-dien-3-one (7). To a
solution of 3 mL of acetone in 10 mL of EtOH, 0.84 g KOH
(15 mmol, dissolved in 5 mL H2O) was added at 0 °C, and the mix-
ture was stirred for 20 min. After that, 2.94 g of 3,4,5-trimetho-
xybenzaldehyde (15 mmol, dissolved in 25 mL of EtOH) was
added dropwise. Then the mixture was brought to room tem-
perature for 2 h and monitored by TLC. Once the 3,4,5-tri-
methoxybenzaldehyde was consumed, the reaction solution was
poured into water, and the resulting solution was neutralized by
2 N HCl. Then the raw product was extracted from water by
acetic ether and purified with column chromatography (petro-
leum ether/acetic ether 5:1) to give 1.23 g of intermediate 11
General Procedure for the Synthesis of 9 and 10. 11 (236 mg,
1 mmol) and 1 mmol of the corresponding benzaldehyde were
dissolved in 5 mL of EtOH, and KOH (56 mg, 1 mmol, dissolved
in 0.1 mL H2O) was added. The reaction mixture was stirred for
1 h at room temperature. Then the solution was poured into
water and the resulting solution was neutralized by 2 N HCl. The
raw product was extracted with ethyl acetate, and the organic
layer was washed with water (10 mL, 2 times), dried (Na2SO4),
and evaporated under vacuum. Finally the raw product was puri-
fied with column chromatography to give the final product.
(1E,4E)-1-(3,4-Dimethoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-
penta-1,4-dien-3-one (9). 3,4-dimethoxybenzaldehyde (166 mg,
1 mmol) was used as the benzaldehyde, and column chroma-
tography (petroleum ether/acetic ether 2:1) gave 324 mg of 9 as a
yellow powder, yield 84%. HPLC tR = 13.5 min; Rf = 0.14
(petroleum ether/acetic ether 2:1). 1H NMR (400 MHz, CDCl3)
δ 7.69 (d, J=15.8 Hz, 1H), 7.64 (d, J=15.8 Hz, 1H), 7.20 (dd, J=
1.9, 8.3 Hz, 1H), 7.13 (d, J=1.9 Hz, 1H), 6.96 (d, J=15.8 Hz,
1H), 6.95 (d, J=15.8 Hz, 1H), 6.88 (d, J=8.3 Hz, 1H), 6.84
(s, 2H), 3.93 (s, 3H), 3.92 (s, 3H), 3.90 (s, 6H), 3.89 (s, 3H). 13
C
NMR (101 MHz, CDCl3) δ 188.60, 153.51, 151.49, 149.33,
143.40, 143.03, 140.41, 130.41, 127.82, 125.00, 123.51, 123.21,
111.20, 110.04, 105.66, 61.02, 56.25, 56.03, 55.99.
(1E,4E)-1-(3-Methylthiophen-2-yl)-5-(3,4,5-trimethoxyphenyl)-
penta-1,4-dien-3-one (10). 3-methylthiophene-2-carbaldehyde
(126 mg, 1 mmol) was used as the benzaldehyde, and column
chromatography (petroleum ether/acetic ether 9:1) gave 266
mg 10 as a yellow powder, yield 77%. HPLC tR=22.9 min; Rf=
0.39 (petroleum ether/acetic ether 3:1). HR-MS calcd for
C
19H20O4S1: 344.1077, found 344.1074. 1H NMR (400 MHz,
CDCl3) δ 7.96 (d, J=15.3 Hz, 1H), 7.63 (d, J=15.9 Hz, 1H),
7.30 (d, J=5.1 Hz, 1H), 6.91 (d, J=5.1 Hz, 1H), 6.88 (d, J=
15.9 Hz, 1H), 6.86 (d, J=15.3 Hz, 1H), 6.85 (s, 2H), 3.92 (s,
6H), 3.90 (s, 3H), 2.40 (s, 3H). 13C NMR (101 MHz, CDCl3)
δ 188.07, 153.48, 142.94, 142.68, 134.48, 134.16, 131.48,
130.34, 127.29, 125.87, 122.85, 105.57, 61.00, 56.20, 14.32.
General Procedure for the Synthesis of 13-17, 19-28, and
30-37. An amount of 1.0 mmol of 1, 2, 3, or 4 and 2 mmol of the
corresponding benzaldehyde as well as 25 mL toluene were
added to a three-neck rounded flask equipped with a water dis-
penser. Pyridine (4.0 mg, 0.05 mmol, in 0.1 mL toluene) and
acetic acid (4.8 mg, 0.08 mmol, in 0.1 mL toluene) were added as
catalysts. The reaction mixture was stirred in 140 °C overnight,
and the generated water was removed by water dispenser during
the whole reaction. Then the reaction mixture was washed with
water (10 mL, twice) to remove pyridine and acetic acid. Next
the organic layer was evaporated under vacuum to get raw pro-
duct. Finally the product was purified by using silica gel column
chromatography.
(35%) and 1.53 g of yellow powder 7 (49%). HPLC tR
=
17.8 min; Rf=0.38 (petroleum ether/acetic ether 2:1). 1H NMR
(400 MHz, CDCl3) δ 7.65 (d, J=15.8 Hz, 2H), 6.97 (d, J=
15.8 Hz, 2H), 6.84 (s, 4H), 3.91 (s, 12H), 3.89 (s, 6H). 13C NMR
(101 MHz, CDCl3) δ 188.48, 153.51, 143.34, 140.52, 130.29,
124.82, 105.72, 61.00, 56.25.
(5E)-3-(3,4,5-Trimethoxybenzylidene)-6-(3,4,5-trimethoxy-
phenyl)hex-5-ene-2,4-dione (8). Compound 8 was synthesized in
two steps. First the intermediate 12 was synthesized with the
following procedures. Acetyl acetone (5.01 g, 50 mmol), boric
anhydride (2.1 g, 30 mmol), and tri-n-butyl borate (23 g,
100 mmol) were mixed in ethyl acetate (100 mL) and stirred at
80 °C for 1 h. Then a solution of 3,4,5-trimethoxybenzaldehyde
(1.96 g, 10 mmol) and n-butylamine (0.5 mL) in ethyl acetate was
added dropwise in 1 h. Next the reaction mixture was stirred at
room temperature for 2 days. The resulting solution was then
neutralized by 0.4 N HCl and stirred for an additional 30 min in
60 °C. Then the raw product was extracted with ethyl acetate,
and the organic layer was washed with water (30 mL, 2 times),
dried (Na2SO4), and evaporated under vacuum. Finally the pro-
duct was isolated by column chromatography (petroleum ether/
acetic ether 9:1) to give 0.93 g intermediate 12 (33%). 1H NMR
(400 MHz, CDCl3) δ 7.53 (d, J=15.8 Hz, 1H), 6.77 (s, 2H), 6.39
(d, J=15.8 Hz, 1H), 5.67 (s, 1H), 3.91 (s, 6H), 3.90 (s, 3H), 2.18
(s, 3H). After this, 557 mg of 12 (2 mmol) and 3,4,5-trimethox-
ybenzaldehyde (1.18 g, 6 mmol) were dissolved in 10 mL of DMF
and stirred in 105 °C overnight. Then the DMF was evaporated
under vacuum. Finally the crude mixture was purified with
column chromatography (petroleum ether/acetic ether 5:1) to
give 280 mg of 8 as a pale yellow powder, yield 31%. HPLC tR=
11.1 min; Rf=0.31 (petroleum ether/acetic ether 2:1). HRMS
(1E,6E)-4-(4-Hydroxy-3-methoxybenzylidene)-1,7-bis(4-methoxy-
phenyl)hepta-1,6-diene-3,5-dione (13). 4-Hydroxy-3-methoxy-
benzaldehyde (304 mg, 2 mmol) and 2 (336 mg, 1 mmol) were
used as reactants and the raw product was purified by column
chromatograph (petroleum ether/acetic ether 2:1) to give 249 mg
of 13 as a yellow powder, yield 53%. HPLC tR=19.5 min; Rf=
0.20 (petroleum ether/acetic ether 2:1). HR-MS calcd for C29-
H26O6: 470.1724, found 470.1727. 1H NMR (500 MHz, CDCl3)
δ 7.81 (s, 1H), 7.77 (d, J=15.4 Hz, 1H), 7.53 (d, J=16.1 Hz, 1H),
7.52 (d, J=8.9 Hz, 2H), 7.42 (d, J=8.8 Hz, 2H), 7.08 (dd, J=2.0,
8.3 Hz, 1H), 7.03 (d, J=2.0 Hz, 1H), 6.99 (d, J=15.4 Hz, 1H),
6.89 (d, J=8.8 Hz, 2H), 6.86 (d, J=8.3 Hz, 1H), 6.86 (d, J=8.9
Hz, 2H), 6.81 (d, J=16.1 Hz, 1H), 3.83 (s, 3H), 3.81 (m, 6H). 13
C
NMR (101 MHz, CDCl3) δ 198.62, 186.81, 162.15, 161.79,
148.14, 146.64, 146.54, 144.48, 140.79, 138.69, 130.49, 130.42,
127.62, 126.90, 125.97, 125.75, 125.42, 120.11, 114.82, 114.50,
114.39, 112.45, 55.94, 55.41.
(1E,6E)-4-(3,4-Dimethoxybenzylidene)-1,7-bis(4-methoxyphenyl)-
hepta-1,6-diene-3,5-dione (14). 3,4-Dimethoxybenzaldehyde
(332 mg, 2 mmol) and 2 (336 mg, 1 mmol) were used as reactants
and the raw product was purified by column chromatography
1
calcd for C25H28O8: 456.1779, found 456.1780. H NMR (400
MHz, CDCl3) δ 7.64 (s, 1H), 7.40 (d, J=16.1 Hz, 1H), 6.75
(d, J=16.1 Hz, 1H), 6.71 (s, 2H), 6.68 (s, 2H), 3.87 (s, 3H), 3.85
(s, 6H), 3.84 (s, 3H), 3.79 (s, 6H), 2.41 (s, 3H). 13C NMR (101
MHz, CDCl3) δ 197.82, 195.75, 153.48, 153.19, 146.41, 141.04,
140.97, 140.36, 139.37, 129.35, 128.23, 126.27, 107.92, 105.91,
60.96, 60.90, 56.20, 56.14, 27.30.

8268 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23
Qiu et al.
(petroleum ether/acetic ether 4:1) to give 312 mg of 14 as a yellow
powder, yield 64%. HPLC tR=27.3 min; Rf=0.33 (petroleum
ether/acetic ether 2:1). HR-MS calcd for C30H28O6: 484.1880,
found 484.1886. 1H NMR (500 MHz, CDCl3) δ 7.82 (s, 1H), 7.78
(d, J=15.4 Hz, 1H), 7.53 (d, J=8.9 Hz, 2H), 7.53 (d, J=15.4 Hz,
1H), 7.43 (d, J=8.7 Hz, 2H), 7.13 (dd, J=8.4, 1.8 Hz, 1H), 7.04
(d, J=1.8 Hz, 1H), 6.99 (d, J=15.4 Hz, 1H), 6.89 (d, J=8.7 Hz,
2H), 6.86 (d, J=8.9 Hz, 2H), 6.83 (d, J=16.1 Hz, 1H), 6.82 (d, J=
chromatography (petroleum ether/acetic ether 2:1) to give
258 mg of 19 as a red powder, yield 49%. HPLC tR=18.1 min;
Rf=0.39 (petroleum ether/acetic ether 1:1). HR-MS calcd for
1
C32H33O6N1: 527.2302, found 527.2301. H NMR (500 MHz,
CDCl3) δ 7.73 (d, J=15.4 Hz, 1H), 7.52 (d, J=16.0 Hz, 1H), 7.42
(d, J=8.9 Hz, 2H), 7.17 (dd, J=8.4, 1.8 Hz, 1H), 7.07 (dd, J=
8.5, 1.9 Hz, 1H), 7.06 (d, J=1.8 Hz, 1H), 7.00 (d, J=1.9 Hz, 1H),
6.99 (d, J=15.4 Hz, 1H), 6.84 (d, J=8.5 Hz, 1H), 6.85 (d, J=
16.0 Hz, 1H), 6.81 (d, J=8.4 Hz, 1H), 6.61 (d, J=8.9 Hz, 2H),
3.90 (s, 3H), 3.90 (s, 3H), 3.88 (s, 3H), 3.86 (s, 3H), 2.99 (s, 6H).
13C NMR (101 MHz, CDCl3) δ 199.52, 186.63, 151.80, 151.69,
151.33, 149.23, 149.20, 146.50, 143.88, 142.14, 135.43, 133.08,
128.14, 127.43, 126.16, 123.47, 123.05, 120.87, 120.72, 111.66,
111.15, 111.06, 110.62, 110.28, 56.06, 56.02, 56.01, 55.93, 39.97.
(1E,6E)-1,7-Bis(3,4-dimethoxyphenyl)-4-(3-fluorobenzylidene)-
hepta-1,6-diene-3,5-dione (20). 3-Fluorobenzaldehyde (248 mg,
2 mmol) and 3 (396 mg, 1 mmol) were used as reactants and the
raw product was purified by column chromatography (petro-
leum ether/acetic ether 3:1) to give 413 mg of 20 as a yellow
powder, yield 82%. HPLC tR=22.5 min; Rf=0.36 (petroleum
ether/acetic ether 3:2). HR-MS calcd for C30H27O6F1: 502.1786,
8.4 Hz, 1H), 3.87 (s, 3H), 3.84 (s, 3H), 3.82 - 3.81 (m, 6H). 13
C
NMR (101 MHz, CDCl3) δ198.73, 186.73, 162.13, 161.77, 151.09,
148.81, 146.81, 144.57, 140.65, 138.80, 130.53, 130.47, 127.52,
126.79, 126.31, 125.35, 125.05, 119.95, 114.47, 114.35, 112.82,
111.02, 55.90, 55.82, 55.43, 55.40.
(1E,6E)-1,7-Bis(4-methoxyphenyl)-4-(2,3,4-trimethoxybenzy-
lidene)hepta-1,6-diene-3,5-dione (15). 3,4,5-Trimethoxybenzal-
dehyde (392 mg, 2 mmol) and 2 (336 mg, 1 mmol) were used
as reactants and the raw product was purified by column chro-
matography (petroleum ether/acetic ether 4:1) to give 283 mg of
15 as a yellow powder, yield 55%. HPLC tR=32.0 min; Rf=0.39
(petroleum ether/acetic ether 2:1). HR-MS calcd for C31H30O7:
514.1986, found 514.1987. 1H NMR (500 MHz, CDCl3) δ 7.78
(d, J=15.4 Hz, 1H), 7.76 (s, 1H), 7.53 (d, J=8.7 Hz, 2H), 7.53 (d,
J=16.1 Hz, 1H), 7.43 (d, J=8.7 Hz, 2H), 6.98 (d, J=15.4 Hz,
1H), 6.90 (d, J=8.8 Hz, 2H), 6.87 (d, J=8.8 Hz, 2H), 6.81 (d, J=
16.1 Hz, 1H), 6.76 (s, 2H), 3.84 (m, 6H), 3.82 (s, 3H), 3.80 (s, 6H).
13C NMR (101 MHz, CDCl3) δ 198.24, 186.77, 162.23, 161.89,
153.13, 146.82, 144.94, 140.46, 140.28, 140.10, 130.54, 128.87,
127.47, 126.76, 125.26, 119.88, 114.54, 114.41, 107.92, 60.93,
56.14, 55.45, 55.43.
(1E,6E)-1,7-Bis(3,4-dimethoxyphenyl)-4-(3,4,5-trimethoxyben-
zylidene)hepta-1,6-diene-3,5-dione (16). 3,4,5-Trimethoxyben-
zaldehyde (392 mg, 2 mmol) and 3 (396 mg, 1 mmol) were used
as reactants and the raw product was purified by column chro-
matography (petroleum ether/acetic ether 2:1) to give 486 mg of
16 as a yellow powder, yield 85%. HPLC tR=17.0 min; Rf=0.39
(petroleum ether/acetic ether 1:1). HR-MS calcd for C33H34O9:
574.2197, found 574.2201. 1H NMR (500 MHz, CDCl3) δ 7.75
(s, 1H), 7.76 (d, J=15.4 Hz, 1H), 7.51 (d, J=16.1 Hz, 1H), 7.18
(dd, J=8.4, 2.0 Hz, 1H), 7.07 (d, J=2.0 Hz, 1H), 7.05 (dd, J=
8.4, 2.0 Hz, 1H), 6.98 (d, J=2.0 Hz, 1H), 6.98 (d, J=15.4 Hz,
1H), 6.86 (d, J=8.4 Hz, 1H), 6.82 (d, J=8.4 Hz, 1H), 6.80 (d, J=
16.1 Hz, 1H), 6.76 (s, 2H) 3.90 (s, 3H), 3.89(s, 3H), 3.88(s, 3H),
3.86 (s, 3H), 3.84 (s, 3H), 3.78 (s, 6H). 13C NMR (101 MHz,
CDCl3) δ 197.96, 186.86, 153.18, 152.10, 151.78, 149.42, 149.33,
146.96, 145.23, 140.46, 140.40, 140.27, 128.84, 127.77, 127.07,
125.48, 123.56, 123.37, 120.21, 111.24, 110.76, 110.33, 108.02,
60.89, 56.17, 56.02, 55.97.
(1E,6E)-1,7-Bis(3,4-dimethoxyphenyl)-4-(4-hydroxy-3-methoxy-
benzylidene)hepta-1,6-diene-3,5-dione (17). 4-Hydroxy-3-meth-
oxybenzaldehyde (304 mg, 2 mmol) and 3 (396 mg, 1 mmol) were
used as reactants and the raw product was purified by column
chromatography (petroleum ether/acetic ether 2:1) to give 320 mg
of 17 as a yellow powder, yield 60%. HPLC tR=11.6 min; Rf=
0.27 (petroleum ether/acetic ether 1:1). HR-MS calcd for C31-
H30O8: 530.1935, found 530.1941. 1H NMR (500 MHz, CDCl3)
δ 7.80 (s, 1H), 7.75 (d, J=15.4 Hz, 1H), 7.50 (d, J=16.1 Hz, 1H),
7.18 (dd, J=8.5, 2.0 Hz, 1H), 7.09 (dd, J=8.5, 2.0 Hz, 1H), 7.06
(dd, J=8.0, 2.0 Hz, 1H), 7.06 (d, J=2.0 Hz, 1H), 7.04 (d, J=
2.0 Hz, 1H), 6.98 (d, J=2.0 Hz, 1H), 6.97 (d, J=15.4 Hz, 1H),
6.87 (d, J=8.5 Hz, 1H), 6.86 (d, J=8.0 Hz, 1H), 6.82 (d, J=
8.5 Hz, 1H), 6.81 (d, J=16.1 Hz, 1H), 3.91 (m, 6H), 3.89 (s, 3H),
3.87 (s, 3H), 3.83 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 198.61,
186.89, 151.96, 151.61, 149.31, 149.24, 148.17, 147.02, 146.54,
144.92, 140.96, 138.58, 127.81, 127.09, 125.90, 125.71, 125.57,
123.60, 123.31, 120.20, 114.84, 112.42, 111.15, 111.12, 110.61,
110.19, 56.04, 56.03, 56.01, 55.94.
1
found 502.1785. H NMR (400 MHz, CDCl3) δ 7.80 (d, J =
15.4 Hz, 1H), 7.78 (s, 1H), 7.46 (d, J = 16.2 Hz, 1H), 7.30 (dd,
J =8.0, 7.5 Hz, 1H), 7.29 (d, J = 8.0 Hz, 1H), 7.21 (d, J =
1.8 Hz, 1H), 7.19 (d, J = 1.8 Hz, 1H), 7.10-7.04 (m, 2H), 7.03
(dd, J = 8.3 Hz, J =1.8 Hz, 1H), 6.99 (s, 1H), 6.95 (d, J = 15.4
Hz, 1H), 6.87 (d, J = 8.3 Hz, 1H), 6.84 (d, J = 8.3 Hz, 1H), 6.80
(d, J = 16.2 Hz, 1H), 3.92 (s, 6H), 3.90 (s, 3H), 3.88 (s, 3H). 13C
NMR (101 MHz, CDCl3) δ 197.47, 186.74, 162.68 (d, J =
247.2 Hz), 152.09, 151.83, 149.29, 149.24, 147.83, 145.92,
141.94, 138.75, 135.71 (d, J=7.9 Hz), 130.39 (d, J=8.3 Hz),
127.54, 126.89, 126.06 (d, J=2.9 Hz), 125.40, 123.85, 123.62,
119.84, 117.16 (d, J=21.4 Hz), 116.60 (d, J=22.3 Hz), 111.10,
111.04, 110.54, 110.04, 56.05, 56.04, 55.93.
(1E,6E)-1,7-Bis(3,4-dimethoxyphenyl)-4-(4-fluorobenzylidene)-
hepta-1,6-diene-3,5-dione (21). 4-Fluorobenzaldehyde (248 mg,
2 mmol) and 3 (396 mg, 1 mmol) were used as reactants and the
raw product was purified by column chromatography (petro-
leum ether/acetic ether 3:1) to give 390 mg of 21 as a yellow
powder, yield 78%. HPLC tR=21.9 min; Rf=0.33 (petroleum
ether/acetic ether 3:2). HR-MS calcd for C30H27O6F1: 502.1786,
1
found 502.1789. H NMR (400 MHz, CDCl3) δ 7.81 (s, 1H),
7.78(d, J=15.4 Hz, 1H), 7.48-7.51 (m, 2H), 7.47 (d, J=16.2 Hz,
1H), 7.19 (dd, J = 8.4, 2.0 Hz, 1H), 7.05-7.06 (m, 2H), 7.03 (d,
J = 8.7 Hz, 1H), 7.01 (d, J = 8.6 Hz, 1H), 6.99 (d, J = 2.0 Hz,
1H), 6.96 (d, J = 15.4 Hz, 1H), 6.87 (d, J = 8.3 Hz, 1H), 6.83 (d,
J = 8.4 Hz, 1H), 6.80 (d, J = 16.2 Hz, 1H), 3.92 (s, 3H), 3.91 (s,
3H), 3.89 (s, 3H), 3.88 (s, 3H). 13C NMR (101 MHz, CDCl3)
δ 197.92, 186.76, 163.73 (d, J=252.5 Hz), 152.09, 151.77, 149.32,
149.25, 147.63, 145.59, 140.57, 139.18, 132.38 (d, J=8.6 Hz),
129.79 (d, J=3.3 Hz), 127.63, 126.93, 125.43, 123.79, 123.52,
119.97, 116.07 (d, J=21.9 Hz), 111.12, 111.07, 110.58, 110.09,
56.06, 56.04, 55.94.
(1E,6E)-1,7-Bis(3,4-dimethoxyphenyl)-4-(4-hydroxybenzylidene)-
hepta-1,6-diene-3,5-dione (22). 4-Hydroxybenzaldehyde (244 mg,
2 mmol) and 3 (396 mg, 1 mmol) were used as reactants and the
raw product was purified by column chromatography (petroleum
ether/acetic ether 2:1) to give 187 mg of 22 as an orange powder,
yield 37%. HPLC tR=11.6 min; Rf=0.30 (petroleum ether/acetic
ether 1:1). HR-MS calcd for C30H28O7: 500.1830, found 500.1826.
1H NMR (400 MHz, DMSO) δ 7.76 (d, J = 15.4 Hz, 1H), 7.50
(d, J = 16.1 Hz, 1H), 7.39 (d, J = 8.7 Hz, 2H), 7.19 (dd, J = 8.4,
1.9 Hz, 1H), 7.06 (d, J = 1.9 Hz, 1H), 7.07 (dd, J = 8.2, 2.1 Hz,
1H), 7.00 (d, J = 2.1 Hz, 1H), 6.97 (d, J = 15.4 Hz, 1H), 6.86 (d,
J = 8.4 Hz, 1H), 6.83 (d, J = 8.4 Hz, 1H), 6.83 (d, J = 16.1 Hz,
1H), 6.78 (d, J = 8.7 Hz, 2H), 3.92 (s, 3H), 3.91 (s, 3H), 3.89 (s,
3H), 3.87 (s, 3H). 13C NMR (101 MHz, DMSO) δ 198.22, 187.83,
159.85, 151.39, 151.14, 148.95, 148.93, 145.93, 143.26, 140.22,
137.93, 132.55, 127.48, 126.78, 125.42, 124.23, 123.51, 123.29,
119.29, 115.81, 111.62, 111.49, 111.16, 110.68, 55.71, 55.54.
(1E,6E)-1,7-Bis(3,4-dimethoxyphenyl)-4-(4-(dimethylamino)-
benzylidene)hepta-1,6-diene-3,5-dione (19). 4-(Dimethylamino)-
benzaldehyde (298 mg, 2 mmol) and 3 (396 mg, 1 mmol) were
used as reactants and the raw product was purified by column

Article
(1E,6E)-4-(2,5-Dimethoxybenzylidene)-1,7-bis(4-hydroxy-3-
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23 8269
140.95, 138.79, 130.24, 129.51, 126.87, 126.27, 125.07, 121.54,
113.04, 111.24, 106.24, 106.13, 60.99, 56.38, 56.27, 55.97, 55.93.
(1E,6E)-4-(2,5-Dimethoxybenzylidene)-1,7-bis(3,4-dimethoxy-
phenyl)hepta-1,6-diene-3,5-dione (27). 2,5-Dimethoxybenzalde-
hyde (332 mg, 2 mmol) and 3 (396 mg, 1 mmol) were used as
reactants and the raw product was purified by column chroma-
tography (petroleum ether/acetic ether 3:1) to give 211 mg of 27
as a yellow powder, yield 39%. HPLC tR=20.4 min; Rf=0.24
(petroleum ether/acetic ether 3:2). HR-MS calcd for C32H32O8:
544.2092, found 544.2096. 1H NMR (400 MHz, CDCl3) δ 8.15
(s, 1H), 7.76 (d, J = 15.5 Hz, 1H), 7.46 (d, J = 16.1 Hz, 1H), 7.20
(dd, J = 8.3, 1.5 Hz, 1H), 7.09 (d, J = 1.5 Hz, 1H), 7.03 (dd, J =
8.0, 1.5 Hz, 1H), 7.04 (d, J = 15.5 Hz, 1H), 6.95-6.96 (m, 2H),
6.87 (d, J = 8.3 Hz, 1H), 6.85-6.87 (m, 1H), 6.82 (d, J = 8.0 Hz,
1H), 6.80 (d, J = 8.8 Hz, 1H), 6.73 (d, J = 16.1 Hz, 1H), 3.92 (s,
6H), 3.89 (s, 3H), 3.87 (s, 3H), 3.84 (s, 3H), 3.67 (s, 3H). 13C
NMR (101 MHz, CDCl3) δ 197.49, 187.78, 153.20, 152.46,
151.70, 151.54, 149.18, 146.47, 144.96, 140.90, 136.31, 127.78,
127.25, 125.53, 123.48, 123.39, 123.30, 120.38, 117.67, 114.94,
111.91, 111.11, 111.02, 110.47, 109.97, 55.98, 55.88, 55.70.
(1E,6E)-4-(2,4-Dimethoxybenzylidene)-1,7-bis(3,4-dimethoxy-
phenyl)hepta-1,6-diene-3,5-dione (28). 2,4-Dimethoxybenzalde-
hyde (332 mg, 2 mmol) and 3 (396 mg, 1 mmol) were used as
reactants and the raw product was purified by column chroma-
tography (petroleum ether/acetic ether 3:1) to give 337 mg of 28
as a yellow powder, yield 62%. HPLC tR=19.7 min; Rf=0.40
(petroleum ether/acetic ether 1:1). HR-MS calcd for C32H32O8:
544.2092, found 544.2094. 1H NMR (400 MHz, CDCl3) δ 8.20
(s, 1H), 7.74 (d, J = 15.5 Hz, 1H), 7.48 (d, J = 16.1 Hz, 1H), 7.36
(d, J = 8.4 Hz, 1H), 7.19 (dd, J = 8.3, 1.8 Hz, 1H), 7.08 (d, J =
1.8 Hz, 1H), 7.05 (dd, J = 8.3, 1.8 Hz, 1H), 7.03 (d, J = 15.5 Hz,
1H), 6.98 (d, J = 1.8 Hz, 1H), 6.87 (d, J = 8.3 Hz, 1H), 6.82 (d,
J=8.3 Hz, 1H), 6.75 (d, J=16.1 Hz, 1H), 6.41 (d, J=2.4 Hz, 1H),
6.40 (dd, J = 8.4, 2.4 Hz, 1H), 3.91 (s, 6H), 3.89 (s, 3H), 3.87
(s, 6H), 3.79 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 198.32,
187.60, 163.17, 159.83, 151.62, 151.37, 149.17, 149.15, 146.10,
144.34, 138.40, 136.26, 131.78, 127.96, 127.37, 125.73, 123.41,
123.10, 120.62, 115.80, 111.09, 111.00, 110.52, 110.04, 105.16,
98.27, 55.99, 55.89, 55.53, 55.43.
(1E,6E)-4-(3,4-Dimethoxybenzylidene)-1,7-bis(4-hydroxy-3-
methoxyphenyl)hepta-1,6-diene-3,5-dione (30). 3,4-Dimethoxy-
benzaldehyde (332 mg, 2 mmol) and 1 (368 mg, 1 mmol) were
used as reactants, and column chromatography (petroleum ether/
acetic ether 3:2) gave 347 mg of 30 as a yellow powder, yield
67%. HPLC tR=9.3 min; Rf=0.20 (petroleum ether/acetic ether
1:1). HR-MS calcd for C30H28O8: 516.1779, found 516.1783. 1H
NMR (400 MHz, CDCl3) δ 7.81 (s, 1H), 7.77 (d, J = 15.4 Hz,
1H), 7.49 (d, J = 16.1 Hz, 1H), 7.16 (dd, J = 8.4, 1.9 Hz, 1H),
7.14 (dd, J = 8.2, 2.0 Hz, 1H), 7.03-7.06 (m, 2H), 7.03 (dd, J =
8.2, 1.9 Hz, 1H), 6.97 (d, J = 1.9 Hz, 1H), 6.96 (d, J = 15.4 Hz,
1H), 6.91 (d, J = 8.2 Hz, 1H), 6.88 (d, J = 8.2 Hz, 1H), 6.82 (d,
J = 8.4 Hz, 1H), 6.81 (d, J = 16.1 Hz, 1H), 5.96 (s, 1H), 5.94 (s,
1H), 3.92 (s, 3H), 3.89 (s, 3H), 3.87 (s, 3H), 3.82 (s, 3H). 13C
NMR (101 MHz, CDCl3) δ 198.79, 186.90, 151.13, 148.98,
148.83, 148.55, 147.49, 146.91, 146.83, 145.25, 140.74, 138.74,
127.31, 126.57, 126.28, 125.20, 125.02, 124.04, 123.59, 119.76,
114.94, 114.91, 112.84, 111.05, 110.44, 109.99, 56.04, 55.95,
55.91, 55.84.
methoxyphenyl)hepta-1,6-diene-3,5-dione (23). 2,5-Dimethoxy-
benzaldehyde (332 mg, 2 mmol) and 1 (368 mg, 1 mmol) were
used as reactants, and column chromatography (petroleum ether/
acetic ether 2:1) gave 177 mg of 23 as a yellow powder, yield
34%. HPLC tR =10.7 min; Rf =0.20 (petroleum ether/acetic
ether 1:1). HR-MS calcd for C30H28O8[M - H]-: 515.1706,
1
found 515.1701. H NMR (400 MHz, CDCl3) δ 8.14 (s, 1H),
7.74 (d, J = 15.5 Hz, 1H), 7.44 (d, J = 16.1 Hz, 1H), 7.17 (dd,
J = 8.2, 1.8 Hz, 1H), 7.05 (d, J = 1.8 Hz, 1H), 7.01 (d, J =
15.5 Hz, 1H), 6.99 (dd, J = 8.1, 1.9 Hz, 1H), 6.94-6.95 (m, 2H),
6.92 (d, J = 8.2 Hz, 1H), 6.84-6.87 (m, 2H), 6.80 (d, J = 9.0 Hz,
1H), 6.70 (d, J = 16.1 Hz, 1H), 5.92 (s, 1H), 5.92 (s, 1H), 3.93 (s,
3H), 3.88 (s, 3H), 3.84 (s, 3H), 3.67 (s, 3H). 13C NMR (400 MHz,
CDCl3) δ197.61, 187.76, 153.21, 152.49, 148.63, 148.41, 146.76,
145.17, 140.88, 136.27, 127.41, 126.87, 125.31, 123.96, 123.58,
123.43, 120.15, 117.70, 114.91, 114.83, 114.76, 111.92, 110.31,
109.72, 56.05, 56.01, 55.95, 55.74.
(1E,6E)-1,7-Bis(3,4-dimethoxyphenyl)-4-(4-ethylbenzylidene)-
hepta-1,6-diene-3,5-dione (24). 4-Ethylbenzaldehyde (268 mg,
2 mmol) and 3 (396 mg, 1 mmol) were used as reactants and
the raw product was purified by column chromatography (petro-
leum ether/acetic ether 4:1) to give 316 mg of 24 as a yellow
powder, yield 62%. HPLC tR=27.9 min; Rf=0.37 (petroleum
ether/acetic ether 3:2). HR-MS calcd for C32H32O6: 512.2193,
1
found 512.2196. H NMR (400 MHz, CDCl3) δ 7.85 (s, 1H),
7.78 (d, J = 15.4 Hz, 1H), 7.49 (d, J = 16.2 Hz, 1H), 7.43 (d, J =
8.2 Hz, 2H), 7.19 (dd, J = 8.5, 1.9 Hz, 1H), 7.16 (d, J = 8.2 Hz,
2H), 7.07 (d, J = 1.9 Hz, 1H), 7.06 (dd, J = 8.3, 1.8 Hz, 1H), 6.99
(d, J = 1.8Hz, 1H), 7.00 (d, J = 15.4 Hz, 1H), 6.86 (d, J = 8.3 Hz,
1H), 6.82 (d, J = 8.5 Hz, 1H), 6.83 (d, J = 16.2 Hz, 1H), 3.91
(s, 6H), 3.88 (s, 3H), 3.87 (s, 3H), 2.62 (q, J = 7.6 Hz, 2H), 1.19
(t, J =7.6 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ 198.33,
187.04, 151.85, 151.60, 149.20, 149.18, 147.21, 147.17, 145.18,
140.76, 139.81, 130.87, 130.61, 130.23, 128.40, 127.92, 127.71,
127.10, 125.67, 123.64, 123.39, 120.08, 111.08, 110.99, 110.51,
110.09, 55.99, 55.88, 28.74, 15.09.
(1E,6E)-4-(2,3-Dimethoxybenzylidene)-1,7-bis(3,4-dimethoxy-
phenyl)hepta-1,6-diene-3,5-dione (25). 2,3-Dimethoxybenzalde-
hyde (332 mg, 2 mmol) and 3 (396 mg, 1 mmol) were used as
reactants and the raw product was purified by column chroma-
tography (petroleum ether/acetic ether 3:1) to give 225 mg of 25
as a yellow powder, yield 41%. HPLC tR=21.0 min; Rf=0.26
(petroleum ether/acetic ether 3:2). HR-MS calcd for C32H32O8:
544.2092, found 544.2090. 1H NMR (400 MHz, CDCl3) δ 8.14
(s, 1H), 7.77 (d, J=15.4 Hz, 1H), 7.43 (d, J=16.1 Hz, 1H), 7.20
(dd, J=8.3, 1.9 Hz, 1H), 7.08 (d, J=1.9 Hz, 1H), 7.02 (dd, J=
8.4, 1.9 Hz, 1H), 7.01 (d, J=15.4 Hz, 1H), 6.98 (dd, J=7.8,
1.8 Hz, 1H), 6.96 (d, J=1.9 Hz, 1H), 6.95 (t, J=7.8 Hz, 1H), 6.90
(dd, J=7.8, 1.8 Hz, 1H), 6.87 (d, J=8.4 Hz, 1H), 6.81 (d, J=
8.3 Hz, 1H), 6.72 (d, J=16.1 Hz, 1H), 3.93-3.90 (m, 9H), 3.89 (s,
3H), 3.86 (s, 3H), 3.84 (s, 3H). 13C NMR (101 MHz, CDCl3)
δ 197.35, 187.65, 152.70, 151.77, 151.65, 149.25, 148.34, 146.66,
145.20, 141.65, 136.09, 128.30, 127.79, 127.27, 125.78, 124.10,
123.51, 123.38, 121.89, 120.42, 114.42, 111.16, 111.06, 110.54,
110.06, 61.35, 56.01, 55.90, 55.86.
(1E,6E)-4-(3,4-Dimethoxybenzylidene)-1,7-bis(3,4,5-trimethoxy-
phenyl)hepta-1,6-diene-3,5-dione (26). 3,4-Dimethoxybenzalde-
hyde (332 mg, 2 mmol) and 4 (456 mg, 1 mmol) were used as
reactants and the raw product was purified by column chroma-
tography (petroleum ether/acetic ether 3:2) to give 323 mg of 26
as a yellow powder, yield 53%. HPLC tR=18.6 min; Rf=0.16
(petroleum ether/acetic ether 3:2). HR-MS calcd for C34H36O10:
604.2303, found 604.2313. 1H NMR (500 MHz, CDCl3) δ 7.82
(s, 1H), 7.72 (d, J = 15.5 Hz, 1H), 7.47 (d, J = 16.1 Hz, 1H), 7.13
(dd, J = 8.5, 2.0 Hz, 1H), 7.04 (d, J = 2.0 Hz, 1H), 7.02 (d, J =
15.5 Hz, 1H), 6.83 (d, J = 8.5 Hz, 1H), 6.83 (d, J = 16.1 Hz, 1H),
6.80 (s, 2H), 6.70 (s, 2H), 3.89 (s, 6H), 3.88 (s, 3H), 3.88 (s, 3H),
3.86 (s, 3H), 3.84 (s, 6H), 3.82 (s, 3H). 13C NMR (101 MHz, CDCl3)
δ 198.25, 186.95, 153.53, 151.43, 149.05, 146.86, 145.03, 141.21,
(1E,6E)-4-(2,4-Dimethoxybenzylidene)-1,7-bis(4-hydroxy-3-
methoxyphenyl)hepta-1,6-diene-3,5-dione (31). 2,4-Dimethoxy-
benzaldehyde (332 mg, 2 mmol) and 1 (368 mg, 1 mmol) were
used as reactants, and column chromatography (petroleum ether/
acetic ether 2:1) gave 268 mg of 31 as a yellow powder, yield
52%. HPLC tR =11.1 min; Rf =0.29 (petroleum ether/acetic
ether 1:1). HR-MS calcd for C30H28O8: 516.1779, found
516.1782. 1H NMR (400 MHz, CDCl3) δ 8.19 (s, 1H), 7.72 (d,
J = 15.4 Hz, 1H), 7.45 (d, J = 16.1 Hz, 1H), 7.35 (d, J = 8.4 Hz,
1H), 7.17 (d, J = 8.4 Hz, 1H), 7.05 (s, 1H), 7.01 (d, J = 8.4 Hz,
1H), 7.00 (d, J = 15.4 Hz, 1H), 6.96 (s, 1H), 6.91 (d, J = 8.2 Hz,
1H), 6.87 (d, J = 8.2 Hz, 1H), 6.73 (d, J = 16.1 Hz, 1H), 6.41

8270 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23
Qiu et al.
(s, 1H), 6.39 (d, J = 8.4 Hz, 1H), 5.89 (m, 2H), 3.93 (s, 3H), 3.88
(s, 3H), 3.87 (s, 3H), 3.79 (s, 3H). 13C NMR (101 MHz, CDCl3) δ
198.45, 187.61, 163.15, 159.85, 148.55, 148.24, 146.76, 146.72,
146.41, 144.56, 138.37, 136.21, 131.77, 127.56, 126.97, 125.46,
123.87, 123.37, 120.34, 115.82, 114.79, 114.75, 110.36, 109.81,
105.15, 98.28, 56.05, 55.95, 55.53, 55.43.
J=8.3, 1.5 Hz, 1H), 7.06 (d, J=1.5 Hz, 1H), 6.98 (dd, J=8.2,
1.5 Hz, 1H), 6.98 (d, J=15.5 Hz, 1H), 6.97 (d, J=1.5 Hz, 1H),
6.88-6.96 (m, 4H), 6.86 (d, J=8.2 Hz, 1H), 6.70 (d, J=16.1 Hz,
1H), 5.92 (s, 1H), 5.90 (s, 1H), 3.93 (s, 3H), 3.92 (s, 3H), 3.88
(s, 3H), 3.84 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 197.56,
187.64, 152.67, 148.72, 148.54, 148.29, 147.05, 146.81, 145.46,
141.56, 136.00, 128.25, 127.30, 126.76, 125.45, 124.11, 123.95,
123.73, 121.82, 120.01, 114.86, 114.80, 114.35, 110.26, 109.81,
61.37, 56.04, 55.93, 55.83.
(1E,6E)-4-(3-Fluorobenzylidene)-1,7-bis(4-hydroxy-3-methoxy-
phenyl)hepta-1,6-diene-3,5-dione (32). 3-Fluorobenzaldehyde
(248 mg, 2 mmol) and 1 (368 mg, 1 mmol) were used as reactants,
and column chromatography (petroleum ether/acetic ether
3:1) gave 329 mg of 32 as a yellow powder, yield 69%. HPLC
tR = 12.2 min; Rf = 0.32 (petroleum ether/acetic ether 1:1).
HR-MS calcd for C28H23O6F1: 474.1473, found 474.1476. 1H
NMR (400 MHz, acetone) δ 7.90 (s, 1H), 7.69 (d, J = 15.5 Hz,
1H), 7.50 (d, J = 16.2 Hz, 1H), 7.39 (m, 2H), 7.39 (d, J = 15.5 Hz,
1H), 7.32 (d, J = 1.8 Hz, 1H), 7.31 (dd, J = 8.2, 1.8 Hz, 1H),
7.28 (dd, J = 8.4, 1.8 Hz, 1H), 7.12-7.17 (m, 1H), 7.11 (dd,
J = 8.2, 1.8 Hz, 1H), 6.90 (d, J = 8.2 Hz, 1H), 6.85 (d, J =
16.2 Hz, 1H), 6.83 (d, J = 8.2 Hz, 1H), 3.90 (s, 3H), 3.88 (s,
3H). 13C NMR (101 MHz, acetone) δ 197.59, 188.25, 163.48
(d, J = 244.6 Hz), 150.86, 150.62, 148.83, 148.77, 148.08,
145.78, 143.93, 138.30, 137.46 (d, J=7.9 Hz), 131.54 (d, J=
8.4 Hz), 127.86, 127.33, 127.00 (d, J=2.8 Hz), 125.68, 124.95,
124.35, 119.85, 117.47 (d, J=21.4 Hz), 117.00 (d, J=22.6 Hz),
116.31, 116.18, 112.43, 111.66, 56.42, 56.35.
(1E,6E)-4-(4-Fluorobenzylidene)-1,7-bis(4-hydroxy-3-methoxy-
phenyl)hepta-1,6-diene-3,5-dione (33). 4-Fluorobenzaldehyde
(248 mg, 2 mmol) and 1 (368 mg, 1 mmol) were used as reactants,
and column chromatography (petroleum ether/acetic ether
3:1) gave 311 mg of 33 as a yellow powder, yield 66%. HPLC
tR = 12.3 min; Rf = 0.31 (petroleum ether/acetic ether 1:1).
HR-MS calcd for C28H23O6F1: 474.1473, found 474.1474. 1H
NMR (400 MHz, CDCl3) δ 7.80 (s, 1H), 7.77 (d, J = 15.4 Hz,
1H), 7.50 (d, J = 8.5 Hz, 1H), 7.48 (d, J = 8.0 Hz, 1H), 7.45
(d, J = 16.2 Hz, 1H), 7.16 (dd, J = 8.2, 1.8 Hz, 1H), 7.04 (d,
J = 2.0 Hz, 1H), 7.03 (dd, J = 8.2, 2.0 Hz, 1H), 7.03 (d, J =
8.2 Hz, 1H), 7.01 (d, J = 8.5 Hz, 1H), 6.97 (d, J = 1.8 Hz, 1H),
6.92 (d, J = 8.0 Hz, 1H), 6.93 (d, J = 15.4 Hz, 1H), 6.88 (d,
J = 8.2 Hz, 1H), 6.77 (d, J = 16.2 Hz, 1H), 5.94 (s, 2H), 3.93
(s, 3H), 3.90 (s, 3H). 13C NMR (101 MHz, acetone) δ 197.89,
188.22, 164.33 (d, J = 249.7 Hz), 150.76, 150.49, 148.78,
148.73, 147.77, 145.41, 142.61, 138.69, 133.26 (d, J =
8.7 Hz), 131.51 (d, J = 3.1 Hz), 127.89, 127.35, 125.74,
124.81, 124.23, 119.89, 116.58 (d, J = 21.9 Hz), 116.27,
116.15, 112.36, 111.63, 56.38, 56.31
(1E,6E)-4-(4-Ethylbenzylidene)-1,7-bis(4-hydroxy-3-methoxy-
phenyl)hepta-1,6-diene-3,5-dione (34). 4-Ethylbenzaldehyde (268 mg,
2 mmol) and 1 (368 mg, 1 mmol) were used as reactants, and column
chromatography (petroleum ether/acetic ether 3:1) gave 288 mg of 34
as a yellow powder, yield 59%. HPLC tR=16.9 min; Rf=0.36 (petro-
leum ether/acetic ether 1:1). HR-MS calcd for C30H28O6: 484.1880,
found 484.1881. 1H NMR (400 MHz, CDCl3) δ7.83 (s, 1H), 7.75 (d,
J = 15.4 Hz, 1H), 7.47 (d, J = 16.2 Hz, 1H), 7.42 (d, J = 8.2 Hz,
2H), 7.13-7.16 (m, 3H), 7.03 (d, J = 1.5 Hz, 1H), 7.01 (dd, J = 8.2,
1.6 Hz, 1H), 6.97 (d, J = 1.6 Hz,1H), 6.96 (d, J = 15.4 Hz, 1H), 6.91
(d, J = 8.2 Hz, 1H), 6.87 (d, J = 8.2 Hz, 1H), 6.80 (d, J = 16.2 Hz,
1H), 3.91(s, 3H), 3.87(s, 3H), 2.61 (q, J= 7.56 Hz, 2H), 1.19 (t, J =
7.59 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ198.43, 187.03, 148.84,
148.54, 147.51, 147.16, 146.82, 146.79, 145.39, 140.69, 139.77, 130.86,
130.61, 128.40, 127.28, 126.66, 125.38, 124.03, 123.67, 119.77, 114.86,
114.83, 110.36, 109.97, 56.06, 55.95, 28.75, 15.10.
(1E,6E)-1,7-Bis(4-hydroxy-3-methoxyphenyl)-4-(3-methoxy-
benzylidene)hepta-1,6-diene-3,5-dione (36). 3-Methoxybenzalde-
hyde (272 mg, 2 mmol) and 1 (368 mg, 1 mmol) were used as
reactants, and column chromatography (petroleum ether/acetic
ether 2:1) gave 285 mg of 36 as a yellow powder, 59%. HPLC
tR=11.6 min; Rf=0.24 (petroleum ether/acetic ether 1:1). HR-
1
MS calcd for C29H26O7: 486.1679, found 486.1674. H NMR
(400 MHz, CDCl3) δ 7.81 (s, 1H), 7.76 (d, J = 15.4 Hz, 1H), 7.45
(d, J = 16.1 Hz, 1H), 7.24 (dd, J =8.0, 8.0 Hz, 1H), 7.16 (dd,
J = 8.2, 1.9 Hz, 1H), 7.0-7.09 (m, 1H), 7.04 (d, J = 1.9 Hz, 1H),
7.02-7.03 (m, 1H), 7.02 (dd, J = 8.2, 1.9 Hz, 1H), 6.96 (d, J =
15.4 Hz, 1H), 6.95 (d, J = 1.9 Hz, 1H), 6.91 (d, J = 8.2 Hz, 1H),
6.87-6.89 (m, 1H), 6.87 (d, J = 8.2 Hz, 1H), 6.77 (d, J = 16.1
Hz, 1H), 5.97 (s, 2H), 3.92 (s, 3H), 3.88 (s, 3H), 3.76 (s, 3H). 13
C
NMR (101 MHz, CDCl3) δ 197.92, 187.01, 159.64, 148.86,
148.59, 147.51, 146.80, 146.77, 145.67, 141.09, 140.31, 134.86,
129.81, 127.25, 126.64, 125.28, 124.07, 123.77, 122.80, 119.74,
116.34, 115.15, 114.85, 114.83, 110.33, 109.90, 56.07, 55.96,
55.27.
(1E,6E)-1,7-Bis(3,4-dimethoxyphenyl)-4-((5-methylfuran-2-yl)-
methylene)hepta-1,6-diene-3,5-dione (37). 5-Methylfuran-2-car-
baldehyde (220 mg, 2 mmol) and 3 (396 mg, 1 mmol) were used
as reactants and the raw product was purified by column chro-
matography (petroleum ether/acetic ether 3:1) to give 276 mg of
37 as a yellow powder, yield 57%. HPLC tR=16.3 min; Rf=0.32
(petroleum ether/acetic ether 1:1). HR-MS calcd for C29H28O7:
488.1830, found 488.1828. 1H NMR (500 MHz, CDCl3) δ 7.74
(d, J = 15.4 Hz, 1H), 7.60 (s, 1H), 7.46 (d, J = 16.1 Hz, 1H), 7.17
(dd, J = 8.4, 1.9 Hz, 1H), 7.11 (dd, J = 8.4, 1.9 Hz, 1H), 7.05 (d,
J = 1.9 Hz, 2H), 6.93 (d, J = 15.4 Hz, 1H), 6.90 (d, J = 16.1 Hz,
1H), 6.84 (d, J = 8.4 Hz, 2H), 6.72 (d, J = 3.4 Hz, 1H), 6.08 (dq,
J = 3.4, 0.9 Hz, 1H), 3.89-3.90 (m, 12H), 2.26 (d, J = 0.9 Hz,
3H). 13C NMR (101 MHz, CDCl3) δ 197.53, 185.99, 157.42,
151.68, 151.55, 149.31, 149.22, 148.45, 146.25, 144.64, 134.83,
127.86, 127.39, 126.67, 126.25, 123.30, 123.25, 120.24, 120.16,
111.13, 110.60, 110.21, 109.74, 56.05, 56.03, 56.00, 55.95, 13.99.
General Procedures for Synthesis of 18 and 29. 3,4-Dimethox-
ybenzaldehyde (831 mg, 5 mmol) or 4-hydroxy-3-methoxyben-
zaldehyde (760 mg, 5 mmol) was mixed with pentane-2,4-dione
(42 mg, 1 mmol) and 25 mL of toluene in a three-neck rounded
flask equipped a water dispenser. Then pyridine (7.9 mg, 0.1 mmol,
in 0.2 mL of toluene) and acetic acid (9.6 mg, 0.16 mmol, in
0.2 mL of toluene) were added in as catalysts. The reaction mix-
ture was stirred at 140 °C overnight, and the generated water was
removed by water dispenser during the whole reaction. After the
mixture was washed with water to remove pyridine and acetic
acid, the solvent was evaporated to get raw product. Finally the
raw product was purified by column chromatography to give
18 and 29, respectively.
(1E,6E)-4-(3,4-Dimethoxybenzylidene)-1,7-bis(3,4-dimethoxy-
phenyl)hepta-1,6-diene-3,5-dione (18). Column chromatography
(petroleum ether/acetic ether 3:2) gave 381 mg of 18 as a yellow
powder, yield 70%. HPLC tR=15.7 min; Rf=0.28 (petroleum
(1E,6E)-4-(2,3-Dimethoxybenzylidene)-1,7-bis(4-hydroxy-3-
methoxyphenyl)hepta-1,6-diene-3,5-dione (35). 2,3-Dimethoxy-
benzaldehyde (332 mg, 2 mmol) and 1 (368 mg, 1 mmol) were
used as reactants, and column chromatography (petroleum ether/
acetic ether 2:1) gave 180 mg of 35 as a yellow powder, yield
35%. HPLC tR =18.8 min; Rf =0.24 (petroleum ether/acetic
ether 1:1). HR-MS calcd for C30H28O8 [M - H]-: 515.1706,
1
ether/acetic ether 1:1). H NMR (500 MHz, CDCl3) δ 7.80 (s,
1H), 7.75 (d, J=15.4 Hz, 1H), 7.51 (d, J=16.1 Hz, 1H), 7.18 (dd,
J=8.4, 2.0 Hz, 1H), 7.13 (dd, J=8.4, 2.0 Hz, 1H), 7.05-7.06
(m, 3H), 6.98 (d, J=2.0 Hz, 1H), 6.99 (d, J=15.4 Hz, 1H), 6.85
(d, J=8.4 Hz, 1H), 6.81 (d, J=8.4 Hz, 2H), 6.81 (d, J=16.1 Hz,
1H), 3.89 (s, 6H), 3.87 (s, 3H), 3.86 (s, 6H), 3.80 (s, 3H). 13C
NMR (101 MHz, CDCl3) δ 198.39, 186.84, 152.01, 151.66,
151.21, 149.37, 149.30, 148.95, 146.90, 144.88, 140.65, 139.00,
1
found 515.1696. H NMR (400 MHz, CDCl3) δ 8.13 (s, 1H),
7.75 (d, J=15.5 Hz, 1H), 7.41 (d, J=16.1 Hz, 1H), 7.16 (dd,

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23 8271
127.86, 127.14, 126.39, 125.61, 124.98, 123.54, 123.27, 120.29,
112.99, 111.24, 111.19, 111.16, 110.75, 110.33, 56.05, 56.01,
55.94, 55.90, 55.87.
viability with Thermo Scientific’s Multiskan MK3 plate reader to
obtain the GI50 values.
Alamar Blue Method. Cells were maintained in RPMI with
10% fetal bovine serum (FBS) and penicillin/streptomycin in a
37 °C incubator with 5% CO2. Cells were plated in 384-well
plates and were treated with test agents on the following day and
further incubated for 72 h before the viability assay was carried
out. The Alamar Blue assay was performed to evaluate cell via-
bility with PerkinElmer’s EnVision multimode plate reader to
obtain the GI50 values.42,43 The GI50 is defined as the concen-
tration of agents that decreases viability by 50% in a total cell
population compared to control cells with solvent vehicle at the
end of the incubation period.
NF-KB Translocation Assay. In brief, A549 cell were seeded in
384-well plates, incubated for 15 h, then incubated with various
concentrations of compounds for 30 min. TNF-R (10 ng/mL,
final) was added to cells to stimulate the NF-κB cell signaling
pathway for 30 min. Cells were then fixed with paraformalde-
hyde (2%, 100 μL) and permeabilized with Triton X-100 (0.1%,
100 μL). Finally, rabbit anti-p65 NF-κB antibody and goat anti-
rabbit IgG with conjugated Alexa Fluor 488 were used to stain
NF-κB, and Hoechst 33342 was used to stain nucleus. Fluores-
cence intensity of NF-κB and nuclear staining were recorded
with an automated imaging system, ImageXpress5000A (Molecular
Devices). The levels of NF-κB translocation were calculated and
expressed as the difference between average fluorescence inten-
sity in the nucleus and in the cytoplasm. After stimulation with
TNF-R, the inhibitory effect of test compounds on TNFR in-
duced NF-κB translocation was expressed as a percentage of
fluorescence intensity difference (in nucleus and in cytoplasm) in
the control wells (TNFR only) after subtracting background (no
TNFR treatment). The IC50 of test compounds in this NF-κB
translocation assay stands for the concentration of a compound
required to induce 50% inhibition. All data are obtained as
average values from triplicate samples, and the experiments
were repeated at least three times.
(1E,6E)-4-(4-Hydroxy-3-methoxybenzylidene)-1,7-bis(4-hy-
droxy-3-methoxyphenyl)hepta-1,6-diene-3,5-dione (29). Column
chromatography (petroleum ether/acetic ether 2:3) gave 223 mg
of 29 as a yellow powder, yield 44%. HPLC tR=7.9 min; Rf=
0.39 (petroleum ether/acetic ether 1:2). HR-MS calcd for C29-
H26O8: 502.1628, found 502.1623. 1H NMR (400 MHz, CDCl3)
δ 7.80 (s, 1H), 7.75 (d, J = 15.4 Hz, 1H), 7.49 (d, J = 16.1 Hz,
1H), 7.16 (dd, J = 8.2, 1.7 Hz, 1H), 7.09 (dd, J = 8.3, 1.8 Hz,
1H), 7.04 (m, 2H), 7.03 (dd, J = 8.2, 1.8 Hz, 1H), 6.96 (d, J = 1.7
Hz, 1H), 6.95 (d, J = 15.4 Hz, 1H), 6.91 (d, J = 8.2 Hz, 1H), 6.88
(d, J = 8.2 Hz, 1H), 6.87 (d, J = 8.3 Hz, 1H), 6.80 (d, J =
16.1 Hz, 1H), 5.95 (s, 1H), 5.93 (s, 1H), 5.89 (s, 1H), 3.92 (s, 3H),
3.89 (s, 3H), 3.83 (s, 3H). 13C NMR (101 MHz, acetone) δ
198.90, 188.17, 150.69, 150.37, 150.05, 148.84, 148.77, 148.37,
147.20, 144.77, 140.74, 140.11, 128.05, 127.48, 126.61, 126.13,
126.01, 124.64, 124.05, 120.15, 116.31, 116.25, 116.23, 114.39,
112.38, 111.75, 56.43, 56.35, 56.25.
General Procedures for the Synthesis of 38-40. Triethoxy-
methane (1.48 g, 10 mmol) and 1 mmol of corresponding sym-
metrical curcuminoids were dissolved in 5 mL of acetic anhy-
dride. The reaction mixture was stirred at 140 °C for 5 h and then
transferred to room temperature, and 10 mL of H2O was added
for an additional 10 min. The raw product was extracted by ethyl
acetate and then purified by column chromatography or recrys-
tallization.
4,40-((1E,6E)-4-(Hydroxymethylene)-3,5-dioxohepta-1,6-diene-
1,7-diyl)bis(2-methoxy-4,1-phenylene) Diacetate (38). 1 (368 mg,
1 mmol) was used as reactant and the raw product was purified
by column chromatography (petroleum ether/acetic ether 4:1)
to give 140 mg of 38 as a yellow powder, yield 29%. HPLC tR=
10.4 min; Rf=0.44 (petroleum ether/acetic ether 3:2). HR-MS
calcd for C26H24O9: 480.1415, found 480.1409. 1H NMR
(400 MHz, CDCl3) δ 10.35 (s, 1H), 7.93 (d, J=15.5 Hz, 2H),
7.76 (d, J=15.5 Hz, 2H), 7.27 (dd, J=8.2, 1.6 Hz, 2H), 7.21
(d, J=1.6 Hz, 2H), 7.10 (d, J=8.2 Hz, 2H), 3.91 (s, 6H), 2.34 (s,
6H). 13C NMR (400 MHz, CDCl3) δ 189.36, 186.95, 168.67,
151.55, 145.09, 142.17, 133.63, 123.44, 122.18, 120.47, 112.77,
111.94, 56.03, 20.67.
Clonogenic Assay. A549 cells were plated in low density in a
12-well plate and treated with test compounds the following day
and every 3 days thereafter. On day 9 after colonies were formed,
cells were fixed with trichloroacetic acid (TCA) (10%) for 30 min
at 4 °C, washed with water, stained with SRB, and then washed
with acetic acid (1%). The images of the plate were scanned, and
colonies were counted using image processing and analysis in
Java (Image J, Research Service Branch, NIH).
(1E,6E)-4-(Hydroxymethylene)-1,7-bis(4-methoxyphenyl)-
hepta-1,6-diene-3,5-dione (39). 2 (336 mg, 1 mmol) was used as
reactant and the raw product was purified by column chroma-
tography (petroleum ether/acetic ether 9:1) to give 255 mg of 39
as a yellow powder, yield 70%. HPLC tR=9.9 min; Rf=0.37
(petroleum ether/acetic ether 3:1). HR-MS calcd for C22H20O5:
364.1305, found 364.1292. 1H NMR (400 MHz, CDCl3) δ 10.35
(s, 1H), 7.94 (d, J = 15.4 Hz, 2H), 7.72 (d, J = 15.4 Hz, 2H), 7.61
IKK Assays. Cell Based IKK Assay. Cells were treated with
various test agents for a defined period of time as described in
each figure and were lysed for detection of phosphorylation
status of IκB, a readout for IKK activity, and total IκB protein.
The NP-40 lysates buffer was used (1.0% NP-40, 10 mM Hepes,
pH 7.4, 150 mM NaCl, 5 mM NaF, 2 mM Na3VO4, 5 mM Na4-
P2O7, 10 μg/mL aprotinin, 10 μg/mL leupeptin, 1 mM PMSF).
For Western blotting, equal volumes of cell lysate were subject
to electrophoresis on SDS-PAGE (12.5%). Proteins were then
electrotransferred to a nitrocellulose membrane (GE Water
and Process Technologies, Trevose, PA) as described previ-
ously.44 Membranes were blocked in a solution of 5% nonfat
dry milk in TBS-T buffer (20 mM Tris, pH 7.6, 500 mM NaCl,
0.5% Tween-20) for 30 min followed by incubation with anti-
pS32-I-κB or I-κB antibodies from Cell Signaling (Beverly,
MA) for at least 2 h. The membrane was then washed and treat-
ed with the corresponding horseradish peroxidase conjugated
anti-mouse immunoglobulin [Ig] or anti-rabbit Ig as indicated.
Immunodetection was performed using West Pico (Pierce,
Rockford, IL) or West Dura (Pierce) followed by imaging on
Kodak’s Image Station 2000R (New Haven, CT).
(d, J = 8.2 Hz, 4H), 6.94 (d, J = 8.2 Hz, 4H), 3.87 (s, 6H). 13
C
NMR (101 MHz, CDCl3) δ 189.48, 187.14, 162.15, 145.44,
130.79, 127.60, 117.94, 114.54, 55.46.
(1E,6E)-1,7-Bis(3,4-dimethoxyphenyl)-4-(hydroxymethylene)-
hepta-1,6-diene-3,5-dione (40). 3 (396 mg, 1 mmol) was used as
reactant and the raw product was purified by recrystallization in
acetic anhydride to give 273 mg of 40 as an orange powder, yield
64%. HPLC tR =14.6 min; Rf =0.40 (petroleum ether/acetic
ether 3:2). HR-MS calcd for C24H24O7, 424.1517; found,
1
424.1518. H NMR (400 MHz, CDCl3) δ 10.37 (s, 1H), 7.93
(d, J=15.4 Hz, 2H), 7.71 (d, J = 15.4 Hz, 2H), 7.26 (d, J=
8.1 Hz, 2H), 7.17 (s, 2H), 6.91 (d, J = 8.1 Hz, 2H), 3.97 (s, 6H),
3.95 (s, 6H). 13C NMR (101 MHz, CDCl3) δ 189.39, 187.23,
151.98, 149.35, 145.80, 127.83, 124.03, 118.04, 111.12, 110.26,
56.04, 56.01.
Biology. Cell Viability Assay. Sulforhodamine B (SRB) Method.
Cells were maintained in RPMI with 5% fetal bovine serum (FBS)
and penicillin/streptomycin in a 37 °C incubator with 5% CO2. Cells
were plated in 96-well plates and were treated with test agents on the
following day and further incubated for 48 h before viability assay
was carried out. The SRB assay was performed to evaluate cell
In Vitro IKK Assay. Activated recombinant IKKβ in MOPS
buffer (8 mM MOPS-NaOH, pH 7.0, 200 μM EDTA, 15 mM
MgCl2) from Upstate Cell Signaling Solutions (Lake Placid, NY)
was used for the in vitro kinase assay. The test compounds were
incubated with IKKβ (40 ng) for 30 min at room temperature.

8272 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23
Qiu et al.
The addition of Mg-ATP mix (15 mM MgCl2, 100 μM ATP,
8 mM MOPS-NaOH, pH 7.0, 5 mM β-glycerophosphate, 1 mM
EGTA, 200 nM sodium orthovanadate, 200 nM DTT), purified
GST- I-κBR (5 μg), and [γ-32P]ATP (0.5 μCi) in a final volume of
25 μL started the reaction, which was allowed to proceed at
30 °C for 30 min. Reactions were terminated by boiling the
kinase reaction solution in a 6ꢀ SDS sample buffer for 5 min.
The samples were resolved on SDS-PAGE (12.5%). The gel
was stained with Coomassie Blue dye (0.05%). 32P incorpora-
tion into GST-I-κBR was assessed by autoradiography. In ad-
dition, the radiolabeled GST-I-κBR protein bands were excised
for quantification with a scintillation counter (Beckman LS 6500,
Beckman Coulter, Fullerton, CA).
Molecular Modeling. Homology Model Building. IKKβ cat-
alytic domain sequence (code O14920) was collected from UniProt
(http://www.uniprot.org/), and C terminal amino acid residues
after G218, which are difficult to be modeled, were deleted since
it is far away from ATP binding pocket. The sequence similarity
search of IKKβ catalytic domain was performed using the BLAST
protocol which is built in Discovery Studio 2.1 (Accelrys Inc.).
Four templates 2JC6, 1A06, 2QNJ, and 2A2A were chosen from
Protein Data Bank (PDB). The C terminal amino acids after a
conserved glycine were deleted from all the templates. The auto-
mated sequence alignment and analysis of template and target
were carried out by Discovery Studio 2.1.
The homology modeling of the IKKβ catalytic domain was
carried out by using the built-in Modeler package of Discovery
Studio 2.1. The four different kinase templates sequences were
aligned together with the IKKβ sequence, and this alignment
was supplied along with the 3D coordinates of templates as an
input to the program. The best model given by the program was
chosen, and energy minimization was done by using the Powell
method for about 1000 iterations, with Tripos force field in
Sybyl 7.3.5. The structural analysis was carried out with the
Ramanchandran plot in Sybyl 7.3.5.
Small Molecular Preparation. The 3D structure of ATP was
extracted from the crystal structures of the phosphorylase
kinase (PDB code 1PHK, 28.6% sequence identity with IKKβ
catalytic domain). The geometries of compound 17 were energy
minimized using density functional theory. These calculations
were performed with the B3LYP hybrid functional and the
6-31G basis set implemented in Gaussian 03, revision E.01, soft-
ware package.45 After energy minimization, it adopts a propel-
ler-shaped conformation.
(3) Bonizzi, G.; Karin, M. The two NF-κB activation pathways and
their role in innate and adaptive immunity. Trends Immunol. 2004,
25, 280–288.
(4) Shukla, S.; MacLennan, G.; Fu, P.; Patel, J.; Marengo, S.; Resnick,
M.; Gupta, S. Nuclear factor-nB/p65 (Rel A) is constitutively
activated in human prostate adenocarcinoma and correlates with
disease progression. Neoplasia 2004, 6, 390–400.
(5) Ni, H.; Ergin, M.; Huang, Q.; Qin, J.; Amin, H.; Martinez, R.;
Saeed, S.; Barton, K.; Alkan, S. Analysis of expression of nuclear
factor κB (NF-κB) in multiple myeloma: downregulation of NF-κB
induces apoptosis. Br. J. Haematol. 2001, 115, 279–286.
(6) Guzman, M.; Neering, S.; Upchurch, D.; Grimes, B.; Howard, D.;
Rizzieri, D.; Luger, S.; Jordan, C. Nuclear factor-κB is constitu-
tively activated in primitive human acute myelogenous leukemia
cells. Blood 2001, 98, 2301–2037.
(7) Tang, X.; Shishodia, S.; Behrens, C.; Hong, W.; Wistuba, I. Nuclear
factor-κB (NF-κB) is frequently expressed in lung cancer and pre-
neoplastic lesions. Cancer 2006, 107, 2637–2646.
(8) Huang, S.; DeGuzman, A.; Bucana, C.; Fidler, I. Nuclear factor-κB
activity correlates with growth, angiogenesis, and metastasis of
human melanoma cells in nude mice. Clin. Cancer Res. 2000, 6,
2573–2581.
(9) Farina, A.; Tacconelli, A.; Vacca, A.; Maroder, M.; Gulino, A.;
Mackay, A. Transcriptional up-regulation of matrix metallopro-
teinase-9 expression during spontaneous epithelial to neuroblast
phenotype conversion by SK-N-SH neuroblastoma cells, involved
in enhanced invasivity, depends upon GT-Box and nuclear factor
κB elements. Cell Growth Differ. 1999, 10, 353–367.
(10) Wang, C.; Mayo, M.; Korneluk, R.; Goeddel, D.; Baldwin, A., Jr.
NF-B antiapoptosis: induction of TRAF1 and TRAF2 and c-IAP1
and c-IAP2 to suppress caspase-8 activation. Science 1998, 281,
1680–1683.
(11) Karin, M. Nuclear factor-κB in cancer development and progres-
sion. Nature 2006, 441, 431–436.
(12) Qiao, L.; Zhang, H.; Yu, J.; Francisco, R.; Dent, P.; Ebert, M.;
Rocken, C.; Farrell, G. Constitutive activation of NF-κB in human
hepatocellular carcinoma: evidence of a cytoprotective role. Hum.
Gene Ther. 2006, 17, 280–290.
€
€
(13) Arlt, A.; Gehrz, A.; Muerkoster, S.; Vorndamm, J.; Kruse, M.-L.;
€
€
Folsch, U. R.; Schafer, H. Role of NF-κB and Akt/PI3K in the
resistance of pancreatic carcinoma cell lines against gemcitabine-
induced cell death. Oncogene 2003, 22, 3243–3251.
(14) Gradilone, A.; Silvestri, I.; Scarpa, S.; Morrone, S.; Gandini, O.;
Pulcinelli, F.; Gianni, W.; Frati, L.; Agliano, A.; Gazzaniga, P.
Failure of apoptosis and activation on NFκB by celecoxib and
aspirin in lung cancer cell lines. Oncol. Rep. 2007, 17, 823–828.
(15) Notarbartolo, M.; Poma, P.; Perri, D.; Dusonchet, L.; Cervello,
M.; D’Alessandro, N. Antitumor effects of curcumin, alone or in
combination with cisplatin or doxorubicin, on human hepatic
cancer cells. Analysis of their possible relationship to changes in
NF-κB activation levels and in IAP gene expression. Cancer Lett.
2005, 224, 53–65.
(16) Wang, X.; Ju, W.; Renouard, J.; Aden, J.; Minsky, S. A.; Lin, Y.
17-Allylamino-17-demethoxygeldanamycin synergistically potenti-
ates tumor necrosis factor-induced lung cancer cell death by block-
ing the nuclear factor-κB pathway. Cancer Res. 2006, 66, 1089–
1095.
Docking. Molecular docking studies of compounds with the
ATP binding pocket were performed with Surflex-Dock in Sybyl
7.3.5. Automatic was chosen as the mode of construction for the
protomol. Threshold is 0.5 and bloat is 0.
(17) Sarkar, F.; Li, Y. Soy isoflavones and cancer prevention. Cancer
Invest. 2003, 21, 744–757.
(18) Holmes-McNary, M.; Baldwin, A., Jr. Chemopreventive proper-
ties of trans-resveratrol are associated with inhibition of activation
of the IκB kinase. Cancer Res. 2000, 60, 3477–3483.
(19) Plummer, S.; Holloway, K.; Manson, M.; Munks, R.; Kaptein, A.;
Farrow, S.; Howells, L. Inhibition of cyclo-oxygenase 2 expression
in colon cells by the chemopreventive agent curcumin involves
inhibition of NF-κB activation via the NIK/IKK signalling com-
plex. Oncogene 1999, 18, 6013–6020.
(20) Maheshwari, R.; Singh, A.; Gaddipati, J.; Srimal, R. Multiple
biological activities of curcumin: a short review. Life Sci. 2006, 78,
2081–2087.
Acknowledgment. We thank Mary Puckett for critical read-
ing of the manuscript. This work was supported in part by grants
from the MOST of China (863 Program 2008AA02Z304) and
NSFC (Grant 30973619) (X.B.) and from the U.S. National
Institutes of Health (Grant P01 CA116676 to H.F.) and Emory
Faculty Distinction Fund (H.F.). H.F. is a GCC Distin-
guished Cancer Scholar and a GRA Distinguished Investiga-
tor. Y.D. is a recipient of Emory Head and Neck Cancer
SPORE Career Development Award (Grant P50 CA128613).
Supporting Information Available:
1H NMR, 13C NMR,
(21) Cheng, A.; Hsu, C.; Lin, J.; Hsu, M.; Ho, Y.; Shen, T.; Ko, J.; Lin,
J.; Lin, B.; Ming-Shiang, W. Phase I clinical trial of curcumin, a
chemopreventive agent, in patients with high-risk or pre-malignant
lesions. Anticancer Res. 2001, 21, 2895–2900.
HRMS, and HPLC spectra; additional cancer cell growth inhibi-
tion data and molecular modeling information. This material is
available free of charge via the Internet at http://pubs.acs.org.
(22) Singh, S.; Aggarwal, B. Activation of transcription factor NF-κB is
suppressed by curcumin (diferuloylmethane). J. Biol. Chem. 1995,
270, 24995–25000.
References
(23) Leu, T.; Maa, M. The molecular mechanisms for the antitumori-
genic effect of curcumin. Curr. Med. Chem.: Anti-Cancer Agents
2002, 2, 357–370.
(1) Ghosh, S.; May, M.; Kopp, E. NF-κB and Rel proteins: evolution-
arily conserved mediators of immune responses. Annu. Rev. Immunol.
1998, 16, 225–260.
(2) Hayden, M.; Ghosh, S. Shared principles in NF-κB signaling. Cell
(24) Milacic, V.; Banerjee, S.; Landis-Piwowar, K.; Sarkar, F.;Majumdar,
A.; Dou, Q. Curcumin inhibits the proteasome activity in human
2008, 132, 344–362.

Article
colon cancer cells in vitro and in vivo. Cancer Res. 2008, 68, 7283–
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23 8273
anti-cancer and anti-angiogenesis agents. Bioorg. Med. Chem. 2004,
12, 3871–3883.
7292.
(25) Anand, P.; Kunnumakkara, A.; Newman, R.; Aggarwal, B. Bio-
availability of curcumin: problems and promises. Mol. Pharma-
ceutics 2007, 4, 807–818.
(26) Ohtsu, H.; Xiao, Z.; Ishida, J.; Nagai, M.; Wang, H.; Itokawa, H.;
Su, C.; Shih, C.; Chiang, T.; Chang, E. Antitumor agents. 217.
Curcumin analogues as novel androgen receptor antagonists with
potential as anti-prostate cancer agents. J. Med. Chem. 2002, 45,
5037–5042.
(27) Lin, L.; Shi, Q.; Nyarko, A.; Bastow, K.; Wu, C.; Su, C.; Shih, C.;
Lee, K. Antitumor agents. 250. Design and synthesis of new cur-
cumin analogs as potential anti-prostate cancer agents. J. Med.
Chem. 2006, 49, 3963–3972.
(37) Kasinski, A.; Du, Y.; Thomas, S.; Zhao, J.; Sun, S.; Khuri, F.; Wang,
C.; Shoji, M.; Sun, A.; Snyder, J. Inhibition of IκB kinase-nuclear
factor-κB signaling pathway by 3,5-bis(2-flurobenzylidene)piperidin-
4-one (EF24), a novel monoketone analog of curcumin. Mol. Phar-
macol. 2008, 74, 654.
(38) Aggarwal, S.; Ichikawa, H.; Takada, Y.; Sandur, S. K.; Shishodia,
S.; Aggarwal, B. B. Curcumin (diferuloylmethane) down-regulates
expression of cell proliferation and antiapoptotic and metastatic
gene products through suppression of IκBR kinase and Akt activa-
tion. Mol. Pharmacol. 2006, 69, 195–206.
(39) Baud, V.; Karin, M. Signal transduction by tumor necrosis factor
and its relatives. Trends Cell Biol. 2001, 11, 372–377.
(28) Zambre, A.; Kulkarni, V.; Padhye, S.; Sandur, S.; Aggarwal, B.
Novel curcumin analogs and proliferation targeting TNF-induced
NF-kappa B activation in human leukemic KBM-5 cells. Bioorg.
Med. Chem. 2006, 14, 7196–7204.
(29) Weber, W.; Hunsaker, L.; Roybal, C.; Bobrovnikova-Marjon, E.;
Abcouwer, S.; Royer, R.; Deck, L.; Vander Jagt, D. Activation of
NFκB is inhibited by curcumin and related enones. Bioorg. Med.
Chem. 2006, 14, 2450–2461.
(30) Adams, B.; Ferstl, E.; Davis, M.; Herold, M.; Kurtkaya, S.;
Camalier, R.; Hollingshead, M.; Kaur, G.; Sausville, E.; Rickles,
F. Synthesis and biological evaluation of novel curcumin analogs
as anti-cancer and anti-angiogenesis agents. Bioorg. Med. Chem.
2004, 12, 3871–3883.
(40) Silverman, N.; Maniatis, T. NF-κB signaling pathways in mam-
malian and insect innate immunity. Genes Dev. 2001, 15, 2321–
2342.
(41) Shoemaker, R.; Wolpert-DeFilippes, M.; Kern, D.; Lieber, M.;
Makuch, R.; Melnick, N.; Miller, W.; Salmon, S.; Simon, R.;
Venditti, J. Application of a human tumor colony-forming assay
to new drug screening. Cancer Res. 1985, 45, 2145–2153.
(42) Rubinstein, L. V.; Shoemaker, R. H.; Paull, K. D.; Simon, R. M.;
Tosini, S.; Skehan, P.; Scudiero, D. A.; Monks, A.; Boyd, M. R.
Comparison of in vitro anticancer-drug-screening data generated
with a tetrazolium assay versus a protein assay against a diverse
panel of human tumor cell lines. J. Natl. Cancer Inst. 1990, 82,
1113–1118.
(31) Padhye, S.; Chavan, D.; Pandey, S.; Deshpande, J.; Swamy, K. V.;
Sarkar, F. H. Perspectives on chemopreventive and therapeutic
potential of curcumin analogs in medicinal chemistry. Mini-Rev.
Med. Chem. 2010, 10, 372–387.
(32) Padhye, S.; Yang, H.; Jamadar, A.; Cui, Q.; Chavan, D.; Dominiak,
K.; McKinney, J.; Banerjee, S.; Dou, Q.; Sarkar, F. New difluoro
Knoevenagel condensates of curcumin, their schiff bases and
copper complexes as proteasome inhibitors and apoptosis inducers
in cancer cells. Pharm. Res. 2009, 1–7.
(33) Padhye, S.; Banerjee, S.; Chavan, D.; Pandye, S.; Swamy, K. V.;
Ali, S.; Li, J.; Dou, Q. P.; Sarkar, F. H. Fluorocurcumins as
cyclooxygenase-2 inhibitor: molecular docking, pharmacokinetics
and tissue distribution in mice. Pharm. Res. 2009, 26, 2438–2445.
(34) Ohtsu, H.; Xiao, Z.; Ishida, J.; Nagai, M.; Wang, H. K.; Itokawa,
H.; Su, C. Y.; Shih, C.; Chiang, T.; Chang, E.; Lee, Y.; Tsai, M. Y.;
Chang, C.; Lee, K. H. Antitumor agents. 217. Curcumin analogues
as novel androgen receptor antagonists with potential as anti-
prostate cancer agents. J. Med. Chem. 2002, 45, 5037–5042.
(35) Qiu, X.; Liu, Z.; Shao, W. Y.; Liu, X.; Jing, D. P.; Yu, Y. J.; An,
L. K.; Huang, S. L.; Bu, X. Z.; Huang, Z. S.; Gu, L. Q. Synthesis
and evaluation of curcumin analogues as potential thioredoxin
reductase inhibitors. Bioorg. Med. Chem. 2008, 16, 8035–8041.
(36) Adams, B. K.; Ferstl, E. M.; Davis, M. C.; Herold, M.; Kurtkaya,
S.; Camalier, R. F.; Hollingshead, M. G.; Kaur, G.; Sausville,
E. A.; Rickles, F. R.; Snyder, J. P.; Liotta, D. C.; Shoji, M.
Synthesis and biological evaluation of novel curcumin analogs as
(43) Skehan, P.; Storeng, R.; Scudiero, D.; Monks, A.; McMahon, J.;
Vistica, D.; Warren, J. T.; Bokesch, H.; Kenney, S.; Boyd, M. R.
New colorimetric cytotoxicity assay for anticancer-drug screening.
J. Natl. Cancer Inst. 1990, 82, 1107–1112.
(44) Zhang, L.; Chen, J.; Fu, H. Suppression of apoptosis signal-
regulating kinase 1-induced cell death by 14-3-3 proteins. Proc.
Natl. Acad. Sci. U.S.A. 1999, 96, 8511–8515.
(45) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb,
M. A.; Cheeseman, J. R.; Montgomery, J. A.; , Jr., Vreven, T.;
Kudin, K. N.; Burant, J. C.; Millam, J. M.; Iyengar, S. S.; Tomasi,
J.; Barone, V.; Mennucci, B.; Cossi, M.; Scalmani, G.; Rega, N.;
Petersson, G. A.; Nakatsuji, H.; Hada, M.; Ehara, M.; Toyota, K.;
Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.;
Kitao, O.; Nakai, H.; Klene, M.; Li, X.; Knox, J. E.; Hratchian,
H. P.; Cross, J. B.; Bakken, V.; Adamo, C.; Jaramillo, J.; Gomperts,
R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.;
Pomelli, C.; Ochterski, J. W.; Ayala, P. Y.; Morokuma, K.; Voth,
G. A.; Salvador, P.; Dannenberg, J. J.; Zakrzewski, V. G.; Dapprich,
S.; Daniels, A. D.; Strain, M. C.; Farkas, O.; Malick, D. K.; Rabuck,
A. D.; Raghavachari, K.; Foresman, J. B.; Ortiz, J. V.; Cui, Q.;
Baboul, A. G.; Clifford, S.; Cioslowski, J.; Stefanov, B. B.; Liu, G.;
Liashenko, A.; Piskorz, P.; Komaromi, I.; Martin, R. L.; Fox, D. J.;
Keith, T.; Al-Laham, M. A.; Peng, C. Y.; Nanayakkara, A.;
Challacombe, M.; Gill, P. M. W.; Johnson, B.; Chen, W.; Wong,
M. W.; Gonzalez, C.; Pople, J. A. Gaussian 03, revision E.01;
Gaussian, Inc.: Wallingford, CT, 2004.