JM 1004545

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-

a sulting in the production of an active p52/RelB dimer and its
Nuclear factor-κB (NF-κB ) proteins are a family of struc-
translocation to the nucleus.
turally related eukaryotic transcription factors with a con-
Recent advances have revealed key roles of NF-κB signal-
served reticuloendotheliosis (Rel) domain.1 They influence
ing in pathological conditions such as oncogenesis and inflam-
gene expression events that impact a large number of physio-
mation. NF-κB is commonly overexpressed and constitutively
logical processes including immune response, cell survival, dif-
activated in different types of hematologic cancers and solid
ferentiation, and proliferation.2 Before cell stimulation, NF-
tumors.4-7 Constitutive activation of NF-κB promotes tumor
κB dimers are inactive in the cytoplasm and mainly regulated
proliferation, invasion, and metastasis;8,9 moreover, NF-κB
by two distinct signaling pathways.3 The canonical NF-κB acti-
activation allows malignant cells to escape apoptosis and there-
vation pathway has been extensively studied. Prior to activa-
fore contributes to radiation and chemotherapy resistance in
tion, NF-κB dimers composed of RelA (also known as p65),
cancer cells.10-12 Noticeably, many chemotherapeutic agents
c-REL, and p50 are retained in the cytoplasm by inhibitory κB
have been found to activate NF-κB via various approaches,
(IκB) proteins. Various extracellular signals, such as those
leading to chemoresistance and subsequent failure of chemo-
from microbial infections, viral infections, and proinflamma-
therapy.13,14 Accumulating evidence suggests that inhibition
tory cytokines, activate the IκB kinase (IKK) complex. The
of NF-κB activation can prevent tumor resistance to chemo-
IKK complex targets NF-κB-bound IκBs by phosphorylating
therapeutic agents, shift the death-survival balance toward
two conserved serine residues, which drives ubiquitin-medi-
apoptosis, and improve the efficacy of current chemothera-
ated degradation of IκB and leads to the liberation and subse-
peutic regimens.15,16
quent nuclear translocation of released NF-κB dimers. Phos-
A large number of compounds have been reported to inhibit
phorylation of IκB mainly depends on the IKKβ catalytic
NF-κB by interacting with key macromolecules in the signal-
subunit of the IKK complex in the canonical pathway. In the
ing pathway. Many natural dietary agents, such as soya iso-
second pathway of NF-κB activation, NF-κB, typically exists
flavone,17 resveratrol,18 and curcumin,19 have been found to
as an RelB/p100 dimer and remains inactivated by p100
inhibit NF-κB and induce apoptosis in tumor cells. Curcumin
instead of IκB. After stimulation by certain signals, such as the
(diferuloylmethane), a yellow spice and pigment isolated from
tumor-necrosis factor (TNF) cytokine family member lym-
the rhizome of Curcuma longa, has been used as a spice in
photoxin B (LTβ), an IKKR homodimer, along with NF-κB
South Asia for centuries. It has attracted much attention
inducing kinase (NIK), induces the phosphorylation and
because of its broad range of bioactivities,20 including anti-
oxidative, anti-inflammatory, and anticancer activities, as
*To whom correspondence should be addressed. For H.F.: phone,
404-727-0365; fax, 404-727-0365; e-mail: [email protected]. For X.B.: well as its pharmacological safety and potency as chemopre-
phone and fax, 020-39942054; e-mail, [email protected]. ventive agent.21 One of the predominant targets of curcumin is
a
Abbreviations: NF-κB, nuclear factor-κB; Rel, reticuloendothelio- the NF-κB cell signaling pathway.22 Curcumin directly inhib-
sis; IκB, inhibitor of κB; IKK, inhibitor of κB kinase; NIK, NF-κB its IKK and the 26S proteasome23,24 to block NF-κB acti-
inducing kinase; TNF, tumor-necrosis factor; LTβ, lymphotoxin B;
GST, glutathione S-transferase; TCA, trichloroacetic acid; SRB, sul- vation. Unfortunately, the clinical potential of curcumin
forhodamine B. remains limited because of its relatively low potency and poor
pubs.acs.org/jmc Published on Web 11/11/2010 r 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- analogues have significantly improved IKK/NF-κB inhibi-
ically modify curcumin in order to increase its activity against tion activity and elevated cytotoxicity over the parent com-
cancer and NF-κB.26-31 However, most of these investiga- pound curcumin. These compounds effectively decreased
tions have focused on design, synthesis, and activity of 1,3- growth and reduced the tumorigenic potential of cancer cells,
diketones and monoketone analogues of curcumin. However, as seen in a clonogenic assay.
curcuminoids, such as Knoevenagel condensates of curcumin
Results and Discussion
analogues, contain a novel skeleton and remain quite less in-
vestigated.28,32,33 In 2006, Ajit P. Zambre et al. reported that Chemistry. To search for potent analogues with favorable
copper(II) conjugates of Knoevenagel condensates of curcu- medicinal properties, we extended the molecular diversity of
min showed potential in inhibiting TNFR induced NF-κB curcuminiods by designing three types of curcuminoids: 1,3-
activation.28 Nevertheless, highly active and clinically promis- diketones curcumin analogues, monoketone curcumin ana-
ing curcuminoids remain to be developed. A systematic in- logues, and 4-arylidene curcumin analogues (4-arylidene-1,
vestigation of current curcumin analogues would greatly 7-bisarylhepta-1,6-diene-3,5-diones) (Figure 1).
facilitate the development of new curcumin analogues for Classical 1,3-diketones (1-6) including curcumin (1) and a
therapeutic interventions. In the present study, we describe the representative 1,3-diketone derivative with known activity
synthesis and identification of new 4-arylidene curcumin (3)34 were synthesized using a previously reported proce-
analogues (Knoevenagel condensates of 1,3-diketones curcu- dure35 with slight modification (Scheme 1). Briefly, to pro-
min analogues with aromatic aldehydes) as a new class of tect the C-3 of acetylacetone from an unwanted Knoevenagel
potential anticancer agents. Screening of the synthesized reaction, a boric acetylacetone anhydride complex was pre-
compounds with high content analysis technology, coupled pared first by refluxing acetylacetone with boric anhydride
with biochemical studies, revealed that 4-arylidene curcumin 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 (38-40, Figure 1), which possess absolute diketone moieties,
as described. were designed as references. 38, 39, and 40 were synthesized
Monoketone curcumin analogues have been extensively by refluxing triethoxymethane and Ac2O with 1, 2, and 3,
investigated because of their potential anticancer activities. respectively (Scheme 3).
7 has been reported to have high activity in antitumor assays36 New 4-Arylidene Crucumin Analogues Exhibit Potent In-
and was therefore considered; 9 and 10 are asymmetric an- hibitory Activity against Caner Cells. In order to evaluate the
alogues of 7. These three compounds were included in our anticancer activities of new curcumin derivatives, we tested
work to represent monoketone curcumin analogues. 7 and the effects of synthesized compounds on the growth of A549
the intermediate 11 were synthesized by treating 3,4,5-tri- lung adenocarcinoma cells. For comparison, dose-response
methoxybenzaldehyde with excess acetone in the presence of experiments were carried out to obtain GI50 values for each
KOH. 9 and 10 were synthesized by condensing 11 with compound as previously described,37 with curcumin (1) as a
veratraldehyde and 3-methylthiophene-2-carbaldehyde, re- control. As summarized in Table 1, all the traditional 1,3-
spectively. Furthermore, another new compound, 8, was pre- diketone cucumin analogues (1-6) showed moderate to
pared by condensing 12 and 3,4,5-trimethoxybenzaldehyde. poor activity against A549 growth, while improved antipro-
The intermediate 12 was obtained using the same conditions liferation activities of monoketone analogues 7 and 9 were
as in the synthesis of 1-6 except the excess protected acet- observed (GI50 of 0.53 and 1.21 μM respectively).
ylacetone (Scheme 2). At the same time, the thiophene ring moiety bearing 10
Compared to the large number of traditional 1,3-dike- (GI50 = 9.31 μM) showed decreased activity compared to
tones and monoketone curcumin analogues that have been 7 and 9, indicating that S replacement of the O atom in the
designed and evaluated, investigation of Knoevenagel con- heterocycle of 9 made a negative contribution to its activity.
densates of curcuminoids remains scarce. In our current As a regioisomer of 4, the Knoevenagel condensate 8 ex-
work, 23 new 4-arylidene curcumin analogues (Knoevenagel hibited much higher activity (GI50 =0.53 μM) than 4. 8 can
condensates 13-17, 19-28, 30-37) were designed and syn- also be regarded as a monoketone curumin analogue with an
thesized by coupling 1-4 with different aromatic aldehydes acetyl substitution. When the activities of 7-9 and 1-6 were
in toluene with AcOH and piperidine as a catalyst. The known compared, it was revealed that the spacing of the two aro-
4-arylidene curcumin analogues 18 and 29 were synthesized matic rings most likely influences the activity of curcumin
under the same conditions except with acetylacetone and analogues against A549 growth. The close activities of 8
aldehydes as reactants (Scheme 1). and 7 indicated that the acetyl group has no apparent effect
The 4-arylidene modification can induce a change of a on activity against A549 growth.
partial enolic diketone, which is universal in 1,3-diketones Remarkably, most of the 4-arylidene curcumin analogues
curcumin analogues, to an absolute diketone moiety, possi- (4-arylidene-1,7-bisarylhepta-1,6-diene-3, 5-diones, 13-37)
bly affecting bioactivity. To explore the effect of this transi- showed potent anticancer activities with a GI50 in the sub-
tion, three new 4-hydroxymethylene curcumin analogues micromolar range (0.23-0.93 μM), except 15, 19, 29, and 37
which exhibited slightly lower activities (GI50 of 1.12-
Scheme 1. Synthesis of Compounds 1-6 and 13-37a 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 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) excess acetone, KOH/H2O, MeOH; (b) pre-mixed acetylacetone and B(n-BuO)3, n-BuNH2, 80 °C to room temperature;
(c) HCl, room temperature; (d) 3,4,5-trimethoxybenzaldehyde, DMF, reflux; (e) veratraldehyde or 3-methylthiophene-2-carbaldehyde, KOH/H2O,
MeOH.
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 15.23 16 0.23 29 2.00
2 >25 17 0.53 30 0.43
3 5.80 18 0.31 31 0.69
4 19.26 19 2.62 32 0.36
5 >25 20 0.58 33 0.78
6 >25 21 0.55 34 0.55
7 0.55 22 0.42 35 0.42
8 0.53 23 0.67 36 0.64
9 1.21 24 0.63 37 1.31
10 9.31 25 0.67 38 >25
13 1.64 26 0.37 39 >25
14 0.93 27 0.53 40 >25
15 1.12 28 0.65
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 Figure 2. Inhibition of lung cancer cell viability by curcumin and its
4-arylidene curcumin analogues. These compounds effec- 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
tively decreased viability of all three major types of lung cancer assessed by the Alamar Blue method and expressed as % control
cells tested. Most selected 4-arylidene analogues potently (DMSO). Results with a panel of lung cancer cells are shown (lung
inhibited the growth of lung cancer cells with GI50 values at adenocarcinoma, A549 H1944, H1792; squamous cell carcinoma,
submicromolar concentrations (low to 0.07 μM) and induced H157; squamous cell carcinoma, H157). Results from one repre-
apoptotic cell death. Data of representative compounds 17, sentative experiment are shown.
21, 35 are shown in Figure 2 (see Supporting Information for
additional data from viability and apoptosis assays). These indicating the disadvantages of thiophene replacement with
studies identified a new class of curcumin analogues with respect to NF-κB inhibition.
significantly improved activity against cancer cell growth. Interestingly, except 19 and 30, the newly designed 4-aryl-
Inhibition of TNFr Induced Nuclear Translocation of NF-KB idene crucumin analogues (13-37) exhibited significant
by New Curcuminoids. One of the major curcumin targets inhibitory activities against NF-κB translocation with IC50
critical for cancer survival is IκB kinase, IKK.38 IKK is a key values in the low micromolar range (1.0-4.9 μM). 17 (IC50=
regulator of NF-κB activation. Activated NF-κB is localized 1.0 μM) and 21 (IC50 =1.1 μM) showed more than 10-fold
at the nucleus to promote transcription, which can be trig- greater potency in blocking the nuclear localization of NF-κB
gered by TNFR.39 Thus, we utilized nuclear translocation of than curcumin. The low activity of 19 may have resulted
NF-κB in response to TNFR as a primary readout to exam- from its dimethylamine moiety, which might be protonized
ine the mechanism of action of new curcuminoids in com- under the test conditions used, leading to unfavorable target
parison to curcumin. An automated imaging system for high interactions. 30, however, remained unclear. Nevertheless,
content analysis was employed to monitor the cytoplasmic/ these synthesized 4-arylidene curcumin analogues generally
nuclear localization of NF-κB in response to TNFR.37 A549 showed superior activity to the classical 1,3-diketones ana-
cells in a 384-well plate format were treated with various test logues (1-6) while the monoketone analogues (7-9) dis-
compounds before TNFR was added to trigger the nuclear played moderate activity, suggesting a strong correlation
translocation of the NF-κB p65 subunit. As shown in Figure 3 between a compound’s molecular skeleton and its activity.
and Table 2, curcumin (1) attenuated TNFR-induced nuclear From a comparison of the chemical structures of all three
redistribution of NF-κB with an IC50 of 11.6 μM, consistent types of curcuminoids, the presence of absolute diketone
with previous reports.37 moieties and 4-arylidene substitutions in 13-37 are unique
The synthesized compounds were tested in the same assay over others (1-7, 9, 10). Both factors may contribute to the
and showed different degrees of activity against TNFR in- increased activities of the tested 4-arylidene curcumin ana-
duced NF-κB activation. IC50 values of the tested curcumi- logues. To explore the nature of these phenomena, we fur-
noids are summarized in Table 2. As shown in Table 2, most ther designed and evaluated the NF-κB inhibitory activity
of the traditional 1,3-diketones curcumin analogues (2-4, 6) of 4-hydroxymethylene curcumin analogues 38-40. 38-40
were less active than curcumin. Three monoketone curcumin showed only poor inhibitory activity against NF-κB activa-
analogues (7-9) showed improved activities (IC50 of 4.8- tion (Table 2), indicating the importance of the 4-arylidene
9.0 μM). Consistent with its cytotoxity profile, 10 was much substitution for activity. On the other hand, although the inhib-
less active (IC50 = 24.3 μM) than 7-9 in NF-κB inhibition, itory activities varied, most of the triarylvinyl condensates
Table 2. Inhibitory Effect of Compounds on NFκB Activation/Trans-

locationa
IC50 (μM) IC50 (μM) IC50 (μM)
compd mean SD compd mean SD compd mean SD
curcumin 9.5 3.00 16 1.8 1.12 29 1.0 1.05
2 >40 17 1.0 0.55 30 >40
3 >40 18 1.5 0.95 31 1.0 1.05
4 >40 19 >40 32 1.4 1.56
5 8.5 2.19 20 1.3 1.01 33 2.7 3.32
6 >40 21 1.1 0.45 34 1.8 2.14
7 7.9 4.26 22 1.4 0.26 35 1.7 1.0
8 4.8 1.85 23 1.4 1.48 36 1.3 1.35
9 9.0 3.44 24 4.9 1.96 37 2.8 1.02
10 24.3 7.01 25 1.5 0.04 38 39.3
13 3.0 2.01 26 1.0 0.05 39 >40
14 3.3 2.37 27 1.5 0.04 40 >40
15 3.8 2.13 28 1.7 0.17
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
Figure 3. Inhibition of the TNFR induced NF-κB activation by were utilized: (i) a cell based IKK activation assay, which
new curcuminoids, 17, 18, 20, and 21. Results from one representa- detects phosphorylated IκB and subsequent IκB degrada-
tive experiment are shown. (A) Example images of NF-κB subcel-
tion, and (ii) a reconstituted in vitro kinase assay which di-
lular localization. A549 cells were treated with compounds or
vehicle (DMSO) for 30 min, followed by stimulation with TNFR rectly measures the transfer of γ-32P from ATP to recom-
(10 ng/mL) for 30 min. In the vehicle (DMSO) treatment, NF-κB is binant IκB. Upon stimulation of A549 cells with TNFR,
located at cytoplasm. Upon TNFR treatment, NF-κB is activated activated IKKβ can catalyze the phosphorylation of IκB at
and translocated to the nucleus. Preincubation of the cells with Ser32 and Ser36, followed by degradation of phosphorylated
increasing concentrations of compounds, with compound 21 as an IκB, leading the release and nuclear translocation of NF-κB.
example, dose-dependently inhibited the TNFR-induced NF-κB Antibodies against both IκB and Ser32 phosphorylated IκB
translocation to the nucleus. (B) Dose-response curves of the
inhibitory effect of test compounds on TNFR-induced NF-κB were used to detect the status of IκB as a readout of IKK
activation. The inhibitory effect of the compound on TNFR induced activity.
NF-κB translocation (activation) was expressed as % of control = Figure 4 shows the Western blotting results of IκB phos-
ΔFI(compound)/ΔFI(TNFR only control). ΔFI is the measured NF-κB fluo- phorylation (A) and subsequent degradation (B). Without
rescence (green) intensity difference between nucleus and cytoplasm. the stimuli of TNFR, IκB showed a basal level of phosphor-
Data shown are average values from triplicate samples with SD. ylation and no degradation (the first column). TNFR induced
significant IκB phosphorylation and subsequent degradation
with different substituted groups (except 19 and 30) were of the protein (the second column). After treatment of cells
active at low micromolar concentrations. It might also be with different compounds for 4 h, curcumin could block IκB
concluded that the triarylvinyl scaffolds of 4-arylidene cur- phosphorylation and degradation when high concentrations
cumin analogues play a key role in their NF-κB inhibition were used, whereas 17, 21, and 35 potently block the IκB
activities as a pharmacophore, though further investigation phosphorylation and degradation in a lower concentration.
is still needed to confirm that hypothesis. Dose-response curves for IκB phosphorylation suggested
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. inhibition assay was performed with recombinant IKKβ and
A549 cells were pretreated with test compounds for 30 min prior to its substrate GST-IκB.37 The addition of curcumin in the range
adding TNFR (10 ng/mL). Whole cell lysates were prepared after of tested concentrations had no obvious effect on IKKβ
7 min of TNFR treatment and analyzed for the phosphorylation state
of IκB with anti-pS32 antibody via Western blotting. Then anti- catalyzed incorporation of 32P into its substrate GST-IκB
bodies on the membrane were stripped and the membrane was re- (Figure 5). On the other hand, incubation of compounds 17,
probed for total IκB with antiserum against IκB as indicated. (B) IκB 21, and 35 induced a dose-dependent inhibition of IKKβ.
stability assay. A549 cells were pretreated with test compounds at Thus, the structural modifications of these novel curcumin
indicated dosage prior to the addition of TNFR. Cells were cultured analogues led to drastically enhanced inhibitory activity over
in the presence of TNFR (10 ng/mL) for an additional 20 min, lysed, curcumin in this defined in vitro IKK kinase assay.
and analyzed for total IκB levels by Western blot.
Inhibition of Lung Cancer Clonogenic Activity by 4-Aryl-
idene Curcumin Analogues. To compare the anticancer effi-
IC50 values of 2.8 (17), 2.2 (21), and 5.0 μM (35), respectively, cacy of curcumin and its new analogues, we investigated the
with a similar trend for IkB degradation. Those results sug- effect of these compounds on colony formation properties of
gest that 17, 21, and 35 block TNF-R induced NF-κB acti- lung cancer cells, a readout for tumorigenesis.41 A549 ade-
vation at least in part by inhibiting IκB phosphorylation and nocarcinoma cells readily formed colonies in a solid matrix.
degradation. Again, the 4-arylidene curcumin analogues show The addition of the new triarylvinyl curcumin analogues 17,
much higher activities than curcumin. 21, and 35 drastically inhibited the number of colonies formed
The phosphorylation of IκB mainly depends on the IKKβ by A549 cells (Figure 6). At concentrations less than 0.2 μM,
catalytic subunit of the IKK complex in the canonical path- 17 and 21 reached ∼50% inhibition, while 35 required ap-
way. It is possible that the inhibitory activities of curcumin proximately 0.4 μM to reach the same level of inhibition. For
and derivatives may be due to direct inhibition to the IKKβ comparison, curcumin inhibited colony forming activity at
kinase. To test this model, an in vitro reconstituted IKK much higher concentrations, with detectable inhibition at
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- Consistent with their enhanced activity against a major
cumin analogues have significantly improved anticancer ac- molecular target, the representative 4-arylidene curcumin ana-
tivity over the parental curcumin. logues effectively inhibited the viability of three types of lung
Molecular Modeling of the IKKβ/17 Complex. To gain cancer cells and attenuated the clonogenic activity of A549
insights into the potential binding pose of the 4-arylidene cur- cells. On the basis of our finding, it can be concluded that the
cumin analogues in IKKβ, a molecular docking analysis was triarylvinyl skeleton of 4-arylidene curcumin analogues acts
carried out. Since three-dimensional structural data are un- as a phamacophore, and the 4-arylmethylene modification is
available for IKKβ, a homology model of the IKKβ catalytic an effective strategy to improve the NF-κB inhibitory activ-
domain was built based on four templates (PDB codes 2JC6, ity of curcumin analogues.
1A06, 2QNJ, and 2A2A) from the Protein Data Bank (PDB) Although presented data support an important role of
(see Experiments for details). Discovery Studio 2.1 (Accelrys IKKβ in the NF-κB pathway in mediating the effect of re-
Inc.) and Sybyl 7.3.5 (Tripos Inc.) were used for the homol- ported compounds, some observed differences in compound
ogy model building and energy minimization of the IKKβ potency between cell viability assay and the IKKβ/NF-κB
catalytic domain, respectively. The docking of the selected assays could be due to a number of factors. For example,
compound, 17, to the IKKβ homology model suggests that (i) the status of compounds in cell culture and in vitro con-
17 could potentially bind to the ATP pocket of IKKβ (Figure 7). ditions could be different. The employed compounds could
Compound 17 adopts a propeller-shaped conformation in be further metabolized in cells to generate multiple active
the pocket. The 4-arylidene moiety of 17 occupied the main species, while in vitro kinase assays only measure the activity
hydrophobic adenine binding pockets (Figure 7, region A). of the test compounds. (ii) Although IKKβ could be an
The 40 -OH and 30 -OMe in 4-arylidene moiety of 17 have important target of the tested curcumin analogues as pro-
hydrogen bonds respectively with hinge region residues Glu97 posed, the efficacy of the test compounds on cell survival is
and Cys99, which are the same residues targeted by the also determined by the cell’s genetic background. Cells har-
adenine moiety during the interaction of ATP with IKKβ boring different oncogene mutations, deletions, or amplifi-
catalytic domain. Furthermore, one of the 1,7-diaryl moieties cations could show different sensitivities to test compounds
of compound 17 lies along the phosphate binding pockets that target IKKβ. Our data in Figure 2 strongly support this
(Figure 7, region P) with a hydrogen bond between a 30 -OMe model. (iii) The suppression of cell growth by these curcumin
and residue Thr180. The other side of 1,7-diaryl moieties analogues could be due to their inhibition of other targets in
extends from the ribose binding pockets (Figure 7, region R) addition to IKKβ. These potential targets include the cyto-
to the hydrophilic solvent-exposed entrance (Figure 7, region protective antioxidant system. Whether and how these
E), keeps orthogonal with the 4-arylidene moiety of 17, and 4-arylidene curcumin analogues inhibit other targets in
addition to IKKβ in cancer cells requires further investiga-
has a hydrogen bond between a 40 -OMe and residue Asp103
tion. Regardless, because of their potent IKKβ inhibitory
in the entrance region of the catalytic domain. It should be
activity, these novel triarylvinyl curcuminoids represent new
cautioned that this computer model provides a starting point,
opportunities for therapeutic development against cancer and
and details of the structure-activity relationship (SAR) have
other NF-κB dependent disorders, including various in-
to be experimentally tested in future work.
flammatory diseases. Detailed SAR analysis is still required
In summary, our design and synthesis have led to the gen-
and is being actively pursued by our laboratory.
eration of a novel series of curcumin analogues. Biochemical
and cell biology based studies coupled with high content Experiments
analysis have led to the identification of a novel class of
General. All reagents used were commercially available. Sol-
4-arylidene curcumin analogues as a new generation of highly vents were treated using standard techniques. Reactions were
potent curcuminoids. These compounds exhibited signifi- monitored by TLC on a glass plate coated with silica gel with
cantly improved potency in blocking IKK activity and the fluorescent indicator (GF254). Column chromatography was
NF-κB pathway as revealed by both in vivo and in vitro performed on silica gel (100-200 mesh). 1H NMR and 13C
kinase assays and pathway analysis. NMR spectra were recorded with Varian INOVA 500NB or
Bruker Ultrashield 400 MHz spectrometer using TMS as an in- General Procedure for the Synthesis of 9 and 10. 11 (236 mg,
ternal standard. Purity of target compounds was determined by 1 mmol) and 1 mmol of the corresponding benzaldehyde were
reversed-phase HPLC analyses (Hypersil BDS-C18, 250 μm dissolved in 5 mL of EtOH, and KOH (56 mg, 1 mmol, dissolved
4.6 μm) with a flow rate of 0.5 mL/min. The samples were eluted in 0.1 mL H2O) was added. The reaction mixture was stirred for
with a gradient of constant 70% A (2-4 and 40), 90% A (38 and 1 h at room temperature. Then the solution was poured into
39), or 50% A (others), where solvent A was CH3CN and sol- water and the resulting solution was neutralized by 2 N HCl. The
vent B was 0.1% TFA in water, using UV monitor at 254 nm for raw product was extracted with ethyl acetate, and the organic
detection. Retention times (tR) were calculated in minutes, and layer was washed with water (10 mL, 2 times), dried (Na2SO4),
purity was calculated as % total area. All compounds were >95% and evaporated under vacuum. Finally the raw product was puri-
pure by HPLC. The HR-MS analysis was performed on a Thermo fied with column chromatography to give the final product.
Finnigan MAT95XP-Trap instrument. (1E,4E)-1-(3,4-Dimethoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-
Chemistry. Synthesis of 1-10. 5-Hydroxy-1,7-bis(4-hydroxy penta-1,4-dien-3-one (9). 3,4-dimethoxybenzaldehyde (166 mg,
-3-methoxyphenyl)hepta-1,4,6-trien-3-one (1), 5-hydroxy-1,7- 1 mmol) was used as the benzaldehyde, and column chroma-
bis(4- methoxyphenyl)hepta-1,4,6-trien-3-one (2), 1,7-bis(3,4- tography (petroleum ether/acetic ether 2:1) gave 324 mg of 9 as a
dimethoxyphenyl)-5-hydroxyhepta-1,4,6-trien-3-one (3), 5-hydroxy- yellow powder, yield 84%. HPLC tR = 13.5 min; Rf = 0.14
1,7-bis(3,4,5-trimethoxyphenyl)hepta-1,4,6-trien-3-one (4), (petroleum ether/acetic ether 2:1). 1H NMR (400 MHz, CDCl3)
5-hydroxy-1,7-bis(5-methylfuran-2-yl)hepta-1,4,6-trien-3-one (5), δ 7.69 (d, J=15.8 Hz, 1H), 7.64 (d, J=15.8 Hz, 1H), 7.20 (dd, J=
and 1,7-di(furan-2-yl)-5-hydroxyhepta-1,4,6-trien-3-one (6) were 1.9, 8.3 Hz, 1H), 7.13 (d, J=1.9 Hz, 1H), 6.96 (d, J=15.8 Hz,
synthesized according to our previous report.35 1H), 6.95 (d, J = 15.8 Hz, 1H), 6.88 (d, J = 8.3 Hz, 1H), 6.84
(E)-4-(3,4,5-Trimethoxyphenyl)but-3-en-2-one (11) and (1E,4E)- (s, 2H), 3.93 (s, 3H), 3.92 (s, 3H), 3.90 (s, 6H), 3.89 (s, 3H). 13C
1,5-Bis(3,4,5-trimethoxyphenyl)penta-1,4-dien-3-one (7). To a NMR (101 MHz, CDCl3) δ 188.60, 153.51, 151.49, 149.33,
solution of 3 mL of acetone in 10 mL of EtOH, 0.84 g KOH 143.40, 143.03, 140.41, 130.41, 127.82, 125.00, 123.51, 123.21,
(15 mmol, dissolved in 5 mL H2O) was added at 0 °C, and the mix- 111.20, 110.04, 105.66, 61.02, 56.25, 56.03, 55.99.
ture was stirred for 20 min. After that, 2.94 g of 3,4,5-trimetho- (1E,4E)-1-(3-Methylthiophen-2-yl)-5-(3,4,5-trimethoxyphenyl)-
xybenzaldehyde (15 mmol, dissolved in 25 mL of EtOH) was penta-1,4-dien-3-one (10). 3-methylthiophene-2-carbaldehyde
added dropwise. Then the mixture was brought to room tem- (126 mg, 1 mmol) was used as the benzaldehyde, and column
perature for 2 h and monitored by TLC. Once the 3,4,5-tri- chromatography (petroleum ether/acetic ether 9:1) gave 266
methoxybenzaldehyde was consumed, the reaction solution was mg 10 as a yellow powder, yield 77%. HPLC tR=22.9 min; Rf=
poured into water, and the resulting solution was neutralized by 0.39 (petroleum ether/acetic ether 3:1). HR-MS calcd for
2 N HCl. Then the raw product was extracted from water by C19H20O4S1: 344.1077, found 344.1074. 1H NMR (400 MHz,
acetic ether and purified with column chromatography (petro- CDCl3) δ 7.96 (d, J=15.3 Hz, 1H), 7.63 (d, J=15.9 Hz, 1H),
leum ether/acetic ether 5:1) to give 1.23 g of intermediate 11 7.30 (d, J=5.1 Hz, 1H), 6.91 (d, J=5.1 Hz, 1H), 6.88 (d, J=
(35%) and 1.53 g of yellow powder 7 (49%). HPLC tR = 15.9 Hz, 1H), 6.86 (d, J=15.3 Hz, 1H), 6.85 (s, 2H), 3.92 (s,
17.8 min; Rf=0.38 (petroleum ether/acetic ether 2:1). 1H NMR 6H), 3.90 (s, 3H), 2.40 (s, 3H). 13C NMR (101 MHz, CDCl3)
(400 MHz, CDCl3) δ 7.65 (d, J = 15.8 Hz, 2H), 6.97 (d, J = δ 188.07, 153.48, 142.94, 142.68, 134.48, 134.16, 131.48,
15.8 Hz, 2H), 6.84 (s, 4H), 3.91 (s, 12H), 3.89 (s, 6H). 13C NMR 130.34, 127.29, 125.87, 122.85, 105.57, 61.00, 56.20, 14.32.
(101 MHz, CDCl3) δ 188.48, 153.51, 143.34, 140.52, 130.29, General Procedure for the Synthesis of 13-17, 19-28, and
124.82, 105.72, 61.00, 56.25. 30-37. An amount of 1.0 mmol of 1, 2, 3, or 4 and 2 mmol of the
(5E)-3-(3,4,5-Trimethoxybenzylidene)-6-(3,4,5-trimethoxy- corresponding benzaldehyde as well as 25 mL toluene were
phenyl)hex-5-ene-2,4-dione (8). Compound 8 was synthesized in added to a three-neck rounded flask equipped with a water dis-
two steps. First the intermediate 12 was synthesized with the penser. Pyridine (4.0 mg, 0.05 mmol, in 0.1 mL toluene) and
following procedures. Acetyl acetone (5.01 g, 50 mmol), boric acetic acid (4.8 mg, 0.08 mmol, in 0.1 mL toluene) were added as
anhydride (2.1 g, 30 mmol), and tri-n-butyl borate (23 g, catalysts. The reaction mixture was stirred in 140 °C overnight,
100 mmol) were mixed in ethyl acetate (100 mL) and stirred at and the generated water was removed by water dispenser during
80 °C for 1 h. Then a solution of 3,4,5-trimethoxybenzaldehyde the whole reaction. Then the reaction mixture was washed with
(1.96 g, 10 mmol) and n-butylamine (0.5 mL) in ethyl acetate was water (10 mL, twice) to remove pyridine and acetic acid. Next
added dropwise in 1 h. Next the reaction mixture was stirred at the organic layer was evaporated under vacuum to get raw pro-
room temperature for 2 days. The resulting solution was then duct. Finally the product was purified by using silica gel column
neutralized by 0.4 N HCl and stirred for an additional 30 min in chromatography.
60 °C. Then the raw product was extracted with ethyl acetate, (1E,6E)-4-(4-Hydroxy-3-methoxybenzylidene)-1,7-bis(4-methoxy-
and the organic layer was washed with water (30 mL, 2 times), phenyl)hepta-1,6-diene-3,5-dione (13). 4-Hydroxy-3-methoxy-
dried (Na2SO4), and evaporated under vacuum. Finally the pro- benzaldehyde (304 mg, 2 mmol) and 2 (336 mg, 1 mmol) were
duct was isolated by column chromatography (petroleum ether/ used as reactants and the raw product was purified by column
acetic ether 9:1) to give 0.93 g intermediate 12 (33%). 1H NMR chromatograph (petroleum ether/acetic ether 2:1) to give 249 mg
(400 MHz, CDCl3) δ 7.53 (d, J=15.8 Hz, 1H), 6.77 (s, 2H), 6.39 of 13 as a yellow powder, yield 53%. HPLC tR=19.5 min; Rf=
(d, J=15.8 Hz, 1H), 5.67 (s, 1H), 3.91 (s, 6H), 3.90 (s, 3H), 2.18 0.20 (petroleum ether/acetic ether 2:1). HR-MS calcd for C29-
(s, 3H). After this, 557 mg of 12 (2 mmol) and 3,4,5-trimethox- H26O6: 470.1724, found 470.1727. 1H NMR (500 MHz, CDCl3)
ybenzaldehyde (1.18 g, 6 mmol) were dissolved in 10 mL of DMF δ 7.81 (s, 1H), 7.77 (d, J=15.4 Hz, 1H), 7.53 (d, J=16.1 Hz, 1H),
and stirred in 105 °C overnight. Then the DMF was evaporated 7.52 (d, J=8.9 Hz, 2H), 7.42 (d, J=8.8 Hz, 2H), 7.08 (dd, J=2.0,
under vacuum. Finally the crude mixture was purified with 8.3 Hz, 1H), 7.03 (d, J=2.0 Hz, 1H), 6.99 (d, J=15.4 Hz, 1H),
column chromatography (petroleum ether/acetic ether 5:1) to 6.89 (d, J=8.8 Hz, 2H), 6.86 (d, J=8.3 Hz, 1H), 6.86 (d, J=8.9
give 280 mg of 8 as a pale yellow powder, yield 31%. HPLC tR= Hz, 2H), 6.81 (d, J=16.1 Hz, 1H), 3.83 (s, 3H), 3.81 (m, 6H). 13C
11.1 min; Rf = 0.31 (petroleum ether/acetic ether 2:1). HRMS NMR (101 MHz, CDCl3) δ 198.62, 186.81, 162.15, 161.79,
calcd for C25H28O8: 456.1779, found 456.1780. 1H NMR (400 148.14, 146.64, 146.54, 144.48, 140.79, 138.69, 130.49, 130.42,
MHz, CDCl3) δ 7.64 (s, 1H), 7.40 (d, J = 16.1 Hz, 1H), 6.75 127.62, 126.90, 125.97, 125.75, 125.42, 120.11, 114.82, 114.50,
(d, J=16.1 Hz, 1H), 6.71 (s, 2H), 6.68 (s, 2H), 3.87 (s, 3H), 3.85 114.39, 112.45, 55.94, 55.41.
(s, 6H), 3.84 (s, 3H), 3.79 (s, 6H), 2.41 (s, 3H). 13C NMR (101 (1E,6E)-4-(3,4-Dimethoxybenzylidene)-1,7-bis(4-methoxyphenyl)-
MHz, CDCl3) δ 197.82, 195.75, 153.48, 153.19, 146.41, 141.04, hepta-1,6-diene-3,5-dione (14). 3,4-Dimethoxybenzaldehyde
140.97, 140.36, 139.37, 129.35, 128.23, 126.27, 107.92, 105.91, (332 mg, 2 mmol) and 2 (336 mg, 1 mmol) were used as reactants
60.96, 60.90, 56.20, 56.14, 27.30. and the raw product was purified by column chromatography
(petroleum ether/acetic ether 4:1) to give 312 mg of 14 as a yellow chromatography (petroleum ether/acetic ether 2:1) to give
powder, yield 64%. HPLC tR =27.3 min; Rf =0.33 (petroleum 258 mg of 19 as a red powder, yield 49%. HPLC tR =18.1 min;
ether/acetic ether 2:1). HR-MS calcd for C30H28O6: 484.1880, Rf =0.39 (petroleum ether/acetic ether 1:1). HR-MS calcd for
found 484.1886. 1H NMR (500 MHz, CDCl3) δ 7.82 (s, 1H), 7.78 C32H33O6N1: 527.2302, found 527.2301. 1H NMR (500 MHz,
(d, J=15.4 Hz, 1H), 7.53 (d, J=8.9 Hz, 2H), 7.53 (d, J=15.4 Hz, CDCl3) δ 7.73 (d, J=15.4 Hz, 1H), 7.52 (d, J=16.0 Hz, 1H), 7.42
1H), 7.43 (d, J=8.7 Hz, 2H), 7.13 (dd, J=8.4, 1.8 Hz, 1H), 7.04 (d, J=8.9 Hz, 2H), 7.17 (dd, J=8.4, 1.8 Hz, 1H), 7.07 (dd, J=
(d, J=1.8 Hz, 1H), 6.99 (d, J=15.4 Hz, 1H), 6.89 (d, J=8.7 Hz, 8.5, 1.9 Hz, 1H), 7.06 (d, J=1.8 Hz, 1H), 7.00 (d, J=1.9 Hz, 1H),
2H), 6.86 (d, J=8.9 Hz, 2H), 6.83 (d, J=16.1 Hz, 1H), 6.82 (d, J= 6.99 (d, J=15.4 Hz, 1H), 6.84 (d, J=8.5 Hz, 1H), 6.85 (d, J=
8.4 Hz, 1H), 3.87 (s, 3H), 3.84 (s, 3H), 3.82 - 3.81 (m, 6H). 13C 16.0 Hz, 1H), 6.81 (d, J=8.4 Hz, 1H), 6.61 (d, J=8.9 Hz, 2H),
NMR (101 MHz, CDCl3) δ 198.73, 186.73, 162.13, 161.77, 151.09, 3.90 (s, 3H), 3.90 (s, 3H), 3.88 (s, 3H), 3.86 (s, 3H), 2.99 (s, 6H).
148.81, 146.81, 144.57, 140.65, 138.80, 130.53, 130.47, 127.52, 13
C NMR (101 MHz, CDCl3) δ 199.52, 186.63, 151.80, 151.69,
126.79, 126.31, 125.35, 125.05, 119.95, 114.47, 114.35, 112.82, 151.33, 149.23, 149.20, 146.50, 143.88, 142.14, 135.43, 133.08,
111.02, 55.90, 55.82, 55.43, 55.40. 128.14, 127.43, 126.16, 123.47, 123.05, 120.87, 120.72, 111.66,
(1E,6E)-1,7-Bis(4-methoxyphenyl)-4-(2,3,4-trimethoxybenzy- 111.15, 111.06, 110.62, 110.28, 56.06, 56.02, 56.01, 55.93, 39.97.
lidene)hepta-1,6-diene-3,5-dione (15). 3,4,5-Trimethoxybenzal- (1E,6E)-1,7-Bis(3,4-dimethoxyphenyl)-4-(3-fluorobenzylidene)-
dehyde (392 mg, 2 mmol) and 2 (336 mg, 1 mmol) were used hepta-1,6-diene-3,5-dione (20). 3-Fluorobenzaldehyde (248 mg,
as reactants and the raw product was purified by column chro- 2 mmol) and 3 (396 mg, 1 mmol) were used as reactants and the
matography (petroleum ether/acetic ether 4:1) to give 283 mg of raw product was purified by column chromatography (petro-
15 as a yellow powder, yield 55%. HPLC tR=32.0 min; Rf=0.39 leum ether/acetic ether 3:1) to give 413 mg of 20 as a yellow
(petroleum ether/acetic ether 2:1). HR-MS calcd for C31H30O7: powder, yield 82%. HPLC tR =22.5 min; Rf =0.36 (petroleum
514.1986, found 514.1987. 1H NMR (500 MHz, CDCl3) δ 7.78 ether/acetic ether 3:2). HR-MS calcd for C30H27O6F1: 502.1786,
(d, J=15.4 Hz, 1H), 7.76 (s, 1H), 7.53 (d, J=8.7 Hz, 2H), 7.53 (d, found 502.1785. 1H NMR (400 MHz, CDCl3) δ 7.80 (d, J =
J=16.1 Hz, 1H), 7.43 (d, J=8.7 Hz, 2H), 6.98 (d, J=15.4 Hz, 15.4 Hz, 1H), 7.78 (s, 1H), 7.46 (d, J = 16.2 Hz, 1H), 7.30 (dd,
1H), 6.90 (d, J=8.8 Hz, 2H), 6.87 (d, J=8.8 Hz, 2H), 6.81 (d, J= J =8.0, 7.5 Hz, 1H), 7.29 (d, J = 8.0 Hz, 1H), 7.21 (d, J =
16.1 Hz, 1H), 6.76 (s, 2H), 3.84 (m, 6H), 3.82 (s, 3H), 3.80 (s, 6H). 1.8 Hz, 1H), 7.19 (d, J = 1.8 Hz, 1H), 7.10-7.04 (m, 2H), 7.03
13
C NMR (101 MHz, CDCl3) δ 198.24, 186.77, 162.23, 161.89, (dd, J = 8.3 Hz, J =1.8 Hz, 1H), 6.99 (s, 1H), 6.95 (d, J = 15.4
153.13, 146.82, 144.94, 140.46, 140.28, 140.10, 130.54, 128.87, Hz, 1H), 6.87 (d, J = 8.3 Hz, 1H), 6.84 (d, J = 8.3 Hz, 1H), 6.80
127.47, 126.76, 125.26, 119.88, 114.54, 114.41, 107.92, 60.93, (d, J = 16.2 Hz, 1H), 3.92 (s, 6H), 3.90 (s, 3H), 3.88 (s, 3H). 13C
56.14, 55.45, 55.43. NMR (101 MHz, CDCl3) δ 197.47, 186.74, 162.68 (d, J =
(1E,6E)-1,7-Bis(3,4-dimethoxyphenyl)-4-(3,4,5-trimethoxyben- 247.2 Hz), 152.09, 151.83, 149.29, 149.24, 147.83, 145.92,
zylidene)hepta-1,6-diene-3,5-dione (16). 3,4,5-Trimethoxyben- 141.94, 138.75, 135.71 (d, J = 7.9 Hz), 130.39 (d, J = 8.3 Hz),
zaldehyde (392 mg, 2 mmol) and 3 (396 mg, 1 mmol) were used 127.54, 126.89, 126.06 (d, J = 2.9 Hz), 125.40, 123.85, 123.62,
as reactants and the raw product was purified by column chro- 119.84, 117.16 (d, J=21.4 Hz), 116.60 (d, J=22.3 Hz), 111.10,
matography (petroleum ether/acetic ether 2:1) to give 486 mg of 111.04, 110.54, 110.04, 56.05, 56.04, 55.93.
16 as a yellow powder, yield 85%. HPLC tR=17.0 min; Rf=0.39 (1E,6E)-1,7-Bis(3,4-dimethoxyphenyl)-4-(4-fluorobenzylidene)-
(petroleum ether/acetic ether 1:1). HR-MS calcd for C33H34O9: hepta-1,6-diene-3,5-dione (21). 4-Fluorobenzaldehyde (248 mg,
574.2197, found 574.2201. 1H NMR (500 MHz, CDCl3) δ 7.75 2 mmol) and 3 (396 mg, 1 mmol) were used as reactants and the
(s, 1H), 7.76 (d, J=15.4 Hz, 1H), 7.51 (d, J=16.1 Hz, 1H), 7.18 raw product was purified by column chromatography (petro-
(dd, J=8.4, 2.0 Hz, 1H), 7.07 (d, J=2.0 Hz, 1H), 7.05 (dd, J= leum ether/acetic ether 3:1) to give 390 mg of 21 as a yellow
8.4, 2.0 Hz, 1H), 6.98 (d, J=2.0 Hz, 1H), 6.98 (d, J=15.4 Hz, powder, yield 78%. HPLC tR =21.9 min; Rf =0.33 (petroleum
1H), 6.86 (d, J=8.4 Hz, 1H), 6.82 (d, J=8.4 Hz, 1H), 6.80 (d, J= ether/acetic ether 3:2). HR-MS calcd for C30H27O6F1: 502.1786,
16.1 Hz, 1H), 6.76 (s, 2H) 3.90 (s, 3H), 3.89(s, 3H), 3.88(s, 3H), found 502.1789. 1H NMR (400 MHz, CDCl3) δ 7.81 (s, 1H),
3.86 (s, 3H), 3.84 (s, 3H), 3.78 (s, 6H). 13C NMR (101 MHz, 7.78 (d, J = 15.4 Hz, 1H), 7.48-7.51 (m, 2H), 7.47 (d, J = 16.2 Hz,
CDCl3) δ 197.96, 186.86, 153.18, 152.10, 151.78, 149.42, 149.33, 1H), 7.19 (dd, J = 8.4, 2.0 Hz, 1H), 7.05-7.06 (m, 2H), 7.03 (d,
146.96, 145.23, 140.46, 140.40, 140.27, 128.84, 127.77, 127.07, J = 8.7 Hz, 1H), 7.01 (d, J = 8.6 Hz, 1H), 6.99 (d, J = 2.0 Hz,
125.48, 123.56, 123.37, 120.21, 111.24, 110.76, 110.33, 108.02, 1H), 6.96 (d, J = 15.4 Hz, 1H), 6.87 (d, J = 8.3 Hz, 1H), 6.83 (d,
60.89, 56.17, 56.02, 55.97. J = 8.4 Hz, 1H), 6.80 (d, J = 16.2 Hz, 1H), 3.92 (s, 3H), 3.91 (s,
(1E,6E)-1,7-Bis(3,4-dimethoxyphenyl)-4-(4-hydroxy-3-methoxy- 3H), 3.89 (s, 3H), 3.88 (s, 3H). 13C NMR (101 MHz, CDCl3)
benzylidene)hepta-1,6-diene-3,5-dione (17). 4-Hydroxy-3-meth- δ 197.92, 186.76, 163.73 (d, J=252.5 Hz), 152.09, 151.77, 149.32,
oxybenzaldehyde (304 mg, 2 mmol) and 3 (396 mg, 1 mmol) were 149.25, 147.63, 145.59, 140.57, 139.18, 132.38 (d, J = 8.6 Hz),
used as reactants and the raw product was purified by column 129.79 (d, J = 3.3 Hz), 127.63, 126.93, 125.43, 123.79, 123.52,
chromatography (petroleum ether/acetic ether 2:1) to give 320 mg 119.97, 116.07 (d, J=21.9 Hz), 111.12, 111.07, 110.58, 110.09,
of 17 as a yellow powder, yield 60%. HPLC tR=11.6 min; Rf= 56.06, 56.04, 55.94.
0.27 (petroleum ether/acetic ether 1:1). HR-MS calcd for C31- (1E,6E)-1,7-Bis(3,4-dimethoxyphenyl)-4-(4-hydroxybenzylidene)-
H30O8: 530.1935, found 530.1941. 1H NMR (500 MHz, CDCl3) hepta-1,6-diene-3,5-dione (22). 4-Hydroxybenzaldehyde (244 mg,
δ 7.80 (s, 1H), 7.75 (d, J=15.4 Hz, 1H), 7.50 (d, J=16.1 Hz, 1H), 2 mmol) and 3 (396 mg, 1 mmol) were used as reactants and the
7.18 (dd, J=8.5, 2.0 Hz, 1H), 7.09 (dd, J=8.5, 2.0 Hz, 1H), 7.06 raw product was purified by column chromatography (petroleum
(dd, J=8.0, 2.0 Hz, 1H), 7.06 (d, J=2.0 Hz, 1H), 7.04 (d, J= ether/acetic ether 2:1) to give 187 mg of 22 as an orange powder,
2.0 Hz, 1H), 6.98 (d, J=2.0 Hz, 1H), 6.97 (d, J=15.4 Hz, 1H), yield 37%. HPLC tR=11.6 min; Rf=0.30 (petroleum ether/acetic
6.87 (d, J=8.5 Hz, 1H), 6.86 (d, J=8.0 Hz, 1H), 6.82 (d, J= ether 1:1). HR-MS calcd for C30H28O7: 500.1830, found 500.1826.
8.5 Hz, 1H), 6.81 (d, J=16.1 Hz, 1H), 3.91 (m, 6H), 3.89 (s, 3H), 1
H NMR (400 MHz, DMSO) δ 7.76 (d, J = 15.4 Hz, 1H), 7.50
3.87 (s, 3H), 3.83 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 198.61, (d, J = 16.1 Hz, 1H), 7.39 (d, J = 8.7 Hz, 2H), 7.19 (dd, J = 8.4,
186.89, 151.96, 151.61, 149.31, 149.24, 148.17, 147.02, 146.54, 1.9 Hz, 1H), 7.06 (d, J = 1.9 Hz, 1H), 7.07 (dd, J = 8.2, 2.1 Hz,
144.92, 140.96, 138.58, 127.81, 127.09, 125.90, 125.71, 125.57, 1H), 7.00 (d, J = 2.1 Hz, 1H), 6.97 (d, J = 15.4 Hz, 1H), 6.86 (d,
123.60, 123.31, 120.20, 114.84, 112.42, 111.15, 111.12, 110.61, J = 8.4 Hz, 1H), 6.83 (d, J = 8.4 Hz, 1H), 6.83 (d, J = 16.1 Hz,
110.19, 56.04, 56.03, 56.01, 55.94. 1H), 6.78 (d, J = 8.7 Hz, 2H), 3.92 (s, 3H), 3.91 (s, 3H), 3.89 (s,
(1E,6E)-1,7-Bis(3,4-dimethoxyphenyl)-4-(4-(dimethylamino)- 3H), 3.87 (s, 3H). 13C NMR (101 MHz, DMSO) δ 198.22, 187.83,
benzylidene)hepta-1,6-diene-3,5-dione (19). 4-(Dimethylamino)- 159.85, 151.39, 151.14, 148.95, 148.93, 145.93, 143.26, 140.22,
benzaldehyde (298 mg, 2 mmol) and 3 (396 mg, 1 mmol) were 137.93, 132.55, 127.48, 126.78, 125.42, 124.23, 123.51, 123.29,
used as reactants and the raw product was purified by column 119.29, 115.81, 111.62, 111.49, 111.16, 110.68, 55.71, 55.54.
(1E,6E)-4-(2,5-Dimethoxybenzylidene)-1,7-bis(4-hydroxy-3- 140.95, 138.79, 130.24, 129.51, 126.87, 126.27, 125.07, 121.54,

methoxyphenyl)hepta-1,6-diene-3,5-dione (23). 2,5-Dimethoxy- 113.04, 111.24, 106.24, 106.13, 60.99, 56.38, 56.27, 55.97, 55.93.
benzaldehyde (332 mg, 2 mmol) and 1 (368 mg, 1 mmol) were (1E,6E)-4-(2,5-Dimethoxybenzylidene)-1,7-bis(3,4-dimethoxy-
used as reactants, and column chromatography (petroleum ether/ phenyl)hepta-1,6-diene-3,5-dione (27). 2,5-Dimethoxybenzalde-
acetic ether 2:1) gave 177 mg of 23 as a yellow powder, yield hyde (332 mg, 2 mmol) and 3 (396 mg, 1 mmol) were used as
34%. HPLC tR = 10.7 min; Rf = 0.20 (petroleum ether/acetic reactants and the raw product was purified by column chroma-
ether 1:1). HR-MS calcd for C30H28O8[M - H]-: 515.1706, tography (petroleum ether/acetic ether 3:1) to give 211 mg of 27
found 515.1701. 1H NMR (400 MHz, CDCl3) δ 8.14 (s, 1H), as a yellow powder, yield 39%. HPLC tR =20.4 min; Rf =0.24
7.74 (d, J = 15.5 Hz, 1H), 7.44 (d, J = 16.1 Hz, 1H), 7.17 (dd, (petroleum ether/acetic ether 3:2). HR-MS calcd for C32H32O8:
J = 8.2, 1.8 Hz, 1H), 7.05 (d, J = 1.8 Hz, 1H), 7.01 (d, J = 544.2092, found 544.2096. 1H NMR (400 MHz, CDCl3) δ 8.15
15.5 Hz, 1H), 6.99 (dd, J = 8.1, 1.9 Hz, 1H), 6.94-6.95 (m, 2H), (s, 1H), 7.76 (d, J = 15.5 Hz, 1H), 7.46 (d, J = 16.1 Hz, 1H), 7.20
6.92 (d, J = 8.2 Hz, 1H), 6.84-6.87 (m, 2H), 6.80 (d, J = 9.0 Hz, (dd, J = 8.3, 1.5 Hz, 1H), 7.09 (d, J = 1.5 Hz, 1H), 7.03 (dd, J =
1H), 6.70 (d, J = 16.1 Hz, 1H), 5.92 (s, 1H), 5.92 (s, 1H), 3.93 (s, 8.0, 1.5 Hz, 1H), 7.04 (d, J = 15.5 Hz, 1H), 6.95-6.96 (m, 2H),
3H), 3.88 (s, 3H), 3.84 (s, 3H), 3.67 (s, 3H). 13C NMR (400 MHz, 6.87 (d, J = 8.3 Hz, 1H), 6.85-6.87 (m, 1H), 6.82 (d, J = 8.0 Hz,
CDCl3) δ197.61, 187.76, 153.21, 152.49, 148.63, 148.41, 146.76, 1H), 6.80 (d, J = 8.8 Hz, 1H), 6.73 (d, J = 16.1 Hz, 1H), 3.92 (s,
145.17, 140.88, 136.27, 127.41, 126.87, 125.31, 123.96, 123.58, 6H), 3.89 (s, 3H), 3.87 (s, 3H), 3.84 (s, 3H), 3.67 (s, 3H). 13C
123.43, 120.15, 117.70, 114.91, 114.83, 114.76, 111.92, 110.31, NMR (101 MHz, CDCl3) δ 197.49, 187.78, 153.20, 152.46,
109.72, 56.05, 56.01, 55.95, 55.74. 151.70, 151.54, 149.18, 146.47, 144.96, 140.90, 136.31, 127.78,
(1E,6E)-1,7-Bis(3,4-dimethoxyphenyl)-4-(4-ethylbenzylidene)- 127.25, 125.53, 123.48, 123.39, 123.30, 120.38, 117.67, 114.94,
hepta-1,6-diene-3,5-dione (24). 4-Ethylbenzaldehyde (268 mg, 111.91, 111.11, 111.02, 110.47, 109.97, 55.98, 55.88, 55.70.
2 mmol) and 3 (396 mg, 1 mmol) were used as reactants and (1E,6E)-4-(2,4-Dimethoxybenzylidene)-1,7-bis(3,4-dimethoxy-
the raw product was purified by column chromatography (petro- phenyl)hepta-1,6-diene-3,5-dione (28). 2,4-Dimethoxybenzalde-
leum ether/acetic ether 4:1) to give 316 mg of 24 as a yellow hyde (332 mg, 2 mmol) and 3 (396 mg, 1 mmol) were used as
powder, yield 62%. HPLC tR =27.9 min; Rf =0.37 (petroleum reactants and the raw product was purified by column chroma-
ether/acetic ether 3:2). HR-MS calcd for C32H32O6: 512.2193, tography (petroleum ether/acetic ether 3:1) to give 337 mg of 28
found 512.2196. 1H NMR (400 MHz, CDCl3) δ 7.85 (s, 1H), as a yellow powder, yield 62%. HPLC tR =19.7 min; Rf =0.40
7.78 (d, J = 15.4 Hz, 1H), 7.49 (d, J = 16.2 Hz, 1H), 7.43 (d, J = (petroleum ether/acetic ether 1:1). HR-MS calcd for C32H32O8:
8.2 Hz, 2H), 7.19 (dd, J = 8.5, 1.9 Hz, 1H), 7.16 (d, J = 8.2 Hz, 544.2092, found 544.2094. 1H NMR (400 MHz, CDCl3) δ 8.20
2H), 7.07 (d, J = 1.9 Hz, 1H), 7.06 (dd, J = 8.3, 1.8 Hz, 1H), 6.99 (s, 1H), 7.74 (d, J = 15.5 Hz, 1H), 7.48 (d, J = 16.1 Hz, 1H), 7.36
(d, J = 1.8 Hz, 1H), 7.00 (d, J = 15.4 Hz, 1H), 6.86 (d, J = 8.3 Hz, (d, J = 8.4 Hz, 1H), 7.19 (dd, J = 8.3, 1.8 Hz, 1H), 7.08 (d, J =
1H), 6.82 (d, J = 8.5 Hz, 1H), 6.83 (d, J = 16.2 Hz, 1H), 3.91 1.8 Hz, 1H), 7.05 (dd, J = 8.3, 1.8 Hz, 1H), 7.03 (d, J = 15.5 Hz,
(s, 6H), 3.88 (s, 3H), 3.87 (s, 3H), 2.62 (q, J = 7.6 Hz, 2H), 1.19 1H), 6.98 (d, J = 1.8 Hz, 1H), 6.87 (d, J = 8.3 Hz, 1H), 6.82 (d,
(t, J =7.6 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ 198.33, J = 8.3 Hz, 1H), 6.75 (d, J = 16.1 Hz, 1H), 6.41 (d, J = 2.4 Hz, 1H),
187.04, 151.85, 151.60, 149.20, 149.18, 147.21, 147.17, 145.18, 6.40 (dd, J = 8.4, 2.4 Hz, 1H), 3.91 (s, 6H), 3.89 (s, 3H), 3.87
140.76, 139.81, 130.87, 130.61, 130.23, 128.40, 127.92, 127.71, (s, 6H), 3.79 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 198.32,
127.10, 125.67, 123.64, 123.39, 120.08, 111.08, 110.99, 110.51, 187.60, 163.17, 159.83, 151.62, 151.37, 149.17, 149.15, 146.10,
110.09, 55.99, 55.88, 28.74, 15.09. 144.34, 138.40, 136.26, 131.78, 127.96, 127.37, 125.73, 123.41,
(1E,6E)-4-(2,3-Dimethoxybenzylidene)-1,7-bis(3,4-dimethoxy- 123.10, 120.62, 115.80, 111.09, 111.00, 110.52, 110.04, 105.16,
phenyl)hepta-1,6-diene-3,5-dione (25). 2,3-Dimethoxybenzalde- 98.27, 55.99, 55.89, 55.53, 55.43.
hyde (332 mg, 2 mmol) and 3 (396 mg, 1 mmol) were used as (1E,6E)-4-(3,4-Dimethoxybenzylidene)-1,7-bis(4-hydroxy-3-
reactants and the raw product was purified by column chroma- methoxyphenyl)hepta-1,6-diene-3,5-dione (30). 3,4-Dimethoxy-
tography (petroleum ether/acetic ether 3:1) to give 225 mg of 25 benzaldehyde (332 mg, 2 mmol) and 1 (368 mg, 1 mmol) were
as a yellow powder, yield 41%. HPLC tR =21.0 min; Rf =0.26 used as reactants, and column chromatography (petroleum ether/
(petroleum ether/acetic ether 3:2). HR-MS calcd for C32H32O8: acetic ether 3:2) gave 347 mg of 30 as a yellow powder, yield
544.2092, found 544.2090. 1H NMR (400 MHz, CDCl3) δ 8.14 67%. HPLC tR=9.3 min; Rf=0.20 (petroleum ether/acetic ether
(s, 1H), 7.77 (d, J=15.4 Hz, 1H), 7.43 (d, J=16.1 Hz, 1H), 7.20 1:1). HR-MS calcd for C30H28O8: 516.1779, found 516.1783. 1H
(dd, J=8.3, 1.9 Hz, 1H), 7.08 (d, J=1.9 Hz, 1H), 7.02 (dd, J= NMR (400 MHz, CDCl3) δ 7.81 (s, 1H), 7.77 (d, J = 15.4 Hz,
8.4, 1.9 Hz, 1H), 7.01 (d, J = 15.4 Hz, 1H), 6.98 (dd, J = 7.8, 1H), 7.49 (d, J = 16.1 Hz, 1H), 7.16 (dd, J = 8.4, 1.9 Hz, 1H),
1.8 Hz, 1H), 6.96 (d, J=1.9 Hz, 1H), 6.95 (t, J=7.8 Hz, 1H), 6.90 7.14 (dd, J = 8.2, 2.0 Hz, 1H), 7.03-7.06 (m, 2H), 7.03 (dd, J =
(dd, J=7.8, 1.8 Hz, 1H), 6.87 (d, J=8.4 Hz, 1H), 6.81 (d, J= 8.2, 1.9 Hz, 1H), 6.97 (d, J = 1.9 Hz, 1H), 6.96 (d, J = 15.4 Hz,
8.3 Hz, 1H), 6.72 (d, J=16.1 Hz, 1H), 3.93-3.90 (m, 9H), 3.89 (s, 1H), 6.91 (d, J = 8.2 Hz, 1H), 6.88 (d, J = 8.2 Hz, 1H), 6.82 (d,
3H), 3.86 (s, 3H), 3.84 (s, 3H). 13C NMR (101 MHz, CDCl3) J = 8.4 Hz, 1H), 6.81 (d, J = 16.1 Hz, 1H), 5.96 (s, 1H), 5.94 (s,
δ 197.35, 187.65, 152.70, 151.77, 151.65, 149.25, 148.34, 146.66, 1H), 3.92 (s, 3H), 3.89 (s, 3H), 3.87 (s, 3H), 3.82 (s, 3H). 13C
145.20, 141.65, 136.09, 128.30, 127.79, 127.27, 125.78, 124.10, NMR (101 MHz, CDCl3) δ 198.79, 186.90, 151.13, 148.98,
123.51, 123.38, 121.89, 120.42, 114.42, 111.16, 111.06, 110.54, 148.83, 148.55, 147.49, 146.91, 146.83, 145.25, 140.74, 138.74,
110.06, 61.35, 56.01, 55.90, 55.86. 127.31, 126.57, 126.28, 125.20, 125.02, 124.04, 123.59, 119.76,
(1E,6E)-4-(3,4-Dimethoxybenzylidene)-1,7-bis(3,4,5-trimethoxy- 114.94, 114.91, 112.84, 111.05, 110.44, 109.99, 56.04, 55.95,
phenyl)hepta-1,6-diene-3,5-dione (26). 3,4-Dimethoxybenzalde- 55.91, 55.84.
hyde (332 mg, 2 mmol) and 4 (456 mg, 1 mmol) were used as (1E,6E)-4-(2,4-Dimethoxybenzylidene)-1,7-bis(4-hydroxy-3-
reactants and the raw product was purified by column chroma- methoxyphenyl)hepta-1,6-diene-3,5-dione (31). 2,4-Dimethoxy-
tography (petroleum ether/acetic ether 3:2) to give 323 mg of 26 benzaldehyde (332 mg, 2 mmol) and 1 (368 mg, 1 mmol) were
as a yellow powder, yield 53%. HPLC tR =18.6 min; Rf =0.16 used as reactants, and column chromatography (petroleum ether/
(petroleum ether/acetic ether 3:2). HR-MS calcd for C34H36O10: acetic ether 2:1) gave 268 mg of 31 as a yellow powder, yield
604.2303, found 604.2313. 1H NMR (500 MHz, CDCl3) δ 7.82 52%. HPLC tR = 11.1 min; Rf = 0.29 (petroleum ether/acetic
(s, 1H), 7.72 (d, J = 15.5 Hz, 1H), 7.47 (d, J = 16.1 Hz, 1H), 7.13 ether 1:1). HR-MS calcd for C30H28O8: 516.1779, found
(dd, J = 8.5, 2.0 Hz, 1H), 7.04 (d, J = 2.0 Hz, 1H), 7.02 (d, J = 516.1782. 1H NMR (400 MHz, CDCl3) δ 8.19 (s, 1H), 7.72 (d,
15.5 Hz, 1H), 6.83 (d, J = 8.5 Hz, 1H), 6.83 (d, J = 16.1 Hz, 1H), J = 15.4 Hz, 1H), 7.45 (d, J = 16.1 Hz, 1H), 7.35 (d, J = 8.4 Hz,
6.80 (s, 2H), 6.70 (s, 2H), 3.89 (s, 6H), 3.88 (s, 3H), 3.88 (s, 3H), 1H), 7.17 (d, J = 8.4 Hz, 1H), 7.05 (s, 1H), 7.01 (d, J = 8.4 Hz,
3.86 (s, 3H), 3.84 (s, 6H), 3.82 (s, 3H). 13C NMR (101 MHz, CDCl3) 1H), 7.00 (d, J = 15.4 Hz, 1H), 6.96 (s, 1H), 6.91 (d, J = 8.2 Hz,
δ 198.25, 186.95, 153.53, 151.43, 149.05, 146.86, 145.03, 141.21, 1H), 6.87 (d, J = 8.2 Hz, 1H), 6.73 (d, J = 16.1 Hz, 1H), 6.41
(s, 1H), 6.39 (d, J = 8.4 Hz, 1H), 5.89 (m, 2H), 3.93 (s, 3H), 3.88 J=8.3, 1.5 Hz, 1H), 7.06 (d, J=1.5 Hz, 1H), 6.98 (dd, J=8.2,
(s, 3H), 3.87 (s, 3H), 3.79 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 1.5 Hz, 1H), 6.98 (d, J=15.5 Hz, 1H), 6.97 (d, J=1.5 Hz, 1H),
198.45, 187.61, 163.15, 159.85, 148.55, 148.24, 146.76, 146.72, 6.88-6.96 (m, 4H), 6.86 (d, J=8.2 Hz, 1H), 6.70 (d, J=16.1 Hz,
146.41, 144.56, 138.37, 136.21, 131.77, 127.56, 126.97, 125.46, 1H), 5.92 (s, 1H), 5.90 (s, 1H), 3.93 (s, 3H), 3.92 (s, 3H), 3.88
123.87, 123.37, 120.34, 115.82, 114.79, 114.75, 110.36, 109.81, (s, 3H), 3.84 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 197.56,
105.15, 98.28, 56.05, 55.95, 55.53, 55.43. 187.64, 152.67, 148.72, 148.54, 148.29, 147.05, 146.81, 145.46,
(1E,6E)-4-(3-Fluorobenzylidene)-1,7-bis(4-hydroxy-3-methoxy- 141.56, 136.00, 128.25, 127.30, 126.76, 125.45, 124.11, 123.95,
phenyl)hepta-1,6-diene-3,5-dione (32). 3-Fluorobenzaldehyde 123.73, 121.82, 120.01, 114.86, 114.80, 114.35, 110.26, 109.81,
(248 mg, 2 mmol) and 1 (368 mg, 1 mmol) were used as reactants, 61.37, 56.04, 55.93, 55.83.
and column chromatography (petroleum ether/acetic ether (1E,6E)-1,7-Bis(4-hydroxy-3-methoxyphenyl)-4-(3-methoxy-
3:1) gave 329 mg of 32 as a yellow powder, yield 69%. HPLC benzylidene)hepta-1,6-diene-3,5-dione (36). 3-Methoxybenzalde-
tR = 12.2 min; Rf = 0.32 (petroleum ether/acetic ether 1:1). hyde (272 mg, 2 mmol) and 1 (368 mg, 1 mmol) were used as
HR-MS calcd for C28H23O6F1: 474.1473, found 474.1476. 1H reactants, and column chromatography (petroleum ether/acetic
NMR (400 MHz, acetone) δ 7.90 (s, 1H), 7.69 (d, J = 15.5 Hz, ether 2:1) gave 285 mg of 36 as a yellow powder, 59%. HPLC
1H), 7.50 (d, J = 16.2 Hz, 1H), 7.39 (m, 2H), 7.39 (d, J = 15.5 Hz, tR=11.6 min; Rf=0.24 (petroleum ether/acetic ether 1:1). HR-
1H), 7.32 (d, J = 1.8 Hz, 1H), 7.31 (dd, J = 8.2, 1.8 Hz, 1H), MS calcd for C29H26O7: 486.1679, found 486.1674. 1H NMR
7.28 (dd, J = 8.4, 1.8 Hz, 1H), 7.12-7.17 (m, 1H), 7.11 (dd, (400 MHz, CDCl3) δ 7.81 (s, 1H), 7.76 (d, J = 15.4 Hz, 1H), 7.45
J = 8.2, 1.8 Hz, 1H), 6.90 (d, J = 8.2 Hz, 1H), 6.85 (d, J = (d, J = 16.1 Hz, 1H), 7.24 (dd, J =8.0, 8.0 Hz, 1H), 7.16 (dd,
16.2 Hz, 1H), 6.83 (d, J = 8.2 Hz, 1H), 3.90 (s, 3H), 3.88 (s, J = 8.2, 1.9 Hz, 1H), 7.0-7.09 (m, 1H), 7.04 (d, J = 1.9 Hz, 1H),
3H). 13C NMR (101 MHz, acetone) δ 197.59, 188.25, 163.48 7.02-7.03 (m, 1H), 7.02 (dd, J = 8.2, 1.9 Hz, 1H), 6.96 (d, J =
(d, J = 244.6 Hz), 150.86, 150.62, 148.83, 148.77, 148.08, 15.4 Hz, 1H), 6.95 (d, J = 1.9 Hz, 1H), 6.91 (d, J = 8.2 Hz, 1H),
145.78, 143.93, 138.30, 137.46 (d, J=7.9 Hz), 131.54 (d, J= 6.87-6.89 (m, 1H), 6.87 (d, J = 8.2 Hz, 1H), 6.77 (d, J = 16.1
8.4 Hz), 127.86, 127.33, 127.00 (d, J=2.8 Hz), 125.68, 124.95, Hz, 1H), 5.97 (s, 2H), 3.92 (s, 3H), 3.88 (s, 3H), 3.76 (s, 3H). 13C
124.35, 119.85, 117.47 (d, J=21.4 Hz), 117.00 (d, J=22.6 Hz), NMR (101 MHz, CDCl3) δ 197.92, 187.01, 159.64, 148.86,
116.31, 116.18, 112.43, 111.66, 56.42, 56.35. 148.59, 147.51, 146.80, 146.77, 145.67, 141.09, 140.31, 134.86,
(1E,6E)-4-(4-Fluorobenzylidene)-1,7-bis(4-hydroxy-3-methoxy- 129.81, 127.25, 126.64, 125.28, 124.07, 123.77, 122.80, 119.74,
phenyl)hepta-1,6-diene-3,5-dione (33). 4-Fluorobenzaldehyde 116.34, 115.15, 114.85, 114.83, 110.33, 109.90, 56.07, 55.96,
(248 mg, 2 mmol) and 1 (368 mg, 1 mmol) were used as reactants, 55.27.
and column chromatography (petroleum ether/acetic ether (1E,6E)-1,7-Bis(3,4-dimethoxyphenyl)-4-((5-methylfuran-2-yl)-
3:1) gave 311 mg of 33 as a yellow powder, yield 66%. HPLC methylene)hepta-1,6-diene-3,5-dione (37). 5-Methylfuran-2-car-
tR = 12.3 min; Rf = 0.31 (petroleum ether/acetic ether 1:1). baldehyde (220 mg, 2 mmol) and 3 (396 mg, 1 mmol) were used
HR-MS calcd for C28H23O6F1: 474.1473, found 474.1474. 1H as reactants and the raw product was purified by column chro-
NMR (400 MHz, CDCl3) δ 7.80 (s, 1H), 7.77 (d, J = 15.4 Hz, matography (petroleum ether/acetic ether 3:1) to give 276 mg of
1H), 7.50 (d, J = 8.5 Hz, 1H), 7.48 (d, J = 8.0 Hz, 1H), 7.45 37 as a yellow powder, yield 57%. HPLC tR=16.3 min; Rf=0.32
(d, J = 16.2 Hz, 1H), 7.16 (dd, J = 8.2, 1.8 Hz, 1H), 7.04 (d, (petroleum ether/acetic ether 1:1). HR-MS calcd for C29H28O7:
J = 2.0 Hz, 1H), 7.03 (dd, J = 8.2, 2.0 Hz, 1H), 7.03 (d, J = 488.1830, found 488.1828. 1H NMR (500 MHz, CDCl3) δ 7.74
8.2 Hz, 1H), 7.01 (d, J = 8.5 Hz, 1H), 6.97 (d, J = 1.8 Hz, 1H), (d, J = 15.4 Hz, 1H), 7.60 (s, 1H), 7.46 (d, J = 16.1 Hz, 1H), 7.17
6.92 (d, J = 8.0 Hz, 1H), 6.93 (d, J = 15.4 Hz, 1H), 6.88 (d, (dd, J = 8.4, 1.9 Hz, 1H), 7.11 (dd, J = 8.4, 1.9 Hz, 1H), 7.05 (d,
J = 8.2 Hz, 1H), 6.77 (d, J = 16.2 Hz, 1H), 5.94 (s, 2H), 3.93 J = 1.9 Hz, 2H), 6.93 (d, J = 15.4 Hz, 1H), 6.90 (d, J = 16.1 Hz,
(s, 3H), 3.90 (s, 3H). 13C NMR (101 MHz, acetone) δ 197.89, 1H), 6.84 (d, J = 8.4 Hz, 2H), 6.72 (d, J = 3.4 Hz, 1H), 6.08 (dq,
188.22, 164.33 (d, J = 249.7 Hz), 150.76, 150.49, 148.78, J = 3.4, 0.9 Hz, 1H), 3.89-3.90 (m, 12H), 2.26 (d, J = 0.9 Hz,
148.73, 147.77, 145.41, 142.61, 138.69, 133.26 (d, J = 3H). 13C NMR (101 MHz, CDCl3) δ 197.53, 185.99, 157.42,
8.7 Hz), 131.51 (d, J = 3.1 Hz), 127.89, 127.35, 125.74, 151.68, 151.55, 149.31, 149.22, 148.45, 146.25, 144.64, 134.83,
124.81, 124.23, 119.89, 116.58 (d, J = 21.9 Hz), 116.27, 127.86, 127.39, 126.67, 126.25, 123.30, 123.25, 120.24, 120.16,
116.15, 112.36, 111.63, 56.38, 56.31 111.13, 110.60, 110.21, 109.74, 56.05, 56.03, 56.00, 55.95, 13.99.
(1E,6E)-4-(4-Ethylbenzylidene)-1,7-bis(4-hydroxy-3-methoxy- General Procedures for Synthesis of 18 and 29. 3,4-Dimethox-
phenyl)hepta-1,6-diene-3,5-dione (34). 4-Ethylbenzaldehyde (268 mg, ybenzaldehyde (831 mg, 5 mmol) or 4-hydroxy-3-methoxyben-
2 mmol) and 1 (368 mg, 1 mmol) were used as reactants, and column zaldehyde (760 mg, 5 mmol) was mixed with pentane-2,4-dione
chromatography (petroleum ether/acetic ether 3:1) gave 288 mg of 34 (42 mg, 1 mmol) and 25 mL of toluene in a three-neck rounded
as a yellow powder, yield 59%. HPLC tR=16.9 min; Rf=0.36 (petro- flask equipped a water dispenser. Then pyridine (7.9 mg, 0.1 mmol,
leum ether/acetic ether 1:1). HR-MS calcd for C30H28O6: 484.1880, in 0.2 mL of toluene) and acetic acid (9.6 mg, 0.16 mmol, in
found 484.1881. 1H NMR (400 MHz, CDCl3) δ 7.83 (s, 1H), 7.75 (d, 0.2 mL of toluene) were added in as catalysts. The reaction mix-
J = 15.4 Hz, 1H), 7.47 (d, J = 16.2 Hz, 1H), 7.42 (d, J = 8.2 Hz, ture was stirred at 140 °C overnight, and the generated water was
2H), 7.13-7.16 (m, 3H), 7.03 (d, J = 1.5 Hz, 1H), 7.01 (dd, J = 8.2, removed by water dispenser during the whole reaction. After the
1.6 Hz, 1H), 6.97 (d, J = 1.6 Hz,1H), 6.96 (d, J = 15.4 Hz, 1H), 6.91 mixture was washed with water to remove pyridine and acetic
(d, J = 8.2 Hz, 1H), 6.87 (d, J = 8.2 Hz, 1H), 6.80 (d, J = 16.2 Hz, acid, the solvent was evaporated to get raw product. Finally the
1H), 3.91 (s, 3H), 3.87 (s, 3H), 2.61 (q, J = 7.56 Hz, 2H), 1.19 (t, J = raw product was purified by column chromatography to give
7.59 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ 198.43, 187.03, 148.84, 18 and 29, respectively.
148.54, 147.51, 147.16, 146.82, 146.79, 145.39, 140.69, 139.77, 130.86, (1E,6E)-4-(3,4-Dimethoxybenzylidene)-1,7-bis(3,4-dimethoxy-
130.61, 128.40, 127.28, 126.66, 125.38, 124.03, 123.67, 119.77, 114.86, phenyl)hepta-1,6-diene-3,5-dione (18). Column chromatography
114.83, 110.36, 109.97, 56.06, 55.95, 28.75, 15.10. (petroleum ether/acetic ether 3:2) gave 381 mg of 18 as a yellow
(1E,6E)-4-(2,3-Dimethoxybenzylidene)-1,7-bis(4-hydroxy-3- powder, yield 70%. HPLC tR =15.7 min; Rf =0.28 (petroleum
methoxyphenyl)hepta-1,6-diene-3,5-dione (35). 2,3-Dimethoxy- ether/acetic ether 1:1). 1H NMR (500 MHz, CDCl3) δ 7.80 (s,
benzaldehyde (332 mg, 2 mmol) and 1 (368 mg, 1 mmol) were 1H), 7.75 (d, J=15.4 Hz, 1H), 7.51 (d, J=16.1 Hz, 1H), 7.18 (dd,
used as reactants, and column chromatography (petroleum ether/ J=8.4, 2.0 Hz, 1H), 7.13 (dd, J=8.4, 2.0 Hz, 1H), 7.05-7.06
acetic ether 2:1) gave 180 mg of 35 as a yellow powder, yield (m, 3H), 6.98 (d, J=2.0 Hz, 1H), 6.99 (d, J=15.4 Hz, 1H), 6.85
35%. HPLC tR = 18.8 min; Rf = 0.24 (petroleum ether/acetic (d, J=8.4 Hz, 1H), 6.81 (d, J=8.4 Hz, 2H), 6.81 (d, J=16.1 Hz,
ether 1:1). HR-MS calcd for C30H28O8 [M - H]-: 515.1706, 1H), 3.89 (s, 6H), 3.87 (s, 3H), 3.86 (s, 6H), 3.80 (s, 3H). 13C
found 515.1696. 1H NMR (400 MHz, CDCl3) δ 8.13 (s, 1H), NMR (101 MHz, CDCl3) δ 198.39, 186.84, 152.01, 151.66,
7.75 (d, J=15.5 Hz, 1H), 7.41 (d, J= 16.1 Hz, 1H), 7.16 (dd, 151.21, 149.37, 149.30, 148.95, 146.90, 144.88, 140.65, 139.00,
127.86, 127.14, 126.39, 125.61, 124.98, 123.54, 123.27, 120.29, viability with Thermo Scientific’s Multiskan MK3 plate reader to
112.99, 111.24, 111.19, 111.16, 110.75, 110.33, 56.05, 56.01, obtain the GI50 values.
55.94, 55.90, 55.87. Alamar Blue Method. Cells were maintained in RPMI with
(1E,6E)-4-(4-Hydroxy-3-methoxybenzylidene)-1,7-bis(4-hy- 10% fetal bovine serum (FBS) and penicillin/streptomycin in a
droxy-3-methoxyphenyl)hepta-1,6-diene-3,5-dione (29). Column 37 °C incubator with 5% CO2. Cells were plated in 384-well
chromatography (petroleum ether/acetic ether 2:3) gave 223 mg plates and were treated with test agents on the following day and
of 29 as a yellow powder, yield 44%. HPLC tR =7.9 min; Rf = further incubated for 72 h before the viability assay was carried
0.39 (petroleum ether/acetic ether 1:2). HR-MS calcd for C29- out. The Alamar Blue assay was performed to evaluate cell via-
H26O8: 502.1628, found 502.1623. 1H NMR (400 MHz, CDCl3) bility with PerkinElmer’s EnVision multimode plate reader to
δ 7.80 (s, 1H), 7.75 (d, J = 15.4 Hz, 1H), 7.49 (d, J = 16.1 Hz, obtain the GI50 values.42,43 The GI50 is defined as the concen-
1H), 7.16 (dd, J = 8.2, 1.7 Hz, 1H), 7.09 (dd, J = 8.3, 1.8 Hz, tration of agents that decreases viability by 50% in a total cell
1H), 7.04 (m, 2H), 7.03 (dd, J = 8.2, 1.8 Hz, 1H), 6.96 (d, J = 1.7 population compared to control cells with solvent vehicle at the
Hz, 1H), 6.95 (d, J = 15.4 Hz, 1H), 6.91 (d, J = 8.2 Hz, 1H), 6.88 end of the incubation period.
(d, J = 8.2 Hz, 1H), 6.87 (d, J = 8.3 Hz, 1H), 6.80 (d, J = NF-KB Translocation Assay. In brief, A549 cell were seeded in
16.1 Hz, 1H), 5.95 (s, 1H), 5.93 (s, 1H), 5.89 (s, 1H), 3.92 (s, 3H), 384-well plates, incubated for 15 h, then incubated with various
3.89 (s, 3H), 3.83 (s, 3H). 13C NMR (101 MHz, acetone) δ concentrations of compounds for 30 min. TNF-R (10 ng/mL,
198.90, 188.17, 150.69, 150.37, 150.05, 148.84, 148.77, 148.37, final) was added to cells to stimulate the NF-κB cell signaling
147.20, 144.77, 140.74, 140.11, 128.05, 127.48, 126.61, 126.13, pathway for 30 min. Cells were then fixed with paraformalde-
126.01, 124.64, 124.05, 120.15, 116.31, 116.25, 116.23, 114.39, hyde (2%, 100 μL) and permeabilized with Triton X-100 (0.1%,
112.38, 111.75, 56.43, 56.35, 56.25. 100 μL). Finally, rabbit anti-p65 NF-κB antibody and goat anti-
General Procedures for the Synthesis of 38-40. Triethoxy- rabbit IgG with conjugated Alexa Fluor 488 were used to stain
methane (1.48 g, 10 mmol) and 1 mmol of corresponding sym- NF-κB, and Hoechst 33342 was used to stain nucleus. Fluores-
metrical curcuminoids were dissolved in 5 mL of acetic anhy- cence intensity of NF-κB and nuclear staining were recorded
dride. The reaction mixture was stirred at 140 °C for 5 h and then with an automated imaging system, ImageXpress5000A (Molecular
transferred to room temperature, and 10 mL of H2O was added Devices). The levels of NF-κB translocation were calculated and
for an additional 10 min. The raw product was extracted by ethyl expressed as the difference between average fluorescence inten-
acetate and then purified by column chromatography or recrys- sity in the nucleus and in the cytoplasm. After stimulation with
tallization. TNF-R, the inhibitory effect of test compounds on TNFR in-
4,40 -((1E,6E)-4-(Hydroxymethylene)-3,5-dioxohepta-1,6-diene- duced NF-κB translocation was expressed as a percentage of
1,7-diyl)bis(2-methoxy-4,1-phenylene) Diacetate (38). 1 (368 mg, fluorescence intensity difference (in nucleus and in cytoplasm) in
1 mmol) was used as reactant and the raw product was purified the control wells (TNFR only) after subtracting background (no
by column chromatography (petroleum ether/acetic ether 4:1) TNFR treatment). The IC50 of test compounds in this NF-κB
to give 140 mg of 38 as a yellow powder, yield 29%. HPLC tR= translocation assay stands for the concentration of a compound
10.4 min; Rf =0.44 (petroleum ether/acetic ether 3:2). HR-MS required to induce 50% inhibition. All data are obtained as
calcd for C26H24O9: 480.1415, found 480.1409. 1H NMR average values from triplicate samples, and the experiments
(400 MHz, CDCl3) δ 10.35 (s, 1H), 7.93 (d, J=15.5 Hz, 2H), were repeated at least three times.
7.76 (d, J = 15.5 Hz, 2H), 7.27 (dd, J = 8.2, 1.6 Hz, 2H), 7.21 Clonogenic Assay. A549 cells were plated in low density in a
(d, J=1.6 Hz, 2H), 7.10 (d, J=8.2 Hz, 2H), 3.91 (s, 6H), 2.34 (s, 12-well plate and treated with test compounds the following day
6H). 13C NMR (400 MHz, CDCl3) δ 189.36, 186.95, 168.67, and every 3 days thereafter. On day 9 after colonies were formed,
151.55, 145.09, 142.17, 133.63, 123.44, 122.18, 120.47, 112.77, cells were fixed with trichloroacetic acid (TCA) (10%) for 30 min
111.94, 56.03, 20.67. at 4 °C, washed with water, stained with SRB, and then washed
(1E,6E)-4-(Hydroxymethylene)-1,7-bis(4-methoxyphenyl)- with acetic acid (1%). The images of the plate were scanned, and
hepta-1,6-diene-3,5-dione (39). 2 (336 mg, 1 mmol) was used as colonies were counted using image processing and analysis in
reactant and the raw product was purified by column chroma- Java (Image J, Research Service Branch, NIH).
tography (petroleum ether/acetic ether 9:1) to give 255 mg of 39 IKK Assays. Cell Based IKK Assay. Cells were treated with
as a yellow powder, yield 70%. HPLC tR =9.9 min; Rf =0.37 various test agents for a defined period of time as described in
(petroleum ether/acetic ether 3:1). HR-MS calcd for C22H20O5: each figure and were lysed for detection of phosphorylation
364.1305, found 364.1292. 1H NMR (400 MHz, CDCl3) δ 10.35 status of IκB, a readout for IKK activity, and total IκB protein.
(s, 1H), 7.94 (d, J = 15.4 Hz, 2H), 7.72 (d, J = 15.4 Hz, 2H), 7.61 The NP-40 lysates buffer was used (1.0% NP-40, 10 mM Hepes,
(d, J = 8.2 Hz, 4H), 6.94 (d, J = 8.2 Hz, 4H), 3.87 (s, 6H). 13C pH 7.4, 150 mM NaCl, 5 mM NaF, 2 mM Na3VO4, 5 mM Na4-
NMR (101 MHz, CDCl3) δ 189.48, 187.14, 162.15, 145.44, P2O7, 10 μg/mL aprotinin, 10 μg/mL leupeptin, 1 mM PMSF).
130.79, 127.60, 117.94, 114.54, 55.46. For Western blotting, equal volumes of cell lysate were subject
(1E,6E)-1,7-Bis(3,4-dimethoxyphenyl)-4-(hydroxymethylene)- to electrophoresis on SDS-PAGE (12.5%). Proteins were then
hepta-1,6-diene-3,5-dione (40). 3 (396 mg, 1 mmol) was used as electrotransferred to a nitrocellulose membrane (GE Water
reactant and the raw product was purified by recrystallization in and Process Technologies, Trevose, PA) as described previ-
acetic anhydride to give 273 mg of 40 as an orange powder, yield ously.44 Membranes were blocked in a solution of 5% nonfat
64%. HPLC tR = 14.6 min; Rf = 0.40 (petroleum ether/acetic dry milk in TBS-T buffer (20 mM Tris, pH 7.6, 500 mM NaCl,
ether 3:2). HR-MS calcd for C24H24O7, 424.1517; found, 0.5% Tween-20) for 30 min followed by incubation with anti-
424.1518. 1H NMR (400 MHz, CDCl3) δ 10.37 (s, 1H), 7.93 pS32-I-κB or I-κB antibodies from Cell Signaling (Beverly,
(d, J = 15.4 Hz, 2H), 7.71 (d, J = 15.4 Hz, 2H), 7.26 (d, J = MA) for at least 2 h. The membrane was then washed and treat-
8.1 Hz, 2H), 7.17 (s, 2H), 6.91 (d, J = 8.1 Hz, 2H), 3.97 (s, 6H), ed with the corresponding horseradish peroxidase conjugated
3.95 (s, 6H). 13C NMR (101 MHz, CDCl3) δ 189.39, 187.23, anti-mouse immunoglobulin [Ig] or anti-rabbit Ig as indicated.
151.98, 149.35, 145.80, 127.83, 124.03, 118.04, 111.12, 110.26, Immunodetection was performed using West Pico (Pierce,
56.04, 56.01. Rockford, IL) or West Dura (Pierce) followed by imaging on
Biology. Cell Viability Assay. Sulforhodamine B (SRB) Method. Kodak’s Image Station 2000R (New Haven, CT).
Cells were maintained in RPMI with 5% fetal bovine serum (FBS) In Vitro IKK Assay. Activated recombinant IKKβ in MOPS
and penicillin/streptomycin in a 37 °C incubator with 5% CO2. Cells buffer (8 mM MOPS-NaOH, pH 7.0, 200 μM EDTA, 15 mM
were plated in 96-well plates and were treated with test agents on the MgCl2) from Upstate Cell Signaling Solutions (Lake Placid, NY)
following day and further incubated for 48 h before viability assay was used for the in vitro kinase assay. The test compounds were
was carried out. The SRB assay was performed to evaluate cell incubated with IKKβ (40 ng) for 30 min at room temperature.
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tion into GST-I-κBR was assessed by autoradiography. In ad- (6) Guzman, M.; Neering, S.; Upchurch, D.; Grimes, B.; Howard, D.;
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6-31G basis set implemented in Gaussian 03, revision E.01, soft- combination with cisplatin or doxorubicin, on human hepatic
ware package.45 After energy minimization, it adopts a propel- cancer cells. Analysis of their possible relationship to changes in
NF-κB activation levels and in IAP gene expression. Cancer Lett.
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ATP binding pocket were performed with Surflex-Dock in Sybyl 17-Allylamino-17-demethoxygeldanamycin synergistically potenti-
7.3.5. Automatic was chosen as the mode of construction for the ates tumor necrosis factor-induced lung cancer cell death by block-
protomol. Threshold is 0.5 and bloat is 0. ing the nuclear factor-κB pathway. Cancer Res. 2006, 66, 1089–
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Acknowledgment. We thank Mary Puckett for critical read- (17) Sarkar, F.; Li, Y. Soy isoflavones and cancer prevention. Cancer
Invest. 2003, 21, 744–757.
ing of the manuscript. This work was supported in part by grants (18) Holmes-McNary, M.; Baldwin, A., Jr. Chemopreventive proper-
from the MOST of China (863 Program 2008AA02Z304) and ties of trans-resveratrol are associated with inhibition of activation
NSFC (Grant 30973619) (X.B.) and from the U.S. National of the IκB kinase. Cancer Res. 2000, 60, 3477–3483.
Institutes of Health (Grant P01 CA116676 to H.F.) and Emory (19) Plummer, S.; Holloway, K.; Manson, M.; Munks, R.; Kaptein, A.;
Farrow, S.; Howells, L. Inhibition of cyclo-oxygenase 2 expression
Faculty Distinction Fund (H.F.). H.F. is a GCC Distin- in colon cells by the chemopreventive agent curcumin involves
guished Cancer Scholar and a GRA Distinguished Investiga- inhibition of NF-κB activation via the NIK/IKK signalling com-
tor. Y.D. is a recipient of Emory Head and Neck Cancer plex. Oncogene 1999, 18, 6013–6020.
(20) Maheshwari, R.; Singh, A.; Gaddipati, J.; Srimal, R. Multiple
SPORE Career Development Award (Grant P50 CA128613). biological activities of curcumin: a short review. Life Sci. 2006, 78,
Supporting Information Available: 1
H NMR, 13C NMR, 2081–2087.
(21) Cheng, A.; Hsu, C.; Lin, J.; Hsu, M.; Ho, Y.; Shen, T.; Ko, J.; Lin,
HRMS, and HPLC spectra; additional cancer cell growth inhibi- J.; Lin, B.; Ming-Shiang, W. Phase I clinical trial of curcumin, a
tion data and molecular modeling information. This material is chemopreventive agent, in patients with high-risk or pre-malignant
available free of charge via the Internet at http://pubs.acs.org. lesions. Anticancer Res. 2001, 21, 2895–2900.
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8260 J. Med. Chem.

2010, 53, 8260–8273

Received April 13, 2010

A series of curcumin analogues including new 4-arylidene curcumin analogues (4-arylidene-1,7-

Introduction proteasome-mediated processing of p100 to generate p52, re-

Scheme 3. Synthesis of Compounds 38-40a

Scheme 2. Synthesis of Compounds 7-10a

Table 1. Effect of Curcumin and Its Analogues on Growth of A549

the third arylvinyl moiety. Importantly, the cytotoxic activ-

Table 2. Inhibitory Effect of Compounds on NFκB Activation/Trans-

Noticeably, the data in Tables 1 and 2 suggest an impor-

Figure 5. Effect of curcumin analogues on the catalytic activity of

Figure 6. Compounds 17, 21, and 35 inhibit the colony formation

(1E,6E)-4-(2,5-Dimethoxybenzylidene)-1,7-bis(4-hydroxy-3- 140.95, 138.79, 130.24, 129.51, 126.87, 126.27, 125.07, 121.54,

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