Structure - Activity relationship studies of chalcone leading to 3-hydroxy-4,3′,4′,5′-tetramethoxychalcone and its analogues as potent nuclear factor κB inhibitors and their anticancer activities

Article abstract of DOI:10.1021/jm901278z

Chalcone is a privileged structure, demonstrating promising anti-inflammatory and anticancer activities. One potential mechanism is to suppress nuclear factor kappa B (NF-κB) activation. The structures of chalcone-based NF-κB inhibitors vary significantly that there is minimum information about their structure-activity relationships (SAR). This study aims to establish SAR of chalcone-based compounds to NF-κB inhibition, to explore the feasibility of developing simple chalcone-based potent NF-κB inhibitors, and to evaluate their anticancer activities. Three series of chalcones were synthesized in one to three steps with the key step being aldol condensation. These candidates demonstrated a wide range of NF-κB inhibitory activities, some of low micromolar potency, establishing that structural complexity is not required for NF-κB inhibition. Lead compounds also demonstrate potent cytotoxicity against lung cancer cells. Their cytotoxicities correlate moderately well with their NF-κB inhibitory activities, suggesting that suppressing NF-κB activation is likely responsible for at least some of the cytotoxicities. One lead compound effectively inhibits lung tumor growth with no signs of adverse side effects.

Full text of DOI:10.1021/jm901278z

7228 J. Med. Chem. 2009, 52, 7228–7235
DOI: 10.1021/jm901278z
Structure-Activity Relationship Studies of Chalcone Leading to
3-Hydroxy-4,30,40,50-tetramethoxychalcone and Its Analogues as Potent Nuclear
Factor KB Inhibitors and Their Anticancer Activities
Balasubramanian Srinivasan,† Thomas E. Johnson,† Rahul Lad, and Chengguo Xing*
Department of Medicinal Chemistry, College of Pharmacy, University of Minnesota, Minneapolis, Minnesota 55455.
†These authors contributed equally to this research.
Received August 26, 2009
Chalcone is a privileged structure, demonstrating promising anti-inflammatory and anticancer acti-
vities. One potential mechanism is to suppress nuclear factor kappa B (NF-κB) activation. The
structures of chalcone-based NF-κB inhibitors vary significantly that there is minimum information
about their structure-activity relationships (SAR). This study aims to establish SAR of chalcone-based
compounds to NF-κB inhibition, to explore the feasibility of developing simple chalcone-based potent
NF-κB inhibitors, and to evaluate their anticancer activities. Three series of chalcones were synthesized
in one to three steps with the key step being aldol condensation. These candidates demonstrated a wide
range of NF-κB inhibitory activities, some of low micromolar potency, establishing that structural
complexity is not required for NF-κB inhibition. Lead compounds also demonstrate potent cytotoxicity
against lung cancer cells. Their cytotoxicities correlate moderately well with their NF-κB inhibitory
activities, suggesting that suppressing NF-κB activation is likely responsible for at least some of the
cytotoxicities. One lead compound effectively inhibits lung tumor growth with no signs of adverse side
effects.
Introduction
of chalcone-based compounds, such a mechanism cannot
account for their anti-inflammatory activities. On the other
hand, the anti-inflammatory properties of chalcone-based
compounds is probably, at least partially, responsible for their
anticancer activities because of the close linkage between
inflammation and tumorigenesis.31-34 Therefore, the anti-
inflammatory and anticancer activities of chalcone-based
compounds may be a result of its inhibitory activities against
the NF-κB signaling pathways.32-37 In fact, quite a few
chalcone-based compounds have been reported to inhibit
the NF-κB signaling pathway, some of them being shown in
Figure 1.14,38-49 Very interestingly, these chalcone-based
NF-κB inhibitors possess diverse structural properties with
varied substitutions, most of which being electron donating
functionalities, such as hydroxy and methoxy functional
groups, at different positions of both aromatic systems. Many
of these chalcones are easily obtainable through synthesis
(1-7, one-step aldol condensation), while others are com-
paratively more challenging to prepare. For instance, 8 has
never been synthesized before while 12-15 require four to
seven steps of syntheses.46,50-52 It is therefore important
to elucidate the structural requirements for chalcone-based
compounds with respect to NF-κB inhibition to determine
whether potent and easily accessible chalcone-based NF-κB
inhibitors can be developed for future therapeutic evaluation.
In this research, we explored the structure-activity rela-
tionship (SAR) of three series of chalcone-based compounds
regarding their NF-κB inhibitory activities by using a lucife-
rase-based in vitro NF-κB reporter assay with the goal to
determine the essential functionalities on the chalcone core for
Chalcone-based compounds (1,3-diaryl-2-propen-1-ones,
Figure 1), natural or synthetic, have been widely reported to
exhibit various biological activities, especially with regard to
anti-inflammatory and anticancer activities.1-14 For instance,
chalcone (1) and isoliquiritigenin (3) demonstrated significant
chemopreventive activities against lung, breast, prostate, and
colorectal cancers.1,15-18 Flavokawain A (6) suppressed blad-
der tumor growth at a dose of 50 mg/kg of body weight in a
mouse xenograft model.9 Cardamonin (5) inhibited inflam-
mation.19 Chalcones thus comprise a class of compounds with
important therapeutic potential. The ease of preparation, the
potential of oral administration,1,15,16,20 and safety20 also
support the feasibility of chalcone-based compounds as
therapeutic agents. Tremendous effort has been devoted to
elucidate the mechanisms of chalcone-based compounds for
their promising biological activities, including their interfe-
rence in microtubule formation2,11,12,21-28 and cellular signal-
ing pathways,7,29,30 such as nuclear factor kappa B (NF-κB)a
inhibition. Although interfering microtubule formation is one
mechanism potentiallyresponsible forthe anticanceractivities
*To whom correspondence should be addressed. Phone: 612-626-
5675. Fax: 612-624-0139. E-mail: xingx009@umn.edu.
a Abbreviations: NF-κB, nuclear factor kappa B; IKKβ, I kappa B
kinase 2; IRAK4, interleukin-1 receptor-associated kinase 4; SAR,
structure-activity relationship; N-Boc, N-tert-butyloxycarbonyl; TNF-R,
tumor necrosis factor R; IκBR, I kappa B alpha; IKKR, I kappa B kinase
1; IRAK1, interleukin-1 receptor-associated kinase 1; TAK1, Tat-
associated kinase 1; SCID, severe combined immunodeficiency; PBS,
phosphate-buffered saline; PEG, polyethylene glycol.
r
pubs.acs.org/jmc
Published on Web 11/02/2009
2009 American Chemical Society

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22 7229
Figure 1. Structures of representative chalcone-based NF-κB inhibitors.
Scheme 1. Representative Synthetic Schemes of Chalcone-
Based Analogues, Using Series 10 as an Examplea
Table 1. NF-κB Inhibitory Activitiesa of Chalcones with Substituents of
Varied Electron Densities on A and B Rings
a Reagents and conditions: (1) Me2SO4, K2CO3, acetone, reflux; (2)
substituted aromatic aldehyde, KOH, MeOH.
R1
R2
R3
R4
R5
R6
IC50 ( SD (μM)
NF-κB inhibition. The lead compound with low micromolar
inhibitory activity was evaluated for its inhibitory activities
against kinases upstream of NF-κB and in vitro and in vivo
anticancer activities. One lead compound, 11a, directly in-
hibits the kinase activities of I kappa B kinase 2 (IKKβ) and
interleukin-1 receptor-associated kinase 4 (IRAK4), poten-
tially responsible for its NF-κB inhibitory activities. 11a also
demonstrates potent cytotoxicity against lung cancer cells in
vitro and safely suppresses lung tumor growth in vivo. The
results of these SAR studies also identified positions on the
chalcone core structure that can accommodate modifications
without significant loss of the NF-κB inhibitory activities,
which will guide the future development of probe-based
chalcones to identify their cellular targets responsible for
NF-κB inhibition, to confirm whether IKKβ and IRAK4
are in fact the cellular targets.
1
H
H
H
H
H
H
H
H
H
H
H
F
H
>20
>20
>20
>20
>20
>20
>20
>20
>20
2
H
H
OH
H
H
H
3
OH
OH
H
H
OH
OH
CH3
F
OH
OH
H
4
OH
H
H
9a
9b
9c
9d
9e
H
H
H
H
H
H
H
H
CF3
F
H
H
OH
H
H
H
H
H
H
H
H
a Results are given as the mean of three independent experiments with
triplicates in each experiment.
Results
Synthesis of Chalcone Analogues. Three series of chalcone-
based compounds were synthesized according to procedures
previous described with the condensation step yielding
∼50-90% (Scheme 1).2,6 Compounds of the first series have
substituents of varied electron density on both rings with a
goal to explore whether the electron density on chalcone may
influence the enone’s electrophilicity (Table 1, 9a-e), poten-
tially responsible for its NF-κB inhibition through modifica-
tion of its target(s), such as IKKβ, via Michael addition
(Figure 2). Compounds of the second series have the same
substituents on the A ring as flavokawains A and B and
varied substituents on the B ring (Table 2, 10a-j) with a goal
to determine whether modification of the B ring could
improve NF-κB inhibitory activity, since flavokawains
Figure 2. Potential mechanism of chalcone-based compounds to
inhibit NF-κB via covalent modification of IKKβ, using butein as
the example of chalcone-based compounds.
A and B were reported to be the most potent NF-κB
inhibitors in kava by Folmer et al.43 On the basis of the
observation that most substituents on natural chalcone-
based NF-κB inhibitors are hydroxyl and methoxy func-
tional groups, a third chalcone series was synthesized to have
varied methoxy and hydroxyl substituents on both rings
(Table 3, 11a-m). 11k was prepared from 11a through Zn
dust reduction (Scheme 2). Such a candidate can help

7230 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22
Srinivasan et al.
determine whether the R,β unsaturated carbonyl functional
group is essential for NF-κB inhibition, potentially revealing
the chemical mechanism of NF-κB inhibition. 11l was
synthesized by reacting 11a with 2-bromoethanol. 11m was
synthesized by reacting 11a with N-Boc-2-bromoethanamine
followed by Boc deprotection.
In Vitro Inhibition of TNF-r-Induced NF-KB Activation in
A549 Lung Adenocarcinoma Cell Line. Human lung adeno-
carcinoma A549 cell line stably transfected with NF-κB-luc
allows easy monitoring of the effects of chalcone-based
compounds on tumor necrosis factor-alpha (TNF-R)-
induced NF-κB activation in vitro. Chalcone-based com-
pounds were first evaluated at a single concentration of
20 μM. Candidates with >50% inhibitory activities at
20 μM were further evaluated in a dose-dependent manner
to determine the concentration needed to inhibit 50% of
TNF-R-induced NF-κB activation (IC50). Disappointingly,
none of the chalcones in series 9, including 1, 2, 3, and 4, the
reported NF-κB inhibitors, showed significant NF-κB inhi-
bitory activities (IC50 > 20 μM). Among the chalcones
in series 10, only two candidates (10f and 10g) demonstrate
moderate NF-κB inhibitory activities (10 μM < IC50 < 20 μM).
The rest of the candidates, including 6 and 7, the most potent
NF-κB inhibitors identified from kava, have minimum inhibi-
tory activities with IC50 values higher than 20 μM. However,
11a was found to potently inhibit NF-κB activation with an
IC50 of 5.1 μM (Table 3). SAR studies revealed that removal
of methoxy functional groups (11b-d) or demethylation (11e)
on the A ring leads to a 2- to 3-fold decrease in activities.
Removal of 30-hydroxy on the B ring also leads to a slight to
moderate decrease in activities (11g, 11i, and 11j), while de-
methylation or removal of 40-methoxy leads to 2- to 3-fold
increase in activities (11f and 11h). Reducing the R,β-unsaturated
carbonyl double bond in 11a leads to significant loss of activity
(11k more than 4-fold less active). Lastly, modification of
30-hydroxy of 11a with 2-hydroxyethyl (11l) or 2-aminoethyl
functional (11m) groups leads to analogues of similar NF-κB
inhibitory activities. Both of these compounds can be further
modified by affinity tags, such as biotin, that can be used to
identify the cellular targets of series 11 that are responsible for
their NF-κB inhibition.
Inhibitory Activities of 11a against Kinases Modulating
NF-κB Activation. The NF-κB pathway can be activated
by multiple signaling pathways, most probably through
upstream kinase-based phosphorylation of IκBR (an inhi-
bitory protein of the NF-κB pathway), leading to IκBR
ubiquitination and proteasome degradation, p65 enrichment
in nucleus, and gene transcription. Chalcone 11a and its
analogues may induce NF-κB inhibition through inhibiting
kinases upstream of IκBR, such as IKKβ, like 4 does. We
therefore evaluated the effect of 11a, the initial lead com-
pound, against six upstream kinases that have been reported
to activate the NF-κB pathway, including I kappa B alpha
(IKKR), IKKβ, interleukin-1 receptor-associated kinase
1 (IRAK1), IRAK4, Tat-associated kinase 1 (TAK1), and
cSrc (Figure 3).53,54 It was found that 11a at 10 μM sig-
nificantly inhibits IKKβ and IRAK4 by 46% and 41%,
respectively, while it has no inhibitory activities against the
other four kinases.
Table 2. NF-κB Inhibitory Activitiesa of Chalcones with the Same A-
Ring as Flavokawains
R1
R2
R3
R4
IC50 ( SD (μM)
6
H
H
OCH3
H
OCH2CH2CH3
H
>20
>20
7
H
H
H
10a
10b
10c
10d
10e
10f
10g
10h
10i
10j
H
H
H
>20
>20
H
H
F
Cl
H
H
H
H
>20
>20
Br
CH3
H
H
H
H
H
Br
H
H
>20
H
H
12.3 ( 2.8
17.0 ( 2.9
>20
H
CH3
OH
CH3
OCH3
H
H
H
OCH3
OCH3
OCH3
H
H
H
OCH3
>20
>20
H
a Results are given as the mean of three independent experiments with
triplicates in each experiment.
Table 3. NF-κB Inhibitory Activitiesa of Chalcones with Varied
Methoxy and Hydroxyl Substituents on A and B Rings
In Vitro Cytotoxicities of Chalcone Series 11 against H2009
and A549 Lung Cancer Cells. As chronic lung inflammation
induced by tobacco usage through NF-κB activation is one
potential mechanism of lung cancer development, NF-κB
inhibitors will have therapeutic potential for lung cancer
treatment.55 Since chalcones in series 11 have potent NF-κB
inhibitory activities, these chalcones were chosen from
the three chalcone series and evaluated for their in vitro
cytotoxicities against two lung cancer cell lines, H2009
(Figure 4) and A549. The concentrations to inhibit the
cancer cell growth by 50% (GI50) are reported in Table 4.
Chalcones in series 11 demonstrated significant cytoto-
xicities, with GI50 mainly in the single-digit micromolar
range, similar to their in vitro NF-κB inhibitory potencies.
Their in vitro cytotoxicities also correlate positively with
their NF-κB inhibitory activities (Figure 5). Representa-
tive chalcones from series 9 and 10 were evaluated for
a Results are given as the mean of at least three independent experi-
ments with triplicates in each experiment.

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22 7231
Scheme 2. Synthesis of Chalcone Analogues (Series 11)a
a Reagents and conditions: (1) KOH, MeOH, room temp; (2) DMF, K2CO3, room temp; (3) trifluoroacetic acid, CH2Cl2, room temp; (4) Zn,
NH4OAc, EtOH, H2O, room temp.
their cytotoxicity in these two cell lines as well with IC50
20 μM (data not shown).
>
Anticancer Activities of 11a against H2009 Lung Cancer
Cell Induced Tumor Growth in Nude Mouse Xenograft Model.
To determine whether chalcone-based NF-κB inhibitors can
be potentially used to treat lung cancer, 11a, the first lead
compound, was evaluated for its effect on lung tumor growth
in vivo in a xenograft model. H2009 lung cancer cells were
inoculated in the right flank of severe combined immunode-
ficiency (SCID) nude female mice. 11a was administered to
mice at a dose of 1 mg/mouse/day 6 days a week through
intraperitoneal injection. Bodyweights of mice were mea-
sured twice a week. At the end of week 5, the mice were
weighed and euthanized by CO2 and the tumor dissected and
weighed. All animals tolerated the treatments well with no
observable signs of toxicity, and increase in body weight
occurred at a similar rate among control mice and 11a
treated mice. No gross pathologic abnormalities were noted
at the time of necropsy as well (data not shown). 11a
significantly suppressed H2009-induced lung tumor growth
by 57%.
Figure 3. Inhibitory activities of chalcone 11a at 10 μM against six
kinases upstream of the NF-κB pathway.
Discussion and Conclusion
Figure 4. Effect of ip 11a at 1 (mg/mouse)/day on the growth of
H2009 tumors in nude mice. Each mouse was inoculated sc in the
right flank with 2 ꢀ 106 H2009 cells suspended in 0.1 mL of PBS/
metrigel (v/v 1:1). Mice were given 11a at 1 (mg/mouse)/day in a
carrier made of PEG 400/EtOH (v/v 2:1, 50 μL) with intraperitoneal
administration for 5 weeks, 6 days per week. Control mice received
carrier alone. (A) Tumor volumes: columns, mean; bars, SE (n = 5;
(//) p < 0.01 compared with the control group). (B) Average body
weight: columns, mean; bars, SE (n = 5; p > 0.05 compared with the
control group).
We have synthesized and evaluated three series of chalcone-
based compounds with different substitutions on both aro-
matic rings and reduction of the alkene linkage with the goal
to determine the structural requirements for chalcone to
inhibit the NF-κB pathway, potentially leading to candidates
with anti-inflammatory and anticancer activities. Disappoin-
tingly, many of the reported simple chalcone-based NF-κB
inhibitors, such as 1-7, were found to have rather weak
inhibitory activities (IC50 > 20 μM). Varying electron density
on chalcone also has no effect with respect to NF-κB inhibi-
tion. However, several simple chalcone candidates with only
hydroxyl and methoxy substitutions in series 11 demonstrate
single-digit micromolar NF-κB inhibitory activities. The re-
duction of the alkene functionality in chalcone completely
abolishes this activity, supporting the notion that the mechan-
ism of inhibition is likely through a covalent Michael addition
of nucleophiles (such as SH from cysteine) from protein
candidate(s) to chalcones. Despite its simple structure and
ease of synthesis, 11a in fact inhibits the kinase activity of
IKKβ of similar potency compared to 15.46 11a also inhibits
the kinase activity of IRAK4. Chalcones in series 11 also
demonstrate potent cytotoxicity against lung cancer cells in
vitro with most GI50 in single-digit micromolar range, con-
sistent with their NF-κB inhibitory potency, which also
correlates positively with each other. These results suggest
that inhibiting NF-κB is likely responsible for the in vitro
cytotoxicity of chalcones 11. The nonperfect correlation may
be due to potential experimental errors, or more likely there
are other mechanisms involved for the cytotoxicities of chal-

7232 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22
Srinivasan et al.
cones in series 11. 11a also effectively suppresses lung cancer
growth in vivo at a dose of 1 mg/mouse/day via ip admin-
istration. Such treatment reveals no signs of adverse side
effect.
These data collectively demonstrate that chalcones as sim-
ple as those in series 11 can potently inhibit NF-κB activation
via inhibiting the kinase activities of IKKβ and/or IRAK4.
These simple chalcones are promising anticancer candidates
against lung cancer, warranting further investigations.
on a Varian Mercury 300 spectrometer. Proton chemical shifts
are reported in ppm from an internal standard of residual CHCl3
(7.26 ppm), and carbon chemical shifts are reported using an
internal standard of residual CHCl3 (77.16 ppm). Proton che-
mical data are reported as follows: chemical shift, multiplicity,
coupling constant, and integration. High resolution mass spec-
tra were obtained at the University of Minnesota, Department
of Chemistry Mass Spectrometry Facility, on a Bruker BioTOF
II ESI-TOF/MS utilizing polyethylene glycol (PEG) as an
internal high resolution calibration standard. All compounds
synthesized were at least 95% pure as analyzed by HPLC.
General Procedure for the Synthesis of Chalcones. A mixture
of the corresponding acetophenone (1 equiv) and the corre-
sponding aldehyde (1 equiv) in anhydrous EtOH (3 mL for
1 mmol of acetophenone) was stirred at room temperature for
5 min. Then NaOH (3 equiv) was added. The reaction mixture
was stirred at room temperature until aldehyde was consumed
(usually 6-12 h). After that, HCl (10%) was added until pH 5
was obtained. In the case of the chalcones that precipitated, they
were filtered and crystallized from MeOH. In the other cases,
the product was purified by using silica gel chromatography.
The physical data for the prepared chalcone compounds 1-4, 6,
7, 9a-e, 10a-d, 10f, 10h, 10j, 11a, 11c, 11d, 11f-j nicely match
literature reported physical data (see Supporting Information).
(E)-1-(2-Hydroxy-4,6-dimethoxyphenyl)-3-m-tolylprop-2-en-
1-one (10g). 10g was recrystallized from MeOH. Yield: 32%.
Materials and Methods
Chemistry. All commercial reagents and anhydrous solvents
were purchased from vendors and were used without further
purification or distillation, unless otherwise stated. Analytical
thin-layer chromatography (TLC) was performed on EM
Science silica gel 60 F254 (0.25 mm). Compounds were visualized
by UV light and/or stained with either p-anisaldehyde, potas-
sium permanganate, or cerium molybdate solutions followed by
heating. Flash column chromatography was performed on
Fisher Scientific silica gel (230-400 mesh). HPLC purity ana-
lyses were performed on a Beckman Coulter system Gold 126
solvent module with a 168 detector. A Clipeus C-18 column
(5 μm, 250 mm ꢀ 4.6 mm) was used for the analyses. The
flow rate used was 0.5 mL/min. The mobile phase A was water,
while B was acetonitrile. The time program used for the ana-
lyses was 55% B (0-5 min) and 55-85% B (5-25 min) and
99% B (25-40 min). Melting points were determined on a
Thomas-Hoover Unimelt melting point apparatus 6406-K and
were uncorrected. IR spectra were recorded on a Nicolet Portege
460 FT-IR instrument. 1H and 13C NMR spectra were recorded
1
HPLC tR =19.6 min; Rf = 0.23 (10% EtOAc/hexanes). H
NMR (CDCl3, 300 MHz) δ 2.39 (s, 3H), 3.83 (s, 3H), 3.92 (s,
3H), 5.96 (d, J = 2.7 Hz, 1H), 6.11 (d, J = 2.7 Hz, 1H),
7.19-7.43 (ovlp m, 4H), 7.75 (d, J = 15.6 Hz, 1H), 7.88 (d, J =
15.3 Hz, 1H), 14.33, (s, 1H). 13C NMR (CDCl3, 75 MHz) δ 21.6,
55.8, 56.1, 91.5, 94.0, 106.6, 125.6, 127.6, 129.0, 129.5, 131.2,
135.7, 138.7, 142.8, 162.7, 166.4, 168.6, 192.9. HRMS calcd
(C18H18O4 þ Naþ): 321.1097, found 321.1119.
Table 4. GI50a of Chalcones against Two Lung Cancer Cells
H2009 (μM)
A549 (μM)
(E)-1-(2-Hydroxy-4,6-dimethoxyphenyl)-3-o-tolylprop-2-en-
1-one (10e). 10e was recrystallized from MeOH. Yield: 16%.
HPLC tR = 19.8 min; Rf = 0.23 (10% EtOAc/hexanes). H
GI50
SE
GI50
SE
1
11a
11b
11c
11d
11e
11f
11g
11h
11i
5.2
4.6
2.3
8.1
7.5
4.2
4.5
1.1
8.4
6.6
47
0.7
0.3
0.6
0.9
1.7
0.8
0.9
0.3
0.9
0.8
7.6
0.8
0.04
2.1
5.2
0.5
1.7
0.3
1.2
0.7
0.2
0.5
0.4
0.6
0.4
2.0
0.5
0.5
NMR (CDCl3, 300 MHz) δ 2.49 (s, 3H), 3.84 (s, 3H), 3.91 (s,
3H), 5.96 (d, J = 2.1 Hz, 1H), 6.11 (d, J = 2.7 Hz, 1H),
7.21-7.31 (m, 3H),7.64 (d, J = 7.2 Hz, 1H), 7.81 (d, J = 15.9
Hz, 1H), 8.07 (d, J = 15.6 Hz, 1H), 14.31 (s, 1H). 13C NMR
(CDCl3, 75 MHz) δ 20.2, 55.8, 56.1, 91.5, 94.0, 106.6, 126.5,
126.8, 128.8, 130.0, 131.1, 134.8, 138.4, 140.2, 162.8, 166.5,
168.6, 193.0. HRMS calcd (C18H18O4 þ Naþ): 321.1097, found
321.1126.
3.3
10.0
4.0
1.7
8.5
2.6
12.4
8.8
(E)-1-(2-Hydroxy-4,6-dimethoxyphenyl)-3-(4-methoxy-3-methyl-
phenyl)prop-2-en-1-one (10i). 10i was recrystallized from MeOH.
Yield: 20%. HPLC tR = 19.3 min; Rf = 0.12 (10% EtOAc/
11j
11k
11l
17.6
4.7
3.3
3.6
1
hexanes). H NMR (CDCl3, 300 MHz) δ 2.25 (s, 3H), 3.83 (s,
11m
5.6
3H), 3.87 (s, 3H), 3.92 (s, 3H), 5.96 (d, J= 2.4 Hz, 1H), 6.11 (d, J =
2.4 Hz, 1H), 6.84 (d, J = 8.1 Hz, 1H), 7.42-7.44 (m, 2H), 7.78, (s,
2H), 14.46, (s, 1H). 13C NMR (CDCl3, 75 MHz) δ 16.6, 55.7, 55.8,
a Results are given as the mean of at least three independent experi-
ments with triplicate in each experiment.
Figure 5. Correlation of the in vitro NF-κB inhibitory activities and cytotoxicities of chalcones in series 11 in A549 and H2009 cells. The mean
values of the GI50 (the concentration to inhibit cancer cell growth by 50%) of each chalcone compounds were analyzed for their linear
relationship with their mean IC50 of NF-κB inhibition (the concentration to inhibit the luciferase-based NF-κB activities by 50%) by using a
linear regression model in GraghPad.

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22 7233
56.1, 91.4, 94.0, 106.6, 110.2, 125.0, 127.4, 128.0, 128.5, 130.7, 143.2,
159.9, 162.7, 166.2, 168.6, 192.9. HRMS calcd (C19H20O5 þ Naþ):
351.1203, found 351.1197.
4 h. The reaction mass was basified to pH 8.5 using saturated
aqueous NaHCO3 solution and extracted using methylene chloride.
The organic layer was dried over anhydrous MgSO4 and concen-
trated under reduced pressure. The crude product was subjected to
column chromatography using 10% methanol in methylene chloride
to afford pure 11m as a yellow solid in 84% yield. TLC (MeOH/
hexane = 0.1:1), Rf = 0.21. 1H NMR (400 MHz, MeOH-d4):δ 7.74
(d,J=15.5Hz,1H),7.63(d,J= 15.5 Hz, 1H), 7.46 (m, 2H), 7.39 (s,
2H), 7.09 (d, J = 8.4 Hz, 1H), 4.30 (t, J = 4.9 Hz, 2H), 3.94 (s, 3H),
3.93 (s, 6H), 3.87 (s, 3H), 3.36 (t, J = 4.9 Hz, 2H). 13C NMR
(100 MHz, MeOH-d4): δ 191.1, 154.6, 153.6, 149.6, 146.3, 143.9,
135.0, 129.6, 125.6, 120.8, 115.1, 113.0, 107.5, 107.0, 70.2, 61.2, 56.9,
56.5, 41.3. MS (ESI, positive) m/z 388.11. Calcd for C21H25NO6H:
388.17
(E)-1-(3,4-Dimethoxyphenyl)-3-(3-hydroxy-4-methoxyphenyl)-
prop-2-en-1-one (11b). Yellow solid. Yield: 52%. TLC (acetone/
hexane = 1:1), Rf = 0.35. 1H NMR (400 MHz, CDCl3): δ 7.75
(d, J = 15.5 Hz, 1H), 7.69 (dd, J = 8.4, 2.0 Hz, 1H), 7.64 (d, J =
1.96 Hz, 1H), 7.44 (d, J = 15.5 Hz, 1H), 7.32 (d, J = 2.1 Hz, 1H),
7.15 (dd, J = 2.0, 8.4 Hz, 1H), 6.95 (d, J = 8.4 Hz, 1H), 6.89 (d,
J = 8.3 Hz, 1H), 5.71 (s, 1H), 3.99 (s, 3H), 3.98 (s, 3H), 3.96 (s,
3H). 13C NMR (100 MHz, CDCl3): δ 188.6, 153.1, 149.2, 148.7,
145.9, 143.9, 131.5, 128.7, 122.9, 122.7, 119.8, 112.9, 110.7, 110.6,
109.9, 76.7, 56.1, 56.0. MS (ESI, positive) m/z 337.05. Calcd for
C18H18O5Na: 337.12.
(E)-1-(4-Hydroxy-3,5-dimethoxyphenyl)-3-(3-hydroxy-4-meth-
oxyphenyl)prop-2-en-1-one (11e). Yellow solid. Yield: 48%. TLC
(acetone/hexane= 1:1), Rf = 0.31. 1H NMR (400 MHz, CDCl3):
δ 7.74 (d, J = 15.5 Hz, 1H), 7.37 (d, J = 15.5 Hz, 1H), 7.33 (s,
2H), 7.31 (d, J = 2.1 Hz, 1H), 7.13 (dd, J = 2.1, 8.4 Hz, 1H), 6.88
(d, J = 8.3 Hz, 1H), 5.95 (s, 1H), 5.68 (s, 1H), 3.99 (s, 6H), 3.95 (s,
3H). 13C NMR (100 MHz, CDCl3): δ 188.5, 148.7, 146.8, 145.8,
144.2, 119.5, 129.9, 128.7, 122.9, 119.8, 112.6, 110.5, 105.8, 76.7,
56.56, 56.1. MS (ESI, positive) m/z 353.05. Calcd for C18H18-
O6Na: 353.11.
In Vitro Inhibition of TNF-r-Induced NF-KB Activation in
A549 Lung Adenocarcinoma Cell Line. Human lung adenocar-
cinoma A549 cell line stably transfected with NF-κB-luc was
purchased from Panomics (catalog no. RC0002, Fremont, CA).
This cell line was obtained by cotransfecting A549 cells with
pNFκB-luc (Panomics P/N LR0051) and pHyg followed by
hygromycin selection, which can monitor NF-κB activity in
vitro. This cell line was cultured using DMEM medium supple-
mented with 10% FBS, penicillin (100 units/mL), streptomycin
(100 μg/mL), and hygromycin (100 μg/mL) at 37 °C with
5% CO2. Cells were plated in a 96-well plate at a density of
5000 cells/well. Twenty-four hours after plating, the cell medium
was replaced with fresh medium and incubated at 37 °C with 5%
CO2 for 1 h to minimize the potential stimulation to the cells
induced by medium change. Cells were then treated with TNF-R
(15 ng/mL) and chalcone-based compounds simultaneously for
7 h. Cells treated with TNF-R alone served as positive controls,
while cells without TNF-R treatment served as negative con-
trols. Luciferase activities from these cells were then measured
by using the Bright-Glo luciferase assay kit from Promega
(catalog no. E2620, Madison, WI), following the manufac-
turer’s protocol (catalog no. TM052). The relative NF-κB
activities of the cells treated by chalcone candidates were
obtained as the ratio of its luciferase activity to that from the
positive controls, both of which have been corrected with back-
ground (signals from negative controls) and cell viability.56
Under these experimental conditions, none of the chalcone
compounds induced significant toxicity to A549 cells (<5%
reduction of cell viability). The IC50 of each fraction was
determined by fitting the relative NF-κB activity to the drug
concentration by using a sigmoidal dose-response model of
varied slope in GraphPad. The IC50 reported herein is the
average of at least three replicates with standard error, in most
cases, of no more than 20% of the average IC50.
In Vitro Cytotoxicity Assay. Both cell lines were cultured in
RPMI medium with 10% FBS, penicillin (100 units/mL), and
streptomycin (100 μg/mL) at 37 °C with 5% CO2. The in vitro
cytotoxicity of the small molecules was assayed by determining
the GI50 (the concentration of the small molecules to reduce the
cell growth by 50%). In brief, the cells were plated in a 96-well
plate (2.5 ꢀ 103 cells/well). The cells were treated with the small
molecules with a series of 3-fold dilution with 0.5% DMSO in
the final cell media (cells treated with media containing 0.5%
DMSO served as a control). After 48 h of treatment, the relative
cell viability in each well was determined by using CellTiter-Blue
cell viability assay kit (a fluorescence assay that measures the
reduction of a dye (resazurin) into a fluorescent end product
(resorufin) by metabolically active cells - viable cells, Promega,
CA). The GI50 of each agent was determined by fitting the
relative viability of the cells to the drug concentrations by using a
sigmoidal dose-response model of varied slope in GraphPad.
The GI50 reported herein is the average of at least three
replicates with standard error, in most cases, of no more than
20% of the average GI50.
3-(3-Hydroxy-4-methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)-
propan-1-one (11k). A 0.01 M solution of 11a in 95% EtOH
(10 mL) was added to 50 mM ammonium acetate aqueous
solution (2 mL) and stirred vigorously with 60 mg of zinc
powder added in three equal intervals of 15 min. Stirring was
continued for further 15 min (monitored by TLC). The sus-
pended material was removed by filtration and washed with
ethanol, and the filtrate was evaporated to dryness under
reduced pressure. The crude product was subjected to column
chromatography on silica gel using 2:3 acetone/hexanes to
afford pure 11k as faint yellow solid in 75% yield. TLC
(acetone/hexane = 1:1), Rf = 0.40. 1H NMR (400 MHz,
CDCl3): δ 7.29 (s, 1H), 6.87 (d, J = 2 Hz, 1H), 6.82 (d, J =
8 Hz, 1H), 6.76 (dd, J = 8 Hz, 2 Hz, 1H), 5.62 (s, 1H), 3.93 (s,
3H), 3.93 (s, 6H), 3.90 (s, 3H), 3.25 (t, J = 8 Hz, 2H), 3.00 (t, J =
8 Hz, 2H). 13C NMR (100 MHz, CDCl3): δ 198.1, 153.1, 145.6,
145.0, 142.5, 134.6, 132.2, 119.8, 114.5, 110.7, 76.7, 60.9, 56.0,
40.4, 29.8. MS (ESI, positive) m/z 369.06. Calcd for
C19H22O6Na: 369.14.
(E)-3-(3-(2-Hydroxyethoxy)-4-methoxyphenyl)-1-(3,4,5-tri-
methoxyphenyl)prop-2-en-1-one (11l). Yellow solid. Yield: 98%.
TLC (acetone/hexane = 1:1), Rf = 0.2. H NMR (400 MHz,
1
CDCl3): δ 7.67 (d, J = 15.6 Hz, 1H), 7.25 (d, J = 15.6 Hz, 2H),
7.16-7.21 (m, 3H), 6.86 (d, J = 8.4 Hz, 1H), 4.12 (t, J = 4.3 Hz,
2H), 3.90 (t, J = 4.3 Hz, 2H), 3.82 (s, 6H), 3.87 (s, 3H), 3.85 (s,
3H), 2.42 (t, J = 5.8 Hz, 1H). 13C NMR (100 MHz, CDCl3):
δ 189.3, 153.1, 152.2, 148.4, 144.6, 142.4, 133.7, 128.0, 123.9,
119.9, 114.1, 111.7, 106.1, 76.7, 71.6, 61.3, 60.9, 56.5, 55.9. MS
(ESI, positive) m/z 411.08. Calcd for C21H24O7Na: 411.15.
(E)-3-(3-(2-Aminoethoxy)-4-methoxyphenyl)-1-(3,4,5-trimethoxy-
phenyl)prop-2-en-1-one (11m). To a mixture of 11a (50 mg,
0.145 mmol) and K2CO3 (30 mg, 0.22 mmol) in dry DMF,
N-Boc-2-bromoethanamine (33 mg, 0.145 mmol) was added and
stirred under N2 atmosphere at room temperature overnight. Solvent
was evaporated under reduced pressure. The reaction mass was
dispersed in ethyl acetate and washed by saturated aqueous solution
of NaCl. The organic layer was dried over anhydrous MgSO4,
filtered, and concentrated under reduced pressure to afford the crude
product. The crude was subjected to column chromatography using
hexanes/ethyl acetate (1:1) to afford the pure 11n in 96% yield. TLC
(MeOH/hexane = 0.1:1), Rf = 0.21. 1H NMR (400 MHz, MeOH-
d4): δ 7.74 (d, J = 15.6 Hz, 1H), 7.32 (d, J = 15.6 Hz, 1H), 7.25 (m,
4H), 6.92 (d, J = 8.3 Hz, 1H), 4.13 (t, J = 4.8 Hz, 2H), 3.95 (s, 6H),
3.93 (s, 3H), 3.91 (s, 3H), 3.52 (t, J = 4.8 Hz, 2H), 1.44 (s, 9H). A
mixture of compound 11n (50 mg) and 10% trifluoroacetic acid in
dry methylene chloride (2 mL) was stirred at room temperature for
Upstream Kinase Inhibitory Screening. Compound 11a was
evaluated by Millipore Kinase Profiler service (Dundee, U.K.)

7234 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22
Srinivasan et al.
for its inhibitory activities against six upstream kinases of the
NF-κB pathway, including IKKR, IKKβ, IRAK1, IRAK4,
TAK1, and cSrc that have been reported to regulate the activa-
tion of the NF-κB signaling pathway. 11a was evaluated at a
single concentration of 10 μM in duplicate with ATP at a
concentration of 10 μM.
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Xenograft Tumor Growth and 11a Treatment. Female athymic
nude mice obtained from Harland were maintained in a laminar
airflow cabinet under pathogen-free conditions and used at
6-12 weeks of age. All facilities were approved by the American
Association for Accreditation of Laboratory Animal Care in
accordance with the current regulations and standards of the
U.S. Department of Agriculture, U.S. Department of Healthand
Human Services, and NIH. Compound 11a was reconstituted in
a carrier made of PEG400/EtOH (v/v 2:1) to a concentration of
20 mg/mL. H2009 cells (60-70% confluent) were used for
injection. Viable tumor cells (2 ꢀ 106 in 0.1 mL of PBS/metrigel
(v/v 1:1)) were implanted subcutaneously into the right flank.
Formation of a bulla indicated a satisfactory injection. Begin-
ning on the same day of implantation, one group of mice (5 mice
per group) were treated with the carrier (50 μL) through
ip. injection and the other group of mice (5 mice per group) were
treated with 11a at 1 (mg/mouse)/day in the carrier (50 μL)
through ip injection for 5 weeks, 6 days per week. The body
weights of mice were monitored twice a week. Mice were
euthanized by CO2 at the end of week 5. Tumor mass and mouse
final body weight were determined.
Statistical Analysis. All biological experiments, including the
NF-κB inhibitory activities and the in vitro cytotoxicity, were
performed at least three times with triplicates in each experi-
ment. Average results are depicted in this report. Data were
analyzed and presented using the GraphPad software. Student’s
t-test with a 95% confidence was applied for comparison
between groups using the GraphPad software. Significance
was set at P < 0.05. The correlation study was performed using
a linear fitting model in GraphPad.
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Studies on cancer chemoprevention by traditional folk medicines.
XXV. Inhibitory effect of isoliquiritigenin on azoxymethane-induced
murine colon aberrant crypt focus formation and carcinogenesis.
Biol. Pharm. Bull. 2002, 25, 247–250.
Acknowledgment. NIH Grant CA114294 and NIH Chem-
istry-Biology interface training grant are gratefully acknow-
ledged. T.E.J. also gratefully acknowledges a 3M Science &
Technology Fellowship.
(17) Kanazawa, M.; Satomi, Y.; Mizutani, Y.; Ukimura, O.; Kawauchi,
A.; Sakai, T.; Baba, M.; Okuyama, T.; Nishino, H.; Miki, T.
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Supporting Information Available: NMR spectra of new
compounds and literature information of known chalcone
compounds used for the study. This material is available free
of charge via the Internet at http://pubs.acs.org.
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