Novel chalcone derivatives as potent Nrf2 activators in mice and human lung epithelial cells

Article abstract of DOI:10.1021/jm2002348

Nrf2-mediated activation of antioxidant response element is a central part of molecular mechanisms governing the protective function of phase II detoxification and antioxidant enzymes against carcinogenesis, oxidative stress, and inflammation. Nrf2 is sequestered in the cytoplasm by its repressor, Keap1. We have designed and synthesized novel chalcone derivatives as Nrf2 activators. The potency of these compounds was measured by the expression of Nrf2 dependent antioxidant genes GCLM, NQO1, and HO1 in human lung epithelial cells, while the cytotoxicity was analyzed using MTT assay. In vivo potency of identified lead compounds to activate Nrf2 was evaluated using a mouse model. Our studies showed 2-trifluoromethyl-2′-methoxychalone (2b) to be a potent activator of Nrf2, both in vitro and in mice. Additional experiments showed that the activation of Nrf2 by this compound is independent of reactive oxygen species or redox changes. We have discussed a quantitative structure-activity relationship and proposed a possible mechanism of Nrf2 activation.

Full text of DOI:10.1021/jm2002348

ARTICLE
pubs.acs.org/jmc
Novel Chalcone Derivatives as Potent Nrf2 Activators in Mice and
Human Lung Epithelial Cells
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||
Vineet Kumar,†, Sarvesh Kumar,‡, Mohammad Hassan,† Hailong Wu,‡ Rajesh K. Thimmulappa,‡
Amit Kumar,§ Sunil K. Sharma,§ Virinder S. Parmar,§ Shyam Biswal,*,‡ and Sanjay V. Malhotra*,†
†Laboratory of Synthetic Chemistry, SAIC Frederick, Inc., National Cancer Institute at Frederick, Frederick, Maryland 21702,
United States
‡Department of Environmental Health Sciences, Johns Hopkins School of Public Health, Johns Hopkins University, Baltimore,
Maryland 21205, United States
§Bioorganic Laboratory, Department of Chemistry, University of Delhi, Delhi 110007, India
ABSTRACT: Nrf2-mediated activation of antioxidant response
element is a central part of molecular mechanisms governing
the protective function of phase II detoxification and antiox-
idant enzymes against carcinogenesis, oxidative stress, and
inflammation. Nrf2 is sequestered in the cytoplasm by its
repressor, Keap1. We have designed and synthesized novel
chalcone derivatives as Nrf2 activators. The potency of these
compounds was measured by the expression of Nrf2 dependent
antioxidant genes GCLM, NQO1, and HO1 in human lung epithelial cells, while the cytotoxicity was analyzed using MTT assay. In
vivo potency of identified lead compounds to activate Nrf2 was evaluated using a mouse model. Our studies showed
2-trifluoromethyl-20-methoxychalone (2b) to be a potent activator of Nrf2, both in vitro and in mice. Additional experiments
showed that the activation of Nrf2 by this compound is independent of reactive oxygen species or redox changes. We have discussed
a quantitative structureꢀactivity relationship and proposed a possible mechanism of Nrf2 activation.
’ INTRODUCTION
gene expression have been shown to exert chemopreventive
activities, e.g., sulforaphane,4,20ꢀ22 dithiolethione,23ꢀ25 curcumin,26
and caffeic acid phenethyl ester (CAPE).26 It is notable that both
curcumin and CAPE bear an R,β-unsaturated ketone moiety and
can therefore act as Michael acceptors that are able to modify
cysteine thiols present in Keap1.
Nuclear factor erythroid 2 p45-related factor 2 (Nrf2) is a
basic-leucine zipper (b-ZIP) transcription factor present in the
cytoplasm of normal cells. Upon activation in response to
inflammatory stimulus and environmental toxicant, oxidative,
and electrophilic stress, Nrf2 detaches from its cytosolic inhibi-
tor, Kelch-like ECH-associated protein 1 (Keap1), and translo-
cates to the nucleus and binds to the antioxidant response
element (ARE) of target genes along with other binding partners
leading to their transcriptional induction.1ꢀ4 The Keap1ꢀNrf2
system is the major regulatory pathway of cytoprotective gene
expression against oxidative and/or electrophilic stresses. Keap1
acts as a stress sensor protein in this system. While Keap1
constitutively suppresses Nrf2 activity under unstressed condi-
tions, oxidants or electrophiles provoke the repression of Keap1
activity, inducing the Nrf2 activation.5ꢀ7 In addition to Keap1,
the activation of different protein kinases has been shown to
activate Nrf2.8ꢀ12 The Nrf2-regulated genes include almost all
of the relevant antioxidants and cytoprotective genes such as
heme oxygenase-1 (HO-1), NAD(P)H/quinone oxidoreductase
1 (NQO1), glutamateꢀcysteineligase modifier subunit (GCLM),
γ-glutamyl cysteine synthase, glutathione peroxidase (GPx), and
several members of the glutathione S-transferase family6,13ꢀ18 that
express an ARE in their promoter.19 Small molecules that activate
Nrf2 signaling are being investigated as potential anticancer or
anti-inflammatory agents. A wide variety of dietary and synthetic
compounds that function as potent inducers of ARE-regulated
Chalcones or 1,2-diphenyl-2-propen-1-ones are Michael ac-
ceptors and constitute an important group of natural products
belonging to the flavonoid family.27,28 Theyhavebeen reported to
possess many biological properties including anticancer,29,30
antimalarial,31,32 anti-inflammatory,33ꢀ35 antileishmanial,33ꢀ35
antituberclulosis,36 nitric oxide inhibition,37,38 antimitotic,39 an-
algesic, antipyretic, antioxidant,40ꢀ43 antibacterial, anti-HIV,44
antifungal,45 and antiprotozoal activities.46ꢀ48 They are also
reported to be gastric protectant,49 antimutagenic, and
antitumorogenic.50ꢀ52 Natural and synthetic chalcones have been
reported to possess strong antiproliferative effects in primary and
established ovarian cancer cells53 and in gastric cancer cells.52
Chalcones contain two aromatic rings separated by R,β-unsatu-
rated ketone, and this unique structure is responsible for various
activities of these molecules.27 It is well-known that the R,β
unsaturated carbonyl entity in chalcones is a soft electrophile and
would attract soft nucleophiles like thiols rather than hard
nucleophiles like amino and hydroxyl groups. Chalcones are
Received: February 28, 2011
Published: May 03, 2011
r
2011 American Chemical Society
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Journal of Medicinal Chemistry
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Table 1. Synthesis of Chalcone Derivatives by ClaisenꢀSchmidt Condensation and Their Nrf2 Induction Activity
substituents on ring A
R3 R4
substituents on ring B
R7
relative fold changea
entry
chalcones
1a
R1
R2
R5
R6
R8
GCLM
NQO1
1
H
H
H
H
H
H
H
H
0.7
3.6
1.5
1.9
2.7
2.5
3.0
2.3
3.0
0.7
2.4
3.1
5.0
4.5
5.6
5.4
5.4
5.7
4.4
4.3
4.0
3.0
5.4
4.4
3.1
3.9
4.6
2.7
4.6
1.8
1.8
4.1
3.8
1.7
3.6
1.4
3.1
4.3
2.9
3.5
5.0
1.4
4.1
3.5
1.8
3.2
2.4
2.8
2.8
1.9
2.6
2.5
2.4
0.7
2.4
1.8
5.3
4.6
4.3
4.6
4.5
5.3
2.7
2.7
3.7
3.1
4.1
4.3
2.8
2.9
3.5
2.8
4.0
0.6
0.8
2.9
3.9
0.8
3.2
0.7
2.9
5.4
3.1
4.6
4.8
0.6
4.0
2.7
2
1b
1c
1d
1e
1f
OMe
H
H
H
H
H
H
H
H
3
OMe
H
H
H
H
H
H
H
4
H
OMe
OMe
H
H
H
H
H
H
5
OMe
OMe
OMe
H
H
H
H
H
H
H
6
H
H
OMe
H
H
H
H
7
1g
1h
1i
H
H
OMe
H
H
H
H
8
OMe
OMe
OMe
OMe
H
OMe
H
H
H
H
H
9
H
OMe
OMe
H
H
H
H
H
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
1j
H
OMe
OMe
OMe
H
H
H
H
H
1k
1l
OMe
OMe
H
H
H
H
H
H
OMe
H
H
H
H
2a
2b
2c
2d
2e
2f
H
H
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
H
H
H
OMe
H
H
H
H
H
H
H
OMe
H
H
H
H
H
H
H
OMe
OMe
H
H
H
H
H
OMe
OMe
OMe
H
H
H
H
H
H
H
H
OMe
H
H
H
2g
2h
2i
H
H
OMe
H
H
H
OMe
OMe
OMe
OMe
H
OMe
H
H
H
H
H
OMe
OMe
H
H
H
H
2j
H
OMe
OMe
OMe
H
H
H
H
2k
2l
OMe
OMe
H
H
H
H
H
OMe
H
H
H
3a
3b
3c
3d
3e
3f
H
H
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
H
H
OMe
H
H
H
H
H
H
H
OMe
H
H
H
H
H
H
H
OMe
OMe
H
H
H
H
H
OMe
OMe
OMe
H
H
H
H
H
H
H
H
OMe
H
H
H
3g
3h
3i
H
H
OMe
H
H
H
OMe
OMe
OMe
OMe
H
OMe
H
H
H
H
H
OMe
OMe
H
H
H
H
3j
H
OMe
OMe
OMe
H
H
H
H
3k
3l
OMe
OMe
H
H
H
H
H
OMe
H
H
H
4a
4b
4c
4d
4e
4f
H
H
H
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
OMe
H
H
H
H
H
H
H
OMe
H
H
H
H
H
H
H
OMe
OMe
H
H
H
H
H
OMe
OMe
OMe
H
H
H
H
H
H
H
H
OMe
H
H
H
4g
4h
H
H
OMe
H
H
H
OMe
OMe
H
H
H
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ARTICLE
Table 1. Continued
substituents on ring A
substituents on ring B
R7
relative fold changea
entry
chalcones
4i
R1
R2
R3
R4
R5
R6
R8
GCLM
NQO1
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
H
OMe
OMe
H
OMe
OMe
OMe
H
OMe
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
CF3
CF3
CF3
H
4.1
4.1
2.4
2.1
1.9
2.9
4.6
2.7
4.1
1.7
2.3
3.4
1.2
2.9
2.6
1.0
2.7
3.7
3.9
2.4
1.6
1.4
2.7
4.3
3.3
3.7
0.7
2.7
4.1
0.7
3.3
2.0
1.0
3.6
4j
OMe
OMe
H
H
H
4k
H
OMe
H
H
5a
H
H
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
5b
OMe
H
H
H
H
H
H
5c
OMe
H
H
H
H
5d
H
OMe
OMe
H
H
H
H
5e
OMe
OMe
OMe
H
H
H
H
H
5f
H
H
OMe
H
H
5g
H
H
OMe
H
H
5h
OMe
OMe
OMe
OMe
H
OMe
H
H
H
5i
H
OMe
OMe
H
H
H
5j
H
OMe
OMe
OMe
H
H
5k
OMe
OMe
H
H
5l
H
OMe
H
DMSO
sulforaphane
a Data presented are representative of three independent experiments. Values shown are mean of quadruplicate wells.
unlikely to react with the amino and hydroxyl groups on nucleic
acids and thus would unlikely induce mutagenicity and carcino-
genicity commonly associated with alkylating agents used in
cancer chemotherapy.28
AlCl3,70 BF3,71 dry HCl,72 Zn(bpy)(OAc)2,73 Cp2ZrH2/
NiCl2,74 and RuCl3 (for cyclic and acyclic ketones).75 Suzuki
coupling has also been employed for the synthesis of chalcone
derivatives.76 Several disadvantages of these procedures include
long reaction time, high reaction temperature, complex reaction
conditions, and the use of expensive and noncommercial
reagents. Recently, an efficient and facile synthesis of chalcones
by condensation of aldehydes and ketones has been reported
The remarkable biological potential of chalcones is due to
their possible interactions with various proteins related to cell
apoptosis and proliferation.54,55 A number of recent studies have
indicated that the anti-inflammatory effect of chalcones is due to
the inhibition of the NF-kB pathway, which is mediated by IkB
degradation and the phosphorylation of c-Jun N-terminal kinase
(JNK) and c-Jun.56ꢀ58 It has been reported that the electrophilic
R,β-unsaturated carbonyl moiety on a chalcone resulted in the
activation of the Nrf2/ARE pathway and the induction of phase
II detoxifying enzyme expression.59,60 This moiety acts as an
electrophile and reacts with free sulfhydryl groups of thioredoxin
and cysteine residues in proteins.58,59,61 It is also reported that
electrophilic phytochemicals could give rise to thiyl radicals,
which could also interact with sulfhydryl residues of intracellular
targets, including Nrf2.62 These studies demonstrate that the
endogenous electrophilic activity, through its R,β-unsaturated
carbonyl moiety, is involved in the antioxidant and anti-inflam-
matory properties of chalcone. In the present study, we synthe-
sized novel chalcone derivatives and tested their Nrf2 activating
activity in human bronchial epithelial cells. Furthermore, to draw
a structureꢀactivity relationship, a possible mechanism for Nrf2
activation is proposed.
using LiOH H2O as a dual activation catalyst under mild
3
conditions.77 We employed similar conditions for the synthesis
of all chalcone derivatives used in the present study. In short, the
appropriately substituted acetophenone was dissolved in ethanol
followed by addition of a catalytic amount of LiOH H2O. The
3
reaction mixture was stirred at room temperature for 15 min, and
the desired substituted benzaldehyde was added. The reaction
was carried out at room temperature until completion, and the
corresponding chalcone derivative (1aꢀ5l) was isolated by
crystallization or by silica gel flash chromatography (Table 1).
All chalcone derivatives were characterized by 1H, 13C NMR, and
HRMS. To the best of our knowledge, compounds 2b, 2f, 2i, 2k,
2l, 3b, 3c, 3f, 3i, 3k, 5f, 5g, and 5i have never been reported
before, whereas compounds 2e, 2g, 2h, 3g, and 3h are commer-
cially available as per the SciFinder search; however, there is no
reporton these compounds in mice andhuman lungepithelial cells.
Biology. Potency of Chalcone Derivatives To Activate the
Expression of Nrf2-Regulated Cytoprotective Genes in Human
Lung Epithelial Cells. To investigate the potency of chalcone
derivatives to activate Nrf2, we measured the expression of
antioxidant genes GCLM and NADPH-NQO1, two well char-
acterized transcriptional targets of Nrf2, as surrogate markers.
Previously, we and others have shown that oxidants or small
molecule activators of Nrf2 increase GCLM and NQO1 in cells
or tissues of wild-type but not in Nrf2-deficient mice.78 In this study,
to screen novel Nrf2 activators, we treated normal human bronchial
epithelial cells (Beas-2B) with chalcone derivatives (10 μM) for
16 h and analyzed the expression of GCLM and NQO1 by
’ RESULTS AND DISCUSSION
Chemistry. Chaclones can be readily synthesized by the base-
catalyzed ClaisenꢀSchmidt condensation of an aldehyde and
ketone in a polar solvent like ethanol or methanol. The tradi-
tional synthesis of chalcones involves the use of strong bases such
as NaOH,31,35,39,63 KOH,16,64 Ba(OH)2,65,66 hydrotalcites,67 and
LiHMDS,68 calcined NaNO3/natural phosphate.69 They can
also be synthesized by acid-catalyzed aldol condensations, e.g.,
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Table 2. List of Positive Leads from Primary Screening and
Their Effect on Cell Viability
relative fold changea
entry
compd
DMSO
GCLM
NQO1
% cell viability
1
1
1
100
2
sulforaphane
2.7
5
3.6
5.3
4.6
4.3
4.6
4.5
5.3
3.7
4.1
4.3
3.5
3.9
5.4
4.6
4.8
4
97.3
144.8
100.9
96.6
96
3
2a
2b
2c
2d
2e
2f
4
4.5
5.6
5.4
5.4
5.7
4
5
6
7
105
8
101.1
87.1
101.6
145.3
95.3
108.2
91
Figure 1. Structureꢀactivity relationship of chalcone derivatives.
9
2i
10
11
12
13
14
15
16
17
18
19
20
21
2k
2l
5.4
4.4
4.6
3.8
4.3
3.5
5
3c
3i
4b
4d
4e
4g
4j
104.1
92.6
91.6
100.1
93.2
75.8
90.9
4.1
4.1
4.6
4.1
3.4
3.7
4.3
3.7
4.1
5d
5f
5i
a Data presented are representative of three independent experiments.
Values shown are mean of quadruplicate wells.
Figure 2. Expression of Nrf2-regulated genes in small intestine after
treatment with chalcone derivatives. Mice (n = 4) were fed with vehicle
(DCP (10% DMSO þ 10% Cremophor þ 80% phosphate buffered
saline)) or chalcone derivatives or sulforaphane (50 mg/kg body
weight) by gavage, and the small intestines were harvested 24 h later.
The expression of Nrf2-regulated genes GCLM and NQO1 was
analyzed in the tissues by qRT-PCR as a surrogate marker of Nrf2
activity. β-Actin was used for normalization. Data are representative of
three independent experiments (P e 0.05).
quantitative RT-PCR (qRT-PCR). We included sulforaphane, a
well-known potent activator of Nrf2, as a positive control. We
identified 59 chalcone derivatives that induce the expression of
GCLM and NQO1 (Table 1). Concurrent with the gene
expression analysis, we analyzed the cytotoxicity of the chalcone
derivatives using the MTT assay. A total of 20 chalcones showed
a higher induction of Nrf2-regulated transcriptional targets than
the positive control, i.e., sulforaphane (Table 2).
In Vivo Potency of Identified Lead Compounds To Activate
Nrf2 Using Mouse Models. Next, we evaluated the potency of the
eight lead chalcones identified in the in vitro screening to activate
the Nrf2 pathway in mouse models. First, we tested various
formulations to dissolve the compounds, and the DCP (10%
DMSO þ 10% Cremophor EL þ 80% phosphate buffered
saline) formulation offered the maximum solubility for delivery
of these compounds by oral route. Mice (C57BL/6) were
administrated with a single dose of vehicle or test compound(s)
or sulforaphane as the positive control at a dose of 50 mg/kg
body weight by gavage, and small intestines were harvested 24 h
later. The expression of Nrf2-regulated genes GCLM and NQO1
was analyzed in the tissue by qRT-PCR. All eight lead com-
pounds increased the expression of GCLM and NQO1 in the
small intestine. However, 2b (Figure 1) was the most potent
inducer of Nrf2 activity (Figure 2). The expression of GCLM and
NQO1 in the small intestine of mice treated with 2b was 6-fold
and 10-fold higher compared to vehicle, respectively. Similarly,
the expression of GCLM and NQO1 in the small intestine
treated with 2b was 3-fold and 5-fold higher compared to
sulforaphane, respectively. Taken together, we selected 2b as
the most potent activator of Nrf2 for further studies.
Preliminary StructureꢀActivity Relationship. The structure
ꢀactivity relationship analysis showed that the chalcone deriva-
tives 1aꢀl without any substitution on ring B were not active.
The activity of similar derivatives with trifluoromethyl (CF3)
substitution on ring B enhanced the activity dramatically. The
position of CF3 substitution was also crucial for the activity and
cytotoxicity of these compounds. In general, the chalcone
derivatives with CF3 substitution at ortho position on ring B
were the most active compounds (entries 13ꢀ24, Table 1),
followed by para (entries 37ꢀ47, Table 1) and meta (entries
25ꢀ36, Table 1) substitution. Also, the cytotoxicity data show
that the ortho CF3-substituted chalcones were noncytotoxic.
This shows that four-bond separation between carbonyl and CF3
is crucial for the induction activity. Interestingly, with nitro
(NO2) substitution at the ortho position on ring B, the activity
decreased and the toxicity increased significantly (entries 48ꢀ59
of Table 1 and entries 19ꢀ21 of Table 2). A brief description of
SAR is presented in Figure 1. Thus, on the basis of these data, we
selected only those chalcone derivatives that showed >4-fold
induction of GCLM and NQO1 genes and >95% cell viability. On
the basis of these stringent criteria, of the 20 compounds that
showed potency to increase Nrf2 activity, we selected com-
pounds 2aꢀf, 2k, and 2l for further analysis.
Nrf2 Is Essential for Induction of Antioxidant Genes by
Compound 2b. We further characterized Nrf2 induction by 2b
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Journal of Medicinal Chemistry
ARTICLE
Figure 3. Levels of NQO1-ARE luciferase activity after treatment with
compound 2b. NQO1-ARE luciferase activity was measured by using
stably transfected Beas-2B cells after treatment with compound 2b or
sulforaphane (SFN) or dimethylsulfoxide (DMSO). The exposure to
compound 2b resulted in a significant concentration-dependent increase
in luciferase activity as relative luminescence intensity (RLI). Data are
representative of three independent experiments. Values shown are the
mean ( SD of quadruplicate wells (P e 0.05).
Figure 5. Time-dependent increase in Nrf2-regulated genes after
treatment with compound 2b. Human bronchial epithelial cells (Beas-
2B) were treated with compound 2b (10 μM) at various time points.
The expression of Nrf2-regulated genes GCLM, HO1, and NQO1 was
analyzed in the tissues by qRT-PCR. β-Actin was used for normalization.
Data are representative of three independent experiments. Values shown
are mean ( SD of triplicate wells (P e 0.05).
Figure 4. Expression of Nrf2-regulated genes after treatment with
compound 2b. Human bronchial epithelial cells (Beas-2B) were treated
with compound 2b at the indicated concentrations for 16ꢀ20 h. The
expression of Nrf2-regulated genes GCLM, HO1, and NQO1 was
analyzed in the tissues by qRT-PCR as a surrogate marker of Nrf2
activity. β-Actin was used for normalization. Data are representative of
three independent experiments. Values shown are mean ( SD of
triplicate wells (P e 0.05).
Figure 6. Activation of Nrf2 genes by compound 2b is independent of
ROS generation. Human bronchial epithelial cells (Beas-2B) were
treated with compound 2b (10 μM) in the presence of an antioxidant,
N-acetylcystiene (10 mM, NAC). Cells were harvested 24 h after the
treatment, and the Nrf2-driven expression of NQO1, HO-1, and GCLM
was quantified. β-Actin was used for normalization. Data are represen-
tative of three independent experiments. Values shown are the mean (
SD of triplicate wells (P e 0.05).
using cell-based assays. Nrf2 increases the expression of NQO1
and GCLM by binding to the ARE present in the promoter
region of these genes.79 We determined whether the ARE
mediates the transcriptional regulation of NQO1 by 2b. We
measured the expression of the luciferase gene under the control
of NQO1-ARE sequence using stably transfected Beas-2B cells
treated with 2b. The exposure to compound 2b resulted in a
significant concentration-dependent increase in luciferase activity
as measured by the chemiluminescence-based assay (Figure 3).
These results implicate the ARE element in the induction of
NQO1 gene by compound 2b. The transcriptional activation of
antioxidant genes through an ARE is largely dependent upon
Nrf2, suggesting that 2b up-regulates antioxidant genes via Nrf2
activation.
Concentration and Time-Course Studies. Next, we examined
the concentration-dependent effect of 2b on the mRNA levels of
Nrf2-driven antioxidant genes GCLM, NQO1, and HO1. We
measured the expression of these genes at 24 h after treatment
with various concentrations (2.5, 5, 10, 20 μM) of 2b in Beas-2B
cells. As shown in Figure 5, compound 2b significantly increased
the Nrf2-regulated gene expression in a concentration-depen-
dent manner. There was ∼5- and 10-fold increase in the
expression of GCLM and NQO1, respectively, at the highest
concentration (20 μM) with no cytotoxicity. Interestingly, we
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found a dramatic concentration-dependent activation of Nrf2
genes. At 10 μM 2b, the expression of HO-1 was 6-fold higher
compared to that of sulforaphane (Figure 4).
General Procedure for Synthesis of Chalcones. In a 14 mL vial, the
substituted acetophenone (1.25 mmol) and lithium hydroxide mono-
hydrate (0.25 mmol) were dissolved in ethanol (5 mL), and the mixture
was stirred at room temperature for 10 min followed by addition of
substituted benzaldehyde (1.27 mmol). The reaction mixture was then
stirred at room temperature and monitored by TLC using 25% ethyl
acetate/hexanes as the solvent system. The reaction was quenched after
2 h by pouring into 50 mL of stirring ice cold water. If the product
precipitated out after quenching with cold water, it was filtered off and
crystallized with hot ethanol. In some examples, a sticky mass was
observed in the aqueous solution after quenching. In those cases, the
product was extracted by ethyl acetate (3 ꢁ 50 mL), dried over sodium
sulfate, and concentrated under vacuum. The crude product was purified
by flash chromatography using ethyl acetate/hexanes as the solvent
system in increasing order of polarity.
(E)-2,3-Diphenylprop-2-en-1-one (1a). It was obtained as a light
yellow solid in 80% yield. 1H NMR (400 MHz, DMSO) δ 8.16ꢀ8.14
(m, 1H), 8.13 (t, J = 1.71, 1.71 Hz, 1H), 7.92 (d, J = 15.67 Hz, 1H),
7.89ꢀ7.85 (m, 2H), 7.74 (d, J = 15.68 Hz, 1H), 7.69ꢀ7.63 (m, 1H),
7.59ꢀ7.53 (m, 2H), 7.47ꢀ7.42 (m, 3H). 13C NMR (101 MHz, DMSO)
δ 189.67, 144.48, 137.99, 135.08, 133.60, 131.09, 129.37, 129.34, 129.24,
128.96, 122.51. LCꢀMS (ESI-TOF): m/z 209.0963 ([C15H12O þ H]þ
calcd 209.0961). Purity 100.00% (rt 7.39 min).
For the time-course studies, we measured the expression of
antioxidant genes (GCLM, NQO1, and HO-1) at 6, 12, 24,
and 48 h after treatment with 2b (10 μM) in Beas-2B cells. The
time-course studies showed the highest induction of GCLM
(∼7-fold) and HO-1 (∼150-fold) at 6 h after treatment with 2b
(Figure 5). The expression of GCLM and HO-1 decreased at 6 h
after treatment with 2b and was comparable to vehicle by 48 h.
The expression of NQO1 was highest at 24 h and remained
significantly elevated even at 48 h after treatment with compound
2b compared to vehicle. Taken together, these results suggest
that 2b is a potent activator of Nrf2-regulated antioxidant
defenses.
Activation of Nrf2 by Compound 2b Is Independent of ROS
Generation. The activation of Nrf2 by various electrophiles and
compounds that are Michael acceptors is attributed to changes in
ROS production and or redox environment and or direct cysteine
modification in Keap1.80,81 We examined whether 2b activates
Nrf2 by generating ROS or redox changes. Beas-2B cells were co-
incubated with compound 2b and with or without N-acetylcys-
teine (NAC, 10 mM), and the expression of GCLM, NQO1, and
HO1 was measured 24 h later. We found that 2b potentially
increases the expression of Nr2-regulated antioxidant genes in
the presence of NAC (Figure 6). NAC alone showed no
induction of Nrf2-regulated genes. Taken together, these results
suggest that the activation of Nrf2 by 2b is independent of ROS
or redox changes. Further studies are required to determine
whether compound 2b activates Nrf2 by direct thiol modification
of Keap1.
(E)-1-(2-Methoxyphenyl)-3-phenylprop-2-en-1-one (1b). It was ob-
1
tained as a yellow oil in 71% yield. H NMR (400 MHz, DMSO) δ
7.76ꢀ7.67 (m, 2H), 7.56ꢀ7.46 (m, 3H), 7.44ꢀ7.37 (m, 4H), 7.18 (d,
J = 7.9 Hz, 1H), 7.05 (td, J = 7.5, 0.9 Hz, 1H), 3.85 (s, 3H). 13C NMR
(101 MHz, DMSO) δ 192.60, 158.17, 142.97, 135.01, 133.53, 130.94,
129.98, 129.45, 129.23, 128.97, 127.41, 121.01, 112.79, 56.27. LCꢀMS
(ESI-TOF): m/z 239.1072 ([C16H14O2 þ H]þ calcd 239.1067). Purity
98.02% (tR = 7.21 min).
(E)-1-(3-Methoxyphenyl)-3-phenylprop-2-en-1-one (1c). It was ob-
tained as a yellow oil in 54% yield. 1H NMR (400 MHz, DMSO) δ 7.95ꢀ
7.85 (m, 3H), 7.78ꢀ7.70 (m, 2H), 7.62ꢀ7.58 (m, 1H), 7.49 (d, J = 8.0 Hz,
1H), 7.47ꢀ7.42 (m, 3H), 7.23 (ddd, J = 8.2, 2.6, 0.8 Hz, 1H), 3.84 (s, 3H).
13C NMR (101 MHz, DMSO) δ 189.36, 159.99, 144.57, 139.42, 135.07,
131.12, 130.40, 129.42, 129.36, 122.51, 121.52, 119.69, 113.41, 55.83. LCꢀ
MS (ESI-TOF): m/z 239.1071 ([C16H14O2 þ H]þ calcd 239.1067).
Purity 98.52% (tR = 7.84 min).
(E)-1-(4-Methoxyphenyl)-3-phenylprop-2-en-1-one (1d). It was ob-
tained as a white solid in 76% yield. 1H NMR (400 MHz, DMSO) δ 8.16
(d, J = 9.0 Hz, 2H), 7.94 (d, J = 15.6 Hz, 1H), 7.90ꢀ7.83 (m, 2H), 7.69
(d, J = 15.6 Hz, 1H), 7.44 (dd, J = 5.1, 1.9 Hz, 3H), 7.07 (d, J = 9.0 Hz,
2H), 3.85 (s, 3H). 13C NMR (101 MHz, DMSO) δ 187.79, 163.68,
143.60, 135.24, 131.39, 130.88, 130.85, 129.33, 129.24, 122.43, 114.48,
56.03. LCꢀMS (ESI-TOF): m/z 239.1068 ([C16H14O2 þ H]þ calcd
239.1067). Purity 100.00% (tR = 7.36 min).
(E)-3-Phenyl-1-(2,4-dimethoxyphenyl)prop-2-en-1-one (1e). It was
obtained as a yellow oil in 40% yield. 1H NMR (400 MHz, CDCl3) δ =
7.76 (d, J = 8.6 Hz, 1H), 7.68 (d, J = 15.8 Hz, 1H), 7.60 (dd, J = 7.3, 1.8
Hz, 2H), 7.52 (d, J = 15.8 Hz, 1H), 7.43ꢀ7.34 (m, 3H), 6.57 (dd, J = 8.6,
2.2 Hz, 1H), 6.50 (d, J = 2.1 Hz, 1H), 3.91 (s, 3H), 3.87 (s, 3H). 13C
NMR (101 MHz, DMSO) δ = 189.73, 164.44, 160.71, 141.62, 135.32,
132.51, 130.65, 129.43, 128.78, 127.52, 121.79, 106.46, 99.07, 56.42,
56.06. LCꢀMS (ESI-TOF): m/z 269.1171 ([C17H16O3 þ H]þ calcd
269.1172). Purity 96.00% (tR = 7.25 min).
’ CONCLUSIONS
We have identified a novel chalcone 2b as a potent activator of
Nrf2 signaling pathway after screening a series of chalcone
derivatives using cell-based and mouse models. Further studies
are needed to evaluate and develop chalcone 2b as a potential
drug for the treatment of inflammatory disorders.
’ EXPERIMENTAL SECTION
Chemistry. General Methods. TLCs were run on precoated Merck
silica gel 60F254 plates and observed under UV light. The products were
isolated and purified by crystallization or using a Teledyne ISCO Rf flash
chromatography system with hexanes and ethyl acetate as eluents. The
1H (400 MHz), 13C (101 MHz), gCOSY, and gHSQC NMR spectra
were taken on a Varian 400-MR spectrophotometer using TMS as an
internal standard. Chemical shifts (δ) are expressed in ppm. Coupling
constants (J) are expressed in Hz, and splitting patterns are described as
follows: s = singlet; d = doublet; t = triplet; q = quartet; m = multiplet;
dd = doublet of doublets; dt = doublet of triplets; td = triplet of doublets;
ddd = doublet of doublet of doublets. For the verification of the product
and purity analysis, the LCꢀMS results were taken on an Agilent 1200
series system with an Agilent 6210 time-of-flight (TOF) mass detector
using Agilent Eclipse XDB-C-18 column (5 mm, 4.6 mm ꢁ 150 mm)
using a flow rate of 0.9 mL/min and solvent system water (with 0.1%
formic acid)/acetonitrile (ACN) (gradient, 50% ACN at 0 min, 80%
ACN at 7 min, 80% ACN at 10 min, and 50% ACN at 15 min). All
synthesized compounds were >95% pure (exact purity is given in the
subsequent sections with the characterization data). All chemicals were
purchased from Sigma-Aldrich (St. Louis, MO) and were used without
further purification.
(E)-3-Phenyl-1-(2,6-dimethoxyphenyl)prop-2-en-1-one (1f). It was
obtained as a white solid in 60% yield. 1H NMR (400 MHz, DMSO) δ =
7.72ꢀ7.58 (m, 2H), 7.45ꢀ7.31 (m, 4H), 7.17 (d, J = 16.2 Hz, 1H), 6.97
(d, J = 16.2 Hz, 1H), 6.74 (d, J = 8.4 Hz, 2H), 3.70 (s, 6H). 13C NMR
(101 MHz, DMSO) δ = 194.53, 157.29, 144.91, 134.61, 131.35, 131.11,
129.43, 129.04, 129.01, 118.25, 104.86, 56.24. LCꢀMS (ESI-TOF):
m/z 269.1175 ([C17H16O3 þ H]þ calcd 269.1172). Purity 100.00%
(tR = 6.19 min).
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(E)-3-Phenyl-1-(2,5-dimethoxyphenyl)prop-2-en-1-one (1g). It was
obtained as a yellow oil in 62% yield. 1H NMR (400 MHz, DMSO) δ =
7.76ꢀ7.67 (m, 2H), 7.50 (d, J = 16.0 Hz, 1H), 7.43 (d, J = 2.7 Hz, 3H),
7.40 (d, J = 12.2 Hz, 1H), 7.14ꢀ7.09 (m, 2H), 7.03 (dd, J = 2.6, 0.8 Hz,
1H), 3.80 (s, 3H), 3.73 (s, 3H). 13C NMR (101 MHz, DMSO) δ =
192.17, 153.47, 152.36, 143.16, 135.00, 130.98, 129.67, 129.46, 128.99,
127.25, 119.02, 114.36, 114.33, 56.80, 56.00. LCꢀMS (ESI-TOF):
m/z 269.1170 ([C17H16O3 þ H]þ calcd 269.1172). Purity 98.59%
(tR = 7.31 min).
7.45ꢀ7.36 (m, 2H), 7.22 (d, J = 15.7 Hz, 1H), 6.97 (td, J = 7.5 Hz, 0.8
Hz, 1H), 6.92 (d, J = 8.4 Hz, 1H), 3.82 (s, 4H). 13C NMR (101 MHz,
DMSO) δ = 192.21, 158.40, 136.83 (d, J = 2.1 Hz), 134.05, 133.58,
133.29 (d, J = 1.6 Hz), 131.30, 130.82, 130.15, 128.79, 128.58, 127.81 (q,
J = 29.2 Hz), 126.68 (q, J = 5.2 Hz), 124.55 (q, J = 274.5 Hz), 121.07,
112.76, 56.31. LCꢀMS (ESI-TOF): m/z 304.0940 ([C17H13 F3O2 þ
H]þ calcd 307.0940). Purity 96.40% (tR = 8.69 min).
(E)-1-(3-Methoxyphenyl)-3-(2-(trifluoromethyl)phenyl)prop-2-en-
1-one (2c). It was obtained as a yellow solid in 26% yield. 1H NMR (400
MHz, DMSO) δ = 8.35 (d, J = 7.9 Hz, 1H), 8.06ꢀ7.94 (m, 2H),
7.88ꢀ7.76 (m, 3H), 7.72ꢀ7.63 (m, 2H), 7.52 (t, J = 7.9 Hz, 1H), 7.28
(ddd, J = 8.2 Hz, 2.7 Hz, 0.8 Hz, 1H), 3.87 (s, 3H). 1H NMR (400 MHz,
CDCl3) δ = 8.13 (d, J = 15.6 Hz, 1H), 7.83 (d, J = 7.8 Hz, 1H), 7.74 (d,
J = 7.8 Hz, 1H), 7.60 (dd, J = 13.7 Hz, 7.1 Hz, 2H), 7.55ꢀ7.47 (m, 2H),
7.41 (dd, J = 15.9 Hz, 8.5 Hz, 2H), 7.15 (dd, J = 8.2 Hz, 1.9 Hz, 1H), 3.89
(s, 3H). 13C NMR (101 MHz, DMSO) δ = 189.12, 160.04, 138.92,
138.33 (d, J = 2.2 Hz), 133.41, 133.21 (d, J = 1.7 Hz), 131.00, 130.47,
129.32, 127.96 (d, J = 29.2 Hz), 126.71, 126.62 (d, J = 6. 0 Hz), 124.62
(d, J = 273.5 Hz), 121.69, 120.04, 113.64, 55.86. LCꢀMS (ESI-TOF):
m/z 304.0945 ([C17H13 F3O2 þ H]þ calcd 307.0940). Purity 100.00%
(tR = 9.13 min).
(E)-1-(4-Methoxyphenyl)-3-(2-(trifluoromethyl)phenyl)prop-2-en-
1-one (2d). It was obtained as a yellow solid in 37% yield. 1H NMR (400
MHz, DMSO) δ = 8.34 (d, J = 7.9 Hz, 1H), 8.24ꢀ8.17 (m, 2H), 8.04 (d,
J = 15.3 Hz, 1H), 7.95 (dd, J = 15.4 Hz, 2.2, 1H), 7.87ꢀ7.76 (m, 2H),
7.67 (t, J = 7.6 Hz, 1H), 7.16ꢀ7.07 (m, 2H), 3.89 (s, 3H). 13C NMR
(101 MHz, DMSO) δ = 187.42, 164.00, 137.46 (d, J = 2.2 Hz), 133.45
(d, J = 1.6 Hz), 133.38, 131.63, 130.78, 130.40, 129.21, 127.87 (d, J =
29.2 Hz), 126.67, 126.58 (d, J = 6.0 Hz), 124.64 (d, J = 274.5 Hz),
114.58, 56.08. LCꢀMS (ESI-TOF): m/z 304.0944 ([C17H13 F3O2 þ
H]þ calcd 307.0940). Purity 100.00% (tR = 8.75 min).
(E)-1-(2,4-Dimethoxyphenyl)-3-(2-(trifluoromethyl)phenyl)prop-2-
en-1-one (2e). It was obtained as a yellow solid in 50% yield. 1H NMR
(400 MHz, DMSO) δ = 8.07 (d, J = 7.6 Hz, 1H), 7.79 (dt, J = 14.8 Hz,
7.9 Hz, 3H), 7.69ꢀ7.60 (m, 3H), 6.71 (d, J = 2.3 Hz, 1H), 6.67 (dd, J =
8.6 Hz, 2.3 Hz, 1H), 3.92 (s, 3H), 3.87 (s, 3H). 13C NMR (101 MHz,
DMSO) δ = 189.18, 164.88, 161.01, 135.47 (d, J = 2.0 Hz), 133.64 (d, J =
1.3 Hz), 133.59, 132.74, 131.55, 130.55, 128.69, 127.74 (d, J = 29.2 Hz),
126.64 (d, J = 6.0 Hz), 124.62 (d, J = 274.5 Hz). 121.23, 106.69, 99.04,
56.50, 56.13. LCꢀMS (ESI-TOF): m/z 337.1050 ([C18H15 F3O3 þ
H]þ calcd 337.1046). Purity 100.00% (tR = 8.71 min).
(E)-1-(2,6-Dimethoxyphenyl)-3-(2-(trifluoromethyl)phenyl)prop-2-
en-1-one (2f). It was obtained as a white solid in 67% yield. 1H NMR
(400 MHz, DMSO) δ 8.07 (s, 1H), 8.01 (d, J = 7.9 Hz, 1H), 7.74 (d, J =
7.8 Hz, 1H), 7.61 (t, J = 7.8 Hz, 1H), 7.38 (t, J = 8.4 Hz, 1H), 7.30 (d, J =
16.3 Hz, 1H), 7.14 (d, J = 16.2 Hz, 1H), 6.74 (d, J = 8.5 Hz, 2H), 3.70 (s,
6H). 13C NMR (101 MHz, DMSO) δ 194.45, 157.36, 142.84, 135.92,
132.43, 131.45, 130.69, 130.41, 130.26 (q, J = 31.1 Hz), 127.17 (q, J = 3.6
Hz), 125.92 (q, J = 3.8 Hz), 124.38 (q, J = 272.5 Hz), 118.22, 104.90,
56.27. LCꢀMS (ESI-TOF): m/z 337.1045 ([C18H15 F3O3 þ H]þ calcd
337.1046). Purity 100.00% (tR = 7.68 min).
(E)-1-(2,5-Dimethoxyphenyl)-3-(2-(trifluoromethyl)phenyl)prop-2-
en-1-one (2g). It was obtained as a yellow solid in 80% yield. 1H NMR
(400 MHz, DMSO) δ = 8.09 (d, J = 7.9 Hz, 1H), 7.83 (d, J = 8.0 Hz,
1H), 7.80ꢀ7.72 (m, 2H), 7.65 (t, J = 7.6 Hz, 1H), 7.51 (d, J = 15.7 Hz,
1H), 7.16 (d, J = 1.8 Hz, 2H), 7.09 (t, J = 1.8 Hz, 1H), 3.83 (s, 3H), 3.76
(s, 3H). 13C NMR (101 MHz, DMSO) δ = 191.72, 153.50, 152.64,
136.95 (d, J = 2.1 Hz), 133.59, 133.28 (d, J = 1.7 Hz), 131.14, 130.86,
128.96, 128.79, 127.82 (q, J = 29.2 Hz), 126.69 (q, J = 16.6 Hz), 124.56
(d, J = 273.5 Hz), 119.72, 114.41, 114.33, 56.80, 56.04. LCꢀMS (ESI-
TOF): m/z 337.1049 ([C18H15 F3O3 þ H]þ calcd 337.1046). Purity
100.00% (tR = 8.76 min).
(E)-3-Phenyl-1-(3,4-dimethoxyphenyl)prop-2-en-1-one (1h). It was
obtained as a yellow oil in 62% yield. 1H NMR (400 MHz, DMSO) δ
7.95 (d, J = 15.6 Hz, 1H), 7.93ꢀ7.85 (m, 3H), 7.70 (d, J = 15.6 Hz, 1H),
7.60 (d, J = 2.0 Hz, 1H), 7.44 (dd, J = 1.9, 5.1 Hz, 3H), 7.09 (d, J = 8.5 Hz,
1H), 3.86 (s, 3H), 3.84 (s, 3H). 13C NMR (101 MHz, DMSO) δ 187.77,
153.67, 149.23, 143.53, 135.25, 130.89, 130.87, 129.32, 129.27, 123.87,
122.36, 111.31, 111.11, 56.23, 56.02. LCꢀMS (ESI-TOF): m/z
269.1173 ([C17H16O3 þ H]þ calcd 269.1172). Purity 97.87% (tR
=
6.33 min).
(E)-3-Phenyl-1-(3,5-dimethoxyphenyl)prop-2-en-1-one (1i). It was
obtained as a yellow oil in 66% yield. 1H NMR (400 MHz, DMSO) δ
7.94ꢀ7.86 (m, 3H), 7.73 (d, J = 15.6 Hz, 1H), 7.44 (dd, J = 2.6, 3.8 Hz,
3H), 7.25 (d, J = 2.3 Hz, 2H), 6.78 (t, J = 2.3 Hz, 1H), 3.82 (s, 6H). 13C
NMR (101 MHz, DMSO) δ 189.16, 161.14, 144.70, 140.05, 135.06,
131.12, 129.48, 129.33, 122.46, 106.71, 105.53, 56.01. LCꢀMS (ESI-
TOF): m/z 269.1176 ([C17H16O3 þ H]þ calcd 269.1172). Purity
100.00% (tR = 8.09 min).
(E)-3-Phenyl-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (1j). It
was obtained as a white solid in 68% yield. 1H NMR (400 MHz,
DMSO) δ 7.99ꢀ7.85 (m, 3H), 7.73 (d, J = 15.5 Hz, 1H), 7.52ꢀ7.36 (m,
5H), 3.89 (s, 6H), 3.75 (s, 3H). 13C NMR (101 MHz, DMSO) δ 188.34,
153.37, 144.31, 142.44, 135.15, 133.39, 131.03, 129.42, 129.32, 122.35,
106.62, 60.64, 56.67. LCꢀMS (ESI-TOF): m/z 299.1284 ([C18H18O4
þ H]þ calcd 299.1278). Purity 100.00% (tR = 6.97 min).
(E)-3-Phenyl-1-(2,3,4-trimethoxyphenyl)prop-2-en-1-one (1k). It
was obtained as a white solid in 66% yield. 1H NMR (400 MHz,
DMSO) δ = 7.73 (dd, J = 6.8, 2.8 Hz, 2H), 7.54 (d, J = 15.9 Hz, 1H),
7.48ꢀ7.39 (m, 4H), 7.37 (d, J = 8.7 Hz, 1H), 6.93 (d, J = 8.8 Hz, 1H),
3.86 (s, 3H), 3.83 (s, 3H), 3.77 (s, 3H). 13C NMR (101 MHz, DMSO)
δ = 190.50, 157.16, 153.34, 142.86, 142.07, 135.08, 130.88, 129.47,
128.89, 126.98, 126.58, 125.60, 108.34, 62.16, 60.96, 56.54. LCꢀMS
(ESI-TOF): m/z 299.1275 ([C18H18O4 þ H]þ calcd 299.1278). Purity
100.00% (tR = 7.19 min).
(E)-3-Phenyl-1-(2,4,6-trimethoxyphenyl)prop-2-en-1-one (1l). It
was obtained as a yellow oil in 71% yield. 1H NMR (400 MHz, DMSO)
δ = 7.64 (dd, J = 6.6, 3.1 Hz, 2H), 7.39 (dd, J = 5.1, 1.8 Hz, 3H), 7.19 (d,
J = 16.1 Hz, 1H), 6.94 (d, J = 16.1 Hz, 1H), 6.30 (s, 2H), 3.82 (s, 3H), 3.70
(s, 6H). 13C NMR (101 MHz, DMSO) δ = 193.70, 162.41, 158.53,
143.97, 134.81, 130.90, 129.46, 129.40, 128.91, 111.45, 91.53, 56.26,
55.91. LCꢀMS (ESI-TOF): m/z 299.1276 ([C18H18O4 þ H]þ calcd
299.1278). Purity 100.00% (tR = 6.23 min).
(E)-1-Phenyl-3-(2-(trifluoromethyl)phenyl)prop-2-en-1-one (2a). It
was obtained as a yellow solid in 64% yield. 1H NMR (400 MHz,
DMSO) δ = 8.35 (d, J = 7.8 Hz, 1H), 8.25ꢀ8.15 (m, 2H), 8.05 (d, J =
15.3 Hz, 1H), 7.98 (dd, J = 15.5 Hz, 2.0 Hz, 1H), 7.89ꢀ7.76 (m, 2H),
7.76ꢀ7.65 (m, 2H), 7.61 (t, J = 7.7 Hz, 2H). 13C NMR (101 MHz,
DMSO) δ = 189.33, 138.24 (d, J = 1.8 Hz), 137.47, 134.01, 133.42,
133.22 (d, J = 1.5 Hz), 130.99, 129.32, 129.26, 129.16, 127.96 (q, J = 29.2
Hz), 126.64, 126.62 (q, J = 6.0 Hz), 124.61 (q, J = 274.5 Hz). LCꢀMS
(ESI-TOF): m/z 277.0833 ([C16H11F3O þ H]þ calcd 277.0835).
Purity 100.00% (tR = 6.97 min).
(E)-1-(2-Methoxyphenyl)-3-(2-(trifluoromethyl)phenyl)prop-2-en-
1-one (2b). It was obtained as a yellow oil in 72% yield. 1H NMR (400
MHz, CDCl3) δ = 7.90ꢀ7.82 (m, 1H), 7.71 (d, J = 7.8 Hz, 1H), 7.63 (d,
J = 7.8 Hz, 1H), 7.55 (dd, J = 7.6 Hz, 1.8, 1H), 7.50 (t, J = 7.6 Hz, 1H),
(E)-1-(3,4-Dimethoxyphenyl)-3-(2-(trifluoromethyl)phenyl)prop-2-
en-1-one (2h). It was obtained as a yellow solid in 72% yield. 1H NMR
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(400 MHz, DMSO) δ = 8.33 (d, J = 7.8 Hz, 1H), 8.04 (d, J = 15.3 Hz,
1H), 7.96 (dd, J = 6.2 Hz, 2.1 Hz, 1H), 7.94 (d, J = 2.0 Hz, 1H), 7.84 (d,
J = 7.8 Hz, 1H), 7.80 (t, J = 7.6 Hz, 1H), 7.67 (t, J = 7.7 Hz, 1H), 7.63 (d,
J = 2.0 Hz, 1H), 7.13 (d, J = 8.5 Hz, 1H), 3.89 (s, 3H), 3.87 (s, 3H). 13C
NMR (101 MHz, DMSO) δ = 187.44, 154.02, 149.31, 137.42 (d, J = 2.0
Hz), 133.49 (d, J = 1.0 Hz), 133.37, 130.76, 130.44, 129.26, 127.85 (q,
J = 29.2 Hz), 126.66, 126.58 (q, J = 5.2 Hz), 124.65 (q, J = 273.5 Hz),
124.22, 111.40, 111.28, 56.28, 56.07. LCꢀMS (ESI-TOF): m/z
337.1048 ([C18H15 F3O3 þ H]þ calcd 337.1046). Purity 100.00%
(tR = 7.77 min).
(E)-1-(3,5-Dimethoxyphenyl)-3-(2-(trifluoromethyl)phenyl)prop-2-
en-1-one (2i). It was obtained as a yellow oil in 72% yield. 1H NMR (400
MHz, DMSO) δ = 8.36 (d, J = 7.9 Hz, 1H), 7.98 (s, 2H), 7.85 (d, J = 7.8
Hz, 1H), 7.80 (t, J = 7.6 Hz, 1H), 7.68 (t, J = 7.6 Hz, 1H), 7.30 (d, J = 2.2
Hz, 2H), 6.83 (t, J = 2.2 Hz, 1H), 3.85 (s, 6H). 1H NMR (400 MHz,
CDCl3) δ = 8.12 (d, J = 13.9 Hz, 1H), 7.82 (d, J = 7.8 Hz, 1H), 7.73 (d,
J = 7.8 Hz, 1H), 7.61 (t, J = 7.6 Hz, 1H), 7.51 (t, J = 7.7 Hz, 1H), 7.35 (d,
J = 15.6 Hz, 1H), 7.14 (d, J = 2.2 Hz, 2H), 6.69 (t, J = 2.1 Hz, 1H), 3.86 (s,
7H). 13C NMR (101 MHz, DMSO) δ = 188.97, 161.20, 139.53, 138.45
(d, J = 2.2 Hz), 133.39, 133.18 (d, J = 1.6 Hz), 131.00, 129.40, 127.96 (q,
J = 29.2 Hz), 126.67, 129.60 (q, J = 5.2 Hz), 124.62 (q, J = 273.5 Hz),
106.93, 105.88, 56.05. LCꢀMS (ESI-TOF): m/z 337.1047 ([C18H15
F3O3 þ H]þ calcd 337.1046). Purity 100.00% (tR = 9.37 min).
(E)-1-(3,4,5-Trimethoxyphenyl)-3-(2-(trifluoromethyl)phenyl)prop-
2-en-1-one (2j). It was obtained as a yellow solid in 80% yield. 1H NMR
(400 MHz, DMSO) δ = 8.31 (d, J = 7.8 Hz, 1H), 8.02 (d, J = 15.2 Hz,
1H), 7.95 (dd, J = 15.4 Hz, 2.1 Hz, 1H), 7.83 (d, J = 8.0 Hz, 1H), 7.78 (d,
J = 7.7 Hz, 1H), 7.66 (t, J = 7.6 Hz, 1H), 7.44 (s, 2H), 3.88 (s, 6H), 3.76
(s, 3H). 13C NMR (101 MHz, DMSO) δ = 188.07, 153.41, 142.77,
138.14 (d, J = 2.0), 133.37, 132.86, 130.91, 129.40, 127.77 (q, J = 29.2
Hz), 126.61, 126.61 (q, J = 5.0 Hz), 124.63 (q, J = 274.5 Hz), 106.86,
60.66, 56.68. LCꢀMS (ESI-TOF): m/z 367.1160 ([C19H17 F3O4 þ
H]þ calcd 367.1152). Purity 100.00% (tR = 8.38 min).
(E)-1-(2,3,4-Trimethoxyphenyl)-3-(2-(trifluoromethyl)phenyl)prop-
2-en-1-one (2k). It was obtained as a yellow solid in 58% yield. 1H NMR
(400 MHz, DMSO) δ = 8.10 (d, J = 7.7 Hz, 1H), 7.80 (dt, J = 22.4, 7.7
Hz, 3H), 7.66 (t, J = 7.6 Hz, 1H), 7.56 (d, J = 15.5 Hz, 1H), 7.46 (d, J =
8.8 Hz, 1H), 6.97 (d, J = 8.9 Hz, 1H), 3.89 (s, 3H), 3.86 (s, 3H), 3.80 (s,
3H). 13C NMR (101 MHz, DMSO) δ = 189.74, 157.66, 153.66, 142.08,
136.44 (d, J = 2.1 Hz), 133.60, 133.46 (d, J = 2.0 Hz), 131.02, 130.73,
128.71, 127.78 (d, J = 29.2 Hz), 126.66 (d, J = 5.0 Hz), 125.95 (d, J = 7.0
Hz), 124.59 (d, J = 274.5 Hz), 108.48, 62.21, 60.98, 56.60. LCꢀMS
(ESI-TOF): m/z 367.1152 ([C19H17 F3O4 þ H]þ calcd 367.1152).
Purity 96.17% (tR = 8.70 min).
(E)-1-(2,4,6-Trimethoxyphenyl)-3-(2-(trifluoromethyl)phenyl)prop-
2-en-1-one (2l). It was obtained as a yellow solid in 72% yield. 1H NMR
(400 MHz, DMSO) δ = 8.04 (d, J = 7.8 Hz, 1H), 7.77 (d, J = 7.7 Hz,
1H), 7.71 (t, J = 7.5 Hz, 1H), 7.60 (t, J = 7.6 Hz, 1H), 7.48 (dd, J = 15.9,
2.2 Hz, 1H), 7.01 (d, J = 15.8 Hz, 1H), 6.30 (s, 2H), 3.82 (s, 3H), 3.70 (s,
6H). 13C NMR (101 MHz, DMSO) δ = 193.37, 162.78, 162.78, 158.71,
138.33 (d, J = 2.2 Hz), 133.51, 133.07, 130.80, 128.83, 127.58 (q, J = 29.2
Hz), 126.61 (q, J = 6.0 Hz), 124.46 (q, J = 274.5 Hz), 110.73, 91.34,
56.23, 55.96. LCꢀMS (ESI-TOF): m/z 367.1157 ([C19H17 F3O4 þ
H]þ calcd 367.1152). Purity 96.17% (tR = 7.58 min).
MHz, DMSO) δ 8.57 (t, J = 1.9 Hz, 1H), 8.29ꢀ8.19 (m, 2H), 7.73 (t, J =
8.0 Hz, 1H), 7.69ꢀ7.61 (m, 2H), 7.61ꢀ7.57 (m, 1H), 7.57ꢀ7.52 (m,
1H), 7.22 (d, J = 7.9 Hz, 1H), 7.08 (td, J = 7.5, 0.9 Hz, 1H), 3.89 (s, 3H).
LCꢀMS (ESI-TOF): m/z 304.0941 ([C17H13 F3O2 þ H]þ calcd
307.0940). Purity 96.00% (tR = 8.88 min).
(E)-1-(3-Methoxyphenyl)-3-(3-(trifluoromethyl)phenyl)prop-2-en-
1-one (3c). It was obtained as a white solid in 21% yield. 1H NMR (400
MHz, DMSO) δ 8.35 (s, 1H), 8.21 (d, J = 7.8 Hz, 1H), 8.11 (d, J = 15.7
Hz, 1H), 7.87ꢀ7.77 (m, 3H), 7.70 (t, J = 7.78 Hz, 1H), 7.66 (dd, J = 1.6,
2.5 Hz, 1H), 7.52 (t, J = 7.94 Hz, 1H), 7.27 (ddd, J = 0.8, 2.6, 8.2 Hz, 1H),
3.87 (s, 3H). 13C NMR (101 MHz, DMSO) δ 189.28, 160.03, 142.68,
139.18, 136.27, 133.32, 130.41, 130.36, 130.25 (q, J = 31.2 Hz), 127.15
(q, J = 3.7 Hz), 125.72 (q, J = 3.7 Hz), 124.50 (q, J = 272.5 Hz), 124.46,
121.70, 119.76, 113.68, 55.85. LCꢀMS (ESI-TOF): m/z 304.0945
([C17H13 F3O2 þ H]þ calcd 307.0940). Purity 100.00% (tR = 9.35 min).
(E)-1-(4-Methoxyphenyl)-3-(3-(trifluoromethyl)phenyl)prop-2-en-
1-one (3d). It was obtained as a white solid in 59% yield. 1H NMR (400
MHz, DMSO) δ 8.33 (s, 1H), 8.24ꢀ8.20 (m, 2H), 8.18 (d, J = 7.8 Hz,
1H), 8.13 (d, J = 15.7 Hz, 1H), 7.82ꢀ7.74 (m, 2H), 7.69 (t, J = 7.8 Hz,
1H), 7.14ꢀ7.07 (m, 2H), 3.88 (s, 3H). 13C NMR (101 MHz, DMSO) δ
187.69, 163.85, 141.74, 136.45, 133.23, 131.57, 130.67, 130.34, 130.24
(q, J = 32.7 Hz), 126.93 (q, J = 3.8 Hz), 125.49 (q, J = 3.7 Hz), 124.52 (q,
J = 272.5 Hz), 124.45, 114.49, 56.05. LCꢀMS (ESI-TOF): m/z
304.0951 ([C17H13 F3O2 þ H]þ calcd 307.0940). Purity 100.00% (tR
= 8.99 min).
(E)-1-(2,4-Dimethoxyphenyl)-3-(3-(trifluoromethyl)phenyl)prop-2-
en-1-one (3e). It was obtained as a white solid in 52% yield. 1H NMR
(400 MHz, DMSO) δ 8.11ꢀ8.03 (m, 2H), 7.77 (d, J = 7.8 Hz, 1H),
7.72ꢀ7.58 (m, 4H), 6.70 (d, J = 2.3 Hz, 1H), 6.66 (dd, J = 2.3, 8.6 Hz,
1H), 3.90 (s, 3H), 3.86 (s, 3H). 13C NMR (101 MHz, D2O) δ 189.57,
164.52, 160.77, 139.58, 136.52, 132.49, 131.97, 130.39, 130.18 (q, J =
31.2 Hz), 129.38, 126.69 (q, J = 3.7 Hz), 125.52 (q, J = 3.8 Hz, H),
124.37 (q, J = 272.5 Hz), 121.55, 106.44, 99.03, 56.38, 56.02. LCꢀMS
(ESI-TOF): m/z 337.1044 ([C18H15 F3O3 þ H]þ calcd 337.1046).
Purity 100.00% (tR = 8.93 min).
(E)-1-(2,6-Dimethoxyphenyl)-3-(3-(trifluoromethyl)phenyl)prop-2-
en-1-one (3f). It was obtained as a light yellow solid in 72% yield. 1H
NMR (400 MHz, DMSO) δ = 8.07 (d, J = 7.8 Hz, 1H), 7.76 (d, J = 7.8,
1H), 7.71 (dd, J = 11.4 Hz, 4.0 Hz, 1H), 7.61 (t, J = 7.6 Hz, 1H), 7.45 (d,
J = 2.1 Hz, 1H), 7.40 (dd, J = 11.1 Hz, 5.7 Hz, 1H), 7.02 (d, J = 15.9 Hz,
1H), 6.75 (d, J = 8.4 Hz, 2H), 3.70 (s, 6H). 13C NMR (101 MHz,
DMSO) δ = 194.52, 157.33, 139.62 (d, J = 2.2 Hz), 133.52, 132.81 (d, J =
1.6 Hz), 132.57, 131.77, 131.02, 128.90, 127.59 (q, J = 30.2 Hz), 126.64
(q, J = 5.7 Hz), 124.38 (q, J = 274.5 Hz), 117.44, 104.69, 56.22. LCꢀMS
(ESI-TOF): m/z 337.1050 ([C18H15 F3O3 þ H]þ calcd 337.1046).
Purity 98.65% (tR = 7.83 min).
(E)-1-(2,5-Dimethoxyphenyl)-3-(3-(trifluoromethyl)phenyl)prop-2-
en-1-one (3g). It was obtained as a yellow oil in 71% yield. 1H NMR
(400 MHz, DMSO) δ 8.12 (s, 1H), 8.08 (d, J = 7.8 Hz, 1H), 7.79 (d, J =
7.8 Hz, 1H), 7.67 (t, J = 7.8 Hz, 1H), 7.62 (d, J = 16.1 Hz, 1H), 7.55 (dd,
J = 0.9, 16.0 Hz, 1H), 7.18ꢀ7.12 (m, 2H), 7.09ꢀ7.03 (m, 1H), 3.82 (d,
J = 0.8 Hz, 3H), 3.76 (d, J = 0.9 Hz, 3H). 13C NMR (101 MHz, DMSO)
δ 192.15, 153.49, 152.47, 141.17, 136.27, 132.29, 130.48, 130.27 (q, J =
31.2 Hz), 129.50, 129.14, 127.06 (q, J = 3.7 Hz), 125.83 (q, J = 3.3 Hz),
124.42 (d, J = 272.5 Hz), 119.20, 114.42, 114.37, 56.84, 56.04. LCꢀMS
(ESI-TOF): m/z 337.1048 ([C18H15 F3O3 þ H]þ calcd 337.1046).
Purity 96.60% (tR = 8.96 min).
(E)-1-Phenyl-3-(3-(trifluoromethyl)phenyl)prop-2-en-1-one (3a). It
was obtained as a white solid in 68% yield. 1H NMR (400 MHz, DMSO)
δ 8.36 (s, 1H), 8.26ꢀ8.18 (m, 3H), 8.15 (d, J = 15.7 Hz, 1H), 7.89ꢀ7.78
(m, 2H), 7.75ꢀ7.67 (m, 2H), 7.64ꢀ7.57 (m, 2H). 13C NMR (101
MHz, DMSO) δ 189.51, 142.61, 137.72, 136.28, 133.83, 133.36, 130.38,
130.26 (q, J = 28.1 Hz), 127.14 (q, J = 3.8 Hz), 125.62 (q, J = 3.7 Hz),
124.15 (q, J = 272.5 Hz), 124.38. LCꢀMS (ESI-TOF): m/z 277.0840
([C16H11F3O þ H]þ calcd 277.0835). Purity 100.00% (tR = 9.20 min).
(E)-1-(2-Methoxyphenyl)-3-(3-(trifluoromethyl)phenyl)prop-2-en-
1-one (3b). It was obtained as a yellow oil in 45% yield. 1H NMR (400
(E)-1-(3,4-Dimethoxyphenyl)-3-(3-(trifluoromethyl)phenyl)prop-2-
en-1-one (3h). It was obtained as a white solid in 64% yield. 1H NMR
(400 MHz, d2o) δ 8.11 (dd, J = 11.7, 8.7 Hz, 3H), 7.98ꢀ7.91 (m, 1H),
7.79 (dd, J = 19.6, 12.0 Hz, 3H), 7.63 (d, J = 1.9 Hz, 1H), 7.13 (d, J = 8.5
Hz, 1H), 3.89 (s, 3H), 3.87 (s, 3H). 13C NMR (101 MHz, d2o) δ 187.59,
153.86, 149.24, 141.43, 139.26 (d, J = 1.3 Hz), 130.59, 130.23 (d, J = 31.2
Hz), 129.74, 126.02 (q, J = 3.7 Hz), 125.06, 124.45 (d, J = 272.5 Hz),
4154
dx.doi.org/10.1021/jm2002348 |J. Med. Chem. 2011, 54, 4147–4159

Journal of Medicinal Chemistry
ARTICLE
124.08, 111.29, 111.13, 56.21, 56.00. LCꢀMS (ESI-TOF): m/z
337.1041 ([C18H15 F3O3 þ H]þ calcd 337.1046). Purity 98.47% (tR
= 8.03 min).
125.20, 121.65, 119.90, 113.58, 55.86. LCꢀMS (ESI-TOF): m/z
304.0943 ([C17H13 F3O2 þ H]þ calcd 307.0940). Purity 100.00%
(tR = 9.52 min).
(E)-1-(3,5-Dimethoxyphenyl)-3-(3-(trifluoromethyl)phenyl)prop-2-
en-1-one (3i). It was obtained as a light yellow solid in 72% yield. 1H
NMR (400 MHz, DMSO) δ 8.34 (s, 1H), 8.22 (d, J = 7.8 Hz, 1H), 8.08
(d, J = 15.7 Hz, 1H), 7.88ꢀ7.77 (m, 2H), 7.70 (t, J = 7.8 Hz, 1H), 7.32
(d, J = 2.3 Hz, 2H), 6.83 (t, J = 2.2 Hz, 1H), 3.85 (s, 6H). 13C NMR (101
MHz, DMSO) δ 189.08, 161.18, 142.83, 139.77, 136.24, 133.33, 130.33,
130.24 (q, J = 32.2 Hz), 127.17 (q, J = 3.6 Hz), 125.85 (q, J = 3.7 Hz),
124.50 (q, J = 273.5 Hz), 124.36, 106.98, 105.52, 56.03. LCꢀMS (ESI-
TOF): m/z 337.1049 ([C18H15 F3O3 þ H]þ calcd 337.1046). Purity
100.00% (tR = 9.55 min).
(E)-1-(4-Methoxyphenyl)-3-(4-(trifluoromethyl)phenyl)prop-2-en-
1-one (4d). It was obtained as a white solid in 56% yield. 1H NMR (400
MHz, DMSO) δ 8.23ꢀ8.01 (m, 5H), 7.76 (dd, J = 21.4, 11.6 Hz, 3H),
7.08 (d, J = 8.2 Hz, 2H), 3.86 (s, 3H). 13C NMR (101 MHz, DMSO) δ
187.70, 163.89, 141.58, 139.30, 131.55, 130.60, 130.32 (d, J = 32.2 Hz),
129.78, 126.11 (dd, J = 3.8 Hz), 125.18, 124.5 (d, J = 272.5 Hz), 114.55,
56.06. LCꢀMS (ESI-TOF): m/z 304.0940 ([C17H13 F3O2 þ H]þ calcd
307.0940). Purity 100.00% (tR = 9.13 min).
(E)-1-(2,4-Dimethoxyphenyl)-3-(4-(trifluoromethyl)phenyl)prop-2-
1
en-1-one (4e). It was obtained as an off-white solid in 40% yield. H
NMR (400 MHz, DMSO) δ 7.92 (d, J = 7.4 Hz, 2H), 7.76 (d, J = 7.5 Hz,
2H), 7.70ꢀ7.50 (m, 3H), 6.74ꢀ6.57 (m, 2H), 3.89 (s, 3H), 3.84 (s, 3H).
13C NMR (101 MHz, DMSO) δ 189.45, 164.74, 160.94, 139.41, 132.65,
130.12) q, J = 32.2 Hz), 130.10, 129.33, 126.21 (dd, J = 3.5 Hz), 124.50
(d, J = 272.5 Hz), 121.48, 106.62, 99.07, 56.48, 56.10. LCꢀMS (ESI-
TOF): m/z 337.1046 ([C18H15 F3O3 þ H]þ calcd 337.1046). Purity
96.35% (tR = 9.13 min).
(E)-1-(3,4,5-Trimethoxyphenyl)-3-(3-(trifluoromethyl)phenyl)prop-
2-en-1-one (3j). It was obtained as a white solid in 60% yield. 1H NMR
(400 MHz, DMSO) δ 8.28ꢀ8.19 (m, 2H), 8.06 (d, J = 15.7 Hz, 1H),
7.86ꢀ7.75 (m, 2H), 7.69 (t, J = 7.8 Hz, 1H), 7.44 (s, 2H), 3.89 (s, 6H),
3.76 (s, 3H). 13C NMR (101 MHz, DMSO) δ 188.34, 153.39, 142.69,
142.48, 136.33, 133.16, 132.95, 130.33, 130.24 (q, J = 32.2 Hz), 127.13
(q, J = 3.7 Hz), 126.02 (q, J = 3.7 Hz), 124.49 (q, J = 272.5 Hz), 124.34,
106.90, 60.66, 56.75. LCꢀMS (ESI-TOF): m/z 367.1155 ([C19H17
F3O4 þ H]þ calcd 367.1152). Purity 96.17% (tR = 8.61 min).
(E)-1-(2,3,4-Trimethoxyphenyl)-3-(3-(trifluoromethyl)phenyl)prop-
2-en-1-one (3k). It was obtained as a yellow oil in 46% yield. 1H NMR
(400 MHz, DMSO) δ 8.13 (s, 1H), 8.09 (d, J = 7.8 Hz, 1H), 7.78 (d, J =
7.7 Hz, 1H), 7.71ꢀ7.56 (m, 3H), 7.42 (d, J = 8.7 Hz, 1H), 6.96 (d, J = 8.9
Hz, 1H), 3.89 (s, 3H), 3.85 (s, 3H), 3.80 (s, 3H). 13C NMR (101 MHz,
DMSO) δ 190.46, 157.30, 153.41, 142.11, 140.87, 136.36, 132.23,
130.49, 130.27 (q, J = 31.2 Hz), 128.92, 126.96 (q, J = 3.7 Hz),
126.42, 125.69, 125.66 (q, J = 5.0 Hz), 124.43 (q, J = 272.5 Hz),
108.33, 62.14, 60.97, 56.57. LCꢀMS (ESI-TOF): m/z 367.1157
([C19H17 F3O4 þ H]þ calcd 367.1152). Purity 97.99% (tR = 8.83 min).
(E)-1-(2,4,6-Trimethoxyphenyl)-3-(3-(trifluoromethyl)phenyl)prop-
2-en-1-one (3l). It was obtained as a light yellow solid in 69% yield. 1H
NMR (400 MHz, DMSO) δ 8.05 (s, 1H), 8.00 (d, J = 7.8 Hz, 1H), 7.73
(d, J = 7.8 Hz, 1H), 7.61 (t, J = 7.8 Hz, 1H), 7.32 (d, J = 16.2 Hz, 1H),
7.11 (d, J = 16.2 Hz, 1H), 6.30 (s, 2H), 3.82 (s, 3H), 3.70 (s, 6H). 13C
NMR (101 MHz, DMSO) δ 193.58, 162.54, 158.63, 141.94, 136.11,
132.29, 130.39, 130.24 (q, J = 30.2 Hz), 127.00 (d, J = 4.0 Hz), 125.81 (d,
J = 4.0 Hz), 124.40 (q, J = 272.5 Hz), 111.35, 91.55, 56.27, 55.93.
LCꢀMS (ESI-TOF): m/z 367.1154 ([C19H17 F3O4 þ H]þ calcd
367.1152). Purity 100.00% (tR = 7.86 min).
(E)-1-(2,6-Dimethoxyphenyl)-3-(4-(trifluoromethyl)phenyl)prop-2-
en-1-one (4f). It was obtained as a light yellow solid in 64% yield. 1H
NMR (400 MHz, DMSO) δ 7.92 (d, J = 8.3 Hz, 2H), 7.75 (d, J = 8.2 Hz,
2H), 7.41 (t, J = 8.4 Hz, 1H), 7.29 (d, J = 16.2 Hz, 1H), 7.13 (d, J = 16.2
Hz, 1H), 6.77 (d, J = 8.5 Hz, 2H), 3.73 (s, 6H). 13C NMR (101 MHz,
DMSO) δ 194.25, 157.40, 142.53, 138.76 (d, J = 1.4 Hz), 131.59, 131.29,
130.55 (q, J = 31.2 Hz), 126.15 (q, J = 3.8 Hz), 124.42 (q, J = 272.5 Hz),
118.12, 104.93, 56.29. LCꢀMS (ESI-TOF): m/z 337.1047 ([C18H15
F3O3 þ H]þ calcd 337.1046). Purity 100.00% (tR = 7.97 min).
(E)-1-(2,5-Dimethoxyphenyl)-3-(4-(trifluoromethyl)phenyl)prop-2-
en-1-one (4g). It was obtained as a yellow oil in 45% yield. 1H NMR
(400 MHz, DMSO) δ 7.95 (d, J = 8.1 Hz, 2H), 7.77 (d, J = 8.2 Hz, 2H),
7.59 (d, J = 16.0 Hz, 1H), 7.54 (d, J = 16.0 Hz, 1H), 7.17ꢀ7.12 (m, 2H),
1
7.08ꢀ7.04 (m, 1H), 3.82 (s, 3H), 3.74 (s, 3H). H NMR (400 MHz,
CDCl3) δ 7.67 (dd, J = 17.3, 8.7 Hz, 5H), 7.51 (d, J = 15.9 Hz, 1H), 7.23
(d, J = 3.1 Hz, 1H), 7.06 (dd, J = 9.0, 3.2 Hz, 1H), 6.96 (d, J = 9.0 Hz,
1H), 3.88 (s, 3H), 3.82 (s, 3H). 13C NMR (101 MHz, DMSO) δ 191.85,
153.50, 152.62, 140.78, 139.13 (d, J = 1.4 Hz), 130.37 (d, J = 32.2 Hz),
129.72, 129.54, 129.28, 126.22 (q, J = 3.7 Hz), 124.46 (d, J = 272.5 Hz),
119.51, 114.44, 114.37, 56.83, 56.01. LCꢀMS (ESI-TOF): m/z
337.1048 ([C18H15 F3O3 þ H]þ calcd 337.1046). Purity 96.42%
(tR = 9.16 min).
(E)-1-Phenyl-3-(4-(trifluoromethyl)phenyl)prop-2-en-1-one (4a). It
was obtained as a white solid in 59% yield. 1H NMR (400 MHz, DMSO)
δ 8.20ꢀ8.11 (m, 3H), 8.08 (d, J = 15.7 Hz, 2H), 7.83ꢀ7.75 (m, 3H),
7.72ꢀ7.65 (m, 1H), 7.62ꢀ7.54 (m, 2H). 13C NMR (101 MHz, DMSO)
δ 189.53, 142.43, 139.13 (d, J = 1.3 Hz), 137.68, 133.87, 130.49 (q, J =
32.2 Hz), 129.91, 129.30, 129.11, 126.14 (q, J = 3.8 Hz), 125.13, 124.44
(q, J = 272.5 Hz). LCꢀMS (ESI-TOF): m/z 277.0834 ([C16H11F3O þ
H]þ calcd 277.0835). Purity 100.00% (tR = 9.35 min).
(E)-1-(2-Methoxyphenyl)-3-(4-(trifluoromethyl)phenyl)prop-2-en-
1-one (4b). It was obtained as a yellow oil in 79% yield. 1H NMR (400
MHz, DMSO) δ 7.97 (d, J = 8.1 Hz, 2H), 7.79 (d, J = 8.2 Hz, 2H), 7.72
(d, J = 8.2 Hz, 1H), 7.61ꢀ7.48 (m, 6H), 7.22 (d, J = 8.0 Hz, 1H), 7.08
(td, J = 7.5, 0.9 Hz, 1H), 3.88 (s, 3H), 3.86 (s, 2H). LCꢀMS (ESI-TOF):
m/z 304.0948 ([C17H13 F3O2 þ H]þ calcd 307.0940). Purity 94.00%
(tR = 9.09 min).
(E)-1-(3-Methoxyphenyl)-4-(4-(trifluoromethyl)phenyl)prop-2-en-
1-one (4c). It was obtained as a white solid in 61% yield. 1H NMR (400
MHz, DMSO) δ 8.14 (d, J = 8.1 Hz, 2H), 8.07 (d, J = 15.7 Hz, 1H),
7.85ꢀ7.80 (m, 3H), 7.79 (d, J = 4.3 Hz, 1H), 7.67ꢀ7.62 (m, 1H), 7.52
(t, J = 7.9 Hz, 1H), 7.27 (dd, J = 8.2, 2.6 Hz, 1H), 3.87 (s, 3H). 13C NMR
(101 MHz, DMSO) δ 189.29, 160.04, 142.49, 139.13, 13.50 (d, J = 31.2
Hz), 130.45, 129.95, 126.12 (q, J = 3.7 Hz), 124.5 (d, J = 272.46 Hz),
(E)-1-(3,4-Dimethoxyphenyl)-3-(4-(trifluoromethyl)phenyl)prop-2-
en-1-one (4h). It was obtained as a white solid in 64% yield. 1H NMR
(400 MHz, DMSO) δ 8.31 (s, 1H), 8.20 (d, J = 7.8 Hz, 1H), 8.12 (d, J =
15.6 Hz, 1H), 7.99 (dd, J = 2.0, 8.5 Hz, 1H), 7.83ꢀ7.75 (m, 2H), 7.70 (t,
J = 7.8 Hz, 1H), 7.62 (d, J = 2.0 Hz, 1H), 7.12 (d, J = 8.5 Hz, 1H), 3.89 (s,
3H), 3.87 (s, 3H). 13C NMR (101 MHz, DMSO) δ 187.70, 153.90,
149.31, 141.68, 136.46, 133.10, 130.72, 130.33, 130.24 (d, J = 32.2 Hz),
126.93 (q, J = 3.6 Hz), 125.64 (q, J = 3.6 Hz), 124.52 (d, J = 272.4 Hz),
124.39, 124.24, 111.30, 111.21, 56.27, 56.09. LCꢀMS (ESI-TOF): m/z
337.1042 ([C18H15 F3O3 þ H]þ calcd 337.1046). Purity 95.94% (tR =
8.19 min).
(E)-1-(3,4,5-Trimethoxyphenyl)-3-(4-(trifluoromethyl)phenyl)prop-
2-en-1-one (4i). It was obtained as a white solid in 77% yield. 1H NMR
(400 MHz, DMSO) δ 8.11 (d, J = 8.2 Hz, 2H), 8.06 (d, J = 15.6 Hz, 1H),
7.83ꢀ7.74 (m, 3H), 7.43 (s, 2H), 3.89 (s, 6H), 3.76 (s, 3H). 13C NMR
(101 MHz, DMSO) δ 188.24, 153.40, 142.69, 142.27, 139.19 (d, J = 1.3
Hz), 133.08, 130.44 (q, J = 31.2 Hz), 129.95, 126.08 (q, J = 3.7 Hz),
125.04, 124.50 (q, J = 271.4 Hz), 106.80, 60.65, 56.70. LCꢀMS (ESI-
TOF): m/z 367.1158 ([C19H17 F3O4 þ H]þ calcd 367.1152). Purity
96.17% (tR = 8.78 min).
(E)-1-(2,3,4-Methoxyphenyl)-3-(4-(trifluoromethyl)phenyl)prop-2-
en-1-one (4j). It was obtained as a white solid in 63% yield. 1H NMR
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Journal of Medicinal Chemistry
ARTICLE
(400 MHz, DMSO) δ 7.95 (d, J = 6.8 Hz, 2H), 7.77 (d, J = 7.0 Hz, 2H),
7.58 (s, 2H), 7.40 (d, J = 8.3 Hz, 1H), 6.94 (d, J = 8.6 Hz, 1H), 3.86 (s,
3H), 3.83 (s, 3H), 3.77 (s, 3H). 1H NMR (400 MHz, CDCl3) δ 7.68
(dd, J = 23.7, 8.2 Hz, 5H), 7.59 (d, J = 15.9 Hz, 1H), 7.53 (d, J = 8.8 Hz,
1H), 6.78 (d, J = 8.8 Hz, 1H), 3.94 (s, 4H), 3.92 (s, 6H). 13C NMR (101
MHz, DMSO) δ 190.22, 157.48, 153.53, 142.08, 140.54, 139.21, 130.30
(q, J = 32.2 Hz), 126.26, 126.23, 125.79, 124.48 (q, J = 271.5 Hz),
108.41, 62.17, 60.97, 56.58. LCꢀMS (ESI-TOF): m/z 367.1158
([C19H17 F3O4 þ H]þ calcd 367.1152). Purity 100.00% (tR = 9.05 min).
(E)-1-(2,4,6-Methoxyphenyl)-3-(4-(trifluoromethyl)phenyl)prop-2-
en-1-one (4k). It was obtained as a yellow oil in 86% yield. 1H NMR
(400 MHz, DMSO) δ 7.91 (d, J = 8.2 Hz, 2H), 7.75 (d, J = 8.3 Hz, 2H),
7.31 (d, J = 16.2 Hz, 1H), 7.11 (d, J = 16.2 Hz, 1H), 6.32 (s, 2H), 3.84 (s,
3H), 3.73 (s, 6H). 13C NMR (101 MHz, DMSO) δ 193.27, 162.66,
158.73, 141.53, 138.97 (d, J = 1.4 Hz), 130.33 (q, J = 32.2 Hz), 126.14 (q,
J = 3.8 Hz), 124.45 (q, J = 271.4 Hz), 111.28, 91.57, 91.40, 56.29, 55.94.
LCꢀMS (ESI-TOF): m/z 367.1152 ([C19H17 F3O4 þ H]þ calcd
367.1152). Purity 96.54% (tR = 8.03 min).
(E)-1-Phenyl-3-(2-nitrophenyl)prop-2-en-1-one (5a). It was ob-
tained as an off-white solid in 46% yield. 1H NMR (400 MHz, DMSO)
δ = 8.24ꢀ8.20 (m, 1H), 8.20ꢀ8.15 (m, 2H), 8.11 (dd, J = 8.1, 1.2 Hz,
1H), 8.00 (d, J = 15.5 Hz, 1H), 7.91 (d, J = 15.5 Hz, 1H), 7.88ꢀ7.81 (m,
1H), 7.76ꢀ7.68 (m, 2H), 7.60 (t, J = 7.6 Hz, 2H). 13C NMR (101 MHz,
DMSO) δ = 189.52, 149.24, 139.03, 137.48, 134.22, 133.98, 131.53,
130.19, 129.98, 129.33, 129.18, 126.83, 125.16. LCꢀMS (ESI-TOF):
m/z 254.0819 ([C15H11NO3 þ H]þ calcd 254.0812). Purity 100.00%
(tR = 6.68 min).
136.03, 134.33, 132.76, 131.63, 131.12, 130.48, 129.55, 125.14, 121.20,
106.68, 99.03, 56.48, 56.13. LCꢀMS (ESI-TOF): m/z 314.1027
([C17H15NO5 þ H]þ calcd 314.1023). Purity 97.57% (tR = 6.62 min).
(E)-1-(2,6-Dimethoxyphenyl)-3-(2-nitrophenyl)prop-2-en-1-one
(5f). It was obtained as a white solid in 82% yield. 1H NMR (400 MHz,
DMSO) δ = 8.06 (dd, J = 8.1, 1.2 Hz, 1H), 7.98 (dd, J = 7.8, 1.3 Hz, 1H),
7.78 (ddd, J = 7.8, 1.2, 0.6 Hz, 1H), 7.70ꢀ7.64 (m, 1H), 7.52 (d, J = 16.0
Hz, 1H), 7.40 (t, J = 8.4 Hz, 1H), 6.96 (d, J = 16.0 Hz, 1H), 6.77 (d, J =
8.4 Hz, 2H), 3.75 (s, 7H). 13C NMR (101 MHz, DMSO) δ = 194.37,
157.44, 148.75, 140.06, 134.33, 132.66, 131.72, 131.52, 129.86, 129.66,
125.21, 117.59, 104.80, 56.26. LCꢀMS (ESI-TOF): m/z 314.1024
([C17H15NO5 þ H]þ calcd 314.1023). Purity 100.00% (tR = 5.74 min).
(E)-1-(2,5-Dimethoxyphenyl)-3-(2-nitrophenyl)prop-2-en-1-one
(5g). It was obtained as a yellow solid in 49% yield. 1H NMR (400 MHz,
D2O) δ = 8.09 (dd, J = 8.1, 1.1 Hz, 1H), 7.97 (d, J = 7.7 Hz, 1H),
7.85ꢀ7.76 (m, 2H), 7.73ꢀ7.65 (m, 1H), 7.41 (d, J = 15.8 Hz, 1H), 7.16
(d, J = 1.8 Hz, 2H), 7.09 (t, J = 1.8 Hz, 1H), 3.84 (s, 3H), 3.76 (s, 3H).
13C NMR (101 MHz, D2O) δ = 191.59, 153.41, 152.61, 137.66, 134.31,
131.31, 131.11, 130.19, 129.56, 128.86, 125.14, 119.67, 114.33, 114.32,
56.74, 55.96. LCꢀMS (ESI-TOF): m/z 314.1029 ([C17H15NO5 þ H]þ
calcd 314.1023). Purity 100.00% (tR = 6.63 min).
(E)-1-(3,4-Dimethoxyphenyl)-3-(2-nitrophenyl)prop-2-en-1-one
(5h). It was obtained as a yellow solid in 17% yield. 1H NMR (400 MHz,
DMSO) δ = 8.20 (d, J = 7.8 Hz, 1H), 8.10 (dd, J = 8.1, 1.0 Hz, 1H),
7.99ꢀ7.88 (m, 3H), 7.84 (t, J = 7.6 Hz, 1H), 7.74ꢀ7.67 (m, 1H), 7.62
(d, J = 2.0 Hz, 1H), 7.13 (d, J = 8.5 Hz, 1H), 3.89 (s, 3H), 3.87 (s, 3H).
1H NMR (400 MHz, CDCl3) δ = 8.06ꢀ 8.12 (m, 2H), 7.74 (d, J = 7.7
Hz, 1H), 7.72ꢀ7.65 (m, 2H), 7.62 (d, J = 1.7 Hz, 1H), 7.56 (t, J = 7.2 Hz,
1H), 7.31 (d, J = 15.7 Hz, 1H), 6.95 (d, J = 8.4 Hz, 1H), 3.99 (s, 3H),
3.98 (s, 3H). 13C NMR (101 MHz, D2O) δ = 187.54, 153.92, 149.23,
149.15, 137.91, 134.06, 131.25, 130.35, 130.26, 129.89, 126.80, 125.02,
124.14, 111.33, 111.21, 56.22, 56.00. LCꢀMS (ESI-TOF): m/z
314.1027 ([C17H15NO5 þ H]þ calcd 314.1023). Purity 100.00%
(tR = 5.63 min).
(E)-1-(3,5-Dimethoxyphenyl)-3-(2-nitrophenyl)prop-2-en-1-one
(5i). It was obtained as an off-white solid in 57% yield. 1H NMR (400
MHz, DMSO) δ = 8.21 (dd, J = 7.8, 1.3 Hz, 1H), 8.10 (dd, J = 8.1, 1.2
Hz, 1H), 7.98 (d, J = 15.5 Hz, 1H), 7.89ꢀ7.80 (m, 2H), 7.74ꢀ7.68 (m,
1H), 7.28 (d, J = 2.3 Hz, 2H), 6.82 (t, J = 2.3 Hz, 1H), 3.85 (s, 6H). 13C
NMR (101 MHz, DMSO) δ = 189.21, 161.19, 149.24, 139.53, 139.25,
134.18, 131.52, 130.15, 130.09, 126.89, 125.13, 106.91, 105.89, 56.05.
LCꢀMS (ESI-TOF): m/z 314.1024 ([C17H15NO5 þ H]þ calcd
314.1023). Purity 100.00% (tR = 7.21 min).
(E)-1-(3,4,5-Trimethoxyphenyl)-3-(2-nitrophenyl)prop-2-en-1-one
(5j). It was obtained as an off-white solid in 31% yield. 1H NMR (400
MHz, DMSO) δ = 8.13 (dd, J = 27.6, 8.0 Hz, 2H), 7.97 (d, J = 15.4 Hz,
1H), 7.92ꢀ7.79 (m, 2H), 7.70 (t, J = 7.7 Hz, 1H), 7.42 (s, 2H), 3.88 (s,
6H), 3.76 (s, 3H). 13C NMR (101 MHz, DMSO) δ = 188.38, 153.39,
149.18, 142.64, 139.01, 134.20, 132.84, 131.45, 130.35, 130.12, 126.80,
125.16, 106.80, 60.66, 56.65. LCꢀMS (ESI-TOF): m/z 344.1130
([C18H17NO6 þ H]þ calcd 344.1129). Purity 99.00% (tR = 6.25 min).
(E)-1-(2,3,4-Trimethoxyphenyl)-3-(2-nitrophenyl)prop-2-en-1-one
(5k). It was obtained as a light white solid in 32% yield. 1H NMR (400
MHz, DMSO) δ = 8.07 (d, J = 8.0 Hz, 1H), 7.97 (d, J = 7.6 Hz, 1H),
7.85ꢀ7.75 (m, 2H), 7.67 (t, J = 7.7 Hz, 1H), 7.43 (dd, J = 12.2 Hz, 3.2
Hz, 2H), 6.95 (d, J = 8.8 Hz, 1H), 3.87 (s, 3H), 3.84 (s, 3H), 3.77 (s, 3H).
LCꢀMS (ESI-TOF): m/z 344.1127 ([C18H17NO6 þ H]þ calcd
344.1129). Purity 100.00% (tR = 6.52 min).
(E)-1-(2-Methoxyphenyl)-3-(2-nitrophenyl)prop-2-en-1-one (5b).
1
It was obtained as a light yellow solid in 49% yield. H NMR (400
MHz, DMSO) δ = 8.09 (dd, J = 8.2, 1.2 Hz, 1H), 7.99 (dd, J = 7.9, 1.2
Hz, 1H), 7.84ꢀ7.76 (m, 2H), 7.73ꢀ7.66 (m, 1H), 7.61ꢀ7.57 (m, 1H),
7.55 (dd, J = 7.7, 1.7 Hz, 1H), 7.42 (d, J = 15.7 Hz, 1H), 7.22 (d, J = 8.0
Hz, 1H), 7.08 (td, J = 7.5, 0.9 Hz, 1H), 3.90 (s, 3H). 13C NMR (101
MHz, DMSO) δ = 192.08, 158.46, 149.07, 137.47, 134.36, 134.07,
131.33, 130.24, 130.22, 129.62, 128.56, 125.20, 121.06, 112.82, 56.32.
LCꢀMS (ESI-TOF): m/z 284.0916 ([C16H13NO4 þ H]þ calcd
284.0917). Purity 97.28% (tR = 6.52 min).
(E)-1-(3-Methoxyphenyl)-3-(2-nitrophenyl)prop-2-en-1-one (5c). It
1
was obtained as an off-white solid in 21% yield. H NMR (400 MHz,
DMSO) δ = 8.18 (d, J = 7.7 Hz, 1H), 8.08 (d, J = 8.0 Hz, 1H), 7.97 (d, J =
15.5 Hz, 1H), 7.83 (dd, J = 17.6, 11.4 Hz, 2H), 7.75 (d, J = 7.5 Hz, 1H),
7.69 (t, J = 7.6 Hz, 1H), 7.61 (s, 1H), 7.50 (t, J = 7.9 Hz, 1H), 7.25 (d, J =
6.4 Hz, 1H), 3.84 (s, 3H). 13C NMR (101 MHz, DMSO) δ = 189.36,
160.02, 149.23, 139.13, 138.91, 134.21, 131.53, 130.50, 130.16, 130.02,
126.91, 125.14, 121.70, 120.06, 113.60, 55.87. LCꢀMS (ESI-TOF): m/
z 284.0918 ([C16H13NO4 þ H]þ calcd 284.0917). Purity 100.00% (tR =
6.91 min).
(E)-1-(4-Methoxyphenyl)-3-(2-nitrophenyl)prop-2-en-1-one (5d).
It was obtained as an off-white solid in 32% yield. 1H NMR (400
MHz, DMSO) δ = 8.17 (t, J = 7.9 Hz, 3H), 8.07 (d, J = 8.1 Hz, 1H),
7.97ꢀ7.85 (m, 2H), 7.81 (t, J = 7.6 Hz, 1H), 7.68 (t, J = 7.7 Hz, 1H), 7.09
1
(d, J = 8.4 Hz, 2H), 3.86 (s, 3H). H NMR (400 MHz, CDCl3) δ =
8.15ꢀ8.00 (m, 3H), 7.74 (d, J = 7.6 Hz, 1H), 7.68 (t, J = 7.6 Hz, 1H),
7.56 (t, J = 7.1 Hz, 1H), 7.32 (d, J = 15.6 Hz, 1H), 7.00 (d, J = 8.9 Hz,
2H), 3.90 (s, 3H). 13C NMR (101 MHz, DMSO) δ = 187.58, 163.98,
149.23, 138.00, 134.15, 131.63, 131.36, 130.38, 130.26, 129.91, 126.89,
125.09, 114.59, 56.08. LCꢀMS (ESI-TOF): m/z 284.0919
([C16H13NO4 þ H]þ calcd 284.0917). Purity 100.00% (tR = 6.55 min).
(E)-1-(2,4-Dimethoxyphenyl)-3-(2-nitrophenyl)prop-2-en-1-one
(5e). It was obtained as a yellow solid in 57% yield. 1H NMR (400 MHz,
DMSO) δ = 8.06 (dd, J = 8.2 Hz, 1.2 Hz, 1H), 7.94 (d, J = 7.7 Hz, 1H),
7.82ꢀ7.73 (m, 2H), 7.69ꢀ7.61 (m, 2H), 7.51 (d, J = 15.6 Hz, 1H), 6.68
(d, J = 2.2 Hz, 1H), 6.64 (dd, J = 8.6, 2.3 Hz, 1H), 3.90 (s, 4H), 3.84 (s,
4H). 13C NMR (101 MHz, DMSO) δ = 189.17, 164.87, 161.02, 149.09,
(E)-1-(2,4,6-Trimethoxyphenyl)-3-(2-nitrophenyl)prop-2-en-1-one
(5l). It was obtained as a yellow solid in 61% yield. 1H NMR (400 MHz,
DMSO) δ = 8.03 (dd, J = 8.1 Hz, 1.0, 1H), 7.93 (d, J = 7.7 Hz, 1H), 7.75
(t, J = 7.6 Hz, 1H), 7.64 (t, J = 7.8 Hz, 1H), 7.53 (d, J = 15.9 Hz, 1H), 6.92
(d, J = 15.9 Hz, 1H), 6.30 (s, 2H), 3.82 (s, 3H), 3.72 (s, 6H). 13C NMR
(101 MHz, DMSO) δ = 193.25, 162.76, 158.80, 148.78, 138.79, 134.30,
4156
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Journal of Medicinal Chemistry
ARTICLE
133.17, 131.33, 130.06, 129.61, 125.18, 110.82, 91.42, 56.26, 55.94.
LCꢀMS (ESI-TOF): m/z 344.1141 ([C18H17NO6 þ H]þ calcd
344.1129). Purity 100.00% (tR = 5.77 min).
Biology. Cell Culture and Treatment. Human bronchial epithelial
(Beas-2B) cells were cultured in DMEM/F12 (pH 7.4) supplemented
with 10% (v/v) FBS, 100 mg/L gentamicin, and genetisin. Beas-2B cells
were grown in 48-well plates for 24 h and then treated with a series of
chalcone derivatives dissolved in DMSO for various time points. The
concentration of DMSO did not exceed 0.1%. RNA was isolated, and
gene expression was measured after 16 h.
Cell Viability Assay. The cytotoxicity of chalcone derivatives was
analyzed by using trypan blue exclusion test and was further confirmed
by colorimetric methylthiazolydiphenyltetrazolium bromide (MTT)
assay as described.82 Briefly, Beas-2B cells were treated with chalcone
analogues or DMSO alone (0.1%, as vehicle) for 24 h. Four hours before
the end of incubation, the medium was removed and 100 μL of MTT
(5 mg/mL in serum free medium) was added to each well. The MTT was
removed after 4 h. Cells were washed with PBS, and 100 μL of DMSO
was added to each well to dissolve the water-insoluble MTT-formazan
crystals. The absorbance was recorded at 570 nm in a plate reader
(Molecular Devices, Sunnyvale, CA).
Author Contributions
These authors contributed equally.
’ ACKNOWLEDGMENT
V.K., M.H., and S.V.M. thank the National Cancer Institute
(NCI) Developmental Therapeutics Program. This project has
been funded in whole or in part with funds from the NCI, National
Institutes of Health, Grant HSN261200800001E. This work was
also supported by National Institutes of Health Grant HL081205
(S.B.), National Heart, Lung, and Blood Institute Specialized
Centers of Clinically Oriented Research Grant P50HL084945,
the Flight Attendant Medical Research Institute (S.B. and
R.K.T.), National Cancer Institute Grants 5P50CA058184 and
5R01CA140492, NIH Phase 2 grant 1U01HL105569, and Na-
tional Institute on Environmental Health Sciences Grants
P50ES015903, P01 ES018176, and ES03819 (S.B.). V.S.P. and S.
KS. thank the University of Delhi and the Department of Scientific
and Industrial Research (DSIR, New Delhi) for financial support.
’ ABBREVIATIONS USED
Generation of Stable Transfectants. Beas-2B cells overexpressing
ARE luciferase reporter plasmid were obtained by transfecting Beas-2B
cells with 3 μg of NQO1-ARE reporter plasmid and 0.3 μg of pUB6
empty vector (Invitrogen, Carlsbad, CA). Stable transfectants were
selected using blasticidin at 6 μg/mL. Stable clones were expanded
and screened for the expression of ARE luciferase.83
Nrf2, nuclear factor erythroid 2 p45-related factor 2; b-ZIP, basic-
leucine zipper; Keap 1, Kelch-like ECH-associated protein 1; ARE,
antioxidant response element; HO-1, heme oxygenase 1; NQO1,
NAD(P)H/quinone oxidoreductase 1; GCLM, glutamateꢀcysteine
ligase modifier subunit
ARE Reporter Assay. Beas-2B cells stably expressing NQO1-ARE
luciferase were seeded onto a 96-well plate at a density of 10 000 cells/
well for 16 h before incubation with test compounds. Next day, cells were
treated with the indicated concentrations of compound 2b. Cells were
also treated with DMSO, which was used as the solvent. The reporter
activity was measured after 16 h of exposure using the luciferase assay kit
(Promega, Madison, WI). The level of increase in luciferase activity
reflects the degree of Nrf2 activity.83
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Mice in Vivo Study. All experiments in mice were performed in
accordance with the standards established by the U.S. Animal Welfare
Acts, set forth in NIH guidelines and the Policy and Procedures Manual
of the Johns Hopkins University Animal Care and Use Committee.
C57BL/6 mice (male, 7 weeks) were maintained on AIN 76A diet
(Harlan Tekland, Madison, WI) and water ad libitum and housed at a
temperature (range, 20ꢀ23 °C) under 12 h light/dark cycles. The mice
were treated with chalcone analogues (50 mg/kg body weight) or vehicle
or sulforaphane as a positive control by gavage. After 24 h of treatment,
the small intestines were harvested and stored at ꢀ80 °C until analysis.
RNA Extraction and Gene Expression Analysis. Total RNA was
extracted from cells/tissue using Qiagen RNeasy kit (Qiagen Corpora-
tion, Valencia, CA), and reverse transcription was performed by using
random hexamers and MultiScribe reverse transcriptase according to the
manufacturer’s recommendations (Applied Biosystems, Foster City,
CA, U.S.). Quantitative real-time RT-PCR analyses of Nrf2, NQO1,
HO1, and GCLM were performed by using Assay-on-Demand primers
and probe sets from Applied Biosystems. Assays were performed using
the ABI 7000 Taqman system (Applied Biosystems, Foster City, CA).
β-Actin was used for normalization.
Statistics. The values were determined as the mean ( SE and analyzed
by student’s t test. Differences were considered significant at P e 0.05.
’ AUTHOR INFORMATION
(8) Niture, S. K.; Jain, A. K.; Jaiswal, A. K. Antioxidant-induced
modification of INrf2 cysteine 151 and PKC-delta-mediated phosphor-
ylation of Nrf2 serine 40 are both required for stabilization and nuclear
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Corresponding Author
*For S.B.: phone, 410-502-1946; fax, 410-955-1946; e-mail,
sbiswal@jhsph.edu. For S.V.M.: phone, þ1-301-846-5151;
fax, 1-301-846-5206; e-mail, malhotrasa@mail.nih.gov.
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