Synthesis and bioevaluation of a series of α-pyrone derivatives as potent activators of Nrf2/ARE pathway (part I)

Article abstract of DOI:10.1016/j.ejmech.2013.06.007

When exposed to electrophiles, human colorectal cancer cells (HCT116) counteract oxidative stress through activating NF-E2-related factor 2 (Nrf2)/antioxidant response element (ARE) pathway. To identify new activators, luciferase reporter gene assay was used to screen in-house database of our laboratory, leading to a novel α-pyrone compound 1 as a hit. 2 with 2-fluoro phenyl group exhibited the strongest ARE inductive activity in the first round structure-activity relationship (SAR) study. Biological studies showed the compound induced nuclear translocation of Nrf2 preceded by phosphorylation of ERK1/2. The data encouraged us to use 2 as lead and 20 derivatives were synthesized to discuss a more detailed SAR, leading to a more potent compound 9, which can be the starting compound for further modification.

Full text of DOI:10.1016/j.ejmech.2013.06.007

European Journal of Medicinal Chemistry 66 (2013) 364e371
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and bioevaluation of a series of
a-pyrone derivatives
as potent activators of Nrf2/ARE pathway (part I)
,
d
,
,
,
b
,
d
,
b,d
Mei-yang Xi b 1, Zhong-ying Sun c 1, Hao-peng Sun a
, Jian-min Jia
,
**
Zheng-yu Jiang b d, Lei Tao c, Ming Ye c, Xi Yang c, Ya-jing Wang c, Xin Xue b
,
,
,d
Jing-jie Huang b d, Yuan Gao c, Xiao-ke Guo b d, Sheng-lie Zhang b d, Ying-rui Yang b
***
,
,
,
,
,d
Qing-long Guo c, Rong Hu c , Qi-dong You
,
a,b,
*
a State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, China
b Jiang Su Key Laboratory of Drug Design and Optimization, China Pharmaceutical University, Nanjing 210009, China
c Department of Physiology, School of Pharmacy, China Pharmaceutical University, Nanjing, China
d Department of Medicinal Chemistry, China Pharmaceutical University, Nanjing 210009, China
a r t i c l e i n f o
a b s t r a c t
Article history:
When exposed to electrophiles, human colorectal cancer cells (HCT116) counteract oxidative stress
through activating NF-E2-related factor 2 (Nrf2)/antioxidant response element (ARE) pathway. To
identify new activators, luciferase reporter gene assay was used to screen in-house database of our
Received 3 April 2013
Received in revised form
22 May 2013
Accepted 5 June 2013
Available online 14 June 2013
laboratory, leading to a novel a-pyrone compound 1 as a hit. 2 with 2-fluoro phenyl group exhibited the
strongest ARE inductive activity in the first round structureeactivity relationship (SAR) study. Biological
studies showed the compound induced nuclear translocation of Nrf2 preceded by phosphorylation of
ERK1/2. The data encouraged us to use 2 as lead and 20 derivatives were synthesized to discuss a more
detailed SAR, leading to a more potent compound 9, which can be the starting compound for further
modification.
Keywords:
Nrf2
Keap1
ARE
Ó 2013 Elsevier Masson SAS. All rights reserved.
a
-Pyrone derivative
Chemopreventive agents
1. Introduction
the well-defined high-risk groups [2,10]. Thus, chemoprevention
deserves further attention.
Despite the enormous progress in the early detection and
treatment of cancer, the high mortality for most cancers has not
decreased in the past three decades [1]. As a new therapeutic
strategy, chemoprevention is proved to be attractive in the modu-
lation of the carcinogenesis process in recent years [2e4]. Many
data from animal models have demonstrated that chemopreven-
tion hinders the development of cancer [5e9]. Additionally, it is
also confirmed to be effective in reducing the incidence of cancer in
Molecular biological investigations have shown that NF-E2-
related factor (Nrf2)/Kelch-like ECH-associated protein
2
1
(Keap1)/ARE pathway plays a crucial role in cancer chemopreven-
tion [11]. It serves as the core in the complicated and efficient
cyto-protective machinery in response to a variety of oxidative or
electrophilic challenges in eukaryotic cells [12,13] and demonstrate
that Nrf2 is a potential molecular target for cancer chemopreven-
tion [14e16].
Nrf2 is a 66-kDa transcription factor which contains a Cap ‘n’
Collar (CNC) structure. It belongs to basic-leucine zipper (bZIP)
transcription factor family (Fig. 1A). Nrf2 is considered to be one of
the most important regulators during the chemoprevention pro-
cess. In quiescent condition, Nrf2 is sequestered in cytoplasm by
Keap1, a 69-kDa cytosolic protein usually existing as a dimer
(Fig. 1B) [17], promoting its ubiquitination and degradation (Fig. 1C)
[18]. However, under stress such as irritant, some mercapto groups
of cysteine (cys) in Keap1 were oxidized or alkylated, changing the
binding conformation of Keap1-Nrf2 complex. The degradation
* Corresponding author. China Pharmaceutical University, Tongjiaxiang 24,
Nanjing 210009, China. Tel./fax: þ86 25 83271351.
** Corresponding author. China Pharmaceutical University, Tongjiaxiang 24,
Nanjing 210009, China. Tel./fax: þ86 25 83271216.
*** Corresponding author. China Pharmaceutical University, Tongjiaxiang 24,
Nanjing 210009, China. Tel./fax: þ86 25 83271126.
E-mail addresses: sunhaopeng@163.com (H.-p. Sun), michelhu@hotmail.com
(R. Hu), youqidong@gmail.com (Q.-d. You).
1
These two authors contributed equally to this work.
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.06.007

M.-y. Xi et al. / European Journal of Medicinal Chemistry 66 (2013) 364e371
365
Fig. 1. Structure of Nrf2, Keap1; the “hinge and latch” two-site binding model is proposed and mercapto group of cys in Keap1 is modified, in favor of DLG dissociation. (A) The Neh2
contains DLG and ETGE for binding Keap1. Neh4 and 5 bind a coactivator CBP. Neh1 binds Maf and DNA. (B) The BTB mediates homodimerization. In BTB and IVR, Cys151, 273 and
288 are important for Nrf2 dissociating from Keap1. DGR consists of 6 Kelch motifs for Nrf2 binding. (C) Without cellular stress, Nrf2 is bound by Keap1. Homodimer DGR domain of
Keap1 interacts with DLG and ETGE motifs of Nrf2, enabling ubiquitination and proteasomal degradation. (D) Under some oxidative stress, the low-affinity DLG “latch” is
disconnected. Nevertheless, the high-affinity ETGE “hinge” is kept. As a result, non-degradable Nrf2 and saturated Keap1 cause that any new Nrf2 is capable of translocating to the
nuclei.
process of Nrf2 is then prohibited, leading to the accumulation of
newly synthesized Nrf2. It then translocates to the nuclei and di-
merizes with Maf. The dimerized protein binds to ARE promotors,
recruits CREB-binding protein (CBP), induces transcription and
upregulates the expression of phase-II enzymes such as heme
2. Results
2.1. Chemistry
In order to identify the action pathway of the hit 1, we designed
and synthesized 25 derivatives 2e26 (Table 1). The synthetic route
oxygenase-1 (HO-1) and NAD(P)H:quinone oxidoreductase
(NQO-1) (Fig. 1D) [19].
1
Due to the significant role of Nrf2 in the chemoprevention
process, compounds inducing ARE activation through Nrf2/Keap1
pathway are attractive and considered to be chemopreventive
agents [11,20,21]. These compounds are blocking agents for the
tumor initiation phase and suppressing agents for the tumor pro-
motion and progression phase [22,23]. Till now, there are several
classes of ARE inducers reported. Most of them are separated from
dietary food, such as curcumin, EGCG, resveratrol, genistein and
sulforaphane (Fig. 2) [24]. A common structure of a, b-unsaturated
ketone plays a key role in the chemopreventive activity of these
compounds. With this information in hand, in the present study, a
structural similarity screening of our in-house natural product
database was performed in order to discover novel chemo-
preventive agents. A compound with a-pyrone scaffold, exhibiting
strong ARE inducer activity, was confirmed as lead compound
through several bioevaluations. Two rounds of molecular optimi-
zation were carried out to study the structureeactivity relationship
(SAR), leading to more potent derivatives.
Fig. 2. The structure of chemopreventive agents.

366
M.-y. Xi et al. / European Journal of Medicinal Chemistry 66 (2013) 364e371
Table 1
The activity of compounds 1e26.
Compd. ID
R/X
Inductivity (concentration/
m
M)
IC50
/m
M
PKa
Log P
Log D7.4
50
20
Predicted
1
2
3
4
5
6
7
8
H
2-F
S
O
NH
2-NO2
3-F
4-F
3,4-F
6.84 ꢀ 0.89
3.04 ꢀ 0.34
5.96 ꢀ 0.08
5.48 ꢀ 0.03
3.58 ꢀ 0.37
0.65 ꢀ 0.22
3.46 ꢀ 0.58
5.79 ꢀ 1.08
3.68 ꢀ 0.47
6.98 ꢀ 0.55
4.51 ꢀ 1.22
6.28 ꢀ 1.37
3.27 ꢀ 0.30
1.20 ꢀ 0.37
7.37 ꢀ 0.95
5.50 ꢀ 0.06
6.81 ꢀ 0.31
5.25 ꢀ 0.08
2.17 ꢀ 0.23
3.60 ꢀ 0.54
8.09 ꢀ 0.23
2.48 ꢀ 0.34
2.07 ꢀ 0.24
0.95 ꢀ 0.15
2.08 ꢀ 0.16
2.55 ꢀ 0.13
5.08 ꢀ 0.06 (80
1 ꢀ 0.05
4.21 ꢀ 0.29
7.08 ꢀ 0.17
4.53 ꢀ 0.68
3.80 ꢀ 0.32
1.66 ꢀ 0.04
2.74 ꢀ 0.52
7.04 ꢀ 0.29
6.33 ꢀ 0.47
9.65 ꢀ 0.82
6.93 ꢀ 1.11
6.46 ꢀ 1.06
6.97 ꢀ 1.07
5.49 ꢀ 0.96
8.00 ꢀ 0.44
6.48 ꢀ 0.24
4.66 ꢀ 1.13
8.21 ꢀ 0.86
8.58 ꢀ 0.43
1.37 ꢀ 0.72
5.67 ꢀ 0.05
4.55 ꢀ 0.50
4.77 ꢀ 0.14
3.68 ꢀ 0.76
5.08 ꢀ 0.25
5.86 ꢀ 0.67
4.27 ꢀ 0.42
>100
>100
>100
>100
>100
>100
5.18
5.11
5.11
2.39
2.45
2.20
2.01
0.92
1.94
2.51
2.40
2.43
2.49
2.99
2.91
3.31
3.39
2.33
2.87
2.22
1.98
3.62
4.15
2.90
2.17
1.96
2.77
2.39
3.36
2.33
0.19
0.18
ꢁ0.06
ꢁ0.25
ꢁ1.34
ꢁ0.34
0.25
0.15
0.15
0.22
0.73
0.66
1.02
1.12
0.09
5.11/16.55/ꢁ0.48
5.11
5.10
5.12
5.13
5.09
5.10
5.12
5.12
5.09
5.10
5.13
5.14
5.12
5.11
5.13
5.12
5.16/5.11
5.09
81.18 ꢀ 6.11
>100
9
74.87 ꢀ 8.04
80.33 ꢀ 6.05
66.35 ꢀ 4.05
87.41 ꢀ 6.55
88.72 ꢀ 7.09
>100
>100
>100
>100
>100
>100
>100
>100
65.02 ꢀ 3.90
66.03 ꢀ 6.92
>100
>100
>100
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
tBHQ
DMSO
2,4-F
3-Cl
4-Cl
2,4-Cl
3,4-Cl
4-OCH3
4-OCH2CH3
2,4-OCH3
3,4,5-OCH3
2-Naphthyl
4-Phenyl
4-N(CH3)2
4-NO2
4-CN
0.63
ꢁ0.04
ꢁ0.29
1.37
1.89
0.67
ꢁ0.11
ꢁ0.31
0.50
0.13
1.09
5.11
5.11
5.12
5.11
2-Cl
2-OCH3
2-CF3
m
M)
>100
12.99/10.80
2.33
HepG2-ARE-C8 cells were treated with the compounds or tBHQ or DMSO for 12 h followed by luciferase assays. The ARE inducer activity was shown as a ratio to the DMSO
control. Predicted values of PKa, Log P, Log D7.4 were used ACDLABS PRO software.
is summarized in Scheme 1. Briefly,
a-pyrone 27 (4-hydroxy-6-
2.2.2. First round SAR and mechanism of a-pyrone derivatives
methyl-2-pyrone) was acetylated with acetic acid in the presence
of DCC, DMAP to obtain 28 in 86.4% yield. Addition of intermediate
28 with substituted aromatic aldehyde produced the target com-
pounds in about 62% yield.
For the preliminary SAR study (Scheme 2), five compounds (2e6)
with different groups were synthesized. They were tested at 20,
50 mM in their ability to elevate ARE levels in HepG2-ARE-C8 cells
(12 h treatments, Table 1). When the phenyl in compound 1 was
substituted by the furan (4) or pyrrole group (5), the activity was
decreased. The thienyl substituted compound (3) exhibited equal
potency as 1. The activity of these compounds was concentration
dependent. Interestingly, while substituted with 2-fluoro phenyl ring
2.2. Screening results
2.2.1. ARE inductivity
(2), the compound remarkably enhanced ARE activation at 20
compared to 1. However, the ARE inductivity decreased at 50
According to above results, 2 was used to do biological mechanism
study.
We determined the ARE activation by 2 at the concentration of
2.5, 5, 10, 20 and 30 mM, and no apparent cytotoxicity was observed.
mM
For the screening, we explored ARE inductivity of the compounds
mM.
using luciferase reporter gene assays at 20 and 50
plate in HepG2-ARE-C8 cells. Compound 1 with a
m
M on a 96-well
a-pyrone scaffold
was identified as a hit. It showed >4-fold induction of ARE compared
to the control group. The cytotoxic activity of the hit was determined
through the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazol-ium-
bromide (MTT) method in HCT116 cells (Table 1).
2 increased ARE inductivity in a concentration dependent manner
(Fig. 3).
We tested the expression of Nrf2 and phase II enzymes such as
HO-1 and NQO-1 after treatment with 2. The compound maximized
the expression of HO-1 and NQO-1 at 8 h and 24 h, respectively, while
induction of Nrf2 reached a stable state after 2 h (Fig. 4). As Nrf2 could
activate the expression of downstream phase II enzymes after nu-
clear translocation, we isolated nuclear and cytoplasmic fractions
after treatment with 2 and determined Nrf2 levels. Nrf2 nuclear
localization was first seen at 1 h, while decreasing at 24 h (Fig. 5).
The activities of several kinases, including MAPK, PI3K, PKC,
CK2, and PERK, are implicated in the regulation of Nrf2 activity.
Scheme 1. The synthetic route of compound 1e26.

M.-y. Xi et al. / European Journal of Medicinal Chemistry 66 (2013) 364e371
367
a Two rounds SAR study is shown above via totally synthesizing 25 compounds 2-26.
Scheme 2. The SAR of
a
-pyrone derivatives.a
It has been also shown that the kinases involved in phosphory-
lation and activation of Nrf2 appear to be cell type dependent [19].
To investigate whether some signal transduction is required for 2
to activate the ARE levels, 4 kinase inhibitors were chosen to
separately cotreat with 2 to during the determination of ARE
inductivity (Fig. 6). While cotreated with a MEK1/2 inhibitor
PD98059, the ARE inductive activity of 2 was blocked obviously
[25]. Therefore, subsequent experiments were implemented with
PD98059.
2.3. Second round SAR of
activation
a-pyrone derivatives that induce ARE
After the above mechanism research, we designed and synthe-
sized another 20 derivatives 7e26 to study the detailed SAR (Scheme
2) and identify a more potent ARE inducer. The IC50 value and ARE
inductivity of 7e26 are shown in Table 1. In general, the optimal
compound was 9 with an approximately 10-fold increase at 20
ARE levels as compared to control followed by 18, 17, 14 and 2.
mM in
Our study indicated that the compound 2 can affect the activity
of ERK1/2 signal transduction pathway by regulating phosphory-
lation process of ERK1/2. Exposure to the compound 2 resulted in a
rapid and sustained activation (phosphorylation) of ERK1/2. Acti-
vation of ERK1/2 was obvious as early as 30 min and maximized at
1 h. On the other hand, the expression of ERK1/2 protein was not
altered. The compound mediated activation of ERK1/2 was addi-
tionally confirmed by determining phosphorylation of the ERK1/2
downstream substrates (Fig. 7A).
For substituted groups on benzene ring, we firstly considered the
steric effect. With regard to naphthyl substitution (19), the inductivity
decreased sharply. This may due to the binding pocket was not large
enoughfor naphthylbinding. Thedataalsosuggested thatthebinding
pocket was a long and narrow area, because the biphenyl compound
(20) showed acceptable activity. The preferable binding mode may
need phenyl substituted with some spatially small groups, such as
halogen, methoxyl or hydroxyl group. As a result, we synthesized a
series of compounds with these substituted groups. According to the
biological results, the compounds with halogens (eF or eCl) and e
OCH3 substitution had preferable inductivity compared to other
compounds. The substituted position at the benzene ring was not key
element for the higher activity. Specially, it was worth paying atten-
tion to compounds with eF substitution (2, 7, 8, 9 and 10) because of
their strong ARE enhancement. The data also indicated that eF sub-
stitutionwas a privileged molecular optimization strategy to enhance
the Nrf2 inductive activity for these compounds. Furthermore, the
activity with di-halogens substitution (9,14) was prior to those single
substituted compounds.
Additionally, cotreatment with a MEK1/2 inhibitor, PD98059
(20
mM), blocked activation of ERK1/2 but not ERK1/2 itself,
significantly attenuated Nrf2, HO-1 and NQO-1 protein upregu-
lation with addition of 2 (Fig. 7B), suggesting that Nrf2 activation
partly attribute to transient activation of ERK1/2 by the com-
pound 2.
Based on immunofluorescence experiment, we obtained that
the compound 2 activated the transcription factor Nrf2 signaling
pathway 1 h later. Subsequently, Nrf2 was continuously enhanced
for 2 h. The data indicated an increased translocation of Nrf2 into
the nuclei after the treatment of 2. After blocking ERK1/2 with
PD98059 in 1 and 2 h respectively, we found that Nrf2 nuclear
translocation was decreased at both indicated times (Fig. 7C). These
For eOCH3 substitution, theactivitywasasfollows:tri-substitution
(18) > di-substitution (17) > single substitution (15, 25). The higher
activity of 15 vs 16 showed a potential steric hindrance at 4-position.
It also indicated that the steric effects of the substituted groups at the
benzene ring need to be carefully considered.
phenomena also demonstrated that the compound
2 which
exhibited Nrf2 inductivity was proceded by activating ERK1/2
signal pathway.
Fig. 3. The ARE inductivity of the compound 2. HepG2-ARE-C8 cells were treated with
the compounds or DMSO for 12 h. The activity was shown as a ratio to the DMSO
control (con). Data are expressed as mean ꢀ SD (n ¼ 3).
Fig. 4. Time-dependent expression of HO-1, NQO-1 and Nrf2 affected by the com-
pound 2. After treatment with 2 (20
mM), cell lysates were prepared from HCT116 cells
and subjected to Western blot analysis.

368
M.-y. Xi et al. / European Journal of Medicinal Chemistry 66 (2013) 364e371
Secondly, we considered the electrostatic effect of the substituted
groups. With eNO2 (6), eOCH3 (25) or eCF3 (26) substitution at
2-position and with eOCH3 (15), eNO2 (22) or eCN (23) substitution
at 4-position, the activity (at 20 mM) decreased dramatically, indi-
cating the electrostatic state of the substituted groups affected crit-
ically to the Nrf2 inductive activity. Additionally, electron-
withdrawing groups also showed toxicity at a high concentration,
which also explained the decrease of the activity. On the contrary,
electron-donating groups showed promising activity except for e
N(CH3)2 (21) substitution. This may be due to the steric effect. Be-
sides, electron-donating groups such as eOCH3 (15) and eN(CH3)2
(21) substitution showed much lower cytotoxicity, as the activity was
concentration dependent obviously.
In a word, 9 exhibited the highest ARE inductivity. 015314, 015317
and 015318 are also excellent ARE inducers. To identify physico-
chemical property, lipo-hydro partition coefficient was predicted.
High Log P value of 14 and low Log D7.4 value of 17, 18 was disad-
vantages to druggability. To improve it, some hydrogen bond ac-
ceptors or donors can be added in the following years. Thus, on the
basis of these data, we can select compound 9 for further analysis.
Fig. 5. Effects of 2 on nuclear translocation of Nrf2. After treatment with 2 (20 mM),
nuclear and cytoplasmic cell extracts were prepared from HCT116 cells and subjected
to western blot analysis for Nrf2, histone and
within 1 h and reached a plateau after 2 h.
b-actin. nuclear translocation initiated
3. Discussion
Expression of the Nrf2/ARE-induced enzymes by some chemical
molecules, which detoxify potential electrophiles and oxidants,
indicates a promising strategy for cancer chemoprevention. In this
point, many potent chemopreventive agents with various types of
structures have been identified [26]. In the present study, we
searched our in-house compound database for novel Nrf2/ARE
activators by using cytotoxity assay and luciferase measurement. A
Fig. 6. The activity of 2 (20
Cotreatment with inhibitors of either PI3K (LY294002, 10
20 M), P38 (SB203580, 5 M) or JNK (SP600125, 5 M) for 12 h, HepG2-ARE-C8 cells
were followed by luciferase assay. The activity was shown as a ratio to the DMSO
control (con). Data are expressed as mean ꢀ SD (n ¼ 3). Differences are statistically
significant at *p < 0.05, **p < 0.01.
m
M) is inhibited when the ERK1/2 pathway is blocked.
potent compound 1 with a-pyrone chemical skeleton was identi-
m
M), MEK1 (PD098059,
fied and five derivatives were synthesized for the first round SAR
study. These compounds were tested for their Nrf2/ARE induction
activity using HepG2-ARE-C8 cells. The potent compound 2, with e
F substituted at ortho-position, was much more potent than the
m
m
m
Fig. 7. Effects of 2 (20
western blot analysis for phosphorylated and total ERK1/2. (B) Cotreatment with the MEK1/2 inhibitor, PD98059 (20
phosphorylated ERK1/2, total ERK1/2, Nrf2, HO-1 and NQO-1. (C) Immunofluorescence staining of Nrf2 in indicated treatment in HCT116 cells. Nrf2 and nuclei was labeled with Cy3
and DAPI, respectively. Red: Nrf2; Blue: nuclei. 1 h, 2 h: treatment with 2 (20 M). Bars inserted indicate magnification (10 m). (For
M); 1 hþ, 2 hþ: cotreatment with PD98059 (20
interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
mM) on ERK1/2 signal transduction. (A) At indicated times after treatment with 2 (20
mM), cell lysates were prepared from HCT116 cells and subjected to
mM), HCT116 cells were subjected to western blot analysis for
m
m
m

M.-y. Xi et al. / European Journal of Medicinal Chemistry 66 (2013) 364e371
369
t
positive control BHQ. Meanwhile, the toxicity of the compounds
40.0 mmol) were added. The reaction mixture was stirred at 100 ꢂC
for 2 h, then the organic solution was evaporated. The crude
product was purified by using column chromatography
(PE:EtOAc ¼ 5:1) to afford 5.8 g 28 as light yellow solid. Yield:
86.4%.
was also evaluated by MTT method. The IC50 values of these
compounds were much higher than the concentration that led to
Nrf2/ARE induction, proving the safety of our compounds.
In the following mechanism study, we found that 2 up-regulates
the expression of NQO-1, HO-1 and promotes Nrf2 translocation
into the nuclei. NQO-1 plays an important role in protecting cells
against ROS [27]. HO-1 possesses anti-inflammatory, anti-oxidative
and anti-apoptotic activity [28,29]. The induction of the HO-1 and
NQO-1 gene is regulated mostly via the Nrf2/ARE pathway as it
includes a variety of ARE sequences in its promoter. Additionally,
the expression of Nrf2 can be induced through some signaling
pathways, such as mitogen-activated protein kinases (MAPKs) and
PI3K/Akt [25,30e33]. Based on these facts, we tested four upstream
kinases by cotreated 2 with the corresponding kinase inhibitor
respectively. We found the expression of Nrf2 and downstream
enzymes induced by 2 was mainly mediated by ERK1/2. Moreover,
covalent modification of the sulfhydryl of Keap1 is proposed as
another molecular mechanism in Nrf2/ARE activation. For example,
sulforaphane and CAPE are speculated to interact with the sulfhy-
dryl and activate the Nrf2/ARE pathway [34,35]. As an electrophilic
5.1.2. The general synthetic procedure of target compound 1e26
In a two-necked flask, the ortho-substituted benzaldehyde
(3.0 mmol), piperidine (2 d) and the intermediate 2 (3.0 mmol)
were dissolved in chloroform (4.0 mL), and the mixture was stirred
for reflux. After 12 h, the mixture was monitored by TLC using 20%
petroleum ether/ethyl acetate as the developing solvent system.
The reaction was stopped and then the mixture was evaporated and
crystallized with ethanol.
5.1.2.1. (E)-1-(3-Acetyl-4-hydroxy-6-methyl-2-pyrone)-3-(3,4-
difluoro-phenyl)-2-en-1-one (9). It was obtained as a yellow solid in
43.5% yield. 1H NMR (300 MHz, CDCl3):
d
2.30 (s, 3H), 5.98 (s, 1H),
7.12e7.51 (m, 3H), 7.82 (d, J ¼ 15.8 Hz, 1H), 8.22 (d, J ¼ 15.8 Hz, 1H);
13C NMR (CDCl3):
191.97, 182.46, 168.54, 160.65, 153.13, 151.87,
d
142.92, 125.34, 123.67, 117.49, 117.25, 116.88, 116.66, 101.72, 20.15.;
m/z (EI-MS): 292 [M]þ; HRMS (ESI): calcd. for C15H11F2O4 [M þ H]þ
293.062, found 293.0634. m.p. 160e161 ꢂC.
agent possessing a, b-unsaturated carbonyl groups, it is required to
testify whether 2 can interact with the sulfhydryl group in later
experiments. Twenty derivatives of 2 were further synthesized and
evaluated for a second round SAR study. Among them, 9 exhibits
much better potency in the induction of ARE activity than 2.
Further analysis can be developed around the promising com-
pound 9.
5.1.2.2. (E)-1-(3-Acetyl-4-hydroxy-6-methyl-2-pyrone)-3-(2,4-
difluoro-phenyl)-2-en-1-one (10). It was obtained as a yellow solid
in 51.2% yield. 1H NMR (300 MHz, CDCl3):
d 2.22 (s, 3H), 5.91 (s, 1H),
6.77e6.90 (m, 2H), 7.66e7.71 (m, 1H), 7.99 (d, J ¼ 15.9 Hz, 1H), 8.23
(d, J ¼ 15.9 Hz, 1H); m/z (EI-MS): 292 [M]þ; HRMS (ESI): calcd. for
C
15H11F2O4 [M þ H]þ 293.062, found 293.0632. m.p. 173e174 ꢂC.
4. Conclusion
We screened in-house database of our laboratory by luciferase
reporter gene assay aimed at identifying a novel small molecule
which is a potential activator in Nrf2/ARE pathway. 1 was chosen
as a hit. Structural modification and preliminary SAR investiga-
tion was in progress, as well as molecular mechanism. The results
presented herein support the hypothesis that 2 is a potent che-
mopreventive agent. Furthermore, up-regulation in the level of
phosphorylated ERK1/2 is shown to be associated with tran-
scription factor Nrf2 activation accompanied by HO-1, NQO-1
induction. By using the first round of SAR information in coop-
erate with biological assay to estimate the inductivity of second
round of synthesized compounds, we discovered the promising
compound 9, planning to optimize the SAR, study underlying
mechanism for chemoprevention and explore the target as a
chemopreventive agent. Accordingly, the compound 9 may pro-
tect cells against oxidative stress. We conclude that ARE activa-
tors described here may be valuable in preventing cells from
irritants.
5.1.2.3. (E)-1-(3-Acetyl-4-hydroxy-6-methyl-2-pyrone)-3-(2- naph-
thalene)-2-en-1-one (19). It was obtained as a yellow solid in 47.3%
yield. 1H NMR (300 MHz, CDCl3):
d 2.05 (s, 3H), 5.56 (s, 1H), 7.54e
8.01 (m, 7H), 8.07 (d, J ¼ 15.7 Hz, 1H), 8.36 (d, J ¼ 15.7 Hz, 1H); m/z
(EI-MS): 306 [M]þ; HRMS (ESI): calcd. for C19H15O4 [M þ H]þ
307.0965, found 307.0974. m.p. 176e178 ꢂC. The data of known
compounds were presented in the Supporting information.
5.2. Biology
5.2.1. Cell culture conditions
HepG2 cells stably transfected with ARE luciferase reporter
(HepG2-ARE-C8) were kindly provided by Professor Dr. A. N. Tony
Kong (Rutgers University, Piscataway, NJ). Cells were maintained in
modified RPMI-1640 medium (GiBco, USA) with 10% fetal bovine
serum (FBS) (GiBco, USA) in a humidified atmosphere of 5% CO2 and
95% air at 37 ꢂC. HCT116 cells (Cell Bank of Shanghai Institute of
Biochemistry and Cell Biology, Shanghai Institutes for Biological
Sciences, Chinese Academy of Sciences) were cultured in McCoy’s
5A (SigmaeAldrich, USA) supplemented with 10% (v/v) FBS.
5. Experimental procedures
5.1. Chemistry
5.2.2. MTT analysis
Cell viabilities were determined by using MTT assay. MTT was
purchased from Sigma (St. Louis, MO). It was dissolved in phos-
phate buffered saline (PBS) to a concentration of 5 mg/mL as the
stock solution and stored at ꢁ20 ꢂC. After cells were exposed with
All reagents were from commercial sources. With tetrame-
thylsilane (TMS) as internal standard, the 1H NMR was recorded on
Bruker AV-300 apparatus by using deuterated solvents. EI-MS was
collected on Shimadzu GCMS-2010 instruments. Every target
compound was purified via silica gel (60 A, 70e230 mesh) column
chromatography. Melting points were measured by XT-4 melting
point apparatus.
different concentrations of test compounds, 20 mL of MTT solution
ꢀ
was added into 96-well plates for 4 h. Then the solution was
removed and 100 L of DMSO was added into each well, The OD
m
values were determined at 570 nm by Elx800 Absorbance Micro-
plate Reader (BioTek, Vermont, USA).
5.1.1. 3-Acetyl-4-hydroxy-6-methyl-2-pyrone (28)
To a solution of 27 (5.0 g, 40.0 mmol) in toluene (60.0 mL),
DMAP (1.0 g, 8.0 mmol), acetic acid (2.4 g, 40.0 mmol), DCC (8.2 g,
IC50 ¼ ½1 ꢁ ðODtest ꢁ ODblankÞ=ðODcontrol ꢁ ODblankÞꢃ*100%:

370
M.-y. Xi et al. / European Journal of Medicinal Chemistry 66 (2013) 364e371
5.2.3. ARE-luciferase activity assay
Laboratory of Natural Medicines, China Pharmaceutical University
(No. JKGQ201103), 2012AA020301 of 863 program, 2010ZX09401-
401 and 2009ZX09501-003 of the National Major Science and
Technology Project of China (Innovation and Development of New
Drugs). The authors declare no other conflicts of interest.
HepG2-ARE-C8 cells were plated in 96-well plates at a density of
4 ꢄ 104 cells/well and incubated overnight. The cells were exposed
with different concentrations of test compounds, with tBHQ serving
as a positive control DMSO as a negative control and the luciferase
cell culture lysis reagent as a blank. In some experiments, cells were
cotreated with inhibitors of either MEK1 (PD098059, 20
m
M), PI3K
M) (Beyotime, China) or JNK
M) (SigmaeAldrich, USA). After 12 h of treatment,
L of cold PBS was added into
each well. Then the cells were harvested in the luciferase cell cul-
ture lysis reagent. After centrifugation, 20 L of the supernatant
Appendix A. Supplementary data
(LY294002, 10
(SP600125, 5
mM), P38 (SB203580, 5 m
m
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2013.06.007.
the medium was removed and 100
m
m
was used for determining the luciferase activity according to the
protocol provided by the manufacturer (Promega, Madison, WI).
The luciferase activity was measured by a luminoskan ascent
(Thermo scientific, USA). The data were obtained in triplicates and
expressed as fold induction over control.
References
[1] W.K. Hong, M.B. Sporn, Recent advances in chemoprevention of cancer, Science
278 (1997) 1073e1077.
[2] G.J. Kelloff, R. Lieberman, V.E. Steele, C.W. Boone, R.A. Lubet, L. Kopelovich,
W.A. Malone, J.A. crowell, H.R. Higley, C.C. Sigma, Agents, biomarkers, and
cohorts for chemopreventive agent development in prostate cancer, Urology
57 (2001) 46e51.
[3] T. Yamadori, Y. Ishii, S. Homma, Y. Morishima, K. Kurishima, K. Itoh,
M. Yamamoto, Y. Minami, M. Noguchi, N. Hizama, Molecular mechanisms for
the regulation of Nrf2-mediated cell proliferation in non-small-cell lung
cancers, Oncogene 31 (2012) 4768e4777.
Inductivity ¼ ðRLUtest ꢁ RLUblankÞ=ðRLUcontrol ꢁ RLUblankÞ: RLU
¼ relative light unit:
[4] S. Magesh, Y. Chen, L. Hu, Small molecule modulators of Keap1eNrf2eARE
pathway as potential preventive and therapeutic agents, Med. Res. Rev. 32
(2012) 687e726.
5.2.4. Western blot
[5] D.E. Corpet, F. Pierre, Point: from animal models to prevention of colon cancer.
Systematic review of chemoprevention in min mice and choice of the model
system, Cancer Epidemiol. Biomarkers Prev. 12 (2003) 391e400.
[6] B.S. Reddy, Studies with the azoxymethane-rat preclinical model for assessing
colon tumor development and chemoprevention, Environ. Mol. Mutagen. 44
(2004) 26e35.
[7] C. Neufert, C. Becker, M.F. Neurath, An inducible mouse model of colon
carcinogenesis for the analysis of sporadic and inflammation-driven tumor
progression, Nat. Protoc. 2 (2007) 1998e2004.
Anti-HO-1 (sc-136960) and -NQO1 (sc-271116) antibodies were
from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-b-actin
(AP0060) and -Nrf2 (BS1258) were purchased from Bioworlde
(Bioworlde, USA). Antibodies against p-erk1/2 (#4370) and erk1/2
(#4695) were obtained from Cell Signaling Technology (Beverly,
MA, USA). Cells were washed once with ice-cold PBS and driven
down with 2 mL of EDTA. Cells were centrifuged at 2500 rpm and
[8] C. Stolfi, A. Rizzo, E. Fraze, A. Rotondi, M.C. Fantini, M. Sarra, R. Caruso,
I. Monteleone, P. Sileri, L. Franceschilli, F. Caprioli, S. Ferrero, T.T. MacDonald,
F. Pallone, G. Monteleone, Involvement of interleukin-21 in the regulation of
colitis-associated colon cancer, J. Exp. Med. 208 (2011) 2279e2290.
[9] C.W. Barrett, B. Fingleton, A. Williams, W. Ning, M.A. Fischer,
M.K. Washington, R. Chaturvedi, K.T. Wilson, S.W. Hiebert, S.W. Hiebert,
C.S. Williams, MTGR1 is required for tumorigenesis in the murine AOM/DSS
colitis-associated carcinoma model, Cancer Res. 71 (2011) 1302e1312.
[10] T. Kakizoe, Chemoprevention of cancer-focusing on clinical trials, Jpn. J. Clin.
Oncol. 33 (2003) 421e442.
[11] C.R. Zhao, Z.H. Gao, X.J. Qu, Nrf2eARE signaling pathway and natural products
for cancer chemoprevention, Cancer Epidemiol. 34 (2010) 523e533.
[12] T. Ishii, K. Itoh, H. Sato, S. Bannai, Oxidative stress-inducible proteins in
macrophages, Free Radic. Res. 31 (1999) 351e355.
[13] M.K. Kwak, P.A. Eqner, P.M. Dolan, M. Ramos-Gomez, J.D. Groopman, K. Itoh,
M. Yamamoto, T.W. Kensler, Role of phase 2 enzyme induction in chemo-
protection by dithiolethiones, Mutat. Res. 305 (2001) 480e481.
[14] W.S. Jeong, M. Jun, A.N. Kong, Nrf2: a potential molecular target for cancer
chemoprevention by natural compounds, Antioxid. Redox Sign. 8 (2006)
99e106.
[15] H.K. Na, Y.J. Surh, Transcriptional regulation via cysteine thiol modification: a
novel molecular strategy for chemoprevention and cytoprotection, Mol. Car-
cinog. 45 (2006) 368e380.
[16] J.S. Lee, Y.J. Surh, Nrf2 as a novel molecular target for chemoprevention,
Cancer Lett. 224 (2005) 171e184.
[17] J.H. Wu, W. Miao, L.G. Hu, G. Batist, Identification and characterization of
novel Nrf2 inducers designed to target the intervening region of Keap1, Chem.
Biol. Drug Des. 75 (2010) 475e480.
[18] E. Balogun, M. Hoque, P. Gong, E. Killeen, C.J. Green, R. Foresti, J. Alam,
R. Motterlini, Curcumin activates the haem oxygenase-1 gene via regulation
of Nrf2 and the antioxidant-responsive element, Biochem. J. 371 (2003)
887e895.
resuspended in 45
mL of Lysis buffer, which was composed of
50 mM TriseHCl, 150 mM NaCl, NP-40, 1 mM EDTA, PMSF, NaF, Leu
and DTT for 1 h. Then cells were centrifuged again at 12,000 rpm for
20 min at 4 ꢂC. The supernatant was retained, and the protein
concentration was determined by the BCA assay with Varioskan
flash (Thermo, Waltham, MA) at 562 nm. Samples were stored
at ꢁ80 ꢂC until use. Nuclear and cytosol were isolated using the
nuclear-cytosol extraction kit (KeyGEN, NJ, China). Then the sepa-
rate extracts were stored at ꢁ80 ꢂC until use. The extracts were
separated by SDSePAGE and then transferred onto PVDF mem-
branes (Perkin Elmer, Northwalk, CT, USA). After blocking with 1%
BSA for 2 h, membranes were incubated at 37 ꢂC for 1 h and then at
4
ꢂC overnight with a primary antibody. After that, they were
reacted with a DyLight 800 labeled secondary antibody at 37 ꢂC for
1 h. The membranes were screened through the odyssey Infrared
Imaging System (LI-COR, Lincoln, Nebraska, USA).
5.2.5. Immunofluorescence
Cells were cotreated with the compound 2 (20
hibitors of MEK1 (PD098059, 20
mM) and in-
m
M), then incubated at 4 ꢂC
overnight with Nrf2 primary antibodies (abcam, UK). After washing
with PBS, cells were incubated at 37 ꢂC for 1 h with cy3-labeled
secondary sheep anti-rabbit IgG antibody (BOSTER, Wh, China).
Cells were then stained with fluorochrome dye DAPI (Santa Cruz
Biotechnology, Santa Cruz, CA) to visualize the nuclei and observed
under a laser scanning confocal microscope (Olympus Fluoview
FV1000, Japan) with a peak excitation wave length of 570 nm and
340 nm.
[19] W. Hur, Z. Sun, T. Jiang, D.E. Mason, E.C. Peters, D.D. Zhang, H. Luesch,
P.G. Schultz, N.S. Gray, A small-molecule inducer of the antioxidant response
element, Chem. Biol. 17 (2010) 537e547.
[20] J.D. Hayes, M. McMahon, NRF2 and KEAP1 mutations:permanent activation of
an adaptive response in cancer, Trends Biochem. Sci. 34 (2009) 176e188.
[21] I.M. Copple, C.E. Goldring, N.R. Kitteringham, B.K. Park, The Nrf2-Keap1
defence pathway: role in protection against drug-induced toxicity, Toxicology
246 (2008) 24e33.
Acknowledgments
[22] I. Berenblum, V. Armuth, Two independent aspects of tumor promotion,
Biochim. Biophys. Acta 651 (1981) 51e63.
[23] C. Heidelberger, A.E. Freeman, R.J. Pienta, A. Sivak, J.S. Bertram, B.C. Casto,
V.C. Dunkel, M.W. Francis, T. Kakunaga, J.B. Little, L.M. Schechtman, Cell
transformation by chemical agents-a review and analysis of the literature.
This work was supported by the project 81230078 (Key Pro-
gram), 81202463 (Youth Foundation) and 91129732 of National
Natural Science Foundation of China, Program of State Key

M.-y. Xi et al. / European Journal of Medicinal Chemistry 66 (2013) 364e371
371
A report of the U.S. Environmental Protection Agency Gene-Tox Program,
Mutat. Res. 114 (1983) 283e385.
[24] R. Hu, C.L. Saw, R. Yu, A.N. Kong, Regulation of NF-E2-related factor 2 signaling
for cancer chemoprevention antioxidant coupled with antiinflammatory,
Antioxid. Redox Signal. 13 (2010) 1679e1698.
[25] T.O. Khor, S. Yu, A.N. Kong, Dietary cancer chemopreventive agents e tar-
geting inflammation and Nrf2 signaling pathway, Planta. Med. 74 (2008)
1540e1547.
[26] R. Kachadourian, B.J. Day, S. Pugazhenti, C.C. Franklin, E. Genoux-Bastide,
G. Mahaffey, C. Gauthier, A. Di Pietro, A. Boumendjel, A synthetic chalcone as a
potent inducer of glutathione biosynthesis, J. Med. Chem. 55 (2012) 1382e1388.
[27] M.S. Yates, M. Tauchi, F. Katsuoka, K.C. Flanders, K.T. Liby, T. Honda,
G.W. Gribble, D.A. Johnson, J.A. Johnson, N.C. Burton, T.R. Guilarte,
M. Yamamoto, M.B. Sporn, T.W. Kensler, Pharmacodynamic characterization
of chemopreventive triterpenoids as exceptionally potent inducers of Nrf2-
regulated genes, Mole. Cancer Ther. 6 (2007) 154e162.
[28] A.K. Jaiswal, Regulation of genes encoding NAD(P)H: quinone oxidoreduc-
tases, Free Radic. Bio. Med. 29 (2000) 254e262.
[29] D. Willis, A.R. Moore, R. Frederick, D.A. Willoughby, Heme oxygenase: a novel
targetforthemodulationoftheinflammatoryresponse,Nat. Med.2(1996)87e93.
[30] A. Prawan, J.K. Kundu, Y.J. Surh, Molecular basis of heme oxygenase-1 induction:
implications for chemoprevention and chemoprotection, Antioxid. Redox Sign 7
(2005) 1688e1703.
[31] Y. Zhang, L. Guan, X. Wang, T. Wen, J. Xing, J. Zhao, Protection of chlorophyllin
against oxidative damage by inducing HO-1 and NQO1 expression mediated
by PI3K/Akt and Nrf2, Free Radic. Res. 42 (2008) 362e371.
[32] M.H. Li, Y.N. Cha, Y.J. Surh, Peroxynitrite induces HO-1 expression via PI3K/
Akt-dependent activation of NF-E2-related factor 2 in PC12 cells, Free Radic.
Biol. Med. 41 (2006) 1079e1091.
[33] A. Tanaka, N. Hamada, Y. Fujita, T. Itoh, Y. Nozawa, M. Iinuma, M. Ito, A novel
kavalactone derivative protects against H2O2-induced PC12 cell death via
Nrf2/ARE activation, Bioorg. Med. Chem. 18 (2010) 3133e3139.
[34] J.H. Lim, K.M. Kim, S.W. Kim, O. Hwang, H.J. Choi, Bromocriptine activates
NQO1 via Nrf2-PI3K/Akt signaling: novel cytoprotective mechanism against
oxidative damage, Pharmacol. Res. 57 (2008) 325e331.
[35] A.T. Dinkova-Kostova, W.D. Holtzclaw, R.N. Cole, K. Itoh, N. Wakabayashi,
Y. Katoh, M. Yamamoto, P. Talalay, Direct evidence that sulfhydryl groups of
Keap1 are the sensors regulating induction of phase 2 enzymes that protect
against carcinogens and oxidants, Proc. Natl. Acad. Sci. U. S. A. 99 (2002)
11908e11913.