Structure-Activity and Structure-Property Relationship and Exploratory in Vivo Evaluation of the Nanomolar Keap1-Nrf2 Protein-Protein Interaction Inhibitor

Article abstract of DOI:10.1021/acs.jmedchem.5b00185

Directly disrupting the Keap1-Nrf2 protein-protein interaction (PPI) is an effective way to activate Nrf2. Using the potent Keap1-Nrf2 PPI inhibitor that was reported by our group, we conducted a preliminary investigation of the structure-activity and structure-property relationships of the ring systems to improve the drug-like properties. Compound 18e, which bore p-acetamido substituents on the side chain phenyl rings, was the best choice for balancing PPI inhibition activity, physicochemical properties, and cellular Nrf2 activity. Cell-based experiments with 18e showed that the Keap1-Nrf2 PPI inhibitor can activate Nrf2 and induce the expression of Nrf2 downstream proteins in an Nrf2-dependent manner. An exploratory in vivo experiment was carried out to further evaluate the anti-inflammatory effects of 18e in a LPS-challenged mouse model. The primary results indicated that 18e could reduce the level of circulating pro-inflammatory cytokines induced by LPS and relieve the inflammatory response. (Chemical Equation Presented).

Full text of DOI:10.1021/acs.jmedchem.5b00185

Article
pubs.acs.org/jmc
Structure−Activity and Structure−Property Relationship and
Exploratory in Vivo Evaluation of the Nanomolar Keap1−Nrf2
Protein−Protein Interaction Inhibitor
Zheng-Yu Jiang,†,‡ Li−Li Xu,†,‡ Meng-Chen Lu,†,‡ Zhi-Yun Chen,†,‡ Zhen-Wei Yuan,†,‡ Xiao-Li Xu,†,‡,§
Xiao-Ke Guo,†,‡,§ Xiao-Jin Zhang,†,‡,∥ Hao-Peng Sun,*,†,‡,§ and Qi-Dong You*,†,‡
†State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, China
‡Jiang Su Key Laboratory of Drug Design and Optimization, China Pharmaceutical University, Nanjing 210009, China
§Department of Medicinal Chemistry, School of Pharmacy, China Pharmaceutical University, Nanjing 210009, China
∥Department of Organic Chemistry, School of Science, China Pharmaceutical University, Nanjing 210009, China
S
* Supporting Information
ABSTRACT: Directly disrupting the Keap1−Nrf2 protein−
protein interaction (PPI) is an effective way to activate Nrf2.
Using the potent Keap1−Nrf2 PPI inhibitor that was reported
by our group, we conducted a preliminary investigation of the
structure−activity and structure−property relationships of the
ring systems to improve the drug-like properties. Compound
18e, which bore p-acetamido substituents on the side chain
phenyl rings, was the best choice for balancing PPI inhibition
activity, physicochemical properties, and cellular Nrf2 activity.
Cell-based experiments with 18e showed that the Keap1−Nrf2
PPI inhibitor can activate Nrf2 and induce the expression of
Nrf2 downstream proteins in an Nrf2-dependent manner. An
exploratory in vivo experiment was carried out to further
evaluate the anti-inflammatory effects of 18e in a LPS-challenged
mouse model. The primary results indicated that 18e could reduce the level of circulating pro-inflammatory cytokines induced by
LPS and relieve the inflammatory response.
the dissociation of the Cul3−Keap1−Nrf2 complex and inhibit
the Keap1-dependent depression of Nrf2. Consequently, Nrf2
accumulates and Keap1−Nrf2−ARE pathway is activated. The
traditional electrophilic Nrf2 activators can mimic this
endogenous process of Nrf2 activation. The most successful
Nrf2 activator, dimethyl fumarate (DMF), has been approved
by the FDA for the treatment of patients with relapsing forms
of multiple sclerosis (MS) as a new first-line oral drug. The
phase 3 study of another promising candidate, CDDO-Me, was
terminated for safety reasons. The molecular mechanism of
these activators is covalent binding to the thiol of the cysteine.7
Thus, the risk of “off-target” effects may be associated with
these Nrf2 activators due to their potential to react with other
cysteine-containing proteins and enzymes.5
INTRODUCTION
■
Nrf2 (nuclear factor erythroid 2-related factor 2), a basic
leucine zipper (bZIP) transcription factor, is the primary master
of the inducible cell defense system. It can mediate the
transcription of a battery of cytoprotective genes, including
antioxidant proteins, phase I and II detoxification enzymes,
transport proteins, proteasome subunits, chaperones, growth
factors, and their receptors, as well as some transcription
factors.1−3 These cytoprotective genes all contain the enhancer
sequence ARE (antioxidant response element, 5′-
GTGACnnnGC-3′) in their promoter regulatory region,
which is the binding target of Nrf2. Modulators regulating
Nrf2 activity are of interest for their promising clinic
applications in treating inflammatory diseases.4−6
Recently, directly and competitively interrupting the Keap1−
Nrf2 protein−protein interaction has been shown to be an
alternative method for enhancing Nrf2 activity. Peptides that
mimic Nrf2 can disrupt the Keap−Nrf2 interaction.8 Addition-
ally, the ETGE motif of Nrf2 was conjugated to a cell-
Nrf2 is mainly regulated by Kelch-like ECH associated
protein 1 (Keap1), which is an adaptor component of CUL3-
based E3 ligase (Figure 1). Keap1 can mediate the
ubiquitination of Nrf2, which leads to its subsequent
degradation by proteasomes. The rapid turnover of Nrf2 can
sustain the low activity of Nrf2 under normal conditions. Under
stressed conditions, the cysteine residues in Keap1 can be
covalently modified by ROS or electrophiles, which can lead to
Received: February 1, 2015
© XXXX American Chemical Society
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Figure 1. Keap1−Nrf2 regulation model and mechanism of action of electrophilic Nrf2 activators.
penetrating peptide, which is a trans-activating transcriptional
activator (TAT) peptide derived from HIV, and this
conjugation resulted in a fused peptide. It was shown to
activate Nrf2 and its downstream target gene, heme-oxygenase-
1 (HO-1), in a dose-dependent manner in intact human THP-1
monocytes.9 However, inhibition with small molecules is a
more attractive proposal because of the pharmacological
advantages of small molecule drugs including enhanced stability
and oral bioavailability.10 Inspired by the successful develop-
ment of peptide inhibitors, two high throughput screenings for
small molecular PPI inhibitors of Nrf2−Keap1 have been
reported by different groups. Fluorescence polarization (FP)
based screening of the MLPCN library gave compound 1, with
an IC50 of 3 μM.11 The detailed binding mode and structure−
activity relationship (SAR) of compound 1 has also been
investigated.12 Homogeneous confocal fluorescence anisotropy
assay (two-dimensional fluorescence intensity distribution
analysis, 2D-FIDA) screening of an Evotec Lead Discovery
library screened out compounds 2 and 3, with an EC50 of 118
and 2.7 μM, respectively.13 In additional to high-throughput
screening, different groups have used a virtual screening
method to identify novel Keap1−Nrf2 PPI inhibitors. Our
group used a receptor−ligand binding model of Nrf2−Keap1 to
carry out a hierarchical structure-based virtual screening. We
successfully discovered compound 4, which can effectively
disrupt the Nrf2−Keap1 interaction with an in vitro EC50 of
9.80 μM in the FP assay.14 Compound 4 can enhance Nrf2
transcriptional activity in the cellular ARE-luciferase reporter
assay in a dose-dependent manner. In another study,15 the
docking method known as Schrodinger’s Glide was used to
discover three classes of novel inhibitors that are capable of
disrupting Keap1−Nrf2 (compound 5, 6, and 7). The most
potent compound had a KD2 of 2.9 μM in the fluorescence
anisotropy assay. These active hits had a Keap1 binding IC50 in
the sub-μM range, which is much less potent than the natural
substrate. Thus, the potency of these compounds on Nrf2−
ARE activation was limited. Recently, on the basis of a
molecular binding determinants analysis of Keap1, we
successfully designed and characterized the most potent PPI
inhibitor of Keap1−Nrf2, compound 8, with a KD value for
binding to Keap1 in the single-digit nanomolar (Figure 2).16
Compound 8, containing two aliphatic carboxylic acid side
chains, can mimic Glu79 and Glu82 in the Nrf2 ETGE motif.
This result suggests that the proper occupation of the polar
subpockets, P1 and P2, should be addressed in the rational
design of the PPI inhibitors of Keap1. Until now, the direct PPI
inhibitors of Keap1−Nrf2 have been effective only at the
protein and cellular level. Whether these inhibitors can exert
their effects in vivo has not been validated.
In this study, on the basis of the potent inhibitor 8
discovered by our group, the structure−activity and structure−
property relationships were investigated. The optimized
compound 18e showed a similar inhibition activity in the in
vitro FP assay, but significant advantages in physicochemical
properties, especially solubility. The cell-based ARE luciferase
reporter assay also showed that 18e has a better Nrf2-inducing
B
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Figure 2. Chemical structures of Keap1−Nrf2 PPI inhibitors.
a
Scheme 1. Synthesis of Compounds with a Phenyl Ring As the Scaffold
a
Reagents and conditions: (a) Pd/C, H2, rt, 4 h; (b) substituted benzenesulfonyl chloride, THF, pyridine, 40 °C, 4 h, 54%; (c) DMF, K2CO3, ethyl
bromoacetate, rt, 3 h, 74%; (d) R3 = ethyl, NaOH, MeOH, H2O, 65 °C, 2 h, 71%; (e) R3 = methyl, LiOH, MeOH, H2O, RT, overnight. The yields
were shown using 12a as an example.
activity. This compound was subsequently used for the
exploratory in vivo evaluation of the anti-inflammatory effects
in the LPS (lipopolysaccharide) challenge model. Pretreat-
ments with 18e markedly changed the overall response profiles
induced by LPS, especially for the circulating pro-inflammatory
cytokines. These results suggested that Keap1−Nrf2 PPI
inhibitor pretreatment can confer protective effects against
LPS challenge and relieve the overall inflammatory response.
This result encourages the further evaluation of the therapeutic
effects on inflammatory diseases.
to evaluate the physicochemical properties of 8 in the present
study. The results showed that 8 is of low solubility (388 μg/
mL), which may restrict the accurate assessment of in vivo
effects. Thus, the subsequent research program mainly focused
on the investigation of the SAR and the structure−property
relationship (SPR) of 8.
According to previous research, the N-acetic acid substituted
sulfonamide groups of 8 play an important role in Keap1
binding. These groups can form multiple polar interactions,
including hydrogen bonds and salt bridges with polar residues
in the P1 and P2 subpockets of Keap1. Thus, we mainly
focused on the aromatic rings of 8 to improve its
physicochemical properties.
Our initial task was to evaluate the requirement of
naphthalene for activity because the planarity and hydro-
phobicity of the core naphthalene ring may restrict the
solubility. Simplifying the naphthalene to a phenyl ring resulted
in 12a (Scheme 1). The FP results showed that such
simplification led to a dramatic decrease in activity, which
RESULTS AND DISCUSSIONS
■
The Core Naphthalene Ring Is the Key Feature for
Nrf2−Keap1 PPI Inhibitor. Our previously reported Nrf2−
Keap1 PPI inhibitor 8 had a good Keap1 binding affinity (9.91
nM) and displayed potent inhibition effects in the in vitro
Keap1−Nrf2 FP assay (28.6 nM). It can also significantly
elevate Nrf2 activity in cell-based experiments. With the aim of
identifying a Keap1−Nrf2 inhibitor active in vivo, we continued
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Figure 3. Docking study of the binding mode differences between 8 and 12a. (A) Structure analysis of 8. (B,C) Binding mode of 8 and 12a from
docking. The hydrogen bonds are represented as green dashed lines, and the electrostatic interactions are represented as yellow dashed lines. The
carbon atoms of the ligands and Keap1 residues are colored cyan and purple, respectively.
was unacceptable for PPI inhibition. To fully confirm this
result, 12b with 4-methyl substituents was synthesized, and the
experimental results were similar to those obtained for 12a.
We further investigated the binding mode differences
between 8 and 12a using a docking method. As shown in
Figure 3, the two compounds did not show great differences in
binding to Keap1. The interacting pattern analysis gave two
possible reasons for the decrease in activity: one possibility is
that the phenyl ring replacement of the naphthalene reduced
the hydrophobic interactions inner the subpocket P3. Ring B of
the naphthalene can form hydrophobic interactions with the
side carbon chain of Arg415 and the side chain of Ala556. The
other possibility could be that the removal of one phenyl ring
reduced the π system and the polarizability of the aromatic ring.
It may weaken the cation−π interaction between the guanidyl
group in Arg415 and the core structure of the ligand.
Therefore, 12a was chosen as the template and the hydro-
phobic substituents were added to the core phenyl ring to
investigate whether the complement of the hydrophobic
interactions could compensate for the decrease in activity
(12c−12e). Only 12e with 2-methoxyl substituents showed a
slight improvement in activity. These results indicated that the
aliphatic hydrophobic groups cannot replace the core
naphthalene ring. The planar aromatic ring with an extended
π system is the key pharmacophore of the Keap1−Nrf2 PPI
inhibitor, which should be retained for activity.
Substituent Transformation on the Side Chain Phenyl
Ring Is Tolerable for Keap1−Nrf2 PPI Inhibition Activity.
Considering that the two N-acetic acid substituted sulfonamide
groups play an important role in Keap1 binding,17 the
substituents on the phenyl groups (ring B) of 8 were chosen
as the next optimization sites. First, we compared the
substituent position effects of the core naphthalene. The 1,5-
substituted analogue, 15, was synthesized. The FP experiment
gave a negative result (IC50 = 1.86 μM), indicating the necessity
of the 1,4-substitued core naphthalene (Table 1).
Then, we systematically evaluated the effects of different
substituents on PPI inhibition activity. Generally, ring B can
tolerate a variety of substituents (Table 2). The electron-
withdrawing substituents are not preferred for Keap1−Nrf2 PPI
inhibition activity, whereas the electron-donating groups can
exhibit better performance. Among the electron-donating
groups, the substituents with steric bulk are not appropriate.
Both 18f with 4-tertiary-butyl substitutes and 18j containing
naphthalene sulfonyl fragments are less active (Scheme 2). The
2,4,6-trimethyl substituted compound 18g showed a dramatic
decrease in activity, indicating that the ortho position was
Table 1. IC50 of Compounds Bearing the Phenyl Ring
Scaffold for the Inhibition of Keap1 Kelch Domain−Nrf2
a
ETGE Interaction
compd
R1
R2
IC50 (nM) 95% CI
8
30.8
27.6−34.4
1448
12a
12b
12c
12d
12e
H
H
4-OCH3
4-CH3
1325−1671
966
900−1036
1666
2-CH3
4-OCH3
4-OCH3
4-OCH3
1262−2620
a
2,5-CH3
2-OCH3
10.17
a
8.46−12.24
602
477−759
a
The unit is μM.
sensitive to steric hindrance. The potency of 3-methoxyl
substituted compound 18l was lower, thus further confirming
that the electron-donating groups were preferable in the para
position. The p-methyl substituent was slightly more potent
than p-methoxyl. Interestingly, the phenyl ring without any
substituent also exhibited high potency, which indicated that
the electron-donating group was not the determinant for
activity. Although the overall performance of electron-with-
drawing was less potent, the p-acetamido substitutes can give
excellent performance.
Substituent Transformation on the Ring Has Dramat-
ic Effects on Solubility and Cellular Nrf2 Inducing
Activity. After the SAR study of the substitutes on ring B,
the physicochemical properties were determined for further
analysis of SPR. Unlike the SAR results, the electron-
withdrawing and halogen substituents showed better results
than the electron-donating groups. The electron-donating
groups, especially those hydrophobic groups without polar
atoms, decreased the solubility significantly. The electron-
withdrawing substituents can improve the solubility. Halogen
substituents had dramatic differences in solubility: F- and Cl-
were beneficial for solubility, whereas Br- was disadvantageous.
Attractively, 18e with the p-acetamido substitutes also showed
advantages in solubility.
The cellular activities of these compounds were further
evaluated using the ARE-luciferase reporter assay in HepG2−
ARE−C8 cells. 18e with p-acetamido substitutes, which is
optimal in both PPI inhibition activity and solubility, gave the
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Table 2. IC50, Physicochemical Properties, and ARE Induction Fold Results of the Compounds Containing Various Substituents
a
on Ring B
induction fold
compd
R1
IC50 (nM) 95% CI
solubility
log D, pH 7.4
0.1 μM
1 μM
5 μM
8
4-OCH3
30.8
27.6−34.4
18.1
388 μg/mL
1.71
1.79 0.43
3.49 0.09
5.93 1.27
18a
18b
18c
18d
18e
18f
4-CH3
248 μg/mL
3.8 mg/mL
1.2 mg/mL
5.7 μg/mL
5.0 mg/mL
1.6 μg/mL
20.0 μg/mL
183 μg/mL
12.3 μg/mL
5.0 μg/mL
12.3 μg/mL
16.0 μg/mL
1.1 mg/mL
6.0 ng/mL
0.93
1.16
2.11
4.29
1.02
5.2
1.49 0.21
1.42 0.14
1.66 0.07
1.83 0.55
2.81 0.67
1.19 0.15
1.51 0.19
1.47 0.08
1.76 0.11
1.92 0.02
1.60 0.02
1.88 0.66
1.59 0.50
1.81 0.03
3.19 0.24
1.79 0.17
1.99 0.09
2.16 0.48
6.69 1.56
1.51 0.23
1.67 0.14
1.67 0.14
1.89 0.55
2.57 0.12
1.69 0.01
2.22 0.02
1.40 0.10
1.77 0.43
5.51 0.18
3.19 0.40
2.52 0.91
2.40 0.10
10.61 1.21
2.18 0.31
1.99 0.08
6.25 0.82
1.45 0.11
4.46 0.49
2.03 0.40
3.99 1.02
2.79 0.19
2.58 0.28
15.0−21.8
54.3
4-F
49.9−59.1
42.3
4-Cl
34.8−51.4
36.5
4-Br
29.7−44.8
14.4
4-acetamido
4-C(CH3)3
2,4,6-trimethyl
H
12.4−16.7
50.2
42.8−58.9
979
18g
18h
18i
3.44
0.45
0.93
3.9
672−1425
34.3
30.3−38.9
179
4-OCF3
5,6-C4H4
4-CF3
144−222
47.4
18j
37.9−59.2
122
18k
18l
2.81
0.89
0.65
1.89
98.1−151
66.1
3-OCH3
4-CN
55.9−78.3
118
18m
18n
84.0−165
42.2
4-n-butyl
37.5−47.4
a
Induction fold was shown as a ratio to the DMSO control, and data are presented as mean SEM of three separate experiments.
a
Scheme 2. Synthesis of Compounds with Various Substitutes on Ring B
a
Reagents and conditions: (a) NH2OH·HCl, 95% ethanol, MeOH, 60 °C, 2 h; (b) Pd/C, H2, rt, 4 h; (c) 4-methoxybenzenesulfonyl chloride,
toluene, pyridine, 100 °C, 2 h, 67%; (d) DMF, K2CO3, ethyl bromoacetate, rt, 3 h, 58%; (e) R2 = ethyl, NaOH, MeOH, H2O, 65°, 2 h, 64%; (f) R2 =
methyl, LiOH, MeOH, H2O, RT, overnight; (g) R2 = benzyl, Pd/C, H2, 40 °C, 6 h. The yields were shown using 18a as an example.
best performance among these compounds. Therefore, it was
chosen for additional cell-based and in vivo evaluation.
Although the electron-donating hydrophobic substituted
analogues (18f and 18n) exhibited potent PPI inhibition
activity, their low solubility restricts their cellular potency in
Nrf2−ARE activation. Only these compounds with proper in
vitro Keap1−Nrf2 PPI inhibition activity and acceptable
solubility simultaneously can be potent Nrf2−ARE activators.
The apparent shortcoming on either sides would abolish the
cellular activity. We also further evaluated the ligand efficiency
(LE), ligand efficiency dependent lipophilicity (LELP), and
lipophilic efficiency (LipE) of these compounds (detailed
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results can be found in the Supporting Information). Overall,
the LE did not correlate with the cellular activity, whereas
LELP and LipE were closely related to the Nrf2−ARE inducing
activity. The compounds with low LELP and high LipE were
more potent in the cellular assay. These results indicated that
affinity changes depend on the molecule that interact directly
with the protein, and enthalpy-driven binding profiles18 are
more advantageous for Nrf2−ARE inducing activity, which can
satisfy the expectation for the molecular optimization of
Keap1−Nrf2 PPI inhibitor.
The Elevation of the Nrf2-Regulated Enzymes
Induced by 18e Depends on Nrf2. An Nrf2 knockdown
strategy was used to further confirm that 18e activates Nrf2
downstream genes through Nrf2. HCT116 cells were treated
with Nrf2 siRNA, 18e, or Nrf2 siRNA plus 18e. The untreated
HCT116 cells were used as the blank control. Nrf2 siRNA
induced a significant inhibition of the mRNA level of Nrf2
together with its transcription target genes NQO1, GCLM, and
HO-1 (Figure 5). The induction of HO-1, GCLM, and NQO-1
Keap1−Nrf2 PPI Inhibitor Can Elevate the Protein
Level of Nrf2-Regulated Genes. We further examined the
concentration-dependent effect of 18e on the protein level of
Nrf2-driven genes including heme oxygenase-1 (HO-1),
NAD(P)H/quinone oxidoreductase (NQO1), and γ-glutamyl-
cysteine synthetase (γ-GCS). As shown in Figure 4A, 18e can
Figure 5. Expression of Nrf2 and Nrf2-regulated genes after treatment
with Nrf2 siRNA and 18e. HCT116 cells were treated with Nrf2
siRNA (50 nM), 18e (20 μM), or Nrf2 siRNA (50 nM) plus 18e (20
μM). The expression of Nrf2, GCLM, NQO-1, and HO-1 genes were
quantified using qRT-PCR. The values shown are the mean SD (n =
3). ***p < 0.001.
by 18e sharply decreased as a result of Nrf2 knockdown. The
addition of compound 18e into the Nrf2 siRNA group only up-
regulated these genes to the normal level, similar to the blank
control. This result is consistent with the well-known Nrf2
activator sulforaphane.19 These results indicated that 18e can
elevate the expression of Nrf2 downstream genes in an Nrf2-
dependent manner.
Keap1−Nrf2 PPI Inhibitor Can Relieve Mouse Inflam-
matory Responses Induced by LPS Challenge. After the
validation of the potency of 18e in the cell-based experiments,
the in vivo anti-inflammatory effects of 18e in the context of the
LPS challenge was evaluated. LPS can lead to both MyD88-
dependent early phase NF-κB transcription of pro-inflamma-
tory cytokines, such as TNF-α, and IL-6 and MyD88-
independent, late phase NF-κB transcription of IFN-β.20 It
can also promote inflammation by inducing the production of
reactive oxygen species.21 It is commonly used to induce the
inflammatory response, which has been successfully used in the
Nrf2 activator.22 The production of pro-inflammatory cytokines
is a critical marker for inflammation, and these inflammatory
cytokines are an important cause of the inflammatory damage.
Thus, the circulating inflammatory cytokines were chosen as
the main marker for monitoring anti-inflammatory effects.
C57BL/6 mice were pretreated with 18e (low dose 10 mg/kg/
day or high dose 80 mg/kg/day) for 3 days (day −3, −2, −1)
and then challenged with LPS (300 μg IP) 24 h after the last
dose of compound (day 0). The untreated group received
saline (day −3, −2, −1) and then the LPS challenge (day 0).
The blank group only received saline during the experiment.
Dexamethasone, the widely used steroid anti-inflammatory
drug, was used as positive control (10 mg/kg/day, the same
procedure as 18e). Sera were collected from all groups 5 h post-
LPS challenge. The level of the cytokines in sera was measured
using ELISA.
Figure 4. (A) Dose-dependent increase of Nrf2-regulated proteins
after treatment with compound 18e. HCT116 cells were treated with
18e at different concentrations (1−20 μM). HO-1, NQO1, and γ-GCS
were determined using β-actin as the loading control after 6 h of
treatment with 18e. (B) Time course study of Nrf2-regulated proteins
after treatment with 18e (20 μM) at various time points. NQO1, HO-
1, and γ-GCS were determined using β-actin as loading control.
dose dependently increase the protein level of HO-1 and γ-
GCS in the HCT116 cells. The elevation of NQO-1 was not so
obvious. The basal level of NQO-1 in HCT116 cells was high,
which was consistent with previously published results using
HCT116 cells.16 For the time course studies, the compound
maximized the expression of HO-1, NQO1, and GCS at 16−24
h, respectively (Figure 4B). The result was consistent with the
result for 8 reported previously and showed a distinct behavior
toward electrophilic Nrf2 activators.14,16 These data further
confirmed that 18e can elevate the downstream proteins of
Nrf2 at the cellular level.
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Figure 6. Quantification of various inflammatory cytokines, including IL-6, Il-12, IL-17, TNF-α, and IFN-γ, in the serum of C57BL/6 female mice
after 5 h of LPS challenge. Dexamethasone (10 mg/kg) was used as the positive control. The data were shown as the mean SEM, *p < 0.05, **p <
0.01, ***p < 0.001, ****p < 0.0001, indicating the statistically significant difference from the untreated blank control group.
During the pretreatment of 18e, no acute toxicity was
observed and the body weights of the mice did not show
significant changes (the detailed body weight data can be found
in Supporting Information). As shown in Figure 6, both high
dose and low dose compound 18e can significantly and dose
dependently reduce the levels of pro-inflammatory cytokines,
including TNF-α and IFN-γ, as well as IL-6, IL-12, and IL-17,
relative to LPS-challenged mice. Furthermore, 18e had a more
potent inhibitory activity against the production of IL-6, IL-17,
and IFN-γ and comparable effects on IL-12 and TNF-α at the
same concentration as the positive control dexamethasone (10
mg/kg/day). In general, these results suggested that 18e
pretreatment can reduce inflammatory cytokines and confer
protection against LPS challenge. Thus, 18e potentially
possesses promising anti-inflammatory effects in vivo and
should be further evaluated in LPS-mediated inflammatory
disease models.
ring systems to improve the physicochemical properties,
especially the solubility. The key structural features for Keap1
binding were retained, and the feasible optimization sites on the
side chain phenyl ring were identified. We systematically
evaluated the effects of different substituents on PPI inhibition
activity, physicochemical properties, and cellular Nrf2 activity.
18e with p-acetamido substituents was the best choice for
balancing all of these aspects and was used for further
investigations. Cell-based experiments using 18e showed that
the Keap1−Nrf2 PPI inhibitor can activate Nrf2 and induce the
expression of Nrf2 downstream protein in an Nrf2-dependent
manner. Encouraged by these results, an exploratory in vivo
experiment was carried out to further confirm the anti-
inflammatory effects of 18e in the LPS-challenged mouse
model. The primary results indicated that 18e can reduce the
circulating pro-inflammatory cytokines induced by LPS and
relieve the inflammatory responses. This pioneering work
advances the target validation of Keap1−Nrf2 PPI using small
molecular agents.
CONCLUSIONS
■
The Keap1−Nrf2−ARE pathway protects humans from
chemical and oxidative insults by regulating the expression of
various cytoprotective proteins. It plays an important role in the
homeostatic regulation of the microenvironment. The
dysregulation of the microenvironment, for example, an
elevated oxidative state, is closely related to inflammatory
disease. The development of anti-inflammatory agents has not
made much progress in recent years. Nrf2 activation is an
innovative strategy to treat inflammatory diseases through
enhancing the innate defense mechanism.
MATERIALS AND METHODS
■
1. Chemistry. Reactions were monitored by thin-layer chromatog-
raphy (TLC) on 0.25 mm silica gel plates (GF254) and visualized
under UV light. Melting points were determined with a Melt-Temp II
1
apparatus. The H NMR and 13C NMR spectra were measured on a
Bruker AV-300 instrument using deuterated solvents with tetrame-
thylsilane (TMS) as the internal standard. ESI-mass and high-
resolution mass spectra (HRMS) were recorded on a Water Q-Tof
micro mass spectrometer. The purity (≥95%) of the compounds was
verified by HPLC performed on an Agilent C18 (4.6 mm × 150 mm,
3.5 μm) column using a mixture of solvent methanol/water 70:30 with
1‰ TFA at the flow rate of 0.5 mL/min and peak detection at 254
nm.
On the basis of the potent Keap1−Nrf2 PPI inhibitor
reported by our group previously, we investigated the
structure−activity and structure−property relationships of the
G
DOI: 10.1021/acs.jmedchem.5b00185
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Target compounds 12a−e were synthesized using similar
procedures. The detailed procedures for the synthesis of 12a are
listed as an example; the others can be found in the Supporting
Information.
δ) 169.876, 162.728, 138.040, 136.451, 131.634, 129.850, 129.712,
114.154, 55.673, 52.164, 17.304. HRMS (ESI): found 615.1066
(C26H28N2NaO10S2, [M + Na]+, requires 615.1078). HPLC (70:30
methanol:water with 1‰ TFA): tR = 5.9 min, 98.3%.
N,N′-(1,4-Phenylene)bis(4-methoxybenzenesulfonamide) (10a).
4-Methoxybenzenesulfonyl chloride (3.75 g, 22 mmol) and pyridine
(2.37 g, 30 mmol) was added to the solution of THF (20 mL) and 1,4-
diaminobenzene (1.08 g, 10 mmol). The reaction mixture was stirred
at 40 °C for 2 h. After the completion of the reaction monitored by
TLC, the solvent was removed and 20 mL of 1 M hydrochloric acid
was added, resulting in a lot of precipitation. After filtration, the solid
was collected and washed with water. Recrystallization from 95%
2,2′-((2-Methoxy-1,4-phenylene)bis(((4-methoxyphenyl)sulfonyl)-
azanediyl))diacetic Acid (12e). The same procedure as 12c, yield
1
81%; mp 247−248 °C. H NMR (300 MHz, DMSO, δ) 12.78 (br,
2H), 7.60 (d, 2H, J = 8.43 Hz), 7.48 (d, 2H, J = 8.67 Hz), 7.31 (d,1H,
J = 8.25 Hz), 7.10−7.02 (m,4H), 6.80 (d, 1H, J = 8.28 Hz), 6.59 (s,
1H), 4.40 (s, 2H), 4.19 (s, 2H), 3.84 (s, 6H), 3.17 (s, 3H). 13C NMR
(75 MHz, DMSO, δ) 170.322, 169.983, 162.759, 162.466, 155.316,
141.011, 133.114, 131.138, 129.735, 129.621, 129.165, 125.327,
119.149, 114.239, 113.832, 110.920, 55.681, 55.631, 55.164, 51.752,
50.640. HRMS (ESI): found 612.1309 (C25H30N3O11S2, [M + NH4]+,
requires 612.1316). HPLC (70:30 methanol:water with 1‰ TFA):
tR= 5.1 min, 95.4%.
1
ethanol gave the white solid, yield 54%; mp 225−227 °C. H NMR
(300 MHz, DMSO, δ) 9.90 (s, 2H), 7.58−7.51 (m, 4H), 7.01−6.97
(m, 4H), 6.94−6.84 (m, 4H), 3.74 (s, 6H). HRMS (ESI): found
471.0687 (C20H20N2NaO6S2, [M + Na]+, requires 471.0655).
Target compounds 18a−o were synthesized according to the
previous reported method16 for synthesis of 8. Detailed procedures of
18a are listed as an example; the others can be found in the Supporting
Information.
Diethyl 2,2′-(1,4-Phenylenebis(((4-methoxyphenyl)sulfonyl)-
azanediyl))diacetate (11a). To a solution of 10a (896 mg, 2
mmol) in DMF (5 mL) was added K2CO3 (830 mg, 6 mmol) followed
by ethyl bromoacetate (836 mg, 5 mmol). After 3 h stirring at room
temperature, the reaction mixture was then diluted in 30 mL of water
and quenched with 2 M hydrochloric acid to pH 5. The crude product
was obtained through filtration. Recrystallization from ethyl acetate/n-
hexane gave the 11a as a light-pink solid, yield 74%; mp 139−141 °C.
1H NMR (300 MHz, DMSO, δ) 7.55 (d, 4H, J = 8.82 Hz), 7.11−6.99
N,N′-(Naphthalene-1,4-diyl)bis(4-methylbenzenesulfonamide)
(16a). To a solution of 4-nitronaphthalen-1-amine (1.81 g, 10 mmol)
in THF was added Pd/C. The reaction mixture was stirred under
hydrogen for 4 h. The solution was filtered to remove the catalyst. The
filtrate was concentrated under reduced pressure to give the light-
yellow oil. The crude product, naphthalene-1,4-diamine, was used
without further purification. 4-Methylbenzenesulfonyl chloride (4.54g,
22 mmol) and pyridine (2.37g, 30 mmol) was added to the solution of
toluene (20 mL) and naphthalene-1,4-diamine. The reaction mixture
was stirred at 100 °C for 2 h under nitrogen. After cooling to room
temperature, reaction mixture was then diluted in 20 mL of petroleum
ether. After filtration, the solid was collected and washed with 1 M
hydrochloric acid. Recrystallization from acetonitrile gave the gray
(m, 8H), 4.46 (s, 4H), 4.06 (q, 4H, J = 7.08 Hz), 3.85 (s, 6H), 1.11(t,
6H, J = 7.08 Hz). HRMS (ESI): found 643.1408 (C28H32N2NaO10S2,
[M + Na]+, requires 643.1391).
2,2′-(1,4-Phenylenebis(((4-methoxyphenyl)sulfonyl)azanediyl))-
diacetic Acid (12a). To a solution of NaOH (2 g) in MeOH/H2O
(10/10 mL) was added 11a (545 mg, 1 mmol). The reaction mixture
was heated in an oil bath maintained at 65 °C for 2 h, then quenched
with 2 M hydrochloric acid to pH 2 and diluted with 75 mL water.
Precipitate removed by filtration and washed with 5 × 10 mL water,
then dried overnight in a vacuum desiccator, yielding 12a as a white
1
solid, yield 67%; mp 252−254 °C. H NMR (300 MHz, DMSO, δ)
10.10 (s, 2H), 7.98−7.95 (m, 2H), 7.53−7.50 (m, 4H), 7.41−7.39 (m,
2H), 7.29−7.26 (m, 4H), 6.99 (s, 2H), 2.32 (s, 6H). HRMS (ESI):
found 489.0943 (C24H22N2NaO4S2, [M + NH4]+, requires 489.0913).
Diethyl 2,2′-(Naphthalene-1,4-diylbis(tosylazanediyl))diacetate
(17a). To a solution of 16a (932 mg, 2 mmol) in DMF (5 mL)
was added K2CO3 (830 mg, 6 mmol), followed by ethyl bromoacetate
(836 mg, 5 mmol). After 3 h stirring at room temperature, the reaction
mixture was then diluted in 30 mL of water and quenched with 2 M
hydrochloric acid to pH 5. The crude product was obtained through
filtration. Recrystallization from ethyl acetate/n-hexane gave the 17a as
1
solid, yield 71%; mp 232−234 °C. H NMR (300 MHz, DMSO, δ)
12.89 (br, 2H), 7.53 (d, 4H, J = 8.91 Hz), 7.14−7.04 (m, 8H), 4.36 (s,
4H), 3.84 (s, 6H). 13C NMR (75 MHz, DMSO, δ) 169.870, 162.667,
138.552, 129.647, 129.433, 127.674, 114.249, 55.652, 51.659. HRMS
(ESI): found 587.0774 (C24H24N2NaO10S2, [M + Na]+, requires
587.0765). HPLC (70:30 methanol:water with 1‰ TFA): tR = 5.1
min, 99.3%.
2,2′-(1,4-Phenylenebis(tosylazanediyl))diacetic Acid (12b). Light-
pink solid, yield 68%; mp 222−224 °C. 1H NMR (300 MHz, DMSO,
δ) 7.47 (d, 4H, J = 7.92 Hz), 7.35 (d, 4H, J = 7.50 Hz), 7.07 (s, 4H),
4.33 (s, 4H), 2.38 (s, 6H). 13C NMR (75 MHz, DMSO, δ) 169.711,
143.667, 140.182, 135.209, 129.604, 129.193, 127.211, 126.760,
126.027, 51.770, 20.992. HRMS (ESI): found 550.1333
(C24H28N3O8S2, [M + NH4]+, requires 550.1312). HPLC (70:30
methanol:water with 1‰ TFA): tR = 6.8 min, 96.2%.
1
a light-pink solid, yield 54%; mp 138−140 °C. H NMR (300 MHz,
DMSO, δ) 8.31−8.29 (m, 1H), 8.17−8.16 (m, 1H), 7.59−7.54 (m,
6H), 7.45−7.42 (m, 2H), 7.37−7.35 (m, 2H), 7.08 (s, 1H), 6.83 (s,
1H), 4.59−4.53 (m, 4H), 4.05−3.95 (m, 4H), 2.46(s, 3H), 2.39 (s,
3H), 1.10−1.03 (m, 6H). HRMS (ESI): found 656.2116
(C32H38N3O8S2, [M + NH4]+, requires 656.2095).
2,2′-(Naphthalene-1,4-diylbis(tosylazanediyl))diacetic Acid (18a).
To a solution of NaOH (2 g) in MeOH/H2O (10/10 mL) was added
17a (638 mg, 1 mmol). The reaction mixture was heated in an oil bath
maintained at 65 °C for 2 h, then quenched with 2 M hydrochloric
acid to pH 2 and diluted with 75 mL of water. Precipitate removed by
filtration and washed with 5 × 10 mL water, then dried overnight in a
vacuum desiccator, yielding 18a as a white solid, yield 68%; mp 240−
2,2′-((2-Methyl-1,4-phenylene)bis(((4-methoxyphenyl)sulfonyl)-
azanediyl))diacetic Acid (12c). To a solution of LiOH (2 g) in
MeOH/H2O (15/5 mL) was added 11c (606 mg, 1 mmol). The
reaction mixture was stirring at room temperature overnight, then
quenched with 2 M hydrochloric acid to pH 2 and diluted with 50 mL
of water. Precipitate removed by filtration and washed with 5 × 10 mL
water, then dried overnight in a vacuum desiccator, yielding 12c as a
white solid, yield 73%; mp 231−232 °C. 1H NMR (300 MHz, DMSO,
δ) 12.86 (s, 2H), 7.59−7.51 (m, 4H), 7.10−7.07 (m, 5H), 6.82−6.79
(m, 2H), 4.37 (s, 2H), 4.22 (s, 2H), 3.85 (s, 6H), 2.17 (s, 3H). 13C
NMR (75 MHz, DMSO, δ) 169.862, 162.707, 139.609, 137.233,
129.794, 129.655, 129.517, 124.511, 114.229, 55.672, 55.246, 51.644,
17.949. HRMS (ESI): found 601.0914 (C25H26N2NaO10S2, [M +
Na]+, requires 601.0921). HPLC (70:30 methanol:water with 1‰
TFA): tR = 5.4 min, 99.0%.
1
241 °C. H NMR (300 MHz, DMSO, δ) 12.80 (br, 2H), 8.28−8.25
(m, 1H), 8.15−8.11 (m, 1H), 7.58−7.51 (m, 6H), 7.42−7.39 (m, 2H),
7.35−7.32 (m, 2H), 7.10 (s, 1H), 6.85 (s, 1H), 4.50−4.33 (m, 4H),
2.44 (s, 3H), 2.37 (s, 3H). 13C NMR (75 MHz, DMSO, δ) 169.814,
143.775, 136.905, 135.046, 134.374, 132.915, 132.714, 129.580,
127.872, 127.611, 126.753, 126.659, 126.192, 124.656, 53.226,
53.010, 21.067, 21.001. HRMS (ESI): found 600.1480
(C28H30N3O8S2, [M + NH4]+, requires 600.1469). HPLC (70:30
methanol:water with 1‰ TFA): tR = 8.1 min, 99.5%.
2,2′-((2,5-Dimethyl-1,4-phenylene)bis(((4-methoxyphenyl)-
sulfonyl)azanediyl))diacetic Acid (12d). The same procedure as 12c,
yield 81%; mp 253−255 °C. 1H NMR (300 MHz, DMSO, δ) 12.80 (s,
2H), 7.55 (d, 4H, J = 8.58 Hz), 7.10 (d, 4H, J = 8.46 Hz), 6.77 (s, 2H),
4.20 (s, 4H), 3.86 (s, 6H), 2.02 (s, 6H). 13C NMR (75 MHz, DMSO,
2,2′-(Naphthalene-1,4-diylbis(((4-fluorophenyl)sulfonyl)-
azanediyl))diacetic Acid (18b). The same procedure as 18a, gray
1
solid, yield 38%; mp 250−252 °C. H NMR (300 MHz, DMSO, δ)
8.21−8.19 (m, 1H), 8.11−8.09 (m, 1H), 7.72−7.70 (m, 4H), 7.61−
H
DOI: 10.1021/acs.jmedchem.5b00185
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
7.50 (m, 2H), 7.46−7.32 (m, 4H), 7.17 (s, 1H), 7.02 (s, 1H), 4.53−
4.37 (m, 4H). 13C NMR (75 MHz, DMSO, δ) 169.919, 136.769,
134.430, 133.896, 132.773, 132.612, 131.011, 130.886, 130.758,
130.634, 130.086, 127.045, 126.688, 124.496, 116.385, 116.108,
53.419, 14.377. HRMS (ESI): found 608.0949 (C26H24F2N3O8S2,
[M + NH4]+, requires 608.0967) HPLC (70:30 methanol:water with
1‰ TFA): tR = 8.6 min, 95.2%.
2,2′-(Naphthalene-1,4-diylbis(((4-chlorophenyl)sulfonyl)-
azanediyl))diacetic Acid (18c). The same procedure as 18a, light-
brown solid, yield 36%; mp 225−227 °C. 1H NMR (300 MHz,
DMSO, δ) 8.29−8.20 (m, 1H), 8.18−8.08 (m, 1H), 7.77−7.56 (m,
10H), 7.20 (s, 1H), 6.92 (s, 1H), 4.51−4.29 (m, 4H). 13C NMR (75
MHz, DMSO, δ) 170.028, 138.099, 137.149, 136.675, 132.795,
129.809, 129.533, 127.007, 126.610, 124.536, 53.918. HRMS (ESI):
found 640.0388 (C26H24Cl2N3O8S2, [M + NH4]+, requires 640.0376).
HPLC (70:30 methanol:water with 1‰ TFA): tR = 14.5 min, 95.8%.
2,2′-(Naphthalene-1,4-diylbis(((4-bromophenyl)sulfonyl)-
azanediyl))diacetic Acid (18d). The same procedure as 18a, light-
brown solid, yield 42%; mp 243−244 °C. 1H NMR (300 MHz,
DMSO, δ) 8.24−8.23 (m, 1H), 8.22−8.18 (m, 1H), 7.77−7.72 (m,
4H), 7.62−7.60 (m, 4H), 7.53−7.52 (m, 2H), 7.29−7.18 (m, 1H),
6.95−6.87 (m, 1H), 4.31−4.19 (m, 2H), 4.03−3.97 (m, 2H). HRMS
(ESI): found 727.9358 (C26H2Br2N2NaO8S2, [M + NH4]+, requires
727.9366). HPLC (70:30 methanol:water with 1‰ TFA): tR = 8.7
min, 99.5%.
(m, 4H), 7.57−7.50 (m, 6H), 7.29 (s, 1H), 7.01 (s, 1H), 4.64−4.35
(m, 4H). HRMS (ESI): found 740.0827 (C28H24F6N3O10S2, [M +
NH4]+, requires 740.0802). HPLC (70:30 methanol:water with 1‰
TFA): tR = 6.5 min, 97.1%.
2,2′-(Naphthalene-1,4-diylbis((naphthalen-2-ylsulfonyl)-
azanediyl))diacetic Acid (18j). The same procedure as 18i, gray solid,
1
yield 68%; mp 243−245 °C. H NMR (300 MHz, DMSO, δ) 8.38−
8.33 (m, 3H), 8.19−7.93 (m, 7H), 7.79−7.48 (m, 6H), 7.47−7.46 (m,
2H), 7.12 (s, 1H), 6.78 (s, 1H), 4.46−4.33 (m, 4H). HRMS (ESI):
found 677.1023 (C34H26N2NaO8S2, [M + Na]+, requires 677.1023).
HPLC (70:30 methanol:water with 1‰ TFA): tR = 17.1 min, 95.2%.
2,2′-(Naphthalene-1,4-diylbis(((4-(trifluoromethyl)phenyl)-
sulfonyl)azanediyl))diacetic Acid (18k). The same procedure as 18i,
white solid, yield 44%; mp > 300 °C. 1H NMR (300 MHz, DMSO, δ)
8.23−8.13 (m, 2H), 8.03−7.89 (m, 8H), 7.58−7.41 (m, 2H), 6.86 (s,
2H), 4.35−4.10 (m, 4H). HRMS (ESI): found 708.0916
(C28H24F6N3O8S2, [M + NH4]+, requires 708.0904). HPLC (70:30
methanol: water with 1‰ TFA): tR = 6.3 min, 99.9%
2,2′-(Naphthalene-1,4-diylbis(((3-methoxyphenyl)sulfonyl)-
azanediyl))diacetic Acid (18l). The same procedure as 18i, white
1
solid, yield 48%; mp 149−150 °C. H NMR (300 MHz, DMSO, δ)
8.13 (s, 2H) 7.63−7.50 (m, 4H) 7.26−7.13 (m, 7H) 7.02 (s, 1H)
4.56−4.39 (m, 4H) 3.76 (s, 3H) 3.67 (s, 3H). 13C NMR (75 MHz,
DMSO, δ) 173.551, 164.125, 163.034, 143.636, 141.502, 137.348,
135.114, 134.995, 131.641, 131.389, 129.079,124.639, 124.502,
124.420, 116.871, 116.623, 60.266, 57.795, 35.380. HRMS (ESI):
found 637.0931 (C28H26N2NaO10S2, [M + Na]+, requires 637.0921).
HPLC (70:30 methanol:water with 1‰ TFA): tR = 8.1 min, 96.2%
2,2′-(Naphthalene-1,4-diylbis(((4-cyanophenyl)sulfonyl)-
azanediyl))diacetic Acid (18m). The same procedure as 12c, gray
2,2′-(Naphthalene-1,4-diylbis(((4-acetamidophenyl)sulfonyl)-
azanediyl))diacetic Acid (18e). The same procedure as 18a, light-pink
1
solid, yield 33%; mp 288−290 °C. H NMR (300 MHz, DMSO, δ)
10.74 (s, 1H), 10.47 (s, 1H), 8.35 (dd, 1H, J = 6.23, 3.08 Hz), 8.18
(dd, 1H, J = 6.36, 3.06 Hz), 7.90 (d, 2H, J = 8.61 Hz), 7.74 (d, 2H, J =
8.73 Hz), 7.58−7.55 (m, 6H), 7.11 (s, 1H), 6.82 (s, 1H), 4.48−4.28
(m, 4H), 2.17(s, 3H), 2.09 (s, 3H). HRMS (ESI): found 686.1614
(C30H32N5O10S2, [M + NH4]+, requires 686.1585). HPLC (70:30
methanol:water with 1‰ TFA): tR = 3.3 min, 98.5%.
1
solid, yield 69%; mp 265−267 °C. H NMR (300 MHz, DMSO, δ)
12.95 (br, 2H), 8.22−7.96 (m, 6H), 7.87−7.71 (m, 4H), 7.57−7.55
(m, 2H), 7.18−7.10 (m, 2H), 4.60−4.35 (m, 4H). 13C NMR (75
MHz, DMSO, δ) 169.743, 166.490, 166.125, 138.247, 136.729,
132.603, 130.022, 128.271, 127.885, 127.788, 127.600, 126.878,
124.385, 53.085. HRMS (ESI): found 627.0564 (C28H20N4NaO8S2,
[M + Na]+, requires 627.0615). HPLC (70:30 methanol:water with
1‰ TFA): tR = 3.6 min, 97.3%.
2,2′-(Naphthalene-1,4-diylbis(((4-(tert-butyl)phenyl)sulfonyl)-
azanediyl))diacetic Acid (18f). The same procedure as 18a, gray solid,
1
yield 67%; mp 290−291 °C. H NMR (300 MHz, DMSO, δ) 8.11−
8.06 (m, 1H), 8.05−7.95 (m, 1H), 7.60−7.55 (m, 8H), 7.49−7.35 (m,
2H), 7.23 (s, 1H), 7.01(s, 1H), 4.33−4.13 (m, 4H), 1.33−1.28 (m,
18H). 13C NMR (75 MHz, DMSO, δ) 170.129, 156.113, 136.863,
135.364, 132.771, 127.575, 127.372, 126.540, 126.099, 125.775,
124.489, 53.561, 34.843, 30.776. HRMS (ESI): found 689.1931
(C34H38N2NaO8S2, [M + Na]+, requires 689.1962). HPLC (70:30
methanol:water with 1‰ TFA): tR = 9.2 min, 96.3%.
2,2′-(Naphthalene-1,4-diylbis(((4-(tert-butyl)phenyl)sulfonyl)-
azanediyl))diacetic Acid (18n). The same procedure as for 18i, white
1
solid, yield 62%; mp 222−224 °C. H NMR (300 MHz, DMSO, δ)
11.82 (s, 2H), 8.32−8.30 (m, 1H), 8.19−8.17 (m, 1H), 7.56−7.53 (m,
6H), 7.12−7.02 (m, 5H), 6.85 (s, 1H), 4.46−4.30 (m, 4H), 4.19−4.05
(m, 4H), 1.80−1.67 (m, 4H), 1.51−1.46 (m, 4H), 0.99−0.94 (m, 6H).
HRMS (ESI): found 682.6418 (C34H42N3O8S2, [M + NH4]+, requires
682.2408). HPLC (70:30 methanol:water with 1‰ TFA): tR = 7.2
min, 96.8%.
2,2′-(Naphthalene-1,4-diylbis((mesitylsulfonyl)azanediyl))-
diacetic Acid (18g). The same procedure as 18a, white solid, yield
1
61%; mp 116−118 °C. H NMR (300 MHz, DMSO, δ) 7.87−7.84
2,2′-(Naphthalene-1,5-diylbis(((4-methoxyphenyl)sulfonyl)-
(m, 2H), 7.58 (s, 1H), 7.51 (s, 1H), 7.40−7.37 (m, 2H), 6.96 (s, 2H),
6.89 (s, 2H), 4.70−4.64 (m, 2H), 4.34−4.28 (m, 2H), 2.25−2.16 (m,
18H). HRMS (ESI): found 656.2113 (C32H38N3O8S2, [M + NH4]+,
requires 656.2095). HPLC (70:30 methanol: water with 1‰ TFA): tR
= 7.0 min, 96.6%.
azanediyl))diacetic Acid (15). 15 was synthesized according to the
1
procedure of 18m, gray solid, yield 71%; mp 267−269 °C. H NMR
(300 MHz, DMSO, δ) 12.82 (s, 2H), 8.29−8.26 (m, 2H), 7.61−7.57
(m, 4H), 7.46−7.34 (m, 2H), 7.22−6.98 (m, 6H), 4.42−4.39 (m, 4H)
3.87 (s, 3H) 3.85 (s, 3H). 13C NMR (75 MHz, DMSO, δ) 169.989,
169.884, 162.826, 136.680, 133.135, 129.937, 129.281, 127.376,
125.648, 114.304, 55.668, 53.294. HRMS (ESI): found 632.1349
(C28H30N3O10S2, [M + NH4]+, requires 632.1367). HPLC (70:30
methanol:water with 1‰ TFA): tR = 5.6 min, 97.2%.
2,2′-(Naphthalene-1,4-diylbis((phenylsulfonyl)azanediyl))diacetic
Acid (18h). The same procedure as 18a, white solid, yield 44%; mp
1
265−266 °C. H NMR (300 MHz, DMSO, δ) 12.90 (br, 2H), 8.25−
8.22 (m, 1H), 8.10−8.07 (m, 1H), 7.77−7.51 (m, 12H), 7.15 (s, 1H),
6.90 (s, 1H), 4.57−4.36 (m, 4H). 13C NMR (75 MHz, DMSO, δ)
169.818, 137.956, 137.297, 136.794, 133.468, 132.764, 132.584,
129.203, 129.142, 127.732, 127.512, 127.032, 126.644, 126.508,
124.500, 124.291, 53.185. HRMS (ESI): found 572.1133
(C26H26N3O8S2, [M + NH4]+, requires 572.1156). HPLC (70:30
methanol:water with 1‰ TFA): tR = 5.4 min, 98.1%.
2. Molecular Docking. The Ligandfit docking tool in Discovery
Studio, which has been validated for this target previously,14,16 was
used to predict the binding mode of the small molecular inhibitor. The
docking site was derived from the position of the small molecular
ligand cocrystallized in the binding site of Keap1 (PDB code 4IQK).
3. Physicochemical Properties. The pKa and partition coefficient
(log D, pH 7.4) were determined according to the methods of Avdeef
and Tsinman23 on a Gemini Profiler instrument (pION) by the “gold
standard” Avdeef−Bucher potentiometric titration method.24 The
experimental procedures were carried out as previously reported.25
The pH-metric method was used to determine the intrinsic solubility.
This is a new potentiometric acid−base titration method. The
potentiometric solubility data were obtained with the pSOL model 3
2,2′-(Naphthalene-1,4-diylbis(((4-(trifluoromethoxy)phenyl)-
sulfonyl)azanediyl))diacetic Acid (18i). To a solution of 17i in
methanol was added Pd/C. The reaction mixture was heated to 40 °C
under hydrogen for 4 h. The solution was filtered to remove the
catalyst. The filtrate was concentrated under reduced pressure to give
the crude product. The crude product was washed by ether to give the
1
gray solid, yield 68%; mp 291−293 °C. H NMR (300 MHz, DMSO,
δ) 12.91 (s, 2H), 8.16−8.14 (m, 1H), 8.04−8.01 (m, 1H), 7.81−7.77
I
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Article
instrument (pION INC., Cambridge, MA, USA) and subsequently
processed with the accompanying computer program, pS. The detailed
mechanism and experimental procedures can be found in the refs
26−28.
(PerkinElmer, Northwalk, CT, USA). After blocking with 1% BSA for
2 h, the membranes were incubated at 37 °C for 1 h and then at 4 °C
overnight with a primary antibody. After that, they were reacted with a
DyLight 800 labeled secondary antibody at 37 °C for 1 h. The
membranes were screened through the Odyssey infrared imaging
system (LI-COR, Lincoln, Nebraska, USA).
4.4. RNA Extraction and qRT-PCR Analysis. Total RNA of
HCT116 cells was isolated using TRIzol (Invitrogen). The
quantification and purity of RNA samples was assessed by A260/
A280 absorption, and RNA samples with ratios above 1.8 were stored
at −80 °C for further analysis. The RNA was reverse transcribed by
the PrimeScrpt RT reagent kit following the manufacturer’s
instructions. The sequence of primers used for PCR can be found in
the Supporting Information. Quantitative real-time RT-PCR analysis
of Nrf2, NQO1, HO-1, and GCLM was performed by using the
StepOne System Fast Real Time PCR system (Applied Biosystems).
The values are expressed as the fold change from the control. β-Actin
was used for normalization. Each cycle consisted of denaturation at 95
°C for 5 s and combined annealing and extension at 60 °C for 30 s. A
total of 55 cycles was performed.
4.4. Transfection of Small Interfering RNA (siRNA). Predesigned
siRNA against human Nrf2 (catalogue no. 115762) and control
scrambled siRNA (catalogue no. 4611) were purchased from Biomics
(Biomics, China). HCT116 cells were plated at a density of 7 × 105
cells per 60 mm dish. The cells were transfected with 50 nM siRNA
against Nrf2 or 50.0 nM scrambled duplex using Lipofectamine 2000
(Invitrogen). After a 24 h incubation, fresh medium was added, and
the cells were cultured for another 48 h. The cells were then treated
with 20.0 μM compound 18e for an additional 6 h and lysed for use in
qRT-PCR.
4.5. LPS Challenge Mouse Acute Inflammation Model. Animal
studies were conducted according to protocols approved by Institu-
tional Animal Care and Use Committee of China Pharmaceutical
University. All animals were appropriately used in a scientifically valid
and ethical manner. Female C57BL/6 mice (12−16 weeks) were
randomly divided into five groups (n = 10): control group,
dexamethasone (Sigma-Aldrich, St. Louis, no. D4902) group (10
mg/kg/day), LPS (Sigma-Aldrich, St. Louis, no. L4130) group (300
μg/kg/day), compound 18e low-dose (10 mg/kg/day) group, and
compound 18e high-dose (80 mg/kg/day) group. The 18e and
dexamethasone pretreated animals received a single IP injection (500.0
μL) containing the desired dose (day −3, −2, −1). All LPS-challenged
mice (blank control, dexamethasone pretreated, 18e pretreated)
received 300.0 μg IP LPS 24 h after the last dose of dexamethasone
or 18e (day 0). Organs and sera were collected 5 h after the LPS
challenge on day 0. Individual serum samples (n = 10) were placed
immediately on ice after collection and were centrifuged at 12000g
before plasma was obtained and frozen at −20 °C for ELISA. Plasma
was assayed for murine IL-6 (EK0411, IL-6 (m) ELISA kit 1200,
Boster, China), TNF-α (EK0527, TNFα (m) ELISA kit 1200, Boster,
China), IFN-γ (EK0375, IFNγ (m) ELISA kit 1000, Boster, China),
IL-17 (EK0431, IL-17 (m) ELISA kit 1200, Boster, China), and IL-12
(EK0422, IL-12 (P70) (m) ELISA kit 1200, Boster, China) using
double-sandwich ELISA techniques.
4. Biology. 4.1. FP Competition Assays to Determine the
Inhibitory Potency of the Keap1−Nrf2 Interaction. Generally, the FP
assay was carried out as previously reported.16 The experiments were
performed on a SpectraMax multimode microplate reader (Molecular
Devices) using the excitation and emission filters appropriate for the
FITC. The plates used for the FP measurements were black
nonbinding surface Corning 3676 384-well plates, loaded with 40
μL of assay solution per well, consisting of 10 μL of 4 nM FITC-9mer
Nrf2 peptide amide and 10 μL of 12 nM Keap1 Kelch domain protein,
10 μL of HEPES buffer, and 10 μL of an inhibitor sample of varying
concentrations. The plate was covered and rocked for 30 min at room
temperature prior to FP measurements. FP was determined by
measuring the parallel and perpendicular fluorescence intensity (F∥
and F⊥) with respect to the linearly polarized excitation light. We
elected to use polarization in our quantitative analysis. The percentage
inhibition of the competitor at each concentration point was
determined by using the equation %inhibition = 1 − (Pobs − Pmin)/
(Pmax − Pmin). The values of Pmax, Pmin, and Pobs in the equations refer
to the polarization of the wells containing Keap1 and the probe, the
polarization of the free probe, and the observed polarization for the
wells containing the inhibitors at a range of concentrations under the
assay conditions, respectively. The IC50 of an inhibitor was determined
from the plot of %inhibition against inhibitor concentration as
analyzed by GraphPad Prism 6.0 software.
4.2. Cell Culture Conditions and ARE-Luciferase Activity Assay.
HepG2 cells stably transfected with a luciferase reporter (HepG2−
ARE−C8) were kindly provided by Professor Dr. A. N. Tony Kong
(Rutgers University, Piscataway, NJ) and Prof. Rong Hu (China
Pharmaceutical University, Nanjing). The cells were maintained in
modified RPMI-1640 medium (GiBco, Invitrogen Corp., USA) with
10% fetal bovine serum (FBS) (Gibco, Invitrogen Corp., USA) in a
humidified atmosphere of 5% CO2 and 95% air at 37 °C. 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 medium (Sigma-Aldrich,
no. M4892, USA) supplemented with 10% (v/v) FBS.
The experimental procedures were carried out as reported
previously.16,24 Generally, HepG2−ARE−C8 cells were plated in 96-
well plates at a density of 4 × 104 cells/well and incubated overnight.
The cells were exposed to different concentrations of test compounds,
with 8 serving as a positive control, DMSO as a negative control, and
the luciferase cell culture lysis reagent as a blank. After 12 h of
treatment, the medium was removed and 100 μL of cold PBS was
added to each well. Then, the cells were harvested in the luciferase cell
culture lysis reagent. After centrifugation, 20 μL of the supernatant was
used to determine 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 triplicate and expressed as fold
induction over the control. Induction fold = (RLUtest − RLUblank)/
(RLUDMSO control − RLUblank). RLU = relative light unit.
4.3. Western Blotting. Anti-NQO1 (sc-271116) antibodies and γ-
GCS (sc-22755) antibodies were from Santa Cruz Biotechnology
(Santa Cruz, CA, USA). Anti-β-actin (AP0060) and anti-Nrf2
(BS1258) were purchased from Bioworlde (Bioworlde, USA). Anti-
HO-1 (no. 5853S) was purchased from Cell Signaling Technology
(USA). The cells were washed once with ice-cold PBS and driven
down with 1 mL of 1× pancreatic enzymes. The cells were centrifuged
at 2500 rpm and resuspended in 45.0 μL of lysis buffer, which was
composed of 50.0 mM Tris-HCl, 150.0 mM NaCl, NP-40, 1 mM
EDTA, PMSF, NaF, Leu, and DTT for 1 h. Then, cells were
centrifuged again at 12000 rpm for 20 min at 4 °C. The supernatant
was retained, and the protein concentration was determined by the
BCA assay with Varioskan flash (Thermo, Waltham, MA) at 562 nm.
The samples were stored at −80 °C until use. The extracts were
separated by SDS-PAGE and then transferred onto PVDF membranes
ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jmed-
chem.5b00185.
Characterization of the other intermediates, detailed
results of SPR study, binding mode predication of 15,
mice body weight records of the in vivo experiments, and
sequences used for PCR (PDF)
J
DOI: 10.1021/acs.jmedchem.5b00185
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Journal of Medicinal Chemistry
Article
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AUTHOR INFORMATION
Corresponding Authors
*For H.-P.S.: Phone/Fax: +86 025 83271216. E-mail:
sunhaopeng@163.com.
*For Q.-D.Y.: Phone/Fax: +86 025 83271351. E-mail:
youqidong@gmail.com.
■
(11) Hu, L.; Magesh, S.; Chen, L.; Wang, L.; Lewis, T. A.; Chen, Y.;
Khodier, C.; Inoyama, D.; Beamer, L. J.; Emge, T. J.; Shen, J.;
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Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work is supported by the projects 81230078 (key
program), 81202463 (youth foundation), 81173087 and
91129732 of National Natural Science Foundation of China,
2014ZX09507002-005-015, 2013ZX09402102-001-005, and
2010ZX09401-401 of the National Major Science and
Technology Project of China (Innovation and Development
of New Drugs), and Specialized Research Fund for the
Doctoral Program of Higher Education (SRFDP,
20130096110002). HepG2 cells stably transfected with a
luciferase reporter (HepG2-ARE-C8) were kindly provided by
Professor Dr. A. N. Tony Kong (Rutgers University, Piscat-
away, NJ) and Prof. Rong Hu (China Pharmaceutical
University, Nanjing).
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ABBREVIATIONS USED
■
Keap1, Kelch-like ECH-associated protein-1; Nrf2, nuclear
factor erythroid 2-related factor 2; LPS, lipopolysaccharide;
bZIP, basic leucine zipper; TAT, trans-activating transcrip-
tional; ARE, antioxidant response element; PPI, protein−
protein interaction; NQO1, NAD(P)H/quinone oxidoreduc-
tase; HO-1, heme oxygenase-1; GCLM, glutamate−cysteine
ligase modifier subunit; GCS, glutamylcysteine synthetase
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