Discovery of 2-oxy-2-phenylacetic acid substituted naphthalene sulfonamide derivatives as potent KEAP1-NRF2 protein-protein interaction inhibitors for inflammatory conditions

Article abstract of DOI:10.1016/j.ejmech.2020.112734

Nuclear factor erythroid 2-related factor 2 (NRF2) is a pleiotropic transcription factor which regulates the constitutive and inducible transcription of a wide array of genes and confers protection against a variety of pathologies. Directly disrupting Kelch-like ECH-associated protein 1 (KEAP1)-NRF2 protein-protein interaction (PPI) has been explored as a promising strategy to activate NRF2. We reported here the first identification of a series of 2-oxy-2-phenylacetic acid substituted naphthalene sulfonamide derivatives as potent KEAP1-NRF2 inhibitors. Our efforts led to the potent small molecule KEAP1-NRF2 inhibitor, 20c, which exhibited a K_d of 24 nM to KEAP1 and an IC₅₀ of 75 nM in disrupting KEAP1-NRF2 interaction. Subsequent biological studies provided consistent evidence across mouse macrophage cell-based and in vivo models that 20c induced NRF2 target gene expression and enhanced downstream antioxidant and anti-inflammatory activities. Our study not only demonstrated that small molecule KEAP1-NRF2 PPI inhibitors can be potential preventive and therapeutic agents for diseases and conditions involving oxidative stress and inflammation but also enriched the chemical diversity of the KEAP1-NRF2 inhibitors.

Full text of DOI:10.1016/j.ejmech.2020.112734

European Journal of Medicinal Chemistry 207 (2020) 112734
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Discovery of 2-oxy-2-phenylacetic acid substituted naphthalene
sulfonamide derivatives as potent KEAP1-NRF2 protein-protein
interaction inhibitors for inflammatory conditions
,
,
b
,
, b, **
Meng-Chen Lu a b, Hong-Li Shao a, Tian Liu a, Qi-Dong You a *, Zheng-Yu Jiang a
a State Key Laboratory of Natural Medicines and Jiang Su Key Laboratory of Drug Design and Optimization, China Pharmaceutical University, Nanjing,
210009, China
b Department of Medicinal Chemistry, School of Pharmacy, 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:
Nuclear factor erythroid 2-related factor 2 (NRF2) is a pleiotropic transcription factor which regulates the
constitutive and inducible transcription of a wide array of genes and confers protection against a variety
of pathologies. Directly disrupting Kelch-like ECH-associated protein 1 (KEAP1)-NRF2 protein-protein
interaction (PPI) has been explored as a promising strategy to activate NRF2. We reported here the
first identification of a series of 2-oxy-2-phenylacetic acid substituted naphthalene sulfonamide de-
rivatives as potent KEAP1-NRF2 inhibitors. Our efforts led to the potent small molecule KEAP1-NRF2
inhibitor, 20c, which exhibited a Kd of 24 nM to KEAP1 and an IC50 of 75 nM in disrupting KEAP1-NRF2
interaction. Subsequent biological studies provided consistent evidence across mouse macrophage cell-
based and in vivo models that 20c induced NRF2 target gene expression and enhanced downstream
antioxidant and anti-inflammatory activities. Our study not only demonstrated that small molecule
KEAP1-NRF2 PPI inhibitors can be potential preventive and therapeutic agents for diseases and condi-
tions involving oxidative stress and inflammation but also enriched the chemical diversity of the KEAP1-
NRF2 inhibitors.
Received 17 April 2020
Received in revised form
26 July 2020
Accepted 4 August 2020
Available online 22 August 2020
Keywords:
KEAP1-NRF2 pathway
Protein-protein interaction inhibitors
Oxidative stress
Inflammation
© 2020 Elsevier Masson SAS. All rights reserved.
1. Introduction
oxidative stress has been closely associated with a number of
chronic and inflammatory disease states, such as neurogenerative
The human body is constantly exposed to various reactive in-
sults, represented by the electrophilic and oxidative chemicals [1].
On one hand, redox reactions and reactive species, including
reactive oxygen species (ROS) and reactive nitrogen species (RNS),
are important for many physiological processes [2], on the other
hand, the imbalance between the production and the removal of
these reactive species could disturb the cellular redox homeostasis
[3]. Excess oxidative chemicals can directly damage nucleic acids,
lipids and proteins and induce the oxidative stress. Constant
diseases, chronic kidney disease, obstructive pulmonary disease,
atherosclerosis and cancer [4e6]. There are multiple antioxidative
and cytoprotective mechanisms in cells, and enhancing cellular
defense system is a possible way to develop therapeutic agents of
such diseases.
Nuclear factor (erythroid-derived 2)-like 2 (NRF2), a transcrip-
tion factor protein with a basic leucine zipper DNA binding motif, is
the predominant regulator of the cellular defensing system. NRF2
induces transcription of various xenobiotic metabolizing enzymes
and antioxidant proteins to maintain a stable internal environment
[7]. Moreover, nuclear NRF2 can directly antagonize transcription of
inflammatory factors [8,9]. The crucial role of NRF2 in anti-
inflammation has also been intensively linked to its interplay
with the redox-regulated transcription factor nuclear factor kappa
* Corresponding author. State Key Laboratory of Natural Medicines and Jiang Su
Key Laboratory of Drug Design and Optimization, China Pharmaceutical University,
Nanjing, 210009, China.
** Corresponding author. State Key Laboratory of Natural Medicines and Jiang Su
Key Laboratory of Drug Design and Optimization, China Pharmaceutical University,
Nanjing, 210009, China.
E-mail addresses: youqd@163.com (Q.-D. You), jiangzhengyucpu@163.com
(Z.-Y. Jiang).
B (NF-
counterbalance each other’s activity via PPIs or through secondary
messenger effects. The NRF2-ARE pathway antagonizes NF-
mediated transcription by inhibiting I degradation. Conversely,
kB) [10]. The NRF2 and NF-kB pathways are proposed to
kB
kBa
https://doi.org/10.1016/j.ejmech.2020.112734
0223-5234/© 2020 Elsevier Masson SAS. All rights reserved.

2
M.-C. Lu et al. / European Journal of Medicinal Chemistry 207 (2020) 112734
Abbreviations
qRT-PCR quantitative real-time PCR
HO-1 Heme oxygenase 1
NRF2
KEAP1
PPI
ARE
ROS
RNS
CUL3
LPS
Nuclear factor erythroid 2-related factor 2
NQO-1
GCLM
SOD
GSH-Px
MPO
NAD(P)H dehydrogenase (quinone) 1
glutamate-cysteine ligase regulatory subunit
superoxide dismutase
glutathione peroxidase
myeloperoxidase
Kelch-like ECH-associated protein 1
protein-protein interaction
antioxidant response element
reactive oxygen species
reactive nitrogen species
Cullin3
IL-1
b
interleukin-1b
TNF-
NO
a
tumor necrosis factor
nitric oxide
a
lipopolysaccharide
FP
ITC
BLI
Fluorescence Polarization
Isothermal Titration Calorimetry
biolayer interferometry
DXM
IFN-
dexamethasone
interferon-g.
g
NF-
k
B can suppress transcription of NRF2-ARE dependent genes by
_ENREF_43In this study, we rationally designed a series of 2-oxy-2-
phenylacetic acid substituted naphthalene sulfonamide derivatives
as potent KEAP1-NRF2 inhibitors and identified that the 1,4-
diaminonaphthalene substituted pattern is unnecessary for inhib-
iting KEAP1-NRF2 interaction. The represented compound 20c
exhibited a Kd of 24 nM to KEAP1 and an IC50 of 75 nM in disrupting
KEAP1-NRF2 interaction. 20c induced the transcription activity of
NRF2-antioxidant response element (ARE) in the ARE luciferase
reporter assay and elevated both the mRNA and protein level of
NRF2 and NRF2-regulated genes in macrophage cells. The activa-
tion of NRF2-regulated cyto-protective system by 20c significantly
antagonized the lipopolysaccharide (LPS) induced inflammatory
conditions in cellular and in vivo models.
preventing CREB binding protein (CBP) from binding to NRF2 and
facilitating recruitment of histone deacetylase 3 (HDAC3) to MafK
[11,12]. Besides, NRF2 activation also enhances antioxidant defense
system, increases HO-1 expression and alters GSH homeostasis,
which eliminate ROS and therefore inhibits ROS mediated NF-kB
activation [13,14]. Thus, NRF2 activation could be a viable way to
antagonize stress and inflammation related disorders [15e18].
Cellular content of NRF2 is mainly controlled by the E3 ligase
adaptor protein, Kelch-like ECH-associated protein 1 (KEAP1) in a
redox sensitive manner. Under normal conditions, KEAP1 functions
as a substrate adaptor for a Cul3-based E3 ubiquitin ligase,
constantly targeting Nrf2 for ubiquitin-dependent degradation.
Upon oxidative and electrophilic stresses, KEAP1 functions as a
sensor for the stress signals and loses its activity to interact with
NRF2, resulting in reduced degradation of NRF2 [19e21]. Then,
newly synthesized NRF2 protein can translocate to the nucleus and
regulate the transcription. This manner of NRF2 activation inspires
the development of electrophilic NRF2 activators, and some elec-
trophiles have been proven to covalently modify sensitive cysteine
residues of KEAP1 [22]. However, the targets of these electrophiles
could be promiscuous, which makes it difficult to clarify the
selectivity and the intact mechanism of action. Therefore, directly
disrupting the KEAP1-NRF2 protein-protein interaction (PPI) have
been proposed as a new avenue for NRF2 activation therapy
[23e25].
In recent years, the development of KEAP1-NRF2 PPI inhibitors
have made much progress [26e30]. Various screening approaches
have been identified [31] and peptide inhibitors with diverse amino
acid residues have been developed to elucidate the interface of the
KEAP1-NRF2 PPI [32e38]. Moreover, small-molecular KEAP1-NRF2
PPI inhibitors have also been reported [39e46] and some molecules
have achieved high potency [47e52]. The therapeutic potential of
KEAP1-NRF2 inhibitors has also been explored in several in vivo
disease models, including colitis [53], retinal ischemia-reperfusion
injury [54], Alzheimer’s disease [55] and inflammatory kidney
disease [56].
2. Results and discussion
2.1. Rational design and synthesis of 2-oxy-2-phenylacetic acid
substituted naphthalene sulfonamide derivatives as KEAP1-NRF2
PPI inhibitors
In recent years, the KEAP1ꢀNRF2 PPI inhibitors with a 1,4-
diaminonaphthalene core have been extensively reported
(Fig. 1A). The X-ray structure of the diacetamide-substituted com-
pound 5 with KEAP1 fully confirmed the binding pattern of this
scaffold (Fig. 1B and C) [60]. Compound 6 (IC50 ¼ 0.14
mM), with
only one sulfonamide group, was the first asymmetric compound of
this scaffold [61]. Very recently, we reported the first identification
of amino acids as preferred substituents to design potent
KEAP1ꢀNRF2 inhibitors, obtaining 7 with an IC50 of 43 nM [62].
Taken together, these studies clearly revealed the five sub-pockets
model of the KEAP1 cavity [63] and it is worthwhile to seek out
novel agents suitable for the KEAP1-NRF2 interface.
Our previous studies have demonstrated the necessity of mak-
ing multiple polar interactions with key Arg residues in the P1 and
P2 polar sub-pockets of KEAP1. In this study, we retained the di-
acetic acid moiety but replaced one of the phenyl sulfonamide
group with the hydroxyl group, resulting in compound 8. The
docking simulation showed that the oxyacetic acid group could
occupy the P1 sub-pocket and form electrostatic interactions with
Arg483 and Arg415. Then, 8 was synthesized and tested for KEAP1-
NRF2 interaction inhibitory activity. Compound 8 showed moder-
Our group reported the first small molecule inhibitor, 1, with
nanomolar potency for KEAP1 binding and KEAP1-NRF2 inhibition
[47]. Based on the crystallographic information of the reported
naphthalene-1,4-sulfonamide based hit 2 [57], we introduced N-
acetic acid groups to mimic the key Glu79 and Glu82 in the NRF2
ETGE domain, successfully obtaining compound 1 (IC50 ¼ 28.6 nM
in a fluorescence polarization (FP) assay) [47] that fulfills all the five
sub-pockets of KEAP1. Further drug-like property optimization of 1
gave analogs represented by 3 and 4 [58,59]. These efforts inspired
the development of various 1,4-diaminonaphthalene-based
KEAP1-NRF2 inhibitors reported in recent years [59e61].
ate KEAP1-NRF2 inhibition activity (IC50 ¼ 8.19
mM), which
confirmed the suitability of the oxyacetic acid group in KEAP1-
NRF2 inhibitor design. Of note, in the structure of compound 1,
the sulfonamide group can restrict the conformation of the acetic
acid group; however, in the structure of 8, the oxyacetic acid group
is free to rotate and unable to occupy the P4 sub-pocket. In order to
interact with the P4 sub-pocket, a hydrophobic benzene ring was

M.-C. Lu et al. / European Journal of Medicinal Chemistry 207 (2020) 112734
3
Fig. 1. Published 1,4-diaminonaphthalene-based KEAP1-NRF2 PPI inhibitors. (A) Chemical structures of known 1,4-diaminonaphthalene-based KEAP1-NRF2 PPI inhibitors. (B)
Binding surface depiction of 5 with KEAP1 (PDB code: 4XMB). (C) Interaction mode of 5 with KEAP1 (PDB code: 4XMB). Hydrogen bonds are represented by green dashed lines, the
electrostatic interactions are represented by orange dashed lines, and the hydrophobic interactions are represented by pink dashed lines. The carbon atoms of small molecules and
KEAP1 residues are colored cyan and purple, respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
introduced to the methylene group in the oxyacetic acid group and
resulted in 14a with a significant increase in KEAP1-NRF2 inhibition
optimization sites. We synthesized 14 compounds (14b ~14o) and
systematically evaluated the effects of the different substituents on
KEAP1-NRF2 PPI inhibition activity.
As shown in Table 1, the substituents on the sulfonamide-
substituted phenyl ring had significant effects both on the KEAP1-
NRF2 PPI inhibitory activity and the cellular NRF2-ARE induction
activity. Generally, removal of the terminal methoxy group resulted
activity (IC50 ¼ 2.28
mM). The docking study showed that 14a could
fulfill all the five sub-pockets of KEAP1: the oxyacetic acid group in
the P1 sub-pocket retained the original strong polar interactions
with Arg415 and Arg483 and the other carboxyl group in the P2
sub-pocket could make polar interactions with Arg415 and Arg380;
the two hydrophobic benzene rings formed the
teractions with Tyr525 and Tyr334 in the P4 and P5 sub-pockets;
the naphthalene ring scaffold kept the cation- interactions with
Arg415 and Ala556 in the P3 sub-pocket to stabilize the binding
conformation and the other part of the molecule can interact with
KEAP1 in a similar way as the lead compound 1. However, it cannot
be ignored that this structure modification led to a significant
decrease in activity compared to compound 1, further compre-
hensive structure-activity relationship studies should be conducted
to improve the activity.
p
-
p
stacking in-
in 14b with a dramatic decrease in activity (IC50 ¼ 17.78
mM); the
electron-donating substituents (14a, 14c ~14f) exhibited better
performance compared to electron-withdrawing (14g ~14k) and
halogen substituents (14l ~14n).
p
In the exploration of the electron-donating substituents, we
found that the p-methoxyl substituent (14a, IC50 ¼ 2.28
slightly more potent than p-methyl (14c, IC50 ¼ 2.69 M). Intro-
M),
mM) was
m
duction of the meta-methoxyl group gave 14e (IC50 ¼ 2.30
m
which is less potent than para-methoxyl, indicating that the hy-
drophobic groups were unfavorable in the meta-position. Note-
worthily, the 2,4,6-trimethyl substituted compound 14f exhibited
2.2. Structure-activity relationships studies of 14a
the most excellent performance (IC50 ¼ 0.58
mM), likely because the
ortho-methyl introduction enhanced the hydrophobic interactions
in the P5 sub-pocket, which is supported by the docking simulation
(Fig. 3A). An increase in potency by 2-naphthyl substitution (14o,
2.2.1. Substituent transformation on the side chain phenyl ring in
the P5 sub-pocket
As shown in Fig. 2B, 14a might not fully occupy the P5 sub-
IC50 ¼ 0.78
mM) also indicated that large hydrophobic group in this
site is preferable for the KEAP1-NRF2 PPI activity.
pocket, with only the hydrophobic benzene ring forming the
p
-p
stacking interaction with Tyr334. Therefore, the substituents on the
sulfonamide-substituted phenyl group of 14a were chosen as the
On the whole, in the exploration of the substituents in the P5
sub-pocket, we obtained the most potent compound 14f with the

4
M.-C. Lu et al. / European Journal of Medicinal Chemistry 207 (2020) 112734
Fig. 2. Discovery of 2-oxy-2-phenylacetic acid substituted naphthalene sulfonamide derivatives. (A) The schemes of structure modification of 14a. (B) Docked poses of 1, 8 and 14a
with KEAP1. (C) Interaction modes of 1, 8 and 14a with KEAP1. Hydrogen bonds are represented by green dashed lines, the electrostatic interactions are represented by orange
dashed lines, and the hydrophobic interactions are represented by pink dashed lines. The carbon atoms of small molecules and KEAP1 residues are colored cyan and purple,
respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
2,4,6-trimethyl substitution, which is approximately 4-fold
increased potency than that of 14a. However, compared with our
first reported small molecule inhibitor 1 (IC50 ¼ 28.6 nM), further
structure optimization of 14f may be necessary to afford gains in
potency.
to micromolar level (IC50 ¼ 1.19
may decrease the polarization of the benzene ring, thus impairing
the stacking interactions with Tyr525 in the P4 sub-pocket.
Comparatively, an increase in potency by the phenolic hydroxyl
M), which is the preferred substituent in the
study of peptide inhibitors, may be attributed to a hydrogen
bonding interaction between the phenolic hydroxyl and Tyr525 in
the P4 sub-pocket. Attractively, 20c with the methoxyl substituent
exhibited dramatic increase in PPI inhibitory activity
mM). The trifluoromethyl group
p-p
(20b, IC50 ¼ 0.16
m
2.2.2. Substituent transformation on the benzene sulfonamide
fragment in the P4 sub-pocket
The docking study showed that 14f can form the
p-p stacking
interactions with Tyr525 in the P4 sub-pocket, therefore we turned
our attention to the modification of the benzene acetic acid frag-
ment in the P4 sub-pocket of this scaffold. Based on our previous
study of this pocket and the docking pose of 14f, a substituent is
preferable in the para position of the phenyl ring. As shown in
Fig. 3A, the benzene ring extended toward outside of the binding
pocket, which closely approximated the outer solvent region.
Therefore, a series of compounds (20a ~20j) containing substituted
benzene sulfonamide fragment were synthesized.
(IC50 ¼ 0.075
in this position. Replacing the methoxyl group with the ethyoxyl
M), indicating the
possible steric hindrance, and it was further proved by that the
isopropoxyl substituent induced the further activity decrease (20e,
mM), indicating that hydrophobic group is preferable
group diminished the activity (20d, IC50 ¼ 0.15
m
IC50 ¼ 0.50
m
M) and the tertiary butyl substituent hugely dimin-
M). The carboxamide group did
M). It
ished the activity (20f, IC50 ¼ 19
m
not give good performance in activity (20g, IC50 ¼ 0.98
m
suggested that the hydrophilic groups may be disadvantageous in
this position. Halogen substituents (20h ~20j) all gave better per-
formance than hydrogen substituent but had differences in activity:
F and Cl displayed equal inhibition activity and both better than Br.
As shown in Table 2, it appears as though strong electron-
withdrawing groups are not well tolerated at this position, as the
inhibitory activity of the trifluoromethyl substituent 20a decreased

M.-C. Lu et al. / European Journal of Medicinal Chemistry 207 (2020) 112734
5
Table 1
KEAP1-NRF2 inhibitory activities and ARE induction fold results of the compounds 14a ~14o.a.
No.
R
IC50 SE (
m
M)
ARE induction fold
0.01
m
M
0.1
m
M
1
mM
5
m
M
10
m
M
20 mM
14a
14b
14c
14d
14e
14f
14g
14h
14i
14j
14k
14l
14m
14n
14o
4-OCH3
-H
4-CH3
4-C(CH3)3
3,4-OCH3
2,4,6-CH3
4-NHCOCH3
4-NO2
4-CONH2
4-CF3
4-OCF3
4-F
4-Cl
4-Br
2.28 0.11
1.02 0.02
1.11 0.55
1.11 0.07
1.01 0.14
1.12 0.17
1.03 0.54
1.09 0.10
1.00 0.44
1.12 0.01
1.02 0.08
1.01 0.02
1.13 0.55
1.14 0.02
1.09 0.02
1.10 0.19
1.00 0.14
1.12 0.15
1.03 0.48
1.04 0.24
1.09 0.11
1.11 0.15
1.08 0.77
1.01 0.03
1.02 0.02
1.34 0.11
1.01 0.08
1.09 0.56
1.04 0.19
1.09 0.02
1.13 0.02
1.12 0.48
1.09 0.19
1.12 0.03
1.11 0.01
1.08 0.91
1.34 0.23
1.01 0.17
1.02 0.27
1.33 0.04
1.21 0.12
1.33 0.14
1.19 0.17
1.09 0.14
1.23 0.40
1.28 0.02
1.24 0.09
1.17 0.91
1.32 0.09
2.38 0.92
1.15 0.66
3.66 1.21
1.12 0.11
1.23 0.02
1.33 0.14
1.17 0.23
1.23 0.08
1.12 0.34
1.23 0.28
1.11 0.41
3.44 0.02
1.97 0.24
1.98 0.12
2.11 0.08
3.45 1.01
2.34 0.19
4.78 1.12
1.34 0.18
1.33 0.66
1.54 0.31
1.25 1.21
1.14 0.23
1.45 0.11
1.55 0.31
1.67 0.14
4.33 0.12
2.89 0.14
2.21 0.10
3.11 0.19
4.76 0.49
3.23 0.27
6.58 0.18
1.98 0.31
1.45 0.51
2.55 0.15
2.09 0.10
1.76 0.82
2.11 1.01
1.91 0.34
2.38 0.15
6.11 0.33
17.78 1.06
2.69 0.12
0.98 0.01
2.30 0.12
0.58 0.01
5.23 1.03
4.79 1.03
2.69 0.08
6.66 1.05
5.84 1.01
5.16 1.11
7.64 1.03
2.97 0.12
0.78 0.01
5,6-C4H4
a
Values shown are the means SEM (n ¼ 3).
Fig. 3. Docked poses of 14f (A) and 20c (B) with KEAP1. Hydrogen bonds are represented by green dashed lines, the electrostatic interactions are represented by orange dashed
lines, and the hydrophobic interactions are represented by pink dashed lines. The carbon atoms of small molecules and KEAP1 residues are colored cyan and purple, respectively.
(For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
We speculated that the weak electron-withdrawing ability of the
halogen substituents might enhance the stacking interactions
were performed to analyze the most active compound 20c bound
to KEAP1. As illustrated in Fig. 3B, compared to the template
compound 14a, introduction of the 2,4,6-trimethyl substitution
increased the interactions with Tyr 334, Phe577 and Tyr572 in
the P5 sub-pocket. In the exploration of the P4 sub-pocket, visual
inspection of the docking poses revealed that introduction of the
hydrophobic para-methoxyl increased the interactions with
Tyr525 and probably formed a new electrostatic interaction with
Gln530, which might in some extent contribute to the enhanced
affinity to KEAP1.
p-p
between the phenyl ring and Tyr525 in P4. Though Br can make
stronger hydrophobic interactions than F and Cl, the size of Br
might be too large for this pocket to gain the superiority in activity.
Taken together, modification of the benzene acetic acid frag-
ment afforded 20c with stronger affinity for the P4 sub-pocket
and approximately 8-fold increased PPI inhibitory activity
compared to 14f. In order to deeply understand the binding
mode of our compound with KEAP1, the docking simulations

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M.-C. Lu et al. / European Journal of Medicinal Chemistry 207 (2020) 112734
Table 2
KEAP1-NRF2 inhibitory activities and ARE induction fold results of the compounds 20a ~20j.a.
No.
R
IC50 SE (
m
M)
ARE induction fold
0.01
m
M
0.1
m
M
1
m
M
5
m
M
10
m
M
20 mM
20a
20b
20c
20d
20e
20f
20g
20h
20i
-CF3
-OH
1.19 0.12
0.16 0.01
0.075 0.001
0.15 0.01
0.50 0.01
19 1.09
0.98 0.01
0.17 0.01
0.19 0.01
0.43 0.01
1.01 0.14
1.02 0.55
1.02 0.43
1.11 0.07
1.13 0.02
1.01 0.02
1.00 0.08
1.10 0.14
1.04 0.02
1.00 0.21
1.00 0.24
1.11 0.11
1.23 0.17
1.04 0.14
1.05 0.28
1.02 0.15
1.12 0.67
1.07 0.14
1.08 0.43
1.23 0.15
1.09 0.19
1.56 0.76
2.87 0.21
1.61 0.05
1.24 0.02
1.03 0.11
1.10 0.02
1.55 0.02
1.42 0.67
1.34 0.22
2.24 0.54
4.58 0.41
5.78 1.56
4.24 0.84
3.38 0.17
1.12 0.34
3.10 0.11
4.48 1.11
4.51 0.64
3.38 1.10
3.12 1.12
5.34 0.54
7.83 1.98
5.27 1.11
4.89 1.18
1.24 0.01
4.08 1.10
5.02 0.43
5.99 0.78
4.89 1.12
4.28 1.19
8.29 1.32
13.38 1.32
8.78 1.12
6.91 1.14
2.13 0.11
5.11 0.98
8.33 1.13
9.58 1.45
6.91 0.49
-OCH3
-OCH2CH3
-OCH(CH3)2
-C(CH3)3
-NHCOCH3
-F
-Cl
-Br
20j
a
Values shown are the means SEM (n ¼ 3).
2.3. Binding characterization of 20c
transcription of NRF2-driven genes. Quantitative real-time PCR
(qRT-PCR) analysis was performed to measure the mRNA levels of
three selected downstream genes: HO-1 (Heme oxygenase 1),
NQO1 (NAD(P)H: Quinone Oxidoreductase 1) and GCLM (gluta-
mate-cysteine ligase, modifier subunit). As shown in Fig. 5B, after
12 h treatment, 20c strongly increased the transcription of NRF2
regulated genes in RAW264.7 cells at a concentration-dependent
manner. In a time-course study, NRF2 activation effects were
Then, the KEAP1 binding affinity and the KEAP1 binding char-
acters of the most potent compound 20c were investigated using
both an isothermal scanning calorimetry (ITC) assay and biolayer
interferometry (BLI). As shown in Fig. 4A, the ITC profile is typical of
a reversible 1:1 binding stoichiometry, indicating that one 20c
molecule bound to one molecule of KEAP1 and curve-fitting anal-
ysis parameters were appropriately configured. By use of these
parameters, 20c had a Kd of 24.0 nM for KEAP1, with a strong
observed after 4 h treatment of 20c (10
mM) and reached the
maximal effects at 16 h (Fig. 5C). Consistent with these results,
western blotting experiments demonstrated that the treatment of
the RAW264.7 cells with 20c induced NRF2, HO-1, NQO-1 and
GCLM protein expression in both concentration- and time-
dependent manner (Fig. 5D and E).
enthalpy to binding (
unfavorable entropic component (T
D
H ¼ ꢀ26.25 kcal molꢀ1) and a relatively
D
S ¼ ꢀ15.85 kcal molꢀ1), which
suggested a putative enthalpy-driven process in the binding of 20c
to KEAP1. Then the BLI assay, which allows for the real time
monitoring of the interactions between molecules, was carried out
to measure the binding kinetics of 20c to KEAP1. Consistent with
the ITC result, the BLI experiment gave a Kd of 36.5 nM, and the
dissociation curves showed that 20c is a long-acting compound
with prolonged residence time (Fig. 4B). Taken together, the above
results demonstrated that 20c can directly bind to KEAP1 in vitro
with a strong affinity.
2.5. 20c enhanced the antioxidant capacity in macrophage RAW
264.7 cells
After the validation of high cellular NRF2 activation effects of
20c, we then explored the effects of 20c on antioxidant capacity of
RAW 264.7 cells against cellular oxidative stress. Firstly, the effect
of 20c on the LPS induced augment of ROS level in RAW264.7 cells
was detected through the living cell microscopy using the redox-
sensitive fluorescent probe c-H2DCF-DA. As shown in Fig. 6A, af-
2.4. 20c effectively activated NRF2-ARE regulated cytoprotective
defense system
ter treating RAW264.7 cells with 1 mg/mL LPS for 6 h, a remarkably
In view of the promising character of 20c, we first investigated
the cellular activities of these compounds using the ARE-luciferase
reporter assay in HepG2-ARE-C8 cells. As shown in Tables 1 and 2,
20c, which is optimal in PPI inhibition activity, gave the best per-
formance among these compounds in the NRF2-ARE inducing ac-
tivity. Therefore, it was chosen for further cell-based evaluation.
Translocation of NRF2 into the nucleus is essential for the trans-
activation of ARE-regulated genes. Therefore, we examined the
nuclear and cytoplasmic NRF2 levels after treatment of the
strong living cell fluorescence microscopic signal was observed,
which was resulted from the high oxidation of the incorporated c-
H2DCF-DA. Treatment with 10 mM of 20c significantly reduced ROS
generation to nearly normal level (Fig. 6A and B). Subsequently, the
activities of two important antioxidant enzymes, superoxide dis-
mutase (SOD) and glutathione peroxidase (GSH-Px), were deter-
mined. Cells were treated with LPS (1
combination with different concentrations (1, 10
exposure induced sharp decrease in the activities of SOD and GSH-
Px, while the thereafter treatment with 10 M of 20c notably
mg/mL) alone or in
mM) of 20c. LPS
RAW264.7 cells with 20c. A time response study with 20c (10
mM)
m
demonstrated that nuclear translocation of NRF2 began within 2 h,
maximized at 8 h and subsequently declined after 16 h (Fig. 5A). We
then investigated whether 20c is potent in promoting the
restored the SOD and GSH-Px levels (Fig. 6C and D), indicating the
restoration of the antioxidant capacity of RAW 264.7 cells. The ratio
of reduced glutathione (GSH) to oxidized glutathione (GSSG),

M.-C. Lu et al. / European Journal of Medicinal Chemistry 207 (2020) 112734
7
Fig. 4. Compound 20c exhibited tight KEAP1 binding in vitro. (A) ITC profile of the titration of the KEAP1 Kelch domain with 20c. Calculated values are stoichiometry (N), affinity
(Kd), enthalpy change ( H), and entropy change ( S). Total Gibbs free energy ( G) is calculated according to the equation H - T S, with T the temperature set up for the ITC
D
D
D
D
G ¼
D
D
experiment. (B) BLI concentration-response curves reflecting the direct binding of 20c to KEAP1. Concentrations: 250 nM, 50 nM, 20 nM, 1 nM.
Fig. 5. 20c activated NRF2-ARE regulated cytoprotective defense system. (A) Effect of 20c on the nuclear translocation of the NRF2 protein (n ¼ 3 independent observations). (B)
Expression of NRF2-regulated genes after treatment with 20c in RAW264.7 cells. The mRNA levels of NRF2-targeted genes were measured at 12 h after treatment of RAW264.7 cells
with various concentrations of 20c. (C) The mRNA levels of NRF2-targeted genes were measured at various time points after treatment with 10 mM 20c. Values shown are the
means SEM (n ¼ 3). ***P < 0.001, **P < 0.01, and *P < 0.05, which were calculated with one-way ANOVA. (D) Expression of NRF2-regulated proteins after treatment with 20c in
RAW264.7 cells. The protein levels of NRF2 and its downstream proteins were measured at 12 h after treatment of RAW264.7 cells with various concentrations of 20c (n ¼ 3
independent observations). (E) The protein levels of NRF2 and its downstream proteins were measured at various time points after treatment with 10
observations).
mM 20c (n ¼ 3 independent

8
M.-C. Lu et al. / European Journal of Medicinal Chemistry 207 (2020) 112734
which is interpreted as evidence of redox balance and reducing
power, and the oxidative-related marker, myeloperoxidase (MPO)
activity, were also assessed (Fig. 6E and F). LPS treatment induced
the concomitant decline in the ratio of GSH to GSSG, while treat-
2.7. 20c reduced the LPS-induced production of the pro-
inflammatory factors in vivo
After confirming the cellular NRF2 activation effects of 20c, we
finally investigated the in vivo curative effect of 20c. To explore the
suitability of 20c for in vivo administration, the ADME properties
and pharmacokinetics of 20c were evaluated. As shown in Table 3,
20c was relatively stable in co-incubation with rat liver microsomes
with half-life of 10.5 h, indicating high metabolic stability. More-
over, it was observed that 20c has no CYP inhibition on 1A2, 2C9,
ment with 20c at the concentration of 10 mM almost restored the
ratio back to normal. As expected, treatment with 20c also showed
a concentration-dependent reversal in LPS-induced increase of
MPO activity. These results clearly demonstrated that 20c enhanced
the capacity of the antioxidant system in the RAW264.7 cells under
inflammatory conditions.
2C19, 2D6 and 3A4 when tested at 10 mM. Then we evaluated the
pharmacokinetics of 20c for IV. 20c showed appropriate overall
pharmacokinetic profile in rats as 1 mg/kg IV dosing led to half-life
of 1.72 0.42 h.
2.6. 20c attenuates LPS-induced production of inflammation factors
Finally, the in vivo therapeutic effect of 20c in LPS-induced
inflammation mouse model was also evaluated. Female C57BL/6
in RAW 264.7 cells
mice (20 2 g) were treated with LPS (300
received an IP injection (500 L) containing the desired dose of 20c
for 3 days. The control group only received saline (IP, 500 L) during
mg/kg, IP) and 4 h later
Following activation by inflammatory stimuli such as LPS,
macrophages secrete various inflammatory mediators such as
interleukin-1b (IL-1b), IL-6, tumor necrosis factor a (TNF-a) and
m
m
the whole experiment. As a positive control, another group
received dexamethasone (DXM), which is the widely used steroid
anti-inflammatory drug. As shown in Fig. 8AeD, LPS administration
resulted in the markedly elevation of the inflammatory cytokines
nitric oxide (NO). Activation of NRF2 has been proven to be effective
on relieving inflammatory conditions. Therefore, we investigated
whether 20c can alleviate LPS induced inflammatory conditions in
mouse RAW264.7 cells. As shown in Fig. 7AeD, LPS exposure
including interferon-g (IFN-g), IL-1b, IL-6 and TNF-a in the model
resulted in sharp rise of the levels of the inflammatory factors IL-1
b,
group. In contrast, administration of 20c diminished inflammatory
response, showing comparable therapeutic effects with DXM at the
same dose (10 mg/kg). Noteworthily, a significant dose-effect
relationship was observed as 20c at high dose (10 mg/kg) showed
much more potent anti-inflammatory activities. These results
IL-6, TNF- and NO in RAW264.7 cells. However, this expression
was markedly attenuated in macrophages by treatment with 20c in
a concentration-dependent manner, and treatment with 20c at the
a
high concentration (10
basal level.
mM) almost reduced these cytokines to the
Fig. 6. 20c increased the expression of the antioxidant enzymes SOD (C) and GSH-PX (D), the ratio of GSH/GSSG (E) and scavenged ROS (A) and MPO (F) induced by LPS in mouse
RAW 264.7 cells. (B) Mean fluorescence intensity analysis of ROS level in mouse RAW 264.7 cells analyzed by ImageJ v1.8.0. The RAW 264.7 cells were pretreated with LPS (1 g/mL)
m
for 6 h, and then treated with 20c for another 12 h. The results are expressed as the means SEM (n ¼ 3 independent observations). *p < 0.05, **p < 0.01, ***p < 0.001, which were
calculated with one-way ANOVA.

M.-C. Lu et al. / European Journal of Medicinal Chemistry 207 (2020) 112734
9
Fig. 7. Quantification of the inflammatory cytokines IL-1
with LPS (1 g/mL) for 6 h, and then treated with 20c for another 12 h. The concentration of the above inflammatory cytokines secreted in the culture media was measured by Elisa
kits. The results are expressed as the means SEM (n ¼ 3 independent observations). ***p < 0.001, which were calculated with one-way ANOVA.
b (A), IL-6 (B), TNF-a (C) activities and NO production (D) in mouse RAW 264.7 cells. The RAW 264.7 cells were pretreated
m
indicated that the small molecule KEAP1-NRF2 inhibitor 20c had
profound therapeutic effect on oxidative stress related inflamma-
tory conditions in vivo.
compound 22. Compound 23 was obtained by nucleophilic sub-
stitution of NH by bromoacetate in the presence of K2CO3 in DMF.
Deprotection of the benzyl group with Pd/C in THF solution pro-
duced compound 24. The reaction of compounds 15a, b, e-g with
NBS in the presence of AIBN as a catalyst gave compounds 16a, b, e-
g, and the subsequent Williamson ether synthesis with compound
24 obtained compounds 19a, b, e-g. Hydrolysis of the ester groups
of compounds 19a, b, e-g yielded the target compounds 20a, b, e-g.
2.8. Chemistry
The synthesis of compound 14a-o are shown in Scheme 1. Per-
oxidation of commercially available 1-nitronaphthalene afforded 4-
nitro-1-naphthol (9). The nitro group of 9 was reduced by hydrogen
and Pd/C, and the subsequent N-Boc protection of the amino group
gave compound 10. Using the conventional Williamson ether syn-
thesis, compound 11 was then prepared. Deprotection of the N-Boc
and the subsequent condensation with arylsulfonyl chloride gave
compounds 12a-o, respectively. Compounds 13a-o were obtained
by nucleophilic substitution of NH by bromoacetate in the presence
of K2CO3 in DMF. Hydrolysis of the ester groups of compounds 13a-
o yielded the target compounds 14a-o.
The synthesis of compound 20c, d, h-j are shown in Scheme 2.
The reaction of compounds 15c, d, h-j with N-bromosuccinimide
(NBS) in the presence of 2,2-Azobisisobutyronitrile (AIBN) as a
calatyst gave compounds 16c, d, h-j, and the subsequent Wil-
liamson ether synthesis with compound 10 obtained compounds
17c, d, h-j. Deprotection of the N-Boc and the subsequent
condensation with 2-Mesitylenesulfonyl chloride gave compounds
18c, d, h-j, respectively. Compounds 19a-e were obtained by
nucleophilic substitution of NH by bromoacetate in the presence of
K2CO3 in DMF. Hydrolysis of the ester groups of compounds 19c, d,
h-j yielded the target compounds 20c, d, h-j.
3. Conclusion
The transcription factor NRF2 allows adaptation and survival
under stressed conditions via regulating the gene expression of
diverse networks of cytoprotective proteins. Modulation of NRF2
activation by directly disrupting KEAP1-NRF2 PPI has emerged as a
rational strategy to discover preventive and therapeutic agents for
diseases involving oxidative stress and inflammation. Recent years
have seen a surge in interest in the development of KEAP1ꢀNRF2
PPI inhibitors with a 1,4-diaminonaphthalene core. In this study, a
series of 2-oxy-2-phenylacetic acid substituted naphthalene sul-
fonamide derivatives were rationally designed and synthesized as
potent KEAP1-NRF2 inhibitors and the 1,4-diaminonaphthalene
substituted pattern has been identified as unnecessary for inhib-
iting KEAP1-NRF2 interaction.
On the basis of the potent KEAP1-NRF2 PPI inhibitor 1 reported
by our group previously, we introduced the oxyacetic acid group to
substitute one of the N-acetic acid substituted sulfonamide group.
We suggested that the preferred oxyacetic acid group may occupy
the P1 sub-pocket of KEAP1 and a series of compounds containing
the oxyacetic acid group were synthesized and evaluated. The
acetic acid group and the core naphthalene ring, which are the key
features for KEAP1 binding, have been retained, and the structure-
The synthesis of compound 20a, b, e-g are shown in Scheme 3.
The O-Bn protection of the hydroxyl group in compound 10 resulted
in compound 21. Deprotection of the N-Boc in compound 21 and
subsequent condensation with 2-Mesitylenesulfonyl chloride gave
Table 3
Detailed in vitro/in vivo profile for 20c.a
Rat liver microsomes
T1/2 (hr)
Clint (in vitro)
L/min/mg)
ER
CLH (mL/min/kg)
2.69
Clint (in vivo)
(mL/min/kg)
(
m
10.5
1.57
4.89%
2.83
CYP inhibition IC50
CYP1A2
(mM)
CYP2C9
CYP2C19
CYP2D6
CYP3A4
>10
>10
>10
>10
>10
Rat Pharmacokinetic properties (1 mg/kg i.v. Dosing)
AUC0-t (ng$hr/mL)
AUC0-∞ (ng$hr/mL)
CL_F (mL/min/kg)
6.38 1.01
T1/2 (hr)
1.72 0.42
Cmax (ng/mL)
Vz_F (mL/kg)
953 290
2546 348
2649 401
15100 2326
a
The data were measured in triplicates.

10
M.-C. Lu et al. / European Journal of Medicinal Chemistry 207 (2020) 112734
Fig. 8. 20c reduced the LPS-induced production of the pro-inflammatory factors in vivo. Levels of serum IFN-
g
(A), IL-1
b (B), IL-6 (C) and TNF-a (D) and were measured by Elisa kits.
Mice were randomly divided into five groups: control group (n ¼ 3), LPS group (300
m
g/kg, n ¼ 8), LPS þ 20c (10 mg/kg) group (n ¼ 8), LPS þ 20c (40 mg/kg) group (n ¼ 8),
LPS þ DXM (10 mg/kg) group (n ¼ 8). ***p < 0.001, which were calculated with one-way ANOVA.
Scheme 1. Synthesis of target compounds 14a-o.
aConditions and reagents: (a) (CH3)3COOH, KOH, DMSO/H2O, r. t. 4 h, yield 82%; (b) H2, Pd/C, MeOH, 6 h; (c) (Boc)2O, TEA, MeOH, 5 h, yield 83%; (d) methyl 2-bromo-2-
phenylacetate, K2CO3, DMF, r. t. 2 h, yield 46%; (e) CF3COOH, CH2Cl2, r. t. 30 min; (f) substituted arylsulfonyl chloride, pyridine, THF, r. t. 10 h, yield 50e70%; (g) BrCH2COOCH3,
K2CO3, DMF, 2 h, yield 80e85%; (h) LiOH, MeOH/H2O, 10 h, yield 85e90%.
Scheme 2. Synthesis of target compounds 20c, d, h-j.
aConditions and reagents: (a) NBS, AIBN, CHCl3, 60 ꢁC, yield 70e80%; (b) K2CO3, DMF, r. t. 2 h, yield 46%; (c) CF3COOH, CH2Cl2, r. t. 30 min; (d) 2-Mesitylenesulfonyl chloride,
pyridine, THF, r. t. 10 h, yield 50e70%; (e) BrCH2COOCH3, K2CO3, DMF, 2 h, yield 80e85%; (f) LiOH, MeOH/H2O, 10 h, yield 85e90%.
activity and structure-property relationships of the substitutions in
the P4 and P5 sub-pockets have been investigated. Intensive
structural modifications led to the identification of 20c as the most
potent KEAP1-NRF2 PPI inhibitor with an IC50 of 75 nM in the FP
competition assay. As revealed by the binding mode analysis, 20c is
likely to fulfill all the five sub-pockets of KEAP1; the oxyacetic acid
group in the P1 sub-pocket could retain the original strong polar
interactions; the 2,4,6-trimethyl substitution increased the hydro-
phobic interactions in the P5 sub-pocket; the hydrophobic para-
methoxyl increased the interactions with Tyr525 and probably
formed a new polar interaction with Gln530 in the P4 sub-pocket
and the other part of the molecule could interact with KEAP1 in a
similar way as the lead compound 1.
and prolonged residence time with KEAP1. Cell-based experiments
showed that the KEAP1-NRF2 PPI inhibitor 20c can activate the
NRF2-ARE pathway and induce the expression of Nrf2 downstream
cytoprotective genes and proteins in macrophage cells. 20c can also
enhance the antioxidant capacity in macrophage RAW 264.7 cells
and therefore antagonized the LPS induced cellular inflammatory
conditions. Encouraged by the above results, the in vivo evaluation
was carried out to further confirm the anti-inflammatory effects of
20c in the LPS-induced inflammation mouse model. The decrease
of the LPS induced circulating inflammatory markers, including
IFN-g, IL-1b, IL-6 and TNF-a proved that 20c could effectively
relieve the LPS induced inflammatory responses.
In this study, we identified the 2-oxy-2-phenylacetic acid
substituted naphthalene sulfonamide derivative, 20c, as a potent
KEAP1-NRF2 inhibitor and proved the therapeutic potential of
The subsequent binding characterization assays demonstrated
that 20c had strong binding affinity with a Kd of 24 nM for KEAP1

M.-C. Lu et al. / European Journal of Medicinal Chemistry 207 (2020) 112734
11
Scheme 3. Synthesis of target compounds 20a, b, e-g.
aConditions and reagents: (a) PhCH2Br, K2CO3, DMF, r. t. yield 46%; (b) CF3COOH, CH2Cl2, r. t. 30 min; (c) 2-Mesitylenesulfonyl chloride, pyridine, THF, r. t. 10 h, yield 75%; (d)
BrCH2COOCH3, K2CO3, DMF, r. t. 2 h, yield 90%; (e) H2, Pd/C, THF, 60 ꢁC, yield 95%; (f) NBS, AIBN, CHCl4, 60 ꢁC, yield 70e80%; (g) K2CO3, DMF, r. t. yield 50e60%; (h) CF3COOH, CH2Cl2,
r. t. 1 h; acetic anhydride, TEA, r. t. 5 h; (i) LiOH, MeOH/H2O, r. t. 10 h, yield 90e95%.
KEAP1-NRF2 inhibitor in LPS induced inflammatory conditions. Our
study provided a new chemical probe with which to further study
the biology of the KEAP1-NRF2-ARE pathway and demonstrated
that activation of NRF2 by small molecule KEAP1-NRF2 PPI in-
hibitors could be a viable way to antagonize stress and inflamma-
tion related disorders.
4.1.2. 2-((4-(N-(carboxymethyl)phenylsulfonamido)naphthalen-1-
yl)oxy)-2-phenylacetic acid (14b)
The same procedure as 14a, white solid, yield 76.6%, m. p.
160.0e162.5 ꢁC. 1H NMR (300 MHz, DMSO‑d6)
d: 4.25e4.36 (m, 1H),
4.48e4.56 (m, 1H), 6.08e6.09 (m, 1H), 6.82e6.85 (m, 1H), 7.12e7.15
(m, 1H), 7.39e7.69 (m, 12H), 7.92e7.97 (m, 1H), 8.29e8.32 (m, 1H),
12.93 (br, 2H); IR (KBr, cmꢀ1): 3467, 1732, 1596, 1350, 1163, 1091; EI-
MS HRMS (ESI): found 492.1109 (C26H22NO7S, [MþH]þ requires
4. Experimental section
492.1111); HPLC (85:15 methanol: water with
1‰ TFA):
tR ¼ 4.1 min, 98.6%.
4.1. Chemical synthesis for all target compounds
4.1.3. 2-((4-(N-(carboxymethyl)-4-methylphenylsulfonamido)
naphthalen-1-yl)oxy)-2-phenylacetic acid (14c)
4.1.1. 2-((4-(N-(carboxymethyl)-4-methoxyphenylsulfonamido)
naphthalen-1-yl)oxy)-2-phenylacetic acid (14a)
The same procedure as 14a, gray solid, yield 73.2%, m. p.
To a solution of 13a (61.0 mg, 111.0 mmol) in MeOH/H2O (3 mL/
179.8e181.3 ꢁC. 1H NMR (300 MHz, DMSO‑d6)
d
: 2.27 (s, 3H),
3 mL) was added LiOH$H2O (300.0 mg, 12.5 mmol). After 10 h
stirring at room temperature, the reaction mixture was then diluted
in 30 mL water and quenched with 2 M hydrochloric acid to pH 4.
The crude product was obtained through filtration. Recrystalliza-
tion from ethyl acetate/n-hexane gave compound 14a as a white
solid, yield 53.4%, m. p. 128.5e130.5 ꢁC. 1H NMR (300 MHz,
4.25e4.50 (m, 2H), 6.08e6.09 (m, 1H), 6.82e6.84 (m, 1H), 7.09e7.11
(m, 1H), 7.32e7.55 (m, 9H), 7.64e7.70 (m, 2H), 7.99e8.05 (m, 1H),
8.29e8.30 (m, 1H), 12.87e13.21 (m, 2H); IR (KBr, cmꢀ1): 3468, 1735,
1340, 1234, 1155; HRMS (ESI): found 506.1278 (C27H24NO7S,
[MþH]þ requires 506.1268); HPLC (85:15 methanol: water with 1
TFA): tR ¼ 4.37, 4.42 min, 96.7%.
‰
DMSO‑d6) d: 3.83e3.85 (m, 3H), 4.23e4.47 (m, 2H), 6.05 (s, 1H),
6.81e6.84 (m, 1H), 7.02e7.12 (m, 3H), 7.38e7.70 (m, 9H), 8.00e8.06
(m, 1H), 8.27e8.30 (m, 1H) 13.07 (br, 2H); IR (KBr, cmꢀ1): 3464,
1736, 1340, 1263, 1154; HRMS (ESI): found 522.1225 (C27H24NO8S,
4.1.4. 2-((4-(4-(tert-butyl)-N-(carboxymethyl)phenylsulfonamido)
naphthalen-1-yl)oxy)-2-phenylacetic acid (14d)
[MþH]þ, requires 522.1217); HPLC (85:15 methanol: water with 1
TFA): tR ¼ 4.2 min, 96.8%.
‰
The same procedure as 14a, gray solid, yield 89.0%, m. p.
101.7e103.3 ꢁC. 1H NMR (300 MHz, DMSO‑d6)
d: 1.30e1.32 (m, 9H),

12
M.-C. Lu et al. / European Journal of Medicinal Chemistry 207 (2020) 112734
4.20e4.32 (m, 1H), 4.48e4.56 (m, 1H), 6.12e6.13 (m, 1H), 6.87e6.89
(m, 1H), 7.19e7.23 (m, 1H), 7.44e7.70 (m, 9H), 7.72e7.79 (m, 2H),
7.81e7.87 (m, 1H), 8.29e8.32 (m, 1H), 12.98 (br, 2H); IR (KBr, cmꢀ1):
3442, 1735, 1390, 1348, 1163; EI-MS HRMS (ESI): found 548.1743
(C30H30NO7S, [MþH]þ requires 548.1737); HPLC (85:15 methanol:
4.1.10. 2-((4-(N-(carboxymethyl)-4-(trifluoromethyl)
phenylsulfonamido)naphthalen-1-yl)oxy)-2-phenylacetic acid (14j)
The same procedure as 14a, white solid, yield 84.4%, m. p.
166.7e168.9 ꢁC. 1H NMR (300 MHz, DMSO‑d6)
d: 4.24e4.36 (m, 1H),
4.58e4.65 (m, 1H), 6.08 (s, 1H), 6.85e6.87 (m, 1H), 7.20e7.23 (m,
1H), 7.36e7.58 (m, 5H), 7.63e7.69 (m, 2H), 7.83e7.95 (m, 5H),
8.28e8.32 (m, 1H), 13.12 (br, 2H); IR (KBr, cmꢀ1): 3475, 1729, 1352,
1323, 1167, 1130; EI-MS HRMS (ESI): found 582.0809
(C27H20F3NNaO7S, [MþNa]þ requires 582.0805); HPLC (85:15
water with 1
‰
TFA): tR ¼ 5.9 min, 95.6%.
4.1.5. 2-((4-(N-(carboxymethyl)-3,4-dimethoxyphenylsulfonamido)
naphthalen-1-yl)oxy)-2-phenylacetic acid (14e)
The same procedure as 14a, gray solid, yield 78.9%, m. p.
methanol: water with 1
‰
TFA): tR ¼ 5.0 min, 96.2%.
68.7e71.1 ꢁC. 1H NMR (300 MHz, CDCl3)
d 3.67 (s, 3H), 3.92 (s, 3H),
4.10e4.16 (m, 1H), 5.05e5.11 (m, 1H), 5.74 (s, 1H), 6.59e6.62 (m,
1H), 6.82e6.85 (m, 1H), 7.13e7.19 (m, 2H), 7.30e7.37 (m, 1H),
7.49e7.64 (m, 5H), 7.66e7.73 (m, 3H), 8.38e8.40 (m, 1H); IR (KBr,
cmꢀ1): 3415, 1734, 1508, 1390, 1332, 1264, 1154; EI-MS HRMS (ESI):
found 552.1331 (C28H26NO9S. [MþH]þ requires 552.1323); HPLC
4.1.11. 2-((4-(N-(carboxymethyl)-4-(trifluoromethoxy)
phenylsulfonamido)naphthalen-1-yl)oxy)-2-phenylacetic acid (14k)
The same procedure as 14a, white solid, yield 71.7%, m. p.
215.0e216.8 ꢁC. 1H NMR (300 MHz, DMSO‑d6)
d: 4.24e4.35 (m, 1H),
4.59e4.66 (m, 1H), 6.10e6.11 (m, 1H), 6.87e6.90 (m, 1H), 7.25e7.28
(m, 1H), 7.37e7.59 (m, 7H), 7.65e7.72 (m, 2H), 7.77e7.88 (m, 3H),
8.26e8.37 (m, 1H), 13.10e13.16 (m, 2H); IR (KBr, cmꢀ1): 3416, 1736,
1595, 1350, 1260, 1162; HRMS (ESI): found 598.0778
(C27H20F3NNaO8S, [MþNa]þ requires 598.0754); HPLC (85:15
(85:15 methanol: water with 1
‰
TFA): tR ¼ 3.8,3.9 min, 98.8%.
4.1.6. 2-((4-(N-(carboxymethyl)-2,4,6-
trimethylphenylsulfonamido)naphthalen-1-yl)oxy)-2-phenylacetic
acid (14f)
methanol: water with 1
‰
TFA): tR ¼ 5.3 min, 96.9%.
The same procedure as 14a, white solid, yield 76.9%, m. p.
144.6e146.9 ꢁC. 1H NMR (300 MHz, DMSO‑d6)
d: 2.12e2.20 (m, 9H),
4.1.12. 2-((4-(N-(carboxymethyl)-4-fluorophenylsulfonamido)
naphthalen-1-yl)oxy)-2-phenylacetic acid (14l)
4.28e4.38 (m, 1H), 4.64e4.72 (m, 1H), 6.11e6.14 (m, 1H), 6.83e6.89
(m, 3H), 7.42e7.48 (m, 5H), 7.62e7.79 (m, 4H), 8.27e8.29 (m, 1H),
The same procedure as 14a, white solid, yield 81.5%, m. p.
12.88 (br, 2H); 13C NMR (75 MHz, DMSO‑d6)
d: 20.29, 22.88, 53.08,
97.1e99.3 ꢁC. 1H NMR (300 MHz, DMSO‑d6)
d: 4.25e4.35 (m, 1H),
78.16, 105.48, 122.08, 122.92, 125.48, 126.71, 127.11, 127.61, 128.37,
130.30, 131.58, 131.80, 133.30, 136.73, 139.11, 141.93, 153.05, 170.40,
170.54, 170.72, 170.92; IR (KBr, cmꢀ1): 3415, 1729, 1389, 1328, 1235,
1156; EI-MS HRMS (ESI): found 534.1569 (C29H27NO7S, [MþH]þ
4.50e4.58 (m, 1H), 6.07 (s, 1H), 6.83e6.86 (m, 1H), 7.15e7.18 (m,
1H), 7.37e7.73 (m, 11H), 7.92e7.95 (m, 1H), 8.28e8.29 (m, 1H),
12.96 (br, 2H); IR (KBr, cmꢀ1): 3466, 1731, 1390, 1237, 1155; HRMS
(ESI): found 527.1295 (C26H24FN2O7S, [M
527.1283); HPLC (85:15 methanol: water with
þ
NH4]þ, requires
requires 534.1581; HPLC (85:15 methanol: water with 1
‰ TFA):
1‰ TFA):
tR ¼ 5.8 min, 96.2%.
tR ¼ 4.3 min, 96.6%.
4.1.7. 2-((4-(4-acetamido-N-(carboxymethyl)phenylsulfonamido)
naphthalen-1-yl)oxy)-2-phenylacetic acid (14g)
4.1.13. 2-((4-(N-(carboxymethyl)-4-chlorophenylsulfonamido)
naphthalen-1-yl)oxy)-2-phenylacetic acid (14m)
The same procedure as 14a, white gray solid, yield 73.9%, m. p.
The same procedure as 14a, white gray solid, yield 69.4%, m. p.
68.6e71.0 ꢁC. 1H NMR (300 MHz, DMSO‑d6)
d: 2.10 (s, 3H),
190.1e192.5 ꢁC. 1H NMR (300 MHz, DMSO‑d6)
d
: 4.25e4.36 (m, 1H),
4.25e4.49 (m, 2H), 6.08e6.09 (m, 1H), 6.82e6.85 (m, 1H), 7.09e7.11
(m, 1H), 7.42e7.76 (m, 11H), 8.02e8.05 (m, 1H), 8.28e8.29 (m, 1H),
10.37 (s, 1H), 12.83e13.31 (m, 2H); IR (KBr, cmꢀ1): 3412, 1735, 1591,
1330, 1263, 1156; HRMS (ESI): found 571.1170 (C28H24N2NaO8S,
[MþNa]þ requires 571.1146); HPLC (85:15 methanol: water with
4.52e4.59 (m, 1H), 6.08 (s, 1H), 6.85 (d, 1H, J ¼ 8.49 Hz), 7.18 (d, 1H,
J ¼ 8.10 Hz), 7.38e7.71 (m, 11H), 7.94e8.00 (m, 1H), 8.29e8.30 (m,
1H),12.8e13.4 (br, 2H); IR (KBr, cmꢀ1): 3467, 1735,1343, 1235,1157;
HRMS (ESI): found 526.0714 (C26H21ClNO7S, [MþH]þ, requires
526.0722); HPLC (85:15 methanol: water with
1‰ TFA):
1
‰TFA): tR ¼ 3.5, 3.8 min, 97.7%.
tR ¼ 4.9 min, 96.9%.
4.1.8. 2-((4-(N-(carboxymethyl)-4-nitrophenylsulfonamido)
naphthalen-1-yl)oxy)-2-phenylacetic acid (14h)
4.1.14. 2-((4-(4-bromo-N-(carboxymethyl)phenylsulfonamido)
naphthalen-1-yl)oxy)-2-phenylacetic acid (14n)
The same procedure as 14a, gray solid, yield 56.0%, m. p.
179.0e180.9 ꢁC. 1H NMR (300 MHz, DMSO‑d6)
d: 4.28e4.37 (m, 1H),
The same procedure as 14a, white gray solid, yield 63.3%, m. p.
180.0e182.3 ꢁC. 1H NMR (300 MHz, DMSO‑d6)
d
4.22e4.33 (m, 1H),
4.59e4.66 (m, 1H), 6.07 (s, 1H), 6.82e6.86 (m, 1H), 7.18e7.21 (m,
1H), 7.41e7.48 (m, 3H), 7.55e7.58 (m, 2H), 7.64e7.70 (m, 2H),
7.89e7.97 (m, 3H), 8.32e8.38 (m, 3H), 13.23 (br, 2H); HRMS (ESI):
found 554.1239 (C26H24N3O9S, [M þ NH4]þ, requires 554.1228); IR
(KBr, cmꢀ1): 3425, 1719, 1352, 1235, 1167; HPLC (85:15 methanol:
4.49e4.56 (m, 1H), 6.05 (s, 1H), 6.83 (d, 1H, J ¼ 8.49 Hz), 7.15 (d, 1H,
J ¼ 8.19 Hz), 7.36e7.70 (m, 11H), 7.93e7.98 (m, 1H), 8.27e8.28 (m,
1H); IR (KBr, cmꢀ1): 3475,1736,1343,1235,1101; HRMS (ESI): found
570.0215 (C26H21BrNO7S, [MþH]þ, requires 570.0217); HPLC (85:15
methanol: water with 1
‰
TFA): tR ¼ 5.1 min, 96.1%.
water with 1
‰
TFA): tR ¼ 4.4, 4.6 min, 97.1%.
4.1.9. 2-((4-(4-carbamoyl-N-(carboxymethyl)phenylsulfonamido)
naphthalen-1-yl)oxy)-2-phenylacetic acid (14i)
4.1.15. 2-((4-(N-(carboxymethyl)naphthalene-2-sulfonamido)
naphthalen-1-yl)oxy)-2-phenylacetic acid (14o)
The same procedure as 14a, white purple solid, yield 74.1%, m. p.
The same procedure as 14a, white solid, yield 92.0%, m. p.
179.2e180.9 ꢁC. 1H NMR (300 MHz, DMSO‑d6)
d: 4.32e4.38 (m, 1H),
190.5e193.0 ꢁC. 1H NMR (300 MHz, DMSO‑d6)
d: 4.34e4.45 (m, 1H),
4.52e4.60 (m, 1H), 6.06e6.07 (m, 1H), 6.83e6.86 (m, 1H), 7.14e7.17
(m, 1H), 7.38e7.59 (m, 4H), 7.64e7.81 (m, 4H), 7.81e8.09 (m, 3H),
8.20e8.28 (m, 1H), 8.30e8.32 (m, 1H); IR (KBr, cmꢀ1): 3489, 1754,
1685, 1353, 1168, 1125; EI-MS HRMS (ESI): found 535.1168
(C27H22N2O8S. [MþH]þ requires 535.1170); HPLC (85:15 methanol:
4.53e4.61 (m, 1H), 6.06e6.08 (m, 1H), 6.78e6.82 (m, 1H), 7.12e7.14
(m, 1H), 7.38e7.55 (m, 5H), 7.63e7.76 (m, 5H), 8.05e8.13 (m, 4H),
8.32e8.37 (m, 2H), 12.83 (br, 2H); IR (KBr, cmꢀ1): 3442, 1730, 1593,
1342, 1153, 1131; EI-MS HRMS (ESI): found 542.1282 (C30H24NO7S,
[MþH]þ requires 542.1268); HPLC (85:15 methanol: water with 1
TFA): tR ¼ 4.9 min, 97.6%.
‰
water with 1
‰
TFA): tR ¼ 3.6 min, 95.2%.

M.-C. Lu et al. / European Journal of Medicinal Chemistry 207 (2020) 112734
13
4.1.16. 2-((4-(N-(carboxymethyl)-2,4,6-
trimethylphenylsulfonamido)naphthalen-1-yl)oxy)-2-(4-
(trifluoromethyl)phenyl)acetic acid (20a)
4.1.20. 2-((4-(N-(carboxymethyl)-2,4,6-
trimethylphenylsulfonamido)naphthalen-1-yl)oxy)-2-(4-
isopropoxyphenyl)acetic acid (20e)
The same procedure as 14a, white solid, yield 58.2%, m. p.
The same procedure as 14a, gray solid, yield 44.1%, m. p.
>250 ꢁC. 1H NMR (300 MHz, CDCl3)
d
2.11e2.19 (m, 9H), 4.10e4.16
115.6e118.0 ꢁC. 1H NMR (300 MHz, CDCl3)
d: 1.39e1.41 (m, 6H), 2.23
(m, 1H), 5.15e5.21 (m, 1H), 5.71 (s, 1H), 6.48e6.51 (m, 1H), 6.68 (s,
2H), 7.37e7.39 (m, 3H), 7.45e7.49 (m, 1H), 7.67e7.69 (m, 2H),
7.80e7.82 (m, 2H), 8.23e8.26 (m, 1H); IR (KBr, cmꢀ1): 3416, 1735,
1637, 1326, 1122, 1068; EI-MS HRMS (ESI): found 624.1268
(C30H26F3NNaO7S, [MþNa]þ requires 624.1274); HPLC (85:15
(s, 3H), 2.30 (s, 6H), 4.21e4.27 (m, 1H), 4.60e4.68 (m, 1H),
5.28e5.33 (m, 1H), 5.68 (s, 1H), 6.56e6.59 (m, 1H), 6.79 (s, 2H),
7.00e7.03 (m, 2H), 7.44e7.46 (m, 3H), 7.61e7.67 (m, 3H), 8.34e8.37
(m, 1H); IR (KBr, cmꢀ1): 3449, 1736, 1492, 1331, 1259, 1156; EI-MS
HRMS (ESI): found 592.2009 (C32H34NO8S. [MþH]þ requires
methanol: water with 1
‰
TFA): tR ¼ 7.0 min, 95.1%.
592.2000); HPLC (85:15 methanol: water with 1‰ TFA):
tR ¼ 10.5 min, 95.1%.
4.1.17. 2-((4-(N-(carboxymethyl)-2,4,6-
trimethylphenylsulfonamido)naphthalen-1-yl)oxy)-2-(4-
hydroxyphenyl)acetic acid (20b)
4.1.21. 2-(4-(tert-butyl)phenyl)-2-((4-(N-(carboxymethyl)-2,4,6-
trimethylphenyl sulfonamido) naphthalen-1-yl)oxy)acetic acid (20f)
The same procedure as 14a, yellow solid, yield 24.5%, m. p.
To a solution of compound 26 (300.0 mg, 443.0
mmol) in CH2Cl2
190.2e192.6 ꢁC. 1H NMR (300 MHz, CDCl3)
d: 1.37 (s, 9H), 2.21e2.27
(5.0 mL), CF3COOH (165.0 L, 2.2 mmol) was added, and the reac-
m
(m, 9H), 4.20e4.26 (m, 1H), 5.27e5.34 (m, 1H), 5.70 (s, 1H),
6.54e6.56 (m, 1H), 6.77 (s, 2H), 7.38e7.54 (m, 3H), 7.51e7.54 (m,
2H), 7.60e7.63 (m, 1H), 7.67e7.69 (m, 2H), 8.35e8.37 (m, 1H); IR
(KBr, cmꢀ1): 3420, 1735, 1638, 1324, 1157, 1093; EI-MS HRMS (ESI):
found 590.2202 (C33H36NO7S, [MþH]þ requires 590.2207); HPLC
tion mixture was stirred for 0.5 h at r. t. After the completion of the
reaction monitored by TLC, saturated Na2CO3 was added to the
reaction mixture until pH 7. Then the reaction mixture was
extracted with EtOAc ( ꢂ 3). The combined organic extracts were
washed with saturated NaHCO3 solution, H2O and saturated NaCl
solution, then dried over Na2SO4, filtered, and concentrated in
vacuo. Purification by silica gel column chromatography provided
compound 20b as a white solid, yield 30.9%, m. p. >250 ꢁC. 1H NMR
(85:15 methanol: water with 1
‰
TFA): tR ¼ 10.6 min, 94.9%.
4.1.22. 2-(4-acetamidophenyl)-2-((4-(N-(carboxymethyl)-2,4,6-
trimethylphenyl sulfonamido) naphthalen-1-yl)oxy)acetic acid
(20g)
(300 MHz, DMSO‑d6) d: 2.16e2.21 (m, 9H), 4.15e4.20 (m, 1H),
4.61e4.67 (m, 1H), 5.70e5.77 (m, 1H), 6.72e6.81 (m, 3H),
6.84e6.87 (m, 2H), 7.36e7.50 (m, 5H), 7.72e7.77 (m, 1H), 8.18e8.23
(m, 1H); IR (KBr, cmꢀ1): 3415, 1638, 1618, 1154, 1122; EI-MS HRMS
(ESI): found 550.1513 (C29H28NO8S, [MþH]þ requires 550.1530);
The same procedure as 14a, light yellow solid, yield 37.3%, m. p.
214.0e216.5 ꢁC. 1H NMR (300 MHz, DMSO‑d6)
d: 2.03e2.05 (m, 3H),
2.11 (s, 3H), 2.16e2.19 (m, 6H), 4.26e4.37 (m, 1H), 5.63e5.70 (m,
1H), 5.99e6.01 (m, 1H), 6.83e6.88 (m, 3H), 7.36e7.42 (m, 2H),
7.47e7.57 (m, 1H), 7.60e7.65 (m, 2H), 7.72e7.80 (m, 2H), 8.24e8.28
(m, 1H), 8.31e8.35 (m, 1H), 10.03e10.07 (s, 1H), 13.12 (br, 2H); IR
(KBr, cmꢀ1): 3416, 1603, 1515, 1318, 1155, 1090; EI-MS HRMS (ESI):
found 613.1604 (C31H30N2O8S, [MþNa]þ requires 613.1615); HPLC
HPLC (85:15 methanol: water with 1
‰
TFA): tR ¼ 4.0 min, 95.5%.
4.1.18. 2-((4-(N-(carboxymethyl)-2,4,6-
trimethylphenylsulfonamido)naphthalen-1-yl)oxy)-2-(4-
methoxyphenyl)acetic acid (20c)
(85:15 methanol: water with 1
‰
TFA): tR ¼ 4.0, 4.1 min, 97.8%.
The same procedure as 14a, gray solid, yield 78.0%, m. p.
4.1.23. 2-((4-(N-(carboxymethyl)-2,4,6-
trimethylphenylsulfonamido)naphthalen-1-yl)oxy)-2-(4-
fluorophenyl)acetic acid (20h)
145.1e147.8 ꢁC. 1H NMR (300 MHz, DMSO‑d6)
d
: 2.12e2.19 (m, 9H),
3.75e3.77 (m, 3H), 4.26e4.36 (m, 1H), 4.64e4.71 (m, 1H),
6.02e6.05 (m, 1H), 6.83e6.91 (m, 3H), 6.94e7.02 (m, 2H), 7.37e7.50
(m, 3H), 7.55e7.58 (m, 2H), 7.71e7.78 (m, 1H), 8.23e8.24 (m, 1H),
The same procedure as 14a, gray solid, yield 71.8%, m. p.
152.3e155.0 ꢁC. 1H NMR (300 MHz, CDCl3)
d: 2.22 (s, 3H), 2.29 (s,
12.86e13.20 (m, 2H); 13C NMR (75 MHz, DMSO‑d6)
d: 25.03, 27.61,
6H), 4.21e4.27 (m, 1H), 5.27e5.33 (m, 1H), 5.73 (s, 1H), 6.56e6.59
(m, 1H), 6.78 (s, 2H), 7.18e7.24 (m, 2H), 7.45e7.48 (m, 3H),
7.59e7.62 (m, 1H), 7.73e7.78 (m, 2H), 8.31e8.35 (m, 1H); IR (KBr,
cmꢀ1): 3464, 1762, 1508, 1333, 1270, 1153; EI-MS HRMS (ESI): found
574.1304 (C29H26FNNaO7S, [MþNa]þ requires 574.1306); HPLC
27.67, 57.63, 59.82, 59.86, 81.71, 110.35, 110.50, 118.78, 118.83,
126.77, 127.65, 127.78, 130.34, 130.40, 131.58, 132.53, 132.61, 132.63,
132.66, 133.42, 133.44, 134.92, 136.35, 136.39, 136.60, 136.64, 137.87,
137.96, 143.93, 143.97, 146.81, 146.84, 157.56, 164.36, 175.02, 175.05,
175.45, 175.50; IR (KBr, cmꢀ1): 3415, 1734, 1512, 1240, 1122; EI-MS
HRMS (ESI): found 586.1519 (C30H29NNaO8S. [MþNa]þ requires
(85:15 methanol: water with 1
‰
TFA): tR ¼ 5.8 min, 96.7%.
586.1506); HPLC (85:15 methanol: water with
1‰
TFA):
4.1.24. 2-((4-(N-(carboxymethyl)-2,4,6-
tR ¼ 5.4 min, 97.3%.
trimethylphenylsulfonamido)naphthalen-1-yl)oxy)-2-(4-
chlorophenyl)acetic acid (20i)
The same procedure as 14a, gray solid, yield 40.7%, m. p.
4.1.19. 2-((4-(N-(carboxymethyl)-2,4,6-
trimethylphenylsulfonamido)naphthalen-1-yl)oxy)-2-(4-
ethoxyphenyl)acetic acid (20d)
169.1e171.3 ꢁC. 1H NMR (300 MHz, CDCl3)
d: 2.20 (s, 3H), 2.28 (s,
6H), 4.20e4.26 (m, 1H), 5.26e5.32 (m, 1H), 5.71 (s, 1H), 6.55e6.58
(m, 1H), 6.77 (s, 2H), 7.45e7.49 (m, 5H), 7.59e7.61 (m, 1H),
7.68e7.71 (m, 2H), 8.31e8.33 (m, 1H), 8.57 (br, 2H); IR (KBr, cmꢀ1):
2983, 1763, 1685, 1334, 1159, 1131; EI-MS HRMS (ESI): found
590.1000 (C29H26ClNNaO7S. [MþNa]þ requires 590.1011); HPLC
The same procedure as 14a, gray solid, yield 35.7%, m. p.
127.8e130.7 ꢁC. 1H NMR (300 MHz, CDCl3)
d
:
1.45 (t, 3H,
J ¼ 6.99 Hz), 2.20e2.27 (m, 9H), 4.08 (q, 2H, J ¼ 6.99 Hz), 4.19e4.25
(m, 1H), 5.24e5.30 (m, 1H), 5.65 (s, 1H), 6.53e6.56 (m, 1H), 6.76 (s,
2H), 6.98e7.01 (m, 2H), 7.38e7.45 (m, 3H), 7.58e7.64 (m, 3H),
8.30e8.33 (m, 1H); IR (KBr, cmꢀ1): 3421, 1740, 1513, 1306, 1254,
1159; EI-MS HRMS (ESI): found 600.1652 (C31H31NNaO8S, [MþNa]þ
(85:15 methanol: water with 1
‰
TFA): tR ¼ 7.2 min, 95.5%.
4.1.25. 2-(4-bromophenyl)-2-((4-(N-(carboxymethyl)-2,4,6-
trimethylphenylsulfonamido)naphthalen-1-yl)oxy)acetic acid (20j)
The same procedure as 14a, gray solid, yield 40.5%, m. p.
requires 600.1663); HPLC (85:15 methanol: water with 1
‰ TFA):
tR ¼ 6.4 min, 96.1%.
114.7e117.1 ꢁC. 1H NMR (300 MHz, CDCl3)
d: 2.21 (s, 3H), 2.29 (s,

14
M.-C. Lu et al. / European Journal of Medicinal Chemistry 207 (2020) 112734
6H), 4.20e4.26 (m, 1H), 5.25e5.31 (m, 1H), 5.70 (s, 1H), 6.55e6.58
(m, 1H), 6.78 (s, 2H), 7.41e7.50 (m, 3H), 7.60e7.64 (m, 5H),
8.31e8.34 (m, 1H); IR (KBr, cmꢀ1): 3415, 1735, 1327, 1235, 1156; EI-
MS HRMS (ESI): found 612.0705 (C29H27BrNO7S, requires [MþH]þ
medium free with FBS. Analysis was done with a fluorescence mi-
croscope (OLYMPUS DP72, Japan) equipped with a U-RFL-T power
supply.
requires 612.0686); HPLC (85:15 methanol: water with 1
‰
TFA):
4.2.6. Detection of SOD, GSH-Px and MPO activities and GSH/GSSG
ratio
tR ¼ 7.9 min, 95.4%.
The RAW 264.7 cells were pretreated with LPS (1 mg/mL) for 6 h,
4.2. Biology
and then treated with compound for another 12 h. The activities of
SOD (Total Superoxide Dismutase Assay Kit with WST-8, S0101,
Beyotime, China), GSH-Px (Total Glutathione Peroxidase Assay Kit,
S0058, Beyotime, China) and MPO (MPO Detection Kit, A044,
Nanjing Jiancheng Bioengineering, China) and the ratio of GSH/
GSSG (GSH and GSSG Assay Kit, S0053, Beyotime, China) were
determined using the corresponding detection kits according to the
manufacturer’s instructions.
4.2.1. ITC assay
ITC was performed at 25 ꢁC using a MicroCal ITC200 system. The
sample cell was loaded with 200
at 0.005 mM in 10 mM HEPES buffer (pH 7.4). The syringe was filled
with purified compound in the same buffer solution. 2 L aliquots
of 0.05 mM compound were injected 19 times with 2.5 min in-
tervals and a stirring speed of 750 rpm into the sample cell. Data
were analyzed with the MicroCal Origin software, version 7.0.
mL of purified Keap1 Kelch domain
m
4.2.7. IL-1
Levels of IL-1
(m) ELISA kit, EK0411, Boster), TNF-
Boster), NO production (Nitrate/Nitrite Assay Kit, S0023, Beyotime,
China) and IFN- (IFN- (m) ELISA kit, EK0375, Boster) were eval-
b
, IL-6, TNF-
a
, NO and IFN-
(m) ELISA kit, EK0394, Boster), IL-6 (IL-6
(TNF- (m) ELISA kit, EK0527,
g production
b
(IL-1
b
4.2.2. BLI assay
a
a
The KEAP1 Kelch domain protein was biotinylated and dissolved
in a buffer of 20 mM HEPES, pH 7.0. The interaction between the
inhibitor and the KEAP1 protein was determined by biolayer
g
g
uated using commercially available kits according to the manu-
ꢀ
interferometry using an Octet Red 96 instrument (ForteBio Inc.).
facturer’s instructions.
The binding data were collected at 30 ꢁC. The association and
ꢀ
dissociation plot and kinetic constants were obtained with ForteBio
4.3. In vitro/in vivo profile assays
data analysis software.
4.3.1. Microsome stability
4.2.2.1. S6. Cell culture 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 mouse RAW 264.7 cell
line was obtained from Cell Bank of Shanghai Institute of
Biochemistry and Cell Biology, Shanghai Institutes for Biological
Sciences, Chinese Academy of Sciences. The cells were maintained
by regular passage in modified RPMI-1640 medium (GiBco, Invi-
trogen Corp. USA) supplied with 10% FBS, 100 units per mL peni-
The in vitro microsome stability of the compound was evaluated
in isolated liver microsomes (from CD-1 male rat). Clozapine was
used as reference compound. A solution of liver microsomes
(20 mg/mL) was added to a microcentrifuge tube containing of PBS
at 37 ꢁC, and the mixture was shaken for 10 min before the actual
assay was started. Then, a DMSO solution of test compound
(0.1 mM) was added. For 0 min, add ice-cold acetonitrile to the
wells of 0 min plate and then add NADPH stock solution (10 mM).
Pre-incubate all other plates at 37 ꢁC for 5 min. Add NADPH stock
solution (10 mM) to the plates to start the reaction and timing. At
5 min, 15 min, 30 min, and 45 min, add ice-cold acetonitrile to the
wells of corresponding plates, respectively, to stop the reaction.
After quenching, shake the plates at the vibrator for 10 min and
then centrifuge at 4000 rpm for 20 min. Transfer the supernatant
from each well into a 96-well sample plate containing ultra pure
water for LC-MS/MS analysis.
cillin and 100 m
g/mL streptomycin, cultured at 37 ꢁC in a water
vapour saturated atmosphere with 5% CO2.
The ARE-luciferase activity assay experimental procedures were
carried out as reported previously [62].
4.2.3. Western blot analysis
Anti-NRF2 (ab62352) was purchased from Abcam (Abcam, UK).
AntieHOe1 (#5853S) were bought from Cell Signaling Technology
(USA). Anti-NQO1 (sc-271116) and anti-GCLM (sc-22755) anti-
bodies were purchased from Santa Cruz Biotechnology (Santa Cruz,
CA, USA). Anti-KEAP1 antibody (10503-2-AP) was bought from
4.3.2. CYP inhibition assay
The in vitro microsome stability of the compound was evaluated
in isolated human liver microsomes (Xenotech). The inhibitors of
1A2, 2C9, 2C19, 2D6 and 3A4 were 7-ethxycoumarin, Sulfaphena-
zole, Omeprazole, Promethazine and Fluconazole, respectively.
Proteintech Group, Inc. Anti-b-action (AP0060) was purchased
from Bioworld (Bioworld, USA). Isolation of cell fractions and
Western blotting were performed as previously reported [53].
150 mL of liver microsome solution/well and 2 mL of 1 mM inhibitor
solution/well were added to a 96 well plate and mixed. The plate
was pre-incubated in water bath at 37 ꢁC for 10 min. Following the
pre-incubation period, the reaction was initiated with the addition
4.2.4. RNA extraction and qRT-PCR analysis
Detailed procedure of qRT-PCR was previously reported [62].
The analysis of HO-1, NQO1 and GCLM were performed by using the
StepOne System Fast Real Time PCR system (Applied Biosystems).
Values are expressed as the fold of the control.
of 50
then mixed and incubated at 37 ꢁC for indicated times. After that,
200 L stop solution was added to the samples to quench the re-
action. The reaction mixture was then centrifuged at 4000 rpm for
20 min at 4 ꢁC. While centrifuging, a new 96-well plate with 300
L/
mL of NADPH/Substrate working solution to the samples and
m
m
4.2.5. Detection of ROS levels
well 50% acetonitrile was loaded. Transfer the supernatant from
each well into a 96-well sample plate to bioanalytical services for
LC-MS/MS analysis.
Mouse RAW 264.7 cells were seeded in 6-well plates at the
density of 70e80% confluence per well for overnight incubation.
Then the cells were treated with compound for indicated time.
After treatment, cells were washed once with 2 mL of 10% PBS and
4.3.3. In vivo pharmacokinetic assay
stained with 10
m
M cH2DCF-DA (S0033, Reactive Oxygen Species
Animals: 6 male SD rats, weight 180e220 g. Compound solu-
tions were prepared by dissolving them in normal saline by
Assay Kit, Beyotime, China) in the dark at 37 ꢁC for 20 min in 1640

M.-C. Lu et al. / European Journal of Medicinal Chemistry 207 (2020) 112734
15
cosolvent before experiment. For the IV study, test compound
References
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centrifuged to obtain plasma sample. The samples were then
centrifuged at 4 ꢁC at 2000 rpm for 10 min. Plasma samples were
transferred and stored at approximately ꢀ80 ꢁC prior to analysis.
The plasma samples were determined by LC-MS/MS assays.
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Author contributions
Qi-Dong You and Zheng-Yu Jiang conceived and designed the
study. Meng-Chen Lu performed most of the biological experiment,
analyzed the data and wrote the draft. Hong-Li Shao and Tian Liu
synthesized the compounds. Zheng-Yu Jiang revised the manu-
script extensively and submitted the manuscript on behalf of other
authors. Qi-Dong You also revised this manuscript. Qi-Dong You
and Zheng-Yu Jiang provided the financial support for this project.
All the authors approved the final version of the manuscript.
Declaration of competing interest
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
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Acknowledgments
This study was supported by Projects 81773639, 81773581,
81803363 and 81930100 of the National Natural Science Founda-
tion of China; National Science & Technology Major Project ‘Key
New Drug Creation and Manufacturing Program’, China (No:
2018ZX09711002 and 2017ZX09302003); the Priority Academic
Program Development of Jiangsu Higher Education Institutions;
China
Postdoctoral
Science
Foundation-funded
Project
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(2017M620231 and 2018T110576); CPU2018GY02 of Double First
Class Innovation Team of China Pharmaceutical University; the
Project Program of State Key Laboratory of Natural Medicines,
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Jiangsu Qing Lan Project and the Young Elite Scientists Sponsorship
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Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2020.112734.

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