Design, Synthesis, and Structure-Activity Relationships of Indoline-Based Kelch-like ECH-Associated Protein 1-Nuclear Factor (Erythroid-Derived 2)-Like 2 (Keap1-Nrf2) Protein-Protein Interaction Inhibitors

Article abstract of DOI:10.1021/acs.jmedchem.0c01116

The Keap1 (Kelch-like ECH-associated protein 1)-Nrf2 (nuclear factor erythroid 2-related factor 2)-ARE (antioxidant response element) pathway is the major defending mechanism against oxidative stresses, and directly disrupting the Keap1-Nrf2 protein-protein interaction (PPI) has been an attractive strategy to target oxidative stress-related diseases, including cardiovascular diseases. Here, we describe the design, synthesis, and structure-activity relationships (SARs) of indoline-based compounds as potent Keap1-Nrf2 PPI inhibitors. Comprehensive SAR analysis and thermodynamics-guided optimization identified 19a as the most potent inhibitor in this series, with an IC50 of 22 nM in a competitive fluorescence polarization assay. Further evaluation indicated the proper drug-like properties of 19a. Compound 19a dose-dependently upregulated genes and protein level of Nrf2 as well as its downstream markers and showed protective effects against lipopolysaccharide-induced injury in both H9c2 cardiac cells and mouse models. Collectively, we reported here a novel indoline-based Keap1-Nrf2 PPI inhibitor as a potential cardioprotective agent.

Full text of DOI:10.1021/acs.jmedchem.0c01116

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Article
Design, Synthesis and Structure-Activity Relationships of Indoline-
Based Kelch-like ECH Associated Protein 1-Nuclear Factor (Erythroid-
derived 2)-like 2 (Keap1-Nrf2) Protein-Protein Interaction Inhibitors
Haishan Zhou, Lvbin Hu, Han Zhang, Wenxin Shan, Yan Wang,
Xue Li, Tian Liu, Jing Zhao, Qi-Dong You, and Zhengyu Jiang
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.0c01116 • Publication Date (Web): 09 Sep 2020
Downloaded from pubs.acs.org on September 10, 2020
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Design, Synthesis and Structure-Activity
Relationships of Indoline-Based Kelch-like ECH
Associated Protein 1-Nuclear Factor (Erythroid-
derived 2)-like 2 (Keap1-Nrf2) Protein-Protein
Interaction Inhibitors
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a,b
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a,b
Hai-Shan Zhou , Lv-Bin Hu , Han Zhang , Wen-Xin Shan , Yan Wang , Xue Li , Tian
Liu , Jing Zhao , Qi-Dong Youa,b*and Zheng-Yu Jianga,b*
a,b
a,b
aState Key Laboratory of Natural Medicines, and Jiang Su Key Laboratory of Drug Design and
Optimization, China Pharmaceutical University, Nanjing 210009, China
bDepartment of Medicinal Chemistry, School of Pharmacy, China Pharmaceutical University,
Nanjing 210009, China
ABSTRACT The Keap1 (Kelch-like ECH-Associating protein 1)-Nrf2 (nuclear factor erythroid
2
-related factor 2)-ARE (antioxidant response element) pathway is the major defending
mechanism against oxidative stresses, and directly disrupting the Keap1-Nrf2 protein-protein
interaction (PPI) has been an attractive strategy to target oxidative stress-related diseases,
including cardiovascular diseases. Here, we describe the design, synthesis, and structure-activity
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relationships (SAR) of indoline based compounds as potent Keap1-Nrf2 PPI inhibitors.
Comprehensive SAR analysis and thermodynamics guided optimization identified 19a as the most
potent inhibitor in this series, with an IC of 22 nM in a competitive fluorescence polarization
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assay. Further evaluation indicated the proper drug-like properties of 19a. Compound 19a dose-
dependently upregulated genes and proteins level of Nrf2 as well as its downstream markers and
showed protective effects against lipopolysaccharide (LPS)-induced injury in both H9c2 cardiac
cells and mouse models. Collectively, we reported here a novel indoline-based Keap1-Nrf2 PPI
inhibitor as potential cardioprotective agent.
Introduction
The human body is surrounded by endogenous and exogenous electrophilic substances,
including reactive oxygen species (ROS) and reactive nitrogen species (RNS).1, 2 Sustained high
levels of ROS and RNS can induce detrimental oxidative stress, which lead to tissue damage.2-4
To resist such damage, cells have evolved the sophisticated cytoprotective system that can up-
regulate cytoprotective factors to maintain body homeostasis.5, 6 Many detoxification and
antioxidant enzymes, such as superoxide dismutase (SOD), NADPH: quinone oxidoreductase 1
(NQO-1), heme oxygenase-1 (HO-1), glutamate-cysteine ligase-modifier subunits (GCLM),
transcriptionally regulated by the upstream antioxidant response element (ARE), are the major
components of body’s defense system.7, 8 Nrf2 (nuclear factor erythroid 2-related factor 2), binding
to ARE and regulating the transcription of these oxidative stress related genes, is the chief regulator
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of this cytoprotective system. Keap1 (Kelch-like ECH-associated protein-1), identified as a
repressor of Nrf2, inhibits the activity of Nrf2 via ubiquitination of Nrf2 under basal conditions.10,
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However, upon stress conditions, modification of several highly sensitive cysteines in Keap1
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leads to the dissociation of Nrf2 from Keap1, thereby preventing ubiquitination of Nrf2.12, 13 As a
result, Nrf2 accumulates and translocates into the nucleus, which then binds to ARE and up-
regulates the expression of cytoprotective factors, ultimately protecting cells (Figure 1).14, 15
Therefore, Keap1-Nrf2-ARE pathway is a key pathway for cells to resist oxidation and maintain
cell homeostasis, and Nrf2 activation agents may be developed as therapeutic drugs for a series of
diseases related to oxidative stress.16, 17
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Figure 1. Schematic diagram for the regulation of Nrf2 activity.
Many electrophilic Nrf2 activators have been developed as potential therapeutic agents (Figure
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2B), such as dimethyl fumarate (DMF, 1), sulforaphane (SFN, 2), bardoxolone methyl (CDDO-
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Me, 3) and RTA-408 (4), which can form covalent bonds to cysteine residues in the target
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Keap1 protein. However, these activators may react with other cysteine-rich proteins to increase
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the uncertainty in further clinical development. Alternatively, directly interfering the Keap1-Nrf2
protein-protein interaction (PPI) is regarded as a more attractive approach to active Nrf2.24-33
Direct inhibitors of Keap1-Nrf2 PPI do not require a covalent group, which may enhance
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selectivity and specificity. The binding between Keap1 and Nrf2 is confined to the interactions
between the Kelch domain of Keap1 and the two Nrf2 motifs with low affinity (DLG) and high
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affinity (ETGE) (Figure 2A). On the basis of co-crystal structures of the Keap1-Nrf2 ETGE
complex, the binding pocket of Keap1 can be artificially subdivided into five sub-pockets, namely,
P1-P5: P1 and P2 contain most of the polar residues (Arg483, Ser508, Ser363, Arg380 and
Arg415); P4 and P5 mainly contain nonpolar residues, including Tyr525, Gln530, Tyr572, Tyr334,
and Phe577; P3 sub-pocket consists of small polar and nonpolar residues (Gly509, Ala556, Ser602
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and Ser555). Benefiting from the availability of these crystal structures of the Kelch domain of
Keap1, various Keap1-Nrf2 PPI inhibitors, including peptides37-42 and non-peptide small
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molecules, have been reported and their therapeutic potential have been explored in several in
vivo models44-46. Among them, the 1,4-diaminonaphthalene core-based compounds represent a
main type of Keap1-Nrf2 PPI inhibitors. Based on the hit compound 5a discovered by Marcotte et
al. (Figure 2C),47 N-acetic acid groups were introduced into the both sulfonamides of 5a and
compound 5b was obtained with significant improvement in activity.48 To improve the
physicochemical and drug-like properties of these naphthalene-based Keap1-Nrf2 inhibitors,
further structure optimizations have been carried out, including bioisosteres replacement
(compounds 5c and 5d),49, 50 asymmetrical substituent (compounds 6a and 6b),51, 52 naphthalene
scaffold replacement (compounds 7a and 7b)53, 54 and prodrug modification . Other potent
inhibitors with 3-phenylpropanoic acid core have been reported by Davies et al.56 The
representative compound 8a, with drug-like physicochemical properties, showed potent Nrf2
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activation effects in cellular and in vivo models. Recently, research group from Biogen disclosed
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a novel series of non-covalent Nrf2 activators with benzotriazole moiety. The representative
analogue 8b showed impressive oral PK and good Nrf2-dependent gene inductions in kidney.
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Figure 2. Chemical structures of representative Nrf2 activators. (A) Sub-pockets of the Keap1
binding cavity based on the Keap1-Nrf2 ETGE complex (PDB 1X2R); (B) Electrophilic activators
of Nrf2; (C) Representative non-electrophilic Keap1-Nrf2 PPI inhibitors.
Myocarditis is classified as an inflammatory disease of the heart muscle and is an important
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cause of sudden death, especially in children and younger patients. It has been reported that viral
infection, bacterial infection, rheumatic carditis, and autoimmunity are the major contributors of
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myocarditis. In etiological consideration, myocarditis is characterized by the overexpression of
inflammatory cytokines and oxidative mediators.60 By controlling the release of inflammatory
cytokines and regulating the transcription of antioxidant enzymes, such as HO-1 and NQO1, the
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Nrf2-ARE pathway plays a critical role in the regulation of oxidative stress and inflammation in
tissue damage.61 Recent studies have reported that Keap1-Nrf2 inhibitors could exert
cardioprotective effects through anti-inflammatory and antioxidant effects,62, 63 and the Keap1-
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Nrf2 signaling may be a potential target for developing new cardioprotective drugs.64
Here, we describe the design, synthesis, and structure-activity relationship (SAR) of a series of
new indoline-based Keap1-Nrf2 PPI inhibitors. Replacing the acetic acid groups with
acylsulfonamide moieties retained the high Keap1-Nrf2 inhibitory activity. The representative
analogue 19a (structure seen in the Abstract) exhibited potent Keap1 inhibitory activity, favorable
drug-like properties, high Nrf2 activation effects in cell-based assays, and effective
cardioprotective effects against lipopolysaccharide (LPS)-induced injury both in vitro and in vivo.
This study provides a potent Keap1-Nrf2 PPI inhibitor with the novel indoline scaffold and without
acetic acid groups as potential cardioprotective agent.
RESULTS AND DISCUSSION
Discovery of Initial Hit Compound 10a as the Keap1-Nrf2 Inhibitor. The Keap1-Nrf2-ARE
signaling has emerged as a potential target to mediate some diseases related to oxidative stress and
inflammation, and numerous Nrf2 activators have been identified, including electrophilic and non-
covalent compounds. Particularly, more and more non-covalent Keap1-Nrf2 PPI inhibitors have
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been developed and some of them exhibit therapeutic potential in multiple animal models. In an
effort to discover a new scaffold for Keap1-Nrf2 inhibitors, we replaced the naphthalene ring with
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a benzene ring based on the structure reported previously by our group. Then a small hydrophobic
methyl group was added to the core phenyl ring to compensate for the loss of hydrophobic effects
on binding activity to some extent and fragment 9 was designed and synthesized. 9 showed weak
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inhibitory activity in Keap1-Nrf2 PPI determined by a fluorescence polarization (FP) assay (IC50
=
450 μM). Despite the weak activity, this fragment contained a phenyl ring other than naphthalene
ring and the potency of such fragment can be further enhanced by means of structure optimization.
By analyzing the reported potent inhibitors, we thought that introduction of hydrophobic groups
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(such as methoxy group substituted aniline, shown in the compounds 5a-5c) may increase
interactions with the Keap1 cavity. Inspired by the potent inhibitor 8a, the meta position of core
phenyl ring may be a favorable site to elicit a new substituent group. Then we introduced a 3-
methoxyaniline moiety into compound 9 and compound 10a was obtained with enhanced activity
(
assessed by a FP assay, IC = 102 μM, Figure 3).
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Figure 3. The design of compound 10a.
SAR of Core Phenyl Ring of Phenylenediamine Compounds. To improve the potency of
Keap1-Nrf2 inhibition, we explored the effects of various substituents in the core phenyl ring of
compound 10a on its activity using a FP assay, as shown in Table1. First, the methyl group was
introduced at different position of the phenyl ring and resulted in target compounds 10b-10e.
Substitutions at the 4- and 5-position (10b and 10c) as well as analogue 10e without methyl
substituent showed similar activity compared with 10a, while 2-position substitution (10d) gave a
nearly 3-fold increase in inhibitory activity. Then trifluoromethyl, fluorine and methoxy were
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introduced at 2-position and compounds 10f-10h were obtained. The electron-donating
substituents were more suitable than electron-withdrawing substituents at this position and all of
them showed decreased potency compared with 10d. Moreover, the 1,4-phenylenediamine
analogue 10i gave an abolished activity, indicating the necessity of the 1,3-substitued core phenyl
ring.
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Table 1. Effects of Core Phenyl Modifications on Keap1-Nrf2 Inhibition
O
S
N
COOH
O
2
6
R
5
HN
4
O
IC ± SE
5
0
Cpd.
R
Cpd.
R
IC ± SE (μM)
50
(μM)
1
0a
10b
0c
0d
0e
0f
6-CH3
5-CH3
4-CH3
102±4.49
93.3±2.87
95.6±2.49
30.1±1.21
101±5.82
>500
10g
10h
2-F
80.1±3.83
55.3±3.12
2-OCH3
1
O
S
1
2-CH3
N
COOH
O
10i
>500
1
H
HN
O
1
2-CF3
Design of Compounds with Indoline Core. We hypothesized that the core phenyl ring of 10d
may occupy the P3 sub-pocket to stabilize the binding conformation just like the naphthalene ring
of compound 5. Therefore, we supposed the cyclization of the secondary amine with the methyl
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moiety of the core phenyl ring may obtain beneficial conformation restriction, resulting in
compound 11a. Inspiringly, as shown in Figure 4, indoline 11a showed approximately 2-fold
improvement in IC compared with 10d. Subsequent replacement of indoline core with indole
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ring (12) and 1,2,3,4-tetrahydroquinoline ring (13) both decreased activity, indicating the
rationality of the five-membered alkyl ring. Then we explored the effects of substituents on the
benzene sulfonamide fragment on inhibitory activity and compounds 11b-11f were synthesized
(Figure 4). Unfortunately, these common substituents exhibited abolished activity, manifesting the
importance of the 2,4,6-trimethyl substitution on the benzene in this scaffold. The effects of the
linker X between indoline ring and phenyl ring were investigated next. We chose amide, sulfamide
and methylene as the linker and compounds 14a-c were synthesized. The sulfonyl (14b) and
methylene (14c) analogues exhibited comparable inhibitory activity to 11a, while the amide
derivative 14a showed significant loss of potency, indicating the requirement of certain orientation
for the linker (Figure 4). Finally, we chose the methylene analogue 14c as a hit for further
optimization in consideration of synthetical accessibility and diversity.
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Figure 4. The design of indoline compound 11a and initial SAR investigation of this structure.
Since the exact binding mode of this chemotype with Keap1 was not available, the effects of
different substituents on benzyl moiety of 14c on Keap1-Nrf2 PPI inhibition were systematically
evaluated and derivatives 14d-14z were obtained. The SAR data of these compounds were
summarized in Table 2. Generally, most substituents led to abolished activity. Substitutions at
para- and ortho-positions (14d-14i) resulted in decreased inhibitory activity with respect to 14c.
Then the 3-position substituted compounds (14k-14s) and disubstituted analogues (14t-14z) also
exhibited no improvement in potency, implying that original 3-methoxy is optimal.
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Table 2. Effects of Benzyl Substituents on Keap1-Nrf2 Inhibition
O
S
N
COOH
O
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
N
R
IC ± SE
IC ± SE
(μM)
5
0
50
Cpd.
R
Cpd.
R
(μM)
1
4c
4d
4e
4f
4g
14h
4i
14j
4k
4l
4m
4n
3-OCH3
4-OCH3
16.4±0.26
98.0±1.49
14o
14p
3-CN
24.8±0.86
1
3-Ph
>100
>100
1
4-CH3
4-CF3
4-Br
>100
>100
14q
14i
3-OCH CH
2
3
1
3-OCH(CH3)2
3-NO2
>100
1
>100
14s
14t
27.2±0.84
22.2±0.97
98.2±2.36
>100
4-Cl
>100
3,5-CH3
1
2-OCH3
H
>100
14u
14v
14w
14x
14y
14z
2,3-CH3
42.0±1.03
>100
3,5-OCH3
3-F-5-OCH3
1
3-CH3
>100
1
3-CF3
>100
3,4-OCH3
>100
1
3-Br
>100
3-Br-5-OCH3
>100
1
3-Cl
>100
3-OCH -5-CH
>100
3
3
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1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Then we investigated the binding mode of this chemotype with Keap1 using a docking study of
4
8
1
4c to the Keap1 Kelch domain. On the basis of the SAR data obtained above, we hypothesized
that the 3-methoxy substituted benzyl moiety probably occupied a narrow pocket. As shown in
Figure 5A, the carboxylic acid moiety of 14c can insert into the P2 sub-pockets and interact with
Arg415 and Arg380 by hydrogen bonds and electrostatic interactions. The 2,4,6-trimethyl
substituted phenyl ring moiety can occupy the hydrophobic pocket P5, interacting with Tyr572,
Tyr334, and Phe577. The indoline core can occupy the P3 sub-pocket similar to the naphthalene
core. Interestingly, the 3-methoxyl substituted benzyl moiety is inserted into the narrow and polar
P1 sub-pocket other than P4 hydrophobic pocket. This predicated binding mode also explained
why most substituents led to abolished activity.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Design and Optimization of Indoline Compounds with Acyl Sulfonamides. Inspection of the
binding mode of 14c suggested that the benzylic carbon is close to Arg415 and carboxyl group can
be introduced at this position. Thus, compound 15 was designed and synthesized (Figure 5B and
Figure S1). 15 showed approximately 7-fold improvement in IC . Considering the undesired
5
0
effect of carboxy group on physicochemical properties and penetrability of Keap1-Nrf2 inhibitors,
we performed a bioisosteric replacement method to improve the drug-like properties of indoline
compounds. It is reported that acyl sulfonamide with chemical and enzymatic stability is a
6
6
favorable replacement of carboxyl group. Thus, N-acylsulfonamide moiety was built by direct
coupling of the carboxylic acid with different substituted sulfonamides and resulted in analogues
16a-16z (Figure 5B).
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3
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3
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3
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9
0
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0
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9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 5. Proposed binding mode of compound 14c with Keap1 using molecular docking and
design of acyl sulfonamide derivatives. (A) The binding mode of 14c with Keap1 using a docking
method. The docking site was derived from the position of the small molecular ligand
cocrystallized in the binding site of Keap1 (PDB 4XMB). The ligand is represented as sticks.
Hydrogen bonds are represented by green dashed lines, and electrostatic interactions are
represented by orange dashed lines. The π-π interactions are represented by pink dashed lines. The
carbon atoms of small molecules and Keap1 residues are colored cyan and purple, respectively.
(B) Design of acyl sulfonamide derivatives.
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
As shown in Table 3, the alkyl analogues (16b and 16c) and aromatic heterocycles (16d and
6e) showed no improvement compared with 15, whereas phenyl substituent 16a exhibited slightly
1
better potency, indicating that the acyl-sulfonamide phenyl is a favorable substitute of carboxylic
acid. In order to confirm the rationality of this optimization, the thermodynamic data of compounds
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
6
7
1
5 and 16a were determined using a tracer displacement assay. As shown in Table 4, the value
of binding enthalpy of 15 is positive (ΔH = 0.69 kJ/mol), which is compensated by significantly
large negative values for -TΔS, indicating that the binding is solely entropy driven, whereas the
negative values of ΔH and -TΔS of 16a suggested the favorable enthalpy and entropy contributions
in the binding of 16a to Keap1.68-71 Encouraged by these results, an exhaustive SAR exploration
of the aromatic ring was performed and target compounds 16f-16z were synthesized. Generally,
as shown in Table 5, the phenyl ring can tolerate a variety of substituents. The substitutions at
para- and ortho-positions showed better performance, whereas the substituent at meta-position was
not preferred for Keap1 inhibition (16g, 16m and 16n). Among them, the analogues 16g with 4-
methyl substitute and 16l with 2-methoxy substitution exhibited the most potent inhibitory activity.
Then disubstituted analogues were designed and synthesized based on the results obtained above
(
16v-16y). Interestingly, derivatives 16w and 16x exhibited sub-micromolar potency. The most
potent analogue 16w with 2-methoxy-4-methyl substitutions gave an IC value as low as 0.23
5
0
μM. The naphthalene 16z showed slightly decreased potency. The thermodynamic data of
representative compounds showed that most of these compounds exhibited favorable binding
enthalpy and entropy in the binding to Keap1 (Table 4).
Table 3. Effects of Sulfonamide Substitutions on Keap1-Nrf2 Inhibition
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O
S
O
N
COOH
N
O
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
H
O
N
R
S
O
O
IC ± SE
IC ± SE
(μM)
5
0
50
Cpd.
R
Cpd.
16c
R
(μM)
CF3
1
5
/
2.37±0.06
1.82±0.09
4.89±0.24
1
6a
16d
16e
10.4±0.45
5.17±0.15
N
S
1
6b
5.72±0.28
a
Table 4. Thermodynamics Data for Representative Compounds
Cpd. IC ± SE (μM) ΔG (kJ/mol)
ΔH (kJ/mol)
0.69
-TΔS (kJ /mol)
-32.6
5
0
1
5
2.37±0.06
1.82±0.09
1.39±0.11
2.86±0.21
1.32±0.10
1.79±0.11
0.23±0.03
-31.9
-33.0
-33.7
-31.2
-34.0
-32.8
-37.5
16a
6g
16h
6l
6m
6w
-19.4
-13.6
1
-23.8
-9.95
4.36
-35.6
1
-22.3
-11.7
1
-3.58
-29.2
1
-23.6
-13.8
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
6x
0.64±0.08
-34.4
-18.8
-15.6
aThe thermodynamic data of compounds were determined by a FP assay as described in
Supporting Information. ΔH, ΔS, and ΔG refer to the changes in binding enthalpy, entropy, and
total Gibbs free energy, respectively.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 5. Effects of Benzene Side Chain Sulfonamide Substituents (R) on Keap1-Nrf2
Inhibition
O
S
N
COOH
O
N
O
H
O
N
R
S
O
O
IC ± SE
IC ± SE
5
0
50
Cpd.
R
Cpd.
R
(μM)
(μM)
1
6a
16f
6g
16h
6i
6j
6k
6l
6m
6n
H
1.82±0.09
3.39±0.12
1.39±0.11
2.86±0.21
2.92±0.16
2.95±0.24
44.5±2.35
1.32±0.10
1.79±0.11
3.81±0.22
16q
16r
16s
16t
2-F
2-Cl
12.0±0.32
4-OCH3
4-CH3
4-F
2.76±0.13
2.34±0.11
16.7±0.98
9.69±0.56
2.65±0.32
0.23±0.03
0.64±0.08
6.75±0.23
1.81±0.16
1
2-Br
2-CF3
2-OCF3
1
4-Cl
16u
16v
16w
16x
16y
16z
1
4-Br
2,4-CH3
1
4-CF3
2-OCH3
2-CH3
3-CH3
2-OCH -4-CH
3
3
1
2,4-OCH3
3,5-CH3
1
1
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1
6o
2-OCH CH
2.56±0.16
2
3
O
S
N
COOH
O
O
N
H
N
O
1
6p
2-CH CH
3.58±0.14
2
3
S
O
O
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Then the binding mode of 16w was elucidated by docking study and the result gave the reason
for the increased activity of compound 16w (Figure 6A). As expected, the NH of the acyl
sulfonamide located at the P1 subsite and formed crucial electrostatic interaction with Arg483 of
Keap1. Furthermore, the carbonyl and sulfonyl fragments increased the hydrogen bonding
interactions with Ser508 and Ser555, respectively. Moreover, the phenyl ring can form a π-π
stacking interaction with Tyr525 and the rest of compound 16w can bind to Keap1 in a similar
way as the compound 14c. Interestingly, the 2-methoxy-4-methyl substitutions of the phenyl ring
can probably locate the acyl sulfonamide moiety at an appropriate position for binding.
Reinspection of the binding mode of 16w gave an idea that the carboxy group can also be
replaced with the bioisostere to increase additional interactions with Keap1. Encouraged by the
favorable result of the introduction of acyl sulfonamide moiety, we replaced the carboxy group of
1
6w with acyl sulfonamide isostere by the similar method described above and resulted in the
derivative 17a with diacyl sulfonamide groups (structure shown in Table 6). The FP assay gave
the positive result that 17a exhibited comparable activity with the highly potent Keap1-Nrf2
48
inhibitor 5b reported previously. We then installed several common substituents in the phenyl
ring of 17a and analogues 17b-17h were synthesized (Table 6). The 4-methoxy 17b showed
comparable potency to 2,4,6-trimethyl 17a. The thermodynamic data exhibited that both 17a and
17b are more potent than 16w (ΔG ~ -40.1 kJ/mol for 17a and ΔG ~ -40.0 kJ/mol for 17b, whereas
ΔG ~ -37.5 kJ/mol for 16w at 296 K, Table 7).
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 6. Effects of Benzene Sulfonamide Substituents (R) on Keap1-Nrf2 Inhibition
R
O
H
S
N
N
S
O
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
O
O
O
N
O
H
N
O
S
O
O
O
IC ± SE
IC ± SE
(μM)
5
0
50
Cpd.
R
Cpd.
R
(μM)
1
7a
7b
7c
7d
2,4,6-CH3
0.058 ± 0.001
0.064 ± 0.001
0.11 ±0.01
17e
17f
4-NHCOCH3
0.23 ± 0.01
1
4-OCH3
2-OCH3
0.16 ± 0.02
0.15 ± 0.01
0.24 ± 0.05
1
4-CH3
17g
17h
3-OCH3
1
4-F
0.58 ± 0.08
H
a
Table 7. Thermodynamics Data of Compounds 17a, 17b and 19a
IC ± SE
(μM)
△H
(kJ/mol)
5
0
Cpd.
ΔG (kJ/mol)
-T△S (kJ /mol)
1
7a
7b
19a
0.058 ± 0.001
-40.1
-40.0
-43.1
-31.3
-28.2
-26.5
-8.84
-11.8
-16.6
1
0.064 ± 0.001
0.022 ± 0.001
aThe thermodynamic data of compounds were determined from a FP assay as described in
Supporting Information. ΔH, ΔS, and ΔG refer to the changes in binding enthalpy, entropy, and
total Gibbs free energy, respectively.
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The impact of sulfonamide substitutions (Table 8) was subsequently examined based on
structure of 17b. Alkyl (18a-18c) and heterocycles (18d and 18e) substitutions exhibited decreased
activity compared with 17b, indicating the priority of aromatic ring at this position.
Table 8. Effects of Sulfonamide Substitutions (R) on Keap1-Nrf2 Inhibition
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
O
O
H
R
S
N
N
S
O
O
O
O
N
O
H
O
N
S
O
O
O
IC ± SE
IC ± SE
(μM)
50
50
Cpd.
17b
R
Cpd.
18c
R
(μM)
CF3
0.064 ± 0.001
15.1± 2.35
1
8a
0.61 ± 0.09
1.74± 0.14
18d
18e
0.77 ± 0.10
5.16± 0.45
N
S
1
8b
Thus, further modifications of the side chain phenyl ring were performed (Table 9). The SAR
data revealed that the para-position can tolerate several substituents, while the meta- and ortho-
positions are not suitable for substitution (19a-19i). Among them, the 4-methoxy analogue 19a
showed potent Keap1-Nrf2 inhibitory activity with an IC50 of 22 nM in the FP assay. The
thermodynamic data of 19a also gave a satisfactory character that the binding of 19a to Keap1 is
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
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primarily enthalpy driven (Table 7). The docking study of compound 19a to Keap1 gave a similar
binding mode as 16w (Figure 6B), while the 4-methoxyphenyl substituted acyl sulfonamide moiety
afforded sufficient interactions with P2 sub-pocket.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 9. Effects of side chain phenyl ring modifications (R) on Keap1-Nrf2 Inhibition
O
O
H
N
R
S
O
N
S
O
O
O
N
O
H
O
N
S
O
O
O
IC ± SE
IC ± SE
(μM)
50
50
Cpd.
17b
R
Cpd.
R
(μM)
H
0.064 ± 0.001
0.022 ± 0.001
0.12 ± 0.01
0.13 ± 0.02
0.40 ± 0.06
19e
19f
19g
19h
19i
4-CH CH
0.19 ± 0.01
2
3
1
9a
19b
9c
9d
4-OCH3
4-CH3
4-F
4-OH
0.51 ± 0.03
0.048 ± 0.001
> 100
4-OCH CH
2
3
1
2-OCH3
1
4-CF3
3-OCH3
> 100
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Figure 6. Proposed binding mode of compounds 16w (A) and 19a (B) with Keap1 using molecular
docking. The docking site was derived from the position of the small molecular ligand
cocrystallized in the binding site of Keap1 (PDB 4XMB). The ligand is represented as sticks.
Hydrogen bonds are represented by green dashed lines, and electrostatic interactions are
represented by orange dashed lines. The π-π interactions are represented by pink dashed lines. The
carbon atoms of small molecules and Keap1 residues are colored cyan and purple, respectively.
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Biophysical Characterization of Selected Compounds Binding to Keap1. To recheck
thermodynamic parameters obtained from the tracer displacement assay, the isothermal titration
calorimetry (ITC) assay was used to test representative compounds 15, 16w and 19a. Consistent
with the data obtained by the tracer displacement assay, compound 15 was dominated by entropic
effects, while compounds 16w and 19a were are primarily enthalpy driven (Figure 7A). Meanwhile,
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the ITC assay gave a K of 58.4 nM of 19a to Keap1 Kelch domain, similar with the potent diacetic
d
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compounds. To further inspect the Keap1 binding properties of 19a, the biolayer interferometry
BLI) technique was applied to monitor the binding kinetics of 19a and gave a K of 26.4 nM
(
d
consistent with the ITC data (Figure 7B). The binding curves and the association rate constant (kon)
indicated a fast binding process of 19a to Keap1, while a relatively slow dissociation rate constant
(
k ) gave the low K value. These binding assays together confirmed the direct and tight binding
off d
of 19a to Keap1.
Figure 7. The Keap1 Binding Properties of selected compounds. (A) ITC titration profiles of
Keap1 Kelch Domain with compounds 15, 16w and 19a (15: 0.02 mM Keap1 Kelch domain was
titrated with 0.2 mM compound 15; 16w: 0.02 mM Keap1 Kelch domain was titrated with 0.2 mM
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compound 16w; 19a: 0.02 mM Keap1 Kelch domain was titrated with 0.2 mM compound 19a).
The thermodynamic parameters of the interaction of Keap1 with compounds as determined by the
ITC assay were listed in the table. N is the stoichiometric coefficient. K is the binding constant.
d
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ΔH, ΔS, and ΔG refer to the changes in binding enthalpy, entropy, and total Gibbs free energy,
respectively. ΔG is calculated according to the equation ΔG = ΔH − TΔS, where T is the absolute
temperature used for the ITC experiment. (B) BLI dose-response curve reflecting the direct
binding of 19a to Keap1. The binding kinetics parameters were listed in the table. K value is
d
calculated by k /k . Concentrations: 100, 50, 20, and 5 nM.
dis on
Drug-like Properties of Selected Compounds. Metabolic stability is a prior consideration of
5
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developing non-electrophilic Keap1-Nrf2 inhibitors. On the basis of their good in vitro potency,
we evaluated the stability of compounds 17a, 17b and 19a in human liver microsomes. As shown
in Table 10, compounds 17b and 19a exhibited good human microsomal stability, while 17a was
relatively metabolically unstable in human liver microsomes.
Mainly due to the polar binding pocket of the Keap1 binding domain, most of the Keap1-Nrf2
PPI inhibitors contain carboxyl usually meaning unfavorable penetrability and affecting the
performance in cellular and in vivo assay (such as 5b). Compounds 17a, 17b and 19a were selected
to profile in a parallel artificial membrane permeability assay (PAMPA). Compared to bis-
carboxylic acid inhibitor, 5b, acyl sulfonamides analogues 17a, 17b and 19a showed
comparatively better PAMPA permeability (Table 10).
Table 10. In Vitro Metabolic Stability in Human Liver Microsomes and PAMPA
a
Permeability Data of Selected Compounds
Parameters
Cpd.
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7a
17b
19a
5b
4
5 min remaining (%)
t1/2 (min)
48.7±3.35
44.0±5.10
39.4±3.14
3.79±0.43
85.7±1.07
74.4±8.45
/
226±10.1
7.64±0.28
4.51±0.91
151±11.6
11.4±3.17
5.27±0.37
/
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Clint (mL/kg)
/
PAMPA Permeability
0.19±0.01
a
Average of two runs ± standard deviation (SD). PAMPA permeability values were determined
-6
at pH 7.4 (10 cm/s).
Further cytochrome P450 (CYP) inhibition determination showed that 19a had no serious
inhibition on the five major isozymes (Table 11). We then studied the in vivo PK profiles of
compounds 19a. As shown in Table 12, intravenous (iv) administration (5 mg/kg) of 19a displayed
favorable PK parameters (4 h of half-life and high exposure in plasma). With the potent inhibitory
activity in FP assay and drug-like properties both in vitro and in vivo, compound 19a was selected
for further biological evaluation.
a
Table 11. CYP450 Isozymes Inhibition of 19a
Isozymes
%inhibition
CYP450 isozymes: CYP1A2, CYP2C9, CYP2C19, CYP2D6,
CYP3A4
8.75, 30.26, 19.80, 19.89, 46.86
aPerformed at 10 μM concentration.
a
Table 12. In Vivo PK Profiles of 19a
Parameters
Cmax (ug/L)
19a (5 mg/kg iv)
78160.00
AUC(0-t) (h*ug/L)
AUC(0-∞) (h*ug/L)
47032.50
47041.77
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t1/2 (h)
4.02
1.10
0.64
0.11
MRT(0-∞) (h)
Vz (L/kg)
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CLz (L/h/kg)
aCmax = maximum plasma concentration; Tmax = time to reach maximum plasma concentration;
AUC = area under plasma concentration-time curve; t1/2 = half-life; MRT = mean residence time;
Vz = apparent volume of distribution; CLz = apparent clearance.
Compound 19a Significantly Activated Nrf2-Regulated Cytoprotective Defense System in
Vitro. As mentioned above, Keap1-mediated negative regulation of Nrf2 can be antagonized by
Keap1-Nrf2 inhibitors, followed by activation of Nrf2-regulated cytoprotective defense system.
1
9a with potent Keap1 binding affinity was chosen to evaluate cellular Nrf2 activation effects.
First, we evaluated the relative potency of 19a in comparison to the known Nrf2 activator SFN
using an ARE-luciferase reporter assay. HepG2-ARE cells that had been stably transfected with a
luciferase reporter were treated with different concentrations (0.01, 0.1, 1, 5, 10 and 20 μM) of
1
9a or SFN for the indicated times. As shown in Table 13, 19a increased the induction of ARE in
a concentration-dependent manner and induced the luciferase activity by 8.3-fold over the DMSO
control at a maximal concentration of 20 μM, much more potent than SFN. We then investigated
the effects of 19a on the Nrf2-regulated genes expression in H9c2 cardiac cells, including NEFL2L
(
encoding Nrf2 protein), HO-1, NQO-1, and GCLM. The quantitative real-time PCR (qRT-PCR)
results showed that treatment with 0.1-10 μM 19a for 12 h strongly increased the transcription of
Nrf2 and the Nrf2-regulated genes in a concentration-dependent manner (Figure 8A). Moreover,
the time-course study showed that the Nrf2 activation effects started after 4 h of 19a treatment and
continued for more than 24 h (Figure 8B). In line with these results, the evaluation of the Nrf2 and
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Nrf2-downstream proteins by western blotting demonstrated that treatment of the H9c2 cells with
0
.1-10 μM 19a elevated protein level of Nrf2, HO-1, NQO1 and GCLM in a concentration-
dependent manner (Figure 8C and 8D).
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Table 13. Induction of ARE Activity in HepG2-ARE-C8 Cellsa
ARE Induction Folds
Cpd.
0
.01 μM
0.1 μM
1 μM
5 μM
10 μM
20 μM
1
9a
1.53±0.18
1.18±0.05
1.85±0.24
2.39±0.14
4.97±0.56
6.93±0.63
8.35±0.56
SFN
1.28±0.11
1.50±0.12
1.93±0.29
2.47±0.22
3.56±0.26
aValues shown are mean ± SD (n = 3, 12 h of treatment). Cells were exposed to compounds
tested for their ability to increase levels of relative luciferase units.
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Figure 8. The Nrf2 activation effects of 19a on H9c2 cells. (A) Cells were exposed to 19a at
various concentrations for 12 h, (B) Cells were exposed to 10 μM 19a at various time points, the
mRNA levels of Nrf2 and Nrf2-targeted genes were determined by qRT-PCR analysis. GAPDH
was used to normalize the expression of these genes. Additional H9c2 cells were treated with
DMSO for use as the blank control. The values shown are the means ± SEM (n = 3 independent
observations). ***P < 0.001, **P < 0.01, and *P < 0.05, one-way ANOVA with Tukey-Kramer
posttest. (C) Cells were exposed to 19a at various concentrations for 12 h, (D) Cells were exposed
to 10 μM 19a at various time points, the protein levels of Nrf2 and Nrf2-regulated proteins were
determined by western blot analysis.
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Compound 19a Showed Cytoprotective Effects against LPS-induced Injury in H9c2 Cells.
Lipopolysaccharide (LPS) has been proven to be a key stimulus for sepsis models, and that sepsis-
induced myocarditis models have been widely used for screening of drugs for the treatment and
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prevention of myocarditis in vitro and in vivo. Therefore, we evaluated the effects of 19a on LPS-
induced cardiac injury in both cellular and in vivo models. First, we used the MTT assay to examine
the protective effects of 19a against the LPS-induced cell damage. No apparent cytotoxicity was
observed toward H9c2 cells with compound 19a at concentrations up to 100 μM (Supporting
Information, Figure S3). As shown in Figure 9A, the viability of cardiac cells was reduced to
approximately 70% after treatment of LPS (1 μg/mL), while pretreatment with 19a showed
favorable cytoprotective effects in a concentration-dependent manner. Inflammatory cytokines,
such as tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6, facilitated the development
of sepsis-induced myocarditis. As shown in Figure 9B-D, exposure to LPS could cause an obvious
increase of inflammatory factors (including IL-1β, IL-6, and TNF-α) compared with vehicle
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control, while pretreatment with 19a remarkably decreased the secretion of these inflammatory
factors.
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Figure 9. Effects of 19a against LPS-induced injury in H9c2 cells. (A) Concentration-dependent
protective effects of 19a against the LPS-induced cell damage. Cells were pretreated with 0.1-10
μM 19a for 12 h then exposed to 1 μg/mL LPS for an additional 12 h. The cell viability was
determined by the MTT assay. (B-D) Cells were pretreated with 1-10 μM 19a for 12 h then exposed
to 1 μg/mL LPS for an additional 12 h, and the ratio of IL-1β (B), IL-6 (C), and TNF-α (D) were
determined. The values shown are the means ± SEM (n = 3 independent observations). **p < 0.01,
and ***p < 0.001, one-way ANOVA with Tukey-Kramer posttest.
We further investigated the antioxidative activity of compound 19a. The Glutathione (GSH)-
based antioxidant system is mediated by Keap1-Nrf2-ARE pathway, thus the GSH/GSSG ratio
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was measured. As shown in Figure 10A, the ratio was decreased after LPS treatment, while
pretreatment with 19a could up-regulate this ratio obviously. Then we investigated the effect of
19a on the LPS-induced augment of ROS level in H9c2 cells. The fluorogenic cellular ROS
indicator c-H2DCF-DA was used to stain the cells. As shown in Figure 10B, the living cell
fluorescence microscopic signal was significantly enhanced after treatment with 1 μg/ml LPS and
this increase could be remarkably suppressed by 10 μM 19a pretreatment, which indicated its
protective effect against the oxidative stress in the H9c2 cells.
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Figure 10. Antioxidative effects of 19a against LPS-induced injury in H9c2 cells. (A) The
GSH/GSSG ratio in H9c2 cells was measured. The H9c2 cells were pretreated with 0.1-10 μM 19a
for 12 h then exposed to 1 μg/mL LPS for an additional 12 h. The values shown are the means ±
SEM (n = 3 independent observations). *p < 0.05, **p < 0.01, one-way ANOVA with Tukey-
Kramer posttest. (B) Living Cell Microscopy. The H9c2 cells were pretreated with 10 µM 19a for
12 h and then exposed to 1 μg/mL LPS for an additional 12 h. After this treatment, the H9c2 cells
were stained with 10 µM cH2DCF-DA for 20 min at 37 °C and living cell fluorescence microscopy
was performed.
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