Synthesis and Evaluation of Noncovalent Naphthalene-Based KEAP1-NRF2 Inhibitors

Article abstract of DOI:10.1021/acsmedchemlett.9b00631

The oxidative stress response, gated by the protein-protein interaction of KEAP1 and NRF2, has garnered significant interest in the past decade. Misregulation in this pathway has been implicated in disease states such as multiple sclerosis, rheumatoid arthritis, and diabetic chronic wounds. Many of the known activators of NRF2 are electrophilic in nature and may operate through several biological pathways rather than solely through the activation of the oxidative stress response. Recently, our lab has reported a nonelectrophilic, monoacidic, naphthalene-based NRF2 activator which exhibited good potency in vitro. Herein, we report a detailed structure-activity relationship of naphthalene-based NRF2 activators, an X-ray crystal structure of our monoacidic KEAP1 inhibitor, and identification of an underexplored area of the NRF2 binding pocket of KEAP1.

Full text of DOI:10.1021/acsmedchemlett.9b00631

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Letter
Synthesis and Evaluation of Non-covalent
Naphthalene-Based KEAP1-NRF2 Inhibitors
Phillip R. Lazzara, Atul D. Jain, Amanda C. Maldonado, Benjamin Richardson,
Kornelia J Skowron, Brian P David, Zamia Siddiqui, Kiira M Ratia, and Terry W. Moore
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.9b00631 • Publication Date (Web): 19 Feb 2020
Downloaded from pubs.acs.org on February 22, 2020
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Synthesis and Evaluation of Non-covalent Naphthalene-Based KEAP1-NRF2 Inhibitors
†
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‡†
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Phillip R. Lazzara , Atul D. Jain , Amanda C. Maldonado , Benjamin Richardson , Kornelia J.
†
†
†
†§
†§#
Skowron , Brian P. David , Zamia Siddiqui , Kiira M. Ratia , Terry W. Moore *
†Department of Pharmaceutical Sciences, College of Pharmacy, University of Illinois at Chicago,
§
#
833 S. Wood Street, Chicago, IL, USA 60612; UICentre for Drug Discovery; University of
Illinois Cancer Center; University of Illinois at Chicago, 833 S. Wood Street, Chicago, IL, USA
¤
6
0612; current address: Northwestern Center for Molecular Innovation and Drug Design, 2145
‡
Sheridan Road Evanston, IL, USA 60208. current address: Department of Chemistry, The
Scripps Research Institute, 110 Scripps Way, Jupiter, FL USA 33458.
*
to whom correspondence should be addressed: twmoore@uic.edu
Abstract: The oxidative stress response, gated by the protein-protein interaction of KEAP1 and
NRF2, has garnered significant interest in the past decade. Misregulation in this pathway has
been implicated in disease states such as multiple sclerosis, rheumatoid arthritis, and diabetic
chronic wounds. Many of the known activators of NRF2 are electrophilic in nature and may
operate through several biological pathways, rather than solely through the activation of the
oxidative stress response. Recently, our lab has reported a non-electrophilic, monoacidic,
naphthalene-based NRF2 activator which exhibited good potency in vitro. Herein, we report a
detailed structure-activity relationship of naphthalene-based NRF2 activators, an X-ray crystal
structure of our monoacidic KEAP1 inhibitor, and identification of an underexplored area of the
NRF2 binding pocket of KEAP1.
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Keywords: KEAP1, NRF2, Protein-protein interaction, Oxidative stress
Chronic oxidative stress is implicated in a number of disease states, such as chronic obstructive
pulmonary disease (COPD), multiple sclerosis, diabetic chronic wounds, and chronic kidney
disease.1-6 Upregulating cellular defenses against oxidative stress may be a viable pathway for
treatment or management of such diseases.7-9 NRF2 (Nuclear factor (erythroid-derived 2)-like 2),
a basic leucine zipper protein, regulates transcription of many antioxidant proteins. This
oxidative stress response is gated primarily by the protein KEAP1 (Kelch-like ECH-associated
protein 1), which sequesters NRF2 and, through a multi-protein assembly, poly-ubiquitinates it,
marking it for proteosomal degradation.10 If the KEAP1-NRF2 protein-protein interaction is
inhibited, NRF2 can no longer be sequestered and tagged for degradation. Inhibiting KEAP1 in
this manner allows cytoplasmic NRF2 concentrations to increase, translocate into the nucleus,
and promote the transcription of genes associated with the antioxidant response, such as NADPH
quinone oxidoreductase 1 (NQO1), heme oxygenase 1 (HO1), and glutamyl cysteine ligases-C
and -M. (Figure 1).10-14
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Figure 1. Top: KEAP1-NRF2 interaction under basal conditions. Bottom: Mechanism of NRF2
via electrophilic and non-electrophilic pathways.
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The KEAP1-NRF2 interaction is inhibited in the presence of electrophiles, reactive oxygen
1
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species, or reactive nitrogen species, leading to a cytoprotective response in the cell. Some
therapies which inhibit the KEAP1-NRF2 interaction utilize KEAP1’s sensitivity to electrophiles
to increase cellular NRF2 levels. Some electrophiles may be promiscuous binders, and their lack
of selectivity may make identification of mechanism of action more challenging.16-17
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Me
Et
OMe
OMe
Me
N
N
O
O
O
N
COOH
S
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S
Me
HN
HN
HN
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Me
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Me
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O
HN
HN
N
Me
Me
Me
O
O
COOH
OMe
OMe
Me
1g
1
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a: R =R = H IC = 2.7 M
1e
1f (RA-839)
IC50 = 140 nM
50
1
2
b: R =R = CH COOH IC = 29 nM
IC50 = 6.2 M
KEAP1 Kd = 0.7 nM
2
50
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c: R = H R = CH COOH IC = 61 nM
2
50
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d: R =R = CH CONH IC = 63 nM
2
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OH
OMe
O
O
Me
Me
S
N
F3C
N
O
O
N
N
O
S
O
O
N
O
N
S
N
N
c[GQLDPETGEFL]
N
HOOC
N
COOH
O
3
Me
O
H C
3
Me
OMe
1h
1i
1j
1k
IC50 = 73 nM
IC50 = < 15 nM
IC50 = 63 nM
IC50 = 18 nM
Chart 1. Representative examples of known KEAP1 inhibitors18-27
There have been multiple reports in recent years of non-electrophilic KEAP1-NRF2 inhibitors
with significant structural diversity, including various small molecules (1a-1j) and peptides (1k)
(Chart 1). Most of these molecules possess anionic character at physiological pH. Due to the
relative ease of modifying compounds such as naphthalene 1a, we and others have developed an
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SAR of these compounds via scaffold-hopping approaches and modification to the flanking
benzenesulfonamide arms; however, comparatively little investigation has been done to probe
variations in the regions that link the naphthalene core to the benzensulfonamides.20, 28 In this
letter, we present structural modifications, informed by a crystal structure of mono-acid inhibitor
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c (Figure 2), that provide valuable insights into the key interactions governing the potency and
binding affinities of these 1,4-disubstituted naphthalene inhibitors.
Previously, we were unable to obtain a suitable co-crystal structure of 1c with the KEAP1 Kelch
domain, so we analyzed the potential binding mode of mono-acidic inhibitor 1c in silico.20
Docking experiments predicted that the carboxylate would likely interact with R483 and R415.
We have now achieved success in co-crystallization of mono-acidic inhibitor 1c with the Kelch
domain of KEAP1 from a sodium formate solution. The co-crystal structure that we obtained
contained a unit cell comprised of four Kelch domains, each possessing 1c in slightly different
orientations. Two Kelch domains contained a formate ion interacting with the unsubstituted
sulfonamide, while the remaining two displayed water molecules in this position. While these
two variations contained slightly different orientations, the overall interactions between 1c and
the Kelch domain remained similar (Figure 2a and 2b). Interestingly, we observed that the
carboxymethyl functionality is engaged in a hydrogen bond network and dipolar interactions
with R415, N414, N382, S363, and a water molecule, which was contradictory with our docking
experiments, which showed interactions with R415, R483.20 In the crystal structure, key
interactions appear to be made between the sulfonamide oxygen atoms and S363, S508, Y525,
S555, S602 and two water molecules. With these data in hand, we set forth to determine which
of these interactions are critical for inhibitor binding.
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Figure 2. Structure of KEAP1 Kelch domain bound to compound 1c. A, B) Diagram of
interactions between KEAP1 Kelch residues (depicted as violet circles) and compound 1c. Of the
four KEAP1 Kelch:1c complexes crystallized in the asymmetric unit, two subunits contain a
formate ion (FMT, shown in teal) within hydrogen bonding distance of 1c (A), and two subunits
contain a water molecule (B). 2f -f electron density of 1c and formate (A) and 1c and bridging
o
c
water (B) is shown in blue mesh contoured at 1. C) Superposition of KEAP1 Kelch:1c complex
with the structures of KEAP1 bound to two other naphthalene-based compounds (1d, orange and
1
e, teal) previously reported in the literature. Associated PDB codes (6V6Z, 4XMB, 4ZY3) are
shown at right. Amino acids in close proximity to bound ligands are labeled on the protein
surface.
Our investigation into the structure-activity relationship began by probing the necessity of the
bis-sulfonamide motif. In previous work, we showed that mono-acidic analogs of 1b still
maintained significant potency; however, no di-carboxyl-mono-sulfonamide analogs were
synthesized. We sought to determine if protein binding was driven more by the sulfonamide
oxygens or the interactions of the carboxylates. A series of these mono-sulfonamide compounds
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were easily accessed through a Heck reaction of 1-bromo-4-aminonaphthalene (2) and
subsequent derivatization (Scheme 1). Although these mono-sulfonamide compounds are
structurally similar to reported mono-sulfonamide compound RA-839 (1f), none of these
compounds retained nanomolar affinities for the Kelch-domain of KEAP1, as determined by
fluorescence anisotropy (FA).21, 29 This difference in affinity may be due to the rigidity of the
acid in RA-839, versus the more freely rotating carboxylate groups in 3 and 4. Even though
compounds 5 and 6 contained an electrophilic α,β-unsaturated carbonyl, the activity of these
compounds in the FA assay would be solely dependent on non-electrophilic inhibition since we
used a truncated version of the KEAP1 protein which does not contain the previously mentioned
electrophilic cysteine residues.
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Scheme 1. Synthesis of mono-sulfonamide analogs of 1b and 1ca
O
OR
O
OH
Br
a, b
c,d
72%
two
steps
74%
two
steps
O
O
d
O
NH2
NH
N
S
S
OH
O
O
2
7 (R = Et)
MeO
MeO
6
IC50: 13 M
5
(R = H) 79%
IC50: > 25 M
O
OR
O
OH
e
c,d
99.5%
83%
two
steps
O
O
O
NH
N
S
S
OH
O
O
MeO
MeO
8
3
(R = Et)
(R = H) 79%
4 IC50: 9.3 M
d
IC50: > 25 M
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a(a) Pd(OAc) , P(o-tolyl) , K CO , ethyl acrylate, dioxane, 100 °C 16h; (b) 4-methoxybenzenesulfonyl
2
3
2
3
chloride, pyridine; (c) ethyl bromoacetate, K CO , MeCN, rt, 16h; (d) 15% NaOH(aq), MeOH, rt, 4h; (e)
2
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1
0 wt. % Pd/C, H (40 PSI), EtOH, rt.
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Next, we sought to determine the extent to which the affinity of these compounds was driven by
interactions stemming from the aryl groups appended to the sulfonamides. Alkynyl sulfonamide
9
was obtained via a Sonogashira coupling of 1-bromo-4-aminonaphthalene (2) and 4-
ethynylanisole, followed by sulfonylation of the amine. Hydrogenation of the alkyne yielded the
saturated analog 10, and subsequent alkylation of the sulfonamide and saponification of the
resulting ester produced phenethyl derivative 11 (Scheme 2). The affinity of this compound was
determined by an FA assay, and it displayed reduced affinity for the Kelch-domain of KEAP1,
which indicated that the benzenesulfonamide groups may be a major contributor to the activities
of these compounds.
Scheme 2. Synthesis of phenethyl analog 11a
OMe
OMe
OMe
Br
a, b
c
d,e
31%
two
steps
89%
65%
two
steps
O
O
S
O
S
O
NH
N
NH2
NH
OH
S
O
O
O
MeO
MeO
MeO
2
9
10
11
IC50: 1.5 M
a(a) Pd(PPh ) , CuI, 4-ethynylanisole, NEt , DMF, 80 °C, 20 h; (b) 4-methoxybenzenesulfonyl chloride,
3
4
3
pyridine, rt, 18 h; (c) 5 wt. % Pd/C, H (40 PSI), EtOAc, rt, 18 h; (d) ethyl bromoacetate, K CO , MeCN,
2
2
3
rt, 18 h; (e) 15% NaOH(aq), MeOH, rt, 5 h
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To test the hypothesis of the sulfonamide groups’ key interactions, we synthesized analog 12, in
which a sulfone rather than a sulfonamide moiety was installed. Synthesis of these analogs began
with a nitration of 1-methylnaphthalene to yield 1-nitro-4-methylnaphthalene 13. Benzylic
bromination with azabisisobutyronitrile/N-bromosuccinimide, followed by substitution with 4-
methoxybenzenethiol, yielded the thioether, which was oxidized with H O to the corresponding
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sulfone 14. Reduction of the nitro group and sulfonylation produced the sulfone analog of 1a,
which then underwent alkylation, and saponification to yield the desired monoacidic sulfone
analog 12 (Scheme 3).
Scheme 3.a
MeO
MeO
MeO
O
O
O
S
S
S
S
S
Me
O
O
O
O
O
a,b,c
6%
three
steps
d,e
f,g
4
87%
two
steps
59%
two
steps
O
NO2
NO2
NH
N
OH
O
O
MeO
MeO
13
14
15
12
IC : 678 nM
5
0
a(a) N-bromosuccinimide, azabisisobutyronitrile, MeCN, rt, 6 h; (b) 4-methoxybenzenethiol, 1M
NaOH(aq), dioxane, rt, 18 h; (c) 30% H O , AcOH, Ac O, rt, 5 h; (d) 10 wt. % Pd/C, H (40 PSI), EtOAc;
2
2
2
2
(
e) 4-methyoxybenzenesulfonamide, pyridine, rt, 18 h; (f) ethyl bromoacetate, K CO , MeCN, rt, 18 h; (g)
2 3
1
5% NaOH(aq), MeOH, rt, 5 h.
Additionally, we attempted to synthesize analogs in which the carboxymethyl group was
attached to the carbon alpha to the sulfone, and where both sulfonamides were replaced with
sulfones; however, we were unable to successfully isolate the desired products due to β-
elimination of a sulfinate to give α,β-unsaturated compounds. Comparison of intermediates 15
and 16 to bis-sulfonamide 1a showed that monosulfone 15 had comparable activity to 1a, yet
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bis-sulfone 16 was inactive (Chart 2). When comparing the sulfone monoacid 12 and diamino
monoacid 1c, sulfone monoacid 12 showed about a 10-fold drop in affinity. This loss of binding
affinity may be due to an unfavorable increase of hydrophobicity in the Arg-rich area of the
binding pocket.
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MeO
MeO
O
O
O
S
S
S
S
NH
O
O
O
O
O
O
N
N
R
S
R
S
O
O
O
MeO
a R = H;
MeO
15 R = H;
12 R = CH COOH; IC : 0.678 M
MeO
16 IC : > 25 M
1
1
IC : 2.7 M
IC : 3.0 M
5
0
50
50
c R = CH COOH; IC : 0.06 M
2
50
2
50
Chart 2. Sulfone Analogs of Compounds 1a and 1c
We were also interested in probing the ability of the binding pocket to accommodate
substitutions alpha to the carboxylate group. Recently, a report by Lu et al., outlined an
exploration of various alpha substituents based on amino acid side chains producing compounds
28
of structure 17 (Chart 3). Due to the lack of activity of our mono-sulfonamide analogs, we
opted to explore the activity of bis-sulfonamide compounds with structure 18. We hypothesized
that the more congested nature of the substituents in 18 may constrain the three-dimensional
structure, leading to enhanced interactions and binding affinity compared to the less constrained
compounds with structure 17.
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X
S
R2
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17
18
This work
Lu et al.
Chart 3. Structural variations explored by Lu et al. vs. this work
Not only would α-substituents potentially lead to the development of an SAR for an
underexplored area of these molecules, substitution in this position introduces chirality into this
framework. Due to 12 being the best inhibitor in the series we synthesized methyl analog 19 and
phenyl analog 20 by substituting ethyl bromoacetate with ethyl-2-bromopropionate and benzyl-
2-bromo-2-phenylacetate, respectively, for the alkylation step. Use of benzyl-2-bromo-2-
phenylacetate was required because basic saponification of the α-phenyl carbonyl resulted in
decomposition of the material.
MeO
MeO
MeO
MeO
O
S
O
S
O
S
O
S
NH
O
O
O
O
O
O
O
O
O
O
O
O
N
N
N
N
S
O
OH
S
OH
S
OH
MeO
S
OH
O
O
O
Me
Me
R
Me
MeO
MeO
MeO
21
IC50 = 126 nM
19 R = Me IC50 = 189 nM
20 R = Ph IC50 = 14000 nM
22
IC50 = 151 nM
22
IC50 = 241 nM
Chart 4. Propionate and phenylacetic acid analogs of 1c and 12
The addition of an R group alpha to the carbonyl did indeed constrain the orientation of the
substituents; the constraint is evident from this modification due to the formation of
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atropisomers, which displayed a 2:1 ratio of the major vs. minor conformer. A variable
temperature NMR experiment, from 20 °C to 70 °C, was performed in an effort to observe the
interconversion of the atropisomers; however, no substantial shift or coalescence of the signals
was observed. Attempts to separate the atropisomers were unsuccessful. Interestingly, the
addition of the methyl group increased binding affinity of sulfone 19 5-fold compared to the
unsubstituted sulfone 12, while addition of the phenyl ring severely diminished the activity.
These results can be rationalized by examination of the crystal structure of 1c (Figure 1c), in
which there is a small hydrophobic pocket where the methyl group may fit. It is likely that this
pocket is unable to accommodate much larger substituents, like phenyl. When comparing the α-
methyl compound 21 and unsubstituted mono-acid 1c, we observe a 2-fold reduction in binding
affinity. The reason for this slight decrease may be due to the methyl group inducing a less
favorable binding pose for this series of compounds.
1
1
1
1
1
1
1
1
1
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2
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0
Because of the relatively high binding affinity of racemic propionate 19, we moved to determine
whether the individual enantiomers of 19 displayed a preference for the binding pocket. To
synthesize each of the enantiomers, it was necessary to preclude basic saponification to avoid
racemization of the stereocenter (See SI). We utilized enantiopure benzyl esters, which were
subsequently deprotected by Pd/C-catalyzed hydrogenation to afford the individual enantiomers
with >95% e.e. (determined by chiral HPLC). Testing each of the enantiomers for their binding
affinity in the FA assay indicated that there was a negligible eudysmic ratio (i.e., the ratio of the
IC50 values of the high-affinity and low-affinity binder), indicating that further probing is
warranted to determine the proper substituent for this area of the molecule. The results of the
structural modifications detailed above are summarized in Table 1.
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Table 1. IC ± standard deviation, retention times (see SI for HPLC method), and cLogD of
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tested compounds.
a
cLogD7.0b
Compound IC ± standard deviation (nM)
Retention Time (min)
n.d.
50
c
0
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3.56
c
1
c
n.d.
5.184
5.008
5.254
5.071
6.737
5.886
6.072
6.184
5.995
6.312
5.910
5.995
5.995
-3.81
-0.39
-3.15
0.25
-3.30
2.07
0.13
3.07
4.08
1.19
2.08
0.44
1.19
1.19
3
>25000
9300 ± 560
>25000
13000 ± 3700
1500 ± 18
678 ± 29
4
5
6
1
1
2
5
6
1
1
1
3000 ± 120
>25000
19
189 ± 9
2
2
2
2
0
1
2
3
14000 ± 1060
126 ± 20
151 ± 20
241 ± 8
a
b
Standard deviation of two independent experiments. Values calculated with ChemAxon’s
LogD Predictor.30 cvalues taken from Jain et al. and compounds were run in parallel as standards
in the FA assay.20
Overall, using a combination of X-ray crystallographic analysis and experimental structure-
activity relationship development, we have determined key features of naphthalene-based
KEAP1–NRF2 inhibitors that are responsible for the binding affinity to the Kelch domain of
KEAP1 (Chart 5).
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Chart 5. Key interactions and sites for optimization identified.
Key interactions are formed between serine residues within the Kelch domain and the oxygen
atoms of either the sulfonamide or sulfone functionalities present within these inhibitors. Pairing
the previously described serine–oxygen interactions with dipolar interactions between the
carboxylate, asparagine, and arginine within the binding pocket appeared to be crucial for
establishing strong protein–inhibitor interactions. Furthermore, we showed that one of the
nitrogen atoms on the naphthalene ring could be replaced with a carbon atom and still maintain
reasonable binding affinities. This substitution allows for the investigation of different
geometries around this prochiral carbon, instead of being confined to an achiral nitrogen linker.
Lastly, our observations agree with Lu et al. in having identified a region, alpha to the carbonyl
of theses inhibitors where additional exploration may yield inhibitors with higher affinity.
Further experiments are underway to determine the role these α-substituents play in inhibitor
binding.
Associated Content:
Supporting Information
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Experimental procedures of syntheses, biochemical assay protocols, and crystal structure
parameters.
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Corresponding Author
Email: twmoore@uic.edu
ORCID
Terry W. Moore: 0000-0002-5410-306X
Phillip R. Lazzara: 0000-0003-3709-2749
Atul D. Jain: 0000-0002-3281-9729
Benjamin Richardson: 0000-0001-6003-3021
Amanda C. Maldonado: 0000-0002-0381-7337
Kornelia J. Skowron: 0000-0002-0846-6134
Brian P. David: 0000-0001-5328-5394
Zamia Siddiqui: 0000-0002-0488-1306
Kiira M. Ratia: 0000-0002-4834-9207
Present Address
A.D.J.: Northwestern Center for Molecular Innovation and Drug Design, 2145 Sheridan Road
Evanston, IL, USA 60208.
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B.G.R.: Department of Chemistry, The Scripps Research Institute, 110 Scripps Way, Jupiter, FL
USA 33458.
Author Contributions
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The manuscript was written through contributions of all authors. All authors have given
approval to the final version of the manuscript.
Notes
The authors declare no competing financial interest.
Acknowledgments:
This research used resources of the Advanced Photon Source, a U.S. Department of Energy
(DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne
National Laboratory under Contract No. DE-AC02-06CH11357. Use of the LS-CAT Sector 21
was supported by the Michigan Economic Development Corporation and the Michigan
Technology Tri-Corridor (Grant 085P1000817).
Funding Sources:
The authors would like to thank the following funders for their support of this work: National
Institute of Arthritis, Musculoskeletal and Skin Diseases to T.W.M. (1R01 AR069541-01A1)
Abbreviations:
KEAP1, Kelch-like ECH-associated protein 1; NRF2, Nuclear factor (erythroid-derived 2)-like
2; FA, Fluorescence anisotropy; SAR, Structure activity relationship; COPD, chronic obstructive
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pulmonary disorder; DMF, dimethyl formamide; NQO1, NADPH quinone oxidoreductase 1;
HO1, heme oxygenase 1
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M.; Yamamoto, M.; Imamura, R.; Kojima, H.; Okabe, T.; Nagano, T.; Mashino, T., Synthesis of Keap1-
phosphorylated p62 and Keap1-Nrf2 protein-protein interaction inhibitors and their inhibitory activity.
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fluorescently labeled Nrf2 peptide probes and the development of a fluorescence polarization assay for
the discovery of inhibitors of Keap1-Nrf2 interaction. J Biomol Screen 2012, 17 (4), 435-447.
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For Table of Contents Only
R1
MeO
O
R3
Z
R1
S
N
Y
O
Z
Y
1
1
1
1
1
1
1
1
1
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2
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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
O
O
N
N
S
OH
S
OH
R2
O
O
R2
MeO
Previously Explored SAR
This Work
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MeO
MeO
MeO
O
O
O
S
S
S
S
S
Me
O
O
O
O
O
a,b,c
6%
three
steps
d,e
f,g
4
87%
two
steps
59%
two
steps
O
0
1
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9
0
1
2
3
4
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9
0
1
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9
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1
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9
0
NO2
NO2
NH
N
OH
O
O
MeO
MeO
13
14
15
12
IC : 678 nM
50
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