Replacement of a Naphthalene Scaffold in Kelch-like ECH-Associated Protein 1 (KEAP1)/Nuclear Factor (Erythroid-derived 2)-like 2 (NRF2) Inhibitors

Article abstract of DOI:10.1021/acs.jmedchem.8b01133

Activators of nuclear factor-erythroid 2-related factor 2 (NRF2) could lead to promising therapeutics for prevention and treatment of oxidative stress and inflammatory disorders. Ubiquitination and subsequent degradation of the transcription factor NRF2 is mediated by Kelch-like ECH-associated protein-1 (KEAP1). Inhibition of the KEAP1/NRF2 interaction with small molecules leads to NRF2 activation. Previously, we and others described naphthalene-based NRF2 activators, but the 1,4-diaminonaphthalene scaffold may not represent a drug-like scaffold. Paying particular attention to aqueous solubility, metabolic stability, potency, and mutagenicity, we modified a previously known, naphthalene-based nonelectrophilic NRF2 activator to give a series of non-naphthalene and heterocyclic scaffolds. We found that, compared to previously reported naphthalene-based compounds, a 1,4-isoquinoline scaffold provides a better mutagenic profile without sacrificing potency, stability, or solubility.

Full text of DOI:10.1021/acs.jmedchem.8b01133

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Article
Replacement of a Naphthalene Scaffold in Kelch-like ECH-Associated
Protein 1 (KEAP1)/Nuclear factor (erythroid-derived 2)-like 2 (NRF2)Inhibitors
Benjamin Richardson, Atul D. Jain, Haranatha Potteti, Phillip R. Lazzara, Brian
P David, Chandra Tamatam, Ewelina Choma, Kornelia Skowron, Katherine Dye,
Zamia Siddiqui, Yue-Ting Wang, Aleksej Krunic, Sekhar Reddy, and Terry W. Moore
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01133 • Publication Date (Web): 20 Aug 2018
Downloaded from http://pubs.acs.org on August 20, 2018
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Replacement of a Naphthalene Scaffold in Kelchꢀ
like ECHꢀAssociated Protein 1 (KEAP1)/ Nuclear
factor (erythroidꢀderived 2)ꢀlike 2 (NRF2) Inhibitors
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‡
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Benjamin G. Richardson , Atul D. Jain , Haranatha Potteti , Phillip R. Lazzara , Brian P.
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†
§
David , Chandra Tamatam , Ewelina Choma , Kornelia Skowron , Katherine Dye , Zamia
†
§
†
‡#
†§#
Siddiqui , YueꢀTing Wang , Aleksej Krunic , Sekhar P. Reddy , Terry W. Moore *
†
Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of
‡
Illinois at Chicago, 833 S. Wood Street, Chicago, IL, 60612; Department of Pediatrics, College
§
#
of Medicine; UICentre for Drug Discovery; University of Illinois Cancer Center; University of
Illinois at Chicago, 833 S. Wood Street, Chicago, IL, 60612
Abstract:
Activators of Nuclear factorꢀerythroid 2ꢀrelated factor 2 (NRF2) could lead to promising
therapeutics for prevention and treatment of oxidative stress and inflammatory disorders.
Ubiquitination and subsequent degradation of the transcription factor NRF2 is mediated by
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Kelchꢀlike ECHꢀassociated proteinꢀ1 (KEAP1). Inhibition of the KEAP1/NRF2 interaction with
small molecules leads to NRF2 activation. Previously, we and others described naphthaleneꢀ
based NRF2 activators, but the 1,4ꢀdiaminonaphthalene scaffold may not represent a drugꢀlike
scaffold. Paying particular attention to aqueous solubility, metabolic stability, potency, and
mutagenicity, we modified a previously known, naphthaleneꢀbased nonꢀelectrophilic NRF2
activator to give a series of nonꢀnaphthalene and heterocyclic scaffolds. We found that,
compared to previously reported naphthaleneꢀbased compounds, a 1,4ꢀisoquinoline scaffold
provides a better mutagenic profile without sacrificing potency, stability, or solubility.
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Introduction:
Detoxification enzymes act as the cell’s main defense mechanism to counter oxidative and
electrophilic stress. Detoxification enzymes include, but are not limited to, NAD(P)H quinone
oxidoreductase 1 (NQO1), heme oxygenase 1 (HMOX1), glutamateꢀcysteine ligaseꢀmodifier and
–
catalytic subunits (GCLM and GCLC), and glutathione Sꢀtransferases (GST). The transcription
of these and other genes is regulated by the transcription factor, NRF2 (nuclear factorꢀ
erythroid2ꢀrelated factor 2), a member of the cap ‘n’ collar family of basic leucine zipper
proteins. Endogenous NRF2 levels are maintained by KEAP1 (Kelch like ECH associated
protein 1), which acts as a negative regulator of NRF2. KEAP1 is a 69 kDa protein that exists as
a homodimer, and it contains 27 cysteine residues, which act as sensors for oxidative or
1, 2, 3
electrophilic stressors.
In the absence of oxidative or electrophilic stressors, NRF2 is
degraded by the E3 ubiquitin ligase Cullin 3 (CUL3), which binds to KEAP1’s BTB domain
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(
Figure 1A). KEAP1 functions as a substrate adaptor protein, by bridging NRF2 to CUL3. A
lysineꢀrich region located between ETGE and DLG motifs of NRF2 is wellꢀpositioned for
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ubiquitination, and NRF2 is subsequently degraded through the ubiquitin/proteasome system.
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Inhibition of the KEAP1/NRF2 interaction prevents this ubiquitination and degradation, leading
to an increase in NRF2 levels. NRF2 then translocates to the nucleus, heterodimerizes with the
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small MAF (sMAF) transcription factors and binds to antioxidant response elements (AREs) in
2, 5, 10ꢀ15
the promotor regions of many detoxification genes;
interaction serve as NRF2 activators.
thus, inhibitors of the KEAP1/NRF2
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Figure 1: (A) Transcription factor NRF2, when bound to substrate adaptor KEAP1, is
ubiquitinated by E3 ligase CUL3, leading to proteosomal degradation. Two mechanisms
explain activation of NRF2 by electrophiles. (B) In the CUL3 dissociation mechanism,
cysteine modification by an electrophile causes dissociation of CUL3, leading to
accumulation of NRF2. (C) In the hingeꢀandꢀlatch mechanism, cysteine modification causes
nonꢀoptimal placement of NRF2 for degradation. (D) In the conformational cycling model,
cysteine modification locks KEAP1 and NRF2 in a conformation not ideal for ubiquitination,
preventing KEAP1 from binding nascent NRF2, leading to accumulation of NRF2 (E) The
nonꢀelectrophilic mechanism aims to create a nonꢀcovalent inhibitor of the proteinꢀprotein
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Oxidative and electrophilic stressors induce transcription of detoxification enzymes by
disrupting the interaction of NRF2 and KEAP1, but they do so indirectly. Three prevailing
mechanisms exist to explain this disruption, namely: 1) the CUL3 dissociation model, 2) the
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, 8, 9, 16, 17
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hinge and latch model, and 3) the conformational cycling model.
In all three models, a
KEAP1 homodimer binds to a single NRF2 molecule through high affinity ETGE and low
affinity DLG motifs within the Neh2 domain of NRF2. Under electrophilic stress, modification
of specific cysteine sulfhydryl groups leads to loss of CUL3, displacement of NRF2’s DLG
motif, or a nonꢀoptimal conformation for ubiquitination. In the CUL3 dissociation model, a
cysteine in the BTB domain, Cys151, of KEAP1 is modified, leading to dissociation of CUL3
from KEAP1, preventing NRF2 ubiquitination (Figure 1B). The hinge and latch model is
triggered through electrophilic modification of cysteine residues in Keap1’s IVR region, rather
than the BTB domain of KEAP1. This modification changes KEAP1’s conformation causing
release of the DLG motif, while NRF2 remains bound at the ETGE motif “hinge.” According to
this model, release of the DLG motif “latch” results in a binding pose of NRF2 that is
inaccessible for ubiquitination (Figure 1C). In the conformational cycling model, all of the
partners are still bound to one another, but covalent modification of KEAP1 brings about
conformational changes in KEAP1 and NRF2 that disfavor ubiquitination of NRF2 (Figure
17
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D). All of these models result in an inability to ubiquitinate and degrade NRF2 in the cytosol,
leading to NRF2 translocation into the nucleus. NRF2 then complexes with small MAF proteins,
binds to antioxidant response elements, and transcribes the above mentioned detoxification
2, 10, 11, 14
enzymes.
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Due to the role of NRF2 in cytoprotection,
several NRF2 activators have been tested in
the clinic against a number of disease
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states.
All of these clinical candidates
have utilized the same mechanism of action:
an electrophile is introduced to interact with
the sulfhydryl groups of KEAP1 and activate
NRF2. Several NRF2 activators have
Chart 1: Electrophilic activators of NRF2
proceeded as far as phase III clinical trials with a few making it into clinical use; however, many
of these electrophilic compounds may have selectivity issues. For instance, both sulforaphane (3)
21, 22
and a bardoxolone (1) may have hundreds of molecular targets (Chart 1).
Similarly,
SchulzeꢀTopphoff et al. have shown that the mechanism of action of dimethyl fumarate 2
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(Tecfidera®, Chart 1) in multiple sclerosis may be independent of the KEAP1/NRF2 axis. A
selective, nonꢀelectrophilic molecule could be useful to
disentangle the contribution of NRF2 activation against a
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particular pathophysiological state.
The binding sites of NRF2’s ETGE and DLG motifs on
KEAP1 represent “hot spots” that can be targeted with
nonꢀelectrophilic small molecules and peptides. This route
differs from the electrophilic mechanism of NRF2
activation, and may provide advantages in terms of onꢀ
Chart 2: Naphthalene 4 (RAꢀ
839) is active in vivo only if
Phase 1 metabolism is inhibited.
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target selectivity.
We and others have previously
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1, 16, 28ꢀ30
reported several NRF2 activators that use a 1,4ꢀsubstituted naphthalene core.
These
compounds show low nanomolar potencies in vitro and increase transcription of NRF2 target
genes in cells; however, the 1,4ꢀdiaminonaphthalene core imparts properties that may negatively
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impact the development of these molecules. For instance, Winkel et al. showed that naphthalene
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(RAꢀ839) was active in vivo only if Phase 1 metabolism were inhibited (Chart 2).
Naphthalene itself is metabolically activated to naphthalene oxide and naphthoquinones, and
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others have shown that it can covalently react with proteins in vitro and in vivo.
Additionally,
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some aminonaphthalenes show carcinogenic and mutagenic properties.
Taken together, these
findings suggest that the central naphthalene scaffold may not be an optimal core for this series
of compounds.
To compare novelꢀscaffold NRF2 activators to extant naphthaleneꢀbased activators, we were
interested in studying the physical properties of these molecules beyond potency. There have
been some recent perspectives that suggest
that the emphasis of medicinal chemists,
particularly those in academia, on potency
often leads to highly potent, but poorly
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drugꢀlike, molecules. With these concerns
in mind, we designed and synthesized
compounds with scaffolds to replace the
naphthalene as a means to study potency,
aqueous solubility, metabolic stability and
mutagenicity of NRF2 activators. Our
results indicate that a 1,4ꢀsubstituted
Figure 2: Nonꢀfused (red), acyclic (blue)
and heterocyclic (magenta) analogs were
designed to replace the naphthalene core of
nonꢀcovalent NRF2 activators.
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isoquinoline presents a new scaffold for NRF2 activators that shows similar potency, aqueous
solubility, and metabolic stability to the 1,4ꢀsubstituted naphthalene. Additionally, a miniꢀAmes
assay shows that the 1,4ꢀsubstituted isoquinoline displays an improved mutagenic profile relative
to the 1,4ꢀsubstituted naphthalene. These results have important implications for the design of
future NRF2 activators.
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Results:
Analog Design. NRF2 activators displaying a large set of different cores were synthesized
based off the naphthalene compound 6a. In previous work, we and others reported compounds
varying the terminal rings, the sulfonamide R groups, the sulfonamide linker, and possible
1, 29, 30
isosteric replacements for the carboxylates.
Additionally, we reported that the 1,4ꢀ
substitution pattern was optimal, as other substitution patterns showed complete loss of activity.
In the current work of designing nonꢀnaphthalene derivatives, we maintained the sulfonamides,
either unsubstituted or with bisꢀcarboxymethyl substitutions, and we varied the connecting
1
scaffold. Previously, we showed that bisꢀcarboxamide 6b showed high potency. During the
course of the current studies, we determined that the amides of 6b hindered aqueous solubility
and caused rapid degradation in liver microsomes. As a result, we did not pursue this substituent
in the current study.
We sought to synthesize compounds that maintained a display of substituents that was similar
to that of the naphthalenes. To accomplish this, we synthesized molecules that featured a
retention of the topology of the naphthalene that displayed different electronics (i.e., 1,4ꢀ
isoquinoline; 5,8ꢀquinoline; 1,4ꢀphthalazine; 4,7ꢀindole) (Figure 2). We also synthesized
compounds that no longer contained the 6,6ꢀfused ring system to examine the distal ring and
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possibilities of altering it (i.e., 1,4ꢀbenzene; 2,5ꢀpyridine; oꢀxylene; 3,5ꢀbiphenyl; phthalamide).
Lastly, we synthesized compounds that no longer contained an aromatic scaffold to fully
determine what was necessary for activity (i.e., butene).
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Analog Synthesis. Compounds 5, 6a, and 6b were synthesized as previously described.
a
Scheme 1. Synthesis of compounds 11a and 11b
a
Reagents: (a) pyridine, rt, 18ꢀ76% yield; (b) RBr, K CO , DMF, rt, 80% yield; (c) NaOH,
2
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MeOH, reflux. 67ꢀ80% yield; rt: room temperature
Benzenes 9b and 11b and diꢀsubstituted pyridine 11a were synthesized (Scheme 1) starting
from the reaction of phenyleneꢀ1,4ꢀdiamine 7b or 2,5ꢀdiamino pyridine 7a and pꢀ
methoxybenzenesulfonyl chloride 8 in pyridine to obtain the corresponding sulfonamides 9a and
9
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b. Reaction with ethyl bromoacetate in the presence of K CO followed by saponification gave
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1a and 11b.
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a
Scheme 2. Synthesis of 1,4ꢀisoquinolines 15 and 17
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a
Reagents: (a) PBr , 140 °C, 89% yield; (b) CuI, N,N’ꢀdimethylethylenediamine, K CO ,
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MeCN, 70 °C, 60% yield; (c) RBr, K CO , DMF, rt, 77% yield; (d) NaOH, MeOH, reflux,
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6% yield. rt: room temperature
Analogs with a 1,4ꢀisoquinoline core were synthesized according to Scheme 2. 1ꢀ
hydroxyisoquinoline 12 was converted to 1,4ꢀdibromoisoquinoline 13 using PBr . An Ullmannꢀ
5
type coupling with pꢀmethoxybenzenesulfonamide 14, followed by alkylation and saponification
yielded isoquinoline 17. In the synthesis of isoquinoline 17, undesired alkylation of the
isoquinoline ring nitrogen, rather than the desired sulfonamide alkylation, could have occurred
while installing the carboxymethyl functionalities (i.e. 15 → 16). 2DꢀNMR experiments
confirmed the assigned sites of alkylation (Figure S8).
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3
4
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
Scheme 3. Synthesis of quinolines 21 and 23
a
Reagents: (a) aniline, NaNO , HCl, AcOH, NaOAc, H O, 30% yield; (b) SnCl , HCl, reflux,
2
2
2
5
0% yield; (c) pꢀmethoxybenzenesulfonyl chloride, pyridine, rt, 77% yield; (d) RBr, K CO ,
2 3
DMF, rt; (e) NaOH, MeOH, reflux, 67% yield over 2 steps. rt: room temperature
Compounds with a quinoline core were synthesized as shown in Scheme 3. 5ꢀAminoquinoline
8 was reacted with HONO in the presence of aniline to form the diazo compound 19, which was
1
39
further reduced in the presence of stannous chloride to yield 5,8ꢀdiaminoquinoline 20.
Sulfonylation followed by alkylation and saponification yielded quinoline 23.
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5
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5
5
5
5
5
6
Analogs containing an indole core were synthesized as shown in Scheme 4. 2,4ꢀ
Difluoronitrobenzene 24 was sequentially aminated at the 2ꢀ and 4ꢀpositions to yield a
differentially bisꢀaminated intermediate 25. Cyclization was carried out in the presence of TFA
to yield 4ꢀbenzylaminoꢀ7ꢀnitroindole. Reduction and hydrogenolysis yielded 4,7ꢀdiaminoindole
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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7
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9
0
1
2
3
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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
a
Scheme 4. Synthesis of indoles 27 and 29
a
Reagents: (a) aminoacetaldehyde dimethyl acetal, iꢀPr NEt, MeOH, reflux, 86% yield; (b)
2
benzylamine, 1,4ꢀdioxane, reflux, 93% yield; (c) F CCO H, CH Cl , rt, 85% yield; (d) Pd/C,
3
2
2
2
EtOH/HCl, H , 35 psi, 100% yield; (e) pꢀmethoxybenzenesulfonyl chloride, pyridine, rt, 60%
2
yield; (f) RBr, K CO , DMF, rt, 82% yield; (g) TFA, CH Cl , rt, 20% yield. rt: room
2
3
2
2
temperature
4
0
2
6, which was further sulfonylated, alkylated, and saponified to yield indole 29. HNMR results
confirmed that compound 27 was sulfonylated at the 1ꢀ and 4ꢀpositions. Singlets at 10.69, 9.79,
and 9.61, each integrating to 1H, correspond to the indole NH, and two sulfonamide NH groups.
Phthalazine 34 was prepared beginning from phthalonitrile 30, which was condensed with
hydrazine in methanol to give 1,4ꢀdiaminophthalazine 31, which yielded the sulfonamide 32.
Alkylation of the sulfonamide gave only the monoꢀalkylated products 33 and 34, regardless of
the reaction conditions employed.
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Page 13 of 70
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a
Scheme 5. Synthesis of phthalazines 32 and 34
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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3
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0
1
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4
5
6
7
8
9
0
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9
0
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2
3
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8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
Reagents: (a) NH NH , MeOH, rt, reflux, 65% yield; (b) pyridine, 1,4ꢀdioxane, reflux, 31%
2
2
t
yield; (c) ethyl bromoacetate, Li BuO, DMF, rt, 100% yield; (d) NaOH, MeOH/H O, 54%
2
yield. rt: room temperature
a
Scheme 6. Synthesis of compounds 38, 40, and 42
a
Reagents: (a) pyridine, rt 55ꢀ58% yield; (b) RBr, K CO , DMF, rt, 70ꢀ80% yield; (c) NaOH,
2
3
MeOH, reflux, 70ꢀ74% yield; (d) F CCO H, CH Cl , rt, 55% yield. rt: room temperature
3
2
2
2
Analogs of 6a that did not feature a fused ring system were synthesized using the appropriate
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6
electrophilic starting materials (Scheme 6). Substituted sulfonamides were used as nucleophiles
Scheme 7. Synthesis of biphenyl 46
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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7
8
9
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9
0
1
2
3
4
5
6
7
8
9
0
a
Reagents: (a) phenylboronic acid, Pd(OAc) , K PO , H O, reflux, quant. yield; (b) SnCl ,
2
3
4
2
2
MeOH, reflux, 99% yield; (c) pꢀmethoxybenzenesulfonyl chloride, pyridine, THF, reflux; (d)
ethyl bromoacetate, K CO , DMF; (e) NaOH, MeOH, reflux, 51% yield over 3 steps. rt: room
2
3
temperature
to yield xylene 38, phthalamide 40, and butene 42 after saponification.
Biphenyl 46 was synthesized as shown in Scheme 7. 1ꢀbromoꢀ3,5ꢀdinitrobenzene 43 was
reacted with phenylboronic acid via a Suzuki coupling to create the biphenyl core. The nitro
groups were reduced to amines to yield a diamino intermediate 44, which was then sulfonylated,
alkylated, and saponified to arrive at the analog 46.
In vitro assays. The IC50 of each compound for inhibiting the interaction of a fluorescent
41
NRF2 peptide and the Kelch domain of KEAP1 was determined using fluorescence anisotropy.
The results are shown in Table 1. The heterocyclic cores ranged in potency: the 1,4ꢀisoquinoline
17 (IC = 60 nM) retained much of the potency of the parent naphthalene 6a, the 5,8ꢀquinoline
5
0
2
3 was well tolerated (IC = 101 nM), the phthalazine 34 was tolerated (IC = 1000 nM), and
50 50
the indole 29 was tolerated (IC = 1300 nM). Unfortunately, the indole 29 was found to be
5
0
unstable, which precluded its further consideration. The nonꢀfused systems all showed little to no
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Page 15 of 70
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activity: the phenyl 11b was tolerated (IC = 980 nM), whereas the xylene 38, pyridine 11b,
5
0
phthalamide 40, and biphenyl 46 were all inactive (>25 ꢀM). The removal of the core to give
butene 42 also produced an inactive compound.
1
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1
1
1
1
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1
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0
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5
6
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8
9
0
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2
3
4
5
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8
9
0
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1
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3
4
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7
8
9
0
Compounds with low IC values in the FP assay were confirmed using an orthogonal Surface
5
0
Plasmon Resonance (SPR) assay (Table 1). 1,4ꢀIsoquinoline 17 exhibited a Kd of 102 nM, 5,8ꢀ
quinoline 24 exhibited a K of 103 nM, and phthalazine 34 exhibited a K of >10 ꢁM, in good
d
d
agreement with the results of the FP assay.
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IC (nM)
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0
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0
Compound
Core
R
R’
K (nM)
d
(
95% CI)
940
b
b
5
ꢀH
ꢀH
1700 ± 120
(
850 – 1300)
b
2
5
b
6a
ꢀCH
2
COOH
ꢀCH
2
COOH
20 ± 3
(
19–31)
b
6
3
b
6
b
ꢀCH
2
CONH
2
ꢀCH
2
CONH
2
44 ± 8
(
52 – 85)
>25000
80
9b
1b
ꢀH
ꢀH
ND
ND
9
1
ꢀCH
2
COOH
COOH
ꢀCH
2
COOH
COOH
(600–1600)
1
1a
ꢀCH
2
ꢀCH
2
>25000
ND
a
1
5
7
ꢀH
ꢀH
ND
ND
102 ± 16
ND
6
0
1
ꢀCH
ꢀCH
ꢀCH
2
COOH
ꢀCH
ꢀCH
ꢀCH
2
COOH
(
30–110)
>25000
01
80–130)
21
ꢀH
ꢀH
1
2
2
2
3
3
3
7
9
2
4
2
COOH
2
COOH
103 ± 2
ND
(
ꢀH
ꢀH
>25000
1300
(390–5400)
COOH
ꢀH
COOH
ꢀH
ND
2
2
>25000
ND
1
100
ꢀCH
ꢀCH
COOH
COOH
ꢀH
> 10 ꢁM
2
(435–2500)
38
40
2
2
2
2
ꢀCH
ꢀCH
ꢀCH
ꢀCH
2
COOH
COOH
COOH
COOH
>25000
ND
ND
ND
ND
ꢀCH
ꢀCH
ꢀCH
COOH
COOH
COOH
>25000
>25000
>25000
2
2
2
4
4
2
6
Table 1: IC and K values. IC values, shown in nM units with 95% confidence intervals,
5
0
d
50
were determined by fluorescence anisotropy. K values, shown in nM units ± standard
d
a
deviation, were determined by surface plasmon resonance. Fluorescent interference from 15
b
1
impeded accurate determination of IC . IC and K values reported in Jain et al. . ND = Not
5
0
50
d
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Page 17 of 70
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9
0
Figure 3: HaCaT cells were treated with 10 ꢁM compound (6a, 6b, 17, 23; positive control CDDOꢀ
Im) or vehicle (DMSO) for 6 h (A) or 24 h (D) as shown in the immunoblots. Cell lysates were
prepared, equal amount of protein was probed with antibodies specific for NRF2, NQO1 or GCLM.
GAPDH was used as a reference. Quantification of band intensities of NRF2 (B, E), NQO1 (C, F)
and GCLM levels (G) are shown. DMSOꢀtreated sample values were used as one arbitrary
unit.*:p<0.05; **:p<0.01; ***:p<0.001; if no * is indicated, p>0.05.
Aqueous Solubility. The aqueous solubility of all compounds at pH 7.4 was determined as
shown in Table 2. Heterocycles 23 and 17, phthalamide 40, and butane 42 all showed solubility
comparable to the naphthalene parent compound 6a. Xylene 38 and benzene 11b showed poor
solubility. Pyridine 11b and biphenyl 46 both showed moderate solubility. In all cases except
phthalazine 32, unsubstituted sulfonamides were poorly soluble (solubility < 10 ꢁM). The bisꢀ
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Page 18 of 70
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6
amide 6b that we previously reported as a potent NRF2 activator was shown to have poor
solubility, which, in part, led us to focus our
attention on the carboxymethyl analogs.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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7
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9
0
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9
0
Aqueous
Solubility
NRF2 Stabilization and NRF2 Target Gene
Activation.
Compound
(ꢁM)
5
6a
5.4 ± 0.9
440 ± 3
22 ± 9
1.2 ± 0.2
1.2 ± 0.2
190 ± 0.8
3.8 ± 0.1
380 ± 3
To see the effects of KEAP1 inhibitors on
NRF2 activation, we carried out a screening assay
to determine the effects of naphthalene acid 6a,
naphthalene amide 6b, isoquinoline 17, quinoline
23, and phthalazine 34 on NRF2 protein stability
and its downstream target gene activation in nonꢀ
transformed immortalized human keratinocytes
(HaCat cells). Cells were treated with compounds
for 6 h to determine the stabilization of NRF2 and
the protein expression levels of NQO1, a wellꢀ
known target of NRF2 (Figure 3). Treatment of
HaCaT cells with 6a, 6b, and 23 for 6 h caused a
modest increase in protein levels of NRF2 and
6
9
b
b
1
1b
11a
1
1
2
5
7
1
6.1 ± 0.1
390 ± 4
23
27
29
32
0.86 ± 0.3
b
N.D.
370 ± 7
380 ± 7
1.3 ± 0.3
490 ± 90
420 ± 12
270 ± 3
34
38
40
42
46
Table 2: Aqueous solubility, shown in
M units ± standard deviation.
ꢁ
NQO1 expression in a manner comparable to CDDOꢀIm 1, a wellꢀknown potent electrophilic
activator of NRF2. Phthalazine 34 caused no increase in either NRF2 protein levels or NQO1
protein levels in this assay.
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Journal of Medicinal Chemistry
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6
7
8
9
0
Figure 4: The effects of nonꢀelectrophilic activators 17 (orange) and 23 (purple) and positive
control CDDOꢀIm on the nuclear accumulation of NRF2 in HaCaT Cells. (A) HaCaT cells
were treated with DMSO (vehicle) or indicated compound (10 ꢁM) for 6 hours and 24 hours,
Nuclear (N) and cytosolic (C) fractions were prepared, equal amount of protein was
immunoblotted using antibodies specific for NRF2. βꢀactin / Lamin A/C was used as loading
control. (B) Quantification of Nrf2 band intensity using βꢀactin as reference. DMSOꢀtreated
sample value was used as one arbitrary unit. Error bars represent the standard error from four
independent experiments. *:p<0.05; **:p<0.01; ***:p<0.001; if no * is indicated, p>0.05.
We next analyzed NRF2 and NQO1 protein expression levels in HaCat cells treated with
compounds for a prolonged period (i.e., 24 h). Increased protein levels of NRF2 and NQO1 were
noted in cells treated with naphthalene acid 6a, naphthalene amide 6b, or isoquinoline 17 for 24
h compared to vehicleꢀtreated counterparts (Figure 3). Likewise, both NRF2 and NQO1 protein
levels were higher in cells treated with CDDOꢀIm 1. Quinoline 23 had no effect on either NRF2
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5
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5
5
6
stability or NQO1 expression. We also analyzed the expression of GCLM, a wellꢀestablished
target of NRF2. GCLM protein levels were significantly higher in cells treated with naphthalene
acid 6a, naphthalene amide 6b, isoquinoline 17, or CDDO 1 compared to vehicle controls.
Unlike NRF2 protein stabilization or NQO1 protein expression, GCLM protein expression was
modestly increased in cells treated with quinoline 23.
0
1
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8
9
0
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Positive results in the screeening assay seen with isoquinoline 17 and quinoline 23 led us to
examine nuclear accumulation of NRF2 (immunoblot) and expression of other NRF2 target
genes (qPCR) in HaCaT cells treated with isoquinoline 17 and quinoline 23 at both 6 and 24
hour time points. NRF2 accumulation in the nucleus was increased twoꢀfold when treated with
isoquinoline 17 compared to DMSO control at both time points analyzed (Figure 4). A small
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
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2
2
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3
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4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
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5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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8
9
0
1
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0
1
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0
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significant increase in NRF2 nuclear accumulation was observed in cells treated with quinoline
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3 at both time points. Analysis of mRNA levels in the HaCaT cells at 6 hours showed
isoquinoline 17 produced a significant increase in HMOX1, GCLM, and GCLC mRNA
expression (Figure 5). Isoquinoline 17 also produced an increase in NQO1 mRNA expression at
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4 hours. Quinoline 23 produced a small increase in NQO1, GCLM, and GCLC mRNA
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expression at 6 and 24 hours, with no increase observed for HMOX1 mRNA expression. mRNA
expression for SOD was also measured. SOD is not an NRF2 target gene, so, as expected, neither
isoquinoline 17 nor quinoline 23 produced a significant increase in SOD expression. Western
blot analysis of NQO1 protein levels corroborated the mRNA expression levels by showing a
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Journal of Medicinal Chemistry
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modest increase in protein expression in comparison to DMSO for both isoquinoline 17 and
quinoline 23 (SI Figure S11).
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Figure 5: The effects of nonꢀelectrophilic activators 17 (orange) and 23 (purple) on NRF2
target gene expression in HaCaT Cells. (A) HaCaT cells were treated with DMSO (vehicle)
or indicated compound (10 ꢁM) for 6 hours and 24 hours. cDNA were prepared from mRNA
and analyzed for the target gene expression (A: HMOX1; B: NQO1; C: GCLC; D: GCLM; E:
SOD) using qPCR with specific primers. Relative fold change was calculated with βꢀactin as
reference. DMSOꢀtreated sample value was used as one unit. Error bars represent the
standard error from 3ꢀ4 independent samples. *:p<0.05; **:p<0.01; ***:p<0.001; if no * is
indicated, p>0.05.
Metabolic Stability in Liver Microsomes. Active and moderately active compounds that
showed good solubility were subjected to human and mouse liver microsomes for 20 minutes to
ascertain the metabolic stability of the compounds. 1,4ꢀIsoquinoline 17, naphthalene 6a, and 5,8ꢀ
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quinoline 23 were stable at 20 minutes in both human and mouse liver microsomes (Figure 6).
Phthalazine 34 and 5,8ꢀquinoline 23 showed reduced stability in human liver microsomes, but
were comparable to the other compounds in mouse liver microsomes. The naphthalene amide 6b
was metabolically unstable, showing 0% compound remaining after 20 minutes in both mouse
and human liver microsomes, providing further evidence that amide analogs may be rapidly
hydrolyzed. The unsubstituted naphthalene 5 was metabolically unstable in mouse liver
microsomes, showing only 15% remaining after 20 minutes.
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Mutagenicity in a MiniꢀAmes Assay.
Figure 6: Stability to Liver Microsomes.
Compounds were incubated with mouse liver microsomes (A) and human liver microsomes
(B) for 20 min. Percentage of compound remaining after incubation was determined by
LCꢀMS.
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To probe our hypothesis that the naphthalene may prove to be mutagenic, we submitted
naphthalene 6a and isoquinoline 17 to a miniꢀAmes assay in Salmonella typhimurium and
Escherichia coli. The compounds were tested for their ability to induce reverse mutations at the
histidine locus of strains of Salmonella typhimurium (TA98, TA100, TA1535, TA1537) and
Escherichia coli (WP2 uvrA (pKM101)) in the presence and absence of exogenous metabolic
activation (Aroclor 1254 induced rat liver S9). Both naphthalene 6a and isoquinoline 17 were
screened at concentrations of 1.5, 4, 10, 25, 64, 160, 400 and 1000 ꢁg /well. No cytotoxicity was
observed in either naphthalene 6a or isoquinoline 17 at any dose level. Naphthalene 6a induced >
3ꢀfold increases (for TA1535) and > 2ꢀfold increases (for TA98 and TA100) that were
statistically significant at higher dose levels in the presence of S9 mix, compared to vehicle
control (Figure 7). Dose response was also observed in 6a for TA98 and TA100; therefore,
naphthalene 6a was judged to be positive for mutagenicity. On the other hand, isoquinoline 17
induced a > 2ꢀfold increase in only one strain (for TA98) at the highest dose level compared to
the vehicle control. No dose response was observed for isoquinoline 17 (Figure 7). Taken
together, these data lead us to conclude that isoquinoline 17 has a better mutagenic profile than
naphthalene 6a.
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Figure 7: MiniꢀAmes Assay Results. Compounds 6a (turquoise) and 17 (orange) and vehicle
control DMSO (white) were tested for their ability to induce reverse mutations at the histidine
locus of strains TA98 (A), TA100 (B), TA1535 (C), and TA1537 (D) of Salmonella
typhimurium and strain WP2 uvrA(pKM101) (E) of Escherichia coli in the presence of
exogenous rat liver S9 fractions. Individual revertant colonies were counted and graphed as a
function of compound concentration (400 and 1000 ꢁg/well). Foldꢀchange for those conditions
that were >2.0× greater than DMSO vehicle control are shown above each bar, along with pꢀ
values determined from a twoꢀtailed tꢀtest.
Discussion:
Previously, our lab investigated the structureꢀactivity relationship of the distal substitutions on
the naphthaleneꢀbased NRF2 activator 5. We found that an electronꢀdonating group on the
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terminal phenyl groups resulted in the most potent inhibitors. We also found that the sulfonamide
linkages were superior to similar linkages and that substituting anything other than a
carboxymethyl group or carboxamidomethyl group off the sulfonamide also resulted in an
inactive compound.
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Having explored the rest of the molecule, we turned our attention to the naphthalene scaffold.
While there are drugs, including propranolol, that contain a naphthalene core, naphthalenes have
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been identified as a minor mutagenic scaffold in the Ames Mutagenicity Tree. A related
naphthalene 4, RAꢀ839, showed in vivo activity only if Phase 1 metabolism were blocked.
Although the identity of the RAꢀ839 metabolites are unknown, they may include reactive
intermediates, as naphthalenes are known to be metabolically activated to reactive naphthalene
32ꢀ35
31
oxides and naphthoquinones.
With these points in mind, we explored alternative scaffolds.
Our previous results suggested that compounds with amideꢀcontaining sidechains (e.g., 6b)
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were comparable in potency to those containing carboxylic acidꢀcontaining sidechains. During
the course of the present study, however, we found that the amides are both relatively insoluble
in water and metabolically unstable in liver microsomes, compared to the acids. This is in good
agreement with work reported by Lu et al., who showed that amideꢀcontaining compounds were
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less soluble than acidꢀcontaining compounds at pH 7.4. Because we were interested in
compounds that would show good solubility and metabolic stability, we synthesized scaffolds
that were decorated with Nꢀcarboxymethyl groups.
To probe the necessity of a 6,6ꢀfused ring system, we synthesized nonꢀfused rings and
substitutions that might afford a disposition of substituents similar to the naphthalene scaffold.
These substitutions included butene 42, 1,4ꢀsubstituted pyridine 11a, 1,4ꢀsubstituted benzene
11b, 1,3,5ꢀsubstituted benzene 46, oꢀxylene 38, and phthalamide 40. Of these, only the benzene
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1b showed activity in the fluorescence anisotropy assay, albeit at values ca. 40ꢀfold higher than
the parent naphthalene 6a. These compounds displayed varying levels of solubility that did not
divulge any discernible pattern. The butene 42 and phthalamide 40 showed solubilities similar to
the parent naphthalene 6a, the pyridine 11a and 1,3,5ꢀsubstituted benzene 11a showed slight
solubility, and the oꢀxylene and 1,4ꢀsubstituted benzene 11b proved insoluble. Because these
compounds showed poor potency and solubility comparable to other scaffolds, we did not carry
them into liver microsome stability studies.
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To probe the necessity of the allꢀcarbon framework of the naphthalene scaffold, we replaced
the naphthalene core with nitrogenꢀcontaining heterocycles, including 1,4ꢀphthalazine 34, 1,4ꢀ
isoquinoline 17, 5,8ꢀquinoline 23, and 4,7ꢀindole 29. Of these, 5,8ꢀquinoline 23 and 1,4ꢀ
isoquinoline 17 retained good potency in both FP (IC = 101 and 60 nM, respectively) and SPR
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assays (K = 103 and 102 nM, respectively). The 1,4ꢀphthalazine 34 scaffold was less potent
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(IC = 1000 nM; K >10 ꢁM). Previously, we reported that a naphthalene monoꢀacid showed
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good affinity for KEAP1 (IC = 61 nM). Phthalazine 34, which is also a monoꢀacid, is less
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potent than the naphthalene monoꢀacid, but this is in agreement with the affinity of the
unsubstituted cores: unsubstituted naphthalene 5 has an IC of 0.96 ꢁM, while the unsubstituted
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phthalazine 32 has an IC of > 25ꢁM. Taken together, these data suggest that the allꢀcarbon
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framework of the naphthalene is not necessary for binding to KEAP1, and other 6,6ꢀfused
systems can be used to replace the naphthalene scaffold.
The binding of this class of compounds is dictated, in part, by a πꢀcation interaction between
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the πꢀsystem of the inhibitor and Arg415, first observed by Marcotte et al. Dougherty and
coworkers have comprehensively studied the binding energy of cations to a number of different
πꢀsystems. Their work suggests that electronꢀrich systems, like indole, should be better cation
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