Probing the structural requirements of non-electrophilic naphthalene-based Nrf2 activators

Article abstract of DOI:10.1016/j.ejmech.2015.08.049

Activation of the transcription factor Nrf2 has been posited to be a promising therapeutic strategy in a number of inflammatory and oxidative stress diseases due to its regulation of detoxifying enzymes. In this work, we have developed a comprehensive structure-activity relationship around a known, naphthalene-based non-electrophilic activator of Nrf2, and we report highly potent non-electrophilic activators of Nrf2. Computational docking analysis of a subset of the compound series demonstrates the importance of water molecule displacement for affinity, and the X-ray structure of di-amide 12e supports the computational analysis. One of the best compounds, acid 16b, has an IC₅₀ of 61 nM in a fluorescence anisotropy assay and a K_d of 120 nM in a surface plasmon resonance assay. Additionally, we demonstrate that the ethyl ester of 16b is an efficacious inducer of Nrf2 target genes, exhibiting ex vivo efficacy similar to the well-known electrophilic activator, sulforaphane.

Full text of DOI:10.1016/j.ejmech.2015.08.049

European Journal of Medicinal Chemistry 103 (2015) 252e268
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Probing the structural requirements of non-electrophilic naphthalene-
based Nrf2 activators
,
,
Atul D. Jain a, Haranatha Potteti b 1, Benjamin G. Richardson a 1, Laura Kingsley d,
Julia P. Luciano c, Aya F. Ryuzoji c, Hyun Lee a, Aleksej Krunic a, Andrew D. Mesecar c
,
,
d
Sekhar P. Reddy b e, Terry W. Moore a
,
, e, *
a Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago, Chicago, IL 60612, USA
b Department of Pediatrics, College of Medicine, University of Illinois at Chicago, Chicago, IL 60612, USA
c Department of Biological Sciences Purdue University, West Lafayette, IN 47907, USA
d Center for Cancer Research, Purdue University, West Lafayette, IN 47907, USA
e University of Illinois Cancer Center, University of Illinois at Chicago, Chicago, IL 60612, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Activation of the transcription factor Nrf2 has been posited to be a promising therapeutic strategy in a
number of inflammatory and oxidative stress diseases due to its regulation of detoxifying enzymes. In
this work, we have developed a comprehensive structureeactivity relationship around a known,
naphthalene-based non-electrophilic activator of Nrf2, and we report highly potent non-electrophilic
activators of Nrf2. Computational docking analysis of a subset of the compound series demonstrates
the importance of water molecule displacement for affinity, and the X-ray structure of di-amide 12e
supports the computational analysis. One of the best compounds, acid 16b, has an IC50 of 61 nM in a
fluorescence anisotropy assay and a Kd of 120 nM in a surface plasmon resonance assay. Additionally, we
demonstrate that the ethyl ester of 16b is an efficacious inducer of Nrf2 target genes, exhibiting ex vivo
efficacy similar to the well-known electrophilic activator, sulforaphane.
Received 24 April 2015
Received in revised form
22 August 2015
Accepted 25 August 2015
Available online 4 September 2015
Keywords:
Nrf2
Keap1
Proteineprotein interaction
Transcription factor
Cul3
© 2015 Elsevier Masson SAS. All rights reserved.
Keap1/Nrf2 interaction
1. Introduction
regulatory subunit, glutathione S-transferase, GST) [3]. These genes
are regulated by the transcription factor, Nrf2 (nuclear factor-
During periods of oxidative or electrophilic stress, one of the
body's main defenses is induction of cytoprotective proteins,
including detoxification enzymes, such as those that reduce qui-
nones (e.g., NAD(P)H quinone oxidoreductase 1, NQO1) [1], those
that degrade heme (heme oxygenase 1, HMOX1) [2], and those
involved in glutathione synthesis and transfer (e.g., glutamate-
cysteine ligase catalytic subunit, glutamate-cysteine ligase
erythroid2-related factor 2), which belongs to a cap ‘n’ collar family
of basic leucine zipper transcription factors that comprise seven
highly conserved domains (Neh1 to Neh7) [4]. In the absence of
electrophilic or oxidative stressors, Nrf2 is negatively regulated by
Keap1 (Kelch like ECH associated protein 1), a 69 kDa sensor protein
that contains 27 cysteine residues [5]. Keap1 is a tightly-associated
dimer that acts as an adaptor protein that simultaneously binds
both Nrf2, through Keap1's Kelch domain, and the E3 ubiquitin
ligase Cul3 [6], through Keap1's BTB domain. Cul3 poly-
ubiquitinates Nrf2, and Nrf2 is subsequently degraded through the
ubiquitin/proteasome system [4]. Nrf2 is activated by inhibiting its
degradation (see below). Nrf2 is then translocated to the nucleus,
where it heterodimerizes with the small MAF (sMAF) transcription
factors and binds to antioxidant response elements in the promoter
regions of many detoxification genes, such as NQO1, GST, HMOX1
and glutathione peroxidase [7].
ꢀ
Abbreviations: BTB, Broad complex, Tramtrack and Bric-a-Brac; LE, Ligand effi-
ciency; DMEM, Dubecco's modified Eagle's medium; FBS, fetal bovine serum;
HMOX1, heme oxygenase 1; Keap1, Kelch-like ECH-associated protein 1; NQO1,
NAD(P)H quinone oxidoreductase 1; MLE, mouse lung alveolar epithelial cell line;
Nrf2, nuclear factor (erythroid-derived 2)-like 2; sMAF, small musculoaponeurotic
fibrosarcoma oncogene homolog.
* Corresponding author. Department of Medicinal Chemistry and Pharmacog-
nosy, College of Pharmacy, University of Illinois at Chicago, Chicago, IL 60612, USA.
E-mail address: twmoore@uic.edu (T.W. Moore).
As shown in Fig. 1, there are two prevailing mechanisms that
1
These authors have contributed equally to this work.
http://dx.doi.org/10.1016/j.ejmech.2015.08.049
0223-5234/© 2015 Elsevier Masson SAS. All rights reserved.

A.D. Jain et al. / European Journal of Medicinal Chemistry 103 (2015) 252e268
253
Fig. 1. A model of Nrf2 activation by both electrophilic and non-electrophilic inhibition of Keap1-Nrf2 complex.
explain how Nrf2 degradation is inhibited: 1) the Cul3 dissociation
model [8,9] and 2) the hinge and latch model [10]. In the Cul3
dissociation model, Keap1 recognizes and binds Nrf2 through the
ETGE and DLG motifs of the Neh2 region of Nrf2. Keap1 brings Nrf2
into close proximity with a ubiquitin-conjugating enzyme (Cul3),
which then transfers ubiquitin to target lysine residues on Nrf2. If
electrophilic inhibitors bind to Keap1 so that Cul3 is dissociated
from Keap1, Nrf2 is not degraded, but it is also not released from
Keap1 [8,9,11]. Newly formed Nrf2 is then available for regulating
expression of cytoprotective enzymes. The alternative hinge and
latch model is governed by modifications affecting Nrf2 [10,12e14].
In this model, upon electrophilic modification of key cysteine res-
idues in the IVR region of Keap1, the protein undergoes a slight
conformational modification and releases the DLG motif, while
maintaining binding to the ETGE motif [12,13]. Once the DLG motif
is released, Nrf2 swings out from its ideal position, becoming
inaccessible for ubiquitination [13]. With this change, Nrf2 does not
get degraded, and nascent Nrf2 production gives rise to Nrf2
accumulation and activation. A recent structural study [15] lends
support for the Cul3-dissociation model, but there is still an active
dialogue in the literature as to which model dominates in Nrf2
activation or whether both are operative [8e10,16e18].
sulforaphane (1), an isothiocyanate derived from cruciferous veg-
etables like broccoli; and dimethyl fumarate, a new therapeutic for
multiple sclerosis (i.e., Tecfidera® (2); see Chart 1). The therapeutic
benefit of each of these electrophilic molecules is thought to arise,
in part, from Nrf2 activation. These compounds covalently react
with the sensor cysteines of Keap1, particularly Cys 151 [24e28],
which results in Nrf2 activation (see above). These covalent acti-
vators are obviously efficacious, but, because they are electrophiles,
they may not be selective for Keap1. This point is exemplified by a
proteomics study done with the oleanic triterpenoid bardoxolone
imidazole (3) (See Chart 1) [29]. That study found that this potent
electrophilic activator of Nrf2 interacts with at least 577 different
proteins in whole cells [29]. It acts as a Michael acceptor for reactive
Cys residues on Keap1 en route to activating Nrf2. A related com-
pound, bardoxolone methyl (4), proceeded as far as a phase III
clinical trial in patients with type 2 diabetes and chronic kidney
disease before adverse cardiovascular events derailed its develop-
ment [30]. Although their cause is unknown, these adverse events
may be attributable to off-target toxicity.
Recently, there has been great interest in developing reversible
covalent drugs to activate Nrf2 [31,32]; however, we and others
have taken a different approach and have begun to develop non-
covalent compounds that might be more selective Nrf2 activators
[31,33e37]. Non-covalent compounds may serve two important
functions: first, as tool compounds that can help to disentangle the
rather complicated pharmacology of Nrf2 activation, and, second,
as lead compounds for eventual therapeutic development.
Given its central importance in regulating cytoprotective en-
zymes, Nrf2 activation has been proposed as a promising phar-
macotherapeutic strategy in
a number of inflammatory and
oxidative stress disorders, including chronic kidney disease [19],
multiple sclerosis [20], pulmonary fibrosis [21], cancer chemopre-
vention [22], and chronic obstructive pulmonary disorder [23]. As
alluded to above, electrophilic Nrf2 activators are known (Chart 1).
Two of the more well-known electrophilic Nrf2 activators are
In developing non-covalent activators of Nrf2,
a sensible
approach is to inhibit the interaction of Nrf2 with its negative
regulator, Keap1. In this case, an inhibitor would occupy the site on

254
A.D. Jain et al. / European Journal of Medicinal Chemistry 103 (2015) 252e268
Keap1's Kelch domain where the ETGE motif of Nrf2 is bound,
which is distal from the binding site of known electrophilic acti-
vators [38]; thus, if a molecule binds in the ETGE pocket, Nrf2 is
displaced and activated [29,34].
Several hit compounds have been identified through high-
throughput screening (Chart 2). These hits include tetrahy-
Buffer C (20 mM Tris, pH 7.5, 5 mM DTT). The concentrated protein
was loaded onto a size-exclusion Superdex75 HR 26/60 column
(Amersham Biosciences) equilibrated with Buffer C. Protein was
used fresh for co-crystallization with the compounds and aliquots
were flash frozen on liquid nitrogen and stored at ꢁ80 ꢀC for further
experimentation. Protein purity at all steps was analyzed by SDS-
PAGE.
droisoquinoline 5 (IC50 ¼ 1.0
[36], naphthalene (IC50
(IC50 ¼ 29 nM [34]; Kd ¼ 9.9 nM), thiopyrimidine 9 (IC50 ¼ 118
m
M) [35], carbazone 6 (IC50 ¼ 9.8
m
M)
8
7
¼
2.7
m
M) [33], naphthalene
mM)
2.2. Co-crystallization and X-ray structure determination of the
Kelch-compound 12e complex
[39,40] and urea 10 (affinity unknown) [33]. Limited SAR and mo-
lecular modeling studies with some of these scaffolds have been
carried out in hopes of uncovering potent non-electrophilic in-
hibitors of the Keap1/Nrf2 complex [7,34,35]. These studies have
revealed that many of these non-electrophilic activators behave
similarly to the ETGE motif; significant interactions seem to occur
between acidic portions of the inhibitors and Arg 415 in the binding
pocket, with other contributions stemming from hydrogen bonding
Co-crystallization of the Kelch-12e complex was carried out by
sitting drop vapor diffusion using a 1:1 (Kelch:reservoir) drop ratio.
The Kelch-12e complex was generated by adding Kelch at 5 mg/mL
(160
compound 12e (320
PEG 3350. Crystals were grown at 4 ꢀC and reached an average size
of 200 m ꢂ 20 m in 11 days. Crystals were soaked
m ꢂ 150
mM) to a reservoir solution containing 2-fold molar excess of
mM), 0.2 M potassium chloride and 20% (w/v)
and
p
-stacking [34]. These studies represent a powerful starting
m
m
m
point in developing more comprehensive SARs of non-electrophilic
activators of Nrf2. In this work, we have expanded the SAR around 7
and 8. We detail here a more complete understanding of the
structural requirements for binding of non-electrophilic molecules
to Keap1 via computational modeling and X-ray crystallography,
and we report a compound with greater ligand efficiency, equi-
potent in vitro activity, and enhanced ex vivo activity.
briefly in reservoir solution supplemented with 20% PEG 400 and
then flash-cooled in liquid nitrogen for X-ray data collection. A
complete X-ray dataset was collected at 100 K and at a X-ray
wavelength of 1.547 Å at distance of 200 mm from our Raxis 4þþ
detector. The X-ray dataset was processed and scaled using
HKL2000 to a resolution of 2.47 Å. The Kelch-12e complex crys-
tallized as a monomer in the asymmetric unit and in space group P2
with unit cell dimensions of a ¼ 38.47 Å b ¼ 51.52 Å c ¼ 67.24 Å and
2. Methods and materials
b
¼ 101.27ꢀ. Molecular replacement using the Kelch:compound 7
structure (4IQK) as a search model was conducted to obtain initial
phases. The structure was then refined using the program Phenix.
The final coordinates for the Kelch:12e complex have been
deposited under PDB code 4XMB.
2.1. Kelch domain expression and purification [24]
The gene for the Kelch domain (amino acids 321e609) of human
Keap1 was codon-optimized for expression in Escherichia coli,
synthesized and cloned into pET15b (Bio Basic Inc.). For crystallo-
graphic work, the codon-optimized gene was PCR amplified and
subcloned via ligation-free cloning into the vector pEV-L8 to
incorporate a TEV-cleavable, N-terminal (His)8-tag. For expression
and purification of both Kelch proteins from the vectors described
above, BL21 (DE3) cells (Novagen) were transformed with each
construct via electroporation. These cells were plated on LB agarose
2.3. Cell culture and treatment
Mouse alveolar epithelial cells (MLE-12) [41,42] (kindly pro-
vided by Dr. Jeff Whitsett, University of Cincinnati) were grown in
DMEM (Dubecco's modified Eagle's medium) with 10% FBS (fetal
bovine serum). Cells were cultured at 37 ꢀC under 5% CO2 in a
humidified incubator. Cells were treated with the indicated com-
plates containing 50
picked and then used to inoculate small starter cultures (LB me-
m
g/mL carbenicillin. Single colonies were
pounds at 10
medium.
mM concentration for 6 h in the presence of complete
dium containing 50
mg/mL carbenicillin). Cells were allowed to
grow overnight at 37 ꢀC and were then used to inoculate 1 L of fresh
media (LB:carbenicillin) and grown at 37 ꢀC until OD600 of 0.6. At
this point, protein expression was induced by the addition of 1 mM
2.4. Immunoblot analyses
Total protein was extracted in lysis buffer consisting of 20 mM
Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-
isopropyl-b-D
-galactopyranoside for 4 h at 37 ꢀC. After expression,
cells were harvested by centrifugation at 8000g at 4 ꢀC for 25 min.
Cell pellets were frozen at ꢁ80 ꢀC. The frozen cell pellets were
resuspended in Buffer A (50 mM Tris, pH 7.5, 500 mM NaCl, 10 mM
imidazole, 3 mM DTT) containing one EDTA-free protease inhibitor
pellet (Roche), 7.5 mM MgSO4 and minimal amounts of DNaseI and
lysozyme and the cells were lysed via sonication. The lysed cells
were pelleted by centrifugation at 28,960g for 25 min at 4 ꢀC. The
supernatant was applied to a 5 mL HisTrap affinity column charged
with Ni2þ (GE Healthcare Life Sciences) equilibrated with Buffer A.
The protein was eluted in a gradient of 0e60% Buffer B (50 mM Tris,
pH 7.5, 500 mM NaCl, 500 mM imidazole, 3 mM DTT) in 5 mL
fractions. The final purity of the proteins was analyzed by SDS/
PAGE.
100, 2.5 mM sodium pyrophosphate, 1 mM Na3VO4, 5 mM
erophosphate, and 1 g/mL leupeptin. Comparable amount of total
protein (~40 g) from each sample were separated on a 10% SDS-
b-glyc-
m
m
PAGE and the membranes were probed with antibodies specific
for Nrf2 (Santa Cruz Biotech, Santa Cruz, CA), HMOX1 (Santa Cruz
Technologies, Santa Cruz, CA), NQO1 (Abcam Company Cambridge,
MA).
b-actin (Sigma, St. Louis, MO) antibody was used as the
loading control. The blots were developed using an HyGlo-Chemi
antibody detection kit (Denville Scientific Inc, Metuchen, NJ) and
images were visualized on ChemiDoc (Biorad) and bands were
quantified using image J software.
2.5. Fluorescence anisotropy assays
To generate purified Kelch protein for crystallization experi-
ments, pooled fractions from the HisTrap column were dialyzed
overnight at 4 ꢀC against Buffer A in the presence of TEV protease
(1:5 (w/w)). Separation of the cleaved tag was achieved by reverse
HisTrap chromatography following the same procedure described
above. Pooled fractions were concentrated and dialyzed against
Fluorescence anisotropy assays were performed on a Biotek
Microplate Reader. The plates used for the fluorescence anisotropy
assay were nonbinding Corning 3686 96-well black, half area, flat
bottom plate polystyrene plates. The assay solution was 40
mL per
well consisting of 10 L of modified HEPES buffer (10 mM) pH 7.4
m

A.D. Jain et al. / European Journal of Medicinal Chemistry 103 (2015) 252e268
255
containing 50 mM EDTA, 150 mM NaCl, and 0.005% Tween-20 (final
concentrations), 10 L of 100 nM Keap1 Kelch domain protein (final
concentration) [43], 10 L of ligand sample of varying concentra-
tions, and 10 L of FITC-9mer Nrf2 peptide (10 nM final concen-
tration) [43]. The DMSO concentration in the final assay solution
was not more than 5%. The experiments were performed in tripli-
2.7. Ensemble docking
m
m
An ensemble containing four structures of the Keap domain was
generated based on the crystal structure 4IQK containing different
patterns of waters 866 and 807. To construct the ensemble the
waters were 1) unaltered, 2) water 866 (water 1) was removed, 3)
water 807 (water 2) was removed, or 4) both waters were removed.
Docking was performed using the Virtual Screening Workflow
m
cate with initial concentration of the ligand set at 25
mM and were
further diluted to 20, 15, 10, 5, 3, 1, 0.3, 0.1, 0.03, 0.01, and 0.003
m
M.
€
The plate was protected from light, centrifuged for 2 min at
2250 rpm, rocked for 1 h, and centrifuged again for 2 min at
2250 rpm to remove any bubbles formed during the rocking stage.
The fluorescence anisotropy measurement was performed at
lex ¼ 480 20 nm and lem ¼ 520 20 nm emission filters. Fluo-
rescence anisotropy was determined by measuring the parallel (Frr)
and perpendicular (F⊥) fluorescence intensity with respect to the
linearly polarized excitation light. The IC50 of the ligand was
calculated by plotting the normalized fluorescence anisotropy
values vs. the log concentration of the ligand, and the relationships
were analyzed by Prism 6.1 software.
implementation of the Glide docking package from Schrodinger
(Small-Molecule Drug Discovery Suite 2014-4: Glide, version 6.5,
Schrodinger, LLC, New York, NY, 2014) [44e46]. Docking was per-
formed using the Standard Precision (SP) settings, and up to two
poses per compound state were allowed.
€
2.8. Hydration site analysis
The docking results obtained in the previous section were used
as input for the hydration site analysis program, WATsite [47],
which is freely available from http://people.pharmacy.purdue.edu/
~mlill/software/. All settings of WATsite used were standard, with
the exception that the production molecular dynamics simulation
was extended from 1 ns (default) to 5 ns and the number of frames
used was 5000.
2.6. Determination of dissociation equilibrium constant (Kd) by
surface plasmon resonance (SPR)
Purified Keap1 protein was diluted to 80
sodium acetate (pH 5.0) and injected for 7 min at a 10
m
g/mL with 10 mM
2.9. Chemistry
mL/min flow
rate on a CM5 sensor chip for immobilization at 25 ꢀC with running
buffer PBS-P (10 mM phosphate pH 7.4, 2.7 mM KCl, 137 mM NaCl,
and 0.05% surfactant P-20) using a Biacore T200 instrument. Blank
surface was used as a control on flow channels 1 and 3. Keap1 was
immobilized to flow channels 2 and 4 after sensor surface activa-
tion by a mixture of 1-ethyl-3-(3-dimethylaminopropyl) carbodii-
mide hydrocholoride (EDC)/N-hydroxysuccinimide (NHS) followed
by ethanolamine blocking on unoccupied surface area. Keap1
immobilization levels of flow channels 2 and 4 were ~6560 RU and
~5700 RU, respectively. Compound solutions with a series of
increasing concentrations were applied to all four channels at a
1H and 13C NMR spectra were recorded on a Bruker 400 MHz
spectrometer using tetramethylsilane (TMS) as an internal stan-
dard. Peak positions are given in parts per million (d). Purity of each
of the final, tested compounds was determined by HPLC on a Shi-
madzu LC-20AB (Solvent system; 55% MeCN/40% H2O, isocratic;
Column: Shimadzu C18, 50
m
m, 50 ꢂ 4.6 mm) and was ꢃ95% (UV,
254 nm). The HRMS spectra were recorded on a Shimadzu LCMS-IT-
TOF, and the molecular weight of the compounds was within 0.05%
of calculated values. Flash chromatography was performed using
silica gel (230e400 mesh). All reactions were monitored by thin-
layer chromatography (TLC) on silica gel GHLF plates (250
mm,
30 mL/min flow rate with assay buffer (10 mM phosphate, pH 7.4,
MachereyeNagel, Inc., Bethlehem, PA). Compounds 7, 8, and 12a
were synthesized according to previously reported literature
[33,34,39]. It should be noted that aminonaphthalenes are gener-
ally carcinogenic in nature. Caution should be used while handling
them.
2.7 mM KCl, 137 mM NaCl, 0.05% surfactant P-20, 2 mM TCEP, and
2% DMSO), and real-time response units (RU) were monitored.
Sensorgrams were analyzed using the Biacore T200 evaluation
software 2.0. Data were referenced with blank channel RU values,
and the Kd values were determined by fitting the reference sub-
tracted data to a single rectangular hyperbolic curve equation (Eq.
(1)) where y is the RU, ymax is the maximum RU, and x is the
compound concentration. Kinetic fittings were done by 1 to 1
binding equation embedded in the Biacore T200 evaluation soft-
ware 2.0.
2.9.1. General method for synthesis of 11aej, and 20 (Method A)
These previously unreported compounds were synthesized ac-
cording to a known, similar procedure [48]. A solution of 1,4-
diaminonaphthalene dihydrochloride (1 equivalent) and
substituted-benzenesulfonyl chloride (2.2 equivalent) in pyridine
(3e5 mL) was allowed stir at room temperature for 24 h. On
completion, the reaction mixture was diluted with EtOAc (40 mL),
washed with H2O (2 ꢂ 50 mL) and HCl (2 N, 2 ꢂ 50 mL), dried
(Na2SO4) and evaporated to yield crude products, which were
y
max$x
y ¼
(1)
KD þ x
Chart 1. Known electrophilic Nrf2 activators that react with cysteine residues in the BTB and IVR domains of Keap1.

256
A.D. Jain et al. / European Journal of Medicinal Chemistry 103 (2015) 252e268
Chart 2. Known Non-electrophilic Nrf2 Activators that Inhibit the Keap1/Nrf2 Interaction in the Kelch domain.
recrystallized or washed with appropriate solvents to yield pure
compounds.
the organic solvent was evaporated, and the residue was suspended
in H2O (40 mL), acidified with HCl (2 N, to pH 1e2), and extracted
with EtOAc (3 ꢂ 30 mL). The combined organic portion was washed
with H2O (3 ꢂ 50 mL) and brine (50 mL), dried (Na2SO4) and
evaporated to yield crude product which, upon recrystallization
from EtOAc/MeOH, yielded 0.01 g (51% yield) of 11d as a buff-
2.9.1.1. Synthesis of N,N0-(naphthalene-1,4-diyl)dibenzenesulfona-
mide (11a). The crude product was recrystallized from MeOH to
yield 0.14 g (51% yield) of 11a as pink needles. 1H NMR (400 MHz,
colored solid. 1H NMR (400 MHz, DMSO-d6,
d): 13.46 (br s, 2H),
DMSO-d6, d) 10.20 (s, 2H), 7.92e7.89 (m, 2H), 7.63e7.34 (m, 10H),
7.24 (d, J ¼ 8.0 Hz, 2H), 7.03 (s, 2H); 13C NMR (100 MHz, DMSO-d6,
d)
10.36 (s, 2H), 8.30 (s, 2H), 8.11 (d, J ¼ 7.6 Hz, 2H), 7.89 (dd, J ¼ 6.4,
3.2 Hz, 2H), 7.74 (d, J ¼ 7.6 Hz, 2H), 7.57 (t, 2H), 7.37 (dd, J ¼ 6.0,
139.8, 132.8, 131.1, 130.1, 129.2, 126.7, 126.2, 123.3, 123.0; HRMS-ESI
(ꢁ) (m/z): [MꢁH]ꢁ calcd for C20H19N2O6S2, 437.0655; found,
437.0635.
2.8 Hz, 2H), 6.99 (s, 2H); 13C NMR (100 MHz, DMSO-d6,
d): 165.9,
140.3, 133.3, 131.8, 130.9, 130.7, 130.2, 129.8, 127.4, 126.4, 123.5;
HRMS-ESI (ꢁ) (m/z): [MꢁH]ꢁ calcd for C24H17N2O8S2, 525.0432;
found, 525.0456.
2.9.1.2. Synthesis of N,N0-(naphthalene-1,4-diyl)bis(3-methoxy
benzenesulfonamide) (11b). The crude product was recrystallized
from CH2Cl2/acetone to yield 0.08 g (25% yield) of 11b as a buff-
2.9.1.5. Synthesis of N,N0-(naphthalene-1,4-diyl)bis(4-trifluoromethyl
benzenesulfonamide) (11e). The crude product was washed with
Et2O to afford 0.06 g (16% yield) of 11e as buff-colored solid. 1H NMR
colored solid. 1H NMR (400 MHz, DMSO-d6,
d
) 10.32 (s, 2H),
7.95e7.93 (m, 2H), 7.38 (t, J ¼ 8.0 Hz, 2H), 7.20e7.06 (m, 9H), 3.67 (s,
6H); 13C NMR (100 MHz, DMSO-d6,
) 159.3, 141.1, 131.1, 130.1, 130.4,
(400 MHz, DMSO-d6,
d
) 10.49 (s, 2H), 7.83e7.81 (m, 10H), 7.22 (s,
d
2H), 7.09 (s, 2H); 13C NMR (100 MHz, DMSO-d6,
d) 143.9, 132.9 (q,
126.3, 123.1, 123.4, 118.8, 118.9, 111.5, 55.5; HRMS-ESI (ꢁ) (m/z):
[MꢁH]ꢁ calcd for C24H21N2O6S2, 497.0887; found, 497.0864.
JCF ¼ 32 Hz), 131.2, 130.6, 128.1, 126.8 (d, JCF ¼ 3 Hz), 126.7, 124.2,
123.8 (d, JCF ¼ 270 Hz),123.5; HRMS-ESI (ꢁ) (m/z): [MꢁH]ꢁ calcd for
2.9.1.3. Synthesis of N,N0-(naphthalene-1,4-diyl)bis(4-chlorobenzene
sulfonamide) (11c). The crude product was washed with Et2O to
afford 0.12 g (37% yield) of 11c as a buff-colored solid. 1H NMR
C24H15N2O6F6S2, 573.0383; found, 573.0393.
2.9.1.6. Synthesis of N,N0-(naphthalene-1,4-diyl)bis(3-trifluoromethyl
benzenesulfonamide) (11f). The crude product was recrystallized
from i-PrOH to afford 0.10 g (50% yield) of 11f as a gray-colored
(400 MHz, DMSO-d6, d) 10.33 (s, 2H), 7.92 (br s, 2H), 7.69e7.41 (m,
10H), 7.06 (m, 2H); 13C NMR (100 MHz, DMSO-d6,
d) 138.6, 137.7,
solid; 1H NMR (400 MHz, DMSO-d6,
d): 10.48 (s, 2H), 7.98 (d,
130.9, 130.2, 129.3, 128.7, 126.4, 123.3; HRMS-ESI (ꢁ) (m/z): [MꢁH]ꢁ
calcd for C22H15N2O4S2Cl2, 504.9874; found, 504.9874.
J ¼ 8.0 Hz, 2H), 7.90 (d, J ¼ 8.0 Hz, 2H), 7.86e7.83 (m, 4H), 7.46e7.71
(m, 2H), 7.36e7.34 (m, 2H), 7.06 (s, 2H); 13C NMR (100 MHz, DMSO-
2.9.1.4. Synthesis of 3,30-(((4-(hydrosulfonylamino)naphthalen-1-yl)
amino)sulfonyl)dibenzoic acid (11d). The crude methyl ester was
washed with Et2O to afford 0.21 g (58% yield) of the methyl ester of
d6,
d
): 140.8,130.9,130.9,130.6,130.1,129.8 (d, JCF ¼ 33 Hz),129.6 (d,
JCF ¼ 5 Hz) 126.4, 123.7, 123.3 (d, JCF ¼ 4 Hz), 123.3 (d, JCF ¼ 271 Hz),
123.2; HRMS-ESI (ꢁ) (m/z): [MꢁH]ꢁ calcd for C24H15F6N2O4S2,
573.0383; found, 573.0370.
11d as a purple solid. 1H NMR (400 MHz, DMSO-d6,
d): 10.41 (s, 2H),
8.28 (s, 2H), 8.13 (d, J ¼ 8.0 Hz, 2H), 7.89 (dd, J ¼ 6.4, 3.2 Hz, 2H), 7.80
(d, J ¼ 8.0 Hz, 2H), 7.61 (t, 2H), 7.37 (dd, J ¼ 6.4, 3.0 Hz, 2H), 6.98 (s,
2H), 3.85 (s, 6H). The methyl ester was further hydrolyzed using
NaOH (15%, 5 mL) in MeOH (15 mL) at reflux for 3 h. On completion,
2.9.1.7. Synthesis of N,N0-(naphthalene-1,4-diyl)bis(3,4-dimethoxy
benzenesulfonamide) (11g). The crude product was recrystallized
from i-PrOH to afford 0.15 g (65% yield) of 11g as a buff-colored

A.D. Jain et al. / European Journal of Medicinal Chemistry 103 (2015) 252e268
257
solid. 1H NMR (400 MHz, DMSO-d6,
d): 10.03 (s, 2H), 8.00e7.93 (m,
4.49e4.40 (m, 2H), 4.06e3.85 (m, 8H); 13C NMR (100 MHz, DMSO-
d6, ): 162.9, 135.9, 133.8, 133.7, 132.6, 132.4, 130.1, 129.9, 128.9,
128.7, 126.9, 125.6, 125.2, 124.2, 119.7, 119.5, 114.5, 55.8, 54.6, 54.4;
HRMS-ESI (ꢁ) (m/z): [MꢁH]ꢁ calcd for C30H29N2O6S2, 577.1473;
found, 577.1202.
2H), 7.42e7.40 (m, 2H), 7.18 (d, J ¼ 8.0 Hz, 2H), 7.10e7.05 (m, 4H),
d
6.98 (d, J ¼ 8.0 Hz, 2H), 3.77 (s, 6H), 3.61 (s, 6H); 13C NMR (100 MHz,
DMSO-d6, d): 152.1, 148.5, 131.4, 131.2, 130.2, 126.2, 123.5, 122.9,
120.5, 110.9, 109.4, 55.8, 55.6; HRMS-ESI (ꢁ) (m/z): [MꢁH]ꢁ calcd
for C26H25N2O8S2, 557.1052; found, 557.1051.
2.9.2.3. Synthesis of N,N0-(naphthalene-1,4-diyl)bis(4-methoxy-N-
methylbenzenesulfonamide) (12c). The crude product was recrys-
tallized from EtOH to yield 0.07 g (89% yield) of 12c as a buff-
2.9.1.8. Synthesis of N,N0-(naphthalene-1,4-diyl)bis(4-methylbenzene
sulfonamide) (11h). The crude product was recrystallized from i-
PrOH to afford 0.092 g (53% yield) of 11h as a white-colored solid;
1H NMR (400 MHz, DMSO-d6) d 10.12 (s, 7H), 7.95 (dd, J ¼ 3.3,
6.4 Hz, 2H), 7.51 (d, J ¼ 8.2 Hz, 4H), 7.39 (dd, J ¼ 3.4, 6.9 Hz, 2H), 7.27
(d, J ¼ 8.1 Hz, 4H), 6.99 (s, 2H), 2.32 (s, 6H); 13C NMR (100 MHz,
colored solid; 1H NMR (400 MHz, DMSO-d6,
d): 8.24 (s, 1H), 8.19
(s, 1H), 7.69 (dd, J ¼ 6.4, 3.2 Hz, 2H), 7.63 (d, J ¼ 8.8 Hz, 4H), 7.20 (d,
J ¼ 8.0 Hz, 2H), 7.14 (d, J ¼ 8.0 Hz, 2H), 6.87 (s, 1H), 6.65 (s, 1H), 3.90
(s, 3H), 3.86 (s, 3H), 3.21 (s, 3H), 3.16 (s, 3H); 13C NMR (100 MHz,
DMSO-d6,
d
): 143.5, 137.5, 131.4, 130.5, 129.9, 127.2, 126.6, 123.8,
DMSO-d6, d): 162.9, 138.4, 138.3, 130.2, 130.0, 127.6, 127.3, 124.1,
123.1, 21.4; HRMS-ESI (þ) (m/z): [MþH]þ calcd for C24H23N2O4S2,
114.6, 114.6 55.8; HRMS-ESI (þ) (m/z): [MþNa]þ calcd for
467.1099; found, 467.1105.
C26H26N2O6S2Na, 549.1130; found, 549.1115.
2.9.1.9. Synthesis of N,N0-(naphthalene-1,4-diyl)bis(4-(N,N-dimethy-
lamino)benzenesulfonamide) (11i). The crude product was washed
with H2O (50 mL) and Et2O (20 mL) to obtain a gray solid which was
recrystallized from MeOH to afford 0.13 g (65% yield) of 11i as a
2.9.2.4. Synthesis of N,N0-(naphthalene-1,4-diyl)bis(N-ethyl-4-
methoxybenzenesulfonamide) (12d). The crude product was puri-
fied by column chromatography (silica gel; hexanes/EtOAc; 1:0 to
1:1) to afford 0.06 g (77% yield) of 12d as a buff-colored solid; 1H
gray-colored solid; 1H NMR (400 MHz, DMSO-d6,
d): d 9.76 (s, 2H),
NMR (400 MHz, DMSO-d6,
d
): 8.25 (dd, J ¼ 7.6, 4.0 Hz, 1H), 8.16 (dd,
8.03e8.02 (m, 2H), 7.42e7.40 (m, 6H), 7.01 (s, 2H), 6.64 (d,
J ¼ 6.4, 3.2 Hz, 1H), 7.69e7.60 (m, 6H), 7.20 (d, J ¼ 8.0 Hz, 2H), 7.12
(d, J ¼ 8.0 Hz, 2H), 6.92 (s, 1H), 6.68 (s, 1H), 3.98e3.81 (m, 2H), 3.87
(s, 3H), 3.82 (s, 3H), 3.45 (app. sextet, J ¼ 7.2 Hz, 1H), 0.96 (t,
J ¼ 7.0 Hz, 3H), 0.88 (t, J ¼ 7.0 Hz, 3H); 13C NMR (100 MHz, DMSO-d6,
J ¼ 8.4 Hz, 4H), 2.94 (s, 12H); 13C NMR (100 MHz, DMSO-d6,
d):
152.9, 131.6, 130.3, 128.8, 126.3, 125.4, 123.9, 122.4, 111.1.
2.9.1.10. Synthesis of N,N0-(naphthalene-1,4-diyl)bis(4-fluorobenzene
sulfonamide) (11j). The crude product was recrystallized from i-
PrOH/Et2O to afford 0.12 g (57% yield) of 11i as pink-colored nee-
d): 162.9, 136.3, 134.3, 129.8, 126.5, 125.1, 124.7, 113.8, 54.9, 54.8,
46.6, 13.1, 12.9; HRMS-ESI (þ) (m/z): [MþNa]þ calcd for
C28H30N2O6S2Na, 577.1437; found, 577.1405.
dles; 1H NMR (400 MHz, CDCl3,
d
): 7.80e7.78 (dd, J ¼ 2.8, 3.2 Hz,
1H), 7.75e7.72 (m, 2H), 7.48e7.46 (dd, J ¼ 2.8, 3.2 Hz, 1H), 7.07 (t,
2.9.2.5. Synthesis of 2,20-(naphthalene-1,4-diylbis(((4-methoxy
phenyl)sulfonyl)azanediyl))diacetamide (12e). The crude product
was recrystallized from i-PrOH to afford 0.04 g (65% yield) of 12e as
J ¼ 8.4 Hz, 2H); 13C NMR (100 MHz, DMSO-d6,
d): 164.7 (d,
JCF ¼ 250 Hz), 136.5 (d, JCF ¼ 3 Hz), 131.4, 130.6,130.2 (d, JCF ¼ 10 Hz),
126.7, 123.7, 123.7, 116.7 (d, JCF ¼ 23 Hz).
a brown-colored solid; 1H NMR (400 MHz, DMSO-d6,
d
): 8.19e7.99
(m, 2H), 7.64e7.41 (m, 6H), 7.13e6.93 (m, 6H), 4.48e4.32 (m, 4H),
3.92e3.82 (m, 6H); 13C NMR (100 MHz, DMSO-d6,
): 169.1, 163.2,
2.9.2. General method for synthesis of 12aef, and 21 (Method B)
These previously unreported compounds were synthesized ac-
cording a published procedure [34]. Potassium carbonate (3
equivalents) and suitable bromo-substituted electrophiles (2.5
equivalents) were added to a solution of 7 (1 equivalents) in
anhydrous DMF (2 mL), and the reaction was stirred at room
temperature until complete as monitored by TLC. On completion
the reaction was quenched with H2O (25 mL) and acidified with HCl
(2 N, to pH 5). The precipitate was collected by filtration and pu-
rified by column chromatography or recrystallized with appro-
priate solvents to yield pure compounds.
d
162.9, 134.9, 133.7, 133.1, 132.0, 130.4, 130.3, 129.9, 129.4, 126.9,
126.8, 126.6, 125.1, 123.7, 121.7, 114.7, 114.6, 56.2, 56.1, 54.4; HRMS-
ESI (þ) (m/z): [MþNa]þ calcd for C28H28N4O8S2Na, 635.1241; found,
635.1294.
2.9.2.6. Synthesis of N,N0-bis-propargyl-(naphthalene-1,4-diyl)bis(4-
methoxybenzenesulfonamide) (12f). Yield: 50 mg (62% yield) of
12f as a white solid. 1H NMR (400 MHz, DMSO-d6,
d): 8.21 (dd,
J ¼ 6.6, 3.0 Hz, 1H), 8.17e8.11 (m, 1H), 7.70e7.62 (m, 6H), 7.17 (d,
J ¼ 8.9 Hz, 2H), 7.11 (d, J ¼ 8.8 Hz, 2H), 7.01 (s, 1H), 6.82 (s, 1H),
4.60e4.46 (m, 4H), 3.90 (s, 3H), 3.85 (s, 3H), 3.25 (br s, 1H), 3.16 (s,
2.9.2.1. Synthesis of diethyl 2,20-(naphthalene-1,4-diyl)bis(((4-
1H); 13C NMR (400 MHz, DMSO-d6,
d):163.10, 163.05, 136.60,
methoxyphenyl)sulfonyl)azanediyl))diacetate
product was recrystallized from EtOH to yield 0.26 g (65% yield) of
12a as a brown-colored solid; 1H NMR (400 MHz, DMSO-d6,
): 8.34
(dd, J ¼ 6.3, 3.3 Hz, 1H), 8.20 (dd, J ¼ 6.4, 3.4 Hz, 1H), 7.63e7.57 (m,
6H), 7.16e7.06 (m, 5H), 6.84 (s, 1H), 4.59e4.40 (m, 4H), 4.07e3.96
(m, 4H), 3.89 (s, 3H), 3.84 (s, 3H), 1.05 (m, 6H); 13C NMR (100 MHz,
(12a). The
crude
133.55, 133.39, 130.30, 130.03, 129.55, 128.93, 127.28, 126.03,
125.66, 124.09, 114.55, 114.48, 78.41, 76.83, 76.65, 5.86, 41.69, 41.48;
HRMS-ESI (þ) (m/z): [MþNa]þ calcd for C30H26N2O6S2Na, 597.1158;
found, 597.1130.
d
2.9.3. Synthesis of 4-methoxy-N-(4-nitronaphthalen-1-yl)
benzenesulfonamide
DMSO-d6, d): 168.9, 163.4, 137.5, 103.6, 103.4, 127.1, 114.8, 61.3, 56.2,
25.9, 14.3; HRMS-ESI (þ) (m/z): [MþNa]þ calcd for
This previously unreported compound was synthesized ac-
cording to a similar procedure [48]. In a round-bottomed flask, 4-
nitronaphthalen-1-amine (synthesized according to a reported
procedure) [49] (5.0 g, 26.6 mmol) and 4-methoxybenzenesulfonyl
chloride (6.6 g, 31.9 mmol) was dissolved in THF (30 mL) and pyr-
idine (9 mL). The stirring mixture was brought to reflux and stirred
for 48 h. Upon reaction completion, monitored by TLC (1:1 Hex:-
EtOAc), the reaction mixture was concentrated under vacuum,
taken up in EtOAc (50 mL), and washed with HCl (2 N, 50 mL) and
H2O (2 ꢂ 50 mL). The organic fraction was dried over sodium
C
34H32N2O10S2Na, 693.1547; found, 693.1588.
2.9.2.2. Synthesis of N,N0-(naphthalene-1,4-diyl)bis(N-allyl-4-
methoxybenzenesulfonamide) (12b). The crude product was puri-
fied by column chromatography (silica gel; hexanes/EtOAc; 1:0 to
3:1) to afford 0.09 g (90% yield) of 12b as a buff-colored solid. 1H
NMR (400 MHz, DMSO-d6,
d
): 8.19e8.14 (m, 2H), 7.63 (d, J ¼ 8.0 Hz,
4H), 7.53 (d, J ¼ 8.0 Hz, 2H), 7.19 (d, J ¼ 12 Hz, 2H), 7.08 (d, J ¼ 8.0 Hz,
2H), 6.78 (s, 1H), 6.64 (s, 1H), 5.70e5.61 (m, 2H), 5.06e4.83 (m, 4H),

258
A.D. Jain et al. / European Journal of Medicinal Chemistry 103 (2015) 252e268
sulfate, filtered, and dried under vacuum. The resulting solid was
washed with ether (2 ꢂ 25 mL) and recrystallized from toluene to
yield 7.19 g (76% yield) of 4-methoxy-N-(4-nitronaphthalen-1-yl)
benzenesulfonamide as an off white solid; 1H NMR (400 MHz,
naphthalen-1-yl)-4-methoxybenzenesulfonamide hydrochlo-
ride (0.20 g, 0.55 mmol) was added to a solution of anhydrous THF
(10 mL), pyridine (1 mL) and p-trifluoromethylbenzenesulfonyl
chloride (0.16 g, 0.70 mmol), and the reaction was allowed to stir at
room temperature for 24 h. On completion, the organic solvent was
evaporated and the residue was dissolved in CH2Cl2 (40 mL),
washed with H2O (2 ꢂ 30 mL) and HCl (2 N, 2 ꢂ 20 mL), dried
(Na2SO4) and evaporated to yield crude product, which was
recrystallized from MeOH to yield 0.15 g (55% yield) of 14b as a buff-
DMSO-d6,
d
): 10.82 (br s, 1H), 8.38 (d, J ¼ 8.7 Hz, 1H), 8.28 (t,
J ¼ 8.4 Hz, 2H), 7.70e7.83 (m, 3H), 7.66 (t, J ¼ 7.2 Hz, 1H), 7.43 (d,
J ¼ 8.5 Hz, 1H), 7.05 (d, J ¼ 8.9 Hz, 2H), 3.77 (s, 3H).
2.9.4. Synthesis of N-(4-aminonaphthalen-1-yl)-4-
methoxybenzenesulfonamide hydrochloride
colored solid. 1H NMR (400 MHz, DMSO-d6,
d): 10.45 (s, 1H), 10.45
This previously unreported compound was synthesized ac-
cording to a known, similar procedure [50]. Pd/C (10%, 0.05 g) was
(s, 1H), 7.96 (d, J ¼ 8.0 Hz, 2H), 7.87e7.79 (m, 5H), 7.56 (d, J ¼ 8.0 Hz,
2H), 7.40e7.33 (m, 2H), 7.07e7.02 (m, 2H), 6.98 (d, J ¼ 8.0 Hz, 2H),
added to
a
suspension of previously prepared N-(4-nitro
(0.50 g,
3.76 (s, 3H); 13C NMR (100 MHz, DMSO-d6,
d): 162.4, 143.7, 132.5 (q,
naphthalen-1-yl)-4-methoxybenzenesulfonamide
JCF ¼ 32 Hz), 131.8, 131.5, 130.3, 130.2, 130.01, 128.9, 127.7, 126.4 (d,
JCF ¼ 3 Hz), 126.3, 126.2, 123.8, 123.5, 123.4 (q, JCF ¼ 271 Hz), 123.1,
122.6, 55.6; HRMS-ESI (ꢁ) (m/z): [MꢁH]ꢁ calcd for C23H19N2O5S2,
535.0615; found, 535.0636.
1.4 mmol) in EtOH (30 mL) and HCl/EtOH (1.25 M, 5.0 mL), and the
reaction was hydrogenated using a Parr shaker apparatus (35 psi)
for 5 h. On completion the reaction mixture was filtered over Cel-
ite®, and the filter bed was washed with EtOH (20 mL). The com-
bined organic portions were evaporated and dried to yield 0.50 g
2.9.8. Synthesis of N-(4-(phenylsulfonamido)naphthalen-1-yl)-4-
(trifluoromethyl)benzenesulfonamide (14c)
This previously unreported compound was synthesized ac-
cording to a similar procedure [48]. N-(4-Aminonaphthalen-1-yl)
benzenesulfonamide hydrochloride (0.20 g, 0.59 mmol) was
added to a solution of anhydrous THF (10 mL) and pyridine (1 mL)
(98%
yield)
of
N-(4-aminonaphthalen-1-yl)-4-methoxy
buff-colored solid
benzenesulfonamide hydrochloride as
a
which was utilized for the synthesis of 14a, 14b and 14d without
further purification or characterization due to the relative insta-
bility of the aniline salt.
and
p-trifluoromethylbenzenesulfonyl
chloride
(0.22
g,
2.9.5. Synthesis of N-(4-aminonaphthalen-1-yl)
benzenesulfonamide hydrochloride
This previously unreported compound was synthesized ac-
cording to a known, similar procedure [50]. Pd/C (10%, 0.45 g) was
0.89 mmol), and the reaction was allowed to stir at room tem-
perature for 20 h. On completion, the organic solvent was evap-
orated and the residue was dissolved in CH2Cl2 (40 mL), washed
with H2O (2 ꢂ 30 mL) and HCl (2 N, 2 ꢂ 20 mL), dried (Na2SO4)
and evaporated to yield crude product, which was recrystallized
from EtOH to afford 0.10 g (37% yield) of 14c as a buff-colored
added to
a
suspension of N-(4-nitronaphthalen-1-yl)-4-
benzenesulfonamide (0.40 g, 1.2 mmol) in EtOH (15 mL) and
HCl/EtOH (1.25 M, 5 mL), and the reaction mixture was hydroge-
nated using a Parr shaker apparatus (40 psi) for 5 h. On completion
the reaction was filtered over Celite®, and the filter bed was washed
with EtOH (20 mL). The combined organic portions were evapo-
solid. 1H NMR (400 MHz, DMSO-d6,
d): 10.47 (s, 1H), 10.25 (s,
1H), 7.92e7.80 (m, 6H), 7.63 (d, J ¼ 8.0 Hz, 2H), 7.58e7.54 (m, 2H),
7.47 (d, J ¼ 8.0 Hz, 2H), 7.36e7.32 (m, 2H), 7.05 (s, 2H); 13C NMR
(100 MHz, DMSO-d6,
d
): 144.1, 140.2, 133.2, 132.9 (q, JCF ¼ 32 Hz),
rated and dried to yield 0.40
g
(100% yield) of N-(4-
131.9, 130.8, 130.6, 130.5, 129.6, 128.1, 127.1, 126.9 (d, JCF ¼ 3 Hz),
126.7, 126.7, 124.1, 123.8 (d, JCF ¼ 270 Hz), 123.8, 123.5, 123.4;
HRMS-ESI (þ) (m/z): [MꢁH]ꢁ calcd for C23H16F3N2O4S2, 507.0628;
found, 507.0631.
aminonaphthalen-1-yl)benzenesulfonamide hydrochloride as
a buff-colored solid which was utilized for the synthesis of 14c
without further purification or characterization due to the insta-
bility of the aniline salt.
2.9.9. Synthesis of 3-methoxy-N-(4-((4-methoxyphenyl)
sulfonamido)naphthalen-1-yl)benzenesulfonamide (14d)
This previously unreported compound was synthesized ac-
2.9.6. Synthesis of 4-methoxy-N-(4-(phenylsulfonamido)
naphthalen-1-yl)benzenesulfonamide (14a)
This previously unreported compound was synthesized ac-
cording to
a
known, similar procedure [48]. N-(4-
hy-
cording to
a known, similar procedure [48]. N-(4-Amino
Aminonaphthalen-1-yl)-4-methoxybenzenesulfonamide
naphthalen-1-yl)-4-methoxybenzenesulfonamide hydrochlo-
ride (0.10 g, 0.31 mmol) was added to a solution of anhydrous THF
(10 mL), pyridine (0.040 mL, 0.46 mmol) and benzenesulfonyl
chloride (0.070 g, 0.37 mmol), and the reaction mixture was
allowed to stir at room temperature for 24 h. On completion, the
organic solvent was evaporated, and the residue was dissolved in
CH2Cl2 (40 mL), washed with H2O (2 ꢂ 30 mL) and HCl (2 N,
2 ꢂ 20 mL), dried (Na2SO4) and evaporated to yield crude product,
which was washed with hot acetone and Et2O to afford 0.14 g (89%
drochloride (0.10 g, 0.31 mmol) was added to a solution of pyridine
(2 mL) and 3-methoxybenzenesulfonyl chloride (0.070 g,
0.35 mmol), and the reaction mixture was allowed to stir at room
temperature for 24 h. On completion, the organic solvent was
evaporated, and the residue was dissolved in CH2Cl2 (40 mL),
washed with H2O (2 ꢂ 30 mL) and HCl (2 N, 2 ꢂ 20 mL), dried
(Na2SO4) and evaporated to yield crude product, which was
recrystallized with EtOH to afford 0.10 g (63% yield) of 14d as light
brown-colored needles. 1H NMR (400 MHz, DMSO-d6,
d): 10.19 (s,
yield) of 14a as a purple solid. 1H NMR (400 MHz, DMSO-d6,
d):
1H), 10.05 (s, 1H), 7.98e7.92 (m, 2H), 7.57e7.55 (AA0XX0, J ¼ 8.8 Hz,
2H), 7.41e7.37 (m, 3H), 7.21e7.10 (m, 3H), 7.04 (s, 2H), 6.99e6.97
(AA0XX0, J ¼ 8.8 Hz, 2H), 3.78 (s, 3H), 3.67 (s, 3H); 13C NMR
10.19 (s, 1H), 10.04 (s, 1H), 7.97e7.89 (m, 2H), 7.63e7.34 (m, 9H),
7.02 (s, 2H), 6.98 (d, J ¼ 8.8 Hz, 2H), 3.77 (s, 3H); 13C NMR (100 MHz,
DMSO-d6,
d): 162.4, 139.8, 132.8, 131.4, 130.8, 130.1, 129.0, 126.7,
(100 MHz, DMSO-d6, d): 162.7, 159.6, 141.5, 131.9, 131.7, 131.2, 130.7,
126.2, 123.3, 123.1, 122.7, 114.3, 55.7; HRMS-ESI (ꢁ) (m/z): [MꢁH]
130.5, 130.4, 129.3, 126.6, 123.8, 123.7, 123.4, 123.1, 119.2, 119.2,
114.6, 111.9, 56.1, 55.9.
calcd for C23H19N2O5S2, 467.0741. Found, 467.0755.
2.9.7. Synthesis of 4-methoxy-N-(4-((4-(trifluoromethyl)phenyl)
sulfonamido)naphthalen-1-yl)benzenesulfonamide (14b)
2.9.10. Synthesis of ethyl N-((4-methoxyphenyl)sulfonyl)-N-(4-
nitronaphthalen-1-yl)glycinate (15)
This previously unreported compound was synthesized ac-
This previously unreported compound was synthesized ac-
cording to a similar procedure [34]. In a round-bottomed flask,
cording to
a known, similar procedure [48]. N-(4-Amino

A.D. Jain et al. / European Journal of Medicinal Chemistry 103 (2015) 252e268
259
4-methoxy-N-(4-nitronaphthalen-1-yl)benzenesulfonamide
DMSO-d6, d): 170.0, 162.9, 162.5, 134.5, 133.4, 132.6, 131.6, 129.9,
(1.0 g, 2.8 mmol) and potassium carbonate (1.16 g, 8.4 mmol) was
129.9, 129.4, 129.0, 126.8, 126.6, 126.3, 124.6, 123.3, 121.4, 114.4,
114.3, 59.8, 55.8, 55.7, 53.2, 20.8, 14.2; HRMS-ESI (þ) (m/z): [MþH]þ
calcd for C26H25N2O8S2, 557.1052; found, 557.1055.
dissolved in N-methylpyrrolidinone (4 mL) and stirred for 30 min
ꢀ
at 85 C. Ethyl bromoacetate (1.17 g, 6.98 mmol) was added to the
stirring mixture and stirred for an additional 1 h. Upon reaction
completion, as determined by TLC, the reaction mixture was
poured into stirring ice water (75 mL). The aqueous mixture was
extracted with EtOAc (3 ꢂ 50 mL), and the combined organic
phases were washed with HCl (2 N, 30 mL), H2O (2 ꢂ 50 mL), and
saturated brine (50 mL). The organic fraction was dried over so-
dium sulfate, filtered, and evaporated under vacuum to yield 1.21 g
(98% yield) 15 as a yellow-colored solid. 1H NMR (400 MHz,
2.9.13. Synthesis of 4-methoxy-N-(naphthalen-1-yl)
benzenesulfonamide (17)
This previously unreported compound was synthesized ac-
cording to a known, similar procedure [48]. A solution of 1-
aminonaphthalene hydrochloride (0.50 g, 2.2 mmol) and p-
methoxybenzenesulfonyl chloride (0.55 g, 2.7 mmol) in pyridine
(5 mL) was allowed to stir at room temperature for 19 h. On
completion, the reaction mixture was quenched with H2O (30 mL),
acidified with HCl (2 N, to pH 6) and the precipitate was collected
by filtration. The collected precipitate was washed with H2O
(2 ꢂ 50 mL), dried and the crude product was recrystallized from i-
PrOH to afford 0.50 g (73% yield) of 17 as a buff-colored solid. 1H
DMSO-d6,
d
): 8.39 (d, J ¼ 8.3 Hz, 1H), 8.23 (d, J ¼ 8.2 Hz, 1H), 8.26
(d, J ¼ 8.6 Hz, 1H), 7.83 (t, J ¼ 7.2 Hz, 1H), 7.76 (t, J ¼ 7.3 Hz, 1H),
7.68e7.59 (m, J ¼ 8.9 Hz, 2H), 7.34 (d, J ¼ 8.2 Hz, 1H), 7.15e7.08 (m,
J ¼ 8.9 Hz, 2H), 4.58 (s, 2H), 4.18e4.10 (m, 2H), 3.86 (s, 3H),
1.11e1.03 (m, 3H).
NMR (400 MHz, DMSO-d6,
d
): 10.01 (s, 1H), 8.05 (d, J ¼ 8.3 Hz, 1H),
2.9.11. Synthesis of ethyl N-(4-((4-methoxyphenyl)sulfonamido)
naphthalen-1-yl)-N-((4-methoxyphenyl) sulfonyl)glycinate (16a)
This previously unreported compound was synthesized ac-
cording to a known procedure [48]. In a round bottomed flask,
previously prepared 15 (1.21 g, 2.72 mmol) and tin chloride
dihydrate (4.91 g, 21.8 mmol) were dissolved in EtOH (35 mL). The
solution was stirred at reflux for 3 h. Upon completion, as deter-
mined by TLC, the reaction mixture was concentrated under vac-
uum. The mixture was basified with NaHCO3 to pH 9 and extracted
with EtOAc (200 mL). The organic phase was then washed with
H2O (3 ꢂ 100 mL) and brine (100 mL), dried over sodium sulfate,
and filtered. 2 M HCl in EtOH (10 mL) was then added to the
organic layer, the precipitate was collected and washed with
EtOAc to yield 0.9 g (70% yield) of aniline salt as a fluffy yellow-
7.88 (d, J ¼ 7.3 Hz,1H), 7.77 (d, J ¼ 8.3 Hz,1H), 7.61 (d, J ¼ 8.8 Hz, 2H),
7.50e7.38 (m, 3H), 7.15 (d, J ¼ 7.1 Hz, 1H), 7.01 (d, J ¼ 8.8 Hz, 2H),
3.78 (s, 3H); 13C NMR (100 MHz, DMSO-d6,
d): 162.4, 133.9, 132.6,
129.4, 128.9, 128.0, 126.6, 126.2, 126.1, 125.6, 123.2, 122.9, 114.3,
55.7; HRMS-ESI (ꢁ) (m/z): [MꢁH]ꢁ calcd for C17H14NO3S, 312.0700;
found, 312.0655.
2.9.14. Synthesis of N-((4-methoxyphenyl)sulfonyl)-N-(naphthalen-
1-yl)glycine (18)
This previously unreported compound was synthesized ac-
cording to a published procedure [34]. Potassium carbonate (0.01 g,
0.72 mmol) and ethyl bromoacetate (0.09 g, 0.58 mmol) were
added to a solution of 17 (0.15 g, 0.48 mmol) in anhydrous DMF
(2 mL), and the reaction mixture was stirred at room temperature
overnight. On completion the reaction was quenched with H2O
(50 mL) and extracted with EtOAc (2 ꢂ 20 mL). The combined
organic portion was washed with H2O (5 ꢂ 50 mL), brine (30 mL),
dried (Na2SO4) and evaporated to yield the crude which was sus-
pended in NaOH (2.5 g, 37 mmol), MeOH (12 mL) and H2O (12 mL)
and allowed to reflux for 4 h. On completion the reaction mixture
was cooled, diluted with H2O (20 mL) and extracted with EtOAc
(2 ꢂ 25 mL). The aqueous portion was acidified with HCl (2 N, to pH
2) and extracted with EtOAc (2 ꢂ 50 mL). The combined organic
portion was washed with H2O (3 ꢂ 50 mL) and brine (50 mL), dried
(Na2SO4) and evaporated to yield the crude product which, upon
recrystallization from i-PrOH/Hexanes, yielded 0.13 g (71% yield) of
colored solid, of which 0.5
g
(1.1 mmol), and 4-
methoxybenzenesulfonyl chloride (0.298 mg, 1.44 mmol) were
dissolved in pyridine (3 mL). The solution was stirred at room
temperature for 24 h. Upon completion, determined by TLC, the
reaction mixture was diluted with EtOAc (100 mL) and washed
with HCl (2 N, 2 ꢂ 75 mL), H2O (2 ꢂ 75 mL), and brine (75 mL).
The organic section was dried over sodium sulfate, filtered, and
evaporated to yield 0.59 g (90% yield) of 16a as an off-white solid;
1H NMR (400 MHz, DMSO-d6,
d
): 10.21 (s, 1H), 8.15 (d, J ¼ 8.3 Hz,
1H), 8.09 (d, J ¼ 7.8 Hz, 1H), 7.65 (d, J ¼ 8.8 Hz, 2H), 7.55 (d, J ¼ 8.7,
2H), 7.53e7.46 (m, 2H), 7.10e7.01 (m, 4H), 7.02e6.96 (m, 2H), 4.46
(s, 2H), 3.99 (q, J ¼ 7.5 Hz, 2H), 3.87 (s, 3H), 3.80 (s, 3H), 1.04 (t,
J ¼ 7.1 Hz, 3H); 13C NMR (400 MHz, DMSO-d6,
d): 168.6, 163.0,
18 as a buff-colored solid. 1H NMR (400 MHz, DMSO-d6,
d): 12.80
162.5, 134.4, 133.5, 132.7, 131.6, 130.0, 129.9, 129.2, 129.0, 126.7,
126.6, 126.4, 124.6, 123.3, 121.3, 114.4, 60.9, 59.8, 55.8, 55.7, 53.3,
20.8, 14.1, 13.9; HRMS-ESI (þ) (m/z): [MþH]þ calcd for
(br s, 1H), 8.17e8.15 (m, 1H), 7.96e7.93 (m, 2H), 7.60 (d, J ¼ 9.1 Hz,
2H), 7.55e7.51 (m, 2H), 7.43 (t, J ¼ 7.8 Hz, 1H), 7.18 (d, J ¼ 7.3 Hz,1H),
7.09 (d, J ¼ 9.1 Hz, 2H) 4.49e4.36 (m, 2H), 3.85 (s, 3H); 13C NMR
C
28H29N2O8S2, 585.1365; found, 585.1360.
(100 MHz, DMSO-d6, d): 170.4, 163.2, 136.9, 134.6, 132.2, 130.3,
129.4, 128.4, 128.3, 126.9, 125.8, 124.7, 114.8, 56.2, 53.6; HRMS-ESI
(ꢁ) (m/z): [MꢁH]ꢁ calcd for C19H16NO5S, 370.0755; found,
370.0711.
2.9.12. Synthesis of N-(4-((4-methoxyphenyl)sulfonamido)
naphthalen-1-yl)-N-((4-methoxyphenyl)sulfonyl) glycine (16b)
This previously unreported compound was synthesized ac-
cording to a known similar procedure [34]. In a round-bottomed
flask ethyl 16a (0.04 g, 0.06 mmol) and NaOH (0.24 g, 5.99 mmol)
were dissolved in MeOH (2 mL) and H2O (2 mL). The solution was
stirred at reflux for 3 h. Upon completion, as determined by TLC, the
reaction mixture was acidified with HCl (2 N, to pH 3) and extracted
with EtOAc (25 mL). The organic phase was washed with H2O
(3 ꢂ 30 mL), dried over Na2SO4, filtered, and evaporated to yield
0.03 g (90% yield) of 16b as a white-colored solid; 1H NMR
2.9.15. Synthesis of N,N0-(naphthalene-1,3-diyl)bis(4-
methoxybenzenesulfonamide) (19)
This previously unreported compound was synthesized ac-
cording to a known, similar procedure [51]. A solution of 1,3-
dinitronaphthalene (0.15 g, 0.70 mmol) and tin chloride dihydrate
(2.3 g, 10 mmol) in EtOAc (15 mL) was heated at reflux for 30 min.
On completion the reaction was cooled to room temperature and
quenched in ice, basified with sat. NaHCO3 (to pH 7e8), and
extracted with EtOAc (3 ꢂ 25 mL). The combined organic portion
was washed with H2O (3 ꢂ 30 mL), brine (30 mL), dried (Na2S2O4)
and evaporated to yield crude 0.10 g (100% yield) of naphthalene-
1,3-diamine, which was utilized in the synthesis of 19 without
(400 MHz, DMSO-d6,
d
): 10.21 (s, 1H), 8.13 (d, J ¼ 9.4 Hz, 1H), 8.06
(d, J ¼ 9.6 Hz, 1H), 7.64 (d, J ¼ 8.9 Hz, 2H), 7.52 (d, J ¼ 8.8 Hz, 2H),
7.50e7.45 (m, 2H), 7.04 (dd, J ¼ 9.0, 2.8 Hz, 4H), 6.99 (d, J ¼ 3.1 Hz,
2H), 4.35 (s, 2H), 3.84 (s, 3H), 3.79 (s, 3H); 13C NMR (400 MHz,

260
A.D. Jain et al. / European Journal of Medicinal Chemistry 103 (2015) 252e268
further purification or characterization due to the instability of the
free base [52]. Naphthalene-1,3-diamine (0.10 g, 0.70 mmol) was
dissolved in anhydrous CH2Cl2 (10 mL) and pyridine (0.20 mL,
2.1 mmol) at 0 ꢀC. p-Methoxybenzenesulfonyl chloride (0.40 g,
2.1 mmol) was added and the reaction was allowed to stir at room
temperature for 24 h. On completion, the precipitate was collected
by filtration, washed with CH2Cl2 (20 mL), washed with H2O
(3 ꢂ 40 mL), dried (Na2SO4), and evaporated to yield a brown oil
which was purified by column chromatography (silica gel; hexanes/
EtOAc; 4:1 to 2:1) to afford 0.30 g (96% yield) of 19 as a yellow-
2.9.19. Synthesis of 2-(naphthalene-2,3-diylbis(((4-methoxyphenyl)
sulfonyl)azanediyl))acetamide (23)
This previously unreported compound was synthesized ac-
cording to a known, similar procedure [34]. Potassium carbonate
(0.08 g, 0.6 mmol) and bromoacetamide (0.06 g, 0.4 mmol) were
added to a solution of 20 (0.1 g, 0.20 mmol) in anhydrous DMF
(2 mL), and the reaction mixture was stirred at room temperature
overnight. On completion the reaction was quenched with H2O
(70 mL) and extracted with EtOAc (2 ꢂ 30 mL). The combined
organic portions were washed with H2O (4 ꢂ 100 mL), brine
(50 mL), dried (Na2SO4) and evaporated to yield the crude product
which was purified using preparative TLC (Hexanes:EtOAc 1:2) to
afford 0.06 g (50% yield) of 22 as a buff-colored solid; 1H NMR
colored solid. 1H NMR (400 MHz, DMSO-d6,
d) 10.40 (s, 1H), 10.12
(s,1H), 7.88 (d, J ¼ 8.0 Hz,1H), 7.68e7.56 (m, 5H), 7.39e7.25 (m, 4H),
6.99 (t, J ¼ 8.0 Hz, 4H), 3.76 (s, 3H), 3.74 (s, 3H); 13C NMR (100 MHz,
DMSO-d6,
d) 162.5, 162.4, 135.3, 133.9, 133.7, 131.5, 130.3, 128.9,
(400 MHz, DMSO-d6,
6H), 4.48e4.32 (m, 4H), 3.92e3.82 (m, 6H); 13C NMR (100 MHz,
DMSO-d6, ): 169.1, 163.2, 162.9, 134.9, 133.7, 133.1, 132.0, 130.4,
d): (m, 2H), 7.64e7.41 (m, 6H), 7.13e6.93 (m,
127.4, 126.9, 124.8, 122.8, 115.9, 114.4, 111.3, 113.7, 55.7, 55.6; HRMS-
ESI (ꢁ) (m/z): [MꢁH]ꢁ calcd for C24H21N2O6S2, 497.0887; found,
497.0864.
d
130.3,129.9,129.4,126.9,126.8,126.6,125.1,123.7,121.7,114.7,114.6,
56.2, 56.1, 54.4.
2.9.16. Synthesis of N,N0-(naphthalene-2,3-diyl)bis(4-
methoxybenzenesulfonamide) (20)
(Method A) 2,3-Diaminonaphthalene (0.40 g, 2.5 mmol) was
used as the amine source. Crude product was recrystallized from
EtOAc/MeOH to afford 1.3 g (92% yield) of 20 as yellow flakes. 1H
2.9.20. Synthesis of N,N0-(1,4-Phenylene)bis(4-
methoxybenzenesulfonamide) (24)
This previously unreported compound was synthesized ac-
cording to a known, similar procedure [51]. 1,4-Phenylenediamine
(0.5 g, 4.6 mmol) was added to a solution of anhydrous CH2Cl2
(10 mL) and pyridine (1.2 mL, 13.9 mmol) at 0 ꢀC. p-Methox-
ybenzenesulfonyl chloride (2.4 g, 11.6 mmol) was added, and the
reaction was allowed to stir at room temperature for 24 h. On
completion, the precipitate was collected by filtration, washed with
CH2Cl2 (30 mL), and recrystallized from EtOH to yield 1.6 g (76%) of
NMR (400 MHz, DMSO-d6,
d
): 9.40 (s, 2H), 7.67 (d, J ¼ 8.8 Hz, 6H),
7.50 (s, 2H), 7.37 (dd, J ¼ 6.4, 3.2 Hz, 2H), 7.03 (d, J ¼ 8.8 Hz, 4H), 3.77
(s, 6H); 13C NMR (100 MHz, DMSO-d6,
d): 162.8, 130.3, 129.4, 128.7,
127.1, 126.3, 120.7, 114.5, 55.7; HRMS-ESI (þ) (m/z): [MþH]þ calcd
for C24H23N2O6S2, 499.0992; found, 499.0973.
24 as pink-colored flakes. 1H NMR (400 MHz, DMSO-d6,
2H), 7.58 (t, J ¼ 8.8, 1.6 Hz, 4H), 7.01 (d, J ¼ 8.8 Hz, 4H), 6.90 (s, 4H),
d): 9.93 (s,
2.9.17. Synthesis of 2,20-(naphthalene-1,3-diylbis(((4-
methoxyphenyl)sulfonyl)azanediyl))diacetamide (21)
3.79 (s, 6H); 13C NMR (100 MHz, DMSO-d6,
d): 162.4, 134.1, 131.1,
128.9, 121.5, 114.3, 55.7; HRMS-ESI (ꢁ) (m/z): [MꢁH]ꢁ calcd for
(Method B) Crude product was recrystallized with EtOH/iPrOH
yielded 0.09 g (71% yield) of 21 as buff-colored needles; 1H NMR
C20H20N2O6S2, 447.0696; found, 447.0690.
(400 MHz, DMSO-d6, d): 8.32e8.29 (m, 1H), 7.84e7.81 (m, 1H), 7.78
(s, 1H), 7.56e7.52 (m, 6H), 7.36 (br s, 1H), 7.27 (br s, 1H), 7.17 (br s,
1H), 7.06 (dd, J ¼ 1.6, 8.8 Hz, 4H), 6.97 (d, J ¼ 2 Hz, 2H), 4.27e4.18 (m,
2H), 4.02e3.95 (m 2H), 3.86 (s, 3H), 3.84 (S, 3H); 13C NMR
(100 MHz, DMSO-d6, d): 169.1, 168.8, 163.2, 137.9, 137.3, 133.9, 131.7,
130.5, 130.1, 129.5, 129.4, 128.2, 127.4, 127.3, 126.6, 125.3, 114.9,
114.8, 56.5, 56.1, 54.4, 53.3.
2.9.21. Synthesis of N,N0-(Naphthalene-1,4-diyl)bis(4-
methoxybenzamide) (25)
This previously unreported compound was synthesized ac-
cording to
a known, similar procedure [48]. 1,4-Diamino
naphthalene (0.10 g, 0.63 mmol) was added to a solution of anhy-
drous CHCl3 (10 mL) and pyridine (0.15 mL, 1.9 mmol) at 0 ꢀC. p-
Methoxybenzoyl chloride (0.21 mL, 1.6 mmol) was added in a
dropwise manner and the reaction was allowed to stir at room
temperature for 24 h. On completion the reaction mixture was
diluted with H2O (25 mL), and the precipitate was collected by
filtration, washed with EtOH (10 mL), and recrystallized with EtOH
to yield 0.15 g (56% yield) of 22 as pink-colored solid. 1H NMR
2.9.18. Synthesis of 2,20-(naphthalene-1,3-diylbis(((4-
methoxyphenyl)sulfonyl)azanediyl))acetic acid (22)
This previously unreported compound was synthesized ac-
cording to a known, similar procedure [34]. Potassium carbonate
(0.1 g, 0.7 mmol) and ethyl bromoacetate (0.09 g, 0.5 mmol) were
added to a solution of 19 (0.12 g, 0.2 mmol) in anhydrous DMF
(2 mL), and the reaction mixture was stirred at room temperature
overnight. On completion the reaction was quenched with H2O
(30 mL), and the yellow-colored residue was collected by filtration
to yield 0.14 g (90% yield) of yellow-colored intermediate. Further,
the intermediate (0.08 g, 0.1 mmol) was suspended in NaOH (15%,
3 mL) and MeOH (10 mL) and the reaction mixture was allowed to
reflux overnight. On completion the reaction mixture was cooled
and MeOH was removed by evaporation. The solid was dissolved
with H2O (20 mL) and the aqueous portion was acidified with HCl
(2 N, to pH 2). The solid was collected by filtration which, upon
recrystallization from MeOH, yielded 0.05 g (68% yield) of 22 as a
(400 MHz, DMSO-d6,
d
) 10.31 (s, 2H), 8.11 (d, J ¼ 8.0 Hz, 2H),
8.02e8.01 (m, 2H), 7.60e7.57 (m, 4H), 7.11 (d, J ¼ 8.0 Hz, 4H), 3.87
(s, 6H); 13C NMR (100 MHz, DMSO-d6,
d) 165.7, 162.1, 132.4, 130.0,
129.8, 126.6, 125.9, 123.8, 113.7, 55.5; HRMS-ESI (þ) (m/z): [MþH]þ
calcd for C26H23N2O4, 427.1649; found, 427.1652.
3. Results
3.1. Chemistry
7 and 8 were synthesized as described previously [34]. Briefly,
1,4-diaminonaphthalene was subjected to sulfonylation with p-
methoxybenzenesulfonyl chloride in pyridine to yield 7. This route
was also used to synthesize various substituted analogs (i.e., 11aej;
Scheme 1). 7 was further alkylated with ethyl bromoacetate in the
presence of a base to obtain the di-ethyl carboxylate (i.e., 12a),
which yielded the di-acid analog 8 after saponification.
brown-colored solid; 1H NMR (400 MHz, DMSO-d6,
d): 8.26 (d,
J ¼ 8.8 Hz, 1H), 7.89 (d, J ¼ 6.8 Hz, 1H), 7.80 (s, 1H), 7.69e7.52 (m,
6H), 7.08e7.01 (m, 4H), 6.88 (s, 1H), 3.86 (s, 3H), 4.44e4.09 (m, 4H),
3.83 (s, 3H); 13C NMR (100 MHz, DMSO-d6,
d): 170.1, 169.9, 163.3,
163.2, 137.9, 137.1, 134.1, 131.5, 130.4, 130.1, 129.9, 129.4, 128.4, 127.6,
127.5, 127.3, 126.6, 124.9, 114.8, 114.8, 56.1, 53.5, 52.2.
As reported previously by Jiang et al. [34], the 1H NMR spectrum

A.D. Jain et al. / European Journal of Medicinal Chemistry 103 (2015) 252e268
261
of 8 showed a doubling of signals; for instance, there were two
3.2. In vitro assays
singlets of 3H each at 3.83 and 3.88 ppm, corresponding to the
methyl ethers, and a multiplet of 4H centered around 4.39 ppm,
To test the potency of the analogs in vitro, a fluorescence
anisotropy assay was carried out according to a previously reported
procedure [43]. Briefly, inhibition of the Keap1-Nrf2 interaction
with small molecules was assayed using both the Kelch domain of
Keap1 and an N-terminally fluorescein-labeled 9-mer peptide
containing the ETGE motif derived from the Neh2 domain of Nrf2
[43]. The experiments were performed in triplicate. Sigmoidal
concentrationeresponse curves were fitted to the data using
Graphpad Prism 6.1 software (see Supplementary information and
Tables 1 and 2).
A subset of the compounds with demonstrated ability to bind to
the Kelch domain via the FP assays was tested in a surface plasmon
resonance (SPR) assay. Briefly, the Kelch domain of Keap1 was
immobilized on a CM5 sensor chip. Compound solutions of
increasing concentrations were applied to the protein-bound chip,
and response units were measured (see Supplementary
information and Table 1). The experiments were conducted three
separate times. The Nrf2 peptide (FITC-LDEETGEFL-NH2) alone was
tested by SPR as a control. The determined Kd value was 68.9 nM,
which is slightly weaker than the reported Kd (25 nM). Approxi-
mately 60% of captured Keap1 was active based on the comparison
corresponding to the methylene groups
a to the ethyl esters. This
effect was not seen with the unsubstituted sulfonamides (e.g., 7)
and was surprising, given the elements of symmetry suggested by
the planar structure. The same effect was observed in the 1D/2D
spectra of other di-substituted sulfonamides collected at room
temperature. In DMSO, 8 and congeners may adopt one or more
solution conformations where the flanking p-methox-
ybenzenesulfonamide substituents are oriented in opposite di-
rections, based on 2D ROESY data collected at room temperature.
We believe this phenomenon is due to restricted rotation about the
NsulfonamideeCnaphthalene bond that arises with increasing steric bulk
on the substituted sulfonamide nitrogen. The coalescence tem-
perature was found to be 313 K, which translates into a low acti-
vation barrier for this motion that can be easily overcome upon
binding. When NMR studies were performed at temperatures
higher than 313 K (e.g., 353 K) each set of peaks appeared as one
signal (see supplementary information). On the other hand, crystals
of this molecule obtained from slow evaporation of methanol
showed the “expected” form with a mirror plane of symmetry (see
supplementary information). The methodology used to synthesize
7 and 8 was also used to prepare various di-substituted analogs
using the appropriate electrophiles (i.e., 12bef). 1,4-
diaminobenzene was used to synthesize 24. Compound 25 was
synthesized from p-anisoyl chloride and 1,4-diaminonaphthalene.
Analogs bearing unsymmetrical substitutions were synthesized as
shown in Scheme 2. 1-Nitronaphthalene was aminated in the
between calculated RUmax and achieved RUmax
:
Analyte binding capacityðRUmax
Þ
analyte MW
¼
ꢂ immobilization levelðRUÞ
ligand MW
presence of hydroxylamine and
a base to yield 4-amino-1-
nitronaphthalene [49], which upon sulfonylation yielded 13.
Further reduction of the nitro group afforded a mono-sulfonylated
amine, which was further sulfonylated using various benzene-
sulfonyl chlorides to yield unsymmetrical sulfonamides (i.e.,
14aed). In an alternate route, sulfonamide 13 was alkylated before
reduction and sulfonylation to give mono-alkylated analogs 16aeb.
1-Aminonaphthalene was sulfonylated to yield 17, which upon
alkylation and saponification yielded 18. Similar to the synthesis of
7, 1,3- and 2,3-diaminonaphthalene analogs were used as starting
materials to synthesize 19 and 20. Further alkylation of 19 and 20
with 2-bromoacetamide yielded 21 and 23, respectively. Similar to
the synthesis of 8, alkylation of 19 with ethyl bromoacetate, fol-
lowed by saponification, yielded 22.
3.3. Docking and hydration site analysis
Inspection of the previously determined X-ray structure of 7
bound to the Kelch domain of Keap1 (4IQK) reveals an interesting
binding mode wherein the ligand is flanked by two co-crystallized
water molecules (water 1 and water 2, shown in red spheres (in the
web version) in Fig. 2A) that are separated by Arg 415. We hy-
pothesized that substitutions at the R2 and R3 positions in Region C
(Fig. 2B; see Discussion section) may potentially utilize the pockets
that these waters occupy, displacing the water molecules in the
process. To investigate this hypothesis and further elucidate
structureeactivity relationships and the role of these water mole-
cules in ligand binding, ensemble docking and hydration site
Fig. 2. A) Co-crystal structure of 7 with Keap1 Kelch domain (4IQK). B) Schematic of the regions of modification on 7.

262
A.D. Jain et al. / European Journal of Medicinal Chemistry 103 (2015) 252e268
Fig. 3. Docking and WATsite results for compounds (A) 8, (B) 12e, (C) 16b, (D) 16a, (E) 12a and (F) 7. For each molecule, the Glide docking score is shown, and the measured IC50 is
noted in parentheses. The template selected by the ensemble docking procedure is shown below the docking score for each compound. The locations of the waters (shown as red
spheres in Fig. 2A) in the crystal structure 4IQK are shown as gray spheres in each frame. High occupancy (>80%) hydration sites predicted by WATsite that were within 2.0 Å of
crystal waters are shown as red spheres. The DG of the hydration sites are shown in kcal/mol next to each site; a negative value indicates a favorable hydration site, and a positive
value indicates an unfavorable site. The crystal structures of compound 12e (PDB: 4XMB; Panel B) and 7 (PDB: 4IQK; panel F) are shown in smaller orange sticks (For interpretation
of the references to color in this figure legend, the reader is referred to the web version of this article.).
analyses were performed on a subset of six ligands (7, 8, 12a, 12e,
16a, 16b) with substitutions at the R2 and/or R3 positions. The top-
ranked docking pose for each molecule along with the results of the
WATsite analysis are shown in Fig. 3.
(Rfree ¼ 22.8%, Rwork ¼ 17.3%). Compound 12e was chosen due to its
nanomolar potency and computational prediction that one water
(water 1) would be displaced (shown in Fig. 3B). The X-ray structure
of 12e and its associated electron density in the Kelch binding site
are shown in Fig. 4.
As predicted, the X-ray structure confirms that water 1 was
displaced by one of the acetamide functional groups and that water
2 was shifted forward in comparison to its location in the co-crystal
structure of 7 (shown in gray spheres in Fig. 3B). To accommodate
the di-acetamide functional groups, the guanidinium side chain
portion of Arg 380 flipped approximately 180ꢀ to pack against one
of the di-acetamides (Fig. 4). The result of Arg 380 adopting this
position is a significant structural shift of Asn 381 to Thr 388 into a
position that alters crystal packing and hence crystallization of the
Kelch-12e complex in the P21 space group.
3.4. X-ray structure of the Kelch-compound 12e complex
To help support the computational docking analysis, we co-
crystallized Keap1 Kelch domain as a complex with compound
12e and determined the X-ray structure of the complex to 2.47 Å
3.5. Immunoblot analysis
Six non-electrophilic compounds that were either active in the
primary fluorescence anisotropy assay or that were esters of active
compounds were tested for their ability to regulate Nrf2 and its
target genes in MLE12 (mouse lung alveolar epithelial cell line) cells
[42,43]. Western blots were used to demonstrate the ability of the
compounds to transactivate Nrf2 target genes heme oxygenase 1
(HMOX1) and NAD(P)H quinone oxidoreductase 1 (NQO1) (Fig. 5).
Nrf2 protein levels were also measured to demonstrate stabiliza-
tion by the activators, and compared to the electrophilic positive
control sulforaphane 1. The experiments were carried out three
separate times.
Fig. 4. X-ray structure of the Kelch domain from human Keap1 in complex with
compound 12e (PDB ID: 4XMB). The structure is shown as a ball-and-stick represen-
tation with atoms colored as follows: oxygen (red), sulfur (green), nitrogen (bright
blue) and hydrogen (light blue). Carbon atoms for the 12e and residues in loop regions
colored white. Carbon atoms of residues in beta sheets are colored steel blue. A final
simulated annealing Fo-Fc electron density omit map, with 12e removed from the map
4. Discussion
calculations, is shown as a gray mesh contoured at 2.7
s. The figure was generated
with CueMOl2 (For interpretation of the references to color in this figure legend, the
reader is referred to the web version of this article.).
In 2013, Marcotte et al. reported one of the first non-

A.D. Jain et al. / European Journal of Medicinal Chemistry 103 (2015) 252e268
263
around 7, supplemented with a confirmatory SPR assay. We have
focused our studies around three regions of the molecule: A) the
methoxyphenyl groups, B) the naphthalene core, and C) the sul-
fonamide linkers (see Fig. 2B).
In our exploration of Region A, we found that removal of both
terminal methoxy groups (i.e., 11a) resulted in an inactive com-
pound. We replaced the methoxy groups with an electron-donating
eCH3 or eN(CH3)2 group (i.e., 11h and 11i) or an electron-
withdrawing substituent (e.g., 11c, 11e). The replacement of the
p-OCH3 substituents with p-CH3 retained activity with an
IC50 ¼ 890 nM, while the activity increased by almost 5-fold in the
presence of a strongly electron donating eN(CH3)2 (IC50 ¼ 190 nM).
An increase in potency by a stronger electron-donating group may
be attributed to stronger cation-p interactions between the central
naphthalene ring and Arg 415. We are investigating this possibility
further and will report in due course. It appears as though large
electron-withdrawing groups are not well tolerated at this position,
as the IC50 values of 11c and 11e are beyond the highest concen-
tration assayed (i.e., 25 mM), although the p-F substituent retained
activity (i.e., 11j; IC50 ¼ 660 nM), which could be used towards
synthesis of metabolically stable analogs of 7.
The crystal structure of 7 bound to the Kelch domain of Keap1
showed that the para-methoxy groups were both solvent-exposed
[33]. It appeared as though substitution at the meta-position of the
phenyl rings might interact with the surface of the protein.
Replacing both p-OCH3 substituents with m-OCH3 groups resulted
in a similar potency analog (i.e., 11b: IC50 ¼ 630 nM). When only
one of the methoxy groups was moved to a meta position (i.e., 14d),
there was a slight improvement in potency (IC50 ¼ 410 nM).
Combining both meta- and para-substitutions into one compound
(i.e., 11g) did little to increase the affinity, and created a rather
insoluble compound. Replacement of the m-methoxy groups with
electron-withdrawing groups (-CF3 in 11f and eCOOH in 11d) gave
inactive compounds, in agreement with substitution at the p-po-
sition. We further desymmetrized the molecule by replacing one or
both of the methoxy groups with a eCF3 or eH substituent. The
only compound that retained activity within this series (i.e., 14aec)
was the one that replaced one methoxy with a hydrogen (14a), with
an IC50 value of 2.5 mM. Although there was a loss of activity by 2.5-
fold, it did reduce the molecular weight to improve ligand effi-
ciency. Binding efficiency of a ligand, which is defined as binding
energy per non-hydrogen atom of a molecule, provides a useful
consideration for hit-to-lead optimization in a drug discovery
program. Ligand efficiency can provide a meaningful comparison
between multiple scaffolds and series of analogs [53].
To explore the naphthalene core region B of 7 (Fig. 2B), we
changed the substitution pattern on the naphthalene from 1,4 to
1,3 (19, 21 and 22) and 2,3 (20, 23). The loss of activity in each
analog demonstrates the requirement of the 1,4-substitution
pattern. The necessity of the fused system was apparent with the
lack of activity seen when the naphthalene core was replaced with
a phenyl ring (24). Additionally, we removed an entire benzene-
sulfonamide to give the 1-substituted analog (17, 18), which was
inactive, demonstrating the need for the core and both of its
flanking substituents. We are currently undertaking a more
comprehensive replacement of the naphthalene scaffold with
other heterocycles.
Fig. 5. The effects of non-electrophilic activators on the expression levels of Nrf2 and
its targets in lung alveolar epithelial cells. (A) MLE 12 cells were treated with DMSO
(vehicle) or indicated compound (10
amount of protein was immunoblotted using antibodies specific for Nrf2, NQO1 or
HMOX1. -actin was used as loading control. (B) Quantification of target gene band
intensity using -actin as reference. DMSO-treated sample value was used as one
mM) for 6 h, cell lysates were prepared, equal
b
b
arbitrary unit. Error bars represent the standard deviation from three independent
experiments.
electrophilic Nrf2 activators, 7, with an IC50 of 2.7 mM. In 2014, Jiang
et al. reported 8, based on modification of 7, as one of the first sub-
nanomolar non-electrophilic Nrf2 activators, with an IC50 in a
fluorescence anisotropy assay of 29 nM and a Kd of 9.9 nM in a
biolayer interferometry assay [34]. We observe similar IC50 values
for both compounds in our assays: Fluorescence anisotropy (7:
IC50 ¼ 1.0
m
M; 8: IC50 ¼ 24 nM) and SPR (7: Kd ¼ 1.7
mM; 8:
Kd ¼ 20 nM; see Table 1). The crystal structure (PDB ID: 4IQK) of 7
bound to the Kelch domain of Keap1 (Fig. 2A) was reported previ-
ously by Marcotte et al. [33]. It highlighted key molecular in-
We also explored the SAR around the sulfonamide region C
(Fig. 2B), which connects the naphthalene core to the flanking aryl
rings. The sulfonamide was replaced with an amide (i.e., 25), which
was inactive. The SeN bond in a sulfonamide usually exhibits lower
sp2 character than the corresponding CeN bond in a carboxamide
[54], so that the topology of the two compounds is likely quite
different.
teractions of 7 bound to Kelch, including a
p-cation interaction of
the naphthalene core with the guanidinium group of Arg 415,
pep
stacking interactions of the flanking phenyl rings of 7 with Tyr 334
and Phe 577, and a hydrogen bond between Ser 508 and one of the
sulfonamide oxygen atoms. In the present study, we have used a
fluorescence anisotropy assay as a primary assay to explore the SAR
In the native interaction of Nrf2 and Keap1, a type I
b-turn

264
A.D. Jain et al. / European Journal of Medicinal Chemistry 103 (2015) 252e268
Reagents: (a) pyridine, rt; (b) R’Br, K2CO3, DMF, rt; (c) NaOH, MeOH, H2O, reflux.
Scheme 1. Synthesis of symmetrically substituted N,N0-dialkylated sulfonamidesa.
comprising an ETGE motif binds to the Kelch domain of Keap1. In
crystal structures of a Nrf2 peptide bound to the Kelch domain [55],
Glu 79 and Glu 82 of the ETGE motif form ionic interactions with
Arg 415 and 483 of Keap1 [55]. In an illuminating study, Jiang, et al.
[34] showed that incorporating two acetic acid moieties into the
structure of 7 (i.e., 8) improved the affinity by almost 100-fold. This
effect was attributed to ionic interactions with Arg 415 and Arg 483
and H-bond interactions with Ser 508 residues. We observed a
similar result in our assay upon synthesizing this compound
(fluorescence anisotropy IC50 ¼ 24 nM vs. literature value of 29 nM;
SPR Kd ¼ 20 nM vs. literature biolayer interferometry Kd ¼ 9.9 nM)
[34]. We also obtained further evidence for the importance of the
polar nature of the interaction, as we saw that a set of di-alkyl
substituted analogs (i.e., 12aed and 12f) were inactive; however,
it does not seem that the polar interactions need to be ionic, as the
di-acetamide 12e was also quite active (IC50 ¼ 63 nM). Our X-ray
structure of 12e in complex with Kelch shows clearly that one of the
di-acetamides makes two strong hydrogen bonds with the amide
Scheme 2. Synthesis of unsymmetrical and mono-alkylated p-substituted sulfonamidesa.

A.D. Jain et al. / European Journal of Medicinal Chemistry 103 (2015) 252e268
265
Table 1
Inhibition of association of Nrf2 peptide with Keap1 (Kelch domain) using fluorescence anisotropy assay and dissociation equilibrium constant (Kd) by surface plasmon
resonance (SPR) assay.
Cmpd.
R
R1
R2
R3
IC50 (nM) (95% CL)b
Kd (nM) SDc
7
8
p-OCH3
p-OCH3
H
p-OCH3
p-OCH3
H
H
H
940 (850e1300)
24 (19e31)
>25,000
1700 120
CH2COOH
H
CH2COOH
H
20
3
11a
11b
11c
11d
11e
11f
11g
11h
11i
11j
12a
12b
12c
12d
12e
12f
14a
14b
14c
14d
16a
16b
NDa
m-OCH3
p-Cl
m-COOH
p-CF3
m-CF3
m,p-di OCH3
p-CH3
p-N(CH3)2
p-F
p-OCH3
p-OCH3
p-OCH3
p-OCH3
p-OCH3
p-OCH3
H
p-OCH3
H
p-OCH3
p-OCH3
p-OCH3
m-OCH3
p-Cl
m-COOH
p-CF3
m-CF3
m,p-di OCH3
p-CH3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
630 (400e1000)
>25,000
>25,000
>25,000
>25,000
1100 (800e1500)
890 (480e1650)
190 (84e444)
660 (172e2565)
>25,000
>25,000
>25,000
>25,000
63 (52e85)
>25,000
>25,000
>25,000
>25,000
2300 110
NDa
NDa
NDa
NDa
NDa
NDa
p-N(CH3)2
p-F
NDa
H
H
NDa
p-OCH3
p-OCH3
p-OCH3
p-OCH3
p-OCH3
p-OCH3
p-OCH3
p-CF3
CH2COOEt
CH2CH]CH2
CH3
Et
CH2CONH2
CH2C^CH
H
H
H
H
H
H
CH2COOEt
CH2CH]CH2
CH3
Et
CH2CONH2
CH2C^CH
H
H
>250,000
NDa
NDa
NDa
44
8
NDa
NDa
NDa
NDa
NDa
p-CF3
H
H
m-OCH3
p-OCH3
p-OCH3
410 (150e1100)
85 (45e160)
61 (46e82)
CH2COOEt
CH2COOH
400 84
110 11
a
ND: Not determined.
b
c
95% confidence level, as calculated by Graphpad Prism 6.1; fluorescence anisotropy experiments were performed in triplicate.
Standard deviation from three independent experiments.
Table 2
Inhibition of association of Nrf2 peptide with Keap1 (Kelch domain) using fluorescence anisotropy assay.3
Cmpd.
Substitution pattern
R1
R2
X
IC50 (nM) (95% CL)a
17
18
19
20
21
22
23
24
25
1-
1-
eH
eCH2COOH
eH
eb
eSO2e
eSO2e
eSO2e
eSO2e
-SO2-
eSO2e
eSO2e
eCOe
eSO2e
>25,000
>25,000
>25,000
>25,000
>25,000
5500 (1400e22,000)
>25,000
>25,000
>25,000
eb
1,3-
2,3-
1,3-
1,3-
2,3-
1,4-
1,4-
eH
eH
-CH2CONH2
eCH2COOH
eCH2CONH2
eH
eH
eCH2CONH2
eCH2COOH
eH
eH
eH
eH
a
95% confidence level, as calculated by Graphpad Prism 6.1; fluorescence anisotropy experiments were performed in triplicate.
Compounds 17 and 18 lack the second arylsulfonamide substituent.
b

266
A.D. Jain et al. / European Journal of Medicinal Chemistry 103 (2015) 252e268
side chain of Asn 414 and the other makes a hydrogen bond with
the backbone carbonyl of Ile 461. Similarly, attempts were made to
prepare analogs of the 1,3- and 2,3-disubstituted naphthalenes
containing acids or amides. The 1,3-substituted di-acid (i.e., 22)
resulted in a gain of activity (IC50 ¼ 5500 nM) compared to 19,
although it is still almost 180-fold less potent than 8. We also
attempted to prepare the disubstituted analog of 20, but repeated
attempts at synthesizing this compound were unsuccessful and
yielded either an unknown product upon basic saponification of the
ethyl ester or degradation of the ethyl ester under acid-catalyzed
hydrolysis. Our standard alkylation conditions, when applied to
compound 20 using 2-bromoacetamide, gave a mono-acetamide
analog 23 instead of the di-acetamide; the reasons for this are
not clear.
amide substituent allows for hydrogen bonding to nearby Arg 483,
Ser 508, Asn 414, and Ser 363. In the case of compounds 8 and 16b,
there may also be chargeecharge interactions of the carboxylates
with Arg 483 and Arg 415, as has been previously modeled [34]. It is
likely that these additional hydrogen bonds contribute to the
increased IC50 values in comparison to the parent compound 7.
These additional hydrogen bonds are made possible by substituents
that protrude into the nearby water pockets resulting in the
removal of one or both water molecules.
Modifications in Region C have the potential to protrude into
two pockets near the bottom of the active site that are occupied by
water molecules in the 7:Kelch domain crystal structure (Fig. 2A).
These water molecules are also present in the apo structure (1ZGK),
but have been shifted by about 2.0 Å, and in a related ligand-bound
structure (PDB ID: 4IN4), only water 1 is present. The different
water patterns in the crystal structures suggest that the positions of
these water molecules vary depending on the type of ligand bound
and may contribute to ligand binding.
We were curious whether both acetic acid groups of 8 were
necessary for binding to the Kelch domain, so we prepared the
singly substituted analogs 16a and 16b. We were gratified to find
that the affinity of 16b (i.e., IC50 ¼ 61 nM in the fluorescence
anisotropy assay and Kd ¼ 110 nM in SPR assay) is almost equivalent
to that of the di-acid 8. The singly substituted acetic acid of 16b
improves the ligand efficiency of the molecule and decreases the
total charge density on the molecule, which may be important for
more advanced assays (see immunoblot studies below). Intrigu-
ingly, the mono-ethyl ester 16a is much more active (i.e.,
IC50 ¼ 85 nM and Kd ¼ 400 nM) than the di-ester 12a (i.e., IC50 and
WATSite was used to evaluate the thermodynamic profile (DG)
of binding site water molecules with the different compounds
bound (Fig. 3). In all cases, excluding 7, the WATsite analysis agreed
with the ensemble docking for water selection (e.g. in 8 the docking
routine selected a template where both water 1 and water 2 were
removed, and the WATsite analysis also showed no waters in the
nearby vicinity of water 1 or water 2). Interestingly, removal of
these waters does not appear to have a high energetic penalty, and
in the case of water 2 even seems to be slightly favorable. The low
energetic cost of removing these waters coupled with the addi-
tional interactions that the compounds gain by occupying these
pockets suggests that displacing at least one of these water mole-
cules with substituents that protrude into these pockets and
interact with nearby residues is more favorable than not doing so
(e.g., 16b vs. 7).
Kd > 25 mM). This marked change in affinity may hint at an upper
limit for the size of one of these substituents (see docking studies
below). Based on the above results, we synthesized 1-substituted
naphthalene 18, the acetic acid analog of 17, to see whether the
acid functionality might be able to rescue the lack of a second aryl
ring. Similarly, we prepared the di-acetamides of the 1,3- and 2,3-
disubstituted naphthalenes (i.e., 21 and 23). All of these com-
pounds were inactive, further confirming the importance of the
1,4-disubstitution pattern of the scaffold
inhibition.
7
for Keap1-Nrf2
We compared the ability of 7, 8, 11g, 12a, 16a and 16b to induce
the expression of Nrf2 target genes HMOX1 and NQO1 in mouse
lung type-II alveolar epithelial cells ex vivo (see Fig. 5), and we
compared these values to that of the well-studied electrophilic
activator sulforaphane 1.
Docking studies were performed to further investigate the in-
fluence of substitutions at the R2 and R3 positions in Region C for
compounds 8, 12a, 12e, 16aeb and 7. In general, the docking results
were found to be highly consistent with the measured IC50 values.
The top-3 predicted compounds by docking were indeed those
with the highest affinity (compound 8, 12e and 16b); furthermore,
these compounds scored significantly better than those com-
pounds which performed poorly (7) or not at all (12a), demon-
strating a >3 unit difference in docking scores in the best case (8)
(see Fig. 3).
Although the differences between 7, 8, and 16a are not statis-
tically significant, the data may imply that the di-acetic acid func-
tionality of 8 is not essential for induction of HMOX1 and NQO1 and
stabilization of Nrf2. Previously, Jiang et al. [34] showed activation
of NQO1, GCLM, and HMOX1 in HCT116 cells treated with 8 for
16e32 h. In the present study, we examined the induction of Nrf2
target gene expression 6 h post-treatment with the activators,
mainly in order to avoid indirect effects, such as metabolic activa-
tion of the small molecules to electrophilic species or interference
at other pathways. We saw that the mono-carboxylic acid 16b was a
rather poor inducer of HMOX1 and NQO1 after six hours; however,
16a, the ethyl ester of the mono-carboxylic acid, potently induced
HMOX1 and stabilized Nrf2 levels after six hours in a manner
similar to that seen with 7 and 8 (see Fig. 5B). This is interesting
because the acid 16b is more potent than the ester 16a in vitro. We
believe 16a may facilitate membrane traversal, and, once in the cell,
may be hydrolyzed by esterases to give the mono-carboxylic acid
16b. This may further imply that the carboxylic acids may be
replaced to provide further leads to achieve induction of HMO1,
NQO1 and Nrf2.
Nearly all of the compounds, with the exception of 12a, were
predicted to bind in a mode similar to that of the parent compound
7 in the crystal structure 4IQK (Fig. 2A). Analysis of the crystal
structure suggests that binding of these compounds may rely
heavily on
pep stacking and p-cation interactions. With the
exception of the low-affinity inhibitor 12a, at least two of these
three pep/p-cation interactions were maintained in all compound
poses. In 12a, the bulky ethyl carboxylate substituents at the R2 and
R3 positions enforce an alternative binding conformation wherein
the phenyl rings of Region A and the ethyl carboxylate groups of
Region C is inverted in comparison to the crystallized ligand in
4IQK, resulting in loss of
pep stacking interactions between the
phenyl rings of Region A and residues Phe 577 and Tyr 525 (Fig. 3E).
The steric bulk of this compound, along with the loss of these in-
teractions may, in part, account for the poor IC50 value observed for
12a.
5. Conclusions
While
p
e
p
stacking appears to be one of the main types of in-
In conclusion, we have conducted an SAR study focusing on the
structures of naphthalenes 7 and 8 in three regions (see Fig. 2B).
Region A shows optimum potency when substituted in the meta or
para position with an electron-donating group. Region B is optimal
teractions influencing the binding of 7, the best performing com-
pounds also utilize extensive hydrogen bonding to the groups
substituted in the R2 and R3 positions. Adding a carboxylic acid or

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tion of the core may be necessary and will be the focus of future
studies. Region C provided the most interesting results, revealing
new compounds that show promise as non-electrophilic activators
of Nrf2: di-acetamides (i.e.,12e) and compounds containing a single
acetic acid group on one of the N-substituents (i.e., 16b), a com-
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substituents in Region C may contribute to their high affinity, and
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stood out as a compound with high potency in vitro, with an IC50 of
61 nM in a fluorescence anisotropy assay, but the potency was lost
ex vivo as we looked at the induction of Nrf2 target genes; however,
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was capable of inducing the expression of native Nrf2 target genes
at levels similar to that of the well-known electrophilic activator
sulforaphane 1. This data obtained with ester 16a may suggest that
neutrally charged groups may be used to replace negatively
charged carboxylates that have previously been shown to give high
affinity compounds.
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The authors thank the University of Illinois at Chicago College of
Pharmacy and the University of Illinois Cancer Center (toTWM), the
National Institutes of Health (grants HL66109 and ES11863 to SPR)
and the Walther Cancer Foundation (to ADM) for their support of
this work. We thank Dr. Bernard Santarsiero for solving the crystal
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Research which is supported by P30 CA023168.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2015.08.049.
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