Discovery and SAR studies of 3-amino-4-(phenylsulfonyl)tetrahydrothiophene 1,1-dioxides as non-electrophilic antioxidant response element (ARE) activators

Article abstract of DOI:10.1016/j.bioorg.2020.104614

The transcription factor NRF2 controls resistance to oxidative insult and is thus a key therapeutic target for treating a number of disease states associated with oxidative stress and aging. We previously reported CBR-470-1, a bis-sulfone which activates NRF2 by increasing the levels of methylglyoxal, a metabolite that covalently modifies NRF2 repressor KEAP1. Here, we report the design, synthesis, and structure activity relationship of a series of bis-sulfones derived from this unexplored chemical template. We identify analogs with sub-micromolar potencies, 7f and 7g, as well as establish that efficacious NRF2 activation can be achieved by non-toxic analogs 7c, 7e, and 9, a key limitation with CBR-470-1. Further efforts to identify non-covalent NRF2 activators of this kind will likely provide new insight into revealing the role of central metabolism in cellular signaling.

Full text of DOI:10.1016/j.bioorg.2020.104614

Bioorganic Chemistry 108 (2021) 104614
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Short communication
Discovery and SAR studies of 3-amino-4-(phenylsulfonyl)tetrahydrothio-
phene 1,1-dioxides as non-electrophilic antioxidant response element
(ARE) activators
,
,
Jeyun Joa †, Lara Ibrahimb †, Jonathan Iaconellib, Jinsook Kwaka, Manoj Kumarc,
Yunjin Junga, Luke L. Lairsonb, Arnab K. Chatterjeec, Peter G. Schultzb c, Michael J. Bollongb
,
,
,*
Hwayoung Yuna
,b,*
a College of Pharmacy, Pusan National University, Busan 46241, Republic of Korea
b Department of Chemistry, The Scripps Research Institute, 10550, North Torrey Pines, La Jolla, CA 92037, United States
c California Institute for Biomedical Research, 11119 North Torrey Pines Road, Suite 100, La Jolla, CA 92037, United States
A R T I C L E I N F O
A B S T R A C T
Keywords:
The transcription factor NRF2 controls resistance to oxidative insult and is thus a key therapeutic target for
treating a number of disease states associated with oxidative stress and aging. We previously reported CBR-470-
1, a bis-sulfone which activates NRF2 by increasing the levels of methylglyoxal, a metabolite that covalently
modifies NRF2 repressor KEAP1. Here, we report the design, synthesis, and structure activity relationship of a
series of bis-sulfones derived from this unexplored chemical template. We identify analogs with sub-micromolar
potencies, 7f and 7g, as well as establish that efficacious NRF2 activation can be achieved by non-toxic analogs
7c, 7e, and 9, a key limitation with CBR-470-1. Further efforts to identify non-covalent NRF2 activators of this
kind will likely provide new insight into revealing the role of central metabolism in cellular signaling.
ARE activator
NRF2
PGK1 inhibitor
Structure-activity relationship
Reactive metabolites
1. Introduction
regulated gene expression have been identified to date [6], including
phenolic and flavonoid antioxidants [7], isothiocyanates [8], Michael
Persistent oxidative stress and exposure to electrophilic xenobiotics
promote aging and age-related diseases such as cancer, chronic
inflammation, cardiovascular disease, and neurodegeneration [1,2].
Consequently, the mammalian cell has evolved an inducible transcrip-
tional program orchestrated by the transcription factor nuclear factor
erythroid 2-related factor 2 (NRF2) to protect against oxidative insult
[3]. In unstressed conditions, NRF2 is continually sent for proteasomal
degradation by its cytoplasmic repressor and Cullin 3 adaptor protein,
Kelch-like ECH-associated protein 1 (KEAP1) [4]. In response to
oxidative injury or electrophilic challenge, cysteine ‘sensor’ residues in
KEAP1 are oxidized or alkylated, a modification which promotes accu-
mulation of NRF2 in the nucleus [4]. Licensed activation of NRF2 pro-
motes upregulation of cytoprotective gene products containing
conserved antioxidant response element (ARE) sites within their
genomic loci [5].
acceptors [9], and organosulfur compounds [10]. As such, the majority
of these molecules are electrophiles or are transformed in cells to form
electrophiles, which promote ARE activation by covalent modification
of KEAP1 [6]. Although electrophilic compounds such as dimethyl
fumarate [11], bardoxolone methyl [12], and sulforaphane [8], have
demonstrated clinical benefit or are FDA approved medications [13,14],
electrophilic small molecules are typically not considered useful drug
candidates because they broadly react with cellular nucleophiles
causing cytotoxicity. Indeed, late stage clinical candidate bardoxolone
methyl interacts with hundreds of cellular targets and displays cyto-
toxicity [15,16], despite impressive clinical efficacy in chronic kidney
disease trials. These observations have prompted the development of
non-covalent binders of the Kelch domain of KEAP1, molecules which
inhibit KEAP1-mediated degradation of NRF2 [17]. While molecules
like KI696 [18,19] and other KEAP1-NRF2 interaction inhibitors have
shown protective activity in preclinical animal models, the comparative
A wide variety of small molecules capable of promoting ARE-
* Corresponding authors.
E-mail addresses: mbollong@scripps.edu (M.J. Bollong), hyun@pusan.ac.kr (H. Yun).
†
These authors contributed equally to this work.
https://doi.org/10.1016/j.bioorg.2020.104614
Received 11 September 2020; Received in revised form 13 November 2020; Accepted 28 December 2020
Available online 5 January 2021
0045-2068/© 2021 Elsevier Inc. All rights reserved.

J. Jo et al.
Bioorganic Chemistry 108 (2021) 104614
Fig. 1. Optimization strategy used in this work to identify novel 3-amino-4-(phenylsulfonyl)tetrahydrothiophene 1,1-dioxide derivatives.
efficacy of these series relative to covalent NRF2 activators has not been
evaluated and the clinical potential of this compound class has not yet
been realized.
approach in which chemical libraries might be broadly interrogated for
non-electrophilic small molecule activators of ARE transcription. In
principle, such an approach might identify compounds that favorably
modify metabolism to augment levels of KEAP1-reactive metabolites.
Recently, we reported the discovery of CBR-470-1 (1, Fig. 1), a molecule
derived from an unexplored 3-amino-4-(phenylsulfonyl) tetrahy-
drothiophene 1,1-dioxide skeleton, which activates NRF2 by inhibiting
the glycolytic enzyme phosphoglycerate kinase 1 (PGK1) [24]. Phar-
macological PGK1 inhibition promotes buildup of the triose phosphate
degradation product methylglyoxal (MGO), a dicarbonyl we found
intermolecularly inactivates KEAP1 by crosslinking proximal cysteine
and arginine residues through a novel methylthioimidazole-based
As a central sensor and integrator of cellular redox status, KEAP1
relays metabolic information to the transcriptional activation of NRF2
through modification of KEAP1′s sensor cysteines. Several endogenous
electrophilic metabolites have been demonstrated as covalent modifiers
of KEAP1 by S-alkylation, driving NRF2-driven cellular adaptations in
response to increased metabolic pathway flux. Key examples include
fumarate [20,21] and itaconate [22,23], mitochondrial metabolites
derived from the citric acid cycle that promote NRF2 activation in
various physiological settings. Given this, we envisioned an alternative
Scheme 1. Synthesis of analogs 1 and 6b-p. Reagents and conditions: (a) Ar-SH, NBS, CH2Cl2, rt, 20–61%; (b) pyridine, CH2Cl2, 70 ◦C, 33–82%; (c) mCPBA, CH2Cl2,
rt; (d) i-butylamine, CH3CN, rt, 14–56% for 2 steps.
2

J. Jo et al.
Bioorganic Chemistry 108 (2021) 104614
Table 1
ARE-LUC inductive activities of compounds 1 and 6b–6p.
Comp.
ARE-LUC fold induction at 20 µMa
Comp.
ARE-LUC fold induction at 20 µMa
1
6.7
6i
7.9
6b
6c
6d
6e
6f
6.1
6j
12.7
7.1
12.0
17.1
10.1
0.8
6k
6l
4.3
6m
6n
6o
6p
20.0
0.0
6g
1.7
2.1
6h
8.5
0.6
a
Values are the mean of three experiments.
modification, termed MICA [24].
afforded the desired β-bromosulfides 3a–p [25]. Exposure of 3a–p to
pyridine in refluxing CH2Cl2 readily provided the dehydrobrominated
thiophenes 4a–p in good yield. Finally, sequential m-chloroperox-
ybenzoic acid (mCPBA) oxidations of 4a–p and conjugate additions of
the resulting bis-sulfones 5a–p with isobutylamine furnished the desired
β-aminosulfones 1 and 6b–p in a one-pot process.
As the molecular architecture of this chemical scaffold is largely
unexplored and there are no other reported cell permeable inhibitors of
PGK1, we engaged in the structural optimization of 1. Herein, we
describe the design, synthesis, and biological evaluation of this novel
series of non-electrophilic ARE activators and a brief structure–activity
relationship (SAR).
With this set of compounds, we investigated the effects of phenyl
substitution using a NRF2-dependent ARE-LUC reporter assay in IMR32
cells. Eight of these analogs (6c–6e, 6h-6k, and 6m) displayed a greater
ARE-activating response than compound 1 at 20 µM (Table 1). Removal
of the chloro substituent on the benzene ring, as in compound 6b,
negatively affected ARE-LUC induction, while chloro substitution at the
meta-position (6k) led to a slight increase in the ARE-LUC activation. In
contrast, moving the substituent to the ortho-position (6l) resulted in a
2. Result and discussion
The efficient and concise synthesis of derivatives of the phenyl-
sulfone moiety (Part A) was carried out in four steps as depicted in
Scheme 1. Bromosulfenylation of commercially available 3-sulfolene (2)
and various thiophenols in the presence of N-bromosuccinimide
Scheme 2. Synthesis of analogs 7a-g, 8 and 9. Reagents and conditions: (a) mCPBA, CH2Cl2, rt; then various amines (except for d), i-Pr2NEt, CH3CN, rt, 17–41% for 2
steps; (b) Ce(NH4)2(NO3)6, CH3CN/H2O, rt, 54% from 7h (c) mCPBA, CHCl3, rt, 81%.
3

J. Jo et al.
Bioorganic Chemistry 108 (2021) 104614
identification experiments in medicinal chemistry campaigns, given the
divergent activities on cytotoxicity and ARE-LUC activity of identified
metabolites in this work (i.e., 8 vs. 9).
Table 2
ARE-LUC inductive activities and cytotoxicity of compounds 6m, 7a-7g, 8h, and
9.
Comp.
ARE-LUC EC50
ARE-LUC Emax
IMR32 cytotoxicity IC50
[µM]a
[FI]b
[µM]c
3. Conclusion
1
1.0
3.6
1.0
>20
1.0
1.0
1.1
0.8
0.7
2.4
1.8
31.9
20.0
52.6
6.9
5.4
6m
7a
7b
7c
7d
7e
7f
7g
8
5.6
In summary, we report the design, synthesis, and biological evalua-
tion of a series of bis-sulfone ARE activator molecules, based on CBR-
470–1 (1). Our SAR results suggest that the phenylsulfone region (Part
A) of the scaffold can be substituted with a number of groups to improve
the magnitude of ARE induction relative to 1. We also demonstrate that
Part B, the i-butylamino moiety of 1, can be substituted to improve
potency of the series below 1 µM, as demonstrated by 7f and 7g. Lastly,
we show that 7c, 7e, and presumptive metabolite 9 efficaciously acti-
vate NRF2 transcriptional activity without cytotoxicity below 20 µM.
Importantly, this result suggests that PGK1 can be engaged in cells
without obligate cytotoxicity, a current limitation with the use of 1 for
studies involving protective NRF2 activation. Together these results
provide a roadmap for the future modification of this scaffold to identify
non-toxic molecules with enhanced potencies and the requisite physi-
cochemical features for in vivo studies, a result we will report in future
work.
7.0
>20
>20
9.9
55.0
56.0
45.6
36.7
30.5
74.0
99.7
>20
12.7
9.0
0.3
9
a
>20
EC50 values are the mean of three experiments and correspond to the con-
centration resulting in half-maximal induction for each compound.
b
c
FI, fold induction relative to a DMSO neutral stimulation control.
IC50 values are the mean of three experiments and correspond to the con-
centration of each compound which results in 50% cytotoxicity.
small decrease in the activity. Among derivatives monosubstituted at
position 4 of this ring (6c–6j), those possessing an electron-donating
group (6e and 6h–6j) displayed more efficacious ARE-inducing activ-
ities compared to those bearing an electron-withdrawing group (6f and
6g), except for halide analogs 6c and 6d. The most active compound was
3,4-dichloro substituted analog 6m, which displayed about three-fold
increase in ARE-LUC-inducing activity relative to 1. Low ARE activa-
tion was observed with other disubstituted analogs 6n–6p at the tested
concentration, which is possibly derived from enhanced cytotoxicity at
this concentration.
4. Experimental
4.1. Chemistry
Unless noted otherwise, all starting materials and reagents were
obtained from commercial suppliers and were used without further
purification. Reaction flasks were dried at 100 ◦C. Air- and moisture-
sensitive reactions were performed under an argon atmosphere. All
solvents used for routine isolation of products and chromatography were
reagent-grade. Flash column chromatography was performed using sil-
ica gel 60 (230–400 mesh, Merck) with the indicated solvents. Thin-
layer chromatography was performed using 0.25 mm silica gel plates
(Merck). 1H and 13C{1H} NMR spectra were recorded on Bruker AMX-
400 (400 MHz), AVANCE NEO 500, and Unity-Inova 500 (500 MHz)
instruments and calibrated using residual solvent peaks as internal
reference. Chemical shifts were expressed in parts per million (ppm, δ)
downfield from tetramethylsilane and calibrated to the deuterated sol-
vent reference peak. 1H NMR data were reported in the order of chem-
ical shift, multiplicity (s, singlet; d, doublet; t, triplet; q, quartet; m,
multiplet and/or multiple resonance), number of protons, and coupling
constant quoted in hertz (Hz). Preparative high-pressure liquid chro-
matography (prep-HPLC) was operated on Agilent Technologies 1200
Infinity Series. High-resolution mass spectrometry (HR-MS) data were
obtained with an Agilent LC/MSD TOF mass spectrometer by electro-
spray ionization-time of flight (ESI-TOF) reflectron experiments.
We next turned our attention to modification of the i-butylamino
group of 6m. Our established synthetic procedure allowed for the design
and synthesis of analogs modified on part B of the scaffold as in 7a–g, 8,
and 9 (Scheme 2). Sulfide oxidation of 4m, followed by conjugate ad-
ditions of the resulting bis-sulfone 5m with the indicated amines pro-
duced the final β-aminosulfones 7a–g (except for the glycine derivative
7d [26]) and the advanced intermediate 7h in a one-pot process.
Oxidative cleavage of the PMB protected bis-sulfone 7h provided the N-
dealkylated analog 8 in moderate yield. The hydroxylamine derivative 9
was obtained by mCPBA oxidation of 6m in good yield.
From our initial investigation of SAR and preliminary metabolite
identification profiling (Figs. S1 and S2) we were encouraged to probe
the influence of the aminoalkyl substitutions on part B of the bis-sulfone
scaffold. We evaluated the maximal fold-induction (Emax) and the EC50
of analogs in concentration-dependent ARE-LUC reporter assays and
assessed their cytotoxic activity (Table 2). Generally, analogs with
modified substituents on the amine showed enhanced potency to 6 m,
except for the O-isopropylhydroxylamino compound 7b. N-methylation
(7a) or introduction of an amino group on the tertiary carbon (7c)
improved both potency and transcriptional efficacy. A similar increase
in ARE-LUC activity was also observed for analogs in which the i-buty-
lamino group was replaced with glycine (7d) or glycine methyl ester
(7e). It is noteworthy that 7c and 7e had no cytotoxic potential toward
IMR32 cells, suggesting that PGK1 activity and likely thus glycolytic flux
can be modulated without obligate cytotoxicity. The most potent com-
pounds evaluated were amide substituted 7f and 7g, which displayed
submicromolar potency with similar Emax values to 1. N-dealkylated
analog 8, a major expected metabolite of 6m, was found to be most toxic
although it exhibited an approximate 3.5-fold enhancement of the effi-
cacy relative to the parent compound. In contrast, putative N-hydroxyl
metabolite 9 had the highest Emax value (99.7-FI) with no evidence of
toxicity below 20 µM. These results suggest that the ARE-inducing ac-
tivity and cytotoxicity of this scaffold can be manipulated by single
modifications to the aminoxy moiety (Part B) of the bis-sulfone scaffold.
These results also underscore the effectiveness of using metabolite
4.1.1. 3-Bromo-4-((3,4-dichlorophenyl)thio)tetrahydrothiophene 1,1-di-
oxide (3m) [24]
To a stirred solution of N-bromosuccinimide (4.26 g, 25.4 mmol) in
CH2Cl2 (60.0 mL) was added dropwise a solution of 3,4-dichlorobenze-
nethiol (3.23 mL, 25.4 mmol). After stirred for 30 min, to the reaction
mixture was added dropwise a solution of 3-sulfolene 2 (3.00 g, 25.4
mmol). After stirring for 2 h, the reaction mixture was quenched with
H2O. The aqueous layer was extracted with CH2Cl2 and the combined
organic layers were dried over MgSO4 and concentrated in vacuo. The
residue was purified by flash column chromatography on silica gel
(EtOAc : n-hexane = 1 : 5) to afford 4.75 g (50%) of 3m as white solid: 1H
NMR (CDCl3, 500 MHz) δ 7.60 (d, 1H, J = 2.1 Hz), 7.47 (d, 1H, J = 8.3
Hz), 7.34 (dd, 1H, J = 8.3, 2.1 Hz), 4.34 (q, 1H, J = 7.1 Hz), 4.04 (q, 1H,
J = 7.4 Hz), 3.93 (dd, 1H, J = 14.1, 7.1 Hz), 3.73 (dd, 1H, J = 13.7, 7.4
Hz), 3.51 (dd, 1H, J = 14.1, 6.9 Hz), 3.14 (dd, 1H, J = 13.9, 7.4 Hz); 13
C
{1H} NMR (CDCl3, 100 MHz) δ 135.3, 134.2, 133.8, 133.0, 131.6, 130.4,
59.4, 55.9, 51.9, 43.4; LR-MS (ESI+) m/z 375 [M + H]+; HR-MS (ESI+)
4

J. Jo et al.
Bioorganic Chemistry 108 (2021) 104614
calculated for C10H10BrCl2O2S2 [M + H]+ 374.8677; found 374.8679.
ARE-LUC, was transferred to each well in 10 µL of Opti-MEM medium
(Gibco), diluted from a master stock composed such that 1 µg of reporter
plasmid was complexed with 4 µL of FugeneHD (Promega). The next
day, serial DMSO dilutions of compounds in a 384-well source plate
were transferred to the above assay plate using a PerkinElmer FX in-
strument outfitted with a 100 nL pintool head (VP Scientific). After 24-
hour incubation, ARE-LUC luminance values were recorded on an
Envision plate reader (PerkinElmer) after the addition of 30 µL of
BrightGlo (diluted 1:3 in water) with shaking.
4.1.2. 3-((3,4-Dichlorophenyl)thio)-2,3-dihydrothiophene 1,1-dioxide
(4m) [24]
To a stirred solution of 3m (487 mg, 1.29 mmol) in CH2Cl2 (10.0 mL)
was added pyridine (0.260 mL, 3.23 mmol). After stirring for 1 h at 70
℃, the reaction mixture was cooled to rt and quenched with saturated
aq. NH4Cl. The aqueous layer was extracted with CH2Cl2 and the com-
bined organic layers were dried over MgSO4 and concentrated in vacuo.
The residue was purified by flash column chromatography on silica gel
(EtOAc : n-hexane = 1 : 2) to afford 310 mg (81%) of 4m as white solid:
1H NMR (CDCl3, 400 MHz) δ 7.55 (d, 1H, J = 2.1 Hz), 7.44 (d, 1H, J =
8.3 Hz), 7.28 (dd, 1H, J = 8.4, 2.2 Hz), 6.71 (m, 1H), 6.69 (m, 1H), 4.47
(m, 1H), 3.66 (dd, 1H, J = 14.1, 8.2 Hz), 3.21 (dd, 1H, J = 14.1, 4.5 Hz);
13C{1H} NMR (CDCl3, 100 MHz) δ 138.8, 135.2, 134.1, 133.7, 133.6,
132.9, 131.4, 130.7, 54.4, 45.0; LR-MS (ESI+) m/z 295 [M + H]+; HR-
MS (ESI+) calculated for C10H9Cl2O2S2 [M + H]+ 294.9416; found
294.9416.
4.3. Cellular viability assay
For miniaturized viability assays, 5000 IMR32 cells were plated per
well in white 384-well plates in 50 µL of growth medium. The next day,
cells were treated with compounds via pintool-based transfer, as above.
After a 24-hour incubation, cellular viability measurements were
recorded after the addition of 30 µL of CellTiterGlo solution (Promega,
diluted 1:6 in water) on an Envision plate reader.
4.1.3. 3-((3,4-Dichlorophenyl)sulfonyl)-4-(isobutylamino)
Declaration of Competing Interest
tetrahydrothiophene 1,1-dioxide (6m) [24]
To a stirred solution of 4m (310 mg, 1.05 mmol) in CH2Cl2 (10.0 mL)
was added mCPBA (648 mg, 2.63 mmol, 70 ~ 75%). After stirring for 1
h, the reaction mixture was filtered and quenched with saturated aq.
NaHCO3. The aqueous layer was extracted with CH2Cl2 and the com-
bined organic layers were dried over MgSO4 and concentrated in vacuo.
The crude mixture was used for next step without further purification.
To a stirred solution of the sulfone in CH3CN (10.0 mL) was added i-
butylamine (0.104 mL, 1.05 mmol). After stirring for 1 h, the reaction
mixture was concentrated in vacuo. The residue was purified by pre-
parative HPLC and lyophilized to afford 215 mg (51%) of 6m as white
solid: 1H NMR (DMSO‑d6, 400 MHz) δ 8.21 (d, 1H, J = 2.0 Hz), 7.96 (d,
1H, J = 8.4 Hz), 7.90 (dd, 1H, J = 8.4, 2.1 Hz), 4.38 (q, 1H, J = 8.5 Hz),
3.66 (m, 3H), 3.42 (dd, 1H, J = 14.1, 9.0 Hz), 3.12 (dd, 1H, J = 12.2, 6.0
Hz), 2.17 (m, 1H), 2.03 (m, 1H), 1.87 (q, 1H, J = 7.7 Hz), 1.31 (m, 1H),
0.67 (d, 3H, J = 3.9 Hz), 0.65 (d, 3H, J = 3.9 Hz); 13C{1H} NMR
(DMSO‑d6, 100 MHz) δ 138.5, 137.6, 132.4, 131.6, 130.6, 128.7, 63.5,
55.4, 55.1, 54.4, 49.4, 28.0, 20.3; LR-MS (ESI+) m/z 400 [M + H]+; HR-
MS (ESI+) calculated for C14H20Cl2NO4S2 [M + H]+ 400.0205; found
400.0204.
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgments
This work was supported by the Skaggs Institute for Chemical
Biology at Scripps Research and the Bio & Medical Technology Devel-
opment Program of the National Research Foundation (NRF) funded by
the Korean government (MSIT) (NRF-2017M3A9G7072568). We thank
Kristen Williams for technical support with manuscript preparation. We
are especially grateful for the assistance of the Compound Management
Group (CMG) at Calibr, namely Alonzo Davila and Emily Roddy.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bioorg.2020.104614.
4.1.4. 3-((3,4-Dichlorophenyl)sulfonyl)-4-(hydroxy(isobutyl)amino)
tetrahydrothiophene 1,1-dioxide (9)
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