Synthesis of Keap1-phosphorylated p62 and Keap1-Nrf2 protein-protein interaction inhibitors and their inhibitory activity

Article abstract of DOI:10.1016/j.bmcl.2016.10.083

The Keap1-Nrf2 system is involved not only in biological defense but also in malignancy progression and chemoresistance. The ubiquitin-binding protein p62/Sqstm1 (p62), which is highly expressed in several cancers, competes with Nrf2 for Keap1 binding, leading to activation of Nrf2-mediated gene expression and survival of cancer cells. We had previously identified an inhibitor for the Keap1-phosphorylated-p62 (p-p62) protein-protein interaction (PPI), the acetonyl naphthalene derivative K67. In this study, we established facile synthetic routes for K67 and derivatives with various side chains on the C-2 position of naphthalene ring. K67 possessed high selectivity in the inhibition of Keap1-p-p62. Other derivatives showed potent Keap1-Nrf2 and Keap1-p-p62 PPI inhibitory activities, though the selectivity between the two activities was lower than K67.

Full text of DOI:10.1016/j.bmcl.2016.10.083

Accepted Manuscript
Synthesis of Keap1-phosphorylated p62 and Keap1-Nrf2 protein-protein inter-
action inhibitors and their inhibitory activity
Daisuke Yasuda, Mao Nakajima, Akihiro Yuasa, Rika Obata, Kyoko Takahashi,
Tomoyuki Ohe, Yoshinobu Ichimura, Masaaki Komatsu, Masayuki Yamamoto,
Riyo Imamura, Hirotatsu Kojima, Takayoshi Okabe, Tetsuo Nagano, Tadahiko
Mashino
PII:
S0960-894X(16)31131-3
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.10.083
BMCL 24386
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
27 September 2016
25 October 2016
27 October 2016
Please cite this article as: Yasuda, D., Nakajima, M., Yuasa, A., Obata, R., Takahashi, K., Ohe, T., Ichimura, Y.,
Komatsu, M., Yamamoto, M., Imamura, R., Kojima, H., Okabe, T., Nagano, T., Mashino, T., Synthesis of Keap1-
phosphorylated p62 and Keap1-Nrf2 protein-protein interaction inhibitors and their inhibitory activity, Bioorganic
& Medicinal Chemistry Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.2016.10.083
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Synthesis of Keap1-phosphorylated p62 and Keap1-Nrf2 protein-protein
interaction inhibitors and their inhibitory activity
Daisuke Yasudaa, Mao Nakajimaa, Akihiro Yuasaa, Rika Obataa, Kyoko Takahashia, Tomoyuki
Ohea,*, Yoshinobu Ichimurab, Masaaki Komatsub, Masayuki Yamamotoc, Riyo Imamurad,
Hirotatsu Kojimad, Takayoshi Okabed, Tetsuo Naganod and Tadahiko Mashinoa,*
aDepartment of Pharmaceutical Sciences, Faculty of Pharmacy, Keio University, 1-5-30
Shibakoen, Minato-ku, Tokyo, Japan
bDepartment of Biochemistry, School of Medicine, Niigata University, 1-757 Asahimachidori,
Chuo-ku, Niigata-shi, Niigata, Japan
cDepartment of Medical Biochemistry, Tohoku University School of Medicine, 2-1
Seiryomachi, Aoba-ku, Sendai-shi, Miyagi, Japan
dDrug Discovery Initiative, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, Japan
* Corresponding authors. Tel.: +81 3 5400 2691; fax +81 3 5400 2691 (T.O.); tel.: +81 3
5400 2694; fax +81 3 5400 2691 (T.M.). E-mail addresses: ohe-tm@pha.keio.ac.jp (T.O.),
mashino-td@pha.keio.ac.jp (T.M.)
Keywords
Nrf2; Keap1; p62/Sqstm1; protein-protein interaction inhibitor
Abbreviations
p62: p62/Sqstm1, p-p62: phosphorylated-p62, PPI: protein-protein interaction, Nrf2: nuclear
factor erythroid 2 related factor-2, Keap1: Kelch-like ECH-associated protein 1, HCC: human
hepatocellular carcinoma, CAN: cerium ammonium nitrite, FP: fluorescent polarization,
FAM: carboxyfluorescein
1

Abstract
The Keap1-Nrf2 system is involved not only in biological defense but also in
malignancy progression and chemoresistance. The ubiquitin-binding protein p62/Sqstm1
(p62), which is highly expressed in several cancers, competes with Nrf2 for Keap1 binding,
leading to activation of Nrf2-mediated gene expression and survival of cancer cells. We had
previously identified an inhibitor for the Keap1-phosphorylated-p62 (p-p62) protein-protein
interaction (PPI), the acetonyl naphthalene derivative K67. In this study, we established
facile synthetic routes for K67 and derivatives with various side chains on the C-2 position of
naphthalene ring. K67 possessed high selectivity in the inhibition of Keap1-p-p62. Other
derivatives showed potent Keap1-Nrf2 and Keap1-p-p62 PPI inhibitory activities, though the
selectivity between the two activities was lower than K67.
2

The nuclear factor erythroid 2 related factor-2 (Nrf2) and Kelch-like
ECH-associated protein 1 (Keap1) system is one of the biological defense mechanisms
against oxidative stress and xenobiotics.1 Nrf2 is a transcription factor that induces various
antioxidant and detoxification enzymes.2 Keap1, a binding partner of Nrf2 is an adaptor
protein of cullin 3 ubiquitin ligase for Nrf2, and thus Nrf2 is constantly ubiquitinated and
degraded by the 26S proteasome.3 Consequently, the transcription activity of Nrf2 is
suppressed to a very low level under physiological conditions. However, under stressed
conditions, Nrf2 is dissociated from Keap1, and the ubiquitination of Nrf2 is arrested.4
Subsequently, free Nrf2 accumulates, translocates to nuclei, and induces a variety of
biological defensive enzymes.4 For this reason, Nrf2 activators have attracted much
attention as novel drug targets for diseases such as diabetic nephropathy and multiple
sclerosis.5 Especially, non-covalent Keap1-Nrf2 protein-protein interaction (PPI) inhibitors
are of particular interest due to lower potential for off-target effects.6
Nrf2 is also involved in the onset of tumor progression and anticancer drug
resistance in certain types of cancerbecause Nrf2 target genes contain enzymes related to not
only anti-oxidative stress but also cancer metabolism.7 The ubiquitin-binding protein
p62/Sqstm1 (p62), also forms a complex with Keap1.8 S349 phosphorylated p62 (p-p62)
binds to the Kelch domain of Keap1 and competes with the binding of Nrf2, leading to
attenuated Nrf2 ubiquitination. In this way, p-p62 activates Nrf2-mediated gene expression.9
The array of p-p62-Keap1-Nrf2 is thought to be involved in tumor development in
autophagy-deficient mouse livers or human hepatocellular carcinoma (HCC). 9b, 10
Our previous report showed that p-p62 accumulates in hepatitis C virus positive
HCC, and we identified a non-covalent type inhibitor of the Keap1- p-p62 interaction that we
called K67 (Fig. 1).11 K67 is an analog of Cpd16, a compound previously identified by
another group as a non-covalent Keap1-Nrf2 PPI inhibitor (Fig. 1).6a Treatment of Huh1 cell
lines with K67 suppressed proliferation and reduced tolerance to cisplatin or sorafenib; no
such effects were observed in the case of Cpd16 despite its structural similarity to K67.11
Thus, the acetonyl side chain of K67 seemed to play a key role in the selective inhibition of
the Keap1-p-p62 PPI. These results suggest that K67 has potential as a lead compound for
novel anticancer agents, particularly in overcoming chemoresistance and as a chemical tool to
3

investigate biological events associated with the p-p62-Keap1-Nrf2 interaction.
Figure 1. Structures of Keap1-Nrf2 inhibitors K67 and Cpd16
Herein, we report facile synthetic routes for K67 and its derivatives possessing
various side chains on the naphthalene ring. We also evaluate their inhibitory activities
against the Keap1-p-p62 and Keap1- Nrf2 PPIs.
The known inhibitors Cpd16 and K67 were prepared as shown in Scheme 1.12-14
Commercially available 4-nitro-1-naphthylamine was reduced to 1,4-diaminonaphtalene with
palladium-catalyzed
hydrogenation.
Condensation
of
two
equivalents
of
4-alkoxybenzenesulfonyl chloride with the amino groups afforded 2 and Cpd16 (3). The
yield of 3 was improved to 95% compared to the literature method (64%).15 Compounds 2
and 3 were oxidized to quinone-imines 4 and 5 with cerium ammonium nitrite (CAN).
Following oxidation, the pure product was precipitated in a high yield. Other oxidants, such
as 2,3-dichloro-5,6-dicyanobenzoquinone, manganese dioxide and lead tetraacetate, were
inferior to CAN with respect to yield and ease of purification. The addition of benzyl
acetoacetate to 4 and 5 via Michael-type reaction gave re-aromatized compounds 6 and 7
with the appended side chain. Removal of the benzyl protecting groups from 6 and 7 under
palladium-catalyzed hydrogenation condition with concurrent decarboxylation afforded K67
(8) and 9.
4

Scheme 1. Preparation of K67 and its derivatives
(a) H2, Pd-C, EtOH, rt, 4h, 98%. (b) p-Alkoxybenzensulfonyl chloride, pyridine, DCM, rt, 10 min, 95% (2), 2
h, 95% (3). (c) CAN, MeCN, rt, 10min, 99% (3 and 4). (d) Benzyl acetoacetate, Et3N, toluene, rt, 2h, 58% (6),
73% (7). (e) H2, Pd-C, EtOH, 24h, 96% (8), 16h, 95% (9).
Carboxylates have been shown to increase the binding inhibitory activity of known
Keap1-Nrf2 PPI inhibitors,6d, 15 so several derivatives containing a carboxyl group with various
side-chain lengths were designed. Compound 10 was prepared from the quinone-imine 4
using a similar synthetic method to that which gave 6, followed by removal of the two benzyl
groups to afford 11 (Scheme 2). Compounds 17 and 18 were synthesized via bromination of
4-nitro-1-naphthylamine followed by reduction of the nitro group using hydrazine
monohydrate and osmium-charcoal to give 13 (Scheme 3). After sulfonamidation of the
two amino groups, a Mizoroki-Heck reaction with two different vinyl compounds gave
compound 15 and 16; subsequent hydrogenation afforded 17 and 18. As shown in Scheme
4, thiol compounds were added to quinone-imine 4 to afford 19 and 20 with corresponding
aryl sulfide side chains. The triazole compound 19 was designed as an analog of a known
Nrf2 activator.6b Hydrolysis of 20 with lithium hydroxide gave carboxylate derivative 21.
All of the synthesized compounds used in the biological assay were recrystallized from
suitable solvents.
5

Scheme 2. Preparation of acetate derivative
(a) Dibenzyl malonate, Et3N, toluene, rt, 6h, 79%. (b) H2, Pd-C, EtOH, rt, 15h, 90%.
Scheme 3. Preparation of carboxylate derivatives
(a) NBS, NH4OAc, MeCN, rt, 90%. (b) (N2H4)•H2O, Os-C, MeOH, 70˚C, 14h, 90%. (c)
p-Etoxybenzensulfonyl chloride, pyridine, rt, 67%. (d) Pd(OAc)2, XPhos, DIEA, DMF, 100˚C, 24h, 28% (15),
5h, 32% (16). (e) H2, Pd-C, EtOH, rt, 16h, 27% (17), 15h, 55% (18).
Scheme 4. Preparation of sulfide derivatives
(a) R-SH, MeOH, rt, 24h, 59% (19), 6h, 57% (20). (b) LiOH, MeOH, rt, 1h, 93%.
6

The inhibition of the Keap1-p-p62 and Keap1-Nrf2 PPIs was evaluated using a
fluorescent polarization (FP) assay with recombinant Keap1-DC domain and
6-carboxyfluorescein
(6-FAM)-labelled
Nrf2
peptide
or
6-FAM-labelled
S349-phosphorylated p62 peptide, according to our previous method (Table 1).11, 16 K67 (8)
exhibited a strong Keap1-p-p62 PPI inhibitory activity; this activity was 4-fold higher than
the activity against the Keap1-Nrf2 PPI, indicating high selectivity. Its methoxy analog 9
also inhibited both PPIs, but with greatly diminished selectivity. Cpd16 (3) and its ethoxy
analog 2 demonstrated relatively low selectivity. In our previous study, K67 (8) inhibited
the interaction between p-p62 and Keap1 and activated ubiquitination and degradation of
Nrf2, resulting in the suppression of proliferation and anticancer agent tolerance of HCC,
while Cpd16 (3) showed less efficacy.11 Thus, the 4-fold selectivity observed in K67 (8)
seems to be critical for cellular activities. Compounds 11, 17 and 18, which contain
carboxyl groups with varying side-chain lengths, exhibited potent PPI inhibitory activities but
showed little selectivity for the Keap1-p-p62 PPI. The PPI inhibitory activities of the
carboxylate compounds tended to increase with extension of the side chain lengths. Sulfide
compounds 19, 20 and 21 were also potent inhibitors against both Keap1-Nrf2 and
Keap1-p-p62 PPI. It is noted that 20 had quite high activities whereas it does not contain a
carboxylic group which has been expected to increase the inhibitory activity against
Keap1-Nrf2 PPI according to the previous reports. 6d, 15 This result shows that the ester side
chain can be also used for p-p62-Keap1-Nrf2 inhibitor under certain circumstances. The all
sulfide compounds exhibited low selectivity for the Keap1-p-p62 PPI inhibition.
In this study, we established facile synthetic routes for K67 and its derivatives and
investigated the PPI inhibitory activities of the derivatives. The newly synthesized
derivatives demonstrated inhibition of the Keap1-Nrf2 and Keap1-p-p62 PPIs, though the
selectivity of derivatives toward Keap1-p-p62 PPI was lower than that of K67. Though the
introduction of a carboxylic side chain resulted in much greater inhibition activities compared
with Cpd16 and K67, the resulting compounds exhibited greatly reduced selectivity. These
structure-activity relationships for inhibition activity and selectivity should be investigated
further. Our synthetic methods for derivatives with varying side chains can be useful for
drug discovery of both Nrf2 activators and suppressors.
7

Table 1
Keap1-p-p62 and Keap1-Nrf2 protein-protein interaction inhibition activities of the
naphthalene derivatives
Compound
p-p62 IC50 (M)
Selectivity index (Nrf2/p-p62)
Nrf2 IC50 (M)
2
317
1.4
2.8
2.0
2.0
4.1
1.3
1.4
1.2
1.0
1.3
1.9
1.1
0.91
1.5
1.8
8
6.2
9
2.4
3.2
11
17
18
19
20
21
0.23
0.19
0.15
1.0
0.32
0.23
0.15
1.3
0.37
0.18
0.72
0.20
8

Acknowledgments
This work was supported by the Platform for Drug Discovery, Informatics, and
Structural Life Science from the Ministry of Education, Culture, Sports, Science and
Technology (MEXT), and Japan Agency for Medical Research and Development (AMED),
2012-2016.
Supplementary data
Supplementary data associated with this article can be found in the online version
at http://dx.doi.org/xxxx/xxxxxxx.
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12. Synthesis of 2: 4-Ethoxybenzenesulfonyl chloride (1.87 g, 8.48 mmol, 1.1 equiv.) and
pyridine (2 mL) were added to the solution of 1 (610 mg, 3.86 mmol) in
dichloromethane (10 mL) and stirred at room temperature for 10 minutes. The reaction
mixture was acidified with 2 M hydrochloric acid and extracted twice with
dichloromethane. The combined organic layer was washed with brine and dried over
anhydrous sodium sulfate. The solvent was evaporated under reduced pressure to
afford a light-purple solid (1.90 g, 95%).
13. Synthesis of 4: Cerium ammonium nitrite (200 mg, 0.38 mmol, 2.0 equiv.) was added to
a solution of 2 (100 mg, 0.19 mmol) in acetonitrile (20 mL) and stirred at room
temperature for 10 minutes. The yellow solid (100 mg, 99% yield) was collected by
filtration.
14. Synthesis of K67 (8): (a) Benzyl acetoacetate (352 mg, 1.82 mmol, 1.1 equiv.) and
triethylamine (1.2 mL, 864 mg, 8.50 mmol, 5.0 equiv.) was added to a solution of 4 in
toluene (30 mL) and stirred at room temperature for 2 hours. The reaction was
quenched with 2 M hydrochloric acid and the reaction mixture was extracted twice with
ethyl acetate. The combined organic layer was washed with brine and dried over
anhydrous sodium sulfate.
Purification with silica-gel column chromatography
(n-hexane/ethyl acetate=1:1) afforded 6 as a brown solid (621 mg, 58% yield). (b)
Compound 6 (621 mg, 0.86 mmol) was dissolved into ethanol (30 mL) and then
palladium on charcoal (5%, 50 mg) was added. The reaction mixture was stirred under
hydrogen atmosphere for 24 hours. The catalyst was removed by filtration through a
Celite® pad and the solvent was concentrated under reduced pressure. Purification with
silica-gel column chromatography (n-hexane/ethyl acetate=1/1) afforded the light-yellow
solid (484 mg, 96% yield).
15. Jiang, Z. Y.; Lu, M.; Xu, L.; Yang, T.; Xi, M.; Xu, X.; Guo. X.; Zhang, X.; You. Q.; Sun,
H. J. Med. Chem. 2014, 57, 2736.
11

16. Five microlitres of 20 nM peptide (FAM-labeled phosphorylated-p62 peptide or
FAM-labeled Nrf2 peptide) solution and 5 L of 500 nM Keap1-DC protein solution
diluted with FP assay buffer (10 mM HEPES, pH 7.4, 0.5 mM EDTA, 150 mM NaCl,
0.5 mM TCEP and 0.005% Tween-20) were dispensed to each well of 384-well
non-binding black plates where 100 nL of 1 mM tested compound solution in dimethyl
sulfoxide was transferred in advance by a liquid handler. Subsequently, the plates were
incubated for 30 min at room temperature. The FP signal was measured at 520 nm with
excitation at 485 nm.
17. Compound 3 used in the FP assay was purchased from Princeton BioMolecular Research,
Inc. (Princeton, NJ).
12

Figure captions
Figure 1. Structures of Keap1-Nrf2 inhibitors K67 and Cpd16
Scheme 1. Preparation of K67 and its derivatives
(a) H2, Pd-C, EtOH, rt, 4.5 h, 96%. (b) p-Alkoxybenzensulfonyl chloride, pyridine, DCM, rt, 10 min, 95% (2),
2 h, 95% (3). (c) CAN, MeCN, rt, 10 min, 99% (4 and 5). (d) Benzyl acetoacetate, Et3N, toluene, rt, 2 h, 58%
(6), 2.5 h, 73% (7). (e) H2, Pd-C, EtOH, 24 h, 96% (8), 16 h, 95% (9).
Scheme 2. Preparation of acetate derivative
(a) Dibenzyl malonate, Et3N, toluene, rt, 6 h, 79%. (b) H2, Pd-C, EtOH, rt, 15 h, 90%.
Scheme 3. Preparation of carboxylate derivatives
(a) NBS, NH4OAc, MeCN, rt, 10 min 90%. (b) (N2H4)•H2O, Os-C, MeOH, 70˚C, 14 h, 90%. (c)
p-Etoxybenzensulfonyl chloride, pyridine, rt, 24 h, 67%. (d) Pd(OAc)2, XPhos, DIEA, DMF, 100˚C, 24 h, 28%
(15), 5 h, 32% (16). (e) H2, Pd-C, EtOH, rt, 16 h, 27% (17), 15 h, 55% (18).
Scheme 4. Preparation of sulfide derivatives
(a) R-SH, MeOH, rt, 24 h, 59% (19), 6 h, 57% (20). (b) LiOH, MeOH, rt, 1 h, 93%.
13

Graphical Abstract
14