Inhibitors of the protein–protein interaction between phosphorylated p62 and Keap1 attenuate chemoresistance in a human hepatocellular carcinoma cell line

Article abstract of DOI:10.1080/10715762.2020.1732955

Resistance to anticancer agents has been an obstacle to developing therapeutics and reducing medical costs. Whereas sorafenib is used for the treatment of human hepatocellular carcinoma (HCC), resistance limits its efficacy. p62, a multifunctional protein, is overexpressed in several HCC cell lines, such as Huh-1 cells. Phosphorylated p62 (p-p62) inhibits the protein–protein interaction (PPI) between Keap1 and Nrf2, resulting in the Nrf2 overactivation that causes drug resistance. We have found a unique Nrf2 inactivator, named K67, that inhibited the PPI between Keap1 and p-p62 and attenuated sorafenib resistance in Huh-1 cells. Herein, we designed and synthesised novel K67 derivatives by modification of the substituent at the 4-position of the two benzenesulfonyl groups of K67. Although these new derivatives inhibited the Keap1-p-p62 PPI to a level comparable to or weaker than that of K67, the isopropoxy derivative enhanced the sensitivity of Huh-1 cells to sorafenib to a greater extent than K67 without any influence on the viability of Huh-7 cells, which is a non-resistant HCC cell line. The isopropoxy derivative also increased the sensitivity of Huh-1 cells to regorafenib, which suggests that this derivative has the potential to be used as an agent to overcome chemoresistance based on Nrf2 inactivation.

Full text of DOI:10.1080/10715762.2020.1732955

Free Radical Research
ISSN: 1071-5762 (Print) 1029-2470 (Online) Journal homepage: https://www.tandfonline.com/loi/ifra20
Inhibitors of the protein–protein interaction
between phosphorylated p62 and Keap1 attenuate
chemoresistance in a human hepatocellular
carcinoma cell line
Daisuke Yasuda, Tomoyuki Ohe, Kyoko Takahashi, Riyo Imamura, Hirotatsu
Kojima, Takayoshi Okabe, Yoshinobu Ichimura, Masaaki Komatsu, Masayuki
Yamamoto, Tetsuo Nagano & Tadahiko Mashino
To cite this article: Daisuke Yasuda, Tomoyuki Ohe, Kyoko Takahashi, Riyo Imamura, Hirotatsu
Kojima, Takayoshi Okabe, Yoshinobu Ichimura, Masaaki Komatsu, Masayuki Yamamoto,
Tetsuo Nagano & Tadahiko Mashino (2020): Inhibitors of the protein–protein interaction between
phosphorylated p62 and Keap1 attenuate chemoresistance in a human hepatocellular carcinoma
cell line, Free Radical Research, DOI: 10.1080/10715762.2020.1732955
To link to this article: https://doi.org/10.1080/10715762.2020.1732955
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Inhibitors of the protein-protein interaction between phosphorylated
p62 and Keap1 attenuate chemoresistance in a human hepatocellular
carcinoma cell line
Daisuke Yasudaa, Tomoyuki Ohea*, Kyoko Takahashia, Riyo Imamurab, Hirotatsu
Kojimab, Takayoshi Okabeb, Yoshinobu Ichimurac, Masaaki Komatsuc, Masayuki
Yamamotod, Tetsuo Naganob and Tadahiko Mashinoa*.
aDepartment of Pharmaceutical Sciences, Faculty of Pharmacy, Keio University, 1-5-30 Shibakoen,
Minato-ku, Tokyo, Japan
bDrug Discovery Initiative, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, Japan
cDepartment of Physiology, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo,
Japan
dDepartment of Medical Biochemistry, Tohoku University School of Medicine, 2-1 Seiryo-machi, Aoba-ku,
Sendai-shi, Miyagi, 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.)

Abstract
Resistance to anticancer agents has been an obstacle to developing therapeutics and reducing
medical costs. Whereas sorafenib is used for the treatment of human hepatocellular carcinoma
(HCC), resistance limits its efficacy. p62, a multifunctional protein, is overexpressed in several
HCC cell lines, such as Huh-1 cells. Phosphorylated p62 (p-p62) inhibits the protein-protein
interaction (PPI) between Keap1 and Nrf2, resulting in the Nrf2 overactivation that causes drug
resistance. We have found a unique Nrf2 inactivator, named K67, that inhibited the PPI between
Keap1 and p-p62 and attenuated sorafenib resistance in Huh-1 cells. Herein, we designed and
synthesized novel K67 derivatives by modification of the substituent at the 4-position of the two
benzenesulfonyl groups of K67. Although these new derivatives inhibited the Keap1-p-p62 PPI
to a level comparable to or weaker than that of K67, the isopropoxy derivative enhanced the
sensitivity of Huh-1 cells to sorafenib to a greater extent than K67 without any influence on the
viability of Huh-7 cells, which is a nonresistant HCC cell line. The isopropoxy derivative also
increased the sensitivity of Huh-1 cells to regorafenib, which suggests that this derivative has the
potential to be used as an agent to overcome chemoresistance based on Nrf2 inactivation.
Keywords: Keap1, Nrf2, p62, protein-protein interaction, chemoresistance

Introduction
Hepatocellular carcinoma (HCC) is the most common liver cancer that develops through
liver cirrhosis and is induced by various factors, such as hepatitis B and C viruses, alcohol,
mycotoxicins and non-alcoholic fatty liver disease [1]. Hepatectomy, radiofrequency ablation
and transarterial chemoembolization are often selected as therapeutics for early-stage HCC[2]. In
addition, sorafenib (Figure 1), an oral multikinase inhibitor, is approved as a molecular targeted
drug against advanced HCC in more than 70 countries [3]. Sorafenib inhibits the Ras/Raf/MEK/
ERK serine/threonine kinase cascade and the tyrosine kinases involved in angiogenesis, such as
VEGFR-1/2/3, PDGFR, Flt3 and RET, to exert its antitumor activity [4]. However, primary
or acquired resistance to sorafenib is sometimes observed in HCC patients [5]. Recently, as a
second-line treatment for sorafenib-ineffective patients, regorafenib (Figure 1), an alternative
multikinase inhibitor, has been approved. Regorafenib improves the outcomes of highly advanced
HCC patients [6], but due to its adverse events and high structural similarity to sorafenib, a novel
type of anti-HCC medicine is strongly desired.
p62/sequestosome1 (hereafter referred to as p62) is a stress-induced multifunctional protein
that is involved in selective autophagy as a receptor protein and in cell survival or death via the
activation of signaling cascades, such as the TRAF6/NF-B pathway [7]. In a selective autophagy
under normal conditions, p62 is localized to ubiquitin-positive aggregated proteins and damaged
mitochondria and bacteria via its ubiquitin-associated domain on the C-terminal side, resulting
in degradation by autophagosomes [8]. However, p62 is abnormally accumulated in autophagy-
deficient cancer cells, including some types of human HCC [9].
p62 also has an interaction site that binds to Kelch-like ECH associated protein 1 (Keap1),
which is an adapter protein for Cullin 3 ubiquitin ligase and forms a complex with nuclear
factor erythroid 2-related factor 2 (Nrf2) [10]. Nrf2 is one of the transcription factors that
induce a variety of antioxidant, phase II detoxification, multidrug resistance-related and cellular
proliferation proteins in response to oxidative stress or xenobiotics [11]. Keap1 homodimers
suppress Nrf2 activity in physiological conditions by forming a protein-protein interaction
(PPI) between the DC domains of Keap1 and ETGE or the DLGex motif of Nrf2, resulting in
ubiquitination of the Keap1-Nrf2 complex and, ultimately, degradation of the protein by the
26S proteasome [12]. However, once the sensor thiol group on a specific cysteine residue of
Keap1 is modified by oxidative stress or electrophiles, Keap1 partly dissociates from Nrf2, which
causes the accumulation of free form Nrf2 followed by the translocation of Nrf2 to the nucleus
[13]. The translocated Nrf2 binds to antioxidant response element in a complex with small Maf
factor and induces various cellular defensive proteins [13]. Moreover, small molecules that
inhibit the PPI between Keap1 and Nrf2 can directly activate Nrf2. This type of small molecule
is expected to be a therapeutic agent against oxidative stress-related disorders without off-target
effects, and various Keap1-Nrf2 PPI inhibitors have been discovered through a high-throughput
screening or fragment-based drug design [14]. The interaction region on p62 to Keap1 has a
relatively low affinity, but phosphorylation of Ser351 on murine p62 (corresponding to Ser349
in human) increases its affinity more than 30-fold in comparison with nonphosphorylated p62
[15]. The phosphorylated p62 (p-p62) competes with Nrf2 for the Keap1-DC domain just like
Keap1-Nrf2 PPI inhibitors, resulting in Nrf2 activation [15]. In autophagy-deficient tumor cells,
overaccumulation of p-p62 causes constitutive Nrf2 activation and induction of cellular defensive
proteins, which leads to tumor progression and gain of chemoresistance [16].

In a previous study, we demonstrated that p-p62 promotes chemoresistance to sorafenib and cell
growth in HCV-positive HCC via the reprogramming of glucose and glutamine metabolism due
to the constitutive Nrf2 activation associated with the overaccumulation of p62 and acceleration
of its phosphorylation [16]. This phenomenon was also observed in a human HCC cell line,
Huh-1, that is resistant to sorafenib[16]. Additionally, we discovered a selective Keap1-p-p62
PPI inhibitor, called K67 (Figure 1), from high-throughput screening using a chemical library
that possesses 155,000 compounds [16]. Co-administration of K67 and sorafenib or cisplatin
increased the sensitivity of Huh-1 cells to these drugs, whereas Cpd16 (Figure 1), a Keap1-Nrf2
PPI inhibitor that has a similar structure to K67, did not show such effects [16]. X-ray co-crystal
analysis of the PPI between K67 and the Keap1-DC domain suggested that the acetonyl group
at the 2-position of the naphthalene ring of K67 is important for selective Keap1-p-P62 PPI
inhibition [16]. We also demonstrated that substitution of the acetonyl group with hydrophilic
carboxylates decreased the selectivity towards the PPI between p-p62 and Keap1 [17].
Herein, we reported the development of more effective agents to overcome drug resistance
in HCC. To this end, we designed and synthesized novel K67 derivatives in which the
4-ethoxybenzenesulfonamide group of K67 was replaced with various 4-substituted
benzenesulfonamides. Then, the potential of these derivatives to attenuate chemoresistance in
Huh-1 cells was also evaluated.
Materials and Methods
General
1H NMR spectra (500 MHz) were measured on a Varian-500 FT-NMR (Agilent Technologies,
USA) with tetramethylsilane as an internal standard (=0.00). 1H NMR spectra (600 MHz)
were measured on a JEOL JNM-ECP600 FT-NMR (JEOL, Japan) with tetramethylsilane as an
internal standard (=0.00). 13C NMR spectra (150 MHz) were measured on a JEOL JNM-ECP600
FT-NMR, and the chemical shifts were compared to the signals of DMSO-d6 (=39.50). Mass
spectra were recorded on a JEOL JMS-T100LP AccuTOF LC-plus 4G mass spectrometer (ESI-
MS). Palladium-catalyzed hydrogenation was performed at ordinary pressure using a balloon.
Benzenesulfonyl chloride, 4-chlorobenzensulfonyl chloride, 4-methylbenzensulfonyl chloride,
4-isopropoxybenzenesulfonyl chloride, 4-nitro-1-naphtylamine and 4-propylbenzenesulfonyl
chloride were purchased from Tokyo Chemical Industry Co., Ltd. (Japan). Anhydrous sodium
sulfate, cerium(IV) ammonium nitrite, 4-ethylbenzensulfonyl chloride and triethylamine were
purchased from FUJI FILM Wako Pure Chemical Corp. (Japan). Palladium on charcoal was
purchased from Kokusan Chemical Co., Ltd. (Japan).
Chemistry
1 Preparation of 1,4-bis-[(benzenesulfonyl)amino]naphthalenes ( - )
2c m
1-1 General procedure
Commercially available 4-nitro-1-naphthylamine was reduced by palladium-catalyzed
hydrogenation to 1 according to our previous method [17]. To a solution of 1 (500 mg, 3.16
mmol) in dichloromethane (6 mL), the corresponding benzensulfonyl chloride (2.0 mol equiv.)
and pyridine (1 mL) were added. The reaction mixture was stirred at room temperature for 2

hr, and then acidified with 2 M hydrochloric acid and extracted twice with ethyl acetate. The
combined organic layer was washed twice with water and twice with brine, dried over anhydrous
sodium sulfate and concentrated under reduced pressure.
1-2 1,4-bis-[(4-n-propoxybenzenesulfonyl)amino]naphthalene ( )
2c
The pink solid (1.69 g, 97% yield) was obtained. 1H NMR (DMSO-d6, 500 MHz, ) 0.95 (t, -
OCH2CH2CH3 x2, J=7.3 Hz, 6H), 1.70 (m, -OCH2CH2CH3 x2, 4H), 3.94 (t, -OCH2CH2CH3 x2,
J=6.6 Hz, 4H), 6.97 (d, Ar H, J=9.0 Hz, 4H), 7.02 (s, Ar H, 2H), 7.39 (dd, Ar H, J=6.6, 3.2 Hz,
4H), 7.53 (d, Ar H, J=9.0 Hz, 4H), 7.96 (dd, Ar H, J=6.6, 3.2 Hz, 2H), 10.02 (brs, 2H, -NH-).
LRMS-ESI (m/z): 553 [M–H]–. HRMS-ESI (m/z): [M–H]– calcd for C28H29N2O6S2, 553.1467;
found, 553.1498 (+3.1 mmu).
1-3 1,4-bis-[(4-isopropoxybenzenesulfonyl)amino]naphthalene ( )
2d
The pink solid (1.77 g, quantitative yield) was obtained. 1H NMR (DMSO-d6, 500 MHz, ) 1.15
(d, -OCH(CH3)2, J=6.8 Hz, 12H), 2.90 (sept, -OCH(CH3)2, J=6.8 Hz, 2H), 7.09 (s, Ar H, 2H),
7.29 (dd, Ar H, J=6.6, 3.2 Hz, 2H), 7.32 (d, Ar H, J=8.3 Hz, 4H), 7.53 (d, Ar H, J=8.3 Hz, 4H),
7.81 (dd, Ar H, J=6.6, 3.2 Hz, 2H), 10.13 (brs, 2H, -NH-). LRMS-ESI (m/z): 521 [M–H]–. HRMS-
ESI (m/z): [M–H]– calcd for C28H29N2O4S2, 521.1569; found, 521.1557 (–1.2 mmu).
1-4 1,4-bis-[(4-n-butoxybenzenesulfonyl)amino]naphthalene ( )
2e
The pink solid (1.77 g, 96% yield) was obtained. 1H NMR (DMSO-d6, 500 MHz, ) 0.98 (t, -
OCH2CH2CH2CH3, J=7.3 Hz, 6H), 1.42-1.52 (m, -OCH2CH2CH2CH3, 2H), 1.79-1.83 (m, -
OCH2CH2CH2CH3, 2H), 4.10 (d, -OCH2CH2CH2CH3, J=6.7 Hz, 6H), 7.04 (s, Ar H, 2H), 7.33 (d,
Ar H, J=8.3 Hz, 4H), 7.36 (dd, Ar H, J=6.6, 3.2 Hz, 2H), 7.53 (d, Ar H, J=8.3 Hz, 4H), 7.90 (dd,
Ar H, J=6.6, 3.2 Hz, 2H), 10.10 (brs, 2H, -NH-). LRMS-ESI (m/z): 581 [M+H]+. HRMS-ESI (m/
z): [M+H]+ calcd for C30H33N2O6S2, 581.1780; found, 581.1781 (+0.1 mmu).
1-5 1,4-bis-[(4-isobutoxybenzenesulfonyl)amino]naphthalene ( )
2f
The pink solid (1.78 g, 97% yield) was obtained. 1H NMR (DMSO-d6, 500 MHz, ) 1.04 (d, -
OCH2CH (CH3)2, J=6.8 Hz, 6H), 2.14 (sept, -OCH2CH (CH3)2, J=6.8 Hz, 1H), 3.88 (d, -OCH2CH
(CH3)2, J=6.8 Hz, 6H), 7.03 (s, Ar H, 2H), 7.21 (d, Ar H, J=8.3 Hz, 4H), 7.34 (dd, Ar H, J=6.6,
3.2 Hz, 2H), 7.55 (d, Ar H, J=8.3 Hz, 4H), 7.94 (dd, Ar H, J=6.6, 3.2 Hz, 2H), 10.12 (brs, 2H,
-NH-). LRMS-ESI (m/z): 581 [M+H]+. HRMS-ESI (m/z): [M+H]+ calcd for C30H33N2O6S2,
581.1780; found, 581.1777 (–0.4 mmu).
1-6 1,4-bis-[(benzenesulfonyl)amino]naphthalene (
)
2h
The pink solid (1.35 g, 97% yield) was obtained. 1H NMR (DMSO-d6, 500 MHz, ) 7.03 (s,
Ar H, 2H), 7.35 (dd, Ar H, J=6.6, 3.3 Hz, 2H), 7.47 (dd, Ar H, J=7.8, 7.6 Hz, 4H), 7.58 (dd, Ar
H, J=7.6, 7.3 Hz, 2H), 7.61-7.63 (m, Ar H,4H), 7.90 (dd, Ar H, J=6.6, 3.3 Hz, 2H), 10.22 (brs,

2H, -NH-). LRMS-ESI (m/z): 437 [M–H]–. HRMS-ESI (m/z): [M–H]– calcd for C22H17N2O4S2,
437.0630; found, 437.0647 (+1.7 mmu).
1-7 1,4-bis-[(4-methylbenzenesulfonyl)amino]naphthalene ( )
2i
The pink solid (1.40 g, 96% yield) was obtained. 1H NMR (DMSO-d6, 500 MHz, ) 2.32 (s, -CH3,
6H), 6.99 (s, Ar H, 2H), 7.28 (d, Ar H, J=7.8 Hz, 4H), 7.50 (dd, Ar H, J=6.6, 3.2 Hz, 2H), 7.52 (d,
Ar H, J=7.8 Hz, 4H), 7.96 (dd, Ar H, J=6.6, 3.2 Hz, 2H), 10.11 (brs, 2H, -NH-). LRMS-ESI (m/z):
465 [M–H]– HRMS-ESI (m/z): [M–H]– calcd for C24H21N2O4S2, 465.0943; found, 465.0932 (–1.1
mmu).
1-8 1,4-bis-[(4-ethylbenzenesulfonyl)amino]naphthalene ( )
2j
The pink solid (1.52 g, 97% yield) was obtained. 1H NMR (DMSO-d6, 500 MHz, ) 1.13 (t, -
CH2CH3, J=7.6 Hz, 6H), 2.61 (q, -CH2CH3, J=7.6 Hz, 4H), 7.04 (s, Ar H, 2H), 7.30 (d, Ar H,
J=8.3 Hz, 4H), 7.33 (dd, Ar H, J=6.6, 3.2 Hz, 2H), 7.53 (d, Ar H, J=8.3 Hz, 4H), 7.89 (dd, Ar H,
J=6.6, 3.2 Hz, 2H), 10.12 (brs, 2H, -NH-). LRMS-ESI (m/z): 493 [M–H]–. HRMS-ESI (m/z): [M–
H]– calcd for C26H25N2O4S2, 493.1256; found, 493.1235 (–2.1 mmu).
1-9 1,4-bis-[(4-n-propylbenzenesulfonyl)amino]naphthalene ( )
2k
The pink solid (1.32 g, 70% yield) was obtained. 1H NMR (DMSO-d6, 500 MHz, ) 0.19 (t, -
CH2CH2CH3, J=7.3 Hz, 6H), 1.53 (sext, -CH2CH2CH3,, J=7.3 Hz, 4H), 2.56 (t, -CH2CH2CH3,,
J=7.3 Hz, 4H),7.04 (s, Ar H, 2H), 7.27 (d, Ar H, J=8.1 Hz, 4H), 7.31 (dd, Ar H, J=6.6, 3.2 Hz,
2H), 7.51 (d, Ar H, J=8.3 Hz, 4H), 7.85 (dd, Ar H, J=6.6, 3.2 Hz, 2H), 10.13 (brs, 2H, -NH-).
LRMS-ESI (m/z): 521 [M–H]–. HRMS-ESI (m/z): [M–H]– calcd for C28H29N2O4S2, 521.1569;
found, 521.1560 (–0.9 mmu).
1-10 1,4-bis-[(4-isopropylbenzenesulfonyl)amino]naphthalene ( )
2l
The pink solid (1.65 g, quantitative yield) was obtained. 1H NMR (DMSO-d6, 500 MHz, ) 1.15
(d, -OCH(CH3)2, J=6.8 Hz, 12H), 2.90 (sept, -OCH(CH3)2, J=6.8 Hz, 2H), 7.09 (s, Ar H, 2H),
7.29 (dd, Ar H, J=6.6, 3.2 Hz, 2H), 7.32 (d, Ar H, J=8.3 Hz, 4H), 7.53 (d, Ar H, J=8.3 Hz, 4H),
7.81 (dd, Ar H, J=6.6, 3.2 Hz, 2H), 10.13 (brs, 2H, -NH-). LRMS-ESI (m/z): 521 [M–H]–. HRMS-
ESI (m/z): [M–H]– calcd for C28H29N2O4S2, 521.1569; found, 521.1557 (–1.2 mmu).
1-11 1,4-bis-[(4-chlorobenzenesulfonyl)amino]naphthalene (
2m
)
The pink solid (1.77 g, 96% yield) was obtained. 1H NMR (DMSO-d6, 500 MHz, ) 7.04 (s, Ar
H, 2H), 7.41 (dd, Ar H, J=6.6, 3.2 Hz, 2H), 7.56 (dd, Ar H, J=6.8, 2.2 Hz, 4H), 7.62 (dd, Ar H,
J=6.8, 2.2 Hz, 4H), 7.92 (dd, Ar H, J=6.6, 3.2 Hz, 2H), 10.33 (brs, 2H, -NH-). LRMS-ESI (m/
z): 504, 506 [M–H]–. HRMS-ESI (m/z): [M–H]– calcd for C22H1535Cl2N2O4S2, 504.9850; found,
504.9836 (–1.4 mmu).

2 Preparation of 2-acetonyl-1,4-bis-[(benzenesulfonyl)amino]naphthalenes (
2-1 General procedure
and
5h-5m)
5c-5f
(1) To a suspension of the obtained product (2c-2f and 2h-2m, 600 mg) in methanol (30 mL),
cerium(IV) ammonium nitrite (2.2 equiv.) was added, and the reaction mixture was stirred at room
temperature for 30 min. Then, the precipitates were collected by filtration and dried in vacuo. (2)
To a suspension of the obtained product in toluene (30 mL), para-methoxybenzyl acetoacetate
(1.1 equiv.) and triethyamine (5.0 equiv.) were added. The reaction mixture was stirred at room
temperature for 30 min. Then, the reaction was quenched with 2 M hydrochloric acid and was
extracted twice with ethyl acetate. The combined organic layer was washed twice with water and
twice with brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure.
(3) The obtained compound was dissolved in trifluoroacetic acid and stirred at room temperature
for 30 min. Then, the reaction mixture was poured into water (ca. 200 mL), and the precipitates
were collected by filtration and dried in vacuo. The crude product in the residue was purified with
MPLC (n-hexane/ethyl acetate gradient).
2-2 2-Acetonyl-1,4-bis-[(4-n-propoxybenzenesulfonyl)amino]naphthalene ( )
5c
The yellow solid (162 mg, 32% yield over 3 steps) was obtained. Recrystallization from n-hexane/
ethyl acetate gave colorless needles. 1H NMR (DMSO-d6, 600 MHz, ) 0.94 (t, J=7.1 Hz, -
OCH2CH2CH3, 3H), 0.96 (t, J=7.1 Hz, -OCH2CH2CH3, 3H), 1.66-1.74 (m, -OCH2CH2CH3 x2,
4H), 2.02 (s, -COCH3, 3H), 3.79 (brs, -CH2CO-, 2H), 3.93-3.96 (m, -OCH2CH2CH3 x2, 4H), 6.92
(d, J=8.8 Hz, Ar H, 2H), 6.97 (d, J=8.8 Hz, Ar H, 2H), 7.06 (s, Ar H, 1H), 7.15 (dd, J=8.2, 7.2
Hz, Ar H, 1H), 7.29 (dd, J=8.2, 7.2 Hz, Ar H, 1H), 7.39 (d, J=8.8 Hz, Ar H, 1H), 7.50 (d, J=8.3
Hz, Ar H, 1H), 7.91 (d, J=8.3 Hz, Ar H, 1H), 9.74 (brs, -NH-, 1H), 10.09 (brs, -NH-, 1H). 13C
NMR (DMSO-d6, 150 MHz, ) 10.21, 21.72, 21.75, 29.66, 46.01, 69.40, 69.42, 114.54, 114.66,
122.92, 124.01, 125.37, 125.61, 125.69, 128.55, 128.73, 128.83, 128.91, 131.22, 131.77, 131.98,
132.65, 161.75, 161.85, 204.74. LRMS-ESI (m/z): 609 [M–H]–. HRMS-ESI (m/z): [M–H]– calcd
for C31H33N2O7S2, 609.1729; found, 609.1742 (+1.2 mmu).
2-3 2-Acetonyl-1,4-bis-[(4-isopropoxybenzenesulfonyl)amino]naphthalene ( )
5d
The yellow solid (254 mg, 43% yield over 3 steps) was obtained. Recrystallization from n-hexane/
ethyl acetate gave colorless needles. 1H NMR (DMSO-d6, 600 MHz, ) 1.22-1.25 (m, -(CH3)2
x2, 12H), 3.80 (brs, -CH2CO-, 2H), 4.65-4.66 (m, -CH- x2, 2H), 6.89 (dd, J=8.8, 2.2 Hz, Ar H,
2H), 6.94 (dd, J=8.8, 2.2 Hz, Ar H, 2H), 7.06 (s, Ar H, 1H), 7.09-7.12 (m, Ar H, 1H), 7.24-7.27
(m, Ar H, 1H), 7.35 (dd, J=8.8, 2.2 Hz, Ar H, 2H), 7.47 (d, J= 8.8 Hz, Ar H, 1H), 7.55 (dd, J=8.8,
2.2 Hz, Ar H, 2H), 7.88 (d, J= 8.8 Hz, Ar H, 1H), 9.73 (brs, -NH-, 1H), 10.07 (brs, -NH-, 1H).
13C NMR (DMSO-d6, 150 MHz, ) 21.45, 21.48, 29.63, 46.15, 69.79, 69.82, 115.39, 115.56,
122.89, 123.95, 125.27, 125.59, 125.79, 128.60, 128.75, 128.86, 128.93, 130.92, 131.41, 131.94,
132.68, 160.64, 160.72, 204.72. LRMS-ESI (m/z): 609 [M–H]–. HRMS-ESI (m/z): [M–H]– calcd
for C31H33N2O7S2, 609.1729; found, 609.1736 (+0.7 mmu).

2-4 2-Acetonyl-1,4-bis-[(4-n-butoxybenzenesulfonyl)amino]naphthalene ( )
5e
The yellow solid (394 mg, 52% yield over 3 steps) was obtained. Recrystallization from n-
hexane/ethyl acetate gave colorless needles. 1H NMR (DMSO-d6, 600 MHz, ) 0.90 (t, J=7.1
Hz, -OCH2CH2CH2CH3, 3H), 0.93 (t, J=7.1 Hz, -OCH2CH2CH2CH3, 3H), 1.36-1.44 (m, -
OCH2CH2CH2CH3 x2, 4H), 1.64-1.70 (m, -OCH2CH2CH2CH3 x2, 4H), 2.01 (s, -COCH3, 3H),
3.78 (brs, -CH2CO-, 2H), 3.96-4.00 (m, -OCH2CH2CH2CH3, 4H), 6.92 (d, J=8.8 Hz Ar H, 2H),
6.96 (d, J=8.8 Hz Ar H, 2H), 7.05 (s, Ar H, 1H), 7.12-7.15 (m, Ar H, 1H), 7.26-7.29 (m, Ar H,
1H), 7.38 (d, J=8.8 Hz Ar H, 2H), 7.49 (d, J=8.3 Hz, Ar H, 1H), 7.57 (d, J=8.8 Hz Ar H, 2H),
7.91 (d, J=8.3 Hz, Ar H, 1H), 9.72 (brs, -NH-, 1H), 10.08 (brs, -NH-, 1H). 13C NMR (DMSO-d6,
150 MHz, ) 13.60, 13.61, 18.59, 29.64, 30.38, 30.43, 46.16, 67.67, 114.52, 114.65, 122.95,
123.99, 125.27, 125.64, 128.72, 128.83, 128.88, 131.79, 132.00, 132.66, 161.71, 161.86, 204.77.
LRMS-ESI (m/z): 637 [M–H]–. HRMS-ESI (m/z): [M–H]– calcd for C33H37N2O7S2, 637.2042;
found, 637.2031 (–1.1 mmu).
2-5 2-Acetonyl-1,4-bis-[(4-isobutoxybenzenesulfonyl)amino]naphthalene ( )
5f
The yellow solid (426 mg, 38% yield over 3 steps) was obtained. Recrystallization from n-hexane/
ethyl acetate gave colorless needles. 1H NMR (DMSO-d6, 600 MHz, ) 0.94 (d, J=6.6 Hz, -
OCH2CH(CH3)2, 6H), 0.96 (d, J=6.6 Hz, -OCH2CH(CH3)2, 6H), 1.95-2.03 (m, -OCH2CH(CH3)2,
2H), 2.01 (s, -COCH3, 3H), 3.76 (d, J=6.6 Hz, -OCH2CH(CH3)2, 2H), 3.76 (brs, -CH2CO-, 2H),
3.77 (d, J=6.6 Hz, -OCH2CH(CH3)2, 2H), 6.93 (d, J=8.8 Hz, Ar H, 2H), 6.98 (d, J=8.8 Hz, Ar H,
2H), 7.06 (s, Ar H, 1H), 7.14 (dd, J=8.2, 7.2 Hz, Ar H, 1H), 7.28 (dd, J=8.2, 7.2 Hz, Ar H, 1H),
7.38 (d, J=8.8 Hz, Ar H, 2H), 7.50 (d, J=8.2 Hz, Ar H, 1H), 7.58 (d, J=8.8 Hz, Ar H, 2H), 7.92
(d, J=8.2 Hz, Ar H, 1H), 9.74 (brs, -NH-, 1H), 10.09 (brs, -NH-, 1H). 13C NMR (DMSO-d6, 150
MHz, ) 18.86, 27.46, 27.51, 29.65, 46.14, 74.06, 74.10, 114.58, 114.71, 122.92, 124.00, 125.33,
125.50, 125.68, 128.49, 128.71, 128.81, 128.91, 131.27, 131.80, 132.00, 132.65, 161.83, 161.97,
204.72. LRMS-ESI (m/z): 637 [M–H]–. HRMS-ESI (m/z): [M–H]– calcd for C33H37N2O7S2,
637.2042; found, 637.2030 (–1.2 mmu).
2-6 2-Acetonyl-1,4-bis-[(benzenesulfonyl)amino]naphthalene (
)
5h
The yellow solid (255 mg, 38% yield over 3 steps) was obtained. Recrystallization from n-hexane/
ethyl acetate gave colorless needles. 1H NMR (DMSO-d6, 600 MHz, ) 2.01 (s, -COCH3, 3H),
3.78 (brs, -CH2CO-, 2H), 7.08 (s, Ar H, 1H), 7.11 (dd, J=8.2, 7.2 Hz, Ar H, 1H), 7.26 (dd, J=8.2,
7.2 Hz, Ar H, 1H), 7.44-7.60 (m, Ar H, 9H), 7.68 (d, J=7.7 Hz, 2H), 7.87 (d, J=8.8 Hz, Ar H, 1H),
9.95 (brs, -NH-, 1H), 10.29 (brs, -NH-, 1H). 13C NMR (DMSO-d6, 150 MHz, ) 29.71, 46.08,
122.85, 123.87, 125.51, 125.82, 126.04, 126.54, 126.67, 128.61, 128.87, 129.06, 129.18, 131.51,
131.96, 132.76, 132.78, 139.76, 140.29, 204.63. LRMS-ESI (m/z): 493 [M–H]–. HRMS-ESI (m/z):
[M–H]– calcd for C25H21N2O5S2, 493.0892; found, 493.0913 (+2.1 mmu).
2-7 2-Acetonyl-1,4-bis-[(4-methylbenzenesulfonyl)amino]naphthalene ( )
5i
The yellow solid (306 mg, 43% yield over 3 steps) was obtained. Recrystallization from n-hexane/
ethyl acetate gave colorless needles. 1H NMR (DMSO-d6, 600 MHz, ) 1.99 (s, -COCH3, 3H),

2.31 (s, -CH3, 3H), 2.30 (s, -CH3, 3H), 3.74 (brs, -CH2CO-. 2H), 7.05 (s, Ar H, 1H), 7.13-7.16
(m, Ar H, 1H), 7.24-7.31 (m, Ar H, 5H), 7.39 (d, J=8.3 Hz, Ar H, 2H), 7.50 (d, J=8.8 Hz, Ar H,
1H), 7.56 (d, J=8.3 Hz, Ar H, 2H), 7.91 (d, J=8.8 Hz, Ar H, 1H), 9.82 (brs, -NH-, 1H), 10.19
(brs, -NH-, 1H). 13C NMR (DMSO-d6, 150 MHz, ) 20.93, 29.05, 29.62, 46.08, 122.89, 124.01,
125.45, 125.77, 126.59, 126.73, 128.47, 128.78, 129.45, 129.52, 131.62, 132.05, 132.65, 137.03,
137.58, 142.98, 143.04, 204.68. LRMS-ESI (m/z): 521 [M–H]–. HRMS-ESI (m/z): [M–H]– calcd
for C27H25N2O5S2, 521.1205; found, 521.1182 (–2.3 mmu).
2-8 2-Acetonyl-1,4-bis-[(ethylbenzenesulfonyl)amino]naphthalene ( )
5j
The yellow solid (466 mg, 70% yield over 3 steps) was obtained. Recrystallization from n-hexane/
ethyl acetate gave colorless needles. 1H NMR (DMSO-d6, 600 MHz, ) 1.11-1.15 (m, -CH2CH3
x2, 6H), 1.98 (s, -COCH3, 3H), 2.59-2.64 (m, -CH2CH3 x2, 2H), 3.75 (brs, -CH2CO-, 2H), 7.04
(s, Ar H, 1H), 7.09 (dd, J=7.7, 7.2 Hz, Ar H, 1H), 7.23-7.27 (m, Ar H, 3H), 7.30 (d, J=8.3 Hz,
Ar H, 2H), 7.40 (d, J=8.3 Hz, Ar H, 2H), 7.43 (d, J=8.2 Hz, Ar H, 1H), 7.57 (d, J=8.3 Hz, Ar
H, 2H), 7.85 (d, J=8.2 Hz, Ar H, 1H), 9.82 (brs. -NH-, 1H), 10.17 (brs, -NH-, 1H). 13C NMR
(DMSO-d6, 150 MHz, ) 15.14, 15.49, 27.95, 28.03, 29.63, 46.09, 122.87, 123.88, 125.35, 125.66,
125.76, 126.68, 126.83, 128.33, 128.48, 128.58, 128.87, 131.66, 131.99, 132.70, 137.12, 137.59,
149.07, 149.28, 204.64. LRMS-ESI (m/z): 549 [M–H]–. HRMS-ESI (m/z): [M–H]– calcd for
C29H29N2O5S2, 549.1518; found, 549.1530 (+1.2 mmu).
2-9 2-Acetonyl-1,4-bis-[(4-n-propylbenzenesulfonyl)amino]naphthalene ( )
5k
The light-yellow solid (303 mg, 46% yield over 3 steps) was obtained. Recrystallization from n-
hexane/ethyl acetate gave colorless needles. 1H NMR (DMSO-d6, 600 MHz, ) 0.82 (t, J=7.7 Hz,
-CH2CH2CH3, 3H), 0.85 (t, J=7.7 Hz, -CH2CH2CH3, 3H), 1.50-1.58 (m, -CH2CH2CH3 x2, 4H),
2.56 (t, J=7.7 Hz, -CH2CH2CH3, 2H), 2.57 (t, J=7.7 Hz, -CH2CH2CH3, 2H), 3.74 (brs, -CH2CO-,
2H), 7.02 (s, Ar H, 1H), 7.05-7.08 (m, Ar H, 1H), 7.20-7.23 (m, Ar H, 1H), 7.24 (d, J=8.3 Hz,
Ar H, 2H), 7.27 (d, J=8.3 Hz, Ar H, 2H), 7.39 (d, J=8.3 Hz, Ar H, 2H), 7.42 (d, J=8.8 Hz, Ar H,
1H), 7.55 (d, J=8.3 Hz, Ar H, 2H), 7.84 (d, J=8.8 Hz, Ar H, 1H), 9.82 (brs, -NH-, 1H), 10.16 (brs.
-NH-, 1H). 13C NMR (DMSO-d6, 150 MHz, ) 13.36, 13.37, 23.64, 23.83, 29.62, 36.82, 36.87,
46.15, 122.89, 123.83, 125.24, 125.60, 126.59, 126.69, 128.85, 128.94, 129.01, 131.96, 132.71,
137.70, 147.29, 147.51, 204.69. LRMS-ESI (m/z): 577 [M–H]–. HRMS-ESI (m/z): [M–H]– calcd
for C31H33N2O5S2, 577.1831; found, 577.1803 (–2.8 mmu).
2-10 2-Acetonyl-1,4-bis-[(4-isopropylbenzenesulfonyl)amino]naphthalene ( )
5l
The yellow solid (152 mg, 23% yield over 3 steps) was obtained. Recrystallization from n-
hexane/ethyl acetate gave colorless needles. 1H NMR (DMSO-d6, 600 MHz, ) 1.15 (d, J=7.1
Hz, -CH(CH3)2, 6H), 1.17 (d, J=7.1 Hz, -CH(CH3)2, 6H), 1.98 (s, -COCH3, 3H) 2.88-2.96 (m, -
CH(CH3)2, 2H), 3.74 (brs, -CH2CO-, 2H), 7.01 (s, Ar H, 1H), 7.05 (dd, J= 8.2, 7.7 Hz, Ar H, 1H),
7.21 (dd, J= 8.2, 7.7 Hz, Ar H, 1H), 7.29 (d, J=8.2 Hz, Ar H, 2H), 7.33 (d, J=8.2 Hz, Ar H, 2H),
7.37 (d, J=8.8 Hz, Ar H, 1H), 7.40 (d, J=8.2 Hz, Ar H, 2H), 7.56 (d, J=8.2 Hz, Ar H, 2H), 7.80
(d, J=8.8 Hz, Ar H, 1H), 9.82 (brs, -NH-, 1H), 10.14 (brs, -NH-, 1H). 13C NMR (DMSO-d6, 150

MHz, ) 23.42, 23.54, 29.61, 33.33, 33.40, 46.12, 122.86, 123.79, 125.23, 125.55, 125.88, 126.69,
126.83, 126.91, 127.03, 128.95, 131.95, 132.74, 137.21, 137.63, 153.56, 153.81, 204.62. LRMS-
ESI (m/z): 577 [M–H]–. HRMS-ESI (m/z): [M–H]– calcd for C31H33N2O5S2, 577.1831; found,
577.1824 (–0.7 mmu).
2-11 2-Acetonyl-1,4-bis-[(4-chlorobenzenesulfonyl)amino]naphthalene (
5m
)
The yellow solid (372 mg, 67% yield over 3 steps) was obtained. Recrystallization from n-hexane/
ethyl acetate gave colorless needles. 1H NMR (DMSO-d6, 600 MHz, ) 2.05 (s. -COCH3, 3H),
3.85 (brs, -CH2CO-, 2H), 7.09 (s, Ar H, 1H), 7.17 (dd, J=8.2, 7.2 Hz, Ar H, 1H), 7.31 (dd, J=8.2,
7.2 Hz, Ar H, 1H), 7.45 (d, J=8.8 Hz, Ar H, 1H), 7.48-7.52 (m, Ar H, 4H), 7.56 (d, J=8.8 Hz, Ar
H, 2H), 7.66 (d, J=8.8 Hz, Ar H, 2H), 7.87 (d, J=8.8 Hz, Ar H, 1H), 10.09 (brs, -NH-, 1H), 10.39
(brs. -NH-, 1H). 13C NMR (DMSO-d6, 150 MHz, ) 29.75, 46.09, 122.92, 123.74, 125.69, 125,98,
126.41, 128.50, 128.57, 128.67, 128.94, 129.25, 129.28, 131.34, 131.78, 132.96, 137.66, 137.70,
138.58, 139.97, 204.58. LRMS-ESI (m/z): 561, 563 [M–H]–. HRMS-ESI (m/z): [M–H]– calcd for
C25H1935Cl2N2O5S2, 561.0112; found, 561.0145 (+3.2 mmu).
3Synthesis of 2-acetonyl-1,4-bis-[4-(hydroxybenzenesulfonyl)amino]naphthalene (
5g)
3-1 Synthesis of 1,4-bis-[4-(benzyloxy)benzenesulfonyl)amino]-naphthalene (
2g)
To a solution of 1 (520 mg, 3.29 mmol) in dichloromethane (10 mL), 4-
(benzyloxy)benzenesulfonylcholide (1.91 g, 6.77 mmol, 1.0 mol equiv.) and pyridine (2 mL) were
added. The reaction mixture was stirred at room temperature for 2 hr and then acidified with 2 M
hydrochloric acid, and the precipitates were collected by filtration, washed with dichloromethane,
and dried in vacuo. A colorless solid (1.97 g, 92% yield) was obtained. 1H NMR (CDCl3, 500
MHz, ) 5.04 (s, -CH2- x2, 4H), 6.62 (s, Ar H, 2H), 6.89 (d, J=8.6 Hz, Ar H, 4H), 7.20 (s, Ar H,
2H), 7.41 (s, Ar H,2H), 7.36-7.44 (m, Ar H,10H), 7.61 (d, J=8.6 Hz,Ar H, 2H), 7.80-7.82 (m,
Ar H, 4H,). LRMS-ESI (m/z): 650 [M–H]–. HRMS-ESI (m/z): [M–H]– calcd for C36H30N2O6S2,
650.1545; found, 650.1528 (–1.7 mmu).
3-2 Preparation of 2-acetonyl1,4-bis-[4-(hydroxybenzenesulfonyl)amino]-naphthalene (
5g)
(1) To a suspension of the 2g (1.97 g, 3.03 mmol) in methanol (50 mL), cerium(IV) ammonium
nitrite (3.32 g, 6.05 mmol, 2.0 mol equiv.) was added and the reaction mixture was stirred at room
temperature for 30 min. Then, the precipitates were collected by filtration and dried in vacuo. (2)
To a suspension of the obtained product in toluene (40 mL), benzyl acetoacetate (1.0 equiv.) and
triethylamine (5.0 equiv.) were added. The reaction mixture was stirred at room temperature for
30 min. Then, the reaction was quenched with 2 M hydrochloric acid and was extracted twice
with toluene. The combined organic layer was washed twice with water and twice with brine,
dried over anhydrous sodium sulfate, and concentrated under reduced pressure. (3) The obtained
compound was dissolved in ethanol, and 5% palladium on charcoal was added to the solution.
The reaction mixture was stirred under a hydrogen atmosphere at room temperature for 14 hr. The
catalyst was removed by filtration through a Celite® pad, and the solvent was concentrated under
reduced pressure. A brown solid (1.26 g, 71% yield over 3 steps) was obtained. Recrystallization

from ethyl acetate gave colorless needles. 1H NMR (DMSO-d6, 600 MHz, ) 2.01 (s, -COCH3,
3H), 3.77 (brs, -CH2CO-, 2H), 6.75-6.77 (m, Ar H, 4H), 7.06 (s, Ar H, 1H), 7.15-7.18 (m, Ar H,
1H), 7.28-7.31 (m, Ar H, 3H), 7.48 (dd, J= 7.2, 2.2Hz, Ar H, 2H), 7.54 (d, J=8.3 Hz, Ar H, 1H),
7.92 (d, J=8.3 Hz, Ar H, 1H), 9.64 (brs, -NH-, 1H), 10.00 (brs, -NH-, 1H), 10.38 (brs, 2H, -OH).
13C NMR (DMSO-d6, 150 MHz, ) 29.69, 46.17, 115.33, 115.45, 122.94, 124.17, 125.37, 125.45,
125.68, 128.62, 128.83, 128.92, 129.09, 129.73, 130.39, 131.86, 132.08, 132.62, 161.13, 161.23,
204.88. LRMS-ESI (m/z): 549 [M+Na]+. HRMS-ESI (m/z): [M+H]+ calcd for C25H22N2Na1O7S2,
631.1297; found, 631.1304 (+0.7 mmu).
4 Preparation of 2-acetonyl-1,4-bis-{[4-(acethylamino)benzenesulfonyl]amino}naphthalene
(
5n)
4-1 Preparation of 1,4-bis-{[4-(acethylamino)benzenesulfonyl)amino]naphthalene (
2n)
To a solution of 1 (422 mg, 2.67 mmol) in dichloromethane (20 mL), 4-
acetoaminobenzenesulfonyl chloride (1.40 g, 5.98 mmol, 1.1 mol equiv.) and pyridine (1 mL)
were added. The reaction mixture was stirred at room temperature for 3 hr, and then acidified with
2 M hydrochloric acid and extracted twice with ethyl acetate. The combined organic layer was
washed twice with brine, dried over anhydrous sodium sulfate and concentrated under reduced
pressure. The residues were purified by silica-gel column chromatography yielding a light-purple
solid (513 mg, 35% yield). 1H NMR (DMSO-d6, 500 MHz, ) 2.06 (s, -CH3 x2, 6H), 6.97 (s, Ar
H, 2H), 7.38 (dd, J=6.5, 3.0 Hz, Ar H, 2H), 7.54 (d, J=8.8 Hz, Ar H, 4H), 7.67 (d, J=8.8 Hz, Ar
H, 4H), 7.98 (dd, J=6.5, 3.0 Hz, Ar H, 2H), 10.05 (brs, 2H, -NH-), 10.23 (brs, 2H, -NH-). LRMS-
ESI (m/z): 551 [M–H]–. HRMS-ESI (m/z): [M–H]– calcd for C26H23N4O6S2, 551.1059; found,
551.1065 (+0.6 mmu).
4-2 Preparation of 2-acetonyl-1,4-bis-{[4-(acethylamino)benzenesulfonyl]amino}naphthalene
(
5n)
(1) To a suspension of 2n (331 mg, 0.599 mmol) in methanol (50 mL), cerium(IV) ammonium
nitrite (1.2 equiv.) was added and the reaction mixture was stirred at room temperature for 30
min. Then, the precipitates were collected by filtration and dried in vacuo. (2) To a suspension
of the obtained product in toluene (30 mL), para-methoxybenzyl acetoacetate (1.1 equiv.) and
triethylamine (5.0 equiv.) were added. The reaction mixture was stirred at room temperature for
30 min. Then, the reaction was quenched with 2 M hydrochloric acid and was extracted twice
with ethyl acetate. The combined organic layer was washed twice with water and twice with
brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. (3) The
obtained compound was dissolved in trifluoroacetic acid and stirred at room temperature for 30
min. Then, the reaction mixture was poured into water (ca. 200 mL), and the precipitates were
collected by filtration and dried in vacuo. Purification of the crude product with MPLC (n-hexane/
ethyl acetate gradient) produced an off-white solid (64 mg, 18% yield). Recrystallization from
ethyl acetate gave colorless plates. 1H NMR (DMSO-d6, 600 MHz, ) 2.00 (s, -COCH3, 3H), 2.05
(s, -COCH3, 3H), 2.08 (s, -COCH3, 3H), 3.76 (brs, -CH2CO-, 2H), 7.07 (s, Ar H, 1H), 7.17 (dd,
J=7.7, 7.1 Hz, Ar H, 1H), 7.30 (dd, J=7.7, 7.1 Hz, Ar H, 1H), 7.45 (d, J=8.8 Hz, Ar H, 2H), 7.55
(d, J=8.2 Hz, Ar H, 1H), 7.60 (d, J=8.8 Hz, 2H), 7.63-7.65 (m, Ar H, 4H), 7.92 (d, J=8.2 Hz, Ar

H, 1H), 9.80 (brs, -NHSO2-, 1H), 10.15 (brs, -NHSO2-, 1H), 10.25 (s, -NHCO-, 1H), 10.28 (s,
-NHCO-, 1H). 13C NMR (DMSO-d6, 150 MHz, ) 24.14, 24.17, 29.69, 46.12, 118.32, 118.48,
122.94, 124.13, 125.53, 125.83, 127.79, 127.91, 128.60, 128.84, 131.71, 132.09, 132.69, 133.04,
134.11,143.04, 169.00, 204.78. LRMS-ESI (m/z): 631 [M+Na]+. HRMS-ESI (m/z): [M+H]+ calcd
for C29H28N4Na1O7S2, 631.1297; found, 631.1304 (+0.7 mmu).
Determination of the PPI inhibitory activities
FAM-labeled Nrf2 peptide (FAM-LDEETGEFL-NH2) and FAM-labeled p-p62 peptide (FAM-
VDP-pS-TGELQ-NH2) were purchased from was purchased from Toray Research Center (Tokyo,
Japan). GST-His-Keap1-DC (amino acids 321–609) were expressed in Escherichia coli and
purified by chromatography on glutathione–Sepharose 4B resin (GE Healthcare, USA). GST-
His-Keap1-DC was cleaved to His-Keap1-DC with PreScission Protease (GE Healthcare, USA)
before use in this assay. Fluorescence polarization assays was performed in 384-well nonbinding
black plates (784900 Greiner Bio-One, Austria). Five microliters of 20 nM peptide solution and 5
L of 500 nM protein solution were diluted with fluorescence polarization (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 where 100 nL of 1 mM tested compound solution in dimethyl sulfoxide
was transferred in advance by an Echo 550 liquid handler (Labcyte, USA). Subsequently, the
plates were incubated for 30 min at room temperature. The FP signal was measured at 520 nm
with excitation at 485 nm by a PHERAstar plate reader (BMG Labtech, Germany).
Cell culture
Both the Huh-1 and Huh-7 cell lines were purchased from the JCRB cell bank (Japan) and
cultured in DMEM (4.5 g/mL glucose) supplemented with 10% fetal bovine serum (Life
Technologies, USA) and 1% penicillin-streptomycin (Sigma, USA) at 37°C, 5% CO2 and a
humidified atmosphere.
Cell viability assay
Both Huh-1 and Huh-7 cells were seeded on 96-well flat bottom plates (AGC Techno Glass,
Japan) at a concentration of 5,000 cell/well (total medium volume was 200 L/well). After 24
hr incubation until adhesion, drug samples diluted in DMSO (1 L) were added (n=3), and the
cells were incubated for 72 hr. Then, sorafenib or regorafenib diluted in DMSO (1 L) were added
to the cells, and the cells were incubated for another 72 hr. Next, Cell Counting Kit-8 (Dojindo,
Japan) solution (10 L) was added to each well and incubated for 4 hr, and the absorbance at 450
nm (reference: 600 nm) was measured to calculate the cell viability.
Results
Compound design and synthesis
As mentioned above, the acetonyl group on 2-position of K67 seemed to be essential for selective
inhibition of the Keap1-p-p62 PPI [17]. However, the effect of the alkoxy group on the 4-position

of the two benzenesulfonyl groups of K67 on the PPI inhibition has been rarely investigated.
Therefore, we designed new K67 derivatives that have various lengths of alkoxy or alkyl groups at
this position. Additionally, hydroxy, chloro and acetamide derivatives were also designed.
The new K67 derivatives were synthesized from commercially available 4-nitro-1-naphthylamine
via 5 steps according to our previous method with a slight modification (Scheme 1) [18].
Benzenesulfonyl chlorides were purchased or synthesized from the corresponding substituted
benzenes according to the method reported in the literature [18]. para-Methoxybenzyl
acetoacetate was prepared from methyl acetoacetate and para-methoxybenzylalcohol [19].
Compound 1 was prepared from 4-nitro-1-naphthylamine and was condensed with the
corresponding benzenesulfonyl chlorides (2a-n) followed by oxidation using ammonium
cerium(IV) nitrite to give compounds 3a-n. The diimine-type compounds 3a-f and 3h-n were
added to the para-methoxybenzyl acetoacetate to obtain 4a-f and 4h-n, and then deprotection of
the para-methoxybenzyl group with trifluoroacetic acid yielded compounds 5a-f and 5h-n with
moderate overall yields (Scheme 1). In the case of 5g, benzyl acetoacetate was added instead of
para-methoxybenzyl acetoacetate to the diimine compound 3g, and then, three benzyl groups were
deprotected by hydrogeneration (Scheme 2). The synthetic compounds used for biological assays
were recrystallized from suitable solvents.
Inhibitory activity against protein-protein interactions between Keap1 and p-p62 or Nrf2
The PPI inhibitory activities of the K67 derivatives were evaluated using a FP assay according to
our previous method[17]. Compounds 5c, 5e, 5f, 5j, 5k and 5l had ClogP values over 4.5 (Table)
and were too insoluble to measure their precise IC50 values. Other derivatives, except 5h, showed
IC50 values in the low M range against both of the PPIs (Table). There was not much difference
among the derivatives in terms of their PPI inhibitory activities (Keap1-p-p62/Keap1-Nrf2,
the range was >1.3~2.2). Compound 5h, which possesses nonsubstituted benzenesulfonamide,
demonstrated the lowest inhibitory activity among the tested compounds.
Effect of the K67 derivatives on the sensitivity to anticancer agents in human hepatocellular
carcinoma cell lines
In our previous study, K67 increased the sensitivity to sorafenib in Huh-1 cells that accumulate
high levels of p-p62 [16]. Therefore, the effect of the newly synthesized K67 derivatives on the
sensitivity to sorafenib was examined using Huh-1. For the purpose of comparison, the effect
on Huh-7 cells in which p62 is not overexpressed was also evaluated. According to the previous
method [16], after pre-incubation with the K67 derivatives (10 M) for 72 hr, sorafenib (2.5 M)
was added to the medium, and the cells were incubated for another 72 hr. Then, cell viability was
determined using a WST-8 assay.
Sorafenib (2.5 M) decreased cell viability in both Huh-7 and Huh-1 cells compared to the
control, but Huh-1 cells were more resistant to sorafenib than Huh-7 cells (Figure 2A). Co-
incubation with the derivatives, except 5c and 5f, and sorafenib had no effect on viability of
the Huh-7 cells. Under this condition, the propoxy derivative 5c and the isopropoxy derivative
5d decreased cell viability to approximately 40% in Huh-1 cells, whereas K67 (5b) and the
methoxy derivative 5a did not affect the sensitivity to sorafenib in this case (Figure 2A). It should

be noted that compounds 5a-5d showed no toxicity against both Huh-7 and Huh-1 cells when
these compounds were added without sorafenib (Figure 3). Both the butoxy derivative 5e and
the isobutoxy derivative 5f decreased the viability of Huh-1 cells by co-treatment with sorafenib
(Figure. 2A); however, these derivatives decreased viability in both Huh-7 and Huh-1 cells
even in the absence of sorafenib (Figure 3). The hydroxy derivative 5g and the nonsubstituted
derivative 5h did not increase sorafenib sensitivity against Huh-1 cells (Figure. 2A). For the
alkyl derivatives, compounds 5j, 5k and 5l demonstrated significant effects, although the methyl
derivative 5i did not affect the sensitivity to sorafenib (Figure 2A). The chloro derivative 5m
and the acetamide derivative 5n also increased the sensitivity to sorafenib (Figure 2A). Single
administration of compounds 5g-5n to both of the cell lines did not suppress cell viability (Figure
3).
Next, compound 5d, which showed the most potent effect for overcoming sorafenib resistance
in Huh-1 cells without any influence on Huh-7 cells, was evaluated for dose-dependency and
compared with the original compound K67 (5b). K67 showed a significant effect against Huh-1
cells at over 50 M, but the effect was weak (Figure 2B). On the other hand, 5d tended to suppress
Huh-1 cell proliferation at 1 M and showed a significant effect at over 5 M (Figure 2C). No
synergistic effect was observed when Huh-7 cells were exposed to K67 or 5d in combination with
sorafenib (Figure 2B and C).
Moreover, we examined the effect of the K67 derivatives (10 M) on the sensitivity to
regorafenib, an alternative multikinase inhibitor, of Huh-1 cells (Figure 4). Compounds 5c, 5d, 5e
and 5f significantly decreased viability of Huh-1 cells by co-treatment with regorafenib, similar
to sorafenib, while other compounds, including K67 (5b), did not show any effect. Considering
that 5e and 5f decreased cell viability in both Huh-7 and Huh-1 cells without regorafenib (Figure
3), it was suggested that only compounds 5c and 5d can overcome the resistance to regorafenib in
Huh-1 cells.
Discussion
Overcoming chemoresistance is one of the key aspects in future cancer therapy not only from a
perspective of improving prognosis but also from a perspective of reducing medical expenses
[20]. In a previous study, we found K67 (5b) as the first-in-class compound that inhibits the PPI
between p-p62 and Keap1 and increases the sorafenib-sensitivity of Huh-1 cells overexpressing p-
p62 [16]. Therefore, the development of more effective anti-chemoresistance agents based on the
structure of K67 is of great significance.
Although we had previously established the synthetic route for K67 in our previous report[17],
a modification was made in the present study to efficiently prepare derivatives that have various
substituents at the 4-position of the two benzensulfonamide groups. In particular, instead of benzyl
acetoacetate, para-methoxybenzyl acetoacetate was added to the diimines, and then the para-
methoxybenzyl group was deprotected with trifluoroacetic acid.
The Huh-1 cell line intrinsically accumulates phospho-S349 p62 [16]. Therefore, p-p62 competes
with Nrf2 in binding to the Keap1 DC-domain and activates Nrf2 in Huh-1 cells, which induces
cell progression and gain of chemoresistance. K67 (5b) increased the sensitivity to sorafenib in
Huh-1 at 50 M (Figure 2B) in agreement with our previous work[16]; however, 10 M of K67
failed to decrease the Huh-1 cell viability under the conditions used in the present study (Figure

2A and 2B). On the other hand,the alkoxy derivatives 5c, 5d, 5e and 5f (10 M) significantly
increased the sensitivity of Huh-1 cells to sorafenib. These results suggest that elongation of
the alkyl chain of the alkoxy groups could increase the sensitivity to sorafenib. The methoxy
derivative 5a had no effect, supporting the importance of alkyl chain length. However, 5e and 5f
decreased the viability of both Huh-7 and Huh-1 cells in the absence of sorafenib, which means
that the observed synergistic effects of sorafenib in combinationwith 5e or 5f in the Huh-1 cells
were due to the toxicity of the compounds themselves and not due to increasing the sensitivity to
sorafenib.
As for other derivatives, including the alkyl derivatives, compounds 5j, 5k and 5l showed
significant effects, while the methyl derivative 5i did not affect the sensitivity to sorafenib. The
chloro derivative 5m and the acetamide derivative 5n also increased the sensitivity to sorafenib.
Thus, the 4-position of the benzenesulfonyl rings seemed to be a very important moiety to increase
the sensitivity to sorafenib in Huh-1 cells.
The results of the current study showed that the selectivity between Keap1-p-p62 and the
Keap-Nrf2 PPI inhibitory activities of the K67 derivatives had no correlation with the effect of
increasing the sorafenib sensitivity in cellular assays. The comparison of the IC50 values evaluated
by a simple FP assay using peptide fragments is probably inadequate to estimate the chemo-
sensitizing effect in a cell-based assay that involves interactions at multiple levels. Although K67
(50 M) clearly decreased the mRNA level of Nrf2-target proteins in our previous study [16], it is
also possible that there is a difference between K67 (5b) and 5c-f in regard to the mechanism of
increasing sensitivity to sorafenib in Huh-1 cells. Thus, further investigations are required in order
to clarify the mechanism of action of the new derivatives.
Compounds 5c, 5d, 5e and 5h also increased the sensitivity of Huh-1 cells to regorafenib, a
second-line treatment agent for sorafenib-refractory HCC, which suggests that regorafenib-
resistance in Huh-1 cells occurs via a similar mechanism to sorafenib-resistance, and patients
with HCC in which p62 is overexpressed cannot achieve remarkable outcomes from treatment
with regorafenib. Thus, the K67 derivatives newly synthesized in the present study may be novel
therapeutics for highly advanced HCC.
Sorafenib not only inhibits various kinases but also induces ferroptosis, a novel type of regulated
cell death[21]. Ferroptosis is characterized by iron-dependent lipid peroxidation-induced cell
death that is not inhibited by the apoptosis inhibitor ZVAD-FMK[22]. In a ferroptotic cell,
iron accumulation generates reactive oxygen species followed by lethal lipid peroxidation [23].
Sorafenib induces ferroptosis by the inhibition of cysteine/glutamate antiporters in the same way
as erastin, a known ferroptosis inducer [24]. The activation of Nrf2 is one of the factors involved
in ferroptosis-resistance, and indeed, Nrf2 suppression using shRNA enhanced the growth
inhibitory activity of sorafenib against HCC cell lines, and this phenomenon was attenuated by
ferrostatin-1, a selective ferroptosis inhibitor [25]. K67 derivatives may alter the response to
sorafenib in Huh-1 cells by Nrf2-inactivation via the inhibition of the p-p62-Keap1 PPI followed
by the induction of ferroptotic cell death. To the best of our knowledge, it has never been reported
that regorafenib induces ferroptosis, but considering the structural similarity between sorafenib
and regorafenib, a ferroptotic response might also be involved in the effect of K67 derivatives
on the sensitivity to regorafenib. Further investigation of the possible molecular mechanisms
underlying the anti-chemoresistance effect of K67 derivatives is needed.

In conclusion, we designed and synthesized novel K67 derivatives that possess p-p62-Keap1
PPI inhibitory activities and revealed their anti-chemoresistance effect in HCC. Among the
derivatives, 5d showed the strongest effect for a p62-overexpressing Huh-1 cell line without
showing any synergistic effect in Huh-7, suggesting that 5d might be a lead-compound for epoch-
making anticancer agents. Our prospects for the future are to develop a more drug-like compound
based on 5d.
Funding
This research was supported by the Platform Project for Supporting in Drug Discovery and Life
Science Research (Platform for Drug Discovery, Informatics, and Structural Life Science) from
the Japan Agency for Medical Research and Development (AMED) and JSPS KAKENHI Grant
Number 19K16322 and 18H02611.
Acknowledgement
We thank Taichi Iwai, Taketo Yoshida, Akihiro Yuasa and Mao Nakajima for their cooperation in
the experiments.

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Table The PPI inhibitory activities and ClogP values of the K67 derivatives
Compound
PPI inhibitory activity
Selectivity index ClogPb
(Nrf2/p-p62)
Keap1-p-
95% CI
Keap1-Nrf2
95% CI
p62 IC50 (M)
IC50 (M)
5a
5b (K67)
5c
1.4
1.2
>3
1.3-1.5
1.1-1.3
–
2.0-2.5
–
2.0
2.6
>3
1.9-2.2
2.1-3.3
1.4
2.2
N.D.
1.8
N.D.
N.D.
1.7
1.5
1.6
>1.3
N.D.
N.D.
2.1
2.9
3.6
4.6
4.3
5.4
5.4
2.4
3.2
4.2
5.0
5.8
5.7
4.3
1.0
–
5d
5e
5f
5g
5h
5i
5j
5k
2.2
3.9
3.2-4.7
N.D.a
N.D.
7.1
13
1.4
N.D.
N.D.
12
20
2.3
–
–
–
10.7-12.9
17.0-22.7
2.0-2.7
–
6.7-7.6
12.0-13.8
1.3-1.5
2.0-2.6
–
2.3
>3
>10
>10
4.3
>10
>10
8.9
–
–
5l
5m
5n
–
3.9-4.6
0.97-1.1
7.7-10.3
1.4-1.7
1.0
1.5
1.5
aNot detected. bCalculated by ChemDraw® Professional 15.1

Schemes and Figures
Scheme 1
Scheme 2
Figure 1

Figure 2

Figure 3
Figure 4

Scheme and Figure Captions
Scheme 1 Preparation of derivatives 5a-f and 5h-n (a) H2, Pd/C, DCM, rt, 24 hr, 89%, (b)
corresponding sulfonyl chloride, pyridine, DCM, rt, 2 hr, 80%-quant. (c) CAN, MeOH, rt, 30 min,
(d) p-methoxybenzyl acetoacetate, TEA, toluene, 2 hr, (e) TFA, rt, 30 min, 34~69% for 3 steps.
Scheme 2 Preparation of derivative 5g. Compound 3g was prepared by CAN oxidation of a
precursor compound synthesized from 1 and para-(benzyloxy)benzenesulfonyl chloride. (f)
Benzyl acetoacetate, TEA, toluene, rt, 30 min, (g) H2, Pd/C, DCM, rt, 24 hr, 38% yield for 2
steps.
Figure. 1 Structures of sorafenib, regorafenib, K67 and Cpd16
Figure 2 Effect of K67 derivatives on the sensitivity to anticancer agents. (A) Co-incubation with
K67 derivatives (10 M) and sorafenib (2.5 M). Huh-1 (resistant) or Huh-7 (nonresistant) cells
(5,000 cells/mL) were seeded onto 96-well microplates and precultured until adherent. After 24 hr,
test compounds were added and incubated for 72 hr. Then, sorafenib was added and incubated for
another 72 hr. Cell viability was determined using a WST-8 assay. (B, C) Dose-dependent effect
of K67 (B) and 5d (C) on the sensitivity to sorafenib of Huh-7 and Huh-1. Experiments were
performed in a similar manner to that described for Figure. 2A. #; P<0.05 vs control (Huh-7),
*P<0.05 vs control (Huh-1), **P<0.01 vs control (Huh-1). n=3.
Figure 3 Single treatment with K67 derivatives for Huh-1 and Huh-7. Huh-1 or Huh-7 cells
(5,000 cells/mL) were seeded onto 96-well microplates and pre-cultured until adherent. After 24
hr, test compounds were added and incubated for 144 hr. Then, cell viability was determined using
a WST-8 assay (n=3).
Figure 4 Co-incubation with K67 derivatives (10 M) and regorafenib (2.5 M). Experiments


were performed in a similar manner to that described for Figure. 2A. #; P<0.05 vs control
(Huh-7), *P<0.05 vs control (Huh-1), **P<0.01 vs control (Huh-1). n=3.
Table The PPI inhibitory activities and ClogP values of the K67 derivatives

Compound
PPI inhibitory activity
95% CI
Selectivity index ClogPb
(Nrf2/p-p62)
Keap1-p-
Keap1-Nrf2
95% CI
p62 IC50 (M)
IC50 (M)
5a
5b (K67)
5c
1.4
1.2
>3
1.3-1.5
1.1-1.3
–
2.0-2.5
–
2.0
2.6
>3
1.9-2.2
2.1-3.3
1.4
2.2
N.D.
1.8
N.D.
N.D.
1.7
1.5
1.6
>1.3
N.D.
N.D.
2.1
2.9
3.6
4.6
4.3
5.4
5.4
2.4
3.2
4.2
5.0
5.8
5.7
4.3
1.0
–
5d
5e
5f
5g
5h
5i
5j
5k
2.2
3.9
3.2-4.7
N.D.a
N.D.
7.1
13
1.4
N.D.
N.D.
12
20
2.3
–
–
–
10.7-12.9
17.0-22.7
2.0-2.7
–
6.7-7.6
12.0-13.8
1.3-1.5
2.0-2.6
–
2.3
>3
>10
>10
4.3
>10
>10
8.9
–
–
5l
5m
5n
–
3.9-4.6
0.97-1.1
7.7-10.3
1.4-1.7
1.0
1.5
1.5
aNot detected. bCalculated by ChemDraw® Professional 15.1
-24-