2-Substituted 3,7,8-trimethylnaphtho[1,2-b]furan-4,5-diones as specific L-shaped NQO1-mediated redox modulators for the treatment of non-small cell lung cancer

Article abstract of DOI:10.1016/j.ejmech.2017.06.028

Based on the scaffold of 3,7,8-trimethylnaphtho[1,2-b]furan-4,5-dione, a series of L-shaped derivatives with substituted side chains at the position of C2 were designed by analyzing the binding mode with NQO1. The drug-like compound 6q (named as DDO-7178) emerged as the most specific substrate of the two-electron oxidoreductase NQO1 and could hardly be reduced by one-electron oxidoreductases CPR (NQO1/CPR = 20.8). In addition, compound 6q showed much improved physicochemical properties such as water solubility than the control β-lap. The follow-up studies indicated that 6q showed a NQO1-expressing cancer-cell-selective killing property. Preliminary mechanism studies on the anticancer effect indicated that 6q induced ROS production in an NQO1 dependent manner and activated Akt/MAPK pathways in a ROS-dependent fashion, thereby inducing apoptosis. In addition, emphasized compound 6q showed more significant antitumor efficacy than β-lap without producing obvious toxic effects in vivo, which gave us a new tool for further investigation of NQO1-mediated redox modulators as anticancer drugs for the treatment of NQO1-overexpressed NSCLC.

Full text of DOI:10.1016/j.ejmech.2017.06.028

Accepted Manuscript
2-Substituted 3,7,8-trimethylnaphtho[1,2-b]furan-4,5-diones as specific L-shaped
NQO1-mediated redox modulators for the treatment of non-small cell lung cancer
Xiaojin Zhang, Jinlei Bian, Xiang Li, Xingsen Wu, Yanan Dong, Qidong You
PII:
S0223-5234(17)30474-9
10.1016/j.ejmech.2017.06.028
EJMECH 9523
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 23 April 2017
Revised Date: 10 June 2017
Accepted Date: 14 June 2017
Please cite this article as: X. Zhang, J. Bian, X. Li, X. Wu, Y. Dong, Q. You, 2-Substituted 3,7,8-
trimethylnaphtho[1,2-b]furan-4,5-diones as specific L-shaped NQO1-mediated redox modulators for the
treatment of non-small cell lung cancer, European Journal of Medicinal Chemistry (2017), doi: 10.1016/
j.ejmech.2017.06.028.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
2-Substituted 3,7,8-trimethylnaphtho[1,2-b]furan-4,5-diones as specific
L-shaped NQO1-mediated redox modulators for the treatment of
non-small cell lung cancer
Xiaojin Zhang a,c,1, Jinlei Bian a,b,1, Xiang Li a, Xingsen Wu a,c, Yanan Dong a, Qidong You a,b*
a
State Key Laboratory of Natural Medicines, Jiangsu Key Laboratory of Drug Design and
Optimization, China Pharmaceutical University, Nanjing 210009, China
b
Department of Medicinal Chemistry, School of Pharmacy, China Pharmaceutical University, Nanjing
210009, China
c
Department of Organic Chemistry, School of Science, China Pharmaceutical University, Nanjing
211198, China
1 These two authors contributed equally to this work.
*Corresponding author. E-mail: youqd@163.com (Q. You)
Abstract
Based on the scaffold of 3,7,8-trimethylnaphtho[1,2-b]furan-4,5-dione, a series of
L-shaped derivatives with substituted side chains at the position of C2 were designed
by analyzing the binding mode with NQO1. The drug-like compound 6q (named as
DDO-7178) emerged as the most specific substrate of the two-electron
oxidoreductase NQO1 and could hardly be reduced by one-electron oxidoreductases
CPR (NQO1/CPR = 20.8). In addition, compound 6q showed much improved
physicochemical properties such as water solubility than the control β-lap. The
follow-up studies indicated that 6q showed a NQO1-expressing cancer-cell-selective
killing property. Preliminary mechanism studies on the anticancer effect indicated that
6q induced ROS production in an NQO1 dependent manner and activated Akt/MAPK
pathways in a ROS-dependent fashion, thereby inducing apoptosis. In addition,
emphasized compound 6q showed more significant antitumor efficacy than β-lap
without producing obvious toxic effects in vivo, which gave us a new tool for further
investigation of NQO1-mediated redox modulators as anticancer drugs for the
treatment of NQO1-overexpressed NSCLC.
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1. Introduction
Non-small cell lung cancer (NSCLC) is one of the most common cancer types in
both male and female population worldwide [1-2]. Treatments for NSCLC include
surgery, radiation therapy. However, surgery is usually the treatments of choice for
early stage NSCLC, for patients with unrespectable disease, local control can be
achieved with radiation therapy combined with chemotherapy, molecular-targeted
drugs, or some combination [1-4]. As for current gene-targeting therapeutic strategies,
some challenges still existed owing to acquired drug resistance and the genomic
instability of cancer cells [5]. Therefore, new and safe drugs for effective treatment of
late stage and resistant NSCLC is an urgent need to develop. In recent years, there is
renewed attention paid to target the unique biochemical alterations in cancer cells that
might be a feasible approach to achieve therapeutic activity and selectivity, and
perhaps prevent the development of drug resistance [6]. Due to most cancer cells
exhibit increased oxidative stress, then it could be an important target in the
development of new anticancer strategies. Oxidative stress is developed by reactive
oxygen species (ROS) that accumulate as a result of an imbalance between ROS
generation and elimination [7]. Higher basal ROS level makes cancer cells more
susceptible to exogenously induced ROS-mediated cell death [8]. Excessive ROS
levels irreversibly damage DNA and lipids which would ultimately lead to apoptosis
of cancer cells [7]. Potentiating ROS in cancers employing small molecules has been
regarded as a rational drug design strategy [8-11]. Many drugs exert their anticancer
activity dependent on the high basal ROS levels uniquely present in cancer cells
[12-14]. Generally, they can be divided into two types: 1) ROS inducers; 2) agents
decreasing cellular oxidative-buffering capacity [6]. Combination of these two types
of ROS modulators is considered to be taken full advantage of oxidative stress to
exert efficient and selective antitumor activity. In addition to the combination
treatments, compounds known as ‘redox cyclers’ could exploit the dual effect to
enhance ROS levels in cancer cells through reacting with flavoprotein reductase such
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as NAD(P)H:quinine oxidoreductase (NQO1) in the presence of reduced NADH or
NADPH [6].
NQO1 is a cytosolic flavoenzyme that uses the reduced pyridine nucleotide
NADH or NADPH as cofactor to catalyze the direct two-electron reduction of a wide
variety of quiones [15-17]. In multiple tumor types with higher levels of ROS, NQO1
is constitutively overexpressed 5- to 100-fold greater than in correlative normal
tissues [18]. It has been proved that the NSCLC showed up to 100-fold
over-expression of NQO1 [19]. Recently, many efficient NQO1 substrates have been
reported to be potential antitumor drug through NQO1-mediated ROS production by
redox cycling [20-23]. In addition, as we previously reported, NQO1-targeting
ROS-generating agents should pay attention to the selectivity between NQO1/CPR
(NADPH-cytochrome P450 reductase) [24]. The CPR-mediated one-electron
reduction of compounds would induce potential hepatotoxicity. However, most of
currently reported NQO1 substrates, such as Phase II clinical drug β-lapachone
(β-lap), can also be catalyzed by CPR. Our research group has recently discovered
that compounds DDO-7134 (2) (Fig.1A) showed good selectivity towards NQO1
(selectivity ratio = 6.37), which is much better than β-lap (selectivity ratio = 1.36)
[24]. The potential therapeutic window of 2 seems superior to the control β-lap,
making this scaffold a potentially safer candidate for use as an anticancer drug.
However, compound 2 exhibited a less ideal profile as a NQO1-targeting antitumor
drug, with poor drug-like properties and moderate reduction rate catalyzed by NQO1,
which greatly limit its application for further preclinical study. And the selectivity
between NQO1/CPR need to be further improved to obtain drugs with superior safety
profile.
Our previous publication highlighted the identification of an L-shaped pocket for
NQO1 substrates occupation. The designed L-shaped ortho-quinone derivatives
resulted in better catalytic efficiency and specificity to NQO1 than the planner
structure [25]. Combining with the previous work and other group reported strategy
focus on the modification on quinone scaffold [26-28], in this work, we were
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promoted to introduce nitrogen-containing side chains attached aromatic rings at the
C2 position of 2 using different linkers to obtain L-shaped molecules that fitted well
into the NQO1-binding pocket and to develop novel and efficient NQO1-targeting
antitumor drug (Fig.1B). The designed L-shaped substrates are expected to have
higher specificity for NQO1 and better drug-like properties than the hit compound 2.
In addition, through improving the matching accuracy of substrates toward NQO1
pocket, compounds with better NQO1/CPR are also expected to be obtained by
introducing L-shaped chains.
Fig. 1. (A) Compounds DDO-7134 (2) with high NQO1/CPR selective ratio was discovered in our
previous reported work. (B) The L-shaped catalytic pocket of NQO1 (PDB ID: 2F1O). (C)
Designing of novel L-shaped ortho-quinone substrates for NQO1 based on the scaffold of 2.
2. Chemistry
The L-shaped ortho-naphthoquinones (6a-6q and 7a-7d) were synthesized from the
compound 2. The synthetic route for compound 2 was reported in previous work [24].
Briefly, treatment of 1,4-benzoquinone 2c with enamine 2b, prepared from aldehyde
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2a and morpholine, provided the adduct product 2d. Compound 2e was obtained by
heating intermediate 2e in the presence of diluted hydrochloric acid. The oxidation of
2e using the reagent 2-iodoxybenzoic acid (IBX) provided the intermediate quinone
2f. Then, compound 2 was prepared by reacting intermediate 2f and
2,3-dimethylbuta-1,3-diene
in
the
presence
of
2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) according to Scheme 1.
The target compounds 6a-6q were synthesized by stepwise that involved a
chloromethylation of 2 to form compound 3 and followed by a nucleophilic
substitution with corresponding amines. Compounds 7a-7d were synthesized from the
intermediate aldehyde 4. Compound 4 was synthesized through Vilsmeier reaction by
treatment of 2 with DMF and POCl3. Then, in the presence of hydrogen peroxide and
sodium chlorite, oxidation of compound 4 afforded the carboxylic acid compound 5.
Finally, the coupling reaction of intermediate 5 with different amines was carried out
using
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide
hydrochloride
(EDCI)/1-hydroxybenzotriazole (HOBt) in CH2Cl2 and afforded the target compounds
7a-7d (Scheme 2).
Scheme 1. Reagents and conditions: (i) morpholine, K2CO3, 0 oC to rt, 3 h; (ii) CH2Cl2, 0 oC to rt,
o
12 h; (iii) dil. HCl, 75 C, 3 h, 54% over three steps; (iv) IBX, CH3CN/H2O, rt, 3 h; (v)
2,3-dimethylbuta-1,3-diene, toluene, DDQ, 110 oC, 6 h, 15% over two steps.
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O
O
O
O
O
O
(i)
(ii)
H3C
H3C
H3C
H3C
H3C
H3C
Cl
R
O
O
O
6a-6q
2
3
(iii)
O
O
O
O
O
O
(v)
(iv)
H3C
H3C
H3C
H3C
H3C
H3C
R
O
O
CHO
COOH
O
4
5
7a-7d
O
o
Scheme 2. Reagents and conditions: (i) (HCHO)n, gaseous HCl, dioxane, 65 C, 1 h, 65%; (ii)
amines, K2CO3, KI, DMF, 45 oC, 2-6 h, 30-54%; (iii) DMF, POCl3, 45 oC, 5 h, 44%; (iv) NaClO2,
H2O2, 90%; (v) EDCI, HOBT, amines, rt, 4-12 h, 34%-67%. For the detailed R groups of
compounds 6a-6q and 7a-7d, see Table 1.
3. Results and Discussion
3.1. Measurement of reduction rates of the L-shaped derivatives of 2 by NQO1.
Table 1 summarizes the L-shaped substrates from the optimization of compound
2. The reduction rate was recorded as µmol NADPH oxidized min-1•µmol-1 NQO1
protein. Generally, most of these derivatives (6a-6e, 6g-6k, 6m-6q, 7a-7d) achieved
equivalent if not superior efficient to compound 2 (905 ± 53 µmol NADPH/min/µmol
NQO1). In fact, compounds 6a-6c, 6p-6q and 7b-7c exhibited equivalent to or higher
reduction rates than β-lap (1258 ± 46 µmol NADPH/min/µmol NQO1). According to
our experimental results, compounds with reduction rates more than 1100 (µmol
NADPH/min/µmol NQO1) at 10 µM could be regarded as good substrates for NQO1.
For the efficient substrates, it was observed that each substrate utilized greater than
one equiv of NADPH over the course of the assay for NQO1 catalytic, indicating the
ability of the reduced hydroquinones to be oxidized back to quinones. It suggested
that these efficient NQO1 substrates had antitumor potential by ROS generation
through redox cycling (Supplementary Information, Fig. S1).
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Table 1
The reduction rates of L-shaped ortho-quinone derivatives catalyzed by NQO1.
reduction rate
reduction rate
(µmol
(µmol
cpd
R
cpd
R
NADPH/min/µmol
NADPH/min/µmol
NQO1)
NQO1)
1332 ± 23
1340 ± 69
1315 ± 48
898 ± 64
1199 ± 87
1046 ± 90
6a
6b
6c
6l
6m
6n
F
1177 ± 28
1057 ± 92
847 ± 48
1194 ± 85
1462 ± 48
1520 ± 85
6d
6e
6f
6o
6p
6q
HN
F
940 ± 46
1149 ± 83
1042 ± 43
1257 ± 99
1294 ± 110
1363 ± 81
6g
6h
6i
7a
7b
7c
955 ± 63
1187 ± 64
6j
7d
1123 ± 83
6k
2
-
-
905 ± 53
β-lap
1258 ± 46
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Representative compounds 6a-6c, 6p-6q and 7c that showed high reduction rates
at the concentration of 10 µmol•L-1 (reduction rates faster than 1300 µmol
NADPH/min/µmol NQO1) were selected to calculate their catalytic efficiency. The
detailed reduction rates by NQO1 of these compounds under various concentrations
(0.1-50 µmol•L-1) were determined and the NQO1 kinetic parameters (kcat/Km) were
shown in Table 2, together with β-lap and compound 2 as references. It was observed
that these highly efficient redox cyclers (6p and 6q) processed much higher catalytic
efficiency than the control β-lap. These two compounds were supposed to be efficient
substrates for NQO1. Among them, compound 6q (kcat/Km = 15 ± 3.8 × 106 M-1s-1)
was the most efficient substrate, which possessed much better kinetic parameters than
β-lap (kcat/Km = 4.4 ± 0.9 × 106 M-1s-1) and 2 (kcat/Km = 3.1 ± 0.4 × 106 M-1s-1).
Furthermore, these representative compounds were also assessed to determine
their reduction rates by CPR and the selectivity ratio (NQO1/CPR) with the purpose
of identifying their possibility of inducing CPR-related hepatotoxicity. As shown in
Table 2, these compounds could hardly be reduced by CPR (73-159 µmol
NADPH/min/µmol CPR). The selectivity ratio (NQO1/CPR) of these compounds
were ranging from 9.19 to 20.8, which were much better than compound 2 and β-lap.
Among them compound 6q showed the best selectivity with a selectivity ratio of 20.8.
It indicated that the introduction of L-shaped chain would greatly improve the NQO1
activity while not affect the CPR activity as compared with compound 2, and thus
greatly increase their selectivity toward NQO1 over CPR and potentially improve
their safety profiles.
Table 2
NQO1 kinetic parameters and selectivity ratio (NQO1/CPR) of the selected compounds.
reduction rate
NQO1 kcat/Km
(106 M-1·s-1)
(µmol NADPH/min/µmol enzyme)
cpd
Selectivity ratio (NQO1/CPR)
NQO1
CPR
2.8 ± 0.5
1332±23
105±12
12.7
12.0
6a
6b
2.5 ± 0.9
1340±69
112±24
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3.4 ± 1.0
6.2 ± 1.1
15 ± 3.8
3.2 ± 0.7
3.1 ± 0.4
4.4 ± 0.9
1315±48
1462±48
1520±85
1363±81
905±53
127±29
159±39
73±9
10.3
9.19
20.8
15.1
6.37
1.36
6c
6p
6q
90±21
142±25
923±49
7c
2
1258±46
β-lap
3.2. Physicochemical properties of the efficient substrates.
In an effort to discover potential NQO1-targeting antitumor drugs with good
drug-like properties prior to the time-consuming and costly development and
optimization of derivatives, next we determined the physicochemical properties of the
selected compounds which exhibited high NQO1 catalytic efficiency. The
“goldstandard” Avdeef-Bucher potentiometric titration method was employed to
determine the intrinsic aqueous solubility and Log D7.4 were determined on a Gemini
Profiler instrument (pION) [29]. The solubility of these compounds under acidic
condition were also determined (compounds were dissolved in diluted hydrochloric
acid buffer at pH ≥ 3). A standard parallel artificial membrane permeability assay
(PAMPA) on a PAMPA Explorer instrument (pION) was used to determine the
Permeability coefficients. As shown in Table 3, compound 6q
(morpholino-substituted in the aromatic amine) showed much improved
physicochemical properties (Log D7.4 3.10, intrinsic solubility 360.2 µg/mL) than the
other aromatic amines series.
In addition, we determined the permeability of the derivatives to inspect the
ability of diffusing through the cell membrane. As shown in Table 3 the Pe values of
all compounds were more than that of the negative control compound, Ketoprofen.
Among them, compound 6q with good solubility also displayed good membrane
permeability (Pe = 46.5 × 106 cm/s), indicating the balanced properties between
hydrophilicity and hydrophobicity of this compound. Taking into consideration of the
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physicochemical properties and reduction rate by NQO1, compound 6q was selected
for further pharmacological evaluation.
Table 3
NQO1 kinetic parameters and physicochemical properties of the selected compounds.
intrinsic
solubility
(µg/mL)
solubility c
(pH = 3)
(µg/mL)
kcat/Km
(106 M-1·s-1)
Log D
Pe, pH = 7.4
(10-6 cm/s)a
cpd
pH = 7.4
2.8 ± 0.5
2.5 ± 0.9
3.4 ± 1.0
6.2 ± 1.1
15 ± 3.8
3.2 ± 0.7
3.1 ± 0.4
4.4 ± 0.9
4.03
4.21
3.31
2.89
3.10
4.82
3.65
2.85a
48.5
51.2
100-500
100-500
< 100
42.0
66.2
NRb
28.3
46.5
11.9
43.1
46.4
6a
6b
33.1
6c
151.1
360.2
41.1
500-1000
> 1000
100-500
< 100
6p
6q
7c
28.6
2
38.0a
< 100
β-lap
aDue to the log D and solubility of β-lap could not be determined by the Gemini Profiler
instrument (pION), thus the Log D of β-lap was calculated Log D, and the solubility was
according to previously reported [18]. Ketoprofen (1.93 × 10-6 cm/s) bNR represents no result was
c
obtained from three independent experiments. Determined in diluted hydrochloric acid buffer at
pH = 3.
3.3. Molecular modeling
Utilizing the crystal structure of NQO1 (PDB ID: 2F1O), computational study
was performed to elucidate the possible binding mode of NQO1 with 2 and the
representative L-shaped compound 6q (Fig. 2). Both of the two compounds oriented
with the quinone ring above the isoalloxazine ring of the bound cofactor FAD as for
hydride transfer by π-stacking interaction. The carbonyl groups in the planar ring of
compound 2 and 6q could bind with Tyr126 and the 7,8-dimethyl groups in the planar
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ring can form C-H…π interactions with the adjacent Phe178 and Trp105 residues. In
addition, for compound 6q, the substituted side chains could fit deep into the
additional pocket and make π-stacking interactions with Tyr128 and Phe232 in the
side binding pocket of NQO1. It supported the fact that compound 6q was excellent
substrate for NQO1 from one hand.
Fig. 2. Docked conformation of compound 2 (A) and 6q (B) into active site of NQO1. The
interaction mode was obtained through molecular docking and depicted using MOE 2013.08. The
carbon atoms of the compounds and the key residues in the active site of NQO1 were colored in
yellow and purple, respectively. The H-bonds were shown as blue dot lines, the C-H…π
interactions were shown as purple dot lines.
3.4. Pharmacological evaluation of the 6q.
In the purpose of discovering compounds with safety profile, we need to verify
that compound 6q could not or weakly initiate the redox cycling through one-electron
reduction by CPR and some other one-electron oxidoreductase, such as thioredoxin
reductase (TrxR) and carbonyl reductase 1 (CBR1), which are expressed in normal
tissues [30]. Therefore, 6q was examined whether can be reduced through
one-electron reduction by not only CPR but other oxidoreductases. As shown in Fig.
3, compound 6q was worse substrate for these one-electron oxidoreductases
compared to β-lap, which showed the best properties of high reduction rate by NQO1
and low reduction rate by all of these one-electron oxidoreductases, especially, the
selectivity ratio (NQO1/CPR) of this compound reached 20.8-fold (Fig. 3). Therefore,
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compound 6q would avoid generating excessive quantities of ROS through the
normal one-electron oxidoreductase activities of the cell and thus exerted superior
safety profile than β-lap.
Fig. 3. The reduction rates of 6q and β-lap catalyzed by oxidoreductase.
3.4. In vitro selective antitumor activity of 6q.
In an effort to obtain antitumor drugs with efficacy and safety, compound 6q was
assessed versus various NQO1-expressing cancer cells (A549, H1299, MCF-7,
HepG2, HCT116 and HEK293), NQO1-deficient cells (A549 cells pretreated with
NQO1 inhibitor dicoumarol (DIC), H596) and normal hepatic cells (L02) (Fig. 4 and
Table S1). Compound 6q was assessed as before: cells were incubated with
compound for 24 h followed by analysis of cell death at 72 h and IC50 values are
listed in Fig. 4. This quinone 6q treatment markedly induced cell death in
NQO1-expressing cancer cells (IC50 = 2.1~4.9 µM), while with weaker toxic effects
towards NQO1-deficient cell lines (A549 + DIC and H596, IC50 > 50 µM and 30 µM,
respectively) and the normal cell line L02 (IC50 = 35 µM). The results confirmed that
compound 6q had a NQO1-expressing cancer-cell-selective killing property, which
showed a superior profile to that of β-lap. Moreover, 6q also showed potent cytotoxic
activity towards A549 Taxol-resistant cell lines (IC50 = 2.2 ± 0.8 µM), indicated that
our compound may overcome drug resistance through NQO1-mediated cell death.
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Fig. 4. Cell death of NQO1-expressing cancer cells (A549, H1299, MCF-7, HepG2, HCT116,
HEK293 and A549 Taxol-resistant cell lines), NQO1-deficient cells (A549 cells pretreated with
NQO1 inhibitor dicoumarol, H596) and normal hepatic cells (L02) treated with compound 6q.
The IC50 value of Taxol for A549 Taxol-resistant cell lines was 28 ± 8.8 µM.
3.5. Induction of ROS accumulation in A549 cells by 6q.
It is supposed that 6q exerted its antitumor activity by accumulating ROS through
futile redox cycles catalyzed by intracellular NQO1. 2’,7’-Dichlorfluorescein
diacetate (DCFH-DA) is a cell-permeable, well-established probe to detect
intracellular production of ROS. When the probe was diffused into cells, DCFH-DA
is
deacetylated
by
cellular
esterases
to
nonfluorescent
2’,7’-dichlorodihydrofluorescin, which is rapidly converted to highly fluorescent
2’,7’-dichlorofluorescein upon reacting with ROS. The probe is a leading approach to
measuring highly reactive species that may be most likely to initiate cancer cell death
[13-14]. As depicted in Fig. 5, the effect of 6q on cellular ROS levels in cancer cells
(A549) and normal cells (L02) was detected through flow cytometry using the
redox-sensitive fluorescent probe DCFH-DA. After treating A549 cells with 6q for 1
h and 3 h, a marked increase in ROS levels was caused. As shown in Fig. 5A, 6q
generated excessive ROS even higher than the control β-lap in A549 cells but lower
in DIC and N-acetyl cysteine (NAC, a typical ROS scavenger) pretreated A549 cells.
In contrast to the results in cancer cell A549, 6q did not cause an increase in ROS
level in normal cell L02 (Fig. 5B). However, the control β-lap increased ROS level
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obviously in the normal cell L02, which may be induced by the one-electron
oxidordeuctase CPR, and explained the potential toxicity of β-lap after prolonged
treatment. This selective induction of ROS in cancer cells indicates that 6q-induced
ROS elevation is a consequence of cell transformation. The results indicated that 6q
induced ROS production in a NQO1-dependent and specific manner and showed
selective towards cancer cells. The property of better selective induction of ROS in
cancer cells than β-lap was promised to have a superior safety profile. In addition, we
also performed the electrochemistry studies on 6q, two cathodic peaks and
corresponding back ward peaks are displayed (Supplementary Information, Fig. S2),
which further indicated that the efficient NQO1 substrate could be potential ROS
generator [31-34]. Lipid peroxidation can be used as an intracellular indicator of
ROS-induced oxidative stress. Further data obtained by TBARS assay [35], as seen in
Table S2, regarding lipid peroxidation (MDA production), also provided the evidence
for the generation of ROS by 6q in A549 cancer cells.
Fig. 5. Induction of ROS in A549 or L02 cells. (A) 6q-Induced ROS elevation and reversion by
DIC or NAC. A549 cells were treated with 6q (10 µM), β-lap (10 µM) or DMSO for 1 h and 3 h.
Cells were also pretreated with 10 µM DIC or 3 mM NAC for 1 h, followed by 10 µM 6q for 3 h.
(B) 6q (10 µM, 3 h) does not increase ROS levels in normal cells (L02). ROS levels were
measured by flow cytometry and shown by quantitative bar graph measured as the fold change
over DMSO-treated levels. All values are mean ± SD of three independent experiments.
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To further confirm the antitumor activity of compound 6q was mediated through
generation of ROS, we examined the expression level of the proteins which would be
activated in response to ROS. It has been reported that Akt/MAPK pathways such as
Akt, JNK, p38 and ERK1/2 were one of the main pathways that responded to the ROS
[36-38]. As compound 6q induced prominent ROS generation, A549 cancer cells
were treated with it (3 h) at various concentration points. The phosphorylation of Akt
and JNK, p38, and ERK was increased in a dose-dependent manner (Fig. 6A). When
cells were pretreated with 10 µM DIC or 3 mM NAC prior to treatment with
compound 6q, the increase of phospho-levels of both Akt and MAPKs were examined
to be negated. It indicated that 6q activated Akt and MAPK pathways in a
NQO1-dependent and ROS-mediated pathways. It is worth noting that increased Akt
and MAPK phosphorylation sensitize cells can aid in ROS-directed cell death by
inducing cellular oxygen consumption, promoting ROS generation, and impairing
ROS degeneration [39-40]. Hence, both of the increased ROS levels and the enhanced
kinase activity might be exerted in A549 cancer cell lines, mediated apoptosis.
Therefore, we next examined apoptosis in A549 cells following treatment with
6q. When A549 cells were incubated with different concentrations of 6q for 24 h
followed by DAPI staining, an increasing number of cells displayed condensed nuclei,
a characteristic morphology of cells undergoing apoptosis (Fig. 6B). A specific
analysis revealed nuclei with dose-dependent chromatin condensation and
characteristic morphological changes of apoptosis, in cells cultures with 6q. PARP
and cleaved PARP are the main apoptotic markers, and the activation of PARP is a
central event in the process of apoptosis. Western blot analysis showed that 6q
increased the expression of cleaved PARP, and likewise, pretreatment with 10 µM
DIC or 3 mM NAC attenuated the escalation of cleaved PARP (Fig. 6C), indicating
that 6q induced apoptosis through NQO1-dependent and ROS-mediated pathways.
15

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Fig. 6. Compound 6q activated Akt and multiple MAPK pathways in a ROS-dependent fashion
and induced apoptosis in A549 cells in a dose-dependent manner. (A) Treatment with 6q for 3 h
enhanced phosphorylation of Akt, JNK, p38 and ERK in A549 cells in a dose-dependent manner.
A549 cells were pretreated with DIC (10 µM) or NAC (10 µM) prior to treatment with 10 µM 6q
for 3 h. NAC and DIC attenuated the activation of Akt, JNK and ERK. (B) Analysis of apoptosis
by nuclear morphology changes. Cells were treated with 0, 1, 2.5, 3, 4 and 5 µM 6q for 24 h. Data
shown are representative of at least three repeats. (C) Treatment with 6q for 24 h caused apoptosis
in A549 cells in a dose-dependent manner. The levels of apoptotic marker cleaved PARP was
increased. Pretreatment with NAC and DIC prior to 6q blocked the generation of PARP cleavage
in A549 cells.
3.6. In vivo evaluations of compound 6q.
Compound 6q was investigated for their in vivo antitumor potency, and the A549
tumor xenografts mouse model was employed. When the tumor grew to a volume of
100-150 mm3, the nude mice bearing A549 tumor xenografts were randomly injected
with vehicle, β-lap (ARQ 501, 30 mg/kg or 50 mg/kg) and compound 6q (15 mg/kg
or 30 mg/kg or 50 mg/kg) every other day for three weeks. At all doses given, the
16

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compound markedly inhibited the tumor growth. Notably, as shown in Fig. 7A and
7B, the tumor growth in the mice treated with 30 mg/kg and 50 mg/kg 6q was
efficaciously inhibited with complete tumor stasis. During the same period, the tumor
volume in the vehicle-treated group was more than quadrupled. In addition, the body
weights of 6q-treated animals and vehicle-treated ones were indistinguishable from
each other during the treatment period even in the high concentration of 50 mg/kg
(Fig. 7C), indicating no overt toxicity with this quinone treatment. As for the control
β-lap, during the treatment period, an obvious decrease in body weight (18%
decreased after treatment) occurred in the mice treated with the concentration of 50
mg/kg. Furthermore, compound 6q and β-lap were also evaluated for general
hepatotoxicity studies using zebrafish larvae [41]. The larvae were grown in the
presence of compound 6q for 48 h. The control group was incubated in 0.1% DMSO.
After compound 6q treatment, the larvae were observed employing visible light at
regular intervals to document the liver. As shown in Fig. 8, compound 6q
demonstrated no obvious hepatotoxic effect at 10 µM while β-lap exerted obvious
hepatotoxicity at a relative lower concentration (1 µM) on the zebrafish. Such
hepatotoxicity of β-lap may be induced by the high expression of one-electron
oxidoreductases, as the poor substrate of the one-electron oxidoreductases 6q did not
show significant toxicity. The results further supported that the concept of designing
selective substrates towards NQO1 over the one-electron oxidoreductases to discover
drugs with low toxicity and improved safety was reasonable. Taken together, these
data clearly demonstrated 6q was more effective and safe drug than β-lap and
supported the notion that this new class of quinone redox modulators could be applied
in new cancer therapeutics.
17

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Fig. 7. Compound 6q suppressed the tumor growth in vivo in A549 tumor xenografts nude model.
A549 tumor-bearing mice were treated with vehicle and 6q (15 mg/kg and 30 mg/kg). β-Lap was
selected as the compared drug. The relative tumor volume (A) and body weight (B) of the treated
mice (C) tumor weight (D) the pictures of the tumor are shown.
Fig. 8. Zebrafish larvae (72 hpf) exposed to 6q (at 10 µM) and the control β-lap (at 1 µM) for 48
hour postexposure. (A) Control (0.5% DMSO). (B) 10 µM 6q. (C) 1 µM β-lap. Green fluorescence
in the images represents the liver.
To better understand the mechanism of antitumor activities in vivo, the abilities of
compound 6q to induce the production of ROS and its effect on the antioxidant
system in tumor tissues were also evaluated using tumor tissues isolated from A549
tumor xenograft models. Compound 6q treated groups showed obvious increase in
ROS levels and decrease of intracellular NADPH and GSH (glutathione), which play
a crucial role in maintaining biological redox homeostasis as important parts of
antioxidant system (Fig. 9). In conclusion, our compound 6q would play a dual role
by also induce ROS generation and suppress the cellular antioxidant capacity so that it
can maximally exploit the ROS-mediated cancer cell death.
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Fig. 9. The effect of 6q on antioxidant system in tumor tissues were also evaluated using
tumor tissues isolated from A549 tumor xenograft models. NADPH depletion (A) GSH
depletion (B). **p < 0.001 versus control.
4. Conclusions
Based on the scaffold of 3,7,8-trimethylnaphtho[1,2-b]furan-4,5-dione (2), a
series of L-shaped derivatives with substituted side chains at the position of C2 were
designed and synthesized. Among them, the drug-like compound 6q (named as
DDO-7178) emerged as the most specific substrate of NQO1 and could hardly be
reduced by the one-electron oxidoreductases. The follow-up studies indicated that 6q
showed a NQO1-expressing cancer-cell-selective killing property. Preliminary
mechanism studies on the anticancer effect indicated that 6q induced ROS production
in an NQO1 dependent manner and active Akt/MAPK pathways in a ROS-dependent
fashion, thereby inducing apoptosis. In addition, emphasized compound 6q showed
more significant antitumor efficacy than β-lap without producing obvious toxic
effects in vivo, which gave us a new tool for further investigate NQO1-mediated
redox modulators as anticancer drugs.
5. Experimental Section
5.1. Chemistry
5.1.1. General Experimental Methods
All chemicals and reagents and were purchased from commercial sources. Organic
o
solutions were concentrated in a rotary evaporator (Büchi Rotavapor) below 55 C
19

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under reduced pressure. Silica gel thin-layer chromatography was performed on
precoated plates GF-254 and visualized under UV light. Melting points were
determined with a Melt-Temp II apparatus. 1H NMR and 13C NMR spectra were
recorded on a Bruker AV-300 instrument using deuterated solvents with
tetramethylsilane (TMS) as internal standard. Chemical shifts are given in ppm (δ).
The multiplicities are denoted as follows: s, singlet; d, doublet; t, triplet; q, quartet; m,
multiplet; br s, broad singlet. IR spectra were recorded on a Nicolet iS10 Avatar
FT-IR spectrometer using KBr film. ESI-mass and high resolution mass spectra
(HRMS) were recorded on a Water Q-Tofmicro mass spectrometer. Analytical results
are within 0.40 % of the theoretical values. Purity of the compounds was analyzed by
HPLC (Agilent Technologies 1260 Infinity) using 80:20 MeOH/H2O as the mobile
phase with a flow rate of 0.8 mL/min on a C18 column. All tested compounds
exhibited greater than 95% purity unless otherwise noted..
5.1.2. Synthesis
2-(Chloromethyl)-3,7,8-trimethylnaphtho[1,2-b]furan-4,5-dione
(3).
Gaseous
hydrogen chloride was filled into a solution of quinone 2 (500 mg, 2.08 mmol) and
paraformaldehyde (624 mg, 20.8 mmol) in dioxane (30 mL). The reaction mixture
was stirred at 65 oC for 1 h. Then, the mixture was poured into ice water and obtained
the crude product, which was purified through column chromatography over silica gel
o
1
to give the red product 3 (390 mg, 65 %). m.p. 161-162 C. H NMR (300 MHz,
CDCl3) δ: 7.84 (s, 1H), 7.50 (s, 1H), 4.64 (s, 2H), 2.37 (d, J = 4.4 Hz, 3H), 2.32 (d, J
= 4.4 Hz, 3H), 2.28 (s, 3H).
2-(((4-Fluorophenyl)amino)methyl)-3,7,8-trimethylnaphtho[1,2-b]furan-4,5-dione
(6a). Following the above procedure, treatment of 3 with 4-fluorophenylamine for 3 h
at 60 oC, gave the crude product, which was purified through column chromatography
(eluent: petroleum ether/EtOAc 1 : 2) to afford 6a as a dark red: yield 30%. m.p.
162-164 oC. 1H NMR (300 MHz, CDCl3) δ: 7.80 (s, 1H), 7.40 (s, 1H), 6.93 (t, J = 8.7
Hz, 2H), 6.68-6.64 (m, 2H), 4.29 (s, 2H), 2.32 (s, 3H), 2.31 (s, 3H), 2.29 (s, 3H). IR
(v, cm-1): 3421.43, 2923.83, 2361.40, 2344.58, 1670.01, 1512.68, 1438.09, 1395.87,
20

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1246.86, 1221.25, 823.11. ESI-HRMS m/z calculated for C22H19FNO3 [M + H]+
364.1349, found 364.1331. HPLC (tR = 11.705 min, purity = 94.5 %).
2-(((3-Fluorophenyl)amino)methyl)-3,7,8-trimethylnaphtho[1,2-b]furan-4,5-dione
(6b). Following the above procedure, treatment of 34 with 3-fluorophenylamine for 3
h at 60 oC, gave the crude product, which was purified through column
chromatography (eluent: petroleum ether/EtOAc 1 : 2) to afford 6b as a dark red solid:
o
1
yield 26%. m.p. 178-180 C. H NMR (300 MHz, CDCl3) δ: 7.83 (s, 1H), 7.46 (s,
1H), 7.22-7.19 (m, 1H), 6.65-6.62 (m, 3H), 4.37 (s, 2H), 2.38 (s, 3H), 2.33 (s, 3H),
2.28 (s, 3H). ESI-HRMS m/z calculated for C22H18FNNaO3 [M + Na]+ 386.1168,
found 386.1148.
2-(((4-Fluoro-2-methylphenyl)amino)methyl)-3,7,8-trimethylnaphtho[1,2-b]furan-4
,5-dione (6c). Following the above procedure, treatment of
3
with
o
4-fluoro-2-methylphenylamine for 2 h at 55 C, gave the crude product, which was
purified through column chromatography (eluent: petroleum ether/EtOAc 1 : 5) to
afford 6c as a dark red solid: yield 30%. m.p. 205-207 oC. 1H NMR (300 MHz, CDCl3)
δ: 7.81 (s, 1H), 7.41 (s, 1H), 6.88-6.75 (m, 3H), 4.36 (s, 2H), 2.35 (s, 3H), 2.31 (s,
3H), 2.27 (s, 3H), 2.22 (s, 3H). IR (v, cm-1): 304.49, 2963.30, 2364.14, 234.63,
1670.36, 1519.14, 1490.65, 1432.02, 793.91. ESI-HRMS m/z calculated for
C23H20FNNaO3 [M + Na]+ 400.1325, found 400.1310. HPLC (tR = 17.938 min, purity
= 94.6 %).
2-(((2,4-Difluorophenyl)amino)methyl)-3,7,8-trimethylnaphtho[1,2-b]furan-4,5-dio
ne (6d). Following the above procedure, treatment of 3 with 2,4-difluorophenylamine
o
for 3 h at 60 C, gave the crude product, which was purified through column
chromatography (eluent: petroleum ether/EtOAc 1 : 8) to afford 6d as a dark red solid:
yield 35%. m.p. 173-175 oC. 1H NMR (300 MHz, CDCl3) δ: 7.82 (s, 1H), 7.43 (s, 1H),
6.85-6.72 (m, 3H), 4.3 (s, 2H), 2.37 (s, 3H), 2.32 (d, J = 2.7 Hz, 6H). IR (v, cm-1):
3385.71, 2359.88, 232.18, 1670.71, 1532.27, 1432.48, 960.14, 840.59, 789.50.
ESI-HRMS m/z calculated for C22H17F2NNaO3 [M + Na]+ 404.1074, found 404.1055.
HPLC (tR = 15.884 min, purity = 97.1 %).
21

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2-(((2,4-Dichlorophenyl)amino)methyl)-3,7,8-trimethylnaphtho[1,2-b]furan-4,5-dione
(6e). Following the above procedure, treatment of 3 with 2,4-dichlorophenylamine for
o
3 h at 55 C, gave the crude product, which was purified through column
chromatography (eluent: petroleum ether/EtOAc 1 : 5) to afford 6e as a dark red solid:
o
1
yield 38%. m.p. 285-287 C. H NMR (300 MHz, CDCl3) δ: 7.83 (s, 1H), 7.43 (s,
1H), 7.27 (s, 1H), 7.17 (d, J = 7.1 Hz, 1H), 6.77 (brs, 1H), 4.41 (s, 2H), 2.38 (s, 3H),
2.38 (s, 3H), 2.33 (s, 6H). IR (v, cm-1): 316.99, 2360.32, 231.72, 1675.72, 1637.45,
1532.95, 1507.70, 1211.84, 841.05, 668.91. ESI-HRMS m/z calculated for
C22H17Cl2NNaO3 [M + Na]+ 436.0483, found 436.0468. HPLC (tR = 4.731 min, purity
= 95.2 %).
2-(((3,4-Dichlorophenyl)amino)methyl)-3,7,8-trimethylnaphtho[1,2-b]furan-4,5-dione
(6f). Following the above procedure, treatment of 3 with 3,4-dichlorophenylamine for
o
3 h at 60 C, gave the crude product, which was purified through column
chromatography (eluent: petroleum ether/EtOAc 1 : 5) to afford 6f as a dark red solid:
o
1
yield 24%. m.p. 202-204 C. H NMR (300 MHz, CDCl3) δ: 7.82 (s, 1H), 7.41 (s,
1H), 7.24 (s, 1H), 6.84 (d, J = 2.5 Hz, 1H), 6.57-6.54 (m, 1H), 4.31 (s, 2H), 2.37 (s,
3H), 2.33 (s, 6H) ppm. 13C NMR (75 MHz, CDCl3) δ: 180.3, 175.4, 159.5, 150.4,
147.1, 144.9, 138.5, 132.8, 131.0, 129.1, 126.3, 125.6, 123.1, 121.8, 120.5, 117.8,
116.3, 112.9, 38.4, 19.7, 19.1, 8.5 ppm. ESI-HRMS m/z calculated for
C22H17Cl2NNaO3 [M + Na]+ 436.0483, found 436.0465. HPLC (tR = 4.743 min, purity
= 96.8 %).
2-(((3-Chloro-4-fluorophenyl)amino)methyl)-3,7,8-trimethylnaphtho[1,2-b]furan-4,
5-dione (6g). Following the above procedure, treatment of
3
with
o
3-chloro-4-fluorophenylamine for 3 h at 55 C, gave the crude product, which was
purified through column chromatography (eluent: petroleum ether/EtOAc 1 : 5) to
o
afford 6g as a red solid: yield 28%. m.p. 192-194 C. 1H NMR (300 MHz, CDCl3) δ:
7.82 (s, 1H), 7.42 (s, 1H), 7.01 (t, J = 8.8 Hz, 1H), 6.79-6.76 (m, 1H), 6.57-6.55 (m,
1H), 4.30 (s, 2H), 2.37 (s, 3H), 2.33 (s, 6H). IR (v, cm-1): 4322.70, 2360.69, 231.97,
1668.85, 1508.61, 1242.15, 1228.95, 801.73. ESI-HRMS m/z calculated for
22

ACCEPTED MANUSCRIPT
C22H17ClFNNaO3 [M + Na]+ 420.0779, found 420.0770. HPLC (tR = 3.920 min,
purity = 95.3 %).
2-(((4-Bromophenyl)amino)methyl)-3,7,8-trimethylnaphtho[1,2-b]furan-4,5-dione
(6h). Following the above procedure, treatment of 3 with 4-bromophenylamino for 3
h at 60 oC, gave the crude product, which was purified through column
chromatography (eluent: petroleum ether/EtOAc 1 : 8) to afford 6h as a dark red solid:
o
1
yield 32%. m.p. 203-205 C. H NMR (300 MHz, CDCl3) δ: 7.78 (s, 1H), 7.37 (s,
1H), 7.30 (d, J = 8.8 Hz, 2H), 6.60 (d, J = 8.8 Hz, 2H), 4.29 (s, 2H), 2.3 (s, 3H), 2.29
(d, J = 2.6 Hz, 6H). IR (v, cm-1): 321.37, 2360.33, 231.69, 1669.51, 1437.39, 1387.88,
1243.29, 1162.38, 742.13, 668.96, 480.89. ESI-HRMS m/z calculated for
C22H18BrNNaO3 [M + Na]+ 446.0368, found 446.0349. HPLC (tR = 7.267 min, purity
= 98.5 %).
2-(((2-Methoxyphenyl)amino)methyl)-3,7,8-trimethylnaphtho[1,2-b]furan-4,5-dione
(6i). Following the above procedure, treatment of 3 with 2-methoxyphenylamine for 3
h at 60 oC, gave the crude product, which was purified through column
chromatography (eluent: petroleum ether/EtOAc 1 : 5) to afford 6i as a dark red solid:
yield 38%. m.p. 170-171 oC. 1H NMR (300 MHz, CDCl3) δ: 7.80 (s, 1H), 7.45 (s, 1H),
6.90-6.81 (m, 4H), 4.40 (s, 2H), 3.86 (s, 3H), 2.35 (s, 3H), 2.31 (s, 3H), 2.25 (s, 3H).
IR (v, cm-1): 341.42, 1668.97, 1618.3, 1513.32, 1247.67, 1226.67, 1029.89, 729.35.
ESI-HRMS m/z calculated for C23H21NNaO4 [M + Na]+ 398.1368, found 398.1355.
HPLC (tR = 16.141 min, purity = 96.2 %).
2-(((3-Methoxyphenyl)amino)methyl)-3,7,8-trimethylnaphtho[1,2-b]furan-4,5-dione
(6j). Following the above procedure, treatment of 3 with 3-methoxyphenylamine for 3
h at 60 oC, gave the crude product, which was purified through column
chromatography (eluent: petroleum ether/EtOAc 1 : 5) to afford 6j as a red solid:
yield 41%. m.p. 198-200 oC. 1H NMR (300 MHz, CDCl3) δ: 7.73 (s, 1H), 7.35 (s, 1H),
7.06 (t, J = 8.0 Hz, 1H), 6.29-6.21 (m, 3H), 4.25 (s, 2H), 4.04 (brs, 1H), 3.72 (s, 3H),
2.28 (s, 3H), 2.24 (s, 6H). IR (v, cm-1): 3377.57, 2920.35, 2360.94, 232.35, 1666.93,
1616.08, 1521.03, 1496.76, 1210.80, 1162.54, 688.24, 482.68. ESI-HRMS m/z
23

ACCEPTED MANUSCRIPT
calculated for C23H21NNaO4 [M + Na]+ 398.1368, found 398.1358. HPLC (tR =
12.530 min, purity = 95.7 %).
2-(((4-Methoxyphenyl)amino)methyl)-3,7,8-trimethylnaphtho[1,2-b]furan-4,5-dione
(6k). Following the above procedure, treatment of 3 with 4-methoxyphenylamine for
o
3 h at 60 C, gave the crude product, which was purified through column
chromatography (eluent: petroleum ether/EtOAc 1 : 5) to afford 6k as a red solid:
o
1
yield 32%. m.p. 148-150 C. H NMR (300 MHz, CDCl3) δ: 7.82 (s, 1H), 7.47 (s,
1H), 6.88-6.80 (m, 4H), 4.33 (s, 2H), 3.78 (s, 3H), 2.35 (s, 3H), 2.33 (s, 3H), 2.26 (s,
3H). IR (v, cm-1): 341.42, 1668.97, 1618.3, 1513.32, 1247.67, 1226.67, 1029.89,
729.35. ESI-HRMS m/z calculated for C23H21NNaO4 [M + Na]+ 398.1368, found
398.1357. HPLC (tR = 10.258 min, purity = 96.0 %).
2-(((4-Cyanophenyl)amino)methyl)-3,7,8-trimethylnaphtho[1,2-b]furan-4,5-dione (6l).
Following the above procedure, treatment of 3 with 4-cyanophenylamine for 3 h at 60
oC, gave the crude product, which was purified through column chromatography
(eluent: petroleum ether/EtOAc 1 : 10) to afford 6l as a dark red solid: yield 28%. m.p.
o
1
148-150 C. H NMR (300 MHz, CDCl3) δ: 7.80 (s, 1H), 7.49 (d, J = 6.8 Hz, 2H),
7.47 (s, 1H), 6.70 (d, J = 6.8 Hz, 2H), 4.37 (d, J = 5.3 Hz, 2H), 2.35 (s, 3H), 2.32 (s,
6H). IR (v, cm-1): 314.59, 2211.67, 1669.63, 1606.39, 1526.40, 1173.63, 825.30.
ESI-HRMS m/z calculated for C23H19N2O3 [M + H]+ 371.1396, found 371.1375.
HPLC (tR = 8.264 min, purity = 95.3 %).
2-(((4-Fluorobenzyl)amino)methyl)-3,7,8-trimethylnaphtho[1,2-b]furan-4,5-dione
(6m). Following the above procedure, treatment of 3 with 4-fluorobenzylamine for 3 h
at 60 oC, gave the crude product, which was purified through column chromatography
(eluent: petroleum ether/EtOAc 1 : 5) to afford 6m as a red solid: yield 37%. m.p.
o
1
208-210 C. H NMR (300 MHz, CDCl3) δ: 7.81 (s, 1H), 7.46 (s, 1H), 7.35-7.28 (m,
2H), 7.04 (t, J = 8.5 Hz, 2H), 3.81 (s, 4H), 2.37 (s, 3H), 2.32 (s, 3H), 2.21 (s, 3H)
ppm. 13C NMR (75 MHz, CDCl3) δ: 181.8, 179.1, 161.2, 159.3 (1JCF = 225 Hz),
145.0, 139.7, 138.7, 135.0, 130.8, 130.4 (2JCF = 22.5 Hz), 126.2, 125.2, 122.4 120.9,
119.0, 114.4 (3JCF = 7.5 Hz), 55.2, 48.8, 19.9, 19.2, 8.4 ppm. ESI-HRMS m/z
24

ACCEPTED MANUSCRIPT
calculated for C23H21FNO3 [M + H]+ 378.1500, found 378.1120; HPLC (tR =6.454
min, purity = 96.9 %).
2-(4-Phenylpiperazin-1-ylmethyl)-3,7,8-trimethyl-naphtho[1,2-b]furan-4,5-dione
(6n). Following the above procedure, treatment of 3 with 1-phenylpiperazine for 3 h
at 55 oC, gave the crude product, which was purified through column chromatography
(eluent: petroleum ether/EtOAc 2 : 7) to afford 6n as a red solid (29 mg, 20%). m.p.
o
1
193-195 C. H NMR (300 MHz, CDCl3) δ: 7.84 (s, 1H), 7.53 (s, 1H), 7.31 (s, 1H),
7.26 (s, 1H), 6.96-6.89 (m, 3H), 3.72 (s, 2H), 3.31 (s, 4H), 2.78 (s, 2H), 2.38 (s, 3H),
13
2.3 (s, 3H), 2.32 (s, 3H). C NMR (75 MHz, CDCl3) δ: 180.2, 175.2, 159.7, 150.6,
149.0, 145.0, 138.7, 131.0, 128.6, 126.2, 125.7, 123.2, 120.4, 119.4, 119.1, 115.7,
52.3, 51.7, 48.6, 19.8, 19.2, 8.6. IR (v, cm-1): 341.40, 2360.58, 232.33, 1691.02,
1636.07, 1540.54, 1495.26, 1228.18, 1133.88, 1106.93, 742.45. ESI-HRMS m/z
calculated for C26H27N2O3 [M + H]+ 415.2022, found 415.2002. HPLC (tR = 4.31 min,
purity = 96.7 %).
2-((4-Benzylpiperazin-1-yl)methyl)-3,7,8-trimethylnaphtho[1,2-b]furan-4,5-dione
(6o). Following the above procedure, treatment of 3 with 1-benzylpiperazine for 3 h at
o
55 C, gave the crude product, which was purified through column chromatography
(eluent: petroleum ether/EtOAc 2 : 1) to afford 6o as a red solid: yield 20%. m.p.
o
1
158-160 C. H NMR (300 MHz, CDCl3) δ: 7.82 (s, 1H), 7.50 (s, 1H), 7.33-7.32 (m,
5H), 3.61 (s, 2H), 3.55 (s, 2H), 2.59 (s, 8H), 2.37 (s, 3H), 2.32 (s, 3H), 2.26 (s, 3H).
IR (v, cm-1): 4322.16, 2944.32, 2924.68, 2808.65, 2360.36, 231.84, 1698.12, 1669.50,
1641.50, 1451.72, 1008.20, 744.17, 698.07. ESI-HRMS m/z calculated for
C27H29N2O3 [M + H]+ 429.2178, found 429.2148; HPLC (tR = 16.926 min, purity =
97.7 %).
2-((4-(4-Fluorophenyl)piperazin-1-yl)methyl)-3,7,8-trimethylnaphtho[1,2-b]furan-4,5
-dione (6p). Following the above procedure, treatment of
3
with
o
4-fluorophenylpiperazin for 3 h at 55 C, gave the crude product, which was purified
through column chromatography (eluent: petroleum ether/EtOAc 1 : 5) to afford 6p as
25

ACCEPTED MANUSCRIPT
a red solid: yield 26%. m.p. 216-219 oC. 1H NMR (300 MHz, CDCl3) δ: 7.82 (s, 1H),
7.51 (s, 1H), 6.96 (t, J = 8.7 Hz, 2H), 6.91-6.85 (m, 2H), 3.67 (s, 2H), 3.17 (s, 4H),
2.73 (s, 4H), 2.36 (s, 3H), 2.32 (s, 3H), 2.30 (s, 3H). IR (v, cm-1): 339.55, 2820.84,
2361.30, 234.28, 1668.60, 1601.13, 1236.81, 1005.05, 75.99, 686.69, 482.29.
ESI-HRMS m/z calculated for C26H26FN2O3 [M + H]+ 433.1927, found 433.1921.
HPLC (tR = 4.099 min, purity = 91.6 %).
2-((4-(Morpholinomethyl)phenyl)amino)-3,7,8-trimethyl-naphtho[1,2-b]furan-4,5-dio
ne (6q). Following the above procedure, treatment of
3
with
o
4-(morpholinomethyl)phenylamine for 2.5 h at 55 C, gave the crude product, which
was purified through column chromatography (eluent: petroleum ether/EtOAc 1 : 2)
to afford 6q as a red solid (51 mg, 33 %). 1H NMR (300 MHz, CDCl3) δ: 7.82 (s, 1H),
7.43 (s, 1H), 7.18 (d, J = 8.4 Hz, 2H), 6.69 (d, J = 8.4 Hz, 2H), 4.3 (s, 2H), 3.71 (t, J =
13
4.6 Hz, 4H), 3.42 (s, 2H), 2.44 (s, 4H), 2.36 (s, 3H), 2.32 (s, 3H), 2.28 (s, 3H). C
NMR (75 MHz, CDCl3) δ: 180.4, 175.4, 159.8, 150.5, 146.6, 145.3, 139.6, 131.5,
130.4, 129.8, 127.0, 126.2, 123.2, 121.0, 117.9, 112.9, 66.9, 63.0, 53.5, 39.2, 20.2,
19.2, 8.9. ESI-HRMS m/z calculated for C27H29N2O4 [M + H]+ 445.2127, found
445.2104. HPLC (tR = 5.698 min, purity = 96.9 %).
2-Formyl-3,7,8-trimethylnaphtho[1,2-b]furan-4,5-dione (4). To a solution of N,N-
dimethylformamide (0.37 mL, 4.72 mmol) was dropwise added phosphorus
oxychloride (0.43 mL, 4.72 mmol) at 0 oC over 30 min. Then, 2 (113 mg, 0.47 mmol)
in DMF was added and the reaction temperature was rise to 65 oC over 4 h. After that,
the mixture was poured into ice water and the saturated sodium hydroxide solution
was used to adjust the pH to 9, afterwards, adjusted to neutral by the addition of
hydrochloric acid. The precipitate was filtered and was purified by silica gel column
chromatography (eluent: Petroleum ether/EtOAc 10 : 1) to afford 4 (55 mg, 44%) as
1
orange red solid. m.p. 192-194 oC. H NMR (300 MHz, CDCl3) δ: 9.92 (s, 1H), 7.83
(s, 1H), 7.52 (s, 1H), 2.37 (d, J = 4.4 Hz, 3H), 2.32 (d, J = 4.4 Hz, 3H), 2.28 (s, 3H).
26

ACCEPTED MANUSCRIPT
2-Carboxyl-3,7,8-trimethylnaphtho[1,2-b]furan-4,5-dione (5). A mixture of 4 (45 mg,
0.17 mmol), 30% H2O2 (0.07 mL, 0.68 mmol), Na2HPO4 buffer (96 mg, 0.68 mmol, 2
mL H2O, pH = 2) and sodium chlorite (15 mg, 0.17 mmol) in acetonitrile (10 mL)
was stirred at room temperature for 8 h. Then the solvent was partly distilled and the
precipitate was filtered. The crude product was ready for the next step without further
purification.
2-(4-Fluorobenzylcarbamoyl)-3,7,8-trimethylnaphtho[1,2-b]furan-4,5-dione (7b). To
a stirred solution of 5 (30 mg, 0.106 mmol) in dry dichloromethane (15 mL) was
added 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 67 mg,
0.4 mmol) and 1-hydroxybenzotriazole (HOBT, 47 mg, 0.4 mmol) at room
temperature for 1 h. Then 4-fluorobenzylamine (26.5 mg, 0.212 mmol) was added and
stirred for another 4 h at room temperature. After being cooled, the mixture was
poured into ice water and extracted with EtOAc. The combined organic layer was
washed with NaHCO3 solution and brine solution, dried over Na2SO4, and
concentrated under vacuum. The residue was purified by silica gel column
chromatography (eluent: Petroleum ether/EtOAc 4 : 1) to afford 7b (18 mg, 45%) as
red solid. m.p. 232-233 oC. 1H NMR (300 MHz, CDCl3) δ: 7.88 (s, 1H), 7.50 (s, 1H),
7.40 (dd, J1 = 8.4 Hz, J2 = 5.4 Hz, 2H), 7.10 (t, J = 8.6 Hz, 2H), 6.71 (s, 1H), 4.66 (d,
J = 6.1 Hz, 2H), 2.71 (s, 3H), 2.40 (s, 3H), 2.36 (s, 3H). IR (v, cm-1): 316, 2360, 231,
1675, 1507, 1211, 841, 668. ESI-HRMS m/z calculated for C23H19FNO4 [M + H]+
392.1293, found 392.1299; HPLC (tR = 12.988 min, purity = 95.4 %).
2-(4-Fluorophenylcarbamoyl)-3,7,8-trimethylnaphtho[1,2-b]furan-4,5-dione
(7a).
Following the general procedure with 7b, treatment of 5 with p-fluoroaniline for 4 h
at room temperature, gave the crude product, which was purified through column
chromatography (eluent: petroleum ether/EtOAc 2 : 1) to afford 7a as a red solid:
o
1
yield 44%. m.p. 229-231 C. H NMR (300 MHz, CDCl3) δ: 8.03 (s, 1H), 7.86 (s,
1H), 7.67-7.63 (m, 2H), 7.09 (t, J = 8.4 Hz, 2H), 2.69 (s, 3H), 2.41 (s, 3H), 2.4 (s, 3H)
ppm. IR (v, cm-1): 316.99, 2360.32, 231.72, 1675.72, 1507.70, 1211.84, 841.05,
27

ACCEPTED MANUSCRIPT
668.91. ESI-HRMS m/z calculated for C22H17FNO4 [M + H]+ 378.1142, found
378.1120. HPLC (tR = 12.988 min, purity = 95.4 %).
2-(4-Benzylpiperazine-1-carbonyl)-3,7,8-trimethylnaphtho[1,2-b]furan-4,5-dione (7c).
Following the general procedure with 7b, treatment of 5 with 1-benzylpiperazine for 3
h at room temperature, gave the crude product, which was purified through column
chromatography (eluent: petroleum ether/EtOAc 2 : 1) to afford 7c as a red solid:
yield 31%. m.p. 230-233 oC. 1H NMR (300 MHz, CDCl3) δ: 7.79 (s, 1H), 7.39 (s, 1H),
7.29-7.23 (m, 5H), 3.68 (s, 4H), 3.52 (s, 2H), 2.49 (s, 4H), 2.37 (s, 3H), 2.31 (s, 3H),
2.27 (s, 3H). IR (v, cm-1): 317.3, 2918.4, 2802.67, 1674.95, 1613.17, 1455.75, 143.19,
126.67, 742.96, 699.54. ESI-HRMS m/z calculated for C27H27N2O4 [M + H]+
443.1965, found 443.1955. HPLC (tR = 12.438 min, purity = 96.3 %).
2-(4-(4-Fluorophenyl)piperazine-1-carbonyl))-3,7,8-trimethylnaphtho[1,2-b]furan-4,
5-dione (7d). Following the general procedure with 7b, treatment of 5 with
4-fluorophenylpiperazine for 3 h at room temperature, gave the crude product, which
was purified through column chromatography (eluent: petroleum ether/EtOAc 2 : 1)
to afford 7d as a red solid: yield 40%. m.p. 225-227 oC. 1H NMR (300 MHz, CDCl3)
δ: 7.89 (s, 1H), 7.49 (s, 1H), 7.06-6.88 (m, 4H), 3.91 (s, 4H), 3.22 (s, 4H), 2.50 (s,
3H), 2.40 (s, 3H), 2.36 (s, 3H). IR (v, cm-1): 321.58, 2918.03, 2360.73, 1674.4,
1621.48, 1510.07, 1458.46, 1441.93, 1226.36, 823.36, 481.02. ESI-HRMS m/z
calculated for C26H24FN2O4 [M + H]+ 447.1715, found 447.1704. HPLC (tR = 11.046
min, purity = 96.0 %).
5.2. Biological experiments.
5.2.1. In vitro enzyme reduction assay.
Compounds were monitored as NQO1 or other one-electron oxidoreductases
substrates using UV-visible spectroscopy by adapting a previous assay [42] to well
plate reader and 96-well quartz plate. The reduction rates were determined using an
NADPH recycling assay and the enzymes (NQO1, CPR, Trx and CBR1, purchased
from Sigma), in which NADPH oxidation to NADP+ was monitored by absorbance
28