Development of novel amino-quinoline-5,8-dione derivatives as NAD(P)H:quinone oxidoreductase 1 (NQO1) inhibitors with potent antiproliferative activities

Article abstract of DOI:10.1016/j.ejmech.2018.05.025

Fourteen novel amino-quinoline-5,8-dione derivatives (6a-h and 7a-h) were designed and synthesized by coupling different alkyl- or aryl-amino fragments at the C6- or C7-position of quinoline-5,8-dione. All target compounds showed antiproliferative potency in the low micromolar range in both drug sensitive HeLaS3 and multidrug resistant KB-vin cell lines. Compounds 6h, 6d, 7a, and 7d exhibited more potent antiproliferative effects than the other compounds. Especially, compounds 6d and 7d displayed NQO1-dependent cytotoxicity and competitive NQO1 inhibitory effects in both drug sensitive HeLaS3 and multidrug resistant KB-vin cell lines. Furthermore, compounds 6h, 6d, 7a, and 7d induced a dose-dependent lethal mitochondrial dysfunction in both drug sensitive HeLaS3 and multidrug resistant KB-vin cells by increasing intracellular reactive oxygen species (ROS) levels. Notably, compound 7d selectively inhibited cancer cells, but not non-tumor liver cell proliferation in vitro, and significantly triggered HeLaS3 cell apoptosis by regulating apoptotic proteins of Bcl-2, Bax, and cleaved caspase-3 in a dose-dependent manner. Our findings suggest that these novel C6- or C7-substituted amino-quinoline-5,8-dione derivatives, such as 7d, could be further developed in the future as potent and selective antitumor agents to potentially circumvent multi-drug resistance (MDR).

Full text of DOI:10.1016/j.ejmech.2018.05.025

Accepted Manuscript
Development of novel amino-quinoline-5,8-dione derivatives as NAD(P)H:quinone
oxidoreductase 1 (NQO1) inhibitors with potent antiproliferative activities
Yong Ling, Qiu-Xing Yang, Yu-Ning Teng, Shi Chen, Wei-Jie Gao, Jing Guo, Pei-Ling
Hsu, Yue Liu, Susan L. Morris-Natschke, Chin-Chuan Hung, Kuo-Hsiung Lee
PII:
S0223-5234(18)30435-5
10.1016/j.ejmech.2018.05.025
EJMECH 10436
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 3 April 2018
Revised Date: 16 May 2018
Accepted Date: 16 May 2018
Please cite this article as: Y. Ling, Q.-X. Yang, Y.-N. Teng, S. Chen, W.-J. Gao, J. Guo, P.-L. Hsu, Y.
Liu, S.L. Morris-Natschke, C.-C. Hung, K.-H. Lee, Development of novel amino-quinoline-5,8-dione
derivatives as NAD(P)H:quinone oxidoreductase 1 (NQO1) inhibitors with potent antiproliferative
activities, European Journal of Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2018.05.025.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Development of novel amino-quinoline-5,8-dione derivatives as NAD(P)H:quinone
oxidoreductase 1 (NQO1) inhibitors with potent antiproliferative activities
Yong Linga,b,1, Qiu-Xing Yanga,1, Yu-Ning Tengb,c, Shi Chena, Wei-Jie Gaoa, Jing Guoa, Pei-Ling Hsub, Yue Liua, Susan
L. Morris-Natschkeb, Chin-Chuan Hungc,*, Kuo-Hsiung Leeb,d*
aSchool of Pharmacy and Jiangsu Province Key Laboratory for Inflammation and Molecular Drug Target, Nantong
University, Nantong 226001, PR China.
bNatural Products Research Laboratories, UNC Eshelman School of Pharmacy, University of North Carolina, Chapel
Hill, North Carolina 27599, United States
cDepartment of Pharmacy, College of Pharmacy, China Medical University, Taichung 40402, Taiwan
dChinese Medicine Research and Development Center, China Medical University and Hospital, Taichung 40402,
Taiwan
Abstract: Fourteen novel amino-quinoline-5,8-dione derivatives (6a-h and 7a-h) were designed and
synthesized by coupling different alkyl- or aryl-amino fragments at the C6- or C7-position of
quinoline-5,8-dione. All target compounds showed antiproliferative potency in the low micromolar
range in both drug sensitive HeLaS3 and multidrug resistant KB-vin cell lines. Compounds 6h, 6d, 7a,
and 7d exhibited more potent antiproliferative effects than the other compounds. Especially,
compounds 6d and 7d displayed NQO1-dependent cytotoxicity and competitive NQO1 inhibitory
effects in both drug sensitive HeLaS3 and multidrug resistant KB-vin cell lines. Furthermore,
compounds 6h, 6d, 7a, and 7d induced a dose-dependent lethal mitochondrial dysfunction in both
drug sensitive HeLaS3 and multidrug resistant KB-vin cells by increasing intracellular reactive
oxygen species (ROS) levels. Notably, compound 7d selectively inhibited cancer cells, but not
non-tumor liver cell proliferation in vitro, and significantly triggered HeLaS3 cell apoptosis by
regulating apoptotic proteins of Bcl-2, Bax, and cleaved caspase-3 in a dose-dependent manner. Our
findings suggest that these novel C6- or C7-substituted amino-quinoline-5,8-dione derivatives, such as
7d, could be further developed in the future as potent and selective antitumor agents to potentially
circumvent multi-drug resistance (MDR).
Keywords: Antiproliferative activity; Quinone oxidoreductase 1 (NQO1); Amino-quinoline-5,8-dione;
Reactive oxygen species (ROS); Multidrug resistance; Apoptosis
1These authors contributed equally to this work.
*Corresponding author.
E-mail addresses: khlee@unc.edu (K.H. Lee), cc0206hung@gmail.com (C.C. Hung).

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1. Introduction
Due to aberrant metabolic events and abnormal proliferative growth phenotypes, reactive oxygen
species (ROS) levels are commonly elevated in cancer cells [1-3]. To cope with an increase in
intrinsic oxidative stress, cancer cells augment their antioxidant capacity through upregulation of
antioxidant enzymes [4]. In doing so, cancer cells become more dependent on the elevated antioxidant
capacity for growth and survival, which makes them susceptible when oxidative stress is further
elevated through production of more ROS and/or inhibition of elevated antioxidant enzymes [4,5].
Therefore, a feasible strategy in cancer chemotherapy is to selectively target cancer cells based on the
different redox states between normal and tumor cells [1,6-8], particularly when such targeting
involves a redox adaptive mechanism that confers drug resistance [2].
In this context, inhibition of NAD(P)H:quinone oxidoreductase 1 (NQO1) has emerged as a
promising redox-modulating strategy for anticancer therapy. NQO1 is a ubiquitous flavoenzyme that
catalyzes the two-electron reduction of quinones to hydroquinones. It is intimately involved with
cancer and upregulated in several human tumor cells, such as pancreas, colon, breast, lung, liver,
stomach, kidney, and ovaries, at levels of up to 50-fold greater than in normal tissue [9,10]. Moreover,
NQO1 is found in the cytosol, Golgi complex, nucleus, mitochondria, cellular membrane, and
endoplasmic reticulum, as well as extracellularly [11–14]. The NQO1 and glutathione systems are the
two major cellular thiol antioxidant systems that can be targeted for anticancer therapy [15-18]. Many
human cancers can reportedly be killed selectively through rapid ROS generation mediated by NQO1
bioreduction (Fig. 1).
Fig. 1. NQO1-mediated redox cycling of quinolinediones.
Recent findings suggest that some quinolinediones are excellent substrates of NQO1 and are
selectively cytotoxic to cancer cell lines that overexpress NQO1, but not to normal cell lines [17−23].
These findings form the basis for the development of novel quinoline-5,8-dione derivatives as
potential NQO1-directed cytotoxicity agents, which efficiently regulate the intracellular human NQO1
dependent redox cycling and thus the ROS generated in the progress (Fig. 1). Moreover, small
substituents such as amine groups at the C-6 or C-7 positions of the quinoline-5,8-dione moiety
favorably impacted binding to the active site of NQO1 [16,17,24,25]. In this study, we coupled
different alkyl- or aryl-amino fragments to the C6- or C7-position of quinoline-5,8-dione to design
novel amino-quinoline-5,8-dione derivatives, and compared the cytotoxic effects of these compounds.
In addition, because most quinolinedione derivatives usually have poor water solubility, we introduced
hydrophilic groups at the end of arylamino fragments to improve the lipid-aqueous partition
coefficient. Herein, we report our synthesis of novel amino-quinoline-5,8-dione derivatives (6a‒6h
and 7a‒7h, Table 1), as well as evaluation of their cytotoxicity effects and mechanisms in multiple

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cancer cell lines.
2. Results and discussion
2.1 Chemistry
Scheme 1. Reagents and conditions: (a) iodobenzene diacetate, CH3CN:H2O = 2:1, 0 , 2h, 86%; (b) MeOH, rt, 6 h,
31-43%; (c) NaN3, DMF, rt, 2 h; (d) PPh3, anhydrous THF, rt, 2 h; (e) quinolinedione 4, MeOH, rt, 6 h, 22-41%.
Scheme 1 summarizes the syntheses of the quinolinedione derivatives 6a‒6h and 7a‒7h.
Quinoline-5,8-dione (4) was prepared from 5-hydroxyquinoline (3) by oxidation with iodobenzene
diacetate (PIDA) in a mixture of CH3CN:H2O (2:1) according to Barret’s method [26]. Compound 4
was subsequently treated with different substituted 2-(1H-indol-3-yl)ethanamines (5a‒5c) or anilines
(5d‒5f) in MeOH to obtain the target compounds 6a‒6c and 7a‒7c or 6d‒6f and 7d‒7f, respectively.
This is a non-regioselective Michael addition to give the hydroquinone followed by an autoxidation
back to quinone. Often, only catalytic base and air are needed for such oxidations [27]. Fortunately,
these two regioisomers were easy separated by column chromatography (CH2Cl2/MeOH= 20/1), and
the major isomers were 6a‒6f. In addition, treatment of (E)-methyl 3-(4-(bromomethyl)phenyl)
acrylate 8a or (E)-methyl 3-(3-(bromomethyl)phenyl) acrylate 8b in DMF with NaN3 produced
intermediate 9a,b. Then intermediate 9a,b was treated with PPh3 in THF to furnish compounds 10a,b,
which were further reacted with quinoline-5,8-dione 4 in the presence of MeOH to produce target

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compound 6g, 7g or 6h, 7h. All target compounds were purified by column chromatography, and their
structures were confirmed by 1H NMR, 13C NMR, MS, and HRMS. Each target compound was >95%
pure as determined by high-performance liquid chromatography and could be used for subsequent
experiments.
2.2 Antiproliferative activity against both sensitive and resistant human cervical cancer cells
To evaluate the antiproliferative activity of the target compounds, we initially tested their effects on
the proliferation of parental sensitive cervical epithelioid carcinoma cells HeLaS3 and multi-drug
resistant human cervical cancer cells KB-vin (both cell lines are used to investigate the cytotoxicity
effect and antidrug resistance of potential drugs in vitro) by the SRB assay using paclitaxel as a
positive control. The IC50 values of individual compounds against each tumor cell line are presented in
Table 1. The results showed that all target compounds displayed antiproliferative potency in the low
micromolar range against both drug sensitive HeLaS3 and multidrug resistant KB-vin cell lines. Some
compounds, such as 6h, 6d, 7a, and 7d, exhibited significant antiproliferative effects with IC50 values
between 0.80 and 1.52 µM. These four compounds displayed comparable or greater potency than
paclitaxel (IC50 = 1.01 µM) against multidrug resistant KB-vin cells. Structurally, the addition of a
4-(4-methylpiperazin-1-yl)phenylamino group to position-6 (6d) or -7 (7d) of quinoline-5,8-dione led
to the greatest antiproliferative potency against both HeLaS3 (IC50 0.80 and 0.59 µM, respectively)
and KB-vin (IC50 1.52 and 0.97 µM, respectively) cell lines. Compounds 6h (6-amino-(3-
methylphenyl)acrylic ester) and 7a (7-aminoethylindole) showed comparable potency to 7d against
KB-vin, but not HeLasS3, cells. Indeed, all other compounds were significantly less potent (IC50
3.40−6.24 µM) against the two tested cancer cell lines, even those with only slight structural
differences (e.g., 6e and 7e have a 2-methoxy group in addition to the 4-(4-methylpiperazin-1-yl)
group found on the phenylamino side chain of 6d and 7d.
Table 1. In vitro antiproliferative data of 6a-h and 7a-h against HeLaS3 and KB-vin cells.
IC50a (µM)
HeLaS3b KB-vinc
6.24 1.12 5.12 0.23
IC50 (µM)
Compounds
Compounds
HeLaS3
KB-vin
6a
6b
6c
6d
6e
6f
±
±
7a
7b
7c
7d
7e
7f
4.53 ± 0.35
6.12 ± 0.33
6.21 ± 0.08
0.59 ± 0.03
4.37 ± 0.36
5.07 ± 0.10
5.67 ± 0.62
4.99 ± 0.16
1.10 ± 0.12
4.00 ± 0.39
5.98 ± 0.58
0.97 ± 0.01
4.69 ± 0.29
6.19 ± 0.59
5.62 ± 0.26
4.27 ± 0.29
1.01 ± 0.11
5.50 0.61
6.20 0.08
±
±
6.64 ± 0.04
4.92 ± 0.62
0.80 0.02
1.52 0.04
±
±
5.26 0.34
4.03 0.26
±
±
5.16 0.22
5.23 0.36
±
±
5.84 0.49
3.40 0.35
±
6g
6h
±
7g
7h
5.36 0.22
0.91 0.03
±
±
Paclitaxel 0.013 ± 0.0014
a
The inhibitory effects of individual compounds on the proliferation of cancer cell lines were
determined by the SRB assay. The data are expressed as the mean ± SD of three independent
experiments data.
b Human cervical epithelioid carcinoma cell line.
c Multi-drug resistant human cervical cancer cell line

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2.3 NQO1-dependency assessments
120
100
80
60
40
20
0
120
100
80
60
40
20
0
HelaS3
KB-vin
66dd
6d
6d
6d + 10 µM Dicumarol
6d + 10 µM DIC
6d + 10 µM Dicumarol
6d + 10 µM DIC
6d + 40 µM N-Acetylcys
6d + 40 µM NAc
6d + 40 µM N-Acetylcyste
6d + 40 µM NAc
0.001
0.01
0.1
1
0.001
0.01
0.1
1
Concentrations(µM)
Concentrations (µM)
120
100
80
60
40
20
0
120
100
80
60
40
20
0
7d
7d
7d
7d
77dd++101µ0MµDMicuDmIaCrol
7d + 40 µM N-Acetylcys
7d + 40 µM NAc
7d + 10 µM Dicumarol
7d + 10 µM DIC
7d + 40 µM N-Acetylcystein
7d + 40 µM NAc
0.001
0.1
1
0.01 Concentrations (µM)
0.001
0.01
0.1
1
Concentrations (µM)
Lapachone
Lapachone
120
100
80
60
40
20
0
120
100
80
60
40
20
0
Lapachone
Lapachone
Lapachone + 10 µM Dicumaro
Lapachone + 10 µM DIC
Lapachone + 10 µM Dicuma
Lapachone + 10 µM DIC
Lapachone + 40 µM N-Acetylc
Lapachone + 40 µM NAc
Lapachone + 40 µM N-Acety
Lapachone + 40 µM NAc
0.001
0.01
0.1
1
0.001
0.01
1
Concentrat0io.1ns (µM)
Concentrations (µM)
Fig. 2. Evaluations of NQO1-dependent cytotoxicity of testing compounds. The cell viability was evaluated by
combination treatment of 10 µM DIC or 40 µM NAc with testing compounds, using SRB assay after 72 h treatment.
Within the drug concentration range tested, the compounds 6d and 7d showed the most potent
cytotoxicities against both drug sensitive HeLaS3 and multidrug resistant KB-vin cell lines. To deepen
our knowledge about the mechanism of action of compounds 6d and 7d, we examined their potential
NQO1-dependent cytotoxicities, with or without dicoumarol (DIC, an NQO1 inhibitor) and
N-acetylcysteine (NAc, a free radical scavenger). Based on the survival curves (Fig. 2), the cell
viabilities of compound-treated HeLaS3 and KB-vin cells were significantly increased by the addition
of DIC and NAc. The cytotoxic IC50 values were greater than 3 µM against both cell lines in the
presence of 6 or 7, DIC, and NAc, but much lower in the presence of compound alone (6d only: 0.8

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µM, HeLaS3 and 1.52 µM, KB-vin; 7d only: 0.59 µM HeLaS3 and 0.97 µM, KB-vin). These results
suggested that the cytotoxic effects of 6d and 7d were NQO1-specific in both drug sensitive HeLaS3
and multidrug resistant KB-vin cell lines.
2.4 NQO1 inhibitory activities and kinetics
Fig. 3. NQO1 activity of (a) HeLa cells (b) KB-vin cells after compound treatment. The HeLa and KB-vin cells were
treated with serial concentrations of 6h, 6d, 7a, and 7d for 24 h. NQO1 activity was detected by the NQO1 activity
assay kit (catalog number: ab184867), and was significantly altered after treatment with 6h, 6d, 7a, and 7d for 24 h.
Table 2. Effects of 6h, 6d, 7a, and 7d on NQO1 activity in HeLaS3 (parental) and KB-vin (MDR)
cancer cell lines.
IC50 (µM)
Compound
HeLaS3
KB-vin
6.25
±
±
±
±
1.25
0.90
0.07
0.08
0.55
0.53
0.56
0.57
±
±
±
±
0.01
0.05
0.01
0.02
7a
6h
6d
7d
5.25
0.47
0.44
Based on the encouraging antiproliferative activity of 6a-h and 7a-h against HeLaS3 and KB-vin
cells, we further tested a selected group of compounds (6h, 6d, 7a, and 7d) for in vitro inhibitory
activity against NQO1. As shown in Fig. 3, the NQO1 activities in both drug sensitive HeLaS3 and
multidrug resistant KB-vin cell lines were inhibited dose-dependently after 24 h treatment with 6h, 6d,
7a, and 7d. Particularly, compounds 6d and 7d were more potent NQO1 inhibitors than 6h and 7a in
drug sensitive HeLaS3 cells. On the other hand, 6h, 6d, 7a, and 7d showed similar NQO1 inhibitory
effects in multidrug resistant KB-vin cells (Table 2). Therefore, these four compounds displayed
dose-dependent NQO1 inhibitory activity.

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A
B
10
8
10
Control
Control
7d 1 µM
6d 1.0 µM
7d 2 µM
6d 2.0 µM
Control
Control
7d 1 µM
7d 1.0 µM
7d 2 µM
7d 2.0 µM
8
6
4
2
0
6
4
2
0
-0.10
-0.05
0.00
0.05
0.10
0.15 -0.10
-0.05
0.00
0.05
0.10
0.15
1/menadione (µM)-1
1/menadione (µM)-1
Fig. 4. Enzyme kinetics for compound 6d and 7d using recombinant human NQO1. Lineweaver-Burk plot for NQO1
inhibition demonstrated that 6d and 7d competitively inhibited NQO1 activity.
The NQO1 inhibitory kinetics of 6d and 7d indicated competitive inhibition as demonstrated by the
Lineweaver-Burk plots (Fig. 4). As the concentration of 6d increased, the maximum rate (Vmax
)
remained the same (12.5±1.2 nmol/mg protein/20s) and the affinity (Km) increased (no treatment
control: 30.22 ± 2.5 µM; addition of 1.0 µM 6d: 45.27 ± 5.33 µM; addition of 2.0 µM 6d: 60.15 ±
3.51 µM). In terms of 7d, as the concentration of 7d increased, the maximum rate (Vmax) remained the
same (11.03±1.5 nmole/mg protein/20s) and the affinity (Km) increased (no treatment control: 20.30 ±
0.97 µM; addition of 1.0 µM 7d: 40.17 ± 2.31 µM; addition of 2.0 µM 7d: 55.17 ± 1.49 µM). These
results indicated that 6d and 7d are competitive inhibitors of NQO1.
2.5 Treatment with quinolinedione derivatives increases ROS levels
We next examined whether the killing effect of quinolinedione derivatives on cervical cancer cells
involved increasing ROS levels. ROS levels in both drug sensitive HeLaS3 and multidrug resistant
KB-vin cell lines were assessed by flow cytometry using the redox-sensitive fluorescent probe
2′,7′-dichlorofluorescein diacetate (DCFH-DA). As shown in Fig. 5, the ROS production increased
dose-dependently in both drug sensitive HeLaS3 and multidrug resistant KB-vin cell lines after 24 h
treatment with 6h, 6d, 7a, and 7d. These findings suggested that treatment with 6h, 6d, 7a, and 7d
lead to increased oxidative stress in both drug sensitive HeLaS3 and multidrug resistant KB-vin cell
lines, which causes significant cytotoxicity in tumor cells.

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Fig. 5. Reactive oxygen species (ROS) generation of (a) HeLa cells (b) KB-vin cells after compound treatment. The
HeLa and KB-vin cells were treated with serial concentrations of PL, 6h, 6d, 7a, and 7d for 24 h. The generated ROS
were detected by the ROS-GloTM H2O2 assay kit (catalog number: G8821), and increased significantly after treatment
with 6h, 6d, 7a, and 7d for 24 h.
2.4 Quinolinedione derivatives induce mitochondrial dysfunction
It is well known that mitochondria are central to the regulation of apoptosis. Loss of mitochondrial
membrane potential (△ψm) is catastrophic for cells and leads to the release of cytochrome C into the
cytosol [28]. The mitochondrial membrane potential of both drug sensitive HeLaS3 and multidrug
resistant KB-vin cell lines were significantly decreased in a dose-dependent manner after 24 h
treatment with 6h, 6d, 7a, and 7d (Fig. 6).
Fig. 6. Changes of mitochondrial membrane potential (MMP) in (a) HeLa cells (b) KB-vin cells after compound
treatment. The HeLa and KB-vin cells were treated with serial concentrations of PL, 6h, 6d, 7a, and 7d for 24 h. The
MMP was detected by the MITO-ID® membrane potential cytotoxicity kit (catalog number: ENZ-51019-KP002), and
decreased significantly after treatment with 6h, 6d, 7a, and 7d for 24 h in drug-resistant KB-vin cells.
2.5 The selectivity of quinolinedione derivatives to tumor cells
Given the strong antiproliferative and NQO1 inhibitory effects of 7d in vitro, we investigated the
selectivity profile by examining inhibitory effects on the growth of both cancer HeLaS3 cells and
normal endometrial epithelial cells (EECs). Treatment with increasing doses of 7d had no significant

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effect on the survival of non-tumor EECs, while the same treatment regimen induced major HeLaS3
cell death (Fig. 7), suggesting that 7d might possess selective antiproliferative activity against tumor
cells.
EEC
70
HeLaS3
60
50
40
30
20
10
0
0
0.1
0.3
1
3
Concentrations of 7d (µM)
Fig. 7. Inhibitory effects of 7d on the proliferation of HeLaS3 and EEC cells. Cells were incubated with the indicated
concentrations of 7d for 72 h. Cell proliferation was assessed using the MTT assay. Data are expressed as means ± SD
of the inhibition (%) from three independent experiments.
2.6 Quinolinedione derivatives induce apoptosis in HeLaS3 cells.
To determine whether the inhibitory effects of 7d on cervical carcinoma cell proliferation are
accompanied by enhanced cancer cell apoptosis, HeLaS3 cells were incubated with different
concentrtions of 7d for 72 h, and the percentages of apoptotic cells were determined by FITC-Annexin
V/PI double staining and flow cytometry. As shown in Fig. 8, the percentage of annexin V + apoptotic
HeLaS3 cells gradually increased for those cells exposed to increasing concentrations of 7d (12.8%
for 0.2 µM; 27.6% for 0.6 µM; and 66.3% for 1.8 µM), demonstrating that incubation with 7d induced
HeLaS3 cell apoptosis in a dose-dependent manner.

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Fig. 8. Compound 7d induces HeLaS3 cell apoptosis in vitro. HeLaS3 cells were incubated with the indicated
concentrations of 7d for 72 h, and the cells were stained with FITC-Annexin V/PI, followed by flow cytometry
analysis. (A) Flow cytometry analysis. (B) Quantitative analysis of apoptotic cells. Data are expressed as means ± SD
of the percentages of apoptotic cells from three independent experiments. *P< 0.01 vs control.
Fig. 9. (A) The expression of Bax, Bcl-2, Caspase 3, and β-actin was examined by western blot analysis. HeLaS3 cells
were incubated with or without 7d at the indicated concentrations for 72 h and the levels of protein expression were
detected using specific antibodies. Data shown are representative images of each protein for three separate experiments.
(B) Quantitative analysis of Bax, Bcl-2, and Caspase 3. The relative levels of each protein compared to control β-actin
were determined by densimetric scanning. Data are expressed as means ± SD from three separate experiments. *P <
0.01 vs respective control.
To further investigate the induction of apoptosis by 7d, we examined the expression of apoptotic
proteins Bax, Bcl-2, and the cleavage states of caspase-3, in response to 7d treatment (Fig. 9).
Sub-confluent HeLaS3 cervical carcinoma cells were treated with or without 7d for 72 h, and then
lysed and analyzed by western blot. β-Actin expression was used as an internal control. The treatment
with 7d dramatically increased the relative levels of pro-apoptotic Bax expression but reduced the
levels of anti-apoptotic Bcl-2 expression in a dose-dependent manner. Furthermore, compound 7d
treatment also induced more cleavage of caspase-3 than the control group (Fig. 8A and 8B). Taken
together, these results confirm that 7d treatment induced apoptosis in HeLaS3 cells.
3. Conclusion
In summary, we have designed and synthesized novel amino-quinoline-5,8-dione derivatives 6a-h and
7a-h by coupling different alkyl- or aryl-amino fragments at the C6- or C7-position of
quinoline-5,8-dione, and then evaluated their biological activities in various in vitro assays. All target
compounds showed antiproliferative potency in the low micromolar range in both drug sensitive
HeLaS3 and multidrug resistant KB-vin cell lines. Among them, compounds 6d and 7d exhibited
NQO1-dependent antiproliferative effects with IC50 values between 0.59 and 1.52 µM, as well as
competitive NQO1 inhibitory effects in both drug sensitive HeLaS3 and multidrug resistant KB-vin
cell lines. Furthermore, compounds 6h, 6d, 7a, and 7d induced a dose-dependent lethal mitochondrial
dysfunction in both drug sensitive HeLaS3 and multidrug resistant KB-vin cells by increasing
intracellular ROS levels. Notably, compound 7d selectively inhibited cancer cells but not non-tumor
liver cell proliferation in vitro, and significantly induced HeLaS3 cell apoptosis by reducing the

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anti-apoptotic protein levels of Bcl-2 and up-regulating the pro-apoptotic proteins of Bax and cleaved
caspase-3 in a concentration dependent manner. Taken together, these results suggest that these novel
C6- or C7-substituted amino-quinoline-5,8-dione derivatives, such as 7d, could be further developed
as potent and selective antitumor agents to potentially circumvent MDR.
4. Experimental section
The synthesized compounds were purified by column chromatography using silica gel (300–400
mesh) except for recrystallization and thin-layer chromatography (TLC) using silica gel 60 F254 plates
(250 mm; Qingdao Ocean Chemical Company, China). 1H NMR and 13C NMR spectra were recorded
with a Bruker Avance 400 MHz spectrometer at 300 K, using TMS as an internal standard. MS spectra
were recorded on a Mariner Mass Spectrum (ESI). High resolution mass spectra were recorded with a
hybrid LTQ FT (ICR 7T) (ThermoFisher, Bremen, Germany) mass spectrometer coupled with a
Waters Acquity H-class liquid chromatograph system. All solvents were reagent grade and, when
necessary, were purified and dried by standard methods. Solutions after reactions and extractions were
concentrated using a rotary evaporator operating at a reduced pressure of ca. 20 Torr. Organic
solutions were dried over anhydrous sodium sulfate.
4.1. General procedure for synthesis of quinoline-5,8-dione (4)
A solution of iodobenzene diacetate (10.35 mmol, 3.3 g) in 12 mL CH3CN:H2O (2:1) was added to
quinolin-5-ol (3) (3.45 mmol, 0.50 g) at 0
under N2 and stirred for 1h. After the reaction was
completed, water (40 mL) was added, and the solution was extracted with EtOAc (3 × 20 mL). The
organic phase was washed by brine and concentrated in vacuo to give 4 as a yellow solid, yield 82%.
MS (ESI) m/z = 160 [M+H]+ .
4.2. General procedure for synthesis of 10a, b
4.2.1. (E)-Methyl 3-(4-(aminomethyl)phenyl)acrylate (10a)
To a solution of (E)-methyl 3-(4-(bromomethyl)phenyl)acrylate 8a (0.1 g, 0.39 mmol) in 4 mL
DMF, NaN3 (38.2 mg, 0.59 mmol) was added and the mixture was stirred at rt for 2 h. After the
reaction was completed, 20 mL water were added and the solution was extracted with EtOAc (3 × 20
mL). The organic phase was washed by brine, dried over anhydrous sodium sulfate, and evaporated in
vacuo to give 9a, which was then dissolved in 3 mL THF. Triphenylphosphine was added and the
mixture was stirred at rt for 2h. Then, 1 mL water was added and the mixture was stirred for 1 h. The
solution was extracted with EtOAc (3 × 5 mL). The organic phase was combined and washed with
brine, dried over anhydrous sodium sulfate, and evaporated in vacuo. The residue was purified by
column chromatography (EA/PE = 2/1) on silica gel to afford 10a as a clear translucent oil in 52%
yield. MS (ESI) m/z = 192 [M+H]+.
4.2.1. (E)-Methyl 3-(3-(aminomethyl)phenyl)acrylate (10b)
Compound 10b was synthesized from (E)-methyl 3-(3-(bromomethyl)phenyl)acrylate 8b, according
to the synthetic procedure for 10a in a yield of 52%. MS (ESI) m/z = 192 [M+H]+ .

ACCEPTED MANUSCRIPT
4.3. General procedure for synthesis of 6a, 7a
Quinoline-5,8-dione (4) (149.1 mg, 0.94 mmol) was added to a solution of 2-(1H-indol-3-yl)
ethanamine (5a) (100.0 mg, 0.63 mmol) in MeOH (4 mL) at rt. The mixture was stirred at rt for 16 h
and evaporated in vacuo. The residue was purified by column chromatography (CH2Cl2/MeOH= 20/1)
to give 6a and 7a in yields of 40% and 26%, respectively.
4.3.1. 6-((2-(1H-Indol-3-yl)ethyl)amino)quinoline-5,8-dione (6a)
1
Analytical data for 6a: H NMR (d6-DMSO, 400 MHz): δ 10.87 (s, 1H, NH), 8.91 (m, 1H, Ar-H),
8.31 (m, 1H, Ar-H), 7.82 (m, 2H, NH, Ar-H), 7.78 (d, 1H, J = 8.0 Hz, Ar-H), 7.36 (m, 2H, Ar-H), 7.28
(m, 2H, Ar-H), 5.80 (s, 1H, CH), 3.53 (m, 2H, CH2), 3.06 (m, 2H, CH2). 13C NMR (d6-DMSO, 100
MHz): δ 180.8, 180.2, 153.0, 149.4, 147.1, 136.7, 133.9, 130.7, 128.9, 127.5, 123.6, 123.5, 121.5,
118.6, 111.9, 111.6, 99.1, 43.2, 23.7. MS (ESI) m/z =318 [M+H]+; MS (ESI) m/z = 318 [M+H]+;
HRMS (ESI): m/z calcd for C19H16N3O2: 318.1243; found: 318.1231 [M+H]+.
4.3.2. 7-((2-(1H-Indol-3-yl)ethyl)amino)quinoline-5,8-dione (7a)
1
Analytical data for 7a: H NMR (d6-CD3OD, 400 MHz): δ 8.83 (m, 1H, NH), 8.42 (m, 1H, Ar-H),
7.79 (dd, 1H, J = 4.0 Hz, J = 4.0 Hz, Ar-H), 7.60 (s, 1H, Ar-H), 7.31 (m, 2H, NH, Ar-H), 6.99-7.15 (m,
4H, Ar-H), 5.78 (s, 1H, CH), 3.51 (m, 2H, CH2), 3.04 (m, 2H, CH2). 13C NMR (d6-DMSO, 100 MHz):
δ 180.8, 180.2, 153.1, 149.4, 147.1, 136.7, 133.9, 130.8, 129.1, 127.6, 123.6, 123.5, 121.4, 118.6,
111.9, 111.6, 99.2, 43.1, 24.0. MS (ESI) m/z =318 [M+H]+; MS (ESI) m/z = 318 [M+H]+; HRMS
(ESI): m/z calcd for C19H16N3O2: 318.1243; found: 318.1241 [M+H]+.
4.4. General procedure for synthesis of 6b, 7b
Compounds
6b
and
7b
were
synthesized
from
quinoline-5,8-dione
(4),
2-(5-methoxy-1H-indol-3-yl)ethanamine (5b), according to the synthetic procedure for compounds 6a
and 7a in yields of 41% and 23%, respectively.
4.4.1. 6-((2-(5-Methoxy-1H-indol-3-yl)ethyl)amino)quinoline-5,8-dione (6b)[29]
1
Analytical data for 6b: H NMR (d6-DMSO, 400 MHz): δ 10.71(s, 1H, NH), 8.96 (m, 1H, Ar-H),
8.32 (d, 1H, J = 4.0 Hz, Ar-H), 7.72 (m, 2H, Ar-H, NH), 7.22 (m, 2H, Ar-H), 7.06 (d, 1H, J = 4.0 Hz,
Ar-H), 6.71 (m, 1H, Ar-H), 5.87 (s, 1H, CH), 3.76 (s, 3H, OCH3), 3.48 (m, 2H, CH2), 3.00 (m, 2H,
CH2). 13C NMR (d6-DMSO, 100 MHz): δ 182.0, 180.3, 155.0, 153.5, 149.2, 148.4, 134.3, 131.8,
128.0, 127.9, 127.9, 126.9, 124.2, 112.5, 111.6, 111.4, 101.1, 100.4, 55.8, 43.1, 23.8. MS (ESI) m/z =
348 [M+H]+; HRMS (ESI): m/z calcd for C20H18N3O3: 348.1348; found: 348.1344 [M+H]+.
4.4.2. 7-((2-(5-Methoxy-1H-indol-3-yl)ethyl)amino)quinoline-5,8-dione (7b)
Analytical data for 7b: 1H NMR (d6-CD3OD, 400 MHz): δ 8.79 (m, 1H, Ar-H), 8.36 (d, 1H, J = 4.0
Hz, Ar-H), 7.76 (m, 1H, Ar-H), 7.18 (m, 3H, J = 6.0 Hz, NH, Ar-H), 6.70 (m, 1H, Ar-H), 5.75 (s, 1H,
CH), 3.75 (s, 3H, OCH3), 3.45 (m, 2H, CH2), 3.08 (m, 2H, CH2). 13C NMR (d6-DMSO, 100 MHz): δ
182.0, 180.3, 155.0, 153.5, 149.3, 148.4, 134.3, 131.8, 128.0, 127.9, 126.9, 124.2, 112.5, 111.6, 111.4,

ACCEPTED MANUSCRIPT
101.1, 100.4, 55.7, 41.1, 23.8. MS (ESI) m/z = 348 [M+H]+; HRMS (ESI): m/z calcd for C20H18N3O3:
348.1348; found: 348.1343 [M+H]+.
4.5. General procedure for synthesis of 6c, 7c
Compounds 6c and 7c were synthesized from quinoline-5,8-dione (4), 2-(2-methyl-1H-indol-3-yl)
ethanamine (5c), according to the synthetic procedure for compounds 6a and 7a in yields of 38% and
25%, respectively.
4.5.1. 6-((2-(2-Methyl-1H-indol-3-yl)ethyl)amino)quinoline-5,8-dione (6c)
1
Analytical data for 6c: H NMR (d6-DMSO, 400 MHz): δ 10.78 (s, 1H, NH), 8.96 (d, 1H, J = 4.0
Hz, Ar-H), 8.33 (d, 1H, J = 8.0 Hz, Ar-H), 7.73 (m, 3H, NH, Ar-H), 7.24 (d, 1H, J = 8.0 Hz, Ar-H),
7.00 (m, 2H, Ar-H), 5.80 (s, 1H, CH), 3.39 (m, 2H, CH2), 2.98 (m, 2H, CH2), 2.34 (s, 3H, CH3). 13C
NMR (d6-DMSO, 100 MHz): δ 182.0, 180.3, 155.0, 149.2, 135.6, 134.4, 134.2, 132.8, 128.6, 127.8,
126.9, 117.6, 117.5, 110.9, 107.2, 101.0, 43.1, 22.8, 11.7. MS (ESI) m/z = 332 [M+H]+; HRMS (ESI):
m/z calcd for C20H18N3O2: 332.1399; found: 332.1390 [M+H]+.
4.5.2. 7-((2-(2-Methyl-1H-indol-3-yl)ethyl)amino)quinoline-5,8-dione (7c)
1
Analytical data for 7c: H NMR (d6-DMSO, 400 MHz): δ 10.78 (s, 1H, NH), 8.90 (d, 1H, J = 4.0
Hz, Ar-H), 8.31 (d, 1H, J = 8.0 Hz, Ar-H), 7.81 (m, 1H, Ar-H), 7.78 (m, 1H, Ar-H), 7.48 (d, 1H, J =
8.0 Hz, Ar-H), 7.24 (d, 1H, J = 8.0 Hz, Ar-H), 6.98 (m, 2H, Ar-H), 5.74 (s, 1H, CH), 3.41 (m, 2H,
CH2), 2.98 (m, 2H, CH2), 2.34 (s, 3H, CH3). 13C NMR (d6-DMSO, 100 MHz): δ 180.7, 180.2, 153.0,
149.3, 147.0, 135.6, 134.0, 132.8, 130.7, 129.0, 128.9, 128.6, 117.7, 117.6, 110.9, 107.3, 99.0, 43.2,
22.8, 11.6. MS (ESI) m/z = 332 [M+H]+; HRMS (ESI): m/z calcd for C20H18N3O2: 332.1399; found:
332.1389 [M+H]+.
4.6. General procedure for synthesis of 6d, 7d
Compounds 6d and 7d were synthesized from quinoline-5,8-dione (4), 4-(4-methyl
piperazin-1-yl)aniline (5d), according to the synthetic procedure for compounds 6a and 7a in yields of
39% and 24%, respectively.
4.6.1. 6-((4-(4-Methylpiperazin-1-yl)phenyl)amino)quinoline-5,8-dione (6d)
Analytical data for 6d: 1H NMR (d6-DMSO, 400 MHz): δ 9.14 (s, 1H, NH), 8.94 (d, 1H, J = 4.0 Hz,
Ar-H), 8.36 (d, 1H, J = 8.0 Hz, Ar-H), 7.72 (m, 1H, Ar-H), 7.19 (d, 2H, J = 8.0 Hz, Ar-H), 6.97 (d, 2H,
J =8.0 Hz, Ar-H), 6.09 (s, 1H, CH), 3.13 (m, 4H, CH2), 2.52 (m, 4H, CH2), 2.18 (s, 3H, CH3). 13C
NMR (d6-DMSO, 100 MHz): δ 182.1, 181.1, 154.9, 149.1, 148.8, 146.6, 134.4, 129.0, 128.0, 127.0,
125.2, 116.0, 102.4, 54.9, 48.3, 46.1. MS (ESI) m/z = 349 [M+H]+; HRMS (ESI): m/z calcd for
C20H21N4O2: 349.1665; found: 349.1660 [M+H]+.
4.6.2. 7-((4-(4-Methylpiperazin-1-yl)phenyl)amino)quinoline-5,8-dione (7d)
1
Analytical data for 7d: H NMR (d6-DMSO, 400 MHz): δ 9.30 (s, 1H, NH), 8.94 (s, 1H, Ar-H),
8.31(s, 1H, J = 8.0 Hz, Ar-H), 7.82 (s, 1H, Ar-H), 7.23 (d, 2H, J = 8.0 Hz, Ar-H), 7.02 (d, 2H, J = 8.0

ACCEPTED MANUSCRIPT
Hz, Ar-H), 5.99 (s, 1H, CH), 3.16 (m, 4H, CH2), 2.40 (m, 4H, CH2), 2.26 (m, 3H, CH3). 13C NMR
(d6-DMSO, 100 MHz): δ 181.6, 180.3, 153.1, 149.2, 147.7, 147.1, 133.7, 130.3, 129.1, 128.9, 125.3,
116.0, 100.5, 54.9, 48.3, 46.1. MS (ESI) m/z = 349 [M+H]+; HRMS (ESI): m/z calcd for C20H21N4O2:
349.1665; found: 349.1658 [M+H]+.
4.7. General procedure for synthesis of 6e, 7e
Compounds 6e and 7e were synthesized from quinoline-5,8-dione (4), 2-methoxy-4-
(4-methylpiperazin-1-yl)aniline (5e), according to the synthetic procedure for compounds 6a and 7a in
yields of 36% and 22%, respectively.
4.7.1. 6-((2-Methoxy-4-(4-methylpiperazin-1-yl)phenyl)amino)quinoline-5,8-dione (6e)
1
Analytical data for 6e: H NMR (d6-CD3OD, 400 MHz): δ 8.89 (d, 1H, J = 4.0 Hz, Ar-H), 8.48 (d,
1H, J = 8.0 Hz, Ar-H), 7.73 (m, 1H, Ar-H), 7.23 (d, 1H, J = 8.0 Hz, Ar-H), 6.68 (s, 1H, Ar-H), 6.62 (d,
1H, J = 8.0 Hz, Ar-H), 6.04 (s, 1H, CH), 4.53 (m, 4H, CH2), 3.88 (s, 3H, OCH3), 3.28 (m, 4H, CH2),
2.35 (s, 3H, NCH3). 13C NMR (d6-DMSO, 100 MHz): δ 182.0, 181.6, 155.0, 148.6, 147.9, 146.3,
141.9, 135.9, 130.3, 130.1, 128.1, 125.2, 124.0, 113.9, 103.4, 56.5, 55.1, 48.3, 46.1. MS (ESI) m/z =
379 [M+H]+; HRMS (ESI): m/z calcd for C21H23N4O3: 379.1770; found: 379.1759 [M+H]+.
4.7.2. 7-((2-Methoxy-4-(4-methylpiperazin-1-yl)phenyl)amino)quinoline-5,8-dione (7e)
Analytical data for 7e: 1H NMR (d6-CD3OD, 400 MHz): δ 8.85 (d, 1H, J = 4.0 Hz, Ar-H), 8.42 (m,
1H, Ar-H), 7.80 (m, 1H, Ar-H), 7.22 (d, 1H, J = 8.0 Hz, Ar-H), 6.68 (m, 2H, Ar-H), 5.94 (s, 1H, CH),
4.62 (m, 4H, CH2), 3.85 (s, 3H, OCH3), 2.61 (m, 4H, CH3), 2.34 (s, 3H, NCH3). 13C NMR (d6-DMSO,
100 MHz): δ 182.0, 181.7, 155.1, 155.0, 148.6, 146.3, 142.3, 134.7, 130.5, 130.0, 128.1, 127.4, 124.0,
114.9, 103.5, 56.8, 55.3, 49.6, 46.2. MS (ESI) m/z = 379 [M+H]+; HRMS (ESI): m/z calcd for
C21H23N4O3: 379.1770; found: 379.1767 [M+H]+.
4.8. General procedure for synthesis of 6f, 7f
Compounds
6f
and
7f
were
synthesized
from
quinoline-5,8-dione
(4),
4-([1,4'-bipiperidin]-1'-ylmethyl)aniline (5f), according to the synthetic procedure for compounds 6a
and 7a in yields of 38% and 23%, respectively.
4.8.1. 6-((4-([1,4'-Bipiperidin]-1'-yl)phenyl)amino)quinoline-5,8-dione (6f)
1
Analytical data for 6f: H NMR (d6-DMSO, 400 MHz): δ 9.30 (s, 1H, NH), 9.00 (m, 1H, Ar-H),
8.43 (m, 1H, Ar-H), 7.80 (m, 1H, Ar-H), 7.37 (m, 2H, Ar-H), 6.72 (d, 2H, J = 8.0 Hz, Ar-H), 6.20 (s,
1H, CH), 3.44 (s, 2H, CH2), 2.51 (m, 8H, CH2), 2.19 (s, 1H, CH), 1.94 (m, 2H, CH2), 1.68 (m, 2H,
CH2), 1.47 (m, 6H, CH2).13C NMR (d6-DMSO, 100 MHz): δ 182.1, 181.5, 155.0, 154.8, 148.7, 146.5,
135.2, 134.5, 132.8, 130.2, 128.1, 127.2, 124.2, 103.2, 62.5, 57.1, 53.0, 51.3, 29.4, 24.9, 22.5. MS
(ESI) m/z = 431 [M+H]+; HRMS (ESI): m/z calcd for C26H31N4O2: 431.2447; found: 431.2433
[M+H]+.
4.8.2. 7-((4-([1,4'-Bipiperidin]-1'-yl)phenyl)amino)quinoline-5,8-dione (7f)

ACCEPTED MANUSCRIPT
1
Analytical data for 6f: H NMR (d6-DMSO, 400 MHz): δ 9.37 (s, 1H, NH), 8.96 (m, 1H, Ar-H),
8.32 (m, 1H, Ar-H), 7.85 (d, 1H, J = 4.0 Hz, Ar-H), 7.42 (m, 2H, Ar-H), 6.46 (d, 2H, J = 8.0 Hz, Ar-H),
6.07 (s, 1H, CH), 3.46 (s, 2H, CH2), 2.50 (s, 8H, CH2), 2.19 (m, 1H, CH), 1.89 (m, 2H, CH2), 1.67 (m,
2H, CH2), 1.43 (m, 6H, CH2). 13C NMR (d6-DMSO, 300 MHz): δ 182.1, 174.9, 153.3, 147.6, 147.2,
135.7, 135.3, 133.8, 130.2, 128.9, 124.2, 101.4, 61.1, 55.9, 53.3, 49.9, 29.4, 24.9, 22.5. MS (ESI) m/z
= 431 [M+H]+; HRMS (ESI): m/z calcd for C26H31N4O2: 431.2447; found: 431.2433 [M+H]+.
4.9. General procedure for synthesis of 6g, 7g
Compounds 6g and 7g were synthesized from quinoline-5,8-dione (4), (E)-methyl
3-(4-(aminomethyl)phenyl)acrylate (10a), according to the synthetic procedure for compounds 6a and
7a in yields of 40% and 23%, respectively.
4.9.1. (E)-Methyl 3-(4-(((5,8-dioxo-5,8-dihydroquinolin-6-yl)amino)methyl)phenyl)acrylate (6g)
Analytical data for 6g: 1H NMR (d6-CD3OD, 400 MHz): δ 8.87 (m, 1H, Ar-H ), 8.43 (m, 1H, Ar-H),
7.59-7.71 (m, 4H, Ar-H), 7.40 (m, 2H, Ar-H), 6.51 (d, 1H, J = 16.0 Hz, CH), 5.77 (s, 1H, CH), 4.52 (s,
2H, CH2), 3.74 (s, 3H, OCH3). 13C NMR (d6-DMSO, 100 MHz): δ 181.9, 180.6, 167.0, 154.9, 149.0,
148.6, 144.8, 138.6, 134.6, 129.6, 128.1, 127.6, 127.5, 126.9, 118.6, 102.1, 52.0, 45.2. MS (ESI) m/z =
349 [M+H]+; HRMS (ESI): m/z calcd for C20H17N2O4: 349.1188; found: 349.1177 [M+H]+.
4.9.2. (E)-Methyl 3-(4-(((5,8-dioxo-5,8-dihydroquinolin-7-yl)amino)methyl)phenyl) acrylate (7g)
1
Analytical data for 7g: H NMR (d6-CD3OD, 400 MHz): δ 8.82 (d, 1H, J = 4.0 Hz, Ar-H), 8.38 (d,
1H, J = 4.0 Hz, Ar-H), 7.67 (m, 1H, Ar-H), 7.66 (s, 1H, Ar-H), 7.58 (s, 1H, CH), 7.52 (d, 1H, J = 6.0
Hz, Ar-H), 7.38 (m, 2H, Ar-H), 6.51 (d, 1H, J = 16.0 Hz, CH), 5.68 (s, 1H, CH), 4.50 (s, 2H, CH2),
3.73 (s, 3H, OCH3). 13C NMR (d6-DMSO, 100 MHz): δ 181.1, 180.1, 167.0, 153.0, 149.6, 147.2,
144.8, 138.5, 134.6, 133.9, 130.5, 129.6, 128.8, 127.5, 118.3, 100.2, 52.0, 45.3. MS (ESI) m/z = 349
[M+H]+; HRMS (ESI): m/z calcd for C20H17N2O4: 349.1188; found: 349.1177 [M+H]+.
4.10. General procedure for synthesis of 6h, 7h
Compounds 6h and 7h were synthesized from quinoline-5,8-dione (4), (E)-methyl
3-(3-(aminomethyl)phenyl)acrylate (10b), according to the synthetic procedure for compounds 6a and
7a in yields of 35% and 22%, respectively.
4.10.1. (E)-Methyl 3-(3-(((5,8-dioxo-5,8-dihydroquinolin-6-yl)amino)methyl)phenyl)acrylate (6h)
Analytical data for 6h: 1H NMR (d6-CD3OD, 400 MHz): δ 8.80 (m, 1H, Ar-H), 8.37 (d, 1H, J = 8.0
Hz, Ar-H), 7.62 (m, 3H, Ar-H, CH), 7.53 (s, 1H, Ar-H), 7.33 (m, 2H, Ar-H), 6.48 (d, 1H, J = 16.0 Hz,
CH), 5.74 (s, 1H, CH), 4.45 (s, 2H, CH2), 3.68 (s, 3H, OCH3). 13C NMR (d6-DMSO, 100 MHz): δ
181.9, 180.5, 167.0, 154.8, 148.9, 148.5, 144.8, 138.5, 134.5, 134.3, 129.6, 129.5, 128.0, 127.5, 127.4,
126.9, 118.4, 102.0, 51.9, 45.2. MS (ESI) m/z = 349 [M+H]+; HRMS (ESI): m/z calcd for C20H17N2O4:
349.1188; found: 349.1179 [M+H]+.
4.10.2. (E)-Methyl 3-(3-(((5,8-dioxo-5,8-dihydroquinolin-7-yl)amino)methyl)phenyl)acrylate (7h)

ACCEPTED MANUSCRIPT
Analytical data for 7h: 1H NMR (d6-CD3OD, 400 MHz): δ 8.81 (m, 1H, Ar-H), 8.35 (d, 1H, J = 4.0
Hz, Ar-H), 7.62 (m, 3H, Ar-H, CH), 7.58 (s, 1H, Ar-H), 7.38 (m, 2H, Ar-H), 6.53 (d, 1H, J = 16.0 Hz,
CH), 5.70 (s, 1H, CH), 4.53 (m, 2H, CH2), 3.74 (s, 3H, OCH3). 13C NMR (d6-DMSO, 100 MHz): δ
181.6, 169.5, 152.1, 149.4, 144.3, 134.8, 134.1, 131.6, 131.5, 129.0, 128.9, 128.5, 128.4, 126.9, 126.6,
117.7, 109.9, 99.8, 50.7, 45.3. MS (ESI) m/z = 349 [M+H]+; HRMS (ESI): m/z calcd for C20H17N2O4:
349.1188; found: 349.1182 [M+H]+.
4.11 Cell Culture
The multidrug resistant cancer cell line, KB-vin, was a generous gift from Dr. Kuo-Hsiung Lee
(University of North Carolina, Chapel Hill, U.S.A) and maintained with vincristine. The drug sensitive
cell line, HeLaS3, was purchased from Bioresource Collection and Research Center (Hsinchu,
Taiwan). Human endometrial epithelial cells (EEC) were purchased from Shanghai Institute of Cell
Biology (Shanghai, China). All cells were cultured in RPMI-1640 containing 10% FBS at 37°C in a
humidified atmosphere of 5% CO2.
4.12 Cell viability assay
The effects of test compounds on cell viability were evaluated by SRB and MTT assays. For the
SRB assay, after 72 h treatment with serial concentrations of test compounds, 50% trichloroacetic acid
(TCA) was added to fix cells for 30 min, and then the cells were washed with water and air-dried.
Subsequently, cells were stained with 0.04% sulforhodamine B (SRB) for 30 min and washed with 1%
acetic acid. The bound stain was solubilized in 10 mM Tris Base and the absorbance was measured by
BioTek Synergy HT Multi-Mode Microplate Reader at 515 nm. For the MTT assay, 100 µL of
different cancer cells were plated in a 96-well flat bottom tissue culture plate at a density of 104
cells/mL in DMEM medium containing 10% fetal bovine serum and allowed to adhere overnight at
37 °C in 5% CO2. The cells were treated by adding 100 µL of different compounds at various
concentrations into the respective well. The reagent DMSO (0.1%) was used as a negative control.
The cell viability assay (MTT assay) was carried out at 72 h after drug treatment. The concentration
that inhibited 50% of cellular growth (IC50 value) was calculated by the following formula: Cell
inhibition rate (%) = (1 − OD of treatment group/OD of control group) × 100%. To evaluate the
NQO1 dependency, cell viabilities were evaluated by combination treatment of 10 µM dicoumarol or
40 µM N-acetylcysteine with test compounds. The cell viability was evaluated by SRB assay after 72
h treatment. The cytotoxic potency of test compounds on cancer cells was expressed as IC50 values or
by a histogram (The bars are the mean ± SD). All data were derived from three independent
measurements.
4.13 NQO1 activity assay
The effects of test compounds (0.1 µM to 10 µM) on endogenous NQO1 activity were evaluated by
the NQO1 activity assay kit (Abcam, Cambridge, MA, USA). Briefly, cell pellets were solubilized in
1X extraction buffer on ice for 20 minutes and then centrifuged at 18,000 × g for 20 minutes at 4°C.
Supernatants were transferred to new tubes and protein concentrations were determined using the
Bio-Rad protein assay method. Samples were diluted with supplemented buffer to 2X the working

ACCEPTED MANUSCRIPT
concentration. Two wells were prepared for each sample (with and without inhibitor) in triplicate. The
reaction buffer and the reaction buffer plus inhibitor were prepared according to the instruction of
manufacturer. Absorbance was measured at 440 nm every 20 seconds for 5 minutes using the BioTek
Synergy HT Multi-Mode Microplate Reader with shaking before reading.
4.14 NQO1 inhibitory kinetic assay
The inhibitory activity of recombinant human NQO1 was measured by the NQO1 activity assay kit
(Abcam, Cambridge, MA, USA). The inhibitory kinetics of NQO1 were analyzed by
Lineweaver-Burk plots and compared to no treatment control. To calculate the kinetic parameters of
inhibition mechanism of NQO1, steady-state rates were obtained at various concentrations of test
compounds and substrate.
4.15 Detection of ROS
The ROS-Glo H2O2 Assay (Promega®, Southampton, UK) was used to measure changes of ROS by
directly detecting H2O2 in cells. The cells were plated in 96-well white tissue culture plates and then
treated with test compounds (0.01 µM to 5 µM) for 24 h. The H2O2 substrate solution was added to
each well and incubated for 6 h at 37 °C in a CO2 incubator. After the incubation with H2O2 substrate,
ROS-Glo Detection Solution was added to each well and incubated at room temperature for 20 min.
The luminescence was detected by BioTek Synergy HT Multi-Mode Microplate Reader.
4.16 Mitochondrial membrane potential assay
The effects of test compounds on changes in the mitochondrial membrane potential (MMP) were
detected by using the Mito-ID membrane potential cytotoxicity and detection kit (Enzo Life Sciences,
Lausen, Switzerland) according to the manufacturer’s instructions. Briefly, 2.5×105/mL cells were
plated in 96-well black wall and clear bottom plates. The cells were treated with test compounds (0.01
µM to 5 µM), CCCP (positive control) and DMSO (vehicle control), respectively. After incubation for
24 hours at 37 °C, each well received 100 µL of the MITO-ID®MP Dye Loading Solution and was
protected from light. Then, the plate was read with a BioTek Synergy HT Multi-Mode Microplate
Reader using excitation at 490 nm and emission at 590 nm after incubation at 37 °C for 30 mins.
4.17 Flow cytometry assay of cell apoptosis
HeLaS3 cells were cultured overnight and incubated in triplicate with different concentrations of
test compounds (0.2, 0.6, and 1.8 µM) or vehicle for 72 h. The cells were harvested and stained with
FITC-Annexin V and PI at room temperature for 15 min. The percentage of apoptotic cells was
determined by flow cytometry (Epics XL-MCL, Beckman Coulter, Indianapolis, USA) analysis. The
FITC signal detector (FL1) and PI staining signal detector (FL3) were used to detect the cells with the
flow cytometer (Ex = 488 nm; Em = 530 nm). Ten thousand cells were counted for three independent
experiments. The data were analyzed using WinList 3D (version7.1) and the histogram was plotted
using Excel 2010.
4.18 Western Blot Analysis

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HeLaS3 cells at 1.5×105/mL were treated with different dosage of test compounds or vehicle
control (0.1% of DMSO) in 2 mL DMEM medium supplemented with 10% FBS for 72 h. After being
harvested and lyzed, the cell lysates (50 µg/lane) were separated by SDS-PAGE (12% gel) and
transferred onto nitrocellulose membranes. After being blocked with 5% fat-free milk, the target
proteins were probed with anti-Bax, anti-Bcl-2, anti-caspase 3, anti-cleaved-caspase 3, and
anti-β-actin antibodies (Cell Signaling Technology, MA, USA), respectively. The bound antibodies
were detected by HRP-conjugated second antibodies and visualized using the enhanced
chemiluminescent reagent. The relative levels of each signaling event to control β-actin were
determined by densimetric scanning.
Acknowledgement
We gratefully acknowledge the financial support from the Natural Science Foundation of China (Grant
Nos. 81302628), the Project of “Jiangsu Six Peaks of Talent” (2014-SWYY-044), China
Pharmaceutical University for the Open Project Program of State Key Laboratory of Natural
Medicines (SKLNMKF201415), China and Jiangsu Province Postdoctoral Science Foundation
(2016M590488 and 1601136B), Jiangsu Government Scholarship for Overseas Studies (JS-2014-212),
and partial support by NIH grant CA177584 from the National Cancer Institute awarded to K.H. Lee,
and also thank a project funded by the Priority Academic Programs Development of Jiangsu Higher
Education Institutions (PAPD).
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Development of novel amino-5,8-quinolinedione derivatives as NAD(P)H: quinone-
oxidoreductase-1 (NQO1) inhibitor with potent antiproliferative activities
Yong Linga,b,1, Qiu-Xing Yanga,1, Yu-Ning Tengb,c, Shi Chena, Wei-Jie Gaoa, Jing Guoa, Pei-Ling Hsub, Yue Liua,
Susan L. Morris-Natschkeb, Chin-Chuan Hungc,*, Kuo-Hsiung Leeb,d*
aSchool of Pharmacy and Jiangsu Province Key Laboratory for Inflammation and Molecular Drug Target, Nantong
University, Nantong 226001, PR China.
bNatural Products Research Laboratories, UNC Eshelman School of Pharmacy, University of North Carolina,
Chapel Hill, North Carolina 27599, United States
cDepartment of Pharmacy, College of Pharmacy, China Medical University, Taichung 40402, Taiwan
dChinese Medicine Research and Development Center, China Medical University and Hospital, Taichung 40402,
Taiwan
Highlights
•
•
•
•
•
Novel amino-5,8-quinolinedione derivatives were synthesized.
All compounds showed significant antiproliferative potency in cancer cells.
Four selected compounds exhibited potent NQO1 inhibitory effects.
Active compounds increased ROS level and induced lethal mitochondrial dysfunction.
The most potent compound triggered cell apoptosis by regulating apoptotic proteins.