Anti-acute myeloid leukemia activity of 2-chloro-3-alkyl-1,4-naphthoquinone derivatives through inducing mtDNA damage and GSH depletion

Article abstract of DOI:10.1016/j.bmc.2018.07.010

2-Chloro-3-alkyl-1,4-naphthoquinone derivatives were synthesized and tested as the anti-acute myeloid leukaemia agents. The compound 9b (2-chloro-3-ethyl-5,6,7-trimethoxy-1,4-naphthoquinone) was the most potent toward HL-60 leukaemia cells. In mechanistic study for 9b, the protein levels of mtDNA-specific DNA polymerase γ (poly-γ) and mtDNA transcription factor A (mt-TFA) were decreased after the 24 h treatment, showing the occurrence of mtDNA damage. And 9b triggered cell cycle arrest at S phase accompanied by a secondary block in G2/M phase which had a direct link to the process of mtDNA damage. The dissipations of mitochondrial membrane potential and ATP also proceeded. On the other hand, 9b promoted the generation of ROS and resulted in the oxidation of intracellular GSH to GSSG. This process was coupled to the formation of adduct between 9b and GSH, detected by the UV–Vis spectrum and HRMS analysis. Depletion of GSH by buthionine sulfoximine enhanced ROS level and produced higher cytotoxicity, suggesting GSH was involved in the anti-leukemic mechanism of 9b. Together, our results provide new insights on the molecular mechanism of the derivatives of 2-chloro-1,4-naphthoquinone and 9b might be useful for the further development into an anti-leukemia agent.

Full text of DOI:10.1016/j.bmc.2018.07.010

Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Anti-acute myeloid leukemia activity of 2-chloro-3-alkyl-1,4-
naphthoquinone derivatives through inducing mtDNA damage and GSH
depletion
Kun Lia,1, Kun Yanga,1, Lifang Zhenga,⁎, Yuanyuan Lia, Qi Wangb, Ruili Lina, Dian Hea,⁎
a
Materia Medica Development Group, School of Pharmacy, Lanzhou University, Lanzhou 730000, China
State Key Laboratory of Applied Organic Chemistry, Lanzhou University, Lanzhou 730000, China
b
A R T I C L E I N F O
A B S T R A C T
Keywords:
2-Chloro-3-alkyl-1,4-naphthoquinone derivatives were synthesized and tested as the anti-acute myeloid leu-
kaemia agents. The compound 9b (2-chloro-3-ethyl-5,6,7-trimethoxy-1,4-naphthoquinone) was the most potent
toward HL-60 leukaemia cells. In mechanistic study for 9b, the protein levels of mtDNA-specific DNA poly-
merase γ (poly-γ) and mtDNA transcription factor A (mt-TFA) were decreased after the 24 h treatment, showing
the occurrence of mtDNA damage. And 9b triggered cell cycle arrest at S phase accompanied by a secondary
block in G2/M phase which had a direct link to the process of mtDNA damage. The dissipations of mitochondrial
membrane potential and ATP also proceeded. On the other hand, 9b promoted the generation of ROS and
resulted in the oxidation of intracellular GSH to GSSG. This process was coupled to the formation of adduct
between 9b and GSH, detected by the UV–Vis spectrum and HRMS analysis. Depletion of GSH by buthionine
sulfoximine enhanced ROS level and produced higher cytotoxicity, suggesting GSH was involved in the anti-
leukemic mechanism of 9b. Together, our results provide new insights on the molecular mechanism of the
derivatives of 2-chloro-1,4-naphthoquinone and 9b might be useful for the further development into an anti-
leukemia agent.
1,4-Naphthoquinone
Acute myeloid leukemia
Mitochondrial DNA
Mitochondria
GSH
1. Introduction
because of the lack of protection by histones and the relatively weak
DNA repair capacity in mitochondria.8 Failure to repair mtDNA damage
Acute myeloid leukaemia (AML) is a malignant hyperplasia and
heterogeneous disease characterized by continuous proliferation, in-
hibition of apoptosis and genetic aberrations, ultimately resulting in the
inhibition of normal hematopoiesis.1–3
has demonstrated to initiate cell death by apoptosis.9In the case of anti-
leukaemia agents, some drugs have a 1,4- naphthoquinone (1,4-NQ)
pharmacophore. The representative compounds are shown in Fig. 1.
Daunorubicin and doxorubicin have been used clinically for the treat-
ment of AML.10,11 Plumbagin (5-hydroxy-2-methyl-1,4-naphthoqui-
none), a naturally occurring naphthoquinone, was much efficient in
killing a broad spectrum of leukaemias, with IC50 value around
2.0 μM.12–14 Additionally, the synthetic compounds TW-92 (2-chloro-3-
amino-phenyl-1,4-NQ derivative),15,16 C2 (1,4-naphthoquinone-1,2,3-
triazole),17 FNQ3 (2-methyl-naphtho[2,3-b]furan-4,9-dione),18 and
BiQ3 (dimeric naphthoquinone)19 have been documented showing the
high cytotoxic activity against leukaemic cells, especially towards AML
cells. Recently, we found that the introduction of chlorine atom at C-2
of 1,4-NQ led to the significant enhancement of cytotoxicity.20
During the development of novel drugs against AML, mitochondria
have emerged as a drug target which has manifested the clinical ef-
fectiveness in combating relapsed or refractory AML.3 Cancer mi-
tochondria are structurally and functionally different from their normal
counterparts, so cancer cells are more susceptible to mitochondrial
perturbations.4 More importantly, a high frequency of mitochondrial
DNA (mtDNA) mutations exists in the AML cells.5,6 The human mi-
tochondrial genome contains 16.5 kb DNA which encode 13 respiratory
chain subunits. As respiratory chain play an important role in mi-
tochondrial ATP generation, drugs that target mtDNA probably will
remarkably affect cell viability and cellular physiological function.7
Besides, mtDNA is more susceptible to the DNA damaging agents,
Obviously, as the anti-AML drugs, 1,4-NQ derivatives have different
active functional groups on the quinone scaffold. Hence, cytotoxic
⁎
Corresponding authors.
E-mail addresses: zhenglf@lzu.edu.cn (L. Zheng), Hed@lzu.edu.cn (D. He).
These two authors contributed equally to this article.
1
https://doi.org/10.1016/j.bmc.2018.07.010
Received 18 May 2018; Received in revised form 4 July 2018; Accepted 6 July 2018
0968-0896/©2018ElsevierLtd.Allrightsreserved.
Pleasecitethisarticleas:Li,K.,Bioorganic&MedicinalChemistry(2018),https://doi.org/10.1016/j.bmc.2018.07.010

K. Li et al.
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Fig. 1. Quinoid compounds display anti-leukaemia activity.
effects might be mediated by a variety of mechanisms: induce cellular
oxidative stress,12,20 activate mitochondrial apoptotic pathway,16,18
interact with DNA topoisomerase II,11 phosphorylate ERK, p38, and
JNK kinase,16,13 regulate thioredoxin reductase14 and others. On the
other hand, cytotoxic effects of 1,4-NQ might be attributed to the al-
kylation of essential protein thiol or amine groups and/or the oxidation
of thiols by reactive oxygen species (ROS).21 In spite of its undoubted
anti-AML activity, the mode of action of 1,4-NQ in mitochondria has
been little investigated.
As a continuous study on synthesis and biological evaluation of 1,4-
NQ derivatives,20 we decided to use 2-chloro-1,4-NQ as a core structure
and modify the C-3 position with the alkyl side chain while introducing
the trimethoxyl substituent on benzene ring. To our knowledge, the
methoxyl substituent is an active fragment present in a number of an-
ticancer drugs. Herein, 2-chloro-3-alkyl- 5,6,7-trimethoxy- 1,4-
NQ derivatives were synthesized. All targeted compounds were
evaluated for their cytotoxicity against acute myeloid leukemia HL-60
cells and fetal lung fibroblast WI-38 cells using plumbagin and 5-
fluorouracil (5-FU) as the positive reference compounds. The anti-AML
mechanism was evaluated for the most potent compound 9b, showing
that 9b induced mtDNA damage, mitochondrial dysfunction, and re-
duced the intracellular glutathione (GSH) amount.
naphthoquinones 8a-8n after aerial oxidation and acidic workup. The
chlorination of 8a-8n on C-2 hydroxyl group gave 9a-9e.
2.2. Evaluation of cytotoxicity
We employed 2,5-diphenyltetrazolium bromide (MTT) colorimetric
assay to assess the cytotoxicity of the compounds (9a-9e, 10, 11) to-
ward acute myeloid leukemia HL-60 cells and the normal cells, fetal
lung fibroblast WI-38 cells. The results are listed in Table 1. All the
compounds displayed potent cytotoxic activity to HL-60 cells and low
toxicity to the normal WI-38 cells. However, they showed less potent or
similar cytotoxicity than plumbagin. The selective index (SI) is calcu-
lated as the ratio of cytotoxic activity between WI-38 and HL-60 cells
after the 48 h treatment. The results revealed that the most active
compound 9b had the higher SI with value of 8.35.
For the compounds (9a-9e) bearing tri-methoxyl moiety, they dis-
played anti-leukemia activity, with the values of IC50/48 h ranging from
2.85 to 12.89 μM. With the elongation of the alkyl side tail at C-3, the
activities of 9a-9e increased in a parabola fashion. Compound 9b dis-
played the highest activity, indicating two carbon atoms were the op-
timum side chain.
Interestingly, it was found that the introduction of methoxyl sub-
stituents on benzene ring slightly decreased the cytotoxic activity. After
the 48 h treatment, compound 10 containing m-dimethoxyl substituent
2. Result
on benzene ring (IC50 = 3.81
more active than the corresponding compound 9d with trimethoxyl
substituent (IC50 = 7.22 0.74 μM). It was also the case in the com-
parison of IC50 values of compound 9a (IC50 = 10.80 1.66 μM) and
compound 11 (IC50 = 6.32 0.36 μM), showing compound 11 with
0.31 μM) was approximately 1.9 times
2.1. Chemistry
According to our reported synthetic methods,20 the title compounds
(9a-9e) were obtained (Table 1). The compounds (9a-9e) were reported
herein for the first time and fully characterized by 1H, 13C NMR, and
ESI/HRMS. Compound 10 has been reported in our published article.20
Compound 11 was from J&K Scientific Ltd.
The synthesis of target compounds (9a-9e) is illustrated in Scheme
1. Briefly, the 3,4,5-trimethoxybenzaldehyde (1) was treated with so-
dium borohydride to give the 3,4,5-trimethoxybenzyl alcohol (2) in
excellent yields. Reaction of alcohol 2 with thionyl chloride afforded
the 3,4,5-trimethoxybenzyl chloride (3), which was further treated
with sodium cyanide to yield 3,4,5-trimethoxybenzyl cyanide (4). The
benzyl cyanide 4 was then hydrolyzed under acidic conditions to give
the corresponding phenylacetic acid 5. Subsequent esterification of 5
gave methyl phenylacetate 6. The compounds 7a-7e were obtained
through acylating 6 with acyl chloride, in dichloromethane containing
AlCl3 as the Lewis acid catalyst. A Claisen condensation reaction was
then developed in refluxing sodium methoxide to afford
none substituent on benzene ring was more active than the corre-
sponding compound 9a with trimethoxyl substituent. The same ob-
servation also has been found after the 24 h treatment.
Finally, the cytotoxicity of 9b against other cancer cell lines was
also tested (Table 2). The cell lines used were: hepatoma (HepG2),
cervical carcinoma (HeLa), and lung carcinoma (A549). The results of
this screening suggested 9b might have a broad spectrum of anticancer
activity and showed excellent cellular selectivity toward HL-60 cells.
Therefore, compound 9b was selected for the further mechanistic study.
2.3. Arrest cell cycle and damage mtDNA by 9b
The perturbation of cell cycle is closely related to the dysfunction of
mitochondria and cytotoxicity.22,23 To explore whether 9b might cause
the arrest of cell cycle, a flow-cytometric analysis of HL-60 cells stained
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Table 1
Cytotoxicity of compounds against HL-60 and WI-38 cells.
Compd.
Structure
IC50 (μM)a
HL-60
SIb
WI-38
48 h
24 h 48 h
48 h
9a
24.74
0.51
10.80
1.54
15.02
23.81
31.12
40.24
1.31
0.54
0.98
2.12
1.39
8.07
8.38
5.54
9b
9c
9d
7.65
0.21
2.85
3.70
7.22
0.29
13.61
15.43
0.72
0.15
1.12
0.33
9e
10
11
21.04
10.19
11.48
0.91
0.86
0.67
12.89
3.81
6.32
1.46
33.01
23.21
21.58
1.14
0.45
0.73
2.56
6.04
3.32
0.31
0.21
Plumbagin
8.25
0.81
3.67
0.53
26.71
0.74
7.28
a
Values represent mean
SI = IC50 WI-38 normal cells/IC50 HL-60 cancer cells after the 48 h treatment.
SE from at least three independent experiments.
b
with propidium iodide (PI) was performed. After the cells were treated
with various concentrations of 9b (3, 6 and 9 μM) for 24 h, 9b increased
the number of cells in S-phase accompanied by a secondary block in
G2/M progression in a dose-dependent manner (Fig. 2A). At 48 post-
treatment, the lower concentrations of 9b (3 μM, 6 μM) enhanced the
obvious arrest at G2/M phase along with the moderate arrest at S phase,
while the higher concentration of 9b at 9 μM exclusively caused a sig-
nificant increase in population of G2/M cells along with loss of cells in
G0/G1 and S phases (Fig. 2A). Besides, the percentage of apoptotic cells
was also determined using Annexin V-FITC/PI double staining assy.
There were the time-dependent and dose-dependent increases in po-
pulation of early-apoptotic cells (right low section) together with late-
apoptotic or necrotic cells (right upper section) (Fig. 2B). We might
infer that 9b mainly induced apoptotic cell death in HL-60 cells.
Specifically, it has reported that the occurrence of S phase arrest
along with a moderate G2/M blockade might accompany the damage to
mtDNA.23,24 Hence, we were motivated to investigate whether 9b could
damage mtDNA after the incubation for 24 h in HL-60 cells. The da-
mage to mtDNA may start at transcription and replication levels. The
mitochondrial transcription factor A (mt-TFA) is the key activator of
mitochondrial transcription as well as a participant in mtDNA replica-
tion, mitochondrial biogenesis and cellular metabolism in cells.25–27
Besides, mitochondrial DNA polymerase gamma protein (poly-γ) is re-
sponsible for mtDNA synthesis, replication, and repair of mtDNA da-
mage.28 We examined poly-γ and mt-TFA expression as the markers of
mitochondrial DNA (mtDNA) damage by Western blots (Fig. 2C), ex-
pecting that treatment with 9b would inhibit the protein levels of poly-γ
and mt-TFA. Upon the treatment with the various doses (3, 6 and 9 μM)
of 9b for 24 h, there were the significant decreases in mt-TFA and poly-
γ levels: the decreases were 71% for mt-TFA level and 32% for poly-γ
level at 9 μM over the control groups (Fig. 2D). Together, the inhibition
of the protein levels of mt-TFA and poly-γ is consistent with a marked
cell cycle arrest in the S phase and a minor arrest in the G2/M phase at
24 h post -treatment, indicating the damage of mtDNA may be
3

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Scheme 1. Reagents and conditions: (i)
NaBH4, THF, MeOH, 0 °C, 2 h, 94%; (ii)
SOCl2, CH2Cl2, rt, 1.5 h, 89%; (iii) NaCN,
DMSO, rt, 36 h, 85%; (iv) H2SO4, AcOH,
H2O, reflux, 22 h, 82%; (v) MeOH, H2SO4,
reflux, 3 h, 78%; (vi) RCOCl, AlCl3, Ar2, 0-rt,
2 h, 70–81%; (vii) MeONa, MeOH, reflux,
40 min; air bubbler, rt, 24 h, 45–58%; (viii)
SOCl2, toluene, reflux, 4 h, 40–58%.
neutralize intracellular ROS accumulation, so they have an effect on the
cytotoxicity of the 1,4-NQ derivatives.21 Quantitative quantification of
the GSH and glutathione disulfide (GSSG) levels was based on the en-
zymatic recycling method.30 Firstly, the effect of 9b on the amount of
intracellular GSH was determined. The results of total glutathione
(GSH + GSSG), GSH, and GSSG are shown in Fig. 4A–D. In comparison
with the control, the amount of total GSH as well as GSH decreased,
while the level of GSSG increased obviously. For example, after ex-
posure to 9 μM of 9b for 24 h, we observed that (i) GSH was 2.7 nm/mg
protein, showing that 9b decreased the amount of GSH around 6.9 nm/
mg protein contrasting to the control; (ii) in particular, the level of
GSSG was 0.71 nm/mg protein, showing that only a small fraction (ca.
20%) of GSH lost was converted to GSSG.
This result let us to investigate the possible mechanisms related to
GSH depletion. First of all, we tested whether 9b could trigger cellular
ROS over-accumulation and then oxidize GSH to GSSG. The level of
ROS was examined by 2′,7′-dichlorofluorescein (DCF) fluorescence
assay after the incubation of cells with 9b for 6 h. 9b promoted ROS
generation in cells remained higher than the control, with evidence of
an increase in the green fluorescence intensity (Fig. 4E). In order to test
whether a decrease in cellular GSH level could in turn augment ROS
level, HL-60 cells were pretreated with buthionine sulfoximine (BSO) to
lower the level of GSH.14 Contrasting to the BSO-untreated cells, ex-
posure to BSO enhanced the level of ROS by 9b, supporting GSH may
act as an antioxidant to scavenge ROS (Fig. 4E). Hence, we might
conclude that 9b could elicit the oxidative stress which might oxidize
GSH to GSSG.
Table 2
Cytotoxicity of compound 9b against three human cancer cell lines.
Compd.
IC50 (μM)a
HepG2
Hela
A549
9b
5-FU
15.37
36.62
0.49
0.44
28.17
30.21
0.54
0.27
17.91
55.73
0.74
0.59
a
Values represent mean
SE from at least three independent experiments.
implicated in the cytotoxicity.
Next, we evaluated the cytotoxic activity of 9b in the mtDNA-de-
ficient HL-60 cells (termed ρ0 cells). Obviously, HL-60 ρ0 cells were
more resistant than HL-60 cells to the growth suppressive induction
after exposure to 9b, suggesting that mtDNA damage is at least in part
involved in the cytotoxic mechanism of action of 9b (Fig. 2E).
2.4. Induce mitochondrial dysfunction by 9b
Building upon the above results, we sought to determine the impacts
of 9b on mitochondrial functions. The dissipation of mitochondrial
membrane potential (MMP) can be detected by the fluorescent probe
tetramethylrhodamine methyl ester (TMRE) which accumulates in the
mitochondrial matrix in proportion to MMP.29 As expected, 9b de-
creased MMP in a dose and time-dependent manner, as evidenced by
the decrease of the mean fluorescence intensity (MFI) (Fig. 3A). The
attenuation of MMP was more significant after the 24 h of incubation.
In comparison to the control, the treatment of cells with 9b for 24 h
decreased MMP by about 75%, 86% and 87% for 3 μM, 6 μM and 9 μM,
respectively.
Next, using the luciferase-luciferin assay for the determination of
total amount of cellular ATP, there was an obvious decrease in ATP
level in both a dose and time-dependent manner (Fig. 3B). For example,
in comparison to the control, the treatment of cells with 9b for 24 h
depleted the level of ATP by about 23%, 38% and 46% for 3 μM, 6 μM
and 9 μM, respectively. Therefore, this body of work demonstrates that
9b caused mitochondrial impairment.
Alternatively, the depletion of GSH promoted by 9b might be partly
due to the formation of corresponding conjugate. The interaction be-
tween 9b and GSH was analyzed using the UV–visible absorption
spectrum. Addition of GSH induced rapid disappearance of the ab-
sorption peak of 9b centered at 294 nm, accompanied by development
of two peaks around 274 and 355 nm and appearance of two isosbestic
points at 285 and 317 nm (Fig. 4F). This was further supported by
HRMS analysis showing formation of only the 1:1adduct ([M+H]+
:
582.1744) between 9b and GSH (Fig. 4G). We inferred that the for-
mation of adduct involved a direct substitution reaction with lose of
HCl, similar to the case of 2,3-dichloro-1,4-naphthoquinone.31
Finally, the effect of GSH on the cytotoxicity of 9b was determined.
Depletion of GSH by BSO remarkably augmented the cytotoxicity of 9b
(Fig. 4H), indicating that GSH displayed a protective role against the
cytotoxicity of 9b.
2.5. Induce GSH depletion by 9b
Cellular thiols can either interact with the 1,4-NQ derivatives or
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Fig. 2. (A) Analysis of cell cycle arrest by PI
staining assay after 24 h and 48 h incuba-
tion. (B) Determination of apoptotic percent
by Annexin V/PI double-staining assay after
24 h and 48 h incubation. (C) Protein levels
of mtDNA transcription factor A (mt-TFA)
and mtDNA polymerase gamma (poly-γ) in
HL-60 cells after 24 h incubation. The blots
shown are representative of three in-
dependent experiments demonstrating si-
milar results. (D) The relative protein levels
of mt-TFA and poly-γ were determined by
IMAGE J software. (E) Concentration-de-
pendent cytotoxicity of 9b towards HL-60
and HL-60 ρ0 cells for 48 h. Cell viability
was assessed by the MTT method. Data are
expressed as mean
SE from triplicates.
***P < 0.001, **P < 0.01 vs. the control
groups.
3. Discussion
9b was identified as a promising candidate. The structure-cytotoxic
activity relationships (SARs) for the tested compounds were obtained:
(i) with the elongation of alkyl tail at C-3, two carbon atoms were the
optimum side chain for activity; (ii) introduction of methoxyl sub-
stituents on benzene ring slightly decreased the cytotoxicity. Pal et al.
In the present study, we reported on the anti-ALM effect and pos-
sible mechanism of 2-chloro-5,6, 7-trimethoxy-1,4-NQ analogues con-
taining the alkyl side chain at C-3. Among these compounds, compound
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Fig. 3. Induction of mitochondrial dysfunction by 9b in HL-60 cells after 12 h and 24 h incubation. (A) Determination of MMP by flow cytometry. The data
represented as mean fluorescence intensity (MFI) of TMRE probe. (B) Determination of ATP by ATP Assay Kit. Values are expressed as the means
SE; n = 3,
***P < 0.001 vs. the control groups.
proposed that the increase in side chain length of the alkyl groups might
restrict the interactions of 1,4-NQ derivatives with DNA or other bi-
molecular, which in turn might decrease cytotoxicity activity.32 Be-
sides, methoxyl substituent would decrease the redox potential of
naphthoquinone/naphthosemiquinone redox couple, which had a ne-
gative effect on ROS generation and cytotoxicity activity. We have
found that the more positive is the redox potential of the naphthoqui-
none, the stronger is the cytotoxicity activity.20 Thus, the cytotoxic
effects might be mediated by more than one mechanism.
The promotion of DNA strand scission, alkylation, and the inter-
calation into DNA, inhibition on DNA topoisomerase I and II are re-
cognized as the prominent mechanisms underlying the anticancer ac-
tivity of 1, 4-NQ.33,34 However, few studies have looked into the effect
of 1, 4-NQ on mtDNA. Here, we found that 9b might have an effect on
mtDNA. Firstly, after 24 h-post treatment, 9b increased the number of
cells in S-phase accompanied by a secondary block in G2/M progres-
sion, indicating the occurrence of damage to mtDNA (Fig. 2A). Koczor
et al. reported that damage to mtDNA was shown to activate the cell
cycle regulatory kinase, Chk-2, trigger S-phase arrest, and cause a
secondary block in G2/M phase.23 Secondly, as a mark of mtDNA
damage, the protein levels of mtDNA-specific DNA poly-γ and mt-TFA
were detected by western blots. Exposure of cells to 9b inhibited the
protein levels of poly-γ and mt-TFA, proving the damage of mtDNA
(Fig. 2C). Further, involvement of mtDNA damage to the cytotoxicity by
9b was demonstrated by the mtDNA-deficient HL-60 ρ0 cells. HL-60 ρ0
cells were more resistant than HL-60 cells to the growth suppressive
induction after exposure to 9b. Moreover, 9b also triggered the depo-
larization of MMP and depletion of ATP in a dose and time-dependent
manner, confirming the occurrence of mitochondrial dysfunction
(Fig. 3). Taken together, we concluded that 9b might target mi-
tochondria by damaging mtDNA and causing dysfunction.
The damage to mtDNA may elicit the dysfunction of electron
transport chain, which will in turn lead to an increase in mitochondria-
generated ROS.7 ROS are involved in cell death by imposing an oxi-
dative stress. Cellular redox homeostasis can be maintained by the GSH
which scavenge excessive production of ROS.35 GSH also is a major
thiol nucleophile in cells, which is important in quinone inactivation by
conjugate formation.21 It was also the case in menadione. In rat pla-
telets, 85% of intracellular GSH was reported to deplete as menadione-
GSH conjugate, whereas in hepatocytes, 75% of GSH was depleted by
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Fig. 4. (A-D) The intracellular concentrations of
GSH, total GSH, GSH/GSSG ration and GSSG after
12 h and 24 h incubation. (E) Enhancement of accu-
mulation of ROS in the cells by GSH depletion. HL-60
cells were incubated with 50 μM BSO for 24 h to re-
duce the intracellular GSH level, followed by 9b
treatment for an additional 6 h. The level of ROS was
probed by DCFH-DA. (F) UV–visible absorption
spectrum changes for 9b (50 μM) in the presence of
GSH (50 μM) in PBS buffer (pH7.4) at 37 °C. (G)
HRMS spectrum of GSH-adduct after mixing 9b with
1 eq. of GSH at 37 °C for 2 h. (H) Enhancement of the
cytotoxicity of 9b by GSH depletion for 48 h. The cell
viability was measured by the MTT assay. Data are
expressed as mean
SE of three experiments.
***P < 0.001, **P < 0.01, *P < 0.05 vs. the control
groups.
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each compound was ≥ 95% in this analysis.
menadione due to formation of GSSG.36
We observed that 9b promoted accumulation of ROS and decreased
the level of intracellular GSH and total GSH, including the generation of
GSSG (Fig. 4A–E). And 20% of GSH was depleted by 9b due to forma-
tion of GSSG. Importantly, this process was coupled to the formation of
adduct between 9b and GSH, showing by a rapid change of the ab-
sorption curve of 9b (Fig. 4F) and the result of HRMS analysis (Fig. 4G).
Thus, we might be sure that the depletion of GSH was due to the for-
mations of GSSG and thioether conjugate. The conjugates might then be
degraded to other substances or actively removed from the intracellular
media by using an ATP-dependent pump toward thioether conjugate.37
Additionally, using BSO to lower the level of intracellular GSH aug-
mented the generation of ROS (Fig. 4E) and sensitized the cytotoxicity
by 9b (Fig. 4H), underpinning that GSH may play a detoxifying role.
In summary, we have synthesized 2-chloro-1,4-NQ derivatives (9a-
9e) and identified compound 9b as a potent cytotoxic agent against HL-
60 cells. Additionally, 9b also exhibited the moderate cytotoxicity
against three selected tumor cell lines (HepG2, Hela, A549) with the
4.2.1. 2-Chloro-5,6,7-trimethoxy-1,4-naphthoquinone (9a)
Yellow solid, M.P. 152.7–153.6 °C, yield 58%; 1H NMR (400 MHz,
CDCl3): δ 7.54 (s, 1H), 7.06 (s, 1H), 4.03 (s, 3H), 3.98 (s, 3H), 3.94 (s,
3H); 13C NMR (100 MHz, CDCl3): δ 180.22, 176.87, 156.58, 153.52,
147.72, 142.75, 136.73, 127.55, 118.32, 106.41, 60.87, 60.61, 55.73;
HRMS (ESI): found 305.0191 for [M+Na]+ (calcd. For C13H11ClNaO5,
305.0187).
4.2.2. 2-Chloro-3-ethyl-5,6,7-trimethoxy-1,4-naphthoquinone (9b)
Yellow solid, M.P. 88.7–90.2 °C, yield 43%; 1H NMR (400 MHz,
CDCl3): δ 7.53 (s, 1H), 4.02 (s, 3H), 3.97 (s, 3H), 3.95 (s, 3H), 2.80 (q,
J = 7.5 Hz, 2H), 1.18 (t, J = 7.5 Hz, 3H); 13C NMR (100 MHz, CDCl3)::δ
180.60, 177.68, 157.39, 154.61, 150.98, 148.44, 140.45, 128.61,
119.37, 106.81, 61.65, 61.46, 56.57, 22.27, 12.25; HRMS (ESI): found
311.0682 for [M + H]+ (calcd. For C15H15ClO5, 310.0681).
IC50 values ranging from 15.37
0.49 μM to 28.17
0.54 μM. The
4.2.3. 2-Chloro-3-propyl-5,6,7-trimethoxy-1,4-naphthoquinone (9c)
Yellow solid, M.P. 64.9–66.2 °C, yield 40%; 1H NMR (400 MHz,
CDCl3): δ 7.51 (s, 1H), 3.99 (s, 3H), 3.95 (s, 3H), 3.92 (s, 3H), 2.73 (t,
J = 8.0 Hz, 2H), 1.63 – 1.52 (m, 2H), 1.01 (t, J = 7.4 Hz, 3H); 13C NMR
(100 MHz, CDCl3): δ 180.66, 177.49, 157.29, 154.52, 149.72, 148.35,
140.68, 128.53, 119.27, 106.71, 61.54, 61.32, 56.47, 30.58, 21.46,
14.35; HRMS (ESI): found 325.0842 for [M+H]+ (calcd. for
SARs have been summarized, suggesting that the structural modifica-
tion focused on the introduction of electron-withdrawing substituents
on the benzene ring may merit exploratory attempt in the future. The
mechanistic studied disclosed that the anti-AML action of 9b was re-
lated to its ability to target the mitochondria by inducing mitochondrial
dysfunction and damaging mtDNA. Further investigation revealed that
9b could induce ROS production, GSH depletion and promote GSH-
mediated cell death. Clarification of the interaction of 9b with GSH
unveiled the formation of GSH-conjugate, underpinning 9b might react
with tissue nucleophiles to modify proteins covalently. The present
results present the possible additional mechanisms underlying the anti-
AML effect of naphthoquinone derivative and shed light on considering
the development of 9b as a potential cancer chemotherapeutic agent.
C
16H17ClO5, 3245.0837).
4.2.4. 2-Chloro-3-butyl-5,6,7-trimethoxy-1,4-naphthoquinone (9d)
Yellow solid, M.P. 74.7–76.3 °C, yield 42%; 1H NMR (400 MHz,
CDCl3):δ 7.53 (s, 1H), 4.01 (s, 3H), 3.96 (s, 3H), 3.95 (s, 3H), 2.77 (t,
J = 7.8 Hz, 2H), 1.57 – 1.42 (m, 4H), 0.95 (t, J = 7.0 Hz, 3H); 13C NMR
(100 MHz, CDCl3): δ 180.67, 177.53, 157.30, 154.55, 150.02, 148.36,
140.53, 128.56, 119.30, 106.72, 61.56, 61.34, 56.49, 30.09, 28.54,
23.07, 13.82; HRMS (ESI): found 339.0988 for [M+H]+ (calcd. for
4. Experimental
C
17H19ClO5, 339.0994).
4.1. Materials and instruments
4.2.5. 2-Chloro-3-hexyl-5,6,7-trimethoxy-1,4-naphthoquinone (9e)
Yellow solid, M.P. 46.5–47.6 °C, yield 58%; 1H NMR (400 MHz,
CDCl3): δ 7.53 (s, 1H), 4.01 (s, 3H), 3.97 (s, 3H), 3.95 (s, 3H), 2.76 (t,
J = 7.8 Hz, 2H), 1.58–1. 50 (m, 2H), 1.46–1. 40 (m, 2H), 1.34–1. 30 (m,
4H), 0.89 (t, J = 7.0 Hz, 3H); 13C NMR (100 MHz, CDCl3):δ 180.91,
177.79, 157.51, 154.75, 150.24, 148.54, 140.74, 128.77, 119.49,
106.93, 61.80, 61.60, 56.73, 31.75, 29.85, 29.04, 28.21, 22.77, 14.31;
HRMS (ESI): found 367.1297 for [M+H]+ (calcd. for C19H23ClO5,
367.1307).
Reduced glutathione (GSH), 2′,7′-dichlorodihydrofluoresceindiacetate
(DCFH-DA), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide (MTT) were obtained from Sigma (Beijing, China). Dulbecco's
modified Eagle medium (DMEM), fetal bovine serum (FBS) were from
Hyclone (Shanghai, China). All antibody were obtained from Santa
Cruz Biotechnology (Santa Cruz, CA)). Tetramethylrhodamine ethyl
ester (TMRE) were from Biolite Biotech (Tianjin, China). 3,8-diamino-
5-ethyl-6-phenylphenanthridinium bromide (EtBr), uridine were
obtained from J&K Scientific (Beijing, China). Pyruvate and l-buthio-
nine-(S,R)-sulfoximine (BSO) were from Solarbio (Beijing, China).
1H NMR and 13C NMR spectra were recorded using a Varian
Mercury spectrometer operating at 400 MHz for 1H NMR and 100 MHz
for 13C NMR. ESI/HRMS spectra were obtained on an Orbitrap Elite
(Thermo Scientific) mass spectrometer Bruker APEX II 47e mass spec-
trometer.
4.3. Biological experimental
4.3.1. Cell culture
The cells (HL-60, HepG2, A549, HeLa, and WI-38) were obtained
from the Shanghai Institute of Biochemistry and Cell Biology, Chinese
Academy of Science. The cells were cultured in DMEM growth medium
supplemented with 10% FBS in a humidified 5% CO2 incubator at 37 °C.
The HL-60 ρo cells were prepared by treating the cells with 50 ng/ml of
EtBr for 14 doublings and documented by lack of mitochondrial EtBr
fluorescence.38
4.2. Chemistry
The target compounds and intermediates were synthesized ac-
cording to our previously reported procedures.20 The products were
purified by flash column chromatography, using petroleum ether and
ethyl acetate as the eluent. The chemical structures were confirmed by
1H NMR, 13C NMR and ESI/HRMS (supplementary data). The chemical
purity of the compounds was determined using a Waters 600 system
equipped with a Waters 2998 photodiode array detector, including a
2707 automatic injector and a computer integrating apparatus. The
column was a Diamonsil C18 (150 mm x 4.6 mm, 5 μm). The injection
volume was 10.0 μL, with detection at 260 nm. The mobile phase was
used: methanol: H2O (80:20, v/v), flow rate of 1 mL/min. The purity of
4.3.2. MTT assay
1 × 104 cells were incubated with the compounds in triplicate in a
96-well plate for the 48 h at 37 °C in a final volume of 100 μL. At the end
of the treatment, MTT assay was performed to assess the cell viability.
The absorbance was measured at 570 nm using a microplate reader
(Thermo Scientific Multiskan GO, Finland).
4.3.3. Apoptosis and cell cycle analysis
5 × 105 HL-60 cells were treated with compound 9b a in a 6-well
8

K. Li et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
plate for 24 or 48 h.
independent experiments. Statistical differences between two groups
were measured by one-way factorial analysis of variance (ANOVA),
using Duncan’s post hoc test (SPSS 22.0). P < 0.05 versus control was
used as the criterion for statistical significance.
After that, apoptosis and cell cycle was determined by FITC/PI kit or
cell cycle detection kit (Beyotime, Jiangsu, China) using a FACS Canto
flow cytometer (Canto, Becton Dickinson, USA).
4.3.4. Western blots
Acknowledgments
After 5 × 105 HL-60 cells were treated with compound 9 a in a 6-
well plate for 24 or 48 h, equal amounts of denatured proteins (20 –
40 μg) were separated by 12% SDS PAGE followed by electroblotting
onto polyvinylidene difluoride membranes. Targeted proteins were
detected with specific primary antibodies. Corresponding secondary
antibodies were then utilized and immunoreactive bands were visua-
lized by fully automatic chemiluminescence analysis system (Tanon,
Shanghai, China).
This work was supported by the Fundamental Research Funds for
the Central Universities (Grant No. lzujbky-2018-131), the National
Natural Science Foundation of China (Grant No. 21302079), and the
Lanzhou Science and Technology Bureau Program Funds (Grant No.
2016-3-108, 2018-1-110).
A. Supplementary data
4.3.5. Mitochondrial membrane potential (MMP) detection
Supplementary data associated with this article can be found, in the
online version, at https://doi.org/10.1016/j.bmc.2018.07.010.
After 5 × 105 HL-60 cells were treated with compound 9b in a 6-
well plate for 12 or 24 h, the medium was replaced with fresh medium
containing 100 nM of TMRE and incubated for another 30 min at 37 °C.
Cells were then washed with PBS buffer (pH 7.4) three times. The
fluorescence intensity was quantitatively measured using a FACS Canto
flow cytometer at 549 nm excitation and 574 nm emission.
References
1. Rowe JM. AML in 2017: advances in clinical practice. Best Pract Res Clin Hae-matol.
2017;30:283–286.
2. Wang ZH, Li DD, Chen WL, et al. Targeting protein–protein interaction between
MLL1 and reciprocal proteins for leukemia therapy. Bioorg Med Chem.
2018;26:356–365.
3. Basak NP, Banerjee S. Mitochondrial dependency in progression of acute myeloid
leukemia. Mitochondrion. 2015;21:41–48.
4. Weinberg SE, Chandel NS. Targeting mitochondria metabolism for cancer therapy.
4.3.6. Intracellular ATP detection
Cellular ATP was detected by ATP Assay Kit (Beyotime, Shanghai,
China) following the manufacturer’s instruction. HL-60 cells were
treated with compound 9b for 12 or 24 h, lysed with ice-cold lysis
buffer, and centrifuged at 12000g for 10 min at 4 °C. The supernatant
was used to assay the ATP level by the detection buffer, and lumines-
cence was measured using a Thermo Varioskan Flash microplate reader.
Nat Chem Biol. 2015;11:9–15.
5. Silkjaer T, Norgaard JM, Aggerholm A, et al. Characterization and prognostic sig-
nificance of mitochondrial DNA variations in acute myeloid leukemia. Eur J
Haematol. 2013;90:385–396.
6. Sharawat SK, Bakhshi R, Vishnubhatla S, Bakhshi S. Mitochondrial d-loop variations
in paediatric acute myeloid leukaemia: a potential prognostic marker. Br J Haematol.
2010;149:391–398.
7. Fliss MS, Usadel H, Caballero OL, et al. Facile detection of mitochondrial DNA mu-
tations in tumors and bodily fluids. Science. 2000;287:2017–2019.
8. Singh KK, Russell J, Sigala B, et al. Mitochondrial DNA determines the cellular re-
sponse to cancer therapeutic agents. Oncogene. 1999;18:6641–6646.
9. Grishko V, Rachek L, Musiyenko S, et al. Involvement of mtDNA damage in free fatty
acid-induced apoptosis. Free Radic Biol Med. 2005;38:755–762.
10. Kostrzewa-Nowak D, Paine MJ, Wolf CR, Tarasiuk J. The role of bioreductive acti-
vation of doxorubicin in cytotoxic activity against leukaemia HL60-sensitive cell line
and its multidrug-resistant sublines. Br J Cancer. 2005;93:89–97.
11. Monneret C. Recent developments in the field of antitumor anthracyclines. Eur J Med
Chem. 2001;36:483–493.
4.3.7. Intracellular GSH and GSSG detection
The GSH assay is based on the oxidation of GSH by the sulfhydryl
reagent 5,5′-dithio-bis(2-nitrobenzoic acid) (DTNB) to form the yellow
derivative 5′-thio-2-nitrobenzoic acid (TNB).The rate of formation of
TNB, measured at 412 nm, is proportional to the concentration of GSH
in the sample. The GSSG formed can be recycled to GSH by glutathione
reductase in the presence of NADPH.30 Briefly, 1 × 106 HL-60 cells
were treated with compound 9b in a 6-well plate for 12 or 24 h. After
that, the assay was performed using the GSH and GSSG Assay Kit (Be-
yotime, Shanghai, China) according to the manufacturer's instructions
using a Thermo Varioskan Flash microplate reader.
12. Xu KH, Lu DP. Plumbagin induces ROS-mediated apoptosis in human promyelocytic
leukemia cells in vivo. Leuk Res. 2010;34:658–665.
13. Bae KJ, Lee Y, Kim SA, Kim J. Plumbagin exert an Immunosuppressive effect on
human T-cell acute lymphoblastic leukemia MOLT-4 cells. Biochem Biophys Res
Commun. 2016;473:272–277.
4.3.8. Intracellular ros assay
14. Zhang J, Peng S, Li X, et al. Targeting thioredoxin reductase by plumbagin con-
tributes to inducing apoptosis of HL-60 cells. Arch Biochem Biophys. 2017;619:16–26.
15. Hallak M, Thakur BK, Winn T, et al. Induction of death of leukemia cells by TW-74, a
novel derivative of chloro-naphthoquinone. Anticancer Res. 2013;3:183–190.
16. Hallak M, Win T, Shpilberg O, et al. The anti-leukaemic activity of novel sy-nthetic
naphthoquinones against acute myeloid leukaemia: induction of cell death via the
triggering of multiple signaling pathways. Br J Haematol. 2009;147:459–470.
17. Coulidiati TH, Dantas BB, Faheina-Martins GV, et al. Distinct effects of novel naph-
thoquinone-based triazoles in human leukaemic cell lines. J Pharm Pharmacol.
2015;67:1682–1695.
18. Desmond JC, Kawabata H, Mueller-Tidow C, et al. The synthetic furanonaphtho-
quinone induces growth arrest, apoptosis and differentiation in a variety of leukae-
mias and multiple myeloma cells. Br J Haematol. 2005;131:520–529.
19. Carter-Cooper BA, Fletcher S, Ferraris D, et al. Synthesis, characterization and an-
tineoplastic activity of bis-aziridinyl dimeric naphthoquinone – A novel class of
compounds with potent Activity against acute myeloid leukemia cells. Bioorg Med
Chem Lett. 2017;27:6–10.
20. Li K, Wang B, Zheng L, et al. Target ROS to induce apoptosis and cell cycle ar- rest by
5,7-dimethoxy- 1,4-naphthoquinone derivative. Bioorg Med Chem Lett.
2018;28:273–277.
21. O'Brien PJ. Molecular mechanisms of quinone toxicity. Chem Biol Interact.
1991;80:1–41.
22. Antico Arciuch VG, Elguero ME, Poderoso JJ, Carreras MC. Mitochondrial regulation
of cell cycle and proliferation. Antioxid Redox Signal. 2012;16:1150–1180.
23. Koczor CA, Shokolenko IN, Boyd AK, et al. Mitochondrial DNA damage initiates a cell
cycle arrest by a Chk2-associated mechanism in mammalian cells. J Biol Chem.
2009;284:36191–36201.
After treatment of 5 × 105 HL-60 cells with compound 9b in a 6-
well plate for 6 h, the incubation medium was replaced with the fresh
FBS-free medium containing ROS indicator DCFH-DA (10 μM) and in-
cubated for another 30 min at 37 °C in the dark. When necessary, the
cells were pretreated with BSO (50 μM) for 24 h to lower the amount of
intracellular GSH before adding compound 9b. The cells were visua-
lized and photographed under an inverted fluorescence microscopy
(DMI 4000B, Leica, Germany).
4.3.9. Glutathione conjugate detection
The UV-visible absorption spectrum changes of compound 9b
(50 μM) were recorded in the presence of the equivalent amount of GSH
in PBS buffer (pH 7.4) at 37 °C. The formation of conjugate was further
determined by ESI/HRMS spectrum. Briefly, 9b (0.5 mM) was dissolved
in methanol (5 mL), and to this solution was added 0.5 mM of GSH
dissolved in 5 mL of PBS buffer (pH 7.4). After stirring for 2 h under Ar2
at 37 °C, the solvent was evaporated and then the molecular mass of
conjugate was assayed by HRMS.
4.4. Statistical analysis
24. Martinez-Diez M, Santamaria G, Ortega Å.D, Cuezva JM. Biogenesis and dynamics of
mitochondria during the cell cycle: significance of 3′UTRs. PLoS One. 2006;1:e107.
The data are expressed as the means
SE of at least three
9

K. Li et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
25. Larsson NG, Wang JM, Wilhelmsson H, et al. Mitochondrial transcription factor A is
necessary for mtDNA maintenance and embryogenesis in mice. Nat Genet.
1998;18:231–236.
alkylamino)-1,4-napthoquinone. J Mol Struct. 2013;1049:355–361.
33. Wellington KW. Understanding cancer and the anticancer activities of naphtho-
quinones – a review. RSC Adv. 2015;5:20309–20338.
26. Ekstrand MI, Falkenberg M, Rantanen A, et al. Mitochondrial transcription factor A
regulates mtDNA copy number in mammals. Hum Mol Genet. 2004;13:935–944.
27. Kang D, Kim SH, Hamasaki N. Mitochondrial transcription factor A (TFAM): roles in
maintenance of mtDNA and cellular functions. Mitochondrion. 2007;7:39–44.
28. Kaguni LS. DNA polymerase gamma, the mitochondrial replicase. Annu Rev Biochem.
2004;73:293–320.
34. Kumar BS, Ravi K, Verma AK, et al. Synthesis of pharmacologically important
naphthoquinones and anticancer activity of 2-benzyllawsone through DNA topoi-
somerase-II inhibition. Bioorg Med Chem. 2017;25:1364–1373.
35. Schafer FQ, Buettner GR. Redox environment of the cell as viewed through the redox
state of the glutathione disulfide/glutathione couple. Free Radic Biol Med.
2001;30:1191–1212.
29. Scaduto Jr RC, Grotyohann LW. Measurement of mitochondrial membrane potential
using fluorescent rhodamine derivatives. Biophys J. 1999;76:469–477.
30. Rahman I, Kode A, Biswas SK. Assay for quantitative determination of glutath one
and glutathione disulfide levels using enzymatic recycling method. Nat Protoc.
2006;1:3159–3165.
31. Nickerson WJ, Falcone G, Strauss G. Studies on quinone-thioethers. I. mechanism of
formation and properties of thiodione. Biochemistry. 1963;2:537–543.
32. Pal S, Jadhav M, Weyhermüller T, et al. Molecular structures and antiproliferative
activity of side-chain saturated and homologated analogs of 2-chloro-3-(n-
36. Seung SA, Lee JY, Lee MY, et al. The relative importance of oxidative stress versus
arylation in the mechanism of quinone-induced cytotoxicity to platelets. Chem Biol
Interact. 1998;113:133–144.
37. Mauzeroll J, Bard AJ. Scaning electrochemical microscopy of menadione-
Glutathione conjugate export from yeast cells. Proc Natl Acad Sci USA.
2004;101:7862–7867.
38. Wang XF, Witting PK, Salvatore BA, Neuzil J. Vitamin E analogs trigger apoptosis in
HER2/erbB2-overexpressing breast cancer cells by signaling via the mitochondrial
pathway. Biochem Biophys Res Commun. 2005;326:282–289.
10