Discovery and structure–activity relationship of plastoquinone analogs as anticancer agents against chronic myelogenous leukemia cells

Article abstract of DOI:10.1002/ardp.201900170

Two series of amino-1,4-benzoquinones (AQ1–18) based on the structural analogs of plastoquinones were synthesized and the structure–activity relationship against chronic myelogenous leukemia activity was examined. All of the synthesized compounds were tested for their cytotoxic effects on different leukemic cell lines. Of interest, AQ15 exhibited a better selectivity than the reference drug imatinib on cancer cells. Owing to this, AQ15 was selected for a further apoptosis/necrosis evaluation where AQ15-treated K562 cells demonstrated similar apoptotic effects like imatinib-treated cells at their IC₅₀ values. The inhibitory effects of AQ15 and the other three compounds with various activities against eight tyrosine kinases, including ABL1, were investigated. AQ15 showed weak activity against ABL1, and a correlation was observed between the anti-K562 and anti-ABL1 activities. The binding mode of AQ15 into the ATP binding pocket of ABL1 kinase was predicted in silico, showing the formation of some key interactions. In addition, AQ15 was shown to suppress the downstream signaling of BCR-ABL in K562 cells. Finally, AQ15 obviously cleaved DNA in the presence of an iron(II) complex system, indicating that this can be the major mechanism of its antiproliferative action, whereas the mild inhibition of ABL kinase is just in-part mechanism of its overall outstanding cellular activity.

Full text of DOI:10.1002/ardp.201900170

Received: 13 June 2019
Revised: 3 September 2019
Accepted: 7 September 2019
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DOI: 10.1002/ardp.201900170
F U L L P A P E R
Discovery and structure–activity relationship of
plastoquinone analogs as anticancer agents against
chronic myelogenous leukemia cells
Halil I. Ciftci1,2
Mohamed O. Radwan1,2,5
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Nilüfer Bayrak3
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Hatice Yıldırım3
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Mahmut Yıldız4
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Masami Otsuka1,2
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Mikako Fujita2
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Amaç F. Tuyun6
1Department of Drug Discovery, Science Farm
Ltd, Kumamoto, Japan
Abstract
2Medicinal and Biological Chemistry Science
Farm Joint Research Laboratory, Faculty of
Life Sciences, Kumamoto University,
Kumamoto, Japan
Two series of amino‐1,4‐benzoquinones (AQ1–18) based on the structural analogs of
plastoquinones were synthesized and the structure–activity relationship against
chronic myelogenous leukemia activity was examined. All of the synthesized
compounds were tested for their cytotoxic effects on different leukemic cell lines.
Of interest, AQ15 exhibited a better selectivity than the reference drug imatinib on
cancer cells. Owing to this, AQ15 was selected for a further apoptosis/necrosis
evaluation where AQ15‐treated K562 cells demonstrated similar apoptotic effects
like imatinib‐treated cells at their IC50 values. The inhibitory effects of AQ15 and the
other three compounds with various activities against eight tyrosine kinases,
including ABL1, were investigated. AQ15 showed weak activity against ABL1, and
a correlation was observed between the anti‐K562 and anti‐ABL1 activities. The
binding mode of AQ15 into the ATP binding pocket of ABL1 kinase was predicted in
silico, showing the formation of some key interactions. In addition, AQ15 was shown
to suppress the downstream signaling of BCR‐ABL in K562 cells. Finally, AQ15
obviously cleaved DNA in the presence of an iron(II) complex system, indicating that
this can be the major mechanism of its antiproliferative action, whereas the mild
inhibition of ABL kinase is just in‐part mechanism of its overall outstanding cellular
activity.
3Department of Chemistry, Faculty of
Engineering, Istanbul University‐Cerrahpasa,
Istanbul, Turkey
4Department of Chemistry, Gebze Technical
University, Kocaeli, Turkey
5Department of Chemistry of Natural
Compounds, Pharmaceutical and Drug
Industries Research Division, National
Research Centre, Cairo, Egypt
6Department of Engineering Sciences,
Engineering Faculty, Istanbul
University‐Cerrahpasa, Istanbul, Turkey
Correspondence
Mikako Fujita, Medicinal and Biological
Chemistry Science Farm Joint Research
Laboratory, Faculty of Life Sciences,
Kumamoto University, 5‐1 Oe‐honmachi,
Chuo‐ku, Kumamoto 862‐0973, Japan.
Email: mfujita@kumamoto-u.ac.jp
Amaç Fatih Tuyun, Engineering Sciences
Department, Engineering Faculty, Istanbul
University‐Cerrahpasa, Avcilar, 34320
Istanbul, Turkey.
K E Y W O R D S
aminoquinone, apoptosis, chronic myelogenous leukemia, DNA cleavage, kinase inhibitor,
plastoquinone, structure–activity relationship
Email: aftuyun@gmail.com and
aftuyun@istanbul.edu.tr
Funding information
Scientific Research Projects Coordination Unit
of Istanbul University, Grant/Award Number:
FBA‐2016‐20662,FBA‐2017‐24559;
Grant‐in‐Aid for Challenging Exploratory
Research, Grant/Award Number: 24659048
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1
INTRODUCTION
activities. In particular, 1,4‐quinones constitute an important group of
anticancer drugs.[1] DNA often becomes a major target for anticancer
Quinone structure is found in many medicinally important compounds
quinones, that is, mitomycin C—which crosslinks DNA[2]; streptonigrin—
possessing anticancer, anti‐inflammatory, antimicrobial, and antiviral
which cleaves DNA[3]; and quinone enediyne antibiotic dynemicin
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Arch Pharm Chem Life Sci. 2019;e1900170.
wileyonlinelibrary.com/journal/ardp © 2019 Deutsche Pharmazeutische Gesellschaft
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https://doi.org/10.1002/ardp.201900170

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FIGURE 1 Biologically important molecules containing the 1,4‐quinone moiety
A—which cleaves DNA, as shown in Figure 1.[4] Natural or synthetic 1,4‐
quinones take part in a wide range of redox processes in organisms and
plants, such as plastoquinones and ubiquinones, which are two
important prenylquinones.[5] Idebenone, a structural analog of plasto-
quinones and ubiquinones (Figure 1), is a drug for the treatment of
emotional disturbances associated with cerebrovascular diseases.[6]
Further investigation about the synthesis and in vitro studies has shown
that cationic plastoquinones serve as powerful inhibitors of ROS‐
induced apoptosis and necrosis in HeLa cells and human fibroblasts.[7]
The recent development in the design of a novel agent is SkQ1
(Figure 1), which suppresses the growth of fibrosarcoma HT1080
and rhabdomyosarcoma RD tumor cells in vitro.[8] In 2010, novel
analogs containing 1,4‐quinone moiety were reported as effective in
combination with chemotherapeutic agents against pancreatic cancer
cell lines.[9] 1,4‐Quinone compounds are also reported as potential
anticancer agents against ovarian cancer cell lines.[10] Recently, a new
series of 1,4‐quinones designed on the basis of the natural product
hybrid approach was proven to be active against two different leukemia
cell lines (CCRF‐CEM and CEM/ADR5000).[11] In addition, a quinone‐
containing natural product shikonin is currently a center of interest due
to its potent effect for multiple myeloma owing to its unique dual
activities, proteasome inhibition and necroptosis induction.[12]
we synthesized some sulfanyl 1,4‐naphthoquinone compounds contain-
ing aryl amines with different substituents to investigate antimicrobial
activities against Gram‐positive and ‐negative bacteria and anticancer
activities against human tumor cell lines.[14] Some compounds having
strong antibacterial efficiency were reported as promising antibacterial
agents.[14a] Furthermore, some tested compounds showed important
selectivity on peripheral blood mononuclear cells (PBMCs), though they
exhibited considerable anticancer activities against colon and leukemia
cancer cell lines.[14b] Herein, we focus on chronic myelogenous leukemia
(CML) among various cancers.
CML is caused by a reciprocal translocation between chromo-
some 9 and chromosome 22, known as the Philadelphia chromosome
or Philadelphia translocation.[15] This translocation gives rise to a
BCR‐ABL kinase made up of two genes BCR and ABL.[16] The ABL
kinase family has been reported to play important roles in normal
cells, for example, cell adhesion and motility, DNA damage response,
and microbial pathogen response that may occur in the cytosol and
cell membranes. ABL kinase is also present in the nucleus.[17]
Deregulation and aberrant expression of ABL kinases are associated
with many types of cancer, such as breast[18] and colon cancers.[19]
Okabe et al.[20] presented the investigation of the combination
therapy with an ABL tyrosine kinase inhibitor (TKI) and alisertib
against Ph+ cells; thus, a strategy for the treatment of Ph+ leukemia
patients could be Aurora A inhibition. Since the overactivity of the
protein tyrosine kinase (PTK) has been implicated in a number of
diseases, a variety of PTKs has been targeted for the screening of
In our ongoing studies on the chemical modification of 1,4‐quinones
aiming at biologically useful compounds, our research group is working
on the synthesis of dimethyl‐1,4‐benzoquinone analogs by using
different pathways reported previously in the literature.[13] Previously,

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antitumor drugs. TKIs, as rivals of ATP for the ATP‐binding site of
PTK reducing tyrosine kinase phosphorylation, have made progress
although resistance has been restricted by the treatment of
cancer.[21] Imatinib is a highly specific inhibitor of BCR‐ABL binding
to the ATP‐binding site of the protein.[22] More recently, highly
potent inhibitors dasatinib and nilotinib have been developed and
used as a standard remedy together with imatinib. Although these
anti‐CML drugs have a combinatorial structure comprising various
compounds (AQ1–18). Among these compounds, AQ1, AQ6,[28a]
AQ8,[29] and AQ10[30] have been reported in the literature, while
the other AQ analogs are novel molecules. Structural character-
ization of the AQ analogs (AQ1–18, Table 1) was complemented by
Fourier‐transform infrared spectroscopy (FTIR), nuclear magnetic
resonance (1H NMR, 13C NMR), and high‐resolution mass spectro-
scopy (HRMS). In addition, the structures of AQ3 (1892191), AQ9
(1892192), AQ11 (1892193), AQ17 (1892194), and AQ18
(1892197) were further elucidated by single‐crystal X‐ray diffrac-
tion analysis (Figure S56).
components, we considered
a possibility of 1,4‐benzoquinone
variants to serve as anti‐CML drugs.
In this study, a new series of 1,4‐quinone compounds was
synthesized, and its activity against CML cell lines was examined.
Finally, some compounds were found to have unique activity based
on the dual mechanism of kinase inhibition and DNA cleavage.
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2.2
Evaluation of anticancer activity
The cytotoxicity and selectivity of the synthesized 18 AQ analogs
(AQ1–18) against three cancer cell lines, K562 (chronic myelogenous
leukemia), Jurkat, and MT‐2 (other two human T‐cell leukemias),
were evaluated by 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazo-
lium bromide (MTT) assay using imatinib as a control drug.
IC50 values are summarized in Table 1. The compounds AQ1–10
showed IC50 values higher than 15 μM against K562, MT‐2, and
Jurkat cells. Among them, AQ7 was the most effective against the
chronic myelogenous leukemia K562 cell line. Introduction of a
trifluoromethyl group at R1 (AQ2), R2 (AQ3), or R3 (AQ4) resulted in
a decreased activity. The activity was retained by the introduction of
the methyl group at R2 (AQ5), R3 (AQ6), and isopropyl group at R1
(AQ7).
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2
RESULTS AND DISCUSSION
Chemical synthesis
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2.1
We designed our molecules based on the plastoquinone scaffold,
replacing either prenyl from plastoquinone or decyl‐triphenylphos-
phonium cation appendage from SkQ1 by aliphatic and aromatic
amines, and named them AQ analogs (Figure 2). We also introduced the
chloro group that appears to be favorable for biological activity.[23]
The reaction of primary and secondary amines with 1,4‐
quinones to give AQs has already been reported.[24] As shown in
Scheme 1, initially, the 2,3‐dimethyl hydroquinone (1) was
smoothly oxidized to the corresponding 1,4‐benzoquinones (2
and 3). The 2,3‐dimethyl‐1,4‐benzoquinone (2) was obtained
On the contrary, the introduction of a chloro group into the
quinone moiety led to an increase in anticancer activity against all
three cell lines. Thus, for the chloro‐substituted series of AQ
analogs (AQ11–18), AQ11, AQ12, AQ14–15, and AQ17 exerted the
best cytotoxic activity against K562 cell line better than imatinib.
These compounds also exhibited strong anticancer activities against
[25]
[26]
according to the reported methods using KBrO3
or MnO2
in 98% or 86% yield, respectively. Another precursor, 2,3‐dichloro‐
5,6‐dimethyl‐1,4‐benzoquinone (3) was synthesized by oxidizing
with the HNO3/HCl variation of the reported method.[27] Subse-
quently, AQ analogs (AQ1–18) were prepared by the reactions of
the key 1,4‐benzoquinones (2 and 3) with the corresponding
substituted amines according to the literature procedure but
omitting the use of CeCl3.[13a] Heating the mixture in refluxing
absolute ethanol at higher temperature gave the target
other two leukemia cell lines (Figure S1). Introduction of
a
trifluoromethyl group at R2 (AQ12) retained the activity, but
introduction at R3 (AQ13) led to a decrease in activity. Introduction
of the methyl group at R2 (AQ14) or R3 (AQ15) retained the
activity. AQ10 and AQ18 with 2‐picolylamine led to a decrease in
activity in all cell lines.
FIGURE 2 Design of new
amino‐1,4‐benzoquinones based on
plastoquinones and SkQ1, AQ analogs

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hr
hr
SCHEME 1 Synthesis of AQ analogs (AQ1–18): (i) KBrO3, H2SO4/H2O, 10 min, 80°C; (ii) HNO3/HCl, 10 min, 90°C; (iii) substituted amines
(1.2 equiv), EtOH, reflux, 6–12 hr
Table 2 shows the cytotoxicity results of selected AQ analogs (AQ11,
AQ12, and AQ15), with submicromolar IC50 against K562, compared
with PBMC. As the selectivity index (SI) is calculated as the ratio of
cytotoxicity of the IC50 between the PBMC and K562 cells, the greater
the SI value, the more selective it is for cancer cells. Among them, AQ15
exhibited a better selectivity than imatinib against K562 cell line.
Among this series, AQ15, the most selective anticancer agent,
was chosen to investigate its apoptotic activity in CML. Thus, the
annexin V/ethidium homodimer III and Hoechst 33342 staining
methods were carried out with K562 cell line treated with AQ15 at
IC50 concentration and then observed by a fluorescence microscope
(Figure 3). In the control experiment, all cells were stained with blue
(healthy cells) at 6 hr after treatment of dimethyl sulfoxide (DMSO;
Figure 3a). On the contrary, K562 cells treated with AQ15 and imatinib
were stained mostly with healthy cells (blue), then with apoptotic cells
(green), late apoptotic or necrotic cells (both green and red), and
necrotic cells (red; Figure 3a), suggesting that AQ15 and imatinib
induced apoptosis mainly in an earlier time. The results indicated that
AQ15 had 66% apoptotic, 22% late apoptotic/necrotic, and 12%
necrotic effects at 6 hr, as shown in Figure 3b. The response of K562
cells upon 6‐hr imatinib treatment was 58% apoptosis, 23% late
apoptosis/necrosis, and 19% necrosis (Figure 3b). The results revealed
that AQ15 induced cell apoptosis similar to imatinib in CML (Figure 3c).
We speculated that significant anticancer activity of AQ15
against CML (BCR‐ABL positive leukemia) may be due to its potential
inhibitory activity on ABL (the kinase portion of BCR‐ABL). Thus, a
panel of kinases including ABL1 was selected. In this activity‐based
kinase system, the inhibitory effects of AQ11, AQ15, AQ16, and
AQ18 with various activities were tested against eight kinases (ABL1,
BRK, BTK, CSK, FYN A, LCK, LYN B, and SRC) using multipoint
dose–response experiments and the results are shown in Table 3. In
this series, AQ15 was found as the most potent ABL1 kinase inhibitor
with the IC50 value of 17.92 2.45 μM, followed by AQ11 with the
IC50 value of 23.60 1.94 μM when compared with imatinib (0.27
0.04 μM). On the contrary, AQ16 and AQ18 were found to be
inactive against ABL1 at 100 μM concentration. A correlation was
observed between anti‐K562 and anti‐ABL1 activities. Notably,
AQ15 significantly inhibited BRK, BTK, CSK, and SRC stronger than
compounds AQ11, AQ16, AQ18, and imatinib with IC50 values in the
micromolar range. Furthermore, all compounds were found to be

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TABLE 1 Structure and cytotoxicity of AQ analogs (AQ1–18) in K562, Jurkat, and MT‐2 cell lines by MTT assay
Substitution groups
Cell type (IC50, μM)a
K562b
ID
R1
R2
H
R3
Jurkatb
MT‐2b
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
AQ1
H
H
20.07 2.95
15.30 3.21
>30
13.38 1.02
15.89 4.03
>30
AQ2
CF3
H
H
AQ3
H
CF3
H
H
AQ4
H
CF3
>30
>30
AQ5
H
CH3
H
H
19.08 1.17
20.49 2.45
15.07 0.89
>30
>30
AQ6
H
CH3
16.55 1.56
15.59 3.21
>30
AQ7
CH(CH3)2
H
H
AQ8
H
H
CH(CH3)2
N(CH2CH3)2
AQ9
H
H
24.84 1.72
>30
17.53 2.19
>30
AQ10
AQ11
AQ12
AQ13
AQ14
AQ15
AQ16
AQ17
AQ18
Imatinibc
Shown above
H
H
H
0.75 0.05
0.88 0.06
17.82 1.87
1.85 0.11
0.76 0.04
7.38 0.57
1.89 0.09
22.15 2.19
5.58 1.83
3.01 0.62
2.92 0.41
5.48 0.95
2.29 0.53
2.34 0.88
8.58 1.34
2.46 0.38
>30
2.99 1.02
2.55 0.83
13.84 2.44
3.17 0.95
5.09 1.11
11.77 1.47
3.51 0.12
>30
H
CF3
H
H
H
CF3
H
CH3
H
H
H
CH3
CH(CH3)2
H
H
H
H
CH(CH3)2
Shown above
9.65 2.17
20.75 1.55
Abbreviations: MTT, 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide; SD, standard deviation.
aThe reported values represent the mean SD for each compound based on three independent experiments.
bCell lines include chronic myelogenous leukemia (K562) and other leukemias (Jurkat and MT‐2).
cUsed as the reference.
TABLE 2 Cytotoxicity of selected AQ analogs (AQ11, AQ12, and AQ15) and selectivity index (SI)
Substitution group
Cell type (IC50, μM)
K562a
R1
H
R2
H
R3
H
PBMCa
SIb
ID
AQ11
0.75 0.05
0.88 0.06
0.76 0.04
5.58 1.83
5.14 1.76
6.85
3.41
10.05
6.08
AQ12
H
CF3
H
H
3.00 1.22
7.64 1.58
33.92 4.19
AQ15
H
CH3
Imatinibc
aCell lines include chronic myelogenous leukemia (K562) and peripheral blood mononuclear cells (PBMCs).
bThe SI values are calculated as the ratio of the IC50 between PBMC and chronic myelogenous leukemia (K562) cells.
cUsed as the reference.

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inactive against FYN A and LYN B. These results pointed out that
AQ15 could be defined as a promising lead multitargeted kinase
inhibitor with different kinase inhibitory profile than imatinib.
Afterward, we tried to explore the potential binding mode of
AQ15 into the ATP‐binding site of ABL kinase by computational
approaches using MOE software. Despite the distinct stereoelec-
tronic features and a molecular weight from imatinib, AQ15 could
form some critical interactions that granted it an acceptable
inhibition effect (IC50 = 17.92 2.45 μM). AQ15 NH forges a key
H‐bond with the gatekeeper amino acid Thr315. Moreover, the
dimethyl benzoquinone is sandwiched between Val256 and Leu370
forming two CH–π interactions. The compound is further anchored
by another CH–π interaction with Phe317. The chlorine atom does
not seem to contribute to the interaction through hydrogen bond or
FIGURE 3 Alteration in K562 cells at
IC50 concentrations of AQ15 and imatinib
(a) for 6 hr. (b) A total of approximately
100 stained cells was selected randomly in
each experiment of (a) and was classified
into three types “apoptosis” (green),
“necrosis or late apoptosis” (both green
and red), and “necrosis” (red). (c)
Quantification of the effect of AQ15 and
imatinib on apoptosis. Data from three
independent experiments are shown as
means standard deviations and p values
were determined using Student’s test

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TABLE 3 The inhibition profile of tested compounds and imatinib in the panel of eight kinases
IC50 (μM)
Kinase
ABL1
BRK
AQ11
AQ15
AQ16
AQ18
>100
Imatinib
23.60 1.94
>100
17.92 2.45
18.53 2.13
12.01 0.94
24.00 1.78
>100
>100
0.27 0.04
20.56 1.41
>100
>100
>100
BTK
26.46 3.12
>100
29.27 3.85
>100
>100
CSK
>100
25.45 2.30
13.34 0.93
0.68 0.06
7.16 0.83
>100
FYN A
LCK
>100
>100
>100
82.52 5.91
>100
>100
49.70 6.43
>100
87.61 9.72
>100
LYN B
SRC
>100
39.85 5.68
13.82 1.29
>100
>100
halogen bond formation.[31] Less affinity of the AQ15, compared to
the native ligand imatinib, may be attributed to the missing crucial
bonding with Met318, Asp381, and Glu286 (Figure S58).
the sole underlying mechanism for its pronounced cytotoxicity. As
described in the Introduction, anticancer quinones target DNA.[1–4] To
explain high toxicity of AQ15 on CML, we next examined another
mechanism of its action with some other compounds (AQ11, AQ16,
and AQ18), involving metal‐binding/oxygen‐activating site of qui-
nones, that is, oxidative cleavage of DNA strand, which was reported
by many papers.[32] Thus, the DNA‐cleaving capability of AQ15 at 1
and 3 μM and AQ11, AQ16, and AQ18 at 3 μM concentrations was
studied using pUC19 DNA with and without the iron(II), hydrogen
peroxide (H2O2), and ascorbic acid complex as shown in Figure 6. The
reaction solution was incubated at 37°C for 2 hr and electrophoresis
was performed at 100 V for 30 min. The DNA was stained with
ethidium bromide and the gel image was captured by an electronic
camera under ultraviolet radiation (UV). AQ15 exhibits the strongest
DNA‐cleaving activity at 3 μM (Figure 6a), followed by AQ11, AQ16,
and AQ18, respectively (Figure 6b). These results demonstrated that
tested compounds may generate activated oxygen and cleaved DNA in
a cell. In addition, these compounds may activate oxygen in the
cytoplasm. It also points out that the induction of DNA may correlate
with the cytotoxic activity against K562.
As shown, AQ15 could be roughly superimposed on the pyridine‐
pyrimidine‐amino‐benzene segment of imatinib and lacks the
piperazine‐benzamide fragment (Figure 4). Presumably, AQ15 is less
active against ABL1, FYN A, LCK, and LYN B due to the lack of the
piperazine‐benzamide fragment and more potent against BTK and
SRC owing to the lack of the piperazine‐benzamide fragment.
The BCR‐ABL and its downstream signaling pathway such as
rapidly accelerated fibrosarcoma (Raf)/mitogen‐activated protein
kinase/extracellular signal‐regulated kinase (MEK)/extracellular
signal‐regulated kinase (ERK) play important roles in CML K562
cells. The K562 cells treated with AQ15 or imatinib were evaluated
for their inhibitory effects at 20 μM concentration after 6 hr of drug
treatment (Figure 5). AQ15 showed stronger inhibitory activity on
phosphorylation of ERK than imatinib. It can be concluded that AQ15
suppresses signaling downstream of tyrosine kinases.
Overall, the docking output is matched with the experimental data
and both confirm that the mild inhibition of ABL kinase by AQ15 is not
To assess the drug‐likeness of AQ15, we used computational
calculations of ADMET Predictor software to predict its potential
risks and physicochemical properties. AQ15 toxicity risk (TOX‐Risk)
value is 1 (acceptable value is up to 3.3). Mutagenic risk (MUT‐Risk)
FIGURE 5 The effect of AQ15 and imatinib on ERK signaling.
K562 cells were incubated with tested compounds at 10 and 20 μM
for 6 hr, and then immunoblot analysis was conducted. ERK,
extracellular signal‐regulated kinase
FIGURE 4 Comparison of the structure between imatinib and
AQ15 is shown below

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FIGURE 6 The DNA‐cleaving activity of AQ15 in the presence and absence of FeSO4, H2O2, and ascorbic acid system (a) and comparison
experiments using AQ11, AQ16, and AQ18 (b)
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value is 1.5, slightly exceeding the standard value 1. Risk related to
metabolism by or inhibition of major cytochrome P450s (CYP‐Risk) is
0.0 (standard value does not exceed 2.5). Noticeably, AQ15 has an
adequate safety profile. AQ15 exhibits zero violation of Lipinski rule
of 5 in terms of molecular weight, Log P, tPSA, number of H‐bond
donors and acceptors, and number of rotatable bonds. Moreover,
absorption risk (Absn‐Risk) is 1.0 (desirable value does not exceed
3.5) reflecting a high likelihood of good oral activity. In general, the
overall ADMET‐Risk value is 2.0 (acceptable value is up to 7.5). In
conclusion, AQ15 is predicted to have both favorable pharmacoki-
netic and safety profiles (Figure S57).
4
EXPERIMENTAL
Chemistry
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4.1
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4.1.1
General
Melting points (mp) were uncorrected and recorded on a Buchi B‐540
melting point apparatus. Thin‐layer chromatograph (TLC) was
purchased from Merck KGaA (Silica gel 60 F254) based on Merck DC‐
plates (aluminum‐based). Compound visualization for TLC was achieved
by UV light (254 nm). For column chromatography, silica gel 60 (63–200‐
µm particle‐sized, 60–230 mesh; Merck) was used as the stationary
phase. FTIR spectra were recorded as ATR on either a Thermo Scientific
Nicolet 6700 spectrometer, an Alpha T FTIR spectrometer or a Perkin
Elmer Spectrum 100 Optical FTIR spectrometer. NMR spectra were
obtained as CDCl3 solutions using either Bruker spectrometers with 400‐
MHz frequency for 1H and 100‐MHz frequency for 13C NMR in ppm (δ)
or VarianUNITY INOVA spectrometers with 500 MHz frequency for 1H
and 125 MHz frequency for 13C NMR in ppm (δ). 1H NMR and 13C NMR
signals were reported relative to the solvent signal at δ 7.19 and δ 76.0
ppm, respectively. Chemical shifts (δ) were reported in parts per million
(ppm). Coupling constants (J) are reported in Hz. Multiplicities were
described using the following abbreviations: s (singlet), br s (broad singlet),
d (doublet), t (triplet), q (quartet), hept (heptet), m (multiplet), and td
(triplet of doublets). Mass spectra were recorded using a Thermo
Finnigan LCQ Advantage MAX MS/MS spectrometer equipped with an
ESI (electrospray ionization) source. HRMS were recorded by a JEOL
JMSDX303HF using positive fast atom bombardment (FAB) with
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3
CONCLUSION
In this study, we found a new amino‐1,4‐benzoquinone compound
AQ15 with high antiproliferative activity against CML cell line K562
among various plastoquinone analogs. The activity of AQ15 (IC50
=
0.76 μM) surpassed imatinib (IC50 = 5.58 μM), and its selectivity
index between K562 and PBMC of AQ15 (10.05) was higher than
imatinib (6.08), indicating a potentially enhanced safety profile. The
results proved that the main mechanism of AQs anticancer activity is
DNA cleavage but not inhibition of BCR‐ABL kinase. DNA cleavage
causes apoptosis of K562 via downstream signaling of the kinase,
showing the unique activity of AQ15 distinct from imatinib. Further
investigation of plastoquinone analogs and their potential anti‐CML
activity is in progress.

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3‐nitrobenzyl alcohol as a matrix. The purity of the AQ analogs was
analyzed by HPLC (Shimadzu/DGU‐20A5 HPLC apparatus fitted with a
25‐cm Chiralpac AD‐H chiral column) using hexane/2‐propanol = 95:5 as
the mobile phase with a flow rate of 1.0 ml/min. The purity of all analogs
was ≥95%. Data for the single‐crystal compounds were obtained with a
Bruker APEX II QUAZAR three‐circle diffractometer. Indexing was
performed using APEX2.[33] Data integration and reduction were carried
out with SAINT.[34] Absorption correction was performed by a multiscan
method implemented in SADABS.[35] The Bruker SHELXTL[36] software
package was used for structures solution and structures refinement.
Aromatic C‐ and N‐bound hydrogen atoms were positioned geometrically
and refined using a riding mode. Crystal structure validations and
geometrical calculations were performed using the Platon software.[37]
Mercury software[38] was used for visualization of the .cif files. The
precursors (2[25] and 3[27]) were synthesized using the reported method
in the literature. All substituted amines were commercially obtained from
the commercial supplier and used without further purification unless
specified otherwise.
(0.710 g, 1.2 equiv, 4.41 mmol), the crude residue was purified by
column chromatography using petroleum ether/CHCl3 (1:2) eluent to
furnish AQ2 as a red solid. Yield: 324 mg, 30%, mp 111–112°C. FTIR
(ATR) υ (cm−1): 3,330 (NH), 2,959, 2,926 (CHaliphatic), 1,639 (>C═O).
1H NMR (400 MHz, CDCl3) δ (ppm): 1.98 (q, J = 1.1 Hz, 3H, CH3),
1.99 (q, J = 1.1 Hz, 3H, CH3), 5.91 (s, 1H, CH), 7.22 (t, J = 7.7 Hz, 1H,
CHaromatic), 7.38–7.42 (m, 2H, [NH and CHaromatic]), 7.50
(t, J = 7.7 Hz, 1H, CHaromatic), 7.62 (d, J = 7.8 Hz, 1H, CHaromatic).
13C NMR (100 MHz, CDCl3) δ (ppm): 12.1, 12.7 (CH3), 101.6, 123.6
1
2
(q, JCF = 273.0 Hz, CF3), 123.9 (q, JCF = 30.0 Hz, Cq), 125.0, 125.4,
3
127.2 (q, JCF = 5.2 Hz), 132.9, 136.0, 137.0, 143.0, 143.6 (Caromatic
and Cq), 183.4, 186.6 (>C═O). MS (+ESI) m/z (%): 297 (17,
[M+2H]+), 296 (100, [M+H]+). HRFABMS: Calcd. for C15H13F3NO2
[M+H]+: 296.0898; Found: 296.0889.
2,3‐Dimethyl‐5‐((3‐(trifluoromethyl)phenyl)amino)‐1,4‐benzoquinone
(AQ3)
The title compound was synthesized according to the general method
from compound 2 (1 equiv, 3.67 mmol) and 3‐trifluoromethylaniline
(0.710 g, 1.2 equiv, 4.41 mmol), the crude residue was purified by
column chromatography using petroleum ether/CHCl3 (1:2) eluent to
furnish AQ3 as a red solid. Yield: 271 mg, 25%, mp 142–144°C. FTIR
(ATR) υ (cm−1): 3,297 (NH), 3,054 (CHaromatic), 2,963 (CHaliphatic),
1,644 (>C═O). 1H NMR (500 MHz, CDCl3) δ (ppm): 1.97 (q, J = 1.0 Hz,
3H, CH3), 1.98 (q, J = 1.0 Hz, 3H, CH3), 6.07 (s, 1H, CH), 7.32 (d, J =
7.8 Hz, 2H, CHaromatic), 7.36 (s, 1H, CHaromatic), 7.38 (br s, 1H, NH),
7.42 (t, J = 7.8 Hz, 1H, CHaromatic). 13C NMR (125 MHz, CDCl3) δ
(ppm): 11.0, 11.7 (CH3), 100.6, 117.6, 120.4, 122.6 (q, 1JCF = 272.7 Hz,
The InChI codes of the investigated compounds are provided as
Supporting Information.
|
4.1.2
General procedure for the preparation of the
AQ analogs[13a]
A mixture of the corresponding substituted amines (1.2 equiv) and
quinone ([2, 0.50 g, 3.67 mmol] or [3, 0.50 g, 2.44 mmol]) in ethanol (25
ml) was stirred at reflux for 6–12 hr until consumption of the quinone.
The reaction mixture was cooled to ambient temperature. After ethanol
was evaporated under reduced pressure, the residue was dissolved with
CH2Cl2 (50 ml), and the solution was washed sequentially with water (3
× 30 ml). The organic layer was dried over CaCl2, filtered, and
concentrated under reduced pressure, and the residue was purified by
means of column chromatography on silica gel to give AQ analogs.
2
CF3), 123.7, 129.2, 131.2 (q, JCF = 33.0 Hz, Cq), 135.9, 137.7, 141.1,
142.9 (Caromatic and Cq), 182.7, 185.4 (>C═O). MS (−ESI) m/z (%): 295
(15, [M]−), 294 (100, [M–H]−). HRFABMS: Calcd. for C15H13F3NO2
[M+H]+: 296.0898; Found: 296.0894.
2,3‐Dimethyl‐5‐((4‐(trifluoromethyl)phenyl)amino)‐1,4‐benzoquinone
(AQ4)
2,3‐Dimethyl‐5‐(phenylamino)‐1,4‐benzoquinone (AQ1)
The title compound was synthesized according to the general method
from compound 2 (1 equiv, 3.67 mmol) and aniline (0.410 g, 1.2 equiv,
4.40 mmol), the crude residue was purified by column chromatography
using petroleum ether/CHCl3 (1:2) eluent to furnish AQ1 as a dark red
solid. Yield: 191 mg, 23%, mp 109–110°C. FTIR (ATR) υ (cm−1): 3,304
(NH), 3,058 (CHaromatic), 2,962, 2,922 (CHaliphatic), 1,642 (>C═O). 1H NMR
(500 MHz, CDCl3) δ (ppm): 1.95 (q, J = 1.5 Hz, 3H, CH3), 1.96
(q, J = 1.0 Hz, 3H, CH3), 6.05 (s, 1H, CH), 7.06 (t, J = 7.8 Hz, 1H,
CHaromatic), 7.10 (d, J = 7.8 Hz, 2H, CHaromatic), 7.26–7.29 (m, 3H,
[CHaromatic and NH]). 13C NMR (125 MHz, CDCl3) δ (ppm): 11.0, 11.7
(CH3), 99.8, 120.9, 124.1, 128.5, 135.6, 136.9, 141.6, 142.8 (Caromatic and
Cq), 183.0, 185.4 (>C═O). MS (+ESI) m/z (%): 229 (14, [M+2H]+), 228
(100, [M+H]+). Anal. calcd. for C14H13NO2 (227.26).
The title compound was synthesized according to the general method
from compound 2 (1 equiv, 3.67 mmol) and 4‐trifluoromethylaniline
(0.710 g, 1.2 equiv, 4.41 mmol), the crude residue was purified by
column chromatography using petroleum ether/CHCl3 (1:1) eluent to
furnish AQ4 as a red solid. Yield: 311 mg, 29%, mp 179–181°C. FTIR
(ATR) υ (cm−1): 3,243 (NH), 3,058 (CHaromatic), 2,962 (CHaliphatic),
1,645 (>C═O). 1H NMR (500 MHz, CDCl3) δ (ppm): 1.98–2.01 (m, 6H,
CH3), 6.18 (s, 1H, CH), 7.22 (d, J = 8.8 Hz, 2H, CHaromatic), 7.39 (br s,
1H, NH), 7.56 (d, J = 8.3 Hz, 2H, CHaromatic). 13C NMR (125 MHz,
CDCl3) δ (ppm): 11.1, 11.7 (CH3), 101.5, 119.9, 122.9 (q, 1JCF = 271.6
3
2
Hz, CF3), 125.4 (q, JCF = 33.1 Hz, Cq), 125.8 (q, JCF = 3.8 Hz, CH),
136.0, 140.5, 142.8 (Caromatic and Cq), 182.6, 185.5 (>C═O). MS (−ESI)
m/z (%): 295 (15, [M]−), 294 (100, [M–H]−). HRFABMS: Calcd. for
C
15H13F3NO2 [M+H]+: 296.0898; Found: 296.0901.
2,3‐Dimethyl‐5‐((2‐(trifluoromethyl)phenyl)amino)‐1,4‐benzoquinone
(AQ2)
2,3‐Dimethyl‐5‐(m‐tolylamino)‐1,4‐benzoquinone (AQ5)
The title compound was synthesized according to the general method
from compound 2 (1 equiv, 3.67 mmol) and 2‐trifluoromethylaniline
The title compound was synthesized according to the general method
from compound 2 (1 equiv, 3.67 mmol) and m‐toluidine (0.472 g, 1.2

10 of 14
CIFTCI ET AL.
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equiv, 4.40 mmol), the crude residue was purified by column
chromatography using petroleum ether/CHCl3 (1:2) eluent to furnish
AQ5 as a red solid. Yield: 227 mg, 26%, mp 98–100°C. FTIR (ATR) υ
(cm−1): 3,311 (NH), 3,047 (CHaromatic), 2,920 (CHaliphatic), 1,641
(>C═O). 1H NMR (500 MHz, CDCl3) δ (ppm): 1.96 (q, J = 1.0 Hz,
3H, CH3), 1.98 (q, J = 1.0 Hz, 3H, CH3), 2.27 (s, 3H, CH3), 6.06 (s, 1H,
CH), 6.88–693 (m, 3H, CHaromatic), 7.17 (t, J = 7.8 Hz, 1H, CHaromatic),
7.23 (br s, 1H, NH). 13C NMR (125 MHz, CDCl3) δ (ppm): 11.1, 11.7,
20.4 (CH3), 99.6, 117.9, 121.4, 124.8, 128.3, 135.6, 136.8, 138.6,
141.6, 142.9 (Caromatic and Cq), 183.0, 185.5 (>C═O). MS (+ESI) m/z
(%): 243 (14, [M+2H]+), 242 (100, [M+H]+). HRFABMS: Calcd. for
C15H16NO2 [M+H]+: 242.1181; Found: 242.1121.
υ (cm−1): 3,337 (NH), 2,957, 2,922, 2,870 (CHaliphatic), 1,646 (>C═O). 1H
NMR (400 MHz, CDCl3) δ (ppm): 1.17 (s, 3H, CH3), 1.19 (s, 3H, CH3),
1.98 (q, J = 1.2 Hz, 3H, CH3), 1.99 (q, J = 1.2 Hz, 3H, CH3), 2.83 (hept, J
= 6.9 Hz, 1H, CH), 6.03 (s, 1H, CH), 7.05 (d, J = 8.3 Hz, 2H, CHaromatic),
7.16 (d, J = 8.4 Hz, 2H, CHaromatic), 7.21 (br s, 1H, NH). 13C NMR (100
MHz, CDCl3) δ (ppm): 12.1, 12.8, 24.0 (CH3), 33.7 (CH), 100.3, 122.2,
127.5, 135.4, 136.5, 142.9, 144.0, 146.1 (Caromatic and Cq), 184.2, 186.5
(>C═O). MS (+ESI) m/z (%): 271 (27, [M+2H]+), 270 (100, [M+H]+). Anal.
calcd. for C17H19NO2 (269.34).
5‐((4‐(Diethylamino)phenyl)amino)‐2,3‐dimethyl‐1,4‐benzoquinone
(AQ9)
The title compound was synthesized according to the general
method from compound 2 (1 equiv, 3.67 mmol) and N,N‐diethyl‐p‐
phenylenediamine (0.723 g, 1.2 equiv, 4.40 mmol), the crude residue
was purified by column chromatography using petroleum ether/
CHCl3 (1:2) eluent to furnish AQ9 as a black solid. Yield: 155 mg,
14%, mp 125–126°C. FTIR (ATR) υ (cm−1): 3,294 (NH), 2,967, 2,922
(CHaliphatic), 1,640 (>C═O). 1H NMR (400 MHz, CDCl3) δ (ppm): 1.09
(t, J = 7.1 Hz, 6H, CH3), 1.96 (q, J = 1.2 Hz, 3H, CH3), 1.98 (q, J = 1.2
Hz, 3H, CH3), 3.28 (q, J = 7.0 Hz, 4H, NCH2), 5.88 (s, 1H, CH), 6.58 (d,
J = 8.5 Hz, 2H, CHaromatic), 6.98 (d, J = 8.8 Hz, 2H, CHaromatic), 7.13 (br
s, 1H, NH). 13C NMR (100 MHz, CDCl3) δ (ppm): 12.0, 12.5, 12.9
(CH3), 44.5 (NCH2), 98.9, 112.2, 124.3, 125.5, 136.2, 143.7, 144.3,
145.8 (Caromatic and Cq), 184.4, 186.1 (>C═O). MS (+ESI) m/z (%): 300
(20, [M+2H]+), 299 (100, [M+H]+), 298 (4, [M]+). HRFABMS: Calcd.
for C18H22N2O2 [M]+: 298.1681; Found: 298.1676.
2,3‐Dimethyl‐5‐(p‐tolylamino)‐1,4‐benzoquinone (AQ6)[28a]
The title compound was synthesized according to the general method
from compound 2 (1 equiv, 3.67 mmol) and m‐toluidine (0.472 g, 1.2
equiv, 4.40 mmol), the crude residue was purified by column
chromatography using petroleum ether/CHCl3 (1:2) eluent to furnish
AQ6 as a dark purple solid. Yield: 185 mg, 21%, mp 110–111°C. FTIR
(ATR)
υ
(cm−1): 3,301 (NH), 3,054 (CHaromatic), 2,962, 2,919
(CHaliphatic), 1,641 (>C═O). 1H NMR (500 MHz, CDCl3) δ (ppm):
1.98 (q, J = 1.0 Hz, 3H, CH3), 1.99 (q, J = 1.0 Hz, 3H, CH3), 2.27 (s, 3H,
CH3), 6.00 (s, 1H, CH), 7.01 (d, J = 8.8 Hz, 2H, CHaromatic), 7.11 (d, J =
8.3 Hz, 2H, CHaromatic), 7.19 (br s, 1H, NH). 13C NMR (125 MHz,
CDCl3) δ (ppm): 11.1, 11.8, 20.0 (CH3), 99.3, 121.1, 129.1, 134.0,
134.2, 135.5, 141.9, 143.0 (Caromatic and Cq), 183.1, 185.4 (>C═O).
MS (+ESI) m/z (%): 243 (16, [M+2H]+), 242 (100, [M+H]+). Anal. calcd.
for C15H15NO2 (241.29).
2,3‐Dimethyl‐5‐((pyridin‐2‐ylmethyl)amino)‐1,4‐benzoquinone
(AQ10)[30]
5‐((2‐Isopropylphenyl)amino)‐2,3‐dimethyl‐1,4‐benzoquinone (AQ7)
The title compound was synthesized according to the general method
from compound 2 (1 equiv, 3.67 mmol) and 2‐isopropylaniline (0.595
g, 1.2 equiv, 4.40 mmol), the crude residue was purified by column
chromatography using petroleum ether/CHCl3 (1:2) eluent to furnish
AQ7 as a purple oil. Yield: 326 mg, 33%. FTIR (ATR) υ (cm−1): 3,357
(NH), 3,063 (CHaromatic), 2,963, 2,926, 2,870 (CHaliphatic), 1,641
(>C═O). 1H NMR (400 MHz, CDCl3) δ (ppm): 1.23 (s, 3H, CH3),
1.24 (s, 3H, CH3), 2.08 (s, 6H, CH3), 3.07 (hept, J = 6.8 Hz, 1H, CH),
5.70 (s, 1H, CH), 7.14 (br s, 1H, NH), 7.20–7.29 (m, 3H, CHaromatic),
7.35–7.40 (m, 1H, CHaromatic). 13C NMR (100 MHz, CDCl3)
δ (ppm): 12.1, 12.9, 23.2 (CH3), 28.2 (CH), 99.9, 125.4, 126.6,
126.8, 127.2, 134.4, 136.4, 143.7, 144.1, 144.8 (Caromatic and Cq),
184.2, 186.3 (>C═O). MS (+ESI) m/z (%): 271 (20, [M+2H]+), 270
(100, [M+H]+). HRFABMS: Calcd. for C17H20NO2 [M+H]+: 270.1494;
Found: 270.1467.
The title compound was synthesized according to the general method
from compound 2 (1 equiv, 3.67 mmol) and 2‐picolylamine (0.477 g,
1.2 equiv, 4.41 mmol), the crude residue was purified by column
chromatography using initially petroleum ether and subsequently
petroleum ether/CHCl3 (1:2) eluent to furnish AQ10 as a red solid.
Yield: 89 mg, 10%, mp 126–128°C. FTIR (ATR) υ (cm−1): 3,344 (NH),
3,055 (CHaromatic), 2,918 (CHaliphatic), 1,640 (>CυO). 1H NMR (500
MHz, CDCl3) δ (ppm): 1.94 (q, J = 1.5 Hz, 3H, CH3), 1.96 (q, J = 1.5 Hz,
3H, CH3), 4.3 (d, J = 5.4 Hz, 2H, CH2), 5.39 (s, 1H, CH), 6.71 (br s, 1H,
NH), 7.14–7.17 (m, 2H, CHaromatic), 7.60 (td, J = 7.8 and 2.0 Hz, 1H,
CHaromatic), 8.51–8.53 (m, 1H, CHaromatic). 13C NMR (125 MHz,
CDCl3) δ (ppm): 10.9, 11.8 (CH3), 46.1 (CH2), 97.7, 120.5, 121.6,
135.4, 135.8, 143.0, 145.1, 148.4, 154.0 (Caromatic and Cq), 182.7,
184.6 (>C═O). MS (+ESI) m/z (%): 243 (100, [M+H]+), 242 (3, [M]+),
241 (12, [M–H]+). Anal. calcd. for C14H14N2O2 (242.27).
5‐((4‐Isopropylphenyl)amino)‐2,3‐dimethyl‐1,4‐benzoquinone
(AQ8)[29]
2‐Chloro‐5,6‐dimethyl‐3‐(phenylamino)‐1,4‐benzoquinone (AQ11)
The title compound was synthesized according to the general method
from compound 3 (1 equiv, 2.44 mmol) and aniline (0.273 g, 1.2 equiv,
2.93 mmol), the crude residue was purified by column chromato-
graphy using petroleum ether/CHCl3 (1:2) eluent to furnish AQ11 as
a claret red oil. Yield: 451 mg, 71%. FTIR (ATR) υ (cm−1): 3,237 (NH),
3,064 (CHaromatic), 2,961, 2,923 (CHaliphatic), 1,660 (>C═O). 1H NMR
The title compound was synthesized according to the general method
from compound 2 (1 equiv, 3.67 mmol) and 4‐isopropylaniline (0.595 g,
1.2 equiv, 4.40 mmol), the crude residue was purified by column
chromatography using petroleum ether/CHCl3 (1:2) eluent to furnish
AQ8 as a purple solid. Yield: 107 mg, 11%, mp 104–105°C. FTIR (ATR)

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(500 MHz, CDCl3) δ (ppm): 2.00 (q, J = 1.5 Hz, 3H, CH3), 2.06 (q, J =
1.5 Hz, 3H, CH3), 6.95 (d, J = 7.8 Hz, 2H, CHaromatic), 7.10 (t, J = 7.8
Hz, 1H, CHaromatic), 7.25 (t, J = 7.8 Hz, 2H, CHaromatic), 7.30 (br s, 1H,
NH). 13C NMR (125 MHz, CDCl3) δ (ppm): 11.3, 12.4 (CH3), 110.4,
123.1, 124.2, 127.4, 135.9, 136.5, 138.4, 142.9 (Caromatic and Cq),
178.7, 181.7 (>C═O). MS (−ESI) m/z (%): 262 (30, [M+H]−), 261 (10,
[M]−), 260 (100, [M–H]−). HRFABMS: Calcd. for C14H12ClN2O2 [M]+:
262.0635; Found: 262.0634.
3H, CH3), 2.04 (q, J = 1.5 Hz, 3H, CH3), 2.26 (s, 3H, CH3), 6.73–6.75 (m,
2H, CHaromatic), 6.90 (d, J = 7.8 Hz, 1H, CHaromatic), 7.11 (t, J = 7.8 Hz,
1H, CHaromatic), 7.28 (br s, 1H, NH). 13C NMR (125 MHz, CDCl3) δ
(ppm): 11.2, 12.4, 20.4 (CH3), 110.2, 120.1, 123.6, 125.0, 127.0, 135.9,
136.3, 137.3, 138.4, 142.8 (Caromatic and Cq), 178.6, 181.7 (>C═O). MS
(−ESI) m/z (%): 277 (4, [M+2H]−), 276 (31, [M+H]−), 275 (13, [M]−), 274
(100, [M–H]−); MS (+ESI) m/z (%): 276 (100, [M+H]+). HRFABMS:
Calcd. for C15H15ClNO2 [M+H]+: 276.0791; Found: 276.0787.
2‐Chloro‐5,6‐dimethyl‐3‐((3‐(trifluoromethyl)phenyl)amino)‐1,4‐
benzoquinone (AQ12)
2‐Chloro‐5,6‐dimethyl‐3‐(p‐tolylamino)‐1,4‐benzoquinone (AQ15)
The title compound was synthesized according to the general method
from compound 3 (1 equiv, 2.44 mmol) and p‐toluidine (0.314 g, 1.2
equiv, 2.93 mmol), the crude residue was purified by column
chromatography using petroleum ether/CHCl3 (1:2) eluent to furnish
AQ15 as a dark purple solid. Yield: 251 mg, 37%, mp 148–149°C. FTIR
(ATR) υ (cm−1): 3,239 (NH), 3,023 (CHaromatic), 2,961, 2,923 (CHaliphatic),
1,664 (>C═O). 1H NMR (500 MHz, CDCl3) δ (ppm): 1.97 (q, J = 1.0 Hz,
3H, CH3), 2.04 (q, J = 1.0 Hz, 3H, CH3), 2.26 (s, 3H, CH3), 6.83 (d, J =
8.3 Hz, 2H, CHaromatic), 7.03 (d, J = 8.3 Hz, 2H, CHaromatic), 7.27 (br s,
1H, NH). 13C NMR (125 MHz, CDCl3) δ (ppm): 11.2, 12.4, 20.0 (CH3),
109.6, 123.2, 127.8, 133.9, 134.2, 135.8, 138.5, 142.9 (Caromatic and
Cq), 178.6, 181.7 (>C═O). MS (−ESI) m/z (%): 276 (40, [M+H]−), 275
(15, [M]−), 274 (100, [M–H]−). HRFABMS: Calcd. for C15H15ClNO2
[M+H]+: 276.0791; Found: 276.0781.
The title compound was synthesized according to the general method
from compound 3 (1 equiv, 2.44 mmol) and 3‐trifluoromethylaniline
(0.472 g, 1.2 equiv, 2.93 mmol), the crude residue was purified by
column chromatography using petroleum ether/CHCl3 (1:2) eluent to
furnish AQ12 as a claret red solid. Yield: 449 mg, 56%, mp 141–142°C.
FTIR (ATR) υ (cm−1): 3,230 (NH), 3,050 (CHaromatic), 2,918 (CHaliphatic),
1,661 (>C═O). 1H NMR (500 MHz, CDCl3) δ (ppm): 2.01–2.02 (m, 3H,
CH3), 2.07–2.08 (m, 3H, CH3), 7.09 (d, J = 7.3 Hz, 1H, CHaromatic), 7.17
(s, 1H, CHaromatic), 7.30 (br s, 1H, NH), 7.33–7.39 (m, 2H, CHaromatic). 13
C
NMR (125 MHz, CDCl3) δ (ppm): 11.3, 12.4 (CH3), 112.1, 119.3, 120.5
(q, 3JCF = 3.9 Hz), 122.7 (q, 1JCF = 272.4 Hz), 125.5, 127.8 (q, 3JCF = 5.3
2
Hz), 130.0 (q, JCF = 32.7 Hz), 136.3, 137.1, 137.9, 142.8 (Caromatic and
Cq), 178.6, 181.4 (>C═O). MS (−ESI) m/z (%): 331 (69, [M+2H]−), 330
(58, [M+H]−), 329 (100, [M]−). HRFABMS: Calcd. for C15H12ClF3NO2
[M+H]+: 330.0509; Found: 330.0507.
2‐Chloro‐3‐((2‐isopropylphenyl)amino)‐5,6‐dimethyl‐1,4‐benzoquinone
(AQ16)
2‐Chloro‐5,6‐dimethyl‐3‐((4‐(trifluoromethyl)phenyl)amino)‐1,4‐
benzoquinone (AQ13)
The title compound was synthesized according to the general method
from compound 3 (1 equiv, 2.44 mmol) and 2‐isopropylaniline (0.396 g,
1.2 equiv, 2.93 mmol), the crude residue was purified by column
chromatography using petroleum ether/CHCl3 (1:2) eluent to furnish
AQ16 as a dark red oil. Yield: 403 mg, 54%. FTIR (ATR) υ (cm−1): 3,325
(NH), 2,962, 2,926, 2,867 (CHaliphatic), 1,656 (>C═O). 1H NMR (400
MHz, CDCl3) δ (ppm): 1.15 (s, 3H, CH3), 1.17 (s, 3H, CH3), 2.00 (q, J =
1.2 Hz, 3H, CH3), 2.06 (q, J = 1.2 Hz, 3H, CH3), 3.08 (hept, J = 6.8 Hz,
1H, CH), 6.89 (d, J = 7.8 Hz, 1H, CHaromatic), 7.07 (td, J = 7.6, 1.7 Hz, 1H,
CHaromatic), 7.12–7.17 (m, 3H, CHaromatic and NH). 13C NMR (100 MHz,
CDCl3) δ (ppm): 12.3, 13.5, 22.9 (CH3), 28.5 (CH), 109.5, 125.5, 125.8,
126.9, 127.1, 135.1, 136.6, 140.3, 143.8, 144.2 (Caromatic and Cq),
179.7, 182.7 (>C═O). MS (+ESI) m/z (%): 305 (18, [M+2H]+), 304 (100,
[M+H]+). HRFABMS: Calcd. for C17H19ClNO2 [M+H]+: 304.1104;
Found: 304.1090.
The title compound was synthesized according to the general method
from compound 3 (1 equiv, 2.44 mmol) and 4‐trifluoromethylaniline
(0.472 g, 1.2 equiv, 2.93 mmol), the crude residue was purified by
column chromatography using petroleum ether/CHCl3 (1:2) eluent to
furnish AQ13 as a claret red oil. Yield: 331 mg, 41%. FTIR (ATR) υ
(cm−1): 3,233 (NH), 2,962, 2,918, 2,849 (CHaliphatic), 1,661 (>C═O). 1H
NMR (500 MHz, CDCl3) δ (ppm): 2.02 (q, J = 1.5 Hz, 3H, CH3), 2.07 (q,
J = 1.5 Hz, 3H, CH3), 6.97 (d, J = 8.3 Hz, 2H, CHaromatic), 7.29 (br s, 1H,
NH), 7.50 (d, J = 8.8 Hz, 2H, CHaromatic). 13C NMR (125 MHz, CDCl3) δ
1
(ppm): 11.3, 12.4 (CH3), 113.0, 118.0, 121.7, 120.0 (q, JCF = 271.7
3
2
Hz), 124.6 (q, JCF = 3.7 Hz), 124.6 (q, JCF = 32.7 Hz), 136.5, 137.7,
139.6, 142.7 (Caromatic and Cq), 178.6, 181.4 (>C═O). MS (−ESI) m/z
(%): 330 (35, [M+H]−), 329 (29, [M]−), 328 (100, [M–H]−). HRFABMS:
Anal. calcd. for C15H11ClF3NO2 [M+Na]+: 352.0328; Found:
352.0335.
2‐Chloro‐3‐((4‐isopropylphenyl)amino)‐5,6‐dimethyl‐1,4‐benzoqui-
none (AQ17)
2‐Chloro‐5,6‐dimethyl‐3‐(m‐tolylamino)‐1,4‐benzoquinone (AQ14)
The title compound was synthesized according to the general method
from compound 3 (1 equiv, 2.44 mmol) and m‐toluidine (0.314 g, 1.2
equiv, 2.93 mmol), the crude residue was purified by column
chromatography using petroleum ether/CHCl3 (1:2) eluent to furnish
AQ14 as a purple solid. Yield: 277 mg, 41%, mp 141–143°C. FTIR
(ATR) υ (cm−1): 3,236 (NH), 3,022 (CHaromatic), 2,922, 2,857 (CHaliphatic),
1,661 (>C═O). 1H NMR (500 MHz, CDCl3) δ (ppm): 1.98 (q, J = 1.5 Hz,
The title compound was synthesized according to the general method
from compound 3 (1 equiv, 2.44 mmol) and 4‐isopropylaniline (0.396 g,
1.2 equiv, 2.93 mmol), the crude residue was purified by column
chromatography using petroleum ether/CHCl3 (1:2) eluent to furnish
AQ17 as a dark purple solid. Yield: 292 mg, 39%, mp 105–106°C. FTIR
(ATR) υ (cm−1): 3,224 (NH), 2,959, 2,922, 2,870 (CHaliphatic), 1,668
(>C═O). 1H NMR (400 MHz, CDCl3) δ (ppm): 1.16 (s, 3H, CH3), 1.18 (s,
3H, CH3), 1.97 (q, J = 1.2 Hz, 3H, CH3), 2.04 (q, J = 1.2 Hz, 3H, CH3),

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2.82 (hept, J = 6.9 Hz, 1H, CH), 6.87 (d, J = 8.3 Hz, 2H, CHaromatic), 7.09
(d, J = 8.3 Hz, 2H, CHaromatic), 7.31 (br s, 1H, NH). 13C NMR (100 MHz,
CDCl3) δ (ppm): 12.3, 13.5, 24.0 (CH3), 33.6 (CH), 110.6, 124.1, 126.3,
135.1, 136.8, 139.5, 143.9, 146.2 (Caromatic and Cq), 179.7, 182.8
(>C═O). MS (+ESI) m/z (%): 305 (19, [M+2H]+), 304 (100, [M+H]+).
HRFABMS: Calcd. for C17H19ClNO2 [M+H]+: 304.1104; Found:
304.1064.
DMSO was added to each well. The absorbance at 550 nm was
measured using
a microplate reader Infinitive M1000 (Tecan,
Mannedorf, Switzerland) with background subtraction at 630 nm. All
experiments were run in triplicate and IC50 values were estimated
from the results of the MTT test described as the drug concentrations
that reduced absorbance to 50% of control values.
|
4.2.3
Detection of cell death
2‐Chloro‐5,6‐dimethyl‐3‐((pyridin‐2‐ylmethyl)amino)‐1,4‐
benzoquinone (AQ18)
Apoptotic/necrotic/healthy detection kit (PromoKine, Heidelberg,
Germany) was performed according to PromoKine’s instructions with
the modifications.[41] Briefly, K562 cells were treated with AQ15 and
imatinib at IC50 concentrations for 6 hr. Then, the cells were
harvested and washed with phosphate‐buffered saline (PBS) and
stained with 4 μl of FITC–Annexin V, 4 μl of ethidium homodimer III
and 4 μl of Hoechst 33342 in 1× binding buffer for 30 min at room
temperature in the dark. The cells were analyzed by a fluorescence
microscope Biorevo Fluorescence BZ‐9000 (Keyence, Osaka, Japan).
The number of apoptotic cells (Annexin V), late apoptotic or necrotic
cells (Annexin V and ethidium homodimer III), and necrotic cells
(ethidium homodimer III) were counted as previously described.[42]
The title compound was synthesized according to the general method
from compound 3 (1 equiv, 2.44 mmol) and 2‐picolylamine (0.317 g,
1.2 equiv, 2.93 mmol), the crude residue was purified by column
chromatography using initially petroleum ether and subsequently
petroleum ether/CHCl3 (1:2) eluent to furnish AQ18 as a dark purple
solid. Yield: 287 mg, 35%, mp 143–144°C. FTIR (ATR) υ (cm−1): 3,242
(NH), 3,055 (CHaromatic), 2,962, 2,916 (CHaliphatic), 1,667 (>C═O). 1H
NMR (500 MHz, CDCl3) δ (ppm): 1.95 (q, J = 1.0 Hz, 3H, CH3), 2.03 (q,
J = 1.0 Hz, 3H, CH3), 5.00 (d, J = 5.4 Hz, 2H, CH2), 7.16–7.20 (m, 2H,
CHaromatic), 7.31 (br s, 1H, NH), 7.63 (td, J = 7.3 and 2.0 Hz, 1H,
CHaromatic), 8.55 (d, J = 4.4 Hz, 1H, CHaromatic). 13C NMR (125 MHz,
CDCl3) δ (ppm): 11.1, 12.4 (CH3), 47.4 (CH2), 120.8, 121.6, 135.2,
135.9, 141.3, 142.8, 148.1, 154.5 (Caromatic and Cq), 178.1, 181.6
(>C═O). MS (−ESI) m/z (%): 278 (11, [M+2H]−), 277 (24, [M+H]−), 276
(43, [M]−), 275 (100, [M–H]−); MS (+ESI) m/z (%): 278 (14, [M+2H]+),
277 (100, [M+H]+). HRFABMS: Calcd. for C14H14ClN2O2 [M+H]+:
277.0744; Found: 277.0769.
|
4.2.4
Tyrosine kinase assay
The kinase inhibition assay system (TK‐2; Promega Corporation,
Madison, WI) was performed as previously described with some
modification.[43] In this system, eight kinase strips (ABL1, BRK, BTK,
CSK, FYN A, LCK, LYN B, and SRC) and their substrates were diluted
with 2.5× kinase reaction buffer (95 µl) and 100 µM ATP (15 µl)
solution, respectively. The reaction of kinases was performed in the
384‐well plate using 2 µl of the compound solution at multiple
concentrations in a buffer, 4 µl of kinase working stock and 4 µl of
ATP/substrate working stock. After 1 hr of incubation at room
temperature, the ADP‐Glo Kinase Assay (Promega Corporation)
protocol was employed and inhibitory kinase activity of the test
compounds was determined as previously described.[44]
|
4.2
Biological assays
|
4.2.1
Cell culture and drug treatment
Briefly, the K562, Jurkat, and MT‐2 leukemia cell lines were cultured in
Rosewell Park Memorial Institute (RPMI) 1640 (Wako Pure Chemical
Industries, Osaka, Japan) medium with 10% fetal bovine serum (FBS;
Sigma‐Aldrich, MO). PBMCs (Precision Bioservices, Frederic, MD) were
incubated in RPMI 1640 medium with 10% FBS. All media were
supplemented with 89 μM/ml streptomycin (Meiji Seika Pharma, Tokyo,
Japan) in a humid atmosphere at 37°C and 5% CO2. In experiments, the
leukemia cells and PBMCs were incubated in 24‐well and 96‐well culture
plates (Iwaki brand Asahi Glass Co., Chiba, Japan) at 2 × 104 and 1 × 106
cells/ml concentration, respectively, for 24 hr. The stock solution of
compounds and Imatinib (Wako Pure Chemical Industries) in concentra-
tions between 0.3–3 mM were prepared in DMSO (Wako Pure Chemical
Industries) and then were added to the fresh culture medium. The
concentration of DMSO in the final culture medium was 1%.[39]
|
4.2.5
Immunoblot analysis
The K562 cells were incubated in the presence of 10 and 20 μM of
AQ15 and imatinib for 6 hr and then lysed in PBS‐Laemmli sample
buffer. Immunoblot analysis using phosphospecific‐p44/42 MAPK
(Erk1/2; Thr202/Tyr204; D13.14.4E) XP rabbit mAb (1:1,000; Cell
Signaling Technology, Danvers, MA) or anti‐β‐actin clone AC‐15
(Sigma‐Aldrich) was conducted. For immunoreactivity detection,
chemiluminescence method was performed.[45]
|
4.2.2
Cytotoxicity assay
|
4.2.6
DNA‐cleaving activity
The MTT test was performed as previously described in the literature
with small modifications.[40] The tested compounds were cultured with
cells in different concentrations (0.3–30 µM) for 24 hr and then, MTT
(Dojindo Molecular Technologies, Kumamoto, Japan) was added to
cells. After 4 hr of incubation, the medium was taken out and 100 µl
The DNA cleavage activities of the compounds on supercoiled plasmid
pUC19 DNA were studied by gel electrophoresis. pUC19 DNA (2 μg)
was dissolved in water and Tris/boric acid (Nacalai Tesque, Kyoto,
Japan) buffer (10 mM, pH 8.5) in the presence and absence of iron(II)

CIFTCI ET AL.
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sulfate heptahydrate (FeSO4·7H2O; 30 μM; Wako Pure Chemical
Industries), H2O2 (30 μM; Tokyo Chemical Industry, Tokyo, Japan) and
ascorbic acid (30 μM; Tokyo Chemical Industry) as an activator and
mixed with the different concentration of compounds. The reaction
was incubated at 37°C for 2 hr and then mixed with the loading buffer
(Takara, Kyoto, Japan). Agarose gel electrophoresis was undertaken
for 30 min at 100 V in Tris‐acetate/ethylenediaminetetraacetic acid
buffer. The gel (1% slab) was stained with ethidium bromide (Wako
Pure Chemical Industries) and then DNA was visualized by photo-
graphing the fluorescence of intercalated ethidium bromide under a
UV illuminator (Nippon Genetics, Tokyo, Japan).
Mahmut Yıldız
http://orcid.org/0000-0001-6317-5738
Mohamed O. Radwan
http://orcid.org/0000-0002-9220-2659
Amaç F. Tuyun
http://orcid.org/0000-0001-5698-1109
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ACKNOWLEDGMENTS
We thank Sadık Metin Ceyhan and Yunus Zorlu for helpful discussions
on crystal structure analyses. This study was financially supported by
the Scientific Research Projects Coordination Unit of Istanbul
University (project numbers: FBA‐2016‐20662 and FBA‐2017‐
24559) and the Grant‐in‐Aid for Challenging Exploratory Research
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http://orcid.org/0000-0002-0777-4012
http://orcid.org/0000-0003-3988-6120
Nilüfer Bayrak
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