A novel series of chlorinated plastoquinone analogs: Design, synthesis, and evaluation of anticancer activity

Article abstract of DOI:10.1111/cbdd.13651

Herein, we report the synthesis and cytotoxic effects of novel chlorinated plastoquinone analogs (ABQ1–17) against different leukemic cells. Compounds ABQ3, ABQ11, and ABQ12 demonstrated a pronounced antiproliferative effect against chronic myelogenous leukemia (CML) K562 cell line with IC₅₀ values of 0.82 ± 0.07, 0.28 ± 0.03, and 0.98 ± 0.22 μM, respectively. Among them, ABQ11 showed approximately three times higher selectivity than imatinib on CML. ABQ11-treated CML cells induced significant apoptosis at low concentration. Inhibitory effect of ABQ11 against eight different tyrosine kinases, including ABL1, was investigated. ABQ11 inhibited ABL1 with IC₅₀ value of 13.12 ± 1.71 μM, indicating that the moderate inhibition of ABL1 kinase is just an in-part mechanism of its outstanding cellular activity. Molecular docking of ABQ11 into ABL1 kinase ATP-binding pocket revealed the formation of some key interactions. Furthermore, DNA cleavage assay showed that ABQ11 strongly disintegrated DNA at 1 μM concentration in the presence of iron (II) complex system, assuming that the major mechanism for the anticancer effects of ABQ11 is DNA cleavage. In silico ADMET prediction revealed that ABQ11 is a drug-like small molecule with a favorable safety profile. Taken together, ABQ11 is a potential antiproliferative hit compound that exhibits unique cytotoxic activity distinct from imatinib.

Full text of DOI:10.1111/cbdd.13651

A Novel Series of Chlorinated Plastoquinone Analogs: Design,
Synthesis, and Evaluation of Anticancer Activity
Nilüfer Bayrak,[a] Hatice Yıldırım,[a] Mahmut Yıldız,[b] Mohamed O. Radwan,[c, d, e] Masami
Otsuka,[c, d] Mikako Fujita,*[d] Halil I. Ciftci,*[c, d] and Amaç Fatih Tuyun*[f]
[a]
Chemistry Department, Engineering Faculty, Istanbul University-Cerrahpasa, Avcilar, 34320,
Istanbul, Turkey
[b]
Chemistry Department, Gebze Technical University, Gebze, 41400, Kocaeli, Turkey
[c]
Department of Drug Discovery, Science Farm Ltd., Kumamoto 860-0802, Japan
[d]
Medicinal and Biological Chemistry Science Farm Joint Research Laboratory, Faculty of
Life Sciences, Kumamoto University, Kumamoto, Japan
[e]
Chemistry
of
Natural
Compounds
Department,
Pharmaceutical
and
Drug
Industries Research Division, National Research Centre, Dokki 12622, Cairo, Egypt
[f]
Department of Chemistry, Faculty of Science, Istanbul University, Vezneciler, 34134, Istanbul,
Turkey
* Author to whom correspondence should be addressed; E-Mail: aftuyun@gmail.com,
aftuyun@istanbul.edu.tr (A.F.T.), hiciftci@kumamoto-u.ac.jp (H.I.C.), and
mfujita@kumamoto-u.ac.jp (M. F.)
Tel.: +90212 440 0000.
Abstract
This article has been accepted for publication and undergone full peer review but has not been
through the copyediting, typesetting, pagination and proofreading process, which may lead to
differences between this version and the Version of Record. Please cite this article as doi:
10.1111/cbdd.13651
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Herein, we report the synthesis and cytotoxic effects of novel chlorinated Plastoquinone
analogs (ABQ1-17) against different leukemic cells. Compounds ABQ3, ABQ11, and
ABQ12 demonstrated a pronounced antiproliferative effect against chronic myelogenous
leukemia (CML) K562 cell line with IC50 values of 0.820.07 μM, 0.280.03 μM, and
0.980.22 M, respectively. Among them, ABQ11 showed approximately three times
higher selectivity than imatinib on CML. ABQ11-treated CML cells induced significant
apoptosis at low concentration. Inhibitory effect of ABQ11 against eight different tyrosine
kinases, including ABL1 was investigated. ABQ11 inhibited ABL1 with IC50 value of
13.121.71 M, indicating that the moderate inhibition of ABL1 kinase is just an in-part
mechanism of its outstanding cellular activity. Molecular docking of ABQ11 into ABL1
kinase ATP binding pocket revealed the formation of some key interactions. Furthermore,
DNA cleavage assay showed that ABQ11 strongly disintegrated DNA at 1 M
concentration in the presence of iron (II) complex system, assuming that the major
mechanism for the anticancer effects of ABQ11 is DNA-cleavage. In silico ADMET
prediction revealed that ABQ11 is a drug-like small molecule with a favorable safety
profile. Taken together, ABQ11 is a potential anti-proliferative hit compound that exhibits
unique cytotoxic activity distinct from imatinib.
Keywords:
Plastoquinone; SAR; CML; Apoptosis; DNA-cleavage
Graphical Abstract
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O
O
O
CH3
H
N
Modifications
H3C
H3C
H3C
H3C
H
n
Cl
R
O
ABQ3
ABQ11
ABQ12
R =
: 4-OCH3
: 2,5-OCH3
: 3,4-OCH3
Plastoquinones
1. Introduction
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The skeleton of a great number of the biologically active compounds is represented by
cyclic structure (Gribble and Joule 2008). Among them, quinone moiety is one of the most
important structure in drug discovery (Wellington et al. 2018). The studies of 1,4-quinones have
drawn a great deal of attention since their scaffold is an important pharmacophore (Jin, Ryu,
Moon, Cho, & Yun, 2004; Lien, Huang, Teng, Wang, & Kuo, 2002; Ravichandiran, Maslyk, et al.,
2019; Ravichandiran, Sheet, Premnath, Kim, & Yoo, 2019), and amply found in numerous natural
products. The first model from nature, prenylquinones are an important class of compounds
occurring in all living cells that contain characteristic 1,4-quinone and isoprenyl moieties. Based
on the structure, the major classes of prenylquinones are Plastoquinones (PQ), Ubiquinones,
Menaquinones, and Rhodoquinones. Among them, the discovery of the PQ (Figure 1) in 1946 by
Barr for electron transfer in photosynthesis was the beginning of an inevitable role in plant growth
and development (Kawamukai, 2018). Nigella sativa, commonly named as the black seed, is well
recognized by its various medicinal uses (Khan, Tania, Fu, & Fu, 2017). Thymoquinone (TQ, 2-
methyl-5-isopropyl-1,4-benzoquinone, Figure 1), the main bioactive compound of the volatile oil
of black seed, has been investigated for its numerous promising biological activities (Mostofa,
Hossain, Basak, & Bin Sayeed, 2017) including anticancer activity at both in vitro and in vivo
models (Attoub et al., 2013), anti-inflammatory (Chehl, Chipitsyna, Gong, Yeo, & Arafat, 2009),
antioxidant (Mansour, Nagi, El-Khatib, & Al-Bekairi, 2002), and antineoplastic effects (Worthen,
Ghosheh, & Crooks, 1998), after its first isolation from Nigella sativa by El-Dakhakhany (El-
Dakhakhany, 1963). Another model from nature is Geldanamycin (Figure 1) that was isolated
from Streptomyces hygrocopicus in 1970 as the first 1,4-benzoquinone Ansamycin (DeBoer,
Meulman, Wnuk, & Peterson, 1970). Numerous studies reveal its biological abilities, especially
anticancer activity through the inhibition of chaperone protein and Hsp90 (Le Brazidec et al.,
2004; Whitesell, Shifrin, Schwab, & Neckers, 1992). Looking at uses of quinones for cancer
therapy from a historical perspective, in 1958 Wakaki et al. (Wakaki et al., 1958) drew attention to
Mitomycin C (Figure 1) which was isolated from Streptomyces caespitosus. During the 1960s,
Mitomycin C was introduced as an anticancer agent in Japan then its usage has declined owing to
its toxicity issues. After realizing the appreciable anticancer activity of Mitomycin C, other
quinone compounds like Porfiromycin, Carbazilquinone, Triaziquone, and EO9 have been
obtained and some of them have been introduced into clinical applications (Baker, Izbicki, &
Vaitkevicius, 1976; Begleiter, 2000; Okada & Arakawa, 1976; Sukie, Takeishi, & Noguchi, 1957).
Nevertheless, Mitomycin C, in combination with other chemotherapeutic drugs, can show
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acceptable anticancer activities (Alkis et al., 2011; Kornek et al., 2004). Quinones have
demonstrated a wide spectrum of activity in cytotoxic activity via numerous mechanisms of
action, specifically reactive oxygen species (ROS) generation, DNA damage, uncoupling of
oxidative phosphorylation protein, and inhibition of electron transporters (da Silva et al., 2011;
Freitas et al., 2012; Wellington, Kolesnikova, Nyoka, & McGaw, 2019). Among them, versatile
studies have shown that the generation of semiquinone free radical after one-electron reduction of
the quinones and active oxygen species during oxidation-reduction reactions involved in the
mechanism of action (Bachur, Gordon, & Gee, 1977; Begleiter, 1985; Glazer, Hartman, &
Richardson, 1982; Lown, Begleiter, Johnson, & Morgan, 1976; Lown, Sim, & Chen, 1978; Myers
et al., 1977; Bayrak et al., 2019). Another mechanism is their interaction with the thiol group of
glutathione (Castro, Mariani, Panek, Eleutherio, & Pereira, 2008). An additional mechanism is
their ability to intercalate into DNA, thus inhibiting the synthesis of DNA and RNA (Cristofanilli
et al., 1999; Lenk, Muller, & Tanneberger, 1987).
O
O
H3C
O
O
O
CH3
CH3
O
O
CH3
CH3
O
O
OCONH2
H3C
H3C
H
N
n
H
CH3
H2N
H3C
O
O
H3C
H3C
CH3
O
H3C
OH
N
NH
O
CH3
H3C
OCONH2
Plastoquinones
Thymoquinone
Geldanamycin
Mitomycin C
Figure 1. Chemical structures of biologically important molecules containing 1,4-quinone moiety in
natural products.
Some key structural features for the antitumor action like substituents on the quinone ring,
the presence of good leaving groups, the number, and position of nitrogen atoms substituted in the
cyclic ring seem to be important from the literature (Hendriks et al., 1993). In light of these
findings, the next goal of scientists has been to introduce new quinone-containing compounds by
offering new opportunities for the development of selective agents in the treatment of cancer.
Recent studies from our research group (Bayrak et al., 2017; Bayrak et al., 2019; Yildirim et al.,
2017) and that of Ferreira (Dias et al., 2018), Ryu (Ryu et al., 2010; Ryu et al., 2012), Tandon
(Tandon, Maurya, Kumar, Rashid, & Panda, 2014; Tandon et al., 2010), and Valderrama
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(Valderrama, Ibacache, Rodriguez, Theoduloz, & Benites, 2011; Valderrama, Leiva, Rodriguez,
Theoduloz, & Schmeda-Hirshmann, 2008) have shown that electrophilic character of 1,4-quinones
can undergo with neutral and/or anionic nucleophiles in different conditions in versatile reactions,
thus leading to various biologically active (hetero)cyclic quinone structures for their
pharmacological properties. In this regard, as a result of the immense value of quinone compounds
in organic and medicinal chemistry, 1,4-quinones have been studied for over a half-century as an
important class of building blocks and still continue to attract considerable attention due to the
broad range of synthetic and medicinal applications (Bayrak et al., 2019; Johnson-Ajinwo et al.,
2018; Pingaew et al., 2015; Ravichandiran, Kannan, Ramasubbu, Muthusubramanian, & Samuel,
2016; Ravichandiran, Subramaniyan, et al., 2019; Tandon & Kumar, 2013; Valderrama et al.,
2016; Wellington, 2015).
Within this framework, in this article, to gain a deeper insight into the biological profile,
the target PQ analogs (ABQ1-17) were rationally designed as follows: (i) use of 2,3-dimethyl-1,4-
benzoquinone moiety of PQ as a core structure; (ii) introduction of chlorine atom to the 2-position
of the 1,4-benzoquinone moiety to increase the cytotoxic activity against CML cells (Ciftci et al.,
2019), since recent studies show that chlorine atom is essential for high anti-cancer activity;
(Lawrence et al., 2010; Li et al., 2018); (iii) substitution of the side chain with substituted amines.
The PQ analogs (ABQ1-17) were synthesized within this concept and investigated in terms of the
structure-activity relationship (SAR) for anticancer activity against three leukemic cancer cell
lines (K562, Jurkat, and MT-2) and human peripheral blood mononuclear cell line (PBMC) using
imatinib as a positive control. In addition, the most potent anticancer agent was further
investigated for their apoptotic effects on CML cell line and inhibitory potencies against different
tyrosine kinases, including ABL1. Furthermore, molecular docking studies with the ATP-binding
sites of ABL1, DNA cleavage capability, and in silico ADMET prediction were also performed
using the most potent compound.
2. Results and Discussion
2.1. Chemical Synthesis
A general synthetic approach to the target PQ analogs (ABQ1-17) started with the quinone
synthesis from commercially available 2,3-dimethylhydroquinone (1) by HNO3/HCl variation
according to the reported method previously (Scheme 1) (Ryu & Lee, 2006). The target PQ
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analogs (ABQ1-17) were synthesized by the reaction of the key intermediate (2) with
corresponding substituted amines in absolute ethanol at relatively high temperatures but in the
absence of CeCl3 (Ryu, Shin, Seo, & Kim, 2002). All synthetic analogs were elucidated by FTIR,
1H NMR, 13C NMR, and mass spectroscopy. Additionally, the structures of a number of PQ
analogs were characterized by X-ray diffraction analysis of a single crystal obtained by slow
evaporation of the ethyl alcohol solution of ABQ10 (1896464), ABQ14 (1896465), and ABQ17
(1896466) (Figure 2) (For details, please see the Supplementary file).
ABQ10
ABQ14
ABQ17
Figure 2. ORTEP drawings of ABQ10, ABQ14, and ABQ17.
O
O
R1
R4
OH
OH
O
O
H
N
H3C
H3C
R2
R3
H3C
H3C
H3C
H3C
Cl
Cl
(i)
(ii)
Cl
1
2
ABQ1-17
Scheme 1. Synthesis of the chlorinated PQ analogs (ABQ1-17): (i) HNO3/HCl, 10 min, 90oC; (ii)
Substituted amines, EtOH, reflux, 4-12 h.
2.2. Anticancer Activity
All newly obtained target PQ analogs (ABQ1-17) were screened for the cytotoxic activity
against three cancer cell lines including K562 (chronic myelogenous leukemia), Jurkat, MT-2
(other leukemias), and peripheral blood mononuclear cells (PBMCs) employing a 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT assay) by using imatinib as control
(Table 1 and Figure 3). The anticancer activity of each analog was presented as the concentration
of compound that completed 50% inhibition (IC50) of cancer cell growth. PQ analogs were
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synthesized in order to discover the effect of the different alkoxy group(s) (methoxy, ethoxy,
butoxy, hexyloxy, and octyloxy) and positions to investigate the effect of the alkyl chain on
cytotoxicity. The values were listed in Table 1. The tested PQ analogs except ABQ9 showed
better cytotoxicity than imatinib against K562 cell lines. The results for ABQ8-10 revealed that
these compounds possessed IC50 values higher than 10 μM against MT-2 cells. In the case of
Jurkat, all compounds (ABQ1-17) exhibited better anticancer activity than imatinib. The analogs
(ABQ7-9) with a long chain alkoxy group led to a decrease the inhibitory activity dramatically
against three cancer cell lines, especially against MT-2 cell line. Among the analogs containing
only one alkoxy group, ABQ3 with one alkoxy group (methoxy) at the 4-position showed the best
anticancer activity with the IC50 value of 0.82±0.07 μM and 1.51±0.29 μM against K562 and
Jurkat cell lines, respectively. In the case of all of the three cancer cell lines, the analogs, ABQ2,
ABQ3, ABQ11, ABQ12, ABQ13, and ABQ15 exhibited strong anticancer activity compared with
the control drug imatinib.
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Table 1. Structure and Cytotoxicity of PQ Analogs (ABQ1-17) in K562, Jurkat, and MT-2 Cell Lines by MTT Assay
O
R1
H
N
H3C
H3C
R2
R3
Cl
O
R4
ABQ1-17
Substitution Groups
Cell Type (IC50, μM)a
ID
SId
R1
R2
R3
R4
H
PBMCb
K562c
Jurkatc
MT-2c
7.50±1.44
4.15±1.23
5.41±0.95
7.67±0.71
7.60±1.56
9.34±0.38
6.30±0.76
>10
ABQ1
ABQ2
ABQ3
ABQ4
ABQ5
ABQ6
ABQ7
ABQ8
ABQ9
ABQ10
ABQ11
ABQ12
ABQ13
ABQ14
ABQ15
ABQ16
OCH3
H
H
3.06±0.09
2.27±1.01
0.82±0.07
4.42±0.39
1.36±0.27
1.90±0.55
3.09±1.05
5.23±1.47
>10
2.78±0.37
2.01±0.88
1.51±0.29
2.81±0.28
2.84±0.73
2.01±0.62
2.44±0.53
4.95±0.85
5.47±1.19
4.86±0.89
0.92±0.08
1.99±0.22
2.85±0.98
3.00±0.32
1.73±0.08
2.60±0.15
H
OCH3
H
H
H
H
OCH3
H
8.37±1.33
10.21
OCH2CH3
H
H
H
H
OCH2CH3
H
OCH2CH3
O(CH2)3CH3
O(CH2)5CH3
O(CH2)7CH3
OCH3
H
H
H
H
H
H
H
H
H
H
H
H
H
>10
OCH3
H
H
5.82±0.32
0.28±0.03
0.98±0.22
4.72±1.21
5.85±1.07
2.14±0.85
2.23±0.59
>10
OCH3
H
H
OCH3
H
4.42±1.18
8.49±1.75
2.50±0.29
5.75±0.81
5.12±1.18
8.37±1.85
6.60±1.79
7.96±0.94
15.78
8.66
H
OCH3
OCH3
H
OCH3
H
H
OCH3
OCH2CH3
H
OCH2CH3
H
H
OCH2O
OCH2CH2O
H
H
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ABQ17
H
OCH3
OCH3
OCH3
4.12±0.76
6.02±0.92
2.62±0.54
8.65±1.35
7.92±2.27
Imatinibe
35.56±2.95
19.30±3.41
5.90
a The reported values represent the mean ±SD for each compound based on three independent experiments.
b Normal blood cells (PBMCs).
c Cell lines include chronic myelogenous leukemia (K562) and other leukemias (Jurkat and MT-2).
d The SI values are calculated as the ratio of the IC50 between the PBMCs and chronic myelogenous leukemia (K562) cells.
e Used as reference.
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120
100
80
60
40
20
0
PBMC
ABQ3
ABQ11
ABQ12
Imatinib
0
3
10
30
100
300
Concentration (µM)
Figure 3. Cytotoxic effects of the compounds and imatinib against PBMC cells after 24 h incubation.
The cytotoxicity results of selected analogs (ABQ3, ABQ11, and ABQ12) on PBMCs were
shown in Figure 3. The selective index (SI) is calculated by dividing the IC50 of the PBMC growth
inhibition with the IC50 of the K562 cell growth inhibition. The presence of two methoxy groups at
2,5-positions (ABQ11) resulted in a not only 21-fold increase in cytotoxicity against K562 cell line
but also an almost 3-fold increase in the SI compared with the control drug imatinib (Table 1). These
selected PQ analogs (ABQ3, ABQ11, and ABQ12) have better potency and SI than imatinib against
K562 cell line.
The SAR studies of the tested analogs were analyzed by modification of various positions
(mono- or polysubstituted) with the different alkoxy group(s) in the substituted amino group. Our
recently published results reveal that the introduction of the chlorine atom at the 2-position of the
quinone moiety has a positive effect on its anti-cancer activity on CML (Ciftci et al., 2019). The
analogs containing bulky alkoxy group in the amino moiety such as butoxy, hexyloxy, and octyloxy
are the least active. With the elongation of the bulky alkyl tail in the monosubstituted alkoxy group at
the 4-position, the cytotoxic activities decreased in the PQ analogs (ABQ3, ABQ6, ABQ7, ABQ8,
and ABQ9). Furthermore, leading a decrease in inhibitory activity has also been noticed for
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polysubstituted analogs at 2,5-positions (ABQ11 vs. ABQ14) against all three cancer cell lines with
the elongation of the alkyl tail in the alkoxy group. The analogs with monosubstituted with alkoxy
group containing not bulky alkyl chain at the 4-position had relatively higher potency against K562
and Jurkat cell lines.
Due to the selective anticancer potential of the ABQ11 against CML, it was further
investigated for the evaluation of apoptosis in K562 cell line using the annexin V/ethidium
homodimer III and Hoechst 33342 staining method. K562 cell-treated ABQ11 was incubated at the
IC50 concentration and then, stained and observed by a fluorescence microscope (Figure 4). In the
control experiment, all cells were stained with blue (healthy cells) at 6 h after the treatment of DMSO.
On the other hand, K562 cell line was incubated with ABQ11 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) (Fig. 4a). This result indicated that ABQ11 induced apoptosis mainly at
low concentration and in earlier time with 71% when compared with imatinib (62%), as shown in
Figure 4b.
(a)
Control
ABQ11
Imatinib
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(b)
K562 Cell Line
120
100
80
60
40
20
0
Necrosis
Late apoptosis/necrosis
Apoptosis
ABQ11
Imatinib
Figure 4. Apoptotic effects of ABQ11 and imatinib at their IC50 concentrations on CML cell line (a)
after 6 h treatment. (b) A total of approximate 100 stained cells were selected randomly in each
experiment of (a) and were classified into 3 types “apoptosis” (green), “necrosis or late apoptosis”
(both green and red), and “necrosis” (red).
In order to explore the inhibition profile of ABQ11 on tyrosine kinase enzymes, multipoint
dose-responses experiments were performed using a panel of kinases (ABL1, BRK, BTK, CSK, FYN
A, LCK, LYN B, and SRC). According to the results given in Table 2, ABQ11 was found as the most
potent analog against ABL1 with IC50 values of 13.12±1.71 μM, when compared with imatinib
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(0.27±0.004 μM). On the other hand, the activity of ABQ11 was stronger than that of imatinib on
BRK, BTK, and SRC with the IC50 values of 12.47±1.22 μM, 54.98±8.19 μM, and 67.46±7.57 μM,
respectively. Furthermore, ABQ11 was found to be inactive against CSK, FYN A, LCK, and LYN B.
The results indicated that ABQ11 inhibited multiple kinases in the panel of eight kinases with a
distinct inhibitory profile than imatinib.
Table 2. The inhibition profile of ABQ11 and imatinib in the panel of eight kinases.
IC50 Value (µM)
Kinases
ABQ11
Imatinib
ABL1
BRK
13.12±1.71 0.27±0.04
12.47±1.22 20.56±1.41
BTK
54.98±8.19
>100
>100
CSK
25.45±2.30
13.34±0.93
0.68±0.06
7.16±0.83
>100
FYN A
LCK
>100
>100
LYN B
SRC
>100
67.46±7.57
Thereafter, a molecular docking study was carried out to explore the possible binding mode of
the most promising antiproliferative lead compound ABQ11 in the ATP-binding site of ABL1 kinase.
It grabbed our attention that ABQ11 is smaller and has different stereo-electronic features from the
standard imatinib. Surprisingly, ABQ11 was able to form some key interactions that granted it a
moderate inhibitory activity, as mentioned above. It adopts a similar, in part, the binding mode of
imatinib’s amino pyrimidyl pyridine moiety and imitates some of its intermolecular bonds with the
amino acid residues. ABQ11 NH forges a key interaction with the gatekeeper amino acid Thr315. The
chlorine substitution on the dimethyl benzoquinone makes a halogen bond (Ibrahim et al., 2017) with
Thr315 as well. The dimethyl benzoquinone forms one more significant hydrogen bond with Met318.
Furthermore, the dimethyl benzoquinone is sandwiched between Val256 and Leu370 making two
CH-π interactions that play a role in stabilizing the complex. ABQ11 methyl group interacts with the
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Phe382 phenyl group through the CH-π bond. ABQ11’s lower activity, compared to imatinib, is
attributed to lacking important interactions with His361, Asp381, and Glu286 (For details, please see
the Supplementary file).
The computational predictions matched the experimental data and confirmed that the mild
inhibition of ABL kinase by ABQ11 is not the sole underlying mechanism for its prominent
cytotoxicity. To explain such cellular activity of ABQ11, we studied another mechanism of action
using metal-binding/oxygen-activating site of quinones, i.e., oxidative cleavage of DNA strand by
ABQ11. The DNA-cleaving capability of ABQ11 at 1 μM concentration was studied using pUC19
DNA in the presence and absence of the iron (II), H2O2, and ascorbic acid complex, as shown in
Figure 5. The reaction solution was incubated at 37 °C for 2 h 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). The results showed that ABQ11 strongly cleaved
the DNA through the activation of the iron (II) complex. Those results pointed out that ABQ11 may
activate oxygen in the cytoplasm. It also indicated that the DNA cleavage might correlate with the
outstanding cytotoxic activity against K562.
Figure 5. The DNA-cleaving activity of ABQ11 with and without of FeSO4, H2O2, and ascorbic acid
system.
To assess the drug-likeness of ABQ11, we applied computational calculations of ADMET
Predictor 9.0 to predict its risks and physicochemical properties (Figure 6). ABQ11 toxicity risk
(TOX-Risk) value is 1 (acceptable value is up to 3.3). Mutagenic risk (MUT-Risk) value is 1.5,
slightly higher than the standard value 1. Risk related to metabolism by or inhibition of major
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cytochrome P450s (CYP-Risk) is 1.0 (standard value does not exceed 2.5). Noticeably, ABQ11
exhibits a potentially adequate safety profile. ABQ11 demonstrated 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). Concomitantly, absorption risk (Absn-Risk) is 0.0, indicating a high possibility of
good oral activity. Taken together, the overall ADMET-Risk value is 2.0 (acceptable value is up to
7.5). Obviously, ABQ11 is likely to combine both favorable pharmacokinetic and safety profiles.
ADMET-Risk 2.0
TOX-Risk 1.0
MUT-Risk 1.5
Absn-Risk 0.0
CYP-Risk 1.0
Figure 6.
Star plots depiction
for different risk descriptors associated with ABQ11 as calculated by ADMET Predictor 9.0.
3. Conclusions
New PQ analogs (ABQ1-17) were synthesized, characterized, and evaluated for their
anticancer activities against different leukemia cell lines (K562, Jurkat, and MT-2). Having a chlorine
atom at the 2-position of the 1,4-benzoquinone moiety enhanced the cytotoxic activity against the
designated cell lines at low micromolar concentrations. ABQ3, ABQ11, and ABQ12 displayed the
highest anticancer activity against all cancer cell lines compared to imatinib. More remarkably,
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ABQ11 showed approximately 21-fold better cytotoxicity on CML cells with approximately 2.5-fold
high SI than imatinib. ABQ11 was further estimated for its apoptotic induction, kinase profiling,
DNA cleavage, and safety profile analysis. The results demonstrated that ABQ11 has significant
apoptotic effects on K562 CML cells in comparison to imatinib. Moreover, ABQ11 showed a far less
ABL1 kinase inhibitory activity than imatinib. Molecular docking revealed the possible binding mode
of ABQ11 into the ATP site of ABL1 kinase showing the formation of some crucial interactions
which are in accordance with the in vitro biological assay data. Most importantly, DNA cleavage
assay demonstrated that ABQ11 efficiently disintegrated DNA at 1 μM concentration in the presence
of the iron (II) complex. These results showed that the central mechanism for the antiproliferative
action of ABQ11 is DNA-cleavage, not ABL1 kinase inhibition. Finally, in silico ADMET prediction
highlighted that ABQ11 has desirable drug-like properties and favorable safety profile.
4. Experimental Section
4.1. Chemistry
All corresponding substituted amines, reagents, and solvents were commercially obtained from the
suppliers and used in research as such without further purification. The precursor (2) was synthesized
using the reported method in the literature (Ryu & Lee, 2006). Melting points (mp) were determined
by the open capillary method and uncorrected. General reaction visualization was achieved by thin
layer chromatography (TLC) purchased from Merck KGaA (silica gel 60 F254) based on Merck DC-
plates (aluminum based) by using UV light (254 nm). Chromatographic separations were carried out
using silica gel 60 (Merck, 63-200 µm particle sized, 60-230 mesh). FTIR spectra were recorded as
ATR on either a Thermo Scientific Nicolet 6700 spectrometer or a Perkin Elmer Spectrum 100
Optical FTIR spectrometer. All the nuclear magnetic resonance spectra of the analogs were obtained
on either a VarianUNITY INOVA spectrometers (500 MHz for 1H NMR and 125 MHz for 13C NMR) or
Bruker spectrometers (400 MHz for 1H NMR and 100 MHz for 13C NMR) in ppm (δ) in CDCl3 refer
to the solvent signal center at δ 7.19 and δ 76.0 ppm. Chemical shifts (δ) are reported in ppm.
Coupling constants (J) are reported in Hz. Standard abbreviations indicating multiplicity were used as
follows: br s (broad singlet), s (singlet), d (doublet), t (triplet), q (quartet), p (pentet), m (multiplet),
and dd (doublet of doublets). Mass spectra were measured using a Thermo Finnigan LCQ Advantage
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MAX MS/MS spectrometer equipped with an ESI (Electrospray ionization) sources. Data for the
single crystal compounds were obtained with Bruker APEX II QUAZAR three-circle diffractometer.
Indexing was performed using APEX2 (APEX2, version 2014.1-1, Bruker, 2014, Bruker AXS
Inc.,Madison, WI). Data integration and reduction were carried out with SAINT (SAINT, version
8.34A, Bruker, 2013, Bruker AXS Inc., Madison, WI). Absorption correction was performed by
multi-scan method implemented in SADABS (SADABS, version 2012/2, Bruker, 2012, Bruker AXS
Inc.,Madison, WI.). The Bruker SHELXTL (SHELXTL, version 6.14, Bruker,2000, Bruker AXS Inc.,
Madison, WI.) software package was used for structures solution and structures refinement. Aromatic
C-bound 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 (Spek, 2009) Mercury software (Macrae et al., 2006) was used for visualization of the .cif
files.
4.2. General Procedure for the Preparation of PQ analogs (Ryu et al., 2002)
A solution of the corresponding substituted amines (1.2 equiv) in ethanol (10 mL) was added
dropwise to a stirred solution of quinone (2, 0.50 g, 2.44 mmol) in ethanol (25 mL). The reaction
mixture was stirred at reflux for 6-12 h 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 or CHCl3 (50 mL) and distilled water (30 mL) was added, and the solution was
washed sequentially with water (3 x 30 mL). The organic layer was dried over anhydrous CaCl2,
filtered, and evaporated to dryness under reduced pressure. The crude product was further purified by
means of column chromatography on silica gel to give the corresponding PQ analogs.
4.3. Biochemistry
4.3.1. Cell Culture and Drug Treatment
K562, Jurkat and MT-2 were cultured in RPMI 1640 (Wako Pure Chemical Industries, Osaka, Japan)
medium with 10% fetal bovine serum (FBS) (Equitech-Bio, Texas, U.S.). PBMCs (Precision
Bioservices, Frederic, MD) were incubated in RPMI 1640 medium with 10% human serum AB (HS)
(Gemini, Woodland, CA). All media were supplemented with 89 M/mL streptomycin (Meiji Seika
Pharma, Tokyo, Japan) in a humid atmosphere at 37oC and 5% CO2. In experiments, the leukemia and
PBMC cells were incubated in 24-well and 96-well culture plates (Iwaki brand Asahi Glass Co.,
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Chiba, Japan) at 2x104 and 106 cells/mL concentration respectively for 24 h. The stock solution of
compounds and imatinib (Wako Pure Chemical Industries, Osaka, Japan) in concentrations between
3-30 mM were prepared in dimethyl sulfoxide (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% (Sever et al., 2019).
4.3.2. Cytotoxicity Assay
The MTT test was performed as previously described in the literature with small modifications (Ali et
al., 2019; Ciftci et al., 2019). Cells were cultured in the presence of various concentrations (0.3-100
µM) of the tested compounds for 24 h and then, stained with MTT solution and incubated for an
additional 4 h at 37 °C. The solution was removed and the formazan crystals were solubilized by
adding 100 µL DMSO. The absorbance at 550 nm was measured using a microplate reader Infinitive
M1000 (Tecan, Groding, Austria) with background subtraction at 630 nm. All experiments were run
in triplicate and cell viability was calculated as the percentage of the viable control cells. IC50 values
of compounds were estimated from the results of the MTT test described as the drug concentrations
that reduced absorbance to 50% of control values.
4.3.3. Detection of Cell Death
After treatment of K562 cells with ABQ11 and imatinib at IC50 concentrations for 6 h,
apoptotic/necrotic/healthy detection kit (PromoKine, Heidelberg, Germany) was performed according
to PromoKine`s instructions with the modifications (Karabacak et al., 2015). Briefly, cells were
harvested and washed with PBS, then were suspended with binding buffer (1). After that, 50 μL of
binding buffer, 4 μL of FITC-Annexin V solution, 4 μL of ethidium homodimer III solution and 4 μL
of Hoechst 33342 solution were added into the cells 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 (Tateishi et al., 2017).
4.3.4. Tyrosine Kinase Profiling Assay
The kinase selectivity profiling assay TK-2 from Promega was performed as previously described
with some modification (Ciftci et al., 2018). In this system, multipoint dose-response experiments
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were carried out using eight kinases, namely ABL1, BRK, BTK, CSK, FYN A, LCK, LYN B, and
SRC. Briefly, the kinase and substrate strips were diluted with 95 µL 2.5x kinase reaction buffer and
15 µL of 100 µM ATP solution respectively. The reaction of kinases was performed in the 384-well
plate using 2 µL of compound solution at multiple concentrations in a buffer, 4 µL of kinase working
stock and 4 µL of ATP/substrate working stock. After 1h of incubation at room temperature, the
ADP-Glo Kinase Assay (Promega) protocol was employed and inhibitory kinase activity of the test
compounds was determined as previously described (Ciftci, 2019).
4.3.5. Molecular Modeling and ADMET Prediction
The crystal structure of the ABL kinase domain in complex with imatinib was obtained from Protein
Data Bank (PDB: 1IEP); ABQ11 was built by ChemDraw Professional 15.1. Before docking
simulations, ABQ11 and 1IEP preparation included the addition of hydrogens, the assignment of
bond order, and assessment of the correct protonation as previously described (Radwan et al., 2019;
Radwan et al., 2019). MOE 2018.01 software (Chemical Computing Group, Montreal, Canada) was
employed for preparation, interactive docking, visualization and analysis procedures using its default
parameters (Koga et al., 2017; Tanaka et al., 2017). The ADMET properties of ABQ11 were
calculated in silico using ADMET predict 9 from Simulation Plus, Inc.; the risk models were
previously explained in detail (Mehanna et al., 2017).
Acknowledgments
This work was financially supported by the Scientific Research Projects Coordination Unit of Istanbul
University (Project number: FBA-2016-20662) for supplying the equipment and materials. The
crystallographic data have been deposited at the Cambridge Crystallographic Data Centre, and CCDC
reference numbers are 1896464 for the title compound ABQ10, 1896465 for the title compound
ABQ14, and 1896466 for the title compound ABQ17. The data can be obtained available free of
charge from http://www.ccdc.cam.ac.uk/conts/retrieving.html or from the Cambridge
Crystallographic Data Centre (CCDC), 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44
(0)1223336033; email: deposit@ccdc.cam.ac.uk. We also thank Sadık Metin Ceyhan for helpful
discussions on crystal structure analyses.
Data Availability Statement
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The data that supports the findings of this study are available in the supporting information of this
article.
Conflicts of Interest
The authors declare no conflict of interest.
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