Structure based design, synthesis, and evaluation of anti-CML activity of the quinolinequinones as LY83583 analogs

Article abstract of DOI:10.1016/j.cbi.2021.109555

Quinone-based small molecules are the promising structures for antiproliferative drug design and can induce apoptosis in cancer cells. Among them, one of the quinolinequinones, named as 6-anilino-5,8-quinolinequinone, LY83583 has the ability to inhibit the growth of cancer cells as an inhibitor of cyclase. The biological potential of all synthesized compounds as the analogs of the identified lead molecule LY83583 that possessed the antiproliferative efficiency was determined. The two series of the LY83583 analogs containing electron-withdrawing or electron-donating group(s) were synthesized and subsequently in vitro evaluated for their cytotoxic activity against K562, Jurkat, MT-2, and HeLa cell lines using MTT assay. All the LY83583 analogs showed antiproliferative activity with good IC₅₀ values (less than positive control imatinib). Four analogs from each series were also selected for the determination of selectivity against human peripheral blood mononuclear cells (PBMCs). The analog AQQ15 showed high potency towards all cancer cell lines with almost similar selectivity of imatinib. In order to get a better insight into cytotoxic effects of the analog AQQ15 in K562 cells, further apoptotic effects due to annexin V/ethidium homodimer III staining, ABL1 kinase inhibition, and DNA cleaving ability were examined. The analog AQQ15 induced apoptotic cell death in K562 cells with 34.6% compared to imatinib (6.5%). This analog showed no considerable ABL1 kinase inhibitory activity but significant DNA cleavage activity indicating DNA fragmentation-induced apoptosis. Besides, molecular docking studies revealed that the analog AQQ15 established proper interactions with the deoxyribose sugar attached with the nucleobases adenine and guanidine respectively, in the minor groove of the double helix of DNA. In silico predicted pharmacokinetic parameters of this analog were found to comply with the standard range making it an efficient anticancer drug candidate for further research.

Full text of DOI:10.1016/j.cbi.2021.109555

Chemico-Biological Interactions 345 (2021) 109555
Contents lists available at ScienceDirect
Chemico-Biological Interactions
journal homepage: www.elsevier.com/locate/chembioint
Research paper
Structure based design, synthesis, and evaluation of anti-CML activity of the
quinolinequinones as LY83583 analogs
a
b
,
c
d
a
c,e
Nilüfer Bayrak , Halil I. Ciftci , Mahmut Yıldız , Hatice Yıldırım , Belgin Sever
,
c
b
,
c
c,
**
f,*
Hiroshi Tateishi , Masami Otsuka , Mikako Fujita
, Amaç Fatih Tuyun
a
Department of Chemistry, Faculty of Engineering, Istanbul University-Cerrahpasa, Avcilar, Istanbul, Turkey
Department of Drug Discovery, Science Farm Ltd., Kumamoto, Japan
b
c
d
e
f
Medicinal and Biological Chemistry Science Farm Joint Research Laboratory, School of Pharmacy, Kumamoto University, Kumamoto, Japan
Chemistry Department, Gebze Technical University, Gebze, Kocaeli, Turkey
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Anadolu University, Eskisehir, Turkey
Department of Chemistry, Faculty of Science, Istanbul University, Fatih, Istanbul, Turkey
A R T I C L E I N F O
A B S T R A C T
Keywords:
Quinone-based small molecules are the promising structures for antiproliferative drug design and can induce
apoptosis in cancer cells. Among them, one of the quinolinequinones, named as 6-anilino-5,8-quinolinequinone,
LY83583 has the ability to inhibit the growth of cancer cells as an inhibitor of cyclase. The biological potential of
all synthesized compounds as the analogs of the identified lead molecule LY83583 that possessed the anti-
proliferative efficiency was determined. The two series of the LY83583 analogs containing electron-withdrawing
or electron-donating group(s) were synthesized and subsequently in vitro evaluated for their cytotoxic activity
against K562, Jurkat, MT-2, and HeLa cell lines using MTT assay. All the LY83583 analogs showed anti-
proliferative activity with good IC50 values (less than positive control imatinib). Four analogs from each series
were also selected for the determination of selectivity against human peripheral blood mononuclear cells
LY83583
Quinolinequinones
Aminoquinone
Apoptosis
Chronic myelogenous leukemia (CML)
DNA-Cleavage
Structure-activity relationship (SAR)
(
PBMCs). The analog AQQ15 showed high potency towards all cancer cell lines with almost similar selectivity of
imatinib. In order to get a better insight into cytotoxic effects of the analog AQQ15 in K562 cells, further
apoptotic effects due to annexin V/ethidium homodimer III staining, ABL1 kinase inhibition, and DNA cleaving
ability were examined. The analog AQQ15 induced apoptotic cell death in K562 cells with 34.6% compared to
imatinib (6.5%). This analog showed no considerable ABL1 kinase inhibitory activity but significant DNA
cleavage activity indicating DNA fragmentation-induced apoptosis. Besides, molecular docking studies revealed
that the analog AQQ15 established proper interactions with the deoxyribose sugar attached with the nucleobases
adenine and guanidine respectively, in the minor groove of the double helix of DNA. In silico predicted phar-
macokinetic parameters of this analog were found to comply with the standard range making it an efficient
anticancer drug candidate for further research.
1
. Introduction
An immense number of natural products (generally defined as small-
interest [2,4]. The anthracyclines doxorubicin (Doxils; Adriamycins),
daunorubicin (Cerubidines), and epirubicin (Ellences), for example, are
typically well-known drugs used in cancer treatment [4,5]. Most of the
clinically approved anticancer drugs in the market have been inspired by
natural products which constitute a broad biodiversity of molecules [6,
7].
molecule secondary metabolites) serving as drugs for the treatment of
many life-threatening diseases that exist in nature originating from
plants, bacteria, fungi, and marine life forms and play a fundamental
role in chemical biology [1–4]. In recent years, the use of natural
products or natural product-like small molecules which enable the
success of privileged structures is of both biological and medicinal
Streptonigrin (1) shown in Fig. 1, a bioactive alkaloid, was first
isolated from Streptomyces flocculus in 1959; afterwards, the following
year (in 1960), the first member of quinolinequinones (QQs) was
*
Corresponding author.
* Corresponding author.
E-mail addresses: mfujita@kumammot-u.ac.jp (M. Fujita), aftuyun@gmail.com, aftuyun@istanbul.edu.tr (A.F. Tuyun).
*
https://doi.org/10.1016/j.cbi.2021.109555
Received 25 March 2021; Received in revised form 22 May 2021; Accepted 10 June 2021
Available online 16 June 2021
0
009-2797/© 2021 Elsevier B.V. All rights reserved.

N. Bayrak et al.
Chemico-Biological Interactions 345 (2021) 109555
originally introduced as an antitumor agent [8] against cancer cell lines
present work, we have designed and synthesized new QQs for cancer
drug research by further realization since LY83583 is used as the lead
compound for the design. As shown in Fig. 1, the LY83583 structure
consists of three fragments (aminophenyl, 1,4-quinonyl, and pyridyl
moieties). Thus, we have decided to merge these important points from
our recent reports with LY83583 both by replacing the methyl groups
with a pyridyl ring (to form a QQ moiety) and inserting the different
group(s) within the aminophenyl ring (Fig. 2). In addition to the design
and synthesis of QQs, these two series of QQs were first evaluated in vitro
against four cancer cell lines (K562 (myeloid leukemia), HeLa (human
cervical carcinoma), Jurkat, and MT-2 (other two human T-cell leuke-
mia)). Then, the most promising analogs were tested against human
peripheral blood mononuclear cells (PBMCs) to determine the selec-
tivity index (SI). The most selective and efficient anticancer agent to-
wards K562 cells was further searched for its apoptotic effects based on
Hoechst 33342/annexin V/ethidium homodimer III staining, ABL1 ki-
nase inhibition, and DNA cleavage capacity. Molecular docking studies
were also carried out for the analogs with the most significant DNA
cleavage efficiency to detect its binding potential to DNA. In silico Ab-
sorption, Distribution, Metabolism, and Excretion (ADME) studies were
also assessed for all synthesized analogs to understand the compliance of
compounds with particular pharmacokinetic determinants.
[
9,10] and an antiviral agent against viruses including HIV-1 [11–13].
The discovery of streptonigrin (1) in the 1970s for anticancer activity
against leukemia cell lines attracted the attention of researchers [14]. In
the human clinical phase (for oral and parenteral application), the
studies showed that the streptonigrin (1) was found effective against
malignant lymphomas, mycosis fungoides, and Hodgkin’s disease [15].
Besides, the use of streptonigrin (1) was limited due to the toxicity,
whereas it is still one of the most important templates for the design of
new drugs in the future [16,17]. The researchers continued to find an
ample ground between the therapeutic efficacy and reduced toxicity. As
a next step, lavendamycin (2) [18,19] and streptonigron (3) [19,20], the
structurally analogs of streptonigrin (1), were originally isolated from
the Streptomyces species (Fig. 1). Recent evidences have suggested that a
significant number of lavendamycin analogs have displayed strong
anticancer activity with low animal toxicity [21,22]. Additionally,
streptonigron (3) was also proposed for its high anticancer, antibacte-
rial, and antiviral activities [14]. The structure-activity relationship
(
SAR) studies of QQs by Kremer (1967) pointed out that the replacement
of carbonyl group with NH group led to a decrease in activity, as in
azastreptonigrin (4) [23]. Thus, what we know about pharmaceutical
activity for streptonigrin (1), lavendamycin (2), or streptonigron (3) are
mainly based on p-quinone moiety [24]. Since the streptonigrin (1) is
composed of four rings designated A (p-quinone), B (pyridine), C
2. Results and discussion
(
substituted pyridine), and D (substituted phenyl), a considerable
amount of literature has been published on examining the structures of
the most potent compounds [25,26]. The extensive SAR studies on
structural modification particularly conducted by Johnson [24] have
revealed that the QQ moiety (AB ring system) has played a critical role in
the anticancer potency [26–28]. In addition to this finding, rings C
2.1. Design and chemical synthesis
The attention of our research group has long focused on the in-
teractions of different types of 1,4-quinones as electron-deficient com-
pounds with electron-rich reagents (nucleophiles), obtaining
compounds with promising biological (antimicrobial and/or anticancer)
activities [32–38]. The synthetic pathway of a series of LY83583 analogs
substituted with different group(s) on aminophenyl ring is shown in
Table 1. For the synthesis of a series of LY83583 analogs (QQs),
following a literature procedure reported by Shaikh et al. [24], the
starting compound (6,7-dichloro-5,8-quinolinequinone, 2) was obtained
by the oxidation of commercially available 8-hydroxyquinoline (1) with
sodium chlorate in concentrated HCl solution. Within this field of
research, a considerable amount of literature has been published on the
reactions of QQs with different nucleophiles [39–43]. These data clearly
indicate that QQs are very reactive towards nucleophiles such as thiols
[41,44], amines [45,46], and alcohols [47,48]. The product distribution
is highly sensitive to modification of the reaction conditions such as
molar ratios of substrates, reagents, and polarity of reaction medium
(
pyridyl moiety) and D (the substituted phenyl moiety) are not essential
for the activity in murine cancer cell lines, whereas these moieties have
enhanced the anticancer activity against the human cancer cell lines
[
15,24]. Commercially available anticancer drug LY83583 (6-anilino-5,
-quinolinequinone) outlined in Fig. 1, which acts also as an inhibitor of
8
guanylyl cyclase, prevents cell proliferation in cancer cells that pro-
duced through over production of reactive oxygen species [29–31]. We
have recently reported the discovery of Plastoquinone analogs (based on
dimethyl amino-1,4-benzoquinones) that are responsible for their se-
lective anticancer efficacy which induces the apoptosis in cancer cell
leaving the normal cells safe [32–34]. In these works, SAR studies of the
Plastoquinone analogs revealed that (a) the existence of the group(s)
within the aminophenyl ring and (b) the presence of a chlorine atom
were important. Taking into account the aforementioned data, in the
Fig. 1. Discovery of LY83583 based on QQ moiety starting from natural products.
2

N. Bayrak et al.
Chemico-Biological Interactions 345 (2021) 109555
Fig. 2. Design of QQs.
Table 1
Preparation of two subseries (AQQ1-19) of the LY83583 as its analogs.
ID
EWG
ID
EWG
ID
EDG
ID
EDG
AQQ1
AQQ2
AQQ3
AQQ4
AQQ5
2-COOCH
3-COOCH
4-COOCH
2-COOCH
3-COOCH
3
3
3
2
2
AQQ6
AQQ7
AQQ8
AQQ9
AQQ10
4-COOCH
2
CH
3
3
3
3
AQQ11
AQQ12
AQQ13
AQQ14
AQQ15
2,3-diCH
2,4-diCH
2,5-diCH
3,4-diCH
3,5-diCH
3
3
3
3
3
[55]
[53]
AQQ16
AQQ17
AQQ18
AQQ19
2-CH(CH
3-CH(CH
4-CH(CH
3
3
3
)
)
)
2
2
2
2-COOC(CH
3-COOC(CH
4-COOC(CH
4-CN
3
)
3
)
3
)
CH
CH
3
2 3 2
4-N(CH CH )
3
[
14,49,50]. First of all, discussing QQs’ nucleophilic reactivity, it must
depicted in Fig. 3 [50,54]. Besides, polar solvents like water or ethanol
are also known to increase the formation of C-6 substituted QQs [50].
The regioselective insertion of aromatic amines was conducted into
the corresponding C-6 substituted QQs (a series of LY83583 analogs) by
nucleophilic substitution of aromatic amines to electrophilic 1,4-
quinone according to the procedure under Ce(III) catalysis (Table 1)
[53]. For the synthesis of the first subseries of a series of LY83583 an-
alogs, AQQ1-10 was obtained by the amination reaction of 6,
be remarked that the two chlorine atoms are not equivalent in it and that
mostly one linked at C-6 and/or C-7 is “selectively” substituted by
amines depending on the reaction medium and/or reagents [51,52]. The
chemistry and SAR of the C-6 and/or C-7 substituted QQs are the major
research areas of previous reports. Therefore, the substituents linked at
C-6 and/or C-7 are mainly versatile groups such as amino, ether, and
thioether, as well as nitro, alkyl, and halogen groups. Furthermore,
previous studies from other groups have also proved that C-6 substituted
QQs have a very significant effect on bioactivity [43,45]. In the litera-
ture concerning the product distribution, the presence of the Lewis acids
3 2
7-dichloro-5,8-quinolinequinone (2) in the presence of CeCl .7H O
with aryl amines containing electron-withdrawing groups (EWGs) such
as ester and cyano groups (-COOR and –CN) at different positions. The
second subseries of the target analogs (a series of LY83583 analogs)
were synthesized in the same method. The reaction of 6,7-dichloro-5,
8-quinolinequinone (2) with aryl amines containing electron-donating
2 2 3 2
such as NiCl .6H O [45] or CeCl .7H O [53] as a catalyst in a reaction
media promotes the substitution with the chlorine atom linked at C-6
since the electron density of C-6 is reduced via the chelation of Ce(III)
between nitrogen atom in B ring and oxygen atom linked at C-8 as
groups (EDGs) such as alkyl and amino groups (-R and –NR
2
) at
3

N. Bayrak et al.
Chemico-Biological Interactions 345 (2021) 109555
3 2
Fig. 3. Preferential nucleophilic substitution with the addition of CeCl .7H O at C-6.
◦
◦
different positions in ethanol resulted in other LY83583 analogs
C13–C16–O4 bond angles of AQQ3 are 122.3 and 124.6 , respectively.
◦
◦
(
AQQ11–19). After the synthesis and purification, the structures of a
The C–O–C bond angle of the ester group is 116.1 for AQQ3, and 123.3
series of LY83583 analogs were evaluated by using diverse modern
analytical techniques like Fourier-transform infrared spectroscopy
for AQQ7. One reason for the different bond angles is that AQQ3 has a
methyl group in the ester group, whereas in AQQ7 the ester group
contains tert-butyl. As known, the tert-butyl group is bulkier than the
methyl group. Therefore, the steric effect of tert-butyl results that a
larger bond angle of the C–O–C bond. In terms of bond distances and
angles, the molecular geometry of AQQ derivatives is compatible with
the expected data. The details of all crystallographic data of AQQ3,
AQQ7, AQQ13, and AQQ15 are summarized in the Supplementary file.
(
FTIR), 1H nuclear magnetic resonance (NMR), 13C NMR, and
high-resolution mass spectrometry (HRMS).
2
.2. Crystal structures
X-ray crystallographic studies have also shown that the nucleophilic
substitution actually has taken place at C-6 or C-7 [43,53]. The repre-
sentative molecular structures of AQQ3, AQQ7, AQQ13, and AQQ15
have been characterized by single crystal X-ray crystallography tech-
nique to verify both structural skeleton of presented novel products and
the regioselectivity of the substitution reaction as described in the
experimental section. The ORTEP diagrams are depicted in Fig. 4. The
target analogs (AQQ3, AQQ7, AQQ13, and AQQ15) were dissolved in
ethanol and kept for crystallization to afford good quality diffraction
crystal for one week.
2.3. Biological evaluation
The antiproliferative activity of the LY83583 analogs (AQQ1-19)
was investigated in vitro against K562 (myeloid leukemia), Jurkat, MT-2
(other two human T-cell leukemia) cell lines, and HeLa (human cervical
carcinoma) cell line as summarized in Table 3 by employing 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.
Imatinib was used as a positive control. We used imatinib as a positive
control because it has been proven to specifically inhibit ABL, which is
expressed in nearly all cases of and has increased the overall survival of
patients with CML from 4 years to an estimated 19–25 years [56–58].
The inhibitory concentrations that completed 50% inhibition (IC50) of
cancer cell growth of all analogs are listed in Table 3. Having better
antileukemia activity in the K562 cell line (suspension cells) in our
previous studies [32–34], we added the HeLa cell line (adherent cells)
for the determination of the further antiproliferative activity of the an-
alogs. Results indicated that some analogs (AQQ2, AQQ3, AQQ5,
AQQ7, AQQ14, AQQ15, AQQ18, and AQQ19) showed better anti-
proliferative activity against leukemia cells than HeLa cells (Table 3).
These analogs were also found as more effective anti-leukemic agents
against K562 and Jurkat cells than MT-2 cells. The analogs (AQQ2,
AQQ14, AQQ15, AQQ18, and AQQ19) demonstrated the most robust
antiproliferative activity towards the K562 cell line with the IC50 values
AQQ3 and AQQ7 crystallize in the monoclinic crystal system (space
group Cc and P 1 21/c 1, respectively), AQQ13 crystallizes in a triclinic
crystal system (space group P1), and AQQ15 crystallizes in an ortho-
rhombic crystal system (space group Ibca) as shown in Table 2. The
ORTEP drawings of the AQQ3, AQQ7, AQQ13, and AQQ15 were re-
ported at a 50% probability level in Fig. 4. The crystallographic data of
these analogs indicate that the C–O bond lengths of the carbonyl groups
marked as C6–O1 and C9–O2 are, on average, 1.218 Å. This value is
–
O) bond length of the quino-
linequinones [43]. When the ester groups attached to the benzene ring
compatible with the carbonyl group (C
–
–
of AQQ3 and AQQ7 are examined; the C O double bond lengths are
–
1
.199 Å and 1.218 Å, while the C–O single bond lengths between the
carbonyl carbon and alkoxide oxygen are 1.329 Å and 1.327 Å,
respectively. The difference between these two C–O bond lengths sup-
ports that there is not any resonance form. The esters having a carbonyl
◦
2
center gives to 120 C–C–O and O–C–O bond angles due to sp hybrid-
ization. In accordance with this situation; O3–C16–O4 and O3–C16–C15
bond angles of AQQ7 are 124.0 and 123.9 ; O4–C16–O3 and
of 1.27 ± 0.16
0.67 ± 0.04
5.71 ± 1.02
μ
M, 0.74 ± 0.08
M, respectively when compared with imatinib (IC50
M). In a similar manner, the three analogs (AQQ3, AQQ5,
μ
M, 0.52 ± 0.06
μM, 0.62 ± 0.07
μ
M, and
μ
=
◦
◦
μ
Fig. 4. ORTEP drawings of AQQ3, AQQ7, AQQ13, and AQQ15 at 50% probability level.
4

N. Bayrak et al.
Chemico-Biological Interactions 345 (2021) 109555
Table 2
Crystallographic data for the LY83583 analogs (AQQ3, AQQ7, AQQ13, and AQQ15).
ID Code
AQQ3
AQQ7
AQQ13
AQQ15
Chemical formula
C
17
H
11ClN
2
O
4
C
20
H
17ClN
O
2 4
C
17
H
13ClN
2
O
2
C
17
H
13ClN
2 2
O
ꢀ
1
Formula weight (g mol
)
342.74
384.80
312.74
312.74
Crystal system, space group/Z
Monoclinic, Cc/4
Monoclinic,
P 1 21/c 1/2
296(2)
Triclinic, P-1
Orthorhombic, Ibca/16
Temperature (K)
Wavelength (Å)
a, b, c (Å)
273.15
296(2)
273(2)
0.71073
0.71073
0.71073
0.71073
a = 4.6642 (12)
b = 27.835 (7)
c = 11.840 (3)
β = 98.088 (4)
a = 17.579(13)
b = 13.345(10)
c = 8.074(6)
a = 8.1605(14)
b = 9.3384(15)
c = 10.5442(17)
a = 13.98(2)
b = 16.39(2)
c = 25.48(4)
α
, β, γ (◦)
β = 98.042(13)
α
= 76.593(2)
α, β, γ = 90
α, γ = 90
α, γ = 90
β = 67.276(2)
γ = 81.824(3)
719.7(2)
3
V (Å )
1521.9 (7)
0.27 × 0.14 × 0.11
1.496
1875.(2)
0.04 × 0.07 × 0.41
1.363
5838.(14)
0.05 × 0.08 × 0.20
1.423
3
Crystal sizes (mm )
0.06 × 0.09 × 0.25
1.443
ꢀ
3
Dcal (g cm
)
ꢀ
1
Absorption coefficient,
μ
(mm
)
0.280
0.232
0.274
0.270
2
Goodness-of-fit on F
0.894
1.006
1.042
1.012
Table 3
General Formula and Cytotoxicity of LY83583 Analogs (AQQ1-19) in K562, Jurkat, MT-2, and HeLa Cell Lines for 24 h.
a
d
e
LY83583 Analogs
General Formula
Subseries (X)
Substituent(s)
Cell Type (IC50
,
μ
M)
SI
SI
b
b
b
b
ID
K562
Jurkat
MT-2
HeLa
AQQ1
EWG
2-COOCH
3-COOCH
4-COOCH
2-COOCH
3-COOCH
4-COOCH
3
3
3
2
2
2
2.13 ± 0.38
1.27 ± 0.16
1.87 ± 0.27
2.18 ± 0.27
1.48 ± 0.13
2.40 ± 0.13
1.71 ± 0.22
2.67 ± 0.46
2.76 ± 0.27
2.98 ± 0.51
2.48 ± 0.45
1.99 ± 0.27
1.65 ± 0.32
0.74 ± 0.08
0.52 ± 0.06
1.68 ± 0.23
1.73 ± 0.19
0.62 ± 0.07
0.67 ± 0.04
5.71 ± 1.02
1.64 ± 0.22
1.45 ± 0.25
1.44 ± 0.31
1.47 ± 0.18
1.29 ± 0.30
1.57 ± 0.36
1.53 ± 0.42
1.86 ± 0.39
1.61 ± 0.27
1.27 ± 0.24
2.11 ± 0.35
2.02 ± 0.15
1.57 ± 0.17
1.35 ± 0.14
0.78 ± 0.07
2.00 ± 0.36
1.46 ± 0.28
0.74 ± 0.09
1.30 ± 0.16
6.45 ± 1.34
2.57 ± 0.18
2.78 ± 0.34
2.74 ± 0.25
2.92 ± 0.33
2.86 ± 0.26
2.70 ± 0.16
2.66 ± 0.42
3.81 ± 0.53
2.35 ± 0.44
3.38 ± 0.45
5.44 ± 0.66
8.44 ± 1.54
5.13 ± 0.97
5.58 ± 0.72
3.13 ± 0.35
3.92 ± 0.50
4.39 ± 0.83
2.14 ± 0.49
2.77 ± 0.56
13.27 ± 5.15
8.25 ± 2.36
6.51 ± 1.22
7.86 ± 1.47
7.61 ± 1.64
7.76 ± 2.54
8.88 ± 1.58
7.91 ± 1.08
8.17 ± 1.74
9.01 ± 2.15
9.05 ± 2.93
6.43 ± 1.20
3.78 ± 0.51
2.57 ± 0.23
6.62 ± 0.84
2.77 ± 0.31
3.90 ± 0.61
6.68 ± 0.95
6.86 ± 1.15
5.39 ± 1.34
16.35 ± 4.96
0.77
1.14
0.77
0.67
0.87
0.65
0.89
0.70
0.58
0.43
0.85
1.02
0.95
1.82
1.50
1.19
0.84
1.19
1.94
1.13
3.87
5.13
4.20
3.49
5.24
3.67
4.63
3.06
3.26
3.04
2.59
1.90
1.56
8.95
5.33
2.32
3.86
11.06
8.04
2.86
AQQ2
AQQ3
AQQ4
CH
CH
CH
3
3
3
3
3
3
AQQ5
AQQ6
AQQ7
2-COOC(CH
3-COOC(CH
4-COOC(CH
4-CN
3
)
3
)
3
)
AQQ8
AQQ9
AQQ10
AQQ11
AQQ12
AQQ13
AQQ14
AQQ15
AQQ16
AQQ17
AQQ18
AQQ19
Imatinib
EDG
2,3-diCH
2,4-diCH
2,5-diCH
3,4-diCH
3,5-diCH
3
3
3
3
3
2-CH(CH
3-CH(CH
4-CH(CH
3
3
3
)
)
)
2
2
2
2 3 2
4-N(CH CH )
c
a
The reported values represent the mean ± SD for each analog based on three independent experiments.
b
c
Cell lines include myeloid leukemia (K562), other two human T-cell leukemia (Jurkat and MT-2), and human cervical carcinoma (HeLa).
Used as the positive control.
d
e
SI = IC50 for Jurkat cell line/IC50 for K562 cell line.
SI = IC50 for HeLa cell line/IC50 for K562 cell line.
and AQQ7) exhibited the lowest IC50 values (1.44 ± 0.31
.30 M, respectively) towards Jurkat cells as
M, and 1.53 ± 0.42
compared to imatinib (IC50 = 6.45 ± 1.34 M) (Table 3).
μ
M, 1.29 ±
These results suggested that these two series of LY83583 analogs could
effectively suppress cancer growth since all of the tested LY83583 ana-
logs showed better cytotoxicity than the positive control (imatinib)
against all tested cancer cell lines.
0
μ
μ
μ
LY83583 analogs were designed to analyze the SARs using synthetic
analogs in order to clarify the crucial functional group(s) and its position
in the aminophenyl moiety for the antiproliferative activity as two series
possessing an EWG or EDG group in the aminophenyl moiety to assess
the importance of substituents. Collectively, the present two series of
LY83583 analogs displayed a range of potencies against the four tested
cancer cell lines. Among them, the analogs (AQQ15 and AQQ18)
exhibited high potency against K562 and Jurkat cell lines, with IC50
In order to evaluate the selectivity of the most active LY83583 an-
alogs (AQQ2, AQQ3, AQQ5, AQQ7, AQQ14, AQQ15, AQQ18, and
AQQ19), we examined their cytotoxic effects on PBMCs and calculated
the SI (Table 4) by dividing the IC50 of the PBMC growth inhibition with
the K562 cell growth inhibition for each. Amongst them, the analog
(AQQ15) deserved special importance, because AQQ15, presenting the
most favorable selectivity between PBMCs and K562 cells with a 4.06 SI
value. Besides, the SI, which was calculated between Jurkat and K562
cells, was found as 1.50 for AQQ15 showing its high selective anti-
leukemic effects against K562 cells. Based on the aforementioned find-
ings, AQQ15 was subsequently selected for further mechanism research
for providing deep insights into its notable anti-leukemic effects against
K562 cells (Table 4).
value less than 1
prominent growth inhibitory activities against these four cell lines with
IC50 values ranging from 0.5 to 3.2 M. In the case of Jurkat cells, all
analogs (except AQQ11, AQQ12, and AQQ16) exhibited better anti-
proliferative activity than imatinib with IC50 value less than 2 M. The
analog (AAQ13) exhibited the strongest activity (IC50 ranging from 2.5
to 4.0 M) followed by the analog (AAQ15) against the HeLa cell line.
μM (Table 3). Notably, AQQ15 displayed the most
μ
μ
μ
Based on the biological data obtained so far, SAR correlations were
5

N. Bayrak et al.
Chemico-Biological Interactions 345 (2021) 109555
Table 4
The cytotoxicity of selected LY83583 analogs and selectivity.
b
LY83583 Analogs
General Formula
Subseries (X)
Substituent(s)
Cell Type (IC50
,
μ
M)
SI
a
a
ID
K562
PBMC
AQQ2
EWG
EDG
3-COOCH
4-COOCH
3-COOCH
3
3
2
1.27 ± 0.16
1.87 ± 0.27
1.48 ± 0.13
1.71 ± 0.22
0.74 ± 0.08
0.52 ± 0.06
0.62 ± 0.07
0.67 ± 0.04
5.71 ± 1.02
1.58 ± 0.18
3.78 ± 0.25
3.39 ± 0.52
1.52 ± 0.21
2.69 ± 0.27
2.11 ± 0.16
0.97 ± 0.13
0.90 ± 0.08
27.14 ± 5.37
1.24
2.02
2.29
0.89
3.64
4.06
1.56
1.34
4.75
AQQ3
AQQ5
CH
3
AQQ7
3
2-COOC(CH )
3
AQQ14
AQQ15
AQQ18
AQQ19
Imatinib
3,4-diCH
3,5-diCH
3
3
4-CH(CH
3 2
)
4-N(CH CH )
2
3 2
c
a
Cell lines include myeloid leukemia (K562) and peripheral blood mononuclear cells (PBMC).
b
c
The selectivity index (SI) values are calculated by dividing the IC50 of the PBMC growth inhibition with the K562 cell growth inhibition.
Used as the positive control.
analyzed. Firstly, we evaluated the effects of the EWG group within the
LY83583 analogs on the cytotoxic activity. Generally, with the changing
the alkyl moiety in the ester group (from methyl to tert-butyl) at the para
position, the cytotoxic activities decreased in the obtained analogs
against K562, Jurkat, and HeLa cell lines. The alteration of the position
of the ester group displayed no any remarkable change in the activity
against any cancer cell line. On the other hand, the replacement of the
ester group with the cyano group at the para position in the first sub-
series did not show any significant effect. Regarding the second subse-
ries, the target analogs with EDG were more potent than the analogs
containing EWG. The analogs with an alkyl group(s) as an EDG group at
the para position showed relatively higher potency. The analog
proteases [61].
Subsequent mechanism including the relationship between DNA
cleavage potency and apoptosis induction was explored using the elec-
trophoresis in agarose gel. This method explains the three distinct pat-
terns of plasmid DNA entitled Form I (supercoiled), Form II (single
break), and Form III (double break or linear form) [62]. Results sug-
gested that the analog AQQ15 exerted its apoptotic functions by dis-
rupting pUC 19 DNA. Of note, the analog AQQ15 was found to possess
4 2 2
the capacity to cleave DNA in the presence and absence of FeSO , H O ,
and ascorbic acid. Ultimately, the analog AQQ15 exhibited augmented
DNA-cleaving activity with 41% intensity of Form II band and 4% in-
tensity of Form III band (Fig. 7c) within the iron (II) complex system
after 40 min (Figs. 7a) and 60 min (Fig. 7b).
(
AQQ15) led to a considerable increase in potency against K562 and
Jurkat cell lines. The use of isopropyl or diethylamino with substitution
at para position for target analogs (AQQ18 and AQQ19) clearly indi-
cated that these donor groups improved the activity compared to the
ester group.
2.4. Molecular docking and ADME prediction
After confirming that the analog AQQ15 was able to cleave DNA
significantly, we next turned our attention to assessing molecular
docking study to warrant the experimental data of biological experi-
ments conjecturing the binding orientation of the analog AQQ15 with
DNA. In accordance with this purpose, the crystal structure of DNA was
retrieved from the Protein Data Bank server (PDB entry: 1BNA) [62,63].
The analog AQQ15 exhibited significantly higher binding affinity to
Based on the promising MTT results of the analog AQQ15, initially
apoptotic effects of this analog in the K562 cell line were investigated
compared to imatinib using Hoechst 33342/annexin V/ethidium
homodimer III staining assay monitored by fluorescence microscope
(
Fig. 5a). This method expresses the difference between necrosis and
apoptosis related to the staining with ethidium homodimer III (red) and
annexin V (green), respectively. The analog AQQ15 was detected to
have a substantial influence on apoptotic cell death in K562 cells with
DNA with corresponding
π-
π
stacking interactions with DA-17 and
DG-16 via its quinoline and 3,5-dimethyl phenyl moieties, respectively
in the minor groove of the double helix of DNA. However, the analog
AQQ15 also established a hydrogen bonding with DA-18, which led to a
reduction in its strong binding capacity to DNA (Fig. 8a and b).
A number of crucial physicochemical properties of the analogs
AQQ1-19 such as predicted aqueous solubility (QPlogS), human serum
albumin binding (QPlogKhsa), predicted octanol/water partition coef-
ficient (QPlogPo/w), predicted human oral absorption, compatibility
with Lipinski’s rule of 5 and Jorgensen’s rule of 3 were in silico postu-
lated. The results were found coherent with acceptable ranges of all
these specified parameters (Table 7). These properties were particularly
were preferred due to their substantial direct or indirect effects in
membrane permeability, tissue penetration, metabolism, clearance,
bioavailability, and toxicity of drugs. The analogs of LY83583 (AQQ1-
19) displayed notable QPlogS and QPlogPo/w values ranging from
(ꢀ 5.55 to ꢀ 3.75) and (1.76–3.75), respectively, which were within the
acceptable ranges in order (ꢀ 6.5 to 0.5) and (ꢀ 2 to 6.5). The QPlogKhsa
values of all analogs (ꢀ 0.32 to 0.31) were also determined at the
appropriate range (ꢀ 1.5 to 1.5). Moreover, the analogs of LY83583
(AQQ1-19) presented significant human oral absorption (79.91–100%)
on a 0–100% scale (>80% is high; <25% is poor) and no violation of
Lipinski’s and Jorgensen’s parameters [64,65].
3
4.6% compared to imatinib (6.5%). Moreover, it can be deduced that
the analog AQQ15 was more effective (6.7%) than imatinib (3.6%) at
the late apoptotic/necrotic phase of the K562 cell line (Fig. 5b, Table 5).
These observations accentuated that in CML cells, the analog AQQ15
underwent apoptosis, an important pathway for cancer prevention [59].
The BCR-ABL1 tyrosine kinase has been reported to constitute an
attractive target for CML therapy related to its critical roles in the sur-
vival and proliferation of CML cells. However, the role of ABL1 tyrosine
kinase in the regulation of apoptosis has remained unlightened so far
due to the fusion of the c-abl gene with the bcr gene in the formation of
BCR-ABL1 hybrid protein making confusion about the exact biological
functions of ABL1 kinase [60].
In this work, based on the aforementioned data, how the ABL1 kinase
inhibitory activity of the analog AQQ15 contributed to the apoptosis in
the K562 cells compared to imatinib was subsequently speculated. As
depicted in Fig. 6 and Table 6, the analog AQQ15 showed no consid-
erable ABL1 kinase inhibitory activity as compared to imatinib at 1
and 10 M concentrations implying that the analog AQQ15 induced
apoptosis in K562 cells without inhibiting ABL1 kinase.
μ
M
μ
Oligonucleosomal DNA degradation and chromatin condensation
attract great attention in the apoptotic program. Furthermore, the dis-
covery of DNA cleavage during apoptosis with the evidence of discrete
ladder (apoptosis) of bands instead of smear (necrosis) also changed the
beliefs about the only digestion of the cells to death and release of
6

N. Bayrak et al.
Chemico-Biological Interactions 345 (2021) 109555
Fig. 5. Changes in K562 cell line at 1
μ
M concentration of control, AQQ15 and imatinib for 12 h (a). A total of approximately 1000 stained cells were selected
randomly in each experiment of (a) and were classified as 4 groups, namely, “alive” (blue), “apoptosis” (green), “necrosis or late apoptosis” (yellow) and “necrosis”
(
red) (b). Quantification of the effect of the analog AQQ15 and imatinib on apoptosis. Data from three independent experiments are shown as means ± standard
deviations and p values were determined using Student’s test (c).
3
. Conclusion
Table 5
The percentage of apoptotic cells treated with the analog AQQ15 and Imatinib.
In our continuing investigations of 1,4-quinones, two subseries of the
Compound
K562 cell line
LY83583 analogs (AAQ1-19) were designed and synthesized. Enthused
by the biological significance of QQs, the 1,4-quinone and aminophenyl
moieties are retained in target analogs, but the 1,4-quinone moiety has
been extended by the addition of a pyridine moiety, in order to increase
the activity. As per the results obtained from in vitro antiproliferative
evaluation, the analog (AAQ15) was identified as the most potent
analog with the highest growth inhibition against the K562 cell line and
acceptable SI value compared with the positive control. This analog
further induced apoptosis in the K562 cell line associated with its
Alive
Apoptosis%
Late Apoptosis/Necrosis%
Necrosis%
Control
AQQ15
Imatinib
96.4
53.4
86.8
1.2
0.0
6.7
3.6
2.4
5.3
3.1
34.6
6.5
7

N. Bayrak et al.
Chemico-Biological Interactions 345 (2021) 109555
using APEX2 [67]. Data integration and reduction were carried out with
SAINT [68]. Absorption correction was performed by multi-scan method
implemented in SADABS [69]. The Bruker SHELXTL [70] software
package was used for structures solution and structure refinement. Ar-
omatic C-bound and N-bound hydrogen atoms were positioned
geometrically and refined using a riding mode. Crystal structure vali-
dations and geometrical calculations were performed using the Platon
software [71]. Mercury software [72] was used for visualization of the.
cif files. 6,7-Dichloro-5,8-quinolinequinone (2) was synthesized using
the reported method by Shaikh et al. [24] in the literature.
4
.2. Procedure for the synthesis of the quinolinequinone (2) [24]
6
,7-Dichloro-5,8-quinolinequinone (2) was prepared according to
the literature from 8-hydroxyquinoline and sodium chlorate in
◦
◦
concentrated HCl in 29% yield. Mp 219–220 C (Lit [24]. 221–223 C).
Fig. 6. The ABL1 kinase inhibition of the analog AQQ15 and Imatinib at 1 M
μ
1
and 10
μ
M concentrations.
H NMR (500 MHz, CDCl
3
) δ (ppm): 9.40 (d, J = 4.39 Hz, 1H, CHar-
omatic), 8.44–8.48 (m, 1H, CHaromatic), 7.67–7.72 (m, 1H, CHaromatic). 3C
1
–
–
NMR (125 MHz, CDCl
3
) δ (ppm): 174.6, 173.3 (>C
O), 154.4, 145.8,
Table 6
The ABL1 kinase inhibition of the analog AQQ15 and Imatinib.
1
43.4, 142.1, 134.6, 127.3, 127.2 (Caromatic and C
q
). MS (+ESI) m/z (%):
+
+
2
48 (100, [M + Na–2H] ), 250 (93, [M+Na] ); Anal. Calcd. for
Concentration (
μ
M)
Relative Activity (%)
AQQ15
C
9
3
H Cl
2 2
NO (226.95).
Imatinib
4
.3. General procedure for the synthesis of the amino substituted
1
19.4
35.1
64.1
84.6
quinolinequinones (AQQ1-19) [53]
1
0
3 2
A suspension of the 2 (0.285 g, 1.25 mmol) and CeCl .7H O (0.512 g,
significant DNA cleavage potential. In addition, the above results pro-
vide a useful correlation between the original natural streptonigrin (1),
described in Fig. 1, and AQQ15 in DNA cleavage activity and confirm
that QQ moiety (AB ring system) is the responsible part for activity [24,
1.375 mmol, 1.1 equiv) in ethanol was stirred at room temperature for 1
h. Then, a suspension of the corresponding amines (1.375 mmol, 1.1
equiv) in ethanol was added to that solution and refluxed for 3–6 h until
consumption of the 2. The reaction mixture was cooled to room tem-
perature. After evaporation of the solvent, the residue was dissolved
6
6]. Molecular docking studies also accentuated that the analog AAQ15
formed important stacking interactions in the minor groove of the
π
-
π
with CH Cl2 (50 mL), and the solution was washed sequentially with
2
double helix of DNA. Furthermore, results of in silico ADME studies
revealed that the analog AAQ15 was found coherent with all the ranges
of specified crucial pharmacokinetic determinants suggesting that this
analog possessed anticancer drug-like properties for future studies.
water (3 x 30 mL). The organic layer was dried over anhydrous CaCl2,
filtered, and concentrated under reduced pressure, and the residue was
purified by means of column chromatography on silica gel to give the
corresponding AQQ.
4
. Experimental section
4.3.1. Methyl 2-((7-chloro-5,8-dioxo-5,8-dihydroquinolin-6-yl)amino)
benzoate (AQQ1)
4
.1. Chemistry
3 2
By subjecting the mixture (2 and CeCl .7H O) and methyl 2-amino-
benzoate (0.208 g, 1.375 mmol) to the general procedure for the syn-
Melting points (Mp) were determined with a Buchi B-540 melting
thesis of the AQQ1, the title compound (AQQ1) was purified by column
◦
point apparatus and are uncorrected. All reagents were commercially
obtained from a commercial suppliers and used without further purifi-
cation unless specified otherwise. Thin layer chromatography (TLC) was
conducted on Merck KGaA (silica gel 60 F254) based on Merck DC-plates
chromatography as a dark red solid. Yield: 33%, mp 250–251 C. FTIR
ꢀ 1
(ATR)
1687, 1672 (>C
1142, 1088, 1058, 1021. H NMR (500 MHz, CDCl
υ
(cm ): 3248 (NH), 3052 (CHaromatic), 2988, 2915 (CHaliphatic),
–
–
O), 1590, 1558, 1506, 1457, 1363, 1321, 1251, 1228,
1
3
) δ (ppm): 10.18 (s,
(
aluminum based). Compound visualization for TLC during the chro-
1H, CHaromatic), 9.09 (s, 1H, CHaromatic), 8.49 (d, J = 7.7 Hz, 1H, CHar-
omatic), 8.04 (d, J = 7.7 Hz, 1H, CHaromatic), 7.75–7.60 (m, 1H, NH),
7.60–7.45 (m, 1H, CHaromatic), 7.18 (t, J = 7.6 Hz, 1H, CHaromatic), 6.86
matographic separation was accomplished by UV irradiation at 254 nm.
Chromatography refers to column chromatography performed on silica
1
3
gel 60 (Merck, 63–200
μm particle sized, 60–230 mesh) with eluent.
(d, J = 8.2 Hz, 1H, CHaromatic), 3.99 (s, 3H, OCH
3
). C NMR (125 MHz,
–
FTIR spectra were recorded on a PerkinElmer Spectrum 100 Optical
FTIR spectrometer by applying ATR Sampling Accessory for both oil and
solid compounds. HRMS was performed on either a Waters SYNAPT G1
MS or an Agilent 6530 Accurate-MassQ-TOF LC/MS. Mass spectra were
obtained on a BRUKER Microflex LT by MALDI (Matrix Assisted Laser
Desorption Ionization)-TOF technique via addition of 1,8,9-anthracene-
triol (DIT, dithranol) as matrix. Proton and carbon NMR spectra were
CDCl
140.66, 138.97, 135.07, 132.40, 130.96, 127.41, 127.22, 123.25,
123.06, 119.71, 118.83 (Caromatic and C ), 52.51 (OCH
). HRMS(+ESI)
[M + H] : 343.0486; found: 343.0486 and
[M + H] : 345.0456; found: 345.0464.
3
) δ (ppm): 179.77, 175.83, 167.86 (>C O), 155.20, 148.00,
–
q
3
3
5
+
2 4
m/z calcd for C17H12ClN O
3
7
+
17 2 4
C H12ClN O
4.3.2. Methyl 3-((7-chloro-5,8-dioxo-5,8-dihydroquinolin-6-yl)amino)
benzoate (AQQ2)
1
13
UNITY
recorded at 500 MHz/125 MHz ( H NMR/ C NMR) on Varian
1
INOVA spectrometers in parts per million (ppm, δ). H NMR spectra and
3 2
By subjecting the mixture (2 and CeCl .7H O) and methyl 3-amino-
1
3
C NMR spectra in CDCl
3
and DMSO‑d
6
refer to the solvent signal
benzoate (0.208 g, 1.375 mmol) to the general procedure for the syn-
center. Coupling constants (J) were reported in Hz. Standard abbrevia-
tions indicating multiplicity were used as follows: s (singlet), br s (broad
singlet), d (doublet), t (triplet), m (multiplet), and dd (doublet of dou-
blets). Data for the single crystal compounds were obtained with Bruker
APEX II QUAZAR three-circle diffractometer. Indexing was performed
thesis of the AQQ2, the title compound (AQQ2) was purified by column
◦
chromatography as a light brown solid. Yield: 55%, mp 197–198 C.
ꢀ
1
FTIR (ATR)
υ
(cm ): 3343 (NH), 3081 (CHaromatic), 2956 (CHaliphatic),
–
1716, 1670 (>C
–
O), 1610, 1576, 1501, 1439, 1295, 1214, 1146, 1102,
1
1064. H NMR (500 MHz, CDCl ) δ (ppm): 9.07 (s, 1H, CHaromatic), 8.45
3
8

N. Bayrak et al.
Chemico-Biological Interactions 345 (2021) 109555
Fig. 7. The DNA-cleaving activity of AQQ15 in the presence of FeSO
4
, H
2
O
2
, and ascorbic acid system after 40 min (a) after 60 min (b). Relative intensity of form I,
form II and form III bands.
(
d, J = 7.8 Hz, 1H, CHaromatic), 7.91 (d, J = 7.7 Hz, 1H, CHaromatic), 7.76
found: 345.0471.
(
s, 1H, CHaromatic), 7.72 (s, 1H, NH), 7.70–7.65 (m, 1H, CHaromatic), 7.46
(
t, J = 7.8 Hz, 1H, CHaromatic), 7.28–7.30 (m, 1H, CHaromatic), 3.93 (s, 3H,
4.3.3. Methyl 4-((7-chloro-5,8-dioxo-5,8-dihydroquinolin-6-yl)amino)
1
3
OCH
3
). C NMR (125 MHz, CDCl
–
–
3
) δ (ppm): 179.89, 175.79, 166.22
benzoate (AQQ3)
(
>C
O), 155.44, 148.21, 140.91, 137.36, 134.87, 130.70, 128.60,
3 2
By subjecting the mixture (2 and CeCl .7H O) and methyl 4-amino-
1
5
3
28.42, 127.06, 126.95, 126.93, 125.06, 116.92 (Caromatic and C
q
),
benzoate (0.208 g, 1.375 mmol) to the general procedure for the syn-
3
5
+
2.39 (OCH
3
). HRMS(+ESI) m/z calcd for C17
H
12ClN
2
O
4
+
[M + H] :
thesis of the AQQ3, the title compound (AQQ3) was purified by column
3
7
◦
43.0486; found: 343.0487 and C17
H
12ClN
2
O
4
[M + H] : 345.0456;
chromatography as a red solid. Yield: 68%, mp 256–258 C. FTIR (ATR)
9

N. Bayrak et al.
Chemico-Biological Interactions 345 (2021) 109555
Fig. 8. Docking pose (a) and interactions (b) of the analog AQQ15 (The ligand is highlighted in the orange colored stick) in the minor groove of the double helix of
DNA (PDB code: 1BNA).
Table 7
Some ADME features of the analog of LY83583 (AQQ1-19).
a
a
a
a
b
The Analog
QPlogS
QPlogPo/w
QPlogKhsa
Human Oral
Absorption%
Rule of Five
Rule of Three
AQQ1
ꢀ 3.75
ꢀ 4.07
ꢀ 4.09
ꢀ 4.27
ꢀ 4.63
ꢀ 4.66
ꢀ 5.05
ꢀ 5.53
ꢀ 5.55
ꢀ 4.37
ꢀ 4.35
ꢀ 4.68
ꢀ 4.48
ꢀ 4.59
ꢀ 4.61
ꢀ 4.58
ꢀ 4.86
ꢀ 4.88
ꢀ 4.96
2.57
2.50
2.49
2.88
2.80
2.80
3.63
3.55
3.54
1.76
3.25
3.28
3.28
3.26
3.30
3.57
3.75
3.72
3.63
ꢀ 0.32
ꢀ 0.21
ꢀ 0.21
ꢀ 0.17
0.02
92.06
88.22
88.01
94.16
90.43
90.22
96.33
91.24
91.23
93.87
79.91
100
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
AQQ2
AQQ3
AQQ4
AQQ5
AQQ6
0.02
AQQ7
0.25
AQQ8
0.16
AQQ9
0.17
AQQ10
AQQ11
AQQ12
AQQ13
AQQ14
AQQ15
AQQ16
AQQ17
AQQ18
AQQ19
ꢀ 0.25
0.17
0.10
0.19
100
0.19
95.81
96.82
100
0.22
0.26
0.30
100
0.31
100
0.14
100
a
Rule of Five: Number of violations of Lipinski’s rule of five. The rules are: mol_MW (Molecular weight of the molecule) < 500, QPlogPo/w (Predicted octanol/
water partition coefficient) < 5, donorHB (hydrogen-bond donor atoms) ≤ 5, accptHB (hydrogen-bond acceptor atoms) ≤ 10. Compounds that provide these rules are
considered drug-like. (The “five” refers to the limits, which are multiples of 5).
b
Rule of Three: Number of violations of Jorgensen’s rule of three. The three rules are: QPlogS (Predicted aqueous solubility) > ꢀ 5.7, QPPCaco (Predicted apparent
Caco-2 cell permeability in nm/s) > 22 nm/s, # Primary Metabolites < 7. Compounds with fewer (and preferably no) violations of these rules are more likely to be
orally available agents (Schr o¨ dinger Release 2016-2: Schr o¨ dinger, LLC, New York, NY, USA).
ꢀ
).13C NMR (125 MHz, CDCl
υ
(cm 1): 3207 (NH), 3093 (CHaromatic), 2993 (CHaliphatic), 1711, 1678
2H, CHaromatic), 3.95 (s, 3H, OCH
3
3
) δ (ppm):
O), 155.52, 148.15, 141.08, 140.48,
134.92, 130.12, 127.18, 127.00, 126.93, 122.90 (Caromatic and C ),
–
–
(
>C
–
O), 1655, 1596, 1564, 1518, 1411, 1277, 1219, 1195, 1169, 1149,
179.89, 175.79, 166.30 (>C
–
1
1
145, 1102, 1064. H NMR (500 MHz, CDCl
3
) δ (ppm): 9.10 (s, 1H,
q
+
3
5
CHaromatic), 8.48 (d, J = 7.9 Hz, 1H, CHaromatic), 8.07 (d, J = 8.4 Hz, 2H,
52.20 (OCH
3
). HRMS(+ESI) m/z calcd for C17
H
12ClN
2
O
4
] [M + H] :
3
7
+
CHaromatic), 7.77–7.58 (m, 2H, NH and CHaromatic), 7.11 (d, J = 8.4 Hz,
343.0486; found: 343.0485 and C17
H
2 4
12ClN O
[M + H] : 345.0456;
1
0

N. Bayrak et al.
Chemico-Biological Interactions 345 (2021) 109555
found: 345.0474.
FTIR (ATR)
liphatic), 1697, 1681 (>C
3
1296, 1269, 1221, 1160, 1144, 1083, 1019. H NMR (500 MHz, CDCl ) δ
υ
(cm 1): 3269 (NH), 3048 (CHaromatic), 2970, 2930 (CHa-
ꢀ
–
–
O), 1607, 1594, 1567, 1510, 1449, 1367,
1
4
.3.4. Ethyl 2-((7-chloro-5,8-dioxo-5,8-dihydroquinolin-6-yl)amino)
benzoate (AQQ4)
(ppm): 10.27 (s, 1H, CHaromatic), 9.12 (s, 1H, CHaromatic), 8.62–8.43 (m,
1H, CHaromatic), 8.07–7.92 (m, 1H, CHaromatic), 7.78–7.64 (m, 1H, NH),
7.63–7.42 (m, 1H, CHaromatic), 7.23–7.12 (m, 1H, CHaromatic), 6.93–6.74
3 2
By subjecting the mixture (2 and CeCl .7H O) and ethyl 2-aminoben-
zoate (0.227 g, 1.375 mmol) to the general procedure for the synthesis of
1
3
the AQQ4, the title compound (AQQ4) was purified by column chro-
(m, 1H, CHaromatic), 1.65 (s, 9H, CH
3
). C NMR (125 MHz, CDCl
3
) δ
O), 155.17, 148.11, 140.68,
138.63, 135.11, 131.76, 131.18, 127.42, 127.16, 123.14, 123.06,
◦
–
matography as a dark purple solid. Yield: 79%, mp 206–207 C. FTIR
(ppm): 179.96, 175.84, 166.77 (>C
–
ꢀ
1
(
ATR)
υ
(cm ): 3255 (NH), 3081 (CHaromatic), 2963, 2922 (CHaliphatic),
–
–
1
1
1
690, 1665 (>C
226, 1139, 1086, 1021. H NMR (500 MHz, CDCl
H, CHaromatic), 9.08 (s, 1H, CHaromatic), 8.49 (d, J = 7.8 Hz, 1H, CHar-
O), 1605, 1560, 1509, 1453, 1435, 1315, 1296, 1246,
120.65, 119.36 (Caromatic and C
q
), 82.69 (OC), 28.24 (CH
3
). HRMS
1
35
+
3
) δ (ppm): 10.21 (s,
(+ESI) m/z calcd for C20
H
18ClN
2
O
4
+
[M + H] : 385.0955; found:
3
7
385.0955 and C20
H
18ClN
2
O
4
[M + H] : 387.0926; found: 387.0944.
omatic), 8.05 (d, J = 7.8 Hz, 1H, CHaromatic), 7.68–7.70 (m, 1H, NH), 7.52
(
d, J = 7.7 Hz, 1H, CHaromatic), 7.17 (t, J = 7.7 Hz, 1H, CHaromatic), 6.85
), 1.46 (t, J
) δ (ppm): 179.80, 175.84,
O), 155.18, 148.04, 140.66, 138.90, 135.03, 132.25,
4.3.8. tert-Butyl 3-((7-chloro-5,8-dioxo-5,8-dihydroquinolin-6-yl)amino)
benzoate (AQQ8)
(
d, J = 7.8 Hz, 1H, CHaromatic), 4.45 (q, J = 7.1 Hz, 2H, OCH
2
1
3
=
7.1 Hz, 3H, CH
3
). C NMR (125 MHz, CDCl
3
3 2
By subjecting the mixture (2 and CeCl .7H O) and tert-butyl 3-ami-
–
1
1
67.41 (>C
–
nobenzoate (0.266 g, 1.375 mmol) to the general procedure for the
30.97, 127.40, 127.18, 123.23, 123.04, 119.57, 119.18 (Caromatic and
synthesis of the AQQ8, the title compound (AQQ8) was purified by
◦
C
C
q
), 61.61 (OCH
2
), 14.25 (CH
3
). HRMS(+ESI) m/z calcd for
column chromatography as a red solid. Yield: 83%, mp 162–163 C.
3
5
+
37
ꢀ 1
18
H
14ClN
2
O
4
[M + H] : 357.0642; found: 357.0641 and C18
H
14ClN
2
O
4
FTIR (ATR)
υ
(cm ): 3304 (NH), 3035 (CHaromatic), 2970 (CHaliphatic),
+
–
[
M + H] : 359.0613; found: 359.0632.
1707, 1675 (>C
–
O), 1656, 1592, 1563, 1508, 1432, 1370, 1299, 1158,
111. H NMR (500 MHz, CDCl ) δ (ppm): 9.13 (s, 1H, CHaromatic), 8.50
1
1
3
4
.3.5. Ethyl 3-((7-chloro-5,8-dioxo-5,8-dihydroquinolin-6-yl)amino)
(s, 1H, CHaromatic), 7.88 (s, 1H, CHaromatic), 7.74 (s, 2H, NH and CHar-
benzoate (AQQ5)
omatic), 7.69 (s, 2H, CHaromatic), 7.45 (s, 1H, CHaromatic), 1.62 (s, 9H,
By subjecting the mixture (2 and CeCl
3
.7H
2
O) and ethyl 3-aminoben-
CH
3
–
–
). 13C NMR (125 MHz, CDCl
3
) δ (ppm): 179.95, 175.77, 164.80
O), 148.31, 141.06, 137.10, 135.15, 132.71, 128.41, 128.35,
zoate (0.227 g, 1.375 mmol) to the general procedure for the synthesis of
(>C
the AQQ5, the title compound (AQQ5) was purified by column chro-
126.94, 125.04, 116.88 (Caromatic and C
q
), 81.66 (OC), 28.18 (CH
3
).
◦
35
+
matography as an orange solid. Yield: 74%, mp 152–154 C. FTIR (ATR)
HRMS(+ESI) m/z calcd for C20
H
18ClN
2
O
4
[M + H] : 385.0955; found:
ꢀ
1
37
+
υ
1
1
(cm ): 3332 (NH), 3093 (CHaromatic), 2981, 2933, 2907 (CHaliphatic),
385.0955 and C20
H
18ClN
2
O
4
[M + H] : 387.0926; found: 387.0948.
721, 1664 (>C–
O), 1600, 1578, 1568, 1506, 1444, 1363, 1287, 1272,
–
1
211, 1184, 1138, 1103, 1067, 1032. H NMR (500 MHz, CDCl
3
) δ
4.3.9. tert-Butyl 4-((7-chloro-5,8-dioxo-5,8-dihydroquinolin-6-yl)amino)
benzoate (AQQ9)
(
ppm): 9.05 (s, 1H, CHaromatic), 8.43 (s, 1H, CHaromatic), 7.89 (s, 1H,
CHaromatic), 7.76 (s, 2H, CHaromatic), 7.66 (s, 1H, NH), 7.43 (s, 1H,
CHaromatic), 7.27 (s, 1H, CHaromatic), 4.49–4.21 (m, 2H, OCH ), 1.47–1.26
) δ (ppm): 179.88, 175.76,
O), 155.44, 148.18, 140.96, 137.34, 134.90, 131.05,
28.50, 128.43, 127.10, 126.94, 126.91, 125.04, 116.78 (Caromatic and
3 2
By subjecting the mixture (2 and CeCl .7H O) and tert-butyl 4-ami-
2
nobenzoate (0.266 g, 1.375 mmol) to the general procedure for the
1
3
(
m, 3H, CH
3
). C NMR (125 MHz, CDCl
3
synthesis of the AQQ9, the title compound (AQQ9) was purified by
–
◦
1
1
65.72 (>C
–
column chromatography as a red solid. Yield: 76%, mp 332–334 C.
ꢀ 1
FTIR (ATR)
1699, 1683 (>C
1215, 1157, 1102. H NMR (500 MHz, CDCl ) δ (ppm): 9.08 (s, 1H,
υ
(cm ): 3244 (NH), 3100 (CHaromatic), 2981 (CHaliphatic),
–
O), 1660, 1592, 1568, 1525, 1408, 1368, 1290, 1260,
C
C
q
), 61.34 (OCH
2
), 14.30 (CH
3
). HRMS(+ESI) m/z calcd for
–
3
5
+
37
1
18
H
14ClN
2
O
4
[M + H] : 357.0642; found: 357.0639 and C18
H
14ClN
2
O
4
3
+
[
M + H] : 359.0613; found: 359.0593.
CHaromatic), 8.46 (d, J = 7.7 Hz, 1H, CHaromatic), 8.00 (d, J = 8.1 Hz, 2H,
CHaromatic), 7.77–7.56 (m, 2H, NH and CHaromatic), 7.08 (d, J = 8.1 Hz,
1
3
4
.3.6. Ethyl 4-((7-chloro-5,8-dioxo-5,8-dihydroquinolin-6-yl)amino)
3 3
2H, CHaromatic), 1.61 (s, 9H, CH ). C NMR (125 MHz, CDCl ) δ (ppm):
–
O), 155.47, 148.16, 140.62, 140.58,
benzoate (AQQ6)
179.90 175.78, 164.96 (>C
–
3 2
By subjecting the mixture (2 and CeCl .7H O) and ethyl 4-aminoben-
134.90, 129.89, 128.90, 127.13, 126.99, 122.89, 118.03 (Caromatic and
3
5
zoate (0.227 g, 1.375 mmol) to the general procedure for the synthesis of
C
q
), 81.26 (OC), 28.19 (CH
3
). HRMS(+ESI) m/z calcd for C20
2 4
H18ClN O
+
37
+
the AQQ6, the title compound (AQQ6) was purified by column chro-
[M + H] : 385.0955; found: 385.0955 and C20
H
18ClN
2
O
4
[M + H] :
◦
matography as a red solid. Yield: 91%, mp 226–227 C. FTIR (ATR)
υ
387.0926; found: 387.0953.
ꢀ
1
(
(
cm ): 3212 (NH), 3089 (CHaromatic), 2984 (CHaliphatic), 1703, 1677
–
>C
170, 1139, 1101, 1064, 1024. H NMR (500 MHz, CDCl
s, 1H, CHaromatic), 8.46 (d, J = 7.7 Hz, 1H, CHaromatic), 8.05 (d, J = 7.9
Hz, 2H, CHaromatic), 7.75 (s, 1H, CHaromatic), 7.69 (s, 1H, NH), 7.10 (d, J
–
O), 1656, 1594, 1562, 1515, 1498, 1411, 1372, 1296, 1275, 1217,
4.3.10. 7-Chloro-6-(4-cyanophenyl)amino-5,8-quinolinequinone
(AQQ10)
1
1
3
) δ (ppm): 9.08
(
3 2
By subjecting the mixture (2 and CeCl .7H O) and 4-aminobenzoni-
trile (0.162 g, 1.375 mmol) to the general procedure for the synthesis of
=
7.9 Hz, 2H, CHaromatic), 4.39 (dd, J = 13.8, 6.8 Hz, 2H, OCH
2
), 1.41 (t,
) δ (ppm): 179.88,
O), 155.51, 148.14, 140.99, 140.52, 134.91,
),
the AQQ10, the title compound (AQQ10) was purified by column
1
3
◦
J = 7.0 Hz, 3H, CH
3
). C NMR (125 MHz, CDCl
3
chromatography as a red solid. Yield: 11%, mp 261–262 C. FTIR (ATR)
75.79, 165.83 (>C–
υ
(cm ): 3341 (NH), 3070 (CHaromatic), 2956, 2922, 2853 (CHaliphatic),
ꢀ 1
1
1
6
–
–
30.06, 127.29, 127.18, 126.99, 122.91, 118.29 (Caromatic and C
q
2224 (CN), 1732, 1672, 1660 (>C
–
O), 1592, 1563, 1507, 1491, 1459,
1
3
5
1.11 (OCH
2
), 14.35 (CH
3
). HRMS(+ESI) m/z calcd for C18
H
14ClN
2
O
4
6
1413, 1291, 1252, 1219, 1169, 1141. H NMR (500 MHz, DMSO‑d ) δ
+
37
+
[
M + H] : 357.0642; found: 357.0637 and C18
H
14ClN
2
O
4
[M + H] :
(ppm): 9.63 (s, 1H, CHaromatic), 9.01 (s, 1H, CHaromatic), 8.40 (dd, J = 7.8
3
59.0613; found: 359.0621.
and 1.7 Hz, 1H, CHaromatic), 7.82 (dd, J = 7.8 and 4.9 Hz, 1H, CHaromatic),
7
.74 (d, J = 8.7 Hz, 2H, CHaromatic), 7.23 (d, J = 8.7 Hz, 2H, CHaromatic).
1
3
–
–
O),
4
.3.7. tert-Butyl 2-((7-chloro-5,8-dioxo-5,8-dihydroquinolin-6-yl)amino)
C NMR (125 MHz, DMSO‑d
6
) δ (ppm): 180.19, 176.14 (>C
benzoate (AQQ7)
154.81, 147.99, 144.30, 142.45, 134.94, 132.76, 128.46, 128.03,
By subjecting the mixture (2 and CeCl
3
.7H
2
O) and tert-butyl 2-ami-
122.63, 121.68, 119.66 (Caromatic and C
q
), 105.05 (CN). HRMS(+ESI) m/
+
nobenzoate (0.266 g, 1.375 mmol) to the general procedure for the
z calcd for C16
H
9
3
ClN O
2
[M + H] : 310.0383; found: 310.0379.
synthesis of the AQQ7, the title compound (AQQ7) was purified by
◦
column chromatography as an orange solid. Yield: 13%, mp 153–154 C.
1
1

N. Bayrak et al.
Chemico-Biological Interactions 345 (2021) 109555
–
6
–
DMSO‑d ) δ (ppm): 180.45, 175.68 (>C O), 154.86, 148.27, 143.28,
136.77, 136.13, 134.85, 133.13, 129.34, 127.96, 127.63, 125.68,
4
.3.11. 7-Chloro-6-(2,3-dimethylphenyl)amino-5,8-quinolinequinone
(
AQQ11) [55]
By subjecting the mixture (2 and CeCl
3
.7H
2
O) and 2,3-dimethylani-
122.14, 115.14 (Caromatic and C
q
), 19.90, 19.34 (CH
3
). HRMS(+ESI) m/z
3
5
+
line (0.167 g, 1.375 mmol) to the general procedure for the synthesis of
calcd for C17
H
14ClN
2
O
2
[M + H] : 313.0744; found: 313.0740 and
3
7
+
the AQQ11, the title compound (AQQ11) was purified by column
C
17
H14ClN
2
O
2
[M + H] : 315.0714; found: 315.0730.
◦
chromatography as an orange solid. Yield: 22%, mp 197–198 C. FTIR
ꢀ
1
(
ATR)
υ
(cm ): 3327 (NH), 3089 (CHaromatic), 2921, 2856 (CHaliphatic),
4.3.15. 7-Chloro-6-(3,5-dimethylphenyl)amino-5,8-quinolinequinone
(AQQ15)
–
1
1
8
664 (>C
–
O), 1608, 1579, 1568, 1497, 1460, 1310, 1269, 1220, 1138,
077. H NMR (500 MHz, DMSO‑d ) δ (ppm): 9.12 (s, 1H, CHaromatic),
.98 (s, 1H, CHaromatic), 8.38 (d, J = 7.5 Hz, 1H, CHaromatic), 7.96–7.65
), 2.12 (s,
) δ (ppm): 180.19, 175.56
O), 154.92, 148.29, 144.33, 138.17, 137.06, 134.84, 133.65,
28.46, 127.75, 127.54, 125.44, 125.22, 112.70 (Caromatic and C ),
1
6
3 2
By subjecting the mixture (2 and CeCl .7H O) and 3,5-dimethylani-
line (0.167 g, 1.375 mmol) to the general procedure for the synthesis of
(
m, 1H, NH), 7.32–6.88 (m, 3H, CHaromatic), 2.28 (s, 3H, CH
3
the AQQ15, the title compound (AQQ15) was purified by column
1
3
◦
3
H, CH
3
). C NMR (125 MHz, DMSO‑d
6
chromatography as a brown solid. Yield: 72%, mp 224–226 C. FTIR
–
–
ꢀ 1
(
>C
(ATR)
υ
(cm ): 3306 (NH), 3056 (CHaromatic), 2911 (CHaliphatic), 1677
–
1
2
q
(>C
–
O), 1654, 1586, 1559, 1510, 1424, 1297, 1265, 1202, 1141, 1067.
3
5
1
0.48, 14.84 (CH
3
). HRMS(+ESI) m/z calcd for C17
H
14ClN
2
O
2
[M +
H NMR (500 MHz, CDCl
3
) δ (ppm): 9.12–8.97 (m, 1H, CHaromatic),
+
37
+
H] : 313.0744; found: 313.0741 and C17
H
14ClN
2
O
2
[M + H] :
8.49–8.34 (m, 1H, CHaromatic), 7.71–7.53 (m, 2H, NH and CHaromatic),
6.92–6.80 (m, 1H, CHaromatic), 6.77–6.61 (m, 2H, CHaromatic), 2.34–2.27
m, 6H, CH ). C NMR (125 MHz, CDCl ) δ (ppm): 180.16, 175.88
3 3
3
15.0714; found: 315.0727.
1
3
(
–
–
4
.3.12. 7-Chloro-6-(2,4-dimethylphenyl)amino-5,8-quinolinequinone
(>C O), 155.28, 148.37, 141.07, 138.25, 136.71, 134.77, 127.78,
(
AQQ12)
By subjecting the mixture (2 and CeCl
126.85, 122.02, 115.82 (Caromatic and C
q
), 21.28, 21.21 (CH
3
). HRMS
3
5
+
3
.7H
2
O) and 2,4-dimethylani-
(+ESI) m/z calcd for C17
H
14ClN
2
O
2
+
[M + H] : 313.0744; found:
3
7
line (0.167 g, 1.375 mmol) to the general procedure for the synthesis of
313.0745 and C17
H
14ClN
2
O
2
[M + H] : 315.0714; found: 315.0725.
the AQQ12, the title compound (AQQ12) was purified by column
◦
chromatography as an orange solid. Yield: 79%, mp 191–192 C. FTIR
4.3.16. 7-Chloro-6-(2-isopropylphenyl)amino-5,8-quinolinequinone
(AQQ16)
ꢀ
1
(
ATR)
υ
(cm ): 3328 (NH), 3085 (CHaromatic), 2915, 2856 (CHaliphatic),
–
1
664 (>C
–
O), 1605, 1578, 1568, 1508, 1483, 1461, 1308, 1263, 1216,
3 2
By subjecting the mixture (2 and CeCl .7H O) and 2-isopropylaniline
1
1
139, 1121. H NMR (500 MHz, CDCl
3
) δ (ppm): 9.12–9.01 (m, 1H,
(0.186 g, 1.375 mmol) to the general procedure for the synthesis of the
CHaromatic), 8.51–8.37 (m, 1H, CHaromatic), 7.74–7.59 (m, 1H, CHar-
omatic), 7.53–7.42 (m, 1H, NH), 7.14–6.89 (m, 3H, CHaromatic), 2.37 (s,
AQQ16, the title compound (AQQ16) was purified by column chro-
ꢀ
1
matography as a red oil. Yield: 48%. FTIR (ATR)
–
3067 (CHaromatic), 2962, 2867 (CHaliphatic), 1675 (>C
–
υ
(cm ): 3318 (NH),
1
3
3
1
1
H, CH
3
), 2.27 (s, 3H, CH
3
). C NMR (125 MHz, CDCl
O), 155.38, 148.46, 141.93, 137.21, 134.75,
33.77, 133.35, 131.12, 131.06, 126.82, 126.61, 126.33, 114.44 (Car-
3
) δ (ppm):
O), 1593, 1560,
1500, 1308, 1259, 1218, 1144, 1084, 1025. H NMR (500 MHz, CDCl ) δ
80.08, 175.69 (>C–
1
–
3
(ppm): 9.06 (br s, 1H, CHaromatic), 8.44 (s, 1H, CHaromatic), 7.66 (s, 1H,
omatic and
C
q
), 21.04, 18.11 (CH
3
). HRMS(+ESI) m/z calcd for
CHaromatic), 7.53 (s, 1H, NH), 7.33–7.26 (m, 2H, CHaromatic), 7.18 (s, 1H,
3
5
+
37
C
17
H
14ClN
2
O
2
[M + H] : 313.0744; found: 313.0740 and C17
H
14ClN
2
O
2
CHaromatic), 7.03 (d, J = 7.0 Hz, 1H, CHaromatic), 3.27–3.11 (m, 1H, CH),
+
13
[
M + H] : 315.0714; found: 315.0738.
1.26 (d, J = 5.4 Hz, 6H, CH
3 3
). C NMR (125 MHz, CDCl ) δ (ppm):
–
80.03, 175.68 (>C O), 155.30, 148.36, 143.94, 142.00, 134.85,
134.70, 127.73, 126.99, 126.85, 125.97, 125.60, 114.66 (Caromatic and
1
–
4
.3.13. 7-Chloro-6-(2,5-dimethylphenyl)amino-5,8-quinolinequinone
+
(
AQQ13)
By subjecting the mixture (2 and CeCl
C
q
), 28.62 (CH), 22.92 (CH
Calcd. for 15ClN
3
). MS (MALDI TOF) m/z: 327 [M+H] . Anal.
3
.7H
2
O) and 2,5-dimethylani-
C
18
H
2
+
O
2
(326.08). HRMS(+ESI) m/z calcd for
3
5
37
line (0.167 g, 1.375 mmol) to the general procedure for the synthesis of
C
18
H16ClN
2
O
2
[M + H] : 327.0900; found: 327.0898 and C18
2 2
H16ClN O
+
the AQQ13, the title compound (AQQ13) was purified by column
[M + H] : 329.0871; found: 329.0888.
◦
chromatography as a red solid. Yield: 32%, mp 175–176 C. FTIR (ATR)
ꢀ
1
υ
(cm ): 3287 (NH), 3070 (CHaromatic), 2922 (CHaliphatic), 1679
–
–
4.3.17. 7-Chloro-6-(3-isopropylphenyl)amino-5,8-quinolinequinone
(AQQ17)
(
>C O), 1651, 1594, 1560, 1501, 1461, 1374, 1311, 1274, 1197, 1130,
1
1
064. H NMR (500 MHz, CDCl
3
) δ (ppm): 9.13–8.98 (m, 1H, CHar-
3 2
By subjecting the mixture (2 and CeCl .7H O) and 3-isopropylaniline
omatic), 8.49–8.37 (m, 1H, CHaromatic), 7.76–7.56 (m, 1H, CHaromatic),
(0.186 g, 1.375 mmol) to the general procedure for the synthesis of the
7
1
.56–7.38 (m, 1H, NH), 7.22–6.94 (m, 2H, CHaromatic), 6.94–6.78 (m,
AQQ17, the title compound (AQQ17) was purified by column chro-
1
3
◦
H, CHaromatic), 2.44–2.14 (m, 6H, CH
3
). C NMR (125 MHz, CDCl
O), 155.39, 148.44, 141.68, 135.72,
35.61, 134.73, 130.58, 130.13, 127.93, 126.81, 114.79 (Caromatic and
3
) δ
matography as a dark purple solid. Yield: 48%, mp 177–178 C. FTIR
–
–
ꢀ 1
(
ppm): 180.06, 175.77 (>C
(ATR)
1677 (>C
1203, 1145, 1101, 1060. H NMR (500 MHz, CDCl
υ
(cm ): 3303 (NH), 3056 (CHaromatic), 2955, 2930 (CHaliphatic),
–
1
C
–
O), 1655, 1560, 1514, 1479, 1464, 1429, 1308, 1291, 1224,
3
5
1
q
), 20.88, 17.77 (CH
3
). HRMS(+ESI) m/z calcd for C17
H
14ClN
2
O
2
[M +
3
) δ (ppm): 9.15–9.01
+
37
+
H] : 313.0744; found: 313.0724 and C17
H
14ClN
2
O
2
[M + H] :
(m, 1H, CHaromatic), 8.52–8.39 (m, 1H, CHaromatic), 7.85–7.56 (m, 2H,
NH and CHaromatic), 7.38–7.20 (m, 1H, CHaromatic), 7.18–7.04 (m, 1H,
CHaromatic), 7.02–6.86 (m, 2H, CHaromatic), 3.03–2.81 (m, 1H, CH),
3
15.0714; found: 315.0726.
1
3
4
.3.14. 7-Chloro-6-(3,4-dimethylphenyl)amino-5,8-quinolinequinone
1.34–1.19 (m, 6H, CH
3
). C NMR (125 MHz, CDCl
O), 155.30, 149.46, 148.30, 140.97, 136.81, 134.92,
128.43, 126.94, 124.33, 122.60, 121.72, 115.66 (Caromatic and C ),
3
) δ (ppm): 180.14,
–
–
(
AQQ14) [53]
175.79 (>C
By subjecting the mixture (2 and CeCl
3
.7H
2
O) and 3,4-dimethylani-
q
3
5
line (0.167 g, 1.375 mmol) to the general procedure for the synthesis of
33.93 (CH), 23.81 (CH
3
). HRMS(+ESI) m/z calcd for C18
H
16ClN
2
O
2
[M
+
+
37
the AQQ14, the title compound (AQQ14) was purified by column
+ H] : 327.0900; found: 327.0864 and C18
H
16ClN
2
O
2
[M + H] :
◦
chromatography as an orange solid. Yield: 23%, mp 194–195 C. FTIR
329.0871; found: 329.0869.
ꢀ
1
(
ATR)
669 (>C
NMR (500 MHz, DMSO‑d
υ
(cm ): 3341 (NH), 3085 (CHaromatic), 2978, 2919 (CHaliphatic),
–
–
1
O), 1605, 1575, 1507, 1311, 1293, 1222, 1145, 1064. 1H
) δ (ppm): 9.25 (s, 1H, CHaromatic), 8.98 (s, 1H,
4.3.18. 7-Chloro-6-(4-isopropylphenyl)amino-5,8-quinolinequinone
(AQQ18)
6
CHaromatic), 8.37 (d, 1H, CHaromatic), 7.77–7.80 (m, 1H, CHaromatic), 7.07
3 2
By subjecting the mixture (2 and CeCl .7H O) and 4-isopropylaniline
(
d, J = 7.4 Hz, 1H, CHaromatic), 6.94 (s, 1H, NH), 6.88 (d, J = 7.5 Hz, 1H,
(0.186 g, 1.375 mmol) to the general procedure for the synthesis of the
1
3
CHaromatic), 2.50 (s, 3H, CH
3
), 2.20 (s, 3H, CH
3
). C NMR (125 MHz,
AQQ18, the title compound (AQQ18) was purified by column
1
2

N. Bayrak et al.
Chemico-Biological Interactions 345 (2021) 109555
◦
chromatography as a dark red solid. Yield: 12%, mp 160–161 C. FTIR
buffer and staining solution, were analyzed by all-in-one fluorescence
microscope Biorevo Fluorescence BZ-9000 (Keyence, Osaka, Japan).
Based on our previous research [81], the number of healthy and necrotic
cells was quantified related to the staining with annexin V and ethidium
homodimer III.
ꢀ 1
(
ATR)
υ
(cm ): 3336 (NH), 3070 (CHaromatic), 2957, 2870 (CHaliphatic),
–
O), 1606, 1577, 1520, 1497, 1463, 1312, 1281, 1217, 1144,
1
672 (>C
–
1
1
066, 1019. H NMR (500 MHz, CDCl
3
) δ (ppm): 8.97 (dd, J = 4.7, 1.6
Hz, 1H, CHaromatic), 8.35 (dd, J = 7.8, 1.7 Hz, 1H, CHaromatic), 7.62 (s,
1
H, NH), 7.57 (dd, J = 7.8, 4.7 Hz, 1H, CHaromatic), 7.14 (d, J = 8.4 Hz,
2
H, CHaromatic), 6.95 (d, J = 8.4 Hz, 2H, CHaromatic), 2.93–2.78 (m, 1H,
4.4.4. Kinase inhibitory activity
1
3
CH), 1.19 (d, J = 6.9 Hz, 6H, CH
3
). C NMR (125 MHz, CDCl
3
) δ (ppm):
Manufacturer’s directions (Promega Corporation, Madison, WI,
USA) were carried out with small amendments for the ABL1 kinase
profiling assay application [82,83].
1
1
2
80.15, 175.84 (>C–
–
O), 155.37, 148.43, 147.07, 141.21, 134.85,
34.65, 126.91, 126.51, 124.45, 115.53 (Caromatic and C
q
), 33.68 (CH),
+
3.97 (CH
15ClN
3
). MS (MALDI TOF) m/z: 326 [M] . Anal. Calcd. for
3
5
C
18
H
2
O
2
(326.08). HRMS(+ESI) m/z calcd for C18
H
16ClN
2
O
2
[M +
4.4.5. DNA cleavage activity
+
37
+
H] : 327.0900; found: 327.0900 and C18
H
16ClN
2
O
2
[M + H] :
The DNA cleavage assay was applied associated with previously
described method with some modification [84]. The most effective
cytotoxic agents were investigated for determination of the
DNA-cleaving activity by means of supercoiled pUC19 DNA and agarose
(Takara, Kyoto, Japan) gel electrophoresis Mupid-2x (Mupid, Tokyo,
3
29.0871; found: 329.0885.
4
.3.19. 7-Chloro-6-(4-(diethylamino)phenyl)amino-5,8-quinolinequinone
(
AQQ19)
By subjecting the mixture (2 and CeCl
3
.7H
2
O) and N,N-diethyl-p-
Japan). Compounds were subject to pUC19 DNA (2
μ
g) in water and
phenylenediamine (0.226 g, 1.375 mmol) to the general procedure for
the synthesis of the AQQ19, the title compound (AQQ19) was purified
by column chromatography as a dark green solid. Yield: 91%, mp
Tris/boric acid (Nacalai Tesque, Kyoto, Japan) buffer (10 mM, pH 8.5)
with and without iron (II) sulfate heptahydrate (FeSO
4
.7H
2
O; 30
μ
μ
M)
M)
(Wako Pure Chemical Industries), hydrogen peroxide (H
O ; 30
2 2
◦
ꢀ 1
1
46–147 C. FTIR (ATR)
υ
(cm ): 3315 (NH), 3052 (CHaromatic), 2956
(Tokyo Chemical Industry Co., Tokyo, Japan) and ascorbic acid (as an
–
(
CHaliphatic), 1670 (>C
–
O), 1596, 1566, 1524, 1502, 1355, 1304, 1265,
activator) (30 M) (Tokyo Chemical Industry Co., Tokyo, Japan). The
μ
1
1
195. H NMR (500 MHz, CDCl
3
) δ (ppm): 9.06 (s, 1H, CHaromatic), 8.43
steps were followed in order: 1) incubation of the reaction mixture at
◦
(
d, J = 6.8 Hz, 1H, CHaromatic), 7.69 (s, 1H, CHaromatic), 7.63 (br s, 1H,
37 C for 2 h, 2) addition of EDTA (Dojindo Molecular Technologies) and
NH), 6.99–6.98 (m, 2H, CHaromatic), 6.64–6.63 (m, 2H, CHaromatic),
loading buffer (Takara, Kyoto, Japan), 3) application of agarose gel
electrophoresis of pUC19 DNA at 100 V for 30 min in 1% slab gels
carrying ethidium bromide (Wako Pure Chemical Industries) in Tris/-
Boronic acid/EDTA buffer. Lastly, the fluorescence of intercalated
ethidium bromide under a UV illuminator (Nippon Genetics Co., Tokyo,
Japan) was photographed.
1
3
3
.49–3.30 (m, 4H, NCH
2
), 1.26–1.13 (m, 6H, NCH
2
CH
3
). C NMR (125
–
–
MHz, CDCl
3
) δ (ppm): 180.28, 175.59 (>C
O), 155.26, 148.73, 146.41,
1
41.23, 134.68, 126.82, 126.57, 126.21, 124.90, 110.99 (Caromatic and
+
C
q
), 44.47 (NCH
2
), 12.54 (NCH
2
CH
3
). MS (MALDI TOF) m/z: 355 [M] .
Anal. Calcd. for C19
H
18ClN
3
O
2
(355.11). HRMS(+ESI) m/z calcd for
3
5
+
37
C
19
H
19ClN
3
O
2
[M + H] : 356.1166; found: 356.1164 and C19
3 2
H19ClN O
+
[
M + H] : 358.1136; found: 358.1112.
4
.5. Molecular docking studies
4
4
.4. Biochemistry
Ligands were prepared in Maestro molecular modeling workspace
.4.1. Cell culture and drug treatment
followed by energy minimization in ligand preparation program of
Schr
o¨ dinger’s Maestro molecular modeling package (Schr o¨ dinger
In the current study, HeLa cell line was cultured in Dulbecco’s
Release 2016-2: Schr
o¨ dinger, LLC, New York, NY, USA) under Opti-
Modified Eagle Medium (DMEM) (Wako Pure Chemical Industries),
while leukemia (K562, MT-2, and Jurkat) cell lines and PBMCs (Preci-
sion Bioservices, Frederic, MD) were cultured in RPMI 1640 (Wako Pure
Chemical Industries) medium. Besides, 10% fetal bovine serum (FBS)
mized Potential Liquid Simulations (OPLS_2005) force field at physio-
logical pH (pH = 7.4). The X-ray crystallographic structure of DNA was
obtained from the PDB server (PDB entry: 1BNA) [62,63] and optimized
for docking studies in protein preparation module of Schr
o¨ dinger soft-
(
Sigma Aldrich, MO, USA) and 89
μ
g/mL streptomycin (Meiji Seika
Pharma, Tokyo, Japan) were involved in DMEM and RPMI 1640 me-
ware. In molecular docking simulations: Grid Generation and Glide/XP
◦
docking protocols were applied (Schr
o¨ dinger Release 2016-2:
dium. All these cells were incubated at 37 C in humidified 5% CO
2
4
Schr
o¨ dinger, LLC, New York, NY, USA) [85].
atmosphere and implemented into 24-well (4 × 10 cells/mL) and 96-
6
well (1 × 10 cells/mL) tissue plates (Iwaki brand Asahi Glass Co.,
Chiba, Japan) and then incubated again for 24 h (the optimum cell
number was verified relevant to previous study) [73,74]. The stock so-
lution of compounds AQQ1-19 and imatinib in concentrations ranging
from 0.1 to 10 mM were prepared in DMSO (Wako Pure Chemical In-
dustries) and further diluted with fresh culture medium. The final DMSO
concentration was left as 1% to hamper its any effect on the cell viability
4
.6. In silico absorption, distribution, metabolism, and excretion (ADME)
prediction
Some ADME characteristics of the analogs AQQ1-19 were screened
by QikProp module of Schr o¨ dinger software (Schr o¨ dinger Release 2016-
2
: QikProp, Schr o¨ dinger, LLC, New York, NY, 2016) [85].
[
75].
4
.4.2. MTT assay
Author statement
Based on the previously described procedures in literature [76,77],
MTT (Dojindo Molecular Technologies, Kumamoto, Japan) assay was
carried out with small modifications [78].
The corresponding authors are responsible for ensuring that the de-
scriptions are accurate and agreed by all authors.
4
.4.3. Detection of cell death
Apoptotic/necrotic/healthy/detection kit (PromoKine, Heidelberg,
Declaration of competing interest
Germany) was administered with some modifications on manufacturer’s
directions [79,80], after K562 cell line was incubated with the most
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
effective antiproliferative agents in this series at 1
μM concentration for
1
2 h. K562 cells, which were subject to proper content including binding
1
3

N. Bayrak et al.
Chemico-Biological Interactions 345 (2021) 109555
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