Highly selective anthraquinone-chalcone hybrids as potential antileukemia agents

Article abstract of DOI:10.1016/j.bmcl.2018.06.048

A series of 23 novel anthraquinone-chalcone hybrids containing amide function was synthesized and structurally characterized. Sixteen compounds exerted strong cytotoxic activities against K562, Jurkat and HL-60 leukemia cell lines and significantly lower cytotoxic effects against normal MRC-5 cells, indicating very high selectivity in their anticancer action. The compounds 6g, 6u and 6v activate apoptosis in K562 cells through the extrinsic and intrinsic apoptotic pathway. The compound 6e triggered apoptosis in K562 cells only through the extrinsic apoptotic pathway. Treatment of K562 cells with each of these four compounds caused decrease in the expression levels of MMP2, MMP9, and VEGF, suggesting their anti-invasive, antimetastatic and antiangiogenic properties. The compounds 6g and 6v downregulated expression levels of miR-155 in K562 cells, while compounds 6e and 6u upregulated miR-155 levels in treated cells, in comparison with control cells. The structure-based 3-D QSAR models for 6f, 6e, 6i and 6l describe pro-apoptotic activity against caspase-3.

Full text of DOI:10.1016/j.bmcl.2018.06.048

Bioorganic & Medicinal Chemistry Letters xxx (xxxx) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Highly selective anthraquinone-chalcone hybrids as potential antileukemia
agents
a
b
a
b
a,c
Tatjana Stanojković , Violeta Marković , Ivana Z. Matić , Milan P. Mladenović , Nina Petrović ,
a
d
b,⁎
Ana Krivokuća , Miloš Petković , Milan D. Joksović
Institute of Oncology and Radiology of Serbia, Pasterova 14, 11000 Belgrade, Serbia
Faculty of Science, Department of Chemistry, University of Kragujevac, R. Domanovica 12, 34000 Kragujevac, Serbia
Laboratory for Radiobiology and Molecular Genetics, “Vinča” Institute of Nuclear Sciences, University of Belgrade, 11000 Belgrade, Serbia
Faculty of Pharmacy, Department of Organic Chemistry, University of Belgrade, Vojvode Stepe 450, 11221 Belgrade, Serbia
a
b
c
d
A R T I C L E I N F O
A B S T R A C T
Keywords:
Anthraquinone-chalcone
Leukemia
Gene expression
miR-155
A series of 23 novel anthraquinone-chalcone hybrids containing amide function was synthesized and structurally
characterized. Sixteen compounds exerted strong cytotoxic activities against K562, Jurkat and HL-60 leukemia
cell lines and significantly lower cytotoxic effects against normal MRC-5 cells, indicating very high selectivity in
their anticancer action. The compounds 6g, 6u and 6v activate apoptosis in K562 cells through the extrinsic and
intrinsic apoptotic pathway. The compound 6e triggered apoptosis in K562 cells only through the extrinsic
apoptotic pathway. Treatment of K562 cells with each of these four compounds caused decrease in the ex-
pression levels of MMP2, MMP9, and VEGF, suggesting their anti-invasive, antimetastatic and antiangiogenic
properties. The compounds 6g and 6v downregulated expression levels of miR-155 in K562 cells, while com-
pounds 6e and 6u upregulated miR-155 levels in treated cells, in comparison with control cells. The structure-
based 3-D QSAR models for 6f, 6e, 6i and 6l describe pro-apoptotic activity against caspase-3.
3
-D QSAR
Anticancer chalcones inhibit various molecular targets like ABCG2,
α-reductase, histone deacetylases, p53 degradation, angiogenesis, tu-
bulin and kinases. Human ABCG2, a member of ATP-binding cassette
ABC) transporters having a protecting role in cancer stem cells was
as daunorubicin, doxorubicin, mitoxantrone and ametantrone that are
9
10
5
widely used for the treatment of cancer. They act as intercalators,
inhibitors of telomerase,11 DNA topoisomerase II and ROS inducers in
12
1
3
(
a redox cycle system by the presence of quinone intermediates. The
natural anthraquinone emodin induces apoptosis in human acute pro-
myelocytic leukemia HL-60 cells through activation of the caspase-3
cascade, but independent of ROS production.14
1
inhibited by chalcones bearing quinoxaline unit. Shimizu et al. isolated
geranylated chalcones from Artocarpus incises for effective blocking
intracellular 5α-reductase to prevent proliferation of prostate cancer by
2
arresting male androgen biosynthesis. Coumarin-chalcone hybrids
Although anthracyclines are effective against a wide variety of
cancer cells, their clinical use is reduced due to low selectivity and high
showed histone-deacetylase inhibitory activity when tested against
3
9
leukemia K562 and U-937 cell lines. Boronic-chalcones act as putative
cardiotoxicity. Incorporating anthraquinone ring in a chalcone system
MDM2 antagonists interfering with p53-MDM2 interaction.4 4′-Hy-
droxy chalcone suppressed several steps of angiogenesis, including
endothelial cell proliferation, migration and tube formation.5 Matrix
metalloproteinases-2/9, a family of enzymes that have ability to de-
grade many molecules of the extracellular matrix can be inhibited by
combined with an aromatic amide function, we tried to obtain hybrid
molecules with better selectivity and synergistic or additive pharma-
cological properties. Thus, a series of new anthraquinone-chalcone
hybrids having amide function were synthesized and screened for their
anticancer and antidiabetic activity.
6
prenylated chalcones containing hydroxyl groups on A or B ring. 3,4,5-
The synthesis of novel anthraquinone-chalcone hybrids containing
amide function 6a–w is presented in Scheme 1, while experimental
details are provided in Supplementary material. The commercially
available anthracene 1 was acylated using acetyl chloride to 1-acet-
Trimethoxychalcones bind with microtubular protein tubulin and pre-
vent its polymerization, which is essential phase for mitosis.7 Chro-
menylchalcones successfully inhibit aurora kinase, a family of serine/
8
15
threonine kinases, responsible for cell cycle control.
ylanthracene 2 which was then oxidized by chromium (VI) oxide in
16
Anthraquinone is well known scaffold in anthracycline drugs such
glacial acetic acid yielding 1-acetylanthraquinone 3. The ketone 3
⁎
Corresponding author.
E-mail address: mjoksovic@kg.ac.rs (M.D. Joksović).
https://doi.org/10.1016/j.bmcl.2018.06.048
Received 14 May 2018; Received in revised form 25 June 2018; Accepted 27 June 2018
0960-894X/ © 2018 Elsevier Ltd. All rights reserved.
Please cite this article as: Stanojkovic, T., Bioorganic & Medicinal Chemistry Letters (2018), https://doi.org/10.1016/j.bmcl.2018.06.048

T. Stanojković et al.
Bioorganic & Medicinal Chemistry Letters xxx (xxxx) xxx–xxx
Scheme 1. Reagents and conditions: a) CH
3
COCl, AlCl
3 2
, CH Cl
2
, 2 h, 0 °C, 5 M HCl; b) CrO
3
, CH
3
COOH, 5 min reflux, H
2
O; c) 4-formylbenzoic acid, NaOH, MeOH,
2
h, reflux, HCl; d) SOCl , DMF, CH Cl , 2 h, rt; e) primary amine, THF, 6 h, reflux, H O.
2 2 2 2
was recrystallized from ethanol and condensed with 4-formylbenzoic
acid in the presence of NaOH as a base catalyst, affording anthraqui-
none-chalcone carboxylic acid 4. The obtained intermediate 4 was then
Table 1
The cytotoxic activity of the investigated compounds 6a–w.
a
Compd.
IC50
± SD (μM)
2
converted into acyl chloride using SOCl , and, without isolation, re-
acted with selected primary amines in dry tetrahydrofuran giving the
K562
Jurkat
HL-60
MRC-5
final hybrids 6a-w in moderate to good yields (51–88%).
The structure of all compounds was confirmed by means of 1H and
6a
10.93 ± 1.70
29.01 ± 1.64
25.03 ± 1.87
5.39 ± 0.59
3.87 ± 0.17
3.69 ± 0.21
5.99 ± 0.24
5.20 ± 0.39
78.18 ± 1.56
31.84 ± 3.41
4.67 ± 0.35
95.11 ± 6.92
8.53 ± 0.41
23.14 ± 2.19
25.52 ± 0.90
5.24 ± 0.92
7.14 ± 0.66
12.84 ± 1.03
3.96 ± 0.95
4.40 ± 0.41
4.45 ± 0.73
4.35 ± 0.47
5.08 ± 0.66
0.14 ± 0.03
4.83 ± 0.77
15.83 ± 1.69
14.62 ± 1.99
3.81 ± 0.05
3.23 ± 0.04
3.22 ± 0.14
3.66 ± 0.41
4.22 ± 0.51
27.64 ± 3.27
28.14 ± 2.39
3.37 ± 0.15
5.73 ± 0.06
4.24 ± 0.63
3.81 ± 0.16
5.15 ± 0.51
3.29 ± 0.02
3.15 ± 0.51
5.21 ± 1.30
2.84 ± 0.56
3.14 ± 0.11
4.25 ± 0.67
4.25 ± 0.78
3.85 ± 0.60
0.11 ± 0.03
6.81 ± 1.16
17.53 ± 2.04
17.56 ± 0.36
7.06 ± 1.03
1.89 ± 0.22
1.68 ± 0.08
3.50 ± 0.67
3.73 ± 1.43
44.51 ± 5.18
22.82 ± 2.42
4.22 ± 0.70
36.66 ± 2.81
5.71 ± 0.15
4.06 ± 1.59
3.57 ± 1.25
3.55 ± 0.69
2.81 ± 1.13
4.24 ± 0.69
3.85 ± 0.75
2.40 ± 0.69
3.16 ± 1.12
4.43 ± 0.78
5.39 ± 0.85
0.05 ± 0.01
187.52 ± 6.90
101.62 ± 4.46
99.84 ± 3.80
> 200
60.36 ± 4.17
17.07 ± 3.01
190.74 ± 7.73
126.84 ± 20.52
191.12 ± 2.23
> 200
133.50 ± 2.64
183.24 ± 23.70
86.71 ± 10.20
197.12 ± 4.08
175.62 ± 10.72
124.93 ± 3.51
166.36 ± 23.77
105.75 ± 11.71
48.99 ± 4.16
8.68 ± 0.24
6
6
b
c
1
3
C NMR spectroscopy, IR and elemental analysis (see Supplementary
material). The olefinic protons of the chalcone double bond in hybrid
compounds 6a–w, as well as their precursor 4, appeared as an AB
system. On the basis of coupling constant values (J = 16.0–16.8 Hz), all
of the compounds were isolated and characterized in E-isomeric form.
The signal for amide proton appears as a sharp singlet for compounds
6d
6
6
6
6
e
f
g
h
6i
6j
6
6
a–r at the highest ppm values (9.70–10.82 ppm), while for compounds
s–w it exists as a triplet in slightly lower ppm range (8.53–9.20 ppm)
6
6
6
6
k
l
m
n
due to the coupling with protons from adjacent methylene group.
The cytotoxicity of anthraquinone-chalcone hybrids containing
amide function 6a-w was evaluated against three human leukemia
cancer cell lines (K562, Jurkat and HL-60), and normal human lung
fibroblasts MRC-5 using MTT cell survival test. The obtained IC50 values
are shown in Table 1. Sixteen of the tested compounds showed good
cytotoxic potential against all three cancer cell lines. As it can be seen
from Table 1, there is no significant difference between the influence of
electron-donating and electron-withdrawing groups of the aniline
scaffold on the cytotoxic action against cancer cells. The derivatives 6e
and 6f containing ortho- and meta-hydroxyphenyl group showed the
best cytotoxic activity against K562 and HL-60 cell lines, with IC50
values of 3.87 and 3.69 μM for K562 cells and 1.89 and 1.68 μM for HL-
6o
6
6
6
6
p
q
r
s
6t
6u
161.01 ± 6.57
188.28 ± 11.20
> 200
6
6
v
w
b
DOX
0.29 ± 0.05
a
Results are mean values ± SD of three independent experiments.
Doxorubicin.
b
60 cells, respectively. The compounds containing aromatic or hetero-
cyclic ring linked to methylene group (6s–v), as well as derivative 6w
containing alkyl group, also exerted potent antiproliferative activities
against tested cell lines. The derivatives 6i and 6 l containing electron-
withdrawing groups (F and Cl, respectively) at meta-position of the
aniline moiety, showed significantly lower activity against K562 cell
line, compared to other tested compounds.
All tested compounds, except 6f and 6t, showed significantly lower
toxicity towards normal MRC-5 cells, compared to our previously
2

T. Stanojković et al.
Bioorganic & Medicinal Chemistry Letters xxx (xxxx) xxx–xxx
presented anthraquinone derivatives containing two chalcone moi-
observed in the cell samples pretreated with Z-LEHD-FMK, an inhibitor
of caspase-9 and treated with compounds 6g, 6u and 6v when com-
pared with those percentages in cell samples which were not pretreated
with the inhibitor. Pretreatment with caspase-9 inhibitor did not cause
changes in the percentage of cells in subG1 phase incubated with the
compound 6e. Our results demonstrate that compounds 6g, 6u and 6v
also activate intrinsic or mitochondrial apoptotic signaling pathway.
The cell migration and angiogenesis in myeloid leukemia (ML) cells
are accompanied by an increased expression of MMP2, MMP9, VEGFA
and miR-155. MiR-155 was first proposed to be oncogenic but later,
miR-155 has also been reported to be downregulated in various hae-
matological malignancies suggesting its different roles depending on
1
7
eties, and especially in comparison with anthraquinone based chal-
1
8
cone analogues containing imine fragment. Moreover, all tested de-
rivatives showed lower toxicity against normal MRC-5 cells comparing
to doxorubicin as a referent drug. The selectivity index (SI = IC50(MRC-
5)/IC50(cancer cells)) of a number of the tested compounds was found
to be over 50.0, with compound 6q, containing meta-tri-
fluoromethylphenyl group, showing the highest SI of 59.2 in case of HL-
60 cells. In light of these facts, compounds 6e, 6g, 6u and 6v were
selected for all further analyses due to their prominent cytotoxic ac-
tivity and excellent selectivity in the antiproliferative action against
K562 cell line.
2
0,21
Apoptosis is one of the main types of programmed cell death, and
many anticancer agents induce apoptosis as a result of their cytotoxic
action.1 To test the ability of the investigated compounds 6e, 6g, 6u,
and 6v to induce cell death, the cell cycle analysis was performed. The
distribution of the cell cycle was investigated on K562 cells, which were
selected because of a somewhat weaker sensitivity to the tested com-
pounds in comparison with other two lines of malignant cells. For the
compounds 6e, 6g, 6u, and 6v, the changes in cell cycle phase dis-
tribution of K562 cells treated with IC50 and 2IC50 concentrations after
the type of malignancy and its ability to inhibit many target genes.
The K562 cells treated with compounds 6g and 6v had lower levels of
miR-155 compared with control cells, while the cells treated with
compounds 6e and 6u showed higher levels of miR-155 compared with
the control cells (Fig. 3). The K562 cells treated with 6e showed the
highest level of miR-155 expression and the lowest expression level of
miR-155 was detected for 6g. Evidently, the different position of the
same hydroxyl group attached to aromatic ring of 6e and 6g made a
great difference in miR-155 expression. The targeting miR-155 with
anti-miR-155 resulted in a significant decrease of miR-155 expression
compared to negative control in the human acute promyelocytic leu-
kemia HL-60 cells, downregulated Slug and upregulated PUMA ex-
pression, decreased HL-60 cell growth and increased HL-60 cell apop-
tosis.22 On the other hand, the overexpression of miR-155 together with
myeloid maturation was found to induce apoptosis in HL-60 cells
through caspase-3 activation.23 Although miR-155 has been postulated
as oncogenic in various types of cancer including hematological ma-
9
24 h were assessed. The effects of the selected compounds on cell cycle
distribution of K562 myelogenous leukemia cells are shown in Fig. 1.
The subG1 population represents dead cells containing only frac-
tional DNA content. Our results showed an increase in the DNA frag-
mentation in K562 cells in a dose-dependent manner after treatment
with given compounds, as reflected by the increase in the percentages
of cells in the subG1 phase. This treatment caused a concomitant de-
crease in the proportion of cells in G2/M phase of the cell cycle com-
pared to the control cells. Thus, treatment of the K562 cells with the
compounds 6e, 6g, 6u, and 6v resulted in a pronounced accumulation
of cells in the subG1 phase of the cell cycle, pointing out an ability of
the examined compounds to induce cell death in chronic myelogenous
leukemia cells.
To get an insight into possible mechanisms of cell death type trig-
gered by the compounds 6e, 6g, 6u and 6v, we explored their ability to
induce apoptosis in K562 cells in presence of specific inhibitors of
caspase-3, caspase-8 and caspase-9. As it could be seen in Fig. 2, the
decrease in the percentage of cells in subG1 cell cycle phase was found
in K562 cell samples which were pretreated with Z-DEVD-FMK, an in-
hibitor of caspase-3 and afterwards exposed to compounds 6e, 6g, 6u
and 6v. The comparison was made with K562 cell samples which were
not pretreated with inhibitor before addition of the selected compound.
These results point out the pro-apoptotic activity of all the tested
compounds. Pretreatment of K562 cells with Z-IETD-FMK, inhibitor of
caspase-8 and a treatment with each of the four compounds induced
remarkable decrease in the percentage of subG1 cells in comparison
with cell samples which were only incubated with these compounds,
indicating the ability of our compounds to activate apoptosis in K562
cells through extrinsic apoptotic signaling pathway via death receptors.
In addition, lower percentage of K562 cells within subG1 phase was
lignancies, it was reported to be downregulated in chronic myelogenous
leukemia K562 cells.2
4,25
The downregulation of miR-155 could be at-
24
tributed to BCR-ABL tyrosine kinase activity. It was showed that
overexpression of miR-155 in K562 cells caused upregulation of
p27kip1 and induction of apoptosis.25 Our results showed induction of
apoptosis upon miR-155 overexpression in K562 cells treated with
compounds 6e and 6u. This behavior might be attributed to high
complexity of miRNA target selection and regulation in different types
of leukemia cells.24 Furthermore, miR-155 might target both oncogene
and tumor suppressors to varying degrees within the same cells, as it
was demonstrated for miR-196b26 and finally, miR-155 may exhibit
different function depending on its expression level.27
All K562 cell samples treated with examined anthraquinone-chal-
cones 6e, 6g, 6u and 6v showed reduced levels of MMP2, MMP9, and
VEGFA. Angiogenesis is an important step in the development and
progression of various types of malignancies, including leukemia.
VEGFA plays a crucial role in regulation of angiogenesis via binding to
the vascular endothelial growth factor receptor 2 (VEGFR2), which
leads to the activation of downstream VEGF signaling cascade, resulting
in in vitro cell proliferation, migration, and angiogenesis.2 It has been
shown that activation of VEGF downstream signaling induces angio-
genesis in ML cells.29 Additionally, upregulation of VEGFA promotes
8
Fig. 1. Changes in the cell cycle phase distribution of K562 cells treated with IC50 (A) and 2IC50 concentrations (B) of the compounds 6e, 6g, 6u and 6v for 24 h.
3

T. Stanojković et al.
Bioorganic & Medicinal Chemistry Letters xxx (xxxx) xxx–xxx
Fig. 2. Effects of the specific caspase inhibitors (Z-DEVD-FMK – caspase-3 inhibitor, Z-IETD-FMK – caspase-8 inhibitor, Z-LEHD-FMK – caspase-9 inhibitor) on the
percentages of K562 cells within subG1 phase treated with 2IC50 concentrations of the compounds 6e (A), 6g (B), 6u (C), and 6v (D).
Fig. 3. Changes in expression levels of MMP2 gene (A), MMP9 gene (B), VEGFA gene (C), and miR-155 in K562 cells exposed to subtoxic IC20 concentrations of the
compounds 6e (1.5 µM), 6g (2 µM), 6u (1.5 µM) and 6v (1.5 µM) for 24 h.
ML cell proliferation, survival and resistance to chemotherapy.30 Fur-
thermore, it has been shown that several anthraquinone derivatives
including Emodin have inhibitory effect on tumor angiogenesis,
through silencing VEGFA signaling cascade in leukemic cell line
models.31,32 Our study showed that K562 cells treated with anthra-
quinone-chalcone hybrids 6e, 6g, 6u and 6v had noticeable reduced
expression levels of VEGFA, indicating their potential antiangiogenic
properties.
4

T. Stanojković et al.
Bioorganic & Medicinal Chemistry Letters xxx (xxxx) xxx–xxx
The MMP2 and MMP9 overexpression in leukemia is associated with
crossing of leukemic cell blasts into the blood and can promote invasion
The compounds’ 6a–w bioactive conformations are established at
chain A-chain C dimer interface, within the capase-3 tetramer structure,
inside the hydrophobic pocket compiled of Thr166, Leu168, Tyr204,
Trp206, Thr255, and Phe256, at the edge of the active site. As the most
active pro-apoptotic agent, derivative 6f (Fig. 4A) forms several strong
hydrophobic interactions with the unsubstituted aromatic part of an-
thraquinone core: with the side chain methyl group of Thr166 from
chain A (further on Thr166.A or similar abbreviation), with Tyr204.A,
and with the side chain methyl group of Thr255.A. Particular attrac-
tions are verified by the appearance of negative 3-D QSAR O]CPLS-
coefficients (yellow maps, indicators of repulsive hydrophobic interac-
tions). Similar interactions were a characteristic of the second ranked
compound, 6e (Fig. 4B), as well. Other important steric clash, also
confirmed by negative O]CPLS-coefficients, is formed between the sub-
stituted aromatic moiety of the anthraquinone core and the side chain
of Glu167.A. Comparable alignment of the anthracene-9,10-dione core
is seen for the remaining compounds, too. The slight drop in the po-
tency of 6e, related to 6f, can be initially attributed to the 6e anthra-
quinone core rotation in comparison to 6f, where rings are orthogonal
to each other. Carbonyl groups within the anthracene-9,10-dione give
significant contribution to the activity: the C9 carbonyl oxygen is, ac-
cording to the alignment of negative ELEPLS-coefficients (blue maps) in-
volved in electrostatic interactions with Thr166.A and Thr166.C.
Moreover, with the progress in anthraquinone-chalcone hybrid
structures, the chalcone phenyl ring, common to all compounds, is at-
tracted by Phe142.A or Leu168.A (negative O]CPLS-coefficients as proof).
Precise attraction is facilitated by the antraquinone-chalcone carbonyl
linker, a function that is within the bioactive conformation of 6f or-
iented towards the hydroxyl group of Thr166.C, whereas for 6e is di-
rected to Thr255.A. Each of the interactions is described by positive
ELEPLS-coefficients (red maps), implying electron-withdrawing potential of
carbonyl linker as important for the activity. With over 5 Å in distance
between the electron-withdrawing center and Thr255.A(C)eOH group
as potential HB donor, the possibility of hydrogen bonding is excluded.
The true difference in the activity of evaluated derivatives lies in the
contribution of R(Ar) substituents. They are introduced into the
3
3
and metastasis into distant organs. Furthermore, all K562 cell samples
treated with 6e, 6g, 6u and 6v showed decreased levels of MMP2 and
MMP9 indicating their potential anti-invasive and antimetastatic
properties. The simultaneous effect of examined anthraquinone-chal-
cone hybrids on decrease of VEGF, MMP2 and MMP9 expression in
K562 cells compared with control, untreated cells might have a pro-
mising effect in future therapies for ML. These results might indicate
that future therapeutics in the treatment of ML may be focused on
3
4
antiangiogenic therapy, and on the introduction of additional antic-
ancer agents such as anthraquinone-chalcone conjugates in combina-
tion with standard therapeutics.
Herein results, considering the miR-155 expression, imply that the
elaborated compounds act as pro-apoptotic agents at signal transduc-
tion extrinsic pathway level in the form of caspase-3 efectors.35 To the
best of our knowledge, there are no deposited crystallographic data at
Protein Data Bank, concerning the co-crystallized 6a–w-caspase-3
complexes. Therefore, compounds’ 6a–w bioactive conformations were
precluded by means of molecular docking, by exploring their binding
modes in caspase-3 co-crystallized with isoquinoline-1,3,4-trione deri-
3
6
vatives (PDB code: 3DEI), utilizing the search algorithms and scoring
functions as implemented in AutoDock Vina.3 Following, the biophy-
sical rationale leading to the compounds’ 6a–w pro-apoptotic activity
was revealed by virtue of generating the structure-based 3-D QSAR
7
3
8
models, as instructed in Open3DQSAR methodology. The bi-probe 3-
D QSAR model (Supplementary Tables S1 and S2), derived from car-
bonyl oxygen as hydrogen bond (HB) acceptor (i.e. the O]C probe form
MMFF94 force filed) and electrostatic charge (i.e. the ELE filed), ex-
plained compounds’ 6a-w biopotential by cause of steric/HB and
electrostatic interactions (Fig. 4, Supplementary material Figs. S49A
and S49C). The molecular docking and 3-D QSAR studies external va-
lidation were performed on a set of anthraquinones from Morinda ci-
trifolia, as leukaemia cell line K562 inhibitors (Supplementary material
3
9
Figs. S49B and S49D). Molecular docking and 3-D QSAR experi-
mented details are provided in Supplementary material.
Fig. 4. Structure-based 3-D QSAR models for 6f (A), 6e (B), 6i (C), 6l (D) describing pro-apoptotic activity against caspase-3. Caspase-3 is depicted in white. Positive
O]CPLS-coefficients are illustrated in green, negative O]CPLS-coefficients are drawn in yellow, positive ELEPLS-coefficients are colored in red, negative ELEPLS-coefficients are
presented in blue.
5

T. Stanojković et al.
Bioorganic & Medicinal Chemistry Letters xxx (xxxx) xxx–xxx
caspase-3 active site via the amide carbonyl linker between the chal-
cone aromatic scaffold and the R(Ar) substituents. The distinct linker
contributes to the pro-apoptotic potency by making electrostatic in-
teractions with Thr255.A and Tyr204.C, respectively. Consequently,
phenolic aromates of 6f and 6e are involved in the weak steric hin-
drance with His 121.C (as verified by the superposition of negative
O]CPLS-coefficients maps as attractive interactions indicators). According
the alignment of negative ELEPLS-coefficients (blue maps), the meta-hy-
droxyphenyl function of 6f is engaged in hydrogen bonding with
Thr255.A (dHB = 2.314 Å), indicating that the particular hydroxyl
group serves as an HB acceptor to the active site residue. On the other
hand, the ortho-hydroxyphenyl function of 6e is facing Thr166.A to
create only electrostatic attraction (negative ELEPLS-coefficients as proof),
and, consequently, the activity of 6e is diminished. The establishment
of hydrogen bond/electrostatic interactions with Thr166.A/Thr255.A is
of the highest importance for the pro-apoptotic activity, inasmuch as
the substitution of –OH group with other electron-withdrawing groups
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(
like halogens, 6i, and 6l, Figs. 4C and D, respectively), alleviates the
activity in significant manner.
In conclusion, we have synthesized a series of twenty-three amide
derivatives combining anthraquinone-chalcone carboxylic acid and
corresponding primary amines. Four examined anthraquinone-chalcone
hybrids exerted strong cytotoxic activity against different human leu-
kemia cell lines with remarkably lower toxicity against normal MRC-5
cells. The compounds showed the ability to trigger apoptotic cell death
in K562 cells through activation of extrinsic and intrinsic signaling
pathway, with the exception of compound 6e which activated only
extrinsic pathway. Treatment of K562 cells with each of the four se-
lected compounds decreased the expression levels of MMP2, MMP9 and
VEGFA genes, indicating their anti-angiogenic, anti-invasive and anti-
metastatic activities. MiR-155 expression strongly depends on the
nature and position of substituents attached to aromatic ring.
Heteroaromatics, furan and thiophene also cause great difference of
miR-155 expression in K562 cells. As the most active pro-apoptotic
agents, derivatives 6f and 6e form several strong hydrophobic inter-
actions with the unsubstituted aromatic part of anthraquinone core. In
conclusion, our research may suggest promising antileukemic potential
of newly synthesized anthraquinone-chalcone hybrids.
1
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The authors are grateful to the Ministry of Science and
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support (Grant Nos. 172016 and 175011).
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