Multi-target weapons: diaryl-pyrazoline thiazolidinediones simultaneously targeting VEGFR-2 and HDAC cancer hallmarks

Article abstract of DOI:10.1039/d1md00125f

In anticancer drug discovery, multi-targeting compounds have been beneficial due to their advantages over single-targeting compounds. For instance, VEGFR-2 has a crucial role in angiogenesis and cancer management, whereas HDACs are well-known regulators o

Full text of DOI:10.1039/d1md00125f

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Multi-target weapons: diaryl-pyrazoline
thiazolidinediones simultaneously targeting
Cite this: DOI: 10.1039/d1md00125f
VEGFR-2 and HDAC cancer hallmarks†
c
Neha Upadhyay,a Kalpana Tilekar,a Sabreena Safuan,b Alan P. Kumar,
a
Markus Schweipert,d Franz-Josef Meyer-Almes
d and Ramaa C S
*
*
In anticancer drug discovery, multi-targeting compounds have been beneficial due to their advantages over
single-targeting compounds. For instance, VEGFR-2 has a crucial role in angiogenesis and cancer
management, whereas HDACs are well-known regulators of epigenetics and have been known to
contribute significantly to angiogenesis and carcinogenesis. Herein, we have reported nineteen novel
VEGFR-2 and HDAC dual-targeting analogs containing diaryl-pyrazoline thiazolidinediones and their
in vitro and in vivo biological evaluation. In particular, the most promising compound 14c has emerged as a
dual inhibitor of VEGFR-2 and HDAC. It demonstrated anti-angiogenic activity by inhibiting in vitro HUVEC
proliferation, migration, and tube formation. Moreover, an in vivo CAM assay showed that 14c repressed
new capillary formation in CAMs. In particular, 14c exhibited cytotoxicity potential on different cancer cell
lines such as MCF-7, K562, A549, and HT-29. Additionally, 14c demonstrated significant potency and
selectivity against HDAC4 in the sub-micromolar range. To materialize the hypothesis, we also performed
molecular docking on the crystal structures of both VEGFR-2 (PDB ID: 1YWN) and HDAC4 (PDB-ID: 4CBY),
which corroborated the designing and biological activity. The results indicated that compound 14c could
be a potential lead to develop more optimized multi-target analogs with enhanced potency and selectivity.
Received 13th April 2021,
Accepted 10th June 2021
DOI: 10.1039/d1md00125f
rsc.li/medchem
(VEGFs) and their kinase receptors remain the most
prominent ones. Tumor cells secrete VEGFs under various
Introduction
Cancer is the most dreaded disease and a major contributor
of death worldwide. To battle against cancer, it is imperative
to develop an agent with target specificity and potency.
Clinical observations suggest that the use of a single-targeting
agent might fail to produce the desired therapeutic effects
due to development of resistance to the target or mutation.1
Moreover, the progression of cancer relies on multiple
receptors or signaling pathways, and thus, an alternative
strategy would be to develop multi-targeting agents.2
Angiogenesis is known to be involved in cancer
progression;3,4 a variety of factors are involved in regulation
of angiogenesis, in which vascular endothelial growth factors
conditions such as oncogenic mutations, inflammatory
stimuli, hypoxia, stromal and inflammatory cells etc. VEGFs
interact with their respective VEGF receptors (VEGFRs) which
initiates the critical downstream signaling pathway that
brings about tumor angiogenesis; thus, targeting VEGFR
would be a reasonable therapeutic approach.5 There are many
FDA-approved agents that target VEGFRs (sorafenib,
sunitinib, pazopanib, vandetanib, axitinib, regorafenib etc.).
However, drug resistance is the major concern associated
with the monotherapy of these agents;6,7 hence, over decades
researchers have been directing the focus on the potential
treatment of resistance.8 Various dual-targeting agents have
been reported in which one target is VEGFR-2 along with
other therapeutic targets.2,9–12 For example, in 2011, using a
multitarget approach, cabozantinib was developed as a dual
inhibitor of VEGFR-2 and c-Met and used for the treatment of
renal cell carcinoma.13–15
a Department of Pharmaceutical Chemistry, Bharati Vidyapeeth's College of
Pharmacy, Navi Mumbai, India. E-mail: sinharamaa@yahoo.in
b Universiti Sains Malaysia School of Health Sciences, Health Campus Universiti
Sains Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia
c Cancer Science Institute of Singapore, Department of Pharmacology, Yong Loo Lin
School of Medicine, National University of Singapore, Singapore
d Department of Chemical Engineering and Biotechnology, University of Applied
Sciences, Darmstadt, Germany. E-mail: franz-josef.meyer-almes@h-da.de
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
d1md00125f
Though genetics is crucial in cancer progression,
epigenetics has become equally important in this field. In the
process of epigenetic alterations, histone deacetylases
(HDACs) play a key role and therefore become therapeutic
targets for anticancer drug discovery.16,17 HDACs are
subdivided into three zinc-dependent classes. Considering
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only human enzymes, class I contains HDACs 1, 2, 3 and 8; Results and discussion
class II is subdivided into class IIa consisting of HDACs 4,
5, 7 and 9 and class IIb containing HDAC6 and HDAC10;
Designing
class IV has only one member, HDAC11. The zinc-
dependent HDACs have in common a sequentially and
structurally highly conserved active site including a catalytic
zinc ion. For the sake of completeness, HDAC class III has
also to be mentioned. This special class consists of 7
human members, also named sirtuins, which exploit a
The main challenge in designing dual VEGFR-2 and HDAC
inhibitors was to achieve a unique framework which satisfied
the binding requirements of both targets. In our previous
reports, a distinct pharmacophoric drug design was used to
achieve both classes of compounds.46,47 Thus, based on our
previous experience with both targets, a multi-targeting
strategy was developed by considering VEGFR-2 and HDAC
pharmacophore properties.
different
NAD+-dependent
deacetylation
mechanism.
Overexpression of HDACs can cause various types of
cancers, e.g., colorectal, pancreatic, lung, breast and colon
as well as leukemia and hepatocellular carcinoma.18–22
There is emerging evidence that class IIa HDACs and
particularly HDAC4 are involved in cancer.23,24 One of the
best investigated pathways which controls the proliferation
of mammary epithelial cells is the MEF2-HDAC axis.
Consequently, targeting class IIa HDACs was suggested as a
therapeutic strategy for the treatment of breast cancer.25
Currently, there are five HDAC inhibitors clinically approved
as novel antitumor drugs: vorinostat (SAHA), panobinostat
(LBH-589), belinostat (PXD-101), romidepsin (FK228) and
chidamide. However, their monotherapy fails to display any
significant effectiveness against solid tumors1 and they are
The pharmacophoric requirement of VEGFR-2 inhibitors
are (a) a “hinge-binding” moiety that interacts with the ATP
pocket, (b) a “linker” usually comprising 3–5 bond lengths
and a hydrogen bond acceptor donor system and (c) a
hydrophobic moiety occupying the allosteric site.48 With
these design considerations, we had previously reported
5-benzylidene-2,4-thiazolidinedione analogs as VEGFR-2
inhibitors (Fig. 1;47 VEGFR-2 IC50
– 0.5 μM). It was
hypothesized that a bulky hydrophobic moiety was found to
be essential to fill the large empty pocket of the DFG
motif;49,50 thus, we modified the 5-benzylidene-2,4-
thiazolidinedione analogs by replacing the terminal aryl
group with the bulkier substituted diaryl-pyrazoline (ring A
and ring B) and retained the remaining structural features to
occlude the basic VEGFR-2 inhibitory activity. Since,
pyrazoline-containing compounds have been reported to
possess excellent VEGFR-2 inhibition capability51–55 and
antiproliferative potential,56–59 they were thought to impart
antiangiogenic activity to the molecule.
associated
with
various
side
effects
such
as
thrombocytopenia, leukopenia, diarrhoea, fatigue and
potential mutagenicity.26,27 Mutagenicity is suspected to be
associated with hydroxamate zinc binding groups
(ZBGs),28–31
rearrangement,
the
a
mechanism
reaction in which hydroxamates are
being
the
Lossen
transformed into the corresponding isocyanate.29 Thus, the
best alternative strategy to circumvent mutagenicity would
be the development of agents containing non-hydroxamate
ZBGs. In addition, there are several mechanisms by which
HDAC inhibitors also exhibit anti-angiogenesis activity such
as: (1) hypoxia causes expression of HIF-1α which initiates
the transcription of downstream pathways such as
overexpression of VEGF,32 HDAC inhibitors cause
suppression of HIF-1α by initiating the expression of
noncoding RNAs that target HIF-1α33 and by regulating the
transforming growth factor-β (TGF-β) pathway,34–36 leading
to anti-angiogenic effects; (2) regulating the acetylation of
many non-histone proteins such as P53 which is known to
promote degradation of HIF-1α;37,38 (3) tie2-expressing
macrophages (TEMs) are known to promote angiogenesis
and HDAC inhibitors suppress the M2 polarization of TEMs,
thus causing anti-angiogenesis;39,40 (4) arresting the cell
cycle; and (5) apoptosis.41 Interestingly, one of the
mechanisms by which HDACs exert their antitumor effects
is downregulation of VEGF32,42 and suppression of
neovascularization through alteration of genes directly
involved in angiogenesis.38,43–45 Thus, using a multitarget
approach, selecting VEGFR-2 and HDAC as therapeutic
targets for the development of novel dual-targeting agents
would offer benefits such as reduced side effects and
increased potency.
Our above designed structural framework also fits
perfectly into the pharmacophoric requirements of HDAC
inhibitors. Typical HDAC inhibitors share
a
broad
pharmacophore as a surface recognition cap (SRC) which
binds with amino acids of the active site, a hydrophobic
linker which occupies the active site channel, and a zinc-
binding group (ZBG) which chelates the catalytic zinc ion.16
The diaryl pyrazoline moiety of our designed molecule was
assumed to occupy the cap portion of HDAC, as recent
literature highlights pyrazolines as an appropriate surface
recognition motif in HDAC inhibitor design.60–63
Additionally, the literature reports non-classical HDAC
inhibitors containing a cyclic ring as a linker rather than a
straight hydrocarbon chain;16,64–66 thus, the central phenyl
ring was assumed as a cyclic linker. Several recently reported
HDAC inhibitors, the so-called non-hydroxamates, have been
an interesting part of the HDAC inhibitors' discovery. In this
regard, several non-hydroxamic ZBGs have been identified
and reported. On a similar line, in our previously reported
HDAC inhibitors, we had incorporated thiazolidinedione
(TZD) as a non-hydroxamate ZBG which was found to be an
effective antitumor agent (Fig. 1; Mohan et al. 2012);46,66–69
hence, the TZD portion of the currently designed series was
assumed as ZBG.
This rational design led to a unique structural framework
that could be considered in accordance with the
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Fig. 1 Rationale of designing novel VEGFR-2 and HDAC dual inhibitors.
pharmacophoric model of both the targets (Fig. 1). The
substituted diaryl-pyrazoline group would correspond to the
hydrophobic tail of VEGFR-2 and CAP for HDAC. The central
phenyl ring with a CO–CH2–O– framework would be the
hydrophobic linker for VEGFR-2 and will also function as a
cyclic linker of HDAC. The terminal TZD ring would be the
hinge binding moiety for VEGFR-2 and ZBG for HDAC.
Therefore, we were fairly confident that these compounds
could have the capacity to target simultaneously VEGFR-2
and HDAC.
10g) and 5-(4-hydroxybenzylidene)-2,4-thiazolidenedione (12).
The first step was the synthesis of chalcones (3a, 3b, 3d–3g,
3j, 3k, 6a–6g, 6j, 9c, 9e, and 9g) which occurred via the
renowned Claisen–Schmidt condensation reaction, where
different acetophenones (1, 5 and 8) were reacted with
substituted aromatic aldehydes (2a–2g, 2j, and 2k) in basic
medium to obtain the respective chalcones. The second step
involved
synthesis
of
chloroacetylated
pyrazoline
intermediates (4a, 4b, 4d–4g, 4j, 4k, 7a–7g, 7j, 10c, 10e, and
10g), which proceeded via refluxing different chalcones (3a,
3b, 3d–3g, 3j, 3k, 6a–6g, 6j, 9c, 9e, and 9g) with hydrazine
hydrate and chloroacetyl chloride in chloroform. The third
step was Knoevenagel condensation of 2,4-thiazolidinedione
(11) with 2-hydroxybenzaldehyde in the presence of
Chemistry
All the final compounds were synthesized by Schemes 1 and
2. The detailed procedure and spectral observations are
presented in the Materials and methods. The purity of all the
final compounds, which was >95%, was confirmed by HPLC.
piperidine
benzoate
in
toluene
to
afford
5-(4-
hydroxybenzylidene)-2,4-thiazolidenedione (12). Scheme
2
describes the synthesis of the final compounds (13a, 13b,
13d–13g, 13j, 13k, 14a–14g, 14j, 15c, 15e, and 15g). Reaction
of chloroacetylated pyrazolines (4a, 4b, 4d–4g, 4j, 4k, 7a–7g,
7j, 10c, 10e, and 10g) and 5-(4-hydroxybenzylidene)-2,4-
thiazolidenedione (12) in DMF and K2CO3 by stirring at RT
for 24 h afforded the final compounds (13a, 13b, 13d–13g,
13j, 13k, 14a–14g, 14j, 15c, 15e, and 15g).
1
All the intermediates were structurally confirmed by H NMR
and FTIR spectroscopy, and the final compounds by 1H
NMR, 13C NMR, FTIR and mass spectrometry. Scheme 1
outlines the synthesis of variously substituted chalcones (3a,
3b, 3d–3g, 3j, 3k, 6a–6g, 6j, 9c, 9e, and 9g), chloroacetylated
pyrazolines (4a, 4b, 4d–4g, 4j, 4k, 7a–7g, 7j, 10c, 10e, and
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Scheme 1 Synthesis of intermediates. Reagents and conditions: (a) aqueous NaOH, EtOH, RT, 5–6 h; (b) NH2NH2·H2O, CHCl3, 80 °C, 12 h, then
K2CO3, ClCH2COCl, rt, 12 h; (c) 4-hydroxybenzaldehyde, piperidine benzoate, toluene, reflux, 4 h.
In vitro HDAC enzyme inhibition assay on HDAC4 and
HDAC8
appear very promising for the development of HDAC
inhibitors with presumably less unwanted toxic effects in
later pharmaceutical applications. It is noteworthy that the
overall results suggest that the novel diaryl-pyrazoline TZDs
exhibit more selectivity and potency towards HDAC4 as
compared to HDAC8. Based on the in vitro HDAC enzyme
inhibition assay results, representative compounds with the
most potent inhibitory activity were selected to further
determine their anti-angiogenesis capability.
To evaluate the HDAC inhibitory potential of all the newly
synthesized compounds, an initial in vitro screening was
performed at 50 μM concentration on HDAC4 (class II) and
HDAC8 (class I) and all the compounds showed satisfactory
inhibition against both isoforms (Table 1). Compounds with
less than 50% residual enzyme activity were evaluated further
to determine their inhibitory concentration (IC50); the best
compound 13j showed activity in the sub-micromolar range
against HDAC4. Most of the analogs were essentially inactive
(IC50 >50 μM) or exhibited 2-digit micromolar IC50 values
against HDAC8. “Cpd16” and “Cpd31” were used as HDAC4
selective reference inhibitors, and the TZD analogs exhibited
less potency against HDAC4 than the reference
inhibitors.70,71 In contrast to these reference compounds, the
presently reported TZD inhibitors did not contain the zinc-
chelating hydroxamate warhead, which mostly contributes to
the affinity of classical HDAC inhibitors. Therefore, sub-
micromolar activities without typical zinc-chelating groups
HDAC selectivity profiling on a panel of HDACs
From the results of the primary evaluation on HDAC4 and
HDAC8, compounds 13j, 14c, and 14j were selected for
further screening on a panel of HDACs (Table 2) to establish
their selectivity profiling on different HDAC isoforms. The
assay revealed that 13j and 14j were active towards most of
the HDAC isoforms; whereas 14c was more selective towards
HDAC4 and its IC50 was found to be in the sub-micromolar
range (Fig. 2). Thus, 14c could be used as a lead for the
development of more optimized molecules and rigorous
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Scheme 2 Synthesis of final compounds. Reagents and conditions: (d) K2CO3, DMF, stirred at RT, 24 h.
Table 1 Preliminary screening and IC50 determination of pyrazoline-based TZD on HDAC4 and HDAC8 isoforms
Sr. no. Compounds
Residual HDAC4 activity at 50 μM (%) Residual HDAC8 activity at 50 μM (%) HDAC4 IC50 (μM) HDAC8 IC50 (μM)
1
2
3
4
5
6
7
8
13a
13b
13d
13e
13f
13g
13j
13k
14a
14b
14c
14d
14e
14f
14g
14j
11
14
12
2.5 0.5
17
4.6 0.2
5.9 0.3
7.0 0.6
10
3.3 1.3
36
2
1
1
39
27
8.2 0.2
26
33
36
1
3
1.6 0.2
1.2 0.3
1.0 0.2
1.7 0.4
2.2 0.2
1.2 0.6
0.7 0.3
1.2 2.0
4.2 0.2
1.1 0.9
20
29
16
26
34 10
31
1
1
1
1
2
3
2
1
1
1
1
9.4 1.6
10
35
21
58
28
45
81
2
1
3
9
3
9
1
>50
>50
>50
>50
>50
>50
>50
25
>50
>50
>50
6.7
10
11
12
13
14
15
16
17
18
19
20
21
2
0.88
1.3
4
3
9.2 1.5
3.3 0.
61 12
1.4 0.4
1.6 0.5
1.8 0.4
0.8 0.2
10
10
13
22
10
9.4 1.8
—
—
2
1
2
3
4
63
81
22
100
69
69
—
4
6
3
8
5
3
7
15c
15e
15g
15
2
1.5 0.7
1.5 0.2
0.039
Cpd 16 (ref. 72)
Cpd 31 (ref. 73)
—
0.02
0.36
structural modifications could provide analogs with
enhanced potency and efficacy towards HDAC4 isoforms in
the future.
Thermal shift assay on HDAC4. Thermal shift is used to
demonstrate target engagement (TE) by a ligand. Binding of
compared to free protein, thus producing a thermal shift.
Test compounds with HDAC4 inhibitory potential (13j and
14j) were subjected to this assay with the HDAC4 isoform to
examine the thermal stabilization of the ligands. The melting
temperature of free HDAC4 (DMSO control) was 57.3 °C, and
those of the protein in the presence of the test compounds
were 56.7 °C and 57.3 °C for 13j and 14j, respectively (Fig. 3).
a
ligand to the target protein can shift the melting
temperature of the protein to the higher or lower side as
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Table 2 Selectivity profile of pyrazoline-based TZD inhibitors against a panel of human HDACs. IC50 values are means, and errors were obtained from
statistical analysis of non-linear regression fits to sets of 10 data points
IC50 (μM)
Compound
HDAC1
HDAC2
HDAC3
HDAC4
HDAC6
HDAC7
HDAC8
13j
14c
14j
4.9
>50
11
13
>50
21
9.1
>50
11
0.7 0.3
39
7.6
13
>50
>50
6.7
10 1.0
>50
25 7.0
0.88
4
0.8 0.2
Fig. 2 Dose–response curve of compounds 13j, 14c, and 14f on different HDAC isoforms.
These minor changes in comparison with the melting point
of the unbound enzyme were within the error range of the
method. Altogether, even though the compounds were able
to inhibit HDAC4 in sub-micromolar concentrations, they
contribute nothing or only to a minor degree to protein
stabilization.
could be measured by the MTT assay,74–77 in which the
number of viable cells is determined. Nine compounds with
the highest HDAC4 inhibition potential (13b, 13d, 13j, 14a,
14c–14e, 14j, and 15g) were selected to determine their
in vitro cytotoxicity effects on HUVEC proliferation. STS was
used as a positive control. Five different concentrations (10,
1, 0.1, 0.01, and 0.001 μM) of test compounds as well as
the positive control were used to determine their inhibition
potential. Results showed that compounds 13b, 14c, 14d
and 14j exhibited satisfactory inhibition effects on HUVEC
proliferation (IC50 <10 μM), while 13d, 13j, 14a, 14e and
15g displayed poor inhibition (>10 μM). Moreover, 14c
showed inhibitory activity comparable to that of STS
(Table 3); thus, we assume that it may have potential anti-
angiogenic activity.
Cytotoxicity assessment on HUVEC proliferation
Human umbilical vein endothelial cells (HUVECs) are the
most commonly used human endothelial cells for in vitro
angiogenesis assays.74,75 In the development of sprouting
angiogenesis, endothelial cells undergo proliferation, which
is related to survival of new vessels; thus, the effects of
anti-angiogenic agents on the proliferation of HUVECs
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Table 4 VEGFR-2 inhibitory activity of 13a, 14c, 14d, and 14j
Sr. no.
Code
% Inhibitiona (at 10 μM)
IC50 (μM)
1
2
3
4
5
13b
14c
14d
14j
48.95
56.66
48.71
22.36
87.85
NDb
5
NDb
NDb
0.5
STSc
a
b
c
Assays were performed in replicate (n ≥ 2). Not determined. STS
represents staurosporine.
at ring A and 2-furyl at ring B, while the other two
compounds with 4-fluorophenyl at ring A (14d and 14j) and
other substituents at ring B (14d: 4-trifluoromethylphenyl;
14j: 3,4-dichlorophenyl) displayed <50% inhibition (Table 4).
However, compound 14j exhibited a remarkable decrease in
% VEGFR-2 inhibitory activity as compared to 14c and 14d,
which suggested that dichlorophenyl at ring B is of least
preference as compared to the other substituents.
Fig. 3 Thermal stabilization of 3 μM HDAC4 upon binding of 100 μM
13j and 14j.
VEGFR-2 inhibition assay
Test compounds with inhibitory capability on HUVEC
proliferation (13a, 14c, 14d, and 14j) were selected to
determine their effects on the activity of VEGFR-2
phosphorylation, which is known to activate other signalling
cascades associated to endothelial cells and bring about
Molecular docking study
A complete docking study was performed to predict the
binding pose of the ligand and determine the putative
molecular determinants of protein–ligand binding. The
docking procedure was validated by redocking of the ligand
in the crystal structure of VEGFR-2 (PDB-ID: 1YWN) and
HDAC4 (PDB-ID: 4CBY).70 The ligands were docked into the
protein receptor which yielded an almost perfect overlap with
the X-ray pose (ESI†). Thus, by assuming that all parameters
were well configurated for docking of pyrazoline TZD
analogs, the same crystal structures were used throughout
the docking study. The GBVI/WSA dG docking score of the
redocked ligand was −11.2 on VEGFR-2 and −13.6 on HDAC4
proliferation,
migration,
invasion,
and
cell
differentiation.78,79 An in vitro cell-based ELISA method was
used in which all four test compounds along with the
positive control STS were preliminarily tested at 10 μM
concentration against pVEGFR-2. The test compounds with
noticeable structural differences displayed poor to good %
VEGFR-2 inhibition, wherein 14c showed >50% inhibition,
and the IC50 of 14c further confirmed its pVEGFR-2
inhibition potential. Structurally, 14c contains 4-fluorophenyl
Table 3 IC50 of HUVECs by compounds 13b, 13d, 13j, 14a, 14c–14e, 14j, and 15g
a
Sr. no.
Code
IC50 (μM)
A ring
B ring
1
2
3
4
5
6
7
8
9
10
13b
13d
13j
14a
14c
14d
14e
14j
2
Phenyl
Phenyl
Phenyl
4-F phenyl
4-F phenyl
4-F phenyl
4-F phenyl
4-F phenyl
2,4-DiF phenyl
—
2-Cl phenyl
4-CF3 phenyl
3,4-DiCl phenyl
Phenyl
>10
>10
>10
0.7
6
>10
2
>10
0.5
2-Furyl
4-CF3 phenyl
4-F phenyl
3,4-DiCl phenyl
4-Tolyl
15g
STS
—
a
Assays were performed in replicate (n ≥ 2).
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(ESI†). Docking of the complementary stereoisomers of 14c
and 14j showed similar binding poses of (S)-14c, (R)-14j and
(S)-14j having the same absolute configuration at the pyrazole
ring (ESI†). The binding mode of (S)-14c to VEGFR-2
resembles that of previously reported TZD analogs.47 The
TZD warhead forms two hydrogen bonds with L838 and
A1048. Multiple hydrophobic pi-alkyl and alkyl interactions
contribute further to affinity (Fig. 4).
growth factors.80,81 To measure the extent of endothelial cell
motility against test compounds, scrape wound healing
assay was performed. The migration capability of HUVECs
was determined for the most active compound 14c along
with positive control STS, which was measured in percent
wound healing. As compared to untreated HUVECs, STS-
treated HUVECs suppressed the endothelial cell migration
to a greater extent (Fig. 6). The inhibitory effect of 14c on
the mobility of HUVECs was less than that of STS but
greater than that of untreated, evident by percent wound
healing. Thus, with the results it was clear that 14c has the
potential to interfere with the migration capability of
endothelial cells.
Furthermore, the docking study of compounds 14c and
14j with HDAC4 gained more insight into the mode of
molecular interaction. The results for the two enantiomers of
these analogs show rather similar docking scores, which were
considerably lower than that obtained from redocking the
reference compound “Cpd 31” into the crystal structure of its
complex with the catalytic domain of HDAC4 (Fig. 5).71 This
appears realistic, since “Cpd 31” is about 45-fold more active
against HDAC4 than 13d and 14c (Table 1). Looking closer at
the binding pose of (S)-14c, it was revealed that the TZD
group was able to replace the hydroxamate warhead of classic
HDAC inhibitors, since the TZD group not only interacted
with the catalytic zinc ion in the catalytic site but also
showed amide-pi stacking with G811 and pi-alkyl interaction
with L943 (Fig. 5). The binding was also strengthened by
parallel pi-stacking interactions with F871. The branched
pyrazoline group serving as capping group was largely
exposed to the surrounding solvent but showed an amide-pi
interaction with F870. Therefore, it is not unexpected that
different substitution patterns at the aromatic rings of the
capping group have no big influence on the enzyme
inhibitory activity (ESI†). Thus, the docking results confirmed
that the pyrazoline TZD analogs of this study were
biologically active against both target proteins VEGFR-2 and
HDAC4.
Capillary tube formation assay
The process of angiogenesis requires capillary-like tube
formation of endothelial cells for ease of blood flow, and it is
considered as representative of later stages of angiogenesis.
Thus, the tube formation assay is extensively used to
determine the in vitro anti-angiogenic effects of test
compounds.74–77 Compound 14c exhibited substantial
inhibition of HUVEC proliferation, VEGFR-2 phosphorylation,
and migration; thus, it was further evaluated to determine its
effect on the tube formation capability of HUVECs. After 24
h, STS-treated HUVECs reduced the number of capillaries to
a greater extent as compared to untreated which formed
hollow capillary-like structures (Fig. 7A and B). However, 14c
did not significantly decrease the capillary formation in
comparison with STS (Fig. 7C and Table S2† ( p = 0.31)). Thus,
we could state that 14c has less potential to decrease the tube
formation activity of HUVECs as compared to STS.
MTT cytotoxicity assay
Considering in vitro results of anti-angiogenic assays, 14c was
evaluated for its effects on the cytotoxicity of four different
cancer cell lines: MCF-7 (human breast cancer), K562
(leukaemia), A549 (human lung cancer), and HT-29 (human
Endothelial cell migration assay
Endothelial cell mobility is significant for tumor
angiogenesis which is known to be initiated by different
Fig. 4 Docking of (S)-14c into VEGFR-2. (A) 3D binding pose showing hydrogen bonds (dotted green lines) to L838 and A1048. (B) Detailed 2D
protein–ligand interactions.
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Fig. 5 Docking pose of (S)-14c in the active pocket of HDAC4 (PDB-ID: 4CBY). (A) 3D binding pose showing hydrogen bonds with F871 and Zn2+
interaction. (B) Detailed 2D protein–ligand interactions.
Fig. 6 (A–C) Representative images of cellular migration assay in HUVECs after 8 h of untreated, STS (10 μM mL−1), and 14c (10 μM mL−1),
respectively. (D) Graphical representation of percent wound healing. Error bars represent SEM, n = 3, *p ≤ 0.01; ***p ≤ 0.0001 (compared to
untreated; calculated with unpaired Student's t-test).
Fig. 7 (A–C) Images of HUVEC capillary-like tube formation assay after 48 h of untreated, STS (10 μM mL−1), and 14c (10 μM mL−1), respectively.
(D) Graphical representation of intersection counts with different treatments. Error bars represent SEM, n = 3, ***p ≤ 0.0001 (STS compared to
untreated; calculated with unpaired Student's t-test).
colorectal adenocarcinoma). Results revealed that 14c
exhibited moderate to good cytotoxicity on these four cancer
cell lines (Table 5). The positive control paclitaxel was several
fold more potent than 14c, whereas cisplatin showed
variation in activity. HT-29 cell lines appeared to be most
affected by 14c followed by A549, MCF-7 and K562 (ESI†).
Thus, we assume that 14c has potential cytotoxicity against
these cancer cell lines.
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Table 5 Cancer cell viability of 14c
IC50a (μM)
apparatus and those of the final compounds were obtained
using a DSC1 STAR system differential scanning calorimeter
from Mettler Toledo. The purity of all final compounds was
determined using an Agilent 1200 high-performance liquid
chromatography (HPLC) system with the software EZ chrome
Compound
MCF-7
K562
A549
HT-29
14c
Paclitaxel
Cisplatin
28.41 4.2
0.35
10.57 1.1
46.27 1.6
0.29
58.4 1.4
19.52 1.44
0.32
16.68 1.74
18.84 1.1
0.28
10.6 1.2
Elite.
The
chromatographic
column
used
was
HemochromIntsil A31 C18 5U 150 mm × 4.6 mm Sn-
B180127, detection at 300 nm. A UV-visible detector was used
with a flow rate of 1 mL min−1. The oven temperature was
maintained at 30 °C; gradient elution with a run time of 10
min using methanol : formic acid (1%) (formic acid: in 1000
mL double-distilled water 1 mL formic acid was added) in an
80 : 20/90 : 10 ratio. The structures of intermediates were
confirmed by FTIR and 1H NMR and that of the final
compounds by FTIR, 1H NMR, 13C NMR and mass
spectrometry. IR analysis was performed using a JASCO FT/
IR-4100 type A spectrometer using a manual sampling
method. 1H NMR spectra were recorded using a Bruker
Avance 400 MHz spectrometer with DMSO-d6. All shifts are
reported in δ (ppm) units relative to the signals for solvent
DMSO (δ – 2.50 ppm). All coupling constants (J values) are
reported in hertz (Hz). NMR abbreviations are bs, broad
singlet; s, singlet; d, doublet; t, triplet; q, quartet; m,
multiplet; and dd, doublet of doublets. 13C NMR was
recorded on a Bruker Avance spectrometer at 100 MHz with
DMSO-d6. The mass spectrum was determined on an LC-MS
Agilent Technologies 1260 Infinity instrument. All final
compounds were synthesized by Schemes 1 and 2. Scheme 1
represents the synthesis of intermediates and Scheme 2 of
the final compounds.
a
Assays were performed in replicate (n ≥ 2).
In vivo chick chorioallantoic membrane (CAM) assay. The
CAMs are easy to access outside the embryo and represent a
reliable approach to study the anti-angiogenic effects of
compounds in vivo.77 Compound 14c was chosen to evaluate
its inhibitory effects on growing CAMs to confirm the in vivo
anti-angiogenic activity. After 12 days of implanting sponges
loaded with the test compound into the CAMs, the average
number of blood vessels was determined. It was observed
that untreated CAMs were surrounded by allantoic vessels
with newly formed capillaries (Fig. 8A), whereas 14c-treated
(Fig. 8B) CAMs demonstrated significant reduction in the
number of branching of capillaries, further supporting its
anti-angiogenic effects.
Materials and methods
Chemistry
All the reagents, solvents, chemicals etc. were procured from
sources, viz. Sigma Aldrich, S.D. Fine Chem. Ltd., and
Himedia, and utilized without any further purification. The
reactions were monitored at each step by thin layer
chromatography (TLC) using Merck precoated silica gel 60
F-254 plates under short wave UV light (254 nm) to identify
the UV absorbing spots for completion of the reaction and
also to trace any impurities. All intermediates were purified
by a recrystallization method using suitable solvents such as
chloroform, methanol etc. All the final molecules were
purified by a column chromatography technique on silica gel
60 (60 to 120 mesh) using a suitable combination of different
solvents. The melting points of all the intermediates were
obtained by using a VEEGO model VMP-DS melting point
Procedure for synthesis of chalcone intermediates
Different chalcones were synthesized using
a previously
reported procedure82,83 with some minor modifications. In
short, to ethanolic NaOH solution (10%, 20 mL), different
acetophenones (0.04 mol-acetophenone, 4-fluoroacetophenone,
and 2,4-difluoroacetophenone) were added followed by
aldehydes (0.04 mol 2a–2g, 2j, and 2k) in an ice bath and
stirred at room temperature (RT) for 5–6 h. The solid
precipitate was filtered and washed with cold water. It was then
Fig. 8 Representative images of untreated (A) and 14c-treated (B) CAMs. (C) Graphical representation of CAM assay. Error bars represent SEM, n =
4, ***p ≤ 0.0001 (STS compared to untreated; calculated with unpaired Student's t-test).
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recrystallized with ethanol to obtain the appropriate chalcones
(3a, 3b, 3d–3g, 3j, 3k, 6a–6g, 6j, 9c, 9e, and 9g) (Scheme 1).
DE3 pLysS cells using a pET14b vector containing codon-
optimized human HDAC8. The expression of recombinant
cHDAC4 was performed according to another recently
published procedure.87 Recombinant cHDAC4 was expressed
in E. coli (BL21) DE3 pLysS using a pET14b vector (Novagen,
EMD Millipore) containing the codon-optimized catalytic
domain of human HDAC4, fused to an N-terminal His6-
SUMO tag and a C-terminal SII tag and auto-induction
medium. Enzyme activity assays were performed in a two-
step procedure as described in detail previously.87 The
fluorogenic activity assay relies on the transformation of Boc-
Lys(trifluoroacetyl)-AMC (Bachem) as substrate for HDAC4, 7
and 8 and Boc-Lys(acetyl)-AMC as substrate for HDAC1, 2, 3
and 6. Afterwards, the deacetylated substrates are converted
into a fluorescent product by trypsin.
Procedure for synthesis of chloroacetylated pyrazoline
intermediates
Various pyrazole derivatives were synthesized and previously
reported by us.82,83 With minor modifications in the previous
procedure, we synthesized pyrazoline derivatives from their
respective chalcones. To a solution of chalcones (3a, 3b,
3d–3g, 3j, 3k, 6a–6g, 6j, 9c, 9e, and 9g) (0.02 mol) in
chloroform, hydrazine hydrate (0.04 mol) was added and
refluxed for 12 h. In this reaction mixture K2CO3 (0.05 mol)
was added and stirred for 15 min followed by chloroacetyl
chloride (0.03 mol) with stirring under an ice bath and
stirred at RT. After 12 h the reaction was stopped, and the
chloroform layer was washed with water to remove excess
K2CO3 and evaporated to obtain the solid. The crude was
purified by extracting with diethyl ether to obtain the
appropriate chloroacetylated pyrazoline intermediates (4a,
4b, 4d–4g, 4j, 4k, 7a–7g, 7j, 10c, 10e, and 10g) (Scheme 1).
Thermal shift assay of HDAC4
HDAC4 was tested in the absence (DMSO control) and in the
presence of the compounds. For the thermal shift assay, 3
μM HDAC4 was mixed with 100 μM of compound (dissolved
in DMSO) and incubated for 15 min at 30 °C. Ten aliquots of
this mix were transferred into PCR strips (25 μl per well) and
further incubated for 60 min at 30 °C in a PCR cycler (T
Gradient, Biometra). Each indicated temperature was held for
10 min followed by a temperature increase of 2 °C. After 9.5
min, a sample from the respective well was stored on ice.
After the sample collection for all indicated temperatures, the
samples were centrifuged at 4 °C and 18 000g for 15 min.
After centrifugation the supernatant was treated with
Laemmli buffer and denatured via heat followed by SDS-
PAGE. By using ImageJ program, the relative band intensity
at each indicated temperature was calculated, and the
GraphPad Prism program was used for plotting intensities
against respective temperatures and fitting the data to a
logistic function.88 The melting point of the protein is the
x-value of the point of infliction, which is the IC50 value of
the logistic function.
Procedure for synthesis of 5-(4-hydroxybenzylidene)-2,4-
thiazolidenedione (12)
Intermediate 12 was synthesized using a previously reported
procedure,84,85 in which 2,4-thiazolidinedione (4.68 g, 0.04
mmol) and 4-hydroxy benzaldehyde (4.88 g, 0.04 mmol) were
dispersed in toluene followed by a catalytic amount of
piperidinium benzoate and this reaction mixture was
refluxed in a Dean Stark apparatus for 4 h. The reaction
mixture was cooled to RT and the solid precipitated out was
collected by vacuum filtration to obtain 12 (Scheme 1). Yellow
solid. Yield 11 g (89%). M. P. 300–302 °C.
Procedure for synthesis of final compounds
The target compounds were synthesized using a similar
synthetic
route
mentioned
previously.66,85
5-(4-
Hydroxybenzylidene)-2,4-thiazolidenedione (12) (1.0 mol)
and potassium carbonate (K2CO3) (1.5 mol) were added to
dimethyl formamide (DMF) (10 mL) and stirred for 5 min at
RT. To this solution chloroacetylated pyrazolines (4a, 4b,
4d–4g, 4j, 4k, 7a–7g, 7j, 10c, 10e, and 10g) (1.5 mol) were
Antiproliferative assay on HUVECs
HUVECs were procured from Vinod Nursing Home, Bhopal,
India; umbilical cord cells were isolated by collagenase
treatment of the umbilical cord and HUVECs were further
cultured and maintained for all further experiments. [(3-(4,5-
added and stirred for 24
h at RT. The reaction was
quenched by adding water (50 mL); the precipitated solid
was collected by filtration and washed with water. The crude
was purified by column chromatography with hexane : ethyl
acetate (1 : 1 to 1.5 : 0.5) solvent to obtain the final products
(13a, 13b, 13d–13g, 13j, 13k, 14a–14g, 14j, 15c, 15e, and
15g) (Scheme 2).
Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide)]
or
MTT, a pale-yellow substrate, is known to be cleaved by living
cells to form a dark blue formazan product. The process
involves active mitochondria, and freshly dead cells do not
cleave a significant amount of MTT. Hence, the amount of
MTT cleaved is in direct proportion with the number of
viable cells; this is quantified by colorimetric methods. Test
compounds and positive control staurosporine (STS) were
solubilized in DMSO and diluted with complete medium to
obtain 5 different ranges of test concentrations (10, 1, 0.1,
0.01, and 0.001 μM). DMSO concentration was maintained at
<0.1% in all the samples. HUVECs maintained in suitable
The ESI† contains all the characterization data.
HDAC enzyme inhibition assay
Recombinant HDAC1, 2, 3, 6 and 7 were purchased from BPS
Bioscience. Recombinant HDAC8 was produced as described
recently.86 In short, HDAC8 was produced in E. coli (BL21)
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environments were seeded in 96-well plates and treated with
different concentrations of all test compounds and incubated
at 37 °C in 5% CO2 for 96 h. MTT reagents were added to the
well plates and kept for incubation for 4 h. The formazan
products (dark blue) formed by the living cells were dissolved
in DMSO under a safety cabinet and read at 550 nm. The %
inhibitions were calculated and IC50 values for test
compounds were determined by plotting the different
concentrations used.
cells were seeded in 96-well flat-bottom micro plates and
maintained at 37 °C in 95% humidity and 5% CO2 overnight.
Different concentrations (100, 75, 50, 25, 10, 2.5 μm ml−1) of
test compound 14c and positive controls (paclitaxel and
cisplatin) were treated. The cells were incubated for 48 h. The
wells were washed twice with PBS, 20 μL of MTT staining
solution was added to each well and the plate was incubated
at 37 °C. After 4 h, 100 μL of DMSO was added to each well
to dissolve the formazan crystals, and absorbance was
recorded at 570 nm using a microplate reader. The IC50 of
compounds was calculated by using Graph Pad Prism Version
5.1.
VEGFR-2 inhibition assay
HUVECs were seeded at 4000–5000 cells per well by using
DMEM and 10% FCS for 12 h. Post-incubation, the cells were
incubated in serum for 24 h. To the test compound and STS
(10 μM), serum was added and incubated for 30 min at 37
°C. The medium was removed, and cells were fixed with 4%
formaldehyde in PBS. The cells were washed thrice with PBS.
The cells were incubated with anti-phospho VEGF Tyr 1175
antibody (1 : 1000) for 1 h. The cells were washed thrice with
PBS T and incubated with secondary antibody labelled with
HRP (1 : 5000) for 1 h. The cells were washed and incubated
with TMB. The reaction was immobilized with 2N H2SO4 and
Formula:
Surviving cells (%) = Mean OD of test compound/Mean OD of
Negative control × 100
Molecular docking
Preparation, visualization of structural data and molecular
docking were performed using MOE 2019 software (Chemical
Computing Group ULC, Canada). The crystal structures of
VEGFR-2 (PDB-ID: 1YWN) and HDAC4 (PDB-ID: 4CBY) were
obtained from the RCSB Protein Data Bank. The structure
files were loaded into the program and subjected to structure
preparation including 3D protonation for subsequent
docking. The partial charges of all protein and ligand atoms
were calculated using the implemented Amber14 force field.
Molecular docking was performed choosing the triangle
matcher for placement of the ligand in the binding site and
ranked with the London dG scoring function. The best 50
poses were passed to the refinement and energy
minimization in the pocket using the induced fit method
and then rescored using the GBVI/WSA dG scoring function.
The protein–ligand complexes were subsequently energy
minimized within a radius of 10 Å around the ligand using
the Amber14 force field.
read at 450 nM. The
% inhibition was calculated by
normalizing with the control.
Migration assay
HUVECs were grown to 90% confluency in 6-well plates in
DMEM and 10% FCS. A scratch was made with a sterile
pipette tip to create a wound. Test compound 14c and
positive control STS 10 μM were added immediately and the
cells were imaged. The cells were splashed with 1 mL of
DMEM and then with 2 mL of DMEM. They were further
incubated at 37 °C in a 5% CO2 atmosphere. Images were
acquired again after 8 h. Cell migration was determined by
wound healing (%), which was the distance the cells
migrated starting from the original wound margin. Wounds
were measured using ImageJ software and % wound closure
was calculated and recorded.
CAM assay
Test sample preparation. Test sample 14c was submitted
in airtight glass vials and stored at 4 °C in a light-controlled
environment. A 10 or 1 μg μl−1 solution of test samples was
prepared in PBS and sterilized by passing through a syringe
filter (0.22 μm). hVEGF (SIGMA) 50 ng μl−1 was prepared in
sterile PBS.
Grafting. Gelatin sponges (Abogel) were cut in
approximately 2 mm3 pieces and loaded with 2 μl of a 1 : 1
mixture of test substance solution and VEGF solution. The
graft was placed on the CAM.
Capillary tube formation assay
In order to perform capillary-like tube formation assay,
HUVECs were trypsinized at 80% confluency and seeded in a
6-well plate at 50 000 cells and DMEM + 10% FCS per well
coated with collagen gel. Test compound 14c and STS in 10
μM concentrations were added at 3 h post seeding and PBS
with 0.01% DMSO was used as a control. The plates were
incubated at 37 °C in 5% CO2 for 48 h. The intersections of
the tubes were measured in a given field and recorded.
Eggs. Fertile hen eggs were procured from a hatchery and
were cleaned and decontaminated using alcohol. 1 ml of
albumin was removed using a syringe and incubated for 8
days. Grafts were placed on developing CAMs and further
incubated to day 12. On day 12, CAMs were fixed with
formaldehyde and dissected.
MTT cytotoxicity assay
In vitro antiproliferative assays were conducted using a
procedure previously described.66,89–91 All the cell lines (MCF-
7, K562, A549, and HT-29) were obtained from National
Centre for Cell Sciences (NCCS), Pune. To summarize, the
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Imaging. Fixed CAMs were observed and scored under
Docking results showed essential binding interactions such
as F1045 and D1044 on VEGFR-2, and H842 and F871 on
HDAC4.
constant illumination and magnification under
stereomicroscope by two independent experts.
a
Statistical analysis. Data were analyzed on MS Excel 2007.
The results obtained were in agreement with our
hypothesis that diaryl-pyrazoline thiazolidinedione analog
14c holds both anti-angiogenic and HDAC inhibitory activity.
To conclude, this study unfolds that 14c has the potential to
simultaneously target both VEGFR-2 and HDAC4. Based on
these findings, we put forward that 14c could be used as a
lead for the discovery of more optimized dual inhibitors.
Conclusions
In the anticancer drug discovery field, single-targeting agents
have failed to show the desired pharmacological effects due
to several factors such as intrinsic and acquired resistance.
Detailed cellular reports suggest that cancer cells obey
Darwin's law of evolution and follow alternative pathways for Conflicts of interest
their survival. Thus, the new strategy to combat drug
There are no conflicts of interest to declare.
resistance would be to develop multi-targeting agents
endowed with anti-tumor efficacy. Angiogenesis is the
fundamental process significant for overall survival of solid
tumors; therefore, the search for anti-angiogenic agents has
become the primary line of investigation in this field. The
epigenetic modulator histone deacetylase has been known to
contribute significantly in the progression and metastasis of
different cancers. Thus, selecting VEGFR-2 and HDAC would
be a rational multi-target approach for the drug discovery of
novel chemical weapons to combat cancer.
Herein, we have rationally designed, synthesized, purified,
and structurally characterized diaryl-pyrazoline TZD
derivatives (nineteen compounds). All the synthetic molecules
were primarily screened at 50 μM on two different HDAC
isoforms (HDAC4 and HDAC8), which revealed their
selectivity towards class II HDAC isoform, i.e., HDAC4, as
Acknowledgements
This research work was supported by an “Indo-ASEAN
Collaborative Research Project Grant” from the ASEAN-India
S&T Development Fund (AISTDF), Department of Science and
Technology (DST), Government of India, Project Reference
Number IMRC/AISTDF/CRD/2018/000001 (to CSR); the
National Medical Research Council of Singapore (to APK);
and the National Medical Research Council of Singapore and
the Singapore Ministry of Education under its Research
Centres of Excellence Initiative to the Cancer Science Institute
of Singapore, National University of Singapore (to APK). We
also acknowledge funding through the LOEWE priority
program TRABITA, State of Hesse, Germany (to FJMA).
compared to class
I HDAC8. All compounds displayed
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the mechanism of binding mode of 14c at the VEGFR-2 (PDB
ID: 1YWN) and HDAC4 (PDB-ID: 4CBY) receptor sites.
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