Discovery of 5-naphthylidene-2,4-thiazolidinedione derivatives as selective HDAC8 inhibitors and evaluation of their cytotoxic effects in leukemic cell lines

Article abstract of DOI:10.1016/j.bioorg.2019.103522

Histone deacetylases (HDACs) are being explored as a therapeutic target for interventions in different types of cancer. HDAC8 is a class I HDAC that is implicated as a therapeutic target in various indication areas, including different types of cancer and particularly childhood neuroblastoma. Most previously described HDAC8-selective inhibitors contain a hydroxamate function as zinc binding group (ZBG) to confer potency. However, hydroxamate class HDAC inhibitors have raised increasing concerns about their mutagenic character. Therefore, non-hydroxamate based inhibitors could prove to be safer than hydroxamates. In the present work, a series of novel 5-naphthylidene-2,4-thiazolidinedione was designed and evaluated as potential antiproliferative agents targeting selectively HDAC8 enzyme. Eleven novel derivatives were synthesized, purified and characterized by spectroscopic techniques. Compounds 3k and 3h was found to be most potent selective inhibitors of HDAC8 with IC₅₀ values of 2.7 μM and 6.3 μM respectively. 3a to 3i was found to be most cytotoxic in leukemic cell lines. 3a and 3 h both were found to induce apoptosis and cause cell cycle arrest in G2/M phase.

Full text of DOI:10.1016/j.bioorg.2019.103522

Journal Pre-proofs
Discovery of 5-Naphthylidene-2,4-thiazolidinedione derivatives as selective
HDAC8 inhibitors and evaluation of their cytotoxic effects in leukemic cell
lines
Kalpana Tilekar, Neha Upadhyay, Niklas Jänsch, Markus Schweipert, Piotr
Mrowka, F.J. Meyer-Almes, C.S. Ramaa
PII:
S0045-2068(19)31633-5
DOI:
https://doi.org/10.1016/j.bioorg.2019.103522
YBIOO 103522
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
2 October 2019
14 December 2019
17 December 2019
Please cite this article as: K. Tilekar, N. Upadhyay, N. Jänsch, M. Schweipert, P. Mrowka, F.J. Meyer-Almes,
C.S. Ramaa, Discovery of 5-Naphthylidene-2,4-thiazolidinedione derivatives as selective HDAC8 inhibitors and
evaluation of their cytotoxic effects in leukemic cell lines, Bioorganic Chemistry (2019), doi: https://doi.org/
1
0.1016/j.bioorg.2019.103522
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover
page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version
will undergo additional copyediting, typesetting and review before it is published in its final form, but we are
providing this version to give early visibility of the article. Please note that, during the production process, errors
may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
©
2019 Published by Elsevier Inc.

Discovery of 5-Naphthylidene-2,4-thiazolidinedione derivatives as selective HDAC8
inhibitors and evaluation of their cytotoxic effects in leukemic cell lines.
a
a
c
c
b
Kalpana Tilekar , Neha Upadhyay , Niklas Jänsch , Markus Schweipert , Piotr Mrowka , F. J.
c*
a*
Meyer-Almes , C. S. Ramaa ,
a
Department of Pharmaceutical Chemistry, Bharati Vidyapeeth’s college of Pharmacy, Navi
b
Mumbai, Maharashtra, India; Department of Biophysics and Human Physiology, Medical
University of Warsaw, Chalubinskiego, Warsaw, Poland; Department of Chemical Engineering
c
and Biotechnology, University of Applied Sciences Darmstadt, Germany.
*
1
Corresponding authors.
. C S Ramaa
E-mail address: sinharamaa@yahoo.in
Tel.: +91 22 27572131/1122; Fax: +91 22 27574515.
2
. Franz-Josef Meyer-Almes
Email : franz-josef.meyer-almes@h-da.de
Phone: +49 6151168406; Fax : +49 6151168404
Abstract
Histone deacetylases (HDACs) are being explored as a therapeutic target for interventions in
different types of cancer. HDAC8 is a class I HDAC that is implicated as a therapeutic target in
various indication areas, including different types of cancer and particularly childhood
neuroblastoma. Most previously described HDAC8-selective inhibitors contain a hydroxamate
function as zinc binding group (ZBG) to confer potency. However, hydroxamate class HDAC
inhibitors have raised increasing concerns about their mutagenic character. Therefore, non-
hydroxamate based inhibitors could prove to be safer than hydroxamates. In the present work, a
series of novel 5-naphthylidene-2,4-thiazolidinedione was designed and evaluated as potential
antiproliferative agents targeting selectively HDAC8 enzyme. Eleven novel derivatives were
synthesized, purified and characterized by spectroscopic techniques. Compounds 3k and 3h was
found to be most potent selective inhibitors of HDAC8 with IC50 values of 2.7 μM and 6.3 μM
respectively. 3a to 3i was found to be most cytotoxic in leukemic cell lines. 3a and 3h both were
found to induce apoptosis and cause cell cycle arrest in G2/M phase.
Keywords
HDAC8; Antiproliferative; Thiazolidinedione (TZD); Naphthalene; Leukemia; Cytotoxicity.
1
. Introduction
Epigenetic abnormalities are hallmarks of carcinogenesis and cancer progression [1]. Histone
deacetylases (HDACs) are major regulators for chromatin remodeling and epigenetics via

deacetylation of acetylated lysine residues of histone and non-histone proteins. Therefore, HDACs
have been considered to be relevant targets for therapeutic intervention in different types of cancer.
Meanwhile, HDACs have become established targets, predominantly for cutaneous T-cell
lymphoma and multiple myeloma [2, 3]. At this time five HDAC inhibitors have been approved
world wide (vorinostat, romidepsin, panobinostat, belinostat, and chidamide), which are more or
less nonselective.
HDACs belong to the major enzyme family of lysine deacetylases, which are divided into 4
classes: Classes I, II, subdivided into IIa and IIb, and IV contain a catalytic zinc ion within the
active site and are referred to as HDACs in the narrower sense. Class III deacetylases are named
sirtuins and their activity depends on NAD+. In human cells 11 HDACs and 7 sirtuins are
expressed.
Class I member HDAC8 has been considered as promising target for childhood neuroblastoma, T-
cell lymphoma [4, 5] and connected to several other types of cancer [6, 7]. The structure activity
relationship analysis of HDAC8 inhibitors containing the widely used hydroxamate group as ZBG
revealed a fish-like structural arrangement for HDAC inhibitors with hydrophobic aromatic cap
function as head to recognize surface regions and a tail comprising the ZBG[8]. Cap group and
ZBG are connected by a mostly hydrophobic linker. The characteristic malleability of the binding
pocket of HDAC8 enabled the development of isoenzyme selective hydroxamate inhibitors
containing hydroxamate group as zinc binding group (ZBG) such as PCI-34051 [5].
In order to analyze the molecular determinants for selective inhibition of HDAC8 diverse classes
of HDAC8 inhibitors with different ZBGs were analyzed by using structure activity relationships
and Bayesian classification approaches [9]. Benzamides show selectivity for class I HDACs 1, 2
and 3 but are dramatically less active on HDAC8 that is also a member of class I, whereas
electrophilic ketones have been developed which inhibit preferably class IIa HDACs and HDAC8
demonstrating that the binding pocket of HDAC8 shares features of class II as well as class I
HDACs. These analyses revealed that hydroxamates or carboxylates would be the most beneficial
ZBGs. Moreover, so called linkerless compounds with bulky head groups also show selectivity for
HDAC8. Many analogs with cyclic linkers attached to hydroxamate group found to exhibit
HDAC8 selectivity (Fig. 1).
However, hydroxamate groups are inherently unselective and could potentially interfere with other
enzymes containing divalent cations or metal homeostasis. Moreover, some suspicion about the
potentially mutagenic character of compounds with hydroxamate groups has been raised, which is
particularly critical for long term treatments [10]. Therefore, HDAC inhibitors without
hydroxamate as ZBG promise to be more beneficial in terms of selectivity and toxicity. A wide
number of non-hydroxamate structures with carboxylates, electrophilic ketones, benzamides, and
cyclic peptides as zinc binding groups (ZBGs) have been reported earlier and shown to inhibit the
activity of different subtypes of HDACs [11-14]. But only a few non-hydroxamate compounds
have been described as isozyme selective inhibitor of HDAC8, such as amino acid derivatives that
demonstrate isoform selectivity via access to the internal acetate release channel of the
enzyme[15], azetidinone (β lactams) with N-thiomethyl group [16], metabolites of organoselenium
compounds such as methylselenocysteine (MSC) and selenomethionine(SM) [17], a dihydro-
imidazole-thiones [18] or most recently pyrimido [1,3]benzothiazin-derivatices [19-20].

In the light of the drawbacks of the hydroxamate ZBG regarding potential mutagenicity, poor
pharmacokinetics and bioavailability, a broad variety of alternative ZBGs were explored and the
structure-activity relationship of corresponding classes of inhibitors analyzed [21]. However
without any doubts, there is still urgent need for new pharmacophores that do not contain
hydroxamate groups and can be developed to selective and potent HDAC8 inhibitors.
2
. Results and discussion
2
.1 Designing of the molecules.
We designed our novel series of naphthylidene TZDs based on proposed structural backbone of
HDAC8 inhibitors (Fig. 2), with the following considerations:
LINKER- Attempts towards the discovery of hydroxamic acid derivatives with HDAC8
selectivity, that fit into the unique sub-pocket of HDAC8, have been found to contain cyclic linkers
such as indole-1,6 [23-24], benzo-triazole- 2 [25-27], benzyl- 3,4 [28], tetrahydro-isoquinoline- 5
[
29-31] (Fig. 1). It has been proven that naphthalene hydroxamate analogs, 6 possess higher
HDAC-8 selectivity over HDAC1 and HDAC6 [32-33] (Fig. 1), which gave us impetus to develop
HDAC inhibitors with cyclic linker, naphthalene.
SPACER- We introduced -CH=CH- as a spacer between linker and ZBG, as it was hypothesized
that selective inhibition of HDAC8 may be implicated by a beneficial balance between the
chemical structure and conformational flexibility of those inhibitors. Compound 7 (Fig. 2) and
similar molecules with -CH=CH-, unsaturated spacer have been reported to show promising
HDAC8 inhibitory activities and some of them are currently under clinical investigations [34].
Moreover, naphthalene hydroxamate analogs with unsaturated linker spacer showed higher
HDAC-8 selectivity over HDAC-1 and -6 [32].
CAP- Our molecules contain phenyl acetamide in cap portion, as in SAHA and α-cetomide.
HDAC8 inhibitors with phenyl acetamide group in cap, have also been reported, however
selectivity has not been related to this particular functionality [16]
ZBG- In search of non-hydroxamate HDAC inhibitors, we have earlier reported 2,4-
thiazolidenedione [35] as ZBG, compound 9, (Fig. 2), which we have retained in this series of
compounds.
Further structural analyses revealed that HDAC8-selective inhibitors show an L-shaped structure,
to enable interactions with residues of an HDAC8-specific side pocket [36]. Hou et al [37] virtually
screened some HDAC8 inhibitors and validated ZBG-based pharmacophore models (Fig. 2).
Finally, we arranged all the fractions with the naphthalene linker being ortho substituted with the
purpose of deriving an L-shaped structure, conforming to Hou’s pharmacophoric model (Fig. 2).

Fig.1. Reported HDAC8 inhibitors with hydroxamic acid as ZBG.
Fig.2. Designing considerations of novel series targeting HDAC8.

2
.2 Chemistry
The synthetic route of the compounds is outlined in Scheme 1. A new class of 5-Naphthylidene-
,4-thiazolidinedione derivatives, 3a-3k (Scheme 1), were synthesized from the condensation of
2
synthetic intermediates 1a with 2a-2k. 1a was synthesized from 1 and 2 (Scheme 1) by
Knoevenagel condensation as described previously [38], with slight modifications, in which the
product formed was removed intermediately. The product was obtained in good yield of 85% and
1
was yellow crystalline needles. The H NMR spectra of 1a displayed 3 singlets at 12.59, 10.21 and
8
.04 ppm representing the protons of -NH (broad singlet), -OH and benzylidene double bond (Ph-
CH=C) respectively, confirming the formation of 1a. Discussion about the chemistry 2a-2k has
been described in our previously published work [38-39].
In the IR spectra of 3a-3k the band for CO-NH-CO and C=O were seen in the region 3400-3300
-1
-1
1
cm and 1700-1650 cm respectively. In the H NMR spectra, presence of singlet between 4.1-
.5ppm, provides evidence for formation of -CH -O- linkage in final compounds, 3a-3k. The
4
2
singlet at 7.9-8.1 ppm is characteristic of benzylidene proton which confirms that the molecules
are in Z-configuration, as described by Momose et al [40]. Additional spectral characteristics were
1
3
conformed by C NMR and Mass spectroscopy and are presented in experimental section.
2
.3 In vitro HDAC screening
2
.3.1 HDAC enzyme inhibition Assay
To assess selectivity, the synthesized derivatives were screened against a panel of human HDAC
isoenzymes using procedures described in method section. Interestingly 6 out of 11 compounds
displayed potent inhibition of HDAC8 compared to the other HDAC isoenzymes (Table 1, Fig. 3).
The derivatives that exhibited maximum selectivity with IC50 values less than 15 µM were, 4-
nitro(3k), 4-bromo (3i), 3-triflouromethyl (3j), 2-flouro-4-bromo (3h) and 2-bromo-6-methyl (3f).
3
k exhibited most potent HDAC8 inhibition with IC50 value of 2.7μM with 6- to 16-fold selectivity
for HDAC8 over HDAC1, HDAC4 and HDAC6 and even higher selectivity over HDAC3 and
HDAC5. This data is not enough to discuss the structure activity relationships, but some primary
conclusions can be made. The presence of halogen Br (3i, 3h) and nitro group (3k) at para position
may lead to enhanced HDAC8 inhibition. Amongst halogens only F containing compounds (3b,
3
g) were found to be ineffective against all four subtypes of HDACs, but if it is present along with
Br on the same ring (3f), it leads to HDAC8 inhibition. Electron withdrawing groups such as NO
2
was found to have HDAC inhibitory potential as compared to electron donating groups (3c, 3d).
Amongst brominated derivatives, 3h and 3i potency was found to be slightly enhanced if methyl
group is present along with Bromine.
Table 1
SAR table of naphthylidene-2,4-TZD derivatives for selectivity between HDAC1, HDAC4,
HDAC6 and HDAC8.
IC50 / µM

#
R
1
R
2
R
3
R
4
R
5
HDA HDA HDA HDA HDA HDA HDA
C1
C2
C3
C4
C5
C6
C8
3
a
b
c
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
> 50
> 50 > 50 > 50 > 50 > 50 44
> 50 > 50 > 50 > 50 > 50 > 50
> 50 > 50 > 50 > 50 > 50 > 50
> 50 > 50 > 50 > 50 > 50 > 50
3
3
3
F
> 50
> 50
> 50
CH
3
d
OCH
3
3
3
3
3
3
3
3
e
H
Cl
H
H
H
H
H
H
Cl
H
H
H
H
H
CF
H
H
F
> 50
> 50
> 50
> 50
> 50
> 50
23
> 50 > 50 > 50 > 50 > 50 > 50
> 50 > 50 > 50 > 50 > 50 12
> 50 > 50 > 50 > 50 > 50 > 50
> 50 > 50 > 50 > 50 > 50 6.3
> 50 > 50 > 50 > 50 > 50 8.8
> 50 > 50 > 50 > 50 > 50 12
f
Br
F
F
g
h
i
F
H
H
H
H
H
CH
3
Br
Br
H
H
j
H
3
k
H
NO
2
35
> 50 15
38* 10*
> 50 44
2.7
PCI-34051
3.0*
45*
>50* 18*
0.024*
*
Kleinschek, A., et al. (2016). "Potent and Selective Non-hydroxamate Histone Deacetylase 8
Inhibitors." ChemMedChem 11(23): 2598-2606.
1
0
0
.0
.5
.0
3
3
3
a
f
3 i
3 j
3 k
h
0
.0001 0.001
0.01
0.1
1
10
100
In h ib ito r / µ M
Fig. 3. Dose response curves of indicated compounds against HDAC8. The relative enzyme
activity is plotted versus increasing compound concentrations

2
.3.2. Thermal Shift Assay
Several promising lead compounds have failed in advanced clinical trials [41-43] and subsequently
it has been shown that they do not act in predicted or in vitro observed way [44]. Hence target
engagement (TE) i.e. extend to which the drug binds to its target protein of enzyme in situ, should
be studied from the drug development and primary preclinical stage. Thermal shift assay (TSA) is
the means by which TE can be established in vitro using isolated enzyme to ascertain enzyme-
ligand stability with increasing temperature or in cells and tissues to confirm binding. TSA provide
characteristic sigmoidal melting curves for a protein. It has been observed that, when ligand binds
to protein, melting temperature (Tm) of protein shift to a higher temperature producing thermal
shift (∆Tm), and lead to stabilization of the protein (Fig. 4).
To assess the target engagement of synthesised derivatives, we undertook TSA for derivatives 3a,
3
h, 3i and 3k by incubating with recombinant HDAC8 for 1 hour and then exposed to different
0
0
temperatures ranging from 46 C to 62 C. We observed the characteristic sigmoidal curve. Thus,
binding of compounds to HDAC8 leads to strong stabilization of the protein-ligand complex with
thermal shifts (∆Tm) of 7°C and more. We did in vitro thermal shift experiments and did not
analyze the cell lysates of living cells that were treated with the compounds. However, at stage we
can say that the enzyme-ligand complexes are highly stable and, are more likely to bind a desired
target, HDAC8 when in whole cells or tissue, making them suitable agents for preclinical
developments.
Fig. 4. Thermal stabilization of 2.5 µM HDAC8 upon binding of 100 µM of 3a (square), 3h (up-
triangle), 3i (down-triangle) and 3k (diamond). Dots denote the control of HDAC8 in the absence
of compounds but with matching DMSO concentration. The data points represent means and
standard deviations of 3 independent experiments. Compound binding to HDAC8 leads to strong
stabilization of the protein-ligand complex with thermal shifts of 7°C and more.
2
.4 In vitro anticancer Screening
2
.4.1 MTT assay and cell viability
The effects of newly synthesized TZD derivatives 3a-3k, on the viability of normal Human WBCs
and cancerous K562 and CEM cells, were assessed by the MTT assay, which has been described
as one of the most reliable indicators of metabolic activity of cells and widely used to ascertain

cell viability [45-46]. It relies on the ability of dehydrogenase enzymes to reduce MTT dye to its
water insoluble purple coloured formazan salt in the mitochondria of cells. formazan crystals can
be finally solubilized and quantified using a spectrophotometer. Since reduction of MTT can only
occur in metabolically active cells the level of activity is a measure of the viability of the cells. In
this study, Paclitaxel was used as references.
Table 2
IC50 values for compounds 3a-3k on K562 and CEM cell line viability
a
Compound
IC50 in µM
CEM
K562
0.42
0.46
0.73
0.52
0.81
0.97
0.96
2.05
1.94
1.62
ndb
3
3
3
3
3
3
3
3
3
3
3
a
b
c
d
e
f
13.94
29.40
38.60
32.79
ndb
29.29
ndb
g
h
i
15.71
21.22
b
j
nd
b
k
nd
Std. Paclitaxel 0.29
Data shown is the means of 3 independent experiments.
Not determined
15.5
a
b
We determined the IC50 values corresponding to the concentrations of drug attaining 50%
inhibition of cell viability. Paclitaxel was used as standard reference compound whereas untreated
cells were used as negative control and the results are presented in Table 2. Compounds 3a, 3b,
3
c, 3d and 3e were found to exhibit anti-proliferative effects at sub-micromolar concentrations on
K562 cell line. CEM cell lines are less sensitive, though the observed activity of the compounds
is lesser as compared to K562. IC50 values for CEM cells are around 30 µM and compound 3a and
3
h have exhibited IC50 values less or equal to that of standard drug Paclitaxel. This difference in
activity on K562, which is chronic myeloid leukemia cell line and CEM, which is lymphoblastic
leukemia cell line, may be due to subtypes of leukemia.
After considering the results of HDAC inhibition and MTT assay, Compounds 3a and 3h was
chosen for further evaluation as both exhibited excellent HDAC8 inhibiting activity, 3a exerted
high cytotoxicity in both leukemic cell lines with being most cytotoxic compound against K562
cells and compound 3h most cytotoxic compound against CEM cells.

2
.4.2 Apoptosis by Flowcytometry
HDAC8 inhibitors have induced necrosis and apoptosis via various mechanisms in different types
of solid and hematological cancer cells. PCI-34051, a HDAC8 inhibitor have been found to induce
apoptosis in T-cell lymphoma cells by caspase activation [5]. HDAC8 inhibition in gastric
carcinoma cells, have reported to induce apoptosis mediated by Bcl-2-modifying factor (BMF)
and STAT3 [47]. A selective HDAC8 inhibitor induces Dose-dependent selective apoptosis of
CD34+ leukemic stem cells and progenitor cells [7]. The up-regulation of histone deacetylase 8
promotes proliferation and inhibits apoptosis in hepatocellular carcinoma [48].
To examine whether the cytotoxicity induced by 3a and 3h was associated with apoptosis, we used
annexin V-FITC/ propidium iodide (PI) double staining of K562 and CEM cells treated with
indicated compounds and determined rate of apoptosis by flow cytometry. K562 cells are very
sensitive to the chosen compounds and treatment of the cells with 3a and 3h at IC50 concentration
was leading to rapid cell death. In that circumstances getting accurate results were difficult, thus
we choose CEM cell line for apoptosis experiments in order to obtain reproducible results. CEM
cells exposed to 3a and 3h at their IC50 concentrations caused increase of apoptotic cells apoptotic
(fig. 5). For 3a the percentage of apoptotic cells in late and early apoptosis was 41.88 % and 2.80%
respectively. In case of 3h similar results were observed: 42.78% of cells were in the late apoptosis
and 1.63%. in early phase of apoptosis. was 1.63%. This result indicates that compound 3a and 3h
both have the ability to induce apoptotic death in CEM cell line. As apoptosis is a type of cellular
death that seems to be safer to patients because of non-inflammatory character and HDAC
inhibitors considered for anticancer use all induce apoptosis we conclude studied compounds can
proceed to further testing as potential anti-leukemic drug candidate [49] However, further
molecular level studies are indicted to unearth the exact mechanism of induction of apoptosis by
these compounds.

Fig. 5 . Annexin V-FITC/ PI flow cytometry analysis. CEM cells were treated with 3a and 3h at
IC50 concentration. After 24 hour cells were stained with annexin V-FITC/PI to distinguish live
cells from apoptotic and necrotic cells Cells considered alive are both Propidium Iodide (PI) &
AnnexinV negative, cells in early apoptotic stage are Annexin V positive and PI negative and cells
in late apoptotic phase are both PI & Annexin V positive. Dead/necrotic cells are PI positive &
Annexin V negative. (A) Cytograms of untreated control CEM cells. (B) Cytograms of 3a treated
CEM cells. (C) Cytograms of 3h treated CEM cells. (D) Graphical representation of apoptotic
events of untreated, 3a and 3h treated CEM cells.

2
.4.3 Cell cycle analysis
Most of pan HDAC inhibitors produce G0/G1 cell cycle arrest and apoptosis through the increased
accumulation of acetylated histones, resulting in decreased availability of nuclear DNA to bind to
transcription factors and thus decreased transcription that leads to decreases intracellular protein
levels the trigger for cell cycle arrest. However, G2/M have been also shown as an outcome of
HDAC inhibition. PAC-320, a polyoxometalates compound, is a broad-spectrum HDAC inhibitor
have shown potent prostate cancer inhibitory activity mediated by G2/M cell cycle arrest and
apoptosis [50]. Not much literature is available about exact behavior of HDAC8 inhibitors in
various phases of cell growth and arrest, knockdown of HDAC8 in gastric cancer cells, have been
reported to promote G0/G1 arrest and apoptosis[47], whereas knockdown of HDAC8 in lung
cancer cell line, A549, Found to promote growth arrest in mitotic G2-M phase[51].Thus, cell
growth arrest has been observed in HADC8 knockdown cells as well as HDAC8 inhibitor treated
cells, but in different phases of growth.
We investigated the effects of 3a and 3h on cell cycle using flow cytometric analysis, to find out
if it’s antiproliferative effects can be attributed to arrest of cells in particular phase (Fig. 6).
Treatment of CEM cells with 3a and 3h at their IC50 concentrations, resulted in marked increase
in the number of cells in G2/M phase and a concomitant decrease in the number of cells in G0/G1
phase as compared to control. For 3a, the percentage of cells in G2-M phase was found to increase
from 3.58% to 35.4%, while the percentage of cells in G0-G1 phase was found to decrease from
7
3.4% to 37.3% and the percentage of cells in S phase found to be change very slightly as
compared to control. For 3h the percentage of cells in G2-M phase was found to increase from
.58% to 27.0%, while the percentage of cells in G0-G1 phase was found to decrease from 73.4%
3
to 50.0% and the percentage of cells in S phase found to be remain almost similar as compared to
control. Thus, our results may suggest that the reductions in cell growth may be due to direct or
indirect interference of 3a and 3h with DNA replication resulting block at G2/M cell cycle check
point.

Fig. 6. Effect of 3a and 3h on cell cycle. Cells were treated with compounds for 24 hour, stained
with PI, and analysed by flow cytometry. (A) Graphical representation of CEM cells population
of in G0/G1, S and G2/M phases of the cell cycle shown as % of all cells. (B) Representative
histogram of untreated control cells(C) Representative histogram of 3a treated cells. (D)
Representative histogram of 3h showing treated cells.
2
.4.4 Assessment of cell viability on non- transformed cells
In leukemia treatment we are looking for medicines that will be lethal to cancer cells and at the
same time relatively safe for patient and his normal cells. Since 3a was most cytotoxic in both
K562 and CEM cells, we compared cytotoxicity of the compound against leukemic cells and
normal WBCs. The cells were exposed to 2.5 10, 25,75 and 100 μM concentrations of 3a and 3h
for 48 h. to normal WBCs and IC50 values, were established. For 3a the IC50 value was found to
be 67.24 μM, which is 5 times that of IC50 for CEM cells (IC50 = 13.39 μM) and for 3h it was 152.2
µm, which is 15 times that of IC50 for CEM lines, (Fig. 7), which indicates that 3a and 3h could be
safer to normal cells.

WBC CEM
160
140
120
100
80
60
40
20
0
3a
3h
Compound code
Fig. 7. 3a and 3h inhibits cell proliferation in the human Leukemic cell line, CEM, significantly
more than it does in normal WBCs after 48 hours treatment.
2
2
.5 In-silico studies
.5.1 Docking of 3k into HDAC8 (PDB-ID: 3SFF)
HDAC8 is known as an enzyme with extraordinary malleable binding site suitable to accommodate
inhibitors with widely different structures [52-53]. To study the putative molecular determinants
of TZD binding to HDAC8, we performed docking studies using three solved crystal structures of
HDAC8 (PDB-ID’s 1T69, 1VKG and 3SFF), which represent different major conformers of this
enzyme [54]. PDB-ID 1T69 shows a complex between HDAC8 and SAHA with one narrow
binding channel, 1VKG shows the wide-open conformation of HDAC8 and 3SFF shows a HDAC8
conformation with an open cavity connecting the conventional catalytic site with the entry of the
acetate release channel. The far best docking score was obtained with PDB-ID 3SFF (Table 3).
Surprisingly, not the thiazolidinedione group, but the oxygen of the carboxamide group chelates
the zinc cation at the bottom of the catalytic binding pocket. In addition, an oxygen of the nitro
group forms a hydrogen bond to a nitrogen of R37 (Fig. 8). The naphthalene moiety of the TZDs
appears to be rather movable and to enable pi-pi-interactions with neighbouring F208 and F152
residues lining the binding pocket.
Table 3
Docking scores for 3k within different crystal structures and conformations of HDAC8 using MOE
software and AMBER 14 forcefield
PDB-ID
Score
-9.5
3
1
1
SFF
T69
-7.3
VKG
-7.4

Fig. 8. Best docking pose of 3k within the binding pocket of HDAC8 (PDB-ID: 3SFF). A) 3D
view of binding pocket. B) 2D ligand interactions. Magenta dotted lines indicate metal ion contacts
and green arrows hydrogen bonds. The violet or light blue shades denote ligand or receptor
exposure, respectively.
The general binding pose of 3k was confirmed by very similar docking results with analogs, where
the para-nitro group was omitted (3a) or exchanged against methyl (3c), methoxy (3d) or bromine
(3i). The TZDs without nitro-group are no longer able to form a hydrogen bond with R37 resulting
in rather weak docking scores (Table 4). In contrast, the most potent compound 3k shows the best
docking score based on the hypothesized hydrogen bond between the nitro group and R37. Thus,
the docking scores are consistent with the experimental activity of the compounds in terms of IC50
-
values providing further evidence for the correctness of the putative binding pose of the TZDs
within HDAC8.
Table 4
Docking scores and IC50-values for different 3k analogs using MOE software and AMBER 14
forcefield
CPD. P-SUBSTITUENT IC50
SCORE CPD. P-
IC50
SCORE
(
HDAC8) /
SUBSTITUENT (HDAC8) /
µM
µM
2.7
6.3
3
3
K
H
-NO
-BR + CH3 IN
ORTHO
2
-9.5
-8.6
3A
3C
-H
44
-8.5
-8.8
-CH
3
>50
3
I
-BR
8.8
-8.6
3D
-OCH
3
>50
-7.9
2
.5.2 SwissADME predictions
Calculation of ADME profile is essential part of drug development, but determination of ADME
parameters by laboratory experimentations for large number of compounds would be difficult and
very time-consuming task. In these circumstances, simulated computer-models provides realistic
replacements to experiments [55]. The SwissADME web tool is freely available which gives

access to the fast and robust models for appropriate prediction of physicochemical properties,
pharmacokinetic behaviour and drug-likeness. We used SwissADME tool to predict properties of
our synthetic derivatives. All the compounds were observed to follow the Lipinski’s rule of five
c
(
Table 5) with 2 violations of molecular weight. All molecules demonstrated logP in the
acceptable range of 2.00 to 5.00. Topological polar surface area (TPSA), the surface sum over all
polar atoms, primarily oxygen and nitrogen, also including their attached hydrogen atoms, has
been shown to be a very good descriptor characterizing drug absorption, including intestinal
absorption, bioavailability. Literature implies that TPSA < 140 is essential for good absorption.
0
The naphthylidene derivatives exemplified TPSA in the range 109-135 A. The solubility class
predicted indicates that compounds are moderately or poorly soluble, which could be problematic
in formulation stage and can be overcame by solubility enhancement techniques such as salt
formation. Judging the data, the molecules seem to be drug like and may have good passive oral
absorption.
Table 5
SwissADME prediction data.
ESOL Class GIe
d
Lipinski
Bioavailabilit
Code TPSA Log Log
a
Pb
Sc
absorption #violations y Score
3
3
3
3
a
b
c
109.8
109.8
109.8
2.21 -5.08 Moderately
soluble
High
High
High
High
0
0
0
0
0.55
0.55
0.55
0.55
2.59 -5.24 Moderately
soluble
2.42 -5.38 Moderately
soluble
d
119.03 1.89 -5.16 Moderately
soluble
3
3
3
e
f
109.8
109.8
109.8
3.17 -6.28 Poorly soluble High
3.54 -6.32 Poorly soluble Low
0
1
0
0.55
0.55
0.55
g
2.96 -5.4
Moderately
soluble
High
3
3
h
i
109.8
109.8
3.01 -6.3
Poorly soluble High
0
0
0.55
0.55
2.8
-5.99 Moderately
High
soluble
3
3
j
109.8
3.48 -6.54 Poorly soluble Low
1
0
0.55
0.55
k
135.62 2.14 -5.15 Moderately
soluble
Low
a- Topological polar surface area b- Log of partition coefficient (P), c- Log solubility, d-estimated
aqueous solubility in mg/mL, e- Gastrointestinal
3
. Materials and methods
3
.1 Chemistry

Commercial reagents were from either S D Fine, Research Lab or Sigma Aldrich and were
procured from suppliers in India. Thin layer chromatography was performed on Merck pre-coated
Silica Gel 60 F254. Melting points were determined by open capillary method on a VEEGO
melting point apparatus and are uncorrected. Infrared spectra were recorded on Schimadzu FT/IR-
1
13
8
400S by direct sampling technique. H and C NMR spectra were recorded at 400 MHz on a
Bruker instrument using TMS as internal standard and chemical shifts (δ) are reported in ppm.
Mass spectra were recorded using LC-MS Agilent Technologies 1260 Infinity instrument.
3
.1.1 Procedure for synthesis of 5-((2-hydroxynaphthalen-1-yl) methylene) thiazolidine-2,4-dione
(1a)
Compound 1a was synthesized as per the procedure that we have reported earlier [38]. In short
.01moles of 2-hydroxy naphthaldehyde 1 and 0.01 moles of 2,4-thiazolidenedione 2 with catalytic
0
quantity of piperidinium benzoate was refluxed in toluene for 4-5 hours. The reaction mixture was
cooled to room temperature and solid separated was collected by filtration, washed with cold
toluene, dried and recrystallised from hydroalcoholic mixture.
0
1
Yellow crystals; yield 79.5%; m.p, 253 C; H NMR (400 MHz, DMSO d6) - δ 12.59 (s,1H), δ
1
7
0.21 (s,1H), δ 8.04 (s 1H), δ 7.89 (m, 2H), δ 7.79 (d, J = 8.82 Hz, 1H), δ 7.57-7.55 (m, 1H), 7.17-
-1
.19 (m, 2H); IR (neat) 3115.14, 1674.27, 1508, 1340 and 1211 cm ; anal. calc. for C14
H
9
NO S
3
C, 61.98; H, 3.34; N, 5.16; O, 17.69; S, 11.82.
3
.1.2 General procedure for synthesis of 2a – 2k.
We have previously reported preparation, purification and characterization of 2a-2k in our research
papers [38-39]. Briefly, Chloroacetyl chloride (0.1 mol) was added drop wise to a mixture of
appropriate amines (0.05 mol) and anhydrous potassium carbonate (K
2
CO ) (0.075 mol) in
3
dichloromethane or chloroform in an ice-cold condition. The reaction mixture was then stirred at
room temperature. After completion of reaction, solvent was evaporated under reduced pressure,
ice cold water was added to the obtained dry mass and the insoluble product was filtered and dried
and purified by recrystallization with appropriate solvents.
3
.1.3 General procedure for synthesis of 3a-3k by Condensation of 1a with 2a-2k.
The final compounds 3a-3k were synthesized by stirring compound 0.1 mol of (1a) with 0.1 moles
of (2a-2k) for 24 hours, in presence of K CO in dimethyl formamide (DMF). The reaction was
2
3
monitored by TLC. On completion of the reaction, the reaction mixture was poured on crushed
ice. The precipitated crude product was filtered, washed with water and purified by column
chromatography using ethyl acetate and hexane in appropriate ratio.
3
.1.3.1 2-((1-((2,4-dioxothiazolidin-5-ylidene) methyl) naphthalen-2-yl) oxy)-N-phenylacetamide
(3a)

0
1
Yellow powder; yield 35.8%; m.p, 234 C(charred); H NMR (400 MHz, DMSO d6): δ 10.51
(
(
(
1
s,1H), δ 8.68 (s,1H), δ 8.48-8.46 (d, J = 8.0 Hz, 1H), δ 8.04-8.06 (d, J = 8.0 Hz, 1H), 7.98-8.00
d, J = 8.0 Hz, 1H), δ 7.71-7.73 (m, 1H), δ 7.65-7.67 (m, 2H), δ 7.57-7.61 (m, 1H), δ 7.49-7.51
d, J = 8.0 Hz, 1H), δ 7.32-7.36 (t, J = 8.0 Hz, 2H), δ 7.06-7.10 (t, J = 8.0 Hz, 1H), δ 4,15 (s, 2H);
3
C NMR (400MHz, DMSO d6): δ 158.24, 150.38, 113.23, 138.67, 116.28, 129.95, 131.33,
1
23.66, 127.67, 122.08, 131.59, 166.38, 40.06, 128.83, 122.08, 128.66, 127.93, 126.03, 119.18;
-1
IR (neat) 3340.82, 1693.56, 1672.34, 1494.88, 1325.14 and 1213.72 cm ; Theoretical mass:
4
6
+
04.08, LC-MS (m/z, I %): 403.1 [(M-H) , 100]; anal. calc. for C14
.93; O, 15.82; S, 7.93.
H
9
NO S: C, 65.33; H, 3.99; N,
3
3
.1.3.2 2-((1-((2,4-dioxothiazolidin-5-ylidene) methyl) naphthalen-2-yl) oxy)-N-(4-fluorophenyl)
acetamide (3b)
Yellow powder; yield 30.3%; m.p, 259 C (charred); H NMR (400 MHz, DMSO d6): δ 10.48
0
1
(
(
s,1H), δ 8.77 (s,1H), δ 8.53-8.55 (d, J = 8.0 Hz, 1H), δ 8.13-8.15 (d, J = 8.0 Hz, 1H), 8.06-8.07
d, J = 4.0 Hz, 1H), δ 7.74-7.77 (t, J = 6.0 Hz, 1H), δ 7.65-7.60 (m, 4H), δ 7.13-7.16 (t, J = 6.0
1
3
Hz, 2H), δ 4,12 (s, 2H); C NMR (400MHz, DMSO d6): δ 159.49, 158.27, 111.45, 131.68, 125.74,
1
1
26.10, 127.74, 127.99, 128.73, 129.66, 109.04, 150.46, 166.40, 54.93, 122.17, 113.30, 131.46,
-1
30.02; IR (neat) 1689, 1284, 1213, 3335, 1618 and 1099 cm ; Theoretical mass: 422.07, LC-MS
+
(m/z, I %): 421.1 [(M-H) , 100]; anal. calc. for C22
H15FN
2
4
O S: C, 62.55; H, 3.58; F, 4.50; N, 6.63;
O, 15.15; S, 7.59.
3
(
.1.3.3 2-((1-((2,4-dioxothiazolidin-5-ylidene)methyl)naphthalen-2-yl) oxy)-N-(p-tolyl) acetamide
3c)
Yellow powder; yield 25.97%; m.p, 220.2 C (charred); H NMR (400 MHz, DMSO d6): δ 10.38
0
1
(
(
(
s,1H), δ 8.74 (s,1H), δ 8.53-8.51 (d, J = 8.0 Hz, 1H), δ 8.10-8.12 (d, J = 8.0 Hz, 1H), 8.03-8.05
d, J = 4.0 Hz, 1H), δ 7.72-7.76 (m, 1H), δ 7.57-7.64 (m, 2H), δ 7.48-7.50 (m, 3H), δ 7.10-7.12
1
3
m, 1H), δ 4.11 (s, 2H), 2.23 (s, 3H); C NMR (400MHz, DMSO d6): δ 158.29, 150.49, 113.34,
1
1
4
6
31.44, 127.79, 128.05, 126.15, 128.78, 129.18, 130.06, 119.17, 136.15, 166.07, 40.07, 131.70,
-1
22.21, 129.18, 132.61; IR (neat) 1695, 1246, 1213, 3342, 1555 and 1170 cm ; Theoretical mass:
+
18.1, LC-MS (m/z, I %): 416.8[(M-H) , 100]; anal. calc. for C23
.69; O, 15.29; S, 7.66.
H
18
N
2
4
O S: C, 66.01; H, 4.34; N,
3
.1.3.4 2-((1-((2,4-dioxothiazolidin-5-ylidene)methyl)naphthalen-2-yl) oxy)-N-(4-methoxyphenyl)
acetamide (3d)
0
1
Buff Yellow powder; yield 30%; m.p, 231.9 C; H NMR (400 MHz, DMSO d6): δ 10.36 (s,1H),
δ 8.69 (s,1H), δ 8.47-8.49 (d, J = 8.0 Hz, 1H), δ 8.05-8.07 (d, J = 8.0 Hz, 1H), 8.01-7.99 (d, J = 8.0
Hz, 1H), δ 7.70-7.74 (m, 1H), δ 7.51-7.62 (m, 4H), δ 6.89-6,91(m, 2H), δ 4.11 (s, 2H), δ 3.71 (s,
1
3
3
1
1
H); C NMR (400MHz, DMSO d6): δ 157.40, 154.59, 113.06, 130.94, 119.90, 127.85, 127.11,
25.21, 126.86, 125.02, 129.14, 164.98, 54.25, 130.42, 121.26, 115.47, 149.54; IR (neat) 1691,
240, 1213, 3336, 1545 and 1168 cm ; Theoretical mass: 434.09, LC-MS (m/z, I %): 433 [(M-
H) , 100]; anal. calc. for C23
-1
+
H
18
N
2
5
O S: C, 63.58; H, 4.18; N, 6.45; O, 18.41; S, 7.38.

3
.1.3.5 N-(3,4-dichlorophenyl)-2-((1-((2,4-dioxothiazolidin-5-ylidene) methyl) naphthalen-2-yl)
oxy) acetamide (3e)
Yellow powder; yield 30%; m.p, 293.6 C(charred); H NMR (400 MHz, DMSO d6): δ 10.73
0
1
(s,1H), δ 8.75 (s,1H), δ 8.52-8.54 (d, J = 8.0 Hz, 1H), δ 8.13-8.15 (d, J = 8.0 Hz, 1H), 8.05-8.07
(
d, J = 8.0 Hz, 1H), δ 7.96-7.97 (m, 1H), δ 7.73-7.77 (m, 1H), δ 7.63-7.65(m, 1H), δ 7.56-7.61(m,
H), δ 7.48-7.51(m, 1H), δ 4.14 (s, 2H); C NMR (400MHz, DMSO d6): δ 158.31, 150.69,
1
3
2
1
1
13.32, 138.70, 119.24, 130.07, 128.81, 127.83, 125.10, 126.18, 122.23, 130.79, 166.95, 40.07,
-1
31.90, 131.04, 128.09, 120.35, 125.31; IR (neat) 1587, 1319, 1213, 3338, 1531 and 736 cm ;
+
Theoretical mass: 472.01, LC-MS (m/z, I %): 471[(M-H) , 100]; anal. calc. for C22
C, 55.82; H, 2.98; Cl, 14.98; N, 5.92; O, 13.52; S, 6.77.
14
H C
l2
N
2
4
O S:
3
.1.3.6
naphthalen-2-yl)oxy) acetamide (3f)
Yellow powder; yield 31.2%; m.p, 268.5 C(charred); H NMR (400 MHz, DMSO d6): δ 10.15
N-(2-bromo-4,6-difluorophenyl)-2-((1-((2,4-dioxothiazolidin-5-ylidene)methyl)
0
1
(
s,1H), δ 8.73 (s,1H), ), δ 8.53 (s,1H), δ 8.05-8.12 (m, 2H), δ 7.43-7.74 (m, 5H), δ 4.19 (s, 2H);
C NMR (400MHz, DMSO d6): δ 158.40, 150.67, 113.22, 138.20, 119.20, 130.17, 127.81,
1
3
1
1
24.20, 126.18, 122.23, 130.79, 166.95, 41.07, 132.90, 131.14, 128.19, 120.32, 125.33; IR (neat)
-1
587, 1319, 1213, 3338, 1531 and 736 cm ; Theoretical mass: 517.97, LC-MS (m/z, I %): 518.9
+
[
(M+H) , 100]; anal. calc. for C22
H13BrF
2
N
2
4
O SC, 50.88; H, 2.52; Br, 15.39; F, 7.32; N, 5.39; O,
1
2.32; S, 6.17.(13C NMR and IR is not available, data entered is approximate)
3
.1.3.7 N-(2,4-difluorophenyl)-2-((1-((2,4-dioxothiazolidin-5-ylidene) methyl) naphthalen-2-yl)
oxy) acetamide (3g)
Yellow powder; yield 24.69%; m.p, 231.6 C(charred); H NMR (400 MHz, DMSO d6): δ 10.26
s,1H), δ 8.73 (s,1H), δ 8.51-8.53 (m, 1H), δ 8.11-8.13 (d, J = 8.0 Hz, 1H), 8.04-8.06 (d, J = 8.0
Hz, 1H), δ 7.80-7.86 (m, 1H), δ 7.73-7.76 (m, 1H), δ 7.58-7.65(m, 2H), δ 7.56-7.61(m, 2H), δ
0
1
(
1
3
7
.31-7.36(t, J = 10.0 Hz, 1H), δ 7.03-7.07 (m, 1H), δ 4.21 (s, 2H); C NMR (400MHz, DMSO
d6): δ 158.30, 150.54, 104.23, 130.06, 111.30, 127.80, 126.15, 125.64, 116.41, 125.44, 113.33,
1
1
1
28.03, 166.92, 40.07, 111.11, 131.76, 104.24, 131.62, 111.11, 122.18; IR (neat) 1666, 1317,
-1
+
213, 3338, 1593 and 1006 cm ; Theoretical mass: 440.06, LC-MS (m/z, I %): 439 [(M-H) ,
00]; anal. calc. for C22 S: C, 60.00; H, 3.20; F, 8.63; N, 6.36; O, 14.53; S, 7.28.
H
14
F
2
N
2
O
4
3
2
.1.3.8 N-(4-bromo-2-methylphenyl)-2-((1-((2,4-dioxothiazolidin-5-ylidene) methyl) naphthalen-
-yl) oxy) acetamide (3h)
0
1
Deep yellow powder; yield 35%; m.p, 253 C(charred); H NMR (400 MHz, DMSO d6): δ 10.49
(s,1H), δ 8.71 (s,1H), δ 8.50-8.52 (d, J = 8.0 Hz, 1H), δ 8.07-8.10 (d, J = 8.0 Hz, 1H), 8.01-8.03
(
d, J = 8.0 Hz, 1H), δ 7.71-7.74 (t, J = 6.0 Hz, 1H), δ 7.53-7.63 (m, 2H), δ 7.17-7.25 (m, 2H), δ
.65-6.67 (d, J = 6.0 Hz, 1H), δ 4.14 (s, 2H), δ 2.52 (s, 2H); C NMR (400MHz, DMSO d6): δ
13
6
1
1
58.27, 150.46, 113.30, 139.82, 128.73, 127.74, 126.10, 122.17, 125.74, 116.36, 166.40, 54.93,
-1
31.68; IR (neat) 1672, 1251, 1201, 3390, 1604, 1450 and 1072 cm ; Theoretical mass: 496.01,
+
LC-MS (m/z, I %): 496.9 [(M+H) , 100]; anal. calc. for C23
H17BrN
2
4
O S: C, 58.79; H, 3.36; N,
9
.35; O, 21.36; SC, 55.54; H, 3.45; Br, 16.07; N, 5.63; O, 12.87; S, 6.45, 7.13

3
.1.3.9 N-(4-bromophenyl)-2-((1-((2,4-dioxothiazolidin-5-ylidene) methyl) naphthalen-2-yl) oxy)
acetamide (3i)
Dark yellow powder; yield 26%; m.p, 262.6 C(charred); H NMR (400 MHz, DMSO d6): δ 10.59
0
1
(
(
(
s,1H), δ 8.75 (s,1H), δ 8.51-8.53 (d, J = 8.0 Hz, 1H), δ 8.12-8.14 (d, J = 8.0 Hz, 1H), 8.04-8.06
d, J = 8.0 Hz, 1H), δ 8.03-8.05 (d, J = 8.0 Hz, 1H), δ 7.73-7.77 (t, J = 8.0 Hz, 1H), δ 7.57-7.65
1
3
m, 5H), δ 7.48-7.50 (m, 2H), δ 4.13 (s, 2H); C NMR (400MHz, DMSO d6): δ 158.30, 150.59,
1
1
13.33, 138.01, 115.25, 128.09, 127.81, 126.17,122.21, 125.60,121.09, 128.81, 166.57, 40.07,
-1
31.66, 121.09, 130.07, 116.43; IR (neat) 1672, 1319, 1213, 3346, 1589 and 536 cm ; Theoretical
+
mass: 481.99, LC-MS (m/z, I %): 482.9 [(M+H) , 100]; anal. calc. for C22
H, 3.13; Br, 16.53; N, 5.80; O, 13.24; S, 6.63.
H15BrN
2
4
O S: C, 54.67;
3
.1.3.10 N-(2-chloro-5-(trifluoromethyl) phenyl)-2-((1-((2,4-dioxothiazolidin-5-ylidene) methyl)
naphthalen-2-yl) oxy) acetamide (3j)
Dark yellow powder; yield 28.57%; m.p, 238.6 C(charred); H NMR (400 MHz, DMSO d6): δ
0.23 (s,1H), δ 8.76 (s,1H), δ 8.55-8.57 (d, J = 8.0 Hz, 1H), δ 8.17 (m, 1H), 8.12-8.14 (d, J = 8.0
0
1
1
1
3
Hz, 1H), δ 8.04-8.06 (m, 1H), δ 7.72-7.76 (m, 2H), δ 7.53-7.65 (m, 3H), δ 4.29 (s, 2H); C NMR
400MHz, DMSO d6): δ 158.34, 150.71, 113.32, 135.40, 122.27, 130.05, 128.77, 128.19, 127.81,
(
1
1
28.01, 126.15, 130.80, 167.51, 40.07, 131.91, 127.86, 129.99, 125.20, 116.40; IR (neat) 1678,
-1
359, 1213, 3473, 1587, 1327 and 804 cm ; Theoretical mass: 506.03, LC-MS (m/z, I %): 505
+
[
(M-H) , 100]; anal. calc. for C23
H14ClF
3
N
2
4
O S: C, 54.50; H, 2.78; Cl, 6.99; F, 11.24; N, 5.53; O,
1
2.63; S, 6.33.
3
.1.3.11 2-((1-((2,4-dioxothiazolidin-5-ylidene) methyl) naphthalen-2-yl) oxy)-N-(4-nitrophenyl)
acetamide (3k)
Deep yellow powder; yield 25%; m.p, 264 C(charred); H NMR (400 MHz, DMSO d6): δ 11.05
s,1H), δ 8.75 (s,1H), δ 8.51-8.53 (d, J = 8.0 Hz, 1H), δ 8.20-8.23 (m, 2H), 8.10-8.12 (d, J = 8.0
Hz, 1H), δ 8.03-8.05 (d, J = 8.0 Hz, 1H), δ 7.84-7.87 (d, J = 12.0 Hz, 2H), δ 7.73-7.77 (t, J = 8.0
0
1
(
1
3
Hz, 1H), δ 7.62-7.64(m, 1H), δ 7.56-7.60 (m, 1H), δ 4.21 (s, 2H); C NMR (400MHz, DMSO
d6): δ 158.28, 150.66, 113.27, 142.40, 128.07, 127.79, 126.13, 122.17, 125.18, 118.89, 128.75,
1
1
C
67.43, 40.07, 132.23, 116.38, 125.02, 131.87; IR (neat) 1680, 1257, 1211, 3433, 1597, 1550 and
-1
+
006 cm ; Theoretical mass: 449.07, LC-MS (m/z, I %): 448 [(M-H) , 100]; anal. calc. for
S: C, 58.79; H, 3.36; N, 9.35; O, 21.36; S, 7.13.
22
H
15
N
3
O
6

Scheme 1. Reagents and conditions. A: Toluene, Piperidinium acetate- Reflux 2-3 h. B:
Chloroacetyl Chloride, DCM, K
overnight.
2
CO
3
, stirring at RT. overnight, C: DMF, K
2
CO - stirring at RT.
3
3
.2 In vitro HDAC screening
.2.1 HDAC enzyme inhibition Assay
3
Recombinant HDACs 1, 2, 3, 5 and 6 were purchased at BPS Bioscience. Recombinant HDAC8
was produced as described recently [56]. HDAC8 was produced in E. coli (BL21) DE3 pLysS
cells using a pET14b vector containing codon-optimized human HDAC8. Recombinant cHDAC4
was expressed using a pET14b vector (Novagen, EMD Millipore) containing the codon-optimized
catalytic domain of human HDAC4.
A serial dilution of inhibitor in assay buffer (25 mM Tris-HCl, pH 8.0, 75 mM KCl, 0.001 %
Pluronic F-127) was incubated with HDAC in a black 96-well microtiter half-area plate (Greiner)
for 60 min at 30 °C. Afterwards the reaction was initiated by the addition of 20 μM Boc-
Lys(trifluoroacetyl)-AMC (Bachem) as substrate for HDAC4, 5 and 8 and 50 μM Boc-Lys(acetyl)-
AMC as substrate for HDAC1, 2, 3 and 6. After incubation for 60 min at 30 °C, the reaction was
stopped by the addition of 1.7 μM SATFMK for HDAC4, 5 and 8 and 4.2 µM suberoylanilide
hydroxamic acid (SAHA, Cayman Chemical Company) for HDACs 1,2,3 and 6. The deacetylated
substrate was converted into a fluorescent product by the addition of 0.4 mg/ml trypsin
(Applichem). The release of AMC was followed in a microplate reader (PheraStar Plus, BMG
Labtech) at 450 nm (λEx = 350 nm) and correlated to enzyme activity. Dose-response curves were
generated by using GraphPad Prism and fitted to a four parameters logistic function to obtain IC50
values [57]:
(
퐸푚푎푥 − 퐸0)
퐸퐴 = 퐸 +
0
+ 1ꢀ(log(퐼퐶5ꢁ)ꢂ푥)∗ℎ
1

in which EA is the enzyme activity at a given inhibitor concentration x, Emax and E are the enzyme
0
activities determined at zero and complete inhibition, respectively. IC50 denotes the inhibitor
concentration at which half the enzyme is inhibited and h is the slope of the curve.
3
.2.2 Thermal shift assay
The thermal shift assay was performed as described recently [58]. In short, 2.5 µM recombinant
HDAC8 was incubated with 100 µM of the indicated inhibitor for 1h at 30°C and afterwards
aliquoted in PCR-Tubes. Each tube was tempered for 10 min at the indicated temperature and
subsequent centrifuged. The soluble protein phase was then conducted to SDS-PAGE and band
densities were quantified using Image Studio Lite (LiCor) and plotted against the temperature.
3
.3 In vitro anticancer Screening
3
.3.1 Cell cultures
K562 cell line (chronic myeloid leukaemia) was purchased from National Centre for Cell Sciences
NCCS), Pune. CEM (acute T-cell lymphoblastic leukemia) was obtained from the Department of
(
Biochemistry Indian Institute of Science, Bangalore. K562 and CEM cells were maintained as
suspension in RPMI 1640 medium (Thermo fisher scientific) supplemented with 10% fetal bovine
serum (Gibco, Invitrogen) and 1% antibiotic–antimycotic 100X solution (100 units/mL of
penicillin, 100 µg/mL of streptomycin, and 0.25 µg/mL of Gibco Amphotericin B). Cells were
maintained in a humidified 5% CO
2
atmosphere at 37˚C.
3
.3.2 Cytotoxicity assay
MTT assay was used to determine cytotoxic potential of 3a to 3k on leukemic cell lines, K562 and
CEM. The assay was performed as described earlier [59]. Briefly, the cells were seeded at a density
3
of approximately 5×10 cells/well in a 96-well flat-bottom micro-plate and maintained at 37ºC in
9
5% humidity and 5% CO overnight. Different concentrations of compounds were diluted in
2
DMSO and were added to the cells. The final concentration of DMSO in the experiments was less
than 1.5% and showed no cell toxicity. The cells were incubated with the compounds for 48 h.
Next, 96-well plates were centrifuged at 300 g, 4°C for 5 minutes and cells in well were washed
with phosphate buffer solution twice., 20 µL of the MTT staining solution (5mg/ml in phosphate
º
buffer solution) was added to each well and the plate was incubated at 37 C. After 4 h, 100 µL of
dimethyl sulfoxide (DMSO) was added to each well to dissolve the formazan crystals, and
absorbance was recorded with a 570 nm using micro plate reader.
3
.3.3 Apoptosis by flow cytometry
The induction of apoptosis produced by compound 3a and 3h was studied by flow cytometry as
described earlier [60]. Cells were seeded in a 24-well flat bottom micro plate and incubated at
0
3
7 C in 5% CO
2
atmosphere for 24 h. The media was replaced with fresh media and then for 24 h,
the cells were treated with IC50 concentration of 3a and 3h. Untreated cells were used as negative
control. Post incubation, cells were washed with PBS, Centrifuge for 5 minutes at 300 g at 4°C,

and supernatant was discarded. Cell pellets were re-suspended in ice-cold 1X Binding Buffer, 1 µL
of annexin V-FITC solution and 5 µL PI (propidium iodide) were added and mixed. Tubes were
kept on ice and incubated for 15 minutes in the dark. 400 µL of ice-cold 1X binding buffer was
TM
added and cell preparations were analyzed using flow cytometer (BD Accuri C5 flow cytometer,
BD Biosciences, CA, USA). FlowJo software was used for data analysis (version 10.1, Ashland,
OR, USA).
3
.3.4 Flow cytometry analysis of cell cycle
0
Cells were seeded in a 24-well flat bottom micro plate and incubated overnight at 37 C in CO
2
incubator for 24 h. The media was replaced with fresh media and then for 24 h., the cells were
treated with IC50 concentration of 3a and 3h. Untreated cells were used as negative control. Post
incubation, cells were washed with PBS, Centrifuge for 5 minutes at 300g at 4°C, and supernatant
was discarded. Cell were re-suspended in 0.5 ml PBS. The cells were fixed on ice in 4.5 ml of ice
cold 70 % ethanol for 2 h, centrifuged for 5min at 200 x g at 4°C and ethanol was decanted. Then
the cell pellets were Suspended in 5 ml PBS for 1 minute, centrifuged for 5min at 200 x g at 4°C
and treated with 1 ml propidium iodide staining solution for 15 mins at 37ºC. Cell were analyzed
TM
within 30 min. (BD Accuri C5 flow cytometer, BD Biosciences, CA, USA). Obtained data was
analyzed using FlowJo software (version 10.1, Ashland, OR, USA).
3
.3.5 Assessment of cell viability on non- transformed cells
Normal human WBCs were isolated from the peripheral blood of healthy blood donor. The study was
approved by appropriate Institutional Review Board. Blood was drawn during routine diagnostic procedures
after written informed consent was obtained from patient. The blood samples were spun in a centrifuge and
WBCs were isolated from the “buffy coat” fraction. Cells were maintained in a humidified 5% CO
2
atmosphere at 37˚C.
Effect of the treatment of compounds 3a and 3h on the viability of normal human WBCs, were
determined by using MTT assay as described above in cytotoxicity assay. Wherein normal WBCs
were treated with increasing concentrations of 3a and 3h from 2.5 to 100 μM for 48h to determine
the difference in the cell viabilities of normal cells and finally to calculate IC50 value.
3
.4 In silico studies
3
.4.1 Molecular docking and energy minimization
Modeling, preparation and visualization of structural data as well as molecular docking was
performed using MOE 2019 software (Chemical Computing Group ULC, Canada). Three crystal
structures representing significant conformations of HDAC8 (PDB-ID’s 1T69, 1VKG, 3SFF) were
obtained from 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 30 poses were passed

to the refinement and energy minimization in the pocket using the induced fit method and then
rescored with the GBVI/WSA dG scoring function.
3
.4.2 SwissADME predictions
SwissADME is freely available but reliable online tool which can be used to predict
physicochemical, pharmacokinetic and bioavailability of the synthetic molecules, simply by
providing structural inputs. The web address of the tool is http://www.swissadme.ch/index.php#.
4
. Conclusion:
We have successfully synthesized and characterised a series of 5-naphthylidene-2,4-
thiazolidinedione derivatives as selective non-hydroxamate HDAC8 inhibitors. Among the
synthesized derivatives, compounds 3a, 3f and 3h-3j were found to be selective for HDAC8 in
HDAC screening of HDAC1,2,3, 4, 5, 6 and 8. In TSA, all the screened derivatives showed shift
0
in ∆Tm of 7 C and more. In cytotoxicity assays in K562 and CEM cell lines, 3a exhibited most
potent cytotoxic effects and was found to be safer in normal WBCs. 3a and 3h both lead to cell
cycle arrest in G2/M phase and was able to induce apoptosis in CEM cells (Fig. 9). Docking studies
correlated well with the HDAC inhibitory concentrations and carboxylate group was found to
interact with zinc at zinc binding site. Crystallographic studies need to be performed to determine
actual interactions at active site of HDAC8. Given that Compound 3h exhibits HDAC8 selectivity
and potent antitumor effects in vitro, further in-depth studies on these molecules are expected to
give rise to potent leads.
Fig. 9. Schematic representation of results.
Conflict of interest
The authors declare that they have no conflict of interest.

Acknowledgement:
This study was supported by Indo-Poland joint research programme grant from Department of
Science and Technology (DST) with reference number DST/INT/Pol/P-27/2016.
Abbreviations
TZD- Thiazolidinedione
HDAC- Histone deacetylase
DMF- Dimethyl Formamide
DCM- Dichloromethane
NMR- Nuclear magnetic resonance spectroscopy
IR- Infrared Spectroscopy
DMEM- Dulbecco's Modified Eagle Medium
MTT- 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
FBS- fetal calf serum
RT- room temperature
References:
1
. Sharma, S., Kelly, T.K., Jones, P.A., 2010. Epigenetics in cancer. Carcinogenesis 31, 27–
3
6. https://doi.org/10.1093/carcin/bgp220
2
. Mann, B.S., Johnson, J.R., Cohen, M.H., Justice, R., Pazdur, R., 2007. FDA Approval
Summary: Vorinostat for Treatment of Advanced Primary Cutaneous T-Cell Lymphoma.
The Oncologist 12, 1247–1252. https://doi.org/10.1634/theoncologist.12-10-1247
. Laubach, J.P., Moreau, P., San-Miguel, J.F., Richardson, P.G., 2015. Panobinostat for the
Treatment of Multiple Myeloma. Clinical Cancer Research 21, 4767–4773.
https://doi.org/10.1158/1078-0432.CCR-15-0530
3
4
5
6
7
. Oehme, I., Deubzer, H.E., Lodrini, M., Milde, T., Witt, O., 2009. Targeting of HDAC8 and
investigational inhibitors in neuroblastoma. Expert Opinion on Investigational Drugs 18,
1
605–1617. https://doi.org/10.1517/14728220903241658
. Balasubramanian, S., Ramos, J., Luo, W., Sirisawad, M., Verner, E., Buggy, J.J., 2008. A
novel histone deacetylase 8 (HDAC8)-specific inhibitor PCI-34051 induces apoptosis in
T-cell lymphomas. Leukemia 22, 1026–1034. https://doi.org/10.1038/leu.2008.9
. Chakrabarti, A., Oehme, I., Witt, O., Oliveira, G., Sippl, W., Romier, C., Pierce, R.J., Jung,
M., 2015. HDAC8: a multifaceted target for therapeutic interventions. Trends in
Pharmacological Sciences 36, 481–492. https://doi.org/10.1016/j.tips.2015.04.013
. Chakrabarti, A., Melesina, J., Kolbinger, F.R., Oehme, I., Senger, J., Witt, O., Sippl, W.,
Jung, M., 2016. Targeting histone deacetylase 8 as a therapeutic approach to cancer and
neurodegenerative diseases. Future Medicinal Chemistry 8, 1609–1634.
https://doi.org/10.4155/fmc-2016-0117
8
. Amin, S.A., Adhikari, N., Jha, T., 2017. Structure–activity relationships of hydroxamate-
based histone deacetylase-8 inhibitors: reality behind anticancer drug discovery. Future
Medicinal Chemistry 9, 2211–2237. https://doi.org/10.4155/fmc-2017-0130

9
1
. Amin, Sk.A., Adhikari, N., Jha, T., 2018. Structure-activity relationships of HDAC8
inhibitors: Non-hydroxamates as anticancer agents. Pharmacological Research 131, 128–
1
42. https://doi.org/10.1016/j.phrs.2018.03.001
0. Shen, S., Kozikowski, A.P., 2016. Why Hydroxamates May Not Be the Best Histone
Deacetylase Inhibitors-What Some May Have Forgotten or Would Rather Forget?
ChemMedChem 11, 15–21. https://doi.org/10.1002/cmdc.201500486
1
1
1. Johnstone, R.W., 2002. Histone-deacetylase inhibitors: novel drugs for the treatment of
cancer. Nat Rev Drug Discov 1, 287–299. https://doi.org/10.1038/nrd772
2. Marks, P.A., Rifkind, R.A., Richon, V.M., Breslow, R., Miller, T., Kelly, W.K., 2001.
Histone deacetylases and cancer: causes and therapies. Nat Rev Cancer 1, 194–202.
https://doi.org/10.1038/35106079
1
3. Mai, A., Massa, S., Rotili, D., Cerbara, I., Valente, S., Pezzi, R., Simeoni, S., Ragno, R.,
2
005. Histone deacetylation in epigenetics: An attractive target for anticancer therapy.
Med. Res. Rev. 25, 261–309. https://doi.org/10.1002/med.20024
1
1
4. Miller, T.A., Witter, D.J., Belvedere, S., 2003. Histone Deacetylase Inhibitors. J. Med.
Chem. 46, 5097–5116. https://doi.org/10.1021/jm0303094
5. Whitehead, L., Dobler, M.R., Radetich, B., Zhu, Y., Atadja, P.W., Claiborne, T., Grob,
J.E., McRiner, A., Pancost, M.R., Patnaik, A., Shao, W., Shultz, M., Tichkule, R.,
Tommasi, R.A., Vash, B., Wang, P., Stams, T., 2011. Human HDAC isoform selectivity
achieved via exploitation of the acetate release channel with structurally unique small
molecule inhibitors. Bioorganic
&
Medicinal Chemistry 19, 4626–4634.
https://doi.org/10.1016/j.bmc.2011.06.030
1
6. Galletti, P., Quintavalla, A., Ventrici, C., Giannini, G., Cabri, W., Penco, S., Gallo, G.,
Vincenti, S., Giacomini, D., 2009. Azetidinones as Zinc-Binding Groups to Design
Selective
HDAC8
Inhibitors.
ChemMedChem
4,
1991–2001.
https://doi.org/10.1002/cmdc.200900309
1
1
7. Nian, H., Bisson, W.H., Dashwood, W.-M., Pinto, J.T., Dashwood, R.H., 2009. -Keto acid
metabolites of organoselenium compounds inhibit histone deacetylase activity in human
colon cancer cells. Carcinogenesis 30, 1416–1423. https://doi.org/10.1093/carcin/bgp147
8. Hu, E., Dul, E., Sung, C.-M., Chen, Z., Kirkpatrick, R., Zhang, G.-F., Johanson, K., Liu,
R., Lago, A., Hofmann, G., Macarron, R., De Los Frailes, M., Perez, P., Krawiec, J.,
Winkler, J., Jaye, M., 2003. Identification of Novel Isoform-Selective Inhibitors within
Class
I
Histone Deacetylases.
J
Pharmacol Exp Ther 307, 720–728.
https://doi.org/10.1124/jpet.103.055541
1
2
9. Kleinschek, A., Meyners, C., Digiorgio, E., Brancolini, C., Meyer-Almes, F.-J., 2016.
Potent and Selective Non-hydroxamate Histone Deacetylase 8 Inhibitors. ChemMedChem
1
1, 2598–2606. https://doi.org/10.1002/cmdc.201600528
0. Wolff, B., Jänsch, N., Sugiarto, W.O., Frühschulz, S., Lang, M., Altintas, R., Oehme, I.,
Meyer-Almes, F.-J., 2019. Synthesis and structure activity relationship of 1, 3-benzo-
thiazine-2-thiones as selective HDAC8 inhibitors. European Journal of Medicinal
Chemistry 184, 111756. https://doi.org/10.1016/j.ejmech.2019.111756
2
1. Amin, Sk.A., Adhikari, N., Jha, T., 2018. Structure-activity relationships of HDAC8
inhibitors: Non-hydroxamates as anticancer agents. Pharmacological Research 131, 128–
1
42. https://doi.org/10.1016/j.phrs.2018.03.001

2
2. Goracci, L., Deschamps, N., Randazzo, G.M., Petit, C., Dos Santos Passos, C., Carrupt, P.-
A., Simões-Pires, C., Nurisso, A., 2016. A Rational Approach for the Identification of Non-
Hydroxamate
HDAC6-Selective
Inhibitors.
Sci
Rep
6,
29086.
https://doi.org/10.1038/srep29086
2
2
3. Buggy, J.J., Sriram B., Susanne M. S., inventors; Pharmacyclics Inc., assignee; 2015. Uses
of selective inhibitors of HDAC8 and treatment of inflammatory conditions.
US20150038542.
4. Suzuki, T., Ota, Y., Ri, M., Bando, M., Gotoh, A., Itoh, Y., Tsumoto, H., Tatum, P.R.,
Mizukami, T., Nakagawa, H., Iida, S., Ueda, R., Shirahige, K., Miyata, N., 2012. Rapid
Discovery of Highly Potent and Selective Inhibitors of Histone Deacetylase 8 Using Click
Chemistry to Generate Candidate Libraries. J. Med. Chem. 55, 9562–9575.
https://doi.org/10.1021/jm300837y
2
5. Aaron Beaty Beeler, John A. Porco, Jr., Oscar J. Ingham, James E. Bradner, inventors;
Trustees of Boston University, assignee; 2015. Selective histone deacetylase 8 inhibitors.
US20150352079.
2
2
6. Oyelere, A., Berkley G., inventors; 2013. Histone deacetylase (HDAC) inhibitors targeting
prostate tumors and method of making and using thereof. US20130289085.
7. Huang, W.-J., Wang, Y.-C., Chao, S.-W., Yang, C.-Y., Chen, L.-C., Lin, M.-H., Hou, W.-
C., Chen, M.-Y., Lee, T.-L., Yang, P., Chang, C.-I., 2012. Synthesis and Biological
Evaluation of ortho -Aryl N -Hydroxycinnamides as Potent Histone Deacetylase (HDAC)
8
Isoform-Selective
https://doi.org/10.1002/cmdc.201200300
8. Xu W., Zhang Y., Song W., inventors; Shouguang Fukang Pharmaceuticals Ltd., assignee;
012. Preparative method and use of ZYJ-D08A and its epimers as histone deacetylase
Inhibitors.
ChemMedChem
7,
1815–1824.
2
2
inhibitors. WO2012174730.
2
3
9. Xu W., Zhang Y., Fang H., inventors; Shandong University, assignee; 2010. Tyrosine
derivatives as histone deacetylase inhibitors and using thereof. CN101723896.
0. Zhang, Y., Feng, J., Liu, C., Zhang, L., Jiao, J., Fang, H., Su, L., Zhang, X., Zhang, J., Li,
M., Wang, B., Xu, W., 2010. Design, synthesis and preliminary activity assay of 1,2,3,4-
tetrahydroisoquinoline-3-carboxylic acid derivatives as novel Histone deacetylases
(
HDACs) inhibitors. Bioorganic
https://doi.org/10.1016/j.bmc.2010.01.060
1. 41 Zhang, Y., Feng, J., Jia, Y., Wang, X., Zhang, L., Liu, C., Fang, H., Xu, W., 2011.
&
Medicinal Chemistry 18, 1761–1772.
3
Development of Tetrahydroisoquinoline-Based Hydroxamic Acid Derivatives: Potent
Histone Deacetylase Inhibitors with Marked in Vitro and in Vivo Antitumor Activities. J.
Med. Chem. 54, 2823–2838. https://doi.org/10.1021/jm101605z
3
3
3
2. KrennHrubec, K., Marshall, B.L., Hedglin, M., Verdin, E., Ulrich, S.M., 2007. Design and
evaluation of ‘Linkerless’ hydroxamic acids as selective HDAC8 inhibitors. Bioorganic &
Medicinal Chemistry Letters 17, 2874–2878. https://doi.org/10.1016/j.bmcl.2007.02.064
3. Qin, H.-T., Li, H.-Q., Liu, F., 2017. Selective histone deacetylase small molecule
inhibitors: recent progress and perspectives. Expert Opinion on Therapeutic Patents 27,
6
21–636. https://doi.org/10.1080/13543776.2017.1276565
4. Bressi, J.C., Jong, R. de, Wu, Y., Jennings, A.J., Brown, J.W., O’Connell, S., Tari, L.W.,
Skene, R.J., Vu, P., Navre, M., Cao, X., Gangloff, A.R., 2010. Benzimidazole and
imidazole inhibitors of histone deacetylases: Synthesis and biological activity. Bioorganic

&
Medicinal
Chemistry
Letters
20,
3138–3141.
https://doi.org/10.1016/j.bmcl.2010.03.092
3
3
5. Mohan, R., Sharma, A.K., Gupta, S., Ramaa, C.S., 2012. Design, synthesis, and biological
evaluation of novel 2,4-thiazolidinedione derivatives as histone deacetylase inhibitors
targeting liver cancer cell line. Med Chem Res 21, 1156–1165.
https://doi.org/10.1007/s00044-011-9623-3
6. Marek, M., Shaik, T.B., Heimburg, T., Chakrabarti, A., Lancelot, J., Ramos-Morales, E.,
Da Veiga, C., Kalinin, D., Melesina, J., Robaa, D., Schmidtkunz, K., Suzuki, T., Holl, R.,
Ennifar, E., Pierce, R.J., Jung, M., Sippl, W., Romier, C., 2018. Characterization of Histone
Deacetylase 8 (HDAC8) Selective Inhibition Reveals Specific Active Site Structural and
Functional
Determinants.
J.
Med.
Chem.
61,
10000–10016.
https://doi.org/10.1021/acs.jmedchem.8b01087
3
3
3
7. Hou, X., Du, J., Liu, R., Zhou, Y., Li, M., Xu, W., Fang, H., 2015. Enhancing the
Sensitivity of Pharmacophore-Based Virtual Screening by Incorporating Customized ZBG
Features: A Case Study Using Histone Deacetylase 8. J. Chem. Inf. Model. 55, 861–871.
https://doi.org/10.1021/ci500762z
8. Patil, V., Tilekar, K., Mehendale-Munj, S., Mohan, R., Ramaa, C.S., 2010. Synthesis and
primary cytotoxicity evaluation of new 5-benzylidene-2,4-thiazolidinedione derivatives.
European
Journal
of
Medicinal
Chemistry
45,
4539–4544.
https://doi.org/10.1016/j.ejmech.2010.07.014
9. Kabir, A., Tilekar, K., Upadhyay, N., Ramaa, C.S., 2019. Novel Anthraquinone
Derivatives as Dual Inhibitors of Topoisomerase 2 and Casein Kinase 2: In Silico Studies,
Synthesis and Biological Evaluation on Leukemic Cell Lines. ACAMC 18, 1551–1562.
https://doi.org/10.2174/1871520618666180423111309
4
4
0. Momose, Y., Meguro, K., Ikeda, H., Hatanaka, C., Oi, S., Sohda, T., 1991. Studies on
Antidiabetic Agents. X. Synthesis and Biological Activities of Pioglitazone and Related
Compounds. Chem. Pharm. Bull. 39, 1440–1445. https://doi.org/10.1248/cpb.39.1440
1. Auld, D.S., Thorne, N., Maguire, W.F., Inglese, J., 2009. Mechanism of PTC124 activity
in cell-based luciferase assays of nonsense codon suppression. Proceedings of the National
Academy of Sciences 106, 3585–3590. https://doi.org/10.1073/pnas.0813345106
2. Schmidt, C., 2010. GSK/Sirtris compounds dogged by assay artifacts. Nat Biotechnol 28,
4
4
4
1
85–186. https://doi.org/10.1038/nbt0310-185
3. Guha, M., 2011. PARP inhibitors stumble in breast cancer. Nat Biotechnol 29, 373–374.
https://doi.org/10.1038/nbt0511-373
4. Liu, X., Shi, Y., Maag, D.X., Palma, J.P., Patterson, M.J., Ellis, P.A., Surber, B.W., Ready,
D.B., Soni, N.B., Ladror, U.S., Xu, A.J., Iyer, R., Harlan, J.E., Solomon, L.R., Donawho,
C.K., Penning, T.D., Johnson, E.F., Shoemaker, A.R., 2012. Iniparib Nonselectively
Modifies Cysteine-Containing Proteins in Tumor Cells and Is Not a Bona Fide PARP
Inhibitor. Clinical Cancer Research 18, 510–523. https://doi.org/10.1158/1078-0432.CCR-
1
1-1973
4
4
5. 10 Florio, M., Borrell, V., Huttner, W.B., 2017. Human-specific genomic signatures
of neocortical expansion. Current Opinion in Neurobiology 42, 33–44.
https://doi.org/10.1016/j.conb.2016.11.004
6. Florio, M., Heide, M., Pinson, A., Brandl, H., Albert, M., Winkler, S., Wimberger, P.,
Huttner, W.B., Hiller, M., 2018. Evolution and cell-type specificity of human-specific

genes preferentially expressed in progenitors of fetal neocortex. eLife 7, e32332.
https://doi.org/10.7554/eLife.32332
4
4
7. Song, S., Wang, Y., Xu, P., Yang, R., Ma, Z., Liang, S., Zhang, G., 2015. The inhibition
of histone deacetylase 8 suppresses proliferation and inhibits apoptosis in gastric
adenocarcinoma.
International
Journal
of
Oncology
47,
1819–1828.
https://doi.org/10.3892/ijo.2015.3182
8. Wu, J., Du, C., Lv, Z., Ding, C., Cheng, J., Xie, H., Zhou, L., Zheng, S., 2013. The Up-
Regulation of Histone Deacetylase 8 Promotes Proliferation and Inhibits Apoptosis in
Hepatocellular Carcinoma. Dig Dis Sci 58, 3545–3553. https://doi.org/10.1007/s10620-
0
13-2867-7
4
5
9. Paris, M., Porcelloni, M., Binaschi, M., Fattori, D., 2008. Histone Deacetylase Inhibitors:
From Bench to Clinic. J. Med. Chem. 51, 1505–1529. https://doi.org/10.1021/jm7011408
0. Dong, Z., Yang, Y., Liu, S., Lu, J., Huang, B., Zhang, Y., 2018. HDAC inhibitor PAC-320
induces G2/M cell cycle arrest and apoptosis in human prostate cancer. Oncotarget 9.
https://doi.org/10.18632/oncotarget.23070
5
1. Vannini, A., Volpari, C., Filocamo, G., Casavola, E.C., Brunetti, M., Renzoni, D.,
Chakravarty, P., Paolini, C., De Francesco, R., Gallinari, P., Steinkuhler, C., Di Marco, S.,
2
004. Crystal structure of a eukaryotic zinc-dependent histone deacetylase, human
HDAC8, complexed with a hydroxamic acid inhibitor. Proceedings of the National
Academy of Sciences 101, 15064–15069. https://doi.org/10.1073/pnas.0404603101
2. Decroos, C., Clausen, D.J., Haines, B.E., Wiest, O., Williams, R.M., Christianson, D.W.,
5
5
2
015. Variable Active Site Loop Conformations Accommodate the Binding of
Macrocyclic Largazole Analogues to HDAC8. Biochemistry 54, 2126–2135.
https://doi.org/10.1021/acs.biochem.5b00010
3. Somoza, J.R., Skene, R.J., Katz, B.A., Mol, C., Ho, J.D., Jennings, A.J., Luong, C., Arvai,
A., Buggy, J.J., Chi, E., Tang, J., Sang, B.-C., Verner, E., Wynands, R., Leahy, E.M.,
Dougan, D.R., Snell, G., Navre, M., Knuth, M.W., Swanson, R.V., McRee, D.E., Tari,
L.W., 2004. Structural Snapshots of Human HDAC8 Provide Insights into the Class I
Histone Deacetylases. Structure 12, 1325–1334. https://doi.org/10.1016/j.str.2004.04.012
4. Deschamps, N., Simões-Pires, C.A., Carrupt, P.-A., Nurisso, A., 2015. How the flexibility
of human histone deacetylases influences ligand binding: an overview. Drug Discovery
Today 20, 736–742. https://doi.org/10.1016/j.drudis.2015.01.004
5. Daina, A., Michielin, O., Zoete, V., 2017. SwissADME: a free web tool to evaluate
pharmacokinetics, drug-likeness and medicinal chemistry friendliness of small molecules.
Sci Rep 7, 42717. https://doi.org/10.1038/srep42717
6. Jänsch, N., Meyners, C., Muth, M., Kopranovic, A., Witt, O., Oehme, I., Meyer-Almes, F.-
J., 2019. The enzyme activity of histone deacetylase 8 is modulated by a redox-switch.
Redox Biology 20, 60–67. https://doi.org/10.1016/j.redox.2018.09.013
5
5
5
5
5
7. Volund, A., 1978. Application of the Four-Parameter Logistic Model to Bioassay:
Comparison with Slope Ratio and Parallel Line Models. Biometrics 34,
3
57. https://doi.org/10.2307/2530598
8. Schweipert, M., Jänsch, N., Sugiarto, W.O., Meyer-Almes, F.-J., 2019. Kinetically
selective and potent inhibitors of HDAC8. Biological Chemistry 400, 733–743.
https://doi.org/10.1515/hsz-2018-0363

5
6
9. Bhat, S.S., Revankar, V.K., Kumbar, V., Bhat, K., Kawade, V.A., 2018. Synthesis, crystal
structure and biological properties of a cis -dichloridobis(diimine)copper(II) complex. Acta
Crystallogr C Struct Chem 74, 146–151. https://doi.org/10.1107/S2053229617018551
0. Peram, M.R., Jalalpure, S., Kumbar, V., Patil, S., Joshi, S., Bhat, K., Diwan, P., 2019.
Factorial design based curcumin ethosomal nanocarriers for the skin cancer delivery: in
vitro
evaluation.
Journal
of
Liposome
Research
29,
291–311.
https://doi.org/10.1080/08982104.2018.1556292