Synthesis, biological evaluation, and molecular docking analysis of novel linker-less benzamide based potent and selective HDAC3 inhibitors

Article abstract of DOI:10.1016/j.bioorg.2021.105050

A series of novel linker-less benzamides with different aryl and heteroaryl cap groups have been designed, synthesized, and screened as potent histone deacetylase (HDAC) inhibitors with promising anticancer activity. Two lead compounds 5e and 5f were foun

Full text of DOI:10.1016/j.bioorg.2021.105050

Bioorganic Chemistry 114 (2021) 105050
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Synthesis, biological evaluation, and molecular docking analysis of novel
linker-less benzamide based potent and selective HDAC3 inhibitors
,
,
Ganesh Routhollaa 1, Sravani Pulyaa 1, Tarun Patela, Sk. Abdul Aminb, Nilanjan Adhikarib,
Swati Biswasa, Tarun Jhab *, Balaram Ghosha
,
,*
a Epigenetic Research Laboratory, Department of Pharmacy, BITS-Pilani, Hyderabad Campus, Shamirpet, Hyderabad 500078, India
b Natural Science Laboratory, Division of Medicinal and Pharmaceutical Chemistry, Department of Pharmaceutical Technology, P. O. Box 17020, Jadavpur University,
Kolkata 700032, West Bengal, India
A R T I C L E I N F O
A B S T R A C T
Keywords:
A series of novel linker-less benzamides with different aryl and heteroaryl cap groups have been designed,
synthesized, and screened as potent histone deacetylase (HDAC) inhibitors with promising anticancer activity.
Two lead compounds 5e and 5f were found as potent and highly selective HDAC3 inhibitors over other Class-I
HDACs and HDAC6. Compound 5e bearing a 6-quinolinyl moiety as the cap group was found to be a highly
potent HDAC3 inhibitor (IC50 = 560 nM) and displayed 46-fold selectivity for HDAC3 over HDAC2, and 33-fold
selectivity for HDAC3 over HDAC1. The synthesized compounds possess antiproliferative activities against
different cancer cell lines and significantly less cytotoxic to normal cells. Molecular Docking studies of com-
pounds 5e and 5f reveal a similar binding mode of interactions as CI994 at the HDAC3 active site. These ob-
servations agreed with the in vitro HDAC3 inhibitory activities. Significant enhancement of the endogenous
acetylation level on H3K9 and H4K12 was found when B16F10 cells were treated with compounds 5e and 5f in a
dose-dependent manner. The compounds induced apoptotic cell death in Annexin-V/FITC-PI assay and caused
cell cycle arrest at G2/M phase of cell cycle in B16F10 cells. These compounds may serve as potential HDAC3
inhibitory anticancer therapeutics.
Linker-less benzamides
HDAC3 inhibitor
Anticancer agent
Histone acetylation
Apoptotic cell death
Cell cycle analysis
1. Introduction
€-amino group of lysine residues present in the N-terminal end of histone
proteins. The imbalance between the activities of HATs and HDACs
often results in aberrant gene expression leading to several epigenetic
disorders [5]. Moreover, HDACs play a prominent role also in cancer cell
proliferation, invasion, and metastasis [6]. It has also been reported that
HDAC inhibitors are well-implicated in apoptosis or programmed cell
death as well as cell cycle arrest [7–9], whereas abnormal HDAC
expression lead to various cancers such as cancers of colon, breast, liver,
lung, pancreas, prostate, along with melanoma, lymphoma, multiple-
myeloma and leukaemia [10]. There are 18 different HDACs catego-
rized into four classes according to their homology with yeast proteins,
subcellular localization, and mechanism of action. Basically, Class I
(HDAC1, 2, 3 and 8), Class II (IIa: HDAC4, 5, 7 and 9; IIb: HDAC6 and
10) and Class IV (HDAC 11) HDACs are Zn+2-dependent whereas Class
III HDACs are NAD+-dependent also known as Sirtuins (SIRT 1–7)
[6,11].
According to WHO, cancer is the second leading cause of death
worldwide, claiming about 9.6 million deaths (approximately 1 in 6) in
2018 [1]. “Cancer is caused by the mutations in tumor suppressor genes
and oncogenes due to epigenetic manipulations that lead to cell prolif-
eration and differentiation” and this is considered the most acceptable
hypothesis in cancer pathogenesis [2]. It is also well-established that
genetic and epigenetic modifications contribute to the development of
cancer [3]. Epigenetic modifications leading to structural changes in
chromatin include DNA methylation, and post-translational modifica-
tions of histone proteins. Among the post-translational modifications,
histone acetylation and deacetylation have been found to play a pivotal
role in the regulation of expression of genes involved in cancer patho-
genesis [4]. Histone acetyl transferases (HATs) and histone deacetylases
(HDACs) are the two key enzymes involved in the regulation of gene
expression through acetylation and deacetylation of histone proteins at
HDAC3 has garnered lot of attention due to its implication in various
* Corresponding authors.
E-mail address: balaram@hyderabad.bits-pilani.ac.in (B. Ghosh).
1
Authors have equal contribution.
https://doi.org/10.1016/j.bioorg.2021.105050
Received 2 April 2021; Accepted 30 May 2021
Available online 4 June 2021
0045-2068/© 2021 Elsevier Inc. All rights reserved.

G. Routholla et al.
Bioorganic Chemistry 114 (2021) 105050
life-threatening disease conditions namely cancers [9], cardiovascular
diseases [12], memory and learning disorders [13,14], neurodegenera-
tive diseases [15], diabetes [16] and rheumatoid arthritis [17]. Most
importantly, HDAC3 has been validated and well-studied as a potential
target for cancer therapy, due to its crucial role in transcriptional
repression through hypoacetylation of histones in different cancers [18].
HDAC3 has been found to modulate various cancers such as colon cancer
[19], breast cancer [20], multiple myeloma [21], melanoma [22],
prostate cancer [23], gastric cancer [24] and leukemia [25], and se-
lective HDAC3 inhibitors have been extensively studied in recent years
for various applications [9,26]. However, still lot of work needs to be
carried out to identify highly potent and selective HDAC3 inhibitors to
combat such disease states with minimum off target side effects. HDAC3,
a well-explored Class I HDAC, is structurally characterized by a unique
C-terminal domain and it forms a stable complex with NCoR and SMRT
for carrying out the deacetylase activity [27]. Several amino acid resi-
dues (such as Tyr198, Asp92, Phe199, Tyr107) at the active site of
HDAC3 contribute to the substrate specificity over other Class I HDAC
isoforms. Nevertheless, the interactions of Ins(1,4,5,6)P4 and DAD with
HDAC3 contribute to the activation of the enzyme [28,29]. The general
pharmacophore model of HDAC active site includes a surface binding
domain that interacts with the cap group, the hydrophobic channel that
recruits the linker region, and the catalytic zinc binding domain where
the Zn+2 ion interacts with the zinc binding group (ZBG) of the HDAC
inhibitors (Fig. 1). There is also an internal cavity adjacent to the zinc
binding domain in case of HDAC3 isoform that might contribute its
substrate specificity [30].
[13,18,37,38]. Since last 10 years, a number of research works have
been carried out on this scaffold to design compounds reflecting potent
HDAC3 inhibition along with selectivity over other HDACs
[39,40,40–47]. Interestingly, these compounds also comprise the typical
features as HDAC inhibitors, i.e., the cap group, the linker moiety and
the ZBG. However, only a few group of researchers tried to design linker-
less atypical selective HDAC3 inhibitors [20,47]. Minami et al. [21] first
reported BG45, which is a linker-less benzamide based HDAC3 selective
inhibitor, showed promising efficacy against multiple myeloma.
Another series of linker-less HDAC3 selective compounds have been
reported by McClure et al. [48]. They used benzofuran scaffold instead
of pyrazine scaffold of BG45 to obtain potent and selective HDAC3 in-
hibitors. Our group has been actively working on the development of
HDAC3 selective inhibitors with an emphasis on benzamides involving
the structural modifications of the cap and linker region [18,49–53].
We, hereby, report a series of linker-less benzamide compounds with
different cap groups derived from the basic pharmacophore scaffold of
CI994 which has been studied most extensively in preclinical and clin-
ical applications [35](Fig. 2) . In-order to enhance the HDAC3 selec-
tivity, modification of the cap region with different aromatic or
heteroaromatic functions of the linker-less benzamides have been
reported.
Herein, we report the synthesis schemes, HDAC3 isoform selectivity
study and detailed biological characterization of the synthesized small
molecule HDAC3 inhibitors.
2. Results
Quite a few number of HDACis have been reported so far and they are
widely studied in pre-clinical and clinical phases as anticancer agents
[9]. Based on various ZBGs, different classes of HDACis have been re-
ported such as hydroxamates, benzamides, short chain fatty acids, cyclic
tetrapeptides, hydrazides and thiols [9]. Six HDACis have been clinically
approved so far namely vorinostat, belinostat, panobinostat, romidep-
sin, chidamide and pracinostat for the treatment of different cancers
such as cutaneous T-cell lymphoma, peripheral T-cell lymphoma, mul-
tiple myeloma and acute myeloid leukemia [31]. With the multiple side-
effects and dose-limiting toxicities associated with these pan-HDACis,
the need for developing novel isoform specific inhibitors has been
realised urgently to overcome these limitations and to improve the po-
tency and specificity towards individual HDAC isoforms.
2.1. Chemistry
2.1.1. Design and synthesis of novel benzamides
Keeping the benzamide scaffold intact as the ZBG, several aryl (such
as phenyl, naphthyl) and heteroaryl (such as quinolinyl, indolyl, thienyl
and pyrazinyl-aminophenyl) cap groups have been incorporated. Most
of these benzamides (5a–5h) were synthesized as per Scheme 1 whereas
compound 5i was synthesized following the Scheme 2.
As per Scheme 1, the aromatic or heteroaromatic carboxylic acids
(3a–3h) were purchased commercially and were coupled with tert-butyl
(2-aminophenyl) carbamate synthesized as per protocol reported pre-
viously [49]. Under the conditions, the acid–amine coupling reactions
were done using EDCI as coupling agent to obtain (4a-4h) as the in-
termediates, which upon deprotection of carbamate group in acidic
medium afforded the final compounds (5a-5h).
The above mentioned clinically approved HDAC inhibitors belong to
hydroxamate class except chidamide which is the only benzamide
compound approved by Chinese FDA for the treatment of relapsed pe-
ripheral T-cell lymphoma [32]. Interestingly, several studies have been
carried out and are still continuing on benzamide class of compounds to
come up with selective HDAC inhibitors with minimum or no off target
side effects [9]. Notably, the benzamide based class-I HDAC selective
inhibitors such as entinostat (MS-275, 1) [33] and tacedinaline (CI994,
4) [35,36] and HDAC3 selective inhibitors, RGFP109 (2)[34], and BG45
(3) [21] have been studied extensively as promising anticancer agents
(Fig. 2). Recently, several other benzamides designed with varying cap
groups have been reported with enhanced HDAC3 selectivity and inhi-
bition potential [9].
Considering the contribution of pyrazine scaffold to the enhanced
HDAC3 selectivity as in the case of BG45, another molecule with ami-
nophenyl as the cap group attached to the pyrazine scaffold at its 2nd
position with the benzamide has been designed and synthesized. Scheme
2 describes the synthesis of compound 5i. There 6-chloro pyrazine 2-
carboxylate (1i) was converted to methyl 6-(phenylamino) pyrazine-2-
carboxylate (2i) using aniline in the presence of NMP as a solvent and
DIPEA as a base. The 6-(phenylamino) pyrazine-2-carboxylic acid (3i)
was obtained through alkaline hydrolysis of the carboxylate (2i). The
acid 3i was coupled with tert-butyl (2-aminophenyl) carbamate syn-
thesized as per our previous report [49]. Under the conditions, acid –
amine coupling was conducted using EDCI as coupling agent to obtain
(4i) as the intermediate, which upon deprotection of carbamate group in
acidic medium resulted in the final compound 5i.
Benzamide has been established as a promising moiety responsible
for chelating the Zn2+ ion for various HDACs and thereby responsible for
potent inhibition along with selectivity towards specific HDACs
The structures of the designed and synthesized compounds along
with their % yield and physicochemical properties are listed in Table 1.
Physicochemical characterisations of the synthesized molecules were
done using 1H NMR, 13C NMR, HRMS analysis and the spectral data is
given in the supporting information section (Spectra S1 – S36).
Fig. 1. General pharmacophoric structure of HDAC inhibitors.
2

G. Routholla et al.
Bioorganic Chemistry 114 (2021) 105050
Fig. 2. Structures of benzamide derivatives and design of our new benzamides derivatives.
Scheme 1. Reagents and conditions: (a) tert-butyl 2-amino phenyl carbamate, EDC, DMAP, DCM:pyridine (1:1), RT (b) 4 M HCl in dioxane, 0 ◦C, 2 h.
2.2. Biological evaluation
but the selectivity factor towards HDAC3 is less when compared to that
of compound 5e which is highly potent with 59% inhibition at 1 µM. It
was noteworthy that the pyrazine scaffold containing benzamide com-
pound, 5i neither exhibited any significant pan-HDAC or HDAC3 inhi-
bition when compared to other compounds in the series.
2.2.1. Pan-HDAC and HDAC3 inhibition
All the synthesized compounds (5a-5i) were screened for enzyme
inhibitory activity towards pan-HDAC (HeLa Nuclear extract) and re-
combinant human HDAC3 enzymes initially (supporting Figure S1 and
S2). All the compounds exhibited effective % inhibition of pan HDAC
activity at 10 µM compound concentration and effective % inhibition
HDAC3 activity at 1 µM compound concentration are depicted in
Table 2.
2.2.2. HDAC isoform inhibition
From the result of initial screening based on their inhibition potency,
we have further selected compounds 5e and 5f for the determination of
´
IC50 values which will give more precise potency and selectivity towards
It was interesting to note that all these compounds (5a-5i) exhibited
comparatively less pan-HDAC inhibitory activity compared to the
reference molecule CI994 and compound 5e showed least pan HDAC
inhibition in the series. However, in case of HDAC3 inhibition, com-
pound 5e (59%) was found be most active and even better inhibitor than
CI994 (53.65%). Further, it can be inferred from the % inhibition
values, that though 5a exhibited considerable HDAC inhibitory activity
HDAC3. The selected compounds 5e and 5f along with the standard
reference CI994 were subjected to their IC50 determination towards
HDAC1, HDAC2, HDAC3, HDAC6 and HDAC8 isoforms (Fig. 3, Table 3).
Compounds 5e and 5f displayed IC50 values of 0.560 µM and 2.077
µM against HDAC3 respectively and that are the highest potency to-
wards HDAC3 compare to that of other HDAC isoforms (Table 3).
Interestingly, compound 5e was found be more effective towards
3

G. Routholla et al.
Bioorganic Chemistry 114 (2021) 105050
Scheme 2. Reagents and conditions: (a) Aniline, N-methyl 2-pyrrolidone (NMP), DIPEA, 160 ◦C, 20 h, reflux (b) NaOH, MeOH, H2O, 1 h (c) tert-butyl 2-amino
phenyl carbamate, EDCI, DMAP, DCM: pyridine (1:1), RT (d) 4 M HCl in dioxane, 0 ◦C, 2 h.
Table 1
Table 2
Structures of the designed compounds along with their % yield and physico-
The % inhibition values of enzymatic activity by the synthesized compounds as
well as the reference molecule CI994 against Hela nuclear extract (Pan-HDAC
activity) and recombinant HDAC3 enzymes. Data represents mean ± SD (n = 2).
chemical properties.
Compound
Pan-HDAC
HDAC3
% Inhibition (at 10 µM)
% Inhibition (at 1 µM)
Compound
R
% yield
87.00
HBD
2
HBA
2
Log P
2.04
5a
38.46 ± 1.68
15.11 ± 2.27
32.88 ± 0.95
22.40 ± 3.12
12.9 ± 0.85
31.67 ± 1.07
22.42 ± 4.15
27.86 ± 0.56
17.78 ± 0.61
53.8 ± 0.91
19.35 ± 1.17
22.74 ± 0.24
33.06 ± 1.88
21.83 ± 0.83
59 ± 0.46
5a
5b
5c
5d
5e
5b
70.00
2
2
3.03
5f
21.13 ± 2.67
21.38 ± 2.75
34.45 ± 2.81
7.93 ± 1.25
53.65 ± 1.21
5g
5h
5i
5c
5d
71.85
55.00
2
2
2
3
3.03
1.87
CI994
other HDAC isoforms tested. Conclusively, compound 5e is the most
potent HDAC3 inhibitor and has been considered to be a lead molecule
from this series with excellent selectivity towards HDAC3 while
compared to other compounds including reference compound CI994.
5e
5f
80.00
70.00
2
3
3
3
1.87
1.58
2.2.3. Anti-proliferative assay against cancer cell lines
5 g
57.30
3
3
1.58
All these final compounds (5a-5i) along with the reference molecule
CI994 were evaluated for their antiproliferative activity against human
triple-negative breast cancer cell line (MDA-MB-231), mouse breast
cancer cell line (4 T1) and murine melanoma cancer cell line (B16F10)
by MTT assay (Fig. 4A-4C).
5 h
5i
86.00
88.80
2
3
2
5
1.96
1.97
Initially, all the synthesized compounds were tested at two different
concentrations (100
μM and 10 μM) in duplicate taking CI994 as
reference molecule (supporting Figure S3 – S6) to have an idea about
the cell growth inhibition potential of the compounds. Based on the
results from initial two dose screen the IC50 values of all the compounds
were determined with a wider range of compound concentrations. The
compounds displayed effective anticancer efficacies against the tested
cancer cell lines with good selectivity for cancer cell lines over normal
cell lines (Table 4).
HDAC3 than even the reference molecule CI994 (IC50 = 0.902 µM). The
IC50 determining dose response curves of the selected compounds 5e and
5f against different HDAC isoforms are shown in the Fig. 3. It was
interesting to observe that the reference molecule CI994 was nonse-
lective towards HDAC3 over HDAC1 and HDAC2 (Table 3). However,
Compound 5e bearing a 6-quinolinyl moiety as the cap group was found
to be highly potent HDAC3 inhibitor (IC50 = 560 nM) and displayed 46-
fold selectivity for HDAC3 over HDAC2 and 33-fold selectivity for
HDAC3 over HDAC1. Interestingly, there was about 4-fold reduction of
HDAC3 inhibition (IC50 = 2.077 µM) compared to the former one when
the quinolone cap group of 5e was replaced by 6-indolyl cap in com-
pound 5f but still retained a minimum of 5-fold HDAC3 selectivity over
The data signifies that the antiproliferative activity of all the com-
pounds was comparable to the reference CI994 in all the cancer cell lines
tested. In fact, some of these compounds (Compounds 5a, 5b, 5d and 5i)
resulted in more or less similar anticancer efficacy to CI994 (Table 4).
Regarding the cytotoxicity against mouse breast cancer cell line 4T1,
only compounds 5b and 5f exhibited better cytotoxicity (IC50 of 13.25
and 14.30 µM, respectively) than CI994 (IC50 = 16.74 µM). However, in
case of cytotoxicity against murine melanoma cancer cell line B16F10,
several compounds (compounds 5a, 5b, 5c, 5i and 5 h) also yielded
better cytotoxicity compared to CI994 (IC50 = 14.34 µM) (Table 4). The
4

G. Routholla et al.
Bioorganic Chemistry 114 (2021) 105050
Fig. 3. The dose response curve for IC50 determination of compounds 5e and 5f using CI994 as positive control. (A) Using kit based human recombinant HDAC1
enzyme assay. (B) Using kit based human recombinant HDAC2 enzyme assay. (C) Using kit based human recombinant HDAC3/NCOR1 enzyme assay. (D) Using kit
based human recombinant HDAC8 enzyme assay. (E) Using kit based human recombinant HDAC6 enzyme assay [IC50 determinations were carried out in the
concentration range of 0.061 – 80 µM of the compounds in duplicate as per the protocol given in the HDAC enzyme assay kits purchased from bio nova. The IC50
values of these compounds were determined using nonlinear regression analysis method using Graph Pad Prism 5. Data represent mean ± SD (n = 2)].
Table 3
The IC50 values of compounds 5e and 5f using CI994 as positive control on human recombinant HDAC1, 2, 3, 8 and 6 isoforms and their HDAC3 selectivity profile. Data
represent mean ± SD (n = 2).
Cpd
IC50 (µM)
Selectivity for HDAC3 over other HDACs
HDAC1
HDAC2
HDAC3
HDAC8
HDAC6
HDAC1/3
HDAC2/3
HDAC8/3
HDAC6/3
CI994
5e
0.929±0.06
18.75±1.98
11.36±0.87
0.93±0.09
26.03±1.2
11.43±0.89
0.902±0.14
0.560±0.11
2.077±0.69
19.57±2.1
10.47±0.71
10.34±1.01
40.58±3.2
44.42±2.31
29.37±1.32
1.03
33.48
5.47
1.03
46.48
5.5
21.7
18.7
4.98
44.99
79.32
14.14
5f
β-naphthyl derivative (compound 5c) and pyrazine derivative (com-
pound 5i) resulted in potent cytotoxicity in B16F10 cell line with an IC50
of 9.26 and 9.39 µM, respectively (Table 4). Again, in case of cytotox-
icity in breast cancer cell line, MDA-MB-231, compounds 5b, 5c, 5d and
5g displayed better efficacy than CI994 (IC50 = 15.14 µM) (Table 4).
Among these molecules, compound 5c was the most cytotoxic one (IC50
2.2.5. Induction of histone hyper-acetylation in B16F10 cells: Western blot
analysis
The cellular HDAC inhibitory activity of compounds 5e and 5f along
with reference molecule CI994 was carried out to find the compounds
ability to induce histone acetylation in B16F10 cells by western blot
analysis. The histone acetylation level was measured in a dose-
dependent manner with compounds 5e and 5f on H3K9 and H4K12 as
endogenous histone substrates. Treatment of compounds 5e, 5f and
CI994 was carried out for 12 h at concentrations of 5 µM and 20 µM that
induced significant acetylation of H3K9 and H4K12 in a dose-dependent
manner (Fig. 5 and Fig. 6). The upregulation of histone acetylation was
in consistent with the in vitro HDAC inhibitory activity and also with the
cellular antiproliferative activity.
= 7.92 µM). Interestingly, compound 5b (IC50 values 11.81
MB-231 and 12.77 M in B16F10 cells) and compound 5c (IC50 values
7.92 M in MDA-MB-231 and 9.26 M in B16F10 cells) exhibited higher
IC50 values than CI994 (IC50 values 15.14 M in MDA-MB-231 and
14.34 M in B16F10 cells) but showed least enzyme inhibitory potency.
μM in MDA-
μ
μ
μ
μ
μ
Though compounds 5e and 5f exhibited higher HDAC3 inhibitory po-
tency and selectivity than remaining compounds, they were found to be
moderately active against MDA-MB-231, 4T1 and B16F10 cells. All the
remaining compounds exhibited comparable IC50 values against MDA-
MB-231 cells and B16F10 cells (Fig. 4A and 4C). However, in case of
4T1 cells, the IC50 values for the remaining compounds were found to be
higher than that of the reference compound CI994 (Fig. 4B).
2.2.6. Nuclear staining assay
Further to study the phenotype effect of compounds in cells, nuclear
staining assay was performed using DAPI and AO as staining dyes. It was
observed that the treatment of B16F10 cells with 5e, 5f and CI994 for
48 h has shown distinct difference in cell morphology when compared to
the untreated cells as evident from the Fig. 7. These observations indi-
cate nuclear disintegration of treated cells and suggested apoptotic cell
death mechanism.
2.2.4. In vitro cytotoxicity against normal HEK293 cell lines
Furthermore, the cellular toxicity of all the compounds were tested
against normal human embryonic kidney (HEK293) cell line for their
IC50 determination (Fig. 4D). The compounds were found to be less
cytotoxic towards HEK293. Interestingly, compounds 5e and 5f with the
IC50 values of 1.08 mM and 753.3 µM were found to be significantly less
toxic than all the other compounds tested and consequently possessed
higher selectivity towards all the cancer cell lines over normal HEK293
cells (Table 4).
2.2.7. Apoptosis assay
Several reports have established that HDAC inhibitor-mediated cell
death follows apoptotic pathway [54]. In order to determine the extent
of apoptosis induced by the lead compounds 5e and 5f, Annexin-V/
FITC–PI apoptotic assay was performed. B16F10 cells were treated with
compounds 5e, 5f and CI994 as reference compound for 72 h with the
concentrations at their respective IC50 values. The cells were then
5

G. Routholla et al.
Bioorganic Chemistry 114 (2021) 105050
Fig. 4. Dose response curve and IC50 values of all novel compounds (5a-5i) along with reference molecule CI994 as positive control on (A) MDA-MB-231, (B) 4T1,
(C) B16F10 cells when treated with the compounds at concentration range of 0.7–200 µM (n = 2) for 72 h. Figure D represents in vitro cytotoxicity data on HEK293
cells when treated with the compounds at concentration range of 7–2000 µM (n = 2) for 72 h. Data represent mean ± SD. IC50 values were calculated using nonlinear
regression analysis method using Graph Pad Prism 5.
Table 4
The tabular presentation of IC50 (µM) values of these novel benzamides against cancer cell lines 4T1, B16F10 and MDA-MB-231 and normal cell line HEK293 and their
selectivity profile for cancer cell lines over normal cell line. Data represent mean ± SD (n = 2).
Compound
IC50 (µM)
Selectivity for cancer cells over normal cell line
4T1
B16F10
MDA-MB-231
HEK293
HEK293/4T1
HEK293/B16F10
HEK293/ MDA-MB-231
5a
18.92 ± 1.2
13.25 ± 0.87
36.73 ± 1.54
20.56 ± 1.35
24.15 ± 0.89
14.30 ± 0.65
51.83 ± 2.35
50.62 ± 3.45
22.61 ± 1.65
16.74 ± 1.65
11.5 ± 0.98
12.77 ± 1.23
9.26 ± 0.69
22.04 ± 0.86
17.89 ± 1.31
26.40 ± 2.14
15.49 ± 1.03
11.58 ± 0.78
9.39 ± 0.68
14.34 ± 0.99
16.58 ± 1.54
11.81 ± 0.87
7.92 ± 0.89
13.27 ± 1.68
24.14 ± 3.54
32.93 ± 3.68
11.07 ± 1.56
19.03 ± 2.34
18.82 ± 1.98
15.14 ± 1.41
279.7 ± 8.76
441 ± 7.69
14.78
33.28
5.09
24.32
34.53
20.20
20.24
60.59
28.53
48.44
35.31
26.69
15.07
16.87
37.34
23.62
33.61
44.90
22.87
67.78
21.48
13.32
14.28
5b
5c
187.1 ± 4.54
446.1 ± 8.65
1084 ± 10.6
753.3 ± 6.8
750.4 ± 10.8
408.9 ± 2.4
250.7 ± 6.9
216.2 ± 8.9
5d
21.69
44.88
52.67
14.47
8.07
5e
5f
5 g
5 h
5i
11.08
12.92
CI994
processed and the analysis was carried out using flow cytometry anal-
ysis. Fig. 8 displays the obtained results which suggest a significant
enhanced apoptotic activity in treated cells with compounds 5e and 5f
when compared to CI994. Compound 5e displayed the total apoptotic
percentage as 20.65% ± 0.49 (Q2 and Q4), whereas increased apoptotic
population was observed for compound 5f with 35.35% ± 2.89 (Q2 and
Q4) when compared to CI994 with 16.8% ± 0.21 of apoptosis. These
results suggest the programmed cell death mechanism induced by
compounds 5e and 5f leading to significant apoptosis in cancer cells.
These results (Table 5) indicated the increased cell population in G2/M
phase of compound 5e (45.14%) and compound 5f (48.66%) when
compared to control and a similar tendency was observed with CI994
(31.74%).
The study also suggested that the cell cycle arrest at G2/M phase of
the cell cycle with increased cell population containing 4n of DNA
content. It was observed that compounds 5e and 5f showed a decrease in
G1 population (31.11% and 26.28%) with no significant change in S
phase (23.75% and 25.06%) when compared to reference molecule
CI994 (G1 = 43.83% and S = 24.43%). These results further emphasize
the promising anticancer activity of the lead compounds (5e and 5f)
which might be guided through cell cycle arrest at G2/M phase. It also
supports the apoptotic assay data of programmed cell death.
2.2.8. Cell cycle analysis
In continuation to the results obtained in the apoptosis assay, the cell
cycle progression was studied with the compounds 5e, 5f and CI994
treatment of B16F10 cells and the cell population at different cell cycle
stages was analysed using flow cytometry analysis (Fig. 9).
In the cell cycle study, B16F10 cells were treated with compounds 5e
and 5f and reference compound CI994 at 15 µM concentration for 72 h.
2.3. Molecular docking study
In order to understand the probable binding mode of interactions of
6

G. Routholla et al.
Bioorganic Chemistry 114 (2021) 105050
Fig. 5. Western blot of Ac-H3K9 in whole cell lysates of B16F10 murine melanoma cells after treatment with (A) compound 5e, (B) compound 5f and (C) CI994 at 5
µM and 20 µM for 12 h. Results were normalized with respect to β-actin as a housekeeping control.
Fig. 6. Western blot of Ac-H4K12 in whole cell lysates of B16F10 murine melanoma cells after treatment with (A) compound 5e, (B) compound 5f and (C) CI994 at
5 µM and 20 µM for 12 h. Results were normalized with respect to β-Actin as housekeeping control.
the promising HDAC3 inhibitors (compounds 5e and 5f) with HDAC3
enzyme (PDB: 4A69), molecular docking studies were performed by
using Schrodinger software [55]. The docked structures of these com-
pounds (compounds 5e and 5f) along with the reference molecule
(CI994) are found to be almost superimposed with the each other on the
active site of the HDAC3 as depicted in Fig. 10.
better HDAC3 inhibitory property of compound 5e over compound 5f
can be explained in terms of binding mode of interactions of these
compounds with HDAC3. The orientation of the 6-quinolinyl moiety
(compound 5e) at the HDAC3 active site makes it better suitable for
stronger binding interaction compared to the binding orientation of 6-
indolyl moiety (compound 5f). It is important to note that the position
or orientation of the heterocyclic nitrogen atom of 6-quinolinyl moiety is
more or less, closer to the amide nitrogen of CI994. However, it is
noticed that the heterocyclic nitrogen atom of 6-indolyl moiety is ori-
ented completely opposite direction of the former ones. Therefore, 6-
quinolinyl moiety is favourable than the 6-indolyl scaffold as far as
the HDAC3 inhibitory potency and selectivity is concerned.
These inhibitors snugly bind to the binding groove and occupy the
pocket as shown in Fig. 10. Interestingly, the docking scores are corre-
lated with our in vitro HDAC3 assay results of compound 5e (glide score:
ꢀ 6.109; HDAC3 IC50 = 0.560 µM), 5f (glide score: ꢀ 5.652; HDAC3 IC50
= 2.077 µM) and reference compound CI994 (glide score: ꢀ 5.977;
HDAC3 IC50 = 0.902 µM).
The carbonyl group of both compounds form a hydrogen bonding
interaction with Tyr298 of HDAC3 (Fig. 11).
3. Conclusion
Another hydrogen bonding interaction is noticed between the –NH
function of benzamide and Gly143 of HDAC3. As seen in Fig. 11, both
A series of linker-less benzamides with different aryl/heteroaryl cap
moieties were designed and synthesized as promising HDAC3 inhibitors.
All the compounds were studied for their pan-HDAC and HDAC3
these inhibitors form a π-π stacking interaction with Phe144. Though
there are similar binding modes of interactions of these compounds, the
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G. Routholla et al.
Bioorganic Chemistry 114 (2021) 105050
Fig. 7. Nuclear staining of B16F10 cells using DAPI (4′,6-diamidino-2-phenylindole, a fluorescent stain) and acridine orange (AO) following treatment by (A) Control
(B) CI994 (C) compound 5e and (D) compound 5f. The stained nuclei are visualised using fluorescence microscope (Leica microsystems, Germany) on 20x
magnification.. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 8. Induction of apoptosis in B16F10 cells quantified by Annexin V/PI assay using flow cytometry. (A) Vehicle control (B) CI994 (C) 5e (D) 5f (Q1 – Necrotic cells,
Q2 - late apoptosis, Q3 – Live cells, Q4 – early apoptotic cells, X-axis: Annexin V intensity, Y-axis: Propidium iodide intensity).
inhibitory activity. Further, the selected lead compounds 5e and 5f were
studied for their HDAC 1, 2, 3, 8 and 6 enzyme inhibitory profiles to
judge the selectivity towards HDAC3. Two lead compounds 5e and 5f
were found to be potent and highly selective HDAC3 inhibitors over
other Class-I HDACs and HDAC6. Compound 5e bearing a 6-quinolinyl
moiety as the cap group was found to be a highly potent HDAC3
inhibitor (IC50 = 560 nM) and displayed 46-fold selectivity for HDAC3
over HDAC2, and 33-fold selectivity for HDAC3 over HDAC1. Moreover,
the HDAC3 selectivity of the lead molecules found to be much better
than the reference compound CI994. All these compounds exhibited
effective antiproliferative activity against various cancer cell lines (4T1,
B16F10 and MDA-MB-231) with less cytotoxicity against normal cells
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Bioorganic Chemistry 114 (2021) 105050
Fig. 9. Cell cycle arrest induced in murine melanoma cell B16F10 treated with (A) Control (B) CI994 as positive control (C) compound 5e and (D) compound 5f for
72 h. After the indicated treatment times, cell cycle analysis was performed and analysed by flow cytometry analysis (BD Aria III) ®.
Table 5
The % cell population in different phases of cell cycle. Data was calculated and
analysed by FCS express plus software from DNA content histograms.
Cell cycle Phase
% Cell population
Control
CI994
5e
5f
G1
54.27
28.20
17.53
43.83
24.43
31.74
31.11
23.75
45.14
26.28
25.06
48.66
S
G2/M
(HEK293) when compared with reference compound CI994 and inter-
estingly the most promising molecule (5e) showed least toxicity towards
normal cells. Further, the acetylation levels of cellular histone (H3K9
and H4K12) were examined and both the lead compounds were able to
enhance the acetylation level significantly in a dose-dependent manner
in B16F10 cells. Moreover, compounds 5e and 5f were found to cause
apoptotic cell death and were causing G2/M cell cycle phase arrest in
B16F10 cells. The molecular docking study revealed similar binding
interactions of the lead molecules and reference compound at the active
site of HDAC3. The higher HDAC3 inhibitory potency along with
selectivity for HDAC3 of compound 5e over compound 5f was also
justified by the molecular docking analysis. Based on the findings, it can
be inferred that most potent and HDAC3 selective lead molecule 5e
might serve as a potential therapeutic as anticancer agent.
Fig. 10. The docked conformations of compounds (5e, 5f and CI994) super-
imposed with each other at the active site of HDAC3 (PDB: 4A69).
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Bioorganic Chemistry 114 (2021) 105050
Fig. 11. Molecular docking interactions of (A) compound 5e and (B) compound 5f with HDAC3 (PDB: 4A69)
4. Experimental
as per the experimental protocol given in the enzyme kits of pan-HDAC
(cat# BML-AK501), HDAC1 (cat# BML-AK511), HDAC2 (cat# BML-
AK512), HDAC3/NCoR1 (cat# BML-AK531-0001), HDAC8 (cat# BML-
AK518) from Enzo life sciences ltd. and HDAC6 (cat# K465-100) from
Biovision Ltd. that were purchased from Bionova suppliers, Hyderabad.
The absorbance for MTT assay and HDAC enzyme inhibition was
measured using a microplate reader (Spectramax™, Molecular Devices).
4.1. General information on materials and instrumentation
All starting materials, chemicals and reagents were commercially
available and were purchased from various chemical suppliers. These
were used without further purification. All reactions were monitored by
thin layer chromatography (TLC) using precoated plates with Merck 60
F254 silica gel plates purchased from Merck Millipore Co., USA and the
reaction components were visualised under ultraviolet light (254 nm).
Column chromatography was performed on silica gel (100 – 200 or 230
– 400 mesh size). 1H and 13C NMR spectrum were recorded in deuterated
NMR solvents DMSO–d6 and CDCl3 using Bruker, ASCEND™ 400 MHz
spectrometer and the chemical shifts (δ) values are given in parts per
million (ppm), and are internally referenced to tetramethylsilane (TMS),
residual solvents peak (DMSO‑d6; 2.50 ppm 1H, 39.51 ppm 13C, CDCl3;
7.2 ppm 1H, 77.6 ppm 13C). Peak multiplicities are abbreviated as fol-
lows: s (singlet), d (doublet), t (triplet), q (quartet) and m (multiplet)
while coupling constants (J) are reported in Hz. NMR data were pro-
cessed using MestReNova Software version 6.0.2–5475.
4.2. Chemistry
All starting materials and reagents were commercially available and
used without further purification. All reactions were monitored by thin
layer chromatography (TLC) using pre-coated plates with silica gel F254
from Merck Millipore Co., USA. 1H and 13C NMR spectrum were recor-
ded in DMSO–d6 and CDCl3 using Bruker-400 MHz and chemical
shifts were reported in ppm using tetramethylsilane (TMS) as inter-
nal standard. Mass spectroscopy was performed in HRMS (6545 Q-TOF
LC/MS, Agilent) at Bits-Pilani, Pilani campus.
4.2.1. Preparation of tert-butyl 2-(benzamido) phenyl carbamate (4a)
Compound (3a) (100 mg; 0.819 mmol) was dissolved in the solution
of dichloromethane and pyridine (1:1) stirred at room temperature in
nitrogen environment. To this reaction mixture, 1-ethyl-3-dimethyl
amino propyl carbodiimide (228 mg; 1.47 mmol) and catalytic
amount of 4-dimethyl amino pyridine were added. This mixture was
stirred at room temperature for 20 min. After 20 min, tert-butyl 2-amino
phenyl carbamate (187 mg; 0.90 mmol) was added into the reaction
mixture and reaction was continued to 12 h. After completion of the
reaction, pyridine was then evaporated under vacuum. The mixture was
then dissolved in ethyl acetate and washed with sodium bicarbonate.
The organic layer was then separated and dried with Na2SO4. The dried
solvent was evaporated under vacuum. The crude product was then
purified using column chromatography (solvent system – hexane and
ethyl acetate (70:30)) in silica 230 – 400 mesh to obtain the final
compound 4a, in its pure form (Yield 58%).1H NMR (400 MHz, CDCl3) δ
9.15 (s, 1H), 7.95 (d, J = 8.4 Hz, 2H), 7.76 (d, J = 7.6 Hz, 1H), 7.55 (t, J
Three different cell lines were used for the determination of anti-
cancer activity of the novel compounds synthesized. MDA-MB-231
(human breast cancer cell line), 4T1 (murine mammary carcinoma cell
line), B16F10 (Murine melanoma cell line) and HEK293 (Human em-
bryonic kidney cell line) were procured from National Centre for Cell
Science (NCCS), Pune, India. B16F10, MDA-MB-231 and HEK293 cell
lines were cultured in DMEM (high glucose media: AL007S, Dulbecco’s
modified eagle medium) and 4 T1 cell line was cultured in MEM (AT154,
Minimum essential medium). All these cell lines were used for cell-
culturing with 10% fetal bovine serum (FBS) and 1% antibiotic (Pen
strep: A001) and were incubated at 37 ◦C and 5% CO2 atmosphere.
Dulbecco’s phosphate buffered saline (PBS), foetal bovine serum (FBS),
antibiotic solution 100 × liquid with 10,000 U penicillin and 10 mg
streptomycin/ml, trypsin, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl
tetrazolium bromide (MTT) were supplied by Himedia Laboratories Pvt.
Ltd., (Mumbai, India). HDAC enzyme inhibition assays were performed
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Bioorganic Chemistry 114 (2021) 105050
= 7.2 Hz 1H), 7.47 (t, J = 7.6 Hz, 2H), 7.23 (m, 3H), 6.84 (s, 1H), 1.51 (s,
9H). C18H20N2O3 [M]: 312.36; MS (ESI) m/z: [M + H]+:313.24
0.955 mmol) was added into the reaction mixture and reaction was
continued to 12 h. The reaction was monitored with TLC. After
completion of the reaction, pyridine was then evaporated under vac-
uum. The mixture was then dissolved in sodium bicarbonate and
extracted with ethylacetate. The organic layer was then separated and
dried with Na2SO4. The dried solvent was evaporated under vacuum.
The crude product was then purified using column chromatography
(solvent system – hexane and ethylacetate (50:50)) in silica 230 – 400
mesh to obtain the final compound 4c in its pure form. (yield: 91%).1H
NMR (400 MHz, CDCl3) δ 9.36 (s, 2H), 8.49 (s, 1H), 8.01 (d, J = 10.0
Hz,1H), 7.93 (dd, J = 8, 8.8 Hz,3H), 7.83 (s, 1H), 7.56 (m, 2H), 7.25 (t, J
= 4.5 Hz, 2H), 7.16 (m, 1H), 6.99 (t, J = 7.6 Hz, 1H), 6.77 (dd, J =
8.0,8.4 Hz,1H), 1.52 (s, 9H). C22H21FN2O3[M]: 362.42; MS (ESI) m/z:
[M + H]+: 363.24.
4.2.2. Preparation of N-(2-amino phenyl) benzamide (5a)
The compound tert-butyl-2-(benzamido) phenyl carbamate (4a)
(120 mg; 0.384 mmol) was dissolved in dichloromethane (5 ml) and the
solution of 4 M dioxane in HCl was added to it at 0 ◦C. This mixture was
then allowed to react under constant stirring at room temperature for 2
h. The dioxane was then evaporated under vacuum. The resulting
compound was then dissolved in ethyl acetate and washed with water.
The organic layer was then separated, dried with Na2SO4 and solvent
was evaporated under vacuum. The mixture was then washed with
pentane to obtain boc-deprotected final compound 5a. (yield: 87%).1H
NMR (400 MHz, CDCl3) δ 8.01 (s, 1H), 7.88 (d, J = 7.88 Hz 2H), 7.54 (d,
J = 7.6 Hz 1H), 7.45 (t, J = 7.6 Hz 2H), 7.28 (d, J = 7.6 Hz 1H), 7.07 (t, J
= 7.6 Hz 1H), 6.83 (t, J = 5.2 Hz 2H), 3.69 (s, 2H).13C NMR (101 MHz,
4.2.6. Preparation of N-(2-amino phenyl)-2-naphthamide (5c)
CDCl3)
δ
165.87, 140.83, 134.17, 131.93, 128.75, 128.73,
The compound tert-butyl-2-(2-naphthamido) phenyl carbamate (4c)
(100 mg; 0.276 mmol) was dissolved in dichloromethane (5 ml) and
treated with the solution of 4 M Dioxane in HCl at 0 ◦C. This mixture was
then allowed to react under constant stirring at room temperature for 2
h. The reaction completion was monitored by TLC. After completion of
the reaction, solvent was then evaporated under vacuum. The resulting
compound was then dissolved in water and extracted with ethyl acetate.
The organic layer was then separated, dried with Na2SO4 and solvent
was evaporated under vacuum. The mixture was then washed with
pentane to obtain boc-deprotected final compound 5c. (yield:
71.85%).1H NMR (400 MHz, DMSO‑d6) δ 9.86 (s, 1H), 8.64 (s, 1H), 8.05
(m, 4H), 7.63 (m, 2H), 7.26 (d, J = 7.6 Hz, 1H), 7.01 (t, J = 8.0 Hz, 1H),
6.84 (d, J = 8.0 Hz, 1H), 6.64 (t, J = 6.8 Hz, 1H), 5.04 (s, 2H). 13C NMR
(101 MHz, DMSO‑d6) δ 165.90, 143.65, 134.70, 132.55, 129.40, 128.60,
128.33 – 127.83, 127.51 – 126.70, 125.11, 123.85, 116.69. C17H14N2O
[M]: 262.30; MS (ESI) m/z: [M + H]+: 263.24.
127.32,127.29, 125.39, 124.72, 119.73, 118.34.
C13H12N2O[M]:
212.24; MS (ESI) m/z: [M + H]+ :213.24
4.2.3. Preparation of tert-butyl 2-(1-naphthamido) phenyl carbamate (4b)
1-Naphthoic acid (3b) (150 mg; 0.872 mmol) was dissolved in the
solution of dichloromethane and pyridine (1:1) followed by the addition
of 1-ethyl-3-dimethyl amino propyl carbodiimide (243.28 mg; 1.569
mmol) and catalytic amount of 4-dimethylamino pyridine. The reaction
mixture was stirred at room temperature for 20 min. After 20 min, tert-
butyl 2-amino phenyl carbamate (199 mg; 0.959 mmol) was added into
the reaction mixture and reaction was continued to 12 h. The reaction
mixture was monitored by TLC. After completion of the reaction, pyri-
dine was then evaporated under vacuum. The mixture was then dis-
solved in ethyl acetate and washed with sodium bicarbonate. The
organic layer was then separated and dried with Na2SO4. The dried
solvent was evaporated under vacuum that was purified on a silica gel
column to give compound as a solid. (yield: 74%).1H NMR (400 MHz,
CDCl3) δ 8.58 (s, 1H), 8.47 (d, J = 8.0 Hz, 1H), 7.95 (d, J = 7.94 Hz 1H),
7.89 (d, J = 8.8 Hz 1H), 7.77 (d, J = 6.8 Hz 2H), 7.57 (m, 2H), 7.48 (t, J
= 7.6 Hz 1H), 7.39 (d, J = 9.2 Hz 1H), 7.24 (m, 2H), 6.92 (s, 1H), 1.43 (s,
9H). C22H22N2O3[M]: 361.43; MS (ESI) m/z: [M + H]+:362.24.
4.2.7. Preparation of tert-butyl 2-(quinolone-3-carboxamido) phenyl
carbamate (4d)
3-Quinoline carboxylic acid (3d) (50 mg; 0.289 mmol) was dissolved
in dichloromethane and pyridine (5 ml) at 0 ◦C. To this mixture, 1-ethyl-
3-dimethylaminopropylcarbodiimide (80 mg; 0.520 mmol) and 3 mg of
4-dimethylaminopyridine were added. The mixture was allowed to react
under constant stirring for 15 min before tert-butyl 2-amino phenyl
carbamate (65.936 mg; 0.317 mmol) was added into the reaction
mixture. This was allowed to react under constant stirring at room
temperature for 12 h. The pyridine was then evaporated under vacuum.
The mixture was then dissolved in ethyl acetate and washed with water.
The organic layer was then separated and concentrated under vacuum.
The crude product was then purified using column chromatography
(solvent system - hexane and ethylacetate (70: 30)) in silica 60–120
mesh to obtain the final compound 4d in its pure form. (yield 76%). 1H
NMR (400 MHz, CDCl3) δ 9.73 (s, 1H), 9.48 (s, 1H), 8.75 (s, 1H), 8.17 (d,
J = 8.4 Hz, 1H), 7.92 (m, J = 8.4 Hz, 3H), 7.62 (t, J = 7.2 Hz, 1H), 7.26
(m, J = 1.2 Hz, 3H), 6.95 (s, 1H), 1.26 (s, 9H). C21H21N3O3 [M]: 363.15;
MS (ESI) m/z: [M + H]+: 364.245.
4.2.4. Preparation of N-(2-amino phenyl)-1-naphthamide (5b)
The compound tert-butyl-2-(1-naphthamido) phenyl carbamate (4b)
(70 mg; 0.269 mmol) was dissolved in dichloromethane (5 ml). The
◦
mixture was stirred at 0 C. To the reaction mixture solution of 4 M
dioxane in HCl was added to it at same temperature. This mixture was
then allowed to react under constant stirring at room temperature for 2
h. After completion of the reaction, dioxane was evaporated under
vacuum. The resulting compound was quenched with water and
extracted with ethyl acetate. The organic layer was then separated, dried
with Na2SO4 and solvent was evaporated under vacuum. The mixture
was then washed with pentane to obtain boc-deprotected final com-
pound 5b. (yield: 70%).1H NMR (400 MHz, DMSO‑d6) δ 9.84 (s, 3H),
8.33 (d, J = 7.6 Hz,1H), 8.09 (dd, J = 8.4,8.8 Hz,2H), 7.86 (d, J = 6.8
Hz,1H), 7.62 (m, 3H), 7.38 (d, J = 7.6 Hz,1H), 7.01 (t, J = 7.2 Hz,1H),
6.84 (d, J = 7.6 Hz,1H), 6.68 (t, J = 7.6 Hz,1H), 5.05 (s,2H).13C NMR
(101 MHz, DMSO‑d6) δ: 167.84, 142.82, 135.16, 133.64, 130.39,
128.75, 127.33, 127.10 – 126.39, 126.16, 126.16, 125.98, 125.50,
4.2.8. Preparation of N-(2-aminophenyl) quinolone-3-carboxamide (5d)
The compound tert-butyl 2-(quinolone-3-carboxamido) phenyl
carbamate (4d) (100 mg; 0.275 mmol) was dissolved in 5 ml DCM and 4
M dioxane HCl was added to it at 0 ◦C. The mixture was then allowed to
react under constant stirring at room temperature for 2 h. The dioxane
was then evaporated under vacuum. The mixture was then dissolved in
ethylacetate and washed with water. The organic layer was then sepa-
rated and concentrated under vacuum. The mixture was then washed
with pentane to obtain final compound 5d. (yield 55%) 1H NMR (400
MHz, DMSO‑d6) δ 10.00 (s, 1H), 9.40 (s, 1H), 9.00 (s, 1H), 8.13 (t, J =
8.4 Hz 2H), 7.90 (d, J = 7.2 Hz, 1H), 7.74 (s, J = 7.2 Hz, 1H), 7.24 (d, J =
7.2 Hz, 1H), 7.02 (s, J = 7.2 Hz, 1H), 6.83 (d, J = 7.6 Hz, 1H), 6.64 (s, J
123.91, 116.84. C17H14N2O [M]: 262.30; MS (ESI) m/z: [M
+
H]+:263.24.
4.2.5. Preparation of tert-butyl 2-(2-naphthamido) phenyl carbamate (4c)
To a solution of dichloromethane and pyridine (1:1) was added to 2-
naphthoic acid (3c) (100 mg; 0.581 mmol) to this reaction mixture 1-
ethyl-3-dimethyl amino propyl carbodiimide (243 mg; 1.56 mmol)
and catalytic amount of 4-di methyl amino pyridine were added in ni-
trogen environment. The mixture was stirred at room temperature for
15 min. After 15 min tert-butyl 2-amino phenyl carbamate (216.80 mg;
=
7.6
Hz,
1H).13
C
NMR
(101
MHz,
DMSO‑d6)
δ:
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G. Routholla et al.
Bioorganic Chemistry 114 (2021) 105050
164.5,149.79,149.02,148.93,143.20, 136.60, 131.70, 129.64, 129.25,
127.89, 127.33 ,127.38,126.99,123.3,116.78, 116.56, 40.19.
C16H13N3O [M]: 263.10; MS (ESI) m/z: [M + H]+:264.14.
carbamate (4f) (140 mg; 0.398 mmol) was dissolved in 5 ml DCM and 4
M dioxane HCl was added to it at 0 ◦C. This mixture was then allowed to
react under constant stirring at room temperature for 4 h. The dioxane
was then evaporated under vacuum. The mixture was then dissolved in
ethyl acetate and washed with water. The organic layer was then
separated and the excess solvent was evaporated under vacuum. The
mixture was then washed with pentane to obtain boc-deprotected final
compound. (yield: 70%) 1H NMR (400 MHz, DMSO‑d6) δ 11.68 (s, 1H),
9.20 (s, 1H), 8.25 (s, 1H), 8.18 (d, 1H), 7.46 (dd, J = 3.6,3.6 Hz 1H),
7.13 (dd, J = 2.4,3.6 Hz 3H), 6.95 (t, 1H), 6.81 (d, 1H), 6.62 (t, 1H), 4.89
(s, 2H). 13C NMR (101 MHz, DMSO‑d6) δ: 163.74, 143.32, 136.61,
134.79, 129.11, 126.80, 126.23, 124.48, 122.46, 121.53, 120.98,
116.82, 112.34, 110.81. C15H13N3O [M]: 251.10; MS (ESI) m/z: [M +
H]+:252.14.
4.2.9. Preparation of tert-butyl 2-(quinolone-6-carboxamido) phenyl
carbamate (4e)
6-Quinoline carboxylic acid (3e) (100 mg; 0.578 mmol) was dis-
solved in dichloromethane: pyridine in the ratio of 1 : 1 (5 ml). To this
mixture, 1-ethyl-3-dimethyl amino propyl carbo di imide (83.23 mg;
1.04 mmol) and 3 mg of 4-dimethylaminopyridine were added. This
mixture was allowed to react under constant stirring for 15 min before
tert-butyl 2-amino phenyl carbamate (132.08 mg; 0.635 mmol) was
added into the reaction mixture. This was allowed to react under con-
stant stirring at room temperature overnight. The pyridine was then
evaporated under vacuum. The mixture was then dissolved in ethyl
acetate and washed with sodium bicarbonate. The organic layer was
then separated and the excess solvent was then evaporated under vac-
uum. The crude product was then purified using column chromatog-
raphy (solvent system- hexane and ethylacetate (60:40)) in silica
230–400 mesh to obtain the final compound 4e in its pure form. (yield:
45%).1H NMR (400 MHz, CDCl3) δ 9.49 (s, 1H), 8.94 (d, J = 2.8 Hz 1H),
8.44 (s, 1H), 8.18 (d, J = 8 Hz 2H), 8.13 (d, J = 8.8 Hz 1H), 7.82 (d, J =
7.6 Hz 1H), 7.42 (dd, J = 4.4, 4.2 Hz 1H), 7.14 (dd, J = 4.8 ,4.4 Hz 2H),
6.76 (s, 1H), 1.46 (s, 9H).C21H21N3O3[M]: 363.15; MS (ESI) m/z: [M +
H]+:364.245.
4.2.13. Preparation of tert-butyl 2-(1H-indole-3-carboxamido) phenyl
carbamate (4 g)
Indole-3-carboxylic acid (3 g) (100 mg; 0.621 mmol) was dissolved
in dichloromethane and added Pyridine (5 ml) under 0 ◦C. To this
mixture, 1-ethyl-3-dimethyl amino propyl carbodiimide, (173.25 mg;
1.11 mmol) and 5 mg of 4-dimethyl aminopyridine were added. This
mixture was allowed to react under constant stirring for 15 min before
tert-butyl 2-amino phenyl carbamate (142.08 mg; 0.683 mmol) was
added into the reaction mixture. This was allowed to react under con-
stant stirring at room temperature overnight. The pyridine was then
evaporated under vacuum. The mixture was then dissolved in ethyl-
acetate and washed with sodium bicarbonate. The organic layer was
then separated and the excess solvent was then evaporated under vac-
uum. The crude product was then purified using column chromatog-
raphy (solvent system – hexane and ethylacetate (70:30)) in silica 230 –
400 mesh to obtain the final compound 4 g in its pure form. (yield:
17%)1H NMR (400 MHz, DMSO‑d6) δ 11.81 (s, 1H), 9.47 (s, 1H), 8.59 (s,
1H), 8.19 (s, 1H), 8.13 (d J = 7.6 Hz, 1H), 7.57 (dd, J = 7.2, 7.6 Hz, 3H),
7.15 (dd, J = 6.8, 6.8 Hz 4H), 1.44 (s, 9H).C20H21N3O3[M]: 351.15; MS
(ESI) m/z: [Mꢀ H]+: 350.250.
4.2.10. Preparation of N-(2-aminophenyl) quinolone-6-carboxamide (5e)
The compound tert-butyl 2-(quinolone-6-carboxamido) phenyl
carbamate (4e) (32 mg; 0.088 mmol) was dissolved in 5 ml DCM and 4
M dioxane HCl was added to it at 0 ◦C. This mixture was then allowed to
react under constant stirring at room temperature for 4 h. The dioxane
was then evaporated under vacuum. The mixture was then dissolved in
ethylacetate and washed with sodium bicarbonate. The organic layer
was then separated and the excess solvent was evaporated under vac-
uum. The mixture was then washed with pentane to obtain boc-
deprotected final compound 5e (yield: 80%).1H NMR (400 MHz,
DMSO‑d6) δ 11.01 (s, 1H), 9.28 (s, 1H), 9.08 (s, 1H), 9.00 (d, J = 8 Hz,
1H), 8.65 (d, J = 8.4 Hz, 1H), 8.42 (d, J = 8.8 Hz, 1H), 7.99 (dd, J = 4.4,
4.4 1H), 7.66 (d, J = 7.6 Hz 1H), 7.56 (d, J = 7.2 Hz 1H), 7.46 (dd, J =
7.4 ,7.6 Hz 2H).13C NMR (101 MHz, DMSO‑d6) δ: 165.03, 148.93,
144.78, 142.44, 133.89, 131.84, 130.19, 128.58, 128.12, 127.80,
127.33, 124.66, 123.69, 123.33 – 122.99. C16H13N3O [M]: 263.10; MS
(ESI) m/z: [M + H]+: 264.14.
4.2.14. Preparation of N-(2-amino phenyl)-1H-indole-3-carboxamide (5
g)
The compound tert-butyl-2-(1H-indole-6-carboxamido) phenyl
carbamate (4 g) (32 mg; 0.09 mmol) was dissolved in 5 ml DCM and 4 M
dioxane HCl was added to it at 0 ◦C. This mixture was then allowed to
react under constant stirring at room temperature for 2 h. Dioxane was
then evaporated under vacuum. The mixture was then dissolved in
ethylacetate and washed with water. The organic layer was then sepa-
rated and excess solvent was evaporated under vacuum. The mixture
was then washed with pentane to obtain boc-deprotected final com-
pound 5 g. (yield: 57.3%).1H NMR (400 MHz, DMSO‑d6) δ 11.51 (s, 1H),
9.04 (s, 1H), 8.07 (d, J = 7.1 Hz, 2H), 7.29 (d, J = 8.0 Hz,1H), 7.04 (d, J
= 3.8 Hz, 3H), 6.78 (t, J = 6.4 Hz,1H), 6.64 (dd, J = 1.2, 1.2 Hz 1H), 6.47
(dd, J = 1.6, 1.2 Hz 1H), 4.72 (s, 2H).13C NMR (101 MHz, DMSO‑d6) δ
164.09, 136.61, 128.93, 126.82, 126.37, 124.54, 122.48, 121.52,
121.00, 116.68, 112.35, 111.17 – 110.98. C15H13N3O [M]: 251.105; MS
(ESI) m/z: [M + H]+:252.200.
4.2.11. Preparation of tert-butyl 2-(1H-indole-6-carboxamido) phenyl
carbamate (4f)
Indole-6-carboxylic acid (3f) (100 mg; 0.621 mmol) was dissolved in
dichloromethane and added Pyridine (5 ml) under 0 ◦C. To this mixture,
1-ethyl-3-dimethyl amino propyl carbodiimide (107 mg, 0.690 mmol)
and 5 mg of 4-dimethyl aminopyridine were added. This mixture was
allowed to react under constant stirring for 15 min before tert-butyl 2-
amino phenyl carbamate, (142.08 mg; 0.683 mmol) was added into
the reaction mixture. This was allowed to react under constant stirring at
room temperature overnight. Pyridine was then evaporated under vac-
uum. The mixture was then dissolved in ethylacetate and washed with
sodium bicarbonate. The organic layer was then separated and the
excess solvent was then evaporated under vacuum. The crude product
was then purified using column chromatography (solvent system –
hexane and ethylacetate (70:30)) in silica 60 – 120 mesh to obtain the
4.2.15. Preparation of tert-butyl 2-(thiophene-3-carboxamido) phenyl
carbamate (4 h)
Thiophene-3-carboxylic acid (3 h) (100 mg; 0.900 mmol) was dis-
◦
solved in dichloromethane and added pyridine (5 ml) at 0 C. To this
mixture, 1-ethyl-3-dimethyl amino propyl carbo di imide, (251 mg; 1.62
mmol) and 5 mg of 4-dimethylamino pyridine were added. This mixture
was allowed to react under constant stirring for 15 min before tert-butyl
2-amino phenyl carbamate (206 mg; 0.990 mmol) was added into the
reaction mixture. This was allowed to react under constant stirring at
room temperature overnight. Pyridine was then evaporated under vac-
uum. The mixture was then dissolved in ethylacetate and washed with
sodium bicarbonate. The organic layer was then separated and excess
1
final compound 4f in its pure form. (yield: 84%) H NMR (400 MHz,
CDCl3) δ 9.25 (s, 2H), 8.11 (s, 1H), 7.71 (d, 3H), 7.45 (dd, J = 3.6,3.6 Hz
1H), 7.33 (dd, J = 3.6, 3.6 Hz 1H), 7.22 (d, J = 2.4 Hz 2H), 6.61 (s, 1H),
1.57 (s, 9H).
4.2.12. Preparation of N-(2-amino phenyl)-1H-indole-6-carboxamide (5f)
The compound tert-butyl ꢀ 2-(1H-indole-6-carboxamido) phenyl
12

G. Routholla et al.
Bioorganic Chemistry 114 (2021) 105050
solvent was then evaporated under vacuum. The crude product was then
purified using column chromatography (solvent system – DCM and ethyl
acetate (5:95)) in silica 230 – 400 mesh to obtain the final compound 4 h
in its pure form. (yield: 43%)1H NMR (400 MHz, CDCl3) δ 9.12 (s, 1H),
8.05 (s, 1H), 7.72 (d, J = 8 Hz 1H), 7.57 (d, J = 1.2 Hz 1H), 7.38 (dd, J =
2.8, 2.8 Hz 1H), 7.21 (dd, J = 1.2, 1.6 Hz 3H), 6.84 (s, 1H), 1.52 (s, 9H).
bicarbonate and extracted with ethyl acetate. The organic layer was then
separated and dried with Na2SO4. The dried solvent was evaporated
under vacuum. The crude product was then purified using column
chromatography (solvent system – hexane and ethylacetate (50:50)) in
silica 230 – 400 mesh to obtain the final compound 4i in its pure form.
(Yield: 89%).1H NMR (400 MHz, CDCl3) δ 1H NMR (400 MHz, DMSO‑d6)
δ 9.86 (s, 2H), 9.04 (s, 1H), 8.56 (s, 1H), 8.45 (s, 1H), 7.96 (d, J = 7.6 Hz
1H), 7.77 (d, J = 8 Hz 2H), 7.42 (t, J = 7.6 Hz 2H), 7.29 (dd, J = 7.6,7.6
Hz 2H), 7.18 (t, J = 6.4 Hz 1H), 7.02 (t, J = 7.6 Hz 1H), 1.20 (s, 9H).
C
16H20N2O3S [M]: 318.10; MS (ESI) m/z: [Mꢀ H]+ : 317.200.
4.2.16. Preparation of N-(2-amino phenyl)-thiophene-3-carboxamide: (5
h)
C
22H23N5O3 [M]: 405.45; MS (ESI) m/z: [M + H]+: 406
The compound tert-butyl-2-(thiophene-3-carboxamido) phenyl
carbamate (4 h) (100 mg; 0.314 mmol) was dissolved in 5 ml DCM and
4 M dioxane HCl was added to it at 0 ◦C. This mixture was then allowed
to react under constant stirring at room temperature for 2 h. Dioxane
was then evaporated under vacuum. The mixture was then dissolved in
ethylacetate and washed with water. The organic layer was then sepa-
rated and excess solvent was evaporated under vacuum. The mixture
was then washed with pentane to obtain boc-deprotected final com-
pound 5 h. (yield: 86%).1H NMR (400 MHz, DMSO‑d6) δ 9.58 (s, 1H),
8.37 (s, 1H), 7.68 (d, J = 2.0 Hz 2H), 7.18 (d, J = 0.8 Hz 1H), 7.03 (t, J =
1.6 Hz 1H), 6.85 (d, J = 1.2 Hz 1H), 6.67 (t, J = 0.8 Hz 1H), 4.94 (s,
2H).13C NMR (101 MHz, DMSO‑d6)δ : 161.46, 143.96, 138.18, 129.97,
127.77, 127.260,127.14,127.01,126.72, 123.49, 116.64 C16H18N2O3S
[M]: 318.103; MS (ESI) m/z: [Mꢀ H]+: 317.200. C11H10N2OS [M]:
218.05; MS (ESI) m/z: [M + H]+: 219.1500.
4.2.20. Preparation of N-(2-aminophenyl)-6-phenylamino pyrazine-2-
carboxamide (5i)
The compound tert-butyl 2-(amino phenyl)-6-phenylamino pyrazine-
2-carbamate (4i) (100 mg; 0.276 mmol) was dissolved in dichloro-
methane (5 ml) and treated with the solution of 4 M dioxane in HCl at
0 ◦C. This mixture was then allowed to react under constant stirring at
room temperature for 2 h. The reaction was monitored by TLC. After
completion of the reaction, solvent was then evaporated under vacuum.
The resulting compound was then dissolved in water and extracted with
ethyl acetate. The organic layer was then separated, dried with Na2SO4
and excess solvent was evaporated under vacuum. The mixture was then
washed with pentane to obtain boc-deprotected final compound (Yield:
88.88%). 1H NMR (400 MHz, CDCl3) δ 9.42 (s, 1H), 8.87 (s, 1H), 8.40 (s,
1H), 7.54 (d, J = 7.6 Hz 1H), 7.43 (m, 4H), 7.18 (t , J = 6.8 Hz, 1H), 7.09
(t, J = 7.2 Hz, 1H), 6.87 (dd, J = 11.5, 7.8 Hz, 2H), 6.77 (s, 1H), 3.95 (s,
2H)13C NMR (101 MHz, CDCl3) δ 161.12, 150.14, 139.82, 138.40,
136.30, 134.25, 129.56, 126.88, 124.54, 124.13, 121.03, 119.95,
118.29.C17H14N2O [M]: 305.33; MS (ESI) m/z: [M + H]+: 306.
4.2.17. Preparation of N-phenyl-6-(phenyl amino) pyrazine-2-
carboxamide (2i):
Aniline was added to a solution of methyl 6-chloro pyrazine-2-
carboxylate (1i) in N-methylpyrrolidine at 0 ◦C. To the reaction
mixture diisopropyl amine was added at the same temperature and the
reaction was heated to 160 ◦C for 20 h. The completion of the reaction
was monitored by TLC. Water was added to the reaction mixture and the
crude compound was extracted with ethyl acetate, dried using anhy-
drous sodium sulphate, solvent was evaporated using rotaevaporator.
The obtained crude product was purified by column chromatography.
1H NMR (400 MHz, DMSO -d6) δ : 10.08 (s, 1H), 9.89 (s, 1H), 8.50 (s,
1H), 8.49 (s, 1H), 7.80 (d, J = 9.8 Hz 4H), 7.47 (dd, J = 5.2, 12 Hz 4H),
7.20 (t, J = 8.0 Hz, 1H),7.12(t, 6.4 Hz 1H). C17H14FN4O [M]: 290.32; MS
(ESI) m/z: [M + H]+: 291.07.
4.3. Biology
4.3.1. Cell culture
Three different cell lines were used for the determination of anti-
cancer activity of the novel compounds synthesized. For the purpose,
MTT assay was carried out on Mouse breast cancer cell line (4 T1),
Murine melanoma cancer cell line (B16F10) and human breast cancer
(MDA-MB-231) cell line that were procured from National Centre for
Cell Science (NCCS, Pune, India). B16F10 and MDA-MB-231 cell lines
were cultured in DMEM (high glucose media: AL007S, Dulbecco’s
modified eagle medium) and 4 T1 cell line was cultured in MEM (AT154,
Minimum essential medium) with 10% fetal bovine serum (FBS) and 1%
antibiotic (Pen strep: A001) and were incubated at 37 ◦C and 5% CO2
4.2.18. Preparation of 6-(phenyl amino) pyrazine-2-carboxylic acid (3i):
N-phenyl-6-(phenyl amino) pyrazine-2-carboxamide (2i) was dis-
solved in methanol and aqueous sodium hydroxide was added to the
reaction mixture and the reaction was stirred at 80 ◦C for 1 h. The re-
action was cooled at room temperature, added ice-cold water and neu-
tralised with 1 N HCl. The compound was extracted with ethylacetate,
dried using sodium sulphate and the excess solvent was evaporated
using rotaevaporator. The obtained crude mixture was purified by col-
umn chromatography (solvent system – dichloromethane and methanol
(7:3)). 1H NMR (400 MHz, DMSO‑d6) δ: 9.79 (s, 1H), 8.47 (s, 2H), 7.71
(d, 2H), 7.34 (t, 2H), 7.03 (t, 1H). C11H9FN3O2[M]: 215.21; MS (ESI) m/
z: [M + H]+: 216.18.
atmosphere.
MTT
[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte-
trazolium bromide], a yellow dye was used for the assay. All reagents
were purchased from Himedia Laboratories Pvt. Ltd., Mumbai, India.
4.3.2. Chemicals and anti-bodies
All the compounds were synthesized as described above. The com-
pounds were dissolved in DMSO stock solution and were stored in
ꢀ 20 ◦C. Primary antibodies - Rabbit mAb H3K9 acetylated histone H3
(catalogue #9649), Rabbit mAb H4K12 acetylated histone H4 (cata-
logue #13944), mouse mAb beta-Actin primary antibodies (catalogue
#58169) and secondary antibodies – anti-rabbit HRP linked antibody
and anti - mouse IgG HRP-Linked antibody were purchased from cell
signalling technology. DAPI (4′,6-diamidino-2- phenylindole) and acri-
dine orange, propidium iodide and RNase were purchased from Sigma.
TACs Annexin-V/FITC – PI assay kit was purchased from Biolegend and
was used as per the protocol given.
4.2.19. Preparation of tert-butyl 2- (amino phenyl)-6-phenylamino
pyrazine-2-carbamate (4i):
o a solution of dichloromethane and pyridine (1:1) was added to 6-
(phenyl amino) pyrazine ꢀ 2-carboxylic acid (3i) (50 mg; 0.232 mmol).
To this reaction mixture, 1-ethyl-3-dimethyl amino propyl carbo di
imide (64.72 mg; 0.417 mmol) and catalytic amount of 4-dimethyla-
mino pyridine were added in nitrogen environment. The mixture was
stirred at room temperature for 15 min. After 15 min, tert-butyl 2-amino
phenyl carbamate (53.209 mg; 0.255 mmol) was added into the reaction
mixture and reaction was continued for 12 h. The reaction was moni-
tored with TLC. After completion of the reaction, pyridine was then
evaporated under vacuum. The mixture was then dissolved in sodium
4.3.3. MTT assays
As per the protocol, 96 well plate was seeded with 100 µL/well of cell
suspension with the cell density of 1 × 104 per well and were incubated
for overnight. Subsequently, the medium was aspirated, and the cells
were treated with the synthesized novel compounds along with CI994 as
positive control at a concentration of 100 µM and 10 µM in 150 µL of
13

G. Routholla et al.
Bioorganic Chemistry 114 (2021) 105050
their respective media in duplicate and further incubated for 72 h.
Following incubation, the culture medium was aspirated and subse-
quently, 50 µL of 5 mg/ml concentrated solution of MTT (3-(4,5-dime-
thylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) in phenol red free
DMEM media was prepared and added in each well and further incu-
bated for 3 h for the formation of formazan crystals that are formed as a
result of cellular enzymatic activity. Subsequently, 150 µL of DMSO was
added to the culture after aspirating media in the wells to dissolve the
formazan crystals and the absorbance was measured using multi-well
plate reader Spectramax (Molecular Devices, USA) at two different
wavelengths of 570 nm and 650 nm. The % cell viability was calculated
as a fraction of absorbance obtained from the treated cells from the
absorbance of untreated control cells. The same procedure was followed
for all the three cell lines.
(Molecular Devices, USA). Initially, all the selected promising com-
pounds 5e and 5f along with CI994 were screened at 10 μM concen-
tration in duplicate. Further, all the compoundsat the concentration
range of 1.25 μM – 80 μM were tested in duplicate to find out the IC50
values following the same procedure as described above. The IC50 values
of these compounds were calculated using nonlinear regression analysis
method using Graph Pad Prism 5.
4.3.4.3. HDAC2 inhibition assay. The HDAC 2 enzyme inhibition assay
was performed using HDAC 2 fluorimetric drug discovery assay kit
(BML-AK512, ENZO life sciences). To the 96 well microtiter plate pro-
vided in the kit, 10 μL of test sample solution and 15 μL diluted HDAC2
complex solution (BML-KI575-0030) were added per well and 25 μL
Fluor de Lys® substrate solution (BML-KI572-0050) was added. The
◦
For the IC50 measurement of all the compounds in the series along
with CI994, the same procedure was followed as described above. The
DMSO solutions of the selected compounds were prepared and they were
further diluted to 200 µM, 100 µM, 50 µM, 25 µM, 12.5 µM, 6.25 µM,
3.125 µM, 1.562 µM and 0.781 µM with the DMEM complete media and
MEM media respectively for the determination of IC50 values along with
a blank control containing DMSO in medium and CI994 as positive
control and were incubated for 72 h. The experiment was repeated
following the same protocol on all the 3 cell lines and the cell viability
was measured by MTT assay as discussed. IC50 determination was also
performed for the selected compounds to evaluate their cytotoxicity
using Human embryonic kidney (HEK293) cell line sub-cultured in
DMEM (high glucose media: AL007S, Dulbecco’s modified eagle me-
dium) with 10% fetal bovine serum (FBS) and 1% antibiotic (Pen strep:
A001) and were incubated at 37 ◦C and 5% CO2 atmosphere. MTT [3-
(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide], a yellow
dye was used for the assay. All reagents were purchased from Himedia
Laboratories Pvt. Ltd., Mumbai, India. Cytotoxicity assay was performed
to study their selectivity over cancer cell lines. The DMSO solutions of
the selected compounds were prepared and they were further diluted to
plate was incubated for 30 min at 37 C for the reaction to occur. To
terminate the reaction, 50 μL of mixture of Fluor de Lys® developer II
(BML-KI105-0300) and Trichostatin A ((BML-GR309-9090) was added
per well and incubated for 15 min at 37 ◦C as per the protocol given in
the kit. The fluorescence intensity was measured at Excitation wave-
length 485 nm, Emission wavelength 530 nm using Spectramax M4
(Molecular Devices, USA). Initially, all the selected promising com-
pounds 5e and 5f along with CI994 were screened at 10
μ
M concen-
tration in duplicate. Further, all the compoundsat the concentration
range of 0.625 μM – 80 μM were tested in duplicate to find out the IC50
values following the same procedure as described above. The IC50 values
of these compounds were calculated using nonlinear regression analysis
method using Graph Pad Prism 5.
4.3.4.4. HDAC3/NCOR1 inhibition assay. The HDAC 3 enzyme inhibi-
tion assay was performed using HDAC3/NCOR1 fluorimetric drug dis-
covery assay kit (BML-AK531-0001, ENZO life sciences). To the 96 well
microtiter plate provided in the kit, 10
μ
L of test sample solution and 15
μ
L diluted HDAC3/NCOR1 complex solution (BMLKI574-0030) were
added per well and 25
μ
L Fluor de Lys® substrate solution (BML-KI177-
0005) was added. The plate was incubated for 15 min at 37 ◦C for the
2 mM, 1 mM, 500
and 7.81
μM, 250 μM, 125 μM, 62.5 μM, 31.25 μM, 15.62 μM
μ
M with the DMEM complete media for the determination of
reaction to occur. To terminate the reaction, 50 μL of mixture of Fluor de
IC50 values along with a blank control containing DMSO in medium and
were incubated for 72 h.
Lys® developer II (BML-KI176-1250) and Trichostatin A ((BML-GR309-
9090) was added per well and incubated for 45 min at 37 ◦C as per the
protocol given in the kit. The fluorescence intensity was measured at
Excitation wavelength 360 nm, Emission wavelength 460 nm using
Spectramax M4 (Molecular Devices, USA). Initially, all the synthesized
4.3.4. HDAC inhibition assays
4.3.4.1. HDAC inhibition assay. The enzyme inhibition assay was per-
compounds along with CI994 were screened at 1
μ
M concentration in
formed using HDAC colorimetric assay kit (BML-AK501, ENZO life sci-
duplicate. The promising test compounds 5e and 5f along with CI994 as
ences). Briefly, 5 μL of HeLanuclear extract (BML-KI137–0500), 10 μL of
positive control at the concentration range of 0.25
μ
M – 8 M were
μ
assay buffer (BMLKI143–0020), 10 μL of sample solution was added per
tested in duplicate to find out the IC50 values following the same pro-
cedure as described above. The IC50 values of these compounds were
calculated using nonlinear regression analysis method using Graph Pad
Prism 5.
well in amicrotiter plate. The reaction was started with addition of 25 μL
Colorde Lys® substrate solution (BML-KI138–0050). The reaction was
then incubated for 30 min at 37 ◦C, which was terminated by addition of
a 50 μL mixture of developer (BML-KI139-0300) plus stop solution. The
plate was incubated for 15 min at 37 ◦C and absorbance was measured at
4.3.4.5. HDAC6 inhibition assay. The HDAC 6 enzyme inhibition assay
405 nm. All synthesized compounds were screened at 10
tration in duplicate.
μM concen-
was performed using HDAC 6 fluorimetric inhibitor screening kit (K465-
100, Biovision). To the 96 well microtiter plate provided in the kit, 2 μL
of test sample solution and 50
(K465-100–2) were added per well and incubated for 15 min at 37 ◦C. To
this 48 L Fluor de Lys® substrate solution (K465-100–3) was added.
The plate was incubated for 30 min at 37 ◦C for the reaction to occur. To
μL diluted HDAC2 complex solution
4.3.4.2. HDAC1 inhibition assay. The HDAC 1 enzyme inhibition assay
was performed using HDAC 1 fluorimetric drug discovery assay kit
(BML-AK511, ENZO life sciences). To the 96 well microtiter plate pro-
μ
vided in the kit, 10 μL of test sample solution and 15 μL diluted HDAC1
terminate the reaction, 10 μL of developer II (K465-100–4) was added
per well and incubated for 10 min at 37 ◦C as per the protocol given in
the kit. The fluorescence intensity was measured at Excitation wave-
length 380 nm, Emission wavelength 490 nm using Spectramax M4
(Molecular Devices, USA). Initially, all the selected promising com-
complex solution (BML-SE456-0050) were added per well and 25 μL
Fluor de Lys® substrate solution (BML-KI177-0005) was added. The
◦
plate was incubated for 15 min at 37 C for the reaction to occur. To
terminate the reaction, 50 μL of mixture of Fluor de Lys® developer II
(BML-KI176-1250) and Trichostatin A ((BML-GR309-9090) was added
per well and incubated for 45 min at 37 ◦C as per the protocol given in
the kit. The fluorescence intensity was measured at Excitation wave-
length 360 nm, Emission wavelength 460 nm using Spectramax M4
pounds 5e, 5f and CI994 were screened at 20
μM concentration in
duplicate. Further, all the compoundsat the concentration range of 10
M – 320 M were tested in duplicate to find out the IC50 values
following the same procedure as described above. The IC50 values of
μ
μ
14

G. Routholla et al.
Bioorganic Chemistry 114 (2021) 105050
these compounds were calculated using nonlinear regression analysis
method using Graph Pad Prism 5.
4.3.7. Apoptosis assay
B16F10 cells were seeded with the cell density of 0.5 × 106/well in
12 well tissue culture plates and left overnight. Next day cells were
treated with 5e (17.89 µM), 5f (26.40 µM) and CI994 (14.59 µM) for 72
h and the cells were incubated at 37 ◦C in CO2 incubator to assess the
apoptosis. The study was carried out as per the manufacturer’s protocol
(BioLegend, US). The cells were washed with ice cold PBS, trypsinized
and centrifuged to get cell pellet. The pellet was resuspended in 100 µL
reagent containing AnnexinV buffer, FITC (1 µL) and PI (10 µL) and kept
for incubation for 30 min at room temperature. AnnexinV binding
buffer, 1X (400 µL) was added to each sample and characterized by flow
cytometer (BDAriaTM III). The cells with no treatment were considered
as controls. FITC versus PI with quadrant gating was done as dot plot
which represents (Q1 – Necrotic cells, Q2 - late apoptosis, Q3 – Live
cells, Q4 – early apoptotic cells). To determine the extent of apoptosis,
early and late apoptotic events were taken.
4.3.4.6. HDAC8 inhibition assay. The HDAC 8 enzyme inhibition assay
was performed using HDAC 8 fluorimetric drug discovery assay kit
(BML-AK518, ENZO life sciences). To the 96 well microtiter plate pro-
vided in the kit, 10 μL of test sample solution and 15 μL diluted HDAC8
complex solution (BML-SE145-0100) were added per well and 25 μL
Fluor de Lys® substrate solution (BML-KI178-0005) was added. The
◦
plate was incubated for 10 min at 37 C for the reaction to occur. To
terminate the reaction, 50 μL of mixture of Fluor de Lys® developer II
(BML-KI176-1250) and Trichostatin A ((BML-GR309-9090) was added
per well and incubated for 45 min at 37 ◦C as per the protocol given in
the kit. The fluorescence intensity was measured at Excitation wave-
length 360 nm, Emission wavelength 460 nm using Spectramax M4
(Molecular Devices, USA). Initially, all the selected promising com-
pounds 5e and 5f along with CI994 were screened at 5
μM concentration
4.3.8. Cell cycle analysis
in duplicate. Further, all the compoundsat the concentration range of
The cell cycle analysis was performed by using flow cytometry. The
cells B16F10 cells were seeded with density of 0.5 × 106 cells per well.
After overnight incubation, 15 µM dose of 5e, 5f and CI994 were added
to cells and incubated for another 48 h. Then the cells were harvested
with trypsin and the cell pellet was washed with ice cold PBS. The cells
were fixed with 70% ethanol by dropwise addition into the cell sus-
pension under gentle vortex. The clumping of cell was avoided and
single cell fixation was visualized under microscope for cross-
verification. The samples were kept in ꢀ 20 ◦C for overnight. The next
day fixed samples were centrifuged at 1000 rpm, 4 ◦C for 7 min to obtain
cell pellet. Finally, the cells were re-suspended in 500 µL of PI and RNAse
staining solution. The staining solution was prepared by addition of 20%
w/v RNAse and 2% w/v PI in 0.1% v/v of Triton X-100 solution in PBS.
The samples were incubated in dark for 30 min at room temperature and
analyzed by flow cytometry (BDAriaTM III). The dot plot of PI width
against PI area was recorded and histogram of PI area on X axis and
counts on Y axis was plotted. The percentage of cells in each phase of the
cell cycle was evaluated using the FCS express software.
0.625 μM – 40 μM were tested in duplicate to find out the IC50 values
following the same procedure as described above. The IC50 values of
these compounds were calculated using nonlinear regression analysis
method using Graph Pad Prism 5.
4.3.5. Western blot analysis
For western blotting of acetylated Histone H3(H3K9) and acetylated
Histone H4 (H4K12) B16F10 murine melanoma cells were plated in flat
bottom 96 well plate and allow to grow overnight, and then they treated
with the 5e, 5f and CI994 at 5 µM, 20 μM final concentrations for 12 h.
After the treatment, the cells were harvested by Trypsinization and
centrifuge at 1250 rpm for 5 min. The cells pallet was washed by ice cold
PBS, and the total protein was extracted using 100 μL, 1X RIPA lysis
buffer (Millipore, Billerica, MA, USA), supplemented with 0.5 mM
phenylmethylsulfonyl fluoride (PMSF) and protease inhibitor. After
lysing, the suspension was vertex and centrifuged at 14000 rpm for 15
min at 4 ◦C. The whole-cell lysates 20
μL and 5 μL of loading buffer (4X)
was heated at 95 ◦C for 5 min and subjected to sodium dodecyl sulfa-
te–polyacrylamide gel electrophoresison 15% Bis-Tris 10-well gels at 60
V for approximately 180 min in SDS Running Buffer. Gels were trans-
ferred to the polyvinylidene fluoride membranes (Bio-Rad, Laboratones,
Inc.) and run at 60 V for 80 min. Membranes were blocked in 5% non-fat
skimmed milk (Bio-Rad, Laboratones, Inc.) in tris-buffered saline with
1% Tween 20 (TBST), and incubated with Rabbit mAb H3K9 acetylated
histone H3, Rabbit mAb H4K12 acetylated histone H4 and Mouse mAb
beta-Actin primary antibodies overnight at 4 ◦C, which were diluted up-
to 1:7000 in 5% (w/v) milk. The membranes were then incubated with
Horseradish peroxidase (HRP)-conjugated anti-rabbit secondary anti-
body and anti-mouse secondary antibody and then visualized with a
chemiluminescence kit (Bio-Rad, Laboratories, Inc.) and exposed using a
Fusion plus 6 Imaging System (Vilber Lourmat, France). Beta-Actin was
used as an internal control.
4.4. Molecular docking
Molecular docking study was performed to predict binding in-
teractions of promising HDAC3 inhibitors with the HDAC3 enzyme using
the Glide module of Schrodinger Maestro software [55]. The protein
structure (PDB: 4A69) was selected [28] and prepared by using the Glide
module of Maestro software[55]. On the other hand, ligands were pre-
pared as per our earlier mentioned protocol [28]. A grid box was created
on the centroid of the HDAC3 binding site. Finally, the prepared ligands
and the protein were used to execute the extra precision (XP) docking
[28,56].
CRediT authorship contribution statement
GR, Compound synthesis and spectral analysis; SP, in vitro biological
evaluation assays, Manuscript drafting and editing; TP, western blot
analysis and nuclear staining assay; SAA, molecular docking studies; NA,
writing the manuscript draft and designing molecular docking study, SB,
validation and Supervision; TJ, validation and supervision; BG,
Conceptualization, Validation, Writing- Reviewing and Supervision.
4.3.6. Nuclear staining assay
The Nuclear staining were performed to investigate the status of
nuclear disintegration of cancerous cells after treatment of 5e, 5f and
CI994 as standard by staining with DAPI (4′,6-diamidino-2- phenyl-
indole) and acridine orange. For nuclear staining, B16F10 murine mel-
anomacells were plated in flat bottom 12 well plate and allow to grow
overnight, and then they treated with the 5e (17.89 µM), 5f (26.40 µM)
and CI994 (14.59 µM) concentrations and incubate for 48 h. After 48 h
of the treatment, control and compounds 5e, 5f and CI994 treated group
were fixed with 4% paraformaldehyde solution, thereafter both control
and compounds treated cells were stained with DAPI and acridine or-
ange. The nuclear staining of both control and treated cells was visual-
ized under fluorescence microscope (Leica microsystems, Germany) on
20x Magnification.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
15

G. Routholla et al.
Bioorganic Chemistry 114 (2021) 105050
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Acknowledgements
The research has been supported by the research fund provided by
Science and Engineering Research Board-Department of Science and
Technology (SERB-DST), New Delhi, India (EMR/2016/001411) and
Council of Scientific and Industrial Research (CSIR-37(1722)/19/EMR-
II) to Dr. Balaram Ghosh, and DST-SERB, New Delhi, India to Dr. Swati
Biswas (CRG/2018/001065). Sravani acknowledges CSIR for providing
senior research fellowship (SRF - 09/1026(0024)/2018-EMR-I). Au-
thors sincerely acknowledge the Department of Pharmacy, BITS-Pilani,
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Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bioorg.2021.105050.
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