Design, synthesis, biological evaluation and molecular docking study of arylcarboxamido piperidine and piperazine-based hydroxamates as potential HDAC8 inhibitors with promising anticancer activity

Article abstract of DOI:10.1016/j.ejps.2019.105046

HDAC8 has been established as one of the vital targets as far as the cancer is concerned. Different compounds having potential HDAC inhibitory activity have been approved by USFDA. However, none of these compounds are selective towards specific HDAC isoform. In this current study, some new hydroxamate derivatives with alkylpiperidine and alkylpiperazine linker moieties have been designed, synthesized and biologically evaluated. All these compounds are effective HDAC8 inhibitors comprising more or less similar cytotoxic potential against different cancer cell lines. It is observed that the piperazine scaffold containing compound is more active than the compound with piperidine scaffold for exerting HDAC8 inhibitory activity. Moreover, the 4-quinolyl cap group is better than the biphenyl group which is better than the benzyl group for producing higher HDAC8 inhibition as well as cytotoxicity. These compounds displayed selective HDAC8 inhibition over HDAC3. Moreover, these compounds showed an increased caspase3/7 activity suggesting their anticancer potential through modulation of apoptotic pathways. Molecular docking study with three potent compounds was performed with both HDAC3 and HDAC8 enzymes to understand the selectivity profile of these compounds. Compound containing 4-quinolyl cap group with alkyl piperazinyl urea linker moiety has been emerged out as the lead molecule that may be further modified to design more effective and selective HDAC8 inhibitors in future.

Full text of DOI:10.1016/j.ejps.2019.105046

EuropeanJournalofPharmaceuticalSciences138(2019)105046
Contents lists available at ScienceDirect
European Journal of Pharmaceutical Sciences
journal homepage: www.elsevier.com/locate/ejps
Design, synthesis, biological evaluation and molecular docking study of
arylcarboxamido piperidine and piperazine-based hydroxamates as
potential HDAC8 inhibitors with promising anticancer activity
Prakruti Trivedia, Nilanjan Adhikarib, Sk. Abdul Aminb, Yamini Bobdea, Routholla Ganesha,
⁎
⁎
Tarun Jhab, , Balaram Ghosha,
a Department of Pharmacy, BITS-Pilani, Hyderabad Campus, Shamirpet, Hyderabad, India, 500078
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:
HDAC8 has been established as one of the vital targets as far as the cancer is concerned. Different compounds
having potential HDAC inhibitory activity have been approved by USFDA. However, none of these compounds
are selective towards specific HDAC isoform. In this current study, some new hydroxamate derivatives with
alkylpiperidine and alkylpiperazine linker moieties have been designed, synthesized and biologically evaluated.
All these compounds are effective HDAC8 inhibitors comprising more or less similar cytotoxic potential against
different cancer cell lines. It is observed that the piperazine scaffold containing compound is more active than
the compound with piperidine scaffold for exerting HDAC8 inhibitory activity. Moreover, the 4-quinolyl cap
group is better than the biphenyl group which is better than the benzyl group for producing higher HDAC8
inhibition as well as cytotoxicity. These compounds displayed selective HDAC8 inhibition over HDAC3.
Moreover, these compounds showed an increased caspase3/7 activity suggesting their anticancer potential
through modulation of apoptotic pathways. Molecular docking study with three potent compounds was per-
formed with both HDAC3 and HDAC8 enzymes to understand the selectivity profile of these compounds.
Compound containing 4-quinolyl cap group with alkyl piperazinyl urea linker moiety has been emerged out as
the lead molecule that may be further modified to design more effective and selective HDAC8 inhibitors in
future.
Cancer
HDAC inhibitor
HDAC8 inhibitor
Piperidine
Piperazine
Cytotoxicity
SAR
1. Introduction
valproic acid, butyric acid), (2) hydroxamic acids such as sub-
eroylanilide hydroxamic acid (SAHA), belinostat, panabinostat, tri-
Histone deacetylases (HDACs) are amidohydrolases. HDACs are
involved in the deacetylation process of the lysine residues of the his-
tone protein during chromatin remodeling. These HDACs take part
crucially for the regulation of epigenetics during gene expression
(Kouzarides, 2007). These epigenetic alterations by the HDACs suppress
transcription of gene and subsequently, are involved in the tumor
suppressor gene silencing (Mottamal et al., 2015). Therefore, inhibition
of HDACs has become a potential strategy for anticancer therapeutics
(Manal et al., 2016). HDAC inhibitors have an immense importance for
the treatment of various cancers as a promising new anticancer ther-
apeutics (Bolden et al., 2006). According to the chemical structure,
there are three main classes of HDAC inhibitors that have been devel-
oped as effective anticancer agents: (1) carboxylic acids (such as
chostatin A (TSA), and (3) 2-aminobenzamides such as mocetinostat
(MGCD-0103), entinostat (MS-275) and tacedinaline (CI-994)
(Bertrand, 2010; Loprevite et al., 2005; Adhikari et al., 2018). Struc-
tures of these compounds are summarized in Fig. 1.
All these three classes of HDAC inhibitors have been found to be
effective for cancer treatment (Dawson and Kouzarides, 2012). Among
these HDAC inhibitors, hydroxamic acid derivatives namely SAHA,
belinostat and panabinostat have been approved by USFDA for the
treatment of Cutaneous T-cell lymphoma (CTCL), Relapsed peripheral
T-cell lymphoma (RPTCL), Relapsed multiple myeloma (RMM), re-
spectively (Mann et al., 2007; Food, Administration D, 2014; Fenichel,
2015). A number of hydroxamic acid derivatives are in different phases
of clinical trials and are effective for treating various types of cancers.
⁎ Corresponding authors.
E-mail addresses: tjupharm@yahoo.com (T. Jha), balaram@hyderabad.bits-pilani.ac.in (B. Ghosh).
https://doi.org/10.1016/j.ejps.2019.105046
Received 10 May 2019; Received in revised form 25 July 2019; Accepted 13 August 2019
Availableonline14August2019
0928-0987/©2019ElsevierB.V.Allrightsreserved.

P. Trivedi, et al.
EuropeanJournalofPharmaceuticalSciences138(2019)105046
Fig. 1. HDAC inhibitors either clinically approved or in clinical trials with their chemical classification.
Apart from hydroxamates, carboxylic acid and benzamide derivatives
are also in clinical trials for cancer treatment (Xie et al., 2017; Amin
et al., 2019).
studies so far showing importance of linker moiety modulating potency
and selectivity towards HDAC8 isoform. Therefore, modifications of the
linker region with different groups interacting with this hydrophobic
channel can be exploited to design selective HDAC8 inhibitors (Wang
et al., 2007).
In spite of therapeutic advantage of HDAC inhibitors especially
hydroxamates, a number of adverse effects or toxicities of these com-
pounds have been noticed during treatment (Mackay et al., 2010). The
off-target effects or toxicity of hydroxamates are poorly understood
even though these are clinically approved (Coiffier et al., 2012; Bishton
et al., 2011; Pili et al., 2012). One of the reasons could be their
panHDAC inhibitory nature of these compounds (Balasubramanian
et al., 2009). Majority of these hydroxamates inhibit number of HDAC
isoforms nonselectively and therefore, are called as panHDAC in-
hibitors. Nevertheless, administration of single drug may also affect
multiple biological pathways and subsequently, may produce toxicity.
Hence, selective HDAC inhibitors should be needed to improve the ef-
ficacy of the compound along with less toxicity (Gupta et al., 2012).
HDAC8, a Zn2+-dependent class I HDAC, is overexpressed in a
number of cancers including cancers of breast, colon, lung and pancreas
(Chakrabarti et al., 2015). It is also implicated in hematological ma-
lignancy. The upregulation of HDAC8 promotes proliferation of cancer
cells and also restricts apoptosis. HDAC8 is implicated not only in
cancer but in many other diseases also (Amin et al., 2017). In current
days, HDAC8 is emerged out as an important target among other
HDACs. Selectivity towards a specific HDAC can be achieved for hy-
droxamates by modifications at the surface recognition site or cap
group as well as by modifications at the linker function. It has been
reported that a rigid and bulky linker region is responsible for in-
creasing the hydrophobic interaction with tunnel residues and there-
fore, the potency and selectivity within class I HDACs is increased
(Vannini et al., 2004).
By considering these views, in this article, we have incorporated
constrained piperidine and piperazine groups attached with methylene
side chain as the linker moiety to reduce the flexibility and to increase
the hydrophobicity. By optimizing the linker group, we modified the
cap region by some bulky and hydrophobic groups and designed a
series of new hydroxamates. These compounds were synthesized and
evaluated for their enzyme inhibitory activity against HeLa nuclear
extract (rich with HDAC1 & 2), human recombinant HDAC3 and human
recombinant HDAC8 enzymes. The anticancer potential of these com-
pounds was evaluated by using five different cancer cell lines followed
by molecular docking studies.
2. Results and discussion
2.1. Design of molecules
Earlier, Neelarapu et al. (2011) designed some potential HDAC8
inhibitors having comparable efficacy over HDAC3 and those inhibitors
contained heterocyclic ring structures (isoxazoles and pyrazoles) as the
linker moiety. However, both the pyrazole and isoxazole rings were
located at the terminal end of the carboxamide group maintaining the
hexamethylene hydroxamate function of SAHA unaltered. Moreover,
Zhang et al. (Zhang et al., 2015) also proposed some potential HDAC8
inhibitors having long chain linear alkyl functions between two car-
boxamide moieties. Again, Tang and co-workers (Tang et al., 2011)
reported several hydroxamate derivatives having some macrocyclic
ring structure in its linker position and these compounds exhibited se-
lective HDAC8 inhibitory activity.
In our previous study, activity of compounds having only linker
region and hydroxamic acid as the zinc binding group (ZBG) was in-
vestigated and it was found that such compounds possess no enzyme
inhibitory activity at the concentration tested (Ghosh et al., 2016).
These results explained crucial role of cap moieties in HDAC inhibitory
efficacy of hydroxamates. The cap group region has been extensively
modified to come up with selective HDAC inhibitors. From various
studies, it was also found that bulky hydrophobic cap group contributes
towards class I HDAC selectivity (Bieliauskas and Pflum, 2008). How-
ever, HDAC8 inhibitors having structural differences in the linker re-
gion have been very few. The probable reason could be the residues in
the hydrophobic channel where the linker region is highly conserved
among these different HDAC isoforms. There are no such exclusive
Here, we tried to modify the hexamethylene linker moiety of SAHA
with alkylpiperidine and alkylpiperazine moieties to judge the HDAC
inhibitory activity as well as the cellular toxicity. Keeping the alkylpi-
peridine and alkylpiperazine rings as the linker function, the cap region
was modified with different bulky hydrophobic aryl groups to design a
series of arylcarboxamido piperidine and piperazine hydroxamate de-
rivatives (Fig. 2).
It was reported earlier for different piperidine and piperazine ring
containing compounds that alkyl side chain attached to those ring as
the linker function exerts optimum HDAC inhibitory activity when it
2

P. Trivedi, et al.
EuropeanJournalofPharmaceuticalSciences138(2019)105046
Fig. 2. Designing of novel piperidine and piperazine derivatives.
has three methylene groups in it (Rossi et al., 2011). In order to check
the effect of alkyl side chain length as per our designing strategy, we
tried two or three methylene moieties having phenyl group as the cap
function at R1 position (Fig. 2) and evaluated the HDAC inhibitory
activity. Our initial study suggested the same difference in accordance
with the reported observation (Trivedi et al., 2018). During further
modifications, we kept the alkyl side chain length unaltered with three
methylene groups and modified the cap region with different hydro-
phobic aryl functions.
of piperidine free to react with aliphatic side chains. Methyl 3-bromo-
propionate or methyl 4-bromobutanoate was exposed to react with
deprotected piperidine moiety in presence of potassium carbonate using
acetonitrile as the solvent. The intermediate esters were then converted
to hydroxamic acid functional groups using 50% (w/v) aqueous hy-
droxylamine and sodium hydroxide to synthesize the desired final
molecules (compound 5a, 5a′, 5b, 5c, 5d and 5e).
Scheme 2 describes the protection of piperazine ring using di-tert-di-
butyl-dicarbonate which is very efficient method to get mono protected
piperazine in good yield (Bala et al., 2015). The compound was used as
the intermediate for the synthesis of piperazine derivatives.
2.2. Chemistry
Scheme 3 describes the synthesis of two final molecules 11a and
11b having piperazine ring attached with aliphatic side chain as linker
function. Phenyl isocyanate was reacted with boc-protected piperazine
(8) and produced intermediate 9 (Keith et al., 2012), which was de-
protected using TFA in dichloromethane and refluxed with methyl 3-
bromopropanoate or methyl 4-bromobutanoate in acetonitrile with
K2CO3 as base to yield intermediates 10a and 10b. These compounds
were reacted with 50% w/v aqueous hydroxylamine in presence of
sodium hydroxide to get the respective hydroxamic acid derivatives
(11a, 11b).
Initially, piperidine derivatives were prepared and the route of
synthesis of these piperidine derivatives (compound 5a, 5a′, 5b, 5c, 5d
and 5e) is depicted in Scheme 1. The amine group of 4-piperidine
carboxylic acid was protected by using di-tert-butyl-dicarbonate
(Trivedi et al., 2018) where, the carboxylic acid was converted to its
salt form using sodium hydroxide and then it was treated with di-tert-
butyl-dicarbonate to react with piperidine nitrogen atom. After com-
pletion of the reaction, it was neutralized with hydrochloric acid to
make the carboxylic acid free from its salt from to yield the desired
intermediate (Nimbarte et al., 2014). This compound was then exposed
to react with different amines using EDC coupling method to obtain the
respective amides. The amide compounds were deprotected with the
help of trifluroacetic acid in dichloromethane to make the amide group
Synthesis of other piperazine derivatives having different aryl
moiety of the cap region using different bulky groups has been de-
scribed in Scheme 4. Here, different arylamines were converted to re-
spective isocyanates using triphosgene in biphasic mixture of DCM and
3

P. Trivedi, et al.
EuropeanJournalofPharmaceuticalSciences138(2019)105046
Scheme 1. Reagents and conditions: (a) di-tert-butyl-dicarbo-
nate, 1 M NaOH, THF, 1 M HCl, 0 °C, 30 min, rt, overnight (b)
R1NH2, EDC.HCl, HOBt, TEA, DCM, rt, overnight (c) i. TFA,
DCM, 0 °C, 2 h ii. methyl 3-bromopropanoate (n = 0), methyl
4-bromobutanoate (n = 1), K2CO3, acetonitrile, 5 h, 90 °C (d)
50% w/v aqueous hydroxylamine, 1 M NaOH, methanol, 1 M
HCl, rt, 2 h.
2.3. Pan HDAC inhibitory activity
The synthesized compounds were subjected to evaluation for their
in vitro HDAC inhibition. The HDAC inhibitory activity was performed
by using color de lys® HDAC assay kit (BML-AK501, ENZO life sciences).
BG45 was taken into consideration as the positive control. BG45 is a
selective HDAC3 inhibitor (IC50 = 289 nM) over HDAC1 (IC50 = 2 μM),
HDAC2 (IC50 = 2.2 μM) and HDAC6 (IC50 > 20 μM) effective against
multiple myeloma (Minami et al., 2014). HeLa nuclear extract (contains
predominantly HDAC1 and HDAC2) (Trivedi et al., 2018) was con-
sidered for the enzyme inhibitory assay. Results obtained from the assay
are listed in Table 1.
Scheme 2. Reagents and conditions: (a) di-tert-dibutyl-dicarbonate, methanol,
NaHCO3, 0 °C, 30 min, rt, 4 h
NaHCO3 (Hammock et al., 2016). The isocyanates were combined with
boc protected piperazine (7) to make respective arylpiperazinyl ureas.
The intermediates were deprotected using TFA in DCM and refluxed
with methyl 3-bromopropanoate or methyl 4-bromobutanoate in acet-
onitrile with K2CO3 as base, yielded compounds 15a–15d. These
compounds were reacted with 50% w/v aqueous hydroxylamine in
presence of sodium hydroxide to get respective hydroxamic acid deri-
vatives (16a–16d).
All these compounds exerted HDAC inhibition at 10 μM concentra-
tion (Table 1, Fig. 3). Most of these compounds exhibit > 50% enzyme
inhibition at 10 μM in HeLa nuclear extract (HDAC1 and HDAC2).
Almost all these compounds exhibited effective HDAC8 inhibitory
activity comparable to pan HDAC inhibition. However, it was noticed
that these compounds showed appreciable HDAC8 selectivity over
HDAC3 at 10 μM concentration. Regarding the alkyl function attached
with the heterocyclic moieties (i.e., piperidine and piperazine), it was
observed that dimethylene group was better than the monomethylene
Scheme 3. Reagents and conditions: (a) Cpd. 7, DCM, 4 h, rt (b) i. TFA, DCM, 0 °C, 2 h ii. methyl 3-bromopropanoate (n = 0), methyl 4-bromobutanoate (n = 1),
K2CO3, acetonitrile, 5 h, 90 °C (c) 50% w/v aqueous hydroxylamine, 1 M NaOH, methanol, 1 M HCl, rt, 2 h.
4

P. Trivedi, et al.
EuropeanJournalofPharmaceuticalSciences138(2019)105046
Scheme 4. Reagents and conditions: (a) triphosgene, NaHCO3, DCM, 0 °C, 1.5 h (b) Cpd.7, DCM, rt, 4 h (c) i. TFA, DCM, 0 °C, 2 h ii. methyl 3-bromopropanoate
(n = 0), methyl 4-bromobutanoate (n = 1), K2CO3, acetonitrile, 5 h, 90 °C (d) 50% w/v aqueous hydroxylamine, 1 M NaOH, methanol, 1 M HCl, rt, 2 h.
piperidine ring. Probably, the piperazine ring may offer better direction
to the associated aryl cap group to enter into the hydrophobic pocket
for proper interaction. The SAR analysis also suggested that the 4-qui-
nolyl moiety at Ar position is important for providing higher HDAC8
inhibition compared to the corresponding biphenyl group at the same
position as cap function. However, naphthyl group at Ar position as the
cap function results in the least HDAC8 inhibitory efficacy among these
Table 1
HDAC inhibitory activity of the synthesized compounds.
Compound % Inhibition at 10 μM
Selectivity
HeLa nuclear
extracta
HDAC3/
NCoR1
HDAC8 HDAC8/HDAC3
5a
35.22
36.30
67.52
66.60
64.03
65.34
56.96
62.63
68.95
57.46
63.07
68.36
73.72
5.93
40.35
44.40
63.49
62.16
67.84
72.44
58.35
67.09
69.28
63.24
71.07
74.16
14.27
6.80
23.0
8.64
5.88
11.21
17.21
6.54
8.44
4.59
6.45
13.28
16.78
0.16
cap
functionalities
(4-quinolyl > biphenyl > benzyl > naphthyl).
5a'
1.93
7.35
10.59
6.05
4.21
8.92
7.95
15.10
9.80
5.35
4.42
85.28
5b
Therefore, it may also be inferred that 4-quinolyl moiety may offer
better interactions with the amino acid residues of the hydrophobic
region compared to other cap groups reported here.
5c
5d
5e
Among these compounds, the most promising HDAC8 inhibitors
(cpd 5e, 16c, 16d) were further evaluated to find out their IC50 values
in HeLa nuclear extract (rich in HDAC1&2) by considering BG45 as
positive control (Minami et al., 2014). These compounds showed higher
HeLa nuclear extract inhibitory activity compared to BG45. Results are
summarized in Fig. 4 and Table 2.
11a
11b
16a
16b
16c
16d
BG45
It was interesting to note that cpd 16d showed the highest HDAC8
inhibition (74.16% at 10 μM) among these compounds that was > 2-
fold HDAC8 selective over HeLa nuclear extract (pan HDACs) whereas
other compounds (cpd 5e and 16d) were nonselective HDAC inhibitors
(Table 2).
a
HeLa nuclear extract (rich in HDAC1 and HDAC2).
group for exerting good HeLa nuclear extract inhibition and HDAC8
inhibition (cpd 11a vs cpd 5a, cpd 11b vs cpd 5a'). However, tri-
methylene moiety yielded far better HDAC8 inhibitory activity. It was
also noticed that compounds containing piperazine ring were more
effective than the corresponding piperidine derivatives (cpd 16a vs cpd
5b, cpd 16b vs cpd 5c, cpd 16c vs cpd 5d, cpd 16d vs cpd 5e).
Therefore, it may be inferred that piperazine ring is preferable than the
2.4. Determination of HDAC3 inhibition through human recombinant
HDAC3/NCoR1
All the synthesized compounds were screened for in vitro HDAC3
Fig. 3. Pan HDAC inhibition assay with the help of HeLa nuclear extract, human recombinant HDAC3 and human recombinant HDAC8 enzyme. All compounds
including BG45 were evaluated at 10 μM dose. All synthesized derivatives exhibited efficient inhibition towards HDAC8 and HeLa nuclear extract, while negligible
inhibition of HDAC3 at 10 μM concentration of the compounds. [Data represents mean
SD (n = 2)].
5

P. Trivedi, et al.
EuropeanJournalofPharmaceuticalSciences138(2019)105046
Fig. 4. Dose response curve of compound 5e, 16c and 16d with BG45 for HDAC inhibitory assay using HeLa nuclear extract. The data indicates mean
SD (n = 2).
Table 2
further considered for determining the IC50 values (Table 2, Fig. 5).
Pan HDAC and HDAC8 inhibitory activity (IC50) of some potential compounds.
Compounds 5e, 16c and 16d exhibited 72.44%, 71.07% and 74.16%
HDAC8 inhibition respectively at 10 μM concentration (Table 1). It was
observed that the selected compounds have marginal difference in their
selectivity towards HDAC8 compared to HeLa nuclear extract (mixture
of HDAC1 and HDAC2). (Table 1).
Cpda
IC50 (μM)
HeLa nuclear extract#
HDAC8
5e
3.57
4.99
3.65
4.42
2.19b
1.82b
2.39b
0.50b
3.14
4.29
1.74
–
1.01
1.42
0.81
16c
16d
BG45
2.6. Cytotoxicity studies
2.6.1. Antiproliferative assay using cancer cell lines
#
a
b
HeLa nuclear extract (rich in HDAC1&2).
Compound number.
Cytotoxicity of these compounds through MTT assay method were
performed using five different cancer cell lines namely B16F10 (murine
melanoma cell line), HeLa (Human cervical cancer cell line), MCF-7
(human breast cancer cell line), A549 (non-small cell lung cancer cell
line) and Jurkat E6 (human acute T cell leukemia). All target com-
pounds were tested at two different concentrations (10 μM and 100 μM)
in triplicate (Trivedi et al., 2018). BG45 were used as the standard
reference compounds in the assay (Supplementary Fig. S1 (i–v)). It was
noticed that most of these compounds displayed effective anti-
proliferative efficacy against all these cancer cell lines in a dose de-
pendent manner.
Included for comparison.
inhibitory activity by using HDAC3/NCoR1 fluorometric drug discovery
kit (BML-AK531, ENZO life sciences). From the results, it was found
that the compounds showed negligible HDAC3 inhibition (Table 1,
Fig. 3) which indicated the selectivity of these compounds towards
other HDACs tested over HDAC3.
2.5. HDAC8 inhibition assay using human recombinant HDAC8 enzyme
All these compounds from the above two dose screening experiment
were further evaluated for determination of their IC50. The cell viability
after 72 h of drug treatment was performed (Fig. 6(i–v)).
To evaluate selectivity of target compounds, these compounds were
further evaluated for HDAC8 inhibitory assay using HDAC8 fluorimetric
drug discovery kit (BML-AK518). All target compounds as well as BG45
were tested at 10 μM concentration on human recombinant HDAC8
enzyme according to manufacturer's protocol. Interestingly, all com-
pounds were found to show good HDAC8 inhibition. Results displayed
higher HDAC8 enzyme inhibitory activity of these compounds com-
pared to HDAC3 and HeLa nuclear extract (Table 1). BG45 showed
negligible HDAC8 inhibition and therefore, it was not further assessed
for IC50 values against HDAC8. To get more precise inhibitory profile,
the potent HDAC8 inhibitors namely compounds 5e, 16c and 16d were
The IC50 values of the compounds on five cell lines are listed in
Table 3.
We have tested these compounds for their anticancer activity on
different solid tumor cell lines as well as a leukemia cell line. It was
found that all these compounds showed good antiproliferative potential
against all these cancer cell lines tested having moderate difference
among them. Almost all these compounds showed better cytotoxicity
than BG45. Compounds with piperidine ring having the phenyl or
naphthyl group showed less activity against all these cell lines (cpd 5a
Fig. 5. Dose response curve of compounds 5e, 16c and 16d in HDAC8 inhibition assay [Data indicates mean
SD (n = 2)].
6

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EuropeanJournalofPharmaceuticalSciences138(2019)105046
Fig. 6. (i–v). Dose response curve and IC50 values of all newly synthesized compounds including BG45. These target molecules were evaluated in ten concentrations
on (i) B16F10, (ii) HeLa, (iii) A-549, (iv) MCF-7 and (v) Jurkat E6 cell lines. These compounds were treated in these respective cell lines for 72 h and subsequently,
the in vitro cellular viability was determined through MTT assay. Data represents mean
SD (n = 2) and was plotted in dose response format. IC50 value of these
compounds was estimated using nonlinear regression analysis method using Graph Pad Prism5 (Trivedi et al., 2018).
and 5c). However, compounds containing piperazine scaffold exhibited
appreciable cytotoxicity against all these cell lines. It was interesting to
note that a similarity of the cytotoxic pattern was noticed for these
compounds. The SAR analysis revealed that the cytotoxicity of these
compounds was mainly dependent on the aryl functionality (Table 3).
For both the piperidine and piperazine series, it was clearly observed
that compound containing the 4-quinolyl group is responsible for ex-
hibiting the higher cytoxicity than the compound containing the bi-
phenyl group. Again, compound containing the biphenyl group may be
responsible for better efficacy than compound containing the benzyl
group as evidenced in the cytotoxicity assay against all the five cell
lines (cpd 5e > cpd 5d > cpd 5b; cpd 16d > cpd 16b > cpd 16a).
The best response among these cell lines was observed for Jurkat E6
leukemia cell line. From the series, compound 5e, 16c and 16d were
found to be more efficient compared to the rest. The same trend was
also found for enzyme inhibition assay. Hence, these three compounds
were further subjected to evaluation for their apoptotic potential using
caspase3/7 assay in Jurkat E6 cells.
2.6.2. Cytotoxicity assay on non-cancerous cell lines
Cytotoxicity study was also conducted on HEK-293 (human em-
bryonic kidney cells) by using MTT assay method (Trivedi et al., 2018).
All the final target molecules were evaluated at the same with two
concentrations (10 μM and 100 μM) in triplicate. BG45 was also used in
the assay (Fig. 7). It was observed that most of these compounds dis-
played very less toxicity on HEK-293 cells indicating its potential to
affect cancer cells without affecting the normal cells.
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EuropeanJournalofPharmaceuticalSciences138(2019)105046
Table 3
IC50 values of target compounds on different cancer cell lines.
Compounds
IC50 (μM)
A-549
HeLa
MCF-7
B16F10
Jurkat E6
5a
˃50
˃50
˃50
˃50
˃50
5a′
39.53
27.49
˃50
42.56
20.91
˃50
39.04
25.48
˃50
31.66
23.80
˃50
35.32
25.34
˃50
5b
5c
5d
17.85
11.55
36.70
30.29
33.84
20.24
12.17
9.12
18.02
11.93
44.79
34.38
25.86
15.45
12.03
10.97
24.53
19.12
12.16
38.19
27.92
25.98
24.43
15.24
11.42
26.01
17.08
9.71
14.96
8.65
5e
11a
11b
16a
16b
16c
16d
BG45
31.29
23.80
27.70
18.31
16.21
9.76
28.49
27.13
30.34
17.95
13.71
7.19
21.43
29.60
17.33
Fig. 8. Caspase3/7 activation level of 5e, 16c, 16d and BG45 in Jurkat E6 cell
line compared to control. Compound treatment shows significant difference
from control indicates increased apoptosis in cells.
2.6.3. Apo-ONE® homogeneous Caspase-3/7 assay
Caspase 3/7 assay was conducted with the help of Apo-ONE®
Homogeneous Caspase-3/7 Assay kit (Promega, USA). Caspase 3 and
caspase 7 are the members of the cysteine-aspartic acid protease (cas-
pase) family. These caspases take part crucially in the execution-phase
of cellular apoptosis (Alnemri et al., 1996). Induction of apoptosis and
activation of caspases can result from a variety of stimuli including
exposure to radiation, growth factor withdrawal, exposure to che-
motherapeutic agents or Fas/Apo-1 receptor-mediated cell death in-
itiation (Nicholson and Thornberry, 1997). Active caspases take part in
the cleavage events those disrupt the homeostasis and repair enzymes
and initiate structural disassembly of dying cells (Vaux and Strasser,
1996; Kumar and Lavin, 1996). Compound 5e, 16c and 16d as well as
BG45 were tested for caspase3/7 activity using Jurkat E6 cells. Results
are displayed in Fig. 8.
perfectly mapped into the active site of HDAC8 whereas none of these
compounds showed any interaction with the active site amino acid
residues of HDAC3 enzyme. Probably, the length, size and bulkiness of
these molecules hinder their entrance into the smaller active site of
HDAC3 and therefore, no such interaction is noticed with HDAC3.
Hence, these molecules exert some HDAC8 selectivity compared to
HDAC3. As far as the ligand binding with HDAC8 is concerned, it is
observed that the hydroxyl group of the hydroxamate moiety for
compound 16c and 16d forms a salt-bridge interaction with active site
Zn2+ ion (Fig. 9B and C).
However, the amide group of the hydroxamate moiety is found to
form the salt bridge interaction with catalytic Zn2+ ion. For compounds
5e and 16c, Tyr306 residues of both chain A and chain B play pivotal
roles during enzyme-ligand interaction. For compounds 5e and 16c,
Tyr306 residue of the B chain forms a π-π stacking interaction with the
quinidine and biphenyl moieties respectively whereas for compound
16d, the quinidine moiety is closely located near the Tyr306 residue of
the B chain. Again, for compound 16c, an additional π-π stacking in-
teraction is noticed between the terminal phenyl group of the biphenyl
moiety and Phe152 of the B chain. Tyr306 of the B chain have π-π
stacking interaction with both the phenyl rings of the biphenyl moiety.
Therefore, it may be inferred that bulky hydrophobic aryl or heteroaryl
groups (such as biphenyl or quinidinyl) should possess a favorable
steric interaction for exerting potential HDAC8 inhibition. On the other
hand, the other Tyr306 residue of chain A is found to form a hydrogen
bond interaction with the carbonyl or amide group of the hydroxamate
The result demonstrates a significant increase in caspase3/7 activity
compared to control cells (vehicle treated cells). Increased caspase3/7
activity indicates the induced apoptosis in Jurkat E6 leukemia cancer
cells by the target compounds. This increased apoptosis level proves the
effective anticancer potential of these newly synthesized compounds.
2.6.4. Ligand-enzyme interactions through docking study
Three compounds (compound 5e, 16c and 16d) are found to be
identified as the effective HDAC8 inhibitors (> 70% inhibition at
10 μM dose) in this series of compounds having a minimum of 10-fold
selectivity over HDAC3 enzyme. These compounds were docked into
the active site of both HDAC8 (PDB: 1T69) and HDAC3 (PDB: 4A69)
enzymes (Fig. 9). It is interesting to note that all these three compounds
Fig. 7. Cytotoxicity study of all target compounds in HEK-293 cells by MTT assay method. Data represents mean
SD (n = 2).
8

P. Trivedi, et al.
EuropeanJournalofPharmaceuticalSciences138(2019)105046
Fig. 9. Docking interactions with HDAC8 (PDB: 1T69) (A) compound 5e (B) compound 16c and (C) compound 16d.
moiety. The carbonyl groups of both compounds 16c and 16d accept
hydrogen bond from Tyr306 residue of the chain A.
residue of the chain A and there is an electron transfer mechanism is
noticed among the groups of hydroxamate moiety and the surrounding
amino acids of the A chain namely His142, His143 and His180. The
carbonyl group becomes positively charged and both the carbonyl and
Interestingly, for compound 5e, the amide group of the hydro-
xamate moiety is found to accept hydrogen bond from the Tyr306
9

P. Trivedi, et al.
EuropeanJournalofPharmaceuticalSciences138(2019)105046
hydroxyl groups of the hydroxamate moiety donate hydrogen bonds to
His143 residue of the chain A. Nevertheless, the carbonyl group be-
comes hydroxylated and positively charged to form a π-cation inter-
action with His180 of chain A. Another π-cation interaction is noticed
for compound 5e between the positively charged amide group of the
piperidine moiety and Phe208. Water molecules also have some im-
portance as far as the enzyme-drug interactions are concerned. For all
these three molecules, carboxamide moiety adjacent to the piperidine
or piperazine nucleus forms hydrogen bonding interaction with the
water molecules at the junction between two chains of the HDAC8
enzyme. For compounds 16c and 16d, the carbonyl group of the car-
boxamide moiety accepts hydrogen bond from the neighborhood water
molecules whereas for compound 5e, the amide group of the carbox-
amide moiety acts as a hydrogen bond donor feature with the water
molecule. Similar type of hydrogen bond donor feature of the amide
group with water molecule is also noticed for compound 16c.
constrained and the bulky piperidine and piperazine linkers with bulky
cap group modifications yielded novel series of analogs with efficient
anticancer potential and preferential HDAC8 inhibition. From the
series, compound 16d having alkyl piperazinyl urea in linker region
and 4-aminoquinoline substituent in surface recognition site emerged
out as the lead compound which can be further modified to design more
effective and selective HDAC inhibitors.
4. Experimental
4.1. Chemistry
All initial raw materials and reagents were commercially available
and used without further purification. All these reactions involved were
monitored by using thin layer chromatography (TLC) using precoated
plates with silica gel F254 from Merck Millipore Co., USA (Trivedi
et al., 2018). Ultraviolet (UV) light as well as staining reagent (ninhy-
drin solution) was used to detect the spots. 1H and 13C NMR spectrums
were recorded in DMSO‑d6 using Bruker-400 MHz. Chemical shifts were
represented in ppm by using tetramethylsilane as the internal standard.
Mass spectroscopy of these compounds was conducted in LCMS-2020,
Schimadzu using ESI mode. Purity of these compounds were measured
by analytical LC/MS (Trivedi et al., 2018). Analysis was performed on a
LCMS-8040 equipped with a photodiode array detector using a Shiseido
C18 4.6 × 150 mm, 5 μ column at a flow rate of 1 mL/min with iso-
cratic flow (40% A: 60%; Solvent A = water + 0.4% TFA, solvent
B = methanol) (Trivedi et al., 2018). The elemental analysis for the
determination % of C, H and N was performed Elemental Analyser
(Vario Micro Cube)-Make-Elementar (Germany) and sulfanilamide (Art
No.:15.00-0062, Lot-No: SZBC3470V) was used as the reference stan-
dard sample.
3. Conclusion
In this article, some new hydroxamic acid-based potential HDAC8
inhibitors with different aryl cap groups linked through alkylpiperidine
and alkylpiperazine linker functions have been designed and synthe-
sized. As far as the design and discovery of potential small molecule
HDAC8 inhibitors is concerned, modification of linker region is some-
what least explored. The long chain methylene linker modification with
piperidine and piperazine thought to be a promising approach to design
potential HDAC inhibitors. Compared to piperidine derivatives, piper-
azine derivatives are found to be more effective may be because of the
urea linkage between cap group and piperazine ring that may offer
favorable hydrogen bonding interaction with the amino acid residues at
the enzyme pocket. Regarding the modification of cap group, it was
observed that increased bulkiness and hydrophobicity of the cap group
increased HDAC inhibitory activity of these compounds. Therefore,
naphthyl or biphenyl derivatives came out as better than the corre-
sponding phenyl or benzyl derivatives. Further activity was enhanced
by the introduction of heterocyclic nitrogen atom by incorporating the
4-aminoquinoline substituent which indicates the scope of heterocyclic
modifications for further design and development of such type of
compounds.
4.1.1. Procedure A. Preparation of tert-butyl 4-(phenylcarbamoyl)
piperidine-1-carboxylate (3a)
Compound 2 (1 g, 4.361 mmol) was dissolved in DCM. To this
mixture, 1-(3-dimethyl aminopropyl)-3-ethylcarbodiimide hydro-
chloride (1.67 g, 8.72 mmol) and 1-hydroxybenzotriazole (1.17 g,
8.72 mmol) was added and stirred for 20 min in nitrogen environment.
Aniline (0.796 mL, 8.72 mmol) and triethylamine (1.8 mL, 13.08 mmol)
was added to above mixture and stirred overnight. Reaction mixture
was diluted with DCM and washed with saturated aqueous solution of
sodium bicarbonate. DCM layer dried over sodium sulphate and eva-
porated. The crude solid was purified by column chromatography
(EtOAc: Hexane; 2.5:7.5) to get 0.9 g compound 3a as white solid (yield
75.35%). 1H NMR (400 MHz, DMSO‑d6) δ 9.97 (s, 1H), 7.62–7.58 (m,
2H), 7.31–7.25 (m, 2H), 7.04–6.99 (m, 1H), 3.99 (d, J = 12.3 Hz, 2H),
3.17–2.95 (m, 1H), 2.77 (s, 2H), 1.80–1.73 (m, 2H), 1.49 (td, J = 12.5,
4.5 Hz, 2H), 1.41 (s, 9H). C17H24N2O3 [M]: 304.18; MS (ESI) m/z:
[M + Na] +: 327.38 [M-H]+: 303.
As far as the biological screening of these compounds was con-
cerned, it was found that the synthesized compounds possessed potent
antiproliferative activity in various cancer cell lines. These compounds
displayed efficient cytotoxicity on human solid tumor cell lines as well
as a leukemia cell line. Moreover, the compounds also displayed neg-
ligible cytotoxicity against human embryonic kidney (HEK) cell line. It
indicated that these compounds were somewhat specific for cancer cell
lines and may produce lower side effects. Nevertheless, these com-
pounds showed an increased caspase3/7 activity which supported the
evidence of their anticancer potential through activation of apoptotic
pathways. All these compounds exhibited significant HDAC enzyme
inhibition in HeLa nuclear extract showing their potential in pan HDAC
inhibitory activity. To evaluate their activity within Class I HDACs, all
these compounds were screened against human recombinant HDAC3/
NCoR1 enzyme and human recombinant HDAC8 enzyme. Significant
HDAC8 inhibition and negligible HDAC3 inhibition proved their se-
lectivity towards HDAC8 compared to HDAC3, though further inhibi-
tion characterization on other HDACs are needed for more specific in-
sights. Modifications of hydroxamate with different linker and cap
group could not increase HDAC inhibitory potency. These newer ana-
logs are more selective towards HDAC8. As far as the cellular toxicity
was concerned, it was noticed that the cytotoxicity was influenced by
the aryl groups. The 4-quinolyl group was better than the biphenyl and
the biphenyl was better than the benzyl group for exerting higher cy-
totoxicity in all these cell lines as well as HDAC8 inhibitory activity.
Moreover, piperazine scaffold was better than the piperidine scaffold.
In a nutshell, the overall efforts to derivatize hydroxamates with
4.1.2. tert-Butyl 4-(benzylcarbamoyl)piperidine-1-carboxylate (3b)
Benzylamine (0.956 mL, 8.72 mmol) and compound
2 (1 g,
4.36 mmol) were reacted according to procedure A which afforded
0.314 g of 3b as creamy solid (yield 23%). 1H NMR (400 MHz,
DMSO‑d6) δ 8.35 (t, J = 5.8 Hz, 1H), 7.31 (dd, J = 10.6, 4.3 Hz, 2H),
7.22 (t, J = 6.4 Hz, 3H), 4.25 (d, J = 5.9 Hz, 2H), 3.95 (d, J = 12.2 Hz,
2H), 2.73 (s, 2H), 2.35 (m, 1H), 1.68 (d, J = 14.7 Hz, 2H), 1.46 (m,
1H), 1.39 (s, 9H). C18H26N2O3 [M]: 318.19; MS (ESI) m/z: [M + Na]+
:
341 [M-H]+: 317.
4.1.3. tert-Butyl
4-(naphthalen-1-ylcarbamoyl)piperidine-1-carboxylate
(3c)
According to procedure A, 1-naphthylamine (0.5 g, 3.49 mmol) and
compound 2 (0.4 g, 1.74 mmol) were reacted to get 0.150 g of product
3c as brown semisolid (yield 33.3%). 1H NMR (400 MHz, DMSO‑d6) δ
8.67 (s, 1H), 7.92 (m, 2H), 7.74 (d, J = 7.9 Hz, 1H), 7.52–7.40 (m, 4H),
10

P. Trivedi, et al.
EuropeanJournalofPharmaceuticalSciences138(2019)105046
3.53–3.49 (m, 4H), 3.43–3.39 (m, 3H), 1.44 (s, 9H). C21H26N2O3 [M]:
4.1.11. Methyl 4-(4-([1,1′-biphenyl]-4-ylcarbamoyl)piperidin-1-yl)butanoate
354.44; MS (ESI) m/z: [M-H]+: 353.
(4d)
Yield 89%, cream color solid, 1H NMR (400 MHz, DMSO‑d6) δ 8.60
(s, 1H), 7.65–7.60 (m, 2H), 7.57 (d, J = 9.0 Hz, 4H), 7.42 (t,
J = 7.6 Hz, 2H), 7.30 (t, J = 7.8 Hz, 1H), 3.59 (s, 3H), 3.44 (s, 5H),
2.39–2.29 (m, 8H), 1.76–1.68 (m, 2H). C23H28N2O3 [M]: 380.48; MS
(ESI) m/z: [M + H]+: 381, [M-H]+: 379.
4.1.4. tert-Butyl 4-([1,1′-biphenyl]-4-ylcarbamoyl)piperidine-1-carboxylate
(3d)
Following procedure A, compound 3d was synthesized from 4-
aminobiphenyl (0.73 mg, 2.18 mmol) and compound
2 (0.5 g,
2.18 mmol) as creamy solid (0.5 g, 60%). 1H NMR (400 MHz, DMSO‑d6)
δ 10.02 (s, 1H), 7.70 (d, J = 8.7 Hz, 2H), 7.65–7.60 (m, 4H), 7.44 (t,
J = 7.7 Hz, 2H), 7.34–7.30 (m, 1H), 4.01 (d, J = 12.2 Hz, 2H), 3.33 (s,
5H), 1.79 (d, J = 13.3 Hz, 2H), 1.41 (s, 9H). C23H28N2O3 [M]: 354.44;
MS (ESI) m/z: [M-H]+: 353.
4.1.12. Methyl 4-(4-([1,1′-biphenyl]-4-ylcarbamoyl)piperidin-1-yl)butanoate
(4e)
Yield 59%, yellow solid, 1H NMR (400 MHz, DMSO‑d6) δ 10.15 (s,
1H), 8.78 (d, J = 5.0 Hz, 1H), 8.36 (d, J = 7.8 Hz, 1H), 8.08 (d,
J = 5.0 Hz, 1H), 7.99 (dd, J = 8.4, 0.8 Hz, 1H), 7.77 (m, 1H), 7.63 (m,
1H), 3.60 (s, 3H), 2.94 (d, J = 10.3 Hz, 2H), 2.77–2.68 (m, 1H), 2.33 (t,
J = 7.2 Hz, 4H), 1.98 (s, 2H), 1.91–1.83 (m, 2H), 1.71 (m,4H).
4.1.5. tert-Butyl 4-(quinolin-4-ylcarbamoyl)piperidine-1-carboxylate (3e)
4-Aminoquinoline (0.5 g, 3.46 mmol) and compound 2 (1.19 g,
5.20 mmol) were reacted by using following procedure A to get 0.237 g
of compound 3e as pale yellow solid (yield 20%). 1H NMR (400 MHz,
DMSO‑d6) δ 10.20 (s, 1H), 8.78 (d, J = 5.0 Hz, 1H), 8.36 (dd, J = 8.5,
0.7 Hz, 1H), 8.08 (d, J = 5.0 Hz, 1H), 8.00 (dd, J = 8.4, 0.8 Hz, 1H),
7.77 (m, 1H), 7.64 (m, 1H), 3.34 (s, 5H), 1.89 (dd, J = 13.7, 2.8 Hz,
2H), 1.78 (dd, J = 13.3, 3.2 Hz, 2H), 1.39 (s, 9H). C20H25N3O3 [M]:
355.43; MS (ESI) m/z: [M + H]+: 356, [M-H]+: 354.
C
20H25N3O3 [M]: 355.43; MS (ESI) m/z: [M + H]+: 356, [M-H]+: 354.
4.1.13. Procedure C. General procedure for synthesis of 5a, 5a′, 5b, 5c, 5d
and 5e
Compound 4a, 4a′, 4b, 4c, 4d and 4e were dissolved in methanol.
50% w/v aqueous hydroxylamine (18.4 equivalents) was added and the
mixture was stirred for 15 min. 1 M NaOH (2 equivalents) was added
and the reaction was stirred for 2 h at room temperature. It was con-
centrated in reduced pressure and the resultant solid was dissolved in
ice cold water. The pH was adjusted to 7.0 with 1 M HCl and the pro-
duct was precipitated. No further purification was required. The pro-
duct was then dried in vacuum oven.
4.1.6. Procedure B: general procedure for synthesis of 4a, 4a′, 4b, 4c, 4d
and 4e
Compound 3a, 3b, 3c, 3d, 3e were dissolved in DCM and kept in ice
bath at 0 °C. Trifluoroacetic acid (50% v/v of total volume of reaction
mixture) was added to it and stirred for 2 h under inert condition (ni-
trogen environment). After completion of the reaction, the solvent was
evaporated and the product was found pure enough to be used further
for next step. To the same compound, methyl 3-bromopropionate (1.5
equivalents) for the synthesis of 4a and methyl 4-bromobutyrate (1.5
equivalents) for the synthesis of 4a′, 4b, 4c, 4d and 4e, potassium
carbonate (3 equivalents) and acetonitrile were added. The reaction
mixture was refluxed at 90 °C for 5 h. The mixture was cooled and
poured in water. The product was extracted in ethyl acetate, as well as
concentrated and purified by column chromatography (EtOAc: me-
thanol; 9.5:0.5).
4.1.14. 1-(3-(Hydroxyamino)-3-oxopropyl)-N-phenylpiperidine-4-
carboxamide (5a)
Yield 40%, brown powder, 1H NMR (400 MHz, DMSO‑d6) δ 10.22 (s,
1H), 7.63 (d, J = 7.7 Hz, 2H), 7.29 (t, J = 7.9 Hz, 2H), 7.04 (t,
J = 7.4 Hz, 1H), 3.52 (d, J = 11.6 Hz, 2H), 3.37 (s, 2H), 3.28 (t,
J = 7.6 Hz, 2H), 2.86 (t, J = 7.6 Hz, 2H), 2.65 (s, 1H), 2.01 (s, 4H). 13
C
NMR (101 MHz, DMSO‑d6) δ 172.27, 172.05, 139.62, 129.11, 123.66,
119.67, 51.93, 51.50, 29.03, 26.15. HPLC Rt: 1.986, LC/MS calculated
for expected C15H21N3O3 [M]: 291.35; Found: [M]+: 291; Anal. calcd.
for C15H21N3O3 (Mr = 291.158): C 61.84, H 7.27, N 14.42; found: C
61.52, H 6.98, N 14.69%.
4.1.7. Methyl 3-(4-(phenylcarbamoyl)piperidin-1-yl)propanoate (4a)
Yield 52.5%, pale brown solid, 1H NMR (400 MHz, DMSO‑d6) δ 9.90
(s, 1H), 7.60 (dd, J = 8.6, 1.0 Hz, 2H), 7.31–7.25 (m, 2H), 7.02 (t,
J = 7.4 Hz, 1H), 3.60 (s, 3H), 3.34 (s, 4H), 2.34 (t, J = 7.1 Hz, 3H),
4.1.15. 1-(4-(Hydroxyamino)-4-oxobutyl)-N-phenylpiperidine-4-
carboxamide (5a′)
Yield 66.6%, yellow oil, 1H NMR (400 MHz, DMSO‑d6) δ 7.61 (d,
J = 7.6 Hz, 2H), 7.30 (t, J = 7.9 Hz, 2H), 7.04 (t, J = 7.4 Hz, 1H), 3.58
(d, J = 36.1 Hz, 3H), 3.09–3.02 (m, 2H), 2.39 (m, 1H), 2.11–1.88 (m,
9H). 13C NMR (101 MHz, DMSO‑d6) δ 173.89, 155.13, 140.67, 128.76,
122.48, 120.18, 55.37, 51.12, 44.17, 41.33, 31.25, 19.16 HPLC Rt:
1.995, LC/MS calculated for expected C16H23N3O3 [M]: 305.37; Found:
[M + H]+: 306; Anal. calcd. for C16H23N3O3 (Mr = 305.173): C 62.93,
H 7.59, N 13.76; found: C 63.14, H 7.22, N 13.99%.
1.83–1.65 (m, 6H). C16H22N2O3 [M]: 290.16; MS (ESI) m/z: [M + H]+
:
291.
4.1.8. Methyl 4-(4-(phenylcarbamoyl)piperidin-1-yl)butanoate (4a′)
Yield 57.8%, pale yellow semi solid, 1H NMR (400 MHz, DMSO‑d6)
δ 9.90 (s, 1H), 7.60 (dd, J = 8.6, 1.0 Hz, 2H), 7.31–7.25 (m, 2H), 7.02
(t, J = 7.4 Hz, 1H), 3.60 (s, 3H), 3.34 (s, 4H), 3.00 (s, 2H), 2.34 (t,
J = 7.1 Hz, 3H), 1.83–1.65 (m, 6H). C17H24N2O3 [M]: 304.18; MS (ESI)
m/z: [M + H]+: 305.
4.1.16. N-benzyl-1-(4-(hydroxyamino)-4-oxobutyl)piperidine-4-
carboxamide (5b)
4.1.9. Methyl 4-(4-(benzylcarbamoyl)piperidin-1-yl)butanoate (4b)
Yield 80%, pale yellow thick oil, 1H NMR (400 MHz, DMSO‑d6) δ
7.25 (m, 5H), 7.06 (t, J = 5.8 Hz, 1H), 4.23 (d, J = 5.8 Hz, 2H), 3.58 (s,
4H), 3.30 (dd, J = 9.7, 5.0 Hz, 4H), 2.29 (m, 8H), 1.69 (m, 2H).
Yield 90%, cream color solid, 1H NMR (400 MHz, DMSO‑d6) δ
7.34–7.24 (m, 5H), 7.20 (m, 1H), 4.23 (d, J = 5.7 Hz, 2H), 3.42 (s, 4H),
2.50 (d, J = 4.2 Hz, 4H), 2.46–2.40 (m, 2H), 2.24–1.99 (m, 2H), 1.71
(m, 2H), 1.30–1.21 (m, 1H). 13C NMR (101 MHz, DMSO‑d6) δ 169.53,
157.83, 141.38, 127.49, 126.85, 57.11, 52.56, 43.82, 43.23, 43.04,
30.44, 22.02 HPLC Rt: 2.025, LC/MS calculated for expected
C
17H24N2O3 [M]: 304.18; MS (ESI) m/z: [M + H]+: 319.
4.1.10. Methyl 4-(4-(naphthalen-1-ylcarbamoyl)piperidin-1-yl)butanoate
(4c)
C
17H25N3O3 [M]: 319.40; Found: [M + H]+: 320; Anal. calcd. for
C17H25N3O3 (Mr = 319.189): C 63.93, H 7.89, N 13.16; found: C 64.04,
H 8.22, N 12.84%.
Yield 60%, pale yellow semi solid, 1H NMR (400 MHz, DMSO‑d6) δ
8.04–8.01 (m, 1H), 7.95–7.92 (m, 1H), 7.76 (d, J = 8.2 Hz, 1H), 7.64
(d, J = 7.4 Hz, 1H), 7.56–7.52 (m, 2H), 7.51–7.46 (m, 1H), 3.60 (s, 3H),
3.59–3.58 (m, 2H), 1.91 (s, 3H), 1.76 (m, 5H), 1.25 (d, J = 9.3 Hz, 5H).
4.1.17. 1-(4-(Hydroxyamino)-4-oxobutyl)-N-(naphthalen-1-yl)piperidine-
4-carboxamide (5c)
C
21H26N2O3 [M]: 354.44; MS (ESI) m/z: [M + H]+: 355.
Yield 92%, beige color solid, 1H NMR (400 MHz, DMSO‑d6) δ 9.03
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EuropeanJournalofPharmaceuticalSciences138(2019)105046
(s, 1H), 7.99–7.90 (m, 2H), 7.76 (d, J = 7.8 Hz, 1H), 7.54–7.42 (m, 5H),
4.32 (d, J = 8.1 Hz, 2H), 3.21–3.09 (m, 5H), 2.37 (m, 4H), 2.14–1.88
(m, 4H). 13C NMR (101 MHz, DMSO‑d6) δ 175.09, 173.92, 158.74,
141.69, 138.78, 134.70, 134.25, 126.60, 124.49, 120.21, 116.42,
102.18, 55.62, 51.22, 31.23, 25.92, 25.55, 19.18.HPLC Rt: 2.467, LC/
compound
9
(0.4 g, 1.31 mmol) and methyl 3-bromopropionate
(0.328 g, 1.968 mmol) afforded 0.156 g of cream color sticky mass
(yield 41%). 1H NMR (400 MHz, DMSO‑d6) δ 7.45 (td, J = 8.7, 0.9 Hz,
2H), 7.23 (m, 2H), 6.96–6.89 (m, 1H), 3.60–3.57 (m, 3H), 3.49 (s, 1H),
3.43–3.39 (m, 3H), 2.36–2.28 (m, 6H), 1.75–1.66 (m, 2H). C15H21N3O3
[M]: 291.35; MS (ESI) m/z: [M + H]+: 292.
MS calculated for expected
C20H25N3O3 [M]: 355.43; Found:
[M + H]+: 356; Anal. calcd. for C20H25N3O3 (Mr = 355.189): C 67.58,
H 7.09, N 11.82; found: C 67.82, H 7.20, N 12.09%.
4.1.23. Methyl 4-(4-(phenylcarbamoyl)piperazin-1-yl)butanoate (10b)
According to procedure B, compound 10b was prepared from
4.1.18. N-([1,1′-biphenyl]-4-yl)-1-(4-(hydroxyamino)-4-oxobutyl)
piperidine-4-carboxamide (5d)
compound 9 (0.330 g, 1.08 mmol) and methyl 4-bromobutyrate
(0.291 g, 1.60 mmol) that afforded 0.187 g of cream color solid (yield
56.6%). 1H NMR (400 MHz, DMSO‑d6) δ 7.45 (td, J = 8.7, 0.9 Hz, 2H),
7.23 (m, 2H), 6.96–6.89 (m, 1H), 3.60–3.57 (m, 3H), 3.48 (s, 2H),
3.43–3.40 (m, 4H), 2.36–2.28 (m, 6H), 1.75–1.66 (m, 2H).C16H23N3O3
[M]: 305.37; MS (ESI) m/z: [M + H]+: 306.
Yield 82%, creamish yellow solid, 1H NMR (400 MHz, DMSO‑d6) δ
7.76 (d, J = 8.7 Hz, 2H), 7.63 (t, J = 8.4 Hz, 4H), 7.44 (t, J = 7.7 Hz,
2H), 7.33 (t, J = 7.3 Hz, 1H), 3.48 (d, J = 12.0 Hz, 2H), 3.05 (m, 8H),
2.75 (m, 1H), 2.35 (t, J = 7.3 Hz, 1H), 1.98 (m, 6H). 13C NMR
(101 MHz, DMSO‑d6)
δ 173.94, 172.44, 140.15, 139.21, 135.19,
129.36, 127.45, 127.25, 126.67, 120.01, 55.73, 51.36, 31.21, 26.09,
19.28. HPLC Rt: 2.689, LC/MS calculated for expected C22H27N3O3
[M]: 381.47; Found: [M + H]+: 382; Anal. calcd. for C22H27N3O3
(Mr = 381.205): C 69.27, H 7.13, N 11.82; found: C 69.68, H 7.49 N
11.53%.
4.1.24. 4-(3-(Hydroxyamino)-3-oxopropyl)-N-phenylpiperazine-1-
carboxamide (11a)
Following procedure C, compound 11a was prepared from compound
10a (0.150 g, 0.51 mmol), produced 0.100 g of product as beige color
powder (yield 66.6%). 1H NMR (400 MHz, DMSO‑d6) δ 8.67 (s, 1H), 7.49
(d, J = 8.2 Hz, 2H), 7.26–7.20 (m, 2H), 6.95 (q, J = 7.3 Hz, 1H), 3.50 (s,
3H), 3.45–3.28 (m, 9H). 13C NMR (101 MHz, DMSO‑d6) δ 155.49, 140.95,
128.79, 128.75, 122.23, 120.15, 51.22, 44.15, 41.38, 28.83. HPLC Rt:
1.957, LC/MS calculated for expected C14H20N4O3 [M]: 292.33; Found:
[M + H]+: 293; Anal. calcd. for C14H20N4O3 (Mr = 292.153): C 57.52, H
6.90, N 19.17; found: C 57.87, H 7.13, N 19.57%.
4.1.19. 1-(4-(Hydroxyamino)-4-oxobutyl)-N-(quinolin-4-yl)piperidine-4-
carboxamide (5e)
Yield 84.3%, yellow color powder, 1H NMR (400 MHz, DMSO‑d6) δ
8.71 (d, J = 6.6 Hz, 1H), 8.61 (d, J = 8.1 Hz, 1H), 8.42 (t, J = 7.7 Hz,
1H), 8.19–8.13 (m, 1H), 8.05 (d, J = 8.5 Hz, 1H), 7.95 (m, 1H), 7.67
(m, 1H), 6.92 (d, J = 6.9 Hz, 1H), 3.15–2.92 (m, 6H), 2.35 (dd,
J = 15.1, 7.3 Hz, 3H), 1.96 (dt, J = 23.8, 13.4 Hz, 7H)·13C NMR
4.1.25. 4-(4-(Hydroxyamino)-4-oxobutyl)-N-phenylpiperazine-1-
carboxamide (11b)
(101 MHz, DMSO‑d6)
δ 175.09, 173.92, 158.74, 141.69, 138.78,
134.25, 126.60, 124.49, 120.21, 116.42, 102.18, 55.62, 51.22, 31.23,
25.92, 25.55, 19.18. HPLC Rt: 2.612, LC/MS calculated for expected
C19H24N4O3 [M]: 356.42; Found: [M + H]+: 357; Anal. calcd. for
According to procedure C, compound 11b was prepared from
compound 10b (0.180 g, 0.59 mmol), produced 0.130 g of product as
cream color powder (yield 72%). 1H NMR (400 MHz, DMSO‑d6) δ 8.97
(s, 1H), 7.50 (d, J = 8.3 Hz, 2H), 7.24 (m, 2H), 6.95 (t, J = 7.3 Hz, 1H),
3.51 (s, 2H), 3.45 (s, 5H), 3.12–3.06 (m, 2H), 2.35 (t, J = 7.3 Hz, 2H),
2.01–1.93 (m, 2H), 1.30–1.22 (m, 1H). 13C NMR (101 MHz, DMSO‑d6) δ
173.89, 155.13, 140.67, 128.76, 122.48, 120.18, 55.37, 51.12, 44.17,
31.25, 19.16. HPLC Rt: 1.980, LC/MS calculated for expected
C
19H24N4O3 (Mr = 356.184): C 64.03, H 6.79, N 15.72; found: C 63.80,
H 7.17, N 15.60%.
4.1.20. tert-Butyl piperazine-1-carboxylate (7)
Anhydrous piperazine (5 g, 58.04 mmol) was dissolved in 30 mL of
methanol. The reaction mixture was bought to 0 °C temperature by
keeping it in an ice bath. Di-tert-butyl-dicarbonate (10.13 g,
46.43 mmol) was mixed with 20 mL of methanol and added to the
above mixture dropwise. The reaction was stirred for 3 h at room
temperature. After completion of reaction, methanol was evaporated
and the resultant solid was dissolved in ice cold water. The product was
extracted in ethyl acetate and washed with saturated solution of sodium
bicarbonate, dried over sodium sulphate and evaporated finally to get
5.9 g of white solid as a product (yield 54.6%). 1H NMR (400 MHz,
DMSO‑d6) δ 3.28 (s, 2H), 3.20 (m, 2H), 2.62–2.57 (m, 4H), 1.39 (d,
C
C
15H22N4O3 [M]: 306.36; Found: [M/Z]+
: 306; Anal. calcd. for
15H22N4O3 (Mr = 306.169): C 58.81, H 7.24, N 18.29; found: C 58.97,
H 7.42, N 18.62%.
4.1.26. Procedure D. General procedure for synthesis of 13a, 13b, 13c,
13d
Aryl amine (12a: benzylamine, 12b: 1-naphthylamine, 12c: 4-ami-
nobiphenyl, 12d: 4-amino quinoline) (1 g) was dissolved in 20 mL of
DCM. To this solution, 20 mL of NaHCO3 solution was stirred at 0 °C.
Stirring was stopped momentarily and triphosgene (0.5 equivalents)
dissolved in 1 mL of DCM was added with syringe to DCM layer and
stirring continued for 1 h. The DCM layer was separated and passed
through bed of celite® and subjected to next reaction immediately.
J = 4.8 Hz, 9H). C9H18N2O2 [M]: 186.25; MS (ESI) m/z: [M + H]+
:
187.
4.1.21. tert-Butyl 4-(phenylcarbamoyl)piperazine-1-carboxylate (9)
tert-Butyl piperazine-1-carboxylate (7) (1 g, 5.36 mmol) was dis-
solved in 10 mL of DCM. Phenyl isocyanate (8) (0.64 g, 5.36 mmol) was
added dropwise to above solution and flushed with nitrogen gas. The
reaction was stirred for 4 h. It was diluted with DCM and washed with
saturated sodium bicarbonate solution. The DCM layer was collected,
dried using sodium sulphate and evaporated to get crude mass. The
crude was purified by column chromatography (EtOAc: Hexane; 3:7) to
get 0.92 g of white powder (yield 56.4%). 1H NMR (400 MHz,
DMSO‑d6) δ 8.56 (s, 1H), 7.46–7.43 (m, 2H), 7.26–7.20 (m, 2H),
6.96–6.91 (m, 1H), 3.42 (m, 4H), 3.35 (m, 4H), 1.42 (s, 9H).
4.1.27. Procedure E. General procedure for synthesis of 14a, 14b, 14c,
14d
To the DCM layer containing 13a, 13b, 13c and 13d, tert-butyl
piperazine-1-carboxylate (7) (1 equivalent) was added and stirred for
4 h in nitrogen environment. Reaction mixture was diluted with DCM
and washed with saturated sodium bicarbonate solution. DCM layer
was dried using sodium sulphate, evaporated and purified by column
chromatography (EtOAc: Hexane; 3:7).
4.1.28. tert-Butyl 4-(benzylcarbamoyl)piperazine-1-carboxylate (14a)
Yield 84%, white solid, 1H NMR (400 MHz, DMSO‑d6) δ 7.33–7.19
(m, 6H), 4.24 (dd, J = 5.7, 3.6 Hz, 2H), 3.33 (s, 1H), 3.29 (d,
J = 7.4 Hz, 7H), 1.40 (s, 9H). C17H25N3O3 [M]: 319.40; MS (ESI) m/z:
[M-H]+: 318.
C
16H23N3O3 [M]: 305.37; MS (ESI) m/z: [M + H]+: 306, [M-H]+: 304.
4.1.22. Methyl 3-(4-(phenylcarbamoyl)piperazin-1-yl)propanoate (10a)
According to procedure B, compound 10a was prepared from
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P. Trivedi, et al.
EuropeanJournalofPharmaceuticalSciences138(2019)105046
4.1.29. tert-Butyl 4-(naphthalen-1-ylcarbamoyl)piperazine-1-carboxylate
(14b)
color sticky solid (Yield 83.3%). 1H NMR (400 MHz, DMSO‑d6) δ
7.34–7.24 (m, 5H), 7.20 (m, 1H), 4.23 (d, J = 5.7 Hz, 2H), 3.42 (s, 4H),
2.50 (d, J = 4.2 Hz, 3H), 2.46–2.40 (m, 2H), 2.24–1.99 (m, 2H), 1.71
(m, 2H), 1.30–1.21 (m, 1H). 13C NMR (101 MHz, DMSO‑d6) δ 169.53,
157.83, 141.38, 127.49, 126.85, 57.11, 52.56, 43.82, 43.23, 30.44,
22.02. HPLC Rt: 1.986, LC/MS calculated for expected C16H24N4O3
Yield 75%, white solid, 1H NMR (400 MHz, DMSO‑d6) δ 8.67 (s,
1H), 7.92 (m, 2H), 7.74 (d, J = 7.9 Hz, 1H), 7.52–7.40 (m, 4H),
3.53–3.49 (m, 4H), 3.43–3.39 (m, 4H), 1.44 (s, 9H). C20H25N3O3 [M]:
355.43; MS (ESI) m/z: [M + H]+: 356, [M-H]+: 354.
[M]: 320.18; Found: [M/Z]+
: 320; Anal. calcd. for C16H24N4O3
4.1.30. tert-Butyl 4-([1,1′-biphenyl]-4-ylcarbamoyl)piperazine-1-carboxylate
(14c)
(Mr = 320.184): C 59.98, H 7.55, N 17.49; found: C 60.33, H 7.78, N
17.17%.
Yield 80%, white solid, 1H NMR (400 MHz, DMSO‑d6) δ 8.69 (s,
1H), 7.65–7.61 (m, 2H), 7.56 (s, 4H), 7.43 (t, J = 7.7 Hz, 2H), 7.30 (t,
J = 7.3 Hz, 1H), 3.48–3.43 (m, 4H), 3.36 (dd, J = 9.2, 5.2 Hz, 4H), 1.43
(s, 9H). C22H27N3O3 [M]: 381.47; MS (ESI) m/z: [M-H]+: 380.
4.1.37. 4-(4-(Hydroxyamino)-4-oxobutyl)-N-(naphthalen-1-yl)
piperazine-1-carboxamide (16b)
According to procedure C, compound 16b was prepared from
compound 15b (0.175 g, 0.49 mmol), produced 0.160 g of product as
beige color solid (yield 91.4%) 1H NMR (400 MHz, DMSO‑d6) δ 9.03 (s,
1H), 7.99–7.90 (m, 2H), 7.76 (d, J = 7.8 Hz, 1H), 7.54–7.42 (m, 5H),
4.32 (d, J = 8.1 Hz, 2H), 3.21–3.09 (m, 5H), 2.37 (m, 4H), 2.14–1.88
(m, 4H). 13C NMR (101 MHz, DMSO‑d6) δ 175.09, 173.92, 158.74,
141.69, 138.78, 134.70, 134.25, 126.60, 124.49, 120.21, 116.42,
102.18, 55.62, 51.22, 31.23, 25.55, 19.18. HPLC Rt: 2.008, LC/MS
4.1.31. tert-Butyl
4-(quinolin-4-ylcarbamoyl)piperazine-1-carboxylate
(14d)
Yield 70%, yellow solid, 1H NMR (400 MHz, DMSO‑d6) δ 10.20 (s,
1H), 8.78 (d, J = 5.0 Hz, 1H), 8.36 (dd, J = 8.5, 0.7 Hz, 1H), 8.08 (d,
J = 5.0 Hz, 1H), 8.00 (dd, J = 8.4, 0.8 Hz, 1H), 7.77 (m, 1H), 7.64 (m,
1H), 3.34 (s, 4H), 1.89 (dd, J = 13.7, 2.8 Hz, 2H), 1.78 (dd, J = 13.3,
3.2 Hz, 2H), 1.39 (s, 9H). C22H27N3O3 [M]: 356.42; MS (ESI) m/z:
[M + H]+: 357, [M-H]+: 355.
calculated for expected C19H24N4O3 [M]: 356.18; Found: [M + H]+
:
357; Anal. calcd. for C19H24N4O3 (Mr = 356.184): C 64.03, H 6.79, N
15.72; found: C 63.81, H 6.92, N 16.03%.
4.1.32. Methyl 4-(4-(benzylcarbamoyl)piperazin-1-yl)butanoate (15a)
Compound 15a was synthesized by following procedure B from
compound 14a (1 g, 3.13 mmol) and methyl 4-bromobutanoate
(0.850 g, 4.69 mmol) yielded 0.310 g 15a as cream color sticky solid
mass (yield 97%). 1H NMR (400 MHz, DMSO‑d6) δ 7.25 (m, 5H), 7.06
(t, J = 5.8 Hz, 1H), 4.23 (d, J = 5.8 Hz, 2H), 3.58 (s, 3H), 3.30 (dd,
J = 9.7, 5.0 Hz, 4H), 2.29 (m, 8H), 1.69 (m, 2H). C17H25N3O3 [M]:
319.40; MS (ESI) m/z: [M + H]+: 320.
4.1.38. N-([1,1′-biphenyl]-4-yl)-4-(4-(hydroxyamino)-4-oxobutyl)
piperazine-1-carboxamide (16c)
According to procedure C, compound 16c was prepared from
compound 15c (0.173 g, 0.45 mmol), produced 0.165 g of product as
beige color solid (Yield 95.3%). 1H NMR (400 MHz, DMSO‑d6) δ 7.76
(d, J = 8.7 Hz, 2H), 7.63 (t, J = 8.4 Hz, 4H), 7.44 (t, J = 7.7 Hz, 2H),
7.33 (t, J = 7.3 Hz, 1H), 3.48 (d, J = 12.0 Hz, 2H), 3.05 (m, 8H), 2.75
(m, 1H), 2.35 (t, J = 7.3 Hz, 1H), 1.98 (m, 6H). 13C NMR (101 MHz,
DMSO‑d6) δ 173.94, 172.44, 140.15, 139.21, 135.19, 129.36, 127.45,
127.25, 126.67, 120.01, 55.73, 51.36, 31.21, 26.09, 19.28 HPLC Rt:
2.513, LC/MS calculated for expected C21H26N4O3 [M]: 382.46; Found:
[M + H]+: 383; Anal. calcd. for C21H26N4O3 (Mr = 382.200): C 65.95,
H 6.85, N 14.65; found: C 66.28, H 7.06, N 15.08%.
4.1.33. Methyl 4-(4-(naphthalen-1-ylcarbamoyl)piperazin-1-yl)butanoate
(15b)
Following procedure B, compound 14b (0.350 g, 0.98 mmol) and
methyl 4-bromobutanoate (0.266 g, 1.47 mmol) reacted together to
produce compound 15b as cream color semisolid (0.200 g, 57%). 1H
NMR (400 MHz, DMSO‑d6) δ 7.94 (m, 2H), 7.54–7.41 (m, 6H), 3.63 (s,
3H), 3.61 (s, 2H), 3.17–3.11 (m, 3H), 2.09–1.81 (m, 7H), 1.23 (s, 2H).
4.1.39. 4-(4-(Hydroxyamino)-4-oxobutyl)-N-(quinolin-4-yl) piperazine-1-
carboxamide (16d)
C
20H25N3O3 [M]: 355.43; MS (ESI) m/z: [M + H]+: 356, [M-H]+: 354.
Following procedure C, compound 16d was prepared from com-
pound 15d (0.160 g, 0.44 mmol), produced 0.120 g of product as
yellow solid (yield 75%). 1H NMR (400 MHz, DMSO‑d6) δ 8.71 (d,
J = 6.6 Hz, 1H), 8.61 (d, J = 8.1 Hz, 1H), 8.42 (t, J = 7.7 Hz, 1H),
8.19–8.13 (m, 1H), 8.05 (d, J = 8.5 Hz, 1H), 7.95 (m, 1H), 7.67 (m,
1H), 6.92 (d, J = 6.9 Hz, 1H), 3.15–2.92 (m, 6H), 2.35 (dd, J = 15.1,
7.3 Hz, 3H), 1.96 (dt, J = 23.8, 13.4 Hz, 7H)·13C NMR (101 MHz,
DMSO‑d6) δ 175.09, 173.92, 158.74, 141.69, 138.78, 134.25, 126.60,
124.49, 120.21, 116.42, 102.18, 55.62, 51.22, 31.23, 25.92, 25.55,
19.18. HPLC Rt: 2.467, LC/MS calculated for expected C18H23N5O3
[M]: 357.41; Found: [M + H]+: 358; Anal. calcd. for C18H23N5O3
(Mr = 357.180): C 60.49, H 6.49, N 19.59; found: C 60.21, H 6.86, N
19.34%.
4.1.34. Methyl
4-(4-([1,1′-biphenyl]-4-ylcarbamoyl)piperazin-1-yl)
butanoate (15c)
Compound 14c (0.565 g, 1.48 mmol) and methyl 4-bromobutanoate
(0.403 g, 2.22 mmol) were reacted by following procedure B afforded
0.350 g pale brown solid (Yield 62%). 1H NMR (400 MHz, DMSO‑d6) δ
8.60 (s, 1H), 7.65–7.60 (m, 2H), 7.57 (d, J = 9.0 Hz, 4H), 7.42 (t,
J = 7.6 Hz, 2H), 7.30 (t, J = 7.8 Hz, 1H), 3.59 (s, 3H), 3.44 (s, 4H),
2.39–2.29 (m, 8H), 1.76–1.68 (m, 2H). C22H27N3O3 [M]: 381.47; MS
(ESI) m/z: [M + H]+: 382, [M-H]+: 380.
4.1.35. Methyl 4-(4-(quinolin-4-ylcarbamoyl) piperazin-1-yl)butanoate
(15d)
Following procedure B, compound 14d (0.300 g, 0.84 mmol) and
methyl 4-bromobutanoate (0.228 g, 1.26 mmol) reacted together to
produce compound 15d as pale yellow solid (0.180 g, 60%). 1H NMR
(400 MHz, DMSO‑d6) δ 10.15 (s, 1H), 8.78 (d, J = 5.0 Hz, 1H), 8.36 (d,
J = 7.8 Hz, 1H), 8.08 (d, J = 5.0 Hz, 1H), 7.99 (dd, J = 8.4, 0.8 Hz,
1H), 7.77 (m, 1H), 7.63 (m, 1H), 3.60 (s, 3H), 2.94 (d, J = 10.3 Hz,
2H), 2.33 (t, J = 7.2 Hz, 4H), 1.98 (s, 2H), 1.91–1.83 (m, 2H), 1.71
(m,4H). C19H24N4O3 [M]: 356.42; MS (ESI) m/z: [M + H]+: 357.
1H NMR, 13C NMR and mass spectra of compound 5a, 5a′, 5b, 5c,
5d, 5e, 11a, 11b, 16a, 16b, 16c and 16d are shown in the
Supplementary material (Fig. S2).
4.2. HDAC inhibition assay using HeLa nuclear extract
The HDAC enzyme inhibition assay was done using HDAC colori-
metric assay kit (BML-AK501, ENZO life sciences) following the ven-
dor's protocol (Trivedi et al., 2018). The assay method was discussed in
our earlier report (Trivedi et al., 2018). As per the protocol, 5 μL of
HeLa nuclear extract (BML-KI137-0500), 10 μL of assay buffer (BML-
KI143-0020), 10 μL of sample solution were added per well in a mi-
crotiter plate. The reaction was initiated when 25 μL Color de Lys®
4.1.36. N-benzyl-4-(4-(hydroxyamino)-4-oxobutyl) piperazine-1-carboxamide
(16a)
Following procedure C, compound 16a was prepared from com-
pound 15a (0.360 g, 1.12 mmol), produced 0.300 g of product as cream
13

P. Trivedi, et al.
EuropeanJournalofPharmaceuticalSciences138(2019)105046
substrate solution (BML-KI138-0050) was added (Trivedi et al., 2018).
The reaction was incubated for 30 min at 37 °C which was terminated
during the addition of a 50 μL mixture of developer and stop solution.
The plate was incubated for 15 min at 37 °C and the absorbance was
recorded at 405 nm. All compounds along with BG45 were evaluated at
10 μM concentration in duplicate (Trivedi et al., 2018). Compound 5e,
16c and 16d were further tested to estimate their IC50 values in the
same way described above. Here, compounds were tested with the
concentrations 0.5 μM, 1 μM, 5 μM, 10 μM, 15 μM.
DMSO at a concentration of 100 mM and stored. The detail cell culture
and drug treatment was conducted as per our earlier observations
(Trivedi et al., 2018).
4.5.1. MTT assay
The in vitro cytotoxicity of all these compounds was evaluated by
MTT assay method. 5 × 103 cells (B16F10, HeLa, A-549, MCF-7 and
HEK-293) were seeded in 96 well plate with overnight incubation
(Trivedi et al., 2018). The cancer cells were treated with BG45 and the
test compounds in two doses (10 μM and 100 μM) in triplicate and in-
cubated for 72 h. 50 μL of 5 mg/mL solution of MTT, i.e., 3-(4,5-di-
methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Himedia La-
boratories Pvt. Ltd., Mumbai, India) was added to it. After 4 h of
incubation, the formazan crystals were dissolved in DMSO and the
absorbance was recorded at 570 nm and 650 nm with the help of
Spectramax M4 (Molecular Devices, USA).
4.3. HDAC3/NCoR1 assay using human recombinant HDAC3 enzyme
HDAC3 enzyme inhibition assay was performed using HDAC3/
NCoR1 fluorometric drug discovery kit (BML-AK531, ENZO life sci-
ences) following the vendor's protocol (Trivedi et al., 2018). Similar
like the HDAC assay, the HDAC3/NCoR1 assay method was discussed in
our earlier report (Trivedi et al., 2018). Initially, 10 μL of sample so-
lution and 15 μL diluted HDAC3/NCoR1 complex solution (BML-KI574-
0030) were added per well of the microtiter plate followed by the ad-
dition of 25 μL Fluor de Lys® substrate solution (BML-KI177-0005)
(Trivedi et al., 2018). The plate was incubated for 15 min at 37 °C. To
stop the reaction, 50 μL of mixture of Fluor de Lys® developer II (BML-
KI176-1250) and trichostatin A (BML-GR309-9090) were added per
well and incubated for 45 min at 37 °C. The fluorescence intensity was
recorded at excitation wavelength 360 nm and emission wavelength
460 nm using Spectramax M4 (Molecular Devices, USA). All synthe-
sized compounds along with BG45 were evaluated at 10 μM con-
centration in duplicate (Trivedi et al., 2018).
For determining the IC50 values of all these synthesized compounds
along with BG45, the same procedure was followed as described above.
The concentration used for IC50 measurement were increased subse-
quently, i.e., 0.625 μM, 1.25 μM, 2.5 μM, 5 μM, 10 μM, 20 μM, 40 μM,
80 μM, 100 μM and 200 μM. The same experiment was also repeated
with other cancer cells.
For Jurkat E6 cells, 2 × 104 cells in 100 μl were seeded in 96 well
plates and incubated overnight. Treatment concentrations of com-
pounds were same for all experiments as described above for other cell
lines. After 72 h of incubation with the compounds, cells were treated
with 22 μl of Prestoblue™ (Invitrogen, life technologies, United States)
reagent. Plate was incubated for 2 h and fluorescence intensity was
recorded at excitation wavelength of 560 nm, emission wavelength of
590 nm using Spectramax M4 (Molecular Devices, USA).
4.4. HDAC8 assay using recombinant HDAC8 enzyme
HDAC8 inhibition assay was conducted using HDAC8 fluorimetric
drug discovery kit (BML-AK518, Enzo life science) following the ven-
dor's protocol (Trivedi et al., 2018). Briefly, 15 μL of diluted human
recombinant HDAC8 enzyme (BML-SE145-0100) was incubated with
10 μL of test inhibitor (10 μM) and 25 μl of Fluor de Lys®- HDAC8,
deacetylase substrate (BML-KI178-0005). After 10 min incubation, re-
action was terminated by adding 50 μL stop solution which contains
Fluor de Lys®- Developer II (BML-KI176-1250) and trichostatin A. The
plate was incubated for 45 min at 37 °C and fluorescence intensity was
measured at excitation wavelength 360 nm, emission wavelength
460 nm using Spectramax M4 (Molecular Devices, USA) (Trivedi et al.,
2018). The promising compound 5e, 16c and 16d were further eval-
uated to find out their IC50 values in the same manner as described
above. Here, compounds were tested in the concentration range from
0.5 μM, 1 μM, 5 μM, 10 μM, 15 μM.
4.5.2. Apo-ONE® homogeneous Caspase-3/7 assay
Caspase 3/7 assay was conducted with the help of Apo-ONE®
Homogeneous Caspase-3/7 Assay kit (Promega, USA) following the
vendor's protocol. Briefly, 2 × 104 Jurkat E6 cells were seeded in white
tissue culture plate followed by incubation for overnight. The cells were
treated with compounds 5e, 16c and 16d at 10 μM dose for 72 h. Apo-
ONE® Homogeneous Caspase-3/7 reagent was prepared by mixing
Caspase substrate Z-DEVD-R110 (100×) and Apo-ONE® Homogeneous
Caspase-3/7 Buffer. 100 μL of reagent was added to the wells and mixed
by shaking it on plate shaker for 30 s at 300 rpm speed. Then the plate
was incubated for 40 min at room temperature and fluorescence in-
tensity was recorded at 485 nm excitation wavelength and 527 nm
emission wavelength using Spectramax M4 (Molecular Devices, USA).
4.5. Cell culture and drug treatment
4.6. Molecular docking study
A panel of cancer cell lines namely murine melanoma (B16F10),
human cervical cancer (HeLa), human breast cancer (MCF-7), human
non-small cell lung cancer (A549), human acute T-cell leukemia (Jurkat
E6) and human embryonic kidney (HEK-293) cell lines were procured
from National Center for Cell Science (NCCS), Pune, India. HeLa,
B16F10, HEK-293 and MCF-7 cells were cultured in Dulbecco's
Modified Eagle Medium (DMEM) (Himedia Laboratories Pvt. Ltd.,
Mumbai, India) whereas A-549 cells were cultured in Dulbecco's
Modified Eagle Medium/Nutrient Mixture F-12 Ham (DMEM/F12,1:1
mixture, (Himedia Laboratories Pvt. Ltd., Mumbai, India), and Jurkat
E6 cells were cultured in Roswell Park Memorial Institute (RPMI-1640,
Himedia Laboratories Pvt. Ltd., Mumbai, India) supplemented with
10% heat inactivated fetal bovine serum (Himedia Laboratories Pvt.
Ltd., Mumbai, India) and 1% of antibiotic solution (10,000 U Penicillin
and 10 mg Streptomycin per mL, Himedia Laboratories Pvt. Ltd.,
Mumbai, India). Cells were cultured at 37 °C in humidified atmosphere
with 5% CO2. Stock solutions of all these compounds were prepared in
Molecular docking study was performed to understand the binding
pattern of few better active inhibitors (compound 5e, 16c and 16d) into
the enzyme active site. This study gave a broad idea about the ligand-
enzyme interactions. These compounds were docked into the active site
of both HDAC8 (PDB: 1T69) and HDAC3 (PDB: 4A69) enzymes.
At first, compounds 5e, 16c and 16d were prepared by Ligprep tool
of Schrödinger (Schrçdinger Suite, 2018). The X-ray crystal structures
of HDAC8 (PDB: 1T69) and HDAC3 (PDB: 4A69) were used for docking
study. The protein was prepared and refined with the help of Protein
Preparation Wizard of Schrödinger software. Depending on the inbound
ligand molecule, a grid box of 15Åx15Åx15Å size was done and this was
defined as binding segment (Baidya et al., 2019). Then the previously
prepared compounds 5e, 16c and 16d were docked into the HDAC8
(PDB: 1T69) and HDAC3 (PDB: 4A69) active site by using the Glide tool
through extra precision (XP) mode. Catalytic zinc ion was used as
constraints during this ligand docking. Finally, the docking poses were
visualized and 2D interaction diagrams were drawn.
14

P. Trivedi, et al.
EuropeanJournalofPharmaceuticalSciences138(2019)105046
Declaration of competing interest
The authors declare no conflict of interests.
Acknowledgments
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Authors sincerely acknowledge Prof. Swati Biswas for her advice in
designing the project and critically analyzing the data. BG sincerely
acknowledge Science and Engineering Research Board-Department of
Science and Technology (SERB-DST), New Delhi, India for research
funding ((EMR/2016/001411)), PT thankfully acknowledges Council of
Scientific and Industrial Research (CSIR), New Delhi, India for pro-
viding research fellowship. NA is grateful to CSIR, New Delhi for pro-
viding RA fellowship [FILE NO.: 09/096(0966)/2019-EMR-I, Dated: 28-
03-2019]. SAA sincerely acknowledges CSIR, New Delhi for awarding
the Senior Research Fellowship [FILE NO.: 09/096(0967)/2019-EMR-I,
Dated: 01-04-2019]. TJ is thankful for the financial support from UPE
Phase II and RUSA 2.0 programs of University Grants Commission
(UGC), New Delhi to Jadavpur University, Kolkata, India. We also thank
the support from Department of Pharmacy, BITS-Pilani, Hyderabad,
India for providing the research facilities and Department of
Pharmaceutical Technology, Jadavpur University, Kolkata, India.
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Appendix A. Supplementary data
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