Green synthesis, antitubercular evaluation, and molecular docking studies of ethyl 3,5-dicyano-6-oxo-2,4-diarylpiperidine-3-carboxylate derivatives

Article abstract of DOI:10.1007/s00044-020-02519-2

A simple and environment friendly one-pot synthesis of ethyl 3,5-dicyano-6-oxo-2,4-diarylpiperidine-3-carboxylate derivatives from aryl aldehydes, ethyl cyanoacetate, and ammonium acetate was developed in aqueous medium without using a catalyst. The signi

Full text of DOI:10.1007/s00044-020-02519-2

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-020-02519-2
ORIGINAL RESEARCH
Green synthesis, antitubercular evaluation, and molecular docking
studies of ethyl 3,5-dicyano-6-oxo-2,4-diarylpiperidine-3-carboxylate
derivatives
1
Thuraka Sekhar1 Pinnu Thriveni
Kolluri Ramesh1 Polu Giri Prasad2 Indla Srihari3 Neelima Gorityala4
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Someswar Rao Sagurthi4 Uday Sankar Allam2
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Received: 26 September 2019 / Accepted: 1 February 2020
© Springer Science+Business Media, LLC, part of Springer Nature 2020
Abstract
A simple and environment friendly one-pot synthesis of ethyl 3,5-dicyano-6-oxo-2,4-diarylpiperidine-3-carboxylate
derivatives from aryl aldehydes, ethyl cyanoacetate, and ammonium acetate was developed in aqueous medium without
using a catalyst. The significant features of this method are easy, inexpensive experimental procedures with short reaction
time and high yield. The use of water as the solvent without catalyst makes the reaction meritorious and further fulfilled
green chemistry protocols. The compounds were screened for antibacterial activity against Gram-positive bacteria
(Staphylococcus aureus 25923) and Gram-negative bacteria (Escherichia coli ATCC25922) by disk diffusion method.
Compounds 4f, 4h, and 4i showed moderate antibacterial activity S. aureus. Intriguingly, compound 4g exhibited very good
antibacterial activity against E. coli. Antitubercular activity assay indicates that the compounds 4(a–c) exhibited activity with
varying MICs against mycobacterium tuberculosis H37RV control strain and multidrug-resistant tuberculosis (MDR-TB)
clinical isolate. Among the three tested compounds, 4c showed an equipotent antitubercular activity against H37Rv and
MDR-TB clinical isolates with MIC 3.13 μg/ml. Further, docking analysis of synthesized piperidinone derivatives with
acetate kinase protein reported that these compounds interact effectively with the catalytic residues that are in the vicinity of
ATP binding and active sites facilitating inhibition of enzyme function. Thus these derivatives can be promising compounds
for antitubercular activity to combat tuberculosis.
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Keywords Antitubercular activity Molecular docking studies One-pot synthesis Piperidinones
Introduction
The synthesis of heterocyclic compounds has been an
Supplementary information The online version of this article (https://
doi.org/10.1007/s00044-020-02519-2) contains supplementary
material, which is available to authorized users.
important area of study since long time, as these molecules
are usually used in various fields such as agriculture,
medicine, pharmacy, and contribute to potent and selective
drugs (Varma 1996). The development of an environmen-
tally benign and viable procedure for the synthesis of
organic compounds is one of the important goals to be
achieved by organic chemists (Ali et al. 2015; Butler and
Coyne 2010; Rao et al. 2015). Synthesizing novel bioactive
compounds with minimum number of synthetic steps and in
a short time is a significant challenge to the scientists (Ali
et al. 2012; Kaur et al. 2015; Saleem et al. 2013). One-pot
reactions have become popular in organic synthesis and
combinatorial chemistry because of their straight forward-
ness, less reaction time, use of green solvent, fewer
byproducts, simple experimentation, and high yield of
* Pinnu Thriveni
pthriveni.vsu@gmail.com
* Uday Sankar Allam
vsuusareddy@gmail.com
1
Department of Chemistry, Vikrama Simhapuri University,
Nellore, Andhra Pradesh 524320, India
2
Department of Biotechnology, Vikrama Simhapuri University,
Nellore, Andhra Pradesh 524320, India
3
Damien Foundation Urban Leprosy & TB Research Centre,
Nellore, Andhra Pradesh 524004, India
4
Molecular Medicine Lab, Department of Genetics, Osmania
University, Hyderabad, Telangana 500007, India

Medicinal Chemistry Research
products (Ali et al. 2018a, 2018b, 2019; Tiwari et al.
2016b). If the multi-component reaction could be carried
out in water, it would be the most ecofriendly and eco-
nomical (Konkala and Dubey 2017; Simona and Li 2012;
Tiwari et al. 2016a).
simple and environment friendly one-pot synthesis of ethyl
3,5-dicyano-6-oxo-2,4-diarylpiperidine-3-carboxylate deri-
vatives 4(a–i).
Tuberculosis (TB) caused by Mycobacterium tubercu-
losis has been a deadly infectious pathogen causing the
death of millions worldwide. The emergence of multidrug-
resistant (MDR) and extensively drug-resistant (XDR) Mtb
isolates created havoc and continues to impede the success
of current TB control programes (Biecker et al. 2019).
There is an urgent need for the design and development of
new anti-TB drugs and many researchers across the world
have been working for the development of novel anti-TB
drugs to combat MDR and XDR TB (Fernandes et al.
2017). Thus, the synthesized piperidinone compounds were
screened for antitubercular activity against mycobacterium
tuberculosis (Mtb) H37RV control strain and multidrug-
resistant tuberculosis (MDR-TB) clinical isolate. Among
the tested ethyl 3,5-dicyano-6-oxo-2,4-diarylpiperidine-3-
carboxylate derivatives 4c showed excellent antitubercular
activity.
In the context of green chemistry, there are many issues
that influence the choice of solvent. Recently, organic
synthesis in water medium without using organic solvents
has become one of the significant approaches in organic
chemistry to fulfill green chemistry needs (Chanda and
Fokin 2009; Hayashi 2006; Klijn and Engbers 2005).
Water as medium has gained importance in organic
synthesis, as it is non-toxic, cost effective, safe, non-
flammable, abundant in nature, and above all ecofriendly
solvent (Li 2005). In view of the significance of water
medium, we have developed a simple and environmentally
friendly one-pot synthesis of ethyl 3,5-dicyano-6-oxo-2,4-
diarylpiperidine-3-carboxylate derivatives 4(a–i) from aryl
aldehydes 1, ethyl cyanoacetate 2, and ammonium acetate 3
employing aqueous medium without using a catalyst
(Scheme 1). The significant features of this method are the
simple inexpensive experimental procedure with short
reaction time and good yield.
Results and discussion
Substituted piperidinones are important class of mole-
cules in the field of synthetic organic, medicinal, and
pharmacological chemistry (Blade Front 1980; Marson and
Fallah 1994). Literature survey reveals several methods for
the preparation of piperidinones such as reaction of ethyl
cyanoacetate with aryl aldehydes in ethanolic ammonia
(Nagai et al. 1974), reaction of phenyl methanimine with
ethyl cyanoacetate in the presence of sodium ethoxide in
ethanol (Ajaykumar and Lokanatharai 2006), reaction of
ethyl cyanoacetate, aryl aldehyde, and mixture of ammo-
nium acetate, ammonia in methanol as solvent (Chakra-
barthy et al. 2007). Although these methods are useful; they
suffer from drawbacks like use of toxic organic solvents,
longer reaction time, cost of the synthesis, and very low
yields. The development of efficient green synthetic meth-
odologies are in demand these days because of the rise in
level of pollutants in the atmosphere. Thus catalyst-free and
replacement of hazardous solvents by environmentally
benign solvents a in chemical process is the core area of
research and development. To fulfill green chemistry needs,
we therefore report in the present study a convenient
An efficient catalyst-free green synthetic protocol has been
developed for the synthesis of piperidinones 4(a–i).
According to literature, the formation of knoevenagel con-
densation intermediate can be accomplished either under
solvent-free condition or inorganic solvent medium. Con-
sidering the significance of green chemistry protocol, we
synthesized piperidinone derivative 4a through a reaction of
benzaldehyde 1, ethyl cyanoacetate 2, and ammonium
acetate 3 in water without using catalyst at room tempera-
ture. It was observed that reaction ensued slowly with the
formation of very low 4a product and high knoevenagel
intermediate. The yield of 4a was very low even after
stirring for 10 h at room temperature but with 80 °C tem-
perature and 6 h stirring the yield of 4a product increased
with low intermediate (unreacted intermediate was recov-
ered). There was no further improvement of yield, beyond
80 °C and 6 h time. The reaction was also carried out in
different solvents such as dichloromethane, ethanol, acet-
onitrile, toluene but the reaction was incomplete even after
10 h and results were given in (Table 1).
Scheme 1 Synthesis of ethyl 3,
5-dicyano-6-oxo-2, 4-
diarylpiperidine-3-carboxylate
derivatives 4(a–i)

Medicinal Chemistry Research
Table 1 Optimization of reaction medium
Biological evaluation
Entry
Solvent
Temp °C
Time (h)
Yielda (%)
Antibacterial screening
1
2
3
4
5
Dichloromethane
Acetonitrile
Ethanol
38
78
80
80
80
10
10
10
10
6
Trace
4
The antibacterial activity screening results of the synthe-
sized piperidinone derivative compounds 4(a–i) are sum-
marized in Table 3. The width of zone of inhibition (ZOI)
represents the potency of piperidinone derivatives. The
synthesized piperidinone derivatives showed varied anti-
bacterial activity. No antibacterial activity was observed
with compounds 4(a–e) at all the tested concentrations
against the Escherichia coli and the Staphylococcus aureus.
However, compound 4f showed moderate inhibition at 600,
800, 1000, and 1500 µg/ml against S. aureus. On the other
hand, compounds 4h and 4i displayed moderate inhibition
at 1500 µg/ml against S. aureus. Intriguingly, compound
4g (Ethyl 2,4-bis(3-bromophenyl)-3,5-dicyano-6-oxopiper-
idine-3-carboxylate) exhibited very good antibacterial
activity against E. coli. Dimethyl sulfoxide (DMSO) served
as a negative control with no inhibition zone. Streptomycin
and ceftazidime served as a positive control with a wider
ZOI against the tested organisms.
Further, the compounds 4(a–c) were evaluated for anti-
tubercular properties using resazurin microtiter assay
(REMA) plate method against Mtb H37Rv control strain
and MDR-TB clinical isolate and the resulting minimum
inhibitory (MIC) values are summarized in Table 4. The
results of REMA plate method indicate that the compounds
4(a–c) exhibited notable antitubercular activity with varying
MICs against tested control and MDR-TB clinical isolates.
Among the three tested compounds, Compound 4c (Ethyl
2,4-bis(4-chlorophenyl)-3,5-dicyano-6-oxopiperidine-3-car-
boxylate) exhibited an excellent antitubercular activity
against control and MDR-TB isolates followed by 4b (Ethyl
3,5-dicyano-2,4-bis(4-fluorophenyl)-6-oxopiperidine-3-car-
boxylate), and 4a (ethyl 3,5-dicyano-6-oxo-2,4-diphe-
nylpiperidine-3-carboxylate) (Fig. 1). Further, 4c showed an
equipotent antitubercular activity against control and MDR-
TB isolates with MIC 3.13 μg/ml. Isoniazid (INH) MICs for
Mtb control and MDR-TB clinical isolates were 0.06 and
0.5 μg/ml, respectively. Similarly, Rifampicin (RIF) MICs
were 0.06 and 0.25 µg/ml for Mtb control and MDR-TB
clinical isolate, respectively.
12
5
Toluene
Water
60
aIsolated yields
Table 2 Synthesis of ethyl 3,5-dicyano-6-oxo-2,4-diarylpiperidine-3-
carboxylate derivativesa 4(a–i)
Entry
R
Product 4
Time (h)
Yieldb (%)
1
2
3
4
5
6
7
8
9
H
4a
4b
4c
4d
4e
4f
6
5
5
6
6
6
6
6
6
60
70
65
34
45
41
31
16
21
4-Fluoro
4-Chloro
2-Bromo
2-Chloro
4-Methoxy
3-Bromo
4-Methyl
2,4-Dichloro
4g
4h
4i
aReaction of Ethyl cyanoacetate 2 (10 mmol), Ammonium acetate 3
(15 mmol) with various Aryl aldehydes 1 (10 mmol) in aqueous
medium
bIsolated yields
The structure of the products 4(a–i) were confirmed by
IR, H NMR, 13C NMR, and high-resolution mass spectra
1
(HRMS). IR spectra of 4a showed prominent sharp NH
stretching in region 3251 cm−1 and showed strong absorp-
tion band at 2358 cm−1 indicating the presence of CN
groups and 1693 (C=O of amide) and 1739 cm−1 (C=O of
ester) absorption bands indicating the presence of two C=O
1
groups. The H NMR spectrum 4a exhibited 8.94 ppm for
NH and a singlet at 5.36 ppm for C2–H, two doublets at
4.55 and 5.01 ppm for C4–H and C5–H of piperidinone.
Moreover, 4a also exhibited quadret at 3.81 ppm for CH2
and triplet at 0.77 ppm for CH3 of ester. The 13C spectra of
4a showed characteristic peaks of C(sp3) at 59.03, 47.32,
39.50, 13.79, and 163.47, 164.50 ppm (C=O) of amide
and ester.
After optimization of reaction conditions, various kinds
of aryl aldehydes were investigated for synthesis of corre-
sponding piperidinone derivatives. The results are given in
(Table 2). Aryl aldehydes having the groups Chlorine (Cl),
Fluorine (F) reacted fast and obtained corresponding
piperidinone with excellent yield and purity. However, we
observed that reaction was slow with aryl aldehyde having
electron releasing groups like CH3, OCH3, and fewer yields
were obtained.
Molecular docking studies
Acetate kinase (AckA) is an essential protein in pathogenic
bacteria including Mycobacterium tuberculosis. It catalyzes
the formation of acetate phosphate from acetate and ATP in
the presence of divalent cation. During chronic infection,
host-derived lipid components act as the major carbon
source at the site of infection. Previous studies reported that
fatty acid consumption by Mycobacterium tuberculosis

Medicinal Chemistry Research
Table 3 Antibacterial activity of
synthesized piperidinone
derivatives 4(a–i)
S. No. Compound
E. coli
S. aureus
A
B
C
D
E
F
A
B
C
D
E
F
1
2
3
4
5
6
7
8
9
11
4a
4b
4c
4d
4e
4f
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
+
++ ++ ++ ++
4g
4h
4i
+++
−
−
−
−
−
−
−
−
−
+
++
++
+ve control (30 μg /ml)
streptomycin
+++
+
+++
+
12
+ve control (30 μg/ml)
+++
+++
ceftazidime
13
14
−ve (DMSO)
−ve (Water)
−
−
−
−
− no antibacterial activity, + inhibition zone 0–5 mm, mild sensitivity, = inhibition zone 5–10 mm,
++ moderate sensitivity, +++ inhibition zone 10–15 mm, highly sensitive, Inhibition zone15–20mm,
A 100 µg/ml, B 400 µg/ml, C 600 μg/ml, D 800 μg/ml, E 1000 μg/ml, F 1500 μg/ml
Table 4 In vitro evaluation of
anti-TB activity of piperidinone
derivatives 4(a–c) against
H37Rv control isolate and
MDR-MTB clinical isolate by
REMA plate method
S. No. Compound Compound name
Anti-TB activity—MIC (μg/mL)
H37RV control
isolate
MDR-TB clinical
isolate
1
2
3
INH
RIF
4a
Isoniazid
0.06
0.06
25
0.5
Rifampicin
0.25
100
Ethyl 2,4-diphenyl-3,5-dicyano-6-
oxopiperidine-3-carboxylate
4
5
4b
4c
Ethyl 3,5-dicyano-2,4-bis(4-fluorophenyl)-6-
oxopiperidine-3-carboxylate
6.25
25
Ethyl 2,4-bis(4-chlorophenyl)-3,5-dicyano-6- 3.13
oxopiperidine-3-carboxylate
3.13
activates metabolic pathway, causing the pathogen to
release acetate as carbon intermediates (Rucker et al. 2015).
AckA is one of the key enzymes of acetate utilization
pathway, regulate flux of metabolites in glycolysis, gluco-
neogenesis, TCA cycle, and fatty acid metabolism resulting
in energy generation, survival, and growth of pathogen
(Chittori et al. 2012). Earlier reports suggested that piper-
idinone derivatives inhibit the growth of pathogenic Mtb by
targeting AckA with equal or higher efficacy than the
clinically proven antibiotics (Tiwari et al. 2018). Therefore
AckA protein of Mycobacterium marinum bearing
PDBID:4DQ8 is chosen as a suitable target for docking
ethyl 3,5-dicyano-6-oxo-2,4-diarylpiperidine-3-carboxylate
derivatives. It is a homodimer protein consisting of Chain
A and Chain B. Each subunit is further divided into
N-terminal and C-terminal domains. Crystal structure of
AckA enzyme of Mycobacterium marinum reveals the fol-
lowing binding sites. ATP binding site residues include 21,
197–201, 271–273, and 319–323. The potential substrate
binding site residue is 80 and active site residue is 137.
Metal binding site residues are 14 and 373.
Analysis of ligand poses demonstrated the binding affi-
nity of the molecules to catalytic subunit of chain A of
AckA protein. All the compounds 4a, 4b, and 4c showed
significant Moldock scores revealing good interaction with
the protein target as shown in Table 5. The MolDock score,
Rerank score, and interacting residues for 4a, 4b, and 4c are
listed in Table 6. 3D and 2D images after docking along
with hydrogen and other bond interactions are depicted in
Figs 2–4. The compounds 4b and 4c reported higher mol-
dock scores of −135.65 and −135.06, respectively. The
compound 4b shows hydrogen bond interaction with

Medicinal Chemistry Research
Fig. 1 Screening of 2-
piperidinone derivatives for
antitubercular properties against
(a) H37Rv control isolate and
(b) MDR-TB clinical isolate
(D2564) by resazurin-based 96-
well plate microdilution method.
Serial twofold dilutions of
compounds were tested at doses
of 800, 400, 200, 100, 50, 25,
12.5, 6.25, 3.125, and 0.0 μg/ml.
RIF at doses of 2.0–0.0156 μg/
ml and INH of 1.0–0.063 μg/ml
doses were used. First and last
columns are sterility and growth
controls, respectively. c DNA
strip patterns of genotype
MTBDR-plus strip of MDR-TB
clinical isolate (D2564)
with Met217 may contribute to the enhanced inhibition of
AckA that resulted in good antitubercular activity. In
compound 4a hydrogen bonds were identified between the
ligand molecule and residues Asn14, Arg80, Asp137, and
Glu373 of protein. In addition to these Val81 and Pro221
formed Pi-alkyl bond, while Met217 displayed Pi–Sulfur
bond interaction. All the compounds displayed optimum
binding with active site residues. Thus these compounds
may serve as potential AckA inhibitors contributing to
antitubercular activity.
Table 5 Molecular docking result of compounds on acetate kinase
chain a protein of Mycobacterium marinum (PDBID:4DQ8)
Ligand
MolDock score
Rerank score
HBond
4a
4b
4c
−126.93
−135.65
−135.06
−95.57
−100.12
−97.351
−4.75
−2.91
−4.20
Asn14, Glu373, Arg80 residues along with pi interactions
with residues like Val82, Pro221, and Met217. A halogen
(fluorine) interaction with Glu373 and His197 is also pre-
sent which may contribute to enhanced antibacterial activ-
ity. In the case of 4c hydrogen bond interactions were seen
with Asn14, Arg80 in addition to carbon hydrogen bonds
with Arg80 and His169. Pi interactions were displayed with
Asp137 (active site residue), Val82, Phe168, His169,
His197, Met217, Pro221, Ala318, and Glu373 residues.
Furthermore, halogen interaction of chlorine substituent
Discussion
In the present study, ethyl 3,5-dicyano-6-oxo-2,4-diarylpi-
peridine-3-carboxylate derivatives were synthesized in
aqueous medium using an efficient environment friendly
one-pot synthesis method and bio assayed for their anti-
bacterial activity. The tested analogs were showing meager

Medicinal Chemistry Research
Table 6 Docking score results of
top compounds with interacting
amino acid residues in the active
site of acetate kinase
Ligand Moldock score Rerank score Interacting residues
4a
4b
4c
−126.93
−135.65
−135.06
−95.57
−100.12
−97.35
Val82, Pro221, Met217, arg80, Asp137, Glu373, Asn14
Val82, Pro221, Met217, Asn14, Glu373, His197, Arg80
(PDBID:4DQ8)
Asn14, Arg80, Val82, Asp137, Phe168, His169, His197, Met217,
Pro221, Ala318, Glu373.
Fig. 2 a 3D structure view of molecular docking of 4a (green sticks)
with Mycobacterium AckA protein (gray lines). b Ligand interaction
diagram of 4a showing hydrogen bond (green dotted lines) interaction
with AckA residues Asn14, Arg80, Asp137, Glu373, and Pi interac-
tion (pink- and yellow-dotted lines) with Val82, Met217, and Pro221
(2D structure view)
antibacterial activity when tested against Gram-negative
bacteria. Intriguingly, piperidinone analogs, in particular,
ethyl 2,4-bis(4-chlorophenyl)-3,5-dicyano-6-oxopiperidine-
3-carboxylate (4c) exhibited excellent antitubercular prop-
erty against the MTB control strain as well as MDR-TB
clinical isolate. MIC values observed in our study were
tested for the first line of drugs using REMA plate method
and were in accordance with the previous report by
(Palomino et al. 2002). Meticulous observation of in vitro
antitubercular activity studies indicates that the presence
of fluorine and chlorine at the fourth position of the
phenyl ring in 4b and 4c is considered to be effective.
Thus, the compounds screened in this study, ethyl 2,4-bis
(4-chlorophenyl)-3,5-dicyano-6-oxopiperidine-3-carbox-
ylate (4c) and ethyl 2,4-bis(2-fluorophenyl)-3,5-dicyano-
6-oxopiperidine-3-carboxylate (4b) were very effective
against TB causing Mycobacterium tuberculosis. Sig-
nificant Moldock scores of molecular docking analysis
demonstrate that all the compounds displayed optimum
binding with active site residues. Affecting AckA might
alter the Pta-AckA pathway which in turn alters the acetate
metabolism, an important pathway of Mycobacterium
tuberculosis for its survival inside the host (Rucker et al.
2015). Thus these compounds may serve as potential AckA
inhibitors contributing to antitubercular activity. Further, in-
depth studies are warranted to know the mechanism of
action of compounds and its synergistic activity with the
existing antitubercular drugs against MDR and XDR-TB. In
addition, these compounds can be used as a source com-
pound to develop novel antitubercular drugs.

Medicinal Chemistry Research
Fig. 3 a 3D representation of molecular docking of compound 4b
(gray sticks) with Mycobacterium AckA protein (gray lines). b 2D
diagram of 4b showing hydrogen bond (green-dotted lines) interaction
with AckA residues Asn14, Arg80, halogen bond (cyan dotted lines)
interaction with His197, and Glu373
Fig. 4 a 3D structure view of molecular docking of compound 4c (green sticks) with Mycobacterium AckA protein (gray lines). b 2D repre-
sentation of 4c interactions at the active site of AckA

Medicinal Chemistry Research
Experimental
with each of the test compounds. Sterile filter paper disks
of about 6 mm are impregnated with six different working
concentrations of the test compounds at 100, 400, 600,
800, 1000, and 1500 µg/ml. A single colony was inocu-
lated into 5 ml of the LB broth and incubated at 37 °C for
8–10 h. After adjusting the turbidity to 0.5 Mac Farland
standard (1.5 × 108 CFU/ml), the culture was swabbed
onto the MH agar media. The plates were left for
5–10 min for drying the excess swabbed culture. The
dried filter paper disks impregnated with test compounds
were kept onto the culture plate not less than 24 mm from
center to center. After gentle pressing of the disks onto the
culture plate, the plates were incubated for about 24 h at
37 °C. Streptomycin sulfate and ceftazidime (30 µg/ml)
served as a positive controls and DMSO and water (10 µl)
as negative controls included in the study. Each of the test
compounds was tested in duplicates.
General consideration
All chemicals were obtained from commercial suppliers and
were used without purification. Infrared spectra were
recorded on a Bruker spectrophotometer with KBr pellets
1H and 13C NMR spectra were recorded in CDCl3 on
500 MHz Broker spectrometer with tetra methyl silane as
the internal standard and resonance (δ) are given in ppm.
Melting points were determined in open capillaries and
were uncorrected. HRMS were recorded using a Bruker
spectrometer. Thin-layer chromatography (TLC) was per-
formed using MERK precoated silica gel and the compo-
nents were visualized under a UV or an iodine chamber.
General procedure for the synthesis of ethyl 3,5-dicyano-6-
oxo-2,4-diarylpiperidine-3-carboxylate derivatives (4)
Resazurin microtiter assay (REMA) plate method
In a 50 ml RBF mixture of aryl aldehyde 1 (10 mmol), ethyl
cyanoacetate
2
(10 mmol) and ammonium acetate
3
A low cost, simple, and rapid calorimetric REMA plate
method was used to study the antitubercular properties of
the synthesized piperidinone derivatives against the
Mycobacterium tuberculosis H37Rv control strain and
multidrug-resistant MTB clinical isolate following the
protocol described by (Palomino et al. 2002). Briefly,
REMA was performed in 7H9-S medium containing
Middlebrook broth, 0.1% Casitone, and 0.5% glycerol
and supplemented with oleic acid, albumin, dextrose, and
catalase. Antitubercular first line of drugs, INH and RIF
were used as positive controls. Stock solutions of RIF and
INH and were prepared at concentrations of 10 mg/ml in
methanol and 1 mg/ml in distilled water, respectively.
Resazurin sodium salt powder was prepared at 0.02%
(wt/vol) in distilled water and filter sterilized and frozen
until used.
Serial twofold dilutions of test compounds and first line of
drugs in 100 μl of 7H9-S medium were prepared directly in
96-well plates at concentrations of 800–3.125, 0 µg/ml for test
compounds, 2.0–0.063 μg/ml for RIF, and 1.0–0.063 μg/ml
for INH. Only solvents without any first lines of drugs and test
compounds were included as negative controls. Mycobacter-
ium tuberculosis H37Rv control strain and multidrug-resistant
MTB clinical isolate was used as reference strain to test the
antitubercular activity of the test compounds. The inoculum
prepared in 7H9-S medium was adjusted to a McFarland tube
no. 1 and then 100 μl of 1:20 diluted culture was used as an
inoculum. The 96-well plates prepared as per the above pro-
tocol were covered and incubated at 37 °C in the normal
atmosphere. After 7–8 days of incubation, 30 μl of 0.02%
resazurin solution prepared in MQ water was added to each
well, incubated at 37 °C for overnight and observed for color
change. Irreversible conversion of blue-colored resazurin to
(15 mmol) were taken in 10 ml water. The reaction mixture
was allowed to stir magnetically at 80 °C. The progress of the
reaction was monitored by TLC. After completion of the
reaction, mass was cooled to room temperature and solid was
filtered. Filtered solid was purified by column chromato-
graphy by using mobile phase (ethyl acetate and hexane).
Bacterial cultures and antibiotics
Antibacterial activity of the synthesized piperidinone deriva-
tives was carried out using the disk diffusion method against
Gram-positive bacteria (S. aureus 25923) and Gram-negative
bacteria (E. coli ATCC25922). Further, antitubercular activity
was tested against Mycobacterium tuberculosis (MTB)
H37Rv and MDR MTB clinical isolate which is resistant to
two key first line of anti-TB drugs (RIF and INH). The
bacterial culture media Luria Bertani (LB) broth,
Mueller–Hinton (MH) Agar, antibiotics streptomycin sulfate,
and ceftazidime were purchased from HiMedia, Mumbai. The
compound screening for antitubercular activity was carried
out at Damien Foundation Urban Leprosy and TB Research
Centre, Nellore, Andhra Pradesh, India. The research center is
a TB culture and drug susceptibility testing (C and DST)
referral laboratory for Revised National Tuberculosis Control
Program and accredited by the National Mycobacteriology
Accreditation System of Central TB Division Ministry of
Health, Government of India.
Antibacterial activity assay
DMSO was used as solvent to dissolve the test com-
pounds. A stock concentration of 20 mg/ml was prepared

Medicinal Chemistry Research
pink-colored resorufin indicates bacterial growth. The MIC
was defined as the lowest concentration of the test compounds
or drugs that prevented color change from blue to pink.
130.09, 129.51 (d, J = 8.6 Hz), 129.45, 129.11, 128.22,
116.76, 116.65, 116.59 (dd, J = 21.9, 14.7 Hz), 116.47,
114.28, 113.70, 63.91, 61.43, 57.99, 48.27, 39.22, and
13.46; MS (m/z): 410.13 [M + H]+.
Molecular docking
ethyl 2,4-bis(4-chlorophenyl)-3,5-dicyano-6-oxopiperidine-
3-carboxylate (4c) Half white solid; Yield: 65%; m.p.:
191–193 °C; IR (KBr) cm−1: 3259 (NH), 2365 (CN), 1743
(C=O), 1702 (C=O); 1H NMR (500 MHz, CDCl3) δ (ppm)
7.26–7.41 (m, 8H, Ar–H), 6.59 (s, 1H, NH), 5.26 (d, J =
4.2 Hz, 1H), 4.34 (dd, J = 13.3, 4.2 Hz, 1H), 4.07 (dd, J =
The interaction of AckA protein with different piper-
idinone derivatives was studied using Molegro Virtual
Docker (MVD 2012.5.5) (CLCbio 2012). Docking was
performed with potential active site detected on AckA
protein with three piperidinone derivatives 4(a–c). The
structures of all the compounds were generated using
Marvin Sketch 5.6.0.2. (1998–2011, Copyright © Che-
mAxon Ltd), cleaned in 3D and saved in.pdb format for
docking studies. The PDB file of the target protein
downloaded from RCSB PDB (www.rcsb.org), AckA
(PDBID:4DQ8). During docking first the protein and
ligands were prepared by assigning bonds, bond orders,
charges, explicit hydrogens, flexible torsions in ligands if
they were missing. The search algorithm is taken as
MolDock SE and numbers of runs are taken as ten and
max iterations were 2000 with population size 50 and with
an energy threshold of 100). Further investigation of the
binding interactions of the most active (4a, 4b, and 4c)
docked compounds were performed using Biovia dis-
covery studio 2017 R2 Client. To obtain more potent
compounds as inhibitors the optimal binding mode of a
molecule (compound) to the active site of macromolecule
is interpreted.
13.7, 4.5 Hz, 1H). 3.87 (q, 2H, CH2), 0.81 (t, 3H, CH3); 13
C
NMR (125 MHz, CDCl3) δ (ppm) 165.57, 163.73, 162.22,
136.81, 136.35, 131.74, 130.75, 129.83, 129.68, 129.56,
129.28, 129.19, 129.12, 128.87, 114.27, 113.59, 64.06,
61.46, 57.66, 48.30, 39.00, and 13.46; MS (m/z): 442.07/
444.069 [M + H]+.
ethyl 2,4-bis(2-bromophenyl)-3,5-dicyano-6-oxopiperidine-
3-carboxylate (4d) Pale yellow solid; Yield: 34%; m.p.:
205–207 °C; 1H NMR (500 MHz, CDCl3)
δ (ppm)
7.63–7.70 (m, 2H, Ar–H), 7.52–7.58 (m, 2H, Ar-H),
7.27–7.44 (m, 4H, Ar–H), 6.10 (s, 1H, NH), 5.86 (s, 1H,
CH), 5.04 (d, J = 12.5 Hz, 1H, CH), 4.27 (d, J = 13.0 Hz,
1H, CH), 3.91 (q, 2H, CH2), and 0.89 (t, 3H, CH3); 13C
NMR (125 MHz, CDCl3) δ (ppm) 162.14, 161.76, 134.27,
133.85, 132.93, 132.48, 132.11, 131.32, 131.11, 129.51,
128.92, 128.78, 127.09, 124.10, 114.93, 113.53, 64.32,
60.33, 56.02, 45.64, 40.40, and 13.32; MS (m/z): 529.97/
531.97 [M + H]+.
Spectral characterization data for 4(a–i)
ethyl 2,4-bis(2-chlorophenyl)-3,5-dicyano-6-oxopiperidine-
ethyl 3,5-dicyano-6-oxo-2,4-diphenylpiperidine-3-carboxy-
late (4a) White solid; Yield: 60%; m.p.: 172–174 °C; IR
(KBr) cm−1: 3251 (NH), 2358 (CN), 1739 (C=O), 1693
3-carboxylate (4e) White solid; Yield: 45%; m.p.:
187–188 °C; 1H NMR (500 MHz, CDCl3)
δ (ppm)
7.28–7.39 (m, 8H, Ar–H), 6.76 (s, 1H, NH), 4.71 (d, J =
2.8 Hz, 1H, CH), 4.64 (d, J = 4.05 Hz, 1H, CH), 4.11 (s,
1H, CH), 3.78 (q, 2H, CH2), and 0.90 (t, 3H, CH3); 13C
NMR (125 MHz, CDCl3) δ (ppm) 162.21, 161.84, 135.56,
133.80, 131.78, 131.22, 130.95, 130.83, 130.79, 130.35,
129.27, 128.26, 128.15, 126.99, 114.78, 113.51, 64.23,
57.78, 56.07, 42.85, 40.26, and 13.22; MS (m/z) = 442
[M + H]+.
1
(C=O); H NMR (500 MHz, DMSO-d6) δ (ppm) 8.94 (s,
1H, NH), 7.29–7.46 (m, 10H, Ar–H), 5.36 (s, 1H, CH), 5.01
(d, J = 13.0 Hz, 1H, CH), 4.55 (d, J = 13.0 Hz, 1H, CH),
3.81 (q, 2H, CH2), and 0.77 (t, 3H, CH3); 13C NMR
(125 MHz, DMSO-d6) δ (ppm) 164.50, 163.47, 135.06,
134.47, 130.01, 129.68, 129.38, 129.06, 128.95, 128.04,
116.98, 114.63, 63.44, 60.95, 59.03, 47.32, 39.50, 13.79;
MS (m/z): 374.14 [M + H]+.
ethyl 3,5-dicyano-2,4-bis(4-methoxyphenyl)-6-oxopiperidine-
3-carboxylate (4f) White solid; Yield: 41%; m.p.:
143–144 °C; 1H NMR (500 MHz, CDCl3) δ (ppm)
7.28–7.35 (m, 4H, Ar–H), 6.93–6.95 (m, 4H, Ar–H), 6.92
(s, 1H, NH), 5.21 (s, 1H, CH), 4.03 (m, 1H, CH), 4.00
(d, J = 13.2 Hz, 1H, CH), 3.83 (q, 2H, CH2), and
1.26–1.27 (6H, s, 2 × CH3); 0.81 (t, 3H, CH3); 13C NMR
(125 MHz, CDCl3) δ (ppm) 167.32, 163.21, 159.43,
158.00, 137.86, 137.72, 137.65, 131.15, 130.22, 128.53,
128.42, 128.40, 128.15, 127.98, 127.90, 127.73, 73.48,
ethyl 3,5-dicyano-2,4-bis(4-fluorophenyl)-6-oxopiperidine-
3-carboxylate (4b) White solid; Yield: 70%; m.p.:
164–165 °C; IR (KBr) cm−1: 3272 (NH), 2377 (CN), 1747
(C=O), 1703 (C=O); 1H NMR (500 MHz, CDCl3) δ (ppm)
7.43–7.44 (m, 4H, Ar–H), 7.12–7.14 (m, 4H, Ar–H), 6.50
(s, 1H, NH), 5.26 (s, 1H, CH), 4.35 (d, J = 12.6 Hz, 1H,
CH), 4.08 (d, J = 12.9 Hz, 1H, CH), 3.84 (q, 2H, CH2), and
0.79 (t, 3H, CH3); 13C NMR (125 MHz, CDCl3) δ (ppm)
164.84, 164.51, 163.85, 162.84, 162.52, 162.19, 130.16,

Medicinal Chemistry Research
73.15, 72.31, 70.28, 67.88, 35.67, 29.5, and 14.20; MS
Research Award Scheme and also acknowledges DST-FIST facilities
of Department of Biotechnology, Vikrama Simhapuri University.
(m/z) = 434 [M + H]+.
Compliance with ethical standards
ethyl 2,4-bis(3-bromophenyl)-3,5-dicyano-6-oxopiperidine-
3-carboxylate (4g) Half white solid; Yield: 31%; m.p.:
197–199 °C; 1H NMR (500 MHz, CDCl3) δ (ppm)
7.57–7.50 (m, 4H, Ar–H), 7.30–7.46 (m, 4H, Ar–H),
6.69 (s, 1H, NH), 5.22 (d, J = 27.7 Hz, 1H, CH), 4.15
(d, J = 12.9 Hz, 1H, CH), 4.05 (d, J = 13.2 Hz, 1H, CH),
3.86 (q, 2H, CH2), and 0.87 (t, 3H, CH3);13C NMR
(125 MHz, CDCl3) δ (ppm) 162.22, 161.92, 154.01,
153.33, 144.38, 130.98, 130.96, 130.10, 130.05, 129.96,
129.01, 128.90, 128.11, 127.99, 127.77, 117.31, 63.54,
61.91, 48.87, 39.20, 29.14, and 13.5; MS (m/z) = 529
[M + H]+.
Conflict of interest The authors declare that they have no conflict of
interest.
Publisher’s note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
References
Ajaykumar A, Lokanatharai KM (2006) A one pot synthesis of ethyl 3,5-
dicyano-4,6-diaryl-piperid-2-one-5-carboxylates. Balgarian Chem
Commun 38:93–96
Ali I, A Wani W, Saleem K, Haque A (2012) Thalidomide: a banned
drug resurged into future anticancer drug. Curr Drug Ther 7
(1):13–23
Ali I, Alharbi OM, Tkachev A, Galunin E, Burakov A, Grachev VA
(2018a) Water treatment by new-generation graphene materials:
hope for bright future. Environ Sci Sci Pollut Res 25
(8):7315–7329
Ali I, Lone MN, Alothman ZA, Badjah AY, Alanazi AG (2019)
Spectroscopic and in silico DNA binding studies on the interac-
tion of some new N-substituted rhodanines with calf-thymus dna:
in vitro anticancer activities. Anti-Cancer Agents Medicinal
Chem 19(3):425–433
Ali I, Mukhtar SD, Hsieh MF, Alothman ZA, Alwarthan A (2018b)
Facile synthesis of indole heterocyclic compounds based micellar
nano anti-cancer drugs. RSC Adv 8(66):37905–37914
Ali I, Nadeem Lone MA, Al-Othman Z, Al-Warthan A, Marsin Sanagi
M (2015) Heterocyclic scaffolds: centrality in anticancer drug
development. Curr Drug Targets 16(7):711–734
Blade Front A (1980) Facile synthesis of γ-, α-, and ε-lactams by
cyclodehydration of ω-amino acids on alumina or silica gel.
Tetrahedron Lett 22:2443–2446
ethyl 3,5-dicyano-6-oxo-2,4-dip-tolylpiperidine-3-carboxy-
late (4h) White solid; Yield: 16%; m.p.: 180–183 °C;
1HNMR (500 MHz, CDCl3) 6.98–7.02 (m, 8H, Ar–H), 6.4
(s, 1H, NH), 5.5 (s, 1H, CH), 4.12 (d, J = 12.8 Hz, 1H, CH),
3.82 (d, J = 11.3 Hz, 1H, CH), 3.6 (q, 2H, CH2), and 0.85
(t, 3H, CH3); MS (m/z) = 402 [M + H]+.
ethyl 2,4-bis(2,4-dichlorophenyl)-3,5-dicyano-6-oxopiperi-
dine-3-carboxylate (4i) White solid; Yield: 21%; m.p.:
215–217 °C; 1HNMR (500 MHz, CDCl3) δ 7.41–7.21
(m, 5H, Ar–H), 6.4 (m, 2H, Ar–H, NH), 5.26 (s, 1H, CH),
4.30 (d, J = 12.9 Hz, 1H, CH), 4.17 (d, J = 11.4 Hz, 1H,
CH), 3.84 (q, 2H, CH2), and 0.82 (t, 3H, CH3); MS (m/z) =
509 [M + H]+.
Biecker AL, Xiaodong L, Jon ST, Zhaoyong Y, Steven, Van Lanen G
(2019) Biosynthetic and synthetic strategies for assembling
capuramycin-type antituberculosis antibiotics. Molecules 24
(3):433–450
Butler RN, Coyne AG (2010) Water: nature’s reaction enforcers
comparative effects for organic synthesis “in-water” and “on-
water”. Chem Rev 110:6302–6337
Chakrabarthy M, Sulakshana K, Yoshihiro SA (2007) One-pot for-
mation of functinalized 2-piperidinones from arylidenecyanoa-
cetates and methanolic ammonia via tandem reactions.
Heterocycles 73:795–804
Chanda A, Fokin VV (2009) Organic synthesis “on water”. Chem Rev
109:725–745
Chittori S, Savithri HS, Murthy MR (2012) Structural and mechanistic
investigations on Salmonella typhimurium acetate kinase (AckA):
identification of a putative ligand Binding pocket at the dimeric
interface. BMC Struct Biol 12:12–24
CLCbio (2012) Molegro virtual docker user manual. MVD 2012.5.5
for Windows, Linux, and Mac OS X, Molegro – A CLC bio
company 1-250.
Fernandes G, Man Chin C, Dos Santos J (2017) Advances in drug
discovery of new antitubercular multidrug-resistant compounds.
Pharmaceuticals 10(2):51–67
Conclusion
In conclusion, we synthesized piperidinone derivatives
from reaction of ethyl cyanoacetate, ammonium acetate
with various aryl aldehydes in aqueous medium using
environment friendly one-pot synthesis. Among the
synthesized ethyl 3,5-dicyano-6-oxo-2,4-diarylpiper-
idine-3-carboxylate derivatives, compound 4c sub-
stituted with Cl moiety exhibited notable antitubercular
activity against Mtb H37RV control strain and MDR-TB
clinical isolate. Molecular docking study shows that the
synthesized piperidinone derivatives are binding effi-
ciently with AckA protein of Mycobacterium. Thus, the
results of the present study suggest that ethyl 3,5-
dicyano-6-oxo-2,4-diarylpiperidine-3-carboxylate deri-
vatives could be promising compounds for antitubercular
activity to combat TB.
Hayashi Y (2006) In water or in the presence of water. Anjewandte
Chem Int Add 45:8103–8104
Kaur A, Kaur M, Singh B (2015) One‐pot regioselective synthesis
of novel 1‐N‐methyl‐spiro[2,3′]oxindole‐spiro[3,3″]‐1″‐N‐
Acknowledgements PT thankful to HCU, Hyderabad and DST-
PURSE, S.V. University, Tirupati for analytical assistance. USA
acknowledges DST SERB, INDIA for support through Early career

Medicinal Chemistry Research
arylpyrrolidine‐2″,5″‐dione‐4‐arylpyrrolidines through multi-
component 1,3‐dipolar cycloaddition reaction of azomethine
ylide. J Het Chem 52:827–833
irradiation and their antibacterial properties. Rsc Adv
5:28958–28964
Rucker N, Billig S, Bucker R, Jahn D, Wittmann C, Bange FC,
Bacteriol (2015) Acetate dissimilation and assimilation in
mycobacterium tuberculosis depend on carbon availability. J Bio
Tech 197(19):3182–3218
Saleem K, Wani WA, Haque A, Lone MN, Hsieh MF, Jairajpuri MA,
Ali I (2013) Synthesis, DNA binding, hemolysis assays and
anticancer studies of copper (II), nickel (II) and iron (III) com-
plexes of a pyrazoline-based ligand. Future Med Chem 5
(2):135–146
Klijn JE, Engbers JB (2005) Efficient reactions in aqueous organic
chemistry do not require soluble reactants, as had been thought, a
newly developed “on-water” protocol is characterized by short
reaction times, and the products are easy to isolate. Nature
435:746–747
Konkala VS, Dubey PK (2017) One‐pot synthesis of 3‐phenyl‐4‐
pyrazolylmethylene‐isoxazol‐(5H)‐ones catalyzed by sodium
benzoate in aqueous media under the influence of ultrasound
waves: a green chemistry approach. J Heterocycl Chem 54
(4):2483–2492
Simona MO, Li CJ (2012) Green chemistry oriented organic synthesis
in water. Chem Soc Rev 41:1415–1427
Li CJ (2005) Organic reactions in aqueous media with a focus on
carbon-carbon bond formations: a decade update. Chem Rev
105:3095–3165
Marson CM, Fallah A (1994) Preparation of γ- and δ-lactams by ring
closure of β, γ-unsaturated amides using trifluoromethanesulfonic
acid. Tetrahedron Lett 35:293–296
Nagai W, Hirata Y, Miwa T (1974) Reactions of benzaldehyde and
analogs with ethyl cyanoacetate in ethanolic ammonia. J Org
Chem 39(25):3735–3739
Palomino JC, Martin A, Camacho M, Guerra H, Swings J, Portaels F
(2002) Resazurin microtiter assay plate: simple and inexpensive
method for detection of drug resistance in mycobacterium
tuberculosis. Antimicrob Agents Chemother 46(8):2720–2722
Rao LC, Kumar NS, Dileepkumar V, Murthy UMS, Meshram HM
(2015) “On water” synthesis of highly functionalized 4H-
chromenes via carbon-carbon bond formation under microwave
Tiwari KN, Bora D, Chauhan G, Yadv D, Sharma K, Thakur A, Singh L,
Tripathi V (2016a) DABCO catalysed synthesis of 3-substituted-
3-hydroxyindolin-2-ones in aqueous media. Synth Commun
46:620–625
Tiwari KN, Pandurang TP, Pant S, Kumar R (2016b) Highly efficient
and regioselective synthesis of spirooxindolo pyrrolizidines by
reaction of isatin, proline and acrylonitrile/methyl acrylate in
water. Tetrahedron Lett 57:2286–2289
Tiwari S, Barh D, Imchen M, Rao E, Kumavath RK, Seenivasan SP,
Jaiswal AK, Jamal SB, Kumar V, Ghosh P, Azevedo V (2018)
Acetate kinase (AcK) is essential for microbial growth and betel-
derived compounds potentially target AcK, PhoP and MDR
proteins in m. tuberculosis, v. cholerae and pathogenic E. coli: an
in silico and in vitro study. Med Chem 18(31):2731–2740
Varma RS (1996) Solvent‐free synthesis of heterocyclic compounds
using microwaves. J Het Chem 36:1565–1571