Synthesis, biological evaluation and molecular docking studies of novel 2-(2-cyanophenyl)-N-phenylacetamide derivatives

Article abstract of DOI:10.1007/s11164-018-3434-9

A series of novel 2-(2-cyanophenyl)-N-phenylacetamide derivatives 3(a-u) were designed and synthesized via selective amidation of methyl-2-(2-cyanophenyl)acetates over amidine formation by using AlMe₃ as catalyst in good yields. All the newly s

Full text of DOI:10.1007/s11164-018-3434-9

Res Chem Intermed
https://doi.org/10.1007/s11164-018-3434-9
Synthesis, biological evaluation and molecular docking
studies of novel 2-(2-cyanophenyl)-N-phenylacetamide
derivatives
Lakshmi Narayana Sharma Konidena1,2 Sathish Kumar Boda3
•
•
Suresh Kumar Chettu1,2 Kumaraswamy Sorra Sreenivas Enaganti
1
4
•
•
•
Praveena Mukkavilli1 N. S. Kameswara Rao5
•
•
P. V. Anantha Lakshmi3 Raghu Babu Korupolu2
•
Received: 21 January 2018 / Accepted: 2 April 2018
Ó Springer Science+Business Media B.V., part of Springer Nature 2018
Abstract A series of novel 2-(2-cyanophenyl)-N-phenylacetamide derivatives 3(a-
u) were designed and synthesized via selective amidation of methyl-2-(2-
cyanophenyl)acetates over amidine formation by using AlMe3 as catalyst in good
1
yields. All the newly synthesized derivatives were well characterized by H NMR,
13C NMR, FTIR and HRMS spectral techniques. All the synthesized title com-
pounds were evaluated for their in vitro anticancer activity against three cancer cell
lines. Among all compounds, 3i (IC50 = 1.20 lM, IC50 = 1.10 lM), 3j (IC50-
= 0.11 lM, IC50 = 0.18 lM), 3o (IC50 = 0.98 lM, IC50 = 2.76 lM) showed
excellent inhibitory activity than the standard Etoposide (IC50 = 2.11 lM, IC50-
= 3.08 lM) against MCF-7 and A-549 cell lines, respectively. Docking analysis of
all the compounds with the human topoisomerase II revealed that the compound 3j
fitted well in the active site pocket, showing the best docking score of
158.072 kcal/mol.
Keywords N-phenylacetamide Á Trimethylaluminum Á Docking studies Á
Anticancer activity
&
&
Lakshmi Narayana Sharma Konidena
lnsharmagvk@gmail.com
Raghu Babu Korupolu
drraghualways@yahoo.co.in
1
2
GVK Biosciences Private Limited, Medicinal Chemistry Laboratory, Hyderabad 500076, India
Andhra University, Department of Engineering Chemistry, Andhra University College of
Engineering (A), Visakhapatnam 530003, India
3
4
Department of Chemistry, Osmania University, Tarnaka, Hyderabad 500007, India
Department of Bioinformatics, Averin Biotech Pvt. Ltd, Windsorplaza, Nallakunta,
Hyderabad 500044, India
5
Koneru Lakshmaiah Education Foundation, Deemed to be University,
Vaddeswaram, Guntur 522502, India
123

L. N. S. Konidena et al.
Introduction
Cancer is one of the most crucial health concerns and forms a critical originator of
human death [1]. The number of new cases of cancer is 454.8 per 100,000 people
per year. The number of cancer deaths (cancer mortality) is 171.2 per 100,000
people per year. From past few decades to date, a plethora of researchers worldwide
have made exceptional effort in the development of novel drugs in the world
market, and the research is heading toward ultimately saving human lives.
Nevertheless, the majority of drugs possess selectivity of action, and hence the
treatment of certain types of tumor are yet challenging due to their aggressive
nature, resistance to chemicals, malignant cell metastasis and the absence of drug
selectivity [2]. Henceforth, the augmentations of new anticancer products that are
effective and safer from the commercial materials are always in demand. With
respect to a recently conducted survey on developing a new pharmacophore model
in order to treat any disease or disorder, a complex of two pharmacophores in a
single form is an effective approach in medicinal chemistry. This approach aids in
the expedition of highly effective novel compounds.
In this regard, researchers worldwide are putting forth significant effort to
discover novel compounds, which help to treat inflammation and cancer.
Heterocyclic compounds were found to be crucial in drug design and development
[3, 4]. During the past few years, many building blocks have been designed,
developed and approved for a range of diseases. Among these, the amide linkage is
one of the most fundamental and widespread chemical bonds, underlying the
properties of a vast array of organic molecules, polymers, and materials, as well as a
wide selection of bioactive natural products including proteins and peptides. Around
25% of amides are present in top-selling medicines and many other pharmaceuticals
[5]. These are versatile intermediates for the synthesis of many pharmacologically
important nitrogen and oxygen containing heterocyclics [6]. Some of the known
drugs containing amide moiety are shown in Fig. 1. Despite the enormous
importance of amides in organic chemistry, most of the established methods for the
synthesis of amides are reported. In the literature number of recent reviews have
covered metal-catalyzed amide bond formation [7, 8] and the use of more traditional
coupling reagents [9]. In general, amides are prepared by the reaction of carboxylic
acid derivatives with amines [10, 11]. Recently, rare earth metal halides and triflates
have been found to be successful Lewis acids for promoting the amination of nitriles
[12]. In 1977 Weinreb et al. [13] published AlMe3 promoted amide synthesis from
esters and amines. After that, the AlMe3-mediated amide synthesis from esters has
found many applications in organic synthesis [14]. These methods suffer from
common limitations such as stoichiometric amounts of coupling reagents, poor
atomic efficiency and formation of a large amount of byproducts [15–18]. To
overcome these problems, novel amide bond forming reactions have been developed
in recent years [18–22]. Herein we report a novel series of 2-(2-cyanophenyl)-N-
phenylacetamide derivatives from methyl-2-(2-cyanophenyl)acetates by selective
amidation using AlMe3 as catalyst. All the newly synthesized derivatives were
123

Synthesis, biological evaluation and molecular docking…
Fig. 1 Some of the known amide moiety containing drugs
evaluated for their in vitro anticancer activity and molecular docking studies were
performed with human DNA topoisomerase II.
Results and discussions
Chemistry
Here we have developed Me3Al promoted synthesis of 2-(2-cyanophenyl)-N-
phenylacetamide derivatives. A series of 2-(2-cyanophenyl)-N-phenylacetamide
derivatives 3(a-u) were synthesized in two steps as shown in Scheme 1. In the first
step, methyl-2-(2-cyanophenyl)acetate derivatives 2(a-b) were obtained by the
reaction of methyl-2-(2-bromophenyl)acetate derivatives with CuCN in DMF
solvent stirring for 10–12 h [23]. In the next step, reaction between methyl-2-(2-
cyanophenyl)acetate derivatives 2(a-b) and various substituted amines in the
presence of AlMe3 as catalyst in toluene stirring at 80 °C for 9–10 h. The
optimization of the reaction conditions is shown in Table 1. We started the reaction
with methyl-2-(2-cyanophenyl)acetate with AlCl3 as catalyst in neat condition. To
our delight, 2-(2-cyanophenyl)-N-phenylacetamide product was observed in 20%
yield (Table 1, Entry 1). Then we tried the reaction in different solvents viz.,
toluene and THF (tetrahydrofuran) resulting in an improved yield of 28% with
toluene and 23% with THF solvents (Table 1, Entries 2–3). We continued our
investigation to improve the yield. We also tried the reaction with other catalytic
systems such as FeCl3, TiCl4, Ti(iPrO)4, SnCl4, ZnCl2 and Me3Al, out of which
Me3Al gave surprising results of amide compound 3a exclusively forming over
amidine formation up to 80% (Table 1, Entries 4–15). Then we increased catalyst
loading with 2.0 mmol resulting in no further remarkable improvement in yield
(82%) (Table 1, Entry 16). Still, we tried to increase catalyst loading to 2.5 mmol,
but no further improvement in yield was observed (Table 1, Entry 17). On the other
hand, we observed that title compound 3a formed over amidine by increasing the
amine to 2.0 mmol, but with no further improvement in yield (Table 1, Entry 18).
Finally, we explored the reaction conditions as methyl-2-(2-cyanophenyl)acetate
(1.0 mmol), aniline (1.5 mmol) and Me3Al (1.5 mmol) in the toluene solvent
system for the conversion reaction of 2-(2-cyanophenyl)-N-phenylacetamide
derivatives. Based on our present investigation, the reaction proceeds through the
formation of intermediate 1. The ester is activated by intermediate 1 and undergoes
a loss of AlMe2OMe to give amide product (Scheme 2).
123

L. N. S. Konidena et al.
Scheme 1 Synthesis of 2-(2-cyanophenyl)-N-phenylacetamide derivatives 3(a-u)
All the products derived from anilines, which includes electron-rich, electron-
deficient and neutral, as well as heteroaromatic and carbocylic amines, participated
well in the reaction with good yields (60–85%). All the newly synthesized
1
compounds were elucidated and were proven using spectral methods such as H
NMR, 13C NMR, FTIR and HRMS. All the newly synthesized compounds were in
good agreement with the proposed structures (Supplemental Material). To the best
of our knowledge, all the newly synthesized derivatives have not yet been reported
in the literature.
Anticancer activity
All the synthesized title compounds 3(a-u) were evaluated for their in vitro
anticancer activity measured by sulforhodamine B (SRB) assay method [24] against
three cancer cell lines colo205 (human colon adenocarcinoma cell line), MCF-7
(human breast adenocarcinoma cell line) and A-549 (human lung adenocarcinoma
cell line). All tested compounds exhibited excellent to potent inhibitory activity as
compared to the standard drug Etoposide and results of anticancer activity are
shown in Table 2. Among the series, compounds 3e (IC50 = 1.17 lM), 3i
(IC50 = 1.20 lM), 3j (IC50 = 0.11 lM), 3o (IC50 = 0.98 lM) and 3s (IC50-
= 1.89 lM) showed excellent inhibitory activity against MCF-7 cancer cell line
compared to the standard Etoposide (IC50 = 2.11 lM). Similarly, compounds 3e
(IC50 = 2.90 lM), 3g (IC50 = 2.99 lM), 3i (IC50 = 1.10 lM), 3j (IC50 = 0.18 lM)
and 3o (IC50 = 2.76 lM) also showed excellent inhibitory activity against A-549
cancer cell line compared to the standard Etoposide (IC50 = 3.08 lM). Compounds
3f (IC50 = 3.81 lM), 3q (IC50 = 4.76 lM) and 3t (IC50 = 4.66 lM) showed good
123

Synthesis, biological evaluation and molecular docking…
Table 1 Optimization conditions for the synthesis of compound 3a
Entry
Catalyst
Solvent
Reaction temp (°C)/time (h)
% of yield
1
AlCl3
Neata
Tolueneb
THFb
Neata
Tolueneb
THFb
Tolueneb
THFb
THFb
Tolueneb
Neata
Tolueneb
Tolueneb
THFb
150 °C, 16 h
90 °C, 14 h
80 °C, 9 h
100 °C, 16 h
90 °C, 14 h
80 °C, 9 h
100 °C, 8 h
80 °C, 9 h
90 °C, 12 h
105 °C, 8 h
100 °C, 12 h
105 °C, 8 h
90 °C, 14 h
80 °C, 9 h
90 °C, 9 h
90 °C, 9 h
90 °C, 9 h
90 °C, 9 h
20
28
23
10
Trace
–
2
AlCl3
3
AlCl3
4
FeCl3
5
FeCl3
6
FeCl3
7
TiCl4
–
8
TiCl4
–
9
Ti(iPrO)4
Ti(iPrO)4
SnCl4
SnCl4
ZnCl2
Me3Al
Me3Al
Me3Al
Me3Al
Me3Al
–
10
11
12
13
14
15
16
17
18
8
5
12
–
40
80
82
82
80
Tolueneb
Toluenec
Toluened
Toluenee
aMethod a: 2a (1 mmol), aniline (1.5 mmol), catalyst (1.5 mmol). bMethod b: 2a(1 mmol), aniline
(1.5 mmol), catalyst (1.5 mmol), in the presence of solvent. cMethod c: 2a (1 mmol), aniline (1.5 mmol),
catalyst (2.0 mmol), in the presence of solvent. dMethod d: 2a (1 mmol), aniline (1.5 mmol), catalyst
(2.5 mmol), in the presence of solvent. eMethod e: 2a (1 mmol), aniline (2.0 mmol), catalyst (1.5 mmol),
in the presence of solvent
inhibitory activity on MCF-7 cancer cell line, compounds 3a (IC50 = 2.90 lM), 3q
(IC50 = 8.34 lM) showed inhibitory good activity on A-549 cancer cell line. The
compounds 3a (IC50 = 3.76 lM), 3b (IC50 = 2.87 lM), 3e (IC50 = 2.18 lM), 3i
(IC50 = 2.89 lM), 3j (IC50 = 2.33 lM), 3k (IC50 = 3.45 lM), 3l (IC50 = 2.89 lM),
3o (IC50 = 2.21 lM) and 3r (IC50 = 3.78 lM) showed good to moderate inhibitory
activity against colo-205 cancer cell line.
Molecular docking studies
To understand the ligand orientation and the inhibitory ability towards human DNA
topoisomerase II, a molecular docking study was carried out by using the LibDock
module in Discovery Studio. The 3D structure of human DNA topoisomerase II
(PDB ID: 1T8I) was retrieved from the Protein Data Bank (http://www.rcsb.org).
We initially carried out docking of the synthesized compounds and later with the
123

L. N. S. Konidena et al.
Table 2 Anticancer activity of
synthesized compounds 3(a-u)
Compound
MCF-7
A-549
Colo-205
3a
6.54
29.50
13.80
–
4.87
–
3.76
2.87
–
3b
3c
–
3d
3e
55.7
2.90
–
–
1.17
3.81
–
2.18
34.7
8.45
–
3f
3g
3h
3i
2.99
10.4
1.10
0.18
11.56
–
–
1.20
0.11
14.3
26.80
–
2.89
2.33
3.45
2.89
–
3j
3k
3l
3m
3n
3o
3p
3q
3r
54.3
9.45
2.76
–
–
17.6
2.21
9.45
13.7
3.78
16.2
–
0.98
12.7
4.78
5.80
1.89
4.66
13.89
2.11
8.34
18.3
31.5
10.4
9.34
3.08
3s
3t
3u
Etoposide
–
0.13
Scheme 2 Plausible mechanism for the formation of amides
known inhibitor/anticancer drug Etoposide. The used docking program LibDock
produces several poses, each producing their corresponding LibDock scores with
different orientations within the defined active site of the protein.
The high LibDock score of the ligand pose was taken into account for the
prediction of the best ligand binding conformation. So, the mentioned pre-validated
analysis was used to sort out the retrieved hit molecules, and then those are further
validated by using the visualization method to find the suitable binding mode of the
inhibitors based on the critical interactions with the active site residues. The docked
ligands were found to have similar binding poses to the co-crystallized ligand, thus
validating the adopted docking methodology. Finally, the Analyze Ligand Poses
subprotocol was performed to count H bonds and close contacts (Van der Waals
123

Synthesis, biological evaluation and molecular docking…
Table 3 Details of LibDock
Compound LibDockScore HBOND count 1:
1t8i
Contacts 1:
1t8i
score of synthesised compounds
3(a-u) and Etoposide on human
DNA topoisomerase II (PDB:
1T8I)
3a
97.333
132.778
117.806
115.2
3
0
4
4
4
2
2
2
2
1
4
0
2
2
3
2
3
2
4
1
2
4
1
4
5
5
5
3
4
2
2
4
2
9
3
1
2
3
4
4
5
8
3
6
3b
3c
3d
3e
122.072
121.139
119.326
120.951
121.559
158.072
150.882
154.208
142.368
121.04
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
3q
3r
133.191
125.042
132.778
130.718
119.605
147.311
140.889
161.438
3s
3t
3u
Etoposide
clashes) between the poses and human topoisomerase II. The summary of docking
information of the top ranked poses of each compound are given in Table 3.
Docking analysis of all compounds with the human topoisomerase II, revealed that
compound 3j fitted well in the active site pocket, showing the best docking score of
158.072 kcal/mol, that is, closest to Etoposide, which has a docking score of
161.438. The best conformation with H-bond interactions obtained for compound 3j
is shown in Fig. 2. From the Fig. 2 it is revealed that three hydrogen bonds and
three close contacts formed between compound 3j with the protein DNA
topoisomerse II. The first hydrogen bond is formed with the amino acid Thr718
of the DNA topoisomerse II. A hydrogen bond is formed between hydrogen atom of
the oxygen molecule of the amino acid Thr418 with the 11th oxygen atom of
˚
compound 3j (A:Thr718:OH-3j:N20) with a hydrogen bond distance of 2.484 A,
and the second hydrogen bond formed in between Arg364:NH2-O10:3j.mol with a
˚
hydrogen bond distance 2.049 A. A third hydrogen bond formed between
˚
A:ARG364:NH22-3j.mol:O10 with a distance of 2.080 A.
123

L. N. S. Konidena et al.
Fig. 2 Receptor-ligand hydrogen bonds (green colour) and bumps (pink colour) of compounds 3j with
active site residues of Human DNA topoisomerase II (PDB: 1T8I)
Conclusion
In conclusion, a series of novel 2-(2-cyanophenyl)-N-phenylacetamide derivatives
3(a-u) were designed as potential anticancer agents. These compounds were
obtained in good yields via methyl-2-(2-cyanophenyl)acetates selective amidation
using AlMe3 as catalyst. All the newly synthesized derivatives were well
characterized by spectral techniques such as 1H NMR, 13C NMR, FTIR and
HRMS. All the synthesized title compounds were evaluated for their in vitro
anticancer activity and molecular docking studies. Among all compounds, 3i
(IC50 = 1.20 lM, IC50 = 1.10 lM), 3j (IC50 = 0.11 lM, IC50 = 0.18 lM), 3o
(IC50 = 0.98 lM, IC50 = 2.76 lM) showed excellent inhibitory activity against
MCF-7 and A-549 cell lines, respectively. Docking analysis of all compounds with
the human topoisomerase II, revealed that the compound 3j fitted well in the active
site pocket, showing the best docking score of 158.072 kcal/mol. The potent activity
of these N-phenylacetamide derivatives suggests that they are potential candidates
for the development of new anticancer drugs. Further work is in progress to improve
the potency of these compounds.
Experimental section
General information
Reagents were commercially available with analytical grade and used as purchased
without further purification. Solvents were purified according to well-known
123

Synthesis, biological evaluation and molecular docking…
laboratory methods. All reactions were performed by using oven-dried glassware
under an inert atmosphere. Reaction mixtures were monitored by thin-layer
chromatography (TLC) using silica gel 60-F254 pre-coated glass plates (Merck,
Italy). Spots on the TLC plates were visualized with a UV lamp (254 nm) and by
spraying with 0.2% ninhydrin in ethanol and charring after elution. Nuclear
magnetic resonance (1H and 13C NMR) spectra were recorded on a 400 MHz and
500 MHz spectrometer (Bruker) in DMSO and CDCl3 using TMS as an internal
standard. Chemical shifts are reported in parts per million (d), coupling constants
(J values) were reported in Hertz (Hz) and spin multiplicities are indicated by the
following symbols: s (singlet), d (doublet), t (triplet), sept (septet), m (multiplet), bs
(broad singlet), bd (broad doublet). 13C NMR spectra were routinely run with
broadband decoupling. Infrared spectra were taken with a Hitachi 260-30
spectrometer. Mass spectra were recorded in LCQ Fleet mass spectrometer,
Thermo Fisher Instruments Limited, US. Electrospray ionisation mass spectrometry
(ESI–MS) analysis was performed in the positive ion and negative ion mode on a
liquid chromatography ion trap.
General procedure for synthesis of compound 2(a-b)
A solution of compound 1(a-b) (1 mmol) in DMF (5 mL) was added to a stirred
solution of CuCN (1.2 mmol) in DMF (10 mL) at room temperature and refluxed
for 12 h. After completion of the reaction (monitored by TLC), the reaction mixture
was quenched with a solution of iron (III) chloride (1.2 mmol) in H2O (5 mL) at
room temperature and stirred vigorously for 15 min. The aqueous layer was
extracted with AcOEt (2 9 15 mL). The combined organic layers were washed
with water, dried over anhydrous sodium sulphate and concentrated in vacuo. Crude
product was purified by flash chromatography (Hexane/AcOEt 9:1 v/v) to afford
compound 2(a-b) as clear oil.
General procedure for synthesis of substituted 2-(2-cyanophenyl)-N-
phenylacetamide 3(a-u) derivatives
Trimethyl aluminium (1.5 mmol, 2.0 M in toluene) was added to a solution of
amine (2 mmol) in toluene (10 mL) at 0 °C and stirred at room temperature for
30 min. Compound 4 (1 mmol) was added at 0 °C and stirred at 80 °C for 9 h. After
the completion of the reaction (by TLC), the reaction mixture was quenched with
water (15 mL), stirred for 10 min, filtered through a pad of Celite, and the Celite
bed washed with AcOEt (10 mL). The organic layer was separated from the filtrate,
and the aqueous layer was extracted with AcOEt (2 9 15 mL). The combined
organic layers were washed with 1 N HCl and brine solution, dried over anhydrous
sodium sulphate and concentrated in vaccuo. Crude product was purified by flash
chromatography (Hexane/AcOEt 7:3 v/v) to afford 2-(2-cyanophenyl)-N-phenylac-
etamide 3(a-u) derivatives.
123

L. N. S. Konidena et al.
2-(2-cyanophenyl)-N-phenylacetamide (3a)
1
Pale brown solid, yield 80%, IR (KBr) cm-1: 3283, 2222, 1655, 1539, 765; H
NMR (400 MHz, DMSO-d6) d: 10.30 (s, 1H), 7.84 (dd, J = 7.82, 0.98 Hz, 1H),
7.66–7.72 (m, 1H), 7.54–7.62 (m, 3H), 7.46–7.52 (m, 1H), 7.32 (t, J = 8.07 Hz,
2H), 7.03–7.09 (m, 1H), 3.96 (s, 2H); 13C NMR (100 MHz, DMSO) d: 167.3, 139.4,
138.9, 133.0, 132.6, 131.0, 128.7, 127.6, 123.3, 119.1, 117.8, 112.6, 41.2; HRMS
(ESI): calcd. for C15H13N2O [M ? H]? 237.1028; found 237.1034.
2-(2-cyanophenyl)-N-(2-fluorophenyl)acetamide (3b)
Off-white solid, yield 75%, IR (KBr) cm-1: 3255, 2224, 1669, 1540, 756; 1H NMR
(400 MHz, DMSO-d6) d: 10.12 (brs, 1H), 7.82–7.87 (m, 2H), 7.69 (td, J = 7.58,
1.47 Hz, 1H), 7.57 (d, J = 7.83 Hz, 1H), 7.46–7.52 (m, 1H), 7.23–7.32 (m, 1H),
7.13–7.20 (m, 2H), 4.02 (s, 2H); 13C NMR (100 MHz, CDCl3) d: 166.8, 153.7,
151.3, 138.0, 133.18, 132.8, 130.8, 128.0, 125.9, 124.8, 122.0, 117.8, 114.9, 113.0,
42.6.; HRMS (ESI): calcd. for C15H12N2OF [M ? H]? 255.0934; found 255.0922.
2-(2-cyanophenyl)-N-(3-fluorophenyl)acetamide (3c)
1
Pale brown solid, yield 73%, IR (KBr) cm-1: 3257, 2226, 1659, 1551, 755; H
NMR (400 MHz, DMSO-d6) d: 10.54 (s, 1H), 7.84 (dd, J = 7.83, 0.98 Hz, 1H),
7.65–7.71 (m, 1H), 7.54–7.60 (m, 2H), 7.48 (td, J = 7.58, 0.98 Hz, 1H), 7.28–7.39
(m, 2H), 6.85–6.92 (m, 1H), 3.97 (s, 2H); 13C NMR (100 MHz, CDCl3) d: 167.2,
164.0, 161.6, 139.12, 138.3, 133.3, 132.7, 130.9, 129.9, 127.9, 118.1, 115.2, 112.6,
111.3, 42.5; HRMS (ESI): calcd. for C15H12N2OF [M ? H]? 255.0934; found
255.0904.
2-(2-cyanophenyl)-N-(4-fluorophenyl)acetamide (3d)
1
Pale brown solid, yield 75%, IR (KBr) cm-1: 3255, 2225, 1654, 1506, 757; H
NMR (400 MHz, CDCl3) d: 7.94 (brs, 1H), 7.64 (d, J = 7.6 Hz, 1H), 7.52–7.59 (m,
2H), 7.36–7.44 (m, 3H), 6.94 (t, J = 8.4 Hz, 2H), 3.87 (s, 2H).; 13C NMR
(100 MHz, CDCl3) d: 167.0, 160.7, 158.3, 138.5, 133.5, 132.7, 130.9, 127.8, 122.0,
118.1, 115.6, 112.6, 42.4; HRMS (ESI): calcd. for C15H12N2OF [M ? H]?
255.0934; found 255.0919.
2-(2-cyanophenyl)-N-(3,5-difluorophenyl)acetamide (3e)
Off-white solid, yield 76%, IR (KBr) cm-1: 3272, 2227, 1662, 1563, 757; 1H NMR
(400 MHz, DMSO-d6) d: 10.71 (s, 1H), 7.84 (d, J = 7.82 Hz, 1H), 7.65–7.72 (m,
1H), 7.56 (d, J = 7.82 Hz, 1H), 7.49 (t, J = 7.58 Hz, 1H), 7.31 (d, J = 7.34 Hz,
2H), 6.93 (t, J = 9.29 Hz, 1H), 3.98 (s, 2H).; 13C NMR (100 MHz, CDCl3) d: 167.0,
164.4, 161.9, 139.57, 137.9, 133.5, 133.8, 131.0, 128.1, 118.2, 112.6, 103.0, 100.1,
42.8; HRMS(ESI): calcd for C15H11N2OF2 [M ? H]? 273.0839; found 273.0832.
123

Synthesis, biological evaluation and molecular docking…
2-(2-cyanophenyl)-N-o-tolylacetamide (3f)
Off-white solid, yield 85%, IR (KBr) cm-1: 3178, 2235, 1680, 1520, 728; 1H NMR
(400 MHz, DMSO-d6) d: 9.65 (s, 1H), 7.83 (d, J = 7.82 Hz, 1H), 7.65–7.72 (m,
1H), 7.58 (d, J = 7.34 Hz, 1H), 7.47 (t, J = 7.58 Hz, 1H), 7.38 (d, J = 7.82 Hz,
1H), 7.21 (d, J = 7.83 Hz, 1H), 7.04–7.11 (m, 1H), 7.13–7.19 (m, 1H), 3.98 (s, 2H),
2.22 (s, 3H); 13C NMR (100 MHz, CDCl3) d: 167.0, 138.7, 135.2, 133.2, 132.7,
130.8, 130.4, 129.6, 127.8, 126.5, 125.5, 123.3, 118.0, 112.7, 42.5, 17.6; HRMS
(ESI): calcd. for C16H15N2O [M ? H]? 251.1184; found 251.1174.
2-(2-cyanophenyl)-N-m-tolylacetamide (3g)
Off-white solid, yield 80%, IR (KBr) cm-1: 3255, 2352, 1659, 1547, 763; 1H NMR
(400 MHz, DMSO-d6) d:10.23 (s, 1H), 7.83 (d, J = 7.34 Hz, 1H), 7.65–7.71 (m,
1H), 7.55 (d, J = 7.82 Hz, 1H), 7.47–7.50 (m, 1H), 7.42–7.46 (m, 1H), 7.36 (d,
J = 8.31 Hz, 1H), 7.18 (t, J = 7.83 Hz, 1H), 6.87 (d, J = 7.34 Hz, 1H), 3.93 (s, 2H),
2.27 (s, 3H); 13C NMR (100 MHz, CDCl3) d: 167.0, 160.0, 138.7, 138.5, 133.2,
132.7, 130.9, 129.5, 127.8, 118.2, 112.7, 112.1, 110.5, 105.6, 55.2, 42.6; HRMS
(ESI):calcd for C16H15N2O [M ? H]? 251.1184; found 251.1183.
2-(2-cyanophenyl)-N-p-tolylacetamide (3h)
Off-white solid, yield 81%, IR (KBr) cm-1: 3294, 2224, 1648, 1525, 759; 1H NMR
(400 MHz, DMSO-d6) d:10.22 (s, 1H), 7.82 (dd, J = 7.6, 0.8 Hz, 1H), 7.65–7.69
(m, 1H), 7.54 (d, J = 7.6 Hz, 1H), 7.45–7.49 (m, 3H), 7.10 (d, J = 8.4 Hz, 2H), 3.92
(s, 2H), 2.25 (s, 3H); 13C NMR (100 MHz, CDCl3) d: 166.8, 138.7, 135.0, 134.2,
133.1, 132.7, 130.8, 129.3, 129.3, 127.73, 120.2, 118.1, 112.7, 42.5, 20.8; HRMS
(ESI): calcd forC16H15N2O [M ? H]? 251.1184; found 251.1180.
2-(2-cyanophenyl)-N-(4-methoxyphenyl)acetamide (3i)
Pale green solid, yield 75%, IR (KBr) cm-1: 3265, 2230, 1635, 1541, 1110, 732; 1H
NMR (400 MHz, DMSO-d6) d: 10.16 (s, 1H), 7.83 (d, J = 7.82 Hz, 1H), 7.68 (td,
J = 7.70, 1.22 Hz, 1H), 7.55 (d, J = 7.34 Hz, 1H), 7.45–7.52 (m, 3H), 6.85–6.91
(m, 2H), 3.90 (s, 2H), 3.72 (s, 3H); 13C NMR (100 MHz, CDCl3) d: 166.67, 156.67,
138.74, 133.23, 132.73, 130.91, 130.61, 127.82, 121.94, 118.18, 114.10, 112.72,
55.44, 42.67; HRMS (ESI): calcd for C16H15N2O2[M ? H]? 267.1134; found
267.1123.
2-(2-cyanophenyl)-N-(3-methoxyphenyl)acetamide (3j)
Pale green solid, yield 75%, IR (KBr) cm-1: 3255, 2352, 1659, 1547, 763; 1H NMR
(400 MHz, DMSO-d6) d: 10.3 (s, 1H), 7.84 (d, J = 7.82 Hz, 1H), 7.65–7.72 (m,
1H), 7.56 (d, J = 7.82 Hz, 1H), 7.49 (t, J = 7.58 Hz, 1H), 7.31 (d, J = 7.34 Hz,
1H), 7.21 (t, J = 8.0 Hz, 2H), 6.93 (t, J = 9.29 Hz, 1H), 3.93 (s, 2H), 3.71 (s, 3H);
13C NMR (100 MHz, CDCl3) d: 167.0, 160.0, 138.8, 138.5, 133.2, 132.7, 130.9,
123

L. N. S. Konidena et al.
129.5, 127.8, 118.2, 112.7, 112.1, 110.5, 105.6, 55.2, 42.6; HRMS (ESI): calcd. for
C16H15N2O2 [M ? H]? 267.1134; found 267.1128.
2-(2-cyanophenyl)-N-(4-phenoxyphenyl)acetamide (3k)
1
Pale brown solid, yield 78%, IR (KBr) cm-1: 3278, 2606, 1653, 1532, 762; H
NMR (400 MHz, DMSO-d6) d: 10.36 (s, 1H), 7.84 (d, J = 7.34 Hz, 1H), 7.66–7.72
(m, 1H), 7.59–7.64 (m, 2H), 7.57 (d, J = 7.82 Hz, 1H), 7.46–7.52 (m, 1H),
7.34–7.40 (m, 2H), 7.11 (t, J = 7.34 Hz, 1H), 6.94–7.03 (m, 4H), 3.95 (s, 2H); 13C
NMR (100 MHz, CDCl3) d: 167.0, 157.4, 153.7, 138.6, 133.2, 133.0, 132.7, 130.9,
129.6, 127.8, 123.0, 121.9, 119.4, 118.4, 118.1, 112.7, 42.4; HRMS (ESI): calcd. for
C21H17N2O2 [M ? H]? 329.1290; found 329.1266.
Methyl 4-(2-(2-cyanophenyl)acetamido)benzoate (3l)
Off-white solid, yield 70%, IR (KBr) cm-1: 3252, 2219, 1715, 1650, 1528, 740; 1H
NMR (300 MHz, DMSO-d6) d: 10.68 (s, 1H), 7.91–7.94 (m, 2H), 7.84 (d,
J = 7.8 Hz, 1H), 7.66–7.74 (m, 3H), 7.56 (d, J = 7.5 Hz, 1H), 7.46–7.51 (m, 1H),
4.0 (s, 2H), 3.82 (s, 3H); 13C NMR (100 MHz, CDCl3) d: 167.1, 166.5, 141.7,
138.1, 133.3, 132.8, 131.0, 130.7, 128.0, 126.0, 119.0, 118.2, 112.7, 52.0, 42.8;
HRMS (ESI):calcd. for C17H15N2O3 [M ? H]? 295.1083; found 295.1082.
2-(2-cyanophenyl)-N-(naphthalen-3-yl)acetamide (3m)
1
Pale brown solid, yield 76%, IR (KBr) cm-1: 3057, 2220, 1614, 1562, 772; H
NMR (400 MHz, DMSO-d6) d: 10.31 (s, 1H), 8.12–8.20 (m, 1H), 8.00–8.00 (m,
1H), 7.96 (dd, J = 6.36, 2.93 Hz, 1H), 7.86 (d, J = 7.34 Hz, 1H), 7.79 (d,
J = 8.31 Hz, 2H), 7.67–7.74 (m, 1H), 7.63–7.66 (m, 2H), 7.45–7.62 (m, 2H), 4.14
(s, 2H); 13C NMR (100 MHz, CDCl3) d: 167.5, 138.6, 134.0, 133.3, 132.8, 131.9,
131.0, 128.6, 127.9, 127.1, 126.4, 126.2, 126.0, 125.5, 121.2, 120.7, 118.2, 112.8,
42.7; HRMS (ESI): calcd. for C19H15N2O [M ? H]? 287.1184; found 287.1188.
2-(2-cyanophenyl)-N-(5-fluoropyridin-2-yl)acetamide (3n)
1
Pale yellow solid, yield 60%, IR (KBr) cm-1: 3252, 2219, 1693, 1547, 759; H
NMR (400 MHz, DMSO-d6) d: 10.98 (s, 1H), 8.35 (d, J = 2.93 Hz, 1H), 8.07 (dd,
J = 9.05, 4.16 Hz, 1H), 7.83 (dd, J = 7.82, 0.98 Hz, 1H), 7.65–7.77 (m, 2H), 7.55
(d, J = 7.34 Hz, 1H), 7.48 (td, J = 7.58, 0.98 Hz, 1H), 4.03 (s, 2H); 13C NMR
(100 MHz, CDCl3) d: 167.0, 157.8, 155.2, 147.1, 137.7, 135.5, 133.2, 130.8, 128.1,
125.3, 117.7, 115.0, 113.2, 42.5; HRMS (ESI): calcd. for C14H11N3OF [M ? H]?
256.0866; found 256.0877.
2-(2-cyanophenyl)-N-cyclohexylacetamide (3o)
1
Off-white solid, yield 70%, IR (KBr) cm-1: 3250, 1645, 1532, 741; H NMR
(400 MHz, DMSO-d6) d: 8.06 (d, J = 8.0 Hz, 1H), 7.77 (dd, J = 7.8, 1.0 Hz, 1H),
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Synthesis, biological evaluation and molecular docking…
7.61–7.65 (m, 1H), 7.43 (q, J = 7.2 Hz, 2H), 3.65 (s, 2H), 3.5–3.53 (m, 1H),
1.66–1.75 (m, 4H), 1.54 (d, J = 12.4 Hz, 1H), 1.11–1.27 (m, 5H); 13C NMR
(100 MHz, CDCl3) d: 167.5, 139.2, 133.1, 132.6, 130.7, 127.5, 118.0, 112.5, 48.6,
42.1, 32.8, 25.3, 24.6; HRMS (ESI): calcd. for C15H19N2O [M ? H]? 243.1497;
found 243.1490.
2-(2-cyanophenyl)-N,2-diphenylacetamide (3p)
Off-white solid, yield 79%, IR (KBr) cm-1: 3225, 1660, 1545, 735;1H NMR
(500 MHz, DMSO-d6) d: 10.59 (s, 1H), 7.85 (dd, J = 7.9, 0.9 Hz, 1H), 7.67–7.73
(m, 1H), 7.58–7.62 (m, 3H), 7.48 (td, J = 7.5, 0.9 Hz, 1H), 7.35–7.41 (m, 2H),
7.29–7.33 (m, 5H), 7.06 (t, J = 7.3 Hz, 1H), 5.55 (s, 1H); 13C NMR (100 MHz,
CDCl3) d: 168.2, 142.9, 137.5, 137.2, 133.1, 132.9, 129.8, 128.9, 128.8, 128.1,
127.7, 124.7, 120.0, 118.0, 112.7, 57.2; HRMS (ESI): Calcd. for C21H17N2O
[M ? H]? 313.1341; found 313.1339.
2-(2-cyanophenyl)-N-(2,4-dimethoxyphenyl)-2-phenylacetamide (3q)
1
Off-white solid, yield 83%, IR (KBr) cm-1: 3230, 1670, 1545, 735; H NMR
(400 MHz, CDCl3) d: 8.26 (d, J = 8.8 Hz, 1H), 7.89 (s, 1H), 7.63–7.69 (m, 2H),
7.55–7.61 (m, 1H), 7.30–7.43 (m, 6H), 6.42–6.47 (m, 2H), 5.47 (s, 1H), 3.78 (s,
3H), 3.74 (s, 3H); 13C NMR (100 MHz, CDCl3) d: 167.6, 156.8, 149.4, 143.1,
137.5, 132.9, 129.7, 129.0, 127.9, 127.6, 120.8, 120.5, 117.8, 113.0, 103.7, 98.6,
57.6, 55.7; HRMS (ESI): Calcd. for C23H21N2O3 [M ? H]? 373.1552; found
373.1562.
2-(2-cyanophenyl)-N-(4-methoxyphenyl)-2-phenylacetamide (3r)
1
Off-white solid, yield 80%, IR (KBr) cm-1: 3230, 1677, 1545, 740; H NMR
(500 MHz, CDCl3) d: 7.81 (brs, 1H), 7.61–7.67 (m, 2H), 7.53–7.57 (m, 1H),
7.31–7.42 (m, 8H), 6.79 (dd, J = 6.75, 2 Hz, 2H), 5.45 (s, 1H), 3.75 (s, 3H); 13C
NMR (125 MHz, CDCl3) d: 168.0, 156.6, 143.0, 137.4, 133.1, 132.8, 130.6, 129.4,
128.8, 127.9, 121.8, 118.0, 114.0, 112.6, 56.9, 55.4; HRMS (ESI): Calcd. for
C22H19N2O2 [M ? H]? 343.1447; found 343.1433.
2-(2-cyanophenyl)-N-(2,3,4-trifluorophenyl)-2-phenylacetamide (3s)
1
Pale brown solid, yield 78%, IR (KBr) cm-1: 3230, 1670, 1535, 745; H NMR
(400 MHz, CDCl3) d: 7.91–7.95 (m, 1H), 7.76 (brs, 1H), 7.74 (d, J = 13.2 Hz, 1H),
7.56–7.63 (m, 2H), 7.32–7.46 (m, 6H), 6.91–6.97 (m, 1H), 5.54 (s, 1H); 13C NMR
(100 MHz, CDCl3) d: 168.7, 146.6, 142.3, 141.4, 141.0, 138.4, 136.5, 133.1, 129.2,
128.2, 125.2, 123.2, 117.7, 116.3, 112.9, 111.6, 57.1; HRMS (ESI): Calcd. for
C21H14N2OF3 [M ? H]? 367.1058; found 367.1060.
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L. N. S. Konidena et al.
2-(2-cyanophenyl)-N-(2-methoxyphenyl)-2-phenylacetamide (3t)
1
Off-white solid, yield 76%, IR (KBr) cm-1: 3230, 1665, 1540, 735; H NMR
(400 MHz, CDCl3) d: 8.44 (d, J = 7.6 Hz, 1H), 8.16 (s, 1H), 7.69 (d, J = 7.3 Hz,
2H), 7.59–7.65 (m, 1H), 7.34–7.47 (m, 6H), 7.05–7.11 (m, 1H), 6.98 (t, J = 7.2 Hz
1H), 6.86 (d, J = 7.8 Hz, 1H), 5.54 (s, 1H), 3.78 (br s, 3H); 13C NMR (100 MHz,
CDCl3) d: 168.0, 148.0, 143.0, 137.3, 133.0, 129.7, 129.0, 128.0, 127.7, 127.2,
124.2, 120.9, 119.6, 117.8, 113.0, 110.0, 57.8, 55.6; HRMS (ESI): Calcd. for
C22H19N2O2 [M ? H]? 343.1447; found 343.1432.
2-(2-cyanophenyl)-N-(3-methoxyphenyl)-2-phenylacetamide (3u)
1
Off-white solid, yield 78%, IR (KBr) cm-1: 3230, 1665, 1540, 740; H NMR
(500 MHz, CDCl3) d: 7.66 (br s, 1H), 7.59–7.64 (m, 2H), 7.56–7.58 (m, 1H),
7.32–7.42 (m, 7H), 7.69 (t, J = 8.0 Hz, 1H), 6.93 (d, J = 8.0 Hz, 1H), 6.65 (d,
J = 6.5 Hz, 1H), 5.46 (s, 1H), 3.76 (s, 3H); 13C NMR (125 MHz, CDCl3) d: 168.3,
160.1, 142.9, 138.8, 137.2, 133.2, 132.9, 129.8, 129.6, 129.2, 128.9, 128.1, 127.8,
118.0, 112.7, 112.0, 110.8, 105.4, 57.3, 55.3; HRMS (ESI): Calcd. for C22H19N2O2
[M ? H]? 343.1447; found 343.1457.
Biological evaluation method
Procedure of the SRB-assay
The synthesized compounds 3(a-u) have been evaluated for their in vitro
cytotoxicity in human cancer cell lines. A protocol of 48 h of continuous drug
exposure has been used and a sulforhodamine B (SRB) protein assay was used to
estimate cell viability or growth. The cell lines were grown in DMEM medium
containing 10% fetal bovine serum and 2 mM L-glutamine and were inoculated into
96 well microtiter plates in 90 mL at plating densities depending on the doubling
time of individual cell lines. The microtiter plates were incubated at 37 °C, 5% CO2,
95% air, and 100% relative humidity for 24 h prior to addition of experimental
drugs. Aliquots of 10 mL of the drug dilutions were added to the appropriate
microtiter wells already containing 90 mL of cells, resulting in the required final
drug concentrations. For each compound four concentrations (0.1, 1, 10 and
100 lM) were evaluated, and each was done in triplicate wells. Plates were
incubated further for 48 h and assay was terminated by the addition of 50 mL of
cold trichloroacetic acid (TCA) (final concentration, 10% TCA) and incubated for
60 min at 4 °C. The plates were washed five times with tap water and air dried.
Sulforhodamine B (SRB) solution (50 mL) at 0.4% (w/v) in 1% acetic acid was
added to each of the cells, and plates were incubated for 20 min at room
temperature. The residual dye was removed by washing five times with 1% acetic
acid. The plates were air dried. Bound stain was subsequently eluted with 10 mM
trizma base, and the absorbance was read on an ELISA plate reader at a wavelength
of 540 nm with 690 nm reference wavelengths. Percent growth was calculated on a
plate by plate basis for test wells relative to control wells. The above determinations
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Synthesis, biological evaluation and molecular docking…
were repeated three times. Percentage growth was expressed as the (ratio of average
absorbance of the test well to the average absorbance of the control wells) 9 100.
Growth inhibition of 50% (GI50) was calculated from [(Ti - Tz)/(C - Tz)] 9 100
1/4 50, which is the drug concentration resulting in a 50% reduction in the net
protein increase (as measured by SRB staining) in control cells during the drug
incubation. Where, Tz 1/4 Optical density at time zero, OD of control 1/4 C, and
OD of test growth in the presence of drug 1/4 Ti.
Acknowledgements The authors express their profound thanks to the management of GVK Biosciences
Private Limited for financial support. We thank Dr. Sudhir Kumar Singh and Dr. Somesh Sharma and Dr.
Krishna Reddy Valluru for their invaluable support and motivation.
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