Novel cyanoacetamide integrated phenothiazines: Synthesis, characterization, computational studies and in vitro antioxidant and anticancer evaluations

Article abstract of DOI:10.1016/j.molstruc.2019.127037

A series of novel phenothiazine based cyanoacrylamides 6a-d have been synthesized from phenothiazine through multistep synthetic strategy. The structure of these novel molecules (6a-d) has been determined by FT-IR, ¹H NMR and ¹³C NMR spectral techniques. Computational studies were carried out for the synthesized compounds 6a-d using DFT method with B3LYP/6-311G(d,p) basis set. The target compounds 6a-d have been screened for their in vitro antioxidant and anticancer activity. The antioxidant data revealed that the compounds 6c and 6d exhibited high radical scavenging activity with IC₅₀ value of 37.32 and 39.07 μM, respectively. Furthermore, all the synthesized compounds displayed significant in-vitro anticancer activity against both the pancreatic tumor cells AsPC1 and SW1990. Particularly, the compound 6c exhibited the highest activity among the molecules tested against both the tumor cells viz., AsPC1 and SW1990.

Full text of DOI:10.1016/j.molstruc.2019.127037

Journal of Molecular Structure 1199 (2020) 127037
Contents lists available at ScienceDirect
Journal of Molecular Structure
journal homepage: http://www.elsevier.com/locate/molstruc
Novel cyanoacetamide integrated phenothiazines: Synthesis,
characterization, computational studies and in vitro antioxidant and
anticancer evaluations
a
a
b
Kannan Gokula Krishnan , Chandran Udhaya Kumar , Wei-Meng Lim ,
b,
c
b
a, *
Chun-Wai Mai , Punniyakoti V. Thanikachalam , Chennan Ramalingan
Department of Chemistry, School of Advanced Sciences, Kalasalingam Academy of Research and Education (Deemed to be University), Krishnankoil, 626
a
1
26, Tamilnadu, India
b
c
School of Pharmacy, International Medical University, 126 Jalan Jalil Perkasa 19, 57000, Bukit Jalil, Kuala Lumpur, Malaysia
Center for Cancer and Stem Cell Research, Institute for Research, Development and Innovation (IRDI), International Medical University, 126, Jalan Jalil
Perkasa 19, 57000, Bukit Jalil, Kuala Lumpur, Malaysia
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 28 April 2019
Received in revised form
A series of novel phenothiazine based cyanoacrylamides 6a-d have been synthesized from phenothiazine
through multistep synthetic strategy. The structure of these novel molecules (6a-d) has been determined
by FT-IR, 1H NMR and C NMR spectral techniques. Computational studies were carried out for the
synthesized compounds 6a-d using DFT method with B3LYP/6-311G(d,p) basis set. The target com-
pounds 6a-d have been screened for their in vitro antioxidant and anticancer activity. The antioxidant
data revealed that the compounds 6c and 6d exhibited high radical scavenging activity with IC50 value of
13
25 August 2019
Accepted 4 September 2019
Available online xxx
3
7.32 and 39.07 mM, respectively. Furthermore, all the synthesized compounds displayed significant in-
Keywords:
Anticancer
Antioxidant
Computation
Cyanoacetamide
Phenothiazine
vitro anticancer activity against both the pancreatic tumor cells AsPC1 and SW1990. Particularly, the
compound 6c exhibited the highest activity among the molecules tested against both the tumor cells viz.,
AsPC1 and SW1990.
© 2019 Published by Elsevier B.V.
1. Introduction
acridine, carbazole, phenothiazine, are significant platforms for a
wide range of drugs which can offer new prospects for controlled
Cancer is a leading cause of death around the globe, which
claims over six million people died a year and still increasing
manipulation of genome [6]. Among these heterocycles, pheno-
thiazine ring systems as one of the most abundant ones and
becoming precious molecular scaffold in medicinal, industrial and
academic fields. Chlorpromazine, a derivative of phenothiazine has
been used to treat psychotic disorders and allergy [7]. Thioridazine,
trifluoperazine, promethazine and azaphenothiazines are some of
the phenothiazine analogues showed very promising clinical trials
for the treatment of patients with different cancers [8]. Further,
remarkable growth has been documented on the synthesis and
applications of these derivatives owing to their medicinal and next-
generation applications [9e18].
On the other hand, nitrile and amide possessing chemical en-
tities are the versatile lead molecules with many potential bio-
activities attributed to pharmacological applications [19]. A large
number of nitrile and amide systems are found in many natural
products [20]. They have been widely used in initial pre-clinical
trials as anti-fungal [21], anti-oxidant [22], anti-viral [23] and
anti-cancer agents [24]. These structural cores with the
(
WHO) [1]. Nearly two hundred types of cancers have been iden-
tified, which affects the major parts of organs [2]. Although various
drugs have been developed, discovery of new drugs to combat
specific cancers is highly essential. On the other hand, to enhance
the anticancer properties of the existing molecules, various syn-
thetic approaches have been established on the basis of structural
modifications [3]. Generally, molecules having heteroatoms are
considered as structural motifs for drug development as they in-
creases the host-guest connectivity between the molecule and the
biological receptors through hydrogen bonding [4,5]. The bi- and
tri-cyclic heterocycles viz. indole, imidazoles, thiazoles, quinolones,
*
Corresponding author.
E-mail addresses:
C. Ramalingan).
ramalinganc@gmail.com,
c.ramalingan@klu.ac.in
(
https://doi.org/10.1016/j.molstruc.2019.127037
022-2860/© 2019 Published by Elsevier B.V.
0

2
K.G. Krishnan et al. / Journal of Molecular Structure 1199 (2020) 127037
encouraging biological activities inspired us to prosecute the
investigation with these molecular analogues. Hence, we designed
phenothiazine based cyanoacetamides as a new class of antioxidant
and anticancer agents. The synthesis of new series of compounds
has been achieved by employing multistep synthetic protocol and
physical, spectroscopic and structural studies have also been car-
ried out. Antioxidant and anticancer activities of the synthesized
molecules have been evaluated using DPPH method and cell
viability assay method, respectively.
using deuterated chloroform and deuterated dimethyl sulfoxide as
solvents. Chemical shift values are reported in parts per million
(ppm) from tetramethylsilane (TMS), an internal standard. H NMR
splitting patterns are designated as singlet (s), broad singlet (br s),
doublet (d), doublet of doublet (dd) and multiplet (m).
1
2.2. Synthesis
The synthetic intermediates, alkylphenothiazines 3a-c and
phenothiazine carbaldehydes 4a-d were synthesized using litera-
ture methods [18].
2
. Experimental
2.1. General
2.3. Synthesis of phenothiazenyl cyanoacrylamide 6a
The melting points reported herein were measured in open-
To a solution of phenothiazine carbaldehyde 4a (0.5 g, 0.2 mmol)
in acetonitrile, were added cyanoacetamide (0.17 g, 0.2 mmol) and
catalytic amount of piperidine. The reaction mixture was refluxed
for 4 h and the reaction progress was monitored by TLC. After
cooling, the reaction mixture was poured into water and extracted
with dichloromethane. The combined organic layers were dried
over anhydrous MgSO , evaporated and purified by column chro-
4
matography on silica gel with hexane-ethyl acetate as the eluent to
afford pure phenothiazenyl cyanoacrylamide 6a as a dark red solid.
capillaries and are uncorrected. Progress and completion of the
reactions were monitored by TLC on pre-coated Silica gel 60 F plates
using hexane and ethyl acetate as eluents. FT-IR spectra of the
synthesized molecules were recorded as KBr pellet form with
number of scans equals 16 using Shimadzu-IR Tracer 100 in the
ꢀ1
range of 400e4000 cm . The spectral features are reported in
ꢀ1
1
13
wavenumber (cm ). H and C NMR spectra were recorded at
00 MHz and 100 MHz, respectively on a Bruker spectrometer
4
Scheme 1. Schematic representation for the synthesis of molecules 6a-d.

K.G. Krishnan et al. / Journal of Molecular Structure 1199 (2020) 127037
3
ꢁ
ꢀ1
Yield (86%); Melting point: 146e148 C; FT-IR (KBr):
3
n
max/cm
1494, 1465, 1442, 1361, 1313, 1288, 1251, 1213, 1143, 1107, 958, 879,
850, 806, 742, 663, 609, 536; H NMR (400 MHz, CDCl ) d 8.12 (1H,
3
1
408, 3153, 2879, 2206, 1691, 1565, 1471, 1440, 1367,1317, 1284,
255, 1213, 1174, 1130, 1107, 956, 806, 742, 678, 655, 605, 534, 460;
1
s), 7.76 (1H, dd, J ¼ 8.8 Hz, 1.6 Hz), 7.65 (1H, d, J ¼ 2.0 Hz), 7.26 (1H,
1
H NMR (400 MHz, DMSO‑d
1H, d, J ¼ 2.0 Hz), 7.66 (1H, br s), 7.20 (1H, m),7.14 (2H, d,
J ¼ 8.4 Hz), 7.07 (1H, d, J ¼ 8 Hz), 6.97 (1H, t, J ¼ 7.6 Hz), 3.96 (2H, q),
6
) d 8.01 (1H, s), 7.77e7.82 (2H, m), 7.71
s), 7.18-6.84 (3H, m), 6.25 (1H, s), 5.72 (1H, s), 3.85 (2H, t, J ¼ 7.2 Hz),
13
(
1.77 (2H, m), 1.42 (2H, m), 1.30 (4H, m), 0.90 (3H, br s); C NMR
3
(100 MHz, CDCl ) 162.7, 152.2, 149.5, 143.1, 131.3, 129.6, 127.6, 127.5,
125.6, 124.8, 123.6, 123.3, 117.7, 115.8, 114.9, 98.9, 48.0, 31.3, 26.7,
26.5, 22.5, 13.9.
13
1
.30 (3H, t, J ¼ 6.8 Hz); C NMR (100 MHz, DMSO‑d
48.1,142.9,131.4,128.6,128.4,127.6,126.2,123.9,123.0,121.8,117.5,
16.3, 115.7, 103.1, 42.1, 12.8.
6
) 163.5, 149.5,
1
1
2.6. Synthesis of phenothiazenyl cyanoacrylamide 6d
2.4. Synthesis of phenothiazenyl cyanoacrylamide 6b
The phenothiazine carbaldehyde 4d (0.72 g, 0.2 mmol), upon
The phenothiazine carbaldehyde 4b (0.56 g, 0.2 mmol), upon
reaction with cyanoacetamide (0.17 g, 0.2 mmol) in acetonitrile
reaction with cyanoacetamide (0.17 g, 0.2 mmol) in acetonitrile
furnished its corresponding phenothiazenyl cyanoacrylamide 6d as
ꢁ
furnished its corresponding phenothiazenyl cyanoacrylamide 6b as
a dark red solid. Yield (81%); Melting point: 130e132 C; FT-IR
ꢁ
ꢀ1
a orange red solid. Yield (89%); Melting point: 125e127 C; FT-IR
(KBr):
n
max/cm 3333, 3190, 2957, 2870, 2208, 1676, 1582, 1493,
ꢀ1
(
KBr):
n
max/cm 3396, 3170, 2873, 2210, 1687, 1568, 1543, 1492,
1462, 1356, 1296, 1271, 1250, 1213, 1177, 1138, 1107, 955, 870, 804,
1
1
6
465, 1440, 1361, 1286, 1255, 1215, 1141, 1107, 956, 869, 800, 738,
3
598, 542; H NMR (400 MHz, CDCl ) d 8.13 (1H, s), 7.77(1H, dd,
1
63, 611, 540; H NMR (400 MHz, CDCl
3
)
d
8.13 (1H, s), 7.77 (1H, dd,
J ¼ 8.8 Hz, 1.6 Hz), 7.63 (1H, d, J ¼ 2.0 Hz), 7.20 (2H, m), 6.86 (1H, d,
J ¼ 8.8 Hz, 1.6 Hz), 7.66 (1H, d, J ¼ 2 Hz), 7.26 (1H, s), 7.18-6.85 (4H,
J ¼ 8.4 Hz), 6.70 (1H, d, J ¼ 8.8 Hz), 6.26 (1H, s), 5.75 (1H, s), 3.83
m), 6.24 (1H, s), 5.65 (1H, s), 3.87 (2H, t, J ¼ 7.2 Hz),1.78 (2H, m),1.44
(2H, t, J ¼ 7.2 Hz), 1.73 (2H, m), 1.43 (2H, m), 0.94 (3H, t, J ¼ 7.2 Hz);
1
3
13
(
1
2H, m), 0.94 (3H, t, J ¼ 7.2 Hz); C NMR (100 MHz, CDCl
3
) 162.8,
3
C NMR (100 MHz, CDCl ) 162.8, 151.9, 149.0, 142.3, 131.3, 130.2,
52.1,149.5,143.1,131.2,129.6,127.6,127.5,125.7,124.8,123.6,123.4,
129.7, 129.6, 126.0, 125.7, 124.1, 117.6, 116.9, 115.8, 115.1, 28.6, 20.0,
13.7.
117.7, 115.8, 114.9, 98.9, 47.6, 28.7, 20.0, 13.7.
2.5. Synthesis of phenothiazenyl cyanoacrylamide 6c
2.7. Computational details
The phenothiazine carbaldehyde 4c (0.62 g, 0.2 mmol), upon
reaction with cyanoacetamide (0.17 g, 0.2 mmol) in acetonitrile
Computations of the target molecules 6a-d have been per-
formed with the Gaussian 09W packages [25]. The density func-
tional theory calculations have been accomplished utilizing the
hybrid functional B3LYP [26] with 6-311G(d,p) basis set. The
theoretically computed frequency values at these levels contain
furnished its corresponding phenothiazenyl cyanoacrylamide 6c as
ꢁ
a dark red solid. Yield (87%); Melting point: 112e114 C; FT-IR
ꢀ
1
(
KBr): nmax/cm 3400, 3165, 2960, 2933, 2872, 2210, 1687, 1568,
Fig. 1. Optimized structure of 6a-d.

4
K.G. Krishnan et al. / Journal of Molecular Structure 1199 (2020) 127037
known systematic errors. To bring the theoretical frequencies in
close proximity to the experimental values, the scaling factor value
of 0.96 was used [27]. Frontier molecular orbitals (FMOs) and
molecular electrostatic potential (MEP) are calculated to enlighten
the possibility of the molecular interaction with other species
around the space by sharing electronic charges.
cells were cultured in RPMI-1640 medium supplemented with 10%
fetal bovine serum, 100 IU/mL of penicillin and 100 g/mL of
m
streptomycin (Sigma-Aldrich, St. Louis, MO, USA). All pancreatic
ꢁ
cancer cells were maintained at an incubator at 37 C in 5% CO
2
,
using the standard in vitro cell culture method established previ-
ously [29e33].
2.8. DPPH scavenging activity
2.10. Cell viability assay
DPPH radical scavenging assay has been carried out using re-
ported procedure [28] with slight modifications. The stock solution
was prepared using DPPH (0.8 mg) in DMSO (25 mL). Various
concentrations such as 10, 20, 30, 40, 50, 100 mg/ml of working
solutions (compounds 6a-d) were prepared to evaluate their
radical scavenging activity. From the working solution, 1.5 ml of
each compound was added with 1.5 ml of DPPH and kept for 30 min
incubation under dark at room temperature. After incubation the
absorbance was measured against the blank DPPH at 517 nm using
UVevisible spectrophotometer. The percentage of DPPH radical
scavenging activity was determined using the following equation
In order to evaluate the anti-tumor effects of the synthetic
compounds, all compounds were reconstituted to 100 mM stock
solution using dimethylsulfoxide, further diluted to sterile phos-
phate buffer saline to the highest effective concentration, 0.1 mM.
The cells were treated based on the previously published cell
viability assay [29,34]. Briefly, all cells were then seeded
(
1500 cells/well) in 384-well plates for 24 h before treated with
compounds for 72 h. Cell viability was quantified using the Cell-
Titre Glo Luminescent Cell Viability Assay (Promega, USA). The
luminescence readings were recorded using SpectraMax M3
microplate reader (Molecular Devices Corporation, USA).
(
1),
3. Results and discussion
%
Scavenging ¼ 100 ꢂ ½ðAcontrol ꢀ AsampleÞ=Acontrol
ꢃ
(1)
3.1. Synthesis
2.9. Cell lines and cell culture
The precursors, phenothiazine carbaldehydes 4a-c have been
synthesized via alkylation of 10H-phenothiazine with respective
alkyl halides in the presence of potassium tert-butoxide in tetra-
hydrofuran at room temperature followed by Vilsmeier-Haack
Two human pancreatic cancer cells (AsPC1 and SW1990) were
procured from the American Type Culture Collection, ATCC, USA. All
Fig. 2. Experimental FT-IR spectra of 6a-d.

K.G. Krishnan et al. / Journal of Molecular Structure 1199 (2020) 127037
5
reaction of alkylphenothiazine with phosphoryl chloride in dime-
thylformamide at cooled to refluxed condition. On the other hand,
the bromo substituted phenothiazine carbaldehyde 4d has been
synthesized by employing phenothiazine carbaldehyde 4b with N-
bromosuccinimide (NBS) at room temperature. Eventually, Knoe-
venagel condensation between respective phenothiazine carbal-
dehydes 4a-d with cyanoacetamide (5) in the presence of
piperidine in acetonitrile afforded the corresponding targets, phe-
nothiazinyl cyanoacrylamides 6a-d in good yields (Scheme 1). The
chemical structures of the newly synthesized compounds 6a-
d have been established on the basis of analytical and spectral
techniques.
by dihedral angle for the compounds 6a-d C2eC3eC8eC9/C7 by
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
4 /-4.7 /-4.6 /-5 and
respectively. Thus, the two units lie on the same plane which in-
dicates strong electron delocalization occurs in the molecule.
ꢀ176.2 /175.3 /175.6 /175.1
[35,36],
ꢁ
ꢁ
ꢁ
Further the phenothiazine ring flattening at S11 (99.4 /99.5 /99.4 /
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
99.4 ) and N18 (122.1 /122.2 /122.1 /122.3 ) makes butterfly
conformation which revealed that the flatted structure could devise
the intramolecular charge transfer. The calculated bond parameters
(viz., bond lengths and angles) associated with the phenothiazine
and acrylamide part are expected in ideal range which is in good
agreement with crystal data of the reported ones [35,36]. From the
observation of computationally investigated molecules 6a-d, a
ꢁ
small deviation about 0.001 Å in bond length and̴ 0.5e1.2 in bond
angle as well as dihedral angle has been found for compounds 6a-d.
3.2. Structural analysis
The synthesized target molecules 6a-d have been optimized in
3.3. FT-IR spectral analysis
ground state achieved by B3LYP/6-311G(d,p) method. The opti-
mized molecular structure of the compounds 6a-d is depicted in
Fig. 1 and their selected bond lengths, bond angles and dihedral
angles are presented in Table S1. Since the target molecules 6a-
d are new ones, it is obviously clear that the exact data resulted
from experiments on the structural parameters of the same would
not be accessible from the literature. Hence, it would not be feasible
to compare the results of computation with the experiment.
Consequently, the crystal information of strongly related molecules
such as 10-ethyl-10H-phenothiazine-3-carbaldehyde [35] and 2-
cyano-N-(furan-2-ylmethyl)-3-(3-nitrophenyl)propanamide [36]
have been compared with that of the title molecules.
The energy minima structures have been utilized for calculating
vibrational frequencies of the synthesized compounds 6a-d at
B3LYP/6-311G(d,p) method and PED assignments have been ach-
ieved using VEDA program [37]. The compounds 6a-d have 38/44/
50/44 atoms, the molecules have normal modes of vibrations 108/
126/144/126 and belong to C1 symmetry. All the 108/126/144/126
vibrational modes are equally disseminated as 40/46/52/46
stretching, 33/39/45/39 bending and 35/41/47/41 torsion vibrations
in the computed spectrum. The experimental FT-IR of the com-
pounds 6a-d and their related computed ones under investigation
are sketched in Fig. 2 and Fig. 3. The experimental and computed
vibrational frequencies in addition to their relative intensities and
plausible assignments of the compounds 6a-d are furnished in
As can be seen from the figure, the orientation of donor and
acceptor units (i.e. phenothiazine and acrylamide) are determined
Fig. 3. Theoretical FT-IR spectra of 6a-d.

6
K.G. Krishnan et al. / Journal of Molecular Structure 1199 (2020) 127037
Fig. 4. (a) 1H NMR spectrum of 6a. (b) 13C NMR spectrum of 6a.

K.G. Krishnan et al. / Journal of Molecular Structure 1199 (2020) 127037
7
Tables S2eS5, respectively.
The unique stretching frequencies of aromatic
emerged from the zone in the region 3100-3000 cm [38]. In the
amino group of amide moiety resulted with strong intensity cen-
ꢀ
1
n
C-H generally
tred on 3500-3100 cm [39]. In the compounds 6a-d, a sharp band
ꢀ
1
ꢀ1
appeared in the region 3408-3332 cm attributed to the presence
experimental spectra of the compounds 6a-d, an absorption bands
of primary
n
N-H vibrations. The computationally calculated fre-
ꢀ1
ꢀ1
exhibited in the region 3190-3153 cm are owing to the aromatic
C-H stretching frequencies. These frequency values show good
conformity with the theoretically computed ones, observed at
quencies of these bands occurred at 3562-3439 cm
by DFT
n
method which is coincide well with the experimental results.
The identification of CeN stretching vibrations is more
complicated ones, since the mixing of several bands are possible in
the region [40]. The sharp intense band resulted in the region 2210-
ꢀ1
3
121-3042 cm in B3LYP calculations. The stretching frequencies
of aliphatic C-H usually arise at reasonably lesser frequencies than
those present in the aromatic rings [39]. In the experimental
spectra, the C-H stretching vibrations of ethyl/butyl/hexyl side
chains are resulted as medium and weak bands at 2960-2870 cm
The calculated alkyl C-H stretching vibrations are found in the re-
n
ꢀ1
2206 cm is due to the characteristics of nitrile group present in
the molecules. The DFT calculations gave the nitrile stretching
n
ꢀ
1
ꢀ1
.
frequencies at 2242-2241 cm . The absorption modes are found in
the region 1471-1361 cm due to the CeN stretching of the com-
pounds 6a-d, while the corresponding computed frequencies lie at
ꢀ1
n
ꢀ
1
gion 3022-2890 cm . Hence, the theoretical results are in accord
with the experimental ones.
ꢀ1
1376-1296/1235-1222/1096-1085 cm . The results reflect that the
computed frequencies are aligned with their experimental ones.
The CeS group is less polar than carbonyl links and has
considerably weaker bands in the infrared [40]. The CeS absorption
band resulted in the FT-IR spectra of compounds 6a-d at 673-
The symmetric and asymmetric stretching frequencies of aro-
matic and heteroaromatic ring systems are commonly arising in the
ꢀ1
area between 1625 and 1400 cm [38]. In the FT-IR spectra of the
compounds 6a-d, the observed C-C symmetric stretching fre-
quencies appeared between 1581 and 1568 cm . The computed
n
ꢀ1
ꢀ1
663 cm and the corresponding computed values are lie at 1029-
ꢀ
1
ꢀ1
frequencies predicted using B3LYP methods are 1593-1517 cm
,
1028/752-718/678-677/436-432 cm
.
respectively. The carbon-carbon out-of-plane and in-plane bending
The vibration frequencies belonging to CX groups (X ¼ Cl, Br and
ꢀ1
vibrations of the compounds 6a-d have been emerged at 1107-
I) usually occurred in the frequency range of 850-500 cm [39]. In
ꢀ
1
6
05 cm
while the corresponding computed ones are also
the experimental spectrum of compound 6d, the absorption zones
ꢀ
1
appeared in a similar area and the results are tabulated in Table S2-
S5, respectively.
at 542 cm
is assigned to CeBr stretching vibrations of the
molecule. The computed frequency of CeBr stretching band arise at
ꢀ1
Generally, the stretching vibrations of carbonyl are expected in
545 cm
by B3LYP/6-311G (d,p) coincided very well with the
ꢀ1
the area from 1750 to 1650 cm [38]. In the experimental case, the
amidic carbonyl group present in the compounds 6a-d gave
an absorption bands at 1691-1676 cm , while in the computational
experimental value.
C O
n ]
ꢀ
1
3
.4. NMR spectral analysis
ꢀ
1
case, the carbonyl stretching bands arise at 1720-1718 cm . These
computed ones (
counterparts.
n
C
]
O
) are in harmony with their experimental
1H NMR and 13C NMR spectra of a representative molecule 6a
are depicted in Fig. 4a and b and the spectra of other molecules
synthesized are given in Figs. S1eS3. The signals exposed in the
In general, asymmetric and symmetric stretching frequencies of
Fig. 5. 3D plots of frontier molecular orbitals of 6a-d.

8
K.G. Krishnan et al. / Journal of Molecular Structure 1199 (2020) 127037
Table 1
and low kinetic stability and, vice versa for compounds possess high
Calculated frontier molecular orbital energies of 6a-d.
energy gap [41]. The pattern of the HOMO and LUMO of the com-
pounds 6a-d, have been displayed in Fig. 5 and their energy values
are summarized in Table 1. In the compound 6a, the electron cloud
of the HOMOs is highly populated in the phenothiazine ring and
very tiny on the cyanoacrylamide motif. In contrast, the electron
transition of the LUMOs is almost positioned in the cyanoacryla-
mide moiety and slightly on the phenothiazine motif. In the case of
compounds 6b-d, the HOMOs is delocalized over whole molecule
except alkyl/amide group; however the LUMOs is mostly located on
cyanoacrylamide group. Thus, the electron distribution profile of
compounds 6a-d clearly states that the electron flow could be
efficiently transferred from phenothiazine ring to cyanoacrylamide
unit.
HOMO
LUMO
DE (eV)
6
6
6
6
a
b
c
ꢀ5.7548
ꢀ5.7488
ꢀ5.7510
ꢀ5.7244
ꢀ3.6604
ꢀ3.6601
ꢀ3.6601
ꢀ3.6425
2.0944
2.0887
2.0909
2.0819
d
shielded region of 0.88e4.01 ppm associated with triplets and
multiplets correspond to the presence of alkyl groups present in the
molecules. Two broad singlets resonated at 5.65e6.25 ppm with
each one proton integral owing to the presence of amidic NeH
protons. Multiplets observed in the region 6.97e7.82 ppm with
seven/six protons integral are due to the presence of phenothiazine
scaffold. A singlet resonated in the region 8.01e8.14 ppm corre-
sponds to methine proton attached to the olefinic carbon. In the
NMR, the signals resulted in the downfield region at
62.7e163.5 ppm are due to the presence of amidic C]O in the
The computed HOMO and LUMO energy of the compound 6a
is ꢀ5.7548 eV and ꢀ3.6604 eV, the resulting band gap energy (
of the compound 6a is 2.0944 eV. Similarly, in the compounds 6b-d,
the energy have been calculated for HOMOs
DE)
13
C
1
as ꢀ5.7488 eV, ꢀ5.7510 eV and ꢀ5.7244 eV while for LUMOs
as ꢀ3.6601 eV, ꢀ3.6601 eV and ꢀ3.6425 eV, which result the band
gap energy of 2.0887 eV, 2.0909 eV and 2.0819 eV.
molecules. The aromatic/olefinic carbons are collectively resonated
in downfield region at 103.1e152.2 ppm. Carbon signals for the
alkyl side chain present in the molecule resonated in the range of
12.8e48.0 ppm.
3.6. Molecular electrostatic potential analysis
3.5. Frontier molecular orbital analysis
Molecular electrostatic potential (MEP) is a powerful tool to
identify the electrophilic and nucleophilic reactive sites of molecule
and it is very valuable in research of molecular structure with its
relationship of physiochemical properties. A model of electrostatic
potential of the compounds 6a-d, computed at the 0.002 a.u iso-
density surface is furnished in Fig. 6. From the MEP image of the
Molecular orbitals of highest occupied and lowest unoccupied
MOs) are used to determine how the molecules interrelate with
other species. Generally, compounds containing small energy gap
exposed higher chemical reactivity, soft in nature, more polarized
(
Fig. 6. Molecular electrostatic potential surfaces of 6a-d.

K.G. Krishnan et al. / Journal of Molecular Structure 1199 (2020) 127037
9
3.8. Anticancer activity
The in vitro anticancer activity of the synthesized molecules 6a-
d have been evaluated against two human pancreatic cancer cell
lines viz., AsPC1 and SW1990. The results of cytotoxic effect of the
compounds 6a-d have been presented in Table 3. All the tested
compounds 6a-d demonstrated moderate to good in vitro anti-
cancer activities towards the tested pancreatic cancer cell lines.
Among them, the compound 6c which holds hexyl group on the
nitrogen of the phenothiazine motif exhibited the highest anti-
cancer activity against SW1990 while the compounds 6b and 6d
displayed good anticancer activity against SW1990. Besides, the
molecule 6a showed moderate anticancer activity against SW1990.
On the other hand, it was also observed that the compounds 6b, 6c
and 6d exhibited significant anticancer activity against AsPC1
pancreatic cancer cell line while the compound 6a was found to be
moderately active against AsPC1 cancer cell line. It could be infer-
red that the anticancer activity of compounds increases with the
increase of alkyl chain length attached at the nitrogen atom of the
phenothiazine ring. Based on the alkyl substitution, the anticancer
activity decreases in the order of ethyl < bromo substituted
butyl < butyl < hexyl substituent.
Fig. 7. Antioxidant activity of molecules 6a-d.
compound 6a, one can observe that the negative potential are
characterized on most electron abundant nitrile and amide
carbonyl part. The positive region is typically distributed on the
4. Conclusion
2
amide NH . Similarly, the MEP image of the compounds 6b-d, the
negative potential is polarized over the nitrile and amide carbonyl
unit. However, the light yellow region spread over the phenothia-
zine ring while the green region covers the whole molecular sur-
face. Thus, these locations provide the information about the area
from where the compound can be devised the intermolecular in-
teractions. Therefore, the most feasible reactive sites of the com-
pounds 6a-d could be involved as an electrophilic reactive site at
C1¼O4 and C22≡N23.
Synthesis of the newly designed phenothiazine based cyanoa-
crylamides 6a-d was achieved from commercially available
phenothiazine using multistep synthetic approach. The structures
of the target compounds (6a-d) were established by physical and
1
13
spectroscopic methods viz, FT-IR, H NMR and C NMR. Compu-
tational studies for the target molecules 6a-d have been performed
to realize the physiochemical properties. DPPH method has been
exploited to study the in vitro radical scavenging activity of the
privileged molecules 6a-d. Of those, the 6b and 6c exhibited high
radical scavenging activity towards DPPH. Further, the synthesized
new compounds have been subjected to in vitro anticancer evalu-
ation against a couple of pancreatic tumor cells AsPC1 and SW1990.
Among them, the molecule 6c has been identified as the most
potent one against both the tumor cells viz., AsPC1 and SW1990.
The results imply that these new compounds could serve as valu-
able intermediates for the construction of efficient radical scav-
enger/anticancer agents. In order to improve the activities further,
structural diversification of this category of molecules is under
progress.
3.7. DPPH radical scavenging activity
The in vitro radical scavenging activity of the synthesized com-
pounds 6a-d has been evaluated using 2,2-diphenyl-1-
picrylhydrazyl (DPPH) method and their results are provided in
Fig. 7 and Table 2. The data of radical scavenging activity reveals
that the synthesized compounds 6a-d exhibited moderate to
excellent activity against the reference drug ascorbic acid. Inter-
estingly, the molecule with butyl substitution on the nitrogen of the
phenothiazine core along with bromo substituent on the sixth
position of the same (6d) has found to be better one when
Table 3
In vitro anticancer activity of 6a-d.
compared to ascorbic acid, the standard antioxidant (IC50 39.07 mM)
while the molecule possessing hexyl substitution on the nitrogen of
the phenothiazine structural unit (i.e., 6c) exhibited slightly lower
S.No.
Compound
Cell viability
SW1990
AsPC1
activity (IC50 value 37.32
Besides, the privileged molecules 6a and 6b exposed comparatively
lower radical scavenging activity along with IC50 value of 32.08 and
m
M) when compared to the standard.
1
2
3
4
6a
6b
6c
6d
44.10
20.70
13.30
22.63
46.23
30.72
25.32
30.44
3
4.55 mM, respectively.
Table 2
Antioxidant activity of 6a-d.
S.No.
Compound
100
mM
50
m
M
40
m
M
30
m
M
20
m
M
10
mM
IC50 mM
1
2
3
4
5
6a
6b
6c
6d
69.49
82.23
80.30
76.44
88.22
61.77
77.41
74.51
68.53
82.62
59.45
71.62
68.91
61.77
78.18
56.37
65.63
60.42
57.14
73.35
49.61
57.91
56.75
51.54
68.14
43.43
54.82
52.12
45.94
65.05
32.21
34.56
37.32
39.08
36.97
Ascorbic acid

10
K.G. Krishnan et al. / Journal of Molecular Structure 1199 (2020) 127037
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