Novel acrylamide/acrylonitrile-tethered carbazoles: Synthesis, structural, biological, and density functional theory studies

Article abstract of DOI:10.1039/c9nj02170a

A series of novel carbazolylacrylamides/acrylonitriles 6a-6h were synthesized and their structures were established using various analytical, spectroscopic, and single-crystal X-ray diffraction techniques. The antioxidant evaluation of the target chemical entities 6a-6h was conducted by the DPPH method. The carbazolylacrylonitrile 6h displayed 65.7% radical scavenging activity (IC₅₀ value, 65.08 μM) with respect to standard ascorbic acid. The in vitro cytotoxic activity studies revealed 6e and 6h as promising anticancer molecules against a human pancreatic cancer cell line, i.e., AsPC1. Density functional theory studies of a model chemical entity 6g were performed and the results obtained were compared with those obtained experimentally. The appropriate structure, the corresponding bonding features and the vibrational frequencies for the molecule 6g were determined by employing the DFT-B3LYP method with the 6-311++G(d,p) basis set. The vibrational frequencies of the carbazolylacrylonitrile 6g calculated theoretically were found to be in good agreement with the corresponding experimental results. In addition, the chemical shifts of ¹H and ¹³C of 6g were computed by the gauge independent atomic orbital (GIAO) method and compared with the experimental ones.

Full text of DOI:10.1039/c9nj02170a

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DOI: 10.1039/C9NJ02170A.
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Chechia Hu, Tzu-Jen Lin et al.
Yellowish and blue luminescent graphene oxide quantum dots
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DOI: 10.1039/C9NJ02170A
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Novel acrylamide / acrylonitrile tethered carbazoles: synthesis,
structural, biological and density functional theory studies
Received 00th January 20xx,
Accepted 00th January 20xx
Krishnaraj Padmavathya, Kannan Gokula Krishnana, Chandran Udhaya Kumara, Ethiraj Sathiyaraja,
Ramar Sivaramakarthikeyana, Wei-Meng Limb, Chun-Wai Maib,c and Chennan Ramalingan*a
DOI: 10.1039/x0xx00000x
A series of novel carbazolylacrylamides / acrylonitriles 6a-6h has been synthesized and the structures of the same have
been established using various analytical, spectroscopic and single crystal X-ray diffraction techniques. Antioxidant
evaluation of the target chemical entities 6a-6h has been determined by DPPH method. The carbazolylacrylonitrile 6h
displayed 65.7% radical scavenging activity (IC50 value 65.08 μM) with respect to the standard ascorbic acid. The in vitro
cytotoxic activity studies revealed 6e and 6h as promising anticancer molecules against pancreatic cancer cell AsPC1.
Density functional theory studies of a model chemical entity 6g have been performed and the results obtained were
compared with experimentally obtained ones. The appropriate structure with corresponding bonding features and
vibrational frequencies for the molecule 6g have been determined by employing DFT-B3LYP method with 6-311++G(d,p)
basis set. The vibrational frequencies of the carbazolylacrylonitrile 6g, calculated theoretically have found to
good synchronization with the corresponding experimental results. In addition, the chemical shifts of 1H and 13C of 6g
have been computed by the Gauge Independent Atomic Orbital (GIAO) method and compared with their experimental
ones.
The raising death rate every year, because of cancer, and
various limitations associated with the same as mentioned has
made obvious that there is an immediate need for
Introduction
development of novel and more potent chemotherapeutic
agents that can abolish cancer cells with no harm to regular
tissues and of course that could overcome the limitations.
In this regard the planar, polycyclic, and aromatic
carbazoles are found to exhibit anticancer activity through
DNA intercalation or inhibition of DNA-dependent enzymes,
such as telomerase and topoisomerase I/II.4 It is also reported
that o-demethylmurrayanine and clausine D that belongs to a
class of carbazole alkaloids possess strong cytotoxicity against
MCF-7 and SMMC-7721 cells, with IC50 values of 2.63–7.59
mg·mL–1.5 It is noteworthy to mention that mahanine, a
carbazole alkaloid from murrayafoline induces ROS-mediated
destabilization of Hsp90 chaperone activity resulting in Hsp90-
Cdc37 disruption leading to apoptosis, signifying its potential
as a specific target in pancreatic cancer.6 Similarly, the
compound[12,13-dihydro-5[2(dimethylamino)ethyl]4Hbenzo
Poor lifestyle and defective genes cause abnormal growth of
malignant cells with high invade potential leading to cancer.
Cancer being the dreadful and most prevalent disease
worldwide which needs to be treated in early stage. It is
anticipated that global death toll from cancer is progressing to
increase (approximately 12 million deaths by 2030). In
particular, the fatal rate due to pancreatic cancer is found to
be reasonably higher due to its chemoresistence and high
invasive nature.1 It has been reported that in USA, the
pancreatic cancer is the fourth leading cause for cancer death.2
Numerous therapies such as surgery, radiation therapy, target
therapy, chemotherapy, hormonetherapy, immunotherapy,
precision medicine and stem cell transplant have been
emerged,3 however, there exist various limitations like
efficacy, high cost, multidrug resistance and side effects
associated with drugs.
[c]pyrimido[5,6,1-jk]carbazole-4,6,10(5H,11H)-trione
hydro
chloride inhibited topoisomerase II activity at 2.5μM
concentration which accounts to be 10 times greater inhibitory
effect than etoposide and the average growth inhibitory effect
against 21 human tumor cell lines were found to be GI50 =59
nm which is twenty times greater potent than etoposide.7 In
fact it has been reported that N-substituted carbazole
imidazolinium salt holding a 2-bromobenzyl substituent at
position-3 of the 5,6-dimethylbenzimidazole, showed powerful
inhibitory activities and was more selective to HL-60, SMMC-
7721, MCF-7 and SW480 cell lines with IC50 values of 0.51–2.48
a.Department of Chemistry, School of Advanced Sciences (SAS), Kalasalingam
Academy of Research and Education (Deemed to be University), Krishnankoil - 626
126, Tamilnadu, India.
E-Mail: ramalinganc@gmail.com; c.ramalingan@klu.ac.in
b.School of Pharmacy, International Medical University, 126 Jalan Jalil Perkasa 19,
57000, Bukit Jalil, Kuala Lumpur, Malaysia
.
c. 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.
Electronic Supplementary Information (ESI) available:SeeDOI: 10.1039/x0xx00000x
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μM.8 The in vitro anticancer activity of compounds 2-[2-{4- functionalities together in target molecules.
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DOI: 10.1039/C9NJ02170A
(dimethylamino)phenyl}-4,5-dihydroimidazol-1-yl
amino]-1-
In this article, synthesis of
a
series of novel
(9H-carbazol-9-yl)ethanone and 2-(9H-carbazol-9-yl)-N'-[{(4- carbazolylacrylonitriles 6a-6h by employing Knoevenagel
hydroxyphenyl)(piperazin-1-yl)]methyl}aceto-hydrazide against condensation as a key step has been explored. Since it is
human breast cancer cell line (MCF 7) by sulphorodamine B inferred that the reactive oxygen species may affect the
(SRB) assay method were found to be more potent with cellular components which cause cancer, the plausible radical
GI<10μg.ml-1.9 Specifically, in pancreatic cancer, over scavenging activities of the synthesized compounds were
expression of heat proteins (Hsp70, Hsp90), constitutive determined first by in vitro DPPH method. Further, the
activation of NFĸB, and BCL-2 family are closely linked with anticancer evaluation of all the synthesized molecules were
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resistance to apoptosis.10
On the other hand, there are several reports describe the and AsPCl by Cell-Titre Glo Luminescent Cell Viability Assay.
nitrile integrated molecules and their biological significances: Besides, the chemical entity 6g, a representative one
also carried out against human pancreatic cancer cells SW1990
for example gossylic nitrile 1,1'-divalerate shows antimalarial among the synthesized targets, has been subjected to Density
profile11 comparable to gossypol itself; several alkene nitriles Functional Theory (DFT) calculations utilizing B3LYP/6-
are already in use for treating maladies namely vidaliptin12; 311++G(d,p) basis set to gain structural parameters of its
trilostane has been fruitfully used for treating breast cancer in optimized structure. In addition, the structural parameters of
post-menopausal women. 13 Lanoconazole14 and luliconazole15 the optimized structure, nuclear magnetic resonance chemical
are also used as topical antifungal drugs. The biocompatibility shift values, and vibrational assignments, acquired from
of the nitrile supports the predominance of the nitrile computational studies, are compared with their experimental
pharmaceuticals and their continued clinical trials.16 They are results.
characterized by their short, polarized triple bond. Only a
minimum steric demand, a cylindrical diameter of 3.6 Å, is
necessary for the same when compared to other groups
Results and discussion
Further, nitriles play a vital role as hydrogen bond acceptors
due to its polarisability / electron richness on the nitrogen
atom of the nitrile core.17
Synthesis
A series of novel bio-pertinent carbazolylacrylonitriles
acrylamides 6a-6h has been constructed by a stepwise
/
synthetic protocol, displayed in Scheme 1.
Condensation reaction between carbaldehydes 4a-4b
synthesized by alkylation of carbazole with respective alkyl
The huge ranges of applications exhibited by carbazole and
nitrile group have stimulated us to design and synthesize a
series of novel chemical entities possessing both
,
halide in the presence of potassium tert-butoxide in
Scheme 1 Synthetic methodology for the construction of carbazolylacrylonitriles 6a-6h.
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tetrahydrofuran followed by Vilsmeier-Haack reaction and, moieties, respectively. The carbazolylacrylonitrileVsiew6Abrt-i6clde Oanlninde
substituted acrylonitriles afforded the target molecules 6a-6h 6f-6h exerted distinct bands in the region 2207~2214 cm ,
in excellent yields.
assigned to CN functionality of the molecules. The 1H-NMR
-1
DOI: 10.1039/C9NJ02170A
To investigate the feasibility and optimization of the spectra of compounds 6a-6h exhibit a set of signals that
protocol, the reaction was performed at various conditions appeared in the downfield region of 7.30-8.79 ppm reveal the
and is listed in Table 1. We initially performed a model presence of carbazole ring and other aryl part in the molecule.
reaction between equimolar amount of carbaldehyde 4a and Four set of signals resonated in the region of 0.83-4.37 ppm
cyanoacetamide 5a in acetonitrile at ambient temperature. with nine and thirteen protons integral correspond to the butyl
When the reaction was performed at ambient temperature, and hexyl side chains of the chemical entities. These key
there was no product obtained. Addition of p-toluenesulfonic characteristic bands/signals undoubtedly substantiate the
acid as a catalyst provided trace amount of the product at structure of the target molecules 6a-6h. In addition, the single
ambient temperature. Increasing the temperature to reflux crystal XRD of the molecule 6g further confirms the proposed
condition offered the product in 65% yield. With a view to structure.
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increase the yield of the product further, the reaction was
performed with other solvents such as dichloromethane, N,N-
Table 2. Products of the target molecules 6a-6h and their yields.
Compound
R1
R2
Yield (%)
95
97
93
93
96
98
94
95
dimethylformamide, and ethanol at reflux conditions. When
the use of ethanol provided comparable (slightly lower) yield,
utilization of dichloromethane and N,N-dimethylformamide
offered considerably lower yields when compared with the use
of acetonitrile. On the other hand, replacing the catalyst p-
toluenesulonic acid by piperidine with the utilization of
acetonitrile as a solvent under reflux condition furnished the
product in excellent yield. In addition, change of solvents from
acetonitrile to dichloromethane, N,N-dimethylformamide and
ethanol by keeping the other conditions as similar provided
the product in good yield although lower than that of the one
obtained with the utilization of acetonitrile. Hence, the
reaction between equimolar amount of carbaldehyde 4a and
cyanoacetamide 5a in the presence of catalytic amount of
piperidine in acetonitrile under reflux condition was found to
be the optimal one.
6a
6b
6c
6d
6e
6f
Butyl
Butyl
Butyl
Butyl
Hexyl
Hexyl
Hexyl
Hexyl
-CO-NH2
4-NO2-C6H4-
-SO2-Ph
-Bmim
-CO-NH2
4-NO2-C6H4-
-SO2-Ph
-Bmim
6g
6h
Structural Analysis
Table 1. Optimization of the model reaction (construction of 6a).
Solvent
MeCN
MeCN
MeCN
DCM
Catalyst (15 mol%)
None
p-TSA
p-TSA
p-TSA
Temp. (°C)
Ambient
Ambient
Reflux
Yield (%)
No reaction
Trace
65
30
Reflux
DMF
p-TSA
Reflux
50
EtOH
MeCN
DCM
DMF
EtOH
p-TSA
Reflux
Reflux
Reflux
Reflux
62
95
70
75
Piperidine
Piperidine
Piperidine
Piperidine
Reflux
85
The scope of the reaction has been examined with
carbaldehydes 4a-4b and substituted acrylonitriles 5a-5d
which gave the desired products with excellent yields. The
aforesaid reactions imply that incorporation of various
structural units such as sulfonyl, benzyl, heteroaryl and amide
on the nitrile reactant with active methylene moiety does not
significantly affect the yields of the products as shown in Table
2. The structure of all the synthesized compounds 6a-6h has
been established using various analytical and spectral
techniques. FT-IR Spectra of molecules 6a and 6e showed
absorption bands at 3298~3416, 2201~2207, and 1653~1683
cm-1, correspond to the presence of NH2, CN and C=O
Figure 1 a) ORTEP drawing of 6g with the atom numbering scheme and
thermal ellipsoids drawn at 50 % probability for every atom other than
hydrogen, b) Optimized molecular structure of 6g.
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Unambiguous confirmation of the structure of indicated by their bond distances [S5-O6 = 1.429 / 1.467 and
carbazolylacrylonitrile 6g has been resulted from a single- S5-O7 = 1.433 / 1.462 Å]. An intramolecular hydrogen bonding
crystal X-ray analysis. Attempts on crystallization of other interactions are observed with C(9)-H(34)· · · N(4)-C(1) = 2.666
synthesized molecules to obtain single crystals failed to Å (Figure 2). Most of the optimized bond lengths were in
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DOI: 10.1039/C9NJ02170A
provide the same. The experimental molecular structure of 6g
,
excellent agreement with experimental values.
obtained by X-ray crystallography with the atom numbering
scheme and the computationally optimized molecular
structure of the same are shown in Figure 1. The packing
diagram of the molecule 6g is depicted in Figure 2. The
carbazolylacrylonitrile 6g crystallized in the monoclinic with
P21/c space group and contains four molecules per unit cell
with a = 7.5621 Å (5), b = 19.4912 Å (15), c = 16.1178 Å (12)
unit cell dimensions. The crystal packing is governed by C-
H••••N≡C bonding interactions. Crystal data and structure
refinement details are listed in Table 3. The selected
theoretical and experimental bond lengths, bond angles and
selected torsion angles of carbazolylacrylonitrile 6g are
presented in Table S1. The chemical entity 6g consists of two
ring structural units, namely benzene and N-alkylated
carbazole. The theoretical values obtained by DFT B3LYP-
6311++G (d,p) method have been compared with the
experimental ones.
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Table 3. Crystal data and structure refinement parameters of 6g.
Empirical formula
Formula weight
C27 H26 N2 O2 S
442.17
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Temperature
296(2) K
Wavelength
0.71073 Å
Crystal system, space group
Monoclinic, P 21/c
a = 7.5621(5) Å α = 90
b = 19.4912(15) Å β = 98.666(3)
c = 16.1178(12) Å γ = 90
2348.6(3) Å3
Unit cell dimensions
Volume
Z, Calculated density
Absorption coefficient
F(000)
4, 1.158 Mg/m3
0.071 mm-1
828
Crystal size
Theta range for data collection
0.250 x 0.160 x 0.120 mm
2.090 to 24.998
-8<=h<=8, -23<=k<=23, -
19<=l<=19
Limiting indices
Reflections collected / unique
Completeness to theta = 25.242
Refinement method
Data / restraints / parameters
Goodness-of-fit on F2
39121 / 4129
97.4 %
Full-matrix least-squares on F2
4129 / 0 / 290
1.053
Final R, RW (obs, data)
0.0563, 0.1322
Vibrational Analysis
The carbazolylacrylonitrile 6g holds 58 atoms and has 168
normal modes of vibrations. All these 168 modes of vibrations
are distributed as 61 stretching, 52 bending and 55 torsional
vibrations. The experimental and computational FT-IR spectra
of carbazolylacrylonitrile 6g are furnished in Figure 3. The
experimental and calculated scaled frequencies as well as the
potential energy distribution (PED) are listed in Table S2. The
PED calculation of the chemical entity 6g has been carried out
using VEDA program.18 The major descriptions of vibrational
assignments for the carbazolylacrylonitrile 6g are given below.
C-H Vibrations. The presence of aromatic rings have
obviously been recognized by the C-H stretching vibrations in
the region 3100-3000 cm-1. As consistent with these, C-H
stretching vibrations of the molecule 6g are resulted at 3176-
3059 cm-1 in the experimental FT-IR spectrum while the same
are observed in the region 3247-3183 cm-1 in the
computational calculations. Consequently, the experimental
and computational C-H stretching vibrations are in good
harmony with each other. In alkyl moieties, the C-H stretching
vibrations generally arise at the frequencies lower than that of
an aromatic region i.e., 2970-2840 cm-1. In the present
experimental study, weak and medium bands originated in the
region 2920-2848 cm-1 are characteristics of C-H stretching
vibrations. These C-H absorption bands are theoretically
resulted in the region 3120-3025 cm-1. The experimental and
computed assignments are in good accord with each other.
Figure 2 Packing diagram of compound 6g.
The torsion angle C1-C2-C3-C18 obtained by X-ray and DFT
are 2.33° and -0.5°, respectively which shows that both
carbazole ring and cyano group are on the same plane. The
bond angle C2-S5-C8 observed from experimental and
theoretical observations are 102.5° and 103.9°, respectively
indicating both phenyl ring and cyano group are nearly
perpendicular to each other. The C-N bond lengths for C1-N4
and C27-N14 are different because of the difference in their
environment and also assume a triple-bond character for the
(XRD/DFT) C1≡N4 = 1.141 / 1.158 Å and single-bond character
for the (XRD/DFT) C27-N14 = 1.448 / 1.457 Å. In addition, the
S-O bonds, S5-O6 and S5-O7 are double bonded in nature as
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C-C Vibrations. Normally, characteristic bands resonate in the case of 6g
,
the C-N stretching vibration resulted
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the region 1650-1400 cm-1 in the FT-IR spectrum are experimentally at 2208 cm-1 while the computationally
correspond to stretching frequencies of molecules owning calculated stretching vibration appeared at 2331 cm-1. The
saturated and unsaturated carbon-carbon bonds. In the pure modes of C-N stretching bands calculated at this level
present experimental case, strong absorption bands found in spectacles 89 % of PED contribution. The experimentally
the region 1577-1514 cm-1 are assigned to carbon-carbon obtained FT-IR results are coherent with their theoretically
stretching frequencies. The computationally calculated carbon- achieved ones.
carbon stretching frequencies fall in the region 1615-1545
cm-1, which are correlated well with the experimental ones.
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UV-Vis Analysis
Also, the carbon-carbon-carbon (C-C-C) in-plane and out-of Theoretical UV-Vis spectra of carbazolylacrylonitrile 6g both in
plane bending modes of vibrations of the examined molecule gas and solvent phase (DMSO) have been acquired by TD-DFT
6g are furnished in Table S2.
method with B3LYP/6-311++G(d,p) basis set. Their appropriate
parameters such as wavelengths, excitation energies, band gap
energies and oscillator strengths are provided in Table 4. The
experimental UV-Vis spectrum of the carbazolylacrylonitrile 6g
has been recorded in DMSO. The UV-Vis spectra of the
carbazolylacrylonitrile 6g both experimental and computed are
displayed in Figure 4.
In the experimental UV-Vis spectrum, three distinctive
absorption bands appeared at 408, 335, 293 nm correspond to
n-π*, intramolecular π-π* and π-π* transitions, respectively.
The experimentally resulted n-π* and π-π* transitions are in
accord with the corresponding transitions obtained from
computations.
Figure 3 Experimental and simulated FT-IR spectra of 6g.
SO2 Vibrations. The symmetric and asymmetric S=O
stretching of SO2 usually occur in the region 1325-1140 cm-1.
The characteristic bands noted at 1232-1197 cm-1 are assigned
to S=O stretching which infact in good agreement with the
calculated frequencies of 1256 and 1088 cm-1. The PED of
these signals reveals that the calculated frequency has 80 and
44 % of S=O stretching vibration.
C-N Vibration. The C-N stretching frequencies of the
majority of nitriles fall in the region around 2300-2200 cm-1. In
Figure 4 Experimental and simulated UV-Vis spectra of 6g.
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Table 4 Electronic transition of compound 6g in gas and solution phase.
unoccupied MOs. The HOMO-LUMO profile clearlVyierweAflrteiccletsOntlhinee
migration of electrons from the carbazole structural motif to
DOI: 10.1039/C9NJ02170A
Expt.
λ (nm)
--
Calculated
E (eV)
Solvent
Gas
λ (nm)
f
the sulfonyl functionality in the molecule. The calculated
energy difference between the HOMO and LUMO is 2.6395 eV
as the energy level of HOMO is -8.0928 eV while the same of
LUMO is -5.4533 eV. The small energy difference between the
HOMO and LUMO reflects the more polarizable nature of the
molecule and is generally associated with low kinetic stability,
high chemical reactivity, and soft in nature.
407.92 (116→118)
365.32 (116→118)
305.80 (115→118)
408.61 (116→118)
365.65 (116→118)
306.16 (115→118)
3.0394
3.3938
4.0544
3.0343
3.3907
4.0496
0.4213
0.2540
0.3997
0.4432
0.2533
0.4064
DMSO
408
335
293
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Further, the lower energy difference would facilitate the
electron density more on the sulfonyl unit in 6g, consequently,
due to the presence of heteroatoms such as sulfur and two
oxygens in the sulonyl group as well as more electron density
on this structural unit (due to lower band gap) would further
enhance the binding affinity with the proteins which intern
would culminate with good biological response.
HOMO-LUMO Analysis
Table 5. Calculated molecular orbitals energies of 6g.
Parameter
HOMO
LUMO
ΔE
Energies (eV)
-8.0928
-5.4533
2.6395
HOMO-1
LUMO+1
ΔE1
-8.8466
-5.0560
3.7906
Molecular Electrostatic Potential Analysis
Figure 5 3D plots frontier molecular orbitals of 6g.
It is known that both HOMO and LUMO are noteworthy
quantum chemical factors to involve in the kinetic stability and
chemical reactivity of a molecule. The HOMO - LUMO energy
difference is a critical factor in deciding the excitability of a
molecule. Normally, molecules possess larger energy
difference imply lower chemical reactivity while smaller energy
difference reveal higher chemical reactivity.
The theoretical findings of highest occupied molecular
orbitals and lowest unoccupied molecular orbitals of the
chemical entity 6g have been obtained utilizing B3LYP/6-
311++G (d,p) basis set. 3D plots of frontier molecular orbitals
are sketched in Figure 5 and the energy values are presented
in Table 5. In the highest occupied MOs, the electron density
over the carbazole structural motif is extremely larger while
the same is larger over the sulfonyl unit in the case of lowest
Figure 6 Molecular surfaces obtained using B3LYP/6-311++G(d,p) level of 6g.
In order to understand the molecular interactions, charge
distributions and charge related properties of molecules, the
MEP analysis has been carried out. Figure 6 represents the 3D
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plots of electrostatic potential distribution, computed at 0.02 ppm with six protons integral and two protons integral,
View Article Online
au isoelectronic surface, of carbazolylacrylonitrile 6g. Regions respectively. The experimentally obtainDeOdI: t1h0.e1s0e39/sCig9nNaJ0ls21a70reA
of negative V(r) are usually associated with the lone pair of matched well with the corresponding signals resulted from the
electronegative atoms. The color code of the map has range computational calculations.
from -0.110 (dark red) to 0.110 (dark blue). A characteristic
In the experimental 13C NMR spectrum, the carbon signals
manifestation of the positive electrostatic potential has been of carbazole and phenyl structural units fall in the downfield
observed around the sulphonyl and nitrile units while the region between 108.9 and 143.7 ppm. These experimentally
negative electrostatic potential spread over the nitrogen atom attained signals are in-line with the computationally resulted
of carbazole structural motif. Further, the light yellow region ones. The methyl carbon signal of the hexyl side chain
extended over the center of the carbazole while the green resonated in the extreme upfield region of 13.9 ppm while the
region covered the molecular surface, reflecting the existence methylene carbon connected to the nitrogen of the carbazole
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of intermolecular interactions.
observed in the extreme downfield region (43.5 ppm) among
the carbons of the hexyl side chain. Besides, the carbons of
other methylene groups exhibit signals between 22.5 and 31.4.
Nuclear Magnetic Resonance Analysis
Theoretical NMR calculations of the representative All these signals obtained from the experimental spectrum are
carbazolylacrylonitrile 6g have been carried out using DFT- in accord with the computationally calculated ones. Further,
1
B3LYP with 6-311++G(2d,p) basis set. The experimental H and the signal for olefinic carbon experimentally resonated in the
13C NMR spectra of 6g and their corresponding chemical shift downfield region 152.7 ppm while the characteristic signal for
values are furnished in Figures 7 & 8 and Table S3, this carbon observed at 149.9 ppm through computational
respectively.
calculation, consequently, the experimental and theoretical
results are in line with each other.
Figure 7 1H NMR spectrum of 6g.
Figure 8 13C NMR spectrum of 6g.
In the experimental 1H NMR spectrum, the two singlets
resonated in high frequency region 8.65 and 8.37 ppm with
each one proton integral correspond to the olefinic proton and
proton (H41) attached at C19 carbon of carbazole unit. The
corresponding computed chemical shifts of these protons are
in good agreement with the experimental ones. The signals for
aromatic protons of carbazole and phenyl structural units are
collectively acquired in the region 7.31-8.13 ppm and these
chemical shift values are in coherence with the computed
ones. A sharp triplet appeared in the extreme upfield region of
0.85 ppm with three protons integral reveals the presence of
methyl protons of terminal methyl moiety of hexyl side chain
and this methyl protons signal is in harmony with the
computed one. Besides, the protons attached to the carbon
connected to the nitrogen of carbazole structural moiety
resonated in the downfield region (4.31 ppm) among the
methylene signals as a triplet with two protons integral and
rest of the protons of four methylene groups of hexyl unit
appeared as multiplets in the regions 1.24-1.36 and 1.83-1.90
Antioxidant Evaluation
It is known that reactive oxygen species has direct relation
with the cellular components: Normally, reactive oxygen
species (ROS) hinders the function of various cellular
components leading to numerous diseases associated with
inflammations and cancer.19,20 Like superoxide dismutase
(SOD), antioxidants are agents scavenging the free radicals
produced in our system. There are plethora of antioxidants
present in nature yet the identification of drug with potential
antioxidant activity becomes indispensable to meet the
requirements.
There are various in-vitro methods being adapted for
determination of radical scavenging activity including Ferric
Reducing Antioxidant Power, hydroxyl radical scavenging
assay, organic radical scavenging assay methods. Among all
methods, organic radical scavenging by DPPH is simplest one.
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Hence, all synthesized carbazolylacrylonitriles/acrylamides 6a- anticancer evaluation against a couple of human pancreatic
6h have been evaluated against a reactive oxygen species, 2,2- cancer cell lines such as AsPC1 and Sw1990, human non-
diphenyl-1-picrylhydrazyl (DPPH). The activity of free radical cancerous lung fibroblast cells (MRC5) and human non-
scavenging has been estimated as percentage of inhibition and cancerous retinal endothelial cells (ARPE19) using Cell-Titre
the results with respect to the standard ascorbic acid are Glo Luminescent Cell Viability Assay.
furnished in Figure 9 and Table 6.
View Article Online
DOI: 10.1039/C9NJ02170A
Table 7. Anticancer activity of target chemical entities 6a-6h
.
IC50 (µM)
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Compound
MRC5
84.20 ±
5.91
ARPE19
>100
SW1990
>100
AsPC1
>100
6a
6b
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
89.37 ±
0.17
>100
6c
75.55 ±
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>100
6d
94.33 ±
5.11
3.52 ±
0.15
83.33 ±
3.09
95.99 ±
9.11
6e
>100
6f
>100
99.12 ±
3.33
89.01 ±
2.22
91.09 ±
7.19
>100
6g
>100
>100
>100
Figure 9 Antioxidant activity of 6a-6h.
6h
6.89 ±
0.69
Table 6. Antioxidant activity of 6a-6h
.
Compound
% inhibition w.r.t AA
IC50 (μM)
94.6
Gemcitabine
35.09 ±
1.78
39.27 ±
4.44
24.17 ±
0.20
17.55 ±
2.26
6a
6b
6c
6d
6e
6f
6g
6h
AA
60.14
61.91
59.73
63.62
60.30
58.60
60.79
65.70
100.00
87.09
101.9
72.36
59.23
37.33
52.74
65.08
169.00
The outcomes obtained against the target chemical entities
6a-6h as well as gemcitabine (positive control) are provided in
Table 7. As shown in Table 7, the IC50 values of all the chemical
entities are greater than 100 µM against the pancreatic cancer
cell line SW1990 irrespective of the substituents / structural
motifs while the same of the standard gemcitabine is ~36 µM.
However, against the cell lines AsPC1, the IC50 values of the
chemical entities possessing primary amide and hexyl
substituents on the nitrogen of the carbazole (6e) and
benzimidazole scaffold along with hexyl substituent on the
nitrogen of the carbazole (6h) are nearly 4 µM and 7 µM,
respectively while the IC50 value of other molecules are greater
than the same. On comparing the inhibition concentration of
6e and 6h against the human pancreatic cancer cell line AsPC1,
the inhibition concentration of the same against the tested
non cancerous cells such as MRC5 and ARPE19 are much
higher. Further, it has to be noted that the IC50 values of the
chemical entities 6e and 6h are much lower than the standard
drug, gemcitabine which reflect that these chemical entities
could serve as better candidates for further evaluation /
development of better drugs. Evaluation of anticancer
activities against other types of cancer cells is under the way.
The carbazolylacrylamide 6a with n-butylcarbazole, primary
amide and cyano structural units showed 60% inhibition of DPPH
radical. In 6a, incorporation of benenesulfonyl (6c) and p-
nitrophenyl as well as hexyl (6f) structural motifs in place of primary
amide / butyl unit slightly reduced the inhibition. On the other
hand, integration of p-nitrophenyl (6b) and benzimidazolyl (6d)
motifs instead of primary amide moiety in 6a enhanced the activity.
Besides, replacing the butyl group by hexyl group along with or
without incorporation of benzenesulonyl and benzimidazolyl
scaffolds in 6a (i.e, in 6e, 6g and 6h) also increased the activity. The
carbazole structure knitted with benzimidazole ring on the
acrylonitrile (6h) exhibited highest inhibition activity of DPPH
radical (65.7%) with IC50 of 65.08 μM.
Anticancer activity
Pancreatic cancer is one of the incurable diseases and the rate
of death is reasonably higher in these days. Since the
molecules synthesized are new ones, we firstly initiated with
evaluating these molecules against pancreatic cancer cells
although those could also be evaluated against other types of
cancer cells. All the synthesized acrylamide / acrylonitrile
tethered carbazoles 6a-6h have been subjected to in vitro
Conclusion
Syntheses of novel carbazolylacrylamides / acrylonitriles 6a-6h
have been accomplished through multistep synthetic strategy.
Structural analysis of 6g, chosen as a representative chemical
entity, has been performed using single crystal X-ray
diffraction technique. Besides, DFT studies of 6g have been
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executed using DFT-B3LYP method with 6-311++G(d,p) basis mixture, alkylcarbazole (0.02 mmol) in DMF (50 mVLi)ewwAartsiclaedOdnelinde
DOI: 10.1039/C9NJ02170A
set and compared with the experimental results. The drop wise. The reaction mixture was then heated at 90 °C with
1
theoretically resulted vibrational frequencies as well as H and stirring for 12 h. The progress of the reaction was checked
13C chemical shifts are in comparable agreement with those of through TLC. After completion of the reaction, the mixture was
the experimentally resulted ones. Of the chemical entities cooled to room temperature and extracted with
synthesized, carbazolylacrylonitrile 6h exerted best radical dichloromethane (3 x 20 mL), washed with water and dried
scavenging profile with an IC50 value of 65.08 μM while over anhydrous sodium sulphate. The crude product thus
adopting DPPH method. Further, among the novel molecules, obtained, after removal of the solvent under reduced
the chemical entities 6e and 6h showed promising anticancer pressure, was purified through column chromatography to
properties with IC50 values of ~4 and 7 µM, respectively against obtain the carbaldehydes as white solids.21
the pancreatic cancer cell AsPC1. Exploration of anticancer
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Synthesis of carbazolylacrylamide / acrylonitriles (6a-6h)
activities against other types of cancer cells as well as
structural diversification of most active molecules with a view
to enhance the anticancer properties against the tested
pancreatic cancer cells are under investigation.
Synthesis of carbazolylcyanoacrylamide 6a
To a stirred solution of carbaldehyde 4a (0.5 g, 2 mmol) and
cyanoacetamide (0.16 g, 2 mmol) in acetonitrile (15 ml), was
added catalytic amount of piperidine (15 mol%). The reaction
mixture was refluxed for 4 hours. The completion of the
reaction was confirmed by TLC. The solid precipitated out
Experimental
Chemistry
upon cooling was filtered and recrystallized from acetonitrile.
The pure compound 6a was obtained as yellow solid. Yield:
0.59 g (95%); M.P 210-211 °C. FT-IR (KBr, cm-1): 3412 (NH),
3300 (NH), 3176-2868 (C-H aromatic & aliphatic), 2207 (CN),
1684 (C=O), 1576, 1499, 1468, 1437, 1381, 1341, 1315, 1261,
1215, 1192, 1155, 1070, 951, 876, 798, 745, 723, 665, 617,
567, 519, 455, 420; 1H NMR (δ, CDCl3, 400 MHz): 8.73 (d, 1H, J=
1.7Hz), 8.51 (s, 1H), 8.15-8.17 (m, 2H), 7.56-7.45 ( m, 3H), 7.31-
7.35 (m, 1H), 6.31 (brs, 1H), 5.69 ( brs, 1H), 4.34 (t, 2H, J=7.2
Hz), 1.46-1.37 (m, 2H), 1.91 -1.84 (m, 2H), 0.96 (t, 3H, J=7.4Hz);
13CNMR (δ, CDCl3, 100 MHz): 163.0, 155.02, 143.2, 141.1,
128.8, 126.9, 125.0, 123.5, 122.7, 122.6, 120.9, 120.5, 118.5,
109.4, 97.7, 43.2, 31.1, 20.5, 13.8; EIMS(m/z): 317.3808 (M+).
Similar procedure was adopted for the synthesis of target
molecules 6b-6h.
The reagents were obtained from viable suppliers and utilized
with no further purifications. All reactions were executed
under standard conditions. Melting points of the products
obtained were checked with the help of SIGMA melting point
instrument and are uncorrected. Progress of the reactions and
completion of the same were observed by TLC on pre-coated
Merck aluminium sheets using hexane and ethyl acetate as
eluents. FT-IR spectra (KBr) were measured using Shimadzu
IRTRACER-100 spectrometer. 1H and 13C NMR spectra were
recorded in CDCl3 on a 400 MHz Bruker NMR spectrometer
using tetramethylsilane (TMS) as an internal standard. Mass
spectra were acquired using JEOL GCMATE II GC-MS and the
data were determined by electron ionization method. Single
crystal XRD was recorded in Bruker X8 KAPPA APEX II. The
structures of known compounds were confirmed with their
melting points reported in the literature.
Synthesis of carbazolylacrylonitrile 6b
The carbaldehyde 4a (0.5 g, 2 mmol) was reacted with nitrile
5b (0.32 g, 2 mmol) to form carbazolylacrylonitrile 6b as
orange solid. Yield: 0.76 g (97%). M.P. 200-202 °C; FT-IR (KBr,
cm-1): 3032-2862 (C-H aromatic & aliphatic), 2207 (CN),1572,
1514, 1466, 1389, 1335, 1231, 1132, 924, 847, 800, 745, 687,
Synthesis of alkylcarbazoles 3a and 3b
In a two necked RB flask (250 mL), carbazole (0.03 mmol) was
dissolved in dry THF (50 mL) and allowed to cool to 0 °C. To
this solution, potassium tert-butoxide (0.03 mmol) was added
and the content was stirred at the same temperature for 20
min. n-Alkyl bromide (0.045 mmol) was then added to the
reaction mixture in drop wise through a pressure equalizer for
20 min. Then it was allowed to attain room temperature and
the reaction was continued at the same temperature for
another 24 hrs. After completion of the reaction, the mixture
was extracted using dichloromethane (3 x 15 mL) and dried
over anhydrous sodium sulphate. Evaporation of the extracted
solution under reduced pressure followed by purification
through column chromatography eventually furnished the
pure products as white solids.21
1
625, 420; H NMR (δ, CDCl3, 400 MHz): 8.70 (d, 1H, J= 1.8 Hz),
8.32-8.15 (m, 4H), 7.88-7.85 (m, 3H), 7.56-7.32 (m, 4H), 4.35 (t,
2H, J=7.2 Hz), 1.93-1.87 (m, 2H), 1.55-1.40 (m, 2H, J=7.6Hz),
0.97 (t, 3H, J=7.4 Hz); 13C NMR (δ, CDCl3, 100 MHz): 147.3,
146.6, 142.3, 141.6, 141.1, 127.6, 126.8, 126.2, 124.3, 124.0,
123.5, 123.4, 122.7, 120.8, 120.2, 118.4, 109.4, 109.3, 104.7,
43.2, 31.1, 20.5, 13.8; EIMS(m/z): 395.4504 (M+).
Synthesis of carbazolylacrylonitrile 6c
The carbaldehyde 4a (0.5 g, 2 mmol) was reacted with nitrile
5c (0.36 g, 2 mmol) to form carbazolylacrylonitrile 6c as
fluorescent green solid. Yield: 0.77 g (93%). M.P. 280-281 °C.
FT-IR (KBr, cm-1): 3076-2874 (C-H aromatic & aliphatic), 2208
(CN), 1627, 1562, 1499, 1468, 1393, 1346, 1321, 1236, 1213,
1152, 1088, 1020, 943, 885, 810, 739, 687, 615, 579, 529, 490,
Synthesis of carbaldehydes 4a and 4b
In a two-necked flask (100 mL), DMF (1.5 mL) was taken under
nitrogen atmosphere. POCl3 (1.9 ml, 20.32 mmol) was then
added drop wise at 0 °C and stirred for half an hour. To this
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(s, 1H), 8.12-8.04 (m, 4H), 7.57-7.52 ( m, 4H), 7.46-7.43 (m, FT-IR (KBr, cm-1): 3059-2851 (C-H aromatic & aliphatic), 2208
View Article Online
DOI: 10.1039/C9NJ02170A
2H), 7.35-7.31 (m, 1H), 4.32 (t, 2H, J=7.2 Hz), 1.89-1.84 (m, 2H), (CN), 1627,1560, 1497, 1468, 1441, 1393, 1331, 1314, 1231,
1.41-1.33 (m, 2H), 0.94 (t, 3H, J=7.3Hz); 13C NMR (δ, CDCl3, 100 1231, 1196, 1155, 1080, 1018, 945, 880, 849, 800, 781, 762,
1
MHz): 152.6, 143.8, 141.1, 139.0, 134.1, 129.5, 128.8, 128.3, 745, 716, 687, 581, 532, 419 ; H NMR (δ, CDCl3, 400 MHz):
127.2, 125.5, 123.6, 122.6, 121.1, 120.9, 120.8, 114.5, 109.6, 8.65 (d, 1H, J= 1.6Hz), 8.37 (s, 1H), 8.13-8.04 (m, 4H), 7.70-7.31
109.0, 43.3, 31.0, 20.5, 13.8; EIMS (m/z): 414.5204 (M+).
(m, 7H), 4.31 (t, 2H, J=7.2 Hz), 1.90-1.83 (m, 2H, J=7.6Hz), 1.36-
1.24 (m, 6H), 0.85 (t, 3H, J=7.0Hz); 13C NMR (δ, CDCl3, 100
MHz): 152.6, 143.7, 141.1, 139.0, 134.1, 129.5, 128.7, 128.3,
Synthesis of carbazolylacrylonitrile 6d
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The carbaldehyde 4a (0.5 g, 2 mmol) was reacted with nitrile 127.1, 125.5, 123.6, 122.6, 121.1, 120.9, 120.8, 114.5, 109.6,
5d (0.31 g, 2 mmol) to form carbazolylacrylonitrile 6d as buff 109.5, 109.0, 43.5), 31.4, 28.9, 26.8 22.5, 13.9; EIMS(m/z):
colored solid. Yield: 0.72 g (93%). M.P. 140-142 °C; FT-IR (KBr, 442.5706 (M+).
cm-1): 3061-2868 (C-H aromatic & aliphatic), 2214 (CN), 1622,
Synthesis of carbazolylacrylonitrile 6h
1535, 1581, 1497, 1470, 1427, 1393, 1350, 1319, 1275, 1231,
1213, 1161, 1130, 1090, 1024, 1005, 966, 924, 810, 743, 766, The carbaldehyde 4b (0.5 g, 1.8 mmol) was reacted with nitrile
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621, 505, 422; H NMR (δ, CDCl3, 400 MHz): 9.48 (s, 1H), 8.79 5d (0.28 g, 1.8 mmol) to form carbazolylacrylonitrile 6h as buff
(d, 1H, J= 1.8Hz), 8.69 (s, 1H), 8.20-8.16 (m, 2H), 7.81-7.31 (m, colored solid. Yield: 0.71 g (95%). M.P 203-204 °C; FT-IR (KBr,
8H), 4.35 (t, 2H, J=7.2 Hz), 1.93-1.85 (m, 2H), 1.57-1.40 (m, 2H), cm-1): 3057-2855(C-H aromatic & aliphatic), 2214 (CN),1622,
0.97 (t, 3H, J=7.4 Hz); 13C NMR (δ, CDCl3, 100 MHz): 155.0, 1582, 1497, 1470, 1445, 1391, 1348, 1319, 1275, 1234,1196,
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149.7, 148.1, 147.5, 143.9, 142.7, 141.1, 138.5, 133.8, 128.2, 1159, 1086, 1007, 962, 924, 847, 806, 743, 619, 503, 420; H
126.8, 123.8, 123.7, 123.3, 122.8, 120.9, 120.3, 119.4, 111.0, NMR (δ, CDCl3, 400 MHz): 9.61 (s, 1H), 8.77 (d, 1H, J= 1.6Hz),
109.4, 94.9, 43.3, 31.0, 20.5, 13.8; EIMS(m/z): 390.4821(M+).
8.67 (s, 1H), 8.19-8.15 (m, 2H), 7.81-7.43 (m, 8H), 4.31 (t, 2H,
J=7.3 Hz), 1.92-1.85 (m, 2H), 1.32-1.30 (m, 6H), 0.87 (t, 3H,
J=7.1 Hz); 13C NMR (δ, CDCl3, 100 MHz): 149.8, 147.5, 144.9,
Synthesis of carbazolylcyanoacrylamide 6e
The carbaldehyde 4b (0.5 g, 1.8 mmol) was reacted with nitrile 143.9, 142.6, 141.1, 133.8, 128.2, 126.8, 124.0, 123.7, 123.8,
5a (0.15 g, 1.8 mmol) to form carbazolylcyanoacrylamide 6e as 123.2, 122.8, 120.8, 120.3, 119.3, 118.0, 111.0, 109.4, 95.0,
yellow solid. Yield: 0.59 g (96%). M.P. 163-165 °C; FT-IR (KBr, 43.4, 31.5, 28.9, 26.9, 22.5, 14.0; EIMS(m/z): 418.2201 (M+).
cm-1): 3416 (NH), 3298 (NH), 3179-2924 (C-H aromatic &
Single Crystal XRD Analysis
aliphatic), 2201 (CN), 1653 (C=O),1562, 1497, 1468, 1439,
1377, 1348, 1314, 1236, 1190, 1159, 1128, 1078, 957, 891, A single crystal of carbazolylacrylonitrile 6g has been grown by
1
804, 772, 748, 725, 675, 619, 573, 527, 455, 420; H NMR (δ, slow evaporation technique in ethanol solution at room
CDCl3, 400 MHz): 8.71 (d, 1H, J= 1.7 Hz), 8.50 (s, 1H), 8.18-8.11 temperature. Diffraction data have been collected on a Bruker,
(m, 2H), 7.63-7.30 (m, 4H), 6.34 ( brs, 1H), 5.88 ( brs, 1H), 4.32 2004 APEX 2 diffractometer using MoKα radiation (Kα =
(t, 2H, J=7.3 Hz), 1.88-1.84 (m, 2H), 1.44-1.26 (m, 6H), 0.86 (t, 0.71073 Å) at 293 K and the structure has been resolved by
3H, J=7.0 Hz);13C NMR (δ, CDCl3, 100 MHz): 163.1, 155.0, direct methods and refined by full-matrix least-squares on F2
143.2, 141.1, 128.8, 126.9, 124.9, 123.5, 122.7, 122.6, 120.9, (SHELXL-97).22 All calculations have been carried out using the
120.8, 118.5, 109.4, 109.3, 97.7, 43.4, 31.5, 28.9, 26.9, 22.5, WinGX suite of programs.23 A total number of 290 parameters
14.0; EIMS(m/z): 345.3733 (M+).
have been refined with 4129 unique reflections which covered
the residuals to R1= 0.0563. The softwares used to prepare the
material for publication are Mercury 3.5.1 and ORTEP-3.
Synthesis of carbazolylacrylonitrile 6f
The carbaldehyde 4b (0.5 g, 1.8 mmol)) was reacted with Crystallographic data of the chemical entity 6g have been
nitrile 5b (0.29 g, 1.8 mmol) to form carbazolylacrylonitrile 6f deposited with the Cambridge Crystallographic Data Centre as
as orange solid. Yield: 0.74 g (98%). M.P. 160-162 °C.; FT-IR supplementary publications number CCDC 1844826. Copies of
(KBr, cm-1): 3051-2862 (C-H aromatic & aliphatic), 2208 (CN), the data can be obtained free of charge via
1627, 1578, 1514, 1466, 1390, 1334, 1232, 1198, 1159, 922, http://www.ccdc.cam.ac.uk/conts/retrieving.html, or from the
849, 799, 746, 687, 627, 596, 420; 1H NMR (δ, CDCl3, 400 MHz): Cambridge Crystallographic Data Centre, 12 Union Road,
8.70 (d, 1H, J= 1.7 Hz), 8.31-8.15 (m, 4H), 7.87-7.84(m, 3H), Cambridge CB2 1EZ, UK; fax: (+44) 1223 336 033: or e-mail:
7.56-7.30 (m, 4H), 4.33 (t, 2H, J=7.3Hz), 1.93-1.86 (m, 2H), deposit@ccdc.cam.ac.uk.
1.42-1.25 (m, 6H), 0.87 (t, 3H, J=7.1Hz); 13C NMR (δ, CDCl3, 100
MHz): 147.2, 146.6, 142.3, 141.6, 141.1, 127.6, 126.8, 126.1,
Computational Studies
124.3, 123.5, 123.4, 122.7,120.8, 120.2, 118.4, 109.4, 109.3, The entire set of calculations was performed in DFT-B3LYP
104.7, 43.4, 31.5, 29.0, 26.9, 22.5, 14.0; EIMS(m/z): 423.5104 method with 6-311++G(d,p) basis set using Gaussian 09
(M+).
program.24 The gradient corrected DFT with the three
parameter hybrid functional Becke3 (B3) for the exchange part
and the Lee-Yang-Parr (LYP) correlation function25 have
Synthesis of carbazolylacrylonitrile 6g
The carbaldehyde 4b (0.5 g, 1.8 mmol) was reacted with nitrile universally been used as a cost effective approach. The
5c (0.32 g, 1.8 mmol) to form carbazolylacrylonitrile 6g as frequency values computed at these levels contain known
fluorescent green solid. Yield: 0.77 g (94%). M.P. 138-140 °C; systematic errors. To bring the theoretical frequencies in close
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proximity to the experimental values, the scaling factor value
of 0.96 was used. NMR chemical shifts were computed at
B3LYP/6-311++G(2d,p) level using the Gauge Independent
Atomic Orbital (GIAO) method.26 The 1H and 13C isotropic
chemical shift values were referenced to the corresponding
values of TMS, which was calculated at the same level of
theory.
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Acknowledgement
View Article Online
Financial assistance provided by the IndiDanOIC: 1o0u.1n0c3i9l/oC9fNMJ0e2d1i7c0aAl
Research, New Delhi (No.58/16/2013BMS), Kalasalingam
Academy of Research and Education (URF to KP) and NMR
facilities offered by BITs Pilani, Pilani campus is gratefully
acknowledged.
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Antioxidant Evaluation
Notes and references
The DPPH radical scavenging assay has been carried out
according to the literature27 with minor modifications. 2,2-
Diphenyl-1-picrylhydrazyl (DPPH) (0.8 mg) was dissolved in
DMSO (25 mL). To 1.5 ml of each compound prepared (10, 20,
30, 40, 50, 100 μg/ml), added 1.5 ml of DPPH solution and kept
for 30 min incubation under dark condition at room
temperature. Control value was determined by preparing
blank solution of DPPH and measuring its absorbance at 517
nm using UV-visible spectrophotometer. After incubation the
changes in the absorbance at 517 nm was measured.
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The DPPH free radical scavenging activity was calculated using
the following formula:
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T. Okada, J. Kamata, J. Niijima, T. Nagasu, N. Koyanagi, H.
% Scavenging = 100 × [(Acontrol – Asample) / Acontrol
]
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The percentage radical scavenging activities were
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Cell lines and cytotoxic assay
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The human pancreatic cancer cells AsPC1 and SW1990 were
purchased from the American Type Culture Collection, ATCC,
USA. All cells were cultured in RPMI-1640 medium
supplemented with 10% fetal bovine serum, 100IU/mL of
penicillin and 100 μg/mL of streptomycin (Sigma-Aldrich, St.
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an incubator maintained at 37 °C in 5% CO2, using the standard
in vitro cell culture method established previously.28-32
In order to assess the anti-tumor effects of the synthetic
compounds, all compounds were reconstituted to 100mM
stock solution using N,N-dimethylsulfoxide, further diluted to
sterile phosphate buffer saline to the highest effective
concentration, 0.1mM. The cells were treated based on the
previously published cell viability assay.28,33 Briefly, all cells
were then seeded (1500 cells/well) in 384-well plates for
24hours before treated with compounds for 72 hours. 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).
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Conflict of interest
The authors declare no conflict of interest.
22 G.M. Sheldrik, SHELXL97, Programs for Crystal Structure
Analysis, University of Gottingen, Germany, 1997.
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DOI: 10.1039/C9NJ02170A
Table of content
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Novel acrylamide / acrylonitrile tethered carbazoles: synthesis, structural, biological and
density functional theory studies
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Krishnaraj Padmavathy, Kannan Gokula Krishnan, Chandran Udhaya Kumar, Ethiraj Sathiyaraj, Ramar
Sivaramakarthikeyan, Wei-Meng Lim, Chun-Wai Mai, Chennan Ramalingan*
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Design and synthesis of novel carbazole based heterocyclic chemical entities have been accomplished as
anticancer agents.