A Simplistic Approach for Preparation of Alkylidenemalononitrile Derivatives: Characterization, In silico Studies, Quantum Chemical Evaluation, Molecular Docking, and In vitro Biological Activity Evaluation

Article abstract of DOI:10.1016/j.molstruc.2020.129451

A new and efficient green grinding-based catalyst free Knoevenagel condensation of aldehydes/ketones and malononitrile for the rapid preparation of twelve malononitrile derivatives (C1-C12) is proposed. Characterization of the derivatives was done by

Full text of DOI:10.1016/j.molstruc.2020.129451

Journal Pre-proof
A Simplistic Approach for Preparation of Alkylidenemalononitrile
Derivatives: Characterization, In silico Studies, Quantum Chemical
Evaluation, Molecular Docking, and In vitro Biological Activity
Evaluation
Iqbal Azad , Tahmeena Khan , Rumana Ahmad , Azhar Kamal ,
Abdul Rahman Khan , Malik Nasibullah
PII:
S0022-2860(20)31766-X
DOI:
Reference:
https://doi.org/10.1016/j.molstruc.2020.129451
MOLSTR 129451
To appear in:
Journal of Molecular Structure
Received date:
Revised date:
Accepted date:
8 May 2020
30 September 2020
13 October 2020
Please cite this article as: Iqbal Azad , Tahmeena Khan , Rumana Ahmad , Azhar Kamal ,
Abdul Rahman Khan , Malik Nasibullah , A Simplistic Approach for Preparation of Alkylidene-
malononitrile Derivatives: Characterization, In silico Studies, Quantum Chemical Evaluation, Molec-
ular Docking, and In vitro Biological Activity Evaluation, Journal of Molecular Structure (2020), doi:
https://doi.org/10.1016/j.molstruc.2020.129451
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Highlights


Twelve different malononitrile derivatives via Knoevenagel condensation were
synthesized
In vitro antibacterial and anticancer activities using the disc diffusion and MTT assay
respectively


Metabolic transformation, molecular parameters, druglikeness, ADMET
Molecular docking was performed with tyrosine-protein kinase and ribonucleoside-
diphosphate reductase
1

A Simplistic Approach for Preparation of Alkylidenemalononitrile Derivatives:
Characterization, In silico Studies, Quantum Chemical Evaluation, Molecular Docking,
and In vitro Biological Activity Evaluation
Iqbal Azad1*, Tahmeena Khan1, Rumana Ahmad2, Azhar Kamal3, Abdul Rahman
Khan1, Malik Nasibullah1
1Department of Chemistry, Integral University, Dasauli, P.O. Bas-ha, Kursi Road, Lucknow-226026, U.P., India
2Department of Biochemistry, Era’s Lucknow Medical College and Hospital, Era University, Lucknow, 226003,
U.P., India
3Department of Bioengineering, Integral University, Dasauli, P.O. Bas-ha, Kursi Road, Lucknow-226026, U.P.,
India
*iqbalazad@iul.ac.in
Abstract
A new and efficient green grinding-based catalyst free Knoevenagel condensation of
aldehydes/ketones and malononitrile for the rapid preparation of twelve malononitrile
derivatives (C1-C12) is proposed. Characterization of the derivatives was done by 1H NMR,
13C NMR, IR, elemental and mass spectral analyses. Quantum chemical calculations were
performed by DFT/B3LYP/6-31G(d,p) method. The experimental and theoretical spectra
were found to be in good agreement with each other. Natural bond order (NBO) calculations
were also performed to calculate the natural atomic charges at atomic sites. The present study
also involved study of the intramolecular charge transfer (ICT) interactions and the non-linear
optical (NLO) properties. Critical drug character assessment parameters like metabolic
transformation, druglikeness, ADMET (absorption, distribution, metabolism and excretion)
and toxicological analyses of the synthesized malononitrile derivatives were also performed.
Molecular docking studies were performed against two target proteins viz. tyrosine-protein
kinase (HCK) and ribonucleoside diphosphate reductase (RR). The synthesized malononitrile
derivatives were also evaluated for their anticancer activity against the triple negative breast
cancer (TNBC) cell line (MDA-MB-231) while their antibacterial potential was tested against
S. aureus and E. coli.
Keywords: Knoevenagel condensation; Grinding; Cancer; Molecular Docking; Nonlinear
Optical Activity; NBO calculations
Introduction
Malononitrile derivatives are multipurpose building blocks used for the synthesis of
different biological and pharmacological molecules [1–4]. In recent investigations,
malononitrile scaffolds have been largely utilized to synthesize novel bioactive motifs such as
2-amino-4-(furan-2-yl)-6-(naphthalen-2-yl)-4,5-dihydropyridine-3-carbonitrile
(3)
and
hexahydrospiro[indeno[2,1-c]pyridazine-9,5'-pyrano[2,3-d]pyrimidine]-4,6'-dicarbonitrile
derivative (6) with significant antibacterial and anticancer potency [5,6].
Many malononitrile derivatives such as (E)-2-(3-(4-aminostyryl)-5,5-dimethylcyclo-
hex-2-enylidene)malononitrile, 2-(4-oxo-3-phenylthiazolidin-2-ylidene) malononitrile and (2-
dicyanomethylene-4,5,5-trimethyl-2,5-dihydrofur-an-3-carbonitrile) are also well known as
organic non-linear optical (NLO) materials, because of their applications in
2

telecommunications and optical information processes. Nowadays, di-2-(2-oxindolin-3-
ylidene)malononitrile (DIM) (2), 2-(2,4-dimethoxybenzylidene) malononitrile (DMM) (1),
(E)-2-(1-(7-(diethylamino)-2-oxo-2H-chromen-3-yl)-3-(4-(diphenylamino)phenyl)allylidene)
malononitrile (4) and (E)-2-(1-(4-aminophenyl)-3-(4-(diphenylamino) phenyl)allylidene)
malononitrile (5) derivatives (Figure 1) are being developed as air-stable n-type organic
field-effect transistors (OFETs), organic semiconductors (OSCs) and NLO crystals material
[7–9]. Zych and Slodek (2020) have proposed an efficient synthetic route for the preparation
of substituted triazole malononitrile derivatives with high yield (65-85%). The synthesized
compounds have been found to display interesting optical, photophysical, biological as well
as fluorescence properties [10].
Organic reactions under catalyst-free and solvent-free conditions have received
considerable attention particularly in green chemistry [11]. The Knoevenagel condensation is
a nucleophilic addition reaction of the carbonyl group (aldehyde/ketone) and active hydrogen
compounds for constructing an α, β unsaturated structure followed by elimination of a water
molecule [12]. This reaction is catalyzed by aliphatic amines such as ethylenediamine,
piperidine and ammonium salts, amino acids like glycine, β-alanine and L-proline etc. and
produces better yield in protic solvents such as water, ethanol and N,N-dimethylformamide
(DMF) etc. [13]. Microwave irradiation has also shown its utility under solvent-free
condition. At room temperature, Knoevenagel condensation reaction has not been achieved
between the aromatic carbonyl group and active hydrogen compounds [14]. Demchuk et al.
(2011) proposed a simple grinding procedure for the synthesis of (2-oxo-1,2-dihydro-3H-
indol-3-ylidene) malononitriles at RT in the presence of 1-5 equivalents of water within 15
min [15].
In the present paper, synthesis of malononitrile derivatives, using the grinding method
via Knoevenagel condensation reaction and investigation of their anticancer and antibacterial
activity has been reported. Density functional theory (DFT) was used to evaluate nonlinear
optical activity (NLO), natural bond order (NBO) and reactivity descriptors. Computer-aided
studies were also performed for the analysis of druglikeness and toxicity of the synthesized
compounds. All the results were compared with standard anticancer and antibiotic drugs viz.
doxorubicin hydrochloride and tetracycline. The synthesized malononitrile derivatives [16]
were docked against two protein targets viz. i. tyrosine-protein kinase (HCK; PDB ID: 5ZJ6),
whose elevated expression is found in several solid carcinomas including breast cancer [17]
which is responsible for aberrant signaling pathways leading to defective cell cycle and
uncontrolled cell division and ii. ribonucleotide diphosphate reductase (RR; PDB ID: 6AUI),
another important enzyme for providing DNA building blocks crucial for cellular propagation
[18].
3

Figure 1. A few examples of recently investigated malononitrile derivatives as n-type
organic field-effect transistors (OFETs), organic semiconductors (OSCs), NLO crystals
material and biological activate motif.
Experimental and computational methods
Chemistry
Synthesis of 2-benzylidenemalononitrile and of 2-(2-oxo-indolin-3-ylidene) malononitrile
derivatives
Malononitrile (66 mg, 1 mmol) and 2,4-dichlorobenzaldehyde (223 mg, 1 mmol) were taken
in pestle-mortar and mixed in 1:1 molar ratio in PEG-400/water as a solvent. After 10 min of
grinding yellowish solid was obtained which was washed with water/ethanol (2×10 mL) and
the reaction was monitored by TLC using hexane and acetyl acetate (8:2) as the eluent and
silica gel as stationary phase. TLC plates were visualized in an iodine chamber. After air
drying and recrystallization in ethanol for 24 h, a white crystalline solid was obtained with
high yield (96-99%) and characterized (Supplementary information) Scheme 1 and Table 1.
Scheme 1. Synthesis of malononitrile derivatives
Characterization details
1.
2-(2,6-dichlorobenzylidene)malononitrile (C1): White crystalline solid, m.p. 89-
91°C, FT-IR (KBr, v, cm-1): 3086 (C-H, alkene), 3024 (C-H, ring), 2238 (CN), 1612 (C=C,
4

1
aromatic), 1558, 1429, 1145, 772 (Cl), 696. H NMR (400 MHz, CDCl3): δ 7.90 (s, 1H,
CH=), 7.37 (d, J = 4.6 Hz, 2H, ArH), 7.21- 7.19 (m, 1H, ArH). 13C NMR (CDCl3): δ 156.7,
134.2, 132.9, 128.9, 111.9, 110.6, 94.3. HRMS (ESI-TOF, m/z) Calcd. for C10H4Cl2N2 (M +
H+): 223.01, Found: 222.98. Anal. Calcd. for C10H4Cl2N2: C, 53.85; H, 1.81; N, 12.56,
Found: C, 53.65; H, 1.67; N, 12.43.
2.
2-(2,4-dichlorobenzylidene)malononitrile (C2): White crystalline solid, m.p. 157-
159°C, FT-IR (KBr, v, cm-1): 3102 (C-H, alkene), 3050 (C-H, ring), 2227 (CN), 1602 (C=C,
1
aromatic), 1573, 1463, 1367, 823, 793 (Cl), 757. H NMR (400 MHz, CDCl3): δ 8.12 (s, 1H,
CH=), 7.51 (s, 1H, ArH), 7.38 (d, J = 1.8 Hz, 1H, ArH), 7.36 (d, J = 1.8 Hz, 1H, ArH). 13C
NMR (CDCl3): δ 154.7, 141.2, 137.3, 130.9, 130.3, 128.5, 127.6, 113.2, 111.9, 86.2. HRMS
(ESI-TOF, m/z) Calcd. for C10H4Cl2N2 (M + H+): 223.01, Found: 222.98. Anal. Calcd. for
C10H4Cl2N2: C, 53.85; H, 1.81; N, 12.56, Found: C, 53.63; H, 1.71; N, 12.46.
3.
2-(furan-2-ylmethylene)malononitrile (C3): Pail yellow crystalline solid, m.p. 74-
76°C, FT-IR (KBr, v, cm-1): 3124 (C-H, alkene), 3043 (C-H, ring), 2227 (CN), 1603 (C=C,
1
aromatic), 1529, 1463, 1397, 1146, 933, 889, 793, 765. H NMR (400 MHz, CDCl3): δ 7.75
(s, 1H), 7.46 (d, J = 3.1 Hz, 1H, furyl), 7.20 (d, J = 2.7 Hz, 1H, furyl), 6.66 (t, J = 5.2 Hz,
1H, furyl). 13C NMR (CDCl3): δ 149.7, 148.1, 143.2, 123.7, 114.6, 113.9, 112.7. HRMS
(ESI-TOF, m/z) Calcd. for C8H4N2 (M + H+): 144.98, Found: 145.13. Anal. Calcd. for
C8H4N2: C, 66.67; H, 2.80; N, 19.44, Found: C, 66.69; H, 2.76; N, 19.44.
4.
2-(4-(dimethylamino)benzylidene)malononitrile (C4): Orange-red crystalline solid,
m.p. 187-189°C, FT-IR (KBr, v, cm-1): 2935 (C-H, alkene), 2905 (C-H, ring), 2815, 2205
1
(C≡N), 1610 (C=C, aromatic), 1558, 1352, 926, 816, 727. H NMR (400 MHz, CDCl3): δ
7.82 (d, J = 9.2 Hz, 2H, ArH), 7.47 (s, 1H, CH=), 6.69 (d, J = 9.3 Hz, 2H, ArH), 3.15 (s, 6H,
CH3). 13C NMR (CDCl3): δ 158.2, 154.4, 133.9, 119.4, 116.1, 115.1, 111.7, 72.0, 40.2.
HRMS (ESI-TOF, m/z) Calcd. for C12H11N3 (M + H+): 198.10, Found: 198.10. Anal. Calcd.
for C12H11N3: C, 73.07; H, 5.62; N, 21.30, Found: C, 73.15; H, 5.71; N, 21.38.
5.
2-benzylidenemalononitrile (C5): Yellowish white crystalline solid, m.p. 83-85°C,
FT-IR (KBr, v, cm-1): 3151 (C-H, alkene), 3028 (C-H, ring), 2227 (C≡N), 1595 (C=C,
aromatic), 1441, 963, 793, 757, 676. 1H NMR (400 MHz, CDCl3): δ 8.07 (s, 1H, CH=), 7.32
– 7.17 (m, 5H, ArH). 13C NMR (CDCl3): δ 160.1, 134.7, 131.0, 130.8, 129.7, 113.8, 112.6,
82.83. HRMS (ESI-TOF, m/z) Calcd. for C10H6N2 (M + H+): 155.08, Found: 155.05. Anal.
Calcd. for C10H6N2: C, 77.91; H, 3.92; N, 18.17, Found: C, 77.87; H, 3.89; N, 18.21.
6.
2-((1H-indol-3-yl)methylene)malononitrile (C6): Dark yellowish powder, m.p. 178-
180°C, FT-IR (KBr, v, cm-1): 3271, 3051 (C-H, alkene), 2927 (C-H, ring), 2220 (C≡N), 1624
1
(C=C, aromatic), 1588, 1565, 1441, 1338, 1235, 1146, 793, 735. H NMR (400 MHz,
CDCl3): δ 10.00 (s, 1H, NH), 8.27 (s, 1H, CH=) 7.79 (s, 1H, ArH), 7.45 (d, J = 2.2 Hz, 1H,
13
ArH), 7.37 (d, J = 1.8 Hz, 1H, ArH), 7.35-7.21 (m, 2H ArH). C NMR (CDCl3): δ 150.6,
136.6, 132.2, 126.5, 123.3, 122.0, 121.0, 117.5, 115.4, 112.6, 111.8, 70.2. HRMS (ESI-TOF,
m/z) Calcd. for C12H7N3 (M + H+): 194.07, Found: 194.06. Anal. Calcd. for C12H7N3: C,
77.91; H, 3.92; N, 18.17, Found: C, 77.97; H, 3.95; N, 18.22.
5

7.
2-(4-hydroxy-3-methoxybenzylidene)malononitrile
(C7):
Reddish-yellow
crystalline solid, m.p. 132-134°C, FT-IR (KBr, v, cm-1): 3403, 3027, 2981, 2227, 1661, 1617,
1565, 1514, 1455, 1382, 1293, 1176, 1022, 955, 816, 793, 727. 1H NMR (400 MHz, CDCl3):
δ 8.03 (s, 1H, CH=), 7.23 (d, J = 1.8 Hz, 1H, ArH), 7.19 (s, 1H, ArH), 6.95 (d, J = 8.0 Hz,
13
1H, ArH), 4.30 (s, 1H, OH), 3.91 (s, 3H, OCH3). C NMR (CDCl3): δ 159.3, 153.7, 147.8,
128.2, 122.8, 115.7, 114.5, 113.7, 111.5, 75.6, 55.5. HRMS (ESI-TOF, m/z) Calcd. for
C11H8N2 (M+ H+): 201.08, Found: 201.06. Anal. Calcd. for C11H8N2: C, 66.00; H, 4.03; N,
13.99, Found: C, 65.34; H, 3.87; N, 14.27.
8.
2-(2-oxo-indolin-3-ylidene)malononitrile (C8): Dark red powder, m.p. 240-242°C,
FT-IR (KBr, v, cm-1): 3256, 3109, 2235, 1712, 1617, 1588, 1338, 793. H NMR (400 MHz,
DMSO-d6): δ 11.43 (s, 1H, NH), 8.14 (d, J = 7.6 Hz, 1H, ArH), 7.57-7.52 (m, 1H, ArH),
7.18-7.14 (m, 1H, ArH), 6.94 (d, J = 8.2 Hz, 1H, ArH). 13C NMR (DMSO-d6): δ 163.7,
150.5, 146.4, 137.8, 125.8, 122.9, 118.5, 113.0, 111.6, 111.5, 80.5. HRMS (ESI-TOF, m/z)
Calcd. for C11H5N3 (M + H+): 196.02, Found: 196.04. Anal. Calcd. for C11H5N3: C, 67.69; H,
2.58; N, 21.53, Found: C 67.49, H 2.71, N 21.54.
1
9.
2-(2-oxo-1-phenylindolin-3-ylidene)malononitrile (C9): Reddish-black powder,
m.p. 195-197°C, FT-IR (KBr, v, cm-1): 3095, 2227, 1712, 1610, 1588, 1463, 1367, 1184, 793.
1H NMR (400 MHz, DMSO-d6): δ 7.78 (d, J = 4.6 Hz, 1H, ArH), 7.53-7.36 (m, 4H, ArH),
13
7.21-7.09 (m, 4H, ArH). C NMR (DMSO-d6): δ 162.1, 149.3, 147.2, 137.9, 132.5, 130.1,
129.3, 127.0, 126.3, 124.5, 118.3, 112.5, 111.1, 110.7, 109.1, 83.0. HRMS (ESI-TOF, m/z)
Calcd. for C17H9N3 (M + H+): 272.13, Found: 272.07. Anal. Calcd. for C17H9N3: C, 75.27; H,
3.34; N, 15.49, Found: C, 75.19; H, 3.42; N, 15.61.
10. 2-(1-methyl-2-oxoindolin-3-ylidene)malononitrile (C10): Reddish-black powder, m.p.
227-229°C, FT-IR (KBr, v, cm-1): 3082, 2919, 2235, 1720, 1611, 1595, 1463, 1331, 1227,
1
786. H NMR (400 MHz, DMSO-d6): δ 7.85 (d, J = 6.7 Hz, 1H, ArH), 7.59-7.52 (m, 1H,
13
ArH), 7.39 (d, J = 6.6 Hz, 1H, ArH), 7.15-7.08 (m, 1H, ArH), 3.39 (s, 3H, CH3). C NMR
(DMSO-d6): δ 181.2, 143.4, 140.6, 129.8, 127.0, 125.9, 123.5, 111.6, 110.6, 92.1, 24.4.
HRMS (ESI-TOF, m/z) Calcd. for C12H7N3 (M + H+): 210.07, Found: 210.06. Anal. Calcd.
for C12H7N3:C, 68.89; H, 3.37; N, 20.09, Found: C 68.96, H 3.25, N 20.10.
11.
2-(5-bromo-2-oxoindolin-3-ylidene)malononitrile (C11): Reddish-black powder,
m.p. 236-238°C, FT-IR (KBr, v, cm-1): 3573, 3507, 3097, 2984, 2235, 1727, 1621, 1595,
1
1463, 1308, 823, 793. H NMR (400 MHz, DMSO-d6): δ 11.33 (s, 1H, NH), 7.93 (s, 1H,
ArH), 7.64 (d, J = 4.5 Hz, 1H, ArH), 6.94 (d, J = 4.3 Hz, 1H, ArH). 13C NMR (DMSO-d6): δ
162.6, 144.9, 139.3, 127.9, 119.3, 114.3, 112.8, 111.4, 109.9, 81.7. HRMS (ESI-TOF, m/z)
Calcd. for C11H4N3 (M + H+): 274.27, Found: 273.95. Anal. Calcd. for C11H4N3: C, 48.21; H,
1.47; N, 15.33, Found: C 48.11, H 1.53, N 15.47.
12.
2-(2-oxo-acenaphthylen-1(2H)-ylidene)malononitrile (C12): Brown powder, m.p.
260-262°C, FT-IR (KBr, v, cm-1): 3086, 2919, 2227, 1720, 1595, 1573, 1485, 1286, 838, 779.
1H NMR (400 MHz, DMSO-d6): δ 8.19 (d, J = 6.7 Hz, 1H, ArH), 8.13 (d, J = 10.8 Hz, 1H,
ArH), 7.99 (d, J = 14.4 Hz, 1H, ArH), 7.79 (d, J = 13.1 Hz, 1H, ArH), 7.63-7.49 (m, 2H,
6

ArH). HRMS (ESI-TOF, m/z) Calcd. for C15H6N2 (M + H+): 231.06, Found: 231.06. Anal.
Calcd. for C15H6N2: C, 78.26; H, 2.63; N, 12.17, Found: C, 78.31; H, 2.52; N, 12.27.
Table 1. Physico-chemical characterization details of compounds (C1-C12) with optimized
and MetaPrint2D structures
Isolate yield
(%)
Melting
point (°C)
Entry
Precursor
Product
Rf
MetaPrint2D Structure
C1
0.67
99
89-91
C2
C3
0.76
0.44
99
71
157-159
74-76
C4
C5
0.31
0.70
98
69
187-189
83-85
C6
0.22
97
178-180
C7
C8
0.25
0.15
98
97
132-134
240-242
C9
0.35
0.19
99
99
195-197
227-229
C10
7

C11
C12
0.18
0.32
99
99
236-238
260-262
Experimental
Spectral assessment
All the chemicals used were of A.R. grade and purchased from Sigma Aldrich. The melting
points of synthesized compounds were determined by open capillary method on digital
melting point apparatus (Model 634, Electronics India (EI), Himachal Pradesh, India). Thin
layer chromatography (TLC) was performed on silica gel G coated plates using ethyl acetate-
hexane (2:8) mixture as eluent and visualization was carried out using iodine vapor.
Elemental analysis (C, H and N) was performed on EuroVector elemental analyzer. UV-
Visible spectra (200-500 nm) were recorded on ELICO SL-160 double beam
spectrophotometer equipped with a 10 mm quartz cell. The IR spectra v (cm-1) (KBr) were
recorded on an Agilent Cary 630 FTIR spectrometer in the frequency range 4000-450 cm-1.
1
The H NMR and 13C NMR spectra were obtained on a BrukerAvance400 (NMR) at 400
MHz, using tetramethylsilane (TMS) as an internal reference and CDCl3/DMSO-d6 as
solvent. Mass spectrometry was performed on Agilent 6520 Q-TOF mass spectrometer with
electro spray ionization (ESI) probe.
Biological Activity Evaluation
All biological evaluation studies were carried out at Cell and Tissue Culture Lab, Dept. of
Biochemistry, Era’s Lucknow Medical College, Era University, Lucknow. Human breast
carcinoma cell line MDA-MB-231 cell line was maintained by sub-culturing and passaging
as monolayers in 25 and 75 cm2 cell culture flasks (Nest, Tarsons) at 37°C in a 5% CO2
incubator at 95% humidity for producing H2CO3 buffering capacity as reported earlier. The
cells were maintained at pH 7.4 in DMEM containing phenol red as a pH indicator and
supplemented with 5% FBS [19]. The medium, prior to being used in cell culture experiments
was vacuum filtered using a Corning filtration system (Corning®, Sigma-Aldrich).
Anticancer Activity
0.1 M solutions of the compounds were prepared in 1.0 mL 50% DMSO solution. The
solutions were diluted 10 times in DMEM to give 0.01 M (10 µM) solutions. In separate
experiments, cells were trypsinized and cultured in 6-well (0.5×105 cells/well) initially for 24
h, to allow the cells to adhere. After 24 h of incubation, the cells were exposed to 20-100 µM
of the synthesized malononitrile derivatives (C1-C12) for the next 48 h. Suitable untreated
controls (containing 50% DMSO as a vehicle) were also concomitantly employed. Each dose
8

was tested in at least 3 replicate wells. For morphological analysis, cells in 6-well plate were
observed under phase contrast microscope (Nikon Eclipse Ti, Japan) & photographed.
Methyl tetrazolium-MTT assay
MTT was performed as per published protocol in 96-well microliter tissue culture plates
(Linbro, MP Biomedicals) [20]. For MTT assay, 104 MDA cells were seeded in 200 µL of the
medium in a 96-well microliter tissue culture plate and cultured in a humidified 5% CO2
incubator at 37°C for 24 h. Defined concentrations (20, 50 and 100 µM) of the compounds in
50% DMSO were freshly prepared in culture media by serial dilution. Serial dilution was
carried out in cell culture media in such a way that the final concentration of DMSO in the
well did not exceed 0.5% (v/v). Three control wells containing medium alone to serve as
blanks were also included. After 24 h of incubation, in separate experiments, cells were
treated with the above-mentioned concentrations of the synthesized compounds C1-C12 in
triplicates for 48 h. Equal volumes of 50% DMSO (in cell culture media) were used as
vehicle controls. At the end of treatment, cell culture medium containing varying amounts of
synthesized compounds was removed and 20 μL of MTT (stock made in PSS at 5.0 mg/mL)
reagent was added to each well and incubated for 4 h. Thereafter, MTT was removed and
formazan crystals were dissolved in 200 μL of DMSO. The plates were read in a Bio-Rad
PW41 ELISA plate reader at a wavelength of 570 nm with a reference wavelength of 630 nm.
Percentage cell viability (Y-axis) was calculated from absorbance and plotted against
concentration in μM (X-axis).
% Cell survival was calculated as = A  A  A  A 100
B  
(1)




T
c
B
where,
T = Absorbance of treatment well
A
A
B = Absorbance of blank
A
C = Absorbance of control well
% cell inhibition = 100-Cell Survival
IC50
values were obtained from the graph as the concentration which decreased cell
by viability 50%.
Antibacterial Activity
The in vitro antibacterial activity of the compounds was evaluated against
Staphylococcus aureus (Gram positive) and Escherichia coli (Gram-negative) bacteria by
disc diffusion method [21] using Mueller-Hinton agar (MHA) medium. The bacteria were
sub-cultured in the agar medium and were incubated for 24 h at 37°C. The discs (Sterile filter
paper discs, Whatman No. 1.0) having a diameter of 5 mm, were then soaked in the test
solutions with the appropriate equivalent amounts of the synthesized compounds dissolved in
sterile 50% DMSO at concentrations of 2-10 mg/disc and placed on lawn culture of the
respective microbial organism and stored in an incubator for the above mentioned period of
time [22]. Formation of inhibition zone (if any) around each disc was measured and the
results recorded in the form of inhibition zones as a function of diameter (mm). To clarify
9

any effect of DMSO (used as a vehicle for the dissolution of the synthesized compounds) on
biological screening, 50% DMSO was used as a negative control where it showed no activity
against any bacterial strains. Tetracycline was used as a positive control [21].
Computational details
Density Functional Theory (DFT) calculations
The geometries of the synthesized compounds, were determined and explored using DFT
with Becke's three-parameter functional and Lee-Yang-Parr functional hybrid model
(B3LYP) combined with 6-31G(d,p) basis set. DFT based calculations were done by Gauss
View v6.0 and Gaussian 09W program package [23,24]. The frequency analysis and
optimization of the alkylidene malononitrile derivatives were executed at the same level of
theory and were scaled by a factor of 0.9679. Potential energy distribution (PED) analysis
was performed by VEDA4 program. Optimized geometries were further used for the DFT
based global reactivity descriptor analysis including EHOMO (energy of highest occupied
molecular orbital), ELUMO (energy of lowest unoccupied molecular orbital energy), EGAP (band
gap energy), absolute softness (σ), absolute hardness (η), optical softness (σ0), chemical
potential (CP), absolute electronegativity (χ), additional electronic charges (∆Nmax
)
nucleophilicity index (N) and electrophilicity index (ω) [25]. Molecular electrostatic potential
surface (MEP) analysis was also done. Gauge independent atomic orbital (GIAO) method
1
was used to evaluate H and 13C NMR spectra. The UV-Vis absorption spectra were
calculated using the Time-Dependent Density Functional Theory (TD-DFT) calculations and
also used for the calculation of oscillator strengths (OS). Non-linear optical (NLO)
parameters such as total static dipole moment (μ), the mean polarizability (α0), the anisotropy
of the polarizability (∆α) and the first hyperpolarizability (β0) were also calculated and
natural bond order (NBO) analysis was done via NBO v6.0 program to identify inter and
intra-molecular delocalization strength and interaction [26].
In-silico bioactivity analysis
Molecular Docking
The synthesized compounds were subjected to molecular docking using the AutoDock Tools
(ADT) v1.5.6 and Auto Dock v4.0.1 (interactive molecular graphics programs) to understand
the drug-receptor interaction with the target receptor enzymes. The results were further
validated with iGEMDOCK v2.1.
Preparation of the receptor for docking (AutoDock)
The X-ray crystal structures of tyrosine-protein kinase HCK (PDB ID: 5ZJ6, UniProtKB ID:
P08631, HCK_HUMAN) and ribonucleoside diphosphate-reductase (PDB ID: 6AUI,
UniProtKB ID: P23921, RIR1_HUMAN) were obtained from the Protein Data Bank in PDB
format [27].
10

Figure 2. Electrostatic potential surface structure of receptor generated by PyMol v2.0.6 (1 =
5ZJ6 and 2 = 6AUI)
Preparation of ligands for docking
The ligand preparation involved building of 2D structure using Chem-Draw professional
v15.1 and energy minimization through Chem3D professional v15.1 MMFF (Merck
Molecular Force Field), using the job type: Minimum RMS Gradient of 0.010 kcal/mol and
RMS distance of 0.1 Å, and saved as a .mol file. Structure validation and optimization was
also performed with the help of Gaussian 9W and Gauss View v6.0.
Docking and Display of the results
Collaborative docking was performed to dock one ligand at a time through the following
steps-(1) Click on menu run/run AutoGrid/run AutoDock, check job status after completion
of docking a .dlg file is automatically generated.
(2) Click Docking/Browse/.dlg file, click Conformations/Play/&/Show info, the algorithm
searches the overall lowest energy function of ligand from the saved minimum energy
conformations with the help of genetic and stochastic global optimization.
iGEMDOCK
Optimized PDB files of the receptor and ligands were prepared, ligand conformations and
orientation comparative to the binding site of receptor based on generic algorithm (GA)
method were analyzed to produce receptor-ligand interactions [28]. Accurate docking (very
slow docking) was performed and the docking parameters were set as follows: population
size: 800, generations: 80, number of solutions: 10. After completion of docking, the best
docking pose was generated having minimum binding energy [29]. The observed scoring
function of iGEMDOCK is assessed as:
(2)
Fitness  vdW  Hbond  Elec
where,
vdW = van der Waals energy, H bond = hydrogen bonding energy and Elec =
electrostatic energy, respectively. Finally, iGEMDOCK post-analysis tool was used to
visualize the docked poses.
11

Computer-aided pharmacokinetics studies
SwissADME was used for pharmacokinetic studies and OSIRIS data warrior v4.6.2 was used
to estimate the toxicity of the compounds. The metabolic sites in the compounds were
predicted using MetaPrint2D in Bioclipse v2.6.2. Molinspiration v2016.03 was used to
calculate the bioactivity score and admetSAR was used to analyze the ADMET properties.
Metabolic transformation prediction
MetaPrint2D is used to predict the metabolic sites and analyzes the results by computing
normalized occurrence ratio (NOR) [30]. A high NOR value specifies a common site found
in the metabolite database. The metabolized sites are shown by different colors; Red: High,
Orange: Medium, Green: Low, White: Very low and Grey: No data, as shown in Table 1.
Molecular parameters
The molecular properties including LogP, topological polar surface area (TPSA), number of
hydrogen bond donors (OHNH) and hydrogen bond acceptors (ON), rotatable bonds (RB),
molar refractivity (MR) molecular weight (MW) and percentage of absorption (% of ABS)
were computed using SwissADME.
Assessment of Druglikeness
Druglikeness of the proposed compounds was assessed using the following filtering rules:
Lipinski’s rule-of-five
According to the rule-of-five, for good oral absorbance, a compound must have hydrogen
bond acceptors (10≥ON), hydrogen bond donors (5≥OHNH), calculated LogP (5≥cLogP),
rotatable bonds (10≥RB) and molecular weight (500≥MW) with a maximum of one violation.
Leadlikeness
According to Teague et al., (1999) compounds with MW in the range 250-350, a XLOGP3
value of <3.5 and <7 rotatable bonds satisfy the criteria for lead-likeness. When the lead is an
agonist, the ED50 is taken to be a maximal value for the affinity of the molecule having the
following desirable properties: MW<350 and cLogP<3 [31].
ADMET prediction
ADMET properties including (i) Brain/blood penetration coefficient (ii) Human Intestinal
Absorption (iii) Caco-2 cell permeability (iv) Prediction of IC50 values for rat model (v)
Ames toxicity and (vi) Carcinogenic effect of the proposed compounds were studied using
the admetSAR server [32].
Toxicity potential assessment
12

The toxicity risk of the proposed compounds was assessed by means of a pre-computed set of
structural fragments. Toxic parameters of the proposed compounds were predicted by
OSIRIS Data Warrior Software [33].
Bioactivity score prediction
Using Molinspiration, the bioactivity scores of the proposed compounds against regular
human receptors were calculated [34]. Larger the bioactivity score, greater is the probability
of the compound to be active. A molecule having a bioactivity score more than 0.0 is most
likely to be biologically active, while values ranging from -5.0 to 0.0 are for moderately
active compounds and if the score is less than -5.0, the compound is expected to be inactive
[35].
Results and Discussion
Chemistry
The synthetic procedure is illustrated in scheme 1. Grinding malononitrile with the
appropriate 2,4-dichlorobenzaldehyde/Isatin derivative in the presence of PEG-400 as a
solvent afforded the 2-benzylidene/2-(2-oxo-indolin-3-ylidene) malononitrile derivative
(C1/C8) as previously reported [36–40].
Grinding of 2,4-dichlorobenzaldehyde with malononitrile at room temperature for 10 minutes
in the absence of solvent gave product in low yield in the case of C1 and C8. When the
Knoevenagel condensation was carried out in PEG-400 with grinding for 10 minutes the
adduct C1 was obtained in 75% yield. High viscosity of PEG-400 caused the product to be
obtained in low yield. If grinding was done using a mixture of PEG-400 and water (1:1) as
solvents at RT, C1 was obtained in high yield (99%). The yield of the product was dependent
on the solvent used (Table 2).
Table 2. Optimization of synthesis of compound C1-C7 and C8-C12 via the Knoevenagel
condensation using PEG-400 and water as solvents in grinding method
Entry
Time (min)
Solvents
Product
Yield (%)
Trace
75
69
99*
89
Trace
90
1
2
3
4
5
6
7
8
9
10
10
10
10
5
10
10
10
10
5
-
C1
C1
C1
C1
C1
C8
C8
C8
PEG-400
water
PEG-400/water
PEG-400/water
-
PEG-400
water
PEG-400/water
PE400/water
89
99**
88
C8
C8
10
* Most suitable optimized condition for 2-benzylidene malononitrile derivatives
** Most suitable optimized condition for 2-(2-oxo-indolin-3-ylidene) malononitrile derivatives
Experimental investigation revealed that grinding with organic solvents in a glass pestle
and mortar favors condensation and dehydration reaction [13]. To the best of our knowledge,
this is the first report where Knoevenagel condensation took place by grinding in the presence
of PEG-400 at RT and in the absence of a catalyst. The proposed synthetic procedure
involved isaldol addition facilitated by a polar solvent (Figure 3). The proposed reaction
mechanism was also optimized with the help of DFT and also explained in terms of dual
13

descriptor (∆f(r)) [41]. The value of ∆f(r)>0 supports nucleophilic attack while ∆f(r)<0 favors
electrophilic attack (Supplementary Table 1). The reaction mechanism was supported by
∆f(r) [41,42]. Initially, active methylene compound malononitrile (I) undergoes tautomerism,
which is favored by the presence of two adjacent nitrogen atoms (∆f(r) = -0.7157 eV,
electrophilic) and activated hydrogen atoms ((∆f(r) = -0.7374 eV, nucleophilic). A highly
reactive tautomer intermediate 3-iminoacrylonitrile (II) is formed and activated methylene
carbon atom becomes suitable for the electrophilic attack (∆f(r) = -8.4820 eV, electrophilic).
On the other hand, aldehyde (III) α, β di-one or isatin (VI) in the presence of proton (protic
solvent) develops intermediate IV/VII with oxonium ion (C=OH+) and converts into
intermediate V/VIII. Due to instability and high reactivity, it proceeds, through a 1,2-
elimination leading to the formation of a double bond and removal of a water molecule.
Moreover, IR spectra of the compounds (C1-C12) revealed a sharp characteristic stretching
absorption band at 2227-2235 cm-1 corresponding to CN group. Aromatic C=C absorbance
was obtained between 1689-1602 cm-1. The band corresponding to the =CH aromatic group
was found between 3086-3057 cm-1. The 1H NMR spectra revealed a singlet assigned to CH
protons at 8.12-7.75 ppm. 13C NMR and mass spectra also confirmed the structure of
synthesized compounds.
Figure 3. The plausible comparative (aldehyde and ketone) reaction mechanism of Knoevenagel
condensation in the presence of proton and in terms of (eV) of dual descriptor (
DFT calculations
Optimized geometry
)
The thermodynamically favored geometry and optimized structural constraints such as bond
length, bond angle and dihedral angles were also calculated. The optimized structures are
14

shown in Figure 4 and parameters are presented in Supplementary tables 10-11. From the
structural parameters, the calculated distances between different C–C bonds of the ring
carbon and active methylene group carbon atoms were found to be approximately same and
correlated with the available experimental data. Different functional groups attached with the
ring influenced the slight change in the optimized parameters as presented in the
Supplementary tables 10-11.
Figure 4. The ball-and-stick model of optimized structures of alkylidenemalononitrile
derivatives (C1-C12) with atom numbering scheme
Vibrational frequency analysis
The number of normal modes of vibration and their classification into stretching, bending,
torsion and out of plane modes along with total number of atoms and electrons are given in
Tables 3-4. The potential energy distribution (PED) analyses were also performed to evaluate
the vibrational frequencies of each compound [43]. The experimental and theoretical infrared
spectra of all the alkylidenemalononitrile derivatives (C1-C12) are shown in Figure 5. The
theoretical vibrational frequencies were computed in the gaseous phase, whereas the
experimental values were obtained in solid phase. Theoretical spectra were scaled by 0.9679
[44], both the experimental and theoretical spectral studies were done between 4000-450 cm-
1. Good correlation was obtained between the experimental and theoretical infrared spectra.
Table 3. Showing the general findings of vibrational frequency analysis obtained from
DFT/B3LYP/6-31G(d,p) level
Compounds
C1
48
18
112
17
16
11
4
C2
48
18
112
17
16
11
4
C3
39
15
74
14
13
10
2
C4
72
25
104
25
24
19
4
C5
48
18
80
17
16
13
2
C6
60
22
100
21
20
16
3
C7
63
23
104
22
21
16
4
C8
54
20
100
19
18
12
5
C9
84
30
140
29
28
21
6
C10
63
23
108
22
21
14
6
C11
54
20
134
19
18
12
5
C12
66
24
118
23
22
15
6
TMV
TA
TE
STRE
BEND
TORS
OUT
TMV: Total mode of vibrations
TA: Total atoms
TE: Total electrons
STRE: Stretching vibration mode
BEND: Bending vibration mode
TORS: Torsion vibration mode
15

OUT: Out of plane vibration
Ring vibration
C–H stretching vibrations were associated with ring and active methylene group. The C-H
stretching vibrations in the phenyl ring occurred between 3100-3000 cm-1 [45]. Substitution
sites and their properties did not affect the aromatic C–H stretching vibration region while
ortho disubstituted aromatic ring caused a strong absorption band at 750 cm-1. The theoretical
=C-H out of plane absorbance was obtained at 922-690 cm-1 for aromatic ring. Similarly, in-
plane C-H bending was found between 1300-1000 cm-1 and aromatic C=C absorbance bands
were obtained at 1600 and 1475 cm-1 [46] (Table 3). The experimental ring vibrations were
found between 1452-1610 cm-1 whereas the theoretical observations were between 1438-1632
cm-1 as shown in Figure 5. All the theoretical and experimental findings were in good
agreement with each other.
C-H vibrations
The alkene C-H stretching vibration was observed in the range 3300-2750 cm-1, =C-H sp2
absorbance band was found near ~3100 cm-1 [46]. The =C-H out of plane vibration was
observed between 1000-650 cm-1 and used to define the degree of substitution on the double
bond C=C (1660-1600 cm-1). An overtone or combination weak band was obtained at 2000
cm-1 and 1667 cm-1. On the other hand for 1,2,4 substitution, a medium band was observed at
900 cm-1 and at 800 cm-1, a strong band was also obtained [47] (Table 4 and Figure 5).
C–Cl vibration
The stretching vibration mode of ring C–Cl bond was obtained between 760–505 cm-1 [47].
The C-Cl stretching vibrations bands were observed at 759 and 780 cm-1 for compound C1
and C2 theoretically whereas the experimental bands were obtained at 772 and 793 cm-1.
Inductive effect as well as vibrational coupling with other groups such as C=N was
responsible for slightly higher absorption of C–Cl bond.
In general the C≡N stretching vibration is found at 2260-2240 cm-1 in saturated C≡N or in
unsaturated C≡N, where conjugation is not found between the C≡N and the C=C group [46].
In a conjugated system, C≡N stretching vibration band shifts towards lower frequency range
~2232-2215 cm-1 [47]. In malononitrile derivatives, two C≡N groups were linked to the same
C=C group, therefore, the symmetrical C≡N stretching vibration was found at 2235-2205 cm-
1
whereas, the theoretical stretching vibration was obtained between 2282-2263 cm-1. The
variation may be due to the conjugation of the bond. The C≡N out-of-plane torsion was
expected at 454 cm−1 [47]. PED analysis also supported the experimental observations.
C–N vibration
The identification of the C–N stretching vibration mode in fingerprint region is very
challenging, because the vibration band overlaps with other core vibrations. The C–N
stretching vibrations appeared between 1382-1266 cm-1 as reported in literature [25]. The
theoretical and experimental observation showed good correlation with each other. The low
PED values showed that C–N vibration bands overlapped with some other vibrations as well.
O–H vibrations
The hydrogen bonded broad peaks of O–H vibrations were obtained between 3400–3300 cm-
1. While, non-hydrogen bonded O–H stretch vibrations appeared as sharp and weak bands
between 3650-3600 cm-1 [47]. The C-O-H bending band is reportedly observed in the finger
16

printing region 1440-1220 cm-1 along with the C-O stretching band between 1260-1000 cm-1
[46,47]. Only compound C7 possessed hydroxyl group and the theoretical band was
calculated at 3626 cm-1 under gaseous phase whereas the theoretical band in solid phase was
found at 3403 cm-1. Thus, it was concluded that theoretical O–H stretching vibration is non-
hydrogen bonded while experimental findings were influenced by hydrogen bonding. 100%
PED contribution also showed good agreement with the experimental data Figure 5.
C=O vibrations
The characteristic carbonyl group vibration absorption frequencies were observed between
1870-1540 cm-1. The C=O stretching vibration of ketone with five-membered ring is
generally reported at ~1751 cm-1. Presence of α, β double bond shifts the typical C=O
stretching band from 1751 cm-1 to a lower frequency viz. ~1730-1700 cm-1 [46]. The C=O
stretching sharp band was observed in the experimental spectra at 1712, 1712, 1720, 1727
and 1720 cm-1 for compounds C8-C12. While, in the theoretical spectra C=O stretching band
was observed at 1774, 1755, 1755, 1774 and 1755 cm-1. The difference between the
experimental and the theoretical bands may be attributed to the conjugation of C=O bonds,
which is probably responsible for the lower stretching frequency.
Figure 5. The experimental and theoretical IR spectra of all the synthesized compounds C1-
C12
Table 4. Some anharmonic frequencies (cm-1) of all the synthesized compounds C1-C12 at
DFT/B3LYP/6-31G(d,p) level in gaseous phase
Compounds
C1
C2
C3
C4
C5
C6
C7
C8
C9
C10
C11
C12
Theoretical Vibrational Frequencies
υC≡N
2282
3129
3087
-
759
-
2276
3148
3116
-
780
-
2275
3192
3162
-
2268
3060
2975
-
2275
3143
3100
-
2263
3154
3109
-
-
-
2264
3064
3008
3626
-
2272
-
3112
-
2268
-
3109
-
-
1755
1608
-
2274
-
3115
-
-
1755
1608
-
2272
-
3102
-
2275
-
3102
-
-
1755
1596
-
υC−H (ali.)
υC−H (aro.)
υO−H
υC−Cl
υC=O
-
-
-
-
-
-
1608
-
-
1617
-
-
1589
-
-
1774
1576
3549
1774
1614
3549
υC=C (aro.)
υN−H
1603
-
1572
-
1585
3551
1585
-
Experimental Vibrational Frequencies
υC≡N
υC−H (ali.)
2238
3086
2227
3102
2227
3124
2205
2935
2227
3151
2220
3051
2227
3027
2235
-
2227
-
2235
-
2235
-
2227
-
17

υC−H (aro.)
υO−H
υC−Cl
υC=O
υC=C (aro.)
υN−H
3024
3050
3043
-
-
-
1603
-
2905
-
-
-
1610
-
3028
-
-
-
1595
-
2931
-
-
2977
3403
-
-
1617
-
3109
-
-
1712
1617
3256
3094
-
-
1712
1610
-
3028
-
-
1720
1611
-
3097
-
-
1727
1621
3573
3086
-
-
1720
1595
-
-
772
-
1612
-
-
793
-
1602
-
-
1624
3271
NMR spectral analysis
The gauge including atomic orbital (GIAO) method was used to calculate theoretical
13
1
chemical shifts values. The C and H chemical shifts were scaled δ = (intercept - isotropic
magnetic shielding)/slope; where the values of intercept and slope were 188.57 and 0.94,
1
respectively, for 13C spectral calculation and 31.54 and 1.03, respectively for H spectral
calculation [48]. Unscaled chemical shifts were calculated at the same level of theory with
13
reference to tetramethylsilane (TMS). The C chemical shifts were designated in CDCl3 for
1
C1-C7 and DMSO for C8-C12. The experimental 13C and H NMR spectra of synthesized
13
compounds C1-C12 have been shown in Supplementary information. The calculated C and
13
1H scaled chemical shift values have been shown in the Supplementary Table 12 & 13. C
chemical shifts values were found in the range of 110-175 and 100-150 ppm [47]. The
chemical shift of =CH- group was deshielded, due to the presence of adjacent strong
electrophilic carbon in two nitrile groups and observed at ≤100 ppm in the theoretical as well
as experimental spectra. The electrophilic carbon of nitrile (C≡N) group was found near 110-
140 ppm, due to the presence of two adjacent C≡N groups, they were slightly deshielded and
the chemical shift was observed at ~100 ppm. The average correlation (R) of theoretical as
well as experimental 13C chemical shifts for C1-C12 was 0.977. The alkylidenemalononitrile
1
derivatives mainly possessed activated double bonded hydrogen =CH. The H chemical shift
value of =CH was strongly shielded due to the presence of two adjacent nitrile groups and
resonance due to the aromatic ring and found between 6.4-8.5 ppm in the theoretical while
between 6.7-8.1 ppm in the experimental spectra. The theoretical and experimental values for
=CH- chemical shifts showed good correlation supporting the Knoevenagel condensation.
1
The average correlation coefficient value (0.934) of the theoretical and experimental H
chemical shift of all the compounds showed good agreement, except for the NH group. This
variation may be due to the presence of hydrogen bonding that leads to strong shielding of the
NH peak, but experimental chemical shift of NH proton was observed as reported in literature
[25].
Electronic absorption spectral analysis
The UV-Vis spectra are observed due to electronic transition from bonding molecular orbitals
(BMOs) to anti-bonding molecular orbital (ABMOs). The UV-visible spectra of all
compounds viz. C1-C12 were calculated in gaseous as well as in CHCl3 (C1-C7) and DMSO
(C8-C12), by using optimized structures. The comparison of computed UV-Vis spectra in
gaseous and solvent phases in CHCl3 (C1-C7) and DMSO (C8-C12) are represented in
Figure 6. The UV-Vis spectral findings are shown in Supplementary Table 18. The
correlation (0.9707) between theoretical and experimental UV-Vis spectra was in good
correlation with each other (Figure 6). All the synthesized compounds illustrated H→L
transition with major contribution between 59-99% in gaseous phase while 61-99% in solvent
phase. The molecular orbital plots and coefficients showed that the frontier molecular orbitals
had more p character. Therefore, the main electronic transition was π→π*.
18

Figure 6. The computed UV-Vis spectra of all synthesized compounds C1-C12
Thermodynamic properties analysis
The thermodynamic properties such as molar capacity, electronic energy (EE), zero point
vibrational (ZPV) energy, rotational constants (RC) and rotational temperature (RT) were
computed for all the synthesized compounds [49]. The statistical thermochemical
investigation was done at ambient temperature (298.15 K) and one atmospheric pressure. All
the statistical thermodynamic findings were used to calculate other thermodynamic properties
such as entropy (S), zero-point energy (ZPE), heat capacity (C), enthalpy (H) and Gibbs free
energy (G) represented in Table 5.
Table 5. The computed thermodynamic parameters of all synthesized compounds C1-C12
Parameter
C1
C2
C3
C4
C5
C6
C7
C8
C9
C10
C11
C12
ZPVE
(Kcal/Mol)
70.06
70.433
128.57
63.53
82.65
101.30
106.06
85.709
136.27
103.37
79.28
104.38
0.670
0.473
0.320
0.032
0.897
0.296
0.223
0.043
0.014
0.011
1.661
0.259
0.224
0.080
0.012
0.010
2.498
0.695
0.544
0.120
0.033
0.026
2.080
0.542
0.430
0.100
0.026
0.021
1.238
0.389
0.296
0.059
0.019
0.014
1.122
0.348
0.266
0.054
0.017
0.013
1.045
0.512
0.344
0.050
0.025
0.017
0.507
0.237
0.168
0.024
0.011
0.008
0.789
0.494
0.305
0.038
0.024
0.015
0.770
0.230
0.177
0.037
0.011
0.009
0.749
0.366
0.246
0.036
0.018
0.012
RC (GHz)
RT (Kelvin) 0.023
0.015
S (Cal/Mol-Kelvin)
Total
Translational 42.095
Rotational
32.429
Vibrational 39.331
113.86
116.38
42.095
32.962
41.319
122.75
41.741
32.479
48.526
95.85
40.806
30.210
24.839
100.15
110.18
41.68
32.09
36.41
117.57
41.79
32.402
43.39
107.61
41.709
31.836
34.060
131.86
42.691
34.031
55.137
114.88
41.92
32.27
40.70
117.52
42.711
33.590
41.217
114.88
42.201
32.835
39.846
41.006
30.872
28.268
CV (Cal/Mol-Kelvin)
Total
Translational 2.981
Rotational
2.981
Vibrational 39.015
44.98
44.713
2.981
2.981
52.56
2.981
2.981
46.60
32.99
2.981
2.981
27.03
37.01
2.981
2.981
31.04
45.80
2.981
2.981
39.84
50.426
2.981
2.981
44.845
2.981
2.981
63.500
2.981
2.981
49.996
2.981
2.981
49.054
2.981
2.981
51.950
2.981
2.981
38.752
44.464
38.883
57.539
44.034
43.092
45.989
E (KCal/Mol)
Total
Translational 0.889
Rotational
0.889
Vibrational 76.155
μ (Debye)
α (a.u.)
77.93
78.271
0.889
0.889
76.493
4.93
137.67
0.889
0.889
135.91
7.45
69.301
0.889
0.889
67.523
11.05
176.68
88.924
108.77
0.889
0.889
106.99
9.62
114.64
0.889
0.889
112.86
6.31
93.117
0.889
0.889
91.339
8.15
146.60
0.889
0.889
144.83
8.38
111.78
0.889
0.889
109.99
8.25
87.646
0.889
0.889
85.868
7.42
112.73
0.889
0.889
110.95
8.64
0.889
0.889
87.147
6.64
6.42
133.39
150.48
105.48
121.03
151.43
149.51
140.79
206.61
153.32
161.41
178.62
EE (a.u.) -1413.31 -1413.32 -491.91 -628.12 -494.14 -625.72 -683.89 -661.63 -892.69 -700.95 -3232.73 -759.90
S (cal/mol-
113.86
44.98
77.93
116.38
44.71
78.27
95.85
32.99
69.30
122.75
100.15
37.01
88.92
110.18
117.57
107.61
44.85
93.12
131.86
114.88
50.00
11.78
117.52
49.05
87.65
114.88
kelvin)
Cv (cal/mol-
kelvin)
E (Thermal)
Kcal/mol
52.56
45.80
50.43
63.50
51.95
137.69
108.77
114.63
146.61
112.73
19

G (au)
ZPE (au)
0.07
0.11
0.07
0.11
0.07
0.10
0.16
0.21
0.10
0.13
0.12
0.16
0.13
0.17
0.10
0.14
0.17
0.22
0.12
0.17
0.09
0.13
0.13
0.17
FMO (Frontier Molecular Orbital) analysis
The electron donating capability is illustrated by EHOMO, whereas the electron accepting
capability is illustrated by ELUMO and the EGAP between HOMO and LUMO describes the
stability of the molecular system and elucidates the subsequent charge transfer relations
occurring within the molecular system. EHOMO is also associated with the ionization potential
(IP) and ELUMO is related to electron affinity (EA) [25]. The small value of EGAP favors the
biological activity of the compound. The EHOMO and ELUMO were computed in gaseous and
solvent phase (compound C1-C7 in CHCl3 and C8-C12 in DMSO) using IEFPCM model.
The value of EGAP for all the compounds was between 4.812-4-4.671 eV in gaseous and
solvent phase while for C1, it was -2.898 and -2.736 eV which was lowest for C9 in gas and
solvent phase. 2-benzylidene malononitrile derivatives (C1-C7) possessed highest value of
EGAP between 4.812 eV (in gas) and 4.671 eV (in solvent) which was highest for compound
C1, whereas 3.621 eV (in gaseous) and 3.552 eV (in solvent) which was lowest for
compound C4, showing highest stability with least reactivity as compared to 2-(2-oxo-
indolin-3-ylidene) malononitrile derivatives (C8-C12), possessing EGAP between 3.291 eV (in
gaseous) and 3.072 eV (in solvent) and highest for C12, 2.898 eV (in gas) and 2.736 eV (in
solvent) lowest for compound C9 as represented in Table 6 and Figure 7. 2-(2-oxo-indolin-
3-ylidene) malononitrile derivatives (C8-C12) showed least stability with high reactivity.
Experimentally, it was found that 2-(2-oxo-indolin-3-ylidene) malononitrile derivatives (C8-
C12) were formed feasibly with high yield as compared to the 2-benzylidene malononitrile
derivatives (C1-C12). In the HOMO and LUMO plots, positive phase was represented by red
and negative phase was represented by green color as shown in Figure 8.
Table 6. HOMO-LUMO energies with HOMO-LUMO energy gap
(eV)
Phase
C1
C2
C3
C4
C5
C6
C7
C8
C9
C10
C11
C12
Gas
Solvent
Gas
Solvent
Gas
-7.351
-7.161
-2.545
-2.490
4.812
4.671
-7.244
-7.076
-3.052
-2.880
4.192
4.196
-6.941
-6.767
-2.645
-2.527
4.296
4.240
-6.076
-5.955
-2.455
-2.403
3.621
3.552
-7.073
-6.955
-2.916
-2.798
4.157
4.157
-6.152
-6.055
-2.288
-2.220
3.864
3.835
-6.291
-6.168
-2.643
-2.582
3.648
3.586
-6.750
-6.473
-3.591
-3.502
3.165
2.971
-6.442
-6.279
-3.544
-3.543
2.898
2.736
-6.585
-6.358
-3.531
-3.489
3.054
2.869
-6.729
-6.435
-3.791
-3.636
2.938
2.799
-6.786
-6.546
-3.495
-3.474
3.291
3.072
EHOMO
ELUMO
EGAP
Solvent
20

Figure 7. Energy level graph of EHOMO, ELUMO and EGAP for all the synthesized compounds
C1-C12
21

Figure 8. The HOMO and LUMO plots for all the synthesized compounds C1-C12 computed
by TD-DFT/B3LYP/6-31G(d,p) method.
Molecular electrostatic potential (MEP) analysis
The molecular electrostatic potential explores the charge distribution and polarization along
with the hydrogen bonding capability and reactivity of the molecular system. MEP also
elucidates complete information of the electrophilic and nucleophilic sites in the molecular
system, thereby providing statistical polarity of the molecule in a pictorial form, to recognize
the polar and nonpolar sites of the molecule in color variations as shown in the Figure 9. The
red color characterizes electrophilic reactivity (most negative), blue color characterizes
nucleophilic reactivity (most positive) and green color generally illustrates nonreactive sites
(zero electrostatic potential). MEP decreases in the order; blue>green>yellow>orange>red
[52]. The color code (red to blue) of the compounds was found between -4.614 to 4.614 (C1),
-4.640 to 4.640 (C2), -5.079 to 5.079 (C3), -5.909 to 5.909 (C4), -4.967 to 4.967 (C5), -
8.303 to 8.303 (C6), -6.034 to 6.034 (C7), -7.252 to 7.252 (C8), -5.584 to 5.584 (C9), -5.548
to 5.548 (C10), -7.727 to 7.727 (C11) and -5.438 to 5.438 a.u. (C12), respectively. From the
MEP plots it was concluded that nitrile group of alkylidenemalononitrile derivatives
possessed high electropositive potential (red) and the activated double bond hydrogen (=CH-)
possessed slightly low electropositive region (blue). Consequently, nitrile groups of
alkylidenemalononitriles support electrophilicity while the presence of activated =CH- group
favors nucleophilicity (Figure 9).
Figure 9. MEP formed by mapping of total density over electrostatic potential in gas phase
for all the synthesized compounds C1-C12
Non-Linear Optical (NLO) properties
Larger value of hyperpolarizability of a molecular system favors NLO properties which can
be explored for optoelectronics and for optical devices. Statistical evaluation of
hyperpolarizability is quite advantageous for establishing a correlation among the molecular
system as well as NLO properties [50]. Various NLO parameters including static dipole
22

moment (μ), mean polarizability (α0), anisotropy of polarizability (Δα) and first
hyperpolarizability (β0) were calculated.
The values of the mean polarizability α0 and the first hyperpolarizability β0 were expressed in
atomic units (a.u.) and converted into electrostatic units (esu) (α: 1 a.u. = 0.1482 × 10-24 esu
and β: 1 a.u. = 8.6393 × 10-33 esu). The values of μ, α0 Δα and β0 for all the synthesized
compounds are presented in Table 8. The calculated values of μ were 6.3921, 5.0228,
10.9961, 7.3570, 6.5609, 9.3780, 6.2581, 8.0757, 8.8739, 8.2803, 7.8269 and 8.7751 D
(Debye), respectively for C1-C12. The highest value of μ was for C3 (10.9961 D). The
calculated α0 values for all the synthesized compounds were 16.2468 × 10-24 , 19.859 × 10-24,
23.183 × 10-24, 14.060 × 10-24, 16.034 × 10-24, 19.863 × 10-24, 19.063 × 10-24, 18.786 × 10-24
,
25.342 × 10-24, 19.771 × 10-24, 20.220 × 10-24 and 23.734 × 10-24 esu. The highest values of α0
were for C9 and C3 viz. 25.342 × 10-24 and 23.183 × 10-24 esu. The value of first β0 of all the
compounds C1-C12 were 2.620 × 10-30, 1.297 × 10-29, 3.727 × 10-29 , 3.883 × 10-30, 4.061 ×
10-30, 1.067 × 10-29, 1.750 × 10-29, 5.370 × 10-30, 3.553 × 10-30, 3.387 × 10-30, 2.968 × 10-30
and 1.486 × 10-30 esu, respectively. The highest values of β0 were for C3, C7, C2 and C6 viz.
3.727 × 10-29, 1.750 × 10-29, 1.297 × 10-29 and 1.067 × 10-29 esu as compared to other
alkylidenemalononitrile derivatives. Urea (β0 = 0.1947 × 10-30 esu) and para-nitro aniline (β0
= 1.426 × 10-29 esu) were used as reference compounds. The value of the molecular β0 is a
significant factor in an NLO system. Compounds C1-C12 possessed 13, 67, 191, 20, 21, 55,
90, 28, 18, 17, 15 and 8 times greater β0 value as compared to urea and compounds C3 and
C7 had β0 values 2.6 and 1.2 times greater than para-nitro aniline. Therefore, all the
alkylidenemalononitrile derivatives are potential candidates to be used as NLO materials
(Table 7).
Table 7. Total static dipole moment (μ), the mean polarizability (α0), the anisotropy of the
polarizability (∆α) and the first hyperpolarizability (β0) of all the synthesized compounds C1-
C12
NLO
C1
C2
C3
C4
C5
C6
C7
C8
C9
C10
C11
C12
Debye
7.8748
μx
μy
μz
-6.2476
0.3069
-1.3158
6.3921
4.8239 10.8945 -6.9359 -6.3936
6.2546
-7.9867 -5.8061
8.2205
0.9838
0.1385
8.2803
-4.5352 -8.4323
1.3942
0.1240
-1.4831 -2.4517 -1.4686 -5.0911 -0.1597 -1.1903
6.7084
-0.1794
8.8739
6.3774
0.1394
7.8269
2.4248
0.1362
8.7751
0.1551
0.0889
0.0999
6.5609
0.1204
9.3780
0.1341
6.2581
0.1148
8.0757
I
μ0
5.0228 10.9961 7.3570
1024 esu
αxx
αyy
αzz
αxy
αyz
αzx
α0
26.9780 37.4387 48.0172 26.9124 30.2012 36.1097 34.9316 33.6664 42.2311 34.9301 36.4064 40.8088
0.7034 -0.1879 0.0305 0.4881 0.0860 -0.7258 -1.5568 0.4564 -2.3357 -0.7783 -2.9731 -0.2503
21.0589 22.3269 21.5026 14.7793 17.8145 24.2043 22.2881 22.2365 36.1295 25.1599 27.2259 30.6448
-1.1249 -0.0003 -0.0001 -0.0012
0.0004
0.0002
5.7952
16.034
35.942
-0.0009 -0.0002 -0.0000
0.0938
1.6036
0.0000
0.0000
-0.0000 -0.0002
-1.1375
11.2705 7.1430
16.2468 19.859
0.0006
0.0001
9.0384
23.183
56.603
-0.0007
5.2067
14.060
31.825
0.0004
7.0150
19.863
44.112
-0.0005 -0.0000
0.0000
0.0813
20.220
-0.0002
7.9632
23.734
7.7307
19.063
35.236
6.6929 13.5017 8.0772
18.786
22.230
25.342
71.269
19.771
46.961
∆α
53.874
44.653
35.688 107.292
1033 esu
βxxx
βxyy
βxzz
βyyy
βyzz
βyxx
βzzz
βzxx
βzyy
β0
2175.59 -14373.7 -35583.5 6115.47 5440.94 -11504.6 -18668.6 6803.92 -2268.07 -6029.79 4652.64 4103.58
739.47 1266.06 -3229.83 -1870.28 -1063.96 -161.12 693.16 153.78 -383.99 1614.90 -3285.81 -2098.88
228.83 1546.55 -377.01 -317.95 1024.66 514.74 -1621.59 4223.22 1520.48 1454.93 -529.13
-448.71
-732.07 -1547.63 -66.46
-333.54 -112.29 -827.79 -1120.22 635.07 -2800.31 -1696.66 905.10
175.79
1.71
-10.46
-25.49
-42.50
25.06
59.95
-135.31
71.86
0.73
-1.54
-5.50
48.13
32.62
-12.94
-4.66
6.52
12.19
2.51
8.89
-67.36
-6.14
-0.13
-1.43
-10.85
-34.69
-10.29
-7.50
-6.63
-4.96
50.95
-44.11
18.84
4.63
-11.97
-64.34
176.33
-12.08
-26.87
-38.66
-25.85
15.81
81.99
-467.30
197.22
-232.05
-36.56
-2.45
8.14
9.60
276.64
195.17
6.15
3.36
3.36
-108.38
4.97
101.06
184.77
503.20
3.68
2619.682 12971.77 37270.41 3882.61
4061.00 10674.26 17495.43 5369.91 3552.72 3386.55 2967.97 1485.61
Chemical reactivity
23

Global reactivity descriptors
The most commonly utilized quantum chemical parameters for global reactivity descriptors
are EHOMO, ELUMO, EGAP, absolute softness (σ), absolute hardness (η), optical softness (σ0),
chemical potential (CP),absolute electronegativity (χ), additional electronic charges (∆Nmax
nucleophilicity index (N) and electrophilicity index (ω). According to Koopman’s theorem,
global reactivity trends are successfully calculated with the help of EHOMO and ELUMO
)
.
Electrophilicity index ω, a global reactivity index similar to CP and η were also calculated.
Electrophilicity index (ω) and its calculation were described by Parr et al. for the reactivity of
organic molecules. A single ω scale (ω > 1.5 eV (strong), ω = 1.5-0.8 eV (moderate) and ω <
0.8 eV (marginal) electrophiles) was developed showing higher value of ω favors high
electrophilicity. Therefore, ω is a new standard of stabilization in energy when the system
obtains a ∆Nmax from the environment. The values of EHOMO, ELUMO, EGAP, σ, η, S, CP, χ,
∆Nmax, N and ω for all the synthesized alkylidenemalononitrile derivatives have been enlisted
in Table 8.
Table 8. Calculated EHOMO, ELUMO, EGAP, absolute softness (σ), absolute hardness (η), optical
softness (σ0), chemical potential (CP),absolute electronegativity (χ), additional electronic
charges (∆Nmax) nucleophilicity index (N) and electrophilicity index (ω) for all the
synthesized compounds C1-C12 at DFT/B3LYP/6-31G(d,p) level
C1
C2
C3
C4
6.076
2.455
3.621
4.266
-4.266
1.811
0.552
0.276
5.025
0.199
2.356
C5
C6
6.152
2.288
3.864
4.220
-4.220
1.932
0.518
0.259
4.609
0.217
2.184
C7
6.291
2.643
3.648
4.467
-4.467
1.824
0.548
0.274
5.470
0.183
2.447
C8
6.750
3.591
3.165
5.171
-5.171
1.583
0.632
0.316
8.447
0.118
3.257
C9
6.442
3.544
2.898
4.993
-4.993
1.449
0.690
0.345
8.603
0.116
3.446
C10
6.585
3.531
3.054
5.058
-5.058
1.527
0.655
0.327
8.377
0.119
3.312
C11
6.729
3.791
2.938
5.260
-5.260
1.469
0.681
0.340
9.417
0.106
3.581
C12
6.786
3.495
3.291
5.141
-5.141
1.646
0.608
0.304
8.029
0.125
3.124
IPI
EAI
EGAP
휒I
7.357
2.545
4.812
4.951
-4.951
2.406
0.416
0.208
5.094
0.196
2.058
7.244
3.052
4.192
5.148
-5.148
2.096
0.477
0.239
6.322
0.158
2.456
6.941
2.645
4.296
4.793
-4.793
2.148
0.466
0.233
5.348
0187
7.073
2.916
4.157
4.995
-4.995
2.079
0.481
0.241
6.001
0.167
2.403
I
CPI
ηI
σI
II
σ0
ωI
NII
I
∆Nmax
2.231
Local reactivity descriptors
The conventional chemical reactivity and site selectivity of molecular system are recognized
with the help of Fukui function. Fukui functions are calculated for a large number of organic
molecules [44]. Fukui function is denoted by f(r).
Fukui function is mainly associated with the electron density
 r of the system
 


to a change in the total number of electrons N   r dr in the limitation of a constant
 



external potential acting on an electron (v(r)). Finite difference calculation classified three
f  f o r f 
r
r
(for nucleophilic, radical
variables Fukui function for a system
and electrophilic attack) from N, as effect of the discontinuity of the electron density [51].
In a molecular system, high value of f 
for an atom represents most likely nucleophilic
,
and
 
 
 
r
 
attack and high value of f 
r
refers to electrophilic attack. To describe the electrophilic
 
and nucleophilic reactive region for the evaluation of the intermolecular reactivity, Roy et al.
24

S
[52] suggested the relative electrophilic local softness
, the nucleophilic local softness


k
S and the radical local softness So descriptors for a kth atom.


 k 
k
Chattaraj et al. [53] also offered three global local electrophilicity indices o


k
k
k
to recognize the radical, electrophilic and nucleophilic region in reactivity and regio-
selectivity evaluation based on Parr global electrophilicity index (ω) and Fukui functions
f o f  f 
f r
 
Recently, a new dual descriptor
was suggested by Morrel et al. for the




k
k
k
nucleophilicity and electrophilicity [54].
The statistical values of are given on the scale of -1, 0 to 1. If
,
f r
 
f r  0
 




suggesting that the site is appropriate for nucleophilic attack and
shows almost
f r  0
 


no nucleophilic attack and a favorable electrophilic attack.
Local reactivity descriptors based on electron density such as Fukui function
, local electrophilicity indices o , local softness
and dual
f o f  f 
SoSS
k k k






k
k
k
k
k
k
descriptor f r  0 describe a specific region of the chemo- or regio-selectivity of a
 


molecular system. The findings of Fukui function, local electrophilicity, local softness and
dual descriptor indices of atoms of the all the synthesized compounds are given in Table 9.
Table 9. Using MPA, Fukui function, local softness, local electrophilicity and dual descriptor
indices in eV for αC and βC
C1
C2
C3
C4
C5
C6
C7
C8
C9
C10
C11
C12
αC -4.7104 -2.9035 -2.3892 -4.2097 -2.7103 -3.8723 -3.8042 -2.2831 -3.1457 -2.5606 -2.5634 -2.6124
βC -4.2750 -2.3674 -4.0464 -3.2546 -1.8531 -3.2899 -3.0287 -1.4531 -1.4586 -1.4776 -1.5783 -0.7320
αC -9.9977 -10.2099 -10.7242 -10.8984 -10.6072 -11.0127 -10.8957 -9.1187 -8.9908 -9.0861 -8.8820 -9.0752
βC -7.4534 -6.3839 -8.2398 -6.6125 -6.4601 -6.8493 -6.5608 -3.7172 -3.5593 -3.5811 -3.7417 -3.3335
αC -6.2778 -6.8438 -7.1132 -7.9976 -7.1894 -7.9949 -7.7962 -5.4397 -5.4506 -5.4560 -5.2057 -5.5567
βC -2.3402 -2.5008 -4.0328 -2.3212 -2.1851 -2.5008 -2.2096 -0.2803 -0.1333 -0.0844 -0.3510 0.1007
αC 3.7199
βC 5.1131
αC 1.5674
3.3661
3.8859
3.9403
3.6110
4.2043
4.7240
2.8981
4.2913
3.7879
3.4151
4.2750
4.4791
3.0178
4.3485
4.1226
3.0994
4.3512
3.9893
3.6791
3.4369
3.1539
3.5430
3.4260
2.3021
3.6301
3.4967
2.8954
3.6763
3.3906
2.6423
3.5185
3.4342
2.9443
βC -1.9348 0.1333 -0.0109 -0.9334 0.3320 -0.7891 -0.8191 -1.1728 -1.3252 -1.3933 -1.2300 -0.8300
αC 2.6423
βC 1.5892
3.6546
2.0082
4.1689
2.0980
3.3444
1.6790
3.9485
2.3021
3.5702
1.7797
3.5457
1.7661
3.4178
1.1320
2.9226
1.0504
3.2627
1.0504
3.1593
1.0803
3.2328
1.3007
αC 22.2213 23.1792 25.4976 22.7003 22.3411 21.2526 23.1193 31.6884 33.2639 32.3496 34.0531 29.1958
βC 30.5455 26.7576 29.6856 33.6123 27.9658 30.6271 32.4558 29.6039 32.1673 31.1632 31.4054 28.4964
αC 9.3636 27.1331 33.3565 29.6692 29.2992 29.0352 29.7563 27.1657 21.6145 25.8024 24.4745 24.4337
βC -11.5569 0.9170 -0.0762 -7.3119 2.1715 -5.5581 -6.1091 -10.1011 -12.4413 -12.4168 -11.3937 -6.8874
αC 15.7830 25.1657 29.4379 26.1943 25.8296 25.1466 26.4473 29.4379 27.4406 29.0760 29.2638 26.8256
βC 9.4943 13.8291 14.8142 13.1516 15.0591 12.5338 13.1733 9.7501
9.8616
0.2640
0.2531
0.1714
9.3609 10.0059 10.7923
αC 0.2286
βC 0.3157
αC 0.0952
0.2367
0.2721
0.2748
0.2041
0.2367
0.2667
0.1306
0.1905
0.1687
0.2041
0.2558
0.2667
0.1361
0.1959
0.1850
0.1606
0.2259
0.2068
0.2721
0.2558
0.2340
0.2640
0.2558
0.2095
0.2966
0.2721
0.2123
0.2612
0.2531
0.2177
βC -0.1197 0.0082
0.0000 -0.0408 0.0190 -0.0354 -0.0435 -0.0871 -0.0980 -0.1007 -0.0980 -0.0626
αC 0.2884
0.4735
0.4599
0.2912
0.4354
0.3021
0.3565
0.5306
0.4626
0.5034
0.5470
0.4789
βC 0.1714
0.2585
0.2313
0.1442
0.2531
0.1497
0.1769
0.1769
0.1660
1.2409
2.1008
0.1606
0.7347
2.1008
0.1878
1.0313
2.1606
0.1932
0.5742
2.6042
αC 2.1497 -0.5742 -1.1130 -0.0408 -1.0640 -1.1048 -0.8898 0.5252
βC 3.1784 3.7525 4.1934 0.2340 3.9457 3.5593 3.5321 2.2640
The alkylidenemalononitrile derivatives were characterized by the presence of two nitrile
groups and activated double bond (
). The Cα (alpha carbon) which is directly
25

attached with the two nitrile groups, in the presence of high electronegativity nitrogen atom
becomes partially positive and attracts the electrons of the bond and Cβ (beta carbon)
becomes partially positively charged. Thus, Cβ becomes more suitable for the nucleophilic
fk
attack. The value of
Cβ is higher for compound C1-C7 (2-benzylidene malononitrile
derivatives) which was between 3.8859-5.11313 eV as compared to Cα which was between
2.8981-3.7199 eV. But in compounds C8-C12 (2-(2-oxo-indolin-3-ylidene) malononitrile
fk
derivatives), the value of
Cβ is slightly lower (3.3906-3.4967 eV) as compared to Cα
(3.5185-3.6791 eV) as shown in Supplementary Table 11. Thus, in this case it refers to
electrophilic attack. These variations are due to the presence of active hydrogen on Cβ
(compound C1-C7) and Cβ in ring (compound C8-C12). Furthermore, the value of
f r
 


supports nucleophilic attack at Cβ for all the compounds (C1-C12). Finally, it was found that
Cβ principal reactivity nucleophilic site is related to the electrophilic and radical attack
Figure 10 & 11.
Figure 10. Comparison of dual descriptor
values in eV for Cα and Cβ in all
synthesized compounds C1-C12
26

Figure 11. Displayed graphical representation of most favorable site for nucleophilic attack
with dual descriptor and Fukui function
Mulliken population analysis (MPA)
The MPA is used to evaluate the Mulliken charge (MC). The evaluations of MC play a
dynamic role in the statistical calculation of effects of atomic charge in the exact molecular
system that is necessary for the observation of bonding propensities and modification of
structural confirmations [51]. In this study, MPA was calculated by DFT/B3LYP/6-31G(d,p)
method and presented in the Supplementary Table 6. It was found the all the carbon atoms
of all compounds C1-C12 were negatively charged except 2C, 8C, 11C and 13C for C1; 2C,
8C, 11C and 13C for C2; 2C, 5C, 11C and 13C for C3; 2C, 5C, 8C, 12C and 14C for C4; 2C,
8C, 9C and 11C for C5; 2C, 3C, 4C, 9C, 12C and 14C for C6;2C, 4C, 6C, 9C, 12C and 14C
for C7; 4C, 5C, 8C, 9C, 10C and 12C for C8; 4C, 5C, 8C, 9C, 10C and 12C for C9; 4C, 5C,
8C, 9C, 10C, 13C and 15C for C10; 1C, 4C, 5C, 8C, 9C, 10C, 13C and 15C for C11; 4C, 6C,
7C, 8C, 9C, 14C, 15C and 17C for C12, respectively. These differences were observed due to
the bonding with electronegative atoms. The hydrogen atoms had positive charge in the
region 0.09-0.34. Nitrogen atom of nitrile groups of all the synthesized
alkylidenemalononitrile derivatives (C1-C12) possessed negative charge between 0.450-
0.511, respectively. Heterocyclic oxygen of compound C3 and nitrogen of compounds C6,
C8, C9, C10 and C11 possessed negative charges -0.436, -0.604, -0.674, -0.706, -0.602 and -
0.677, respectively and it was observed that negative charge of heteroatoms increased with
the increase of cyclization such as from C3 (-0.436) to C9 (-0.706). Furthermore, it was also
found that exocyclic oxygen also carried high negative charge between -0.431-0.565.
27

Natural population analysis (NPA)
The value of natural charge from NPA denotes statistical stability and defines the electron
distribution in the molecular system. The NPA is listed in Supplementary Table 7. The carbon
atoms possessed negative charge with the exception of 11C and 13C for C1; 3C, 11C and
13C for C2; 2C, 5C, 9C and 11C for C3; 5C, 12C and 14C for C4; 9C and 11C for C5; 2C,
9C, 12C and 14C for C6; 6C, 9C, 12C and 14C for C7; 4C, 5C, 8C, 9C, 10C, 12C and 14 for
C8; 4C, 8C, 12C, 18C and 20C for C9; 4C, 8C, 13C and 15C for C10; 4C, 8C, 13C and 15C
for C11; 8C, 9C, 15C and 17C for C12, respectively. These differences were due to
attachment of electronegative atoms. All carbon atoms of nitrile group were positively
charged between 0.268-0.284 while, nitrogen atoms carried negative charge between -0.237-
0.308, respectively. Exocyclic oxygen atoms also possessed negative charge -0.535, -0.533, -
0.546, -0.531 and -0.485, respectively for compounds C8-C12. Furthermore, heterocyclic
oxygen (-0.430 for C3) and nitrogen (-0.539, -0.634, -0.451, -0.444 and -0.633 for
compounds C6, C8, C9, C10 and C11) atoms also carried negative charges.
NBO analysis
NBO study gives maximum information for the evaluation of hybridization, covalence,
hydrogen-bonding and Van der Waals interactions along with electron density delocalization
from donor (Lewis-type NBO) and acceptor (non-Lewis-type NBO) orbitals [25]. Second-
order perturbation theory is utilized to calculate the strength of interaction between Lewis and
non-Lewis NBO. For each donor (i) and acceptor (j), the stabilization energy (E(2)) related
with electron delocalization among donor and acceptor was calculated. High stabilization
energy represents strong interaction among donor (i) and acceptor (j) orbitals [25]. The
electron density at the conjugated π bonds was (1.64622-1.67852 for C1, 1.62618-1.66606
for C2, 1.74022-1.79581 for C3, 1.57667-1.73786 for C4, 1.60625-1.65897 for C5, 1.61231-
1.71992 for C6, 1.62236-1.72251 for C7, 1.63161-1.65724 for C8, 1.63047-1.67581 for C9,
1.63588-1.65938 for C10, 1.63767-1.65801 for C11 and 1.52388-1.70939 for C12) and π*
bonds (0.31498–0.43276 for C1, 0.27300–0.46569 for C2, 0.26326-0.34597 for C3, 0.26715-
0.44319 for C4,0.27806-0.39711 for C5, 0.28802-0.47344 for C6, 0.33973-0.41917 for C7,
0.33117-0.39311 for C8, 0.31527-0.39339 for C9, 0.32824-0.38727 for C10, 0.36756-
0.38788 for C11 and 0.24583-0.43331 for C12) of conjugated ring robust π-electron
delocalization under the ring system prominent to a highest stabilization of energy 22.85 for
C1, 22.21 for C2, 24.07 for C3, 28.75 for C4, 23.34 for C5, 21.25 for C6, 23.77 for C7, 24.45
for C8, 24.42 for C9, 24.16 for C10, 23.08 for C11 and 20.20 for C12 kcal/mol. Therefore,
the donor (i) aptitude of π bonding NBOs are higher with lowest occupancy as compared to σ
bonding on the other hand highest occupancies of the π* anti-bonding NBOs indicate their
better acceptor (j) aptitude. The alkylidenemalononitrile derivatives (C1-C12) were
characterized by the presence of two nitrile groups, which played a major role the in
stabilization and reactivity of the molecular system. Hence, a significant resonance in the
molecule system was found from the electron donation by nitrogen atom n(1)N (first) and
n(1)N (second) to the adjacent σ*(1)C−C bond with the stabilization energy of between
12.81-13.22 kcal/mol depicted in the Table 10.
Table 10. Second order perturbation theory analysis of the Fork matrix in the NBO basis for
intramolecular interaction for the compounds C1-C12 unit 1
Donor NBO (i)
Occupancy
Acceptor NBO (j)
C1 (within unit 1)
σ* (1)C8−C11 0.03504
Occupancy (E2)a (kcal/mol) E(j)-E(i)b (a.u.) F (i,j)c (a.u.)
σ (1)C11−N12
1.99568
6.15
1.53
0.087
28

n (3)C11−N12
σ (1)C13−N14
n (3)C13−N14
n(1)N12
1.95640
1.99555
1.95332
1.96920
1.96899
π* (2)C1−C8
σ* (1)C8−C13
π* (2)C1−C8
σ* (1)C8−C11
σ* (1)C8−C13
0.13576
0.03834
0.13576
0.13576
0.03834
8.92
6.18
9.54
12.94
13.01
0.35
1.53
0.35
1.00
1.00
0.051
0.088
0.053
0.102
0.102
n(1)N14
C2 (within unit 1)
0.03359
σ (1)C11−N12
n (3)C11−N12
σ (1)C13−N14
n (3)C13−N14
n(1)N12
1.99569
1.95677
1.99505
1.95377
1.96873
1.96810
σ* (1)C8−C11
π* (2)C1−C8
σ* (1)C8−C13
π* (2)C1−C8
σ* (1)C8−C11
σ* (1)C8−C13
6.18
8.27
6.20
8.94
13.06
12.96
1.53
0.34
1.54
0.35
1.00
1.01
0.087
0.049
0.088
0.052
0.102
0.102
0.20078
0.03905
0.20078
0.03359
n(1)N14
0.03905
C3 (within unit 1)
0.03310
σ (1)C9−N10
n (3)C9−N10
σ (1)C11−N12
n (3)C11−N12
n(1)N10
1.99566
1.95830
1.99547
1.95331
1.96898
1.96925
σ* (1)C7−C9
π* (2)C6−C7
σ* (1)C7−C11
π* (2)C6−C7
σ* (1)C7−C9
σ* (1)C7−C11
6.24
8.17
6.30
9.09
12.96
12.96
1.53
0.34
1.54
0.34
1.01
1.01
0.088
0.050
0.089
0.052
0.102
0.102
0.25630
0.03690
0.25630
0.03310
n(1)N10
0.03690
C4 (within unit 1)
0.03312
σ (1)C12−N13
n (3)C12−N13
σ (1)C14−N15
n (3)C14−N15
n(1)N13
1.99568
1.96401
1.99510
1.96041
1.96880
1.96819
σ* (1)C8−C12
π* (2)C1−C8
σ* (1)C8−C14
π* (2)C1−C8
σ* (1)C8−C12
σ* (1)C8−C14
6.20
7.46
6.23
8.08
12.90
12.81
1.54
0.34
1.55
0.35
1.01
1.02
0.088
0.048
0.088
0.050
0.102
0.102
0.26253
0.03793
0.26253
0.03312
n(1)N15
0.03793
C5 (within unit 1)
0.03356
σ (1)C9−N10
n (3)C9−N10
σ (1)C11−N12
n (3)C11−N12
n(1)N10
1.99572
1.95958
1.99513
1.95599
1.96881
1.96825
σ* (1)C8−C9
σ* (1)C1−C8
σ* (1)C8−C11
π* (2)C1−C8
σ* (1)C8−C9
σ* (1)C8−C11
6.16
8.04
6.20
8.73
12.98
12.90
1.53
0.34
1.54
0.35
1.01
1.01
0.087
0.049
0.088
0.051
0.102
0.102
0.20653
0.03852
0.20653
0.03356
n(1)N12
0.03852
C6 (within unit 1)
σ (1)C12−N13
n (3)C12−N13
σ (1)C14−N15
n (3)C14−N15
n(1)N13
1.99475
1.96224
1.99577
1.96515
1.96704
1.96850
σ* (1)C11−C12
σ* (1)C10−C11
σ* (1)C11−C14
π* (2)C10−C11
σ* (1)C11−C12
σ* (1)C11−C14
0.03796
0.27038
0.03279
0.27038
0.03796
0.03279
6.38
8.21
6.13
7.32
12.80
12.94
1.55
0.35
1.53
0.34
1.02
1.01
0.089
0.051
0.087
0.047
0.102
0.102
n(1)N15
C7 (within unit 1)
0.03815
σ (1)C12−N13
n (3)C12−N13
σ (1)C14−N15
n (3)C14−N15
n(1)N13
1.99502
1.96015
1.99567
1.96237
1.96783
1.96880
σ* (1)C2−C12
σ* (1)C2−C3
σ* (1)C2−C14
π* (2)C2−C3
σ* (1)C2−C12
σ* (1)C2−C14
6.18
8.12
6.22
7.68
12.80
12.96
1.55
0.35
1.53
0.34
1.02
1.01
0.088
0.050
0.088
0.048
0.102
0.102
0.24483
0.03325
0.24483
0.03815
n(1)N15
0.03325
C8 (within unit 1)
σ (1)C12−N13
π (2)C12−N13
σ (1)C14−N15
π (2)C14−N15
n(1)N13
1.99526
1.98174
1.99546
1.98181
1.96793
1.96884
σ* (1)C10−C12
π* (2)C9−C10
σ* (1)C10−C14
π* (2)C9−C10
σ* (1)C10−C12
σ* (1)C10−C14
0.03467
0.24355
0.03593
0.24355
0.03467
0.03593
6.12
8.92
6.26
9.87
13.00
13.18
1.54
0.35
1.53
0.34
1.01
1.00
0.087
0.053
0.088
0.054
0.102
0.103
n(1)N15
C9 (within unit 1)
σ (1)C18−N19
π (2)C18−N19
σ (1)C20−N21
π (2)C20−N21
n(1)N19
1.99525
1.98165
1.99545
1.98181
1.96791
1.96880
σ* (1)C10−C18
0.03452
0.24513
0.03589
0.24513
0.03452
0.03589
6.13
8.87
6.25
9.82
12.99
13.18
1.54
0.36
1.53
0.34
1.01
1.00
0.087
0.05
0.088
0.054
0.102
0.103
π* (2)C9−C10
σ* (1)C10−C20
π* (2)C9−C10
σ* (1)C10−C18
σ* (1)C10−C20
n(1)N21
C10 (within unit 1)
σ (1)C13−N14
π (2)C13−N14
σ (1)C15−N16
π (2)C15−N16
n(1)N14
1.99527
1.98177
1.99546
1.98184
1.96794
1.96884
σ* (1)C10−C13
0.03461
0.24212
0.03591
0.24212
0.03461
0.03591
6.11
8.87
6.25
9.82
12.99
13.17
1.54
0.36
1.53
0.34
1.01
1.00
0.087
0.053
0.088
0.054
0.102
0.103
π* (2)C9−C10
σ* (1)C10−C15
π* (2)C9−C10
σ* (1)C10−C13
σ* (1)C10−C15
n(1)N16
C11 (within unit 1)
σ (1)C13−N14
π (2)C13−N14
σ (1)C15−N16
π (2)C15−N16
n(1)N14
1.99545
1.98178
1.99522
1.98155
1.96885
1.96786
σ* (1)C10−C13
0.03599
0.23778
0.03473
0.23778
0.03599
0.03473
6.28
10.10
6.14
9.16
13.22
13.06
1.53
0.34
1.54
0.35
1.00
1.01
0.088
0.055
0.087
0.053
0.103
0.103
π* (2)C9−C10
σ* (1)C10−C15
π* (2)C9−C10
σ* (1)C10−C13
σ* (1)C10−C15
n(1)N16
29