A novel method for the synthesis and characterization of 10-hexyl-3-(1-hexyl-4, 5-diphenyl-1H-imidazol-2-yl)-10H-phenothiazine: DFT computational, in vitro anticancer and in silico molecular docking studies

Article abstract of DOI:10.1007/s11164-020-04297-3

This study aimed to newly synthesized compound 10-hexyl-3-(1-hexyl-4, 5-diphenyl-1H-imidazol-2-yl)-10H-phenothiazine (abbreviated as HHDIP) by direct and thermal condensation methods, which were characterized by different spectral analysis. The optimized geometry completed the theoretical calculation; the Fourier-transform infrared spectroscopy (FTIR) vibrational frequency studies of HHDIP were found by using the (density functional theory) ab initio calculation with B3LYP/6-311++G (d,p) level. The computed and scaled vibrational frequency values were well matched with the experimental FTIR and Fourier-transform Raman spectroscopy (FT-Raman) spectra. A particular understanding of the FTIR spectra of this title compound was performed by the computed potential energy distribution. The UV–Vis, ¹H, and ¹³C NMR investigations were completed as well as observed; these theoretical outcomes have been seen as in good concurrence with the experimental value. The mass spectroscopy investigation was completed to this compound. In addition to this title compound, the computed (HOMO and LUMO) energy values have been revealed with the charge transfer inside the organic molecules. Nonlinear optics and Mulliken population studies were completed for this title compound. Finally, intensive studies on their anticancer properties were studied through in vitro as well as in silico approaches, suggesting our title compound exhibited tremendous anticancer activity at the concentration value of 250?μg/mL. The in silico docking and adsorption, distribution, metabolisms, excretion and toxicity (ADMET) studies were executed through commercial docking software Discovery Studio, version 4.0 against the protein target c-Met kinase (hepatocyte growth factor; PDB ID: 3F66). The results showed a ligand–receptor interaction energy value of ? 64.494 KCal/mol against 3F66 protein (standard anticancer drug tivantinib exhibited ? 57.804 KCal/mol). In the ADMET study, it gives good results of nonmutagenic and noncarcinogenic activities. However, in vitro studies exposed more significant antioxidant activity at the range of hydrogen peroxide (H₂O₂) scavenging activity as 66.31% and 52.877% which showed a vigorous DPPH radical scavenging antioxidant activity concentration value of 500?μg/mL. Further studies are desired to explore its promising anticancer, clinical research and other pharmacological aspects.

Full text of DOI:10.1007/s11164-020-04297-3

Research on Chemical Intermediates
https://doi.org/10.1007/s11164-020-04297-3
A novel method for the synthesis
and characterization of 10‑hexyl‑3‑(1‑hexyl‑4,
5‑diphenyl‑1H‑imidazol‑2‑yl)‑10H‑phenothiazine: DFT
computational, in vitro anticancer and in silico molecular
docking studies
J. Irshad Ahamed, et al. [full author details at the end of the article]
Received: 14 August 2020 / Accepted: 8 October 2020
© Springer Nature B.V. 2020
Abstract
This study aimed to newly synthesized compound 10-hexyl-3-(1-hexyl-4, 5-diphe-
nyl-1H-imidazol-2-yl)-10H-phenothiazine (abbreviated as HHDIP) by direct and
thermal condensation methods, which were characterized by diferent spectral anal-
ysis. The optimized geometry completed the theoretical calculation; the Fourier-
transform infrared spectroscopy (FTIR) vibrational frequency studies of HHDIP
were found by using the (density functional theory) ab initio calculation with
B3LYP/6-311++G (d,p) level. The computed and scaled vibrational frequency val-
ues were well matched with the experimental FTIR and Fourier-transform Raman
spectroscopy (FT-Raman) spectra. A particular understanding of the FTIR spectra
of this title compound was performed by the computed potential energy distribution.
The UV–Vis, 1H, and 13C NMR investigations were completed as well as observed;
these theoretical outcomes have been seen as in good concurrence with the experi-
mental value. The mass spectroscopy investigation was completed to this compound.
In addition to this title compound, the computed (HOMO and LUMO) energy values
have been revealed with the charge transfer inside the organic molecules. Nonlin-
ear optics and Mulliken population studies were completed for this title compound.
Finally, intensive studies on their anticancer properties were studied through in vitro
as well as in silico approaches, suggesting our title compound exhibited tremendous
anticancer activity at the concentration value of 250 µg/mL. The in silico docking
and adsorption, distribution, metabolisms, excretion and toxicity (ADMET) studies
were executed through commercial docking software Discovery Studio, version 4.0
against the protein target c-Met kinase (hepatocyte growth factor; PDB ID: 3F66).
The results showed a ligand–receptor interaction energy value of −64.494 KCal/mol
against 3F66 protein (standard anticancer drug tivantinib exhibited −57.804 KCal/
Electronic supplementary material The online version of this article (https://doi.org/10.1007/s1116
4-020-04297-3) contains supplementary material, which is available to authorized users.
Vol.:(0123456789)
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J. I. Ahamed et al.
mol). In the ADMET study, it gives good results of nonmutagenic and noncarci-
nogenic activities. However, in vitro studies exposed more signifcant antioxidant
activity at the range of hydrogen peroxide (H2O2) scavenging activity as 66.31% and
52.877% which showed a vigorous DPPH radical scavenging antioxidant activity
concentration value of 500 µg/mL. Further studies are desired to explore its promis-
ing anticancer, clinical research and other pharmacological aspects.
Keywords Synthesis of new HHDIP · Spectroscopic analysis · DFT computational ·
Anticancer · Molecular docking
Introduction
Nowadays, chemotherapy is routinely applied to treat the cancer disease although it
has diferent kinds of toxicities such as myelotoxicity, cardiotoxicity, renal toxicity
and pulmonary toxicity. These side efects create serious health problems and some-
times lead to death. Cancer is yet a life-threatening disease due to a lack of efective
anticancer drugs and the side efects of chemotherapy [1]. Therefore, eforts are still
being needed to develop high-active anticancer agents for the successful treatment
for cancer. Heterocyclic class of the chemical compound works for a vital function in
modern drug discovery by a structural modifcation to prepare several compounds.
The imidazole-containing compounds have several biological activities as well as
pharmaceutical signifcance with anti-HIV, antidiabetic and antimalarial activities
[2–8]. Imidazole products are illustrated to be pharmacologically and physiologi-
cally active that are utilized in the treatment for various diseases. Its portion-con-
taining compounds are obtained to be a great number of novel chemotherapeutic
materials based on imidazole nucleus, natural products, clinical active molecules,
and several medicines possess the imidazole ring, comprising ketoconazole utilized
for the treatment for bacterial infections, gastric ulcers and fungal infections [9–11].
The search of a new imidazole agent for the cure of cancer is an essential tool in
medicinal chemistry [12]. A new acidic ionic liquid (AIL) based on imidazolium
cations is used as a catalyst for the one-pot synthesis of 2-aryl- 1H-phenanthro[9,10-
d]imidazole derivatives [13]. The 1,2,4,5-tetrasubstituted imidazole derivatives have
shown enhanced anticancer and antibacterial activities against Huh-7 hepatocellular
carcinoma cell and four bacterial species names such as wild-type Escherichia coli,
wild-type Staphylococcus aureus, Pseudomonas aeruginosa PAM1032, and Escher-
ichia coli-NMD-1 [14]. The preparation of chalcones-imidazole (E)-1-(4-((1H-
imidazol-1-yl) methyl) phenyl)-3-phenylprop-2-en-1-one derivatives has exhibited
superior antibacterial and antifungal activities [15]. Benzimidazole derivatives
have shown superior antiviral and anticancer biological activities [16]. A series of
1-(2-(4-morpholinomethyl)-1H-benzoimidazol-1-yl)propan-2-one oxime-ethers has
excellent antifungal activities [17]. The syntheses of these heterocyclic compounds
are cost-efective, and their initial materials are readily available. Owing to their
signifcance, they have become a suitable goal for synthetic and clinical uses. The
newly synthesized compound of HHDIP has performed the diferent spectroscopic
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A novel method for the synthesis and characterization of…
analyses by using experimental and theoretical density functional theory (DFT)
calculation with B3LYP/6-311++G (d,p) level. Furthermore, in vitro and in silico
methods have been done against cancer studies; it was well known to act as an efec-
tive agent of anticancer in this present work.
Materials and methods
Chemicals and Reagents
The following chemicals and reagents such as benzil, phenothiazine, n-bromohexane
(Sigma-Aldrich, USA), Silica gel with a mesh of 60–120 (Merck, India), precoated
aluminum sheet of silica gel G/UV-254 of 0.2 mm thickness (Merck, Germany),
K2CO3, hexane-1-amine, NH4OAC, ethyl alcohol, methanol, CH3COOH, POCl3,
EtOAc, Na2SO4 and sodium hydroxide (Spectrochem, India) were utilized for the
synthesis of imidazole compound.
Spectral characterization
1
The title compound was recorded by infrared (IR), H and 13C (NMR—nuclear
magnetic resonance) data information, and its melting point was found by a super-
ft (India) capillary melting-point apparatus. The title molecule IR spectrum was
accomplished on a Perkin Elmer spectrometer V 10.03.06 utilizing the potassium
bromide (KBr) disk, and FT-Raman spectrum was also completed in 4000–100 cm−1
range on BRUCKER RFS 27 utilizing Nd:YAG laser (excitation wavelength) with
the resolution of 2 cm−1 at SAIF, IIT, Chennai. The UV–Vis: λmax spectrum of
compounds was recorded using Shimadzu UV–Vis spectrometer. The 1HNMR spec-
tra were confrmed on a Bruker Avance (400 MHz) spectrometer in Chloroform-D
(CDCl3) solvent, and chemical shift values were confrmed in units δ (ppm) relative
to tetramethylsilane (Me4Si) as an internal standard. 13C-NMR spectra were con-
frmed at 100 MHz in CDCl3 with the same internal standard. The mass spectra
were performed on Total Ion Current (TIC) Chromatogram spectrometers. Elemen-
tal analyses were executed on 2400 Series II PerkinElmer CHNS/O Elemental Ana-
lyzer (India). Elemental (CHNS/O) analysis specifed that the calculated and found
values were within the acceptable limits ( 0.4%). Reactions were observed by
means of TLC utilizing precoated plates (Merck, India). Column chromatographic
separation was done by silica gel with a mesh of 60–120.
Synthesis of HHDIP
The 10-hexyl-10H-phenothiazine-3-carbaldehyde (1 g, 3.2 mmol), benzil (0.6 g,
3.2 mmol), hexan-1-amine (0.3 g, 0.0032 mmol) and ammonium acetate (NH4OAc)
(0.4 g, 8 mmol) were mixed to a 100 mL RB fask, and 20 mL of acetic acid
(CH3COOH) was added to this reaction mixture. The reaction was refuxed at
70–90 °C for 3 h. The improvement in the fnal reaction was monitored by TLC.
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J. I. Ahamed et al.
After the reaction ended, the crude mass was permitted to be purifed by 60–120
mesh column chromatography techniques using the mixture of 40% of hexane/
EtOAc as eluent.
Experimental data
Yield: 95%; brown solid; mp:523–524 °C; IR (KBr, ν, cm−1): 3325, 2974, 2881,
1654, 1379,, 1328, 1274, 1087, 1045, 879, 802, 605; (FT-Raman, ν, cm−1); 3239,
3118, 3034, 1667, 1604, 1563, 1509, 1435, 1279, 1038, 984; UV–Vis: λmax
364 nm;1H NMR (400 MHz, CDCl3): δ 0.74–0.70 (t, J=6.9 Hz, 3H), 0.86–0.89
(m, 3H), 0.94–0.99 (m, 4H), 1.03–1.08 (m, 2H), 1.291.32(m, 6H), 1.33–1.48(m,
2H), 1.78–1.86(m, 2H), 3.82–3.89 (m, 4H), 6.88–6.94 (m, 3H), 7.09–7.20 (m, 5H),
7.38–7.51 (m, 9H); 13C NMR (100 MHz, CDCl3): 13.82, 13.99, 22.23, 22.60, 25.89,
26.59, 26.80, 30.36, 30.81, 31.45, 44.76, 47.76, 115.16, 115.46, 122.59, 124.48,
125.16, 126.17, 126.83, 127.47, 127.82, 128.01, 128.11, 128.58, 129.00, 129.03,
129.56, 129.92, 131.05, 131.61, 134.57, 144.91, 145.65, 146.87; Analysis calcd. %
for C39H43N3S: C, 79.96; H, 7.40; N, 7.17; S, 5.47; Found %: C, 79.84; H, 7.34; N,
7.03; S, 5.32; MS (ESI) for C39H43N3S: m/z 586.60 [M+H] +.
Computational details
The DFT-optimized geometry of the HHDIP was computed by B3LYP (Beckee-
3-Lee–Yang–Parr) method, Gaussian 09 program package as well as included
6-311++G (d,p) basis set [18–21]. In DFT study, the ab initio method of molecu-
lar calculating properties has recently enhanced [22–24]. The optimized structural
parameters were utilized for simulating vibrational frequencies, and their frequency
assignments were completed accurately through VEDA program. To fnd visual ani-
mation and to perform verifcation of the (normal modes) assignment, Gauss View
program [25] has been utilized. The (FMOs—Frontier molecular orbitals) energies,
molecular optimization structure, bond parameters, Mulliken population analyses,
NLO, thermodynamic studies have been completed by DFT method. Electronic
properties were completed by DFT time-dependent (TD-DFT) methods [26–28].
1
Proton H and carbon13C NMR spectra results were completed, and molecules
chemical shifts were computed using (GIAO—gauge-independent atomic orbital)
method [29].
In vitro anticancer activity
The liver cancer (HepG2) cell line was used to check the anticancer activity of the
compound HHDIP. Cells were cultured and conserved in Minimal Essential Media
(MEM) (Hi-Media, India) enhanced with 10% fetal bovine serum (FBS) Cistron
Laboratories), Trypsin, MTT (methyl thiazolyl diphenyl-tetrazolium bromide) and
dimethyl sulfoxide (DMSO) HiMedia, India). Cells were incubated at 37 °C in a
5% CO2 atmosphere; they were regularly subcultured and preserved in 2% MEM. A
confuent monolayer of HepG2 cells was trypsinized and suspended in 10% MEM;
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A novel method for the synthesis and characterization of…
100 µL of cells suspension was transferred to 96 well plates, and 10% MEM was
added as well as the plate incubated at 37 °C at 5% CO2 environment. Various
concentrations of compounds were prepared as 100 µg/mL, 50 µg/mL, 25 µg/mL,
12.5 µg/mL, 6.25 µg/mL, 3.125 µg/mL, 1.56 µg/mL and 0.78 µg/mL and transferred
in 96 well plates, and fnally 100 µL of 2% MEM was added. Morphological altera-
tions of the HepG2 cells were monitored every 24 h for the following 3 days, and
fnally the changes were noted after 72 h of period. The highest concentration which
did not show cellular toxicity was considered as maximum nontoxic concentration
of the compound. To more, the cytotoxicity concentration was confrmed by MTT
(3-(4, 5-dimethylthiazol-2-YL)-2, 5-diphenyltetrazolium bromide) assay. In this
assay, after 72 h of incubation of 96 well plate, 20 µL of (MTT) solution (5 mg/mL)
was mixed in all the wells with the plate incubated at 37 °C with 5% CO2 for 4 h.
After 4 h of incubation, the medium in the plate was carefully removed without dis-
turbing the cell monolayer. Finally, a volume of 120 µL of DMSO was mixed to the
wells to dissolve the formazan crystals and read at 510 nm and 650 nm. The formula
utilized for measuring cell viability percentage is as follows: the percentage of cell
viability=treated×100%/untreated.
In vitro antioxidant activity (H2O2 and DPPH radical scavenging activity)
The antioxidant property was estimated by the H2O2 scavenging assay. The syn-
thesized compound HHDIP at diferent concentrations from 100 to 1000 µg/mL
prepared in ethanol was mixed to 0.6 mL of H2O2 solution. The absorbance of the
mixture of reaction was read after 15 min of evolution at 230 nm. The blank solu-
tion possesses an H2O2 solution without any compound. H2O2 scavenging activity of
synthesized compounds was calculated as
(
)
Hydrogen peroxide scavenging (%) = A0 − A1 ∕A0 × 100
where A0 is the absorbance of the control and A1 is the absorbance of the sample.
The antioxidant potential of the synthesized compound was analyzed based on the
scavenging of free radicals using 1,1-diphenyl-2-picrylhydrazyl (DPPH) method. In
this method, 0.1 mM (ethanolic DPPH) was prepared as well as added with the test
solution of synthesized titular compound HHDIP at diferent concentrations ranging
from 100 to 500 µg/mL. After that, it was incubated for 30 min. Its absorbance was
evaluated at 517 nm using (Beckman spectrophotometer). The discolorations were
estimated with accurate blank at 517 nm after 30-min incubation at 30 °C in the
dark. Calculations were performed in triplicate. In this investigation, uses of stand-
ard drug are D-ascorbic acid, and DPPH was absorbed in 517 nm, and also concen-
tration was decreased using the activity of antioxidant. The free-radical scavenging
activity percentage of the HHDIP was measured using the following formula:
1− Asample − Ablank
Scavenging % =
× 100
Acontrol
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J. I. Ahamed et al.
Here, ethanol (3 mL) with the solution sample (0.1 mL) was utilized as the
blank, and 3 ml of DPPH solution with EtOH (0.1 ml) was utilized as the nega-
tive control.
Molecular docking and ADMET properties
Human c-Met Kinase (PDB code: 3F66) in complex with quinoxaline inhibitor was
chosen as a protein target for molecular docking studies. X-ray difraction resolution
of 3F66 was 1.4Å. The preparation of recovered protein was accomplished using the
made protein wizard of Discovery studio 4.0 [30–32]. The HHDIP ligand was pre-
pared by using Discovery studio 4.0 small molecule tools for further use in molec-
ular docking studies. The ADMET properties and toxicity profle outcomes such
as non-mutagen and non-carcinogen in natures were estimated by computational
analysis. Primarily, all the internal ligand, water molecules, ions and metal elements
were removed. The active site was predicted from the prepared protein by the cur-
rent selection method in the protein 3F66 B-chain. The amino acid residues obtained
in the active site of target proteins such as B: ILE1084 B: ALA1108 B: LUE1157
B: PRO1158 B: TYR1159 B: MET1160 B: LYS1161 B: MET1211 were utilized
for the docking with HHDIP. The structure of the synthesized ligand HHDIP was
drawn using ChemDraw12.0 software. The synthesized 2D compound structure was
changed into mol format, and that was further converted into 3D structure by Accel-
rys Discovery Studio V.4.0. This ligand was purifed and prepared by Discovery stu-
dio4.0 small molecule tools for further use in molecular docking studies. The prop-
erties of ADMET and toxicity contours such as non-mutagen and non-carcinogen in
nature of the HHDIP were estimated by computational analysis.
Results and discussion
Chemistry
The innovative method for the newly synthesized protocol of HHDIP was pre-
pared by a three-step reaction. (Step I) 10H-phenothiazine 1 by the reaction with
10-hexyl-10H-phenothiazine 2 with reagents such as n-bromohexane, K2CO3 and
DMF solvents has refuxed at 120 °C for 24 h. K2CO3, was originated to be an
efcient catalyst for this reaction. (Step II) 10-hexyl-10H-phenothiazine-3-car-
baldehyde 3 was prepared by the reaction of POCl3/DMF under N2 atmosphere for
24 h. Here, the POCl3/DMF was utilized as Vilsmeier reagent, which served as an
efective formylating agent. (Step III) The fnal product (HHDIP) was prepared by
the reaction of benzil 4, hexan-1-amine 5 with NH4OAC withCH3COOH refux at
70–90 °C for 3 h. The complete reaction is exhibited in Scheme 1 which obtained
a fnal product with a signifcantly high yield of 95% which is given in Table 1.
The signifcance of this fnal product HHDIP has multidonor and multiacceptor
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A novel method for the synthesis and characterization of…
Scheme 1 General scheme of the synthesis of compound (HHDIP)
groups that are located in imidazole and phenothiazine ring. It also revealed bet-
ter anticancer activity in our research. Some types of biologically important com-
pounds of imidazoles are represented in Fig. 1.
Geometric structure
Thirty-nine optimized step numbers are obtained in the potential energy curve anal-
ysis method. The minimum energy value of −2074.04 a.u. is exhibited in Fig. 2.
The optimized structure of HHDIP is shown in Fig. 3. It has C1 point group symme-
try. The optimized parameter of geometry like bond angles as well as bond lengths
is computed by the DFT method using 6-311G++ (d,p) basis set, which is given in
Tables 2 and 3 [33–35]. The optimized structure showed a mild alteration in the cal-
culated C–C bond distances in the phenyl ring such as C1–C2, C9–C10 and C4–C12,
respectively, for their values of 1.410, 1.407 and 1.393 A0. The excellent outcomes
of various bond lengths, respectively, C35–C36, C16–C21, and C32–C34 bonds are
more signifcantly greater to thereof another C–C bond. This was owing to the pro-
ducing electron in the substituents amino group, which is placed in the hydrogen
atoms. Also, the benzene ring symmetry was distorted because of the replacement
of hydrogen atoms. For example, C22–C19–C23 and C27–C20–C28 bond angles
computed at 118.5 and 118.50 were incredibly matching the reported values [36].
Similarly; C2–C9–C10, C4–C12–C10, C1–C4–C12, C5–C6–C13, C9–C10–C11
and C17–C19–C23 were somewhat superior to 1200 as shown in Table 3.
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J. I. Ahamed et al.
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A novel method for the synthesis and characterization of…
Fig. 1 Biologically important compounds of imidazole
Fig. 2 Potential energy curve using DFT/B3LYP method with 6-311++G (d,p) basis set for HHDIP
Vibrational analysis
In this study HHDIP is detected 86 atoms, belongs to the C1 point group, and
has 252 normal modes of vibration. The fnal assignments of FTIR as well as
FT-Raman spectra were done by experimental and theoretical analysis. Further,
the harmonic vibrational frequencies are calculated for the HHDIP compound by
DFT/B3LYP level with 6-311G++ (d,p) basis of theory. The percentage of (PED)
potential energy distribution values for the experimental and calculated frequen-
cies of the target compound are reported in Table S1 (Supplementary Material).
In FTIR and FT-Raman spectra, to create an assessment between the experi-
mental observation and theoretically calculated correlation coefcient, graphs
are presented in Figs. S1 and S2 (Supplementary Material). Observed from the
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J. I. Ahamed et al.
Fig. 3 Optimized theoretical geometric structure of 10-hexyl-3-(1-hexyl-4,5-diphenyl-1H-imidazol-2-yl)-
10H-phenothiazine (HHDIP)
correlation graphs, the FTIR correlation coefcient (CC) R2 is 0.99809 and the
FT-Raman (CC) R2 value is 0.99999 at B3LYP/6-311G++ (d,p) level. The exper-
imental and calculated FTIR, as well as FT-Raman spectrum images, is demon-
strated in Figs. 4 and 5; the computed frequencies are in good agreement with
the FTIR as well as FT-Raman experimental values. The Gauss Sum 3.0 program
has been used to obtain calculated FTIR and FT-Raman spectrum images [37].
For complete HHDIP, vibrational assignments are important under four heads:
(i) aromatic C–H vibration, (ii) aromatic (C=C) stretching, (iii) C–N stretching
vibration and (iv) C–S vibration.
CH vibration
The aromatic structures of C–H stretching vibrations are revealed in the region
at 3306–3342 cm−1 [38] and 3150–2900 cm−1, which is the characteristic region
utilized for the prediction of C–H stretching vibration bands that are not afected
[39]. In the title compound, FTIR and FT-Raman, the bands discovered at 3290,
3325, 2974, 2881 cm−1, and 3239, 3118, 3036 cm−1, respectively, are assigned to
the C–H stretching vibrations. Diferent bending vibrations such as β HCC were
observed at the region of 1563–125 cm−1, and β HCH was observed in the range at
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A novel method for the synthesis and characterization of…
Table 2 Calculated bonds
Atom no.
B3LYP/6-
Atom no.
B3LYP/6-
311G++
(d,p)
length (Å) of the molecule
with DFT theory employing
6-311G++(d,p) basis set
311G++ (d,p)
C1–C2
1.410
1.413
1.419
1.393
1.835
1.537
1.096
1.097
1.097
1.393
1.08
1.411
1.412
1.423
1.395
1.836
1.398
1.08
C21–C38
C21–H54
C21–H55
C22–C24
C22–H79
C23–C26
C23–H78
C24–C25
C24–H77
C25–C26
C25–H76
C26–H75
C27–C29
C27–H74
C28–C31
C28–H73
C29–C30
C29–H72
C30–C3\)
C30–H71
R(31–70)
C32–C34
C32–H68
C32–H69
C32–N84
C33–C38
C33–H66
C33–H67
C33–N85
C34–C35
C34–H64
C34–H65
C35–C36
C35–H62
C35–H63
C36–C37
C36–H60
C36–H61
C37–H58
C37–H59
C38–H56
C38–H57
1.539
1.101
1.101
1.396
1.084
1.398
1.083
1.401
1.086
1.399
1.086
1.086
1.399
1.086
1.398
1.085
1.400
1.086
1.400
1.085
1.085
1.546
1.096
1.096
1.479
1.539
1.093
1.09
1.476
1.540
1.097
1.097
1.539
1.101
1.101
1.540
1.101
1.101
1.100
1.100
1.098
1.098
C1–C4
C1–N84
C2–C9
C2–S86
C3–C37
C3–H47
C3–H48
C3–H49
C4–C12
C4–H82
C5–C6
C5–C7
C5–N84
C6–C13
C6–S86
C7–C15
C7–H41
C8–C16
C8–C39
C8–H50
C8–H51
C9–C10
C9–H40
C10–C11
C10–C12
C11–N83
C11–N85
C12–H4)
C13–C14
C13–H43
C14–C15
C14–H81
C15–H80
C16–C21
C16–H52
C16–N53
C17–C18
C17–C19
C17–N83
C18–C20
C18–N85
1.54
1.537
1.100
1.100
1.407
1.084
1.467
1.405
1.334
1.394
1.084
1.399
1.085
1.396
1.084
1.085
1.540
1.101
1.101
1.394
1.471
1.394
1.476
1.407
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J. I. Ahamed et al.
Table 2 (continued)
Atom no.
B3LYP/6-
Atom no.
B3LYP/6-
311G++
(d,p)
311G++ (d,p)
C19–C22
C19–C23
C20–C27
C20–C28
1.410
1.409
1.410
1.410
C39–H44
C39–H45
C39–H46
1.096
1.097
1.097
1461–1342 cm−1[40]. In our present study, the experimental FTIR bending β HCC
vibrations are discovered at 1328 and 1030 cm−1 with PED contribution values of
22 and 32%. In FT-Raman, it was obtained experimentally at 1509 cm−1 with a
PED contribution at 21%. Furthermore, in FT-Raman, the bending vibration of β
CNC is observed theoretically at 988 cm−1, and experimentally, it is well matched in
984 cm−1 (mode number 101) with a PED contribution of 17%.
Moreover, some HCN, CCN and CCC bending vibrations are obtained in the
HHDIP compound and HCCH, HCCC, CCCC and HCCH torsion vibrations were
obtained in HHDIP, which have a great deal with the literature [41–43].
Phenyl ring vibrations
The aromatic C=C stretching phenyl group vibrations are predicted in the range
from 1650–1200 cm−1 [44]. Hence, in the FTIR strong vibrational frequency band at
1654 cm−1 is assigned to C=C stretching vibrations experimentally, which matches
well with the calculated vibrational band at 1662 cm−1, respectively; the medium
intensity bands experimentally at 1087 and 1045 cm−1 with the mode numbers of
123 and 115 are also reasoned to C=C stretching and coincide well with the com-
puted frequency values at 1085 cm−1 and 1051 cm−1 with a 62% and 25% of PED
contribution. However, in FT-Raman studies, the strong vibrational frequency bands
were recorded experimentally at 1667 and 1601 cm−1, which is perfectly matched
with calculated vibrational bands at 1666 and 1604 cm−1 with the mode numbers of
208 and 202.
C–N and C–S vibrations
The prediction of C–N stretching frequency is a very challenging task because of
the C–N Composite region falls on the vibrational spectrum; specifcally, mix-
ing of the various bands is the promising assignment of C–N stretching region at
1386–1266 cm−1 for the aromatic compound [45, 46]. In this present analysis, the
C–N stretching vibrations of HHDIP are found at 1274 cm−1 in FTIR and 1272 cm−1
in the FT-Raman spectrum, and also they have presented PED contribution value
of 13 and 10% with the mode numbers of 145 and 144. The carbon–sulfur (C–S)
stretching vibration shows in the region at 570–380 cm−1 [47]. In the present studies,
1 3

A novel method for the synthesis and characterization of…
Table 3 Calculated bond angles (°) of the molecule with DFT theory employing 6-311G++(d,p) basis
set
Structural parameters
B3LYP/6-311G++
(d,p)
Structural parameters
B3LYP/6-
311G++
(d,p)
C2–C1–C4
C2–C1–N84
C1–C2–C9
116.6
122.1
121.8
120.4
121.3
121.7
120.2
122.6
118.4
117.7
121.2
118.4
98.50
111.4
111.1
111.1
113.0
109.5
109.5
107.8
107.8
107.6
118.1
121.3
119.9
117.0
121.9
121.6
120.4
121.1
121.4
120.2
118.4
117.8
120.6
119.0
118.4
120.6
119.0
113.1
C18–C20–C27
C18–C20–C28
C18–N85–C33
C22–C19–C23
C19–C22–C24
C19–C22–H79
C19–C23–C26
C19–C23–H78
C27–C20–C28
C20–C27–C29
C20–C27–H74
C20–C28–C31
C20–C28–H73
C38–C21–H54
C38–C21–H55
C21–C38–C33
C21–C38–H56
C21–C38–H57
H54–C21–H55
C24–C22–C79
C22–C24–C25
C22–C24–H7)
C26–C23–H78
C23–C26–C25
C23–C26–H75
C25–C24–H77
C24–C25–C26
C24–C25–H76
C26–C25–H76
C25–C26–H75
C29–C27–H74
C27–C29–C30
C27–C29–H72
C31–C28–H73
C28–C31–C30
C28–C31–H70
C30–C29–H72
C29–C30–C31
C29–C30–H71
C31–C30–H71
121.0
120.5
124.2
118.5
120.8
118.3
120.7
120.0
118.5
120.7
119.2
120.7
119.1
109.4
109.4
112.0
109.9
109.9
106.4
120.9
120.4
119.6
119.3
120.5
119.5
120.0
119.3
120.4
120.3
120.0
120.0
120.2
119.7
120.2
120.3
119.7
120.1
119.7
120.2
120.2
C1–C2–S86
C4–C1–N84
C1–C4–C12
C1–C4–H82
C1–N84–C5
C1–N84–C32
C9–C2–S86
C2–C9–C10
C2–C9–H40
C2–S86–C6
C37–C3–H47
C37–C3–H48
C37–C3–C49
C3–C37–C36
C3–C37–H58
C3–C37–H59
H47–C3–H48
H47–C3–H49
H48–C3–H49
C12–C4–H82
C4–C12–C10
C4–C12–H42
C6–C5–C7
C6–C5–N84
C5–C6–C13
C5–C6–S86
C7–C5–N84
C5–C7–C15
C5–C7–H41
C5–N84–C32
C13–C6–S86
C6–C13–C14
C6–C13–H43
C15–C7–H41
C7–C15–C14
C7–C15–H80
C16–C8–C39
1 3

J. I. Ahamed et al.
Table 3 (continued)
Structural parameters
B3LYP/6-311G++
(d,p)
Structural parameters
B3LYP/6-
311G++
(d,p)
C16–C8–H50
C16–C8–H51
C8–C16–C21
C8–C16–H52
C8–C16–H53
C39–C8–H50
C39–C8–H51
C8–C39–H44
C8–C39–H45
C8–C39–H46
C50–H8–C51
C10–C9–H40
C9–C10–C11
C9–C10–C12
C11–C10–C12
C10–C11–N83
C10–C11–N85
C10–C12–H42
N83–C11–N85
C11–N83–C17
C11–N85–C18
C11–N85–C33
C14–C13–H43
C13–C14–C15
C13–C14–H81
C15–C14–H81
C14–C15–H80
C21–C16–H52
C21–C16–H53
C16–C21–C38
C16–C21–H54
C16–C21–H55
H52–C16–H53
C18–C17–C19
C18–C17–N83
C17–C18–C20
C17–C18–N85
C19–C17–N83
C17–C19–C22
C17–C19–C23
C20–C18–N85
109.2
109.1
113.3
109.2
109.2
109.5
109.5
111.4
111.0
111.1
106.2
120.4
123.4
117.4
119.1
123.1
126.3
118.8
110.5
107.2
107.0
126.5
120.4
118.8
120.2
120.9
120.3
109.3
109.3
113.0
109.2
109.2
106.3
131.1
109.4
132.0
105.9
119.5
118.7
123.0
122.1
C30–C31–H70
C34–C32–H68
C34–C32–H69
C34–C32–N84
C32–C34–C35
C32–C34–H64
C32–C34–H65
H68–C32–H69
H68–C32–N84
H69–C32–N84
C38–C33–H66
C38–C33–H67
C38–C33–N85
C33–C38–H56
C33–C38–H57
H66–C33–H67
H66–C33–N85
H67–C33–N85
C35–C34–H64
C35–C34–H65
C34–C35–C36
C34–C35–H62
C34–C35–H63
H64–C34–H65
C36–C35–H62
C36–C35–H63
C35–C36–C37
C35–C36–H60
C35–C36–H61
H62–C35–H63
C37–C36–H60
C37–C36–H61
C36–C37–H58
C36–C37–H59
H60–C36–H61
H58–C37–H59
H56–C38–H57
H44–C39–H45
H44–C39–H46
H45–C39–H46
120.1
109.4
109.4
116.9
111.4
110.2
110.2
106.4
107.2
107.1
110.2
109.8
112.6
108.9
109.1
107.6
108.0
108.4
109.6
109.6
113.2
109.4
109.4
105.6
109.2
109.2
113.4
109.3
109.3
106.1
109.2
109.2
109.2
109.2
106.2
106.2
107.0
107.8
107.8
107.6
1 3

A novel method for the synthesis and characterization of…
Fig. 4 Experimental FTIR spectrum of (HHDIP) (a) and theoretical FTIR spectrum of (HHDIP) (b) at
DFT/B3LYP/6-311G++ (d,p)
Fig. 5 Experimental FT-Raman spectrum of (HHDIP) (a) and theoretical FT-Raman spectrum of
(HHDIP) (b) at DFT/B3LYP/6-311G++ (d,p)
the calculated wave numbers show region at 452 cm−1 (mode no. 51) with 11% of
PED contribution which is assigned as C–S stretching vibration.
The experimental C–S stretching bands region was determined at 428 cm−1 for
HHDIP, respectively, which is in good agreement with the computed value.
NLO properties
The improvements in NLO material properties gained an enormous interest in the
past few periods. These materials have revealed various device uses such as an opti-
cal switches, electronics device, telecommunications, optical interconnections and
signal processing [48–51]. These are very important in areas such as linear polar-
izability (α), total molecular dipole moment (µ) and frst-order hyperpolarizabil-
ity (β), which are calculated by B3LYP/6-311G++ (d,p) basis set from Gaussian
09W. The calculation of the magnitude of total molecular dipole moment (µ), linear
1 3

J. I. Ahamed et al.
polarizability (α), the anisotropy of the polarizability (Δα) and frst-order hyperpo-
larizability (β) from complete equations is given below [52, 53]:
ꢀtotal = ꢀ0 + ꢁijEj + ꢂijkEjEk+
(1)
( )
ꢀxx + ꢀyy + ꢀzz
1
3
ꢀtot
=
(2)
(3)
(4)
[
]
1∕2
(
)
(
)
(
)
Δꢀ = 2−1∕2 ꢀxx − ꢀyy 2 + ꢀyy − ꢀzz 2 + ꢀzz − ꢀxx + 6ꢀx2z
.
2
(
)
1∕2
ꢀtot = ꢀx2 + ꢀy2 + ꢀz2
[
]
1∕2
(
)
(
)
(
)
2
ꢀtot
=
ꢀxxx + ꢀxyy + ꢀxzz 2 + ꢀyyy + ꢀyzz + ꢀxxy 2 + ꢀzzz + ꢀxxz + ꢀyyz
(5)
The title molecule NLO properties was calculated with its results of the total
molecular dipole moment value of (µ = 3.3231 Debye) and mean the frst hyper-
polarizability value of (βtot = 6.49 × 10−30 esu), which are specifed in Table 4,
and its total dipole moment of the molecule is approximately two times higher
than that of urea and the frst hyperpolarizability is 15 times higher than that of
Table 4 Calculated electronic dipole moment (µ), linear polarizability (α), anisotropy of polarizability
(Δα), frst-order hyperpolarizability (β) values for HHDIP by DFT/B3LYP/6-311G++ (d,p) level of the-
ory
Parameter Value Parameter a.u.
esu(x10−24
)
Parameter
a.u.
esu(x10−33
)
μx
μy
μz
μ
1.1123 αxx
2.380 αxy
2.035 αyy
3.3231 αxz
αyz
221.255 32.789
βxxx
βxxy
βxyy
βyyy
βxxz
βxyz
βyyz
βxzz
βyzz
βzzz
−25.376 −219.230
−31.934 −275.896
16.774 2.486
267.953 39.710
3.225 3.703
11.224
25.075
96.967
216.630
9.024 1.337
−13.118 −113.330
−11.820 −102.116
αzz
αtot
Δα
260.589 38.619
249.929 37.039
480.558 71.219
11.139
96.493
−19.934 −172.2166
−37.454 −323.5757
−0.433
−3.740
βtot
750.589 6484.564
−31
β
= 64.84×10 esu
tot
The values of the frst-order hyperpolarizability (β) tensors of Gaussian09W output are reported in
atomic units (a.u)
The calculated (DFT) values were converted into electrostatic units (For α: 1a.u.=0.1482×10−24 esu; for
β: 1a.u.=8.6393×10−33 esu)
1 3

A novel method for the synthesis and characterization of…
urea (µ and β values of urea are µ = 1.3732 Debye and βtot = 0.3728 × 10−30esu,
6-311G++ (d,p), respectively [54]. Hence, this molecule has a higher dipole
moment and hyperpolarizability value than urea which shows that the molecule
has a large applicant for nonlinear optics (NLO) material.
Thermodynamic study
The thermodynamics parameter of HHDIP is computed to obtain authentic relations
between structural, energetic and reactivity characteristics of the molecules. Selec-
tive information on the stable (dipole moment) of a molecule permits us to fnd out
the molecule’s confrmation. The calculated thermodynamic properties by using a
basic set of B3LYP/6-311G ++ (d,p) as well as their outcomes values are given
in Table 5. The frequency calculations are utilized to fnd out the Gibbs free ener-
gies, enthalpy, entropy and the heat capacity for a molecular system. Higher values
of Gibbs free energies signify more stable molecules [55]. The enthalpy, entropy,
zero-point vibrational energy (ZPVE) and heat capacity values are found at any tem-
perature and are helpful in evaluating the direction of chemical reactions. The dipole
moment (Debye) parameter value is completed by using this study. The valuable
outcomes of the synthesized HHDIP free energy, entropy, enthalpy and ZPVE are
utilized.
Mulliken’s population analysis
This study plays a vital role of application in quantum chemical approach to the
molecular system by atomic charge efect, molecular polarizability, dipole moment,
electronic structure, and other molecular properties that are afected by the atomic
charge. Mulliken atomic charge distribution for the HHDIP is presented in Fig. S3
(Supplementary Material), and Mulliken plot of charge distribution for the title com-
pound is exhibited in Fig. 6. From Fig. S3 (Supplementary Material) and Fig. 6, it is
distinctly found that the charge of nitrogen atoms N83, N84, and N85 of the HHDIP
compound is highly negative and the corresponding protonated hydrogen H79 atom
possesses higher positive charge compared to other hydrogen atoms. The C5 car-
bon atom is shown highly positive charge which is located phenothiazine group and
thereof nearby located phenothiazine, as well as imidazole rings, are containing
Table 5 Theoretically computed
Thermodynamic parameters
HHDIP
thermodynamic parameters
of HHDIP by B3LYP/6-
311G++(d,p) basis set
Total energy (Hartrees)
Enthalpy (Hartrees)
−2075.0912
0.7816
Gibbs free energy (Hartrees)
0.6600
Zero-point vibrational energy (Kcal mol−1
)
464.6521
153.1701
255.6542
3.3231
Heat capacity (cal mol−1 K−1
)
Entropy (cal mol−1 K−1
Dipole moment (Debye)
)
1 3

J. I. Ahamed et al.
Fig. 6 Mulliken plot of atomic charge distribution for HHDIP
eight carbon atoms namely C3, C39, C13, C16, C19, C21, C34, and C35 have a
higher negative infuence. In this investigation, aromatic rings have negative values
on C15, C37, C22, C23, C24, C26, C27, C25 and C40 atoms which leads to real-
location of the electron density. As an outcome of these strong negative charges, C1
and C11 accommodate large positive charge and are higher acidic.
Moreover, it is also noticed that the sulfur atom S86 attains maximum positive
charge owing to its bonding with electronegative nitrogen atom N84. Table S2 (Sup-
plementary Material) gives the total atomic charges (ground state) of HHDIP which
are found from DFT/B3LYP/6-311++G (d,p) level.
Fig. 7 UV–Vis spectra of HHDIP experimental (a) and theoretical (b) (GAS phase))
1 3

A novel method for the synthesis and characterization of…
UV–Vis spectral analysis
The experimental and computational UV–Vis comparability spectrum investiga-
tion HHDIP is represented in Fig. 7, and its absorption maximum (λmax) is calcu-
lated by TD-DFT/B3LYP method with a 6-311++G (d,p) basis set. The stimulated
molecular orbital geometry indicates a visible absorption maximum of the HHDIP
is matched to the (HOMO–LUMO) electronic transition in (gas phase).
The electronic transition among the transformation from (HOMO–LUMO) trans-
formation can be observed by using UV–Vis spectra absorption values of 364 nm
and band gap 3.3086 (eV) (Experimental). The experimental energy value of band
gap has estimated utilizing the formula, E=hc/λ. Here, h and c are constant; λ is the
cutof wavelength [56, 57]. The title molecule theoretically observed value of 355.70
nm (gas phase), oscillator strength value of (f=0.3073), and band gap energy value
of 3.4856(eV), which are presented in Table 6; the experimental and theoretical UV
data are in good agreement with each other. The calculated UV–Vis spectrum is
plotted with the Gauss Sum 3.0 program [17]. The computed absorption spectrum
reveals that the highest range of UV–Vis absorption wavelength complies from the
HOMO–LUMO electronic transition with (80%) contribution [58].
Frontier molecular orbitals (FMOs) analysis
Analysis of FMOs is utilized to confrm the molecular interactions, which has huge
signifcance in recent biochemistry and molecular biology [59]. It provides evidence
about the properties of ligand molecules as well as their interactions with the biolog-
ical receptor. Analysis of HOMO–LUMO outcome of HHDIP is revealed in Fig. 8.
HOMO (highest occupied molecular orbital) and LUMO (lowest unoccupied molec-
ular orbital) orbitals compute the electron–donor and electron–acceptor character,
respectively. Table 7 gives the values of FMOs associated with molecular properties
[60].
The computed lofty ionization potential value of HHDIP reveals the capacity to
carry out a nucleophilic attack. The band gap energy is an important for the stability
of structures [61, 62]. Energy of HOMO–LUMO is associated with the electronic
afnity as well as ionization potential. The DFT outcome of HOMO–LUMO ener-
gies, the energy gap (∆E), the ionization potential (I), the electron afnity (A), the
absolute electronegativity (χ), the absolute hardness (η), electrophilicity index (ω)
and softness (S) for the HHDIP molecule energies are computed. The title mole-
cule (HOMO)–(LUMO) energy gap values are obtained value of (−0.7992 eV) and
(−4.8195 eV) which are also given lower softness value of 2.2099. The softness
value helps in knowing the toxicity of several pollutants in terms of their reactivity
and site selectivity. Measured band gap value (4.0203 eV) signifes that the HHDIP
compound has a stable structure. The gas phase calculated using the B3LYP/6-
311G++ (d,p) method provides outcomes as HHDIP possessed 157 occupied
and 320 unoccupied molecular orbitals. Besides, the title compound (see Table 7)
1 3

J. I. Ahamed et al.
1 3

A novel method for the synthesis and characterization of…
Fig. 8 Atomic orbital HOMO–LUMO composition of the frontier molecule orbital for HHDIP
Table 7 Calculated quantum
Quantum parameters (eV)
HHDIP
chemical parameters HOMO–
(HOMO–
LUMO)
LUMO results of HHDIP
EHOMO
ELUMO
∆Egap
−4.8195
−0.7992
4.0203
4.8195
0.7992
5.2191
I=−EHOMO
A=−ELUMO
I+A
χ=
2
μ=−(I+A)/2
η=I−A)/2
−5.2191
4.4199
2.2099
1
S=
2ꢀ
ꢀ2
ω=
2ꢁ
3.0815
HOMO–LUMO has a high electrophilicity index value of 3.0815 and hence it is
biologically active [63].
In HOMO–LUMO analysis, red color is represented as a positive phase and green
color is represented as a negative phase. It is evident from the fgure that the HOMO
levels difuse mainly over phenothiazine and partially slightly on the imidazole
group. LUMO levels are difused mostly over imidazole and partially presented on
1 3

J. I. Ahamed et al.
Fig. 9 Experimental 1 H spectrum of the title compound
Fig. 10 Experimental 13 C spectrum of the title compound
the phenothiazine group [64]. The lower softness and higher hardness values signify
molecule stability. The electrophilicity indexes as well as lower chemical potential
values have exhibited the potential bioactive molecules [65].
1 3

A novel method for the synthesis and characterization of…
1H and 13C NMR spectral studies
The NMR assists as a grand resource in deciding the structure of an organic compound
by disclosing the hydrogen and carbon skeleton. To furnish a specifc assignment and
investigation 1H as well as 13C NMR spectra, theoretical chemical shift value of calcu-
lations in the HHDIP was done by gauge-independent atomic orbital (GIAO) manner
1
at B3LYP/6-311++G (d,p) level with vacuum method. The HNMR and 13C NMR
spectral studies of HHDIP showed peaks at diferent chemical shift values as given
in Figs. 9 and 10. Theoretical and experimental chemical shift values are specifed in
Table S3 and S4 (Supplementary Material). The little diference observed among DFT
and experimental spectra values is owing to the solvent used. The 13C NMR chemical
shifts, absolute shielding tetramethylsilane (TMS) values are 192.8847 and 31.8927 for
1
1H NMR spectra. In this investigation, the title compound HNMR spectrum chemi-
cal shifts are interpreted to conform to the promising diferent efects which act on the
shielding constants of protons. Aromatic protons are seemed to be the downfeld sig-
nals at d values of 6.880, 6.940, 7.090 and 7.200 for protons H43, H81, H41 and H75
which give multiplet (Ar–H) signals, which are located in phenothiazine group. Pro-
tons H76 and H77 give multiplet (Ar–H) signals, which are located in the benzil ring.
The upfeld protons are seemed to be from 1 to −5 (ppm), respectively. The methyl
protons signal was presented at δ 0.740 (ppm) for proton H48 which gives a triplet.
Furthermore, the methylene proton signal was shown at δ 0.890, 0.990, 1.030, 1.060,
1.080, 1.290, 1.330, 1.360, 1.380, 1.420, 1.440, 1.460, 1.480, 1.880, 3.820 and 3.890
(ppm) for protons H45, H55, H57, H47, H46, H49, H50, H55, H57, H63, H60, H51,
H64, H56, H68 and H66 giving multiplet, which are located in hexyl group. In this
analysis, the 13C-NMR spectrum of aromatic carbons provides signals with chemical
shift values from 100 to 200 ppm [66, 67]. The HHDIP chemical shifts (experimental)
ranges take place in the range of δ 115.160–131.050 ppm, and phenothiazine carbons
C13 and C1 are detected in the downfeld at δ 145.910 and 146.870 ppm owing to the
presence of the phenothiazine group. Due to the more electronegative sulfur atoms in
the ring, the carbon atoms chemical shifts ranges are shown in the heterocyclic rings,
which are set to the downfeld. Therefore, the chemical shift of imidazole carbon C18 is
also detected at the downfeld at δ 131.610 ppm. The C33 carbon atom at δ 47.590 ppm
is highly shielded which is located in the hexyl group of aliphatic carbon. Furthermore,
the chemical shifts of benzil ring carbon atoms C24, C28, C19 and C20 are assigned to
124.480, 125.160, 127.820 and 128.580 ppm.
The calculated chemical shifts are in good agreement with the experimental values.
As it can be detected from the graph, the correlation coefcient (CC) R2 values are
1HNMR 0.999 and 0.996 for 13C NMR spectra. The correlation between the calculated
and experimental 1H and 13C chemical shifts of HHDIP is represented in Figs. 11 and
12.
Mass spectrum
The mass spectrum range of synthesized compound HHDIP with its molecular ion
peak observed at m/z 586.60 is owing to the presence of (MH+), which coincides with
1 3

J. I. Ahamed et al.
1H-NMR DFT/B3LYP/6-311G++(d,p)
8.0
y = -0.020x2 + 1.177x - 0.191
7.0
(R² = 0.999)
6.0
5.0
4.0
3.0
2.0
1.0
0.0
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
Experimental(ppm)
Fig. 11 Correlation graph between the experimental and simulated 1H NMR chemical shift values of
HHDIP
13C-NMR DFT/B3LYP/-6-311G++(d,p)
160
y = 0.903x1.024
(R² = 0.996)
140
120
100
80
60
40
20
0
0
20
40
60
80
100
120
140
160
Experimental (ppm)
Fig. 12 Correlation graph between the experimental and simulated 13C NMR chemical shift values of
HHDIP
the molecular mass; their m/z scale ranges observed at 200–2000 are presented in Fig.
S4 (Supplementary Material). The obtained molecular ion peaks in the spectra are in
excellent agreement with the proposed molecular structures of the title compound. The
spectrophotometers were run in positive scan mode.
1 3

A novel method for the synthesis and characterization of…
Table 8 Anticancer activity of
% of concentration (μg/ml)
% of cell
viability
HHDIP
synthesized compound HHDIP
measured by MTT assay and
HepG2 liver cancer cell line
15.75
31.25
62.5
78.7
74.4
70.7
60.2
59.1
100
125
250
Control
Fig. 13 HepG2 liver cancer
cell lines—controls compound
HHDIP—250 µg/mL
Biological section
In vitro anticancer activity
Most of the imidazole derivatives have been observed to be successful in vitro cyto-
toxic compounds against a human liver tumor cell line [68, 69]. In this investigation,
new imidazole compound was synthesized for their anticancer activity. The title
compound showed the anticancer activity at the lowest concentration of 62.5 µg/mL.
This compound reduced the cell viability to 70.7%; the cell viability was much more
decreased to 59.1% at 250 µg/mL. The HepG2 liver cancer cells were tested at con-
centrations from 15.75 µg/mL to 250 µg/mL given in Table 8. The (HepG2) liver
cancer cell lines controls image is represented in Fig. 13. The title molecule showed
potent anticancer activity against HepG2 cell line only at the higher concentration
250 µg/mL, which is represented in Fig. 14. The resultant (HepG2) graphical repre-
sentation of HHDIP is given in Fig. 15.
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J. I. Ahamed et al.
Fig. 14 HepG2 liver cancer cell
lines image of HHDIP
Fig. 15 Anticancer activities of
synthesized compound HHDIP
measured by MTT assay and
HepG2 liver cancer cell line
150
100
50
0
l
5
5
5
5
0
.
o
7
2
2
5
r
.
.
2
t
1
2
5
1
6
n
1
3
o
C
Concentrations in µg/ml
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A novel method for the synthesis and characterization of…
In vitro antioxidant activity (H2O2 and DPPH radical scavenging activity)
The H2O2 scavenging assay was evaluated according to the method of some medica-
tions [70]. The outcomes of the antioxidant (H2O2) study have shown that using syn-
thesized compound HHDIP was potentially a good source of hydroxyl radical scav-
enging activity. Hydroxyl radicals (OH˙) are short-lived species having high afnity
toward other molecules. The results of OH˙ scavenging activity of synthesized com-
pounds revealed that the title compound possessed strong antioxidant activity. The
complete results of H2O2 in vitro antioxidant activities are shown in Table 9.
100
% of Inhibition HHDIP
90
Standard Drug D - Ascorbic
80
acid
70
60
50
40
30
20
10
0
100
200
300
400
500
Concentration μg/ml
Fig. 16 In vitro DPPH radical scavenging activity of test compound 10-hexyl-3-(1-hexyl-4,5-diphenyl-
1H-imidazol-2-yl)-10H-phenothiazine (HHDIP)
Table 9 Antioxidant activity of test compound 10-hexyl-3-(1-hexyl-4,5-diphenyl-1H-imidazol-2-yl)-
10-pentyl-10H-phenothiazine (HHDIP) measured by H2O2 assay
Sample name
Control
Test (1 mg/mL)
H2O2 Scavenging (%)
66.31
HHDIP
3.8
1.28
Table 10 Antioxidant activity
of test compound 10-hexyl-
3-(1-hexyl-4,5-diphenyl-1H-
imidazol-2-yl)-10-pentyl-
10H-phenothiazine (HHDIP)
measured by DPPH assay
Concentration (μg/ml) % of inhibition HHDIP Standard drug
D-ascorbic acid
100
200
300
400
500
4.95604
18.5452
32.1743
40.4077
52.8777
47.363
55.3158
69.425
86.45
90.888
*Ascorbic acid was used as a standard at 100, 200, 300, 400 and
500 μg concentrations of compound/mL
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J. I. Ahamed et al.
500.00
450.00
400.00
350.00
300.00
250.00
200.00
150.00
100.00
50.00
0.00
D - Ascorbic acid
98.12
HHDIP
472.79
ic50
Fig. 17 Free-radical scavenging activities of the 10-hexyl-3-(1-hexyl-4,5-diphenyl-1H-imidazol-2-yl)-
10H-phenothiazine (HHDIP) and vitamin C measured in DPPH assay
The synthesized title molecule was estimated for their potential to scavenge the
free-radical formation, which was measured by the DPPH assay method [71, 72].
The HHDIP antioxidant activity is shown in Table 10. The D-ascorbic acid (vita-
min C) was utilized as a standard antioxidant. The IC50 value for each compound
was calculated, and it was found that the antioxidant tested compound HHDIP
possesses IC50 value of 472.79 µg/mL (Fig. 17). When compared to the standards,
HHDIP showed lowest free-radical scavenging activity at 52.8777% of inhibition,
and D-ascorbic acid (Vitamin C) showed the activity of 90.888% of free-radical
scavenging concentration at 500 µg/mL (Table 10). Finally, antioxidant test com-
pound HHDIP has given satisfactory radical scavenging activity (Fig. 16).
In silico section
Molecular docking studies
In this investigation, the title compound showed signifcant in vitro anticancer
activity. Hence, this compound was further studied by in silico molecular dock-
ing investigations against 3F66 (c-Met kinase; a hepatocyte growth factor) protein
target. The receptor tyrosine kinases (RTKs) are a pharmaceutically attractive tar-
get that acted as a vital job in tumor formation and regulated the variety of signal-
transduction pathways within a cell. New therapies are being developed according
to the molecular profle of patients, especially based on the special involvement
of molecular routes in the initiation and progression of cancer. The c-Met acts
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A novel method for the synthesis and characterization of…
Fig. 18 Key fexible docked compound HHDIP in the active site of 3F66 (a) and key fexible docked
compound of standard drug (tivantinib) in the active site of 3F66 (b)
as an (HGF) hepatocyte growth factor and mediates epithelial tissue remodeling,
migration, morphogenesis, cell growth, diferentiation and angiogenesis [73–76].
Moreover, in 2011, c-Met was found a sign of pancreatic cancer stem cell, which
made it a suitable therapeutic target for cancer treatment [77].
In this study, against c-Met kinases protein target, the substrate-binding domain
(SBD) site sphere of 3D fexible docked ligands such as HHDIP and standard
drug, namely tivantinib, with the active site of 3F66 and its outcomes are shown
in Fig. 18. The title molecule exhibited higher binding interaction energy of
− 64.494(KCal/mol) against the target protein (3F66) and formed interaction with
the amino acid of MET1160 (N–H….O; bond angle of 2.7Å). Standard anticancer
drugs, namely tivantinib, interacted with the amino acids of ASN-1167 (N–H…
.O; 2.5Å) and ILE-1084 (C–H…O; 2.5Å) which also showed the binding interac-
tion of -57.804 (KCal/mol) against the target 3F66. These compounds in silico
section outcomes are shown in Table 11, and thereof hydrogen-bond interaction
and 2D interactions are shown in Figs. 19 and 20.
In silico ADMET and toxicity profles
The synthesized title compound from the current study exhibited superior absorp-
tion capacity (>90% absorbed), as located within the ellipse. The graphical repre-
sentation of HHDIP is presented in Fig. 21. In ADMET (adsorption, distribution,
metabolisms, excretion and toxicity) analysis, HIA (human intestinal absorption),
level of aqueous solubility, BBB (brain–blood barrier) penetration levels and
CYP2D6 inhibiting values of this HHDIP were analyzed. The measurement of
hepatotoxicity is able to predict its ADMET properties of molecules [78]. The title
molecule was determined by a minimum range of solubility and exhibited a higher
level of BBB penetration (Table 12). The important prediction of the toxicity pro-
fle of HHDIP using TOPKAT was also utilized in the range of QSTR quantitative
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