a novel synthesis of (e)-n'-((3, 4 diaminophenyl)(phenyl...

A Novel Synthesis of (E)-N'-((3, 4 diaminophenyl)(phenyl)methylene)

nicotinohydrazide, and its Applicable spectral, Cytotoxic activity

And DFT studies

Chinnadurai Anbuselvan* Department of Chemistry, Annamalai University, Annamalainagar − 608 002, India.

*Corresponding author: Address

C. Anbuselvan

Assistant professor, Department of Chemistry, Annamalai University, Annamalainagar 608 002

E-mail: [email protected]

Abstract: The chemical structures of the synthesized compound were characterized by FT-IR, 1H, and 13C

NMR. The in vitro anticancer activity of the synthesized compound tested against HepG2 cancer cell line. The

revealed data showed that the compound has promising anticancer activity against HepG2 cell line at low

concentrations. Finally, Density functional theory (DFT) theory calculations were performed to investigate the

optoelectronic properties. The obtained theoretical results validate with available experimental evidence.

Keywords: Schiff base; HepG2; cytotoxic activity; live cell images; DFT.

INTRODUCTION

Medicinal chemistry against disease was based on natural products and organic compounds, but

nowadays, a developing interest in the synthesis of Schiff bases are important compounds in the chemistry and

biochemistry fields due to their biological activities1-7. Schiff bases which are a class of compounds containing an

azomethine group (-C=N-) as a functional group have focus attention for a long time due to their medicinal and

pharmaceutical activities8. They have many applications in different fields, for example, antibacterial, antifungal,

and antitumor activity10. Schiff bases obtain from different organic heterocyclic compounds chiefly those

consisting of hydrazide molecule; there is an important class of organic heterocyclic compounds because they

have abroad applications for example, anticancer, antioxidant5, antiviral, anti-inflammatory, antibacterial and

antifungal, roperties12. Cancer is a disease that makes cells growth in the body out of control; the most common

types of cancer are breast, lung, colon and prostate cancer13. When it starts in the breast, then it is called breast

cancer14. Breast and lung cancer are one of the most common diagnosed types of cancer in women around the

world an inducing to cancer death with almost 1.67 million new cases of cancer, and more than 500,000 deaths

predicted to have appeared in 201215. Thus, in our current research, we were aim to synthesis new (E)-N'-((3,4-

diaminophenyl)(phenyl)methylene)nicotinohydrazide Schiff bases and tested their cytotoxic activity against

HepG2 cell line. The newly synthesized compounds were analyzed for their characterized by elemental, FT-IR

and NMR analyses, anticancer activity against KB cell line by MTT assay The preferred conformation of the

compound. Also, were studied with the help of DFT analysis.

International Journal of Research

Volume 7, Issue VIII, August/2018

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EXPERIMENTAL SECTION

General information

All chemicals (analytical and spectroscopic grade) were commercially purchased from Sigma-Aldrich and

used without further purification. The spectra were recorded on AVATAR–330 FT–IR spectrometer (Thermo

Nicolet) and only noteworthy absorption levels (reciprocal centimeters) are listed. FT-IR spectra of organic

compounds are measured between the wave number of 4000 and 400 cm-1 by using KBr to make pellets.

Elemental analyses were carried out on VARIOMICRO V2.2.0 CHN analyzer. 1H NMR spectra were recorded

on a BRUKER AVANCE III 400 MHz NMR spectrometer operating at 400.13 MHz. Samples were prepared

by dissolving about 10 mg of the compound in 0.5 mL of DMSO-d6 containing 1% TMS. 13C NMR, spectra

were recorded on a BRUKER AVANCE III 400 MHz NMR spectrometer operating at 100.61 MHz. Samples

were prepared by dissolving about 50 mg of the compound in 0.5 mL of DMSO-d6 containing 1% TMS. TMS

was used as an internal standard. The confocal image captured in a Leica DM3000, Germany with 40X

magnification. All the theoretical values performed by using the Gaussian 03W program package on a personal

computer. Geometry optimization determined by DFT method 6-31G (d,p) basis sets.

Synthesis of the compound DMN

The compound was prepared by the general procedure (Scheme 1) 0.1 mole of 3,4-diaminobenzo phenone

(0.21 g,1 mmol) was added to nicotinohydrazide (0.13 g,1 mmol) were taken and mixed in ethanol (25 ml) added two

drops of glacial acetic acid. The resultant mixture was refluxed for 4 hrs and cooled. The precipitates were poured

into crushed ice. Solids thus obtained were filtered and washed several times with water, followed by ethanol and

then dried in a vacuum. The crude products were crystallized in ethanol.

Cell culture

The human hepatocellular liver carcinoma (HepG2) cell line procured from NCCS (Pune). Cells were

placed on 18 mm glass coverslips and allowed to adhere for 24 h. Then cultured in Dulbecco's Modified Eagle’s

Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS) and antibiotics (100 mg/mL

streptomycin and 100 U/mL penicillin) at 37 ◦C in the humidified atmosphere of 5% CO2.

Cell viability assay

The cytotoxicity of compound DMN was tested against HepG2 cell lines using the 3-(4,5-

dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. The cell was seeded into a 96-well plate at

a density of 1.5 × 104 cells per well and incubated in medium containing compound DMN at concentrations

ranging from 1.5 to 500 μM for 48 hours. Triplicate wells maintained for each treatment. 100 μL of MTT added

to each well, and it was incubated at 37°C for 4 hours to allow MTT to the formation of formazan crystals by

reacting with MTT and metabolically active cells. The medium with MTT was discarded from the wells carefully.

Each well was added 100 μL of DMSO to dissolve Intracellular formazan crystals, and the plates were shaken

for 10 min. Using ELISA (Enzyme-Linked Immuno Sorbent Assay) reader and absorbance was read out at 417

nm, and the cell images were examined using a fluorescence microscope. The percentage of survival was

calculated using the formula: % survival = [live cell number (test)/live cell number (control)] × 100.

Computational details

Theoretical investigations on compound DMN entire calculation were performed at ab initio DFT levels

using the Gaussian 03W, program package.



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RESULTS AND DISCUSSION Chemistry

(E)-N'-((3,4-diaminophenyl)(phenyl)methylene)nicotinohydrazide (DMN).Yield (%): 73; yellow colour solid; m.p

(°C): 195-201; MF: C20H18N4O: Elemental analysis: Calcd (%): C,72.71; H , 5.49; N , 16.96: found (%): C , 72.62;

H , 5.41; N, 16.79: 1H NMR (400 MHz, DMSO-d6, δ, ppm): 3.93-3.87 (t, 1H), 6.07-6.02 (t, 2H), 6.28-6.23 (T,

2H), 7.61-8.50(m, 13H, Ar-H), 8.80-8.78 (d, 1H) shown in Fig 2. 13C NMR (100 MHz, DMSO-d6, δ, ppm)

203.40 (C-O), 122.58-133.25 (Ar-C) 160.62 (C=N) shown in Fig 3. IR (KBr, cm-1): 3061 (υAr-CH), 1656

(υC=O), 159 (υC=N), 3471 (C=NH), 1483, 1436 (υC=C), 1258-926 (βC-H), 830-688(ΓC-H) shown in Fig 1.

Cytotoxicity

The cytotoxicity responses of compound DMN with various concentrations added are evident from the

cellular imaging. Hence, these results give in compound DMN is an efficient candidate for monitoring changes

in the intracellular concentration under certain biological conditions, and it has justified its cytotoxicity, MTT

assay in HepG2 cells treated with various concentrations of compound DMN for up to 5 hours. As shown in

Fig. 5, 20µM levels of compound DMN did show significant cytotoxic effects on HepG2 cancer cells for at least

up to 4 hours. The synthesized compound DMN examined for cytotoxic activity on HepG2 cell line through

MTT test that allows us to assess the effect of complexes on cellular mitochondrial metabolism. Cells tested for

two days with increasing concentrations of the tested compound. Microscopic images of control cancer cells and

apoptotic morphological changes in HepG2 cell line treated with compound DMN given in Fig. 4. The results

showed that the compound DMN have minimum cell death. The compound DMN exhibit broad inhibition on

the HepG2 cell lines with IC50 values of 81.51 respectively. The IC50 values of the compound DMN (Fig. 5)

suggest that DMN possessed a more potent inhibitory effect against the cancer cells. Compound DMN carrying

the =O group in ortho position and shows the highest IC50 value, convincing us to suggest that the electronic

effect may be one of the factors in determining the anticancer activities of compound DMN. The IC50 values of

compound DMN against HepG2 cell lines result given in Table 1.

Scheme 1. Synthesis of compound DMN.



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Table 1. The IC50 values of compound DMN against HepG2 cell lines.

Anticancer effect of compound DMN on

HepG2 cell line

Viability %

Concentration (μM) DMN

1.5 93

2 87

3.8 74

7.6 61

15.3 53

30.6 45

61.25 37

125 19

250 11

500 4

Computational studies

Geometry optimization

Geometry optimization for title compound DMN was carried out in DFT using B3LYP/6-31G (d,p)

basis set. The optimized structure of compound DMN displayed in Fig. 6a. Numbering pattern of the molecule

is shown in Fig 6b.

Fig. 4. Live cell images of compound DMN: (a) before and (b and c) after

treatment with compound DMN examined by fluorescence microscopy.

a b c

Fig. 5. The IC50 values of compound DMN against HepG2 cell lines.



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Mulliken charge analysis

The Mulliken atomic charges are calculated by decisive the electron population of the individual atom as

outlined by the basis function. The Mulliken atomic charges of designed compound molecule evaluated by

B3LYP using 6-31G (d,p) basis set and listed in Table 2. Mulliken atomic charge calculation plays a significant

role in the employment of computation calculation of the chemical compound. In compound DMN, the

positive charge found at C2, C3, C6, C7, C16, and C17. The maximum positive charge for above carbon atoms

than other atoms is due to the presence of mostly electronegative oxygen and nitrogen atoms in the closest

position. The nitrogen (N2’, N3’, N14 and N15) and oxygen (O16’ ) atoms have more negative charges. Some

of the carbon atoms (C1, C4, C5, C6, C7, C8, C13, C9, C10, C11, C12, C16, C17, C18, C19, C20, C21, C22, and

C54) in title compound possess a negative charge. From the above results shows in Fig. 7, we conclude that our

synthesized compound is suitable for the substitution reactions.

Table 2. Mulliken atomic charges of compound DMN.

Atom DMN Atom DMN

C1 -0.153 C12 -0.085

C2 0.243 C13 -0.105

C3 0.265 N14 -0.32

C4 -0.127 N15 -0.394

C5 -0.149 C16 0.538

C6 0.084 O16’ -0.507

N2’ -0.684 C17 0.048

N3’ -0.679 C18 -0.092

C7 0.197 C19 -0.096

C8 -0.008 C20 -0.076

C9 -0.088 C21 -0.104

C10 -0.089 C22 -0.087

C11 -0.079

Fig. 7. Mulliken charges of compound DMN.

a b

Fig. 6 (a) Optimized structure of compound DMN. (b) Numbering pattern of

macromolecule compound DMN.



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MEP is concerned with the electronic density and is a very valuable descriptor in accepting spots for

nucleophilic reaction and electrophilic attack as well as hydrogen bonding interactions. It also gives visual

thoughtful of relative polarity of the molecule.

The electrostatic surface potential of compound DMN calculated by applying B3LYP/6-31G (d,p) level

of theory. The red color part indicates the regions of negative electrostatic potential while the blue site

represents the areas of positive electrostatic potential and the part with a green color show the areas of zero

potential. From Fig. 8, it is evident that the negative regions localized over the oxygen and nitrogen atoms. Also,

C1 and C22 have positive potential owing to the pressure of the oxygen atom. The observed molecule has many

potential sites for electrophilic and nucleophilic attack as evidenced by Fig. 9.

Frontier molecular orbital analysis

HOMO-LUMO band gap plays a genuine necessary for determining the chemical reactivity, stability of

the chemical compound, electrical, chemical reactions, and optical properties. LUMO energy means the aptitude

to recognize an electron while HOMO energy implies the ability to donate an electron. The conjugated

compound is characterized by a HOMO-LUMO gap, which is the result of intermolecular charge transfer from

the end-capping electron-donor groups to the efficient electron-acceptor groups through a pi-conjugated path.

The HOMO energy, the LUMO energy and the energy gap of compound DMN are computed using

B3LYP level with 6-31G (d,p) basis set is listed in Table 3. Fig. 9 depicts the molecular orbitals and energies for

the HOMO-LUMO, where the positive phase and negative phase of orbitals are represented in red and green

color, respectively. It is clear from the figure that, the HOMO lying at -5.08 eV and LUMO lying at -1.19eV.

The HOMO-LUMO gap lies at 3.83 eV for the title compound. The energy gap of HOMO-LUMO resolves the

ultimate charge transfer interactions that take place inside the molecule.

Fig. 8. MEP diagram of compound DMN.

HOMO -5.08eV LUMO -1.19eV

Fig. 9. Molecular orbitals and energies for the HOMO and LUMO diagram of compound DMN.

3.88eV



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Table 3. Calculated energy values (eV) of compound DMN.

Non-linear optical activity

Density functional theory has been used as an efficient methodology to analyze the organic nonlinear

optical (NLO) materials. Recent analysis has exemplified that the organic materials are having high optical non-

linearity than inorganic materials. In the presence of an applied electric field, the energy of a system could be

operating of the electric field. Polarizabilities and hyperpolarizabilities characterize the response of a system in

an applied electric field. They determine not only the strength of molecular interactions as well as the NLO

properties of the system.

Table 4. Dipole moment, Polarisability, Hyperpolarizability of compounds me1 and dm2 calculated using a

B3LYP method using 6-31G (d,p) basis set.

Parameter Dipolemoment (Debye) Parameter Hyperpolarisability (a.u)

DMN

μx 4.03 βxxx 60.70

μy -2.90 βyyy -62.71

μz 0.316 βzzz -3.11

μtotal 4.97 βxyy 78

Parameter Polarisability (a.u) βxxy -90.5

αxx 131.18 βxxz -28.59

αyy 124.53 βxzz -1.3

αzz 143.14 βyzz 16.88

αxy 12.29 βyyz 10.90

αxz 3.14 βxyz 4.35

αyz 2.14 αo (esu)x10-23 1.97

β0 (esu) x10-30 1.68 Δα (esu) x10-24 4.19

For this subject, in this study, the electronic dipole moment, polarizability, anisotropy of polarizability

and molecular first hyperpolarizability of the present compound were investigated. The electric dipolemoment,

polarizability, the hyperpolarizability and first-order hyperpolarizability of the title compound are calculated by

finite field method using B3LYP/6-31(d,p) level of theory.

The calculated mean linear polarizability and the first hyperpolarizability values are 1.68 x10-23 and 4.97

x10-30 esu, listed in Table 4 respectively. From the above result, the first hyperpolarizability of title compound

material is comparatively12.7 times larger than the NLO reference material Urea. The above results show that

DMN can be the best material for NLO applications.

DFT/B3LYP/6-31G(d,p) DMN

EHOMO -5080

ELUOMO -1.196

ELUMO-HOMO (ΔE) 3.883

Electrinegativity(χ) 3.138

Hardness(η) -1.941

Electrophilicity index(ψ) -2.535

Softness(s) 7.006



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CONCLUSION

In summary, we have successfully synthesized and developed a novel Schiff base (E)-N'-((3,4-

diaminophenyl)(phenyl)methylene) nicotinohydrazide compound DMN, were synthesized and characterized

through elemental analysis, FT-IR, 1H NMR,13C NMR. We believe that this work will inspire the development

of a Schiff base multifunctional library by modifiable lateral and terminal groups for many practical applications

in chemical, environmental and biological systems. Moreover, the explained by the theoretical calculations,

Mulliken and MEP analyses were summarized available reactive sites present in the title compounds. NBO

analysis was used to identify inter and intramolecular interaction in the synthesized compounds. The HOMO-

LUMO study also indicated that on modifying the phenyl group with electron donating or electron withdrawing

functionality changes their HOMO-LUMO gap, which in turn is expected to modify the band gap and thereby

may increase its efficiency. The dipole moment, polarisability and hyperpolarizability have been evaluated. This

study is expected to provide valuable information for further development and formulation of such optical

material and their potential for electronic applications.

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Sublimentry Information

Fig. 1 The FT-IR spectrum of compound DMN.

Fig. 2 The 1H NMR spectrum of compound DMN.

Fig. 3 The 13C NMR spectrum of compound DMN



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a novel synthesis of (e)-n'-((3, 4 diaminophenyl)(phenyl...

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