Novel benzildihydrazone based Schiff bases: Syntheses, characterization, thermal properties, theoretical DFT calculations and biological activity studies

Article abstract of DOI:10.1016/j.molstruc.2019.01.104

A novel series of benzildihydrazone derived Schiff base compounds having electron-accepting and donor-donating appendices were synthesized. A combined experimental and theoretical analysis was conducted to elucidate all the characteristic and structural features of the synthesized molecules. The experimental characterizations were performed by using FT-IR, ¹H/¹³C NMR, mass spectroscopic techniques, and X-ray diffraction methods. The thermal stabilities of the molecules were evaluated by thermal gravimetric analysis (TGA) and all them were found to be stable above 300 °C. To support the experimental, structural, and spectroscopic data of the dyes, the quantum chemical parameters were obtained within the density functional theory (DFT) calculations. In addition, some properties based on the HOMO and LUMO energies and the temperature dependence of some thermodynamic quantities (i.e. entropy, heat capacity, and enthalpy) were also studied. According to the antimicrobial activity evaluation results, compound 1f has been found to be potentially effective in suppressing microbial growth of bacteria with variable potency. The DNA cleavage activity of the compounds was analysed by gel electrophoresis assay. The results showed that the compounds have DNA cleaving activity.

Full text of DOI:10.1016/j.molstruc.2019.01.104

Accepted Manuscript
Novel benzildihydrazone based Schiff bases: Syntheses, characterization, thermal
properties, theoretical DFT calculations and biological activity studies
Gökhan Elmacı, Halil Duyar, Burcu Aydıner, Issah Yahaya, Nurgül Seferoğlu, Ertan
Şahin, Süheyla Pınar Çelik, Leyla Açık, Zeynel Seferoğlu
PII:
S0022-2860(19)30122-X
DOI:
https://doi.org/10.1016/j.molstruc.2019.01.104
MOLSTR 26155
Reference:
To appear in: Journal of Molecular Structure
Received Date: 31 August 2018
Revised Date: 12 December 2018
Accepted Date: 30 January 2019
Please cite this article as: Gö. Elmacı, H. Duyar, B. Aydıner, I. Yahaya, Nurgü. Seferoğlu, E.
Şahin, Sü.Pı. Çelik, L. Açık, Z. Seferoğlu, Novel benzildihydrazone based Schiff bases: Syntheses,
characterization, thermal properties, theoretical DFT calculations and biological activity studies, Journal
of Molecular Structure (2019), doi: https://doi.org/10.1016/j.molstruc.2019.01.104.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
Graphical Abstract

ACCEPTED MANUSCRIPT
Novel benzildihydrazone based Schiff bases: Syntheses, characterization,
thermal properties, theoretical DFT calculations and biological activity
studies
Gökhan Elmacıa, Halil Duyarb, Burcu Aydınerb, Issah Yahayab, Nurgül Seferoğluc, Ertan
Şahind, Süheyla Pınar Çelike, Leyla Açıke, Zeynel Seferoğlub,*
aDepartment of Chemistry, Faculty of Science, Adıyaman University, Adıyaman 02040,
Turkey
bDepartment of Chemistry, Faculty of Science, Gazi University, Teknikokullar, Ankara 06500,
Turkey
cDepartment of Advanced Technology, Gazi University, Teknikokullar, Ankara 06500, Turkey
dDepartment of Chemistry, Faculty of Science, Atatürk University, Erzurum 25240, Turkey
eDepartment of Biology, Faculty of Science, Gazi University, Teknikokullar, Ankara 06500,
Turkey
*Corresponding author’s Tel.: +90 312 2021525; fax: +90 312 2122279
*E-mail addresses: znseferoglu@gazi.edu.tr (Z. Seferoğlu).
1

ACCEPTED MANUSCRIPT
Abstract
A novel series of benzildihydrazone derived Schiff base compounds having electron-
accepting and donor-donating appendices were synthesized. A combined experimental and
theoretical analysis was conducted to elucidate all the characteristic and structural features of
the synthesized molecules. The experimental characterizations were performed by using FT-
IR, 1H/13C NMR, mass spectroscopic techniques, and X-ray diffraction methods. The thermal
stabilities of the molecules were evaluated by thermal gravimetric analysis (TGA) and all
o
them were found to be stable above 300 C. To support the experimental, structural, and
spectroscopic data of the dyes, the quantum chemical parameters were obtained within the
density functional theory (DFT) calculations. In addition, some properties based on the
HOMO and LUMO energies and the temperature dependence of some thermodynamic
quantities (i.e. entropy, heat capacity, and enthalpy) were also studied. According to the
antimicrobial activity evaluation results, compound 1f has been found to be potentially
effective in suppressing microbial growth of bacteria with variable potency. The DNA
cleavage activity of the compounds was analyzed by gel electrophoresis assay. The results
showed that the compounds have DNA cleaving activity.
Keywords: Benzil dihydrazone, X-ray, TGA, DFT/TD-DFT, Antimicrobial activity, DNA
binding.
2

ACCEPTED MANUSCRIPT
1. Introduction
In 1864, Hugo Schiff described the preparation of a Schiff base by a condensation reaction
between an aldehyde and an amine [1]. Schiff bases, due to the presence of the imine group,
are employed in the elucidation of the mechanism of transformation of racemization reaction
in biological systems [2–5]. Different biological activities including antitumor, antibacterial,
antifungal, herbicidal, etc., are studied as a result of the presence of the azomethine linkage
(>C=N-) in living systems [6]. Again, Schiff base derivatives are valuable therapeutic drugs
due to their potent antibacterial, antifungal, and antiviral properties [7-9]. Furthermore, Schiff
bases and their analogues are frequently used as model compounds for understanding the
structure-activity relationship between biomolecules and drug molecules [10-13]. Sriram et al.
[14] reported that, antiviral prodrugs modified with Schiff bases, are highly effective against
mouse hepatitis viruses (MHV) at very low concentrations (EC50 of 1.6 ꢀM). Mahmoud and
co-researchers reported that, Cu (II) Schiff base complex, prepared by condensing 2,6-
diaminopyridine with o-benzoyl benzoic acid, had an effect on human breast cancer cell,
according to in vitro cytotoxicity studies they carried out [15].
Hydrazones and their metal analogues usually have biological activity and can display
enzymatic reactions in cells [16-18]. Edward and co-workers have successfully reported that,
the replacement of an aromatic moiety by different groups in some antibiotics may lead to the
improvement of their antibiotic activity [19,20]. Beside their biological importance, due to the
presence of the toxicologically vital moiety (N–C=O), hydrazones can act as potential donors
for varieties of metal ions. These hydrazine based ligands contain an amide bond and have the
ability to undergo a keto-enol tautomerism. Thus, they can coordinate to central metal ions via
nitrogen and/or oxygen [21]. Additionally, their capability to coordinate may depend on the
3

ACCEPTED MANUSCRIPT
pH of the medium, the nature of the substituents, and the metal they are coordinating with
[22].
Understanding the effects of substituent on the properties of compounds has been a vital goal
in chemistry. Libin Zang and Shimei Jiang [23] have shown how substituens have played
significant roles in the sensing and selectivity of Schiff bases, appended with different
substituents, towards some completing anions. Recently, I. Yahaya et al. [24] investigated and
successfully reported the effects of changing substituents on the solubility and the absorption
properties of some Schiff bases they had prepared.
Antimicrobial susceptibility testing can be used for novel drug candidate discovery, and
epidemiology. Antibiotics used to treat bacterial infections are in danger of losing their
efficacy because of the increase in microbial resistance [25]. The multi-drug resistant bacteria
have become a global concern to public health. Therefore, there has been a growing concern
in developing new antimicrobial agents to combat microbial resistance. Schiff base complexes
have been used as drugs and have valuable antibacterial [26], antifungal [27], and antitumor
[28] activities. In recent times, DNA has been intensively studied as the target molecule in
the development of potential antiviral and anti-cancer drugs [29-31]. Non-covalent
interactions between prepared candidate molecules and DNA are crucial for drug design
studies [32,33]. Schiff base compounds based on benzyl dihydrazone have become well-
known ligands in the design and synthesis of compounds in coordination and supramolecular
chemistry, due to the simplicity in their synthesis and their coordination versatility [34,35].
In recent times, and among various applications, DNA has been intensively studied as the
target molecule in the development of potential antiviral and anti-cancer drugs [36,37]. Non-
covalent interactions between prepared candidate molecules and DNA are crucial for drug
design studies [38,39].
4

ACCEPTED MANUSCRIPT
In our previous studies, we synthesized, characterized, and evaluated the molecular structures
of benzilmonohydrazone derived Schiff bases bearing different electro-donating and electro-
accepting substituents [40,41].
Considering all the literature facts mentioned so far, we therefore realized that the synthesis of
novel Schiff bases, especially those appended with benzildihydrazones, is of great importance
for drug development studies and cannot over-emphasized. Therefore, in the present study, we
have prepared a new series of benzildihydrazone derived Schiff bases also having different
electron-accepting and electron-donating groups and conducted spectroscopic
characterization. In our contribution, we focus our attention on the differences between the
mono- and the dihydrazones in terms of biological and other important properties. The
thermal properties of the prepared dihydrazone compounds were determined. The
experimental results obtained in this study were further explained by theoretical calculations.
Finally, in the investigation of the biological properties of the compounds, we focused on the
interaction of the compounds with plasmid-DNA and their antimicrobial properties against
some bacteria and fungi.
2. Experimental
2.1. Materials and instrumentations
The chemicals used in the syntheses of all the compounds were obtained from Aldrich
Chemical Company (USA) and were used without further purification. All reactions were
magnetically stirred and monitored by thin layer chromatography (TLC), using Merck silica
gel (60 F254) plates (0.25 mm) and visualized under Ultraviolet light (UV). FT-IR (ATR)
spectra were recorded on Perkin-Elmer Spectrum 100 FT-IR spectrophotometer. NMR spectra
5

ACCEPTED MANUSCRIPT
were recorded on a Bruker Avance 300 Ultra-Shield in DMSO-d6. Chemical shifts are
expressed in δ units (ppm). Thermal analysis of the prepared compounds was performed using
Hitachi 7300 thermal analysis system at a heating rate of 20 °C min−1 under a nitrogen flow
(100 mL min−1). Mass spectra was performed using a Waters LCT Premier XE (TOF MS)
(Gazi University Laboratories, Department of Pharmacological Sciences) mass spectrometer.
X-ray single crystal structure was determined on Rigaku R-axis Rapid-S IP area detector
diffractometer in the Department of Chemistry, Atatürk University, Erzurum, Turkey.
2.2. Synthesis and characterization of the compounds 1a-h
The benzildihydrazone was prepared based on literature procedures [29,30,40,41] and used as
the starting material. The Schiff base compounds 1a-h were synthesized by mixing
benzildihydrazone (0.001 mol) and corresponding aldehydes a-h (0.002 mol) in 20 mL dry
methanol. Two drops of hydrochloric acid were added to the mixture. The mixture was then
refluxed for 5 h to form the products 1a-h (Scheme 1). The products obtained were then
cooled and the solvent was removed by slow evaporation. All the compounds 1a-h were
recrystallized using ethyl acetate. The characterization data for all the compounds can be seen
in Figs. S1-S32 in the Supplementary Information.
Scheme 1 is here
(1E,2E)-1,2-bis(((E)-benzylidene)hydrazono)-1,2-diphenylethane (1a)
Yield: 70%, m.p. 110 °C. FT-IR (ATR, υmax., cm-l): 2979, 2937 (aromatic and aliphatic C-H
stretch); 1610 (C=N stretch). 1H NMR (300 MHz): δ 8.57 (s, 2H); 7.80 (m, 4H); 7.62-7.30 (m,
6

ACCEPTED MANUSCRIPT
13
16H). C NMR (75 MHz) δ: 164.8; 159.8; 134.2; 130.9; 130.7; 128.7; 128.6; 127.7. HRMS
(m/z) (M-H)+ calculated for C28H23N4: 415.1923; found: 415.1923.
(1E,2E)-1,2-bis(((E)-4-nitrobenzylidene)hydrazono)-1,2-diphenylethane (1b)
Yield: 73%, m.p. 180 °C. FT-IR (ATR, υmax., cm-l): 3078, 2979 (aromatic and aliphatic C-H
stretch); 1615 (C=N stretch); 1317 (N-O asymmetric stretch). 1H NMR (300 MHz): δ 8.72 (s,
1H); 8.38 (d, J: 7.8 Hz, 2H); 7.82 (m, 4H); 7.53 (m, 3H). 13C NMR (75 MHz) δ: 164.8; 15.0;
149.2; 139.8; 133.4; 132.0; 129.6; 127.8; 124.5. HRMS (m/z) (M-H)+ calculated for
C28H21N6O4: 505.1624; found: 505.1621.
4,4'-((1E,1'E)-(((1E,2E)-1,2-diphenylethane-1,2-diylidene)bis(hydrazine-2,1-
diylidene))bis(methanylylidene))dibenzoic acid (1c)
Yield: 68%, m.p. 345 °C. FT-IR (ATR, υmax., cm-l): 3050,2970 (aromatic and aliphatic C-H
stretch); 3250 – 2400 (carboxylic acid O-H stretch); 1687 (C=O stretch); 1612 (C=N stretch).
1H NMR (300 MHz): δ 13.14 (s, 2H); 8.64 (s, 2H); 7.92 (d, J: 7.2 Hz, 4H); 7.78 (m, 4H); 7.68
13
(d, J: 8.2 Hz, 4H); 7.49 (m, 6H). C NMR (75 MHz) δ: 167.1; 164.5; 159.9; 137.8; 133.7;
133.3; 131.8; 130.1; 129.5; 128.7; 127.7. HRMS (m/z) (M-H)+ calculated for C30H23N4O4:
503.1719; found: 505.1621.
(1E,2E)-1,2-bis(((E)-4-chlorobenzylidene)hydrazono)-1,2-diphenylethane (1d)
Yield: 76%, m.p. 170 °C. FT-IR (ATR, υmax., cm-l): 3059, 2979 (aromatic and aliphatic C-H
1
stretch); 1609 (C=N stretch). H NMR (300 MHz): δ 8.58 (s, 2H); 7.77 (m, 4H); 7.59 (d, J:
8.2 Hz, 4H); 7.48 (m, 10H).13C NMR (75 MHz) δ: 164.6; 159.7; 136.4; 133.8; 132.9; 131.7;
120.2; 129.5; 129.4; 127.7. HRMS (m/z) (M-H)+ calculated for C28H20Cl2N4: 483.1103 ;
found: 483.1113.
(1E,2E)-1,2-bis(((E)-4-methylbenzylidene)hydrazono)-1,2-diphenylethane (1e)
Yield: 74%, m.p. 125 °C. FT-IR (ATR, υmax., cm-l): 3061, 2933 (aromatic and aliphatic C-H
1
stretch); 1601 (C=N stretch). H NMR (300 MHz): δ 8.52 (s, 2H); 7.77 (m, 4H); 7.47 (m,
7

ACCEPTED MANUSCRIPT
10H); 7.20 (d, J: 8.0 Hz, 4H); 2.29 (s, 6H). 13C NMR (75 MHz) δ: 164.2; 160.6; 141.9;
134.1; 131.4; 131.4; 129.8; 129.4; 128.7; 127.6; 21.5. HRMS (m/z) (M-H)+ calculated for
C30H26N4: 443.2236; found: 443.2223.
4,4'-((1E,1'E)-(((1E,2E)-1,2-diphenylethane-1,2-diylidene)bis(hydrazine-2,1-
diylidene))bis(methanylylidene))diphenol (1f)
Yield: 65%, m.p. 236-238 °C. FT-IR (ATR, υmax., cm-l): 3300–3000 (O-H stretch); 3062, 2970
1
(aromatic and aliphatic C-H stretch); 1602 (C=N stretch). H NMR (300 MHz): δ 10.07 (s,
1H); 8.47 (s, 1H); 7.75 (m, 2H); 7.45 (m, 8H); 7.40 (d, J: 8.6 Hz, 2H); 6.76 (d, J: 8.6 Hz, 2H).
13C NMR (75 MHz) δ: 169.9; 161.0; 160.8; 134.4; 131.1; 130.7; 129.3; 127.5; 125.2; 116.1.
HRMS (m/z) (M-H)+ calculated for C28H23N4O2: 447.1821; found: 447.1808.
(1E,2E)-1,2-bis(((E)-4-methoxybenzylidene)hydrazono)-1,2-diphenylethane (1g)
Yield: 74%, m.p. 125 °C. FT-IR (ATR, υmax., cm-l): 3061, 2933 (aromatic and aliphatic C-H
1
stretch); 1601 (C=N stretch); 1250 (Ar-O-C). H NMR (300 MHz): δ 8.52 (s, 1H); 7,77 (m,
12H); 7.53 (d, J: 8.8 Hz, 2H); 7,46 (m, 12H); 6.95 (d, J: 8.7 Hz, 2H); 3.76 (s, 3H). 13C NMR
(75 MHz) δ: 165.8; 162.2; 160.5; 134.8; 131.7; 129.3; 127.1; 125.7; 114.4; 55.3. HRMS (m/z)
(M-H)+ calculated for C30H27N4O2: 475.2123; found: 475.2124.
4,4'-((1E,1'E)-(((1E,2E)-1,2-diphenylethane-1,2-diylidene)bis(hydrazine-2,1-
diylidene))bis(methanylylidene))bis(N,N-dimethylaniline) (1h)
Yield: 78%, m.p. 210 °C. FT-IR (ATR, υmax., cm-l): 3060, 2918 (aromatic and aliphatic C-H
1
stretch); 1602 (C=N stretch); 1342 – 1296 (C-N). H NMR (300 MHz): δ 8.41 (s, 1H); 7.39
13
(m, 2H); 7.80-7.35 (m, 5H); 6.66 (d, J: 8.0 Hz, 2H); 2.96 (s, 6H). C NMR (75 MHz) δ:
163.4; 161.1; 152.7; 130.7; 130.3; 129.2; 127.4; 121.4; 111.9; 95.6; 40.0. HRMS (m/z) (M-
H)+ calculated for C32H33N6: 501.2767; found: 501.2776.
8

ACCEPTED MANUSCRIPT
2.3. X-ray crystallography
For the crystal structure analysis, suitable quality of single crystals were selected. When it was
necessary, the crystals were cut to obtain the appropriate size. Crystals, mounted on a Rigaku
R-AXIS RAPID-S diffractometer (with MoKα radiation λ=0.71069 Å) was used for the data
collection. Data collections were carried out at room temperature [273 (2) K]. Integration of
the intensities, correction for Lorentz and polarization effects, and cell refinement were
performed using CrystalClear (Rigaku/MSC Inc.,2005) software [42]. The atomic coordinates
were located using direct methods employed by SHELXS [43]. The successive refinements,
once the atoms were placed in their postulated positions, were made using SHELXL [43] All
non-hydrogen atoms were then refined anisotropically. The hydrogen atoms were placed in
idealized positions in a riding model, after location on a Fourier difference map. An isotropic
refinement was used for all hydrogen atoms and temperature factors of 1.2 or 1.5 times that of
the parent atoms were assigned. The experimental data such as the crystal data, structure
refinement details, etc. can be seen in Table 1.
Table 1 is here
2.4. Computational procedures
The calculations were performed in the framework of density functional theory (DFT) in the
Gaussian 09 program package [45]. The geometric structures on the ground state were
obtained using B3LYP/6-31+g(d,p) methods. The confirmation of the convergence to minima
on the potential energy surface was made from the vibrational analysis for each molecule. The
9

ACCEPTED MANUSCRIPT
electronic spectra of the compounds were also obtained from TD-DFT calculations at the
same level, in DMSO and chloroform, in which the integral equation formalism of the
polarizable continium model (PCM) [42,43] was employed to evaluate the solvent effects.
2.5. Antimicrobial Activity
The antimicrobial potency of the synthesized compounds was done by agar well diffusion
technique against fungi and gram negative and positive bacteria. Compounds 1a, 1b, 1c, 1d,
1e, 1f, 1g and 1h were tested for their inhibitory activities against Candida albicans ATCC
10231, Candida tropicalis NRRL Y-12968, Candida krusei ATCC 6258 fungi and Bacillus
subtilis ATCC 6633, Bacillus cereus NRRLB-3711, Staphylococcus aureus ATCC 25923,
Enterococcus faecalis ATCC 29212, Enterococus hirae ATCC 9790, Escherichia coli ATCC
35218, Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Proteus
vulgaris RSKK 96029, Salmonella typhimurium ATCC 14028, Klebsiella pneumaniae ATCC
13883 bacteria. The microorganisms were acquired from the collections of Gazi University
Molecular Biology Culture Collection, Turkey. Ampicillin (10 µg), chloramphenicol (30 µg)
(antibacterial), and ketoconazole (50 ꢀg) (antifungal) were used for comparison as positive
control. The agar plate surface was inoculated by spreading a volume of the tested strain
inoculum with microbial suspension adjusted to 0.5 McFarland scale, over the agar surface.
Then, a hole with a diameter of 6 to 8 mm was punched, aseptically with a sterile tip, and a
volume (100 µL) of the compound at 4000 µM was introduced into the well. After that, the
o
o
agar plates were incubated at 37 C for bacteria and 30 C for fungi. The antimicrobial agent
diffused in the agar medium and inhibited the growth of the microbial strain tested. The
diameter of the zone of growth inhibition was measured by antibiogram zone measuring scale.
Each bacterial strain was sub-cultured overnight at 37 °C in LB agar. The bacteria were
10

ACCEPTED MANUSCRIPT
processed using different concentrations of the compounds. Compound-free control tubes
were included in each run. All test were replicated in three time.
2.6. DNA binding Studies
The stock solutions of the compounds were disolved in DMSO. Then, the aliquots of the
compounds, ranging from 2000 to 250 µM, were incubated with plazmid DNA in dark at 37
oC for 24 h. The compounds\DNA mixtures were loaded onto the agarose gel. The agarose gel
electrophoresis run under TAE buffer (0.05 M Tris base, 0.05 M glacial acetic acid and 1 mM
EDTA, pH=8.0) for 3 h at 70 V. After that, the gel was stained with ethidium bromide,
visualized under UV light using a transilluminator (BioDoc Analyzer, Biometra) and the
image was photographed with a video camera. Each test was conducted for triplicate and the
mean values were taken.
2.7. DNA Binding with the compounds
The binding experiment of each of the compounds (1a, 1b, 1c, 1d, 1e, 1f, 1g and 1h) with
DNA was carried out by agarose gel electrophoresis. The plasmid-DNA and different
concentrations of the compounds was incubated for 24 hours. Agarose gel electrophoresis was
carried out at 70 V for 3 h in TAE buffer. DNA bands were visualized by using Biometra
Transilluminator.
11

ACCEPTED MANUSCRIPT
2.8. Restriction enzyme digestion
BamH1 and HindIII are restriction endonuclease that recognize sequences 5’-G/GATCC-3’,
5’-A/AGCTT and hydrolyze hosphodiester bond between adjacent guanine and adenine sites
respectively. The plasmid DNA used in this study contains a single restriction site for both
enzymes and these enzymes convert the supercoiled Form I and singly nicked circular Form II
to linear form III DNA. Therefore conformational changes can be observed by treating DNA-
compound mixture with the restriction enzymes. Each of the compound-DNA mixtures was
first incubated for 24 h at 37°C and then subjected to enzyme digestion. The mixtures were
left at 37°C for another 1 h and loaded onto the gel. The gel was then photographed.
3. Results and discussion
3.1. X-ray diffraction analysis
The suitable crystals of the derivatives 1d, 1e, and 1h were obtatained by using ethanol or
methanol or a mixture of ethanol/methanol solution via slow evaporation process. After the
process, suitable crystals that formed analysed using single crystal X-rays diffraction analysis.
The single-crystal structures formed are shown in Figs. 1 and 2. Selected metrical parameters
from the X-ray diffraction are listed in Table S1 (Supplementary Information). The average
bond lengths and bond angle parameters of the ring systems (phenyl), C=N-N=C and –N-CH3
units were noted to be in the normal ranges.
Figs. 1 and 2 are here
12

ACCEPTED MANUSCRIPT
The crystallographic data (Table S2, Supplementary Information) demonstrate that, two
dihydrazon Schiff bases (1d,1e) crystallized in triclinic system with space group P-1 and the
other (1h) crystallized in monoclinic system with space group
P21/c.
Dibenzilidene hydrazine units were found to be nearly planar. The N-N (hydrazine) distances
are in a typical single bond range [1.407(3)-1.430(3) Å]. The C=N double bond units
in hydrazine have been determined to be around 1.264(3)-1.286(3) Å. The torsion
angles involving the C=N-N=C units have values in the range of 137.3-180.0°. These torsion
angles in all the crystals and optimized gas phase geometry were found to be in close
agreement with the expected value due to the sp2 hybridization of the N atoms. The C-Cl bond
lengths are 1.728(3) and 1.729(3)Å. All the results realized herein are similar to those found
in our previous studies [29,30,40,41]. The conformations have been defined by steric effects
which forced a rotation of the N,N'-Dibenzylidene-hydrazine units relative to C7-C8 single
bond axis, and these bonds vary between 1.498(3)-1.508(3)Å. One of the N,N'-dibenzilidene-
hydrazine groups of each molecule was more planar, the other units were significantly twisted
around their molecular axis. For these turn around units, the dihedral angles formed by LSQ-
planes of C16/C21 and C9/C14 phenyl rings have been determined to be 28.11(2)° (for 1d),
23.24(4)° (for 1e), and 61.48( 2)° (for 1h).
The Cl1⋯Cl1 intermolecular interactions were considered as the primary reason for the
layered structure of the dichloride (1d) crystal. The distance d(Cl•••Cl) = 3.383(4) Å (3) was
found to be sufficiently less than the sum of the vdW radii (3.52 Å). The π-π stacking
interactions between the delocalized π-electrons of the phenyl rings have been found to be
relatively weak. The distance between the rings centroids were obtained in the range of 3.9–
4.3ꢁÅ. The crystal packings are also controlled by non-classical weak C-Cl⋯Cg and C-H⋯Cg
(pi-ring) interactions (Table S3, Supplementary Information).
13

ACCEPTED MANUSCRIPT
3.2. Computational analysis
3.2.1. Optimized structures
The optimized geometries of all the studied molecules (1a-1h) are illustrated in Fig. 3. The
selected optimized structural parameters are given in Table 1 and compared with the
experimental data of 1d, 1e, and 1h. The calculated parameters are in good agreement with X-
ray results for 1d, 1e and 1h. The correlation coefficient (R2) obtained from the comparison of
X-ray and computed bond lengths are found as R2= 0.98 for 1d, R2=0.96 for 1e and R2=0.97
for 1h. Comparison of the parameters of the other molecules, without X-ray data, with 1d, 1e
and 1h showed that they have similar geometry with 1d, 1e and 1h. The Ph-C=N-N=C-Ph
moieties have been found to be in the anti-form and a significant conjugation was noticed as a
result of the planarity of the C16, C15, N4, N3, C8, and C9 atoms in one moiety, and the C6,
C7, N1, N2, C22, and C23 atoms, in the other moiety, as seen from the dihedral angles (Table
S1, Supplementary Information). In addition, two Ph-C=N-N=C-Ph moieties are almost
perpendicular to each other with the dihedral angle N3-C8-C7-N1 changes in the range 94.5-
96.1o.
Fig. 3 is here
3.2.2. Absorption spectra of 1a-1h
The absorption spectra were obtained in DMSO (ε = 46.45 Debye) and chloroform (ε = 4.81
Debye) both experimentally and theoretically. The absorption spectra of compounds 1a-h
were recorded over the λ range of 300-700 nm using a variety of solvents with concentration
14

ACCEPTED MANUSCRIPT
range of 10-6-10-8 M (Table 1). Using calculations, the absorption spectra were also obtained
by employing the TD-DFT methods with 6-31+G(d,p) basis set. The experimental and
theoretical results are shown in Table 1. According to the experimental spectra results, all the
molecules have strong absorption band in the range 322-397 nm in DMSO, and 309-380 nm
in chloroform, and are attributed to π-π* transitions. The comparison of the absorption
maxima (λmax) of 1b, 1c and 1d, with that of 1a indicated that, the largest bathochromic shift
(19 nm) has been observed for 1b which is appended with a strong electron withdrawing (-
NO2) group, and there was no significant changes for 1c and 1d, which were appended with -
COOH and –Cl, at the para position on the phenyl rings, respectively. Similarly, the electron-
donating groups at the para position of the phenyl rings affected the absorption spectra. There
was a shift in the range 7-19 nm, relative to 1a, for receptors 1g, 1f, and 1e, having electron-
donating groups –CH3, -OH, -OCH3, respectively. The biggest shift (75 nm) was realized for
1h which the strongest electron-donating group, –N(CH3)2, among the derivatives that have
electron-donating substituents. Similar behaviors were observed for the absorption spectra
recorded in chloroform. On the other hand, the effects of the solvents on the absorption
spectra for each molecule could also be appreciated as shown in Fig. 1 and Table 1. The
smallest bathochromic shifts occurred in the absorption spectra of 1c and 1g while the largest
bathochromic shifts were seen for the other molecules due to the increasing dielectric constant
from chloroform to DMSO. The calculated maximum wavelength values are in agreement
with the experimental values, whereas the solvent effects and substituent effects were not
observed in the calculation results. The biggest deviation of experimental values was obtained
as 84 nm for 1b in chloroform, and the smallest as 10 nm for 1e in DMSO. It could be noted
that, the reason for these discrepancies between the experimental and the theoretical results is
that, the geometries were obtained in the gas phase whereas the experimental parameters refer
to the crystalline form of the molecules.
15

ACCEPTED MANUSCRIPT
Table 1 is here
3.2.3. HOMO-LUMO gaps and global reactivities of 1a-1h
The highest occupied molecular orbital and the lowest unoccupied molecular orbital energies
(i.e. EHOMO and ELUMO) and the energy gap (ꢂE) between the HOMO and the LUMO can be
used to determine the molecular properties of the molecules, such as stability and reactivity.
While the instability and the ability of the electron donating are characterized with high
EHOMO value, the ability of the electron accepting is related to the lower ELUMO value.
However, small ꢂE value refers to a molecule having more polarizable, high chemical
reactivity, and low kinetic stability [48].
The diagram of the HOMO and LUMO of the studied molecules are shown in Fig.S33 and the
EHOMO and ELUMO values obtained from the optimized geometries and ꢂE values of the
molecules are presented in Table S4 (Supplementary Information). The ionization energy
(I), electron affinity (A), electronic chemical potential (ꢀ), global hardness (η), global
electrophilicity index (ω), softness (s), and the expressions used to determine them from
EHOMO and ELUMO are also given in Table S4. Derivative 1h has higher capability of donating
electrons than the rest because it is having the highest EHOMO and the lowest electrophilicity
index (ω). Similarly, 1b has lower ELUMO, the highest ω values, and its electron accepting
ability is higher. The largest energy gap has been estimated to be 3.96 eV for 1a, indicating
that 1a should be a harder molecule with good stability, it is also having the highest chemical
hardness (η=1.98 eV) among the studied molecules.
16

ACCEPTED MANUSCRIPT
3.2.4. Thermodynamic properties
The thermodynamic functions were obtained from the vibrational analysis with 6-31+g(d,p)
basis set in gas phase. The temperature variations of the heat capacity at constant pressure
(Cp), entropy (S), and enthalpy (H), with respect to the zero-temperature (T=0 K) for 1d, 1e
and 1h, are demonstrated in Fig. 4. As illustrated in Fig. 4, the Cp, S, and the ꢂH increase
from 200 K to 1000 K, which is caused by the rise of molecular vibrations with increasing
temperature.
Fig. 4 is here
3.2.5. Thermal Analysis
The TGA curve for the prepared Schiff bases is depicted in Fig. 5. As shown in TGA curve,
the decomposition process starts at about 300 °C and proceeds rapidly with increasing
temperature until about 330 °C (Table S5). The order of thermal stability of samples is: (1g)
(1h) > (1e) (1d) (1a) > (1b) (1c) (1f). No direct connection has been observed
̴
̴
̴
̴
̴
between the thermal stability and the effect(s) substituent(s). Molecules (1f) and (1h) can
exhibit intermolecular hydrogen bonding between the compounds and the substituent groups.
The relatively high melting points of (1f) and (1h) may be related to the strong intermolecular
H-bonding in the crystal structures of the molecules.
Fig. 5 is here
17

ACCEPTED MANUSCRIPT
3.2.6. Antimicrobial activity
All the Schiff base compounds 1a-h were used to evaluate their antibacterial activity against
bacteria and yeast species. Evaluation of antibacterial activity of the compounds was recorded
and is shown in Table 2. The results show that, only compound 1f is potentially effective in
suppressing microbial growth of bacteria with variable potency. Compound 1f also showed
activity towards Bacillus subtilis ATCC 6633 (G+), Staphylococcus aureus ATCC 25923
(G+), Enterococcus faecalis ATCC 29212 (G+). In addition, all the compounds did not show
any antifungal activity against the tested yeast strain.
Table 2 is here
3.2.7. DNA-compound binding
The results show conformational changes due to compounds-DNA interactions as well as
compounds binding sites and sequence preference. The DNA cleavage was determined by the
relaxation of supercoiled circular Form I of plasmid DNA into the linear Form III. If
supercoiled circular plasmid DNA is subjected to agarose gel electrophoresis, the fastest
migration will be observed for supercoiled Form I DNA. If one strand is cleaved, the
supercoils will relax to produce a slower moving open circular Form II DNA. If both strands
are cleaved, a linear Form III DNA is generated which migrates in between Form I and Form
II [49].
When plasmid DNA was interacted with decreasing concentrations of the compound mixture
(compounds 1a, 1b, 1c, 1d, 1e, 1f, 1g and 1h), two DNA bands corresponding to Forms II
and III were observed in the treated plasmid (Fig. 6). In the electrophoregram, the untreated
18

ACCEPTED MANUSCRIPT
plasmid DNA, used as control, has the major supercoiled Form I and minor singly nicked
relaxed circular Form II bands. However, for 1b, 1c, 1e, and 1f, they showed Form II bands of
linear DNA. These results show that the compounds cleave the DNA in both strands.
Fig. 6 is here
3.2.8. Restriction enzyme digestion
To gain further insight into the changes in DNA confirmation, the compounds-DNA
incubation, as shown above, was followed by BamH1 or HindIII digestion. Fig. 7 shows the
electrophoretograms applying to the interaction of plasmid DNA with one concentrations of
each of the compounds.
Fig.7 is here
4. Conclusions
In conclusion, novel series of benzildihydrazone based Schiff bases 1a-h have been prepared
in good yields and successfully characterized by using FT-IR, 1H/13C NMR and HRMS. The
structures of three of the prepared Schiff bases (1d, 1e and 1h) have been verified by using
single crystal X-ray diffraction methods. Again, the experimental results of the molecules
were explained using DFT calculations. For applicability as optic dyes, TGA analyses were
done for each dye. The compounds have been found to be stable and remained undecomposed
o
at 300 C. This means that the compounds can be applicable as optic dyes. Compound 1f has
been noted to be potentially effective in suppressing microbial growth of bacteria with
19

ACCEPTED MANUSCRIPT
variable potency towards Bacillus subtilis ATCC 6633, Staphylococcus aureus ATCC 25923,
Enterococcus faecalis ATCC 29212. Also, 1f has also been found to be a good potential for
therapeutic uses against some Gram positive pathogens. It appears that, a compound with
good antimicrobial activity against Gram-positive bacteria does not have any activity against
Gram–negative bacteria. This may mean that, the activity is related to the differences in cell
wall structure of the bacteria. In the case of compound-DNA interaction, all the compounds
caused DNA fragmentation.
Finally, we believe that, the compounds will have potential application as chemsensors for
any cation or can be precursor ligands for some metals. The investigation of their
chemosensor properties and affinity towards some metal is currently ongoing and results
found scientifically viable will be published sooner.
Acknowledgements
The authors are very grateful to Gazi University Research Fund for providing financial
support for this project (grant No. 05/2009-43). The numerical calculations reported in this
paper were fully performed at TUBITAK ULAKBIM, High Performance and Grid
Computing Center (TRUBA resources).
Appendix A. Supplementary data
Supplementary data related to this article can be found at………….
20

ACCEPTED MANUSCRIPT
References
[1] H. Schiff, The syntheses and characterization of Schiff base, Ann. Suppl., 1864, 3, 343.
[2] E. Keskioglu, A.B. Gunduzalp, S. Cete, F. Hamurcu, B. Erk, Cr (III), Fe (III) and Co (III)
complexes of tetradentate (ONNO) Schiff base ligands: synthesis, characterization, properties
and biological activity. Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy 70.3 (2008): 634-640.
[3] Abu-Hussen, Azza A.A., Synthesis and spectroscopic studies on ternary bis-Schiff-base
complexes having oxygen and/or nitrogen donors. Journal of Coordination Chemistry 59.2
(2006): 157-176.
[4] Singh, Kiran, Manjeet Singh Barwa, and Parikshit Tyagi, Synthesis, characterization and
biological studies of Co (II), Ni (II), Cu (II) and Zn (II) complexes with bidentate Schiff bases
derived by heterocyclic ketone. European Journal of Medicinal Chemistry 41.1 (2006): 147-
153.
[5] P. Panneerselvam, R.R. Nair, G. Vijayalakshmi, E.H. Subramanian, S.K. Sridhar,
Synthesis of Schiff bases of 4-(4-aminophenyl)-morpholine as potential antimicrobial
agents. European journal of medicinal chemistry 40.2 (2005): 225-229.
[6] (a) Chohan, Zahid Hussain, and Samina KAUSAR, Synthesis, structural and biological
studies of nickel (II), copper (II) and zinc (II) chelates with tridentate Schiff bases having
NNO and NNS donor systems. Chemical and Pharmaceutical Bulletin 41.5 (1993): 951-953.;
(b) Thomson, Andrew J., and Harry B. Gray, Bio-inorganic chemistry. Current opinion in
chemical biology 2.2 (1998): 155-158
.
[7] L.H. Abdel-Rahman, A.M. Abu-Dief, R.M. El-Khatib, S.M. Abdel-Fatah, Some new
nano-sized Fe (II), Cd (II) and Zn (II) Schiff base complexes as precursor for metal oxides:
21

ACCEPTED MANUSCRIPT
Sonochemical synthesis, characterization, DNA interaction, in vitro antimicrobial and
anticancer activities, Bioorganic chemistry, 69 (2016) 140-152.
[8] M. Abo-Aly, A. Salem, M. Sayed, A.A. Aziz, Spectroscopic and structural studies of the
Schiff base 3-methoxy-N-salicylidene-o-amino phenol complexes with some transition metal
ions and their antibacterial, antifungal activities, Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy, 136 (2015) 993-1000.
[9] M. Shebl, Synthesis, spectroscopic characterization and antimicrobial activity of binuclear
metal complexes of a new asymmetrical Schiff base ligand: DNA binding affinity of copper
(II) complexes, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, 117
(2014) 127-137.
[10] M.M. Abd-Elzaher, A.A. Labib, H.A. Mousa, S.A. Moustafa, M.M. Ali, A.A. El-
Rashedy, Synthesis, anticancer activity and molecular docking study of Schiff base complexes
containing thiazole moiety, Beni-Suef University Journal of Basic and Applied Sciences, 5
(2016) 85-96.
[11] J.-R. Duan, H.-B. Liu, P. Jeyakkumar, L. Gopala, S. Li, R.-X. Geng, C.-H. Zhou, Design,
synthesis and biological evaluation of novel Schiff base-bridged tetrahydroprotoberberine
triazoles as a new type of potential antimicrobial agents, MedChemComm, 8 (2017) 907-916.
[12] Y. Jia, J. Li, Molecular assembly of schiff base interactions: construction and application,
Chemical reviews, 115 (2014) 1597-1621.
[13] S. Zehra, M. Shavez Khan, I. Ahmad, F. Arjmand, New tailored substituted
benzothiazole Schiff base Cu (II)/Zn (II) antitumor drug entities: effect of substituents on
DNA binding profile, antimicrobial and cytotoxic activity, Journal of Biomolecular Structure
and Dynamics, (2018) 1-17.
22

ACCEPTED MANUSCRIPT
[14] D. Sriram, P. Yogeeswari, N.S. Myneedu, V. Saraswat, Abacavir prodrugs: Microwave-
assisted synthesis and their evaluation of anti-HIV activities, Bioorganic and medicinal
chemistry letters, 16 (2006) 2127-2129.
[15] W.H. Mahmoud, R.G. Deghadi, G.G. Mohamed, Novel Schiff base ligand and its metal
complexes with some transition elements. Synthesis, spectroscopic, thermal analysis,
antimicrobial and in vitro anticancer activity, Applied Organometallic Chemistry, 30 (2016)
221-230.
[16] Bulaov A O, Lukyanov B S, Kogan V A, Lukov V V, Photo- and thermochromic spirans.
New metal chelates based on azomethines and hydrazones containing a spiropyran fragment,
Russ J. Coord. Chem. 28 (2002), 46-49, DOI 10.1023/A:1013715821212.
[17] Al-Assar F, Zelenin K N, Lesiovskaya E E, Bezhan I P, Chakchir B A, Synthesis and
Pharmacological Activity of 1-Hydroxy-, 1-Amino-, and 1-Hydrazino-Substituted 2,3-Dihydro-
1H-pyrazolo[1,2-a]pyridazine-5,8-diones and 2,3-Dihydro-1H-pyrazolo[1,2-b] phtha-lazine-
5,10-diones, Pharm Chem J. 36 (2002), 598-603, DOI 10.1023/A:1022665331722.
[18] Sharma V K, Srivastava S, Synthesis, Spectroscopic and Antifungal Studies of Trivalent
Chromium, Manganese, Iron and Cobalt Complexes with Hydrazones derived from Benzil
–monoxime and various Aromatic Hydrazides, Synt. React. Inorg. Met. Org. Nano. Met.
Chem. 35 (2005), 311-318, DOI10.1081/SIM-200055249.
[19] Murukan B, Mohanan K, Synthesis, Characterization, Electrochemical Properties and
Antibacterial Activity of Some Transiton Metal Complexes with [(2-hydroxy-1-
naphthaldehyde)-3-isatin]-bishydrazone, Transition Met. Chem. 31 (2006), 441-446, DOI
10.1007/s11243-006-0011-7.
[20] Edward E I, Epton R, Marr G, Organometallic derivatives of penicillins and
cephalosporins a new class of semi-synthetic antibiotics, J. Organomet. Chem. 85 (1975),
C23-C25, DOI 10.1016/S0022-328X(00)80708-1.
23

ACCEPTED MANUSCRIPT
[21] Pandey J K, Pandey O P, Sengupta S K, Synthesis and spectroscopic investigations of
oxovanadium (IV) derivatives with 1,1’-diacetylferrocenylbis( hydrazones), Indian J. Chem.
43A (2004), 1906-1910.
[22] Vatsa B G, Pandey O P, Sengupta S K, Synthesis, Spectroscopic andToxicity Studies of
Titanocene Chelates of Isatin-3-Thiosemicarbazones, Bioinorg. Chem. Appl. 3 (2005), 151-
160, DOI 10.1155/BCA.2005.151.
[23] Libin Zang, Shimei Jiang, Substituent effects on anion sensing of salicylidene Schiff base
derivatives: Tuning sensitivity and selectivity. Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy 150 (2015) 814–820.
[24] Issah Yahaya, Meryem Chemchem, Burcu Aydıner, Nurgül Seferoğlu, Fulya Erva Tepe,
Leyla Açık, Nebahat Aytuna Çerçi, Mustafa Türk, Zeynel Seferoğlu, Novel Coumarin-
Thiophene-Derived Schiff Bases: Synthesis, Effects of Substituents, Photophysical Properties,
DFT Calculations, and Biological Activities. Journal of Photochemistry & Photobiology A:
Chemistry 368 (2019) 296–306.
[25] J. R. Anacona, N. Noriega, J. Camus, Synthesis, characterization and antibacterial
activity of a tridentate Schiff base derived from cephalothin and sulfadiazine, and its transition
metal complexes. Spectrochimica Acta - Part A: Molecular and Biomolecular Spectroscopy.
137 (2015) 16–22.
[26] Bagihalli G. B., Avaji P. G., Patil S. A., Badami P. S., Synthesis, spectral
characterization, in vitro antibacterial, antifungal and cytotoxic activities of Co(II), Ni(II) and
Cu(II) complexes with 1,2,4-triazole Schiff bases. European Journal of Medicinal Chemistry.
2008;43(12):2639–2649.
[27] S. Amer, N. El-Wakiel, H. El-Ghamry, Synthesis, spectral, antitumor and antimicrobial
studies on Cu(II) complexes of purine and triazole Schiff base derivatives. Journal of
Molecular Structure, 1049 (2013) 326–335.
24

ACCEPTED MANUSCRIPT
[28] B.-L. Fei, W.-S. Xu, W.-L. Gao, J. Zhang, Y. Zhao, J.-Y. Long, C.E. Anson, A.K.
Powell, DNA binding and cytotoxicity activity of a chiral iron (III) triangle complex based on
a natural rosin product, Journal of Photochemistry and Photobiology B: Biology, 142 (2015)
77-85.
[29] P. Gotwals, S. Cameron, D. Cipolletta, V. Cremasco, A. Crystal, B. Hewes, B. Mueller,
S. Quaratino, C. Sabatos-Peyton, L. Petruzzelli, Prospects for combining targeted and
conventional cancer therapy with immunotherapy, Nature Reviews Cancer, 17 (2017) 286.
[30] C.C. Wang, S.M. Wu, H.W. Li, H.T. Chang, Biomedical Applications of DNA‐
Conjugated Gold Nanoparticles, ChemBioChem, 17 (2016) 1052-1062.
[31] J. Shawon, A.M. Khan, A. Rahman, M.M. Hoque, M.A.K. Khan, M.G. Sarwar, M.A.
Halim, Molecular recognition of azelaic acid and related molecules with DNA polymerase I
investigated by molecular modeling calculations, Interdisciplinary Sciences: Computational
Life Sciences, (2016) 1-13.
[32] H. Sun, J. Ren, X. Qu, Carbon nanomaterials and DNA: From molecular recognition to
applications, Accounts of chemical research, 49 (2016) 461-470.
[33] G. Elmacı, E. Aktan, N. Seferoğlu, T. Hökelek, Z. Seferoğlu, Synthesis, molecular
structure and computational study of (Z)-2-((E)-4-nitrobenzylidene) hydrazone)-1, 2-
diphenylethan-1-one, Journal of Molecular Structure, 1099 (2015) 83-91.
[34] R. Chandra, A. Ghorai, G. K. Patra, A simple benzildihydrazone derived colorimetric and
fluorescent ‘on–off-on’ sensor for sequential detection of copper(II) and cyanide ions in
aqueous solution, Sensors and Actuators B 255 (2018) 701–711.
[35] D.L. Mayers, J.D. Sobel, M. Ouellette, K.S. Kaye, D. Marchaim, Antimicrobial Drug
Resistance C: Clinical and Epidemiological Aspects, vol. 2, Springer Dordrecht Heidelberg,
London (2009) 681–1347.
25

ACCEPTED MANUSCRIPT
[36] C.C. Wang, S.M. Wu, H.W. Li, H.T. Chang, Biomedical Applications of DNA‐
Conjugated Gold Nanoparticles, ChemBioChem, 17 (2016) 1052-1062.
[37] J. Shawon, A.M. Khan, A. Rahman, M.M. Hoque, M.A.K. Khan, M.G. Sarwar, M.A.
Halim, Molecular recognition of azelaic acid and related molecules with DNA polymerase I
investigated by molecular modeling calculations, Interdisciplinary Sciences: Computational
Life Sciences, (2016) 1-13.
[38] H. Sun, J. Ren, X. Qu, Carbon nanomaterials and DNA: From molecular recognition to
applications, Accounts of chemical research, 49 (2016) 461-470.
[39] G. Elmacı, E. Aktan, N. Seferoğlu, T. Hökelek, Z. Seferoğlu, Synthesis, molecular
structure and computational study of (Z)-2-((E)-4-nitrobenzylidene) hydrazone)-1, 2-
diphenylethan-1-one, Journal of Molecular Structure, 1099 (2015) 83-91.
[40] G. Elmacı, H.Duyar, B.Aydıner, N. Seferoğlu, Mir A. Naziri, E. Şahin, Z. Seferoğlu, The
syntheses, molecular structure analyses and DFT studies on new benzil monohydrazone based
Schiff bases, Journal of Molecular Structure, 1162 (2018) 37-44.
[41] I. Rigaku/MSC, 9009 new Trails Drive, TheWoodlands, TX 77381, USA.
[43] G.M. Sheldrick, Program for crystal structure solution and refinement, SHELXS-97 and
SHELXL-97, (1997).
[44] M. Frisch, G. Trucks, H.B. Schlegel, G. Scuseria, M. Robb, J. Cheeseman, G. Scalmani,
V. Barone, B. Mennucci, G. Petersson, Gaussian 09, revision a. 02, gaussian, Inc.,
Wallingford, CT, 200 (2009).
[45] R. Bauernschmitt, R. Ahlrichs, Treatment of electronic excitations within the adiabatic
approximation of time dependent density functional theory, Chemical Physics Letters, 256
(1996) 454-464.
[46] M. Cossi, V. Barone, Time-dependent density functional theory for molecules in liquid
solutions, The Journal of chemical physics, 115 (2001) 4708-4717.
26

ACCEPTED MANUSCRIPT
[47] M. Wieland, W. Seichter, A. Schwarzer, E. Weber, Influence of different aryl
substitution on the crystal structures of benzil monohydrazone and dibenzil azine parent
compounds, Structural Chemistry, 22 (2011) 1267.
[48] X.-H. Li, X.-R. Liu, X.-Z. Zhang, Molecular structure and vibrational spectra of three
substituted 4-thioflavones by density functional theory and ab initio Hartree–Fock
calculations, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, 78
(2011) 528-536.
[49] T.L. Greaves, C.J. Drummond, Protic ionic liquids: properties and applications, Chemical
reviews, 108 (2008) 206-237.
27

ACCEPTED MANUSCRIPT
CAPTIONS
Scheme Captions;
Scheme 1. The synthetic pathway of synthesis of benzildihydrazone derivatives.
Figure Captions;
Fig. 1. (a) Crystal structure of the compound 1d with atom labeling scheme. Thermal
ellipsoids are drawn at the 40% probability level. (b) Unit cell with the layered structure of
the molecule in the crystal.
Fig. 2. Crystal structure of the compounds 1e (a) and 1h (b) with atom labeling scheme.
Thermal ellipsoids are drawn at the 40% probability level.
Fig. 3. The optimized geometry of all studied molecules (1a-1h).
Fig. 4. Temperature dependence of thermodynamic quantities (C, S and ꢂH) for 1d, 1e and 1h.
Fig. 5. TGA-DTA thermograms of 1a-1h samples.
Fig. 6. Electrophotograms applying to the interaction of plasmid DNA with decreasing
concentrations of 1a, 1b, 1c,1d, 1e, 1f, 1g and 1h. Lane P applied untreated plasmid DNA to
serve as a control. Lines 1-5 applied to plasmid DNA interacted with decreasing
concentrations of compounds (4000 µM, 2000 µM, 1000 µM, 500 µM).
Fig. 7. Electrophoretogram applying to incubated mixtures of plasmid DNA and compounds
1a, 1b, 1c,1d, 1e, 1f, 1g and 1h followed by digestion with BamH1 or HindIII. Lane P applied
to the untreated and undigested plasmid DNA. Lane P/B or P/H applied to untreated, but
digestion with restriction enzyme BamH1 or HindIII, respectively. Results shows that all of
the compounds inhibited enzyme digestion indicating compounds binding A/A and G/G
nucleotides on DNA.
Table Captions;
Table 1. The calculated and experimental absorption maxima (λmax) values and oscillator
strength (f), transitions with the absolute CI coefficient values obtained in the calculations.
28