Synthesis, characterization, cytotoxicity, cell cycle analysis of 3-(4-methoxyphenyl)-1-(pyridin-2-ylmethyl)thiourea and quantum?chemical analyses

Article abstract of DOI:10.1016/j.molstruc.2017.03.066

Thiourea derivative,3-(4-methoxyphenyl)-1-(pyridin-2-ylmethyl)thiourea, was synthesized. The structure of the synthesized compound (3) was elucidated by IR, UV–visible, ¹H NMR, mass Spectrometry, and X-ray single crystal structure. The computat

Full text of DOI:10.1016/j.molstruc.2017.03.066

Accepted Manuscript
Synthesis, characterization, cytotoxicity, cell cycle analysis of 3-(4-methoxyphenyl)-1-
(pyridin-2-ylmethyl)thiourea and quantum chemical analyses
Md. Mushtaque, Fernando Avecilla, Md. Shahzad Khan, Zubair Bin Hafeez, M.
Moshahid A. Rezvi, Anurag Srivastava
PII:
S0022-2860(17)30354-X
10.1016/j.molstruc.2017.03.066
MOLSTR 23562
DOI:
Reference:
To appear in: Journal of Molecular Structure
Received Date: 7 August 2016
Revised Date: 15 March 2017
Accepted Date: 17 March 2017
Please cite this article as: M. Mushtaque, F. Avecilla, M.S. Khan, Z.B. Hafeez, M.M.A. Rezvi, A.
Srivastava, Synthesis, characterization, cytotoxicity, cell cycle analysis of 3-(4-methoxyphenyl)-1-
(pyridin-2-ylmethyl)thiourea and quantum chemical analyses, Journal of Molecular Structure (2017), doi:
10.1016/j.molstruc.2017.03.066.
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Synthesis, Characterization, Cytotoxicity, cell cycle analysis of 3-(4-methoxyphenyl)-1-
(pyridin-2-ylmethyl)thioureaand Quantum chemical analyses
a
b
c
d
Md. Mushtaque *, Fernando Avecilla , Md. Shahzad Khan , Zubair Bin Hafeez , M. Moshahid
d
c
A. Rezvi *, Anurag Srivastava
a
School of physical and Molecular Sciences (chemistry), Al-Falah University, Dhauj, Faridabad,
Haryana, 121004, India.
b
Departamento de Química Fundamental, Universidade da Coruña, Campus da Zapateira s/n,
1
5008, ACoruña, Spain.
c
Advanced Material Research Group, CNT Lab, ABV-Indian Institute of Information Technology
and Management, Gwalior 474015, India
d
Department of Bioscience, JamiaMilliaIslamia, New Delhi, 110025, India.
Abstract: Thiourea derivative,3-(4-methoxyphenyl)-1-(pyridin-2-ylmethyl)thiourea, was
synthesized. The structure of the synthesized compound (3) was elucidated by IR, UV-visible,
1
H-NMR, mass Spectrometry, and X-ray single crystal structure. The computational quantum
chemical studies like, IR, UV, NBO analysis were performed by DFT with Becke-3-Lee-Yang-
Parr (B3LYP) exchange-correlation functional in combination with 6-311++G(d,p) basis sets. It
was observed experimentally and theoretically that compound (3) exhibited syn-anti-
6
-1
b
conformation around sulphur atom. The DNA-binding constant K was found 3.3x 10 Lmol .
The docking energy of compound (3) with 1BNA was found -6.2 Kcal/mol. MTT-assay against
HepG2 (IC50 = 140.39) and Siha (IC50 = 119.87 ꢀM) cell lines revealed that compound (3)
wasnon-toxic up to140.39 ꢀM against HepG2 and 119.87ꢀM against Siha cells respectively. It
was also found that compound (3) is non-toxic against normal human cell line HEK-293(IC =
5
0
1
48.67ꢀM). Cell cycle analyses displayed that treated HepG2 cells at 40 ꢀM and 80 ꢀM showed
6
5 % and 70 % arrest in G0/G1with respect to untreated controls (60%) and Siha cells at the
same concentration displayed 59 % and 65 % arrest with respect to G0/G1 as compared to
untreated control (45%).
Keywords: Cytotoxicity, Cell cycle analysis, DNA binding and molecular docking & Quantum
chemical studies.
*
Corresponding Author: Email: mush.chem@gmail.com; Contact No: +91-9891717992
Email: rizvi_ma@yahoo.com; Contact no +91-9911661657
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1
.
Introduction
Molecules possessing thiourea moiety are promising structural units and have great significance
in modern medicinal chemistry. Thiourea is a compound composed of nitrogen and sulphur.
They have been known to display various biological activities vizantibacterial [1], anti-fungal[2]
anti-tubercular[3] anti-thyroid[4], anti-helmintic[5], rodenticidal[6] insecticidal[7], herbicidal[8],
and plant growth regulator [9] activities. Thiourea derivative is one of the most promising groups
of anti-cancer agents associated with a wide range of activities against various leukemia and
solid tumors [10-19]. They exhibit a significant role as anti-cancer agents due to its inhibitory
activity against protein tyrosine kinases (PTKs) [10-13] DNA repair synthesis [18].Recently,
Camacho et.al synthesized a series of novel compounds associated with urea and thiourea of
kynurenamine each with different substituents and screened against neural nitric oxide synthase
(nNOS) and inducible nitric oxide synthase activity (iNOS), and he found that thiourea
derivatives demonstrated the potent activity as shown in Fig.1(a-b)[20]. Moreover Pingaew et.al
synthesized some urea and thiourea derivatives associated with 2-picolyl amine and aromatic
compounds and observed that compounds(2a& 2b) exhibited potent cytotoxic effects against
HepG2 and MOLT-3 cell lines as shown in Fig2.(a-b)[21]. In the view of versatile report on
different biological activities of thiourea derivatives, we here report the synthesis 3-(4-
methoxyphenyl)-1-(pyridin-2-ylmethyl)thiourea derivative, and molecular docking of the
compound with known receptor of DNA (PDBID:IBNA) and DNA-binding constant(Ct-DNA)
by UV-Visible spectrophotometer method, and MTT-assay against HepG2, and Siha cancer cell
linesand normal human(HEK-293) cell line to ensure its cytotoxic efficacy. Additionally,
quantum chemical studies have also been performed in order to explore its physico-chemical
properties.
2
. Result and discussion
Chemistry
Compound (3), 1-(4-methoxyphenyl)-3-(pyridine-2-ylmethyl)thiourea was synthesized by
reported method[22-24]as shown in scheme(1). The compound (3) was characterized by FT-IR,
1
UV-Visible, H-NMR and mass spectrophotometer. Moreover, the structure was also confirmed
0
by X-ray single crystal structure. The melting point was recorded 130 C and is uncorrected.
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The presence and absence of significant bands in the spectra confirm the formation of
compound. The formation of compound (3) was confirmed by IR spectrum. The appearance of
-1
-1
characteristic bands at 3300-3480cm and 748 cm were assigned to N-H and C=S bond groups
1
which confirmed the formation of compound (3). In H-NMR, chemical shift resonated at higher
value the in range 9.651-9.258 ppm and 8.336-8.058 ppm as broad singlet due to presence of (-
NH) and (S=CNH) protons confirmed the formation of compound (3).The compound (3) was
+
also confirmed by mass spectrophotometer. The appearance of m/z; [M+H] peak at 275
confirmed the formation of compounds (3). Furthermore, the structure is confirmed by X-ray
single crystal structure as shown in the Fig.(3), and the values have been given in the
experimental section.
2
.1. Discussion of crystal structure
ORTEP diagram for the compound 1-(4-methoxyphenyl)-3-(pyridine-2-ylmethyl)thiourea (3) is
showed in Fig. (3).The compound (3) crystallizes in the monoclinic system, with P2 /c space
1
group. The asymmetric unit of 3 only contains one molecule of the thiourea derivative. The
thiourea moiety [N (2)-C1(S))-N1] form dihedral angles with the rings of the compound. A
dihedral angle of 40.33(4)º with the benzene ring and of 88.34(5)º with the pyridine ring are
present in the compound. The thiourea group is planar, r.m.s. deviation from plane 0.0040(5) Å.
The distances corresponding to N-C and C-S bonds, in the thiourea moiety, are indicative of the
Presence of amide-type resonance between the bonds N-C(=S), [N-C distances, C(1)-
N(1),1.401(2) Å and C(1)-N(2), 1.354(2) Å, and C-S distance, C(1)-S(1), 1.9040(17) Å], and
they are agree with the values found in literature [25]. The bond angles and bond lengths have
been given in Table (TS1). In the crystal packing, hydrogen atoms of the secondary amine
groups participate in intermolecular interactions, with nitrogen atoms of pyridine groups and
with the sulphur atoms, through the typical N–H···S hydrogen bond in this type of compounds
(see Table (TS2) and Fig.(4).
3
. Computational details
The ground state geometry optimizations have been carried out using DFT with Becke-3-Lee-
Yang-Parr(B3LYP) exchange-correlation functional [25, 26] in combination with 6-311++G(d,p)
basis sets using Gaussian09 package [27] without any symmetry constraint. To have a
comparative data analysis an isolated molecule of thiourea has also been optimized with the
same level of theory. The simulated IR spectrum has been analyzed using GAUSSVIEW and
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VEDA 4 program to assign vibrational frequency with high degree of accuracy. The NBO and
Mullikan population analysis are also reported for the local minima of title molecule. For
electronic excited state calculations TD-DFT/B3LYP method has been employed with same
basis set considered for ground state optimizations. The TD-DFT calculations for titled
compound (3) are made in the background of chloroform using PCM model [28, 29].
3
.1 Conformational analyses
Several thiourea derivatives displayed syn-anti-conformations [30-31]. In quest of stable
structure and configuration, different structures of compound (3) have been optimized and
frequencies were calculated using 6-311++G (d, p) basis sets, and the structures have been
demonstrated in Fig.5 (a-c). From geometry optimization and frequency calculations, it was
found that the configuration of structure 5(a) has local minima at potential energy surface as
evidenced by no imaginary frequency. The relative energy of structure 5(a) was found-740719.21
kcal/mol. Furthermore, the structures 5(b) and 5(c) configurations have local minima at potential
energy surface as displayed no imaginary frequency. The relative energy of structure 5(b) was
found
-740720.93kcal/mol, However, relative energy of structure 5(c) was obtained -
7
40723.94kacl/mol. Thus, it can be concluded that structure 5(c) has minimum energy,
confirming most stable configuration, and exhibited syn-anti-configuration around sulphur atom.
This theoretical configuration was supported by the experimental configuration as obtained by
X-ray single crystal structure and was shown in Fig.(3).
3
.2 Charge analysis
The atomic charges are reported both in terms of NBO and Mulliken’s population analysis.
These charges have been shown in Table (TS3).Qualitatively, electronic charges on
constituent’s atoms are same in nature for different theoretical analyses but, quantitatively, they
are different. NBO analysis is fit to the chemists as it is based on localized orbital and leads to
Lewis concept behavior, however, Mulliken analysis is result of canonical molecular orbital
distribution which is highly delocalized and loses its significance in chemical dynamics of
molecule [32]. So, further analyses have been made in terms of NBO for betterment of
understanding of charge distributions. The NBO and Mulliken charges have demonstrated in the
the supporting information Table (TS3). From the analysis, it was found that For the central part
of thiourea there was a significant change in charge character of dangling sulphur atom, it has
been changed from 0.07e (for isolated thiourea) to -0.286e compound(3). The N-atoms of central
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thiourea region suffer electronic charge decrements from -0.86e (thiourea) to-0.6ecompound
(
3).Consequently, enhancement of polarity of bond length -C=S takes, resulting in the increment
of its bond length.
.3 IR analysis
3
The 1-(4-methoxyphenyl)-3-(pyridine-2-ylmethyl)thiourea consists of 34 atoms which
consequences to 96 normal modes of fundamental vibrations. This molecule belongs to
C1symmetry. Up to the best of our knowledge none have reported the quantum chemical study
for molecular structure and IR-spectrum of 1-(4-methoxyphenyl)-3-(pyridine-2-ylmethyl)
thiourea.The observed and calculated IR-spectra have been presented in Fig. 6(a-b).The rigorous
analysis for fundamental modes of vibration with B3LYP has been demonstrated in Table (TS4)
and Veda 4 Programme[33-36] has been used to assign to vibrational frequency. In our study we
have scaled B3LYPcalculation with 0.98 to correct the theoretical error[37].
C-H vibrations
The theoretical C-H stretching modes of vibration of compound (3) were calculated in the range
-1
of 3000-3229cm . The C-H terminal of methyl group displayed asymmetric vibration at 3058
-1
-1
cm and symmetric vibration at 3000 cm theoretically, however, its experimental counterpart
-1
-1
were observed at 3000 cm and 2931 cm . Theoretical C-H vibrations associated with benzene
-1
-1
-1
-1
were found at 3229 cm , 3202 cm , 3195 cm and 3157 cm with more than 88% contributions.
-1
-1
C-H vibrational peak associated pyridine was majorly found at 3184 cm , 3168 cm and 3163
-1
-1
cm . The in-plane bending vibrations of H-C-C was calculated in the region of 1129-1660 cm
-1
-1
-1
-1
-
and particularly, wave numbers were 1129 cm , 1135 cm , 1171 cm , 1175 cm , 1194 cm
1
-1
-1
-1
-1
-1
-1
,
1250 cm and 1467 cm , 1478 cm , 1491 cm , 1637 cm and 1660 cm . The plane torsional
-1
-1
-1
-1
-1
bending of H-C-C-C was at 751 cm , 840 cm , 949 cm , 953 cm and 1014 cm . Calculated
-1
wave number at 1478 cm was assigned to bending vibration of H-C-H which was of methoxy
-1
group, however, its experimental value was found at1464 cm . Other modes of vibration have
been given in Table (TS4).
O-C vibrations
-1
The theoretical ethereal (-O-C) wave numbers of methoxy group were observed at 1506 cm ,
-
1
-1
-1
-1
1
1
491 cm , 1065 cm , 1171 cm , and 521 cm . But its experimental values were observed at
-1
-1
-1
-1
498 cm , 1171 cm , 841 cm and544 cm . The bending vibration of C-O associated with C15-
-1
O18-C19 was observed at 533 cm .The torsion associated with H33-C19-O18-C15 of C-O with
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-1
5
7% contributions was observed at1171.0 cm . The stretching vibration of C-O associated with
-1
O18-C19 with 75 % contribution was found at 1065 cm . The wave numbers associated with
-1
-1
H32-C19-O18-C15, H34-C19-O18-C15 and H33-C19- O18-C15 at 1506 cm and 1491cm of
C-O were found torsional in nature.
N-H vibrations
-1
Normally, N-H stretching vibrations are observed in the region of 3500-3200cm . The band
-1
observed at 3481 cm in FT-IR spectrum was assigned to N-H and its theoretical values were
-1
-1
assigned at 3602 cm and 3563cm . The rest of vibrations associated with nitrogen have been
displayed given in Table [TS4]. Saeed et.al [38] reported for free -NH group, symmetric and
2
-1
-1
asymmetric stretching were observed at 3324 cm and 3114cm respectively. Lower value of
symmetric stretching was due to intramolecular hydrogen bonding. However, in our reported
-1
compound (3), -NH stretching was observed at 3481 cm .Saeed et.al also reported the intense
-1
-1
peak of -NH deformation at 1619 cm and 1547 cm . But inour case, NH deformation was
-1
observed at 1593 cm .
C-N vibrations
The stretching vibrations and including other vibration modes of C-N were found in the range of
-1
1
062-1407cm . However, the most effective wave numbers associated with C-N were observed
-1
-1
-1
-1
-1
at 982 cm , 1014 cm , 1067 cm , 1296 cm and 1637 cm . The out of plane mode of vibration
-1
of CN associated with C7-C6-N4-C5 was calculated at478 cm . Rest modes have been given in
Table (TS4).
C=S vibrations
-1
The experimental stretching vibration of C=S of compound (3) was at 758 cm , however, its
-1
theoretical values was found at 748 cm .
.4 NBO analysis
3
NBO analysis depicts the role of inter and intramolecular bonding. It is suitable to find charge
transfer or hyper conjugative interactions in molecular systems. The energy of interacting orbital
differences is proportional to the stabilization of orbital interactions. Thus, the prominant
interactions of stabilization occur between dominant donors (i) and acceptors (j). Quantatively,
the interactions between bonding anti-bonding orbitals are interpreted in terms of NBO basis,
and is expressed by means of second order perturbation interaction energy, E(2)[39]. This energy
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describes an estimate of off diagonal NBO Fock matrix element. The stabilization energy is
calculated by following mathematical expression.
ꢀ
(ꢁ,ꢂ)ꢃ
E2=∆Ei,j=qi
(1)
ꢄꢁꢅꢄꢂ
Where qi is donor orbital occupancy, εi and εj are diagonal elements (orbital energy) and F(i, j)
Is off diagonal Fock matrix. In our studied compound, Second lone pair electron of S11 is
strongly disperses into anti-bonding orbital σ*(N8-C9) and σ* (C9-N10) through
hyperconjugation, resulting hyperconjugative stabilization energy 10 and 13 Kcal/mol. Lone pair
of electron of oxygen resonated to anti-bonding molecular orbital π*(C14-C15) and gave
stabilization energy 30 Kcal/mol. Furthermore, Lone pair electrons from N8 and N10 are
strongly interacted to π-anti-bonding orbital of C9-S11 but the electronic charge and hence C9-
S11 loses its double bond character which is also evident in terms of its bond length value
of1.35Å instead of 1.37Å for pristine thiourea. The stabilization energy for such hyper
conjugation is 77 and 68kcal/mol. However, F. Weinhold reported that lone pair of electron of
nitrogen of formamide showed strong hyperconjugation to anti-bonding orbital of π*(N-CO)
which led to conjugative stabilization 60 Kcal/mol[40] which is less than our title compound. It
was also reported that the nitrogen around thiocarbamate, urea and thioester occupied planar
structures[41-42] which are important for the strong resonance and exhibiting conjugative
stabilization energy. Saeed et. al found conjugative energy of urea and thiocarbamate 56 and 41
kcal/mol,due to the intreraction of lone pair of nitrogen to the anti-bonding orbital of π*(CO [43]
and are not greater than our studied compound. Furthermore, lone pair of electron present on
nitrogen of pyridine dispersed into anti-bonding molecular π*(C5-C6) through intramolecular
charge transfer, causing stabilization energy 10 Kcal/mol. However, Saeed et. al found [44] that
lone pair of electron on nitrogen interacted with anti-bonding π* (C=C) orbital through
resonance and conjugative stabilization energy was observed to 35 Kcal/mol which is almost
three times higher than our title compound. Additionally, The bonding molecular orbital of π
(C14-C15) interacted with anti-bonding molecular π*(C12-C13) through intramolecular charge
transfer and gives stabilization energy 22 Kcal/ mol. The stabilization energy has been given in
Table (TS5).
3
.5 UV-Visible analysis
In order to describe the nature of electronic transitions within 1-(4-methoxyphenyl)-3-(pyridine-
2
-ylmethyl)thiourea, TD-DFT calculation was performed in chloroform solvent background. The
7

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theoretical wavelength was obtained from UV-Visible spectra with B3LYP/6-311G++(d,p) level
of theory for twelve lowest singlet state. The experimental and theoretical electronic spectra are
shown in Fig.7(a-b). The comparative theoretical and experimental electronic transitions are
given in Table (TS6). The inter-frontier orbitals obtained from TDFFT calculations at the
contour value of 0.02, as shown in Fig.(8), are used to analyze the nature of electronic
transitions. The first theoretical electronic transition was obtained at 276 nm wavelength (λmax).
However, its experimental wavelength was observed at 278 nm. This transition is majorly
contributed from HOMO-1 to LUMO+3 which is primarily n→π* in nature. This transition takes
place from bonding orbital of suphur to anti-bonding orbital of 4-methyl benzyl ring as
evidenced from Fig.(8). The second theoretical electronic transition was observed at 236 nm
wavelength (λmax) and experimental transition was found at 214 nm. This transition involves
HOMO to LUMO+4 and LUMO+5 molecular orbitals. The nature of transition is π→π* in
nature. The transition takes place from π-bonding orbitals of 4-methyoxy benzyl group to anti-
bonding orbitals of the same group and pyridine ring as is cleared from the Fig.(8).
3
.6 Frontier Molecular Orbital
In order to know the physico-chemical behavior and energy associated with compound(3),
HOMO and LUMO energy and energy gap are calculated using optimized geometry with
B3LYP/6-311++G(d,p) basis set and is represented in Fig.(9). The positive and negative phases
of the molecular orbitals have been shown in red and green color respectively. The chemical
reactivity and biological efficacy depend upon the eigen value of HOMO and LUMO energy and
energy gap [45]. The molecule possessing smaller interfrontier orbitals are chemically more
active and have lower kinetic stability [23-24]. Moreover, the electrical and optical properties
and electronic transition and chemical reactions are described by HOMO and LUMO molecular
orbitals[46,47]. They also signify the electron donating and accepting abilities. They are also
helpful in interpreting the electronic transition from ground state to first excited state [45]. From
Fig.(9), it can be observed that HOMO molecular orbital is comprised of C(17, 14, 13) and C(12,
1
5 & 16), N(10), C(9), S(11) and N(8) and LUMO molecular orbital is spread over pyridine ring.
HOMO-1 orbital is spread over thiourea part of compound (3). However, HOMO-2 is spread
over entire molecule except pyridine ring. But HOMO-1 and HOMO-2 participate in electronic
transition as shown in Fig.(8).
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Some new chemical descriptors have been proposed viz ionization energy (I.E), electron
affinity (E.A), electronegativity(χ), chemical potential(ꢀ), global harness(η), softness(s) and
global electrophilicity index(ω) to understand the various aspects associated with
pharmacological sciences, drug design and possible eco-toxicological features of the drugs. All
these are calculated using HOMO and LUMO energy. The structure and reactivity of a
compound can also be understood by them. The mathematical expression and calculation of the
studied compound (3) are given in Table (1). From the calculation, the ionization energy and
electron affinity were found 5.31 eV and 1.40 eV. However, the chemical potential,
-
electronegativity, softness and global electrophilicity index were found -3.35eV, 3.35eV, 0.26eV
1
and 2.88eV respectively. The energy gap between HOMO and LUMO was found 3.91 eV.
This energy gap explains the charge transfer interactions with the molecule, and affects
biological activity of the compound. The concept of global electrophilicity index was proposed
by Parr et al[48] and is used to calculate the stabilization energy when an extra electronic charge
is transferred to the molecular system from environment. It also explains the ability of an
electron to gain additional electronic charge and resistance of the molecule to exchange the
electronic charge with surroundings. The chemical potential and hardness are useful for global
chemical reactivity. The chemical potential of compound (3) is negative which clearly indicates
that compound is stable and can not decompose spontaneously. Hardness offers resistance to the
molecule so that electron cloud could not deformed under the influence of perturbations
encountered during the chemical reactions. Larger the energy gap between HOMO and LUMO,
greater would be the hardness and would be more stable and less reactive and is less polarizable
and vice-versa.
Aswathy et.al reported 1-(3,4-dichlorophenyl)-3-[3-(trifluoromethyl)phenyl]thiourea
derivative[49]. He found that the chemical potential of the reported compound was -5.934 eV.
On the other, War et.al also reported 1-[3-(1H-imidazol-1-yl)propyl]-3-phenylthiourea derivative
and it was found that chemical potential of compound was -3.3475 eV [50]. Furthermore, in our
previously reported compound, 1-(4-methoxyphenyl)-3-(pyridine-3-ylmethyl)thiourea derivative,
chemical potential was reported -3.37 eV [23]. But in our studied compound (3), chemical
potential is -3.35 eV. As we know that higher the negative chemical potential, greater is the
reactivity. On the on the basis of this fact, order of reactivity can be compared. Thus, studied
compound (3) is less reactive than 1-(3, 4-dichlorophenyl)-3-[3-(trifluoromethyl)phenyl]thiourea
9

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derivative and our previous reported compound1-(4-methoxyphenyl)-3-(pyridine-3-
ylmethyl)thiourea. However, the reactivity of compound (3) is comparable with 1-[3-(1H-
imidazol-1-yl)propyl]-3-phenylthiourea derivative.
4
. Pharmacology
DNA-binding
Nowadays, DNA has become widely used target for a large number of pharmaceutical active and
anti-cancer compounds. Many organic metal complexes and organic compounds are known to
bind with DNA in a various ways [51-52]. So, the study of interactions with new compounds
with DNA is a crucial step for developing new drugs. In this context, UV–visible spectroscopy is
one of the most commonly working techniques [53]. Through, UV-Visible spectrophotometric
titration, hypochromic and hyperchromic shifts with change in wavelength, absence or presence
of isosbestic points are observed, which are the characteristics of interactions of compounds with
DNA[54-55]. Classically, hypochromism and bathochromism display a non-covalent binding,
however, hyperchromism shows covalent binding[53]. In our experiment, the significant peak
appeared in the range of 200-420 nm showed the interactions of a compound (3) with ct-DNA
and has been demonstrated in Fig. 10(a&b). It was also observed that the second band was
shifted slightly, and it was because of intra ligand π→π* transitions [54, 56]. The presence of
band at the range of 218-250 nm indicated that there was π→π* transition. The second band
appeared at 250-350 nm which exhibited n→π* transition. These experimental data suggested
the bathochromic shift, due to interactions of compound (3) with DNA. It was also observed that
-5
with the addition of different concentrations of DNA [0.45-3.8x 10 M], the absorption peaks
underwent hypochromicities for the target compound (3), thus, pointing out the formation of
DNA-compound adducts [57]. Furthermore, the binding constant of compound(3) was found to
6
-1
be(K
b
) 3.3x10 Lmol which is in good agreement with molecular docking.
4
.2 Cell cycle perturbation
Result & discussion
To insure the effects of compound (3) on cell cycle perturbation, the flow cytometry assay
technique was performed against HepG2 and Siha cell lines. In present work, treated HepG2
cells at concentration ranges 40ꢀM and 80 ꢀM exhibited the cell cycle phase distribution
6
5%and 70% respectively arrest in G0/G1 as compared to untreated control (60%) as shown in
Fig. 11 (a-c). However, at concentrations 40 ꢀM, and 80 ꢀM, treated Siha cells displayed cell
1
0

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cycle distribution 59% and65 % respectively arrest in G0/G1 as compared to untreated control
(45%) as in Fig.11 (d-f). Thus, it can be concluded from data that compound (3) induced the cell
cycle arrest G0/G1 in both HepG2 and Siha cell lines.
4
.3 MTT Assay
3
-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) is an important dye
reduced by succinate dehydrogenase enzyme of mitochondrial living cells to produce water
insoluble purple formazan crystals[58-59]. MTT assay has widely been used to find the cytotoxic
effects of the synthetic /Natural compounds/Extract[60-61]. The cytotoxicity of compound (3)
was performed against HepG2, Siha and HEK-293 cells for 48 hours treatment at concentration
range 0-250 ꢀM as shown in Fig.12(a-c). In the present study, it was observed that compound (3)
displayed non-cytotoxic effect against both the concerous cell lines, HepG2 (IC50= 140.39 ꢀM)
and Siha (IC = 119.87ꢀM). Compound (3) was also found non-cytotoxic against HEK-293(IC
5
0
50
=
148.67ꢀM). Hence, compound (3) was non-cytotoxic againstHepG2 Cells and Siha cells at the
concentration ranges 0-140 ꢀM and 0-120 ꢀM respectively, and also non-toxic against HEK-293
up to the concentration range 0-149 ꢀM.
4
.4Molecular docking
Virtual screening and computational study has become most commonly used tool for drug
development [62]. This may be fast outcomes of prediction of binding sites, low expenses and
free availability of much software [63]. In order to examine, our experimental results, DNA
docking study has been carried out through Auto Dock-Vinal tool [64-65]. The docking study of
the compound (3) was performed with DNA dodecamersd(CGCGAATTCGCG)2 (PDB ID:
1
BNA). The binding energy of the docked compound (3) was found to be -6.2 Kcal/mol. It was
observed that molecule preferred to interact with base pair of DNA as shown in Fig. (13), and
made polar and several no-polar interactions and displayed in Fig.(FS1), and data have been
presented in Table (TS7). This molecular orientation of compound (3) confirmed formation of
two H-bonds hydrogen bonds with chain B (B: DC’21/O2::N of thiourea (0.244) and chain
A(A: DA’5/O4:: N of pyridine (0.356). Thus, docking study supported our experimental results.
4
.5 Mechanism of action at supramolecular level
UV-visible spectroscopic data, it was observed that compound (3) formed adducts with DNA due
to covalent and non-covalent bindings. The docking studies also suggested that compound (3)
bound with DNA bases and a compound (3) behaved as intercalating agent. The intense
1
1

ACCEPTED MANUSCRIPT
evaluation and 3-D visualization of compound (3) showed that 4-Methoxy-substituted phenyl
ring and pyridine rings were present in between the chains A and B as shown in Fig. (13)
Nitrogen of pyridine formed one hydrogen bond with Guanine-cytosine base pair of DNA, and
however, nitrogen of thiourea associated with 4-methoxy benzene formed hydrogen bondwith
Adenine-thymine base pairs of DNA as depicted in Table (TS7). It can be said that the major
force for the interactions of compound (3) with DNA is hydrogen bonding. Steric effects and
Vander Waal’s force also played vital role in binding of compound (3) with DNA. Thus,
compound (3) has an affinity towards DNA, which is in accordance with the experimental UV–
visible spectroscopic data [23].
5
. Conclusion
From our experimental results, it can be concluded that compound (3) adopted syn-anti-
conformation. The ionization and electron affinity were found to be 5.31eV and 1.40eV.The
6
-1
DNA-binding constant was found 3.3 x 10 Lmol . Compound (3) displayed binding energy -6.2
Kcal/mol. Cell cycle phase distribution demonstrated that treated HepG2 cells showed 65 % and
7
5
0% arrest in G0/G1in comparison with untreated control (60%). However, Siha cells, displayed
9 % and 65 % arrest in G0/G1 as compared to untreated control (45%). From MTT-assay, it
was found that compound (3) displayed non-cyctotoxic nature against cancerous cell lines, HeG2
(
IC = 140 ꢀM), and Siha(IC = 119.67 ꢀM), and also normal human cell lines HEK-293(IC
50 50 50
=
148.87 ꢀM). Hence, compound (3) was toxic at concentrations greater than 140 ꢀM against
HepG2 cells and against Siha at 120 ꢀM.
. Experimental protocol: All the required chemicals were purchased from Merck and Aldrich
6
Chemical Company (USA). Precoated aluminium sheets (Silica gel 60 F254, Merck Germany)
were used for thin-layer chromatography (TLC) and spots were visualized under UV-light. FT-
IR spectra were recorded on Perkin Elmer model 1600 FT-IR RX1 spectrophotometer as KBr
1
13
discs. H-NMR and C-NMR spectra were recorded on Bruker Spectrospin DPX 300 MHz using
CDCl as a solvent and trimethylsilane (TMS) as an internal standard. Splitting patterns are
3
designated as follows; s,singlet; d, doublet; m, multiplet. Chemical shift values have been given
in ppm. The ESI-MS was recorded on micrOTOF-Q II 10330 Electronspray ionization mass
spectrometer (Bruker). X-ray data were collected on Bruker SMART Apex CCD diffractometer
(SAI, Universidade daCoruña).
6
.1 Synthesis of thiourea derivative
1
2

ACCEPTED MANUSCRIPT
one mole of picolylamine was taken in 10 ml of toluene in round bottom flask. To it, 1 mole of
4
-methoxy phenylisothiocynate was slowly added and reaction mixture was stirred at room
temperature. The Progress of reaction was monitored by TLC (5% Chloroform and Methanol).
Solid white product was obtained with 30 minutes. The product was filtered and washed with
toluene and recrystallized in chloroform which yielded light white crystalline solid.
0
6
.1.1 1-(4-methoxyphenyl)-3-(pyridine-2-ylmethyl)thiourea (m.p. 130 C): (Yield: 90 %): IR
-1
(
λmax) cm ; 3467.14(-NH-Ar), 3341.80(-NH-), 3147.68 (Ar-H), 2989.67(OCH3),
1
1
519.67(CN),1242.69(C=S), H-NMR(DMSO-d6):9.651(bs, 1H, NH-Ar), 8.058(bs, 1H, -NH-
C=S), 8.511(d,1H, J = 3.3 Hz, Ar-H), 7.792(t, 1H, J = 7.5 Hz, Ar-H), 7.339-7.257(m, 4H, Ar-H),
.932 (d, 2H, J= 6.6 Hz, Ar-H), 4.799 (d, 2H, J = 5.4 Hz, CH2N-), 3.745(s, 3H, OCH3);
m/z;[M+H]+= 275
6
6
.2 X-Ray crystal structure determination
Three-dimensional X-ray data were collected on a Bruker Kappa Apex CCD
diffractometer at low temperature for 3 by the
φ
-
ω
scan method. Reflections were measured from
. A total of 28849
a hemisphere of data collected from frames, each of them covering 0.3º in
ω
reflections measured were corrected for Lorentz and polarization effects and for absorption by
multi-scan methods based on symmetry-equivalent and repeated reflections. Of the total, 2314
independent reflections exceeded the significance level ( F /σ F ) > 4.0. After data collection,
in each case an multi-scan absorption correction (SADABS)[66] was applied, and the structure
2
was solved by direct methods and refined by full matrix least-squares on F data using SHELX
suite of programs[67]. Hydrogen atoms were located in difference Fourier map and left to refine
freely, except for C(8), were included in calculation position and refined in the riding mode. A
–
3
final difference Fourier map showed no residual density in the crystal 0.371 and -0.231 e.Å . A
2
2
o
2
weighting scheme w = 1/[σ (F ) + (0.054100 P) + 0.515500 P] was used in the latter stages of
refinement. Further details of the crystal structure determination are given in Table (2). CCDC
1
448611 contains the supplementary crystallographic data for the structure reported in this paper.
These data can be obtained free of charge via http://www.ccdc.cam.ac.uk/conts/retrieving.html,
or from the Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK;
fax: (+44) 1223 336 033; or e-mail: deposit@ccdc.cam.ac.uk. Supplementary data associated
with this article can be found, in the online version, at doi: $$$$$.
6
.3 Cytotoxicity studies (MTT assay)
1
3

ACCEPTED MANUSCRIPT
Cell culture
Human hepatocellular carcinoma cell line (HepG2), Cervical cancer cell line (Siha, Hela), Breast
cancer cell (MCF-7) and Human Embryonic Kidney cell line (HEK-293) were procured from
National Curator of Cell Sciences (NCCS) Pune, India. Cells were cultured in
Dulbecco’smodified Eagle’s medium (DMEM) with 10 % fetal bovine serum with 100 units/mL
penicillin, 100 ꢀg/mL streptomycin, and 2.5 ꢀg/mL amphotericin B, at 37 0C in a relative
humidity 80 % , 5% CO2[68].
MTT assay
Cytotoxicity of compound (3) was evaluated through MTT (3-(4,5-dimethylthiazole-2-yl)-2,5-
diphenyl tetrazolium bromide, M2128 Sigma Aldrich) assay onHepG2, Siha, and HEK-293 cells.
MTT is a validated assay for the in vitro cytotoxicity of any natural, synthetic compounds and
4
extracts [69-70]. The cell count 1.2x10 cells/well was seeded in 96 well plate (150 ꢀL/well).
After overnight incubation, cells were treated with different concentrations of compound (3) for
4
8 hours. After 48 hours treatment, medium was removed and incubated with 20 ꢀL of MTT
solution (5 mg/mL in Phosphate saline buffer) for 4 hours. The formazan crystals were formed
by mitochondrial enzyme reduction, finally solublized in DMSO (150 ꢀL/ well) and absorbance
was recorded at 570 nm through the Microplate reader (iMark, BIORAD, S/N 10321). Percent
viability was defined as the relative absorbance of treated versus untreated control cells.
6
.4DNA-binding
The stock solution of disodium salt of Ct-DNA was prepared in tris-HCl buffer (pH 7.2–7.3) and
0
stored at 4 C temperature. Once prepared, the stock solution was used within 4 days. The
concentration of the solution was determined spectrometrically. The ratio of absorbance at 260
and 280 (≥1.8) indicated that DNA was sufficiently free of protein. The concentration of DNA
-1
-1)
was measured using its extinction coefficient at 260 nm (6600 M cm after dilutions. For the
titration purpose, DNA stock solution was diluted using tris-HCl buffer. The compound (3) was
-4
dissolved in minimum amount of DMSO (2.0 x 10 M). UV-visible absorption spectra were
recorded after each addition of different concentrations of DNA. Absorption titration was
-5
conducted by adding varying concentrations (0.3-1.7 x 10 M) of DNA. The intrinsic binding
constant (K )was determined by Eq. (1), which was originally known as Benessi–Hilderbrand
equation and further modified by Wolfe et al[71].
b
[
DNA]/(ɛ
a
− ɛ
f
) = [DNA]/(ɛ
a
− ɛ
f
)+ 1/Kb (ɛ
b
− ɛ
f
) (1)
1
4

ACCEPTED MANUSCRIPT
a f a
Where the apparent absorption coefficients ɛ , ɛ and ɛ correspond to A obs/[compounds], the
extinction coefficient for compound(3), and the extinction coefficient for compound (3) in the
fully bound form. In plots of [DNA]/(ɛa− ɛf ) versus [DNA], Kb is given in the ratio of the slope
to intercept.
6
.5 DNA docking studies
Docking studies were performed at Intel(R) Core(TM) i3 CPU (2.3 GHz) with XP-based
operating system (Windows 2007). 3D Structure of compound (3) was drawn by Mercury
software using CIF file of crystal structure and saved in pdb file format. The preparation of the
compounds was done by assigning Gastegier charges, merging non-polar hydrogens, and saving
it in PDBQTfile format using AutoDock Tools (ADT)4.2 [64-65]. The X-ray crystal structure of
DNA (PDB ID:1BNA) was obtained from the Protein Data Bank [Protein Data Bank. Available
online at:http://www.rcsb.org/pdb]. Using ADT 4.2, DNA was saved in PDB file format leaving
heteroatoms(water). Gastegier charges were assigned to DNA and saved in PDBQT file format
using ADT. Preparation of parameter files for grid and docking were done using ADT. Docking
was performedwith Auto-Dock4.2 (Scripps Research Institute, USA), considering all the
rotatable bonds of ligandas rotatable and receptor as rigid[72] Grid box size of 80 × 80 × 60 Å
with 0.375 Å spacing was usedthat included whole DNA. In the present work, we describe a plug
in for PyMOL which allow scarrying out molecular docking, virtual screening and binding site
analysis with PyMOL. The plug inrepresents an interface between PyMOL and two popular
docking programs, Autodock [64-65], AutodockVina [74] and also the extensive use of a Python
script collection (Autodock Tools) [73].
6
.6 Methodology of Cell cycle analysis
Flow cytometry was performed for analysis of cell cycle arrest according to the standard
method[74]. The effects of compound (3) on cell cycle on Siha and HepG2 cell lines were
analyzed by flow cytometry. After reaching 70% confluency, cells were serum starved overnight
and treated with compound (3) for 48 h in complete medium. The cells were trypsinised, washed
two times with chilled PBS and centrifuged at 1500 rpm for 5 minutes. Further, 1ml chilled 70%
ethanol was added drop wise to the cell pellet and vortexed gently. The cells were incubated for
1
h at 4ºC. The cells were centrifuged at 2000 rpm for 5 minutes, and the pellet was washed
twice with PBS. The cells were re-suspended in 500 ꢀl PBS, added 10ꢀl ribonuclease(100ꢀg/ml
final concentration) and incubated for30 min at 370C. Cells were stained with propidium iodide
1
5

ACCEPTED MANUSCRIPT
(50 ꢀg/ml final concentrations) and incubated for 15 min. Flow cytometry analysis was
performed with FACS (Becton Dickinson). A minimum of 10,000 cells were acquired. Further,
histogram was analyzed by Flowjo software for cell cycle analysis.
Acknowledgment
One of the authors, Md. Mushtaque, is highly thankful Science and Engineering Research
Board (SERB) Govt. of India, for financial assistance, and Professor Stephen Husbands
(Department of Pharmacy and Pharmacology, University of Bath, Claverton Down, BA2 7YA,
U.K.) and chancellor (Mr. Jawad Ahmed Siddiqui) of Al-Falah University.
References
1
. M. K. Mallath, D. G Taylor, R. A. Badwe, G. K. Rath, V. Shanta, C. S. Pramesh,
R.Digumarti, P. Sebastian, B.B. Borthakur, A. Kalwar, S. Kapoor, S. Kumar, J. L.Gill, M.
A.Kuriakose, H. Malhotra, S.C. Sharma, S. Shukla, L. Viswanath, R. T. Chacko, J. L.
Pautu,K.S. Reddy, K.S. Sharma, A. D. Purushotham, R. Sullivan. Lancet Oncol.15(2014)
e205‒212.
2
3
. F. Bray. B. Moller, Nat. Rev. Cancer. 6 (2006) 63‒74.
. L.A.Shervington, N. Smith, E. Norman. T. Ward, R. Phillips, A. Shervington, Eur. J. Med.
Chem. 44 (2009) 2944‒2951.
4
5
6
7
. K. Loven, L. Stein, K. Furst, S. Levy, ClinTher.24 (2002) 990‒1000.
. A. Skladanowski, J. Konopa. BiochemPharmacol.46 (1993) 375‒382.
. R. Lesyk, B. Zimenkovsky, Curr. Org. Chem.8 (2004) 1547‒1577.
. R. Lesyk, O. Vladzimirska, S. Holota, L. Zaprutko, A. Gzella, Eur. J. Med. Chem.
4
2(2007)641‒648.
8
. D. Havrylyuk, B. Zimenkovsky, O. Vasylenko, L. Zaprutko, A. Gzella, R. Lesyk. Eur. J.
Med. Chem. 44 (2009) 1396‒1404.
9
1
1
1
. D. Kaminskyy, B. Zimenkovsky, R. Lesyk. Eur. J. Med. Chem.44 (2009) 3627‒3636.
0. O. Ates, H. Altıntas, G. Otuk, Arzneim. Forsch. Drug Res. 50 (2000) 569‒575.
1. A. Kocabalkanl, O. Ates, G. Otuk, Arch. Pharm. Med. Chem. 334 (2001) 35‒39.
2. S.G. Kucukguzel, E.E. Oruc, S. Rollas, F. Sahin, A. Ozbek, Eur. J. Med. Chem. 3 (2002)
1
97‒206.
1
3. V. S. Palekar, A. J. Damle, S.R. Shukla, Eur. J. Med. Chem.44 (2009) 5112‒5116.
1
6

ACCEPTED MANUSCRIPT
1
4. N. Cesur, Z. Cesur, N. Ergenc, M. Uzun, M. Kiraz, O. Kasımoglu, D. Kaya, Arch. Pharm.
Weinheim.327 (1994) 271‒273.
1
1
1
5. N. Karali, E. Ilhan, A. Gursoy, M. Kiraz, Farmaco.53 (1998) 346‒349.
6. H. T. Y. Fahmy, Boll. Chim. Farmaco. 140 (2001) 422‒427.
7. M. L. Barreca, A. Chimirri, L. D. Luca, A. M. Monforte, P. Monforte, A. Rao, M. Zappala,
J.Balzarini, E. De Clercq, C. Pannecouque, M. Witvrouw, Bioorg.Med. Chem.
Lett.11(2001)1793‒1796.
1
1
2
2
8. A. Rao, A. Carbone, A. Chimirri, E. D. Clercq, A. M. Monforte, P. Monforte, C.
Pannecouque, M. Zappala, Farmaco.57 (2002) 747‒751.
9. Rao, A. Carbone, A. Chimirri, E.D. Clercq, A.M. Monforte, P. Monforte, C. Pannecouque,
M. Zappala, Farmaco.58 (2003) 115‒120.
0. M. Chayah, M. D. Carriûn, M. A. Gallo, R. Jimnez, J. Duarte, M. E. Camacho.
ChemMedChem. 10(2015)874–882.
1. R. Pingaew, P. Tongraung, A. Worachartcheewan, C. Nantasenamat, S. Prachayasittikul,
S. Ruchirawat, V. Prachayasittikul. Med Chem Res. 22 (2013) 4016–4029.
2. M. Mushtaque, F. Avecilla, A. Azam. Eur. J. Med. Chem. 55 (2012)439‒448.
3. M. Mushtaque, M. Jahan, M. Ali, M. S. Khan, M. Shahid Khan, P. Sahay, A. Kesarwani,
Journal of Molecular Structure. 1122 (2016) 164–174.
2
2
2
4. M. Mushtaque, F. Avecilla, Z. B. Hafeez, M. Jahan, M. S. Khan, M. M. A. Rizvi, M.
Shahid Khan, A. Srivastava, A. Mallik, S. Verma, Journal of Molecular Structure 1127
(2017) 99‒113
2
2
2
2
5. A. D. Becke. Phys. Rev. A38. 6(1988) 3098‒3100.
6. C. Lee, W. Yang, R. G. Parr 1988. Phys. Rev.B37 (1988) 785‒789.
7. M.J. Frisch, et al., Gaussian09, Revision A.02, Gaussian, Inc., Wallingford CT, 2009.
8. Gaussian 03. Revision C.02.Wallingford CT: Gaussian Inc.; 2004.
2
9. S. Miertus, E. Scrocco, J.Tomasi, J.Chem. Phys. 55(1981)117‒129.
3
0. K. Gholivand, C.O. Della V_edova, M.F. Erben, F. Mojahed, A.M. Alizadehgan, J. Mol.
Struct. 840 (2007) 66‒71.
3
3
1. D. Glossman-Mitnik, A. M_arquez-Lucero, J. Mol. Struct. Theochem 548 (2001) 153-163.
2. M. F. Erben, C.O. Della Vedova, R. Boese, H. Willner, C. Leibold, H. Oberhammer, Inorg.
Chem. 42 (2003) 7297‒7303.
1
7

ACCEPTED MANUSCRIPT
3
3. L. J. S. Knutsen, C. J. Hobbs, C. G. Earnshaw, A. Fiumana, J. Gilbert, S. L. Mellor, F.
Radford, N. J. Smith, P. J.Birch, J. R. Burley, S. D. C. Ward, I. F. James, Bioorg. Med.
Chem. Lett. 17 (2007) 662‒667.
3
3
3
3
3
3
4. Jamróz, H. Michał. SpectrochimicaActa Part A: Molecular and Biomolecular Spectroscopy.
1
14 (2013) 220‒230.
5. M. H. Jamro´z, J. Cz. Dobrowolski, R. Brzozowski. Journal of molecular structure. 787
2006) 172‒183.
(
6. C. T. Zeyrek, S. B. Koçak, H. Ünver, S. Pektas, N. S. Basterzi, O. Çelik . Journal of
Molecular Structure 1100 (2015) 570‒581.
7. T. A. de Toledo, L.E. da Silva, A. M. R. Teixeira , P.T.C. Freire, P. S. Pizani. Journal of
Molecular Structure. 1091 (2015) 37‒42.
8. A. A. Prasad, K. Muthu, M. Rajasekar, V. Meenatchi, S. P.Meenakshisundaram.
SpectrochimicaActa Part A: Molecular and Biomolecular Spectroscopy. 135 (2015) 46‒4.
9. A. Saeed, A. Khurshid, M. Bolte, A. C. Fantoni, M. F. Erben. SpectrochimicaActa Part A:
Molecular and Biomolecular Spectroscopy. 143 (2015) 59‒66.
4
4
0. J. P. Foster, F. Weinhold, J. Am. Chem. Soc. 102 (1980) 7211‒7218.
1. F.Weinhold, C. R. Landis. Cambridge University press 2005.
(
www.cambridge.org/9780521831284).
2. S. T. Vallejos, M. F. Erben, O. E. Piro, E. E. Castellano, C. O. Della Védova. Polyhedron.
8 (2009) 937‒946.
4
4
4
4
2
3. M. F. Erben, R. Boese, C. O. Della Ve´dova, H. Oberhammer, H. Willner. Journal of
organic chemistry. 71 (2006) 616‒622.
4. A. Saeed, A. Khurshid, J. P. Jasinski, C. G. Pozzi, A. C. Fantoni, M. F. Erben. Chemical
Physics. 431 (2014) 39‒46.
5. Diego M. Gil, M.E. Defonsi Lestard, O. Estevez-Hernandez, J. Duque , E. Reguera.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 145 (2015) 553–
5
62.
6. E. Kavitha, N. Sundaraganesan, S. Sebastian, M. Kurt. Spectrochim. Acta A. 77(2010)612-
19.
7. R. J. Parr, L.V. Szentpaly, S. Liu, J. Am. Chem. Soc.121 (1999)1922-1924.
4
4
6
1
8

ACCEPTED MANUSCRIPT
4
4
8. A. Saeed, M. I. Arshad, M. Bolte, A. C. Fantoni, Z. Y. Delgado Espinoza, M. F. Erbend.
SpectrochimicaActa Part A: Molecular and Biomolecular Spectroscopy.157 (2016) 138‒
1
45.
9. V.V. Aswathy, Y. S. Mary, P. J. Jojo, C. Y. Panicker, A. Bielenica, S. Armakovi, S. J.
Armakovi, P. Brz, ozka, S. Krukowski, C. V. Alsenoy. J. of Mol. Struc. 1134 (2017) 668-
6
80.
5
5
0. J. A. War, K. Jalaja, Y. S. Mary, C. Y. Panicker, S. Armakovi, S. J. Armakovi, S. K.
Srivastava, C. V. Alsenoy. J. Mol. Struc.1129 (2017) 72-85.
1. K. N. Houk, S. Menzer, S. P. Newton, F. M. Raymo, J. F. Stoddart, D. J. Williams, J. Am.
Chem. Soc.121 (1999) 1479‒1487.
5
5
2. J. J. Novoa, F. Mota, Chem. Phys. Lett.318 (2000) 345‒354.
3. M. N. Patel, P.A. Parmar, D.S. Gandhi, A.P. PatidarSpectrochimicaActa Part A: Molecular
and Biomolecular Spectroscopy. 97 (2012) 54‒59.
5
5
5
5
5
5
4. Eric, Long, K. Jacqueline. Acc. Barton. Chem. Res. 23 (1990) 271‒273.
5. T. A. Koopmans, Physica.1 (1934) 104‒113.
6. S. M. Thom, R.W. Horobin, E. Seidler, M. R. Barer. Appl. Bacteriol. 7 (1993) 433‒443.
7. B. Kitchen, H. Decornez, J. R. FurrBajorath. J. Nat. Rev. Drug Discov. 3 (2004) 935‒949.
8. S. M. Thom, R.W. Horobin, E. Seidler, M. R. Barer. Appl. Bacteriol.74 (1993) 433‒443.
9. S. R. Kim, M. J. Park, M. K. Lee, S. H. Sung, E. J. Park , FreeRadic. Biol. Med. 32(2002)
5
96‒604.
6
6
0. S. H. Kim, J. H. Zo, M.A. Kim, K.K. Hwang, I. H. Chae. Nutr. Res. 23 (2003) 1671‒1683.
1. H.Y. Lina, S.H. Juan, S.C. Shen, F.L. Hsu, Y.C. Chen. Biochem. Pharmacol. 66 (2003)
1
821‒1832.
6
6
2. B. Kitchen, H. Decornez, J. R. FurrBajorath, J.; Nat. Rev. Drug Discov. 3 (2004) 935‒949.
3. G.M. Morris, D.S. Goodsell, D.S.Halliday, R. Huey, WE. Hart R. Belew A.J. Olson J.
Comp. Chem.19(1998) 1639‒1662.
6
6
6
6
4. R. Huey, G.M. Morris, A. J. Olson, D. S. Goodsell, J. Comp. Chem. 28 (2007) 1145‒1152.
5. O. Trott, A. Olson, J. Comp. Chem. 31(2010) 455‒461.
6. G. M. Sheldrick, SADABS, version 2.10, University of Göttingen, Germany, (2004).
7. SHELX, G. M. Sheldrick, ActaCrystallogr., Sect. A 64 (2008) 112–122.
1
9

ACCEPTED MANUSCRIPT
6
6
7
8. M. K. Gupta, T.V. Neelakantan, M. Sanghamitra, R.K. Tyagi, A. Dinda, S. Mualik, C. K.
Mukhopadhyay, Goswami. S.K.; Antioxi. Redox Signal. 8(2006) 1081‒1093.
9. S.J. Mehdi, A. Ahmad, M. Irshad, Cytotoxic effect of Carvacrol on human cervical cancer
cells. BM, 3 (2011) 307‒12.
0. M. Irshad, S. J. Mehdi, A. A. Al-Fatlawi, Phytochemical Composition of Cassia fistula Fruit
Extracts and its Anticancer Activity Against Human Cancer Cell Lines. J Biologically
Active Prod Nat. 4(2014)158‒70.
7
7
7
1. A. Wolfe, G. H. Shimer, Jr. Meehan, T. Biochemistry. 26 (1987)6392‒6396.
2. M. F.; Sanner, J. Mol. Graph Model. 17(1999)57‒61.
3. M. Morris, G. S. Goodsell, R. S. Halliday, R. Huey, W. E. Hart, R. Belew, A. J Olson. J.
Comput.Chem.19(1998)1639‒1662.
7
4. W. W Lai, J. S Yang, K. C Lai, C. L Kuo, C. K Hsu, C. K Wang. Rhein induced apoptosis
through the endoplasmic reticulum stress, caspase-and mitochondria-dependent pathways in
SCC-4 human tongue squamous cancer cells. In Vivo. 23 (2009) 309‒316.
Caption illustrations
Caption figures
Caption tables
Caption figures
Fig.1(a-b): Thiourea derivatives displaying nNOS activity(a) and inducible activity against
iNOS activity
Fig.2(a-b):Thiourea derivatives showing promising cytotoxicity against HepG2 and
MOLT-3 cell lines.
Fig.(3): X-ray single crystal structure of 1-(4-methoxyphenyl)-3-(pyridine-2-
ylmethyl)thiourea
Fig. (4): Part of the crystal packing in the compound 1-(4-methoxyphenyl)-3-(pyridine-2-
ylmethyl)thiourea. Hydrogen bonds between nitrogen atoms of secondary amines with
nitrogen atoms of pyridine groups and sulphur atoms are presented in dark dashes lines
Fig. 5(a-c):Optimized structure of 3-(4-methoxyphenyl)-1-(pyridine-2-ylmethyl) thiourea
Fig. 6(a-b): Experimental and (b)theorical IR-spectra of 3-(4-methoxyphenyl)-1-(pyridin-2-
2
0

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ylmethyl)thiourea
Fig.7: (a-b) Experimental & (b) theoretical UV-visible spectra of 3-(4-methoxyphenyl)-1-
pyridin-2-ylmethyl)thiourea
(
Fig.(8):Frontiermolecular orbitals of 3-(4-methoxyphenyl)-1-(pyridine-2- ylmethyl)thiourea
Fig.(9): Energy gap between HOMO and LUMO molecular orbitals
Fig. 10(a): DNA binding spectra of 3-(4-methoxyphenyl)-1-(pyridine-2-yl methyl)thiourea
Fig.10 (b): DNA binding spectra of compound in the presence of increasing amount of Ct-
-
1
DNA. Inset: plots of [DNA]/(ɛa− ɛf ) (M2 cm ) versus [DNA] for the titration of CT DNA
with compound. Experimental data points; full lines, linear fitting of the data (Compound)
-
4
-5
2
.0 x 10 M, [DNA] 0.3-1.5 x 10 M
Fig.11(a-c): Cell cycles phase distribution against HepG2 cells at 40ꢀM & 80ꢀM.
Fig.11(d-f): Cell cycles phase distribution against Siha cells at 40ꢀM & 80ꢀM
Fig.12(a-c): Cytotoxic effects of 3-(4-methoxyphenyl)-1-(pyridine-2-yl methyl)thiourea
against (a)Siha(b) HepG2 (c) HEK-293 Cell
Fig. (13):3D-image of interactions of between DNA(PDBID: 1BNA) and ligand3-(4-
Caption Tables
Table(1): Frontier molecular orbital descriptors of 3-(4-methoxyphenyl)-1-(pyridin-2-
ylmethyl)thiourea at B3LYP/6-311++G(d,p) level
Table(2): Crystal Data and Structure Refinement for the compound 1-(4-methoxyphenyl)-
3
-(pyridine-3-ylmethyl)thiourea
Caption Figures
O
S
O
S
CH3
Cl
N
N
N
N
CH3
H
H
H
H
NH2
NH2
1
(a)=(IC = 0.18 µM against nNOS)
(1b)=(IC = 0.59 µM against iNOS)
50
5
0
Fig1(a-b): Thiourea derivatives displaying nNOS activity(a) and inducible activity against
NOS activity.
2
1

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Cl
S
NO2
S
N
N
H
H
N
N
H
H
Cl
O N
2
HN
HN
N
S
N
S
H
H
2
(a)
2 (b)
Fig.2(a-b):Thiourea derivatives showing promising cytotoxicity against HepG2 and
MOLT-3 cell lines.
Fig. (3): X-ray single crystal structure of 1-(4-methoxyphenyl)-3-(pyridine-2-
ylmethyl)thiourea
2
2

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Fig. (4): Part of the crystal packing in the compound 1-(4-methoxyphenyl)-3-(pyridine-2-
ylmethyl)thiourea (3). Hydrogen bonds between nitrogen atoms of secondary amines with
nitrogen atoms of pyridine groups and sulphur atoms are presented in dark dashes lines
Fig. 5(a)
Fig.5(b)
Fig.5(c)
Fig.5 :(a-c):Optimized structure of 3-(4-methoxyphenyl)-1-(pyridine-2-ylmethyl) thiourea
2
3

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6
(a)
Fig. 6(a-b): Experimental and (b) theoretical IR-spectra of 3-(4-methoxyphenyl)-1-
pyridin-2-ylmethyl)thiourea
6(b)
(
0
0
0
0
0
0
.5
.4
.3
.2
.1
.0
2
00
300
400
500
600
-
1
Wavelength(cm )
7
(a)
2
4

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7
(b)
Fig. 7: (a) Experimental & (b) theoretical UV-visible spectra of 3-(4-methoxyphenyl)-1-
pyridin-2-ylmethyl)thiourea
(
2
5

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2
6

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Fig.(8): Molecular orbitals of 3-(4-methoxyphenyl)-1-(pyridine-2-ylmethyl)thiourea.
Fig.(9): Energy gap between HOMO and LUMO molecular orbitals
0
.5
-4
2
1
x10 M
0.4
-5
.7 x10 M
-
5
1
.1x10 M
0.3
-5
0
.7x10 M
-
5
0
.3x10 M
0.2
0.1
0.0
200
300
400
500
Wavelength(nm)
Fig.10(a): DNA binding spectra of 3-(4-methoxyphenyl)-1-(pyridine-2-yl methyl)thiourea
2
7

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Fig.10(b): DNA binding spectra of compound in the presence of increasing amount of Ct-
2
-1
DNA. Inset: plots of [DNA]/(ɛa− ɛf ) (M cm ) versus [DNA] for the titration of Ct-DNA
with compound. Experimental data points; full lines, linear fitting of the data (Compound)
-
4
-5
2
.0 x 10 M, [DNA] 0.3-1.5 x 10 M
2
8