Synthesis, crystal structure, computational analysis and biological properties of 1-(4-chlorobenzoyl)-3-[2-(2-{2-[3-(4-chlorobenzoyl)-thioureido]-ethoxy}ethoxy)ethyl]-thiourea and its Ni(II) and Cu(II) complexes

Article abstract of DOI:10.1016/j.molstruc.2018.05.015

A new dianionic ligand, 1-(4-chlorobenzoyl)-3-[2-(2-{2-[3-(4-chlorobenzoyl)-thioureido]-ethoxy}ethoxy)ethyl]-thiourea(CBEDEA), and its Ni(II) and Cu(II) complexes have been synthesized. X-ray single crystal analysis of CBEDEA shows that the molecule cryst

Full text of DOI:10.1016/j.molstruc.2018.05.015

Accepted Manuscript
Synthesis, crystal structure, computational analysis and biological properties of 1-
(4-chlorobenzoyl)-3-[2-(2-{2-[3-(4-chlorobenzoyl)-thioureido]-ethoxy}ethoxy)ethyl]-
thiourea and its Ni(II) and Cu(II) complexes
Ebube Evaristus Oyeka, Jonnie Niyi Asegbeloyin, Ilknur Babahan, Bernard Eboma,
Obinna Okpareke, Joseph Lane, Akachukwu Ibezim, H. Halil Bıyık, Bahadır Törün,
David Chukwuma Izuogu
PII:
S0022-2860(18)30571-4
10.1016/j.molstruc.2018.05.015
MOLSTR 25187
DOI:
Reference:
To appear in: Journal of Molecular Structure
Received Date: 9 March 2018
Revised Date: 4 May 2018
Accepted Date: 5 May 2018
Please cite this article as: E.E. Oyeka, J.N. Asegbeloyin, I. Babahan, B. Eboma, O. Okpareke, J.
Lane, A. Ibezim, H.H. Bıyık, Bahadı. Törün, D.C. Izuogu, Synthesis, crystal structure, computational
analysis and biological properties of 1-(4-chlorobenzoyl)-3-[2-(2-{2-[3-(4-chlorobenzoyl)-thioureido]-
ethoxy}ethoxy)ethyl]-thiourea and its Ni(II) and Cu(II) complexes, Journal of Molecular Structure (2018),
doi: 10.1016/j.molstruc.2018.05.015.
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1
Synthesis, crystal structure, computational analysis and biological properties of 1-(4-
chlorobenzoyl)-3-[2-(2-{2-[3-(4-chlorobenzoyl)-thioureido]-ethoxy}ethoxy)ethyl]-thiourea and its
Ni(II) and Cu(II) complexes
Ebube Evaristus Oyeka1,3, Jonnie Niyi Asegbeloyin1*, Ilknur Babahan2, Bernard Eboma1, Obinna
Okpareke1,6, Joseph Lane6, Akachukwu Ibezim4, H. Halil Bıyık5,Bahadır Törün5 and David Chukwuma
Izuogu1,3,
1Department of Pure and Industrial Chemistry, University of Nigeria, Nsukka, Enugu State,
Nigeria
2 Department of Chemistry, Adnan Menderes University, 09010, Aydin, Turkey
3
Department of Chemistry, Graduate School of Science, Tohoku University, 6-3 Aza-Aoba, Aoba-ku,
Sendai, Miyagi, 980-8578 Japan
4Department of Pharmaceutical Chemistry, University of Nigeria, Nsukka, Enugu State, Nigeria
5Department of Biology, Adnan Menderes University, 09010, Aydin, Turkey
6School of Science, University of Waikato, Private Bag 3105, Hamilton, New Zealand.
Corresponding Author: niyi.asegbeloyin@unn.edu.ng
Abstract
A
new
dianionc
ligand,
1-(4-chlorobenzoyl)-3-[2-(2-{2-[3-(4-chlorobenzoyl)-thioureido]-
ethoxy}ethoxy)ethyl]-thiourea(CBEDEA), and its Ni(II) and Cu(II) complexes have been synthesized.
X-ray single crystal analysis of CBEDEA shows that the molecule crystallized in the triclinic crystal
system, space group P-1, with two molecules per unit cell. The studied compounds were further
characterized by FT-IR, UV-VIS,1H NMR and mass spectroscopy. The metal complexes were isolated
as four coordinated ML(M: Ni(II), Cu(II), L:CBEDEA) molecules. Computational studies on CBEDEA
gave electrostatic potential energyisovalues which showed that there is a higher probability of metal
coordination around the carbonyl and thione groups. Results of non-covalent interaction studies
revealed the presence of significant amount of hydrogen bonding and other weak non-covalent
interactions in the molecule. Docking calculations on CBEDEA and its Ni(II) and Cu(II) metal
complexes revealed that they have affinity for beta-lactamase, a protein implicated in antibiotic drug-
resistant mechanism. Complexation with the metal ion shrank the size of the molecule and enabled the
metal complexes to fit more appropriately within the binding groove of the protein resulting in the
improved affinity over CBEDEA ligand. Target-ligand binding interactions resulted from
hydrophobicity and possibility of hydrogen bonding of the molecules. Furthermore, in vitro screening of
the compounds against 17 bacteria and 4 yeasts confirmed their antimicrobial potency against more
susceptible Gram-positive bacteria. Results of this study suggest that the metal complexes could be
developed into novel antimicrobial compounds.

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Keywords: Thiourea; Complexes; Spectroscopy; Computational; Docking calculations; Antimicrobial
studies
1. Introduction
The coordination chemistry of thioureas and their derivatives have been the subject of investigations in
recent times. Several works exploring the rich coordination geometries of this group of compounds
have been reported in the literature[1-4]. Thiourea derivatives are versatile ligands with the ability to
coordinate various metal centers in different coordination modes to form metal complexes with good
stability [5]. The presence of hard O- and N- and soft S-donor atoms in the backbones of thioureas
enable them to interact with both transition and main group metal ions, yielding metal complexes with
interesting physicochemical properties and significant biological activities [1-2,4,6]. Thiourea
derivatives can act as monodentate, bidentate or polydentate ligands, due to the presence of multi-donor
atoms [7], and can coordinate a range of metal centres as neutral ligands [8], monoanions[3,4] or di-
anions [9]. The introduction of carbonyl functional group to thiourea derivatives to give acylthiourea
increases the binding ability of the compounds to both cations and anions [1], resulting in different
levels of intramolecular and intermolecular hydrogen bonding interactions [10]. Thiourea functionalities
are of great importance in supramolecular chemistry, owing to the strong hydrogen bond donor and
acceptor ability of the N-H and C=S groups. They, therefore, take part in self-association processes such
as dimer formation and binding to guest ions [11,12]. The hydrogen bonding ability of thiourea moieties
has been utilized in the construction of anion receptors [13,14]. The high binding and ion receptive
ability of thioureas are properties being exploited in their use as chemotherapeutics [15,16]. Higher
hydrogen bonding interaction has been associated with increased target specificity to nuclear bases of
DNA and oxidative cleavage of DNA, thereby enhancing anticancer activity [17]. A vast range of
biological activities have been reported for acylthiourea derivatives which include antitumor [8,18],
antibacterial [4,19,20], antifungal [21], herbicidal and insecticidal properties [4,19]. Also Ni(II) and
Cu(II) complexes of thiourea derivatives have been reported to have appreciable biological activity
[22,23].
The isolation of molecules suitable for x-ray studies has remained a challenge despite successes
recorded in the past decades [24]. The design of molecular crystals involves proper understanding and
control of the non-covalent interactions such as hydrogen bonding and Van der Waals forces which play
significant roles in crystal formation. However, the utilization of thiourea functionality for building
crystalline frameworks requires a different approach, so as to prevent them from self-association, and
thereby make them available for binding guest ions [25]. Consequently, the use of quantum mechanical
tools in the elucidation of effects such as non-covalent bond interactions and anion receptor properties
in molecules is imperative.
Recently, we reported the crystal structure, computational and in-silico anticancer studies of N,N-
diethyl-N’-palmitoylthiourea exhibiting strong intermolecular hydrogen bonding [26]. In the present

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contribution, we have explored the synthesis, structure, non-covalent interaction and biological
properties of a bis(acylthiourea) and its Ni(II) and Cu(II) complexes.
2. Experimental Methods
2.1. Chemicals and instrumentation
4-chlorobenzoylchloride, 2,2’-(ethylenedioxy)bis(ethylamine), acetone, dichloromethane, methanol,
distilled water, potassium thiocyanate were obtained from Sigma Aldrich and used without further
purification. Melting point was obtained with a Fisher John melting point apparatus. The infrared
spectra were recorded in the range of 4000 – 400 cm-1 as KBr discs on a Perkin Elmer 100 Infrared
Spectrophotometer.1H NMR spectra were obtained from a MERCURY-300 MHz NMR spectrometer
using DMSO-D6 as a solvent. Mass spectra were obtained using a BruckerDaltonics ESI-MS
spectrometer. The conductivity of the complexes was checked using a WTW LF90 conductivity meter
by dipping the electrode of the meter into the solution of the sample and recording the reading at the
National Centre for Energy Research and Development, University of Nigeria, Nsukka.
2.2. Synthesis of 1-(4-chlorobenzoyl)-3-[2-(2-{2-[3-(4-chlorobenzoyl)-thioureido]-ethoxy}ethoxy)ethyl]-
thiourea (CBEDEA)
Synthesis of 1-(4-chlorobenzoyl)-3-[2-(2-{2-[3-(4-chlorobenzoyl)-thioureido]-ethoxy}ethoxy)ethyl]-
thiourea (CBEDEA) was done by similar literature procedure [10].
A solution of 4-chlorobenzoylchloride (0.02 mol, 2.926 mL) in anhydrous acetone (40 mL) was added
dropwise to a suspension of potassiumthiocyanate (0.02 mol, 1.944 g) in dry acetone (30 mL), and the
reaction mixture was heated under reflux for 30 min and then cooled to room temperature. A solution of
2,2’-(ethylenedioxy)bis(ethylamine) (0.01 mol, 1.462 mL) in anhydrous acetone (30 mL) was added,
and the resulting mixture stirred for 2 h. The solution was filtered to remove inorganic solid. The filtrate
was left for few days and yellow solid was obtained. The solid was then washed with water and ethanol.
Crystals suitable for X-ray crystallographic studies were obtained by dissolution followed by slow
evaporation of the product in methanol/dichloromethane mixture 1:1 at room temperature for few days.
o
Colour: yellow crystals; m.p: 164 C, yield: 82 %, ES-MS : calculated 566.570 [M+Na]+; experimental
1
566.1 [M+Na]+; H NMR (300 MHz, DMSO D6): δ = 11.42 (s, 2H, NH), δ = 10.95 (s, 2H, NH), δ =
7.90 – 7.93 (d, 4H, Ar-H, J = 8.7 Hz), δ = 7.53 – 7.56 (d, 4H, Ar-H, J = 8.7 Hz), δ = 3.78-3.79 (t, 4H,
CH2-CH2, J = 4.4 Hz), δ = 3.69 (t, 4H, -CH2-O-, J = 6.5 Hz), δ = 3.62 (t, 4H, CH2-NH-, J = 6.5 Hz); 13C
NMR (300 MHz, DMSO D6): δ = 178.2 (C=O), 167.7 (C=S), δ = 30.5 – 135.7 (C-1 - C-22); IR (KBr):
ʋ(cm-1): 3411, 3243 (st, ʋN-H), 1667 (st, ʋC=O), 1261 (ʋC=S).

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O
O
Cl
2
NCS
KSCN
2
Cl
Cl
O
NH2
H2N
O
acetone, RT
Cl
O
S
O
NH
NH
NH
NH
O
S
O
Cl
Scheme
1:
Synthesis
of
1-(4-chlorobenzoyl)-3-[2-(2-{2-[3-(4-chlorobenzoyl)-thioureido]-
ethoxy}ethoxy)ethyl]-thiourea
2.3. Synthesis of Ni (II) complex of 1-(4-chlorobenzoyl)-3-[2-(2-{2-[3-(4-chlorobenzoyl)-thioureido]-
ethoxy}ethoxy)ethyl]-thiourea (NiCBEDEA)
An ethanolic solution (20 ml) of Ni(CH3COOH)2.4H2O (1 mmol, 0.249 g) was added dropwise to a
solution of 1-(4-chlorobenzoyl)-3-[2-(2-{2-[3-(4-chlorobenzoyl)-thioureido]-ethoxy}ethoxy)ethyl]-
thiourea (1 mmol, 0.543 g) in ethanol (20 ml) at room temperature. The resulting mixture was stirred for
30 min at room temperature [27]. The solid complex formedwas filtered and recrystallized from
o
methanol and dried in a dessicator. Colour: green; m.p: 212 C; ES-MS : calculated 600.164 [M]+;
experimental 600.1 [M]+1H NMR (300 MHz, DMSOd6): δ = 10.97 (s, 2H, NH), δ = 7.92 (d, 4H, Ar-H),
δ = 7.56 (d, 4H, Ar-H), δ = 3.77 (t, 4H, CH2-CH2), δ = 3.68 (t, 4H, -CH2-O-), δ = 3.61 (t, 4H, CH2-NH-
); IR (KBr): ʋ(cm-1): 3237 (st, ʋN-H), 1667 (st, ʋC=O), 1236 (ʋC=S), 1590 (ʋC=N).
2.4. Synthesis of Cu (II) complex of 1-(4-chlorobenzoyl)-3-[2-(2-{2-[3-(4-chlorobenzoyl)-thioureido]-
ethoxy}ethoxy)ethyl]-thiourea (CuCBEDEA)
A solution of CuCl2.6H2O (1 mmol, 0.171 g) in water (20 mL) was added dropwise to a solution of 1-
(4-chlorobenzoyl)-3-[2-(2-{2-[3-(4-chlorobenzoyl)-thioureido]-ethoxy}ethoxy)ethyl]-thiourea (1 mmol,
0.543 g) in ethanol at room temperature. The resulting mixture was stirred for 30 min at room
temperature [27]. The solid complex formed was filtered and recrystallized from methanol and dried in
o
1
a dessicator. Colour: orange; m.p: 210 C; H NMR(300 MHz, DMSOd6): δ = 11.30 (s, 2H, NH) δ =
7.92 (d, 4H, Ar-H), δ = 7.51 (d, 4H, Ar-H), δ = 3.2 - 3.55 (t, 12H, CH2-CH2, CH2-O, CH2-NH); IR
(KBr): ʋ(cm-1): 3171 (st, ʋN-H), 1634 (st, ʋC=O), 1255 (ʋC=S).

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2.5. X-ray crystallography
The X-ray crystallographic data of 1-(4-chlorobenzoyl)-3-[2-(2-{2-[3-(4-chlorobenzoyl)-thioureido]-
ethoxy}ethoxy)ethyl]-thioureawas obtained on a Bruker SMART APEX II diffractometer equipped with
a CCD area detector using MoKα radiation (λ = 0.71073 Å). The software SMART was used for the
collection of data frames, for indexing reflections, and to determine lattice parameters; SAINT was also
used for the integration of the intensity of the reflections and for scaling. SADABS was used for
empirical absorption correction. The structures were solved using SHELXS-97,which was also used for
structure refinements and reporting. The structures were refined by full-matrix least-squares based on
2
Fo (SHELXL-97) with anisotropic thermal parameters for non-hydrogen atoms.
2.6. Computatonal studies
All theoretical calculations were performed using the Gaussian 09 program suite [28]on the high
performance computing cluster at the University of Waikato. Initial input geometries were constructed
from the crystal structure of CBEDEA. Geometry optimization was completed at the DFT level of
theory with the ωB97XD density functional which includes empirical dispersion and the 6-
311G+(2d,2p) basis set. This level of theory was found to have the highest congruency with the bond
lengths and angles of the CBEDEA crystal structure. Quantum mechanical wave functions files were
generated from the optimized structure of CBEDEA at the same level of theory. Non-covalent
interaction (NCI) calculations were performed using the NCI index software [29].
2.7. Antimicrobial screening
2.7.1. Microorganisms and Condition for Cultivation
Seventeen bacteria and four yeast cultures were used in the study. The bacterial strains and three yeast
strains were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA).
Candida trophicalis were obtained from the Faculty of Medicine, Adnan Menderes University. The
Gram-negative bacteria (G−) were Escherichia coli ATCC 35218, Enterobacteraerogenes ATCC
13048, Salmonella typhimurium ATCC 14028, Klebsiellapneumoniae ATCC 13882,
Pseudomonasaeruginosa ATCC 35032, Proteus vulgaris ATCC 33420, SerratiamarcescensATCC
13880, and the Gram-positive bacteria (G+) were Micrococcus luteus ATCC 9341, Staphylococcus
aureus ATCC 25923, Staphylococcus epidermidis ATCC 12228, Streptococcus pneumoniae ATCC
27336, Corynebacterium xerosis ATCC 373, Listeriamonocytogenes ATCC 19112, Enterococcus
faecalisATCC29212, Bacillus cereus ATCC 11778, Bacillus subtilis ATCC 6633, Also, yeast strains
such as Candida utilisATCC 9950, Candida albicansATCC 10231, Candida tropicalis, and
Saccharomyces cerevisiae ATCC 9763 were used.
The bacterial strains were cultured in Tryptic Soy Agar (TSA) and Brain Heart Infusion Agar (BHIA) at
30-37 oC for 24 h. The yeast strains were cultured in Malt Extract Agar (MEA) at 30 oC for 24 h.

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2.7.2. Antimicrobial Assay
The antimicrobial activities of all the synthesized compounds were determined by the discdiffusion
method [30,31]and the minimum inhibitory concentrations (MIC) were obtained by broth dilution
method [32-34].
2.7.3. Disc Diffusion method
The standard method of Antimicrobial Disc Susceptibility Tests reported by the National Committee for
Clinical Laboratory Standards [30,31]was used. Fresh stock solutions (1000 ꢀg⋅mL−1) of all the
synthesized compounds were prepared in DMSO. The inoculum suspensions of the tested bacteria and
yeasts were prepared from the broth cultures (18–24 h) and the turbidity equivalent adjusted to 0.5
McFarland standard tube to give a concentration of 1 × 108 bacterial cells/mL and 1 × 106 yeast
cells/mL. To test the antimicrobial activity of all the synthesized compounds, a Mueller Hinton Agar
(MHA) plate was inoculated with 0.1mL broth culture of bacteria or yeast. Then a hole of 6 mm in
diameter and depth was made on top with a sterile stick and filled with 50 ꢀL of synthesized
compounds. Plates inoculated with E. coli ATCC 35218, E. aerogenes ATCC 13048, S. typhimurium
ATCC 14028, S. aureus ATCC 25923, S. epidermidis ATCC 12228, E. faecalisATCC 29212, K.
pneumoniae ATCC13882, P. Aeruginosa ATCC 35032, P. vulgaris ATCC 33420, S. arcescensATCC
13880, S. pneumonia ATCC 27336, C. xerosis ATCC 373 and L. monocytogenes ATCC 19112 were
incubated at 37 oC for 24 h,while those with M. luteusATCC 9341, B. subtilisATCC 6633 and B. cereus
o
ATCC 11778 were incubated at 30 C for 24 h. In addition, C. albicansATCC 10231, C. utilisATCC
9950, C. tropicalis, and S. cerevisiae ATCC 9763 were incubated at 27∘C for 24 h. At the end of
incubation time, the diameters of the inhibition zones formed on the MHA were evaluated in
millimeters. Discs containing Chloramphenicol (30 mg Oxoid), Gentamycin (10 mg Oxoid),
Tetracycline (30 mg Oxoid), Ampicillin (10 mg Oxoid), Ofloxacin (5 mg Oxoid), Vancomycin (30 mg
Oxoid), Cefotaxime (30 mg Oxoid), Nystatin (100 mg Oxoid) were used as positive controls. The
measured inhibition zones of the study compounds were compared with those of the reference discs.
2.7.4.Minimum Inhibitory Concentration (MIC)
Minimum inhibitory concentration (MIC) was determined by reported method[32-34].The study
bacteria were inoculated in Tryptic Soy Broth (TSB) and Brain Heart Infusion Broth (BHIB) and
∘
incubated at 30–37 C for 24 h while the yeasts were inoculated in Malt Extract Broth (MEB) and
∘
incubated at 27 C for 24 h. The inoculums were adjusted according to 0.5 McFarland standard tubes.
Initially, 100 ꢀL of Mueller Hinton Broth (MHB) was placed in each well. After, the compounds were
dissolved in DMSO (2 mgmL−1) and transferred into the first well. Two fold serial dilutions of the
compounds were carried out to determine the MIC, within the concentration range 256 to 0.125
−1
6
∘
ꢀgmL . Cultures were grown at 30–37 C (18–20 h) and the final inoculum was approximately 10
cfumL−1.The lowest concentration of the study compounds that resulted in complete inhibition of the

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microorganisms was represented as MIC (ꢀgmL−1). Similar procedure was done on positive controls. In
each case, the test was performed in triplicate and the results were expressed as means.
2.8. Molecular Simulations
X-ray crystal structures of beta lactamase co-crystallized with its inhibitor were retrieved from the
protein databank (PDB code 1ONH) [35]. The protein-ligand complex dimers were treated as described
in our earlier work [36] using Molecular Operating Environment (MOE) software running on Linux
workstation. The three-dimensional chemical structure of CBEDEA and its metal (copper and nickel)
complexes were generated using the graphical user interface of MOE. The proteins and ligands were
energy minimized in Gromacs 4.2.2 using ffG53a6 [37]. The Auto Dock Tool implemented in
AutoDock4.2 software was used to carry out docking after the docking protocol has been validated
though the root mean square deviation method. A grid box size of 16 x 22 x 20 Å3 points (spacing
between the grid points of 0.375 A) was used which centered on the mass center (18.611, 21.867, 6.326)
of the crystallographic macromolecule encompassing all active site atoms.
3. Results and discussion
3.1. Physical properties of the synthesized compounds
The ligand and metal complexes were isolated by recrystallization using suitable solvents. CBEDEA
was sparingly soluble in water, it was soluble in polar organic solvents such ethanol, methanol and
DMF and but highly soluble in dichloromethane and DMSO. The metal complexes are colored, non-
hygroscopic and stable solids. They were soluble in ethanol, methanol, dichloromethane, DMSO and
DMF, however they showed only slight solubility in water and diethylether. The low molar conductivity
values of the complexes indicate that they are non-electrolytes. All the compounds showed satisfactory
mass spectroscopic results. The data from the mass spectra are all in agreement with the expected
molecular masses of the ligand and metal complexes. Some physical parameters of the synthesized
compounds are shown in Table 1.

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Table 1: Some Physical Parameters of the compounds
Compound
Colour %
Melting M+
ES-MS:
(Calculated) [M+Na]+
(Experimental)
Molecular formular Molar
yield point
conductivity
oC
(ohm-1mol-
1cm-2)
CBEDEA
Yellow 82
164
212
210
543.486
600.164
641.047
566.1
C22H24Cl2N4O4S2
-
NiCBEDEA Green 78
CuCBEDEA Orange 81
623.1
664.3
C22H22Cl2N4NiO4S2 0.87
C22H26Cl2CuN4O6S2 0.98
3.2. Structural analysis of CBEDEA
The molecular structure and crystal packing of CBEDEA are shown in Figure 1 and 2 respectively.
Table 2 gives the crystal data and structure refinement. The bond lengths and bond angles are listed in
Table 3. The single x-ray structural analysis shows that CBEDEA crystallized in the triclinic crystal
system with space group P-1 and Z = 2. The molecule possesses a zigzag shape enacted by the N3-C14-
o
o
C13-O3 and N2-C9-C10-O2 torsion angles of 61.93 and 65.98 respectively. Also the chlorophenyl
groups are trans to each other. The molecule is non-planar in relation to the carbonyl and thione
carbons as can be seen from C17-C16-N4-C15, O1-C7-N1-C8, C1-C7-N1-C8 and O4-C16-N4-C15
dihedral angles of 172.04o, -4.19o, 174.70o and -176.21o. However, the acylthiourea group of the C1-C6
ring is more twisted than that of the C17-C22 ring.
The C16-O4, C15-S1, C7-O1 and C8-S2 bonds show typical double bond lengths of 1.227(2) Ǻ,
1.666(2) Ǻ, 1.224(2) Ǻ and 1.670(2) Ǻ respectively [20,38]. The C-N bonds in the vicinity of carbonyl
and thione carbon, C16-N4 (1.372(2) Ǻ), C15-N4 (1.395(2) Ǻ), C15-N3 (1.319(3) Ǻ), C8-N2 (1.320(2)
Ǻ), C8-N1 (1.392(3) Ǻ) and C7-N1 (1.371(3) Ǻ) are less than expected for an average C-N bond length
of 1.48 Ǻ, therefore showing different degrees of double bond character [20,39]. This can be explained
as resulting from the contribution of the thiol tautomer formed by the conjugation of the C=S π-
electrons with the lone pairs of electrons on nitrogen. The shortening of the C-N bonds between the
aliphatic substituent and the thione carbon, C15-N3 (1.319(3) Ǻ) and C8-N2 (1.320(2) Ǻ) compare to
other C-N bonds in the molecule are in agreement with literature reports for other acylthiourea
derivatives [20,26,38,39,40]. The C-C, C-O and C-N bonds of the 2,2’-(ethylenedioxy)bis(ethylamine)
moiety are close to values expected for average single bond length of aliphatic systems; C-C (1.54 Ǻ),
C-O (1.43 Ǻ), C-N (1.48 Ǻ).The C-C bond, C11-C12 (1.492(3) Ǻ), C10-C9 (1.499(3) Ǻ), C13-C14
(1.496(3) Ǻ) are slightly shorter because of the influence of the electronegative oxygen. The two 4-
chlorobenzoylthiourea moieties of CBEDEA are expected to be equivalent, they, however, possess
different bond lengths and bond angles as can be seen in Table 2. The bond angles of the 2,2’-
o
(ethylenedioxy)bis(ethylamine) chain C11-O2-C10 (112.22o), C13-O3-C12 (112.21 ), C14-C13-O3
o
o
(108.41 ), C11-C12-O3 (109.31 ) show single bond character with sp3 hybridization, while those of

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the phenyl ring, carbonyl and thione moieties are essentially sp2 hybridized. The geometry of the
carbonyl and thiocarbonyl groups each of the acylthiourea moiety is planar.
The CBEDEA molecule is stabilized by two equivalent intramolecular hydrogen bonds through N2-
H2…O1 and N3-H3…O4. The thione group was not involved in any hydrogen bonding. No
intermolecular hydrogen bond was observed in the molecule. Thus, CBEDEA is expected to show an
excellent binding ability for guest ions because of the presence of free hydrogen bond donor and
acceptor groups.
Table 2: Crystal data and structure refinement details for CBEDEA
Identification Code
CBEDEA
Formula
C22H24Cl2N4O4S2
Formula weight
543.47
Temperature (K)
100(2)
Wavelength (Å)
0.71073
Crystal system
Triclinic
Space group
P-1
a/Å
7.6280(4)
b/Å
9.1780(5)
c/Å
18.1787(10)
77.754(3)
86.591(3)
79.544(3)
α/o
β/o
γ/o
Volume (Å3)
1222.78(12)
Z
2
Calculated density (g/cm3)
Absorption coefficient (mm-1)
F(000)
1.476
0.474
564.0
Crystal size (mm3)
Index
0.604 × 0.362 × 0.285
-9<=h<=9, -10<=k<=10, -21<=l<=21
2.293 to 25.043
Theta range for data collection
Reflections collected / independent reflections 17703/ 4316 [R(int) = 0.0359]
Completeness to theta (%)
Absorption correction
99.9
SADABS
0.284 and -0.208 e.Å-3
Largest diff. peak and hole
Refinement method
Goodness of fit on F2
Full-matrix least square on F2
1.046
Final R indices [I>2σ(I)]
R indices (all data)
R1 = 0.0321, wR2 = 0.0747
R1 = 0.0412, wR2 = 0.0797

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Table 3: Bond lengths (Ǻ) and bond angles (o)
Bond Lengths
Cl1 C4
1.734(2)
1.378(3)
1.492(3)
1.224(2)
1.371(3)
1.392(3)
1.670(2)
1.320(2)
1.456(2)
1.419(2)
1.666(2)
C11 O2
C12 O3
C13 O3
C13 C14
C14 N3
C15 N3
C15 N4
C16 N4
C16 O4
C17 C22
N3 H3
1.427(2)
1.424(3)
1.418(2)
1.496(3)
1.451(2)
1.319(3)
1.395(2)
1.372(2)
1.227(2)
1.389(3)
0.8800
C5 C6
C1 C7
C7 O1
C7 N1
C8 N1
C8 S2
C8 N2
C9 N2
C10 O2
C15 S1
Bond Angles
C6 C1 C7
H3A C2 C3
C3 C4 Cl1
C4 C5 C6
C1 C7 N1
N1 C7 O1
N1 C8 N2
N1 C8 S2
N2 C8 S2
C9 C10 O2
123.75(18)
119.5
N3 C15 N4
N3 C15 S1
N4 C15 S1
C17 C16 O4
N4 C16 O4
C16 C17 C22
C7 N1 C8
117.02(17)
123.78(15)
119.17(15)
120.42(17)
122.02(18)
124.85(17)
128.28(17)
122.56(17)
118.7
120.38(17)
119.36(19)
116.79(17)
121.22(18)
116.70(18)
118.58(15)
124.72(16)
107.01(16)
C8 N2 C9
C8 N2 H2
C10 O2 C11
112.21(14)

ACCEPTED MANUSCRIPT
11
3.3. Spectroscopic analysis of CBEDEA
1
In the H NMR spectra of CBEDEA, there were characteristic peaks in the aliphatic, aromatic and
hydrogen bonded regions. The triplet peaks at δ3.62 and δ3.69 were assigned to the CH2-N and CH2-O
protons. The -CH2-CH2- protons were assigned to the triplet band at δ3.79. The doublets at δ7.73-7.93
were assigned to the aromatic protons of the 4-chlorophenyl, while the characteristic NH protons
appeared at δ10.95 and δ 11.42. The δ11.42 band was explicitly assigned to the NH adjacent to the C=S
group because of its involvement in N-H…O intramolecular hydrogen bonding as affirmed by the x-ray
crystallographic analysis.
In the FTIR spectra of CBEDEA, the sharp and strong band at 3411 and 3242 cm-1 were ascribed to the
stretching vibration of the N-H groups. This agrees with the report from the 1H NMR spectra. The C=O
stretching vibration was assigned to a strong absorption peak at 1667 cm-1, which is in agreement with
the literature reports [4,10,20,41]. A sharp absorption band with weak intensity observed at 1261 cm-1
was assigned to C=S stretching vibration [4,20,41]. The characteristic C-H stretch of aliphatic groups is
expected to appear in 2850-3000 cm-1 frequency range and this was observed for CBEDEA at 2872 cm-
1. Aromatic C-H stretch was observed at 2931 cm-1. The C-N stretching vibration could not be assigned
because it appeared in the fingerprint zone of the IR. All the peaks observed in the IR spectra of
CBEDEA were consistent with the results from the NMR spectra and X-crystallography and were also
in good agreement with related literature spectra[4,10,20,41]. The UV spectra for CBEDEA was
recorded in ethanol solution and an absorption band was observed at 235 nm which is due to the π*←n
electronic transition of the C=S bond.
The ESI mass spectra of CBEDEA, CuCBEDEA and NiCBEDEA showed parent peak at 566.10,
623.1 and 664.3 respectively for [M+Na]+, and this values are is in close agreement with calculated
values. In the spectra of CBEDEA, adjacent peaks appeared at 567.10 and 568.10 which are due to
contribution of the isotopic carbon-13 and chlorine-37 respectively. The detailed mass fragmentation
patterns of the synthesized compounds can be found in the supplementary information.
3.4. Electrostatic Potential Energy
The strength of nearby charges, nuclei and electrons at any given position (equation 1) can be
ascertained by measuring the electrostatic potential (ESP) energy,
ꢇ
ꢈ
ꢃꢄꢃꢅ
ꢆ
ꢁ
ꢂ
1.
∞
K= Coulomb’s constant q = charge of particles and r = separation between particles or radius.
Electrostatic potential maps or potential energy surface maps are electron density models used in
theoretical chemistry to represent areas of high or low potential energy in a molecule. They are three-
dimensional representations of the charge distribution of a molecule and thus an indication of the
potential energy distribution of that molecule [42]. Areas of low potential are mapped in red and

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12
characterized by an abundance of electrons while areas of high potential are mapped in blue and
characterized by a relative absence of electrons (Figure 3). The regions of intermediary electron
distribution are mapped from yellow to green. The ESP map gives an indication of the polarity or dipole
moment of a molecule and this information can be used in predicting the chemical reactivity of the
molecule of interest towards other molecules. In the case of the CBEDEA ligand, the ESP and its
contour diagram (Figure 3a and 3b respectively) indicate the possible coordination sites of the ligand to
metal ions. The electrostatic potential map for CBEDEA was generated from the optimized structure of
the molecule at DFT level of theory using the Self Consistent Field (SCF) density matrix. The highest
electronegative point of the molecule with the most intense red color was observed to be around the two
carbonyl oxygens with an electrostatic potential isovalue of -0.045 and the most electropositive point on
the molecule with the most intense blue coloration were observed to be around the phenyl rings with
electrostatic potential isovalue of +0.471. The ESP maps show that there is a higher probability of metal
coordination around the carbonyl and thione functional groups of the CBEDEA ligand than any other
part of the molecule.
3.5. Analysis of Non-covalent interaction calculations
A wide range of interactions including hydrogen bonding, dispersion forces, Pauli repulsion, dipole-
dipole attraction, CH---π, CH…..lone pair and lone pair ….π have recently been classified as non-
covalent interactions [43]. Among these interactions, hydrogen bonding isthe most ubiquitous. The
nature and strength of the different non-covalent interactions in a molecule determine the
supramolecular architecture of the compound in the solid state. The relatively recently developed non-
covalent interaction index (NCI index) [29] has been used to visualize real space regions where non-
covalent interactions are located. The NCI index depends on the analysis and pictorial interpretation of
the electron density (ρ) and its derivatives which includes the Hessian eigen value λ2 and its reduced
density gradient(s) which has been defined as
1
|∇ꢌ|
ꢉ =
2.
2(3ꢊꢅ)ꢄ/ꢋ ꢌ(ꢍ)ꢎ/ꢋ
Non-covalent interactions can be identified as regions in three-dimensional space where the reduced
density gradient (s) and the electron density ρ are tending to zero [43]. The NCI index plots the reduced
gradient as a function of the electron density ρ, oriented by the sign λ2 which identifies the interaction
types. Attractive interactions appear at λ <0 while repulsive interactions appear at λ >0 (as in the centre
of rings or cages) where several non-bonded atoms interact resulting in steric crowding [44]. Pictorial
3D representation of these interactions in an RGB (red-green-blue) colouring scheme is used to rank
these interactions. Red is used for repulsive/destabilizing interactions, blue for attractive/stabilizing
interactions and green for delocalized weak interactions. The 2D electron density plots for CBEDEA
and the corresponding 3D isosurface representations are shown in Figure 4. The low density, low
gradient peak at sign(λ2)ρ < -0.03 au is characteristic of an attractive intramolecular hydrogen bonding
interaction on two ends of the CBEDEA molecule and corresponding to the deep blue end of the trophy

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13
shaped bicolored isosurface trough between the thiourea N----H and the adjacent carbonyl C=O
functional group. The red lower end of the isosurface trough corresponds to the low gradient high
density peaks appearing at 0.01 au > sign(λ2)ρ < 0.02 au. This is as a result of non-bondingmulticentric
ring closure interactions in the six membered ring resulting from the intramolecular hydrogen bonding
interaction. The other high density low gradient peaks appearing at sign(λ2)ρ > 0.02 au are as a result of
the non-bonding steric interactions within the benzene rings on each side of the CBEDEA ligand. This
interaction corresponds to the deep red cigarshaped isosurface troughs situated at the center of the
benzene rings. Apart from the hydrogen bonding interactions and the destabilizing steric interactions,
the other type of interactions present in the solid state structure of the CBEDEA ligand are the CH----
O=C intramolecular interactions, the weak CH---S=C intramolecular interactions, and the very weak
CH----HN delocalized Van der Waals interactions. The low gradient low density peak for the attractive
intramolecularCH----O=C interactions appear at sign(λ2)ρ = 0.015 au on the 2D electron density plot.
This corresponds to the blue end of the flat bicolored isosurface inside the five membered ring formed
by the intramolecular CH----O=C interaction. The red end of the flat isosurface trough corressponds to
the electron density peaks at 0.01 au > sign(λ2)ρ < 0.02 au resulting from multicentric ring closure
interactions. Similar multicentric ring closure interactions have been reported for intra-molecular CH----
O=C interactions in N-acetyl phenylalanyl-amide [45]. The low density low gradient peaks at -0.01 au >
sign(λ2)ρ < -0.012 au are as a result of the weakly attractive intramolecular CH---S=C interactions
corresponding to the light blue end of the flat isosurface troughs between the thio-carbonyl functional
group and the adjacent phenyl carbon as shown on 3D isosurface plot in Figure 4. The orange coloured
end of the flat isosurface corresponding to the almost simultaneous critical density value 0.01 au <
sign(λ2)ρ < 0.012 au are as a result of the strain on the ring back bone resulting from non-bonding
multicentric interactions within the ring. This type of simultaneous interactions was also reported in the
solid state structure of a related N,N-diethyl-N’-palmitoylthiourea[26]. The CH----NH delocalized
interactions have been known to appear at critical density values of -0.01 au > sign(λ2)ρ < 0.01 au and
the two simultaneous critical density peaks within this range on the 2D density plots have been assigned
to weak delocalized VanderWaal interactions in the CBEDEA ligand.The large amount of hydrogen
bonding and other weak non-covalent interaction in the molecule is a pointer to the possibility of a high
degree of bioactivity for the CBEDEA ligand.
3.6. Spectroscopic analysis of the metal complexes
The 1H NMR of the diamagnetic nickel complex NiCBEDEA showed the disappearance of two protons
at δ11.42 ppm which were assigned to the NH protons between the thione and carbonyl groups. This
shows that there is deprotonation on metal chelation. The other peaks of the metal complexes shifted
only slightly from the corresponding peaks in the ligand spectra.
The IR peak at 3411 cm-1 which was assignable to the N-H in the vicinity of the thione and the carbonyl
group disappeared in the copper complex while that at 3243 cm-1 was shifted to a lower frequency. This
is as a result of deprotonation of the N-H proton in the acylthiourea moiety on complexation followed
by the delocalization of the C=O and C=S π-electrons. Also the peak at 1261 cm-1 which was assigned

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14
to C=S stretching vibration reduced to lower frequencies in the metal complexes showing its
coordination to the metal center with CuCBEDEA and NiCBEDEA showing 1256 and 1236 cm-1
valuesfor C=S stretching vibration respectively. These results are in agreement with literature reports
[4,19,27,41]. Also, a new weak band appeared at 1589 cm-1 and 1634 cm-1 for NiCBEDEA and
CuCBEDEA respectively. These were assigned to C=N stretching vibration, showing that there was
delocalization of the π-electrons on the thione group to the C-N bond. Thus, it can be concluded that
the ligand coordinated via the thione sulfur and the carbonyl oxygen to give a six- membered ring
system with the metal atom. Therefore the π-electrons are delocalized between the CO-N-CS moiety
during coordination.
In the electronic spectra, CuCBEDEA showed peaks at 28,746 cm-1, 21,435 cm-1 and 15,890 cm-1 which
1
1
1
can be assigned to the A1g→ Eg, A1g→1B1g and A1g→1A2g transitions of the square planar geometry.
Similarly, NiCBEDEA displayed absorption bands at 27,739 cm-1, 20,930 cm-1 and 15,235 cm-1 which
1
1
1
can be assigned to the A1g→ Eg, A1g→1B1g and A1g→1A2g transitions of the square planar geometry.
However, only a few works havebeen reported on the electronic spectra of acylthiourea metal
complexes. It has been mentioned that UV spectroscopy does not find extensive use in structure
elucidation of thioureas [1]. On the basis of the IR, UV/vis spectra and mass spectra, the structure of the
metal complexes was proposed (Figure 5).
3.7. In-vitro antimicrobial assay
The compounds were screened for their antimicrobial activities against 17 bacteria and 4 fungi and were
reported based on their inhibition zone diameter and minimum inhibitory concentration as shown in
Tables 4 and 5 respectively. The minimum inhibitory concentration (MIC) was determined by reported
method [32-35]. Two-fold serial dilutions of the compounds were carried out to determine the MIC,
o
within the concentration range 256 to 0.125 ꢀgmL−1. Cultures were grown at 30 – 37 C (18–20 h) and
the final inoculums were approximately 106cfumL−1. The lowest concentration of the study compounds
that resulted in complete inhibition of the microorganisms was represented as MIC (ꢀgmL−1). Similar
procedures were done on positive controls. In each case, the test was performed in triplicate and the
results were expressed as means.MICs were measured for the compounds that showed appreciable
growth inhibition zones; the study was restricted to microorganisms that were affected by the
compounds. The result on Table 5 showed that the MIC for CBEDEA against the screened bacteria
varied from 32 to 256 µg.mL-1 and was 32 µg.mL-1 for yeast, Candidautilis ATCC 9950.The MICs for
the metal complexes were in the range of 32 - 256 for bacteria and 64 – 128 µg.mL-1 for yeast. The MIC
values suggested that some of the studied compounds showed considerable antimicrobial activity.
CBEDEA and its Ni(II) and Cu(II) complexes demonstrated significant effects against some pathogenic
bacteria and yeasts. CBEDEA showed significant antimicrobial activity against P. aeruginosa ATCC
35032, S. marcescens ATCC 13880, B. Subtilis ATCC 6633 and C. utilis ATCC 9950; CuCBEDEA
revealed considerable antimicrobial effect againstM. luteus ATCC 9341, S. pneumoniae ATCC 27336,
P. aeruginosaATCC 35032, B. subtilisATCC 6633, C. utilisATCC 9950 and C. trophicalis and

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15
NiCBEDEA registered substantial antimicrobial effect against S. pneumoniae ATCC 27336, P.
aeruginosa ATCC 35032, S. marcescensATCC 13880 and B. Subtilis ATCC 6633. CuCBEDEA proved
to be the most effective among the compounds tested. However, there was no significant improvement
on the antimicrobial activity of CBEDEA on complexation. Also, NiCBEDEA did not show any
antimicrobial activity against used yeasts as can be seen in Table 5.
The compounds were found to be more effective against Gram-positive bacteria compared to Gram
negative bacteria. The observed trend is similar to an earlier report [46]. This is attributed to the nature
of the cell walls of theGram-negative bacteria and Gram-positive bacteria. Gram-negative bacteria are
not readily permeated by drugs and dyes because of the protective outer membrane of their cell walls
[47], such protective outer membrane is absent in Gram-positive bacteria.
From the result of the antimicrobial screening, it can be concluded that bis(acylthiourea) ligand and its
copper complex showed promising antimicrobial properties against some tested bacteria and yeast.

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16
Table 4: Inhibition zone diameter (mm) of the synthesized compounds (1000µg/ml) and the reference antibiotics
Test microorganism
Inhibition zone diameter (mm)
Compounds
Reference antibiotics
CN1 TE
CBEDEA CuCBEDEA NiCBEDEA C30
AM OFX5 VA30 CTX3 NS
0
21
30
15
P10
-
0
10
100
Escherichiacoli
ATCC 35218
Enterobacteraerogenes
ATCC 13048
SalmonellatyphimiriumATC
C 14028
Micrococcusluteus,
ATCC 9341
StapylococcusaureusATCC
25923
Stapylococcusepidermidis
ATCC 12228
Klebsiellapneumoniae
ATCC 13882
Streptococcuspneumoniae
ATCC 27336
PseudomonasaeruginosaAT 16
CC 35032
Corynebacteriumxerosis
ATCC 373
MycobacteriumsmegmatisA
TCC 607
Listeriamonocytogenes
ATCC 19112
Serratiamarcescens
ATCC 13880
Proteusvulgaris
ATCC 33420
-
-
-
24
19
17
25
23
22
21
24
22
20
23
19
23
17
16
28
19
25
24
23
22
27
28
29
22
30
29
27
26
28
23
-
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
-
-
-
20
16
15
20
17
19
20
20
17
18
14
19
24
11
14
15
26
22
19
20
25
20
25
26
12
13
17
19
-
20
13
17
12
13
14
15
14
20
12
16
13
18
16
-
-
-
8
21
14
13
12
23
29
18
21
20
25
27
24
20
-
12
9
9
-
-
28
20
17
-
8
8
-
-
-
-
10
13
12
-
14
11
-
14
-
-
27
19
12
19
-
-
-
-
-
-
-
12
-
9
-
13
-
Entereococcusfaecalis
ATCC29212
-
-
-
14

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17
NT
Bacillluscereus
ATCC 11778
-
9
-
23
24
25
-
28
21
17
Bacillussubtilis
ATCC 6633
Candidaalbicans
ATCC 10231
13
-
18
8
13
-
22
20
12
-
27
20
16
NT
22
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
Candidautilis
16
11
-
21
ATCC 9950
Candidatrophicalis
Saccharomycescerevisiae
ATCC 9763
-
-
12
-
-
-
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
20
15
Key: C30: Chloramphenicol (30 mg Oxoid), CN10: Gentamycin (10 mg Oxoid), TE30: Tetracycline (30 mg Oxoid), E15:
Erythromycin (15mg Oxoid), AMP10: Ampicillin (10 mg Oxoid), OFX5: Ofloxacin (5 mg Oxoid), VA30: Vancomycin (30 mg
Oxoid),CTX: Cefotaxime (30 mg Oxoid), NS: Nystatin (100 mg Oxoid),
(-): Zonedid not occur. NT: Not tested.

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18
Table 5: Antimicrobial activities reported as the minimum inhibitory concentration (MIC, µg.mL-1)
Test Microorganisms
CBEDEA
CuCBEDEA
CuCBEDEA
Micrococcusluteus, ATCC 9341
StapylococcusaureusATCC 25923
-
64
-
-
256
256
Stapylococcusepidermidis
ATCC 12228
Pseudomonasaeruginosa
ATCC 35032
256
32
-
256
64
-
-
128
-
Klebsiellapneumoniae
ATCC 13882
Streptococcuspneumoniae
ATCC 27336
128
-
64
-
32
-
Mycobacteriumsmegmatis
ATCC 607
SerratiamarcescensATCC 13880
64
-
256
256
64
-
BacillluscereusATCC 11778
BacillussubtilisATCC 6633
64
-
16
64
-
Candidaalbicans ATCC 10231
256
CandidautilisATCC 9950
32
-
128
64
-
-
-
-
Candidatrophicalis
Saccharomycescerevisiae
ATCC 9763
-
(-) = No effect

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19
3.8.Analysis of Docking Calculations
The use of computational techniques in the design and development of new chemotherapeutic agent
has become a common practice in both academic and industrial field because of their cost and time
efficiencies [48,49]. Due to several kinds of literature that proposed compounds of thiourea series to
demonstrate antibiotic property, we investigated the possibility of CBEDEA, CuCBEDEA and
NiCBEDEA to bind to bacterial β-lactamase [50,51]. Aside from being a validated antibiotic target,
the protein was chosen because its production is a significant resistance-mechanism that impedes the
function of antibiotics [52]. The theoretical free binding energy retrieved from the highest populated
clusters of poses and characterized by the top-ranking score revealed that all the molecules could
inhibit the activity of the enzyme. Moreover, metal complexes of CBEDEA showed improved
affinity for the protein (CBEDEA = -6.7 kcal/mol, CuCBEDEA = -8.7 kcal/mol and NiCBEDEA = -
8.2 kcal/mol).
Thus, the observed increased interaction of CuCBEDEA and NiCBEDEA with the target protein is
an advantage. The best dock poses of the compounds identified unique binding mode toward β-
lactamase (Figure 6). The decrease in the size of the ligand molecule due to complexation with the
metal ion possibly enabled CuCBEDEA and NiCBEDEA to fit more appropriately within the active
site groove of the protein. All the candidates had one of their terminal phenyl rings accommodated
within the enzyme cavity surrounded by the following amino acids with hydrophobic chains: Val124,
Val212, Val214, Leu219 and Ala223. Also, all the compounds made hydrogen bonding interaction
with β-lactamase Ser64, Lys67 and Asn152. The observed π-π contact between the aromatic rings of
CuCBEDEA and Tyr224, which is absent in others, could account for its increased binding affinity
for the protein. The result of the docking simulation shows strong correlation with the in vitro
antimicrobial result, with CuCBEDEA showing the highest activity.
4. Conclusion
A new bis(acylthiourea) ligand and its Ni(II) and Cu(II) metallic derivatives were synthesized. The
ligand behaved as a dianionictetradentate molecule, coordinating through the thione sulfur and the
carbonyl oxygen.Four coordinate square planar geometries have been proposed for the Ni(II) and
Co(II) complexes.The results of the electrostatic potential map for the bis(acylthiourea) predicted the
possibility of metal coordination through the carbonyl and thione functional groups.Results of non-
covalent interation studies revealed the presence of significant hydrogen bonding and other weak
non-covalent interaction in the molecule. Docking calculations revealed that the compounds had an
affinity for a protein notable for its involvement in the drug resistance mechanism of bacteria – beta-
lactamase. Moreover, complexation of the ligand appeared to improve the binding affinity toward the
protein and analysis of their binding poses showed that the compounds adopted unique conformations
within the binding cavity of the studied protein. Furthermore, in-vitro screening of the compounds
against Gram-positive bacteria, Gram-negative bacteria and yeast confirmed the antimicrobial

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20
potencies of the candidates. However, observation suggests the metal complexes failed to improve
permeability across the bacteria cell walls because almost only the Gram-positive bacteria were
susceptible to the compounds. Nonetheless, CuCBEDEA appeared to be most promising compound
because it demonstrated lowest theoretical binding affinity towards beta-lactamase and highest
antimicrobial activity. Therefore, further attention on the compound is necessary, and the predicted
binding poses could guide the structural modification in the activity optimization process.
Acknowledgement
Dr. Christopher J. Ziegler and Briana Schrage' of Department of Chemistry, the University of
Akron, 43325, Akron, OH, USA are acknowledged for the collection of the X-ray crystal data.
Appendix A : Supplementary material
CCDC 1819392 contains the supplementary crystallographic data for 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 336033).
Supplementary data for this publication can be found at….

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References
[1] A. Saeed, U. Florke, M.F. Erben, A review on the chemisty, coordination, structure and biological
properties of 1-(acyl Iaroyl)-3-(substituted) thioureas. J. Sulfur Chem. 35 (2014) 318-355.
[2] H. Liu, W. Yang, W. Zhou, Y. Xu, J. Xie, M. Li, Crystal structures and antimicrobial activities of
copper (II) complexes of fluorine thioureido ligands.Inorg.Chim.Acta 405 (2013) 387-397.
[3] M. Lipowska, B.L. Hayes, L. Hansen, A. Taylor, L.G. Marzilli, Rhenium(v) oxo complexes of
novel N2s2 dithiourea (DTU) chelate ligands: synthesis and structural and structural characterization.
Inorg. Chem. 35 (1996) 4227-4231.
[4] G. Binzet, H. Arslan, U. Florke, N. Kulcu, N. Duran, Synthesis, characterization and
antimicrobial activities of transition metal complexes of N,N-dialkyl-N’-(2-chlorobenzoyl) thiourea
derivatives. J. Coord. Chem. 59 (2006) 1395-1406.
[5] E.J. Waheed, Synthesis and characterization of some metals complexes of [N-[(benzoylamino)-
thioxomethyl] proline]. J. Al-Nahrain Uni. 15 (2012) 1-10.
[6] Y.F. Yuan, J.T. Wang, M.C. Gimeno, A. Laguna, P.G. Jones, Synthesis and characterization of
copper complexes with N-ferrocenoyl-N’(alkyl)thioureas. Inorg.Chim.Acta 324 (2001) 309-317.
[7] T.S. Smith, W. Henderson, B.K. Nicholson,Cycloauratedgold(III) complexes with monoanionic
thiourea ligands. Inorg.Chim.Acta 408 (2013) 27-32.
[8] C. Sacht, M.S. Datt, S. Otto, A. Roodt, Chiral and achiral platinum (II) complexes for potential
use as chemotherapeutic agents: crystal and molecular structures of cis-[Pt(L’)2] and [Pt (L’)
Cl(MPSO)] [HL’= N,N-diethyl-N’-benzoylthiourea]. J. Chem. Soc. Dalton Trans. 5 (2000) 727-733.
[9] R.L. Zuckerman, R.G. Bergman, Structural factors that influence the course of overall [2+2]
cycloaddition reactions between midozirconocene complexes and heterocumulene. Organometallics
19 (2000) 4795-4809.
[10] C.K. Ozer, H. Arslan, D. Van derveer, N. Kulcu, Synthesis and characterization of N-(aryl
carbamothioyl).cyclohexanecarboxamide derivatives: the crystal structure of N-(Naphthalen-1-
ylcarbamothioyl) cyclohexanecarboxamide. Molecules 14 (2009) 655-666.
[11] P. Byrne, D.R. Turner, G.O. Hoyd, N. Clarke, J.W. Steed,Gradual transition from NH---
Pyridylhydrogen bonding to the NH---O tape synthon in pyridyl ureas.Crys.Growth Des. 8 (2008)
3335-3344.
[12] M.W. Hosseinni,Reflection on molecular tectonics. Cryst.Eng. Comm. 6 (2004) 381-322.
[13] P.A. Gale, S.E. Garcia-Garrido, J. Garric, Anion receptors based on Organic frameworks:
highlights from 2005 and 2006. Chem. Soc. Rev. 37 (2008) 151-190.
[14] A.G. Doyle, E.N. Jacobsen, Small molecule H-bond donors in asymmertric catalysis. Chem.
Rev. 107 (2007) 5713-5743.
[15] M. Behray, C.A. Webster, S. Pereira, P. Ghosh, S. Krishnamurthy, W.T. Al-Jamal, Y.
Chao,Synthesis of diagnostic silicon nanoparticles for targeted delivery of thiourea to epidermal
growth factor receptor-expressing cancer cells. Appl. Mat. Int. 8 (2016 8908-8917.
[16] H. Peng, Y. Liang, L. Chen, L. Fu, H. Wang, H. He, Efficient synthesis and biological
evaluation of 1,3-benzenedicarbonyldithioureas.Biorg. Med. Chem. Let. 21 (2012) 1102 – 1104.

ACCEPTED MANUSCRIPT
22
[17] Y. Benabdelouahab, L. Munoz-Moreno, M. Frik, I. Cueva-Alique, M.A. Amrani, M. Contel,
A.M. Bajo, T. Cuenca, E. Royo, Bonding and anticancer properties of water-soluble chiral p-cymene
Ru(II) compounds with amino-oxime ligands.Euro. J. Inorg. Chem. 13 (2015) 2295-2307.
[18] W. Hernandez, E. Spodine, L. Beyer, U. Schroder, R. Richter, J. Ferreira, N. Pavani, Synthesis,
characterization and antitumor activity of copper(II) complexes, [CuL2] [HL1-3 =N,N-Diethy-N’-(R-
benzoyl)thiourea (R=H, o-Cl and p-NO2].Bioinorg. Chem. App. 3 (2005) 299-316.
[19] M.F. Emen, H. Arslan, N. Kuluc, U. Florke, N. Duran, Synthesis, characterization and
antimicrobial activities of some metal complexes with N-(2-chlorobenzoyl)thiourea lignads: the
crystal sstructure of fac-[Co L3] and cis- ]PdL2]. Pol. J. Chem. 79 (2005) 1615-1626.
[20] S. Saeed, N. Rashid, M. Ali, R. Hussan, P.G. Jones, The crystal strycture of 1-(4-chlorophenyl)-
3-(4-methylbenzoyl)thiourea.Euro. J. Chem., 1 (2010) 221-227.
[21] R. del Campo, J.J. Criado, E. Garua, M.R. Hermosa, A. Jimenez-Sanchez, J.L. Manzano, E.
Monte, E. Rodriquez-Fernandez, F. Sanz, Thiourea derivatives and their nickel(II) and platinum(II)
complexes: antifungal activity. J. Inorg. Biochem. 89 (2002) 74-82.
[22] G.Y. Nagesh, K.M. Raj, B.H.M. Mruthyunjayaswamy, Synthesis, characterization, thermal
study and biological evaluation of Cu(II), Ni(II) and Zn(II) complexes of Schiff base ligand
containing thiazole moiety. J. Mol. Struct. 1079 (2015) 423-432.
[23] H. Arslan, N. Duran, G. Boreka, C.K. Ozer, C. Akbay,Antimicrobial activity of some thiourea
derivatives and their nickel and copper complexes. Molecules 14 (2009) 519-527.
[24] R. Custelcean, M.G. Gorbunova, P.V. Beonnesen, Steric control over hydrogen bonding in
crystalline organic solids: a structural study of N,N’-dialkylthioureas. Chem. Euro. J. 11 (2005)
1459-1466.
[25] R. Custelcean, Crystal engineering with urea and thiourea hydrogen-bonding groups. Chem.
Comm., 2008 (2007) 295-307.
[26] J.N. Asegbeloyin, E.E. Oyeka, O. Okpareke, A. Ibezim, Synthesis, structure, computational and
in-silico anticancer studies of N, N-diethyl-N′-palmitoylthiourea. J. Mol. Struc. 1153 (2018) 69-77.
[27] G. Binzet, N. Kulcu, U. Florke, H. Arslan, Synthesis and characterization of Cu(II) and Ni(II)
complexes of some 4-bromo-N-(dialkyl/aryl)carbamothioyl) benzamide derivatives. J. Coord. Chem.
62 (2010) 3454-3462.
[28] M. Frisch, G. Trucks, M. Schlegel, G. Scuseria, M. Robb, J. Cheeseman, G. Scalmani, V.
Barone, B. Mennucci, G. Petersson, 2009. Gaussian, Inc., Wallingford CT.
[29] J. Contreras-García, E.R. Johnson, S. Keinan, R. Chaudret, J.P. Piquemal, D.N. Beratan, W.
Yang, NCIPLOT: a program for plotting noncovalent interaction regions. J. Chem. Theo. Comput. 7
(2011) 625-632.
[30] C.H. Collins, P.M. Lyne, J.M. Grange, J.O. Falkinham, (2004) Collins and Lyne’s
microbiological methods. 8. Ed. London: Butterworths, p. 456.
[31] Clinical and Laboratory Standards Institute (2015) M02-A12: performance standards for
antimicrobial disk susceptibility tests: approved standard. 12. Ed. Wayne: CLSI, p.1-73.

ACCEPTED MANUSCRIPT
23
[32] R.N. Jones, A.L. Barry, T.L. Gavan, J.A. Washington, 1985. Susceptibility tests: microdilution
and macrodilution broth procedures. Manual of Clinical Microbiology. 4. Ed. Washington: Am. Soc.
Microbio. p. 972-977.
[33] J.H. Jorgensen, M.J. Ferraro, Antimicrobial susceptibility testing: a review of general principles
and contemporary practices.J. Med. Microbio., 49 (2009) 1749-1755.
[34] Clinical and Laboratory Standards Institute (2009) M07–A8: Methods for dilution antimicrobial
susceptibility testing for bacteria that grow aerobically: approved standard. 8. Ed. Wayne: CLSI, p.1-
65.
[35] H.M. Berman, J. Westbrook, Z. Feng, G. Gilliland, T.N. Bhat, H. Weissig, I.N. Shindyalov, P.E.
Bourne, The protein data bank, Nucleic Acids Res., 28 (2000) 235e342.
[36] F. Ntie-Kang, J.N. Nwodo, A. Ibezim, C.V. Simoben, B. Karaman, V.F. Ngwa, W. Sippl, M.U.
Adikwu, L.M. Mbaze,Molecular modeling of potential anticancer agents from African medicinal
plants. J. Chem. Inf. Model 54 (2014) 2433e2450.
[37] W.R.P. Scott, P.H. Hunenberger, I.G. Tironi, A.E. Mark, S.R. Billeter, J. Fennen, A.E. Torda, T.
Huber, P. Kruger, W.F. vanGunsteren, The GROMOS biomolecular simulation program package. J.
Phys. Chem. 103 (1999) 3596-3607.
[38] A. Saeed, M. Parvez, The crystal strycture of 1-(4-chlorophenyl)-3-(4-
methylbenzoyl)thiourea.Cent.Euro. J. Chem. 3 (2005) 780-791.
[39] H. Arslan, U. Flörke, N. Külcü,The crystal and molecular structure of 1-(biphenyl-4-carbonyl)-
3-p-tolyl-thiourea.ActaChim.Slov. 51 (2004) 787–792.
[40] G. Kavak, S. Ozbey, G. Binzet, N. Kulcu, Synthesis and single crystal structure analysis of three
novel benzoyl thiourea derivatives.Turk. J. Chem. 33 (2009) 867-868.
[41] G. Binzet, G. Kavak, N. Kulcu, S. Ozbey, V. Florke, H. Arslan, Synthesis and characterization
of novel thiourea derivatives and their nickel and copper complexes. J. Chem. 2013 (2013) 1-9.
[42] I.G. Csizmadia, Theory and practice of MO calculations on organic molecules, Elsevier.
[43] A. Otero-de-la-Roza, E.R. Johnson, J. Contreras-García, Revealing non-covalent interactions in
solids: NCI plots revisited.Phy. Chem. Chem. Phy. 14 (2012) 12165-12172.
[44] Z. Narth, R.A. Maroun, R. Boto, M.L. Chaudret, J.P. Bonnet, J. Piquemal, A. Contreras-García,
(2015) Complete NCI perspective: from new bonds to reactivity, in: R.C.C.L.B.S. Esmail Alikhani
(Ed.), Applications of Topological Methods in Molecular Chemistry, Springer.
[45] R. Chaudret, B. De Courcy, J. Contreras-Garcia, E. Gloaguen, A. Zehnacker-Rentien, M. Mons,
J.P.Piquemal,Unraveling non-covalent interactions within flexible biomolecules: from electron
density topology to gas phase spectroscopy. PCCP 16 (2014) 9876-9891.
[46] J.N. Asegbeloyin, O.T. Ujam, E.C. Okafor, I.Babahan, E.P. Coban, A. Ozman, H.
Biyik,Synthesis, characterization, and biological activity of N’-[(Z)-(3-methyl-5-oxo-1-phenyl-1,5-
dihydro-4H-pyrazol-4-ylidene)(phenyl)methyl]benzohydrazide and its Co(II), Ni(II), and Cu(II)
complexes. Bioinorg. Chem. Appl. 2014 (2014) http://dx.doi.org/10.1155/2014/718175.
[47] H. Nikaido, Prevention of drug access to bacterial targets: permeability barriers and active
efflux.Science 264 (1994) 382-388.

ACCEPTED MANUSCRIPT
24
[48] A. Ibezim, N.J. Nwodo, N. Nnaji, O.T. Ujam, O.O. Olubiyi, C.J. Mbah, In silico investigation of
morpholines as novel class of trypanosomaltriosephosphate isomerase inhibitors. Med. Chem. Res.
26 (2016) 180-189.
[49] A. Ibezim, O. Olujide, A. Olubiyi, C.J. Mbah, N.J. Nwodo, Structure-based design of natural
products as anti-schistosoma drug: virtual screening, structure activity relationship and molecular
dynamic studies.Cur. Comp.-aid. Drug Des. 13 (2017) 91-100.
[50] A. Bielenica, J. Stefanska, K. Stepien, A. Napiorkowska, E. Augustynowicz-Kopec, G. Sanna,
S. Madeddu, S. Boi, G. Giliberti, M. Wrzosek, M. Struga, Synthesis, cytotoxicity and antimicrobial
activity of thiourea derivatives incorporating 3-(trifluoromethyl)phenyl moiety.Euro. J. Med. Chem.
101 (2015) 111-125.
[51] A.N. AbdHalim, Z. Ngaini, Synthesis and bacteriostatic activities of bis(thiourea) derivatives
with variable chain length. J. Chem. 2016 (2016) doi.org/10.1155/2016/2739832.
[52] S. Shaikh, J. Fatima, S. Shakil, S. Mohd, D. Rizvi, M.A. Kamal, Antibiotic resistance and
extended spectrum beta-lactamases: types, epidemiology and treatment.Saudi J. Bio. Sci. 22 (2015)
90-101.

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Figure captions
Figure 1: Molecular structure of CBEDEA showing the atomic numbering scheme at 30% ellipsoid.
a) ORTEP diagram b) showing the zigzag conformation of CBEDEA.
Figure 2: The crystal packing of CBEDEA showing the zig-zag structure of the molecule (dashed
lines denote the intramolecular hydrogen bonding)(grey: C, red: O, yellow: S, blue : N, green: Cl. H
– atoms were omitted for clarity).
Figure 3: Electrostatic potential map and contour diagram for CBEDEA ligand
Figure 4 : 3D isosurface representation (top) and 2D (bottom) NCI plots for CBEDEA. The 3D
isosurface uses a blue-green-red colour scheme from -0.04 a.u.(blue) < sign(λ2
)ρ < +0.04 a.u (red).
Figure5: Proposed structure of Cu(II) and Ni(II) complex of CBEDEA
Figure 6: Predicted binding modes for CBEDEA and its metal complexes toward β-lactamases.
Carbon atoms are coloured grey in CBEDEA, pink in CuCBEDEA and green in NiCBEDEA while
the rest of the atoms retain in their standard colours. Yellow dash lines represent hydrogen bonding.

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a
b
Figure 1: Molecular structure of CBEDEA showing the atomic numbering scheme at 30% ellipsoid.
(a) ORTEP diagram (b) showing the zigzag conformation of CBEDEA.

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Figure 2: The crystal packing of CBEDEA showing the zig-zag structure of the molecule (dashed
lines denote the intramolecular hydrogen bonding)(grey: C, red: O, yellow: S, blue : N, green: Cl. H –
atoms were omitted for clarity).

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+0.471206
-0.0450064
Figure 3: Electrostatic potential map and contour diagram for CBEDEA ligand.