Biologically active acylthioureas and their Ni(II) and Cu(II) Complexes: Structural, spectroscopic, anti-proliferative, nucleolytic and antimicrobial studies

Article abstract of DOI:10.1016/j.ica.2021.120590

The study investigated the effects of morpholine substituent and metal complexation on in vitro anticancer and antimicrobial activities of acylthioureas using two acylthiourea molecules that differ only by the presence of morpholine oxygen; N,N-diethyl-N′-(4-chlorobenzoyl)thiourea (CBDEA) and N-morpholine-N′-(4-chlorobenzoyl)thiourea (CBMOR), and their Ni(II) and Cu(II) complexes (NiCBDEA, CuCBDEA, NiCBMOR, CuCBMOR). All compounds were synthesized and characterized by physicochemical and spectroscopic studies. CBDEA, CuCBDEA, NiCBDEA, CBMOR, and NiCBMOR were structurally elucidated by single-crystal X-ray diffraction. The metal complexes were isolated as neutral four coordinate complexes of the form, ML₂ (M: Ni(II), Cu(II), H.L.: CBDEA/CBMOR) in square-planar geometry. The compounds were screened for DNA binding/cleavage, antimicrobial activity, and anti-proliferative effects on human prostate cancer PC-3 and breast cancer MCF-7 cells. DNA binding interaction studies suggest that the metal complexes bind more strongly to the DNA compared to the ligands. The morpholine derivative CBMOR shows similar activity to CBDEA against PC-3 cell lines but twice as effective against MCF-3 cells at cell death and apoptotic levels. Anticancer activities were enhanced by complexation with Cu(II), as evident in CuCBMOR, which showed the optimal anticancer activity (IC50: 1.76 μM for MCF-7 and 1.97 μM for PC-3), comparable to known anticancer drug paclitaxel. The CuCBMOR apoptosis results show that the cancer cells die by apoptotic mechanisms (Apoptosis rate: 91.53 % in MCF-7 and 85.95 % in PC-3). In vitro screening of the compounds against seventeen bacteria and four yeast strains confirmed antimicrobial potency against more susceptible Gram-positive bacteria strains. The results of the study suggest that some of the compounds could be developed into novel antimicrobial and anticancer agents.

Full text of DOI:10.1016/j.ica.2021.120590

Inorganica Chimica Acta 528 (2021) 120590
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Inorganica Chimica Acta
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Research paper
Biologically active acylthioureas and their Ni(II) and Cu(II) Complexes:
Structural, spectroscopic, anti-proliferative, nucleolytic and
antimicrobial studies
,
Ebube E. Oyekaa b, Ilknur Babahanc, Bernard Ebomaa, Kenechukwu J. Ifeanyiezea,
,
*
d
d
e
e
f
Obinna C. Okparekea , Esin P. Coban , Ali Ozmen , Burak Coban , Mehran Aksel ,
¨
g
h
a
f
¨
Namık Ozdemir , Tatiana.V. Groutso , Jude I. Ayogu , Ufuk Yildiz , Mehmet Dinçer Bilgin ,
H. Halil Biyikd, Briana R. Schragei, Christopher J. Ziegleri, Jonnie N. Asegbeloyina
,
*
a Department of Pure and Industrial Chemistry, University of Nigeria, Nsukka, Enugu State, Nigeria
b Department of Chemistry, Clemson University, Clemson, SC 29634, USA
c Department of Chemistry, Aydin Adnan Menderes University, 09010 Aydin, Turkey
d Department of Biology, Aydin Adnan Menderes University, 09010 Aydin, Turkey
e Department of Chemistry, Zonguldak Bulent Ecevit University, 67100 Zonguldak, Turkey
f Department of Biophysics, Faculty of Medicine, Aydin Adnan Menderes University, 09010 Aydin, Turkey
g Department of Mathematics and Science Education, Faculty of Education, Ondokuz Mayıs University, 55139 Samsun, Turkey
h School of Chemical Sciences, The University of Auckland, New Zealand
i Department of Chemistry, University of Akron, Akron, OH 44325, USA
A R T I C L E I N F O
A B S T R A C T
Keywords:
The study investigated the effects of morpholine substituent and metal complexation on in vitro anticancer and
antimicrobial activities of acylthioureas using two acylthiourea molecules that differ only by the presence of
morpholine oxygen; N,N-diethyl-N′-(4-chlorobenzoyl)thiourea (CBDEA) and N-morpholine-N′-(4-chlorobenzoyl)
thiourea (CBMOR), and their Ni(II) and Cu(II) complexes (NiCBDEA, CuCBDEA, NiCBMOR, CuCBMOR). All
compounds were synthesized and characterized by physicochemical and spectroscopic studies. CBDEA, CuCB-
DEA, NiCBDEA, CBMOR, and NiCBMOR were structurally elucidated by single-crystal X-ray diffraction. The
metal complexes were isolated as neutral four coordinate complexes of the form, ML2 (M: Ni(II), Cu(II), H.L.:
CBDEA/CBMOR) in square-planar geometry. The compounds were screened for DNA binding/cleavage, anti-
microbial activity, and anti-proliferative effects on human prostate cancer PC-3 and breast cancer MCF-7 cells.
DNA binding interaction studies suggest that the metal complexes bind more strongly to the DNA compared to
the ligands. The morpholine derivative CBMOR shows similar activity to CBDEA against PC-3 cell lines but twice
as effective against MCF-3 cells at cell death and apoptotic levels. Anticancer activities were enhanced by
complexation with Cu(II), as evident in CuCBMOR, which showed the optimal anticancer activity (IC50: 1.76 µM
for MCF-7 and 1.97 µM for PC-3), comparable to known anticancer drug paclitaxel. The CuCBMOR apoptosis
results show that the cancer cells die by apoptotic mechanisms (Apoptosis rate: 91.53 % in MCF-7 and 85.95 % in
PC-3). In vitro screening of the compounds against seventeen bacteria and four yeast strains confirmed anti-
microbial potency against more susceptible Gram-positive bacteria strains. The results of the study suggest that
some of the compounds could be developed into novel antimicrobial and anticancer agents.
Anticancer
Antimicrobial
Complexes
DNA binding
Morpholine
Thiourea
X-ray diffraction
Breast cancer (MCF-7)
Prostate cancer (PC-3)
1. Introduction
is only second to lung cancer among other frequent tumors found in men
[1,2]. For decades, several platinum-based drugs have played front-line
Breast cancer is the most frequent malignant cancer among women,
and the second most common cancer worldwide, while prostate cancer
roles in the treatment of different forms of cancer; however, some lim-
itations such as adverse side effects of most anticancer drugs and low
* Corresponding authors.
E-mail addresses: obinna.okpareke@unn.edu.ng (O.C. Okpareke), niyi.asegbeloyin@unn.edu.ng (J.N. Asegbeloyin).
https://doi.org/10.1016/j.ica.2021.120590
Received 31 May 2021; Received in revised form 22 August 2021; Accepted 23 August 2021
Available online 27 August 2021
0020-1693/© 2021 Elsevier B.V. All rights reserved.

E.E. Oyeka et al.
Inorganica Chimica Acta 528 (2021) 120590
clinical efficacy still necessitate a constant search for new drugs. One
approach to a more synergistic anticancer agent is to develop metal
complexes with biologically friendly metals that can mimic the chem-
istry of Pt(II), such as in the formation of square-planar geometry and
soft Lewis acidity [3,4]. Cu(II) and Ni(II) fit well into this category
because of their vast enzymatic activities, tendency to coordinate in
square-planar geometry, and ability to form stable compounds with li-
gands containing hard O- and N- donor atoms and soft S- donor atom
such as thioureas, and acylthioureas [5,6].
water was used to prepare the buffers. The stock solution of CT-DNA
was stored at 4 ◦C and used within 4 days.
2.2. General procedure for the synthesis of ligands
Synthesis of the ligands was done by using similar literature pro-
cedures [22,23]. A solution of 4-chlorobenzoylchloride (0.02 mol) in
anhydrous acetone (40 mL) was added dropwise to a suspension of
potassium thiocyanate (0.02 mol) in anhydrous acetone (30 mL), and
the reaction mixture was heated under reflux for 30 min and then cooled
to room temperature. A solution of diethylamine (0.01 mol) or mor-
pholine (0.01 mol) in anhydrous acetone (30 mL) was added, and the
resulting mixture was stirred for 2 h at room temperature. The reaction
mixture was filtered to remove suspended inorganic solids. The filtrate
was left for few days, and colorless solids of N,N-diethyl-N′-4-chlor-
obenzoylthiourea (CBDEA), or N-morpholine-N′-4-chlorobenzoylth-
iourea (CBMOR) were obtained (Scheme 1). The solid products were
then washed with water and ethanol and dried in the air. Crystals suit-
able for X-ray crystallographic studies were obtained by slow evapora-
tion of a solution of the compounds in a methanol/dichloromethane
(1:1) mixture at room temperature for 7 days.
Literature survey has revealed that thioureas are an excellent pool of
bioactive moieties, with activities such as anticancer [7,8], antimicro-
bial [9,10], analgesic [11], antituberculosis [12], and anti-HIV [13].
Compared to thioureas, acylthioureas have increased coordination
possibility because of the presence of the carbonyl oxygen. The blend
and orientation of the hard O- and soft S- donor atoms in the acylth-
iourea backbone enable them to bond readily to Ni(II) and Cu(II) ions in
a square-planar fashion. Several stable metal complexes of acylthioureas
with interesting physicochemical properties and significant biological
activity have been reported [10-14]. Among the acylthiourea com-
pounds, benzoylthioureas have been well studied as bioactive agents
[14,15]. However, the effect of bioactive substituents has only been
mildly explored. To bridge this gap, we investigate the effect of mor-
pholine in the biological potency of benzoylthioureas and their Ni(II)
and Cu(II) complexes. Morpholine is a known bioactive substituent with
activity including antimicrobial, HIV-protease inhibition, antitubercu-
losis, antitumor, and human neurokinin-1 (hNK-1) receptor antagonism
[7,16,17]. The addition of the morpholine group to ibuprofen and
indomethacin has been reported to lead to increased selective COX-2
inhibitory activity [18]. Recently, ongoing research on morpholine-
directed targeted therapy was reported [19]. This has further aroused
our interest in investigating morpholine-based anticancer drugs. N,N-
diethyl-N′-(4-chlorobenzoyl)thiourea (CBDEA) and its morpholine
analogue N-morpholine-N′-(4-chlorobenzoyl)thiourea (CBMOR), and
their Ni (II) and Cu(II) were synthesized for a comparative anticancer
and antimicrobial study. In contrast with previous reports on the spec-
troscopic and biological studies of CBDEA and its Pt(II) complex [20],
we present the crystal structures of CBDEA, CBMOR, NiCBDEA, CuCB-
DEA, and NiCBMOR. All the compounds were screened for DNA cleav-
age/binding and their antimicrobial potency against seventeen bacteria
and four fungi strains. The anti-proliferative effects of these compounds
on human prostate cancer PC-3 cells and breast cancer MCF-7 cells were
also investigated.
2.2.1. N,N-diethyl-N′-4-chlorobenzoylthiourea (CBDEA)
Color: colorless; m.p: 156 – 158 ◦C, yield: 89 %; C12H15ClN2OS
Calculated %: C 53.23, H 5.58, N 10.35 ; Found %: C 53.77, H 5.12, N
10.11; 1H NMR (300 MHz, DMSO D6) δ: 10.62 (s, 1H, NH), 7.82 – 7.90
(m, 2H, Ar-H), 7.46 – 7.54 (m, 2H, Ar-H), 3.83 – 3.93 (q, 2H, CH2-CH3),
3.44 – 3.50 (q, 2H, -CH2-CH3), 1.20 (t, 3H, CH3-CH2-, J 7.53, 2.26 Hz),
1.14 (t, 3H, CH3-CH2-, J 7.53, 3.81 Hz); 13C NMR (300 MHz, CDCl3) δ:
–
–
–
179.06 (C O), 162.89 (C S), 139.27(C-Cl), 131.00 (Cpara–Ph), 129.31,
–
129.07 (Cmeta–Ph), 128.86, 128.77 (Cortho–Ph), 47.93, 47.68 (CH2),
11.46, 13.28 (CH3); IR (KBr): ʋ(cmꢀ 1): 3271 (st, ʋN-H), 1642 (st, ʋC =
–
O), 1228 (ʋC = S). UV: 281 nm (C S, n
π
*).
–
2.2.2. N-morpholine-N′-4-chlorobenzoylthiourea (CBMOR)
Color: colorless; m.p: 146 – 148 ◦C, yield: 91 %, C12H13ClN2O2S
Calculated %: C 50.61, H 4.60, N 9.84; Found %: C 51.02, H 4.61, N
9.80; 1H NMR (300 MHz, DMSO D6): δ = 10.92 (s, 1H, NH), 7.92 – 7.95
(d, 2H, Ar-H), 7.56 – 7.59 (d, 2H, Ar-H), 4.15 (t, 2H, CH2-CH2), 3.57 –
3.71 (t, 6H, -CH2-CH2); 13C NMR (300 MHz, CDCl ) δ: 178.91 (C O),
–
–
162.31 (C S), 139.64 (C-Cl), 130.57 (Cpara–Ph),3 129.26 (Cmeta–Ph),
–
–
129.23 (Cortho–Ph), 52.47, 51.60 (CH2); IR (KBr): ʋ(cmꢀ 1): 3258 (st, ʋN-
–
H), 1686 (st, ʋC = O), 1246 (ʋC = S).; UV: 275 nm (C S, n→
π
*).
–
2. Experimental methods
2.3. General procedure for the synthesis of metal complexes
2.1. Chemicals and instrumentation
A solution of Ni(CH3COO)2⋅4H2O or Cu(CH3COO)2⋅4H2O (1 mmol)
in ethanol (20 mL) was added dropwise to a solution of CBDEA or
CBMOR (2 mmol) in dichloromethane in a 1:2 ratio at room tempera-
ture. The resulting mixture was refluxed for 30 min at room tempera-
ture, and the solid obtained was filtered (Schemes 2a-b). NiCBDEA and
CuCBDEA crystals suitable for X-ray crystallography were obtained by
slow evaporation from a 1:1 methanol-dichloromethane solution of the
complexes at room temperature for 48 h. NiCBMOR crystals were ob-
tained by slow diffusion of ether into dichloromethane solution of the
metal complex for a week.
4-Chlorobenzoylchloride, morpholine, diethylamine, acetone,
methanol, dichloromethane, and potassium thiocyanate were obtained
from Sigma Aldrich and used without further purification. The melting
points were determined with a Fisher John melting point apparatus. The
infrared spectra were recorded in the range of 4000 – 400 cmꢀ 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. Elemental analyses of C, H, and N, were
performed using a Carlo Erba Elemental analyzer EA1108. The con-
ductivity of the complexes was checked using a WTW LF90 conductivity
meter. UV–Vis spectra of the synthesized compounds were acquired
using a UV-2500PC Series model spectrophotometer. U.V–Vis spectra for
DNA titrations were recorded at room temperature on a Cary 100 Bio U.
V-Vis spectrophotometer. Solutions of calf thymus DNA (CT-DNA; Sigma
D1501) in 100 mM KCl, 10 mM Tris-HCl, and pH-7.5 buffer had a U.V.–
Vis absorbance ratio of 1.8 – 1.9: 1 at 260 and 280 nm (A260/A280 = 1.9),
indicating that the DNA was sufficiently free of protein [21]. The con-
centration of DNA was determined spectrophotometrically using a
molar absorptivity of 6600 Mꢀ 1 cmꢀ 1 (260 nm) [21]. Double-distilled
2.3.1. bis(N,N-diethyl-N′-4-chlorobenzoylthioureato)nickel(II)
(NiCBDEA)
Colour: red crystals; m.p: 222 ◦C, yield: 75 %; C24H28Cl2N4NiO2S2;
Calculated %: C 48.18, H 4.72, N 9.37; Found %: C 47.98, H 4.72, N
9.41; 1H NMR (300 MHz, DMSO D6): 8.50 (d, 2H, Ar-H), 8.06 (d, 2H, Ar-
H), 4.89 (q, 2H, CH2-CH3), 4.39 (q, 2H, -CH2-CH3), 1.32–1.40 (t, 6H,
13
–
CH3-CH2-); C NMR (300 MHz, CDCl ): δ = 178.92, 172.50 (C O),
–
3
–
–
171.29, 162.80 (C S), 139.33, 137.61 (C-Cl), 135.29, 131.01 (C
par-
a–Ph), 130.44, 129.20 (Cmeta–Ph), 129.15, 128.14 (Cortho–Ph), 48.06,
2

E.E. Oyeka et al.
Inorganica Chimica Acta 528 (2021) 120590
Scheme 1. Synthesis of CBDEA and CBMOR.
Scheme 2a. Synthesis of Ni(II) complexes of CBDEA (R = -CH2CH3) and CBMOR (R2 = O(CH2CH2)2).
Scheme 2b. Synthesis of Cu(II) complexes of CBDEA (R = -CH2CH3) and CBMOR (R2 = O(CH2CH2)2).
47.74, 46.23, 45.60 (CH2), 13.25, 13.12, 12.48, 11.49 (CH3); IR (KBr) ʋ
4.62, N 9.31; IR (KBr) ʋ(cmꢀ 1): 1575 (st, ʋC = O), 1250 (ʋC = S); UV:
(cmꢀ 1): 1590 (st, ʋC = O), 1251 (ʋC = S); UV: 274 nm (C S, n→
π
*),
274 nm (C S, n→
π
*), 631 nm (2B1g→2B2g).
–
–
–
–
509 nm (1A1g→1A2u).
2.3.3. bis(N-morpholine-N’-4-chlorobenzoylthioureato)nickel(II)
(NiCBMOR)
2.3.2. bis(N,N-diethyl-N′-4-chlorobenzoylthioureato)copper(II)
(CuCBDEA)
Color: reddish brown; m.p: 270 ◦C (decomposed), yield: 74 %,
Colour: light green crystals; m.p: 176 ◦C, yield: 61 %, C24H28Cl2Cu-
N4O2S2; Calculated %: C 47.80, H 4.68, N 9.29; Found %: C 47.92, H
C24H24Cl2N4NiO4S2; Calculated %: C 46.03, H 3.86, N 8.95; Found %: C
45.97, H 3.93, N 8.96; 1H NMR (300 MHz, DMSO D6): δ = 7.46 (d, 2H,
3

E.E. Oyeka et al.
Inorganica Chimica Acta 528 (2021) 120590
Scheme 3. Proposed delocalization pattern for N,N-diethyl-N’-(4-chlorobenzoyl)thiourea.
Ar-H), 7.40 (d, 2H, Ar-H), 4.36 (t, 2H, CH2-CH2), 3.29–3.69 (t, 6H, –CH2-
idealized positions. Molecular graphics were created by using OLEX2
[28]. The crystallographic data and refinement parameters are sum-
marized in Table 1.
CH2); 13C NMR (100 MHz, DMSO D ) δ: 174.50 (C N), 171.20 (C O),
–
–
–
–
6
–
162.80 (C S), 139.33 (C-Cl), 133.26 (Cpara–Ph), 128.14 (Cmeta–Ph),
–
125.14 (Cortho–Ph), 68.06 (-CH2-CH2- morpholine), 47.74 (-CH2-CH2-
morpholine), IR (KBr): ʋ(cmꢀ 1): 1591 (st, ʋC = O), 1290 (ʋC = S). UV:
2.6. DNA binding studies
–
276 nm (C S, n→π*).
–
2.6.1. UV titrations
2.4. bis(N-morpholine-N′-4-chlorobenzoylthioureato)copper(II)
(CuCBMOR)
The study compounds (about 1 mmol) were dissolved in DMSO (0.5
mL) and diluted in a 100 mM KCl, 10 mM Tris-HCl, and pH-7.5 buffer
solution to the absorption titrations a final concentration of 20 μM [31].
◦
Color: green; m.p: 256 – 258 C, yield: 72 %, C24H24Cl2CuN4O4S2;
Titrations were performed in a 10-mm stoppered quartz cell using a
mixed concentration of each compound. The CT-DNA stock solution was
Calculated %: C 45.68, H 3.83, N 8.88; Found %: C 45.66, H 3.87, N
8.85; IR (KBr): ʋ(cmꢀ 1) 1593 (st, ʋC = O), 1270 (ʋC = S), UV: 274 nm
added in increments of 2 μL, creating 4 μM additions, till no change in
–
(C S, n→
π
*), 633 nm (2B1g→2B2g).
absorption spectrum was observed. Analyses were performed by means
of a UV–Vis spectrophotometer by recording the spectrum after each
addition of DNA. Ligand or complex-DNA solutions were mixed by using
a Pasteur pipette and were incubated for 10 min each time before the
spectra were recorded. Cell compartments were thermostated at 25 ±
0.1 ◦C. The stabilities of the compounds in titration conditions were
tested by measuring the change in absorption for 4 h at 30 min intervals.
–
2.5. X-ray crystallographic analysis
X-ray intensity data of CBMOR and CBDEA were measured at the
University of Akron on a Bruker CCD-based diffractometer with dual Cu/
Mo ImuSmicrofocus optics (Mo Kα radiation, λ = 0.71073 Å). Crystals
were mounted on a cryoloop using Paratone oil and placed under a
stream of nitrogen at 100 K (Oxford Cryosystems). The detector was
placed at 5.00 cm from the crystal. The data were corrected for ab-
sorption with the SADABS program [24]. The structures were refined
using the Bruker SHELXTL Software Package (Version 6.1). NiCBDEA
and CuCBDEA diffraction data were collected at 105.9(4) K on an Oxford
Rigaku four-circle diffractometer equipped with PhotonJet (Cu, λ =
1.54184 Å) X-ray source. Data integration, scaling, and empirical ab-
sorption correction were carried out using the CrysAlis-Pro program
package [25]. The structures were solved with the intrinsic phasing
method in ShelXT [26] and refined by Matrix-least-square against F2 on
ShelXL [27]. The non-hydrogen atoms were refined anisotropically, and
hydrogen atoms were placed at idealized positions and refined using the
riding model. The crystal structure of CuCBDEA had a disorder at the N
(1)-C(1)-N(2) atoms and the adjourning ethyl groups; these were
modeled in two positions with half occupancies on the atoms, with
constraints on the bonds to stabilize the molecule. All calculations were
implemented in OLEX2 program package [28]. X-ray diffraction data of
NiCBMOR were recorded with a STOE IPDS II diffractometer at room
2.6.2. Gel Electrophoresis:
Gel electrophoresis experiments were performed according to the
reported procedure [31], by using pBR322 negatively supercoiled
plasmid DNA on 1 % agarose gels in 100 mM KCl, 10 mM Tris-HCl, and
pH-7.5 buffer solution. Reaction mixtures (10 μL) containing 20 ng of
pBR322 together with different concentrations of the study compounds
(10, 25, 50, 100, 200 µM) were prepared in the same buffer at 0 ◦C, and
then allowed to incubate at 25 ◦C for 1 h in the dark, and then in the
presence of a peroxide solution. Before loading samples onto the gel, 2.5
mL of 0.25 % bromophenol blue loading buffer and sucrose in water (40
% w/v) were added to the reaction mixtures. Gels were obtained at room
temperature by using a Thermo midi horizontal agarose gel electro-
phoresis system and applying a potential of 35 V for 4 h. The resulting
gels were stained in ethidium bromide solution (0.5 mg mLꢀ 1) for 45
min, after which they were further soaked in water for 20 min. Gels were
visualized under U.V. light and photographed.
2.7. Antimicrobial assay
temperature using graphite-monochromated Mo K
α radiation by
applying the -scan method. Data collection and cell refinement were
ω
The biological activities of all the synthesized compounds against
bacteria and fungi were determined by the disc diffusion method [32-
35], and the minimum inhibitory concentrations (MIC) were obtained
by the broth dilution method [36,37]. Seventeen bacteria and four fungi
were tested. To test the antimicrobial activity of all the synthesized
compounds, a Mueller Hinton Agar (MHA) plate was inoculated with a
carried out using X-AREA [29], while data reduction was applied using
X-RED32[29]. The structure was solved by direct methods with SIR2019
[30], and refined by means of the full-matrix least-squares calculations
on F2 using SHELXL-2018[27]. All H atoms were located in a difference
electron-density map and then treated as riding atoms in geometrically
Table 1
Some physical parameters of the compounds.
Compounds
Molecular formula
Color
% yield
Melting point (◦C)
Molar conductivity (ohmꢀ 1molꢀ 1cmꢀ 2
)
CBDEA
C
C
C
C
C
C
12H15ClN2OS
Colorless
Red
89
75
61
90
74
72
156 – 158
222
–
NiCBDEA
CuCBDEA
CBMOR
24H28Cl2N4NiO2S2
24H28Cl2CuN4O2S2
12H13ClN2O2S
1 ± 1
3 ± 1
–
Light green
Colorless
Reddish brown
Green
176
146 – 148
270 (decomposed)
256 – 258
NiCBMOR
CuCBMOR
24H24Cl2N4NiO4S2
24H24Cl2CuN4O4S2
1 ± 1
2 ± 1
4

E.E. Oyeka et al.
Inorganica Chimica Acta 528 (2021) 120590
0.1 mL broth culture of bacteria or yeasts. Detailed procedure of the
antimicrobial assay has been reported elsewhere [32].
3.2. Structural description of CBDEA, CBMOR, NiCBDEA, CuCBDEA,
and NiCBMOR
2.8. Anti-proliferative studies
The crystal structure refinement parameters of CBMOR, NiCBMOR,
CBDEA, NiCBDEA, and CuCBDEA are presented in Table S1 of the sup-
plementary information. Selected bond lengths and angles in the
structures are shown in Tables S2 and S3. The molecular structure and
atom numbering scheme of CBDEA is presented in Fig. 1. CBDEA crys-
tallized in a monoclinic P21/c space group with two molecules per unit
cell. The molecule of CBDEA is non-planar with the N,N-diethyl moiety
twisted out of the plane with the aromatic group. The conformation of
the CBDEA molecule with respect to acylthiourea group was twisted as
reflected by the C7-N1-C8-S1 and C1-C7-N1-C8 dihedral angles of 94.1◦
and ꢀ 177.2◦, respectively. These twist angles are quite low compared to
the 3-chlorobenzoylthiourea derivatives previously reported [43]. The
dihedral angle of CBDEA also reveals that the thiourea moiety is almost
perpendicular to the chlorobenzoyl group. The twisting of conformation
is a result of steric repulsion between the carbonyl C7-O1 group and the
thiocarbonyl C8-S1 group, which was also confirmed by the S1-C8-N2
bond angle of 126.6(2)◦. In CBDEA, the bond lengths of carbonyl (C7-
O1) and thiocarbonyl (C8-S1) are 1.233(3) Å and 1.656(3) Å respec-
tively, which are indicative of double bond character [22,43-45]. The
C8-N2 and C7-N1 bond lengths of 1.327(3) and 1.355(3) Å respectively
2.8.1. Cell culture
Two lines of human cancer cells were used in these experiments.
Human prostate cancer PC-3 cells and human breast cancer MCF-7 cells
were grown in RPMI-1640 and DMEM, respectively, and supplemented
with 10 % FBS, 1 % L-glutamine, and 100 U/mL penicillin and strepto-
mycin (Sigma). Cells were maintained in a humidified atmosphere with
5 % CO2 air at 37 ◦C and incubated overnight. Cells in the exponential
phase of growth were used in each experiment. The cells were seeded in
48 or 96 well plates with 105 cell densities. Apoptosis measurement was
maintained in 24 wells and was carried out at a cell density of 106 cells
per mL. After 24 h, different concentrations of study compounds (5, 10,
20, 40 μM) and 1 μM paclitaxel (Pax) were added to the well plates, and
measurements were performed on the cells [38-40].
2.8.2. Cell survival assay
The toxicity of the compounds was evaluated by using 3-(4,5-dime-
thylthiazol-2-yl)-2,5-diphenyltetrazolium bromide tetrazolium (MTT)
reduction assay on both cancer cells. For this, 5 mg/mL MTT dye was
dissolved in PBS, and 40 µL was added to each well. After 4 h of incu-
bation, formazan crystals were dissolved in DMSO and measured at 540
nm and 620 nm spectrophotometrically [38-40].
–
are shorter compared to the average C N single bond length of 1.48 Å.
–
This shows that the acylthiourea C N single bonds possess partial
–
–
–
–
double bond character, perhaps due to the conjugation of C O and C
S
π
-electrons with lone pairs of electrons on nitrogen (Scheme 3)
2.8.3. HOPI staining
[44,46–48]. The elongation of the C8-N1 (1.431(3) Å) bond relative to
C8-N2 (1.327(3) Å ) and C7-N1 (1.355(3) Å) bonds agrees with litera-
ture reports [46,47]. Previous reports on chlorobenzoyl thiourea de-
Apoptosis was assessed by Hoechst and propidium iodide (HOPI)
staining. The cells were seeded in a 24-well plate at a density of 106
cells/well. The day after, the cells were treated with the studied com-
pounds. After treatments for 24 h, cells were washed with PBS, trypsi-
–
–
–
rivatives show that the longer the C S bond, the shorter the C O bond
–
[43,44,49]. This serves as means to compensate for the repulsion be-
nized, and centrifuged. Pellets were suspended in 50 μL fresh medium
tween the two polar groups.
with addition of 0.25
μ
L of Hoechst and 0.25
μL of propidium iodide and
The molecular structure of CBMOR shown in Fig. 2 is similar to the
structure of CBDEA. The conformation of the CBMOR molecule with
respect to the thiourea moiety is non-planar, as reflected by the C5-N1-
C6-N2 torsion angle of 62.0(2)◦. The carbonyl oxygen is twisted away
from the chlorophenyl plane, as seen from the C1-C4-C5-O2 torsion
angle of ꢀ 151.0◦. The morpholine ring exhibits a chair conformation
while the chlorophenyl ring is almost planar. The carbonyl C5-O2 (1.217
(2) Å) and thiocarbonyl C6-S1 (1.671(2) Å) bond lengths in CBMOR
show a typical double bond character similar to the CBDEA structure
incubated for 30 min at 37 ◦C in the dark. The cells were immediately
analyzed by fluorescence microscopy, and images were taken by Zeiss
axiocam ICc5 camera [38–40].
2.8.4. Statistical analyses
Statistical analysis was determined by using GraphPad Prism
(GraphPad Software, Inc., La Jolla, CA, USA). Cytotoxicity values were
expressed as mean ± SD of at least four independent experiments, the
differences among the groups were analyzed by one-way analysis of
variance (one-way ANOVA), and HOPI data were analyzed by two-way
analysis of variance (two-way ANOVA). p < 0.01 and p < 0.05 were
considered to be significant.
–
[10,45,50]. The C N bonds C5-N1 (1.392(2) Å), C6-N1 (1.409(2) Å),
–
N
and C6-N2 (1.326(2) Å) are all shorter than the average single C
bond lengths of 1.48 Å, thus showing varying degrees of partial double
bond character [50–52]. The C6-N2 bond in the vicinity of the thio-
–
carbonyl has a much shorter bond length than other C N bonds in the
3. Results and discussion
molecule [47,50]. This shows the contribution of the thiol tautomeric
form caused by the conjugation of the nitrogen lone pairs with the
π
–
3.1. Physical properties of the synthesized compounds
electrons of the C S. The bond lengths and angles in Table S2 are
–
similar to those in chlorobenzoylthiourea derivatives reported in the
literature [10,50,51]. Molecular aggregation in CBDEA (Fig. S1) and
CBMOR (Fig. S2) show intermolecular hydrogen bonding between the
The acylthioureas and metal complexes were isolated by recrystal-
lization using suitable solvents. The acylthioureas were soluble in warm
organic solvents such as ethanol, methanol, and acetone, but highly
soluble in dichloromethane, DMF, and DMSO. The metal complexes
were colored non-hygroscopic and stable solids. They were slightly
soluble in ethanol, methanol, and acetone. The nickel complexes are
soluble in dichloromethane, DMSO, and DMF, while copper complexes
are soluble in these solvents when hot. The low molar conductivity
values of the metal complexes indicate that they are non-electrolytes
[34,41,42]. Some physical parameters of the synthesized compounds
are shown in Table 1.
–
thiourea N H donor group and O acceptor of the morpholine [53].
The molecular structure of NiCBDEA is shown in Fig. 3, while
selected bond lengths and angles are presented in Table S3. The NiCB-
DEA molecule crystallized with two independent molecules in the
asymmetric unit. The crystal structure of NiCBDEA consists of a Ni(II)
center coordinated to two anionic chlorobenzoyl thiourea moieties
through S and O atoms in a distorted S2O2Ni square planar geometry
[43,47]. The 6-membered metallacyclic ring formed on each side of the
square plane consists of two carbon atoms, anionic S and O atoms, and
deprotonated imine nitrogen. The S2O2Ni square plane is planar to a root
mean square deviation of ~ 0.01 Å and ~ 0.023 Å in the two structures.
The cis angles subtended at the Ni center for the two independent
structures are slightly different. For example, the S1-Ni1-O1 angles in
5

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Inorganica Chimica Acta 528 (2021) 120590
Fig. 1. Molecular structure of CBDEA showing the atomic numbering scheme at 30 % ellipsoids.
Fig. 2. Molecular Structure of CBMOR, showing the atomic numbering scheme at 30 % ellipsoids.
structures are 93.84(5)◦ and 94.46(5)◦. A similar trend was observed
with the O-Ni-S trans angles ranging from 176.53(5)◦ to 179.23(6)◦. The
magnitude of the trans angles in the structures is an indication that the
coordination environment around the Ni atom is essentially planar. The
CuCBDEA is similar to that in NiCBDEA discussed earlier, with two
CBDEA molecules coordinated to Cu(II) through O and S atoms in a
slightly distorted square-planar geometry. However, unlike in NiCBDEA,
the O and S coordinated to the Cu(II) ion in CuCBDEA in a trans fashion,
a unique geometry in acylthiourea based metal complexes as they often
adopt the cis conformations [43,56,57]. In any case, the synthesis of cis
and trans diethylamine acylthiourea derivatives using different Cu salts
has been reported [43,56-58]. The mechanism of cis–trans isomerization
observed in this group of compounds is not clearly extablished yet,
however a number of factors including electronic properties of the
ligand and solvent polarity have been fingered as the driving forces for
cis–trans isomerization in alkyl-substuted thiourea complexes [59,60].
The O-Cu-S cis angles of 86.30(9)◦ and 93.70(9)◦ are indicative of the
square planar configuration of the complex. The S1-Cu1-S1 and O1-Cu1-
O1 bond angles of approximately 180◦ show the symmetrical nature of
the structure with an inversion center at the Cu atom. The difference in
the Cu–O and Cu-S bond lengths of 1.912(3) Å and 2.2529(10) Å in the
present complex is due to the difference in trans influence of the O and S
donor atoms on the metal d-orbital. These Mꢀ S/O bond lengths in
CuCBDEA are conspicuously longer than the corresponding bonds in
NiCBDEA probably due the occupation of the d(x2-y2) orbital in the Cu
and subsequent Jahn teller distortion. Similar trends have been reported
for related acylthiourea copper(II) complexes [43,56,58].
–
–
C
C
N bonds in the structures are slightly shortened, while the C S and
–
O bonds are elongated when compared to the corresponding bonds
–
N
in the structure of the thiourea ligand CBDEA. For instance, the C
bond length was shortened from 1.431(3) Å in the ligand to 1.340(3) Å
–
–
in the complex. The C O and C S bond lengths in CBDEA are 1.233(3)
Å and 1.656(3) Å, respectively, while the corresponding bonds in
NiCBDEA are 1.272(2) Å and 1.7316(19) Å, respectively. This is an
indication of the delocalization of
π electrons in the six-membered
metallacyclic ring system on coordination [43,54,55].
The crystal structure of CuCBDEA is presented in Fig. 4. The com-
pound crystallized in the monoclinic crystal system P21/n space group
with one-half of the molecule in the asymmetric unit. The symmetry
elements in the molecule include an inversion center on the Cu metal
center. The crystal structure has a disorder at the metallacyclic N1A-CIA
bond, including the adjacent diethylamine moiety, and this was
modeled in two positions with half occupancies for the atoms involved.
A few RIGU constraints were put in place to stabilize the refinement.
Information relating to the geometric parameters, coordination, geom-
etry, and the symmetry of the metal complex was successfully obtained
from one half of the compound. The coordination geometry of the
The molecular structure of NiCBMOR is in Fig. 5, is similar to
6

E.E. Oyeka et al.
Inorganica Chimica Acta 528 (2021) 120590
angle between the planes formed by Ni1/O1/S1 and Ni1/O3/S2 atoms is
11.37(9)◦. In the square-planar coordination, the intraligand S─Ni─O
angles averaging 94.69(6)◦ are considerably larger than the interligand
S─Ni─S and O─Ni─O angles of 86.17(3)◦ and 86.64(8)◦, respectively.
The cis angles varying from 86.17(3)◦ to 94.75(6)◦ and the trans angles
ranging from 171.09(8)◦ to 172.27(7)◦ deviate significantly from their
ideal values of 90◦ and 180◦. These are similar to values found in
NiCBDEA. There is no significant difference between the Ni1─S1 and
Ni1─S2 bond distances of 2.1430(7) and 2.1516(8) Å, respectively. The
Ni1─O1 and Ni1─O3 bond distances of 1.8739(19) and 1.8755(17) Å,
respectively, are equal within the experimental errors. Both Ni─S and
Ni─O lengths are comparable with those in NiCBDEA and other square-
planar NiO2S2 complexes in the literature [61-67]. The C8─S1 and
C20─S2 bond distances of 1.739(3) and 1.738(3) Å, respectively, are
consistent with the single bond character, demonstrating that the li-
gands are bound to nickel in thiolate form. The ligand is monoanionic
and coordinates in its keto form, which can be verified from the C7─O1
and C19─O3 bond lengths of 1.278(3) and 1.275(3) Å, respectively. The
chelate ring bond distances demonstrate the delocalization of
trons. The packing structure of NiCBMOR is shown in Fig. S4.
π-elec-
3.3. Spectroscopic studies
The characteristic I.R. bands, 1H NMR and U.V.-vis are presented in
the experimental section, and they confirm the structures of the com-
Fig. 3. Molecular structure of NiCBDEA showing the atomic numbering scheme
at 30 % ellipsoids. Some phenyl ring atom numbering and one of the two in-
dependent structures are omitted for clarity.
–
pounds. In the I.R. spectra of the ligands, the characteristic N
H
stretching vibration peak was observed at 3271 cmꢀ 1 (CBDEA), 3258
cmꢀ 1(CBMOR) [44]. The N H stretching peak disappeared upon
–
NiCBDEA where CBMOR ligands are coordinated to the central Ni(II) ion
in a monometallic biconnected pattern through two chalcogen atoms,
resulting in a bicyclic system with a cis-square-planar NiO2S2 core. The
complexation. This deprotonation led to the delocalization of the
π
electrons on the OC-N-CS system. This is consistent with literature re-
ports [16,44] and supports the formation of the metal complexes.
Fig. 4. Molecular structure of CuCBDEA showing the atomic numbering scheme at 30 % ellipsoids.
7

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Inorganica Chimica Acta 528 (2021) 120590
Fig. 5. Molecular structure of NiCBMOR showing the atom numbering scheme. Displacement ellipsoids are drawn at the 30 % probability level.
CBDEA and CBMOR showed single peaks at 1642 cmꢀ 1 and 1686 cmꢀ 1,
V. absorbances, which is an indication of DNA interactions. First, the
stability of the compounds in aqueous was studied by using U.V. analysis
against the time frame before the DNA titrations. All compounds, except
NiCBMOR were stable at the interaction conditions for at least 4 h,
giving no change in absorption spectra. The DNA titrations were
monitored by using U.V. spectrophotometry and the results of the li-
gands and the complexes are shown in Fig. 6. CBDEA and CBMOR had U.
V. absorptions at 274 nm and 279 nm, respectively. When DNA was
incrementally added to the ligands the absorption increased, indicating
external interaction with DNA. The acylthiourea functions of ligands
may be protonated under the titration conditions and interact with
negatively charged phosphate groups of DNA. On the other hand,
CuCBDEA, CuCBMOR, and NiCBDEA complexes have an additional
LMCT absorption band around 330 nm which instantly decreased (hy-
pochromic effect) on interaction with the DNA. This is suggestive of
stronger interaction between the metal complexes and DNA. The hy-
pochromic effect without shift at the wavelength generally indicates a
DNA groove binding type of interaction of the compounds [68].
–
respectively which were due to C O stretching vibration [44,45,53].
–
These peaks were observed at lower frequencies in the metal complexes
(CuCBDEA, 1575 cmꢀ 1; NiCBDEA, 1590 cmꢀ 1; CuCBMOR, 1593 cmꢀ 1
;
NiCBMOR, 1591 cmꢀ 1), suggesting coordination through the oxygen
atom [14,44]. The 1H NMR spectra of CBDEA and CBMOR are consistent
with structural information provided by the single-crystal X-ray
–
diffraction studies. The characteristic N H proton signal appeared at
10.62 ppm (CBDEA) and 10.82 ppm (CBMOR). The aromatic protons of
CBDEA were observed in the range of 7.54–7.90 ppm while that of
CBMOR were observed from 7.56 ꢀ 7.93 ppm. The aliphatic proton
signals and CBDEA were found in the upfield region of the spectra in the
range of 1.14–3.93 ppm. The triplet peaks at 1.20 and 1.14 ppm were
assigned to –CH3 protons, while the quartet peaks between 2.44 and
3.93 ppm were assigned to –CH2-N of the diethyl group. In the 1H NMR
spectra of CBMOR, the morpholine ring protons were observed as trip-
lets at 3.71–3.57 ppm. The 13C NMR spectra of CBDEA and CBMOR are
also consistent with structural information provided by the single-
–
crystal X-ray diffraction studies. The C O groups were observed
To quantitatively compare the DNA-binding affinities of the ligand
and the complexes, the intrinsic binding constants Kb of the complexes
–
–
around 179–178 ppm, and the peaks for C S and C-Cl are seen around
–
162–139 ppm. The values of elemental analysis of compounds shown in
the experimental section, are in agreement with their molecular for-
mula. The U.V./vis spectra of CBDEA, CBMOR and metal complexes
were recorded in ethanol/DMSO. The absorption band were observed in
to DNA were obtained by monitoring the changes of the π*←n and LMCT
absorbance at around 300 nm for ligands and metal complexes,
respectively according to the equation below.
[DNA]
[DNA]
1
the range at 274–281 nm were due to the
π
*←n electronic transition of
=
+
εA
ꢀ εf εB ꢀ εf Kb(εB ꢀ εf )
–
the C S bond [22]. Bands assignable to d←d transitions were also
–
observed at 509 nm for NiCBDEA and 633 nm for CuCBMOR.
where [DNA] is the concentration of the nucleic acid in base pairs, εA is
the apparent absorption coefficient obtained by calculating Aobs/[Drug],
and εf and εb are the absorption coefficients for the free and the fully
3.4. DNA binding studies
bound compounds, respectively. In the [DNA]/(εA ꢀ εf ) versus [DNA]
plot, Kb is given by the ratio of the slope to the intercept [68]. The li-
gands have very low affinity while the complexes have moderate af-
finities to DNA (Table 2) comparable to other complexes in the literature
[69-71].
3.4.1. UV titrations of the ligand and the complexes
The DNA interactions were initially monitored by U.V. spectropho-
tometry by using the most common and valid methods [68]. The addi-
tion of DNA to the solutions of the compounds caused changes in the U.
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Inorganica Chimica Acta 528 (2021) 120590
Fig. 6. Absorption spectra of the ligands and complexes in 5-mM Tris-HCl buffer (pH 7.5) upon the addition of CT-DNA. (a) CBDEA, (b) CBMOR, (c) CuCBDEA, and
(d) CuCBMOR. NiCBDEA is presented in Fig. S17 of the Supporting Information. The concentration of the ligand and complexes = 20 μM; [DNA] = 0–5 μM. Arrow
shows the absorbance changing upon the increase of DNA concentration.
the DNA function such as transcription and/or replication. This will in-
turn induce cell death [7,77,79]. However, the nature of reactive in-
termediates involved in the DNA-cleavage by the complexes is not clear
yet. Further studies on the mechanism are currently underway.
Table 2
Binding constants for the ligand and the metal complexes.
Compounds
Binding constants (Kb) 10ꢀ 5 Mꢀ 1
CBDEA
0.031
0.55
4.4
CuCBDEA
NiCBDEA
CBMOR
3.5. In vitro antimicrobial assay
0.015
4.04
CuCBMOR
The results of antimicrobial activities of the study compounds and
the reference antibiotics reported as inhibition zone diameter (mm) are
shown in Table S4 of the Supporting Information. The minimum
inhibitory concentration (MIC) values which suggested that some of the
studied compounds exhibited considerable antimicrobial activity are
shown in Table S5. Fig. 7 shows a comparison of the minimum inhibitory
concentrations (MIC, µgmLꢀ 1) of CBDEA, CBMOR, and the control
drugs, streptomycin and fluconazole.
3.4.2. Cleavage of pBR322 DNA by the metal complexes
The potential of the complexes to cleave DNA was studied by gel
electrophoresis using supercoiled pBR322 DNA in 100 mM KCl, 10 mM
Tris-HCl, pH-7.5 buffer. The plasmid DNA is naturally in intact supercoil
form (I) and a relatively fast migration will be observed when subjected
to gel electrophoresis. Small damage causes the supercoil to relax,
generating an open circular form (II) which moves slower in the gel.
More damage creates a linear form (III) that migrates between (I and II)
[68]. Severe damage causes digestion of DNA creating small pieces
which are not visible in the gel [72-76]. Fig. S17 shows gel electro-
phoresis separation of pBR322 after incubation with each of the ligands
and complexes with increasing concentrations (A) and also with the
addition of peroxide solution (B). CBDEA and CBMOR had no effect on
DNA under both conditions. The complexes alone (A) also did not cause
any damage in the plasmid DNA. However, copper complexes CuCBDEA
and CuCBMOR severely damaged DNA in the presence of peroxide,
creating very small DNA pieces which are not visible in the gel at high
concentrations (lanes 6 and 7; 100 and 200 µM). This is due to Cu(II) to
Cu(I) redox reaction, which generates radical ions from peroxide that
hydrolyze DNA phosphate linkages [7,77-79] CuCBMOR also shows
form III of DNA, indicating a higher degree of DNA damage at the 50 µM
concentration (lane 5) before it disappears at the higher concentrations
(lanes 6–7).
In general, CBDEA exhibited better antimicrobial properties
compared to CBMOR; while CBDEA was active against 67 % of all the
microbial strains studied, CBMOR was active against about 43 %, and
the antimicrobial activity was somewhat diminished on complexation.
The superior antimicrobial activity of CBDEA is further reflected in the
fact that it exhibited better activity against Gram-negative bacterial
strains P. Aeruginosa ATCC 35032, K. pneumoniae ATCC 13882 and
S. marcescens ATCC 13880. Generally, all the studied compounds
showed relatively higher antimicrobial activity against the Gram-
positive bacteria and the fungi strains, which is consistent with previ-
ous report [9,46]. The cell walls of Gram-negative bacteria are noted for
having two membranes, made up of outer lipopolysaccharide (LPS)
molecules and inner anionic phospholipids, while Gram-positive bac-
teria possess only one membrane made up of phospholipids
[7,74,75,77,79]. A good antimicrobial agent is expected to cause cell
leakage by disrupting the membranes or cross the membranes barriers
into the cell to obstruct cellular processes and thereby destroy the bac-
teria [77]. It is therefore envisaged that Gram-negative bacteria mem-
branes will be less accessible to membrane-targeting antimicrobials.
Lipophilicity has been reported as an important factor for the anti-
microbial action of drugs [80,81]. The calculated logP (a measure of
lipophilicity) of CBDEA (2.85 ± 0.60) compared to CBMOR (1.01 ±
0.64) is an indication that CBDEA has a better lipophilic character
NiCBDEA results in a different pattern of damage on DNA structure in
the presence of peroxide. A high amount of Form II and Form III for-
mations resulting from the addition of NiCBDEA at 50–200 µM con-
centrations (lanes 5–7) indicates DNA damage. All the metal complexes
induce DNA damage which would create some degree of inhibition of
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Inorganica Chimica Acta 528 (2021) 120590
Fig. 7. Comparing the minimum inhibitory concen-
tration (MIC, µg/mL) of CBDEA, CBMOR and control
drug (streptomycin for antimicrobial and fluconazole
for antifungal). A lower value of MIC indicates higher
activity. ML = Micrococcusluteus ATCC 9341, SA =
Stapylococcus aureus ATCC 25923, SE = Stapylococcus
epidermidis ATCC 12228, PA = Pseudomonas aerugi-
nosa ATCC 35032, KP = Klebsiella pneumoniae ATCC
13882, SP = Streptococcus pneumoniae ATCC 27336,
MS = Mycobacterium smegmatis ATCC 607, SM =
Serratia marcescens ATCC 13880, BC = Bacilllus cereus
ATCC 11778, BS = Bacillus subtilis ATCC 6633, CA =
Candida albicans ATCC 10231, CU = Candida utilis
ATCC 9950, CT = Candida trophicalis, SC = Saccha-
romyces cerevisiae ATCC 976.
[80,82]. The conformational rigidity of CBDEA and its more effective
interactions with LPS of P. aeruginosa ATCC 35032, K. pneumoniae ATCC
13882 and S. marcescens ATCC 13880 may be essential for its more
potent antimicrobial action against these strains [79]. CBDEA demon-
strated antimicrobial activity against the Gram-positive bacterial strains
in increasing order as follows; S. aureus ATCC 25923, B. cereus ATCC
11778, S. epidermidis ATCC 12228, M. luteus ATCC 9341 and B. subtilis
ATCC 6633. CBDEA was also active against all the fungi. On the other
hand, CBMOR exhibited (though relatively lower) antimicrobial activity
against the microbes in increasing order as follows; M. luteus ATCC
9341, S. aureus ATCC 25923, S. epidermidis ATCC 12228, B. cereus ATCC
11778, P. aeruginosa ATCC 35032 and B. subtilis ATCC 6633. CBMOR
showed antimicrobial activity against 3 of the 4 fungi strains studied.
The Ni complexes of both ligands had better antimicrobial property
compared to the Cu complexes. NiCBDEA was active against 13 microbes
while CuCBDEA was active against only 4 microbes.
Table 3
The IC50 values and apoptotic cell rate of the compounds against the study cell
lines.
Compounds
Cell line/IC50 (µM)
Cell line apoptotic rate
MCF-7
PC-3
MCF-7
PC-3
CBDEA
10 ± 2
9 ± 10
11 ± 6
4 ± 6
2 ± 1
–
15 ± 5
11 ± 5
20 ± 6
18 ± 6
2 ± 2
–
88 ± 5
86 ± 4
85 ± 4
88 ± 2
92 ± 1
6 ± 2
87 ± 4
91 ± 2
83 ± 4
77 ± 4
86 ± 2
4 ± 2
CuCBDEA
NiCBDEA
CBMOR
CuCBMOR
Control
ligands or decreased. Anticancer activity of breast (MCF-7) and prostate
(PC-3) cancer cells of CuCBMOR and CuCBDEA are more pronounced
than NiCBDEA. Ni(II) complexes display cis conformation, while Cu(II)
complexes exhibit trans conformation confirmed by crystal structure
determination in this study. It is, therefore, apparent that the CuCBMOR
and CuCBDEA exhibited better anticancer activity than NiCBDEA.
CBMOR and CuCBMOR were very effective against the MCF-7 cell
lines with IC50 values of 4.1 µM and 1.76 µM, respectively, and CuCB-
MOR showed high activity against the PC-3 cell lines. Judging from the
IC50, the potency of CuCBMOR is comparable to that of the control drug,
paclitaxel. This is a strong effect for a novel compound, and this high
activity for CBMOR and CuCBMOR against MCF-7 and PC-3 cells will
increase interest in the development of morpholine-based anticancer
agents. The higher activity of CBMOR and CuCBMOR can be linked to
the morpholine moiety. Compounds with morpholine moieties have
been well studied as anticancer agents, and commercially available
anticancer drugs aprepitant and gefitinib contain morpholine fragments
in their structure [83,84]. The cytotoxicity of CuCBMOR was ~ 2 fold
and ~ 10 fold higher compared to CBMOR against MCF-7 and PC-3 cell
lines, respectively.
MICs were determined for the compounds that showed appreciable
growth inhibition zones; the study was restricted to microorganisms that
were affected by the compounds. From the results, MIC of selected
compounds against bacterial and yeast pathogens varied from 4 to 256
µgmLꢀ 1. The lowest MICs were observed for CBDEA (4–16 µg mLꢀ 1
)
against M. luteus, ATCC 9341, S. aureus ATCC 25923, S. epidermidis ATCC
12228, K. pneumoniae ATCC 13882, S. pneumoniae ATCC 27336,
B. Cereus ATCC 11778, B. subtilis ATCC 6633, C. albicans ATCC 10231,
C. utilis ATCC 9950, C. tropicalis and S. cerevisiae ATCC 9763. The results
suggest that CBDEA and the Ni(II) complexes of both ligands show
promising antimicrobial activities against the studied bacteria and
yeasts and are most likely good candidates for further investigation as
antimicrobial agents.
3.6. Anti-proliferative studies
The results of the anti-proliferative activities of the study compounds
and control drug MCF-7 cells are presented in Table 3, Figs. 8 – 9. The
results on PC-3 cells are shown in Figs. S18 – S19 of the Supporting
Information. Table 3 shows the values of IC50 on apoptotic cells and
necrotic cells. In the figures, the cytotoxic effects of drugs against cancer
cells and the ratios of the cell death types are presented. All the com-
pounds show moderate to high activities against the tested cell lines.
CBMOR showed much higher potency on MCF-7 cells compared to
CBDEA, while their activity values on PC-3 cells are similar. The anti-
proliferative activities were enhanced in the Cu(II) complexes, while
the activities of the Ni(II) complexes is comparable to those of the free
Copper is a well-known bio-essential metal ion; its redox switching
and tunable coordination geometry ensure DNA-binding and bioactivity
[85-87]. The development of anticancer agents with endogenous metals
such as copper is important as they are less toxic to normal cells
compared to cancer cells. However, copper can also be toxic due to its
affinity for redox reactions and the ability to substitute other essential
metals in the active site of proteins and DNA [67]. Also, Cu(II) ion can
bind to both hard and soft bases, thus enacting its effect, which can be
positive or negative. To further investigate the cytotoxicity of the
studied compounds, the apoptotic rates on MCF-7 and PC-3 cell lines
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Inorganica Chimica Acta 528 (2021) 120590
Fig. 8. The cytotoxicity of study compounds with untreated control groups on MCF-7 cells. The data were represented as mean ± standard error of the mean. A
comparison was performed by the One Way ANOVA test with Dunnett’s multiple comparison test applied as a post-hoc test. p-values equal or<0.05 were considered
as statistically significant (+p < 0.05, ++p < 0.01, +++p < 0.001, ++++p < 0.0001) versus untreated control.
NiCBDEA in cis geometry and CuCBDEA in trans, a similar coordination
geometry observed in Pt(II) anticancer compounds. In-vitro screening of
the compounds against some Gram-positive bacteria, Gram-negative
bacteria, and yeasts confirmed the superior antimicrobial potencies of
the CBDEA and its Ni(II) complex as compared to other studied com-
pounds. Additionally, metal complexation did not lead to improved
antimicrobial properties. CBDEA and CBMOR did not affect DNA under
the experimental conditions; however, the copper complexes, CuCBDEA
and CuCBMOR, severely damaged DNA in the presence of peroxide. The
results of anti-proliferation tests indicated that CuCBMOR has the most
effective anticancer activity with IC50 values of 1.76 µM for MCF-7 and
1.97 µM for PC-3, which is comparable to values obtained for paclitaxel,
a well-known cancer drug. The apoptosis ratios observed for CuCBMOR
(91.53 % in MCF-7 cell line and 85.95 % in PC-3 cell line) is a strong
indication that it is a potential anticancer agent.
Fig. 9. Apoptosis measurement of study compounds on MCF-7 cells. The data
were represented as mean ± standard error of mean. Comparison was per-
Declaration of Competing Interest
formed by One Way ANOVA test with Dunnett’s multiple comparison test was
applied as a post-hoc test. p-values equal or<0.05 were considered as statisti-
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
cally significant (+p < 0.05, ++p < 0.01, +++p < 0.001, ++++ p < 0.0001)
versus untreated control.
were investigated. From the apoptotic studies, a large amount of the
cells died after the application of the compounds via apoptotic mecha-
nisms. (Fig. 9). In Table 3, the apoptosis rate of the compounds and the
control drug is given. The apoptosis ratios observed for the most effec-
tive derivative CuCBMOR was 91.53 % for MCF-7 cells and 85.95 % for
PC-3 cells.
Acknowledgement
The authors are grateful to the Department of Chemistry, Akron
University, for assistance with X-ray crystal structure determination of
CBDEA and CBMOR, School of Chemical Sciences, University of Auck-
land, New Zealand for crystal data of NiCBDEA and CuCBDEA and Sci-
entific Research Projects Unit of Ondokuz Mayıs University (Project No:
PYO.FEN.1906.19.001), Turkey for NiCBMOR. Many thanks to National
Centre for Energy Research and Development, University of Nigeria,
Nsukka for infrastructural support.
4. Conclusion
Two acylthiourea derivatives N,N-diethyl-N′-(4-chlorobenzoyl)thio-
urea (CBDEA), N-morpholine-N′-(4-chlorobenzoyl)thiourea (CBMOR)
and their Ni(II) and Cu(II) complexes were synthesized. The ligands
behaved as monoanionic bidentate molecules, coordinating through the
thiol sulfur and the carbonyl oxygen. Four coordinate square-planar
geometries were observed for the Ni(II) and Cu(II) complexes with
Appendix A. Supplementary data
CCDC 2009344, 2065533, 2009343, 2012502 and 2012503 contain
the supplementary crystallographic data for CBMOR, NiCBMOR, CBDEA,
11

E.E. Oyeka et al.
Inorganica Chimica Acta 528 (2021) 120590
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