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