Design, synthesis, and physicochemical characterization of new aminothiohydantoin Schiff base complexes for cancer chemotherapy

Article abstract of DOI:10.1016/j.ica.2021.120504

A novel aminothiohydantoin Schiff base (ATHSB) ligand and its complexes {M(II)ATHSB; M = Mn(II), Cu(II), Zn(II)} have been successfully synthesized. The coordination modes, stoichiometry, geometry, thermal stability, and morphology of M(II)ATHSB were deduced based upon the outputs of different physicochemical, spectroscopic, and microscopic techniques. The comparative in-vitro anticancer studies revealed that all complexes are more cytotoxic than the parent ligands against human cancer cell lines (liver carcinoma (HepG2), colon carcinoma (HCT116)), whereas, they exhibited lower toxicity as compared to the free ligand against normal liver cells (HL7702). Complex Zn(II)ATHSB seems to be a superlative candidate; as it is the most cytotoxic one against HepG2 concomitantly with lowest toxicity toward the normal human liver cells (HL7702), thus may offering a potential alternative for conventional cancer chemotherapeutic agents.

Full text of DOI:10.1016/j.ica.2021.120504

Inorganica Chimica Acta 526 (2021) 120504
Contents lists available at ScienceDirect
Inorganica Chimica Acta
journal homepage: www.elsevier.com/locate/ica
Research paper
Design, synthesis, and physicochemical characterization of new
aminothiohydantoin Schiff base complexes for cancer chemotherapy
Ahmed R.E. Mahdya, Mohammad Y. Alfaifib, Mohamed S. El-Gareba, Nessma Farouka, Reda F.
,d,*
M. Elshaarawyc
a Department of Chemistry, Faculty of Science, Port Said University, 42526 Port Said, Egypt
b Biology Department, Faculty of Science, King Khalid University, 9004 Abha, Saudi Arabia
c Department of Chemistry, Faculty of Science, Suez University, 43533 Suez, Egypt
d
¨
Institut für Anorganische Chemie und Strukturchemie, Heinrich-Heine Universitat Düsseldorf, Düsseldorf, Germany
A R T I C L E I N F O
A B S T R A C T
Keywords:
A novel aminothiohydantoin Schiff base (ATHSB) ligand and its complexes {M(II)ATHSB; M = Mn(II), Cu(II), Zn
(II)} have been successfully synthesized. The coordination modes, stoichiometry, geometry, thermal stability,
and morphology of M(II)ATHSB were deduced based upon the outputs of different physicochemical, spectro-
scopic, and microscopic techniques. The comparative in-vitro anticancer studies revealed that all complexes are
more cytotoxic than the parent ligands against human cancer cell lines (liver carcinoma (HepG2), colon carci-
noma (HCT116)), whereas, they exhibited lower toxicity as compared to the free ligand against normal liver cells
(HL7702). Complex Zn(II)ATHSB seems to be a superlative candidate; as it is the most cytotoxic one against
HepG2 concomitantly with lowest toxicity toward the normal human liver cells (HL7702), thus may offering a
potential alternative for conventional cancer chemotherapeutic agents.
Thiohydantoin
Metallo-Schiff base
Spectroscopic study
SEM
Cytotoxicity
1. Introduction
metal ions has proved advantageous in improving the biological activ-
ities and stability of these derivatives [8,9].
The limited-spectrum therapy, neurotoxicity, nephrotoxicity,
emetogenesis, and acquired resistance of platinum (Pt)-based anticancer
drugs [1,2] are serious clinical drawbacks that limit their usage as
cancer chemotherapeutic, worldwide. These challenges have motivated
many researchers to search for new non-platinum anticancer agents
[3,4]. However, up to date, there is no non-Pt metal anticancer drugs
have been authorized for today’s cancer therapy, therefore, extensive
trials have been performed for exploring novel metal-based anticancer
candidates [5].
Schiff bases and their metal complexes are very attractive classes of
organic compounds to the scientific research community due to their
ease of preparation, synthetic flexibility, electronic features, sensitivity
and selectivity towards metal ions. Moreover, structural similarities
(mimic) with natural biomolecules in the presence of azomethine group
–
–
–
N CH ), which is the key to the prediction of the mechanism of
–
(
many biochemical reactions in the biological systems [10,11]. In this
respect, they have shown broad-spectrum biological potencies including
antimicrobial [12,13], antiviral [14], anti-biofilm [15,16], antimalarial
[17], anti-inflammatory [18], antioxidant [19], antipyretic, anti-
proliferative, and antitumor activities [20,10,11]. Furthermore, they
have displayed significant effectiveness in diverse fields such as catalysts
[21], chemical sensing [22,23], anti-fouling coatings [24], food, poly-
mer, and dye industries [25], water remediation [26,27], and analytical
chemistry [28,29].
During the last few decades, 2-thiohydantoin (4-oxoimidazolidine-2-
thiones) and its derivatives have received considerable attention in the
drug-discovery community due to their diverse biological properties [6].
In this respect, they have exhibited antimicrobial, anti-thrombotic,
anticonvulsant, and anticancer activities [6]. Structurally, 2-thiohydan-
–
S ↔ N
– –
N C S) that can undergo
–
toin moiety contains a thioamide group (H
–
–
– –
–
– –
N
the thione–thiol tautomerism (H
C
C
S
H) which
Motivated by these aforesaid outstanding facts and in continuation of
our directed program to design and explore new chemotherapeutic
nominees [13,30,31], this study aims to design and synthesize new
–
promote the capacity of this group to chelate with many metal ions [7].
Interestingly, coordination of thiohydantoin derivatives to transition
* Corresponding author at: Department of Chemistry, Faculty of Science, Suez University, 43533 Suez, Egypt.
E-mail address: reda.elshaarawy@suezuniv.edu.eg (R.F.M. Elshaarawy).
https://doi.org/10.1016/j.ica.2021.120504
Received 26 February 2021; Received in revised form 21 June 2021; Accepted 23 June 2021
Available online 26 June 2021
0020-1693/© 2021 Elsevier B.V. All rights reserved.

A.R.E. Mahdy et al.
Inorganica Chimica Acta 526 (2021) 120504
aminothiohydantoin Schiff base (ATHSB) and its transition metal com-
plexes with Mn(II), Cu(II), and Zn(II) ions; targeting anticancer
applications.
3.81 (s, 3H); 13C NMR (DMSO‑d6): 169.51, 153.79, 148.35, 146.68,
142.83, 121.01, 120.00, 119.63, 113.83, 56.31, 49.03. Anal. Calc. for
C9H11N3O2S (225.06 g/mole): C, 48.0; H, 4.9; N, 18.7; S, 14.2. Found: C,
47.9; H, 5.0; N, 18.5; S, 13.9%. EI-MS: m/z = 226 [M]+.
2. Experimental
2.4. Synthesis of ATHSB (3-((1-(2,4-dihydroxyphenyl)ethylidene)
2.1. Materials and methods
amino)-2-thiohydantoin-4-one) (3)
2.1.1. Materials
2,4-Dihydroxyacetophenone thiosemicarbazone (2) (1.13 g, 5.12
mmol) and ethyl chloroacetate (0.62 mL, 5.11 mmol) were dissolved in
hot dry ethanol (20 mL) containing a catalytic amount of anhydrous
sodium acetate (0.75 g, 9.14 mmol). Thereafter, the reaction mixture
was heated under stirring and reflux for 5 h. The reaction progress was
followed by TLC to investigate the disappearance of (2) and conse-
quently reaction completion. After the disappearance of (2), the reaction
mixture was cooled to room temperature and poured into an ice-water
mixture. The solid product was collected by filtration, dried, and crys-
tallized from hot ethanol to afford yellow crystals of the desired ligand
(3). 1.5H2O. Yield 73%; mp 273–275 ◦C. FTIR (KBr, cmꢀ 1): 3571 (s, sh,
phenolic νO H), 3390 (m, br, νO H), 3192 (s, br, νN H), 2937 (s, sh, ,
Chemicals and solvents used in this work were provided by the
following suppliers and used without further purification: resorcinol,
semicarbazide hydrochloride, ethyl chloroacetate (Sigma–Aldrich), so-
dium acetate anhydrous (CH3COONa), anhydrous zinc chloride (ZnCl2),
CuCl2⋅2H2O, MnCl2⋅4H2O (GRÜSSING GmbH).
2.1.2. Instrumentation
Elemental analyses (CHNS) were carried out using a Perkin–Elmer
263 elemental analyzer. A BRUKER Tensor-37 FT-IR spectrophotometer
was utilized to record FT-IR spectra in the wavenumber range 400–4000
cmꢀ 1. The absorption bands were coded according to their intensities as:
vs (very strong), s (strong), m (medium), w (weak), br (broad), sh
(sharp). NMR-spectra were collected from a Bruker Avance DRX400
–
–
–
–
–
ν
H), 2597 (m, sh,
ν
H), 1707 (vs, sh, νC O), 1599 (s, sh, νC N), 1549
S
– –
–
C
–
(s, sh,), 1517 (s, sh, thioamide I, δ(N
+
ν(C N)), 1458 (s, sh), 1360 (s,
–
–
–
sh), 1330 (s, sh), 1296 (s, sh), 1251 (s,Hs)h, ν(Ar O)), 1211 (w, sh), 1149 (s,
1
(400 MHz for H) or Bruker Avance DRX100 (100 MHz for 13C) spec-
–
trometer with calibration to the residual solvent proton signals for
DMSO‑d6 (1H NMR: 2.52 ppm, 13C NMR: 39.5 ppm) and CDCl3 (1H NMR:
7.26 ppm, 13C NMR: 77.16 ppm) against TMS reference. Multiplicities of
the peaks were assigned as singlet (s), doublet (d), triplet (t), quartet (q),
or multiplet (m). The mass spectra were acquired in the linear mode for
positive ions on UHR-QTOF maXis 4G (Bruker Daltonics) and BRUKER
Ultraflex MALDI-TOF instrument equipped with a 337 nm nitrogen laser
pulsing at a repetition rate of 10 Hz. UV–Vis spectra were measured in
sh, thioamide IV, ν(C S)), 1085 (s, sh), 985 (m, sh), 895 (m, sh,
–
–
–
δ(C
H)), 796 (m, sh, δ(C
H)), 744 (m, sh, δ(C S)), 645 (m, sh, δ(C S)),
S
– –
– –
– –
581 S(m, sh, δ(C S)), 528 (s, sh). 1H NMR (400 MHz, DMSO‑d6) δ (ppm):
–
12.81 (s, 1H, OH), 12.05 (s, 1H, OH), 10.03 (s, 1H, NH), 7.48 (d, J = 7.3
Hz, 1H, Ar–H), 6.37 (d, J = 7.3 Hz, 1H, Ar–H), 6.28 (s, 1H, Ar–H), 4.00
(s, 2H, CH2 of thiohydantoin ring), 2.45 (s, 3H, CH3). 13C NMR (100
MHz, DMSO‑d6) δ (ppm): 173.96, 166.27, 164.69, 161.50, 161.26,
131.02, 111.75, 107.79, 103.29, 33.87, 14.61. EI-MS: m/z 292.30
([M]+·, 68%). Anal. Calcd for C11H14N3O4.5S (M = 292.31 g/mol): C,
45.20; H, 4.83; N, 14.38; S, 10.97%. Found: C, 45.08; H, 4.87; N, 14.29;
S, 10.59%.
◦
DMSO (10–3 M) at 25 C on a Shimadzu UV-2450 spectrophotometer
using quartz cuvettes (1 mL). The morphology of new compounds was
investigated using Scanning electron microscopy (SEM, Hitachi S-7400,
Hitachi, Japan). Vibrating Sample Magnetometer (VSM), (Model PAR
155) was used to carry Magnetic measurements.
2.5. Preparation of ATHSB complexes (4a-c)
2.2. 2,4-Dihydroxyacetophenone (1)
The following general procedure was used to prepare ATHSB com-
plexes (4a-c). A hot aqueous ethanolic solution (1:1, 25 mL) of the
divalent metal salt (MCl2⋅xH2O; CuCl2⋅2H2O, MnCl2⋅4H2O, ZnCl2) (1
mmol) was added to a hot ethanolic solution (25 mL) of ATHSB (0.265 g,
1 mmol) containing few drops of NH3/NH4Cl -buffer (pH 9.2). The re-
action mixture was heated under reflux for 2 h. Thereafter, the solvent
was partially evaporated under reduced pressure and then the reaction
mixture was cooled to room temperature to allow the precipitation of
the desired products. These products were collected by filtration,
washed successively with H2O (5 mL × 3), warm EtOH (5 mL × 2), Et2O
(5 mL × 3), and dried in vacuo.
2,4-Dihydroxyacetophenone was prepared according to the previ-
ously reported method [32]. Briefly, a mixture of dry powdered zinc
chloride (0.24 mol) and glacial acetic acid (32 mL) was heated to 140 ◦C
in a sand bath. Thereafter, dry resorcinol (0.2 mol) was added to this
mixture under stirring at the same temperature. Afterward, the tem-
perature of the reaction mixture was raised 150 ◦C until it just begins to
boil and kept for 20 min at this temperature. Then, HCl (18%, 50 mL)
was added and the content was cooled to 5 ◦C. The separated dirty white
product was collected by filtration and washed with dilute HCl. The
product was recrystallized from hot water to give the desired product.
1H-NMR: 300 MHz (CDC13, δ ppm) 12.69 (s, 1H), 9.93 (s, 1H), 7.61 (d, J
= 8.5 Hz, 1H), 6.38 (d, J = 8.6 Hz, 1H), 6.36 (s, 1H), 2.53 (s, 3H).
[Mn(ATHSB)Cl(H2O)2]0.2.25H2O (4a): Pale brown powder
(63%). FTIR (KBr, cmꢀ 1): 3421 (s, sh, water νO H and νN H), 2601 (w,
–
–
–
–
br), 1638 (vs, sh, νC O), 1579 (s, sh, νC N), 1536 (s, sh), 1489 (s, sh),
–
–
1393 (m, sh), 1338 (s, sh), 1276 (m, sh, νAr O), 1243 (s, sh), 1184 (w,
–
–
2.3. 2,4-Dihydroxyacetophenone thiosemicarbazone (2)
sh), 1147 (m, sh, νC S), 1072 (m, sh), 1022 (w, sh), 986 (w, sh), 853 (w,
–
–
sh), 799 (m, sh), 743 (m, sh, δ(C S)), 615 (s, sh), 496 (s, sh), 400 (m, sh).
–
A mixture of semicarbazide hydrochloride (1 g), anhydrous sodium
acetate (0.9 g), and distilled water (5 mL) and was warmed gently until a
clear solution is obtained. Thereafter, a solution of 2, 4 dihydrox-
yacetophenone (1 g) in 5 mL of ethanol was added under stirring. Then
the reaction mixture was warmed gently in a water bath (55 ◦C) for 15
min to crystallizes the desired semicarbazone. Finally, the content was
cooled and the obtained product was filtered off, washed thoroughly
with distilled water, and drained. Recrystallized from ethanol and dried
under vacuum. It was obtained as a white crystalline solid. Yield: 74.6%.
mp: 226 ◦C. IR data (KBr, cmꢀ 1): 3379, 3296, 3159, 1632, 744. 1H NMR
(DMSO‑d6): δ 11.28 (s, 1H), 10.23 (s, 1H), 7.97 (s, 1H), 7.18 (d, J = 7.1
Hz, 1H), 7.02 (d, J = 7.1 Hz, 1H), 6.85 (t, J = 7.9 Hz, 1H), 4.39 (s, 2H),
EI-MS: m/z 431.20 ([M]+·, 35%). Anal. Calcd. for C11H18.5ClMnN3O7.25
S
(M = 431.23 g/mol): C, 30.64; H, 4.32; N, 9.74; S, 7.43%. Found: C,
30.55; H, 4.34; N, 9.71; S, 7.42%.
[Cu(ATHSB)Cl].4H2O (4b): Reddish-brown powder (68%). FTIR
(KBr, cmꢀ 1): 3449 (s, sh, water νO H), 3346 (s, sh, νN H), 1641 (s, sh,
–
–
–
–
νC O), 1584 (s, sh, νC N), 1537 (s, sh), 1437 (m, sh), 1393 (m, sh), 1246
–
–
–
(s, sh, νAr O), 1205 (m, sh), 1150 (m, sh, νC S), 1074 (w, sh), 991 (m,
–
–
–
sh), 896 (m, sh), 848 (w, sh), 748 (m, sh, δ(C S)), 610 (w, sh), 514 (w,
–
sh), 479 (m, sh), 451 (w, sh). EI-MS: m/z 435.30 ([M]+·, 35%). Anal.
Calcd. for C11H18ClCuN3O7S (M = 435.34 g/mol): C, 30.35; H, 4.17; N,
9.65; S, 7.36; Found: C, 30.28; H, 4.21; N, 9.55; S, 7.29%.
[Zn(ATHSB)Cl(H2O)2] (4c): Yellowish-white powder (58%). FTIR
2

A.R.E. Mahdy et al.
Inorganica Chimica Acta 526 (2021) 120504
Scheme 1. Schemeatic digram for synthesis of aminothiohydantoin Schiff base ligand (ATHSB, 3) and its transition metal complexes (M(II)ATHSB, 4a-c).
(KBr, cmꢀ 1): 3506 (s, br, water νO H), 3350 (m, sh), 3301 (m, sh, νN H),
OriginPro 9.1 32. all results were presented as mean ± standard medium
error and significance levels were considered at values of P < 0.05.
–
–
–
–
2927 (m, sh), 2589 (w, br), 1649 (s, sh, νC O), 1574 (s, sh, νC N), 1456
–
–
(s, sh), 1367 (m, sh), 1343 (m, sh), 1256 (s, sh, νAr O), 1206 (m, sh),
–
–
1169 (m, sh), 1144 (m, sh, νC S), 1085 (m, sh), 988 (m, sh), 905 (m, sh),
3. Results and discussion
–
–
853 (m, sh), 801 (m, sh), 744 (m, sh, δ(C S)), 610 (w, sh), 575 (w, sh),
–
548 (m, sh), 519 (m, sh), 461 (m, sh). EI-MS: m/z 401.10 ([M]+·, 35%).
Anal. Calcd. for C11H14ClN3O5SZn (M = 401.14 g/mol): C, 32.94; H,
3.52; N, 10.48; S, 7.99%. Found: C, 32.87; H, 3.64; N, 10.43; S, 7.87%.
3.1. Synthesis
Aminothiohydantoin Schiff base (ATHSB, HL, 3) was prepared from
the resorcinol as a starting substrate through three consecutive steps
outlined in Scheme 1. Initially, acylation of resorcinol with glacial acetic
acid in presence of a catalytic amount of anhydrous ZnCl2 as a Lewis acid
in order to prepare 2,4-dihydroxyacetophenone (2,4-DHA, 1). The sec-
ond step involves the condensation reaction between 2,4-DHA and thi-
osemicarbazide in the presence of an acid catalyst to get the
corresponding thiosemicarbazone (DHATSC, 2), quantitatively. The
main aim of this reaction to protect the terminal amino group of the
thiohydrazide fragment in thiosemicarbazide to avoid the formation of a
nucleophilic substitution product with ethyl chloroacetate, in the next
step. Finally, the cyclization of the thiosemicarbazone (2) into ATHSB
(3) was carried out by the reaction of 2 with ethyl chloroacetate over
reflux in an ethanolic medium containing a catalytic amount of anhy-
drous sodium acetate.
2.6. In vitro cytotoxicity study
2.6.1. Cell cultures
Human colon carcinoma (HCT116), liver hepatocellular carcinoma
(Hepg2), and normal human liver cells (HL7702) cell lines were ob-
tained from VACSERA tissue culture unit, Egypt. These cell lines have
cultivated in RPMI-1640 media supplemented with 10% FBS, 1% L-
glutamine, 50 µg/mL gentamycin, and a HEPES buffer. All cells were
kept at 37 ◦C in a humidified carbon dioxide atmosphere (5%) for 24 h.
2.6.2. Cytotoxic effect assay
The cytotoxic effects of the tested samples toward the cancer cells
were assessed according to the protocol adopted by the regional center
for Mycology & Biotechnology, Egypt [33]. Briefly, 100 µL of the culture
media were seeded in a 96-wells plate (Falcon, NJ, USA) (104 cells per
well). The wells have categorized into groups (three wells per group) for
each sample concentration. The control groups were incubated with and
without DMSO (negative control). Thereafter, serial concentrations
(50.0–1.56 µg/mL) of the tested samples were added to the plate of cells
and incubated at 37 ◦C under a humid CO2 atmosphere (5%) for 48 h.
Manganese(II), copper(II), and zinc(II) complexes of the ATHSB
ligand (M(II)ATHSB, 4a-c) were synthesized by refluxing mixtures of the
deprotonated ATHSB and metal chlorides in aqueous ethanol (Scheme
1). The deprotonation of the ATHSB was performed using ammonium
buffer solution (pH 9.2). Of interest is the fact that the Cu(II) did not
react with the sulfur atom to form a disulfide with the reduction to Cu(I).
The parent ligand and its complexes were obtained in acceptable
yields. The structures of these new compounds were investigated based
upon elemental and spectral analysis (FTIR, UV–Vis, NMR (1H, 13C), EI-
MS) as well as thermal and magnetic measurements. The elemental
analyses (CHNS) and electron impact mass spectra (EI-MS) results
confirm that the complexes have been successfully prepared. All at-
tempts to obtain crystals suitable for X-ray structural analyses were
unsuccessful.
After 48 h incubation, 20
μL of 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyl tetrazolium bromide (MTT) solution (5 mg/mL, Sigma) was
added to each well and re-incubated for further 4 h. Afterward, the
staining medium was cautiously removed followed by supplementing
each well with 200 μL of DMSO. Then the plate was carefully agitated for
10 min before colorimetric examination by measuring the absorbance of
each well at 490 nm using a microplate reader (Multiskan-Thermo
Scientific).
3.2. Structural characterization
2.7. Statistical analyses
3.2.1. FTIR
The results and mathematical calculation accompanied this study
Always the FTIR spectroscopy for new complexes would be the first
informative guide to confirm its successful formation and inspection of
was handled out by independent student’s t-test using SPSS v17 and
3

A.R.E. Mahdy et al.
Inorganica Chimica Acta 526 (2021) 120504
Fig. 1. (A) FTIR spectra of the parent ligand (ATHSB, HL) and its chelates (M(II)ATHSB, M(II)L) for illustrating the changes in the positions and/ or intensities of the
carbonyl, imine, and phenolate stretches in the spectrum of ATHSB owing to coordination with metal(II) ions. (B) UV–Vis spectra of the free HL ligand and its
complexes in DMSO (10–3 M).
modes of binding within them [34]. The FTIR spectra of the free Schiff
base ligand (ATHSB, HL) and its metal M(II)-complexes are shown in
Figs. S1-S4 (Electronic Supplementary Information (ESI†)), Fig. 1A, and
Table S1 (ESI†). In the spectrum of ATHSB, the IR peaks distinctive for
moiety through azomethine nitrogen and oxygen atoms of phenolic and
carbonyl groups, thus ATHSB acts as a monoanionic (ONO) tridentate
ligand [37]. This further confirmed by emergence of new medium in-
tensity peaks in complexes spectra at the range of 496–461 and 575–569
cmꢀ 1 due to the vibrations ν(M O) and ν(M N), respectively [37]. Moro-
–
the phenolic O H and imine groups of the Schiff base segment were
–
–
observed at 3571 and 1599 cmꢀ 1, respectively. On the other hand, the
vere, new broad bands were appeared in the spectra Mn(II) and Zn(II)
–
–
ν(C O), thioamide IV ν(C
,
S)
–
νas(NCS), and νs(NCS) of the thiohydantoin
complexes at regions of wavenumber ranges 3506–3421, 780–739, and
–
moiety were observed at 1707, 1149, 1458, 744 cmꢀ 1, respectively [35].
Moreover, the two strong bands at 1517 and 1330 cmꢀ 1 are assigned to
the thioamide I and II modes, respectively [34]. The emergence of a
weak IR band at 2597 cmꢀ 1 in the spectrum of ATHSB, attributable to
638–628 cmꢀ 1 which may be assigned to
ν(OH), ρr(H2O)), and ρw(H2O),
respectively, of coordinated water molecules [12,37].
3.2.2. UV–Vis spectra
the ν(S H) stretching vibration, confirms the minor contribution of thiol
Fig. 1B shows the electronic absorption spectra for the solutions of
ATHSB and its M(II) complexes in DMSO (10–4 M). It can be noticed that
the spectrum of ATHSB was dominated by two main absorption peaks.
–
form in the thiol ↔ thione tautomeric equilibrium in the solid structure
of ATHSB [24]. Further evidence for confirmation of coexistence of two
tautomeric formes in the molecular structure of ATHSB is the observa-
tion of absorption bands at 1149, 895, 796, 645 cmꢀ 1 characteristic for
The first peak was observed at 267 nm that assigned to π → π* transition
of the heterocyclic and/or phenyl moieties. The second one appeared
around 369 nm which can be ascribed to the n → * transition involving
all electron-possessing groups in the ligand (such as C O, C S, and
–
–
ν(C S), δ(C S), δ(C
H), and δ(C S), respectively [36]. Our findings are
π
–
– –
S
–
–
– –
– –
consistent with the previously reported work [36]. These FTIR spectral
results prove the successful formation of aminothiohydantoin Schiff
base (ATHSB, 3) ligand.
–
C
N) [38].
–
In the electronic spectra of M(II)ATHSB complexes, the main ab-
However, by comparison of the IR spectra of metal complexes with
that of free ligand, we can observe that the band distinctive for phenolic
–
sorption peaks have hypsochromically shifted relative to the corre-
sponding ones in the free ATHSB, supporting the coordination of anionic
form of ATHSB ligand with metal(II) ions [39,40]. The electronic
spectrum of Mn(II)ATHSB complex displays three characteristic elec-
O
H group (3571 cmꢀ 1) of ATHSB has disappeared coupled with a red-
shifting of ν(Aryl O) band by 15–18 cmꢀ 1, indicating the deprotonation of
–
phenolic OH group and participation of the aryl-O in the chelation of
metal ions. Meanwhile, the absorption bands characteristic for carbonyl
and azomethine groups of the free ATHSB have appreciably shifted to
lower wavenumbers by 58–69 and 14–25 cmꢀ 1, respectively (see
experimental section). These downward shifts in wavenumber values
are indicative of the coordination of the carbonyl-O of the thiohydantoin
segment and an imine-N to metal ions. Interestingly, the emergence of
thiol and thione peaks in the spectra of Mn(II) and Zn(II) complexes
around 2600 and 1150 cmꢀ 1, respectively, is indicative of the contri-
bution of thiol and thione forms in coordination to metal ions, whereas,
tronic transition bands due to intraligand transitions (π → π*, n → π*)
[41]. These findings coupled with the value of magnetic moment for Mn
(II) complex (5.81B.M) demonstrate the high spin d5 configuration for
Mn(II) ion and consequently paramagnetism of its complex as well as an
octahedral geometry around Mn(II) ion [41]. Also, the electronic spec-
trum of Cu(II)ATHSB complex shows the same π → π* and n → π* bands
of the parent ligand but with hypsochromic shifts to centered in the
range of 265–375 nm, as well as relatively weak and broad peak
centered at 581 nm and 634 nm which are distinctive for the d →
2
2
2
d electronic transitions of type B1g + A1g and B1g→2Eg transitions,
respectively. Based on these results, the square planar geometry has
proposed for Cu(II) ion in the Cu(II)ATHSB complex. Furthermore, the
magnetic moment of Cu(II) complex was found to be 1.81B.M which in
matching with that assigned for the square planar Cu(II) complexes
the Cu(II) involve thione form only. Notable, the positions of ν(S
,
–
,
νas(NCS), and νs(NCS) peaks kept almost unchanged in the spectraHo)f
–
ν(C
S)
–
complexes, ruling out the sharing of the sulfur atom in coordination to
metal ions. These findings revealed the coordination of ATHSB ligand
4

A.R.E. Mahdy et al.
Inorganica Chimica Acta 526 (2021) 120504
Fig. 2. Thermograms of the free ligand (ATHSB) and its complexes (M(II)-ATHSB); (A) (thermogravimetric analysis (TGA), (B) differential thermogravimetry (DTG),
and (C) differential thermal analysis (DTA) curves.
[37]. As the Zn(II) has fully filled d-orbitals (d10), it is expected that the
Zn(II)ATHSB complex is diamagnetic and does not have any d-d transi-
tions. In this respect, the spectrum of the Zn(II)ATHSB complex displays
presented at δ 33.87 and 14.61 ppm, respectively.
3.2.4. Thermal analysis
only high-energy absorption bands at 263 and 294 nm due to
π
→
π
* and
The thermal stabilities of the M(II)-ATHSB chelates were investi-
gated using thermogravimetric analysis (TGA) (Fig. 2A), differential
thermogravimetry (DTG) (Fig. 2B), and differential thermal analysis
(DTA) (Fig. 2C) techniques at the temperature range from the ambient
temperature up to 800 ◦C under an inert nitrogen atmosphere. The TG
curves of the free ligand (ATHSB) evident that it losses water around
100 ◦C and is thermally stable up to 175 ◦C. The thermogram of Mn(II)-
ATHSB indicates its thermal stability up to 210 ◦C (Fig. 2A) and hy-
drated nature as revealed from loosing of lattice and coordinated water
molecules in the initial thermal degradation stages. The hydrated nature
of Cu(II)-ATHSB complex can be confirmed from its TG curves which
show the volatilization of water molecules in the foremost thermal
decomposition around 100 ◦C (Fig. 2A) with a broad DTG peak around
78 ◦C (Fig. 2B). As shown in Fig. 2A, the Zn(II)-ATHSB complex is
thermally stable up to 179 ◦C and exhibits four thermal events. The first
two events involve dehydration of the complex, while the last two
thermal events involve the decomposition of the complex.
n → π* transitions (shifted by –20 nm comparative to the parent ligand).
Based on the electronic spectral and thermal analysis (will be discussed
later) results, it could be concluded that the Zn(II) complex had an
octahedral geometry [37].
3.2.3. NMR spectrosacopy
The 1H and 13C NMR assignments confirm the successful preparation
of the parent ligand, aminothiohydantoin Schiff base (ATHSB, HL, 3).
The 1H NMR spectrum of ATHSB (Fig. S5, ESI†) shows three singlets at δ
12.81, 12.05, 10.03 ppm attributable for the resonance of thioamide and
phenolic protons, respectively. The protons of the phenyl group are
displayed as two doublets and a singlet at the range of δ 7.48–6.28 ppm.
The methylene (of thiohydantoin moiety) and methyl groups were
represented as two singlets at δ 4.00 and 2.45 ppm, respectively. Further
evidence for the structure of ATHSB was obtained from its 13C NMR
spectrum (Fig. S6, ESI†). Where the three very low-field signals at δ
173.96, 166.27, 164.69 ppm are assigned to the thiocarbonyl, azome-
thine, and carbonyl groups, respectively. The phenolic carbon signals
can be seen at δ 161.50 and 161.26 ppm. A set of carbon peaks observed
at the range of δ 131.02–103.29 ppm are assigned to the resonance of
phenyl carbons. The resonances of methylene and methyl carbons were
The DTA curves of the free ligand and its complexes (Fig. 2C) display
weak and broad endothermic peaks at the temperature zone just prior to
or accompanying the mass loss step in the corresponding TG
thermograms.
5

A.R.E. Mahdy et al.
Inorganica Chimica Acta 526 (2021) 120504
Fig. 3. Concentration – cytotoxicity correlation for the free ligand (ATHSB) and its M(II) complexes (M(II)ATHSB or M(II)L; M = Mn, Cu, Zn) toward three human
cell lines (A) liver carcinoma (HepG2), (B) colon carcinoma (HCT116), (C) and normal liver cells (HL7702), in comparison with the clinical anticancer drug (VBL).
(D) The IC50 values (μg/ mL) of the tested compounds against cancer species.
3.3. Morphological characterization
3.4.1. In vitro cytotoxicity
The viabilities of the treated cancer cells (Fig. 3) demonstrated that
all compounds have the ability to inhibit the growth of both carcinoma
cells (HepG2 and HCT116) with a performance depend upon; type of
cancer cell, structural features of treating agent, and sample
concentration.
The surface morphology of the free ligand and its metal complexes
was investigated using SEM analysis and the obtained micrographs were
collected in Fig. S7 (ESI†). Noteworthy, the SEM micrographs of M(II)
ATHSB complexes are significantly different than that for the ATHSB
ligand due to coordination of M(II) ions with the chelating sites for
ligand [37]. Furthermore, the surface morphology of the M(II) com-
plexes is dependent on the nature of metal ions as revealed from
Fig. S7B-D.
Generally, the colon carcinoma cells (HCT116) were little more
affected than the liver cells (HepG2) by the new chemotherapeutic
agents. For instance, the surviving HCT116 cells have decreased from
100% to ~29% (~71% growth reduction) after treating with 25 μg/mL
of Mn(II)ATHSB (Fig. 3B). However, the HepG2 cells were decreased
only by ~65% under the same remediation conditions (Fig. 3A).
Meanwhile, and further evidence for our conclusion, the IC50 values for
3.4. In vitro anticancer study
The main aim of this work is to design safe and cytotoxic metal(II)
thiohydantoin Schiff base complexes for application in the mitigation of
both liver and colon cancers. To this end, the newly synthesized ami-
nothiohydantoin Schiff base ligand (ATHSB, HL) and its metal com-
plexes (M(II)ATHSB, M(II)L) have in vitro assessed for their anticancer
activity against three human cell lines (liver carcinoma (HepG2), colon
carcinoma (HCT116), and normal liver cells (HL7702)), in comparison
with a clinical anticancer drug (Vinblastine, VBL) (C46H58N4O9, 810.99
g/mol).
the tested compounds were in the ranges of 22.97–2.78 μg/mL and
25.45–3.92 μg/mL against HCT116 and HepG2 cells, respectively.
Noteworthy, the M(II)ATHSB complexes displayed greater cancer-
inhibitory effects than that induced by the free ATHSB ligand. The
sequence of efficacy-pattern against HepG2 cells was as follow; Zn(II)
ATHSB (IC50 3.92 ± 0.85
mL) > VBL (IC50 6.83 ± 0.75
g/mL) > ATHSB (IC50 25.45 ± 1.1
against HCT116 cell lines was as follow; Cu(II)ATHSB (IC50 2.78 ± 0.78
g/mL) > Zn(II)ATHSB (IC50 3.41 ± 0.89 g/mL) > VBL (IC50 4.02 ±
0.77 g/mL) > Mn(II)ATHSB (IC50 6.81 ± 0.96 g/mL) > ATHSB (IC50
μ
g/mL) > Cu(II)ATHSB (IC50 5.19 ± 1.01
g/mL) > Mn(II)ATHSB (IC50 7.83 ± 0.96
g/mL). Whilst the activity order
μg/
μ
μ
μ
μ
μ
μ
μ
6

A.R.E. Mahdy et al.
Inorganica Chimica Acta 526 (2021) 120504
Fig. 4. Structural and geometrical features of the new M(II)ATHSB complexes showing; two types of metallocyclic rings (five-/ six-membered), all possible H-
bonding acceptors (HBA) (N and O atoms of the azomethine, imidazole, and phenolic groups), H-bonding donor (HBD) sites (NH and OH imidazole, phenolic, and
water fragments) and electrostatic interaction site (metal ion); those can enhance the interaction with cancer cell DNA through
teractions, and intercalation.
π-π stacking, hydrophobic in-
22.97 ± 1.3
μ
g/mL).
they exhibited lower toxicity as compared to the free ligand against
normal liver cells (HL7702); signifying the importance of these com-
plexes as new potential candidates to develop and explore novel
chemotherapeutic agents in the field of cancer therapy. Particularly Zn
(II)ATHSB seems to be a superlative candidate as it the most cytotoxic
one against HepG2 concomitantly in lowest toxicity toward the normal
human liver cells (HL7702), thus may offering a probable alternative to
conventional chemotherapeutic agents.
In general, the greater cytotoxic effects of M(II)ATHSB complexes in
comparison to the free ligand may be attributed to an increment in the
planarization of the entire molecule upon chelation of metal ions, more
extended π-electron delocalization system attained with the formation of
the metallacycle (Fig. 4), and additional electrostatic interactions
intrinsic due to the presence of the metal ion [42]. Notable, the planarity
of complexes could influence their cytotoxicity by impacting their
interaction capacity with bioactive targets, namely DNA [43]. This in
turn can lead to promoting the interaction between complex and cancer
cell DNA through non-covalent modes of action such as groove binding,
CRediT authorship contribution statement
π–π stacking, phosphate clamps, or intercalation, resulting in dramatic
Ahmed R.E. Mahdy: Conceptualization, Software, Data curation,
Validation, Writing - original draft. Mohammad Y. Alfaifi: Methodol-
ogy, Software, Data curation, Validation, Visualization, Writing - orig-
inal draft, Writing - review & editing. Mohamed S. El-Gareb:
Conceptualization, Supervision, Validation, Visualization, Writing -
original draft. Nessma Farouk: Methodology, Data curation, Software,
Writing - original draft. Reda F.M. Elshaarawy: Conceptualization,
Methodology, Software, Data curation, Validation, Visualization,
Writing - original draft, Writing - review & editing.
changes in the DNA structure and cell death [42]. Moreover, the metal
ion can selectively bind to multiple active biomolecular targets and in-
duces several cytotoxic mechanisms inside the cancer cell lines, dimin-
ishing their proliferation [5].
The highest antiproliferative effects of Cu(II)ATHSB compared to
others against colon carcinoma cells (HCT116) may be related to the
great planarization of this complex (cf. Fig. 4), owing to its square planer
geometry, which enables it to strongly interact with cancer cellular
DNA. Moreover, the endogenous copper ion can induce several mech-
anisms into the cancer cells including DNA interaction (intercalation,
groove binding), oxidative cleavage, hydrolytic cleavage, Topoisomer-
ase inhibition (TOPO I, II), and Proteasome inhibition [44].
Declaration of Competing Interest
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.
On the other hand, Zn(II) complex displayed the greatest anti-liver
cancer activity (IC50 3.92 ± 0.85 μg/mL) that almost 2-times greater
than the clinical drug (VBL) (IC50 6.83 ± 0.75 μg/mL). This could be
ascribed to the ability of the Zn(II) to block the hemeoxygenase inhibitor
(HMOX1) which is produced in large amounts in solid tumors to help the
cancer cell in defense against oxidative and other cellular stresses [45].
Interestingly, the Zn(II) complex is less cytotoxic than the other com-
plexes and the clinical drug (VBL) (IC50 32.12 ± 1.27 µg/mL) toward the
normal liver cells (HL7702) (Fig. 4C,D). Therefore, the new chemo-
therapeutic agent (Zn(II)ATHSB) may of a promising anticancer candi-
date that can selectively trigger apoptosis in liver and colon carcinoma
cells without harming the normal human cells.
Acknowledgments
The authors extend their appreciation to the Deanship of Scientific
Research at King Khalid University for funding this work through Gen-
eral Group Research Project under grant number (R.G. P. 1 /113 / 42).
Appendix A. Supplementary data
Supplementary data (spectral data) to this article can be found online
at https://doi.org/10.1016/j.ica.2021.120504.
4. Conclusion
A new series of Mn(II), Cu(II) and Zn(II) complexes of novel ami-
nothiohydantoin Schiff base ligand (ATHSB, HL) have been prepared by
reaction of the ligand with metal(II) chlorides in 1:1 M ratio. The co-
ordination profiles of HL with M(II) ions, stoichiometry, and stereo-
chemistry of metal complexes were investigated based upon various
physicochemical and spectroscopic techniques. The thermal stability
and surface morphological features of the free ligand and its coordina-
tion compounds have been studied using TGA, DTA, and SEM analysis.
The comparative in-vitro anticancer studies revealed that all complexes
are more cytotoxic than the parent ligands against human cancer cell
lines (liver carcinoma (HepG2), colon carcinoma (HCT116)), whereas,
References
[1] B. Stordal, N. Pavlakis, R. Davey, Cancer Treat. Rev. 33 (2007) 347.
[2] C. Jia, G.B. Deacon, Y. Zhang, C. Gao, Coord. Chem. Rev. (2020), 213640.
[3] I. Ott, R. Gust, Arch. Pharm. (Weinheim) 340 (2007) 117.
[4] P.V. Simpson, N.M. Desai, I. Casari, M. Massi, M. Falasca, Fut. Med. Chem. 11 (2)
(2019) 119.
[5] U. Ndagi, N. Mhlongo, M.E. Soliman, Drug Des. Devel. Ther. 11 (2017) 599.
[6] S.-H. Cho, S.-H. Kim, D. Shin, Eur. J. Med. Chem. 164 (2019) 517.
[7] E.K. Beloglazkina, A.G. Majouga, I.V. Yudin, N.A. Frolova, N.V. Zyk, V.
D. Dolzhikova, A.A. Moiseeva, R.D. Rakhimov, K.P. Butin, Russ. Chem. Bull. Int.
Ed. 55 (2006) 1015.
[8] L. Vatannavaz, S.J. Sabounchei, A. Sedghi, R. Karamian, S.H.M. Farida,
N. Rahmani, Polyhedron 181 (2020), 114478.
7

A.R.E. Mahdy et al.
Inorganica Chimica Acta 526 (2021) 120504
´
[9] K. Tishchenko, E. Beloglazkina, M. Proskurnin, V. Malinnikov, D. Guk,
M. Muratova, O. Krasnovskaya, A. Udina, D. Skvortsov, R.R. Shafikov,
Y. Ivanenkov, V. Aladinskiy, I. Sorokin, O. Gromov, A. Majouga, N. Zyk, J. Inorg.
Biochem. 175 (2017) 190.
[28] E.N. Oiye, M.F.M. Ribeiro, J.M.T. Katayama, M.C. Tadini, M.A. Balbino, I.
´
˜
C. Eleoterio, J. Magalhaes, A.S. Castro, R.S.M. Silva, J.W.C. Júnior, E.R. Dockal, M.
F. de Oliveira, Crit. Rev. Anal. Chem. 49 (2019) 488.
[29] A.L. Berhanu, G.I. Mohiuddin, A.K. Malik, J.S. Aulakh, V. Kumar, K.-H. Kim, TrAC
Trends Analy. Chem. 116 (2019) 74.
[10] W. Al Zoubi, Int. J. Org. Chem. 3 (2013) 73.
[11] P.M. Jadhav, Int. J. ChemTech Res. 13 (2020) 217.
[12] R.F.M. Elshaarawy, I.M. Eldeen, E.M. Hassan, J. Mol. Struct. 1128 (2017) 162.
[13] R.F.M. Elshaarawy, C. Janiak, Arab. J. Chem. 9 (2016) 825.
[14] A.R. Sofy, A.A. Hmed, N.F. Abd El Haliem, M.A.E. Zein, R.F.M. Elshaarawy,
Carbohydr. Polym. 226 (2019), 115261.
[30] R.F.M. Elshaarawy, T.B. Mostafa, A.A. Refaee, E.A. El-Sawi, RSC Adv. 5 (2015)
68260.
[31] H.K. Ibrahim, S.H. El-Tamany, R.F. El-Shaarawy, I.M. El-Deen, Maced. J. Chem.
Chem. Eng. 27 (2008) 65.
[32] R.B. Patil, S.D. Sawant, P.A. Thombare, Int. J. PharmTech Res. 4 (1) (2012)
375–381.
[15] R.F.M. Elshaarawy, F.H.A. Mustafa, A.R. Sofy, A.A. Hmed, C. Janiak, J. Env. Chem.
Eng. 7 (2019), 102800.
[33] T. Mosmann, J. Immunol. Methods 65 (1983) 55.
[34] R.F.M. Elshaarawy, Y. Lan, C. Janiak, Inorg. Chim. Acta 401 (2013) 85–94.
[35] S.S. Kandil, G.B. El-Hefnawy, E.A. Bakr, A.Z. Abou El-Ezz, Trans. Metal Chem. 28
(2003) 168.
[16] J. Alkabli, W.N. El-Sayed, R.F.M. Elshaarawy, A.I.M. Khedr, Eur. Polym. J. 138
(2020), 109960.
[17] G. Garg, A. Acharya, K. Patel, Int. J. Biomed. Res. 4 (2013) 137.
[18] M. Manjunatha, V.H. Naik, A.D. Kulkarni, S.A. Patil, J. Coord. Chem. 64 (2011)
4264.
´
[36] A. Gomez-Zavaglia, I.D. Reva, L. Frija, M.L. Cristiano, R. Fausto, J. Mol. Struct. 786
(2006) 182.
[19] W. Al Zoubi, A.A.S. Al-Hamdani, M. Kaseem, Appl. Organomet. Chem. 30 (2016)
810.
[37] M. Shakir, S. Hanif, M.A. Sherwani, O. Mohammad, S.I. Al-Resayes, J. Mol. Struct.
1092 (2015) 143.
[20] M.Y. Alfaifi, M.A.-E. Zein, A.A. Shati, M.A. Alshehri, S.E.I. Elbehairi, H.S. Hafez, R.
F.M. Elshaarawy, J. Photochem. Photobiol. A: Chem. 385 (2019), 112083.
[21] H.M.A. Hassan, M.A. Betiha, E.A. El-Sharkawy, R.F.M. Elshaarawy, N.B. El-Assy, A.
A. Essawy, A.M. Tolba, A.M. Rabie, Colloids Surfaces A: Physicochem. Eng. Asp.
591 (2020), 124520.
[38] V. Deval, A. Kumar, V. Gupta, A. Sharma, A. Gupta, P. Tandon, K.-K. Kunimoto,
Spectrochim. Acta Part A: Mol. Biomol. Spect. 132 (2014) 15.
[39] R.S. Bhaskar, C.A. Ladole, N.G. Salunkhe, J.M. Barabde, A.S. Aswar, Arab. J. Chem.
13 (2020) 6559.
[40] S.S. Kandil, G.B. El-Hefnawy, E.A. Baker, Thermochimica Acta 414 (2004) 105.
[41] F. Karipcin, B. Dede, S. Percin-Ozkorucuklu, E. Kabalcilar, Dyes Pigm. 84 (2010)
14.
[22] S.M. Saleh, R. Ali, R.F.M. Elshaarawy, RSC Adv. 6 (2016) 68709.
[23] R.F.M. Elshaarawy, R. Ali, S.M. Saleh, C. Janiak, J. Mol. Liq. 241 (2017) 308.
[24] R.F.M. Elshaarawy, H.R.Z. Tadros, R.M. Abd El-Aal, F.H.A. Mustafa, Y.A. Soliman,
M.A. Hamed, J. Environm, Chem. Eng. 4 (2016) 2754.
[42] B.S. Mendiguchia, I. Aiello, A. Crispini, Dalton Trans. 44 (2015) 9321.
[43] K. Choroba, B. Machura, A. Szlapa-Kula, J.G. Malecki, L. Raposo, C. Roma-
Rodrigues, S. Cordeiro, P.V. Baptista, A.R. Fernandes, Eur. J. Med. Chem. 218
(2021), 113404.
[25] A.M. Abu-Dief, I.M.A. Mohamed, Beni-Suef Univ. J. Basic Appl. Sci. 4 (2) (2015)
119.
[26] R.F.M. Elshaarawy, H.A. El-Azim, W.H. Hegazy, F.H.A. Mustafa, T.A. Talkhan,
Polym. Test. 83 (2020), 106244.
[44] C. Santini, M. Pellei, V. Gandin, M. Porchia, F. Tisato, C. Marzanod, Chem. Rev.
114 (2014) 815.
[27] W.N. El-Sayed, J. Alkabli, K. Althumayri, R.F.M. Elshaarawy, L.A. Ismail, J. Mol.
Liq. (2020), 114525.
[45] R. Huang, A. Wallqvist, D.G. Covell, Biochem. Pharmacol. 69 (2005) 1009.
8