New thiosemicarbazone Schiff base ligands: Synthesis, characterization, catecholase study and hemolytic activity

Article abstract of DOI:10.1016/j.molstruc.2019.127566

Two novel thiosemicarbazones ligands have been synthesized and characterized by FT-IR, ESI-MS, ¹H NMR, and also by single-crystal X-ray diffraction for L1. The crystal structure shows that L1 molecules are planar and are connected via N-H----S

Full text of DOI:10.1016/j.molstruc.2019.127566

Journal Pre-proof
New thiosemicarbazone Schiff base ligands: Synthesis, characterization, catecholase
study and hemolytic activity
Oussama Kheireddine Nehar, Radia Mahboub, Samira Louhibi, Thierry Roisnel,
Mohammed Aissaoui
PII:
S0022-2860(19)31675-8
DOI:
https://doi.org/10.1016/j.molstruc.2019.127566
MOLSTR 127566
Reference:
To appear in: Journal of Molecular Structure
Received Date: 23 September 2019
Revised Date: 5 December 2019
Accepted Date: 9 December 2019
Please cite this article as: O.K. Nehar, R. Mahboub, S. Louhibi, T. Roisnel, M. Aissaoui, New
thiosemicarbazone Schiff base ligands: Synthesis, characterization, catecholase study and hemolytic
activity, Journal of Molecular Structure (2020), doi: https://doi.org/10.1016/j.molstruc.2019.127566.
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New thiosemicarbazone Schiff base ligands:
Synthesis, characterization, catecholase study and hemolytic activity
Oussama Kheireddine Nehar1, Radia Mahboub2, Samira Louhibi1, Thierry Roisnel3 and Mohammed
Aissaoui4
1 Laboratory of inorganic chemistry and environment, Departement of Chemistry, Faculty of Science, BP 119,
Tlemcen University, Tlemcen 13000, Algeria.
2 Department of Chemistry, Faculty of Sciences, University of Tlemcen, B.P. 119, Tlemcen, 13 000, Algeria.
e-mail: radiamahboub@yahoo.com
3 Center of Diffractometry X, UMR 6226 CNRS, University of Rennes 1, 35042 Rennes, France
4 Antibiotics Antifungal Laboratory: Physical-Chemistry, Synthesis and Biological Activity, Faculty of Biology,
Tlemcen University, Tlemcen 13000, Algeria
Corressponding author*: samhibi1@yahoo.fr
Abstract
Two novel thiosemicarbazones ligands have been synthesized and characterized by FT-IR, ESI-
1
MS, H NMR, and also by single-crystal X-ray diffraction for L1. The crystal structure shows
that L1 molecules are planar and are connected via N-H----S and O-H----S interactions. The
catecholase activity of is situ copper and cobalt complexes of this ligands has been investigated
against 3,5-di-tert-butylcatechol. The progress of the oxidation reactions was closely monitored
over time following the strong peak of 3,5-DTBC using UV-Vis. Oxidation rates were
determined from the initial slope of absorbance vs. time plots, then analyzed by Michaelis-
Menten equations. Catechol oxidation reactions were realized using different concentrations of
copper and cobalt acetate and ligands (L/Cu: 1/1, 1/2, 2/1). The results show that all complexes
were able to catalyze the oxidation of 3,5-DTBC. Acetate complexes have the highest activity.
CuL1 and CoL1 complexes act as a catalyst and inhibitor. While copper and cobalt complexes
obtained from ligand L2 illustrate concentration-independent oxidation activation. The hemolysis
study performed by L1 increases as a function of its concentration. However, ligand L2 has no
hemolytic effect.
Keywords: Thiosemicarbazone, Crystal structure, Catechol Oxidation, Catalysis, hemolytic
activity.
1. Introduction
Proteins are the most important building blocks of a living cell. They carry out a large
variety of functions [1], such as: structural stabilization, molecule transport and catalysis.
Metalloproteins are estimated to represent about one third of all proteins [2]. A significant
number of them contain transition metal ions such as iron, copper, nickel, zinc and molybdenum
[3].
Copper Metalloproteins are very interesting because of their biological activities as
catalysts in redox reactions. Studies on copper metalloproteins led to their classification based on
spectroscopic features into 3 types: type-1, type-2 and type-3 active sites [4]. Copper proteins
with a type-3 active site, have an intriguing ability to reversibly bind dioxygen under ambient
conditions [4, 5]. This category of proteins includes catechol oxidase, an enzyme which catalyzes
exclusively the oxidation of catechols to the corresponding quinones [6, 7]. In literature,
considerable amount of studies are investigated on the synthesis of dicopper compounds in order

to mimic the activity of catechol oxidase. Their catalytic activity has been investigated in the
literature as in situ [8, 9] or as isolated complexes [5, 10]. In recent years, mononuclear copper
complexes also were reported to demonstrate catecholase activity [11]. Nevertheless many other
metal complexes are being studied to investigate their catechol oxidase capacities [12–14].
Thiosemicarbazones are one of the main subgroups of hydrazine. They are obtained
through the reaction of thiosemicarbazides with aldehydes or ketones. Thiosemicarbazones are a
group of compounds with numerous pharmacological applications such as antibacterial [15],
antifungal, antiviral [16], anti-inflammatory [17], antituberculosis [18], and antitumoral [19]. The
antitumoral activity of these compounds is due to their capacity to restrain ribonucleotide
reductase (RR), an enzyme necessary for DNA synthesis [20], Moreover, their metal complexes
possess valuable catalytic activities [21, 22], as well as pharmacological properties as drug
candidates, biomarkers and biocatalysts [23, 24]. Thiosemicarbazones are also reported to have
various analytical applications [25, 26]. They are used as corrosion inhibitors [27] and as heavy
metals in removal wastewater [28].
In the present work, we report the synthesis of two novel thiosemicarbazone Schiff base
ligands (L1, L2), their characterizations by spectroscopic methods and by single-crystal X-ray
diffraction for L1. Then, we study the catalytic activity of the in situ metal-thiosemicarbazone
complexes (CuL1, CuL2, CoL1 and CoL2) towards the oxidation of 3,5-di-tert-butylcatechol.
Finally, we present the hemolytic activity of ligands L1 and L2.
2. Experimental
2.1. Materials and Physical measurements
All the chemicals and solvents were reagent grade, and they were used as purchased with
no further purification.
X-ray data were collected on a D8 VENTURE Bruker AXS diffractometer with Mo-Kα
radiation (λ = 0.71073 Å). IR spectra were measured in the 400-4000 cm-1 range on a 9800 FTIR
spectrometer (Perkin–Elmer) where samples were run as KBr pellets. IR spectra were measured
in the 400-4000 cm-1 range on a Perkin-Elmer spectrum two spectrometer. Samples were run as
1
KBr pellets. H nuclear magnetic resonance spectra were realized using Varian Mercury M400
400 MHz spectrometer. Chemical shifts are listed in ppm and are reported relatively to
tetramethylsilane using the acetone-d6 solvent. Mass spectra were done on a spectrometer
LC/MSD-TOF G1969A (ESI-MS). Kinetic measurements were carried on spectrophotometrically
using UV-Vis on Perkin Elmer Lambda 25 and OptizenPOP 3 spectrophotometers.
2.2. Synthesis of (E)-2-((4-hydroxynaphthalen-1-yl)methylene)hydrazinecarbothioamide L1
In a mixture of three solvents: (water, ethanol and acetic acid) in a ratio of (1:3:3), was
dissolved 10 mmol/10ml (1.72 g) of 3-hydroxy-2-naphthaldehyde. Then 10 mmol/10ml (0.91 g)
of thiosemicarbazide was added. The mixture was refluxed for 2h and was left to recrystallize at
room temperature. After slow evaporation, brown crystals suitable for XRD analysis were
formed. Yield: 79%. MP = 256 °C.
1H NMR (400 MHz, Acetone-d6), δ (ppm): 7.40 and 7.70 (2H, NH2), 6.69 and 7.55-8.62 (6H,
CHAr), 8.84 (s, 1H, =CH-Ar), 9.60 (s, 1H, OH), 10.30 (s, 1H, NH). Selected IR bands (KBr
pellet, cm-1): 3420 (OH), 3320a /3200s (NH2), 3177 (NH), 3030 (C-HAr), 1577 (C=C), 1511
(C=N), 1115/ 783 (C=S), 967 (N-N). MS (ESI) (m/z): [M-H]+ 244.05.
2.3. Synthesis of (E)-2-(2,5-dihydroxybenzylidene)hydrazinecarbothioamide L2

To an ethanolic solution of thiosemicarbazide 10 mmol/10ml (0.91 g), was added 10
mmol (1.38 g) of 2,5-dihydroxybenzaldehyde dissolved in 10 ml of ethanol, in the presence of
catalytic amount of glacial acetic acid. The mixture then was refluxed for 2h. The obtained
precipitate was filtrated, rinsed with ethanol, and left to dry overnight in room temperature.
Yield: 86%. MP = 266 °C.
1H NMR (400 MHz, acetone-d6) δ (ppm): 6.30-6.84 (m, 2H, Har), 7.08 (s, 1H, HAr), 7.44 and
7.68 (s, 2H, NH2), 7.92 (s, 1H, NH), 8.39 (s, 1H, =CH-Ar), 8.70 (s, 1H, OH) and 10.38 (s, 1H,
OH). Selected IR bands (KBr pellet, cm-1): 3432 (OH), 3333a /3145s (NH2), 3177 (NH), 3030
(C-HAr), (C=C), 1627 (C=N), 1161/ 797 (C=S), 943 (N-N). MS (ESI) (m/z): [M-H]+ 210.03.
2.4. X-ray crystallography
The data of single crystal L1 were collected on D8 VENTURE Bruker AXS
diffractometer with Mo-Kα radiation of wavelength λ = 0.71073 Å. The ligand L1 crystallizes in
the triclinic space group P-1. The molecular structure of L1 is shown in (Figure 2). The cell
parameters of L1 as well as the structural data are grouped in Table 1. The structure of L1 was
solved by direct methods using the SIR97 program [29] and then refined with full-matrix least-
square methods based on F2 (SHELXL-97) [30]. All non-hydrogen atoms were refined with
anisotropic atomic displacement parameters. Except, nitrogen and oxygen linked hydrogen atoms
that were introduced in the structural model through Fourier difference maps analysis, H atoms
were finally included in their calculated positions. A final refinement on F2 with 2560 unique
intensities and 166 parameters converged at ωR (F2) = 0.0834 (R(F) = 0.0318) for 2305 observed
reflections with I > 2σ(I.).
2.5. Catecholase activity study
In situ catechol oxidase study was performed in DMF. 3,5-di-tert-butylcatechol (3,5-
DTBC) was used as substrate [6, 13]. In a spectrophotometric cell, ligand (L1 and L2 ) (10-4 M)
and metal salt (Cu(AcO)2 and Co(AcO)2) (10-4 M) solutions were mixed together then treated
with 100:1 up to 700:1 substrate to complex ratios (1x10-2 - 7x10-2 M). The progress of oxidation
reactions was regularly monitored over time at room temperature, under aerobic conditions.
Absorbance values were recorded following the strong peak of 3,5-di-tert-butylquinone (3,5-
DTBQ) [7] (400 nm maximum absorbance) using UV-Vis spectroscopy. Oxidation rates were
determined from the initial slope of absorbance vs. time plots, and then analyzed by Michaelis-
Menten equations (Eq.1, Eq.2).
ꢅ ꢇ
ꢂꢃꢄ × ꢆ
ꢅ ꢇ
ꢆ + ꢈꢂ
ꢀ
(1)
ꢀ =
ꢁ
ꢅ ꢇ
ꢀꢂꢃꢄ × ꢆ
ꢀꢁ =
(2)
ꢅ ꢇ
ꢆ
ꢊ
ꢅ ꢇ
ꢆ + ꢈꢂ ꢉ1 +
ꢋ
ꢈ
Were Ki is the inhibition constant, Km is the Michaelis constant, [S] is the concentration of the
substrate, V0 is the velocity and Vmax is the maximum velocity.
2.6. In vitro hemolytic activity
From a breast patient, a blood sample was taken on heparin tube and centrifuged at 4000 rpm at
4 ° C for 5 minutes. The pellet was washed twice with NaCl solution (150 mM) and then
centrifuged at 4000 rpm at 4 ° C for 5 minutes. Subsequently, the pellet was diluted to 2% in PBS
(saline phosphate buffer) at 10 mM, pH 7.4. 2970 ꢀL of the 2% erythrocyte suspension was

placed in contact with 30 ꢀl of ligand at different concentrations and then incubated in a benchtop
incubator (Orbital Shaker Thermo Forma) at 37 ° C. for 60 min. 100 .mu.l was taken every 15
min to be resuspended in 1900 .mu.l of PBS. The tubes are centrifuged at 4000 rpm for 5 min.
Leakage of intracellular hemoglobin was measured by reading the absorbance at 548 nm. Total
hemolytic was achieved by suspending red blood cells with Triton X-100. The buffer alone was
used as a negative control. The hemolytic rate was determined as a percentage by the following
equation (Eq.3) [31]:
Hemolysis rate (%) = [(A sample - A spontaneous hemolysis) / (Atriton X-100 - A spontaneous hemolysis))] ×100 Eq.3
3. Results and discussion
3.1. Synthesis of (E)-1-((4-hydroxynaphthalen-1-yl)methylene)thiosemicarbazide (L1)
In this work, we have modified the condensation reaction of thiosemicarbazide with 4-
hydroxy-1-naphthaldehyde [32]. So, we have prepared the ligand L1using a mixture of three
solvents: (water, ethanol and acetic acid) in a ratio of (1:3:3). As a result, the use of water and an
excess of acetic acid were more advantageous to obtain L1 in crystal form.
The formation of L1 is characterized by the presence of characteristic IR bands. Two main
absorption bands of the thionic function (C=S) appeared at 1115 cm-1 and 783 cm-1 and one band
of the azomethine function (C=N) presents at 1616 cm-1. These results are comparable to those of
literature [33, 34]. The ligand L1 exists in thione form at solid state which is confirmed by the
absence of the (S-H) band in the IR spectra (Figure S1a). In DMSO, the thiol (S-H) band appears
around 2570 cm-1 (Figure S1b). We have not found this result in literature. Furthermore, 1H NMR
spectrum of L1 in deuterated acetone gives signals at 7.4 ppm (s, 1H) and 7.7 (s, 1H) ppm
corresponding to the NH2 function (Figure S1c). In literature, the 1H NMR data for the isomer: 1-
((2-hydroxynaphthalen-1-yl)methylene)thiosemicarbazide shows that NH2 protons are observed
at 7.82 ppm [35]. These chemical shifts depend both on the position of hydroxyl group on the
aromatic rings and the used solvents.
3.2. Crystal Structure of ligand L1
The molecular structure of L1 is illustrated in Figure 1. The main crystal parameters are
reported in Table 1. The ligand L1 crystallizes with two molecules per unit cell. The sulfur and
nitrogen imine (N4) atoms are in trans-position with respect to the C1-N3 bond. This result is in
agreement with the literature [36-38]. The molecule shows an (E) conformation with respect to
the C5=N4 bond and is approximately planar. The maximum deviation from the mean plane
through the 18 non-hydrogen atoms is 0.118 (2) Ǻ for C5. This planarity is due to electron
delocalization along the skeleton of the molecule. In Table 2, we give the selected interatomic
distances and angle values. So, these bond lengths and angles agree very well with to those
observed for similar thiosemicarbazones [39]. In the crystal, the stacking of molecules is in a
zigzag shape along the b axis (Figure 2). The three-dimensional network is established by short
oxygen-sulfur contacts (d(OꢁꢁꢁS) = 3.195 Å) and three types hydrogen bonds (S1ꢁꢁꢁH10, S1ꢁꢁꢁH3 and
S1ꢁꢁꢁH2B) shown in Figure 2 (red color). These parameters are grouped in Table 3. Within this
network, there are also van der Waals interactions (CAr-H12ꢁꢁꢁH12-CAr) and CH..π interactions
(N=HC5ꢁꢁꢁC16-Ar) involving the symmetry-related naphthalene rings. Their respective distances
are 2.227 Å and 3.282 Å (blue color in Figure 2).

Figure 1. ORTEP representation of ligand L1 with 50% thermal ellipsoid plot; T=150K.
Figure 2. Packing and projection of L1 along the b axis showing S---O contacts, hydrogen bonds
and C-Hꢁꢁꢁπ interactions.
Table 1 Crystallographic parameters of L1.
Crystal data
Chemical formula
Mr
L1
C12H11N3OS
245.30
Crystal size (mm)
Crystal system
0.440 x 0.260 x 0.200
Triclinic

Space group
P-1
Unit cell parameters
a
b
c
6.8023 (Å)
8.4470 (Å)
11.1599 (Å)
2
93.353
105.959
111.749
563.33
1.446
Z
α (deg)
β (deg)
γ (deg)
V (Å3)
Dcalc (g cm-3)
T (K)
150
0.273
µ (mm-1)
F(000)
256
R(int)
0.0329
9529
2560
Total reflections
Unique reflections
I>2σ
2305
Number of parameters
R1
166
0.0318
0.0834
0.970
wR2
Goodness-of-fit (GOF)
Table 2 Bonds and angles parameters of L1.
Selected bonds (Å)
Selected angles (°)
C9 - O10 1.3555(15)
N3- C1-S1
N2- C1-S1
N2- C1-N3
C5- N4-N3
N4- C5- C6
119.78(10)
O10 - H10
C15 - H15
C6 - C7
C5 - C6
N4 - C5
N3 - N4
N3 - H3
C1 - N3
C1 - N2
C1 - S1
0.82(2)
0.9500
1.3825(17)
1.4556(17)
1.2872(17) C15-C16-C6 123.53(11)
1.3835(14) C14- C15-C16 120.78(12)
0.850(18) C9- O10- H10 106.2(14)
1.3389(16)
1.3325(17)
1.7006(13)
122.93(10)
117.29(12)
114.58(11)
125.48(11)
-
-
-
-
-
-
Table 3 Hydrogen bonds of L1.
Hydrogen bonds
D-H···
Distances (Å)
Symmetry
d(D-H)
Entry
A
d(H···A)
d(D···A)
1
2
3
2.418
2.696
2.617
0.820(2)
0.845(19)
0.850(18)
3.195
-1+x,y,z
-1+x,y,z
-1+x,y,z
O10-H10···S1
N2-H2B···S1
N3-H3···S1
-
-
3.3. Synthesis of (E)-1-(2,5-dihydroxy benzylidene)thiosemicarbazide (L2)
The ligand L2 was prepared by condensation of thiosemicarbazide with 2,5-
dihydroxybenzaldehyde in ethanol. The formation of L2 is characterized by the presence of
characterized IR bands. Two main absorption bands of the thionic function (C=S) appeared at
1161 cm-1 and at 797 cm-1 and one band of the azomethine function (C=N) presents at 1627 cm-1.
These results are comparable to those of literature [33, 34]. Ligand L1 exists in thione form at

solid state (Figure S2a). While the presence of the (S-H) band in the IR spectra appears around
2570 cm-1 in DMSO solution (Figure S2b). To our knowledge, this result has not been reported in
literature. In addition, 1H NMR spectrum of L2 in deuterated acetone shows signals at: 7.75 ppm
(s, 1H) and at 7.4 (s, 1H) ppm corresponding to the NH2 function (Figure S2c). In literature, the
1H NMR data for the isomer: 1-(2,4-dihydroxybenzylidene)thiosemicarbazide shows that NH2
protons are observed at 9.82 ppm [40]. These chemical shifts depend both on the position of
hydroxyl group on the aromatic rings and the used solvents.
4. Catecholase activities
4.1. UV-Visible studies of the ligands L1 and L2
UV-Vis spectra of L1 show two bands: first at 295 nm corresponding to n ꢀ π transition
in the thiosemicarbazone group, and second one at 365 nm corresponding to π ꢀ π transition of
the naphthalene ring (Fig 3a).
UV-Vis spectra of L2 show two bands: first at 313 nm corresponding to n ꢀπ transition
in the thiosemicarbazone group and second at 360 nm corresponding to π ꢀ π transition of the
benzyl ring (Fig 3b).
DMF
1,0
0,8
0,6
0,4
0,2
0,0
DMF
1,0
0,8
0,6
0,4
0,2
0,0
400
600
Wavelength (nm)
800
400
600
Wavelength (nm)
800
a
b
Figure 3: UV-Vis spectra of: a) ligand L1 and b) ligand L2 at 400-800 nm in DMF.
4.2. Effect of ligands concentration on the catecholase activity
The formation of the in situ complexes of copper and cobalt ions was followed by the
measurement of UV-visible absorption at room temperature in DMF. In order to study the
influence of ligand concentration to obtain the best in situ catalysts during the oxidation reaction
of 3,5-DTBC to 3,5-DTBQ, we have realized this reaction using different concentration of ligand
(L1 and L2) and metal ion (Cu and Co) (L/M: 1/1; 2/1; 1/2).
From the results obtained in Figure 6, we note that the combination of 2 moles of metal
and 1 mole of ligand leads to an excellent result. Therefore, we consider that the proportion (L/
M: 1/2) is a catalytic condition of choice for this reaction.
However, we note a particular situation in the combination of cobalt with ligands. For
ligand L1, the combinations (L1/Co: 1/2) and (L1/Co: 1/1) are similar (Figure 4c). We think that
this specificity is related to the nature and the characteristic of cobalt and may be due to the
coordinating environment.
Based on these results, we can suggest that the oxidation reaction occurs with two metal
ions associated with one ligand molecule.

0,75
0,70
0,65
0,60
0,55
0,50
0,45
0,40
0,35
1,3
1,2
1,1
1,0
0,9
0,8
0,7
0,6
L1 CuAc 1:1
L1 CuAc 2:1
L1 CuAc 1:2
L2 CuAc 1:1
L2 CuAc 2:1
L2 CuAc 1:2
0
10
20
30
40
50
60
0
10
20
30
40
50
60
Time (min)
Time (min)
a
b
2,8
2,6
2,4
2,2
2,0
1,8
1,6
1,4
1,2
1,0
0,8
0,6
1,8
1,6
1,4
1,2
1,0
0,8
0,6
0,4
0,2
L1 CoAc 1:1
L1 CoAc 2:1
L1 CoAc 1:2
L2 CoAc 1:1
L2 CoAc 2:1
L2 CoAc 1:2
0
10
20
30
Time (min)
40
50
60
0
10
20
30
Time (min)
40
50
60
c
d
Figure 4: Oxidation of 3,5-DTBC with different concentration of copper acetate and ligands. a)
L1Cu, b) L2Cu, c) L1Co and d) L2Co.
4.3. UV-Visible studies of the in situ complexes
UV-Vis survey scans were carried out at room temperature in DMF. UV-Vis spectra of in
situ copper complexes (Cu/L1: 2/1) show different bands (Figure 5a). First band at 295 nm
corresponds to n
corresponds to π
ꢀ
π transition of the thiosemicarbazide group. Second one at 365 nm
ꢀ
π transition of the naphthalene ring. In the course of complexation, we note a
hypsochromic effect of the band at 280 nm of the thiosemicarbazide group and bathochromic
effect of the band at 380 nm of the naphthalene ring. The appearance of two bands, one at 400
nm and the other at 620 nm is attributed to d-d transition which can suggests two geometries:
square plane and square pyramidal respectively (Scheme 1a) [41]. In the case of in situ cobalt
complexes (Co/L1: 2/1), hypsochromic and bathochromic effects are not appearance (Figure 5b).
The presence of two bands at 530 nm and 580 nm are attributed to d-d transition that suggests a
pseudo-octahedral geometry (Scheme 1b) [42]. Thus, the cobalt charge is Co+3 because of its
oxidation with air oxygen.
UV-Vis spectra of in situ copper complexes (Cu/L2: 2/1) show different bands (Figure
5c). The band at 315 nm corresponds to n
ꢀ π transition of the thiosemicarbazide group. Another
one at 360 nm corresponds to π π transition of the benzene ring. During complexation, we note
ꢀ
a bathochromic effect of the two bands of thiosemicarbazide group and benzyl group at 330 nm
and 420 nm respectively. The bands at 410 nm and at 610 nm are attributed to d-d transition
suggesting square plane and square pyramidal geometries respectively (Scheme 1c) [43]. In the
case of in situ cobalt complexes (Co/L2: 2/1), the hypsochromic effect appears of benzyl and
thiosemicarbazide groups at 265 nm and 345 nm respectively (Figure 5d). The two bands at 430

nm and 600 nm are attributed to d-d transition that suggests a distorted octahedral geometry
(Scheme 1d) [44]. Similarly, the cobalt charge is Co+3 because of its oxidation with air oxygen.
In addition, by comparing the results given in Fig.5, we note that the characteristics of the
in situ copper(II) and cobalt(III) complexes are similar to those found in the literature [45]. Thus,
we propose complex structures obtained during the oxidation process. The complex structures are
given in Scheme 1.
0 min
5 min
0 min
5 min
0,07
0,06
0,05
0,04
0,03
0,02
0,01
0,00
0,05
0,04
0,03
0,02
0,01
0,00
0,8
0,6
0,4
0,2
0,0
10 min
15 min
20 min
25 min
30 min
35 min
40 min
45 min
50 min
55 min
60 min
L1
10 min
15 min
20 min
25 min
30 min
35 min
40 min
45 min
50 min
55 min
60 min
L2
1,0
0,8
0,6
0,4
0,2
0,0
600
800
600
800
Wavelength (nm)
Wavelength (nm)
Complex
Complex
300
400
500
600
700
800
300
400
500
600
700
800
Wavelength (nm)
Wavelength (nm)
a
b
0 min
0 min
0,12
5 min
5 min
0,14
2,4
1,8
10 min
15 min
20 min
25 min
30 min
35 min
40 min
45 min
50 min
55 min
60 min
L1
10 min
15 min
20 min
25 min
30 min
35 min
40 min
45 min
50 min
55 min
60 min
L2
0,10
0,08
0,06
0,04
0,02
0,00
0,12
0,10
0,08
0,06
0,04
0,02
0,00
2,2
2,0
1,8
1,6
1,4
1,2
1,0
0,8
0,6
0,4
0,2
0,0
1,6
1,4
1,2
1,0
0,8
0,6
0,4
0,2
0,0
600
800
Wavelength (nm)
600
Wavelength (nm)
800
Complex
Complex
300
400
500
600
700
800
300
400
500
600
700
800
Wavelength (nm)
Wavelength (nm)
c
d
Figure 5: UV/Vis analysis of the 3,5-DTBC oxidation by in situ complexes obtained with 1/2
ligand/copper (II) acetate combination over time. a) L1Cu, b) L2Cu, c) L1Co and d) L2Co.
OH2
OH
HO
HO
H
O
N
O
Me
HO
Cu
S
N
N
OH
O
N
O
Cu
Cu OAc
O
N
Cu
O
S
NH
Me
OAc
OH
AcO
OH
a
b

OH2
HO
H
OH2
AcO
N
H2O
Co
S
N
OAc
OAc
O
N
N
OH
O
HO
Me
Co
OH
N
Co
O
Co
H2O
OAc
S
NH
AcO
OH2
AcO
OH2
OH
c
d
Scheme 1. Proposed structures for the in situ complexes (Me/L: 2/1). a) 2Cu(AcO)2.L1.H2O, b)
2Cu(AcO)2.L2, c) 2Co(OH)(AcO)2..L1.2H2O and d) 2Co(OH)(AcO)2.L2.H2O.
4.4. Michaelis-Menten kinetics
4.4.1. Oxidation reactions using in situ copper complexes (L1Cu and L2Cu)
Catechol oxidation reactions were realized using different concentration of copper acetate
and ligands (L/Cu: 1/1, 1/2, 2/1). In Figures 6, we give the absorbance of 3,5-DTBQ for each
proportion. According to the results given in Table 4, we note that the combination obtained by 2
moles of copper and one mole of each ligand gives the best catalytic condition for the oxidation
reaction of 3,5-DTBC (Table 4, entries 2 and 5).
For ligand L1, we note that the kinetic models of two in situ copper complexes (L1/Cu:
1/2 and L1/Cu: 2/1) follow the model of enzyme inhibition by excess of substrate. Thus, the
complexes exhibit an increase in activity as the concentration increases but beyond a certain
concentration of substrate. At the concentration 4.10-2 M of (L1/Cu: 1/2), the activity decreases
due the inhibition effect caused by the excess of substrate by applying the modified Michaelis-
Menten model (Equ.2) while the in situ complex (L1/Cu: 1/1) follow the simple Michaelis-
Menten model (Equ.1) (Figure 8a). The complex obtained from copper (II) acetate (L1/Cu: 1/2)
shows highest activity (Table 4, entry 2, Vmax = 7.57 x 10-5mol. L-1.min-1, Kcat=45.42 h-1) if
compared to those of proportions: (L1/Cu: 1/1) (Table 4, entry 1, Vmax = 1.98 x 10-5 mol. L-1.min-
1, Kcat=11.90 h-1) and (L1/Cu: 2/1) (Table 4, entry 3, Vmax= 1.82 x 10-5 mol. L-1.min-1, Kcat= 10.92
h-1) (Figure 6a). To our knowledge, this study has not been reported in literature.
For ligand L2, we note that the kinetic models of different in situ copper complexes
follow the simple Michaelis-Menten equation (Eq. 1). The kinetic parameters reveal that the
complex obtained from 2 moles of copper (II) acetate and one mole of ligand L2 exhibits the
highest activity (Table 4, entry 5, Vmax = 2.39 x 10-5 mol. L-1.min-1, Kcat= 14.34 h-1) while those
of proportion (L2/Cu: 1/1) presents a moderate catalytic activity (Table 4, entry 4, Vmax = 1.14 x
10-5 mol. L-1.min-1, Kcat= 6.84 h-1) and those of (L2/Cu: 2/1) presents low catalytic activity (Table
4, entry 6, Vmax = 0.738 x 10-5 mol. L-1.min-1, Kcat= 4.43 h-1 respectively) (Figure 6b).
From these results, we conclude that the associations of ligands L1 and L2 with copper
acetate contribute in higher catalytic activity for in situ complexes. We can explain that with the
nature of the acetate ion and its large size, which helps to create enough steric hindrance around
the metallic center allowing the increase in the catalytic activities of the complexes [9].

Table 4 Kinetic parameters of in situ complexes
105 Vmax (mol L-1 min-1) 102 Km(mol L-1) Kcat (h-1) 102 Ki (mol L-1)
Entry
Complex
1
2
3
4
5
6
L1[Cu(CH3COO)2] 1:1
L1[Cu(CH3COO)2] 1:2
L1[Cu(CH3COO)2] 2:1
L2[Cu(CH3COO)2] 1:1
L2[Cu(CH3COO)2] 1:2
1.98
7.57
1.82
1.14
2.39
2.74
1.92
0.99
6.65
0.23
11.90
45.42
10.92
6.84
-
4.02
9.20
-
14.34
-
L2[Cu(CH3COO)2] 2:1
0.74
3.30
4.43
-
7
8
9
10
11
12
L1[Co(CH3COO)2] 1:1
L1[Co(CH3COO)2] 1:2
L1[Co(CH3COO)2] 2:1
L2[Co(CH3COO)2] 1:1
L2[Co(CH3COO)2] 1:2
L2[Co(CH3COO)2] 2:1
0.01
0.15
0.35
0.90
1.03
1.10
0.06
22.60
16.40
8.95
3.32
11.80
0.40
135.60
98.40
53.70
19.92
70.80
2.43
-
-
-
-
-
L1 Cu Act 1:2
L1 Cu Act 2:1
L1 Cu Act 1:1
0,000035
0,000030
0,000025
0,000020
0,000015
0,000010
0,000005
0,000000
-0,000005
L2 Cu Act 1:2
L2 Cu Act 2:1
L2 Cu Act 1:1
0,000025
0,000020
0,000015
0,000010
0,000005
0,000000
-1
0
1
2
3
4
5
6
7
8
-1
0
1
2
3
4
5
6
7
8
[S] (1-2 M)
[S] (1-2 M)
a
b
Figure 6. Kinetic studies of the 3,5-DTBC oxidation with different concentration of copper
acetate and ligands: a) L1Cu and b) L2Cu.
4.4.2. Oxidation reactions using in situ cobalt complexes (L1Co and L2Co)
Many transition metals were elaborated to investigate their catecholase activities such as
Nickel, Zinc and cobalt [7, 12, 13]. In this work, we opted to test the catalytic capacity of in situ
complexes of cobalt using the same ligands. In situ cobalt complexes were prepared in the same
manner using cobalt (II) acetate.
From Figure 7a, we note that in situ L1Co complex (L1/Co: 1/1) follows the model of
inhibition by excess of substrate at the concentration 3.10-2 M and applying the modified
Michaelis-Menten equation (Eq. 2) (Table 4, entry 7, Vmax = 22.6 x 10-5 mol.min-1L-1, Kcat=
135.6). It shows highest activity (7 times) if compared to in situ L1Co (L1/Co: 1/2) and L1Co
(L2/Co: 2/1) complexes which follows the simple Michaelis-Menten model (Eq. 1) (Table 4,
entries 8 and 9, Vmax = 16.4 x 10-4 mol.min-1L-1, Kcat= 98.4 h-1, Vmax = 8.95 x 10-4 mol.min-1L-1,
Kcat= 53.7 h-1 respectively).
For ligand L2, we note that the kinetic models of different in situ cobalt complexes follow
the simple Michaelis-Menten equation (Eq. 1). The kinetic parameters reveal that the complex
obtained from 2 moles of cobalt (II) acetate and one mole of ligand L2 exhibits the highest
activity (Table 4, entry 11, Vmax = 11.8 x 10-5 mol. L-1.min-1, Kcat= 70.8 h-1) while those of

proportion (L2/Cu: 1/1) presents good catalytic activity (Table 4, entry 10, Vmax = 3.32 x 10-5
mol. L-1.min-1, Kcat= 19.92 h-1) and those of (L2/Cu: 2/1) presents low catalytic activity (Table 4,
entry 12, Vmax = 0.404 x 10-5 mol. L-1.min-1, Kcat= 2.43 h-1 respectively) (Figure 7b).
0,00012
0,00018
0,00010
0,00016
L1 Co Act 1:2
L1 Co Act 2:1
L1 Co Act 1:1
0,00014
0,00012
0,00010
0,00008
0,00006
0,00004
0,00002
0,00000
-0,00002
L2 Co Act 1:2
L2 Co Act 2:1
L2 Co Act 1:1
0,00008
0,00006
0,00004
0,00002
0,00000
-1
0
1
2
3
4
5
6
7
8
-1
0
1
2
3
4
5
6
7
8
[S] (1-2 M)
[S] (1-2 M)
a
b
Figure 7. Kinetic studies of the 3,5-DTBC oxidation with different concentration of cobalt
acetate and ligands : a) L1Co and b) L2Co.
As shown in Table 4, the difference of the catalytic activities between the two metal
complexes is apparent. This is due to the nature and the electronic properties of the each metal
ion. Moreover, the steric hindrance occurs between the substrate and the active site of the
complex. Thus, the cobalt atomic radius is larger and it has greater oxygen affinity compared to
copper atom. Furthermore, the difference in geometry of the ligands L1 and L2 have an effect on
the catalytic activity. As shown in Figure 1, L1 geometry is wide which can creates more steric
hindrance around the metal center. As result, the complex geometry is affected and the activity is
rise [46]. In each case, the activities of cobalt complexes are more important than that of copper
one (Figure 8) [12].
L2[Cu(CH3COO)2]2
L2[Co(CH3COO)2]2
L1[Cu(CH3COO)2]2
L1[Co(CH3COO)2]2
1,2x10-4
1,0x10-4
8,0x10-5
6,0x10-5
4,0x10-5
1,8x10-4
1,6x10-4
1,4x10-4
1,2x10-4
1,0x10-4
8,0x10-5
6,0x10-5
4,0x10-5
2,0x10-5
0,0
2,0x10-5
0,0
0
1
2
3
4
5
6
7
0
1
2
3
4
5
6
7
[S] (1-2 M)
[S] (1-2 M)
a
b
Figure 8. Kinetic studies of the 3,5-DTBC oxidation with different concentration of copper
acetate and cobalt acetate and ligands: a) L1Cu / L1Co and b) L2Cu / L2Co. (L1/Co: 1/2) and
(L2/Co: 1/2)

The catalytic oxidation reaction of 3,5-DTBC under the same conditions with ligands and metal
salts alone was performed to evaluate their catalytic efficiency. The results show that there is low
activity comparing with that of in situ complexes. These results are given in Table 5.
Table 5. Comparison of kinetic parameters of in situ complexes (copper and cobalt) with ligands
(L1 and L2) and metallic salts (Cu(CH3COO)2 and Co(CH3COO)2).
Complex/Parameters
L1
Vmax (mol L-1 min-1)
6.1E-6
Linear dependence on [S]
6.56E-6
Km (mol L-1)
6.72E-2
-
L2
Cu(CH3COO)2
Co(CH3COO)2
in situ complex (Cu/L1:2/1)
in situ complex (Cu/L2:2/1)
in situ complex (Co/L1:2/1)
in situ complex Co/L2:2/1)
5,3E-2
3.42E-2
1.92
0.23
0.01
1.4E-5
7.57 E-5
2.39 E-5
22.60 E-5
11.80 E-5
1.03
By comparing the catalytic efficiency of our in situ complexes with other results of the
literature (give these results), we find that our complexes have higher maximum velocity values.
We conclude that the catalytic activity of our in situ copper and cobalt complexes is higher
compared to those prepared by Bouanane et al. And Mouadili et al [47, 48].
5. Hemolysis assay
Figures 9 show the evolution of the rate of hemolysis in percentage during the 60 min.
The Ligand L1 shows an increase in hemolysis rate with concentration as a function of time (15,
30, 45, 60 min). For concentrations of 100 ꢀg/mL and 50 ꢀg/mL, the rate of hemolysis increases
remarkably from the first minutes until reaching 60 min, 116 % and 82 %. However for the 25
ꢀg/mL concentration, hemolysis only begins to evolve after 30 minutes and reaches 42 % after 60
minutes. We also find that there is no hemolysis with the concentration 12.5 ꢀg/mL (Figure 9a).
Further, no hemolytic effect was observed with the L2 ligand whatever the concentration used
(Figure 9b). We can conclude that the L1 ligand is toxic to red blood cells from the concentration
25 ꢀg/mL, while L2 has no toxicity to red blood cells.
a)
b)
Figure 9: Evolution of the hemolysis rate as a function of time for: a) ligand L1, b) ligand L2.

6. Conclusions
Two novel thiosemicarbazone ligands L1 and L2 has been synthesized and characterized
1
by FT-IR, H NMR and mass spectroscopy analysis. L1 structure was also determined by single
crystal X-ray diffraction. In situ catecholase activity of L1 and L2 against 3,5-DTBC was
investigated with copper and cobalt transition metals. Catechol oxidation reactions were realized
using different concentrations of copper and cobalt acetate and ligands (L/Cu; L/Co): 1/1, 1/2,
2/1). The oxidation reaction rates were analyzed with Michaelis-Menten equations, and the
kinetic parameters were calculated. The results show that all in situ complexes were able to
catalyze the oxidation of 3,5-DTBC. However, acetate metal salt complexes always present the
highest catecholase activity. The application of the Michaelis-Menten equations revealed a
difference in the mode of activity between copper and cobalt complexes. Oxidation followed by
inhibition by excess of substrate concenbtration was applied to L1 complexes. Oxidation
activation was applied to L2 complexes. The study also revealed that both the ligand structure
and the metal have a major influence on the catalytic activity and model. Hemolysis tests
revealed that the L1 ligand is toxic to red blood cells from the concentration 25 ꢀg/mL, while L2
has no toxicity to red blood cells.
Supplementary Information
CCDC No. 1451400 can be obtained free of charge via http://www.ccdc.cam.ac.uk/conts/
retrieving.html.
Acknowledgments
Authors acknowledge the Algerian Ministry for Higher Education and Research for supporting
this research.
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[48] Moualdidi et al.

Highlights
• Synthesis of new thiosemicarbazone derivative L1 and L2 ligands
• Crystal structure of L1
• Copper and Cobalt in situ complexes catecholase activity
• In situ complexes of L1 act as catalysts and inhibitors
• In situ complexes of L2 act as a catalysts
• Hemolytic activity
.

Author Contribution Statement
Ousama Nehar: realization of all the experimental part (except hemolysis), tracet graphs,
interpretation, validation of results, writing and correction of a part of the article.
Radia Mahboub: interpretation of part of the results and correction of the writing.
Samira Louhibi: Interpreting the results, writing and correcting the writing of part of the
article.
Thierry Roisnel: Single crystal study
Mohammed Aissaoui: Writing and realization of the experimental hemolysis partwriting and
correction of a part of the article.

Declaration of interests
☒ 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.
☐The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests: