Design and fabrication of novel thiourea coordination compounds as potent inhibitors of bacterial growth

Article abstract of DOI:10.1038/s41429-019-0147-2

A new thiourea ligand (HL), namely N-(4-chlorophenyl)morpholine-4-carbothioamide and its Co(III), Ni(II) and Ag(I) complexes (1a, 1b and 1c) were synthesized and investigated by Fourier-transform infrared, ¹H NMR and UV-visible spectroscopies.

Full text of DOI:10.1038/s41429-019-0147-2

The Journal of Antibiotics
https://doi.org/10.1038/s41429-019-0147-2
ARTICLE
Design and fabrication of novel thiourea coordination compounds
as potent inhibitors of bacterial growth
1 ●
1 ●
Michal Dušek Václav Eigner2
2 ●
Sajjad Rakhshani
Ali Reza Rezvani
Received: 10 October 2018 / Revised: 19 December 2018 / Accepted: 16 January 2019
The Author(s), under exclusive licence to the Japan Antibiotics Research Association 2019
©
Abstract
A new thiourea ligand (HL), namely N-(4-chlorophenyl)morpholine-4-carbothioamide and its Co(III), Ni(II) and Ag(I)
1
complexes (1a, 1b and 1c) were synthesized and investigated by Fourier-transform infrared, H NMR and UV-visible
spectroscopies. The compounds HL and 1c were characterized by single-crystal X-ray crystallography revealing the triclinic
space group P1 for both compounds. The inhibitory effect of HL ligand, 1a, 1b, and 1c complexes was investigated with in vitro
tests on Gram-positive and Gram-negative bacteria. For the 1c complex, the results showed that the coordination of the HL to
Ag(I) ion increased its antibacterial effect especially against E. coli. The assays also indicated that for the same bacteria strains,
the new complexes showed higher activity than the ligand, with the relative activity 1c > 1b > 1a > HL. Moreover, all samples
were more suitable antimicrobial agents against the Gram-negative than those of the Gram-positive bacteria. Eventually, the
relationship between the structure and bactericidal activities of these specimens was examined by calculating frontier molecular
orbital (HOMO and LUMO) energies using density functional theory method at the 6‐31 G*/LANL2DZ level of theory.
Introduction
infectives and is used for the treatment of infections caused
by Gram-positive bacteria [7]. On the other hand, the
complexation capacity of thiourea derivatives and the bio-
logical activities of thiourea complexes are well recognized
for different biological systems [8–10].
Among the transition metal, cobalt, nickel, and silver
ions have been attracted the biologists attention due to their
individual characteristics. A large number of reports on the
antibacterial characteristics of cobalt complexes have
developed in the literature and selecting Co complexes as
the main subjects for the study in order to find effective
antibacterial agents still being continued [8, 11]. Nickel is
an essential trace element for bacteria, plants, animals, and
humans [12]. There is spectroscopic evidence for
nickel–methyl complexes in CODH of anaerobic bacteria
Thiourea compounds are important ligands in coordination
chemistry, indispensable intermediates for the preparation
of organocatalysts or ancillaries in chemosensors, and
important part of anticorrosion compounds [1, 2]. They are
widely recognized for their biological and antibacterial
properties, with increasing use as antitubercular or anti-
tumor agents, insecticides, antiprotozoal agents, and herbi-
cides [3−5]. In addition, morpholine derivatives possess
anti-inflammatory, antimicrobial and central nervous system
activities [6]. The morpholine nucleus incorporated in a
wide variety of therapeutically important drugs like line-
zolid, which belongs to the oxazolidinone class of anti-
[
13]. For instance, the nickel transport system in E. coli
composed of five proteins, NiKABCDE.
Supplementary information The online version of this article (https://
doi.org/10.1038/s41429-019-0147-2) contains supplementary
material, which is available to authorized users.
On the other hand, silver deposition has been found in the
skin, the lungs, the gingiva, mucous membranes, and gas-
trointestinal tract [14, 15]. Essentially complex structures of
silver-protein are take in within the body but have no phy-
siological or biochemical roles [14, 16, 17]. Ag complexes
have a very broad range of antibacterial activity and annihilate
both Gram-positive and Gram-negative bacteria. Silver is a
more lethal element to microorganisms than many other
metals [18–20]. Actually, silver destructs the structure of
*
Ali Reza Rezvani
ali@hamoon.usb.ac.ir
1
2
Department of Chemistry, University of Sistan and Baluchestan,
Zahedan P.O. Box 98135-674, Iran
Institute of Physics, Academy of Sciences of the Czech Republic,
Na Slovance 2, 182 21, Praha 8, Czech Republic

S. Rakhshani et al.
Scheme 1 .Synthetic route to HL ligand, 1a, 1b, and 1c complexes
bacterial cell membrane, prevent the cell respiration, and
increase or diminish the expression abundance of some
enzymes [20]. Indeed, the mechanism for the antibacterial
activity of silver complex is not correctly recognized;
although it is accepted that they intervene with cell growth via
interruption of cell metabolism, restraint of transport actions
in the cell wall, and interaction with DNA [14].
(Scheme 1). This ligand was recrystallized by ether diffu-
sion into the N,N-Dimethylformamide solution of the
ligand.
According to the Scheme 1, the Co(III), Ni(II), and Ag(I)
complexes, 1a, 1b, and 1c were prepared by adding a hot
methanolic solution (50 °C) of the respective metal nitrate
to a solution of the HL ligand in the molar ratio of 1:3 for
1a and 1:2 for 1b and 1c. The synthesized Co(III), Ni(II),
In this research, we report the synthesis of a novel
thiourea derivative, N-(4-chlorophenyl)morpholine-4-car-
bothioamide (HL) and its Co(III), Ni(II), and Ag(I) com-
plexes (1a, 1b, and 1c) and their characterization by FT-IR,
and Ag(I) complexes are soluble in CHCl , DMF, and
3
DMSO. These compounds were characterized by UV-vis,
1
H NMR, FT-IR spectroscopy and the single-crystal X-ray
1
H NMR, and UV-vis spectroscopy. For HL and its 1c
diffraction analysis.
complex, crystal structures were determined using X-ray
single-crystal diffraction methods. We also evaluated the
antibacterial activities of these compounds using three
methods, macro dilution (MIC), colony count and well
diffusion. The relationship between the structures of these
specimens and their antimicrobial activity was prospected
based on density functional theory (DFT) calculations.
FT-IR spectral studies
In the FT-IR spectra of HL ligand, the band at 3159 cm−
could be attributed to the stretching frequencies of the N–H
group. Stretching vibrations of C = S bond was observed at
1
−
1
1323 cm . Symmetric and asymmetric stretching vibra-
−
1
tions of (-CH ) protons appeared at 2861–2908 cm . The
2
−
1
absence of ν(S-H) band at about 2570 cm and the pre-
sence of the ν(N-H) band at about 3159 cm− indicated
formation of the thione tautomer in the solid state (Fig-
ure S1a in the supplementary content). After complexation
of the HL ligand to the Co(III) and Ni(II) the N-H stretching
vibrations disappeared due to the loss of the H bonded to
the N atom of thioamide group (Figure S1b and c, in the
1
Results and discussion
Synthesis
The reaction between 4-chlorophenyl isothiocyanate and
morpholine in acetone resulted in the HL ligand

Design and fabrication of novel thiourea coordination compounds as potent inhibitors of bacterial growth
supplementary content). Also, the vibrational frequencies
corresponding to C = S group in the free ligand were shifted
towards down fields upon the complexation, confirming that
the HL ligand is coordinated to the cobalt and nickel ions
via the nitrogen and sulfur atoms in a form of neutral
complexes (1a and 1b) [21, 22].
Electronic absorption spectroscopy
CHCl solutions of the thiourea ligand HL and its metal
3
complexes 1a, 1b, and 1c were prepared and their electronic
absorption spectra were recorded in the range of
200–800 nm. For HL, the observed absorption bands at 240
and 270 nm were assigned to intra-ligand charge transfer
The FT-IR spectrum of the 1c complex demonstrates
bands similar to those of ligand with a slight shift to the
lower/upper wavenumbers owing to its coordination with
Ag(I) metal center in the complex. For example, the char-
*
*
transitions π–π (phenyl ring) and n–π corresponding to C
= S bond [25, 26].
As expected from the results of FT-IR spectroscopy, no
−
1
*
acteristic band at 3208 and 1304 cm can be assigned to N-
H and C-S stretching vibration of 1c complex. Therefore,
the presence of the ν(N-H) band and shifting of the ν(C-S)
band towards lower wavenumber confirmed that the HL
coordinates to the Ag(I) via S atom, unlike Co(III) and Ni
significant shifts in the π–π transitions were observed after
complexation but a bathochromic shift that corresponding
*
to the n–π transitions of C-S bond were recognized, which
demonstrated that the ligand coordinated to the Co(III) and
Ni(II) through the N and S atoms. Accordingly, in the 1a
and 1b, bands above 340 nm are attributed to charge
transfer processes, probably from the ligand to the metal.
Absorption bands at higher wavelengths in the visible
region (650 and 635 nm for 1a and 1b, respectively) were
due to d–d transitions. In the 1c complex, the absorption
band corresponding to the n–π* transition of C = S is
demonstrating a bathochromic shift (265 nm) due to the
complexation of thione sulfur atoms to metal ion. Generally,
in electronic absorption spectra of 1c complex, no d–d
(
II) complexes. The infrared absorption band of the nitrate
anion in this complex exhibits that the nitrate isn’t coordi-
nated to Ag(I) center. This fact was deduced from the strong
−
1
new band revealed at 1343 cm (Figure S1d in the sup-
plementary content) [14, 23].
NMR spectral studies
1
The H NMR spectra were recorded at room temperature at
1
0
3
00 MHz in DMSO-d for ligand, Co(III) and Ag(I) com-
transition was observed due to d electronic configuration
of Ag(I) ion [27–29].
6
plexes and in CDCl for Ni(II) complex (Figure S2 a-d, in
3
the supplementary content). The formed thione tautomer of
HL instead of thiol tautomer was proved by presence of NH
resonances at δ 9.42 ppm and absence of SH resonances.
The phenyl protons and the diastereotopic hydrogens of
Description of crystal structures
Crystal structure of HL
-
CH (morpholine moiety) were observed at δ 7.32–7.39
2
and δ 3.65–3.92 ppm, respectively.
The structure of HL was affirmed by the results of single-
crystal X-ray structure determination. The molecule of this
compound is shown in Fig. 1, details about crystal data
collection and structure refinement and important bond
lengths and angles are summarized in Table 1. The bond
length of thiocarbonyl (C7-S1 = 1.685(5) Å) lies between
values of a single and a double bond [30, 31]. On the other
hand, due to the delocalization of electron density in the
ligand, the mentioned C–N bonds, C7-N1 = 1.365(1) Å,
Based on the results, complexation of HL to Ag(I)
through S atom is approved by the presence of the N–H
signal at δ 10.01 ppm. Indeed, pursuant to the Lewis acids
and bases theory, the soft Ag(I) metal center tends to
coordinate to the sulfur atom of the ligand than the
nitrogen or oxygen atoms. In the spectrum of 1c, each
peak is shifted toward upfield from its normal peak
position corresponding to HL. The diastereotopic hydro-
gens of -CH (morpholine moiety) and phenyl protons are
2
observed at δ 3.72–3.75, δ 3.91–3.94 and δ 7.19–7.46
ppm, respectively.
On the other hand, the loss of NH resonances in the 1a
and 1b metal complexes confirmed that the chelation of HL
ligand to the Co(III) and Ni(II) occurs via N and S atoms.
The diastereotopic hydrogens of -CH morpholine moiety
2
appeared at δ 3.06–3.78 and δ 3.14–3.86 ppm for 1a and
1
b, respectively. Similarly, the characteristic signal of the
phenyl protons corresponding to 1a and 1b was observed at
δ 6.40–7.64 and δ 6.55–7.26 ppm, respectively. Both FT‐IR
and NMR data are in agreement with the proposed struc-
tures [24].
Fig. 1 ORTEP diagram of HL ligand. CCDC number is 1884234

S. Rakhshani et al.
Table 1 Summary of X-ray diffraction data and selected bond lengths (Å), angles (°), torsion angles (°) for HL ligand and Ag(I) complex (1c)
Compound
HL
1c
Bond lengths (Å) of HL
Bond lengths (Å) of 1c
Empirical formula
Formula weight
Wavelength (Å)
Crystal system
Space group
a (Å)
C14H20Cl1N3O2S1 C22H26Ag1Cl2N5O5.335S2 C7-S1
1.6855(10)
1.3652(17)
1.4178(15)
0.880(4)
Ag1-S1b
S1b-C1b
C1b-N2b
C1b-N1b
N2b-H1n2b
C6b-N2b
Ag1-S1a
S1a-C1a
2.3969(6)
1.717(2)
1.350(2)
1.343(3)
0.86(2)
329.8
688.7
C7-N1
0.71073
Triclinic
P 1
1.54184
Triclinic
P 1
C1-N1
N1-H1n1
C7-N2
1.3523(15)
1.4708(17)
1.4669(14)
1.4217(18)
1.7458(12)
1.234(2)
8.7614(3)
9.8877(4)
10.3554(4)
73.963(4)
84.073(3)
66.620(3)
791.37(6)
2
10.7920(2)
10.8255(2)
13.2124(2)
66.1841(17)
74.4009(14)
75.0505(15)
1340.45(4)
2
C11-N2
C8-N2
1.416(3)
2.3925(5)
1.7197(2)
1.338(2)
1.353(2)
0.86(1)
b (Å)
c (Å)
C9-O1
α (°)
C4-Cl1
C1s-O1s
N1s-C1s
N1s-C2s
N1s-C3s
C1a-N1a
C1a-N2a
N2a-H1n2a
C6a-N2a
β (°)
γ (°)
V (Å3)
1.3265(15)
1.4537(19)
1.455(2)
1.416(2)
Z
Dcalc (g/cm3)
μ (Mo Kα)
F(000)
1.3842
1.7064
Bond angles (°) of 1c
0.381
9.685
S1a-Ag1-S1b
N1b-C1b-N2b
S1b-C1b-N2b
C1b-N2b-H1n2b
N1a-C1a-N2a
S1a-C1a-N2a
S1a-C1a-N1a
C1a-N2a-H1n2a
175.007(2)
117.09(19)
123.67(17)
119.3(15)
115.97(16)
124.08(14)
119.95(12)
114.9(14)
348
697
Bond angles (°) of HL
θ range (°)
Reflection collected
Independent reflection
Rint
3.47 to 29.64
13152
3.72 to 74.47
22315
S1-C7-N1
S1-C7-N2
N1-C7-N2
C7-N1-C1
C7-N2-C11
122.73(8)
122.35(10)
114.88(9)
123.16(9)
122.34(9)
3957
5407
0.0207
0.0284
Data/parameters
GOF on F2
R1 [I > 3σ(I)]
wR2 [I > 3σ(I)]
R1 (all data)
wR2 (all data)
Largest diff. peak/hole
3957/193
1.40a
5407/367
1.35a
0.0296
0.0223
Torsion angles (°) of HL
0.0843
0.0689
S1-C7-N2-C11
S1-C7-N2-C8
S1-C7-N1-C1
4.6(2)
Torsion angles (°) of 1c
0.0381
0.0229
−163.52(12)
Ag1-S1a-C1a-N1a −25.976(164)
0.0905
0.0697
8.6(2)
Ag1-S1b-C1b-N1b
21.522(178)
0.22/−0.16
0.45/−0.42
C1-N1-C7-N2 −173.61(13)
−
3
(
e Å )
aJANA2006 does not refine the weighting scheme. Therefore, the goodness of fit is usually fairly above 1, especially for well-exposed data bearing
information about bonding electrons.
C7-N2 = 1.353(3) Å, become shorter in comparison with
the typical single C–N bond length of 1.479 Å [C8-N2 =
1
.466(9) Å and C11-N2 = 1.470(8) Å] [32, 33]. This
delocalization is also confirmed by values of the angles N1-
C7-N2 = 114.87(7)°, N1-C7-S1 = 122.73(6)° and N2–C7-
2
S1 = 122.35(0)° indicating sp hybridization on the C7
atom [34].
Crystal structure of 1c
Figure 2 shows that the Ag center is coordinated to two
ligands in a neutral monodentate (S) binding mode, giving
rise to a nearly linear geometry around Ag [S1a-Ag1-S1b =
1
75.007(19)°]. The crystal structure also contains a free
disordered NO anion and two partially occupied crystal
3
Fig. 2 ORTEP diagram of 1c complex. CCDC number is 1884216.
(The hydrogens of water molecules and nitrate ion were omitted for
more clarity)
water molecules, one of them being weakly coordinated to
Ag (Ag1-O2w = 2.5115(216) Å). A summary of selected
bond lengths and bond angles for the 1c complex is given in

Design and fabrication of novel thiourea coordination compounds as potent inhibitors of bacterial growth
Table 1. The C–S bond lengths for 1c are longer than that
for HL [35, 36]. Other bond lengths and angles are in good
agreement. For other newly synthesized complexes (1a and
explaining the relationship between the bactericidal activity
and lipophilicity. As we know, the lipid membrane that
envelopes the cell favors the passage of lipid-soluble sub-
stances [41–47]. Furthermore, the presence of the chlorine
moiety in the structure of these reagents may also contribute
to their antibacterial effect due to the hypochlorous acid
formation. This acid crumbles forming hydrochloric acid
and oxygen, and the oxygen damages microorganisms
through oxidizing cellular parts. Chlorine compounds can
also interact and combine with proteins and enzymes of
membranes and as a result, destroy the microbes or prevent
their growth by obstruction of their active sites [48].
Meanwhile, the presence of the chlorine atom and the
morpholine group increases the lipophilicity and the
electron-withdrawing effect on these samples, and this can
also improve the bactericidal effects [49, 50]. Although
antimicrobial activity of all complexes was observed to
have the notable effect versus all tested Gram-negative and
Gram-positive bacteria, they exhibited lower activity com-
pared to standard antibiotics amikacin and gentamicin.
1
b) several attempts failed to obtain a single-crystal suitable
for X-ray crystallography.
Antibacterial activities
The bactericidal properties of HL, 1a, 1b, 1c, and the
reference antibiotics amikacin (AMK-30 µg) and gentami-
cin (GEN-10 µg) were examined against two Gram-nega-
tive; Escherichia coli (ATCC 25922) and Pseudomonas
aeruginosa (ATCC 27853) and two Gram-positive; Sta-
phylococcus aureus (ATCC 25923) and Enterococcus fae-
calis (ATCC11700) bacteria. DMSO was employed as a
negative control while amikacin and gentamicin were uti-
lized as positive controls for Gram-positive and Gram-
negative antibacterial activity, respectively. Silver sulfa-
diazine (AgSD) is regarded as reference Ag(I) specimen for
susceptibility testing [14, 37]. Macro dilution, colony count,
and well diffusion methods were employed to characterize
the antimicrobial activity of these reagents.
Well diffusion assay
Minimum inhibitory concentration (MIC) assay
The results of well-diffusion method for antimicrobial
activity of synthesized samples show the highest zone of
inhibition (19.7 mm) for 1c against E. coli. The results
(Table 2) show the studied compounds possess antibacterial
activity against the above-mentioned Gram-negative and
Gram-positive bacteria, and the activity against the Gram-
negative bacteria is larger. The efficacy of these compounds
is controlled by the cell-wall thickness and the polarity of
the inner cell membrane, so the permeation into the bac-
terial strains of some agents can be facilitated [51–56].
Gram-negative bacterium has a thin peptidoglycan layer
that an outer protein-phospholipid-lipopolysaccharide
membrane covers that, whereas a Gram-positive bacterium
has a thick peptidoglycan layer that contains teichoic and
lipoteichoic acid. As a result, the cell wall of Gram-negative
In our investigation, the metal salts all presented moderate
MIC values against the studied bacteria strains (MIC values
below 0.1 mg/mL are considered significant, between 0.1
and 0.5 mg/mL moderate, between 0.5 and 1.0 mg/mL
weak, and above 1.0 mg/mL negligible) [38–40]. On the
other hand, although HL ligand also showed a moderate
MIC value against E. coli, P. aeruginosa, and S. aureus, its
activity against E. faecalis was weak. As for the metal
complexes, a significant increase of the bactericidal activity
was observed in comparison to the free ligand. The order by
antimicrobial activity was 1c > 1b > 1a > HL. The enhanced
activity of these complexes in comparison with the free
ligand can be explained by the Ovetone’s concept
Table 2 Results of minimal
Samples
E. coli ATCC 25922 P. aeruginosa ATCC S. aureus ATCC
7853 25923
E. faecalis ATCC
11700
inhibitory concentrations (MIC,
2
mg/mL) and zones of inhibition
(
ZI, diameter/mm) of all
MIC
ZI (mm) MIC ZI (mm) MIC
ZI (mm) MIC
ZI (mm)
compounds against the studied
bacterial strains
(mg/mL)
(mg/mL)
(mg/mL)
(mg/mL)
HL
0.256
0.016
0.007
0.001
—
—
0.256
—
0.512
0.032
0.013
0.002
—
—
1.024
0.064
0.021
0.004
—
—
1
1
1
a
b
c
14.3
17.5
19.7
—
0.032
0.009
0.001
—
13.3
16
13
13
15.9
19.5
—
14
19.5
—
18.7
—
DMSO
AgSD
0.022
—
—
0.028
—
—
0.022
0.0025
—
—
0.023
0.003
—
—
Amikacin
—
—
17.9
—
17.2
—
Gentamicin 0.001
20.2
0.0015
19.4

S. Rakhshani et al.
bacteria is more polar [57, 58]. As a result, the bactericidal
properties of HL ligand and its complexes against P. aer-
uginosa and E. coli is higher than against S. aureus and E.
faecalis [59]. On the other hand, for all tested bacterial
strains, the antibacterial activity of complexes was higher
than for the free ligand. Indeed, the coordination of metal
ions to the ligand enhanced the antibacterial activities. This
is maybe caused by a synergistic effect involving Co(III),
Ni(II), and Ag(I) ions and the thiourea ligand.
Overall, the 1c complex has more ability than the other
reagents to penetrate the cell wall or the lipid layer of the
cell membrane. This is caused by relative lability of this
complex compared with the Co and Ni complexes, There-
fore, 1c interacts more adequately with biological ingre-
dients inside the cytoplasm, interlinks to bacterial surface
and as a result, frustrates the microorganism activities by
either changing the cell wall or the membrane function or
barricade replication [60]. On the other hand, the lower
activity of the 1a, and 1b complexes can be attributed to
low lipid solubility. Therefore, the metal ion cannot reach
the desired site of action of the cell wall [61, 62].
Colony count assay
As shown in Fig. 3, the consequences achieved from the
colony count assay corroborate the results of MIC as well as
of well diffusion. The 1c complex demonstrates stronger
activity than the other specimens and inhibits all bacteria
strains, E. coli, and S. aureus, in the lowest time.
Structure-activity relationship
The molecular structures of HL, 1a, 1b, and 1c compounds
were optimized by using the DFT method (B3LYP) at
Fig. 3 Plot of the survival number of a E. coli ATCC 25922 and b S. aureus ATCC 25923 with HL ligand, 1a, 1b, and 1c complexes in colony
count method

Design and fabrication of novel thiourea coordination compounds as potent inhibitors of bacterial growth
Fig. 4 a Frontier molecular orbitals of the studied specimens; b The linear relations of the differences between the band gap and the zone of
inhibitions for the studied bacteria strains in HL, 1a, 1b, and 1c
6
‐31 G* and LANL2DZ basis set level. After that, DFT
enzymes and denaturation of one or more cellular proteins
and ultimately lead to cell death [63, 64].
results were obtained for the frontier molecular orbital
energies. Fig. 4(a) demonstrates the frontier molecular
orbitals (FMOs) of these samples.
FMOs have a substantial role in the advent of optical and
electrical properties. The band gap is also associated with
antimicrobial properties. The narrower band gap facilitates
the electron transfer from the electron-donor groups to the
electron-acceptor groups, that can cause interruption of the
cellular respiration process, deactivation of several cellular
The inverse relationship between the size of the band gap
(ΔEL–H) and the bactericidal activities of these samples
were approved by these conclusions. i.e., if the difference
between HOMO and LUMO energies is smaller, the
greatness of the antibacterial activity gets more [31].
Also, the linear relationship between the thickness of
zone of inhibitions for above-named standard bacterial
strains and band gap values can be found by an accurate

S. Rakhshani et al.
examination of the computational conclusions. Pursuant to
Fig. 4(b), the 1c complex has the narrowest band gap, which
corresponds to its strongest bactericidal activity among the
investigated samples [8].
white precipitate that was filtered off and washed with
distilled water. The colorless crystals were obtained by ether
diffusion into the DMF solution of ligand. Yield (87%).
−
1
Mp: 158–160 °C. Selected FT-IR data (ν, cm ): 3159 (N-
H), 2953–3097 (Ph-H), 2861–2908 (C-H), 1324 (C = S).
1
H NMR (DMSO-d ; δ, ppm): 9.42 (s, 1 H, N-H),
6
Conclusion
7.31–7.41 (m, 4 H, Ph), 3.89–3.92 (t, 4 H, O-CH2),
3.65–3.69 (t, 4 H, N-CH2).
In the present research, we synthetized a novel thiourea
ligand and its Co(III), Ni(II) and Ag(I) complexes. Their
structures were characterized by FTIR, UV-vis, H NMR.
Preparation of Co complex (1a)
1
For HL and 1c, we also performed single-crystal X-ray
diffraction analysis. These characterizations proposed the
HL coordinated to the Co(III), Ni(II) ions through N and S
atoms and constructed a four-membered chelate rings, while
to Ag(I) it coordinated via S atom as a monodentate ligand.
For all of the samples, antimicrobial activity was examined
against Gram-negative (E. coli and P. aeruginosa) and
Gram-positive (S. aureus and E. faecalis) bacteria by three
methods, showing that they have good bactericidal activity
in the order of 1c > 1b > 1a > HL. While the HL ligand did
not demonstrate a remarkable antibacterial activity, its
related complexes, especially 1c, had the best performance
versus all tested strains.
The desired complex was prepared by addition of the
methanolic solution of Co(NO ) .6H O in 50 °C to the
3
2
2
solution of HL ligand in 1:3 molar ratio and the resulting
mixture was refluxed for 3 h. The dark green precipitates of
the complex were filtered off and dried in vacuo. Yield
−
1
(78%). Mp: 138–140 °C. Selected FT-IR data (ν, cm ):
1
2918–2965 (Ph-H), 2848 (C-H), 1233 (C = S). H NMR
(DMSO-d ; δ, ppm): 6.40–7.46 (m, 12 H, Ph), 3.06–3.78
6
(m, 24 H, -CH2).
Preparation of Ni complex (1b)
A similar procedure was carried out using HL (2 mmol) and
Ni(NO ) .6H O (1 mmol) and refluxed for 3 h. The green
3
2
2
precipitates were filtered off and dried in vacuo. Yield
−
1
Materials and methods
(75%). Mp: 280–282 °C. Selected FT-IR data (ν, cm ):
2
1
919–3026 (Ph-H), 2848–2892 (C-H), 1241 (C = S). H
Experimental instruments
NMR (CDCl ; δ, ppm): 6.55–7.26 (m, 8 H, Ph), 3.14–3.86
3
(m, 16 H, -CH2).
Infrared spectra were recorded on KBr pellets using a Per-
1
kinElmer 781 spectrophotometer FTIR. H NMR was
Preparation of Ag complex (1c)
recorded in DMSO-d and CDCl solvents on a BRUKER
6
3
3
00 MHz AVANCE III spectrometer with tetramethylsilane
In a similar procedure, the Ag complex was obtained by the
as internal reference. The melting points were measured on
an Electro-thermal 9100 apparatus. Single-crystal X-ray
data were collected on Gemini diffractometer of Rigaku
Oxford Diffraction using the graphite monochromated
MoKα radiation from a sealed X-ray tube and CCD detector
Atlas S2 at 120 K. For structure plots, Diamond 4.5.2 was
used.
reaction of AgNO with the ligand (molar ratio of 1:2) in
3
methanol. The gray precipitates of the complex were filtered
off and dried in vacuo. Yield (88%). Mp: 170–172 °C.
−
1
Selected FT-IR data (ν, cm ): 3208 (N-H), 3044–3165
-
1
(Ph-H), 2858–2968 (C-H), 1343 (NO ), 1304 (C = S). H
3
NMR (DMSO-d ; δ, ppm): 10.01 (s, 2 H, N-H), 7.19–7.46
6
(m, 8 H, Ph), 3.91–3.94 (t, 8 H, -CH ), 3.72–3.75 (t, 8 H,
2
All solvents and chemical materials were reagent grade
and were purchased from Merck and Fluka, including 4-
chlorophenyl isothiocyanate, Co(NO ) .6H O, Ni(NO )
-CH2).
X-ray crystallographic analysis
3
2
2
3
2.
6H O, AgNO , morpholine, methanol, and acetone.
2 3
The measurement of HL was performed at 120 K on a
Gemini diffractometer of Rigaku Oxford Diffraction using
the graphite monochromated MoKα radiation from a sealed
X-ray tube, and CCD detector Atlas S2. The measurement
of 1c was performed at 95 K on a SuperNova diffractometer
of Rigaku Oxford Diffraction using the mirror-collimated
CuKα radiation from a micro-focus sealed X-ray tube, and
CCD detector Atlas S2. CrysAlis PRO software was utilized
Preparation of the N-(4-chlorophenyl)morpholine-4-
carbothioamide (HL)
The thiourea ligand was synthesized by dissolving 0.01 mol
4
-chlorophenyl isothiocyanate and 0.01 mol morpholine in
acetone. This mixture was refluxed for 1 h. After that, this
solution was poured into 250 mL distilled water, resulting a

Design and fabrication of novel thiourea coordination compounds as potent inhibitors of bacterial growth
for correction of reduction and absorption data [65]. The
structures were solved using charge flipping methods and
refined using Jana2006 [66, 67]. The residual electron
density maps were plotted using MCE [68]. All hydrogen
atoms could have been distinguished in difference Fourier
maps and could have been refined to a reasonable geometry.
According to common practice, H atoms bonded to C were
kept in ideal positions with C–H 0.96 Å while positions of
H atom bonded to N were refined with restrained bond
lengths. In both cases, U (H) was set to 1.2U (C,N). All
non-hydrogen atoms were refined using harmonic
refinement.
Hydrogen atoms of partially occupied crystal water
molecules (0.270(10) for O1w and 0.064(6) for O2w] in 1c
could not be determined because they were not present in a
difference Fourier map. Moreover, 1c contained a dis-
tubes were incubated at 37 °C for 24 h. Then, 100 μL
samples were taken from each tube and were applied on the
specified amount of the Nutrient Agar and incubated at 37 °
C for 24 h. The lowest concentration of test compounds
eliminating bacterial growth indicates the MIC (Minimum
Inhibitory Concentration).
Colony count method
The bacteria strains have been streaked in nutrient broth
medium (NB) contained HL ligand, 1a, 1b, and 1c com-
plexes suspension at the concentration of 20 μg/mL for
measuring the rate of bacteria growth. These samples were
appended into 10 mL NB medium which was added 200 μL
iso
eq
5
bacteria at the concentration of 1.5 × 10 CFU. The same
condition was used for preparing the blank samples by
growth of the culture on the ligand and metal complexes
free medium. Then a shaker platform was used to wiggle
these samples at a speed of 200 rpm. After that, 100 μL of
each bacteria suspension was dispersed on the Nutrient
Agar medium. Finally, after 24 h of incubation at 37 °C the
number of colonies forming units (CFUs) was counted by
considering the dilution factor [33].
ordered NO anion with two close positions occupied 0.774
3
(
12) and 0.226(12). These positions were refined using the
rigid body approach of Jana2006, and their occupancies
were restricted to keep the full total occupancy.
In vitro antibacterial studies
The in vitro antimicrobial tests were examined against P.
aeruginosa, ATCC 27853, and E. coli, ATCC 25922
Agar well diffusion test
(
Gram-negative) and S. aureus, ATCC 25923, and E. fae-
calis, ATCC 11700 (Gram-positive) bacteria. Nutrient broth
NB) medium for both macro dilution (MIC) and colony
In vitro antimicrobial activity of all synthesized HL ligand
and its Co(III), Ni(II), and Ag(I) complexes was investigated
in an agar diffusion test. In a common method, the test bac-
teria were counted by dilution plate method. The cultured
bacteria were spread on the agar medium by an appropriate
sterile swab. After drying the plates, a well was created on the
agar medium by sterilized cork borer. DMSO stock solutions
of all compounds were diluted with sterile water to the test
concentration of 100 µg/mL, were added (100 μL) to these
wells by using a micropipette and then were incubated with
different test bacteria. During this step, the growth of the
inoculated bacteria was impressed by diffusion of the test
solution. Subsequently, antimicrobial activity was assessed by
measuring the diameter of the inhibition zones around the
hole. The size of inhibition zones was determined visually. If
the inhibition zone was greater than 6 mm the compounds
were regarded as active [70–72].
(
count methods and Nutrient agar (NA) medium was used
for the well-diffusion method. The bacterial strains were
retained on agar slant at 4 °C and were sub-cultured on the
fresh appropriate agar plate in incubators for 18–24 h prior
to any antimicrobial analysis. To promote the solubility of
these samples in water, they were dissolved in 10% DMSO
solution. The same solution (without reagents) was used as
negative control. The amikacin and gentamicin were used as
positive control for Gram-positive and Gram-negative bac-
teria, respectively. Comparative investigation of the bac-
tericidal activities of these samples was determined by using
three following methods:
Macro-dilution (tube) broth method
According to the literature, 1 mL of Mueller-Hinton broth
was added to the desired quantity of sample tubes
Acknowledgements We thank the University of Sistan and Baluche-
stan for the financial support. The crystallographic part was supported
by the project 18–10504 S of the Czech Science Foundation using
instruments of the ASTRA lab established within the Operation pro-
gram Prague Competitiveness—project CZ.2.16/3.1.00/24510.
(
10 sample tubes) [69]. Then 1 mL stock solution of any
compounds (HL, 1a, 1b, and 1c) dissolved in 10% DMSO
solution and added to the first test tubes. After that, different
concentration of the ligand and its complexes (93.68, 46.83,
2
3.42,11.71, 5.85, 2.93, 1.46 μM) were prepared from
Compliance with ethical standards
initial solutions. 1 mL of the suspension of bacteria that had
been adjusted to 0.5 McFarland in sterile peptone water,
was added to each sample tubes. After mixing, the sample
Conflict of interest The authors declare that they have no conflict of
interest.

S. Rakhshani et al.
Publisher’s note: Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
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