Synthesis, physico-chemical characterization and bioevaluation of Ni(II), Pd(II), and Pt(II) complexes with 1-(o-tolyl)biguanide: Antimicrobial and antitumor studies

Article abstract of DOI:10.1002/aoc.5807

New complexes of type [M(tbg)₂]Cl₂ [tbg = 1-(o-tolyl)biguanide; M = Ni(II), Pd(II), and Pt(II)] were synthesized and characterized to develop new biologically active compounds. The features of the complexes were assigned from microanalytical and thermal data. The NMR, FT-IR, and UV-Vis spectra were established by comparison with HtbgCl. All complexes exhibit a square-planar geometry resulting from the chelating behavior of tbg. The HtbgCl and [Ni(tbg)₂]Cl₂ complexes were fully characterized by single-crystal X-ray diffraction. The HtbgCl species crystallize in the monoclinic C2/c spatial group, while the Ni(II) complex adopts an orthorhombic Pna2₁ spatial group. The structure is stabilized by a complex hydrogen bonds network. The in vitro antimicrobial assays revealed improved antimicrobial activity for complexes in comparison with the ligand against both planktonic and biofilm embedded microbial cells. The most efficient compound, showing the largest spectrum of antimicrobial activity, including Gram-positive and Gram-negative bacteria, as well as fungal strains, in both planktonic and biofilm growth states was the Pd(II) complex, followed by the Pt(II) complex. The Pt(II) compound exhibited the most significant antiproliferative activity on the human cervical cancer SiHa cell line, inducing a cell cycle arrest in the G2/M phase.

Full text of DOI:10.1002/aoc.5807

Received: 2 March 2020
DOI: 10.1002/aoc.5807
Revised: 6 April 2020
Accepted: 26 April 2020
F U L L P A P E R
Synthesis, physico-chemical characterization and
bioevaluation of Ni(II), Pd(II), and Pt(II) complexes with
1-(o-tolyl)biguanide: Antimicrobial and antitumor studies
ꢀ1
Ileana Nuţa | Mihaela Badea1
| Mariana Carmen Chifiriuc2,3
|
Coralia Bleotu4 | Marcela Popa2 | Constantin-Gabriel Daniliuc5 | Rodica Olar1
1Faculty of Chemistry, University of
New complexes of type [M(tbg)2]Cl2 [tbg = 1-(o-tolyl)biguanide; M = Ni(II),
Pd(II), and Pt(II)] were synthesized and characterized to develop new biologi-
cally active compounds. The features of the complexes were assigned from
microanalytical and thermal data. The NMR, FT-IR, and UV-Vis spectra were
established by comparison with HtbgCl. All complexes exhibit a square-planar
geometry resulting from the chelating behavior of tbg. The HtbgCl and [Ni
(tbg)2]Cl2 complexes were fully characterized by single-crystal X-ray diffrac-
tion. The HtbgCl species crystallize in the monoclinic C2/c spatial group, while
the Ni(II) complex adopts an orthorhombic Pna21 spatial group. The structure
is stabilized by a complex hydrogen bonds network. The in vitro antimicrobial
assays revealed improved antimicrobial activity for complexes in comparison
with the ligand against both planktonic and biofilm embedded microbial cells.
The most efficient compound, showing the largest spectrum of antimicrobial
activity, including Gram-positive and Gram-negative bacteria, as well as fungal
strains, in both planktonic and biofilm growth states was the Pd(II) complex,
followed by the Pt(II) complex. The Pt(II) compound exhibited the most signif-
icant antiproliferative activity on the human cervical cancer SiHa cell line,
inducing a cell cycle arrest in the G2/M phase.
Bucharest, 90-92 Panduri Str., Bucharest,
050663, Romania
2Faculty of Biology, University of
Bucharest, 1-3 Aleea Portocalelor Str.,
Bucharest, 60101, Romania
3Life, Environment and Earth Sciences
Department, Spl. Independentei 91-95,
Research Institute of the University of
Bucharest, Bucharest, Romania
4Stefan S Nicolau Institute of Virology,
285 Mihai Bravu Ave, Bucharest,
Romania
5Organisch-Chemisches Institut,
Westfälische Wilhelms-Universität
Münster, Corrensstrasse 40, Münster,
48149, Germany
Correspondence
Rodica Olar, University of Bucharest,
Faculty of Chemistry, 90-92 Panduri Str.,
050663 Bucharest, Romania.
Email: rodica_m_olar@yahoo.com
K E Y W O R D S
complex, 1-(o-tolyl)biguanide, antimicrobial activity, biofilm, human cervical cancer
1 | INTRODUCTION
complexes bearing a chelate ligand and/or a species that
exhibits anti-adhesive abilities.[8–10]
Antimicrobial drug resistance has become one of the
greatest public health challenges at the global level for
the twenty-first century, highlighting the urgent need for
the discovery of new antibacterial agents.[1–7]
One of the strategies used for developing new and
effective antimicrobial agents is the enhancement of
organic species activity by complexation. Such studies
have indicated promising antimicrobial potential for
Interest in the field of biguanides has been raised by
evidence of the good antibiofilm activity of 1,10-
(1,3-phenylene)bis
(biguanide)[11]
and
the
antimicrobial activity shown by complexes with such
derivatives both on planktonic and biofilm embedded
strains.[8,12–20]
Among these, Cu(II),[8,12,13] Mn(II),[13] and
Zn(II)[12,14] complexes with N,N0-dimethylbiguanide
(dmbg) have been found to be efficient against planktonic
Appl Organomet Chem. 2020;e5807.
wileyonlinelibrary.com/journal/aoc
© 2020 John Wiley & Sons, Ltd.
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https://doi.org/10.1002/aoc.5807

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NUŢĂ ET AL.
Gram-positive and Gram-negative bacteria. Moreover,
species of Mn(II), Ni(II), Cu(II), and Zn(II) with this
ligand showed the ability to inhibit Staphylococcus aureus
and Pseudomonas aeruginosa biofilm development on an
inert substratum.[12,13] Significant inhibitory activity was
observed for Fe(III)[15] and Pd(II)[16] complexes with
dmbg against both planktonic and biofilm embedded
cells of S. aureus, Bacillus subtilis, and Escherichia coli.
The Pd(II) complex showed antibiofilm activity against
Candida albicans,[16] while Cu(II) complexes with 1-(o-
tolyl)biguanide (tbg) against S. aureus and P. aeruginosa
biofilms.[17]
Vis) spectra. The HtbgCl and Ni(II) complexes were fully
characterized through single-crystal X-ray diffraction.
Bioevaluation of the antimicrobial activity of the com-
pounds was performed on a large variety of bacterial and
fungal strains, in both planktonic and biofilm states. The
antiproliferative activity was tested on the human cervi-
cal cancer SiHa cell line by CellTiter Glo and cell cycle
analysis.
2 | EXPERIMENTAL
As for complexes with mixed ligands, Cu(II), Fe(III),
Mn(III),[14] and Ni(II)[15] species with α-ketoglutarate
anion and tbg exhibited good antibiofilm activity against
S. aureus and P. aeruginosa strains. Also,
organoiridium(III) species with cyclopentadienyl and
biguanide derivatives proved to be active against a large
spectrum of microbial strains, including the methicillin-
resistant S. aureus (MRSA), and were also able to disrupt
and eradicate bacterial mature biofilm.[20]
2.1 | Materials and reagents
The high-purity reagents were purchased from Sigma-
Aldrich (Saint-Louis, MO, USA) (NiCl2·6H2O, tbg) and
Fluka (Saint-Louis, MO, USA) (PdCl2, PtCl2) and were
used as received, without further purification.
2.2 | Physical measurements
Furthermore, for Ni(II) complexes with dmbg either
the ability to cleave DNA[21] or cytotoxicity against
HepG2 human liver cancer[22] was evidenced.
Chemical analysis of carbon, nitrogen, and hydrogen was
performed on a Perkin Elmer (Waltham, MA, USA) PE
2400 analyzer. FT-IR spectra were recorded in KBr pellets
with a Bruker (Billerica, MA, USA) Tensor 37 spectrome-
The biological activity of biguanide complexes is not
surprising since, after coordination, the biguanide scaf-
fold can engage a large variety of noncovalent interac-
tions with biological targets, such as hydrogen bonds,
dipole–dipole, lone pair (N)–π and π–π stacking, render-
ing it a powerful pharmacophore. Some of these interac-
tions have been evidenced for several complexes in their
supramolecular arrangement in solid networks.[8,20,23]
The possible interactions for tbg complexes are presented
in Figure 1.
Moreover, both antimicrobial and antiproliferative
activities for Ni(II),[24–26] Pd(II),[27–31] and Pt(II)[30,31]
complexes with Schiff bases, species bearing the same
imine donor groups such biguanides, have recently been
reported.
1
ter in the range 400–4000 cm-1. H NMR and 13C NMR
spectra were recorded on a Bruker Fourier spectrometer
(working frequency 300 MHz) at 25ꢀC. Chemical shifts
were measured in parts per million from internal stan-
dard tetramethylsilane (TMS). Electronic spectra were
established by the diffuse reflectance technique, using
spectralon as standard and were recorded in the range
200–1500 nm, on a Jasco (Easton, MD, USA) V670 spec-
trophotometer. The heating curves (thermogravinetric
(TG), derivative thermogravimetric (DTG) and differen-
tial thermal analysis (DTA)) were recorded using a
Labsys 1200 SETARAM (Caluire, France) instrument,
with a sample mass of 7–12 mg over a temperature range
of 20–900ꢀC, using a heating rate of 10ꢀC/min. The mea-
surements were carried out in a synthetic air atmosphere
(flow rate 17 cm3/min) using alumina crucibles. The X-
ray powder diffraction patterns were collected on a
DRON-3 (St. Petersburg, Russia) diffractometer with
nickel-filtered Cu Kα radiation (λ = 1.5418 Å) in a 2θ
range of 5 70ꢀ, step width 0.05ꢀ, and an acquisition time
of 2 s per step. X-ray single-crystal diffraction data sets
were collected with a Nonius Kappa (Delft, The Nether-
lands) CCD diffractometer. Programs used: data collec-
tion COLLECT,[32] data reduction Denzo-SMN,[33]
absorption correction Denzo,[34] structure solution
SHELXT-2015,[35] structure refinement SHELXL-2015,[36]
and graphics XP.[37] R values are given for observed
Bearing these points in mind, we report here the syn-
thesis of Ni(II), Pd(II), and Pt(II) chloride complexes with
tbg. The compounds were characterized by elemental
and thermal analyses, NMR, FT-IR, and UV–visible (UV-
FIGURE 1 Non-covalent interactions posible for 1-(o-tolyl)
biguanide complexes

NUŢĂ ET AL.
3 of 12
reflections and wR2 values are given for all reflections.
Crystallographic data for HtbgCl and [Ni(tbg)2]Cl2 struc-
tures have been deposited with the Cambridge Crystallo-
graphic Data Centre, CCDC Nos. 1904301 and 1904302,
respectively. Copies of the data can be obtained free of
charge.
inhibitory
concentration
(MIC),
as
previously
reported.[38,39] The antibiofilm activity assay was per-
formed by the violet crystal microtiter method, esta-
blishing of the minimal biofilm eradication concentration
(MBEC), following previously described protocols.[38,39]
All biological experiments were performed in triplicate.
2.3 | Synthesis and analytical data of tbg
hydrochloride and complexes
2.5 | In vitro cytotoxicity assays
2.5.1 | Cell culture conditions
The tbg hydrochloride (HtbgCl) was synthesized by
adding 1 mL of 37% HCl solution to a solution containing
1 mmol (191 mg) of tbg in 20 mL of ethanol. The
obtained solution was heated under magnetic stirring for
2 hr. Single crystals suitable for X-ray diffraction were
obtained after slow evaporation of this solution.
[Ni(tbg)2]Cl2 (1): A solution of 382 mg (2 mmol) tbg
in 40 mL of ethanol was dropwise added to a solution
containing 237 mg (1 mmol) nickel(II) chloride hexahy-
drate in 20 mL of ethanol. The reaction mixture was
refluxed for 2 hr until the color changed to orange. The
sparingly soluble species was obtained after cooling the
solution at room temperature and the volume was
reduced to half by slow evaporation. The resulting solid
product was filtered off, washed with hot ethanol and
diethyl ether, and dried at room temperature. Single crys-
tals suitable for X-ray diffraction were obtained by the
slow diffusion of the tbg ethanol solution in nickel(II)
chloride ethanol solution: yield 464 mg (91%); analysis
found: C 42.32, H 5.03, N 27.44; NiC18H26N10Cl2 requires:
C 42.22, H 5.12, N 27.35%.
The human cervical cancer SiHa cell line (ATCC HTB-
35) was cultivated in DMEM:F12 (GE Life Sciences, Pitts-
burgh, USA) supplemented with 10% heat-inactivated
bovine serum and penicillin/streptomycin at 37ꢀC with
5% CO2.
2.5.2 | Cell viability
The CellTiter-Glo luminescent cell viability assay
(Promega, Madison, WI, USA) was used to quantify the
adenosine triphosphate (ATP) generated by metabolically
active cells. The cells were seeded at a concentration of
104 cell per well in 96-well plates. The compounds were
added in triplicate at concentrations ranging between
400 and 50 μg/ml. The plates were then incubated for
24 hr and the cells were lysed using 100 μl of CellTiter-
Glo reagent for 10 min at room temperature. The lumi-
nescence was recorded in
a TriStar luminometer
(Berthold Technologies GmbH & Co.KG, BW, Bad
Wildbad, Germany). The cell viability was calculated as
the percentage of viable cells from the sample reported to
the total number of cells of control.
The other complexes of the series were obtained by
the same method starting from PdCl2 and PtCl2,
respectively.
[Pd(tbg)2]Cl2 (2): yield 412 mg (74%); analysis found:
C 38.67, H 4.61, N 25.11; PdC18H26N10Cl2 requires: C
38.62, H 4.68, N 25.02%.
[Pt(tbg)2]Cl2 (3): yield 524 mg (81%); analysis found:
C 42.32, H 5.03, N 27.44; PtC18H26N10Cl2 requires: C
33.34, H 4.04, N 21.60%.
The fluorescein diacetate (FDA) and propidium
iodide (PI) dual stain was used as the second method for
toxicity confirmation.[40] Briefly, the cellular monolayer
obtained in each 24-well plate was treated with 100 μg/ml
of the tested compounds. After 24 hr, 10 μg/ml of FDA
and 20 μg/ml of PI were added and the stained cells were
evaluated with an Observer D1 Zeiss fluorescence micro-
scope (Zeiss GmbH, Gottingen, Germany).
2.4 | Antimicrobial assays
The antimicrobial activity of the obtained compounds
was determined on bacterial and fungal reference strains
(Escherichia coli ATCC 25922, Pseudomonas aeruginosa
ATCC 27853, Staphylococcus aureus ATCC 29213, Bacil-
lus subtilis ATCC 6633, Enterococcus faecalis ATCC
29212, Candida albicans 22). The quantitative antimicro-
bial activity assay was performed by the liquid broth mic-
rodilution method, allowing to establish the minimal
2.5.3 | Cell cycle analysis
For the cell cycle analysis, the monolayer obtained from
105 cells was treated with 100 μg/ml of the tested com-
pounds for 24 hr at 37ꢀC in 5% CO2 atmosphere and
humid conditions. The cells were harvested using tryp-
sin:EDTA (Sigma-Aldrich, Saint-Louis, MO, USA),
washed in phosphate saline buffer (PBS), fixed in 70%

4 of 12
NUŢĂ ET AL.
Summary of crystal data for HtbgCl and [Ni(tbg)2]
[Ni (tbg)2]Cl2
ethanol, and maintained at –20ꢀC overnight. Rehydrated
sample were treated with 100 μg/ml RNAse A and
10 μg/ml propidium iodide (Sigma) and incubated at
37ꢀC for 1 hr. An Epics XL Beckman Coulter flow
cytometer (Beckman Coulter, Indianapolis, IN, USA) was
used for sample acquisition and FlowJo 7.2.5 software
(FlowJo, LLC, Ashland, OR, USA) was used for cell cycle
analysis.
TABLE 1
Cl2 (1)
Compound
Htbg
(1)
Color/shape
Colorless/plate
C9H14ClN5
227.70
Orange/plate
C18H26Cl2N10Ni
512.10
Empirical formula
Formula weight
Temperature
223(2) K
Monoclinic
C2/c
223(2) K
Crystal system
Space group
Orthorhombic
Pna21
3 | RESULTS AND DISCUSSION
Unit cell dimensions
a = 18.5936(5)
Å
a = 14.3504(7)
Å
3.1 | Synthesis and physicochemical
characterization of ligand and complexes
b = 13.8288(3)
b = 8.0550(3) Å
Å
Nickel(II), palladium(II), and platinum(II) complexes
with tbg were obtained from refluxing an ethanol solu-
tion of ligand and the metal(II) (M: Ni, Pd, Pt) chloride in
a 1:2 molar ratio as depicted in Scheme 1.
The elemental (experimental part) and thermal analy-
sis data confirmed the 1:2 metal to ligand stoichiometry
for all complexes. The compounds were formulated as
mononuclear species based on single-crystal X-ray dif-
fraction as well as NMR, FT-IR, and UV-Vis spectra.
c = 9.3332(3) Å
β = 99.097(1)ꢀ
c = 19.7390(9) Å
a = 14.3504(7)
Å
2281.68(17) Å3
Volume
2369.63(11) Å3
8
1.277 mg/m3
0.300 mm-1
960
Z
4
Calculated density
Absorption coefficient
F(000)
1.491 mg/m3
3.612 mm-1
1064
Goodness-of-fit on F2
1.099
1.016
Final R indices [I > 2σ
(I)]
R1 = 0.0434
wR2 = 0.0978
R1 = 0.0508
wR2 = 0.1045
R1 = 0.0458
wR2 = 0.1111
R1 = 0.0526
wR2 = 0.1171
3.1.1 | Description of tbg hydrochloride
and [Ni(tbg)2]Cl2 structure
R indices (all data)
The details of the crystal parameters, data collection, and
refinement for complexes are summarized in Table 1.
The compound tbg hydrochloride crystallized in the
monoclinic crystal system C2/c with an ionic structure
containing the protonated tbg unit and the corresponding
chloride anion in the asymmetric unit cell (Figure 2a and
Supporting Information Figure S1). The packing diagram
of HtbgCl compound presents a complex hydrogen bonds
network. Dimeric units are built between two cationic
units involving N–H… N hydrogen bonds (N2–H03… N3
2.993(2) Å and 167(2)ꢀ (Figure 2b and Supporting Infor-
mation Figure S2)). An additional six N–H… Cl hydrogen
bonds found between the cationic and anionic units
(Supporting Information Table S1 and Figure S3) lead to
Largest difference peak
and hole
0.193 and –
0.407 and –
0.241 e Å-3
0.213 e Å-3
the formation of a polymeric chain parallel to the ab
plane (Figure 2c, and Supporting Information Figures S4
and S5). No relevant π… π interactions were found
between the aromatic substituents.
The [Ni(tbg)2]Cl2 complex crystallizes in the ortho-
rhombic space group Pna21 with four molecules in the
unit cell (Figure 3a and Supporting Information
Figure S6). In addition to the classical hydrogen bonds
interactions (N–H… Cl) (Supporting Information Table S2
and Figure S7) noncovalent intermolecular π… π interac-
tions between the biguanide units and the adjacent o-
tolyl substituents were observed in the packing diagram
(Figure 3b and Supporting Information Figure S8). The
combination of these two types of interactions leads to
the formation of a three-dimensional network with a zig-
zag arrangement of cationic complex units (Figure 3c and
Supporting Information Figure S9).
SCHEME 1 Complexes synthesis procedure

NUŢĂ ET AL.
5 of 12
FIGURE 2 Asymmetric unit of
HtbgCl (thermal ellipsoids at the 15%
probability level) (a), dimmer type
formation involving N-H…N interactions
between the cationic units (b) and
excerpt of the packing diagram
representing the formation of a
polymeric chain parallel to the ab-plane
(c)
3.1.2 | FT-IR and NMR spectral data
the band assigned to NH2 group deformation is shifted by
5–10 cm-1 in the same direction. All these aspects result
from tbg coordination and thus from the hydrogen bond
rearrangement in the solid network as otherwise was
shown for Ni(II) complex.
The shifting of the bands assigned to the o-substituted
aromatic ring to lower wavenumbers is the result of both
charge density reorganization in the tbg molecule after
coordination and π… π interactions.
The IR selected bands of tbg and complexes are listed in
Table 2. The spectra of tbg and HtbgCl are similar, with
only small differences in the characteristic range of
amine group vibrations. The band assigned to the imine
moiety stretching vibration ν(C=NH) appears at 1612 cm-
1
in the spectrum of tbg and is shifted to lower
wavenumbers by 2–7 cm-1 in the complexes spectra. This
modification indicates the involvement of the imine
groups in coordination.[17]
The tbg presence in the complexes structure was
1
confirmed by the H NMR and 13C NMR spectra pat-
A
new weak band appears in the range
terns. In the 1H NMR spectrum of tbg the singlet at
2.13 ppm assigned to methyl protons is downfield
shifted, a shift that increases with the increasing polar-
izing action of the metallic ion. The signals of phenyl
ring protons appear as multiplets at 6.84 and 7.13 ppm
and are both upfield and downfield shifted as result of
1300–1305 cm-1 in the complexes spectra; according to
the available literature data this band can be assigned to
the vibration of the six-membered chelate ring formed by
metallic ion coordination at two imine groups from a
biguanide molecule.[41]
The system composed by three bands in the range
3310–3350 cm-1, assigned to asymmetrical and symmetri-
cal stretching vibrations of the NH2 group, as well as to
stretching vibration of NH groups, is shifted in the spec-
tra of the complexes to lower wavenumbers. Moreover,
neighborhood
modification
after
coordination
(Table 3). It is well known that chemical shifts of NH
1
protons can occur virtually anywhere in H NMR spec-
tra.[42] Since for tbg and its combinations the signals
of imine and amine groups are not observed, this

6 of 12
NUŢĂ ET AL.
FIGURE 3 Asymmetric unit of
complex (1) (thermal ellipsoids at the
15% probability level) (a), hydrogen
interaction N-H…Cl between the cations
and anions (b) and intermolecular p…p
interactions between the biguanide
units and adjacent o-tolyl substituents
(c)
TABLE 2
Absorption maxima (cm-1) from FT-IR spectra and assignments for tbg, its hydrochloride, and complexes 1–3
tbg
HtbgCl
3470 m
3434 m
3377 m
3356 m
3110 w
2960 w
2891 w
1612 vs
1578 s
1483 s
–
1
2
3
Assignments
νasym(NH2)
νsym(NH2)
ν(NH)
3464 m
3434 m
3376 m
3352 s
3348 m
3312 m
3344 s
3333 m
3319 m
3344 m
3328 m
3321 m
3108 m
2906 w
2815 w
1612 vs
1525 s
1482 m
–
3069 m
2961 m
2832 w
1605 vs
1517 vs
1486 m
1301 w
1265 m
1100 w
721 m
3061 m
2920 w
2856 w
1608 vs
1535 vs
1482 s
3075 m
2961 m
2898 w
1610 vs
1520 vs
1495m
1305 w
1261 m
1114 w
746 w
ν(CH)aromatic
νas(CH3)
νs(CH3)
ν(C=N)
δ(NH2)
ν(C=C)aromatic
ν(chelate ring)
ν(Caromatic–N)
ν(Caliphatic–N)
γ(CH)aromatic
1305 w
1256 m
1105 w
727 m
1248 m
1115 w
749 m
1250 m
1116 w
750 m
Note. vs, very strong; s, strong; m, medium; w, weak; sym, symmetrical; asym, asymmetrical.
indicates that they appear in the same region as the
aromatic ones and thus are hidden. The 13C NMR
spectra show the signal characteristic for a methyl
group at 39.7 ppm while those for aromatic carbon
atoms appear in the range 127-152 ppm, depending on
the neighboring atoms. The signal assigned to imine
carbon atoms is upfield shifted by 4–5 ppm as result
of coordination of nitrogen atoms of this group.

NUŢĂ ET AL.
7 of 12
TABLE 3
The shifts in 1H and 13C NMR spectra of tbg, HtbgCl, and complexes
δ (ppm)
Compound
tbg/HtbgCl
CH3
CH
CH
C=NH
CH3
CH
CH–CH3
CH–NH
2.13
6.84
7.13
18.1
39.5
126.6
130.4
127.3
131.4
127.6
131.6
127.5
131.6
150.7
151.7
151.6
151.7
151.6
[Ni(tbg)2]Cl2 (1)
[Pd(tbg)2]Cl2 (2)
[Pt(tbg)2]Cl2 (3)
2.14
2.17
2.22
6.67
6.71
6.78
7.20
7.25
7.25
17.6
17.7
17.7
39.7
39.7
39.7
150.6
150.2
150.2
3.1.3 | UV-vis spectra
to the splitting parameter increase in series
3d8 < 4d8 < 5d8.
Solid state UV-Vis spectra provided useful information
concerning the stereochemistry for d8 ions. In both tbg
and HtbgCl spectra one band assigned to intraligand
π ! π* transitions appears at about 34,500 cm-1. This
band is shifted to higher energy in the spectra of the com-
plexes as result of coordination (Supporting Information
Figure S10).
3.1.4 | Thermal behavior of complexes
Thermal analyses data (TG, DTG, and DTA) were used to
obtain information concerning the thermal stability of
the complexes. The thermal decomposition data are sum-
marized in Table 4 and are discussed in the following
paragraphs.
The curves corresponding to complex 1 indicate that
decomposition follows three well-defined steps
(Figure 4). The sharp and intense endothermic signal
The electronic spectra of all complexes show the pat-
tern of the square-planar geometry with the band
1
1
assigned to spin allowed transition A1g ! A2g.[43] This
band is located at 22,990 cm-1 for complex 1, 26,315 cm-1
for complex 2, and 28,990 cm-1 for complex 3 accordingly
TABLE 4
Compound
Thermal behavior data (in air) for complexes
Thermal
Step effect
Temperature
range/ ꢀC
Δmexp
(%)
Δmcalc
(%)
Process
[Ni(tbg)2]Cl2
(1)
1
2
Endothermic
Miscellaneous
206
–
–
Melting
206–420
31.9
31.8
Chloride elimination and oxidative
degradation of 24% tbg
3
4
Exothermic
Exothermic
420–700
700–900
43.3
11.4
41.5
12.1
Oxidative degradation of 56% tbg
Oxidative degradation of 20% tbg and NiO
formation
Residue
14.4
38.3
42.8
18.9
5.5
14.6
38.0
43.0
19.0
5.6
[Pd(tbg)2]
Cl2 (2)
1
2
Endothermic
Exothermic
Residue
200–380
380–830
Chloride and 37% tbg elimination
Oxidative degradation of 63% tbg
[Pt (tbg)2]
Cl2 (3)
1
2
Endothermic
Endothermic
158–290
290–395
Chloride elimination
14.1
13.9
Chloride elimination and oxidative
degradation of 15% tbg
3.
4
Exothermic
395–740
800–860
47.8
47.9
Oxidative degradation of 85% tbg and PtO
formation
Exothermic
2.3
2.5
PtO decomposition
Residue
30.1
30.1

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NUŢĂ ET AL.
the bond-breaking process predominating over reorgani-
zation. According to the DTA curve at least two over-
lapping exothermic processes occur until the Pd is
formed at 830ꢀC (found/calcd overall mass loss:
81.1/81.0%).
Complex 3 decomposition starts with elimination of
one chloride anion according with the mass loss of 5.5%
(calcd 5.6%), a process accompanied by a weak endother-
mic effect (Supporting Information Figure S12). The sec-
ond step is another chloride anion elimination, a process
that overlaps with the elimination of both methyl and
amine groups from tbg. These processes lead to a mass
loss of 14.1% (calcd 13.9%). The oxidative degradation of
the rest of the tbg starts immediately, accompanied by
several strong exothermic effects and a mass loss of 47.8%
(calcd 47.9%). The decomposition continues with PtO
decomposition that leads to Pt as residue at 860ꢀC
(found/calcd overall mass loss: 69.9/69.9%). PtO, similar
to PdO, is a covalent species that decomposes in air with
metal formation.[44]
FIGURE 4 TG, DTG and DTA curves of [Ni(tbg)2]Cl2 (1)
seen at the beginning of decomposition indicates the
complex melting point at 206ꢀC. The DTA curve shows
one endothermic event for the first step that with a mass
loss of 31.9% (calcd 31.8%) corresponds to chloride anions
together with NH=C–NH2 group elimination from each
tbg molecule. The decomposition continues with oxida-
tive degradation of the CH3C6H4NH moieties, accompa-
nied by a mass loss of 43.3% (calcd 41.5%). On the DTA
curve in the 420–700ꢀC range several overlapping exo-
thermic processes can be observed, one of them being
very strong. This strong effect is due to the aromatic moi-
ety burning. In the last step NiO formation occurs
together with further tbg oxidative degradation. The over-
all mass loss of 85.6% fits well with the calculated one of
85.4%.
Complex 2 is also anhydrous and thus stable up to
175ꢀC. As result of higher molar weight both Pd(II) and
Pt(II) complexes decompose without melting. The
decomposition starts with the elimination of chlorine
anions and of CH2 and N=C(N)–NH2 tbg fragments
(Supporting Information Figure S11), processes accompa-
nied by 38.3% (calcd 38.0%) mass loss and several endo-
thermic effects. The endothermic effect is the result of
3.2 | Antimicrobial activity assay
3.2.1 | Antimicrobial efficiency of the
complexes against planktonic cells
The antimicrobial activity of the tested compounds
against planktonic microbial cells is summarized in
Table 5. It can be noticed that complexes 2 and 3 present
superior antimicrobial activity against all tested strains in
the planktonic state, with MIC values 2 to 64 times lower
than those of tbg and between 2 and 128 times lower
than those of HtbgCl. Complexes 2 and 3 exhibited excep-
tional antibacterial activity against the P. aeruginosa
strain, which is well known for its intrinsic and acquired
multiple drug-resistance mechanisms, thus showing very
promising potential for the development of novel tools
for fighting antimicrobial resistance. Complex 3showed
better antibacterial activity against the two Gram-
negative strains (i.e., E. coli and P. aeruginosa), while
TABLE 5
MIC and MBEC (μg/ml) values of the tbg, HtbgCl, and complexes 1–3 against microbial strains in planktonic growth form
tbg
HtbgCl
1
2
3
Strain
MIC
>1000
500
MBEC
1000
500
MIC
MBEC
>1000
>1000
>1000
>1000
>1000
>1000
MIC
>1000
125
MBEC
1000
125
MIC
500
MBEC
15.62
15.62
62.5
MIC
62.5
7.81
125
MBEC
62.5
7.81
125
E. coli
>1000
1000
P. aeruginosa
S. aureus
B. subtilis
E. faecalis
C. albicans
15.62
62.5
31.25
125
500
500
>1000
>1000
>1000
1000
>1000
500
7.81
500
500
62.5
31.25
125
125
125
>1000
>1000
1000
>1000
125
125
125
125
>1000
>1000
62.5
62.5
125
125

NUŢĂ ET AL.
9 of 12
complex 2 exhibited a larger antimicrobial spectrum,
with low MIC values ranging from 15.62 to 62.5 μg/ml
against four of the six tested microbial strains. Complex
1 showed improved antimicrobial activity compared to
tbg and HtbgCl for only two bacterial strains,
P. aeruginosa and E. faecalis, respectively.
A possible mechanism for the antimicrobial activity
of the obtained complexes is their ability to penetrate the
microbial cell wall and to interact and inactivate intracel-
lular targets (proteins or DNA). Considering the ionic
nature of these species, their cytosolic internalization can
occur not only by passive diffusion mediated by the o-
tolyl moiety, but also by using organic cation trans-
porters. Thus, the good activity noticed for Pd(II) and
Pt(II) complexes could be related to their affinity for such
molecules. Bearing in mind that the investigated com-
plexes have the same oxidation state and configuration of
metallic ion, coordination geometry, N4 donor set, che-
late effect, and counterion, the differences in the intensity
of the antimicrobial activity of the complexes can be only
related to complex cation dimension.
FIGURE 5 Assessment of cytotoxicity of tested compounds by
using CellTiter Glo
3.2.2 | Antibiofilm activity of the
complexes
Biofilm development amplifies the antimicrobial resis-
tance phenomenon and 70–80% of all infections are pro-
duced by microbial biofilms.[45,46] Moreover, due to the
FIGURE 6 The cell viability observed using FDA/PI stain (Propidium iodide, the double-stranded DNA dye, cross the plasma
membrane of compromised/permeabilized cells. Stained altered cells can be observed in red in fluorescent microscopy. Fluorescein
diacetate, a cell-permeant esterase substrate, cross the plasma membrane of viable cells. The integrity of cell-membrane is required for
intracellular retention of the product associated with green stain in fluorescent microscopy.)

10 of 12
NUŢĂ ET AL.
high density and close proximity of bacterial cells inside
biofilms, the horizontal gene transfer of resistance genes
is facilitated in different habitats.[47] Taking into account
the important contribution of microbial biofilms to the
global threat of antimicrobial resistance, a huge research
effort is presently dedicated to the development of
antibiofilm strategies.[38] The antibiofilm activity of the
tested complexes proved to be much improved compared
to that of the tbg and HtbgCl precursors for all com-
plexes, with two exceptions for complex 1 in the case of
E. coli and C. albicans biofilms. Remarkably, complexes
2 and 3 preserved the same efficiency against planktonic
and biofilm-embedded P. aeruginosa cells, as revealed by
the similar values of MIC and MBEC. Also, complex
1shows better efficiency against the biofilm embedded
S. aureus and B. subtilis cells, in comparison with the
planktonic growth state, the MBEC being 8 to 128 times
lower than the corresponding MIC values. The same
behavior was seen for complex 2 and E. coli biofilm, with
an MBEC value 32 times lower than the corresponding
MIC one.
(Figure 6). It has been observed that TBG and compound
1 exhibit a low toxicity with IC50 values of 270.18 and
226.84 μg/ml, respectively. In contrast, the most cytotoxic
compound was 3, with an IC50 of 59.52 μg/ml. This value
is higher in comparison with those reported in the scien-
tific literature for cisplatin (IC50 8.47 μg/ml), but similar
to that of carboplatin (IC50 39.44 μg/ml).[48]
3.2.4 | Cell cycle analysis
The cytotoxicity data were also confirmed by cell cycle
analysis. The appearance of the sub-G0 fraction peak on
the left of the G1 peak demonstrates the occurrence of
apoptotic cells (Figure 7). At a concentration of 100 μg/ml
tbg induced a slight increase of the G1 fraction and a
decrease in the S fraction. As seen in Figure 8, compound
3 exhibited the most intensive pro-apoptotic effect at a
concentration of 100 μg/ml, where the sub-G0 peak was
very high. This aspect is very important considering that
this type of tumor cell develops resistance to cisplatin
and its analogues. Because the species [Pt(tbg)2]Cl2, have
a different stability, these could probably exhibit a differ-
ent metabolization pathways and/or a different mecha-
nism of action different metabolization pathways and
exhibit a different mechanism of action. At a concentra-
tion of 50 μg/ml, compound 3 induced a cell cycle arrest
in G2/M. This effect is probably due to the presence of
3.2.3 | Cytotoxicity assays
To establish IC50, the cytotoxic activity of the compounds
was tested using CellTiter Glo (Figure 5) and confirmed
by fluorescence microscopy using FDA/PI staining
FIGURE 7 The effect induced by the treatment with 100 μg mL-1 on the cell cycle (Data are expressed as fractions of cells in the
different cell cycle phases.)

NUŢĂ ET AL.
11 of 12
FIGURE 8 The effect induced by the treatment with 50 μg mL-1 on the cell cycle (Data were expressed as fractions of cells in the
different cell cycle phases.)
Pt(II) as it is known that compounds with this ion are
blockers of the G2/M phase.[49,50]
Gram-negative bacteria, as well as fungal strains, in both
planktonic and biofilm growth states, was complex 2,
followed by complex 3.
4 | CONCLUSION
The toxicity of the tested compounds on the SiHa can-
cer line increased in the following order: tbg < 1 <2 < 3.
The ability of 3 to induce cycle arrest in the G2/M phase
is important for its further development as a potential
antitumor agent.
Direct reaction of metal chloride with tbg allowed the
synthesis of new complexes [M(tbg)2]Cl2 (M: Ni, Pd, Pt),
which were further submitted to physicochemical charac-
terization. The FT-IR spectra displayed the characteristic
features of the biguanide derivatives acting as chelate
through imine nitrogen atoms. The complexes displayed
square planar stereochemistry in accordance with the
UV-Vis spectra features.
ORCID
Mihaela Badea https://orcid.org/0000-0002-4018-7610
Rodica Olar https://orcid.org/0000-0002-4208-0972
Complexes were stable up to 150–200ꢀC as result of
their anhydrous nature. Decomposition started with
hydrochloric acid elimination followed by tbg fragmenta-
tion and oxidative degradation.
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SUPPORTING INFORMATION
Additional supporting information may be found online
in the Supporting Information section at the end of this
article.
ꢀ
How to cite this article: Nuţa I, Badea M,
Chifiriuc MC, et al. Synthesis, physico-chemical
characterization and bioevaluation of Ni(II), Pd(II),
and Pt(II) complexes with 1-(o-tolyl)biguanide:
Antimicrobial and antitumor studies. Appl
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