Synthesis, characterisation and biological activity of Co(III) complex with the condensation product of 2-(diphenylphosphino)benzaldehyde and ethyl carbazate

Article abstract of DOI:10.1016/j.ica.2012.09.043

A cobalt(III) complex with the condensation derivative of 2-(diphenylphosphino)benzaldehyde and ethyl carbazate was synthesized. X-ray crystal structure was determined for both the ligand and the complex. In the cobalt(III) complex two deprotonated ligand molecules coordinate the metal atom in a distorted octahedral geometry by chelation through the PNO donor system formed by the phosphorus, the imine nitrogen and the carbonyl oxygen. The complex showed a moderate antibacterial activity and a strong cytotoxic activity, stronger than cisplatin. Based on cell cycle progression, apoptotic assays, spectroscopic and electrophoretic studies, it was shown that high cytotoxicity and moderate potential of induction of apoptosis are not consequence of interactions with DNA.

Full text of DOI:10.1016/j.ica.2012.09.043

Inorganica Chimica Acta 395 (2013) 33–43
Contents lists available at SciVerse ScienceDirect
Inorganica Chimica Acta
journal homepage: www.elsevier.com/locate/ica
Synthesis, characterisation and biological activity of Co(III) complex
with the condensation product of 2-(diphenylphosphino)benzaldehyde
and ethyl carbazate
a
b
b
c
c
´
´
´
´
Milica Milenkovic , Alessia Bacchi , Giulia Cantoni , Siniša Radulovic , Nevenka Gligorijevic ,
c
a
a
a,
⇑
´
Sandra Arandelovic , Dušan Sladic , Miroslava Vujcic , Dragana Mitic , Katarina Andelkovic
ˇ ´ d
-
-
´
´
a Faculty of Chemistry, University of Belgrade, Studentski trg 12-16, 11000 Belgrade, Serbia
b Dipartimento di Chimica Generale ed Inorganica, Chimica Analitica, Chimica Fisica, University of Parma, Parco Area delle Scienze 17 A, I 43100 Parma, Italy
c Institute for Oncology and Radiology of Serbia, Department of Experimental Oncology, Laboratory for Experimental Pharmacology, Pasterova 14, Belgrade, Serbia
d Institute of Chemistry, Technology and Metallurgy, University of Belgrade, Njegoševa 12, P.O. Box 815, 11000 Belgrade, Serbia
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 15 June 2012
Received in revised form 27 September 2012
Accepted 29 September 2012
Available online 2 November 2012
A cobalt(III) complex with the condensation derivative of 2-(diphenylphosphino)benzaldehyde and ethyl
carbazate was synthesized. X-ray crystal structure was determined for both the ligand and the complex.
In the cobalt(III) complex two deprotonated ligand molecules coordinate the metal atom in a distorted
octahedral geometry by chelation through the PNO donor system formed by the phosphorus, the imine
nitrogen and the carbonyl oxygen. The complex showed a moderate antibacterial activity and a strong
cytotoxic activity, stronger than cisplatin. Based on cell cycle progression, apoptotic assays, spectroscopic
and electrophoretic studies, it was shown that high cytotoxicity and moderate potential of induction of
apoptosis are not consequence of interactions with DNA.
Keywords:
Cobalt(III) complex
X-ray
Antitumor activity
Antimicrobial activity
DNA binding
Ó 2012 Elsevier B.V. All rights reserved.
1. Introduction
[17]. The number of papers dealing with biological activity of
derivatives of 2-(diphenylphosphino)benzaldehyde and their me-
The design and synthesis of multidentate ligands which are
derivatives of 2-(diphenylphosphino)benzaldehyde continue to
be an important research area in coordination chemistry. Deriva-
tives with ‘soft’ phosphorus and ‘hard’ nitrogen donor atoms are
of particular interest in catalytic processes [1–3]. The incorporation
of a chiral centre into complexes of these ligands allows enantiose-
lectivity in catalytic transformations mediated by these com-
pounds [4–10]. Complexes of nickel(II) and copper(I) with
condensation derivatives of 2-(diphenylphosphino)benzaldehyde
and semicarbazide, thiosemicarbazide or selenosemicarbazide
were synthesized. These ligands are coordinated as tridentates
via PNO, PNS or PNSe donor atom sets [11–14]. Square-planar
Pd(II) and Ni(II) complexes with condensation derivatives of 2-
(diphenylphosphino)benzaldehyde and different hydrazides coor-
dinated as tridentates via PNO donor atom sets show a significant
catalytic activity [15–17]. The catalytic properties of nickel(II) ace-
tato complex of 2-(diphenylphosphino)benzaldehyde 2-pyr-
idylhydrazone with PNN set of donor atoms were also studied
tal complexes is much smaller.
Complexes of Pt(II), Pd(II), and Ni(II) with the condensation
derivative of 2-(diphenylphosphino)benzaldehyde and semiox-
amazide were synthesized with the aim to obtain biologically ac-
tive compounds. The ligand showed antibacterial and antifungal
activity, which was enhanced upon complexation [18].
Palladium(II) complex with the condensation product of 2-
(diphenylphosphino)benzaldehyde and ethyl hydrazinoacetate
exhibits a similar effect to cisplatin on HeLa cells, inducing apopto-
sis followed by arrest of cells in S phase of cell cycle, while com-
plexes of platinum(II) and palladium(II) with the condensation
product of 2-(diphenylphosphino)benzaldehyde and semioxamaz-
ide induce apoptosis in HeLa cells without significant perturba-
tions of cell cycle distribution. Complexes of platinum(II) and
palladium(II) with the condensation product of 2-(diphenylphos-
phino)benzaldehyde and semioxamazide showed a strong cytotox-
icity to CDDP-resistant U2-OS/Pt cells [19].
The subject of this work was the synthesis, characterization and
preliminary evaluation of the biological activity of Co(III) complex
with a ligand which is the condensation derivative of 2-(diphenyl-
phosphino)benzaldehyde and ethyl carbazate.
⇑
Corresponding author.
E-mail address: kka@chem.bg.ac.rs (K. Andelkovic´).
-
0020-1693/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.ica.2012.09.043

´
M. Milenkovic et al. / Inorganica Chimica Acta 395 (2013) 33–43
34
This is a continuation of the study of complexes with this type
in 1 that were found on difference maps and refined. Anisotropic
of ligands, in which our attention was shifted to complexes with
potential octahedral geometry, with the aim to get information
on influence of structure on activity. Ethyl carbazate was selected
as the hydrazine part of the ligand instead of ethyl hydrazinoace-
tate, since the latter would not be able to form a five-membered
ring suitable for coordination of the ligand as a tridentate.
displacement parameters were refined for all non-hydrogen atoms.
The BF4 anion in 2 is disordered and has been modeled over two
ꢀ
images with 0.7:0.3 population. Hydrogen bonds have been ana-
lyzed with SHELXL97 [22] and PARST97 [23] and extensive use was
made of the Cambridge Crystallographic Data Centre packages
[24] for the analysis of crystal packing. Table 1 summarizes crystal
data and structure determination results.
2. Experimental
2.3. Antimicrobial activity
2.1. Material and methods
The antimicrobial activity was evaluated using seven different
laboratory control strains of bacteria, i.e., the Gram-positive: Staph-
ylococcus aureus (ATCC 25923), Staphylococcus epidermidis (ATCC1
2228), Kocuria rhizophila (ATCC 9341), Bacillus subtilis (ATCC
6633) and the Gram-negative: Escherichia coli (ATCC 25922), Kleb-
siella pneumoniae (ATCC 13883), Pseudomonas aeruginosa (ATCC
27853), and two strains of yeast, i.e., Candida albicans (ATCC
2-(Diphenylphosphino)benzaldehyde (97%) and ethyl carbazate
(97%) were obtained from Aldrich. IR spectra were recorded on a
Perkin–Elmer FT-IR 1725X spectrometer using the ATR technique
in the region 4000–400 cmꢀ1 1H NMR (500 MHz), 13C NMR
.
(125 MHz) and 2D NMR spectra were recorded on a Bruker Avance
500 spectrometer in DMSO-d6 using TMS as internal standard for
1H and 13C. All spectra were measured at 25 °C. 1 and 2 were char-
acterized on the basis of NMR spectroscopy results: 1D (1H, 13C,
DEPT), 2D (COSY, NOESY) as well as 2D 1H–13C heteronuclear cor-
relation spectra (HSQC). Mass spectrum for 1 was taken on a 6210
TOF LC/MS coupled with an Agilent Technologies 1200 Series HPLC
system. Elemental analyses (C, H, and N) were performed by stan-
dard micro-methods using the ELEMENTARVario ELIII C.H.N.S.O
analyzer.
}
10259 and ATCC 10231). All tests were performed in Muller Hinton
broth for the bacterial strains and in Sabouraud dextrose broth for
the yeast. Overnight broth cultures of each strain were prepared,
and the final concentration in each well was adjusted to 2 ꢃ 106
CFU mlꢀ1 for the bacteria and 2 ꢃ 105 CFU mlꢀ1 for the yeast. The
investigated compounds were dissolved in 1% dimethyl sulfoxide
(DMSO) and then diluted to the highest concentration. Twofold se-
rial concentrations of the compounds were prepared in a 96-well
microtiter plate over the concentration range 31.25–500 l .
g mlꢀ1
The microbial growth was determined after 24 h incubation at
37 °C for the bacteria and after 48 h incubation at 26 °C for the fun-
gi. The MIC is defined as the lowest concentration of the compound
at which no visible growth of microorganism is observed.
2.1.1. Synthesis of ethyl (2E)-2-[2-(diphenylphosphino)
benzylidene]hydrazinecarboxylate (1)
A mixture of 0.14 g (0.48 mmol) 2-(diphenylphosphino)benzal-
dehyde and 0.05 g (0.48 mmol) ethyl carbazate was dissolved, by
heating, in 25 mL ethanol. pH of the mixture was adjusted to ꢁ4
with a hydrochloric acid. The mixture was heated at 56 °C for
60 min. The reaction solution was left to stand at room tempera-
ture while the colourless crystals separated from the solution.
Yield 0.15 g (83%). Mp 164–166 °C. IR (vs-very strong, s-strong,
m-medium, w-weak): 3253 (w), 3189 (w), 3049 (m), 2974 (w),
1729 (m), 1707 (s), 1550 (s), 1458 (w), 1435 (w), 1385 (w), 1247
(vs), 1178 (w), 1092 (w), 1055 (m), 763 (w), 744 (w), 696 (m),
657 (w), 499 (w). HRMS (ESI) of C22H21N2O2P found for (M+H+)
377.1384, calcd (m/z) for (M+H+) 377.1414.
2.4. The brine shrimp test
A teaspoon of lyophilized eggs of the brine shrimp Artemia sal-
ina was added to 0.5 L of the artificial sea water containing several
drops of yeast suspension (3 mg of dry yeast in 5 mL distilled
water), and air was passed through the suspension thermostated
at 18–20 °C, under illumination for 48 h. Hatched nauplii were
used in further experiments.
In a glass vial, into 1 mL of artificial sea water 1–2 drops of yeast
extract solution (3 mg in 5 mL of distilled water) and 10–20
hatched nauplii were added, and finally water solution of Co(BF4)2
ꢂ6H2O to the appropriate concentrations. The substances 1 and 2
were dissolved in DMSO and then in various amounts applied to
paper discs 2 cm in diameter. Paper discs were placed on the bot-
tom of the glass vial into which was added 5 mL of artificial sea
water, 1–2 drops of yeast suspension, and about 15–20 hatched
nauplii.
2.1.2. Synthesis of Co(III) complex (2)
A mixture of 0.24 g (0.64 mmol) of the ligand and 0.13 g
(0.38 mmol) Co(BF4)2ꢂ6H2O was dissolved, by heating, in 25 mL
ethanol. The mixture was heated at 56 °C for 6 h. The color of the
solution changed to dark red. The reaction solution was left to
stand at room temperature. Dark red crystals were obtained from
the reaction solution. Yield 0.08 g (23.4%). IR (vs-very strong,
s-strong, m-medium, w-weak): 3079 (w), 2981 (w), 1507 (vs),
1480 (vs), 1429 (vs), 1379 (m), 1337 (vs), 1060 (s), 750 (m), 701
(m), 659 (w), 611 (m). Elemental analysis for C44H40BCoF4N4O4P2.
Anal. Calc. N 6.25, C 58.95, H 4.50%. Found: N 6.05, C 58.82, H 4.63%.
The vials were left at room temperature under illumination for
24 h, and afterwards live and dead nauplii were counted. LC50 was
defined as the concentration of substances that causes death of 50%
nauplii.
2.5. Cytotoxic activity
2.2. Crystallographic structure determination
2.5.1. Cell culture
Single crystal X-ray diffraction data were collected at T = 293 K
Human cervix carcinoma cells (HeLa), human melanoma cells
(FemX), human colon cancer cells (LS-174) and human fetal lung
fibroblast cells (MRC-5) were maintained as monolayer culture in
nutrient medium (the RPMI 1640 medium). Powdered RPMI 1640
medium, was purchased from Sigma Aldrich Co. Nutrient medium
RPMI 1640 was prepared in sterile ionized water, supplemented
using the Mo K
fractometer for 2 and on a Siemens AED diffractometer equipped
with scintillation detector and Cu K radiation (k = 1.54178 Å) for
a radiation (k = 0.71073 Å) on a SMART APEX2 dif-
a
1. Lorentz, polarization, and absorption corrections were applied
[20]. Structures were solved by direct methods using SIR97 [21]
and refined by full-matrix least-squares on all F2 using SHELXL97
[22] implemented in the WinGX package [23]. Hydrogen atoms
were introduced in calculated positions apart from NH hydrogens
with penicillin (192 IU/mL), streptomycin (200
hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES) (25 mM),
-glutamine (3 mM) and 10% of heat-inactivated fetal calf serum
lg/mL), 4-(2-
L

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M. Milenkovic et al. / Inorganica Chimica Acta 395 (2013) 33–43
Table 1
of drug producing 50% inhibition of cell survival. It is determined
from the cell survival diagrams.
Crystal data and structure refinement for 1 and 2.
1
2
2.5.3. Cell cycle analysis
Empirical formula
Formula weight
Temperature (K)
Crystal system
Space group
a (Å)
C
44H42N4O4P2
C44H40N4O4P2CoBF4
896.48
293(2)
monoclinic
P21/n
15.677(1)
13.403(1)
19.949(2)
93.012(1)
4185.9(6)
4
1.423
0.553
1848.0
3.22–63.98°
68322
Quantitative analysis of cell cycle phase distribution was per-
formed by flow-cytometric analysis of the DNA content in fixed
HeLa cells, after staining with propidium iodide (PI) [26].
Cells were seeded at density of 2 ꢃ 105 cells/well at 6-well plate
and grown in nutrition medium. After 24 h cells were continually
exposed to investigated cobalt(III) complex (2) with concentrations
that correspond to 0.5xIC50 and IC50 (determined for 48 h treat-
ment Table 2). Control cells were incubated only in nutrient med-
ium, and as reference compound cisplatin was used. After 24 and
48 h of continual treatment cells were collected by trypsinization,
washed twice with ice-cold PBS, and fixed for 30 min in 70% EtOH.
After fixation cells were washed again with PBS, and incubated
with RNaseA (1 mg/ml) for 30 min at 37 °C. Cells were then stained
752.76
293(2)
monoclinic
I2/a
24.584(3)
9.2487(4)
36.028(2)
98.297(6)
8106(1)
8
1.234
1.347
3168.0
7.26–139.84°
14815
b (Å)
c (Å)
b (°)
Volume (Å3)
Z
qcalc (mg/mm3)
l
(mm-1
)
F(000)
range for data collection
2H
Reflections collected
Independent reflections
7669
13805
with PI (at concentration of 400 lg/ml) 15 min before flow-cyto-
[Rint = 0.0297]
7669/0/498
1.035
0.0425, 0.1219
0.0452, 0.1247
0.51/ꢀ0.28
[Rint = 0.0284]
13805/0/569
1.029
0.0386, 0.1053
0.0535, 0.1150
0.57/ꢀ0.45
metric analysis. Cell cycle phase distribution were analyzed using
a fluorescence activated sorting cells (FASC) Calibur Becton Dickin-
son flow cytometer and Cell Quest Pro computer software.
Data/restraints/parameters
Goodness-of-fit (GOF) on F2
Final R1, wR2 [I > 2
Final R1, wR2 [all data]
Largest F maximum/minimum
(e Åꢀ3
r
(I)]
D
)
2.5.4. Apoptotic assay
Induction of apoptosis by cobalt(III) complex (2) and cispaltin
(CDDP) in HeLa cells was evaluated by Annexin V–FITC apoptosis
detection kit (BD Biosciences Cat. No. 65874x, Pharmingen San
Diego, CA, USA). Briefly, 1 ꢃ 106 HeLa cells/mL were treated with
0.5 ꢃ IC50 and IC50 concentrations of cobalt(III) complex and CDDP
(see Table 2) for 48 h. After treatment cells were washed twice
Table 2
Results of MTT assay after 48 h agent action.
IC50 M)
(l
with cold PBS and then resuspended in 200 lL binding buffer
HeLa
FemX
LS-174
>100
7.42 2.45
94.29 5.05
22.41 7.18
MRC-5
>100
7.42 0.05
77.27 1.29
30.26 2.98
(10 mM HEPES/NaOH pH 7.4, 140 mM NaCl, 2.5 mM CaCl2).
(1)
(2)
58.28 0.23
4.90 0.08
72.04 6.83
7.79 2.32
>100
100
l
L of the solution (1 ꢃ 105 cells) was transferred to a 5 mL cul-
6.45 1.85
82.11 2.95
10.77 0.88
ture tube and 2.5 lL of Annexin V–FITC and 2.5 lL of PI was added.
Co(BF4)2ꢂ6H2O
CDDP
Cells were gently vortexed and incubated for 15 min at 25 °C in the
dark. After that 400 L of binding buffer was added to each tube
l
and then analysed using a FACS Calibur Becton Dickinson flow
cytometer and Cell Quest Pro computer software.
(FCS) (pH 7.2). The cells were grown at 37 °C in 5% CO2 and humid-
ified air atmosphere, by twice weekly subculture.
2.6. DNA binding experiments
2.5.2. MTT assay
Cytotoxicity of the investigated cobalt(III) complex (2), the
ligand (1) and cobalt salt in comparation to cisplatin, was deter-
mined using the 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltet-
razolium bromide (MTT, Sigma) assay [25]. Cells were seeded in
96-well cell culture plates (Linbro), HeLa (3000 c/w), FemX (5000
c/w), LS-174 (7000 c/w) and MRC-5 (5000 c/w) in culture medium
and grown for 24 h. Stock solutions of investigated agents were
made in DMSO at concentration of 10 mM, and afterwards diluted
with nutrient medium to desired final concentrations (in range up
2.6.1. Interaction of the Co(III) complex (2) with double stranded
closed circular plasmid DNA
Plasmid pUC18 was prepared by transformation of a clone from
Escherichia coli RR1 (pUC18, 2686 bp, purchased from Sigma–
Aldrich, USA) into electrocompetent E. coli DH5a strain cells
according to the protocol for growing E. coli culture overnight in
LB medium at 37 °C by electroporation with the ‘‘Gene Pulser’’
(Bio-Rad) [27]. The plasmid DNA from E. coli clones were isolated
by modified method of alkali lysis [28] and purified with the
‘‘JetStar’’ kit (Genomed) using anion-exchange column. After final
washing step with ice-cold 70% ethanol, DNA pellet was air-dried
to 100
at concentration of 10 mM and afterwards diluted with nutrient
medium to desired final concentrations (in range up to 100 M).
lM). Cisplatin (CDDP) stock solution was made in 0.9% NaCl
l
and finally resuspended in 150
ꢀ20 °C. The concentration of plasmid DNA (213 ng/
was determined by measuring the absorbance of the DNA-contain-
l
L sterile H2O and stored at
Solutions of various concentrations of the examined compounds
were added to the wells, except the control wells where only nutri-
ent medium was added. All samples were done in triplicate. Nutri-
ent medium with corresponding agent concentrations but without
target cells, was used as a blank, also in triplicate.
l
L of pUC18)
ing solution at 260 nm. One optical unit corresponds to 50
of double stranded DNA.
lg/mL
Plasmid DNA was incubated for 4 min at 95 °C, followed by
incubation at 4 °C for the next 20 min. Then 213 ng (0.13 pmol)
Cells were incubated for 48 h with the test compounds at 37 °C,
with 5% CO2 in humidified atmosphere. After incubation, 20
l
L of
of pUC18 in a 20 lL reaction mixtures in TS buffer (20 mM Tris
MTT solution, 5 mg/mL in phosphate buffer solution (PBS), pH
7.2, were added to each well. Samples were incubated for 4 h at
37 °C with 5% CO2 in humidified atmosphere. Formazan crystals
20 mM NaCl, pH 7.92), were incubated with different concentra-
tion (5 nmol, 10 nmol or 15 nmol) of the complex at 37 °C, for
1.5 h. The control sample was prepared with 3 lL of DMSO instead
were dissolved in 100
lL 10% sodium dodecyl sulfate (SDS) in
of complex. The reaction mixtures were vortexed from time to
0.01 M HCl. Absorbance was recorded on the ThermoLabsystems
408 Multiskan EX 200–240 V after 24 h at a wavelength of
time. The reaction was terminated by short centrifugation at
10000 rpm and adding 7
lL of loading buffer (0.25% bromophenol
570 nm. Concentration IC50 (lM) was defined as the concentration
blue, 0.25% xylene cyanol FF and 30% glycerol in TAE buffer, pH

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M. Milenkovic et al. / Inorganica Chimica Acta 395 (2013) 33–43
36
8.24 (40 mM Tris–acetate, 1 mM EDTA)). The samples were sub-
jected to electrophoresis on 1% agarose gel (Amersham Pharma-
cia-Biotech, Inc., USA) prepared in TAE buffer pH 8.24. The
electrophoresis was performed at a constant voltage (80 V) for
1.5 h (until bromophenol blue had passed through 75% of the
gel). After electrophoresis, the gel was stained for 30 min by soak-
1507 cmꢀ1 originating from (ꢀO–C@N) of the deprotonated
m
hydrazide moiety, instead of the carbonyl band from uncoordi-
nated ligand at 1707 cmꢀ1. In the bound ligand hydrazide nitrogen
is deprotonated and as a consequence of electron delocalization
C@O bond acquires partial single bond character, and the C–N bond
partial double bond character. The B–F band is located at
1060 cmꢀ1 in the spectrum of 2. This band originates from the tet-
rafluoroborate anion in the outer sphere of the complex.
ing it in an aqueous ethidium bromide solution (0.5
after that was visualized under UV light.
lg/mL), and
3.3. NMR spectra
2.6.2. Interaction of the Co(III) complex (2) with a linear double-
stranded DNA
From the 1H NMR spectrum of 2 (Table 3) it can be seen that the
ligand is coordinated in monodeprotonated form, since the signal
of hydrazide NH at 11.20 ppm is absent. Coordination via the imine
nitrogen can be confirmed from 1H NMR spectrum from the posi-
tion of the signal of the imine carbon at 8.99 ppm which is shifted
downfield in comparison to the corresponding signal in the ligand
(8.71 ppm). In the 1H NMR spectrum of 2 chemical shifts of aro-
matic protons have higher values, due to electron withdrawal by
the coordinated metal ion. The signals of the methylene and
methyl groups of the alcohol part of the ester are at 4.10 and
1.19 ppm, respectively in the ligand, and at 3.65 and 0.91 ppm,
respectively in the complex.
Coordination through N1 results in a downfield shift of the
hydrazone carbon C-3 (141.6 ppm in the spectrum of 1;
155.4 ppm in the spectrum of 2), and of the para-carbon C-7
(129.2 ppm in the spectrum of 1; 133.7 ppm in the spectrum of
2) (Table 4). There is a strong downfield shift of the carbonyl car-
bon C-14 due to coordination to Co(III), the signal is shifted from
153.4 ppm in the spectrum of 1 to 170.3 ppm in the spectrum of
2. The consequence of coordination through phosphorus atom is
the downfield shift of carbons para to phosphorus (C-6, C-13).
Linear double-stranded lambda bacteriophage DNA (cl857
Sam7, 48502 base pairs with
a
molecular weight of
31.5 ꢃ 106 Da) isolated from E. coli W3110 (purchased from Ther-
mo Scientific, USA) was used in the experiments. DNA concentra-
tion and purity were confirmed spectrophotometrically. For
agarose gel electrophoretic analysis, 0.3 lg of lambda DNA
(0.01 pmol) was incubated with 5 nmol, 10 nmol or 15 nmol of
the complex as described previously for plasmid DNA. For UV–
Vis spectrometric analysis 500 lL of solution of lambda-DNA
(0.5 pmol) in TS buffer were incubated at 37 °C for 2 h, vortexing
from time to time, with different concentration of the complex
(5 mM stock solution in DMSO). UV–Vis spectra were recorded at
Shimadzu 1800 UV/Vis spectrometer.
3. Results and discussion
3.1. Synthesis
The reaction of 2-(diphenylphosphino)benzaldehyde and ethyl
carbazate in ethanol at pH ꢁ4 was performed and 1 was obtained.
This ligand was used for preparation of Co(III) complex. Complex of
Co(III) (2) was obtained by direct synthesis starting from Co(BF4)2-
ꢂ6H2O and 1 (Fig. 1).
3.4. X-ray crystallographic analysis
Ligand 1 crystallizes with two independent molecules in the
asymmetric unit of the monoclinic I2/a space group (Fig. 2). The
comparison of their bonding geometry with the average values
found in the literature for similar compounds by using the program
Mogul [29] shows that the molecular structures observed for 1 in
3.2. IR spectra
Comparison of IR spectra of ligand 1 and complex 2 indicates
the complex formation. A new band appears in the complex at
Fig. 1. (a) Synthesis of ligand 1. (b) Synthesis of complex 2.

´
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M. Milenkovic et al. / Inorganica Chimica Acta 395 (2013) 33–43
Structures with Z0 > 1 are in fact supposed to map the distribu-
tion of low energy conformers present in solution and kinetically
frozen in the crystal [30]. The two conformations differ for small
adjustments in the orientation of the phenyl rings, for the orienta-
tion of the terminal methyl group with respect to the NNHCOOCH2
moiety and for the value of the torsion angle C5–C6–C7–N1 = 31°
and C27–C28–C29–N3 = ꢀ6°. In both cases the imine nitrogen is
pointing opposite to the phosphorous atom, but the orientation
may be changed by rotation around the flexible C6–C7 bond in or-
der to create a PN chelating system.
Table 3
1H NMR spectral data of 1 and 2.
Assignment Chemical shift (ppm),
multiplicity, number of
H-atoms, coupling
Assignment Chemical shift (ppm),
multiplicity, number of
H-atoms, coupling
constant J (Hz) for 1
constant J (Hz) for 2
C1
C2
1.19 (t, 3H, J = 7.1 Hz)
4.10 (q, 2H, J = 7.0 Hz)
C1
C2
C20
C3
C5
0.91 (t, 3H, J = 7.0 Hz)
3.47 (m, 1H)
3.69 (m, 1H)
8.99 (s, 1H)
7.17 (m, 1H)
C3
C5
C6
8.71 (s, 1H)
6.80 (m, 1H)
7.31 (dt, 1H, J = 7.5 Hz, C6
J = 1 Hz)
7.51 (m, 1H)
The ligand displays a conformational rearrangement around the
C6–C7 bond upon coordination. In the cobalt(III) complex 2 two
deprotonated ligand molecules coordinate the metal atom in a dis-
torted octahedral geometry by chelation through the PNO donor
system formed by the phosphorus, the imine nitrogen and the car-
bonyl oxygen (Fig. 3). Table 5 reports relevant bonding parameters
for the complex. Apart from the slightly elongated Co–P distances,
the remaining bond distances are not significantly different than
the average observed for similar systems; however the chelation
induces a strain on the bonding angular geometry of the two li-
gands. Namely the angles on the phosphorus atoms and on C7
and on C29 are wider than the average, while the angles N–C@O
are smaller than usual, as evidenced by a comparison with similar
systems performed by Mogul [29]. The C–O distance for the keto
oxygen is now slightly elongated meaning that the negative charge
is spread over more atoms, as in acetonates. Fig. 3, inset, shows the
points of maximum strain in the complex. The octahedral complex
is in the mer configuration with an approximate molecular twofold
symmetry, and the coordination implies the formation of two six-
membered Co–P–C–C–C–N and two five-membered Co–N–N–C–O
rings. These rings are practically planar, with puckering amplitudes
close to zero: 0.03 Å for the ring containing P1, 0.16 for the one
with P2, 0.07 for the ring comprising O3 and 0.19 for the one
including O1. P2 and O1 are the atoms most deviating from planar-
ity. The two chelation planes, comprising the atoms P–N–O–Co are
practically perpendicular (dihedral angle = 87°). The octahedral
complex cation in 2 is comparable with the complex containing a
similar ligand, [bis(2-(diphenylphosphino)benzaldehyde ben-
zoylhydrazone)-iron(III)]3+ (CSD refcode QEVYUM) [31] to which
the structure of the cation in 2 is almost superimposable. Other
two octahedral similar complexes have been described in the liter-
ature, namely bis(2-(diphenylphosphino)benzaldehyde ben-
zoylhydrazone)-iron(II) tetrachloro-iron chloride (CSD refcode
QEVYOG) [31] and bis(2-(diphenylphosphino)benzaldehyde ben-
zoylhydrazone)-ruthenium(II) dimethyl sulfoxide solvate monohy-
drate (CSD refcode DEKJUZ) [32], but in these cases the two
C7
C8
C11
7.41 (m, 1H)
7.94 (m, 1H)
7.20 (m, 4H)
C7
C8
7.86 (m, 1H)
8.05 (m, 1H)
7.22 (m, 2H)
6.96 (m, 2H)
7.33 (m, 2H)
7.11 (m, 2H)
7.51 (m, 1H)
7.41 (m, 1H)
C11
C110
C12
C120
C13
C130
C12
C13
N2
7.41 (m, 4H)
7.41 (m, 2H)
11.20 (s, 1H)
Table 4
13C NMR spectral data of 1 and 2.
Assignment 13C NMR chemical shift
Assignment 13C NMR
(ppm), coupling constant, J in
Hz of 1
chemical shift
(ppm) of 2
C1
C2
C3
C4
C5
C6
C7
C8
C9
C10
C11
14.5
60.5
141.6
135.5, J = 10 Hz
132.9
C1
C2
C3
C4
C5
C6
C7
C8
14.0
65.4
155.4
136.2
135.3
131.8
133.7
136.5
136.1
136.1
133.8
132.9
128.1
129.0
130.9
170.3
129.6
129.2, J = 15 Hz
125.5, J = 3.75 Hz
135.6, J = 18.75 Hz
138.0, J = 18.75 Hz
133.5, J = 20 Hz
C9
C10
C11
C110
C12
C120
C13
C14
C12
128.8, J = 7.5 Hz
C13
C14
129.1
153.4
the solid state are not uncommon and their slightly different con-
formations evidenced in the inset suggest flexibility of the free
ligand.
Fig. 2. Molecular structures and labeling of the two conformers observed in the crystal structures of 1. In the inset the overlay of the two conformations is shown. Thermal
ellipsoids are drawn at the 50% probability level.

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M. Milenkovic et al. / Inorganica Chimica Acta 395 (2013) 33–43
38
Fig. 3. Molecular structure of 2, with labeling and thermal ellipsoids drawn at the 50% probability level. Phenyl groups attached to phosphorous atoms have been omitted for
clarity and only the ipso carbon has been retained, shown in plain style. Inset: thick sticks indicate the main deformations of the molecular skeleton as compared to average
values identified with Mogul [29].
Table 5
negative bacteria was moderate, but again higher than both the li-
gand and the cobalt salt. The complex and the ligand were inactive
Most relevant bond lengths (Å) and angles (°) for 2, with e.s.d.’s in parentheses.
Bond lenghts
Co1–P1
Co1–P2
to both strains of C. albicans (Table 6).
2.2541(4)
2.2405(4)
1.927(1)
Co1–O3
Co1–N1
Co1–N3
1.928(1)
1.914(1)
1.915(1)
3.6. The brine shrimp test
Co1–O1
Bond angles
P1–Co1–P2
P1–Co1–O1
P1–Co1–O3
P1–Co1–N1
P1–Co1–N3
P2–Co1–O1
P2–Co1–O3
P2–Co1–N1
P2–Co1–N3
O1–Co1–O3
O1–Co1–N1
O1–Co1–N3
Co1–N1–C7
Co1–N3–C29
The biological activity of 1 and 2 was tested by the brine shrimp
test (toxicity to A. salina). The results of this test can be extrapo-
lated to cell-line toxicity and anti-tumor activity [33,34]. The brine
shrimp lethality test showed a moderate activity for 2 with LC50
3.20 mM. The ligand and Co(BF4)2ꢂ6H2O showed no activity.
99.42(2)
173.42(3)
87.36(3)
95.85(4)
91.66(4)
87.11(3)
173.10(3)
92.50(4)
95.19(4)
86.14(4)
82.92(4)
88.60(5)
133.4(1)
133.7(1)
O3–Co1–N1
O3–Co1–N3
N1–Co1–N3
Co1–P1–C2
Co1–P1–C11
Co1–P1–C17
Co1–P2–C24
Co1–P2–C33
Co1–P2–C39
Co1–O1–C8
Co1–O3–C30
Co1–N1–N2
Co1–N3–N4
88.08(5)
83.23(5)
168.24(5)
110.94(5)
121.00(5)
111.18(5)
111.42(5)
114.32(5)
115.51(5)
108.43(9)
107.95(9)
112.71(9)
112.47(9)
3.7. Cytotoxic activity
Cytotoxic activity of the investigated cobalt(III) complex (2), the
ligand (1) and Co(BF4)2ꢂ6H2O was determined by MTT assay after
48 h treatment of three tumor cell lines (HeLa, FemX and LS-174)
and one normal cell line (MRC-5). Results are shown in Table 2
in terms of IC50 values. The complex (2) showed a very high cyto-
toxic activity, which was approximately twofold higher than cis-
platin on cervix (HeLa), and melanoma tumor cell lines (FemX),
and almost threefold higher in colorectal tumor cell line (LS-
chelation systems are much less planar and P and O atoms deviate
significantly from the average ligand planes.
The complex cation is counterbalanced by a BF4ꢀ anion orienta-
tionally disordered around the boron atom and the crystal packing
does not reveal any significant structural motif.
174), with IC50 values ranging from 4.90 0.08
lM (HeLa) to
7.42 2.45 M (LS-174). Selectivity to HeLa cells was observed,
l
both in regard to the other tumor cell lines and to the normal cell
line. The ligand (1), when tested in the range of concentrations up
3.5. Antimicrobial activity
to 100
lM, showed activity only to HeLa cells (IC50 value
58.28 0.23
l
M), which was twelve times lower than the activity
The complex showed a strong activity to Gram positive bacteria,
much higher than the activity of both the ligand and Co(BF4)2ꢂ6H2O
and similar to cefotaxime. The activity of the complex to Gram
of the complex. The cobalt(II) salt exhibited cytotoxic activity
although to a much lower extent compared to the cobalt complex.
The results are presented in terms of IC50 values that are deter-

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M. Milenkovic et al. / Inorganica Chimica Acta 395 (2013) 33–43
Table 6
Minimal inhibitory concentration of the investigated compounds to selected microorganisms.
Microorganism
1 (mM) MIC
2 (mM) MIC
Co(BF4)2ꢂ6H2O (mM) MIC
0.734
Cefotaxime (mM) MIC
0.027
Nystatin (
n.t
lM) MIC
Staphylococcus aureus
ATCC 25923
1.328
0.070
Staphylococcus epidermidis
ATCC 12228
Kocuria rhizophila
ATCC 9341
Bacillus subtilis
ATCC 6633
Escherichia coli
ATCC 25922
Klebsiella pneumoniae
ATCC 13883
Pseudomonas aeruginosa
ATCC 27853
Candida albicans
ATCC 10259
Candida albicans
ATCC 10231
1.328
0.070
0.035
0.070
0.279
0.139
0.139
>0.558
>0.558
0.367
0.367
0.183
0.367
0.734
0.367
0.183
0.183
0.027
0.027
0.027
0.007
0.007
0.027
n.t.
n.t.
1.328
n.t.
1.328
n.t.
>1.328
1.328
n.t.
n.t.
>1.328
>1.328
>1.328
n.t.
0.216
0.540
n.t.
n.t.-not tested.
Fig. 4. Cell survival diagrams of HeLa, FemX, LS-174 and MRC-5 cells after 48 h of continual agent action for ligand (1), Co(III) complex (2) and cobalt salt. Data are
representative for one out of three separate experiments with standard deviations.
mined from cell survival diagrams (Fig. 4). Values of IC50 represent
the average of two to three experiments, with each experiment
performed in three replicates.
tinual treatment for 24 and 48 h, using staining with PI [26].
Examination of the histograms indicated that cobalt(III) complex
induced perturbations of cell cycle of HeLa cells, results presented
in Fig. 5. Obtained results show that cobalt(III) complex in HeLa
cells induced decrease of percent of cells in G1 and slight increase
of percent of cells in the S phase of cell cycle, with no important
increase of apoptotic fraction of cells (evaluated as Sub-G1
3.7.1. Cell cycle
The effect of investigated cobalt(III) complex on cell cycle pro-
gression of HeLa cells was examined by flow cytometry after con-

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M. Milenkovic et al. / Inorganica Chimica Acta 395 (2013) 33–43
40
Fig. 5. Effect of Co(III) complex (2) and CDDP on cell cycle progression of HeLa cells following 24 and 48 h incubation with concentrations of investigated complexes
corresponding to 0.5xIC50 and IC50. Control were untreated cells (incubated with nutrient medium only). Histograms presented are representative of three independent
experiment.
fraction) after 24 h of continual agent action. Treatment for 48 h
with the investigated complex indicated that cells that survived
treatment were able to recover to normal cell cycle, indicating that
the investigated complex induced reversible interactions with
DNA, in contrast to cisplatin which induced more permanent dis-
tortions of cell cycle.

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M. Milenkovic et al. / Inorganica Chimica Acta 395 (2013) 33–43
Fig. 6. Representative dot plot diagrams obtained by flow cytometry of Annexin-V-FITC/PI double-stained HeLa cells untreated (control) or treated with Co(III) complex (2)
and CDDP with concentrations corresponding to 0.5xIC50 and IC50. FITC(ꢀ)/PI(ꢀ)(lower-left quadrant) are intact cells, FITC(+)/PI(ꢀ) (lower-right quadrant) are early apoptotic
cells, FITC(+)/PI(+) (upper-right quadrant) are late apoptotic or necrotic cells and FITC(ꢀ)/PI(+) (upper-left quadrant) are necrotic cells.
3.7.2. Apoptotic assay
it is crucial to consider interactions with DNA, through examina-
tion of cell cycle perturbations and potential of induction of apop-
tosis (determination of mode of cell death preferentially induced
by investigated agent).
Potential of cobalt(III) complex (2) and CDDP to induce apopto-
tic cell death was determined by staining treated HeLa cells with
two dyes Annexin V–FITC and propidium iodide and analysis on
flow cytometer. Treatment with 0.5xIC50 and IC50 concentrations
Numerous studies of the molecular mechanisms of antitumor
action of cisplatin pointed out that biochemical mechanisms of
the cisplatin cytotoxicity involve the binding of the drug to DNA
and non-DNA targets and further induction of cell death through
apoptosis, necrosis or both within the heterogeneous population
of cells that form a tumor mass [35,36]. Consequently, in investi-
gating mechanism of action of newly synthesized metal complexes

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M. Milenkovic et al. / Inorganica Chimica Acta 395 (2013) 33–43
42
of cobalt(III) complex and cisplatin for 48 h, induced similar per-
cent of apoptotic cells for both complexes and with no differences
between both concentrations (from 20% to 27%), Fig. 6.
3.8. DNA binding experiments
The interaction of metal complexes with double stranded closed
circular plasmid DNA is usually monitored by agarose electropho-
resis. The ability of Co(III) complexes to mediate DNA cleavage was
well documented [37,38]. The effects of the Co(III) complex (2) on
supercoiled DNA were studied using plasmid pUC 18 in TS buffer
pH 7.9 (Fig. 7). The results showed that no strand scission was ob-
served at increasing concentrations of the complex (Fig. 7, lanes 2,
3 and 4). The cleavage of plasmid DNA does not present a mode of
action for this Co(III) complex, i.e., the complex has no nuclease
activity. The result is in accordance with the previously obtained
ones for dinuclear cobalt(III) complex with the condensation prod-
uct of 2-acetylpyridine and malonic acid dihydrazide [39].
In order to test further cleavage efficiency of Co(III) complex,
the complex was allowed to react with native linear double
stranded bacteriophage lambda DNA for 1.5 h at 37 °C. It can be
seen from the electrophoregram in Fig. 8 that the complex again
did not show a noticeable DNA cleavage activity. Binding of the
complex to the DNA causes the DNA bands to smear in the gel
(lanes 2–4). The changes in DNA bands were observed in a concen-
tration-dependent way.
Fig. 7. Agarose gel electrophoresis showing no changes in electrophoretic mobility
of the superhelicoidal forms FI and the open circular forms FII of plasmid pUC18
DNA (0.13 pmol, lane 1) after incubation (1.5 h at 37 °C) with 5 nmol, 10 nmol and
15 nmol of the Co(III) complex (lanes 2, 3 and 4, respectively). The control sample:
pUC18 (213 ng) with DMSO (3 lL) (lane 5).
Spectroscopic methods were employed to further ascertain
whether there is any binding of the complex with bacteriophage
lambda DNA. Electronic absorption spectra of the complex in aque-
ous buffer media both in the absence and the presence of lambda
DNA are given in Fig. 9.
It can be seen that there are minimal changes in spectroscopic
patterns, indicating no binding of the complex to lambda DNA.
The observations are in accordance with the results of cell cycle
and apoptosis experiments.
Fig. 8. Agarose gel electrophoresis showing damage of linear double-stranded
lambda DNA (0.01 pmol, lane 1) after incubation (1.5 h at 37 °C) with 5 nmol,
10 nmol and 15 nmol of the Co(III) complex (lanes 2, 3 and 4, respectively).
Fig. 9. Electronic absorption spectra of various amounts of the Co(III) complex in TS buffer after incubation (1.5 h at 37 °C) in the absence (lines) and the presence (circles) of
lambda DNA (0.49 pmol).

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M. Milenkovic et al. / Inorganica Chimica Acta 395 (2013) 33–43
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4. Conclusions
ˇ
[12] V.M. Leovac, B. Ribár, G. Argay, A. Kálmán, I. Brceski, J. Coord. Chem. 39 (1996)
The newly synthesized complex showed a strong cytotoxic
activity to the tested cell lines with selectivity to HeLa cells. The
Pt(II) and Pd(II) complexes with similar ligands also showed a
strong cytotoxic activity [19], but with a different mechanism of
action. The investigated Co(III) complex had a different effect on
cell cycle progression of HeLa cells than either of the three previ-
ously studied complexes [19] or cisplatin. Namely, it caused no
important increase of apoptotic (sub-G1) fraction. Contrary to cis-
platin the effects on cell cycle progression were temporary. On the
other hand, in the Annexin V–FITC apoptosis test, for detection of
one marker of early apoptosis, externalization of phosphatidylser-
ine, the newly synthesized complex, cisplatin and the previously
synthesized complexes showed similar effects. High cytotoxicity
of cobalt(III) complex and low perturbations of cell cycle, moderate
potential of induction of externalization of phosphatidylserine and
insignificant spectral and electrophoretical changes indicate that
DNA is not the target responsible for cytotoxicity of this complex.
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Appendix A. Supplementary material
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