Imino-phosphine palladium(II) and platinum(II) complexes: Synthesis, molecular structures and evaluation as antitumor agents

Article abstract of DOI:10.1016/j.jinorgbio.2013.09.010

The imino-phosphine ligands L1 and L2 were prepared via condensation reaction of 2-(diphenylphosphino)benzaldehyde with substituted anilines and obtained in very good yields. An equimolar reaction of L1 and L2 with either PdCl₂(cod) or PtCl₂(cod) gave new palladium(II) and platinum(II) complexes 1-4. The compounds were characterized by elemental analysis, IR, ¹H and ³¹P NMR spectroscopy. The molecular structures of 2, 3 and 4 were confirmed by X-ray crystallography. All the three molecular structures crystallized in monoclinic C2/c space system. The coordination geometry around the palladium and platinum atoms in respective structures exhibited distorted square planar geometry at the metal centers. The complexes were evaluated in vitro for their cytotoxic activity against human breast (MCF-7) and human colon (HT-29) cancer cells, and they exhibited growth inhibitory activities and selectivity that were superior to the standard compound cisplatin.

Full text of DOI:10.1016/j.jinorgbio.2013.09.010

Journal of Inorganic Biochemistry 129 (2013) 112–118
Contents lists available at ScienceDirect
Journal of Inorganic Biochemistry
journal homepage: www.elsevier.com/locate/jinorgbio
Imino-phosphine palladium(II) and platinum(II) complexes: Synthesis,
molecular structures and evaluation as antitumor agents
a
b
b
William M. Motswainyana a, , Martin O. Onani , Abram M. Madiehe , Morounke Saibu ,
⁎
Ntevheleni Thovhogi b, Roger A. Lalancette c
a
Chemistry Department, University of the Western Cape, Private Bag X17, Bellville 7535, South Africa
Department of Biotechnology, University of the Western Cape, Private Bag X17, Bellville 7535, South Africa
Carl A. Olson Memorial Laboratories, Department of Chemistry, Rutgers University, Newark, NJ 07102, USA
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 26 July 2013
Received in revised form 11 September 2013
Accepted 11 September 2013
Available online 18 September 2013
The imino-phosphine ligands L1 and L2 were prepared via condensation reaction of 2-(diphenylphosphino)
benzaldehyde with substituted anilines and obtained in very good yields. An equimolar reaction of L1 and L2
with either PdCl2(cod) or PtCl2(cod) gave new palladium(II) and platinum(II) complexes 1–4. The compounds
were characterized by elemental analysis, IR, 1H and 31P NMR spectroscopy. The molecular structures of 2, 3
and 4 were confirmed by X-ray crystallography. All the three molecular structures crystallized in monoclinic
C2/c space system. The coordination geometry around the palladium and platinum atoms in respective structures
exhibited distorted square planar geometry at the metal centers. The complexes were evaluated in vitro for their
cytotoxic activity against human breast (MCF-7) and human colon (HT-29) cancer cells, and they exhibited
growth inhibitory activities and selectivity that were superior to the standard compound cisplatin.
© 2013 Elsevier Inc. All rights reserved.
Keywords:
Imino-phosphine
Palladium
Platinum
Synthesis
Molecular structures
Antitumor
1. Introduction
gold(I) complexes [10–13]. For instance, triethylphosphine (2,3,4,6-
tetra-O-acetyl-β-1-D-thiopyranosato-S)gold(I), commonly known as
Most of the chemistry of imino-phosphine compounds originated
from the discovery of the ligand 2-(diphenylphosphino)benzaldehyde
by Rauchfuss et al. in 1982 [1]. This compound has since been preferred
as a main precursor in the preparation of many derivatives of imino-
phosphine ligands. Consequently, several metal complexes containing
imino-phosphine ligands started to emerge in the early 1990s [2–4].
Since then, hetero-dentate ligands with hard and soft P–N donor
atoms with interesting properties have been generated and continue
to attract interest to date [5–8]. Upon coordination to transition
metals, the hard and soft combination creates asymmetry in the
metal orbitals, which subsequently regulates the reactivity of a com-
plex bearing these ligands [5]. An illustration of this effect is demon-
strated by the observed difference in the trans-influence of the
phosphorus donor atom compared to either nitrogen or oxygen [5].
Despite the preparation of several phosphine based complexes,
they remain least explored for their potential to inhibit tumor
growth. The likely advanced reason could be their susceptibility to
oxidation, which makes them very difficult to handle in air [9]. The
few existing reports on antitumor studies are based on phosphine
auranofin is the first phosphine complex that showed significant
cytotoxicity about two decades ago [10]. In the auranofin molecule,
the neutral triethylphosphine ligand confers membrane perme-
ability while the mono-anionic tetraacetylthioglucose is displaced
quite rapidly in vivo. The compound was proved to possess in vitro
cytotoxic potency against melanoma (B16) and leukemia (P388) cancer
cell lines [10]. Recently, additional in vitro studies have shown that
auranofin was able to overcome cisplatin resistance in human ovarian
cancer cells [11]. In particular, it was found that auranofin causes an
alteration of the redox state of the cell, leading to an increased produc-
tion of hydrogen peroxide and oxidation of the components of the
thioredoxin system, therefore creating suitable conditions for en-
hanced apoptosis [11]. Other gold(I) phosphine complexes which
have also shown great potential to induce apoptosis on tumor cell
lines include the 1,2-[bis(diphenylphosphino)]ethane gold(I) [12]
and tetrakis[tris(hydroxyl-methyl)]phosphine gold(I) chloride [13].
The work of Bacchi et al. has mechanistically demonstrated that an
imino-phosphine ligand which forms a 6-membered ring around
the metal center stabilizes the complex during biological investiga-
tions, while the steric bulkiness of the ligand manipulates the activ-
ity of the complex against cisplatin resistant cells [14]. In the context
of this background, we report the synthesis and full characterization
⁎
Corresponding author. Tel.: +27 21 959 2621; fax: +27 21 959 3055.
E-mail address: wmotswainyana@uwc.ac.za (W.M. Motswainyana).
0162-0134/$ – see front matter © 2013 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.jinorgbio.2013.09.010

W.M. Motswainyana et al. / Journal of Inorganic Biochemistry 129 (2013) 112–118
113
of sterically congested mononuclear imino-phosphine palladium(II)
2.3.2. Dichloro-[2-diphenylphosphino-benzylidene)-2,6-dimethylphenylen-
amine] palladium(II) (2)
and platinum(II) complexes containing substituted aniline. Antitumor
activity of these complexes has been investigated in vitro against MCF-7
and HT-29 cancer cell lines and the results are herein discussed. This
work could add up to the current drive in the design and development
of platinum group metal (PGM) complexes in anticancer therapy.
The compound was synthesized according to the procedure used
in 1, using PdCl2(cod) (0.0424 g, 0.149 mmol) and L2 (0.0561 g,
0.143 mmol). Suitable crystals for X-ray crystallography were grown
by the slow diffusion of hexane into a solution of the complex in
CH2Cl2. Yield: 0.0567 g (72%); IR (nujol cm−1); ν(C_N imine) 1610,
ν(C_C phenyl) 1586, 1559, 1501; 1H NMR (200 MHz, CDCl3): δ 8.62
(s, 1H, \CH_N); 8.22, 6.89–7.92 (m, 12H, phenyl); 5.75 (d, 6H, J =
1.6, phenyl); 2.15 (d, 6H, J = 2.2, CH3); 31P NMR (161.9 Hz, CDCl3) δ
27.68(s); Anal. Calcd for C27H24Cl2NPPd: C, 56.81; H, 4.24; N, 2.45;
Found: C, 57.01; H, 4.30; N, 2.29.
2. Experimental
2.1. Materials and instrumentation
All reactions were carried out under nitrogen atmosphere using a
dual vacuum/nitrogen line and standard Schlenk techniques unless
stated otherwise. Solvents were dried and purified by heating at reflux
under nitrogen in the presence of a suitable drying agent. All the re-
agents and starting materials were purchased from Sigma-Aldrich and
were used without any further purification. The palladium metal pre-
cursor PdCl2(cod) (cod = cyclooctadiene) was prepared by following
literature method [15].
2.3.3. Dichloro-[(2-diphenylphosphino-benzylidene)-2-methylphenylen-
amine] platinum(II) (3)
The compound was synthesized according to the procedure used
in 1, using PtCl2(cod) (0.0388 g, 0.131 mmol) and L1 (0.0479 g,
0.126 mmol). Suitable crystals for X-ray crystallography were grown
by the slow diffusion of hexane into a solution of the complex
in CH2Cl2. Yield: 0.0618 g (76%); IR (nujol cm−1); 1607, ν(C_C
phenyl) 1588, 1560, 1502; 1H NMR (200 MHz, CDCl3): δ 8.88 (s,
1H, \CH_N); 8.17, 6.95–7.86 (m, 12H, phenyl); 5.75 (s, 6H, phe-
nyl); 2.14 (d, 3H, J = 2.4, CH3); 31P NMR (161.9 Hz, CDCl3) δ
28.41(s); Anal. Calcd for C26H22Cl2NPPt: C, 48.38; H, 3.44; N, 2.17;
Found: C, 48.26; H, 3.19; N, 2.42.
2.2. Synthesis of ligands
2.2.1. (2-Diphenylphosphino-benzylidene)-2-methylphenylen-amine (L1)
To a solution of 2-(diphenylphosphino)benzaldehyde (0.279 g,
0.961 mmol) in CH2Cl2 (10 ml) in a Schlenk tube was added 2-
methylaniline (0.103 g, 0.961 mmol) dropwise. Anhydrous magnesium
sulfate (~0.5 g) was added to the Schlenk tube and the reaction was
stirred at room temperature for 20 h. The resulting yellow mixture
was filtered to obtain a yellow solution, which gave yellow oil upon
evaporation of the solvent. Yield: 0.2990 g (82%); IR (nujol cm−1);
ν(C_N imine) 1622, ν(C_C phenyl) 1593, 1585, 1505; ν(P\Ph)
1435; 1H NMR (200 MHz, CDCl3): δ 8.99 (d, 1H, J = 5.2, \CH_N);
8.27, 6.91–7.67 (m, 12H, phenyl); 6.46 (t, 6H, J = 7.6, phenyl); 1.27
(s, 3H,CH3); 31P NMR (161.9 Hz, CDCl3) δ −13.96(s); Anal. Calcd for
2.3.4. Dichloro-[2-diphenylphosphino-benzylidene)-2,6-dimethylphenylen-
amine] platinum(II) (4)
The compound was synthesized according to the procedure used
in 1, using PtCl2(cod) (0.0476 g, 0.161 mmol) and L2 (0.0609 g,
0.155 mmol). Suitable crystals for X-ray crystallography were
grown by the slow diffusion of hexane into a solution of the com-
plex in CH2Cl2. Yield: 0.0756 g (74%); IR (nujol cm−1); 1603,
ν(C_C phenyl) 1583, 1561, 1503; 1H NMR (200 MHz, CDCl3): δ
8.87 (s, 1H, \CH_N); 8.20, 6.76–7.89 (m, 12H, phenyl); 5.74 (s,
6H, phenyl); 2.13 (d, 6H, J = 2.2, CH3); 31P NMR (161.9 Hz,
CDCl3) δ 26.23(s); Anal. Calcd for C27H24Cl2NPPt: C, 49.18; H,
3.67; N, 2.12; Found: C, 49.40; H, 3.72; N, 2.33.
C26H22NP: C, 82.30; H, 5.84; N, 3.69; Found: C, 82.04; H, 5.93; N, 3.55.
2.2.2. (2-Diphenylphosphino-benzylidene)-2,6-
dimethylphenylen-amine (L2)
The ligand was synthesized according to the procedure described for
L1 using 2-(diphenylphosphino)benzaldehyde (0.2752 g, 0.948 mmol)
and 2,6-dimethylaniline (0.1149 g, 0.948 mmol). Yellow oil was
obtained. Yield: 0.2984 g (80%); IR (nujol cm−1); ν(C_N imine) 1632,
ν(C_C phenyl) 1590, 1562, 1502; ν(P\Ph) 1434; 1H NMR (200 MHz,
CDCl3): δ 8.88 (d, 1H, J = 5.2, \CH_N); 8.30, 6.88–7.94 (m, 12H, phe-
nyl); 6.73 (t, 6H, J = 7.4, phenyl); 1.83 (s, 6H,CH3); 31P NMR (161.9 Hz,
CDCl3) δ −14.00(s); Anal. Calcd for C27H24NP: C, 82.42; H, 6.15; N, 3.56;
Found: C, 82.63; H, 5.98; N, 3.82.
2.4. Molecular structures of 2, 3 and 4
Single crystals of complexes 2, 3 and 4 suitable for X-ray crystallog-
raphy were grown by various crystallization techniques. X-ray diffrac-
tion data for the compound was collected on a Bruker KAPPA APEX II
DUO diffractometer using graphite-monochromated Mo-Kα radiation
(χ = 0.71073 Å). The crystal structures were solved by direct methods
using SHELX [17] and refined by full-matrix least-squares methods
based on F2 [17] using SHELX [17] and using the graphics interface
program ORTEP-3 for Windows [18,19].
2.3. Synthesis of metal complexes
2.5. Cytotoxicity determination
2.3.1. Dichloro[(2-diphenylphosphino-benzylidene)-2-
methylphenylen-amine] palladium(II) (1)
2.5.1. Cell culture
To a suspension of PdCl2(cod) (0.0482 g, 0.169 mmol) in CH2Cl2
(20 ml) was added a solution of L1 (0.0635 g, 0.167 mmol) in CH2Cl2
(5 ml). The reaction was stirred at room temperature for 6 h, resulting
in a yellow solution. The solution was concentrated before excess hex-
ane was added to precipitate out a yellow solid. The precipitate was
filtered, washed with hexane (2 × 5 ml) and dried under vacuum.
Suitable crystals for X-ray crystallography were grown by slow evapora-
tion of CH3CN solution of the complex. [16]. Yield: 0.0697 g (75%); IR
(nujol cm−1); ν(C_N imine) 1614, ν(C_C phenyl) 1585, 1560, 1502;
1H NMR (200 MHz, CDCl3): δ 8.67 (s, 1H, \CH_N); 8.20, 6.92–7.96 (m,
12H, phenyl); 5.77 (d, 6H, J = 1.8, phenyl); 2.14 (d, 3H, J = 2.2, CH3);
31P NMR (161.9 Hz, CDCl3) δ 30.47(s); Anal. Calcd for C26H22Cl2NPPd: C,
56.09; H, 3.98; N, 2.52; Found: C, 55.83; H, 4.22; N, 2.77.
Cancerous cell lines MCF-7 and HT-29 were cultured in DMEM
medium with GlutaMAX-1, 10% (v/v) fetal calf serum, and 0.2% (v/v)
streptomycin–penicillin. All cell culture reagents were supplied by
Invitrogen Ltd. All cell lines were maintained at 37 °C in an atmosphere
of 5% CO2. Cells were plated in 96 well tissue culture plates at a cell den-
sity of 2.4 × 104 cells per well or in 24 well tissue culture plates at a cell
density of 1 × 105 cells per well. After 24 h the medium was replaced
with medium containing the test compounds.
2.5.2. MTT assay
The 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium
bromide (MTT) assay measures the amount of MTT reduction by
the mitochondrial dehydrogenase based on the ability of live cells

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W.M. Motswainyana et al. / Journal of Inorganic Biochemistry 129 (2013) 112–118
PPh2
PPh2
CH2Cl2
H2
N
R
+
rt, 20hr
N
R
O
PdCl2(COD)
PtCl2(COD)
Ph2
P
Cl
M
Cl
R
N
R = 2-MeC6H4, M = Pd(1); M = Pt(3)
R = 2,6-Me2C6H3, M = Pd(2); M = Pt(4)
Scheme 1. Preparation of imino-phosphine palladium(II) and platinum(II) complexes 1–4.
to incorporate and bind the dye. The cells were plated in 96-well
tissue plates at a density of 2.4 × 104 cells/ml per well. Cells were
then treated with various concentrations of the complexes (100 to
10 μM) after which they were incubated for 24 h. Triplicate wells
were established for each concentration. Just 5 h before the elapse
of 24 h, 10 μl of 5 mg/ml MTT solution was added to each well and
the plates were further incubated for 4 h. At the end of the incuba-
tion period, the media was removed from each well and replaced
with 50 μl of DMSO. The plates were shaken on a rotating shaker
for 10 min before taking readings at 560 nm using a microplate
reader.
with substituted anilines (Scheme 1). The ligands were obtained as
light brown oil in good yields of over 80%. Imino-phosphine compounds
are known for their susceptibility to oxidation, therefore an oxygen free
technique was used to prevent the ligands from possible contamination
with phosphine oxide. Ligand L1 is reported for the first time to the best
of our knowledge. L2 has been reported by other research groups
[5,20,21] but it was independently prepared and characterized by us.
The ligands were reacted with either PdCl2(cod) or PtCl2(cod) to give
the corresponding new imino-phosphine palladium(II) and platinum(II)
complexes 1–4 (Scheme 1). The complexes were obtained as yellow
solids in good yields averaging 70% after filtration and evaporation of
solvents.
All the compounds were characterized by a combination of ele-
mental analysis, IR, 31P and 1H NMR spectroscopy. In the IR spectra
of the ligands, imine formation was confirmed by strong absorption
bands at 1622 and 1632 cm−1 for L1 and L2 respectively. These
absorption frequencies are consistent with those of related imino-
phosphine compounds [22–25]. Coordination of the ligands was
further confirmed by lower absorption bands between 1603 and
1614 cm−1, which is a typical characteristic of metal coordinated
imines [22–25].
2.5.3. APOPercentage™ apoptosis assay
This assay helps with detection and quantification of apoptosis. Cells
were treated for 24 h with increasing concentrations (6.25–100 μM) of
the complexes and 10 μM cisplatin (positive control) and incubated for
a further 24 h, after which the cells were harvested. A minimum of
10,000 cells per sample was acquired and analyzed on a FASCan™
(Becton Dickinson) instrument using CELLQuest PRO software (BD
Biosciences Pharmingen). All the experiments were done in triplicate.
In the 1H NMR spectra of the ligands, doublet peaks were observed at
8.99 ppm and 8.88 ppm for L1 and L2 respectively due to (HC_N), thus
confirming a Schiff base condensation reaction. The doublet suggests
that the imine proton had coupled to the phosphorus by pointing
towards the phosphorus lone pair [22,25–28]. The 31P NMR spectra
of the ligands showed an upfield characteristic singlet at around
−14.00 ppm for uncoordinated phosphorus [22]. Coordination of
the ligands was confirmed in the 1H NMR spectra of complexes,
3. Results and discussion
3.1. Synthesis of ligands and metal complexes
The imino-phosphine ligands L1 and L2 were prepared via a Schiff
base condensation reaction of 2-(diphenylphosphino)benzaldehyde
Fig. 1. X-ray crystal structure of complex 2.
Fig. 2. X-ray crystal structure of complex 3.

W.M. Motswainyana et al. / Journal of Inorganic Biochemistry 129 (2013) 112–118
115
Fig. 3. X-ray crystal structure of complex 4.
which showed singlet peaks in the upfield region 8.62–8.88 ppm.
This provided evidence for strong coordination of the imine nitro-
gen to the palladium or platinum metal center. These chemical
shift values generally reflect an upfield shift compared to their
corresponding ligands, a phenomenon which is consistent with co-
ordinated imines [29,30]. The 31P NMR spectra of the complexes
also revealed a downfield shift in the phosphorus peak to around
30.00 ppm, confirming coordination of the phosphorus to the metal
center [25,28].
generating a distorted square planar coordination geometry around
the metal center. The bond angles N(1)\Pd(1)\P(1) (89.14(5)°) and
Cl(2)\Pd(1)\Cl(1) (91.039(18)°) in 2, N(1)\Pt(1)\P(1) (88.14(13)°)
and Cl(2)\Pt(1)\Cl(1) (89.06(5)°) in 3 and N(1)\Pt(1)\P(1)
(89.25(7)°) and Cl(2)\Pt(1)\Cl(1) (89.38()°) in 4 describe the ob-
served distorted square planar geometry, while also indicating some
Table 2
Selected bond lengths and bond angles for complexes 2, 3 and 4.
Complex
Bond length (Å)
Bond angle (°)
3.2. Single crystal X-ray diffraction studies
2
Pd(1)\N(1) 2.0634(18)
Pd(1)\P(1) 2.2213(5)
Pd(1)\Cl(1) 2.3628(5)
Pd(1)\Cl(2) 2.2841(5)
Pt(1)\N(1) 2.041(5)
Pt(1)\P(1) 2.2030(13)
Pt(1)\Cl(1) 2.3631(13)
Pt(1)\Cl(2) 2.2891(14)
Pt(1)\N(1) 2.044(2)
Pt(1)\P(1) 2.2099(8)
Pt(1)\Cl(1) 2.3624(7)
Pt(1)\Cl(2) 2.2889(7)
N(1)\Pd(1)\P(1) 89.14(5)
N(1)\Pd(1)\Cl(1) 89.77(5)
P(1)\Pd(1)\Cl(2) 90.675(19)
Cl(2)\Pd(1)\Cl(1) 91.039(18)
N(1)\Pt(1)\P(1) 88.14(13)
N(1)\Pt(1)\Cl(1) 89.80(13)
P(1)\Pt(1)\Cl(2) 93.19(5)
Cl(2)\Pt(1)\Cl(1) 89.06(5)
N(1)\Pt(1)\P(1) 89.25(7)
N(1)\Pt(1)\Cl(1) 89.32(7)
P(1)\Pt(1)\Cl(2) 92.43(3)
Cl(2)\Pt(1)\Cl(1) 89.38(3)
The molecular structures of complexes 2, 3 and 4 were determined
by X-ray diffraction studies to confirm their structures. The molecular
structures of the complexes are shown in Figs. 1, 2 and 3 respectively.
Crystallographic data and refinement residuals are summarized in
Table 1, while selected bond lengths and bond angles are found in
Table 2.
The three molecules crystallize in the monoclinic space group C2/c.
The palladium and platinum atoms are four coordinated via the P and
N atoms of the imino-phosphine ligand and two chloride anions,
3
4
Table 1
Crystallographic data and refinement for complexes 2, 3 and 4.
Crystallographic data
2
3
4
Empirical formula
Formula weight
Temperature (K)
Crystal system
Space group
Unit cell dimensions
a (Å)
C
27H24Cl2NPPd·CH2Cl2
C26H22Cl2NPPt·CH2Cl2
730.33
100
Monoclinic
C2/c
C28H26Cl4NPPt
744.36
173
Monoclinic
C2/c
655.67
100
Monoclinic
C2/c
33.5076(6)
9.7724(2)
20.1900(3)
123.885(1)
5488.35(17)
8
33.1988(8)
9.7768(2)
20.4397(5)
125.568(1)
5396.5(2)
8
33.532(3)
9.8773(7)
20.2834(15)
123.941(1)
5573.3(7)
8
b (Å)
c (Å)
β (°)
V (Å3)
Z
D
cal (Mg m−3
)
1.587
1.798
1.774
F(000
)
2640
9.73
0.44 × 0.12 × 0.08
4990
2832
14.06
0.37 × 0.10 × 0.10
4915
2896
5.50
0.07 × 0.07 × 0.05
5967
Absorption coefficient (mm−1
Crystal size (mm3)
)
Number of data collected
Number of parameters refined
318
0.023
0.054
308
0.037
0.083
328
0.021
0.043
R[(F2 N 2σ(F2)]
wR(F2)
Theta range for data collection
Goodness-of-fit on F2
Largest diff. peak and hole
2.8 to 71.8°
1.08
4.3 to 71.8°
1.22
1.5 to 26.8°
1.02
0.87 and −0.71 e·Å−3
1.80 and −1.59 e·Å−3
0.51 and −0.52 e·Å−3

116
W.M. Motswainyana et al. / Journal of Inorganic Biochemistry 129 (2013) 112–118
Table 3
3.3. Cytotoxicity studies
Cytotoxic activities and selectivity index for the free ligand and complexes 1–4 tested
against MCF-7 and HT-29 cancer cell lines.
The main aim of this study was to evaluate imino-phosphine
palladium(II) and platinum(II) complexes as possible antitumor agents.
The antitumor investigation of these complexes was motivated by two
reasons: firstly the complexes are bulky, and the steric bulk around
the coordinated metal atom has been perceived as a perfect strategy
to prevent axial approach to the metal atom, which would inhibit the
formation of a five-coordinate intermediate that would lead to a ligand
substitution [36]. The steric hindrance would also permit high selectiv-
ity to DNA binding [37–39]. Secondly, chelating bidentate ligands
have been viewed as important in preventing trans-labilization and
undesired displacement of the ligands by bio-molecules [40]. Fur-
thermore, imino-phosphine complexes have been less explored for
their potential to inhibit tumor growth possibly due to their suscep-
tibility to oxidation.
IC50 (μM)a
Compound
MCF-7
SIb
HT-29
SIb
L2
N100
0.0
N100
0.0
Palladium(II) complexes
1
2
43.5
28.5
0.25
0.21
2.29
2.87
48
29
0.19
0.15
2.08
2.81
Platinum(II) complexes
3
4
87
86
100
0.03
0.38
0.20
1.0
1.1
0.94
55
50
0.31
0.41
1.6
1.8
0.94
Cisplatin
100
0.51
a
The concentration of the complex required to inhibit cell growth by 50%. The experi-
ments were done in triplicate. Data were expressed as the mean of the triplicate.
IC50 N 100 µM is considered to be inactive.
b
Selectivity index. SI N 1 indicates high selectivity.
The free imino-phosphine ligand L2 and palladium(II) and
platinum(II) complexes 1–4 were evaluated for their potential to
exert cytotoxicity on highly invasive human breast (MCF-7) and
human colon (HT-29) tumor cell lines using MTT assay with minor
modifications [41–43]. The compounds were first solubilized in DMSO.
The solvent is regarded as highly effective in accelerating the rate
of chloride displacement from a complex [44]. The results, expressed
as concentration of the complex required to inhibit tumor cell growth by
50% (IC50) are summarized in Table 3. Cytotoxicity of cisplatin was evalu-
ated under the same experimental conditions for comparison. The selec-
tivity index (SI), which gives the cytotoxicity safety of the complexes was
calculated from IC50 values obtained from the induced significant levels of
ring strain induced by the chelating nature of the bidentate ligand. The
Pd(1)\C1(2) bond length of 2.2841(5)Å and Pt(1)\C1(2) bond length
of 2.2891(14)Å are in good agreement with the average Pd\Cl and
Pt\Cl bond distances respectively for known complexes [31–33]. The av-
erage bond lengths Pd(1)\N(1) and Pd(1)\P(1) in 2 and Pt(1)\N(1)
and Pt(1)\P(1) in 3 and 4 also compare well with the literature values
[32–35]. There is a detectable trans-influence as evidenced in bond
lengths Pd(1)\Cl(1) and Pt(1)\Cl(1) trans- to the phosphorus atom
which are significantly longer than their corresponding bond lengths
trans- to the amine. This reflects the stronger trans-influence of the
diphenylphosphino group compared to an amine [28].
MCF-7
Fig. 4. Quantification of pro-apoptotic activities of imino-phosphine complexes against MCF-7.

W.M. Motswainyana et al. / Journal of Inorganic Biochemistry 129 (2013) 112–118
117
apoptosis in all the cell lines. The imino-phosphine ligand L2 was also
evaluated to assess whether free imino-phosphine ligands could exert
cytotoxicity on cancer cells, and the ligand did not show any appreciable
activity (IC50 N 100 μM). Therefore, any observed significant activity
could be attributed to the complexes.
The imino-phosphine palladium(II) and platinum(II) complexes 1–4
proved to possess remarkable antiproliferative activities against MCF-7
and HT-29 cancer cell lines compared to the reference drug cisplatin,
which returned IC50 values around 100 μM. Furthermore, imino-
phosphine palladium(II) complexes showed superior cytotoxic ac-
tivities compared to their platinum(II) counterparts. For instance,
complexes 1 and 2 displayed cytotoxic effectiveness that is almost
three times that of complexes 3 and 4 against MCF-7 cancer cell
lines, and this could be attributed to the higher aqueous solubility
of palladium(II) complexes, which allows them to dissociate readily
in solution, thereby making them bio-available [45].
All the complexes generally exhibited higher cytotoxic activities
against human colon (HT-29) cell line (IC50 29–55 μM) compared to
the human breast (MCF-7) cell line. However, the highest cytotoxic ac-
tivity was observed in complex 2 (IC50 = 29 μM), which was approxi-
mately four times more potent than cisplatin against the two examined
cancer cell lines. The general pattern observed in cytotoxicity values
for the complexes across the two cell lines showed higher activities
for 2 and 4. These complexes contain aniline with two methyl groups
substituted at ortho-positions, making the complexes more sterically
demanding compared to 1 and 3. This trend is supported by the liter-
ature which stresses the importance of steric congestion in preventing
axial approach to the coordinated metal atom, thereby permitting high
selectivity to DNA binding [36–39]. Furthermore, complexes 1 and 2
gave selectivity indices N2 against the two cell lines and their selectivity
towards these cancer cell lines make them promising compounds.
The imino-phosphine ligands L1 and L2 form a 6-membered ring
around the metal center, which stabilized the complexes during biolog-
ical investigations, and probably resulting in the observed antitumor
profiles [14]. The complexes are also square planar with metal chloride
bonds in cis-position, and when considering the proposed mechanism
of action of cisplatin [46,47], it is reasonable to suggest that cytotoxicity
of these imino-phosphine complexes is derived from DNA binding. It is
also well known that square planar metal complexes with aromatic li-
gands bind to DNA by intercalation, implying that the complexes have
potential as intercalation agents [48,49]. Our initial study therefore,
has shown that imino-phosphine complexes are promising antitumor
agents. However, further biological investigations are needed to eluci-
date the intercalation modes and study the structure–activity rela-
tionship of the complexes, especially those that showed remarkable
cytotoxicity profiles in order to qualify them as potential drugs.
APOPercentage was used to evaluate pro-apoptotic activity of the
complexes. The assay was performed following a literature procedure
[50]. In the pro-apoptotic activity of the complexes (Figs. 4 and 5), a
dose dependent increase in the number of apoptotic cells was observed
in HT-29 and MCF-7 cell lines treated with the complexes, suggesting
that the complexes induced apoptosis against the examined cell lines.
4. Conclusions
We have successfully prepared and fully characterized imino-
phosphine palladium(II) and platinum(II) complexes. The complexes
were evaluated for the first time in vitro for their cytotoxic activity
HT-29
Fig. 5. Quantification of pro-apoptotic activities of imino-phosphine complexes against HT-29 cells.

118
W.M. Motswainyana et al. / Journal of Inorganic Biochemistry 129 (2013) 112–118
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against human breast (MCF-7) and human colon (HT-29) cancer cell
lines. The free imino-phosphine ligand did not return any appreciable
activity against the examined cell lines. The complexes exhibited growth
inhibitory activities that were even better than cisplatin, with the highest
cytotoxic activity pronounced in complex 2 (IC50 = 29 μM). Complex 2
also displayed remarkable cytotoxicity and selectivity against the
human breast (MCF-7) cell line (IC50 = 28.5 μM) compared to
complex 4 (IC50 = 86 μM).
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