Synthesis, characterisation and in vitro evaluation of platinum(II) and gold(I) iminophosphine complexes for anticancer activity

Article abstract of DOI:10.1016/j.poly.2012.09.053

The reactions of iminophosphine ligands with [PtCl₂(COD)], [PtCl₂(DMSO)₂], and [Au(tht)Cl] has been investigated. The new platinum(II) and gold(I) complexes were characterised using elemental analysis, electrospray ionisat

Full text of DOI:10.1016/j.poly.2012.09.053

Polyhedron 49 (2013) 29–35
Contents lists available at SciVerse ScienceDirect
Polyhedron
journal homepage: www.elsevier.com/locate/poly
Synthesis, characterisation and in vitro evaluation of platinum(II) and gold(I)
iminophosphine complexes for anticancer activity
Haleden Chiririwa a,b,c, , John R. Moss a,1, Denver Hendricks b, Gregory S. Smith a, Reinout Meijboom c
⇑
a Department of Chemistry, University of Cape Town, Private Bag, Rondebosch 7701, South Africa
b Division of Medical Biochemistry, University of Cape Town, Private Bag X3, Observatory 7935, South Africa
c Research Centre for Synthesis and Catalysis, Department of Chemistry, University of Johannesburg, P.O. Box 524, Auckland Park 2006, Johannesburg, South Africa
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 10 July 2012
Accepted 30 September 2012
Available online 9 October 2012
The reactions of iminophosphine ligands with [PtCl2(COD)], [PtCl2(DMSO)2], and [Au(tht)Cl] has been inves-
tigated. The new platinum(II) and gold(I) complexes were characterised using elemental analysis, electro-
spray ionisation-mass spectrometry (ESI-MS), NMR (1H and 31P) and IR spectroscopy and X-ray diffraction
studies. In vitro cytotoxic study results show that platinum and gold complexes block the proliferation of
WHCO1 and KYSE450 cell lines with an IC50 range of 2.16–9.47
lM.
Keywords:
Ó 2012 Elsevier Ltd. All rights reserved.
Iminophosphines
Platinum(II) complexes
Gold(I) complexes
Anticancer
1. Introduction
pressing blood vessels or organs such as the brain, or disrupting
critical processes.
The rapidly growing field of organometallic chemistry has ex-
panded into the scope of biology and medical applications [1–3].
Metal complexes are also of great interest in bioinorganic chemis-
try. According to the International Agency for Research on Cancer
(IARC), in the year 2005, oesophageal cancer was reported as the
ninth most common cancer in the world, and the sixth leading
cause of cancer-related deaths worldwide [4]. A unique epidemio-
logical feature of oesophageal cancer is its very uneven geographic
distribution, with high incidence found within sharply demarcated
geographic confines [5]. These geographic ‘hot spots’ include areas
in Northern Iran, Kazakhstan, South Africa, and Northern China [6].
On the African continent, a number of reports have documented a
very high incidence of oesophageal cancer in South Africa, particu-
larly in the Transkei districts [7].
Cancer is fundamentally a disease, in which the cells proliferate
indefinitely, due to dysregulated control pathways. Consequently,
cancer cells continue to grow and divide yielding an ever increas-
ing mass referred to as a tumour [8]. The tumour grows invasively,
destroying surrounding body tissues. Cancer cells from this pri-
mary tumour may then spread, or metastasize, to other parts of
the body, where new tumours may begin to grow. Eventually the
tumour load will cause death, often by physically blocking or com-
Cancer makes a contribution to morbidity and mortality world-
wide, with the International Agency for Research on Cancer (IARC)
estimating that for 2008, there were 12.4 million new cases of can-
cer, 7.6 million deaths from cancer and 28 million people alive
with cancer within 5 years from initial diagnosis. They do report
that about half of the cases and about 60% of the mortalities were
from medium and low income countries [9].
Ever since its approval in 1978 for the treatment of testicular
and ovarian cancer, cisplatin has been used extensively in cancer
therapy [10]. Transition metal-based anticancer drugs such as cis-
platin have found widespread use, since they form highly reactive,
charged, platinum complexes that bind to nucleophilic groups such
as GC-rich sites in DNA, inducing DNA cross-links that result in
apoptosis and cell growth inhibition. In SA, like in other countries
worldwide, cisplatin is the drug of choice when treating oesopha-
geal cancer with chemotherapeutic agents. However, cisplatin has
numerous shortcomings, including induced cellular resistance, se-
vere toxicity, and a spectrum of activity limited to a narrow range
of tumor types. This has prompted considerable efforts to develop
novel metal-based anticancer agents that could circumvent the
limitations of cisplatin. As a result, thousands of platinum-based
compounds have been synthesized and screened for toxicity and
anticancer activity, although very few have been approved for clin-
ical use [11–15].
⇑
Corresponding author at: Department of Chemistry, University of Cape Town,
Private Bag, Rondebosch 7701, South Africa. Tel.: +27 11 559 3436; fax: +27 11 559
Auranofin, an orally absorbed gold(I) phosphine complex, had
been used for the treatment of rheumatoid arthritis [16], and early
reports also suggested that this compound and other gold phos-
phine complexes were also active against the growth of cultured
2819.
E-mail address: harrychiririwa@yahoo.com (H. Chiririwa).
Deceased.
1
0277-5387/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.poly.2012.09.053

30
H. Chiririwa et al. / Polyhedron 49 (2013) 29–35
tumour cells [17–20]. Auranofin itself has proved cytotoxic against
HeLa cells in vitro and P388 leukaemia cells in vivo [21,22]. Recent
advances in medicinal inorganic chemistry demonstrate significant
prospects for the utilization of Au(I) and Au(III) and their coordina-
tion complexes as drugs, presenting an interesting arena for inor-
ganic chemistry. Significant progress in gold based anticancer
agents has been achieved, based in part on a mechanistic under-
standing of the pharmacological effects of classical antitumor
drugs. There are quite a number of results indicating that gold
coordination compounds might be developed into future drugs,
but it will be a long time before their pharmacological potential
can be realised. It will take even longer to have a suitable candidate
turned into a clinically acceptable drug. The future development of
medicinal inorganic chemistry of Au(I) and Au(III) and their coordi-
nation complexes requires an understanding of the physiological
processing of gold complexes, to provide a rational basis for the de-
sign of new gold-based drugs.
2.3.2. Spectroscopic and analytical data for 1
Yellow powder. Yield: 76%. M.p.: 201–203 °C. Anal. Calc. for C31-
H32Cl2NPPt: C, 52.03; H, 4.51; N, 1.96. Found: C, 52.19; H, 4.74; N,
1.99%. MS (EI, m/z): 678.34, [MÀCl]+. IR (KBr) (
m
max/cmÀ1): 1624 s
m
(C N). 1H NMR (CDCl3): dH (ppm): 8.95 (d, 1H, J = 111.3 Hz) 8.18
(dd, 1H, J = 4.2 Hz, J = 9.0 Hz) 7.90 (m, 2H) 7.60 (m, 10H) 7.18 (m,
4H) 3.01 (m, 2H) 1.24 (d, 6H, J = 6.8 Hz) 0.83 (d, 6H, J = 6.8 Hz).
31P NMR (CDCl3; ppm) 5.83 [J (195Pt–31P): 3661 Hz].
2.3.3. Spectroscopic and analytical data for 2
Light yellow powder. Yield: 65%. M.p.: 180–183 °C. Anal. Calc.
for C26H22Cl2NPPt: C, 48.38; H, 3.44; N, 2.17. Found: C, 48.19; H,
3.12; N, 2.47%. MS (EI, m/z): 609.36, [MÀCl]+. IR (KBr) (mmax
/
cmÀ1): 1632 s (C N). 1H NMR (CDCl3): dH (ppm): 7.79 (m, 1H)
m
7.60 (t, 1H, J = 7.4 Hz) 7.47 (t, 1H, J = 7.4 Hz) 7.07 (t, 1H,
J = 8.6 Hz) 6.90 (t, 1H, J = 7.7 Hz) 6.49 (d, 1H, J = 7.2 Hz) 4.13 (t,
1H, J = 12.8 Hz). 31P NMR (CDCl3; ppm) 8.09 [J
(
195Pt–31P):
Here we report the synthesis and characterisation of novel plat-
inum(II) and gold(I) complexes with iminophosphine ligands and
the examination of their cytotoxic properties.
3668 Hz].
2.3.4. Spectroscopic and analytical data for 3
Yellow powder. Yield: 76%. M.p.: 168–169 °C. Anal. Calc. for C25-
H21Cl2N2PPt: C, 46.45; H, 3.27; N, 4.33. Found: C, 46.19; H, 3.12; N,
4.72%. MS (EI, m/z): 610.91, [MÀCl]+. IR (KBr) (
m
max/cmÀ1): 1630 s
2. Experimental
m
(C N). 1H NMR (CDCl3): dH (ppm): 9.19 (m, 1H) 8.64 (m, 1H)
8.53 (d, 1H, J = 4.3 Hz) 8.05 (dd, 1H, J = 4.1 Hz, J = 6.9 Hz) 7.92 (t,
1H, J = 7.5 Hz) 7.84 (t, 1H, J = 7.4 Hz) 7.60 (m, 2H) 7.41 (m, 3H)
7.09 (m, 5H) 5.80 (d, 2H, J = 18.7 Hz). 31P NMR (CDCl3; ppm) 8.44
[J (195Pt–31P): 3469.9 Hz].
2.1. Materials
All solvents were purified prior to use by standard literature
procedures and were stored under nitrogen in Teflon sealed stor-
age bottles. All reagents were purchased from Aldrich and used
without further purification. The ligands L1–L5 [23], [PtCl2(COD)]
[24,25], [PtCl2(DMSO)2] [26–28] and [Au(tht)Cl] [29] were pre-
pared as described in the literature.
2.3.5. Spectroscopic and analytical data for 4
Dark yellow powder. Yield: 72%. M.p.: 198–199 °C. Anal. Calc.
for C24H20Cl2NOPPt: C, 48.83; H, 3.77; N, 2.28. Found: C, 48.69;
H, 3.52; N, 2.52%. MS (EI, m/z): 599.91, [MÀCl]+. IR (KBr) (mmax
/
cmÀ1): 1633 s (C N). 1H NMR (CDCl3): dH (ppm): 9.06 (m, 1H)
m
8.02 (m, 1H) 7.88 (td, 2H, J = 7.4 Hz, J = 19.3 Hz) 7.60 (t, 2H,
J = 7.2 Hz) 7.49 (t, 4H, J = 6.4 Hz) 7.22 (ddd, 6H, J = 15.4 Hz,
J = 23.5 Hz, J = 30.5 Hz) 6.55 (m, 2H) 5.80 (d, 2H, J = 13.0 Hz). 31P
NMR (CDCl3; ppm) 5.66 [J (195Pt–31P): 3777.6 Hz].
2.2. Physical measurements
1H NMR spectra were recorded in DMSO-d6 at room tempera-
ture on either a Varian Mercury-300 or a Varian Unity-400 spec-
trometer operating at 300.13 and 75.5 MHz or 400.13 and
100.6 MHz. Chemical shifts are reported relative to TMS on delta
(d) scale in parts per million (ppm). The splitting of proton reso-
nances in the reported 1H NMR spectra are defined as s = singlet,
d = doublet, t = triplet, m = multiplet, w = weak and br broad. Melt-
ing points were determined on a Reichert-Jung Thermovar hot-
stage microscope and are uncorrected. Infrared spectra (KBr
pellets) were recorded on a Thermo Nicolete FT-IR instrument
and are reported using the following abbreviation: s = strong,
m = medium, strong and br = broad. Microanalyses were deter-
mined using a Fisons EA 1108 CHNO-S instrument. Mass spectra
(MS) were recorded on a Waters atmosphere pressure ionization
quadruple time-of-flight (API Q-TOF) Ultima (electronspray ioniza-
tion (ESI), 70 eV) and/or SA VG70-SEQ (Fast atom bombardment
(FAB), 7 kV) instrument.
2.3.6. Spectroscopic and analytical data for 5
Pale yellow powder. Yield: 72%. M.p.: 201–203 °C. Anal. Calc. for
C
24H20Cl2NPPtS: C, 44.25; H, 3.09; N, 2.15; S, 4.92. Found: C, 44.19;
H, 3.12; N, 2.42; S, 5.03%. MS (EI, m/z): 616.01, [MÀCl]+. IR (KBr)
(m m
max/cmÀ1): 1631 s (C N). 1H NMR (CDCl3): dH (ppm): 8.73 (s,
1H) 7.86 (m, 1H) 7.60 (m, 1H) 7.44 (m, 1H) 7.24 (dd, 1H,
J = 1.2 Hz, J = 5.1 Hz) 7.06 (ddd, 1H, J = 1.6 Hz, J = 7.2 Hz,
J = 10.6 Hz) 6.77 (dd, 1H, J = 3.4 Hz, J = 5.1 Hz) 6.50 (dd, 1H,
J = 1.0 Hz, J = 3.4 Hz) 4.77 (t, 1H, J = 7.4 Hz). 31P NMR (CDCl3;
ppm) 8.06 [J (195Pt–31P): 3758.5 Hz].
2.3.7. General procedure for the preparation of gold(I) complexes (6–
9)
To the appropriate ligand (L1, L3–L5) in dry CH2Cl2 (10 ml) was
added [Au(tht)Cl] also in dry CH2Cl2 (10 ml) in a ratio of 1:0.9. The
reaction was allowed to stir at room temperature for ca 2 h before
reducing the solvent to ca 5 ml and precipitating out the products
with hexane, filtering under gravity and washing the precipitate
with dry Et2O and drying under vacuum for 4 h.
2.3. Synthesis
2.3.1. General procedure for the preparation of platinum(II) complexes
(1–5)
2.3.8. Spectroscopic and analytical data for 6
To a solution of the appropriate ligand (L1–L5) in dry CH2Cl2
(10 ml) was added an equimolar amount of [PtCl2(COD)] or PtCl2
(DMSO)2] dissolved in dry CH2Cl2 (10 ml). The reaction was allowed
to stir at room temperature for 4 h before reducing the solvent to ca
5 mland precipitating theproductsusinghexane. Theproducts were
filtered, washed with Et2O and dried under vacuum.
Pale yellow powder. Yield: 73%. M.p.: 200–201 °C. Anal. Calc. for
C
31H32AuClNP: C, 54.60; H, 4.73; N, 2.05. Found: C, 54.88; H, 4.57;
N, 1.97%. MS (EI, m/z): 449.31, [MÀClÀAu]+. IR (KBr) (
m
max/cmÀ1):
1628 s m
(C N). 1H NMR (CDCl3): dH (ppm): 8.90 (s, 1H) 8.41 (dd,
1H, J = 4.5 Hz, J = 7.2 Hz, ArH) 7.60 (t, 1H, J = 7.5 Hz, ArH) 7.41 (m,
11H, ArH) 6.94 (m, 3H, ArH) 6.79 (dd, 1H, J = 7.8 Hz, J = 13.2 Hz)

H. Chiririwa et al. / Polyhedron 49 (2013) 29–35
31
2.47 (td, 2H, J = 6.8 Hz, J = 13.7 Hz, –CHMe2) 0.89 (d, 12H, J = 6.8 Hz,
–CHMe4). 31P NMR (CDCl3; ppm) 26.64.
by Western blot. p38 was used as a loading control. Treatment of
cells with 10 M doxorubicin served as a positive control. These re-
l
sults are representative of two independent experiments.
2.3.9. Spectroscopic and analytical data for 7
Off white powder. Yield: 53%. M.p.: 215–217 °C. Anal. Calc. for
2.5.3. Cell cycle analysis
C
25H21AuClN2P: C, 49.00; H, 3.45; N, 4.57. Found, C, 49.22; H,
Cells were seeded in 60 mm dishes at 0.25 Â 106 cells per dish.
Following an overnight settling period, compounds were added to
culture media (to avoid the loss of cycling cells that had detached
during division). At appropriate time points, cells were harvested
by trypsinisation (taking care to collect all washes to avoid discard-
ing floating cells), resuspended in 1 ml PBS, and counted. 9 ml of
ice cold 100% EtOH was then added to each sample, and samples
were stored at À20 °C for up to 2 weeks. Cells were then centri-
fuged out of EtOH, rinsed several times in PBS, and 5 Â 106 cells/
3.23; N, 4.54%. IR (KBr) (
m
max/cmÀ1): 1630 s (C N). 1H NMR
m
(CDCl3): dH (ppm): 8.64 (s, 1H) 8.33 (d, 1H, J = 4.5 Hz) 8.28 (d,
1H, J = 5.8 Hz) 8.04 (m, 1H, ArH) 7.78 (m, 1H) 7.55 (m, 1H, ArH)
7.33 (m, 1H) 7.17 (m, 1H, ArH) 7.02 (dd, 1H, J = 5.0 Hz, J = 7.9 Hz,
ArH) 6.79 (dt, 1H, J = 8.3 Hz, J = 13.6 Hz, ArH) 4.70 (s, 1H, ArH)
4.60 (s, 2H). 31P NMR (CDCl3; ppm) 31.38.
2.3.10. Spectroscopic and analytical data for 8
Pale yellow powder. Yield: 59%. M.p.: 220–221 °C. Anal. Calc. for
ml were incubated in 50 lg/ml RNAse A in PBS for 30 min at room
C
24H20AuClNOP: C, 46.90; H, 3.35; N, 2.33. Found: C, 46.77; H, 3.48;
temperature. Cell Cycle Staining solution was added to bring the
cell concentration to 1 Â 106 cells/ml, and following a 30 min incu-
bation in the dark, cells were analysed on a Beckman Coulter FACS-
calibur flow cytometer. Analysis of cell cycle results was carried
out using ModFit 3.0 (Verity Software House).
N, 2.09%. MS (EI, m/z): 563.87, [MÀCl]+. IR (KBr) (
m
max/cmÀ1): 1634
s m
(C N). 1H NMR (CDCl3): dH (ppm): 8.54 (d, 1H, J = 1.4 Hz) 7.82
(dd, 2H, J = 4.8 Hz, J = 7.1 Hz, ArH) 7.37 (m, 3H, ArH) 7.17 (m, 2H,
ArH) 7.13 (dd, 4H, J = 0.8 Hz, J = 1.8 Hz, ArH) 6.75 (dd,2H,
J = 7.8 Hz, J = 13.1 Hz) 6.13 (dd, 1H, J = 1.9 Hz, J = 3.1 Hz) 5.91 (d,
1H, J = 3.2 Hz) 4.53 (s, 2H). 31P NMR (CDCl3; ppm) 30.51.
3. Results and discussion
2.3.11. Spectroscopic and analytical data for 9
Off-white powder. Yield: 55%. M.p.: 230–232 °C. Anal. Calc. for
3.1. Synthesis and characterisation of Pt(II) and Au(I) complexes
C
24H20AuClNPS: C, 46.65; H; 3.26; N, 2.27; S, 5.19. Found: C,
46.80; H, 3.42; N, 2.45; S, 4.97%. MS (EI, m/z): 581.38, [MÀCl]+. IR
The platinum(II) iminophosphine complexes 1–5 were obtained
in reasonable yields (65–76%) by reacting [PtCl2(COD)] or [PtCl2
(DMSO)2] with L1–L5 ligands in a 1:1 molar ratio at room temper-
ature in CH2Cl2 (Scheme 1). The platinum compounds were iso-
lated as pale to dark yellow powders and are air stable.
The gold(I) iminophosphine complexes were obtained by react-
ing [Au(tht)Cl] with L1 and L3–L5 at room temperature (Scheme 2).
These were isolated as off white to pale yellow powders which are
also air stable.
(KBr) (m m
max/cmÀ1): 1635 s (C N). 1H NMR (CDCl3): dH (ppm):
8.59 (d, 1H, J = 1.4 Hz) 8.35 (m, 1H, ArH) 7.84 (m, 2H, ArH) 7.68
(dd, 3H, J = 4.6 Hz, J = 7.2 Hz, ArH) 7.39 (m, 2H, ArH) 7.17 (m, 4H,
ArH) 7.04 (dd, 2H, J = 1.1 Hz, J = 5.1 Hz, ArH) 6.96 (dd, 1H,
J = 1.1 Hz, J = 5.1 Hz) 6.77 (m, 1H) 6.55 (m, 1H) 4.74 (s, 2H). 31P
NMR (CDCl3; ppm) 31.93.
2.4. X-ray crystallography
Both platinum and gold complexes are soluble in CHCl3, CH2Cl2
and DMSO, less soluble in MeCN and insoluble in MeOH or EtOH.
The platinum complexes 1–5 displayed sharp singlets flanked
by platinum satellites in their 31P NMR spectra. The 31P NMR spec-
tra of the platinum complexes provides a sensitive probe for the
structures of complexes. The one-bond coupling 1J(195Pt–31P) is
characteristic of these complexes and those which contain Cl
atoms trans to phosphorus atoms have been found to possess cou-
pling constants greater than 3500 Hz [32]. 31P NMR data for the
complexes 6–10 show a downfield shift of between 19.76 and
21.86 ppm on complexation of the ligands to the metal centre,
with the most significant shift arising from formation of complex
10 indicating that the P centre of the ligand is coordinated to the
metal. The coordination chemical shift observed for complexes
6–10 is comparable to that reported previously [33]. There is a
strong similarity in chemical shift observed among all the com-
plexes e.g dp values for the platinum complexes are almost identi-
cal, indicating that the substituents on the aldehyde constituent of
the iminophosphine ligand have little influence on the value of the
shift. This observation is also consistent with previous reports of
similar types of ligands [33]. The signal due to the P centre in the
platinum complexes is shifted ca 20 ppm upfield. This trend was
also noted previously for platinum complexes of the [P, N, O] ligand
2-[Ph2PC6H4CH@N]C6H3OH [33]. The presence of Pt satellites in
the spectra of complexes 1–5 is further evidence of coordination
by the P centre.
Single crystals for X-ray data collections regarding 1 and 6 were
grown by the slow evaporation of CH2Cl2 solutions of their com-
plexes at room temperature. X-ray intensity data were collected
on a Nonius Kappa-CCD diffractometer with 1.5 kW graphite
monochromated Mo K
a radiation. The structures were solved by
direct methods using SHELXS-97 and refined employing full-matrix
least-squares with the program SHELXL-97 [30,31] refining on F2.
2.5. Biological studies
2.5.1. MTT assay
1500 cells were seeded per well in 90
plates. Cells were incubated for 24 h, then test samples were plated
at a range of concentrations in 10 l media, with a final concentra-
tion of 0.2% DMSO. After 48 h of incubation, the cells were ob-
served under phase contrast microscope and the general
appearance of the cells together with confluency status and pres-
ence of precipitate if any was recorded. 10 l of MTT reagent was
added per well at the end of the experiment, and the plates incu-
bated for 4 hours at 37 °C. 100 l of Solubilisation solution was
ll DMEM in 96 well
l
a
l
l
then added to each well, and plates were incubated at 37 °C over-
night. After 16 h, the plates were read at 595 nm on an Anthos
microplate reader 2001.
2.5.2. Western blotting
Cells prepared for western blotting were routinely plated at
500 000 cells/60 mm dish. Cells were incubated for 24 h, then trea-
ted with the indicated concentrations of compounds for the indi-
cated times. Cell lysates from cells treated with 2Â IC50 for
complexes 1, 4, 6, 8 were harvested at 12, 24 and 48 h and analysed
The most informative peak in the IR spectra of iminophosphine
complexes of platinum is that of the C@N group occurring in the
1600 cmÀ1 region. Comparison of the position of this peak in com-
plexes 1–5 with that of the corresponding ligands L1–L5 shows a
shift to lower wavenumbers on complexation of between 5 and

32
H. Chiririwa et al. / Polyhedron 49 (2013) 29–35
R = (CH3)4(CH)2C6H4
R = (CH2)C6H5
1
2
3
4
5
R
R
N
N
R = (CH2)C5H4N
R = (CH2)C4H3O
R = (CH2)C4H3S
/ P t(DMS O)2Cl2
Pt(CO D)Cl2
Cl
Cl
Pt
DCM , RT
P
P
Ph
Ph
Ph
Ph
-
L1 L5
Scheme 1. Synthesis of platinum(II) dichloride complexes.
R = (CH3)C6H3
6
7
R = (CH2)C5H4N
R
R
R = (CH2)C4H3O 8
R = (CH2)C4H3S 9
N
N
Au(tht)Cl
DCM, RT
Cl
P
P
Ph
Ph
Ph
Ph
,
-
L1 L3 L5
Scheme 2. Synthesis of gold(I) chloride complexes.
9 cmÀ1. This shift is in agreement with previous observations on
metal complexes of similar types of ligands [35,37,39].
The positive-ion EI-MS spectra of all complexes contained
molecular ion peaks at m/z 678.34 (1), 609.36 (2), 610.92 (3),
599.91 (4) and 616.01 (5). The fragmentation patterns arising from
the loss of a Cl was evident in each spectrum of the proposed
structures.
free ligand the length is 1.2668 (19) Å. The ligand backbone car-
bon-carbon distances for the complex are comparable with that
of the free ligand. The average Pt–N and Pt–P bond lengths of
2.0421(18) and 2.2128(6) Å, respectively are in the range expected
for iminophosphine platinum(II) complexes [40]. The torsion angle
Pt(1)–P(1)–C(15)–C(14) = À35.5(2)° indicates that the @CHC6H4–
unit lies below the PtCl2(P,N) plane.
The crystallographic asymmetric unit contains two independent
molecules which are essentially identical structurally. The main
differences concern the arrangement of the phenyl groups. No unu-
sual bond angles or distances were observed and the complex 6
3.2. X-ray diffraction studies
To further confirm the integrity of these complexes, crystals
suitable for X-ray structure determination were obtained for com-
plexes 1 and 6. Crystals were grown from dichloromethane at room
temperature. Details of the crystal parameters, data collection and
refinements were listed in Table 1.
showed
a virtually linear P–Au–Cl system (bond angle of
177.17°). Williams et al. report the preparation and characterisa-
tion of three new complexes of gold(I) with bidentate P–N ligands
where the ligand binds to gold(I) either in a mono- or bi-dentate
fashion to form the corresponding gold(I) complexes [41]. For the
The molecular structures for complexes 1 and 6 with atomic
numbering scheme are shown in Figs. 1 and 2, respectively. Se-
lected bond lengths and angles are given in Tables 2 and 3.
The platinum complex is of distorted square planar geometry
with the P–Pt–N angle at 89.80(5)° and the corresponding angle
between the chloride ligands has also been reduced to 87.92(2)°.
As phosphorus ligands have higher trans influence than amine li-
gands, the Pt–Cl bond trans to P is slightly elongated compared
to the chloride bound cis to P [36].
C
23H24AuClNP complex they report bond lengths of 2.2916 and
2.2380 Å for the Cl–Au and P–Au bonds and the complex had a lin-
ear P–Au–Cl geometry with a bond angle of 177°.
3.3. Biological evaluation
A preliminary investigation into the antitumour activity of sev-
eral metal-containing complexes prepared during the course of
this study was carried out in the form of cytotoxicity assays on
two separate cancer cell lines–human oesophageal cancer cell lines
WHCO1 and KYSE450.
A similar geometrical arrangement of the ligands was observed
by McIsaac et al. [33] for novel iminophosphino rhodium(I) com-
plexes including [Rh(K2-o-Ph2PC6H4CH@N-2,6-iPr2C6H3)(
l-Cl)]2.
In comparison to the related compound o-Ph2PC6H4CH@NC6H4-
OMe-o [36], both iminophosphines have a very similar arrange-
ment with planar C(14)–C(19)@N(20)AC(21) imino units with an
E configuration with respect to the diphenylphosphine group.
These structures are in contrast to o-Ph2PC6H4CH@NC6H4OH-o
[38] where the nitrogen is closer to the phosphorus atom as the
molecule assumes a Z configuration. The N(1)–C(13) double bond
of complex 1 is 1.287(2) Å is well within the range observed for
these types of compounds [34]. There is little variation observed
in the bond lengths of the ligand upon chelation to Pt(II), for the
3.3.1. In vitro anticancer activity
The in vitro anticancer activity data of the platinum and gold
complexes (1–9) against WHCO1 and KYSE450 cell lines is pre-
sented in Table 4. The cytotoxicity of several platinum complexes
was examined. In WHCO1 cell lines complex 3 had the highest
activity with an IC50 value of 5.49
complex 1 with an IC50 value of 9.47
l
M and the least active was
lM. In comparison to cisplatin
which has an IC50 of 13–15 lM in WHCO1 cell lines, all the novel

H. Chiririwa et al. / Polyhedron 49 (2013) 29–35
33
Table 1
the oesophageal cancer cells, we investigated the effect of these
complexes on DNA damage and the induction of apoptosis in the
cancer cells. Phosphorylation of H2AX was used as a marker of
Double Strand Breaks (DNA). H2AX by itself cannot repair the dam-
age, but it acts as a recruiter, and collects other repair site of dam-
age to initiate repair. The results show that complexes 1 and 6 very
strongly induce the phosphorylation of H2AX, suggesting that
these complexes induce DNA damage in the form of DSB’s. Our
Western blot shows that complexes 4 and 8 also induce DNA dam-
age although to a lesser extent than in complexes 1 and 6.
Considering that treatment with these complexes resulted in
DSB’s we determined whether these compounds also induced
apoptosis in treated cells. PARP cleavage was investigated by Wes-
tern blot analysis in order to determine the mode of cell death in-
duced by the complexes 1, 4, 6, and 8. From the results, there is
very little evidence that treatment of the WHCO1 cells with com-
plexes 1, 4, 6 and 8 resulted in PARP cleavage and hence apoptosis.
The system that we used to detect PARP cleavage was clearly work-
ing since we could detect the PARP cleavage in cells treated with
Doxorubicin.
Selected crystallographic and refinement data for complexes 1 and 6.
Complex
1
6
Empirical formula
Formula weight
T (K)
C
31H32Cl2NPPt
C32H35AuClNP
705.00
173(2)
0.7103
triclinic
715.54
173(2)
k (Å)
0.71073
monoclinic
P21/n
12.0686(7)
13.4007(7)
17.8182
90
105.8190(10)
90
2772.6(3)
4
1.714
5.333
1408
0.22 Â 0.11 Â 0.09
1.83–29.60
À16 6 h 6 16,
À18 6 k 6 18,
À24 6 1 6 24
40326
Crystal system
Space group
a (Å)
b (Å)
c (Å)
P1
13.0315(3)
13.3638(2)
19.3489(5)
96.358(2)
99.2290(10)
116.1910(10)
2921.15(11)
4
1.603
5.205
1396
0.21 Â 0.18 Â 0.12
2.31–26.38
À16 6 h 6 16,
À16 6 k 6 16,
À24 6 1 6 24
119370
a
(°)
b (°)
c
(°)
V (Å3)
Z
Dcalc (mg/ml)
Absorption coefficient (mmÀ1
F(000)
)
Crystal size (mm)
h (°)
Limiting indices
Reflections collected
Independent reflections (Rint) 7788 (0.0000)
Completeness of theta
maximum and minimum
transmission
3.3.3. Effects of complexes 1, 4, 6 and 8 on the cell cycle
11941 (0.0534)
26.38 (99.8%)
The complexes 1, 4, 6 and 8 exhibited significant DNA damage
as evidenced by the phosphorylation of histone H2AX we then fur-
ther explored the effect of the complexes on the cell cycle as it is
well documented that DNA damage triggers cell cycle arrest in
cells. From the results it appears that the compounds do not affect
the cell cycle significantly except for complex 6. Cattaruzza et al re-
port that gold(III)-dithiocarbamato derivatives cause cell damage,
slightly affecting the cell cycle, thus suggesting a different mecha-
nism of action from established platinum-based compounds [42].
The lack of a sub-G1 peak in the cell analysis is most likely due
to the extended processing of cells for this assay (multiple washes
and spins), during which the population may have been lost. The
cell-cycle disturbances were associated with inhibition of cell pro-
liferation in agreement with results reported by Martin et al [43] in
their cell-cycle disturbance studies. This suggests that the reduc-
tion in G1-phase cells was as a result of the accumulation of cells
a fact that is attributed to induction of apoptosis by compounds
in test. The cells were trapped in G1-phase and therefore paused
29.6 (99.9%)
Refinement method
full-matrix least-
squares on F2
7788/0/326
1.059
full-matrix least-
squares on F2
11941/9/604
1.050
Data/restraints/parameters
Goodness-of-fit (GOF) on F2
platinum complexes described here displayed significant activity
against oesophageal cancer cell lines.
The gold(I) complexes exhibited very good activity against both
cancer cell lines. Complexes 8 and 9 were the most active with IC50
values of 3.41 and 3.61 lM.
3.3.2. Effect of compounds on DNA damage and induction of apoptosis
Since the morphology of the WHCO1 cells treated with com-
plexes 1, 4, 6 and 8 clearly showed that the complexes were killing
Fig. 1. The ORTEP plot of the molecular structure of 1 showing the atomic numbering. All the H atoms are omitted for clarity and are presented with ellipsoidal model with
probability level 35%.

34
H. Chiririwa et al. / Polyhedron 49 (2013) 29–35
Fig. 2. The ORTEP plot of the molecular structure of 6 showing the atomic numbering. There are two crystallographic independent molecules labelled as A and B in the
asymmetric unit. All non-hydrogen atoms were presented with ellipsoidal model with probability level 25%. There is one ethanol solvent molecule and all hydrogen atoms are
omitted for clarity.
Table 2
Table 4
Selected bond distances and angles for complex 1.
IC50 determination for Pt(II) and Au(I) complexes (1–9) evaluated for anticancer
activity in WHCO1 and KYSE450 cell lines.
Bond distances (Å)
Bond angles (°)
Compound
IC50 in
WHCO1 (
95% CI
IC50 in
KYSE450 (lM)
95% CI
Pt(1)–N(1)
Pt(1)–P(1)
Pt(1)–Cl(2)
Pt(1)–Cl(1)
P(1)–C(15)
P(1)–C(26)
N(1)–C(13)
2.0421(18)
2.2128(6)
2.2901(6)
2.3512(6)
1.809(2)
N(1)–Pt(1)–P(1)
N(1)–Pt(1)–Cl(2)
P(1)–Pt(1)–Cl(2)
N(1)–Pt(1)–Cl(1)
P(1)–Pt(1)–Cl(1)
Cl(2)–Pt(1)–Cl(1)
89.80(5)
178.85(5)
91.25(2)
90.98(5)
174.20(2)
87.92(2)
lM)
1
2
3
4
5
6
7
8
9
9.47
8.45
5.49
7.24
6.66
8.42
4.15
3.41
3.61
8.96–10.01
7.99–8.94
3.97–7.60
5.89–8.90
5.81–7.63
8.28–8.58
3.35–5.13
2.39–4.88
2.92–4.47
2.16
6.00
3.11
7.58
7.17
5.23
6.89
5.87
5.40
1.96–2.38
4.91–7.35
2.83–3.41
6.29–9.12
6.24–8.25
4.73–5.79
5.87–8.51
5.29–6.51
4.88–5.98
1.816(2)
1.287(3)
Table 3
Selected bond distances and angles for complex 6.
All experiments were done three times and all experimental points within an
experiment were done in triplicate.
Bond distances (Å)
Bond angles (°)
Molecule 1
selenite-induced apoptosis involves DNA damage. Selenite has also
been shown to stimulate phosphorylation of H2AX [44,45].
Au(1A)–P(1A)
Au(1A)–Cl(1A)
P(1A)–C(26A)
P(1A)–C(20A)
P(1A)–C(15A)
N(1A)–C(13A)
2.2350(13)
2.2783(14)
1.805(6)
1.825(6)
1.834(6)
1.231(7)
P(1A)–Au(1A)–Cl(1A)
C(26A)–P(1A)–C(20A)
C(26A)–P(1A)–C(15A)
C(26A)–P(1A)–Au(1A)
C(20A)–P(1A)–Au(1A)
177.17(5)
105.2(2)
105.3(2)
110.80(18)
116.84(17)
4. Conclusions
New neutral Pt(II) and Au(I) complexes containing iminophos-
phine ligands were prepared and fully characterised. The complexes
discussed were studied for their cytotoxicity on oesophageal cancer
cells. The platinum and gold complexes showed moderate activity
and block the proliferation of WHCO1 cells with an IC50 range of
Molecule 2
Au(1B)–P(1B)
Au(1B)–Cl(1B)
P(1B)–Cl(26B)
P(1B)–Cl(20B)
P(1B)–C(15B)
N(1B)–C–(13B)
2.2272(13)
2.2709(15)
1.810(5)
1.814(6)
1.828(5)
1.259(7)
P(1B)–Au(1B)–Cl(1B)
C(26B)–P(1B)–C(20B)
C(26B)–P(1B)–C(15B)
C(26B)–P(1B)–Au(1B)
C(20B)–P(1B)–Au(1B)
178.55(6)
106.4(2)
106.4(2)
110.01(17)
115.51(19)
2.5–9.4 lM, and IC50 range of 2.2–7.6 lM for the KYSE450 cell lines.
The morphological changes observed showed that the complexes
induce cell death, however apoptosis was not observed and cell
death is occurring by some other mechanism. It might therefore
be interesting to investigate whether the compounds trigger cell
death in WHCO1 cells by necrosis, induce senescence or autophagy.
Further analysis of cytotoxicity activities of these complexes by
FACS analysis showed that the complexes do not affect the cell cycle
significantly. These studies provide an insight into the action of
platinum and gold complexes and demonstrates potential use of
these compounds in the development of new anticancer agents.
their progress in the cycle and subsequently entered an indefinite
phase. This is attributed to the complex binding to DNA in
G1-phase and therefore disrupting its programme of passing the
DNA for replication in the S-phase. This then caused the cell recep-
tors to detect the anomaly hence triggering apoptosis. Selenium
compounds are the most extensively studied cancer chemopreven-
tive agents, and induce apoptotic death of tumor cells and the

H. Chiririwa et al. / Polyhedron 49 (2013) 29–35
[15] D. Wang, S.J. Lippard, Nat. Rev. Drug Discov. 4 (2005) 307.
35
Acknowledgements
[16] N.J. Wheate, J.G. Collins, Curr. Med. Chem: Anti-Cancer Agents 5 (2005) 267.
[17] B.K. Bhuyan, B.E. Loughman, T.J. Fraser, K. Day, J. Exp. Cell Res. 97 (1996) 275.
[18] T.M. Simon, D.H. Kunishima, G.J. Vilbert, A. Lorber, Cancer 44 (1979) 1965.
[19] C.K. Mirabelli, R.K. Johnson, L. Faucette, C.M. Sung, Proc. Am. Assoc. Cancer Res.
25 (1984) 367.
[20] C.K. Mirabelli, R.K. Johnson, L. Faucette, C.M. Sung, Cancer Res. 45 (1985) 32.
[21] P.J. Sadler, R.E. Sue, Met.-Based Drugs 1 (1994) 107.
[22] C.F. Shaw, Chem. Rev. 99 (1999) 2589.
[23] M.M. Mogorosi, T. Mahamo, J.R. Moss, S.F. Mapolie, J.C. Slootweg, K.
Lammertsma, G.S. Smith, J. Organomet. Chem. 696 (2011) 3585.
[24] J.X. McDermott, J.F. White, G.M. Whitesides, J. Am. Chem. Soc. 98 (1976) 6521.
[25] D. Drew, J.R. Doyle, Inorg. Synth. 28 (1990) 346.
[26] T.A.K. Al-Allaf, L.J. Rashan, A.S. Abu-Surrah, R. Fawzi, M. Steimann, Transition
Met. Chem. 23 (1998) 403.
[27] J.J. Price, A.N. Williamson, R.F. Schramm, B.B. Wayland, Inorg. Chem. 11 (1972)
1280.
[28] K.A. Jensen, Z. Anorg. Allg. Chem. 229 (1936) 265.
[29] R. Uson, A. Laguna, Organometallic Synthesis, in: R.B. Lang, J.J. Eish (Eds.),
Elsevier, Amsterdam, 1986, p. 324.
[30] G.M. Sheldrick, SHELX97, Programme for Solving Crystal Structures, University
of Gottingen, Germany, 1997.
[31] G.M. Sheldrick, SHELX97, Programme for the Refinement of Crystal Structures,
University of Gottingen, Germany, 1997.
Financial support from the University of Cape Town, the Na-
tional Research Foundation of South Africa, CANSA, Project AuTEK
(Mintek and Harmony Gold, South Africa) and a donation of
metal salts from the Anglo Platinum Corporation is gratefully
acknowledged.
Appendix A. Supplementary material
Supplementary data and figures for complex 1 are available
from the IUCR electronic archives (Reference: GO2029). CCDC
872768 contains the supplementary crystallographic data for com-
plex 6. These data can be obtained free of charge via http://
www.ccdc.cam.ac.uk/conts/retrieving.html, or from the Cambridge
Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ,
UK; fax: (+44) 1223-336-033; or e-mail: deposit@ccdc.cam.ac.uk.
[32] G. Petocz, G. Rangits, M. Shaw, H. de Bod, D.B.G. Williams, J. Organomet. Chem.
694 (2009) 219.
References
[33] D.I. McIsaac, S.J. Geier, C.M. Vogels, A. Decken, S.A. Westcott, Inorg. Chim. Acta
359 (2006) 2771.
[34] P. Bhattacharyya, M.L. Loza, J. Parr, A.M. Z Slawin, J. Chem. Soc., Dalton Trans.
17 (1999) 2917.
[35] J. Parr, A.M. Z Slawin, Inorg. Chim. Acta 303 (2000) 116.
[36] M.L. Clarke, A.M.Z. Slawin, J.D. Woolins, Polyhedron 22 (2003) 19.
[37] G. Bandoli, A. Dolmella, L. Crociani, S. Antonetta, B. Crociani, Transition Met.
Chem. 25 (2000) 17.
[38] J.R. Dilworth, S.D. Howe, A.J. Hutson, J.R. Miller, J. Silver, R.M. Thompson, M.
Harman, M.B. Hursthouse, J. Chem. Soc., Dalton Trans. (1994) 3533.
[39] H.A. Ankersmit, B.H. Loken, H. Kooijman, A.L. Spek, K. Vrieze, G. van Koten,
Inorg. Chim. Acta 252 (1996) 141.
[40] S.I. Pascu, K.S. Coleman, A.R. Cowley, M.L.H. Green, N.H. Rees, New J. Chem. 29
(2005) 385.
[41] D.B.G. Williams, T. Traut, F.H. Kriel, W.E. van Zyl, Inorg. Chem. Comm. 10
(2007) 538.
[42] L. Cattaruzza, D. Fregona, M. Mongat, L. Ronconi, A. Fassina, A. Colombatti, D.
Aldinucci, Int. J. Cancer 126 (2010) 1.
[43] B. Martin, S.A. Oleksiewicz, J. Virol. 71 (1997) 1386.
[44] T. Kim, U. Jung, D.Y. Cho, A.S. Chung, Carcinogenesis 22 (2001) 559.
[45] N. Zhou, H. Xiao, T. Kunli, A.N. E-Kamal, L.F. Liu, J. Biol. Chem. 278 (2003)
29532.
[1] G. Jaouen, A. Vessieres, I.S. Butler, Acc. Chem. Res. 26 (1993) 361.
[2] R.H. Fish, G. Jaouen, Organometallics 22 (2003) 2166.
[3] N. Metzler-Nolte, Angew. Chem., Int. Ed. 40 (2001) 1040.
[4] D.M. Parkin, S.L. Whelan, J. Ferlay, H. Storm, Cancer incidence in five
continents, IARC CancerBase No. 7, 2005.
[5] N. Ahmed, P. Cook, Br. J. Cancer 23 (1969) 302.
[6] D.G. Gatei, P.A. Odhiambo, D.A. Orinda, F.J. Muruka, Cancer Res. 38 (2) (1978)
303.
[7] J. Roth, A. Putman, S.L. Allen, A.F. Aslene, Cancer: Principles and Practise of
Oncology, fourth ed., J.B. Lippincott Co., Philadelphia, PA, 1993.
[8] C.W. Evans, The Metastatic Cell Behaviour and Biochemistry, second ed.,
Chapman and Hill, London, 1991.
[9] P. Boyle, B. Levin, World Cancer, Report 15 (2008).
[10] P. Andre Ferreira, L. Joao Ferreira da Silva, M. Teresa Duarte, M. Fatima Minas
da Piedade, M. Paula Robalo, G. Shrika Harjivan, Cristina Marzano, Valentina
Gandin, M. Matilde Marques, Organometallics 28 (2009) 5412.
[11] E. Wong, C.M. Giandomenico, Chem. Rev. 99 (1999) 2451.
[12] L.R. Kelland, S.Y. Sharp, C.F. O’Neill, F.I. Raynaud, P.J. Beale, I.R. Judson, J. Inorg.
Biochem. 77 (1999) 111.
[13] J.M. Perez, M.A. Fuertes, C. Alonso, C. Navarro-Ranninger, Crit. Rev. Oncol.
Hematol. 35 (2000) 109.
[14] Y.-P. Ho, S.C.F. Au-Yeung, K.K.W. To, Med. Res. Rev. 23 (2003) 633.