Synthesis and DNA-binding studies of a dinuclear gadolinium(iii)-Platinum(ii) complex

Article abstract of DOI:10.1071/CH14572

The synthesis and characterisation of a new dinuclear Gd^III-Pt^II complex (1·PF₆) containing a functionalised macrocyclic 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid derivative linked to a PtII-terpy (terpy≤2,2′:6′,

Full text of DOI:10.1071/CH14572

RESEARCH FRONT
CSIRO PUBLISHING
Aust. J. Chem. 2015, 68, 576–580
Full Paper
http://dx.doi.org/10.1071/CH14572
Synthesis and DNA-Binding Studies of a Dinuclear
Gadolinium(III)–Platinum(II) Complex*
A B
,
A B
,
A C
,
Jacob M. Fenton,
Madleen Busse,
and Louis M. Rendina
A
School of Chemistry, The University of Sydney, Sydney, NSW 2006, Australia.
These authors contributed equally to the project.
B
C
Corresponding author. Email: lou.rendina@sydney.edu.au
The synthesis and characterisation of a new dinuclear GdIII–PtII complex (1ꢀPF6) containing a functionalised macrocyclic
1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid derivative linked to a PtII-terpy (terpy ¼ 2,20 : 60,200-terpyridine) unit
by means of a short thiolato linker are reported. The complex was synthesised in six steps from cyclen by means of a
modular synthetic strategy. A preliminary DNA-binding study with calf-thymus DNA (ct-DNA) was performed on 1ꢀPF6
by means of linear dichroism (LD). The observed changes in the DNA LD signal in the presence of the metal complex are
fully consistent with an intercalative binding mode. Furthermore, an induced negative LD signal in the ultraviolet
absorption region of the complex provides strong evidence of a strong DNA-binding interaction. The in vitro cytotoxicity
of 1ꢀPF6 towards a human glioblastoma cell line (T98G) was also determined.
Manuscript received: 16 September 2014.
Manuscript accepted: 9 October 2014.
Published online: 27 January 2015.
with the prototype GdIII–PtII complex: (1) improved hydrolytic
stability by the introduction of the macrocyclic 1,4,7,10-tetra-
azacyclododecane-1,4,7-triacetic acid (DO3A) ligand instead of
the acyclic diethylenetriaminepentaacetato (DTPA) ligand;
(2) decreased cytotoxicity in the absence of externalX-ray photons
or thermal neutrons by reducing the number of [PtII(terpy)]2þ
(terpy ¼ 2,20 : 60,200-terpyridine) units from two to one, and
(3) increased cellular uptake by lowering the overall charge of
the complex from þ2 to þ1. The modular synthetic strategy
reported herein will also allow for the preparation of a family of
related potential GdIII agents for potential application in GdNCT
and GdPAT.
Introduction
GdIII complexes are widely used in medicine. They are exten-
sively used as diagnostic agents in magnetic resonance imaging
(MRI) as water relaxation agents to improve the image con-
trast,[1–3] but GdIII complexeshavemorerecentlyshownpotential
as therapeutic agents. The archetypal example of a therapeutic
GdIII agent is Motexafin-Gd (MGd), which underwent Phase I/II
clinical trials as a radiosensitiser in the treatment of lung tumour
metastases to the brain by means of whole-brain radiotherapy
until the FDA stopped further clinical trials with this agent in late
2007.[4–9] However, GdIII complexes can indeed offer intriguing
new avenues for cancer research with respect to binary therapies.
Notable examples of binary cancer therapies include neutron
capture therapy (NCT)[10–14] and photon activation therapy
(PAT).[7,9] Both GdNCT and GdPAT are dependent on the pro-
duction of high linear-energy transfer Auger Coster–Kro¨nig
(ACK) electrons which result from thermal neutron capture or
photon activation reactions of the f-block element (or more spe-
cifically, in the case of NCT, the non-radioactive and naturally
occurring 157Gd isotope). Due to the extremely short path length
(,12nm) exhibited by ACK electrons,[15] the development of
tumour-selective Gd agents which are localised near important
macromolecules such as DNA or sub-cellular organelles such as
mitochondriaisa non-trivial matter.[10–14] Recently, both types of
sub-cellular components have been targeted successfully by
GdIII.[10,16] The first example of a GdIII–PtII DNA metallointer-
calator that was found to selectively deliver GdIII to A459 lung
tumour cell nuclei was reported in 2010 by Crossley et al.[10]
Herein, we present the next generation of DNA metallointerca-
lators that addresses three unfavourable key factors associated
Results and Discussion
Synthesis
The target complex 1ꢀPF6 was successfully synthesised as out-
lined in Scheme 1.
t
The Bu-protected macrocycle DO3A-tBu3 was synthesised
according to the method developed by Moore and Patterson.[17]
The synthesis of the linker unit first involved the protection of the
thiol group in cysteamine with a triphenylmethane (trityl) group,
as described by Gale et al.[18] The synthesis of N-(2-bromoacetyl)-
triphenylmethyl-cysteamine was carried out using a modified
procedure developed by O’Neil et al.,[19] whereby bromoacety-
lation was conducted at ꢁ788C in a dry ice/acetone bath with
very slow dropwise addition of trityl-cysteamine.
The macrocycle DO3A-tBu3 was then functionalised using
the trityl-protected thiol linker group under basic conditions in
acetonitrile whilst maintaining the reaction temperature of the
*Dedicated to Sir John ‘Kappa’ Cornforth, FRS for his many outstanding contributions to Organic and Biological Chemistry.
Journal compilation Ó CSIRO 2015
www.publish.csiro.au/journals/ajc

Gadolinium(III)–Platinum(II) Complex
577
O
O
O
O
O
O
N
O
N
N
N
O
O
O
K2CO3
Br
HBr
SCPh3
HN
Ph3CS
N
N
H
N
H
CH3CN, Δ
N
O
O
N
O
O
2
TFA
N
Et3SiH, CH3CH2CH2SH
ϩ
HO
O
O
O
PFϪ6
HO
N
O
·
1) [Pt(terpy)Cl]Cl 2H2O, GdCl3
N
O
N
Pt S
N
N
2) Saturated KPF6
O
O
Gd
N
HS
N
N
O
N
H
N
H
H2O
N
O
N
O
O
OH
1
3
Scheme 1. The synthesis of 1ꢀPF6.
reaction mixture at 708C for 5 h. Under these optimised reaction
conditions, the yield of the protected trityl-DO3A-tBu3 (2) was
significantly improved in comparison with that obtained using
the reported synthetic protocol.[20]
The global deprotection of 2 was carried out in a 1 : 1 solution
of TFA/propanethiol in the presence of triethylsilane to yield the
thiol-DO3A (3). Deprotection of a trityl group to a thiol
functionality is often reversed by re-substitution of the trityl
group, resulting in low yields of the desired product.[21] In order
to improve the yield of the desired products, triethylsilane and
excess propanethiol were used, acting as a trityl group deactiva-
tor and tert-butyl scavenger, respectively.[21,22]
significantly lower cytotoxicity when compared with that of the
prototype DNA-targeted GdIII–PtII agent or MGd.[8,10] Interest-
ingly, the relative cell viability in the presence of 1ꢀPF6 did not
significantly decrease further; even at the highest concentration
(250mM), a relative cell viability of 43.1 ꢂ 0.5 % was observed
(Fig. S2, Supplementary Material). This unusual trend is possibly
the result of strong non-covalent (p–p) stacking interactions
between 1þ cations inaqueous solution thatmay leadto a reduced
cellular uptake at higher concentrations of complex due to the
formation of dimers and higher-order aggregates, as observed
previously with other PtII-terpy complexes.[24]
The final step of the synthesis involved complexation to the two
metal ions, PtII and GdIII. The chlorido ligand in [Pt(terpy)Cl]þ
is a reasonable leaving group and can be readily substituted by
thiolato ligands in aqueous solution.[23] A one-pot synthesis
employing both GdCl3 and [Pt(terpy)Cl]Clꢀ2H2O in the pres-
ence of the deprotected macrocycle 3 was successfully carried
out to afford 1ꢀCl. A metathesis reaction employing aqueous
KPF6 afforded the pure microcrystalline target complex 1ꢀPF6.
Preliminary DNA-Binding Study
Linear dichroism (LD) is a measure of the absorption difference
between linearly and perpendicularly polarised light.[25–28] Unless
the system in question has an inherent anisotropy orientation,
certain techniques are required to introduce anisotropy in samples
in order to measure their LD spectrum. Orientation techniques
include squeezed gel methods, electric field orientation, and flow
techniques. Large macromolecules such as DNA (.1000 base-
pairs) can be easily oriented by flow techniques using a flow
Couette cell, which was exclusively used in this study.[25]
Characterisation of 1 ꢀ PF6
High-resolution electrospray ionisation–mass spectrometry
(ESI–MS) was used to confirm the identity of 1ꢀPF6. Due to
several naturally occurring isotopes of both Pt and Gd, the iso-
topic envelope is quite pronounced and the molecular ion peak is
very distinctive. The theoretically calculated and experimen-
tally determined ESI–MS (Fig. S1, Supplementary Material)
were in very good agreement with the most intense peak
attributed to [MþH]þ observed at m/z 1044.1629 (Calc. m/z
1044.1616). The purity of 1ꢀPF6 was confirmed by elemental
analysis and full details are given in the experimental section.
Unfortunately, despite repeated attempts, crystals of 1ꢀPF6 that
were suitable for X-ray diffraction could not be obtained.
The non-covalent binding interaction between 1ꢀPF6 and calf-
thymus DNA (ct-DNA) was investigated by recording the LD
spectrum of a ct-DNA solution with increasing concentrations of
1ꢀPF6. The concentration of ct-DNA was kept constant in order to
avoid alterations in the LD signals due to viscosity changes. The
experiments were carried out in phosphate-buffered saline (PBS;
1.0 mM PO34ꢁ and 2.0 mM NaCl; pH 7.0). Separate solutions
were prepared for each measurement in order to minimise the
errors associated with a single solution being continuously
titrated. The LD titration curves are presented in Fig. 1.
The addition of 1ꢀPF6 to ct-DNA caused marked changes in
the LD signal of the DNA in solution, consistent with significant
structural changes in the ct-DNA when 1ꢀPF6 was present. An
important trend observed was the increase in the negativity of
the LD signal at 260 nm, thereby providing evidence that the ct-
DNA was becoming more oriented in flow upon addition of
1ꢀPF6. This result is consistent with an intercalative binding
mode by 1ꢀPF6,[26] whereby the non-covalent insertion of the
PtII-terpy intercalator between the DNA base-pairs causes
In Vitro Cytotoxicity Study
The in vitro cytotoxicity of 1ꢀPF6 was assessed by means of a
standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) assay protocol using a human glioblastoma
(T98G) cell line. The half-maximal inhibitory concentration
(IC50) was determined to be 31.3ꢂ 0.7 mM which demonstrates a

578
J. M. Fenton, M. Busse, and L. M. Rendina
lengthening and stiffening of ct-DNA. It should also be noted
that an induced LD signal was observed in the 310–360 nm
region. Thisnegativesignalwas found toincreasewithincreasing
concentrations of 1ꢀPF6. The negative LD signal qualitatively
provides an indication of the angle of binding of 1ꢀPF6 to
ct-DNA, indicating a perpendicular transition moment of the
complex to the axis of polarisation. The signal in this range is
most likely associated with an electronic transition within the
terpy ligand (p-p*, 306–350 nm).[29] As p-p* transition
moments are polarised along the plane of an aromatic system,
the LD spectra in Fig. 1 show that the terpy ligand of 1ꢀPF6 must
be co-planar with the ct-DNA bases and approximately perpen-
dicular to the ct-DNA axis. These preliminary DNA binding
studies provide strong evidence of an intercalative binding
interaction between 1ꢀPF6 and ct-DNA, and therefore DNA
intercalation is the dominant binding mode at a complex-to-
base pair ratio of 1 : 1.
according to the method developed by Moore and Patterson.[17]
The synthesisoftriphenylmethyl cysteamine hydrochloride(trityl
cysteamine hydrochloride) was carried out as described by Gale
et al.[18] All precursor chemicals were commercially available.
Triethylenetetraamine (TETA) was purchased from Alfa Aesar
and all other chemicals were purchased from the Sigma–Aldrich
Chemical Co. An exceptions is [Pt(terpy)Cl]Clꢀ2H2O that was
prepared by the method described by Lowe and Vilaivan.[31]
Human glioblastoma multiforme (T98G) cells were maintained
as monolayers in minimum essential medium supplemented
with 10 % fetal bovine serum, penicillin (100 units mLꢁ1),
streptomycin (100 mg mLꢁ1), and L-glutamine (2.5 mM) at 378C
in a humidified 5 % CO2 atmosphere. All LD measurements
were carried out at room temperature in 1.0 mM phosphate
buffer (pH 7) containing 2.0 mM NaCl. Duplicates of each
reading were taken. LD spectra of ct-DNA (2 ꢃ 10ꢁ4 M) were
performed in the presence of 1ꢀPF6 (ratio of complex/DNA
ranged from 0 to 1, using increments of 0.125, except for one
reading at 0.0625). The DNA in the samples was oriented by a
flow Couette cell (path length ¼ 0.1 cm) rotating at a velocity
generated by a 2-V power supply. The data were collected with
six accumulations between 200 and 400 nm at a rate of 500 nm
minꢁ1 using a spectral band width of 1 nm, a response time of
Conclusion
A new dinuclear GdIII–PtII complex 1ꢀPF6 was synthesised by
means of a six-step synthesis starting from cyclen and was
characterised by high-resolution ESI–MS and elemental anal-
ysis. The synthesis protocol developed in this work allows for
the preparation of a family of potential GdIII agents for potential
use in binary therapies. The in vitro cytotoxicity of 1ꢀPF6
towards a human glioblastoma cell line (T98G) was also
determined and it displayed a significantly reduced cytotoxicity
when compared with the previously reported prototype,[10] i.e.
a trinuclear GdIII–PtII complex. Complex 1ꢀPF6 was found to
bind to ct-DNA by using the LD technique and the results are
fully consistent with a non-covalent, intercalative interaction.
Investigations regarding the cellular uptake and biodistribution
using synchrotron X-ray fluorescence spectroscopy of 1ꢀPF6 are
planned. Exploration of the structure–activity relationships of
this family of complexes represents a new avenue of research
for GdNCT and GdPAT.
1 s, a step resolution of 0.5 nm, and a N2 flow rate of 5 L minꢁ1
.
Instrumentation
1H NMR spectra were recorded at 300 K on either a Bruker
AVANCE 200 spectrometer at 200 MHz or a Bruker AVANCE
300 spectrometer at 300 MHz. All 13C{1H} NMR spectra were
recorded at 300 K on a Bruker AVANCE 300 spectrometer
1
at 75 MHz. All NMR signals (d) are reported in ppm. H and
13C{1H} NMR spectra were referenced to tetramethylsilane
(TMS) at 0 ppm or their residual solvent peaks. Low-resolution
ESI–MS was conducted on a Finnigan LCQ mass spectrometer.
High-resolution electrospray ionisation Fourier transform ion
cyclotron resonance mass spectrometry (ESI–FT–ICR–MS)
data were recorded on a Bruker 7.0 T mass spectrometer. Cell
viability was determined by measuring the absorbance at
600 nm using a Victor3V microplate reader (Perkin–Elmer). LD
studies were performed on a Jasco J-710 spectropolarimeter.
Elemental analyses were performed by the Campbell Micro-
analytical Laboratory, Otago, New Zealand.
Experimental
Materials and Methods
Distilled water was used in all experiments requiring this sol-
vent. Anhydrous CH2Cl2 was dried by freshly distilling from
calcium hydride according to the procedures reported by
Armarego and Chai.[30] All other solvents were used without
prior purification. The macrocycle DO3A-tBu3 was synthesised
Biological Assays
The in vitro cytotoxicity of 1ꢀPF6 was assessed using the MTT
assay.[32,33] Briefly, cells were harvested with trypsin (0.1 %
v/v), and cell pellets were isolated by centrifugation (3000 rpm
for 3 min). Cells were then re-suspended to a single cell sus-
pension, cell numbers were counted using a haemocytometer
(Weber), and then cells were seeded (density 1 ꢃ 104 cells per
well) in growth medium (100 mL) using 96-well plates and were
allowed to adhere overnight at 378C in a humidified atmosphere
of 5 % CO2. Cells were incubated at 378C in a humidified
atmosphere of 5 % CO2 in the presence of 1ꢀPF6 or the vehicle
(control). The stock solution of 1ꢀPF6 (10 mM) was prepared in
Milli-Q water and filter-sterilised before use. The maximum
concentration (MaxC) of 250 mM (and further serial dilutions)
was diluted with complete minimum essential medium. Milli-Q
water was used for the vehicle control, reflecting the amount of
water used for MaxC. Serial dilutions of 1ꢀPF6 were added to
wells with six repeats per concentration. The maximum con-
centration (MaxC) for the experiments was 250 mM and the
Complex/DNA
ratio (rb)
200
Ϫ0.01
250
300
350
400
0
Ϫ0.03
Ϫ0.05
Ϫ0.07
Ϫ0.09
Ϫ0.11
Ϫ0.13
0.0625
0.1250
0.2500
0.3750
0.5000
0.6250
0.7500
0.8750
1
Wavelength [nm]
Fig. 1. Linear dichorism spectra of ct-DNA (200 mM bpꢁ1) at 258C
(phosphate-buffered saline: PO34ꢁ, 1.0 mM; NaCl, 2.0 mM; pH 7.0) in the
presence of 1ꢀPF6 (rb ¼ 0–1.0, 0–200 mM).

Gadolinium(III)–Platinum(II) Complex
579
minimum concentration was 1.95 mM (N ¼ 4, where N is the
number of independent experiments). After 72 h, MTT solution
in PBS (30 mL, 0.17 % w/v) was added and the incubation was
continued. After a further 4 h, the culture medium and excess
MTT solution were removed and the resulting MTT–formazan
crystals were dissolved by addition of 200 mL DMSO. Cell
viability was determined by measuring the absorbance at
600 nm and all readings were blank-corrected before they were
normalised to wells containing the absolute control (cells only),
and the level of MTT was expressed relative to the corre-
sponding treated cells as % viability. A vehicle control was used
to investigate the influence of the vehicle on the cells and no
significant changes were observed when compared with the
absolute control. The corresponding IC50 value for 1ꢀPF6 was
determined at the dose required to induce a 50 % decrease in cell
viability. All experiments were conducted in quadruplicate and
the IC50 value is reported with a standard error.
Synthesis of 2,20,200-(10-(2-((2-Mercaptoethyl)amino)-2-
oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)
triacetic acid (3)
The protected macrocycle 2 (350 mg, 0.40 mmol) was added to a
solution of propanethiol (4 mL) and triethylsilane (0.15 mL, d
0.733, 0.94 mmol). To this solution was added TFA (4 mL)
dropwise with stirring, and the reaction mixture was allowed to
stir overnight at room temperature. The volatiles were removed
under vacuum, and the remaining colourless solid was parti-
tioned between water (30 mL) and chloroform (30 mL). The
aqueous layer was collected and washed further with chloroform
(2 ꢃ 20 mL). The solution was then concentrated to a volume of
,1 mL under reduced pressure. The solution was then lyophi-
lised overnight to yield the deprotected macrocycle 3 as a very
hygroscopic, colourless solid in quantitative yield (184 mg). dH
(D2O, 300 MHz) 4.16–2.70 (28H, b m, CH2). m/z (ESI–FT–
ICR) 464.2180; [M þ H]þ requires 464.2174.
Synthesis of (2,20,200-(10-(2-((2-Mercaptoethyl)amino)-2-
oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)
triacetato-N,N’,N0,N00,O,O’,O0)-gadolinium(III)-2,20 : 60,200-
terpyridineplatinum(II) hexafluorophosphate (1ꢀPF6)
Synthesis of N-(2-Bromoacetyl)-triphenylmethyl-
cysteamine
The compound was synthesised using a modified procedure
reported by O’Neil et al.[19] Trityl cysteamine hydrochloride
(1.50 g, 4.21 mmol) and triethylamine were dissolved in CH2Cl2
(10 mL). The solution was added dropwise to a stirred solution
of bromoacetyl bromide (0.4 mL, 4.59 mmol) in CH2Cl2 (4 mL)
at ꢁ788C. The reaction mixture was then allowed to warm to
room temperature before quenching with water (15 mL).
CH2Cl2 (20 mL) was added and the organic layer was extracted
and washed with 1 M HCl (1 ꢃ 20 mL), water (1 ꢃ 20 mL),
NaHCO3 (saturated, 1 ꢃ 20 mL), and brine (1 ꢃ 20 mL). The
organic layer was dried over Na2SO4 and concentrated to a
volume of 10 mL. Addition of n-hexane (45 mL) resulted in the
formation of colourless crystals which were filtered off and
washed with cold n-hexane (3 ꢃ 5 mL) to yield the desired
product (1.55 g, 84 %). dH (CDCl3, 200 MHz) 7.52–7.40 (6H, m,
phenylH), 7.41–7.21 (12H, m, phenylH), 6.56 (1H, s, NH), 3.81
(2H, s, CH2Br), 3.11 (2H, q, 3JHH 6.2, CH2NHC¼O), 2.43 (2H, t,
3JHH 6.4, CH2SCPh3).
[Pt(terpy)Cl]Clꢀ2H2O (45.9 mg, 0.09 mmol) and GdCl3ꢀ6H2O
(25.7 mg, 0.07 mmol) were dissolved in water (5 mL). To this
solution was added the deprotected macrocycle 3 (90.1 mg,
0.19 mmol) in water (5 mL) in a dropwise manner. An imme-
diate colour change from orange to red–purple was observed.
The reaction was allowed to stir overnight at room temperature.
The reaction mixture was concentrated under vacum and ace-
tone was added to precipitate 1ꢀCl as a purple solid. The solid
was washed with acetone (2 ꢃ 30 mL) and then dried under
vacuum. The purple solid was dissolved in a minimum amount
of water and a saturated solution of KPF6 was added dropwise to
afford a purple precipitate. The solid was filtered off and washed
with water (2 ꢃ 5 mL). Recrystallisation of the solid from
water afforded 1ꢀPF6 (67.5 mg, 66 %). m/z (ESI–FT–ICR)
1044.1629; [M – PF6]þ requires 1044.1616. Anal. Calc. for
C33H40F6GdN8O7PPtSꢀ6H2O: C 30.53, H 4.04, N 8.63. Found:
C 30.65, H 3.80, N 8.38 %.
Synthesis of Tri-tert-butyl-2,20,200-(10-(2-oxo-2-
((2-(tritylthio)ethyl)amino)ethyl)-1,4,7,
Supplementary Material
10-tetraazacyclododecane-1,4,7-triyl)triacetate (2)
The calculated and experimentally determined high resolution
ESI-MS of 1ꢀPF6 and a dose-response curve of 1ꢀPF6 with the
T98G cell line are available on the Journal’s website.
A mixture of N-(2-bromoacetyl)-triphenylmethyl-cysteamine
(435 mg, 1.22 mmol), DO3A-tBu3ꢀHBr (720 mg, 1.21 mmol)
and K2CO3 in MeCN (20 mL) were heated at 708C for 5 h. The
mixture was allowed to cool to room temperature and KBr was
removed by filtration. The filtrate was evaporated to dryness and
the residue dissolved in CH2Cl2 (30 mL). The solution was
washed with water (30 mL), NaHCO3 (saturated, 1 ꢃ 30 mL)
and brine (1 ꢃ 30 mL). The organic layer was dried over Na2SO4
and then concentrated under reduced pressure to ,10 mL. To
this solution was added diethyl ether (65 mL) and the product
was allowed to crystallise out of solution over 3 days. Filtration
yielded product 2 as colourless crystals (732 mg, 69 %). dH
Acknowledgements
We thank Dr Ian Luck for assistance with the NMR studies and Dr Nick
Proschogo for the ESI–MS studies. We also thank the ARC for funding.
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