Preparation, characterization and evaluation of novel 1,3,5-triaza-7-phosphaadamantane (PTA)-based palladacycles as anti-cancer agents

Article abstract of DOI:10.1016/j.jorganchem.2017.09.005

A series of novel mononuclear 1,3,5-triaza-7-phosphaadamantane (PTA)-based palladacycles were prepared by cleaving μ-Cl binuclear orthopalladated dimers of substituted benzylidene-2,6-diisopropylphenylamines. All complexes were fully characterized using IR and NMR spectroscopy, mass spectrometry as well as elemental analysis. In-vitro evaluation of the complexes as anti-cancer agents against the breast-cancer cell lines MCF7 and MDA-MB 231 as well the melanoma cell line ME1402 shows that four of the five complexes tested are active. These palladacycles exhibit their cytotoxicity by inducing DNA damage which subsequently triggers apoptosis. DNA binding studies using electrophoresis and spectroscopic techniques, such as UV-Vis and circular dichroism spectroscopy, confirms that the palladacycle, C2 definitely interacts with DNA. Results from these DNA binding experiments seem to rule out co-valent and intercalative binding, pointing rather to a non-covalent interaction, with electrostatic binding being the most likely possibility. It is envisioned that this would probably involve a hydrolysed or solvated derivative of C2.

Full text of DOI:10.1016/j.jorganchem.2017.09.005

Accepted Manuscript
Preparation, characterization and evaluation of novel 1,3,5-triaza-7-
phosphaadamantane (PTA)-based palladacycles as anti-cancer agents
A. Blanckenberg, S. Aliwaini, S.W. Kimani, A. van Niekerk, A. Neumann, S. Prince,
S.F. Mapolie
PII:
S0022-328X(17)30516-8
10.1016/j.jorganchem.2017.09.005
JOM 20080
DOI:
Reference:
To appear in: Journal of Organometallic Chemistry
Received Date: 13 June 2017
Revised Date: 29 August 2017
Accepted Date: 2 September 2017
Please cite this article as: A. Blanckenberg, S. Aliwaini, S.W. Kimani, A. van Niekerk, A. Neumann,
S. Prince, S.F. Mapolie, Preparation, characterization and evaluation of novel 1,3,5-triaza-7-
phosphaadamantane (PTA)-based palladacycles as anti-cancer agents, Journal of Organometallic
Chemistry (2017), doi: 10.1016/j.jorganchem.2017.09.005.
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ACCEPTED MANUSCRIPT
Preparation, characterization and evaluation of novel 1,3,5-triaza-7-
phosphaadamantane (PTA)-based palladacycles as anti-cancer agents
a
b,c
b
a
b
b
*a
A. Blanckenberg, S. Aliwaini, S. W. Kimani, A. van Niekerk, A. Neumann , S. Prince and S. F. Mapolie
a
Department of Chemistry and Polymer Science, University of Stellenbosch, Private Bag X1, Matieland 7602, South Africa
b
Department of Human Biology, Faculty of Health Sciences, University of Cape Town, Observatory, 7925, Cape Town, South Africa
c
Department of Biology, Islamic University of Gaza, P.O. Box 108, Gaza Strip, Palestine
A series of novel mononuclear 1,3,5-triaza-7-phosphaadamantane (PTA)-based palladacycles were prepared by
cleaving µ-Cl binuclear orthopalladated dimers of substituted benzylidene-2,6-diisopropylephenylamines. All
complexes were fully characterized using IR and NMR spectroscopy, mass spectrometry as well as elemental
analysis. In-vitro evaluation of the complexes as anti-cancer agents against the breast-cancer cell lines MCF7
and MDA-MB 231 as well the melanoma cell line ME1402 shows that four of the five complexes tested are
active. These palladacycles exhibit their cytotoxicity by inducing DNA damage which subsequently triggers
apoptosis. DNA binding studies using electrophoresis and spectroscopic techniques, such as UV-Vis and
circular dichroism spectroscopy, confirms that the palladacycle, C2 definitely interacts with DNA. Results from
these DNA binding experiments seem to rule out co-valent and intercalative binding, pointing rather to a non-
covalent interaction, with electrostatic binding being the most likely possibility. It is envisioned that this would
probably involve a hydrolysed or solvated derivative of C2.
Key words: palladacycles; metallacycles, DNA binding studies, anti-cancer agents, metallodrugs, 1,3,5-triaza-
7
-phosphaadamantane complexes.
*Corresponding author:
Prof. Selwyn F. Mapolie
Department of Chemistry and Polymer Science
Private Bag X1, Matieland 7602, South Africa
Telephone: +72-21-8082722
E-mail: smapolie@sun.ac.za
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1
. Introduction
Over the past couple of years there have been several reports on cyclometallated
complexes showing the potential to be applied as anti-cancer agents [1-5]. Amongst
these metallacycles are the so-called palladacycles which have shown great promise
in-vitro as having potent anti-cancer activities [6-9]. In some cases, these
palladacycles have also successfully been tested in-vivo using mouse models [10-12].
Several of these previously reported complexes were found to be more effective than
cisplatin [13-15]. Many of the palladacycles referred to above have tertiary
phosphines as co-ligand with the majority of these being based on triphenylphosphine
or its bidentate analogue, 1,2-bis(diphenylphosphino)ethane, (dppe).
To our
knowledge there are no examples of palladacycles in which 1,3,5-triaza-7-
phosphaadamantane (PTA) has been employed as co-ligand. Drawing inspiration
from the well-known ruthenium/arene/PTA (RAPTA) complexes [16-17], the water-
soluble tertiary phosphine, 1,3,5-triaza-7-phosphaadamantane (PTA) was selected to
cleave the chloro bridged dimeric palladacycle precursors in order to obtain the
mononuclear palladacycles as per the general methodology (Scheme 1) usually
adopted in the literature [18].
Cl
N
C
Cl
C
N
C
N
C
(
MeCN) PdCl
2
2
L
Pd
Pd
Pd
NaOAc
N
Cl
L
R
N
(
C1): R = H
Cl
N
(C2): R = Cl
iPr
N
N
P
Pd
Cl
(
(
(
C3): R = Br
C4): R = F
=
Pd
N
C
L
iPr
C5): R = Me
Scheme 1. General synthetic scheme for dimeric and monomeric palladacycles L = tertiary phosphine
The mononuclear palladacycle targets are shown in (Scheme 1). PTA is well known to be
a non-toxic and biocompatible tertiary phosphine [19]; hence its complexes would be ideal to
2

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be employed in biological applications. Additionally, also we set out to investigate the
effects of various ortho substituents (R) on the N-containing aromatic ring on the efficacy of
these novel mononuclear PTA-based palladacycles as anti-cancer agents (Scheme 1). This
paper thus reports on the synthesis and biological evaluation of these novel
palladacycles as anti-cancer agents against two breast cancer cell lines, MCF7 and
MDA-MB-231 as well as the human melanoma cell line, ME1402.
2
.
Results and Discussion
2
.1 Synthesis and characterization of mononuclear palladacycles, C1-C5
Similar to previously reported palladacycles with phosphine auxiliary ligands, the
PTA-based palladacycles were prepared in a three step process. The known imine
ligands [20] were synthesised by Schiff base condensation of 2,6-diisopropylaniline
with various ortho-substituted benzaldehydes. The Schiff base benzylidene ligands
(
L1-L5), were isolated as yellow crystalline solids. The formation of the product was
monitored by FT-IR spectroscopy, where the absence of the carbonyl C=O stretch and
-1
the appearance of a new imine C=N stretch in the region of 1626-1638 cm confirmed
the correct product, as reported in the literature [20].
The ligands were then cyclopalladated by reacting them with the palladium
precursor, bis(acetonitrile)dichloropalladium(II), in the presence of excess sodium
acetate which acts as a base. The µ-chloro complexes (B1
-B5) were isolated as yellow
crystalline or amorphous solids. FT-IR spectroscopy was used to determine whether
-1
the cyclopalladation was successful. An approximately 30-40 cm shift of the νC=N
band to lower wavenumbers confirmed coordination of the imine nitrogen to
palladium [20, 21].
The µ-chloro binuclear palladacycles were then readily cleaved with PTA in a 1:2
mole ratio, producing the mononuclear complexes, C1-C5, the structures of which are
shown in Scheme 1. For details of the synthetic approach taken, see Scheme S1† in the
Supplementary Information section. The final products were isolated as light yellow
amorphous or crystalline solids and were fully characterized by a range of analytical
1
13
31
techniques including FT-IR and NMR ( H, C and P) spectroscopy as well as mass
spectrometry and melting point determinations.
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The characterisation data for the various complexes together with examples of
representative spectra are included in the Electronic Supplementary Information (ESI).
The FT-IR spectra (solid state) of the complexes showed a distinctive νC=N band in
-1
the region 1599 –1606 cm . This is slightly higher than the corresponding imine band
I
of the
µ
-Cl binuclear precursors. The H NMR spectra of the compounds in CDCl
3
confirm successful cleavage of the
µ–Cl bridged dimers to form the orthometallated
I
monocuclear complexes C1–C5. The principal features of the H NMR spectra
include the upfield shift of the imine proton to the region between 7.99–8.48 ppm. In
4
addition, the imine occurs as a doublet which is a result of the JH–P coupling of this
proton with the phosphorous atom of the tertiary phosphine ligand, PTA. The protons
of the PTA ligand are also clearly observed as multiplets integrating for 12 protons in
the region 4.52 – 4.64 ppm. The methyls signals of the isopropyl substituents on the
benzylidene ligand are observed between 1.07 and 1.40 ppm while the methine signals
occur between 3.12 and 3.29 ppm.
13
C NMR spectra of the complexes show the imine carbon appearing in the region
1
around 175 ppm. Once again as is the case for the H NMR spectra, the signal
indicates heteronuclear coupling with the phosphorous of the tertiary phosphine
ligand. The coupling is in the order of 3.0 – 4.5 Hz. The signals for the carbons of the
PTA ligand occur as two doublets at around 70 ppm and 52 ppm. These represent the
N–CH –N and N–CH –P carbons of the PTA respectively. The fine structure of the
2
2
31
signals is due to coupling to the P atom. The P NMR spectra of the complexes show
singlets at approximately – 47 ppm. This represents a significant shift when compared
1
13
31
to the signal for the free PTA ligand which occurs at –102 ppm. The H, C and P
NMR spectra as well as representative mass spectra of the complexes are also shown
in the electronic supporting information.
2
.2 In vitro evaluation of the anti-cancer activity of the novel mononuclear PTA-based
palladacycles.
2
.2.1 MTT assays of the complexes against breast cancer cell lines, MCF7 and
MDA-MB-231.
The mononuclear, PTA-based palladacycles were subjected to preliminary in vitro
evaluation for their efficacy as anti-cancer agents against two mammalian breast
cancer cell lines, MCF7 and MDA-MB-231 as well as the melanoma cell line ME1402
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by means of a MTT assay. Following treatment of the cells with the metallodrug at
various concentrations, the half maximal inhibitory concentration (IC ) values were
50
calculated. These values are reported in Table 1.
Table 1: IC50 values of mononuclear PTA-based palladacycles
IC ± SEM (µM)
IC ± SEM (µM)
IC ± SEM (µM)
50
5
0
50
Complex
MCF7
MDA-MB-231
54 ± 3.5
ME1402
C1
C2
46.2 ± 1.4
18.8 ± 1.0
11.2 ± 1.9
28.3 ± 4.3
>100
21.02 ± 3.5
6.03 ± 1.3
8.37 ± 1.4
>100
17 ± 1.6
a
C3
7.5 ± 1.0
24.4 ± 1.3
>100
C4
C5
>100
b
cisplatin
19 ± 4.5
6.5 ± 2.4
5.63 ± 0. 9
a
C3 was found to be unstable, showing palladium black formation. Hence no further studies were performed using this complex.
b
cisplatin as a reference standard [22].
From the initial screening and based specifically on the IC values obtained
50
from MTT assays it was found that four of the new mononuclear complexes (C1-C4)
show reasonable activity against the breast cancer cell line MCF7, while the same four
were active against the hormone independent breast cancer cell line MDA-MB-231.
Only complexes C1-C3 showed activity against the melanoma cell line ME1402.
The IC values for the various complexes against the different cell lines are listed in
50
Table 1. In terms of the breast cancer cell lines, the complexes appear to be more
active against MDA-MB-231, a more aggressive cell-line. This might be an indication
of some degree of selectivity. C1-C3 also showed relatively good activity against the
melanoma cell line ME1402.
The complexes tested exhibit moderate to good
cytotoxicity against the above-mentioned cell-lines. In some cases, the activity is
greater than that of cisplatin against the same cell-lines.
The nature of the various ortho substituents appeared to have quite a significant
influence on the anti-cancer activity. The best anti-cancer activity was observed for the
bromo analogue (C3), followed by the chloro analogue (C2) and then the fluoro and
unsubstituted analogues (C4 and C1 respectively). In general complexes with an
electron withdrawing substituent appears to more active against the cell lines tested.
The IC values of the complexes when tested against the MCF7 cell line varied from
50
1
1 µM to 46 µM. Against the MDA-MB-231 cells the IC values were generally
50
lower ranging from 7.4 to 54 µM.
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The IC50 values compare well with related palladacycles reported in the
literature. For example an analogous cyclopalladated complex in which
triphenylphosphine is employed as an auxiliary ligand and in which the N-donor
chelate ligand is a substituted benzylamine, shows IC values of 41 µM and 13 µM
50
against MCF7 and MDA-MB 231 cells respectively [23]. Similarly, our complexes
are also more effective than palladacycles having an ethylene glycol oligomer as a
substituent at the methanimine N of the imine ligand in conjunction with
triphenylphosphine as co-ligand. In this case, this particular complex had IC values
50
of 25 µM against MCF7 and 5.5 µM against MDA-MB 231 cells [24]. The activities
of two of the complexes, C2 and C3 compare favourably with that of cisplatin against
the same cell-lines (Table 1). In the case of the activity against the melanoma cell line
ME1402, C2 shows comparable activity when compared to cisplatin.
Although C3 showed the best activity of the complexes evaluated, it
unfortunately was also found to be highly unstable in solution showing extensive
decomposition over relatively short periods. Solutions of this compound in solvents
such as dichloromethane, chloroform and DMSO turn significantly darker over time
and show extensive formation of colloidal Pd when left overnight at room
temperature. Given the instability of C3, it was thus decided that it would be futile to
study this species any further as its long term instability would impact on its
usefulness as an anti-cancer agent. In light of this it was decided to investigate the next
best candidate, viz. C2 in more detail since it only had a slightly lower activity than
C3, when tested against MCF7 and MDA-MB 231, while it had superior activity
against ME1402 cells (see Table 1). C2 when tested against fibroblast (FGO) cells,
which is a model cell-line for normal cells, shows an IC value of 23.25± 1.02. C2
50
thus show greater selectivity for the tumour cells as opposed to normal cells.
2
.2.2 Evaluation of DNA damage induced by C2
Given that C2 exhibited reasonable activity and also had reasonable long term
stability, it was decided to explore its biological activity in more detail. This included
an investigation into the interaction of C2 with DNA, with the aim of ascertaining its
mode of action in comparison with the known metallodrug, cisplatin. Initially western
blot assays were conducted on cells treated with C2 to ascertain if it induces any DNA
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damage. It is known from studies of other palladacycles [6, 11], that these types of
complexes induce a cascade process which involves initial DNA damage followed by
apoptosis. These are considered two important traits of effective anti-cancer agents.
The results of the western blot assays obtained for C2, show the presence of the
proteins γ-H2AX and cleaved PARP, which are markers for DNA damage and
evidence for the induction of apoptosis, respectively (Figure 1). Thus from the western
blot results it is clear that C2 also induces DNA damage and apoptosis, one of main
mechanisms of cell death. We therefore decided to perform DNA binding studies on
this complex in order to probe its interaction with the hope of gaining further insight
into its biological activity.
Figure 1: Western blot analyses with anti-bodies to γ-H2AX and cleaved PARP show that C2 treatment
of MCF7 cells induces a DNA damage response and apoptosis. Total p38 was used as a loading control.
2
.2.3 DNA binding studies using C2
Usually DNA binding studies involve the investigation of the interaction of the
drug molecule with DNA at various concentrations and probing this using various
analytical techniques such as gel electrophoresis and spectroscopic techniques
especially ultraviolet-visible (UV-Vis) spectroscopy and circular dichroism (CD)
spectroscopy. We thus conducted a preliminary investigation into the DNA binding
ability of one of our most active complexes, C2
.
2
.2.3.1 DNA migration studies using electrophoresis
Electrophoresis has commonly been employed as a technique to probe DNA
binding of metal complexes. Essentially changes in electrophoretic mobility of the
various forms of plasmid DNA (oc–open circular plasmid, –linear plasmid and ccc
superhelical plasmid) have been considered as confirmation of metal-DNA interaction
l
–
[
25]. Previous reports seem to indicate that the interaction of cisplatin with plasmid
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DNA results in the uncoiling of DNA at low metallodrug concentrations. This is
usually observed as retardation of the oc and ccc electrophoretic bands. However, at
higher cisplatin concentrations, supercoiling of the DNA is induced, leading to a
situation where the DNA undergoes recoiling, but with an inverse handedness of the
DNA helix. This phenomenon results in an increase in the electrophoretic mobility of
the ccc band [24, 25]. Given the apparent simplicity of this method, we decided to
employ a similar approach to evaluate whether the, C2 would induce similar changes
in the electrophoretic behaviour of DNA when the latter is treated with a range of
concentrations of the metal complex. The protocols reported by Frik et al. and Albert
et al. [24, 25] were employed to carry out this study. The electrophoresis experiments
were conducted using pBluescript plasmid DNA at a concentration range of 10-75 µM
of the metallodrugs. The results of these experiments are shown in Figure 2. Untreated
plasmid DNA was employed as positive control. Furthermore the impact of C2 on the
electrophoretic behaviour of DNA was compared to that of cisplatin.
From Figure 2 it can be seen that as expected cisplatin induces definite changes
in the electrophoretic behaviour of the DNA. Lanes 5-8 in Figure 2 show the changes
induced by cisplatin. On treatment with cisplatin, the oc bands are retarded while the
l
-bands (just below the oc bands) as well as the ccc bands migrate slightly faster. In
the case of the latter, the band appears to completely disappear as only streaking of the
band is observed at higher concentrations. The results obtained for cisplatin correlate
well with those previously reported for its interaction with DNA at relatively high
drug concentrations [24, 25]. It is also clear from the results obtained that C2 behaves
distinctly differently to cisplatin. Unlike the case for cisplatin, the DNA solutions
treated with the palladacycle showed signs of precipitation of material in the wells at
higher complex concentrations. This is likely due to the formation of insoluble DNA-
metal complex aggregates, in which electrostatic interactions are the driving force
inducing the formation of these aggregates [26, 27].
The impact of C2 on the electrophoretic mobility of the various forms of DNA is
quite pronounced when compared to cisplatin. C2 initially induces an increase in the
migration rate of the oc band at low concentrations (Figure 2, lanes 9 and 10).
Similarly the ccc bands are observed to migrate faster with increasing concentration of
C2. At relatively higher concentrations (50 µM and 75 µM), C2 shows dramatic
retardation of the oc band, accompanied with a fair degree of streaking of the band as
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the drug concentration is increased (Figure 2, lanes 11 and 12). This streaking seems
to point to the onset of significant DNA damage at the higher concentrations [28-30].
This observation would tie in with the results obtained from our western blotting
experiments carried out using C2, where increased levels of γ-H2AX, a marker of
DNA damage, were observed.
The electrophoresis results clearly indicate that C2 interacts with DNA in a
manner which is different to that of cisplatin. To further probe this interaction, we
employed UV-Vis spectroscopy as technique to further understand the DNA binding
behaviour of the C2. The aim was to try and further elucidate the mode of action of C2
in the presence of DNA.
Lane content
lane 1:
λ DNA marker
lane 3:
DNA in buffer - control
DNA in 33% DMSO/buffer – control
10 µM metal with DNA
25 µM metal with DNA
50 µM metal with DNA
75 µM metal with DNA
lane 4:
lanes 5 and 9:
lanes 6 and 10:
lanes 7 and 11:
lanes 8 and 12:
Figure 2: Gel electrophoresis comparing the interactions of cisplatin and C2 with pBluescript plasmid
DNA.
2
.2.3.2 Probing the DNA binding ability using UV-Vis spectroscopy
UV-Vis spectroscopy can be used to determine the interaction between drug
molecules and DNA, by monitoring the changes in the absorbance spectrum of DNA
or that of the drug. DNA has a characteristic absorbance maximum at approximately
2
60 nm. This absorbance is due to the purine and pyrimidine groups in the nucleoside
bases which are chromophores. Comparison of the free drug and the drug-DNA
complex should show the changes due to the interaction of the drug with the DNA
[
31]. The changes in the spectra could include bathochromic shifts in the absorption
maxima of DNA and hypo- or hyper-chromic effects. Weaker interacting drugs may
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show only hypo- or hyper-chromic effects and no significant shifts in the absorbance
maxima [32, 33].
Often, DNA binding studies using UV-Vis spectroscopy are carried out as
quantitative absorbance titrations, where either the metal or DNA concentrations are
kept constant while the other component is varied. Such assays also allow for the
determination of a DNA binding constant if the titration is carried out until a point of
binding saturation is achieved. We started out attempting to employ such assays for
the evaluation of DNA binding abilities of the complexes. However, these proved to
be problematic in the case of our compounds as precipitation of DNA-metal complex
conjugates were observed before the point of binding saturation could be achieved,
thus interfering with the expected absorbance trends [32, 33-34]. Furthermore, these
interactions are usually monitored at a unique wavelength where only the DNA or the
compound absorbs. Unfortunately, such an unique wavelength did not exist for C2 as
the spectrum of the complex overlaps with that of DNA. For this reason, many of our
initial attempts to monitor the DNA binding of the metal complex proved to be futile.
Additionally, the relatively low solubility of the C2 necessitated the use of at least
1
0% DMSO to keep the complex in solution which impacted on the quality of the
spectra obtained. Using the above protocol, we obtained inconclusive results which
seem to indicate no apparent DNA binding was taking place. However, this was in
contradiction of the results obtained for the western blot assays as well as the
electrophoresis studies discussed earlier, since these techniques pointed to extensive
DNA damage occurring in the presence of the complex tested. We thus set out to
explore other DNA binding protocols which could possibly be used to probe DNA
binding of the metal complex.
A less commonly reported protocol previously employed by Aslanoğlu and Öge
for the investigation of the interaction of norepinephrine with DNA was attempted
next [35]. This method involves the comparison of the absorbance spectrum of the
DNA and the compound with the summed absorbance of the respective compounds
(
(DNA+drug) vs (DNA)+(drug)) [26]. Calf thymus DNA was used for these assays in
a 10% DMSO in Tris-HCl buffer (pH 7.2) solution. The incorporation of the DMSO
was to ensure that the C2 remains in solution in the aqueous medium. Cisplatin was
included as a control for covalent binding. The results of this study are shown in
Figure 3a-b. Note that the samples containing both DNA and compound are denoted in
1
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the legends as DNA_Compound x, where x is the µM metal concentration. The spectra
which represent the summed absorbance of pure compound and DNA solutions
measured separately, are denoted in the legends as (DNA + Compound x).
For cisplatin, a hypochromic effect was observed. The summed spectra showed
no change with increasing metal concentration, as cisplatin does not absorb in this
region, whilst the samples containing both DNA and cisplatin showed a hypochromic
effect. As expected, the sample with a higher concentration of cisplatin showed a
larger hypochromic effect than that containing a lower concentration of cisplatin. The
extent of interaction with DNA (hypochromism) is greater than the change in
absorbance due to varying metal concentration, as can be seen by comparing the
change in absorbance between the 2.5 and 5.0 µM samples and the summed spectra.
The sample experiences a small change in absorbance due to increasing metal
concentration (0.005 AU) compared to the overall hypochromic effect relative to the
summed spectra (0.013 AU). This correlated with the strength of DNA-binding rather
than the type, as it is well known that cisplatin forms covalent bonds with DNA.
C2 (solid lines) appears to behave differently in its interaction with DNA as
determined by changes in its UV-Vis spectrum, viz. it exhibits hypochromism at
approximately 260 nm (the absorbance maximum of DNA), relative to the sum of
absorbances of the complex and pure DNA (dashed lines). In comparison with
cisplatin, the degree of hypochromism shown by C2 is far less than the change in
absorbance due to simple changes in metal concentration. This suggests that the
complex does not bind as strongly to DNA as cisplatin. Thus the type of binding
observed for the complex, C2 seems to suggest the existence of weaker non-covalent
interaction rather than pure covalent binding usually observed for cisplatin. Based on
reports in the literature, hypochromism could be regarded an indication of either
groove or electrostatic binding [32, 36-38].
Given the fact that in the case of C2 there is no strong evidence for covalent binding to
DNA, the next step in our investigation entailed an attempt to determine whether the
interaction with DNA involved groove- or electrostatic binding. For this, CD spectroscopy
was employed, as it has been reported that this technique is more sensitive to changes in the
DNA structure than simple UV-Vis spectroscopy [39].
1
1

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(
a)
(
b)
Figure 3a-b: UV-Vis spectroscopic DNA binding studies showing hypochromic effects for the
interaction of cisplatin, C2 with calf thymus DNA (―), relative to the summed absorbance for the pure
DNA and the pure compounds (– –). Note that the numbers indicated after the compound names in the
legend are the µM metal concentrations used
.
2
.2.3.3 Circular dichroism spectroscopy studies to probe DNA binding of C2
Circular dichroism (CD) spectroscopy is a very powerful technique for studying
drug-DNA interactions. The CD spectrum of DNA shows two bands, a positive band
between 260-280 nm due to base stacking and a negative band at approximately
2
45 nm due to the right-handed helicity of the double helical structure [24, 39].
Achiral complexes (like most drug molecules) do not display a CD spectrum;
1
2

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however, drug-DNA interactions cause changes in the conformation of the DNA helix
and the drug molecule. These changes often reflect as changes in the CD spectrum of
the DNA, as the absorbance of light will differ relative to the unaltered DNA
molecule. Changes in the CD spectra are usually observed as variations in the
intensities of the positive and negative bands as well as shifts in the wavelengths of the
bands with varying drug or DNA concentrations.
A similar approach as employed for the UV-Vis spectroscopic studies was used
for the CD spectroscopic studies. Both C2 and cisplatin were studied using CD
spectroscopy. It was first confirmed that the metal complexes on their own do not
have CD spectra. This was not unexpected, since these complexes are inherently
achiral. In light of this, the summed spectra are not shown as these spectra are the
same as that of pure DNA. The results for this study are shown in Figure 4-b. The
samples containing both DNA and compound are denoted in the legends as
DNA_Compound x, where x is the micro-molar metal concentration.
For cisplatin, there is an overall decrease in the intensity of the positive band
(
284 nm), which corresponds to a decrease in the extent of base stacking. The negative
band (244 nm) shows an increased intensity which is probably due to an increase in
the helicity of the DNA [40; 41]. The observed changes in the DNA signal intensities
are usually related to a transformation of the DNA from the B→C form which has a
greater degree of helicity, as previously reported for cisplatin [41].
Upon addition of C2 to DNA, a decrease in the intensity of both the positive and
negative bands is observed. There was also a clear shift in the negative band of C2, to
a higher wavelength. The decreased intensity of both the positive and negative bands
is an indication of decreased base stacking and helicity. At higher metal concentrations
C2 showed CD spectra which have almost indiscernible positive and negative bands.
When considered in conjunction with the lower concentration data, the results could
possibly be associated with a number of different possible transformations that the
DNA could be involved in. Included amongst these are: the formation of ssDNA, a
transformation in the DNA from the B→Z form or the formation of achiral aggregates
[
34]. In addition the UV-Vis spectral results also indicate the presence of
hypochromism which is often suggestive of electrostatic binding.
1
3

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(
a)
(
b)
Figure 4a-c: CD spectra of cisplatin and C2 interaction (―) with calf thymus DNA (– –), respectively,
at various metal concentrations. Note that the numbers indicated after the compound names in the
legend are the µM metal concentrations used.
However, it should be noted that the synthesised palladacycles are neutral
complexes, thus for electrostatic binding to occur, it would require the neutral
complexes to undergo a prior transformation to a charged species. This transformation
is likely to occur during the preparation of stock solutions of metal complex. These
stock solutions made up in DMSO are used to prepare the test solutions employed the
various biological assays.
The possibility thus exists that these neutral palladacyclic compounds are only
precursors rather than the active anti-cancer agents.
One of the possible
1
4

ACCEPTED MANUSCRIPT
transformations that these complexes could be involved in would be aquation or
solvolysis of the Pd-Cl bond to leading a cationic species, an example of which is
shown in Figure 5. Similar processes have been reported for several platinum
complexes which have shown activity as anti-cancer agents [41].
-
+
Cl
Cl
N
N
N
iPr
N
P
Pd
S
iPr
S= H O or (CH ) SO
2
3 2
Figure 5 Proposed aquated species which is cationic in nature.
Unfortunately, attempts to detect such species in-situ have thus far not been
successful. A NMR experiment in which C2 was dissolved in DMSO-d and then
6
allowed to stand for several days at room temperature showed the species to be stable
in solution with hardly any significant changes being observed in the spectrum after
several weeks.
1
5

ACCEPTED MANUSCRIPT
T 0 min
T = 3 weeks
Figure 6 C2 stability in DMSO-d over 3 week period.
6
Thus, under the NMR conditions employed no discernible changes were detected. We can
thus conclude that under these conditions C2 is quite stable in solution. This however does
preclude the possibility that in the cellular environment, transformation to a cationic species
does not occur. We will thus be embarking on a wider investigation to explore the solution
properties of complexes of the type reported in this article in the presence of various
biological targets. This will be reported on in due course.
3
.
Conclusions
Five novel
palladacycles
based
on
substituted
benzylidene-2,6-
diisopropylphenylamine ligands in conjunction with 1,2,5-triaz-7-phosphaadamantane
as auxiliary ligand were synthesized and fully characterized by a range of
spectroscopic techniques. Four of the five complexes were found to exhibit activity
against the breast cancer cell lines MCF and MDA-MB-231 as well as against the
melanoma cell-line ME1402. Two of the complexes viz. C2 and C3 exhibit activities
which exceed that of cisplatin. These complexes were also relatively more active than
analogous orthopalladated complexes of aryl imines/amines in which other tertiary
phosphines are employed as auxiliary ligands. The most active of the new complexes,
C2 was studied in some detail. Western blot assays clearly indicates that C2 induces
DNA damage. Furthermore clear evidence for apoptosis exists in the light of the
detection of the proteins γ-H2AX and cleaved PARP. The interaction of C2 with DNA
was confirmed by gel electrophoresis as well as by circular dichroism and UV-vis
1
6

ACCEPTED MANUSCRIPT
spectroscopy. These studies indicate that unlike the known anti-cancer metallodrug,
cisplatin, C2’s mode action with DNA does not involve intercalation. Initial results
point to possible electrostatic interaction with DNA. We are currently exploring the
exact mode of interaction of C2 and other palladacycles with similar ligand scaffolds.
4
.
Experimental section
4
.1 Materials and methods
Reagents were obtained from Sigma Aldrich, Kimix, Strem Chemicals and
Merck. The palladium precursor, bis(acetonitrile)dichloropalladium (II) was prepared
by refluxing PdCl in excess acetonitrile for 3-4 hours, followed by filtration and
2
drying. Benzylidene ligands and chloro-bridged palladacycles were prepared
according to the methods reported by Mungwe et al. [44]. Reactions were performed
in sealed Schlenk tubes. All solvents for syntheses were distilled before use or dried
over ZANTECH Alusorb ZT200 2-5 mm sieves in Innovative Technology PS-Micro
solvent purifiers.
1
13
31
All new complexes were fully characterised by FT-IR-, NMR ( H, C and P)
spectroscopy and mass spectrometry, as well as melting points and elemental analysis.
FT-IR spectra were recorded on a Thermo Nicolet AVATAR 330 instrument with a
1
smart performer ATR accessory. Samples were recorded neat. NMR ( H: 300 and 400
13
31
and 600 MHz; C: 300, 400 and 600 MHz, P: 300 and 600 MHz) spectra were
recorded on 300 MHz; 400 MHz and 600 MHz Varian NMR spectrometers at 273 K
and chemical shifts are referenced to the residual protons of the deuterated solvents
1
13
31
and external tetra-methyl silane (TMS) in H and C NMR spectra, whilst P NMR
chemical shifts are referenced relative to external 85% phosphoric acid (H PO ).
3
4
Chemical shifts (δ) and coupling constants (J) are reported in ppm and Hertz (Hz),
respectively. The data in bracket is as follows: multiplicity, number of atoms (as per
normalised integration), coupling constants, assignment of atoms (where ‘,’ denotes
‘
and’ and ‘/’denotes ‘or’).The following abbreviations were used to describe
multiplicity of signals: s = singlet, d = doublet, t = triplet, q = quartet, sept = septet, m
multiplet and (br) = broad signals. ESI-MS (positive ion mode) analyses were
=
performed on either a Waters API Quattro Micro or a Waters API Q-TOF Ultima
instrument by direct injection of sample with acetonitrile as solvent. Microanalysis
was done at the University of Cape Town, Department of Chemistry on a Thermo
1
7

ACCEPTED MANUSCRIPT
Elemental Analyser CHNS-O instrument. Melting points were determined using a
Stuart Scientific SMP3 melting point apparatus and are reported as uncorrected. UV-
Vis spectra were recorded on a SHIMADZU UV-1800 with a CPS-100 cell positioner.
4
.2 General procedure for the preparation of the substituted benzylidene-2,6-
diisopropylphenylamine ligands, L1-L5
The substituted benzylidene-2,6-diisopropylphenylamine ligands L1-L5 are known and
were prepared employing procedures reported in the literature [42-44]
The IR and NMR spectra of the complexes compare favourably with those reported
previously
4
.3. Synthesis of µ-Cl binuclear complexes (B1-B5)
The µ-Cl bridged binuclear complexes B1-B3, were prepared as previously reported by
group using bis(acetonitrile)dichloropalladium(II) as palladium precursor [43]. The new µ-Cl
bridged dimers, B4 and B5 were prepared in a similar way with slight modification. The
preparation of B4 is described below to illustrate the procedure followed.
i
4
.3.1 [PdCl(2-F-C H )CH=N{2,6- Pr -C H }] (B4)
6
3
2
6
3
2
B4 was synthesised by stirring a solution of bis(acetonitrile)dichloropalladium(II)
0.100 g, 0.386 mmol) in dichloromethane (10 mL) in a Schlenk tube. L4 (0.109 g, 0.386
(
mmol) and sodium acetate (0.063 g, 0.772 mmol) were added to the solution. The resulting
orange solution was stirred for 24 hours at 25 °C. The solvent was removed to obtain yellow
solid residue. The residue was dissolved in dichloromethane (20 mL). The solution was
filtered through celite. The solvent was removed from the yellow filtrate to obtain a yellow
oily residue. The product was recrystallized from the residue by layering with hexane at room
temperature. The solution was kept at room temperature overnight. A yellow solid was
isolated by vacuum filtration and rinsed with hexane. The solid was air dried. Yield: 0.053 g,
-
1
1
3
2
6
6
1
2%. FT-IR (νC=N, cm ) 1604. m.p.: 330-332 °C. H NMR (299.74 MHz, CDCl ): δ 8.02 (s,
3
H, CH=N); δ 7.31-7.36 (m, 2H, Ph); δ 7.19-7.22 (m, 4H, Ph); δ 7.03-7.11 (m, 2H, Ph); δ
i
.94-6.97 (m, 2H, Ph); δ 6.71-6.77 (m, 2H, Ph); δ 3.42-3.55 (m, 4H, Pr-CH); δ 1.39 (d, 12H,
i
i
13
1
.8 Hz, Pr-CH ); δ 1.18 (d, 12H, 6.8 Hz, Pr-CH ). C{ H} NMR (CDCl , 75.38 MHz): δ
3
3
3
71.30 (CH=N); δ 161.35 (C ); δ 157.87 (C ); δ 156.21 (C ); δ 144.25 (C ); δ 141.45
Ar
Ar
Ar
Ar
(
C ); δ 132.91-133.12 (m, C ); δ 129.56 (C ); δ 128.00 (C ); δ 123.31(C ); δ 111.32 (d,
Ar Ar Ar Ar Ar
2
i
i
i
+
JC-F 19.1 Hz, C ); δ 28.25 ( Pr-CH); δ 24.45 ( Pr-CH ); δ 22.97 ( Pr-CH ). ESI-MS: [M-Cl]
Ar
3
3
1
8

ACCEPTED MANUSCRIPT
2+
8
13.11, [(M/2)-Cl+MeCN] 429.01. Anal. Found: C, 53.4; H, 4.97; N, 2.48. Calc. for
C H N F Cl Pd : C, 53.8; H, 4.99; N, 3.30.
38
42
2
2
2
2
i
4
.3.2 [PdCl(2-Me-C H )CH=N{2,6- Pr -C H }] (B5)
6 3 2 6 3 2
B5 was synthesized according to the method above using L5 instead of L1 and
-1
1
reacting for 24 hours. Yield: 0.090 g, 56%. FT-IR (νC=N, cm ) 1594. m.p.305-307°C: H
NMR (299.74 MHz, CDCl ): δ 8.01 (s, 2H, CH=N); δ 7.31-7.33 (m, 2H, Ph); δ 7.20-7.22 (m,
3
i
4
1
7
1
H, Ph); δ 6.80-7.03(m, 6H, Ph); δ 3.53 (m, 4H, Pr-CH); δ 2.28 (s, 6H, Ar-CH ); δ 1.39 (d,
3
3
i
3
i
13
1
2H, J 6.6 Hz, Pr-CH ); δ 1.15 (d, 12H, J 7.0 Hz, Pr-CH ). C{ H} NMR (CDCl ,
H-H
3
H-H
3
3
5.38 MHz): .): δ 174.17 (CH=N), 158.84 (C ), 143.95 (C ), 141.59 (C ), 138.37 (C ),
Ar
Ar
Ar
Ar
31.80 (C ), 131.53 (C ), 127.76 (C ), 121.71 (C ), 126.50 (C ), 123.26 (C ), 123.00
Ar
Ar
Ar
Ar
Ar
Ar
i
i
i
(
C ), 28.13 ( Pr-CH), 24.56 and 22.97 ( Pr-CH ) 19.85 ( Pr-CH ).
Ar
3
3
4
.4. Synthesis of mononuclear complexes (C1-C5)
i
4
.4.1 [Pd(PTA)(C H )CH=N{2,6- Pr -C H }Cl] (C1)
6
4
2
6
3
C1 was synthesised by stirring a solution of B1 (0.060 g, 0.074 mmol) in
dichloromethane (5 mL) in a Schlenk tube. PTA (0.023 g, 0.15 mmol) was added. The
solution was stirred for 2 hours at 25 °C. The solvent was removed on a rotary evaporator to
obtain a yellow oily residue. The product was recrystallized by layering a dichloromethane
solution with ether at room temperature. The solution was kept at room temperature
overnight. The yellow crystals formed were isolated by vacuum filtration and rinsed with
-1
ether. The crystals were air dried. Yield: 0.118 g, 89%. FT-IR (νC=N, cm ) 1606. m.p.: 222-
1
4
2
1
1
26 °C. H NMR (299.74 MHz, CDCl ): δ 7.99 (d, 1H, J 7.6 Hz, CH=N); δ 7.46-7.48 (m,
3 H-P
i
H, Ph); δ 7.16-7.35 (m, 6H, Ph); δ 4.53-4.64 (m, 12H, PTA); δ 3.16-3.29 (m, 2H, Pr-CH); δ
i
13
1
3
.07-1.39 (br s, 12H, Pr-CH ). C{ H} NMR (CDCl , 75.38 MHz): δ 176.91 (d, J 4.5
3
3
C-P
Hz, CH=N); δ 157.99 (d, JC-P 4.5 Hz, C ); δ 147.81 (C ); δ 144.23 (C ); δ 140.81 (C ); δ
Ar
Ar
Ar
Ar
1
1
36.27 (d, JC-P 9.5 Hz, C ); δ 131.91 (d, J 4.5 Hz, C ); δ 130.05 (C ); δ 127.19 (C ); δ
Ar C-P Ar Ar Ar
3
1
24.97 (C ); δ 122.86 (C ); δ 73.25 (d, J 6.4 Hz, N-CH -N); δ 52.43 (d, J 16.5 Hz, P-
Ar
Ar
C-P
2
C-P
i
i
i
31
1
CH -N); δ 28.36 ( Pr-CH); δ 24.43 ( Pr-CH ); δ 23.00 ( Pr-CH ). P{ H} NMR (121.34 MHz,
2
3
3
+
+
CDCl ): δ -47.02 (s). ESI-MS: [M+H] 564.1; [M-Cl] 527.2. Anal. Found: C, 53.4; H, 6.08;
3
N, 9.29. Calc. for C H N PClPd: C, 53.3; H, 6.08; N, 9.94.
2
5
34
4
1
9

ACCEPTED MANUSCRIPT
Details of the preparation of the other mononuclear complexes (C2-C5) can be found in
the Supplementary information (SI) section.
4
.5 Cell culture
The human breast adenocarcinoma MDA-MB-231 (triple negative) cells were
maintained in DMEM (Highveld Biological, Lynd- Hurst, United Kingdom (UK) and the
human breast adenocarcinoma MCF7 (oestrogen receptor positive) cells were maintained in
RPMI 1640 (Highveld Biological, Lyndhurst, UK). All media were supplemented with 10%
fetal bovine serum (FBS), 100 U/ml penicillin and 100 µg/ml streptomycin. Cells were
maintained at 37 °C in a 5% CO2– 95% air-humidified incubator.
4
.6 MTT assay protocol
The complex solutions were prepared by dissolution in DMSO to obtain 5 mM
stock solutions which were stored at room temperature for no more than a week. The
-3
MCF7 and MDA-MB-231 cells were seeded in 96-well plates at 3-6 x 10 cells per
well. After 48 hours, the cells were treated with various concentrations of complex (0-
5
0 µM) or the vehicle for 48 hours. The 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyyltetrazolium bromide (MTT) assay was then used, as described by the
manufacturer’s instructions. Briefly, 10 µL of MTT solution was added to each well
and the plates were then incubated at 37 °C for 4 h. This was followed by addition of
1
00 µL solubilisation buffer (10% SDS in 0.01 M HCl) and incubation overnight at 37
°
C. Absorbance at 585 nm was then determined for each well and the mean cell
viability was calculated as a percentage of the mean vehicle control. The experiments
were performed in triplicate and the data were used to determine the half maximal
inhibitory concentration values (IC ).
50
4
.7 Western blot protocol
The complex solutions were prepared by dissolution in DMSO to obtain 0.1 and
0
.2 µM stock solutions. Solutions were stored at room temperature for no more than a
5
week. MCF7 cells were plated in 6 cm dishes in RPMI 1640 medium at 6 x 10 cells
per plate and treated with the complex solutions for 24 or 48 hours. Cells were then
lysed using whole cell lysis buffer (0.5 M tris-HCl, pH 6.8, 2% SDS, 10% glycerol,
1
1
% β-mercaptoethanol and 0.02% bromophenol blue) and the samples were boiled for
0 minutes. The proteins were resolved using SDS/PAGE (8-15% gels) as required.
They were then transferred to Hybond ECL membranes which were then incubated
2
0

ACCEPTED MANUSCRIPT
with primary antibodies against PARP 1/2 (sc-7150) (Santa Cruz, California, USA),
phospho-H2AX (#2577) (Cell Signalling Boston, MA, USA) and p38 (M0800) from
Sigma. Thereafter, the membranes were incubated with HRP-conjugated secondary
antibodies (1:5000) and the antibody-reactive proteins were visualised with a
chemiluminescence reaction (ECL) detection system.
4
.8 Agarose gel electrophoresis protocols
pBluescript plasmid DNA was isolated with a Zyppy plasmid isolation kit,
following the product protocol. DNA concentration was determined and adjusted to 32
µg/mL with 1xTBE buffer. For the high concentration gels, stock solutions (33, 83,
1
66 and 249 M) of the complexes were prepared in DMSO and stock solutions of
cisplatin were prepared in physiological saline solution and kept in the dark. Samples
20 µL) were prepared with constant DNA volume (12.5 µL) and the metal
(
concentration was varied by adding the different stock solutions (6.25 µL) to obtain
samples with final metal concentrations of 10, 25, 50 and 75 µM. DNA controls were
prepared with DNA stock (12.5 µL) and DMSO or saline (6.25 µL) to determine the
effect of these reagents on the DNA. All samples were incubated in the dark for 22 hrs
at 37⁰C.
Agarose gel (0.800 g/100 mL) was prepared in 1xTBE buffer. The gel was allowed to
set at room temperature for at least 1 hr. Loading dye (2 µL) was added to each
sample. Samples were loaded and a λ marker was included. Gels were run for 3 hrs at
8
0 V in 1xTBE buffer. The gel was then stained in 1xTBE buffer containing ethidium
bromide stain (15 µL) for 15 min, followed by destaining in water for 5 min. Gels
were then photographed under UV light (254 nm).
4
.9 UV-Vis spectroscopy protocols
Calf thymus DNA was reconstituted in tris-HCl buffer (pH 7.2) for 48 hours at
room temperature. DNA concentration was determined spectrophotometrically, as
described by Sigma, and adjusted to 0.5 mM with tris-HCl buffer. Stock solutions (0.5
mM or 1.25 mM) of the complex were prepared in DMSO and stock solutions of
cisplatin were prepared in physiological saline solution and kept in the dark. Samples
(
4.0 mL) were prepared with constant DNA volume (0.4 mL; DNA concentration of
0 µM) and the metal concentration was varied by adding different volumes of the
stock solution (0.0 - 0.4 mL) to obtain samples with final metal concentrations of 2.5-
.5 µM (cisplatin) and 10-50 µM (C2). Solubilising agents were added in various
5
7
2
1

ACCEPTED MANUSCRIPT
quantities (0.0 – 1.0 mL) to ensure a constant percentage in all samples. Finally, buffer
was added to bring the sample volume to a total of 4 mL. Compound control samples
were prepared in the same way, the only difference being that no DNA was added. All
samples were incubated in the dark for 1 hour at 25 °C. Samples were scanned and the
data recorded for further processing.
4
.10 CD spectroscopy protocols
Calf thymus DNA was reconstituted and the concentration was determined as for the
UV-Vis spectroscopy assay. Samples (1.0 mL) were prepared as described above; however,
the quantities were adjusted to obtain a final sample volume of 1 mL. All samples were
incubated in the dark for 1 hour at 25 °C. Samples were scanned at 25 °C and the data
recorded for further processing.
Acknowledgements
The authors would like to acknowledge financial support received from the Medical Research
council of South Africa, National Research Foundation of South Africa and the Research
committees of Stellenbosch University and the University of Cape Town
Supplementary data related to this article can be found at http://
2
2

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ACCEPTED MANUSCRIPT
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This work highlights the synthesis of a series of novel mononuclear palladacycles with 1,3,5-
triaza-7-phosphaadamantane (PTA) as co-ligand.
Screening of the target complexes against the human breast tumour cell lines (MCF-7) and MDA-
MB231 as well as the melanoma cell line, ME1402
Several of the complexes tested showed cytotoxicity comparable to and in two cases better than
cisplatin against the same cell-lines.
Studying of the interaction of the palladacycles with DNA shows that they behave differently to
cisplatin in terms of their mode of action, with the results from the DNA binding studies ruling
out co-valent and intercalative mode of binding for the new palladacycles