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Geometry matters: inverse cytotoxic relationship for cis/trans-Ru(ii) polypyridyl complexes from cis/trans-[PtCl2(NH3)2].
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Zamoria, D. K. Heidary, J. Ruiz and E. Glazer, Chem. Commun., 2016, DOI: 10.1039/C6CC04813G.
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COMMUNICATION
Geometry matters: inverse cytotoxic relationship for cis/trans-
Ru(II) polypyridyl complexes from cis/trans-[PtCl (NH ) ]
2 3 2
Received 00th January 20xx, Erin Wachter,a,† Ana Zamora,b,† David K. Heidary,a José Ruiz,b and Edith C. Glazera,*
Accepted 00th January 20xx t
p
DOI: 10.1039/x0xx00000x
i
www.rsc.org/ r
c
Two thermally activated ruthenium(II) polypyridyl s
complexes, cis-Ru(bpy) Cl and trans-Ru(qpy)Cl were u
2 2 2
investigated to determine the impact of the geometric n
arrangement of the exchangable ligands on the potential of
a
the compounds to act as chemotherapeutics. In contrast to
the geometry requirements for cisplatin, trans-Ru(qpy)Cl M
2
was 7.1–9.5x more cytotoxic than cis-Ru(bpy) Cl . This
2 2
discovery could open up a new area of metal-based d
chemotherapeutic research. e
t
Cisplatin, cis-[PtCl (NH ) ] has received worldwide 1a; R = Cl 2a; R = Cl p
2 32 Cisplatin Transplatin
acceptance as a clinical drug for the treatment of various 1b; R = 2b; R = e
neoplastic diseases,1 while its isomer transplatin, trans-
c
[PtCl 2 (NH 3 ) 2 ] was found to be therapeutically inactive.2 This inhibit metastases of solid invasive cancers and successfully c
observation was considered a paradigm for the structure- completed phase I clinical trials but ultimately failed in phase II
A
activity relationships (SAR) of Pt-based antitumor compounds, clinical trials.6 Generally, antitumor Ru(II) complexes can be
according to which the antitumor activity requires a neutral divided in two primary families, i.e. the half-sandwich “piano-
m
square-planar platinum center with two ammine ligands and stool” and polypyridyl-types.5b The later family has been
two leaving groups in cis-geometry.2 The lack of antitumor gaining attention due to their appealing physicochemical
m
activity in transplatin has been associated with the formation properties, which offer the possibility to use them in
of intrastrand cross-links between purine-pyrimidine3 residues photodynamic therapy (PDT) and photoactivated o
instead of purine-purine (major DNA adducts formed by chemotherapy (PACT).7 However, all the Ru(II) compounds
C
cisplatin)4 due to stereochemical constraints. able to form covalent bonds to biomolecules exhibit a cis
In addition to Pt based compounds, other metal complexes geometry, and no examples of trans polypyridyl Ru(II) isomers m
have been shown to have biological activity in vitro, including with biological activity are yet known, in spite of their
potency in cisplatin-resistant tumor cells.5 Ruthenium has interesting photophysical and catalytic properties.8 But, does e
received particular attention in the present search for geometry matter in the design of anticancer Ru(II) complexes? h
therapeutic agents, and ruthenium compounds exhibit Here we report that geometry appears to play a very C
antitumor effects as well as antibiotic, antiviral, and important role, and moreover, a Ru(II) polypyridyl complex
antimalarial activity.5b Two anionic Ru(III) coordination with exchangeable ligands with trans geometry exhibits in vitro
compounds, NAMI-A and KP1019, possess a strong ability to anticancer activity significantly superior to the cis compound.
In order to determine the behavior of trans Ru(II)
polypyridyl complexes, trans-Ru(qpy)Cl (2a, qpy =
2
2,2’:6’,2”:6”,2’”-quaterpyridine) was synthesized and the Cl-
ligand exchange rate, DNA binding, cytotoxicity, and cellular
uptake were investigated in comparison to cis-Ru(bpy) Cl (1a,
2 2
bpy = 2,2’-bipyridine) (Chart 1). The qpy ligand was chosen to
generate a complex with exchangeable sites only in the trans
arrangement; this was required as trans-Ru(bpy) Cl is known
2 2
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A B Table 1. Cytotoxicity IC50 values for HL-60 and A549 cell lines.
HL-60 A549
Compound
IC (μM) IC (μM)
50 50
Cisplatin 1.5 ± 0.1 1.7 ± 0.5
Transplatin >100 >100
1a 96 ± 1 73 ± 1
1b >100 98 ± 1
2a 10.1 ± 1.1 10.3 ± 1.2
2b >100 93 ± 1
Figure 1. Cytotoxicity of cisplatin (black, (cid:1)), transplatin (blue, (cid:2)), 1a (red, (cid:1)), 1b
(purple, (cid:3)), 2a (green, (cid:4)), and 2b (grey, Δ) in (A) HL-60 and (B) A549. complex with exchangeable ligands in the trans geometry was
7.1–9.5x more cytotoxic than the cis geometry. For 1b and 2b,
to be highly insoluble.9 Alternatively, attempting a comparison
the control compounds that are incapable of ligand exchange,
of the cis- and trans-Ru(bpy) 2 (H 2 O) 2 complexes is not viable as the cytotoxicity was eliminated in HL-60 cells, and only t
the two systems photoisomerize.10 The qpy ligand was p
minimal toxicity was observed at 100 μM in A549 cells. Two
synthesized via an oxidative coupling reaction with 6-chloro- i
2,2’-bipyridine; coordination to RuCl then yielded 2a.8d hypotheses were proposed to rationalize the differences in r
3 cytotoxicity for the complexes: 1) the trans geometry interacts c
The qpy ligand and derivatives have been reported as a
tetradentate ligand in mononuclear Ru(II) complexes,8d, 8e, 11 with different in vivo targets from the cis geometry; and 2) s
thermally exchangeable ligands are required for the cytotoxic u
but some derivatives may act as a bridging ligand in dinuclear
effect to occur.
Ru(II) complexes.8a, 12 Thus, the mono-metallic structure of the n
The DNA binding behavior for each compound was
complex 2a was confirmed by the further synthesis and a
assessed using agarose gel electrophoresis to compare cis and
characterization of trans-[Ru(qpy)(py) ]2+ (2b, py = pyridine)
2 trans Pt(II) versus Ru(II). Dose responses were performed with M
from 2a.8d As complex 2b is not capable of ligand exchange, it
cisplatin, transplatin, 1a, 1b, 2a, and 2b, with pUC19 plasmid
also served as a control compound to assess if the addition of
DNA after reaction at 37 °C for 12 hours (Figure 2). Cisplatin d
the qpy ligand itself to the Ru(II) center was responsible for the
interacts with plasmid DNA at very low concentrations (15 µM)
observed biological activity. Similarly, cis-[Ru(bpy) (py) ]2+ (1b) e
2 2 and effectively crosslinks the DNA. The adducts were visualized
was used as a nonexchanging control for the cis geometry. t
by the reduced mobility of the DNA at low concentrations of p
To determine the efficacy of 1a, 1b, 2a, and 2b in cancer
cisplatin, followed by increased mobility of the DNA at higher
cells, cytotoxicity studies were performed in HL-60 leukemia e
concentrations. In contrast, transplatin has minimal interaction
cells and A549 lung cancer cells and compared to cisplatin and c
with the DNA, even at high concentrations, where the
transplatin (Figure 1 and Table 1). For square planar platinum migration of the DNA is only effected at ≥125 µM transplatin. c
compounds with exchangeable Cl- ligands, the cis geometry is Surprisingly, when incubated with plasmid DNA, 1a and 2a A
known to be more potent. Here, cisplatin was cytotoxic with
only showed minimal perturbation of DNA mobility, suggesting
IC values of 1.5 and 1.7 µM in HL-60 and A549 cells. As
50 that either they do not interact strongly with plasmid DNA or m
anticipated, transplatin had no effect on either cell line.
the interaction does not cause significant changes to the
In marked contrast, for octahedral ruthenium complexes supercoiled plasmid structure. Replacement of the Cl- ligands m
with exchangeable Cl- ligands, the opposite relationship
with py ligands resulted in an even smaller effect, with only a
between geometry and cytotoxicity was observed. The o
slight decrease in mobility at the highest concentration of 1b
and 2b. C
1 2 34 5 6 7 8 91011 12 1 2 34 5 6 78 9101112
Given that both 1a and 2a contain thermally labile chloride m
A B
ligands, their ligand exchange rates were monitored using
UV/Vis absorption spectroscopy. Compounds 1b and 2b were e
also studied, and were anticipated to be less susceptible to h
thermal exchange. Initially, the thermal exchange of 1a, 1b, 2a,
C
C D and 2b was determined under different buffer and media
conditions (Figures 3, S1–S4). Full spectrum absorbance was
measured in 96 well plates incubated at 37 °C over the course
of 15 hours. To determine the half-life (t ) of ligand
E F
1/2
exchange, the change in absorbance was plotted as a function
of time. While a t could be determined for 1a, compound 2a
1/2
underwent a very slow ligand exchange and the reaction never
Figure 2. Agarose gel electrophoresis showing the dose response of (A) cisplatin, (B)
transplatin, (C) 1a, (D) 2a, (E) 1b, and (F) 2b with 40 μg mL-1 pUC19 DNA incubated reached completion; therefore, the half-life could not be
at 37 °C. Lanes 1 and 12: DNA ladder; Lane 2: EcoRI; Lane 3: Cu(OP)2; Lane 4–11: 0, accurately determined. Not surprisingly, 1b and 2b show
7.8, 15.6, 31.3, 62.5, 125, 250, and 500 μM compound. EcoRI and Cu(OP)2 were minimal changes in UV/Vis spectra following 15 hour
used as controls to represent linear DNA and relaxed circle DNA, respectively. incubation in aqueous solutions, confirming that they are
essentially kinetically inert.
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To compare the rates of ligand exchange under different A B
conditions for 1a, 1b, 2a, and 2b, the spectral change at 500,
450, 345 and 325 nm, respectively, was determined. These
wavelengths represent the maximal signal change for the
majority of conditions tested. Striking differences were seen
for 1a and 2a, where 1a exhibited the fastest exchange in Opti-
MEM supplemented with 1% FBS (fetal bovine serum, t =
1/2
12.8 min; used as a control for the cell cytotoxicity
C D
experiments), and slowest in water (t = 53 min, Figures 3
1/2
and S1, Table S2). On the other hand, 2a had the largest
spectral change in water (Δabs = 0.19) yet the smallest change
in Opti-MEM with 1% FBS (Δabs = 0.011, Figure 3 and S3, Table
S4). Compounds 1b and 2b were studied under the same
conditions, but once more only showed minimal change (∆ < t
abs p
0.08) over the course of 15 hrs due to the thermally stable
i
pyridine ligands. Figure 3. Thermal exchange studies of 40 μM 1a and 2a at 37 °C showing rapid r
The fast exchange for 1a and minimal exchange for 2a in exchange for 1a and slow exchange for 2a. (A) 1a in water, (B) 2a in water, (C) 1a in c
Opti-MEM, 1% FBS, and (D) 2a in Opti-MEM, 1% FBS. Insets show the change in
Opti-MEM with 1% FBS may help to explain the drastic s
absorbance fit to a one phase decay equation. Note: 2a undergoes incomplete
differences in cytotoxicity. The slow/minimal reaction of 2a
conversion over the course of 15 hours. u
with cell culture media would potentially allow the complex to
n
enter the cell without reaction with media components, death for 2a occurs via apoptosis with no visible sign of
whereas the fast reaction of 1a could essentially deactivate the necrosis.13 Thus, the damage induced by compound 2a triggers a
complex prior to entering the cell. In addition to aqueous the programmed cell death pathway. M
media, the thermal exchange in the presence of duplex DNA It is possible that the number of exchangeable ligands
and small molecules, used to mimic the side chains of amino differed for the cis and trans complexes, and this also could d
acids, was tested.13 The results from these studies revealed contribute to the disparate biological activities. As both 1a and
e
differences in the reactivity profiles for 1a compared to 2a, 2a react with imidazole, the complexes were incubated with
t
supporting that fast exchange reactions for 1a prevents it from this heterocycle until there was no further change in the p
either entering the cell, or enables unintended side reactions absorption spectra, and then samples were analyzed by HPLC
e with other biomolecules upon entering the cell.13 and mass spectrometry. Both complexes produced new
c
One of the causes for transplatin’s inactivity is its high species with longer retention times than the products that
chemical reactivity, where it becomes deactivated through form in buffer alone. The reaction of complex 1a with c
reactions with plasma and tissue proteins before entering a imidazole resulted in full conversion to cis- A
cell.14 In a recent publication, transplatin was successfully [Ru(bpy) (imidazole) ]2+ with the same retention time and
2 2
internalized as the inactive molecule by encapsulation into absorption spectrum of the molecule produced by chemical m
nanocapsules, essentially preventing deactivation; following synthesis (Figure S8). Mass spectrometry also confirmed that
intracellular release it was able to induce a cytotoxic effect.15 two imidazole ligands replaced the chloride ligands in both the m
Likewise, we have previously reported Ru(II) complex prodrugs cis complex 1a (Figure S6) and the trans complex 2a (Figure
o
that are inactive, but when irradiated with light produce a cis- S7). Thus, both the cis and trans complexes can form
Ru(bpy) L (L = H O or Cl-) and are quite cytotoxic.16 It appears biadducts, and should be capable of crosslinks, either between C
2 2 2
that caging the “inactive” compound to allow uptake into DNA bases, DNA and proteins, or within a protein.
m
cancer cells renders these compounds “active”. SAR studies for cytotoxic metal compounds rarely address
In order to test our deactivation hypothesis, cellular uptake the impact of geometry. The history of research in platinum e
of 1a and 2a was determined in HL-60 cells. The HL-60 cells compounds and the inactivity of transplatin were interpreted h
were incubated in the presence of 20 μM 1a or 2a for 12 as a demonstration that a particular geometric arrangement
C
hours; following this time 90–95% of cells remained viable. The was required for efficacy. However, replacement of the NH
3
cells were then harvested and the ruthenium content was ligand in ineffective transplatin by planar N-heterocyclic
determined using graphite furnace atomic absorption amines produced trans-platinum complexes with significantly
spectrometry (GFAAS) for the media and cells separately. The improved cytotoxicity due to enhanced rate of bifunctional
total uptake for 2a (475.8 ng) was 49x greater than the uptake interstrand adduct formation and altered sequence
for 1a (9.8 ng); this represents 15% cellular uptake for 2a specificity.17 Not only the geometry requirements for platinum
(Figure S5). These results provide support for the deactivation species have been lifted; many compounds with “non-
hypothesis, where the fast reaction of 1a in Opti-MEM renders conventional” structures, including polynuclear,18
the complex unable to accumulate in cells. On the other hand, monofunctional,19 Pt(IV)20 and organometallic21 complexes
the slow reaction of 2a correlated to significant cellular have displayed anticancer potential. These findings highlighted
accumulation, ultimately leading to the cytotoxic effect. that more chemical space is available for exploration among
Furthermore, flow cytometry confirmed the mechanism of cell platinum compounds than previously thought. The same
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S. -M. Ng, S. -M. Yiu, W. W. Y. Lam, X. -G. Wei, K. -C. Lau and T. -
complexes and studied their binding interactions and
C. Lau, Angew. Chem. Int. Ed. Engl., 2014, 53, 14468-14471; (e)J.
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complex containing exchangeable ligands is 7.1–9.5x more
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This work was supported by the National Institutes of 16. B. S. Howerton, D. K. Heidary and E. C. Glazer, J. Am. Chem. Soc.,
2012, 134, 8324-8327. Health (5R01GM107586). We would like to thank the d
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