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Half-sandwich organometallic Ru and Rh complexes of (N,N) donor compounds: effect of ligand methylation on solution speciation and anticancer activity.
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ARTICLE
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
Half-sandwich organometallic Ru and Rh complexes of (N,N)
donor compounds: effect of ligand methylation on solution
speciation and anticancer activity
a,b,
c,d
János P. Mészáros, * Veronika F.S. Pape, Gergely Szakács,
f,g
f
a,b,
Tamás Holczbauer, Nóra V. May, Éva A. Enyedy *
c,e
a
Gábor Németi, Márk Dénes,
f
A series of half-sandwich polypyridyl complexes was synthesized and compared focusing on structural, cytotoxic and
aqueous solution behaviour. The formula of the synthesized complexes is [M(arene)(N,N)Cl]Cl, where M: Ru or Rh, arene:
p-cymene, toluene or C5Me5‒, (N,N): 2,2’-bipyridine (bpy), 4,4’-dimethyl-2,2’-bipyridine (dmb), 1,10-phenanthroline (phen)
or 2,9-dimethyl-1,10-phenanthroline (neo). The structures of five half-sandwich complexes were determined by X-ray
crystallography. It was found that introducing methyl groups next to the coordinating nitrogen atoms of the bidentate
ligand causes steric congestion around the metal centre which changes the angle between ligand planes. The ligands and
the Rh complexes showed significant cytotoxicity in A2780 and MES-SA cancer cell lines (IC50 = 0.1–56 μM) and in the
cisplatin-resistant A2780cis cells. Paradoxically, phen and dmb as well as their half-sandwich Rh complexes showed
increased toxicity against multidrug resistant MES-SA/Dx5 cells. In contrast, coordination to Ru caused loss of toxicity.
Solution equilibrium constants showed that the studied metal complexes have high stability, and no dissociation was
found for Ru and Rh complexes even at micromolar concentrations in a wide pH range. However, in case of Ru complexes
a slow and irreversible decomposition, namely arene loss was also observed, which was more pronounced in light
exposure in aqueous solution. In case of neo, the methyl groups next to the nitrogen atoms significantly decrease the
stability of complexes. For Rh complexes, the order of the stability constants corrected with ligand basicity (log K*): 9.78
(phen) > 9.01 (dmb) > 8.89 (bpy) > 3.93 (neo). The coordinated neo resulted in an enormous decrease in the chloride ion
affinity of Ru compounds. Based on the results, a universal model was introduced for the prediction of chloride ion
capability of half-sandwich Rh and Ru complexes. It combines the effects of the bidentate ligand and the M(arene) part
using only two terms, performing multilinear regression procedure.
1
Introduction
Based on the success of cisplatin, complexes of other platinum
group metal ions were developed and introduced into clinical trials.
Ru(III) complexes, namely NAMI-A and BOLD-100 (formerly known
a.
Department of Inorganic and Analytical Chemistry, Interdisciplinary Excellence
Centre, University of Szeged, Dóm tér 7, H-6720 Szeged, Hungary; E-mail:
meszaros.janos@chem.u-szeged.hu; enyedy@chem.u-szeged.hu
b.
MTA-SZTE Lendület Functional Metal Complexes Research Group, University of
Szeged, Dóm tér 7, H-6720 Szeged, Hungary
c.
Institute of Enzymology, Research Centre for Natural Sciences, Magyar Tudósok
körútja 2, H-1117 Budapest, Hungary
d.
Department of Physiology, Semmelweis University, Tűzoltó utca 37-47, H-1094
Budapest, Hungary
e.
Institute of Cancer Research, Medical University of Vienna, Borschkegasse 8a, A1090 Vienna, Austria
f.
Centre for Structural Science, Research Centre for Natural Sciences, Magyar
tudósok körútja 2, H-1117 Budapest, Hungary
g.
Institute of Organic Chemistry, Research Centre for Natural Sciences, Magyar
tudósok körútja 2, H-1117 Budapest, Hungary
†Electronic Supplementary Information (ESI) available: 1H and 13C NMR spectra of
complexes, list of NMR peaks, ESI-MS spectra, crystallographic data and additional
spectra about aqueous stability (PDF). See DOI: 10.1039/x0xx00000x The
crystallographic data files for the complexes have been deposited with the
Cambridge Crystallographic Database as CCDC 2038629-2038634.
as KP-1339) and the Ru(II) containing TLD1433 entered clinical
1
trials. The proposed mechanism is that they are activated by
reduction. Based on this idea several Ru(II) complexes have been
synthesized, possessing an organometallic half-sandwich structure
having a bidentate ligand and a monodentate leaving group. Early
examples contained ethylenediamine and halide ion as ligands (the
1,2
so-called RAED complexes), which were followed by the Os(II),
Ir(III) and Rh(III) analogues and with different bidentate ligands.
Some of these half-sandwich complexes showed remarkable
cytotoxic activity and several structure-activity relationship analyses
1,3
were conducted to identify the key chemical parameters. The
mechanism of action of these compounds show a wide variety, as
4
the RAED complexes are capable of DNA-binding, enzyme
5,6
inhibition (cathepsin B, thioredoxin reductase),
and the
6
complexes
of
phenylazopyridines
with
[Ru/Os( -p2+
7,8
cymene)(H2O)3] show catalytic GSH oxidation. The latter is the
9
primary mechanism of the ‘catalytic metallodrugs’. The group of
Sadler proved the occurrence of intracellular catalysis by halfsandwich complexes of (N,N) donor ligands, such as
ethylenediamine, 2,2’-bipyridine (bpy) and 1,10-phenanthroline
10
(phen). The reaction of these organometallic complexes with the
+
NAD /NADH pair was found in the presence of formate ions.
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Journal Name
Polypyridines (phen and bpy) and their myriad derivatives are
common ligands for half-sandwich complexes. Detailed solution
3,11-13
14
behaviour and/or biological data were reported with Ru,
Rh,
14-16
16,17
Ir
and Os
. These ligands usually show similar or higher
12
cytotoxic activity than their metal complexes. However, complex
formation in general might have advantageous effects on the
selective cytotoxicity on cancer cells, since it can change the overall
charge, lipophilicity and size, which affect the pharmacokinetics and
can also result in altered mechanism of action. Different biological
effects were found for Ru/Rh polypyridyl complexes. DNA
intercalation was reported in the coordinatively saturated and
kinetically inert tris-polypyridyl Ru and Rh complexes, in which
18,19
mostly phen and its derivatives are the ligands.
On the other
hand, taking into consideration the viscosity measurements,
cytotoxicity data and ultrafiltration measurements, DNAintercalation is not likely to occur and DNA is not believed to be the
target
molecule
of
half-sandwich
complexes
2+ 20-22
[Ru/Rh(arene)(phen)(H2O)] .
There are also examples for
23
topoisomerase I and II inhibitors, octahedral and half-sandwich
24
cholinesterase inhibitors.
In the field of cancer treatment, one of the major impediments is
the appearance of resistance to chemotherapeutic agents. Cellular
mechanisms promoting multidrug resistance (MDR) often rely on
the elevated expression of ATP-binding cassette proteins, which
25-27
pump a wide variety of drug molecules from the cell.
Drug
resistant cells resort to further mechanisms in the case of
compounds that are not recognized as transported substrates. In
the case of cisplatin, cells become resistant as a result of elevated
26
glutathione concentrations
and
increased
DNA-repair.
Interestingly, the majority of RAED compounds seemed to
3
overcome cisplatin resistance in the A2780cis cell lines model.
Similarly, a Ru cyclopentadienyl complex containing 4,4’-dimethyl2,2’-bipyridine (dmb) showed comparable activity in parental and in
28
cisplatin-resistant cancer cells. In our former studies, we reported
half-sandwich complexes with high stability, in which the ligands
29,30
were 8-hydroxyquinoline derivatives.
The ligands substituted at
th
the 7 position showed preferential toxicity in otherwise multidrug
resistant MES-SA/Dx5 and Colo320 cell lines, and this characteristic
persisted after combination with the half-sandwich organometallic
5
2+
29,30
[Rh( -C5Me5)(H2O)3]
triaqua cation.
However, complex
6
2+
formation with [Ru( -arene)(H2O)3] resulted in a decrease in
29,30
cytotoxicity and the loss of preferential toxicity.
In this study, we selected four polypyridines and their half-sandwich
Ru and Rh complexes to reveal possible relationships between their
Scheme 1 Synthesis procedure of the complexes.
structure, aqueous solution behaviour and anticancer activity
against parental and drug resistant cancer cell lines. We synthesized
6
novel Ru( -toluene) to investigate the effects of the exchanges of
p-cymene to toluene, and 2,9-dimethyl-1,10-phenanthroline
(neocuproine, neo) complexes were also prepared to reveal the
effect of methylation close to the coordinating nitrogens.
Results and discussions
Synthesis and characterization of complexes
As illustrated in Scheme 1, synthesis of complexes with the general
formula [M(arene)(N,N)Cl]Cl, where M: Ru or Rh, arene: p-cymene
‒
(p-cym), toluene (tol), C5Me5 , (N,N): bpy, dmb, phen and neo, was
11,31-33
performed according to previously described methods.
6
Among the listed complexes, Ru( -tol) complexes and neo
complexes are new compounds. In this work, all the
[M(arene)(N,N)Cl]Cl complexes were obtained as orange solids with
moderate-to-excellent yields (45-95%) using methanol (MeOH) as
solvent. Notably, the neo complexes were isolated in the lowest
yields, which could be improved by using an excess of the ligand.
6
[Ru( -p-cym/tol)(ethylenediamine)Cl]Cl complexes are well-known
2
compounds, they were also synthesized with the same method for
further solution chemical and comparative purposes.
1
13
H and C NMR spectra recorded in CD3OD confirmed complex
formation, as shown in Figures S1-S14. Deuteration of the C5Me5
1
13
ligand (-CH2D and -CHD2 groups) was detected in the H and C
NMR spectra, as three peaks are shown in Figure S7 with the same
intensity next to the peak of the methyl groups. ESI-MS spectra
were recorded only for the novel complexes, and the results
confirmed the stoichiometry of each complex (Figures S15-S21).
Stability and photosensitivity of the complexes were investigated in
water (at pH 7.4). Notably, the Rh complexes were stable in water
1
for at least 7 days, as the yellow colour and the H NMR spectra
remained unchanged (see Figure S22 as an example). In order to
investigate the photostability of the Ru complexes parallel samples
6
2+
6
2+
6
of [Ru( -tol)(bpy)(H2O)] , [Ru( -p-cym)(bpy)(H2O)] and [Ru( 2+
p-cym)(phen)(H2O)] were prepared and followed in time by UVvisible (UV-Vis) spectrophotometry. One of the parallel samples was
protected from light, while the other was exposed to diffuse solar
irradiation. The starting solutions had yellow colour, which is typical
for half-sandwich complexes. Spectra of the complexes (shown in
Figure S23) remained unchanged in dark after one day, except for
6
2+
[Ru( -tol)(bpy)(H2O)] , which showed signs of decomposition after
18 h (Figure S23.a). Namely, the sample turned blue-green, and a
new band developed in the UV-visible (UV-Vis) spectra, first after
18 h with max = 588 nm, and after 6 days another band appeared
with max = 644 nm. When exposed to light for a longer period of
time, all samples showed signs of this process, which is most
probably linked to the irreversible decomposition of the halfsandwich structure (also known as arene loss). The decomposition
6
2+
of [Ru( -p-cym)(bpy)(H2O)] is slower than that of the toluene
analogue, which appears only after more than 1 day. The product
has the same max at 588 nm (Figure S23.b), which is indicative of
6
2+
the loss of arene. The product of [Ru( -p-cym)(phen)(H2O)] has a
max = 658 nm, where a tiny amount (Amax~0.03) appears only in
light after 4 days (Figure S23.c). These experiments suggest that the
2 | J. Name., 2012, 00, 1-3
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metal−carbon bond in the organometallic moiety can break and
both the type of the metal ion and the arene have important roles
in the stabilization: the bond between Ru(II) and p-cymene is more
stable than between Ru(II) and toluene, while the bond between
Rh(III) and C5Me5 is the strongest in this set of complexes. In case
of Ru complexes formed with other (N,N) bidentate ligands, the loss
34
of the arene ligand was reported earlier. In our previous studies,
arene loss induced by ligand excess or by another coordinating
29,30
bidentate ligand was observed.
Stability studies of the bpy
complexes was followed in the cell culture medium Roswell Park
Memorial Institute Medium 1640 (RPMI 1640) completed with fetal
bovine serum (FBS) in dark. Under these conditions no arene ligand
dissociation was detected (Figure S24).
Reactions of the complexes in the cell culture medium Dulbecco’s
Modified Eagle Medium (DMEM) was also followed for a week by
1
H NMR spectroscopy. This medium provides physiological pH and
contains inorganic salts, amino acids, sugar and vitamins, which
may interact with these complexes. Figure S25 shows the important
ranges of the recorded spectra, focused on the interactions with
5
2+
biomolecules. In the case of [Rh( -C5Me5)(phen)(H2O)] and
6
2+
[Ru( -p-cym)(phen)(H2O)] complexes after 5 h new peaks
appeared showing the formation of mixed ligand complexes. After
this period, no big changes in the spectra could be observed after
one week, except in the case of neo complexes, which showed a
6
slow reaction and changed continuously. The complex of [Ru( -p2+
cym)(neo)(H2O)] partially dissociated even in the phosphate
buffer, indicating its lower stability at pH = 7.40. The measurements
were repeated in RPMI 1640 medium completed with FBS (Figure
5
S26). New sets of peaks were observed only for the [Rh( 2+
C5Me5)(phen)(H2O)] complex compared to the spectra measured
in DMEM.
The structures of (N,N) donor bidentate ligands complexes formed
6
5
with Ru( -arene) and Rh( -C5Me5) organometallic cations are
well-known and several examples show the complexes in
3,12,13,20
35,36
chlorinated
or in aqua form
in the solid structures. The
different arene ligands did not change the piano-stool shaped
structure. However, Ru−N and Ru−ring centroid distances can vary
in these complexes. Generally, the structures represent mono
complexes, in which the ligands are bound to the metal centre
through two nitrogen atoms.
-
After counter ion exchange (Cl to CF3SO3), single crystals for
5
6
complexes [Rh( -C5Me5)(dmb)Cl](CF3SO3) (crystal I), [Ru( tol)(dmb)Cl](CF3SO3) (crystal II) and [Ru(dmb)3](CF3SO3)2×2 H2O
(crystal III) were obtained, which were subjected to X-ray
crystallographic structure determination. Crystal data and structure
refinement parameters are collected in Table S1. The results proved
5
the presence of half-sandwich structure of the [Rh( +
6
+
C5Me5)(dmb)Cl] and [Ru( -tol)(dmb)Cl] complexes, similarly to
6/5
+
6
the other [Ru( -arene)(polypyridyl)Cl] complexes, e.g. [Ru( -p+ 37
cym)(dmb)Cl] . However, a dark red crystal was also isolated that
showed evidence of arene loss and the formation of
[Ru(dmb)3](CF3SO3)2×2 H2O with octahedral structure. This
irreversible reaction occurred in organic solvents and in water as
well (vide supra), as the colour of solutions turned to green-blue
5
over time. The [Rh( -C5Me5)(dmb)Cl](CF3SO3) complex crystallized
a)
b)
c)
d)
e)
f)
Fig. 1 Molecular structures of a) [Rh(5-C5Me5)(dmb)Cl](CF3SO3) (I), b)
[Ru(6-tol)(dmb)Cl](CF3SO3) (II) c) [Ru(dmb)3].2(CF3SO3)×2 H2O (III), d)
[Ru(6-tol)(neo)Cl]Cl×2 MeOH (IV), e) [Ru(6-p-cym)(neo)Cl](CF3SO3) (V) and
f) [Rh(5-C5Me5)(neo)Cl](CF3SO3) (VI). Hydrogen atoms, solvent molecules
and counter ions are omitted for clarity. Displacement ellipsoids are drawn
at 50% probability level.
in the orthorhombic and both Ru complexes in the monoclinic
crystal systems in Pbcn, P21/n and P21/c space groups, respectively,
with the inclusion of a CF3SO3 counter ion (and two water
molecules in the latter) per asymmetric unit. The ORTEP
representation of the compounds is depicted in Figure 1.a,b,c.
6
Crystal structures of [Ru( -tol)(neo)Cl]Cl×2 MeOH (crystal IV),
6
5
[Ru( -p-cym)(neo)Cl](CF3SO3)
(crystal
V)
and
[Rh( C5Me5)(neo)Cl](CF3SO3) (crystal VI) could be also obtained
(Figure 1.d,e,f). Crystal data and structure refinement parameters
for neo complexes are collected in Table S2. Crystal IV and V
crystallized in the monoclinic and crystal VI in the triclinic crystal
systems in P21/c (IV, V) and P-1 (VI) space groups. The asymmetric
unit contains one complex and one counter ion in crystal V, two
extra MeOH molecules in crystal IV and two complexes with two
counter ions in crystal VI. Selected bond lengths and angles are
collected in Tables S3-S4. In the the neo complexes, the neo ligand
is not fully planar. The angle enclosed by the planes of the outer
ring is bended for IV and V (12.7° and 9.8°, respectively) and the
most bent ring was found in crystal VI where this angle is 17.2° and
17.7° for molecules 1 and 2. The bar chart in Figure S27 shows the
metal ion−N atom distances, which are longer for neo complexes
and are the shortest in the tris-dmb complex.
6
6
The structures of [Ru( -tol)(dmb)Cl](CF3SO3) and [Ru( -p37
cym)(dmb)Cl](BF4) are practically identical as the average Ru−N
bond lengths are 2.088 Å vs. 2.085 Å, the Ru−ring centroid distances
are 1.686 Å vs. 1.685 Å. The two Ru complexes of neo also show a
strong similarity, the change of arene has no effect on the
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J. Name., 2013, 00, 1-3 | 3
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geometrical parameters (Figure S28.a). The geometrical changes are
negligible when p-cymene is substituted to toluene ligand.
complexes exhibited in all cases weaker cytotoxicity than the Rh
congeners.
All neocuproine containing structures differ from the analogous
13,20
phen complexes,
as the steric congestion around the metal ion
nd
th
is caused by the methyl groups in the 2 and 9 positions. For
instance the Rh−N bond length changed from 2.11 Å to 2.13 Å, in
case of exchanges of phen to neo change. A spectacular proof of the
steric hindrance is the finding that the planes of the arene ligand
and of the bidentate ligand are not the same. The difference
6
+
6
+
between [Ru( -p-cym)(neo)Cl]
and [Ru( -p-cym)(phen)Cl]
13
complexes is shown in Figure S28.b. In this example there is a 19°
alteration between the plane of phen and neo. The best
visualization for this steric congestion is provided by the ligand solid
38,39
angles calculated by the Olex2 software.
Figure S29 shows the
6
+
ligand solid angles in the complex of [Ru( -p-cym)(neo)Cl] , from
two different views. Less overlap is present between the ligands
around Rh(III) than in the two Ru(II) complexes (see more details in
the legend of Figure S29). For crystallization of all neocuproine
complexes we also tried to perform anion exchange using
Ag(CF3SO3) salt. However, during the crystallization procedure, red
crystals of the precursor [M(arene)Cl2]2 dimer and colourless
crystals of [Ag(neo)2](CF3SO3) appeared in these samples, most
probably as a consequence of the low stability of the neocuproine
complexes (notably they were also characterized by the lowest
yields in synthesis). Scheme S1 shows side reactions of neocuproine
complexes.
Table 1 In vitro cytotoxic effects (72 h) (IC50 values in M) in parental (MESSA) and multidrug resistant (MES-SA/Dx5) cell lines treated with
polypyridine ligands and their half-sandwich complexes in addition to the
corresponding organometallic precursors. Resistance ratio (RR = IC50(MESSA/Dx5)/IC50(MES-SA)) values are also represented. N. d. = not determined
(in cases where both IC50 > 100 M).
In vitro anticancer activity
The anticancer activity of four related polypyridyl ligands (phen,
6
6
neo, bpy, dmb) and their half-sandwich Ru( -p-cym), Ru( -tol)
5
and Rh( -C5Me5) complexes was investigated against the uterine
sarcoma cell line MES-SA and its doxorubicin resistant counterpart
MES-SA/Dx5, as well as against the ovarian cancer cell line A2780
and its cisplatin resistant counterpart A2780cis. A2780cis cells show
an increased ability to repair DNA and have higher intracellular
26
concentrations of glutathione,
while MES-SA/Dx5 cells
26,40
overexpress P-gp,
which results in multidrug resistance.
The paradoxical toxicity of phen against P-glycoprotein (P-gp)expressing MDR cells (MDR-selective toxicity) was reported
41
earlier. A summary of the literature data on the in vitro toxicity of
the half-sandwich complexes of the selected ligands on other
cancer cell lines is shown in the Electronic Supplementary
13,14,42,43
Information.
The obtained IC50 values are shown in Tables 1-2 and in Figure S30.
The relative toxicity of the complexes against parental and drug
resistant cancer cell lines was compared, and the selective toxicity
was expressed as resistance ratio (RR = IC50 (resistant cell) / IC50
(sensitive cell)). Based on the determined IC50 values (Tables 1-2),
the ligands phen, neo and dmb displayed significant toxicity,
reaching submicromolar IC50 values in some cases. Neocuproine has
a superior cytotoxic effect, it is comparable with doxorubicin (and
10 times higher than phen) in MES-SA cells, while 20 times more
5
active than cisplatin in A2780 cells. The toxicity of the [Rh( +
C5Me5)(N,N)Cl] complexes were similar or slightly lower compared
to that of their corresponding ligands. Surprisingly, the Ru
phen
[Rh(5-C5Me5)(phen)Cl]Cl
[Ru(6-tol)(phen)Cl]Cl
[Ru(6-p-cym)(phen)Cl]Cl
neo
[Rh(5-C5Me5)(neo)Cl]Cl
[Ru(6-tol)(neo)Cl]Cl
[Ru(6-p-cym)(neo)Cl]Cl
bpy
[Rh(5-C5Me5)(bpy)Cl]Cl
[Ru(6-tol)(bpy)Cl]Cl
[Ru(6-p-cym)(bpy)Cl]Cl
dmb
[Rh(5-C5Me5)(dmb)Cl]+
[Ru(6-tol)(dmb)Cl]+
[Ru(6-p-cym)(dmb)Cl]+
4±1
8±2
> 100
24 ± 12
0.37 ± 0.08
1.4 ± 0.1
2.1 ± 0.2
4±1
66 ± 19
> 100
> 100
> 100
46 ± 7
41 ± 6
> 100
63 ± 11
MESSA/Dx5
1.30 ± 0.01
2.0 ± 0.9
> 100
> 100
0.30 ± 0.04
2.66 ± 0.09
4.2 ± 0.7
7±1
50 ± 15
69 ± 25
> 100
> 100
15 ± 2
16 ± 6
> 100
> 100
[Rh(5-C5Me5)Cl2]2
[Ru(6-tol)Cl2]2
[Ru(6-p-cym)Cl2]2
doxorubicin
> 100
> 100
> 100
0.35 ± 0.06
> 100
> 100
> 100
3 ± 0.9
MES-SA
RR
0.33
0.25
n. d.
>4.2
0.81
1.90
2.00
1.75
0.75
<0.7
n. d.
n. d.
0.33
0.39
n. d.
>1.59
n. d.
n. d.
n. d.
8.57
Table 2 In vitro cytotoxic effect (72 h) (IC50 values in M) of polypyridine
ligands and their half-sandwich complexes in addition to the organometallic
precursors in sensitive (A2780) and cisplatin resistant (A2780cis) human
ovarian cancer cell lines. Resistance ratio (RR = IC50(A2780cis)/IC50(A2780))
values are also represented. N. d. = not determined (in cases where both
IC50 > 100 M).
phen
[Rh(5-C5Me5)(phen)Cl]Cl
[Ru(6-tol)(phen)Cl]Cl
[Ru(6-p-cym)(phen)Cl]Cl
neo
[Rh(5-C5Me5)(neo)Cl]Cl
[Ru(6-tol)(neo)Cl]Cl
[Ru(6-p-cym)(neo)Cl]Cl
bpy
[Rh(5-C5Me5)(bpy)Cl]Cl
[Ru(6-tol)(bpy)Cl]Cl
[Ru(6-p-cym)(bpy)Cl]Cl
dmb
[Rh(5-C5Me5)(dmb)Cl]+
[Ru(6-tol)(dmb)Cl]+
[Ru(6-p-cym)(dmb)Cl]+
[Rh(5-C5Me5)Cl2]2
[Ru(6-tol)Cl2]2
4 | J. Name., 2012, 00, 1-3
A2780
0.14 ± 0.03
0.28 ± 0.09
38 ± 12
11 ± 2
A2780cis
2.5 ± 0.2
10 ± 3
> 100
> 100
RR
17.9
35.7
>2.6
>9.1
0.13 ± 0.03
0.4 ± 0.2
0.6 ± 0.1
2±1
9±2
1.2 ± 0.3
1.5 ± 0.3
6±4
69.2
3.0
2.5
3.0
2.4 ± 0.8
7±3
11.2 ± 0.9
19 ± 5
39 ± 20
> 100
> 100
> 100
16.3
>14.3
>8.9
>5.3
0.13 ± 0.07
0.4 ± 0.2
~100
13 ± 3
35 ± 6
57 ± 10
> 100
57 ± 9
269
143
>1.0
4.4
45 ± 6
> 100
> 100
> 100
>2.2
n. d.
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[Ru(6-p-cym)Cl2]2
cisplatin
> 100
> 100
n. d.
2.5 ± 0.8
17 ± 8
6.8
+TQ
6.0
phen
+TQ
+TQ
Rh-phen
doxorubicin
Rh-dmb
dmb
+TQ
5.0
+TQ
pIC50(MES-SA/Dx5)
7.0
4.0
4.0
5.0
6.0
7.0
pIC50 (MES-SA)
Fig. 2 Effect of the P-gp inhibitor TQ on pIC50 values in MES-SA and MESSA/Dx5 cells. Doxorubicin and compounds with paradoxical cytotoxicity are
shown. ‘Rh-dmb’ and ‘Rh-phen’ represent the corresponding [Rh(5C5Me5)(N,N)(H2O)]2+ complexes. Points above the diagonal represent
paradoxical behaviour; points on the diagonal show compounds, whose
cytotoxic effect is the same on both cell lines; points under the diagonal
display lower toxicity in MES-SA/Dx5 than in MES-SA. △: without TQ; ●: coincubated with TQ. Arrows show the effect of TQ on the activity, which is
decreased in the case of compounds with paradoxical toxicity (red)
potentiated by P-gp, while increased for non-selective compounds (green).
As compared to A2780 parental cells, the cisplatin-resistant
A2780cis cells were found to be cross-resistant to all investigated
compounds (c.f. IC50 values in Table 2). However, the RR values are
smaller for the neocuproine complexes unlike the ligand or the
cisplatin itself. Interestingly, ligands phen, dmb and their Rh
complexes showed higher cytotoxicity in MES-SA/Dx5 cell lines than
in the MES-SA cells (RR < 0.5 in Table 1), suggesting that these
25
compounds may show MDR-selective activity. However, despite
the similar structures of neo and phen or bpy and dmb, neo and bpy
showed no preferential toxicity against MES-SA/Dx5 cells. The
toxicity of MDR-selective compounds is exacerbated by the function
41,44
of P-gp.
To test the relevance of P-gp function in the selective toxicity of
phen, dmb and their Rh complexes, the in vitro toxicity assays were
repeated in the presence of the P-gp inhibitor tariquidar (TQ) in
MES-SA and MES-SA/Dx5 cell lines (Figure 2, Table S5). As expected,
the IC50 of doxorubicin significantly decreased (pIC50 increased), in
line with the inhibition of drug efflux. In contrast, phen, dmb and
their Rh complexes show decreased toxicity in the presence of TQ,
proving that their activity is potentiated by P-gp. The most
prominent example is dmb, where the IC50 in MES-SA/Dx5 cells
changes from 15 M to 72 M upon addition of TQ, respectively.
As a conclusion, phen, dmb and their Rh complexes were found to
be cytotoxic, and showed preferential cytotoxicity in the MESSA/Dx5 cell-line. Exchange of Rh to Ru results in the loss of activity
and selectivity, as it was found in our previous publications for
29,30
ligands with (N,O) donor set.
In line with the results reported for
14,15
these 8-hydroxyquinoline complexes
irreversible arene loss was
found for Ru complexes, and a somewhat higher cell accumulation
was detected in the case of Rh. These findings might contribute to
the different anticancer properties of the Ru and Rh polipyridyl
complexes as well. In case of the Ru complexes, which were
characterized in this work, arene loss was demonstrated even
without an extra ligand (vide supra), which might be responsible for
the observed loss of cytotoxicity in parental and/or resistant cell
lines. Since the methylation of ligands (phen and bpy to neo and
dmb), and the type of the metal centre (Ru or Rh) strongly affect
the toxicity in the MES-SA and the MDR counterpart MES-SA/Dx5
cell lines, further studies were performed in order to better
understand the differences in the solution behaviour of these
complexes. The A2780cis cells showed cross-resistance for these
compounds, however, cells were less cross-resistant for the
complexes of neocuproine, as the RR values indicate.
Deprotonation of ligands and hydrolysis of half-sandwich cations
Hydrolysis of the organometallic cations and the protonation
constants of the ligands influence the solution speciation of metal
complexes. The hydrolytic processes of half-sandwich cations
5
2+
6
2+
6
([Rh( -C5Me5)(H2O)3] , [Ru( -p-cym)(H2O)3]
and [Ru( 2+
45,46
tol)(H2O)3] ) were already investigated in detail earlier.
Proton
dissociation constants of bpy, phen and ethylenediamine (Table 3)
47,48
are also known under various conditions,
and were redetermined herein by pH-potentiometry (see detailed description
of this method in ESI). Due to the limited water solubility of dmb
and neo, only spectrophotometric titrations were feasible for these
ligands in chloride-free medium (I = 0.20 M KNO3) using lower
concentrations.
Table 3 Proton dissociation constants (Ka (H2L) and Ka (HL)) for (N,N) ligands, stability (K [M(arene)(L)]), proton dissociation (Ka [M(arene)(L)]) and waterchloride exchange (K’ (H2O/Cl-)) constants of complexes of phen, neo, bpy, dmb and en. {T = 25.0 °C; I = 0.20 M (KNO3)}
M(arene)
Constant
pKa(HL)
Rh(5-C5Me5)
Ru(6-p-cym)
Ru(6-tol)
log K [M(arene)(L)]
pKa [M(arene)(L)]
log K’ (H2O/Cl-)
log K [M(arene)(L)]
pKa [M(arene)(L)]
log K’ (H2O/Cl-)
log K [M(arene)(L)]
pKa [M(arene)(L)]
log K’ (H2O/Cl-)
phen
4.92
neo
a
bpy
5.77(1)
c
14.70(3)
8.58a
2.92a
>12.8
7.59(1)f
1.79(1)g
>13.0
7.39(1)f
1.68(1)g
d
9.70(3)
8.88(1)e
2.76(1)g
8.21(4)e
7.62(1)e
0.93(1)g
8.19(8)e
7.55(1)e
0.87(1)g
4.41
dmb
b
5.31(1)
c
13.30(2)
8.61b
2.58b
>12.5
7.48(1)f
1.83(1)g
>13.0
7.39(1)f
1.62(1)g
This journal is © The Royal Society of Chemistry 20xx
14.32(2)c
8.40(1)f
2.36(1)g
>12.8
7.55(1)f
2.02(1)g
>13.2
7.47(1)f
1.88(1)g
en
H2L: 7.25,
HL: 10.01b
15.04b
9.58b
2.14b
14.85(5)h
8.14(2)e
1.51(5)g
14.90(6)h
8.04(2)e
1.69(5)g
J. Name., 2013, 00, 1-3 | 5
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a
Journal Name
b
c 1
d1
e1
f
See Ref. 22. See Ref. 49. H NMR, displacement measurements. H NMR, pH = 0.7-2.1. H NMR titrations, pH = 2.0-11.5. UV-Vis titration, pH = 2g
h1
11.5. UV-Vis, c(Cl )=0.0-0.2 M. H NMR, c(M(arene)) = 100 M, c(ethylenediamine) = 0-7.3 mM.
Based on the single-crystal X-ray structures and previous solution
3,13,22,49
equilibrium studies on similar complexes,
these bidentate
(N,N) donor ligands form mono complexes with half-sandwich
organometallic triaqua cations. Scheme S2 shows the occurring
equilibrium processes in aqueous solutions for these complexes.
Although the complex formation equilibrium is reached with (O,O)
donors within minutes, hours or days are needed in case of the
22
(N,N) donors. Complex formation rates at pH = 0.7 and 2.1 are
6
compared in Figures S31-S33, which show that the [Ru( 2+
5
arene)(H2O)3] triaqua cations react much slower than [Rh( 2+
C5Me5)(H2O)3] with the same ligand (h versus min). Generally, it
can be concluded that the reaction rate is lower at more acidic pH.
However, the pH cannot be increased beyond a certain limit to
accelerate the reaction, since the hydrolysis of the organometallic
cations produces inert hydroxido complexes.
Complexes of bpy, dmb and phen are present in aqueous solutions
at pH = 2 and 0.7 exclusively, there is no sign of unbound
organometallic cation or ligand under the given conditions (I =
1
0.20 M KNO3, aqueous solution) based on the H NMR spectroscopic
measurements. (Notably, pH 0.7 is the lowest pH where the ionic
strength can be kept constant.) With decreasing concentrations
6
2+
6
(from 1.2 mM to 20 M) of [Ru( -tol)(bpy)(H2O)] and [Ru( -p2+
cym)(phen)(H2O)] , the molar UV-Vis spectra remain unchanged
(Figure S34), which also confirmed the high stability of the
complexes. Since measurements with decreasing pH (down to 0.7)
or concentration (down to 20 M) do not give any information
about the stability constants owing to the lack of complex
dissociation, displacement studies were performed with
6
ethylenediamine. For this purpose, stability constants for [Ru( 2+
arene)(ethylenediamine)(H2O)] were determined (the constant of
5
2+
49
[Rh( -C5Me5)(ethylenediamine)(H2O)] was reported earlier).
Samples were prepared at pH = 3.0 using different
ethylenediamine-to-Ru ratios (up to 14-fold ligand excess) and
1
measured by H NMR spectroscopy. Figure S35 shows extremely
slow complex formation under the conditions used.
When ~80-fold excess of ethylenediamine was used, signals of
1
mixed ligand complexes appeared in the H NMR spectra instead of
5
the free polypyridine ligand. Figure S36 shows examples for [Rh( 2+
6
2+
C5Me5)(bpy)(H2O)] – ethylenediamine and [Ru( -tol)(bpy)(H2O)]
systems, in which two peaks indicated the monodentate
coordination of ethylenediamine. This ternary complex formation
process hindered the determination of the stability constant.
HQCl-Pro/bpy
22.5
15.5
9.2
4.2
1.1
I.
II.
III.
9.1
b)
Ratio of bound
Determination of formation constants for complexes
2+
[M(arene)(N,N)(H2O)]
a)
of bound HQClRatio HQCl-Pro
(%)
Considering the different ionic strengths, values are in good
47,48
agreement with literature data.
We find that methylation in
ortho and in para positions increases the pKa. Based on the pKa
values it can be concluded that the polypyridyl ligands are mainly in
their neutral form at physiological pH.
* *
* *
* *
* *
8.6
8.1
(ppm)
7.6
1.7
75
50
25
0
0
10
20
c(HQCl-Pro)/c(bpy)
Fig. 3 a) Selected 1H NMR spectra of the [Rh(5-C5Me5)(bpy)(H2O)]2+-HQClPro (1:x) system at different HQCl-Pro concentrations (x = 0-22.5). Intensity
is decreased in < 2.0 ppm region for the sake of clarity. I.: spectrum of
[Rh(5-C5Me5)(bpy)(H2O)]2+; II.: spectrum of unbound bpy at pH 6.19; III.:
spectrum of [Rh(5-C5Me5)(L)(H2O)]+, where L is the deprotonated
(coordinated) form of HQCl-Pro. Peaks signed with * belong to the unbound
HQCl-Pro; ■ shows the peak of bpy used for calculations; dashed rectangle
shows
[Rh(5-C5Me5)(L)(H2O)]+;
solid
rectangle
shows
[Rh(52+
C5Me5)(bpy)(H2O)] . b) Measured and fitted (dashed line) percentages of
[Rh(5-C5Me5)(L)(H2O)]+; ■ is calculated from integrals of unbound-bound
bpy peaks, ◊ is calculated from C5Me5 peaks. {c([Rh(5-C5Me5)(H2O)3]2+) =
c(bpy) = 200 M; c(HQCl-Pro) = 0-4.53 mM; pH = 6.51; solvent: 90% H2O /
10% D2O; T = 25.0 °C; I = 0.20 M (KNO3)}
An
8-hydroxyquinoline
derivate
((S)-5-chloro-7-((proline-130
yl)methyl)8-hydroxyquinoline (HQCl-Pro))
was used for the
competition experiments, however, its use was limited only to the
Rh-containing compounds, as the Ru complexes seem to lose the
arene ligands when a competitor ligand is added. A successful
displacement of bpy from its Rh complex is shown in Figure 3, at
22.5-fold excess of HQCl-Pro ~70% of the liberated bpy. From this
data a log K = 13.30(2) was calculated (Table 3). Bpy was employed
for the displacement of dmb and phen ligands. In these
1
measurements H NMR spectroscopy was used, since the peak
separation of these similar compounds is satisfactory on NMR
spectra (Figures S37 and S38). To determine the stability constants
of the Ru complexes, we attempted to study a potential
displacement between the organometallic cations. Unfortunately,
5
2+
there was no reaction between [Rh( -C5Me5)(polypyridine)(H2O)]
6
2+
and [Ru( -p-cym)(H2O)3] , even at 10-fold excess of the latter. In
all, only minimum values could be provided (Table 3).
6 | J. Name., 2012, 00, 1-3
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The behaviour of neocuproine complexes is different due to the
steric effect of the methyl groups next to the donor atoms, namely
the complex formation reaction is much slower as compared with
6
2+
phen. Samples containing [Ru( -arene)(H2O)3] and neocuproine
1
in 1:1.2 ratio were followed by H NMR spectroscopy for 15 days
(Figure 4.a). An extra set of peaks belongs to an intermediate
complex which is most probably a sandwich-type complex is
(Figure 4.b,c). The solution stability of metal complexes also
changed compared to phen. Free neocuproine occurs next to its Rh
complex (pH = 0.7, ~50%, Figure S39.a) in the equilibrium, as well as
next to the Ru complexes (pH = 2.5, ~15%, Figures 5.a and S40.a).
Based on the samples measured for samples with acidic pH, molar
fractions for bound and unbound neocuproine can be calculated
and used for the determination of stability constants (Figure S39
shows this in case of Rh). The Ru-containing complexes have smaller
stability constants (Table 3). The lower complex stability constants
2+
and the stronger bias of [Ru(arene)(H2O)3] towards hydrolysis
2+
ensure the lower aqueous stability of [Ru(arene)(neo)(H2O)]
5
2+
complexes compared to [Rh( -C5Me5)(H2O)3] . The lower stability
may originate from the shorter distance between the Ru metal ion
and arene ligand compared with the Rh‒C5Me5 distance (1.697 Å vs.
1.780 Å), which may cause greater steric repulsion around the Ru
centre. To quantify this steric repulsion, the overlaps between
38
ligand solid angles (calculated by Olex2 ) were listed in the legend
of Figure S29. The bigger the overlap, the less the aqueous stability
of complex.
a)
15 d
8d
4.5 d
9h
8.7
8.4
8.1
7.8
7.5
(ppm)
c)
b)
0.04
0.26
0.51
0.55
Fig. 4 a) Time-dependence of complex formation of [Ru(6-tol)(H2O)3]2+ with
neo at pH = 6.0 followed by 1H NMR spectroscopy (only region of ligand is
shown). Time of reaction is shown on the left. Assignment: bidentate metal
complex: ■, free neo: ♠, ‘sandwich intermediate’: ♥. Arrows show the
unusually big shifts of ‘sandwich intermediate’ from free ligand, which
differences are shown in b) (in ppm). c) Proposed structure of the ‘sandwich
intermediate’. {c([Ru(6-tol)(H2O)3]2+) = 200 M; c(neo) = 245 M; pH= 6.0
(20 mM phosphate); I = 0.20 M (KNO3); T = 25.0°C}
Comparison of the stability constants of Rh complexes is not
feasible due to the different basicity of the ligands. The derived
stability constants are calculated as the equation shows below:
2+
log K* = log K [M(arene)(L)(H2O)] ‒ pKa(HL)
5
2+
With this transformation, the log K* [Rh( -C5Me5)(N,N)(H2O)]
values show the following trend: 9.78 (phen) > 9.01 (dmb) > 8.89
(bpy) > 3.93 (neo). While methylation far from the coordinating
nitrogen atoms (bpy vs. dmb) causes slight difference, the
methylation next to the coordinating nitrogen atoms results in a
huge difference. However, except neocuproine, there are no
significant differences in the stability of these Rh complexes. The
5
loss of preferential cytotoxicity in MES-SA/Dx5 cells of [Rh( 2+
C5Me5)(neo)(H2O)] can be partly explained by the probable
dissociation of neo-complex in the cell. However, the small stability
difference between bpy and dmb cannot be the reason of the
different behaviour against MDR cell lines. Moreover, the question
arises whether the reactivity of the coordinated water molecule can
be tuned using steric control of a bulky bidentate ligand.
Reactions of the coordinated water molecule: deprotonation and
substitution to chloride ion
In half-sandwich complexes, next to the arene hapto ligand and a
bidentate (N,N) donor ligand, the coordination sphere is completed
by a water molecule, which can either lose a proton or can be
substituted by another ligand (for example Cl in Scheme S2). Chen
and co-workers found that the reaction of RAED complexes with
nucleobases is slower at higher pH or in the presence of chloride or
50
phosphate anions. At higher pH, deprotonation occurs, which is
characterized by the pKa[M(arene)(L)] constant. With the
knowledge of pKa values one can calculate not only the actual
average charge (between +2 and +1) of the compound at a given
pH, which has an effect also on lipophilicity, but also the molar
+
fraction of the generally less reactive [M(arene)(L)(OH)] mixed
hydroxido complex.
Increasing the pH, the effect of the deprotonation process on the
UV-Vis spectra is unambiguous, as the pH-dependent spectra
6
2+
(Figure 5) of the example ([Ru( -tol)(dmb)(H2O)] ) shows. From
the absorbance change pKa values can be computed. Determination
1
of this constant is also feasible by performing H NMR titrations,
where the changes of the chemical shifts are used for calculations.
6
In a previously reported article the pKa of [Ru( -p2+
42
cym)(phen)(H2O)] was determined as 7.32 in pure D2O, which is
in good agreement with the constant reported here. This type of
6
2+
measurement was used in the case of [Ru( -p-cym)(neo)(H2O)]
and interestingly, in the region of aromatic p-cymene protons a
singlet appears, which belongs to the arene in the complex
(Figure 6). This peak belongs to protons, which are generally not
magnetically equivalent in other complexes. As the pH increases,
this singlet splits into two doublets.
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 7
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-
significantly low constants: log K’(H2O/Cl ) = 1.79→0.93 and
6
1.68→0.87 (Table 3). The Cl -dependent spectra of [Ru( -p2+
cym)(neo)(H2O)] show smaller spectral changes (A~0.05) in
a)
pH = 9.9
0.4
0.2
pH = 5.1
pH
Absorbance
a) 0.6
0.0
330
380
480
0.45
*
**
*
7.6
*
8.7
b)
0.35
*
**
8.2
5.8
5.4
(ppm)
7.7
6.1 5.8 5.5 5.2
(ppm)
0.15
5.0
6.5
8.0
9.5
pH
Fig. 5 a) UV-Vis spectra of the [Ru(6-tol)(dmb)(H2O)]2+ recorded at pH = 5.19.9. b) Absorbance change at 364 nm in dependence of the pH. {c([Ru(6tol)(dmb)(H2O)]2+) = c(dmb) = 200 M; I = 0.20 M (KNO3); ℓ = 1 cm; T=25.0°C}
As shown in Table 3, methylation of the polypyridine ligands has an
influence on the deprotonation of the coordinated water molecule.
Namely, the methyl groups increase the pKa values of the
complexes in all cases. Ru complexes have stronger OH affinity than
the Rh analogues, as the lower pKa values indicate it. Due to the
+
lower complex stability, at physiological pH [(Ru(arene))2(OH)3]
2+
also appears in the case of [Ru(arene)(neo)(H2O)] complexes.
Chloride ions can potentially replace the coordinating water
molecule, as shown in the single-crystal XRD structures (Figure 1),
and this process is characterized by the water-chloride exchange
constant (K’(H2O/Cl )). Cl is present in aqueous solutions, e.g. in
biofluids, where the concentration drops from 103 mM to 24 mM
and 4 mM, entering from the blood serum to cytoplasm and
51
nucleus. The coordination of Cl affects the actual charge and the
presence of chloride ion in medium can suppress the deprotonation
49
process of the complexes to a more basic pH region. The Cl
5
2+
5
affinity
of
[Rh( -C5Me5)(phen)(H2O)]
and
[Rh( 2+
22,49
C5Me5)(bpy)(H2O)] was already reported by our group.
Upon
addition of chloride ions, changes similar to deprotonation can be
6
observed in the UV-Vis spectra (as shown for [Ru( 2+
tol)(phen)(H2O)] in Figure 7.a). From the spectral changes the
water-chloride exchange constants were calculated (Table 3).
Methylation of the ligands has an effect on the water-chloride
exchange constant as well. While for Rh complexes only a slight
decrease can be seen, the effect for Ru complexes is remarkable.
The Cl affinity of dmb complexes is higher than that of the bpy
complexes. The Ru(II)-arene complexes of neocuproine have
Ratio of bound
M(arene) (%)
100
0.25
95
90
85
2.5
3.5
4.5
5.5
pH
c)
(CH, p-cym) (ppm)
364
Absorbance
nm
Absorbance
atat
364
430
(nm)
b)
11.40
9.90
8.97
7.91
7.47
7.18
6.68
5.82
3.08
2.04
6.3
6.0
5.7
2
6
10
pH1
Fig. 6 a) Aromatic region of selected H NMR spectra of the [Ru(6-pcym)(neo)(H2O)]2+ system recorded at pH = 2.0-11.4 after 34 days. Peaks of
complex I are signed with *. Appearance of ‘sandwich intermediate’ is
shown in the inset. b) Measured (neo H at position 5 and 6: □; all protons of
ligand: ×) and fitted (dashed line) ratio of formed complex. c) Measured (○)
and fitted (dashed line) chemical shift values of aromatic p-cymene protons
at different pH-values. {c([Ru(6-p-cym)(H2O)3]2+) = c(neo) = 500 M; solvent:
90% H2O / 10% D2O; T = 25.0°C; I = 0.20 M (KNO3); c(phosphate) = 20 mM}
Figure S40.b. Most probably a steric repulsion is the reason. The
methyl groups of neo hinder the strong interaction between the
chloride ion and Ru.
2+
For [Ru(arene)(ethylenediamine)(H2O)]
complexes, a larger
uncertainty can be seen in the constants. This is in connection with
the phosphate ion coordination to RAED complexes based on the
8 | J. Name., 2012, 00, 1-3
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ARTICLE
abovementioned work of Chen et al.,
showed in details in Figures S41-S42.
50
and on our measurements
constants even for Ru(arene) complexes were integrated (except
for neo complexes because of the strong steric effect on this
Figure 7.c shows the ratio of chlorinated and aqua forms of
complexes in the solution at different chloride ion concentrations.
a)
2.5
1.2
log K'(H2O/Cl-)
c(Cl-)=0.08 M
0.6
c(Cl-)=0.00 M
0
300
350
R² = 0.6659
0.67
Ru(6-p-cym)
1.5
R² = 0.86
Ru(6-tol)
R² = 0.58
0.60
0.5
7
0.50
0
mM
44mM
44mM
mM
0.03
0.06
24
24mM
mM
24
mM
c(Cl-) (M)
c)
8
9
pKa[M(arene)(L)]
10
b)
6-tol)(neo)(H O)]2+
0.40
[Ru(
2
0.09
5-C
++ +
5-C
5-C
[Rh(
[Rh(
[Rh(
5Me
5)(phen)Cl]
5Me
5)(phen)Cl]
5Me
5)(phen)Cl]
5-C
2+
5-C
5-C
2+2+
[Rh(
[Rh(
[Rh(
5Me
5)(phen)(H
2O)]
5Me
5)(phen)(H
2O)]
5Me
5)(phen)(H
2O)]
100mM
mM
100
mM
Chlorinated form 100
100
mM
-)- -)
c(Cl
c(Cl
c(Cl
)
c
c(Cl---))
c(Cl
c(Cl )
Aqua species
[Rh(5-C5Me5)(phen)Cl]+
100%
450
(nm)
b)
nm
Absorbance
346
346
Absorbanceatat
400
Rh(5-C5Me5)
75%
50%
25%
Distribution of M(arene)(L)
mM
44mM
44mM
mM
2+
6-tol)(neo)(HO)]
2+ 2+
66-tol)(neo)(H
[Ru(
-tol)(neo)(H
[Ru(
[Ru(
2 2O)]
2O)]
24mM
mM
24
24
mM
++
6-tol)(neo)Cl]
[Ru(6-tol)(neo)Cl]
100mM
mM [Ru(
100
mM
Chlorinated
form
100
mM
100
0%
0%
0%
0%
25% 50%
50% 75%
75% 100%
100%
25%
50%
75%
100%
25%
Distribution
of
M(arene)(L)
Distribution
ofM(arene)(L)
M(arene)(L)
Distribution
of
Calculated log K'(H2O/Cl-)
Absorbance
a) 1.8
-0.24 log [(M(arene))2H-3]−0.63 pKa+4.56=log K’(H2O/Cl-)
2.5
R2=0,76
neo complexes
1.5
0.5
0.5
Fig. 7 a) UV-Vis spectra of [Ru(6-tol)(phen)(H2O)]2+ recorded at various
chloride ion concentrations. b) Absorbance change at 346 nm plotted
against the concentration of the chloride ion. c) Ratio of the aqua and
chlorinated forms of complexes (c = 200 M) with the highest and lowest
chloride affinity measured in the studied group of compounds. Chloride
concentrations are representing the different biofluids. Constants from
Table 3 were used for calculation. {c([Ru(6-tol)(H2O)3]2+) = c(phen)
= 182 M; pH = 6.0; ℓ = 1 cm; T = 25.0°C}
Our calculations show the contrasting behaviour of two complexes,
which possess the highest and the lowest chloride ion affinity
5
2+
6
2+
([Rh( -C5Me5)(phen)(H2O)]
and
[Ru( -tol)(neo)(H2O)] ,
respectively). As seen in Figure 7.c, the fraction of the chlorinated
form of the neo complex is minimal at c(Cl ) = 4 mM, and it
5
increases to 43% at c(Cl ) = 100 mM. On the contrary, the [Rh( 2+
C5Me5)(phen)(H2O)] complex shows an opposite behaviour, as the
chlorinated form predominates under all considered chloride ion
concentrations.
The importance of the knowledge of chloride ion affinity was
51
already mentioned above and in previous reviews. In our earlier
work, a linear relationship between the pKa[M(arene)(L)] and the
5
log K’(H2O/Cl ) constants was found for Rh( -C5Me5) complexes
with (O,O), (O,N), (N,N) and (O,S) donor bidentate ligands. To
complete this model, already published and the newly determined
1.5
2.5
Measured log K'(H2O/Cl-)
Fig. 8 a) The measured log K’(H2O/Cl-) values of [M(arene)(L)] complexes in
the function of their pKa[M(arene)(L)] constants (I = 0.20 M (KNO3)). b)
Multilinear regression for calculating log K’(H2O/Cl-) based on the shown
equation. Used data are collected in Table S6.22,29,30,45,46,49,52-56 Outlier points
are circled.
constant; all used data are summarized in Table S6). The displayed
constants (Figure 8.a) are in three different groups with their own
fitting lines. However, applying multilinear regression and the
log [(M(arene))2H-3] constants arranged the values in one group
(Figure 8.b). The equation of this regression is:
-0.24×log [(M(arene))2H-3] − 0.63×pKa[M(arene)(L)] + 4.56 = log K’(H2O/Cl-)
The first term of this equation takes the M(arene) part into
consideration, while the second term describes the effect of
bidentate ligand. This model is universal, as it can predict the
chloride ion affinity of different half-sandwich M(arene) complexes,
if the log β[(M(arene))2H‒3] and pKa[M(arene)(L)] constants are
known. The main drawback is that only chloride ion free
pKa[M(arene)(L)] values are acceptable in this model.
After determining those constants, which govern the speciation in
solution, we can compare them and find connections with
cytotoxicity. In Figure 9 the basicity corrected stability constant, the
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 9
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log K*
pKa[M(arene)(L)] values, the chloride ion affinity, the cytotoxicity
against MES-SA cell line and resistance ratios in the same cell lines
are shown for all complexes.
10
5
pKa
7
3
2
1
0
6
pIC50
RR
(MES-SA) (MES-SA)
8
log K’
0
9
5
4
neo
bpy
neo
neo
phen
bpy
dmb
dmb
<4
<4
bpy dmb phen
phen
<4
<4
<4
n.d.
n.d.
n.d.
4.2
4
2
1.9
0.7
0.39
0.25
2
1.75
1.59
n.d.
0
Rh( 5-C5Me5) Ru( 6-p-cym)
Ru( 6-tol)
Fig. 9 Comparison of the determined constants: basicity corrected stability
constants (log K*), proton dissociation constants of coordinated water
molecules (pKa), water-chloride exchange constants (log K’), the cytotoxicity
in MES-SA cell lines (pIC50) and resistance ratio in MES-SA cell lines. N.d.=not
determined.
It is clearly seen that methylation of ligand improves anticancer
activity. Although neocuproine complexes have the highest
cytotoxicity, they have lower stability as compared to the other
complexes, suggesting that they might be ligand carriers. High6
stability complexes of Ru( -tol) are likely to lose their arene
ligands. This process can explain the loss of cytotoxicity and may
6
occur for Ru( -p-cym) complexes as well. The MDR-selective
complexes show the highest complex stability from all, combined
with high pKa and high chloride ion binding capability. However, the
difference between these MDR-selective complexes and the non5
2+
selective [Rh( -C5Me5)(bpy)(H2O)]
complex is not evident
because of the slight differences in the respective constants.
Together these data suggest that further factors should be
considered, such as lipophilicity, redox potential or a specific
44
interaction with a bio-ligand.
Conclusions
6
2+
6
Complexes of the half-sandwich [Ru( -p-cym)(H2O)3] , [Ru( 2+
5
2+
tol)(H2O)3] and [Rh( -C5Me5)(H2O)3] organometallic cations
formed with polypiridyl ligands were compared regarding their
differences in structural, cytotoxic and aqueous solution behaviour.
The structurally related bpy, dmb, phen and neo and their
complexes were examined to investigate the effect of methylation
and complexation of the ligands. Synthesis of complexes was
performed in moderate-to-excellent yields. Based on the X-ray
crystallographically determined structures, the change from pcymene to toluene has only a negligible effect on the complex
structure, while in the investigated Rh complexes, longer bonds
between the bidentate ligands and the metal centre could be
found. The methyl groups distorted the structure, when they are
present next to the coordinating atoms, namely there is an angle
between the plane of the arene and the plane of bidentate ligand.
The polypyridines and their complexes show anticancer activity in
A2780 and MES-SA cancer cell lines; however, in A2780cis cell lines
they have reduced effect. Phen and dmb and their half-sandwich Rh
complexes showed paradoxical toxicity against multidrug resistant
MES-SA/Dx5 cells. Interestingly, in all cases coordination to Ru
caused a loss of activity and selectivity of these ligands.
Generally,
metal
complexes
with
the
structure
2+
[M(arene)(N,N)(H2O)] show high stability, although stability
constants could be determined only for Rh complexes by ligand
competition studies. In case of neocuproine, the methyl groups next
to the nitrogen atoms lower the stability of complexes due to steric
congestion with the arene. This is clearly shown, when log K*
(stability constant in which the different basicity of the ligand is
taken into account) of complexes are compared with each other:
9.78 (phen) > 9.01 (dmb) > 8.89 (bpy) > 3.93 (neo). The pKa value of
coordinated water molecule is between 7.39-7.62 for Ru and 8.408.88 for Rh compounds; thus, methylation has a small effect on this
constant. Methyl groups have a great effect on the K’(H2O/Cl )
constants of Ru compounds containing neocuproine, decreasing
them with one order of magnitude compared to 1,10-phenantroline
(log K’(H2O/Cl ) = 1.79 vs. 0.93 for p-cymene and 1.68 vs. 0.87 for
toluene complexes), which is also in connection with steric
hindrance.
A correlation was made between the hydrolytic properties and the
water-chloride exchange constants, which is universal for Rh and Ru
complexes as well. No direct correlation was seen between the
determined equilibrium constants (stability, proton dissociation and
chloride/water exchange constants) of these half-sandwich
complexes and cytotoxicity. The studied complexes are highly
stable, thus the liberation of the bidentate ligands is not likely
(except for the neocuproine complexes). The loss of the cytotoxicity
of the Ru complexes is suggested to be connected to the slow and
irreversible decomposition processes, in which the half-sandwich
structure of these Ru(II) complexes is destroyed due to the arene
loss, while the bidentate ligand remains coordinated.
Experimental
Chemicals
All solvents were of analytical grade and used without further
purification. 1,10-phenanthroline, neocuproine, 2,2’-bipyridine,
5
4,4’-dimethyl-2,2’-bipyridine, ethylenediamine, [Rh( -C5Me5)(6
Cl)Cl]2, [Ru( -p-cym)(-Cl)Cl]2, RuCl3 × 3 H2O, 1-fmethyl-1,4cyclohexadiene, doxorubicin, cisplatin, KCl, AgNO3, Ag(CF3SO3),
HNO3, KOH, 4,4-dimethyl-4-silapentane-1-sulfonic acid (DSS),
10 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
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ARTICLE
Dulbecco’s Modified Eagle Medium (DMEM), RPMI 1640 with FBS,
NaH2PO4, Na2HPO4 and KH2PO4 were purchased from Sigma-Aldrich
in puriss quality. The P-gp inhibitor tariquidar is from Dr. S. Bates
(NCI NIH). Ultrapure Milli-Q water was used for sample preparation.
[M(arene)(N,N)Cl]Cl complexes were synthesized as previously
11,31-33
described.
Synthesis, yields and characterization is described
in the Electronic Supplementary Information as well as
characterization methods in details (ESI-MS, NMR and single crystal
57-62
6
X-ray crystallography
). [Ru( -tol)(-Cl)Cl]2 was prepared
63
according to literature procedures, as well as 5-chloro-7-(1-L30
prolinylmethyl)-8-hydroxyquinoline.
The exact concentration of the ligand stock solutions together with
the proton dissociation constants were determined by pHpotentiometric titrations with the use of the computer program
64
65
Hyperquad2013, , Irving method and the water ionization
66
constant (Kw = 13.76). Detailed description of pH-potentiometry
5
can be found in ESI. The aqueous [Rh( -C5Me5)(H2O)3](NO3)2,
6
6
[Ru( -p-cym)(H2O)3](NO3)2 and [Ru( -tol)(H2O)3](NO3)2 stock
solutions were obtained by dissolving an exact amount of the
dimeric precursor in water followed by addition of equivalent
amounts of AgNO3 and filtration of AgCl precipitate. The exact
concentrations of chloride-free metal ion stock solutions were
determined by pH-potentiometric titrations employing stability
5
(4-i)+
6
(4-i)+
constants for [(Rh( -C5Me5))2(-OH)i] , [(Ru( -tol))2(-OH)i]
6
(4-i)+
45,46
and [(Ru( -p-cym))2(-OH)i]
(i = 2 or 3) complexes.
Stock
solutions of dmb and neo were prepared with HNO 3 to increase
solubility, exact concentrations were calculated from the weight-involume basis.
The buffered samples were prepared in 20 mM phosphate buffer or
in a modified phosphate buffered saline (PBS’) at pH 7.40. PBS’
contains 12 mM Na2HPO4, 3 mM KH2PO4, 1.5 mM KCl and 100.5
+
+
mM NaCl; and the concentration of the K , Na and Cl ions
corresponds to that of the human blood serum. Phosphate is the
best choice for the pH range 6.0-7.4 because mostly it does not
coordinate to these metal ions (except RAED complexes vide supra).
1
UV-Vis spectrophotometric and H NMR specroscopy
An Agilent Cary 8454 diode array spectrophotometer was used to
record the UV-Vis spectra in the interval 200–800 nm. The path
length was 0.5 or 1 cm. Only one of the proton dissociation
constants of neo and dmb could be determined by
spectrophotometric titrations. Complex formation kinetics was
investigated with the use of tandem cuvette. Deprotonation of
coordinated water molecule in complexes was followed by
spectrophotometry. UV-Vis spectra were used to investigate the
−
H2O/Cl exchange processes of complexes at 200 M concentration
(500 M for en complexes), around pH 6.0 (20 mM phosphate
buffer) as a function of chloride concentrations (0–310 mM).
NMR spectroscopic studies were carried out on a Bruker Avance III
1
HD Ascend 500 Plus instrument. For aqueous samples H NMR
spectra were recorded with the WATERGATE water suppression
pulse scheme using DSS internal standard. Deprotonation of
coordinated water molecule in complexes was also followed by
1
H NMR. Samples were made in a 10% (v/v) D2O/H2O mixture and
were titrated at 25.0 °C, at I = 0.20 M (KNO3) at 1:1 metal-to-ligand
ratio. The slower kinetic measurements were checked by NMR to
see the endpoint of complex formation. Stability constants for the
complexes of ethylenediamine and neocuproine were calculated by
67
1
the computer program PSEQUAD based on H NMR spectra.
In vitro cell studies
Cell lines and culture conditions
The human ovarian cancer cell line A2780 and its cisplatin resistant
(A2780cis) counterpart, human uterine sarcoma cell lines MES-SA
and the doxorubicin selected MES-SA/Dx5 were obtained from
ATCC (American Type Culture Collection) (MES-SA: No. CRL-1976™,
MES-SA-MES-SA/Dx5: No. CRL-1977™). The phenotype of the
resistant cells was verified using cytotoxicity assays (Tables 1, 2 and
S5, doxorubicin and cisplatin). Cells were cultivated in Dulbecco’s
Modified Eagle Medium (DMEM, Sigma-Aldrich) and supplemented
with 10% fetal bovine serum, 5 mM glutamine, and 50 units per mL
penicillin and streptomycin (Life Technologies). All cell lines were
cultivated at 37 °C under a humidified atmosphere containing 95%
air and 5% CO2.
Cell viability assay
Cytotoxic effects were determined by the colorimetric microculture
68
MTT assay.
Cells were harvested from culture flasks by
trypsinization, seeded in 100 μL aliquots into 96-well microculture
plates (Sarstedt, Newton, USA) at 5000 cells per well and allowed to
settle and resume exponential growth in drug-free complete culture
medium for 12 h to 24 h. Ligands and complexes were diluted in
complete culture medium and added to the plates. The complexes
of the ligands were prepared in situ by mixing the ligand with an
equimolar concentration of the organometallic cations using their
5
stock solutions containing known amounts of [Rh(η 2+
6
2+
6
C5Me5)(H2O)3] ,
[Ru(η -p-cymene)(H2O)3]
and
[Ru(η 2+
toluene)(H2O)3] . Following the addition of the serial dilutions of
ligands and complexes and an incubation period of 72 h, the
supernatant was removed and fresh medium supplemented with
the MTT reagent (0.83 mg/mL) was added. Incubation with MTT at
37 °C was terminated after 1 h by removing the supernatants and
lysing the cells with 100 μL DMSO per well. Viability of the cells was
measured spectrophotometrically by absorbance at 540 nm using
an EnSpire microplate reader. Data were background corrected by
subtraction of the signal obtained from unstained cell lysates and
normalized to untreated cells. Curves were fitted with the Prism
69
software using the sigmoidal dose–response model (comparing
variable and fixed slopes). Curve fit statistics were used to
determine the concentration of the test compound that resulted in
50% toxicity (IC50). Evaluation is based on means from three
independent experiments, each comprising three replicates per
each concentration. Co-incubation experiments were also
performed in the presence of the P-gp inhibitor tariquidar.
Doxorubicin and cisplatin were used as positive controls.
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 11
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Journal Name
This work was supported by the National Research, Development
and Innovation Office-NKFIA through projects GINOP-2.3.2-152016-00038, FK 124240, PD 128504, KH129588, 2018-1.2.1-NKP2018-00005 (financed under the 2018-1.2.1-NKP funding scheme)
and Ministry of Human Capacities, Hungary grant, TKP-2020. GS and
EAE were supported by a Momentum Grant of the Hungarian
Academy of Sciences.
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