Multinuclear ruthenium(II) complexes as anticancer agents

Article abstract of DOI:10.1039/c4nj00545g

A series of dinuclear ruthenium(ii) complexes that contain labile chlorido ligands, [{Ru(tpy)Cl}₂{μ-bb_n}]²⁺ {designated Cl-Rubb_n; tpy = 2,2′:6′,2′′-terpyridine, bb_n = bis[4(4′-methyl-2,2′-bipyridyl)]-1,n-alkane (n = 7, 10, 12, 14 or 16)} and derivatives containing nitro substituents on the tpy ligand and/or secondary amines within the bb_n linking chain have been synthesised and their potential as anticancer agents examined. Some of the Cl-Rubb_n species showed good anticancer activity against MCF-7 and MDA-MB-231 breast cancer cell lines, with the Cl-Rubb₁₂ complex being four-times more active than cisplatin. Inclusion of nitro substituents on the tpy ligands of Cl-Rubb₁₂ resulted in significantly decreased anticancer activity. The incorporation of amine groups into the linking ligand did not increase the anticancer activity of the Cl-Rubb_n complexes. The Cl-Rubb_n complexes and those containing amine groups in the linking chain aquated at approximately the same rate, with 50% aquation within 120 minutes. By comparison, the complexes containing nitro substituents on the tpy ligand aquated extremely slowly, with 60% of the chlorido complex remaining 24 hours after they were dissolved in water. Cyclic voltammetry with the model mononuclear complex [Ru{(NO₂)₃tpy}(Me₂bpy)Cl] ⁺ {(NO₂)₃tpy = 4,4′,4′′- trinitro-2,2′:6′,2′′-terpyridine} showed that the nitro substituents exerted a strong effect on the ruthenium centre, with the anodic peak corresponding to the Ru(iii/ii) couple shifted positively by 300 mV compared to that from the non-nitrated parent complex [Ru(tpy)(Me ₂bpy)Cl]⁺. ¹H NMR studies of the reaction of the Cl-Rubb_n complexes with GMP indicated that the ruthenium complexes covalently bound the nucleotide slowly, with 33% bound in 24 hours. However, the results of this study suggest that the cytotoxicity of the dinuclear ruthenium complexes is a combination of covalent and reversible binding with DNA. the Partner Organisations 2014.

Full text of DOI:10.1039/c4nj00545g

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Multinuclear ruthenium(II) complexes as
anticancer agents
Cite this:DOI: 10.1039/c4nj00545g
Anil K. Gorle,a Alaina J. Ammit,b Lynne Wallace,*a F. Richard Keene*cde and
J. Grant Collins*a
A series of dinuclear ruthenium(II) complexes that contain labile chlorido ligands, [{Ru(tpy)Cl}2{m-bbn}]2+
{designated Cl-Rubbn; tpy
= =
2,20:60,200-terpyridine, bbn bis[4(40-methyl-2,20-bipyridyl)]-1,n-alkane
(n = 7, 10, 12, 14 or 16)} and derivatives containing nitro substituents on the tpy ligand and/or secondary
amines within the bbn linking chain have been synthesised and their potential as anticancer agents
examined. Some of the Cl-Rubbn species showed good anticancer activity against MCF-7 and MDA-MB-
231 breast cancer cell lines, with the Cl-Rubb12 complex being four-times more active than cisplatin.
Inclusion of nitro substituents on the tpy ligands of Cl-Rubb12 resulted in significantly decreased
anticancer activity. The incorporation of amine groups into the linking ligand did not increase the
anticancer activity of the Cl-Rubbn complexes. The Cl-Rubbn complexes and those containing amine
groups in the linking chain aquated at approximately the same rate, with 50% aquation within 120 minutes.
By comparison, the complexes containing nitro substituents on the tpy ligand aquated extremely slowly,
with 60% of the chlorido complex remaining 24 hours after they were dissolved in water. Cyclic
voltammetry with the model mononuclear complex [Ru{(NO2)3tpy}(Me2bpy)Cl]+ {(NO2)3tpy = 4,40,400-
trinitro-2,20:60,200-terpyridine} showed that the nitro substituents exerted a strong effect on the ruthenium
centre, with the anodic peak corresponding to the Ru(III/II) couple shifted positively by 300 mV compared
Received (in Victoria, Australia)
10th April 2014,
Accepted 25th May 2014
1
to that from the non-nitrated parent complex [Ru(tpy)(Me2bpy)Cl]+. H NMR studies of the reaction of the
Cl-Rubbn complexes with GMP indicated that the ruthenium complexes covalently bound the nucleotide
slowly, with 33% bound in 24 hours. However, the results of this study suggest that the cytotoxicity of the
dinuclear ruthenium complexes is a combination of covalent and reversible binding with DNA.
DOI: 10.1039/c4nj00545g
www.rsc.org/njc
Multinuclear platinum complexes, where two or more plati-
num coordination units are linked by a variety of organic ligand
Introduction
Although cisplatin has been in clinical use for over 30 years, bridges, represent a genuinely new class of anticancer drug.2
its toxicity and natural/acquired resistance to many cancers While complexes with bi-functional platinum centres have
has considerably limited its application.1 While some second- been reported, those containing mono-functional coordinating
generation platinum complexes are less toxic than cisplatin, spheres on the terminal platinum atoms (e.g. BBR 3005,
and others can partially overcome acquired resistance, there see Fig. 1) gave the most encouraging results.8–11 Furthermore,
has been little success in developing drugs that are active in complexes bearing a cationic charge and hydrogen-bonding
cancer cell lines resistant to cisplatin. Consequently, there has capacity (e.g. amine groups or inert am(m)ineplatinum(II)
been considerable interest in the development of ‘‘non-classical’’ centres) in the linking ligand were shown to be the most active
platinum complexes – complexes that can bind DNA in a in both cisplatin-sensitive and -resistant cell lines.12–20 The
different manner than cisplatin and its analogues.2–7
trinuclear complex BBR 3464, [trans-{PtCl(NH3)2}2-{m-trans-
Pt(NH3)2(H2N(CH2)6NH2)2}]4+, has undergone Phase II clinical
trials,21–23 while dinuclear complexes linked by spermidine
(BBR 3571, see Fig. 1) and spermine (BBR 3610 and BBR
3611) are cytotoxic at nanomolar concentrations.2
While the multinuclear platinum complexes are highly
cytotoxic, they are also highly toxic.13,23–26 Furthermore, upon
administration they bind thiol-containing plasma proteins in
the bloodstream, and are subsequently degraded to non-active
a School of Physical, Environmental and Mathematical Sciences, University of New
South Wales, Australian Defence Force Academy, Canberra, ACT 2600, Australia.
E-mail: l.wallace@adfa.edu.au, g.collins@adfa.edu.au
b Faculty of Pharmacy, The University of Sydney, Sydney, NSW, 2006, Australia
c Centre for Biodiscovery and Molecular Development of Therapeutics,
James Cook University, Townsville, QLD 4811, Australia
d School of Pharmacy and Molecular Sciences, James Cook University, Townsville,
QLD 4811, Australia. E-mail: richard.keene@jcu.edu.au
e School of Chemistry and Physics, University of Adelaide, Adelaide, SA 5005, Australia metabolites. Although BBR 3464 has been withdrawn from
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[{Ru(bpy)2Cl}2{m-BL}]2+ complexes {where bpy = 2,20-bipyridine
and BL = 1,6-diaminohexane or 1,12-diaminododecane} and
examined their cytotoxicity.28 While the chlorido complexes
showed little activity, replacement of the chlorido ligand by DMSO
in the 1,12-diaminododecane-bridged complex resulted in good
activity against L1210 cells.
Corral et al. have recently demonstrated that the mono-
nuclear ruthenium(II) complexes [Ru(apy)(tpy)X]n+ (where apy =
azobis(2-pyridine), tpy = 2,20:60,200-terpyridine and X = a labile
ligand such as Clꢀ or H2O) had good activity against a variety
of cancer cell lines, but were significantly less active than
cisplatin.29 In an attempt to increase the activity of mononuclear
[Ru(tpy)(L)(Cl)]+ complexes (where L = a non-labile bidentate
ligand), we previously synthesised the dinuclear ruthenium
complexes [{Ru(tpy)Cl}2{m-bbn}]2+ {Cl-Rubbn, see Fig. 2; where
bbn = bis[4(40-methyl-2,20-bipyridyl)]-1, n-alkane, for n = 7, 10, 12,
and 14}.30 The Cl-Rubbn complexes showed good activity against
the highly sensitive L1210 cell line (IC50 E 5–10 mM) and were
ten-times more active than the corresponding mononuclear
Fig. 1 Cisplatin, and the structure of
complex (top right) with the linking ligands (Y) shown for BBR 3464, BBR
3005 and BBR 3571.
a generic dinuclear platinum
clinical trials, there has been recent interest in ‘‘transferring the complex [Ru(tpy)(Me2bpy)Cl]+ {Me2bpy
=
4,40-dimethyl-2,20-
concept of multinuclearity to ruthenium complexes’’.27 Mendoza- bipyridine}.30 In this present study we sought to extend the
Ferri et al. synthesised a series of dinuclear ruthenium(II)–arene family of Cl-Rubbn dinuclear complexes by using a similar
compounds containing a bis(pyridinone)alkane linking ligand approach to that of Farrell and co-workers for the multinuclear
that incorporated 3, 6 or 12 methylene groups in the alkane platinum complexes.2,9–11 Consequently, we have synthesised
chain.27 The ruthenium–arene complexes showed good activity in and examined the anticancer activities, rates of hydrolysis, and
a variety of cancer cell lines, with the activity increasing with the binding ability to guanosine 50-monophosphate (GMP) of a
length of the alkane linker, and were more active than a similar series of Cl-Rubbn complexes that contain cationic groups
+
mononuclear analogue. In addition, Yamada et al. synthesised (NH2 ) in the chain of the bbn linking ligand (Cl-RubbNn).
Fig. 2 Chlorido-containing dinuclear ruthenium(II) complexes, top Cl-Rubbn for X = H and Cl-RubbnNO2 for X = NO2, and bottom Cl-RubbNn for
X = H and Cl-RubbNnNO2 for X = NO2.
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Furthermore, in order to determine the effect of changes in Cytotoxicity
charge distribution (and hence, the rate of ligand exchange)
The in vitro cytotoxicities of the ruthenium complexes and the
control platinum complexes cisplatin and carboplatin were
determined against the MCF-7 and MDA-MB-231 breast cancer
cell lines, and the results are summarised in Table 1. Cisplatin
showed moderate cytotoxicity against both cell lines, while
carboplatin was essentially inactive. Although IC50 values
reported for cisplatin against MCF-7 cells can vary consider-
ably, the results obtained for both control platinum complexes
against both cell lines are consistent with previous studies.29,33–35
on the ruthenium(II) complexes, we have prepared several
Cl-Rubbn and Cl-RubbNn complexes that contain three electron-
withdrawing NO2 groups on the tpy ligands (Cl-RubbnNO2 and
Cl-RubbNnNO2).
Results
Synthesis
The synthesis of the mononuclear [Ru(tpy)(bpy)Cl]+ and the The dinuclear ruthenium complexes Cl-Rubbn, for n = 10, 12 and
dinuclear complexes [{Ru(tpy)Cl}2(m-bbn)]2+ (Cl-Rubbn for n = 7, 14 were more active than cisplatin against both cell lines. Inter-
10, 12, 14 and 16) have been previously reported.30,31 In this estingly, Cl-Rubb12 was the most active, with the ruthenium
study, we have extended the family of dinuclear complexes complexes having the shortest linking chain (Cl-Rubb7) and long-
through the synthesis of Cl-RubbnNO2, Cl-RubbNn, and est linking chain (Cl-Rubb16) being the least active. Addition of
Cl-RubbNnNO2 complexes, as shown in Schemes 1–3. For the nitro substituents onto the tpy rings of Cl-Rubb12 and Cl-Rubb16
Cl-RubbNn complexes, the procedure used for the synthesis of decreased the activity of the ruthenium complexes, particularly in
the Cl-Rubbn complexes resulted in poor yield and purity for the case of the highly active Cl-Rubb12. The replacement of two
the Cl-RubbNn complexes. To obtain satisfactory yields the methylene groups by two amine groups in the ligand bridge for
bbNn ligand was dissolved in ethanol–water and heated to Cl-Rubb7 (giving Cl-RubbN7) and Cl-Rubb16 (Cl-RubbN16) decreased
60 1C before the [Ru(tpy)Cl3] was added, and then the mixture the activity of the former but had no effect on the latter complex
refluxed for a longer time period than was necessary for the that contained the longer linking chain. However, it was also noted
synthesis of Cl-Rubbn. [Ru{(NO2)3tpy}Cl3] was prepared in a that the replacement of the Me2bpy ligand in [Ru{(NO2)3tpy}-
similar manner to that previously reported for [Ru(tpy)Cl3],32 (Me2bpy)Cl]+ by the bbN16 ligand to form the mononuclear
and upon addition of 4,40-dimethyl-2,20-bipyridine yielded complex Cl-RubbN16NO2-mono did significantly increase the
[Ru{(NO2)3tpy}(Me2bpy)Cl]Cl in good yield. The synthesis of the activity in both cancer cell lines. In the one example examined,
new chlorido-containing dinuclear complexes Cl-RubbnNO2 and the combination of amine groups in the linking ligand and nitro
Cl-RubbNnNO2 were achieved using similar procedures.
substituents on the tpy ligands for Cl-RubbN16NO2 had little
Scheme 1
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Scheme 2
Scheme 3
Table 1 The IC50 values of the metal complexes against the MCF-7 and
MDA-MB-231 breast cancer cell lines, defined as the concentration (mM) of
the complex required to inhibit cell growth by 50%
in the binding to GMP, a simple model for DNA, is aquation.
Consistent with previous studies,30 aquation of [Ru(tpy)(Me2bpy)Cl]+
was found to be relatively fast, with 50% of the ruthenium complex
being converted to the corresponding aqua form in approximately
60 minutes. Similarly, 50% aquation of each ruthenium centre in
the dinuclear complexes Cl-Rubbn and Cl-RubbNn was shown by
1H NMR spectroscopy to occur in approximately 120 minutes
(see Fig. 3). The aquation then proceeds to equilibrium, where
approximately 90% of the ruthenium complex exists in the aqua
form. The inclusion of amine groups into the linking ligand had no
significant effect on the rate or equilibrium position of aquation.
Fig. 4 shows the 1H NMR spectrum of Cl-RubbN16 as a function
of time after dissolution in D2O and the addition of 2 equivalents
of GMP. After 120 minutes, the spectrum of the Cl-RubbN16 is
essentially identical to that in the absence of GMP, as shown in
Fig. 3, with approximately 50% of the dinuclear complex aquated
but with no covalent binding to GMP observed. As evidenced by
the increasing intensity of the resonance at 5.36 ppm, assigned to
the sugar H10 of GMP bound to a ruthenium centre, the aquated
form of Cl-RubbN16 slowly reacts with GMP, reaching an equili-
brium of approximately 33% bound in 24 hours. Similar results
were obtained with the Cl-Rubbn complexes (results not shown).
Fig. 5 shows the 1H NMR spectrum of [Ru{(NO2)3tpy}-
IC50 (mM)
Metal complex
MCF-7
MDA-MB-231
Cisplatin
Carboplatin
Cl-Rubb7
Cl-Rubb10
Cl-Rubb12
Cl-Rubb14
34 ꢁ 2
273 ꢁ 7
29 ꢁ 4
8 ꢁ 3
31 ꢁ 3
451 ꢁ 8
24 ꢁ 5
14 ꢁ 3
9 ꢁ 4
8 ꢁ 4
7 ꢁ 4
13 ꢁ 1
24 ꢁ 6
105 ꢁ 7
35 ꢁ 4
31 ꢁ 4
35 ꢁ 4
32 ꢁ 2
36 ꢁ 2
26 ꢁ 2
Cl-Rubb16
27 ꢁ 5
48 ꢁ 4
68 ꢁ 3
27 ꢁ 2
42 ꢁ 5
36 ꢁ 2
31 ꢁ 2
27 ꢁ 2
[Ru{(NO2)3tpy}(Me2bpy)Cl]+
Cl-RubbN7
Cl-RubbN16
Cl-Rubb12NO2
Cl-Rubb16NO2
Cl-RubbN16NO2
Cl-RubbN16NO2-mono
effect on the cytotoxicity with the MCF-7 cells but decreased the
activity against the MDA-MB-231 cell line.
Aquation and GMP binding
Previous studies with mononuclear ruthenium(II) complexes
that contain a chlorido ligand have shown that the first step (Me2bpy)Cl]+ at various time points after the ruthenium complex
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Fig. 3 Aromatic region of the 1H NMR spectrum of Cl-RubbN16 in D2O as a function of time, after 5 minutes (A), 120 minutes (B) and 27 hours (C).
The asterisk indicates the decrease in the H6-Me2bpy resonances of the Cl-RubbN16 complex, while the arrow shows the increase in the H6-Me2bpy
resonances from the D2O–RubbN16 complex.
Fig. 4 Aromatic region of the 1H NMR spectrum of the Cl-RubbN16 + GMP in D2O as a function of time, after 10 minutes (A), 120 minutes (B),
450 minutes (C), 25 hours (D) and 76 hours (E). The asterisk indicates the decrease in H6-Me2bpy resonances of the Cl-RubbN16 complex, while the
arrows shows the increase of the peak for the H6-Me2bpy protons of the GMP bound ruthenium complex (8.76 ppm) and the sugar H10 of the bound
GMP (5.36 ppm).
was dissolved in D2O. Unlike the corresponding non-nitrated complex was converted into the aqua form well within 24 hours,
complex [Ru(tpy)(Me2bpy)Cl]+, where 495% of the ruthenium 60% of the [Ru{(NO2)3tpy}(Me2bpy)Cl]+ remained unchanged
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Fig. 5 Aromatic region of the 1H NMR spectrum of [Ru{(NO2)3tpy}(Me2bpy)Cl]+ in CD3OD (A) and in D2O as a function of time, after 30 minutes (B),
4 hours (C) and 24 hours (D). NO2–Cl indicates the non-aquated complex {H30 and H50 of (NO2)3tpy} and NO2–D2O represents the aquated form, while
NH2–Cl {H3 and H300 of (NO2)3tpy} and NH2–D2O represent the putative ‘‘(NO2)2(NH2)-tpy’’ complexes.
Table
2
Electrode potentials for [Ru(L)(Me2bpy)Cl]Cl in acetonitrile
after 24 hours. This indicates that the incorporation of the nitro
substituent on the tpy ligand significantly slowed the aquation
reaction. Even after 216 hours, 25% of the original [Ru{(NO2)3tpy}-
(Me2bpy)Cl]+ remained in the chlorido form. Interestingly how-
ever, 10% of the [Ru{(NO2)3tpy}(Me2bpy)Cl]+ was rapidly converted
(in V vs. Ag/AgCl; working electrode = glassy carbon)
Processa
L = tpy
L = (NO2)3tpy
Oxidation Ea
0.94b
1.28
1.24 (sh)
1.33
into another form after being dissolved. This new complex then Reduction Ec
appeared to slowly aquate. Based upon the observations of
Fallahpour et al.,36 it is proposed that one of the three nitro
substituents on the tpy ligand is reduced to an amine. This new
ꢀ1.36
ꢀ0.40 (sh)
ꢀ0.52 (sh)
ꢀ0.66
ꢀ1.54
ꢀ1.17 (sh)
ꢀ1.45
‘‘(NO2)2(NH2)-tpy’’ complex then slowly aquates.
a
All peaks irreversible unless otherwise stated; potentials are given for
forward peaks; anodic (Ea) for oxidations and cathodic (Ec) for reduc-
tions. Reversible; DEp = 0.90 V.
Cyclic voltammetry of [Ru{(NO2)3tpy}(Me2bpy)Cl]+
b
Electrochemical measurements were carried out on the
[Ru{(NO2)3tpy}(Me2bpy)Cl]+ and [Ru(tpy)(Me2bpy)Cl]+ complexes
to assess the electronic effect of the nitro substituents on the peaks at more negative potentials. Previous work on the electro-
ruthenium centre, and the electrode potentials are listed in chemical behaviour of nitrated bipyridines and their platinum
Table 2.
complexes has shown analogous cathodic behaviour: for example
The electrochemical response of the [Ru(tpy)(Me2bpy)Cl]+ [Pt{4,40-(NO2)2bpy}Cl2] displayed two closely-spaced reductions, and
complex as a hexafluorophosphate salt has previously been the LUMOs for that complex were shown to be localised largely on
investigated;37 the results here are consistent with that report: the ‘‘NO2-py’’ units.38 Further reduction of the complex occurred at
two ligand-based reductions are observed in the cathodic ꢀ1.05 V,39 very close to the potential of ꢀ1.06 V observed for the
region (tpy/tpyꢀ followed by Me2bpy/Me2bpyꢀ), while the ano- first reduction (bpy/bpyꢀ) of the non-nitrated complex [Pt(bpy)Cl2]
dic region shows a reversible Ru(III/II) peak at +0.90 V. In the under the same conditions.38 Based on these observations, the first
present case, an irreversible peak is also seen at +1.28 V, three cathodic peaks for [Ru{(NO2)3tpy}(Me2bpy)Cl]Cl are assigned
corresponding to oxidation of the chloride counter-ion. The here to reductions involving the NO2-py moieties. The next two
[Ru{(NO2)3tpy}(Me2bpy)Cl]+ complex shows several important peaks are assigned to further reduction of the (NO2)3tpy ligand and
changes compared to the non-nitrated parent complex. Three reduction of the Me2bpy ligand, probably in that order.
closely-spaced reductions appear at low potentials in the catho-
Most importantly, the nitro substituents are observed to
dic region (ꢀ0.4 to ꢀ0.7 V), followed by further irreversible exert a strong effect on the ruthenium centre, as the anodic
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peak corresponding to the Ru(III/II) couple is shifted positively Cl-Rubb12 but had a relatively small effect with the less cyto-
by at least 300 mV, to the point where it coincides with toxic Cl-Rubb16. It was determined that the [Ru{(NO2)3tpy}-
oxidation of the chloride counter-ion (and is irreversible). This (Me2bpy)Cl]+ complex aquated significantly more slowly than
large positive shift indicates that the nitro substituents cause a the non-nitrated parent complex [Ru(tpy)(Me2bpy)Cl]+. This
significant decrease in the electron density on the ruthenium observation is consistent with the results from the cyclic
centre, making oxidation to Ru(III) more difficult.
voltammetry study, from which it was concluded that there
was a significant reduction in the electron density on the
ruthenium centre for the trinitrated complex, compared to
the non-nitrated parent complex. The reduced electron density
on the ruthenium centre of [Ru{(NO2)3tpy}(Me2bpy)Cl]+ would
Discussion
The results of this study show that the dinuclear ruthenium(II) increase the energy barrier for the removal of the chlorido
complexes Cl-Rubbn have potential as drugs against breast ligand from the metal centre, thereby decreasing the rate of
cancer. The most active complex, Cl-Rubb12, was almost four- the aquation reaction. Aquation was shown to be the first step
times more active than cisplatin. Furthermore, Cl-Rubb12 is more in the coordination of the ruthenium complexes with DNA.
active than the mononuclear [Ru(apy)(tpy)Cl]+ and dinuclear Consequently, the Cl-RubbnNO2 complexes would not form as
[{Ru(bpy)2Cl}2{m-BL}]2+ complexes previously reported by other many covalent adducts with DNA over the time period of the
groups,28,29 and of similar activity to the most active dinuclear cytotoxicity assays, compared to their non-nitrated parent com-
ruthenium–arene complex linked by a bis(pyridinone)alkane plexes. This suggests that the observed cytotoxicity of the
chain reported by Mendoza-Ferri et al.27 Interestingly, the Cl-RubbnNO2 complexes would largely be due to their reversible,
Cl-Rubbn complexes with the shortest (Cl-Rubb7) or the longest non-covalent, binding to DNA. Furthermore, it is reasonable to
linking chain (Cl-Rubb16) were the least active against both expect that the chlorido form of the complex would more easily
breast cancer cell lines. Insertion of three nitro substituents cross a cellular membrane than the more highly positively-
onto the tpy ligand of Cl-Rubb12 significantly decreased the charged aquated species. Based upon these assumptions, it
activity against both breast cancer cell lines. Incorporation of could be tentatively concluded that the activity of Cl-Rubb16
amine groups into the linking bridging ligand of Cl-Rubb7 was predominantly due to reversible binding to DNA, while the
decreased the activity, whereas it had little effect on the activity activity of Cl-Rubb12 was due to a combination of covalent and
of Cl-Rubb16
.
reversible binding to DNA.
In previous studies with chlorido-containing dinuclear
Although the inclusion of one or more secondary amines
ruthenium(II) complexes,27,28,30,40 the cytotoxicity has always into the bridging ligand of multinuclear platinum complexes
increased as the number of methylene groups in the flexible significantly increases their cytotoxicity,2 the incorporation of
alkane chain increased. Interestingly, in the present study the amine groups into the ligand bridge of Cl-Rubbn did not
Cl-Rubb16 complex was the least active of the Cl-Rubbn com- increase the cytotoxicity. For the multinuclear platinum com-
plexes. The decreased activities of Cl-Rubb7 and Cl-Rubb16
,
plexes, incorporation of an amine group or an inert am(m)-
compared to Cl-Rubb12 suggest two competing factors govern ineplatinum(II) centre into the bridge enhances cellular accumu-
the anticancer activity. While it is yet to be confirmed, it is lation and increases the affinity for DNA.2,14,42 The corresponding
assumed that the major mechanism of anticancer activity is inert Rubbn dinuclear ruthenium complexes (that do not contain
related to DNA binding, analogous to the corresponding dinuclear labile chlorido ligands) enter L1210 murine leukaemia cells by
platinum complexes. Increasing the number of methylene groups passive diffusion, with a minor contribution from protein-
in the linking chain should increase the lipophilicity of the mediated active transport.41 Consequently, incorporation of
dinuclear complex, and hence the ease with which it can pass amine groups into the ligand bridge could decrease the cellular
through the cellular membrane. While aquation is the necessary uptake of the Cl-Rubbn complexes, and thereby result in the
first step in DNA binding, as determined by the GMP binding observed lower activity for Cl-RubbN7 relative to Cl-Rubb7. How-
experiments, all the Cl-Rubbn complexes exhibited similar rates ever, it was also noted that Cl-RubbN16 was equally as active
of aquation and percentage of the aqua form at equilibrium. (albeit weakly) as Cl-Rubb16. This could suggest that the inclusion
Consequently, the relative cytotoxicity results could imply that of an amine in the bridging ligand of a Cl-Rubbn complex does
the range of possible DNA cross-linked adducts formed have increase the reversible binding affinity for DNA, thereby compen-
significantly different biological outcomes, and/or the anticancer sating for the lower cellular uptake.
activity is controlled by both covalent and reversible binding to
DNA. For the corresponding inert Rubbn complexes, the DNA
binding affinity decreases with increasing methylene groups in
Conclusions
the linking chain.41 Furthermore, based purely upon polycation
condensation of polyanionic DNA, it would also be expected that In conclusion, the results of this study support the idea of devel-
the cytotoxicity of the Cl-Rubbn complexes would decrease with oping a new class of anticancer agent by transferring from plati-
increasing chain length.
num to ruthenium the concept of gaining advantages in efficacy
The inclusion of three nitro substituents on the tpy ligand through the use of multinuclear complexes, as proposed by
significantly increased the IC50 value for the more cytotoxic Mendoza-Ferri et al.27 Dinuclear ruthenium complexes – containing
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a single chlorido ligand on each metal centre – were synthesised Synthesis of ligands
and found to be significantly more active than cisplatin against two
breast cancer cell lines. The anticancer activity appears to be due to
Trinitro-terpyridine
2,20:60,200-Terpyridine trioxide.
A
solution of 2,20:60,200-
a combination of covalent and reversible binding with DNA. The
IC50 results indicated that the Cl-Rubb12 complex was the most
active of the dinuclear complexes. The superior activity of
Cl-Rubb12 might be due to the best compromise between
lipophilicity (for cellular uptake) and the cytotoxic effects of
the covalent adducts formed with DNA. Given the vast array of
ligands that can be utilised for the Cl-Rubbn complexes, it
should be possible to optimise cellular uptake and the kinetics
of DNA binding, and thereby produce dinuclear ruthenium(II)
complexes with significant clinical potential.
terpyridine (4.0 g, 17.1 mmol) in glacial acetic acid (21 mL)
and 30% hydrogen peroxide (14 mL) was heated for 2 h at 80 1C
after addition of further hydrogen peroxide (14 mL) the tem-
perature was raised to 90 1C and maintained for 18 h. The
mixture was then poured into acetone (200 mL). After standing
for 4–6 h, the precipitate was filtered and washed with acetone
(2 ꢃ 40 mL) to obtain 4.2 g of pure product (yield 88%). 1H NMR
(400 MHz, CDCl3): d 8.35 (t, J = 9.3 Hz, 2H); 7.81 (d, J = 6.9 Hz,
2H); 7.77 (t, J = 10.4 Hz, 2H); 7.45 (t, J = 14.5 Hz, 1H);
7.36 (m, 4H).
4,40,400-Trinitro-2,20:60,200-terpyridine trioxide. Fuming nitric
acid (90%, 7.2 mL) was added slowly to a cooled mixture of
2,20:60,200-terpyridine trioxide (4.2 g, 15.1 mmol), conc. sulfuric
acid (15 mL) and fuming sulfuric acid (30%, 3.6 mL) at 0–5 1C.
The mixture was then stirred at 100 1C for 1 h and at 120 1C for
4 h. The contents of the flask were then poured into ice water
and filtered. The precipitate, after washing first with sodium
bicarbonate solution (40 mL) and then with water (40 mL), was
dried and crystallised from 50% aqueous pyridine (50 mL) to
yield 1.3 g of a light yellow coloured product (yield 21%).
1H NMR (400 MHz, CDCl3): d 8.66 (s, 2H); 8.55 (d, J = 3.0 Hz,
2H); 8.39 (d, J = 7.4 Hz, 2H); 8.25 (dd, J = 2.9 Hz, 3.2 Hz, 2H).
Experimental
Physical measurements
1D and 2D 1H NMR spectra were recorded on a Varian Advance
400 MHz spectrometer at room temperature in D2O {99.9%,
Cambridge Isotope Laboratories (CIL)}, CDCl3 (99.8%, CIL), or
CD3CN (499.8%, Aldrich). Microanalyses were performed by
the Microanalytical Unit, Research School of Chemistry,
Australian National University, Canberra.
Materials and methods
4,40-Dimethyl-2,20-bipyridine (Me2bpy), 2,20:60,200-terpyridine
(tpy), sodium borohydride, phosphorus trichloride, 1,3-diamino-
propane, 1,12-diaminopropane, guanosine 50-monophosphate dis-
odium salt (GMP), ammonium hexafluorophosphate (NH4PF6),
potassium hexafluorophosphate (KPF6) and Amberlites IRA-400
(chloride form) anion-exchange resin were purchased from Aldrich
and used as supplied; Sephadexs LH-20 was obtained from GE
Health Care Bioscience, RuCl3ꢂ3H2O was obtained from American
Elements, SeO2 was obtained from Ajax Chemicals. The syntheses
of ligands bbn (n = 7, 10, 12, 14 and 16)31 and [Ru(tpy)Cl3]32 were
performed according to reported literature methods.
4,40,400-Trinitro-2,20:60,200-terpyridine. A mixture of 4,40,400-
trinitro-2,20:60,200-terpyridine trioxide (1.3 g) and phosphorus
trichloride (15 mL) was refluxed for 18 h under an Ar atmo-
sphere, and the hot solution was then poured on ice and made
alkaline with 40% ammonium hydroxide solution. The preci-
pitate was filtered, dried under vacuum, and crystallised from
benzene to obtain 0.64 g of the pure product (yield 56%).
1H NMR (400 MHz, CDCl3): d 9.30 (s, 2H); 9.28 (d, J = 2.0 Hz,
2H); 9.08 (d, J = 5.2 Hz, 2H); 8.18 (dd, J = 2.0 Hz, 1.9 Hz, 2H).
bbNn ligands
4-Formyl-40-methyl-2,20-bipyridine. 4,40-Dimethyl 2,20-bipyridine
(2.0 g, 10.8 mmol) and SeO2 (1.8 g, 16.7 mmol) were refluxed in
1,4-dioxane (45 mL) under a N2 atmosphere for 24 h. The solution
was filtered while hot to remove the solid selenium and the filtrate
allowed to stand at room temperature for 1 h and then evaporated
to obtain a pale pink powder. This crude product was redissolved
in ethyl acetate (150 mL), the undissolved solid was removed by
filtration and the filtrate was evaporated to obtain pale yellow
solid. The crude product was dissolved in minimal volume of
DCM and impregnated with silica gel (230–400 mesh, 5 g) the
impregnated mixture was then loaded on a silica gel column
Cyclic voltammetry
Cyclic voltammetry was carried out using an eDAQ EA161
potentiostat operated via an eDAQ ED401 e-corder. A glassy
carbon working electrode, platinum wire counter electrode and
Ag/AgCl reference electrode were used. HPLC grade acetonitrile was
used as solvent and the supporting electrolyte was 0.1 mol Lꢀ1
tetra-n-butyl ammonium hexafluorophosphate (Aldrich).
Cytotoxicity assays
Cytotoxicity data was obtained using the mitochondrial- (230–400 mesh; 3 cm diam. ꢃ 15 cm), the unreacted Me2bpy was
dependent reduction of 3-(3,4-dimethylthiazol-2yl)-5-diphenyl eluted with 5% (v/v) ethyl acetate in n-hexane and the product
tetrazolium bromide (MTT) to formazan as described by Guh was eluted using 20–30% (v/v) ethyl acetate in n-hexane. The
et al.43 Metal complex solutions, including the control platinum purity of each fraction was monitored by TLC, using 30% (v/v)
complexes cisplatin and carboplatin, were made to the required ethyl acetate in n-hexane as the mobile phase. The purest
concentrations in warm Milli-Q water. Growth inhibition assays fractions were combined and the solvent was evaporated in vacuo
were carried out over a 72 h continuous exposure period.
to obtain white solid. A final recrystallisation with n-pentane
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gave 0.82 g of the pure product as a white powder (yield 38%). dissolved in a minimum amount of acetone and loaded onto a
1H NMR (400 MHz, CDCl3): d 10.17 (s, 1H); 8.89 (d, J = 5.1 Hz, column of Sephadex LH-20 (2 cm diam. ꢃ 30 cm), and using
1H); 8.85 (s, 1H); 8.57 (d, J = 4.9 Hz, 1H); 8.28 (s, 1H); 7.72 acetone as the eluent, the major first band was collected and
(d, J = 5.0 Hz, 1H); 7.20 (d, J = 4.2 Hz, 1H); 2.46 (s, 3H).
acetone was evaporated to obtain [Ru{(NO2)3tpy}(Me2bpy)Cl]PF6
complex as a dark violet-brown material and was crystallised
using acetonitrile–toluene. Anal. calcd for [Ru{(NO2)3tpy}-
(Me2bpy)Cl]PF6: C, 38.9%; H, 2.42%; N, 13.4%. Found: C,
39.0%; H, 2.22%; N, 13.2%. 1H NMR (400 MHz, CD3CN):
d 9.90 (d, J = 5.4 Hz, 1H); 9.57 (s, 2H); 9.35 (t, J = 2.5 Hz, 2H);
8.56 (s, 1H); 8.23 (s, 1H); 8.07–8.05 (m, 4H); 7.92 (d, J = 5.7 Hz,
1H); 6.88 (d, J = 5.9 Hz, 1H); 6.80 (d, J = 6.0 Hz, 1H); 2.82 (s, 3H);
2.34 (s, 3H). 13C NMR (CD3CN): d 160.8, 159.9, 157.7, 155.6,
154.9, 154.4, 152.35, 152.28, 152.20, 151.3, 150.9, 129.4, 128.2,
125.6, 125.4, 122.2, 118.9, 118.7, 21.4 and 20.8.
bbN7. A mixture of 4-formyl-40-methyl-2,20-bipyridine (0.74 g
3.76 mmol) and the 1,3-diaminopropane (0.16 mL, 1.88 mmol)
was stirred in methanol (50 mL) at room temperature under N2
atmosphere for 4 h. Sodium borohydride (0.57 g, 15.07 mmol)
was then added to the reaction mixture and stirred at 65 1C for
1–2 h. The solvent was evaporated from the reaction mixture
and water (10 mL) added to the crude residue. The organic
component was extracted with dichloromethane (3 ꢃ 50 mL),
and the organic phase was then washed with water (20 mL) and
brine (20 mL). After removing the solvent, the crude residue
was purified by column chromatography using silica gel, the
unreacted starting material and other impurities were eluted
with 1–2% (v/v) MeOH in DCM and the bbN7 was eluted with
5–8% (v/v) MeOH and 0.1% (v/v) triethylamine in DCM. Yield:
ꢀ
The chloride salt was obtained by stirring the PF6 salt in
water with Amberlite IRA-400 (chloride form) anion-exchange
resin. The resin was removed by filtration, and the dark violet-
brown solution was freeze-dried to obtain a fluffy dark violet-
brown [Ru{(NO2)3tpy}(bpy)Cl]Cl. Yield: 65 mg, 51%.
1
0.38 g, 23%. H NMR (400 MHz, CDCl3): d 8.59 (d, J = 6.2 Hz,
2H); 8.53 (d, J = 7.9 Hz, 2H); 8.32 (s, 2H); 8.22 (s, 2H); 7.30
[{Ru{(NO2)3tpy}(Cl)}2(m-bbn)]Cl2. The syntheses of [{Ru{(NO2)3tpy}-
(bs, 2H); 7.13 (d, J = 3.9 Hz, 2H); 3.89 (s, 4H); 2.74 (t, J = 10.9 Hz, (Cl)}2(m-bbn)]Cl2 (n = 12, 16) complexes were adapted from
4H); 2.44 (s, 6H); 1.66–1.52 (m, 2H).
literature methods.30,32 [Ru{(NO2)3tpy}Cl3] (70 mg, 0.12 mmol)
was dissolved in EtOH/H2O (4 : 1; 15 mL), the appropriate bbn
ligand (0.06 mmol) added and the mixture was refluxed under
an N2 atmosphere for 5–6 h. After cooling, the solvent from the
reaction mixture was evaporated to approximately half of the
original volume and then cooled, after which a saturated
aqueous NH4PF6 solution was slowly added until no further
precipitation occurred. The dark violet-purple precipitate was
then filtered and washed with ethanol (2 ꢃ 20 mL) followed by
diethyl ether (2 ꢃ 20 mL). The crude product was dissolved in a
minimum amount of acetone and loaded onto a column of
Sephadex LH-20 (2 cm diam. ꢃ 30 cm); on elution with acetone
the major first band collected. The pure [{Ru{(NO2)3tpy}Cl}2-
(m-bbn)](PF6)2 complex was isolated as dark violet-purple
material.
bbN16. This compound was prepared analogously to the
above method from 4-formyl-40-methyl-2,20-bipyridine (0.81 g
4.10 mmol) and 1,12-diaminopropane (0.41 g, 2.05 mmol).
Yield: 0.56 g, 24%. 1H NMR (400 MHz, CDCl3): d 8.61 (d, J =
5.0 Hz, 2H); 8.52 (d, J = 4.9 Hz, 2H); 8.30 (s, 2H); 8.21 (s, 2H);
7.36 (bs, 2H); 7.12 (d, J = 5.1 Hz, 2H); 3.90 (s, 4H); 2.63 (t, J =
14.3 Hz, 4H); 2.42 (s, 6H); 1.33–1.21 (m, 20H).
Synthesis of metal complexes
[{Ru(tpy)Cl}2{m-bbn}]2+ (Cl-Rubbn). The ruthenium(II) com-
plexes Cl-Rubbn were synthesised using a slight modification
of methods previously described.30
[Ru{(NO2)3tpy}Cl3]. 4,40,400-Trinitro-2,20:60,200-terpyridine (0.44 g,
1.7 mmol) was stirred in absolute ethanol (220 mL) with gentle
heating until dissolution. RuCl3ꢂ3H2O (0.63 g, 1.7 mmol) was
added and the solution refluxed for 3 h with stirring under
nitrogen atmosphere. After the mixture was cooled to room
temperature, the violet brown precipitate was filtered, washed
with excess of ethanol and ether, and dried under vacuum to yield
0.58 g of the product (yield 59%). 1H NMR (400 MHz, DMSO-d6):
d 9.91 (s, 2H); 9.73 (d, J = 2.5 Hz, 2H); 9.70 (d, J = 6.3 Hz, 2H); 8.30
(dd, J = 2.5 Hz, 2.4 Hz, 2H). 13C NMR (DMSO-d6): d 160.0, 158.1,
157.3, 154.2, 153.2, 120.8, 117.9, 117.5, 56.4, 19.0.
[{Ru{(NO2)3tpy}(Cl)}2(m-bb16)](PF6)2.
Anal.
calcd
for
[{Ru(NO2terpy)(Cl)}2(m-bb16)](PF6)2ꢂC3H6O: C, 44.4%; H,
3.78%; N, 11.7%. Found: C, 44.3%; H, 3.67%; N, 11.3%.
1H NMR (400 MHz, CD3CN): d 9.91 (d, J = 5.8 Hz, 2H); 9.56
(s, 4H); 9.37–9.33 (m, 4H); 8.56 (dd, J = 3.8 Hz, 3.5 Hz, 2H); 8.24
(dd, J = 3.3 Hz, 4.7 Hz, 2H); 8.08–8.06 (m, 8H); 7.93–7.90
(m, 2H); 6.88 (m, 2H); 6.83–6.79 (m, 2H); 3.08–3.07 (m, 2H);
2.82 (s, 3H); 2.61–2.60 (m, 2H); 2.34 (s, 3H); 1.60–1.10 (m, 28H).
13C NMR (CD3CN): d 160.8, 159.9, 157.90, 157.85, 156.7, 155.8,
154.96, 154.92, 154.5, 152.4, 152.33, 152.29, 152.25, 151.3,
150.9, 129.4, 128.7, 128.2, 127.5, 125.7, 125.4, 124.9, 124.7,
122.2, 119.0, 118.7, 36.0, 35.4, 31.1, 30.7, 30.6, 30.46, 30.42,
30.39, 30.30, 30.1, 29.96, 29.92, 29.6, 21.5 and 20.9.
[Ru{(NO2)3tpy}(Me2bpy)Cl]Cl. A solution of [Ru{(NO2)3tpy}Cl3]
(0.10 g, 0.17 mmol) and Me2bpy (0.032 g, 0.17 mmol) in EtOH/
H2O (4 : 1; 20 mL) was refluxed under an N2 atmosphere for 5 h.
After cooling, the solvent mixture was evaporated to approxi-
[{Ru{(NO2)3tpy}(Cl)}2(m-bb12)](PF6)2.
Anal.
calcd
for
mately half of the original volume and saturated aqueous [{Ru{(NO2)3tpy}Cl}2(m-bb12)](PF6)2: C, 42.6%; H, 3.24%; N,
1
NH4PF6 was added slowly to precipitate a dark violet-purple 12.4%. Found: C, 42.8%; H, 3.33%; N, 12.2%. H NMR (400 MHz,
material, which was filtered and washed with ethanol (2 ꢃ 15 mL) CD3CN): d 9.91 (d, J = 5.3 Hz, 2H); 9.56 (s, 4H); 9.36–9.32 (m, 4H);
followed by diethyl ether (2 ꢃ 15 mL). The crude product was 8.55 (dd, J = 5.8 Hz, 6.6 Hz, 2H); 8.23 (dd, J = 5.4 Hz, 7.0 Hz, 2H);
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8.07 (m, 8H); 7.92–7.89 (m, 2H); 6.88 (d, J = 5.6 Hz, 2H); 6.81 51.2, 50.7, 49.5, 49.3, 30.0, 29.9, 29.6, 29.5, 27.3, 27.0, 26.9, 21.4
(m, 2H); 3.07–3.06 (m, 2H); 2.81 (s, 3H); 2.60–2.59 (m, 2H); 2.34 and 20.9.
(s, 3H); 1.61–1.08 (m, 20H). 13C NMR (CD3CN): d 160.8, 159.9,
The chloride salt was obtained by stirring the PF6 salt in
ꢀ
157.8, 156.6, 155.7, 154.95, 154.90, 154.4, 152.4, 152.33, 152.29, water with Amberlite IRA-400 (chloride form) anion-exchange
152.24, 151.3, 150.9, 129.4, 128.7, 128.2, 127.5, 125.6, 125.4, resin. The resin was removed by filtration, and the solution was
124.9, 124.7, 122.2, 118.9, 118.7, 36.0, 35.3, 31.1, 30.76, 30.73, freeze-dried to obtain a fluffy dark purple-brown powder of
30.4, 30.3, 30.19, 30.13, 30.09, 29.99, 29.96, 29.89, 29.72, 29.66, [{Ru(tpy)Cl}2(m-bbH2Nn)]Cl4. Yield: 20–25%.
21.4 and 20.9.
[{Ru{(NO2)3tpy}(Cl)}2(m-bbH2N16)]Cl4. The synthesis of
[{Ru{(NO2)3tpy}(Cl)}2(m-bbH2N16)]Cl4 complex was prepared as
described for [{Ru(tpy)Cl}2(m-bbH2Nn)]Cl4. Typical yield B20%.
The chloride salts were obtained by stirring the PF6 salt in
water using Amberlite IRA-400 (chloride form) anion-exchange
resin. The resin was removed by filtration, and the solution was
freeze-dried to obtain a fluffy dark violet-purple powder of pure
[{Ru(NO2terpy)(Cl)}2(m-bbn)]Cl2 in 30–35% yield.
[{Ru{(NO2)3tpy}(Cl)}2(m-bbH2N16)](PF6)2Cl2. Anal. calcd for
[{Ru(NO2tpy)(Cl)}2(m-bbH2N16)](PF6)2Cl2ꢂ1.5C3H6O: C, 41.8%;
H, 3.73%; N, 12.5%. Found: C, 41.7%; H, 3.48%; N, 12.1%.
[{Ru(tpy)Cl}2(m-bbH2Nn)]Cl4. To the bbN7 ligand (53 mg,
1H NMR (400 MHz, CD3CN): d 9.92–9.91 (m, 2H); 9.57 (s, 2H);
0.122 mmol) dissolved in EtOH/H2O (4 : 1; 15 mL), solid
9.48 (s, 1H); 9.36 (s, 2H); 9.31 (dd, J = 1.6 Hz, 2.1 Hz, 2H); 8.71
(m, 1H); 8.56–8.55 (m, 2H); 8.40–8.37 (m, 1H); 8.26–8.25
(m, 1H); 8.17 (d, J = 2.5 Hz, 1H); 8.07–8.05 (m, 8H); 7.43 (t, J =
10.2 Hz, 1H); 6.97–6.95 (m, 4H); 4.28 (dd, J = 5.8 Hz, 5.0 Hz, 2H);
[Ru(tpy)Cl3] (108 mg, 0.245 mmol) was added at 60 1C and
the reaction mixture was refluxed under an N2 atmosphere for
5–6 h. After cooling, half of the solvent was evaporated from the
reaction mixture and saturated aqueous NH4PF6 was added to
3.84–3.82 (m, 2H); 2.96–2.95 (m, 2H); 2.82 (s, 3H); 2.79–2.77
obtain the PF6ꢀ salt as a dark purple-brown material, which was
(m, 2H); 2.35 (s, 3H); 1.74–1.05 (m, 20H). 13C NMR (CD3CN):
filtered and washed with ethanol (2 ꢃ 20 mL) followed by
d 160.79, 160.74, 159.8, 159.7, 155.4, 154.9, 154.4, 153.0, 152.2,
diethyl ether (2 ꢃ 20 mL). The crude product was dissolved in a
151.6, 151.1, 129.5, 128.44, 128.32, 126.9, 125.6, 125.0, 124.3,
minimum amount of acetone and loaded onto a column of
122.2, 119.3, 118.9, 118.6, 115.5, 112.8, 51.0, 49.4, 29.8, 29.6,
Sephadex LH-20 (2 cm diam. ꢃ 30 cm); and eluted with acetone,
28.1, 27.2, 21.4, 20.9 and 14.4.
the major first band (dark purple coloured) was collected, the
acetone evaporated to obtain [{Ru(tpy)Cl}2(m-bbH2Nn)]Cl4
complex as a dark purple-brown material.
[Ru{(NO2)3tpy}(Cl)(bbH2N16)]Cl3. The mononuclear complex
was prepared using an analogous method to that reported for
[{Ru(tpy)Cl}2(m-bbH2Nn)]Cl4 from [Ru{(NO2)3tpy}Cl3] (50 mg,
0.086 mmol) and the bbN16 ligand (49 mg, 0.086 mmol) to
obtain [Ru{(NO2)3tpy}(Cl)(bbH2N16)]Cl3 as dark violet-brown
solid. Typical yield B24%.
[{Ru(tpy)Cl}2(m-bbH2N7)](PF6)2Cl2. Anal. calcd for [{Ru(tpy)Cl}2-
(m-bbH2N7)](PF6)2Cl2: C, 44.4%; H, 3.53%; N, 10.9%. Found: C,
44.6%; H, 3.75%; N, 10.6%. 1H NMR (400 MHz, CD3CN): d 10.16
(dd, J = 5.0 Hz, 5.4 Hz, 1H); 10.00 (d, J = 5.7 Hz, 1H); 8.50–8.46
(m, 4H); 8.37–8.29 (m, 6H); 8.12–8.05 (m, 4H); 7.82 (m, 6H); 7.66–
7.62 (m, 4H); 7.24–7.6 (m, 6H); 7.05 (d, J = 6.3 Hz, 1H); 6.80
(bs, Hz, 1H); 4.50 (bs, 2H); 4.07 (bs, 2H); 3.10–3.03 (m, 2H); 2.93–
2.87 (m, 2H); 2.68 (s, 3H); 2.33 (s, 3H); 1.70–1.64 (m, 2H). 13C NMR
(CD3CN): d 159.5, 158.8, 158.6, 153.5, 153.1, 152.6, 152.2, 149.8,
149.1, 137.9, 134.6, 128.9, 128.1, 127.2, 125.4, 124.4, 123.4, 51.3,
46.0, 21.5 and 20.8.
[Ru{(NO2)3tpy}(Cl)(bbH2N16)](PF6)Cl2. Anal. calcd for
[Ru{(NO2)3terpy}(Cl)(bbH2N16)](PF6)Cl2ꢂ0.5C3H6O: C, 47.9%; H,
4.67%; N, 12.5%. Found: C, 47.7%; H, 4.47%; N, 12.6%.
1H NMR (400 MHz, CD3CN): d 9.95 (m, 1H); 9.56 (d, J =
3.4 Hz, 1H); 9.46 (m, 1H); 9.33–9.30 (m, 2H); 8.66 (m, 1H);
8.50–8.41 (m, 2H); 8.33 (m, 1H); 8.26–8.22 (m, 2H); 8.17
(m, 1H); 8.03–7.96 (m, 5H); 7.44–7.41 (m, 2H); 7.27–7.24
(m, 1H); 6.97–6.89 (m, 3H); 4.28–4.26 (m, 2H); 3.76–3.71
(m, 2H); 2.84–2.78 (m, 4H); 2.46–2.39 (m, 3H); 2.34 (s, 3H);
1.65–1.08 (m, 20H). 13C NMR (CD3CN): d 168.0, 161.5, 161.0,
160.0, 159.3, 158.4, 158.1, 157.9, 156.1, 155.1, 154.7, 153.5,
153.3, 153.1, 152.8, 152.5, 152.1, 151.8, 150.1, 129.4, 128.4,
127.6, 127.3, 126.6, 125.6, 123.9, 122.8, 122.5, 122.2, 121.9,
115.8, 113.0, 66.8, 50.1, 49.6, 30.4, 29.8, 28.0, 27.6, 21.5, 20.0
and 14.7.
[{Ru(tpy)Cl}2(m-bbH2N16)](PF6)2Cl2ꢂ3H2O. Anal. calcd for
[{Ru(tpy)Cl}2(m-bbH2N16)](PF6)2Cl2ꢂ3H2O: C, 46.0%; H, 4.57%;
N, 9.8%. Found: C, 45.6%; H, 4.28%; N, 9.4%. 1H NMR
(400 MHz, CD3CN): d 10.27 (d, J = 5.2 Hz, 1H); 10.03 (d, J =
5.6 Hz, 1H); 8.59 (bs, 1H); 8.50 (dd, J = 1.1 Hz, 1.8 Hz, 4H); 8.43
(bs, 1H); 8.39 (dd, J = 1.7 Hz, 2.6 Hz, 4H); 8.30 (bs, 1H); 8.15
(bs, 1H); 8.11 (t, J = 16.2 Hz, 2H); 7.98–7.95 (m, 1H); 7.92–7.87
(m, 4H); 7.85 (dd, J = 2.5 Hz, 2.3 Hz, 1H); 7.67–7.63 (m, 4H); 7.37
(bs, 1H); 7.31–7.28 (m, 4H); 7.17 (d, J = 5.7 Hz, 1H); 6.95 (d, J =
5.8 Hz, 1H); 6.86 (d, J = 5.2 Hz, 1H); 4.49 (bs, 2H); 4.06 (bs, 2H);
3.23–3.16 (m, 2H); 2.94–2.86 (m, 2H); 2.78 (s, 3H); 2.36 (s, 3H);
1.61–1.20 (m, 20H). 13C NMR (CD3CN): d 160.1, 159.6, 159.5,
158.9, 158.7, 158.4, 157.5, 156.2, 153.6, 153.1, 153.0, 152.9,
152.6, 152.2, 149.9, 149.1, 137.9, 134.7, 134.5, 128.9, 128.2,
128.1, 127.8, 126.8, 125.1, 124.45, 124.42, 124.1, 123.4, 123.3,
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