Functionalization of Ruthenium(II) Terpyridine Complexes with Cyclic RGD Peptides To Target Integrin Receptors in Cancer Cells

Article abstract of DOI:10.1002/ejic.201601094

The lack of selectivity for cancer cells and the resulting negative impact on healthy tissue is a severe drawback of actual cancer chemotherapy. Tethering of cytotoxic drugs to targeting vectors such as peptides, which recognize receptors overexpressed on the surface of tumor cells, is one possible strategy to overcome such a problem. The pentapeptide cyc(RGDfK) targets the integrin receptor α_vβ₃, important for tumor growth and metastasis formation. In this work, two terpyridine-based Ru^II complexes were prepared and for the first time conjugated to cyc(RGDfK) through amide bond formation, which resulted in a monomeric and a dimeric bioconjugate. Both Ru^II complexes were found to bind strongly and selectively to integrin α_vβ₃, and the dimeric molecule displayed a 20-fold higher affinity to the receptor than the monomeric one. However, the cytotoxicity of the complexes and related bioconjugates against human A549 and SKOV-3 cell lines is still not sufficient for application as anticancer agents. Nevertheless, considering the high selectivity for integrin receptor α_vβ₃, the synthesis of Ru-based bioconjugates with cyc(RGDfK) paves a promising way towards the design of effective targeted anticancer agents.

Full text of DOI:10.1002/ejic.201601094

A Journal of
Accepted Article
Title: Functionalization of Ruthenium(II) terpyridine complexes with cyclic RGD pep-
tides to target integrin receptors in cancer cells
Authors: Eva M. Hahn; Natalia Estrada-Ortiz; Jiaying Han; Vera F. C. Ferreira; Tobi-
as Kapp; Joao D. G. Correia; Angela Casini; Fritz E. K u¨ hn
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To be cited as: Eur. J. Inorg. Chem. 10.1002/ejic.201601094
Link to VoR: https://doi.org/10.1002/ejic.201601094

European Journal of Inorganic Chemistry
10.1002/ejic.201601094
COMMUNICATION
Functionalization of Ruthenium(II) terpyridine complexes with
cyclic RGD peptides to target integrin receptors in cancer cells
[
a,b]
[c]
[c]
[d]
[e]
Eva M. Hahn,
Natalia Estrada-Ortiz, Jiaying Han, Vera F. C. Ferreira, Tobias G. Kapp, João D.
[
d]
[b,c,e]
[a]
G. Correia, Angela Casini,*
and Fritz E. Kühn*
Abstract: The lack of selectivity for cancer cells and the resulting
negative impact on healthy tissue is a severe drawback of actual
cancer chemotherapy. Tethering of cytotoxic drugs to targeting
vectors such as peptides, which recognize receptors overexpressed
on the surface of tumor cells, is one possible strategy to overcome
such a problem. The pentapeptide cyc(RGDfK) targets the integrin
become a priority in the field, together with the design of new
chemical scaffolds.
Within this framework, an increasing number of reports on
tethering metal complexes to
a wide range of functional
molecules or nanoparticles with or without targeting groups has
[
6]
appeared in recent years. Specifically, the functionalization of
metallodrugs is aimed at improving the tumor selectivity and/or
minimizing the systemic toxicity to enhance their cellular
accumulation and overcome tumor resistance. Moreover, a
synergistic anticancer effect of different therapeutic modalities
would also be welcome. In some cases, the use of imaging tags
conjugated to the metal compounds allow to visualize the drug
molecules in vitro or in vivo, thus leading to the design of
v 3
receptor α β , important for tumor growth and metastasis formation.
In this work, two terpyridine based Ru(II) complexes were prepared
and for the first time conjugated to cyc(RGDfK) via amide bond
formation resulting in a monomeric and a dimeric bioconjugate. Both
v 3
Ru(II) complexes bind strongly and selectively to integrin α β , with
the dimeric molecule displaying a 20-fold higher affinity to the
receptor than the monomeric one. However, the cytotoxicity of the
complexes and related bioconjugates against human A549 and
SKOV-3 cell lines is still not sufficient for application as anticancer
agents. Nevertheless, considering the high selectivity for integrin
[
7]
theranostic agents.
Among the various strategies explored so far to actively target
cytotoxic metallodrugs to cancer cells, tumor-targeting peptides
(TTPs) that are specific for tumor related surface markers, such
receptor
v 3
α β , the synthesis of Ru-based bioconjugates with
[
8]
cyc(RGDfK) paves a promising way towards the design of effective
targeted anticancer agents.
as membrane receptors, can be used.
Integrin receptors have been largely explored as drug targets
since they are heterodimeric, transmembrane receptors that
function as mechanosensors, adhesion molecules and signal
[
9]
Introduction
transduction platforms in a multitude of biological processes.
Integrins interact with the extracellular matrix (ECM) thereby
regulating many cellular functions, such as proliferation,
migration, and survival. Integrins are also involved in the cell-to-
cell interactions. Through cell–cell and cell–ECM contacts, the
integrins transduce the information from the external
environment into the cell and vice-versa, to promote cell
Platinum anticancer drugs are widely used for chemotherapy of
various cancers. However, indiscriminate distribution or poor
selectivity often results in severe side effects and drug
[
1]
resistance. Therefore, enhancing the tumor selectivity has
become a major goal for the development of platinum-based
cytotoxic agents. Similar issues are encountered with the new
generation of experimental anticancer metal complexes,
[
10]
adhesion, spreading and motility. One common feature of the
integrin family is a heterodimeric structure that consists of  and
[11]
[
2]

subunits.
These structures form 24 different subtypes in
including, among others, compounds based on ruthenium,
[
3]
[4]
[5]
mammals, which can be classified according to their binding
partners (e.g. laminin, collagen). Different integrins are also
gold, iron and copper . Thus, the development of so-called
targeting and drug delivery strategies of metallodrugs has
[
12]
associated with tumor angiogenesis and metastasis,
upregulated in tumor cells compared to low levels in normal
endothelial cells. The integrin receptor  plays a crucial role in
and became an attractive target for
being
[
a]
Molecular Catalysis, Catalysis Research Center and Department of
Chemistry, Technische Universität München, Lichtenbergstr. 4,

v 3
85747 Garching bei München, Germany.
[
13],[14]
these processes
pharmaceutical research.
E-mail: fritz.kuehn@ch.tum.de
[
15]
[b]
[c]
[d]
School of Chemistry, Cardiff University, Park Place, CF103AT
Cardiff, United Kingdom. E-mail: casinia@cardiff.ac.uk
Groningen Research Institute of Pharmacy, University of Groningen,
Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands.
Centro de Ciências e Tecnologias Nucleares, Instituto Superior
Técnico, Universidade de Lisboa, CTN, Estrada Nacional 10 (km
In 1984, Pierschbacher and Ruoslahti discovered that the amino
acid sequence Arg-Gly-Asp-Ser (RGDS) is essential for binding
[
16]
integrin receptors.
In fact, eight of the above mentioned
[
17]
integrin subtypes form the RGD-binding class. Since then, a
wide screening of peptide libraries has been carried out to
discover ligands including the RGD sequence, and targeting the
integrin receptors with even higher selectivity. Interestingly, the
139.7), 2695-066 Bobadela LRS, Portugal.
[
e]
Institute for Advanced Study, Technische Universität München,
Lichtenbergstr. 2a, 85748 Garching, Germany.
cyclic pentapeptide cyc[RGDfK] (Figure 1) was found to have
Supporting information for this article is given via a link at the end of
the document.
[18]
increased selectivity for integrin 
v 3
 .
This article is protected by copyright. All rights reserved

European Journal of Inorganic Chemistry
10.1002/ejic.201601094
SHORT COMMUNICATION
As far as it concerns RGD-type peptides, only a few examples
are described. Thus, Sadler et al. reported the synthesis of a
[
28]
Ru(II) arene complex attached to the linear RGD tripeptide
,
which dissociates from the peptide by irradiation with visible light
to form an aqua complex that generates monofunctional adducts
with guanine bases of the DNA. Furthermore, Adamson et al.
designed luminescent Ru(II) polypyridyl complexes attached to
the linear RGD tripeptide, acting as molecular probes for
[
29]
reporting on the presence and conformation of integrins. Live
cell studies with confocal microscopy confirmed the selective
binding to an integrin receptor, but no cytotoxicity studies were
described.
Finally, fluorescent ruthenium polypyridyl complexes have been
attached
nanoparticles,
to
RGD-functionalized
whose uptake and sub-cellular distribution
mesoporous
silica
[
30]
could be followed by fluorescence microscopy. Interestingly, the
RDG peptide on the nanoparticle surface induces an increased
selectivity for cancer cells. After internalization of the
nanoparticle, the ruthenium species are released and induce
changes in the phosphorylation pathway of certain protein
kinases leading to apoptosis.
To the best of our knowledge, besides the above mentioned
publications, there are no further studies about conjugation of
ruthenium complexes to RGD-type peptides. Therefore, in this
work the successful bioconjugation of two Ru(II) terpyridine
complexes to the cyclic RGD peptide cyc[RGDfK] for targeting
Figure 1. Cyclic pentapeptide cyc[RGDfK] (A) and two representative targeted
[
19]
Pt constructs: a Pt(IV) conjugate
cisplatin prodrug (C).
(B) and nanoparticles encapsulating a
[
20]
Among the metal-based radiopharmaceuticals tethered to cyclic
RGD peptides, the majority of the examples reported in the
literature have been evaluated as SPECT and PET radiotracers
[
7a, 21]
for tumor imaging.
potential theranostic
E(cyc[RGDfK]) compound has been reported.
Recently, the preclinical evaluation of the
66
radiopharmaceutical
Ga-DOTA-
[
22]
2
integrin 
v 3
is reported. The two Ru(II) compounds were

As an example of targeted anticancer metal complexes, recent
reports describe the synthesis and biological evaluation of Pt(IV)
prodrugs, whose axial positions could be functionalized with
cyclic RGD tripeptides that bind selectively to the integrin
designed to feature carboxylic acid groups for conjugation to the
lysine residue of the RGD peptide via amide bond formation.
Thus, the compounds were tethered to one or two peptides. In
the latter case, anchoring to two cyc[RGDfK] were intended to
[
[19, 23]
receptor 
v

3
.
In a more elaborated approach, Lippard et al.
[
13]
enhance the binding affinity to the 
binding affinities of the ruthenium-RGD conjugates for both the
and  integrin receptors have been evaluated by integrin
v

3
integrin receptor. The
synthesized a cisplatin prodrug encapsulated into poly(D,L-
lactic-co-glycolic acid)-block-polyethylene glycol (PLGA-PEG)
nanoparticles tethered to cyc[RGDfK]. The prodrug shows a

v

3

5 1
binding assays. The anticancer effects of the “free” ruthenium
complexes and their respective conjugates were evaluated in
vitro against human cancer cell lines with different expression
significant increase in cytotoxicity towards
expressing cancer cell lines, comparable to cisplatin. In vivo
studies also revealed equivalent tumor growth inhibition (ca.

v

3
integrin–
levels of integrin 
v
3
 , namely human lung cancer A549 cells
6
0%) by both the prodrug and cisplatin in mice bearing ovarian
[
20]
(scarce v3 integrin expression) and human mammary
cancer xenografts.
[31]
v 3
carcinoma SKOV3 cells (moderate   integrin expression).
Concerning anticancer ruthenium complexes coupled to
peptides, some examples have been already reported in the
[
8]
literature,
including luminescent Ru(II) complexes linked
[
24]
through the mitochondrial penetrating peptide (MPP), as well Results and Discussion
[
25]
as to the nuclear localization sequence (NLS),
the latter
enabling the active transport of drugs into the cell nucleus as
confirmed by fluorescence microscopy studies. Interestingly,
Keyes et al. developed ruthenium(II) polypyridyl luminophores
anchored to peptide sequences as a new class of stimulated
emission depletion (STED) microscopy probes for imaging of
The experimental procedures can be found in the Supporting
Information. The two ligands used in this work are 2,2':6',2''-
terpyridine (terpy, 1a) and [2,2':6',2''-terpyridine]-4'-carboxylic
acid (terpy*, 1b). For the synthesis of 1b a reported two step
procedure has been followed. In the first step, 2-acetylpyridine
and furfural were combined in ethanol under basic conditions to
yield 4'-(furan-2-yl)-terpyridine, which was oxidized in the
[
32]
[
26]
key cell organelles.
Ueyama et al. also described a peptide
labeling approach using Ru(II) terpyridine complexes, to
implement the mass spectrometry detection of proteolytic
4
following step with KMnO to obtain [2,2':6',2''-terpyridine]-4'-
carboxylic acid (terpy*, 1b) (see Scheme 1).
[
27]
peptides.
This article is protected by copyright. All rights reserved

European Journal of Inorganic Chemistry
10.1002/ejic.201601094
SHORT COMMUNICATION
Scheme 1. Synthesis of ligand 1b, [2,2':6',2''-terpyridine]-4'-carboxylic acid.
Scheme 3. Synthesis of the bioconjugate products 4a and 4b (PG = protecting
group).
Characterization of the ligand 1b complexes 3a,b and 4a,b
The complexes 3a and 3b were prepared by a novel synthetic
[
33]
route based on literature procedures
(Scheme 2). Heating
RuCl 3H O with 1a or 1b in dry ethanol yields the brown
3
2
complexes 2a and 2b, respectively, after one hour in the dark.
Afterwards, the complexes reacted with 1b, triethylamine and
LiCl for chloride abstraction and reduction of Ru(III) to Ru(II).
Upon addition of
Ru(terpy)(terpy*)](PF
1
M
KPF
6
solution, the complexes
](PF (3b)
[
6
)
2
(3a) and [Ru(terpy*)
2
6 2
)
bearing one or two carboxylic acid groups, respectively,
precipitate.
1
Figure 2. H NMR spectra of 1b and complexes 3a and 3b (in DMSO-d6).
The ligands and the corresponding complexes were
1
13
31
characterized by H-, C- and P-NMR spectroscopy and ESI-
MS.
1
Comparing the H NMR spectra of ligand 1b with 3a, several
Scheme 2. Two step procedure for the synthesis of [Ru(terpy)(terpy*)](PF
6 2
)
signal shifts are observed due to complex formation (Figure 2).
(
3a) and [Ru(terpy*) ](PF (3b).
2
6 2
)
3
’,5’
3,3’’
The signals of H
+
the signals of H
and H
are shifted downfield around ∆δ =
remains and
show a strong upfield shift of ∆δ =
4
,4’’
0.61 or +0.49 ppm. In contrast, the signal of H
6
,6’’
5,5’’
and H
The conjugation of 3a and 3b to the cyclic peptide
cyc[R(Pbf)GD(tBu)fK] was accomplished by reaction of the free
carboxylic acid groups of the complexes with the primary amine
of the lysine side chain in the presence of a mixture of the
activating agents HATU and HOAt (Scheme 3). The success of
the bioconjugation reaction was confirmed by Electrospray
Ionisation Mass Spectrometry (ESI-MS), which allowed to
−1.25 and −0.28 ppm. Nearly the same values are observed for
complex 3b containing two ligands 1b. The downfield shift of
3
’,5’
3,3’’
H
and H
is about ∆δ = +0.62 and +0.47 ppm, whereas the
4
,4’’
6,6’’
5,5’’
signal of H
remains and the signals of H
and H
are
shifted upfield about ∆δ = −1.20 and −0.26 ppm. For these
observations, two effects have to be taken into account: first, the
deshielding effect of the carboxylic acid group and second, the
increase of electron density in the aromatic system through
coordination of ruthenium. The remaining signals in the
spectrum of 3a can be assigned to coordinated ligand 1a. In the
identify the intermediate products at m/z
=
752.78 for
and 1221.58 for
2
] , respectively. Afterwards,
2+
[
[
Ru(terpy)(terpy-cyc(R(Pbf)GD(tBu)fK))]
Ru(terpy-cyc(R(Pbf)GD(tBu)fK))
2+
the remaining protection groups of Arg and Asp were cleaved
using a cleavage cocktail as detailed in the experimental section.
For purification of the crude product, size exclusion
3
1
-
P NMR spectra the presence of the PF
6
counter ions in
complexes 3a and 3b is confirmed by the characteristic septet.
The complexes 3a and 3b and their conjugation derivatives 4a
and 4b were characterized by ESI-MS, where the characteristic
isotopic patterns are consistent with the assigned structures
®
chromatography with Sephadex G-15 was used since the
compounds decomposed during reverse phase (RP)-HPLC.
Finally, the products were precipitated by addition of solid KPF
to give [Ru(terpy)(terpy-cyc(RGDfK)](PF (4a) and [Ru(terpy-
cyc(RGDfK)) ](PF (4b) as red solids.
6
(
Figure S3-S14 in the supplementary material). The ESI-MS
6 2
)
spectra of the complexes show signals at 757.05 and 306.04
2
6 2
)
m/z for 3a and 801.04 and 328.04 m/z for 3b, which indicate the
-
loss of one or two PF
6
anions, leading to a single or double
positive charge cationic species. Similarly, for the coupling
-
products 4a and 4b, the loss of PF
6
anions is observed. The
characteristic isotopic patterns of the signals match perfectly
with the calculated ones, which can be seen in the supporting
information.
Integrin binding assay
This article is protected by copyright. All rights reserved

European Journal of Inorganic Chemistry
10.1002/ejic.201601094
SHORT COMMUNICATION
The impact of the conjugation of Ru(II) complexes to
4b
>100
>100
cyc[RGDfK] on the binding affinity to the integrin receptors α
and α was evaluated. The binding affinities for 4a, 4b and
benchmark Cilengitide are shown in Table 1.
a exhibits an IC50 value of 49 ± 4.3 nM, 90 fold higher than
Cilengitide (0.54 ±0.06 nM). However, the selectivity for α is
reasonably high reflecting the fact that the bioconjugate does not
bind the α receptor at all (IC50  1000 nM), while Cilengitide
v 3
β
5 1
β
[
a] The reported values are the mean ± SD of at least three determinations.
[
34]
4
v 3
β
Conclusions
5 1
β
In summary, two novel ruthenium(II) polypyridyl complexes
coupled to the cyclic pentapeptide cyc[RGDfK] with monomeric
or dimeric character have been prepared in order to deliver
anticancer metallodrugs directly to tumors cells overexpressing
has still an affinity of 15.4 ± 0.2 nM. Considering bioconjugate 4b,
enhanced binding affinities are predicted due to its dimeric
character. Indeed, the binding affinity for integrin α
.3 nM, presenting a 20 times higher affinity than that observed
for the monomeric product and nearly approaching the value of
Cilengitide. Since the affinity for the α receptor shows merely
v 3
β is 2.5 ±
0
the α
The preparation of the terpy-based ruthenium complexes 3a and
b bearing one or two carboxylic acid groups, respectively, was
v 3
β integrin receptor.
5 1
β
3
v 3
a value about 595 ± 67 nM, the high selectivity of 4b for α β is
carried out using a novel synthetic strategy. The compounds
were coupled to a protected derivative of the cyclic pentapeptide
via amide bond formation between the carboxylic acid of the
complex and the amine group of the lysine side chain.
Purification of the resulting monomeric (4a) or dimeric (4b)
bioconjugates and was achieved by Size Exclusion
demonstrated.
Table 1. Results of integrin binding assays for the bioconjugates 4a and 4b, in
comparison to the benchmark Cilengitide.
[
a]
IC50 [nM] ± SD
Chromatography followed by precipitation as PF
Considering the binding affinities of the bioconjugates towards
the integrin receptors, a high selectivity for the α integrin
receptor and a negligible impact on the α receptor was
6
-salt.
Compound



3
5 1
 
v 3
β
5 1
β
[
34]
Cilengitide
0.54 ± 0.06
49 ± 4.3
15.4 ± 0.2
>1000
observed. Still, the cytotoxicity of all the reported bioconjugates
was low, most likely due to still scarce uptake in cancer cells.
Hence, while the reported strategy holds promise to achieve
targeted metallodrugs, future studies have to focus on the
tethering to the RGD peptide of ruthenium complexes with an
intrinsically higher cytotoxic potency, such as similar types of
4a
4b
2.5 ± 0.3
595 ± 67
[
a] The reported IC50 values were determined using a solid-phase binding
assay (see the Supporting Information for details).
[
35]
ruthenium complexes with terpyridine-type ligands
.
Antiproliferative activity
Acknowledgements
The ruthenium compounds (3a and 3b) and their respective
cyc[RGDfK] bioconjugates (4a and 4b) were evaluated for their
antiproliferative properties on two human cancer cell lines with
scarce (A549) or moderate (SKOV3) expression of integrins
A.C. acknowledges support from Cardiff University, the August-
Wilhelm Scheer Visiting Professorship at the Technical
University of Munich (TUM) and
a Hans Fischer Senior
[31]
α
v
β
3
.
Unfortunately, both the ruthenium(II) complexes and
Fellowship of TUM IAS (Institute for Advanced Study) funded by
the German Excellence Initiative and the European Union
Seventh Framework Program under grant agreement n° 291763.
E.M.H. acknowledges Prof. H. Kessler for helpful discussions
and is grateful for the financial support of the TUM Graduate
School of Chemistry and the Short Term Scientific Mission
supported by COST action CM1105. V.F.C.F. and J.D.G.C.
gratefully acknowledge the FCT (Portugal) support through the
UID/Multi/04349/2013 project.
their targeted derivatives show similarly very low cytotoxic
effects against both cell lines, independent of the presence of
the RGD domains (Table 2). This could be attributed to the
intrinsic limited anticancer effects of the selected Ru(II)
derivatives. Therefore, although their cell uptake should be
favored by the presence of cyc[RGDfK] domains, in the end no
toxic effects are observed.
Table 2. IC50 values of Ru complexes and their RGD bioconjugates against
human A549 and SKOV-3 cell lines.
Keywords: tumor targeting peptide • ruthenium • antitumor
[
a]
IC50 [µM]
agent • bioconjugation • RGD
Compound
A549
SKOV-3
74.5 ± 13.7
85.2 ± 18.7
>100
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European Journal of Inorganic Chemistry
10.1002/ejic.201601094
SHORT COMMUNICATION
Entry for the Table of Contents
COMMUNICATION
Two terpyridine-based ruthenium(II)
complexes were designed to be
coupled to a cyclic peptide targeting
Targeting drugs
Eva M. Hahn, Natalia Estrada, Jiaying
Han, Vera F. C. Ferreira, Tobias G.
Kapp, João D. G. Correia, Angela
Casini* and Fritz E. Kühn*
v 3
integrin receptor   in order to
achieve models for targeted
anticancer agents.
Page No. – Page No.
Functionalization of Ruthenium(II)
terpyridine complexes with cyclic
RGD peptides to target integrin
receptors in cancer cells
This article is protected by copyright. All rights reserved