Synthesis and biological evaluation of a Platinum(II)-c(RGDyK) conjugate for integrin-targeted photodynamic therapy

Article abstract of DOI:10.1016/j.ejmech.2017.09.058

A cancer-targeting conjugate 4 of a cyclometalated [N,C,N-Pt(II)] complex bearing a N?C?N 1,3-di(2-pyridyl)-benzene with c(RGDyK) peptide as guiding molecule was designed and synthesized for real-time drug delivery monitoring in cancer cells and photodynamic therapy (PDT). This conjugate demonstrates a mild cytostatic effect to six cancer cell lines expressing integrins at different extent, while possessing promising features for PDT. Conjugate 4 demonstrated rapid cell uptake by receptor-mediated endocytosis and efficient generation of ¹O₂ upon irradiation. Incubation of rat bladder cancer cells AY27 with conjugate 4 (50 μΜ) prior to blue light exposure (5 min) resulted in significant reduction (50%) of cell survival compared to control cells as measured by MTT assay post 24 h after treatment.

Full text of DOI:10.1016/j.ejmech.2017.09.058

Accepted Manuscript
Synthesis and biological evaluation of a Platinum(II)-c(RGDyK) conjugate for integrin-
targeted photodynamic therapy
T. Chatzisideri, S. Thysiadis, S. Katsamakas, P. Dalezis, I. Sigala, T. Lazarides, E.
Nikolakaki, D. Trafalis, O.A. Gederaas, M. Lindgren, V. Sarli
PII:
S0223-5234(17)30773-0
10.1016/j.ejmech.2017.09.058
EJMECH 9773
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 20 June 2017
Revised Date: 25 September 2017
Accepted Date: 27 September 2017
Please cite this article as: T. Chatzisideri, S. Thysiadis, S. Katsamakas, P. Dalezis, I. Sigala, T.
Lazarides, E. Nikolakaki, D. Trafalis, O.A. Gederaas, M. Lindgren, V. Sarli, Synthesis and biological
evaluation of a Platinum(II)-c(RGDyK) conjugate for integrin-targeted photodynamic therapy, European
Journal of Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.09.058.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Synthesis and Biological Evaluation of a Platinum(II)-c(RGDyK) Conjugate for
Integrin-Targeted Photodynamic Therapy
T. Chatzisideri,a S. Thysiadis,a S. Katsamakas,b P. Dalezis,c I. Sigala,a T. Lazarides,a E. Nikolakaki,a D. Trafalis,c O.
A. Gederaas,d M. Lindgrene and V. Sarli*,a
a
Department of Chemistry, Aristotle University of Thessaloniki, University Campus, 54124, Thessaloniki,
Greece, sarli@chem.auth.gr
b
Department of Pharmaceutical Chemistry, School of Pharmacy, Aristotle University of Thessaloniki,
University Campus, 54124, Thessaloniki, Greece
c
Laboratory of Pharmacology, Medical School National and Kapodistrian University of Athens 75 Mikras
Asias Street, Athens, 11527, Greece, dtrafal@med.uoa.gr
d Department of Cancer Research and Molecular Medicine, Norwegian University of Science and Technology,
NTNU, N-7491 Trondheim, Norway, odrun.gederaas@ntnu.no
e
Department of Physics, Norwegian University of Science and Technology, NTNU, N-7491 Trondheim,
Norway, mikael.lindgren@ntnu.no
Highlights
•
•
•
•
•
A cancer-targeting conjugate of a cyclometalated [N,C,N-Pt(II)] complex has been synthesised
The conjugate exhibited a mild cytostatic effect to six different cancer cell lines
The conjugate demonstrated rapid cell uptake by receptor-mediated endocytosis
The conjugate efficiently generated 1O2 upon irradiation
Conjugate 4 resulted in reduction of cell survival of AY27 cells after blue light exposure
Abstract
A cancer-targeting conjugate 4 of a cyclometalated [N,C,N-Pt(II)] complex bearing a N^C^N 1,3-di(2-pyridyl)-
benzene with c(RGDyK) peptide as guiding molecule was designed and synthesized for real-time drug
delivery monitoring in cancer cells and photodynamic therapy (PDT). This conjugate demonstrates a mild
cytostatic effect to six cancer cell lines expressing integrins at different extent, while possessing promising
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features for PDT. Conjugate 4 demonstrated rapid cell uptake by receptor-mediated endocytosis and
efficient generation of 1O2 upon irradiation. Incubation of rat bladder cancer cells AY27 with conjugate 4 (50
μΜ) prior to blue light exposure (5 min) resulted in significant reduction (50%) of cell survival compared to
control cells as measured by MTT assay post 24 h after treatment.
Keywords: Targeted drug delivery, RGD peptide, integrin avβ3, Angiogenesis, Tumor therapy, Pt(II)-RGD
conjugate, Photodynamic therapy, theranostic agent
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Introduction
Photodynamic therapy (PDT) is a clinically approved method in oncology, by which photosensitizers (PSs)
generate cytotoxic reactive oxygen species (ROS) for the efficient killing of cancer cells under visible light
illumination via oxidative damage [1]. PDT has been applied for the treatment of various cancers such as
melanoma, head and neck, bladder, breast, and pulmonary carcinomas [2,3,4,5]. Main factors that
determine PDT efficacy include the PS's ability to produce ROS, as well as its selectivity and targeting ability
[6]. Presently, scientific endeavors have focused on the development of targeted drug delivery systems for
effective delivery of PSs [7].
Targeted drug delivery offers an attractive strategy to achieve the most of the drug's potential therapeutic
benefit and minimize any undesirable side effects at the lowest dose [8,9]. Peptides such as Arg-Gly-Asp
(RGD) peptides [10,11], octreotide [12], bombesin [13] and poly-Arg peptides [14] have been extensively
used as carriers to selectively direct drugs to cancer cells with upregulated receptors [15, 16]. A variety of
linear as well as cyclic RGD peptide delivery systems with cytotoxic drugs have been developed to target
integrin receptors αvβ3 and αvβ5 that are overexpressed in several cancer types such as melanoma,
glioblastoma, ovarian, prostate and breast cancer [17, 18, 19, 20, 21]. RGD conjugates for theranostic
applications are also available for monitoring of drug release to ensure efficient delivery [22]. Commonly
employed labels that allow effective drug monitoring by the use of various fluorescence microscopy
techniques include luminescent complexes of transition metals [23,24]. These complexes have been proved
effective for live cell imaging and in vivo detection of target molecules [25].
Scheme 1. Schematic illustration of [N,C,N-Pt(II)]-c(RGDyK) conjugate 4 for integrin-targeted PDT
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In particular, cyclometalated Pt(II) complexes display useful photochemical properties and have been
extensively studied for their use as agents for biological labeling [26], chemosensors [27], phosphorescent
emitters in organic light-emitting diodes (OLEDs) [28], photocatalysts [29], anticancer agents [30], optical
power limiting [31] and photovoltaic devices [32]. Another important application of platinum (II) complexes
is in cancer chemotherapy [33]. Cisplatin, carboplatin, and oxaliplatin are among the most widely used
antitumor agents. However, their clinical application is limited by acquired or intrinsic resistance and/or their
high toxicity mainly due to their nonselective mode of action [34]. Platinum complexes exhibit cytostatic
effects by interacting with DNA, thus inhibiting the DNA synthesis.
In this study, we aimed to develop a novel delivery system of a Pt(II) complex with c(RGDyK) peptide suitable
for targeted tumor therapy and theragnostic applications. Toward this direction, [N,C,N-Pt(II)] complex 3 was
attached to c(RGDyK) via a stable amide bond. Complex 3 bearing a N^C^N 1,3-di(2-pyridyl)-benzene was
employed based on its reported advantageous photophysical and antiproliferative properties (Scheme 1).
De Cola and coworkers have previously reported the activities of a series of [N,C,N-Pt(II)] complexes as well
as their application as bio-imaging probes [35]. They have shown that these complexes display wide emission
color tunability, a very large Stokes shift and long lived luminescent excited states. In another study, the
water soluble, cyclometalated [N,C,N-Pt(II)] complex 2 has been used for imaging of live cells and histological
tissues with high resolution by “two-photon time-resolved emission imaging microscopy” (TP-TREM) [36].
In addition, Weinstein and Bryant showed that complex 1 induces photodynamic killing in a range of cancer
cells including a cisplatin resistant cell line [37]. Herein, we show that the [N,C,N-Pt(II)]-c(RGDyK) conjugate 4
exhibits a moderate cytostatic activity in a panel of integrin expressing cancer cells in vitro and possesses
promising photochemical properties for PDT applications in rat bladder cancer cells (AY27).
Results and discussion
Synthesis of [N,C,N-Pt(II)]-c(RGDyK) conjugate 4
The synthesis of conjugate 4 is outlined in Scheme 2. Methyl 3,5-dibromobenzoate 5 was subjected to
reaction with pinacolborane in the presence of Pd(dppf)Cl2 under microwave irradiation to give aryl boronic
ester 6 [38]. A Suzuki–Miyaura coupling of 6 with 2-chloropyridine using Pd(OAc)2, PPh3 and Na2CO3 in THF
and H2O at 85 °C yielded methyl 3,5-di(pyridin-2-yl)benzoate in 63% yield. Cycloplatination with K2PtCl4 in
CH3CN as previously described [39], followed by methyl ester hydrolysis afforded the desired acid 3 [40].
Subsequently, [N,C,N-Pt(II)] complex 3 was activated using EDCI and NHS and the resulting N-
hydroxysuccinimidyl ester was coupled with the c(RGDyK) peptide lysine amino group to give conjugate 4 in
excellent yield. The cycloplatinated structures of 3 and 4 were confirmed by 1H-NMR and mass spectrometry
(ESI-MS for C44H51ClN11O9Pt+ [M+H]+=1107.32, was found 1107.95, Figure S2).
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After HPLC purification of 4, ESI-LCMS analysis revealed that the purity of 4 was greater than 93%. When the
same sample after HPLC purification was lyophilized, dissolved in MeOH/DMF and analyzed again, one more
peak was observed in the chromatograph, which was attributed probably to platinum(II) aqua complex 7.
When this product was isolated by HPLC and analyzed again the chromatograph indicated the presence of 4.
Thus, it seems that the two compounds are in equilibrium and interconvert under certain conditions possibly
as a consequence of reversible ligand exchange reactions. The molecular ions at m/z 556, 1071, 1186 were
interpreted as [M-Cl+H+CH3CN]2+/2, [M-Cl]+, [M+H+2CH3CN]+ species (Figure S2). A number of experiments
were performed involving the ionization of 4 with or without chromatographic separation diluted in
different solvents. The study of the hydrolysis of 4 by the LC-MS method was very challenging, as the
intermediate platinum(II) aqua complexes were highly reactive with organic molecules, solvents and ions
and the complexes observed via ESI-MS did not always match with the species in solution (Figure S2, S3 S4).
For example the peak at m/z 1097.75 was attributed to [M-Cl+N2]+ arising from the clustering of [M-Cl]+ with
dissolvation gas, a phenomenon previously observed during the ESI-MS ionization of cis-platin [41]. It should
also be noted that 4 displays poor solubility in water and it was dissolved in DMSO prior to biological
experiments.
Scheme 2. Synthesis of [N,C,N-Pt(II)]-c(RGDyK) conjugate 4
The UV-vis absorption spectrum of conjugate 4 in DMSO solution is dominated by strong absorption bands in
1
the UV region (λ < 300 nm), which are attributed to π-π* transitions of the ligand and the phenol moiety of
tyrosine. The lowest energy absorption band of conjugate 4 lies in the region between 350 and 440 nm and
1
is attributed to overlapping ligand based π-π* and singlet metal-to-ligand-charge-transfer transitions
(1MLCT) [39]. Selective excitation of the platinum chromophore of conjugate 4 at 400 nm, gives rise to a
structured emission band with maximum at ca. 487 nm attributed to a transition of predominantly ligand
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based 3π-π* character [39]. The good match between the absorption and excitation profiles further confirms
that the emission of conjugate 4 originates from the platinum(II) chromophore. These spectroscopic
observations are in excellent agreement with those made in the cases of similar platinum(II) complexes [39]
indicating that the cyclometallated chromophore is virtually unaffected by conjugation to the peptide. It is
worth noting that in more concentrated solutions (>5 × 10-6 M) in aqueous solvent mixtures, the emission
spectra of 4 are dominated by a featureless excimer band centred at ca. 660 nm (Figure S2) indicating that
even in the presence of the bulky peptide moiety, the platinum chromophores are still able to stack and
form excimers under certain conditions.
Figure 1: Normalized absorption, emission and excitation profiles of conjugate 4 in DMSO solution (5 × 10-6
M).
Reactivity of [N,C,N-Pt(II)]-c(RGDyK) conjugate 4 toward amino acids and water
In order to study the behavior of conjugate 4 in a biological system, the reactivity of model complex 3 in
aqueous solution was also investigated. The hydrolysis and the reversibility of the hydrolysis of 3 were
1
studied by analyzing the H-NMR spectra of (∼8.5 mM) solutions of 3 in d6-DMSO/D2O (9/1 v/v) at 25 °C,
1
both without NaCl and in the presence of 10 mM NaCl. The H NMR spectra of complex 3 (∼8.5 mM) in d6-
DMSO, and in d6-DMSO/D2O (9/1 v/v) are shown in Figure 2A and Figure 2B, indicating the fast hydrolysis of
3 after 10 min in the aqueous medium. The reversibility of the hydrolysis in the presence of NaCl (10 mM) is
shown in Figure 2C indicating that the hydrolyzed sample of 3 most probably returns quantitatively to the
chlorido complex. In addition, the ESI-MS data of 3 (dissolved in DMSO/H2O=9/1) revealed a set of peaks at
m/z 510.65 showing the Pt isotope pattern, which is attributed to hydroxido platinum(II) complex [M-
Cl+OH+Na]+ (Figure S7).
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1
Figure 2. H NMR (500 MHz) spectra of complex 3 in (A) d6-DMSO and (B) d6-DMSO (90%) /D2O (10%) after
10 min. C) Addition of NaCl 10 mM to sample of (B) after 30 min.
To evaluate the stability of conjugate 4 at different pH values, it was dissolved in DMSO/MeOH and diluted
with phosphate or acetate buffers at 37 °C. The LC-MS analysis data indicated the hydrolysis of 4 after 24h at
both physiological pH 7.4 buffer and acidic pH 5.2 (Figure S8, S9). The reactivity of conjugate 4 toward small
biomolecules was also assessed by studying the reaction of 4 with selected amino acids such as L-histidine
(His), L-cysteine (Cys), and glycine (Gly). Conjugate 4 was incubated with an equimolar mixture of Gly, Cys,
and His and the reaction mixture was analyzed by ESI-LCMS. After 6 h, amino acid adduct peaks started to
form preferably with Cys [(M-Cl+2Cys+2H)2+]/2=656 and His [(Μ-Cl+His+2Na)2+/2= 636]. It should be noted
that precipitation of insoluble platinum compounds were largely observed.
Photosensitising properties of [N,C,N-Pt(II)]-c(RGDyK) conjugate 4
In order to confirm the production of singlet oxygen, triplet excited state absorption recorded for UV
excitation (λ = 355 nm, Figure 3A) and the subsequent formation of singlet oxygen, was measured from the
direct luminescence at 1275 nm (Figure 3B). Firstly, solutions of compounds 3 and 4 in DMSO with the same
concentration corresponding to an absorbance of approximately 0.15, were prepared (Figure 3C). In order to
prove the existence of triplet states in 3 and 4, excited state absorption was measured by exciting Argon
flushed samples with ns laser pulse at 355 nm, and recording the full spectrum with a broad band flash lamp
and a gated detector synchronized with the excitation pulse. These spectra (Figure 3A) show depleted
absorption bands in the 350 -420 nm region due to bleaching of the ground states of 3 and 4 (view
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comparison with ground state absorption spectra in Figure 3C) accompanied by the emergence of broad
3
low-energy absorption signals in the 400-500 nm region. The latter are attributed to ligand-based π-π*
absorptions [42]. Repeating the procedure for a sample containing oxygen the triplet absorption signal are
strongly quenched and diminishes (data not shown). Next, the transient singlet oxygen luminescence was
verified by exciting the sample using a 420 nm laser and recording the transient luminescence at 1275 nm.
Here, an oxygen containing sample was used, and the background subtraction was carried out using the
Argon flushed sample for each case. A weak transient signal was observed in both samples using DMSO as
solvent (Figure 3B). Complex 3 (but not 7) exhibited solubility in THF and it was possible to record a much
stronger signal from the singlet oxygen (Figure 3B) to confirm the energy transfer from the triplet state to
proximate dissolved oxygen.
Figure 3. A) Excited state absorption with solutions of 3 and 4 in DMSO, B) Singlet oxygen luminescence at
1275 nm with solutions of 3 and 4 in THF and DMSO, C) Absorption of solutions of 3 and 4 in DMSO used for
excited state absorption and singlet oxygen luminescence measurements
Cellular uptake of [N,C,N-Pt(II)]-c(RGDyK) conjugate 4
To demonstrate the role of the RGD moiety in targeting conjugate 4 to αvβ3 integrin-rich tumor cells,
compounds 3 and 4 were incubated with U87 and Hela cells. The reason for choosing these two cell lines is
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because U87 glioblastoma cells contain considerably higher levels of αvβ3 integrin receptors than HeLa cells
[43, 44, 45, 46]. When U87 cells were treated with 4, a strong fluorescence signal was observed, indicative
of the efficient cellular uptake of conjugate 4, whereas the fluorescence intensity in HeLa cells was much
weaker (Figure 4). The strong fluorescence signal seen in U87 cells is due to αvβ3 integrin receptor-mediated
endocytosis, since the uptake of an analogous compound 3, which however lacks an RGD-targeting group, is
significantly less.
Figure 4. Confocal microscopy images of U87M and HeLa cells after incubation with 10 μΜ of 4 and 3 for 0
and 2 h, respectively. Cells were then washed twice with PBS to remove floating compounds and were
visualized in a Nikon confocal microscope using the EZ-C1 3.20.
To provide further evidence that the uptake of conjugate 4 was mediated through endocytosis, we repeated
the experiment using an endocytosis inhibitor, okadaic acid [47, 48, 49]. As it is clearly shown in Figure 5,
when U87 cells were treated with 4 the fluorescence signal markedly decreased following addition of 100
nΜ okadaic acid, whereas, when 3 which lacks the RGD-targeting group was added to U87 cells the
fluorescence intensity remained practically unaffected in the presence of okadaic acid.
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Figure 5. Confocal microscopy images of U87 cells treated with 10 μΜ of 4 and 3 for 2h. Cells were
preincubated, prior to addition of 4 and 3, with medium containing DMSO (control) or okadaic acid (100 nM)
for 30 min. The floating conjugate was removed by washing twice with PBS and cells were then visualized in
a Nikon confocal microscope using the EZ-C1 3.20.
Cytotoxicity of [N,C,N-Pt(II)]-c(RGDyK) conjugate 4
The antiproliferative activities of [N,C,N-Pt(II)]-c(RGDyK) conjugate 4 in comparison with c(RGDyK) and
complex [N,C,N-Pt(II)] 3 were studied by MTT assays in 6 cell lines of different tumour types. The PC3 [50],
SCOV-3 [51], A549 [52], MDA-MB-231 [53,54], MCF-7 cells [55] and U87M [56] cell lines all express integrin
αvβ3 at different extent. Results regarding the cytostatic and cytotoxic activity of the compound 4 are
presented in Table 1. Treatment with c(RGDyK), 3 and 4 reduced the cell growth of all six cell lines at
different extent and their IC50 values were estimated to be higher than 100 μM. In general, it was
demonstrated that the tested compounds exhibit mostly cytostatic activity rather than cytotoxic (Figure
6,7,8). Compounds c(RGDyK), 3 and 4 exhibited a moderate cytostatic effect against PC3 and A549 and a
very good cytostatic effect against MDA-MB-231 cell lines. It is worth noting that SKOV-3 are more sensitive
to 4 (GI50= 12 μM) than its precursor 3 (GI50>100 μM). The growth inhibitory effect against MDA-MB-231
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cells is slightly increased for conjugate 4 GI50= 10 μM, TGI=58 μΜ versus GI50 =26 μΜ, TGI=78 μΜ for
complex 3.
Table 1. Growth inhibition/cytostatic (GI50 and TGI) and cytotoxic/cytocidal (IC50) anticancer effects induced
by c(RGDyK), complex 3 and conjugate 4 on six human cancer cell lines
Cell Lines
c(RGDyK)
TGI
Complex 3
TGI
Conjugate 7
TGI
GI50
>100
74,5
61
IC50
GI50
>100
18
IC50
GI50
12
IC50
SKOV-3
PC-3
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
49
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
58
>100
>100
>100
>100
>100
>100
22
A549
28
>100
>100
78
76
MCF-7
>100
72
>100
26
>100
10
MDA-MB-231
U87M
40
>100
>100
>100
>100
Figure 6. Growth inhibition (sensitivity curves) of PC-3, SKOV-3, A549, MCF7 and U87 cells, produced after
treatment with cRGDyK and complex 3. GI50 represents the 50% growth inhibition; IC50 the half maximal
inhibitory concentration and TGI the total growth inhibition.
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Figure 7. Growth inhibition (sensitivity curves) of PC-3, SKOV-3, A549, MCF7 and U87 cells, produced after
treatment with conjugate 4. GI50 represents the 50% growth inhibition; IC50 the half maximal inhibitory
concentration and TGI the total growth inhibition.
Figure 8. Growth inhibition (sensitivity curves) of MDA-MB-231 cells, produced after treatment with
c(RGDyK), complex 3 and conjugate 4.
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To evaluate the potential of conjugate 4 for PDT, the effect of blue light exposure on survival of rat bladder
cancer cells (AY27) was determined after treatment with complex 3 and conjugate at different
concentrations (Figure 9). This cell line has been previously used in an orthotopic rat bladder cancer model
for PDT studies and is chosen to be further applied to in vivo experiments [57].
Preliminary PDT experiments show a clear dose dependent reduction of cell viability after blue light
exposure. Incubation with conjugate 4 (50 μΜ) prior to light exposure (5 min) resulted in significant
reduction (50%) of cell survival compared to control cells as measured by MTT assay post 24 h after
treatment. The experimental data clearly showed cell death in the dark (“dark toxicity”) compared to control
cells. A reduction of 31% and 43% in cell viability was observed at 0 min of light exposure after treatment
with 50 μΜ of complex 3 and conjugate 4, respectively. Importantly, it was found that the cell viability effect
of 4 is about 10% higher compared to compound 3 (5 min). This finding indicates the important contribution
of c(RGDyK) peptide to the anticancer action of conjugate 4.
Conclusion
Taken together, the results indicate that the combination of complex [N,C,N-Pt(II)] 3 with c(RGDyK) leads to
a new delivery system with drug monitoring capabilities and promising anticancer activities for PDT
applications. The synthesis, characterization, photophysics, cellular uptake and toxicity after blue light
exposure of conjugate 4 has been presented, indicating a great potential of such type of conjugates in
targeted photodynamic therapy. Based on these encouraging results, further in vitro and vivo experiments
are in progress and will be reported in due course.
Figure 9. Survival of cancer cells AY27 post 24 h after treatment with complex 3 or conjugate 4.
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Acknowledgements
This programme was implemented within the framework of the IKY/SIEMENS Excellence Research Grants
2016 (Synthesis of anticancer peptide derivatives). S. Katsamakas is thankful to Aristotle University of
Thessaloniki Research Committee for financial support through an excellence Post-doctoral Scholarship. The
authors declare to have no competing financial interests.
Experimental procedures
General Experimental Details. All reactions were carried out under an atmosphere of Ar unless otherwise
specified. Commercial reagents of high purity were purchased and used without further purification, unless
otherwise noted. Reactions were monitored by TLC and using UV light as a visualizing agent and aqueous
ceric sulfate/phosphomolybdic acid, ethanolic p-anisaldehyde solution, potassium permanganate solution,
1
13
and heat as developing agents. The H and C NMR spectra were recorded at 500 and 125 MHz (Agilent)
with tetramethylsilane as an internal standard. Chemical shifts are indicated in δ values (ppm) from internal
1
reference peaks (TMS 1H 0.00; CDCl3 H 7.26, 13C 77.00; DMSO-d6 1H 2.50, 13C 39.51). Melting points (mp) are
uncorrected. High-resolution mass spectra (HRMS) were recorded on a Waters Synapt G2 HDMS (Waters,
UK) mass spectrometer with an electrospray source acquired positive and negative ion data over a mass
range 50-2000 m/z units. Microwave reactions were carried out in a Biotage Initiator microwave system 2.0
(400W, operating at 2.45 GHz). HPLC purifications using C18 analytical (Gracesmart RP 18, 4.6 mm × 250
mm) column was performed with Scientific Systems Inc. instrumentation comprising of 4-Q Grad Pumps
connected to diode array (UV-Vis Thermo Finnigan Spectra system UV6000LP Detector, Lab Alliance (New
York, USA). The LC-MS spectra were recorded on a LC-20AD Shimadzu connected to Shimadzu LCMS-2010EV
equipped with C18 analytical column (Reprospher 100 C18-DE, 5μm 250 x 4.6mm, Dr Maisch GmbH). UV/Vis
spectra were recorded with a Hitachi 2001 UV/Vis spectrometer (quartz 1cm). Fluorescence spectra were
obtained on a Hitachi F7000 fluorimeter equipped with a Hamamatsu red sensitive R928 photomultiplier
tube (Measurements were performed on air equilibrated samples, quartz 1cm3). For PDT experiments a Lumi
Source lamp (PCI Biotech, Oslo, Norway), blue light (λ = 435 nm, 13 mW/cm2) was used.
Synthetic procedures
Methyl 3,5-bis(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoate, 6: Pd(dppf)Cl2 (22 mg, 6 mol-%) and
methyl 3,5-dibromobenzoate 5 (150 mg, 0.51 mmol) were placed in a dry screw-cap vessel with a septum.
Then, dry toluene (2.6 mL) was added, and the mixture was degassed with argon (1 min). Dry triethylamine
(0.6 mL) and 4,4,5,5-tetramethyl-1,3,2-dioxaborolane (0.25 mL, 1.53 mmol) were successively added to the
mixture. The reaction was ultrasonicated (1-2 min) to affect solution. The vial was transferred to the
microwave synthesizer and after pre-stirring 15 s, irradiated for 8 h at 140 oC using a power setting of "High".
After cooling, the reaction vessel was uncapped and the mixture was filtered through Celite. The filtrate was
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concentrated and the residue was purified by flash chromatography (eluent; hexane/ethyl acetate = 10/1) to
1
obtain 87 mg of 6 (total yield= 45%). 6: H NMR (500 MHz, CDCl3) δ 8.55 (s, 2H), 8.43 (s, 1H), 3.91 (s, 3H),
13
1.35 (s, 24H); C NMR (126 MHz, CDCl3) δ 167.2, 145.4, 138.6, 132.3, 128.9, 84.0, 51.9, 24.9; FT-IR (KBr):
2980, 2952, 2929, 1720, 1595, 1450, 1401, 1372, 1333, 1320, 1248, 1167, 1143, 988 cm-1; ESI-MS, positive
mode (M=C20H30B2O6): m/z 389 [M+H]+.
Methyl 3,5-di(pyridin-2-yl)benzoate, S1: A mixture of 1,3-diboronic acid dipinacol ester derivative 6 (275
mg, 0,708 mmol) and 2-chloropyridine (0.13 mL, 1.417 mmol) were dissolved in 7.5 mL THF. Then, Na2CO3
(601 mg, 5.67 mmol) in H2O (1.4 mL), PPh3 (111 mg, 0.425 mmol) and Pd(OAc)2 (32 mg, 0.142 mmol) were
added and the mixture was degassed with argon. The mixture was heated at 85 °C overnight. Subsequently,
after cooling to rt the product was extracted with dichloromethane. The organic layers were dried over
Na2SO4, filtered and concentrated under vacuum. The residue was purified by flash chromatography (eluent;
hexane/ethyl acetate = 5/1) to afford 130 mg of compound S1 (yield = 63%). Characterization was in
1
agreement with literature data [39]. S1: H NMR (500 MHz, CDCl3) δ 8.92 (t, J = 1.8 Hz, 1H), 8.78 – 8.73 (m,
13
2H), 8.72 (d, J = 1.7 Hz, 2H), 7.91 (d, J = 7.8 Hz, 2H), 7.81 (m, 2H), 7.29 (m, 2H), 3.99 (s, 3H); C NMR (126
MHz, CDCl3) δ 166.8, 156.1, 149.7, 140.3, 136.9, 131.3, 129.8, 128.4, 122.7, 120.8, 52.3.
[4-(Methoxycarbonyl)-2,6-bis(pyridin-2-yl)phenyl]platinum(II)
chloride,
S2:
Methyl-3,5-di(2-
pyridyl)benzoate S1 (130 mg, 0.448 mmol) was dissolved in acetonitrile (15 mL) and a solution of K2[PtCl]4
(186 mg, 0.448 mmol) in water (5 mL) was added, giving a yellow-orange solution. The reaction mixture was
heated at vigorous reflux for 3 days, and then allowed to cool, giving a yellow precipitate. The precipitate
was separated, washed with water (4 x 5 mL), ethanol (4 x 5 mL), acetonitrile (4 x 5 mL), and diethylether
(2x5 mL), and then dried under reduced pressure. The product was isolated in 90% yield (208 mg).
1
Characterization was in agreement with literature data [39]. S2: H NMR (500 MHz, DMSO-d6) δ 9.17 (d, J =
5.0 Hz, 2H), 8.35 (m, 4H), 8.30 – 8.19 (m, 2H), 7.71-7.54 (m, 2H), 3.91 (s, 3H); ESI-MS, positive mode (M=
C18H14N2NaO3Pt): m/z 524.95 [M-Cl+OH+Na]+.
4-Carboxy-2,6-bis(pyridin-2-yl)phenyl]platinum(II) Chloride, 3: KOH (755 mg, 13.46 mmol) was dissolved in
MeOH (3.5 mL) and then [4-(methoxycarbonyl)-2,6-bis(pyridin-2-yl)phenyl]platinum(II) chloride (70 mg,
0.135 mmol) was suspended in this solution. The suspension was stirred at 40 °C for 24 h. After that, a 4M
HCl (6.7 mL, 26.92 mmol) solution was added to the mixture. The yellow precipitate was washed with water
(3 x 15 mL) and MeOH (2 x 15 mL) and the solvents evaporated to dryness. A yellow solid was obtained in
83% yield (80 mg). Characterization was in agreement with literature data [40]. 3: 1H NMR (500 MHz, DMSO-
d6) δ 12.98 (s, 1H), 9.15 (d, J = 5.5 Hz, 2H), 8.30 (m, 4H), 8.27-8.18 (t, J= 7.5 Hz, 2H), 7.61 (t, J= 7.5 Hz, 2H); 13C
NMR (126 MHz, DMSO-d6) δ 167.7, 166.8, 165.6, 151.2, 140.6, 140.5, 125.9, 125.8, 124.5, 120.9; ESI-MS,
positive mode (M= C17H12N2NaO3Pt): m/z 510.65 [M-Cl+OH+Na]+.
15

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4-(2,5-dioxopyrrolidin-1-yl)carbonyl-2,6-bis(pyridin-2-yl)phenyl]platinum(II) chloride, S3: To a solution of 3
(0.06 mmol, 30 mg) in DMF (0.6 mL), EDCI (0.07 mmol, 12.5 mg) and NHS (0.07 mmol, 7.5 mg) were added.
The reaction mixture was left stirring overnight. A yellow precipitate was formed. The yellow solid was
1
washed with DMF (3 X 0.5 mL) and dried to give 28 mg of S3 in 77% yield. S3: H NMR (500 MHz, DMSO-d6)
δ 9.02 (d, J = 5.5 Hz, 2H), 8.32-8.18 (m, 6H), 7.58 (t, J = 6.4 Hz, 2H), 2.97 (s, 4H); 13C NMR (126 MHz, DMSO-d6)
δ 170.5, 170.1, 164.7, 162.3, 151.2, 141.2, 140.5, 125.9, 124.9, 121.4, 118.8, 25.6; FT-IR (ΚBr): 2958, 2923,
1736, 1608, 1588, 1479, 1425, 1364, 1342, 1242, 1202, 1069, 1036, 1007, 862; ESI-MS, positive mode (M=
C21H15N3NaO5Pt): m/z 607.75 [M-Cl+OH+Na]+.
Conjugate 4: To a solution of cRGDyK (3.2 μmol, 2 mg) in dry DMF (0.8 mL), DIPEA was added (3.8 μmol, 0.7
μL), following the addition of the activated ester S3 (1.6 μmol, 0.96 mg). The reaction was left stirring at
room temperature for 48 h and monitored by LC-MS. The solvent was evaporated in vacuo and the product
was purified by reverse-phase HPLC (linear gradient from 0 to 100% B in 32 min, flow rate: 1 mL/min, Rt = 18
min). After chromatography 1.72 mg of conjugate 4 were obtained in 98% yield. The purity of the conjugate
was confirmed by LC-MS and mass spectrometry (linear gradient from 90% H2O to 10% acetonitrile, HCOOH
0.1%, over 30 min, flow rate: 0.4 mL/min, Rt = 13.5 min). ESI-MS with chromatographic separation, positive
mode: m/z calcd mass for C48H57ClN13O9Pt+ [M-Cl+2ACN+H]+= 1189.37, was found 1189.56; calcd mass for
C44H50N11O9Pt+ [M-Cl]+= 1071.35, was found 1071.15
calc mass for C44H52N11NaO10Pt2+ [M-
Cl+H+CH3CN]2+/2=556.68, was found 556.75; ESI-MS direct ionization, positive mode: m/z calcd mass for
C44H51ClN11O9Pt+ [M+H]+=1107.32, was found 1107.95; calcd mass for C44H50ClN11NaO9Pt+
[M+Na]+=1129.3027, calcd mass for C46H54ClN12O9Pt+ [M+H+CH3CN]+=1148.34 was found 1149.20; HRMS
(ESI) with chromatographic separation m/z calcd for C44H51N11O9Pt+ [M-Cl]2+/2 =536.1756; was found
536.1739, C46H54N12O9Pt+ [M-Cl+H+CH3CN]2+/2 =556.6890; was found 556.6889; 1H NMR (500 MHz, DMSO-d6
with 20 equiv NaCl) δ 9.16 (s, 1H), 9.15 (s, 1H), 8.63 (t, J = 5.3 Hz, 1H), 8.35 (s, 1H), 8.34 (s, 1H), 8.31 – 8.18
(m, 8H), 7.94 (d, J = 7.7 Hz, 1H), 7.61 (m, 3H), 6.89 (d, J = 8.2 Hz, 1H), 6.59 (d, J = 8.2 Hz, 1H), 6.52 (s, 1H), 4.67
(m, 1H), 4.43 (d, J = 14.0, 7.2 Hz, 1H), 4.22 (m, 1H), 4.10 (m, 2H), 3.09 (m, 2H), 2.87 (m, 1H), 2.83 – 2.70 (m,
1H), 2.64 (m, 2H), 2.41 – 2.32 (m, 2H), 2.08 (d, J =6.0 Hz, 1H), 2.00 (m, 1H), 1.75 (m, 1H), 1.65 (m, 1H), 1.52
(m, 2H), 1.40 (m, 1H), 1.25 – 1.16 (m, 4H) (1 proton is missing due to overlapping to DMSO-d6); 13C NMR (126
MHz, DMSO-d6) δ 172.2, 171.7, 171.2, 170.9, 169.9, 169.5, 165.2, 156.6, 155.7, 150.6, 141.5, 141.3, 140.6,
129.9, 127.2, 124.9, 124.6, 123.8, 120.9, 114.9, 54.6, 51.9, 48.9, 43.2, 36.7, 34.9, 30.7, 29.1, 29.0, 28.7, 28.5,
26.6, 25.2, 22.8, 22.1 (2 carbons are missing due to overlapping to DMSO-d6).
Spectroscopic characterization and singlet oxygen production of complex 3 and conjugate 4: UV quartz
cuvettes with 1 cm path length were used. DMSO and THF were used as solvents with Teflon caps allowing
flushing with Argon gas to remove oxygen from the solvent. Absorbance spectra were obtained using a U-
16

ACCEPTED MANUSCRIPT
3010 spectrophotometer (Hitachi, Japan), and the software UV Solution. Steady state luminescence spectra
were obtained using a Fluoromax II spectrometer.
The excited triplet state absorbance was measured using a NT 342B-SH-10-WW laser (Ekspla, Lithuania),
array detector (Applied Photophysics, United Kingdom), and a Xenon flash lamp module (Model L9456-01,
5W, Hamamatsu) with BWSpec software (B&W Tek, USA). Transient absorption was obtained by firing the
excitation OPO laser and flashlamp using DG535 Delay/Pulse generator (Stanford Research Systems) for
control of triggers and detectors. Prior to measurement the sample was flushed with Ar-gas for 10 minutes
in order to remove oxygen that quench the triplet signal. The recorded spectra was background corrected by
subtracting the spectrum of pure solvent. Since the fluorescence was negligible, no need for fluorescence
emission was needed. For details of the set-up and the procedure of analyzing similar triplet state absorption
data, see Glimsdal et al [42].
The singlet oxygen production was demonstrated as the transient singlet oxygen luminescence (1275 nm) of
a solution with the sample in a standard 90o configuration. A tunable OPO laser, NT 342A-SH-10-WW (Ekspla,
Lithuania) was used for excitation. For transient recording a PMT (R5509, Hamamatsu) and wavelength
selective filters (interference filter with maximum transmission at 1272.5 nm and a long pass filter
transmitting above 780 nm) were used, an Infiniium BDSU Oscilloscope (Keysight, United States) was used to
collect data. A DG535 Delay/Pulse generator (Stanford Research Systems) was used to control the time
between the laser excitation and the recording of the luminescence transient. The transients were
background corrected by subtracting with signal of the same sample flushed with Argon for 10 min. A similar
procedure for confirming singlet oxygen production of Ruthenium complexes for PDT was recently presented
in Bogoeva et al [58].
Stability Evaluation: Aqueous stability of the conjugate was determined at pH 7.4 and 5.2. Conjugate 4 (0.32
mg, 0.289 μmol) was diluted in 30 μL DMSO and 16 μL solution of MeOH/H2O (1/1). Next, 250 μL of buffer
solution (phosphate buffer pH 7.4 or acetate buffer pH 5.2) was added and the solution was incubated at 37
°C. Mass spectra were recorded after 1, 3, 6, 24, and 48 h of incubation at 37 °C.
Reactivity toward aminoacids: Conjugate 4 (0.33 mg, 1.007 μmol) was diluted in 30 μL DMSO and 16 μL
mixture of aminoacids L-histidine (1.007 μmol)/L-cysteine (1.007 μmol)/glycine (1.007 μmol) in MeOH/H2O
(1/1). Next, 250 μL of double distilled water was added and the solution was incubated at 37 °C. Mass
spectra were recorded after 1, 3, 6, 24, and 48 h of incubation at 37 °C.
Cell lines: Three compounds conjugate 4, compound 3 and c(RGDyK) were tested for anticancer activity
against six well established human cancer cell lines (1 ovarian, 1 breast, 1 prostate cancer, 1 lung and 2
glioblastoma). SKOV-3 ovarian cancer cells, MCF7 breast adenocarcinoma cells, PC-3 prostate
17

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adenocarcinoma cells, A549 lung adenocarcinoma cells, U251M and U87M, astrocytoma cells were tested
for cytostatic (growth inhibition: IC50, TGI) and cytotoxic/cytocidal (IC50) activity against the three compounds
at concentrations of 1-100 μΜ. The cell lines were obtained from the American Type of Culture collection
(ATCC) and were grown in different culture medium according to the instructions. Cultures were maintained
at 37 oC in 5% CO2 and 95% air. Rat bladder transitional carcinoma cells (AY27) were maintained using RPMI-
1640 culture medium (BioWhittaker) supplemented with 5% L-glutamine, 1% penicillin/streptomycin, 0.1%
o
fungizone and 10% fetal bovine serum (FBS) in a humidified atmosphere of 37 C in 5% CO2 and 95% air as
described in our earlier study [59].
Photodynamic Treatment: A number of 0.5 x 106 cells were seeded per Petri dish (diameter: 6 cm) and
cultured for one day. Subsequent to wash cells with PBS twice, RPMI-1640 culture medium (FBS-free)
containing different concentrations of the 3 or 4, was added to cells and kept in the dark for 3.5 h. The
medium was then replaced with PBS, and the cells were exposed to blue light (435 nm, 13 mW/cm2) at
different intervals (Lumi Source, PCI Biotech AS, Norway), given a total light dose of 7.8 J/cm2 on the cell
level during a 10 min illumination period, see Bogoeva et al [58]. The light was detected near the bottom of
the cell dish and before passing the dishes. Subsequent to 18 h incubation, the viability of cultured cells were
determined by the MTT assay [60, 61].
Fluorescence assays: U87 glioma and HeLa cervical cancer cells were maintained in DMEM medium
supplemented with 10% fetal bovine serum (FBS) at 37 °C in 5% CO2. For fluorescence assays, cells were
cultured on coverslips overnight (24 well plate-7x104 cells per well) and the day after they were exposed to
10 μΜ of conjugate 4 and compound 3 for 0 and 2 h. To test the effect of the endocytosis inhibitor, okadaic
acid, cells were preincubated, prior to addition of 4 and 3, with medium containing DMSO (control) or
okadaic acid (100 nM) for 30 min. Cells were then washed twice with PBS to remove floating conjugate and
visualized in a Nikon confocal microscope using the EZ-C1 3.20 software.
MTT Assay: Cells were plated in 96-well plate at a density of 1x104cells/mL per well and maintained for 72 h
o
at 37 C in a 5% CO2 incubator and grown as monolayers. After 24 hours, cells were treated with 0.1–100
μmol/L of the compounds for 48 h. The viability of cultured cells was estimated by an (3-(4,5-imethylthiazol-
2-yl)-2,5-diphenyltetrazolium bromide (MTT) metabolic assay as described previously [60,61].
MTT (Sigma, St Louis, Missouri, USA) was dissolved in PBS in a concentration of 5 mg/mL, filter sterilized, and
o
o
stored at 4 C. MTT (50 μL of stock solution) was added to each culture and incubated for 3 h at 37 C to
allow metabolization. Formazan crystals were solubilized by DMSO (100 μL). Absorbance of the converted
dye was measured at a wavelength of 540 nm on ELISA reader (Versamax, Orleans, USA). The mean
concentrations of each drug that generated 50% or total (100%) growth inhibition (GI50 and TGI, respectively)
18

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as well as the drug concentrations that produced cytotoxicity against 50% of the cultured cells [(half maximal
inhibitory concentration (IC50)] were calculated using the linear regression method [62].
Using seven absorbance measurements [time 24 h (Ct24), control growth 72 h (Ct72), and test growth in the
presence of drug at five concentration levels (Tt72x)], the percentage of growth was calculated at each level
of the drug concentrations. The percentage growth inhibition was calculated according to National Cancer
Institute (NCI) as:
[(Tt72x)-(Ct24) / (Ct72)-(Ct24)] x 100 for concentrations for which Tt72x>Ct24 and
[(Tt72x)-(Ct24)/Ct24] x 100 for concentrations for which Tt72x<Ct24
GI50 was calculated from [(Tt72x)-(Ct24)/(Ct72)-(Ct24)] x 100=50,
TGI from [(Tt72x)-(Ct24)/(Ct72)-(Ct24)] x 100=0, and
IC50 from [(Tt72x)-(Ct24)/Ct24] x 100= 50
All the experiments were carried out in triplicate.
References
[1] D. Van Straten, Mashayekhi, V., de Bruijn, H. S., Oliveira, S., Robinson. D. J. Oncologic Photodynamic Therapy: Basic
Principles, Current Clinical Status and Future Directions, Cancers (Basel), 9 (2017) pii: E19.
[2] K. Lu, C. He, W. Lin. Nanoscale Metal–Organic Framework for Highly Effective Photodynamic Therapy of Resistant
Head and Neck Cancer. J. Am. Chem. Soc. 136 (2014) 16712-16715.
[3] X. Wang, J. Hu, P. Wang, S. Zhang, Y. Liu, W. Xiong, Q. Liu, Analysis of the In Vivo and In Vitro Effects of
Photodynamic Therapy on Breast Cancer by Using a Sensitizer, Sinoporphyrin Sodium. Theranostics 5 (2015) 772-786.
[4] R. K. Horlings, J. B. Terra, M. J. Witjes, mTHPC mediated, systemic photodynamic therapy (PDT) for nonmelanoma
skin cancers: Case and literature review, Lasers Surg. Med. 47 (2015) 779–787.
[5] Z. Huang, A review of progress in clinical photodynamic therapy, Technol Cancer Res Treat. 4 (2005) 283-293.
[6] J. P. Celli, B. Q. Spring, I. Rizvi, C.L. Evans, K. S. Samkoe, S. Verma, B. W. Pogue, T. Hasan, Imaging and photodynamic
therapy: mechanisms, monitoring, and optimization, Chem. Rev. 110 (2010) 2795e838.
[7]S. A. Sibani, P. A. McCarron, A. D. Woolfson, R. F. Donnelly, Photosensitiser delivery for photodynamic therapy. Part
2: systemic carrier platforms, Expert Opin Drug Deliv. 5 (2008) 1241-1254.
[8] R. V. Chari, M. L. Miller, W. C. WiddisonAntibody-drug conjugates: an emerging concept in cancer therapy, Angew.
Chem. Int. Ed, Engl. 53, (2014) 3796-3827.
[9] M. A. Firer, G. Gellerman, Targeted drug delivery for cancer therapy: the other side of antibodies, J Hematol Oncol.
5, (2012) 70.
[10] S. Katsamakas, T. Chatzisideri, S. Thysiadis, V. Sarli. RGD-mediated delivery of small-molecule drugs, Future Med
Chem. 9(6), (2017) 579-604.
19

ACCEPTED MANUSCRIPT
[11] A. Dal Corso, L. Pignataro, L. Belvisi, C. Gennari, αvβ3 Integrin-Targeted Peptide/Peptidomimetic-Drug Conjugates:
In-Depth Analysis of the Linker Technology, Curr. Top. Med. Chem. 16, (2016) 314-329.
[12] M. Huo, Q. Zhu, Q. Wu, T. Yin, L. Wang, L. Yin, J. Zhou, Somatostatin receptor-mediated specific delivery of
paclitaxel prodrugs for efficient cancer therapy, J. Pharm. Sci. 104, (2015) 2018-2028.
[13] A. Nagy, A. V. Schally, G. Halmos, P. Armatis, R. Z. Cai, V. Csernus, M. Kovács, M. Koppán, K. Szepesházi, Z. Kahán
Proc. Natl. Acad. Sci. U S A. 95, (1998) 1794-1799.
[14] B. P. Pemmaraju, S. Malekar, H. K. Agarwal, R. K. Tiwari, D. Oh, G. F. Doncel, D. R. Worthen, K. Parang, Design,
synthesis, antiviral activity, and pre-formulation development of poly-L-arginine-fatty acyl derivatives of nucleoside
reverse transcriptase inhibitors, Nucleosides Nucleotides Nucleic Acids 34, (2015) 1-15.
[15] M. Soler, L. Feliu, M. Planas, X. Ribas, M. Costas, Peptide-mediated vectorization of metal complexes: conjugation
strategies and biomedical applications, Dalton Trans 45(33) (2016) 12970-12982.
[16] F. Danhier, A. Le Breton, V. Préat, RGD-based strategies to target alpha(v) beta(3) integrin in cancer therapy and
diagnosis, Mol Pharm. 9(11) (2012) 2961-2973.
[17] S. Mukhopadhyay, C. M. Barnés, A. Haskel, S. M. Short, K. R. Barnes, S. J. Lippard, Conjugated platinum(IV)-peptide
complexes for targeting angiogenic tumor vasculature, Bioconjug Chem. 19(1) (2008) 39-49.
[18]W. Cai, X. Chen, Anti-angiogenic cancer therapy based on integrin alphavbeta3 antagonism, Anticancer Agents
Med. Chem. 6 (2006) 407-428.
[19] Y. Yuan, R. T. Kwok, B. Z. Tang, B. Liu, Targeted theranostic platinum(IV) prodrug with a built-in aggregation-
induced emission light-up apoptosis sensor for noninvasive early evaluation of its therapeutic responses in situ, J. Am.
Chem. Soc. 136(6) (2014) 2546-2554.
[20] A. Massaguer, A. González-Cantó, E. Escribano, S. Barrabés, G. Artigas, V. Moreno, V. Marchán, Integrin-targeted
delivery into cancer cells of a Pt(IV) pro-drug through conjugation to RGD-containing peptides, Dalton Trans. 44(1),
(2015) 202-212.
[21] A. Gandioso, E. Shaili, A. Massaguer, G. Artigas, A. González-Cantó, J. A. Woods, P. J. Sadler, V. Marchán, An
integrin-targeted photoactivatable Pt(IV) complex as a selective anticancer pro-drug: synthesis and photoactivation
studies. Chem. Commun (Camb) 51(44) (2015) 9169-9172.
[22] K. Chen, X. Chen, Integrin targeted delivery of chemotherapeutics, Theranostics. 1 (2011) 189-200.
[23] W. B. Edwards, W. J. Akers, Y. Ye, P. P. Cheney, S. Bloch, B. Xu, R. Laforest, S. Achilefu, Mol Imaging. 8 (2009) 101-
110.
[24] D. Fan, X. Zhang, L. Zhong, X. Liu, Y. Sun, H. Zhao, B. Jia, Z. Liu, Z. Zhu, J. Shi, F. Wang (68)Ga-labeled 3PRGD2 for
dual PET and Cerenkov luminescence imaging of orthotopic human glioblastoma Bioconjug Chem. 26 (2015) 1054-1060.
[25]F. L. Thorp-Greenwood, R. G. Balasingham, M. P. Coogan, Organometallic complexes of transition metals in
luminescent cell imaging applications J. Organometal. Chem. 714 (2012) 12-21.
[26] C. K. Koo, L. K. So, K. L. Wong, Y. M. Ho, Y. W. Lam, M. H. Lam, K. W. Cheah, C. C. Cheng, W. M. Kwok. A
triphenylphosphonium-functionalised cyclometalated platinum(II) complex as a nucleolus-specific two-photon
molecular dye, Chemistry 16 (2010) 3942-3950.
20

ACCEPTED MANUSCRIPT
[27] S. W. 3rd Thomas, K. Venkatesan, P. Müller, T.M. Swager, Dark-field oxidative addition-based chemosensing: new
bis-cyclometalated PtII complexes and phosphorescent detection of cyanogen halides J. Am. Chem. Soc. 128, (2006)
16641-16648.
[28] J. A. G. Williams, S. Develay, D. L. Rochester, L. Murphy, Optimising the luminescence of platinum(II) complexes and
their application in organic light emitting devices (OLEDs). Coordination Chem. Rev. 252, (2008) 2596–2611.
[29] K. Feng, R. Y. Zhang, L. Z. Wu, B. Tu, M. L. Peng, L. P. Zhang, D. Zhao, C. H. Tung, Photooxidation of olefins under
oxygen in platinum(II) complex-loaded mesoporous molecular sieves J. Am. Chem. Soc. 128 (2006) 14685-14690.
[30] R. W. Sun, D. L. Ma, E. L. Wong, C. M. Che, Some uses of transition metal complexes as anti-cancer and anti-HIV
agents. Dalton Trans. 43, (2007) 4884-4892.
[31] E. Glimsdal, M. Carlsson, T. Kindhal, M. Lindgren, C. Lopes, Eliasson. Luminescence, Singlet Oxygen Production, and
Optical Power Limiting of Some Diacetylide Platinum(II) Diphosphine Complexes, J Phys. Chem. A 114 (2010) 3431-3442.
[32] A. Bossi, A. F. Rausch, M. J. Leitl, R. Czerwieniec, M. T. Whited, P. I. Djurovich, H. Yersin, M. E. Thompson
Photophysical properties of cyclometalated Pt(II) complexes: counterintuitive blue shift in emission with an expanded
ligand π system, Inorg Chem. 52 (2013) 12403-12415.
[33] B. Rosenberg, L. Vancamp, J. E. Trosko, V. H. Mansour, Platinum compounds: a new class of potent antitumour
agents, Nature 222 (1969) 385-386.
[34] T. Boulikas, A. Pantos, E. Bellis, P. Christofis, Designing platinum compounds in cancer: structures and mechanisms.
Cancer Therapy 5 (2007) 537-583.
[35] A. Colombo, F. Fiorini, D. Septiadi, C. Dragonetti, F. Nisic, A. Valore, D. Roberto, M. Mauro, L. De Cola, Neutral
N^C^N terdentate luminescent Pt(II) complexes: their synthesis, photophysical properties, and bio-imaging
applications, Dalton Trans, 44 (2015) 8478-8487.
[36] E. Baggaley, S. W. Botchway, J. W. Haycock, H. Morris, I. V. Sazanovich, J. A. Gareth Williams, J. A. Weinstein, Long-
lived metal complexes open up microsecond lifetime imaging microscopy under multiphoton excitation: from FLIM to
PLIM and beyond. Chem. Sci. 5, (2014) 879-886.
[37] R. E. Doherty, I. V. Sazanovich, L. K. McKenzie, A. S. Stasheuski, R. Coyle, E. Baggaley, S. Bottomley, J. A. Weinstein,
H. E. Bryant, Photodynamic killing of cancer cells by a Platinum(II) complex with cyclometallating ligand. Sci Rep. 6,
(2016) 22668.
[38] T. Ishiyama, M. Murata, N. Miyaura, Palladium(0)-Catalyzed Cross-Coupling Reaction of Alkoxydiboron with
Haloarenes: A Direct Procedure for Arylboronic Esters, J. Org. Chem. 60 (1995) 7508–7510.
[39] J. A. G., Williams, A. Beeby, E. S. Davies, J. A. Weinstein, Wilson C. An Alternative Route to Highly Luminescent
Platinum(II) Complexes:ꢀ Cyclometalation with N∧C∧N-Coordinating Dipyridylbenzene Ligands. Inorg. Chem. 42, (2003)
8609-8611.
[40] B. Wieczorek, B. Lemcke, H. P. Dijkstra, M. R. Egmond, R. J. M. Klein Gebbink, G. van Koten Site-Selective Ser-
Hydrolase Labelling with a Luminescent Organometallic NCN–Platinum Complex, Eur. J. Inorg. Chem. 13 (2010) 1929-
1938.
[41] X. Feifan, C. Pieter, V. B. Jan. Electrospray ionization mass spectrometry for the hydrolysis complexes of cisplatin:
implications for the hydrolysis process of platinum complexes. J Mass Spectrom. 52(7) (2017) 434-441.
21

ACCEPTED MANUSCRIPT
[42] E. Glimsdal, I. Dragland, M. Carlsson, B. Eliasson, T. B. Melø, M. Lindgren Triplet Excited States of Some Thiophene
and Triazole Substituted Platinum(II) Acetylide Chromophores, J. Phys. Chem. A, 113 (14) (2009) 3311–3320.
[43] W. Cai, Y. Wu, K. Chen, Q. Cao, D.A. Tice, X. Chen, In vitro and in vivo characterization of 64Cu-labeled Abegrin, a
humanized monoclonal antibody against integrin alpha v beta 3. Cancer Res. 66, (2006) 9673-9681
[44] M. H. Lee, J. Y. Kim, J. H. Han, S. Bhuniya, J. L. Sessler, C. Kang, J. S. Kim, Direct fluorescence monitoring of the
delivery and cellular uptake of a cancer-targeted RGD peptide-appended naphthalimide theragnostic prodrug. J. Am.
Chem. Soc. 134 (2012) 12668-12674.
[45] M. S. Maginnis, J. C. Forrest, S. A. Kopecky-Bromberg, S. K. Dickeson, S. A. Santoro, M. M. Zutter, G. R. Nemerow, J.
M. Bergelson, T. S. Dermody, Beta1 integrin mediates internalization of mammalian reovirus. J. Virol. 80 (2006) 2760-
2770.
[46] X. Zhang, Z., Xiong, Y. Wu, W. Cai, J. R. Tseng, S. S. Gambhir, X. Chen, Quantitative PET imaging of tumor integrin
alphavbeta3 expression with 18F-FRGD2. J Nucl Med. 47 (2006) 113-121.
[47] K. A. Hinchliffe, R. F. Irvine, N. DivechaAggregation-dependent, integrin-mediated increases in cytoskeletally
associated PtdInsP2 (4,5) levels in human platelets are controlled by translocation of PtdIns 4-P 5-kinase C to the
cytoskeleton. EMBO J. 15 (1996) 6516−6524.
[48] M. Higashihara, K. Takahata, K. Kurokawa, M. Ikebe, The inhibitory effects of okadaic acid on platelet function,
FEBS Lett. 307 (1992) 206−210.
[49] I. Holen, P.B. Gordon, P. E. Stromhaug, T. O. Berg, M. Fengsrud, A. Brech, N. Roos, T. Bergs, P. O. Seglen, Inhibition
of asialoglycoprotein endocytosis and degradation in rat hepatocytes by protein phosphatase inhibitors Biochem. J. 311
(1995) 317−326.
[50] C. M. Witkowski, I. Rabinovitz, R. B. Nagle, K. S. Affinito, A. E. Cress, Characterization of integrin subunits, cellular
adhesion and tumorgenicity of four human prostate cell lines, J. Cancer Res. Clin. Oncol. 119 (1993) 637-644.
[51] S. A. Cannistra, C. Ottensmeier, J. Niloff, B. Orta, J. DiCarlo, Expression and function of beta 1 and alpha v beta 3
integrins in ovarian cancer, Gynecol. Oncol. 58 (1995) 216-225.
[52] L. Guo, F. Zhang, Y. Cai, T. Liu, Expression profiling of integrins in lung cancer cells, Pathol. Res. Pract. 205 (2009)
847-853.
[53] R. Czarnomysy, A. Surażyński, B. Popławska, E. Rysiak, N. Pawłowska, A. Czajkowska, K. Bielawski, A. Bielawska,
Synergistic action of cisplatin and echistatin in MDA-MB-231 breast cancer cells. Mol. Cell Biochem. 427 (2017)13-22.
[54] P. M. Kazmierczak, A. Todica, F. J. Gildehaus, H. Hirner-Eppeneder, M. Brendel, R. S. Eschbach, M. Hellmann, K.
Nikolaou, M. F. Reiser, H. J. Wester, S. Kropf, A. Rominger, C. C. Cyran. 68Ga-TRAP-(RGD)3 Hybrid Imaging for the In
Vivo Monitoring of αvß3-Integrin Expression as Biomarker of Anti-Angiogenic Therapy Effects in Experimental Breast
Cancer. PLoS One. 11 (2016) e0168248.
[55] H. A. Kim, K. Nam, S. W. Kim, Tumor targeting RGD conjugated bio-reducible polymer for VEGF siRNA expressing
plasmid delivery. Biomaterials. 35(26) (2014) 7543-7452.
[56] E. Delamarre, S. Taboubi, S. Mathieu, C. Berenguer, V. Rigot, J. C. Lissitzky, D. Figarella-Branger, L. Ouafik, J. Luis,
Expression of integrin alpha6beta1 enhances tumorigenesis in glioma cells, Am. J. Pathol. 175 (2009) 844-855.
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[57] E. L. Larsen, L. L. Randeberg, O. A. Gederaas, C. J. Arum, A. Hjelde, C. M. Zhao, D. Chen, H. E. Krokan, L. O. Svaasand,
Monitoring of hexyl 5-aminolevulinate-induced photodynamic therapy in rat bladder cancer by optical spectroscopy. J
Biomed Opt. 13 (2008) 044031.
[58] V. Bogoeva, M. Siksjø, K. G. Sæterbø, T. B. Melø, A. Bjørkøy, M. Lindgren, O. A. GederaasRuthenium porphyrin-
induced photodamage in bladder cancer cells, Photodiagnosis Photodyn Ther. 14 (2016) 9-17.
[59] Y. Baglo, M. M. Sousa, G. Slupphaug, L. Hagen, S. Håvåg, L. Helander, K. A. Zub, H. E. Krokan, O. A. Gederaas,
Photodynamic therapy with hexyl aminolevulinate induces carbonylation, posttranslational modifications and changed
expression of proteins in cell survival and cell death pathways. Photochem Photobiol Sci. 10(7) (2011) 1137-1145.
[60] G. J. Finlay, W. R. Wilson, B. C. Baguley Comparison of in vitro activity of cytotoxic drugs towards human carcinoma
and leukaemia cell lines, Eur. J. Cancer Clin. Oncol. 22 (1986) 655–662.
[61] D. T. Trafalis, C. Camoutsis, P. Karamanakos, A. Arvanitis, E. Tegou, N. Ziras et al. Preclinical evaluation of the
homo-aza-steroid ester 13-beta-hydroxy-13alpha-amino-13,17-seco-5alpha-androstan-17-oic-13,17-lactam-p-bis(2-
chloroethyl)amino phenoxy acetate for the treatment of malignant melanoma, J BUON. 8 (2007) 333–339.
[62] M. C. Alley, D. A. Scudiero, A. Monks, M. L. Hursey, M. J. Czerwinski, D. L. Fine, B. J Abbott, J. G. Mayo, R. H.
Shoemaker, M. R. Boyd Feasibility of drug screening with panels of human tumor cell lines using a microculture
tetrazolium assay, Cancer Res. 48 (1988) 589–601.
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