An in vivo active 1,2,5-oxadiazole Pt(II) complex: A promising anticancer agent endowed with STAT3 inhibitory properties

Article abstract of DOI:10.1016/j.ejmech.2017.03.017

New Pt(II) complexes (Pt-1-3) bearing 1,2,5-oxadiazole ligands (1-3) were synthesized, characterized and evaluated for their ability to disrupt STAT3 dimerization. Ligand 3·HCl showed cytotoxic effects on HCT-116 cells (IC₅₀ = 95.2 μM) and a se

Full text of DOI:10.1016/j.ejmech.2017.03.017

Accepted Manuscript
An in vivo active 1,2,5-oxadiazole Pt(II) complex: A promising anticancer agent
endowed with STAT3 inhibitory properties
Federica Porta, Giorgio Facchetti, Nicola Ferri, Arianna Gelain, Fiorella Meneghetti,
Stefania Villa, Daniela Barlocco, Daniela Masciocchi, Akira Asai, Nao Miyoshi, Silvia
Marchianò, Byoung-Mog Kwon, Yena Jin, Valentina Gandin, Cristina Marzano,
Isabella Rimoldi
PII:
S0223-5234(17)30174-5
10.1016/j.ejmech.2017.03.017
EJMECH 9277
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 8 February 2017
Revised Date: 9 March 2017
Accepted Date: 10 March 2017
Please cite this article as: F. Porta, G. Facchetti, N. Ferri, A. Gelain, F. Meneghetti, S. Villa, D. Barlocco,
D. Masciocchi, A. Asai, N. Miyoshi, S. Marchianò, B.-M. Kwon, Y. Jin, V. Gandin, C. Marzano, I.
Rimoldi, An in vivo active 1,2,5-oxadiazole Pt(II) complex: A promising anticancer agent endowed
with STAT3 inhibitory properties, European Journal of Medicinal Chemistry (2017), doi: 10.1016/
j.ejmech.2017.03.017.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
An in vivo active 1,2,5-oxadiazole Pt(II) complex: a promising
anticancer agent endowed with STAT3 inhibitory properties
Federica Porta,a± Giorgio Facchetti,a± Nicola Ferri,b* Arianna Gelain,a* Fiorella Meneghetti,a Stefania Villa,a
Daniela Barlocco,a Daniela Masciocchi,a Akira Asai,c Nao Miyoshi,c Silvia Marchianò,d Byoung-Mog
Kwon,e Yena Jin, e Valentina Gandin,b Cristina Marzano,b Isabella Rimoldi.a*
a Dipartimento di Scienze Farmaceutiche, Università degli Studi di Milano, Via L. Mangiagalli 25, 20133
Milano, Italy
b
Dipartimento di Scienze del Farmaco, Università degli Studi di Padova, Via Marzolo 5, 35131, Padova,
Italy
c
Center for Drug Discovery, Graduate School of Pharmaceutical Sciences, University of Shizuoka, 52-1
Yada, Suruga-ku, Shizuoka, 422-8526, Japan
d Dipartimento di Scienze Farmacologiche e Biomolecolari, Università degli Studi di Milano, Via Balzaretti
9, 20133 Milano, Italy
e
Korea Research Institute of Bioscience and Biotechnology, University of Science and Technology, 125
Gwahakro Yoosunggu, Daejeon 34141
KEYWORDS: DNA-interaction, cytotoxic activity, platinum diamine complex, antitumor agents, protein–
protein interactions.
ABSTRACT. New Pt(II) complexes (Pt-1-3) bearing 1,2,5-oxadiazole ligands (1-3) were
synthesized, characterized and evaluated for their ability to disrupt STAT3 dimerization. Ligand
.
3
HCl showed cytotoxic effects on HCT-116 cells (IC50 = 95.2 µM) and a selective ability to
interact with STAT3 (IC50 = 8.2 µM) versus STAT1 (IC50> 30 µM). Its corresponding platinum
complex Pt-3 exhibited an increased cytotoxicity (IC50 = 18.4 µM) and a stronger interaction with
STAT3 (IC50 = 1.4 µM), leading to inhibition of its signaling pathway. Pt-3 was also evaluated in
cell-based assays for its action on p53 expression and on STAT3 phosphorylation. In syngeneic
murine Lewis lung carcinoma (LLC) implanted in C57BL/6 mice, Pt-3 showed a higher antitumor
activity with fewer side effects than cisplatin.

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1. INTRODUCTION
The development of cisplatin and other cytotoxic metal complexes have contributed to
tremendous advances in the field of chemotherapy based on metallorganic entities over
the past few decades.[1-3] The Food and Drug Administration (FDA)-approved Pt-
based anticancer drugs cisplatin, carboplatin and oxaliplatin are still among the most
effective drugs used for the treatment of solid cancers.[4-6] These Pt-based anticancer
agents typically form bifunctional intra- and interstrand DNA cross-links through
covalent bonds with purine nucleobases, inhibiting transcription and resulting in cell-
death. Unfortunately, the strong side effects and the increasing resistance are
limitations to their use, thus evoking the need for new Pt-based chemotherapeutics.[7-
10]
Literature data highlighted that cisplatin, besides interacting with DNA, was able to
modulate mitogen-activated protein kinase (MAPK) family and the phosphoinositide 3
(PI-3)-kinase/Akt pathway.[11, 12] Moreover, many platinum complexes displayed an
effective inhibition of signal transducer and activator of transcription 3 (STAT3)
cascade,[13, 14] leading to significant clinical benefits. Indeed STAT3 is constitutively
activated in a variety of human cancers, including solid tumors, such as breast, prostate
and cervical cancers, as well as many leukemias and lymphomas.[15]
In this regard, kinetically liable Pt(IV) complexes inducing tumor regression in mouse
colon cancer model, were established to inhibit STAT3 activity in living cells through an
irreversible interaction with the STAT3 DNA-binding domains.[13] The involvement of
STAT3 activated cellular responses in cisplatin resistant tumors was highlighted by the
ability of Stattic, an effective STAT3 inhibitor, to restore the sensitivity to Pt-based drugs
both in vitro and in vivo.[16, 17] Complexes endowed with these dual-targeting features
could lead to enhanced therapeutic benefits, as we expect tumor cell apoptosis with
minimal side effects.[18]
The effect on STAT3 signaling cascade has been often related to platinum
complexes[13] and small molecules (Stattic and S3I-201)[19] capability to interact with
thiol moiety of cysteines, leading to STAT3 dimerization disruption. Moreover, the
disulphide bond-mediated STAT3-dimer formation might be due to the presence of

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several cysteine residues or vicinal polar residue serving to activate the target cysteine
while analogous aggregates through disulphide bond formation haven’t been observed
related to STAT1.[20]
Among the molecules disclosed as potential antitumor agents, oxadiazoles have
received considerable attention in recent years because of their interesting activity.[21,
22] In this respect, the oxadiazole ring emerged as a key molecular feature for STAT3
inhibition, amongst others, the 1,3,4-oxadiazole characterizes STX-0119,[23, 24] while
the 1,2,5-oxadiazole isomer is present in AVS-0288,[25] both known as potent STAT3
inhibitors. All these findings supported our ongoing research focusing on the design of
new platinum complexes possibly able to simultaneously interact with DNA and to
interrupt STAT3 signaling.[18] Taking advantage of our experience in the chemistry and
biology of the oxadiazole ring[26-28] and of platinum compounds,[29, 30] we planned
the synthesis of 3-aminomethyl-1,2,5-oxadiazole derivatives as ligands in Pt(II)
complexes (Figure 1).[31]
Figure 1. New Pt(II) complexes.
2. RESULTS AND DISCUSSION
In this study, we designed 1,2,5-oxadiazole derivatives and their Pt(II) complexes with
the aim to obtain a dual mechanism of action: interfering with DNA replication and
inhibiting STAT3 signaling pathway. We devised an aminomethyl moiety as the pivotal
feature to be introduced in the 3 position of the oxadiazole ring in order to realize a
planar-coordinated Pt(II) drug. Therefore, since our previous research on 1,2,5-
oxadiazole derivatives[27] allowed us to identify p-chlorophenyl and benzoyl as useful
moieties for STAT3 interaction, starting from the commercially available 3-aminomethyl-
4-methyl-1,2,5-oxadiazole (1), compound 2 (bearing a p-chlorophenyl system at 4-
position of the heterocycle) and compound 3 (presenting also a benzoyl group on the
aminomethyl chain) were designed and synthesized. Hence, compounds 1, 2 · HCl and

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3 · HCl were used in coordination with Pt(II) centre. Both ligands and metal complexes
(Figure 2) underwent biological evaluation.
Figure 2. Ligands and their corresponding Pt(II) complexes.
a.
Synthetic Strategy. The synthesis of compounds 2, 3, Pt-1, Pt-2 and Pt-3 are
shown in Schemes 1-3.
Intermediate 4 was synthesized starting from the commercially available p-
chlorocinnamic acid, according to the literature.[32] Azide 5 was prepared by a one pot
reaction[33] in presence of NaN3 and Ph3P in CCl4/DMF, and converted to the
corresponding amine 2 by catalytic hydrogenation (Scheme 1). The final product was
isolated as hydrochloride salt (2 . HCl).
Scheme 1. Reagents and conditions: i) NaN3, Ph3P, CCl4/DMF (1:4), 90°C, 5 h; ii) Pd/C, H2, MeOH, 20
atm, 4 h; iii) 2 M HCl in Et2O.

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The synthesis of compound 3 (Scheme 2) started from the commercially available p-
chloropropiophenone which was treated with tert-butyl nitrite and chlorotrimethylsilane
to obtain the correspondent α-ketooxime 6. This was converted to the bis-oxime 7, in
presence of hydroxylamine hydrochloride and sodium hydroxide,[34] and then cyclized
with potassium hydroxide and cesium carbonate at high temperature to give
intermediate 8. Treatment with n-butyl lithium and methyl benzoate gave ketone 9 which
underwent an α-oximation with tert-butyl nitrite and chlorotrimethylsilane. The reduction
of compound 10 with zinc in trifluoroacetic acid led to product 3, purified as
hydrochloride salt.
Cl
O
O
NOH
iv
i
iii
ii
N
N
NOH
NOH
Cl
Cl
Cl
Cl
O
6
7
8
Cl
Cl
OH
N
NH2
vi
vii
v
O
O
O
3 . HCl
N
N
N
O
N
N
N
O
O
9
10
3
Scheme 2. Reagents and conditions i) t-BuONO, TMSCl, dry THF/DCM, -20°C to rt, 12 h; ii) NH2OH·HCl,
NaOH, H2O, reflux, 3 h; iii) KOH, Cs2CO3, ethylen glycol, 160°C, 1.5 h; iv) 2.5 M n-BuLi in hexane, methyl
benzoate, dry THF, -55°C for 1 h, then rt for 12 h; v) t-BuONO, TMSCl, dry THF, -20°C to rt, 12 h; vi) Zn,
TFA, rt, 40 min; vi) 2 M HCl in Et2O.
A classical methodology was employed for the preparation of the corresponding Pt(II)
complexes.[35] Accordingly, 1, 2 . HCl and 3 . HCl were treated with K2PtCl4 in water in
presence of 4 M HCl affording the target compounds Pt-1, Pt-2 and Pt-3 (Scheme 3).

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Scheme 3. Reagents and conditions: i) K2PtCl4, 4 M HCl, H2O, reflux, 2 h.
2.2
Characterization. The identity and purity of all synthesized compounds and their
1
Pt(II) complexes were ascertained by H NMR, 195Pt NMR, ESI-MS, or elemental
analysis and IR (see spectra in SI). The observed chemical shifts (around -2070 ppm)
for 195Pt NMR spectra are in the range expected for a Pt(II) core with a N2Cl2
coordination environment,[36]. In the infrared spectra, the strong absorption around
3200-3100 cm-1 corresponding to the N-H stretching vibration resulted significantly red
shifted in comparison to the corresponding free ligands. The coordination geometry was
confirmed by single-crystal X-ray diffraction on the complex Pt-1. The ORTEP diagram
of Pt-1 is shown in Figure 3 with selected bond distances and angles. This compound
crystallized with two independent molecules in the asymmetric unit, which are almost
planar and parallely oriented.
Figure 3: ORTEP[37] view of one of the two molecules present in the asymmetric unit of Pt-1, showing
the arbitrary atom labeling scheme. Ellipsoids are drawn at 40% probability level and H atoms are shown
as spheres of arbitrary radius. Selected averaged bond distances (Å): Pt-N1, 1.956(1); Pt-N3, 2.046(1);
Pt-Cl1, 2.274(1); Pt-Cl2, 2.294(1); N1-C2, 1.291(1); N3-C3, 1.497(1). Angles (°): N1-Pt-N3, 80.8(1); N3-
Pt-Cl1, 90.7(1); Cl1-Pt-Cl2, 91.6(1); Cl2-Pt-N1, 96.8(1); C2-N1-Pt, 119.9(1); C3-N3-Pt, 112.2(1); C2-C3-
N3, 110.5(1).
The complex Pt-1 crystallized with two independent molecules in the asymmetric unit
with very similar configurations, which are almost planar and parallel oriented. The
ORTEP diagram of one molecule is shown in Figure 3, with selected bond distances
and angles.

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The determined lengths and angles values are in agreement with the literature for
similar Pt(II) complexes.[38] The Pt(II) atom is coordinated by N atoms of the bidentate
ligand and Cl atoms. The coordination environment around the central Pt(II) in both
molecules are slightly distorted from an ideal square planar configuration, and the
chlorine atoms are cis to each other. The angles around Pt atom are close to the
expected 90° and 180°. The mean deviation of the PtNNClCl coordination plane is ca.
0.030(6) Å (N1) and the methyl groups lie almost in the same plane of the oxadiazole
ring.
In the unit cell, two non-equivalent dimers head-to-tail are formed by π–π interactions
between individual complexes. These latter are stacked forming molecular layers,
parallel to the ab plane, where consecutive molecules interact through Pt…Pt contacts at
3.612 and 3.3364 Å,[39] developing chains along the c axis[40] also consolidated by N-
H…Cl type interactions.
2.3
Aqueous stability and lipophilicity. The aqueous stability for Pt(II) complexes
were performed using 0.9 % w/v NaCl solution in DMF by monitoring UV-vis spectra at
their respective λmax during a period of 48 h. Each compound was first dissolved in DMF
and then diluted with physiological solution to 100 µM final concentration (1% v/v DMF).
Pt-1, Pt-2 and Pt-3 demonstrated good stability in physiological solution with negligible
variation of their UV peak profile after 48 h. (see spectra in SI)
The values of log Pow were determined using RP-HPLC technique.[41-43] The
lipophilicity increased along the series (-0.123 and 2.28 for 1 and 2 . HCl, respectively),
.
reaching the maximum value of 1.09 and 3.01 with 3
HCl. The same trend
characterized Pt(II) compounds (-0.773, 2.00 and 2.99 for Pt-1, Pt-2 and Pt-3
respectively) still satisfying the Lipinski’s lipophilicity criterion for druglikeness. Ligand 3
showed a keto-enolic equilibrium responsible for the racemization at the chiral
centre,[44, 45] as confirmed by the two values of log Pow. Its tautomerism was evaluated
at different pH conditions by RP-HPLC (Figure 4): the peaks showed the different ratio
between the ketone and the enol species confirming the stability of the compound in all
cases.

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Figure 4: pH dependent ligand 3 enolization ratio (keto /enol) using RP-HPLC Partisil C-18 ODS.
2.4
In vitro biological evaluation. The ligands and their corresponding complexes
were tested by MTT assay for cytotoxic activity and AlphaScreen-based assay to
evaluate the effect on STAT3 dimerization.
MTT assay on HCT-116. The cytotoxic effect of the new ligands and their
corresponding Pt(II) complexes was initially assessed in cultured colorectal cancer cells
HCT-116. The cell viability was estimated by MTT assay after 48 h incubation with
increasing concentrations (10 ÷ 150 µM) of the compounds. As reported in Table 1, the
.
.
ligands generally expressed a negligible (1 and 2 HCl) or weak (3 HCl) cytotoxic
activity (IC50 = 95.2 µM). However, the coordination to the platinum centre resulted in an
increased cytotoxicity with respect to the free ligand. In particular, Pt-3 displayed the
lowest IC50 value (18.4 µM), being about 5 times more effective than the uncoordinated
3 . HCl ligand.
AphaScreen-based assay. This test is an in vitro competitive binding test used to
identify compounds able to inhibit the binding of SH2-containing proteins to their
correspondent phosphopeptides (5-carboxyfluorescein (FITC)-GpYLPQTV for STAT3
and FITC-GpYDKPHVL for STAT1). Compounds characterized by an interesting affinity
were also investigated for their selectivity versus STAT1 having a high degree of
sequence homology to STAT3 (78%) but an opposite physiological role.[46, 47] Ligands
.
1 and 2 HCl did not disrupt STAT3 dimerization whereas their corresponding Pt(II)
complexes Pt-1 and Pt-2, possessed high inhibitory properties. On the other hand,

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ligand 3 . HCl was shown to selectively interfere with STAT3 (IC50 = 8.2 ± 0.1 µM) over
STAT1 (IC50 value >30 µM); also its coordination to the Pt(II) center, similarly to ligands
1 and 2, improved STAT3 inhibitory activity (IC50 = 1.4 ± 0.1 µM) but reduced selectivity
versus STAT1 (IC50 5.9 ± 0.4 µM). (Table 1).
Table 1. Biological properties of the reference (Stattic) and of the oxadiazole derivatives.
AlphaScreenb (IC50, µM)
MTT assay on HCT116a
(IC50, µM)
Compound
STAT3
STAT1
＞30
Stattic
1
N.E.
N.E.
15
＞30
N.T.
1.2 ± 0.1
N.T.
Pt-1
122.9±12.3
N.E.
0.4 ± 0.04
＞30
2 · HCl
Pt-2
150±16.9
95.2±4.41
18.4±6.9
0.3 ± 0.01
8.2 ± 0.1
1.4 ± 0.1
1.9 ± 0.1
＞30
3 · HCl
Pt-3
5.9 ± 0.4
a IC50 values measured by MTT assay on HCT-116 cell lines. N.E.: not effective. b The ability to disrupt the
binding of STATs to the cognate pTyr–peptide (5-carboxyfluorescein (FITC)-GpYLPQTV for STAT3 and
FITC-GpYDKPHVL for STAT1) is expressed as IC50. N.T.: not tested.
2.5
In vivo tumor growth inhibition. In the light of MTT assay results, the in vivo
antitumor activity of Pt-3 and its uncoordinated ligand was evaluated in a model of solid
tumor, the syngeneic murine Lewis lung carcinoma (LLC) implanted i.m. in C57BL/6
mice.
Tumor growth inhibition induced by Pt-3 was compared with that promoted by the
reference metallo-drug, cisplatin. From day 7 after tumor inoculation, when tumors
.
became visible, tumor-bearing mice received daily i.p. doses of Pt-3 (30 mg·kg−1), 3
HCl (75 mg·kg−1) or cisplatin (1.5 mg·kg−1). Cisplatin treatment schedule was selected
according to standard protocols designed to optimize its efficacy and minimize the

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occurrence of adverse events.[48] For the assessment of the adverse side effects,
changes in the body weights of tumor-bearing mice were monitored at day 1 and every
two days from day 7 to day 15 (Figure 5).
Figure 5. Body weight changes. The body weight changes of LLC-bearing C57BL mice treated with
vehicle or tested compounds. Body weight was measured at day 1 and every two days from day 7 and
was taken as a parameter of systemic toxicity. The error bars indicate the SD.
The time course of body weight changes, depicted in Figure 5, highlights that
treatment with Pt-3 did not induce any adverse effect, including significant body weight
loss, throughout the therapeutic experiment. On the contrary, mice treated with ligand 3
. HCl showed a roughly 15% body weight loss and those treated with cisplatin appeared
prostrate and showed substantial (about 27%) weight loss.
.
As shown in Table 2, uncoordinated compound 3 HCl ligand tested at 75 mg·kg−1
induced a significant tumor growth inhibition that was, however, lower than that
promoted by cisplatin. Chemotherapy with Pt-3 tested at 30 mg·kg−1 reduced the tumor
mass by roughly 85%, only very slightly lower compared with the reference metallo-
drug.
Table 2. In vivo antitumor activity toward LLC.
Daily dose
Average tumor
Inhibition of tumor
weight (mean ± SD,

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i.p. (mg·kg-1)
g)
growth (%)
Controla
Pt-3
-
0.582±0.16
0.086±0.06**
0.101±0.08**
0.073±0.03**
-
30
75
1.5
85.2
82.6
87.5
3 . HCl
Cisplatin
avehicle (20% Cremophor EL (v/v), 20% PEG400 (v/v) and 60% saline solution (v/v)); ** p<0.01
Overall, these in vivo results confirmed that the coordination of STAT3 inhibitor 3 . HCl
to a platinum moiety, with the aim to develop a dual action agent, led to the obtainment
.
of an effective antitumor metallo-drug. In addition, Pt-3 and 3 HCl appeared to be
better tolerated than cisplatin and could be administered at a higher dose.
2.6
Cytotoxic effect of Pt-3 and 3 . HCl on cell lines poorly sensitive to cisplatin.
The cytotoxicity of Pt-3 was assessed on two additional cancer cell lines less sensitive
to cisplatin as compared with HCT-116 cells,[49] namely the breast cancer cell line
MCF-7 and the colorectal cancer cell line DLD-1,. As shown in Figure 6, Pt-3 did not
affect cell viability of MCF-7 at the concentration range of 1÷25 µM, while a significant
inhibition of cell viability was observed for DLD-1 cells at 25 µM concentration. As
expected, cisplatin showed a limited cytotoxic efficacy on all three cell lines and with no
concentration-dependent action (Figure 6). MCF-7 cells resulted particularly resistant to
both Pt-3 and cisplatin.
Figure 6. Cytotoxic effects of Pt-3 complex and cisplatin on DLD-1, HCT-116 and MCF-7 cell lines. Cells
were seeded at the density of 40,000/well in a 48 well tray. The day after the medium was replaced with

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one containing 10% FCS and the reported concentrations of Pt-3 and cisplatin. After 48 h the cell viability
was estimated by MTT assay. Each bar represents the mean ± SD of three determinations. Pt-3 and
cisplatin vs control *P < 0.05;**P < 0.01; ***P < 0.001.
2.7
Intracellular and nuclear accumulation of Pt-3 and induction of p53. To
explore the molecular mechanism of action of Pt-3, HCT-116 cell line were incubated
with the oxadiazole Pt(II) complex (25 and 50 µM) or cisplatin (10 and 25 µM) for 3h and
the 195Pt content was determined from both total cell lysates and nuclear DNA
preparations. As shown in Figure 7A, a concentration-dependent accumulation of
platinum was observed in the DNA extract after incubation with Pt-3 and cisplatin.
However, the efficiency of Pt-3 to reach the DNA was significantly lower than cisplatin:
at 25 µM the amount of Pt measured was 0.48±0.30 ng Pt / µg DNA and 9.0±0.1 ng Pt /
µg DNA for Pt-3 and cisplatin, respectively. By contrast, the 195Pt concentrations were
threefold higher in the total cell lysates of HCT-116 incubated with 25 µM of Pt-3 as
compared to 25µM of cisplatin (126.6 ng Pt / µg protein and 42.3 ng Pt / µg protein for
Pt-3 and cisplatin, respectively) (Figure 7B).[4] These data suggest that Pt-3 has a
different subcellular distribution than cisplatin, potentially due to an interaction with a
cytoplasmic protein.
Figure 7. DNA and total intracellular concentrations of 195Pt in HCT-116 cell line incubated with Pt-3 and
cisplatin. Cells were seeded (250,000/35 mm petri dish) and incubated with McCoy’s supplemented with
10% FCS; 24 h later the medium was replaced with one containing 10% FCS and indicated concentrations
of Pt-3 or cisplatin. After 3 h, the nuclear DNA and total cell homogenates were prepared and 195Pt
concentrations determined by ICPMS and normalized with total DNA (A) and protein contents (B).
Although Pt-3 interacts less efficiently than cisplatin with nuclear DNA, the incubation
of HCT-116 cell line with increasing concentrations of platinum complex for 24 h led to a

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significant induction of p53, as determined by Western blot analysis (Figure 8). These
results suggest that Pt-3 elicited its cytotoxic effect by interacting with nuclear DNA and
thus inducing the expression of p53, similarly to cisplatin.
Figure 8. Effect of Pt-3 on p53 expression in HCT-116 cell line. Cells were seeded (250,000/35 mm petri
dish) and incubated with McCoy’s supplemented with 10% FCS; 24 h later the medium was replaced with
one containing 10% FCS and the reported concentrations of Pt-3 and cisplatin. The incubation was
continued for a further 24 h at 37°C and Western blot analysis was performed from total cell lysates. α-
tubulin antibody was utilized as loading control.
2.8
DNA binding study. To verify the ability of Pt-3 to interact with deoxyguanosine
rich region of DNA, binding studies were performed with the model nucleobase 9-
ethylguanine (9-EtG) by using 1H-NMR.[50, 51] The N7of guanine is the putative binding
1
position for the metal centre, thus a downfield chemical shift of the H8 next to N7 in H-
NMR should be observed after binding. The experiment was realized using 0.9 % w/v
NaCl/D2O solution in DMF-d7 in order to mimic physiological conditions. In the free 9-
EtG, the H8 is characterized by a singlet at 7.68 ppm; however, after 12 h it was
possible to evidence the presence of a new singlet at 8.32 ppm, thus confirming the
binding between Pt-3 and 9-EtG. (see spectra in SI)
2.9
Glutathione binding study. Glutathione (GSH), a thiol containing tripeptide, is
considered to be responsible of the most drug resistance, especially towards platinum
drugs.[8, 52, 53] The GSH binding study with Pt-3 was conducted using 0.9 % w/v

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1
NaCl/D2O solution in DMSO-d6. The H-NMR spectrum showed the appearance of two
multiplets at 3.04-3.09 and 3.10-3.14 ppm that may be assigned to the methylene
protons next to SH group which are set at 2.60-2.70 and 2.74-2.82 ppm in the unbound
form. These signals underlined the interaction between Pt-3 and GSH thus limiting the
bioavailability of the metal compound. (see spectra in SI)
2.10 Effect of Pt-3 on STAT3 expression and phosphorylation. The data obtained
.
with the AlphaScreen-based assay clearly demonstrated the capacity of compounds 3
HCl and Pt-3 to inhibit the binding between the STAT3-SH2 domain and the
phosphotyrosine of the other monomer and, therefore, the STAT3 dimerization. In order
to deeply explore the pharmacological activity, we employed a series of experiments
aimed at investigating the expression and the phosphorylation state of STAT3. HCT-116
cells were incubated with 25 µM concentration of compounds 3 . HCl and Pt-3 for 24 h
and both total protein extracts were prepared. As shown in Figure 9A, both compounds
did not significantly affect the STAT3 tyrosine phosphorylation state, with a 7.2% and
10.4% inhibition after incubation with 3 . HCl and Pt-3, respectively.
.
Figure 9. Effect of ligand 3 HCl and Pt-3 on STAT3 activity in cultured cells. (A and B) HCT-116 cells
were seeded and incubated with McCoy’s supplemented with 10% FCS; 24 h later the medium was
.
replaced with one containing 10% FCS and the reported concentrations of 3 HCl and Pt-3. The
incubation was continued for a further 24 h at 37°C and Western blot analysis (A) were performed from
total cell lysates. The ratio of phosphoSTAT3/STAT3 signals are shown in the histogram plot. (B)
Luciferase reporter assay for STAT3 transcriptional activity was performed in HeLa stable cells pre-

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.
incubated for 2 h with reported concentrations of 3 HCl and Pt-3. The luciferase activity was the
stimulated by adding Oncostatin M for 4 h. ** p < 0.01; *** p < 0.001 vs control (Cnt).
The direct measurements of the STAT3 promoter activity by luciferase assay in HeLa
cells, showed that Pt-3 significantly interfered with STAT3 function with an IC50 value
equal to 16.7 µM, close to the value observed in the MTT cytotoxicity assay (Figure 9C
and Table 1). On the contrary, compound 3 . HCl, did not show any significant activity at
the maximal concentration tested (30 µM), confirming its lower cytotoxic effect.
2.11 AlexaFluor488-maleimide labeled STAT3 competition assay. To suggest a
possible mechanism by which dimerization disruption occurred in the presence of
.
compound 3
HCl and Pt-3, competitive experiments were performed using
AlexaFluor488-maleimide labeled STAT3, able to discriminate the interaction site on
STAT3 for both the compounds. The results obtained in two different types of
experiments are reported in Figure 10: a pre-treatment with test compounds assay (A)
.
and post-treatment with test compound one (B). AlexaFluor488-maleimide reduced 3
HCl and Pt-3 ability to interact with cysteine residues resulting in lower inhibitory effect.
We can speculate that the partial residual activity shown by Pt-3 might be due to the
coordination of platinum metal centre to the thiol groups as expected also in the case of
Pt-1 and Pt-2. On the contrary, the lower affinity of compound 3 . HCl for cysteines than
AlexaFluor488-maleimide led to a total loss of effect. The AlphaScreen-based assay
.
results confirmed 3 HCl and Pt-3 capability to disrupt STAT3 dimerization without
influencing the phosphorylation as reported before by our experimental data (see
Figure 9A).

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Figure 10. AlexaFluor488-maleimide labeled STAT3 competition assay: a pre-treatment with test
compounds assay (A) and post-treatment with test compound one (B)
3. CONCLUSIONS
Our study aimed to disclose a Pt(II) complex able to interfere with DNA replication and
to inhibit STAT3 signaling cascade. In order to achieve this goal, we selected a 3-
aminomethyl-1,2,5-oxadiaxole scaffold as ligand for the preparation of Pt(II) complexes.
This based-structure was differently substituted to allow the modulation of cytotoxicity
and affinity/selectivity for STAT3.
Regarding the in vitro experiments, Pt-1 and Pt-2 showed a higher inhibitory effect in
AlphaScreen-based assay in comparison with Pt-3 but a negligible cytotoxicity. The 9-
EtG results, the induction of p53 determined by Western blot analysis and the presence
of 195Pt in DNA extracted from treated HCT-116 cells suggested an interaction with
nuclear DNA, although the molecular mechanism of cytotoxicity still needs to be
determined. In addition, Pt-3 did not significantly inhibit the phosphorylation state of
STAT3 in HCT-116 cells, but significantly affected the STAT3 promoter activity and thiol
modification. All these results supported a possible dual targeting mechanism of Pt-3.
Digestive LC-MS/MS fragmentation spectroscopy and other biochemical approaches
.
will be employed to fully understand the interaction of 3 HCl and Pt-3 with specific
cysteines of STAT3 domain.
In vivo studies carried out in the highly aggressive Lewis Lung Carcinoma (LLC)
.
model confirmed that the coordination of STAT3 inhibitor 3 HCl to a platinum moiety
led to the obtainment of an effective and safe antitumor metallo-drug.
4. EXPERIMENTAL SECTION:
4.1
Materials and Methods. Reagents and solvents were purchased from Sigma-
Aldrich and used without further purification. Some reactions involving air-sensitive
reagents were performed under nitrogen atmosphere and anhydrous solvents were
used when necessary. Reactions were monitored by thin layer chromatography analysis
on aluminum-backed Silica Gel 60 plates (70-230 mesh, Merck), using an ultraviolet
fluorescent lamp at 254 nm and 365 nm. Visualization was aided by opportune staining
reagents. Purification of intermediates and the final compounds was performed by flash

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chromatography using Geduran® Si 60 (40-63 µm, Merck). HCT-116 cell line (colorectal
cancer) were cultured in McCoy’s media, the DLD-1 (colorectal cancer) and MCF-7
were cultured in Dulbecco’s modified eagle’s medium (DMEM) with glutamine. All the
media were supplemented with penicillin (10,000 U/mL), streptomycin (10 mg/mL),
nonessential amino acid and 10% Fetal Calf Serum (FCS). The same media was
utilized for the experiments where cells were incubated with newly synthesized
compounds dissolved in DMSO. The same volume of solvent was added to control
conditions and did not exceed 0.5% v/v.: HCT 116 ATCC® number CCL 247TM; MCF7:
ATCC® number HTB-22TM, DLD1: ATCC® number CCL-221TM, HELA: ATCC® number
CCL-2TM and STAT3-HeLa SL-0003 (from Signosis).
1
4.2
Physical Measurements. H and 13C NMR spectra were recorded in CDCl3,
CD3OD, DMSO-d6 or DMF-d7 on Bruker DRX Avance 300 MHz or on a Variant 300
MHz Oxford equipped with a non-reverse probe at 25° C. Chemical shifts (in ppm) were
referenced to residual solvent proton/carbon peak. Chemical shifts are expressed as δ
(ppm). Multiplicity is reported as s (singlet), br s (broad singlet), d (double), t (triplet), q
(quartet), m (multiplet), dd (double of doublets), dt (doublet of triplets). The coupling
constants (J-values) are given in Hertz (Hz). All spectroscopic data match the assigned
structures. FTIR spectra were collected by using a Perkin Elmer (MA, USA) FTIR
Spectrometer “Spectrum One” in a spectral region between 4000 and 450 cm-1 and
analysed by transmittance technique with 32 scansions and 4 cm-1 resolution. ESI-MS
analyses were performed by using a Thermo Finnigan (MA, USA) LCQ Advantage
system MS spectrometer with an electronspray ionisation source and an ‘Ion Trap’
mass analyser. The MS spectra were obtained by direct infusion of a sample solution in
MeOH under ionisation, ESI positive. Log Pow’s values and purity were evaluated with
Partisil C18-ODS reversed-phase HPLC column with Merck-Hitachi L-7100 equipped
with Detector UV6000LP. Elemental analyses were performed using a Perkin Elmer
SeriesII/CHNS/O 2400 Analyzer. ICP-MS data were recorded with BRUKER aurora
M90 ICP-MS (MA, USA).
4.3
CHEMISTRY

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4.3.1 General procedure for the synthesis of platinum complexes Pt-1, Pt-2 and
Pt-3
An amount of K2PtCl4 (1 mmol) was dissolved in 10 mL of distilled water. Ligand (1.1
mmol) when available in dihydrochloride form was preventively neutralized with
NaHCO3 aqueous solution and was dropped into palatinate solution. 4 M HCl (10
equivalents) was added and the mixture was refluxed under nitrogen atmosphere for 12
h. After cooling to room temperature, the product was filtered as a solid and washed
extensively with water. The precipitate was then dissolved in diethyl ether and
crystallized for slow diffusion of hexane.
Pt-1: pale-yellow solid, 62% yield. Chemical Formula: C4H7Cl2N3OPt. Molecular
Weight: 377.96 g/mol. 1H NMR (300 MHz, DMF-d7) δ 6.72 (br, 2H), 4.34 (t, J = 6.3 Hz,
2H), 2.61 (s, 3H) ppm. 13C NMR (75 MHz, DMF-d7) 163.18, 153.94, 42.79, 8.64 ppm.
195Pt NMR (300 MHz, DMF-d7) δ -2065 ppm. Elemental analysis for C4H7Cl2N3OPt:
calculated C, 12.67; H, 1.81; N, 11.09; found C, 12.72; H, 1.78; N, 10.98, IR ν = 3217,
3188, 3103, 1586, 1567, 1426, 1376, 1201, 1050, 970 cm-1, MS (ESI) m/z 365.9 [M-
Cl+Na]+.
Pt-2: brown solid, 45% yield. Chemical Formula: C9H8Cl3N3OPt. Molecular Weight:
1
473.94 g/mol. H NMR (300 MHz, DMF-d7) δ 7.98 (d, J = 8.8 Hz, 2H), 7.76 (d, J = 8.8
Hz, 2H), 6.94 (br, 2H), 4.61 (t, J = 6.0 Hz, 2H) ppm. 13C NMR (75 MHz, DMF-d7) 137.78,
130.49, 130.19, 125.25, 123.31, 116.47, 44.35 ppm. 195Pt NMR (300 MHz, DMF-d7) δ -
2075 ppm. Elemental analysis for C9H8Cl3N3OPt: calculated C, 22.93; H, 1.70; N, 8.84;
found C, 23.01; H, 1.75; N, 8.89, IR ν = 3243, 3204, 2962, 2919, 1602, 1567, 1464,
1261, 1096, 1016, 802 cm-1, MS (ESI) m/z 461.9 [M-Cl+Na]+.
Pt-3: olive-green solid, 61% yield. Chemical Formula: C16H12Cl3N3O2Pt. Molecular
Weight: 577.96 g/mol. 1H NMR (300 MHz, CDCl3 ) δ 7.77 (d, J = 8.5 Hz, 2H), 7.50-7.44
(m, 5H), 7.25-7.21 (m, 2H), 6.20 (t, J = 5.8Hz, 1H), 5.46-5.41 (m, 1H), 4.88-4.85 (m, 1H)
ppm. 13C NMR (75 MHz, CDCl3) 191.27, 152.84, 149.45, 138.06, 135.09, 132.73,
130.44, 130.35, 130.23, 130.19, 129.60, 129.51, 129.47, 129.32, 129.23, 128.93, 55.15
ppm. 195Pt NMR (300 MHz, CDCl3 ) δ -2068 ppm. Elemental analysis for
C16H12Cl3N3O2Pt: calculated C, 33.35; H, 2.13; N, 7.45; found C, 33.58; H, 2.18; N,

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7.54, IR ν = 3145, 3078, 2931, 1697, 1597, 1448, 1261, 1216, 1094, 995, 960, 834, 688
cm-1,MS (ESI) m/z 578.5 [M+H]+.
4.3.2 Crystallization, data collection and structural determination.
Single crystals of Pt-1 suitable for X-ray structure analysis were obtained by slow
evaporation of a solution solution 33% MeOH:water at room temperature. The crystals
were small yellow needles. Intensity data were collected at room temperature on a
Bruker Axs CCD-based three cycle diffractometer, using graphite-monochromatized
Mo-K radiation (λ= 0.71073 Å).
X-ray diffraction data in the θ range 2-25° were collected acquiring 4 sets of 600
bidimensional CCD frames with the following operative conditions: omega rotation axis,
scan width 0.3°, acquisition time 40 s, sample-to-detector distance 50 mm, phi angle
fixed at four different values (0°, 90°, 180°, 270°) for the four different sets.
Omega-rotation frames were processed with the SAINT software[54] for data
reduction (including intensity integration, background, Lorentz and polarization
corrections) and for determination of accurate unit-cell dimensions, obtained by least-
squares refinement of the positions of 8049 independent reflections with I > 10σ(I) in the
θ range 2-24°. Absorption effects were empirically evaluated by the SADABS
software[55] and absorption correction was applied to the data (0.367 and 0.602 min
and max transmission factor).
The structure was solved by direct methods (SIR-97[56]) and completed by iterative
2
cycles of full-matrix least squares refinement on Fo and
∆F synthesis using the
SHELXL-97[57] program (WinGX suite).[37] All non-H-atoms were refined
anisotropically. The H-atoms positions were introduced in calculated positions in their
described geometries and allowed to ride on the attached carbon atom with fixed
isotropic thermal parameters. Geometrical calculations were carried out with the
program PARST.[58] The crystal data are given below. CCDC-1478539 contains the
supplementary crystallographic data for this paper. These data can be obtained free of
charge via www.ccdc.cam.ac.uk/conts/retrieving.html (or from the Cambridge
Crystallographic Data Centre, 12, Union Road, Cambridge CB21EZ, UK; fax: ++44
1223 336 033; or deposit@ccdc.cam.ac.uk ).

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Crystal data: C4 H7 O1 Pt1 Cl2 N3, Mr = 379.01 g/mol, Trigonal, Space group R-3 a=
39.836(2) Å, c = 6.724(3) Å, V = 9240.8(5) Å3, Z = 12, Dcalc = 2.439 Mg/m3, F(000) =
6120, R = 0.026 (reflections collected/unique = 3614/3228), wR2 = 0.031, T = 293(2)K,
GOF = 1.057. The reflections were collected in the range 1.02° ≤ θ ≤ 25.03° (limiting
indices = -47≤h≤47, -47≤k≤47, -8≤l≤7) employing a 0.01 x 0.02 x 0.01 mm3 crystal. The
residual positive and negative electron densities in the final map were 0.950 and –0.540
eÅ-3.
4.3.3 Stability test. The stability study of complexes (Pt-1, Pt-2 and Pt-3) was
performed under physiological conditions through UV-vis spectroscopy at their
respective λmax. Stock solutions were diluted to a final concentration of 100 µM with 1 %
DMF. The UV-vis peak profile of the sample was monitored for 48 h at 4 h intervals.
4.3.4 Log Pow determination. RP-HPLC analysis were performed to correlate the
hydrophobicity of the compounds and the Pt(II) complexes with their retention time. The
chromatograms were registered using Partisil C18-ODS reversed-phase HPLC column
at 25 °C and water/acetonitrile (60:40) as mobile phase with KI as internal standard
(flow rate: 1 mL/min; λ= 230 nm). For platinum complexes the same column was used
and water (15 mM HCOOH)/MeOH (70:30) mobile phase with KI as internal standard
(flow rate of 1 mL/min, λ= 210 nm).[41-43, 59] The calibration curve was realized in
comparison with reference compounds chosen in OECD guideline.[54]
4.4
BIOLOGY
4.4.1 Cell culture. The following human cancer cell lines have been utilized in the
study: HCT-116 (colorectal cancer), DLD1 (colorectal cancer), and MCF-7 (breast
cancer). All cultured media were supplemented with penicillin (10,000 U/mL),
streptomycin (10 mg/mL), non-essential amino acid and 10% Fetal Calf Serum (FCS).
Dulbecco’s modified eagle’s medium (DMEM) with glutamine was utilized for MCF-7,
and DLD-1 cell lines and McCoy’s for HCT-116. Cells were incubated with newly
synthesized compounds dissolved in DMSO. The same volume of solvent were added
to control conditions and did not exceed 0.5% v/v.

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4.4.2 MTT-assay. The MTT assay was performed according to literature data.[29, 49]
4.4.3 AlphaScreen-based assay. It is a bead-based nonradioactive assay system for
detecting biomolecular interactions in a microtiter plate format. Binding of biological
partners brings donor and acceptor beads into close proximity and as a result, a
fluorescent signal between 520 and 620 nm is produced. The AlphaScreen-based
assays[23, 60] were performed in a final reaction volume of 25 µL of the assay buffer
containing 10 mM HEPES–NaOH (pH 7.4), 50 mM NaCl, 1 mM EDTA (pH 8.0), 0.1%
NP-40, and 10 ng/µL BSA in a 96-well microtiter plate at 25 °C. Phospho-Tyr (pTyr)
peptide probes used in this study were 5-carboxyfluorescein (FITC)-GpYLPQTV for
STAT3 and (FITC)-GpYDKPHVL for STAT1. Firstly, 75 nM of each SH2-containing
protein was incubated with the test compound for 15 min. Each protein sample was then
incubated for 90 min with 50 nM of its corresponding FITC-pTyr peptide, and mixed with
streptavidin coated donor beads and anti-FITC acceptor beads simultaneously before
detection at 570 nm using EnVison Xcite (PerkinElmer).
4.4.4 In vivo antitumor activity in Lewis Lung Carcinoma. All experiments were
performed according to D.L.G.S. 26/2014 which warrants care of experimental animals
in Italy. The research project was approved by the Italian Health Department according
to the art. 7 of above mentioned D.L. The LLC cell line was purchased from ECACC,
United Kingdom. The LLC cell line was maintained in DMEM (Euroclone) supplemented
with 10% heat-inactivated fetal bovine serum (FBS; Euroclone), 10 mM L-glutamine,
100 U mL−1 penicillin, and 100 µg mL−1 streptomycin in a 5% CO2 air incubator at 37 °C.
The LLC was implanted intramuscularly (im) as a 2 × 106 cell inoculum into the right
hind leg of 8 week old male and female C57BL mice (24 ± 3 g body weight). After 24 h
from tumor implantation, mice were randomly divided into five groups (8 animals per
group, 10 controls). From day 7 after tumor inoculation (visible tumor) animals were
.
treated daily with a single dose of compound 3 HCl and of Pt-3 complex (75 mg kg−1
and 30 mg kg−1, respectively, dissolved in a vehicle solution composed of 20%
Cremophor EL (v/v), 20% PEG400 (v/v) and 60% saline solution (v/v)) or with a daily ip

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dose of cisplatin (1.5 mg kg−1 in 0.9% NaCl solution). At day 15, animals were
sacrificed, the legs were amputated at the proximal end of the femur, and the inhibition
of tumor growth was determined according to the difference in weight of the tumor-
bearing leg and the healthy leg of the animals expressed as a percentage referred to
the control animals. Body weight was measured at day 1 and every two days from day
7, and was taken as a parameter for systemic toxicity. All the values are the means ±
SD of not less than three measurements.
4.4.5 Determination of intracellular and DNA-bound Pt concentrations. For the
determination of total intracellular 195Pt concentrations, cells were homogenates with
lysis buffer containing 0.5% NP-40, 150 mM NaCl and 50 mM Tris-HCl at pH 7.5. The
cell monolayer was three times with PBS before the incubation with the lysis buffer for
30 min at 4°C. Cell lysates were treated then cleared by centrifugation at 14,000g for 10
min, and the 195Pt concentrations determined by ICP-MS. The data were normalized
with the protein concentrations were determined using the BCA protein assay (Thermo
scientific, Rockford, IL USA). The 195Pt concentration bound to DNA was estimated from
nuclear DNA extracted as previously described.[29] The data were normalized with
DNA concentrations determined by spectrophotometer determination with NanoDrop
system.
4.4.6 Binding study with 9-ethylguanine and GSH. The binding studies were directly
conducted in NMR tube preparing 1 eq. of Pt-3 in 20 % of 0.9 % w/v NaCl-D2O solution
in DMSO-d6. 1eq. of 9-ethylguanine or GSH was added to a solution. The samples were
evaluated at different times by 1H-NMR analysis.
4.4.7 Western blot analysis. Cells were washed twice with PBS and lysed by
incubation with a solution of 50 mM Tris pH 7.5, 150 mM NaCl, 0.5% Nonidet-P40,
containing a protease inhibitor cocktails (Sigma Aldrich, Milan, Italy) for 30 min on ice.
Cell lysates were then cleared by centrifugation at 14,000g for 10 min.[61] Protein
samples were separated by SDS-PAGE under reducing conditions, transferred to
nitrocellulose membrane (GE Healthcare Little Chalfont, Buckinghamshire, UK) and

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subsequently immunoblotted with primary antibody following appropriate secondary
fluorescently labeled antibody and acquired with the Odissey FC system (LI-COR).
Quantitative densitometric analyses were performed with Image Studio software (LI-
COR). The following antibodies were utilized: monoclonal antibody anti p53 and β-
tubulin (SIGMA), anti-pSTAT3 (Abcam) and anti-STAT3 (Santa-Cruz Biotechnology).
4.4.8 Luciferase reporter gene assay. STAT3 reporter HeLa stable cells (Signosis
Inc) were incubated in a 96-well microplate for 24 h. Cells were pretreated with test
compounds for 2 h, and 10 ng/ml (w/v) of Oncostatin M were applied and incubated for
4 h. Cells were washed with medium not supplemented with phenol red, and Steady-
Glo® reagent (Promega) was applied. After 15 min incubation, the signals were
detected by ARVO Light 1420 (PerkinElmer Life Sciences). The relative signal intensity
was calculated in each well as the ratio for the mean signal of vehicle. In parallel with
the procedure, CellTiter-Glo (CTG) Luminescent Cell Viability Assay (Promega) was
performed for checking the viability of the cells.
4.4.9 AlexaFluor488-maleimide labeled STAT3 competition assay.
Pre-treatment: The recombinant human STAT3 protein (0.3 µM) was incubated with
the test compound in 100 mM HEPES for 2 h at 25 . Alexa Fluor 488 C5 maleimide (50
µM) was added to the mixture and incubated for further 2h at 25 . Samples were
diluted in SDS-PAGE sample buffer without 2-mercaptoethanol and run on an SDS-
PAGE gel. Fluorescence of Alexa-labeled STAT3 was analyzed with FUJIFILM LAS-
3000 image analyzer (excitation/emission: 493/516 nm)
Post-treatment: The recombinant human STAT3 protein (0.3 µM) was incubated with
Alexa Fluor 488 C5 maleimide (50 µM) in 100 mM HEPES for 2 h at 25 . The test
compound was added to the mixture and incubated for further 2h at 25 . Samples were
diluted in SDS-PAGE sample buffer without 2-mercaptoethanol and run on an SDS-
PAGE gel. Fluorescence of Alexa-labeled STAT3 was analyzed with FUJIFILM LAS-
3000 image analyzer (excitation/emission: 493/516 nm).
4.4.10 Statistical analysis

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All data shown are representative of at least three replicate experiments. Data are
expressed as mean ± SD. Statistical analyses were performed using the unpaired
Student’s t test. P values <0.05 were considered significant. The concentration of
compounds required to reduce by 50% the cell viability (EC50) was calculated by
nonlinear regression curve (SigmaPlot software; Systat Software, Inc., Point Richmond,
CA).
AUTHOR INFORMATION
Corresponding Author
*isabella.rimoldi@unim.it, +39 0250314609; nicola.ferri@unipd.it, +39 0498275080;
arianna.gelain@unimi.it, +39 0250319369.
Author Contributions
±These authors contributed equally.
Acknowledgments
This study was supported by funds from PRIN 2010-2011. Authors also thank
Fondazione Confalonieri (Fellowship 2015) for the financial support. BM Kwon was
supported by the Bio-Synergy Research Project (2012M3A9C4048777).
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LISTS OF CAPTIONS
Figure 1. New Pt(II) complexes.
Figure 2. Ligands and their corresponding Pt(II) complexes.
Scheme 1. Reagents and conditions: i) NaN3, Ph3P, CCl4/DMF (1:4), 90°C, 5 h; ii) Pd/C, H2, MeOH, 20
atm, 4 h; iii) 2 M HCl in Et2O.
Scheme 2. Reagents and conditions i) t-BuONO, TMSCl, dry THF/DCM, -20°C to rt, 12 h; ii) NH2OH·HCl,
NaOH, H2O, reflux, 3 h; iii) KOH, Cs2CO3, ethylen glycol, 160°C, 1.5 h; iv) 2.5 M n-BuLi in hexane, methyl
benzoate, dry THF, -55°C for 1 h, then rt for 12 h; v) t-BuONO, TMSCl, dry THF, -20°C to rt, 12 h; vi) Zn,
TFA, rt, 40 min; vi) 2 M HCl in Et2O.
Scheme 3. Reagents and conditions: i) K2PtCl4, 4 M HCl, H2O, reflux, 2 h.
Figure 3: ORTEP[37] view of one of the two molecules present in the asymmetric unit of Pt-1, showing
the arbitrary atom labeling scheme. Ellipsoids are drawn at 40% probability level and H atoms are shown
as spheres of arbitrary radius. Selected averaged bond distances (Å): Pt-N1, 1.956(1); Pt-N3, 2.046(1);
Pt-Cl1, 2.274(1); Pt-Cl2, 2.294(1); N1-C2, 1.291(1); N3-C3, 1.497(1). Angles (°): N1-Pt-N3, 80.8(1); N3-
Pt-Cl1, 90.7(1); Cl1-Pt-Cl2, 91.6(1); Cl2-Pt-N1, 96.8(1); C2-N1-Pt, 119.9(1); C3-N3-Pt, 112.2(1); C2-C3-
N3, 110.5(1).