New trans dichloro (triphenylphosphine)platinum(II) complexes containing N-(butyl),N-(arylmethyl)amino ligands: Synthesis, cytotoxicity and mechanism of action

Article abstract of DOI:10.1016/j.bmc.2016.04.067

Some new platinum(II) complexes have been prepared, of general formula trans-[PtCl₂(PPh₃){NH(Bu)CH₂Ar}], where the dimension of the Ar residue in the secondary amines has been varied from small phenyl to large pyrenyl group. The obtained complexes, tested in vitro towards a panel of human tumor cell lines showed an interesting antiproliferative effect on both cisplatin-sensitive and -resistant cells. For the most cytotoxic derivative 2a the investigation on the mechanism of action highlighted the ability to induce apoptosis on resistant cells and interestingly, to inhibit the catalytic activity of topoisomerase II.

Full text of DOI:10.1016/j.bmc.2016.04.067

Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
New trans dichloro (triphenylphosphine)platinum(II) complexes
containing N-(butyl),N-(arylmethyl)amino ligands: Synthesis,
cytotoxicity and mechanism of action
a
b
c
c
Lisa Dalla Via a, , Aída Nelly García-Argáez , Enzo Agostinelli , Daniela Belli Dell’Amico , Luca Labella ,
⇑
Simona Samaritani c
a Dipartimento di Scienze del Farmaco, Università degli Studi di Padova, Via F. Marzolo 5, 35131 Padova, Italy
b Dipartimento di Scienze Biochimiche, Università degli Studi di Roma ‘La Sapienza’, Piazzale Aldo Moro 5, 00185 Roma, Italy
c Dipartimento di Chimica e Chimica Industriale, Università di Pisa, Via G. Moruzzi 13, 56124 Pisa, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Some new platinum(II) complexes have been prepared, of general formula trans-[PtCl2(PPh3){NH(Bu)
CH2Ar}], where the dimension of the Ar residue in the secondary amines has been varied from small phe-
nyl to large pyrenyl group. The obtained complexes, tested in vitro towards a panel of human tumor cell
lines showed an interesting antiproliferative effect on both cisplatin-sensitive and -resistant cells. For the
most cytotoxic derivative 2a the investigation on the mechanism of action highlighted the ability to
induce apoptosis on resistant cells and interestingly, to inhibit the catalytic activity of topoisomerase II.
Ó 2016 Elsevier Ltd. All rights reserved.
Received 23 February 2016
Revised 26 April 2016
Accepted 30 April 2016
Available online xxxx
Keywords:
Transplatinum(II) complexes
Antiproliferative activity
DNA topoisomerases
1. Introduction
complexes, of general structure trans-[PtCl2(PPh3){NH(Bu)CH2Ar}],
where the dimension of the aromatic residue on the secondary
Since the discover of cis-[PtCl2(NH3)2] (cisplatin) anticancer
properties,1 hundreds of new platinum(II) complexes have been
tested for their antiproliferative activity.2 Among them, trans com-
plexes also containing phosphorous based ligands,3 are promising,
and in some cases they have shown a good activity towards cis-
platin resistant cell lines.3a,h We have recently prepared a series
of [PtCl2(PPh3)(L)] complexes,4 were L was a neutral ligand. Most
of them were tested in vitro4c,d against HeLa, H460 and A549
human tumor cell lines and our results showed that a higher
antiproliferative activity could be observed in the case of trans
compounds and L = R2NH.4d In particular, the highest
antiproliferative activity was observed for L = NH(CH2CH2OH)2
and the investigation about its mechanism of action highlighted
the capacity to affect the mitochondrial functions,4c with a mode
of action different from cisplatin. Moreover, its interaction with a
phospholipid bilayer, used as a model of the cellular membrane,
proved to be different from that of cisplatin,5 suggesting that even
the mode of crossing the cellular membrane could be different.
In this contest and with the aim of expanding knowledge about
structure–activity relationship, we prepared four new platinum(II)
amines was varied (Ar = Phenyl, 1-Naphthyl, 9-Anthracenyl,
2-Pyrenyl).
In addition, trans-[PtCl2(PPh3)(p-Tolu)] (p-Tolu = 4-methylani-
line) was prepared, as a model devoid of the CH2 spacer between
the aromatic moiety and the coordinated nitrogen.
In the present work, we report the synthesis and characteriza-
tion of the secondary amines ArCH2NHBu and of the corresponding
trans-platinum(II) complexes, together with the antiproliferative
activity towards a panel of tumor cell lines, both sensitive and
resistant to cisplatin. For the most interesting complex, the study
on the mechanism of action is reported. In particular, the death
pathway on resistant cells was analyzed by flow cytometry and
moreover, the binding to salmon testes DNA and the effect on cell
cycle were determined in comparison with cisplatin. Finally, the
ability to interfere with the catalytic activity of the nuclear
enzymes topoisomerase I and II was investigated.
2. Materials and methods
2.1. General experimental conditions
All manipulations were performed under a dinitrogen atmo-
sphere, if not otherwise stated. Solvents and liquid reagents were
⇑
Corresponding author. Tel.: +39 0498275712; fax: +39 0498275366.
E-mail address: lisa.dallavia@unipd.it (L. Dalla Via).
http://dx.doi.org/10.1016/j.bmc.2016.04.067
0968-0896/Ó 2016 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Dalla Via, L.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.04.067

2
L. Dalla Via et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
dried according to reported procedures.6 1H, 13C, 31P and 195Pt NMR
spectra were recorded with a Bruker ‘Avance DRX400’ spectrome-
ter, in CDCl3 solution if not otherwise stated. Chemical shifts were
measured in ppm (d) from TMS by residual solvent peaks for 1H
and 13C, from aqueous (D2O) H3PO4 (85%) for 31P and from aqueous
(D2O) hexachloroplatinic acid for 195Pt NMR. A sealed capillary
containing C6D6 was introduced in the NMR tube to lock the spec-
trometer to the deuterium signal when non-deuterated solvents
were used. FTIR spectra in solid phase were recorded with a
Perkin–Elmer ‘Spectrum One’ spectrometer, equipped with an
ATR accessory. IR data are expressed in cmꢀ1. Elemental analyses
(C, H, N) were performed at Dipartimento di Scienze e Tecnologie
Chimiche, Università di Udine. cis-[PtCl2(NCMe)(PPh3)]4a was
prepared according to a reported procedure.
(br s, 1H, NH), 1.56 (m, 2H, NHCH2CH2), 1.33 (m, 2H, CH2CH2CH3),
0.88 (t, 3H, J = 7.3 Hz, CH3); 13C NMR: 131.6, 131.1, 130.4, 129.2,
127.4, 126.2, 125.0, 124.1, 50.1, 45.6, 32.0, 20.5, 14.0.
2.2.4. N,N-(2-Pyrenylmethyl)butylamine (2-PyrCH2NHBu, 1d)9
Ar = 2-Pyr; the sample was used in the following synthesis
without further purification; colorless solid; 98%. Anal. Calcd for
C21H21N: C, 87.4; H, 7.4; N, 4.8. Found: C, 87.7; H, 7.3; N, 4.8. IR
(ATR): 3380 (b), 3039, 2952, 2927, 2894, 2859, 2814, 1601, 1586,
1475, 1443, 1415, 1393, 1369, 1331, 1299, 1183, 1117, 1094,
839, 824, 801, 743, 710, 679; 1H NMR: 8.34–7.96 (m, 9H, HAr),
4.45 (s, 2H, ArCH2), 2.76 (t, 2H, J = 7.0 Hz, NHCH2CH2), 1.54 (m,
3H, NHCH2CH2), 1.35 (m, 2H, CH2CH2CH3), 0.89 (t, 3H, J = 7.0 Hz,
CH3); 13C NMR: 134.2, 131.4, 130.9, 130.6, 129.1, 127.6, 127.5
(2C), 127.0, 126.9, 125.9, 125.1, 125.0 (2C), 124.7, 123.2, 51.9,
49.8, 32.3, 20.6, 14.1.
2.2. Synthesis of ArCH2NHBu
In a round-bottomed flask equipped with magnetic stirrer, con-
denser and dropping funnel, a solution of ArCHO in ethanol
([ArCHO] ꢁ 0.37 mol/L) was treated with butylamine ([BuNH2]/
[ArCHO] = 2.1 mol/mol). The mixture was refluxed until the com-
plete conversion of ArCHO into ArCH@NHBu was reached [IR spec-
2.3. Synthesis of trans-[PtCl2(PPh3){NH(Bu)CH2Ar}] (2a–d)
In a round-bottomed flask equipped with magnetic stirrer, con-
denser and dropping funnel, a suspension of cis-[PtCl2(CH3CN)
(PPh3)] in 10 mL of 1,2-DCE was refluxed (84 °C) and treated with
a solution containing the secondary amine (ArCH2)BuNH in 2.0 mL
of the same solvent (cis-[PtCl2(CH3CN)(PPh3)]/[R2NH] molar
ratio = 1:1.2). The progress of the reaction was monitored by 31P
NMR spectroscopy until the complete conversion of the precursor
into the desired product was observed (3 h). Solvent was elimi-
nated under vacuum (0.01 mmHg), the residue was dissolved in
CH2Cl2 (5 mL) and treated with heptane (10 mL). Solid products
were recovered by filtration and dried under vacuum
(0.01 mmHg). The same procedure was followed for the
preparation of trans-[PtCl2(PPh3)(p-Tolu)] (3). For each of
the trans-[PtCl2(PPh3)(amine)] complexes the amine used, the
troscopy, mCHO (ꢁ1680 cmꢀ1) versus mCH@ (ꢁ1640 cmꢀ1)]. The
~
~
N
solution was cooled (0 °C) and an excess of NaBH4 ([NaBH4]/
[ArCHO] = 2.5/1 mol/mol) was added in small portions. When the
vigorous evolution of dihydrogen ceased, the mixture was warmed
(25 °C) and stirring was continued (10 h). Ethanol and excess
BuNH2 were evaporated under reduced pressure, water was added
and the aqueous solution was extracted in Et2O (3 ꢂ 100 mL).
Organic extracts were washed with water (3 ꢂ 30 mL) and dried
over anhydrous Na2SO4, then solvents were evaporated under
reduced pressure. For each amine prepared the purification
method, the percentage isolated yield and the spectroscopic char-
acterization are reported.
isolated
% yield and the spectroscopic characterization are
indicated.
2.2.1. N,N-Benzylbutylamine (PhCH2NHBu, 1a)7
Ar = Ph; filtration over a short package of anhydrous Al2O3; col-
orless liquid; 73%. Anal. Calcd for C11H17N: C, 80.9; H, 10.5; N, 8.6.
Found: C, 80.8; H, 10.3; N, 8.6. IR (ATR): 3340 (b), 3087, 3064, 3028,
2957, 2928, 2872, 2815, 1495, 1454, 1120, 1076, 1029, 730, 696;
1H NMR: 7.27–7.18 (m, 5H, HArom), 3.73 (s, 2H, PhCH2), 2.58 (t,
2H, J = 7.3 Hz, NHCH2), 2.50 (br s, 1H, NH), 1.46 (quint, 2H,
J = 7.3 Hz, NHCH2CH2), 1.28 (sest, 2H, J = 7.5 Hz, CH2CH3), 0.87 (t,
3H, J = 7.5 Hz, CH3); 13C NMR: 139.8, 128.4, 128.3, 127.0, 53.8,
48.9, 31.9, 20.4, 13.9.
2.3.1. trans-[PtCl2(PPh3){NH(Bu)(CH2Ph)}] (2a)
PhCH2NHBu; 50%; Anal. Calcd for C29H32Cl2NPPt: C, 50.4; H, 4.7;
N, 2.0. Found: C, 50.3; H, 4.4; N, 2.0. IR (ATR): 3195 m, 2964, 2929,
2871, 2792, 2358, 1811, 1588, 1496, 1480, 1384, 1312, 1097 s,
1010, 977, 911, 866, 707 m, 691 s; 1H NMR: 7.66–7.32 (m, 20H,
3
H
arom), 4.50 (dd, [2JH–H = 12.4 Hz, JH–H = 6.7 Hz], 1H, PhCHH),
4.03–3.81 (br s [2JH–Pt = 73.0 Hz]+m, 2H, NH + PhCHH), 3.25 (m,
1H, NHCHH), 2.66 (m, 1H, NHCHH), 2.24 (m, 1H, NHCH2CHH),
1.86 (m, 1H, NHCH2CHH), 1.38 (m, 2H, CH2CH3), 0.92
(t, 3H, CH3); 13C NMR: 148.0, 134.7 (d, [JC–P = 10.5 Hz]), 130.7 (d,
[JC–P = 2.0 Hz]), 130.0, 129.8, 127.9 (d, [JC–P = 56.5 Hz]), 127.9 (d,
[JC–P = 11.2 Hz]), 56.3, 50.7, 31.0, 20.4, 13.9; 31P NMR: 5.25
(1JP–Pt = 3576 Hz); 195Pt NMR: ꢀ3669 (1JPt–P = 3576 Hz).
2.2.2. N,N-(1-Naphtylmethyl)butylamine (1-NpCH2NHBu, 1b)7
Ar = 1-Np; distillation under reduced pressure (130 °
C/0.5 Torr); colorless oil; 98%. Anal. Calcd for C15H19N: C, 84.5; H,
9.0; N, 6.6. Found: C, 85.0; H, 9.2; N, 7.0. IR (ATR): 3350 (b),
3048, 2956, 2928, 2870, 2828, 1590, 1576, 1509, 1457, 1396,
1377, 1333, 1236, 1225; 1H NMR: 8.11–7.41 (m, 7H, HAr), 4.22 (s,
2H, ArCH2), 2.73 (t, 2H, J = 7.0 Hz, NHCH2CH2), 1.73 (br s, 1H,
NH), 1.53 (m, 2H, NHCH2CH2), 1.35 (m, 2H, CH2CH2CH3), 0.91 (t,
3H, J = 7.0 Hz, CH3); 13C NMR: 136.0, 133.9, 131.8, 128.8, 127.7,
126.1, 126.0, 125.6, 125.4, 123.6, 51.6, 49.8, 32.2, 20.6, 14.1.
2.3.2. trans-[PtCl2(PPh3){NH(Bu)(CH2Np)}] (2b)
1-NpCH2NHBu; 86%; Anal. Calcd for C33H34Cl2NPPtꢃ0.5(CH2Cl2):
C, 51.3; H, 4.5; N, 1.8. Found: C, 52.3; H, 4.5; N, 1.9. IR (ATR): 3250,
1481, 1433, 1265, 1098, 1014, 800, 726, 690; 1H NMR: 8.24 (d, 1H,
HNp), 7.87 (m, 2H, 2HNp), 7.64–7.32 (m, 19H, 4HNp + Ph), 5.05 (d,
[2JH–H = 12.0 Hz], 1H, NpCHH), 4.37–4.17 (br s [2JH–Pt = 81.0 Hz]+d,
[2JH–H = 12.0 Hz], 2H, NH + NpCHH), 3.29 (m, 1H, NHCHH), 2.51
(m, 1H, NHCHH), 2.20 (m, 1H, NHCH2CHH), 1.77 (m, 1H,
NHCH2CHH), 1.31 (m, 1H, CHHCH3), 1.22 (m, 1H, CHHCH3), 0.82
(t, 3H, CH3); 13C NMR: 134.8 (d, [2JC–P = 10.2 Hz]), 134.0, 131.7,
131.4, 130.8, 129.4, 129.0, 128.9 (d, [1JC–P = 62 Hz]), 127.9
(d, [2JC–P = 11.1 Hz]), 127.9, 127.1, 126.1, 125.2, 123.3, 53.7, 49.9,
31.1, 20.0, 13.8; 31P NMR: 4.09 (1JP–Pt = 3580 Hz); 195Pt NMR:
ꢀ3620 (1JPt–P = 3580 Hz).
2.2.3. N,N-(9-Anthracenylmethyl)butylamine (9-AnthCH2NHBu,
1c)8
Ar = 9-Anth; the sample was used in the following synthesis
without further purification; yellow, low melting solid; 88%. Anal.
Calcd for C19H21N: C, 86.6; H, 8.0; N, 5.3. Found: C, 86.3; H, 8.0;
N, 5.2. IR (ATR): 3330 (b), 3052, 2954, 2926, 2858, 1623, 1524,
1445, 1376, 1337, 1320, 1158, 1107, 952, 882, 839, 788, 727; 1H
NMR: 8.37–8.31 (m, 3H, HAr), 7.96 (d, 2H, HAr), 7.53–7.41 (m, 4H,
HAr), 4.71 (s, 2H, ArCH2), 2.84 (t, 2H, J = 7.3 Hz, NHCH2CH2), 2.37
Please cite this article in press as: Dalla Via, L.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.04.067

L. Dalla Via et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
3
2.3.3. trans-[PtCl2(PPh3){NH(Bu)(CH2Anthr)}] (2c)
GI50 values, i.e., the concentration of the test agent inducing 50%
reduction in cell number compared with control cultures.
9-AnthrCH2NHBu; 57%; Anal. Calcd for C37H36Cl2NPPt: C, 56.1;
H, 4.6; N, 1.8. Found: C, 55.7; H, 4.5; N, 1.8. IR (ATR): 3250, 3047,
2921, 1622, 1525, 1481, 1433, 1310, 1186, 1156, 1099, 1H NMR:
8.49 (m, 2H, 2HAnth), 8,02 (m, 2H, 2HAnth), 7.72–7.32 (m, 20H,
5HAnth + Ph), 5.42 (d, [2JH–H = 13.5 Hz], 1H, AnthCHH), 5.27
(d, [2JH–H = 13.5 Hz], 1H, AnthCHH), 4.50 (br s, [2JH–Pt = 80.0 Hz],
1H, NH), 3.46 (m, 1H, NHCHH), 2.30 (m, 1H, NHCHH), 2.16 (m,
1H, NHCH2CHH), 1.73 (m, 1H, NHCH2CHH), 1.21 (m, 1H, CHHCH3),
1.08 (m, 1H, CHHCH3), 0.71 (t, 3H, CH3); 13C NMR: 134.8
(d, [2JC–P = 10.2 Hz]), 131.5, 131.1, 130.8, 129.4, 128.9
(d, [1JC–P = 62 Hz]), 128.6, 127.9 (d, [2JC–P = 11.0 Hz]), 127.0, 126.7,
125.3, 124.0, 49.5, 48.0, 31.3, 19.8, 13.7; 31P NMR: 4.16
(1JP–Pt = 3573 Hz); 195Pt NMR: ꢀ3618 (1JPt–P = 3573 Hz).
2.5. Evaluation of apoptotic cell death by Annexin V-FITC and
propidium iodide staining
To detect phosphatidylserine translocation from the inner face
to the outer surface of plasma membrane, a FITC Annexin V Apop-
tosis Detection Kit I (BD Pharmigen) was used.
A2780cis cells (2.0 ꢂ 105) were seeded into each cell culture
plate in complete growth medium. After incubation for 24 h the
test agents were added at the indicated concentrations and cells
were incubated for a further 18 h. After treatment, cells were cen-
trifuged and resuspended at 106 cells/mL in binding buffer. Cell
suspensions (100 lL) were added with Annexin V-FITC and propid-
2.3.4. trans-[PtCl2(PPh3){NH(Bu)(CH2Pyren)}] (2d)
ium iodide (PI) as indicated by the supplier’s instructions, and
incubated for 15 min at room temperature in the dark. The popu-
lations of Annexin V-negative/PI-negative cells (viable), Annexin
V-positive/PI-negative cells (early apoptosis), Annexin V-positive/
PI-positive cells (late apoptosis) and Annexin V-negative/PI-posi-
tive cells (necrosis) were evaluated by FACSCanto II flow cytometer
(Becton–Dickinson, Mountain View, CA).11
2-PyrenCH2NHBu; 63%; Calcd for C39H36Cl2NPPtꢃCH2Cl2: C,
53.3; H, 4.2; N, 1.6. Found: C, 54.1; H, 4.1; N, 1.6. IR (ATR): 3229,
3042, 2957, 2929, 1590, 1482, 1433, 1185, 1100, 1078, 999, 850;
1H NMR: 8.49 (d, 1H, HPyren), 8,20–8.00 (m, 7H, 7HPyren), 7.98 (t,
1H, HPyren) 7.59–7.13 (m, 15H, Ph), 5.28 (br s, 1H, PyrenCHH),
4.63 (br s, 1H, PyrenCHH), 4.39 (br s, [2JH–Pt = 79.0 Hz], 1H, NH),
3.43 (m, 1H, NHCHH), 2.60 (m, 1H, NHCHH), 2.22 (m, 1H, NHCH2
CHH), 1.81 (m, 1H, NHCH2CHH), 1.33 (m, 1H, CHHCH3), 1.23 (m,
1H, CHHCH3), 0.82 (t, 3H, CH3); 13C NMR: 134.8 (d, [2JC–P = 10.2
Hz]), 131.7, 131.4, 130.9, 130.7, 129.7, 128.9 (d, [1JC–P = 61 Hz]),
127.9 (d, [2JC–P = 10.7 Hz]), 127.5 (2C), 126.2 (2C), 125.5 (2C),
125.2, 124.9, 124.8, 123.0 (2C), 53.9, 50.4, 31.2, 20.1, 13.9; 31P
NMR: 4.05 (1JP–Pt = 3590 Hz); 195Pt NMR: ꢀ3618 (1JPt–P = 3590 Hz).
2.6. Cell cycle analysis
A2780 cells (2.0 ꢂ 105) were seeded into each cell culture plate
in complete growth medium. After incubation for 24 h the test
agents were added to the complete medium at the indicated con-
centrations and cells were incubated for a further 18 h. After treat-
ment, 300–500 ꢂ 103 cells were fixed in 70% iced-cold ethanol at
ꢀ20 °C for 20 min and then washed twice with phosphate buffered
2.3.5. trans-[PtCl2(PPh3)(p-Tolu)] (3)
4-Methylaniline (p-toluidine); 95%; mp: 251–254 °C (lit.
247–249 °C);10 Anal. Calcd for C25H24Cl2NPPt: C, 47.3; H, 3.8; N,
2.2. Found: C, 47.0; H, 3.7; N, 2.3. IR (ATR): 3212, 3128, 3052,
1559, 1509, 1480, 1433, 1097, 814, 754, 745, 691; 1H NMR:
7.72–7.67 (m, 6H, Harom Phosphine), 7.46–7.38 (m, 9H, Harom Phos-
phine), 7.32 (d, 2H, J = 8.2 Hz, Harom Toluidine), 7.14 (d, 2H,
saline. Cells were incubated with 0.1 mg/mL RNAse and 36 lg/mL
PI at room temperature for 20 min and the analysis was performed
using a FACSCanto II flow cytometer (Becton–Dickinson, Mountain
View, CA).
2.7. Nucleic acids
2
J = 8.2 Hz, Harom Toluidine), 5.31 (br s, 2H, JH–Pt = 46.0 Hz, NH2),
2.35 (s, 3H, CH3); 13C NMR: 136.8, 135.2, 134.8 (d, [2JC–P = 10.5 Hz]),
130.9, 129.9, 128.3 (d, [1JC–P = 64.0 Hz]), 128.0 (d, [2JC–P = 11.3 Hz]),
121.5, 20.9; 31P NMR: 3.63 (1JP–Pt = 3773 Hz); 195Pt NMR: ꢀ3614
(1JPt–P = 3773 Hz).
Salmon testes DNA was purchased from Sigma Chemical Com-
pany. The DNA concentration was determined using extinction
coefficient 6600 Mꢀ1 cmꢀ1 at 260 nm. pBR322 DNA was purchased
from Fermentas Life Sciences.
2.4. Inhibition growth assay
2.8. Quantitative platinum and phosphorus analysis
HeLa (human cervix adenocarcinoma) were grown in Nutrient
Mixture F-12 [HAM] (Sigma Chemical Co.); H460 (large cell lung
carcinoma) and MSTO-211H (human biphasic mesothelioma) were
grown in RPMI 1640 (Sigma Chemical Co.) supplemented with
2.38 g/L Hepes, 0.11 g/L pyruvate sodium and 2.5 g/L glucose;
A2780 (human ovarian carcinoma) and A2780cis (human ovarian
carcinoma cisplatin-resistant) were grown in RPMI 1640 (Sigma
Chemical Co.). 1.5 g/L NaHCO3, 10% heat-inactivated fetal calf
The quantitative platinum and phosphorus analyses were per-
formed according to a previous established method.12 Briefly, an
aqueous solution of salmon testes DNA (9 ꢂ 10ꢀ4 M) was incu-
bated at 37 °C with test compound (stock solution 5 mM in DMSO)
or cisplatin (stock solution 2 mM in 0.9% NaCl) to reach a [DNA]/
[drug] = 10. At fixed incubation time, aliquots of exact volume
were collected and DNA was precipitated with Na-acetate (up to
0.3 M concentration) and cold ethanol (2 vol). The precipitated
DNA was washed with 70% ethanol, dried and then dissolved in a
measured volume of milliQ water. The samples were then miner-
alized by treating with HNO3 (65%) at 90 °C for 20 min. Finally
the samples were re-suspended in diluted HCl to determine the P
and Pt content.
The analyses of P and Pt were performed by inductively coupled
plasma atomic emission spectrometry (ICP-AES) at emission lines k
(P) = 178.290 nm and k(Pt) = 214.423 nm. A Spectroflame Modula
sequential and simultaneous ICP-spectrometer (ICP SPECTRO Arcos
with EndOnPlasma torch) equipped with a capillary cross-flow
Meinhard nebulizer was used (Spectro Analytical). Analytical
determinations were performed using a plasma power of 1.2 kW,
a radiofrequency generator of 27.12 MHz and an argon gas flow
serum (Invitrogen), 100 U/mL penicillin, 100
and 0.25 g/mL amphotericin B (Sigma Chemical Co.) were added
to the media. The cells were cultured at 37 °C in a moist atmo-
lg/mL streptomycin,
l
sphere
of
5%
carbon
dioxide
in
air.
Cells
(2.5–3 ꢂ 104) were seeded into each well of a 24-well cell culture
plate. After incubation for 24 h, various concentrations (from
0.25 to 20 lM) of the test agents were added to the complete med-
ium and incubated for a further 72 h. Stock solutions of new com-
plexes were made in dimethylsulfoxide at 5 mM concentration and
then diluted with complete medium in such a way that the final
amount of solvent in each well did not exceed 0.5%. Cisplatin
was dissolved in 0.9% NaCl. A Trypan blue assay was performed
to determine cell viability. Cytotoxicity data were expressed as
Please cite this article in press as: Dalla Via, L.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.04.067

4
L. Dalla Via et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
with nebulizer, auxiliary, and coolant set at 1, 0.5 and 14 L minꢀ1
respectively.
,
Table 1
Some significant spectroscopic data for complexes 2
31P NMRb
(ppm)
195Pt NMRb,c
(ppm)
1JP–Pt
(Hz)
a
Compound
~
mN—H
(cmꢀ1
)
2.9. DNA topoisomerase relaxation assay
2a
2b
2c
2d
3195
3205
3250
3229
3219
5.25
4.09
4.16
4.05
4.30
ꢀ3669
ꢀ3620
ꢀ3618
ꢀ3618
ꢀ3612
3576
3580
3573
3590
3560
Supercoiled pBR322 plasmid DNA (0.25
30 min at 37 °C in 20 L reaction buffer containing test compound
dissolved in DMSO (0.5 L) at indicated concentrations, then 1 U
topoisomerase II (USB Corporation) or 2 U topoisomerase I (Topo-
gen) were added and reactions performed for further 60 min at
37 °C.
lg) was incubated for
l
l
trans-[PtCl2(PPh3)
(NHEt2)]d
a
b
c
FTIR (ATR).
Solvent = CDCl3.
Doublet.
Reactions were stopped by adding 4
lL stop buffer (5% SDS,
d
0.125% bromophenol blue and 25% glycerol), 50
l
g/mL proteinase
See Ref. 4b.
K (Sigma) and incubating for a further 30 min at 37 °C. The samples
were separated by electrophoresis on 1% agarose gel at room tem-
purification procedures, amines ArCH2NH(CH2)3CH3 1a–d were
obtained in very good yields (Scheme 1). Their spectroscopic char-
acterization (IR, 1H and 13C NMR) is reported in the experimental
part and is in agreement with the expected structures.
perature. The gels were stained with ethidium bromide 1 lg/mL in
TAE buffer, transilluminated by UV light, and fluorescence emis-
sion was visualized by a CCD camera coupled to a Bio-Rad Gel
Doc XR apparatus.
As for the synthesis of platinum complexes, they were prepared
by reacting the described secondary amines with cis-[PtCl2(PPh3)
(NCMe)],4a under experimental conditions allowing the precursor
isomerization and the following substitution of coordinated
nitrile,4b to afford trans-[PtCl2(PPh3){NH(Bu)(CH2Ar)}] (Scheme 2).
The reactions, monitored by 31P NMR spectroscopy to detect the
disappearing of the precursor signal, were completed in 2–3 h
and afforded, after the usual work-up procedures, crystalline
samples of the desired complexes 2a–d in good yields. The same
procedure was followed to prepare trans-[PtCl2(PPh3)(p-Tolu)] (3)
in 95% isolated yield.
2.10. Topoisomerase II-mediated DNA cleavage
Reaction mixtures (20
(pH = 7.9), 50 mM NaCl, 50 mM KCl, 5 mM MgCl2, 0.1 mM EDTA,
15 g/mL bovine serum albumin (BSA), 1 mM ATP, 0.25
lL) containing 10 mM Tris–HCl
l
lg
pBR322 plasmid DNA, 10 U topoisomerase II (human recombinant
topoisomerase II, USB), and test compounds dissolved in DMSO
(0.5
stopped by adding 4
nol blue and 25% glycerol) and 50
l
L) were incubated for 60 min at 37 °C. Reactions were
L of stop buffer (5% SDS, 0.125% bromophe-
g/mL proteinase K (Sigma) and
l
l
New prepared complexes 2 and 3 were insoluble in water and
ethanol, but soluble ([complex] > 5 mM) in dimethylsulfoxide.
The new platinum complexes were characterized by elemental
analysis, IR and NMR (1H, 13C, 31P and 195Pt) spectroscopies. The
most significant IR, as well as 31P and 195Pt NMR data are reported
in Table 1. In the IR spectrum of each complex a narrow, medium-
weak band was observed around 3200 cmꢀ1, attributable to coor-
dinated amine N–H stretching, while both aromatic and saturated
C–H stretching vibrations were observed between 3050 and
2900 cmꢀ1. In 31P NMR spectra, a single signal with satellites was
observed, thus confirming the chemio-4b and stereoselectivity of
the process; this datum was also confirmed by the presence, in
incubating for a further 30 min at 37 °C. The samples were sepa-
rated by electrophoresis on 1% agarose gel containing ethidium
bromide 0.5 lg/mL at room temperature in TBE buffer (0.09 M
Tris–borate and 0.002 M EDTA).
3. Results and discussion
3.1. Chemistry
The preparation of secondary amines ArCH2NH(CH2)3CH3 was
carried out starting from the corresponding aromatic aldehydes
ArCHO and according to a well known procedure13 reported in
Scheme 1.
the 195Pt NMR spectra, of
a single signal with a doublet
multiplicity. Moreover, both the observed chemical shifts and
1JP–Pt coupling constants were in agreement with data previously
collected for analogous complexes4b–d where the trans
stereochemistry had been structurally determined.4b For ease of
comparison, data for trans-[PtCl2(PPh3)(NHEt2)] are included in
Table 1.
Briefly, aromatic aldehydes were reacted with n-butylamine in
refluxing ethanol, affording the corresponding imines, which were
not isolated, but easily identified by IR spectroscopy, where C@O
stretching disappeared and C@N stretching was observed. The fol-
lowing, smooth reduction of the intermediates by sodium borohy-
dride was carried out in situ and, after the usual work-up and
n-Bu
H
n-Bu
O
n-BuNH2
NaBH4
Ar = Ph (1a, 73%)
1-Npa (1b, 98%)
9-Anthb (1c, 88%)
2-Pyrc (1d, 98%)
N
N
Ar
H
EtOH, Δ
Ar
H
Ar
1
Scheme 1. Synthesis of ArCH2NH(CH2)3CH3. Reagents and conditions: (a) Np = Naphthyl; (b) Anth = Anthracenyl; (c) Pyr = Pyrenyl.
Scheme 2. Synthesis of trans-[PtCl2(PPh3){NH(Bu)(CH2Ar)}]. Reagents and conditions: (a) Np = Naphthyl; (b) Anth = Anthracenyl; (c) Pyr = Pyrenyl.
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L. Dalla Via et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
5
Table 2
Chemical shifts of diastereotopic hydrogens in 1H NMR spectra for trans-[PtCl2(PPh3){NH(Bu)CH2Ar}]
Ph3P
Cl
Pt
Ha Hd
Hd'
N
Hb; Hb0
(
D
d, ppm)
Hc; Hc0
(
D
d, ppm)
Hd; Hd0
(D
d, ppm)
He; He0
(Dd, ppm)
Cl
Me
Hb
Hb'
Hc
Hc'
He
Ar
He'
2a (Ar = Ph)
4.50; 3.81 (0.69)
5.05; 4.17 (0.88)
5.42; 5.27 (0.15)
5.28; 4.63 (0.65)
3.25; 2.66 (0.59)
3.29; 2.51 (0.78)
3.46; 2.30 (1.16)
3.43; 2.60 (0.83)
2.24; 1.86 (0.38)
2.20; 1.77 (0.43)
2.16; 1.73 (0.43)
2.22; 1.81 (0.41)
1.38; 1.38 (0)
2b (Ar = 1-Np)
2c (Ar = 9-Anth)
2d (Ar = 2-Pyr)
1.31; 1.22 (0.09)
1.21; 1.08 (0.13)
1.33; 1.23 (0.10)
The coordination of amines to platinum was also evident in 1H
NMR spectra, recorded in CDCl3 solution. In each sample, NH orig-
inated a broad singlet signal around 4.1–4.4 ppm, where a further
Taking into consideration the chemical structures of the novel
2a–c complexes, it is possible to observe an opposite relationship
between cytotoxicity and the dimension of Ar residue in the sec-
ondary amines although the differences in GI50 values are quite
small. Indeed, the benzyl-substituted 2a exerts the highest cell
effect, which decreases of about 1.7 and 2.6 times for the naphthyl
(2b) and the anthracenyl derivative (2c), respectively. Otherwise,
the pyrenyl derivative 2d shows an antiproliferative effect some-
what dependent on cell line.
The well-known drug cisplatin, taken as reference compound, is
more cytotoxic than the new trans-platinum(II) complexes in all
sensitive cell lines, showing GI50 values low micromolar or submi-
cromolar. Moreover, as expected, in A2780cis cells a decrease in
cytotoxicity of about one order of magnitude with respect to the
sensitive A2780 cells, is attained. In this connection, it is interest-
ing to note that unlike cisplatin, 2a–d induce a comparable effect
on both sensitive and resistant A2780cis cells. In particular, 2a
exerts on resistant cells an antiproliferative effect significantly
higher with respect to that induced by the drug, thus suggesting
the involvement of cell pathways different from those of cisplatin.
2
broadening near the baseline allowed to measure a JH–Pt coupling
constant of ꢄ80 Hz. Moreover, due to the presence of the chiral
nitrogen (Table 2), the hydrogen nuclei on the amine moiety are
diastereotopic and, hence, non equivalent. Thus, Hb and Hb0
(Table 2) originate two, well separated doublet signals, with a
2JH–H coupling constant of ꢄ13 Hz, typically observed for coupling
between geminal hydrogen nuclei.14 Two distinct signals of multi-
plet were observed, as expected, also for Hc and Hc0 (Table 2). The
non equivalence of geminal hydrogen atoms was observed along
the whole butyl chain, with Hd,d0 and He,e0 (Table 2) generating
well separated sets of multiplets, despite their growing distance
from the chiral center.
3.2. Antiproliferative activity
The antiproliferative activity of the new complexes 2a–d and 3
was evaluated on a panel of human tumor cell lines: HeLa (human
cervix adenocarcinoma), H460 (large cell lung carcinoma), MSTO-
211H (human biphasic mesothelioma), A2780 (human ovarian
carcinoma) and A2780cis (human ovarian carcinoma cisplatin-
resistant). The cisplatin was taken as reference drug.
The results have been expressed as GI50 values, i.e., the concen-
tration of compound able to induce the death of 50% of the cells
with respect to a control culture, and have been reported in
Table 3.
3.3. Determination of apoptosis
To investigate the mechanism of cytotoxicity induced by 2a on
A2780cis cells, flow cytometry analysis was performed in the pres-
ence of Annexin V-FITC and DNA-specific dye propidium iodide.
The results are shown in Figure 1A as dot plots and the percentage
data are reported as histograms in Figure 1B. By increasing the con-
centration of 2a a notable decrease in viable cells is obtained,
accompanied by a concurrent increase in apoptotic (both early
and late apoptosis) cells. Actually, the percentage of viable cells
is reduced from 88.2% in the absence of complex (control) to
The obtained results indicate for all complexes belonging to the
2 series the ability to exert a significant antiproliferative effect
towards all cell lines taken into consideration. In particular, 2a–d
show in all cases GI50 values lower than 20 lM and interestingly,
2a, characterized by the less bulky Ar residue, appears the most
active. Otherwise, complex 3, which differs from 2a for the absence
of the n-butyl residue and of the methylene spacer at the level of
the secondary amine, shows a low cytotoxicity profile with GI50
values detectable only on A2780 and A2780cis cell lines.
43.2% after incubation for 18 h in the presence of 2a at 25 lM. In
the same experimental conditions, the percentage of apoptotic
cells increases from 9.3% to 47.9%. Otherwise, the percentage of
necrotic cells varies only scarcely, reaching a percentage of 8.9%
at the maximum concentration of 2a taken into account. These
Table 3
Cell growth inhibition values in the presence of examined complexes and cisplatin
Complex
Cell line
a
GI50
(
lM)
HeLa
H460
MSTO-211H
A2780
A2780cis
2a
2b
2c
2d
5.31 0.65
9.35 1.28
16.3 1.4
19.0 1.4
>20
7.33 0.86
13.3 0.7
17.7 1.2
9.03 0.98
>20
8.93 0.27
14.9 1.3
18.5 1.4
17.5 1.2
>20
3.55 0.26
6.41 0.65
7.97 0.45
4.90 0.75
17.7 0.4
3.63 0.21
5.91 1.14
12.6 1.1
6.95 1.45
18.7 1.4
7.23 0.26
3
Cisplatinb
1.60 0.37
0.83 0.11
1.3 0.22
0.80 0.06
a
Values are the mean SD of at least three independent experiments.
Taken as reference drug.
b
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6
L. Dalla Via et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
Control
μ
μ
M
A
25
M
15
A
DNA
100
B
cisplatin
90
80
70
60
50
40
30
20
10
0
[Pt] ppb
) [P] ppb
viable
apoptotic
necrotic
(
tim e (h)
B
DNA
control
15
25
complex 2a (μM)
Figure 1. Flow cytometry analysis of cell death induced by 2a. (A) Dot plots of the
flow cytometry analysis performed on A2780cis cells labeled with Annexin V-FITC
and propidium iodide after 18 h of treatment with different concentrations of 2a.
(B) The histograms show the percentage of viable (Q3), early and late apoptotic (Q4
+ Q2) and necrotic (Q1) cells. Values are the mean SD of four independent
experiments.
2a
tim e (h)
Figure 2. Binding of cisplatin (A) and 2a (B) to salmon testes DNA (9 ꢂ 10ꢀ4 M) as a
results confirm the capacity of 2a to overcome the resistance of
A2780cis, cisplatin-resistant ovarian cancer cells, and highlight
its ability to induce the apoptotic pathway in a dose-dependent
manner pointing to a different mechanism of action or to an addi-
tional molecular target with respect to cisplatin. On the basis of the
above results, we investigated more in depth the biological effects
of 2a and in particular, its ability to interact with some
biomacromolecules.
function of incubation time. [DNA]/[drug] = 10.
3.5. Effect on cell cycle distribution
Most anticancer drugs block the cell cycle in either the S or
G2/M phase and also cisplatin inhibits cancer cell growth by arrest-
ing the cell cycle progression through these phases.17,18
In order to compare the effect of 2a with that of cisplatin, flow
cytometric analysis was performed on A2780 cells, stained with PI
and analyzed for DNA/cell content. The cells were incubated in the
absence (control) or in the presence of different concentrations
(from 1 to 15 lM) of cisplatin or 2a, and the results are shown in
Figure 3A and B, respectively.
In detail, for the reference drug a dose-dependent arrest in S
phase is highlighted (Fig. 3A), as previously reported by some
authors.18 Otherwise, complex 2a induces a notable increase in
subG0 population (Fig. 3B). Interestingly, the occurrence of a hypo-
diploid DNA peak can be retained indicative of apoptosis,19 and it
appears in accordance with the ability of 2a to induce apoptosis
on cells.
3.4. Binding to DNA
It was already widely demonstrated that DNA constitutes the
main target responsible for the antiproliferative activity of
cisplatin.15 Moreover, for some trans-diiodophosphine Pt(II) com-
plexes the interaction with DNA was demonstrated and a correla-
tion of such ability with cytotoxicity was also supposed.16 In this
connection, the capacity of the most active complex 2a to coordi-
nate DNA was investigated by ICP-AES technique, in comparison
with cisplatin (Fig. 2). In detail, salmon testes DNA platination
was determined for both 2a and the drug, at incubation time from
0 to 48 h. Furthermore, in each sample also the total amount of
phosphorus was estimated, as internal standard for DNA content.12
The performed quantitative evaluation indicates for complex 2a
an ability to bind to DNA clearly lower with respect to that
obtained for cisplatin. In particular, after 48 h of incubation in
the presence of the reference drug, about 700 ppb of Pt is bound
to DNA, while for 2a, an amount of about 200 ppb is detected in
the same experimental conditions. These results appear in agree-
ment with the data reported in Table 3, showing for the novel com-
plex, GI50 values higher than those of the drug in all sensitive cells
taken into consideration, i.e., a lower antiproliferative activity.
Then, for both 2a and cisplatin a similar mechanism of action,
involving the platination of DNA, could be assumed. Nevertheless,
due to the ability of 2a to exert a comparable cytotoxic effect on
both A2780 and A2780cis cells, the involvement of further intracel-
lular targets was hypothesized and investigated.
3.6. Effect on topoisomerases
Topoisomerases are nuclear enzymes able to solve topological
problems that arise during replication, transcription and chromo-
some segregation, as a consequence of the double-stranded helix
nature of DNA.20 Moreover, due to their catalytic activity, which
introduces into DNA single (topoisomerase I) or double (topoiso-
merase II) strand breaks, a role of these enzymes in the resolution
of intermediates that are found in recombinational repair, was also
postulated.21 Furthermore, it is to underline that a significant
decrease in the rate of repair of cisplatin-induced DNA interstrand
cross-links was demonstrated in human glioblastoma cells after
exposure to novobiocin, a known topoisomerase II inhibitor.22
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L. Dalla Via et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
7
Figure 3. Cell cycle distribution of A2780 cells cultured in the presence of cisplatin (A) or complex 2a (B) for 18 h at indicated concentrations.
Moreover, an increase in topoisomerase I and II activity was
detected in cisplatin-resistant ovarian cancer and murine leukemia
cell line, respectively23,24 and interestingly, a number of topoiso-
merase inhibitors showed notably cytotoxicity on cisplatin-resis-
supercoiled DNA (lane DNA) is relaxed by the enzyme into a series
of topoisomers (lane topo I), characterized by lower elec-
trophoretic mobility with respect to the supercoiled form. The
complex 2a, tested at 5, 10 and 20 lM concentration, appears
tant cells.25–27 Overall, these results suggest
a
role of
unable to affect the catalytic activity of topoisomerase I also at
the higher concentration taken into consideration, as demon-
strated by the occurrence of the same pattern of topoisomers as
in the control (lane topo I). Similar experiments were also per-
formed, by incubating topoisomerase II (lane topo II) in the pres-
ence of the same concentrations of 2a (Fig. 4B). The obtained
results show that 2a prevents the relaxation of DNA catalyzed by
topoisomerases in the occurrence of cisplatin resistance and, in
this connection, the possible effect of 2a on catalytic activity of
both topoisomerase
respectively).
I and II was evaluated (Fig. 4A and B,
Figure 4A shows the effect of test complex on the relaxation of
supercoiled plasmid DNA mediated by topoisomerase I. The
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8
L. Dalla Via et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
Figure 4. Effect of 2a on relaxation of supercoiled pBR322 DNA by human recombinant topoisomerase I (A) and II (B).
Figure 5. Effect of 2a–d on relaxation of supercoiled pBR322 DNA by human recombinant topoisomerase II.
the enzyme and notably, the inhibition is complete already at
10 M concentration, pointing out this complex as an effective
inhibitor of topoisomerase II.
The structurally related 2b–d were also evaluated, in compar-
ison with 2a, for the capacity to inhibit the relaxation activity of
topoisomerase II and all test complexes showed a significant effect
(Fig. 5).
by the enzyme (lane topo II) and the addition of m-AMSA induces,
as expected, the formation of linear DNA. Also complex 2a appears
able to stabilize the cleavable complex, even if the amount corre-
sponding to the linear DNA appears lower and detectable at higher
concentrations with respect to that induced by the reference drug.
l
4. Conclusions
Some important clinically used antitumor anti-topoisomerase II
agents, interfere with the enzyme activity by stabilizing a covalent
intermediate, the cleavable complex, into a lethal damage for the
cell.28–30 These drugs are called topoisomerase II poisons and their
mechanism of action and role in the anticancer activity has been
widely discussed.31 The occurrence of the cleavable complex can
be highlighted experimentally by the enzyme-dependent forma-
tion of linear from supercoiled DNA. The ability of 2a to inhibit sig-
nificantly the relaxation activity mediated by topoisomerase II
prompted us to investigate if a poisoning effect could be demon-
strated for this complex. Figure 6 shows the results of the cleavable
complex assay performed with topoisomerase II in the presence of
Platinum(II) complexes containing triphenylphosphine and N
(butyl),N(arylmethyl)amino ligands were synthesized and studied.
The evaluation of the cytotoxicity on a panel of human tumor cell
lines showed the ability of the complexes to induce an interesting
antiproliferative activity with GI50 values in most cases in the low
micromolar range. In particular, it is noteworthy the ability of the
new complexes to exert a comparable cytotoxicity on both sensi-
tive and resistant cell lines, with complex 2a as the most effective.
The ability to overcome resistance prompted an investigation on
the mechanism of action that revealed a different effect on cell
cycle and, in particular, an interesting capacity to interfere with
the catalytic activity of topoisomerase II. Based on the role played
by this enzyme on the occurrence of resistance, it could be
assumed that the inhibitory effect exerted by 2a–d could be
responsible for overcoming the resistance.
50 and 100
l
M concentrations of 2a. The m-AMSA, a well known
M con-
topoisomerase II poison,32 was taken as reference at 10
l
centration. The supercoiled pBR322 DNA (lane DNA) was relaxed
Considering the results obtained in this study, it may be con-
cluded that the new trans platinum(II) complexes could represent
a model for the development of novel trans-Pt(II) based drugs able
to exert an antiproliferative effect on cisplatin resistant cells.
Acknowledgements
L. Dalla Via and A.N. García Argáez are grateful to the financial
support provided by Università degli Studi di Padova—Progetti di
Ricerca di Ateneo 2010—‘Attività antitumorale di nuovi composti
di coordinazione nel carcinoma del polmone non a piccole cellule
(NSCLC)’ (CPDA109877). S. Samaritani is grateful to the financial
support provided by Università di Pisa—Progetti di Ricerca di Ate-
neo 2015—‘Sintesi e studio delle proprietà di composti di metalli di
transizione come agenti Antitumorali’ (PRA_2015_0055).
Figure 6. Effect of 2a on the stabilization of the covalent DNA–topoisomerase II
complex.
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L. Dalla Via et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
9
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Belli Dell’Amico, D.; Dalla Via, L.; García-Argáez, A. N.; Labella, L.; Marchetti, F.;
Samaritani, S. Polyhedron 2015, 85, 685.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2016.04.067.
5. Nierzwicki, L.; Wieczor, M.; Censi, V.; Baginski, M.; Calucci, L.; Samaritani, S.;
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ˇ
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Please cite this article in press as: Dalla Via, L.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.04.067