Synthesis and pharmacological evaluation of modified adenosines joined to mono-functional platinum moieties

Article abstract of DOI:10.3390/molecules19079339

The synthesis of four novel platinum complexes, bearing N⁶-(6- Aminohexyl) adenosine or a 1,6-di(adenosin-N⁶-yl)-hexane respectively, as ligands of monofunctional cisplatin or monochloro(ethylendiamine)platinum(II), is reported. The chemistry exploits the high affinity of the charged platinum centres towards the N7 position of the adenosine base system and a primary amine of an alkyl chain installed on the C6 position of the purine. The cytotoxic behaviour of the synthesized complexes has been studied in A549 adenocarcinomic human alveolar basal epithelial and MCF7 human breast adenocarcinomic cancer cell lines, in order to investigate their effects on cell viability and proliferation.

Full text of DOI:10.3390/molecules19079339

Molecules 2014, 19, 9339-9353; doi:10.3390/molecules19079339
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis and Pharmacological Evaluation of Modified
Adenosines Joined to Mono-Functional Platinum Moieties
Stefano D’Errico 1, Giorgia Oliviero 1,*, Nicola Borbone 1, Vincenzo Piccialli 2, Brunella Pinto 1,
Francesca De Falco 1, Maria Chiara Maiuri 1,3, Rosa Carnuccio 1, Valeria Costantino 1,
Fabrizia Nici 1 and Gennaro Piccialli 1
1
Dipartimento di Farmacia, Università degli Studi di Napoli “Federico II”, via D. Montesano, 49,
80131 Napoli, Italy; E-Mails: stefano.derrico@unina.it (S.D.); borbone@unina.it (N.B.);
brunella.pinto87@gmail.com (B.P.); francesca.defalco@unina.it (F.D.F.);
mariachiara.maiuri@unina.it (M.C.M.); rosa.carnuccio@unina.it (R.C.);
valeria.costantino@unina.it (V.C.); fabrizia.nici@unina.it (F.N.); picciall@unina.it (G.P.)
2
Dipartimento di Scienze Chimiche, Università degli Studi di Napoli “Federico II”, via Cintia, 21,
80126 Napoli, Italy; E-Mail: vinpicci@unina.it
3
INSERM U848, IGR, 39 rue C. Desmoulins, 94805 Villejuif, France
* Author to whom correspondence should be addressed; E-Mail: golivier@unina.it;
Tel.: +39-081-679-896.
Received: 26 March 2014; in revised form: 23 June 2014 / Accepted: 25 June 2014 /
Published: 3 July 2014
Abstract: The synthesis of four novel platinum complexes, bearing N6-(6-amino-
hexyl)adenosine or a 1,6-di(adenosin-N6-yl)-hexane respectively, as ligands of mono-
functional cisplatin or monochloro(ethylendiamine)platinum(II), is reported. The chemistry
exploits the high affinity of the charged platinum centres towards the N7 position of the
adenosine base system and a primary amine of an alkyl chain installed on the C6 position
of the purine. The cytotoxic behaviour of the synthesized complexes has been studied in A549
adenocarcinomic human alveolar basal epithelial and MCF7 human breast adenocarcinomic
cancer cell lines, in order to investigate their effects on cell viability and proliferation.
Keywords: platinum complexes; nucleosides; cisplatin; chemotherapy

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1. Introduction
The discovery of the anti-proliferative properties of cisplatin [1] marked the beginning of modern
chemotherapy based on the use of metal complexes capable of blocking the replication of cancer cells
targeting the nuclear DNA [2,3]. In fact, it is widely accepted that cisplatin, once inside the cell, may
form a highly reactive species [4,5] that can react with DNA through the formation of intra-strand
linkages [6,7]. Such linkages alter the secondary structure of DNA, resulting in an inhibition of
transcription and replication, ultimately leading to cell death [4]. However, the poor solubility in
biological fluids [8], the serious side effects [9,10], and, more importantly, the intrinsic and acquired
resistance of many types of tumors [5], have limited its use in the clinic. Carboplatin and oxaliplatin,
second and third generation anti-neoplastic agents, respectively, are able to enhance the quality of life
of patients in terms of dosage and drug administration. However, they can still trigger mechanisms of
resistance (intrinsic and/or acquired) during repeated cycles of oncological treatment [5]. It was then
discovered that the presence of more charged metal centres separated by unbranched alkylamine chains
leads to complexes capable of being uptaken by cells via active import [11] and of overcoming the
intrinsic and/or acquired resistance in some tumors [6,12–15]. Based on the understanding that they
may exert anti-tumoral activities through inter-strand linkages with DNA that cannot be repaired by
enzymes [16], many multinuclear platinum complexes have recently been synthesized, by varying the
diamine backbone chain length, or by introducing modified linkers [8,10–12,17,18]. In addition, new
Pt-based compounds have been obtained by conjugating biologically important substances or drugs to
Pt-containing subunits [19–24] and novel drug-delivery based methodologies have been explored to
vehiculate platinum-based anti-cancer complexes directly against tumors [25,26].
Nucleoside and nucleotide anti-metabolites and their base analogues are able to inhibit specific
pathways of the cancer cell metabolism by blocking the biosynthesis or the function of nucleic acids.
For example, the combination of cisplatin and 5-fluorouracil, a chemotherapeutic agent that inhibits
thymidylate synthase, has been extensively used in clinical practice to treat various types of cancer.
Acyclovir, a guanosine nucleoside analogue containing an open-chain sugar surrogate, has been used
as a ligand for platinum and the corresponding complex exhibits high in vitro activity against various
herpes viruses [27]. Furthermore, the synthesis and the preliminary pharmacological activity of novel
modified adenosines and thymidines, employed as N-donor ligands of platinum(II) dichloride
complexes, have been recently reported [28,29].
Recently, we have reported the solid-phase synthesis and the pharmacological activity of the first
examples of bis-platinated nucleoside complexes in which the mono-functional metal is linked both to
N-7 of the purine nucleus of inosine and to the terminal amino-group of a hexylamine side chain
installed on N-1 (compounds 1–3, Figure 1) [30]. The amino-alkyl chain was introduced on the purine
base system through a chemical strategy recently developed by us [30–36]. These complexes showed
very good water solubility thanks to the charged platinum centres and to the ribose hydroxyl groups.
They were tested against four different human tumor cell lines and, in particular, the complex bearing
two monofunctional cisplatin units was revealed to be more cytotoxic than cisplatin against the MCF7
cancer cell line in a short-term exposure assay [30].

Molecules 2014, 19
Figure 1. Bis-platinated nucleoside complexes synthesized starting from inosine [30].
9341
-
-
HCO3
H2
N
HCO3
Cl
-
-
HCO3
Cl
HCO3
NH3
-
Cl
+
H3N
H3N
HCO3
-
HCO3
NH3
NH3
+
Pt
O
H
2N
Pt
O
NH3
+
Pt
O
H3N
N
+
N
H2
N
Pt Cl
N
N
+
4
Pt
Cl
N
H
2
H2
N
N
N
N
Pt
4
N
4
H2
H2
N
R
N
Cl
N
R
NH3
N
N
R
N
1
2
3
O
HO
R =
HO
OH
Ribose
In the light of the above results, to investigate the importance of nucleoside scaffolds in the
construction of novel platinum complexes we have speculated about the antitumoral activity of
cisplatin-adenosine complexes. As a result, in this paper we report on the synthesis of four dinuclear
platinum complexes 8a,b and 10a,b (Scheme 1) carrying N6-(6-aminohexyl)adenosine (compound 5,
Scheme 1) or a 1,6-di-(adenosin-N6-yl)-hexane (compound 9, Scheme 1) respectively, as ligands of
mono-functional cisplatin or monochloro(ethylene diamine)platinum(II) (compounds 7a and 7b,
respectively, Scheme 1).
Scheme 1. Synthesis of complexes 8 and 10.
-
-
HCO3
Cl
HCO3
L
L
L
L
+
+
Pt
HN
NH2
iv
HN
Cl
N
H2
Pt
Cl
4
4
N
i
N
N
7
6
N
N
1N
5
4
8
2
9
3
N
R
N
R
N
R
N
N
N
7a,b
4
5
8a,b
4
ii
-
-
HCO3
HCO3
L
L
O
Cl
Cl
+
Pt
HO
+
Pt
R =
HN
N
NH
N
HN
N
NH
4
4
L
HO
OH
N
N
N
L
N
iv
N
N
N
N
Ribose
N
R
N
R
N
R
N
R
N
7a,b
9
10a,b
-
-
NO3
H3N
NH3
H3N
NH3
+
iii
iii
a
b
L = L = NH3,
L = L = NH2(CH2)2NH2
Pt
Pt
Cl
Cl Cl
DMF
7a
6a
NO3
NH2
H2N
NH2
H2N
+
Pt
Pt
Cl Cl
Cl
DMF
6b
7b
Reagents and conditions: (i) NH2(CH2)6NH2 (7 equiv.), TEA, EtOH, 3 h, reflux; (ii) 4 (1.5 equiv.),
TEA, EtOH, 5 h, reflux; (iii) AgNO3 (0.9 equiv.), DMF, 16 h, r.t.; (iv) 7a,b, 16 h, r.t..

Molecules 2014, 19
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In the case of complexes 8a,b the monofunctional platinum centres are linked to the N-7 of the
purine base and to the terminal amino group of a hexylamine side chain bonded to N-6, whereas for the
complexes 10a,b they are both linked to N-7.
Platinum complexes, endowed with particular structural strains, have been designed to evaluate
alternative interactions with DNA strands [8,12,17,37,38]. The novel prepared complexes 8a,b and
10a,b are characterized by a rigid planar purine substructure joined to a flexible hexylamine chain. On
this basis, it could be expected that they may alter the binding mode with DNA through, for example,
base-stacking or by the formation of multiple hydrogen bonds. Therefore, we have investigated the
preliminary effects of these compounds on viability and proliferation in two different human cancer
cell lines.
2. Results and Discussion
2.1. Synthesis and Characterization
The synthesis of the novel modified adenosine-based platinum complexes 8a,b and 10a,b is
depicted in Scheme 1. 6-Cloropurine riboside 4 proved to be a good candidate to obtain the key
intermediate 5 from which the construction of complexes 8a,b and 10a,b has been accomplished. As
reported by Schammells et al. [39] seven equivalents of 1,6-diaminohexane have proved sufficient to
convert 4 into 5 in a very good yield (86%), avoiding the formation of dimeric species, which would
complicate the purification procedure. In fact, the recovery of 5 from the reaction mixture was easily
performed thanks to its insolubility in EtOH upon cooling. Compound 9 was prepared by reacting 4
with a slight excess of 5. During the reaction, this substance precipitated from the reaction mixture as a
white solid which was then collected in 93% yield by filtration. This represents a very good
improvement on the reported yield for 9 (12%) [40].
Next, the platinum-containing moieties were installed. In particular, treatment of 5 and 9 with a
seven-fold excess of the suitable platinating complex 7a,b, activated by overnight reaction with
AgNO3 (0.9 equiv.) in DMF, furnished the bisplatinated compounds 8a,b and 10a,b. Purification
of these substances could be accomplished by reverse-phase HPLC only using a gradient of CH3CN
in 0.1 M triethylammonium bicarbonate buffer (TEAB) as the solvent mixture, whereas complex
chromatographic patterns were observed in the absence of the TEAB.
The structures of complexes 8a,b and 10a,b (yields 58%–62%) were supported by 2D-NMR and
positive mode high resolution mass spectrometry (HRMS) data; whereas their purity was ascertained
by CHN analyses.
In Figure 2 (panel A) the representative HRMS spectrum of complex 8a is reported; in particular,
the isotopic pattern of the base peak (expansion, panel B) perfectly fits with that of the calculated one
(expansion, panel C), confirming the presence of two Pt and two Cl atoms and a net 2+ charge.
In the1H-NMR spectra of 8a,b and 10a,b the downfield shift of H-8, compared with the resonance
of the same proton in 5 and 7 (∆δ = 0.6 and 0.5, respectively), confirmed the presence of the
N(7) → Pt bond in all these substances [41,42]. In Table 1 the differences between the 13C-NMR shifts
of the Pt-coordinated and not-coordinated purine carbon atoms (∆δ = δcomplex − δligand, ppm) are listed:
in the case of complexes 8a,b significant coordination shifts were found for the C-5 and C-8 atoms,

Molecules 2014, 19
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whereas for the complexes 10a,b the C-5 atoms underwent the major shifts. Further evidence of
metallation at N-7 come from the increased acidity of the H-8 as indicated by the reduced intensity of
the pertinent signal in the 1H-NMR spectra of 8a,b and 10a,b, when these complexes were dissolved in
D2O and the spectra acquired after several hours [43]. 1H-NMR analyses excluded also the presence of
equilibria involving migrations of the platinum moieties from N-7 to N-1 of the nucleobases; such
migrations would shift the H-2 resonances of complexes 8a,b and 10a,b to higher frequences in
comparison to the same signals of compounds 5 and 9, respectively [44]. The resonances of the
methylene protons belonging to the CH2-NH2-Pt moieties were seen as broad partly overlapped triplets
in the range 2.3–2.7 ppm, in the proton spectra of 8b and 10b, likewise the protons of the methylene
13
group geminal to platinum in 8a resonated as a triplet at δ 2.6. In C-NMR spectra of 8a and 8b the
5 ppm downfield shift of the ω-methylene carbon could be a consequence of the NH2 platination.
Figure 2. HRMS spectrum of complex 8a (panel A). Expansion of the isotopic pattern of
the base peak (panel B) and of the corresponding calculated one (panel C).
AdenosinaN6ammesano_biscisPt #122 RT: 0.72 AV: 1 NL: 3.57E7
T: FTMS + p ESI Full ms [150.00-1300.00]
447.0870
AdenosinaN6ammesano_biscisPt
100
AdenosinacisPt_o447
448.0869
447.0870
95
90
85
80
75
70
65
60
55
50
45
40
35
30
25
20
15
10
5
100
50
0
448.5872
449.0872
449.5874
450.5864
450 452
446.5870
A
B
C
446.0860
445.0852
444 446
448
m/z
448.5872
449.0872
447.0858
446.5859
446.0849
445.5850
100
50
0
446.5870
448.5864
449.0863
449.5864
450.5870
444
446
448
m/z
450
452
449.5874
450.0876
459.1093
381.0662
380.0647
893.1682
891.1647
932.1432
615.1407
333.1891
1086.0939
0
200
400
600
800
1000
1200
m/z
In the UV spectra of the complexes 8a,b and 10a,b the maxima of absorption appeared red-shifted
(∆λ = 10–13 nm) in respect to those of the free ligands, in accordance with literature data [45].
Comparison of the IR spectra of the ligands and complexes did not provide evidence about the bonding
mode of the ligands in the complexes; in fact in the 4000–2700 cm−1 region very little information
could be obtained about the nature of metal-ligand interaction, because of the presence of broad and
intense bands belonging to hydrogen-bonded hydroxyls of the ribose moieties. The 1800–400 cm−1
region, where vibration frequencies of purine and ribose skeletons fall, did not furnish significant
differences between platinated and not-platinated nucleosides. Furthermore, in the region 550–400 cm−1,
where the resonances of Pt-N could be expected [45,46], no distinctive bands were observed.

Molecules 2014, 19
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Table 1. Differences between the 13C-NMR shifts of the Pt-coordinated and not-coordinated
purine carbons (∆δ = δcomplex − δligand, ppm).
13C-NMR (∆δ, ppm)
Entry
C-2
−1.0
−1.0
−1.9
−2.0
C-4
−1.4
−1.3
−2.3
−2.5
C-5
−2.9
−2.8
−4.2
−4.2
C-6
0.9
C-8
2.1
2.1
0.9
1.1
8a [39]
8b [39]
10a
0.9
−0.1
0.1
10b
2.2. Cytotoxicity Studies
The synthesized complexes were subjected to preliminary cell viability and proliferation assays.
In particular, the cytotoxic behaviour was studied in A549 adenocarcinomic human alveolar basal
epithelial and MCF7 human breast adenocarcinomic cancer cell lines by MTT and BrdU assays, to
investigate the potential effects of platinum complexes on cell viability and proliferation, respectively.
The incubation of A549 cells with 8a,b and 10a,b (50, 100 and 200 µM) for 72 h caused a
concentration-dependent reduction of cell survival (Figure 3, panel A) as well as an inhibition of cell
proliferation at the higher concentrations (Figure 3, panel B) compared to untreated cells. Indeed,
comparable results were obtained when the cell number was directly determined by cell counting (data
not shown).
Figure 3. Effect of 8a, 8b, 10a and 10b on cell viability and proliferation. A549 cells were
incubated with cisplatin, 8a, 8b, 10a and 10b (50, 100 and 200 µM) for 72 h. Thereafter,
cell viability (panel A) and proliferation (panel B) were determined, respectively, by MTT
and BrdU assay as described in the Experimental Section (*** p < 0.001, ** p < 0.01,
* p < 0.05 vs. untreated cells).

Molecules 2014, 19
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Figure 3. Cont.
The compounds 8b, 10a and 10b (50, 100 and 200 µM) modified the viability and proliferation of
MCF7 cells at higher concentrations, according to the published evidence that these cells have been
associated with cisplatin resistance [47]. The compound 8a proved to reduce the proliferation of the
MCF7 cell line with a greater ability than the other compounds. Strikingly, compound 8a was able to
inhibit the cell proliferation slightly better than cisplatin, but only at 50 µM concentration (Figure 4).
Figure 4. Effect of 8a, 8b, 10a and 10b on cell viability and proliferation. MCF7 cells
were incubated with cisplatin, 8a, 8b, 10a and 10b (50, 100 and 200 µM) for 72 h.
Thereafter, cell viability (panel A) and proliferation (panel B) were determined, respectively,
by MTT and BrdU assay as described in the Experimental Section (*** p < 0.001,
** p < 0.01, * p < 0.05 vs. untreated cells).

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Figure 4. Cont.
3. Experimental Section
3.1. General Methods
All the reagents and solvents were obtained from commercial sources and used without further
1
13
purification. H- and C-NMR spectra were acquired on Varian Mercury Plus 400 MHz instrument
using D2O or (CD3)2SO as solvents. Chemical shifts were reported in parts per million (δ) relative to
13
the residual solvent signal (1H: HDO 4.80; C: (CD3)(CD2H)SO 40.4) and assigned by 2D-NMR
experiments. UV spectra were recorded on a Jasco V-530 UV spectrophotometer. IR spectra were
recorded on a Jasco FT-IR 430 spectrophotometer. High-resolution MS spectra were recorded on a
Thermo Orbitrap XL mass spectrometer using the electrospray ionization (ESI) technique in positive
mode. Elemental analyses were performed on a Thermo Finnigan Flash EA 1112 CHN analyzer.
RP-HPLC analyses and purifications were carried out on a Jasco UP-2075 Plus pump, equipped with a
Jasco UV-2075 Plus UV detector, using a C-18 reverse-phase column (5 µm, 4.8 × 150 mm), eluted
with a linear gradient of CH3CN in 0.1 M triethylammonium bicarbonate (TEAB) buffer (System A:
from 0% to 50% in 90 min, flow 1.3 mL/min, or System B: from 0% to 100% in 90 min, flow
1.3 mL/min). Human alveolar basal carcinoma epithelial cells (A549) and human breast cancer cells
(MCF7) were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10%
Foetal bovine serum (FBS), 100 U/mL penicillin and 100 U/mL streptomycin at 37 °C under 5% CO2.
The cell lines were all from the ATCC catalogue. All media and supplements for cell culture were
purchased from Gibco-Invitrogen (GE Healthcare).
N6-(6-Aminohexyl)adenosine (5). Compound 5 (326 mg) was obtained by reaction of 4 (297 mg,
01.04 mmol) in accordance to the procedure by Schammells et al. [37]. Spectroscopic data and yields
were in agreement with those reported by the authors.

Molecules 2014, 19
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1,6-Di-(adenosin-N6-yl)-hexane (9). Compound 5 (100 mg, 0.27 mmol), 6-chloropurine riboside (4,
116 mg, 0.40 mmol), Et3N (41 µL, 0.30 mmol) were refluxed in EtOH (1 mL). During the reaction, a
colourless solid precipitated from the reaction. After 5 h it was filtered, washed with boiling EtOH and
dried, giving 9 as a colourless powder (164 mg, 93%). El. An. Calcd. for C26H36N10O8: C, 50.64; H,
1
5.88; N, 22.72. Found: C, 50.68; H, 5.90; N, 22.69. H-NMR and UV data were in agreement with
13
those reported in literature [38]. C-NMR (100 MHz, (CD3)2SO) δ 155.6 (C-2), 153.3 (C-6), 149.1
(C-4), 140.5 (C-8), 120.7 (C-5), 88.9 (C-1'), 86.9 (C-4'), 74.4 (C-2'), 71.6 (C-3'), 62.6 (C-5'), 40.8
(CH2NH, covered by residual solvent signal), 30.0 (CH2), 27.1 (CH2); IR (KBr pellet) 3336, 2935,
2861 1630, 1587, 1537, 1474, 1418, 1369, 1334, 1307, 1221, 1185, 1130, 1097, 1058, 985, 866, 822,
793, 641, 554 cm−1; m/z 639.2620 (HRESIMS) ([M + Na]+, C26H36N10NaO8, requires 639.2615).
3.2. General Procedure for the Preparation of Dinuclear Platinum Complexes 8a,b and 10a,b
In a representative experiment, cisplatin (57 mg, 0.19 mmol) was activated by treatment with
AgNO3 (29.0 mg, 0.17 mmol) in DMF (2 mL) in the dark (16 h, r.t.). AgCl was removed by filtration
and the resulting solution of [Pt(NH3)2(Cl)DMF]+(NO3)− in DMF was added to compound 5 (10 mg,
0.027 mmol). The solution was shaken in the dark (16 h, r.t.) and then filtered over a GHP Acrodisc
13 mm syringe filter (0.45 µm GHP membrane). The filter was washed with the minimal amount
of DMF and the clarified solution was then subjected to HPLC purification [system A for 8a,b (tR:
38.7 min and 38.4 min respectively) and system B for 10a,b (tR: 31.0 min and 31.5 min respectively),
see General Methods]; the fractions containing the title compound were evaporated under reduced
pressure and then lyophilized affording pure 8a.
Compound 8a. Bis-bicarbonate salt (17 mg, 60%). El. An. Calcd. for C18H40Cl2N10O10Pt2: C, 21.24; H,
1
3.96; N, 13.76. Found: C, 21.21; H, 3.98; N, 13.79. H-NMR (400 MHz, D2O) δ 8.84 (s, 1H, H-8),
8.33 (s, 1H, H-2), 6.12 (bs, 1H, H-1'), 4.78–4.76 (m, 1H, H-2', partially covered by solvent signal),
4.43–4.36 (m, 1H, H-3'), 4.31–4.25 (m, 1H, H-4'), 3.97–3.76 (m, 2H, Ha,b-5'), 3.67–3.55 (m, 2H, CH2N),
2.62 (t, J = 6.4 Hz, 1H, CH2NH2Pt), 1.86–1.72 (m, 2H, CH2), 1.71–1.59 (m, 2H, CH2), 1.57–1.34
13
−
(complex signal, 4H, 2 × CH2); C-NMR (100 MHz, D2O) δ 160.2 (2 × HCO3 ), 153.7 (C-2), 153.2
(C-6), 146.8 (C-4), 141.6 (C-8), 116.8 (C-5), 89.1 (C-1'), 85.9 (C-4'), 73.9 (C-2'), 70.1 (C-3'), 61.0
(C-5'), 45.7 (CH2NH2), 40.7 (CH2NH), 29.8 (CH2), 28.0 (CH2), 25.4 (CH2), 25.2 (CH2). IR (KBr
pellet) 3246, 2930, 1626, 1589, 1492, 1339, 1084, 1058, 835, 790, 553 cm−1; UV (H2O) λmax = 277 nm;
HRESI-MS: (m/z) 447.0870, calcd. [M]2+ 447.0859.
Compound 8b. Bis-bicarbonate salt (17 mg, 58%). El. An. Calcd. for C22H44Cl2N10O10Pt2: C, 24.70; H,
1
4.15; N, 13.09. Found: C, 24.73; H, 4.13; N, 13.06. H-NMR (400 MHz, D2O) δ 8.85 (s, 1H, H-8),
8.39 (s, 1H, H-2), 6.16 (d, J = 5.0 Hz, 1H, H-1'), 4.80 (m, 1H, H-2', covered by solvent signal),
4.48–4.41 (m, 1H, H-3'), 4.37–4.30 (m, 1H, H-4'), 4.00–3.84 (m, 2H, Ha,b-5'), 3.78–3.67 (m, 2H,
CH2N), 2.85–2.50 (complex signal, 10H, CH2NH2Pt and 4 × CH2 ethylene diamine moieties)
1.91–1.78 (m, 2H, CH2), 1.77–1.62 (m, 2H, CH2), 1.61–1.36 (complex signal, 4H, 2 × CH2); 13C-NMR
−
(100 MHz, D2O) δ 160.2 (2 × HCO3 ), 153.7 (C-2), 153.2 (C-6), 146.9 (C-4), 141.6 (C-8), 116.9
(C-5), 89.1 (C-1'), 85.9 (C-4'), 73.9 (C-2'), 70.1 (C-3'), 61.0 (C-5'), 48.3, 48.0, 47.1 (4 × CH2 ethylene
diamine moieties), 45.9 (CH2NH2), 40.7 (CH2NH), 29.9 (CH2), 28.1 (CH2), 25.5 (CH2), 25.2 (CH2); IR

Molecules 2014, 19
9348
(KBr pellet) 3406, 3208, 2931, 1627, 1589, 1470, 1336, 1303, 1232, 1079, 1054, 834, 790, 555,
472 cm−1; UV (H2O) λmax = 278 nm; HRESI-MS: (m/z) 473.1037, calcd. [M]2+ 473.1031.
Compound 10a. Bis-bicarbonate salt (12 mg, 60%). El. An. Calcd. for C28H50Cl2N14O14Pt2: C, 26.53;
H, 3.97; N, 15.47. Found: C, 26.50; H, 3.95; N, 15.45. 1H-NMR (400 MHz, D2O) δ 8.85 (s, 2H, 2 × H-8),
8.26 (s, 2H, 2 × H-2), 6.14 (bs, 2H, 2 × H-1'), 4.80 (m, 2H, 2 × H-2', covered by solvent signal),
4.50–4.40 (m, 2H, 2 × H-3'), 4.38–4.29 (m, 2H, 2 × H-4'), 4.03–3.76 (complex signal, 6H, 2 × Ha,b-5'
and 2 × CHaN), 3.67–3.53 (m, 2H, 2 × CHbN), 1.94–1.75 (m, 4H, 2 × CH2), 1.73–1.51 (m, 4H, 2 × CH2);
−
13C-NMR (100 MHz, D2O) δ 160.2 (HCO3 ), 153.7 (C-2), 153.2 (C-6), 146.8 (C-4), 141.4 (C-8), 116.5
(C-5), 89.1 (C-1'), 85.8 (C-4'), 74.0 (C-2'), 70.0 (C-3'), 61.0 (C-5'), 40.5 (CH2NH), 27.8 (CH2), 25.2
(CH2); IR (KBr pellet) 3280, 2933, 1626, 1589, 1492, 1415, 1339, 1306, 1230, 1085, 1059, 985, 864,
790, 552 cm−1; UV (H2O) λmax = 277 nm; HRESI-MS: (m/z) 572.1213, calcd. [M]2+ 572.1226.
Compound 10b. Bis-bicarbonate salt (13 mg, 62%). El. An. Calcd. for C32H54Cl2N14O14Pt2: C, 29.12;
H, 4.12; N, 14.86. Found: C, 29.15; H, 4.10; N, 14.83. 1H-NMR (400 MHz, D2O) δ 8.81 (s, 2H, 2 × H-8),
8.24 (s, 2H, 2 × H-2), 6.14 (bs, 2H, 2 × H-1'), 4.80 (m, 2H, 2 × H-2', covered by solvent signal),
4.49–4.41 (m, 2H, 2 × H-3'), 4.37–4.31 (m, 2H, 2 × H-4'), 4.04–3.83 (m, 4H, 2 × Ha,b-5'), 3.81–3.60
(m, 4H, 2 × CH2N), 2.84–2.50 (complex signal, 8H, 4 × CH2, ethylene diamine moieties), 1.95–1.74
13
−
(m, 4H, 2 × CH2), 1.72–1.52 (m, 4H, 2 × CH2); C-NMR (100 MHz, D2O) δ 160.1 (HCO3 ), 153.6
(C-2), 153.2 (C-6), 146.6 (C-4), 141.6 (C-8), 116.5 (C-5), 89.1 (C-1'), 85.7 (C-4'), 74.1 (C-2'), 70.1
(C-3'), 61.2 (C-5'), 48.1, 47.3 (2 × CH2 ethylene diamine moieties) 40.5 (CH2NH), 27.7 (CH2), 25.1
(CH2); IR (KBr pellet) 3284, 2930, 1625, 1592, 1490, 1417, 1338, 1310, 1233, 1083, 1061, 980, 860,
791, 554 cm−1; UV (H2O) λmax 276 nm; HRESI-MS: (m/z) 598.1391, calcd. [M]2+ 598.1382.
3.3. MTT Viability Assay
The cells were plated in 96 culture wells (20 × 104 cells/well) and allowed to adhere overnight.
Thereafter, the medium was replaced with fresh medium and the cells were incubated in the absence
or presence of cisplatin, 8a, 8b, 10a and 10b (50, 100 and 200 µM). After 72 h, the cell viability
was determined by using 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl-2H-tetrazoliumbromide (MTT)
conversion assay [48]. Briefly, 10 µL of MTT (5 mg/mL) were added to the cells and incubated for
an additional 3 h. After this time point, the cells were lysed and the dark blue crystals solubilized
with 150 µL of a solution containing 50% (v:v) DMF, 20% (w:v) SDS with an adjusted pH of 4.5.
The optical density (OD) of each well was measured with a microplate spectrophotometer (Multiskan
MCCC/340, Titertek, Huntsville, AL, USA) equipped with a 620 nm filter. The experiment was
performed twice in triplicate. The vitality inhibition, induced by each compound at the indicated
concentrations, was expressed as a percentage versus the untreated cells (the control). The viable cells
were also counted by the trypan blue exclusion assay and light microscopy.
3.4. BrdU Cell Proliferation Assay
The cells were plated onto 96-well plates (2 × 104 cells/well) overnight. Then, the medium was
replaced with fresh medium and the cells were incubated in the absence or presence of cisplatin (50,

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9349
100 and 200 µM), 8a, 8b, 10a and 10b (50, 100 and 200 µM). After 72 h, 5-bromo-2'-deoxyuridine
(BrdU; 10 µM) was added and the cells were cultured for a further 12 h. The mitogenic activity was
determined according to the manufacturer’s instructions (BrdU cell proliferation assay kit, Cell
Signaling). The experiment was performed three times in triplicate. The proliferation inhibition,
induced by each compound at the indicated concentrations, was expressed as a percentage versus the
untreated cells (the control).
3.5. Statistical Analysis
The results are expressed as the means ± SEM of n experiments. The statistical significance was
calculated by one-way analysis of variance (ANOVA) and Bonferroni-corrected p-value for multiple
comparison testing. The level of statistically significant difference was defined as p < 0.05.
4. Conclusions
In summary, in this paper we have reported the synthesis of four novel platinum complexes embodying
a N6-(6-aminohexyl)adenosine or a 1,6-di-(adenosin-N6-yl)-hexane as ligands of monofunctional
cisplatin or monochloro(ethylene diamine)platinum(II) moieties, starting from commercially available
6-chloropurine riboside. The designed synthetic route allowed us to obtain the desired platinum
complexes without blocking the ribose hydroxyl groups and through easy purification steps.
The effects of these platinum complexes on cell viability and proliferation have been studied in
A549 and MCF7 cell lines. Our data demonstrate that these compounds are able to inhibit the survival
and proliferation of sensitive cancer cells. Nevertheless, cisplatin remains the most active molecule
under our experimental conditions. Further experiments are necessary to understand the mechanism by
which 8a penetrates into the cells and inhibits cell proliferation in the MCF7 cell line.
Acknowledgments
This work was financially supported by “Progetto FARO 2011” (Finanziamento per l’Avvio di
Ricerche Originali). We are grateful to Luisa Cuorvo for her technical assistance and to CSIAS
(Centro di Servizio Interdipartimentale di Analisi Strutturale) for the NMR facilities.
Author Contributions
Stefano D’Errico, Brunella Pinto and Fabrizia Nici designed and synthesized the compounds shown
in Scheme 1, furthermore Stefano D’Errico wrote the initial draft of the paper and edited the final
draft. Vincenzo Piccialli wrote an intermediate draft of the paper and helped write and edit the final
draft of the manuscript. Nicola Borbone and Valeria Costantino characterized all the novel platinum
complexes, analyzed spectroscopic data from the experiments and wrote the pertinent section in
Results and Discussion and in the Experimental Section. Francesca De Falco, Maria Chiara Maiuri and
Rosa Carnuccio performed the in vitro biological assays, analyzed and processed the data obtained
from the experiments and wrote the pertinent section in the Results and Discussion and in the
Experimental Section. Giorgia Oliviero and Gennaro Piccialli supervised all experiments, reanalyzed

Molecules 2014, 19
9350
all data for accuracy, and wrote the final draft of the manuscript. All authors reviewed and approved
the final version of the manuscript.
Conflicts of Interest
The authors declare no conflict of interest.
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© 2014 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
(http://creativecommons.org/licenses/by/3.0/).