Cytotoxic effect of (1-methyl-1H-imidazol-2-yl)-methanamine and its derivatives in PtII complexes on human carcinoma cell lines: A comparative study with cisplatin

Article abstract of DOI:10.1016/j.bmc.2013.01.063

The synthesis and pharmacological characterisation of (1-methyl-1H- imidazol-2-yl)-methanamine and its derivatives in Pt^II complexes are described. Six out of eleven new Pt^II complexes showed a significant cytotoxic effect on NCI-H460 lung cancer cell line with EC₅₀ values between 1.1 and 0.115 mM, determined by MTT assay. Compound Pt-4a showed a particularly more potent cytotoxic effect than the previously described Pt ^II complex with 2,2′-bipyridine, [Pt(bpy)Cl₂], with an EC₅₀ value equal to 172.7 μM versus 726.5 μM respectively, and similar potency of cisplatin (EC₅₀ = 78.3 μM) in NCI-H460 cell line. The determination of the intracellular and DNA-bound concentrations of ¹⁹⁵Pt, as marker of the presence of the complexes, showed that the cytotoxic compound Pt-4a readily diffused into the cells to a similar extent of cisplatin and directly interacted with the nuclear DNA. Pt-4a induced both p53 and p21^Waf expression in NCI-H460 cells similar to cisplatin. A direct comparison of the cytotoxic effect between compound Pt-4a and cisplatin on 12 different cancer cell lines demonstrated that compound Pt-4a was in general less potent than cisplatin, but it had a comparable cytotoxic effect on non-small-cell lung cancer NCI-H460 cells, and the colorectal cancer cells HCT-15 and HCT-116. Altogether, these results suggested that the Pt^II complex with 1-methyl-1H-imidazol-2-yl)-methanamine (compound Pt-4a), displayed a significant cytotoxic activity in cancer cells. Similarly to cisplatin this compound interacts with nuclear DNA and induces both p53 and p21^waf, and thus it represents an interesting starting point for future optimisation of new Pt^II complexes forming DNA adducts.

Full text of DOI:10.1016/j.bmc.2013.01.063

Bioorganic & Medicinal Chemistry 21 (2013) 2379–2386
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Cytotoxic effect of (1-methyl-1H-imidazol-2-yl)-methanamine
and its derivatives in PtII complexes on human carcinoma cell lines:
A comparative study with cisplatin
Nicola Ferri a, Stefano Cazzaniga a, Luca Mazzarella b, Giuseppe Curigliano b, Giorgio Lucchini c,
Daniele Zerla d, Raffaella Gandolfi d, Giorgio Facchetti d, Michela Pellizzoni d, Isabella Rimoldi d,
⇑
a Dipartimento di Scienze Farmacologiche e Biomolecolari, Università degli Studi di Milano, Via Balzaretti 9, 20133 Milano, Italy
b Division of Medical Oncology, Department of Medicine, Istituto Europeo di Oncologia, Milano, Italy
c Dipartimento di Scienze Agrarie e Ambientali-Produzione, Territorio, Agroenergia, Università degli Studi di Milano,Via Celoria 2, 20133 Milano, Italy
d Dipartimento di Scienze Farmaceutiche, Università degli Studi di Milano, Via Venezia 21, 20133 Milano, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis and pharmacological characterisation of (1-methyl-1H-imidazol-2-yl)-methanamine and
its derivatives in PtII complexes are described. Six out of eleven new PtII complexes showed a significant
cytotoxic effect on NCI-H460 lung cancer cell line with EC50 values between 1.1 and 0.115 mM, deter-
mined by MTT assay. Compound Pt-4a showed a particularly more potent cytotoxic effect than the pre-
Received 19 October 2012
Revised 18 January 2013
Accepted 24 January 2013
Available online 5 February 2013
viously described PtII complex with 2,20-bipyridine, [Pt(bpy)Cl2], with an EC50 value equal to 172.7
lM
versus 726.5 lM respectively, and similar potency of cisplatin (EC50 = 78.3 lM) in NCI-H460 cell line.
Keywords:
Cytotoxicity
Cisplatin
Cancer cell line
Platinum(II) complex
Imidazole
The determination of the intracellular and DNA-bound concentrations of 195Pt, as marker of the presence
of the complexes, showed that the cytotoxic compound Pt-4a readily diffused into the cells to a similar
extent of cisplatin and directly interacted with the nuclear DNA. Pt-4a induced both p53 and p21Waf
expression in NCI-H460 cells similar to cisplatin. A direct comparison of the cytotoxic effect between
compound Pt-4a and cisplatin on 12 different cancer cell lines demonstrated that compound Pt-4a
was in general less potent than cisplatin, but it had a comparable cytotoxic effect on non-small-cell lung
cancer NCI-H460 cells, and the colorectal cancer cells HCT-15 and HCT-116. Altogether, these results sug-
gested that the PtII complex with 1-methyl-1H-imidazol-2-yl)-methanamine (compound Pt-4a), dis-
played a significant cytotoxic activity in cancer cells. Similarly to cisplatin this compound interacts
with nuclear DNA and induces both p53 and p21waf, and thus it represents an interesting starting point
for future optimisation of new PtII complexes forming DNA adducts.
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
and programmed cell death/apoptosis. Tumor suppressor genes
also influence cisplatin-induced apoptosis.
The great impact of cisplatin, discovered in 1969 by Rosenberg
et al., for the treatment of cancer opened the era of platinum-based
anticancer drugs.1,2 Cisplatin, carboplatin and oxaliplatin are
highly polar molecules that do not readily diffuse across lipid
membranes. Although a variety of pumps and transporters can
influence the cellular uptake of the platinum-containing drugs;
the copper transporter 1 (CTR1) plays a major role.3 Once inside
the cells, cisplatin becomes activated by the acquation of one of
the two chloride leaving groups, it covalently binds to DNA, form-
ing DNA adducts and realising its cytotoxic effect.4,5
Protein p53 is considered a ‘guardian of the genome’ and facil-
itates DNA repair before DNA replication. Cisplatin DNA damage
leads to expression of p53 protein that subsequently induces both
expression of downstream p21waf protein and G1 phase cell cycle
arrest.6 Cisplatin and its derivatives are still used for more than
50% of cancer diseases and a request for an alternative is highly
needed especially when their toxicity and inefficiency against plat-
inum-resistant tumors are considered.7–10 The resistance to cis-
platin and carboplatin could be mediated through three broad
mechanisms: first, a failure of a sufficient amount of platinum to
reach the target DNA; second, a failure to achieve cell death after
platinum-DNA adduct formation; and third the cellular efflux of
PtII complexes through the ATP7A and ATP7B channels.11
This effect triggers a variety of signal-transduction pathways in-
volved in DNA-damage recognition and repair, cell-cycle arrest,
Chemical modifications of platinum containing drugs that could
lead to better cell-membrane permeability and facilitate the acqua-
tion of the chloride leaving group, may generate a new class of
⇑
Corresponding author. Tel./fax: +39 0250314609.
E-mail address: isabella.rimoldi@unimi.it (I. Rimoldi).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.01.063

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N. Ferri et al. / Bioorg. Med. Chem. 21 (2013) 2379–2386
chemotherapic agents suitable also as antiproliferative agents.
Regarding these aspects organometallic compounds based on com-
plexes of different transition metals have been proven to be a valid
alternative,12–14 even if platinum still furnishes the best results.15
In order to improve the selectivity, one possible strategy could
be the introduction of changes to the ligands’ properties.16–23 In
the last decade bipyridines, histamines and pyrrolidines have been
used as bidentate ligands in PtII complexes and their antineoplastic
activity has been known for years.24–27
to facilitate the departure of a second ligand trans to the first (chlo-
ride) and its replacement or substitution by an external ligand
(water).28 By increasing this effect the intracellular concentration
of the activated aqua ion might be raised, making the drug much
more available for binding to DNA.
The aim being to study the influence of substitution, initially the
change only occurred on NH2 moiety maintaining fixed N-methyl-
1H-imidazole scaffold. The synthesis of ligands and their corre-
sponding PtII complexes are recorded in Scheme 1.
Based on these premises, in the present study the PtII complexes
with bidentate diamines derived from imidazol moiety are de-
scribed. The choice of different substituents, as proposed easily
functionalised methylaminoimidazolic ligands, is carried out with
the aim to modulate the properties of the considered complexes
affecting intracellular distribution.
At first, N-Methyl-1H-imidazole-2-carbaldehyde 2 was synthes-
ised,29 then a condensation with selected amines occurred obtain-
ing the corresponding Schiff bases 3a–l.30 For imines 3a–f a
reduction was realised by hydrogenation with Pd/C. The diamines
4a–f thus obtained were purified by recrystallisation; finally the
PtII complexes Pt-4a/Pt-4f and Pt-3g/Pt-3i were formed by react-
31
ing with PtCl4K2 (Scheme 1).
The cytotoxic effect of all the PtII complexes was then studied in
cultured non-small lung carcinoma (NSLC) NCI-H460 cell line in a
concentration range from 0.25 to 1 mM. After 48 h incubation with
tested compounds, the MTT assay was performed and the cell via-
bility was compared to the control condition. A significant cyto-
toxic effect was observed for all the compounds tested at 1 mM
concentration, except for compound Pt-3g and Pt-4f. Being the
more efficient compound Pt-4a, the free ligand 4a was tested
and did not demonstrate a significant cytotoxic effect.
These results indicate that these platinum complexes are cyto-
toxic and that the coordination with PtII is required for such effect.
Compounds Pt-4b, Pt-3a, Pt-3i and Pt-4a showed a significant ef-
fect starting from 0.25 mM concentration (Fig. 1). Beyond cisplatin,
a second reference compound utilised was the PtII complexes of the
2,20-bipyridine, [Pt(bpy)Cl2] with known cytotoxic effect.24 Under
our experimental conditions, this compound showed a lower cyto-
toxic potency compared to compounds Pt-4b, Pt-3a, Pt-3i, and Pt-
4a (Fig. 1).
2. Results and discussion
Imidazole belongs to the p-electronrich heteroaromatic family,
with 6 electrons arranged on 5 atoms, even if they are mainly cen-
tred on N-atoms. Various substitute imidazoles form complexes
with many metal ions in which the donation is realised by the
pyridinic N-atom. The presence in variously substituted 2-meth-
ylaminoimidazoles of another aminic group, that can coordinate
to the metal centre, leads to the formation of bidentate ligands.
The starting point of the present report was the preparation of
these types of ligands derived from variously substituted 2-meth-
ylaminoimidazoles, because of imidazolic moiety’s chemical fea-
tures: functionalised easily with hydrophobic or hydrophilic
groups of various nature and length influencing the complex prop-
erties. The second important target was the modification of trans
effect that arises from the trans-activation mechanism in the cell,
where the active complex is the corresponding aqua species (acti-
vated form of the complex of PtII).
In order to study a possible correlation between the cytotoxic
For a metal complex, the so-called ‘trans effect’ is intended as
the ability of a ligand coordinated to a metal (imidazole ligand),
effect and the cellular uptake of the new PtII complexes, we
Scheme 1. Synthesis of ligands and their corresponding PtII complexes.

N. Ferri et al. / Bioorg. Med. Chem. 21 (2013) 2379–2386
2381
Figure 1. Cytotoxic effects of PtII complexes on NCI-H460 cell line. Cells were seeded (35,000/well of 48 well tray) and incubated with DMEM supplemented with 10% FCS;
48 h later the medium was changed with one containing 0.4% FCS to stop cell growth and the cultures were incubated for 48 h. At this time the medium was replaced with
one containing 10% FCS and the reported concentrations of compounds and cisplatin and the incubation was continued for a further 48 h at 37 °C. At the end of this incubation
period the cell viability was determined by MTT assay. Each bar represents the mean SD of three independent experiments. Inhibitors versus control: ⁄P <0.05; ⁄⁄P <0.001;
⁄⁄⁄P <0.0001.
determined the 195Pt concentrations from total cell homogenates.
This analysis was conducted after 3 h incubation of NCI-H460 cell
lines with 1 mM concentration of compounds using ICP-MS spec-
troscopy (Table 1).
The low concentrations of 195Pt observed after incubation with
compounds Pt-4d (0.11 M), Pt-4e (0.45 M) and Pt-4f (0.42 M)
l
l
l
may explain their lower cytotoxic effect on NCI-H460 cell line
(Fig. 1). Indeed, a significant linear correlation between the intra-
cellular levels of 195Pt and EC50 values was found (R2 = 0.648)
(Fig. 2). From this analysis it is interesting to note that Pt-4a is
the complex that more closely mimics the cisplatin’s properties
(Fig. 2), recently showing a very potent cytotoxic activity without
Cells were seeded (250,000/35 mm petri dish) and incubated
with RPMI 1640 supplemented with 10% FCS; 24 h later the med-
ium was changed with one containing 0.4% FCS to stop cell growth
and the cultures were incubated for 48 h. At this point, the medium
was replaced with one containing 10% FCS and 1 mM of com-
pounds or cisplatin and the incubation continued for 3 h at 37 °C.
At the end of this incubation period the total cell lysates were pre-
pared and 195Pt concentrations determined by ICP-MS. The cyto-
toxic effect was determined under the same experimental
conditions described in Fig. 1 and the EC50 values calculated by
nonlinear regression curve.
a particularly high intracellular accumulation (1.55
Finally, compounds Pt-(S)4c and Pt-(R)4c, although accumu-
lated efficiently into the cells (4.11 and 2.20 M, respectively),
lM).
l
were less cytotoxic than other compounds, suggesting an impaired
formation of active species (aquation) and thus interaction with
DNA. Considering the above, compound Pt-4a was chosen as the
leading compound for further investigations.
To determine whether compound Pt-4a is capable as efficiently
diffusing into the cells and reaching the nuclear DNA, the presence
of 195Pt was measured from DNA extracts of NCI-H460 cell line
incubated with increasing concentrations of compound Pt-4a or
cisplatin for 3 h (Fig. 3).
The concentrations of 195Pt from total cell lysates after incuba-
tion with the two reference drugs, cisplatin and compound
[Pt(bpy)Cl2], were 1.55 and 3.80 lM, respectively. Considering
the compounds that inhibited the cell viability by more than
50%, similar 195Pt levels were observed for compounds Pt-3i
(2.98
l
M) and Pt-4a (1.98
l
M). Compound Pt-3a showed a lower
Cisplatin and compound Pt-4a appears to have a similar capac-
ity to accumulate into NCI-H460 cell line and form DNA adducts.
The accumulation of platinum complex was linearly dependent
on the concentration of the compound in the cultured media, sug-
gesting a diffusion process across the membrane as a major route
of cellular uptake. In this regard it is worth mentioning that the
cellular uptake of cisplatin is partially regulated by the copper
transporter CTR1,33 while the effect of this cell membrane channel
on compound Pt-4a is still unknown. In agreement with previous
evidence, the amount of platinum observed in the DNA extract
intracellular concentration (0.7
l
M) possibly due to the presence
of oxyme moiety32 that probably decreases the diffusion across
the cellular membranes. Thus, the lower cytotoxic effect of Pt-3a
compared to Pt-4a may be due to an unfavourable pharmacoki-
netic property, since ligand 3a contains an oxymic group hydroly-
sed to corresponding 4a that is the real molecule interacting with
DNA. For complex Pt-3i, in which the ligand is a long alkylic chain
substituted oxyme, the intracellular concentration of platinum is
very high thanks to the presence of a hydrophobic group.
Table 1
Intracellular concentration of 195Pt and cytotoxic potency of newly synthesised
complexes in NCI-H460 cell line
Compound
[Pt]
lM
EC50
78.3
(lM)
Cisplatin
[Pt(bpy)Cl2]
Pt-4d
Pt-4e
Pt-4f
Pt-(S)4c
Pt-(R)4c
Pt-4b
Pt-3g
Pt-3h
1.55
3.80
0.11
0.45
0.42
4.11
2.20
3.13
0.70
0.13
0.70
2.98
1.98
736.5
1110.1
540.3
N/A
N/A
N/A
250.3
N/A
N/A
437.4
115.2
172.7
Pt-3a
Pt-3i
Pt-4a
Figure 2. Correlation between 195Pt concentrations and EC50 values of cytotoxic
effect.
N/A: Not applicable.

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N. Ferri et al. / Bioorg. Med. Chem. 21 (2013) 2379–2386
Table 2
Cytotoxic effect of compound Pt-4a and cisplatin on different tumor cell lines. Cells
were seeded (35,000/well of 48 well tray) and incubated with appropriate medium
supplemented with 10% FCS; 24 h later the medium was changed with one containing
0.4% FCS to stop cell growth and the cultures were incubated for 48 h. At this time, the
medium was replaced with one containing 10% FCS and compounds or cisplatin (from
0.01 mM to 0.5 mM concentrations) and the incubation was continued for a further
48 h at 37 °C. At the end of this incubation period the cell viability was determined by
MTT assay. The EC50 values were then calculated by nonlinear regression curve.
Cell line
EC50 cisplatin (
l
M) EC50 Pt-4a (
l
M) EC50 cisp/EC50 Pt-4a
H460
78.3 1.8
88.5 36.1
31.5 4.9
107.8 27.2
197.0 161.2
238.0 62.2
170.5 27.6
107.5 2.8
203.0 15.6
NE
172.7 20.5
145.5 9.2
265.5 2.1
NE
NE
NE
0.45
0.61
0.12
—
—
—
0.85
—
—
—
—
HCT-15
A2780
IGROV-1
HEY
A549
HCT-116
A431
KYSE-150
MCF-7
MDA-MB-
231
Figure 3. DNA-bound concentration of 195Pt after incubation of NCI-H460 cell line
with compound Pt-4a and cisplatin. Cells were seeded (250,000/35 mm petri dish)
and incubated with DMEM supplemented with 10% FCS; 24 h later the medium was
changed with one containing 0.4% FCS to stop cell growth and the cultures were
incubated for 48 h. At this time, the medium was replaced with one containing 10%
FCS and compound Pt-4a and cisplatin (0.25 and 1 mM) and the incubation was
continued for a further 3 h at 37 °C. At the end of this incubation period the nuclear
DNA was extracted and 195Pt concentration determined by ICP-MS.
201.3 37.8
NE
NE
NE
NE
NE
DLD-1
NE
NE
—
NE: Not effective.
was approximately 3–4% of that measured in the cells from a total
cell homogenate (0.06
lM vs 2 lM after incubation with 1 mM of
compound Pt-4a or cisplatin).6,34
The cytotoxic effect of compound Pt-4a was statistically signif-
icant in four cell lines tested, the non-small lung carcinoma NCI-
H460, the epithelial ovarian cancer A2780, the colorectal cancer
Thus, compound Pt-4a showed a significant cytotoxic effect be-
cause it is able to reach the nuclear DNA, the intracellular target to
cisplatin. To investigate whether this compound was capable of
activating the same cellular responses as cisplatin, the intracellular
levels of p53 and p21waf were determined by Western blot analysis
from total protein extracts.
As shown in Fig. 4, 24 h incubation of NCI-H460 cell line with
0.1 mM cisplatin determined a significant induction of both p53
and p21waf compared to control cells. Compound Pt-4a had a sim-
ilar effect to cisplatin on p21waf at 0.25 and 0.5 mM, while a less
pronounced effect was observed on p53 levels. These data showed
that compound Pt-4a had a cytotoxic effect on tumor cell line NCI-
H460 with a similar mechanism of action to cisplatin, although a
more detailed investigation will be needed to determine the for-
mation of the Pt-4a-DNA adducts.
cells HCT-15 and HCT-116, with EC50 values between 145.5
lM
and 265.5 M (Table 2). In all these cell lines cisplatin was more
l
potent than compound Pt-4a, although comparable EC50 values
were observed for the NCI-H460. HCT-116 and HCT-15, with EC50
ratio of cisplatin and compound Pt-4a equal to 0.45, 0.85 and
0.61, respectively. Thus compound Pt-4a was not active on five
cancer cell lines that were effect by cisplatin: the epithelial ovarian
cancer IGROV-1, the ovarian cancer HEY, the lung adenocarcinoma
A549, the cutaneous squamous carcinoma A431 and the esopha-
geal squamous cell carcinoma KYSE-150. The breast cancer cell
lines MCF-7, MDA-MB-231 and colorectal cancer DLD-1 were
shown to be resistant to both chemical entities.
Finally, the cytotoxicity of compound Pt-4a was compared to
that of cisplatin in 12 different cancer cell lines with a range of con-
centration between 0.01 and 0.5 mM.
3. Conclusions
This study describes PtII complexes, where the ligands are var-
iously substituted bidentate diamines derived from imidazol moi-
ety and revealed that same complexes showed a significant
cytotoxic effect on the non-small-cell lung cancer NCI-H460 cell
line. The PtII complex with (1-methyl-1H-imidazol-2-yl)-methan-
amine Pt-4a showed the most potent cytotoxic effect with
comparable activity to cisplatin on three cancer cell lines: the non-
small-cell lung cancer cells NCI-H460 and the colorectal cancer
cells HCT-15 and HCT-116. This compound was shown to interact
with the nuclear DNA and to activate similar intracellular response
to cisplatin inducing the expression of p53 and p21waf. In order to
improve both pharmacokinetic and pharmacodynamic properties
of the lead compound Pt-4a additional substitutions on N1 of the
imidazole ring will be performed.
Figure 4. Effect of compound Pt-4a on p53 and p21Waf in NCI-H460 cell line. Cells
were seeded (250,000/35 mm petri dish) and incubated with DMEM supplemented
with 10% FCS; 24 h later the medium was changed with one containing 0.4% FCS to
stop cell growth and the cultures were incubated for 48 h. At this time, the medium
was replaced with one containing 10% FCS and the reported concentrations of
compound Pt-4a, and cisplatin at 0.1 mM, and the incubation was continued for a
further 24 h at 37 °C. At the end of this incubation period the total cell lysates were
prepared and protein expression evaluated by Western blotting analysis with a
specific antibody anti p53 or p21waf. The same membrane was then probed with
4. Experimental section
4.1. Chemistry
4.1.1. General
All manipulations involving air sensitive materials were carried
out in an inert atmosphere glove box or using standard Schlenk
line techniques under an atmosphere of nitrogen or argon in
oven-dried glassware. All solvents were used anhydrous. 1H
anti-
a-tubulin antibody (Sigma–Aldrich) as a loading control. Cis. stands for
cisplatin and Cnt for control.

N. Ferri et al. / Bioorg. Med. Chem. 21 (2013) 2379–2386
2383
NMR, 13C NMR and 195Pt NMR spectra were recorded on a Bruker
DRX Avance 300 MHz equipped with a non-reverse probe and also
on a Bruker DRX Avance 400 MHz. 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 infu-
sion of a sample solution in MeOH under ionisation, ESI positive.
FT-IR spectra were collected by using a Perkin–Elmer (MA, USA)
FT-IR Spectrometer ‘Spectrum One’ in a spectral region between
4000 and 450 cmꢀ1 and analysed by transmittance technique with
32 scansions and 4 cmꢀ1 resolution. Elemental analyses were per-
formed using a Perkin–Elmer SeriesII/CHNS/O 2400 analyzer. ICP-
MS data were recorded with BRUKER aurora M90 ICP-MS (MA,
USA)
4.1.2.2.4. 4-((1-Methyl-1H-imidazol-2-yl) methyleneamino)phenol
(3d). 1H NMR (300 MHz, CDCl3) dH 8.47 (1H, s, N@CH), 7.21–7.26
(2H, s, aromatic), 7.11 (1H, d, imz), 7.07 (2H, m, aromatic), 6.89
(1H, d, imz), 4.13 (3H, s, CH3). 13C NMR (300 MHz, CDCl3) dc
154.3 (aromatic), 147.5 (CH@N), 145.3 (aromatic), 141.6 (imz),
131.6 (imz), 122.8 (aromatic), 120.0 (imz), 116.3 (aromatic), 35.3
(CH3). Elemental analysis for C11H11N3O: calcd C, 65.66; H, 5.51;
N, 20.88; found C, 65.43; H, 5.60; N, 20.12. MS (ESI) m/z 202.1
[M+H]+, 224.3 [M+Na]+. IR
m
cmꢀ1 3431w, 3013w, 2671w, 2613w,
1622w, 1581w, 1458w, 1440w, 1271w, 1246w, 1143w, 837w,
756w. Yield = 0.359 g (40%).
4.1.2.2.5. 4-Methoxy-N-((1-methyl-1H-imidazol-2-yl)methylene)
benzenamine (3e). 1H NMR (300 MHz, CDCl3) dH 8.50 (1H, s, N@CH),
7.20–7.23 (2H, m, aromatic), 7.14 (1H, s, imz), 6.98 (1H, s, imz), 6.89–
6.92 (2H, m, aromatic), 4.10 (3H, s, OCH3), 3.80 (3H, s, CH3). 13C NMR
(300 MHz, CDCl3) dc 154.9 (aromatic), 147.0 (CH@N), 146.3 (aro-
matic), 141.6 (imz), 133.6 (imz), 120.8 (aromatic), 119.5 (imz),
117.3 (aromatic), 61.4 (OCH3), 34.3 (CH3). Elemental analysis for
C11H13N3O: calcd C, 65.01; H, 6.45; N, 20.68; found C, 64.92; H,
4.1.2. Preparation of ligands
4.1.2.1. N-Methyl-1H-imidazole-2-carbaldehyde (2). The synthesis
of this compound was realised according with literature procedure.29
Yield = 0.400 g (73%). 1H NMR (300 MHz, CDCl3) dH 9.80 (1H, s,
HC@O), 7.25 (1H, s, H5 imz), 7.09 (1H, s, H4, imz), 4.00 (3H, s, CH3).
13C NMR (300 MHz, CDCl3) dc 182.5 (C@O), 144.0 (imz), 131.6
(imz), 129.1 (imz), 35.1 (imz). Elemental analysis for C5H6N2O: calcd
C, 54.54; H, 5.49; N, 25.44; found C, 55.08; H, 5.97; N, 24.23.
6.02; N, 20.54. MS (ESI) m/z 194.1 [M+H]+, 216.2 [M+Na]+. IR
m
cmꢀ1 3026w, 2711w, 2608w, 1632w, 1586w, 1464w, 1440w,
1270w, 1248w, 1138w, 827w, 766w. Yield = 0.468 g (32%).
4.1.2.2.6.
N-(4-((1-Methyl-1H-imidazol-2-yl)methyleneamino)
phenyl)acetamide (3f). 1H NMR (300 MHz, CDCl3) dH 8.49 (1H, s,
N@CH), 7.63 (1H, s, imz), 7.52–7.55 (2H, m, aromatic), 7.17–7.26
(2H, m, aromatic), 7.02 (1H, s, imz), 4.13 (3H, s, OCH3), 2.19 (3H,
s, CH3). 13C NMR (300 MHz, CDCl3) dc 169.3 (C@O), 147.0 (CH@N),
141.6 (imz), 138.3 (aromatic), 133.6 (imz), 126.5 (aromatic), 118.9
(imz), 116.3 (aromatic), 36.3 (CH3), 25.7 (CH3). Elemental analysis
for C13H16N4O: calcd C, 63.91; H, 6.60; N, 22.93; found C, 64.46; H,
4.1.2.2. (1-Methyl-1H-imidazol-2-yl)methanimines (3). To a solu-
tion of N-Methyl-1H-imidazole-2-carbaldehyde (2) (0.400 g,
3.47 mmol) in anhydrous CH3OH (40 mL), K2CO3 (0.483 g, 3.5 mmol)
was added at 0 °C. A solution of amine (3.5 mmol) was added drop-
wise. The temperature was maintained at 0 °C for 20 min, then
wormed to room temperature and stirred overnight. The mixture
was filtered and the product was obtained as yellow oil.
6.14; N, 22.48. MS (ESI) m/z 245.1 [M+H]+, 267.2 [M+Na]+. IR
m
4.1.2.2.1. 1-Methyl-1H-imidazole-2-carbaldehyde O-benzyloxime
(3a). 1H NMR (300 MHz, CDCl3) dH 8.19 (1H, s, N@CH), 7.26–7.42
(5H, m, aromatic), 7.07 (1H, s, imz), 6.88 (1H, s, imz), 5.23 (2H, d,
CH2), 3.79 (3H, s, CH3). 13C NMR (300 MHz, CDCl3) dc 142.5 (N@CH),
139.7 (imz), 138.0 (aromatic), 129.7 (aromatic), 129.1 (imz), 129.0
(imz), 128.5–128.8 (aromatic), 76.4 (CH2), 35.3 (CH3). Elemental
analysis for C12H13N3O: calcd C, 66.96; H, 6.09; N, 19.52; found
C, 68.80; H, 5.56; N, 18.98. MS (ESI) m/z 216.1 [M+H]+, 238.2
cmꢀ1 3427w, 3105w, 1680w, 1626w, 1541w, 1437w, 1309w,
1265w, 1107w, 858w, 790w. Yield = 0.500 g (55%).
4.1.2.2.7. 1-Methyl-1H-imidazole-2-carbaldehydeoxime (3g). 1H
NMR (300 MHz, CDCl3) dH 8.27 (1H, s, N@CH), 7.11 (1H, s, imz),
6.92 (1H, s, imz), 3.88 (3H, s, CH3). 13C NMR (300 MHz, CDCl3)
dc 144.5 (N@CH), 139.7 (imz), 131.1 (imz), 119.4 (imz), 35.3
(CH3). Elemental analysis for C5H7N3O: calcd C, 47.99; H, 5.64;
N, 33.58; found C, 47.78; H, 5.56; N, 33.08. MS (ESI) m/z 148.2
[M+Na]+. IR
m
cmꢀ1 3248w, 2875w, 1617w, 1519w, 1443w,
[M+Na]+. IR cmꢀ1 3429w, 3106w, 2939w, 1677w, 1510w,
m
1365w, 1288w, 1015w, 943w, 813w, 748w, 698w. Yield = 0.715 g
(95%).
1475w, 1445w, 1270w, 1131w, 1036w, 913w, 763w, 720w.
Yield = 1.716 g (74%).
4.1.2.2.2. N-((1-Methyl-1H-imidazol-2-yl) methylene)(phenyl)meth-
anamine (3b). 1H NMR (300 MHz, CDCl3) dH 8.42 (1H, s, N@CH),
7.32–7.34 (5H, m, aromatic), 7.11 (1H, s, imz), 6.92 (1H, s, imz),
4.78 (2H, d, CH2), 4.00 (3H, s, CH3). 13C NMR (300 MHz, CDCl3) dc
145.9 (imz), 145.5 (CH@N), 139.6 (aromatic), 132.4 (imz), 126.1–
129.0 (aromatic), 120.0 (imz), 64.3 (CH2), 34.5 (CH3). Elemental
analysis for C12H13N3: calcd C, 72.33; H, 6.58; N, 21.09; found C,
71.58; H, 6.12; N, 20.78. MS (ESI) m/z 200.1 [M+H]+, 223.3
4.1.2.2.8. 1-Methyl-1H-imidazole-2-carbaldehyde O-methyloxime
(3h). 1H NMR (300 MHz, CDCl3) dH 8.12 (1H, s, N@CH), 7.00 (1H,
s, imz), 6.90 (1H, s, imz), 3.95 (3H, s, OCH3), 3.86 (3H, s, CH3). 13C
NMR (300 MHz, CDCl3) dc 143.7 (N@CH), 141.1 (imz), 131.4
(imz), 119.8 (imz), 56.8 (OCH3), 33.3 (CH3). Elemental analysis for
C6H9N3O: calcd C, 51.79; H, 6.52; N, 30.20; found C, 50.99; H,
6.11; N, 29.60. MS (ESI) m/z 162.2 [M+Na]+. IR
m
cmꢀ1 3111w,
2941w, 1674w, 1520w, 1469w, 1445w, 1288w, 1147w, 1057w,
922w, 758w, 711w. Yield = 0.718 g (65%).
[M+Na]+. IR
m
cmꢀ1 3109w, 2932w, 2876w, 1608w, 1520w,
1496w, 1444w, 1365w, 1288w, 1016w, 943w, 815w, 748w,
700w. Yield = 0.663 g (83%).
4.1.2.2.9. 1-Methyl-1H-imidazole-2-carbaldehyde O-hexyloxime
(3i). 1H NMR (300 MHz, CDCl3) dH 8.14 (1H, s, N@CH), 7.09 (1H,
s, imz), 6.91 (1H, s, imz), 4.15 (2H, t, OCH2), 3.87 (3H, s, CH3),
1.70 (2H, m, CH2), 1.30 (6H, m, (CH2)3), 1.00 (3H, t, CH3). 13C
NMR (300 MHz, CDCl3) dc 144.7 (N@CH), 143.1 (imz), 131.4
(imz), 120.1 (imz), 69.5 (OCH2), 32.3 (CH3), 31.6 (CH2), 29.1
(CH2), 26.8 (CH2), 24.5 (CH2), 14.6 (CH3). Elemental analysis for
4.1.2.2.3. (R)- or (S)-Methyl 2-((1-methyl-1H-imidazol-2-yl)meth-
yleneamino)-3-phenylpropanoate (3c). 1H NMR (300 MHz, CDCl3)
dH 8.04 (1H, s, N@CH), 7.14–7.26 (5H, m, aromatic), 7.08 (1H, s,
imz), 6.92 (1H, s, imz), 4.18 (1H, t, CH), 3.98 (3H, s, OCH3), 3.73
(3H, s, CH3), 3.71 (1H, m, CHH), 3.33 (1H, m, CHH). 13C NMR
(300 MHz, CDCl3) dc 168.5 (C@O), 148.8 (CH@N), 145.3 (imz),
135.4 (aromatic), 131.5 (imz), 126.3–129.6 (aromatic), 119.7
(imz), 70.6 (CH), 52.2 (OCH3), 37.4 (CH2), 35.7 (CH3). Elemental
analysis for C15H17N3O2: calcd C, 66.40; H, 6.32; N, 15.49; found
C, 65.86; H, 5.98; N, 15.01. MS (ESI) m/z 272.1 [M+H]+, 294.2
C
11H19N3O: calcd C, 63.13; H, 9.15; N, 20.08; found C, 62.98; H,
8.99; N, 19.83. MS (ESI) m/z 209.3 [M+Na]+. IR cmꢀ1 2932w,
m
2870w, 1714w, 1467w, 1444w, 1288w, 1059w, 943w, 748w,
711w. Yield = 0.543 g (51%).
[M+Na]+. IR
m
cmꢀ1 3014w, 2957w, 2673w, 1622w, 1581w,
4.1.2.3. (1-Methyl-1H-imidazol-2-yl)methanamines (4).
In
1529w, 1510w, 1479w, 1438w, 1270w, 1248w, 1140w, 814w,
760w. Yield = 0.288 g (71%).
a stainless steel autoclave (20 mL), equipped with temperature
control and magnetic stirrer, was purged 5 times with hydrogen,

2384
N. Ferri et al. / Bioorg. Med. Chem. 21 (2013) 2379–2386
a solution of imine (3) (3.0 mmol) in CH3OH with 5% of Pd/C was
transferred, the autoclave was pressurised at 20 atm and maintain-
ing at room temperature. After 24 h, the mixture was filtered on
celite and the solvent was evaporated with vacuum.
cmꢀ1 3483w, 3383w, 3138w, 3036w, 1664w, 1608w, 1564w,
1520w,1496w, 1313w, 1263w, 1078w, 829w, 750w.
Yield = 0.488 g (97%).
4.1.2.3.1. (1-Methyl-1H-imidazol-2-yl) methanamine (4a). The
product was purified by crystallization in CH2Cl2 at ꢀ18 °C. 1H
NMR (300 MHz, DMSO) dH 6.93 (1H, s, H5 imz), 6.81 (1H, s, H4,
imz), 3.87 (2H, s, CH2), 3.63 (3H, s, CH3). 13C NMR (300 MHz,
DMSO) dc 145.2 (imz), 129.7 (imz), 125.0 (imz), 40.2 (CH3), 36.6
(CH2). Elemental analysis for C5H9N3: calcd C, 54.03; H, 8.16; N,
37.81; found C, 53.98; H, 8.01; N, 37.56. MS (ESI) m/z 111.08
4.1.3. General procedure for preparation of PtII(diamine)
dichloride complexes
To a solution of K2PtCl4 (0.208 g, 0.500 mmol) in 16 mL of H2O/
HCl 4 M (ratio 15/1), the diamine (0.500 mmol) was added. The
mixture was stirred for 6 h and heated under reflux, then it was
warmed to room temperature and stirred overnight. The complex
was filtered off and washed with water, methanol and diethyl
ether. The solid was dried under vacuum at room temperature.
[M+H]+, 95.2 [MꢀNH2]. IR
m
cmꢀ1 3154w, 2033w, 1632w, 1607w,
1529w, 1466w, 1404w, 1119w, 1182w, 863w, 828w, 768w,
744w. Yield = 0.410 g (98%).
4.1.3.1. (1-Methyl-1H-imidazol-2-yl)methanamine dichloro-
4.1.2.3.2. N-((1-Methyl-1H-imidazol-2-yl)methyl)(phenyl)meth-
anamineꢁ2HCl (4b). 1H NMR (300 MHz, DMSO) dH 7.30–7.32
(5H, m, aromatic), 6.91 (1H, s, imz), 6.79 (1H, s, imz), 4.55 (2H, s,
CH2imz), 4.33 (2H, d, CH2Ph), 3.93 (3H, s, CH3). 13C NMR
(300 MHz, DMSO) dc 148.2 (imz), 146.5 (aromatic), 130.0 (aro-
matic), 126.8 (imz), 122.3 (imz), 118.5 (aromatic), 113.5 (aro-
matic), 53.8 (CH2), 39.0 (CH2), 35.0 (CH3). Elemental analysis for
platinum(II) (Pt-4a).
Light grey solid. Yield = 0.164 g (87%).
1H NMR (300 MHz, DMSO) dH 7.33 (1H, s, H5 imz), 7.07 (1H, s,
H4, imz), 6.09 (2H, br, NH2), 3.90 (2H, dd, CH2), 3.66 (3H, s, CH3).
195Pt NMR (300 MHz, DMSO) d ꢀ2079. Elemental analysis for
C5H9Cl2N3Pt: calcd C, 15.92; H, 2.41; N, 11.14; found C, 15.32; H,
2.73; N, 10.54. MS (ESI) m/z 400.3 [M+Na]+.
C
12H15N3ꢁ2HCl: calcd C, 52.57; H, 6.25; N, 15.33; found C, 52.54;
4.1.3.2. N-((1-Methyl-1H-imidazol-2-yl)methyl) (phenyl)metha-
H, 6.15; N, 15.20. MS (ESI) m/z 202.1 [M+H]+, 224.3 [M+Na+]. IR
m
naminedichloroplatinum(II) (Pt-4b).
Pale yellow solid.
cmꢀ1 3337w, 3111w, 2677w, 2376w, 1601w, 1560w, 1498w,
1433w, 1035w, 752w, 688w. Yield = 0.670 g (97%).
Yield = 0.191 g (82%). 1H NMR (300 MHz, DMSO) dH 7.65 (1H, d,
NH), 7.32–7.40 (5H, m, aromatic), 7.25 (1H, d, imz), 6.90 (1H, d,
imz), 4.33 (2H, dd, CH2imz), 4.03 (2H, d, CH2Ph), 3.57 (3H, s,
CH3). 195Pt NMR (300 MHz, DMSO) d ꢀ2098. Elemental analysis
for C12H15Cl2N3Pt: calcd C, 30.85; H, 3.24; N, 8.99; found C,
31.01; H, 3.76; N, 8.83. MS (ESI) m/z 490.3 [M+Na]+.
4.1.2.3.3. (R)- or (S)-Methyl 2-((1-methyl-1H-imidazol-2-yl)meth-
ylamino)-3-phenylpropanoate (4c). 1H NMR (300 MHz, DMSO) dH
7.11–7.26 (5H, m, aromatic), 6.88 (1H, d, imz), 6.75 (1H, d, imz),
3.89 (1H, s, CHH), 3.88 (1H, s, CHH), 3.84 (1H, m, CH), 3.68 (3H, s,
OCH3), 3.42 (3H, s, CH3), 2.98 (1H, m, CHH), 2.76 (1H, m, CHH).
13C NMR (300 MHz, DMSO) dc 168.3 (C@O), 143.2 (imz), 137.1 (aro-
matic), 129.0–129.3 (aromatic), 127.1 (imz), 115.5 (imz), 59.3 (CH),
53.8 (OCH3), 48.1 (CH2), 39.2 (CH2), 35.0 (CH3). Elemental analysis
for C15H19N3O2: calcd C, 65.91; H, 7.01; N, 15.37; found C, 65.73; H,
4.1.3.3. (S)-Methyl 2-((1-methyl-1H-imidazol-2-yl)methylamino)-
3-phenylpropanoatedichloro platinum(II) (Pt-(S)4c). Green solid.
Yield = 0.223 g (88%). 1H NMR (300 MHz, DMSO) dH 7.03–7.24 (5H,
m, aromatic), 6.98 (1H, d, imz), 6.89 (1H, d, imz), 3.79 (1H, s, CHH),
3.71 (1H, s, CHH), 3.64 (1H, m, CH), 3.61 (3H, s, OCH3), 3.43 (3H, s,
CH3), 3.26 (1H, m, CHH), 3.17 (1H, m, CHH). 195Pt NMR (300 MHz,
DMSO) d ꢀ2122. Elemental analysis for C15H19Cl2N3O2Pt: calcd C,
33.41; H, 3.55; N, 7.79; found C, 33.08; H, 3.77; N, 7.84. MS (ESI)
m/z 562.2 [M+Na]+.
6.98; N, 15.22. MS (ESI) m/z 274.1 [M+H]+, 296.3 [M+Na+]. IR
m
cmꢀ1 3343w, 3122w, 2689w, 2356w, 1608w, 1610w, 1510w,
1489w, 1423w, 1070w, 762w, 696w. Yield = 0.290 g (97%).
4.1.2.3.4. 4-((1-Methyl-1H-imidazol-2-yl)methyl amino)phenol
(4d). 1H NMR (300 MHz, DMSO) dH 6.97 (1H, s, imz), 6.80 (1H,
s, imz), 6.63–6.70 (4H, m, aromatic), 4.26 (2H, d, CH2), 3.70 (3H,
s, CH3). 13C NMR (300 MHz, DMSO) dc 151.7 (aromatic, HO–C),
146.2 (imz), 143.3 (aromatic), 126.7 (imz), 122.2 (imz), 114.2–
115.2 (aromatic), 39.4 (CH2), 32.9 (CH3). Elemental analysis for
4.1.3.4. (R)-Methyl 2-((1-methyl-1H-imidazol-2-yl)methylamino)-
3-phenylpropanoatedichloro
platinum(II)
(Pt-(R)4c). Moss-
green solid. Yield = 0.230 g (85%). 1H NMR (300 MHz, DMSO) dH
7.03–7.24 (5H, m, aromatic), 6.98 (1H, d, imz), 6.89 (1H, d, imz),
3.79 (1H, s, CHH), 3.71 (1H, s, CHH), 3.64 (1H, m, CH), 3.61 (3H, s,
OCH3), 3.43 (3H, s, CH3), 3.26 (1H, m, CHH), 3.17 (1H, m, CHH).
195Pt NMR (300 MHz, DMSO) d ꢀ2124. Elemental analysis for
C11H13N3O: calcd C, 65.01; H, 6.45; N, 20.68; found C, 64.51; H,
6.39; N, 20.22. IR
1249w, 1109w, 821w, 738w. Yield = 0.344 g (95%).
m
cmꢀ1 3416w, 2934w, 2592w, 1516w, 1439w,
4.1.2.3.5. 4-Methoxy-N-((1-methyl-1H-imidazol-2-yl)methyl)ben-
zenamine (4e). 1H NMR (300 MHz, DMSO) dH 6.98 (1H, s, imz),
6.97 (1H, s, imz), 6.79–6.86 (2H, m, aromatic), 6.68–6.71 (2H, m,
aromatic), 4.28 (2H, d, CH2), 3.75 (3H, s, OCH3), 3.66 (3H, s, CH3).
13C NMR (300 MHz, DMSO) dc 151.8 (aromatic), 146.0 (imz),
143.7 (aromatic), 126.9 (imz), 122.5 (imz), 114.1–115.6 (aromatic),
55.93 (OCH3), 39.6 (CH2), 31.9 (CH3). Elemental analysis for
C
15H19Cl2N3O2Pt: calcd C, 33.41; H, 3.55; N, 7.79; found C, 32.73;
H, 3.82; N, 7.86. MS (ESI) m/z 562.3 [M+Na]+.
4.1.3.5. 4-((1-Methyl-1H-imidazol-2-yl)methyl amino)phenoldi-
chloroplatinum(II) (Pt-4d). Light green solid. Yield = 0.180 g
(76%). 1H NMR (300 MHz, DMSO) dH 9.50 (1H, s, OH), 9.04 (1H, d,
NH), 7.40 (1H, s, imz), 6.97 (1H, s, imz), 6.96 (2H, d, aromatic),
6.68 (2H, d, aromatic), 4.49–4.47 (1H, dd, CHH), 4.26 (1H, d,
CHH), 3.71 (3H, s, CH3). 195Pt NMR (300 MHz, DMSO) d ꢀ2052. Ele-
mental analysis for C11H13Cl2N3OPt: calcd C, 28.16; H, 2.79; N,
8.96; found C, 27.42; H, 2.96; N, 8.59. MS (ESI) m/z 492.2 [M+Na]+.
C
12H15N3O: calcd C, 66.34; H, 6.96; N, 19.34; found C, 66.47; H,
7.21; N, 19.5. MS (ESI) m/z 218.1 [M+H]+, 240.3 [M+Na+]. IR
m
cmꢀ1 3246w, 3119w, 2993w, 1861w, 1510w, 1466w, 1286w,
1236w, 1033w, 981w, 823w, 748w. Yield = 0.449 g (96%).
4.1.2.3.6. N-(4-((1-Methyl-1H-imidazol-2-yl)methylamino)phenyl)
acetamide (4f). 1H NMR (300 MHz, DMSO) dH 7.26–7.43 (2H, m,
aromatic), 6.97 (1H, s, imz), 6.86 (1H, s, imz), 6.63–6.67 (2H, m,
aromatic), 4.28 (2H, d, CH2), 3.65 (3H, s, OCH3), 2.17 (3H, s, CH3).
13C NMR (300 MHz, DMSO) dc 168.3 (C@O), 146.4 (imz), 145.7 (aro-
matic), 130.1 (aromatic), 127.0 (imz), 122.7 (imz), 113.3–121.8
(aromatic), 39.9 (CH2), 31.6 (CH3), 24.7 (CH3). Elemental analysis
for C13H16N4O: calcd C, 63.91; H, 6.60; N, 22.93; found C, 64.07;
4.1.3.6.
4-Methoxy-N-((1-methyl-1H-imidazol-2-yl)methyl)
benzenaminedichloroplatinum(II) (Pt-4e). Light yellow solid.
Yield = 0.197 g (83%). 1H NMR (300 MHz, DMSO) dH 9.15 (1H, d,
NH), 7.42 (1H, s, imz), 6.90 (1H, s, imz), 7.05 (2H, d, aromatic),
6.88 (2H, d, aromatic), 4.55 (1H, dd, CHH), 4.28 (1H, d, CHH),
3.73 (3H, s, OCH3), 3.72 (3H, s, CH3). 195Pt NMR (300 MHz, DMSO)
d ꢀ2058. Elemental analysis for C12H15Cl2N3OPt: calcd C, 29.82; H,
H, 6.89; N, 23.6. MS (ESI) m/z 245.1 [M+H]+, 267.3 [M+Na+]. IR
m

N. Ferri et al. / Bioorg. Med. Chem. 21 (2013) 2379–2386
2385
3.13; N, 8.69; found C, 29.87; H, 3.28; N, 8.49. MS (ESI) m/z 506.2
mented with penicillin (10,000 U/mL), streptomycin (10 mg/mL),
nonessential amino acid and 10% Fetal Calf Serum (FCS) unless
otherwise indicated. Dulbecco’s modified eagle’s medium (DMEM)
with glutamine was utilised for HEY, A2780, A549, MDA-MB-231,
MCF-7, A431, DLD-1 cell lines; RPMI 1640 for NCI-H460, IGROV-1
e HCT-15; McCoy’s for HCT-116; 49% Ham’s plus 49% RPMI with 2%
FCS for KYSE-150. 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.
[M+Na]+.
4.1.3.7.N-(4-((1-Methyl-1H-imidazol-2-yl)methylamino)phenyl)
acetamidedichloro platinum(II) (Pt-4f). Pale yellow solid.
Yield = 0.206 g (81%). 1H NMR (300 MHz, DMSO) dH 9.06 (1H, d,
NH), 7.26 (1H, d, imz), 7.06–6.93 (4H, m, aromatic), 6.84 (1H, d,
imz), 4.63 (1H, dd, CHH), 4.36 (1H, d, CHH), 3.75 (3H, s, CH3), 2.67
(3H, s, CH3). 195Pt NMR (300 MHz, DMSO) d ꢀ2063. Elemental anal-
ysis for C13H16Cl2N4OPt: calcd C, 30.60; H, 3.16; N, 10.98; found C,
29.97; H, 3.22; N, 10.86. MS (ESI) m/z 533.2 [M+Na]+.
4.2.3. MTT-assay
The determination of the conversion of MTT (MTT = 3-(4,5-di-
methyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) to for-
mazan was determined by using a commercially available kit
(Millipore, Billerica, MA, USA), according to the manufacturer’s
instructions.35
4.1.3.8. 1-Methyl-1H-imidazole-2-carbaldehyde O-benzyloxi-
medichloroplatinum(II) (Pt-3a). Deep yellow solid. Yield =
0.208 g (87%). 1H NMR (300 MHz, CDCl3) dH 8.84 (1H, d, N@CH),
7.50–7.38 (7H, m, aromatic+ imz), 5.30 (2H, d, CH2), 3.87 (3H, s,
CH3). 195Pt NMR (300 MHz, DMSO) d ꢀ2943. Elemental analysis
for C12H13Cl2N3OPt: calcd C, 29.95; H, 2.72; N, 8.73; found C,
29.67; H, 2.98; N, 8.62. MS (ESI) m/z 504.1 [M+Na]+.
4.2.4. Determination of intracellular and DNA-bound Pt
concentration
For the determination of total intracellular 195Pt concentra-
tions, cells were washed twice with PBS and lysed by incubation
with 1% Triton X100/0.1% SDS for 5 min at room temperature.
Cell lysates were then cleared by centrifugation at 14,000g for
10 min, and the 195Pt concentrations determined by ICP-MS.
The data were normalised with the protein concentrations deter-
mined using the BCA protein assay (Thermo scientific, Rockford,
IL USA). For the DNA-bound 195Pt concentrations, the nuclear
DNA was extracted by incubating cell monolayers with digest
buffer (50 mM Tris–HCl 1 M, 100 mM NaCl, 100 mM EDTA, 1%
SDS) then transferred to 1.5 mL microcentrifuge tubes and satu-
rated NaCl solution added. The samples were then clear by cen-
trifugation for 15 min at 13,000 rpm and the supernatant
transferred to new microcentrifuge tubes and DNA precipitated
by isopropanol. DNA was then washed by centrifugation two
times with 70% ethanol and resuspended in TE buffer (10 mM
Tris pH 8.0; 0.1 mM EDTA). The 195Pt concentrations were then
determined by ICP-MS.
4.1.3.9. 1-Methyl-1H-imidazole-2-carbaldehydeoximedichloro-
platinum(II) (Pt-3g). Orange solid. Yield = 0.150 g (77%). 1H NMR
(300 MHz, CDCl3) dH 8.97 (1H, s, OH), 8.73 (1H, d, N@CH), 7.77
(1H, d, imz), 7.69 (1H, d, imz), 3.91 (3H, d, CH3). 195Pt NMR
(300 MHz, DMSO) d ꢀ2904. Elemental analysis for C5H7Cl2N3OPt:
calcd C, 15.35; H, 1.80; N, 10.74; found C, 15.73; H, 2.03; N,
10.74. MS (ESI) m/z 393.3 [M+H]+, 804.8 [2M+Na]+.
4.1.3.10. 1-Methyl-1H-imidazole-2-carbaldehyde O-methyloxi-
medichloroplatinum(II) (Pt-3h). Pale orange solid. Yield =
0.159 g (79%). 1H NMR (300 MHz, CDCl3) dH 8.80 (1H, d, N@CH),
7.57 (1H, d, imz), 7.50 (1H, d, imz), 4.03 (3H, d, OCH3) 3.91 (3H,
d, CH3). 195Pt NMR (300 MHz, DMSO) d ꢀ2910. Elemental analysis
for C6H9Cl2N3OPt: calcd C, 17.79; H, 2.24; N, 10.37; found C, 17.80;
H, 2.28; N, 10.32. MS (ESI) m/z 428.1 [M+Na]+
.
4.1.3.11. 1-Methyl-1H-imidazole-2-carbaldehyde O-hexyloxi-
medichloroplatinum(II) (Pt-3i). Yellow solid. Yield = 0.194 g
(82%). 1H NMR (300 MHz, CDCl3) dH 8.79 (1H, d, N@CH), 7.55
(1H, d, imz), 7.49 (1H, d, imz), 4.25 (2H, t, OCH2), 3.82 (3H, d,
CH3), 1.73 (2H, m, CH2), 1.36 (6H, m, (CH2)3), 1.00 (3H, t, CH3).
195Pt NMR (300 MHz, DMSO) d ꢀ2960. Elemental analysis for
C11H19Cl2N3OPt: calcd C, 27.80; H, 4.03; N, 8.84; found C, 28.33;
H, 4.13; N, 8.99 . MS (ESI) m/z 497.3 [M+Na]+, 973.0 [2 M+Na]+.
4.2.5. 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% Non-
idet-P40, containing a protease and phosphatase inhibitor cock-
tails (Sigma–Aldrich, Milan, Italy) for 30 min on ice. Cell lysates
were then cleared by centrifugation at 14,000 g for 10 min, and
protein concentrations were determined using the BCA protein
assay (Thermo scientific, Rockford, IL USA). Equal amount of total
protein per sample were separated by SDS-PAGE under reducing
conditions, transferred to Immobilon PVDF (GE Healthcare Little
Chalfont, Buckinghamshire, UK) and subsequently immunoblot-
ted with primary antibody following appropriate secondary anti-
body, prior to visualization by enhanced chemiluminescence
(LiteAblot Extend Long Lasting Chemiluminescent Substrate,
EuroClone).
4.2. Biological evaluation procedures
4.2.1. Reagents and antibodies
DMEM, trypsin-EDTA, penicillin, streptomycin, non-essential
amino acid solution, fetal calf serum (FCS), disposable culture
flasks and petri dishes were purchased from Euroclone S.p.A. (Pero,
Milan, Italy). For western blot analysis, the following antibodies
were used: mouse monoclonal anti-p53 and p21Waf (Santa-Cruz
Biotechnology Inc., Santa Cruz, CA, USA), mouse monoclonal anti-
Quantitative densitometric analyses were performed using Gel
Doc acquisition system and Quantity One software (BIO-RAD Lab-
oratories, Hercules, CA, USA).36
a-tubulin (Sigma–Aldrich, Milan, Italy), anti-mouse peroxidase-
conjugated secondary antibodies (Jackson ImmunoResearch Lab;
Cambridgeshire, UK).
4.2.6. Statistical analysis
4.2.2. Cell culture
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 re-
quired to reduce by 50% the cell viability (EC50) was calculated
by nonlinear regression curve (SigmaPlot software; Systat Soft-
ware, Inc., Point Richmond, CA).
The following human cancer cell types have been utilised in the
study: NCI-H460 (non-small cell lung cancer) A2780 and IGROV-1
(epithelial ovarian cancer), HEY (ovarian cancer), A431 (cutaneous
squamous carcinoma), KYSE510 (esophageal squamous cell
carcinoma) HCT15, KM12, SW620, HCT116, DLD1, and COLO205
(colorectal cancer), MCF-7 and MDA-MB-231 (breast cancer), and
A549 (lung adenocarcinoma). All cultured media were supple-

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