N6-benzyladenosine derivatives as novel n-donor ligands of platinum(ii) dichlorido complexes

Article abstract of DOI:10.3390/molecules18066990

The platinum(II) complexes trans-[PtCl2(Ln)2]·xSolv 1-13 (Solv = H2O or CH₃OH), involving N6-benzyladenosine-based N-donor ligands, were synthesized; Ln stands for N6-(2-methoxybenzyl)adenosine (L1, involved in complex 1), N6-(4-methoxybenzyl) adenosine (L2, 2), N6-(2-chlorobenzyl)adenosine (L3, 3), N6-(4-chlorobenzyl)- adenosine (L4, 4), N6-(2-hydroxybenzyl)adenosine (L5, 5), N6-(3-hydroxybenzyl)- adenosine (L6, 6), N6-(2-hydroxy-3-methoxybenzyl) adenosine (L7, 7), N6-(4-fluorobenzyl) adenosine (L8, 8), N6-(4-methylbenzyl) adenosine (L9, 9), 2-chloro-N6-(3-hydroxybenzyl) adenosine (L10, 10), 2-chloro-N6-(4-hydroxybenzyl)adenosine (L11, 11), 2-chloro- N6-(2-hydroxy-3- methoxybenzyl)adenosine (L12, 12) and 2-chloro-N6-(2-hydroxy-5- methylbenzyl)adenosine (L13, 13). The compounds were characterized by elemental analysis, mass spectrometry, IR and multinuclear (1H-, 13C-, 195Pt- and 15N-) and two-dimensional NMR spectroscopy, which proved the N7-coordination mode of the appropriate N6-benzyladenosine derivative and trans-geometry of the title complexes. The complexes 1-13 were found to be non-toxic in vitro against two selected human cancer cell lines (HOS and MCF7; with IC50 > 50.0 μM). However, they were found (by ESI-MS study) to be able to interact with the physiological levels of the sulfur-containing biogenic biomolecule L-methionine by a relatively simple 1:1 exchange mechanism (one Ln molecule was replaced by one L-methionine molecule), thus forming a mixed-nitrogen/sulfur-ligand dichlorido-platinum(II) coordination species.

Full text of DOI:10.3390/molecules18066990

Molecules 2013, 18, 6990-7003; doi:10.3390/molecules18066990
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
N6-Benzyladenosine Derivatives as Novel N-Donor Ligands of
Platinum(II) Dichlorido Complexes
Pavel Štarha, Igor Popa, Zdeněk Trávníček * and Ján Vančo
Regional Centre of Advanced Technologies and Materials, Department of Inorganic Chemistry,
Faculty of Science, Palacký University, 17. listopadu 12, CZ-77146 Olomouc, Czech Republic;
E-Mails: pavel.starha@upol.cz (P.S.); igor.popa@upol.cz (I.P.); jan.vanco@upol.cz (J.V.)
* Author to whom correspondence should be addressed; E-Mail: zdenek.travnicek@upol.cz;
Tel.: +420-585-634-352; Fax: +420-585-634-954.
Received: 3 May 2013; in revised form: 7 June 2013 / Accepted: 8 June 2013 /
Published: 14 June 2013
Abstract: The platinum(II) complexes trans-[PtCl2(Ln)2]·xSolv 1–13 (Solv = H2O or
CH3OH), involving N6-benzyladenosine-based N-donor ligands, were synthesized; Ln
stands for N6-(2-methoxybenzyl)adenosine (L1, involved in complex 1), N6-(4-methoxy-
benzyl)adenosine (L2, 2), N6-(2-chlorobenzyl)adenosine (L3, 3), N6-(4-chlorobenzyl)-
adenosine (L4, 4), N6-(2-hydroxybenzyl)adenosine (L5, 5), N6-(3-hydroxybenzyl)-
adenosine (L6, 6), N6-(2-hydroxy-3-methoxybenzyl)adenosine (L7, 7), N6-(4-fluoro-
benzyl)adenosine (L8, 8), N6-(4-methylbenzyl)adenosine (L9, 9), 2-chloro-N6-(3-hydroxy-
benzyl)adenosine (L10, 10), 2-chloro-N6-(4-hydroxybenzyl)adenosine (L11, 11), 2-chloro-
N6-(2-hydroxy-3-methoxybenzyl)adenosine (L12, 12) and 2-chloro-N6-(2-hydroxy-5-
methylbenzyl)adenosine (L13, 13). The compounds were characterized by elemental analysis,
mass spectrometry, IR and multinuclear (1H-, 13C-, 195Pt- and 15N-) and two-dimensional NMR
spectroscopy, which proved the N7-coordination mode of the appropriate N6-benzyladenosine
derivative and trans-geometry of the title complexes. The complexes 1–13 were found to
be non-toxic in vitro against two selected human cancer cell lines (HOS and MCF7; with
IC50 > 50.0 µM). However, they were found (by ESI-MS study) to be able to interact with
the physiological levels of the sulfur-containing biogenic biomolecule L-methionine by
a relatively simple 1:1 exchange mechanism (one Ln molecule was replaced by one
L-methionine molecule), thus forming a mixed-nitrogen/sulfur-ligand dichlorido-platinum(II)
coordination species.

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Keywords: platinum(II) complexes; transplatin derivatives; N6-benzyladenosine derivatives;
multinuclear NMR; antitumor activity
1. Introduction
Despite a formidable intensity of research and development in the field of metallopharmaceuticals,
the group of platinum(II) complexes still leads in the number of treated patients, as well as in the
number of marketed drugs and yearly sales [1]. The success of this group began with the discovery of
anticancer activity of the cis-isomer of Peyrone’s salt [diammine-dichloridoplatinum(II)] [2], known as
cisplatin, by the group of Rosenberg et al. [3], while the other, biologically nearly inactive trans-isomer
(transplatin) remained just a scientific oddity for a long time. The conundrum of the inactivity of the
trans-isomer was unravelled by the discoveries during the latest 20 years (for comprehensive reviews
see [4,5] and the references therein), which showed that trans-platinum inspired complexes, such as
those involving the aromatic heterocyclic ligands [6], aliphatic amines [7], or iminoether ligands [8],
could exhibit cytotoxicity comparable to that of cisplatin, and also that some of these biologically
active species could circumvent the mechanisms of resistance to cisplatin [9,10]. These discoveries
fired up the recent development in the area and proved that trans-platinum complexes must be
considered as biologically relevant, even if the mechanisms of their action are much more complex
than in the case of cisplatin.
N6-benzyladenine and its substituted-benzyl derivatives represent the plant hormones from the
group of aromatic cytokinins [11]. The free bases are responsible for their physiological action, while
the ribosides, i.e., N6-benzyladenosine and its substituted-benzyl derivatives, are one of the transport
forms of the mentioned phytohormones. The quoted N6-benzyladenosines have been shown as in vitro
antitumor active substances against various types of human cancer cell lines [12,13]. However, from
the coordination chemistry point of view, only two papers dealing with the transition metal complexes
with N6-benzyladenosine and its substituted-benzyl derivatives acting as N-donor ligands have been
published to date. Concretely, Waysbort et al. studied the dinuclear rhenium acetato-bridged
complexes with N6-benzyladenosine [14], while Trávníček et al. described a series of iron mixed-
valence complexes involving N6-(substituted-benzyl)adenosine derivatives and their in vitro antitumor
effect on four human cancer cell lines [15]. The [FeCl3(L8)2] complex, which involves N6-(4-
fluorobenzyl)adenosine symbolized as L8 in this work, was found to be moderately effective against
human osteosarcoma (HOS; IC50 = 8.0 µM), human myelogenous leukemia (K562; IC50 = 9.0 µM) and
human adenocarcinoma (MCF7; IC50 = 16.0 µM) cancer cells. These findings led us to study the
utilization of in vitro anticancer active organic molecules (N6-benzyladenosine derivatives) as N-donor
ligands of the coordination compounds of the biologically more perspective transition metal (i.e.,
platinum). We chose thirteen synthetically relatively easily obtainable N6-benzyladenosine derivatives,
which represent two types of compounds, i.e., N6-(substituted-benzyl)adenosine derivatives (L1–L9)
and 2-chloro-N6-(substituted-benzyl)adenosine derivatives (L10–L13), which act as N-donor ligands
within the structures of trans-platinum(II) dichlorido complexes (1–13). The complexes were screened
for their in vitro antitumor activity against two human cancer cell lines (HOS and MCF7) to find out

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whether the complex formation may enhance the cytotoxic effect of the ligands involved in the
prepared complexes.
2. Results and Discussion
2.1. Characterizations and General Properties of the Complexes
The trans-[PtCl2(Ln)2]·xSolv complexes 1–13 (Solv = H2O or CH3OH) were prepared as white
powders. Although, the preparation of cis-isomers was the main goal of this work, only the pure
trans-isomers were isolated. This was quite surprising because two series of cis-[PtCl2(L)2] complexes
with differently substituted N6-benzyladenine derivatives [16] or with 7-azaindole derivatives [17]
were formerly prepared by the same procedure, i.e., the reaction of K2[PtCl4] with the corresponding
N-donor ligand. This unexpected behaviour may be connected with very low thermodynamic stability of
the cis-isomers, which probably transform instantly into the trans-isomers. We tried to detect the
cis-to-trans transformation process by means of 195Pt-NMR experiments, however, although the NMR
spectra were measured immediately after the complex preparation, only the presence of pure trans-isomer
was confirmed (see below for more details).
It has been found that the chlorination at the C2 atom on the purine moiety of the Ln ligand is
important for final complex solubility, and thus the complexes 10–13, which involve 2-chloro-N6-
benzyladenosine derivatives, are more soluble in DMF, ethanol, methanol and acetone than complexes
1–9, whose structures contain N6-benzyladenosines (see Scheme 1). A limited solubility in distilled
water was observed for all the complexes. The obtained results of the elemental analysis for the
prepared complexes are in good agreement with the calculated ones.
Scheme 1. Schematic representations of the N6-benzyladenosine-based N-donor ligands
(L1–L13) involved in structures of complexes 1–13.
All the complexes behave as non-electrolytes in DMF solutions (1.2–3.5 S·cm2·mol−1) [18].
Thermal decomposition of 1–13 started right after the beginning of the experiment and is accompanied
by a loss of the solvate molecules (½CH3OH for 1, 5, 8 and 9, CH3OH for 2, 4, 6, 7, 11 and 12, ½H2O
for 3 and ¾CH3OH for 10 and 13). The calculated weight losses of the desolvation process differ from
the calculated ones by no more than 0.3%. The desolvated complexes decay to PtO without the
formation of any thermally stable intermediates from 100–186 °C to 476–595 °C.

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The ESI– mass spectra of 1–13 dissolved in methanol were found to be similar in terms of the
detected types of species (see Supplementary Material). The pseudomolecular peaks corresponding to
[PtCl2(Ln)2-H]− were detected in the ESI– mass spectra of 1–13. Typically, the most intense peaks
belonged to the [PtCl3(Ln)2]− species. The species whose composition corresponded to the other
detected peaks, involved the appropriate adenine derivative without ribose (symbolized as Ln`).
Concretely [PtCl2(Ln)(Ln`)-H]−, [PtCl(Ln)(Ln`)-2H]− and [PtCl(Ln`)-2H]− were found in the spectra.
Detection of the characteristic bands of the appropriate N6-benzyladenosine derivatives
(L1–L13; [19,20]) in the IR spectra of the complexes 1–13 proved the presence of the mentioned
organic ligands in the structure of the studied complexes. The bands were found and assigned as
follows: the medium to strong bands at 3408–3584 cm−1 can be attributed to ν(O–H) of the ribose
moiety, the medium to strong bands between 3308 and 3359 cm−1 are assignable to ν(N–H) and the
medium bands between 3121 and 3133 cm−1 belong to ν(C–H)ar. It should be pointed out that the
interpretation of the bands in the region of 3100–3600 cm−1 is markedly influenced by overlapping of
the vibrations. The majority of the bands found in the 660–900 cm−1 and 1450–1590 cm−1 region are
assignable to the skeletal vibrations of the purine moiety, while the bands at 1605–1616 cm−1 can be
attributed ν(C=N)ar purine skeletal vibrations [20]. The substitutions at the aromatic rings were proved
by the presence of the stretching vibrations of ν(C–Cl)ar (3, 4, 11–13), ν(C–F)ar (8) and ν(C–O)ar (1, 2,
5–7, 11–13) in the regions of 1152–1182, at 1215, and 1214–1248 cm−1, respectively, in the IR spectra.
The bands of medium to strong intensity at 1053–1116 cm−1 can be attributed to the ν(C–O)aliph vibrations,
which belong to the hemiacetal groups of the ribose moiety. The bands between 326 and 349 cm−1 are
assignable to ν(Pt–Cl), while those detected between 510 and 545 cm−1 belong to ν(Pt–N) [21].
1
The H-, 13C- and 15N-NMR signals of free N6-benzyladenosines L1–L13 were detected in the
13
spectra of the complexes (except for N3 in 13). The 1H-, C- and 15N-NMR coordination shift values
(Δδ = δcomplex − δligand, ppm; see Table 1) clearly showed the N7-coordination mode of the L1–L13
molecules to the central Pt(II) atom, similarly as reported for the platinum(II) dichlorido complexes
involving N9-substituted N6-benzyladenine (analogous NMR studies in combination with
crystallography; [16]) or adenine [22] derivatives. The greatest coordination shifts in the 1H-NMR and
13C-NMR spectra were found for the atoms situated close to the coordination site represented by the
1
N7 atom, i.e. N6H, C8H, C5 and C8 (Table 1). The 15N signals found by H–15N gs-HMBC
experiments proved beyond all doubt the coordination via the N7 atom. It is obvious that the signals of
the N7 atom are shifted much more as compared with other nitrogen atoms involved in the structure of
the studied complexes (Table 1) that means the N1, N3, N6 and N9 atoms are not significantly
influenced by the ligand coordination to Pt(II) atom. It is well known that conclusions about the isomer
types can be obtained from 195Pt-NMR experiments, where significant differences between chemical
shifts of cis- (δcis) and trans- (δtrans) complexes are expected, as was already proved in our previous
papers regarding NMR and crystallographic study of the cis-to-trans isomerisation of platinum(II)
dichlorido complexes with differently substituted N6-benzyladenine derivatives, with δcis ≈ −2020 ppm
and δtrans ≈ −2065 ppm [16]. As can be seen from the chemical shifts listed in Table 1 and depicted in
Figure 1, those ones assignable to trans-isomers 1–13 correlated well with the previously reported
values. Moreover, the number, position and intensity of the signals detected in the 195Pt-NMR spectra
of 1–13 did not change in time (up to eight weeks), which is typical feature of trans-isomers of
platinum(II) dichlorido complexes, while being highly improbable for the cis-isomers.

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Figure 1. 195Pt-NMR spectra of the selected complexes 4 and 8 (involving N6-benzyladenosine
derivatives) and 10 and 12 (involving 2-chloro-N6-benzyladenosine derivatives).
Table 1. The 1H-, 13C- and 15N-NMR coordination shifts (∆δ = δcomplex − δligand; ppm) of the
selected atoms and 195Pt chemical shifts (δ; ppm) of the prepared complexes.
1H-NMR
13C-NMR
C5
15N-NMR
N6
195Pt
C2H N6H C8H
C2
C4
C6
C8
N1
N3
N7
N9
1
2
−0.09 0.82 0.84 1.35 −0.82 −4.07 −2.36 2.76 3.82 1.17 8.47 −105.03 5.60 −2077.10
0.18 0.52 0.84 1.41 −0.85 −4.03 −2.28 2.92 5.03 2.00 6.67 −105.02 6.17 −2072.08
0.17 0.60 0.88 1.37 −0.80 −4.01 −2.27 2.96 3.24 0.82 6.57 −106.00 4.96 −2074.81
0.19 0.51 0.92 1.27 −0.98 −4.14 −2.37 2.88 3.61 1.55 4.96 −106.69 5.45 −2071.38
0.12 0.69 0.72 1.61 −0.82 −3.90 −1.91 2.62 7.11 0.70 5.05 −105.70 5.09 −2076.80
0.18 0.59 0.84 1.31 −0.88 −4.07 −2.29 2.93 3.85 1.91 5.51 −105.78 6.21 −2074.68
0.15 0.75 0.69 1.49 −0.92 −3.97 −4.06 2.73 6.98 0.34 5.82 −106.22 4.78 −2078.30
0.19 0.52 0.90 1.31 −0.93 −4.13 −2.29 2.90 3.67 0.33 6.52 −105.37 6.32 −2071.68
0.19 0.55 0.83 1.34 −0.88 −4.07 −2.32 2.89 4.46 1.76 4.89 −106.10 5.86 −2073.15
3
4
5
6
7
8
9
10
11
12
13
−
−
−
−
0.26 0.83 −0.53 −1.19 −3.76 −0.66 3.03 3.13 −0.31 6.15 −106.52 5.02 −2084.94
0.60 0.83 1.30 −1.01 −3.68 −2.18 3.14 3.45 0.26 3.30 −106.24 5.00 −2083.47
0.81 0.69 1.34 −0.90 −3.66 −2.31 3.04 3.92 0.16 4.90 −105.72 5.03 −2088.28
0.85 0.78 0.12 −0.83 −3.27 −0.60 1.81 4.89
–
4.96 −105.76 5.33 −2091.62
2.2. In Vitro Cytotoxic Activity
The trans-complexes 1–13 were screened for their in vitro antitumor effect against two human
cancer cell lines, namely breast adenocarcinoma (MCF7) and osteosarcoma (HOS). We also tested
K2[PtCl4] and cisplatin to compare potential antitumor activity of the prepared complexes with the
precursors and with commercially used platinum-based drug (N6-benzyladenosine derivatives
involved in the structures of 1–13 were not tested, since their in vitro antitumor activity against HOS
and MCF7 was previously reported [12]). Unfortunately, although the complexes 1–13 were found to
be well soluble (>50.0 µM) in the medium used (0.1% DMF in distilled water), in other words they

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should be quite bioavailable, they did not show any cytotoxic effects up to the highest concentration
tested (50.0 µM concentration, IC50 > 50.0 µM). As for K2[PtCl4] it was found to be inactive up to 50.0
µM. The IC50 values determined for cisplatin equal 18.1 ± 5.1 µM (MCF7) and 25.4 ± 8.5 µM (HOS).
Although the results of in vitro antitumor activity of 1–13 against HOS and MCF7 do not seem
promising, it should be noted that situation regarding the free ligands Ln is completely different as
shown in a Czech patent [12], where it is demonstrated that some of the ligands L1–L12 (L13 was not
reported) are moderately to highly effective against HOS (IC50 = 2.8 µM for L5, 20.0 µM for L8, 21.2
µM for L1, >166.7 µM for L2–4,6,7,9–12), MCF7 (IC50 = 11.4 µM for L5, 14.0 µM for L8, 23.0 µM for
L12, 27.0 µM for L7, >166.7 µM for L1–4,6,9–11), and human T-lymphoblastic (CEM; IC50 = 3.2 µM for
L1, 14.5 µM for L3, 10.2 µM for L4, 0.7 µM for L5, 51.9 µM for L6, 0.3 µM for L7, 1.3 µM for L8,
47.8 µM for L10, 39.7 µM for L11, 0.2 µM for L12) and promyelocytic (HL-60; IC50 = 2.3 µM for L1,
1.6 µM for L3, 1.7 µM for L4, 0.4 µM for L5, 23.7 µM for L6, 0.2 µM for L7, 1.2 µM for L8, 15.9 µM
for L10, 9.5 µM for L11, 0.1 µM for L12) leukemic cell lines. As already mentioned in the Introduction,
one of the main reasons for the present study was to utilize these organic molecules as N-donor ligands
in platinum(II) complexes and find out whether the complex formation may influence the resulting
cytotoxic effect of the prepared complexes. Therefore, two human cancer cell lines (HOS and MCF7)
were chosen for a biological screening. Unfortunately, it has been found that incorporation of the
N6-benzyladenosine derivatives L1–L13 into trans-platinum(II) dichlorido complexes 1–13 did not
produce the expected goal. However, it is possible that the prepared complexes may show a selective
cytotoxicity against human cancer cell lines and thus, next time-consuming experiments on diverse
types of human cancer cell lines will be necessary to prove this presumption. Anyway, it is also known
that initially inactive platinum complexes may serve as pro-drugs [23,24] owing to their metabolic
activation, e.g. by interactions with diverse biomolecules such as reducing amino acids or proteins.
2.3. Interactions of the Selected Complexes 7 and 12 with L-Methionine, Evaluated by
Mass Spectrometry
In an effort to identify the actual active species which might be biologically relevant in terms of
long-lasting biological activity (due to anticipated slow kinetics of Ln-Pt(II)-S-Met interaction) and to
mechanistically describe the basic modes of interactions of two representative complexes 7 and 12
with relevant sulfur-containing biomolecule (L-methionine), the electrospray-ionization mass-spectrometry
(ESI-MS) experiments were carried out in both negative and positive ionization modes. To simulate at
least one aspect of the physiological conditions, the solution of L-methionine was used containing the
physiological levels (20 μM) of this significant sulfur-containing biomolecule [25].
The corresponding interaction systems were formed by mixing the two freshly prepared working
solutions: (solution 1) containing L-methionine (at the final concentration of 20 μM) and (solution 2)
containing the solutions of 7 or 12 in methanol, to give the final concentration of 10 μM (the final ratio
of H2O:MeOH was 1:1 v/v). The resulting interacting solutions were measured immediately after the
preparation and 12 h after preparation either. The interacting systems stayed clear during the whole
12 h of the observation.
The ESI-MS experiments revealed a relatively simple 1:1 exchange mechanism (one Ln molecule
was replaced by one L-methionine molecule) in the measured interacting systems (Figure 2). In the

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control samples, composed only by the solutions of the complexes 7, and 12, respectively, the
following main ions were identified in the negative ionization mode for 12 (see Figure 2a),
top spectrum; m/z, [the corresponding pseudomolecular ion]−, sorted by the decreasing relative
abundance]: 1175.19, [PtCl3(L12)2]−; 1140.04, [PtCl2(L12)2-H]−; 435.95, [L12-H]−; and in positive
ionization mode for 7 (see Figure 2b), top spectrum; m/z, [the corresponding pseudomolecular ion]+,
sorted by the decreasing relative abundance]: 404.28, [L7+H]+; 1111.03, [PtCl2(L7)2+K]+; 1073.01,
[PtCl2(L7)2+H]+; 1092.85, [PtCl2(L7)2+H+H2O]+. An analogous set of pseudomolecular ions, as
presented in Figure 2, was identified in the corresponding mass spectra of 7 measured in the negative
ionization mode and 12 measured in the positive ionization mode (Figure S1). No significant
qualitative differences, apart from the appearance of pseudomolecular ions (see Figure 2a), middle
spectrum; m/z, [the corresponding pseudomolecular ion]−]: 148.03 [(L-Met)-H]−; and Figure 2b,
middle spectrum; m/z, [the corresponding pseudomolecular ion]+): 150.02 [(L-Met)+H]+ corresponding
to the presence of L-methionine in the interacting systems, were observed in the measured mass spectra
just after mixing the solutions of 7 and 12 with L-methionine in both the negative and positive modes
of electrospray-ionization.
In accordance with the established slower kinetics of interactions between the dichlorido-
platinum(II) complexes involving the N-donor co-ligands and methionine [26], we identified the
significant changes in both qualitative and quantitative aspects of the mass spectra of the interacting
systems measured 12 h after the mixing of their principal components. The mass spectra measured in
both ionization modes (see Figures 2a,b), lower spectra, proved the formation of the analogous
coordination species—see Figure 2a, lower spectrum; m/z, [the corresponding pseudomolecular ion]−):
851.00 [PtCl2(L12)(L-Met)-H]−; and Figure 2b, lower spectrum; m/z, [the corresponding pseudomolecular
ion]+): 782.99 [PtCl(L7)(L-Met)]+], a (di)chlorido-platinum(II) complex containing one ligand of
adenosine derivative and one L-methionine ligand in its coordination sphere. The corresponding
cleavage of one adenosine ligand from 7, and 12, respectively, manifested itself quantitatively by the
increased ratio between the relative abundance of pseudomolecular ions of the free adenosine ligands
and the relative abundance of pseudomolecular ions of 7 or 12.
As some of the transplatin derived complexes involving the sulfur-containing ligands proved their
remarkable cytotoxic effects [27], the obtained results of interaction studies suggest that the slow
kinetics of the ligand exchange between the studied complexes and sulfur-containing biomolecules
might represent the essential step for their bio-activation, thus offering the explanation for the
insignificant cytotoxicity found by the standardized screening models and opening the new horizons
for them to be studied more deeply by means of advanced biological models, which would further
consider them as pro-drugs.

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Figure 2. (a) The ESI− mass spectra of 12 (top spectrum), and interacting systems with L-
6997
methionine measured immediately after the preparation (middle spectrum) and 12 h after
the preparation (lower spectrum); (b) the ESI+ mass spectra of 7 (top spectrum), and
interacting systems with L-methionine measured immediately after the preparation (middle
spectrum) and 12 h after the preparation (lower spectrum).

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3. Experimental
3.1. Materials
K2[PtCl4], 6-chloropurine-9-riboside, 2,6-dichloropurine-9-riboside, 2-methoxybenzylamine, 3-
methoxybenzylamine, 4-methoxybenzylamine, 2,3-dimethoxybenzylamine, 2,5-dimethoxybenzylamine,
2-chlorobenzylamine, 4-chlorobenzylamine, 4-fluorobenzylamine, 4-methylbenzylamine, triethylamine
and solvents used were purchased from Sigma-Aldrich Co. (Prague, Czech Republic), Acros Organics
Co. (Pardubice, Czech Republic), Lachema Co. (Brno, Czech Republic) and Fluka Co. (Prague, Czech
Republic) and used without further purification. 2-hydroxybenzylamine, 3-hydroxybenzylamine,
4-hydroxybenzylamine, 2-hydroxy-3-methoxybenzylamine and 2-hydroxy-5-methylbenzylamine were
obtained from the respective methoxy derivatives by the action of the concentrated HBr or HI acids in
the presence of acetylacetate.
3.2. Methods
Elemental analysis (C, H, N) was performed on a Flash 2000 CHNS Elemental Analyzer (Thermo
Scientific, Waltham, MA, USA). Conductivity measurements of 10–3 M DMF solutions of the
prepared complexes were carried out using a conductometer 340i/SET (WTW) at 25 °C. Simultaneous
thermogravimetry (TG) and differential thermal (DTA) analysis was carried out using an Exstar
TG/DTA 6200 thermal analyzer (Seiko Instruments Inc., Torrance, CA, USA); dynamic air
atmosphere (100 mL min–1), 25–900 °C (5.0 °C min–1). Infrared spectra (150–600 cm–1 region by nujol
technique and 400–4,000 cm–1 region by ATR technique) were recorded on a Nexus 670 FT-IR
(Thermo Nicolet, Waltham, MA, USA). 1H, 13C and 195Pt NMR spectra and 2D correlation experiments
1
1
1
1
(1H–1H gs-COSY, H–1H gs-ROESY, H–13C gs-HMQC, H–13C gs-HMBC and H–15N gs-HMBC;
gs = gradient selected, COSY = correlation spectroscopy, ROESY = rotating frame Overhauser effect
spectroscopy, HMQC = heteronuclear multiple quantum coherence and HMBC = heteronuclear
multiple bond coherence) of the DMF-d7 solutions were measured at 300 K on a Varian 400 device
(Varian, Santa Clara, CA, USA) at 400.00 MHz (1H), 100.58 MHz (13C), 40.53 MHz (15N) and 85.78
MHz (195Pt). H and 13C spectra were adjusted against the tetramethylsilane (Me4Si) signals. 195Pt spectra
were calibrated against K2[PtCl6] in D2O (0 ppm). H–15N gs-HMBC experiments were obtained at
1
1
natural abundance and calibrated against the residual signals of the DMF adjusted to 8.03 ppm (1H)
and 104.7 ppm (15N). Splitting of the proton resonances is defined as s = singlet, d = doublet,
t = triplet, sx = sextuplet, sep = septuplet, br = broad band, dd = doublet of doublets, tt = triplet of
triplets, m = multiplet. ESI− mass spectra (methanol solutions) were obtained by an LCQ Fleet Ion
Trap mass spectrometer (Thermo Scientific; QualBrowser software, version 2.0.7, Waltham, MA, USA).
3.3. Synthesis
N6-(2-methoxybenzyl)adenosine (L1), N6-(4-methoxybenzyl)adenosine (L2), N6-(2-chlorobenzyl)-
adenosine (L3), N6-(4-chlorobenzyl)adenosine (L4), N6-(2-hydroxybenzyl)adenosine (L5), N6-(3-
hydroxybenzyl)adenosine (L6), N6-(2-hydroxy-3-methoxybenzyl)adenosine (L7), N6-(4-fluoro-
benzyl)adenosine (L8), N6-(4-methylbenzyl)adenosine (L9), 2-chloro-N6-(3-hydroxybenzyl)adenosine

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(L10), 2-chloro-N6-(4-hydroxybenzyl)adenosine (L11), 2-chloro-N6-(2-hydroxy-3-methoxybenzyl)-
adenosine (L12) and 2-chloro-N6-(2-hydroxy-5-methylbenzyl)adenosine (L13) (see Scheme 1) were
synthesized according to the general procedure, which involves the interaction of appropriate purine-
based precursor with an excess of amine in an excess of triethylamine [28]. In our case, 1 mmol of
6-chloropurine-9-riboside (for N6-benzyladenosine derivatives L1–L9) or 2,6-dichloropurine-9-riboside
(for 2-chloro-N6-benzyladenosine derivatives L10–L13) reacted with 1.33 mmol of the appropriate
benzylamine derivative in n-butanol (20 mL) containing triethylamine (1.67 mmol). The reaction
mixture was stirred at 90 °C for 4 h and then it was left at -5 °C in the freezer overnight. The white
solid was collected by filtration and washed with cold n-butanol (2 × 5 mL), cold distilled water
(2 × 5 mL) and diethyl ether (2 × 5 mL). In case that not sufficient purity of the sample was achieved
(controlled by thin-layer chromatography and elemental analysis), the product was recrystallized from
ethanol. The results of elemental analyses, IR and NMR spectroscopy can be found in the
Supplementary Material.
The solution 1.0 mmol of L1–L13 in 50 mL of methanol was slowly poured in the solution of
0.5 mmol of K2[PtCl4] in a minimum volume of distilled water. The reaction mixture was stirred at room
temperature for several days, followed by filtration of the obtained precipitate. The products of the
composition trans-[PtCl2(L1)2]·½CH3OH (1), trans-[PtCl2(L2)2]·CH3OH (2), trans-[PtCl2(L3)2]·½H2O
(3), trans-[PtCl2(L4)2]·CH3OH (4), trans-[PtCl2(L5)2]·½CH3OH (5), trans-[PtCl2(L6)2]·CH3OH (6),
trans-[PtCl2(L7)2]·CH3OH (7), trans-[PtCl2(L8)2]·½CH3OH (8), trans-[PtCl2(L9)2]·½CH3OH (9),
trans-[PtCl2(L10)2]·¾CH3OH (10), trans-[PtCl2(L11)2]·CH3OH (11), trans-[PtCl2(L12)2]·CH3OH (12)
and trans-[PtCl2(L13)2]·¾CH3OH (13), generally expressed as trans-[PtCl2(Ln)2]·xSolv 1–13
(Solv = H2O or CH3OH), were washed by distilled water, methanol and diethyl ether and dried in
desiccator over KOH. The physical measurement results are given in the Supplementary Material.
3.4. In Vitro Cytotoxic Activity Testing
In vitro cytotoxicity was studied by an MTT assay [MTT = 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide] against osteosarcoma (HOS; ECACC No. 87070202) and breast
adenocarcinoma (MCF7; ECACC No. 86012803) human cancer cell lines supplied from European
Collection of Cell Cultures (ECACC). The cell lines were cultured according to the ECACC
instructions, maintained at 37 °C and 5% CO2 in a humidified incubator (100% humidity). The studied
complexes 1–13, K2[PtCl4], and cisplatin (0.01, 0.1, 1.0, 5.0, 25.0 and 50.0 μM concentration), as well
as vehicle (DMF, 0.1%, v/v; positive control to assess the minimal cell damage, i.e., 100% viability)
and Triton X-100 (1%, v/v; negative control to assess the maximal cell damage, i.e., 0% viability) were
added to the cell suspensions in 96-well culture plates and incubated for 24 h. The MTT assay was
measured spectrophotometrically at 540 nm (TECAN, Schoeller Instruments LLC, Prague, Czech
Republic). The data from the respective cancer cells were acquired from three independent
experiments (conducted in triplicate) using cells from different passages. The resulting IC50 values
(µM) were calculated from viability curves and the results are presented as arithmetic mean ± SD.

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3.5. Interactions of the Selected Complexes 7 and 12 with L-Methionine, Evaluated by
Mass Spectrometry
The electrospray-ionization mass spectrometry (ESI-MS) is widely used method for studies of
covalent [29] or non-covalent interactions ([30,31] and references therein) of small molecules or ions
with biologically relevant molecules. Therefore, we performed the interaction experiments between the
two complexes (7 and 12), one selected from the group of 2-chloro-substituted derivatives and the
second one selected from the 2-unsubstituted group of compounds (dissolved in methanol, at the final
concentration of 10 μM) with the physiological concentration of L-methionine (dissolved in water, at
the final concentration of 20 μM), using an LCQ Fleet Ion Trap mass spectrometer, in both the
negative and positive ionization modes. The measured solutions were infused directly to the
spray-needle by linear pump with the rate of 50 μL/min, and the measured range of ions was set from
m/z 50 to 1500. No additional tuning was needed to perform the analyses.
4. Conclusions
We prepared and thoroughly characterized thirteen platinum(II) dichlorido complexes of the general
composition trans-[PtCl2(Ln)2]·xSolv 1–13 (Solv = water or methanol), which represent the first
examples of the platinum complexes with N6-benzyladenosine derivatives acting as N-donor ligands.
13
The composition and constitution of the complexes were proved by the multinuclear (1H-, C-, 195Pt-
15
and N-) and two-dimensional NMR spectroscopy. The screening of antitumor activity showed that
the complexes 1–13 are inactive against the MCF7 and HOS human cancer cell lines (IC50 > 50.0 µM).
The selected complexes 7 and 12 showed their ability to interact with the physiological levels of
sulfur-containing biogenic biomolecule (L-methionine) by a relatively simple 1:1 exchange mechanism
(one Ln molecule was replaced by one L-methionine molecule), thus forming a mixed-nitrogen/sulfur-
ligand dichlorido-platinum(II) coordination species, which could be responsible for bio-activation of
complexes during the longer time-period. From this point of view, the results of interaction studies
support an idea that the studied complexes might be considered as possible pro-drugs. However,
to validate such an assumption, the more sophisticated and advanced mechanistic studies have to
be undertaken.
Supplementary Materials
Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/18/6/6990/s1.
Acknowledgments
The authors gratefully thank the Czech Science Foundation (GAČR P207/11/0841), Operational
Program Research and Development for Innovations - European Regional Development Fund
(CZ.1.05/2.1.00/03.0058), Operational Program Education for Competitiveness - European Social
Fund (CZ.1.07/2.3.00/20.0017) of the Ministry of Education, Youth and Sports of the Czech Republic
and Palacký University in Olomouc (PrF_2013_015).

Molecules 2013, 18
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Conflict of Interest
The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 1–13 are available from the authors.
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distributed under the terms and conditions of the Creative Commons Attribution license
(http://creativecommons.org/licenses/by/3.0/).