Pt(II) complexes with (N,N′) or (C,N,E)- (E = N,S) ligands: Cytotoxic studies, effect on DNA tertiary structure and structure-activity relationships

Article abstract of DOI:10.1016/j.bmc.2013.05.005

The cytotoxic activity of two series of platinum(II) complexes containing the polyfunctional imines R¹-CHN-R² [R¹ = phenyl or ferrocenyl unit and R² = (CH₂) _n-CH₂-NMe₂ where n = 1 or 2) (1 and 2) or C₆H₄-2-SMe (3)] acting as a bidentate (N,N′) (4-7) or terdentate [C(phenyl or ferrocenyl),N,N′]^- (8-10) or [C(ferrocenyl),N,S]^- ligand (11) in front of A549 lung, MDA-MB231 breast and HCT116 colon human adenocarcinoma cell lines is reported. The results reveal that most of the platinum(II) complexes are active against the three assayed lines and compounds 6, 7 and the platinacycles 10 and 11 exhibit a remarkable antiproliferative activity, even greater than cisplatin itself, in the cisplatin resistant HCT116 human cancer cell line. Electrophoretic DNA migration studies showed that most of them modify the DNA tertiary structure in a similar way as the reference cisplatin. Solution studies of a selection of the most relevant complexes have also been performed in order to test: (a) their stability in the aqueous biological medium and/or the formation of biologically active species and (b) their proclivity to react with 9-methylguanine (9-MeG), as a model nucleobase. Computational studies at DFT level have also been performed in order to explain the different solution behaviour of the complexes and their proclivity to react with the nucleobase.

Full text of DOI:10.1016/j.bmc.2013.05.005

Bioorganic & Medicinal Chemistry 21 (2013) 4210–4217
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Bioorganic & Medicinal Chemistry
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Pt(II) complexes with (N,N0) or (C,N,E)ꢀ (E = N,S) ligands: Cytotoxic
studies, effect on DNA tertiary structure and structure–activity
relationships
Joan Albert a,b, Ramon Bosque a,b, Margarita Crespo a,b, Jaume Granell a,b, Concepción López a,
,
⇑
Roldán Cortés b,c, Asensio Gonzalez b,d, Josefina Quirante b,d, , Carme Calvis e, Ramon Messeguer e,
⇑
Laura Baldomà b,f, Josefa Badia b,f, Marta Cascante b,c
a Departament de Química Inorgànica, Facultat de Química, Universitat de Barcelona, Diagonal 645, E-08028 Barcelona, Spain
b Institut de Biomedicina (IBUB), Universitat de Barcelona, 08028 Barcelona, Spain
c Department of Biochemistry and Molecular Biology, Faculty of Biology, IDIBAPS, Unit Associated with CSIC, Diagonal 643, E-08028 Barcelona, Spain
d Laboratori de Química Orgànica, Facultat de Farmàcia, Universitat de Barcelona, Av. Joan XXIII, s/n, E-08028 Barcelona, Spain
e Biomed Division LEITAT Tecnological Center, Parc Científic de Barcelona, Edifici Hèlix, Baldiri i Reixach, 15-21, E-08028 Barcelona, Spain
f Departament de Bioquímica i Biologia Molecular, Facultat de Farmàcia, Universitat de Barcelona, Av. Joan XXIII, s/n, E-08028 Barcelona, Spain
a r t i c l e i n f o
a b s t r a c t
The cytotoxic activity of two series of platinum(II) complexes containing the polyfunctional imines R1–
CH@N–R2 [R1 = phenyl or ferrocenyl unit and R2 = (CH2)n–CH2–NMe2 where n = 1 or 2) (1 and 2) or
C6H4-2-SMe (3)] acting as a bidentate (N,N0) (4–7) or terdentate [C(phenyl or ferrocenyl),N,N0]ꢀ (8–10)
or [C(ferrocenyl),N,S]ꢀ ligand (11) in front of A549 lung, MDA-MB231 breast and HCT116 colon human
adenocarcinoma cell lines is reported. The results reveal that most of the platinum(II) complexes are
active against the three assayed lines and compounds 6, 7 and the platinacycles 10 and 11 exhibit a
remarkable antiproliferative activity, even greater than cisplatin itself, in the cisplatin resistant HCT116
human cancer cell line. Electrophoretic DNA migration studies showed that most of them modify the
DNA tertiary structure in a similar way as the reference cisplatin. Solution studies of a selection of the
most relevant complexes have also been performed in order to test: (a) their stability in the aqueous bio-
logical medium and/or the formation of biologically active species and (b) their proclivity to react with 9-
methylguanine (9-MeG), as a model nucleobase. Computational studies at DFT level have also been per-
formed in order to explain the different solution behaviour of the complexes and their proclivity to react
with the nucleobase.
Article history:
Received 1 March 2013
Revised 26 April 2013
Accepted 3 May 2013
Available online 14 May 2013
Keywords:
Platinum(II)
Cycloplatinated complexes
Cytotoxicity
Anticancer drugs
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
severe dose-limiting toxicity, such as neuro-, hepato-, and nephro-
toxicity6–9 and/or (b) inherent or acquired resistance.10
The potential of metal-based anticancer agents has been widely
explored and recognised1–3 since the landmark discovery of the
biological activity of cisplatin.4 To date this prototypical anticancer
drug remains one of the most effective chemotherapeutic agents in
clinical use.5 However, its utility against cancer is limited by (a)
A better understanding of the cellular response to cisplatin and
how tumours are or become resistant,11–14 will contribute to the
design of novel platinum compounds15 with improving anticancer
properties. Cisplatin enters cells by passive diffusion16 and also, as
discovered later, by active transport mediated by the copper trans-
porter Ctr1p in yeast and mammals.17,18 Once inside the cell, the
low chloride concentration (4–20 mM) results in drug aquation
with the loss of one or both of the chlorido ligands. The resulting
aquo-platinum complex binds to its target, DNA, by the formation
of covalent cross-links; being the 1,2-intrastrand d(GpG) cross-link
the major adduct. Binding of cisplatin to DNA causes significant dis-
tortion of the helical structure and results in inhibition of DNA
replication and transcription,16,19 which ultimately leads to cell cy-
cle arrest and cellular apoptosis. The distorted, platinated DNA
Abbreviations: DMEM, Dulbecco’s modified eagle medium; MTT, 3-(4,5-dimeth-
ylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; G, guanine; 9-MeG, 9-methyl-
guanine; 9-AA, 9-aminoacridine; ELISA, enzyme-linked immunosorbent assay; DFT,
density functional theory; B3LYP, Becke 3-parameter (exchange)—Lee, Yang and
Parr (correlation) functional; LANL2DZ, Los Alamos National Laboratory 2 double
zeta basis set; C-PCM, conductor polarized continuum model.
⇑
Corresponding authors. Tel.: +34 934039134; fax: +34 934907725 (C.L.), tel.:
+34 934035849; fax: +34 934024539 (J.Q.).
E-mail addresses: conchi.lopez@qi.ub.es (C. López), quirantese@ub.edu (J.
Quirante).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.05.005

J. Albert et al. / Bioorg. Med. Chem. 21 (2013) 4210–4217
4211
Figure 1. Chemical formulae of the free ligands (1–3) and the platinum(II) complexes (4–11) selected for this study.
Table 1
Cytotoxic activities (IC50 valuesa) on A549 lung, MDA-MB231 breast human and HCT116 colon cancer cell lines for the free ligands (1a–1c and 3)b the platinum complexes (4–11)
and cisplatin
Type of product
Mode of binding of the ligand
IC50(lM) for the cell lines:
A549
MDA-MB231
HCT116
Ligandb
1a
1b
1c
3
—
—
—
—
>100
>100
>100
173 25
>100
>100
>100
nd
>100
>100
>100
44 nd
Platinum(II) complexes with bidentate ligands
4
5
6a
6b
7
(N,N0)
(N,N0)
(N,N0)
(N,N0)
(N,N0)
26 12
>100
47 nd
57 nd
14.1 2.4
10.7 4.6
28 0.3
35 2.2
55 6.8
11.9 9 2
10.6 1.7
30 4.2
95
7
>200
178 32
Platinum(II) complexes with terdentate ligands
8a
8b
8c
9
10a
10b
11
[C(phenyl), N,N0]ꢀ
95
7
10.7 4.6
42.2 9.5
30 10.2
87 nd
28 2.4
17 1.1
32 6.2
6.5 2.4
60 1.7
>100
[C(phenyl), N,N0]ꢀ
[C(phenyl), N,N0]ꢀ
[C(ferrocenyl), N,ꢀN0]ꢀ
[C(phenyl), N,N0]ꢀ
[C(phenyl), N,N0]
[C(ferrocenyl), N,S0]ꢀ
(N)
38 18
27.0 10
75 14
40 14
23
23
44 8.5
45 7.8
28 6.7
18 1.9
20.7 3.8
40 4.4
4
8
Cisplatin
9.3 3.0
a
Data are shown as the mean values of two or more experiments performed in triplicate with the corresponding standard deviation (SD).
Ref. 32.
b
structure also serves as a recognition binding site for cellular pro-
teins,11,12 such transcription factors, histones, high-mobility group
(HMG)-domain proteins, and nucleotide excision repair (NER) pro-
teins, which may modulate the cisplatin effectiveness and/or
resistance.
Taking these findings into account, one of the strategies for
developing new anticancer agents include the design of platinum
complexes that bind to DNA in a fundamentally different manner
than cisplatin. One of them consists in the synthesis of new organic
compounds with one or more N atoms with donor abilities (i.e.,
amines, oximes, thiosemicarbazones, functionalized azoles, etc.)
and their use as ligands for Pt(II).20–23 Despite the great number
of studies on this field and the variety of complexes reported in re-
cent years, to the best of our knowledge, none of these studies
deals with sets of platinum(II) complexes derived from closely
related ligands, exhibiting different coordination modes. Struc-
ture–activity relationship (SAR) upon this sets of platinum(II) com-
pounds may provide interesting information of how their cytotoxic
activity could be influenced by several structural factors such as (a)
the donor atoms of the ligand, (b) its mode of binding [i.e., (N),

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J. Albert et al. / Bioorg. Med. Chem. 21 (2013) 4210–4217
(N,E), (C,N,E)ꢀ, E = additional heteroatom], (c) the chelate size, (d)
the presence of substituents or even (e) the nature of additional
ancillary ligands.
During the last decade, we have prepared different families of
polyfunctional imines (1–3, Fig. 1)24–28 and used them as ligands
to build up a wide variety of platinum(II) complexes (4–11),24,26–
31
in which the Schiff bases behave as neutral and bidentate
(N,N0) ligands (in comꢀpounds 4–7 of Fig. 1) or as monoanionic
and terdentate (C,N,N0) (in 8–10) or (C,N,S)ꢀ (in 11) ligands.
Now we report comparative studies of the cytotoxic activity
of the platinum(II) complexes (4–11) in front of A549 lung,
MDA-MB231 breast and HCT116 colon (cisplatin resistant) human
adenocarcinoma cell lines, together with the investigation of
their effect on the electrophoretic mobility of DNA. The results
presented here provide conclusive evidences of the influence of
the type of ligands and their binding mode on their biological
effectiveness.
2. Results and discussion
2.1. Biological studies
2.1.1. Antiproliferative assay
Human lung, breast and colon cancer cell lines (A549, MDA-
MB231 and HCT116, respectively) were used to test the cytotoxic
activity of the Pt(II) complexes with (N,N0) ligands (4–7) and the
platinacycles (8–11). For comparison purposes cisplatin (as positive
control) and the free ligands 1–3 were evaluated under the same
experimental conditions.32
Data presented in Table 1 shows that the investigated com-
pounds exhibit variable selectivity in front of the adenocarcinoma
cell lines tested (Table 1, Fig. 2). Several complexes showed greater
cytotoxicity effectiveness than that of cisplatin versus the platinum
resistant HCT116 cancer cell line selected, namely compounds 4,
6a, 6b, 7, 10a, 10b, and 11 (Fig. 3).
Figure 3. Comparison of the IC50 (lM) values obtained for the platinum(II)
complexes with bidentate (N,N0) ligands (3–7), the cyclometallated compounds
(8–11) and cisplatin in front of the HCT-116 cell line.
similar variable effect, depending of the assayed cancer cell line,
has also been observed for Pt(II) complexes with 2,3-diaminopro-
panoate or 2,4-diaminobutiroate ligands towards A431, Hela and
HL-60 cell lines.33 Furthermore the ferrocene derivative 7 (six
membered) exhibited greater cytotoxic effectiveness than the cor-
responding five membered ferrocene derivative 5 in the three can-
cer cells lines assayed. No increase in potency is observed when the
ferrocenyl compounds 5 and 7 are compared with the phenyl com-
plexes 4 and 6a in pairs: 5–4 and 7–6a.
Considering the cyclometallated complexes 8 (8a–8b) having a
tricyclic [6.5.5] system, the non-substituted compound 8a exhibits
a slight greater cytotoxic activity than the 3,5-dichlorido derivative
8b in A549 lung and HCT116 colon cancer cell lines; and the oppo-
site situation is given for MDA-MB231 breast cancer cell line. Sur-
prisingly, the platinacycle 8c, having a methyl ligand instead of the
Clꢀ ligand showed lower IC50 values in the three human cancer cell
lines than the corresponding monofuntional complex 8a.
Finally, the cyclometallated complexes 10 (10a and 10b) and 11
having a tricyclic [6.5.6] and tetracyclic [5.5.5.6] system, respec-
tively, showed a remarkable cytotoxic effectiveness, although not
so high as the seven-membered platinacycles previously investi-
gated in our research group.34,35 In cisplatin resistant HCT116 colon
adenocarcinoma cell line, platinacycle 10b exhibited a low IC50 va-
lue, being approximately 2.2-fold more potent than cisplatin. Con-
sidering the two cycloplatinated ferrocene derivatives 9 and 11, it
is noteworthy that complex 11, with Pt–S bond exhibits lower IC50
than compound 9 with a Pt–N(amine) bond.
Regarding the chelated (N,N0) Pt(II) complexes (4–7), it has been
reported that the five membered 1,2-ethanediamine complexes
showed greater antiproliferative activity than the six membered
1,3-propanediamine complexes in front of L1210 cell line.20,33 In
our study, the chelated five-membered complex 4 exhibited great-
er potency than compound 6a (six-membered chelate ring) in
A549 lung cancer cell line. However, for the other cancer cell lines
tested (MDA-MB231 breast and HCT116 colon) complex 4 showed
higher IC50 values than that of complex 6a. In cisplatin resistant
HCT116 colon adenocarcinoma cell line, the six-membered com-
plexes 6a and 6b exhibited a cytotoxic effectiveness approximately
fourfold greater than that of cisplatin. It should be mentioned that a
2.1.2. DNA migration studies
The effect of binding of the compounds investigated in this
study on supercoiled DNA was determined by their ability to alter
the electrophoretic mobility of pBluescript plasmid DNA: super-
coiled closed circular (ccc) and open circular (oc) forms. The ccc
form usually moves faster due to its compact structure, so it
showed almost one band in gel. When the test compounds (1–
11) were incubated with plasmid DNA at 37 °C, most of them could
coordinate to the DNA molecule, which in some extend was
Figure 2. Inhibition of cell growth proliferation in HCT116 colon cancer cells line,
after 72 h of exposure to the compounds 6 (6a–6b), 10 (10a–10b), 11 and cisplatin.

J. Albert et al. / Bioorg. Med. Chem. 21 (2013) 4210–4217
4213
cleaved into fragments, and the brightness of band was diminished
in gel.
positive supercoiled DNA was displayed in the electrophoretogram
(Fig. 4B). The same effect was observed for cisplatin36 (Fig. 4D, lines
5 and 6). For the ferrocene complex 7 a slight decrease in plasmid
DNA mobility was observed in a dose dependent manner.
Platinacycles 8 (8a–8c) greatly alter the mobility of plasmid
DNA. For these three compounds (Fig. 4C) the coalescence point,
Figure 4 shows the electrophoretic mobility of native pBlue-
script DNA (40
(1–11) ranging from 2.5 to 200
l
g/mL) incubated with the selected compounds
M concentrations. To provide a
l
basis for comparison, incubation of DNA with cisplatin and 9-AA
was also performed using the same concentrations and conditions.
obtained in the titration assay occurs at 10
lM concentration of
As expected, at the lowest concentration (2.5
lM) assayed, cisplatin
8a and 8b, and at 50 M concentration of 8c. The lower efficiency
l
greatly altered the electrophoretic mobility of pBluescript DNA.
For the free ligands 1 (1a–1c) an increase in the presence of
nicked relaxed forms without shifting the plasmid DNA mobility
of supecoiled form was observed when increasing the concentra-
tion of the compounds. For the ferrocenyl ligand 3 almost no de-
crease in mobility was observed at any assayed concentration
(Fig. 4A).
The chelated complexes having five (4 and 5) and six (6a and
6b) membered rings greatly alter the electrophoretic mobility of
plasmid DNA (Fig. 4B). When increasing the concentration of the
platinum complex, the migration rate of the supercoiled band de-
creases until it comigrates with the nicked relaxed band. In these
titration experiments, the coalescence point (defined as the
amount of platinum complex that is necessary for complete re-
of complex 8c than that of 8a in removing the supercoils from DNA
could be related to the nature of the ancillary ligand {Cl (in 8a) or
Me (in 8c)}. It is well known that Pt–Cl bonds are more labile than
Pt–C bonds, thus metallacycle 8a is expected to be more reactive
than the methyl derivative 8c. At high concentration of complexes
8a–8b (lanes 5, 6 and 7) and complex 8c (lanes 7, 8 and 9), the
strong unwinding of negative supercoiled DNA to positive super-
coiled DNA was also observed. Regarding the ferrocene platinacy-
cle 9 a weaker effect on the migration rate of supercoiled closed
circular plasmid DNA in a dose dependent manner was displayed.
Paradoxically, compound 9 (ferrocenylimine) exhibits a similar
structure as complex 8a (benzylimine), which is able to produce
a great unwinding effect on DNA.
The Pt(II) complexes 10a and 10b greatly alter the electropho-
retic mobility of plasmid DNA (Fig. 2D). In the unwinding
experiment the coalescence point for complexes 10a and 10b takes
moval of all supercoils from DNA) occurs at 75
lM concentration
of 4 and 5 and at 10 M concentration of 6a and 6b. At high con-
l
centration of complexes 4 and 5 (lanes 8 and 9) and 6 (6a–6b, lanes
place with 5 and 25
lM, respectively. Interestingly these two
5, 6 and 7), a strong unwinding of negative supercoiled DNA to
compounds are even more potent than cisplatin in HCT116 colon
(A)
3
6
1a
5
1b
4 5
1c
5
1
8
9
1
3
4
6 8 9
1
3
3
2
2
7
8 9
1
3 4
6
7
8
oc
ccc
(B)
4
5 7
5
5
6a
4
6b
4
7
7
1
3
4
8
9
1
4
3
3
7
8 9
1
2
3
5
6 7
1
2
3
5
6
7
1 6
8 9
oc
ccc
8a
8b
4
8c
6
9
4
(C)
(D)
1
2
3
4
5
6
7
1
5
6
7
1
4
5
7
8 9
1
3
5
6
8 9
oc
ccc
10a
10b
4
11
cisplatin
9-AA
6
1
2
3
5
1
8
9
1
5 6 7
1
2
3
4
5
6
7
1
6
8 9
oc
ccc
Figure 4. Interaction of pBluescript SK+ plasmid DNA (40
ligands (4–7), and (C–D) 5-membered metallacycles (8–11) and cisplatin and 9-AA used for comparison purposes. Lanes 1, DNA only; lanes 2, 2.5
compound; lanes 4, 10 M compound; lanes 5, 25 M compound; and lane 6, 50 M compound; lanes 7, 75 M compound; lanes 8, 100 M compound; and lane 9, 200
compound. [Abbreviations: ccc = supercoiled closed circular DNA form and oc = open circular DNA form].
l
g/ml) with increasing concentrations of: (A), free ligands (1–3), (B) their platinum(II) derivatives with (N,N0)
l
M compound; lanes 3, 5
lM
l
l
l
l
l
lM

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J. Albert et al. / Bioorg. Med. Chem. 21 (2013) 4210–4217
cancer cell line (cisplatin resistant). With regard to the ferrocenyl-
containing platinacycle 11, although it exhibits a strong antiprolif-
erative activity versus HCT116 adenocarcinoma cell line, no
changes on the plasmid DNA migration rate were observed in the
electrophoretogram. Compound 11, with a polycyclic [5.5.5.6] sys-
tem, is hypothesized to behave as 9-AA, which is used as intercala-
tor reference in the experiment, or through another target/
mechanism.
In general, the five-membered platinacycles under study exhibit
a greater effect on DNA migration than that of the non-planar se-
ven-membered platinacycle previously investigated in our
group.34,35 In recent studies centered on five-membered cyclomet-
allated complexes, the metallacycle showed a planar five-mem-
bered ring structure37–44 and hence it was hypothesized that
they may act by intercalating into DNA.41–44 However, the five-
membered platinacycles 8 (8a–8c) and 10 (10a–10b) investigated
in this study greatly alter the superhelicity of the DNA molecules in
a similar way as the model cisplatin.
2.2. Solution studies
Scheme 1. Sequences of reactions chosen in the computational studies undertaken
to the evaluate the aquation processes and the subsequent formation of the Pt(II)–
Given that the assayed compounds were insoluble in water,
their stabilities in the aqueous biological media were evaluated,
recording the 1H NMR spectra of the solubilized compounds (6a,
8a, 8c, and 11) in a mixture of DMSO-d6 and D2O (1:1). Then, the
1H NMR spectra of the freshly prepared samples was compared
with those obtained after different storage periods (Figs. S1–S4).
As shown in Figures S2 and S3 the solution behavior of 8a and
8c with identical terdentate ligands were markedly different.
While 8c was stable in aqueous solution for 48 h, for 8a, a new
compound was formed upon addition of D2O. An analogous behav-
ior to that of 8a in D2O was observed for the coordination com-
pound 6a, while for complex 11 no significant changes in the 1H
NMR spectra were detected after four days.
guanine complexes {the mono-(III) or V) or bis (VI) aduct. The calculated
DG value
(in kcal/mol) of each one of the reactions, (in H2O at 298 K) is presented next or
close to the arrows and the values in itallics correspond to complexes with n = 2.
Figure 5. Guanine–platinum(II) monoadducts (VII) used in the computational
studies.
In a further step we also studied the effect produced by the
presence of equimolar amounts of 9-methylguanine on the com-
plexes solutions (Figs. S5–S7). In contrast with the results obtained
above, in the presence of the nucleobase, complex 11 degradated
(Fig. S7); whereas for compound 10a (Fig. S6) the spectral changes
detected were consistent with the coordination of 9-methylgua-
nine to platinum. For 8c, the quality of the 1H NMR spectra de-
creased with time, but new signals were detected (in the range
7.7 < d < 8.4 ppm) after 24 h of storage (Fig. S5-C).
2.3. Theoretical interpretation
It is well-known that the mechanism of action of cisplatin in-
volves aquation prior to the binding to DNA12–14 and we have
proved that the length of the aliphatic chain (in 8a and 10a) and
the nature of the ancillary ligand {Clꢀ (in 8a) or Me (in 8c)} affects
their cytotoxic activity, their effect on the electrophoretic mobility
of DNA and their solution behavior. In view of this, and in order to
compare the effect produced by the size of the chelate (in 8a and
10a) in the aquation process and the subsequent formation of
the mono- or bis (adducts) with a nucleobase such as guanine,
we undertook computational studies to determine the variations
Scheme 2. Sequences of reactions choosen for the computational studies for the
evaluation of the aquation processes of the cycloplatinated complex 8c. Transfor-
mation of the species formed (I and II0) into the mono- or bis-Pt(II)-guanine adducts
would proceed as shown in Scheme 1, {from I: steps; c or (c and h); from II or II0: e
and f to the monoadducts aducts II, and V, or step g for the bis(guanine) derivative}.
The calculated
DG for these processes (in kcal/mol) are presented next to the arrow.
of the free energy (DG) of the process involving the incorporation
of a H2O molecule in the coordination sphere of the Pt(II), by either
a simple ligand exchange reaction (Scheme 1, a) or the cleavage of
the Pt–N(amine) bond and the coordination of the H2O molecule
(Scheme 1, b) to give the neutral species (II).
In a first stage the geometries of the complexes were optimized,
afterwards the free energy and solvation corrections were calcu-
and 8.5 kcal/mol (for 10a). These values indicate that from a ther-
modynamic point of view the aquation is expected to proceed
through path b. In addition, for 8a the
DG (for the formation of type
II complex) is (ca. 2.7 kcal/mol) greater than for 10a; thus suggest-
ing that 10a is more prone to give the aquo-complex II than 8a.
It should be noted that for complex II with n = 2 the
DG value
lated and finally we determined the
one of the reactions under study. Data presented in Scheme 1,
show that differences G (path a)— G (path b) are 5.4 (for 8a)
D
G values in water of each
for the aquation of the Pt–Cl bond and the formation of the mono-
adduct with guanine (V) (steps e and f), is greater than for its ana-
logue with n = 1. These differences could be attributed to the
D
D

J. Albert et al. / Bioorg. Med. Chem. 21 (2013) 4210–4217
4215
presence of a larger and more flexible pendant arm. Furthermore,
the computational studies also showed that the reactions of 8a
or 10a with the guanine to give the monoadducts VII, shown in Fig-
ure 5, are even less favored than steps a and b of Scheme 1.
In order to clarify the role of the ancillary ligand {Cl (in 8a) or
Me (in 8c)}, a parallel study on the aquation process of 8c was also
performed. As shown in Scheme 2, (steps a and b), the formation of
I (in a) is less likely to occur than that of II0 (by a ring opening pro-
characterized by NMR spectroscopy and X-ray crystallography (3–
4 and 7, 8a, 9–11). The synthesis of the ligands 1–3 was accom-
plished by condensation reaction of equimolar amounts of the
appropriate aldehyde and the corresponding amine in refluxing
benzene. The synthesis was usually carried out using a Dean–Stark
apparatus to remove the benzene–water azeotrope formed in the
course of the reaction. Ligand 1a24 and 1b–1c25 were synthesized
by reaction between the corresponding benzaldehide and the dia-
mine Me2N(CH2)2NH2 or Me2N(CH2)3NH2. Ligands 2 (2a–2b)26,28
were prepared by reaction between ferrocenecarbaldehyde and
the appropriate diamine. Finally ligand 327 was obtained by reac-
tion of ferrocenecarbaldehyde with 2-mercaptoaniline. Complex
4,29 5,26 6 (6a–6b)30 and 728 were prepared by reaction of the cor-
responding ligand with cis[PtCl2(DMSO)2] in MeOH at reflux tem-
perature. Complexes 8a,29 9,26 10 (10a–10b),30 by reaction of the
corresponding ligand with cis[PtCl2(DMSO)2]/NaOAc in the same
reaction conditions. Compound 8c,24 by reaction of ligand 1a with
cess and coordination of water in step b). The
DG for the following
step c is more than four times higher than for the chlorido-com-
plexes (8a). A similar result was obtained for the formation of
the mono- or bis(guanine) adducts. These findings suggest that in
II0 the ancillary Me ligand inhibits the binding of the nucleobase.
It should be noted that for the direct conversion of 8c into the
guanine monoadduct (VIII in Scheme 2, d)
DG (13 kcal/mol) is
smaller than that of the transformation of II0 into the aquo-guanine
derivative V—shown in Scheme 1—(44.7 kcal/mol).
[Pt2Me4(l
-SMe2)2] in acetone at reflux temperature. Complex 8b31
by reaction of a methyl precursor with CH3COCl in CH2Cl2/MeOH at
room temperature. Finally complex 11,27 by reaction of ligand 3
with cis-[PtCl2(PhCN)2] in toluene at reflux temperature.
3. Conclusions
The evaluation of the in vitro cytotoxic activity of five Pt(II)
complexes with bidentate (N,N0) ligands (4–7) and eight 5-mem-
bered platinacycles with (C,N,E)ꢀ pincer ligands (8–11) revealed
that all of them exhibit growth inhibitory activity against lung
(A549), colon (HCT116) and breast (MDA-MB231 and MCF7) hu-
man cancer cell lines. For SAR analysis some preliminary conclu-
sions could be highlighted when comparing the framework and
the substitution pattern of the tested compounds: (1) the investi-
gated compounds showed a variable selectivity towards the se-
lected human adenocarcinoma cell lines assayed. For instance the
chelated 1,3-propanediamine Pt(II) complexes 6 (6a–6b) exhibited
four-times greater antiproliferative activity than that of cisplatin in
HCT116 colon (cisplatin resistant) cancer cell line and no activity in
A549 lung cancer cell line; (2) the [6.5.6] fused polycylic com-
plexes 10 (10a–10b) and the [5.5.5.6] fused polycyclic complex
11 showed greater cytotoxic effectiveness than the corresponding
[6.5.5] fused polycylic complexes 8 and [5.5.5] fused polycylic
complex 9; in particular complex 10b inhibits cell growth prolifer-
ation at the level two times higher to that of cisplatin in HTC116
(cisplatin resistant) colon cancer cell line; (3) the substitution of a
phenyl by a ferrocenyl group in compounds 4, 6a and 8a to give
complexes 5, 7 and 9, respectively did not show any increase in
cytotoxicity; (4) the presence of a non-labile methyl ligand in 8c,
instead of the labile chlorido-ligand in 8a interestingly give rise
to an increase in potency.
Most of the complexes evaluated in this study show an effect on
DNA electrophoretic mobility. Complexes 6, 8 and 10 are those
exhibiting the strongest interaction with DNA and display moder-
ate to good cytotoxic activities towards the three cancer cell lines
assayed. Solution studies and DFT calculations suggest that the
replacement of the Me in (8c) by a Cl (in 8a) is important as to
modify the mode of action of the two products. Further studies
on these areas are in progress centered on both (1) the mechanistic
elucidation (cell cycle arrest, induction of apoptosis, etc) of the
investigated complex, and (2) the development of more potent
complexꢀcontaining bidentate [(N,N0) or (N,S)], and terdentate
[(C,N,N0) or (C,N,S)ꢀ] donor ligands based on SAR analysis.
4.1.2. Study of the stability of the platinum(II) complexes in
solution
These studies were performed using the following methodol-
ogy: the corresponding complex [5.0 mg (of 6a), 4.4 mg (of 8a),
4.6 mg (of 8c), or 5 mg (of 11)] was introduced in a NMR tube, then
0.7 mL of a DMSO-d6/D2O (1:1) mixture were added, the tubes
were sealed and the mixtures were shaken at 298 K to complete
homogenization. The stability of the compounds in this solvent
was studied by comparison of the 1H NMR spectra of these freshly
prepared solutions and those obtained after different periods of
storage (t) at 298 K.
4.1.3. Study of the effect produced by 9-methylguanine on the
platinum(II) complexes
A solution formed by the selected compound [4.4 mg (of 8c), or
5.8 mg (of 10a) or 4 mg (of 11)] and 0.7 mL of a DMSO-d6/D2O (1:1)
mixture was introduced in a NMR-tube. Then, the equimolar
amount of 9-methylguanine was added, the resulting solution
was shaken at 298 K to complete the homogenization and the tube
was sealed. 1H NMR spectra of these freshly prepared solutions
were recorded at 298 K and the progress of the reaction was mon-
itored by NMR after different periods of storage at 298 K.
4.2. Biological studies
4.2.1. Cell culture
Human lung A549, colon HCT116 cells and MBA-MD231 breast
adenocarcinoma cells were grown as a monolayer culture in min-
imum essential medium (DMEM with
cose and without sodium pyruvate) in the presence of 10% heat-
inactivated fetal calf serum, 10 mM -glucose and 0.1% streptomy-
L-glutamine, without glu-
D
cin/penicillin, in standard culture conditions (humidified air with
5% CO2 at 37 °C).
4.2.2. Cell viability assay
For A549 cell viability assays, compounds were suspended in
high purity DMSO at 20 mM as stock solution. To obtain final assay
concentrations, they were diluted in DMEM (final concentration of
DMSO was the same for all conditions and was always lower than
1%). The assay was performed by a variation of the MTT assay de-
scribed by Mosmann45 as specified by Matito et al.46 which is
based on the ability of live cells to cleave the tetrazolium ring of
the MTT thus producing formazan, which absorbs at 550 nm. In
brief, 3 ꢁ 103 A549 cells/well were cultured in 96 well plates for
4. Experimental
4.1. Chemistry
4.1.1. Synthesis
The free ligands 1–3 and the platinum(II) complexes 4–11 were
prepared according to procedures developed in our group24–31 and

4216
J. Albert et al. / Bioorg. Med. Chem. 21 (2013) 4210–4217
24 h prior to the addition of the different compounds at different
concentrations, in triplicate. After incubation for 72 h more, the
Acknowledgments
supernatant was aspirated and 100 lL of filtered MTT (0.5 mg/
mL) was added to each well. Following 1 h of incubation with the
MTT, the supernatant was removed and the precipitated formazan
was dissolved in 100 lL DMSO. Relative cell viability, compared to
This work was supported by the Ministerio de Ciencia y Tec-
nología (Projects CTQ2009-11501 and CTQ2009-07021/BQU) and
the AGAUR, Generalitat de Catalunya (Grants 2009-SGR-1111,
2009SGR01308, 2006ITT-10007 and 2009CTP-00026). This study
was also supported by the project SAF2011-25726 and by RD06/
0020/0046 from Red Temática de Investigación Cooperativa en
Cáncer (RTICC), Instituto de Salud Carlos III, both funded by the
Ministerio de Ciencia e Innovación-Spanish government and Euro-
pean Regional Development Funds (ERDF) ‘Una manera de hacer
Europa’. Marta Cascante acknowledges the support received
through the prize ‘ICREA Academia’, funded by ICREA foundation-
Generalitat de Catalunya.
the viability of untreated cells, was measured by absorbance at
550 nm on an ELISA plate reader (Tecan Sunrise MR20-301, TECAN,
Salzburg, Austria). Concentrations that inhibited cell growth by
50% (IC50) after 72 h of treatment were subsequently calculated.
For MDA-MB231 and HCT116 cell viability assays, compounds
were dissolved in high purity DMSO at 50 mM as stock solution.
Then, serial dilutions were made with DMSO (1:1). In this way
DMSO concentration in cell media was always the same. Finally,
1:500 dilutions of the serial dilutions of compounds on cell media
were prepared. The assay was performed as described by Givens
et al.47 In brief, HCT116 and MDA-MB231 cells were plated at
Supplementary data
5000 cells/well, respectively, in 100
well plates (Cultek). After 24 h, media was replaced by 100
l
L media in tissue culture 96
Supplementary data (stability studies of the complexes (6a, 8a,
8c, and 11) in solution, by comparison of the 1H NMR spectra of
freshly prepared samples and those obtained after different storage
periods (Figs. S1–S4). Effect produced by the presence of equimolar
amounts of 9-methylguanine on the complexes solutions (Figs. S5–
S7)) associated with this article can be found, in the online version,
at http://dx.doi.org/10.1016/j.bmc.2013.05.005.
lL/well
of serial dilution of drugs. Control wells did not contain com-
pounds. Each point concentration was run in triplicate. Reagent
blanks, containing media and colorimetric reagent without cells
were run on each plate. Blank values were subtracted from test val-
ues and were routinely 5–10% of uninhibited control values. Plates
were incubated 72 h. Hexosaminidase activity was measured
according to the following protocol: the media was removed and
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