Versatile Route to trans-Platinum(II) Complexes via Manipulation of a Coordinated 3-(Pyridin-3-yl)propanoic Acid Ligand

Article abstract of DOI:10.1021/acs.inorgchem.9b00126

We describe the direct coupling of alcohols and amines to a 3-(pyridin-3-yl)propanoic acid ligand coordinated to a Pt(II) to afford ester and amide derivatives. Using this approach, a family of trans-Pt(II) compounds with amine ligands bearing long perfluorinated chains was prepared, as these chains potentially endow the complexes with thermoactivatable properties. Related compounds with alkyl chains in place of the perfluorinated chains were also prepared as controls using the same direct coupling method. The stability of the complexes in solution, their reactivity with DNA and proteins, and their antiproliferative activity evaluated in tumorigenic (A2780 and A2780cisR) and nontumorigenic (HEK293) cells at 37 °C and following exposure to elevated temperatures (that mimic the temperatures employed in thermotherapy) were also studied to assess their utility as putative (thermoactivated) anticancer agents.

Full text of DOI:10.1021/acs.inorgchem.9b00126

Article
pubs.acs.org/IC
Cite This: Inorg. Chem. XXXX, XXX, XXX−XXX
Versatile Route to trans-Platinum(II) Complexes via Manipulation of
a Coordinated 3‑(Pyridin-3-yl)propanoic Acid Ligand
Silvia Cabrera,†,‡ Francisco Navas,†,# Ana I. Matesanz,† Marta Maroto,†,# Tina Riedel,§
Paul J. Dyson,§ and Adoracion G. Quiroga*,†,‡
†
‡
́
Inorganic Chemistry Department and Institute for Advanced Research in Chemical Sciences (IAdChem), Universidad Autonoma
de Madrid, Madrid 28049, Spain
§Institute of Chemical Sciences and Engineering, Swiss Federal Institute of Technology Lausanne (EPFL), CH-1015 Lausanne,
Switzerland
S
* Supporting Information
ABSTRACT: We describe the direct coupling of alcohols and
amines to a 3-(pyridin-3-yl)propanoic acid ligand coordinated to
a Pt(II) to afford ester and amide derivatives. Using this
approach, a family of trans-Pt(II) compounds with amine ligands
bearing long perfluorinated chains was prepared, as these chains
potentially endow the complexes with thermoactivatable proper-
ties. Related compounds with alkyl chains in place of the
perfluorinated chains were also prepared as controls using the
same direct coupling method. The stability of the complexes in
solution, their reactivity with DNA and proteins, and their
antiproliferative activity evaluated in tumorigenic (A2780 and A2780cisR) and nontumorigenic (HEK293) cells at 37 °C and
following exposure to elevated temperatures (that mimic the temperatures employed in thermotherapy) were also studied to
assess their utility as putative (thermoactivated) anticancer agents.
combination is considerably more effective than the two
monotherapies alone. However, carboplatin was not designed
for application in combination with heat, and a heat-activated
platinum drug could further enhance the efficacy of the
combination.
1. INTRODUCTION
The limitations associated with traditional platinum agents,
combined with improved knowledge of tumor biology, and the
fact that cisplatin still is the most used metallodrug in the
clinic, supports the further development of platinum drugs.
Nonconventional platinum anticancer agents are particularly
promising due to their selective activity against specific cancer
cells types.1
It has previously been shown that the action of nonconven-
tional platinum drugs with aliphatic amines in the trans
configuration can be modulated by replacing one of the trans
aliphatic amines. As examples, a steroid-functionalized amine
increases the selectivity of the complex,7,8 phosphines
modulate drug uptake,9,10 and functionalizing the ligand with
a DNA intercalator affords a dual-action platinum com-
plex.11,12 In the present study, we developed an alternative
synthetic methodology that affords perfluorinated chain-
modified platinum(II) complexes by directly coupling the
perfluorinated chain to a 3-(pyridin-3-yl)propanoic acid
already coordinated to a Pt(II) center, which to the best of
our knowledge is the first time such an approach has been
reported for platinum(II) complexes. Perfluorinated chains are
known to endow compounds with thermoresponsive proper-
ties,13,14 and related complexes with long hydrocarbon chains
were also prepared for comparison purposes. The ability of the
complexes to bind to DNA and proteins was evaluated at 37
°C and following a hyperthermia treatment. Their cytotoxicity
DNA is the main pharmacological target for platinum drugs,
although many studies have shown that proteins are also
involved in their therapeutic effect, such as proteins that
remove Pt-DNA adducts.2,3 However, specific interactions with
proteins are difficult to control due to the relative structural
simplicity of square-planar platinum(II) compounds. Since
DNA is the main target of platinum drugs and many additional
nonselective interactions with proteins take place, these
compounds have high general toxicities resulting in severe
side effects. In order to improve the efficacy of platinum drugs
and reduce their side effects, they tend to be applied in
combination therapies, either with other drugs or with other
types of therapies, e.g., radiotherapy, thermotherapy, etc.4,5
In general, clinically approved platinum drugs are used in
different combinations without adapting their structure to
improve their performance in the given combination. For
example, carboplatin is extensively used in the treatment of
retinoblastoma in combination with thermotherapy.6 Here,
following administration of carboplatin, the tumor is selectively
heated with a laser through the pupil of the eye, and the
Received: January 14, 2019
© XXXX American Chemical Society
A
DOI: 10.1021/acs.inorgchem.9b00126
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
was also evaluated in vitro under both normal conditions and
hyperthermia.
(pyridin-3-yl)propanoic acid ligand coordinated to the Pt(II)
center with the appropriate perfluorinated precursors shown in
Scheme 1, employing the same coupling conditions used in the
synthesis of ligands L1, L2, and L3 (see Scheme 1). The
desired complexes, 1−6, were obtained as pure compounds
after flash chromatography.
2. RESULTS AND DISCUSSION
2.1. Synthesis and Characterization of the Com-
pounds. The synthesis of the platinum compounds with
ligands modified with fluorinated and/or aliphatic chains is
illustrated in Scheme 1 (ligands are named L1, L2, and L3).
Using this approach, we prepared a family of trans-Pt(II)
compounds with ligands L1, L2, and L3. The synthesis of
trans-[PtCl2(ipa)(L3)] 6, was more complicated than ex-
pected. The established route (Scheme 2) led to the formation
of the complex together with other byproducts. Moreover,
after several attempts to purify 6 by flash chromatography, the
resulting yield was low. Therefore, we decided to activate the
acid using SOCl2, which in spite of generating SO2, does not
afford any platinated byproducts, allowing the successful
synthesis and isolation of complex 6 (see Scheme 3).17
a
Scheme 1. Synthesis of the Ligands L1, L2, and L3
1
Complexes 1−6 were characterized by H, 13C, and 195Pt
nuclear magnetic resonance (NMR) spectroscopy and
elemental analysis (see Experimental Section for full details).
The NMR spectra confirm the expected structures and are
essentially as expected following coordination of the ligand to
the Pt(II) center. In brief, the 195Pt NMR spectra of each trans
complex contains only one signal between −2100 and −2000
ppm, which is consistent with a trans-PtN2Cl2 unit.18,19,11 The
1H NMR confirms the presence of dma and ipa and ligands L1
a
EDCI (N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydro-
chloride) and DMAP (4-(dimethylaminopiridine).
The method comprises a coupling reaction using 1-(3-
(dimethylamino)propyl)-3-ethylcarbodiimide (EDCI) to acti-
vate the carboxylic acid group and dimethylaminopyridine
(DMAP) in catalytic amounts. The synthesis of L3 involves
the formation of an amide bond from the O-acyl-isourea
intermediate and perfluorinated fragment.
The traditional method used to prepare trans-platinum
complexes involves the direct reaction of cis-[PtCl2(amine)2]
compounds with the desired ligand.15,16 However, applying
this method for the synthesis of trans-platinum complexes
containing ligands L1, L2, and L3 resulted in complicated
mixtures due to the harsh conditions required. Consequently,
an alternative synthetic route was required in order to obtain
the target complexes in good yield. First, we prepared a trans-
platinum complex which incorporates a picolinic-type ligand
giving a general formula trans-[PtCl2(amine)(3-(pyridin-3-
yl)propanoic acid)] (amine = dimethylamine, dma and
isopropylamine, ipa). These starting complexes are named
Pdma and Pipa, respectively, and are depicted in Scheme 2.
Second, the trans-Pt(II) compounds bearing the perfluorinated
chains were obtained from a coupling reaction between the 3-
1
to L3 in 1−6. Following coordination, the H NMR signals
corresponding to the pyridine moiety move to a lower
frequency relative to the free ligands (ΔδHα = 0.3 to ΔδHβ
= 0.1), in agreement with the coordination of the N of the
pyridine to the platinum center. The signals in the 13C NMR
spectra corresponding to the C atoms adjacent to the donor N
atom are also shifted to lower values. This kind of shielding
produced by the platination has been reported previously with
other complex examples.7,12,20−22 The 19F NMR spectra
contain resonances that are readily assigned to the perfluori-
nated chains, and since they are far from the site of
coordination, do not show significant differences in chemical
shifts compared to the free ligands.
2.2. Stability of the trans-Pt(II) Complexes in DMSO
and Aqueous (1% DMSO) Solution. The stability of Pdma,
Pipa, and 1−6 was studied in DMSO using 1H NMR
spectroscopy (spectra were recorded after 0, 2, 6, and 24 h on
samples maintained at 37 °C in a thermoshaker in the dark).
a
Scheme 2. Route Used to Obtain the trans-[PtCl2(amine)L] Complexes 1−6
a
L = L1, L2, or L3.
B
DOI: 10.1021/acs.inorgchem.9b00126
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
Scheme 3. Synthesis of trans-[PtCl2(ipa)(L3)] 6 Using SOCl2 and Et3N
1
Figure 1. H NMR spectra of 3 in DMSO as a function of time.
Table 1. kobs (× 10−5 s−1) Values of 1−6 and Cisplatin
The spectra remain unchanged demonstrating the stability of
the compounds (see Figure 1 for the H NMR spectra of 3,
1
complex
kobs (10−5 s−1
)
other spectra are provided in the Supporting Information).
Note that DMSO was selected as the solvent for the stability
studies as the compounds are predissolved in DMSO prior to
dilution with water for the cytotoxicity studies.
In order to confirm that no other species with similar
chemical shifts (possible speciation with DMSO) are present
in solution, we performed stabilities studies of the complexes in
DMSO solution by reverse-phase HPLC using mixtures of
water/acetonitrile as the mobile phase. The stability of the
complexes was also monitored by HPLC as a function of time
(see Supporting Information), which confirms their high
stability in DMSO solution, except for 1, which precipitates
after 6 h under the experimental conditions.
The stability of 1−6 in aqueous solution (containing 1%
DMSO) was also evaluated using UV−vis spectroscopy over
24 h, as it has previously been shown that water can induce
reactions of complexes with DMSO (see in Supporting
Information).23 The stability constants (kobs, Table 1) were
estimated by assuming a pseudo-first-order reaction, expressing
the absorption as a function of time. Applying the equation
Abs = Abs0·e−kt, we obtained the kobs values collected in Table
1. The nonfluorinated complex 1 (kobs: 7.53 × 10−5 s−1) is less
stable in aqueous solution than its ipa analogue 2 (kobs: 4.04 ×
10−5 s−1), which exhibits a stability similar to cisplatin.24
Complexes with perfluorinated ligands (3−6) display variable
stability, with 3 being less stable than its ipa counterpart 4. In
general, the complexes with perfluorinated ligands (3−6) are
more stable in aqueous solution than the complexes with the
alkyl ligands (1 and 2).
a
trans-[PtCl2(dma)(L1)], 1
trans-[PtCl2(ipa)(L1)], 2
trans-[PtCl2(dma)(L2)], 3
trans-[PtCl2(ipa)L2)], 4
trans-[PtCl2(dma)(L3)], 5
trans-[PtCl2(ipa)(L3)], 6
cisplatin
11.6
2.92
7.04
4.04
9.41
8.28
4.45
a
Calculated value after 6 h in aqueous solution containing 1% DMSO.
2.3. Interaction of the trans-Pt(II) Complexes with
Supercoiled Plasmid DNA. Supercoiled pBR322 plasmid
DNA is a broadly used model for evaluating the interaction of
platinum compounds with a secondary structure DNA
model.11,25 Figure 2 shows the agarose gels of the DNA
incubated with 1−6 at 37 and 42 °C (the latter temperature
representing hyperthermia). For comparison, the Pdma and
Pipa starting materials were included in the study. Pdma
interacts with the ccc form of the supercoiled, migrating along
with the oc form at ri of 0.1, and Pipa shows the same pattern
but at a higher ri (0.2). The elevated temperature leads to only
a slight enhancement in the interaction of these complexes
with DNA.
Complex 1 with the aliphatic branched chain (L1) ligand
and dma does not interact with the plasmid even at 42 °C,
presumably due to its poor solubility (see above). Complex 2,
trans-[PtCl2(ipa)(L1)], on the other hand, shows a similar
interaction profile to its precursor trans-[PtCl2(ipa)(3-(pyr-
idin-3-yl)propanoic acid)] Pipa. Complexes 1 and 2 and their
C
DOI: 10.1021/acs.inorgchem.9b00126
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
Figure 2. Agarose gels of pBR322 plamid DNA incubated with trans-[PtCl2(ipa)(L)] (top) and trans-[PtCl2(dma)(L)] (bottom) at 37 and 42 °C
for 24 h at ri values of 0.05, 0.1, and 0.2. ri = [complex]/[no. nucleotide].
Figure 3. Cytotoxic activity of the trans-Pt(II) complexes in A2780, A2780cisR, and HEK293 cell lines at 37 °C and with a 2 h period at 42 °C
(total incubation time = 72 h).
precursors do not contain a perfluorinated chain, so the low
impact in their reactivity with plasmid at higher temperatures is
as expected.
the series, which is indeed the case (see below). The data are
also in agreement with the kobs values obtained in solution (see
Table 1).
Notably, the trans-Pt(II) complexes with perfluorinated
chains (3−6) show a different and interesting interaction with
DNA compared to the aliphatic counterparts (1−2) and the
precursor compounds (Pdma and Pipa). Complex 3 exhibits
greater interactions with DNA than its counterpart, 4, which
seems to be in agreement with the stability data obtained in
solution in which 3 is less stable than 4 and therefore more
reactive with the DNA. Notably, complexes 5 and 6 with the
longest perfluorinated chains show more extensive reactivity
with the plasmid DNA at 42 °C relative to that observed at 37
°C. They reach comigration at even lower ri values, indicating
that they should be more cytotoxic than the other complexes of
2.4. Cytotoxicity of the trans-Pt(II) Complexes. The
cytotoxicity of Pdma, Pipa, and complexes 1−6 was
determined at 37 °C over 72 h and additionally at 37 °C
over 72 h with a 2 h period at 42 °C at the start of the
incubation period (Figure 3). The study was carried out in two
cancer cell lines, i.e., A2780 (human ovarian carcinoma) and
A2780cisR (Human ovarian carcinoma with acquired resist-
ance to cisplatin) and also in the nontumorigenic HEK293
(human embryonic kidney) cell line. The two precursors
(Pdma and Pipa) do not show significant cytotoxic activity or
are inactive (at values up to 100 μM). Regarding the
complexes with alkyl ligands, complex 1 is also inactive (at
D
DOI: 10.1021/acs.inorgchem.9b00126
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
values up to 100 μM), whereas 2 is more cytotoxic but to a
lesser extent than cisplatin.
synthetic route used allows alcohols and amines to be directly
coupled to a platinum-coordinated 3-(pyridin-3-yl)propanoic
acid (pi) ligand, which in principle allows a wide range of
groups to be added to the Pt(II) scaffold. Such a synthetic
approach opens the way to rapidly generate small libraries of
Pt(II) complexes for cancer cell screening and other
applications. Concerning the specific compounds prepared
here, complexes 5 and 6, with perfluorinated ligands, are able
to overcome cisplatin resistance in the A2780cisR cell line, but
they are not endowed with cancer cell selectivity, and their
thermoresponsive behavior is rather limited. Nonetheless, their
cytotoxicity correlates reasonably well to the observed
complex−DNA binding interactions.
In general, the inclusion of hyperthermia did not lead to
large changes in the cytotoxicity of the complexes, although
slight increases in the cytotoxicity of the fluorinated complexes
at higher temperatures was observed. Moreover, the most
active complexes, 3, 5, and 6, contain ligands with
perfluorinated chains, but they are not endowed with cancer
cell selectivity.
2.5. Interaction Studies with Model Proteins. Although
DNA is probably the main target for these trans-Pt(II)
complexes, with more extensive binding observed at the higher
temperature, the interaction of the complexes with proteins
cannot be discarded. For this reason, we studied the
interaction of the complexes with a model protein at 37 and
42 °C, employing bovine pancreatic ribonuclease A (RNase
A), often used as a model to study metallodrug binding
interactions.26−30 The rate constants (kobsR) of the complexes
were calculated by assuming a pseudo-first-order reaction, and
the values obtained at 37 and 42 °C are listed in Table 2.
4. EXPERIMENTAL SECTION
4.1. General Methods. NMR spectra were acquired on a Bruker
300 spectrometer, running at 300, 75, and 64.5 MHz for 1H, 13C, and
195Pt, respectively. Chemical shifts (δ) are reported in ppm relative to
residual solvent signals (CDCl3: 7.26 ppm for 1H NMR, 77.0 ppm for
1
13C NMR; DMSO-d6: 2.50 ppm for H NMR, 39.52 for 13C NMR;
1
acetone-d6: 2.05 ppm for H NMR, 29.84 for 13C NMR). 13C NMR
spectra were acquired on a broad band decoupled mode. 195Pt NMR
spectra were obtained with chemical shifts reported in ppm downfield
relative to the external reference 1.0 M Na2PtCl6 in D2O. Elemental
analyses were performed on a PerkinElmer 2400 Series II micro-
analyzer. UV−vis in a Thermo Fisher Scientific Evolution 260 Bio
spectrophotometer. Analytical thin layer chromatography (TLC) was
performed using precoated aluminum-backed plates (Merck Kieselgel
60-F254) and visualized by ultraviolet irradiation or iodine dip. All
reagents and materials were purchased from commercial sources and
used without further purification. Cytotoxicity studies were performed
as described previously.34
4.2. Synthesis of the Starting Materials. 4.2.1. Ligands.
Method (a): Synthesis of Ligands L1 and L2. To a suspension of
picolinic acid (py) (300 mg, 1 equiv) in 10 mL of CH2Cl2, 1 equiv of
1-octadecanol or 1H,1H,2H,2H-perfluorooctanol (for L1 or L2
ligand, respectively), 1 equiv of EDCI and DMAP (10 mol %) were
added, and the mixture was stirred at rt for 14 days. The mixture was
washed with a saturated solution of NaHCO3 (3 × 10 mL), brine (3
× 10 mL), and then the organic phase was dried over anhydrous
MgSO4. Finally, the organic solvent was eliminated under reduced
pressure. Final purification by flash column chromatography using
silica gel and eluting with hexane and ethyl acetate (5:1) afforded the
desired compounds.
Table 2. Calculated kobsR Values for the Interaction of 1−6
and Cisplatin with Rnase at 37 and 42 °C
K (× 10−5s−1
)
complex
k
obsR (37 °C)
kobsR (42 °C)
1
2
3
4
5
6
11.5
17.5
9.23
7.79
7.93
8.07
18.8
10.2
26.6
a
13.2
0.03
7.00
a
44.4
cisplatin
a
Calculated values after 6 h in solution.
Caution should be applied when comparing the values at the
two different temperatures, as the structure of the protein
probably differs, as hyperthermia is known to lead to protein
denaturation.31,32
The kobsR values of the nonfluorinated complexes 1 and 2 at
37 °C indicate a higher affinity of these complexes for the
protein than the other complexes in the series, exhibiting
similar protein affinities to cisplatin.33 Notably, 1 and 2 are the
least reactive with DNA. The long aliphatic chain in 1 and 2
presumably leads to interactions with the lipophilic residues of
RNase. However, this effect is not as strong with the complexes
bearing perfluorinated ligands, although the values are
indicative of interactions.
At the elevated temperature of 42 °C, the trend is less clear,
reflecting the lower stability and increased dynamic properties
of the complexes at this temperature. Moreover, at 42 °C the k
observed values have very short detection times, making it
increasingly difficult to obtain good-quality absorptions, as the
sample shows little differences with the control. Complexes 2
and 6 have very high KobsR values at the elevated temperature,
leading to precipitation after 6 h. In contrast, the KobsR value of
4 is very low indicating that it interacts differently with the
model protein.
L1: Yield: 64%. 1H NMR (CDCl3, 300 MHz): δ 8.43 (s, 1H, ArPy),
8.41 (d, 1H, J = 4.8 Hz, ArPy), 7.47 (d, 1H, J = 7.1 Hz, ArPy), 7.14 (dd,
1H, J = 7.1, 4.8 Hz, ArPy), 4.00 (t, 2H, J = 6.4 Hz, O−CH2), 2.90 (t,
2H, J = 6.4 Hz, Py-CH2), 2.58 (t, 2H, J = 6.4 Hz, CH2−CO), 1.55−
1.51 (m, 2H, O- CH2−CH2), 1.21 (s, 30H, CH2), 0.83 (t, 3H, J = 6.4
Hz, CH3). 13C NMR (CDCl3): δ 172.3 (COO−), 149.9 (Npy-CH-C),
147.7 (Npy-CH), 135.8 (Npy-CH-C), 135.7 (Npy-CH-C-CH), 123.3
(Npy-CH-CH), 64.8 (O−CH2), 35.4 (CH2−COO−), 31.9 (CH2−
CH2−CO−), 29.7 (11C, (CH2)n-), 29.3 (1C, (CH2)−), 28.6 (1C,
(CH2)-), 28.1 (1C, (CH2)n-), 25.8 (1C, (CH2)−), 22.7 (-CH2−
CH3), 14.1 (CH2−CH3). Anal. Calcd for C26H45NO2: C, 77.37%; H,
11.24%; N, 3.47%. Found: C, 77.70%; H, 11.48%; N, 3.26%.
L2: Yield: 62%. 1H NMR (CDCl3, 300 MHz): δ 8.44 (s, 1H, ArPy),
8.42 (d, 1H, J = 4.9 Hz, ArPy), 7.48 (d, 1H, J = 7.6 Hz, ArPy), 7.16 (dd,
1H, J = 7.6, 4.9 Hz, ArPy), 4.33 (t, 2H, J = 6.4 Hz, O−CH2), 2.92 (t,
2H, J = 6.4 Hz, Py-CH2), 2.64 (t, 2H, J = 6.4 Hz, CH2−CO), 2.49−
2.37 (m, 2H, CH2−CF2). 13C NMR (CDCl3): δ 171.9 (COO−),
149.8 (Npy-CH−C), 147.9 (Npy-CH), 135.7 (Npy-CH-C-CH), 135.6
(Npy-CH-C-CH), 123.3 (Npy-CH-CH), 105.8−121.5 (6 C−F), 56.4
(O−CH2), 35.0 (CH2−COO−), 30.6 (CH2−CH2−O−), 28.1
(CH2−CH2−CO−). Anal. Calcd for C16H12F13NO2: C, 38.65%; H,
2.43%; N, 2.82%. Found C, 39.01%; H, 2.58%; N, 2.77%.
3. CONCLUSIONS
We have synthesized a new family of trans-Pt(II) complexes
with long-chain aliphatic or perfluorinated ligands. The
Method (b): Synthesis of Ligand L3. To a suspension of picolinic
acid (py) (300 mg, 1 equiv) in 10 mL of CH2Cl2, 1 equiv of 1H,1H-
E
DOI: 10.1021/acs.inorgchem.9b00126
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
perfluorooctilamine, 1 equiv of EDCI, and 1 equiv of Et3N were
added, and the mixture stirred at rt for 14 days. The mixture was
washed with a saturated solution of NaHCO3 (3 × 10 mL) and brine
(3 × 10 mL), and then the organic phase was dried over anhydrous
MgSO4. Finally, the organic solvent was eliminated under reduced
pressure. Final purification by flash column chromatography afforded
the desired compounds. L3: Yield: 52%. H NMR (acetone-d6, 300
MHz): δ 8.46 (s, 1H, ArPy), 8.39 (d, 1H, J = 4.4 Hz, ArPy), 7.74 (s,
1H, NH), 7.62 (d, 1H, J = 7.8 Hz, ArPy), 7.24 (dd, 1H, J = 7.8, 4.4 Hz,
acid)] complex (250 mg, 1 equiv) in 8.25 mL of CH2Cl2, 1 equiv of 1-
octadecanol or 1H,1H,2H,2H-perfluorooctanol (for the formation of
trans-[PtCl2(amine)(L1)] or trans-[PtCl2(amine)(L2)], respectively),
1 equiv of EDCI and DMAP (10 mol %) were added, and the mixture
was stirred at rt for 14 days. The mixture was washed with a saturated
solution of NaHCO3 (3 × 10 mL) and brine (3 × 10 mL), and then
the organic phase was dried over anhydrous MgSO4. Finally, the
organic solvent was eliminated under reduced pressure. Final
purification by flash column chromatography afforded the desired
compounds, using 90 active neutral alumina Merck 0.063−0.200 mm
(70−230 mesh ASTM), dichloromethane/ethyl acetate (10:1 for
complex 1 and 2), and dichloromethane/EtOH (8:1 for complexes
3−6) as eluent.
1
ArPy), 4.15−4.02 (m, 2H, Py-CH2), 2.95 (td, 2H, JH,H = 6.2 Hz, JF,H
=
16.0 Hz, NH-CH2), 2.63 (t, 2H, J = 6.4 Hz, CH2−CO). 13C NMR
(CDCl3): δ 172.7 (CON-), 150.7 (Npy-CH−C), 148.3 (Npy-CH),
137.4 (Npy-CH-C-CH), 136.5 (Npy-CH-C), 124.0 (Npy-CH-CH),
118.0−109.2 (7 C−F), 56.5 (CON-CH2), 39.5 (CH2−CON), 29.8
(CH2−CH2−NH−). Anal. Calcd for C16H11F15N2O: C, 36.11%; H,
2.08%; N, 5.26%. Found: C, 36.39%; H, 2.36%; N, 5.12%.
1
trans-[PtCl2(dma)(L1)], 1. Yield: 45%. H NMR (DMSO-d6, 300
MHz): δ 8.56 (s, 1H, ArPy), 8.51 (d, 1H, J = 6.0 Hz, ArPy), 7.84 (d,
1H, J = 7.8 Hz, ArPy), 7.36 (dd, 1H, J = 7.7, 6.0 Hz, ArPy), 5.50 (bs,
1H, NH), 4.06 (t, 2H, J = 6.7 Hz, CH2−O), 2.88 (t, 2H, J = 7.6 Hz,
Py-CH2), 2.62 (t, 2H, J = 7.4 Hz, CH2−CO), 2,68 (6 H, d, J 5.7, NH-
CH3), 2.42 (d, 2H, J = 6.4 Hz), 1.52 (m, 2H), 1.23 (bs, 34H), 0.85 (t,
3H, J = 6.9 Hz, CH3). 13C NMR (CDCl3): δ 171.9 (COO−), 153.4
(Npy-CH−C), 151.5 (Npy-CH), 138.3 (Npy-CH-C-CH), 129.9 (Npy-
CH-C-CH), 124.8 (Npy-CH-CH), 65.1 (O−CH2), 43.4
((CH3)2NH), 34.7 (CH2−COO−), 31.9 (CH2−CH2−COO−),
29.7 (1C, (CH2)n-), 29.5 (1C, (CH2)n-), 29.3 (9C, (CH2)n-), 28.6
(1C, (CH2)−), 27.7 (1C, (CH2)n-), 25.9 (1C, (CH2)−), 22.7 (CH2−
CH3), 14.1 (CH2−CH3). 195Pt-NMR (DMSO-d6, 64 MHz): δ
−2046. MS (ESI) m/z: 737.29 [M + Na]+. Anal. Calcd for
C28H52Cl2N2O2Pt[(CH3)2CO]0,5: C, 47.75%; H, 7.48%; N, 3.74%.
Found: C, 48.05%; H, 7.39%; N, 3.73%.
4.2.2. Starting cis-Platinum Complexes. cis-[PtCl2(amine)2]
Complexes. The synthesis of the cis compounds was performed
following the protocol published by our group.35,36 cis-[PtCl2(dma)2]
was synthesized by treating K2PtCl4 (500 mg; 1.2 mmol) with 4 equiv
of dimethylamine (dma) or isopropylamine (ipa) (4.8 mmol) in 1 mL
of H2O. The reaction was stirred at room temperature in dark
conditions for 24 h. The yellow residue paste was filtered off and
washed with water, acetone, and chloroform. Finally the solid was air-
dried. Characterization data agree with the data reported.
1
cis-[PtCl2(ipa)2]. Yield: 82%. H NMR (DMSO-d6, 300 MHz): δ
4.74 (s, 2H, NH2), 3.09 (sept, J = 6.3 Hz, 1H, CH); 1.20 (d, J = 6.5
Hz, 6H, CH3). 13C NMR (DMSO-d6, 125 MHz): δ 47.6 (CH), 23.5
(2C, CH3). Anal. Calcd for PtC6H18Cl2N2: C, 18.75%; H, 4.72%; N,
7.29%. Found PtC6H18N2Cl2: C, 18.10%; H, 4.56%; N, 7.30%.
trans-[PtCl2(ipa)(L1)], 2. Yield: 31%. 1H NMR (CDCl3, 300
MHz): δ 8.69 (s, 1H, ArPy), 8.67 (d, 1H, J = 6.7 Hz, ArPy), 7.62 (d,
1H, J = 6.7 Hz, ArPy), 7.17 (t, 1H, J = 6.9 Hz, ArPy), 4.05 (t, 2H, J =
6.4 Hz, CH2−O), 3.46 (bs, 2H, NH2), 3.40 (m, 1H, CH-(CH3)2),
2.94 (t, 2H, J = 6.4 Hz, Py-CH2), 2.62 (t, 2H, J = 6.4 Hz, CH2−CO),
1.38 (d, 6H, J = 6.4 Hz, CH-(CH3)2), 1.21 (s, 32H, CH2), 0.86 (t,
3H, J = 6.4 Hz, CH2-CH3). 13C NMR (CDCl3): δ 171.9 (COO−),
153.2 (Npy-CH−C), 151.3 (Npy-CH), 138.3 (Npy-CH-C-CH), 129.5
(Npy-CH-C-CH), 124.7 (Npy-CH-CH), 65.1 (O−CH2), 49.1
((CH3)2CHNH2), 34.8 (CH2−COO−), 29.7 (CH2−CH2−
COO−), 29.6 (1C, (CH2)n-), 29.2 (9C, (CH2)n-), 28.6 (1C,
(CH2)−), 27.7 (1C, (CH2)n-), 25.9 ((CH3)2CHNH2), 24.0 (1C,
(CH2)n-), 22.7 (CH2−CH3), 14.1 (CH2−CH3). 195Pt-NMR (DMSO-
d6, 64 MHz): δ −2071. MS (ESI) m/z: 751.4 [M + Na]+. Anal. Calcd
for C29H54Cl2N2O2Pt: C, 47.80%; H, 7.47%; N, 3.84%. Found C,
48.12%; H, 7.69%; N, 3.56%.
1
cis-[PtCl2(dma)2. Yield: 60%. H NMR (DMSO-d6, 300 MHz): δ
5.69 (s, 1H, NH), 2.52 (s, 6H, CH3). 13C NMR (DMSO-d6, 125
MHz): δ 43.6. Anal. Calcd for PtC4H14N2Cl2: C, 13.48%; H, 3.96%;
N, 7.86%. Found PtC4H14Cl2N2: C, 13.66%, H, 3.93%, N, 7.90%.
trans-[PtCl2(amine)(L)] Complexes (P). To a suspension of the
corresponding cis-[PtCl2(amine)2] (500 mg, 1 equiv) in 5 mL of
H2O, 2.5 equiv of 3-(pyridin-3-yl)propanoic acid was added, and the
mixture was stirred at 80 °C for 24 h. The colorless solution was
cooled and then concentrated slowly until the minimum solvent. To
this solution, 20 equiv of HCl (36% aqueous solution) was added, and
the mixture was maintained at the reflux temperature for 24 h. The
final yellow solid was isolated, washed with water, and dried under a
vacuum.
trans-[PtCl2(ipa)(3-(pyridin-3-yl)propanoic acid)], Pipa Yield:
1
71%. H NMR (DMSO-d6, 300 MHz): δ 12.23 (s, 1H, COOH),
1
trans-[PtCl2(dma)(L2)], 3. Yield: 52%. H NMR (DMSO-d6, 300
8.55 (s, 1H, ArPy), 8.53 (d, 1H, J = 6.5 Hz, ArPy), 7.83 (d, 1H, J = 6.5
Hz, ArPy), 7.36 (t, 1H, J = 6.7 Hz, ArPy), 4.60 (s, 2H, NH2), 3.10 (m,
1H, CH-(CH3)2), 2.86 (t, 2H, J = 6.4 Hz, Py-CH2), 2.55 (t, 2H, J =
6.4 Hz, CH2−CO), 1.22 (d, 6H, J = 6.4 Hz, CH-(CH3)2).13C NMR
(DMSO-d6, 100 MHz): δ 173.3 (COOH), 154.0 (Npy-CH−C), 151.9
(Npy-CH), 139.5 (Npy-CH-C-CH), 139.3 (Npy-CH-C), 125.7 (Npy-
CH-CH), 49.2 (CH(CH3)2), 34.8 (Py-CH2), 28.2 (Py-CH2−CH2),
24.5 (CH(CH3)2). 195Pt-NMR (DMSO-d6, 64 MHz): δ −2066. Anal.
Calcd for C11H18Cl2N2O2Pt: C, 27.74%; H, 3.81%; N, 5.88%. Found:
C, 27.97%; H, 3.62%; N, 5.62%.
MHz): δ 8.57 (s, 1H, ArPy), 8.51 (d, 1H, J = 5.7 Hz, ArPy), 7.85 (d,
1H, J = 7.9 Hz, ArPy), 7.37 (dd, 1H, J = 5.7, 4.9 Hz, ArPy), 5.50 (bs,
1H, NH), 4.31 (t, 2H, J = 6.0 Hz, CH2−O), 2.95 (t, 2H, J = 7.4 Hz,
Py-CH2), 2.72−2.62 (m, 4H, CH2−CO and CH2−CF2), 2.40 (d, 2H,
J = 5.7 Hz). 13C NMR (CDCl3) δ 171.3 (COO−), 153.3 (Npy-CH−
C), 151.6 (Npy-CH), 138.3 (Npy-CH-C-CH), 137.7 (Npy-CH-C-CH),
136.5 (Npy-CH-CH), 124.8−108.1 (6 C−F), 56.1 (O−CH2), 43.4
(CH3)2NH), 34.4 (CH2−COO−), 30.4 (CH2−CH2−O), 27.5
(CH2−CH2−COO). 195Pt-NMR (DMSO-d6, 64 MHz): δ −2046.
MS (ESI) m/z: 831.0 [M + Na]+. 19F-NMR (acetone-d6, 282.40
MHz): δ −82.80 (t, 3F, JF,F = 8.5 Hz), −115.04 (m, 2F), −123.50 (m,
2F), −124.51 (m, 2F), −125.20 (m, 2F), −127.86 (m, 2F). Anal.
Calcd for C18H19Cl2F13N2O2Pt: C, 26.75%; H, 2.37%; N, 3.47%.
Found C, 26.82%; H, 2.58%; N, 3.45%.
trans-[PtCl2(dma)(3-(pyridin-3-yl)propanoic acid)], Pdma Yield:
81%. 1H NMR (DMSO-d6, 300 MHz): δ 11.78 (s, 1H, COOH), 8.11
(s, 1H, ArPy), 8.06 (d, 1H, J = 6.4 Hz, ArPy), 7.38 (d, 1H, J = 6.4 Hz,
ArPy), 6.91 (t, 1H, J = 6.8 Hz, ArPy), 5.06 (bs, 1H, NH), 2.39 (t, 2H, J
= 6.4 Hz, Py-CH2), 2.15 (t, 2H, J = 6.4 Hz, CH2−CO), 1.96 (d, 6H, J
trans-[PtCl2(ipa)(L2)], 4. Yield: 41%. 1H NMR (CDCl3, 300
MHz): δ 8.76 (m, 2H, ArPy), 7.61 (d, 1H, J = 6.8 Hz, ArPy), 7.19 (dd,
1H, J = 6.9, 5.7 Hz, ArPy), 4.37 (t, 2H, J = 6.4 Hz, CH2−O), 3.53 (bs,
2H, NH2), 3.41 (m, 1H, CH-(CH3)2), 2.97 (t, 2H, J = 6.4 Hz, Py-
CH2), 2.66 (t, 2H, J = 6.4 Hz, CH2−CO), 2.47−2.39 (m, 2H, CH2−
CF2) 1.30 (d, 6H, J = 6.4 Hz, CH-(CH3)2).13C NMR (CDCl3): δ
170.4 (COO−), 152.2 (Npy-CH−C), 150.5 (Npy-CH), 137.2 (Npy-
CH-C-CH), 136.8 (Npy-CH-C-CH), 136.2 (Npy-CH-CH), 123.9−
105.1 (6 C−F), 55.7 (O−CH2), 48.0 ((CH3)2CHNH2), 33.5 (CH2−
COO), 29.5 (CH2−CH2−O), 26.5 (CH2−CH2−COO), 23.0
= 6.4 Hz, CH3). 13C NMR (DMSO-d6, 100 MHz): δ 173.2 (COOH),
152.7 (Npy-CH−C), 150.9 (Npy-CH), 138.6 (Npy-CH-C-CH), 138.4
(Npy-CH-C), 125.0 (Npy-CH-CH), 43.2 (2C, NH(CH3)2), 34.1 (Py-
CH2), 26.9 (Py-CH2−CH2). 195Pt-NMR (DMSO-d6, 64 MHz): δ
−2044. Anal. Calcd for C10H16Cl2N2O2Pt: C, 25.98%; H, 3.49%; N,
6.06%. Found: C, 26.43%; H, 3.40%; N, 6.05%.
4.3. Synthesis of the trans-Platinum Complexes 1−6.
Method (a): Synthesis of complexes trans-[PtCl2(amine)(L1)] (1−
2), trans-[PtCl2(amine)(L2)] (3−4) and trans-[PtCl2(dma)(L3)] (5).
To a suspension of trans-[PtCl2(amine)(3-(pyridin-3-yl)propanoic
F
DOI: 10.1021/acs.inorgchem.9b00126
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
(CH3)2CHNH2). 195Pt-NMR (DMSO-d6, 64 MHz): δ −2079. 19F-
NMR (acetone-d6, 282.40 MHz): δ −82.80 (t, 3F, JF,F = 8.5 Hz),
−115.04 (m, 2F), −123.49 (m, 2F), −124.49 (m, 2F), −125.18 (m,
2F), −127.83 (m, 2F). MS (ESI) m/z: 844,0 [M + Na]+. Anal. Calcd
for C19H21Cl2F13N2O2Pt: C, 27.75%; H, 2.57%; N, 3.41%. Found: C,
28.10%; H, 2.67%; N, 3.52%.
4.6. Sample Preparation and UV−vis Kinetic Experiments of
the Complex Solutions. The desired concentration of complexes
was achieved by dilution of the stock DMSO solution with aqueous
tris buffer. All the solutions and buffers were previously tempered to
37 °C for the stability in solution and at 37 °C. The freshly prepared
complex solutions were mixed in a thermoshaker at the desired
temperature with Milli-Q water (for stability k).The electronic spectra
of trans-Pt complexes were recorded from fresh to 24 h in DMSO/
buffer solution monitoring the changes in the absorbance of one
characteristic charge transfer band. The absorbance was then
represented versus time for a typical kinetic run, and the data were
1
trans-[PtCl2(dma)(L3)], 5. Yield: 40%. H NMR (DMSO-d6, 300
MHz): δ 8.58−8.49 (m, 3H, ArPy and CO-NH), 7.82 (d, 1H, J = 7.9
Hz, ArPy), 7.35 (dd, 1H, J = 7.9, 5.7 Hz, ArPy), 5.53 (bs, 1H, NH),
4.01−3.92 (td, 2H, JH,H = 6.1 Hz, JF,H = 17.2 Hz, CH2−CF2), 2.87 (t,
2H, J = 7.6 Hz, Py-CH2), 2.58 (m, 2H, CH2−CO), 2.40 (d, 6H, J =
5.6 Hz, CH3). 13C NMR (CDCl3,): δ 171.1 (CON-), 153.2 (Npy-
CH−C), 151.5 (Npy-CH), 138.7 (Npy-CH-C-CH), 137.9 (Npy-CH-C-
CH), 124.9 (Npy-CH-CH), 118.1−109.0 (7 C−F), 43.4 (CH2−
CON), 39.3 (m, CON-CH2C−F), 36.7 (CH3)2NH), 27.9 (CH2−
CH2−CO). 195Pt-NMR (DMSO-d6, 64 MHz): δ −2044. 19F-NMR
(acetone-d6, 282.40 MHz): δ −82.78 (t, 3F, JF,F = 11.3 Hz), −119.45
(m, 2F), −123.43 (m, 2F), −123.68 (m, 2F), −124.34 (m, 2F),
−125.12 (m, 2F), −127.81 (m, 2F). MS (ESI) m/z: 866.0 [M + Na]+.
Anal. Calcd for C18H18Cl2F15N3OPt: C, 25.59%; H, 2.21%; N, 4.95%.
Found: C, 25.15%; H, 2.15%; N, 4.98%.
Method (b) trans-[PtCl2(ipa)(L3)], 6. On a flask containing 70 mg
(1 equiv) of trans-[PtCl2(ipa)(3-(pyridin-3-yl)propanoic acid)], 0.25
mL of SOCl2 was added, and the mixture was stirred at rt for 90 min.
The yellow solution was dried under a vacuum, and then a solution
containing 1 equiv of 1H,1H-perfluorooctilamine with 1 equiv of Et3N
in 5 mL of CH2Cl2 anhydrous was added, and then stirred at rt
overnight. Finally, the yellow solution was dried with MgSO4 and then
filtered, and the final solution was dried under reduced pressure.
trans-[PtCl2(ipa)(L3)], 6. Yield: 29%. 1H NMR (CDCl3, 300
MHz): δ 8.72 (s, 1H, ArPy), 8.67 (d, 1H, J = 5.7 Hz, ArPy), 7.86 (d,
1H, J = 8.1 Hz, ArPy), 7.33 (dd, 1H, J = 8.1, 5.7 Hz, ArPy), 5.95 (bs,
1H, CO-NH), 3.95 (td, 2H, JH,H = 6.3 Hz, JF,H = 15.9 Hz, CH2−CF2),
3.57 (bs, 1H, NH), 3.41 (m, 1H, CH-(CH3)2), 3.00 (t, 2H, J = 6.4
Hz, Py-CH2), 2.58 (t, 2H, J = 6.4 Hz, CH2−CO), 1.37 (d, 6H, J = 6.4
Hz, CH-(CH3)2). 13C NMR (acetone-d6) δ 171.4 (COO−), 152.9
(Npy-CH−C), 150.8 (Npy-CH), 139.5 (Npy-CH-C-CH), 138.4 (Npy-
CH-C-CH), 125.0 (Npy-CH-CH), 118.0−109.2 (7 C−F), 48.6
((CH3)2CHNH2), 46.1 (CH2−CON), 39.7 (m, CON-CH2C−F),
27.0 (CH2−CH2−CO), 22.9 ((CH3)2CHNH2). 19F-NMR (acetone-
d6, 282.40 MHz): δ −82.77 (m, 3F), −119.46 (m, 2F), −123.45 (m,
2F), −123.67 (m, 2F), −124.36 (m, 2F), −125.13 (m, 2F), −127.83
(m, 2F). MS (ESI) m/z: 880,0 [M + Na]+. Anal. Calcd for
C19H20Cl2F15N3OPt: C, 30.10%; H, 3.06%; N, 4.68%. Found
C19H20Cl2F15N3OPt[(CH3)2CO]0.5: C, 29,68%; H, 2,55%; N, 4,61%.
4.4. Stability Studies of the trans-Pt(II) Complexes in DMSO
by 1H NMR. We dissolved 1 mg of each compound in DMSO-d6 (0.5
mL), placed it into an NMR tube, and analyzed it several times from 0
to 24 h by 1H NMR using a Bruker AMX-300 (300 MHz)
spectrometer at room temperature (25 °C).
4.5. Stability Studies of the trans-Pt(II) Complexes in DMSO
by HPLC. For the stability studies of the trans-Pt(II) complexes by
HPLC, stock solutions were prepared fresh in DMSO (5 mM). Then,
1 mL of a 100 μM solution in DMSO of the test compound was
incubated at 37 °C, and aliquots of 20 μL were taken at various time
points (0, 1, 2, 5, and 24 h) and analyzed by RP-HPLC on an Agilent
1200 system using a Zorbax Eclipse Plus C18 column (4.6 × 100 mm,
3.5 μm): flow rate, 1 mL/min; detection, UV 254 nm; gradient
solvent system A/B (acetonitrile/water): for complexes trans-
[PtCl2(dma)(3-(pyridin-3-yl)propanoic acid)], trans-[PtCl2(ipa)(3-
(pyridin-3-yl)propanoic acid)], trans-[PtCl2(dma)(L2)], trans-
[PtCl2(ipa)(L2)], and trans-[PtCl2(dma)(L3)], initial 30% A +
70% B; 10 min linear gradient to 100% A; for complexes trans-
[PtCl2(dma)(L1)], trans-[PtCl2(ipa)(L1)] and trans-[PtCl2(ipa)-
(L3),] initial 50% A + 50% B; 30 min linear gradient to 100% A.
The disappearance of the compound over time was expressed as the
remaining percentage in comparison to the initial amount. The
different time point alicuots were analyzed by RP_HPLC monitored
using a Detector photodiode array.
fit to an exponential decay function: A = A0·e−kt
.
4.7. Interaction Studies of the trans Pt(II) versus pBR322
Plasmid DNA. The plasmid pBR322 DNA stock was purchased from
Gencust at a concentration of 0.5 μg/μL in phosphate buffer 50 mM
(pH 7.4). Stock solutions of the complexes were prepared in DMSO
at 5 mM and then diluted with water to the desired concentration.
The DNA interaction studies were performed with a total volume of
20 μL. A 20 μL portion containing 0.125 μg/μL of DNA in 10 mM
Tris-HCl (pH 7.6) and 1 mM EDTA was incubated with the platinum
compounds at ri values ranging from 0.05 to 0.2 (ri is defined ri =
[complex]/[no. nucleotide]). The samples were incubated at 37 °C
for 24 h, after which 2 μL of a loading dye buffer containing 50%
glycerol, 0.25% bromophenol blue, and 0.25% xylene cyanol was
added. The total amount of the sample (20 μL) was loaded in the
agarose gel (1.2% w/v), and electrophoresis was carried out for a
period of 150 min at approximately 70 V in TAE 1× (Tris-acetate/
EDTA) buffer. After electrophoresis, the gel was immersed in 200 mL
of Millipore water containing 10 μL from a 10 mg/mL stock solution
of ethidium bromide for 30 min to stain the DNA. Finally, the stained
gel was analyzed with a UVITEC Cambridge UVIDOC HD2
instrument.
4.8. Cell Culture and Cytotoxicity Measurements. Human
A2780 and A2780cisR ovarian carcinoma cells were obtained from the
European Centre of Cell Cultures (ECACC, Salisbury, UK).
Nontumorigenic HEK-293 cells were obtained from ATCC (Sigma,
Switzerland). A2780 and A2780cisR cells were routinely grown in
RPMI 1640 medium with GlutaMAX containing 5% fetal bovine
serum (FBS) and 1% antibiotics (penicillin and streptomycin) at 37
°C and 5% CO2. HEK-293 cells were grown in DMEM medium
containing 5% fetal bovine serum (FBS) and 1% antibiotics (penicillin
and streptomycin) at 37 °C and 5% CO2. In order to keep the
A2780cisR cells resistant to cisplatin, the cells were monthly treated
with 2 μM cisplatin for one passage. Cytotoxicity was determined
using the MTT assay (MTT = 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyl-2H-tetrazolium bromide). Briefly, the cells were seeded in
96-well plates (10 000 cells per well) and grown for 24 h in complete
medium. For each testing, compounds were freshly prepared as
DMSO stock solution and then dissolved in the culture medium and
immediately serially diluted to the appropriate concentration, to give a
final DMSO concentration of 0.5% v/v. Drug solution (100 μL) was
added to each well, and the plates were incubated at 37 °C for 72 or 2
h at 41.5 °C followed by a 70 h incubation at 37 °C. Following drug
exposure, 20 μL of MTT (5 mg/mL solution in PBS) was added to
the cells and incubated for 4 h, then the culture medium was
aspirated, and the violet formazan crystals were dissolved in DMSO
(100 μL). The optical density of each well (96 well plates) was
quantified at 590 nm using a multiwell plate reader (Molecular
Devices, UK), and the percentage of surviving cells was calculated
from the ratio of absorbance of treated to untreated cells. The IC50
values for the inhibition of cell growth were determined by fitting the
plot of the logarithmic percentage of surviving cells against the
logarithm of the drug concentration using a linear regression function.
Mean values and standard deviations computed from two
independent experiments, each comprising four microcultures per
concentration level, are reported.
4.9. Sample Preparation for RNaseA Interaction Studies by
UV−vis Spectroscopy. trans-Pt complexes were initially dissolved in
DMSO to a concentration of 5 mM. The desired concentration of
complexes was achieved by dilution of the stock DMSO solution with
G
DOI: 10.1021/acs.inorgchem.9b00126
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
aqueous tris buffer. Cisplatin stock solutions were prepared in 5 mM
NaClO4 and diluted in Milli-Q water. All the solutions and buffers
were previously tempered to 37 °C for the stability in solution, and at
37 and 42 °C for the interaction with Rnase. Afterward, the freshly
prepared complex solutions were mixed in a thermoshaker at the
desired temperature aqueous buffer tris with the RNase solutions. The
final concentration used for these studies never exceeded 1% DMSO
(v/v) in the final solution. Control experiments with DMSO were
performed, and no changes in the spectra of the model protein RNase
were observed.
(4) Guchelaar, H. J.; Uges, D. R. A.; de Vries, E. G. E.; Oosterhuis, J.
W.; Mulder, N. H. Combination therapy with cisplatin: Modulation of
activity and tumour sensitivity. Clinic. Oncology 1992, 4 (6), 388−393.
(5) Dasari, S.; Bernard Tchounwou, P. Cisplatin in cancer therapy:
Molecular mechanisms of action. Eur. J. Pharmacol. 2014, 740, 364−
378.
(6) Dimaras, H.; Corson, T. W.; Cobrinik, D.; White, A.; Zhao, J.;
Munier, F. L.; Abramson, D. H.; Shields, C. L.; Chantada, G. L.;
Njuguna, F.; Gallie, B. L. Retinoblastoma. Nature Rev. Disease Primers
2015, 1, 15021−15021.
4.9.1. UV−vis Kinetics Experiments with Proteins. The electronic
spectra of the model protein RNase A (bovine pancreatic ribonuclease
A) were recorded at 10−5 M, monitoring the changes in the
absorbance at λmax = 280 nm, and after the addition of the Pt
complexes (trans-Pt(II) complexes and cisplatin) from fresh to 24 h
both at 37 and 42 °C. The assays were performed first using pseudo-
first-order conditions (ratios of 10:1 metal to protein) obtaining poor
absorbance signals (values range from 0.10 to 0.05 au); then to
increase the absorbance, we repeated the experiment using 3:1 ratios.
The experimental time dependence spectra profiles (1:10 and 1:3
ratios) were analyzed as a pseudo-first-order reaction by plotting the
variation of the absorbance as a function of time obtaining the same
results. The data were fit to a exponential decay function: A = A0·e−kt
expected for a pseudo-first-order reaction.
(7) Sanchez-Cano, C.; Huxley, M.; Ducani, C.; Hamad, A. E.;
Browning, M. J.; Navarro-Ranninger, C.; Quiroga, A. G.; Rodger, A.;
Hannon, M. J. Conjugation of testosterone modifies the interaction of
mono-functional cationic platinum(ii) complexes with DNA, causing
significant alterations to the DNA helix. Dalton Trans. 2010, 39 (47),
11365−11374.
(8) Huxley, M.; Sanchez-Cano, C.; Browning, M. J.; Navarro-
Ranninger, C.; Quiroga, A. G.; Rodger, A.; Hannon, M. J. An
androgenic steroid delivery vector that imparts activity to a non-
conventional platinum(ii) metallo-drug. Dalton Trans. 2010, 39 (47),
11353−11364.
(9) Medrano, A.; Dennis, S. M.; Alvarez-Valdes, A.; Perles, J.;
McGregor Mason, T.; Quiroga, A. G. Synthesis, cytotoxicity, DNA
interaction and cell cycle studies of trans-diiodophosphine Pt(ii)
complexes. Dalton Trans. 2015, 44 (8), 3557−3562.
(10) Medrano, M. A.; Alvarez-Valdes, A.; Perles, J.; Lloret-Fillol, J.;
Munoz-Galvan, S.; Carnero, A.; Navarro-Ranninger, C.; Quiroga, A.
G. Oxidation of anticancer Pt(ii) complexes with monodentate
phosphane ligands: towards stable but active Pt(iv) prodrugs. Chem.
Commun. 2013, 49 (42), 4806−4808.
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.inorg-
chem.9b00126.
(11) Navas, F.; Mendes, F.; Santos, I.; Navarro-Ranninger, C.;
Cabrera, S.; Quiroga, A. G. Enhanced Cytotoxicity and Reactivity of a
Novel Platinum(IV) Family with DNA-Targeting Naphthalimide
Ligands. Inorg. Chem. 2017, 56 (11), 6175−6183.
HPLC spectra gradient conditions and 1H NMR spectra
for all the complexes. UV−vis spectra for all the
complexes (PDF)
(12) Herrera, J. M.; Mendes, F.; Gama, S.; Santos, I.; Navarro
Ranninger, C.; Cabrera, S.; Quiroga, A. G. Design and Biological
Evaluation of New Platinum(II) Complexes Bearing Ligands with
DNA-Targeting Ability. Inorg. Chem. 2014, 53, 12627.
(13) Clavel, C. M.; Zava, O.; Schmitt, F.; Halamoda Kenzaoui, B.;
Nazarov, A. A.; Juillerat-Jeanneret, L.; Dyson, P. J. Thermoresponsive
Chlorambucil Derivatives for Tumour Targeting. Angew. Chem., Int.
Ed. 2011, 50 (31), 7124−7127.
AUTHOR INFORMATION
Corresponding Author
*E-mail: adoracion.gomez@uam.es.
ORCID
Silvia Cabrera: 0000-0002-4907-2932
Ana I. Matesanz: 0000-0002-6113-0656
Paul J. Dyson: 0000-0003-3117-3249
Adoracion G. Quiroga: 0000-0002-9261-9542
■
(14) Renfrew, A. K.; Scopelliti, R.; Dyson, P. J. Use of Perfluorinated
Phosphines to Provide Thermomorphic Anticancer Complexes for
Heat-Based Tumor Targeting. Inorg. Chem. 2010, 49 (5), 2239−2246.
(15) Quiroga, A. G.; Ranninger, C. N. Contribution to the SAR field
of metallated and coordination complexes. Studies of the palladium
and platinum derivatives with selected thiosemicarbazones as
antitumoral drugs. Coord. Chem. Rev. 2004, 248 (1−2), 119−133.
(16) Quiroga, A. G.; Perez, J. M.; Alonso, C.; Navarro-Ranninger,
C.; Farrell, N. Novel transplatinum(II) complexes with [N2O2]
donor sets. Cellular pharmacology and apoptosis induction in Pam
212-ras cells. J. Med. Chem. 2006, 49 (1), 224−231.
Present Address
#Industry.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This research was supported by the Spanish MINECO
(CTQ2015-68779-R).
(17) Higa, T.; Krubsack, A. J. Oxidations by thionyl chloride. VI.
Mechanism of the reaction with cinnamic acids. J. Org. Chem. 1975,
40 (21), 3037−3045.
REFERENCES
(18) Priqueler, J. R. L.; Butler, I. S.; Rochon, F. D. An Overview of
195Pt Nuclear Magnetic Resonance Spectroscopy. Appl. Spectrosc.
Rev. 2006, 41 (3), 185−226.
■
(1) Johnstone, T. C.; Suntharalingam, K.; Lippard, S. J. The Next
Generation of Platinum Drugs: Targeted Pt(II) Agents, Nanoparticle
Delivery, and Pt(IV) Prodrugs. Chem. Rev. 2016, 116 (5), 3436−
3486.
(2) Brabec, V.; Kasparkova, J.; Kostrhunova, H.; Farrell, N. P.
Inhibition of nuclear factor kappaB proteins-platinated DNA
interactions correlates with cytotoxic effectiveness of the platinum
complexes. Sci. Rep. 2016, 6, 28474.
(19) Still, B. M.; Kumar, P. G. A.; Aldrich-Wright, J. R.; Price, W. S.
195Pt NMR theory and application. Chem. Soc. Rev. 2007, 36 (4),
665−686. and references therein.
(20) Ramos-Lima, F. J.; Vrana, O.; Quiroga, A. G.; Navarro-
Ranninger, C. N.; Halamikova, A.; Rybnickova, H.; Hejmalova, L.;
Brabec, V. Structural characterization, DNA interactions, and
cytotoxicity of new transplatin analogues containing one aliphatic
and one planar heterocyclic amine ligand. J. Med. Chem. 2006, 49 (8),
2640−2651.
(3) Pinato, O.; Musetti, C.; Farrell, N. P.; Sissi, C. Platinum-based
drugs and proteins: reactivity and relevance to DNA adduct
formation. J. Inorg. Biochem. 2013, 122, 27−37.
H
DOI: 10.1021/acs.inorgchem.9b00126
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
(21) Quiroga, A. G.; Cubo, L.; de Blas, E.; Aller, P.; Navarro-
Ranninger, C. Trans platinum complexes design: One novel water
soluble oxime derivative that contains aliphatic amines in trans
configuration. J. Inorg. Biochem. 2007, 101 (1), 104−110.
(22) Cubo, L.; Quiroga, A. G.; Zhang, J.; Thomas, D. S.; Carnero,
A.; Navarro-Ranninger, C.; Berners-Price, S. J. Influence of amine
ligands on the aquation and cytotoxicity of trans-diamine platinum(ii)
anticancer complexes. Dalton Trans 2009, No. 18, 3457−66.
̈
(23) Varbanov, H. P.; Ortiz, D.; Hofer, D.; Menin, L.; Galanski, M.;
Keppler, B. K.; Dyson, P. J. Oxaliplatin reacts with DMSO only in the
presence of water. Dalton Trans. 2017, 46 (28), 8929−8932.
(24) Miller, S. E.; Gerard, K. J.; House, D. A. The hydrolysis
products of cis-diamminedichloroplatinum(II) 6. A kinetic compar-
ison of the cis- and trans-isomers and other cis-di(amine)di(chloro)-
platinum(II) compounds. Inorg. Chim. Acta 1991, 190 (1), 135−144.
(25) Martin Santos, C.; Cabrera, S.; Rios-Luci, C.; Padron, J. M.;
Lopez Solera, I.; Quiroga, A. G.; Medrano, M. A.; Navarro-Ranninger,
C.; Aleman, J. Novel clioquinol and its analogous platinum
complexes: importance, role of the halogen substitution and the
hydroxyl group of the ligand. Dalton Trans. 2013, 42 (37), 13343−
13348.
(26) Messori, L.; Marzo, T.; Michelucci, E.; Russo Krauss, I.;
Navarro-Ranninger, C.; Quiroga, A. G.; Merlino, A. Interactions
between Anticancer trans-Platinum Compounds and Proteins: Crystal
Structures and ESI-MS Spectra of Two Protein Adducts of trans-
(Dimethylamino)(methylamino)dichloridoplatinum(II). Inorg. Chem.
2014, 53 (15), 7806−7808.
(27) Picone, D.; Donnarumma, F.; Ferraro, G.; Russo Krauss, I.;
Fagagnini, A.; Gotte, G.; Merlino, A. Platinated oligomers of bovine
pancreatic ribonuclease: Structure and stability. J. Inorg. Biochem.
2015, 146, 37−43.
(28) Marzo, T.; Navas, F.; Cirri, D.; Merlino, A.; Ferraro, G.;
Messori, L.; Quiroga, A. G. Reactions of a tetranuclear Pt-
thiosemicarbazone complex with model proteins. J. Inorg. Biochem.
2018, 181, 11−17.
(29) Bettaieb, A., Wrzal, P. K., Averill-Bates, D. A. Hyperthermia:
Cancer Treatment and Beyond. In Cancer Treatment - Conventional
and Innovative Approaches; Rangel, L., Ed.; IntechOpen, 2013; Ch. 12.
(30) Sisken, J. E.; Morasca, L.; Kibby, S. Effects of temperature on
the kinetics of the mitotic cycle of mammalian cells in culture. Exp.
Cell Res. 1965, 39 (1), 103−116.
(31) Ritchie, K. P.; Keller, B. M.; Syed, K. M.; Lepock, J. R.
Hyperthermia (heat shock)-induced protein denaturation in liver,
muscle and lens tissue as determined by differential scanning
calorimetry. Int. J. Hyperthermia 1994, 10 (5), 605−618.
(32) Freeman, M. L.; Borrelli, M. J.; Meredith, M. J.; Lepock, J. R.
On the path to the heat shock response: destabilization and formation
of partially folded protein intermediates, a consequence of protein
thiol modification. Free Radical Biol. Med. 1999, 26 (5), 737−745.
́
́
(33) Echeverri, M.; Alvarez-Valdes, A.; Navas, F.; Perles, J.; Sanchez-
́
Perez, I.; Quiroga, A. G. Using phosphine ligands with a biological
role to modulate reactivity in novel platinum complexes. R. Soc. Open
Sci. 2018, 5 (2), 171340.
(34) Stibal, D.; Riedel, T.; Dyson, P. J.; Suss-Fink, G.; Therrien, B.
Dinuclear arene ruthenium thiolato complexes with fluorous side-
chains. Inorg. Chim. Acta 2016, 444, 51−55.
́
́
́
(35) Pantoja, E.; Alvarez-Valdes, A.; Perez, J. M.; Navarro-
Ranninger, C.; Reedijk, J. Synthesis and characterization of new cis-
[PtCl2(isopropylamine)(amine′)] compounds: cytotoxic activity and
reactions with 5′-GMP compared with their trans-platinum isomers.
Inorg. Chim. Acta 2002, 339, 525−531.
(36) Cubo, L.; Thomas, D. S.; Zhang, J.; Quiroga, A. G.; Navarro-
Ranninger, C.; Berners-Price, S. J. [1H,15N] NMR studies of the
aquation of cis-diamine platinum(II) complexes. Inorg. Chim. Acta
2009, 362 (3), 1022−1026.
I
DOI: 10.1021/acs.inorgchem.9b00126
Inorg. Chem. XXXX, XXX, XXX−XXX