Synthesis and evaluation of the cytotoxic activity of water-soluble cationic organometallic complexes of the type [Pt(η1-C2H4OMe)(L)(phen)]+ (L = NH3, DMSO; phen = 1,10-phenanthroline)

Article abstract of DOI:10.3390/pharmaceutics13050642

Starting from the [PtCl(η¹-C₂H₄OMe)(phen)] (phen = 1,10-phenanthroline, 1) platinum(II) precursor, we synthesized and characterized by multinuclear NMR new [Pt(η¹-C₂H₄OMe)(L)(phen)]⁺ (L = NH₃, 2; DMSO, 3) complexes. These organometallic species, potentially able to interact with cell membrane organic cation transporters (OCT), violating some of the classical rules for antitumor activity of cisplatin analogues, were evaluated for their cytotoxicity. Interestingly, despite both complexes 2 and 3 resulting in greater cell uptake than cisplatin in selected tumor cell lines, only 3 showed comparable or higher antitumor activity. General low cytotoxicity of complex 2 in the tested cell lines (SH-SY5Y, SK-OV-3, Hep-G2, Caco-2, HeLa, MCF-7, MG-63, ZL-65) appeared to depend on its stability towards solvolysis in neutral water, as assessed by NMR monitoring. Differently, the [Pt(η¹-C₂H₄OMe)(DMSO)(phen)]⁺ (3) complex was easily hydrolyzed in neutral water, resulting in a comparable or higher cytotoxicity in cancer cells with respect to cisplatin. Further, both IC₅₀ values and the uptake profiles of the active complex appeared quite different in the used cell lines, suggesting the occurrence of diversified biological effects. Nevertheless, further studies on the metabolism of complex 3 should be performed before planning its possible use in tissue- and tumor-specific drug design.

Full text of DOI:10.3390/pharmaceutics13050642

pharmaceutics
Article
Synthesis and Evaluation of the Cytotoxic Activity of
Water-Soluble Cationic Organometallic Complexes of the Type
[Pt(η1-C2H4OMe)(L)(Phen)]+ (L = NH3, DMSO;
Phen = 1,10-Phenanthroline)
Federica De Castro , Erika Stefàno, Danilo Migoni, Giorgia N. Iaconisi, Antonella Muscella
,
Santo Marsigliante , Michele Benedetti * and Francesco P. Fanizzi
Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali, Università del Salento, Prov.le Lecce-Monteroni,
Centro Ecotekne, 73100 Lecce, Italy; federica.decastro@unisalento.it (F.D.C.); erika.stefano@unisalento.it (E.S.);
danilo.migoni@unisalento.it (D.M.); giorgianatalia.iaconisi@studenti.unisalento.it (G.N.I.);
antonella.muscella@unisalento.it (A.M.); santo.marsigliante@unisalento.it (S.M.); fp.fanizzi@unisalento.it (F.P.F.)
* Correspondence: michele.benedetti@unisalento.it
Abstract: Starting from the [PtCl(
precursor, we synthesized and characterized by multinuclear NMR new [Pt(
(L = NH3, ; DMSO, ) complexes. These organometallic species, potentially able to interact with
cell membrane organic cation transporters (OCT), violating some of the classical rules for antitumor
activity of cisplatin analogues, were evaluated for their cytotoxicity. Interestingly, despite both
η
1-C2H4OMe)(phen)] (phen = 1,10-phenanthroline,
1-C2H4OMe)(L)(phen)]+
1) platinum(II)
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
η
ꢀꢁꢂꢃꢄꢅꢆ
2
3
Citation: De Castro, F.; Stefàno, E.;
Migoni, D.; Iaconisi, G.N.; Muscella,
A.; Marsigliante, S.; Benedetti, M.;
Fanizzi, F.P. Synthesis and Evaluation
of the Cytotoxic Activity of
complexes
2
and
3
resulting in greater cell uptake than cisplatin in selected tumor cell lines, only
3
showed comparable or higher antitumor activity. General low cytotoxicity of complex
2
in the tested
cell lines (SH-SY5Y, SK-OV-3, Hep-G2, Caco-2, HeLa, MCF-7, MG-63, ZL-65) appeared to depend
on its stability towards solvolysis in neutral water, as assessed by NMR monitoring. Differently, the
1-C2H4OMe)(DMSO)(phen)]+
[Pt(η (3) complex was easily hydrolyzed in neutral water, resulting in a
comparable or higher cytotoxicity in cancer cells with respect to cisplatin. Further, both IC50 values
and the uptake profiles of the active complex appeared quite different in the used cell lines, suggesting
the occurrence of diversified biological effects. Nevertheless, further studies on the metabolism
Water-Soluble Cationic
Organometallic Complexes of the
Type [Pt(η1-C2H4OMe)(L)(Phen)]+
(L = NH3, DMSO; Phen =
1,10-Phenanthroline). Pharmaceutics
2021, 13, 642. https://doi.org/
10.3390/pharmaceutics13050642
of complex
3 should be performed before planning its possible use in tissue- and tumor-specific
drug design.
Academic Editors: Wukun Liu and
Damiano Cirri
Keywords: cisplatin; coordination compounds; platinum complex; cationic complex; square planar
complex; organometallic complex; antitumor drug; cytotoxicity; antitumor activity
Received: 30 March 2021
Accepted: 27 April 2021
Published: 30 April 2021
1. Introduction
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
Cisplatin was synthesized for the first time in 1845, together with its trans isomer, by
the Italian chemist Michele Peyrone. Its antitumor properties were highlighted only after
1960, thanks to the work of Barnett Rosenberg et al. [1,2]. Interestingly, a long time was
required for its approval as an anticancer drug (in 1979) due to its relevant side effects,
notwithstanding the generally observed increase of life expectancy or the complete recov-
ery of oncological patients [3,4]. Another critical aspect of cisplatin was identified in its
intrinsic low solubility, strongly limiting the possible pharmacological application with
resistant tumors, probably requiring higher dosages. In any case, due to the progresses
made in cisplatin therapeutic protocols, to date it remains one of the most widely used
anticancer drugs [4,5]. For this reason, research on new platinum-based antitumor drugs
has been mainly focused on the development of new complexes violating the classical rules
Copyright:
© 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
early defined for antitumor activity of platinum compounds (i.e., cis-isomers, presence of
two medium labile ligands and two inert N-donors different from tertiary amines) [4–10]
.
About cisplatin mechanism of action, it is well known that passive diffusion is not the only
Pharmaceutics 2021, 13, 642. https://doi.org/10.3390/pharmaceutics13050642
https://www.mdpi.com/journal/pharmaceutics

Pharmaceutics 2021, 13, 642
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mechanism of cellular uptake, as several experiments showed that cisplatin is also imported
by cell membrane carriers normally involved in the transport of different substrates. In this
context, the two cis chlorido ligands of cisplatin, characterized by a medium lability, seem
necessary to achieve the equilibrium formation of the antitumor active cationic solvento
species cis-[PtCl(NH3)2(OH2)]+, cis-[Pt(NH3)2(OH2)2]2+, and cis-[Pt(NH3)2(OH)(OH2)]+.
The peculiar ability of cisplatin to produce such cationic species once inside the cytoplasm,
due to the relatively low chloride concentration with respect to blood plasma (extracellular
plasma [Cl−] > 100 mM; cell cytoplasm [Cl−]
one of the serendipity aspects that have determined the success of this drug. In fact, the
formation of reactive charged platinum solvento species inside the blood plasma seems
≈
23 mM; cell nucleus
≈
4 mM) [4,5,11] is
related to the severity of side effects observed elsewhere in the body [12,13]. Indeed,
the unselective binding of such reactive hydrolysis products to serum proteins, etc., is
considered responsible for the observed cisplatin toxicity and side effects. Moreover, the
reactivity of a platinum drug in the blood serum is a key factor determining how much of
the administered drug actually reaches the cell membranes to begin uptake via passive dif-
fusion or active transport (involving hCtr1, OCT, etc.) [14,15]. Furthermore, the hydrolyzed
drug, when formed inside the cell cytoplasm, constitutes the essential active species for
the desired antitumor activity. These reactive forms seem capable to interact with suitable
nucleophilic targets present in cells, such as nucleic acids, proteins, thiols, phospholipids,
cytoskeleton, etc. Among them, DNA is considered the main pharmacological target, as
its functionality results strongly altered by interaction with cisplatin [4,14–16]. Since its
approval, to solve some of the problems occurred in clinical applications of cisplatin, many
attempts have been made to develop new different antitumor active platinum-based drugs
with a greater spectrum of efficacy, a different mechanism of action, lower side effects, and
enhanced water solubility [4,5,17–19]. In particular, recent studies on the action mecha-
nism of cisplatin and oxaliplatin showed that cell membrane organic cation transporters
(OCT) can be involved in the uptake of platinum drugs in the form of cationic solvento
species [20–25]. This was also confirmed by recent studies on new cationic Pt(II)-complexes
showing a relevant antitumor activity [5,19,26,27].
It should be also underlined that there is a limited number of studies on platinum
organometallic compounds tested as antitumor drugs [28
–
35]. In this regard, the Zeise’s
−
anion, [PtCl3(
η
2-C2H4)] , showing an extreme lability of the trans to olefin chlorido ligand
and a peculiar reactivity of the coordinated ethene, resulted to be a suitable precursor
for the synthesis of new antitumor organometallic complexes [19]. In fact, it allows the
formation of platinum(II) organometallic complexes by the promotion of coordination
in the platinum(II) sphere of specific carrier ligands and the exploitation of the intrinsic
π
-acid character of platinum-coordinated η –39]. We already explored the
2-ethene [36
synthetic possibilities offered by the peculiar reactivity of the Zeise’s anion in strongly basic
oxydrilated solvents, demonstrating that in these conditions, anionic species of the type
−
η
2-C2H4)(OR)] (R = H, alkyl) are easily formed. Interestingly, the last product
trans-[PtCl2(
shows a quite different reactivity with respect to that of the Zeise’s anion, allowing the
synthesis of [PtCl(
1-C2H4OR)(N-N)] (N-N = dinitrogen ligand) complexes. These include,
besides the here considered [PtCl( ),
1-C2H4OMe)(phen)] (phen = 1,10-phenanthroline,
also other complexes bearing even more hindered dinitrogen ligands [39].
η
η
1
The aim of this work was to synthesize and characterize new cationic Pt(II) complexes
able to cross the cell membrane and exhibiting antitumor activity with mechanisms po-
tentially different from that of cisplatin. In this way, we planned to change and possibly
improve the selectivity and efficacy toward tumor cells, with eventual reduction of side
effects. In particular, we focused on Pt(II)-phen derivatives, which can also be synthesized
in high yields, Ref. [39] targeting new water-soluble cationic coordination compounds of
the type [Pt(
The specific interest toward this type of complexes relies on their potential ability to interact
with organic cation transporters [20 25] and to intercalate DNA and other nucleic acids, as
η
1-C2H4OR)(L)(phen)]+ (L = medium labile or not labile substituent; R = alkyl).
–

Pharmaceutics 2021, 13, 642
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generally observed in the case of phen derivatives [40
,41]. These complexes were obtained
and tested as new potential Pt-based antitumor drugs, as described in the present work.
2. Materials and Methods
2.1. Reagents and Methods
Commercially available reagents and solvents were used as received, without further
purification. Zeise’s salt, K[PtCl3(
η
2-C2H4)]
H2O, was synthesized with a previously
·
reported procedure, as indicated in ref. [39]. Elemental analyses were performed with a
CHN Eurovector EA 3011. NMR spectra were recorded with Bruker Avance III 400 and
600 NMR spectrometers, equipped with probes for inverse detection and with z gradient
for gradient-accelerated NMR spectroscopy. 1H NMR monodimensional spectra and
[1H,195Pt]-HETCOR bidimensional experiments were recorded by using CDCl3 or D2O
1
as solvents. H NMR spectra were referenced to TMS; the residual proton signal of the
solvent [CDCl3; δ ; D2O; δ
(1H) = 7.24 ppm (1H) = 4.7 ppm] was used as the internal standard.
195Pt NMR chemical shifts were referenced to H2[PtCl6] [
external reference.
δ(
195Pt) = 0 ppm] in D2O, as the
2.2. Synthesis of Pt(II) Complexes
[PtCl(
η
1-C2H4OMe)(phen)] (
1
). Na (81 mg, 3.5 mmol) was dissolved in 3.5 mL of
◦
MeOH (
−
20 C, ice-NaCl bath). After evolution of H2, the temperature was increased
to 0 ◦C (ice bath), then the Zeise’s salt, K[PtCl3(
η
2-C2H4)]
H2O, (100 mg, 0.26 mmol) was
·
added under magnetic stirring. A white precipitate of KCl was immediately formed, and
the color of the solution changed from bright to pale yellow. After about 2 min from
dissolution, 1,10-phenanthroline (47 mg, 0.26 mmol) was added to the reaction mixture,
maintaining a vigorous stirring. After a few more minutes, the formation of a yellow
precipitate, poorly soluble in cold methanol, started. The reaction was then followed
by 1H NMR spectroscopy, by analyzing 10
dissolution in an NMR tube containing 500
signals of the final product , the free phen ligand, and the trans-[PtCl2(
intermediate species, could be observed. The three specie’s relative amount changed
with time in favor of complex , which was the only remaining precipitate at the end
µ
L aliquots of the rection suspension after
1
µ
L of CD3OD. In this way, the H NMR
1
η
2-C2H4)(OMe)]−
1
of the reaction, after about 4 h. The final product was first isolated by filtration, then
washed with abundant water (to eliminate excess base and formed NaCl), and finally dried
under vacuum. Complex
C15H15ClN2OPt: C, 38.3; H, 3.2; N, 6.0%. Found: C, 38.0; H, 3.1; N, 6.1%. NMR (400 MHz,
CDCl3, 300 K).
(1H) 2.41 (m, 2H, Pt-CαH2, 2JPt–H = 90 Hz), 3.42 (s, 3H, OCH3), 3.70 (m,
1 yield 115 mg, 95% referred to platinum. Anal. Calcd. for
δ
2H, CβH2-O), 7.81 (d.d., 1H, CH), 7.95 (m, 3H, CH), 8.55 (d, 1H, CH), 8.65 (d, 1H, CH), 9.53
(d, 1H, CH, 3JPt-H = 57 Hz) and 9.81 (d, 1H, CH) ppm; δ(195Pt) −3243 ppm.
[Pt(η 2). In a typical reaction, the solid complex 1 (50 mg,
1-C2H4OMe)(NH3)(phen)]Cl (
0.11 mmol) was suspended under stirring in 2.5 mL of 30% NH3 in water (m/m), at room
temperature. After about two days, a colorless solution was obtained, which provided the
final [Pt(
to eliminate water and excess NH3. Complex
C15H18ClN3OPt: C, 37.0; H, 3.7; N, 8.6%. Found: C, 36.9; H, 3.9; N, 8.5%. NMR (400 MHz,
D2O/H2O = 10/90, 300 K):
(1H) 1.46 (m, 2H, Pt-CαH2, 2JPt–H = 50 Hz), 3.38 (s, 3H, OCH3),
η
1-C2H4OMe)(NH3)(phen)]Cl (
2
) complex salt, after evaporation under vacuum
2
yield 52 mg, 99.9%. Anal. Calcd. for
δ
3.37 (m, 2H, CβH2-O), 4.20 (s, 3H, Pt-NH3), 7.31 (d, 1H, CH), 7.37 (d, 1H, CH), 7.47 (d.d.,
1H, CH), 7.66 (d.d., 1H, CH), 8.19 (d, 1H, CH), 8.27 (d, 1H, CH), 8.32 (d, 1H, CH), 8.38 (d,
1H, CH); δ(195Pt) −3322 ppm.
[Pt(η 3). In a typical reaction, the solid complex 1
1-C2H4OMe)(DMSO)(phen)]Cl (
(50 mg, 0.105 mmol) was suspended in a DMSO (10 mL) under stirring. The reaction
was nearly quantitative, and after 2 days at room temperature, a yellow DMSO solution
containing the final pure [Pt(η 3) complex salt was obtained.
1-C2H4OMe)(DMSO)(phen)]Cl (
Complex 3·2DMSO could be isolated as a microcrystalline precipitate by adding a mixture
of ethanol/diethylether = 1:1 to a concentrated DMSO solution, followed by filtration.

Pharmaceutics 2021, 13, 642
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The obtained complex was then washed with the same non-solvent mixture and dried.
Isolated yield 42 mg, 57%. Anal. Calcd. for C21H33ClN2O4PtS3: C, 35.8; H, 4.7; N, 4.0%.
Found: C, 35.9; H, 4.8; N, 3.9%. NMR (400 MHz, D2O, 300 K): δ
(1H) 1.74 (m, 2H, Pt-
CαH2, 2JPt–H = 78 Hz), 3.37 (s, 3H, OCH3), 3.59 (m, 2H, CβH2-O), 3.68 (s, 6H, Pt-DMSO,
3JPt–H = 31 Hz), 8.00 (d, 1H, CH), 8.06 (s, 1H, CH), 8.07 (s, 1H, CH), 8.09 (d, 1H, CH), 8.75
(d.d., 1H, CH), 8.83 (d.d., 1H, CH), 9.03 (d, 1H, CH), 9.70 (d, 1H, CH);
δ(
195Pt)
−
3988 ppm.
2.3. Cell Cultures
The human cancer cell lines used for the evaluation of the Pt compounds cytotoxicity
(see Table 1) were cultured in different culture media, as reported below:
•
•
•
•
•
Caco-2: DMEM (low glucose) medium (Sigma-Aldrich, St. Louis, MO, USA), 10%
FBS (Sigma-Aldrich, St. Louis, MO, USA), glutamine 2 mM, penicillin (100 U/mL),
streptomycin (100 mg/mL), 1% non-essential amino acids.
MG-63 and SK-OV-3: DMEM (high glucose) medium (EuroClone, Pero, MI, Italy), 10%
FBS (Sigma-Aldrich, St. Louis, MO, USA), glutamine 2 mM, penicillin (100 U/mL),
streptomycin (100 mg/mL).
HeLa, MCF-7 and ZL-55: RPMI 1640 (EuroClone, Pero, MI, Italy), 10% FBS (Sigma-
Aldrich, St. Louis, MO, USA), glutamine 2 mM, penicillin (100 U/mL), streptomycin
(100 mg/mL).
Hep-G2: DMEM (low glucose) medium (Sigma-Aldrich, St. Louis, MO, USA), 10%
FBS (Sigma-Aldrich, St. Louis, MO, USA), glutamine 2 mM, penicillin (100 U/mL),
streptomycin (100 mg/mL).
SH-SY5Y: 1:1 mixture of DMEM (high glucose) and Ham’s F-12 Nutrient Mixture
(Sigma-Aldrich, St. Louis, MO, USA), 10% FBS (Sigma-Aldrich, St. Louis, MO, USA),
glutamine 2 mM, penicillin (100 U/mL), streptomycin (100 mg/mL).
Table 1. Cell cultures.
Cell Lines
Disease
Caco-2
HeLa
Colorectal adenocarcinoma
Endocervical adenocarcinoma
Hepatocellular carcinoma
Breast adenocarcinoma
Osteosarcoma
Neuroblastoma
Ovarian adenocarcinoma
Pleural epithelioid mesothelioma
Hep-G2
MCF-7
MG-63
SH-SY5Y
SK-OV-3
ZL-55
2.4. Cytotoxicity Assays
We evaluated the IC50 with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenoltetrazolium bro-
mide (MTT) and sulforhodamine B (SRB) assays. The SRB assay and the conversion of
MTT by cells were both used as an indicator of cell number, as described previously [42].
Cells, at 70–80% confluency, were trypsinized (0.25% trypsin with 1 mM EDTA), washed,
and resuspended in growth medium. Then, 100
µ
L of a cell suspension (8
×
104 cells/mL)
was added to each well of a 96-well plate. After overnight incubation, cells were treated
with different Pt compounds for 12–72 h. The percentage cell survival was calculated as the
absorbance ratio between treated and untreated cells (medium-treated control cell). Viable
cells were also counted by the Trypan blue exclusion assay and light microscopy. The data
presented are means standard deviation (S.D.) from eight replicate wells per microliter
plate, repeated three times.
2.5. Analysis by ICP-AES (Inductively Coupled Plasma Atomic Emission Spectroscopy)
For the determination of platinum concentration, each sample (consisting of cellular
pellet) was previously treated with 0.5 mL of 67% super-pure nitric acid at room tem-
perature for 24 h, to obtain a clear solution. Acid digestion was required to destroy the

Pharmaceutics 2021, 13, 642
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complex organic matrix and obtain the complete mineralization of the samples, essential
for subsequent determinations. The samples were then diluted to a final volume of 5 mL, in
order to obtain a suitable concentration of the acid used in the mineralization process and
avoid damage to the system. Each sample was filtered before analysis (0.45 µm) to prevent
the entry of any remaining suspension in the measuring instrument. The platinum concen-
tration in the analyzed samples was determined by a Thermo iCAP 6000 spectrometer. The
spectrophotometer was calibrated with a calibration line consisting of four points, each
corresponding to a concentration of the element: 1 µg/L, 10 µg/L, 100 µg/L, 1000 µg/L.
3. Results and Discussion
3.1. PtCl(η1-C2H4OMe)(phen)] (1), phen = 1,10-phenanthroline
We obtained the solid [PtCl(
by following a previously reported synthetic procedure [39
C2H4)] H2O, was dissolved in strongly basic methanol ([NaOMe]
of the stoichiometric amount of phen. In this way, the neutral [PtCl2(
η
1-C2H4OMe)(phen)] (
43]. The Zeise’s salt, K[PtCl3(η2
1M) before addition
2-C2H4)(phen)] pen-
1) complex as a precipitate,
,
-
·
≈
η
tacoordinate complex, normally produced when the phen ligand is added to a methanol
solution of the Zeise’s salt [44], was not observed. The absence of pentacoordinate species
in the presence of excess NaOMe was due to both its high reactivity in the adopted exper-
imental conditions and the preliminary formation of the trans-[PtCl2(η
2-C2H4)(OMe)]−
anionic complex which is characterized by a lower reactivity with respect to N-donors [39].
This reaction mechanism (Scheme 1) was also confirmed in the present case by 1H NMR
monitoring of complex 1 formation reaction.
Scheme 1.
in strongly basic MeOH, and the phen ligand. (
from 1 and NH3. (C) Synthesis of the [Pt(η1-C2H4OMe)(DMSO)(phen)]+ (3) complex starting from 1 and DMSO.
(A
) Synthesis of the [PtCl(
η
1-C2H4OMe)(phen)] (
) Synthesis of the [Pt(
1
) complex precursor, starting from the Zeise’s anion, dissolved
B
η
1-C2H4OMe)(NH3)(phen)]+ (
2) complex starting
3.2. Synthesis of [Pt(η 2) and [Pt(η
1-C2H4OMe)(NH3)(phen)]Cl ( 1-C2H4OMe)(DMSO)(phen)]Cl
(3) Complexes
In this work, we evaluated the possibility to synthesize water-soluble phenanthroline
cationic complexes suitable for exhibiting antitumor activity, as previously observed in
the case of similar [PtCl(η
2-C2H4)(N-N)]+, N-N = (R,R) and (S,S)-1,2-diaminocyclohexane,
cationic species [19]. These latter compounds were also suggested as possible prodrugs
introducing the active Pt(1R,2R-diaminocyclohexane) moiety in the cells. Unfortunately,
due to the extremely high tendency to lose the
ophilic agents (as the simple Cl− ligand), the analogous [PtCl(
results unsuitable for a slow release of the Pt(phen) moiety in physiological conditions.
On the other hand, also the direct use of [PtCl( ), as precursor of
1-C2H4OMe)(phen)] (
the olefin cationic species [39 43], for antitumor activity evaluations, was prevented due
to its extremely low water solubility. Therefore, we decided to synthesize and test new
types of water-soluble cationic species of the type [Pt(
1-C2H4OMe)(L)(phen)]+ obtained
η
2-ethene ligand in the presence of nucle-
η
2-C2H4)(phen)]+ complex
η
1
,
η

Pharmaceutics 2021, 13, 642
6 of 16
starting from complex
1, after chlorido substitution with a neutral L ligand. Such new
species are expected to be water-soluble prodrugs, possible sources of Pt(L)(phen) and
Pt(phen) active moieties, and suitable for antitumor activity evaluation. For this reason, in
the present study we focused on the synthesis and biological activity evaluation of two
[Pt(
chelated phen and the
η
1-C2H4OMe)(L)(phen)]Cl complexes as prototypes of those containing, besides the
η
1-C2H4OMe, a further nitrogen (L = NH3) or sulfur (L = DMSO)
ligand in the Pt(II) coordination sphere.
3.3. Pt(η1-C2H4OMe)(NH3)(phen)]Cl (2)
Complex
2 could be obtained by reacting the solid [PtCl(η 1)
1-C2H4OMe)(phen)] (
complex suspended in a concentrated water solution of ammonia. Quantitative substi-
tution of the chlorido ligand with NH3 took place, leading to formation of the colorless
water-soluble [Pt(
characterized by NMR spectroscopy.
The observed
195Pt) NMR signal exhibited by complex
shift about 80 ppm lower, with respect to that of complex , as expected on the basis of the
η 2) complex that could be isolated and
1-C2H4OMe)(NH3)(phen)]Cl (
δ(
2 corresponds to a chemical
1
slightly higher NMR shielding ability of a coordinated ammonia with respect to a chlorido
ligand [45]. The bidimensional [1H,195Pt]-HETCOR spectrum, highlighting the cross peaks,
accounts for the NMR couplings between phenanthroline H9 (close to the N10 which is in
trans to NH3), coordinated NH3, σ
-bonded CαH2, CβH2, and the central 195Pt (evidenced
in red, in the schematic representation of the molecular structure reported in Figure 1). No
cross peak was observed for the phen H(2) proton (close to the N1 cis to NH3), as expected
for the lengthening of the Pt–N bond trans to the σ-alkyl. Complex 2 resulted to be very
stable in water solution for more than 15 days, as demonstrated by the absence of any 1H
NMR signals variation observed in a neutral D2O/H2O = 10:90 solution.
Figure 1. [1H,195Pt]-HETCOR NMR spectrum of the [Pt( 1-C2H4OMe)(NH3)(phen)]Cl (
η 2) complex dissolved in D2O. In the
above-reported schematic molecular structure, the groups whose H atoms 1H NMR signals are coupled with the 195Pt are
evidenced in red. When a H2O/D2O = 90:10 solvent mixture was used, a new cross peak, corresponding to the 1H NMR
signal of the Pt-bonded NH3 group (indicated in red together with the corresponding 1H NMR signal produced in the 1D
1H NMR spectrum) appeared.

Pharmaceutics 2021, 13, 642
7 of 16
3.4. Pt(η1-C2H4OMe)(DMSO)(phen)]Cl (3)
When the solid complex was reacted with excess DMSO, the quantitative substitution of
the coordinated chlorido ligand occurred, producing the [Pt(
1-C2H4OMe)(DMSO)(phen)]Cl
) complex, as observed by 1H NMR monitoring. Complex
could be isolated at room
1
η
(
3
3
temperature by ethanol/diethyl ether addition to a concentrated DMSO solution.
A 195Pt NMR chemical shift decrease of about 800 ppm was observed, going from com-
plex
from the cis-[PtCl2{1,4-(i-Pr)2-dab}] complex,
Pr)2-dab}(DMSO)]Cl complex, 3150 ppm, with substitution of the coordinated
195Pt) =
chlorido ligand with a DMSO [46].
The two-dimensional [1H,195Pt]-HETCOR spectrum of a freshly prepared solution of
complex 0.3 mM in D2O/DMSO = 98:2 mixture, pH
7) with highlighted 1H-195Pt
1 to 3
. Consistently, a similar 195Pt NMR chemical shift decrease was observed, going
δ(
195Pt) =
−
2300 ppm, to the cis-[PtCl{1,4-(i-
δ
(
−
3
(
≈
≈
cross peaks is shown in Figure 2. The observed cross peaks account for the NMR couplings
between phenanthroline H9 (close to the N10 which is in trans to DMSO), coordinated
DMSO, σ
-bonded CαH2, and the central 195Pt (evidenced in red, in the schematic repre-
sentation of the molecular structure reported in Figure 2). No cross peak was observed for
the phen H(2) proton (close to the N1 cis to DMSO), as expected for the lengthening of the
Pt-N bond trans to the σ-alkyl.
Figure 2. [1H,195Pt]-HETCOR NMR spectrum of the [Pt( 1-C2H4OMe)(DMSO)(phen)]Cl (
η 3) complex
dissolved in a D2O/DMSO = 98:2 mixture. In the above-reported schematic molecular structure, the
groups whose H atoms 1H NMR signals are coupled with the 195Pt are evidenced in red.
As observed by 1H NMR monitoring in neutral D2O solution, in sharp contrast with
respect to
dissociation when dissolved in water, even in the presence of DMSO (Figure S1). As a
consequence, besides the hydrolysis of the -C2H4OMe moiety, with release of ethane and
2, complex 3 slowly underwent ethene and methanol release and further DMSO
σ
methanol, the formation of hydrolytic products was also observed. In particular, NMR spec-
tral data and literature evidence of the high acidity of Pt coordinated water in these systems,
suggests the formation of [Pt(DMSO)(OH)(phen)]+ and [{Pt( -OH)(phen)}2]2+ complexes
µ

Pharmaceutics 2021, 13, 642
8 of 16
with an asymmetric and a symmetric set of phenanthroline 1H NMR signals, respectively.
Both the new [Pt(DMSO)(OH)(phen)]+ and the already known [{Pt( -OH)(phen)}2]2+ com-
µ
plexes [47] were spectroscopically identified (Figure S1), in the latter case also by 195Pt NMR
spectroscopy (Figure S2). A mechanism for the observed hydrolysis process is reported in
Scheme 2. During hydrolysis, the formation of slight amounts of the insoluble [PtCl2(phen)]
complex, separating as a precipitate, was also observed. Therefore, for further biological
studies, complex
where it demonstrated a long-term stability (over 2 weeks at room temperature). It should
be noted that, despite the observed decomposition of in D2O, a small amount of water in
the presence of excess DMSO disfavored complex decomposition by chlorido reentering
in the Pt coordination sphere and increased complex solubility in the stock solution. Com-
plex resulted indefinitely stable when stocked at
20 ◦C in DMSO/H2O = 90:10 mother
solutions at a [3] ≈10 mM.
3 was prepared in and used from DMSO/H2O = 90:10 stock solutions
3
3
3
−
Scheme 2.
providing the reactive [Pt(DMSO)(OH)(phen)]+ intermediate, followed by slow DMSO loss and formation of the [{Pt(
OH)(phen)}2]2+ hydroxo-bridged dimer. The last step is disfavored by a high DMSO concentration. (
) Parallel mechanism
of water hydrolysis for complex
, favored by high Cl− concentration, providing the reactive [PtCl(DMSO)(phen)]+
(A) Hydrolysis in water of the [Pt(η 3) complex, involving the σ-C2H4OMe moiety,
1-C2H4OMe)(DMSO)(phen)]+ (
µ-
B
3
intermediate, producing by DMSO loss, the low soluble [PtCl2(phen)] species.
NMR monitoring demonstrated that complex
3
complete decomposition in neutral
water was slow (about three days) and occurred via formation of solvento species of the
type [Pt(DMSO)(OH)(phen)]+ and [{Pt( -OH)(phen)}2]2+, together with slight amounts of
µ
the insoluble [PtCl2(phen)] compound. These latter could therefore be considered as the
possibly involved active species produced when diluting in biological assays DMSO/H2O
= 90:10 stock solutions of complex 3.
3.5. Cytotoxicity of the Potential Antitumor Drugs [Pt(η1-C2H4OMe)(NH3)(phen)]+ (2) and
[Pt(η1-C2H4OMe)(DMSO)(phen)]+ (3)
Cells were treated with various concentrations (0.1–200 µM) of 2 or 3 in comparison
with cisplatin, and viable cell number was determined by MTT metabolic assay and
confirmed by SRB assay 12, 24, 48 and 72 h later.
For all cell lines, viable cell number values obtained after [Pt(η
1-C2H4OMe)(DMSO)(phen)]+
treatment were corrected for a “blank” value (‘blank’ was the mean optical density of the
background control wells containing the incubation medium and 0.725% DMSO, represent-
ing the highest DMSO quantity present in complex
3 solution). Furthermore, cell viability
after 0.725% DMSO treatments of SK-OV-3, Hep-G2, and SH-SY5Y cell lines did not change
significantly over 0–72 h incubation times and is shown in Figure S3.
Of the two new organometallic Pt(II)-complexes containing 1,10-phenantroline, only
complex
fact, it was possible to calculate the IC50 for compound
72 h of incubation (190.9 8.2 M), as shown in Figure 3; in all the other cell lines and
3
proved to be more effective than cisplatin in many of the cell lines used. In
2
only in HeLa cells and after
±
µ

Pharmaceutics 2021, 13, 642
9 of 16
for all the incubation times tested, it was not possible to determine an IC50 value. It is
generally known that the presence of DMSO causes a reduction of cytotoxicity or complete
inactivation of most of the tested antitumor drugs [46
the case of complex , when administered to tumor cells, the presence of DMSO conferred
a level of cytotoxicity comparable to that of cisplatin, rather than inactivating the molecule.
This was probably due to the hydrolysis mechanism involving first the loss of the η1
–49]. Interestingly, we found that in
3
-
C2H4OMe moiety and further the generation of active hydrolytic species exhibiting a
cytotoxicity comparable to that of cisplatin.
Figure 3. IC50 after 12, 24, and 48 h of treatment with [Pt(
and cisplatin at different concentrations (0.1, 1, 10, 100, and 200 µM) obtained in different human cancer cell lines.
η η
1-C2H4OMe)(DMSO)(phen)]+, [Pt( 1-C2H4OMe)(NH3)(phen)]+
Conversely, [Pt(
than cisplatin in six of the eight cell lines treated, with an IC50 evaluable already at 12 h of
incubation (Figure 3 and Table S1). IC50 at 12 h ranged between 56.9 7.7 M (SH-SY5Y
cells) and 139.1 6.8 M (ZL-55 cells), and IC50 at 24 h ranged between 45.8 7.8 M (SH-
SY5Y cells) and 178.5 9.3 M (Hep-G2 cells) (Table S1). Compared to cisplatin, a drastic
reduction of cell viability after treatment with was observed mainly in neuroblastoma
(SH-SY5Y); no significant differences between cisplatin and complex were observed
in Hep-G2 cells. Furthermore, an intermediate sensitivity to was shown by SK-OV-3
cells (Figure 3 and Table S1). The comparisons between the mortality curves of cisplatin,
[Pt( ), and [Pt( ) at various
1-C2H4OMe)(NH3)(phen)]+ ( 1-C2H4OMe)(DMSO)(phen)]+ (
η 3) showed higher cytotoxic effects
1-C2H4OMe)(DMSO)(phen)]+ (
±
µ
±
µ
±
µ
±
µ
3
3
3
η
2
η
3
concentrations and at different incubation times for SH-SY5Y, SK-OV-3, and Hep-G2 cells
are shown in Figures 4–6, respectively.

Pharmaceutics 2021, 13, 642
10 of 16
Figure 4.
Cell viability assay after [Pt(η -
1-C2H4OMe)(DMSO)(phen)]+, [Pt(η1
C2H4OMe)(NH3)(phen)]+, and cisplatin treatments in SH-SY5Y cell line. The viability of
the neuroblastoma cell line was evaluated after treatment with Pt compounds at different
concentrations (0.1, 1, 10, 100, and 200
µ
M) for 12, 24, 48, and 72 h. The data are means
±
S.D. of
three different experiments run in eight replicates and are presented as percent of control. Asterisks
indicate values that are significantly different (* p < 0.05; **** p < 0.0001) from those of cisplatin at the
same concentrations and time points.
As it can be seen, in SH-SY5Y cells, complex
cisplatin up to 24 h of treatment (IC50 between 56.9
much longer times (72 h) the effects were the same (Figure 4). In SK-OV-3 cells, complex
caused a halving of the cell population rather early (12–24 h) (IC50 between 92.1 7.8
and 62.8 6.6 M). By contrast, the IC50 was evaluable with cisplatin starting from 24 h of
3
was significantly more cytotoxic than
±
7.7 M and 45.8 7.8 M), and at
µ
±
µ
3
±
µM
±
µ
treatment (IC50 130
observed between
±
8.9
µM). However, also in this cell line, no significant differences were
and cisplatin at very long incubation times (72 h) (IC50 37.5 ± 3.2 µM
3
and 41.7 ± 4.6 µM, respectively) (Figure 5).

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Figure 5.
Cell viability assay after [Pt(η -
1-C2H4OMe)(DMSO)(phen)]+, [Pt(η1
C2H4OMe)(NH3)(phen)]+, and cisplatin treatments in SK-OV-3 cell line. The viability of
the ovarian adenocarcinoma cell line was evaluated after treatment with Pt compounds at different
concentrations (0.1, 1, 10, 100 and 200
µ
M) for 12, 24, 48, and 72 h. The data are means
±
S.D. of
three different experiments run in eight replicates and are presented as percent of control. Asterisks
indicate values that are significantly different (** p < 0.01; *** p < 0.001; **** p < 0.0001) from those of
cisplatin at the same concentrations and time points.
With regard to Hep-G2 cells, there were no differences between the effects of 3 and
those of cisplatin at any of the times tested and for any concentration in the 0.1–200
range (Figure 6).
µM
Then, we established whether the different sensitivity of the three cell lines mentioned
above (Hep-G2, SH-SY5Y, and SK-OV-3) depended on a different intracellular accumulation
of the Pt(II) compounds.

Pharmaceutics 2021, 13, 642
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Figure 6.
Cell viability assay after [Pt(η -
1-C2H4OMe)(DMSO)(phen)]+, [Pt(η1
C2H4OMe)(NH3)(phen)]+, and cisplatin treatments in Hep-G2 cell line. The viability of the
hepatocellular cell line was evaluated after treatment with Pt compounds at different concentrations
(0.1, 1, 10, 100, and 200
µ
M) for 12, 24, 48, and 72 h. The data are means
±
S.D. of three different
experiments run in eight replicates and are presented as percent of control. Asterisks indicate values
that are significantly different (* p < 0.05; ** p < 0.01; *** p < 0.001) from those of cisplatin at the same
concentrations and time points.
3.6. In-Cell Accumulation of the Tested Pt(II) Compounds
Total intracellular Pt(II) content was evaluated in Hep-G2, SH-SY5Y and SK-OV-3
after treatment with 100
C2H4OMe)(DMSO)(phen)]+ (
tion. In accordance with what has been found for cisplatin [50] and for [Pt(O,O0-acac)(
acac)(DMS)] [42 51 52], the here considered new Pt(II) compounds seemed to enter cells via
a passive or facilitated passive transport very quickly, and the cellular accumulation of
was higher than that of both cisplatin and (Figure 7 and Table S2). Thus, the high cytotoxi-
city of the [Pt(
1-C2H4OMe)(DMSO)(phen)]+ complex may be related to a high intracellular
µM of cisplatin, [Pt(
η 2 -
1-C2H4OMe)(NH3)(phen)],+ ( ) and [Pt(η1
3
) or by ICP-AES after 1.5, 3, 4.5, 6, and 12 h of incuba-
γ
-
,
,
3
2
η
uptake with respect to cisplatin, both in SH-SY5Y (about 4.5-fold) (Figure 7A) and SK-OV-3
cells (about 18-fold) (Figure 7B). However, no correlation between the cytotoxicity and
the intracellular uptake of the [Pt(
fact, this compound did not decrease cell viability, although its intracellular uptake was
η
1-C2H4OMe)(NH3)(phen)]+ complex was found; in

Pharmaceutics 2021, 13, 642
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greater than that of cisplatin in both SH-SY5Y (about four-fold) (Figure 7C) and SK-OV-3
cells (about three-fold) (Figure 7B). In addition, although complex
2 accumulated consider-
ably, it showed no significant cytotoxic effects in Hep-G2 cells (Figure 6). Therefore, these
Pt(II)-based compounds exhibited very different uptake profiles and cytotoxicity levels in
the three considered cell lines, suggesting diversified biological effects, thus making them
potentially tissue- and tumor-specific drugs.
Figure 7. Intracellular uptake after [Pt(
treatments in SH-SY5Y ( ), SK-OV-3 ( ) and Hep-G2 (
by ICP-AES; cells were continuously exposed to 100
means S.D. of three different experiments and is indicated as ng of Pt(II)/mg of protein. Asterisks indicate values that
η
1-C2H4OMe)(DMSO)(phen)]+, [Pt(
) cell lines. The total intracellular accumulation was determined
M of each Pt compound for 1.5–12 h. Each point represents the
η
1-C2H4OMe)(NH3)(phen)]+, and cisplatin
A
B
C
µ
±
are significantly different (* p < 0.05; *** p < 0.001; **** p < 0.0001) from those of cisplatin at the same concentration and
time point.
4. Conclusions
Several strategies have been developed to solve some of the problems related to the
clinical application of cisplatin, since its approval. Greater spectrum of efficacy, lower side
effects, and enhanced water solubility have been constantly targeted by the researchers
in the field [
species and, therefore, possible substrates for organic cation transporters (OCT) were
studied. In particular, two novel water-soluble organometallic cationic species, [Pt(η1
C2H4OMe)(NH3)(phen)]+ ( 1-C2H4OMe)(DMSO)(phen)]+ (
) and [Pt( ), were obtained
from [PtCl(
1-C2H4OMe)(phen)] by clorido substitution, using the Zeise’s anion, [PtCl3(η2
C2H4)]− as starting material. The new cationic complexes (
) were spectroscopically
1–4,16]. In this work, specific platinum drugs in the form of cationic solvento
-
2
η
3
η
-
2, 3
characterized, and their cellular uptake and cytotoxicity evaluated, in comparison with
cisplatin, for a range of tumor cell lines. The Pt(II)-based drugs exhibited very different
uptake profiles and cytotoxicity levels in the considered cell lines, suggesting diversified
biological effects. The DMSO complex (3) showed general higher cytotoxicity, with respect

Pharmaceutics 2021, 13, 642
14 of 16
to the amino species
2, exhibiting also lower IC50 values with respect to cisplatin in almost
all cell lines starting from the first hours of treatment, particularly in neuroblastoma
and adenocarcinoma cell lines. It is also worth pointing out that cellular uptake did not
appear to be directly related to cytotoxicity as (
a
) there was a significant uptake of the
in hepatocarcinoma cells without this compound being more effective
) the uptake of the NH3 complex was elevated in liver cells without
) the uptake of the new complexes was always
DMSO complex
than cisplatin; (
3
b
demonstrating important cytotoxic effects; (
c
greater than that of cisplatin and strain-dependent in the investigated cells, even if their
cytotoxicity was not always greater than that of cisplatin. The different behavior of the two
[Pt(
DMSO derivative with respect to that with NH3, with consequent easier formation of active
solvento species containing the Pt(phen) moiety. This aspect differentiates complex from
cisplatin where the presence of DMSO can inhibit the formation of the antitumor active
aquo species, with a consequent general reduction of the observed cytotoxicity [46 49 53].
More in-depth studies will be conducted in order to determine the mechanism of action
of the [Pt( ) complex and its metabolic alterations in the
1-C2H4OMe)(DMSO)(phen)]+ (
η
1-C2H4OMe)(L)(phen)]+ complexes could be related to the much higher lability of the
3
,
,
η
3
cell lines more sensitive to its antiproliferative activity. Nevertheless, the new Pt(II)-
based compounds exhibited specific uptake profiles and different cytotoxicity levels in the
considered cell lines, suggesting the occurrence of diversified biological effects. Therefore, a
potential use of the obtained species as models for tissue- and tumor-specific drugs design
is also envisaged.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10
.3390/pharmaceutics13050642/s1, Figure S1. Water hydrolysis of the [Pt(
complex
followed by 1H NMR spectroscopy. Figure S2. 195Pt NMR spectrum of the [{Pt(
OH)(phen)}2]2+ solvento species. Figure S3. Cell viability assay after 0.725% DMSO treatments in
SK-OV-3, Hep-G2, and SH-SY5Y cell lines. Table S1. The IC50 after [Pt(
1-C2H4OMe)(DMSO)(phen)]+
and cisplatin treatment in eight human cancer cell lines. Table S2. Pt total intracellular accumulation,
η
1-C2H4OMe)(DMSO)(phen)]+
3
µ-
η
determined by ICP-AES, in SH-SY5Y, SK-OV-3, and Hep-G2 cell lines, exposed to 100 µM of each of
the 2 and 3 Pt compounds for 1.5–12 h.
Author Contributions: F.D.C. and E.S. contributed equally. Conceptualization, M.B. and A.M.;
methodology, F.D.C., E.S.; investigation, F.D.C., G.N.I., D.M. and E.S.; data curation, F.D.C., E.S.,
M.B. and A.M.; writing—original draft preparation, M.B.; writing—review and editing, M.B., F.P.F.
and A.M.; supervision, M.B. and S.M. All authors have read and agreed to the published version of
the manuscript.
Funding: This research received no external funding.
Conflicts of Interest: The authors declare no conflict of interest.
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