Synthesis & antitumor activity of new group 3 Metallocene complexes

Article abstract of DOI:10.3390/molecules22040526

The quest for alternative drugs with respect to the well-known cis-platin and its derivatives, which are still used in more than 50% of the treatment regimens for patients suffering from cancer, is highly needed. In this context, organometallic compounds, which are defined as metal complexes containing at least one direct covalent metal-carbon bond, have recently been found to be promising anticancer drug candidates. A series of new metallocene complexes with scandium, yttrium, and neodymium have been prepared and characterized. Some of these compounds show a very interesting anti-proliferative activity in triple negative breast cancer cell line (MDA.MB231) and the non-hormone sensitive prostate cancer cell line (DU145). Moreover, the interaction of some of them with biological membranes, evaluated using liposomes as bio-membrane mimetic model systems, seems to be relevant. The biological activity of these compounds, particularly those based on yttrium, already effective at low concentrations on both cancer cell lines, should be taken into account with regard to new therapeutic approaches in anticancer therapy.

Full text of DOI:10.3390/molecules22040526

molecules
Article
Synthesis and Antitumor Activity of New Group 3
Metallocene Complexes
Angelamaria Caporale 1,†, Giuseppe Palma 2,†, Annaluisa Mariconda 3, Vitale Del Vecchio 2,
Domenico Iacopetta 4, Ortensia Ilaria Parisi 4, Maria Stefania Sinicropi 4, Francesco Puoci 4,
Claudio Arra 2,*, Pasquale Longo 3 and Carmela Saturnino 1,
*
1
Department of Science, University of Basilicata, Viale dell'Ateneo Lucano 10, Potenza 85100, Italy;
acaporale@unisa.it
2
3
4
SSD Sperimentazione Animale, Istituto Nazionale Tumori, IRCCS, “Fondazione G. Pascale”,
Via Mariano Semmola, Napoli 80131, Italy; palma.giuseppe@icloud.com (G.P.); vitale_84@hotmail.it (V.D.V.)
Department of Biology and Chemistry, University of Salerno, Via Giovanni Paolo II, 132, Fisciano 84084,
Italy; amariconda@unisa.it (A.M.); plongo@unisa.it (P.L.)
Department of Pharmacy, Health and Nutritional Sciences, University Calabria, Via Pietro Bucci,
Arcavacata di Rende 87036, Italy; domenico.iacopetta@unical.it (D.I.); ortensiailaria.parisi@unical.it (O.I.P.);
s.sinicropi@unical.it (M.S.S.); francesco.puoc i@unical.it (F.P.)
*
Correspondence: c.arra@istitutotumori.na.it (C.A.); carmela.saturnino@unibas.it (C.S.);
Tel.: +39-0815-903-645 (C.A.)
†
These authors contributed equally to the manuscript.
Academic Editor: Derek J. McPhee
Received: 25 February 2017; Accepted: 22 March 2017; Published: 28 March 2017
Abstract: The quest for alternative drugs with respect to the well-known cis-platin and its derivatives,
which are still used in more than 50% of the treatment regimens for patients suffering from cancer,
is highly needed. In this context, organometallic compounds, which are defined as metal complexes
containing at least one direct covalent metal-carbon bond, have recently been found to be promising
anticancer drug candidates. A series of new metallocene complexes with scandium, yttrium,
and neodymium have been prepared and characterized. Some of these compounds show a very
interesting anti-proliferative activity in triple negative breast cancer cell line (MDA.MB231) and the
non-hormone sensitive prostate cancer cell line (DU145). Moreover, the interaction of some of them
with biological membranes, evaluated using liposomes as bio-membrane mimetic model systems,
seems to be relevant. The biological activity of these compounds, particularly those based on yttrium,
already effective at low concentrations on both cancer cell lines, should be taken into account with
regard to new therapeutic approaches in anticancer therapy.
Keywords: breast cancer; prostate cancer; metallocene group 3; metal complexes; cytotoxic activity;
cell membrane permeability
1. Introduction
After the remarkable effectiveness demonstrated by cis-platinum [cis(NH3)2PtCl2] as an anticancer
drug, mainly in the clinical treatment of ovarian, testicular, and head and neck cancers [1–3], scientists
have developed considerable studies to obtain new organometallic compounds with less toxic
effects [4].
The cis-platinum and its derived molecules, as oxaliplatin and carboplatin, are still in use in more
than 50% of cancer treatment; on the other hand, these platinum compounds suffer from two major
drawbacks: they are ineffective against platinum-resistant cancer, and have serious side effects, such
as nephrotoxicity and neurotoxicity [5,6].
Molecules 2017, 22, 526; doi:10.3390/molecules22040526
www.mdpi.com/journal/molecules

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Platinum complexes have labile cis-chloride groups or similar ligands that enter into the cells
by diffusion, here they are converted to the active form; this species is not determined with great
sureness. It is believed that the most active species is the monohydrate platinum complex, even if
the most common one is the di-hydrate form. The active cis-platin compound interacts with many
types of proteins involved in DNA replication and cell division, but the main target is the DNA double
strand [7–11].
An important aspect in cancer research is represented by achieving new molecules with lower
toxicity and higher selectivity towards cancer cells with respect to the drugs already used in clinical
treatment [4,12].
In this context, organometallic compounds, defined as metal complexes containing at least one
direct covalent metal-carbon bond including those of group 3, have recently been highlighted as good
anticancer candidates for their significant biological activity [13–28].
Many of the pharmacological properties of the group 3 metals are due to the ability to replace
calcium ions (Ca2+) in biological molecules and strongly bond water molecules; furthermore, they can
also substitute metal ions Mg2+, Mn2+, and Fe3+ in biological effectors involved in cell mechanisms [13
,14].
These features lead to modifying many cellular processes, for example, inhibiting or activating
calcium-dependent enzymes. This different regulation depends on the role of calcium in the native
enzyme: if calcium plays a catalytic role, its substitution by an ion of group 3 leads to the enzyme
deactivation, and the degree of inhibition depends on the ionic radius of the metal. Instead, if Ca2+
plays a structural role, the replacing with a M3+ ion preserves the function of the effector [13
,14]. In this
way the antitumor action of group 3 metal complexes is carried out through the positive or negative
regulation of cell cycle effectors.
The group 3 metal and lanthanide complexes bound with coumarin, show higher activity
in various tumor models than the corresponding inorganic salts [15
lanthanide complexes with monoanionic macrocyclic ligands containing five nitrogen atoms
coordinated to the central core, are approved for clinical trials [19 20], as well as the series of cerium
–18]. Moreover texaphyrins,
,
(III) with bipyridyl or phenanthroline ligands that show anti-proliferative activity against tumor cell
lines [21,22].
Recently, the cytotoxic and anti-proliferative activity of several new group 3 compounds has been
evaluated: lanthanides with heterocyclic ligands (tridentate monoanionic quinoline-phenoxy-amine,
quinoline-phenoxyimine, and ansa-monocyclopentadienyl-immino-pyridine) [23–27], and also
complexes with heteroscorpionate group 3 (i.e., N,N0-dicyclohexyl-2,2 bis-(3,5-dimethyl-pyrazol-1-yl)-
acetamidinate). All of these molecules have proven to be good candidates for the synthesis of a wide
range of stable compounds of group 3 and lanthanide derivatives with anti-proliferative activity [28].
The metals with cyclopentadienyl or cyclopentadienyl-derivative ligands instead need other
detailed studies because they showed a strong anti-proliferative effect on cancer with a high grade of
cytotoxicity [29–34].
The complexes of titanium (Group 4) with those of the third group metals may have different
mechanisms of action, leading to a completely different specificity and selectivity.
The selected ligands have the ability to coordinate the metal center in a bi-dentate or mono-dentate
manner. The latter type of coordination allows the ligand to give additional interactions with target
molecules. Moreover, we synthesized both mono-cyclopentadienyl and bis-cyclopentadienyl group 3
metal complexes because, as observed with titanium derivatives, half-metallocenes and metallocenes
may exert different activities [28–31].
In this paper, we report the synthesis and characterization of several group 3 metal complexes,
selected among those who have already proved ability to stabilize the cation titanium by intramolecular
or intermolecular coordination, and the data about their potential anti-proliferative activity in triple
negative breast cancer and prostate cancer cell lines. Moreover we also show the interaction between
some of the synthesized metal complexes and biological membranes, which often act as barriers in

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the physiological processes of absorption and distribution of therapeutic agents into the body, with a
remarkable effect on the pharmacological effectiveness [35,36].
2. Results and Discussion
2.1. Chemistry
The group 3 metals have significant differences in atomic radius and electronegativity and this
parameter could influence their antitumor activity; therefore, we synthesized scandium, yttrium,
and neodymium complexes.
Synthesis of group 3 and lanthanide metal complexes was carried out in four steps in an inert
nitrogen atmosphere, as reported in the Figure 1.
(i) Cyclopentadiene was obtained by a retro Diels–Alder of dicyclopentadiene at high temperature
(180 ◦C).
(ii) The synthesis of fulvene pro-ligands by reaction of cyclopentadiene with suitable aromatic aldehyde.
(iii) Lithium salts of ligands were synthesized by reacting the appropriate fulvene with LiEt3BH
(super-hydride), in dry diethyl ether. The reaction is a nucleophilic addition of a hydride to the
double bond 5–6 of fulvene. We used a slight excess with respect to the stoichiometric amount of
super-hydride. The choice of the Li(Et)3BH as reducing agent derived from its high selectivity.
In fact, the double bond 5–6 of fulvene had a high polarity due to the inductive effect of the
methoxy group bonded to benzene. By this increase of the polarity, the reducing agent attacked
the double bond 5–6 and not the dienic component of fulvene [28–30].
(iv) Synthesis of group 3 and lanthanide complexes were carried out in THF at
◦
−
78 C, by the reaction
of a chosen lithium salt of ligands with the suitable stoichiometric amount of metal(III) halide,
in order to obtain half- and metallocene derivatives and, consequently, to evaluate the influence
of these lipophilic groups on the biological activity of the compounds.
Figure 1. Synthetic route for the preparation of compounds 1–12.
All of the synthesized complexes were purified following common procedures and isolated in
1
good yields. Elemental analysis (C, H) were in agreement with the proposed formulae. H COSY
experiments allowed the assignment of all the proton resonances of the 1H-NMR spectra, whereas
DEPT experiments were useful for the attribution of 13C-NMR signals. The synthesized compounds
were also characterized by mass spectrometry and elemental analysis. The set of these data (see Result
and Discussion) allowed us to have an unambiguous structural determination, as reported in Figure 1
and Table 1.

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Table 1. Synthesized compounds 1–12.
Compound
R
M
Name
1
2
3
4
5
6
7
8
9
H
H
H
Sc
Y
Nd
Sc
Y
Nd
Sc
Y
Nd
Sc
Y
[(p-methoxybenzyl)cyclopentadienyl]scandium dichloride
[(p-methoxybenzyl)cyclopentadienyl]yttrium dichloride
[(p-methoxybenzyl)cyclopentadienyl]neodymium dichloride
[(3,4-dimethoxybenzyl)cyclopentadienyl]scandium dichloride
[(3,4-dimethoxybenzyl)cyclopentadienyl]yttrium dichloride
[(3,4-dimethoxybenzyl)cyclopentadienyl]neodymium dichloride
bis-[(p-methoxybenzyl)cyclopentadienyl]scandium chloride
bis-[(p-methoxybenzyl)cyclopentadienyl]yttrium chloride
bis-[(p-methoxybenzyl)cyclopentadienyl]neodymium chloride
bis-[(3,4-dimethoxybenzyl)cyclopentadienyl]scandium chloride
bis-[(3,4-dimethoxybenzyl)cyclopentadienyl]yttrium chloride
bis-[(3,4-dimethoxybenzyl)cyclopentadienyl]neodymium chloride
-OCH3
-OCH3
-OCH3
H
H
H
-OCH3
-OCH3
-OCH3
10
11
12
Nd
Hydrolysis Stability
The hydrolysis stability of the six half-metallocene (1–6) and of six metallocene complexes (7–12)
has been determined in aqueous solution, 90% d6-DMSO by 1H-NMR spectroscopy, in order to correlate
the chemical stability and coordination chemistry of these complexes with their observed cytotoxic
activity. Since we can expect that rapid hydrolysis of the leaving group (-Cl) and cyclopentadienyl
ligands could give biologically-inactive species, an active species could be generated if the Cp rings
remain metal-bound. Hydrolysis of aromatic rings of 1–6 and 7–12 complexes was evaluated by
integration of two signals of protons of cyclopentadienyl bonded to metal, as to newly-formed
multiples of substituted cyclopentadiene. All of the complexes show high hydrolytic stability; in fact,
the cyclopentadienyl rings of complexes are hydrolyzed for less than 5% after 24 h. These data
provide sufficient evidence that the presence of coordinating groups on the aryl substituent of the
cyclopentadienyl are effective for the stabilization of the complexes. Therefore, these coordinating
groups might be fundamental to increase their biological effectiveness.
2.2. Biological Activity
In order to assess the cytotoxic effects on cancer cell proliferation of novel synthesized complexes,
we treated MDA.MB231 and DU145 with each compound, testing concentrations in a range from 5
µM
to 100 M. The obtained data have been statistically analyzed in order to assess the concentration
µ
able to inhibit the 50% of cell growth respect to the untreated control (IC50 value). Several nonlinear
functions have been commonly used to estimate the IC50 value, finding a relation between the drug
concentration, on the X axis, and the percentage of survival at this concentration, on the Y axis.
We propose a statistical nonlinear model, for a better evaluation of the dose response relationship
between our variables. In our experiments, we apply a polynomial fitting curve using local regression;
this is a least squares method which minimizes the error between the original data and the values
predicted by the equation. The goodness of fit has been evaluated with the determination of an R2
value which is higher than 0.789 in all models of IC50 assessment of the described compounds, on both
adopted cell lines, (0.789 < R2 < 0.987). In Figure 2 we report the cytotoxic activity and in Table 2 the
IC50 values of active compounds on both cell lines from the MTT assay data processing.
All synthesized compounds show an inhibition growth activity (see Supplementary Materials)
but compounds 10, 2, 9, and 12 show a low efficiency yet in the highest concentrations. The yttrium
complexes seem more efficient already at very low concentrations; in fact, the IC50 value for
5 and 8 is
less than 5 µM.
Our results show an impaired rate of growth inhibition by different compounds on the same cell
line, but also by the same compound on different cell lines. The highest inhibition effect is made by
compound
1, on MDA.MB231, and by 3 and 8 on DU145 (the IC50 of these molecules is under the first
value of the tested concentration and, for this reason, it is not determined numerically). Each of these

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molecules is comprised of a different metal core:
1
has the best activity amongst scandium compounds,
3
amongst neodymium compounds, and 8 amongst yttrium compounds. We can assess that, on the
MDA.MB231 cell line, all of the compounds with yttrium show a good inhibition effect, metallocene
as well as half-metallocene. Furthermore, the scandium-based compounds do not show a significant
effect, except for
1. Finally, the neodymium based compounds have a mild effect. On the DU145 cell
line we observe a wide spectrum of IC50 referring to the same class of compounds.
Figure 2. The cytotoxic activity of compounds was evaluated as IC50
(µM) responses from 5 µM to
100 M of compounds in MDA.MB231, human triple negative breast cancer and DU145, human
µ
1–2
non-hormone sensitive prostate cancer, defined by the MTT assay. Cell growth was measured as the
percentage of cells treated with different concentrations, distinct compounds respect to cells treated for
48 h. The IC50 of
1, 3, and 8 are under the first value of tested concentration and for this reason it is not
determined numerically; we show an approximated value.
Table 2. Effect of complexes 1–12 on MDA.MB231, human triple negative breast cancer cells, and DU145,
prostate cancer cell line.
MDA.MB231
Yttrium
DU145
Scandium
Neodymium
Scandium
Yttrium
Neodymium
Compounds
IC50 (µM)
Compounds
IC50 (µM)
Compounds
IC50 (µM)
Compounds
IC50 (µM)
Compounds
IC50 (µM)
Compounds
IC50 (µM)
1
<10
5
<5
3
12
1
<10
5
<5
3
12
4
<20
8
<5
6
50
4
<20
8
<5
6
50
7
20
11
<50
7
<20
11
<50
The differences between the three series of synthesized complexes are certainly indicative of the
very complex mechanisms of cell growth inhibition, closely related to the molecule structures and
not only to the ligand or to metal ion separately. From a structural point of view the data analysis

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shows that, among the scandium compounds, the most active are the two half-metallocenes with the
1
which shows the best IC50 value on the MDA.MB231, as well as on DU145. The significant activities of
the two half-metallocenes of scandium, compared with two scandiocenes, might be due to the small
atomic radius of metal ion that could allow intramolecular coordination of the methoxy groups of the
ligands in the case of half-scandiocenes, with a gain of stability of the complexes. This coordination
may not be possible in the case of scandiocenes because of steric hindrance of ligands.
The yttrium complexes with better activity, on both tested cell lines, were the half-metallocene 5
and the metallocene
of two methoxy groups: both on one aryl group in
stabilization of yttrium complexes, the coordination of two methoxyl groups to the metal ion core is
required. The complexes of neodymium are not particularly active, except for , which shows a strong
8. In this case, it seems that the inhibitor activity may be related to the presence
5
or one for each aryl group in . Clearly, for the
8
3
activity, probably due to its lower electronegativity, than scandium and yttrium, which do not allow
the intramolecular coordination of the methoxy groups, for which the complexes turn out to be less
stable [37].
2.3. Evaluation of the Drug-Membrane Interaction
In our experiments, we used liposomes as mimetic models able to reproduce the anisotropic
properties of cell membranes. In this way, we investigated the interaction between synthesized
metal complexes and biological membranes, evaluating the cell permeability, also considering the
electrostatic interaction. Based on these considerations, the use of this kind of membrane model is
more reliable than the octanol/water partitioning to evaluate the lipophilicity of a drug.
The ability of a drug to interact with bio-membranes affects its biological properties in terms
of both therapeutic and toxic effects. Therefore, the evaluation of the partitioning behavior between
lipid and water phases, throughout the quantification of the partition coefficient (kp), allows to
predict the ability of the therapeutic agent to pass across the membrane to estimate its in vivo activity.
The partition coefficient (kp), which is considered a useful parameter for the design and development
of new effective antitumor agents, of compounds
After the ultrafiltration process, drug concentrations in the aqueous phase were quantified by UV-VIS
spectrophotometry and the obtained kp values were 1700 for and 1200 for . The experimental results
8 and 4 was evaluated by the ultrafiltration method.
8
4
indicate that both anticancer agents tested have shown a relevant membrane permeability.
3. Materials and Methods
3.1. General Procedure
All manipulations were carried out under oxygen- and moisture-free atmosphere, using Schleck
techniques or working in an MBraun MB 200 glove-box (Daimlerstraße 29-31 D-76316 Malsch, Germany).
3.2. Solvents
All the solvents were thoroughly deoxygenated and dehydrated under argon by refluxing over
suitable drying agents; while NMR deuterated solvents (Euriso-Top products, Parc des Algorithmes,
Bâtiment Homère Route de l'Orme F-91194 Saint-Aubin Cedex, France) were kept in the dark over
molecular sieves.
3.3. Reagents
All chemicals were obtained from Sigma-Aldrich (Sigma Aldrich SRL Via Gallarate. 154 I-20151
Milano, Italy) and used without further purification. Cyclopentadiene was obtained by freshly
cracked dicyclopentadiene.

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3.4. Characterization Techniques
1H-NMR, homodecoupled 1H-NMR, 1H COSY, and 13C-NMR spectra were recorded on a Bruker
Avance 300 Spectrometer (Bruker Co., Billerica, MA, USA) operating at 300 MHz (1H) and 75 MHz
(13C). Chemical shifts referring to tetramethylsilane were used as the internal standard.
3.5. Cell Lines
The MDA-MB-231 tumor cell line of triple negative breast human cancer was purchased from
American Type Culture Collection (ATCC®HTB-26™, Manassas, VA, USA), MDA-MB-231 cells were
cultured in Dulbecco’s Modified Eagle’s Medium (Invitrogen, Carlsbad, CA, USA) supplemented with
10% fetal bovine serum and 1% penicillin/streptomycin, at 37 ◦C in 5% CO2- 95% air. The DU145
tumor cell line of human non-hormone sensitive prostate cancer, was purchased from American Type
Culture Collection (ATCC®HTB-81™). DU145 cells were cultured in RPMI medium supplemented
with 10% fetal bovine serum and 1% penicillin/streptomycin, at 37 ◦C in a humidified atmosphere
containing 5% CO2.
3.6. Biological Assay Kit
The cell viability was assessed using standard MTT assay according to the manufacturer’s
instructions (MTT cell proliferation assay kit, purchased from TREVIGEN, catalog #4890-025-K, 8405
Helgerman Ct, Gaithersburg, MA, USA).
Analysis of cell proliferation was performed in the presence of different concentration of any
synthetized compounds on cell lines seeded in 96-well plates at the density of 1–2
in serum-containing media. After 24 h of incubation at 37 C, cells were treated with increasing
×
103 cells/well
◦
concentrations of compounds or vehicle (DMSO; maximum concentration
≤
0.5%), following which
the cells were cultured in drugs for 48 h, and 10 L MTT solution (TREVIGEN, Helgerman Ct) was
µ
added subsequent to drawing off the medium. The mixture was incubated for an additional 4 h at
37 ◦C, the absorbance was read at 572 nm in a microplate reader (Biocompare, Italy). Cell viability
was expressed as the percentage of proliferated cells compared to cells treated with the different
compounds. The same cells were used as control. All experiments were repeated in triplicate [38
–41].
3.7. Evaluation of the Drug-Membrane Interaction
3.7.1. Liposomes Preparation
Liposomes preparation was carried out following the thin film hydration method [35].
Egg phosphatidylcholine (EPC) was dissolved in chloroform and, then, the obtained solution was
evaporated leading to the formation of a lipid film (final lipid concentration 2 mM), which was left
under vacuum overnight in order to remove solvent traces. The dried film was hydrated with HEPES
buffer (10 mM, I = 0.1 M, pH 7.4) and vortexed at room temperature to get multilamellar vesicles
(MLVs). After 30 min at 25 ◦C, the resultant MLVs suspension was extruded at room temperature
10 times through polycarbonate filters (Nucleopore, Pleasanton, CA, USA, pore size 100 nm) obtaining
large unilamellar vesicles (LUVs).
3.7.2. Determination of the Partition Coefficient (Kp)
The partition coefficient (Kp) of
8 and 4 between liposomes and the aqueous phase was
determined by ultrafiltration and using UV-Vis spectrophotometry to quantify drug concentrations in
the aqueous phase.
The egg phosphatidylcholine stock suspension was diluted to get a LUVs suspension characterized
by an EPC concentration equal to 1000 µM, and contained a constant concentration of the active
molecule (40
µ
M). A reference sample was prepared according to the same experimental protocol but
◦
in the absence of liposomes. The obtained samples were incubated at 25 C for 2 h in dark conditions to

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reach the partition equilibrium state. Then, the samples were placed in diafiltration tubes (Vivaspin 20
centrifugal concentrator, Sigma-Aldrich, Italy, MWCO 3000 Da) and centrifuged in order to separate the
aqueous phase from liposomes. The UV-Vis absorption spectra of the aqueous phases were recorded
with a Jasco V-530 UV/Vis spectrometer (Eppendorf, Milan, Italy). The drug concentrations in lipid
membrane phase were achieved by subtracting the concentration in the water phase of the tested
sample from the concentration in the water phase of the correspondent reference sample prepared in
the absence of liposomes.
Partition coefficients were calculated by the following Equation (1):
nm
nw
Kp =
(1)
where nm and nw are the number of drug moles distributed on the lipid membrane phase and in the
aqueous phase, respectively.
4. Synthesis of Ligands
0
0
0
The pro-ligands: 4 -methoxy-phenylfulvene, 6-(3 ,4 -dimethoxyphenyl) fulvene were synthesized
as reported in the literature and, afterwards, were reduced by a solution of the super-hydride LiBEt3H.
4.1. Synthesis of Group 3 Metal Cyclopentadienyl Complexes
4.1.1. General Procedure
Two separate flasks of the lithium salt of ligands (1.0 mmol) and the appropriate amount of MCl3
(1.0 mmol for half-metallocene complexes or 0.5 mmol for metallocene complexes) were weighed in a
dry-box. The ligand was dissolved in 8 mL of anhydrous THF and introduced in a dropping funnel,
then◦it was slowly added to a solution of metal halide in 32 mL of THF previously thermostated at
−
78 C. The reaction was kept under stirring overnight. The temperature was allowed to rise to room
temperature, then the solvent was removed under vacuum and dichloromethane was added the solid.
The mixture was filtered on celite, to remove the lithium chloride, and then the solution was dried in
vacuum. The products were characterized by NMR analysis.
4.1.2. Yields and Spectral Data of Scandium Complexes
[Cp-CH2-C6H5-OCH3)]ScCl2 [(p-Methoxybenzyl) cyclopentadienyl] scandium dichloride (1) Yield: 95%,
1H-NMR (300 MHz, CDCl3): 2.80 (dd, 1H, CH2), 2.92 (dd, 1H, CH2), 3.60 (s, 3H, OCH3), 5.98–6.37
(m, 4H, C5H4), 6.84 (d, 2H, C6H4), 7.05 (d, 2H, C6H4). 13C-NMR (300 MHz, d6-DMSO): 42.9 (CH2),
55.8 (OCH3), 113.3 (C30 –C50 of C6H4), 128.1(C10 of C6H4), 129.9 (C20 –C60 of C6H4), 131.6 (C1 of C5H4),
132.6–132.9 (C2–C5 of C5H4), 134.5–134.8 (C3–C4 C5H4), 158.0 (C40 -OCH3 of C6H4). ESI-MS (CH3CN,
m/z): 288–290 Dalton [C13H13OClScNa]+. Elemental analysis: found (%): C 51.47; H 4.19. Calc. for
C13H13Cl2OSc (%): C 51.86; H 4.35.
[Cp–CH2–C6H4–(OCH3)2] ScCl2 [(3,4-Dimethoxybenzyl)cyclopentadienyl]scandium dichloride (4). Yield:
33%, 1H-NMR (300 MHz, CDCl3): 2.77 (dd, 1H, CH2), 2.91 (dd, 1H, CH2), 3.80 (s, 6H, OCH3), 5.87–6.33
(m, 4H, C5H4), 6.67 (m, 3H, C6H3). 13C-NMR (300 MHz, d6-DMSO): 33.9 (CH2), 54.8 (OCH3), 110.6
(C20 of C6H3), 111.8 (C50 of C6H3), 112.2 (C60 of C6H3), 117.9 (C10 of C6H3), 119.9 (C1 of C5H4), 130.0
(C2–C5 of C5H4), 134.9 (C3–C4 C5H4), 146.7 (C40 –OCH3 of C6H3), 148.3 (C30 –OCH3 of C6H3). ESI-MS
(CH3CN, m/z): 299.4 Dalton [C14H15O2ScK]+. Elemental analysis: found (%): C 50.52; H 4.76. Calc.
for C14H15Cl2O2Sc (%): C 50.78; H 4.57.
[Cp-CH2-C6H5-OCH3]2ScCl bis-[(p-Methoxybenzyl)cyclopentadienyl]scandium chloride (7). Yield: 24%,
1H-NMR (300 MHz d6-DMSO): 2.80 (dd, 2H, CH2), 2.92 (dd, 2H, CH2), 3.71 (s, 6H, OCH3), 5.76–6.38
(m, 8H, C5H4), 6.83 (d, 4H, C6H4), 7.10 (d, 4H, C6H4). 13C-NMR (300 MHz, d6-DMSO): 40.1 (CH2),
53.9 (OCH3), 112.9 (C30 –C50 of C6H4), 125.7(C10 of C6H4), 129.3 (C20 –C60 of C6H4), 130.2 (C1 of C5H4),

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130.9–131.3 (C2–C5 of C5H4), 132.6–133.7 (C3–C4 of C5H4), 155.1 (COCH3). ESI-MS (CH3CN, m/z):
422.2 Dalton [C26H26O2ScLi]+. Elemental analysis: found (%): C 69.79; H 5.12. Calc. for C26H26ClO2Sc
(%): C 69.26; H 5.81.
[Cp-CH2-C6H5-(OCH3)2]2ScCl bis-[(3,4-Dimethoxybenzyl)cyclopentadienyl]scandium chloride (10). Yield:
1
37%, H-NMR (400 MHz, CD2Cl2): 2.93 (dd, 2H, CH2), 2.99 (dd, 2H, CH2), 3.83 (s, 12H, OCH3),
6.17–6.56 (m, 8H, C6H4), 6.87 (m, 6H, C6H3). 13C-NMR (300 MHz, d6-DMSO): 36.3 (CH2), 55.0 (OCH3),
111.9 (C20 of C6H3), 112.2 (C50 of C6H3), 114.9 (C60 of C6H3), 120.4 (C10 of C6H3), 122.1 (C1 of C5H4),
132.7 (C2–C5 of C5H4), 137.2 (C3–C4 C5H4), 148.9 (C40 –OCH3 of C6H3), 149.2 (C30 –OCH3 of C6H3).
ESI-MS (CH3CN, m/z): 514.6 Dalton [C28H30O4ScK]+. Elemental analysis: found (%): C 65.23; H 5.41.
Calc. for C28H30O4ClSc (%): C 65.82; H 5.92.
4.1.3. Yields and Spectral Data of Yttrium Complexes
[C5H4–CH2–C6H4–OCH3]YCl2 [(p-Methoxybenzyl)cyclopentadienyl]yttrium dichloride (2). Yields: 42%,
1H-NMR (300 MHz, d6-DMSO): 2.80 (dd, 1H, CH2), 2.92 (dd, 1H, CH2), 3.71 (s, 3H, OCH3), 6.22–6.38
(m, 4H, C5H4), 6.82 (d, 2H, C6H4), 7.09 (d, 2H, C6H4). 13C-NMR (300 MHz, d6-DMSO): 42.7 (CH2),
54.9 (OCH3), 113.7 (C30 –C50 of C6H4), 127.1 (C10 of C6H4), 129.4 (C20 –C60 of C6H4), 131.4 (C1 of
C5H4), 132.5–132.6 (C2–C5 of C5H4), 134.0–134.4 (C3–C4 of C5H4), 157.5 (COCH3). ESI-MS (CH3CN,
m/z): 348–350 Dalton [C13H13ClOYK]+. Elemental analysis: found (%): C 45.59; H 3.13. Calc. for
C13H13Cl2OY (%): C 45.25; H 3.80.
[C5H4–CH2–C6H3(OCH3)2]YCl2 [(3,4-Dimethoxybenzyl)cyclopentadienyl]yttrium dichloride (5). Yield: 46%,
1H-NMR (300 MHz, CDCl3): 2.82 (dd, 1H, CH2), 2.94 (dd, 1H, CH2), 3.60 (s, 3H, OCH3), 3.71 (s, 3H,
OCH3), 6.00–6.39 (m, 4H, C5H4), 6.76 (m, 3H, C6H3). 13C-NMR (300 MHz, d6-DMSO): 34.7 (CH2), 53.5
(OCH3), 110.3 (C20 of C6H3), 111.4 (C50 of C6H3), 113.3 (C60 of C6H3), 118.8 (C10 of C6H3), 120.7 (C1 of
C5H4), 130.6 (C2–C5 of C5H4), 135.8 (C3–C4 C5H4), 147.5 (C40 –OCH3 of C6H3), 148.3 (C30 –OCH3 of
C6H3). ESI-MS (CH3CN, m/z): 238.2 Dalton [C14H15O2YNa]+. Elemental analysis: found (%): C 44.21;
H 4.54. Calc. for C14H15Cl2O2Y (%): C 44.83; H 4.03.
[Cp-CH2-C6H5-OCH3]2YCl bis-[(p-Methoxybenzyl)cyclopentadienyl]yttrium chloride (8). Yield: 21%,
1H-NMR (300 MHz, d6-DMSO): 2.80 (dd, 2H, CH2), 2.92 (dd, 2H, CH2), 3.70 (s, 6H, OCH3), 5.99–6.38
(m, 8H, C5H4), 6.84 (d, 4H, C6H4), 7.10 (d, 4H, C6H4). 13C-NMR (300 MHz, d6-DMSO): 40.8 (CH2),
54.3 (OCH3), 113.1 (C30 –C50 of C6H4), 125.9(C10 of C6H4), 129.3 (C20 –C60 of C6H4), 130.4 (C1 of C5H4),
131.1–131.6 (C2–C5 of C5H4), 133.6–134.0 (C3–C4 of C5H4), 156.1 (COCH3). ESI-MS (CH3CN, m/z):
482.9 Dalton [C26H26O2YNa]+. Elemental analysis: found (%): C 62.89; H 5.34. Calc. for C26H26ClO2Y
(%): C 63.11; H 5.30.
[Cp-CH2-C6H5-(OCH3)2]2YCl bis-[(3,4-Dimethoxybenzyl)cyclopentadienyl]yttrium chloride (11). Yield: 36%,
1H-NMR (400 MHz, d6-DMSO): 2.88 (dd, 2H, CH2), 3.02 (dd, 2H, CH2), 3.79 (s, 12H, OCH3), 6.08–6.48
(m, 8H, C6H4), 6.93 (d, 4H, C6H3), 7.19 (d, 2H, C6H3). 13C-NMR (300 MHz, d6-DMSO): 35.7 (CH2), 54.8
(OCH3), 111.3 (C20 of C6H3), 111.8 (C50 of C6H3), 114.2 (C60 of C6H3), 119.0 (C10 of C6H3), 121.9 (C1 of
C5H4) , 131.6 (C2–C5 of C5H4), 136.8 (C3–C4 C5H4), 148.5 (C40 –OCH3 of C6H3), 148.8 (C30 –OCH3 of
C6H3). ESI-MS (CH3CN, m/z): 526.1 Dalton [C28H30O4YLi]+. Elemental analysis: found (%): C 60.11;
H 5.92. Calc. for C28H30O4ClY (%): C 60.61; H 5.45.
4.1.4. Yields and Spectral Data of Neodymium Complexes
[C5H4–CH2–C6H4–OCH3]NdCl2 [(p-Methoxybenzyl)cyclopentadienyl]neodymium dichloride (3). Yield: 49%,
1H-NMR (250 MHz. d6-DMSO): 2.80 (dd, 1H, CH2), 2.93 (dd, 1H, CH2), 3.71 (s, 3H, OCH3), 5.99–6.38
(m, 4H, C5H4), 6.85 (d, 2H, C6H4), 7.11 (d, 2H, C6H4). 13C-NMR (300 MHz, d6-DMSO): 40.6 (CH2),
53.8 (OCH3), 112.7 (C30 –C50 of C6H4)), 126.1(C10 of C6H4), 127.3 (C20 –C60 of C6H4), 130.2 (C1 of C5H4),
131.2–131.9 (C2–C5 of C5H4), 133.9–134.0 (C3–C4 of C5H4), 156.8 (COCH3). ESI-MS (CH3CN, m/z):

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368.2 Dalton [C13H13ONdK]+. Elemental analysis: found (%): C 38.68; H 3.41. Calc. for C13H13Cl2ONd
(%): C 39.00; H 3.27.
[C5H4–CH2–C6H3 (OCH3)2]NdCl2 [(3,4-Dimethoxybenzyl)cyclopentadienyl]neodymium dichloride (6). Yield:
30%, 1H-NMR (250 MHz, CDCl3): 2.79 (dd, 1H, CH2), 2.92 (dd, 1H, CH2), 3.79 (s, 6H, OCH3), 5.93–6.35
(m, 4H, C5H4), 6.68 (m, 3H, C6H3). 13C-NMR (300 MHz, d6-DMSO): 35.7 (CH2), 54.5 (OCH3), 110.1
(C20 of C6H3), 111.3 (C50 of C6H3), 113.2 (C60 of C6H3), 117.9 (C10 of C6H3), 121.6 (C1 of C5H4), 130.5
(C2–C5 of C5H4), 134.6 (C3–C4 C5H4), 146.2 (C40 –OCH3 of C6H3), 147.3 (C30 –OCH3 of C6H3). ESI-MS
(CH3CN, m/z): 418–420 Dalton [C14H15O2ClNdNa]+. Elemental analysis: found (%): C 39.61; H 3.92.
Calc. for C14H15Cl2O2Nd (%): C 39.07; H 3.51.
[Cp-CH2-C6H5-OCH3]2NdCl bis-[(p-Methoxybenzyl) cyclopentadienyl] neodymium chloride (9). Yield: 23%,
1H-NMR (300 MHz, d6-DMSO): 2.86 (dd, 2H, CH2), 2.97 (dd, 2H, CH2), 3.79 (s, 6H, OCH3), 6.14–6.41
(m, 4H, C5H4), 6.85 (d, 4H, C5H4), 7.10 (d, 4H, C5H4). 13C-NMR (300 MHz, d6-DMSO): 41.8 (CH2),
53.3 (OCH3), 112.1 (C30 –C50 of C6H4), 124.9(C10 of C6H4), 128.3 (C20 –C60 of C6H4), 129.4 (C1 of
C5H4), 130.1–130.6 (C2–C5 of C5H4), 132.6–133.0 (C3–C4 of C5H4), 155.1 (COCH3). ESI-MS (CH3CN,
m/z): 521.3 Dalton [C26H26O2NdLi]+. Elemental analysis: found (%): C 56.11; H 4.52. Calc. for
C26H26ClO2Nd (%): C 56.76; H 4.76.
[Cp-CH2-C6H5-(OCH3)2]2NdCl bis-[(3,4-Dimethoxybenzyl)cyclopentadienyl]neodymium chloride (12). Yield:
1
55%, H-NMR (400 MHz, d6-DMSO): 2.79 (dd, 2H, CH2), 2.92 (dd, 2H, CH2), 3.80 (s, 12H, OCH3),
5.94–6.346 (m, 8H, C6H4), 6.69 (m, 6H, C6H3). 13C-NMR (300 MHz, d6-DMSO): 36.7 (CH2), 55.8 (OCH3),
112.3 (C20 of C6H3), 112.8 (C50 of C6H3), 113.2 (C60 of C6H3), 120.0 (C10 of C6H3), 122.9 (C1 of C5H4),
132.6 (C2–C5 of C5H4), 137.8 (C3–C4 C5H4), 149.5 (C40 –OCH3 of C6H3), 150.0 (C30 –OCH3 of C6H3).
ESI-MS (CH3CN, m/z): 613.2 Dalton [C28H30O4NdK]+. Elemental analysis: found (%): C 55.19; H 4.42.
Calc. for C28H30O4ClNd (%): C 55.11; H 4.96.
5. Statistical Analysis
The MTT assay data were statistically analyzed with descriptive statistics software (Graphpad®)
to assess the means and standard deviation (the values are reported in figures as percentage of cell
growth). The IC50 has been evaluated adopting a statistical nonlinear regression model, applying a
polynomial fitting curve on the base of our empiric data. The goodness of fit has been evaluated through
the definition of the coefficient of determination (R2) that results in the range of 0.789 < R2 < 0.987,
considering all of our models.
6. Conclusions
We reported the synthesis process and characterization of new scandium (III), yttrium (III),
and neodymium (III) chloride complexes with substituted cyclopentadienyl ancillary ligands,
highlighting their activity for the treatment of solid tumors, such as triple negative breast cancer
(TN) and prostate cancer. The ligands were chosen among those that showed ability to stabilize
analogous titanium complexes with significant antitumor activity.
The anti-proliferative effect of novel synthesized compounds were evaluated on MDA.MB231,
human triple negative breast cancer cells, and DU145, human non-hormone sensitive prostate cancer
cells. Our results highlight three compounds with the best inhibitory activity on both cell lines:
(1) [(p-methoxybenzyl)cyclopentadienyl]scandium dichloride;
(5) [(3,4-dimethoxybenzyl)cyclopentadienyl]yttrium dichloride;
(8) bis-[(p-methoxybenzyl)cyclopentadienyl]yttrium chloride;
Instead there is only one compound that imparts a strong effect only on the DU145 cell line:
(3) [(p-methoxybenzyl)cyclopentadienyl]neodymium dichloride.

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The compounds based on yttrium (
low concentration (IC50 < 5
complex ( ) is the only truly interesting one among those based on this metal, whereas those based on
5) and (
8) show very interesting results, being active at very
µM), probably due to their ability to induce apoptosis. The scandium
1
neodymium do not show remarkable activity.
According to our data on the inhibitory activity of cisplatin on DU145 cell lines (IC50
≈
2 µM) and
MDAMB231 (IC50
concentration 50 (IC50).
The interaction of the synthesized complexes
≈
5
µM) we can affirm that our best compounds
1
,
8
,
3
have a similar inhibitory
8
and 4 with biological membranes was also
investigated and our results highlighted a significant membrane permeability, this feature could allow
the adoption of these molecules in future studies.
The results have been tentatively rationalized on the basis of electronic and steric considerations.
Supplementary Materials: Supplementary materials are available online.
Acknowledgments: The authors are grateful to “Fondi di Ateneo FARB 2015” e Fondazione Medica Natale
Ferrara (FMNF) for financial support; Claudio Arra is grateful to MIUR-Ricerca Corrente “Modelli animali per studi
traslazionali in oncologia” for financial support and Massimiliano Spinelli from SSD Sperimentazione Animale,
National Cancer Institute-IRCCS-“G. Pascale Foundation for kind help in providing informatics assistance.
Author Contributions: G.P., C.A., M.S.S., and F.P. have conceived and designed the experiments; G.P., C.A., A.M.,
V.D.V., D.I. and O.I.P. have performed the experiments; C.S. and P.L. have analyzed the data; V.D.V. contributed
reagents/materials/analysis tools; G.P., C.S., and P.L. wrote the paper. The authors have contributed substantially
to the work reported.
Conflicts of Interest: The authors declare no conflict of interest.
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©
2017 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 (http://creativecommons.org/licenses/by/4.0/).