Bis(pyrazol-1-yl)- and bis(3,5-dimethylpyrazol-1-yl)-acetates were conjugated with the 2-hydroxyethylester and 2-aminoethylamide derivatives of the antineoplastic drug lonidamine to prepare Cu(I) and Cu(II) complexes that might act through synergistic mechanisms of action due to the presence of lonidamine and copper in the same chemical entity. Synchrotron radiation-based complementary techniques [X-ray photorlectron spectroscopy and near-edge X-ray absorption fine structure (NEXAFS)] were used to characterize the electronic and molecular structures of the complexes and the local structure around the copper ion (XAFS) in selected complexes. All complexes showed significant antitumor activity, proving to be more effective than the reference drug cisplatin in a panel of human tumor cell lines, and were able to overcome oxaliplatin and multidrug resistance. Noticeably, these Cu complexes appeared much more effective than cisplatin against 3D spheroids of pancreatic PSN-1 cancer cells; among these, PPh3-containing Cu(I) complex 15 appeared to be the most promising derivative. Mechanistic studies revealed that 15 induced cancer cell death by means of an apoptosis-alternative cell death.
Bis(pyrazol-1-yl)- and bis(3,5-dimethylpyrazol-1-yl)-acetates were conjugated with the 2-hydroxyethylester and 2-aminoethylamide derivatives of the antineoplastic drug lonidamine to prepare Cu(I) and Cu(II) complexes that might act through synergistic mechanisms of action due to the presence of lonidamine and copper in the same chemical entity. Synchrotron radiation-based complementary techniques [X-ray photorlectron spectroscopy and near-edge X-ray absorption fine structure (NEXAFS)] were used to characterize the electronic and molecular structures of the complexes and the local structure around the copper ion (XAFS) in selected complexes. All complexes showed significant antitumor activity, proving to be more effective than the reference drug cisplatin in a panel of human tumor cell lines, and were able to overcome oxaliplatin and multidrug resistance. Noticeably, these Cu complexes appeared much more effective than cisplatin against 3D spheroids of pancreatic PSN-1 cancer cells; among these, PPh3-containing Cu(I) complex 15 appeared to be the most promising derivative. Mechanistic studies revealed that 15 induced cancer cell death by means of an apoptosis-alternative cell death.
Lonidamine (LND) is
an antineoplastic drug able to sensitize tumors
to radio-, chemo-, and photodynamic therapy. Although its mechanism
of action is not completely clear yet, its use has been reported to
affect the metabolic pathways of cancer cells by inhibiting mitochondrial
respiration and glycolysis.[1−3] It has also been suggested that
LND induces intracellular tumor acidification by inhibiting the efflux
of l-lactic acid from cells mediated by monocarboxylate transporters
and the mitochondrial uptake of pyruvate mediated by the mitochondrial
pyruvate carrier.[4,5] Moreover, this drug induces a
mitochondrial transmembrane potential disruption through a direct
effect on the mitochondrial permeability transition pore.[6,7]Although the antitumor activity of LND as a single agent is
limited,
this drug has great potential in increasing the efficacy of traditional
chemotherapeutic agents, including cisplatin and other platinum-based
drugs.[1,8−10] Platinum complexes conjugated
with LND or its derivatives showed interesting antitumor activity
profiles in vitro, with improved cytotoxic effects compared to that
of cisplatin and other reference drugs.[11−13] In addition, a recent
and promising approach concerned the preparation and biological study
of gold nanoparticles conjugated with LND and aptamer AS1411 as effective
cancer treatments.[14]Cu(I) and Cu(II)
complexes have received great attention for their
both in vitro and in vivo unique properties.[15−27] Copper complexes and copper-based nanomedicines[28,29] are coming out as promising antitumor agents due to the elevated
need for copper in cancer tissues compared to that in normal cells
and its role as a limiting factor for multiple aspects of tumor progression,
including angiogenesis, growth, and metastasis.[22,30−34] While tumor cells, avid of copper to fuel neovascularization in
tumor progression, enrich their copper content, eventually causing
copper overload and thus cell death, normal cells continue to adopt
physiological mechanisms that regulate copper intracellular concentration.
Somewhat reversing the anticancer strategy based on sequestration
of copper to prevent establishment of the tumor blood supply,[35] tumor cells may represent a suitable, selective
target for copper-based antitumor drugs.[36] Actually, anticancer copper-based drugs are endowed with improved
selectivity toward tumor cells. Even if little information is available
on the molecular basis for the mode of action of copper complexes,
several molecular effectors and signalling pathways have emerged as
suitable targets for copper complexes.[36,37]In addition,
endogenous metal ions are generally less toxic than
nonendogenous ones toward normal cells, and for this reason, copper
complexes might represent efficacious alternatives to Pt-based drugs.[34,36,38−42] Copper complexes show broader spectra of activities
and lower toxicity, thereby providing the possibility of circumventing
the problems encountered by platinum drugs, such as dose-limiting
toxicity and inherent/acquired resistance.[43,44] Considering their reactivity to biomolecules other than DNA, there
is increasing evidence that the mechanisms of action of copper complexes
are markedly different from those of platinum drugs.[45,46] In this contest, some classes of copper complexes were found to
exert an effective antiproliferative action by dysregulating mitochondrial
function in cancer cells.[47]To our
knowledge, no copper complexes with LND derivatives have
been reported in the literature to date. Therefore, the aim of this
work was to functionalize LND with species able to coordinate metals
in order to form Cu(I) and Cu(II) complexes potentially capable of
exerting an anticancer activity through synergistic mechanisms of
action.For this purpose, LND was converted into 2-hydroxyethylester
and
2-aminoethylamide derivatives LONES and LONAM, respectively, which
were conjugated to bifunctional species bis(pyrazol-1-yl)acetic acid
[HC(pz)2COOH] and bis(3,5-dimethyl-pyrazol-1-yl)acetic
acid [HC(3,5-Me2pz)2COOH] to form heteroscorpionate
ligands 1–4 (L1–L4, Scheme ). HC(pz)2COOH and HC(3,5-Me2pz)2COOH were selected
as coordinating agents for their κ3-NNO coordination
properties of bis(azol-1-yl)methane[48] and
for the presence of a carboxylic function suitable to be derivatized
with the hydroxyl group of LONES or the primary amino group of LONAM.
In addition, we have recently reported that copper complexes with
heteroscorpionate ligands, obtained via conjugation with nitroimidazole,
glucosamine, and a noncompetitive NMDA receptor antagonist, showed
cytotoxic activity toward a panel of several human tumor cell lines.[49−52]
Scheme 1
Synthesis of Ligands 1–4 and Complexes 5–16; Reagents and Conditions: (a) HOCH2CH2OH, H2SO4, 85 °C,
1 h; (b) H2NCH2CH2NH2,
CDI, THF, 18 h; (c) HC(pz)2COOH or HC(3,5-Me2pz)2COOH, DMAP, EDCI·HCl,16 h; (d) HC(pz)2COOH or HC(3,5-Me2pz)2COOH, HOBT, EDCI·HCl,
DMF, 16 h; (e) PTA, Cu(CH3CN)4PF6, CH3CN, Overnight; (f) PPh3, Cu(CH3CN)4PF6, CH3CN, Overnight; and (g)
CuCl2·2H2O, CH3CN, 24 h
Ligands 1–4 were used for the
preparation of Cu(I) and Cu(II) complexes 5–16 (Scheme ) that might act through synergistic mechanisms of action due to
the presence of LND and copper in the same chemical entity. Concerning
the Cu(I) complexes, to stabilize copper in the +1 oxidation state,
lipophilic triphenylphosphine (PPh3) and hydrophilic 1,3,5-triaza-7-phosphaadamantane
(PTA) were selected as coligands in order to confer different solubility
properties to the corresponding complexes.The molecular structure
of selected coordination compounds was
investigated in the solid state by means of synchrotron radiation-induced
X-ray photoelectron spectroscopy (SR-XPS), near-edge X-ray absorption
fine structure (NEXAFS), and X-ray absorption spectroscopy (XAS);
the multitechnique approach allowed us to properly define the coordination
geometry around the copper ion, as well as to ascertain the molecular
structural stability of the ligands upon interaction with the metal.[53]The new complexes 5–16, the corresponding
uncoordinated ligands 1–4, and LND
were investigated for their cytotoxic potential on a panel of human
cancer cell lines, derived from different solid tumors, by means of
both 2D and 3D cell viability studies. The cell panel also includes
cancer cells selected for their resistance to oxaliplatin or multidrug
resistant (MDR) cells. Furthermore, mechanistic studies were performed
in order to elucidate the multimodal mechanistic effect of the new
Cu(I) and Cu(II) species.
Results and Discussion
Synthesis and Characterization
Starting materials LONES
and LONAM and ligands L1, L2, L3,
and L4 were prepared according to the procedure reported
in Scheme . In particular,
the reaction between LND and ethylene glycol in the presence of sulfuric
acid led to intermediate LONES, which was treated with acids HC(pz)2COOH and HC(3,5-Me2pz)2COOH[54,55] in the presence of 3-(ethyliminomethylideneamino)-N,N-dimethyl-propane-1-amine hydrochloride (EDCI·HCl)
and N,N-dimethylaminopyridine (DMAP)
to give ligands L1 (1) and L2 (2), respectively. Treatment of LND with ethylenediamine in
the presence of carbonyldiimidazole (CDI) led to intermediate LONAM,
which reacted with HC(pz)2COOH or HC(3,5-Me2pz)2COOH in the presence of EDCI·HCl and 1-hydroxybenzotriazole
(HOBT) to give ligands L3 (3) and L4 (4), respectively. After separation and purification
via column chromatography, ligands 1–4 were obtained in a reasonable yield and purity. They are soluble
in CHCl3, CH2Cl2, dimethyl sulfoxide
(DMSO), and CH3OH but insoluble in water. Ligands 1–3 are also soluble in CH3CN. The infrared (IR) spectra obtained for solid samples of ligands 1–4 showed all the bands expected for
these heteroscorpionate ligands. The 1H nuclear magnetic
resistance (NMR) spectra, recorded in CDCl3 and in DMSO-d6 solution at room temperature, show all the
expected signals for the bioconjugated ligands with a single set of
resonances for the pyrazole rings, indicating that the pyrazole protons
are equivalents. The elemental analyses confirm the stoichiometry
and the purity of the products in the solid state.Cu(I) complexes
[(PTA)2Cu(L1)]PF6 (5), [(PTA)2Cu(L3)]PF6 (8), [(PTA)2Cu(L2)]PF6 (11), and [(PTA)2Cu(L4)]PF6 (14) were prepared via the reaction of PTA, Cu(CH3CN)4PF6, and ligands L1, L3, L2, and L4, respectively (Scheme ), following a one-pot synthesis
with CH3CN as the solvent. Analogously, Cu(I) complexes
[(PPh3)2Cu(L1)]PF6 (6), [(PPh3)2Cu(L3)]PF6 (9), [(PPh3)2Cu(L2)]PF6 (12), and [(PPh3)2Cu(L4)]PF6 (15) were prepared
via the reaction of PPh3, Cu(CH3CN)4PF6, and the related ligands (Scheme ). All the compounds are soluble in CH3CN and DMSO; the complexes with PPh3 coligands
(6, 9, 12, and 15) are also soluble in CHCl3; complexes 6, 9, 12, and 14 are soluble in methanol;
complex 15 is soluble in ethanol; while complexes 8, 9, and 11 are soluble in acetone.
The IR spectra obtained for solid samples of the Cu(I) complexes show
all the expected bands for the bioconjugated ligands and the phosphane
coligands. The absorptions due to the C=O stretching of the
ester groups for complexes 5, 6, 11, and 12 do not significantly vary with respect to the
same absorptions of the carbonyl groups detectable in the spectra
of the free ligands. The absorptions due to the C=O stretching
of the amide groups for 8, 9, 14, and 15 are slightly shifted at lower frequencies with
respect to those of the free ligands. The 1H NMR spectra
of the Cu(I) complexes, recorded in DMSO-d6 solution at room temperature, showed a single set of resonances
for the pyrazole rings, indicating that the pyrazole protons are equivalents,
with a slight shift due to the coordination to the metal center. A
significant shift to higher frequencies of the N–H signals is detected only for compounds 14 (δ
8.43 and 9.07 ppm) and 15 (δ 8.38 and 9.35 ppm)
probably due to a secondary interaction between the hydrogen atoms
and the copper center. The PTA and PPh3 coligands show
a characteristic series of peaks at δ 4.04–4.73 and 7.20–7.80
ppm, respectively, with an integration, with respect to the ligand
peaks, that confirms the 1:2 stoichiometric ratio between the ligand
and the phosphane coligands. The room-temperature 31P{H}
NMR spectra of the Cu(I) complexes, recorded in DMSO-d6 and CD3CN solution at room temperature, give
singlets shifted downfield with respect to the value of the free phosphanes
PPh3 and PTA. The characteristic septet centered at about
δ −144 ppm is due to the presence of the PF6– counterion. The electrospray ionization mass
spectroscopy (ESI-MS) studies, performed by dissolving the Cu(I) complexes
in CH3CN and recording the spectra in the positive- and
negative-ion modes, confirm the formation of the PTA and PPh3 complexes and the presence of hexafluorophosphate as counterions.Cu(II) complexes [(L1)CuCl2] (7), [(L3)CuCl2] (10), [(L2)CuCl2] (13), and [(L4)CuCl2] (16) were prepared via the reaction of CuCl2·2H2O with ligands L1, L3, L2, and L4, respectively, in acetonitrile
solution for 7 and 13 and in methanol solution
for 10 and 16 at room temperature (Scheme ). All the compounds
are soluble in DMSO; complexes 7 and 13 are
soluble in CH3OH, CHCl3, and CH3CN;
complexes 10 and 16 are slightly soluble
in CH3CN; complex 16 is soluble in CHCl3; and complexes 13 and 16 are soluble
in CH2Cl2. The IR spectra obtained for solid
samples show all the expected bands for the bioconjugated ligands.
The strong absorptions due to the C=O stretching of the ester
and amide groups do not significantly vary with respect to the absorptions
detectable for the free ligands. These data indicate that the carbonyl
groups are not involved in the coordination of the metal: the copper
center results in a tetracoordinated environment with the ligand chelating
in a bidentate fashion and the other two positions being occupied
by the chlorides. The ESI-MS studies, conducted by dissolving the
Cu(II) complexes in CH3CN and recording the spectra in
the positive- and negative-ion modes, confirm the formation of the
complexes and the presence of the chlorides as counterions.
Investigation
of the Molecular and Electronic Structures
The electronic and molecular
structures of coordination compounds 10, 15, and 16, in comparison with
those of ligand 4, were probed using SR-XPS. SR-XPS spectra
were collected at C 1s, N 1s, O 1s, Cl 2p, P 2p (for 15), F 1s (for 15), and Cu 2p core levels; the detailed
data analysis results [binding energy (BE), full width at half maximum
(FWHM), and assignments], confirming the proposed molecular structures
for the complexes and the stability of the ligand 4 molecular
structure upon coordination to copper, are collected in the Supporting Information (Table S1). In the following,
the most interesting signals are described and compared for the three
analyzed samples, considered representative of Cu(II) and Cu(I) coordination
compounds.The C 1s signal can always be resolved using curve
fitting analysis into several components corresponding to the different
C atoms in the proposed molecular structure. More in detail, in the
order of increasing BE, the contributions are assigned as follows:
aromatic and aliphatic C–C carbons (BE = 284.7 eV), C–N
carbons of the pyrazole-like rings (BE = 286.6 eV), C=O carbonyls
of amide groups and imine-like C=N groups (BE = 288.0 eV),
−COOH impurities always found on the surface of samples deposited
in air (BE = 289.3 eV), and C–Cl groups (BE = 291.5 eV). C
1s spectra of ligand 4 and complexes 10 and 15 are reported in Figure A–C.
Figure 1
C 1s spectra of ligand 4 (A), Cu(II)
complex 10 (B), and Cu(I) complex 15 (C)
and N 1s spectra
of 4 (D) and coordination compounds 10 (E)
and 15 (F).
C 1s spectra of ligand 4 (A), Cu(II)
complex 10 (B), and Cu(I) complex 15 (C)
and N 1s spectra
of 4 (D) and coordination compounds 10 (E)
and 15 (F).N 1s spectra of ligand 4 and complexes 10 and 15 are reported
in Figure D–F,
respectively. For 4, a couple of main signals can be
detected at 399.5 and 401.0 eV
BEs, indicative for the two kinds of nitrogens of the pyrazole rings,
that is, amine-like and imine-like N atoms, respectively.[56] Amide-like N atoms are found at a BE value very
close to that of the amine-like N atoms and cannot be resolved due
to the experimental resolution (0.6 eV). As for the coordination compounds,
it is expected that only the amine-like contribution appears when
the two nitrogen atoms coordinate with a metal ion, as reported in
the literature for heterocycles coordinating metal ions (e.g., porphyrins
or phthalocyanines).[57−59] In excellent agreement with this prediction, in complexes 10, 15 (Figure E,F, respectively), and 16, a single N
1s component at about 400.0 eV, attributed to the symmetrized nitrogen
atoms coordinating to copper ions (and to the indistinguishable amide-like
N), can be observed. Cl 2p BE values observed for both ligand 4 and the three coordination compounds 10, 15, and 16 (Cl 2p3/2 BE = 200 eV)
are compatible with chlorine atoms covalently bonded to carbon in
organic molecules.[60] In addition, in Cl
2p spectra of 10 and 16, a contribution
of slightly higher intensity at a lower BE (198 eV BE) can be observed,
as expected for chlorine atoms bonded to Cu(II) ions in the coordination
compounds.[60,61]Cu 2p spectra collected
for copper complexes 10 and 15 are reported
in Figure S1 in the Supporting Information; both Cu 2p spectra collected for complexes 10 and 16 show a spin–orbit pair with
the Cu 2p3/2 component centered at 936 eV, indicative of
Cu(II) ions in coordination compounds,[60] in excellent agreement with analogous systems.[59] On the other hand, the Cu 2p spectrum collected on complex 15 has the Cu 2p3/2 spin–orbit component
centered at a 932.0 eV BE, as expected for Cu(I) ions in coordination
compounds.[60]
NEXAFS Data Analysis Results
NEXAFS spectroscopy measurements
were carried out at C and N K-edges on ligand 4 and on
coordination compounds 10 and 16, with the
aim to obtain further information about the influence of the metal
coordination on the electronic structure of the ligand. Experimental
spectra of the C K-edge and N K-edge of 4, 10, and 16 samples are reported in Figure . They were collected at the grazing incidence
of the polarized photon beam with respect to the sample surface; no
angular dependence was observed on the NEXAFS spectra of the investigated
compounds when the incidence angle of the impinging radiation was
changed from grazing to magic and normal, indicating the absence of
a preferential orientation of the investigated molecules on the sample
surface.
Figure 2
C K-edge (left) and N K-edge (right) NEXAFS spectra of ligand 4 and Cu(II) complexes 10 and 16.
C K-edge (left) and N K-edge (right) NEXAFS spectra of ligand 4 and Cu(II) complexes 10 and 16.According to the literature,[59,62−65] peak positions and assignments of the main features detected in
the C and N K-edge spectra of the analyzed samples are also shown
in Table .
Table 1
NEXAFS: Peak Position (eV) and Relative
Assignment of the Main Features Appearing in the C and N K-Edge NEXAFS
Spectra of Samples 4, 10, and 16
sample
4
10
16
assignment
C K-edge
284.7
285.1
284.8
πC=C*
286.0
286.2
286.4
πC=N*
287.6
287.8
287.8
σC–H*
288.4
288.4
288.4
πC=O*
293.4
294.3
291.5
σC–C*
301.5
303.5
299.0
σC=N*
N K-edge
396.1
396.1
396.1
397.5
397.9
397.9
π1*
399.4
399.4
399.4
π2*
404.9
403.7
403.7
σC=N*
411.0
411.0
411.0
σC–N*
For the C K-edge spectra, the energy scale is referenced
to the
πC=O* transition of the amide function in the side chain of LND,[62,65] while for the N K-edge spectra, the energy scale is referenced to
the π2* transition of the pyrazole ring.[59,63,64]The C K-edge spectra present the expected πC=C* and πC=N* features
of the pyrazole ring and the πC=O* feature related to the amide group at
288.4 eV. It can be noticed (Table ) that the first two peaks lie at lower energy in 4 and that the πC=N* peak is attenuated in Cu complexes 10 and 16 with respect to that in ligand 4. These effects could be related to the Cu complexation of
the pyrazole nitrogens.All the samples exhibit a σC–H* resonance
originated from the presence
of the aliphatic chains. Above the edge, two large features σC–C* and σC=N* can be
observed.The N K-edge spectra show the N 1s → π*
transitions
(π1* and
π2*) originating
from two distinct nitrogen atoms. As shown in Table , the energy of the π1* peak is higher for Cu complexes 10 and 16 than for ligand 4: this
effect might be further evidence of the complexation with copper.
The weak feature detected at 296.1 eV for all the analyzed samples
is probably related to impurities on the beamline mirrors.
XAFS
Data Analysis Results
X-ray absorption data collected
at the Cu K-edge on selected complexes 10, 15, and 16 were analyzed with the aim of understanding
the average local coordination chemistry and electronic structure
around Cu. The main near-edge features (XANES) originate from the
absorber valence state (edge position) and coordination geometry (edge
shape).[66] The Cu K-edge normalized XANES
spectra measured on complexes 10, 15, and 16 are presented in Figure , together with those measured on Cu metal foil and
reference Cu oxides, for the sake of comparison. The edge energies
of complexes 10 and 16 match the edge energy
of the pure CuO reference compound, in accordance to the Cu(II) valence
state in these complexes.[59] A roughly planar
geometry could be assumed for sample 10 due to the close
similarity with the XANES spectrum of the glycine complex reported
in the literature.[67] The XANES spectrum
measured from complex 16 (Figure ) depicts a higher white line (around 8995
eV) with respect to complex 10 and attenuation of the
pre-edge shoulder, this behavior being compatible with the Cu coordination
geometry changing from square planar in complex 10 to
nearly octahedral in complex 16. The complex 15 edge position matches the edge energy measured on the pure Cu2O reference compound, confirming the Cu(I) valence state.
The pre-edge peak we found at 4 eV above the Cu0 edge is
related to the copper coordination geometry and type of neighbors.
Its amplitude is expected to be the highest (around 1) for the Cu(I)
bidentate coordination,[66] while it decreases
upon increasing the Cu(I) coordination number and/or reducing the
Cu(I) site symmetry.[68,69] We found the pre-edge peak amplitude
of around 0.52 of the edge jump consistent with 3- or 4-coordinated
Cu. The squeezing of the structural oscillations in the XANES region,
with respect to the Cu2O spectrum, suggests averagely longer
neighbor distances in complex 15. Further details about
the local structure are obtained from the quantitative analysis of
the EXAFS region.
Figure 3
Cu K-edge normalized XANES spectra measured on complexes
and reference
compounds, shifted for the sake of clarity. Edge energies of Cu(II)
complexes 10 and 16 match the edge energy
of the CuO reference compound. The edge energy of Cu(I) complex 15 matches the edge energy of Cu2O.
Cu K-edge normalized XANES spectra measured on complexes
and reference
compounds, shifted for the sake of clarity. Edge energies of Cu(II)
complexes 10 and 16 match the edge energy
of the CuO reference compound. The edge energy of Cu(I) complex 15 matches the edge energy of Cu2O.Looking at the k2-weighted EXAFS
signal
in the k space and at its Fourier transform (FT)
in the real space, remarkable differences are evident among the samples
(Figure ). In particular,
looking at complexes 10 and 16, the k2χ(k) signal presents
a prominent first oscillation in sample 16, while in
sample 10, two oscillations of equal and lower intensity
are visible. Moreover, from the first oscillation onward, the signals
become partially out of phase. Such a different behavior corresponds
to differences in the real space for the FT spectra. Noticeably, the
modulus and imaginary part of the FT depict very similar shapes (Figure , dashed lines) except
around 2 Å (Figure , red arrow), where a lack of the structural signal is found in the
sample 16 data with respect to the sample 10 data. This behavior may point out some antiphase structural signal
originating from a different neighbor arrangement around Cu in the
two complexes.[70] Noticeably, complex 16 has two CH3 substituents that may provide such
an additional coordination shell, in agreement with the XANES features
suggesting the nearly octahedral coordination. Moreover, such a different
coordination seems to promote greater rigidity of the structure, providing
larger next neighbor signals in the next neighbor region (highlighted
by dashed lines in Figure B). Looking at the weaker k2χ(k) signal of complex 15, it is evident that
the local structure around Cu(I) is definitively more disordered than
those of Cu(II) complexes.
Figure 4
(A) Cu K-edge EXAFS data analysis of complexes 10, 15, and 16 are reported. At
the top, the experimental k2χexp(k) (dots)
and best fit curves k2χfit(k) (black lines) are presented (vertically shifted
for clarity). The middle curves (orange) represent the partial contributions
used in the analysis of Cu(II) complex 10 (vertically
shifted for clarity) for the sake of the example, and the lowest curve
(gray) is the best fit residual k2(χfit – χth). (B) Corresponding FT moduli
are shown for experimental k2 weighted
EXAFS data (dots) and the best fit (black lines). The FT imaginary
(Imm-FT) part of the experimental spectrum, best fit, and partial
contributions are shown for complex 10 for the sake of
an example. The dashed lines highlight in-phase Imm-FT oscillations
for Cu–N and Cu–Cl contributions in Cu(II) complexes 10 and 16. The red arrow points out the structural
signal lack in the region of the Cu–Cl shell (red dashed line)
likely due to some antiphase structural signals. (C) Local structure
around Cu is shown to highlight the neighbor shells involved in the
analysis, being R = H (complex 10) or R = CH3 (complex 16) (see Scheme ) and Y = Cl [Cu(II) complexes 10 and 16] and Y = P [Cu(I) complex 15].
(A) Cu K-edge EXAFS data analysis of complexes 10, 15, and 16 are reported. At
the top, the experimental k2χexp(k) (dots)
and best fit curves k2χfit(k) (black lines) are presented (vertically shifted
for clarity). The middle curves (orange) represent the partial contributions
used in the analysis of Cu(II) complex 10 (vertically
shifted for clarity) for the sake of the example, and the lowest curve
(gray) is the best fit residual k2(χfit – χth). (B) Corresponding FT moduli
are shown for experimental k2 weighted
EXAFS data (dots) and the best fit (black lines). The FT imaginary
(Imm-FT) part of the experimental spectrum, best fit, and partial
contributions are shown for complex 10 for the sake of
an example. The dashed lines highlight in-phase Imm-FT oscillations
for Cu–N and Cu–Cl contributions in Cu(II) complexes 10 and 16. The red arrow points out the structural
signal lack in the region of the Cu–Cl shell (red dashed line)
likely due to some antiphase structural signals. (C) Local structure
around Cu is shown to highlight the neighbor shells involved in the
analysis, being R = H (complex 10) or R = CH3 (complex 16) (see Scheme ) and Y = Cl [Cu(II) complexes 10 and 16] and Y = P [Cu(I) complex 15].Quantitative details were obtained from EXAFS data
fitting (see
the Supporting Information for details).
The distances and mean square relative displacement (MSRD) σ2 obtained from the refinement are reported in Table for single scattering (SS)
contributions. In complex 10, the distances demonstrate
a good agreement with the expected molecular structure. The Cu–N
nearest neighbor shell is found at 1.96 Å, and Cl is at 2.23
Å from Cu(II). The complex 16 EXAFS data analysis
demonstrates the octahedral distorted geometry for Cu in complex 16 with two closer Cu–N and Cu–CR nearest neighbor shells and a third longer Cu–Cl shell, and
the Cu–CR link would likely originate from the CH3 residue. Noticeably, the bimodal Cu–C/N(2) shell resulting
in a shorter shell at around 2.9 Å and a long one at around 3.3
Å is consistent with the tilting of pyrazole rings.
Table 2
Best Fit Results for Cu K-Edge XAFS
Data Analysis of Complexes 10, 15, and 16a
sample 10 Cu(II)
sample 16 Cu(II)
sample 15 Cu(I)
N
R (Å)
σ2 × 102 (Å2)
R (Å)
σ2 × 102 (Å2)
R (Å)
σ2 × 102 (Å2)
Cu–N
2
1.959(5)
0.68(5)
1.967(7)
0.24(3)
2.001(3)
3.2(2)
Cu–CR
2
1.89(2)
1.62(2)
Cu–Cl(P)
2
2.23(1)
0.64(4)
2.21(2)
2.3(2)
2.16(1)
0.69(3)
Cu–C/N(2)
4
2.97(3)
0.86(2)
2.85(2)
0.75(3)
2.92(2)
1.3(2)
3.35(3)
0.45(2)
Cu–CPh3
9
3.29(3)
1.4(5)
Cu–C(2) (SS + MS)
4
4.08(3)
4.40(3)
0.8
4.05(8)
2.1
The Cu–CR shell
originates from the carbons of R = CH3 groups of the pyrazoles;
Cu–CPh originates from the C atoms of
phenyl rings bonded to P of PPh3. The multiplicity numbers
(N) are constrained to the structural model, and interatomic distances
[R (Å)] and the MSRD (σ2) parameters
are shown. Standard uncertainties on the last digit are reported in
parentheses.
The Cu–CR shell
originates from the carbons of R = CH3 groups of the pyrazoles;
Cu–CPh originates from the C atoms of
phenyl rings bonded to P of PPh3. The multiplicity numbers
(N) are constrained to the structural model, and interatomic distances
[R (Å)] and the MSRD (σ2) parameters
are shown. Standard uncertainties on the last digit are reported in
parentheses.In complex 15, the Cu–N nearest neighbor distance
is slightly longer with respect to the other complexes and largely
more disordered. We found a significatively better fit using 2N +
2P neighbor shells with respect to 1N + 2P or 2N + 1P, but the quite
large σ2 found for the Cu–N suggests more
loosely bound Cu(I) in complex 15 with respect to complexes 10 and 16, likely related to the lower electronegativity
of Cu(I). In summary, the Cu oxidation and the local structure around
Cu obtained via XAFS is consistent with the expected complex structure;
the Cu(I) local structure appears more distorted likely due to the
more loosely bound neighbor.
Stability Studies
The stability of the new complexes
in 0.5% DMSO/RPMI cell culture medium was also evaluated using UV–vis
spectroscopy. Changes observed in the UV–vis spectra of the
complexes over 24 h were insignificant or only minimal for complexes
with L3 and L4 ligands, indicating that these
complexes are stable under physiological conditions (Figure S2 in
the Supporting Information). On the contrary,
for complexes with L1 and L2 ligands, a more
evident change in the UV–vis spectrum was detected.
Cytotoxicity
Studies
The newly developed complexes 5–16, the corresponding uncoordinated
ligands 1–4, and their precursors
were evaluated for their ability to promote cell death in a panel
of human cancer cell lines derived from solid tumors (2008 ovarian,
HCT-15 colon, PSN-1 pancreatic, A431 cervical, and H157 lung carcinoma
cells). The cytotoxicity parameters, expressed in terms of IC50 and obtained after 72 h of drug exposure using a 3-[4,5-dimethylthiazol-2-yl]-2,5
diphenyl tetrazolium bromide (MTT) assay, are reported in Table . For comparison purposes,
the cytotoxicity of cisplatin was assessed under the same experimental
conditions.
Table 3
Cytotoxic Activity of LND, LONES,
LONAM, 1–16, and Cisplatina
IC50 (μM) ± S.D.
compound
2008
HCT-15
PSN-1
H157
A431
LND
24.9 ± 3.3
>25
24.6 ± 2.9
>25
>25
LONES
>25
>25
21.3 ± 2.4
>25
>25
LONAM
18.2 ± 0.9
>25
>25
>25
>25
1 (L1)
11.70 ± 0.02
13.4 ± 5.3
14.3 ± 3.8
16.8 ± 2.4
12.5 ± 2.1
2 (L2)
21.3 ± 3.5
6.2 ± 0.5
2.7 ± 0.3
5.2 ± 0.7
3.2 ± 0.2
3 (L3)
7.9 ± 2.2
4.7 ± 1.6
3.1 ± 0.9
5.6 ± 1.4
3.7 ± 1.6
4 (L4)
2.9 ± 1.1
1.9 ± 1.0
1.5 ± 0.5
1.3 ± 0.6
1.4 ± 0.5
5 [(PTA)2Cu(L1)][PF6]
2.4 ± 0.9
2.5 ± 0.5
1.2 ± 0.1
1.3 ± 0.5
0.4 ± 0.1
6 [(PPh3)2Cu(L1)][PF6]
2.2 ± 0.8
1.7 ± 0.6
1.4 ± 0.1
1.30 ± 0.03
0.6 ± 0.3
7 [(L1)CuCl2]
2.8 ± 0.9
1.6 ± 0.4
1.6 ± 0.4
0.6 ± 0.2
0.70 ± 0.02
8 [(PTA)2Cu(L3)]PF6
0.8 ± 0.3
0.4 ± 0.1
0.6 ± 0.2
0.6 ± 0.1
0.60 ± 0.01
9 [(PPh3)2Cu(L3)]PF6
1.1 ± 0.3
0.30 ± 0.01
0.5 ± 0.1
0.40 ± 0.01
1.0 ± 0.2
10 [(L3)CuCl2]
1.20 ± 0.02
0.2 ± 0.1
0.5 ± 0.2
0.5 ± 0.2
1.6 ± 0.8
11 [(PTA)2Cu(L2)][PF6]
2.2 ± 0.9
2.2 ± 0.4
1.0 ± 0.4
0.5 ± 0.2
0.6 ± 0.1
12 [(PPh3)2Cu(L2)][PF6]
1.4 ± 0.1
3.4 ± 0.1
0.6 ± 0.1
1.2 ± 0.2
0.7 ± 0.1
13 [(L2)CuCl2]
1.3 ± 0.2
4.7 ± 0.9
0.5 ± 0.1
1.0 ± 0.2
1.5 ± 0.6
14 [(PTA)2Cu(L4)]PF6
0.7 ± 0.2
0.3 ± 0.1
1.0 ± 0.3
0.900 ± 0.001
0.5 ± 0.2
15 [(PPh3)2Cu(L4)]PF6
0.6 ± 0.1
0.3 ± 0.1
0.6 ± 0.1
0.3 ± 0.1
0.4 ± 0.2
16 [(L4)CuCl2]
0.8 ± 0.8
0.2 ± 0.1
1.6 ± 0.2
0.60 ± 0.02
1.00 ± 0.01
cisplatin
2.2 ± 1.0
15.3 ± 2.6
12.1 ± 2.8
2.1 ± 0.8
2.1 ± 0.9
Cells (3–8
× 103 mL–1) were treated for 72
h with the tested
compounds. Cell viability was measured by means of an MTT test. The
IC50 values were calculated using a 4-PL logistic model
(P < 0.05). S.D. = standard deviation.
Cells (3–8
× 103 mL–1) were treated for 72
h with the tested
compounds. Cell viability was measured by means of an MTT test. The
IC50 values were calculated using a 4-PL logistic model
(P < 0.05). S.D. = standard deviation.LND and ligand precursors LONAM
and LONES did not induce a significant
reduction of cell viability (the IC50 values were greater
than 25 μM). Uncoordinated ligands 1–4 possessed a moderate cytotoxic potency that was, however,
on average, 3 to 10 times lower than that of the corresponding metal
complexes. Actually, all tested copper complexes showed a promising
cytotoxic potential, with IC50 values in the low or submicromolar
range toward all the human cancer cell lines belonging to the in-house
panel, and proved to be more effective than the reference chemotherapeutic
drug cisplatin. These results suggest that both LND and copper might
contribute to the potent antitumor activity of these complexes.In general, L1 and L2 derivatives were on
average less effective than the corresponding L3 and L4 complexes, and among the series, no significant differences
in terms of in vitro antitumor activities were detected for Cu(I)
and Cu(II) complexes. It is important to note that complexes with
L1 and L2 ligands were less stable in physiological
media compared with complexes bearing L3 and L4. Hence, their lower cytotoxic effectiveness could be attributed,
at least in part, to their instability in physiological conditions.
Among all, complex 15 was the most effective derivative,
eliciting, on average, IC50 values about 16 times lower
than those detected with cisplatin. On the contrary, compound 5 was the weakest among the series, with a cytotoxic potency
that was, however, more than 4 times higher than that of cisplatin.Considering the very promising antiproliferative effects and keeping
in mind that drug resistance represents a key determinant for the
variable efficacy of anticancer therapy, we also assessed the ability
of the newly developed Cu(I) and Cu(II) complexes to bypass the acquired
drug resistance. In particular, complexes were evaluated for their
antiproliferative activity against some cancer cell lines selected
for sensitivity/resistance to oxaliplatin or MDR cells, namely, LoVo,
LoVo-OXP, and LoVo MDR human colon cancer cells.As previously
reported, LoVo OXP cells (derived from LoVo cells
grown in the presence of increasing concentrations of oxaliplatin)
were about 13-fold more resistant to oxaliplatin than parental cells.[23] The main molecular mechanisms involved in oxaliplatin
resistance appear to depend upon (i) the decreased cellular accumulation,
which is thought to be related to a greater activity of the ATP7B
exporter rather than the activity of P-glycoprotein (P-gp) and multidrug
resistance protein 1 (MRP1), and (ii) the more efficient repair of
oxaliplatin-induced DNA damage via nucleotide excision repair (NER).[71] Conversely, in human colon LoVo MDR cancer cells,
the resistance to doxorubicin, a drug belonging to the MDR spectrum,
is associated with an overexpression of drug transporters, such as
the 170 kDa P-gp.[72]Table shows the
degree of resistance in terms of the resistant factor (RF), which
is defined as the ratio between IC50 (obtained using an
MTT assay after 72 h of drug exposure) calculated for the resistant
cells and those arising from the sensitive ones (Table ).
Table 4
Cross-Resistance
Profile of Complexes 5–16, Oxaliplatin,
and Doxorubicina
IC50 (μM) ± S.D.
compound
LoVo
LoVo OXP
RF
LoVo MDR
RF
5 [(PTA)2Cu(L1)][PF6]
2.4 ± 0.3
1.8 ± 0.5
0.8
1.6 ± 0.7
0.7
6 [(PPh3)2Cu(L1)][PF6]
1.4 ± 0.4
1.1 ± 0.3
0.8
1.2 ± 0.4
0.9
7 [(L1)CuCl2]
1.8 ± 0.5
1.7 ± 0.6
0.9
1.0 ± 0.4
0.6
8 [(PTA)2Cu(L3)]PF6
0.9 ± 0.1
0.7 ± 0.2
0.8
0.7 ± 0.3
0.9
9 [(PPh3)2Cu(L3)]PF6
0.20 ± 0.05
0.30 ± 0.04
1.5
0.10 ± 0.03
0.5
10 [(L3)CuCl2]
0.6 ± 0.1
0.7 ± 0.1
1.0
0.8 ± 0.2
1.3
11 [(PTA)2Cu(L2)][PF6]
2.8 ± 0.7
2.2 ± 0.9
0.8
2.5 ± 0.4
0.9
12 [(PPh3)2Cu(L2)][PF6]
2.2 ± 0.4
1.6 ± 0.3
0.7
1.7 ± 0.6
0.8
13 [(L2)CuCl2]
2.8 ± 0.7
2.1 ± 0.6
0.8
2.0 ± 0.5
0.7
14 [(PTA)2Cu(L4)]PF6
0.4 ± 0.1
0.7 ± 0.1
1.8
0.7 ± 0.2
1.0
15 [(PPh3)2Cu(L4)]PF6
0.20 ± 0.02
0.20 ± 0.03
1.0
0.10 ± 0.01
0.5
16 [(L4)CuCl2]
0.8 ± 0.1
0.9 ± 0.3
1.1
1.1 ± 0.3
1.4
oxaliplatin
1.5 ± 0.6
19.6 ± 1.9
13.1
doxorubicin
1.1 ± 0.5
19.4 ± 2.2
17.4
Cells (5 ×
103 mL–1) were treated for 72 h with
the tested compounds.
Cell viability was measured by means of an MTT test. IC50 values were calculated using the 4-PL logistic model (P < 0.05). S.D. = standard deviation. RF = IC50 (resistant
cells)/IC50 (wild-type cells).
Cells (5 ×
103 mL–1) were treated for 72 h with
the tested compounds.
Cell viability was measured by means of an MTT test. IC50 values were calculated using the 4-PL logistic model (P < 0.05). S.D. = standard deviation. RF = IC50 (resistant
cells)/IC50 (wild-type cells).All complexes were equally effective against sensitive
(LoVo) and
resistant (LoVo-OXP) colon cancer cells and possessed RFs much lower
than that of doxorubicin, thus attesting their ability to overcome
the oxaliplatin resistance and the MDR phenomenon and not acting as
P-gp substrates.Clearly, such results make these complexes
promising for further
biological studies aiming at an application in solid tumors refractory
to platinum drug treatment.In an attempt to better appreciate
the antitumor potential of the
new Cu(I) and Cu(II) complexes containing the LND-conjugated ligands,
we compared their cytotoxic profiles with those of similarly unconjugated
or differently conjugated (pyrazolyl)acetate complexes that we had
previously characterized.[49,52,73,74] No significant differences in
terms of antiproliferative activity and overcoming drug resistance
have been detected. The calculated IC50 values, both in
sensitive and resistant cancer cells, were always in the very low
or submicromolar range. The newly developed copper complexes were
also screened against 3D spheroids of pancreatic PSN-1 cancer cells
to further evaluate their anticancer potential. Actually, 3D cell
cultures possess several features that more closely mimic the in vivo
tumor architecture and physiology, being consequently potentially
more predictive for in vivo effectiveness.[75] The cancer spheroids were treated with the tested complexes for
72 h, and cell viability was assessed by means of the acid phosphatase
(APH) assay (Table ). Notably, all complexes were much more effective than cisplatin
against the 3D model. Similarly to 2D studies, L1 and L2 derivatives were less effective than the corresponding L3 and L4 complexes. Differently from cytotoxicity
studies performed on 2D cell cultures, in 3D models, Cu(I) complexes
with PPh3 ligands proved to be much more effective than
Cu(II) derivatives. These results could be related to the more lipophilic
character of PPh3-containing Cu(I) complexes, which makes
them more effective at penetrating across the entire spheroid domain,
including the inner core. Complex 15 again emerged as
the most promising derivative, with an IC50 value roughly
12-fold better than that of cisplatin, and complex 5 as
the less effective derivative of the series.
Table 5
Cytotoxicity
toward Pancreatic PSN-1
Cancer Cell Spheroids of Complexes 5–16 and Cisplatina
IC50 (μM) ± S.D.
compound
PSN-1
5 [(PTA)2Cu(L1)][PF6]
40.4 ± 2.0
6 [(PPh3)2Cu(L1)][PF6]
8.3 ± 1.2
7 [(L1)CuCl2]
33.0 ± 3.0
8 [(PTA)2Cu(L3)]PF6
11.6 ± 1.6
9 [(PPh3)2Cu(L3)]PF6
6.8 ± 0.4
10 [(L3)CuCl2]
7.6 ± 0.3
11 [(PTA)2Cu(L2)][PF6]
31.9 ± 4.0
12 [(PPh3)2Cu(L2)][PF6]
22.0 ± 2.1
13 [(L2)CuCl2]
34.0 ± 2.1
14 [(PTA)2Cu(L4)]PF6
6.6 ± 0.5
15 [(PPh3)2Cu(L4)]PF6
4.5 ± 0.4
16 [(L4)CuCl2]
13.7 ± 3.7
cisplatin
52.6 ± 4.9
Cancer cell spheroids (2.5 ×
103 cells/well) were treated for 72 h with the tested compounds.
Cell viability was evaluated by means of the APH test. IC50 values were calculated from the dose–response curves obtained
using the 4-PL logistic model (P < 0.05). S.D.
= standard deviation.
Cancer cell spheroids (2.5 ×
103 cells/well) were treated for 72 h with the tested compounds.
Cell viability was evaluated by means of the APH test. IC50 values were calculated from the dose–response curves obtained
using the 4-PL logistic model (P < 0.05). S.D.
= standard deviation.As
compound 15 emerged as the most promising derivative
from cytotoxicity studies in both 2D and 3D models, it was selected
for uptake and mechanistic studies. Derivatives 14 and 16, bearing the same ligand but a different coligand or a
different Cu oxidation state, were included in these studies for useful
comparison.
Cellular Uptake
As stated before,
copper accumulation
in cancer cells is one of the most important factors affecting copper
complex cytotoxicity. In an attempt to correlate cytotoxic activity
with cellular accumulation, copper content was evaluated in PSN-1
cells treated for 24 or 36 h with 1 μM of complexes 14–16. The intracellular copper amount was quantified
by means of graphite furnace atomic absorption spectrometry (GF-AAS)
analysis, and the results, expressed as metal parts per billion per
106 cells, are shown in Figure .
Figure 5
Intracellular copper content after treatment
with compounds 14–16. PSN-1 cells
were treated for 24
or 36 h with 1 μM copper complexes, and the intracellular copper
amount was estimated using GF-AAS analysis. Error bars indicate the
standard deviation. *P < 0.1 and **P < 0.01 compared with the control.
Intracellular copper content after treatment
with compounds 14–16. PSN-1 cells
were treated for 24
or 36 h with 1 μM copper complexes, and the intracellular copper
amount was estimated using GF-AAS analysis. Error bars indicate the
standard deviation. *P < 0.1 and **P < 0.01 compared with the control.Although to a different extent, all three copper complexes accumulated
into cancer cells. Notably, the intracellular Cu levels follow the
trend 15 > 14 > 16 after
24
h, thus suggesting that Cu(I) complexes are more effective in crossing
the cancer cell membrane with respect to the Cu(II) derivative. Interestingly,
in the case of the Cu(II) complex 16, the intracellular
copper levels significantly increased with exposure time, whereas
Cu(I) complexes 14 and 15 seemed to accumulate
in a time-independent manner. These results might suggest the involvement
of different internalization mechanisms for Cu(I) and Cu(II) complexes,
whose electronic and molecular structures have been confirmed using
XPS and XAS analysis. Moreover, by comparing uptake and cytotoxicity
data in human pancreatic PSN-1 cancer cells, a direct correlation
between cellular accumulation and cytotoxic potency can be highlighted.
Mechanistic Studies
Copper complexes have been regarded
as redox active modulators as they may catalyze hydrogen peroxide
in the form of Fenton-like reactions inside the cell to produce reactive
oxygen species (ROS), thus altering the cellular redox homeostasis
and driving cells toward oxidative stress.[76] In addition, it has previously been reported that LND acts as an
antitumor drug by inhibiting both mitochondrial respiration and glycolysis,
thus shifting cultured cells to a more oxidized redox state.[77] Moreover, some classes of copper complexes were
found to exert an effective antiproliferative action by dysregulating
the mitochondrial function in cancer cells.[47]On these bases, we evaluated the ability of 14–16 to alter cellular redox homeostasis, in terms
of total cellular sulfhydryl content, ROS production, and perturbation
of the mitochondrial membrane potential in PSN-1 cells (Figure ). For compounds 15 and 16, SR-XPS data analysis provided information about
the electronic and molecular structures, evidencing the molecular
structural stability for both Cu(I) and Cu(II) complexes and confirming
the expected oxidation states.
Figure 6
(A) Sulfhydryl content in PSN1-treated
cancer cells incubated for
24 or 36 h with tested compounds 14–16. The sulfhydryl group amount was determined using the DTNB assay.
Error bars indicate the S.D. **P < 0.01 compared
with the control. (B) Effect of copper compounds on hydrogen peroxide
formation in PSN-1 cells. PSN-1 cells were preincubated in PBS/10
mM glucose medium for 20 min at 37 °C in the presence of 10 μM
CM-DCFDA and then treated with 10 μM of tested compounds.
(A) Sulfhydryl content in PSN1-treated
cancer cells incubated for
24 or 36 h with tested compounds 14–16. The sulfhydryl group amount was determined using the DTNB assay.
Error bars indicate the S.D. **P < 0.01 compared
with the control. (B) Effect of copper compounds on hydrogen peroxide
formation in PSN-1 cells. PSN-1 cells were preincubated in PBS/10
mM glucose medium for 20 min at 37 °C in the presence of 10 μM
CM-DCFDA and then treated with 10 μM of tested compounds.Interestingly, Cu(I) complexes 14 and 15 were completely ineffective in modulating the total thiol
content
in PSN1-treated cancer cells, whereas treatment with 16 induced a substantial time-dependent alteration of the total cellular
sulfhydryl content, determining 18 and 25% reduction of thiol groups
with respect to control untreated cells following 24 or 36 h of exposure,
respectively (Figure A).Consistently, treatment of PSN-1 cells with complex 16 determined a substantial time-dependent increase in cellular
basal
ROS production, whereas treatment with 14 and 15 did not result in an increase in the basal cellular ROS production
(Figure B). Notably,
treatment with 16 determined a substantial increase in
basal hydrogen peroxide formation, which was even higher than that
obtained with antimycin, a classical inhibitor of the mitochondrial
respiratory chain at the level of complex III.Overall, these
results demonstrate that the newly developed Cu(II)
complex 16 induced an oxidative shift in the redox status
of PSN-1 cells. On the contrary, Cu(I) complexes 14 and 15 seemed to act through a mechanism of action that does not
encompass oxidative stress induction.A persistent increase
in the rate of ROS production and the induction
of thiol redox stress can in turn prompt the collapse of the mitochondrial
membrane potential as well as the loss of the mitochondrial shape
and integrity (swelling), possibly leading to the
induction of cell apoptosis.[78] We hence
evaluated the effect determined by treatment with complexes 14–16 in terms of the modification of
mitochondrial pathophysiological characteristics, such as the mitochondrial
membrane potential and induction of cell death.For mitochondrial
membrane potential detection, PSN-1 cells were
treated with IC50 concentrations of the tested complexes,
and the percentage of cells with the altered mitochondrial membrane
potential was determined by means of the Mito-ID membrane potential
kit. As evident by results depicted in Figure A, as expected, complex 16 induced
a 31% decrease in the dye red fluorescence, rather similar to that
induced by the reference compound carbonyl cyanide-m-chlorophenylhydrazone (CCCP), thus attesting a significant increase
in the percentage of hypopolarized cells. On the other hand, 14 and 15 induced a slight (about 12 and 19%,
respectively) increase in the dye red fluorescence, thus indicating
a modest increase in cancer cell population with the hyperpolarized
mitochondrial membrane potential.
Figure 7
(A) PSN-1 cells were treated for 24 h
with IC50 concentrations
of tested complexes or CCCP (3 μM). The mitochondrial membrane
potential was determined using the Mito-ID membrane potential kit.
Data are the means of three independent experiments. Error bars indicate
the S.D. *P < 0.1 and **P <
0.01 compared with the control. (B) Hoechst staining of PSN-1 cells
incubated for 48 h with IC50 doses of 14, 15, or 16.
(A) PSN-1 cells were treated for 24 h
with IC50 concentrations
of tested complexes or CCCP (3 μM). The mitochondrial membrane
potential was determined using the Mito-ID membrane potential kit.
Data are the means of three independent experiments. Error bars indicate
the S.D. *P < 0.1 and **P <
0.01 compared with the control. (B) Hoechst staining of PSN-1 cells
incubated for 48 h with IC50 doses of 14, 15, or 16.Considering these results, it is possible to state that Cu(I) complexes 14 and 15 possess a rather different mechanism
of action compared to Cu(II) complex 16.It has
been widely described that Cu(I) and Cu(II) complexes can
induce different types of cell death. In order to analyze the mechanism
involved in the loss of cancer cell viability, we assessed the ability
of selected complexes to induce cancer cell death by means of apoptosis. Figure B shows the results
obtained upon monitoring the cellular morphological changes in PSN-1
cells treated for 48 h with IC50 doses of 14, 15, and 16 and stained with a Hoechst
33258 fluorescent probe. Compared with control cells, cells treated
with 16 presented brightly stained nuclei and morphological
features typical of cells undergoing apoptosis, such as chromatin
condensation and fragmentation, thus confirming the ability of Cu(II)
complex 16 to induce cancer cell death by means of apoptosis.
Conversely, cells treated with 14 and 15 did not show any classical sign of apoptosis induction and appeared
increased in size and ultrastructural complexity.These data
are consistent with those already obtained with other
series of similarly unconjugated or differently conjugated (pyrazolyl)acetate
complexes and once again point out to the importance of the development
of Cu(I) species able to trigger paraptosis, a strategic approach
toward cancer cells that have become resistant to the apoptosis inducer
drugs.[49,52,73,74]Altogether, these results clearly suggest that
Cu(II) complex 16 triggers cancer cell death via an apoptotic
pathway, whereas
Cu(I) complexes 14 and 15 kill cancer cells
by means of an apoptosis alternative cancer cell death, possibly paraptosis,
that has been recognized as a strategic pathway in cancer cells that
are resistant to apoptotic mechanisms. Paraptosis is a type of programmed
cell death, morphologically distinct from apoptosis and necrosis.
Defining features of paraptosis are cytoplasmic vacuolation and the
lack of an apoptotic morphology (cell shrinkage, apoptotic bodies,
chromatin condensation, and nuclear fragmentation). Like apoptosis
and other types of programmed cell death, the cell is involved in
causing its own death, and gene expression is required.[79]Interestingly, the above discussed EXAFS
data analysis revealed
that in Cu(I) complex 15, the Cu–N nearest neighbor
distance is slightly longer with respect to Cu(II) complexes 10 and 16, suggesting more loosely bound copper
in complex 15, likely related to the lower electronegativity
of Cu(I). This structural feature, potentially leading to a faster
ligand exchange rate and reactivity, might be responsible for the
higher antitumor efficacy of 15.Considering all
the collected data, complex 15 can
be recommended for a more detailed investigation of its biological
properties, such as the assessment of its interactions with important
biomolecules and the evaluation of the in vivo efficacy.
Conclusions
In this study, the known antitumor drug LND was functionalized
with species able to coordinate metals, affording new heteroscorpionate
ligands 1–4, which were used for
the preparation of Cu(I) and Cu(II) complexes 5–16. In the solid state, a multitechnique approach (SR-XPS,
NEXAFS, and XAFS) allowed us to ascertain the molecular stability
of the ligands upon interaction with the copper ions, as well as to
determine the coordination geometry and copper ion oxidation state.From the biological studies, the following promising results emerged:All the
complexes showed an extremely
promising cytotoxic activity in a panel of human tumor cell lines,
eliciting IC50 values in the low or submicromolar range
and being more effective than the reference metallodrug cisplatin.Noteworthily, they were
able to overcome
the oxaliplatin and multidrug resistance.They were also more effective than
cisplatin against the more predictive 3D spheroids of pancreatic PSN-1
cancer cells. In particular, the lipophilic PPh3-containing
Cu(I) complexes proved to be the most active compounds, with complex 15 being the most promising candidate.Representative complexes 14–16 were able to cross the cellular plasmalemma,
and Cu accumulated differently in treated cancer cells with a direct
correlation between cellular accumulation and cytotoxic potency, suggesting
the involvement of different internalization mechanisms for Cu(I)
and Cu(II) complexes.More importantly, Cu(I) complexes proved
to be ineffective in modulating cellular oxidative stress and induced
cancer cell death via an apoptosis-alternative pathway, possibly paraptosis.
On the contrary, Cu(II) complexes caused oxidative stress and triggered
apoptotic cell death.Based on their
interesting biological profile, Cu(I) LND-conjugated
complexes deserve to be further investigated, with the aim to find
application in solid tumors, refractory to platinum-based drug treatment.
Experimental Section
Chemistry
Materials
and General Methods
All the reagents have
been purchased and used without further purification. Melting points
(mps) were performed using an SMP3 Stuart Scientific Instrument (Bibby
Sterilin Ltd., London, UK). Elemental analyses (C, H, N, and S) (EA)
were performed using a Fisons Instruments EA-1108 CHNS–O elemental
analyzer (Thermo Fisher Scientific Inc., Waltham, MA, USA). Fourier
transform infrared (FT-IR) spectra were recorded from 4000 to 700
cm–1 on a PerkinElmer Frontier instrument (PerkinElmer
Inc., Waltham, MA, USA), equipped with an attenuated total reflection
unit using a universal diamond top plate as a sample holder. Abbreviation
used in the analyses of the FT-IR spectra: br = broad, m = medium,
mbr = medium broad, s = strong, sbr = strong broad, sh = shoulder,
vs = very strong, w = weak, and wbr = weak broad. NMR spectra for
nuclei 1H, 13C, and 31P were recorded
using a Bruker 500 Ascend spectrometer (Bruker BioSpin Corporation,
Billerica, MA, USA; 500.1 MHz for 1H, 125 MHz for 13C, and 202.4 MHz for 31P). Tetramethylsilane [Si(CH3)4] was used as an external standard for the 1H and 13C NMR spectra, while 85% H3PO4 was used for the 31P NMR spectra. The chemical
shifts (δ) are reported in parts per million. Abbreviation used
in the analyses of the NMR spectra: br = broad, d = doublet, m = multiplet,
s = singlet, sbr = singlet broad, t = triplet, and tbr = triplet broad.
ESI-MS spectra were recorded in the positive- [ESI-MS(+)] or negative-ion
[ESI-MS(−)] mode on a Waters Micromass ZQ spectrometer equipped
with a single quadrupole (Waters Corporation, Milford, MA, USA), using
a methanol or acetonitrile mobile phase. The compounds were added
to reagent grade methanol or acetonitrile to give approximately 0.1
mM solutions. These solutions were injected (1 μL) into the
spectrometer fitted with an autosampler. The pump delivered the solutions
to the mass spectrometer source at a flow rate of 200 μL/min,
and nitrogen was employed both as a drying gas and as a nebulizing
gas. Capillary voltage was typically 2500 V. The temperature of the
source was 100 °C, while the temperature of the desolvation was
400 °C. In the analyses of ESI-MS spectra, the confirmation of
major peaks was supported by comparison of the observed and predicted
isotope distribution patterns, the latter calculated using IsoPro
3.1 computer software (T-Tech Inc., Norcross, GA, USA).
Synthesis
of 2-Hydroxyethyl-1-(2,4-dichlorobenzyl)-1H-indazole-3-carboxylate
(LONES)
Sulfuric acid
(2 mL) was added to a solution of LND (0.50 g, 1.56 mmol) in ethylene
glycol (35 mL), and the reaction mixture was stirred at 85 °C
for 1 h. After cooling to room temperature, the solution was poured
into the ice water. The aqueous layer (50 mL) was extracted by dichloromethane
(3 × 30 mL). The organic phase was dried over Na2SO4. The evaporation of the solvent under reduced pressure afforded
a residue, which was purified using column chromatography, eluting
with cyclohexane/EtOAc (6:4). A white solid was obtained (65% yield).
mp: 121–122 °C. IR (cm–1): 3480mbr (O–H);
3084wbr, 2949wbr (C–H); 1713s (C=O); 1585m; 1560sh (C=C/C=N);
1479m, 1472s, 1446m, 1436s, 1421m, 1311s, 1275m, 1249s, 1225s, 1167s,
1157s, 1129s, 1096m, 1084s, 1044m, 1025m, 1009m, 990m, 977m, 944m,
898m, 855m, 839m, 807m, 789m, 776m, 747s, 737s. 1H NMR
(CDCl3, 293 K): δ 4.07 (m, 2H, −CH2–OH), 4.65 (m, 2H, −CH2–O), 5.81 (s, 2H, N–CH2-Ph), 6.72 (d, 1H, ArH), 7.10–7.49
(m, 6H, ArH and OH), 8.28 (d, 1H,
ArH). ESI-MS (major positive ions, CH3OH), m/z (%): 365 (20) [LONES +
H]+, 387 (100) [LONES + Na]+, 753 (60) [2LONES
+ Na]+. ESI-MS (major negative ions, CH3OH), m/z (%): 363 (100) [LONES – H]−.
Synthesis of N-(2-Aminoethyl)-1-(2,4-dichlorobenzyl)-1H-indazole-3-carboxamide (LONAM)
CDI (0.61 g, 3.74
mmol) was added to a solution of LND (1.00 g, 3.12 mmol) in anhydrous
tetrahydrofuran (THF) (20 mL), and the mixture was stirred at room
temperature for 1 h. After ethylenediamine (0.94 g, 15.6 mmol) was
added, the solution was stirred at room temperature for 18 h. After
evaporation of the solvent, the oil formed was dissolved in CHCl3 (30 mL) and washed with H2O (2 × 20 mL).
The organic phase was dried over Na2SO4. The
evaporation of the solvent under reduced pressure afforded a residue,
which was purified using column chromatography, eluting with MeOH/EtOAc
(1:9). A white solid was obtained (65% yield). mp: 71–72 °C.
IR (cm–1): 3282br (N–H); 3060w, 3030w, 2937wbr
(C–H); 1657sh, 1642s (C=O); 1620sh, 1588m; 1575m, 1538s
(C=C/C=N); 1491s, 1473s, 1436s, 1406m, 1386m, 1372m,
1357m, 1310sbr, 1282m, 1233s, 1175s, 1155m, 1132m, 1097s, 1048s, 1005m,
968mbr, 942m, 859m, 836s, 788s, 772s, 742s. 1H NMR (CDCl3, 293 K): δ 3.01–3.58 (m, 4H, NH–CH2–CH2–NH),
4.87 (br, 2H, NH2), 5.64 (s, 2H, N–CH2–Ph), 6.63 (d, 1H, ArH), 7.02–7.48 (m, 6H, ArH and NH), 8.46 (d, 1H, ArH). ESI-MS (major positive ions,
CH3OH), m/z (%): 363
(100) [LONAM + H]+, 385 (20) [LONAM + Na]+,
727 (40) [2LONAM + H]+. ESI-MS (major negative ions, CH3OH), m/z (%): 361 (100)
[LONAM – H]−.
Synthesis of L1 (1)
A solution
of HC(pz)2COOH (0.300 g, 1.560 mmol), LONES (0.624 g, 1.710
mmol), and DMAP (0.020 g, 0.157 mmol) in THF (20 mL) was cooled to
0 °C. EDCI·HCl (0.360 g, 1.880 mmol) was added, and the
mixture was stirred for 16 h at room temperature. The reaction was
then quenched with H2O (30 mL), and the aqueous phase was
extracted with EtOAc (2 × 30 mL) The organic phase was washed
with brine (2 × 20 mL) and dried over Na2SO4. The evaporation of the solvent afforded a residue, which was purified
using column chromatography, eluting with cyclohexane/EtOAc (7:3).
A white solid was obtained (72% yield). mp: 95–96 °C.
IR (cm–1): 3121wbr, 2958wbr (C–H); 1762m,
1716mbr (C=O); 1616w; 1590w, 1564w, 1518w (C=C/C=N);
1476m, 1434m, 1387s, 1317m, 1291m, 1259m, 1221s, 1191m, 1156s, 1123s,
1088s, 1046s, 1008m, 967m, 948m, 916m, 884m, 835m, 817m, 801m, 788m,
748s. 1H NMR (DMSO-d6, 293
K): δ 4.52–4.61 (m, 4H, O–CH2–CH2–O), 5.89 (s,
2H, N–CH2–Ph), 6.26 (t,
2H, 4-CH), 6.96 (d, 1H, ArH), 7.37–8.01
(m, 11H, 3-CH, 5-CH, ArH and CHCO). 1H NMR (CDCl3,
293 K): δ 4.72 (m, 4H, O–CH2–CH2–O), 5.81 (s, 2H, N–CH2-Ph), 6.30 (m, 2H, 4-CH),
6.73 (d, 1H, ArH), 7.11–7.81 (m, 10H, 3-CH, 5-CH, ArH and CHCO), 8.15 (d, 1H, ArH). ESI-MS (major
positive ions, CH3OH) m/z (%): 539 (20) [L1 + H]+, 561 (100) [L1 + Na]+. Calcd for C25H20Cl2N6O4: H, 3.74; C, 55.67; N, 15.58%.
Found: H, 3.94; C, 55.33; N, 15.20%.
Synthesis of L2 (2)
This compound
was prepared from HC(3,5-Me2pz)2COOH and LONES
following the procedure described for L1 (1): a white solid was obtained (75% yield). mp: 139–140 °C.
IR (cm–1): 3063wbr, 2958w, 2928w (C–H); 1763s,
1726sbr (C=O); 1616w; 1590m, 1562s (C=C/C=N);
1476s, 1416s, 1378s, 1316s, 1265s, 1218vs, 1202vs, 1156vs, 1123vs,
1102vs, 1048s, 1035vs, 1008s, 970s, 947m, 892m, 862m, 835s, 787vs,
748vs. 1H NMR (DMSO-d6, 293
K): δ 1.96 (s, 6H, CH3), 2.09 (s,
6H, CH3), 4.55–4.63 (m, 4H, O–CH2–CH2–O),
5.79 (s, 2H, N–CH2-Ph), 5.88 (s,
2H, 4-CH), 6.97 (d, 1H, ArH), 7.33–8.05
(m, 7H, ArH and CHCO). 1H NMR (CDCl3, 293 K): δ 2.11 (s, 6H, CH3), 2.14 (s, 6H, CH3), 4.74
(m, 4H, O–CH2–CH2–O), 5.81, (s, 2H, N–CH2-Ph), 5.83 (s, 2H, 4-CH), 6.73 (d, 1H,
ArH), 7.06–8.16 (m, 7H, ArH and CHCO). ESI-MS (major positive ions, CH3OH), m/z (%): 595 (20) [L2 + H]+, 617 (100) [L2 + Na]+, 633 (20) [L2 + K]+, 1213 (10) [2L2 + Na]+. Calcd for C29H28Cl2N6O4: H, 4.74; C, 58.49; N, 14.11%.
Found: H, 4.45; C, 58.71; N, 13.88%.
Synthesis of L3 (3)
A mixture
of HC(pz)2COOH (0.156 g, 0.810 mmol), LONAM (0.330 g, 0.900
mmol), EDCI·HCl (0.230 g, 1.20 mmol), HOBt (0.160 g, 1.18 mmol),
and triethylamine (0.107 mg, 1.06 mmol) in N,N-dimethylformamide (DMF) (10 mL) was stirred at room temperature
for 16 h. The reaction was quenched with H2O (30 mL), and
the aqueous phase was extracted with EtOAc (2 × 20 mL). The organic
phase was washed with brine (3 × 20 mL) and NaHCO3 (3 × 20 mL) and dried over Na2SO4. The
evaporation of the solvent afforded a residue, which was purified
using column chromatography, eluting with cyclohexane/EtOAc (3:7).
A white solid was obtained (68% yield). mp: 179–181 °C.
IR (cm–1): 3275mbr, 3223m (N–H); 3075m, 2951m,
(C–H); 1673vs, 1641vs (C=O); 1587m, 1539vs, 1515s (C=C/C=N);
1493s, 1473s, 1449s, 1433m, 1389vs, 1371s, 1313s, 1285s, 1249s, 1230vs,
1195s, 1177vs, 1154s, 1131m, 1112m, 1099s, 1092s, 1084s, 1064m, 1049vs,
1005m, 985w, 969m, 946m, 935m, 915m, 896w, 883w, 864s, 837vs, 812vs,
788s, 751vs. 1H NMR (DMSO-d6, 293 K): δ 3.30–3.47 (m, 4H, NH–CH2–CH2–NH), 5.83
(s, 2H, N–CH2-Ph), 6.27 (t, 2H,
4-CH), 6.78 (d, 1H, ArH), 7.25–8.64
(m, 13H, 3-CH, 5-CH, ArH, CHCO and NH). 1H NMR
(CDCl3, 293 K): δ 3.69 (m, 4H, NH–CH2–CH2–NH),
5.67 (s, 2H, N–CH2-Ph), 6.26 (m,
2H, 4-CH), 6.68 (d 1H, ArH), 7.00–7.72
(m, 12H, 3-CH, 5-CH, ArH, CHCO and NH), 8.38 (d, 1H, ArH). ESI-MS (major positive ions, CH3OH), m/z (%): 559 (100) [L3 + Na]+, 1097 (30) [2L3 + Na]+. ESI-MS (major
negative ions, CH3OH), m/z (%): 557 (100) [L3 – H]−. Calcd
for C25H22Cl2N8O2: H, 4.13; C, 55.87; N 20.85%. Found: H, 3.97; C, 55.53; N, 20.59%.
This compound was prepared from
PPh3 and L3 (3) following the procedure
described for 5. The obtained residue was filtered and
then purified in diethyl ether (50 mL) to obtain the white complex
[(PPh3)2Cu(L3)]PF6 (9) in 65% yield. mp: 186–189 °C. IR (cm–1): 3399wbr, 3288wbr (N–H); 3128wbr, 3055wbr (C–H);
1702sh, 1674mbr (C=O); 1626m, 1615m; 1588m, 1540mbr (C=C/C=N);
1479m, 1435s, 1403m, 1373m, 1361m, 1289mbr, 1245mbr, 1231mbr, 1181mbr,
1119m, 1095m, 1056m, 1027m, 999m, 981mbr, 919wbr, 834vs, 741vs. 1H NMR (DMSO-d6, 293 K): δ
3.37–3.41 (m, 4H, NHCH2CH2NH), 5.82 (s, 2H, NCH2Ph), 6.36 (sbr, 2H, 4-CH), 6.79 (d, 1H, ArH), 7.31–7.97 (m, 40H, 3-CH, 5-CH, ArH and CHCO), 8.22
(d, 1H, ArH), 8.35 (tbr, 1H, NH),
8.65 (tbr, 1H, NH). 13C NMR (DMSO-d6, 293 K): δ 38.1 (NCH2CH2N); 50.0 (NCH2Ph); 107.1 (4-CH); 110.8, 122.5, 122.7,
123.2, 127.7, 128.3, 129.2, 129.4, 129.5, 130.8, 132.0, 132.5, 132.7,
132.9, 133.4, 133.8, 133.9, 138.5, 141.6, 141.9 (ArH, Ar, CHCO, 3-CH and 5-CH);
162.5, 164.0 (CO)·31P{1H} NMR (DMSO-d6, 293 K): δ −144.00
(septet, J(F–P) = 714 Hz, PF6),
0.35 (sbr). 31P{1H} NMR (CD3CN, 293
K): δ −143.53 (septet, J(F–P)
= 707 Hz, PF6), 0.26 (sbr). ESI-MS (major positive ions,
CH3CN), m/z (%): 587
(100) [Cu(PPh3)2]+, 601 (10) [Cu(L3)]+, 863 (20) [(PPh3)Cu(L3)]+. ESI-MS (major negative ions, CH3CN), m/z (%): 145 (100) [PF6]−. Calcd for C61H52Cl2CuF6N8O2P3: H, 4.13;
C, 57.67; N, 8.82%. Found: H, 4.06; C, 57.38; N, 8.50%.
Synthesis
of [(L3)CuCl2] (10)
This
compound was prepared from L3 (3) following
the procedure described for 7, using CH3OH
as a solvent, to give the light blue complex [(L3)CuCl2] (10) in 68% yield. mp: 216–219
°C dec. IR (cm–1): 3494wbr, 3407m, 3290mbr
(N–H); 3152w, 3121m, 3091m, 3065w, 2977w, 2943w, 2832w (C–H);
1672vs, 1659sh (C=O); 1565sbr, 1539s (C=C/C=N);
1491s, 1471s, 1450m, 1428m, 1406s, 1384s, 1361m, 1330s, 1313s, 1282vs,
1244s, 1230s, 1205m, 1197m, 1172s, 1150m, 1130m, 1095s, 1063vs, 1046m,
1026m, 1007s, 990s, 956m, 942m, 924m, 892m, 861s, 841s, 833vs, 787vs,
765vs, 748vs, 729s. ESI-MS (major positive ions, CH3CN), m/z (%): 537 (80) [L3 + H]+, 559 (100) [L3 + Na]+, 600 (55) [Cu(L3) – H]+, 1097 (40) [2L3 + Na]+, 1137 (20) [Cu(L3)2]+. ESI-MS
(major negative ions, CH3CN), m/z (%): 170 (10) [CuCl3]−, 573
(100) [L3 + Cl]−. Calcd for C25H22Cl4CuN8O2: H, 3.30;
C, 44.69; N, 16.68%. Found: H, 3.52; C, 45.02; N, 16.34%.
Synthesis
of [(PTA)2Cu(L2)]PF6 (11)
This compound was prepared from L2 (2) following the procedure described for 5. The reaction
was filtered, and the solution was dried under
reduced pressure to give the white complex [(PTA)2Cu(L2)]PF6 (11) in 94% yield. mp: 187–191
°C. IR (cm–1): 3080br, 2985sh, 2950sh, 2925wbr,
2893sh (C–H); 1763m, 1719mbr (C=O); 1647wbr, 1616wbr;
1590w, 1563m (C=C/C=N); 1475m, 1449m, 1418m, 1389m,
1316m, 1297mbr, 1268mbr, 1242m, 1218mbr, 1163mbr, 1123m, 1104m, 1043mbr,
1014s, 969sbr, 948s, 895m, 874sh, 834vs, 803sh, 744s, 718m. 1H NMR (DMSO-d6, 293 K): δ 2.05
(s, 6H, CH3), 2.36 (s, 6H, CH3), 4.04 (s, 12H, NCH2P),
4.40–4.73 (m, 16H, NCH2N and OCH2CH2O), 5.79–5.96
(m, 4H, NCH2Ph and 4-CH), 6.90–8.03 (m, 8H, CHCO and ArH). 13C NMR (DMSO-d6, 293 K):
δ 11.0 (CH3); 14.3 (CH3); 50.3 (NCH2Ph); 50.5,
51.8 (CH2P); 72.4 (CH2N); 65.1, 66.7 (OCH2CH2O); 106.2 (4-CH); 111.3, 122.0, 123.2,
123.9, 127.9, 128.2, 129.6, 131.6, 133.5, 135.0, 141.4, 144.3, 148.2,
151.4 (ArH, Ar, CHCO, 3-CH and 5-CH); 161.9, 163.4 (CO). 31P{1H} NMR (DMSO-d6, 293 K): δ
−144.19 (septet, J(F–P) = 711 Hz, PF6), −92.78 (sbr). 31P{1H} NMR
(CD3CN, 293 K): δ −143.52 (septet, J(F–P) = 706 Hz, PF6), −94.0 (br).
ESI-MS (major positive ions, CH3CN), m/z (%): 659 (100) [Cu(L2)]+. ESI-MS (major negative ions, CH3CN), m/z (%): 145 (100) [PF6]−. Calcd for C41H52Cl2CuF6N12O4P3: H, 4.69; C, 44.03; N, 15.03%.
Found: H, 4.80; C, 43.77; N, 14.78%.
Synthesis of [(PPh3)2Cu(L2)]PF6 (12)
This compound was prepared from
PPh3 and L2 (2) following the procedure
described for 5. The reaction was filtered, and the solution
was dried under reduced pressure. Then, the residue in the round-bottom
flask was purified with hot diethyl ether (3 × 30 mL) to give
the white complex [(PPh3)2Cu(L2)]PF6 (12) in 51% yield. mp: 118–122 °C.
IR (cm–1): 3055wbr, 2959wbr, 2924wbr (C–H);
1763m, 1718mbr (C=O); 1635w, 1616w; 1588w, 1563m (C=C/C=N);
1478m, 1435m, 1422sh, 1389m, 1315m, 1265mbr, 1217mbr, 1163m, 1123m,
1095m, 1049m, 1029m, 1008w, 998m, 961w, 896w, 875m, 837vs, 789m, 743s. 1H NMR (DMSO-d6, 293 K): δ
1.92 (s, 6H, CH3), 2.41 (s, 6H, CH3), 3.70, 4.27 (sbr, 4H, OCH2CH2O), 5.88 (s, 2H, NCH2Ph), 6.02 (s, 2H, 4-CH), 7.07
(d, 1H, ArH), 7.28–7.71 (m, 35H, CHCO and ArH), 7.93 (d, 1H, ArH), 8.05 (d, 1H, ArH). 13C NMR (DMSO-d6, 293 K): δ 11.1 (CH3); 14.4 (CH3); 50.7 (NCH2Ph); 64.8, 66.3 (OCH2CH2O); 107.2 (4-CH); 111.3, 122.0, 123.3, 123.9, 128.0, 128.4, 130.0, 131.6, 133.6,
135.2, 141.7, 141.8, 144.2, 151.3 (ArH, Ar, CHCO,
3-CH and 5-CH); 161.8, 164.4 (CO). 31P{1H} NMR (DMSO-d6, 293 K): δ −144.20 (septet, J(F–P) = 710 Hz, PF6), −1.97 (sbr). ESI-MS
(major positive ions, CH3CN), m/z (%): 587 (100) [Cu(PPh3)2]+, 659 (80) [Cu(L2)]+, 921 (10) [(PPh3)Cu(L2)]+. ESI-MS (major negative ions, CH3CN), m/z (%): 145 (100)
[PF6]−. Calcd for C65H58Cl2CuF6N6O4P3: H, 4.40; C, 58.76; N, 6.33%. Found: H, 4.23; C, 58.42; N,
6.06%.
Synthesis of [(L2)CuCl2] (13)
CuCl2·2H2O (0.061 g, 0.359
mmol) was added to an acetonitrile solution (80 mL) of L2 (2, 0.214 g, 0.359 mmol). The reaction was stirred
at room temperature for 24 h and dried under reduced pressure. Subsequently,
the raw product was recrystallized using CH3CN/Et2O in a 1:10 ratio to obtain the dark green complex [(L2)CuCl2] (13) in 60% yield. mp: 148–150
°C. IR (cm–1): 3137mbr, 2960mbr, 2926mbr, 2856mbr
(C–H); 1765m, 1716mbr (C=O); 1590m, 1563m (C=C/C=N);
1472m, 1442m, 1419m, 1386m, 1316m, 1273m, 1217s, 1162s, 1124s, 1105s,
1047s, 1007m, 954mbr, 899m, 858m, 835m, 788s, 752s, 721m, 708m. ESI-MS
(major positive ions, CH3CN), m/z (%): 658 (100) [Cu(L2) – H]+. ESI-MS (major negative ions, CH3CN), m/z (%): 170 (100) [CuCl3]−. Calcd for C29H28Cl4CuN6O4: H, 3.87; C, 47.72; N, 11.51%. Found: H, 3.94; C, 47.48;
N, 11.23%.
Synthesis of [(PTA)2Cu(L4)]PF6 (14)
This compound was prepared
from L4 (4) following the procedure described
for 5. The reaction mixture was filtered, and the solution
was
dried under reduced pressure to obtain the white complex [(PTA)2Cu(L4)]PF6 (14) in 47%
yield. mp: 227–230 °C. IR (cm–1): 3389wbr,
3252wbr (N–H); 2923wbr, 2887wbr (C–H); 1692m, 1658mbr
(C=O); 1590w, 1562m, 1533mbr (C=C/C=N); 1493m,
1472m, 1449m, 1416m, 1388m, 1373m, 1314m, 1295mbr, 1267m, 1242m, 1230sh,
1176m, 1158sh, 1135w, 1104m, 1042mbr, 1013s, 968sbr, 948s, 894m, 874m,
833vs, 806sh, 776sh, 744s, 729s. 1H NMR (DMSO-d6, 293 K): δ 2.19 (s, 6H, CH3), 2.37 (s, 6H, CH3), 3.36–3.48
(m, 4H, NHCH2CH2NH), 4.04–4.66 (m, 24H, NCH2P
and NCH2N), 5.82 (s, 2H, NCH2Ph), 6.10 (sbr, 2H, 4-CH), 6.76 (d,
1H, ArH), 6.92 (s, 1H, CHCO), 7.33–7.79
(m, 5H, ArH), 8.22 (d, 1H, ArH),
8.43 (br, 1H, NH), 9.07 (br, 1H, NH). 13C NMR (DMSO-d6, 293 K):
δ 11.1 (CH3); 14.4 (CH3); 38.3 (NCH2CH2N); 50.0 (NCH2Ph); 50.5,
51.9 (CH2P); 72.4 (CH2N); 107.0 (4-CH); 110.8, 122.5, 122.7, 123.2,
127.7, 128.3, 129.6, 130.6, 133.3, 133.7, 133.9, 138.5, 141.6, 142.3
(ArH, Ar, CHCO, 3-CH and 5-CH); 162.6, 164.2 (CO). 31P{1H}-NMR (DMSO-d6, 293 K): δ
−144.02 (septet, J(F–P) = 714 Hz, PF6), −92.29 (s). ESI-MS (major positive ions, CH3CN), m/z (%): 657 (100)
[Cu(L4)]+, 814 (25) [(PTA)Cu(L4)]+. ESI-MS (major negative ions, CH3CN), m/z (%): 145 (100) [PF6]−. Calcd for C41H54Cl2CuF6N14O2P3: H, 4.88;
C, 44.11; N, 17.57%. Found: H, 4.61; C, 43.80; N, 17.21%.
Synthesis
of [(PPh3)2Cu(L4)]PF6 (15)
This compound was prepared from
PPh3 and L4 (4) following the procedure
described for 5. The reaction mixture was filtered, and
the solution was dried under reduced pressure. Then, the residue in
the round-bottom flask was solubilized with CHCl3 (10 mL)
and filtered, and the solution was dried under reduced pressure to
give the white complex [(PPh3)2Cu(L4)]PF6 (15) in 54% yield. mp: 100–105
°C. IR (cm–1): 3401wbr, 3284wbr (N–H);
3056wbr, 2926wbr, 2869wbr (C–H); 1694sh, 1672mbr (C=O);
1587m, 1562m, 1535m (C=C/C=N); 1492m, 1476m, 1435s,
1388m, 1311m, 1271m, 1230m, 1177m, 1158m, 1096m, 1071m, 1048m, 1028m,
998m, 918w, 835vs, 742vs. 1H NMR (DMSO-d6, 293 K): δ 1.82 (sbr, 6H, CH3), 2.43 (s, 6H, CH3), 3.37–3.41
(m, 4H, NHCH2CH2NH), 5.80 (s, 2H, NCH2Ph), 6.08 (s, 2H,
4-CH), 6.79 (d, 1H, ArH), 7.07 (s,
1H, CHCO), 7.26–7.52 (m, 33H, ArH), 7.73 (d, 1H, ArH), 7.79 (d, 1H, ArH), 8.20 (d, 1H, ArH), 8.38 (t, 1H, NH), 9.35 (tbr, 1H, NH). 13C NMR (DMSO-d6, 293 K): δ 11.0 (CH3); 13.8 (CH3); 37.6 (NCH2CH2N); 50.0 (NCH2Ph); 107.2 (4-CH); 110.8,
122.5, 122.8, 123.2, 127.7, 128.2, 129.4, 129.6, 129.8, 130.8, 131.9,
132.0, 132.5, 133.4, 133.7, 133.8, 138.5, 141.6, 143.0, 150.5 (ArH,
Ar, CHCO, 3-CH and 5-CH); 162.6, 164.5 (CO). 31P{1H} NMR (DMSO-d6, 293 K): δ −144.20
(septet, J(F–P) = 711 Hz, PF6),
4.99 (s). 31P{1H} NMR (CD3CN, 293
K): δ −143.53 (septet, J(F–P)
= 705 Hz, PF6), −1.06 (sbr). ESI-MS (major positive
ions, CH3CN), m/z (%):
587 (100) [Cu(PPh3)2]+, 657 (15)
[Cu(L4)]+, 919 (45) [(PPh3)Cu(L4)]+. ESI-MS (major negative ions, CH3CN), m/z (%): 145 (100) [PF6]−. Calcd for C65H60Cl2CuF6N8O2P3: H, 4.56; C, 58.85; N, 8.45%. Found: H, 4.27; C, 58.51; N, 8.12%.
Synthesis of [(L4)CuCl2] (16)
This compound was prepared from L4 (4)
following the procedure described for 7, using CH3OH as a solvent, to give the light blue complex [(L4)CuCl2] (16) in 83% yield. mp: 248–250
°C. IR (cm–1): 3426wbr (N–H); 3169wbr,
2904mbr, 2799mbr (C–H); 1669s, 1620sh (C=O); 1576m,
1559s (C=C/C=N); 1528s, 1493m, 1467m, 1448m, 1428m,
1415m, 1397m, 1384m, 1375m, 1349m, 1314m, 1268w, 1248m, 1220m, 1194m,
1177m, 1157m, 1133m, 1116m, 1103m, 1064m, 1042m, 1005m, 987m, 949w,
913m, 875m, 861m, 837s, 807m, 787s, 754s, 744s, 708m. ESI-MS (major
positive ions, CH3CN), m/z (%): 1248 (100) [Cu(L4)2 – H]+. ESI-MS (major negative ions, CH3CN), m/z (%): 170 (100) [CuCl3]−. Calcd for C29H30Cl4CuN8O2: H, 4.15; C, 47.85; N, 15.39%. Found: H, 4.43; C, 48.02;
N, 15.09%.
Spectroscopic Methods
SR-XPS measurements were
performed at the Materials Science Beamline
(MSB) at the ELETTRA synchrotron radiation source (Trieste, Italy).
The MSB is placed at the left end of the bending magnet 6.1, and it
is equipped with a plane grating monochromator that provides light
in the energy range of 21–1000 eV. The base pressure in the
UHV end-station is of 2 × 10–10 mbar; the end-station
is equipped with a SPECS PHOIBOS 150 hemispherical electron analyzer,
low-energy electron diffraction optics, a dual-anode Mg/Al X-ray source,
an ion gun, and a sample manipulator with a K-type thermocouple attached
to the rear side of the sample. For this experiment, we detected photoelectrons
emitted by C 1s, N 1s, Cl 2p, Cu 2p, and O 1s core levels at the normal
emission geometry. A photon energy of 650 eV impinging at 60°
was selected for all signals, with the aim to maximize especially
the N 1s signal intensity; Cu 2p spectra were also collected using
the Al Kα anode source (1487.0 eV) so as to maximize its photoemission
signal. Charging correction of BEs was always performed using the
aromatic C 1s signal as a reference (BE 284.70 eV).[80] To fit core-level spectra, we subtracted a Shirley background
and then used Gaussian peak functions as signal components.[81,82] The BE resolution was 0.6 eV for all measured core levels.
NEXAFS
Spectroscopy
NEXAFS spectroscopy experiments
were performed at the ELETTRA storage ring at the BEAR (Bending Magnet
for Emission Absorption and Reflectivity) beamline, installed at the
left exit of the 8.1 bending magnet exit. The apparatus is based on
a bending magnet as a source, a beamline optics delivering photons
from 5 eV up to about 1600 eV with a selectable degree of ellipticity.
The carbon and nitrogen K-edge spectra were collected at grazing (20°),
magic (54.7°), and normal (90°) incidence angles of the
linearly polarized photon beam with respect to the sample surface.
The photon energy and resolution were calibrated and experimentally
tested at the K absorption edges of Ar, N2, and Ne. The
normalization procedure consists of three steps: (i) the energy calibration,
in which the I0 reference current (drain current) of the
sample is shifted on the I0 reference current (drain current)
of the Au clean sample recorded; (ii) the signal is obtained from
the double ratio after
the interpolation of each signal
to the Au reference. The double ratio allows us to correct for variations
of the incident X-ray intensity with time as a function of photon
energy due to instabilities of the electron beam in the storage ring
or to changes of the X-ray optics in the beamline;[62] and (iii) finally, the signal is reduced to the standard
form through a pre-edge and post-edge fit: a linear pre-edge background
is subtracted from the data and a linear post-edge fit is applied
to the post-edge region to evaluate the jump and obtain the normalized
signal.
X-ray Absorption Fine Structure
XAFS Cu K-edge (ECu = 8980 eV) experiments were performed at
the XAFS beamline[83] of the ELETTRA (Trieste)
storage ring (complexes 10 and 16) and the
B18 beamline[84] of the Diamond Light Source
(UK) (complex 15). Complexes 10 and 16 were measured in the transmission geometry at LN temperature.
Samples were prepared in the form of solid homogeneous pellets, in
which the weight ratio between the PVP matrix and the Cu complex sample
was approximately 5:1. Complex 15 was measured in the
fluorescence geometry using a 36 element Ge detector. The sample was
homogeneously mixed with boron nitride, pressed into a pellet, and
measured at 77 K using a liquid nitrogen cryostat. The data were treated
using the standard procedure for data normalization and extraction
of the structural EXAFS signal χexp(k).[85] The quantitative EXAFS data analysis
was carried out using program FitEXA.[85] The least squares method was used to minimize the difference between
the experimental k2-weighted EXAFS function k2χexp(k) and the theoretical one k2χteo(k) in the reciprocal space
3 to 15 k–1. The model function was built as a sum
of Gaussian-shaped shells, representative of the neighbor atoms around
the absorberEach shell is defined by three parameters:
the number of neighbors N (multiplicity), the average
distance R, and the
MSRD parameter σ2, representing the variance of the scattering
(SS or MS) path distribution. The So2 parameter, taking into account many body losses,
was fixed to be 0.9. The same energy shift was used for all the contributions.
The amplitude A, phase
shift φ, and mean free path λ
functions were calculated using FEFF 8.2 program[86] using the molecular geometry around the absorber.
Experiments with Cultured Human Cancer Cells
Cu(I)
and Cu(II) complexes and the corresponding uncoordinated ligands and
precursors were dissolved in DMSO just before the experiment, and
a calculated amount of drug solution was added to the cell growth
medium to a final solvent concentration of 0.5%, which had no detectable
effects on cell viability. Cisplatin (Sigma Chemical Co, St. Louis,
USA) and oxaliplatin (Sigma Chemical Co.) were dissolved in 0.9% sodium
chloride solution.
Cell Cultures
Human lung (H157),
colon (HCT-15 and
LoVo), and pancreatic (PSN-1) carcinoma cells were obtained from the
American Type Culture Collection (ATCC, Rockville, MD, USA). Human
cervical carcinoma A431 cells were kindly provided by Prof. F. Zunino
(Division of Experimental Oncology B, Istituto Nazionale dei Tumori,
Milan). Human ovarian adenocarcinoma 2008 cells were kindly provided
by Prof. G. Marverti (Dipartimento di Scienze Biomediche, Università
di Modena University, Italy). Human colon cancer cells resistant to
doxorubicin (LoVo MDR) were kindly provided by Prof. M. P. Rigobello
(Dipartimento di Scienze Biomediche, Università di Padova,
Italy). The LoVo-OXP cells were derived, using a standard protocol,
by growing LoVo cells in increasing concentrations of OXP and following
17 months of selection of resistant clones, as previously described.[23]Cell lines were maintained in the logarithmic
phase at 37 °C in a 5% carbon dioxide atmosphere using the following
culture media containing 10% fetal calf serum (EuroClone, Milan, Italy),
antibiotics (50 units/mL penicillin and 50 μg/mL streptomycin),
and 2 mM l-glutamine: (i) RPMI-1640 medium (EuroClone) for
A431, PSN-1, H157, HCT-15, and 2008 cells and (ii) Ham’S F-12
(Sigma Chemical Co.) for LoVo, LoVo MDR, and LoVo-OXP cells.
MTT Assay
The growth inhibitory effect toward tumor
cells was evaluated by means of an MTT assay.[23] Briefly, 3–8 × 103 cells/well, dependent
upon the growth characteristics of the cell line, were seeded in 96-well
microplates in growth medium (100 μL). After 24 h, the medium
was removed and replaced with a fresh one containing the compound
to be studied at the appropriate concentration. Triplicate cultures
were established for each treatment. After 72 h, each well was treated
with 10 μL of a 5 mg/mL MTT saline solution, and following 5
h of incubation, 100 μL of a sodium dodecyl sulfate solution
in HCl 0.01 M was added. After overnight incubation, cell growth inhibition
was detected by measuring the absorbance of each well at 570 nm using
a Bio-Rad 680 microplate reader. Mean absorbance for each drug dose
was expressed as a percentage of the control untreated well absorbance
and plotted versus drug concentration. IC50 values, the
drug concentrations that reduce the mean absorbance at 570 nm to 50%
of those in the untreated control wells, were calculated using the
four-parameter logistic (4-PL) model. Evaluation was based on the
mean from at least four independent experiments.
Spheroid Cultures
Spheroid cultures were obtained by
seeding 2.5 × 103 PSN-1 cancer cells/well in a round-bottom
nontreated tissue culture 96-well plate (Greiner Bio-one, Kremsmünster,
Austria) in phenol red free RPMI-1640 medium (Sigma Chemical Co.)
containing 10% fetal calf serum and supplemented with 20% methyl cellulose
stock solution.
APH Assay
An APH modified assay
was used for determining
cell viability in 3D spheroids, as previously described.[87] IC50 values were calculated using
a 4-PL model.
Copper Cellular Accumulation
PSN-1
cells (2.5 ×
106) were seeded in 75 cm2 flasks in growth
medium (20 mL). After 24 h, the medium was replaced, and the cells
were incubated for 24 h with tested complexes. Monolayers were then
washed twice with ice-cold phosphate-buffered saline (PBS), harvested,
and counted. Cell samples were subjected to five freeze–thaw
cycles at −80 °C and then vigorously vortexed. The samples
were treated with highly pure nitric acid and transferred into a microwave
Teflon vessel. Samples were then submitted to standard mineralization
procedures and analyzed for the copper amount by using a Varian AA
Duo graphite furnace atomic absorption spectrometer (Varian, Palo
Alto, CA; USA) at 324 nm. The calibration curve was obtained using
known concentrations of standard solutions purchased from Sigma Chemical
Co.
ROS Production
The production of ROS was measured in
PSN-1 cells (104 per well) grown for 24 h in a 96-well
plate in RPMI medium without phenol red (Sigma Chemical Co.). Cells
were then washed with PBS and loaded with 10 μM 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein
diacetate acetyl ester (CM-H2DCFDA) (Molecular Probes-Invitrogen,
Eugene, OR) for 25 min in the dark. Afterward, cells were washed with
PBS and incubated with increasing concentrations of tested compounds.
Fluorescence increase was estimated utilizing a plate reader (Tecan
Infinite M200 PRO, Männedorf, Switzerland) at 485 nm (excitation)
and 527 nm (emission). Antimycin (3 μM, Sigma Chemical Co),
a potent inhibitor of Complex III in the electron transport chain,
and auranofin were used as positive controls.
Quantification of Thiols
The PSN-1 cells (2 ×
105) were seeded in a six-well plate in growth medium (4
mL). After 24 h, cells were incubated for 24 h with increasing concentrations
of tested compounds. Subsequently, the thiol content was measured
as previously described.[88]
Mitochondrial
Membrane Potential
The mitochondrial
membrane potential (ΔΨ) was assayed using the Mito-ID
membrane potential kit according to the manufacturer’s instructions
(Enzo Life Sciences, Farmingdale, NY). Briefly, PSN-1 cells (8 ×
103 per well) were seeded in 96-well plates; after 24 h,
cells were washed with PBS and loaded with the Mito-ID detection reagent
for 30 min at 37 °C in the dark. Afterward, cells were incubated
with increasing concentrations of tested complexes. Fluorescence intensity
was estimated using a plate reader (Tecan Infinite M200 PRO, Männedorf,
Switzerland) at 490 (excitation) and 590 nm (emission). CCCP (4 μM,
Sigma Chemical Co), a chemical inhibitor of the oxidative phosphorylation,
was used as the positive control.
Apoptosis Induction
PSN-1 cells were seeded into 8-well
tissue culture slides (BD Falcon, Bedford, MA, USA) at 5 × 104 cells/well (0.8 cm2). After 24 h, the cells were
washed twice with PBS, and following 48 h of treatment with IC50 doses of tested compounds, cells were stained for 5 min
with 10 μg/mL Hoechst 33258 [20-(4-hydroxyphenyl)-5-(4-methyl-1-piperazinyl)-2,50-bi-1H-benzimidazole trihydrochloride hydrate, Sigma-Aldrich,
St. Louis, MI, USA] in PBS. Samples were examined at 5× and 40×
magnification in a Zeiss LSM 800 confocal microscope using the Zeiss
ZEN 2.3 software system.
Statistical Analysis
All values
are the means ±
S.D. of no less than three measurements starting from three different
cell cultures. Multiple comparisons were made using ANOVA, followed
by the Tukey–Kramer multiple comparison test (*P < 0.05, **P < 0.01), using GraphPad software.
Authors: Lili Guo; Alexander A Shestov; Andrew J Worth; Kavindra Nath; David S Nelson; Dennis B Leeper; Jerry D Glickson; Ian A Blair Journal: J Biol Chem Date: 2015-10-31 Impact factor: 5.157
Scheme 1
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7