In vitro antitumor activity of the water soluble copper(I) complexes bearing the tris(hydroxymethyl)phosphine ligand

Article abstract of DOI:10.1021/jm701146c

Monocationic hydrophilic complexes [Cu(thp)₄]⁺ 3 and [Cu(bhpe)₂]⁺ 4 were synthesized by ligand exchange reactions starting from the labile [Cu(CH₃CN)₄][PF ₆] precursor in the presence of an excess of the relevant hydrophilic phosphine. Complexes 3 and 4 were tested against a panel of several human tumor cell lines. Complex 3 has been shown to be about 1 order of magnitude more cytotoxic than cisplatin. Chemosensitivity tests performed on cisplatin and multidrug resistance phenotypes suggested that complex 3 acts via a different mechanism of action than the reference drug. Different short-term proliferation assays suggested that lysosomal damage is an early cellular event associated with complex 3 cytotoxicity, probably mediated by an increased production of reactive oxygen species. Cytological stains and flow cytometric analyses indicated that the phosphine copper(I) complex is able to inhibit the growth of tumor cells via G2/M cell cycle arrest and paraptosis accompanied with the loss of mitochondrial transmembrane potential.

Full text of DOI:10.1021/jm701146c

798
J. Med. Chem. 2008, 51, 798–808
In Vitro Antitumor Activity of the Water Soluble Copper(I) Complexes Bearing the
Tris(hydroxymethyl)phosphine Ligand
Cristina Marzano,*,† Valentina Gandin,† Maura Pellei,‡ Davide Colavito,§ Grazia Papini,‡ Giancarlo Gioia Lobbia,‡
Elda Del Giudice,§ Marina Porchia, Francesco Tisato, and Carlo Santini*,‡
Dipartimento di Scienze Farmaceutiche, UniVersità di PadoVa, Via Marzolo 5, 35131 PadoVa, Italy, Dipartimento di Scienze Chimiche,
UniVersità di Camerino, Via S. Agostino 1, 62032 Camerino, Macerata, Italy, Research and InnoVation (R&I) S.p.A.,
Via SVizzera 16, 35127 PadoVa, Italy, and ICIS-C.N.R., Corso Stati Uniti, 4, 35127 PadoVa, Italy
ReceiVed September 13, 2007
Monocationic hydrophilic complexes [Cu(thp)4]+ 3 and [Cu(bhpe)2]+ 4 were synthesized by ligand exchange
reactions starting from the labile [Cu(CH3CN)4][PF6] precursor in the presence of an excess of the relevant
hydrophilic phosphine. Complexes 3 and 4 were tested against a panel of several human tumor cell lines.
Complex 3 has been shown to be about 1 order of magnitude more cytotoxic than cisplatin. Chemosensitivity
tests performed on cisplatin and multidrug resistance phenotypes suggested that complex 3 acts via a different
mechanism of action than the reference drug. Different short-term proliferation assays suggested that lysosomal
damage is an early cellular event associated with complex 3 cytotoxicity, probably mediated by an increased
production of reactive oxygen species. Cytological stains and flow cytometric analyses indicated that the
phosphine copper(I) complex is able to inhibit the growth of tumor cells via G2/M cell cycle arrest and
paraptosis accompanied with the loss of mitochondrial transmembrane potential.
Introduction
Other phosphine rich gold(I) compounds of the type
[Au(dppe)2][Cl] (dppea) 1,2-bis(diphenylphosphino)ethane)
have been reported to show cytotoxic activity against several
cell lines, including B16 melanoma, P388 leukemia, and M5076
reticulum cell carcinoma.6
Phosphine complexes of group 11 metals, especially those
including the gold(I) ion, have been proposed as alternative
anticancer drugs to the cisplatin reference drug.1,2 In general,
the higher drug tolerance profiles of gold-based agents compared
to the severe toxic effects on normal tissues or the occurrence
of intrinsic or acquired resistance induced by cisplatin have
represented a hopeful prospect for the chemotherapeutic ap-
plication of gold-based drugs. Despite this promising outlook,
clinical trials in humans of gold compounds still remain elusive.
A rational extension of diphosphine gold(I) chemistry to the
first row congener copper indicated that analogous copper(I)
complexes of “CuP4” stoichiometry could be readily prepared
and tested in vitro. Among them, [Cu(dppe)2][Cl] and [Cu-
(dppey)2][Cl] (dppey ) 1,2-bis(diphenylphosphino)ethylene)
produced cytotoxic effects on cell metabolism comparable to
those exhibited by Au(I) analogues, which were finally postu-
lated to be the result of the uncoupling of mitochondrial
oxidative phosphorylation.7 However, the presence of several
phenyl groups appended to the phosphorus donors in both
[Cu(dppey)2][Cl] and [Au(dppe)2][Cl] species caused undesired
nephrotoxicity8 and cardiovascular toxicity9 in animal models,
respectively, thus precluding clinical trials in humans.
Partial substitution of the CuP4 coordination sphere was
attempted using heterocyclic thiones10 or acetonitrile,11 produc-
ing “mixed compounds”. Among these derivatives, [Cu2(dppe)3-
(CH3CN)2][ClO4]2 exhibited a potent in vitro cytotoxicity in
H460 human lung carcinoma cells, comparable to that displayed
by adriamycin.11
The introduction of the first metal phosphine complex
auranofin (a thioglucose derivative of triethylphosphine gold(I))
into clinical use as an antiarthritic drug has represented the basis
for a series of investigations focused on the biological chemistry
and anticancer properties of phosphines and their related metal
complexes.3 Auranofin itself was shown to possess in vivo
antitumor activity against P388 murine leukemia.4 More re-
cently, in vitro studies indicated that auranofin is able to
overcome cisplatin resistance in human ovarian cancer cells,5
confirming the early assumption that a mechanism of action
different from the DNA damage induced by cisplatin could
underlie the cytotoxic activity of phosphine Au(I) drugs. It was
found that auranofin, acting as a potent inhibitor of thioredoxin
reductase, determines an alteration of the redox state of the cell
leading to an increased production of hydrogen peroxide and
oxidation of the components of the thyoredoxin system, therefore
creating the conditions for augmented apoptosis.5
Following a similar “mixed ligand” approach, we have
recently coordinated several N2-scorpionate ligands to a vacant
lipophilic CuP2 moiety (P2 ) bidentate dppe or two monodentate
arylphosphines), trying to enhance the hydrophilic character of
the overall phosphine-scorpionate copper species.12 These less
a Abbreviations: bhpe, bis[bis(hydroxymethyl)phosphino]ethane; DAB-
CO, 1,4-diazabicyclo[2.2.2]octane; dppe, 1,2-bis(diphenylphosphino)ethane;
dppey, 1,2-bis(diphenylphosphino)ethylene; DMSO, dimethyl sulfoxide; HE,
hematoxylin-eosin; MDR, multidrug resistance; MI, mitotic index; MTT,
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NR, neutral
red; PBS, phosphate buffered saline; PI, propidium iodide; ROS, reactive
oxygen species; SDS, sodium dodecyl sulfate; TB, trypan blue exclusion;
thp, tris(hydroxymethyl)phosphine; TMRM, tetramethylrhodamine methyl
ester.
* To whom correspondence should be addressed. For C.M.: phone,
+390498275347; fax, +390498275366; e-mail, cristina.marzano@unipd.it.
For C.S.: phone, +390737402213; fax, +390737402213; e-mail,
carlo.santini@unicam.it.
†
Università di Padova.
Università di Camerino.
Research and Innovation (R&I) S.p.A.
‡
§
ICIS-C.N.R.
10.1021/jm701146c CCC: $40.75
2008 American Chemical Society
Published on Web 02/06/2008

In Vitro Antitumor ActiVity of Copper(I) Complexes
Scheme 1. Preparation of bhpe, 2a
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4 799
a Reaction conditions: (i) 180 °C, reflux; (ii) 0 °C, Et2O, LiAlH4; (iii) room temp, EtOH, 40% HC(O)H, 3 M HCl; (iv) room temp, 1 M NaHCO3.
Table 1. Selected IR, 31P{1H} NMR, and ESI-MS Data of thp 1, bhpe 2 Ligands, and Copper(I) Complexes 3 and 4
compd
IR (cm-1 a
)
δ
31P{1H} NMRb
ESI-MS m/z (%)c
thp 1
bhpe 2
[Cu(thp)4][PF6] 3
-24.70 (s)
3458 br (OH)
3360 mbr (OH)
3134 m (CH)
560 s, 477 sbr
455 w
3448 br (OH)
3197 w (CH)
565 m, 452 sbr
376 w
-22.18 (s)
ESI(-): 250 (100) [bhpe + Cl]-
560 (6) [Cu(thp)4]+
-5.35 (q, JPCu ) 780 Hz)
-145.14 (septet, JPF ) 709 Hz)
436 (65) [Cu(thp)3]+
312 (100) [Cu(thp)2]+
[Cu(bhpe)2][PF6] 4
+9.67 (dbr)
-144.05 (septet, JPF ) 709 Hz)
492 (100) [Cu(bhpe)2]+
a In Nujol mull. b In D2O solution at 293 K. c In water/methanol solutions. Positive ions unless otherwise stated.
) bis[bis(hydroxymethyl)phosphino]ethane). These Cu(I) com-
plexes have been characterized by elemental analysis, FTIR,
ESI-MS, and multinuclear (1H, 31P{1H}, 13C{1H}, and 63Cu)
NMR spectral data, and they indeed showed high solubility and
stability in aqueous solutions. We have assessed the antitumoral
properties of these copper complexes in a large panel of human
cancer cell lines belonging to a variety of different tumor types
and also including cisplatin and multidrug resistance (MDR)
phenotypes. Our results showed that complex 3 is endowed with
a remarkable in vitro antitumor activity significantly higher than
that of cisplatin which was used as reference drug in all our
investigations. In particular, chemosensitivity tests performed
on the resistant sublines suggested that [Cu(thp)4][PF6] may act
with a different mechanism compared to cisplatin. The detection
of complex 3 mediated cytotoxicity, by means of different assays
measuring the damage to various subcellular organelles, revealed
that lysosomal damage represents the early cellular event
associated with complex 3 induced cell death. Moreover, this
event may be mediated by an increased production of reactive
oxygen species (ROS) as attested by measuring the tumor cell
viability of complex 3 treated cells in the presence of different
ROS scavengers. Finally, in order to throughly explore the
sequence of cellular events responsible for the antiproliferative
action of complex 3, we have investigated the effects on cell
cycle modulation and the cell death mechanism by means of
flow cytometric analysis. In previous studies, the antitumor
properties of the isostructural gold(I) cation [Au(thp)4]+ had
been ascribed to its ability in selectively elongating (or arresting)
the G1 phase of the cell cycle.16 In the present work, we have
Figure 1. Molecular structure of copper complexes 3 and 4.
hydrophobic Cu(I) compounds were proved to be easier to
handle during the in vitro tests and, more importantly, retained
cytotoxic activity against a panel of human tumor cell lines
including, for instance, A549 carcinoma cells that show poor
sensitivity to cisplatin treatment.12 In a subsequent work, we
established that the substitution of hydrophobic phosphines (e.g.,
dppe or triphenylphosphine) with hydrophilic tris(hydroxy-
methyl)phosphines did not affect the stability of the resulting
mixed Cu(I) complexes and permitted the preparation of water
soluble species with improved cytotoxic properties. In particular,
[HC(CO2)(pzMe2)2]Cu[P(CH2OH)3]2 was found to have a sig-
nificant antiproliferative activity against different human tumor
cells, also including examples of cisplatin resistant cancers. Our
results have suggested that the cytotoxic activity of this copper(I)
complex may be correlated to its ability to induce a nonapoptotic
mechanism of cell death.13
In the present study, we have prepared two copper complexes
of the “CuP4” type,14,15 namely, [Cu(thp)4][PF6] (3) (thp )
tris(hydroxymethyl)phosphine) and [Cu(bhpe)2][PF6] (4) (bhpe

800 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4
Table 2. Cytotoxic Activitya
Marzano et al.
IC50 ( SD (µM)
compd
HL60
A549
MCF-7
Daudi
HepG2
A375
CaCo2
HCT-15
HeLa
[Cu(thp)4][PF6] 3
0.60 ( 0.02 9.11 ( 2.71 11.08 ( 0.52 6.94 ( 0.18 1.26 ( 0.10 4.58 ( 2.41 1.08 ( 0.12 2.00 ( 0.03 8.21 ( 1.50
[Cu(bhpe)2][PF6] 4 47.40 ( 2.92 57.60 ( 2.19 49.71 ( 2.03 65.5 ( 1.22 78.23 ( 1.11 68.21 ( 1.23 52.50 ( 0.81 57.36 ( 1.31 62.41 ( 1.33
thp
bhpe
KPF6
cisplatin
68.63 ( 2.44 72.91 ( 2.44 64.23 ( 4.29 >100
98.71 ( 3.63 88.70 ( 3.88 >100
71.71 ( 1.64 73.21 ( 1.22 84.11 ( 2.22 91.71 ( 4.01 >100
>100 >100 >100 >100 >100
>100
>100
83.72 ( 3.23 >100
>100 >100
>100
>100
>100
>100
15.91 ( 1.51 29.21 ( 1.92 19.04 ( 1.51 23.97 ( 2.51 21.50 ( 1.41 20.33 ( 1.33 35.42 ( 1.40 25.34 ( 1.31 10.50 ( 1.51
a SD ) standard deviation. ND ) not detectable. IC50 values were calculated by probit analysis (P < 0.05, ꢀ2 test). Cells ((5-8) × 104 mL-1) were
treated for 48 h with increasing concentrations of tested compounds. Cytotoxicity was assessed by MTT test.
Table 3. Cross-Resistance Profilesa
Human Ovarian Adenocarcinoma Cells
IC50 ( SD (µM)
compd
2008
C13
RF
[Cu(thp)4][PF6] 3
cisplatin
1.48 ( 0.21
12.69 ( 1.72
2.88 ( 1.07
89.18 ( 4.50
1.9
7.02
Human Cervix Squamous Carcinoma Cells
IC50 ( SD (µM)
compd
A431
A431/Pt
RF
[Cu(thp)4][PF6] 3
cisplatin
14.37 ( 1.41
22.06 ( 1.62
13.26 ( 0.80
57.76 ( 1.81
0.92
2.61
Human Colon Adenocarcinoma Cells
IC50 ( SD (µM)
compd
LoVo
LoVo-MDR
RF
Figure 3. Effects of ROS scavengers on complex 3 cytotoxicity against
2008 cells. Mannitol (1 mM, 2), DABCO (1.2 mM, 9), tocopherol +
ascorbic acid (1 mM, b), and sodium selenite (0.1 µM, [) were added
to cell cultures for 48 h in concomitance with complex 3. Results from
complex 3 alone are represented by the open circle (O). MTT test was
used to measure cell viability. Results are expressed as percentage
viability of control cells.
[Cu(thp)4][PF6] 3
doxorubicin
1.54 ( 0.03
1.46 ( 2.30
2.9 ( 0.1
1.88
30.74
44.89 ( 0.90
a SD ) standard deviation. IC50 values were calculated by probit analysis
(P < 0.05, ꢀ2 test). Cells ((3-5) × 104 mL-1) were treated for 48 h with
increasing concentrations of tested compounds. Cytotoxicity was assessed
by MTT test. Resistant factor (RF) is defined as IC50 resistant/parent line.
gen, using standard Schlenk techniques or a glovebox. All solvents
were dried, degassed, and distilled prior to use. Description of the
instrumentation is reported in the Supporting Information (see also
ref 13).
Synthesis. All reagents were purchased from Aldrich and used
without further purification. The tris(hydroxymethyl)phosphine (thp)
1 was prepared in accordance with the literature methods.17,18 1,2-
Bis[bis(hydroxymethyl)phosphino]ethane (bhpe) 2 was synthesized
using a multistep procedure different from that originally reported
by Katti and co-workers.19
Synthesis of 1,2-Bis[bis(hydroxymethyl)phosphino]ethane
(bhpe) (2). Triethyl phosphite (50 mL, 0.3 mol) and 1,2-dibromo-
ethane (53 mL, 0.6 mol) were refluxed at 180 °C overnight. After
cooling, the solution was distilled under vacuum. The residual
product of distillation was a colorless mobile liquid identified as
1,2-bis(diethylphosphonyl)ethane (2a). Freshly distilled anhydrous
diethyl ether (25 mL) was added to a two-necked round-bottom
flask containing 2a (1.000 g, 3.309 mmol) under a dinitrogen
atmosphere. The flask was cooled to 0 °C, and LiAlH4 (20 mL of
a 1.0 M Et2O solution) was slowly added. The ice bath was
removed, and the whitish cloudy mixture was stirred at room
temperature for 30 min. The flask was cooled again to 0 °C, and a
saturated water solution of Na2SO4 was added dropwise until
evolution of dihydrogen stopped. Anhydrous Na2SO4 was added
to the white suspension. The mixture was then filtered off under a
dinitrogen atmosphere, and the filtrate was washed with diethyl
ether. The ethereal phase was characterized by 31P{1H} NMR
spectroscopy (CDCl3, 293K: δ –132.79 s) as the primary phosphine
H2PCH2CH2PH2 (2b). Degassed EtOH (10 mL), formaldehyde (1.2
mL of a 40% m/v H2O solution), and HCl (2 mL of a 3 M solution)
were added to the ethereal solution of 2b under a dinitrogen
atmosphere. The reaction mixture was stirred at room temperature
Figure 2. Comparison of IC50 values detected by MTT, NR, and TB
tests after incubation of 2008 cells with complex 3 for 3, 12, 24, and
48 h. Error bars indicate standard deviation.
demonstrated that the coordination of monophosphine ligands
to copper(I) gives rise to a metallodrug able to inhibit the growth
of tumor cells via G2/M cell cycle arrest and paraptosis
accompanied by the loss of mitochondrial transmembrane
potential.
Experimental Section
Materials and General Methods. All syntheses and handling
were carried out under an atmosphere of dry, oxygen-free dinitro-

In Vitro Antitumor ActiVity of Copper(I) Complexes
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4 801
Cell Cultures. Human lung (A549), breast (MCF-7), liver
(HepG2), colon (CaCo-2, HCT-15), and cervix (HeLa) carcinoma
cell lines along with melanoma (A375), Burkitt lymphoma (Daudi),
and promyelocytic leukemia (HL60) cell lines were obtained from
ATCC, Rockville, MD. The 2008 and its cisplatin resistant variant,
C13*, are human ovarian cancer cell lines, and they were kindly
provided by Prof. G. Marverti (Department of Biomedical Science
of Modena University, Italy). A431 and A431-Pt are sensible and
resistant human cervix carcinoma cells, respectively; they were
kindly provided by Prof. Zunino (Division of Experimental On-
cology B, Istituto Nazionale dei Tumori, Milan, Italy). LoVo human
colon-carcinoma cell line and its derivative multidrug-resistant
subline (LoVo MDR) were kindly provided by Prof. Franca Majone
(Department of Biology of Padova University, Italy). 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 (Biochrom-Seromed GmbH&Co, Berlin, Germany),
antibiotics (50 units ·mL-1 penicillin and 50 µg·mL-1 streptomy-
cin), and 2 mM L-glutamine: (i) RPMI-1640 medium (Euroclone,
Celbio, Milan, Italy) with 25 mM HEPES buffer for HL60, MCF-
7, HCT-15, Daudi, 2008, C13*, A431, and A431-Pt cells; (ii) F-12
HAM’S (Sigma Chemical Co.) for HeLa, LoVo, and LoVo MDR
cells; (iii) D-MEM medium (Euroclone) for A549, A375, HepG2
cells; (iv) MEM medium (Sigma) for CaCo2 cells. LoVo MDR
culture medium also contained 0.1 µg·mL-1 doxorubicin.
Cytotoxicity Assays. MTT Test. The growth inhibitory effect
toward tumor cell lines was evaluated by means of MTT (tetrazo-
lium salt reduction) assay.20 Briefly, (3–8) × 103 cells/well,
dependent on the growth characteristics of the cell line, were seeded
in 96-well microplates in growth medium (100 µL) and then
incubated at 37 °C in a 5% carbon dioxide atmosphere. 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 3, 12,
24, or 48 h, each well was treated with 10 µL of a 5 mg·mL-1
MTT saline solution, and after 5 h of incubation, 100 µL of a
sodium dodecyl sulfate (SDS) solution in 0.01 M HCl was added.
After overnight incubation, the inhibition of cell growth induced
by the tested complexes 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 vs drug
concentration. IC50 values represent the drug concentrations that
reduced the mean absorbance at 570 nm to 50% of those in the
untreated control wells.
Figure 4. Effect of IC50 concentrations of complex 3 on ROS
production in 2008 cells. Cells were loaded with the fluorescent probes
H2DCFDA (10 µM) (A) and DHR (50 µM) (B) as described in
Experimental Section. Symbols are as follows: b, control; O, complex
3; 1, antimycin (3 µM).
for 1 h to obtain a white solid, which was filtered off under a
dinitrogen atmosphere, and was identified as 1,2-bis[tris(hydroxy-
methyl)phosphonium]ethane chloride (2c). NaHCO3 (1 M solution)
was added to a water solution (3 mL) of 2c to yield a basic solution
(pH 9). The solvent was removed in vacuo, and the residue was
recrystallized from methanol to afford [(HOCH2)2PCH2CH2P-
(CH2OH)2)] · 2NaCl 2 as a white solid. Yield 52%. Anal.
(C6H16Cl2Na2O4P2) C, H.
Synthesis of [Cu(thp)4][PF6] (3). To an acetonitrile solution (40
mL) of [Cu(CH3CN)4][PF6] (0.373 g, 1.0 mmol), a methanol
solution (20 mL) of thp (0.496 g, 4.0 mmol) was added at room
temperature. After the addition, the reaction mixture was stirred
for 24 h. The resulting white solid was filtered off, washed with
acetonitrile, and recrystallized from acetonitrile/ethanol to give
complex 3 in 80% yield. Anal. (C12H36CuF6O12P5) C, H.
Synthesis of [Cu(bhpe)2][PF6] (4). To an acetonitrile solution
(40 mL) of [Cu(CH3CN)4][PF6] (0.373 g, 1.0 mmol), 2 (0.662 g,
2.0 mmol) was added at room temperature. After the addition, the
reaction mixture was stirred for 2 h. The resulting white solid was
filtered off, washed with methanol, and recrystallized from methanol/
acetonitrile to give complex 4 in 78% yield. Anal. (C12H32-
CuF6O8P5) C, H.
Experiments with Human Cells. [Cu(thp)4][PF6] (3) and
[Cu(bhpe)2][PF6] (4) complexes and the corresponding uncoordi-
nated ligands and counteranions were dissolved in purified water
just before the experiment. Cisplatin was dissolved in DMSO just
before the experiment, and a calculated amount of drug solution
was added to the growth medium containing cells to a final solvent
concentration of 0.5%, which had no discernible effect on cell
killing. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide) and cisplatin were obtained from Sigma Chemical Co,
St.Louis, MO.
In MTT experiments with ROS scavengers, 24 h after the
seeding, cells were treated for 48 h with mannitol (1 mM) or
DABCO (1.2 mM) or acid ascorbic plus R-tocopherol (1 mM) or
sodium selenite (0.1 µM) in concomitance with increasing con-
centrations of complex 3. All chemicals were purchased from
Sigma.
Neutral Red uptake (NR) Assay. This assay was performed as
described by Borenfreund and Peuerner21 with slight modification.
Briefly, 2008 cells (3 × 103 cells/well) were seeded in 96-well
microplates in growth medium (90 µL) and then incubated at 37
°C in a 5% carbon dioxide atmosphere. Twenty-four hours later,
following incubation with increasing concentrations of tested
compounds for 3, 6, 12, 24, or 48 h, the medium was removed and
replaced with a fresh one containing 50 µg/mL neutral red dye
(Sigma). After 3 h of incubation, the dye-containing medium was
discarded, each well was washed twice with PBS and 200 µL of
extractant solution (50% ethanol in acetic acid, 1%) were added.
After 20 min the absorbance of each well at 540 nm using a Bio-
Rad 680 microplate reader was detected. Mean absorbance for each
drug dose was expressed as a percentage of the control untreated
well absorbance and plotted vs drug concentration. IC50 values
represent the drug concentrations that reduced the mean absorbance
at 540 nm to 50% of those in the untreated control wells.
Trypan Blue Exclusion (TB) Assay. This assay was performed
as described by Durkin et al.22 The 2 × 104 2008 cells were
incubated for 3, 12, 24, or 48 h in the presence of increasing

802 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4
Marzano et al.
Figure 5. Cell cycle analysis of 2008 cells. Panels A-H represent different time points (3, 12, 24, and 48 h) of control cells (left) and cells treated
with IC50 concentrations of complex 3 (right).
concentrations of tested compound. Cells were then incubated for
4 min with 0.25% trypan blue (Sigma) and 5% fetal calf serum.
Viable cells were identified by their ability to exclude dye, whereas
the dye diffuses into nonviable cells. At least 100 cells were counted
for each experimental point recorded. The controls were triplicate
samples of untreated cells, and their viability ranged from 92% to
98%.
Reactive Oxygen Species (ROS) Production. The production
of ROS was measured in 2008 cells (104 per well) grown for 24 h
in a 96-well plate in RPMI 1640 without phenol red. Cells were
then washed in PBS and loaded with 10 µM 2′,7′-dichlorodihy-
drofluorescein diacetate (H2-DCFDA) or 50 µM dihydrorhodamine
123 (DHR) (Molecular Probes-Invitrogen, Eugene, OR) for 25 min
in the dark. Afterward, cells were washed with the same medium
and incubated with IC50 concentration of complex 3. Fluorescence
increase was estimated utilizing the wavelengths of 485 nm
(excitation) and 527 nm (emission) in a Fluoroskan Ascent FL
(Labsystem, Finland) plate reader. Antimycin (3 µM, Sigma), a
potent inhibitor of complex III in the electron transport chain, was
used as positive control.
Light Microscopy Analyses. Mitotic Index (MI) Determina-
tion. MI was determined as previously described.23 Briefly, 2008
cells were plated at a density of 1 × 105 cells in 5 cm dishes.
Next day, they were treated with with IC50 concentration of
complex 3. After 12 and 24 h incubation, cells were treated with
colchicine (10-4 M) for 4 h, harvested, washed with cold PBS,
and then treated with 1% sodium citrate for 12 min at room
temperature. Cells were fixed with methanol/acetic acid solution
(3:1 v/v), spread on clean slides, air-dried, and stained with a
10% Giemsa solution. At least 150 cells were counted for each
concentration, and MI was calculated as the ratio of the number
of dividing cells to the total number of cells multiplied by
100.
Hematoxylin-Eosin (HE) Staining. The 2008 cells were seeded
into eight-well tissue-culture slides (GIBCO) at 104 or 5 × 104
cells/well (0.8 cm2). After 24 h, cells were washed twice with PBS
and treated for an additional 24 h with IC50 concentration of
complex 3. Then, after being washed with PBS, cells were fixed in
4% freshly prepared, ice-cold paraformaldehyde, postfixed in

In Vitro Antitumor ActiVity of Copper(I) Complexes
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4 803
ethanol, and air-dried. Slides were then stained with hematoxylin
and eosin before being examined by light microscopy (Olympus
BX41).
Flow Cytometric Analysis. Drug-induced cell cycle effects and
DNA fragmentation were analyzed by flow cytometry after DNA
staining with propidium iodide (PI) according to Nicoletti et al.24
Briefly, 2008 cells (5 × 105 cells) were exposed for 1–48 h to
tested compound concentrations corresponding to IC50 values. One
milliliter of a PI solution, containing 50 µg/mL PI, 0.1% m/v Triton
X-100, and 0.01% m/v sodium citrate, was added to cells and then
incubated for 25 min at 4 °C in the dark. Induced cell death was
determined as a percentage of hypodiploid nuclei counted over the
total cell population measured by FACScalibur flow cytometer
(Becton-Dickinson, CA) using a 550–600 nm filter. Analysis was
performed by Cell Quest software (Becton-Dickinson, CA).
Mitochondrial membrane potential depletion was determined by
measuring the fluorescence of cells stained with the dye tetra-
methylrhodamine methyl ester (TMRM, Molecular Probes). The
2008 cells (106), following 24 h of incubation with complex 3,
were washed once with PBS, harvested, and incubated for 15 min
at 37 °C in PBS with TMRM at a final concentration of 10 nM,
freshly prepared from stock solution 10 mM in DMSO. The TMRM
signal was analyzed in the FL2 channel, which was equipped with
a bandpass filter at 560 ( 20 nm.
Results and Discussion
Synthesis and Characterization of Phosphine Ligands
and Cu(I) Complexes. The ligand bhpe was synthesized
following the procedure outlined in Scheme 1, different from
that previously reported.19 Neat triethyl phosphite and 1,2-
dibromoethane were refluxed overnight to obtain a colorless
liquid that was distilled under vacuum. The first three fractions
were discharged, and the remaining colorless liquid, identified
as 1,2-bis(diethylphosphonyl)ethane (2a), was treated with an
excess of LiAlH4 in anhydrous diethyl ether under a dinitrogen
atmosphere at 0 °C. The resulting cloudy mixture was treated
with anhydrous Na2SO4 and filtered. The filtrate containing the
pure primary phosphine (2b) was combined in degassed ethanol
with a slight excess of aqueous formaldehyde in an acidic
medium to obtain a white precipitate of the phosphonium salt
(2c). The addition of an aqueous solution of NaHCO3 to an
aqueous solution of 2c produced the tertiary phosphine 2.
Copper complexes [Cu(thp)4][PF6] (3) and [Cu(bhpe)2][PF6]
(4) have been synthesized according to eqs 1 and 2 by mixing
the [Cu(CH3CN)4][PF6] precursor with an excess of the phos-
phine ligand (4 equiv of thp (1) or 2 equiv of bhpe (2)) in
methanol/acetonitrile solutions.
Cu(CH CN) PF + 4 thp f Cu(thp) PF (3) (1)
4][ 4][
[
]
6
[
]
3
6
Cu(CH CN) PF + 2 bhpe f Cu(bhpe) PF (4) (2)
4][ 2][
[
]
[
]
6
3
6
The choice of the PF6 counteranion was determined primarily
for chemical reasons. The coordination ability of such an anion
is negligible, thus preventing its direct coordination to the metal,
which is common for other anions as, for example, iodide. In
addition, PF6 ensures good aqueous solubility of the copper salt
and displays a distinct septet in the 31P NMR spectrum, allowing
easy characterization of complex 3 in deuterated water (see
below).
The identity of complexes 3 and 4 has been determined by
ESI-MS, IR, and multinuclear NMR spectral studies (1H,
13C{1H}, 31P{1H}, and 63Cu NMR) (see Table 1). The infrared
spectra showed broad peaks in the range 3360–3449 cm-1 due
to the hydroxylic groups of the phosphines. No significant shifts
were observed upon complex formation compared to those
exhibited by uncoordinated phosphine ligands. The room-
temperature 1H NMR spectra of 3 and 4 in D2O solution showed

804 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4
Marzano et al.
Figure 6. Forward scattering vs side scattering as a function of time in 2008 cells untreated (left) and treated with IC50 concentrations of complex
3 (right). Panels A-H represent different time points (3, 12, 24, and 48 h).
rather simple spectra consistent with the formulation (Table 1).
The 31P{1H} NMR spectra gave a four-line signal pattern
centered at δ -5.35 and 9.67 ppm for 3 and 4, respectively,
downfield shifted compared to the signals at δ -24.70 and
-22.18 ppm exhibited by uncoordinated thp and bhpe, respec-
tively. The 63Cu NMR spectrum of 3 displayed a quintet
centered at δ 135.2 ppm due to the coupling of copper with
four equivalent I ) 1/2 phosphorus nuclei (Figure 1S, top). The
multiplicities shown in the 31P{1H} and 63Cu NMR spectra are
consistent with the coordination of four phosphines around
3
due to the coupling with the I ) /2 63Cu and 65Cu nuclei
(Supporting Information Figure 1S, bottom), along with a septet
centered at about δ -145 ppm due to the PF6 counteranion.
The 31P{1H} NMR signals of the complexes were significantly

In Vitro Antitumor ActiVity of Copper(I) Complexes
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4 805
signal becomes a broad doublet, and the 63Cu NMR signal is
no longer detectable.
The full ESI(+) spectrum of complex 3 showed the molecular
ion peak corresponding to the cationic [CuP4]+ species at 560
m/z (6%), along with more intense peaks at 436 m/z (65%) and
312 m/z (100%) due to the [CuP3]+ and [CuP2]+ fragments.
Instead, the ESI(+) spectrum of complex 4 showed only the
molecular ion peak at 492 m/z (100%) without detectable
fragmentation, indicating that the chelation effect provided by
bhpe additionally stabilizes the [CuP4]+ moiety.
Complexes 3 and 4 are very soluble and stable for days when
dissolved in aqueous solutions. Such stability is probably due
to water solvation of the complexes, which prevent them from
both hydrolysis and disproportionation to Cu(II) and Cu(0).
However, although stored under a dinitrogen atmosphere at -20
°C, solid samples of complexes 3 and 4 did not grant
reproducible biological results, owing to partial decomposition
of the original species in the solid state, as assessed by 31P{1H}
NMR control experiments. For example, a month old batch of
complex 3 exhibited a broad singlet at δ -2.0 ppm overlapped
with the original quartet and a significant drop of the PF6 signal.
These data are consistent with the occurrence of partial
decomposition of cationic 3 into the corresponding deprotonated
neutral [Cu(thp)3(thp-H)] complex (3dec) (tph-H ) monodepro-
tonated form of tph),15 via the intramolecular acid–base reaction
reported below.
Figure 7. Phase contrast images of 2008 cells stained with hemeatoxy-
lin and eosin: (A) control cells; (B) cells treated with IC50 concentration
of complex 3 for 24 h (40×), where the inset represents a magnification
(100×).
Cu(thp) PF f Cu(thp) (thp - H) (3dec) + HPF6 (3)
4][
[
]
6
[
]
3
The cytotoxic efficiency of neutral 3dec appears to be drastically
reduced. In contrast, lyophilized samples of 3 and 4 gave
reproducible biological results.
Cytotoxicity Studies. Complexes 3 and 4 and the corre-
sponding uncoordinated ligands and counteranions were exam-
ined for their cytotoxic properties against a panel of human
tumor cell lines containing examples of lung (A549), colon
(CaCo2, HCT-15), cervix (HeLa), and breast (MCF-7) cancer,
leukemia (HL60), lymphoma (Daudi), epatoma (HepG2), and
melanoma (A375). For comparison purposes, the cytotoxicity
of cisplatin was evaluated under the same experimental condi-
tions. IC50 values, calculated from the dose-survival curves
obtained after 48 h of drug treatment from the MTT test, are
shown in Table 2. Complex 4 showed a relatively low cytotoxic
activity against all cancer cell lines, with IC50 values roughly
3-fold higher than those of cisplatin. In contrast, complex 3
showed the greatest antitumor efficacy with an average IC50
over nine cell types that was over 5 times lower than that of
cisplatin. In particular, in HCT-15, HepG2, and HL60 cells IC50
values were about 12-, 17-, and 26-fold lower than those
calculated for cisplatin, respectively. Remarkably, against CaCo2
cells, cytotoxic potency of complex 3 exceeded by over 30-
fold that of the reference drug, thus reflecting a useful selectivity
for this human colon adenocarcinoma cell type.
The uncoordinated thp and bhpe ligands and the PF6
counteranion proved to be hardly effective, even if on certain
tumor cell types detectable IC50 values were collected; in any
case, their values were markedly higher than those displayed
by complex 3. The negligible activity shown by the copper
complex 4 could be associated with the high kinetic inertness
of this molecule, as assessed by ESI-MS studies. This property
determines a low tendency both to dissociate and to play a part
in physiologically mediated transport mechanisms which may
be relevant for the cellular internalization of Cu(I) species.
The encouraging results obtained against the in-house panel
of cell lines prompted us to test the cytotoxic activity of the
Figure 8. Flow cytometric profiles of 2008 cells untreated (A) and
treated with 3.125 (B) or 6.25 (C) µM complex 3 for 24 h and stained
with TMRM (10 nM).
copper, the complex adopting a perfect spherical symmetry
characteristic of the tetrahedral geometry (Figure 1), which is a
common feature in [CuP4]+ type species.25 The steric constraints
generated by the insertion of the ethylene bridge in the bhpe
ligand lower the symmetry in complex 4. Its 31P{1H} NMR

806 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4
Marzano et al.
most potent derivative, complex 3, on three additional cell line
pairs, two of which (2008/C13* ovarian cancer cells and A431/
A431-Pt cervix carcinoma cells) had been selected for their
resistance to cisplatin and one (LoVo/LoVoMDR) for its
resistance to doxorubicin, thus retaining the MDR phenotype.
Cytotoxicity of tested compounds in sensitive and resistant cells
was assessed after a 48 h drug exposure by the MTT test. Data
are accumulated in Table 3. Cross-resistance profiles were
evaluated by means of the resistance factor (RF), which is
defined as the ratio of IC50 values calculated for the resistant
cells to those arising from the sensitive ones. Although cisplatin
resistance is multifactorial, the main molecular mechanisms
involved in resistance in cisplatin resistant sublines have almost
been defined. In C13* cells, resistance is correlated to reduced
cellular drug uptake, high cellular glutathione levels, and
enhanced repair of DNA damage.26 In human squamous cervix
carcinoma A431-Pt cells, resistance is due to defect in drug
uptake and to decreased levels of proteins involved in DNA
mismatch repair (MSH2), causing an increased tolerance to
cisplatin-induced DNA damage.27 Remarkably, complex 3
showed activity levels very similar on both cisplatin sensitive
and resistant cell lines that indicate a different cross-resistance
profile than that of cisplatin. Against the ovarian cancer, the
RF value was about 4 times lower than that calculated for
cisplatin, while against the cervix cell line pair, it was about 3
times lower. The overcoming of cross-resistance phenomena
in both cell line pairs strongly supports the hypothesis of a
different pathway of action of this copper(I) complex from that
of cisplatin.
contrast, the poor permeability to vital dye indicates that plasma
membrane function is still maintained until the late phase of
cell death.
It is well-known that copper represents an excellent catalyst
of redox cycles, and therefore, it can stimulate cellular ROS
formation.29 To evaluate the contribution of oxidative stress in
the execution of tumor cell death, complex 3 mediated cyto-
toxicity against 2008 cells was investigated in the presence of
various ROS scavengers. Mannitol (1 mM) and DABCO (1.2
mM) were respectively used as scavengers of hydroxyl radical
and singlet oxygen, the main damaging oxidative species.30
Ascorbic acid and R-tocopherol (1 mM), working in synergism,
act as direct antioxidants toward singlet oxygen and superoxide
anion and as indirect antioxidants breaking the chain reaction
of lipid peroxidation.31 Furthermore, selenium, as cofactor of
various selenoproteins, has been found to protect, at low
concentrations, tumor ovarian cells from oxidative damage
induced by hydrogen peroxide.32 Chemicals were added to 2008
cell growth medium containing different concentrations of
complex 3, and the treatment was extended to 48 h. The results
presented in Figure 3 clearly indicate the cytoprotective proper-
ties of all ROS scavengers; in particular, the copresence of
R-tocopherol and ascorbic acid totally prevent the cytotoxic
activity of complex 3. Taken together, these data suggest the
involvement of ROS production in copper(I) complex mechanism.
Cellular production of ROS upon treatment with complex 3
was measured in 2008 cells by the fluorogenic probes
H2DCFDA (2′,7′-dichlorodihydrofluorescein diacetate) and DHR
(dihydrorhodamine 123).33 The 2008 cells, preincubated with
H2DCFDA or DHR, were treated with IC50 concentrations of
complex 3 for increasing exposure times. Figure 4 shows that
no significant increase in ROS level with respect to the basal
level of the control cells was detected after either of the exposure
times with both H2DCFDA (Figure 4A) and DHR (Figure 4B)
probes. Moreover, also further increasing complex 3 range
concentrations, ROS were not detectable (data not shown).
Similar negative results had been already obtained by other
authors investigating the cellular effects induced by a thioxo-
1,2,4-triazolecopper(II) complex.34 Most probably, as stated in
that context, the intracellular ROS production associated with
copper complexes needs to be analyzed by other methods. In
our case, the potential presence of free phosphines, which are
well-known reducing agents in chemical reactions, partially
releasedfromthemetalcentermayinterfereinROSdetermination.
Acquired multidrug resistance (MDR), whereby cells become
refractory to multiple drugs, poses a very important challenge
to the success of anticancer chemotherapy. The resistance of
LoVo MDR cells to doxorubicin, a drug belonging to the MDR
spectrum, is associated with an overexpression of the multi-
specific drug transporters, such as the 170 kDa P-glycoprotein
(P-gp).28 Cytotoxicity assays testing complex 3 against this cell
line pair allowed the calculation of an RF value about 16-fold
lower than that obtained with doxorubicin, thus suggesting that
this copper(I) complex is not a potential MDR substrate.
In order to provide information on the antiproliferative effect
of complex 3, we performed a time-dependent evaluation of its
cytotoxicity by using three different standard methods that, at
relatively short exposure times, assess the damage to different
subcellular organelles. The MTT test, detecting the viable
mitochondria that are able to reduce the MTT dye to blue
formazan, mainly reflects damage to mitochondria. The NR
assay, measuring the intracellular accumulation of a weak
cationic dye, indicates damage to lysosomes and Golgi ap-
paratus. Finally, the TB test quantifying dead cells by means
of their staining after a vital dye treatment reveals damage to
the cell membrane. Figure 2 shows the comparison of IC50
values detected by MTT, NR, and TB assays in 2008 cells
treated with complex 3 for different exposure times. At 3 h,
neither of the three methods revealed significant IC50 values;
otherwise, a time dependent increase in cytotoxicity becomes
apparent after longer exposure times. Anyway, the NR test
revealed the strongest and earliest effects among those revealed
by all three assays; in fact, following the 12 h treatment, IC50
values obtained with NR were about one-half and one-third of
those calculated from the dose-response profiles by MTT and
TB test, respectively. These results suggest that lysosomes/Golgi
apparatuses are more sensitive to complex 3 treatment. In
In order to study further the cellular effects induced by
complex 3 in terms of cell cycle modifications and induction
of apoptosis, a time-dependent evaluation of cell cycle profile
was performed by FACS in 2008 human ovarian cancer cells
treated with IC50 concentrations of complex 3 (Figure 5). Cell
cycle analysis revealed a significant time-dependent decrease
of the G1 phase. After 48 h of treatment, with respect to control
cells, about a 1.8-fold reduction of G1 phase and about a 1.5-
fold increase of S, G2, and M phases were observed (Figure 5,
panels G and H; Table 4).
PI cytofluorimetric analysis is not able to discriminate the S
and M phases from the G2 phase because of the equal DNA
cellular content. To better evaluate the influence of complex 3
on the M phase, the mitotic index in 2008 cells treated with
IC50 concentrations of copper(I) complex for 12 and 24 h was
calculated. Mean percentages of mitotic index (42.92 ( 3.11
and 19.33 ( 3.21 at 12 and 24 h, respectively, against an average
value of 68.34 ( 3.01 for control cells) attest to a significant
time-dependent decrease of the number of treated cells able to
complete the mitotic division.

In Vitro Antitumor ActiVity of Copper(I) Complexes
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4 807
Moreover, by the examination of histograms, no DNA
fragmentation was detected following 3, 12, and 24 h exposures
(Figure 5, panels A-F). However, cells treated with complex
3 for 48 h showed a slightly appreciable DNA fragmentation
signal compared to the control samples (15.4%) (Figure 5, panels
G and H, Table 4).
dell’Istruzione dell’Università e della Ricerca. We are grateful
to CIRCMSB (Consorzio Interuniversitario di Ricerca in
Chimica dei Metalli nei Sistemi Biologici).
Supporting Information Available: Description of instrumenta-
tion and complete characterization details of ligand 2 and copper
complexes 3–4. This material is available free of charge via the
Internet at http://pubs.acs.org.
Side scattering and forward scattering based analyses showed
a time dependent increase of cell complexity and size in complex
3 treated cells (Figure 6, panels B, D, F, H). Following 48 h of
treatment, it can be noted that 36% of cytofluorimetric counts
shifted in the high complexity and size fraction of the cytogram
(Figure 6, panel H, upper right quadrant). These remarkable
morphological changes may be related to the ability of complex
3 to cause an extensive cytosolic vacuolization. Hematoxylin
and eosin staining of 2008 cells treated with IC50 concentrations
of complex 3 for 24 h revealed a striking cytoplasmatic
vacuolization (Figure 7), which does not appear if cells were
preincubated with the autophagy inhibitor monensin35 (data not
shown). These cellular effects were previously described in
tumor cells treated with other copper complexes.13,34 Taken
together, all these results confirm the hypothesis that complex
3 may activate the programmed cell death paraptosis as the main
cell death mechanism.36,37
Finally, considering that mitochondria play a key role in the
pathways to cell death, we investigated the mitochondrial
energization of treated tumor cells as the retention of a
mitochondrial selective cationic fluorescent probe tetramethyl
rhodamine methyl ester (TMRM). Figure 8 shows the flow
cytometric profiles of 2008 cells treated with 6.25 and 12.5 µM
of complex 3 for 24 h and stained with 100 nM TMRM. The
results indicate that copper(I) complex induced a massive
increase of the TMRM fluorescence, reflecting a dramatic
alteration of mitochondrial membrane potential. The marked
increase of ∆Ψmt (hyperpolarization) can be attributed to a
variety of mechanisms such as increased mitochondrial mass
and number per cell38 or unregulated mitochondrial matrix
volume.39 In particular, mitochondrial membrane hyperpolar-
ization induced by complex 3 might be correlated with the
induction of a G2/M phase cell cycle arrest, as previously
reported for other anticancer agents.40
In conclusion, the study presented herein on the synthesis of
hydrophilic phosphine copper(I) compounds coupled with their
marked cytotoxic activity against a panel of human cell lines
provides the basic chemical background in view of the attractive
opportunity to use hydrophilic “hot” phosphine copper (64Cu
and 67Cu) complexes as potential therapeutic agents in the
radiopharmaceutical field. Several radioactive copper isotopes
are available nowadays for biomedical purposes both for
radioimaging and for targeted radiotherapy.41,42 The excellent
hydrophilic character of this class of copper(I) complexes may
also help to overcome the undesired cardiotoxicity exhibited
by previously investigated bis(arylphosphine) copper(I) and
gold(I) compounds. Actually, the intravenous administration of
[Au(dppe)2]+ caused myocardial lesions, presumably because
of an elevated uptake of the cationic species in the myocardium.9
It has already been shown that other monocationic lipophilic
drugs, such as the tracers 99mTc-Cardiolite and 99mTc-Myoview,
enter the myocardial cell membrane through a passive diffusion
mechanism.43 The myocardial uptake of hydrophilic copper
complexes should be much less marked, thus precluding
accumulation in the heart and subsequent heart damage.
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