Synthesis and characterization of novel tetrahedral copper(I) complexes comprising tridentate PNP-aminodiphosphines and tetradentate PN(X)P-substituted aminodiphosphines (X = O, S)

Article abstract of DOI:10.1016/j.ica.2012.01.012

Two series of novel tetrahedral copper(I) complexes comprising tridentate PNP-aminodiphosphines and tetradentate PN(X)P-substituted aminodiphosphines (X = O, S) have been prepared and characterized by conventional physico-chemical techniques. The first series includes '3 + 1'-type complexes comprising an aromatic PNP-aminodiphosphine and acetonitrile or triphenylphosphine. In the second series, the central amine function of the PNP-ligand was substituted with functionalized pendant arms containing ether, hydroxyl or thioether groups to enhance the chelation ability of the ligand. Fully coordinated neutral and cationic complexes were isolated. A preliminary study investigating both the labeling of ⁶⁴Cu with the prototype PN(S)P ligand and the potential cytotoxic activity of the 'cold' [Cu(PN(S)P)][BF₄] complex is reported.

Full text of DOI:10.1016/j.ica.2012.01.012

Inorganica Chimica Acta 387 (2012) 163–172
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Inorganica Chimica Acta
journal homepage: www.elsevier.com/locate/ica
Synthesis and characterization of novel tetrahedral copper(I) complexes
comprising tridentate PNP-aminodiphosphines and tetradentate PN(X)P-substituted
aminodiphosphines (X = O, S)
a
a
Valentina Peruzzo a, Cornelia Pretzsch b, Francesco Tisato a, , Marina Porchia , Fiorenzo Refosco ,
⇑
Cristina Marzano c, Valentina Gandin c, Eik Schiller d, Martin Walther b, Hans-Jürgen Pietzsch b
a I.C.I.S. – C.N.R., Corso Stati Uniti, 4, 35127 Padova, Italy
b Helmholtz-Zentrum Dresden-Rossendorf, Institut für Radiopharmazie, Bautzner Landstraße 400, D-01328 Dresden, Germany
c Department of Pharmaceutical Sciences, University of Padova, Via Marzolo, 5, 35131 Padova, Italy
d ROTOP Pharmaka AG, Bautzner Landstraße 400, D-01328 Dresden, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Two series of novel tetrahedral copper(I) complexes comprising tridentate PNP-aminodiphosphines and
tetradentate PN(X)P-substituted aminodiphosphines (X = O, S) have been prepared and characterized by
conventional physico-chemical techniques. The first series includes ‘3 + 1’-type complexes comprising an
aromatic PNP-aminodiphosphine and acetonitrile or triphenylphosphine. In the second series, the central
amine function of the PNP-ligand was substituted with functionalized pendant arms containing ether,
hydroxyl or thioether groups to enhance the chelation ability of the ligand. Fully coordinated neutral
and cationic complexes were isolated. A preliminary study investigating both the labeling of 64Cu with
the prototype PN(S)P ligand and the potential cytotoxic activity of the ‘cold’ [Cu(PN(S)P)][BF4] complex
is reported.
Received 27 September 2011
Received in revised form 27 December 2011
Accepted 5 January 2012
Available online 20 January 2012
Keywords:
Aminodiphosphines
Copper(I) complexes
Cytotoxic activity
Polydentate phosphines
Phosphorus NMR
Ó 2012 Elsevier B.V. All rights reserved.
1. Introduction
Concerning the use of copper in the radiopharmaceutical field,
due to their wide range of half-lives and decay schemes, copper
Copper complexes have been proposed, among others, as inno-
vative chemotherapeutic agents [1–3] and diagnostic and therapeu-
tic radiopharmaceuticals [4,5]. The former application stems from
the observation that the current treatment of various solid malig-
nancies with classic Pt(II) anticancer drugs such as cisplatin, carbo-
platin and oxaliplatin [6], although effective, is often associated
with unfavorable toxicological profiles. Moreover, some common
cancers (e.g. colon adenocarcinoma) are intrinsically refractory to
Pt(II) drugs therapy, and, frequently, resistance is developed in ini-
tially sensitive tumors [7]. The alternative use of copper compounds
offers two main advantages: (i) to employ a physiologically recog-
nized trace element (copper is a micronutrient that plays an essen-
tial role in biology, serving as co-factor for several enzymes) that
may be selective to tumor cells with reduced toxicity to normal
cells, and (ii) to overcome Pt(II) resistance phenomena as it has been
shown that mechanisms of action and biological targets of copper
drugs are different from those utilized by Pt(II) drugs [8,9].
radionuclides have found application in both positron emission
tomography (PET) (60Cu, 61Cu, 62Cu and 64Cu) and targeted radio-
therapy (64Cu and 67Cu) [10,11].
Copper has a well-established coordination chemistry and elec-
trochemistry. The red-ox property of the metal could provide a re-
dox-mediated trapping of copper compounds in cells. Exploitation
of this mechanism has been used with the copper(II) pyruvaldehyde
bis(N4-methylthiosemicarbazone) (64Cu–PTSM) and copper(II) dia-
cetyl bis(N4-methylthiosemicarbazone) (64Cu–ASTM) for the in vivo
measurement of blood flow and tissue hypoxia, respectively [12–
14]. It is interesting to mention that non-radioactive Cu–ASTM com-
plexes, and more generally Cu–thiosemicarbazone complexes, were
initially tested as chemotherapeutic agents [15,16].
Intrigued by the potential double chance offered by the use of
copper compounds in both antitumor and radiopharmaceutical
fields, we have been working on the synthesis and characterization
of copper species including tertiary phosphines [2]. Coordination of
the copper(I) ion with phosphines is a well-documented chemistry
because of the efficient overlap of the empty d orbitals of soft
donor phosphines with the electron-rich d10 full orbitals of the me-
tal ion [17]. Copper(I) complexes with mono-dentate phosphines
alone [9,18,19] or in combination with additional N-based chelate
⇑
Corresponding author. Tel.: +39 0498295958; fax: +39 0498702911.
E-mail address: tisato@icis.cnr.it (F. Tisato).
0020-1693/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved.
doi:10.1016/j.ica.2012.01.012

164
V. Peruzzo et al. / Inorganica Chimica Acta 387 (2012) 163–172
ligands [20–22], have already shown remarkable cytotoxic activity
toward a wide panel of human tumor cell lines [9,21–23], and
examples of similar 64Cu-species that accumulate in cancer tissue
have been reported as well [24].
deuterated solvent employed as internal references (1H and 13C),
and 85% aqueous H3PO4 as external reference (31P). The splitting
of nuclear resonances in the reported NMR spectra is defined as
s = singlet, d = doublet, q = quartet, m = multiplet, and bs = broad
singlet. Mass spectra have been recorded by an electrospray LCQ
ThermoFinnigan mass spectrometer. The purification of the ‘cold’
complex [Cu(PN(S)P)][BF4] and of the corresponding radiolabeled
Taking advantage of long years of experience in the preparation of
aminodiphosphines and corresponding technetium and rhenium com-
plexes [25–29], in this study we report on the synthesis of new poten-
tially tetradentate PN(X)P-substituted aminodiphosphines (X = O, S)
(Scheme 1), and related tetrahedral copper(I) complexes. PN(X)P ligands
were prepared via multi-step reactions (Scheme 2) starting from cheap
precursors and using the promoter lithium perchlorate to afford amin-
odiphosphonate intermediates, which were subsequently reduced to pri-
mary phosphines, then lithiated and alkylated to the final amino
substituted diphosphines PN(OH)P and PN(S)P. From the metal point-
of-view, the first goal was to prepare ‘3 + 1’ [Cu(PNP)(L)]-type complexes
(L = acetonitrile, [Cu(PNP24)(CH3CN)][BF4], or triphenylphosphine,
[Cu(PNP24)(PPh3)][BF4]). In the second step, the central PNP-amine func-
tion was substituted with functionalized pendant arms to enhance the
chelation ability of the ligand. The corresponding cationic copper(I) com-
plexes [Cu(PNP2)][BF4], [Cu(PNP5)][BF4], [Cu(PN(S)P)][BF4] and neutral
[Cu(PN(O)P)] were isolated and characterized. A preliminary study inves-
tigating both the labeling of 64Cu with the tetradentate PN(S)P ligand and
the potential cytotoxic activity of the prototype complex [Cu(PN (S)
P)][BF4] is reported.
[
64Cu(PN(S)P)][BF4] was performed via HPLC on a Beckman System
Gold instrument equipped with a programmable solvent Model
126, a sample injection valve 210A, a scanning detector Module
166, and a radioisotope detector Model 170. HPLC analysis was car-
ried out using
a reverse phase Agilent Zorbax 300 SB-C18
(9.4 ꢁ 250 mm) column at a flow rate of 4 mL/min using a gradient
condition. The chromatographic details were as follows: solvent A
(CH3CN/0.1% TFA), solvent B (H2O/0.1% TFA) 20–60% A in 20 min,
60–80% A in 2 min, 5 min 80% A, 80–20% A in 3 min. Copper-64
was produced on a cyclotron CYCLONE 18/9 via the 64Ni(n,p)64Cu
reaction according to the method of Avila-Rodriguez et al. [31].
2.2. Synthesis of substituted aminodiphosphines
2.2.1. Synthesis of PN(OH)P
2.2.1.1. Synthesis of OH2. In a two-necked round-bottom flask,
diethyl-2-bromoethyl phosphonate (0.90 mL, 5.0 mmol), 2-amino-
ethanol (0.15 mL, 2.5 mmol), acetonitrile (10 mL) and potassium
carbonate (0.346 g, 2.5 mmol) were sequentially added. The reac-
tion mixture was heated under stirring for 3 h at 70 °C in an oil
bath. After cooling, the mixture was filtered and the solid washed
with acetonitrile (3 ꢁ 2 mL). The combined acetonitrile fractions
were concentrated with a rotavapor affording a yellow oil contain-
ing the mono-substituted phosphonate and diethylvinylphospho-
nate, as assessed by 31P NMR. The yellow oil was transferred in a
5 mL vial and lithium perchlorate (300 mg, 2.5 mmol) was added.
The vial was filled with dinitrogen and hermetically capped. It
was completely immersed in an oil bath heated at 75 °C and the re-
agents left to react under stirring for 7 h. After cooling, the vial was
open and the content was treated with chloroform (3 ꢁ 2 mL). The
combined organic phases were poured into a separating funnel and
treated with water (6 mL). The organic phase was separated, and
the aqueous phase was treated again with chloroform (3 ꢁ 3 mL).
The combined organic phases were collected and treated with so-
dium sulfate (200 mg). The mixture was filtered on a G4 frit, the fil-
trate was concentrated with a rotavapor and then left in vacuo for
30 min. A yellow oil was obtained. Yield 70%. 1H NMR (CDCl3, d
ppm): 4.11 (m, 8H, (CH3CH2O)2PO), 3.66 (m, 2H, NCH2CH2OH),
2. Experimental
2.1. General procedures
All solvents and commercially available substances were of
reagent grade and used without further purification. In particular,
diethyl-2-bromoethyl phosphonate and diethylvinylphosphonate
were purchased from Sigma–Aldrich. The Cu(I) starting compound
[Cu(CH3CN)4][BF4] was prepared by reaction of [Cu(H2O)6][BF4]2
with metallic copper in acetonitrile [19]. Aminodiphosphines
PNP24 [30], PNP2 [25] and PNP5 [27] were prepared according to
published procedures. Aromatic aminodiphosphines PNP24 and
PNP2 are white solids stable in air and in solutions, whereas ali-
phatic aminodiphosphine PNP5 is a colorless oil which should be
stored under dinitrogen at ꢀ20 °C to avoid oxidation to the corre-
sponding phosphine oxide. Elemental analyses for new ligands and
copper complexes were performed on a Carlo Erba 1106 Elemental
Analyzer. 1H, 31P and 13C NMR spectra were recorded on a Bruker
AMX-300 instrument, using SiMe4 and residual protons of the
O
O
O
PN(OH)P
P
P
N
R
R
R
R
O
O
P
P
N
OH
R'
R = Ph, R' = CH3;
PNP24
O
O
R = Ph, R' = CH2CH2OCH3;
PNP2
PN(S)P
P
P
N
R = CH2CH2CH2OCH3, R' = CH2CH2OCH2CH3;
PNP5
O
S
Scheme 1. PNP-type ligands utilized in this study.

V. Peruzzo et al. / Inorganica Chimica Acta 387 (2012) 163–172
165
O
P
O
P
O
i)
H
O
O
O
O
O
O
P
N
X
NH2
+
Br
2
+
X
X = OH;
OH1
Br-PO
X = SCH2CH2CH3;
S1
ii)
O
O
P
X = OH;
OH2
O
O
P
N
X = SCH2CH2CH3;
S2
S3
O
O
X
iii)
H
H
X = OH;
OH3
H
H
P
P
P
P
N
X
X = SCH2CH2CH3;
iv)
O
O
Li
Li
Li
Li
v)
N
X
P
P
N
O
O
X
X = OH;
PN(OH)P
X = SCH2CH2CH3;
PN(S)P
Scheme 2. Synthesis of ligands PN(OH)P and PN(S)P. Reaction conditions: (i) acetonitrile, K2CO3; (ii) LiClO4; (iii) diethylether, LiAlH4; (iv) tetrahydrofuran, n-butyllithium; (v)
tetrahydrofuran, (2-chloroethyl)methylether.
3
2.86 (dt, 4H, 3J = 7.5 Hz, JHP = 12.4 Hz, (O)PCH2CH2N), 2.61 (t, 2H,
(H2PCH2CH2)2NCH2CH2OH), 1.59 (m, 4H, (H2PCH2CH2)2NCH2CH2OH).
31P NMR (CDCl3, d ppm): ꢀ146.1 (s). 13C NMR (CDCl3, d ppm): 58.78 (s,
NCH2CH2OH), 55.84 (s, (H2PCH2CH2)2NCH2CH2OH), 55.00 (s, (H 2PCH
2CH2)2NCH2CH2OH), 11.49 (d, 1JCP = 8.7 Hz, H2PCH2CH2)2 NCH2 CH2OH).
3J = 6.2 Hz, NCH2CH2OH), 1.98 (dt, 4H, 3J = 7.5, JHP = 16.0 Hz,
2
(O)PCH2CH2N), 1.37 (t, 12H, 3J = 7.5 Hz, (CH3CH2O)2PO). 31P NMR
(CDCl3, d ppm): 31.7 (s). 13C NMR (CDCl3, d ppm): 61.62 (s,
(CH3CH2O)2P); 58.93 (s, NCH2CH2OH); 55.07 (s, NCH2CH2OH);
1
46.27 (s, (O)PCH2CH2N); 23.44 (d, JCP = 138 Hz; (O)PCH2CH2N);
2.2.1.3. Synthesis of PN(OH)P. In a two-necked round-bottom flask
containing OH3 (0.265 g, 2.19 mmol), freshly distilled anhydrous tet-
rahydrofuran (10 mL) was added. The solution was cooled to ꢀ78 °C
by means of an acetone/liquid dinitrogen bath. A 2.5 M solution of n-
BuLi (2.9 mL, 7.2 mmol) in n-hexane solution was injected through a
rubber cap. The solution became yellow-green, and then it was left
to reach 0 °C (ice bath). A tetrahydrofuran (10 mL) solution containing
(2-chloroethyl)methylether (0.7 mL, 7.2 mmol) was added to the reac-
tion within 30 min by means of a dropwise funnel. The solution was
left to react at room temperature under stirring for 15 h. The solution
was then reduced to 1/3 of the initial volume, and placed into an ice-
bath. Previously degassed water (5 mL) was added by syringe through
a rubber cap. After 10 min, anhydrous diethylether (20 mL) was added
and the mixture was cannuled into a two-neck funnel. Under dinitro-
gen atmosphere, the ethereal phase was transferred into a flask
containing anhydrous sodium sulfate (5.0 g). Additional diethylether
(3 ꢁ 10 mL) was added to the funnel and the ethereal fractions trans-
ferred to the flask. The resulting mixture was filtered and the solvent of
the filtrate was eliminated by a gentle stream of dinitrogen giving a
colorless oil. Yield 90%. 1H NMR (CDCl3, d ppm): 3.55 (m, 8H + 2H,
4ꢁCH3OCH2CH2P, NCH2CH2OH), 3.34 (s, 12H, CH3OCH2CH2P), 2.75–
2.59 (6H, [(CH3OCH2CH2)2PCH2CH2]2NCH2CH2OH), 1.77 (t, 8H,
16.42 (s, (CH3CH2O)2P). ESI-MS (m/z assignment): 402 ([M+Na]+).
Anal. Calc. for C14H33NO7P2: C, 43.18; H, 8.54; N, 3.60. Found: C,
42.89; H, 8.71; N, 3.73%.
2.2.1.2. Synthesis of OH3. OH2 (0.350 g, 1.6 mmol) was placed in a
100 mL two-necked round-bottom flask previously desiccated. Anhy-
drous freshly distilled diethylether (10 mL) was added, under a dinitro-
gen atmosphere, to the flask which was cooled to 0 °C in an ice-bath.
LiAlH4 (4.8 mL, 4.8 mmol) was added to the solution through a rubber
septum within 2 min. When the evolution of dihydrogen finished, the
clear solution was allowed to reach room temperature and then stirred
for 30 min under dinitrogen. A previously degassed saturated solution
of sodium sulfate (4 mL) was added dropwise giving a white suspension.
Further anhydrous sodium sulfate (5 g) was added, and the mixture was
filtered under dinitrogen on a G2 frit. The filtrate was collected in a
100 mL two-necked round-bottom flask. The solid on the frit was washed
with anhydrous diethylether (4 ꢁ 10 mL) and the combined ethereal
phases were left under a gentle dinitrogen stream until the evaporation
of the solvent was completed. A whitish cereous solid was obtained. Yield
87%. 1H NMR (CDCl3, d ppm): 3.50 (t, 2H, 3J = 6.9 Hz, NCH2CH2OH), 2.88
and 2.22 (m + m, 2H + 2H; 1JHP = 198 Hz; (H2PCH2CH2)2N), 2.56 (m, 6H,

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V. Peruzzo et al. / Inorganica Chimica Acta 387 (2012) 163–172
3J = 7.1 Hz, (CH3OCH2CH2)2P), 1.67 (m, 4H, PCH2CH2N). 31P NMR
(CDCl3, d ppm): ꢀ38.6 ppm (s). 13C NMR (CDCl3, d ppm):70.58 (s,
NCH2CH2OH), 70.27 (s, CH3OCH2CH2P), 58.56 (s, CH3OCH2CH2P),
a degassed saturated aqueous solution of Na2SO4 (2 mL) was added
dropwise until no more gas developed. Diethylether (10 mL) was
subsequently added and the mixture was then dried over anhy-
drous sodium sulfate, filtered and the solvent of the filtrate was re-
moved under a dinitrogen stream. The colorless oil residue was
dried in vacuo. Yield 80%. 1H NMR (CDCl3 d ppm): 1.00 (t,
3JHH = 7.3 Hz, 3H, CH3), 1.63 (m, 6H, CH2CH3, PCH2), 2.30 and 2.95
2
54.98 (s, NCH2CH2OH), 50.09 (d, JCP = 23.7 Hz, PCH2CH2N), 27.95 (d,
1
1JCP = 13.9 Hz, (CH3OCH2CH2)2P), 25.13 (d, JCP = 13.7 Hz, PCH2CH2N).
ESI-MS (m/z assignment): 414 ([M+H]+), 436 ([M+Na]+). Anal. Calc.
for C18H41NO5P2: C, 52.28; H, 10.00; N, 3.89. Found: C, 52.79; H,
9.77; N, 3.75%.
1
(m + m, 2H + 2H, JHP = 195 Hz, PH2), 2.62 (m, 10H, SCH2, NCH2),
2.95 (m, 2H, PH2). 13C NMR (CDCl3) d ppm: 13.44 (s, SCH2CH2CH3),
1
2.2.2. Synthesis of PN(S)P
15.48 (d, JCP = 142 Hz, CH2P), 22.52 (s, SCH2CH2CH3), 29.37 (s,
2.2.2.1. Synthesis of S1. NaOH (0.88 g, 22 mmol) was dissolved in
methanol (25 mL). The cloudy solution was filtered and the filtrate
was added dropwise to a solution of 1-propanethiol (0.9 mL,
10 mmol) in methanol (25 mL). Then, 2-chloroethylammonium
chloride (1.16 g, 0.01 mol) dissolved in methanol (15 mL) was
added dropwise. During all steps dinitrogen was flushed through
the reaction apparatus. The reaction mixture was stirred overnight
at r.t. The solvent was then removed in vacuo and the residue dis-
solved in diethylether (25 mL). The mixture was filtered and the
filtrate was concentrated in vacuo giving a pale yellow oil. Yield
42%. 1H NMR (CDCl3, d ppm): 0.95 (t, J = 7.4 Hz, 3H, CH3), 1.57
(m, 2H, CH2CH3), 2.45 (t, J = 7.4 Hz, 2H, SCH2CH2CH3), 2.57 (t,
J = 6.4 Hz, 2H, NCH2CH2S), 2.83 (t, J = 6.4 Hz, 2H, NCH2). 13C NMR
(CDCl3, d ppm): 13.67 (s, CH3), 23.29 (s, CH2CH3), 34.07 (s,
SCH2CH2CH3), 36.50 (s, NCH2CH2S), 41.38 (s, NCH2). Anal. Calc. for
C5H13NS: C, 50.37; H, 10.99; N, 11.75; S, 26.89. Found: C, 49.57;
H, 10.60; N, 11.88; S, 26.65%.
SCH2CH2CH3), 35.85 (s, NCH2CH2S), 50.73 (s, NCH2CH2P), 53.79 (s,
NCH2CH2S). 31P NMR (CDCl3) d ppm: ꢀ145.8 (s).
2.2.2.4. Synthesis of PN(S)P. The colorless oil S3 was weighted in a
two-necked round bottom flask, dissolved in dry THF (7.5 mL),
and cooled to ꢀ78 °C. A 1.4-fold molar excess of n-butyllithium
(2.5 M solution in n-hexane) was added dropwise and the solution
stirred for one hour at r.t. Then, the flask was placed into an ice-
bath and a 4-fold molar amount of (2-chloroethyl)methylether dis-
solved in dry THF (5 mL) was added dropwise. The yellowish solu-
tion was stirred over night at r.t. Degassed water (3 mL) was then
added and the product was extracted twice with diethylether
(2 ꢁ 10 mL). The combined organic phases were dried over anhy-
drous sodium sulfate, the solid was then filtered off and the solvent
of the filtrate was removed under a gentle dinitrogen stream. The
residue was dried in vacuo, giving 5 as pale yellow oil. Yield 22%.
1H NMR (CDCl3) d ppm: 0.96 (m, 3H, SCH2CH2CH3), 1.59 (m, 6H,
CH2CH3 and PCH2CH2N), 1.74 (t, J = 7.2 Hz, 2H, PCH2CH2O), 2.57
(m, 10H, CH2S, NCH2), 3.31 (m, 12H, OCH3), 3.50 (m, 8H, OCH2).
13C NMR (CDCl3, d ppm): 71.06 (s, CH3OCH2CH2P), 58.35 (s,
2.2.2.2. Synthesis of S2. Diethyl-2-bromoethyl phosphonate (0.9 mL,
5 mmol) and 2-propylsulfanyl ethylamine S1 (0.298 g, 2.5 mmol)
were dissolved in acetonitrile (10 mL). Potassium carbonate
(1.38 g, 10 mmol) and acetonitrile (10 mL) were added. The reac-
tion mixture was stirred overnight at 70 °C. A solid was filtered
off and washed with acetonitrile. The filtrate was concentrated in
vacuo affording a clear pale yellow oil. Such oil was found to con-
tain a mixture of the monosubstituted product analogous to OH10
and diethylvinylphosphonate, as assessed by 31P NMR (CDCl3, d
ppm: 18.39 (s, diethylvinylphosphonate), 31.44 (s, monosubsti-
tuted product)). The mixture was transferred into a 10 mL glass
vial. Lithium perchlorate (0.266 g, 2.5 mmol) was added and the
vial was flushed with dinitrogen and tightly sealed. The mixture
was heated for 8 h at 75 °C in an oil bath. After cooling down to
room temperature, the vial was opened and the yellow oil was
treated with chloroform (50 mL). The organic solution was then
treated with water (3 ꢁ 20 mL) and the combined aqueous solu-
tions were re-extracted with chloroform (10 mL). The combined
chloroform solutions were dried over anhydrous sodium sulfate,
filtered and the solvent was removed in vacuo. The remaining yel-
low oil was purified by column chromatography on silica with a
chloroform/ethanol 9:1 mixture as eluant. Yield: 45%. 1H NMR
2
CH3OCH2CH2P), 53.09 (s, NCH2CH2S), 51.88 (d, JCP = 26 Hz,
PCH2CH2N),36.80 (s, NCH2CH2S), 29.90 (s, SCH2CH2CH3), 28.03 (d,
1
1JCP = 15 Hz, (CH3OCH2CH2)2P), 26.24 (d, JCP = 14 Hz, PCH2CH2N),
22.81 (s, SCH2CH2CH3),13.58 (s, SCH2CH2CH3). 31P NMR (CDCl3) d
ppm: ꢀ38.2 (s). ESI-MS (m/z assignment): 472 ([M+H]+). Anal. Calc.
for C21H47P2NO4S: C, 53.48; H, 10.04; N, 2.97; S, 6.80. Found: C,
53.32; H, 9.85; N, 2.8%.
2.2.3. Synthesis of copper complexes
2.2.3.1. [Cu(PNP24)(CH3CN)][BF4]. Solid PNP24 (0.152 g, 0.35 mmol)
was added to [Cu(CH3CN)4][BF4] (0.111 g, 0.353 mmol) dissolved in
dichloromethane (30 mL). The solution was stirred for 1 h at r.t. un-
der dinitrogen. The solvent was then removed by a gentle stream of
dinitrogen, and the residue was treated with methanol (10 mL). The
resulting precipitate was filtered off and washed with diethylether
(2 ꢁ 5 mL) affording a white powder. Yield: 81%. 1H NMR (CDCl3,
ppm): 7.53–7.21 (20H, Ph), 3.10–1.90 (8H, CH2), 2.71 (s, 3H, N-
CH3), 2.04 (s, 3H, NC–CH3). 31P{H} NMR (CDCl3, ppm): ꢀ16.5 (bs).
ESI-MS (m/z assignment, % intensity): 518 ([M–CH3CN]+, 100), 442
([M–CH3CN–Ph]+, 12). Anal. Calc. for CuP2N2C31H34BF4: C, 57.55;
H, 5.30; N, 4.33. Found: C, 57.32; H, 5.44; N, 4.44%.
3
(CDCl3, d ppm): 0.95 (t, JHH = 7.2 Hz, 3H, SCH2CH2CH3), 1.30 (m,
12H, OCH2CH3), 1.58 (m, 2H, SCH2CH2CH3), 1.88 (m, 4H, PCH2),
3
2.47 (t, JHH = 7.2 Hz, 2H, SCH2CH2CH3), 2.54 (m, 2H, NCH2CH2S),
2.62 (m, 2H, NCH2CH2S), 2.76 (m, 4H, NCH2CH2P), 4.07 (m, 8H,
2.2.3.2.
[Cu(PNP24)(PPh3)][BF4]. Triphenylphosphine
(0.055 g,
OCH2CH3).13C NMR (CDCl3) d ppm: 13.4 (s, SCH2CH2CH3) 16.6 (d,
0.21 mmol) was added to [Cu(PNP24)(CH3CN)][BF4] (0135 g,
0.21 mmol) dissolved in dichloromethane (30 mL). The reaction
mixture was stirred overnight at room temperature under a dinitro-
gen atmosphere. The colorless solution was concentrated using a
gentle dinitrogen stream and the residue was treated with diethyl-
ether (10 mL) giving a white powder which was dried under vac-
uum. Yield: 75%. 1H NMR (CDCl3, ppm): 7.69–6.98 (35H, Ph),
3.32–1.87 (8H, CH2), 2.60 (s, 3H, CH3). 31P{H} NMR (CDCl3, ppm):
ꢀ1.1 (d), ꢀ15.9 (t). ESI-MS (m/z assignment, % intensity): 780
([M]+, 100), 518 ([M–PPh3]+, 36), 442 ([M–PPh3–Ph]+, 5. Anal. Calc.
for CuP3NC47H46BF4: C, 65.02; H, 5.34; N, 1.61. Found: C, 64.88; H,
5.21; N, 1.71%.
1
J = 5.8 Hz, OCH2CH3), 23.0 (d, JCP = 137 Hz, CH2P), 24.4 (s,
SCH2CH2CH3), 30.0 (s, SCH2CH2CH3), 34.8 (s, NCH2CH2S), 46.7 (s,
NCH2CH2P) 53.2 (s, NCH2CH2S) 61.8 (s, OCH2CH3). 31P NMR (CDCl3)
d ppm: 31.35 (s). ESI-MS (m/z assignment): 470 ([M+Na]+), 486
([M+K]+). Anal. Calc. for C17H39NO6P2S: C, 45.63; H, 8.78; N, 3.13;
S, 7.17. Found: C, 45.63; H, 8.83; N, 2.96; S, 6.97%.
2.2.2.3. Synthesis of S3. S2 (0.224 g, 0.5 mmol) was dissolved in dry
diethylether (5 mL) and cooled to 0–5 °C. A 1 M diethylether solu-
tion of LiAlH4 (3 mL) was added dropwise and then the reaction
mixture was stirred for one hour at r.t. After cooling again to 0 °C

V. Peruzzo et al. / Inorganica Chimica Acta 387 (2012) 163–172
167
2.2.3.3. [Cu(PNP2)][BF4]. A solution of [Cu(CH3CN)4][BF4] (0.078 g,
0.05 mmol) dissolved in degassed acetonitrile (5 mL) was added
dropwise to a solution of PNP2 (0.027 g, 0.05 mmol) in degassed
dichloromethane (5 mL) at room temperature. The reaction mix-
ture was stirred for 30 min at room temperature under a dinitro-
gen atmosphere. The solvent was removed under a dinitrogen
radiolabeled mixture was purified by HPLC using the following condi-
tions. Column: Agilent Zorbax 300 SB-C18; 5
m; 9.4 ꢁ 250 mm; sol-
l
vent system A: MeCN 0.1% TFA; solvent system B: H2O 0.1% TFA. HPLC
conditions: 20–60% A in 20 min; 60–80% A in 2 min; 5 min 80% A; 80–
20% A in 3 min; flow rate: 4 ml/min. After HPLC purification of the
slightly acidic mixture of HPLC eluate or of the neutralized phosphate
buffered (pH 7.4) eluate, [64Cu(PN(S)P)]Cl was found to be not enough
stable (formation of free Cu(II) as illustrated in Fig. 3 at 1 and 24 h after
purification) to plan further in vitro and in vivo studies. The authenti-
cation of the initial [64Cu(PN(S)P)]Cl complex was performed by com-
parison with the HPLC retention time of the analogous non-radioactive
copper complex.
stream giving
a solid that was washed with diethylether
(2 ꢁ 3 mL) and then dried in vacuo. Yield 51%. 1H NMR (CDCl3) d
(ppm) = 7.53–7.02 (20H, Ph), 3.45–3.02 (5H, –CH2OCH3), 3.02–
1.78 (10H, (other CH2 groups). 31P{H} NMR (CDCl3, ppm): ꢀ6.1
(bs). ESI-MS (m/z assignment, % intensity): 562 ([M]+, 100), 348
([M–CH2CH2P(Ph)2]+, 30), 304 ([M–3Ph–OCH3]+, 11). Anal. Calc.
for CuP2NOC31H35BF4: C, 57.29; H, 5.43; N, 2.15. Found: C, 57.56;
H, 5.54; N, 2.02%.
2.4. Experiments with human cells
2.2.3.4. [Cu(PNP5)][BF4]. This compound was prepared as above re-
ported for [Cu(PNP2)][BF4], but using PNP5 instead of PNP2 under
strictly controlled anaerobic conditions. Yield 54% 3.89–3.60 (12H;
various CH2O–), 3.39 (s, 12H, CH3O–), 2.76 (bs, 6H, NCH2), 1.83–
1.59 (20H, (–OCH2CH2CH2)2PCH2CH2N), 1.25 (t, 3H, –OCH2CH3).
31P{H} NMR (CDCl3, ppm): ꢀ17.6 (bs). ESI-MS (m/z assignment,
% intensity): 546 ([M]+, 100). Anal. Calc. for CuP2NO5C24H53BF4: C,
44.48; H, 8.24; N, 2.16. Found: C, 44.84; H, 8.43; N, 2.08%.
The complex [Cu(PN(S)P)][BF4] was dissolved in purified water
just before the experiment as well as the reference metal-drug cis-
platin. [Cu(PN(S)P)][BF4] was examined for its cytotoxic properties
against a panel of human tumor cell lines containing examples of
lung (U1810), colon (HCT-15), cervix (Hela) and breast (MCF-7)
cancer and leukemia (HL60).
2.5. Cell cultures
2.2.3.5. [Cu(PN(O)P)]. To 1 mmol amount of PN(OH)P dissolved in
anhydrous diethylether (10 mL) an equimolar amount of
[Cu(CH3CN)4][BF4] dissolved in degassed acetonitrile was added. After
15 min stirring under a dinitrogen atmosphere the solvent was re-
moved by a gentle stream of dinitrogen giving a fluid cerous solid,
which was treated with chloroform (3 mL). After filtration, the solvent
of the filtrate was removed again by a gentle stream of dinitrogen and
the cerous residue dried in vacuo overnight. Yield 65%. 1H NMR (CDCl3)
d (ppm): 3.79 (bs, 2H, CH2OH), 3.65 (bs, 8H, CH3OCH2CH2P), 3.34 (s,
12H, CH3OCH2CH2P), 2.85 (bs, 2H, NCH2CH2OH), 2.67 (bs, 4H,
NCH2CH2P), 2.06 (bs, 12H, (CH3OCH2CH2)2PCH2CH2N). 31P{H} NMR
(CDCl3, ppm): ꢀ24.4 (bs). ESI-MS (m/z assignment, % intensity): 476
([M+H]+, 100), 418 ([M–CH2CH2OCH3]+, 9). Anal. Calc. for CuP2-
NO5C18H40: C, 45.42; H, 8.47; N, 2.94. Found: C, 45.84; H, 8.31; N,
3.02%.
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, antibiotics (50 units mLꢀ1 penicil-
lin and 50 l L-glutamine: (i) RPMI-
g mLꢀ1 streptomycin) and 2 mM
1640 medium with 25 mM HEPES buffer for HL60, MCF-7, HCT-15,
U1810, 2008, C13⁄; (ii) F-12 HAM’S for HeLa cells.
2.6. Cytotoxicity assay
The growth inhibitory effect towards tumor cell lines was
evaluated by means of MTT (tetrazolium salt reduction) assay.
Briefly, 3–8 103 cells/well, dependent upon the growth character-
istics of the cell line, were seeded in 96-well microplates in
growth medium (100 lL) 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 72 h, each well was treated
2.2.3.6. [Cu(PN(S)P)][BF4]. PN(S)P (40.0 mg, 0.085 mmol) was dis-
solved in degassed acetonitrile (20 mL). An equimolar amount of
the precursor [Cu(CH3CN)4][BF4] (26.7 mg, 0.085 mmol) was added
at r.t. The clear pale yellow solution was stirred for 1 h at room
temperature. The pure compound was separated via HPLC
(Rf = 14.0 min) according to the method detailed above. Yield
55%. 1H NMR (CDCl3) d ppm: 1.03 (t, J = 7.4 Hz, 3H, SCH2CH2CH3)
1.67 (m, 2H, CH2CH3) 1.92 (m, 4H, 2ꢁPCH2CH2O) 2.68 (m, 2H,
SCH2CH2CH3) 2.97 (m, 2H, NCH2CH2P) 3.31 (m, 12H, 4ꢁOCH3)
3.64 (m, PCH2CH2O). 31P NMR (CDCl3) d ppm: ꢀ20.90 (bs). ESI-
MS (m/z assignment): 534 ([M]+). Anal. Calc. for CuP2-
NO4SC21H47BF4P: C, 40.55; H, 7.61; N, 2.55; S, 5.15. Found: C,
40.87; H, 7.47; N, 2.62%.
with 10
2,5-diphenyltetrazolium bromide) saline solution, and after 5 h
of incubation, 100 L of a sodium dodecylsulfate (SDS) solution
l
L of a 5 mg mLꢀ1 MTT (3-(4,5-dimethylthiazol-2-yl)-
l
in HCl 0.01 M was added. After overnight incubation, the inhibi-
tion of cell growth induced by the tested complexes was de-
tected 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 absor-
bance at 570 nm to 50% of those in the untreated control
wells.IC50 values, calculated from the dose-survival curves ob-
tained after 72 h of drug treatment from the MTT test, are shown
in Table 1.
2.3. Radiolabeling of PN(S)P with 64CuCl2: [64Cu(PN(S)P)]Cl
[
64CuCl2] was produced on a cyclotron CYCLONE 18/9 via the
64Ni(n,p)64Cu reaction according to the method of Avila-Rodriguez
et al. [31]. The specific activity of Cu-64 was 200–400 GBq/ mol. Li-
l
3. Results
gand PN(S)P (1.5 mg) was dissolved in a mixture of degassed acetoni-
trile (1 mL), deionized water (0.8 mL) and 0.2 mL of ammonium
acetate solution (1 M, pH 7). To this combined solution 15 MBq of
3.1. Synthesis and characterization of the ligands
[
64CuCl2] in 70
ll HCl (0.01 M) were added. After 15 min at r.t. the
As specified in the Section 2, aminodiphosphines PNP24, PNP2
and PNP5 were prepared according to published procedures
[25,27,28,30]. The novel hydroxyl- and thioether-substituted
aminodiphoshpines PN(OH)P and PN(S)P were instead synthesized
reaction was completed (no free 64Cu(II)), as can be seen from
Fig. 2(top). Radiochemical yield of [64Cu(PN(S)P)]Cl was about 75%, be-
cause of the presence of a more lipophilic species (Fig. 2(top)). The

168
V. Peruzzo et al. / Inorganica Chimica Acta 387 (2012) 163–172
Table 1
Cytotoxicity.
Compound
IC50
(lM) S.D.
HL60
MCF-7
HCT-15
HeLa
U1810
2008
C13⁄
R.F.
[Cu(PN(S)P)][BF4]
[Cu(thp)4][PF6]
Cisplatin
23.35 2.32
0.42 0.25
9.43 1.32
19.43 3.12
5.19 0.32
7.60 0.21
39.78 1.76
0.54 0.67
13.33 2.16
9.50 1.12
3.02 0.95
2.25 0.73
12.25 1.34
0.55 0.25
3.64 0.81
35.71 1.33
0.44 0.03
3.12 1.03
31.33 1.23
0.62 0.09
22.18 2.01
0.9
1.4
7.0
S.D. = standard deviation. thp = tris-hydroxymethyl phosphine.
IC50 values were calculated by probit analysis (P < 0.05, v2 test). Cells (5–8 ꢁ 104 mLꢀ1) were treated for 72 h with increasing concentrations of tested compounds. Cyto-
toxicity was assessed by MTT test.
as outlined in Scheme 2. The relevant primary amine was alkylated
with diethyl-2-bromoethyl phosphonate and potassium carbonate
in acetonitrile at 70 °C to give the mono-substituted phosphonate
and diethylvinylphosphonate. This mixture was then treated with
the promoter lithium perchlorate at 75 °C for 7 h to afford the
aminodiphosphonate OH2 and S2 after extraction with chloroform.
The bis-phosphonates OH2 and S2 were reduced with lithium alu-
minum hydride to the bis-primary phosphines OH3 and S3, which
were subsequently treated with n-butyllithium followed by 4-fold
molar amount of (2-chloroethyl)methylether to give the final ami-
no substituted diphosphines PN(OH)P and PN(S)P.
The proton NMR spectra of the various intermediate species are in
agreement with the proposed formulation (see Section 2). 31P NMR
spectroscopy was used to check the subsequent steps of the overall
process. As illustrated in Fig. 1 for PN(OH)P, the 31P signal of the start-
ing diethyl-2-bromoethyl phosphonate moves slightly downfield
from 26.4 to 31.7 ppm in the bis-phosphonate OH2, then remarkably
upfield in the primary diphosphine OH3 at ꢀ146.1 ppm, and eventu-
ally downfield at ꢀ38.6 ppm in the final aminodiphosphine PN(OH)P.
Analogously for PN(S)P, in the intermediate bis-phosphonate S2 the
31P signal falls at 31.5 ppm, then upfield at ꢀ145.8 ppm in the pri-
mary phosphine S3, and downfield again at ꢀ38.2 ppm in the final
tertiary phosphine.
3.2. Synthesis and characterization of the copper complexes
All of the copper complexes were prepared by ligand-exchange
reactions treating the labile [Cu(CH3CN)4][BF4] precursor with an
equimolar amount of the relevant aminodiphosphine in dichloro-
methane or acetonitrile solutions at room temperature (see
Scheme 3). After stirring, the solvent was removed giving the
mono-cationic or neutral complexes in moderate to high yields.
Copper compounds were characterized by means of 1H and 31P
NMR spectroscopy. Generally, a slightly downfield shift and broaden-
ing of the aliphatic proton signals of the ligand backbone is observed
upon coordination to copper(I). The broadening is due to the quadru-
polar relaxation induced by I = 3/2 63Cu and 65Cu nuclei onto neigh-
boring protons [9]. Such signal broadening is magnified in the 31P
spectra, in which the half-linewidth is widened to ca. 150 Hz com-
pared to the value of ca. 20 Hz observed in the uncoordinated amin-
odiphosphines. The 31P signal appears as a broad singlet and
experiences a downfield shift of about 5 ppm in [Cu(PNP24)]+-type
complexes compared to the corresponding signal in uncoordinated
PNP24. This shift is enlarged to ca. 15 ppm in all of the other com-
plexes including tetradentate-type ligands, indicating a more robust
metal-phosphine bond. The mixed-ligand complex [Cu(PNP24)PPh3]+
exhibits instead a doublet/triplet pattern in the 31P spectrum,
50
0
-50
-100
-150
ppm (t1)
Fig. 1. 31P NMR spectra of intermediates species obtained during the synthesis of PN(OH)P.

V. Peruzzo et al. / Inorganica Chimica Acta 387 (2012) 163–172
169
Fig. 2. Superimposition of the electronic and radiometric chromatographic traces of the two complexes [64Cu(PN(S)P)][Cl] (top) and cold [Cu(PN(S)P)][BF4] (bottom).
Fig. 3. Stability of HPLC purified [64Cu(PN(S)P)][Cl] at 1 and 24 h after labeling.
consistent with the coupling of the triphenylphosphine-P with two
equivalent aminodiphosphine-P.
[31], in acidic acetonitrile/water mixtures. PN(S)P acted both as
reducing and coordinating agent. In fact, 64Cu(II) chloride was
quickly reduced to Cu(I) with contemporary oxidation of excess
phosphine to phosphineoxide. The radiolabeled complex was puri-
fied via HPLC and identified by superimposition of the radioactive
Electrospray ionization mass spectrometry (ESI-MS) in the posi-
tive ion mode shows the expected molecular ion peak [M]+ for all
the cationic complexes, and the protonated peak [M+H]+ in the
case of the neutral complex [Cu(PN(O)P)]. Only the ‘3 + 1’ complex
[Cu(PNP24)PPh3]+ does not show the molecular ion peak, but the
fragment ion corresponding to the [Cu(PNP24)]+ species.
c-trace with the UV–Vis trace of an authenticated sample of
[Cu(PN(S)P)]+ prepared by conventional coordination chemistry
procedures (Fig. 2). [64Cu(PN(S)P)]+ was found to be stable in the
reaction mixture containing an excess of ligand for at least 48 h.
However, when the complex was separated by chromatography
from the reaction mixture (and, consequently, from the excess of
ligand), the eluate of the purified complex showed the presence
of oxidized copper, and only 80% and 20% of the native complex
was recovered intact after 1 and 24 h, respectively (Fig. 3).
3.3. Radiolabeling of ligand PN(S)P with 64Cu
PN(S)P was selected for preliminary investigations to study the
ability of these polydentate ligands to stabilize radiocopper spe-
cies. It was labeled with [64CuCl2], produced as reported elsewhere

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V. Peruzzo et al. / Inorganica Chimica Acta 387 (2012) 163–172
[Cu(PNP24)(PPh3)][BF4]
PPh3
[Cu(PNP24)(CH3CN)][BF4]
PNP24
CH3CN
PNP2
PNP5
[Cu(CH3CN)4][BF4]
[Cu(PNP2)][BF4]
[Cu(PNP5)][BF4]
PN(S)P
PN(OH)P
[Cu(PN(O)P)]
Scheme 3. Synthesis of copper compounds.
[Cu(PN(S)P)][BF4]
3.4. Cytotoxicity assay
sphere. For the syntheses of new polydentate PN(OH)P and PN(S)P we
have instead developed a simplified procedure that required only two
anaerobic steps and the use of inexpensive reagents (e.g. hydroxyeth-
ylamine, diethyl-2-bromoethyl phosphonate and diethylvinylphosph-
onate in the case of PN(OH)P). During the first step of the reaction with
the primary amines OH1 and S1, diethyl-2-bromoethyl phosphonate
underwent both N-alkylation and b-elimination giving the mono-
substituted phosphonate and diethylvinylphosphonate (detected in
the 31P NMR spectrum at d = 18.39 ppm in the reaction mixture).
The latter promoted the formation of the aminodiphosphonates OH2
and S2 via an aza-Michael addition of the secondary amine under mild
conditions through its activation with lithium perchlorate.
We thought that both electronic and stereochemical properties of
PNP-type ligands could be suitable also for the construction of tetra-
hedral copper(I) complexes, taking advantage of the efficient overlap
of empty d orbitals of soft donor phosphines with the electron-rich
d10 full orbitals of the metal ion supported by the chelation effect pro-
vided by other donors. The first goal of the study was to use PNP24
that worked as a pure tridentate donor. The tetrahedral arrangement
of the coordinated Cu(I) ion was achieved by means of an additional
mono-dentate donor (N-acetonitrile in [Cu(PNP24)(CH3CN)]+ and P-
triphenylphosphine in [Cu(PNP24)PPh3]+, respectively). As expected,
all of the other ligands utilized in this study (Scheme 1) behaved as
tetradentatechelates that fully surrounded the copper(I) ion with
the PNP-donor set supported by an heteroatom of the pendant group
of the substituted aminodiphosphine: an ether oxygen in [Cu(PNP2)]+
and [Cu(PNP5)]+, an hydroxylate oxygen in [Cu(PN(O)P)] and a thio-
ether sulfur in [Cu(PN(S)P)]+ (Scheme 4).
The cytotoxic activity of [Cu(PN(S)P)][BF4] toward a panel of hu-
man tumor cell lines including examples of promyelocytic leuke-
mia (HL60), breast (MCF-7), colon (HCT-15), cervix (HeLa), non-
small cell lung (U1810) is shown in Table 1. We also tested the
cytotoxic activity of the complex onto an additional cell line pair
selected for its resistance to cisplatin (2008/C13⁄ ovarian cancer
cells). [Cu(PN(S)P)][BF4] showed a relatively low cytotoxic activity
against all cancer cell lines, with IC50 values roughly: (i) 3.5-fold
higher than those of cisplatin and (ii) 3- to 80-fold higher than
those displayed by [Cu(thp)4][PF6] (thp = trishydroxymethyl phos-
phine) [9], a further reference standard among the series of
cytotoxic copper phosphine complexes synthesized and tested so
far. Although moderately cytotoxic, the RF value shown by
[Cu(PN(S)P)][BF4] on the 2008/C13⁄ pair was found to be 8 times
lower than that calculated for cisplatin.
4. Discussion
PNP-type aminodiphosphines have been used by our research
group since 2000 showing the capability to stabilize both high-valent
nitride-Tc(V) and –Re(V) compounds [25–29] and low-valent Tc(III)/
Tc(I) and Re(III) complexes. The quite robust metal-phosphine bonds
ensured the metal encapsulation by the ligand(s), a property that
made a class of technetium complexes (i.e. [Tc(N)(PNP5)(DTC)]+ spe-
cies, DTC = various dithiocarbamates) suitable candidates for the
development of myocardial imaging agents [32–34]. An additional
peculiarity shown by PNP-type ligands was their ability to easily rear-
range from meridional to facial configurations in octahedral com-
plexes, depending on the nature on the ancillary co-ligands [35]. The
early method for producing both P-aryl and P-alkyl aminodiphos-
phines was proposed by Sacconi in 1970’s [36,37]. The protocol for
the synthesis of P-alkyl aminodiphosphines was time-consuming
and most of the reactions required the use of strictly controlled atmo-
Despite the great effort devoted to the synthesis of these poly-
dentate ligands, the resulting copper complexes displayed relatively
low red-ox stability both in the solid and in the solution states.
Among the synthesized series, only [Cu(PN(S)P)]+ showed some sta-
bility to justifying further studies toward potential applications. The
higher affinity of the copper(I) ion for the soft thioether sulfur
compared to hard oxygen donors likely supports tetracoordination.
R
X
N
R
CH2CH2OCH3
CH2CH2OCH3
C6H5
SCH2CH2CH3
O-
[Cu(PN(S)P)][BF4]
[Cu(PN(O)P)]
P
R
Cu
X
[Cu(PNP2)][BF4]
OCH3
R
P
OCH2CH3
CH2CH2CH2OCH3
[Cu(PNP5)][BF4]
R
Scheme 4. Proposed structure of copper compounds.

V. Peruzzo et al. / Inorganica Chimica Acta 387 (2012) 163–172
171
The use of tetradentate ligands can help to encapsulate com-
pletely the copper(I) ion, thereby increasing, at least in principle,
stability and kinetic inertness, a necessary pre-requisite for the
use of copper compounds in the radiopharmaceutical field. The
quick radiolabeling of PN(S)P with 64CuCl2 demonstrated the abil-
ity of the ligand to act both as reducing and coordinating agent in
acidic medium, and the high radiochemical yield indicates that
dilution was not a critic issue because reactions conducted at mil-
limolar (‘cold’ copper) and micro/nanomolar (64Cu) levels afforded
the same species. However, when separated and purified from the
reaction mixture, [64Cu(PN(S)P)][BF4] was not sufficiently stable
with the time (see Fig. 3) to plan further studies in vivo. In general,
for any radiopharmaceutical application a metal-complex should
be delivered with a precise formulation and should remain intact
to avoid unspecific uptake. Using this class of potentially tetraden-
tate ligands, the corresponding radiolabeled copper complexes do
not display physico-chemical features suitable for radiopharma-
ceutical application. Considering the well-balanced electronic abil-
ity of the P2NS-donor set toward coordination to the Cu(I) ion, it is
supposed that steric constraints imposed by the complex stereo-
chemistry of the ligand can be the driving force for metal displace-
ment with the time. Consequently, ligand denticity should be
modified and works in this direction have been undertaken. A rel-
ative instability of the copper complex may instead become useful
in the design of a cytotoxic agent.
In fact, it has been demonstrated that copper is delivered from
the extracellular to the intracellular domain through a transmem-
brane protein named hCtr1 (human copper transporter 1) [38–40].
During this intricate path, copper(I) has to be continuously ex-
changed from methionine-, histidine- and cysteine-rich residues
[38,39]. This process requires that the metal should not be tightly
bonded either to our aminodiphosphine or to any aminoacidic do-
nor, but constantly available for a cascade of interactions, eventu-
ally reaching the intracellular target(s). In this view, we have tested
the ‘cold’ complex [Cu(PN(S)P)][BF4] for its cytotoxic activity to-
ward a panel of human tumor cell lines.
The thioether substituted aminodiphosphine ligand PN(S)P has
been labeled with 64Cu with high radiochemical yield under mild
conditions, but the resulting [64Cu(PN(S)P)][Cl] species was not suf-
ficiently stable to plan further studies in vivo.
The kinetic instability of this complex has been exploited as a tool
useful for the design of a copper cytotoxic agent. [Cu(PN(S)P)][BF4]
showed moderately cytotoxic activity, but confirmed the antiprolifer-
ative action observed for other phosphine copper(I) complexes. Stud-
ies focused on the determination of the cytotoxic activity of classes of
‘3 + 1’ complexes including [Cu(PNP24)(X)]-type species are in
progress.
Acknowledgments
This work was financially supported by MIUR(PRIN20078EWK9B).
The authors thank A. Moresco for skilled assistance in the preparation
of the manuscript.
References
[1] F. Tisato, C. Marzano, M. Porchia, M. Pellei, C. Santini, Med. Res. Rev. 30 (2010)
708.
[2] C. Marzano, M. Pellei, F. Tisato, C. Santini, Anti-Cancer Agents Med. Chem. 9
(2009) 185.
[3] S. Tardito, L. Marchio, Curr. Med. Chem. 16 (2009) 1325.
[4] B.M. Paterson, P.S. Donnelly, Chem. Soc. Rev. 40 (2011) 3005.
[5] T.J. Wadas, E.H. Wong, G.R. Weisman, C.J. Anderson, Curr. Pharm. Des. 13
(2007) 3.
[6] M. Kartalou, J.M. Essigmann, Mutat. Res. 478 (2001) 23.
[7] C.X. Zhang, S.J. Lippard, Curr. Opin. Chem. Biol. 7 (2003) 481.
[8] Y. Xiao, D. Chen, X. Zhang, Q. Cui, Y. Fan, C. Bi, Q.P. Dou, Int. J. Oncol. 37 (2010)
81.
[9] C. Marzano, V. Gandin, M. Pellei, D. Colavito, G. Papini, G.G. Lobbia, E. Del
Giudice, M. Porchia, F. Tisato, C. Santini, J. Med. Chem. 51 (2008) 798.
[10] P.J. Blower, J.S. Lewis, J. Zweit, Nucl. Med. Biol. 23 (1996) 957.
[11] P. McQuade, D.J. Rowland, J.S. Lewis, M.J. Welch, Curr. Med. Chem. 12 (2005)
807.
[12] F. Dehdashti, P.W. Grigsby, M.A. Mintun, J.S. Lewis, B.A. Siegel, M.J. Welch, Int.
J. Radiat. Oncol. Biol. Phys. 55 (2003) 1233.
[13] F. Dehdashti, M.A. Mintun, J.S. Lewis, J. Bradley, R. Govindan, R. Laforest, M.J.
Welch, B.A. Siegel, Eur. J. Nucl. Med. Mol. Imaging 30 (2003) 844.
[14] J.S. Lewis, D.W. McCarthy, T.J. McCarthy, Y. Fujibayashi, M.J. Welch, J. Nucl.
Med. 40 (1999) 177.
Although moderately cytotoxic compared to the metal-based
Pt-standard (cisplatin) and our Cu-standard [Cu(thp)4][PF6] (see
Table 1), [Cu(PN(S)P)][BF4] confirmed the antiproliferative action
observed for other phosphine copper(I) complexes [9,22,23], with
[15] M.R. Taylor, E.J. Gabe, J.P. Glusker, J.A. Minkin, A.L. Patterson, J. Am. Chem. Soc.
88 (1966) 1845.
[16] J.A. Crim, H.G. Petering, Cancer Res. 27 (1967) 1278.
[17] P. Schwerdtfeger, H.L. Hermann, H. Schmidbaur, Inorg. Chem. 42 (2003) 1334.
[18] C. Santini, M. Pellei, G. Papini, B. Morresi, R. Galassi, S. Ricci, F. Tisato, M.
Porchia, M.P. Rigobello, V. Gandin, C. Marzano, J. Inorg. Biochem. 105 (2011)
232.
[19] M. Porchia, F. Benetollo, F. Refosco, F. Tisato, C. Marzano, V. Gandin, J. Inorg.
Biochem. 103 (2009) 1644.
[20] C. Marzano, M. Pellei, S. Alidori, A. Brossa, G.G. Lobbia, F. Tisato, C. Santini, J.
Inorg. Biochem. 100 (2006) 299.
[21] S. Alidori, G.G. Lobbia, G. Papini, M. Pellei, M. Porchia, F. Refosco, F. Tisato, J.S.
Lewis, C. Santini, J. Biol. Inorg. Chem. 13 (2008) 307.
[22] C. Marzano, M. Pellei, D. Colavito, S. Alidori, G.G. Lobbia, V. Gandin, F. Tisato, C.
Santini, J. Med. Chem. 49 (2006) 7317.
[23] V. Gandin, M. Pellei, F. Tisato, M. Porchia, C. Santini, C. Marzano, J. Cell Mol.
Med. 16 (2011) 142.
[24] S. Alidori, G. Gioia Lobbia, G. Papini, M. Pellei, M. Porchia, F. Refosco, F. Tisato,
J.S. Lewis, C. Santini, J. Biol. Inorg. Chem. 13 (2008) 307.
[25] C. Bolzati, A. Boschi, L. Uccelli, F. Tisato, F. Refosco, A. Cagnolini, A. Duatti, S.
Prakash, G. Bandoli, A. Vittadini, J. Am. Chem. Soc. 124 (2002) 11468.
[26] C. Bolzati, A. Mahmood, E. Malago, L. Uccelli, A. Boschi, A.G. Jones, F. Refosco, A.
Duatti, F. Tisato, Bioconjug. Chem. 14 (2003) 1231.
[27] C. Bolzati, F. Refosco, A. Cagnolini, F. Tisato, A. Boschi, A. Duatti, L. Uccelli, A.
Dolmella, E. Marotta, M. Tubaro, Eur. J. Inorg. Chem. (2004) 1902.
[28] M. Porchia, G. Papini, C. Santini, G.G. Lobbia, M. Pellei, F. Tisato, G. Bandoli, A.
Dolmella, Inorg. Chem. 44 (2005) 4045.
activities roughly in the 10 lM range against cervix (HeLa) and
non-small cell lung (U1810) human tumor cell lines.
In addition, [Cu(PN(S)P)][BF4] was able to overcome cross-resis-
tance phenomena in cisplatin sensitive and resistant ovarian cell
lines. 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 cor-
related to reduced cellular drug uptake, high cellular glutathione
levels, and enhanced repair of DNA damage [41]. Cross-resistance
profiles were evaluated by means of the resistance factor (RF),
which is defined as the ratio between IC50 values calculated for
the resistant cells and those arising from the sensitive ones.
[Cu(PN(S)P)][BF4] showed activity levels very similar on both cis-
platin sensitive and resistant ovarian cell lines, being the RF value
about eight times lower than that calculated for cisplatin, thus
indicating a different cross-resistance profile than that of cisplatin.
The overcoming of cross-resistance phenomena supports the
hypothesis of a different pathway of action of this copper(I) com-
plex from that of cisplatin.
[29] F. Tisato, M. Porchia, C. Bolzati, F. Refosco, A. Vittadini, Coord. Chem. Rev. 250
(2006) 2034.
[30] M. Porchia, F. Tisato, F. Refosco, C. Bolzati, M. Cavazza-Ceccato, G. Bandoli, A.
Dolmella, Inorg. Chem. 44 (2005) 4766.
5. Conclusions
[31] M.A. Avila-Rodriguez, J.A. Nye, R.J. Nickles, Appl. Radiat. Isot. 65 (2007) 1115.
[32] A. Boschi, C. Bolzati, L. Uccelli, A. Duatti, E. Benini, F. Refosco, F. Tisato, A.
Piffanelli, Nucl. Med. Commun. 23 (2002) 689.
[33] A. Boschi, L. Uccelli, C. Bolzati, A. Duatti, N. Sabba, E. Moretti, G. Di Domenico,
G. Zavattini, F. Refosco, M. Giganti, J. Nucl. Med. 44 (2003) 806.
This work reports on the synthesis and characterization of two
novel, potentially tetradentate substituted aminodiphosphine li-
gands and of the corresponding tetrahedral copper(I) complexes.

172
V. Peruzzo et al. / Inorganica Chimica Acta 387 (2012) 163–172
[34] C. Bolzati, M. Cavazza-Ceccato, S. Agostini, S. Tokunaga, D. Casara, G. Bandoli, J.
Nucl. Med. 49 (2008) 1336.
[38] K.J. Franz, J.T. Rubino, M.P. Chenkin, M. Keller, P. Riggs-Gelasco, Metallomics 3
(2011) 61.
[35] F. Tisato, F. Refosco, M. Porchia, C. Bolzati, G. Bandoli, A. Dolmella, A. Duatti, A.
Boschi, C.M. Jung, H.J. Pietzsch, W. Kraus, Inorg. Chem. 43 (2004) 8617.
[36] L.M. Sacconi, J. Chem. Soc. A (1971) 492.
[39] K.J. Franz, J.T. Rubino, P. Riggs-Gelasco, J. Biol. Inorg. Chem. 15 (2010) 1033.
[40] J.H. Kaplan, E.B. Maryon, S.A. Molloy, A.M. Zimnicka, Biometals 20 (2007) 355.
[41] G. Marverti, P.A. Andrews, G. Piccinini, S. Ghiaroni, D. Barbieri, M.S. Moruzzi,
Eur. J. Cancer 33 (1997) 669.
[37] R.M.L. Sacconi, J. Chem. Soc. A (1969) 2904.