Platinum and ruthenium complexes of new long-tail derivatives of PTA (1,3,5-triaza-7-phosphaadamantane): Synthesis, characterization and antiproliferative activity on human tumoral cell lines

Article abstract of DOI:10.1016/j.ica.2012.04.031

The alkylation of PTA, (1,3,5-triaza-7-phosphaadamantane), with long chain alkyl iodides produced the novel ionic derivatives (PTAC₁₂H ₂₅)I, (PTAC₁₆H₃₃)I and (PTAC₁₈H ₃₇)I. Anion exchange g

Full text of DOI:10.1016/j.ica.2012.04.031

Inorganica Chimica Acta 391 (2012) 162–170
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Inorganica Chimica Acta
journal homepage: www.elsevier.com/locate/ica
Platinum and ruthenium complexes of new long-tail derivatives
of PTA (1,3,5-triaza-7-phosphaadamantane): Synthesis, characterization
and antiproliferative activity on human tumoral cell lines
a
a
a
a
Paola Bergamini a, , Lorenza Marvelli , Andrea Marchi , Francesca Vassanelli , Marco Fogagnolo ,
⇑
Paolo Formaglio a, Tatiana Bernardi a, Riccardo Gavioli b, Fabio Sforza b
a Dipartimento di Chimica dell’Università di Ferrara, Via L. Borsari 46, 44121 Ferrara, Italy
b Dipartimento di Biochimica e Biologia Molecolare dell’Università di Ferrara, Sezione di Biologia Molecolare, Via Fossato di Mortara, 74, 44121 Ferrara, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
The alkylation of PTA, (1,3,5-triaza-7-phosphaadamantane), with long chain alkyl iodides produced the
novel ionic derivatives (PTAC12H25)I, (PTAC16H33)I and (PTAC18H37)I. Anion exchange gave the PF6 ana-
À
Received 16 January 2012
Received in revised form 13 April 2012
Accepted 20 April 2012
logs (PTAC12H25)PF6, (PTAC16H33)PF6 and (PTAC18H37)PF6. The new phosphines were characterized by ESI-
MS, elemental analysis, 1H, 13C, and 31P NMR spectroscopy and their coordination ability was investigated
towards Pt(II) and Ru(II) cores. Complexes cis-[PtCl2(PTAR)](PF6)2 (1–3) and [CpRuCl(PPh3)(PTAR)]I (5–7)
have been isolated and characterized. Antiproliferative activity, given as estimated IC50, was tested for
each complex against appropriate human tumoral lines (T2, SKOV3 and SW480) in comparison with cis-
platin, with the parent complexes [CpRuCl(PPh3)(PTA)] and [CpRuCl(PPh3)(PTAMe)]I and with the free
ligands.
Available online 28 April 2012
Keywords:
PTA
N-alkylation
Long chain alkyl
Platinum
Ó 2012 Elsevier B.V. All rights reserved.
Ruthenium
Antiproliferative activity
1. Introduction
tion in catalysis [5,6] and in medicine (Pt, Ru, Au) [7–10]. In
particular, Ru–PTA complexes called RAPTA-type, studied by Dyson
The phosphine PTA (1,3,5-triaza-7-phosphaadamantane), first
prepared by Daigle in 1974 [1], has been recently rediscovered
and much exploited in coordination chemistry because of its valu-
able properties as a ligand [2]: (i) its coordination to a wide range
of metals occurs in most cases via phosphorus, frequently showing
regioselectivity of this donor-atom, although some examples of N-
coordination have been recently reported [3,4]; (ii) it is remarkably
soluble in water and stable to oxidation in comparison with other
non-aromatic phosphines; (iii) it has a low steric hindrance and a
narrow cone angle; (iv) it is non-toxic and biocompatible.
[11–13], showed a pH dependent action which makes them selec-
tive towards cancer cell.
Ru
Ru
Ru
Cl
Cl
Cl
PTA
Cl
PTA
PTA
Cl
Cl
RAPTA T
RAPTA B
RAPTA C
N
N
N
P
PTA = 1,3,5-triaza-7-phosphaadamantane
Another important feature of PTA is that all the positions of the
cage structure are reactive in specific conditions, allowing
researchers to design and prepare families of PTA derivatives pre-
senting some of the parent compound properties, amplified and
improved. The three carbon atoms bonded to phosphorus are
known as upper rim and their functionalization is particularly rel-
evant for catalysis allowing e.g., the introduction of chiral centers
or of other coordinating groups in proximity to phosphorus, the
All of these characteristics are at the root of the extensive re-
search relating to PTA coordination compounds and their applica-
⇑
Corresponding author. Tel.: +39 532 455129; fax: +39 532 240709.
E-mail address: bgp@unife.it (P. Bergamini).
0020-1693/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.ica.2012.04.031

P. Bergamini et al. / Inorganica Chimica Acta 391 (2012) 162–170
163
primary coordination site [14]. Additionally, the carbon atoms
bridging the nitrogen atoms (lower rim) can also be functionalized
[15,16], and the nitrogen can be easily alkylated with formation of
quaternary ammonium salts where the phosphorus atom main-
tains its coordinative ability.
The positive charge of N-alkylated PTA derivatives can be
advantageous when they are designed as platinum or ruthenium
ligands, with the aim of obtaining new metal-based anticancer
drugs. It is well known that the ultimate target for platinum-con-
taining species is polyanionic DNA, and a positive charge on the li-
gand can then be exploited to improve the speed and efficiency of
drug action through an electrostatic attraction [17].
1.95 mmol, yield 76%). 1H NMR (300 MHz, d6-DMSO, 25 °C): d
0.84 (bt, 3H, CH3), 1.27 (s, 18H, CCH2C), 1.65 (m, 2H, CH2CH2N+),
2.81 (m, 2H, CH2CH2N+), 3.90 (m, 4H, PCH2N), 4.35 (bs, 2H,
PCH2N+), 4.32 (d, 2JHH 14, 1H, NCH2N), 4.53 (d, 2JHH 14, 1H, NCH2N),
4.82 (d, 2JHH 11, 2H, NCH2N+), 4.97 (d, 2JHH 11, 2H, NCH2N+). 13C{1H}
NMR (100.58 MHz, d6-DMSO, 25 °C): d 14.0 (s, CH3), 19.0 (s, CH2),
22.1 (s, CH2), 26.1 (s, CH2), 28.5 (s, CH2), 28.7 (s, CH2), 29.0 (4
CH2), 31.3 (s, CH2), 45.4 (d, JPC 20.1, PCH2N), 51.7 (d, JPC 32.2,
PCH2N+), 61.4 (s, RCH2N+), 69.3 (s, NCH2N), 78.6 (s, NCH2N+).
31P{1H} NMR (121.50 MHz, d6-DMSO, 25 °C): d À84.35 ppm (s).
Anal. Calc. for C18H37IN3P (453): C, 47.66; H, 8.23; N, 9.27. Found:
C, 47.67; H, 8.45; N, 9.11%.
1
1
Several N-alkylated PTA derivatives have been reported in addi-
tion to simple (PTAMe)+ [18,19]. Our group has recently described
some bisPTA2+ ligands obtained by treating dibromoxylenes (orto,
meta and para) with 2 equivalents of PTA [20].
2.2.2. Dodecyl-PTA hexafluorophosphate, (PTAC12H25)PF6
KPF6 (0.163 g, 0.89 mmol) dissolved in 0.5 mL of water was
added to a solution of (PTAC12H25)I (0.4 g, 0.89 mmol) in 35 mL
of MeOH. (PTAC12H25)PF6 immediately precipitated as a white so-
lid, which was collected and washed with 5 mL of MeOH (0.23 g,
0.49 mmol, yield 55%). 1H NMR (300 MHz, d6-DMSO, 25 °C): d
0.84 (bt, 3H, CH3), 1.27 (bs, 18H, CCH2C), 1.68 (bm, 2H, CH2CH2N+),
2.80 (bm, 2H, CH2CH2N+), 3.87 (m, 4H, PCH2N), 4.35 (d, 2JHP 4.7, 2H,
In this paper we present PTA derivatives obtained by function-
alization of nitrogen with long aliphatic chains with the aim to
generate a new class of ligands containing a large lipophilic portion
beside the hydrophilic, positively charged PTA cage. The structure
of these ligands, reminescent of cationic species with amphiphilic
surfactant properties, could influence the cell penetration mecha-
nism and favor the formulation and distribution of their platinum
and ruthenium complexes when they are used as drugs [21].
The coordination to Pt and Ru of these new PTA derivatives and
the antiproliferative activity of the obtained complexes on some
human cancer cell lines are reported in this paper.
2
2
PCH2N+), d 4.35 (d, JHH 14, 1H, NCH2N), 4.52 (d, JHH 14, 1H,
NCH2N), 4.78 (d, JHH 11, 2H, NCH2N+), 4.95 (d, JHH 11, 2H,
NCH2N+). 13C{1H} NMR (100.58 MHz, d6-DMSO, 25 °C): d 14.0 (s,
CH3), 18.9 (s, CH2), 22.1 (s, CH2), 26.1 (s, CH2), 28.5 (s, CH2), 28.7
2
2
1
(s, CH2), 29.0 (s, 2CH2), 29.1 (s, 2CH2), 31.3 (s, CH2), 45.5 (d, JPC
1
20.7, PCH2N), 51.7 (d, JPC 31.2, PCH2N+), 61.5 (s, CH2N+, chain),
69.3 (s, NCH2N), 78.6 (s, NCH2N+). 31P{1H} NMR (121.50 MHz, d6-
1
DMSO, 25 °C): d À84.32 (s), À142.21 ppm (heptet, JPF 707 Hz,
2. Experimental
PF6À). Anal. Calc. for C18H37F6N3P2 (471): C, 45.84; H, 7.91; N,
8.91. Found: C, 45.79; H, 8.01; N 8.88%. Electrospray MS (in
H2O): observed m/z 326 (I = 100%), calcd. 326 for C18H37N3P
(MÀPF6)+; observed m/z 283 (I = 10%) calcd. 283 for C15H30N3P
(MÀ43)+.
2.1. General procedures
All manipulations were carried out under argon atmosphere
with the use of standard Schlenk techniques unless otherwise
noted. Elemental analyses were carried out using a Carlo Erba
instrument model EA1110. The ESI mass spectra were acquired
with a Micromass LCQDuo Finningan. NMR spectra were recorded
2.2.3. Hexadecyl-PTA iodide, (PTAC16H33)I
A 100 mL Schlenk flask was charged with 0.40 g (2.58 mmol) of
PTA and 25 mL of freshly distilled and degassed acetone. After the
addition of 1.62 ml (5.14 mmol) of 1-iodohexadecane C16H33I the
solution was kept under stirring at room temperature under nitro-
gen for 20 h. After this time, the white precipitate of (PTAC16H33)I
was collected on a frit and washed with n-hexane and allowed to
air dry (0.948 g, 1.87 mmol, 72%). 1H NMR (300 MHz, d6-DMSO,
25 °C): d 0.85 (bt, 3H, CH3), 1.23 (s, 26H, CCH2C), 1.62 (m, 2H,
CH2CH2N+), 2.80 (m, 2H, CH2CH2N+), 3.90 (m, 4H, PCH2N), 4.35
on
a
Bruker 200 AM spectrometer (1H at 200 MHz, 13C at
50.29 MHz, 31P at 81.15 MHz) or a Varian Gemini 300 MHz spec-
trometer (1H at 300 MHz, 13C at 75.43 MHz, 31P at 121.50 MHz)
or a Varian Mercury Plus (1H at 400 MHz, 13C at 100.58 MHz, 31P
at 161.92 MHz). The 13C and 31P spectra were run with proton
decoupling and 31P spectra are reported in ppm relative to an
external 85% H3PO4 standard, with positive shifts downfield. 13C
NMR spectra are reported in ppm relative to external tetramethyl-
silane (TMS), with positive shifts downfield. Solvents were distilled
and dried prior to use. Commercial iodides C12H25I, C16H33I and
2
2
(bs, 2H, PCH2N+), 4.35 (d, JHH 14, 1H, NCH2N), 4.55 (d, JHH 14,
2
2
1H, NCH2N), 4.80 (d, JHH 11, 2H, NCH2N+), 4.92 (d, JHH 11, 2H,
NCH2N+). 13C{1H} NMR (100.58 MHz, d6-DMSO, 25 °C): d 13.9 (s,
CH3), 18.9 (s, CH2), 22.1 (s, CH2), 26.1 (s, CH2), 28.4 (s, CH2), 28.7
C18H37I were purchased and used without further purification.
The compounds PTA [22], (PTAMe)PF6 [23] and the metal com-
plexes precursors [PtCl2(1,5-COD)] (1,5-COD = 1,5-cyclooctadiene)
[24] and [CpRuCl(PPh3)(PTA)] [25] were prepared as described in
the literature.
1
(s, CH2), 28.8 (s, CH2), 29.0 (bs, 7 CH2), 31.3 (s, CH2), 45.4 (d, JPC
1
20.6, PCH2N), 51.7 (d, JPC 31.2, PCH2N+), 61.4 (s, RCH2N+), 69.3
(s, NCH2N), d 78.6 (s, NCH2N+). 31P{1H} NMR (121.50 MHz, d6-
DMSO, 25 °C): d À84.27 ppm (s). Anal. Calc. for C22H45IN3P (509):
C, 51.84; H, 8.91; N, 8.25. Found: C, 51.84; H, 8.97; N 8.19%.
2.2. Synthesis of long tail PTA derivatives (PTACnH2n+1)X, (X = IÀ, PF6
n = 12, 16, 18)
;
À
2.2.4. Hexadecyl-PTA hexafluorophosphate, (PTAC16H33)PF6
The phosphine (PTAC16H33)I (0.45 g, 0.89 mmol) was treated
with KPF6 (0.163 g, 0.89 mmol) in the same conditions as described
above for (PTAC12H25)I. The reaction gave (PTAC16H33)PF6 (0.35 g,
0.66 mmol, yield 74%). 1H NMR (300 MHz, d6-DMSO, 25 °C): d
0.86 (bt, 3H, CH3), 1.30 (bs, 26H, CCH2C), 1.67 (bm, 2H, CH2CH2N+),
2.79 (bm, 2H, CH2CH2N+), 3.88 (m, 4H, PCH2N), 4.32 (bs, 2H,
2.2.1. Dodecyl-PTA iodide, (PTAC12H25)I
PTA (0.4 g, 2.57 mmol) was dissolved in 120 mL of acetone with
stirring under continuous bubbling of argon. After the addition of
1.27 mL (5.14 mmol, 2 equiv.) of 1-iodododecane C12H25I the solu-
tion was stirred at room temperature for 20 h, under a continuous
flux of argon, which reduced the volume at ca. 30 mL. In these con-
ditions (PTAC12H25)I precipitated as a white solid which was col-
lected on a frit, washed with ether and allowed to air dry (0.88 g,
2
2
PCH2N+), 4.32 (bd, JHH 14, 1H, NCH2N), 4.57 (bd, JHH 14, 1H,
2
2
NCH2N), 4.78 (d, JHH 10, 2H, NCH2N+), 4.97 (d, JHH 10, 2H,
NCH2N+). 1H NMR (300 MHz, d3-CD3CN, 25 °C): d 0.92 (t, JHH 7,
2
3H, CH3), 1.35 (bs, 26H, CCH2C), 1.72 (bm, 2H, CH2CH2N+), 2.80

164
P. Bergamini et al. / Inorganica Chimica Acta 391 (2012) 162–170
(bm, 2H, CH2CH2N+), 3.92 (m, 4H, PCH2N), 4.23 (d, JHP 4.7, 2H,
PCH2N+), 4.42 (d, 2JHH 14, 1H, NCH2N), 4.58 (d, 2JHH 14, 1H, NCH2N),
4.72 (d, 2JHH 10, 2H, NCH2N+), 4.88 (d, 2JHH 10, 2H, NCH2N+). 13C{1H}
NMR (100.58 MHz, d6-DMSO, 25 °C): d 14.0 (s, CH3), 18.9 (s, CH2),
22.1 (s, CH2), 26.1 (s, CH2), 28.5 (s, CH2), 28.8 (s, CH2), 29.1 (9
with
(PTAC16H33)PF6,
(PTAC18H37)PF6
and
(PTAMe)PF6,
2
respectively.
2.3.1. [PtCl2(PTAC12H25)2](PF6)2 (1)
1H NMR (300 MHz, d6-DMSO, 25 °C): d 0.87 (bs, 3H, CH3); 1.30
(bs, 18H, CCH2C); 1.70–2.00 (bm, 2H, CH2CH2N+); 2.85 (bm, 2H,
CH2CH2N+); 4.2–5.2 (broad signals, 12H, PTA cage). 13C{1H} NMR
(100.58 MHz, d6-DMSO, 25 °C): d 14.0 (s, CH3), 19.3 (s, CH2), 22.1
(s, CH2), 25.9 (s, CH2), 28.2 (s, CH2), 28.7 (s, CH2), 29.0 (4 CH2),
31.3 (s, CH2), 48.3 (bm, PCH2N + PCH2N+), 61.1 (s, CH2N+), 71.2 (s,
NCH2N), 78.5 (s, NCH2N+). 31P{1H} NMR (121.50 MHz, d6-DMSO,
1
CH2), 31.3 (s, CH2), 45.5 (d,1JPC 19.8, PCH2N, Hz), 51.7 (d, JPC
31.3, PCH2N+), 61.5 (s, RCH2N+), 69.3 (s, NCH2N), 78.6 (s, NCH2N+).
31P{1H} NMR (121.50 MHz, d6-DMSO, 25 °C):
d
À84.27 (s),
1
À142.21 ppm (heptet, JPF 707 Hz, PF6À). Anal. Calc. for
C22H45F6N3P2 (527): C, 50.07; H, 8.60; N, 7.97. Found: C, 50.12;
H, 8.75; N, 7.88%. Electrospray (in H2O): observed m/z 382
(I = 100%), calcd. 382 for C22H45N3P (MÀPF6)+; observed m/z 339
(I = 20%), calcd. 339 for C19H38N3P MÀ43)+.
1
1
25 °C): d À40.94 (broad s, JPtP 3340), À142.21 ppm (heptet, JPF
707 Hz, PF6À). Anal. Calc. for C36H74Cl2F12N6P4Pt (1208): C, 35.75;
H, 6.17; N, 9.65. Found: C, 35.76; H, 6.27; N, 9.52%. Electrospray
MS (CH3CN/MeOH): observed m/z 1063 (I = 100%), calcd. 1063 for
2.2.5. Octadecyl-PTA iodide, (PTAC18H37)I
C
36H74Cl2F6N6P3Pt (MÀPF6)+; observed m/z 917 (I = 30%), calcd.
Through the same procedure described for (PTAC16H33)I, using
0.20 g (1.29 mmol) of PTA and C18H37I (0.98 g, 2.57 mmol), (PTA-
C18H37)I was obtained as a white solid (0.57 g yield 83%). 1H
NMR (300 MHz, d6-DMSO, 25 °C): d 0.92 (t, 3H, CH3), 1.24 (s,
30H, CCH2C), 1.62 (m, 2H, CH2CH2N+), 2.80 (m, 2H, CH2CH2N+),
3.90 (m, 4H, PCH2N), 4.33 (d, 2H, PCH2N+), 4.35 (d, 1H, NCH2N),
917 for C36H74Cl2N6P2Pt (MÀ2PF6)+; observed m/z 751(I = 18%),
calcd. 750 for C24H50Cl2N6P2Pt [PtCl2(PTAC12H25)(PTA)]+; observed
m/z 326 (I = 57%), calcd. 326 for C18H37N3P (PTAC12H25)+.
2.3.2. [PtCl2(PTAC16H33)2](PF6)2 (2)
2
2
4.53 (d, JHH 14, 1H, NCH2N), 4.77 (d, JHH 11, 2H, NCH2N+), 4.95
1H NMR (300 MHz, d6-DMSO, 25 °C): d 0.88 (t, 3H, CH3), 1.27
(bs, 26H, CCH2C), 1.70 (bm, 2H, CH2CH2N+), 2.85 (bm, 2H,
CH2CH2N+), 4.2–5.2 (broad signals, 12H, PTA cage). 13C{1H} NMR
(100.58 MHz, d6-DMSO, 25 °C): d 14.4 (s, CH3), 19.7 (s, CH2), 22.6
(s, CH2), 26.3 (s, CH2), 27.9 (s, CH2), 29.2 (s, CH2), 29.5 (8 CH2),
31.8 (s, CH2), 48.4 (bm, PCH2N + PCH2N+), 61.5 (s, CH2N+), 69.1 (s,
NCH2N), 78.9 (s, NCH2N+). 31P{1H} NMR (121.50 MHz, d6-DMSO,
(d, JHH 11, 2H, NCH2N+). 13C{1H} NMR (100.58 MHz, d6-DMSO,
2
25 °C): d 13.9 (s, CH3), 18.9 (s, CH2), 22.1 (s, CH2), 26.1 (s, CH2),
28.4 (s, CH2), 28.7 (s, CH2), 28.7 (s, CH2), 29.0 (9 CH2), 31.3 (s,
1
1
CH2), 45.4 (d, JPC 20.6, PCH2N), 51.7 (d, JPC 32.2, PCH2N+), 61.4
(s, RCH2N+), 69.3 (s, NCH2N), 78.6 (s, 2C, NCH2N+). 31P{1H} NMR
(121.50 MHz, d6-DMSO, 25 °C): d À84.26 ppm (s). Anal. Calc. for
1
1
C
24H49IN3P (537): C, 53.60; H, 9.19; N, 7.82. Found: C, 53.56; H,
25 °C):
d
À41.25 (bs, JPtP 3400), À142.21 ppm (heptet, JPF
707 Hz, PF6À). Anal. Calc. for C44H90Cl2F12N6P4Pt (1320): C, 39.98;
H, 6.87; N, 6.36. Found: C, 39.85; H, 6.92; N, 6.52%. Electrospray
MS (CH3CN/MeOH): observed m/z 1175 (I = 100%), calcd. 1175 for
9.35; N, 7.77%. Electrospray MS (in H2O): observed m/z 410, calcd.
410 for C24H49N3P MÀI)+.
C
44H90Cl2F6N6P3Pt (MÀPF6)+; observed m/z 1029 (I = 15%), calcd.
2.2.6. Octadecyl-PTA hexafluorophosphate, (PTAC18H37)PF6
1029 for C44H90Cl2N6P2Pt (MÀ2PF6)+; observed m/z 807 (I = 30%),
calcd. 805 for C28H57Cl2N6P2Pt [PtCl2(PTAC16H33)(PTA)]+; observed
m/z 382 (I = 87%), calcd. 382 for C22H45N3P (PTAC16H33)+.
The phosphine (PTAC18H37)I (0.28 g, 0.52 mmol) was treated
with KPF6 (0.095 g, 0.52 mmol) in the same conditions as described
above for preparing (PTAC12H25)PF6. The reaction gave
(PTAC18H37)PF6 (0.2 g, 0.36 mmol, yield 69%). 1H NMR (300 MHz,
d6-DMSO, 25 °C): d 0.83 (bs, 3H, CH3), 1.20 (s, 30H, CCH2C), 1.63
(bm, 2H, CH2CH2N+), 2.78 (bm, 2H, CH2CH2N+), 3.88 (m, 4H, PCH2N),
4.30 (bs, 2H, PCH2N+), 4.30 (d, 2JHH 14, 1H, NCH2N), 4.52 (d, 2JHH 14,
2.3.3. [PtCl2(PTAC18H37)2](PF6)2 (3)
1H NMR (300 MHz, d6-DMSO, 25 °C): d 0.87 (bs, 3H, CH3), 1.25
(bs, 30H, CCH2C), 2.30 (bm, 2H, CH2CH2N+), 2.82 (bm, 2H,
CH2CH2N+), 4.2–5.2 (broad signals, 12H, PTA cage). 13C{1H} NMR
(100.58 MHz, d6-DMSO, 25 °C): d 13.9 (s, CH3), 19.3 (s, CH2), 22.1
(s, CH2), 25.9 (s, CH2), 28.4 (s, CH2), 29.0 (bs, 8 CH2), 31.3 (s,
CH2), 48.3 (m, PCH2N + PCH2N+), 61.1 (s, RCH2N+), 68.7 (s, NCH2N),
78.5 (s, NCH2N+). 31P{1H} NMR (121.50 MHz, d6-DMSO, 25 °C): d
2
2
1H, NCH2N), 4.77 (d, JHH 10, 2H, NCH2N+), 4.97 (d, JHH 10, 2H,
NCH2N+). 13C{1H} NMR (100.58 MHz, d6-DMSO, 25 °C): d 14.0 (s,
CH3), 18.9 (s, CH2), 22.1 (s, CH2), 26.1 (s, CH2), 28.5 (s, CH2), 28.7 (s,
1
CH2), 29.0 (10 CH2), 31.3 (s, CH2), 45.4 (d, JPC 20.1, PCH2N), 51.7
(d, JPC 31.4, PCH2N+), 61.5 (s, RCH2N+), 69.3 (s, NCH2N), 78.6 (s,
1
NCH2N+). 31P{1H} NMR (121.50 MHz, d6-DMSO, 25 °C): d À84.24
1
1
À40.32(bs JPtP 3642), À142.21 ppm (heptet, JPF 707 Hz, PF6À).
Anal. Calc. for C48H98Cl2F12N6P4Pt (1376): C, 41.84; H, 7.17; N,
6.10. Found: C, 41.75; H, 7.35; N, 6.10%.
1
(s), d À142.21 ppm (heptet, JPF 707 Hz, PF6À). Anal. Calc. for
C
24H49F6N3P2 (555): C, 51.86; H, 8.89; N, 7.56. Found: C, 51.92; H,
9.10; N, 7.48%. Electrospray MS (in H2O): observed m/z 410
(I = 100%), calcd. 410 for C24H49N3P (MÀPF6)+, observed m/z 367
(I = 10%), calcd. 367 for C21H42N3P MÀ43)+.
Electrospray MS (CH3CN/MeOH): observed m/z 1231 (I = 100%),
calcd. 1231 for C48H98Cl2F6N6P3Pt (MÀPF6)+; observed m/z 1085
(I = 30%), calcd. 1085 for C48H98Cl2N6P2Pt (MÀ2PF6)+; observed
m/z 410 (I = 40%), calcd. 410 for C24H49N3P (PTAC18H37)+.
2.3. Coordination of (PTACnH2n+1)PF6 (n = 12, 16, 18) and PTAMePF6 to
Pt(II)
2.3.4. [PtCl2(PTAMe)2](PF6)2 (4)
1H NMR (300 MHz, d6-DMSO, 25 °C): d 2.83 (s, 3H, CH3), 4.40
(m, 5H, cage), 4.82 (bs, 2H, cage), 5.07 (m, 5H, cage). 13C{1H}
NMR (100.58 MHz, d6-DMSO, 25 °C): d 47.8 (br, PCH2N), 48.6 (s,
MeN), 53.9 (br, PCH2N+), 68.3 (s, NCH2N), 78.4 (s, NCH2N+).
A solution of [PtCl2(1,5-COD)] (0.055 g, 0.15 mmol) in 5 mL of
CH2Cl2 was added dropwise under stirring to a suspension of
(PTAC12H25)PF6 (0.14 g, 0.30 mmol) in 25 mL of CH2Cl2. All the sus-
pended solids dissolved and a clear solution was obtained. After
1 h stirring, the solution was taken to dryness under vacuum,
and the solid residue was washed with ether and dried in vacuum
over P2O5. The product was identified as [PtCl2(PTAC12H25)2](PF6)2
(1) (0.15 g, 0.12 mmol, yield 83%).
31P{1H} NMR (121.50 MHz, d6-DMSO, 25 °C): d À43.51(s, JPtP
1
1
3384), À142.20 ppm (heptet, JPF 707 Hz, PF6À). Anal. Calc. for
C14H30Cl2F12N6P4Pt (900): C, 18.66; H, 3.36; N, 9.33. Found: C,
18.55; H, 3.45; N, 9.42%.
Electrospray MS (CH3CN/MeOH): observed m/z 755 (I = 100%),
In the same way, complexes 2 (yield 83.5%), 3 (yield 62%) and 4
(yield 83%) were obtained from the reaction of [PtCl2(1,5-COD)]
calcd. 755 for C14H30Cl2F6N6P3Pt (MÀPF6)+; observed m/z 305
(I = 60%), calcd. 305 for C14H30Cl2N6P2Pt (MÀ2PF6)2+
.

P. Bergamini et al. / Inorganica Chimica Acta 391 (2012) 162–170
165
2.4. Coordination of (PTACnH2n+1)I, (n = 12, 16, 18) to Ru(II)
2.4.2. Method 2
An orange mixture of [CpRuCl(PPh3)2] (100 mg, 0.140 mmol),
solid (PTACnH2n+1)PF6 (0.15 mmol) and acetone (15 mL) was re-
fluxed for 1 h under nitrogen. The volume was then reduced to
ca. 3 mL. By addition of Et2O (10 mL) [CpRuCl(PPh3)(P-
TACnH2n+1)]PF6 precipitated as a pale yellow solid which was col-
lected on a frit and washed with Et2O (3 Â 2 mL). 5-PF6, yield
70%; 6-PF6, yield 72%; 7-PF6, yield 80%.
2.4.1. Method 1
A large excess (15 equiv.) of C12H25I was added to a solution of
[CpRuCl(PPh3)(PTA)] (0.10 g, 0.13 mmol) in acetone/dichlorometh-
ane 1:1 (12 mL). The orange mixture turned yellow in the course of
18 h with stirring at room temperature. The slow addition of
diethyl ether (20 mL) induced the precipitation of a yellow solid,
which was filtered and washed three times with ether until com-
plete elimination of the excess of the alkyl iodide. Complex
[CpRuCl(PPh3)(PTAC12H25)]I (5) was obtained as a pure and stable
yellow solid (0.024 g, 0.026 mmol, 20% yield). In the filtrate the
presence of the more soluble orange isomer [CpRuI(PPh3)(P-
TAC12H25)]X, (X = Cl, I) was observed by NMR spectra.
In the same way it was possible to obtain 6 and 7 with 35% and
40% yield, respectively.
2.4.2.1. [CpRuCl(PPh3)(PTAC12H25)]PF6 (5-PF6). 1H NMR (300 MHz,
d6-DMSO, 25 °C): d 0.86 (br t, 3H, CH3), 1.28 (s, 18H, CCH2C), 1.53
(m, 2H, CH2CH2N+), 2.82 (m, 2H, CH2CH2N+), 3.70–4.35 (m, 6H, cage
PTA), 4.57 (s, 5H, Cp), 4.58–4.90 (m, 6H, cage PTA), 7.4–7.6 (br s,
15H, Ph). 13C{1H} NMR (100.58 MHz, d6-DMSO, 25 °C): d 14.0 (s,
CH3), 19.2 (s, CH2), 22.1 (s, CH2), 26.0 (s, CH2), 29.0 (m, CH2, 6C),
31.3 (s, CH2), 49.9 (m, PCH2N), 51.8 (m, PCH2N+), 60.9 (s, RCH2N+),
68.7 (s, NCH2N), 78.1 (s, NCH2N+), 79.2 (s, Cp), 128.2–137.8 (Ph).
2
31P{1H} NMR (121.50 MHz, d6-DMSO, 25 °C): d 47.46 (d, JPP
2
43.74, PPh3), À13.78 ppm (d, JPP 43.74 Hz, (PTAR)+). Anal. Calc.
2.4.1.1. [CpRuCl(PPh3)(PTAC12H25)]I (5). 1H NMR (300 MHz, d6-
DMSO, 25 °C): d 0.86 (br t, 3H, CH3), 1.28 (s, 18H, CCH2C), 1.53
(m, 2H, CH2CH2N+), 2.82 (m, 2H, CH2CH2N+), 3.70–4.35 (m, 6H,
PTA), 4.57 (s, 5H, Cp), 4.58–4.90 (m, 6H, cage), 7.4–7.6 (br s, 15H,
Ph). 13C{1H} NMR (100.58 MHz, d6-DMSO, 25 °C): d 14.0 (s, CH3),
19.2 (s, CH2), 22.1 (s, CH2), 26.0 (s, CH2), 29.0 (m, CH2, 6C), 31.3
(s, CH2), 49.9 (m, PCH2N, 2C), 51.8 (m, PCH2N+), 60.9 (s, RCH2N+),
68.7 (s, NCH2N), 78.1 (s, NCH2N+), 79.2 (s, Cp), 128.2–137.8 (Ph).
for C41H57ClF6N3P3Ru (935): C, 52.62; H, 6.14; N, 4.49. Found: C,
52.01; H, 5.98; N, 4.36%. Electrospray MS (H2O): observed m/z
790 calcd. 790 for C41H57ClN3P2Ru (MÀPF6)+.
2.4.2.2. [CpRuCl(PPh3)(PTAC16H33)]PF6 (6-PF6). 1H NMR (300 MHz,
d6-DMSO, 25 °C): d 0.82 (bt, 3H, CH3), 1.23 (bs, 30H, CCH2C), 1.50
(bs, 2H, CH2CH2N+), 2.80 (m, 2H, CH2CH2N+), 3.73–4.20 (m, 6H, cage
PTA), 4.54 (s, 5H, Cp), 4.58–4.97 (m, 6H), 7.39–7.53 (bs, 15H, Ph).
13C{1H} NMR (100.58 MHz, d6-DMSO, 25 °C): d 14.0 (s, CH3), 19.2
(s, CH2), 22.1 (s, CH2), 26.0 (s, CH2), 28.4 (s, CH2), 29.0 (m, 9H,
31P{1H} NMR (121.50 MHz, d6-DMSO, 25 °C): d 47.46 (d, JPP
2
43.74, PPh3), À13.78 ppm (d, JPP 43.74 Hz, (PTAR)+). Anal. Calc.
2
for C41H57ClIN3P2Ru (917): C, 53.66; H, 6.27; N, 4.58. Found: C,
53.58; H, 6.32; N, 4.49%.
1
1
CH2), 31.7 (s, CH2), 49.8 (d, JPC 14.5, PCH2N), 51.8 (d, JPC14.5,
PCH2N+), 60.9 (s, RCH2N+), 68.7 (s, NCH2N), 78.3 (s, NCH2N+), 79.1
(s, Cp), 128.1–137.8 (Ph). 31P{1H} NMR (121.50 MHz, d6-DMSO,
Electrospray MS (H2O): observed m/z 790 (I = 100%), calcd. 790
for C41H57ClN3P2Ru (MÀI)+; observed m/z 326 (I = 50%), calcd. 326
for C18H37N3P (PTAC12H25)+.
2
2
25 °C): d 47.71 (d, JPP 42.89, PPh3), À13.46 ppm (d, JPP 42.89 Hz,
(PTAR)+). Anal. Calc. for C45H65ClF6N3P3Ru (991): C, 54.49; H,
6.61; N, 4.24. Found: C, 54.83; H, 6.98; N, 4.42%. Electrospray MS
(H2O): observed m/z 846, calcd. 846 for C45H65ClN3P2Ru (MÀPF6)+.
2.4.1.2. [CpRuCl(PPh3)(PTAC16H33)]I (6). 1H NMR (300 MHz, d6-
DMSO, 25 °C): d 0.82 (br t, 3H, CH3), 1.23 (br s, 30H, CCH2C), 1.50
(br s, 2H, CH2CH2N+), 2.80 (m, 2H, CH2CH2N+), 3.73–4.20 (m, 6H,
PTA), 4.54 (s, 5H, Cp), 4.58–4.97 (m, 6H), 7.39–7.53 (br s, 15H,
Ph). 13C{1H} NMR (100.58 MHz, d6-DMSO, 25 °C): d 14.0 (s, CH3),
19.2 (s, CH2), 22.1 (s, CH2), 26.0 (s, CH2), 28.4 (s, CH2), 29.0 (m,
9H, CH2), 31.7 (s, CH2), 49.8 (d, JPC 14.5, PCH2N), 51.8 (d, JPC
14.5, PCH2N+), 60.9 (s, RCH2N+), 68.7 (s, NCH2N), 78.3 (s, NCH2N+),
79.1, (s, Cp), 128.1–137.8, (Ph). 31P{1H} NMR (121.50 MHz, d6-
2.4.2.3. [CpRuCl(PPh3)(PTAC18H37)]PF6 (7-PF6). 1H NMR (300 MHz,
d6-DMSO, 25 °C): d 0.83 (bt, 3H, CH3), 1.24 (bs, 30H, CCH2C), 1.51
(bs, 2H, CH2CH2N+), 2.80 (m, 2H, CH2CH2N+), 3.70–4.20 (m, 6H, cage
PTA), 4.55 (s, 5H, Cp), 4.58–4.90 (m, 6H, cage PTA), 7.35–7.60 (br s,
15H, Ph). 13C{1H} NMR (100.58 MHz, d6-DMSO, 25 °C): d 14.4 (s,
CH3), 19.6 (s, CH2), 22.5 (s, CH2), 26.5 (s, CH2), 28.9 (s, CH2), 29.5
1
1
1
1
(s, 11 CH2), 31.7 (s, CH2), 50.1 (d, JPC 10.6, PCH2N), 52.2 (d, JPC
16.1, PCH2N+), 61.4 (s, RCH2N+), 69.3 (s, NCH2N), 78.7 (s, NCH2N+),
79.6 (s, Cp), 128.6–138.2 (Ph). 31P{1H} NMR (121.50 MHz, d6-
2
2
DMSO, 25 °C): d 47.71 (d, JPP 42.89, PPh3), À13.46 ppm (d, JPP
42.89 Hz, (PTAR)+). Anal. Calc. for C45H65ClIN3P2Ru (973): C,
55.50; H, 6.73; N, 4.32. Found: C, 55.51; H, 6.88; N, 4.27%. Electro-
spray MS (H2O): observed m/z 846 (I = 100%), calcd. 846 for
2
2
DMSO, 25 °C): d 47.42 (d, JPP 43.74, PPh3), À13.71 ppm (d, JPP
43.74 Hz, (PTAR)+). Anal. Calc. for C47H69ClF6N3P3Ru (1019): C,
55.35; H, 6.82; N, 4.12. Found: C, 55.33; H, 6.98; N, 4.09%. Electro-
spray MS (in H2O): observed m/z 874, calcd. 873 for
C
45H65ClN3P2Ru (MÀI)+; observed m/z 382 (I = 30%), calcd. 382
for C22H45N3P (PTAC16H33)+.
C
47H69ClN3P2Ru (MÀPF6)+.
2.4.1.3. [CpRuCl(PPh3)(PTAC18H37)]I (7). 1H NMR (300 MHz, d6-
DMSO, 25 °C): d 0.83 (br t, 3H, CH3), 1.24 (br s, 30H, CCH2C), 1.51
(br s, 2H, CH2CH2N+), 2.80 (m, 2H, CH2CH2N+), 3.70–4.20 (m, 6H,
PTA), 4.55 (s, 5H, Cp), 4.58–4.90 (m, 6H, PTA), 7.35–7.60 (br s,
15H, Ph). 13C{1H} NMR (100.58 MHz, d6-DMSO, 25 °C): d 14.4 (s,
CH3), 19.6 (s, CH2), 22.5 (s, CH2), 26.5 (s, CH2), 28.9 (s, CH2), 29.5
(s, 11CH2), 31.7 (s, CH2), 50.1 (d, JPC 10.6, PCH2N), 52.2 (d, JPC
16.1, PCH2N+), 61.4 (s, RCH2N+), 69.3 (s, NCH2N), 78.7 (s, NCH2N+),
79.6 (s, Cp), 128.6–138.2 (Ph). 31P{1H} NMR (121.50 MHz, d6-
2.5. Growth inhibition assays
Cell growth inhibition assays were carried out using the cis-
platin-sensitive T2 human cell line, the cisplatin-resistant SKOV3
cell line and the human tumor cell line SW480. T2 is a cell hybrid
obtained by the fusion of the human lymphoblastoid line 174 (B
lymphocyte transformed by the Epstein–Barr virus) with the CEM
human cancer line (leukemia T), SKOV3 is derived from a human
ovarian tumor and SW480 is derived from a human colorectal ade-
nocarcinoma. The cells were seeded in triplicate in 96-well trays at
1
1
2
2
DMSO, 25 °C): d 47.42 (d, JPP 43.74 PPh3), À13.71 ppm (d, JPP
43.74 Hz, (PTAR)+). Anal. Calc. for C47H69ClIN3P2Ru (1001): C,
56.34; H, 6.95; N, 4.20. Found: C, 56.45; H, 7.12; N, 4.15%. Electro-
spray MS (H2O): observed m/z 874 (I = 100%), calcd. 874 for
a density of 50 Â 103 in 50
l
l of AIM-V medium for T2, 25 Â 103 in
50 ll of AIM-V medium for SKOV3 and SW480. Stock solutions
C
47H69ClN3P2Ru (MÀI)+; observed m/z 410 (I = 25%), calcd. 410
(10 mM) of each compound were made in DMSO and diluted in
for C24H49N3P (PTAC18H37)+.
AIM-V medium to give final concentrations of 2, 10 and 50 M. Cis-
l

166
P. Bergamini et al. / Inorganica Chimica Acta 391 (2012) 162–170
platin was employed as a control for the cisplatin-sensitive T2 cell
line and for the cisplatin-resistant SKOV3. Untreated cells were
placed in every plate as a negative control. The cells were exposed
16, 18) in MeOH gives (PTACnH2n+1)PF6 as immediate precipitates
with a high purity grade.
The molecular formula of compounds (PTACnH2n+1)PF6 (n = 12,
16, 18) were determined from elemental analysis and ESI-MS.
The ESI-MS spectra are very clean and in each case show the M+
to the compounds for 72 h and then 25
ozol-2-yl)2,5-diphenyltetrazolium bromide solution (MTT)
(12 mM) were added. After 2 h of incubation, 100 l of lysing buf-
ll of a 3-(4,5-dimethylthi-
À
l
peak formed by the loss of one counter-ion PF6 as 100% and a
fer (50% DMF + 20% SDS, pH 4.7) were added to convert the MTT
solution into a brown colored formazane. After additional 18 h
the solution absorbance, proportional to the number of live cells,
was measured by spectrophotometer and converted into % of
growth inhibition [26].
small peak (ca 10%) corresponding to
CH3CH2CH2).
a loss of 43 (43 =
In each case the subsequent m/m fragmentation of M+ increases
the intensity of (MÀ43)+ and generates two new species corre-
sponding at MÀ29)+ (29 = CH3CH2) and MÀ114)+ [114 =
CH3(CH2)7], thus confirming the occurrence of C–C fragmentations
in ESI-MS conditions in the uncoordinated ligands.
The NMR characterization is similar to the one previously
3. Results and discussion
described for (PTAC12H25)I, except for the signals of PF6 in 31P of
À
1
(PTACnH2n+1)PF6 (n = 12, 16, 18) (À142 ppm, heptet, JPF = 707 Hz,
3.1. Synthesis and characterization of long-tail PTA derivatives
PF6À).
N-alkyl PTA derivatives (PTACnH2n+1)I (n = 12, 16, 18) have been
prepared in high yields by reacting 1,3,5-triaza-7-phosphaada-
mantane (PTA) with the appropriate alkyl iodide in acetone de-
gassed with argon. Each product precipitated from solution as an
air-stable solid that needed little purification (see Scheme 1).
The phosphines (PTACnH2n+1)I (n = 12, 16, 18) were character-
ized by 1H, 13C, and 31P NMR in d6-DMSO and the data are included
in the Section 2.
3.2. Coordination of (PTACnH2n+1)PF6 (n = 12, 16, 18) to platinum and
ruthenium
The coordination of PTA itself to platinum and other d8 ions has
been explored and the suitability of PTA–Pt complexes for thera-
peutical activity has been suggested and encouraged by the results
of in vitro tests reported by us [27–29] and others [30]. Neverthe-
less the coordination chemistry of N-alkylated PTA, included the
well known (PTAMe)+, to Pt group metals, and the biological activ-
ity of the complexes are nearly unexplored topics. The presence of
positive charges on the complexes can enhance the chemical affin-
ity of these species towards negatively charged DNA, the molecular
target of platinum-based drugs [17]. For this reason we prepared
some Pt complexes of the above described long-tail phosphines,
with the non-coordinating anion PF6À. The use of the iodide forms
(PTACnH2n+1)I would produce Pt iodide complexes, which are gen-
erally considered not suitable for the interaction with DNA, be-
cause iodide on platinum does not undergo substitution.
The presence of the long aliphatic tail has been designed with
the aim of favoring the self-aggregation of the complexes into
localized hydrophobic environments in polar media [31,32]. This
property could improve platinum drugs formulation and distribu-
tion and promote their targeting, exploiting the crossing of the
lipophilic cell membranes and the uptake of Pt via endocytosis.
Complexes [PtCl2(PTAC12H25)2](PF6)2 (1), [PtCl2(PTAC16H33)2]
(PF6)2 (2) and [PtCl2(PTAC18H37)2](PF6)2 (3) were prepared and
characterized (see Scheme 2).
31P NMR is diagnostic of the conversion of PTA into N-alkylated
products, in fact the peak of PTA at À100 ppm is shifted downfield
by about 20 ppm in (PTACnH2n+1)I derivatives (d À84.35, À84.27
and À84.26 ppm for n = 12, 16, 18, respectively).
The 1H NMR of (PTACnH2n+1)I shows a typical pattern, such as
herein described for (PTAC12H25)I: the alkyl tail signals are found
as a triplet of
x-CH3 at 0.84 ppm, a broad signal at 1.27 ppm
including all chain methylene groups, except the CH2
a
(2.81 ppm) and b (1.65 ppm) to N+. The cage signals pattern ap-
pears more complicated than that of free PTA, as observed with
other N-substituted derivatives of PTA [17], because N-alkylation
causes a reduction of the molecular symmetry of the PTA moieties.
The cage pattern in (PTAC12H25)I appears to consist of four signals,
assigned on the basis of the reported spectrum of (PTAMe)(O-
SO2CF3) [18]. The multiplet at 3.90 ppm is due to the four PCH2N
protons and the broad singlet at 4.35 ppm to PCH2N+. Two doublets
at 4.32 ppm and 4.53 ppm belong to NCH2N diastereotopic protons,
2
reciprocally coupled with JHH = 14 Hz and two doublets at 4.82
and 4.97 ppm are due to NCH2N+, also diastereotopic protons,
2
reciprocally coupled with JHH = 11 Hz.
The 13C NMR of (PTAC12H25)I shows the alkyl chain signals due
The Pt(II) complexes 1–3 were obtained by reaction of
[PtCl2(1,5-COD] with 2 equiv of phosphine. The products have been
isolated as white solids and characterized by elemental analysis,
ESI MS and NMR.
The ESI MS showed in each case the presence of the cation mass
peak, produced by the loss of a counter-ion PF6À and presenting the
expected Pt isotopic pattern (Fig. 1).
to
x CH3 at 13.99 ppm and CH2 at 18.98, 22.11 and 26.10 ppm, a
broad intense signal at 29.03 ppm due to internal CH2 groups
and a signal at 31.30 ppm; the PTA cage signals are found at
45.44 ppm as a doublet for PCH2N, coupled with P (1JPC = 20.11 Hz),
a doublet for PCH2N+ at 51.68 ppm (1JPC = 32.20 Hz,) and three
singlets at 61.42 (CH2N+), 69.29 (NCH2N) and 78.56 (NCH2N+)
ppm. The spectroscopic characterization of (PTAC16H33)I and (PTA-
C
18H37)I show closely related patterns (see Section 2).
In order to exclude the involvement of iodide in the planned
(n-1)
coordination reactions of (PTACnH2n+1)I (n = 12, 16, 18), this has
been exchanged with non-coordinating anions. In particular, the
addition of KPF6 in water to solutions of (PTACnH2n+1)I (n = 12,
N
N
-
2PF6
-
Cl
Cl
PF6
P
P
N
N
N
N
[PtCl2(1,5-COD)] + 2
Pt
N
(n-1)
-
COD
I-
PF6
P
N
N
N
-
CnH(2n+1)
I
PF6
N
N
N
N
N
N
N
N
n = 12,16,18
(n-1)
1 ( n = 12 )
2 ( n = 16 )
3 ( n = 18 )
(n-1)
P
P
P
(n-1)
Scheme 1. Synthesis of long tail PTA derivatives (PTACnH2n+1)X, (X = IÀ, PF6
n = 12, 16, 18).
;
À
Scheme 2. Synthesis of platinum complexes 1–3.

P. Bergamini et al. / Inorganica Chimica Acta 391 (2012) 162–170
167
1175.00
100
80
60
40
20
1203.13
1121.00
1212.00
1106.93
1161.00
1191.07
1147.33
1094.53
1127.07
0
100
1175.54
80
60
40
20
0
1173.53
1177.54
1179.54
1181.54
1180
1171.53
1100
1120
1140
1160
m/z
1200
1220
Fig. 1. Detail of ESI MS of 2, cation mass peak (top) and simulated (bottom).
All the ESI-MS spectra of the Pt complexes 1, 2, 3 and 4 show the
(M–PF6)+ peak as the major species (I = 100%), and other minor Pt-
containing species (characteristic isotopic pattern) like MÀ2PF6)+
(I = 20–30%) and [PtCl2(PTACnH2n+1)(PTA)]+ (n = 12, 16, 18)
(I = 15–30%).
CnH(2n+1)
I
Ru
Ru
N
N
N
N
P
Ph3P
n = 12,16,18
Ph3P
P
I-
Cl
Cl
The C–C fragmentations in ESI-MS spectra of the Pt complexes
do not appear relevant.
N
N
(n-1)
5 ( n = 12)
6 ( n = 16)
7 ( n = 18)
The 31P NMR is diagnostic of the phosphines coordination, in
fact the signals of the complexes arise between at À40 Ä À41 ppm,
showing Pt satellites. The Pt–P coupling constants is ca. 3400 Hz,
Scheme 3. Alkylation of Ru-coordinated PTA.
undoubtfully indicating
a cis geometry for the Pt complex
1
Following the reactions by 31P NMR, the formation of side prod-
ucts can be observed, deriving from both the Cl–I exchange on Ru
[33,34]. The same size for JPtP was obtained for the Pt–chloride
complex of (PTAMe)PF6, here reported for the first time, obtained
in the same way described as for the complexes of long tail phos-
phines. In fact [PtCl2(PTAMe)2](PF6)2 shows a signal at À43.51 ppm
and the C –Cb bond breaking, as previously described for (PTAn-
a
Bu)+, (PTAnPr)+ and (PTAEt)+ [35], giving [CpRuX(PPh3)(PTAMe)]X
(X = Cl, I), identified by comparison of its data with those reported
for a genuine sample [18].
1
with JPtP = 3384 Hz. Surprisingly, the 31P NMR spectra of Pt com-
plexes of phosphines (PTACnH2n+1)PF6 show invariably broad sig-
nals with broad satellites, while the PF6 signal is always sharp.
We have studied the dependence of the signal-broadness on many
possible factors: temperature (up to 160 °C in DMSO, down to
À60 °C in CH2Cl2), concentration, solvent, presence of added salt,
phosphine counter-anion, water content, but none seems to clearly
affect the line broadening in a regular way. Because the signals of
[PtX2(PTAMe)2](PF6)2 (X = Cl, I) are sharp in every tested condition,
we believe that the broadening phenomena can be ascribed to a lo-
cal dis-homogeneity due to the presence of long aliphatic chains.
The same lack of resolution is observed in the 1H NMR spectra of
Pt complexes, showing reproducible series of overlapped broad
signals, which do not allow detailed signals analysis.
To minimize the presence of impurities in the final products, it
is crucial to modulate the precipitation conditions. By controlled
addition of Et2O to the reaction mixture, it is possible to isolate
pure Ru–Cl, which is the first yellow precipitate, being this less sol-
uble than the orange Ru–I complex in CH2Cl2. Moreover, low tem-
perature and reduction of reaction time by using a large excess of
alkyliodide are factors favoring the recover of pure Ru–Cl com-
plexes 5–7. The accurate elimination of acidic impurities (e.g., in
CHCl3) allows avoiding the alkyl C –Cb bond breaking process pro-
a
ducing Ru–(PTAMe) side products. Despite these precautions, the
yields of pure products are low (20–40%), and the exchange io-
dide–chloride may be difficult to completely eliminate.
Looking for a more convenient synthetic route to complexes 5–
7, we found that they can be prepared in higher yield (70–80%) by
direct reaction between [CpRuCl(PPh3)2] and (PTACnH2n+1)PF6 in
Scheme 4.
The 13C NMR spectra of Pt complexes show the signals of alkyl
chain carbons, slightly shifted with respect with the relative sig-
nals in the free ligands, while all the P-bonded carbons (d 45.47,
1JPC = 19.81 Hz and d 51.71, JPC = 31.3 Hz in (PTAC16H33)PF6), coa-
1
lesce into a single broad signal at d 48.44.
The proposed structures for 5- to 7-PF6 is consistent with the
spectroscopic data. In the 31P NMR spectra the signals of the re-
agents [CpRuCl(PPh3)2] (s, 40.52 ppm) and (PTACnH2n+1)PF6 (s, ca.
À81 ppm) are gradually replaced by an AM spin system at ca.
47 ppm and at ca. À14 ppm, respectively, according to the presence
of Ru-coordinated PPh3 ligand and alkyl-PTA, reciprocally coupled
Many attempts to prepare the above described alkyl-PTA Pt
complexes by alkylation with alkyl iodide of Pt-coordinated PTA
in cis-[PtCl2(PTA)2] gave mixtures of products containing mainly
Pt-iodide species (1JPtP ca. 2400 Hz).
On the contrary, this synthetic route was found to work for pre-
paring the ruthenium (II) complexes [CpRuCl(PPh3)(PTACnH2n+1)]I,
(5–7, Scheme 3) obtained by treating the known parent complex
2
with JPP = 43.74 Hz.
In the 1H NMR spectra, the signals of the aliphatic chains do not
undergo a significant shift with respect to the free ligand, while for
the cage protons, a broadening and a partial signals overlap is ob-
[CpRuCl(PPh3)(PTA)] with the commercial iodides CnH2n+1
(n = 12, 16, 18) in acetone/dichloromethane (1:1).
I

168
P. Bergamini et al. / Inorganica Chimica Acta 391 (2012) 162–170
-
Table 1
PF6
N
Estimated IC50 (
SKOV3.
lM) on cisplatin-sensitive cell line T2 and on cisplatin-resistant
N
N
(n-1)
P
Ru
Ru
Complex
IC50 T2
IC50 SKOV3
N
N
PPh3
-
Ph3P
Ph3P
P
n = 12,16,18
PF6
Cl
Cl
1
2
3
4
0.53 0.12
0.62 0.13
0.63 0.14
1.54 0.25
ca. 1
51.05 1.52
49.05 2.32
>50
>50
>50
N
(n-1)
5-PF6 ( n = 12)
6-PF6 ( n = 16)
7-PF6 ( n = 18)
Cisplatin
Scheme 4. Synthesis of ruthenium complexes 5–PF6, 6–PF6, 7–PF6.
the cisplatin sensitive T2 cell line and the cisplatin resistant SKOV3
cell line. The results are reported in Table 1. The growth inhibition
activity of the complexes on T2 was found to be high even at low
doses. Indeed, the estimated IC50 for each complex was in the
served. The characteristic singlet of the cyclopentadienyl ligand is
observed between 4.54 and 4.57 ppm.
The 13C NMR spectra of Ru complexes 5–7 are in agreement
with the proposed composition; the singlet of the cyclopentadienyl
ligand is observed in the range 79.15–79.60 ppm and the P-bonded
carbons undergo a small shift with respect to the relative signals in
the free ligands.
range of 1–0.1
contrary, the antiproliferative activity of the Pt complexes on
SKOV3 was low and the IC50 is >50 M (see Table 1).
lM except for 4, which was around 2 lM. On the
l
As previously noticed for other Pt complexes, the antiprolifera-
tive activity on cisplatin-sensitive cell line and the lack of antipro-
The ESI MS of the isolated ruthenium complexes 5, 6 and 7,
showed in each case the presence of the cationic mass peak
MÀI)+, produced by the loss of the counter-ion IÀ as 100% and less
intense peaks (all less than 5% intensity) corresponding to loss of a
chloride MÀIÀCl)+, of PPh3 MÀIÀPPh3)+, of chloride and PPh3
liferative activity on cisplatin-resistant cell line suggest
a
mechanism similar to cisplatin, whose biomolecular target is
known to be the DNA.
MÀIÀClÀPPh3)+, the all of them presenting the Ru isotopic cluster
Moreover, it can be noticed that the less active complex of the
tested group is [PtCl2(PTAMe)2](PF6)2, (4), the only one bearing a
short alkylic chain, while the observed IC50 values are not signifi-
cantly different for [PtCl2(PTAC12H25)2](PF6)2, (1), [PtCl2
(PTAC16H33)2](PF6)2, (2) and [PtCl2(PTAC18H37)2](PF6)2 (3), all bear-
ing long-chain ligands, not so different in length and hindrance and
consequently in solubility and lipophilicity.
+
(Fig 2). The fragment corresponding to the ligand (PTACnH2n+1
also observed in each case.
)
is
3.3. Cell growth inhibition tests
3.3.1. Platinum complexes
The difference in activity between the (PTAMe)+ complex and
long chain phosphines complexes can be explained in two ways:
The antiproliferative activity of the complexes 1–4 was mea-
sured at 50, 10, 2, 1 and 0.1 M on two human tumoral cell lines:
l
874.00
876.00
100
90
80
70
60
50
40
30
20
10
873.00
872.07
871.07
877.00
878.00
868.07
879.00
838.07
833.27
902.07
906.20 918.93
842.20
860.93
881.00
887.93
849.27
0
100
874.37
90
80
70
60
50
40
30
20
10
0
876.37
873.37
872.37
871.37
877.38
878.37
868.37
870
879.38
880.38
830
840
850
860
880
m/z
890
900
910
Fig. 2. Detail of the ESI MS of 7, cation mass peak (top) and simulated (bottom).

P. Bergamini et al. / Inorganica Chimica Acta 391 (2012) 162–170
169
Table 2
Table 5
Estimated IC50 (
l
M): comparison between Pt complexes and free ligands on T2.
Estimated IC50 (lM): comparison between Ru complexes and free ligands on SKOV3.
Complex
IC50 T2
Ligand
IC50 T2
Complex
IC50 SKOV3
Ligand
IC50 SKOV3
1
2
3
4
0.53 0.12
0.62 0.13
0.63 0.14
1.54 0.25
ca. 1
(PTAC12H25)PF6
(PTAC16H33)PF6
(PTAC18H37)PF6
(PTAMe)PF6
5.21 0.75
0.84 0.03
1.57 0.22
6.59 2.19
[CpRuCl(PPh3)(PTA)]
[CpRu(PPh3)(PTAMe)Cl]I
5
6
>100
35.35 1.72
9.07 0.22
9.64 0.19
9.53 1.27
>50
(PTAMe)I
>50
(PTAC12H25)PF6
(PTAC16H33)PF6
(PTAC18H37)PF6
>50
36.09 0.42
42.04 1.92
Cisplatin
7
Cisplatin
Table 3
Estimated IC50 (
lM): comparison between Pt complexes and free ligands on SKOV3.
were re-tested on SKOV3 and SW480 and the results were taken
after 24 h, a time estimated to be sufficient for the detection of
cytotoxicity .
Complex
IC50 SKOV3
Ligand
IC50 SKOV3
1
2
3
4
51.05 1.52
49.05 2.32
>50
(PTAC12H25)PF6
(PTAC16H33)PF6
(PTAC18H37)PF6
(PTAMe)PF6
>50
We found that the ligands did not show any inhibitory effect on
cell growth after 24 h treatment. Nevertheless, we cannot exclude
that the antiproliferative activity observed for Ru complexes could
be due to the coordinated ligands. In fact it has been reported that
in many cases Ru complexes do not express any activity on pri-
mary tumor cell lines, or they do only at very high doses, even in
cases where they have been found active on metastatic tumors
[36,37].
For this reason the results here presented are of interest for the
purpose of finding Ru complexes active on primary tumors, in
addition to hopefully maintaining the anti-metastatic activity ex-
pressed by RAPTA complexes at present under trials.
36.09 0.42
42.04 1.92
>50
>50
Cisplatin
50–10
(i) the long chain phosphines themselves have an antiproliferative
activity; (ii) the lipophilicity of long-chain coordinated ligands
could favor the cellular uptake of platinum, which therefore
reaches its target in a larger amount and consequently expresses
higher activity.
To understand this point, we tested uncoordinated (PTAMe)PF6
and (PTACnH2n+1)PF6 (n = 12, 16, 18) both on T2 and SKOV3 and
compared the obtained values with those of the relative complexes
(Table 2 and 3). We found that the ligands present an activity pro-
file similar to the one of complexes, having low IC50 on T2 and
high IC50 on SKOV3, although in both cases lower than the corre-
sponding Pt complexes.
4. Conclusions
The new long chain PTA derivatives, (PTACnH2n+1)+, (n = 12, 16,
18), have been prepared and characterized both as IÀ and PF6
À
salts. Some Pt(II) and Ru(II) complexes of the new ligands, resem-
bling complexes of known anticancer activity, have been prepared,
characterized and tested on model cell lines.
The platinum complexes [PtCl2(PTAR)2](PF6)2 structure recall
the classical pattern of platinum based drugs, being Pt(II) square
planar species with cis geometry, bearing two neutral donors and
two hydrolysable chlorides as ligands. Although we did not explore
the new complexes behavior in the cell, a cisplatin-like mechanism
and target (DNA) can be reasonably forecasted.
These data suggest that the antiproliferative activity observed
for the complexes is the result of a synergic action of metal and
ligand.
3.3.2. Ruthenium complexes
Also the ruthenium complexes 5, 6 and 7 have been tested for
antiproliferative activity on two human tumoral cell lines, SKOV3
and SW480.
In order to evaluate the contribution of long-chain ligands
(PTACnH2n+1)I to the activity of compounds 5, 6 and 7, the parent
complexes [CpRu(PPh3)(PTA)Cl] and [CpRu(PPh3)(PTAMe)Cl]I have
The Ru complexes [CpRuCl(PPh3)(PTAR)]I someway recall the
complexes deeply investigated by Dyson [11,12] and also in this
case it is reasonable to expect a parallel behavior.
For a fair comparison of the antiproliferative activity, cisplatin
and the known ruthenium parent complexes [CpRuCl(PPh3)(PTA)]
and [CpRuCl(PPh3)(PTAMe)]I have been tested beside the new
Pt and Ru complexes on tumoral human cell lines T2, SKOV3 and
SW480 and the reference complexes have been found less active
than the new complexes whose activity has been found remarkable
been tested in the same experiments at 100, 50, 10, 2 and 1
the estimated IC50 values are reported in Table 4.
On both cell lines the long chain ruthenium complexes 5–7
show high activities, superior to those of the parent complexes
containing PTA or (PTAMe)+ ([CpRuCl(PPh3)(PTA)], [CpRuCl(PPh3)
(PTAMe)]I) or cisplatin.
If the data obtained for Ru complexes are compared with the
data of the uncoordinated ligands on SKOV3, it appears that each
ligand has a lower activity than the relative complex (Table 5).
In order to exclude that the antiproliferative activity of the com-
plexes was due to a generalized cytotoxicity of the ligands, the
antiproliferative activity of both the complexes and the ligands
lM and
even at low doses. This observation indicates that the new ligands
+
(PTACnH2n+1
)
must contribute to the antiproliferative activity of
their Pt and Ru complexes.
Acknowledgments
We acknowledge for support Consorzio Interuniversitario di
Ricerca in Chimica dei Metalli nei Sistemi Biologici and the Italian
MURST.
Table 4
Estimated IC50 (
l
M) of Ru complexes on SKOV3 e SW480 cell lines.
IC50 SKOV3
Complex
IC50 SW480
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[CpRuCl(PPh3)(PTA)]
>100
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[CpRu(PPh3)(PTAMe)Cl]I
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7
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9.64 0.19
9.53 1.27
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56.37 9.01
9.69 0.15
9.73 0.09
9.10 1.27
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