New PTA (1,3,5-triaza 7-phosphaadamantane) derivatives associating zwitterionic structure and coordinative ability

Article abstract of DOI:10.1016/j.ica.2012.12.006

The reactions of PTA (1,3,5-triaza 7-phosphaadamantane) and HMTA (1,3,5,7-tetraazaadamantane) with 1,3-propanesultone or 1,4-butanesultone gave the water soluble zwitterionic derivatives PTA⁺C₃H ₆SO₃^-

Full text of DOI:10.1016/j.ica.2012.12.006

Inorganica Chimica Acta 398 (2013) 11–18
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New PTA (1,3,5-triaza 7-phosphaadamantane) derivatives associating
zwitterionic structure and coordinative ability
a
a
Paola Bergamini a, , Lorenza Marvelli , Andrea Marchi , Valerio Bertolasi a,b, Marco Fogagnolo a,
⇑
Paolo Formaglio a, Fabio Sforza c
a Dipartimento di Scienze Chimiche e Farmaceutiche dell’Università di Ferrara, via Fossato di Mortara 17, 44121 Ferrara, Italy
b Centro di Strutturistica Diffrattometrica dell’ Università di Ferrara, via L. Borsari 46, 44121 Ferrara, Italy
c Dipartimento di Scienze della Vita e Biotecnologie dell’Università di Ferrara, via L. Borsari 46, 44121 Ferrara, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 2 August 2012
Received in revised form 22 November 2012
Accepted 1 December 2012
Available online 13 December 2012
The reactions of PTA (1,3,5-triaza 7-phosphaadamantane) and HMTA (1,3,5,7-tetraazaadamantane) with
1,3-propanesultone or 1,4-butanesultone gave the water soluble zwitterionic derivatives PTA+C3H6SO3
À
À
À
À
(1), PTA+C4H8SO3 (2), HMTA+C3H6SO3 (3) and HMTA+C4H8SO3 (4). The crystal structure of HMTA+C3
H6SO3 is reported. The coordinative ability of 1–4 towards Pt(II) and Ru(II) has been investigated and
the antiproliferative activity of ligands and complexes has been tested in two human ovarian cancer cell
lines, A2780 and SKOV3.
Keywords:
PTA
Ó 2012 Elsevier B.V. All rights reserved.
N-alkylation
Zwitterionic
Platinum
Ruthenium
Antiproliferative activity
1. Introduction
and therefore they are not de-protonable and they are protonable
only at very low pH values.
Hydrophilicity combined with the lack of net charge makes
zwitterionic compounds suitable for many applications: e.g. they
have been exploited as non-denaturating detergents for proteins
[1], metal salt extractants [2], ionic liquids [3], quantum dots com-
ponents [4] and also as drugs with low affinity for blood serum
albumin and high oral biodisponibility, e.g. the antibiotics cipro-
floxacin and amoxicillin [5].
Zwitterionic species, both naturally occurring like phosphocho-
line and synthetically prepared or modified like sulfabetaines, have
also attracted a great deal of attention as functional groups for
polymeric structures with valuable properties [6].
Because of the above described advantageous interactions
with bio-systems, we reasoned that it could be of interest to
produce species where the zwitterionic character is coupled with
the presence of a coordinative site for metal ions, particularly for
those with well-known pharmaceutical activity, like Pt(II) and
Ru(II). Being these soft acidic ions, the introduction of a soft do-
nor like phosphorus is likely to favour the metal coordination
[9].
We designed the new ligands 1 and 2, which are water-soluble
zwitterionic phosphines, easily obtainable through PTA (1,3,5-tri-
aza 7-phosphaadamantane) N-alkylation.
The presence of zwitterionic moieties produces innovative bio-
materials of great potential in biochemistry, and in drugs and diag-
nostics development [7]: conjugation to zwitterionic biomaterials
represents a new strategy for stabilising peptide-based drugs and
for protecting implantable electrochemical glucose biosensors
from biofouling [8]. The zwitterions used for these purposes are
We have recently prepared some cationic PTA derivatives
exploiting the alkylation process which occurs invariably to a
single nitrogen atom, with complete regioselectivity, leaving va-
cant the P-donor, the favourite site for soft metals coordination
[10].
In this paper we describe the synthesis, properties and coordi-
nation to platinum and ruthenium of 1 and 2, two zwitterionic
phosphines where short polymethylene chains connect the PTA
cage to a sulfonated group, joining in the same molecule the two
groups traditionally exploited to make a phosphine water soluble
(PTA and SO3À) [11].
not pH sensitive, bearing quaternary ammonium groups coupled
2À
with anionic groups which are weak bases (e.g. RSO3À, RPO4
)
⇑
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.2012.12.006

12
P. Bergamini et al. / Inorganica Chimica Acta 398 (2013) 11–18
Electrospray MS (in H2O): observed m/z 280 (M+1), 302 (M+23),
calcd 280 for C9H19N3O3PS (MH+}.
N
P
+
N
-
1, n = 1
2, n = 2
SO3
N
n
2.3. Synthesis of PTA+C4H8SO3 (2)
À
The aminic analogues 3 and 4, obtained from HMTA (1,3,5,7-
tetraazaadamantane), have been also prepared and characterised.
0.300 g (1.9 mmol) of PTA were mixed with 780 lL (7.6 mmol,
4 eq) of 1,4-butanesultone for 20 h without any solvent. 5 mL of
ether were then added and the white solid product was separated
by filtration and washed with ether (0.483 g, 1.65 mmol, 86.8%).
Solubility in water (25 °C): 504 mg/mL.
N
+
N
-
3, n = 1
4, n = 2
SO3
N
Data for 2 were as follows: 1H NMR (300 MHz, D2O, 25 °C): d
1.63 (m, 2H, CH2CH2SO3À), d 1.80 (m, 2H, CH2CH2SO3À), d 2.82
(m, 4H, CH2CH2CH2N+), d 3.80 (m, 4H, PCH2N), d 4.22 (s, 1H, PCH2
n
N
2
N+), d 4.23 (s, 1H, PCH2N+), d 4.38 (d, JHH = 14 Hz, 1H, NCH2N), d
4.52 (d, 2JHH = 14 Hz, 1H, NCH2N), d 4.72 (d, 2JHH = 11 Hz, 2H, NCH2N+),
2. Experimental
2.1. General procedures
2
d 4.90 (d, JHH = 11 Hz, 2H, NCH2N+).
13C{1H}NMR (75.43 MHz, D2O, 25 °C): d 18.1 (s CH2SO3À), d 21.2
(s, CH2CH2CH2SO3À), d 30.1 (s, CH2CH2CH2N+), d 45.2 (d, JPC = 21.4
1
1
Hz, PCH2N), d 50.4 (s, CH2CH2CH2N+), d 52.9 (d, JPC = 34 Hz, PCH2
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
N+), d 62.2 (s, CH2CH2N+), d 69.4 (s, NCH2N), d 78.8 (s, NCH2N+).
31P{1H} NMR (121.5 MHz, D2O, 25 °C): d À82.86 ppm (s).
Anal. Calc. for C10H20N3O3PS (293): C, 40.94; H, 6.88; N, 14.33; S,
10.91. Found: C, 41.03; H, 6.97; N, 14.25; S, 10.82%.
Electrospray MS (in H2O): observed m/z (M+H+) 294, (M+Na+)
316, calcd 294 for C10H21N3O3PS (MH+).
on a Varian Gemini 300 MHz spectrometer (1H at 300.23 MHz, 13
C
at 75.43 MHz, 31P at 121.50 MHz) or a Varian Mercury Plus (1H at
399.97 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 rel-
ative to external tetramethylsilane (TMS), with positive shifts
downfield. The solubility in water for 1–4, was determined by pro-
2.4. Synthesis of HMTA+C3H6SO3 (3)
À
0.500 g of HMTA (3.27 mmol) were dissolved in refluxing tolu-
ene and 0.522 g of 1,3-propanesultone (4.28 mmol, 1.2 eq) were
added. The mixture was refluxed for 18 h. The product was sepa-
rated by filtration as a white solid and washed with ether
(0.840 g, 3.21 mmol, 98.2% yield).
gressively adding 50 lL volumes of water to 0.1 g of solid product
until it appears completely dissolved. For Pt and Ru complexes the
concentration of saturated solutions have been measured by a Per-
kin-Elmer atomic absorption spectrometer (Analyst 800) against
the appropriate standards.
Solvents were distilled and dried prior to use. Commercial sul-
tones were purchased and used without further purification. PTA
[12] and the metal complexes precursors [PtCl2(1,5-COD)] (1,5-
COD = 1,5-cyclooctadiene) [13] and [CpRuCl(PPh3)(PTA)] [14] were
prepared as described in the literature.
Solubility in water (25 °C): 180 mg/mL.
Data for 3 were as follows. 1H NMR (300 MHz, D2O, 25 °C): d
2.10 (m, 2H, CH2SO3À), d 2.80 (pst, 2JHH = 7.00 Hz, 2H, CH2CH2CH2),
2
d 2.95 (m, 2H, CH2CH2N+), d 4.44 (d, 3H, JHH = 14 Hz, NCH2N), d
2
4.62 (d, 3H, JHH = 14 Hz, NCH2N), d 5.07 (s, 6H, NCH2N+).
13C{1H} NMR (75.43 MHz, D2O, 25 °C): d 15.6 (s, CH2SO3), d 47.4
(s, CH2CH2CH2), 55.3 (s, CH2CH2N+), d 70.0 (s, NCH2N), d 78.2 (s,
NCH2N+).
Anal. Calc. for C9H18SO3N4 (262): C, 41.20; H, 6.92; N, 21.37; S,
12.20. Found: C, 41.20; H, 6.92; N, 20.80; S, 12.97%.
Electrospray MS (in H2O): observed m/z 263 (M+H+), 285
(M+Na+), 417, 525 (2M+H+), 547 (2M+Na+) 686, calcd 263 for
C9H19SO3N4 = (MH+).
2.2. Synthesis of PTA+C3H6SO3 (1)
0.100 g of PTA (0.64 mmol) were dissolved in AcOEt (12 mL)
and 2.2 eq (0.173 g, 1.42 mmol) of 1,3-propanesultone were added.
The mixture was stirred at room temperature for 18 h. The product
precipitated as a white solid and was separated by filtration and
washed with ether (0.110 g, 0.40 mmol, 62.5%).
2.5. Synthesis of zwitterions HMTA+C4H8SO3 (4)
À
0.230 g of HMTA (2.44 mmol) and 1 mL of 1,4-butanesultone
(1.330 g, 9.76 mmol, 6 eq) were mixed and kept under stirring
at room temperature for 3 days. Diethylether was then added
to the white slurry and the white solid was filtered and washed
Solubility in water (25 °C): 50 mg/mL.
Data for 1 were as follows. 1H NMR (300 MHz, D2O, 25 °C): d
2.10 (m, 2H, CH2SO3À), d 2.85 (pst, HH = 7.22 Hz, 2H, CH2CH2CH2),
d 3.00 (m, 2H, CH2CH2N+), d 3.80 (m, 4H, PCH2N), d 4.24 (s, 1H,
PCH2N+), d 4.25 (s, 1H, PCH2N+), d 4.36 (d, 2JHH = 13.6 Hz, 1H, NCH2-
with ether (0.440 g, 1.59 mmol, 65.2%). Data for
follows.
4 were as
2
2
N), d 4.50 (d, JHH = 13.6 Hz, 1H, NCH2N), d 4.72 (d, JHH = 11.4 Hz,
Solubility in water (25 °C): 385 mg/mL.
2H, NCH2N+), d 4.93 (d, JHH = 11.4 Hz, 2H, NCH2N+).
1H NMR (300 MHz, D2O, 25 °C): d 1.68 (m, 2H, CH2SO3À), d 1.76
2
2
13C{1H} NMR (50.320 MHz, D2O, 25 °C): d 16.7 (s, CH2SO3À), d
(m, 2H, CH2CH2SO3À), d 2.82 (pst, JHH = 7.00 Hz, 4H, N+CH2CH2
1
46.9 (d, JPC = 20.6 Hz, PCH2N), d 49.0 (s, CH2CH2CH2), d 54.0 (d,
CH2), d 4.42 (d, 3H, 2JHH = 14 Hz, NCH2N), d 4.61 (d, 3H, 2JHH = 14 Hz,
NCH2N), d 5.00 (s, 6H, NCH2N+).
1JPC = 33 Hz, PCH2N+), d 62.2 (s, CH2CH2N+), d 70.5 (s, NCH2N), d
80.2 (s, NCH2N+).
13C{1H} NMR (75.43 MHz, D2O, 25 °C): d 18.4 (s, CH2SO3À), d 21.6
(s, CH2CH2SO3), d 50.0 (s, CH2CH2CH2), d 56.7 (s, CH2CH2N+), d 70.3
(s, NCH2N), d 78.5 (s, NCH2N+).
31P{1H} NMR (121.5 MHz, D2O, 25 °C):
(121.5 MHz, DMSO, 25 °C): d À84.76.
d
À83.67 ppm (s).
Anal. Calc. for C9H18N3O3PS (279): C, 38.70; H, 6.50; N, 15.05; S,
Anal. Calc. for C10H20N4O3S (276): C, 43.46; H, 7.30; N, 20.29; S,
11.58. Found: C, 43.56; H, 7.67; N, 20.03; S, 11.75%.
11.46. Found: C, 38.68; H, 6.45; N, 14.90; S, 11.36%.

P. Bergamini et al. / Inorganica Chimica Acta 398 (2013) 11–18
13
Electrospray MS (in H2O): observed m/z 277.1 (M+H+), 553.2
2.7. Coordination reactions of PTA+C3H6SO3À (1) and PTA+C4H8SO3À (2)
(2M+H+), calcd 277 for C10H21N4O3S = (MH+).
2.7.1. Synthesis of cis-[PtCl2(PTA+C3H6SO3À)2], (5)
A solution of K2PtCl4 (0.083 g, 2 Â 10À4 mol) in water (5 mL)
was added to a suspension of PTA+C3H6SO3 (0.111 g, 4 Â 10À4
À
2.6. X-ray crystal structure of 3
mol) in 10 mL of ethanol.
The crystal data for HMTA+C3H6SO3À (3) were collected at room
temperature (T = 295 K) using a Nonius Kappa CCD diffractometer
with graphite monochromated Mo Ka radiation and corrected for
Lorentz and polarisation effects. The structure was solved by direct
methods (SIR97 [15]) and refined using full-matrix least-squares. All
non-hydrogen atoms were refined anisotropically and hydrogens
isotropically. All the calculations were performed using SHELXL-97
The sudden precipitation of a pink-white solid and the decolor-
ation of the solution are observed. The solid was filtered, washed
with water and dried (0.140 g, 1.7 Â 10À4 mol, 85.0% yield).
The product 5 has a very low solubility in water (178 mg/L at
25 °C, measured by atomic absorption), but its solubility in a NaCl
solution or in 5 M HCl, although scarce, allowed spectroscopic
characterization.
1H NMR (300 MHz, D2O sat NaCl, 25 °C): d 2.38 (m, 2H, CH2CH2
CH2SO3À), d 3.15 (m, 3H) + 3.52 (m, 1H) (+NCH2CH2CH2) 4.9 (bm,
6H, PCH2N+ e PCH2N), d 5.20 (bs, 2H, NCH2N), d 5.42 (dd, 4H,
2JHH = 11 Hz, NCH2N+).
Table 1
Crystal data and details of data collection for 3.
31P{1H} NMR (121.5 MHz, D2O sat NaCl, 25 °C): d À37.98 ppm
À
Compound
Formula
M
HMTA+C3H6SO3
C9H18N4O3SÁ3H2O
316.38
(1JPtP 3534 Hz).
31P{1H} NMR (121.5 MHz, HCl 5 M, 25 °C): d À38.27 ppm (1JPtP
3534 Hz).
System
Space group
a (Å)
b (Å)
c (Å)
monoclinic
P21/c
Anal. Calc. for C18H36Cl2N6O6S2P2Pt (824): C, 26.21; H, 4.40; N,
10.19; S, 7.76. Found: C, 25.95; H, 4.42; N, 9.85; S, 7.75%.
10.1098(3)
6.3722(2)
22.8048(6)
96.237(1)
1460.43(4)
4
1.439
295
2.53
2.7.2. Synthesis oÀf cis-[PtCl2(PTA+C4H8SO3À)2] (6)
b (°)
V (Å3)
Z
PTA+C4H8SO3 (0.117 g, 4 Â 10À4 mol) was dissolved in 5 mL of
water and added at 0 °C to a solution of K2PtCl4 (0.083 g, 2 Â 10À4
mol) in water (2.5 mL).
Dcalc (g cmÀ3
T (K)
)
l
(cmÀ1
)
An off-white solid precipitated while the solution shaded. The
solid was separated by centrifugation, washed with water and
dried (0.135 g, 1.6 Â 10À4 mol, 80.0% yield).
h
min–hmax/°
3.67–27.88
8254
3420
Collected reflections
Unique reflections
Rint
The product 6 has a very low solubility in water (36.5 mg/L at
25 °C, measured by atomic absorption), but its solubility in a NaCl
solution or in 5 M HCl allowed spectroscopic characterization.
1H NMR (300 MHz, D2O sat NaCl, 25 °C): d 2.10 (m, 2H, CH2CH2
SO3À), d 2.25 (m, 2H, CH2CH2SO3À), d 3.35 (m, 3H) + 3.60 (m, 1H)
(CH2CH2CH2N+) 4.9 (bm, 6H, PCH2N+ and PCH2N), d 5.20 (bs, 2H,
0.025
Observed reflections [I > 2
R (Observed reflections)
wR (All reflections)
S
r
(I)]
2716
0.0400
0.1129
1.023
D
qmax
;
D
qmin (e ÅÀ3
CCDC Deposit No.
)
0.26; À0.38
CCDC 894267
2
NCH2N), d 5.42 (dd, 4H, JHH = 11 Hz, NCH2N+).
31P{1H} NMR (121.5 MHz, D2O sat NaCl, 25 °C): d À37.99 ppm
(1JPtP 3546 Hz).
Table 2
Electrospray MS (in H2O): observed m/z 875 (M+Na+), calcd 875
for C20H40Cl2N6P2Pt O6S2Na.
Selected structural parameters (Å, °) for compound 3.
Bond distances
Anal. Calc. for C20H40Cl2N6P2PtO6S2 (852): C, 28.16; H, 4.73; N,
9.86; S, 7.50. Found: C, 28.43; H, 4.84; N, 9.73; S, 7.55%.
N1–C1
N1–C2
N1–C4
N1–C7
N2–C1
N2–C3
N2–C5
N3–C2
N3–C3
N3–C6
1.534(2)
1.536(2)
1.533(2)
1.497(2)
1.439(2)
1.474(2)
1.475(2)
1.435(2)
1.471(2)
1.469(2)
N4–C4
N4–C5
N4–C6
C7–C8
C8–C9
C9–S1
S1–O1
S1–O2
S1–O3
1.440(2)
1.471(2)
1.474(2)
1.515(2)
1.519(2)
1.779(2)
1.441(1)
1.450(1)
1.457(1)
2.7.3. Synthesis of [Cp(PPh3)(PTA+C3H6SO3À)RuCl], (7)
Method A: 102 mg of [Cp(PPh3)2RuCl] (0.14 mmol) was mixed
with PTA+C3H6SO3 (39 mg, 0.14 mmol) in 20 mL of ethanol and
À
2 mL of distilled water. The mixture was refluxed for 1 h. Concen-
tration of the solution to ca. half volume and the addition of dieth-
ylether (5 mL) gave [Cp(PPh3)(PTA+C3H6SO3À)RuCl], 7, as a dark
yellow solid, which was filtered off and dried with diethyl ether
(3 Â 2 mL). (50.0% yield).
Bond angles
C1–N1–C2
C1–N1–C4
C1–N1–C7
C2–N1–C4
C2–N1–C7
C4–N1–C7
N1–C7–C8
C7–C8–C9
107.2(1)
107.9(1)
112.3(1)
107.5(1)
108.0(1)
113.6(1)
115.9(1)
107.9(1)
C8–C9–S1
C9–S1–O1
C9–S1–O2
C9–S1–O3
O1–S1–O2
O1–S1–O3
O2–S1–O3
113.3(1)
107.5(1)
106.7(1)
105.0(1)
111.9(1)
112.4(1)
112.9(1)
Method B:
A
solution of [Cp(PPh3)(PTA)RuCl] (0.200 g,
3.2 Â 10À4 mol) in 15 mL of CH2Cl2 was treated with an excess of
1,3-propanesultone (0.314 g, 2.57 Â 10À3 mol) and the mixture
was refluxed for 3 h under nitrogen. The colour slowly turned from
yellow to dark orange; a dark yellow solid precipitated, which was
filtered out and washed with dichloromethane (2 Â 2 mL) and
diethyl ether (3 Â 2 mL) (0.150 g, 2 Â 10À4 mol, 62.5% yield).
Complex 7 has a very low solubility in water (41 mg/L at 25 °C,
measured by atomic absorption), but its solubility in DMSO al-
lowed spectroscopic characterization.
Torsion angles
N1–C7–C8–C9
176.9(1)
C7–C8–C9–S1
À172.4(1)
1H NMR (300 MHz, DMSO d6, 25 °C): d 1.85 (m, 2H, CH2CH2SO3À),
d 2.10 (m, 2H, CH2CH2SO3À), d 2.98 (m, 2H, CH2CH2CH2N+), d 3.65
(bm, 1H, PCH2N), d 3.80 (bm, 1H, PCH2N), d 3.97 (bm, 1H, PCH2N),
[16] and PARST [17] implemented in WINGX [18] system of programs.
The crystal data and refinement parameters are summarized in Ta-
ble 1. Selected structural parameters are given in Table 2.

14
P. Bergamini et al. / Inorganica Chimica Acta 398 (2013) 11–18
d 4.09 (bm, 1H, PCH2N), d 4.22 (bm, 2H, PCH2N+), d 4.47 (m, 5H, Cp), d
4.72 (m, 4H, NCH2N+), d 4.98 (m, 2H, NCH2N).
3. Results and discussion
2
31P{1H} NMR (121.5 MHz, DMSO d6, 25 °C): d 46.56 (d, JPP
3.1. Zwitterionic phosphines PTA+C3H6SO3 (1) and PTA+C4H8SO3 (2)
À
2
43.90 Hz), d À15.35 ppm (d, JPP 43.90 Hz).
Anal. Calc. for C32H38ClN3P2RuO3S (743): C, 51.70; H, 5.16; N,
From the reaction of PTA with 1,3-propane- and 1,4-butanesul-
tone, the zwitterionic phosphines 1 and 2 were obtained in good
yield (Scheme 1).
5.66; S, 4.30. Found: C, 52.12; H, 5.35; N, 5.59; S, 4.25%.
The reaction of PTA with solid 1,3-propanesultone to give 1 was
carried out in AcOEt, while 2 was prepared mixing PTA with liquid
1,4-butanesultone without solvent.
Polialkylation, described with HMTA [20], was never observed
for PTA in these experiments.
2.7.4. Synthesis of [Cp(PPh3)(PTA+C4H8SO3À)RuCl] (8)
Method A: Complex [Cp(PPh3)(PTA+C4H8SO3À)RuCl], 8, was ob-
tained by treating [Cp(PPh3)2RuCl] (0.15 mmol) with 1 eq of PTA+-
C4H8SO3 as above described for [Cp(PPh3)(PTA+C3H6SO3À)RuCl],
À
(0.093 mmol, yield 62.0%).
Compounds 1 and 2 were characterised by spectroscopic tech-
niques. In both cases, the 31P NMR in D2O showed a single peak,
at À83.67 and À82.86, respectively. 1H and 13C data are reported
in the Experimental, the signals have been assigned through COSY
(Fig. 1) and HETCOR (Fig. 2): the multiplet integrating 2H at d 2.10
has been assigned to CH2SO3À, the pseudotriplet (2H) at d 2.85
(2JHH = 7.22 Hz) is due to the central chain CH2, while the CH2 pro-
tons close to PTAN+ give a signal at 3.00 ppm; the cage CH2 have
Method B:
A Schlenk tube was charged with [Cp(PPh3)(-
PTA)RuCl] (0.200 g, 3.2 Â 10À4 mol) and 2 mL of liquid 1,4-butane-
sultone; a dark yellow solid was formed from the reaction mixture
stirred at 50 °C for 2 h. Slow addition of diethyl ether (3 mL) com-
pleted the precipitation of the product, which was filtered and
washed with diethyl ether (3 Â 2 mL). (0.180 g, 2.36 Â 10À4 mol,
73.8% yield).
Complex 8 has a very low solubility in water (35.0 mg/L at
25 °C, measured by atomic absorption), but its solubility in DMSO
allowed spectroscopic characterization.
2
been found at 3.80 ppm (m, 4H, PCH2N), 4.25 (d, JHH = 6.04 Hz,
2H, PCH2N+), at d 4.36 and 4.50 (two doublets due to reciprocally
2
coupled protons of NCH2N JHH = 13.6 Hz), at d 4.72 and 4.93 also
1H NMR (300 MHz, DMSO d6, 25 °C): d 1.70 (m, 4H, CH2CH2SO3À),
d 2.05 (m, 2H, CH2CH2CH2SO3À), d 2.86 (m, 2H, CH2CH2N+), d 3.6–4.3
(6H, PCH2N), d 4.48 (m, 5H, Cp), d 4.78 (m, 4H, NCH2N+), d 4.90
(m, 2H, NCH2N).
two doublets due to reciprocally coupled protons NCH2N+,
(2JHH = 11.4 Hz).
The 13C NMR shows the chain CH2 as singlets at d 16.7 (CH2SO3À),
d 49.0 (CH2CH2CH2) and d 62.2 (CH2CH2N+), while the cage CH2 are
found at d 46.9 as a doublet due to PCH2N (1JPC = 20.6 Hz) and at
31P{1H} NMR (121.5 MHz, DMSO d6, 25 °C): d 46.52 (d, JPP
2
2
43.90 Hz), d À15.03 ppm (d, JPP 43.90 Hz).
1
d 54.0 for PCH2N+, also a doublet with JPC = 33 Hz. At 70.5 and
Anal. Calc. for C33H40ClN3P2RuO3S (757): C, 52.33; H, 5.33; N,
80.2 ppm two singlets due to NCH2N e NCH2N+ are observed.
The ES-MS shows a peak at 280, due to molecular weight plus
an H+, and one at 302, due to the addition of a Na+ ion.
Similarly, it has been carried out the characterization of PTA+C4-
H8SO3À, which presents analogue NMR feature (see Section 2) and,
in ESI-MS, peaks at 294 (M+H+), and 316 (M+Na+).
5.55; S, 4.22. Found: C, 52.02; H, 5.39; N, 5.51; S, 4.18%.
2.8. Growth inhibition assays
Cell growth inhibition assays were carried out using two human
ovarian cancer cell lines, A2780 and SKOV3; A2780 cells are cis-
platin-sensitive and SKOV3 cells are cisplatin-resistant. Cells were
maintained in RPMI 1640, supplemented with 10% newborn bo-
vine serum, penicillin (100 U/mL), streptomycin (100 U/mL) and
glutamine (2 mM); the pH of the medium was 7.2 and the incuba-
tion was at 37 °C in a 5% CO2 atmosphere. Cells were routinely
passed every three days. MTT test was used to study the com-
pounds antiproliferative activity. The cells were seeded in triplicate
3.2. Zwitterionic amines HMTA+C3H6SO3À (3) and HMTA+C4H8SO3À (4)
The aminic analogues of the above described zwitterionic phos-
phines, were obtained by alkylation of HMTA (Scheme 2).
HMTA+C3H6SO3À (3) was prepared refluxing HMTA with a small
excessÀof 1,3-propanesultone in toluene for 18 h, while HMTA+C4-
H8SO3 (4) was obtained by mixing HMTA with pure 1,4-butane-
sultone at room temperature for 3 days.
in 96-well trays at a density of 25 Â 103 in 50
ll of AIM-V medium
for A2780 and SKOV3. Stock solutions (10 mM) of each compound
were made in DMSO and diluted in AIM-V medium to give final
Compounds 3 and 4 have been characterised by NMR and mass.
The 1H NMR Àspectrum of HMTA+C3H8SO3 (3) is similar to that
À
concentrations of 10, 50 and 100
lM. Cisplatin was employed as
a control for the cisplatin-sensitive A2780 cell line and for the cis-
platin-resistant SKOV3. Untreated cells were placed in every plate
as a negative control. The cells were exposed to the compounds, in
of PTA+C3H6SO3 for the alkylic chain pattern, while the aminic
cage, more simmetrical than PTA, is characterised by two signals
at 4.55 ppm (dd, 6H, NCH2N+) and 5.07 ppm (s, 6H, NCH2N) in 1H
and at 70.0 (s, NCH2N) and 78.2 ppm (s, NCH2N+) in 13C NMR. In
ESI mass the peak M+H+ is observed at 263 and M+Na+ at 285, to-
gether with dimeric species at 525 (2M+H+) and 547 (2M+Na+).
The NMR characterization of HMTA+C4H8SO3À (4), Fig. 3, is sim-
ilar that of 3: the peaks M+H+ at m/z 277.1 and 2 M + H+ at m/z
553.2 have been identified in the mass spectrum.
100
thiozol-2-yl)2,5-diphenyltetrazolium bromide solution (MTT)
(12 mM) were added. After 2 h of incubation, 100 l of lysing buf-
ll total volume, for 72 h, and then 25 ll of a 3-(4,5-dimethyl-
l
fer (50% DMF + 20% SDS, pH 4.7) were added to convert the MTT
solution into a violet coloured formazane. After additional 18 h
the solution absorbance, proportional to the number of live cells,
was measured by spectrophotometer at 570 nm and converted
into% of growth inhibition [19].
Suitable crystals of 3 were obtained and the X-ray crystal struc-
ture was determined.
The ORTEP [21] view of the trihydrated zwitterionic compound 3
is shown in Fig. 4. The crystal packing projected down the b axis is
displayed in Fig. 5. The positive charge localised on the quaternary
N1 nitrogen gives rise to significant lengthening of N1–C bonds
(1.52[2] Å on average) with respect to the other N–C bond dis-
tances (1.46[2] Å on average) involving neutral N2, N3 and N4
nitrogens. The three-methylene linker adopts a full extended
conformation (Table 2) as observed in other similar zwitterionic
N
N
O
O
n
+
N
N
+
N
-
SO3
S
N
n
O
P
P
n = 1, 2
n = 1, (1)
n = 2, (2)
Scheme 1. Synthesis of PTA+C3H6SO3 (1) and PTA+C4H8SO3 (2).
À

P. Bergamini et al. / Inorganica Chimica Acta 398 (2013) 11–18
15
À
Fig. 1. COSY of PTA+C3H6SO3 (1).
À
Fig. 2. HETCOR of PTA+C3H6SO3 (PTA cage detail).
compounds [22]. The molecules are packed in the crystal by means
of C–HÁ Á ÁO(sulfonate) and C–HÁ Á ÁN(HMTA+) hydrogen bonds form-
ing channels, parallel to b axis, which include all the molecules of
water linked in infinite chains by means of strong hydrogen bonds
and fixed to the channel walls through Ow–HÁ Á ÁO(sulfonate)
hydrogen bonds (Figs. 5, 6 and Table 3 inserted as Supplementary
material).
3.3. Coordination of PTA+C3H6SO3 (1) and PTA+C4H8SO3 (2) to
À
À
platinum
The coordination of the ligands PTA+C3H6SO3À, PTA+C4H8SO3
,
À
N
N
N
N
O
O
n
+
N
+
N
N
-
SO3
S
HMTA+C4H8SO3À and HMTA+C3H8SO3À to platinum (II) has been at-
tempted in various conditions with different precursors. The most
successful reÀactions were performed dissolving the phosphine
N
n
O
n = 1, 2
n = 1, (3)
n = 2, (4)
PTA+C3H6SO3 or PTA+C4H8SO3 in water and then adding a water
À
Scheme 2. Synthesis of HMTA+C3H6SO3 (3) and HMTA+C4H8SO3À(4).
solution of K2PtCl4 in a 2:1 ligand to metal ratio (Scheme 3).
À

16
P. Bergamini et al. / Inorganica Chimica Acta 398 (2013) 11–18
Fig. 3. 1H NMR of HMTA+C4H8SO3À, 4, in D2O.
Fig. 6. A space filling representation of crystal packing of host–guest compound
HMTA+C3H6SO3ÀÁ3H2O (3) as viewed down the crystallographic b axis. The guest
water molecules, included in the channels built up by the host HMTA+C3H6SO3
À
zwitterionic molecules, are shown using the stick representation.
Table 3
Estimated IC50 (lM) of 1–4 on A2780 and SKOV3 cell lines. Results are presented as a
mean SD of three independent experiments performed in triplicates.
IC50
A2780
SKOV3
PTA+C3H6SO3À, 1
PTA+C4H8SO3À, 2
HMTA+C3H8SO3À, 3
HMTA+C4H8SO3À, 4
83.87 0.78
75.01 1.67
39.58 1.56
38.22 3.18
>100
>100
>100
>100
ion surface charges, which prevent the formation of solute solid
aggregates and stabilise the soluble forms [23].
The 31P NMR of [PtCl2(PTA+C3H6SO3À)2], 5, has been acquired in
HCl 5 M showing the presence of a signal with satellites at
À38.27 ppm (1JPtP = 3534 Hz) typical of a Pt(II) chloride PTA com-
plex with cis geometry [24]. The 31P NMR data in water saturated
with NaCl are À37.98 ppm (1JPtP 3534 Hz), indicating the presence
of the same compound.
Fig. 4. ORTEP view of compound HMTA+C3H6SO3À (3). Thermal ellipsoids are drawn
at 30% probability level.
In both cases the off-white products precipitated in a colloidal
form from the solution, and after 10 min stirring at room tempera-
ture they were isolated by centrifugation and dried under vacuum.
The complexes 5 and 6 are poorly soluble in DMSO, H2O, CH3CN,
CH2Cl2. In order to reach the concentration required by a reason-
ably fast acquisition of NMR spectra, we dissolved them in a 5 M
HCl solution or in an aqueous NaCl saturated solution, where their
solubility is higher then in pure water. In these media, interactions
between Na+ (or H+) and SO3À and between Cl- and N+ are likely to
be established. The observed increase of solubility could be due to
the electrostatic interactions between the salt ions and the zwitter-
Similarly the 31P NMR of complex [PtCl2(PTA+C4H8SO3À)2], 6, in
D2O saturated with NaCl, is a singlet with satellites at À37.99 ppm
(1JPtP = 3546 Hz). The ESI-MS analysis shows the M+Na+ species at
875.
It is worth noticing that 5 and 6 are a rare type of zwitterionic
complex where both the positive and negative charge belong to the
ligand. In fact in the frequently reported so-called platinum group
metal zwitterions the negative charge is due to the ligand and the
positive to the metal [25].
Fig. 5. Compound HMTA+C3H6SO3 (3), crystal packing projected down the b axis.
À

P. Bergamini et al. / Inorganica Chimica Acta 398 (2013) 11–18
17
N
N
N
N
-
SO3
SO3
N
P
P
Cl
Cl
N
2
K2PtCl4
Pt
N
-
SO3
P
-
N
N
Scheme 3. Synthesis of the Pt complex cis-[PtCl2(PTA+C3H6SO3À)2], 5.
N
the free ligand, while for the cage protons, a broadening and a par-
tial signals overlap is observed.
N
EtOH, acetone, H2O
1h reflux
N
-
Ru
SO3
The coordination of ligands 3 and 4 to platinum and ruthenium
P
Ph3P
PPh3
was attempted unsuccessfully in a variety of conditions1.
Cl
3.5. AntiproliferÀative activity of PTA+C3H6SO3 (1), PTA+C4H8SO3À(2),
À
Ru
N
HMTA+C4H8SO3 (3), and HMTA+C3H8SO3À(4)
Ph3P
P
N
Cl
PTA
N
The antiprolÀiferative activity of PTÀA+C3H6SO3À, PTA+C4H8SO3
,
À
toluene, 2h reflux/N2
Yield 89%
O
Ru
HMTA+C3H8SO3 and HMTA+C4H8SO3 have been tested on two
human ovarian cancer cell lines, A2780 and SKOV3 and the results
are reported in Table 3.
S
CH2Cl2
2h reflux/N2
Ph3P
PTA
O
O
Cl
- O3S
These figures indicated that the organic free ligands show some
activity on A2780, higher for the amino HMTA derivatives.
In order to verify if the coordination of 1–4 to Pt or Ru would
have improved the antiproliferative activity, we tried to obtain
their complexes with these metal ions.
Scheme 4. Synthesis of the Ru complex 7.
Table 4
We found thatÀonly the zwitterionic phosphines PTA+C3H6SO3
À
Estimated IC50 (lM) of 1, 5, 7, 2, 6 and cisplatin on A2780 and SKOV3 cell lines.
Results are presented as a mean SD of three independent experiments performed in
and PTA+C4H8SO3 give isolable pure complexes. All the attempts
(see Section 2) to obtain Pt complexes of the zwitterionic amines
HMTA+C3H8SO3À and HMTA+C4H8SO3À, both by direct coordination
and by alkylation of [PtCl2(HMTA)2] failed.
triplicates.
IC50
A2780
SKOV3
PTA+C3H6SO3À, 1
83.87 0.78
37.97 1.52
>100
75.01 1.67
37.33 3.94
10. 85 1.10
>100
>100
>100
>100
>100
>100
[PtCl2(PTA+C3H6SO3À)2], 5
[Cp(PPh3)(PTA+C3H6SO3À)RuCl], 7
PTA+C4H8SO3À, 2
In Table 4 we report the IC50 of Pt complexes of PTA+C3H6SO3
À
À
and PTA+C4H8SO3 compared with the corresponding ligands and
[PtCl2(PTA+C4H8SO3À)2], 6
Cisplatin
cisplatin. As expected, the introduction of Pt does not improve
the activity on cisplatin-resistant SKOV3, while on cisplatin-sensi-
tive A2780, the activity of Pt complexes is higher than that of the
corresponding phosphine.
The coordination to Ru decreases the antiproliferative activity
of ligand 1 on both cell lines.
3.4. Synthesis of Ru complexes of PTA+C3H6SO3À (1) and PTA+C4H8SO3
(2)
À
4. Conclusion
Ru complexes NAMI-A and KP1019 are two anticancer ruthe-
nium drugs which have already entered clinical trials. [26] The
RAPTA-type complexes, ruthenium-arene-PTA organometallic spe-
cies, have shown potential for the further development of antican-
cer remedies [27] and represent the reference for Ru complexes of
new PTA derivatives.
Complexes 7 and 8 have been prepared in two ways (Scheme 4):
(i) by replacing Ru coordinated PPh3 in [CpRuCl(PPh3)2] with 1 or 2
or (ii) by treating the known parent complex [CpRuCl(PPh3)(PTA)]
with the commercial 1,3-propanesultone or 1,4-butanesultone in
dichloromethane.
Phosphines 1 and 2 are the first reported example of PTA zwit-
terionic derivatives. Their antiproliferative activity, found signifi-
cant on two human cancer cell lines, is increased upon
coordination to Pt(II). The amino HMTA derivatives 3 and 4 present
a higher activity than the corresponding PTA derivatives 1 and 2.
These observations indicate that compounds 1–4 deserve further
investigation aimed to exploit their zwitterionic properties and ste-
reospecific coordinative ability for pharmaceutical purposes.
Acknowledgements
The proposed structures for 7 and 8 is consistent with the spec-
troscopic data. In the 31P NMR spectra the products are character-
ised by an AM spin system of two doublets at ca. d 46.56 ppm and
at ca. d À15.35 ppm, respectively, according to the presence of Ru-
coordinated PPh3 ligand and alkyl-PTA, reciprocally coupled with
2JPP = 43.0 Hz.
We acknowledge for support Consorzio Interuniversitario di
Ricerca in Chimica dei Metalli nei Sistemi Biologici.
Appendix A. Supplementary material
In the 1H NMR spectra, the characteristic singlet of the cyclo-
pentadienyl ligand is observed at 4.47 ppm; the signals of the ali-
phatic chains do not undergo a significant shift with respect to
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.ica.2012.12.006.

18
P. Bergamini et al. / Inorganica Chimica Acta 398 (2013) 11–18
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