Novel platinum pyridinehydroxamic acid complexes: Synthesis, characterisation, X-ray crystallographic study and nitric oxide related properties

Article abstract of DOI:10.1016/j.poly.2007.03.011

Herein, we describe the synthesis and characterisation of a novel class of Pt^II and Pt^IV pyridinehydroxamic acid (pyhaH) complexes of general formula cis-[Pt^IICl₂(x-pyhaH)₂] and cis-[Pt^IVCl₄(x-pyhaH)₂], respectively (where x = 3 or 4) in which the pyridinehydroxamic acid is coordinated to the platinum ion via the pyridine nitrogen only leaving the hydroxamic acid free to potentially release cytotoxic nitric oxide (NO). The crystal structure of the Pt^IV derivative, cis-[PtCl₄(4-pyhaH)₂] · 2CH₃OH is reported. To establish the biological effect of the uncoordinated hydroxamic acid moiety in the Pt^II compounds synthesised, the corresponding pyridinecarboxylic acid (pycaH) complexes of general formula cis-[Pt^IICl₂(x-pycaH)₂] (where x = 3 or 4) and the Pt^II pyridine (py) complex, cis-[Pt^IICl₂(py)₂] were synthesised and served as reference standards. The NO-releasing properties of each of the Pt^II compounds, the pyhaH and the pycaH ligands were studied. The Pt^II pyridinehydroxamic acid derivatives were found to induce potent in vitro effects attributable to either NO-release from the hydroxamic acid moiety and/or stimulation of inducible nitric oxide synthase of endothelial cells.

Full text of DOI:10.1016/j.poly.2007.03.011

Polyhedron 26 (2007) 4697–4706
www.elsevier.com/locate/poly
Novel platinum pyridinehydroxamic acid complexes:
Synthesis, characterisation, X-ray crystallographic study and
nitric oxide related properties
a
b
b
c
Darren Griffith , Alberta Bergamo , Sara Pin , Marta Vadori ,
Helge Muller-Bunz , Gianni Sava b,c, Celine J. Marmion
d
a,
*
¨
a
Centre for Synthesis and Chemical Biology, Department of Pharmaceutical and Medicinal Chemistry,
Royal College of Surgeons in Ireland (RCSI), 123 St. Stephen’s Green, Dublin 2, Ireland
b
Fondazione Callerio, Via A. Fleming 22-31, I-34127 Trieste, Italy
Department of Biomedical Sciences, University of Trieste, Via L. Giorgieri 7-9, 34127 Trieste, Italy
c
d
School of Chemistry and Chemical Biology, University College Dublin, Belfield, Dublin 4, Ireland
Received 22 January 2007; accepted 7 March 2007
Available online 15 March 2007
Abstract
Herein, we describe the synthesis and characterisation of a novel class of PtII and PtIV pyridinehydroxamic acid (pyhaH) complexes of
general formula cis-[PtIICl2(x-pyhaH)2] and cis-[PtIVCl4(x-pyhaH)2], respectively (where x = 3 or 4) in which the pyridinehydroxamic
acid is coordinated to the platinum ion via the pyridine nitrogen only leaving the hydroxamic acid free to potentially release cytotoxic
nitric oxide (NO). The crystal structure of the PtIV derivative, cis-[PtCl4(4-pyhaH)2] Æ 2CH3OH is reported. To establish the biological
effect of the uncoordinated hydroxamic acid moiety in the PtII compounds synthesised, the corresponding pyridinecarboxylic acid
(pycaH) complexes of general formula cis-[PtIICl2(x-pycaH)2] (where x = 3 or 4) and the PtII pyridine (py) complex, cis-[PtIICl2(py)2]
were synthesised and served as reference standards. The NO-releasing properties of each of the PtII compounds, the pyhaH and the
pycaH ligands were studied. The PtII pyridinehydroxamic acid derivatives were found to induce potent in vitro effects attributable to
either NO-release from the hydroxamic acid moiety and/or stimulation of inducible nitric oxide synthase of endothelial cells.
ꢀ 2007 Elsevier Ltd. All rights reserved.
Keywords: Platinum; Hydroxamic acids; Nitric oxide; Nitric oxide synthase; Guanylate cyclase; Vascular relaxation
1. Introduction
of Pt drugs is attributed to their ability to bind DNA and
induce apoptosis [1,2]. There are drawbacks however asso-
ciated with their use (such as their limited activity against
many common human cancers, their susceptibility to
acquired drug resistance and their toxicity) which limits
their widespread application and efficacy. The search for
metallodrugs (Pt or non-Pt based) which (i) target the cell
and/or (ii) have a different mode of action to Pt drugs cur-
rently in clinical use remains the subject of intense investi-
gation [3].
Cancer is one of the most complex and deadly diseases
known to man. Although today nearly 50% of all anti-can-
cer therapies are platinum (Pt)-based [1], surprisingly to
date only three Pt drugs have been approved for worldwide
clinical use, namely cisplatin, carboplatin and oxaliplatin,
Fig. 1 while three others, nedaplatin, heptaplatin and
lobaplatin, Fig. 1, have been approved only in Japan,
South Korea and China, respectively [1,2]. The cytotoxicity
Pt complexes having trans geometry [4], polynuclear Pt
complexes [5] and Pt complexes with ligands capable
of intercalating DNA [6] represent an emerging class of
anti-tumour active compounds that circumvent some
*
Corresponding author. Tel.: +353 01 4022161; fax: +353 01 4022168.
E-mail address: cmarmion@rcsi.ie (C.J. Marmion).
0277-5387/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.poly.2007.03.011

4698
D. Griffith et al. / Polyhedron 26 (2007) 4697–4706
O
damage by generating reactive nitrogen oxide species
O
H2
N
(RNOS) such as peroxynitrite and ONOOꢀ, which can
oxidise and nitrate DNA and potentially cause single-
strand DNA breaks via attack on the sugar–phosphate
backbone [10,14]. Dinitrogen trioxide, another RNOS
derived from NO, can nitrosate amines to form carcino-
genic N-nitrosamines, which can then alkylate DNA
[10,15].
O
O
H3N
H3N
Cl
Cl
H3N
H3N
O
O
Pt
Pt
Pt
N
H2
O
O
Oxaliplatin (c)
O
Cisplatin (a)
Carboplatin (b)
O
O
O
H2N
H2N
O
H3N
H3N
O
O
Pt
Pt
O
O
Conflicting reports of pro- and anti-cancer activity
associated with NO clearly illustrate the need for further
research in this area in order to fully elucidate and
understand the precise role of NO in cancer biology.
High concentrations of NO, for example, can promote
tumour cell death by apoptosis whereas lower concentra-
tions of NO can promote tumour cell proliferation, vas-
cularisation or even resistance to apoptosis [10,11]. In
addition to concentration, the cellular effects of NO also
depend on its source, latency, cell type and phenotype
[10].
Nedaplatin (d)
Heptaplatin (e)
H2N
H2N
O
O
H2N
Pt
O
Pt
H2N
O
O
O
Lobaplatin (f)
Fig. 1. Structures of cisplatin, carboplatin, oxaliplatin, nedaplatin,
heptaplatin and lobaplatin.
of the problems associated with clinically used Pt drugs.
They break the cisplatin paradigm by binding to DNA
in other ways and may therefore be active against cis-
platin-resistant cancer cell lines. PtIV complexes are also
under investigation [7] where they are thought to be acti-
vated by reduction in the more reducing environment of
cancer cells, thereby acting as prodrugs of the correspond-
ing anti-tumour active PtII derivatives. An additional
strategy to overcome the drawbacks associated with clin-
ically used Pt drugs is to tether biologically-active ligands
to the Pt which have, in themselves, inherent anti-tumour
properties [1,3].
Nitric oxide (NO) has emerged as one of the most versa-
tile and ubiquitous molecules in the human body with a
diverse range of physiological functions including, amongst
others, gene regulation, cytostasis, platelet function, vascu-
lar smooth muscle relaxation and proliferation, memory,
immune stimulation and apoptosis [8–10]. It is derived
from L-arginine via the catalytic action of a remarkable
class of haem-containing metalloenzymes, the NO syn-
thases (NOS), of which three different isoforms are known
to exist; endothelial NOS (eNOS), neuronal NOS (nNOS)
and inducible NOS (iNOS) [9,10].
Expression of NOS has been demonstrated in a variety
of human and murine tumour types including gynecolog-
ical, breast, head and neck, prostate, bladder and colon
cancers and central nervous system tumours such as glio-
blastomas [11]. The toxic properties of NO are harnessed,
within the immune system, in order to facilitate the con-
trol of infections and tumour cell growth [11,12]. Gener-
ated by iNOS, at high lmol/L concentrations at the site
of infection, NO is thought to attack cancer cells by bind-
ing to metal centres involved in their respiration, thereby
disrupting their function [10,13]. Additionally, NO has
been demonstrated to upregulate p53, the tumour sup-
pressor gene [10]. NO-mediated inhibition of DNA syn-
thesis may also be an important mechanism by which
macrophages induce cytotoxicity. NO can induce DNA
NO donors are currently under investigation as thera-
peutic agents for the treatment of a variety of disease
states including cancer [16,17]. We recently reported that
hydroxamic acids (RC(O)NHOH), a class of organic bio-
ligands with a diverse range of biological functions
[18,19], can act as effective NO donors by virtue of the
fact that they readily transfer NO to ruthenium(III)
(RuIII) to form stable RuII nitrosyls. Moreover, in recent
years, although there has been a surge of interest in devel-
oping novel RuII nitrosyl complexes, capable of releasing
cytotoxic NO [20–23], as yet there is little or no evidence
in the literature of NO-releasing Pt-based chemotherapeu-
tics. The potential of NO delivery systems in the enhance-
ment of platinum cytotoxicity has however been
demonstrated, by pretreating Chinese hamster V79 lung
fibroblast cells with bolus NO or NO delivered from
NONOate NO donors. The tumour cells were markedly
sensitized to subsequent cisplatin treatment [24]. It was
on this premise that we wished to employ a novel syner-
gistic approach, exploiting the well-established anti-cancer
properties of Pt and the NO-releasing ability of hydroxa-
mic acids and, in so doing, advance a new class of
bifunctional metallodrug with dual DNA-binding and
NO-releasing properties. 3- and 4-pyridinehydroxamic
acids were specifically chosen with a view to synthesising
Pt pyridinehydroxamic acid complexes where the pyridine
nitrogen would selectively coordinate PtII or PtIV thereby
leaving the hydroxamic acid moiety uncoordinated and
potentially free to release cytotoxic NO.
Herein, we describe the synthesis, characterisation and
NO-releasing ability of a novel class of 3- and 4-pyr-
idinehydroxamic acid (3- and 4-pyhaH) complexes of PtII
and PtIV. The crystal structure of the PtIV derivative, cis-
[PtCl4(4-pyhaH)2] Æ 2CH3OH is reported. The correspond-
ing 3- and 4-pyridinecarboxylic acid (3- and 4-pycaH) PtII
and PtIV complexes, together with the PtII pyridine ana-
logue were also synthesised and characterised to serve as
reference standards in biological testing.

D. Griffith et al. / Polyhedron 26 (2007) 4697–4706
4699
2. Experimental
2.1.3. Synthesis of cis-[PtIICl2(3-pyhaH)2] Æ 2H2O (6)
3-Pyridinehydroxamic acid (3-pyhaH) (0.146 g, 1.06
mmol) in methanol (7 ml) was added to K2[PtIICl4]
(0.20 g, 0.48 mmol) in deionised water (7 ml). The resulting
solution was stirred without protection from light at room
temperature for 24 h turning yellow in colour. A yellow
precipitate was filtered and dried over P2O5. Yield:
0.18 g, 0.31 mmol, 65%. Anal. Calc. for PtC12H16N4O6Cl2:
C, 24.92; H, 2.79; N, 9.69; Cl, 12.26. Found: C, 24.88; H,
2.65; N, 9.64; Cl, 12.35%. 1H NMR (400 MHz, [d6]-
DMSO, 25 ꢁC): d = 11.48 (s, 2H, OH), 9.31 (s, 2H, NH),
8.98 (s, 2H, aromatic H), 8.76 (d, 2H, aromatic H), 8.14
(d, 2H, aromatic H), 7.47 (dd, 2H, aromatic H).
2.1. General
3-Pyridinecarboxylic acid (nicotinic acid) 3, 4-pyridine-
carboxylic acid (isonicotinic acid) 4, benzohydroxamic acid
5, methylnicotinate, ethylisonicotinate and pyridine, were
all purchased from Aldrich and used without further puri-
fication. K2[PtIICl4] and K2[PtIVCl6] were used as received
from Johnson Matthey. K[RuIII(Hedta)Cl] Æ 2H2O was
synthesised as previously reported [25].
IR and far-IR spectroscopic measurements were
obtained using a Mattson Genesis II CSI FTIR spectrom-
eter. The spectra were analysed using WINFIRST software.
Between 4000 and 400 cmꢀ1, KBr discs were employed
while between 600 and 200 cmꢀ1, polythene discs (Nujol
mull) were used. FTIR grade KBr and nujol were pur-
The PtII and PtIV compounds, 7–14, were synthesised by
reaction of the appropriate pyridine derivative or pyridine
with either K2[PtIICl4] or K2[PtIVCl6], respectively, by the
method described in Section 2.1.3 above.
1
chased from Aldrich. H NMR spectra were recorded on
a Bruker Avance 400 NMR spectrometer and the spectra
analysed using TopSpin 1 software. Chemical shifts were
measured relative to the undeuterated solvent peak. Ele-
mental analysis was performed by the Microanalytical
Laboratory, School of Chemistry & Chemical Biology,
University College Dublin, Belfield, Dublin. Electrospray
ionisation mass spectroscopy (ESI-MS) experiments were
performed on a Quattro Micro quadrupole electrospray
mass spectrometer (Micromass, Waters Corp., USA):
10 lL of the samples was injected in 300 lL of acetoni-
trile:water (60:40, v/v).
2.1.4. cis-[PtIICl2(4-pyhaH)2] Æ H2O (7)
Yield: 70%. Anal. Calc. for PtC12H14N4O5Cl2: C, 25.73;
H, 2.52; N, 10.00; Cl, 12.66. Found: C, 25.94; H, 2.34; N,
9.74; Cl, 12.49%. 1H NMR (400 MHz, [d6]-DMSO,
25 ꢁC): d = 11.53 (s, 2H, OH), 9.43 (s, 2H, NH), 8.78
(dd, 4H, aromatic H), 7.59 (dd, 4H, aromatic H).
2.1.5. cis-[PtIICl2(3-pycaH)2] (8)
Yield: 69%. Anal. Calc. for PtC12H10N2O4Cl2: C, 28.14;
H, 1.97; N, 5.47; Cl, 13.84. Found: C, 28.01; H, 1.90; N,
5.25; Cl, 13.56%. 1H NMR (400 MHz, [d6]-DMSO,
25 ꢁC): d = 9.19 (s, 2H, aromatic H), 8.84 (d, 2H, aromatic
H), 8.33 (d, 2H, aromatic H), 7.45 (dd, 2H, aromatic H).
2.1.1. Synthesis of 3-pyridinehydroxamic acid (3-pyhaH) (1)
Hydroxylamine hydrochloride (5.1 g, 72 mmol) was
added to sodium hydroxide (5.8 g, 146 mmol) in deionised
water (37 ml). The resulting solution was then added
dropwise to methylnicotinate (5.0 g, 36 mmol) in methanol
(55 ml). The solution was stirred at room temperature for
72 h, after which the solution was acidified to pH 5.5 using
5% HCl. The solvent was removed in vacuo yielding a yel-
low solid. Methanol (60 ml) was added and sodium chlo-
ride filtered. The solvent was removed in vacuo yielding a
light pink solid, which was recrystallised from water. Yield:
3.0 g, 22 mmol, 66%. Anal. Calc. for C6H6N2O2: C, 52.17;
H, 4.38; N, 20.28. Found: C, 52.11; H, 4.24; N, 20.14%. 1H
NMR (400 MHz, [d6]-DMSO, 25 ꢁC): d = 11.25 (s, 1H,
OH), 9.13 (s, 1H, NH), 8.77 (s, 1H, aromatic H), 8.55
(d, 1H, aromatic H), 7.93 (d, 1H, aromatic H), 7.33 (dd,
1H, aromatic H).
2.1.6. cis-[PtIICl2(4-pycaH)2] (9)
Yield: 77%. Anal. Calc. for PtC12H10N2O4Cl2: C, 28.14;
H, 1.97; N, 5.47; Cl, 13.84. Found: C, 28.11; H, 1.91; N,
5.27; Cl, 13.96%. 1H NMR (400 MHz, [d6]-DMSO,
25 ꢁC): d = 8.79 (dd, 4H, aromatic H), 7.70 (dd, 4H, aro-
matic H).
2.1.7. cis-[PtIICl2(py)2] (10)
Yield: 75%. Anal. Calc. for PtC10H10N2Cl2: C, 28.31; H,
2.38; N, 6.60. Found: C, 28.21; H, 2.15; N, 6.38%. 1H
NMR (400 MHz, [d6]-DMSO, 25 ꢁC): d = 8.47 (d, 4H, aro-
matic H), 7.49 (t, 4H, aromatic H), 7.32 (m, 2H, aromatic
H).
2.1.8. cis-[PtIVCl4(3-pyhaH)2] Æ 2H2O (11)
Yield: 63%. Anal. Calc. for PtC12H16N4O6Cl4: C, 22.20;
H, 2.48; N, 8.63; Cl, 21.85. Found: C, 22.47; H, 2.41; N,
8.48; Cl, 21.69%. 1H NMR (400 MHz, [d6]-DMSO,
25 ꢁC): d = 11.79 (s, 2H, OH), 9.51 (s, 2H, NH), 9.13 (s,
2H, aromatic H), 8.88 (d, 2H, aromatic H), 8.26 (d, 2H,
aromatic H), 7.48 (dd, 2H, aromatic H).
2.1.2. Synthesis of 4-pyridinehydroxamic acid (4-pyhaH)
(2)
4-pyhaH was synthesised from ethylisonicotinate in line
with the procedure for 3-pyhaH described in Section 2.1.1
above. Yield: 74%. Anal. Calc. for C6H6N2O2: C, 52.17; H,
1
4.38; N, 20.28. Found: C, 52.20; H, 4.26; N, 20.18%. H
NMR (400 MHz, [d6]-DMSO, 25 ꢁC): d = 11.42 (s, 1H,
OH), 9.25 (s, 1H, NH), 8.57 (dd, 2H, aromatic H), 7.54
(dd, 2H, aromatic H).
2.1.9. cis-[PtIVCl4(4-pyhaH)2] Æ 2CH3OH (12)
Yield: 63%. Anal. Calc. for PtC14H20N4O6Cl4: C, 24.83;
H, 2.98; N, 8.27; Cl, 20.94. Found: C, 24.77; H, 2.91; N,

4700
D. Griffith et al. / Polyhedron 26 (2007) 4697–4706
8.22; Cl, 20.78%. 1H NMR (400 MHz, [d6]-DMSO, 25 ꢁC):
d = 11.88 (s, 2H, OH), 9.64 (s, 2H, NH), 8.85 (dd, 4H, aro-
matic H), 7.97 (dd, 4H, aromatic H).
Table 1
Summary of crystal data for 12
Empirical formula
Formula weight
Temperature (K)
C14H20N4O6Cl4Pt
677.23
100(2)
2.1.10. cis-[PtIVCl4(3-pycaH)2] (13)
˚
Wavelength (A)
0.71073
orthorhombic
Pbcn
Yield: 61%. Anal. Calc. for PtC12H10N2O4Cl4: C, 24.72;
H, 1.73; N, 4.80; Cl, 24.32. Found: C, 24.70; H, 1.69; N,
4.63; Cl, 24.11%. 1H NMR (400 MHz, [d6]-DMSO,
25 ꢁC): d = 9.23 (s, 2H, aromatic H), 8.61 (m, 4H, aromatic
H), 7.66 (dd, 2H, aromatic H).
Crystal system
Space group
Unit cell parameters
˚
a (A)
7.3794(5)
11.9791(8)
24.8071(17)
˚
b (A)
˚
c (A)
a (ꢁ)
b (ꢁ)
c (ꢁ)
Volume (A ), Z
Dcalc (Mg/m3)
Absorption coefficient (mmꢀ1
F(000)
Crystal size (mm)
h Range for data (ꢁ)
Index ranges
90
90
90
2.1.11. cis-[PtIVCl4(4-pycaH)2] (14)
Yield: 67%. Anal. Calc. for PtC12H10N2O4Cl4: C, 24.72;
H, 1.73; N, 4.80; Cl, 24.32. Found: C, 24.56; H, 1.91; N,
4.55; Cl, 24.17%. 1H NMR (400 MHz, [d6]-DMSO,
25 ꢁC): d = 8.79 (dd, 4H, aromatic H), 7.98 (dd, 4H, aro-
matic H).
3
˚
2192.9(3), 4
2.051
6.924
1304
0.30 · 0.20 · 0.20
3.21–29.46
ꢀ9 6 h 6 9, ꢀ16 6 k 6 16,
ꢀ33 6 l 6 32
18484
2891 (0.0289)
94.8
semi-empirical from equivalents
0.3381 and 0.2305
)
2.1.12. K[RuII(Hedta)(NO)Cl] Æ H2O (15)
cis-[PtIICl2(3-pyhaH)2] Æ 2H2O (6) (0.29 g, 0.50 mmol)
was added to a yellow aqueous solution (15 ml) of
K[RuIII(Hedta)Cl] Æ 2H2O (0.25 g, 0.50 mmol), resulting in
a reddish solution. The reaction was refluxed for 3 h where-
upon it turned brown in colour. The reaction was concen-
trated in vacuo (ꢁ5 ml), purified on a sephadex column
LH20 using water as eluent to give K[Ru(Hedta)(NO)-
Cl] Æ H2O as a brown solid. Yield: 58%. Anal. Calc. for
RuC10H15N3ClKO10: C, 23.42; H, 2.95; N, 8.19; Cl, 6.91;
K, 7.62. Found: C, 23.45; H, 2.91; N, 8.42; Cl, 6.97; K,
7.46%. 1H NMR (400 MHz, D2O, 25 ꢁC): d = 4.43 (d,
2H, CH2 glycine), 4.34 (s, 2H, CH2 glycine), 4.09 (2H, s,
CH2 glycine), 3.84 (2H, s, CH2 glycine) 3.61 (4H, m, CH2
ethylenediamine); 13C NMR (400 MHz; D2O; 25 ꢁC):
d = 181.8, 178.5 (CO bound glycinate), 177.7, 168.8 (CO
free glycinate), 65.45, 65.4, 64.7, 64.3 (CH2 glycinate),
62.8, 60.4 (CH2 ethylenediamine); mmax/cmꢀ1 3345s (OH),
2986s, 2947s (CH, H2edta), 1894vs (NO), 1726s (CO free
glycine arms) and 1649b (CO bound glycine arms); ESI-
MS m/z: 456 ([MꢀH]ꢀ).
Reflections collected
Independent reflections (Rint
Completeness to h = 29.46ꢁ (%)
Absorption correction
Maximum and minimum
transmission
)
Refinement method
full-matrix least-squares on F2
2891/0/172
Data/restraints/parameters
Goodness-of-fit on F2
Final R indices [I > 2r(I)]
R indices (all data)
1.049
R1 = 0.0217, wR2 = 0.0453
R1 = 0.0277, wR2 = 0.0471
Largest difference in peak and hole 1.713 and ꢀ0.904
ꢀ3
˚
(e A
)
P
P
P
P
2
2 1=2
R1 = iFoj ꢀ jFci/ jFoj, wR2 ¼ ½ wðF 2o ꢀ F c2Þ = wðF o2Þ ꢂ
.
2.3. Pharmacological tests
The NO-releasing properties of the test compounds were
investigated using an isolated organ bath experiment as fol-
lows. Thoracic aorta rings were prepared from male Wistar
Hannover rats weighing about 250–400 g purchased from
the animal facility of the University of Trieste. The animals
were maintained according to the guidelines in force in
Italy (DDL 116, 21/02/92 and subsequent addenda) and
in compliance with the ‘‘Guide for the care and use of lab-
oratory animals’’ DHIIS Publ. No (NHI) 86-23, Bethesda,
MD, NHI (1985). Rat thoracic aortas were carefully
removed, after killing of the animals by CO2 overdose,
put into a Petri dish containing Krebs solution (containing
118.3 mM NaCl, 25 mM NaHCO3, 4.8 mM KCl, 2.5 mM
CaCl2, 1.2 mM MgSO4, 1.2 mM KH2PO4, 11.1 mM glu-
cose, at pH 7.4), and cleaned of fat and of connective tis-
sue. Aorta rings, cut at about 2 mm, were put into a
20 mL organ bath (model 4050, Ugo Basile, Padova Italy)
with constant gas flow (95% O2 + 5% CO2) at 37 0.5 ꢁC.
Two stainless steel hooks were inserted into the aortic
lumen to fix the test sample to the bath bottom and to con-
nect it to a force transducer. Aortic rings were equilibrated
with Krebs solution and maintained under an optimal ten-
K[RuII(Hedta)(NO)Cl] Æ H2O (15) was likewise obtained
by reacting cis-[PtIICl2(4-pyhaH)2] Æ H2O (7) with K[RuIII
(Hedta)Cl] Æ 2H2O in line with the procedure described
above.
2.2. Structural characterisation of cis-[PtIVCl4(4-pyhaH)2] Æ
2CH3OH (12)
Crystal data and experimental details for 12 are summa-
rised in Table 1. The structure of 12 was refined in the Pbcn
space group. Crystal data were collected on a Bruker Smart
Apex CCD diffractometer at 100 K using monochromated
˚
Mo Ka radiation, (k = 0.71073 A) and the / ꢀ x scan
method. The structure was solved by direct method
(SHELXS-97) [26] and refined by full-matrix least squares
using SHELXL 97-2 package [26]. All hydrogen atoms were
localised in the difference fourier map and allowed to refine
freely.

D. Griffith et al. / Polyhedron 26 (2007) 4697–4706
4701
sion of 2 g for 40 min before experiments. Rings were stim-
ulated with 30 mM KCl to assess the integrity of the strips
(rings with abnormal contraction were discarded), and
responsive rings were rinsed with Krebs solution and
allowed to equilibrate for a further 30 min. Contraction
was elicited by adding phenylephrine (dissolved in ultra-
pure water) to the bath (final concentration 3 · 10ꢀ7 M).
Once sample contraction reached a plateau, the relaxa-
tion–response curve was obtained by cumulative additions
of acetylcholine (dissolved in ultrapure water) to the organ
bath (final concentrations ranging between 10ꢀ9 M and
10ꢀ4 M). After their maximal relaxation, aorta rings were
rinsed with Krebs solution and allowed to re-equilibrate
for 30 min. Contraction was elicited again by phenyleph-
rine, and compounds 1–10 were tested in the concentration
interval between 10ꢀ9 and 10ꢀ4 M (stock solutions were
prepared in DMSO with subsequent dilutions in ultra-pure
water in order to keep DMSO in the bath to less than
0.1%); cisplatin was used as control, at the same concentra-
tions and conditions.
The vehicle used for dissolving the test complexes was
also tested for relaxation. Contraction or relaxation were
recorded isometrically via a force–displacement transducer
(model 7003, Ugo Basile, Padova, Italy) connected to a
Unirecord Microdynamometer recorder (model 7050,
Ugo Basile, Padova, Italy). Experiments with compounds
6 and 8 were repeated in the presence of 10 lM methylene
blue (dissolved in ultrapure water), a guanylate cyclase
inhibitor [27]. In this case, methylene blue was added to
the organ bath, for 10 min, every time before inducing con-
traction with phenylephrine. For each compound, experi-
ments were replicated at least three times.
sponding to mC@O are found as only one stretch due to
intramolecular hydrogen bonding, while the broad mO–H
of the hydroxamato may be attributed to intermolecular
hydrogen bonding. These values concur with those previ-
ously reported in the literature [29,30]. Selected IR spectral
data for 1 and 2 are provided in Supplementary data.
The 1H NMR spectra of the hydroxamic acids 1 and 2 in
d6-DMSO show N–H and O–H resonances at ca. 9.2 ppm
and 11.3 ppm, respectively, both of which are concentra-
tion-dependent due to extensive intermolecular hydrogen
1
bonding at high concentrations. The H NMR spectrum
of 1 exhibits 4 resonances at 8.77, 8.55, 7.93 and
7.33 ppm corresponding to the four protons of the meta-
substituted pyridine ring, while that of 2 has two
resonances at 8.57 and 7.54 ppm corresponding to the four
protons of the para-substituted derivative.
3.2. Synthesis and characterisation of PtII and PtIV
complexes 6–14
The dichlorido PtII complexes, 6–10 and their PtIV ana-
logues, 11–14 were prepared by reaction of K2[PtIICl4] or
K2[PtIVCl6], respectively, with the appropriate pyridine
derivative (pyd) in aqueous or aqueous/methanolic solu-
tions (Scheme 1).
The previously reported PtII pyridine analogue, cis-
[PtIICl2(py)2] 10 [31], was also synthesised to serve as a ref-
erence standard in biological testing. In all cases the yellow
PtII and PtIV products were obtained in good yield (60–
80%) and excellent purity and were further characterised
1
by IR and H NMR spectroscopy. The cis-configurations
of the PtII complexes, 6–10 (Fig. 2), were confirmed by
the classic Kurnakov test [32].
2.4. Statistical analysis
The spectroscopic data for the PtII and PtIV pyhaH and
pycaH complexes are consistent with coordination via the
pyridine nitrogen and reveal the free nature of the uncoor-
dinated hydroxamic or carboxylic acid moiety as expected.
Selected IR and far-IR spectral data for the free ligands
and PtII and PtIV complexes are provided in Supplementary
data (Table 1). Briefly the mC@O of the Pt pyhaH and pycaH
complexes are found at ca. 1660 and 1705 cmꢀ1, respec-
tively, and as expected are comparable to those of the cor-
responding free ligands. The hydroxamic acid complexes,
in addition, display sharp mN–H and broad mO–H stretches,
again comparable to those of the corresponding free
ligands. Far-IR data can also be particularly useful in
determining the stereochemistry of the complexes
Data were submitted to computer-assisted statistical
analysis using ANOVA analysis of variance and Tukey–
Kramer multiple comparison post-test or Student t-test.
3. Results and discussion
3.1. Synthesis and spectroscopic characterisation of ligands
1–4
3- and 4-pyridinecarboxylic acid (3- and 4-pycaH) were
used as purchased and a selection of IR spectral data are
provided in Supplementary data.
The hydroxamic acids were synthesised by reacting the
corresponding carboxylic methyl or ethylesters with
hydroxylamine in aqueous methanol in line with literature
methods [28]. They were obtained in good yields (55–85%)
and excellent purity and were characterised by elemental
R
R
R
Cl
Pt
Cl
N
N
Cl
Cl
Cl
Cl
K2[PtIVCl6
]
K2[PtIICl4]
Pt
1
analysis, IR and H NMR spectroscopy. The IR spectra
N
N
N
of 3-pyhaH 1 and 4-pyhaH 2 contain stretches between
3180–3190 cmꢀ1, 2800–2850 cmꢀ1 and 1640–1660 cmꢀ1
which may be assigned to mO–H, mN–H and mC@O, respec-
tively. The symmetric and asymmetric stretches corre-
R
R= H, CONHOH, COOH
R
(a)
(b)
Scheme 1. Synthesis of (a) cis-[PtIICl2(pyd)2] and (b) cis-[PtIVCl4(pyd)2].

4702
D. Griffith et al. / Polyhedron 26 (2007) 4697–4706
OH
OH
O
N
O
H
H
N
N
N
N
N
Cl
Cl
Cl
Cl
Pt
Pt
O
H
N
N
O
H
OH
OH
cis-[PtIICl2(3-pyhaH)2] 6
cis-[PtIICl2(4-pyhaH)2] 7
HO
O
OH
O
N
N
N
N
N
N
Cl
Cl
Cl
Cl
Cl
Pt
Cl
Pt
Pt
O
OH
HO
O
cis-[PtIICl2(4-pycaH)2] 9
cis-[PtIICl2(py)2] 10
cis-[PtIICl2(3-pycaH)2] 8
Fig. 2. Structures of the dichlorido PtII complexes.
synthesised. For a square planar PtII complex of general
formula PtCl2L2, where L is a monodentate ligand, the
trans-isomer (D2h) exhibits only one mPtꢀCl whereas the
cis-isomer (C2v) exhibits two stretches. For PtIV complexes
of type PtCl4L2, the trans-isomer (D4h) exhibits one mPtꢀCl
and the cis-isomer (C2v) exhibits four [33]. The far-IR data
support the cis configuration for 6 and 8–14. Occasionally
the mPt–Cl merge as is the case with cis-[PtIICl2(4-pyhaH)2] 7
and the configuration of the complex cannot be accurately
determined from far-IR data alone.
3.3. Structural characterisation of cis-[PtIVCl4(4-
pyhaH)2] Æ 2CH3OH (12)
Yellow crystals of 12 suitable for a single crystal X-ray
diffraction experiment were obtained by slow evaporation
from a methanol solution. The atomic numbering scheme
and atom connectivity for 12 are shown in Fig. 3 and a
selection of bond lengths and angles reported in Table 2.
In 12, the PtIV complex displays a near perfect octahedral
geometry, not surprisingly given its symmetric nature.
The N(1)–Pt–N(1) and the Cl(2)#1–Pt–Cl(2) bond angles
are 178.42(6)ꢁ and 179.86(4)ꢁ, respectively. The N(1)–Pt–
Cl(1)#1 and N(1)–Pt–Cl(2)#1 bond angles are 89.91(6)ꢁ
and 89.21(7)ꢁ, respectively. The Pt–Cl bond lengths range
1
The H NMR spectra for all the Pt complexes support
the presence of one isomer only. For the pyhaH complexes
6, 7, 11 and 12 in d 6-DMSO, N–H and O–H resonances are
observed at ca. 9.3 ppm and ca.11.5 ppm, respectively, and
are, as expected, comparable to those of the free ligands.
The 1H NMR spectrum of cis-[PtIICl2(3-pyhaH)2] contains
4 resonances at 8.98, 8.76, 8.14 and 7.47 ppm correspond-
ing to the eight protons of the meta-substituted pyridine
rings. A similar overall pattern is observed in the 1H
NMR spectra of the PtII and PtIV complexes with 4 and
2 resonances integrating well for the complexes with coor-
dinated meta and para-substituted pyridine rings, respec-
tively, and a shift consistent with complexation.
˚
from 2.295(1) to 2.308(1) A and the shorter Pt–N bond
˚
lengths are 2.062(2) A. The two pyhaH ligands lie in the
cis-configuration where each is present as a monodentate
ligand coordinated via the pyridine nitrogen only with an
uncoordinated hydroxamic acid functional group.
In addition, hydroxamic acids are known to adopt one
of two tautomeric forms, the enol and the keto, each of
which can exist as E or Z conformers [19], where the stereo-
descriptors E and Z are used to describe the trans and cis

D. Griffith et al. / Polyhedron 26 (2007) 4697–4706
4703
ing Z conformation. The hydroxamic acid NH and OH
each hydrogen-bond to the oxygen acceptor atoms of dif-
ferent methanol solvent molecules with NHꢃ ꢃ ꢃO and
O1
O1
N2
O2
O2
C6
C6
˚
N2
OHꢃ ꢃ ꢃO bond length of approximately 2.00 and 1.81 A,
C3
C2
C4
C3
C2
C4
C5
N1
respectively, while the hydroxamic acid carbonyl oxygen
hydrogen-bonds to a third methanol solvent molecule with
C5
C1
N1
C1
˚
an Oꢃ ꢃ ꢃHO bond length of approximately 1.86 A (Supple-
mentary data, Fig. 1 and Table 2). With the complex
situated on a twofold axis, a three-dimensional hydrogen-
bonded lattice results (Fig. 4). The hydrogen-bond
distances and angles are presented in Supplementary data
(Table 2).
Pt
Cl2
Cl1
Cl2
Cl1
The crystal structure of 12, together with a comparison
of the far-IR spectral data for 12 with those of 11, 13 and
14, assist in the definitive assignment of the cis-configura-
tion for each of the PtIV pyhaH and pycaH complexes syn-
thesised (Fig. 5). Selected IR and far-IR spectral data are
provided in Supplementary data (Table 1).
Fig. 3. ORTEP diagram of 12 with solvent molecules omitted. The atomic
numbering scheme is displayed (thermal ellipsoids at 80% probability level).
Table 2
˚
Selected bond lengths (A) and angles (ꢁ) with estimated standard
deviations for 12
˚
Bond atoms
Distance (A)
Angle atoms
Angle (ꢁ)
3.4. Synthesis and characterisation of
K[RuII(Hedta)(NO)Cl] Æ H2O (15)
Pt–N(1)
2.062(2)
2.062(2)
2.2951(7)
2.2951(7)
2.3084(7)
2.3084(7)
1.497(3)
1.232(3)
1.330(3)
1.394(3)
N(1)–Pt–N(1)#1
N(1)–Pt–Cl(1)#1
N(1)#1–Pt–Cl(1)#1
N(1)–Pt–Cl(1)
N(1)#1–Pt–Cl(1)
Cl(1)#1–Pt–Cl(1)
N(1)–Pt–Cl(2)#1
N(1)#1–Pt–Cl(2)#1
Cl(1)#1–Pt–Cl(2)#1
Cl(1)–Pt–Cl(2)#1
N(1)–Pt–Cl(2)
91.66(12)
89.91(6)
178.42(6)
178.42(6)
89.91(6)
88.51(4)
89.21(7)
90.69(7)
89.18(3)
90.93(3)
90.69(7)
89.21(7)
90.93(3)
89.18(3)
179.86(4)
Pt–N(1)#1
Pt–Cl(1)#1
Pt–Cl(1)
Pt–Cl(2)#1
Pt–Cl(2)
C(3)–C(6)
C(6)–O(1)
C(6)–N(2)
N(2)–O(2)
To establish that the free hydroxamic acid moieties in
cis-[PtIICl2(x-pyhaH)2] (x = 3, 4) are sources of NO, we
reacted cis-[PtIICl2(x-pyhaH)2] with the well-known ruthe-
nium (Ru) NO scavenger, K[RuIII(Hedta)Cl] Æ 2H2O,
which resulted in the facile formation of the RuII-nitrosyl,
K[RuII(Hedta)(NO)Cl] Æ H2O. Reaction of cis-[PtCl2(py)2]
with K[RuIII(Hedta)Cl] Æ 2H2O did not yield a RuII-nitrosyl
product.
N(1)#1–Pt–Cl(2)
Cl(1)#1–Pt–Cl(2)
Cl(1)–Pt–Cl(2)
The RuII–NO+ complex, K[RuII(Hedta)(NO)Cl] Æ H2O
(15) was obtained upon reaction of cis-[PtIICl2(x-pyhaH)2]
(x = 3, 4) i.e. 6 or 7 with the well-known NO scavenger,
Cl(2)#1–Pt–Cl(2)
K[RuIII(Hedta)Cl] Æ 2H2O. Elemental analysis, IR, H and
1
13C NMR and ESI-mass spectra are consistent with the
formulation, K[RuII(Hedta)(NO)Cl] Æ H2O and concur
with previously reported examples of RuII-edta-NO
orientation of the OH and C@O groups of the hydroxamic
acid. The X-ray structural determination of 12 evidenced
the free hydroxamic acid moieties in the keto form, adopt-
c
b
C
H
N
O
Cl
Pt
Fig. 4. Crystal packing of 12. View of three-dimensional hydrogen-bonded lattice along [100] (thermal ellipsoids at 50% probability level).

4704
D. Griffith et al. / Polyhedron 26 (2007) 4697–4706
OH
N
Table 3
The free ligands, benzohydroxamic acid and the PtII compounds and their
OH
O
H
O
corresponding reference numbers
N
H
3-Pyridinehydroxamic acid (3-pyhaH)
4-Pyridinehydroxamic acid (4-pyhaH)
3-Pyridinecarboxylic acid (nicotinic acid) (3-pycaH)
4-Pyridinecarboxylic acid (isonicotinic acid) (4-pycaH)
Benzohydroxamic acid (bhaH)
cis-[PtIICl2(3-pyhaH)2] Æ 2H2O
1
2
3
Cl
Cl
Pt
Cl
N
N
N
N
4
Cl
Cl
Cl
Cl
Pt
Cl
5
6
cis-[PtIICl2(4-pyhaH)2] Æ H2O
7
cis-[PtIICl2(3-pycaH)2]
8
O
cis-[PtIICl2(4-pycaH)2]
9
10
cis-[PtIICl2(py)2]
H
N
N
O
H
OH
OH
ied using thoracic aorta rings isolated from rats. Given the
fact that PtIV compounds are thought to be activated by
reduction to their corresponding PtII analogues, the NO-
releasing properties of the PtIV compounds herein
described were not investigated.
cis-[PtIVCl4(3-pyhaH)2] 11
cis-[PtIVCl4(4-pyhaH)2] 12
HO
O
OH
O
Compounds 6 and 7, as well as their ligands 1 and 2,
induce the same extent (ꢁ80% at the highest concentration
used) of vasorelaxation of aortic rings pre-contracted with
phenylephrine, irrespective of the position in the pyridine
ring of the hydroxamic acid moiety (Fig. 6). Benzohydrox-
amic acid, used as a positive control [18], induces vasore-
laxation to a lesser extent (Fig. 8). Ligands characterised
by the presence of a carboxylic acid group, i.e. compounds
3 and 4, induce mild relaxation of aorta rings, comparable
to that induced by water (the solvent used to dissolve these
compounds), that reaches approximately 30% at concen-
trations P10ꢀ7 M (Fig. 7). In contrast, the corresponding
Pt derivatives 8 and 9 cause vasorelaxation statistically (3
versus 8 p < 0.02 for concentrations P10ꢀ7 M) greater
than that of the free ligands, with a maximum of approxi-
mately 60%, at the highest concentration used (Fig. 7).
Cisplatin and cis-[PtIICl2(py)2] 10, studied in the same
experimental model (Fig. 8), do not induce relaxation of
the aorta rings (relaxation comparable to that induced by
Cl
Pt
Cl
Cl
N
N
Cl
Cl
Cl
Pt
Cl
N
N
Cl
O
OH
HO
O
cis-[PtIVCl4(3-pycaH)2] 13
cis-[PtIVCl4(4-pycaH)2] 14
Fig. 5. Structures of tetrachlorido PtIV complexes.
complexes in the literature [34]. The IR spectra of 15
contains a distinctive mNO at 1894 cmꢀ1. ESI-MS in the
negative mode was used to unequivocally identify the
RuII–NO+ complex, [RuII(Hedta)(NO)Cl]ꢀ; a mass peak
at 456 amu with the correct isotopic abundances was
observed. Previous reported reactions of K[RuIII(Hedta)Cl] Æ
2H2O with hydroxamic acids such as benzohydroxamic
acid gave the RuII–NO+ complex, K2[RuII(edta)(NO)Cl]
[18]. The doubly protonated edta complex Ru(H2edta)
(NO)Cl was previously isolated by reacting K[Ru(Hedta)-
Cl] Æ 2H2O with N-methyl-N-nitroso-p-toluenesulfonamide
or NO gas under argon [34]. Depending on pH, the RuII-
NO+ complexes can be isolated with the free arm of the
ethylenediaminetetraacetate in the protonated or the anio-
nic form.
100
75
50
25
0
3.5. Pharmacological tests
The free ligands 1–4, benzohydroxamic acid 5 and the
PtII compounds 6–10 and their corresponding reference
numbers are listed in Table 3.
-10 -9
-8
-7
log [M]
-6
-5
-4
1
2
6
7
The effects on vasorelaxation of PtII compounds 6–9, the
hydroxamic acid ligands 1 and 2, the carboxylic acid
ligands 3 and 4 and the control compounds, benzohydrox-
amic acid 5, cis-[PtIICl2(py)2] (10), and cisplatin were stud-
Fig. 6. Effects of compounds 3-pyhaH 1, 4-pyhaH 2 and of respective
platinum compounds cis-[PtIICl2(3-pyhaH)2] (6) and cis-[PtIICl2(4-
pyhaH)2] (7) on aortic ring relaxation.

D. Griffith et al. / Polyhedron 26 (2007) 4697–4706
4705
100
75
50
25
0
100
75
50
25
0
-10
-9
-8
-7
log [M]
-6
-5
-4
-10
-9
-8
-7
log [M]
-6
8
-5
-4
6
6+MB
8+MB
3
4
8
9
Fig. 9. Effects of cis-[PtIICl2(3-pyhaH)2] (6) and cis-[PtIICl2(3-pycaH)2] (8)
in the presence or absence of methylene blue (MB).
Fig. 7. Effects of compounds 3-pycaH 3, 4-pycaH 4 and of respective
platinum compounds cis-[PtIICl2(3-pycaH)2] (8) and cis-[PtIICl2-
(4-pycaH)2] (9) on aortic rings relaxation.
release NO. It may therefore be hypothesised that the
mechanism of vasorelaxation of aortic rings is probably
due to stimulation of NOS activity of the aorta endothelial
cells, to which NO-release from compounds 6 and 7 might
also contribute, at least at concentrations higher than
3 · 10ꢀ5 M.
100
75
50
25
0
4. Conclusions
A series of novel PtII and PtIV pyridinehydroxamic acid
complexes were synthesised with a view to advancing a new
class of metallodrug with dual DNA-binding and NO-
releasing properties. We can conclude from our investiga-
tion that the PtII pyridinehydroxamic acid derivatives
induce potent in vitro effects attributable to either NO-
release from the hydroxamic acid moiety and/or stimula-
tion of iNOS of the endothelial cells. Considering the role
of NO and iNOS activation in the process of proliferation
and metastases, the data of the present work suggest fur-
ther in vitro and in vivo testing of these compounds on cell
proliferation and solid metastasising tumours, respectively.
The complexes herein described are not however suffi-
ciently soluble for a full biological investigation. We are
currently in the process of developing analogues with supe-
rior aqueous solubility.
-10 -9
-8
-7
log [M]
-6
-5
Cisplatin
-4
H2O
5
10
Fig. 8. Effects of water, benzohydroxamic acid 5, cis-[PtIICl2(py)2] (10)
and cisplatin on aortic rings relaxation.
water) suggesting the lack of any role of the Pt ion per se in
this phenomenon.
The involvement of NO in the induced vasorelaxation of
compounds 6 and 8 was further investigated in the presence
and absence of the guanylate cyclase inhibitor methylene
blue (Fig. 9). Methylene blue completely abolished the
vasorelaxation induced by both compounds 6 and 8, indi-
cating the involvement of the NO signaling pathway, like-
wise in the case of compound 8, a derivative free of the
hydroxamic acid moiety.
This data show that both hydroxamic acid and carbox-
ylic acid derivatives in the rat aortic ring model cause NO-
dependent releasing properties through a mechanism
involving the NO signaling pathway, as supported by the
inhibitory effects of the guanylate cyclase inhibitor methy-
lene blue. The former exhibited enhanced NO-dependent
releasing properties relative to the latter. It should be
stressed that only hydroxamic acid derivatives, via the
hydroxamic acid functional group, have the potential to
Acknowledgements
The authors thank Enterprise Ireland under its Basic
Research Grant Programme and International Collabora-
tion Scheme, EU COST D20, EU COST D39 and the Pro-
gramme for Research in Third Level Institutions (PRTLI),
administered by the HEA for funding. MADE project at
Callerio Foundation is also acknowledged for support.
We thank Dr. Dilip Rai (School of Chemistry and Chem-
ical Biology, Science Education and Research Centre,
UCD, Belfield, Dublin 4, Ireland) for MS analysis.
C.J.M. gratefully acknowledges Johnson Matthey for the
generous loan of K2[PtIICl4] and K2[PtIVCl6].

4706
D. Griffith et al. / Polyhedron 26 (2007) 4697–4706
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Appendix A. Supplementary material
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[16] G.R. Thatcher, Curr. Top. Med. Chem. 5 (2005) 597.
[17] R. Scatena, P. Bottoni, G.E. Martorana, B. Giardina, Expert Opin.
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[20] E. Alessio, G. Mestroni, A. Bergamo, G. Sava, Curr. Top. Med.
Chem. 4 (2004) 1525.
CCDC 251892 contains the supplementary spectro-
scopic data for 1–4 and 6–14 and the crystallographic data
for 12. These data can be obtained free of charge via http://
www.ccdc.cam.ac.uk/conts/retrieving.html, or from the
Cambridge Crystallographic Data Centre, 12 Union Road,
Cambridge CB2 1EZ, UK; fax: (+44) 1223-336-033; or
e-mail: deposit@ccdc.cam.ac.uk. Supplementary data asso-
ciated with this article can be found, in the online version,
at doi:10.1016/j.poly.2007.03.011.
[21] M.J. Clarke, Coord. Chem. Rev. 236 (2003) 209.
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