New antitumour active platinum compounds containing carboxylate ligands in trans geometry: Synthesis, crystal structure and biological activity

Article abstract of DOI:10.1039/b512357g

New asymmetric trans-platinum(ii) complexes, composed of an isopropylamine, an azole and two carboxylate leaving groups, are presented. The crystal and molecular structures of one of the complexes has been determined and the cytotoxicity and reactivity with 5′-guanosine monophosphate is reported. The complexes show a reduced reactivity, but no decrease in cytotoxic activity compared to their chloro-counterparts. Furthermore the complexes largely overcome cisplatin resistance, they therefore present an interesting class of antitumour active trans-platinum complexes. The Royal Society of Chemistry 2006.

Full text of DOI:10.1039/b512357g

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www.rsc.org/dalton | Dalton Transactions
New antitumour active platinum compounds containing carboxylate ligands
in trans geometry: synthesis, crystal structure and biological activity
Steven van Zutphen,a Elena Pantoja,a Rosario Soriano,a Carlos Soro,a Duncan M. Tooke,b Anthony L. Spek,b
Hans den Dulk,a Jaap Brouwera and Jan Reedijk*a
Received 31st August 2005, Accepted 23rd November 2005
First published as an Advance Article on the web 7th December 2005
DOI: 10.1039/b512357g
New asymmetric trans-platinum(II) complexes, composed of an isopropylamine, an azole and two
carboxylate leaving groups, are presented. The crystal and molecular structures of one of the complexes
has been determined and the cytotoxicity and reactivity with 5ꢀ-guanosine monophosphate is reported.
The complexes show a reduced reactivity, but no decrease in cytotoxic activity compared to their
chloro-counterparts. Furthermore the complexes largely overcome cisplatin resistance, they therefore
present an interesting class of antitumour active trans-platinum complexes.
Introduction
was based on literature procedures.19 These compounds, contain
an azole and an isopropylamine ligand trans to one another, and
have shown high in vitro activities in both cisplatin sensitive and
cisplatin resistant cell lines.18 This activity may be related to the fact
that these complexes react with DNA in a monofunctional manner,
quite unlike platinum complexes with cis geometry.18 Using these
chloro compounds as starting materials in the synthesis of the
target compounds 3 and 4, the Cl− ligands have to be exchanged
for OAc− (see Scheme 1 for the reactions). Stirring the complexes
in aqueous sodium acetate solution over a long period of time
was expected to yield the desired products. However, the rate of
reaction is rather low, due to the slow hydrolysis of the platinum–
chloride bond. In order to increase the reaction rate, Ag+ ions
can be added to the solution.20 In this case, the precipitation
of AgCl moves the equilibrium towards the hydrated species,
thereby greatly increasing the rate of product formation. Treating
the chloro complexes 1 and 2 with AgOAc yielded the desired
complexes 3 and 4 in a simple overnight reaction. This silver salt
was prepared by neutralising acetic acid with NaOH followed by
treatment with AgNO3. Due to the low solubility of this silver salt,
best results were obtained by treating the chloro compounds with
Cisplatin and many other platinum drugs are known DNA-
binding agents.1 The majority of platinum drugs contain two
leaving groups, allowing the complexes to react with DNA in
a bifunctional manner. In early structure–activity relationship
studies both the nature and geometry of these leaving groups
was investigated.2–4 It was concluded that for antitumour activity
platinum complexes must have two labile leaving groups in cis
geometry. Compounds with Cl− or Br− leaving groups display
mostly good activity, however more stable ligands can also yield
active compounds. In fact, the second generation platinum drugs,
carboplatin, nedaplatin and oxaliplatin have all exchanged the Cl−
leaving groups of cisplatin for kinetically less labile chelating di-
carboxylate or glycolate ligands.5–8 As a result patients treated with
these compounds suffer from less severe dose-limiting side effects.
In more recent years, a number of trans compounds with high anti-
tumour activities in both cisplatin-sensitive and cisplatin-resistant
cell lines have been discovered.9–13 Although these compounds have
not yet entered the clinic, it is not unlikely that their treatment will
be obstructed by dose-limiting side effects similar to cisplatin, ow-
ing to the nature of the Cl− leaving groups. It has been shown that
the chlorides on various trans platinum complexes can be replaced
efficiently by acetate ligands.14,15 Furthermore, it was recently
discovered that trans-diaminedicarboxylate complexes can have
greatly increased aqueous solubility compared to their dichloro
counterparts.16,17 In the current study the influence of the acetate
ligand on the reactivity and in vitro cytotoxicity of two recently dis-
covered active trans amine platinum complexes is described in de-
tail, including the crystal and molecular structures of one of them.
Results and discussion
Synthesis and characterisation
The known, earlier reported active trans-platinum complexes 1 and
2 served as a starting point for the current study.18 Their synthesis
aLeiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University,
P.O. Box 9502, 2300, RA Leiden, The Netherlands. E-mail: reedijk@
chem.leidenuniv.nl; Fax: +31 71 527 4671; Tel: +31 71 527 4459
Scheme 1 Synthesis of trans carboxylate complex trans-[Pt(OAc)2-
(isopropylamine)(N-methylimidazole)] 3 and trans-[Pt(OAc)2(isopropy-
lamine)(N-methylpyrazole)] 4 starting from the chloride precursor 1 or
2 respectively. a) AgOAc in H2O.
bCrystal and Structural Chemistry, Utrecht University, Padualaan 8, 3584,
CH Utrecht, The Netherlands
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a small excess of AgOAc overnight at 40 ◦C in water. The platinum
carboxylate complexes formed in solution so that they could be
separated by filtration from the excess AgOAc, unreacted dichloro
complex and AgCl formed during the reaction. Any silver ions
remaining in the solution were reduced by exposing the reaction
to light and the reaction mixture was filtered again. Freeze-drying
the resultant solution yielded compounds 3 and 4 in low, but
acceptable yield (10–15%) of high purity. The structural integrity
of the complexes was verified using 1H NMR, 195Pt NMR and ESI-
MS. For complex 3 crystals where obtained by slow evaporation
of a methanolic solution of the compound and used in a single
crystal X-ray structure determination (Fig. 1). This confirmed the
expected square-planar platinum moiety surrounded by the two
carboxylate ligands in trans position and the two nitrogen donor
ligands trans to one another, together with a single water molecule
of crystallisation.
of the complexes. These data clearly show that these complexes
present a promising class of platinum antitumour agents.
To gain insight into the reactivity of the complexes, directly
related to the toxicity of platinum complexes,21 the reaction
of complex 3 with an excess of the nucleobase 5ꢀ-guanosine
monophosphate (5ꢀGMP) was followed by 1H NMR at physiolog-
ical temperature in buffered solution and compared to carboplatin
(Fig. 2). In this reaction the labile ligands of a platinum complex
are replaced by the nucleobase coordinated via the N7 nitrogen
to give a bis-5ꢀGMP platinum adduct. In 0.1 M NaClO4 in D2O
the bisadduct of cisplatin is known to be formed rapidly and the
reaction is complete within 24 h.22 For carboplatin the reaction
rate is much slower, but the rate can be increased by adding
50 mM NaCl to the reaction.23 Under these conditions, the same
bisadduct is observed after 3 h of reaction, but it takes 6 days
for the reaction to reach completion. In the absence of NaCl
the platinum bis(5ꢀGMP) adduct of complex 3 is detectable only
after 4 days of reaction, and after 11 days only 30% of product
is formed. In the presence of NaCl the bisadduct is detectable
after 15 h and the reaction is complete after 11 days. At this
point a 195Pt NMR, showing a single peak at −2383 ppm, was
used to confirm the completed formation of the bisadduct. In
summary, the carboxylate complexes are kinetically much more
inert than either cisplatin or carboplatin. In contrast, compounds
1 and 2 are known to react with 5ꢀGMP at a rate only slightly
lower than cisplatin, and it takes a mere 48 h for the reaction
to reach completion.18 It appears therefore that the complexes
presented in the current paper are able to retain their cytotoxic
activity compared to their chloro-analogues, whilst their reactivity
towards DNA nucleobases is reduced significantly.
Fig.
1
Displacement ellipsoid plot (50%) of trans-[Pt(OAc)2-
(isopropylamine)(N-methylimidazole)] 3. H and H2O omitted for clarity.
Biological evaluation
To analyse the compounds’ potential as antitumour agents the
IC50 values were determined in two L1210 mouse leukaemia cell
lines, i.e. one sensitive and one resistant to cisplatin treatment. The
results are illustrated in Table 1. The compounds are moderately
active, slightly less active than cisplatin yet more active than
carboplatin in the sensitive cell line. Moreover, the complexes
largely retain their activity in the L1210R cell line, unlike cisplatin
or carboplatin. In fact, with a resistance factor of 1.7 and
1.3 respectively, complexes 3 and 4 show 10-fold less reduction
in activity in the resistant cell line than cisplatin. Although
such behaviour may be expected for asymmetric dichloro trans-
platinum complexes, it is of interest that changing the leaving
groups to acetate does not appear to alter the cytotoxic properties
Fig. 2 Graph showing the percentage of 5ꢀGMP coordinated relative to
the amount of platinum compound present, illustrating the difference in
reaction rate for 3 and carboplatin with or without the addition of NaCl
to the reaction mixture. ᭹: carboplatin (51 mM NaCl), ꢀ: 3 + 5ꢀGMP
(NaClO4), ꢁ: 3 + 5ꢀGMP (51 mM NaCl).
Conclusion
We have synthesised two representative examples of a group of new
platinum complexes for anti-tumour evaluation, based on known
active trans-dichloro compounds and simple carboxylate ligands.
The biological evaluation shows that these compounds display
high cytotoxicity in both cisplatin-sensitive and cisplatin-resistant
cell lines, whilst at the same time showing low reactivity in a
model reaction with 5ꢀGMP monitored by 1H NMR. Their chloro-
counterparts show similar cytotoxic activity but a fast reaction
with 5ꢀGMP. This fundamental differnce is similar to the difference
observed between cisplatin and carboplatin. The main advantage
Table 1 IC50 values (lM) for complexes 3, 4, cisplatin and carboplatin
in two different cell lines, L1210, L1210R mouse leukemia sensitive and
resistant to cisplatin on 72 h incubation
Entry
L1210
L1210R
3
6.43 0.71
5.98 0.52
1.73 0.08
10.2 1.69
11.1 0.34
7.86 0.92
27.5 0.90
>50
4
Cisplatin
Carboplatin
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Table 2 Crystallographic data for complex 3
of carboplatin over cisplatin is the reduced toxic side-effects in the
clinic. The results presented in the current paper may therefore
indicate that the asymmetric trans-carboxylate complexes could
display high antitumour activity in combination with reduced
toxic side effects in vivo, compared to either cisplatin or their
chloro-analogues. Current studies with the complexes aim to
verify this hypothesis. Furthermore, studies aimed at determining
whether our results can be generalised for other known active
trans-platinum(II) compounds are ongoing.
Chemical formula
Formula weight
Crystal system
Space group
PtC11H21N3O4·H2O
472.41
Monoclinic
P21/c
˚
a/A
9.8064(6)
12.5128(7)
15.6712(11)
90
123.406(5)
90
1605.3(2)
4
1.955
˚
b/A
˚
c/A
a/◦
b/◦
c /◦
3
˚
V/A
Z
Experimental
qcalc/g cm−3
l/mm−1
8.762
All NMR measurements were performed on a 300 MHz Bruker
DPX300 spectrometer with a 5 mm multi-nucleus probe. Tem-
perature was kept constant at 298 K or 310 K using a variable
temperature unit. 1H and 195Pt chemical shifts were referenced to
TSP and Na2PtCl4 (d = 0 ppm), respectively. The water signal for
spectra measured in D2O was minimized using a WATERGATE
pulse sequence.
Transmission
Crystal color
Crystal size/mm
No. refl. measured
No. unique refl.
No. parameters
R1 (all refl.)
0.34–0.5
Colourless
0.14 × 0.08 × 0.08
25395
3675
194
0.0604
0.0287
0.0519
0.0459
0.65
0.03
1.01
R1 (obs. refl.)
wR2 (all refl.)
wR2 (obs. refl.)
−1
˚
(sinh/k)max/A
Synthesis of 3
Rint
GoF(goodness of fit)
To trans-[PtCl2(isopropylamine)(N-methylimidazole)] 1 (20 mg,
49 lmol) in water (2 ml) was added AgOAc (147 lmol). The
suspension was stirred overnight in the dark at 40 ◦C. The
suspension was cooled to room temperature, filtered and the
solution was exposed to light for 1 h. The resultant suspension
was filtered and the solution was freeze-dried to yield complex 3
in 15% yield. 195Pt NMR (D2O) d (ppm): −1480. 1H NMR (D2O)
d (ppm): 1.24 (d, J = 6.43, 6H, CH(CH3)2), 1.84 (s, 3H, CH3-
acetate), 2.80 (sept, J = 6.68, 1H, CH(CH3)2), 3.67 (s, 1H, NCH3),
6.74 (d, J = 2.63, 1H, NCH), 7.04 (d, J = 2.63, 1H, NCH), 7.53
−3
˚
Residual electron density/e A
−1.22/0.81
X-Ray crystal structure determination
Nonius KappaCCD diffractometer, rotating anode, MoKa radi-
ation (graphite monocromator), 150(2) K. See Table 2 for crys-
tallographic data. Multi-scan absorption correction applied using
SADABS.25 Structure solved with DIRDIF9926 and refined with
SHELXL-97.27 Drawings, structure calculations, and checking for
additional symmetry performed with PLATON.28 Non-hydrogen
atoms refined freely with anisotropic displacement parameters.
H(5A) and H5(B) of the water molecule were located in a difference
map and freely refined with isotropic displacement parameters.
All other H atoms in geometrically idealized positions [d(C–H) =
0.98 for methyl H and 0.95 for other H] and constrained to ride
on parent atoms, Uiso(H) = 1.5Ueq(C) for methyl H and Uiso(H) =
1.2Ueq(C) for all other H.
(s, 1H, NCHN). ESI-MS: m/z: 454 [M + H]+, 477 [M + Na]+. IR
−1
=
(cm ): 1594 m(C O).
Synthesis of 4
As for 3 where 1 was replaced by trans-[PtCl2(isopropylamine)(N-
methylpyrazole)] 2. Yield 12%. 195Pt NMR (D2O) d (ppm): −1471.
1H NMR (D2O) d (ppm): 1.41 (d, J = 6.43, 6H, CH(CH3)2), 2.14
(s, 3H, CH3-acetate), 2.97 (sept, J = 6.68, 1H, CH(CH3)2), 3.85
(s, 1H, NCH3), 6.94 (t, J = 1.67, 1H, NCH), 7.25 (d, J = 1.39
1H, CHCHCH), 7.71 (d, J = 1.54, 1H, NCH). ESI-MS: m/z: 477
CCDC reference number 279701.
For crystallographic data in CIF or other electronic format see
DOI: 10.1039/b512357g
+
−1
=
[M + Na] . IR (cm ): 1594 m(C O).
Acknowledgements
5ꢀGMP reactions
This research was supported by the Council for Chemical Sciences
of the Netherlands Organisation for Scientific Research (CW-
NWO). Support and sponsorship by COST Action D20 is kindly
acknowledged. The authors thank Johnson & Matthey (Reading,
UK) for their generous loan of K2PtCl4.
A four-fold excess of 5ꢀGMP was reacted with complex 3 and
carboplatin at 310 K in 0.1 M NaClO4 in D2O or 0.1 M NaClO4
and 50 mM NaCl in D2O. All reactions were monitored by
recording a 1H NMR spectrum every 30 min for 16 h then every
24 h until no further changes were observed.
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