Synthesis and characterisation of estrogenic carriers for cytotoxic Pt(II) fragments: Biological activity of the resulting complexes

Article abstract of DOI:10.1039/b507716h

This paper describes the synthesis and the spectroscopic characterisation of cis-dichloro[N-(4-(17αethynylestradiolyl)-benzyl)-ethylenediamine] platinum(II) and cis-diamino[2-(4-(17α-ethynylestradiolyl)benzoylamino)- malonato]platinum(n). These complexes

Full text of DOI:10.1039/b507716h

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A R T I C L E
Synthesis and characterisation of estrogenic carriers for cytotoxic
Pt(II) fragments: biological activity of the resulting complexes†
Elisabetta Gabano,a Claudio Cassino,a Samuele Bonetti,a Cristina Prandi,a
Donato Colangelo,b AnnaLisa Ghigliab and Domenico Osella*a
a Dipartimento di Scienze dell’Ambiente e della Vita, Universita` del Piemonte Orientale
“A. Avogadro”, Spalto Marengo 33, 15100 Alessandria, Italy.
E-mail: domenico.osella@mfn.unipmn.it; Fax: +39 0131 287416; Tel: +39 0131 287429
b Dipartimento di Scienze Mediche, Universita` del Piemonte Orientale “A. Avogadro”, via
Solaroli 17, I-28100, Novara, Italy
Received 1st June 2005, Accepted 1st August 2005
First published as an Advance Article on the web 22nd August 2005
This paper describes the synthesis and the spectroscopic characterisation of cis-dichloro[N-(4-(17a-
ethynylestradiolyl)-benzyl)-ethylenediamine]platinum(II) and cis-diamino[2-(4-(17a-ethynylestradiolyl)-
benzoylamino)-malonato]platinum(II). These complexes were synthesised in good yield according to multi-step
procedures based on the classical and non-classical Sonogashira coupling reaction, respectively. These compounds
retain an acceptable degree of relative binding affinity (RBA) for the a form of estrogen receptor. Combined
treatment of breast cancer cell lines, namely hormone-sensitive MCF-7 and hormone-insensitive MDA-MB-231 cell
lines, indicates that these compounds maintain agonistic activity so that the potential advantage in vehiculation of
the cytotoxic moiety by means of the receptor system is counteracted by the proliferative effect of the estrogenic
component of the entire molecule, especially at low concentrations.
reaction between the resulting estradiol–arm–Pt(II) nucleophile
Introduction
and DNA can be obstructed by the steric encumbrance.7
Cisplatin and its second-generation analogues, namely car-
Alternatively, a [Pt(NH3)2]2+ moiety could be delivered to
boplatin and oxaliplatin, have afforded good results in the
treatment of solid tumours, especially testicular, ovarian and
DNA by forming the same cis-Pt(NH3)2d(GpG) intrastrand
adduct that cisplatin does. This can be achieved by tethering
colon cancer (oxaliplatin only). However, there are several major
the [Pt(NH3)2]2+ moiety to a coordinating arm ending in a
drawbacks to their clinical use, such as poor selectivity between
leaving group, such as the malonate group.8 It has been reported
malignant and normal cells (resulting in severe side effects), and
that dicarboxylate ligands strongly increase the solubility of the
the tendency to provoke chemoresistance. The strategy of drug
corresponding complexes.9 We are aware that the hydrolysis of
targeting and delivery could be applied to tumours that exhibit
malonato complexes at physiological pH is enormously slower
biochemical differences from normal tissue. Tumours possessing
hormone receptors represent a large class of malignancies that
includes breast, endometrium and prostate cancers. Given that
60–70% of mammary carcinomas express estrogen receptors,
in vitro than that of corresponding chloride complexes, however,
enzymatic removal seems to be very efficient in vivo.10–12
17a-Ethynylestradiol represents a good starting molecule by
virtue of its reactive acetylene hydrogen atom, which can easily
at least at the early stage, an estrogen-like ligand might prove
be linked to arms ending with either a diamine or a dicarboxylate
useful in delivering cytotoxic agents, in particular the cisplatin
group. Considerable effort has been devoted to optimizing
[Pt(II)X2] fragment (X = leaving group: halide or carboxylate),
the reactions of organic frameworks bearing alkynyl moieties.
to such estrogen-responsive (ER+) neoplasms.1–4 Provided the
Among these, we have chosen classical Sonogashira13 and non-
relative binding affinity (RBA) for the estrogen receptor of
classical Sonogashira14 metal-catalysed carbon–carbon bond-
the resulting Pt(II)-estrogen complexes is high enough, the
forming reactions with terminal alkynes and aryl halides.
receptor system would selectively recognise it. Furthermore, if
In this paper we report on the synthesis, characterisation
the cytotoxic complexes exhibit agonistic activity, the receptor
and biological activity of two ethynylestradiol-ligands and their
would vehiculate and approximate them to key sequences of
corresponding Pt(II)-complexes (Fig. 1).
genomes in mammary tumour cells.1,5 This would require a
coordinating arm capable of linking the PtX2-unit to the estrogen
carrier. In principle, a suitable arm would consist of a rigid spacer
ending in a diamine functionality (ethylenediamine, piperazine,
Results and discussion
Ligand synthesis
etc.). In order to link the estrogen to the arm, several reactions
are available, but they often require that the –NH2 groups
be protected (e.g. with tert-butyl carbonate, Boc). Thus, the
procedure involves multi-step reactions, which generally afford
low yields. The use of tertiary amines is therefore generally
preferred, since it does not call for protection or deprotection
steps. However, the substitution in the amine carrier groups
diminishes the cytotoxic activity of the cis-Pt(II)X2-moiety.6
Moreover, after hydrolysis of the PtX2-unit, the necessary
Synthesis of N-[4-(17a-ethynylestradiolyl)-benzyl]-ethylenedia-
mine, 4. Originally introduced by Inhoffen and Hohlweg in
1938,15 the majority of the synthetic approaches to ethynylestra-
diol analogues have relied on the addition of acetylide to the
resident ketone at C-17 in estrone. From a retrosynthetic per-
spective, we envisioned installation of the five-carbon spacer late
in the synthesis (Scheme 1), thereby permitting construction of
ligand 4 from the precursor 17a-ethynylestradiol. Accordingly,
assembly of the requisite five-carbon linker would entail cou-
pling of N-(4-iodobenzyl)-N,Nꢀ-diBoc-ethylenediamine (2) with
17a-ethynylestradiol, exploiting a Cassar–Heck–Sonogashira
C(sp2)–C(sp) cross-coupling reaction.16–18 Our starting point
† Electronic supplementary information (ESI) available: NMR spectra
for ligands 4 and 6 and complexes 7 and 8. See http://dx.doi.org/
10.1039/b507716h
T h i s j o u r n a l i s
T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 5
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 3 5 3 1 – 3 5 3 9
3 5 3 1
©

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Protection of the amino groups in N-(4-iodobenzyl)-
ethylenediamine was imperative for the alkyne coupling re-
action in order to avoid competing ortho-palladation of the
substrate.20
Synthesis of 2-[4-(17a-ethynylestradiolyl)-benzoylamino]-
malonic acid diethyl ester, 6. The steroid-malonate ester 6
was prepared starting from commercially available diethyl 2-
aminomalonate hydrochloride. Regarding the connecting arm
between the 17a-ethynylestradiol moiety and aminomalonate,
we chose to insert a rigid, suitably functionalised, aromatic
structure. 4-Iodobenzoic acid proved to be a good starting
material. The first step was the synthesis of a peptide bond
between aminomalonate and 4-iodobenzoic acid. Although it
is generally accepted that carbodiimides are the most efficient
condensing agents, the formation of by-products prompted us
to choose 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT) as a
carboxylic acid activator.21 The best result in the activation stage
was achieved by adding N-methylmorpholine dropwise to a
solution of diethyl 2-aminomalonate and 4-iodobenzoic acid
in CH2Cl2 at −5 ◦C. The pure 2-(4-iodobenzoylamino)-malonic
acid diethyl ester 5 was obtained after recrystallization with
a yield of 71.5%. The following synthetic step was meant to
have been a Sonogashira cross-coupling reaction between the
intermediate 5 and 17a-ethynylestradiol. Unfortunately, under
classical Sonogashira reaction conditions (CuI, alkylamine as
the solvent or in stoichiometric amounts, and Pd-complexes)
only a trace amount of the expected product was recovered.
The literature22 indicates that copper(I) iodide as co-catalyst
in the presence of oxygen induces a homocoupling reaction
(Glaser-type reaction)23 of terminal alkynes; moreover, the
presence of the amine may cause deprotonation of the malonate
acidic proton and generation of palladium enolate species.24
Copper-free methodologies employing amines as solvents have
been described.25 The first described copper- and amine-free
procedure involves the use of stoichiometric amounts of silver(I)
oxide for aryl iodide.14 In a typical procedure, malonate ester 5
(1.5 mmol) and 17a-ethynylestradiol (1.5 mmol) were dissolved
in THF (15 mL). (PPh3)2PdCl2 (5%) and Ag2O (1.5 mmol) were
added, and the reaction mixture was warmed to 60 ◦C. Routine
workup and purification of the crude mixture by column
chromatography afforded the expected coupling product 6 with
a yield of 61.1% (Scheme 2).
Fig. 1 Sketch of structures of the platinum complexes cis-dichloro[N-
(4-(17a-ethynylestradiolyl)-benzyl)-ethylenediamine]platinum(II), 7, and
cis-diamino[2-(4-(17a-ethynylestradiolyl)-benzoylamino)-malonato]-
platinum(II), 8.
Scheme
1 Reaction pathway for the synthesis of N-[4-(17a-
ethynylestradiolyl)-benzyl]-ethylenediamine, 4.
for 4 was the commercially available 4-iodobenzyl bromide.
This reacted with excess ethylenediamine19 in EtOH to
provide the corresponding N-(4-iodobenzyl)-ethylenediamine,
which, by protection with di-tert-butyl dicarbonate (Boc2O)
in CH2Cl2, afforded 2. Sonogashira reaction of 2 with 17a-
ethynylestradiol in the presence of (PPh3)2PdCl2, CuI, Et2NH in
MeCN gave N-(4-(17a-ethynylestradiolyl)-benzyl)-N,Nꢀ-diBoc-
ethylenediamine (3), and subsequent removal of the Boc groups
(HCl in Et2O) gave the required internal alkyne 4 as a dihy-
drochloride with an overall yield of 42% (Scheme 1).
Scheme
2
Reaction pathway for the synthesis of 2-[4-(17a-
ethynylestradiolyl)-benzoylamino]-malonic acid diethyl ester, 6.
3 5 3 2
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Platinum coordination
The purity of the complexes was further verified by 195Pt NMR
spectroscopy. Since the d(Pt) chemical shift depends on the elec-
tronic density on the Pt atom, it is sensitive to the nature of the
bound donor atoms and each species will show a different signal.
Since complex 7 was not soluble in water, the spectrum was
measured in (CD3)2SO. The only signal detectable after accumu-
lation was observed at −3307 ppm; this signal was attributable
to the chloro(dimethylsulfoxide)[N-(4-(17a-ethynylestradiolyl)-
benzyl)-ethylenediamine]platinum(II) species. This substitution
of DMSO for chloride in 7 is in tune with MS results, and is
typical of all Pt(II)Cl2 moieties.30
As far as 8 is concerned, as previously described by Gibson
et al.,33 the presence of the nitrogen atom in the a position
relative to the malonato unit promotes a coordination isomerism
of the (O,Oꢀ)-chelate 8 to the (N,O)-chelate 8 (see Fig. 2). Indeed,
Gibson described the reaction between cis-[Pt(NH3)2(H2O)2]2+
and 2-aminomalonate in water and showed that the kinetic
product (O,Oꢀ)-chelate was first formed and then quickly
isomerised to the thermodynamically stable (N,O)-chelate.
Complex 8 is barely soluble in water: when its 195Pt spectrum
was run in (CD3)2SO at room temperature, it exhibited only
one signal at −1718 ppm. The spectrum in CD3OD at rt
shows two peaks at −1737 ppm and −2171 ppm attributable
to the (O,Oꢀ)-chelate 8 and the (N,O)-chelate 8, respectively,
in a 6 : 1 ratio. With the hormonal ligand, the conversion is
slower because the nitrogen responsible for this isomerisation
is less suitable as an amidic nitrogen for platinum coordination
than the amine one. The steric hindrance further inhibits the
isomerisation. Thus, the ratio between the (O,Oꢀ)-chelate 8 and
the (N,O)-chelate 8 is still in favour of the former.
Synthesis of cis-dichloro[N-(4-(17a-ethynylestradiolyl)-benzyl)-
ethylenediamine]platinum(II), 7. Compound 7 was obtained in
good yield by reaction of K2[PtCl4] and 4 according to Miller’s
procedure for platinum coordination.26
K2[PtCl4] was added to an aqueous solution of N-(4-(17a-
ethynylestradiolyl)-benzyl)-ethylenediamine dihydrochloride at
about 60 ◦C. During the reaction, the pH was kept at ca. 6.0
by the dropwise addition of 0.1 N NaOH. The pH value is
important at this stage of the reaction: it must be high enough
to deprotonate the diamine ligand, thus making it suitable for
platinum coordination, but not so high as to cause the formation
of inactive hydroxo-Pt species.
Synthesis of cis-diamino[2-(4-(17a-ethynylestradiolyl)-
benzoylamino)-malonato]platinum(II), 8. The synthesis of
the hormone–malonato platinum(II) complex 8 is shown in
Scheme 3. The complex was prepared by reacting NH3 with
K2[PtI4], generated in situ according to Dhara’s method,27,28
after which iodide ions were replaced with water by means of
Ag2SO4. The treatment of the estradiol-substituted malonic
ester 6 with aqueous barium hydroxide gave the corresponding
barium salt (6a). The reaction of this barium salt with an
aqueous solution of cis-diaminodiaquoplatinum(II) sulfate
yielded the final complex 8.29
Scheme
3 Platinum coordination of 2-(4-(17a-ethynylestradiolyl)-
benzoylamino)-malonato barium salt (6a).
Fig. 2 Coordination isomers of 8.
Spectroscopic characterisation
A similar behaviour has also been reported for some macro-
molecular platinum complexes having the same coordination
geometry.34,35
The molecular formula of each compound was verified by com-
paring the molecular ion peak in the DCI or ESI mass spectrum
with the correct isotope distribution. The ESI-MS spectrum of
carboxylato complex 8, run in water–methanol (1 : 1), showed
the molecular ion peak [M + H]+. The dichloride complex 7
featured an ESI-MS spectrum in water–DMSO (3 : 1) whose
main signal was attributable to the [M–Cl + DMSO]+ ion.30
This kind of isomerisation is solvent-dependent. According to
the data reported by Lee et al.,36 the coordination mode of the an-
ionic ligand depends on the hydrogen bonding ability of the sol-
vent. Since the (N,O)-chelate has a zwitterionic form, the hydro-
gen bonds between protic solvent molecules and the zwitterionic
dipole seem to be responsible for its stabilisation. Methanol has
a hydrogen bonding ability that allows the two chelation modes
to coexist at room temperature. The above (N,O)-chelate is a tri-
amine cationic platinum complex usually considered inactive as
an antitumour agent. Hollis et al.,37 however, showed a non neg-
ligible antitumour activity for triamine cationic Pt(II) complexes.
1
As a general trend, the H and 13C chemical shifts in the
complexes were shifted to lower field when compared to free
ligands, and atoms closer to the Pt centre were more strongly
affected. 18-CH3 appeared between 0.4 and 0.9 ppm in the
1H NMR spectra of the complexes as well as of the parent
hormones. The aliphatic skeleton protons were spread between
1.0 and 2.8 ppm, whereas the aromatic protons 1-H, 2-H and
Structure optimization by molecular mechanics (MM)
4-H formed an AMX pattern (3J1,2 8.4, J2,4 2.6 Hz) between
4
8.0 and 6.1 ppm and the aromatic protons of the linking arm
Although we have not obtained X-ray quality crystals of 7 and
8, a reasonable model of their structures was obtained using
the HyperChemTM Package. It was constructed by exploiting
the published X-ray structures of the steroidal skeleton and of
platinum(II) complexes having the same coordination sphere.
The starting structure of 7 was constructed by replacing the
17a proton of 17b-estradiol38 with the –C≡C-pPh–CH2-spacer,
whose bond distances and angles were adjusted to typical values.
appeared between 7.6 and 7.2 ppm.31,32
In the 13C spectra the aliphatic carbons were spread between
12 and 52 ppm; whereas aromatic carbons appeared between
112 and 157 ppm. The signals of C-20, C-21 and C-17 appeared
from 98 to 74 ppm. The assignment of resonances due to C-8
and C-12 in (CD3)2SO was not possible owing to their partial
overlap with the carbon signals of the solvents.
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Table 1 RBA and log Po/w values
The structure of the coordination sphere was derived from that
of cis-[dichlorideethylenediamineplatinum(II)],39 replacing one
of the –NH2 protons with the methylene group of the spacer. The
resulting structure was energy-minimized in the Amber force
field using a steepest descent algorithm, leaving the platinum
coordination geometry unchanged.
RBA (%)a (ERa)
Log Po/w
a
Compounds
17b-Estradiol
17a-Ethynylestradiol
100b
3.14 0.07
3.25 0.09
2.21 0.04
4.97 0.08
70c
4
6
1.0 0.1
5.1 0.4
The starting point for the structure of (O,Oꢀ)-chelate 8 was the
same. For the platinum-containing moiety the structure of cis-
[diaminomalonatoplatinum(II)]40 was chosen as a model. One of
the –CH2 protons was replaced with the amidic group of the –
C≡C-pPh–CO–NH-spacer. After adjusting bond distances and
angles to the normal value, the resulting structure was energy-
minimized as described above.
a The experimental values are means SD of 3 independent experiments.
b By definition. c Ref. 41
Although this procedure is rather approximate, given that the
metal centre is a fixed unit, the method provides a reasonable
model of the overall molecular geometry. The resulting struc-
tures are shown in Fig. 3.
Fig. 4 Effect on MCF-7 cell viability. Cells were challenged with
compounds under study at 1 lM concentration for 5 day continuous
treatment.
Fig. 3 Optimized structures of 7 and 8.
As immediately visible in the optimized structures, the rigid
spacer keeps the platinum core well away from the two –OH
groups responsible for binding to the receptors. Interestingly
enough, the MM model of the (N,O)-chelate 8 again shows that
the Pt unit is still far away from the estrogen domain.
Biochemical and biological results
We have measured the relative binding affinity (RBA) of estra-
diol bio-ligands 4 and 6 for the a form of the estrogen receptor
(ERa), which indicates whether or not these compounds could
still be recognized by this receptor (these measurements were
performed in a competitive radioreceptor binding assay using
lamb uterine cytosol as the source of ERa and [3H]-estradiol
as a tracer) (Table 1). We have also determined the lipophilicity
of 4 and 6 (octanol/water partition coefficient, log Po/w: this
value, which is determined by means of HPLC, is a rough
measure of the drug lipophilicity, related to its ability to cross
cell membranes) (Table 1).
Finally, we have studied the effects of the bio-ligands 4 and
6, and the corresponding Pt(II) complexes 7 and 8, on the
proliferation of a human mammary adenocarcinoma cell line
(MCF-7), that expresses estrogen receptor (ER+) and responds
to estrogen activity. As control for estrogen activity we have
employed a hormone-insensitive human mammary MDA-MB-
231 cell line (Figs. 4 and 5).
Fig. 5 Effect on MDA-MB-231 cell viability. Cells were challenged with
compounds under study at 1 lM concentration for 5 day continuous
treatment.
The RBA values indicate that compounds 4 and 6 do
interact with the receptor, although their affinity is moderate
as compared to 17a-ethynylestradiol, thus suggesting that they
may behave as carriers for Pt delivery to the nucleus.
The RBA value for 4 was somewhat disappointing (only 1%).
However, this result finds an explanation in the modelling study
by Foy et al.42 on the estrogen receptor binding site. This study
considered the affinity of a series of 17a-arylestradiol derivatives,
demonstrating that only the p-NH2 substituent dramatically
reduced the RBA value. The protonated amino hydrophilic
group underwent an electrostatic repulsion, likely due to a
methionine group, in the active site of the estrogen receptor.
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We have recently shown for a similar series of 17a-
ethynylestradiol-carriers ending in ethylenediamine or piper-
azine, that their coordination by a PtX2-moiety, which engages
the lone pair present on nitrogen ligands, thus preventing their
protonation, leads to a significant increase in the RBA.43 On
the contrary, the replacement for 8 of two Et groups with the
[Pt(NH3)2]2+ moiety should lead to a drop in RBA.
In this context, an estimated RBA value of both compounds
7 and 8 is about 2%, in the same order of magnitude as the
potent selective estrogen receptor modulator (SERM) tamoxifen
(Tam).44 Thus, we have indirectly evaluated the affinity of
complexes 7 and 8 for the receptor on the basis of their prolif-
eration effect on ER+ and ER-cell lines, along with that of the
corresponding ligands and reference compounds. Interestingly
enough, the estimated RBA values as well as the effect on cell
growth are higher for complexes 7 and 8 with respect to those
of previously reported 17a-ethynylestradiol-ethylenediamine-
Pt(II)-malonato complexes.43 The presence of an aromatic spacer
in the arm between estradiol and ethylenediamine moieties
improves their biological activity. This is in tune with previ-
ous studies from Be´rube´ et al.45–47 on triphenylethylene- and
estradiol-Pt(II) complexes having 6–10 aliphatic carbon atom
spacers.
The effect of compounds 4–8 on MCF-7 growth was com-
pared to that of 17b-estradiol (E2) and 17a-ethynylestradiol
(EE), the reference estrogens, tamoxifen (Tam) and hydrox-
ytamoxifen (OH–Tam), two SERMs, cis-[Pt(en)Cl2] (en =
ethylenediamine) and cis-[Pt(NH3)2mal] (mal = malonato), the
model complexes, and cisplatin as a reference antiproliferative
agent, all at a concentration of 1 lM (Fig. 4). The bars in
the histogram show the inhibitory (for Tam and OH–Tam)
or proliferative (for E2, EE, 4 and 6) effect of the compounds
relative to the control.
Fig. 6 Effect on MCF-7 cell viability. Cells were challenged with
compounds under study at 0.1 lM concentration for 5 day continuous
treatment.
We treated both cell lines with a mixture at equimolar
concentrations of estradiol (the natural ligand for ERa+) and
cisplatin, the most active platinum drug. The data obtained
from this combination indicate that the cytotoxic effect of
cisplatin is overcome by the growth stimulation of estradiol
at concentrations lower than 10−6 M (Fig. 7). We are aware
that the two compounds probably differ in pharmacokinetics
and cellular uptake, but the gross effect appears nonetheless to
be incontrovertible. It is important to recall that the treatment
of cells with the mixture of both compounds at 10−5 M (data
not shown) had a negative affect on cell viability (all the
cells died within 5 days). While this concentration corresponds
to the optimal activity for cisplatin, the same concentration
represents an unphysiological condition for E2, leading to strong
interference of growth.50
As expected, all the compounds under study, except those
containing the cytotoxic Pt(II) moieties, have no significant effect
on the MDA-MB-231 cell line (Fig. 5). The proliferative effect
observed on MCF-7 cells is therefore clearly a hormonal effect
mediated by the estrogen receptor.48
As far as non-hormonal Pt(II) complexes are concerned,
the cytotoxic effect of cis-[Pt(en)Cl2], cis-[Pt(NH3)2mal], and
cisplatin is lower for MDA-MB-231 with respect to that
of the MCF-7 cell line, as previously reported in the lite-
rature.49
Returning to the question of viability of the MCF-7 cell line,
it is disappointing that complexes 7 and 8 showed no advantage
in terms of inhibition of proliferation over the corresponding
model complexes cis-[Pt(en)Cl2], cis-[Pt(NH3)2mal] or cisplatin
itself. This failure might be attributable to several factors: i)
the decrease in cytotoxicity due to the steric effect of chelating
arms necessary for linking the Pt(II)-moiety to the estrogen;6 ii)
the isomerisation from (O,Oꢀ) to (N,O)-chelate forms in 8; iii)
the limited number of receptors able to act as a shuttle for the
estrogen–platinum intracellular adduct (see bio-stoichiometry
in the experimental section) and iv) most importantly, the
estrogenic (agonistic) activity of the overall assembly. Indeed
comparison of the effects of the panel of compounds on ER+
cells clearly indicates that complexes 7 and 8 are recognised
by the receptor as well as the ligands 4 and 6, and all of
them produce an overall proliferative effect. This is because
the estrogenic effect generally operates at a nanomolar level,50
whereas a classical cytotoxic effect for cisplatin-like complexes
is usually comprised in the range 1–10 lM.
Indeed, at 0.1 lM concentration the hormonal Pt(II) com-
plexes, namely 7 and 8 had an overall proliferative effect (Fig. 6).
In a hypothetical in vivo model, it is reasonable to suppose
that, due to pharmacokinetic reasons, the cancer cells are
exposed to optimal cytotoxic levels of these compounds only
for short periods, whereas, until the plasma concentration drops
to 0, the stimulatory effects of the compounds (due to lower
concentration) will be prevalent.
Fig. 7 Effect of the equimolar combination of E2 and cisplatin on
MCF-7 cell viability (5 day continuous treatment).
Seminal investigations on the action of both steroidal hor-
mones and cisplatin on breast cancer cells have been made by
Lippard et al.51–53 The aim of these investigations, as well as the
concentrations and treatment times involved, were profoundly
different from those of the present study.
Short exposure periods of MCF-7 cells to low concentrations
of E2 (i.e. 10−7 M for 4 h) were shown to induce high mobility
group (HMG) protein overexpression and, hence, protection
of the cisplatin-DNA adduct by the nucleotide excision repair
(NER) system. In this short time span, it was impossible for E2 to
induce significant growth stimulation, and the overall result was
an increased sensitivity of the hormone-treated cells to cisplatin.
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CH2–CH2–N), 3.20 (2H, m, N–CH2–CH2–N), 1.45 (9H, s, 3 ×
BocCH3), 1.40 (9H, s, 3 × BocCH3); dC (100.5 MHz; (CD3)2CO)
Experimental
General procedures
=
=
156.07 (BocC O), 155.76 (BocC O), 139.43 (PhCa), 137.80
(PhCb), 130.02 (2 × PhCc), 91.97 (PhCd), 79.57 (BocC), 78.15
(BocC), 50.42 (Ph–CH2–N), 46.69 (N–CH2–CH2–N), 39.27 (N–
CH2–CH2–N), 28.12 (3 × BocCH3), 28.02 (3 × BocCH3). DCI-
MS: m/z 477.1241 ([M + H]+, 100%), 478.1238 (22.02), 479.1252
(3.15) (M + H)+; calcd for C19H30IN2O4 477.1250 ([M + H]+,
100%), 478.1250 (21.89), 479.1250 (3.11). Elemental analysis
calcd for C19H29IN2O4: C 47.9; H 6.1; N 5.9; found: C 48.0;
H 6.15; N 5.9%.
K2[PtCl4] was purchased from Johnson Matthey and Co. 17a-
Ethynylestradiol was obtained from ICN Biomedicals. Deuter-
ated solvents were bought from Euriso-top, France. All other
chemicals were obtained from Aldrich and used without further
purification. The NMR spectra were recorded at 25 ◦C on
a JEOL Eclipse Plus spectrometer operating at 400 MHz
(1H), 100.5 MHz (13C) and 85.9 MHz (195Pt with a spectral
window of 1200 ppm). H and 13C NMR chemical shifts were
1
iii) Synthesis of N-(4-(17a-ethynylestradiolyl)-benzyl)-
N,Nꢀ-diBoc-ethylenediamine, 3. In a 100 ml three-necked round-
bottomed flask, 17a-ethynylestradiol (1.25 g, 4.23 mmol) and 2
(2 g, 4.19 mmol) were dissolved in MeCN (28 ml) and Et2NH
(40 ml). CuI (3% mol/mol respect of the halide and the alkyne,
25 mg, 0.13 mmol) was added to this pale yellow mixture and the
mixture turned orange-pink). When (PPh3)2PdCl2 (2%, 59 mg,
0.084 mmol) was added, the mixture turned brown-orange.
Nitrogen was blown through the solution for 30 min. After
being stirred for 5 days at 40 ◦C, during which time the solution
remained a dark brown-orange, the mixture was cooled at rt
and dried under vacuum, resulting in an oil that was purified via
water–CH2Cl2 extraction. The organic phase was dried under
vacuum to produce a thick dark brown-orange oil which was
purified by silica gel column chromatography with n-hexane–
acetone 2 : 1 to get 3 (2.7 g, 63% from 2). dH (400 MHz;
reported in parts per million referenced to solvent resonances;
for D2O measurements, 1% methanol was added as a 13C internal
reference. 195Pt NMR spectra were recorded using a solution
of K2[PtCl4] with KCl as an external reference. The shift for
K2[PtCl4] was adjusted to −1628 ppm from Na2[PtCl6] (d =
0 ppm).
DCI-MS spectra of the ligands were recorded on a Finnigan-
MAT 95Q instrument with magnetic and electrostatic analysers.
Isobutane was used as the reagent gas at 0.5 mbar pressure. The
ion source temperature was kept at 50 ◦C, the electron emission
current at 0.2 mA, and the electron energy at 200 eV. Positive
ion spectra were collected. ESI mass spectrometry, due to the
soft ionisation, was chosen as an efficient method for the char-
acterisation of platinum complexes in solution.30 These mass
experiments were conducted by means of Finnigan Thermo-
quest LCQ DUO ion trap mass spectrometer equipped with an
ESI ion source. High purity nitrogen was used as a nebuliser
(operating pressure at 80 of the arbitrary scale 0–100 of the
instrument). The ESI probe tip and capillary potential were set
at 4.50 kV◦and 10.00 V, respectively. The heated capillary was
set at 270 C. The mass spectrometer was operated in positive
ion full-scan mode. Molecular ion peaks were assigned from the
m/z values and from the simulated isotope distribution patterns.
3
(CD3)2CO; Me4Si) 8.27 (1H, s, 3-H), 7.38 (2H, d, J 7.9, 23-
3
H and 27-H), 7.23 (2H, d, J 7.9, 24-H and 26-H), 7.08 (1H,
d, 3J1,2 8.4, 1-H), 6.58 (1H, dd, 3J1,2 8.4 and 4J2,4 2.6, 2-H), 6.51
4
(1H, d, J2,4 2.6, 4-H), 6.03 (1H, s, NH), 4.61 (1H, s, 17-H),
4.45 (2H, s, 28-H), 3.21 (4H, m, 29-H and 30-H), 2.74 (2H, m,
6-H and 6ꢀ-H), 2.40–2.20 (2H, m, 11-H and 12-H), 2.20–1.90
(3H, m, 16-H, 12ꢀ-H and 9-H), 1.90–1.50 (4H, m, 16ꢀ-H, 15-H,
14-H, 7-H), 1.50–1.30 (4H, m, 15ꢀ-H, 11ꢀ-H, 8-H, 7ꢀ-H), 1.46
(9H, s, 3 × BocCH3), 1.38 (9H, s, 3 × BocCH3), 0.92 (3H, s,
Ligand synthesis
=
18-H); dC (100.5 MHz; (CD3)2CO; Me4Si) 156.14 (BocC O),
155.72 (BocC O), 155.30 (C-3), 139.20 (C-25), 137.64 (C-5),
Synthesis of N-(4-(17a-ethynylestradiolyl)-benzyl)-ethylene-
diamine, 4.
=
131.65 (C-23 and C-27), 131.11 (C-10), 127.91 (C-24 or C-26),
127.47 (C-26 or C-24), 126.32 (C-1), 122.55 (C-22), 115.30 (C-4),
112.96 (C-2), 94.34 (C-20), 84.81 (C-21), 79.91 (BocC), 79.55
(BocC), 78.11 (C-17), 50.87 (C-28), 49.96 (C-14), 47.75 (C-13),
46.38 (C-29 or C-30), 44.03 (C-9), 39.97 (C-8), 39.30 (C-12),
38.94 (C-30 or C-29), 33.31 (C-16), 29.67 (C-6), 27.99 (3 ×
BocCH3), 27.88 (3 × BocCH3), 27.45 (C-7), 26.67 (C-11), 22.90
(C-15), 12.71 (C-18). DCI-MS: m/z 645.3895 ([M + H]+, 100%),
646.3911 (43.86), 643.3907 (10.53), 649.3888 (1.85) (M + H)+;
calcd for C39H53N2O6 645.3903 ([M + H]+, 100%), 646.3903
(43.94), 647.3903 (10.66), 649.3903 (1.86). Elemental analysis
calcd for C39H52N2O6: C 72.6; H 8.1; N 4.3; found: C 72.8;
H 8.15; N 4.3%.
i) Synthesis of N-(4-iodobenzyl)-ethylenediamine, 1. 4-
Iodobenzyl bromide (2.86 g, 9 mmol) was dissolved in EtOH
(29 ml). A great excess of ethylenediamine 99% (47 mmol, 3.2 ml)
was dissolved in the same solvent (10 ml) and added to the
first solution while being stirred under a nitrogen atmosphere
in the dark. After 24 h the resulting pale yellow mixture was
filtered and dried under vacuum, and the oily residue was parti-
tioned between benzene and water. The organic phase was
dried over Na2SO4 and concentrated to yield N-(4-iodobenzyl)-
ethylenediamine as a light yellow oil which was used in a
subsequent step without further purification. dH (400 MHz;
3
CDCl3; Me4Si) 7.49 (2H, d, Jb,c 8.2, 2 × PhHb), 6.95 (2H,
3
d, Jb,c 8.2, 2 × PhHc), 3.59 (2H, s, Ph–CH2–N), 2.63 (2H, t,
iv) Synthesis of N-(4-(17a-ethynylestradiolyl)-benzyl)-
ethylenediamine dihydrochloride, 4. The above compound (1.5 g,
2.33 mmol) was treated at room temperature with 0.1 N
HCl in diethyl ether (12 ml). After 5 days stirring we got
a brown precipitate of N-(4-(17a-ethynylestradiolyl)-benzyl)-
ethylenediamine dihydrochloride (506 mg, 42%). dH (400 MHz;
D2O; Me4Si) 7.22 (2H, d, 3J 7.3, 23-H and 27-H), 7.17 (2H, d,
3J 7.3, 24-H and 26-H), 6.72 (1H, d, 3J1,2 5.5, 1-H), 6.40 (1H, d,
3J1,2 5.5, 2-H), 6.17 (1H, s, 4-H), 3.96 (2H, s, 28-H), 3.40–3.07
(4H, m, 29-H and 30-H), 2.22 (2H, m, 6-H), 2.10–0.60 (13H, m,
7-H, 8-H, 9-H, 11-H, 12-H, 14-H, 15-H and 16-H), 0.46 (3H, s,
18-H); dC (100.5 MHz; D2O; Me4Si) 153.36 (C-3), 137.96 (C-5),
133.62 (C-25), 132.38 (C-23 or C-27), 132.16 (C-27 or C-23),
131.25 (C-10), 130.39 (C-24 or C-26), 130.25 (C-26 or C-24),
126.59 (C-1), 124.11 (C-22), 115.45 (C-4), 112.89 (C-2), 94.09
(C-20), 85.22 (C-21), 80.15 (C-17), 51.40 (C-28), 49.82 (C-14),
47.47 (C-13), 44.07 (C-29 or C-30), 43.39 (C-9), 39.22 (C-8),
35.68 (C-30 or C-29), 33.04 (C-12), 29.23 (C-6), 27.89 (C-16),
27.32 (C-7), 26.22 (C-11), 22.84 (C-15), 12.86 (C-18). DCI-MS:
3J 5.8, N–CH2–CH2–N), 2.52 (2H, t, 3J 5.8, N–CH2–CH2–N),
1.42 (3H, s, NH and NH2); dC (100.5 MHz; CDCl3; Me4Si)
140.25 (PhCa), 137.34 (2 × PhCb), 130.12 (2 × PhCc), 92.23
(PhCd), 53.20 (Ph–CH2–N), 51.82 (N–CH2–CH2–N), 41.71 (N–
CH2–CH2–N). DCI-MS: m/z: 277.0207 ([M + H]+, 100%),
278.0200 (10.70); calcd for C9H14IN2 277.0202 ([M + H]+,
100%), 278.0202 (10.68). Elemental analysis calcd for C9H13IN2:
C 39.15; H 4.75; N 10.15; found: C 39.2; H 4.8; N 10.2%.
ii) Synthesis of N-(4-iodobenzyl)-N,Nꢀ-diBoc-ethylenedia-
mine, 2. A solution of di-tert-butyl dicarbonate (Boc2O, 2.8 g,
12.8 mmol) in CH2Cl2 (20 ml) was added to a solution of the
above compound in CH2Cl2 (3 ml). The reaction mixture was
milky at first and then turned a clear yellow. After 24 h stirring
at rt, the mixture was dried under vacuum to obtain an oil.
Precipitation was induced by n-hexane, resulting in an abundant
white precipitate 2 (2 g, 42%). dH (400 MHz; (CD3)2CO; Me4Si)
7.69 (2H, d, 3Jb,c 8.2, 2 × PhHb), 7.10 (2H, d, 3Jb,c 8.2, 2 × PhHc),
5.84 (1H, s, NH), 4.44 (2H, s, Ph–CH2–N), 3.30 (2H, m, N–
3 5 3 6
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 3 5 3 1 – 3 5 3 9

View Article Online
m/z 223.6505 ([M–2Cl−]2+, 100%), 224.1499 (32.63), 224.6507
(5.67) (M–2Cl−)2+; calcd for C29H38N2O2 223.6503 ([M–2Cl−]2+,
100%), 224.1503 (32.75), 224.6503 (5.60). Elemental analysis
calcd for C29H38Cl2N2O2: C 67.3; H 7.4; N 5.4; found: C 67.4;
H 7.4; N 5.4%.
577.2804 (1.39). Elemental analysis calcd for C34H39NO7: C 71.2;
H 6.85; N 2.4; found: C 71.3; H 6.9; N 2.4%.
Platinum coordination
Synthesis of cis-dichloro[N -(4-(17a-ethynylestradiolyl)-
benzyl)-ethylenediamine]platinum(II), 7. The ligand–dihydro-
chloride 4 (132 mg, 0.255 mmol) was dissolved in water (5 ml)
at 65 ◦C. A solution of K2[PtCl4] (127 mg, 0.306 mmol) in water
(5 ml) was added and the pH value was adjusted to 5–6 with
0.1 N aqueous NaOH. Initially, the pH of the mixture was 4–5
decreasing towards 1 because of the progressing reaction; the pH
had to be kept from the region of hydroxocomplexes (basic pH).
The mixture was stirred while protected from light for 2 days
at rt. The resulting light brown precipitate of 7 was washed
with cold water, ethanol and ether and dried, resulting in pure
7 (160 mg, 88% from 4). dH (400 MHz; (CD3)2SO; Me4Si) 8.99
(1H, s, 3-H), 7.58 (2H, d, 3J 7.9, 24-H and 26-H), 7.45 (2H, d,
3J 7.9, 23-H and 27-H), 7.06 (1H, d, 3J1,2 8.4, 1-H), 6.70 (1H, brs,
NH), 6.51 (1H, d, 3J1,2 8.4, 2-H), 6.44 (1H, s, 4-H), 5.45 (1H, s,
17-H), 5.43–5.23 (2H, m, NH2), 4.60–3.60 (6H, m, 28-H, 29-H
and 30-H), 2.71 (2H, m, 6-H), 2.45–1.13 (13H, m, 7-H, 8-H,
9-H, 11-H, 12-H, 14-H, 15-H and 16-H), 0.82 (3H, s, 18-H);
dC (100.5 MHz; (CD3)2SO; Me4Si) 155.01 (C-3), 137.91 (C-5),
134.32 (C-10), 131.49 (C-23 or C-27), 130.52 (C-27 or C-23),
130.35 (C-24 or C-26), 130.26 (C-26 or C-24), 128.39 (C-25),
126.18 (C-1), 123.09 (C-22), 115.00 (C-4), 112.82 (C-2), 95.90
(C-20), 83.74 (C-21), 78.72 (C-17), 53.95, 53.16, 45.32 (N–CH2,
C-28, C-29, C-30), 49.50 (C-14), 47.33 (C-13), 43.49 (C-9), 33.02
(C-16), 29.27 (C-6), 27.08 (C-7), 26.30 (C-11), 22.66 (C-15), 12.97
(C-18); dPt (85.9 MHz; (CD3)2SO; K2[PtCl4]) −3307 ppm (where
a chloride is replaced by DMSO). ESI-MS (water–DMSO (3 :
1)): m/z 749.221 (1.52%), 751.221 (64.40), 752.220 (88.58),
753.220 ([M–Cl + DMSO]+, 100), 754.220 (54.73), 755.221
(46.76), 756.221 (14.95), 757.220 (8.66), 758.221 (2.44); calcd
for C31H42ClN2O3PtS 749.220 (1.53), 751.220 (64.46%), 752.220
(88.53), 753.220 ([M–Cl + DMSO]+, 100), 754.220 (54.77),
755.220 (46.74), 756.220 (14.97), 757.220 (8.63), 758,220 (2.45).
Elemental analysis calcd for C29H36Cl2N2O2Pt: C 49.0; H 5.1; N
3.9; Pt 27.45; found: C 49.1; H 5.1; N 3.9; Pt 27.4%.
Synthesis of 2-[4-(17a-ethynylestradiolyl)-benzoylamino]-
malonic acid diethyl ester, 6.
i) Synthesis of 2-[4-iodobenzoylamino]-malonic acid diethyl
ester, 5. To a stirred solution of CDMT (0.88 g, 5 mmol) and
4-iodobenzoic acid (1.26 g, 5 mmol) in CH2Cl2 (7 mL), we added
N-methylmorpholine (0.52 g, 5 mmol) dropwise at −5 ◦C. After
2 h a TLC control indicated the consumption of CDMT. To
the crude solution obtained as described above, a mixture of
diethyl 2-aminomalonate hydrochloride (1.06 g, 5 mmol) and
N-methylmorpholine (0.52 g, 5 mmol) in CH2Cl2 (7 mL) was
added at −5 ◦C. After 2 h at 0 ◦C the temperature was brought
to rt and the reaction stirred for 14 h. Evaporation of the solvent
resulted in a residue that was suspended in ethyl acetate (30 mL).
The organic layer was successively washed with H2O (10 mL),
a 10% citric acid solution (10 mL), H2O (10 mL), saturated
NaHCO3 solution (10 mL) and H2O (10 mL). Anhydrification
of the organic phase with MgSO4 and evaporation of the solvent
produced a residue that was recrystallised from ethyl acetate,
resulting in 1.45 g (71.5% yield) of pure 5. dH (400 MHz;
(CD3)2CO; Me4Si) 8.24 (1H, d, 3J 7.2, NH), 7.89 (2H, d, 3Jb,c 8.6,
2 × PhHc), 7.74 (2H, d, 3Jb,c 8.6, 2 × PhHb), 5.36 (1H, d, 3J 7.2,
N–CH), 4.23 (4H, m, 2 × OCH2CH3), 1.26 (6H, t, 3J 7.1, 2 ×
OCH2CH3); dC (100.5 MHz; (CD3)2CO; Me4Si) 166.55 (2 × C-
=
=
C
O), 166.06 (N–C O), 137.95 (2 × PhCc), 133.35 (PhCa),
129.62 (2 × PhCb), 98.63 (PhCd), 62.13 (2 × OCH2CH3),
57.08 (N–CH), 13.67 (2 × OCH2CH3). DCI-MS: m/z 406.0149
([M + H]+, 100%), 407.0146 (15.84), 408.0156 (2.18); calcd
for C14H17INO5 406.0151 ([M + H]+, 100%), 407.0151 (15.96),
408.0151 (2.22). Elemental analysis calcd for C14H16INO5:
C 41.5; H 4.0; N 3.5; found: C 41.6; H 4.0; N 3.5%.
ii) Synthesis of 2-[4-(17a-ethynylestradiolyl)-benzoylamino]-
malonic acid diethyl ester, 6. In a 50 ml three-necked round-
bottomed flask, anhydrous THF (10 mL) was degassed with N2
for 10 min. Then, Ag2O (0.35 g, 1.5 mmol), 17a-ethynylestradiol
(0.44 g, 1.5 mmol), 5 (0.61 g, 1.5 mmol) and (PPh3)2PdCl2
(52.6 mg, 0.075 mmol) were subsequently added. The reaction
mixture was heated to 60 ◦C and stirred under nitrogen
overnight. The reaction was then cooled to rt, filtered through a
filter agent, and the solid washed with diethyl ether. The organic
phase was washed with NaHCO3 5% (10 mL) and brine (10 mL),
dried over MgSO4 and the solvent was evaporated. The residue
was purified by column chromatography using petroleum ether–
diethyl ether 50 : 50 as eluant. Appropriate fractions were
combined giving 0.53 g (61.1% yield) of pure 6. dH (400 MHz;
(CD3)2CO; Me4Si) 8.24 (1H, d, 3J 7.3, NH), 7.99 (1H, s, 3-H),
Synthesis of cis-diamino[2-(4-(17a-ethynylestradiolyl)-
benzoylamino)-malonato]platinum(II), 8. K2[PtCl4] (500 mg,
1.21 mmol) was dissolved in water (2 ml) and an aqueous
solution of KI (1.20 mg, 7.23 mmol of KI in 2 ml of water) was
added in the dark to the mixture. After about 30 min it was
filtered to remove some solid impurities. An aqueous solution
(2 ml) containing 4 mmol of NH3 was added to the filtrate.
Fine yellow-brown crystals of cis-[(NH3)2PtI2] immediately
precipitated. After 15 min the compound was separated
by centrifugation and washed with cold water, ethanol and
diethyl ether (567 mg, 97.5% yield). cis-[(NH3)2PtI2] (300 mg,
0.62 mmol) was then suspended in an aqueous solution (30 ml)
of Ag2SO4 (190 mg, 0.61 mmol) and the mixture was stirred
for 20 h in the dark; after that the silver iodide precipitate
was removed by filtration. The barium salt 6a [prepared by
adding 0.11 M aqueous Ba(OH)2 (5.65 ml) to a solution of 6
(400 mg, 0.70 mmol) in methanol (10 ml)] was added to the
filtrate. The mixture was stirred for 24 h and then centrifugated
to separate the BaSO4 and 8 that coprecipitated. The solid
obtained was washed several times with methanol to dissolve
8. Methanol was evaporated to dryness, yielding 8 (250 mg,
54% yield from cis-[(NH3)2PtI2]). dH (400 MHz; (CD3)2SO;
Me4Si) 9.01 (1H, s, 3-H), 7.89 (1H, d, 3J 7.7, NH), 7.86 (2H, d,
3
3
7.94 (2H, d, J 8.4, 24-H and 26-H), 7.54 (2H, d, J 8.4, 23-
3
3
H and 27-H), 7.10 (1H, d, J1,2 8.4, 1-H), 6.60 (1H, dd, J1,2
8.4 and 4J2,4 2.6, 2-H), 6.53 (1H, d, 4J2,4 2.6, 4-H), 5.38 (1H, d,
3J 7.3, 29-H), 4.61 (1H, s, 17-H), 4.24 (4H, m, 2 × OCH2CH3),
2.75 (2H, m, 6-H and 6ꢀ-H), 2.38–2.31 (2H, m, 11-H and 12-H),
2.24–1.92 (3H, m, 16-H, 12ꢀ-H and 9-H), 1.92–1.72 (4H, m,
16ꢀ-H, 15-H, 14-H, 7-H), 1.52–1.30 (4H, m, 15ꢀ-H, 11ꢀ-H, 8-H,
3
7ꢀ-H), 1.26 (6H, t, J 7.1, 2 × OCH2CH3), 0.94 (3H, s, 18-
=
H); dC (100.5 MHz; (CD3)2CO; Me4Si) 166.46 (C O, C-30 and
C-31), 165.98 (C O, C-28), 155.15 (C-3), 137.65 (C-5), 132.67
=
(C-25), 131.48 (C-23 and C-27), 131.12 (C-10), 127.78 (C-24 and
C-26), 127.24 (C-22), 126.34 (C-1), 115.21 (C-4), 112.87 (C-2),
97.09 (C-20), 84.19 (C-21), 79.54 (C-17), 62.03 (2 × OCH2CH3),
56.95 (C-29), 50.00 (C-14), 47.76 (C-13), 43.90 (C-9), 39.86 (C-8),
39.21 (C-12), 33.32 (C-16), 29.58 (C-6), 27.43 (C-7), 26.64 (C-
11), 22.87 (C-15), 13.57 (2 × OCH2CH3), 12.67 (C-18). DCI-MS:
(m/z (relative intensity)): 574.2808 ([M + H]+, 100%), 575.2797
(38.09), 576.2796 (8.47), 577.2812 (1.38); calcd for C34H40NO7
574.2804 ([M + H]+, 100%), 575.2804 (38.01), 576.2804 (8.46),
3
3J 8.4, 24-H and 26-H), 7.49 (2H, d, J 8.4, 23-H and 27-H),
7.06 (1H, d, 3J1,2 8.4, 1-H), 6.49 (1H, dd, 3J1,2 8.4 and 4J2,4 2.6,
4
3
2-H), 6.42 (1H, d, J2,4 2.6, 4-H), 5.72 (1H, d, J 7.7, 29-H),
5.55 (1H, s, 17-H), 4.31 (6H, s, 2 × NH3), 2.70 (2H, m, 6-H and
6ꢀ-H), 2.40–1.20 (13H, m, 7-H, 8-H, 9-H, 11-H, 12-H, 14-H,
15-H and 16-H), 0.80 (3H, s, 18-H); dC (100.5 MHz; (CD3)2SO;
=
=
Me4Si) 171.73, 170.48 (C O, C-30 and C-31), 164.39 (C O,
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 3 5 3 1 – 3 5 3 9
3 5 3 7

View Article Online
C-28), 155.00 (C-3), 137.28 (C-5), 133.51 (C-10), 131.31 (C-23
and C-27), 130.39 (C-25), 127.78 (C-22), 127.64 (C-24 and
C-26), 126.24 (C-1), 115.03 (C-4), 112.86 (C-2), 97.17 (C-20),
83.78 (C-21), 78.80 (C-17), 59.47 (C-29), 49.56 (C-14), 47.38
(C-13), 43.47 (C-9), 33.08 (C-16), 29.32 (C-6), 27.09 (C-7), 26.34
(C-11), 22.72 (C-15), 13.01 (C-18); dPt (85.9 MHz; (CD3)2SO;
K2[PtCl4]) −1718 ppm. ESI-MS (water–methanol = 50 : 50):
m/z 742.214 (1.78%), 744.215 (73.08), 745.215 ([M + H]+,
100), 746.214 (86.88), 747.215 (25.31), 748.216 (20.76), 749.215
(6.19), 750.216 (1.22); calcd for C30H36N3O7Pt 742.215 (1.73%),
744.215 (73.11), 745.215 ([M + H]+,100), 746.215 (86.85),
747.215 (25.38), 748.215 (20.80), 749.215 (6.18), 750.215 (1.23).
Elemental analysis calcd for C30H35N3O7Pt: C 48.4; H 4.7;
N 5.6; Pt 26.2; found: C 48.5; H 4.7; N 5.6; Pt 26.15%.
of the compound under analysis (final concentration 1 lM).
The organic compounds Tam, OH–Tam, E2, EE, 4 and 6,
were dissolved in EtOH, whereas Pt(II)-complexes 7, 8, cis-
[Pt(en)Cl2] and cis-[Pt(NH3)mal] were dissolved in DMSO. The
final concentration of the cosolvent in culture media never
exceeded 0.5%. Cisplatin was dissolved in culture media. Cell
monolayers grown with culture media containing the cosolvent
at the maximum concentration employed were assumed as
reference controls (100% of the growth).
Cell monolayers were treated continuously for 120 h. Vi-
ability was measured using an MTT test. Cells were incu-
bated with 0.5 mg ml−1 MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-
diphenyltetrazolium bromide) under standard culture condi-
tions for 1 h. Wells were dried, and formazan crystals were
soluted with 750 lL of acidic isopropanol (0.1 N HCl in absolute
isopropanol). Absorbance was measured by spectrophotometry
at 570 nm with background subtraction at 630 and 690 nm.
Log Po/w determination
Log Po/w values were estimated from values of log kꢀw (kꢀw
=
capacity ratio in absence of methanol), determined by HPLC
chromatography according to Minick et al.54 and Pomper et al.55
using a Macherey-Nagel EC250/3 Nucleosil 100-5C18HD col-
umn. The UV detector was set to 277 nm. The organic portion of
the mobile phase was composed of methanol containing 0.25%
(v/v) 1-octanol. The aqueous portion was an octanol-saturated
buffer prepared from 0.02 M 3-morpholinopropanesulfonic acid
(MOPS) and 0.15% (v/v) n-decylamine; the pH was adjusted to
7.4 with NaOH. The flow rate was 0.5 ml min−1. The steroids
were dissolved at 1 mM concentration in ethanol and 20 ll were
injected. Column void volume was estimated from the retention
time of uracil (t0), which was included as an unretained internal
reference with each run. kꢀ values were obtained from the steroids
retention time (tR) according to kꢀ = (tR − t0)/t0. The log kꢀw was
determined by linear extrapolation of log kꢀ vs. φ methanol (φ =
volume fraction of methanol); data acquired with 0.55 ≤ φ ≤
0.85. Applying Pomper’s relationship, log kꢀ returned the log Po/w
value.
Bio-stoichiometry
Rough calculations allowed us to assess the order of magnitude
of platinum atoms potentially bound to the receptor or necessary
to exert cytotoxic effects. The number of estrogen-receptor (ER)
adducts in hormone dependent (ER+) breast cancer cells is in
the range of 10 000–30 000 (20 000 on average).57 The level of
cellular uptake in the human mammary adenocarcinoma MCF-
7 cell line with cisplatin, evaluated by means of ICP-MS and
extrapolated at IC50 level (24 h continuous treatment), is about
47 ng Pt/106 cells.58 The corresponding DNA platination is
about 1% of the uptake, i.e. 470 pg Pt/106 cells.59,60 Provided
that the 20 000 ER adducts transport 20 000 Pt atoms directly to
the DNA (vehiculation perfectly efficient), the DNA platination
would correspond to 6.4 pg Pt/106 cells. Thus, the platination
produced by the receptorial system proves to be sorely inade-
quate for cytotoxicity, with a deficit of at least 73-fold.
Acknowledgements
Determination of the relative binding affinity (RBA) for the a
form of the estrogen receptor (ERa)
This work received financial support from MIUR (Roma) as
part of the COFIN2004 project, and from C. I. R. C. M. S. B.
(Bari). We are indebted to Johnson Matthey (Reading, UK) for
a generous loan of K2[PtCl4]. Research was carried out within
the framework of the European Cooperation COST D20 action
(Metal compounds in the treatment of cancer and viral diseases)
and COST B16 action (Multidrug resistance reversal). Thanks
are due to Dr A. Vessie`res-Jaouen and to Ms A. Cordaville (EN-
SCP) for RBA and log Po/w measurements, and to Dr L. McLean
for her assistance in writing the manuscript.
Lamb uterine cytosol prepared in buffer A (0.05 M Tris-HCl,
◦
0.25 M sucrose, 0.1% b-mercaptoethanol, pH 7.4 at 25 C) as
described in the literature56 was used as a source of ERa.
Aliquots (200 lL) of diluted ERa (in glass tubes) were
incubated for 3.5 h at 0 ◦C with 2 × 10−9M of [6,7-3H]-estradiol
(specific activity 1.96 TBq mmol−1) in the presence of nine
different concentrations of the hormone derivative to be tested.
At the end of the incubation period, the free and bound fractions
of the tracer were separated by protamine sulfate precipitation.42
The percentage reduction of [3H]-estradiol binding (Y) was cal-
culated using the logit transformation of Y (logit Y = ln[Y/1 −
Y]) versus the logarithm of the mass of the competing steroid.
The unlabelled steroid concentration, requiring displacement of
50% of the bound [3H]-estradiol, was calculated for each steroid
tested, and the results are expressed as RBA. The RBA value of
estradiol is by definition equal to 100%.
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