Highly selective metal mediated ortho-alkylation of phenol. First platinum containing organometallic chromane analogues

Article abstract of DOI:10.1039/b712248a

We were able, for the first time, to synthesize and characterize Pt derivatives with a structural shape similar to vitamin E, having a metalla-chromane core. The formation reaction mechanism includes an unexpected highly selective ortho aromatic electrophilic substitution on phenol, operated by [PtCl(η¹-C₂H₄OR)(N-N)], R = Me or Ph, and a final cyclization step. The X-ray structure of one of the new metalla-chromane complexes [Pt(EtPh)(phen)], 1a, (EtPh = 2-(ethan-2′-yl- kC¹)-1-phenolato-kO¹, phen = 1,10-phenanthroline) is reported. Cytotoxicity and Pt uptake measurements, performed on HeLa cancer cells, show an interesting structure-activity correlation for the new metalla-chromane analogues 1a and [Pt(MeOEtPh)(phen)], 1b, (MeOEtPh = 2-(ethan-2′-yl-kC¹)-4-(methoxy)-1-phenolato-kO¹), being the structurally closest to vitamin E and also the most active. This journal is The Royal Society of Chemistry.

Full text of DOI:10.1039/b712248a

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PAPER
www.rsc.org/dalton | Dalton Transactions
Highly selective metal mediated ortho-alkylation of phenol. First platinum
containing organometallic chromane analogues†‡
Vita M. Vecchio,a Michele Benedetti,*a Danilo Migoni,a Sandra A. De Pascali,a Antonella Ciccarese,a
Santo Marsigliante,a Francesco Capitellib and Francesco P. Fanizzi*a
Received 9th August 2007, Accepted 1st October 2007
First published as an Advance Article on the web 16th October 2007
DOI: 10.1039/b712248a
We were able, for the first time, to synthesize and characterize Pt derivatives with a structural shape
similar to vitamin E, having a metalla-chromane core. The formation reaction mechanism includes an
unexpected highly selective ortho aromatic electrophilic substitution on phenol, operated by
1
[PtCl(g -C2H4OR)(N–N)], R = Me or Ph, and a final cyclization step. The X-ray structure of one of the
new metalla-chromane complexes [Pt(EtPh)(phen)], 1a, (EtPh = 2-(ethan-2ꢀ-yl-kC1)-1-phenolato-kO1,
phen = 1,10-phenanthroline) is reported. Cytotoxicity and Pt uptake measurements, performed on
HeLa cancer cells, show an interesting structure–activity correlation for the new metalla-chromane
analogues 1a and [Pt(MeOEtPh)(phen)], 1b, (MeOEtPh = 2-(ethan-2ꢀ-yl-kC1)-4-(methoxy)-
1-phenolato-kO1), being the structurally closest to vitamin E and also the most active.
Introduction
Results and discussion
Bioorganometallic chemistry,
a
rapidly emerging area of
In this work we report the synthesis of [Pt(EtPh)(phen)], 1a, and
[Pt(MeOEtPh)(phen)], 1b, the first Pt containing organometal-
lic species having the same structural core of vitamin E, a
remarkable family of biologically active chromane derivatives,
Scheme 1. Compounds 1a–b are characterized by substitution of
the carbon atom in the 2 position within the chromane pseudo-
pyran heterocycle with a square planar Pt(II) centre, which also
carries a chelate nitrogen ligand, Scheme 1. Compounds 1a–
b, are unprecedented in Pt chemistry and represent, together
with previously reported [Ni(Me2EtPh)(N–N)], Me2EtPh = 2-
(ethan-1ꢀ,1ꢀ-dimethyl-2ꢀ-yl-kC1)-1-phenolato-kO1, N–N = phen,
dip = 2,2ꢀ-dipyridyl, and [Ni(Me2EtPh)(dmpe)], 2, dmpe = 1,2-bis-
(dimethylphosphino)ethane complexes,9 all the available metalla-
chromanes, of d8 metals, at present, Scheme 1.
organometallic chemistry, has recently provided significant ad-
vancements in structural diversity and molecular recognition
studies.1 Complex systems, characterized by the presence of both
coordination and organometallic bonds, considerably increase
the number of available structures for biomimetic and molec-
ular recognition investigations. The presence of a metal in a
biomimetic molecule may be useful, in principle, for a number
of reasons. Among them, one should consider the possibility
to send a particular metal to a specific target for therapeutic
(platinum derivatives are currently used in the chemotherapy
of many cancer forms2) and/or imaging purposes. Moreover,
metal containing biomimetic molecules, allow a wider range of
structural modification and screening possibilities (with respect
to plane organic compounds) in host guest related Quantitative
Structure–Activity Relationships (QSAR) analysis. In particular,
cyclometallated Pt(II) and Pd(II) compounds have been recently
synthesized and tested for their cytotoxic activity due to possible
interactions with DNA.3–8
Among cyclometallated complexes organometallic metalla-
chromane systems, of many metals,9–19 are widely reported in
the literature. It should be noted that, due to the structural
analogies with vitamin E, such complexes, could also be used to
address a metal to specific targets. Here we discuss the synthesis,
characterization and biological activity of the first Pt(II) metalla-
chromane complexes obtained by a novel highly selective metal
mediated ortho-alkylation of phenols.
aDipartimento di Scienze e Tecnologie Biologiche ed Ambientali, Universita`
del Salento, Via Provinciale Lecce-Monteroni, I-73100 Lecce, Italy. E-mail:
fp.fanizzi@unile.it; Fax: +39 0832 298867; Tel: +39 0832 298626
bIstituto di Cristallografia, IC-CNR, Via Amendola 122/o, I-70126, Bari,
Italy
† CCDC reference number 603625. For crystallographic data in CIF or
other electronic format see DOI: 10.1039/b712248a
‡ Electronic supplementary information (ESI) available: Selected bond
lengths and angles for complex 1a. See DOI: 10.1039/b712248a
Scheme 1 Structure of chromane, vitamin E (tocopherols and to-
cotrienols; R1, R2, R3 = H or Me; R4 = phytol side chain) and the chromane
derivatives 1a, 1b (N–N = 1,10-phenanthroline), 2 and 6. The conventional
numbering scheme of chromane is also reported.
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Step-by-step synthesis of 1a suggests that the formation reaction
mechanism occurs according to Scheme 2, eqn (a). The cationic
2
species [PtCl(g -C2H4)(phen)]+, 3,20 undergoes a fast nucleophilic
1
attack of phenolate oxygen, to give [PtCl(g -C2H4OC6H5)(phen)],
4, which can be obtained as the sole reaction product (iso-
lated yield 27%, based on 3), if only phenolate is used (see
Experimental). In the presence of a phenol/phenolate mixture,
1
compound 4 undergoes a transposition which gives [PtCl{o-(g -
C2H4)-phenol}(phen)], 5, via the concerted mechanism, depicted
in Scheme 2, eqn (a). This requires that the same phenol which
protonates the ether oxygen of 4 undergoes electrophilic attack
on the ortho position by the incipient carbocation formed at
the b carbon of 4. The overall transposition can therefore be
considered as an electrophilic substitution at the ortho position
of the phenol which occurs in unusually very mild conditions.
The phenol/phenolate ratio must be carefully set in the reaction
since excess acidity inhibits cyclization of 5 to give 1a. Indeed
compound 5 can be obtained (isolated yield 43% based on 3)
if the phenol/phenolate ratio is strongly increased in favour of
phenol. The possibility of an alternative direct intramolecular
rearrangement of 4, to give 5, has been excluded since: (i) the
rate of formation of 5, starting from isolated 4, strongly depends
on the concentration of the phenol, according to Scheme 2, eqn
(a); (ii) in the presence of an acid, different from phenol, but with
a similar pKa (i.e. 2,6-dimethylphenol or 2,4,6-trimethylphenol),
4 does not give any observable reaction (after 48 h), Scheme 3,
eqn (a); (iii) compound 5 could also be obtained in the reaction
Scheme 3
experimental conditions for the synthesis of 1 require that
phenolate should be almost twice the stoichiometric amount with
respect to the Zeise’s salt or 3. This is because phenolate is also
needed to neutralize excess HCl produced in the final cyclization
of 5. Phenol/phenolate ratios in the range 5–10 gave both high
yields and reaction rates.
According to organic chemistry rules, alkylation on phenols
should take place at the activated both ortho and para positions.
This is the case for the reported formation of ring alkylated prod-
ucts, from alkyl phenyl ethers, in the presence of Brønsted/Lowry
acids, although the reaction conditions are considerably more
drastic in that case.21 The total absence of para alkylated derivatives
in the reaction of 4 with phenol, which selectively gives 5, further
supports the concerted mechanism of Scheme 2, eqn (a). The high
concentration of phenol, required only to speed up the reaction, is
consistent with the need of such a suitable acid in the isomerization
step which gives 5. This excludes a priori a possible mechanism in-
volving an intramolecular Claisen-type rearrangement, previously
reported for the formation of Si and Ge chromane derivatives.19
Noteworthy the present highly selective metal mediated ortho-
alkylation of the phenol also represents a new synthetic appli-
1
of [Pt(g -C2H4OCH3)Cl(phen)] with phenol, Scheme 3, eqn (b);
(iv) in the presence of excess phenol-d6, 4 gives 5-d5 and therefore
1a-d4, fully deuterated at the phenolate ring, Scheme 3, eqn (c).
cation of olefin containing cationic species,22 [PtCl(g -olefin)(N–
N)]+, since in their derivatives [Pt(g -CR1R2-CR3R4-Nu)Cl(N–
2
1
N)], R1–4 = H or alkyl, one end of the former olefin proved to
1
be characterized (at least in the cases of [Pt(g -C2H4OR)Cl(phen)],
R = Me or Ph) by an incipient carbo-cationic character.
The molecular structure of compound 1a, obtained by single
crystal X-ray diffraction is reported in Fig. 1. Relevant crystal-
lographic parameters are given in Table 1, while selected bond
lengths and angles are given in Table 1s.‡
Comparison of the X-ray structure data of 2,2-dimethyl-6-
chromanol, 6, (X2 = C)23 with the previously described 2 (X2 =
Ni)9 and the here reported 1a (X2 = Pt) metalla-chromane
compound shows a progressive size and distortion increase of
the pseudo-pyrane ring on passing from X2 = C to X2 = Pt.
Structural markers for this behaviour are the reduction of O1–
X2–C3 angle and the lengthening of O1–X2 and X2–C3 distances
for the increase of ring size and both h (torsion angle between the
O1–X2 bond and the aromatic ring) and hc (torsion angle between
the C3–C4 bond and the aromatic ring) as a measure of the ring
distortion, Table 2.
Scheme 2 Step-by-step (a) and one-pot (b) synthesis of complex 1a
(N–N = 1,10-phenanthroline). Isolated yields are referenced to Pt.
Interestingly, the metalla-chromanes 1a–b could also be directly
obtained, starting from Zeise’s salt, phenol, phenolate and the
bidentate nitrogen donor, Scheme 2, eqn (b), or 3 and the phenol,
phenolate mixture in a one-pot reaction. The phenol/phenolate
ratio strongly affects the yield of complex 1 also in the one-
pot reactions starting either from Zeise’s salt or 3. Optimized
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Table 1 Crystallographic data† for complex [Pt(EtPh)(phen)], 1a
has been devoted to the synthesis of chromane derivatives,
characterized by the presence of functional groups or heteroatoms
bonded to or inside the chromane skeleton.30
Biological activity evaluation, by standard protocols, of the
metalla chromane complexes 1a and 1b, on HeLa cancer cells, gave
interesting preliminary results. The short term cellular platinum
uptake, measured by atomic absorption,31 resulted clearly in favour
of 1b with respect to 1a, by more than an order of magnitude,
moreover a MTT test32 showed a much higher cytotoxicity of
Formula
Formula weight
Temperature/K
C20H16N2OPt
495.44
293(2)
˚
Wavelength/A
0.71073
Crystal system
Space group
Monoclinic
P21/c
7.506(1)
20.254(2)
10.733(1)
102.473(6)
1593.2(3)
4, 2.066
˚
a/A
˚
b/A
˚
c/A
b/◦
Volume/A
compound 1b, IC50 = 11.3
0.6 lM, with respect to 1a, IC50
≈40 5 lM (IC50 evaluated with a ∼20% cells killed at the
3
˚
Z, calculated density/Mg m−3
Absorption coefficient/mm−1
F(000)
maximal administered dose of 10 lM), Fig. 2. It is well known that
the 6-hydroxo chromane unit plays a key role for the biological
activity of vitamin E derivatives. The 6-hydroxo substituent in
the chromane core of Vitamin E derivatives is required for
their activity as free radical scavengers.33 Moreover, together
with the hydrophobic phytol chain, both the endocyclic and
exocyclic 6-hydroxo chromane’s oxygens are known to constitute
essential binding sites recognized by a range of proteins in living
tissues25,34–44 (i.e. tocopherol transfer proteins,35,38–41 phospholipase
A2,25 tocopherol associated protein,37 protein kinases,44 super-
natant protein factor,36,42 proteins involved in regulation of gene
expression37 and cell signalling34). It is very interesting to note that
the only molecular difference between 1a and 1b responsible for
the higher both cytotoxicity, Fig. 2, and cellular uptake, Fig. 3, of
8.815
944
Red, prism
Crystal colour and shape
Crystal size
0.02 × 0.03 × 0.7 mm
5.04 to 27.52
−9 ≤ h ≤ 9, −26 ≤ k ≤ 24,
−13 ≤ l ≤ 12
9761/3471 [Rint = 0.0377]
94.6% (h = 27.52)
Full-matrix least-squares on F2
2467/0/281
h range for data collection/◦
Limiting indices
Reflections collected/unique
Completeness
Refinement method
Data/restraints/parameters
Goodness-of-fit on F2
Final R indices [I > 2r(I)]
R indices (all data)
1.122
R1 = 0.0484, wR2 = 0.0848
R1 = 0.0800, wR2 = 0.0934
−3
˚
Largest diff. peak and hole
2.287 and −1.020 e A
Fig.
1
Single crystal X-ray diffraction molecular structure of
[Pt(EtPh)(phen)], 1a.
Vitamin E vitamers (a-, b-, d-, and c-tocopherols and to-
cotrienols), Scheme 1, are naturally occurring O-heterocycles, with
a chromane structural core, characterized by useful pharmacolog-
ical properties. These vitamers are involved in processes such as
cellular signalling, gene expression, immune response, apoptosis
and protection of biological systems from oxidation. They have
also been proposed for the treatment of many diseases (i.e.
mental disorders,24 inflammations,25,26 cancer,27 cardiovascular28
and diabetic29 diseases). For this reason, considerable interest
Fig. 2 HeLa cell survival measured by MTT test after 48 h of incubation.
Cisplatin, 1a and 1b were administered in three different concentrations:
0.1, 1 and 10 lM. An ethanol concentration equal to 1a and 1b solutions
was opportunely added to the cisplatin solution. The data were the results
of three different experiments presented as means S.D.
Table 2 Significant structure parameters of 6, 2 and 1a moleculesa
O1–X2–C3/◦
h/◦
hc /◦
b
c
˚
˚
X2
O1–X2/A
X2–C3/A
C (6)
Ni (2)
Pt (1a)
1.456(3)
1.872(3)
2.013(6)
1.507(4)
1.936(5)
2.041(10)
108.5(2)
94.4(2)
92.2(4)
18.0
30.6
50.8
14.1
46.1
60.2
Fig. 3 Platinum intracellular accumulation in HeLa cells after 3 h
continuous exposure to cisplatin, 1a and 1b, administered at the same
concentration (1 lM). An ethanol concentration equal to 1a and 1b
solutions was opportunely added to the cisplatin solution. The data were
the results of three independent experiments presented as means S.D.
a The conventional numbering scheme of chromane is adopted even in the
case of metal complexes. b Torsion angle between the O1–X2 bond and the
aromatic ring. c Torsion angle between the C3–C4 bond and the aromatic
ring.
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1b with respect to 1a, is the presence of the 6-methoxo substituent
on the metalla-chromane core.
10 min, complex 3 is added (525 mg, 1.0 mmol), obtaining a
pale yellow suspension. After 1 h, the temperature is increased
to 25 ◦C. After 3 h stirring, the solution is concentrated under
vacuum until a pale yellow oil is obtained. The residual oil, stirred
at room temperature for a further 8 h, undergoes a colour change
to dark-red. Isolation of pure compounds 1a or 1b from the oil
mixtures was performed with the above described method. Since
the reaction yields of 1a and 1b based on 3 are very similar to those
reported above for the one-pot reaction, this latter was preferred
in our routine synthetic procedure.
Conclusions
We were able, for the first time, to synthesize and characterize Pt
derivatives with a structural shape similar to vitamin E, having a
metalla-chromane core. The new complexes were obtained by a
novel, highly selective, metal mediated ortho-alkylation of phenols
which takes place in unusually very mild conditions, by a concerted
mechanism. Cytotoxicity and Pt uptake measurements, performed
on HeLa cancer cells, showed an interesting structure–activity
correlation for the new metalla-chromane analogues 1a and 1b,
being the structurally closest to vitamin E and also the most active.
(1a) (Found: C, 48.0; H, 3.2; N, 5.6%. C20H16N2OPt requires
C, 48.5; H, 3.3; N, 5.7%) 1H-NMR (400 MHz, CDCl3) d 2.61 (t,
JH–H = 6.4 Hz, 3JPt–H = 51 Hz, 2H, Pt–CH2–CH2–), 2.76 (t, JH–H
=
6.4 Hz, 2JPt–H = 81 Hz, 2H, Pt–CH2–CH2–), 6.56 (m, 1H, CH), 7.02
(m, 2H, CH), 7.10 (d, JH–H = 6.9 Hz, 1H, CH), 7.57 (dd, JH–H
=
5.4, 8.2 Hz, 1H, CH), 7.85 (s, 1H, CH), 7.87 (s, 1H, CH), 8.02 (dd,
JH–H = 4.8, 8.2 Hz, 1H, CH), 8.48 (d, JH–H = 8.2 Hz, 1H, CH), 8.52
(d, JH–H = 8.2 Hz, 1H, CH), 9.31 (d, JH–H = 5.4 Hz, 3JPt–H = 54 Hz,
1H, CH), 9.73 (d, JH–H = 4.8 Hz, 3JPt–H = 13 Hz, 1H, CH) ppm. 13C-
NMR (400 MHz, CDCl3) d 15.8 (Pt–CH2-CH2-), 29.9 (Pt–CH2–
CH2–), 117.0, 119.0, 126.6, 128.7, 134.4 and 163.7 (6C, phenyl),
125.2, 125.5, 126.9, 127.1, 129.6, 130.8, 134.9, 136.5, 145.4, 146.5,
148.3 and 149.6 (12C, phen) ppm. 195Pt–NMR (400 MHz, CDCl3)
d −2948 ppm. (1b) (Found: C, 47.5; H, 3.9; N, 5.4%. C21H18N2O2Pt
requires C, 48.0; H, 3.5; N, 5.3%) 1H–NMR (400 MHz, CDCl3) d
Experimental
All solvents and reagents, except otherwise stated, were purchased
from Aldrich Chemical Company and used as received. Zeise’s salt
was prepared from potassium tetrachloroplatinate and ethylene
gas as previously described.45 NMR classical experiments were
recorded on a Bruker Avance DPX 400 instrument, using deuter-
ated solvents. 1H and 13C chemical shifts were referenced to TMS
by using the residual protic solvent peak as internal reference;
195Pt chemical shifts were referenced to Na2PtCl6, d(Pt) = 0 ppm,
3
2
2.62 (t, JH–H = 6.2 Hz, JPt–H = 62 Hz, 2H, Pt–CH2–CH2–), 2.84
in D2O as an external reference. [PtCl(g -C2H4)(phen)]BF4, 3, and
(t, JH–H = 6.2 Hz, 2JPt–H = 91 Hz, 2H, Pt–CH2–CH2–), 3.74 (s, 3H,
CH3), 6.60 (dd, JH–H = 2.9, 8.5 Hz, 1H, CH), 6.71 (d, JH–H = 2.9,
1H, CH), 6.93 (d, JH–H = 8.5 Hz, 1H, CH), 7.61 (dd, JH–H = 5.4,
7.9 Hz, 1H, CH), 7.90 (m, 1H, CH), 7.91 (m, 1H, CH), 8.05 (dd,
JH–H = 5.1, 8.2 Hz, 1H, CH), 8.54 (d, JH–H = 5.3 Hz, 1H, CH), 8.56
(d, JH–H = 7.7 Hz, 1H, CH), 9.40 (d, JH–H = 5.4 Hz, 3JPt–H = 52 Hz,
1
[PtCl(g -C2H4OCH3)(phen)] were synthesized with a previously
described method.20
[Pt{2-(ethan-2ꢀ-yl-kC1)-1-phenolato-kO1}(phen)],
[Pt(EtPh)(phen)], 1a, and [Pt{2-(ethan-2ꢀ-yl-kC1)-4-methoxy-
1-phenolato-kO1}(phen)], [Pt(MeOEtPh)(phen)], 1b
3
1H, CH), 9.79 (d, JH–H = 4.7 Hz, JPt–H = 14 Hz, 1H, CH) ppm.
In a typical synthetic procedure the phen ligand (90 mg, 0.50 mmol)
is added to a mixture of phenol (470 mg, 5.0 mmol), sodium
phenolate (170 mg, 1.0 mmol) (1a) or 4-methoxyphenol (745 mg,
6.0 mmol), NaOH (40 mg, 1.0 mmol) (1b) and the Zeise’s salt
(195 mg, 0.5 mmol), partially dissolved in CH2Cl2 (2–3 mL)
and kept under stirring at 0 ◦C. After one hour, stirring in the
dark, the colour of the reaction mixture turns from pale yellow
to orange–yellow. The solvent is then evaporated under vacuum
and the residual oil, stirred at room temperature for a further
8 h, undergoes a colour change from orange–yellow to dark-red.
The dark-red oil is then diluted with CH2Cl2 (5 mL) and flash
chromatographed on silica gel (≈1 g) eluting, first with CH2Cl2
to obtain a pale yellow eluate containing intermediate 5, which
is discarded, then with CH2Cl2/acetone = 90/10, to obtain a red
fraction which is collected, concentrated to 2 mL, under vacuum,
and added with Et2O (ca. 100 mL) to obtain a red precipitate. This
is separated from the solution by filtration and recrystallized from
CH2Cl2/Et2O to give the final product 1 (yield 175 mg, 0.35 mmol,
70%, 1a; 160 mg, 0.3 mmol, 60%, 1b, with respect to the starting
Zeise’s salt).
13C-NMR (400 MHz, CDCl3) d 16.1 (Pt–CH2–CH2–), 30.5 (Pt–
CH2–CH2–), 56.2 (OCH3), 111.7, 115.0, 117.0, 135.0, 150.2 and
158.0 (6C, phenyl), 125.2, 125.5, 126.8, 127.0, 129.5, 130.9, 134.8,
136.5, 145.8, 146.5, 148.4 and 149.7 (12C, phen) ppm. 195Pt-NMR
(400 MHz, CDCl3) d −2937 ppm.
[PtCl(ethan-2-yl-1-phenolato-kC1)(phen)],
[PtCl(g1-C2H4OC6H5)(phen)], 4
In a typical synthetic procedure, sodium phenolate (120 mg,
2
0.7 mmol) and [PtCl(g -C2H4)(phen)]BF4, 3, (460 mg, 0.9 mmol)
are reacted, suspended in CH2Cl2 (200 mL) by strongly stirring, for
1 h at 0 ◦C and a further 2 h at room temperature. A yellow–orange
solution is collected by filtration of the suspension. This solution
is concentrated to 20 mL, by evaporation under vacuum. The
yellow precipitate 4 is then collected by filtration after addition of
abundant Et2O (≈400 mL). To obtain a pure product, the solid 4 is
washed with abundant Et2O (≈100 mL) and dried (yield 130 mg,
0.24mmol, 27%). (Found:C, 45.3; H, 3.2;N, 5.4%. C20H17ClN2OPt
1
requires C, 45.2; H, 3.2; N, 5.3%) H-NMR (400 MHz, CDCl3)
2
3
Compounds 1a and 1b were also obtained starting from
d 2.58 (m, JPt–H = 94 Hz, 2H, Pt–CH2–CH2–), 4.32 (m, JPt–H =
2
[PtCl(g -C2H4)(phen)]BF4, 3,20 as a substitute of Zeise’s salt and
28 Hz, 2H, Pt–CH2–CH2–), 6.85 (m, 1H, CH), 6.96 (m, 2H, CH),
phen. In a typical synthetic procedure we partially dissolve phenol
(941 mg, 10.0 mmol), sodium phenolate (170 mg, 1.0 mmol) (1a) or
4-methoxyphenol (745 mg, 6.0 mmol), NaOH (40 mg, 1.0 mmol)
(1b) in CH2Cl2 (20 mL) and kept under stirring at 0 ◦C. After
7.23 (m, 2H, CH), 7.80 (m, 1H, CH), 7.97 (m, 2H, CH), 7.99
(m, 1H, CH), 8.57 (dd, JH–H = 4.8, 8.2 Hz, 1H, CH), 8.66 (dd,
3
JH–H = 5.2, 8.3 Hz, 1H, CH), 9.57 (d, JH–H = 5.2 Hz, JPt–H
=
3
58 Hz, 1H, CH), 9.85 (d, JH–H = 4.8 Hz, JPt–H = 16 Hz, 1H,
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CH) ppm. 13C-NMR (400 MHz, CDCl3) d 4.1 (Pt–CH2–CH2–),
71.2 (Pt–CH2–CH2–), 114.6 (2C), 119.4 (1C), 129.0 (2C), 159.4
(1C) (6C, phenyl), 125.5, 125.6, 127.0, 127.6, 130.0, 131.0, 136.3,
136.9, 146.2, 148.3 (2C), and 149.4 (12C, phen) ppm. 195Pt-NMR
(400 MHz, CDCl3) d −3230 ppm.
and recrystallized from CH2Cl2/Et2O to give the final product
1a-d4 (isolated yield ≈5 mg, 0.01 mmol, 50%).
Atomic absorption determination of cell Pt uptake
Atomic absorption analyses were performed at the central facility
ofCIRCMSB, ConsorzioInteruniversitariodiChimica dei Metalli
nei Sistemi Biologici in Bari, Italy. Pt uptake in HeLa cells
mediated by 1a and 1b was determined by atomic absorption
spectroscopy, according to a previously proposed method.31 After
3 h incubation, treated cells were harvested with a lysis buffer (SDS
0.1%, Na deossicolate 0.1%, Nonidet P40 10 lL mL−1), after which
the pellets were digested in 65% nitric acid at room temperature
in closed vials. The total amount of intracellular platinum was
determined by a Varian SpectrAA-880 Zeeman graphite furnace
atomic absorption spectrometer. A five-point calibration curve
was prepared for our measurements. The data were expressed as
nanogram Pt per milligram of cell proteins (means S.D.) and
were the result of three independent experiments. The Protein
content of the treated cells was determined with the Bio-Rad
protein assay kit 1, using lyophilized bovine serum albumin as
a standard.
[PtCl{2-(ethan-2ꢀ-yl-kC1)-1-phenol}(phen)],
[PtCl{o-(g1-C2H4)-phenol}(phen)], 5
In a typical synthetic procedure the phen ligand (47 mg, 0.26 mmol)
is added to a mixture of phenol (243 mg, 2.6 mmol), sodium
phenolate (44 mg, 0.26 mmol) and the Zeise’s salt (100 mg,
0.26 mmol), partially dissolved in CH2Cl2 (3–4 mL) and kept
under stirring at 0 ◦C. After 1 h, the colour of the reaction mixture
turns from pale yellow to orange–yellow. After evaporation of
the solvent, under vacuum, the residual oil, stirred at room
temperature for a further 4 h, undergoes a colour change from
orange–yellow to dark-red. The dark-red oil is then diluted with
CH2Cl2 (5 mL) and flash chromatographed on silica gel (≈1 g)
eluting, with CH2Cl2 to obtain a pale yellow eluate of 5. This
is separated from the solution by filtration and recrystallized
from CH2Cl2/pentane to give the final product 5 (yield 60 mg,
0.11 mmol, 43%). (Found:C, 45.0;H, 3.4;N, 5.1%. C20H17ClN2OPt
1
requires C, 45.2; H, 3.2; N, 5.3%) H-NMR (400 MHz, CDCl3)
Biological assays
2
3
d 2.40 (m, JPt–H = 85 Hz, 2H, Pt–CH2–CH2–), 2.79 (m, JPt–H
=
47 Hz, 2H, Pt–CH2–CH2–), 6.53 (m, 1H, CH), 6.62 (s, JPt–OH
13 Hz, 1H, OH), 6.84 (m, 1H, CH), 6.93 (m, 1H, CH), 7.29 (m,
=
Stability of 1a and 1b in the presence of water was checked,
before any biological test, recrystallizing the complexes unal-
tered from ethanol/water mixtures. The cytotoxicity of com-
pounds 1a–b was evaluated by the MTT biological test, based
on the metabolic reduction of a soluble tetrazolium salt i.e.
MTT = 3-[4,5-dimethyllthiazol-2-yl]-2,5-diphenyl tetrazolium
bromide (Sigma),32 on HeLa cancer cells. This colorimetric assay
measures cell survival after compound administration. As they
were scarcely water soluble, 1a–b were previously dissolved in
absolute ethanol, obtaining a mother liquor. Aliquots of the
mother liquor were added to PBS (phosphate buffered saline
solution), in order to treat HeLa cell cultures with ethanol
quantities suitably diluted and to avoid the cytotoxic effects of
the alcohol (at least 1 : 100). Moreover, control experiments
where only ethanol was used were performed. HeLa cells from
human tumoral endometrium were grown in DMEM (Euroclone).
Culture medium was supplemented with 10% heat-inactivated
fetal bovine serum (Euroclone), 0.2 mg mL−1 streptomycin,
200 IU mL−1 penicillin. Cells were cultured routinely at 37 ◦C
and 5% CO2 in a humidified incubator. After harvesting, cells
were counted and diluted appropriately with the culture medium;
100 lL containing 3000 cells were seeded in each well of a
96-well microtiter plate (Corning). After 24 h of incubation,
platinum containing compounds were administrated to each well
with a concentration between 0.1 lM and 10 lM. The toxicity
of these compounds was tested for 48 h. At the end of every
incubation, MTT was added at the final concentration of 500 lg
mL−1. After a 4 h incubation, the amount of formazan was
spectrophotometrically measured in isopropanol, k = 550 nm.
The optical density was used to calculate cell growth inhibition, as
a percentage with respect to the control. For the statistical analysis
of the data the Bonferroni–Dunn test was used and a p value < 0.05
was considered significant. All the results are the mean of three
different experiments.
1H, CH), 7.47 (dd, JH–H = 5.4, 8.1 Hz, 1H, CH), 7.97 (dd, JH–H
=
5.1, 8.3 Hz, 1H, CH), 7.90 (dd, JH–H = 8.1 Hz 1H, CH), 7.92 (dd,
JH–H = 8.3 Hz 1H, CH), 8.50 (m, 1H, CH), 8.54 (m, 1H, CH), 8.96
(dd, JH–H = 5.4, 3JPt–H = 62 Hz, 1H, CH), 9.80 (dd, JH–H = 5.1 Hz,
3JPt–H = 26 Hz, 1H, CH), ppm. 13C-NMR (400 MHz, CDCl3) d
7.2 (Pt–CH2–CH2–), 34.1 (Pt–CH2–CH2–), 116.0 (1C), 120.2 (1C),
126.4 (1C), 129.8 (1C), 131.2 (1C), 153.7 (1C) (6C, phenyl), 125.1,
125.6, 126.7, 127.0, 130.2, 130.9, 136.1, 137.0, 145.7, 146.2, 148.4,
and 148.6 (12C, phen) ppm. 195Pt–NMR (400 MHz, CDCl3) d
−3311 ppm.
Synthesis of 5 starting from [Pt(g1-C2H4OCH3)Cl(phen)]
1
In a typical experiment [Pt(g -C2H4OCH3)Cl(phen)] (10 mg,
0.02 mmol) are dissolved in 1 mL of CDCl3 adding a light excess
1
of phenol (3 mg, 0.03 mmol). Formation of 5 is followed by H
NMR spectroscopy and is complete after a week.
Reaction of 4 with phenol-d6 to give 5-d5 and 1a-d4
In a typical experiment complex 4 (10 mg, 0.02 mmol) was
dissolved in 10 mL of CDCl3 adding phenol-d6 (25 mg, 0.25 mmol)
and NaOD (≈2 mg, 0.05 mmol). After 1 h, stirring in the dark,
the colour of the reaction mixture turns from pale yellow to
orange–yellow. The formation of 5-d5, and 1a-d4 is followed by 1H
NMR spectroscopy. After a week, the CDCl3 red solution is flash
chromatographed on silica gel (≈1 g) eluting, first with CH2Cl2, to
obtain a pale yellow eluate containing intermediate 5-d5 (isolated
yield ≈1 mg, 0.002 mmol, 10%) then with CH2Cl2/acetone =
90/10, to obtain a red fraction which is collected, concentrated to
1 mL, under vacuum, and added with Et2O (ca. 10 mL) to obtain
a red precipitate. This is separated from the solution by filtration
5724 | Dalton Trans., 2007, 5720–5725
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X-Ray structure
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(Fig. 1) The structure of 1a was determined by single crystal
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˚
(k = 0.71073 A), in φ and x scans mode. The unit-cell parameters
were obtained by analyzing 173 reflections with 3.87 ≤ h ≤
22.47◦. The systematic absences were uniquely consistent for the
monoclinic space group P21/c. The structure was solved by the
Direct Method procedure of SIR97,46 and refined by a full-matrix
least-squares technique based on F2, SHELXL-97.47 Highest
residual peaks in the difference Fourier map were located close
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Acknowledgements
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The Ministero dell’Universita` e della Ricerca Scientifica e Tecno-
logica (MURST, Roma, Prin 2006031888–004), the University of
Lecce, Italy, (ex 60% funds) and the Consorzio Interuniversitario
di Ricerca in Chimica dei Metalli nei Sistemi Biologici (CIR-
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gratefully acknowledge Dr F. Cannito (CIRCMSB), for the atomic
absorption data collection, and Prof. L. Maresca (University of
Bary, Italy), for helpful discussions.
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The Royal Society of Chemistry 2007
Dalton Trans., 2007, 5720–5725 | 5725
©