Electrophilic attack of [I(py)2]+(NO3-) on three-coordinate Pt0 precursors: Synthesis and in vitro antitumor activity of water-soluble, five- coordinate Pt(II) complexes

Article abstract of DOI:10.1002/1099-0682(200008)2000:8<1717::aid-ejic1717>3.0.co;2-1

[I(py)₂]⁺(NO₃^-) oxidatively adds to the three-coordinate platinum(0) precursors [Pt(N,N-chelate)(olefin)] affording the cationic five-coordinate products [Pt(N,N-chelate)I(py)-(olefin)]⁺(NO₃^-) (1) {N, N-chelate = 2,9- Me₂-1,10-phenanthroline (dmphen), 2-methyl-6-[(phenylimino)methyl]pyridine (pimpy) and 2-methyl-6-[(4-methoxyphenylimino)methyl]pyridine (mpimpy)}. The crystal structure of [Pt(pimpy)I(py)(dimethyl fumarate)]⁺(NO₃^-) is reported. The crystals are tri-clinic, space group P1(bar), with a = 13.511(2), b = 13.754(2), c = 8.678(1) ?, α = 95.2(1)°, β = 92.5(2)°, γ = 64.7(1)°, Z = 2. Type I complexes, which are soluble both in chlorinated solvents and in water, were tested for cytotoxicity against a panel of tumour cell lines of various histotypes including NSCLC H460, leukaemic HL-60, ovarian A 2780, IGROV-1, SKOV-3, and an ovarian carcinoma A 2780/CP selected for resistance to cis-platin [DDP = cis-diamminedichloroplatinum(II)]. [Pt(dmphen)I(py)(methyl acrylate)]⁺(NO₃^-) was the most active in all cell lines tested, its antitumoural activity in vitro being comparable to that of DDP.

Full text of DOI:10.1002/1099-0682(200008)2000:8<1717::aid-ejic1717>3.0.co;2-1

FULL PAPER
Electrophilic Attack of [I(py)2]؉(NO3 ) on Three-Coordinate Pt0 Precursors:
Synthesis and In Vitro Antitumor Activity of Water-Soluble, Five-Coordinate
PtII Complexes
؊
Mario Bigioni,*[a] Paolo Ganis,[b] Achille Panunzi,*[c] Francesco Ruffo,[b]
Carmela Salvatore,[a] and Antonio Vito[d]
Keywords: Platinum / N ligands / Water soluble complexes / Antitumor agents / Electrophilic additions
[I(py)2]+(NO3−) oxidatively adds to the three-coordinate plat-
inum(0) precursors [Pt(N,N-chelate)(olefin)] affording the
cationic five-coordinate products [Pt(N,N-chelate)I(py)-
(olefin)]+(NO3−) (1) {N,N-chelate = 2,9-Me2-1,10-phenanthro-
2. Type I complexes, which are soluble both in chlorinated
solvents and in water, were tested for cytotoxicity against a
panel of tumour cell lines of various histotypes including
NSCLC H460, leukaemic HL-60, ovarian A 2780, IGROV-1,
line (dmphen), 2-methyl-6-[(phenylimino)methyl]pyridine SKOV-3, and an ovarian carcinoma A 2780/CP selected for
(pimpy) and 2-methyl-6-[(4-methoxyphenylimino)methyl]py- resistance to cis-platin [DDP = cis-diamminedichloroplati-
ridine (mpimpy)}. The crystal structure of [Pt(pimpy)I(py)(di-
num(II)]. [Pt(dmphen)I(py)(methyl acrylate)]+(NO3−) was the
methyl fumarate)]+(NO3−) is reported. The crystals are tri- most active in all cell lines tested, its antitumoural activity in
clinic, space group P1(bar), with a = 13.511(2), b = 13.754(2),
c = 8.678(1) A, α = 95.2(1)°, β = 92.5(2)°, γ = 64.7(1)°, Z =
vitro being comparable to that of DDP.
˚
Introduction
and so broaden the clinical spectrum of antitumour activity.
In the last few decades interest in five-coordinate plat-
inum(II) complexes (I) (Figure 1) has increased.[3]
Cisplatin [DDP ϭ cis-diamminedichloroplatinum(II)] is
an extensively investigated anticancer drug used in the treat-
ment of some solid tumours such as ovarian, testicular and
bladder cancers.[1] Its therapeutic efficacy is often limited
by its dose-related nephrotoxicity and by the development
of drug resistance, depending on the increase of intracellu-
lar glutathione,[2] increased tolerance to DNA damage or
increased DNA repair.
The stability of these species is prompted by the presence
of sterically hindered nitrogen chelates, e.g. 2,9-Me2-1,10-
phenanthroline (dmphen). Such ligands inhibit olefin loss
because the resulting square-planar product would suffer
severe constraints in the coordination plane.[3] Some type I
[4]
complexes of formula [PtCl(Me)(dmphen)(olefin)]BF4
and [PtCl(SnMenCl4Ϫn)(dmphen)(dimethyl fumarate)][5]
have been tested in vitro against some tumour cell lines with
promising results. As far as we know, this was the first in-
vestigation on coordinatively saturated platinum(II) derivat-
ives, since a literature search shows that other studies have
been concerned with square-planar platinum(II)[6] or octa-
hedral platinum(IV) species.[7] The insolubility in water of
the above-mentioned species of type I limits a further devel-
opment of these studies. Thus, we were prompted to invest-
igate the feasibility of related compounds with properties
more suitable to pharmacological uses. Keeping in mind
that a charged complex may display a good solubility in
water without loss of pharmacological activity,[8] we aimed
to prepare cationic water-soluble complexes of type I. A
straightforward procedure was found to be the oxidative ad-
Many analogues have been synthesised in an attempt to
either decrease the toxic side effects of the parent drug or
to overcome the intrinsic and induced resistant phenotype
Figure 1. General formula for type I complexes
[a]
Pharmacology Department, Menarini Ricerche S.p.A.,
via Tito Speri 10, I-00040 Pomezia, Italy
[b]
`
Dipartimento di Chimica, Universita di Napoli ‘‘Federico II’’,
via Mezzocannone 4, I-80134 Napoli, Italy
[c]
`
Facolta di Agraria and Dipartimento di Chimica,
dition of the readily available iodonium salt
`
Universita di Napoli ‘‘Federico II’’,
Ϫ [9]
[I(py)2]ϩ(NO3
)
to three-coordinate platinum(0) pre-
via Mezzocannone 4, I-80134 Napoli, Italy
Fax: (internat.) ϩ 39-081/552-7771
E-mail: panunzi@chemna.dichi.unina.it
Algalite,
cursors[10] (Scheme 1).
Herein we describe the synthesis of novel type I species,
[d]
via Leonardo da Vinci 277, I-20090 Trezzano sul Naviglio, Italy their NMR characterisation and the crystal structure of a
Eur. J. Inorg. Chem. 2000, 1717Ϫ1721 1717
WILEY-VCH Verlag GmbH, D-69451 Weinheim, 2000
1434Ϫ1948/00/0808Ϫ1717 $ 17.50ϩ.50/0

M. Bigioni, P. Ganis, A. Panunzi, F. Ruffo, C. Salvatore, A. Vito
FULL PAPER
which contain a pyridine and an iodide in the axial posi-
tions (Scheme 1).
The N,N ligands used in this work are shown in
Scheme 1. The products 1 are listed in Table 1 with their
labels. The reactions were performed in chloroform, which
is a good solvent for both reagents and products. The only
exception was the addition to [Pt(dmphen)(maleic anhyd-
ride)], which required the use of tetrachloroethane due to
the poor solubility of the Pt0 precursor.
Type I complexes could be crystallised by slow addition
of diethyl ether to the reaction mixture. They are soluble
both in water and chlorinated solvents, except for the di-
benzoylethylene derivative, which is only soluble in chlorin-
ated solvents. The products are fairly stable in solution, and
no appreciable decomposition could be seen even after sev-
eral hours.
The possibility that water may substitute iodide in one
of the apical positions has been ruled out by conductivity
measurements. Actually, if hydrolysis occurred, the diposit-
Ϫ
ive cation [Pt(N,N-chelate)(H2O)(py)(olefin)]2ϩ(IϪ)(NO3
)
should form. However, the molar conductivities of type I
complexes in water are in the range 1.0Ϫ1.3 ϫ 102
ohmϪ1 cm2 molϪ1. These values are close to that measured
(1.4 ϫ 102 ohmϪ1 cm2 molϪ1) for an authentic 1:1 salt, i.e.
K[PtCl3(C2H4)]. On the other hand, the conductivity of a
2:1 salt such as K2PtCl4 in water is significantly higher (2.3
ϫ 102 ohmϪ1 cm2 molϪ1).
Scheme 1. Synthesis of type-1 complexes and used N,N ligands
representative complex. This paper reports the pharmacolo-
gical advantages of new modifications introduced in the
type I species on the inhibitory effect of the growth of se-
lected tumoural DDP resistant and sensitive cell lines.
The NMR spectra were recorded in D2O in most cases
(Table 1). It is possible to assign unequivocally a trigonal
bipyramidal geometry to the products on the basis of argu-
ments already thoroughly discussed for related species.[3] As
for the dmphen derivatives 1a-e, the number of isomers the-
oretically predicted depends on the symmetry of the alkene.
Dimethyl maleate and maleic anhydride can afford two rot-
amers, while methyl acrylate could afford two enantiomeric
pairs. In all cases, only one spectral pattern was observed,
indicating that the olefins coordinate with a high degree of
stereoselectivity. Dimethyl fumarate gives rise to a pair of
enantiomers, obviously not distinguishable by NMR spec-
troscopy in the absence of chiral interference.
Results and Discussion
Synthesis, NMR Characterisation and Reactivity of Type I
Complexes
Cationic five-coordinate PtII complexes are known.[11,12]
They were obtained by chloride abstraction starting from a
suitable precursor in the presence of a silver salt [Equa-
tion (1) and Equation (2)]:
[PtCl(R)(N,N-chelate)(olefin)]
[Pt(R)(MeCN)(N,N-chelate)(olefin)](BF4) ϩ AgCl
ϩ
AgBF4
ϩ
MeCN
Ǟ
(1)
(2)
cis-[PtCl2(MeCN)(olefin)] AgBF4 N,N-chelate
ϩ
ϩ
Ǟ
In the case of pimpy (1h), we observed the expected two
diastereomers (each one a pair of enantiomers) for the com-
[PtCl(MeCN)(N,N-chelate)(olefin)](BF4) ϩ AgCl
Route 1 allowed the synthesis of type I derivatives con- plex with dimethyl fumarate. Slow recrystallization of the
taining a hydrocarbyl group R and a neutral ligand (e.g. complex affords only one pair whose X-ray crystal structure
MeCN) in the axial positions. The second procedure af- is described below. Finally, complex 1i with mpimpy and
forded the related chloro derivatives. In the latter case, only dimethyl maleate should also exist as two diastereomers
products with electron-rich olefins could be obtained, since (pairs of enantiomers). The high degree of stereoselectivity
the required precursors cis-[PtCl2(L)(olefin)] are not avail- in olefin coordination allows us to detect only one pair in
able when the olefin bears electron-withdrawing substitu- solution.
ents. The synthetic procedure herein described bypasses this
obstacle and allows the attainment of several cationic halo
Description of the Molecular Structure of 1h
complexes containing electron-acceptor olefins.
A view of the structure of the cation giving the atom
The method is based on the well-known ability of three- numbering scheme is shown in Figure 2. Selected bond
coordinate platinum(0) complexes of general formula lengths and angles and torsion angles are listed in Table 2.
[Pt(N,N-chelate)(olefin)] to undergo electrophilic attack.[10]
Ϫ [9]
Thus, the iodonium(I) salt [I(py)2]ϩ(NO3
)
oxidatively
The bipyramidal trigonal coordination about the metal is
adds to the suitable Pt0 complexes with formation of pyrid- as expected and does not differ substantially from that
ine and of the corresponding five-coordinate complexes, found for similar five-coordinate PtII complexes.[3] The li-
1718
Eur. J. Inorg. Chem. 2000, 1717Ϫ1721

Water-Soluble, Five-Coordinate PtII Complexes
FULL PAPER
Table 1. Selected spectroscopic and analytical data for type I complexes
Compound
1H NMR[a]
Me-chelate
Analysis (%)
found (calcd)
H-olefin[b]
Other selected signals
C
H
N
1a [PtI(py)(dmphen)
5.19 (s, 2 H, 80)
3.53 (6 H)
7.92 (d, 2 H, 2,6-H-py)
3.64 (s, 6 H, OMe)
7.90 (d, 2 H, 2,6-H-py)
3.86 (s, 3 H, OMe)
3.55 (s, 3 H, OMe)
36.61
3.15
6.69
(dimethyl maleate)](NO3)
1b[PtI(py)(dmphen)
(36.82)
36.88
(3.09)
3.18
(6.87)
7.02
5.52 (d, 1 H)
4.95 (d, 1 H)
3.55 (3 H)
3.38 (3 H)
(dimethyl fumarate)](NO3)
(36.82)
(3.09)
(6.87)
1c [PtI(py)(dmphen)
5.16 (s, 2 H, 76)
3.56 (6 H)
35.75
2.60
7.41
(maleic anhydride)](NO3)
1d [PtI(py)(dmphen)
(methyl acrylate)](NO3)
(35.90)
36.28
(2.49)
2.98
(7.28)
7.24
5.12 (dd, 1 H, 65)
4.17 (d, 1 H, 59)
3.82 (d, 1 H, 55)
5.15 (s, 2 H, 78)
3.51(3 H)
3.36 (3 H)
7.98 (d, 2 H, 2,6-H-py)
3.54 (s, 3 H, OMe)
(36.47)
(3.06)
(7.40)
1e[c] [PtI(py)(dmphen)
(dibenzoylethylene)](NO3)
1f [PtI(py)(dmphen)
2.71 (6 H)
3.48 (6 H)
8.20 (d, 2 H, 2,6-H-py)
46.36
3.27
6.29
(46.32)
38.57
(3.22)
3.38
(6.17)
6.78
5.06 (s, 2 H, 76)
7.92 (d, 2 H, 2,6-H-py)
4.05 (m, 4 H, OCH2)
1.14 (t, 6 H, CH2Me)
8.03 (d, 2 H, 2,6-H-py)
4.30 (q, 2 H,OCH2)
3.91 (m, 1 H, OCH2)
3.65 (m, 1 H,OCH2)
1.38 (t, 3 H, CH2Me)
1.14 (t, 3 H, CH2Me)
9.31 (s, 1 H, CHϭN)
3.87 (s, 3 H, OMe)
3.39 (s, 3 H, OMe)
9.19 (s, 1 H, CHϭN)
3.94 (s, 3 H, OMe)
3.55 (s, 3 H, OMe)
3.46 (s, 3 H, OMe)
(diethylmaleate)](NO3)
(38.44)
(3.47)
(6.64)
1g [PtI(py)(dmphen)
(diethyl fumarate)](NO3)
5.51 (d, 1 H, 70)
4.95 (d, 1 H, 68)
3.56 (6 H)
38.22
(38.44)
3.33
(3.47)
6.50
(6.64)
1h [PtI(py)(pimpy)
5.50 (d, 1 H)
4.48 (d, 1 H)
3.41 (3 H)
3.37 (3 H)
36.04
(35.88)
3.11
(3.14)
7.12
(6.97)
(dimethyl fumarate)](NO3)
(more abundant isomer)
1i [PtI(py)(mpimpy)
5.25 (ABq, 2 H)
36.28
(36.03)
3.19
(3.26)
6.57
(6.72)
(dimethylmaleate)](NO3)
[a]
At 250 or 200 MHz and 298 K; in D2O, HDO (δ ϭ 4.8) as internal standard; abbreviations: ABq: AB quadruplet; d: doublet; dd:
[b] 2
[c]
doublet of doublets; m: multiplet; s: singlet; q: quadruplet; t: triplet. Ϫ
CHCl3 (δ ϭ 7.26) as internal standard.
J
(Hz) in parentheses (when measurable). Ϫ In CDCl3,
PtϪH
Table 2. Selected geometrical parameters for 1h
˚
Bond lengths (A)
PtϪI
2.616(2)
2.209(8)
2.21(1)
2.08(1)
2.10(1)
2.116(9)
1.44(1)
PtϪN(1)
PtϪN(2)
PtϪN(3)
PtϪC(17)
PtϪC(14)
C(14)ϪC(17)
Bond angles (°)
C(14)ϪPtϪI
92.1(3)
91.9(4)
93.2(3)
88.4(4)
85.5(3)
122.2(4)
90.5(3)
123.1(4)
90.5(4)
75.1(4)
88.5(5)
177.4(4)
N(1)ϪPtϪN(3)
C(17)ϪPtϪI
N(2)ϪPtϪN(3)
N(1)ϪPtϪI
C(17)ϪPtϪN(1)
N(2)ϪPtϪI
C(14)ϪPtϪN(2)
C(14)ϪPtϪN(3)
N(1)ϪPtϪN(2)
Figure 2. The molecular structure of the cation [PtI(py)(pimpy)(di-
methyl fumarate)]ϩ with the atom numbering scheme; the trigonal
bipyramidal coordination sphere is clearly shown
gands iodide and pyridine occupy the axial positions, the
pimpy ligand and the fumarate double bond are in the C(17)ϪPtϪN(3)
IϪPtϪN(3)
equatorial coordination sites. All the atoms of pimpy, ex-
cept those of the phenyl group bonded to N2, lie in a plane
almost coincidental with the equatorial coordination plane
Torsion angles(°)
of Pt (the corresponding dihedral angle is ca. 3°). Also C14 C(18)ϪC(17)ϪC(14)ϪC(15)
133.6
26.9
Ϫ175.1
177.8
C(1)ϪN(2)ϪC(8)ϪC(9)
and C17 of the olefin bond of dimethyl fumarate are con-
tained in this plane with deviations of less than 0.01Ϫ0.02
˚
C(14)ϪC(15)ϪO(2)ϪC(16)
C(17)ϪC(18)ϪO(4)ϪC(19)
A. The phenyl group bonded to N2 is rotated about the
Eur. J. Inorg. Chem. 2000, 1717Ϫ1721
1719

M. Bigioni, P. Ganis, A. Panunzi, F. Ruffo, C. Salvatore, A. Vito
FULL PAPER
bond C8ϪN2 in order to relieve short contact interactions SKOV-3) and on two leukaemia cell lines (human HL-60
˚
between HC9 and HC1 (here at 2.1 A). The rotation is im- and murine L1210). It exhibited a cytotoxic activity similar
posed by the pyridine group in the apical position on Pt to (SKOV-3 cells) or three times higher (IGROV cells) than
such as to produce the most favourable contact distances DDP, whereas on HL-60 and L1210 leukaemia its potency
between these two groups. The atoms C17 and C14, at dis- was lower (Table 4).
˚
tances of 1.44 A, reveal, as found in several similar com-
plexes,[13,14] a sharp dehybridization sp2Ǟsp3 due to a con-
Table 4. Cytotoxicity (IC50 µ) of 1d in comparison to DDP
sistent platinum-to-olefin back-donation. As
a con-
Cell lines
DDP
1d
sequence, a rotation about the bond C14ϪC17 is allowed
(here it is ca. 132°, i.e. 48° from a trans conformation). This
rotation is imposed by stereochemical requirements in order
to relieve intramolecular interactions, with I on one side
A 2780
A 2780/CP
H460
SKOV-3
IGROV
HL-60
L1210
GLC-4
0.33 ± 0.07
3.3 ± 0.1
0.40 ± 0.07
1.32 ± 0.03
0.09 ± 0.003
1.45 ± 0.33
0.13 ± 0.03
1.08 ± 0.35
1.65 ± 0.07
0.40 ± 0.13
0.67 ± 0.10
4.2 ± 2.5
˚
˚
(I O1 ϭ 3.4 A; I C15 ϭ 3.3 A) and the pyridine group
1.0 ± 0.7
˚
˚
on the other side (N3 C18 ϭ 2.88 A; N3 O3 ϭ 2.95 A).
Repulsions between pimpy and dimethyl fumarate also
cooperate to this effect, in particular C7 with O4, and C18
and C13 with O2. These interactions cannot be avoided
through a rotation about the coordination axis of the olefin
owing to demands imposed by the coordination geometry
about the metal.[14] Dimethyl fumarate in its observed con-
figuration seems to have no influence on the conformation
or on the choice of coordination isomerism by the Schiff
base at Pt (or vice versa, the Schiff base does not appear
to induce any preference for the coordination of dimethyl
0.40 ± 0.10
1.0 ± 0.13
0.53 ± 0.10
In the A2780 sub-line selected for resistance to DDP
(A2780/CP), characterised by an increased ability to repair
drug-induced DNA damage, compound 1d was cross-resist-
ant to DDP with a Resistance Index (RI) value of 3 (vs. 10).
fumarate in either of its prochiral configurations). Thus, we Conclusion
believe that the diastereoisomer examined here is the one
favoured in the crystal only because of crystal field forces.
All the other geometrical parameters are normal and have
been exhaustively discussed in our previous structural
reports.[12Ϫ14]
New cationic, water-soluble, trigonal bipyramidal com-
plexes of platinum(II) were prepared by oxidative addition
of [I(py)2]ϩ(NO3Ϫ) to suitable platinum(0) precursors. The
products were tested for cytotoxicity against a panel of tu-
mour cell lines of various histotypes. The data show that
the new class of PtII five-coordinate complexes is endowed
with promising antitumor properties. The next step will
therefore be the characterisation of the antitumour efficacy
of the compounds in vivo. In the meantime, studies evaluat-
ing the potential nephrotoxic effects of the molecule will
be performed.
Cytotoxic Activity
The cytotoxic activity of six cisplatin (DDP) analogues
were studied on a panel of three human carcinoma cell lines
in comparison with the parent compound. The drugs were
dissolved in DMSO and diluted with 0.9% NaCl solution.
After further dilution in culture, DMSO is present at a con-
centration ranging from 0.01 to 1%. The pattern of cellular
response is shown in Table 3.
Experimental Section
Table 3. Cytotoxicity (IC50 µ) of type I compounds in comparison
to DDP
General: NMR spectra were recorded at 250 or 200 MHz on a
Compound
A-2780
GLC-4
H-460
Bruker AC-250 or a Varian XL-200 spectrometer. The three-coord-
Ϫ [9]
inate precursors [Pt(N,N-chelate)(olefin)][10] and [I(py)2]ϩ(NO3
)
DDP
1a
0.33 ± 0.06
0.49 ± 0.18
4.41 ± 0.02
0.40 ± 0.07
0.25 ± 0.16
1.30 ± 0.12
4.2 ± 0.6
0.53 ± 0.10
1.59 ± 0.12
1.96 ± 0.25
0.40 ± 0.13
0.58 ± 0.21
1.85 ± 0.27
2.39 ± 0.48
0.67 ± 0.10
1.28 ± 0.49
2.69 ± 0.60
0.092 ± 0.003
3.08 ± 0.95
2.96 ± 0.59
4.43 ± 1.44
were prepared according to literature procedures. Solvents and re-
agents were of analytical grade and were used without further puri-
fication.
1b
1d
1f
1g
Type-1 Complexes: To a solution of the appropriate [Pt(N,N-che-
late)(olefin)] precursor (0.20 mmol) in chloroform (1 mL) was ad-
ded [I(py)2]ϩ(NO3Ϫ) (0.20 mmol) previously dissolved in the min-
imum amount of chloroform. Careful addition of diethyl ether
prompted the crystallisation of the product, which was washed with
diethyl ether and dried under vacuum (yield: 80Ϫ90%). In the case
of the precursor [Pt(dmphen)(maleic anhydride)] the reaction was
performed in tetrachloroethane.
1i
Compound 1d showed an activity similar to DDP on
A2780 and GLC-4 cell lines, while on the H460 cell line the
IC50 value was at least three orders of magnitude greater
than that of the parent compound. The other analogues
exhibited a cytotoxic potency lower or, at best, similar to
that of DDP. The cytotoxic activity of 1d was further evalu-
X-ray Data Collection and Refinement of the Structure of 1h: A
ated on two additional ovarian cell lines (IGROV and crystal of 1h suitable for X-ray analysis was obtained from dichloro-
1720 Eur. J. Inorg. Chem. 2000, 1717Ϫ1721

Water-Soluble, Five-Coordinate PtII Complexes
FULL PAPER
Evaluation of Cytotoxic Activity: The cytotoxicity of all compounds
was studied on a variety of human tumour cell lines of different
types, using the SRB assay.[15] Briefly, the A 2780 (1.0 ϫ 104 cells/
mL), A 2780/CP (7.0 ϫ 103 cells/mL), SKOV-3 (4.0 ϫ 104 cells/
mL), IGROV-1 (2.5 ϫ 104 cells/mL), L1210 (5.0 ϫ 103 cells/mL),
HL-60 (5 ϫ 103 cells/mL), H460 (7 ϫ 103 cells/mL) or GLC-4 (7
ϫ 103 cells/mL) cell line was seeded in 96-well microtiter plates and
incubated for 24 h at 37 °C in a 5% CO2 incubator. Drugs were
then added to the wells to achieve final drug concentrations ran-
ging from 0.1 to 100 µ. After 24 h of drug exposure, the cells were
washed twice with PBS and incubated in a drug-free medium for
about three doubling times (72 h). The cells were then fixed with
16% TCA, incubated at 4 °C for 1 h and finally rinsed five times
with tap water and dried thoroughly. The cells were stained with
100µL/well of SRB (0.4% in 1% acetic acid) for 15 min, rinsed five
times in 1% glacial acetic acid and suspended in 10 m TRIS-HCl
(pH ϭ 10.5) buffer solution. The absorbance was measured with
Microplate Reader (BioRad MR 3550) at 570 nm. The results were
expressed as IC50, calculated from dose-response curves, and de-
fined as the drug concentration required for a 50% reduction of
absorbance compared to control cells. Each experiment was re-
peated at least three times.
Table 5. Crystal data and experimental details for 1h
Formula
C24H25IN4O7Pt[a]
M (g/mol)
Cryst. Syst.
Space group
803.5
Triclinic
P1(bar)
13.511(2)
13.754(2)
8.678(1)
95.2(1)
92.5(1)
64.7(1)
2
˚
a, A
˚
b, A
˚
c, A
α (°)
β (°)
γ (°)
Z
D (g/cm3)
µ (Mo-Kα) (cmϪ1
F(000)
1.84
)
56.67
768
Cryst. Size (mm)
0.2ϫ0.2ϫ0.3
2.0 in 2θ scan mode
3.0 Յ 2θ Յ 45
6927
Scan speed (°/min)
2θ range (°)
Measd. reflns.
Scan width (°)
1.2
No. of data used [I Ͼ 2σ(I)]
No. of params. refined
5721
383
R[b], Rw
0.060, 0.064
1.11
[c]
GOF[d]
3
˚
Highest map residues (e/A )
1.2[e]
[a]
The probable presence of water of crystallization has not been
considered. See text. Ϫ [b] R ϭ Σ||Fo| Ϫ |Fc||/Σ|Fo|. Ϫ [c] Rw ϭ [Σw(|Fo|
Acknowledgments
We thank the Consiglio Nazionale delle Ricerche and the Ministero
Ϫ |Fc|)2/Σw|Fo|2]1/2. Ϫ GOF ϭ [Σw(|Fo| Ϫ |Fc|)2/(ND Ϫ NU)2]1/2
.
[d]
[e]
Ϫ
This value is explained in the Experimental Section.
`
dellЈUniversita e della Ricerca Scientifica (Programmi di Ricerca
Scientifica di Rilevante Interesse Nazionale, Cofinanziamento
1998Ϫ1999) for financial support and the Centro Interdipartimen-
`
tale di Metodologie Chimico-Fisiche, Universita di Napoli ‘‘Feder-
methane/diethyl ether. Crystal data, intensity data and processing
ico II’’, for NMR facilities. A. P. and F. R. thank the Menarini for
financial support. We thank also Prof. G. Valle for helpful assist-
ance.
details for 1h are shown in Table 5.
The data were obtained with a Philips PW-100 four-circle auto-
mated diffractometer with graphite monochromator. Intensity data
were collected at 25 °C using the θϪ2θ scan method. Two reference
reflections, monitored periodically, showed no significant vari-
ations in intensity. Data were corrected for Lorentz and polariza-
tion effects and an empirical absorption correction was applied (Ψ
scan). The position of Pt was determined from a three-dimensional
Patterson function. All the remaining atoms, except for the hydro-
gens, were located from successive Fourier maps using SHELX-76.
The hydrogen atoms were located at calculated positions and re-
fined with a fixed geometry with respect to their carrier atoms
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(CϪH ϭ 1.08 A). Anisotropic thermal parameters were used for all
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Ϫ
the atoms except for the anion NO3 and the hydrogens. Blocked-
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Ϫ
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the R index and the highest map residuals (see Table 5). Nonethe-
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here described.
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Crystallographic data (excluding structure factors) for the structure
reported in this paper have been deposited with the Cambridge
Crystallographic Data Centre as supplementary publication
no CCDC-143825. Copies of the data can be obtained free
of charge on application to CCDC, 12 Union Road, Cambridge
[14]
`
M. F. C. Guedes da Silva, J. J. R. Frausto da Silva, A. J. L.
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Received January 25, 2000
[15]
CB2 1EZ, UK [Fax: (internat.)
deposit@ccdc.cam.ac.uk].
ϩ 44-1223/336-003; E-mail:
[I00025]
Eur. J. Inorg. Chem. 2000, 1717Ϫ1721
1721