DOI: 10.1002/cmdc.200900224
The Relevance of Polar Surface Area (PSA) in Rationalizing
Biological Properties of Several cisDiamminemalonatoplatinum(II) Derivatives**
Giulia Caron,[b] Giuseppe Ermondi,[b] Marzia B. Gariboldi,[c] Elena Monti,[c]
Elisabetta Gabano,[a] Mauro Ravera,[a] and Domenico Osella*[a]
A panel of six cis-diamminemalonatoplatinum(II) derivatives
were designed and synthesized, and their physicochemical
properties and in vitro biological activity were experimentally
evaluated and studied in silico. All the complexes showed
higher IC50 values ( 20 mm) than those observed for cisplatin
and its malonato analogue on three different human tumor
cell lines, namely A2780 ovarian carcinoma, A549 lung carcino-
ma, and MCF-7 breast carcinoma. In silico studies revealed that
polar surface area (PSA) is the best descriptor to explain the
poor biological activity observed for this series of new compounds, which in turn is likely due to poor cellular uptake. This
finding is in line with general rules that assign a major role to
PSA in characterizing the transport properties of drugs, in the
actual case of antiproliferative metallopharmaceuticals.
Introduction
For decades cis-diamminedichloroplatinum(II), or cisplatin (1)
(Figure 1), has been among the top-selling anticancer drugs.
The serendipitous discovery of its cytotoxic activity in 1961 by
Rosenberg and co-workers[1] was followed by one of the most
impressive synthetic efforts in research laboratories all over the
world, aimed at finding alternatives to cisplatin in order to
bypass its several side effects (mainly nausea, ototoxicity, and
nephrotoxicity) and the development of drug resistance phenomena. However, besides cisplatin, only few other platinum
complexes have been approved, worldwide or locally, for anticancer therapy, namely carboplatin, oxaliplatin, nedaplatin, lobaplatin, and heptaplatin.[2] In the majority of the new PtII derivatives, chlorides have been replaced by more stable chelating ligands (very often dicarboxylates), resulting in complexes
that have lower systemic toxicity, while retaining cytotoxic activity. A notable example of this strategy is carboplatin, which
has significantly decreased side effects, but is as effective as
cisplatin in the treatment of cancer and is widely employed in
pediatric oncology.
The role of the four ligands around the square planar PtII
core in determining the overall biochemical properties of platinum complexes has been extensively explored, although the
influence of the leaving groups has certainly been investigated
in lesser detail than that of the carrier groups.[3, 4] The reactivity
of the anionic ligands is an important feature of platinum compounds, because rapid substitution reactions with water or
other biological molecules can lead to failure of the complex
to reach its pharmacological target (mainly DNA), while excessive inertia will result in loss of biological activity. The structure–activity relationship (SAR) summarized by Cleare and Hoeschele several years ago[5, 6] identified chlorides in the cis configuration as having optimal lability when present with ammonia or organoamine ligands. Before dissociation, the structural
characteristics of the leaving group play a role, along with
ChemMedChem 2009, 4, 1677 – 1685
those of the carrier groups, in determining water solubility,
transport, and cellular uptake of the overall complexes. The
use of dicarboxylates as leaving ligands introduces enormous
possibilities of structural variation by modifying the carbon
backbone. In particular, oxalic, glycolic, and malonic acids have
been used to replace chloride ions in cisplatin analogues.
Malonate has been used as a leaving group in “traditional”
complexes, that is, in the already cited heptaplatin, approved
in South Korea in 1999 as Sunpla. Moreover, malonate is
widely used as a modifiable linker in drug targeting and delivery strategies applied to platinum complexes, with the aim to
obtain drugs with higher selectivity for malignant tissues.[7–10]
The mechanism of action of cisplatin and its derivatives consists of four key steps:[11, 12] 1) cellular uptake, 2) activation by
hydrolysis in the cytosol, 3) formation of DNA adducts in the
nucleus, and 4) recognition of platinum–DNA adducts by
damage-response proteins, followed by induction of apoptosis.
[a] Dr. E. Gabano, Prof. M. Ravera, Prof. D. Osella
Dipartimento di Scienze dell’Ambiente e della Vita
Universit del Piemonte Orientale “Amedeo Avogadro”
Viale T. Michel 11, 15100 Alessandria (Italy)
Fax: (+ 39) 0131-360250
E-mail: [email protected]
[b] Dr. G. Caron, Prof. G. Ermondi
Dipartimento di Scienza e Tecnologia del Farmaco
Universit di Torino
Via P. Giuria 9, 10125 Torino (Italy)
[c] Dr. M. B. Gariboldi, Prof. E. Monti
Dipartimento di Biologia Strutturale e Funzionale
Universit dell’Insubria
Via A. da Giussano 10, 21052 Busto Arsizio (VA) (Italy)
[**] Based on a lecture given at the 8th Workshop on Pharmaco-Bio-Metallics,
October 24–26, 2008, Ravenna (Italy).
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.200900224.
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Figure 1. Structures of the complexes under study: cis-diamminedichloroplatinum(II), cisplatin (1); cis-diamminemalonatoplatinum(II) (2); derivatives 3–6
based on 2-aminomalonate; and derivatives 7–8 based on the 2-(3-propylamino)malonate leaving group. In the text, the dicarboxylic ligands corresponding
to complexes 2–8 are indicated as L2–L8.
Platinum drugs enter the cell via both passive and active
mechanisms, but the finer details of cellular uptake remain incomplete. This limitation could, in part, explain the problems
encountered in the development of new platinum drugs over
the last 30 years; more than 3000 new platinum compounds
were synthesized, but only 30 derivatives entered clinical trials.
Herein we describe the design, synthesis, physicochemical
characterization, in vitro biological behavior, and in silico results obtained with a panel of PtII derivatives (3–8 in Figure 1)
containing the same cis-[PtACHTUNGRE(NH3)2]2 + core and different malonate backbones, with the aim to identify the parameters that
control their cellular uptake. The amidomalonate ligands were
designed to guarantee variation in the hydrophilic/hydrophobic balance and water solubility of the resulting complexes, as
it is known that an increase in molecular lipophilicity is generally associated with greater ability to permeate cellular membranes,[13] while enhanced water solubility results in a more efficient transport in the extracellular fluids.
Results and Discussion
Design of PtII complexes
For the present study, we designed a series of PtII complexes
spanning a wide range of lipophilicity. This choice is in line
with the results reported by Platts et al., who established a
mathematical relationship between log P and cellular uptake
for a large series of platinum drugs.[14]
Because most lipophilicity calculators are not parameterized
for platinum, the partition coefficients of the free ligands L2–
L8 (as diethyl esters) were calculated, and their respective log P
values are reported in Table 1. A number of different algorithms can be used to estimate log P. In Table 1 we report the
values obtained with ADME Boxes (AB) software, but the trend
described below was verified with all the tools implemented in
the Virtual Computational Chemistry Laboratory (VCCLAB).
L7 is by far the most lipophilic ligand in the series, whereas
L3 is the most hydrophilic. As we assume that the contribution
of the PtII core to the log P values of the complexes will remain
constant along the series, the lipophilicity of the overall complexes under study should span more than two log P units.
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Table 1. Lipophilicity-related data for ligands L2–L8 (as diethyl esters)
and complexes 1–8.
Leaving groups L2–L8
log k’[b]
ACHTUNGRE(log P)AB[a]
L2
L3
L4
L5
L6
L7
L8
1.07
0.22
2.07
1.48
1.65
2.52
1.92
0.93
0.75
1.65
1.52
1.75
1.71
1.60
Complexes 1–8
log k’[b]
AR[c]
1
2
3
4
5
6
7
8
0.66
0.69
0.67
0.12
0.11
0.21
0.19
0.09
8.00 0.50
1.90 0.20
0.40 0.03
0.23 0.02
0.24 0.02
0.20 0.02
0.15 0.01
0.25 0.02
[a] Logarithm of the partition coefficient, log P, estimated with ADME
Boxes (AB) software. [b] Logarithm of the capacity factor, log k’, measured
by RP-HPLC. [c] Accumulation ratio (A2780 cell line): the ratio between intracellular and extracellular PtII complex concentrations (for details, see
the Experimental Section).
Synthesis of the ligands derived from malonate
The synthesis of the ligands required coupling between carboxylic acids and amines. The use of carbodiimide-based coupling agents, such as dicyclohexylcarbodiimide (DCC)[15, 16] and
1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide (EDC),[17] as
well as diphenylphosphoryl azide (DPPA)[18] and diethyl cyanophosphonate (DECP),[19, 20] resulted in a number of byproducts
and/or poor yield. Therefore, we followed the method developed by Kamiński (see Supporting Information), which proved
to be successful in the synthesis of peptide bonds.[21–23]
We carried out the coupling at the NH2 group of commercially available diethyl 2-aminomalonate hydrochloride or ditert-butyl-2-(3-aminopropyl)malonate[24] to the benzoic acid derivatives (Scheme 1). According to the conditions for the coupling developed by Kamiński, subjection of diethyl 2-aminomalonate hydrochloride to 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT) and N-methylmorpholine (NMM) (0 8C!room
temperature, 20 h) in the presence of the above-mentioned
benzoic acid derivatives in DMF, led to diethyl esters L4–L6,
with yields ranging from satisfactory to excellent. Similarly,
condensation of di-tert-butyl-2-(3-aminopropyl)malonate with
the same set of benzoic acid derivatives under identical conditions gave di-tert-butyl esters L7 and L8 in modest to good
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cis-Diamminemalonatoplatinum(II) Derivatives
the tert-butyl esters in L7–L8 with trifluoroacetic acid at room
temperature, followed by complexation of the respective K + or
Ba2 + salts with cis-[PtACHTUNGRE(H2O)2ACHTUNGRE(NH3)2]2 + produced the corresponding platinum complexes 7–8. Notably, when the ligands were
transformed into Ba2 + salts, Ag2SO4 was used for the preparation of the aquaplatinum complexes; BaSO4 then precipitated
and could be separated by centrifugation. This procedure is
useful for water-soluble complexes. Otherwise, the final complex and BaSO4 must be separated by selectively dissolving the
platinum complex, that is, 4 and 5, in DMF. When the ligands
were transformed into K + salts, AgNO3 was used to produce
the water-soluble KNO3 byproduct. This method is well suited
to cases in which the resulting complexes are barely water
soluble and precipitate from solution. All the platinum complexes were highly soluble in DMSO and moderately soluble in
water, and their structural assignments are based on analysis
of their 1H, 13C and 195Pt NMR spectra.
Solution behavior
RP-HPLC and UV/Vis spectroscopy, along with ESIMS and
Pt NMR techniques, were used to evaluate the solution behavior of the complexes in a cell-free system (phosphate buffer
(pH 7.4), 100 mm NaCl, 37 8C).[28] Complexes without amide or
with amide groups at some distance from the metal center (2,
7, and 8) were quite stable over five days. In contrast, complexes 3, 4, 5, and 6, bearing the amide group in the a poACHTUNGREsition to the malonate, rapidly turned into a new species
(t1/2 5–6 h). This species exhibited retention times in RP-HPLC
different from those of the parent compounds, but the same
quasimolecular ion peak in ESIMS analysis. These results are
consistent with the occurrence of (O,O’)/ACHTUNGRE(N,O) coordination isomerism (Figure 2), rather than with an aquation reaction.
195
Scheme 1. Synthesis of ligands L4–L8 (with esterified carboxylic groups) and
their corresponding platinum complexes. Reagents and conditions: a) benzoic acid derivative, CDMT, NMM, DMF, 0 8C!RT; b) KOH or Ba(OH)2, H2O, RT;
c) cis-[PtACHTUNGRE(H2O)2ACHTUNGRE(NH3)2]2 + , H2O, 40 8C; d) TFA (neat), RT. (CDMT = 2-chloro-4,6dimethoxy-1,3,5-triazine; DMF = N,N-dimethylformamide; NMM = N-methylmorpholine; TFA = trifluoroacetic acid).
yields. Synthesis of the diethyl ester derivatives of L7 and L8
using similar procedures resulted in very low yields. For this
reason, these ligands were not used for coordination to PtII,
but only for the determination of log k’ (see below).
Synthesis of the corresponding malonato PtII complexes
Complex 2 was synthesized using the method patented by
Rosenberg and co-workers,[25] whereas complexes 3–8 were
obtained according to the method described by Dhara[26] and
modified by Rochon and Gruia.[27] The method consists of
treating K2ACHTUNGRE[PtCl4] with KI to produce K2ACHTUNGRE[PtI4] in solution; the
latter complex reacts with amines to produce the cis-[PtACHTUNGRE(amine)I2] precipitate. This is a good synthon for obtaining the
dicarboxylato complexes. In fact, upon reaction with Ag2SO4 or
AgNO3, cis-[PtACHTUNGRE(amine)I2] produces the corresponding diaqua
complex, which reacts with dicarboxylate to yield the final
complex.
We found that treatment of commercially available malonic
acid and 2-acetamidomalonic acid diethyl ester and ligands
L4–L6 with aqueous KOH or Ba(OH)2 at room temperature led
to chemoselective hydrolysis without affecting the benzamido
group. K + or Ba2 + salts of 2-acetamidomalonate and ligands
L4–L6 were therefore used for the subsequent complexation
in the presence of cis-[PtACHTUNGRE(H2O)2ACHTUNGRE(NH3)2]2 + in H2O at 40 8C to
obtain the respective complexes 3–6 (Scheme 1). Cleavage of
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Figure 2. General scheme of (O,O’) to (N,O) isomerization.
As previously described,[29, 30] the presence of the nitrogen
atom a to the malonato unit promotes a solvent-dependent
coordination isomerism of the (O,O’) chelate to the (N,O) chelate complex. During the synthesis of such complexes in water,
the kinetically favored (O,O’) chelate is formed first. When kept
in water, the complex isomerizes to the thermodynamically
stable (N,O) chelate. The 195Pt NMR spectra clearly distinguish
the (O,O’) chelate complexes (195Pt chemical shift ranges from
1700 to 1750 ppm) from the (N,O) chelate complexes (195Pt
chemical shift ~ 2100 ppm). Steric hindrance partially inhibits
the isomerization reaction. The coordination mode preference
of the anionic ligand depends on the hydrogen bonding (HB)
ability of the solvent.[31] Because the (N,O) chelate is a zwitterion, the HB between protic solvent molecules and the zwitterionic dipole are responsible for its stabilization. In DMSO the
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(O,O’) chelate is more stable, thus the (N,O) chelate reconverts
into the original (O,O’) form.
When freshly prepared, complexes 3–6 exhibited one retention peak in RP-HPLC and a single 195Pt NMR signal in D2O in
the range of the (O,O’) chelate. After a few hours, the original
RP-HPLC peak disappeared, and a new peak appeared corresponding to a compound having a 195Pt NMR signal in the
range of the (N,O) chelate. No further appreciable alterations
(attributable to the aquation reaction) could be observed up
to five days by means of RP-HPLC, UV/Vis, ESIMS or NMR techniques. The same held true for the non-isomerizable derivatives 7–8. Dicarboxylato PtII complexes are known to have
aquation half-life on the order of days in cell-free systems.[28]
However, in the cellular milieu, activation by enzymatic systems or by other anions such as carbonates may accelerate hydrolysis.[12] Interestingly, when the five-day-old solutions of 3–6
were evaporated to dryness, and the residues were dissolved
in [D6]DMSO, the 195Pt NMR spectra showed complete re-conversion of the isomer into the (O,O’) form (after 5 h in DMSO
solution). This observation was also confirmed in RP-HPLC by
the presence of the original peak alone. On the other hand,
the same five-day-old residues dissolved in D2O revealed the
presence of the (N,O) chelate form in the 195Pt NMR spectra,
again, a finding also corroborated by RP-HPLC.
Biological assays
For cytotoxicity assays, the complexes were first dissolved in
DMSO to prevent isomerization. These stock solutions were diluted with cell culture medium immediately before their addition to cell cultures. From that time on, (O,O’) to (N,O) isomerization was possible. Because the incubation time is three days,
and the isomerization half-life is ~ 5–6 h, the isomerizable derivatives act mainly in the (N,O) form.
All the amidomalonato complexes 3–8 showed poor cytotoxic activity (Figure 3). Surprisingly, no significant differences
in cytotoxicity were observed between 7 and 8 (the non-iso-
merizable derivatives) and the group formed by the isomerizable derivatives 3–6.
The (N,O) chelate is a triamine zwitterionic platinum complex
usually considered inactive as an antitumor agent, even
though some triamine–PtII complexes showed non-negligible
antiproliferative activity.[32] Interestingly, the macromolecular
platinum complexes AP5280 and AP5346 (ProLindac),[33, 34] consist of a platinum diamine unit linked to a water-soluble copolymer N-(2-hydroxypropyl)methacrylamide (HPMA) through an
amidomalonic acid as a (N,O) chelate, which slows the release
of platinum species. Both conjugates reached phase I clinical
trials (AP5346 started phase II in 2006), in which they demonstrated similar or better efficacy than carboplatin and oxaliplatin, with the added benefit of a higher therapeutic index (TI),
due to the stability of the inactive prodrug conjugate in the
systemic circulation.[35, 36]
All the complexes considered in this study showed higher
IC50 values ( 20 mm) than those obtained for the reference
compounds (1 and 2) on all the cell lines tested (A2780 ovarian
carcinoma, A549 lung carcinoma, and MCF-7 breast carcinoma), even though cellular response was quantitatively different
in the three cell lines, with A2780 being the most sensitive to
the action of PtII complexes. The weak cytotoxicity exhibited
by complexes 3–8 may be due to inadequate cellular uptake.
Uptake values were measured on the most sensitive cell line
(A2780), and are expressed as the accumulation ratio (AR, the
ratio between the intracellular and extracellular (i.e., in the culture medium) PtII complex concentration; Table 1).
Uptake (or AR) gives important information on the intracellular accumulation of platinum derivatives, even though the relationship between uptake and cytotoxicity is not straightforward. In contrast, DNA platination is more closely related to
the cytotoxicity of platinum complexes. In this specific case,
however, as the uptake of complexes 3–8 is very low, determination of DNA platination could be a difficult task, with platination generally representing 1–2 % of the overall cellular
uptake.[12, 37]
Structure–activity relationships
Figure 3. IC50 values of the compounds 1–8 on three different cell lines
(light gray = A2780, white = A549, gray = MCF-7).
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Biological assays show that the uptake of compounds 3–8 is
very low and almost constant along the series. This experimental evidence indicates that the initial design aimed at increasing intracellular accumulation based on lipophilicity calculations has failed. To investigate the reasons for this failure, we
decided to measure the lipophilicity of ligands and complexes.
Because evaluation of the n-octanol/water partition coefficient
(Po/w) based on the shake-flask method is time- and compound-consuming, and is affected by severe imprecision when
the compounds under study show very high or very low solubility in water, we performed the measurements by a chromatographic approach.[38, 39]
Because RP-HPLC retention is due to partitioning between
mobile and stationary phases, there is a correlation between
partition coefficients and HPLC capacity factors (k’ = (tRt0)/t0,
where t0 is the retention time for an unretained compound,
and tR is the retention time of the species under investigation).
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Therefore, to obtain an experimental measure of the lipophilicity of the compounds under study, the capacity factors k’ were
estimated using a mixture of methanol and 15 mm aqueous
formic acid (30:70) as eluent on a C18 column (Table 1).[40, 41]
Importantly, a previous study showed that chromatographic
indices (log k’) do not represent a reliable estimate of lipophilicity for compounds bearing aromatic amide groups,[42] as
these groups can interact with residual silanols of the stationary phase and level out differences in retention times. The results of our study support this observation: in fact, the experimental log k’ values of complexes 4–8 were very similar; in addition, the experimental log k’ values of the ligands L4–L8
differ widely from the calculated log P values (Table 1).
To verify the geometric orientation of the aromatic amide
moieties supposedly responsible for the interaction with residual silanols, we analyzed the 3D structure of the complexes.
The amide moieties are exposed on the molecular surface, and
are thus able to undergo HB interactions with the stationary
phase. Figure 4 shows the 3D structure of 6 as an example.
ty). Generally speaking, compounds with PSA 140 2 should
exhibit poor cell penetration ( 10 %), whereas compounds
with PSA 60 2 (such as cisplatin) show high absorption
( 90 %),[44] but few studies have addressed the relevance of
PSA in describing the cell penetration characteristics of antiproliferative drugs.[14, 45] In the present study, we calculated
total surface areas (CPK) and PSA values for all conformers of
derivatives 1–8. Several variants of PSA calculations such as dynamic, topological, and fast PSA are incorporated in various
software packages.[44] We used the method implemented in
Spartan and took the influence of conformation into consideration.[46] The results of this analysis are shown in Figure 5. Even
at first glance, no large variations are associated with conformational variability, as shown by the limited differences observed between maximal (Max), minimum (Min), and averaged
(Avg) values.
Figure 4. 3D structure of compound 6 (PSA profile is also shown).
Figure 5. Calculated total (CPK) and polar (PSA) surface areas for compounds
1–8 (CPK: & minimum, Min, * maximal, Max, ~ averaged, Avg; PSA: & minimum, Min, * maximal, Max, ~ averaged, Avg).
These findings confirm that for this series of complexes,
log k’ is not a good estimate for overall lipophilicity. However,
comparison of the log k’ values of L2–L3 and 2–3 (compounds
not bearing an aromatic amide moiety) seems to confirm that
the contribution of the PtII center to log k’ remains constant
along the series.
Because of the limits of 2D lipophilicity calculators in the
case of PtII complexes, and of the experimental limitations in
measuring lipophilicity descriptors described above, we moved
to molecular modeling to gain a better insight into the lipophilicity pattern of the complexes. Lipophilicity is a complex molecular property resulting from both polar and hydrophobic
components.[43] The polar surface area (PSA, defined as the surface sum over all polar atoms, including attached hydrogens)
could be used as a descriptor for the polar component of lipophilicity.
The role of PSA in governing passive diffusion has been
widely discussed, as PSA seems to optimally recapitulate the
drug properties that play an important role in membrane penetration (i.e., molecular polarity, HB features, and water solubili-
Because of the lack of an amide moiety, 1 and 2 should be
considered separately from 3–8. Figure 5 reveals that 1 and 2
have lower PSA values than 3–8, which show an almost constant value of ~ 140 2. Conversely, a significant and almost
progressive increase in CPK values was observed with molecular weight, both for 1 and 2, and 3–8. Because for 3–8 the
polar component to the overall lipophilicity can be assumed to
be constant, changes in CPK reflect changes in the hydrophobic properties of the complexes.
The similar PSA values observed for complexes 3–8 are consistent with the fact that their biological properties are similar
(i.e., low AR and high IC50 values), whereas compounds 1 and
2 showed lower PSA values, consistent with higher AR and
lower IC50. Thus, PSA seems to be a very relevant descriptor of
the biological profile of complexes, in contrast with hydrophobicity. However, complex 2 seems to deviate from this pattern.
In fact, 2 is only slightly less polar than 7 (PSA Avg: 123 and
138 2, respectively), but the two complexes differ widely in
both cellular uptake and cytotoxic activity. Two main reasons
for this behavior can be hypothesized: 1) the presence of an
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amide group is detrimental for cellular uptake; 2) the threshold
value of PSA set at 140 2 in the literature[44] does not completely hold for the challenging of tumor cell lines with platinum complexes. Studies addressing this topic are ongoing in
our research groups.
Conclusions
A series of cis-diamminemalonatoplatinum(II) derivatives was
synthesized. These complexes were designed to permit simple
lipophilicity-related SAR considerations. Biological tests indicate
that (O,O’)–(N,O) isomerization does not significantly influence
IC50 values. A molecular modeling approach revealed that PSA
seems to be the key descriptor to explain the poor biological
activity of these amidomalonato derivatives, which in turn is
due to their limited cellular uptake.
Experimental Section
Chemistry
K2ACHTUNGRE[PtCl4] and 1 (Johnson Matthey and Co.) and all other chemicals
(Aldrich) were used without further purification. Elemental analyses
for all the compounds were performed routinely in our laboratories, and the experimental values correspond to calculated values
within 0.4 %. NMR spectra were measured on a JEOL Eclipse Plus
spectrometer operating at 400 MHz (1H), 100.5 MHz (13C), and
85.9 MHz (195Pt). 1H NMR and 13C NMR chemical shifts are reported
in parts per million referenced to residual solvent proton resonances. 195Pt NMR spectra were recorded in [D6]DMSO, using a solution
of K2ACHTUNGRE[PtCl4] in aqueous KCl as the external reference. The shift for
K2ACHTUNGRE[PtCl4] was adjusted to 1628 ppm from Na2ACHTUNGRE[PtCl6] (d = 0 ppm).
Electrospray ionization mass spectra (ESIMS) were obtained with a
Micromass ZMD mass spectrometer. Typically, a dilute solution of
compound in H2O/MeOH 20:80 was delivered directly to the spectrometer source at 0.01 cm3 min1 using a Hamilton microsyringe
controlled by a single-syringe infusion pump. The nebulizer tip was
operated at 3000–3500 V and 150 8C, with N2 used both as drying
and nebulizing gas. The cone voltage was usually 30 V. Quasimolecular ion peaks [M+H] + or sodiated [M+Na] + peaks were assigned on the basis of the m/z values, and, in the case of Pt complexes, of the simulated isotope distribution patterns. Ligand
names were generated with ChemDraw software, version 11.
Synthesis of ligands L4–L8. Ligands L4–L8 (Scheme 1) were synthesized according to the Kamiński reaction.[21, 22] Briefly, the benzoic acid derivatives were coupled via an amide bond with the diester of the aminomalonic acids by using 2-chloro-4,6-dimethoxy1,3,5-triazine (CDMT) and N-methylmorpholine (NMM) in DMF as
coupling agents (Scheme 1; see the Supporting Information for the
complete synthetic procedure).
2-benzamidomalonic acid diethyl ester (L4). 80 % yield; 1H NMR
(CDCl3): d = 7.83 (m, 2 H, 2 CH, Ph), 7.52 (m, 1 H, CH, Ph), 7.45 (m,
2 H, 2 CH, Ph), 7.13 (d, J = 7.0 Hz, 1 H, C(O)NH), 5.34 (d, J = 7.0 Hz,
1 H, NHCH), 4.30 (m, 4 H, 2 CH2CH3), 1.31 ppm (t, J = 7.1 Hz, 6 H,
2 CH2CH3); 13C NMR (CDCl3): d = 166.93 (PhC(O)), 166.51 (2 COO),
133.12 (Cquat, Ph), 132.20 (CH, Ph), 128.74 (2 CH, Ph), 127.36 (2 CH,
Ph), 62.81 (2 CH2CH3), 56.95 (CH, malonate), 14.10 ppm (2 CH2CH3);
MS (ESI): m/z (%) [M+H] + calcd for C14H18NO5 : 280.12 (100.00),
281.12 (16.40), 282.12 (2.26), found: 280.10 (100.00), 281.14 (16.43),
282.16 (2.24).
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2-(2-benzamidoacetamido)malonic acid diethyl ester (L5). 52 %
yield; 1H NMR (CD3OD): d = 7.86 (m, 2 H, 2 CH, Ph), 7.54 (m, 1 H, CH,
Ph), 7.46 (m, 2 H, 2 CH, Ph), 4.24 (m, 4 H, 2 OCH2CH3), 4.15 (s, 2 H,
NHCH2), 1.27 ppm (t, J = 7.1 Hz, 6 H, 2 OCH2CH3); 13C NMR (CD3OD):
d = 170.33 (PhC(O)), 169.21 (CH2C(O)NH), 166.31 (2 COO), 133.71
(Cquat, Ph), 131.64 (CH, Ph), 128.29 (2 CH, Ph), 127.14 (2 CH, Ph),
63.91 (NHCH), 62.19 (2-OCH2CH3), 42.30 (NHCH2), 12.99 ppm
(2 OCH2CH3); MS (ESI): m/z (%) [M+Na] + calcd for C16H20N2O6Na:
359.12 (100.00), 360.13 (19.06), 361.13 (2.92), found: 359.16
(100.00), 360.10 (19.02), 361.15 (2.95).
2-(4-benzamidobutanamido)malonic acid diethyl ester (L6). 70 %
yield; 1H NMR (CDCl3): d = 7.78 (m, 2 H, 2CH, Ph), 7.46 (m, 1 H, CH,
Ph), 7.41 (m, 2 H, 2 CH, Ph), 6.95 (m, 2 H, 2 NH), 5.13 (d, J = 7.0 Hz,
1 H, NHCH), 4.25 (m, 4 H, 2 OCH2CH3), 3.52 (m, 2 H, NHCH2), 2.43 (t,
J = 7.1 Hz, 2 H, CH2C(O)), 1.97 (m, 2 H, NHCH2CH2), 1.26 ppm (t, J =
7.1 Hz, 6 H, 2 OCH2CH3); 13C NMR (CDCl3): d = 173.01 (C(O)NHCH),
167.78 (PhC(O)), 166.33 (2 C(O)OEt), 134.47 (Cquat, Ph), 131.44 (CH,
Ph), 128.56 (2 CH, Ph), 127.06 (2 CH, Ph), 62.76 (2 OCH2CH3), 56.64
(NHCH), 39.64 (NHCH2), 33.56 (CH2C(O)), 24.86 (NHCH2CH2),
14.06 ppm (2 OCH2CH3); MS (ESI): m/z (%) [M+Na] + calcd for
C18H24N2O6Na: 387.15 (100.00), 388.16 (21.34), 389.16 (3.37), found:
387.19 (100.00), 388.12 (21.30), 389.14 (3.35).
2-(3-benzamidopropyl)malonic acid di-tert-butyl ester (L7). 76 %
yield; 1H NMR (CDCl3): d = 7.78 (m, 2 H, 2 CH, Ph), 7.48 (m, 1 H, CH,
Ph), 7.42 (m, 2 H, 2 CH, Ph), 6.44 (s, 1 H, C(O)NH), 3.45 (m, 2 H,
NHCH2), 3.17 (t, J = 7.5 Hz, 1 H, CH2CH), 1.89 (m, 2 H, CH2CH), 1.65
(m, 2 H, CH2CH2CH), 1.45 ppm (s, 6 H, CH3, tBu); 13C NMR (CDCl3):
d = 168.95 (2 COO), 167.56 (PhC(O)), 134.76 (Cquat, Ph), 131.41 (CH,
Ph), 128.60 (2 CH, Ph), 126.99 (2 CH, Ph), 81.76 (2 Cquat, tBu), 53.54
(CH2CH), 39.60 (NHCH2), 27.92 (6CH3, tBu), 27.15 (NHCH2CH2),
25.86 ppm (CH2CH); MS (ESI): m/z (%) [M+H] + calcd for C21H32NO5 :
378.23 (100.00), 379.23 (24.40), 380.23 (3.85), found: 378.25
(100.00), 379.20 (24.42), 380.22 (3.87).
2-(3-(2-benzamidoacetamido)propyl)malonic acid di-tert-butyl
ester (L8). 66 % yield; 1H NMR (CD3OD): d = 7.88 (m, 2 H, 2CH, Ph),
7.53 (m, 1 H, CH, Ph), 7.47 (m, 2 H, 2 CH, Ph), 4.00 (s, 2 H, CH2C(O)),
3.24–3.18 (m, 3 H, NHCH2CH2 and CH2CH), 1.79 (m, 2 H, CH2CH),
1.60–1.40 ppm (m, 20 H, CH2CH2CH and 6CH3, tBu); 13C NMR
(CD3OD): d = 170.41 (2 COO), 169.11 (C(O)NH), 169.03 (PhC(O)),
133.77 (Cquat, Ph), 131.57 (CH, Ph), 128.22 (2 CH, Ph), 127.20 (2 CH,
Ph), 81.38 (Cquat, tBu), 53.39 (CH2CH), 48.11 (NHCH2C(O)), 42.50
(NHCH2CH2), 26.87 (6CH3, tBu), 26.70 (NHCH2CH2), 25.67 ppm
(CH2CH); MS (ESI): m/z (%) [M+H] + calcd for C23H35N2O6 : 435.25
(100.00), 436.25 (27.07), 437.26 (4.72), found: 435.24 (100.00),
436.27 (27.04), 437.28 (4.70).
Deprotection of the carboxylic ligands: ligands L4–L8 were transformed into their K + or Ba2 + salts according to the following two
general procedures. Malonic acid, 2-acetylaminomalonate diethyl
ester and ligands L4–L6 were dissolved in H2O, and then a stoichiometric amount of KOH (molar ratio acid/KOH = 1:2) or Ba(OH)2
(molar ratio acid/Ba(OH)2 = 1:1) was added to get the corresponding potassium or barium salt. The solutions were used directly in
reaction with the platinum synthon.
Ligands L7–L8 were treated with excess trifluoroacetic acid (TFA)
for 1 h. Excess TFA was removed under reduced pressure, and the
residue was washed with Et2O. The residue was then dissolved in
H2O, and KOH or Ba(OH)2 (acid/KOH = 1:2, acid/Ba(OH)2 = 1:1) was
added. In this case the solutions were also used directly in the reaction with the platinum synthon cis-[PtACHTUNGRE(H2O)2ACHTUNGRE(NH3)2].
2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2009, 4, 1677 – 1685
cis-Diamminemalonatoplatinum(II) Derivatives
Synthesis of the Pt complexes 2–8. Complexes 3–8 and the precursor cis-[PtI2ACHTUNGRE(NH3)2] were synthesized according to Dhara’s
method,[26, 27] whereas a different procedure was used for complex
2.[25]
Synthesis of cis-[diammine(2-acetamidomalonato)platinum(II)],
3. cis-[PtI2ACHTUNGRE(NH3)2] (0.400 g, 0.83 mmol) was added to a solution of
Ag2SO4 (0.253 g, 0.81 mmol) in H2O (~ 40 mL), and the mixture was
stirred at 40 8C overnight in the dark. It was then filtered to
remove AgI, and barium 2-acetylaminomalonate (obtained from reaction between Ba(OH)2·8 H2O (0.256 g, 0.81 mmol) and the 2-acetamidomalonic acid diethyl ester (0.212 g, 0.93 mmol)) was added
to the filtrate. The mixture was stirred at 40 8C overnight in the
dark, and BaSO4 precipitated. The precipitate was filtered off, and
the filtrate was evaporated to dryness. The residue was washed
with MeOH and Et2O and dried under vacuum. Yield: 0.296 g, 92 %;
1
H NMR ([D6]DMSO): d = 7.62 (d, J = 7.9 Hz, 1 H, NH), 5.57 (d, J =
7.9 Hz, 1 H, CH), 4.27 (s, 6 H, 2 NH3), 1.88 ppm (s, 3 H, CH3); 13C NMR
([D6]DMSO): d = 172.53 (2 COO), 168.87 (CH3C(O)), 59.80 (CH, malonato), 23.09 ppm (CH3); 195Pt NMR ([D6]DMSO): 1729 ppm; MS
(ESI) m/z (%) [M+H] + calcd for C5H12N3O5Pt: 388.04 (91.12), 389.04
(100.00), 390.04 (77.74), 391.05 (6.13), 392.05 (20.86), found: 388.00
(91.19), 389.05 (100.00), 390.10 (77.70), 391.09 (6.16), 392.01 (20.90).
Synthesis of cis-[diammine(2-benzamidomalonato)platinum(II)],
4. cis-[PtI2ACHTUNGRE(NH3)2] (0.353 g, 0.731 mmol) was added to a solution of
Ag2SO4 (0.223 g, 0.716 mmol) in H2O (~ 40 mL), and the mixture
was stirred at 40 8C overnight in the dark. It was then filtered to
remove AgI, and L4 (as Ba2 + salt obtained from stoichiometric reaction between Ba(OH)2·8 H2O and the acid) was added to the filtrate. The mixture was stirred at 40 8C overnight in the dark, and
the product and BaSO4 co-precipitated. The precipitate was isolated by centrifugation and dissolved in DMF to remove insoluble
BaSO4. DMF was removed under reduced pressure, and the residue
was washed with MeOH and Et2O and dried under vacuum. Yield:
0.293 g, 89 %; 1H NMR ([D6]DMSO): d = 7.87 (m, 2 H, 2 CH, Ph), 7.79
(d, J = 7.9 Hz, 1 H, PhC(O)NH), 7.54 (m, 1 H, CH, Ph), 7.49 (m, 2 H,
2 CH, Ph), 5.74 (d, J = 7.9 Hz, 1 H, CH, malonato), 4.33 ppm (s, 6 H,
2 NH3); 13C NMR ([D6]DMSO): d = 172.24 (2 COO), 165.45 (PhC(O)),
134.75 (Cquat, Ph), 131.88 (CH, Ph), 128.96 (2 CH, Ph), 127.72 (2 CH,
Ph), 59.92 ppm (CH, malonato); 195Pt NMR ([D6]DMSO): d =
1732 ppm; MS (ESI) m/z (%) [M+H] + calcd for C10H14N3O5Pt:
450.06 (86.72), 451.06 (100.00), 452.06 (79.42), 453.06 (10.09),
454.06 (20.27), found: 450.03 (86.75), 451.01 (100.00), 452.106
(79.44), 453.08 (10.05), 454.02 (20.30).
Synthesis of cis-[diammine(2-(2-benzamidoacetamido)malonato)platinum(II)], 5. cis-[PtI2ACHTUNGRE(NH3)2] (1.456 g, 0.703 mmol) was added
to a solution of Ag2SO4 (0.445 g, 1.427 mmol) in H2O (~ 50 mL), and
the mixture was stirred at 40 8C overnight in the dark. It was then
filtered to remove AgI, and L5 (as Ba2 + salt obtained from stoichiometric reaction between Ba(OH)2 and the acid) was added to the
filtrate. The mixture was stirred at 40 8C overnight in the dark, and
the product and BaSO4 co-precipitated. The precipitate was isolated by centrifugation and dissolved in DMF to remove insoluble
BaSO4. DMF was removed under reduced pressure, and the residue
was washed with Et2O and dried under vacuum. Yield: 0.328 g,
92 %; 1H NMR ([D6]DMSO): d = 8.88 (t, J = 6.0 Hz, 1 H, PhC(O)NH),
7.88 (m, 2 H, 2 CH, Ph), 7.61 (d, J = 7.5 Hz, 1 H, NHCH), 7.55 (m, 1 H,
CH, Ph), 7.50 (m, 2 H, 2 CH, Ph), 5.51 (d, J = 7.5 Hz, 1 H, NHCH), 4.30
(s, 6 H, 2NH3), 3.97 ppm (d, J = 6.0 Hz, 2 H, CH2C(O)); 13C NMR
([D6]DMSO): 172.17 (2 COO, malonato), 168.50 (CH2C(O)NH), 166.98
(PhC(O)), 134.68 (Cquat, Ph), 131.96 (CH, Ph), 128.93 (2 CH, Ph),
127.83 (2 CH, Ph), 59.66 (NHCH), 43.21 ppm (CH2C(O)); 195Pt NMR
([D6]DMSO): d = 1730 ppm; MS (ESI) m/z (%) [M+H] + calcd for
ChemMedChem 2009, 4, 1677 – 1685
C12H17N4O6Pt: 507.08 (84.76), 508.08 (100.00), 509.08 (80.42), 510.08
(12.16), 511.08 (20.26), 554.13 (3.58), found: 507.03 (84.70), 508.12
(100.00), 509.05 (80.45), 510.11 (12.12), 511.08 (20.32), 554.09 (3.50).
Synthesis of cis-[diammine(2-(4-benzamidobutanamido)malonato)platinum(II)], 6. cis-[PtI2ACHTUNGRE(NH3)2] (0.390 g, 0.807 mmol) was added
to a solution of AgNO3 (0.269 g, 1.581 mmol) in H2O (~ 30 mL), and
the mixture was stirred at 40 8C overnight in the dark. It was then
filtered to remove AgI, and L6 (as K + salt) was added to the filtrate. The mixture was stirred at 40 8C overnight in the dark, and
the white precipitate was isolated by centrifugation. The product
was washed with H2O, EtOH, and Et2O, and dried under vacuum.
Yield: 0.359 g, 83 %; 1H NMR ([D6]DMSO): d = 8.48 (t, J = 6.0 Hz, 1 H,
NHCH), 7.83 (m, 2 H, 2 CH, Ph), 7.70 (d, J = 8.4 Hz, 1 H, PhC(O)NH),
7.51 (m, 1 H, CH, Ph), 7.46 (m, 2 H, 2 CH, Ph), 5.62 (d, J = 8.4 Hz, 1 H,
NHCH), 4.29 (s, 4 H, 2 NH3), 3.27 (m, 2 H, NHCH2), 2.26 (t, J = 7.1 Hz,
2 H, CH2C(O)), 1.74 ppm (m, 2 H, CH2CH2C(O)); 13C NMR ([D6]DMSO):
d = 172.61 (2COO), 171.67 (CH2C(O)NH), 166.76 (PhC(O)), 135.30
(Cquat, Ph), 131.55 (CH, Ph), 128.78 (2 CH, Ph), 127.74 (2 CH, Ph),
59.73 (NHCH), 40.50 (NHCH2), 33.31 (CH2C(O)), 26.18 ppm
(NHCH2CH2); 195Pt NMR ([D6]DMSO): d = 1730 ppm; MS (ESI) m/z
(%) [M+H] + calcd for C14H21N4O6Pt: 535.11 (83.16), 536.11 (100.00),
537.11 (81.12), 538.11 (13.75), 539.11 (20.15), 540.12 (3.40), found:
535.31 (83.40), 536.31 (100.00), 537.23 (81.20), 538.25 (13.80),
539.22 (20.18), 540.20 (3.02).
Synthesis of cis-[diammine(2-(3-benzamidopropyl)malonato)platinum(II)], 7. cis-[PtI2ACHTUNGRE(NH3)2] (0.227 g, 0.471 mmol) was added to a
solution of AgNO3 (0.157 g, 0.923 mmol) in H2O (25 mL), and the
mixture was stirred at 40 8C overnight in the dark. It was then filtered to remove AgI, and L7 (as K + salt) was added to the filtrate.
The mixture was stirred at 40 8C overnight in the dark; complex 7
precipitated and was isolated by centrifugation. The final product
was washed with EtOH and Et2O, and dried under vacuum. Yield:
0.181 g, 78 %; 1H NMR ([D6]DMSO): d = 8.45 (t, J = 5.7 Hz, 1 H,
PhC(O)NH), 7.84 (m, 2 H, 2 CH, Ph), 7.50 (m, 1 H, CH, Ph), 7.45 (m,
2 H, 2 CH, Ph), 4.16 (s, 6 H, 2 NH3), 3.59 (t, J = 7.1 Hz, 1 H, CH, malonato), 3.24 (m, 2 H, NHCH2), 1.82 (m, 2 H, CH2CH), 1.53 ppm (m, 2 H,
CH2CH2CH); 13C NMR ([D6]DMSO): d = 176.33 (2COO), 166.57
(PhC(O)), 135.32 (Cquat, Ph), 131.51 (CH, Ph), 128.78 (2 CH, Ph),
127.67 (2 CH, Ph), 57.99 (CH, malonato), 39.28 (NHCH2), 28.17
(CH2CH2CH), 27.28 ppm (CH2CH2CH); 195Pt NMR ([D6]DMSO): d =
1718 ppm; MS (ESI) m/z (%) [M+H] + calcd for C13H20N3O5Pt:
492.10 (84.23), 493.11 (100.00), 494.11 (80.47), 495.11 (12.48), 496.11
(20.05), 497.11 (3.11), found: 492.15 (84.27), 493.14 (100.00), 494.17
(80.40), 495.08 (12.42), 496.10 (20.02), 497.17 (3.10).
Synthesis of cis-[diammine(2-(3-(2-benzamidoacetamido)propyl)malonato)platinum(II)], 8. cis-[PtI2ACHTUNGRE(NH3)2] (0.386 g, 0.799 mmol)
was added to a solution of Ag2SO4 (0.243 g, 0.780 mmol) in H2O
(25 mL), and the mixture was stirred at 50 8C overnight in the dark.
It was then filtered to remove AgI, and L8 (as Ba2 + salt) was added
to the filtrate. The mixture was stirred at 50 8C overnight in the
dark, and then the precipitate was filtered off. The filtrate was
dried under reduced pressure, and the residue was washed with
MeOH and Et2O, and dried under vacuum. Yield: 0.312 g, 71 %;
1
H NMR ([D6]DMSO): d = 8.75 (t, J = 6.0 Hz, 1 H, PhC(O)NH), 7.95 (t,
J = 5.9 Hz, 1 H, CH2C(O)NH), 7.88 (m, 2 H, 2 CH, Ph), 7.54 (m, 1 H, CH,
Ph), 7.48 (m, 2 H, 2 CH, Ph), 4.19 (s, 6 H, 2 NH3), 3.84 (d, J = 6.0 Hz,
2 H, CH2C(O)), 3.56 (t, J = 7.1 Hz, 1 H, CH, malonato), 3.05 (m, 2 H,
NHCH2CH2), 1,79 (m, 2 H, CH2CH), 1.42 ppm (m, 2 H, CH2CH2CH);
13
C NMR ([D6]DMSO): d = 176.36 (2 COO), 169.20 (PhC(O)), 166.99
(CH2C(O)NH), 133.77 (Cquat, Ph), 131.86 (CH, Ph), 128.81 (2 CH, Ph),
127.93 (2 CH, Ph), 58.02 (CH, malonato), 43.10 (CH2C(O)), 40.69
(CH2C(O)NHCH2), 28.25 (CH2CH2CH), 27.26 ppm (CH2CH2CH);
2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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D. Osella et al.
195
Pt NMR ([D6]DMSO): d = 1720 ppm; MS (ESI) m/z (%) [M+H] +
calcd for C15H23N4O6Pt: 549.12 (82.39), 550.13 (100.00), 551.13
(81.51), 552.13 (14.54), 553.13 (20.12), 554.13 (3.60), found: 549.09
(82.40), 550.15 (100.00), 551.20 (81.54), 552.17 (14.50), 553.19
(20.07), 554.16 (3.65).
Determination of log k’. Compounds were injected onto a C18 reversed-phase HPLC column, and their capacity factors (k’ = (tR/t0)/t0,
where t0 is the retention time for an unretained compound, and tR
is the retention time of the analyzed species) were registered. The
chromatographic conditions were as follows: silica-based C18 gel as
the stationary phase, mobile phase containing 70 % HCOOH
(15 mm) and 30 % MeOH (isocratic elution); flow rate =
0.75 mL min1; UV/Vis detector set at 210 nm; KI was the internal
reference to determine the column dead-time (t0).
Biological assays
Cytotoxicity tests. Three human tumor cell lines, A2780 (ovarian
carcinoma), A549 (lung carcinoma), and MCF-7 (breast adenocarcinoma), were obtained from the American Type Culture Collection
(Manassas, VA, USA) and maintained in RPMI-1640 (A2780 and
A549) or DMEM (MCF-7) (Sigma, Italy) supplemented with 10 %
fetal calf serum (Celbio, Italy), 2 mm l-glutamine, and 1 % antibiotic
mixture (penicillin/streptomycin; Sigma, Italy) under standard culture conditions (95 % air and 5 % CO2 at 37 8C in a humidified atmosphere). Cell survival following exposure to metal complexes
was evaluated using the MTT ([3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]) assay, based on the reduction of MTT
by living cells following exposure to cytotoxic drugs.[47] Briefly, 1 103 cells per well (for A2780 and A549) or 2 103 cells per well (for
MCF-7) were plated onto 96-well sterile plates and allowed to
attach and grow for 24 h. PtII complexes were dissolved in DMSO
to obtain 10 mm stock solutions that were diluted with complete
medium for cell treatment. The final range of PtII concentrations
was 10–500 mm; the co-solvent concentration never exceeded
0.1 %. Three days later, MTT was added to each well (final concentration 0.4 mg mL1), and plates were incubated for 3 h at 37 8C.
Cell viability was determined by measuring the absorbance in individual wells using a Universal Microplate Reader (Bio-Teck Instruments). The cytotoxic effects of PtII complexes were quantified by
calculating the drug concentration that inhibits tumor cell growth
by 50 % (IC50), based on nonlinear regression analysis of dose–response data, performed with Calcusyn software (Biosoft, Cambridge, UK).
Platinum content in tumor cells. The platinum content of cells following treatment with the various platinum complexes was evaluated by inductively coupled plasma mass spectroscopy (ICPMS). To
evaluate the intracellular platinum content of A2780 cells following
in vitro exposure to equitoxic concentrations (corresponding to
their respective IC50 values) of platinum complexes for three days,
cells were seeded and allowed to grow for 24 h before treatment.
At the end of the drug exposure period, the cells were detached,
counted, and washed in PBS; 107 cells were lysed by adding 2 mL
of a 1:1 mixture of distilled H2O and HNO3 (69.5 %), mineralized,
and completely dried at 120 8C. The platinum content in all the
samples was measured with an X5 Series ICPMS instrument from
Thermo Optek (Cinisello Balsamo, Italy). Instrument settings were
optimized to yield maximum sensitivity for platinum. Dry platinumcontaining material was dissolved in 1 mL 69.5 % HNO3 and then
suitably diluted with an aqueous solution of indium, that is, the internal standard. The most abundant isotopes of platinum and
indium were measured at m/z 195 and 115, respectively. Intratumor
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platinum content at the end of the experiment was expressed as
ng Pt per 106 cells (corresponding to ~ 1 mg extracted protein);[48]
each value is the mean SEM of four replicates. The corresponding accumulation ratios (ARs) were calculated based on uptake
data. AR is the nondimensional ratio of intracellular platinum concentration (roughly estimated by considering the volume of
106 cells to be ~ 2 mL[49]) to platinum concentration in the extracellular (culture) medium.[50, 51]
Computational methods
ADME Boxes (Pharma Algorithms, Toronto, Canada) and Virtual
Computational Chemistry Laboratory (VCCLAB: http://www.vcclab.org/) were used to estimate log P values of ligands L2–L8. The
3D structures of investigated PtII complexes were obtained as follows: The structure of 2 (CSD code: DICTOY) was downloaded
from Cambridge Structural Database (CSD; version 5.30) and imported in Spartan ’08 molecular modeling software (Spartan ’08;
Wavefunction Inc. 2009, Irvine, CA, USA). According to the published methods,[52] the geometry of the complex was fully optimized without symmetry constraints using the semiempirical quantum mechanical PM3 Hamiltonian. The minimized structure was
confirmed as minimum via harmonic frequency calculations (absence of imaginary frequencies) and was used as template to build
all the complexes. Because two hydrogen atoms can be substituted in any malonato ligand, a couple of isomers were obtained for
3–8 which were then submitted to a conformational analysis using
the simulated annealing method. This procedure randomly rotates
bonds and bends rings until a preferential (minimum energy) geometry is attained. Briefly, the molecule is considered to be a hightemperature system; this means that it has significant energy and
is flexible enough to move from a low- to high-energy conformation. As more conformers are explored, the temperature of the
system decreases, making the molecule less inclined to move out
of low-energy conformations, thus looking “more closely” at other
minima in the nearby vicinity. The MMFF force field without solvation (the solvation parameters for platinum were missing) was
used, and constrains were applied to avoid distortion in the coordination geometry during conformational analysis. A set of ad hoc
conformers covering extended and folded geometries was selected
for any structure, and minimized as described above to overcome
the absence of solvation. The total (CPK) and the polar surface area
(PSA) were calculated for any conformer, and for any compound,
the maximum, minimum, and average values of both descriptors
were retained.
Acknowledgements
Financial support for this work was from Regione Piemonte
(Turin, Italy) and ATF Association (Alessandria, Italy). The research
was carried out within the framework of the European Cooperation COST D39 (Metallo-Drug Design and Action) and Consorzio
CIRCMSB (Bari, Italy). We are indebted to the anonymous referees,
whose critique and suggestions improved the quality of the
manuscript.
Keywords: cellular uptake · cytotoxicity · lipophilicity ·
platinum · polar surface area
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Received: June 10, 2009
Revised: July 2, 2009
Published online on July 27, 2009
2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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