Influence of Equatorial and Axial Carboxylato Ligands on the Kinetic

Oct 10, 2013 - Inertness of Platinum(IV) Complexes in the Presence of Ascorbate and Cysteine and within DLD‑1 Cancer Cells. Catherine K. J. Chen,. â...
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Influence of Equatorial and Axial Carboxylato Ligands on the Kinetic Inertness of Platinum(IV) Complexes in the Presence of Ascorbate and Cysteine and within DLD‑1 Cancer Cells Catherine K. J. Chen,† Jenny Z. Zhang,† Jade B. Aitken,†,‡,§ and Trevor W. Hambley*,† †

School of Chemistry, The University of Sydney, Sydney, New South Wales, 2006, Australia Australian Synchrotron, Clayton, Victoria, 3168, Australia § Institute of Materials Structure Science, KEK, Tsukuba, Ibaraki 305-0801, Japan ‡

S Supporting Information *

ABSTRACT: The rapid and premature reduction of platinum(IV) complexes in vivo is a significant impediment to these complexes being successfully employed as anticancer prodrugs. This study investigates the influence of the platinum(IV) coordination sphere on the ease of reduction of the platinum center in various biological contexts. In the presence of the biological reductants, ascorbate and cysteine, platinum(IV) complexes with dicarboxylato equatorial ligands were observed to exhibit lower reduction potentials and slower reduction rates than analogous platinum(IV) complexes with dichlorido equatorial ligands. Diaminetetracarboxylatoplatinum(IV) complexes exhibited unusually long half-lives in the presence of excess reductants; however, the complexes exhibited moderate potency in vitro, indicative of rapid reduction within the intracellular environment. By use of XANES spectroscopy, trans-[Pt(OAc)2(ox)(en)] and trans-[PtCl2(OAc)2(en)] were observed to be reduced at a similar rate within DLD-1 cancer cells. This large variability in kinetic inertness of diaminetetracarboxylatoplatinum(IV) complexes in different biological contexts has significant implications for the design of platinum(IV) prodrugs.

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INTRODUCTION Platinum(II)-based chemotherapy is an important component of treatment for many types of cancers, particularly for ovarian and testicular cancers.1 However, platinum(II) complexes readily undergo nonselective ligand substitution en route to the tumor site, with approximately 90% of administered cisplatin estimated to be deactivated in the bloodstream by irreversibly binding to albumin and other plasma proteins and as little as 1% (or less) binding to its intended target, the nuclear DNA.2 Protein binding to cisplatin has been suggested to result in the formation of metabolites that cause harmful side effects such as kidney and nerve damage, nausea, vomiting, and hearing loss.2a,3 Second generation platinum anticancer drugs, carboplatin and oxaliplatin, exhibit less toxicity compared to cisplatin but do not exhibit improvements in terms of activity or selectivity. An attractive strategy for addressing the toxicity of platinum anticancer drugs is to utilize the platinum(IV) oxidation state. Platinum(IV) complexes are more inert than their platinum(II) analogues and have the potential to be more resistant to nonspecific biointeractions. The design of platinum(IV) prodrugs has previously been guided by a set of structure−activity rules4 based on the relationship between the nature of the axial ligands and their influence on the rates of reduction of the complex. Platinum(IV) complexes with axial chlorido and trifluoroacetato ligands were reported to be most readily reduced by © 2013 American Chemical Society

common biological reductants such as ascorbate, cysteine, and glutathione, which is consistent with their relatively positive reduction potentials.5 Platinum(IV) complexes with axial carboxylato ligands have been observed to exhibit intermediate reduction potentials and intermediate reduction rates; complexes with hydroxido axial ligands exhibited both the lowest reduction potentials and the slowest reduction rates. All of the platinum(IV) complexes that have entered clinical trials (tetraplatin, iproplatin, and satraplatin) yield cis-dichlorido cisplatin-like platinum(II) congeners upon reduction. Thus far, in vivo studies have revealed that these complexes exhibit no greater activity than cisplatin, possibly because of the premature reduction of the platinum(IV) complexes.6 Reduction studies of tetraplatin and satraplatin in tissue culture medium and biological reductants have demonstrated that the reduction of these platinum(IV) complexes occurred so rapidly that almost all the platinum(IV) is reduced extracellularly.7 The implication of these results is that tetraplatin and satraplatin are almost entirely reduced to their platinum(II) congeners in the bloodstream, which would likely result in rapid deactivation by biomolecules, and any advantage offered by the platinum(IV) oxidation state would be limited. Received: August 8, 2013 Published: October 10, 2013 8757

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Recently, Zhang et al. reported that platinum(IV) derivatives of oxaliplatin, which had four carboxylato ligands, exhibit unusual kinetic inertness in the presence of excess ascorbic acid despite their relatively positive reduction potentials.8 The authors attributed this unusual stability to the coordination sphere of the platinum(IV) complex; the nitrogen and oxygen donor atoms from the amine and carboxylato ligands were hypothesized to form poor electron transfer bridges between the reductant and platinum center, hindering inner sphere reduction. Keppler et al. has reported similar kinetic inertness in carboplatin-based diaminetetracarboxylatoplatinum(IV) complexes in the presence of ascorbic acid.9 Although the enhanced kinetic inertness exhibited by these platinum(IV) complexes against simple biological reductants indicates that they are less likely to be prematurely reduced before arriving at the target site, an effective platinum(IV) prodrug must be eventually reduced to release the cytotoxic platinum(II) component upon reaching the cancer cells. As yet, the unusual stabilizing effects of the equatorial carboxylato ligands have yet to be tested in the complex intracellular environment. In this study we aimed to explore the kinetic inertness of platinum(IV) complexes with equatorial and axial carboxylato ligands in a range of biological contexts. In particular, the reduction of diaminetetracarboxylatoplatinum(IV) complexes within whole cells was studied using X-ray absorption near edge structure (XANES) spectroscopy, a method that has been previously used to track the reduction of platinum(IV) complexes within cells.10

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RESULTS Synthesis and Characterization. Complexes I−VIII (Figure 1) were prepared following established methods11 and obtained in yields comparable to those previously reported. Characterization was carried out by 1H and 195Pt NMR spectroscopy and elemental analyses, and results were found to be in agreement with calculated values. Reduction Potential. Platinum(IV) complexes undergo irreversible reduction as the result of the loss of the axial ligands, and as such, their electrochemical reduction potentials are reported as cathodic peak potentials (Epc). It was found that platinum(IV) complexes with trifluoroacetato axial ligands, cis,trans-[PtCl2(OTFA)2(en)] (III) and trans-[Pt(OTFA)2(ox)(en)] (VI), have the most positive reduction potentials, followed by complexes with acetato axial ligands, cis,trans-[PtCl2(OAc)2(en)] (II), trans-[Pt(OAc)2(ox)(en)] (V), and trans-[Pt(OAc)2(ox)(R,R-chxn] (VIII), and finally, complexes with hydroxido axial ligands, cis,trans-[PtCl2(OH)2(en)] (I), trans-[Pt(OH)2(ox)(en)] (IV), and trans-[Pt(OH)2(ox)(R,R-chxn] (VII) were found to have the lowest reduction potentials (Table 1). Reduction Rate of Platinum(IV) Complexes. The rates of reduction of platinum(IV) complexes in the presence of excess ascorbate and cysteine (Table 2) were determined using 1 H NMR. Complexes I and II, containing equatorial chlorido ligands, were found to have shorter half-lives compared to their oxalato analogues, complexes IV and V, respectively, in the presence of excess ascorbate and cysteine. This trend was also observed for complexes III and VI, which have highly electron withdrawing trifluoroacetato axial ligands, where complex III was observed to undergo complete reduction in less than 2 min in the presence of excess ascorbate, while complex VI appears to be slightly more resistant to reduction with a half-life of 18 min. Cytotoxicity and Cellular Accumulation. All platinum(II) and platinum(IV) complexes were found to be consistently more cytotoxic against A2780 human ovarian cancer cells than against DLD-1 human colon cancer cells (Table 3).

Figure 1. Platinum(IV) complexes: cis,trans-[PtCl2(OH)2(en)] (I); cis,trans-[PtCl2(OAc)2(en)] (II); cis,trans-[PtCl2(OTFA)2(en)] (III); trans-[Pt(OH)2(ox)(en)] (IV); trans-[Pt(OAc)2(ox)(en)] (V); trans[Pt(OTFA)2(ox)(en)] (VI); trans-[Pt(OH)2(ox)(R,R-chxn)] (VII); trans-[Pt(OAc)2(ox)(R,R-chxn)] (VIII).

Table 1. Cathodic Forward Potentials, Epc, Referenced to Ag+/AgCla

a

complex

axial ligand

Epc vs Ag+/AgCl (mV)

I II III IV V VI VII VIII

OH OAc OTFA OH OAc OTFA OH OAc

−769 −603 −221 −867 −713 −307 −995 −650

Voltammograms were obtained at a scan rate 100 mV/s at 298 K.

The diacetato platinum(IV) complexes II, V, and VIII were found to be consistently more cytotoxic than their dihydroxido analogues, complexes I, IV, and VII in both A2780 and DLD-1 cells, but are less cytotoxic than the platinum(II) complexes with the exception of complex II. Complex VI shows similar cytotoxicity to complex V, despite containing highly electron withdrawing trifluoracetato axial ligands. Platinum(II) and platinum(IV) complexes exhibited comparable cellular accumulation in A2780 and DLD-1 cells (Table 3). Platinum(II) complexes generally showed greater cellular accumulation than platinum(IV) complexes. 8758

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Table 2. Average Half-Lives and Rate Constants of the Reduction of Platinum(IV) Complexes in a 10-Fold Excess of Ascorbate or Cysteine at 37 °Ca ascorbate 1.7 ± 0.2 h 30 ± 8 min ≪2 min 20.6 ± 4 h 2.5 ± 0.1 day 18 ± 2 min

I II III IV V VI a

cysteine k (s−1)

t1/2

complex

k (s−1)

t1/2 −4

(1.1 ± 0.1) × 10 (4.4 ± 0.9) × 10−4

8.7 ± 0.5 h 4.1 ± 0.8 h

(2.0 ± 0.3) × 10−5 (5.1 ± 0.9) × 10−5

(1.0 ± 0.2) × 10−5 (3.3 ± 0.2) × 10−6 (6.6 ± 0.6) × 10−4

1.9 ± 0.1 day 27 ± 1.5 day 5±1h

(4.2 ± 0.4) × 10−6 (3.0 ± 0.2) × 10−7 (4.2 ± 0.9) × 10−5

The average values from at least three independent studies and SE are reported.

Table 3. IC50 Values of Platinum Complexes in A2780 and DLD-1 Cells after 72 h Incubation Periodsa IC50 (μM) complex

A2780

control cis-[PtCl2(NH3)2] (cisplatin) cis-[PtCl2(en)] [Pt(ox)(en)] [Pt(ox)(R,R-chxn)] (oxaliplatin) cis,trans-[PtCl2(OH)2(en)] (I) cis,trans-[PtCl2(OAc)2(en)] (II) cis,trans-[PtCl2(OTFA)2(en)] (III) trans-[Pt(OH)2(ox)(en)] (IV) trans-[Pt(OAc)2(ox)(en)] (V) trans-[Pt(OTFA)2(ox)(en)] (VI) trans-[Pt(OH)2(ox)(R,R-chxn)] (VII) trans-[Pt(OAc)2(ox)(R,R-chxn)] (VIII)

accumulation, Pt (nmol)/protein (mg) DLD-1

3.3 ± 0.6 16 ± 1 27 ± 3 1.3 ± 0.2 54 ± 4 8.6 ± 1.3 4.2 ± 0.1 93 ± 3 45 ± 3 44 ± 2 37 ± 3 11 ± 2

11 ± 2 70 ± 4 58 ± 4 57 ± 5 134 ± 11 70 ± 4 98 ± 5 >200 >200 >200 >200 126 ± 14

A2780 0.04 0.52 0.17 0.30

± ± ± ±

0.01 0.05 0.01 0.06

DLD-1 ± ± ± ± ± ± ±

0.17 ± 0.01 0.11 ± 0.02

0.02 0.42 0.29 0.36 0.09 0.30 0.14

0.01 0.05 0.08 0.11 0.01 0.07 0.02

0.07 ± 0.01 0.12 ± 0.03 0.14 ± 0.01

0.16 ± 0.04 0.14 ± 0.04 0.11 ± 0.06

Platinum accumulation studies in A2780 and DLD-1 cells after 24 h of treatment with platinum complexes (30 μM). Average IC50 and accumulation values from at least three independent experiments with SE are reported. a

Table 4. Experimentally Determined Peak Height Ratios, a/b, for XANES Spectra of Solid Platinum(II) and Platinum(IV) Complexes complex

coordination

ratio a/b

cis-[PtCl2(en)] [Pt(ox)(en)] cis,trans-[PtCl2(OH)2(en)] (I) cis,trans-[PtCl2(OAc)2(en)] (II) trans-[Pt(OH)2(ox)(en)] (IV) trans-[Pt(OAc)2(ox)(en)] (V)

PtIICl2N2 PtIIO2N2 PtIVCl2O2N2 PtIVCl2O2N2 PtIVO4N2 PtIVO4N2

1.52 1.67 2.34 2.53 2.61 2.73

XANES. Calibration with Solid Standards. The peak height ratio, a/b, of platinum complexes (Table 4) have been shown to be dependent on oxidation state12 where the platinum(II) and platinum(IV) complexes were found to have peak height ratios in the ranges 1.5−1.7 and 2.3−2.8, respectively. The peak height ratio, a/b, of the platinum(IV) complexes, their corresponding platinum(II) analogues, and their molar ratio mixtures were found to have a linear relationship (Figures S3 and S4).13 Reduction of Platinum(IV) Complexes in DLD-1 Cells. The reduction of complexes II and V in DLD-1 cells was monitored at 2, 6, and 24 h (Figures 2 and S5). By use of the XANES spectra, the percentage of unreduced platinum(IV) for each time point was calculated from the linear function derived from the peak height ratios of the platinum(II) and platinum(IV) species (Figures S3 (complex V) and S4 (complex II)). For complex V, it was calculated that at 2 h approximately 69% of the platinum(IV) species remained, while at 6 and 24 h the proportion of platinum(IV) decreased to approximately

Figure 2. Normalized XANES spectra of DLD-1 cells incubated with trans-[Pt(OAc)2(ox)(en)] (V) for 2, 6, and 24 h and normalized XANES spectra of solid cis,trans-[Pt(OAc)2(ox)(en)] (V) used as solid platinum(IV) standard.

24% and 10%, respectively (Table 5). For complex II, it was found that at 2 h, 47% of the platinum(IV) species remained, while 14% and 5% of the platinum(IV) remained at 6 and 24 h, respectively (Table 5).

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DISCUSSION The ease of reduction of the diaminetetracarboxylatoplatinum(IV) complex trans-[Pt(OAc)2(ox)(en)] (V) and its dichlorido 8759

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Table 5. Peak Height Ratio, a/b, of Normalized XANES Spectra of DLD-1 Cells Incubated with trans-[Pt(OAc)2(ox)(en)] (V) and cis,trans-[PtCl2(OAc)2(en)] (II) at 2, 6, and 24 h and the Extrapolated Percentage of Unreduced Platinum(IV) Using the Derived Linear Functions (Figure S3 and S4)

analogue cis,trans-[PtCl2(OAc)2(en)] (II) were compared to examine the influence of the equatorial carboxylato ligands on the inertness of the platinum(IV) center. Their dihydroxido and ditrifluoroacetato analogues trans-[Pt(OH)2(ox)(en)] (IV), cis,trans-[PtCl2(OH)2(en)] (I), and trans -[Pt(OTFA)2(ox)(en)] (VI) were also investigated in these studies to provide further insights into the relationship between inertness and the axial ligands. The electrochemistry of these complexes was characterized using cyclic voltammetry, and the reduction half-lives of the complexes in the presence of the simple biological reductants, ascorbate and cysteine, were determined. Cytotoxicity and cellular accumulation studies and XANES spectroscopy were employed to gain insights into the kinetic inertness of these complexes, as well as those previously studied oxaliplatin derivatives (complexes VII and VIII), within more complex intracellular environments. Reduction Potential. For the platinum(IV) complexes I−VIII, the reduction potentials measured (Table 1) were consistent with those previously reported.5a Irrespective of the equatorial leaving group and the nonleaving amine groups, complexes containing acetato axial ligands (II, V, and VIII) were found to have more positive reduction potentials than those containing hydroxido axial ligands (I, IV, and VII). Complexes III and VI have highly electron withdrawing trifluoroacetato axial ligands, and complexes have reduction potentials (Table 1) that are similar to those of platinum(IV) complexes with axial chlorido ligands.5b Rates of Reduction of Platinum(IV) Complexes. Ascorbate and cysteine were chosen as reductants in the platinum reduction studies, since ascorbate is an essential antioxidant in cell metabolism, and cysteine serves as a model for the reduction of platinum(IV) complexes by thiols such as glutathione(GSH) and albumin. The inertness of platinum(IV) complexes with chlorido equatorial ligands, complexes I and II, was compared with that of complexes with carboxylato equatorial ligands, complexes IV, V, and VI. In general, platinum(IV) complexes VI and V, with oxalato equatorial ligands, were found to be reduced significantly more slowly than those with chlorido equatorial ligands, complexes I and II (Table 2). This difference may be due to the slightly lower reduction potentials of the latter class of platinum(IV) complexes (Table 1). The highly polarizable

nature of chlorido donor atoms, which can more readily form electron bridges with the biological reductants than the oxalato ligands, may also facilitate more efficient electron transfer from the platinum to the reductant.14 A correlation between the reduction rates of the dichlorido platinum(IV) complexes and their reduction potentials (Table 1) is evident. This is consistent with previous reports by Choi et al.5b and Hambley et al.5a that platinum(IV) complexes with two chlorido ligands in equatorial sites and with dichlorido, diacetato, or dihydroxido axial ligands exhibited reduction rates and degrees of DNA binding correlating to their reduction potentials. In contrast, in the case of the oxalato platinum(IV) complexes, the reduction of complex V by ascorbate and cysteine is significantly slower than that of its dihydroxido analogue, complex IV (Table 3), despite its more positive reduction potential. The unusual kinetic inertness of complex V is consistent with that of an analogous diaminetetracarboxylatoplatinum(IV) complex, trans-[Pt(OAc)2(ox)(chxn)] (VII) reported previously by Zhang et al.8 The diaminetetracarboxylato complex cis,trans-[Pt(OTFA)2(ox)(en)] (VI) was observed to have unexpectedly large half-lives of 18 min and 5 h in the presence of ascorbate and cysteine, respectively, based on its positive reduction potential. In contrast, its dichlorido platinum(IV) analogue, complex III, was observed to be reduced almost immediately, consistent with studies by Choi et al. which revealed complex III to have a rate constant (k) of 209 M−1 s−1 and a half-life (t1/2) of 6.4 s in the presence of 10-fold excess ascorbate.5b,7 Although complex VI is reduced more rapidly than other tetracarboxylato complexes such as complex V, a greater degree of stabilization is evident when compared with III. The final pH of the solutions, used for reduction studies in cysteine, was found to be between 5.0 and 6.0 with the exception of complex VII, which gave rise to a final pH between 2.5 and 3.5. This may have also contributed to the long half-life of complex VII in cysteine, as the reduction of platinum(IV) complexes by cysteine has been reported to be highly pH dependent.15 Although studies into the reduction of platinum(IV) complexes by small biological reductants yield important insights into the relative stability of platinum(IV) complexes, such model 8760

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by the diaminetetracarboxylato complexes, both complexes V and VIII are more cytotoxic than their dihydroxido analogues, complexes IV and VII in A2780 cells. Although this trend was also observed for complexes VII and VIII in DLD-1 cells, it could not be shown for complexes IV and V, as all platinum complexes were found to be less cytotoxic in DLD-1 cells. It appears that the intracellular environment has redox mechanisms that overcome the stabilization effect of complexes with diaminetetracarboxylato coordination spheres. XANES Spectroscopy. XANES spectroscopy was employed to further monitor the reduction of platinum(IV) complexes within the intracellular environment. Pt L3-edge XANES spectra were obtained for cellular DLD-1 samples incubated with platinum(IV) complexes at 2, 6, and 24 h. XANES spectra of solid platinum complexes were also obtained to serve as standard calibrations to estimate the extent of intracellular reduction of platinum(IV) complexes in DLD-1 cells over time. Calibration with Solid Standards. The XANES spectra show the characteristic “white line” peak at 11 570 eV for platinum(II) and 11 572 eV for platinum(IV) (Figure S2).20 Because of the proximity of the edge energies of the platinum(II) and platinum(IV) XANES spectra, quantitative determination of the proportion of these two oxidation states in mixtures cannot be obtained using the edge energies alone. Previously, Hall et al. established that there is a linear correlation between the average oxidation state and the peak height ratio, a/b, where a is the edge maxima and b is the edge local minima (Figure S2).13a The proportion of intracellular platinum(II) and platinum(IV) was quantitatively estimated in the present study using the same method.13 Reduction of Platinum(IV) Complexes in DLD-1 Cells. The reduction of complex V in DLD-1 cells was monitored at 2, 6, and 24 h (Figure 2 and Table 5). At 2 h complex V appears to be more resistant to intracellular reduction than complex II, but at 6 and 24 h, the proportions of unreduced platinum(IV) are similar (Table 6). Although complex V exhibited

systems are too simple to accurately predict the kinetic inertness of platinum(IV) complexes in complex biological systems, such as those of the intracellular environment where a vast range of biomolecules could act as platinum(IV) reductants. Nemirovski et al. reported that small molecules only account for a small fraction of the total reduction of platinum(IV) complexes within cells, whereas high molecular weight biomolecules (>3000 Da) account for the majority of the reduction.16 As such, in vitro studies are required to shed light on the stability of diaminetetracarboxylatoplatinum(IV) complexes in more realistic biological contexts. Cytotoxicity and Cellular Accumulation. The cytotoxicity and cellular accumulation of the platinum(IV) complexes were determined to provide insight into the extent of reduction of the complexes within the cancer cells. The similar degrees of cellular accumulation of the platinum complexes in the A2780 and DLD-1 cells suggest that the higher degree of cytotoxicity of A2780 cancer cells cannot be attributed to the degree of cellular accumulation but may be due to inherent differences between the cell types. The generally lower extent of cellular accumulation of platinum(IV) complexes observed may be attributed to the lack of active transporters available to platinum(IV) complexes in carcinoma cells17 or to a greater rate of efflux of the platinum(IV) species compared to the rate of reduction of the platinum(IV) and subsequent aquation of the platinum(II) species once intracellular reduction of the platinum(IV) complex has occurred.18 Platinum(II) complexes are more cytotoxic than their platinum(IV) analogues in both A2780 and DLD-1 cell lines; this is consistent with the platinum(IV) complexes being more inert than their platinum(II) analogues, requiring reduction prior to releasing the cytotoxic component. Platinum(IV) complexes with hydroxido axial ligands, complexes I, IV, and VII, are less cytotoxic than their corresponding carboxylato analogues with acetato and trifluoroacetato axial ligands (complexes II, V, VI, and VIII). This result correlates with the observed trend in their reduction potentials, as it is expected that platinum(IV) complexes that are more easily reduced will also be more cytotoxic.18 The cytotoxicity of the complex cis,trans[PtCl2(OTFA)2(en)](III) was not studied. However, it is expected that its cytotoxicity would be similar to that of its platinum(II) analogue, cis-[PtCl2(en)], because of its kinetic lability.5b Complex VI was observed in both cell lines to be significantly less cytotoxic than its platinum(II) congener, [Pt(ox)(en)], and similar in potency to complex V (Table 3) despite its relatively rapid rate of reduction by cellular reductants. This may be attributed to the lower extent of cellular accumulation of VI and to some degree the stabilization effect of the diamintetracarboxylato coordination sphere. Kwon et al. reported similar cytotoxicities for another diaminetetracarboxylatoplatinum(IV) complex containing axial trifluoracetato ligands against several cell lines.19 The cytotoxicities of complexes I and II in A2780 and DLD-1 cells can be explained by their reduction rates, with complex II being more easily reduced and thus exhibiting higher cytotoxicities (Tables 2 and 3). Complex V is more cytotoxic than complex IV in A2780 cells, and although this correlates well with their reduction potentials (Table 1), it does not correlate with their reduction rates in the presence of ascorbate or cysteine. Similarly, complex VIII exhibits higher cytotoxicity than complex VII in both A2780 and DLD-1 cells, despite having a lower reduction rate in ascorbate. Despite the high resistance to reduction by ascorbate and cysteine exhibited

Table 6. Summary of Extrapolated Percentage of Unreduced Platinum(IV) for DLD-1 Cells Incubated with trans[Pt(OAc)2(ox)(en)] (V) and cis,trans-[PtCl2(OAc)2(en)] (II) Using Their Respective Derived Linear Functions (Figure S3 (Complex V) and Figure S4 (Complex II))

an unusual resistance to reduction in a 10-fold excess of ascorbate and cysteine, with half-lives of 2.5 and 27 days, respectively, its rate of reduction within the intracellular environment is similar to that of complex II (Table 6), which is consistent with its relatively high cytotoxicities in DLD-1 and A2780 cells. After an incubation period of 2 h, the proportion of platinum(IV) remaining within cells treated by complex II was 8761

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47 ± 11%, suggesting that its intracellular half-life in DLD-1 cells is ∼2 h. This is comparable with the half-lives of complex II in the presence of ascorbate and cysteine (Table 2). However, complex V appears to be initially more stable with 69 ± 1% platinum(IV) remaining at 2 h in DLD-1 cells and only 24 ± 12% remaining at 6 h. This infers that the intracellular half-life of complex V is approximately 3−5 h. This is significantly shorter than the half-lives of 2.5 h and 27 days observed in simple model systems using only ascorbate or cysteine and is presumably due to intracellular reduction by high molecular weight biomolecules.16 At the incubation period of 24 h, the proportion of unreduced platinum(IV) was found to be 10 ± 7% and 5 ± 4% for complexes V and II, respectively, suggesting that almost complete intracellular reduction in DLD-1 cells occurs after about 24 h. This result is consistent with the hypothesis that reduction mechanisms within the intracellular environments are able to overcome the stabilizing effect of the diaminetetracarboxylato coordination sphere. Previously, Hall et al.13a reported faster rates of intracellular reduction of platinum(IV) complexes in A2780 cells, with only 33% of cis,trans,cis-[PtCl2(OAc)2(NH3)2] remaining intact at 2 h, and complete intracellular reduction was observed at 24 h. The significantly faster rate of reduction in A2780 cells may be due to the intracellular environment of A2780 cells being more reducing than DLD-1 cells, which is consistent with the higher cytotoxicities observed.

were obtained from Cambridge Isotope Laboratories. All substances were used without further purification unless otherwise specified. Preparation of cis,trans-[PtCl 2(OH) 2 (en)] (I), cis,trans[PtCl2(OAc)2(en)] (II), cis,trans-[PtCl2(OTFA)2(en)](III), trans-[Pt(OH) 2 (ox)(en)] (IV), trans-[Pt(OAc)2(ox)(en)] (V), trans-[Pt(OH)2(ox)(R,R-chxn] (VII), and trans-[Pt(OAc)2(ox)(R,R-chxn] (VIII). Complexes I−V, VII, and VIII were synthesized using previously reported methods.6b,11b−d,21 All complexes were characterized using methods including 1H and 195 Pt NMR, ESI-MS, and IR spectroscopy. Purity was determined using microanalysis to confirm purity >95% (Supporting Information) Preparation of trans-[Pt(OTFA)2(ox)(en)] (VI). The method of synthesis was adapted from the methods reported by Khokhar.22 trans[Pt(OH)2(ox)(en)] (0.15 g, 0.4 mmol) was suspended in DMA (7 mL). Trifluoroacetic anhydride (0.5 mL) was added and the mixture stirred at room temperature for 16 h. The solvent was removed and a colorless solid precipitated upon the addition of diethyl ether and was isolated by filtration (yield 0.15 g, 0.27 mmol, 67%). 1H NMR(400 MHz, D2O), δ 3.02(t, 4H).195Pt NMR(400 MHz, DMF), δ 1601. Anal. Calcd for C8H8F6N2O8Pt: C, 16.88; H, 1.42; N, 4.92. Found: C, 17.09; H, 1.54; N, 4.86. Electrochemistry. Electrochemical measurements were performed using a BAS 100B/W Electrochemical analyzer. Cyclic voltammetry was carried out at a scan rate of 100 mV s−1 over the range −1000 to 0 mV, using a glassing carbon working electrode, a platinum wire auxiliary electrode, and an Ag+/AgCl reference electrode. The samples were prepared as 1 mM solutions in 0.1 M KCl and were deoxygenated with a stream of argon through the solution immediately prior to measurement. Cell Lines. Human colon cancer (DLD-1) and ovarian cancer (A2780) cell lines, were obtained from the American Type Culture Collection (ATCC). The cells were maintained in exponential growth as monolayers in advanced Dulbecco’s modified Eagle medium DMEM (Gibco) supplemented with 1% (w/v) glutamine and 2% (v/v) fetal bovine serum at 37 °C in 5% (v/v) CO2. Cytotoxicity Assays. Platinum complexes were prepared as 1, 2, or 5 mM aqueous solutions prior to the assay. Cytotoxicity was determined using the MTT assay method described previously.23 The assay is dependent on the cellular reduction of 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT), by the mitochondrial dehydrogenase of viable cells, to the purple formazan product, which is measured spectrophotometrically. An amount of 100 μL containing approximately 1 × 106 cells/mL cell culture medium (Advanced DMEM + 2% (v/v) fetal bovine serum and 1% (w/v) glutamine) was seeded onto each well of a 96-well plate and allowed to adhere overnight at 37 °C in 5% (v/v) CO2. Platinum complexes were diluted in culture medium such that 10 different concentrations ranging from 0 to 200 μM were obtained. An amount of 100 μL of each drug concentration was added to triplicate wells, and the cells were incubated for 72 h. Following incubation, MTT (20 μL, 2.5 mg/mL in Milli Q water) was added to each well and the plate incubated for a further 4 h. The medium was removed and DMSO (dimethyl sulfoxide) (150 μL) was added to each well. The plate was shaken for 3 s and the absorbance measured immediately at 600 nm using a Victor3V plate reader (Perkin-Elmer). IC50 values were determined as the drug concentration that reduced the absorbance to 50% relative to that of the untreated control wells. Cellular Accumulation. Approximately 4 × 106 cells were seeded onto a 7 cm Petri dish containing 6 mL of cell culture medium (Advanced DMEM with 2% (v/v) fetal bovine serum and 1% (w/v) glutamine added) and allowed to adhere overnight at 37 °C in 5% (v/v) CO2. Platinum complexes were diluted in culture medium to a final concentration of 30 μM. The medium in the Petri dish was replaced with the prepared medium containing the diluted platinum complex, and the cells were incubated for 24 h. The cells were washed with PBS before being trypsinized and centrifuged. The pelleted cells were washed with PBS and centrifuged before resuspending in 500 μL of PBS. Then 100 μL aliquots of the cell sample were taken for protein analysis and platinum analysis. The samples were stored at −80 °C.

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CONCLUSIONS This study has shown that the equatorial ligands in platinum(IV) complexes can have a significant effect on the kinetic inertness of the complex, with bidentate oxalato equatorial groups having a larger stabilizing effect than dichlorido ligands. Diaminetetracarboxylato coordination spheres have the highest stabilizing effect, but the extent of stabilization is dependent on the carboxylato groups; acetato groups give rise to much slower rates of reduction than trifluoroacetato groups. Despite the unusual kinetic inertness of certain diaminetetracarboxylatoplatinum(IV) complexes in simple single reductant model systems, their relatively high cytoxicities and short half-lives within cancer cells suggest that the intracellular environment is able to overcome the stabilizing effects of the tetracarboxylato coordination sphere. The evidently large variation in the kinetic inertness of this type of platinum(IV) complex in different biological contexts can be exploited in prodrug design strategies. Potentially, platinum(IV) complexes can be tailored to be more resistant to reduction in the extracellular environment using the tetracarboxylato coordination sphere, but such complexes have been shown here to undergo rapid reduction following entry into cancer cells. Suppressing reduction of the platinum(IV) complexes until they have gone into the cancer cells would lead to more effective and less toxic complexes.

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EXPERIMENTAL SECTION

Materials. K2[PtCl4] was obtained from Precious Metals Online. Acetic anhydride, KCl, KI, NaOH, AgNO3, oxalic acid, and dimethyl sulfoxide were obtained from Ajax Chemicals. L-Ascorbic acid, L-cysteine, ethane-1,2-diamine, and 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) were obtained from Sigma Aldrich. Trifluoroacetic anhydride and dimethylacetamide (DMA) were obtained from Alfa Aesar, and hydrogen peroxide was obtained from Merck and stored at 4 °C. Deuterated solvents D2O and DMF-d7 8762

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Protein Assays. Protein concentrations were determined using the Bio-Rad protein assay. The protein assay dye (Bio-Rad) was diluted 1 in 5 with Milli-Q water. The standard protein solutions were prepared using bovine serum albumin (in Milli-Q) and diluted to a range of concentrations (0, 0.1, 0.2, 0.3, 0.4, 0.5, and 0.6 mg/mL) and stored at 4 °C prior to use. The protein assay was carried out by first placing 200 μL of diluted protein assay dye (Bio-Rad) into each well of a 96-well plate. Aliquots (10 μL) of the protein standards and the cell samples were added to the protein assay dye in triplicate. The plate was allowed to stand for at least 10 min before the absorbance was measured at 600 nm. The protein concentrations were determined by using the absorbance standard curve derived from the protein standard solutions. GFAAS/ICP-MS. Graphite furnace atomic absorption spectrometry was conducted using an Agilent 240Z AA spectrometer with a PSD120 autosampling system and a GTA120 atomizer. An UltrAA Pt lamp in UltrAA mode was used to detect elemental platinum in samples. ICP-MS was conducted by the National Measurement Institute (NMI, Lindfield, NSW, Australia) Determination of Reduction Kinetics Using 1H NMR. The reduction of the platinum(IV) complexes by the biologically relevant reductants, ascorbate and cysteine, was studied using 1H NMR. Reduction kinetics was recorded as a series of 1H NMR spectra at 310 K using a 10-fold excess of ascorbate and cysteine. The reduction of the Pt(IV) complexes was measured by analyzing the absolute integrals of the 1H peaks over time. The experiments were conducted using a Bruker AMX 400 MHz spectrometer, and the temperature was calibrated to 310 K using ethylene glycol. Sample Preparation. Platinum(IV) complexes were prepared as 3 and 5 mM solutions in deuterated solvents with a 10-fold excess of either ascorbate or cysteine added immediately prior to reduction experiments. Solutions of the platinum(IV) complexes were prepared by dissolving the solid complexes in D2O (400 μL). These solutions were then combined with solutions of ascorbic acid or cysteine (400 μL, 60 mM or 100 mM) to give a final concentration of the platinum(IV) complex of 3 or 5 mM. Stock L-ascorbate solutions were prepared in D2O. The pH was adjusted to 7.10 ± 0.01, at 37 °C, using a concentrated solution of NaOH in D2O. The solution was stored frozen and thawed immediately prior to conducting kinetics experiment. L-Cysteine solutions were prepared immediately prior to conducting kinetics experiments. No adjustments to pH were made, as this solution was found to result in reduction to cystine upon storage. Determination of Platinum(IV) Reduction Using X-ray Absorption Near Edge Structure (XANES) Analysis. Cell Preparation for XANES Spectroscopy. An amount of 2 × 106 DLD-1 cells/mL was plated on 75 cm2 flasks and allowed to establish monolayers for 72 h before dosing. The cells were then treated with platinum complexes (50 μM) and incubated for 2, 6, and 24 h. Following incubation, the medium was removed and the cells were washed thoroughly with PBS. The cells were then trypsinized and centrifuged at 2000 rpm for 2 min. The supernatant was removed, and the cells were resuspended in PBS and centrifuged. This procedure was repeated two more times. The cells were resuspended in a solution of ammonium acetate (0.1 M, 5 mL) before being centrifuged and then washed in isopropanol and centrifuged to yield dry cell pellets. The samples were freeze-dried overnight before storing in a desiccator. The freeze-dried cells were packed into polycarbonate sample holders and secured with Kapton tape (Kapton) for XANES spectroscopy analysis. Solid Standard Preparation for XANES Spectroscopy. Solid platinum complexes were combined with solid boron nitride to a final concentration of 5% w/w platinum complex. Mixtures of platinum(II) and platinum(IV) standards were prepared as 50:50 mixtures by molar ratios before being combined with solid boron nitride to a final concentration of 5% w/w platinum complex. XANES Spectroscopy. Pt L3-edge XANES spectra were obtained at the Australian National Beamline Facility (ANBF) on bending magnet beamline 20B at the KEK Photon Factory, Tsukuba, Japan, using a Si(111) channel-cut monochromator. A Pt foil standard was used to calibrate the energy scale to the first derivative of the Pt edge (11 567.1 eV).

XANES spectra of the solid and cellular samples were collected by measuring the fluorescence over the following data ranges: pre-edge region from 11 354 to 11 545 eV (5 eV steps), XANES edge region from 11 545 to 11 605 eV (0.25 eV steps, and XANES postedge region from 11 605 to 11 789 eV (1.4 eV steps). Four scans were taken per sample, and the spectra were checked, calibrated, and averaged using Average software. Background subtraction and normalization were performed on the averaged spectra using Athena software. The empirical background spline was calculated and subtracted using the AUTOBK algorithm in Athena.24 The edge maximum, a, was divided by the edge local minimum, b, to derive the ratio a/b. Error analysis for the XANES spectra was derived using Graph software to estimate the noise (error) associated with each point on the spectra, a and b. Error propagation was then used to derive the overall error in the peak height ratio, a/b.

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ASSOCIATED CONTENT

S Supporting Information *

Synthesis and characterization of complexes I−V, VII, and VIII; reduction monitored by 1H NMR time course experiments and pseudo-first - fitting; normalized XANES spectra of reduction in DLD-1 cells for complexes III and V; calibration of platinum solid standards. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +61-2-93513320. Fax: +61-2-9351-3329. Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by ARC Discovery Grant DP110100461. We acknowledge scientific and technical support from the Australian Microscopy and Micro-Analysis Research Facility (AMMRF) at the University of Sydney, NSW, Australia, and the Australian National Beamline Facility (ANBF) at the Photon Factory in Japan, operated by the Australian Synchrotron. We acknowledge the Australian Research Council for financial support and the High Energy Accelerator Research Organisation (KEK) in Tsukuba, Japan, for operations support and the travel funding provided by the International Synchrotron Access Program (ISAP) managed by the Australian Synchrotron and funded by the Australian Government.

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ABBREVIATIONS USED R,R-chxn, (R,R)-1,2-diaminocyclohexane; en, ethane-1,2-diamine; OAc, acetato; OTFA, trifluoroacetato; ox, oxalato; DMSO, dimethyl sulfoxide; DMA, dimethylacetamide

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REFERENCES

(1) Siddik, Z. H. Cisplatin: mode of cytotoxic action and molecular basis of resistance. Oncogene 2003, 22, 7265−7279. (2) (a) Galanski, M.; Jakupec, M. A.; Keppler, B. K. Update of the preclinical situation of anticancer platinum complexes: novel design strategies and innovative analytical approaches. Curr. Med. Chem. 2005, 12, 2075−2094. (b) Cepeda, V.; Fuertes, M. A.; Castilla, J.;

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Alsonso, C.; Quevedo, C.; Perez, J. M. Biochemical mechanisms of cisplatin cytotoxicity. Anti-Cancer Agents Med. Chem. 2007, 7, 3−18. (3) Wong, E.; Giandomenico, C. M. Current status of platinumbased antitumor drugs. Chem. Rev. 1999, 99, 2451−2466. (4) (a) Galanski, M.; Keppler, B. K. Searching for the magic bullet: anticancer platinum drugs which can be accumulated or activated in the tumor tissue. Anti-Cancer Agents Med. Chem. 2007, 7, 55−73. (b) Hall, M. D.; Mellor, H. R.; Callaghan, R.; Hambley, T. W. Basis for the design and development of platinum(IV) anticancer complexes. J. Med. Chem. 2007, 50, 3403−3411. (c) Hall, M. D.; Hambley, T. W. Platinum(IV) antitumour compounds: their bioinorganic chemistry. Coord. Chem. Rev. 2002, 232, 49−67. (5) (a) Ellis, L. T.; Er, H. M.; Hambley, T. W. The influence of the axial ligands of a series of platinum(IV) anti-cancer complexes on their reduction to platinum(II) and their reaction with DNA. Aust. J. Chem. 1995, 48, 793−806. (b) Choi, S.; Filotto, C.; Bisanzo, M.; Delaney, S.; Lagasee, D.; Whitworth, J. L.; Jusko, A.; Li, C.; Wood, N. A.; Willingham, J.; Schwenker, A.; Spaulding, K. Reduction and anticancer activity of platinum(IV) complexes. Inorg. Chem. 1998, 37, 2500− 2504. (6) (a) Carr, J. L.; Tingle, M. D.; McKeage, M. J. Rapid biotransformation of satraplatin by human red blood cells in vitro. Cancer Chemother. Pharmacol. 2002, 50, 9−15. (b) Dhara, S. C. A rapid method for the synthesis of cis-[Pt(NH3)2Cl2]. Indian J. Chem. 1970, 8, 193−194. (7) Chaney, S. G.; Wyrick, S.; Till, G. K. In vitro biotransformations of tetrachloro(D,L-trans)-l,2-diaminocyclohexaneplatinum(IV) (tetraplatin) in rat plasma. Cancer Res. 1990, 50, 4539−4545. (8) Zhang, J. Z.; Wexselblatt, E.; Hambley, T. W.; Gibson, D. Pt(IV) analogs of oxaliplatin that do not follow the expected correlation between electrochemical reduction potential and rate of reduction by ascorbate. Chem. Commun. 2012, 48, 847−849. (9) Varbanov, H. P.; Valiahdi, S. M.; Kowol, C. R.; Jakupec, M. A.; Galanski, M.; Keppler, B. K. Novel tetracarboxylatoplatinum (IV) complexes as carboplatin prodrugs. Dalton Trans. 2012, 41, 14404− 14415. (10) (a) Nemirovski, A.; Vinograd, I.; Takrouri, K.; Mijovilovich, A.; Rompel, A.; Gibson, D. New reduction pathways for ctc[PtCl2(CH3CO2)2(NH3)(Am)] anticancer prodrugs. Chem. Commun. 2010, 46, 1842−1844. (b) Hall, M. D.; Foran, G. J.; Zhang, M.; Beale, P. J.; Hambley, T. W. XANES determination of the platinum oxidation state distribution in cancer cells treated with platinum (IV) anticancer agents. J. Am. Chem. Soc. 2003, 125, 7524−7525. (11) (a) Khokhar, A. R.; Deng, Y.; Kido, Y.; Siddik, Z. H. Preparation, characterization, and antitumor activity of new ethylenediamine platinum (IV) complexes containing mixed carboxylate ligands. J. Inorg. Biochem. 1993, 50, 79−87. (b) Giandomenico, C. M.; Abrams, M. J.; Murrer, B. A.; Vollano, J. F.; Rheinheimer, M. I.; Wyer, S. B.; Bossard, G. E.; Higgins, J. D. Carboxylation of kinetically inert platinum(IV) hydroxy complexes. An entree into orally active platinum(IV) antitumor agents. Inorg. Chem. 1995, 34, 1015−1021. (c) Galanski, M.; Yasemi, A.; Slaby, S.; Jakupec, M. A.; Arion, V. B.; Rausch, M.; Nazarov, A. A.; Keppler, B. K. Synthesis, crystal structure and cytotoxicity of new oxaliplatin analogues indicating that improvement of anticancer activity is still possible. Eur. J. Med. Chem. 2004, 39, 707−714. (d) Kizu, R.; Nakanishi, T.; Hayakawa, K.; Matsuzawa, A.; Eriguchi, M.; Takeda, Y.; Akiyama, N.; Tashiro, T.; Kidani, Y. A new orally active antitumor 1R,2R-cyclohexanediamineplatinum(IV) complex: trans-(n-valerato)chloro(1R,2Rcyclohexanediamine)(oxalato)platinum(IV). Cancer Chemother. Pharmacol. 1999, 43, 97−105. (12) Yamamoto, N.; Renfrew, A. K.; Kim, B. J.; Bryce, N. S.; Hambley, T. W. Dual targeting of hypoxic and acidic tumor environments with a cobalt(III) chaperone complex. J. Med. Chem. 2012, 55, 11013−11021. (13) (a) Hall, M. D.; Foran, G. J.; Zhang, M.; Beale, P. J.; Hambley, T. W. XANES determination of the platinum oxidation state distribution in cancer cells treated with platinum(IV) anticancer agents. J. Am. Chem. Soc. 2003, 125 (25), 7524−7525. (b) Hall, M. D.;

Daly, H. L.; Zhang, J. Z.; Zhang, M.; Alderden, R. A.; Pursche, D.; Foran, G. J.; Hambley, T. W. Quantitative measurement of the reduction of platinum(IV) complexes using X-ray absorption nearedge spectroscopy (XANES). Metallomics 2012, 4, 568−575. (14) (a) Shi, T.; Berglund, J.; Elding, L. I. Reduction of transdichloro- and trans-dibromo-tetracyano-platinate(IV) by L-methionine. J. Chem. Soc., Dalton Trans. 1997, 2073−2077. (b) Shi, T.; Berglund, J.; Elding, L. I. Kinetics and mechanism for reduction of transdichlorotetracyanoplatinate(IV) by thioglycolic acid, L-cysterine, DLpenicillamine, and glutathione in aqueous solution. Inorg. Chem. 1996, 35, 3498−3503. (c) Lemma, K.; Sargeson, A. M.; Elding, L. I. Kinetics and mechanism for reduction of oral anticancer platinum(IV) dicarboxylate compounds by L-ascorbate ions. J. Chem. Soc., Dalton Trans. 2000, 1167−1172. (15) Chen, L.; Lee, P. F.; Ranford, J. D.; Vittal, J. J.; Wong, S. Y. Reduction of the anti-cancer drug analogue cis,trans,cis[PtCl2(OCOCH3)2(NH)3)2] by L-cysteine and L-methionine and its crystal structure. Dalton Trans. 1999, 1209−1212. (16) Nemirovski, A.; Kasherman, Y.; Tzaraf, Y.; Gibson, D. Reduction of cis,trans,cis-[PtCl2(OCOCH3)2(NH3)2] by aqueous extracts of cancer cells. J. Med. Chem. 2007, 50, 5554−5556. (17) Hall, M. D.; Okabe, M.; Shen, D.-W.; Liang, X.-J.; Gottesman, M. M. The role of cellular accumulation in determining sensitivity to platinum-based chemotherapy. Annu. Rev. Pharmacol. Toxicol. 2008, 48, 495−535. (18) Hall, M. D.; Amjadi, S.; Zhang, M.; Beale, P. J.; Hambley, T. W. The mechanism of action of platinum(IV) complexes in ovarian cancer cell lines. J. Inorg. Biochem. 2004, 98, 1614−1624. (19) Kwon, Y.-E.; Whang, K.-J.; Park, Y.-J..; Kim, K. H. Synthesis, characterization and antitumor activity of novel octahedral Pt(IV) complexes. Bioorg Med Chem 2003, 11 (8), 1669−1676. (20) Brown, M.; Peierls, R.; Stern, E. White lines in X-ray absorption. Phys. Rev. B 1977, 15, 738. (21) Kohkhar, A. R.; Deng, Y.; Al-Baker, S.; yoshida, M. A.; Siddick, Z. H. Synthesis and antitumor activity of ammine/amine platinum(II) and (IV) complexes. J. Inorg. Biochem. 1993, 51, 677−687. (22) Khokhar, A. H.; Deng, Y.; Kido, Y.; Siddik, Z. H. Preparation, characterization, and antitumor activity of new ethylenediamine platinum(IV) complexes containing mixed carboxylate ligands. J. Inorg. Biochem. 1993, 50, 79−87. (23) Carmichael, J.; DeGraff, W. G.; Gazdar, A. F.; Minna, J. D.; Mitchell, J. B. Evaluation of a tetrazolium-based semiautomated colorimetric assay: assessment of chemosensitivity testing. Cancer Res. 1987, 47, 936. (24) Ravel, B.; Newville, M. ATHENA, ARTEMIS, HEPHAESTUS: data analysis for X-ray absorption spectroscopy using IFEFFIT. J. Synchrotron Radiat. 2005, 12, 537−541.

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