The Effect of Ligand Lipophilicity on the Nanoparticle
Encapsulation of Pt(IV) Prodrugs
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Citation
Johnstone, Timothy C., and Stephen J. Lippard. “The Effect of
Ligand Lipophilicity on the Nanoparticle Encapsulation of Pt(IV)
Prodrugs.” Inorg. Chem. 52, no. 17 (September 3, 2013):
9915–9920.
As Published
http://dx.doi.org/10.1021/ic4010642
Publisher
American Chemical Society
Version
Author's final manuscript
Accessed
Wed May 25 22:40:52 EDT 2016
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http://hdl.handle.net/1721.1/88424
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The Effect of Ligand Lipophilicity on the Nanoparticle Encapsulation of Pt(IV) Prodrugs
Timothy C. Johnstone and Stephen J. Lippard*
Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA, 02139
ABSTRACT: In an effort to further expand the therapeutic range of platinum-based anticancer
agents, several new approaches to platinum-based therapy, including nanodelivery, are under active investigation. To better understand the effect of ligand lipophilicity on the encapsulation of
Pt(IV) prodrugs within polymer nanoparticles, the series of compounds cis,cis,trans[Pt(NH3)2Cl2L2] was prepared, where L = acetate, propanoate, butanoate, pentanoate, hexanoate,
heptanoate, octanoate, nonanoate, and decanoate. The lipophilicities of these compounds, assessed by reversed-phase HPLC, correlate with the octanol/water partition coefficients of their
respective free carboxylic acid ligands, which in turn affect the degree of encapsulation of the
Pt(IV) complex within the hydrophobic core of poly(lactic-co-glycolic acid)-block-poly(ethylene
glycol) (PLGA-PEG-COOH) nanoparticles. The most lipophilic compound investigated,
cis,cis,trans-[Pt(NH3)2Cl2(O2C(CH2)8CH3)2], displayed the best encapsulation. This compound
was therefore selected to evaluate the effect of increased platinum concentration on encapsulation. As the platinum concentration was increased, there was an initial increase in encapsulation
1
followed by a decrease due to macroscopic precipitation. Maximal loading occurred when the
platinum complex was present at a 40% w/w ratio with respect to polymer during the nanoprecipitation step. Particles formed under these optimal conditions had diameters of approximately
50 nm, as determined by transmission electron microscopy.
Introduction
Following the serendipitous discovery of anticancer activity for cis-[Pt(NH3)2Cl2], or cisplatin1,
it has come to be one of the most widely used chemotherapeutic agents for the treatment of testicular, ovarian, bladder, and non-small cell lung cancers (NSCLC), as well as small cell lung
cancer (SCLC), melanoma, lymphomas, and myelomas.2 The advantages of platinum-based
treatment are perhaps most evident in testicular cancer, where, following the introduction of cisplatin into the arsenal of available treatments, cure rates have risen to > 90%.3 Despite the clinical efficacy demonstrated by platinum-based drugs currently approved by the FDA, intense research is underway to discover variants with enhanced efficacy, lower toxicity, and a more diverse spectrum of activity.4 One approach is to use nano-sized drug delivery vehicles, which offers several potential advantages.5 The controlled release provided by these systems can enhance
retention in the bloodstream, their size allows for passive targeting, and their surfaces can be
functionalized for active targeting. Our lab has reported the preparation of Pt(IV) constructs designed for delivery by gold nanoparticles,6 single-walled carbon nanotubes,7,8 and polymeric nanoparticles.9-11
Most platinum complexes investigated for their cytotoxic activity are unsuitable for nanoparticle encapsulation. Different approaches have been used to produce analogues that can be integrated into a nanodelivery device. One strategy that we previously described involved the intro2
duction of hydrophobic axial ligands L into the coordination sphere of a cis,cis,trans[Pt(NH3)2Cl2L2], platinum(IV) complex, allowing it to be encapsulated within the hydrophobic
core of a polymeric micelle.9 The micelle used in this prior study was composed of carboxyterminated amphiphilic poly(lactic-co-glycolic acid)-block-poly(ethylene glycol) (PLGA-PEGCOOH) copolymers, the self-assembly of which has been exploited previously for the construction of other drug delivery vehicles.12 The hexanoate ligands L of the Pt(IV) complex provided
lipophilicity sufficient to promote encapsulation within the PLGA nanoparticle core. The remaining ligands in the coordination sphere are such that, following reduction of this Pt(IV) complex,
an equivalent of cisplatin would be released as the active Pt(II) cytotoxic payload.
These results raised the question, is the addition of two lipophilic carboxylates to a Pt(II) core
complex a readily generalizable strategy for nanodelivery of platinum(IV) anticancer prodrugs?
Before exploring different Pt(II) core structures, however, we wanted to understand the precise
effect of carboxylate ligand choice on the nanoprecipitation process used to form the polymeric
micelles. We therefore undertook the present study of a series of compounds of the form
cis,cis,trans-[Pt(NH3)2Cl2(OOCR)2], where R is a straight chain alkyl (Scheme 1). The complexes comprising this series all contain a cis-diamminedichloroplatinum core as the pharmacophore
Scheme 1. The synthesis and general structure of the Pt(IV) carboxylate species studied here.
3
and two axial ligands that vary systematically in length. In previous related work, a series of
Pt(IV) carboxylates complexes containing either a cis-1,4-diaminocyclohexanedichloroplatinum13 or a trans-1R,2R-diaminocyclohexanedichloroplatinum pharmacophore14 investigated
the influence of axial ligand chain length on their biological properties. The aim of the present
work, however, is to exploit the axial ligands for nanoparticle encapsulation with the aim of enhancing anticancer efficacy. The efficiency of nanoparticle encapsulation was investigated and
correlated with the physical properties of the carboxylate ligands. The effect of relative platinum
complex concentration during the nanoprecipitation procedure on the degree of encapsulation
was also studied. Two competing processes, nanoparticle encapsulation and bulk precipitation,
were correlated with this parameter, and conditions for maximal loading were determined by
finding the optimal balance between these two processes.
Experimental Section
General Procedures and Chemicals for Synthesis. All chemicals were reagent grade and
used as purchased without further purification. Acetic, butanoic, and pentanoic anhydrides were
obtained from Sigma-Aldrich. Propanoic, hexanoic, heptanoic, and octanoic anhydrides were
purchased from TCI America. Nonanoic and decanoic anhydrides were prepared by the DCC
mediated dehydration of the corresponding carboxylic acids,15 which were obtained from TCI
America. Solvents were used as received without further purification. Cis,cis,trans[Pt(NH3)2Cl2(OH)2], compound 1, was prepared as previously described.16 The PLGA-PEGCOOH block co-polymer, prepared as described previously,17 was kindly provided by the
Farokhzad Lab (Suresh Gadde, Harvard Medical School). MilliQ water was used for all work
involving nanoparticles.
4
Physical Measurements. 1H and 13C{1H} NMR measurements were performed on a Varian
Inova-500 spectrometer in the MIT Department of Chemistry Instrumentation Facility with deuterated DMSO as a solvent. NMR chemical shifts (δ) are reported in ppm with respect to tetramethylsilane and referenced to residual solvent peaks. NMR spectra are presented in Figures S1
to S18. Fourier-transform infrared spectra were recorded on a ThermoNicolet Avatar 360 spectrophotometer using the OMNIC software. All IR samples were prepared as KBr pellets and
measurements are reported in cm–1. Electrospray ionization mass spectrometry (ESI-MS) measurements were performed on an Agilent Technologies 1100 series LC/MSD ion trap. Liquid
chromatography mass spectrometry (LC-MS) measurements were performed using the same ion
source and trap.
Synthesis. Compounds of general formula cis,cis,trans-[Pt(NH3)2Cl2(OOCR)2], where R= methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, or nonyl, were all prepared in the same
manner. Compound 1 (200 mg, 0.60 mmol) was suspended in 8 mL of DMF. To this mixture
was added 1.5 mL of the appropriate carboxylic acid anhydride. The reaction mixture was stirred
in the dark at 50 °C for 24 h. The resulting yellow to orange solution was filtered through Celite
and the volume of the filtrate was reduced to 2 mL in vacuo at 50 °C. This concentrated solution
was then added in a dropwise manner to a rapidly stirring volume of diethyl ether (40 mL) forming a pale yellow to white precipitate. The ether suspension was stirred for 30 min, and the solid
was collected by filtration and dried under vacuum for 12 h. The detailed characterization of 2 –
10, including spectroscopic and crystallographic data, is collected in the Supporting Information.
Liquid Chromatography-Mass Spectrometry (LC-MS). Solutions (1:1 methanol/water containing 5% DMF) of 2 through 6 were injected onto an Eclipse XDB-C18 column (3 x 150 mm,
Agilent) and eluted with 1:1 methanol/water at a rate of 0.25 mL/min. The elution was isocratic
5
and the temperature of the mobile phase within the column was maintained at 25 °C. The analytes were detected by monitoring the intensity of the negative ion mode base peak, which in all
cases was the [M–H]– signal. The dead time of the column was evaluated by monitoring the elution of potassium iodide at 210 nm. This dead time was used in the calculation of the capacity
factors of the analyzed Pt(IV) compounds.
Nanoparticle Encapsulation. A 550 µL DMF solution containing 10 mg of PLGA-PEGCOOH and an amount of a platinum complex to give the desired feed, defined as (mg Pt complex/mg polymer) x 100, was prepared. A 500 µL aliquot of this solution was added in a dropwise manner over the course of 10 min to 5 mL of rapidly stirring MilliQ water. The DMF solutions were added by a mechanical pipette and the nanoprecipitations were carried out in 20 mL
glass scintillation vials. The water was stirred magnetically using a 0.5 cm stir bar at approximately 500 rpm. After addition of the DMF solution, the water acquired a milky blue coloration
owing to Tyndall scattering of the nanoparticles that formed. At higher loadings, some macroscopic precipitation was also observed. An aqueous solution of poly(vinyl alcohol) (5 mL, 0.1%
w/w PVA) was then added along the edge of the vial to bring the final volume to 10.5 mL and
the final PVA concentration to approximately 0.05 % w/w. This suspension of nanoparticles was
stirred for an additional 20 min and then passed through a 0.45 µm cellulose acetate syringe filter
(VWR). If macroscopic precipitation was evident, the suspension was first passed through a plug
(0.25 x 1 cm) of Celite in order to avoid excessively clogging of the syringe filter. The filtrate
from the 0.45 µm filtration was loaded into an Amicon Centrifugal Filtration Device (100 kDa
MWCO regenerated cellulose membrane). The loaded device was centrifuged at 1500 x g for 20
min, concentrating the nanoparticle suspension to approximately 1 mL. This concentrated material was suspended in an additional 10 mL of fresh MilliQ water and centrifuged again under
6
identical conditions. Each sample was washed 3 times in this manner. The final concentrated
suspension was diluted to 1.4 mL with MilliQ water for use in further experiments. All nanoprecipitations were carried out in triplicate.
Evaluation of Encapsulation. The metric used to evaluate encapsulation efficacy was the
concentration of platinum present in the final 1.4 mL colloidal suspension. This concentration
was determined by electrothermal atomic absorption spectroscopy (AAS) using a Perkin-Elmer
AAnalyst 600 spectrometer outfitted with a transverse heated graphite atomizer. Platinum absorption was measured at 265.9 nm and a Zeeman background absorption correction was applied. Samples were prepared by diluting nanoparticle suspensions with MilliQ water until the
platinum concentration fell within the linear calibration range (50 – 200 µg Pt/L). All AAS
measurements were carried out in triplicate and averaged.
Transmission Electron Microscopy. PLGA-PEG-COOH nanoparticles containing compound
10 at a 40% feed, the optimal loading conditions as described below, were prepared and an aliquot was allowed to evaporate on a carbon-coated copper TEM grid. Imaging was conducted by
using a JEOL JEM-200CX operated at 120 kV.
Results and Discussion
Synthesis. The nucleophilicity of hydroxo ligands coordinated to Pt(IV) centers provides a
convenient synthetic route for modifying Pt(IV) carboxylate compounds via condensation with
an appropriate acid anhydride.18 In the present work, this chemistry was carried out in DMF. As
the reaction proceeds, the suspension of the dihydroxo compound 1 in DMF is consumed, forming a clear solution of the soluble dicarboxylatoplatinum(IV) product. The differential solubility
of the starting material and products both drives the reaction and provides a convenient way to
7
monitor its progress. To purify the compounds, the reaction volume was reduced in vacuo before
addition to a large excess of diethyl ether. For many of the compounds, addition in the reverse
order yielded only intractable oils. For longer chain carboxylates, the solubility of the products in
DMF decreases. For example, 2, with acetate ligands, is readily soluble at concentrations up to
100 mg/mL (0.24 M) at room temperature, but for 10, which has decanoate ligands, a precipitate
formed when a warm DMF solution of equivalent molar concentration was cooled to room temperature.
Physical characterization of the compounds confirmed their proposed structures. In the IR
spectra of the products, sharp bands corresponding to O–H and Pt–O stretches in the starting material, at 3516 cm–1 and 559 cm–1, respectively, are absent, and new bands in the ranges of 1665
cm–1 to 1620 cm–1 (carbonyl C=O stretch), 1470 cm–1 to 1190 cm–1 (C–H bend) and 2960 cm–1 to
2850 cm–1 (C–H stretch) appear. The remaining notable features of the IR spectra are bands from
1530 cm–1 to 1580 cm–1 (N–H bend) and 3260 cm–1 to 3110 cm–1 (N–H stretch) that are present in
both the starting material and the final products.
The 1H NMR spectra of 2 through 10 all contain a resonance at approximately 6.52 ppm, consistent with protons on an ammine ligand bound to a Pt(IV) center. Features present on this signal arise from unresolved coupling of the protons to both 14N and 195Pt nuclei. For 2 - 5, each additional methylene unit in the carboxylate chain produces a well-resolved resonance in the aliphatic region that displays the expected 1H-1H coupling pattern. As additional methylene groups
are added from 6 to 10, the overall CH2 signal intensity increases, but individual units were not
resolved at 500 MHz. The 13C NMR spectra all display resolved peaks for the expected number
of carbon atoms despite nearly overlapping signals (peak-to-peak separations of as little as 0.005
8
ppm) in some of the longer chains. The mass spectra all show the presence of the desired compound as indicated by [M–H]– peaks in negative ion mode ESI–MS.
Crystallography. The crystal structure of a DMSO solvate of c,c,t-[Pt(NH3)2Cl2(OOCCH3)2],
2·2DMSO, is depicted in Figure 1. Selected crystallographic parameters are presented in Table 1.
Full details including bond lengths and angles are collected in Tables S1 and S3. The room temperature structure of a monohydrate of this compound has been previously reported.19 In the previous structure a two-fold rotation axis passed through the platinum, but 2·2DMSO has no such
Figure 1. Molecular diagrams of 2 (left) and 3 (right). Thermal ellipsoids are drawn at the 50%
probability level.
required symmetry. The orientations of the acetate ligands differ in the two structures, results in
differing intramolecular hydrogen-bonding interactions, but the bond lengths and angles of the
two are comparable. If allowances are made for flexibility of dihedral angles controlling the rota-
9
tion of the acetate ligands, then the structures have a RMSD of 0.098 Å, as calculated with the
program MERCURY.
Table 1. Crystallographic Parameters for 2·2DMSO and 3
Formula
Formula weight
Crystal system
Space group
Color
a, Å
b, Å
c, Å
α, °
β, °
γ, °
V, Å3
Z
T, K
C8H18Cl2N2O6PtS2
568.35
Triclinic
P
Colorless
7.2454(7)
10.2263(10)
14.0657(14)
72.516(2)
81.044(2)
69.485(2)
929.51(16)
2
100(2)
C6H16Cl2N2O4Pt
446.20
Tetragonal
P43
Yellow
9.2357(3)
R1a (%)
wR2b (%)
GOFc
3.42
8.79
1.308
1.68
3.71
1.065
13.9563(11)
1190.45(11)
4
100(2)
a
R1=Σ||Fo| - |Fc||/ Σ|Fo|. bwR2= {Σ[w(Fo2-Fc2)2]/Σ[w(Fo2)2]}1/2. cGOF= {Σ[w(Fo2-Fc2)2]/(np)}1/2, where n is the number of data and p is the number of refined parameters.
The crystal structure of 3 was also determined. The apparent Laue symmetry visible in precession camera pseudo-images is 4/mmm, but no successful solution could be obtained in any of the
space groups consistent with the 00l, l=4n systematically absent reflections. The abnormally low
<E2-1> value of 0.525 and the similarity between Rint(4/mmm) = 0.093 and Rint(4/m) = 0.042
suggested that the crystal may be twinned by merohedry. A Patterson map generated using preliminarily detwinned data revealed the locations of the Pt and Cl atoms. Subsequent refinement
using the appropriate twin law afforded a satisfactory crystal structure (Figure 1). Selected crystallographic parameters are presented in Table 1. Full details including bond lengths and angles
are collected in Tables S2 and S4.
10
Evaluation of Lipophilicity. The traditional measure of the lipophilicity of a molecule is the
log of the water/octanol partition coefficient (log P).20 Such values are typically obtained by the
“shake-flask” method in which the analyte is introduced into a biphasic mixture of n-octanol saturated water and water saturated n-octanol. The compound is allowed to achieve a thermodynamic equilibrium between the two phases and the concentration of analyte in each phase is determined. We find, however, that more reproducible results are obtained by using a reversedphase high performance liquid chromatography (RP-HPLC) method.21 For many compounds,
including platinum complexes, the capacity factor under isocratic elution conditions is linearly
proportional to log P values obtained by the shake-flask method.22 Compounds 2 - 6 were analyzed by RP-HPLC and their capacity factors (k) are presented in Table 2. Chromatograms are
shown in Figure S20.
Table 2. Retention Times and Capacity Factors of 2 - 6
Compound
2
3
4
5
6
Retention time
(min)
2.8
3.4
4.7
9.6
32.7
Capacity factor
(k)
0.077
0.31
0.81
2.70
11.6
The lipophilicity of an organic molecule can be accurately modeled as the sum of the contributions from its individual functional groups.23 When the log of the capacity factors of compounds
2 - 6 were plotted against the log P values of acetic through hexanoic acid, a straight line is obtained (Figure 2). This correlation indicates that the effect of adding additional methylene units
to the carboxylate chain has approximately the same effect on the platinum complex as it does on
the isolated carboxylic acid. Therefore, in subsequent analyses we compare the properties of the
11
platinum complexes with the log P values of the carboxylic acids from which the carboxylate
ligands are derived.
Figure 2. Correlation between the log of the capacity factors for 2 = 6 (abscissa) and the log of
the octanol/water partition coefficients of the carboxylic acids from which the carboxylates of 2 6 are derived (ordinate).
Nanoparticle Encapsulation. The strategy used here to encapsulate compounds 2 - 10 within
a polymeric nanoparticle has been successfully utilized to encapsulate other molecules.12,17,24,25
The process by which the nanoconstruct forms is depicted schematically in Figure 3. An organic
solution containing both an amphiphilic block copolymer and a potential guest molecule is added
to a large excess of water. Provided that the organic solvent has been properly chosen so as to be
readily miscible with water, this process drives self-assembly of the polymer chains into coreshell polymeric micelles. The hydrophobic block is buried within the core and the hydrophilic
block forms the solvent-exposed shell. When the polymer chains self-assemble, the additional
hydrophobic guest molecule present in the initial organic solution can become trapped within the
hydrophobic core of the particle.
12
Figure 3. Schematic depiction of the nanoprecipitation process, whereby the amphiphilic block
copolymer chains self-assemble into polymeric micelles, trapping hydrophobic guest molecules
within.
The encapsulation of complexes for 2 - 10 reflects the lipophilicities of their axial carboxylate
ligands. Increasing the chain length increases the overall lipophilicity of the complex, facilitating
its partition into the hydrophobic nanoparticle core. The series of platinum complexes reported
here was prepared to determine the precise effect of chain length on the encapsulation of platinum complexes within a PLGA-PEG-COOH nanoparticle.
The specific experimental encapsulation procedure used was based upon a method described
previously.12 The polymer and platinum complex are dissolved together in DMF and added in a
dropwise manner to a tenfold excess volume of water. In previous studies using compound 6,9,10
acetonitrile was employed as the organic solvent, but not all of the compounds prepared here are
sufficiently soluble in acetonitrile to allow for a unilateral comparison. When a droplet of the
DMF solution comes into contact with the water, the organic solvent rapidly disperses into the
aqueous phase forcing the copolymer chains to assemble into micelles. In the process of nano-
13
particle assembly, lipophilic platinum complexes that were present in the DMF solution are
trapped within the hydrophobic core of the polymer nanoparticle.
When attempting to work-up nanoparticle suspensions, particularly during the concentration
step in the centrifugal filtration devices, the particles would often aggregate irreversibly. Inclusion of a small amount of a surfactant such as poly(vinyl alcohol) (PVA) during this stage of the
preparation eliminated the problem.
When the encapsulation was carried out at a 10% feed – that is, when the platinum complex
was present at 10% w/w with respect to polymer in the DMF solution – a consistent trend was
observed. As the chain length of the carboxylate ligand increased, the amount of the platinum
complex encapsulated increased (Figure 4). Because log P of the carboxylic acid used as the lig-
Figure 4. Correlation between log of encapsulation efficiency (%EE) for 2 - 10 (ordinate) and
log P of the carboxylic acids from which the carboxylates of 2 - 10 are derived (abscissa).
and correlates with the lipophilicity of the entire platinum complex (vide supra), the log of the
encapsulation efficiency (EE) was plotted against this parameter. The encapsulation efficiency is
defined as the % w/w of platinum complex encapsulated with respect to the amount used in the
14
nanoprecipitation. Figure 4 shows that an excellent linear correlation exists between the aforementioned variables.
This correlation is intuitively satisfying, because one would expect that, as the platinum compounds become more lipophilic, they should more readily encapsulate within the hydrophobic
core of the nanoparticles. Moreover, the linearity of the correlation attests to the reliability and
reproducibility of the procedure.
Because 10 is the most effectively encapsulated at a 10% feed, it was selected to study the effect of feed variation on encapsulation. Initially, as the amount of platinum complex present during the nanoprecipitation was increased, the amount of platinum complex encapsulated increased, as quantified by the loading, the %w/w of platinum compound encapsulated with respect to polymer used in the nanoprecipitation (Figure 5). As the amount of platinum compound
Figure 5. Variation in the platinum loaded into PLGA-PEG-COOH nanoparticles as the feed of
10 was varied.
increased, however, the amount of macroscopic precipitation that occurred during nanoprecipitation also increased. This precipitation became so appreciable that samples had to be passed
through a macroscopic filter aid prior to passage through 0.45 µm cellulose acetate membranes.
15
This macroscopic precipitation also decreased the loading. The competing factors of increased
loading and increased macroscopic precipitation resulted in a maximum loading when the feed
was 40% (Figure 5).
Nanoparticle Characterization. Although a detailed examination of the effect of chain length
of the Pt(IV) carboxylate complex on nanoparticle size and morphology was outside the scope of
this study, measurements were made to confirm that the nanoparticles displayed characteristics
consistent with the formulation provided above. The nanoparticles were imaged by TEM and the
results are depicted in Figure 6. The nanoparticles appear as roughly circular black shapes, approximately 50 nm in diameter.
Figure 6. Transmission electron micrograph of PLGA-PEG-COOH nanoparticles loaded with
10. Scale bar of main image = 500 nm, scale bar of inset = 100 nm.
Conclusion
This work presents a systematic investigation of the effect of ligand lipophilicity on the encapsulation of Pt(IV) prodrugs within PLGA-PEG-COOH nanoparticles. A procedure was optimized for encapsulation of a series of compounds spanning a wide range of lipophilicities. We
find that, as the chain length increases, there is a direct and predictable increase in the degree to
16
which the platinum complex is encapsulated. With the most readily encapsulated compound, 10,
the effect of platinum feed during nanoprecipitation was investigated. With an increase in concentration of platinum used in the nanoprecipitation, encapsulation also increased but so too did
the amount of macroscopic precipitation. At higher feeds, macroscopic precipitation outweighed
the increase in encapsulation. An intermediate feed level of 40% allowed for maximum loading
of platinum complex 10 into the nanoparticles.
ASSOCIATED CONTENT
Supporting Information. Spectroscopic and crystallographic characterization of 2 – 10, detailed
crystallographic parameters for the structure of 2·2DMSO and 3, selected bond lengths and angles of 2·2DMSO and 3, LC-MS chromatograms of 2 – 6. Crystallographic information for
2·2DMSO and 3 are also provided in CIF format. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
* email: lippard@mit.edu
ACKNOWLEDGMENTS
This work was supported by grant CA034992 from the National Cancer Institute. Spectroscopic
instrumentation at the MIT DCIF is maintained with funding from NIH Grant 1S10RR13886-01.
T.C.J. received partial funding from the Harvard/MIT CCNE, NIH grant 5-U54-CA151884. We
17
acknowledge Dr. Suresh Gadde (Farokhzad lab, Harvard Medical School) for preparing the
PLGA-PEG-COOH used in this study.
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Hansch, C.; Fujita, T. J. Am. Chem. Soc. 1964, 86, 1616-1626.
Farokhzad, O. C.; Jon, S. Y.; Khademhosseini, A.; Tran, T. N. T.; LaVan, D. A.; Langer,
R. Cancer Res. 2004, 64, 7668-7672.
Fonseca, C.; Simoes, S.; Gaspar, R. J. Control. Release 2002, 83, 273-286.
19
ToC SYNOPSIS
A series of Pt(IV) carboxylate complexes were prepared to investigate the effect of ligand lipophilicity on encapsulation of Pt(IV) prodrugs within polymeric nanoparticles. A linear relationship exists between these parameters. The effect of increased platinum concentration on the degree of encapsulation afforded a set of optimal conditions that provide a prodrug loading maximum of 7%.
ToC GRAPHIC
20
S1
Supporting Information for
The Effect of Ligand Lipophilicity on the
Nanoparticle Encapsulation of Pt(IV) Prodrugs
Timothy C. Johnstone and Stephen J. Lippard*
Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA, 02139
Contents
Characterization of 2 – 10
Table S1. Refinement parameters of 2·2DMSO.
Table S2. Refinement parameters of 3.
Table S3. Selected bond lengths and angles for 2·2DMSO.
Table S4. Selected bond lengths and angles for 3.
Figures S1 – S18. 1H (500 MHz, DMSO-d6) and 13C (125 MHz, DMSO-d6) NMR Spectra of 2 –
10.
Figure S19. Molecular diagrams of 2·2DMSO and 3.
Figure S20. LC-MS chromatograms for 2 to 6.
S2
Characterization of 2 – 10
Compound 2. cis,cis,trans-[Pt(NH3)2Cl2(OOCCH3)2]: Pale yellow solid, 222 mg (88%), mp. (dec) 245 –
250 °C. 1H NMR (500 MHz, DMSO-d6) δ = 1.89 (s, 6H), 6.52 (s, 6H); 13C NMR (125.7 MHz, DMSO-d6)
δ = 22.94, 178.31; IR (KBr, cm–1) 3270 (m), 3221 (m), 3080 (m), 1653 (s), 1429 (w), 1364 (s), 1300 (s),
1273 (s), 1023 (w), 943 (w), 702 (m); ESI-MS (negative ion mode) m/z = [M–H]– 418.2 (calc. 418.0),
[M+TFA]– 530.8 (calc. 531.0); Anal. Calc. for C4H12Cl2N2O4Pt: C 11.49, H 2.89, N 6.70. Found: C 11.97,
H 2.87, N 6.65.
Compound 3. cis,cis,trans-[Pt(NH3)2Cl2(OOCCH2CH3)2]: Pale yellow solid, 202 mg (76%), mp. (dec)
260 – 265 °C. 1H NMR (500 MHz, DMSO-d6) δ = 0.94 (t, 6H, 7.5 Hz), 2.24 (q, 4H, 7.5 Hz), 6.53 (s, 6H);
13
C NMR (125.7 MHz, DMSO-d6) δ = 10.08, 28.73, 181.45; IR (KBr, cm–1) 3230 (br m), 3101 (m), 2984
(w), 2945 (w), 1648 (s), 1575 (m), 1461 (w), 1416 (w), 1362 (m), 1241 (s), 1077 (w), 1014 (w), 810 (w),
671 (m); ESI-MS (negative ion mode) m/z = [M–H]– 445.0 (calc. 445.0), [M+TFA]– 558.7 (calc. 559.0);
Anal. Calc. for C6H16Cl2N2O4Pt: C 16.15, H 3.61, N 6.28. Found: C 16.17, H 3.56, N 6.22.
Compound 4. cis,cis,trans-[Pt(NH3)2Cl2(OOC(CH2)2CH3)2]: Off-white solid, 201 mg (71%), mp. (dec)
270 – 274 °C. 1H NMR (500 MHz, DMSO-d6) δ = 0.87 (t, 6H, 7.5 Hz), 1.47 (sext, 4H, 7.3 Hz), 2.19 (t,
4H, 7.25 Hz), 6.53 (s, 6H); 13C NMR (125.7 MHz, DMSO-d6) δ = 13.70, 18.86, 37.66, 180.77; IR (KBr,
cm–1) 3335 (m), 3193 (br s), 3092 (m), 2963 (m), 2933 (m), 2873 (m), 1662 (s), 1628 (s), 1567 (m), 1458
(w), 1412 (w), 1382 (m), 1293 (br s), 1216 (m), 1094 (w), 954 (w), 669 (w); ESI-MS (negative ion mode)
m/z = [M–H]– 472.9 (calc. 473.0), [M+TFA]– 585.9 (calc. 586.0); Anal. Calc. for C8H20Cl2N2O4Pt: C
20.26, H 4.25, N 5.91. Found: C 20.04, H 4.01, N 5.93.
Compound 5. cis,cis,trans-[Pt(NH3)2Cl2(OOC(CH2)3CH3)2]: Off-white solid, 220 mg (73%), mp. (dec)
201 – 203 °C. 1H NMR (500 MHz, DMSO-d6) δ = 0.85 (t, 6H, 7.3 Hz), 1.28 (sext, 4H, 7.4 Hz), 1.43
(quin, 4H, 7.5 Hz), 2.21 (t, 4H, 7.5 Hz), 6.53 (s, 6H); 13C NMR (125.7 MHz, DMSO-d6) δ = 13.83, 21.75,
27.60, 35.42, 180.89; IR (KBr, cm–1) 3261 (s), 3213 (s), 3110 (m), 2960 (s), 2934 (s), 2873 (m), 1659 (s),
1623 (s), 1584 (s), 1539 (m), 1464 (w), 1418 (w), 1372 (m), 1327 (s), 1289 (s), 1216 (s), 1103 (w), 946
(m), 890 (w), 798 (w), 762 (m), 715 (m), 443 (w); ESI-MS (negative ion mode) m/z = [M–H]– 501.7
S3
(calc. 501.3), [M+TFA]– 614.7 (calc. 615.1); Anal. Calc. for C10H24Cl2N2O4Pt: C 23.91, H 4.82, N 5.58.
Found: C 23.48, H 4.65, N 5.43.
Compound 6. cis,cis,trans-[Pt(NH3)2Cl2(OOC(CH2)4CH3)2]: Off-white solid, 240 mg (75%), mp. (dec)
194 – 196 °C. 1H NMR (500 MHz, DMSO-d6) δ = 0.85 (t, 6H, 6.8 Hz), 1.25 (m, 8H), 1.45 (quin, 4H, 7.4
Hz), 2.20 (t, 4H, 7.5 Hz), 6.52 (s, 6H); 13C NMR (125.7 MHz, DMSO-d6) δ = 13.90, 21. 96, 25.12, 30.84,
35.65, 180.88; IR (KBr, cm–1) 3257 (m), 3205 (m), 3118 (m), 2954 (m), 2931 (m), 2871 (w), 1658 (s),
1622 (m), 1584 (m), 1540 (w), 1466 (w), 1417 (w), 1373 (m), 1330 (m), 1287 (m), 1255 (m), 1215 (m),
1106 (w), 949 (w), 713 (w); ESI-MS (negative ion mode) m/z = [M–H]– 529.4 (calc. 529.1), [M+TFA]–
642.7 (calc. 643.1); Anal. Calc. for C12H28Cl2N2O4Pt: C 27.18, H 5.32, N 5.28. Found: C 26.94, H 5.19, N
5.27.
Compound 7. cis,cis,trans-[Pt(NH3)2Cl2(OOC(CH2)5CH3)2]: Off-white solid, 197 mg (60%), mp. (dec)
185 – 191 °C. 1H NMR (500 MHz, DMSO-d6) δ = 0.86 (t, 6H, 6.8 Hz), 1.25 (m, 12H), 1.44 (quin, 4H, 7.3
Hz), 2.20 (t, 4H, 7.5 Hz), 6.52 (s, 6H); 13C NMR (125.7 MHz, DMSO-d6) δ = 15.13, 23.18, 26.57, 29.46,
32.31, 36.86, 182.03; IR (KBr, cm–1) 3256 (s), 3201 (s), 2957 (s), 2929 (s), 2857 (m), 1658 (s), 1617 (s),
1582 (s), 1466 (m), 1415 (m), 1372 (s), 1333 (s), 1284 (s), 1242 (s), 1208 (s), 1107 (w), 951 (w), 888 (w),
725 (w), 676 (w); ESI-MS (negative ion mode) m/z = [M–H]– 557.1 (calc. 557.1), [M+TFA]– 670.9 (calc.
671.1); Anal. Calc. for C14H32Cl2N2O4Pt: C 30.11, H 5.78, N 5.02. Found: C 30.60, H 5.76, N 5.19.
Compound 8. cis,cis,trans-[Pt(NH3)2Cl2(OOC(CH2)6CH3)2]: Off-white solid, 226 mg (64%), mp. (dec)
186 – 191 °C. 1H NMR (500 MHz, DMSO-d6) δ = 0.85 (t, 6H, 6.9 Hz), 1.24 (m, 16H), 1.44 (quin, 4H, 7.2
Hz), 2.20 (t, 4H, 7.5 Hz), 6.52 (s, 6H); 13C NMR (125.7 MHz, DMSO-d6) δ = 13.97, 22.09, 25.45, 28.58,
28.59, 31.21, 35.69, 180.87; IR (KBr, cm–1) 3247 (m), 3205 (m), 3110 (m), 2955 (m), 2927 (s), 2855 (m),
1658 (s), 1618 (m), 1583 (m), 1537 (w), 1467 (w), 1414 (w), 1375 (m), 1331 (m), 1287 (m), 1234 (m),
1205 (m), 1109 (w), 724 (w); ESI-MS (negative ion mode) m/z = [M–H]– 585.4 (calc. 585.2), [M+TFA]–
698.9 (calc. 698.2); Anal. Calc. for C16H36Cl2N2O4Pt: C 32.77, H 6.19, N 4.78. Found: C 32.79, H 6.16, N
4.69.
S4
Compound 9. cis,cis,trans-[Pt(NH3)2Cl2(OOC(CH2)7CH3)2]: White solid, 270 mg (73%), mp. (dec) 186
– 191 °C. 1H NMR (500 MHz, DMSO-d6) δ = 0.84 (t, 6H, 6.9 Hz), 1.24 (m, 20H), 1.44 (quin, 4H, 7.2
Hz), 2.19 (t, 4H, 7.5 Hz), 6.51 (s, 6H); 13C NMR (125.7 MHz, DMSO-d6) δ = 13.97, 22.12, 25.45, 28.645,
28.650, 28.89, 31.29, 35.69, 180.86; IR (KBr, cm–1) 3246 (m), 3201 (m), 3114 (w), 2956 (m), 2925 (s),
2854 (m), 1664 (m), 1618 (m), 1577 (w), 1532 (w), 1467 (w), 1377 (m), 1315 (m), 1293 (m), 1227 (m),
1201 (m), 1111 (w), 949 (w), 723 (w); ESI-MS (negative ion mode) m/z = [M–H]– 613.5 (calc. 613.2),
[M+TFA]– 726.9 (calc. 727.2); Anal. Calc. for C18H40Cl2N2O4Pt: C 35.18, H 6.56, N 4.56. Found: C
35.25, H 6.21, N 4.60.
Compound 10. cis,cis,trans-[Pt(NH3)2Cl2(OOC(CH2)8CH3)2]: White solid, 264 mg (69%), mp. (dec)
185 – 190 °C. 1H NMR (500 MHz, DMSO-d6) δ = 0.85 (t, 6H, 6.7 Hz), 1.24 (m, 24H), 1.44 (quin, 4H, 7.0
Hz), 2.19 (t, 4H, 7.5 Hz), 6.52 (s, 6H); 13C NMR (125.7 MHz, DMSO-d6) δ = 13.97, 22.12, 25.46, 28.65,
28.72, 28.95, 28.96, 31.33, 35.70, 180.86; IR (KBr, cm–1) 3244 (m), 3201 (m), 3110 (w), 2955 (m), 2922
(s), 2853 (m), 1661 (m), 1618 (m), 1580 (w), 1532 (w), 1468 (w), 1411 (w), 1376 (m), 1332 (m), 1292
(m), 1223 (m), 1198 (m), 1112 (w), 950 (w), 723 (w); ESI-MS (negative ion mode) m/z = [M–H]– 641.2
(calc. 641.2), [M+TFA]– 759.9 (calc. 755.2); Anal. Calc. for C20H44Cl2N2O4Pt: C 37.38, H 6.90, N 4.36.
Found: C 37.38, H 7.08, N 4.71.
X-ray Crystallography. Diffraction quality crystals of 2 were grown by room temperature vapor
diffusion of diethyl ether into a DMSO solution of the compound. Crystals of 3 were grown by room
temperature vapor diffusion of diethyl ether into a DMF solution of the compound. Suitable crystals were
selected and mounted on a nylon cryoloop in Paratone oil and cooled to 100 K under a stream of nitrogen.
A Bruker APEX CCD X-ray diffractometer controlled by the APEX2 software1 was used to collect data
using graphite-monochromated Mo Kα radiation (λ = 0.71073 Å). The data were integrated with SAINT2
and absorption, Lorentz, and polarization corrections were calculated by SADABS.3 The space group of
the crystal of 2·2DMSO was determined by analyzing the Laue symmetry and systematic absences of the
diffraction pattern with XPREP.4 Using the SHELXTL-97 software package,5,6 the structure was solved by
the heavy atom method and refined against F2 using standard procedures.7 All non-hydrogen atoms were
S5
located on difference Fourier maps and refined anisotropically. Hydrogen atoms were placed at calculated
positions for coordinated NH3 and terminal CH3 groups and refined with their isotropic displacement
parameters (Uiso) set equal to 1.5 times the Uiso of the atom to which they were attached. One of the
DMSO molecules present in the asymmetric unit displays a pyramidal inversion disorder, equivalent to a
180° rotation about the long axis of the molecule, as commonly encountered.8 The hydrogen atoms of this
disordered solvent molecule were not included in the final model. Analysis of the Laue symmetry of the
crystal of 3 revealed that it was twinned by merohedry. An initial solution was obtained with data
preliminarily detwinned using XPREP. Subsequent refinement was performed as described above and the
twin law (0 1 0) (1 0 0) (0 0 -1) was included in the final model. CIF data are provided in the Supporting
Information along with tables of bond lengths and angles (Tables S1-S4). The structures, deposited in the
Cambridge Structural Database, were checked for missed higher symmetry and twinning with PLATON9
and were further validated using CheckCIF.
(1)
(2)
(3)
(4)
(5)
(6)
(7)
(8)
(9)
APEX2, 2008-4.0; Bruker AXS, Inc: Madison, WI, 2008.
SAINT: SAX Area-Detector Integration Program, 2008/1; University of Göttingen: Göttingen,
Germany, 2008.
Sheldrick, G. M. SADABS: Area-Detector Absorption Correction, University of Gottingen:
Gottingen, Germany, 2008.
XPREP, 2008/2; Bruker AXS: Madison, WI, 2008.
Sheldrick, G. M. SHELXTL-97, University of Göttingen: Göttingen, Germany, 2000.
Sheldrick, G. M. Acta Crystallogr. A 2008, 64, 112-122.
Müller, P. Crystallogr. Rev. 2009, 15, 57-83.
Cruz-Cabeza, A. J.; Day, G. M.; Jones, W. Phys. Chem. Chem. Phys. 2011, 13, 12808-12816.
Spek, A. L. PLATON, A Multipurpose Crystallographic Tool, Utrecht University: Utrecht, The
Netherlands, 2008.
S6
Table S1. X-Ray Crystallographic Refinement Parameters of 2·2DMSO.
___________________________________________________________________________________
Empirical formula
Formula weight
Temperature
Wavelength
Crystal system
Space group
Unit cell dimensions
C8H18Cl2N2O6PtS2
568.35
100(2) K
0.71073 Å
Triclinic
P
a = 7.2454(7) Å
α = 72.516(2) °
b = 10.2263(10) Å
β = 81.044(2) °
c = 14.0657(14) Å
γ = 69.485(2) °
3
Volume
929.51(16) Å
Z
2
3
Density (calculated)
2.031 Mg/m
Absorption coefficient
8.081 mm-1
F(000)
544
3
Crystal size
0.11 x 0.04 x 0.04 mm
Theta range for data collection
1.52 to 28.77°
Index ranges
-9≤h≤9, -13≤k≤13, -19≤l≤18
Reflections collected
19028
Independent reflections
4781 [R(int) = 0.0280]
Completeness to theta = 28.77°
99.0 %
Absorption correction
Semi-empirical from equivalents
Max. and min. transmission
0.7381 and 0.4701
2
Refinement method
Full-matrix least-squares on F
Data / restraints / parameters
4781 / 0 / 206
2
Goodness-of-fit on F
1.308
Final R indices [I>2sigma(I)]
R1 = 0.0342, wR2 = 0.0879
R indices (all data)
R1 = 0.0365, wR2 = 0.0912
-3
Largest diff. peak and hole
3.121 and -2.772 e Å
____________________________________________________________________
S7
Table S2. X-Ray Crystallographic Refinement Parameters of 3.
___________________________________________________________________________________
Empirical formula
Pt
Formula weight
Temperature
Wavelength
Crystal system
Space group
Unit cell dimensions
C6H16Cl2N2O4
446.20
100(2) K
0.71073 Å
Tetragonal
P43
a = 9.2357(3) Å
c = 13.9563(11) Å
Volume
1190.45(11) Å3
Z
4
3
Density (calculated)
2.490 Mg/m
Absorption coefficient
12.232 mm-1
F(000)
840
3
Crystal size
0.08 x 0.08 x 0.04 mm
Theta range for data collection
1.46 to 28.80°
Index ranges
-12≤h≤12, -12≤k≤12, -18≤l≤18
Reflections collected
24400
Independent reflections
3098 [R(int) = 0.0322]
Completeness to theta = 28.77°
100.0 %
Absorption correction
Semi-empirical from equivalents
Max. and min. transmission
0.6404 and 0.4411
2
Refinement method
Full-matrix least-squares on F
Data / restraints / parameters
3098 / 1 / 141
2
Goodness-of-fit on F
1.065
Final R indices [I>2sigma(I)]
R1 = 0.0167, wR2 = 0.0371
R indices (all data)
R1 = 0.0168, wR2 = 0.0371
-3
Largest diff. peak and hole
0.889 and -0.531 e Å
___________________________________________________________________
S8
Table S3. Selected Bond Lengths and Angles for 2·2DMSO.
_____________________________________________________
Pt(1)-O(21)
Pt(1)-N(1)
Pt(1)-N(2)
Pt(1)-O(11)
Pt(1)-Cl(2)
Pt(1)-Cl(1)
O(11)-C(11)
O(12)-C(11)
O(21)-C(21)
O(22)-C(21)
O(21)-Pt(1)-N(1)
O(21)-Pt(1)-N(2)
N(1)-Pt(1)-N(2)
O(21)-Pt(1)-O(11)
N(2)-Pt(1)-Cl(2)
O(11)-Pt(1)-Cl(2)
N(2)-Pt(1)-Cl(1)
O(11)-Pt(1)-Cl(1)
Cl(2)-Pt(1)-Cl(1)
C(11)-O(11)-Pt(1)
O(12)-C(11)-O(11)
O(12)-C(11)-C(12)
1.999(5)
2.034(5)
2.038(5)
2.050(4)
2.3211(17)
2.3286(16)
1.285(8)
1.233(9)
1.288(9)
1.229(10)
94.8(2)
90.9(2)
93.5(2)
175.5(2)
87.30(17)
93.64(14)
178.17(17)
87.89(13)
91.48(7)
125.7(4)
126.6(7)
120.8(6)
_____________________________________________________________
S9
Table S4. Selected Bond Lengths and Angles for 3.
_____________________________________________________
Pt(1)-O(11)
Pt(1)-O(21)
Pt(1)-N(2)
Pt(1)-N(1)
Pt(1)-Cl(2)
Pt(1)-Cl(1)
O(11)-Pt(1)-O(21)
O(11)-Pt(1)-N(2)
O(21)-Pt(1)-N(2)
O(11)-Pt(1)-N(1)
O(21)-Pt(1)-N(1)
C(21)-O(21)-Pt(1)
C(11)-O(11)-Pt(1)
O(22)-C(21)-O(21)
O(22)-C(21)-C(22)
2.013(4)
2.028(4)
2.039(6)
2.050(6)
2.2969(16)
2.3203(17)
171.34(14)
97.5(2)
87.6(2)
89.0(2)
97.9(2)
123.8(4)
123.4(4)
124.3(6)
123.8(6)
_____________________________________________________________
S10
Figure S1. 1H NMR Spectrum (500 MHz, DMSO-d6) of 2.
S11
Figure S2. 13C NMR Spectrum (125 MHz, DMSO-d6) of 2.
S12
Figure S3. 1H NMR Spectrum (500 MHz, DMSO-d6) of 3.
S13
Figure S4. 13C NMR Spectrum (125 MHz, DMSO-d6) of 3.
S14
Figure S5. 1H NMR Spectrum (500 MHz, DMSO-d6) of 4.
S15
Figure S6. 13C NMR Spectrum (125 MHz, DMSO-d6) of 4.
S16
Figure S7. 1H NMR Spectrum (500 MHz, DMSO-d6) of 5.
S17
Figure S8. 13C NMR Spectrum (125 MHz, DMSO-d6) of 5.
S18
Figure S9. 1H NMR Spectrum (500 MHz, DMSO-d6) of 6.
S19
Figure S10. 13C NMR Spectrum (125 MHz, DMSO-d6) of 6.
S20
Figure S11. 1H NMR Spectrum (500 MHz, DMSO-d6) of 7.
S21
Figure S12. 13C NMR Spectrum (125 MHz, DMSO-d6) of 7.
S22
Figure S13. 1H NMR Spectrum (500 MHz, DMSO-d6) of 8.
S23
Figure S14. 13C NMR Spectrum (125 MHz, DMSO-d6) of 8.
S24
Figure S15. 1H NMR Spectrum (500 MHz, DMSO-d6) of 9.
S25
Figure S16. 13C NMR Spectrum (125 MHz, DMSO-d6) of 9.
S26
Figure S17. 1H NMR Spectrum (500 MHz, DMSO-d6) of 10.
S27
Figure S18. 13C NMR Spectrum (125 MHz, DMSO-d6) of 10.
S28
Figure S19. Molecular Structures of 2 (left) and 3 (right). Ellipsoids are drawn at the 50%
probability level.
S29
Figure S20. LC-MS Chromatograms for 2 - 6 (conditions are described in the main text).