Bulky N (,N)-(Di)alkylethane-1,2-diamineplatinum(II) compounds as precursors for generating unsymmetrically substituted platinum(IV) complexes

Article abstract of DOI:10.1021/ic400816g

Investigations of the influence of bulky groups in the equatorial ligand sphere of platinum(IV) compounds on the complexes' stability and reaction pattern were performed. Four dihydroxidoplatinum(IV) complexes were reacted with anhydrides, cinnamoyl chloride, and n-propyl isocyanate and yielded the symmetric dicarboxylated products or, if steric hindrance was observed, unsymmetrically substituted monocarboxylated analogues. With the aim of raising the steric demand, the following ligands were chosen: N-cyclohexylethane-1,2- diamine, N,N-dimethylethane-1,2-diamine, N,N-diethylethane-1,2-diamine, and N,N-diisopropylethane-1,2-diamine. All of the novel complexes were characterized by electrospray ionization mass spectrometry (ESI-MS), one- and two-dimensional NMR spectroscopy, elemental analysis, and reversed-phase HPLC; complexes B3, C3, C6, and D4 were also analyzed by X-ray diffraction. Additionally, the cytotoxicities of 10 compounds toward the cisplatin-sensitive cell line CH1 and the intrinsically cisplatin-resistant cell lines A549 and SW480 were investigated, and IC₅₀ values down to the nanomolar range were found. To aid in the interpretation of structure-activity relationships, log k _w values as a measure for the lipophilicity were determined for all of the new complexes, and the rates of reduction of C1, C3, and C4 relative to satraplatin were determined by means of NMR spectroscopy and ESI-MS.

Full text of DOI:10.1021/ic400816g

Article
pubs.acs.org/IC
Bulky N(,N)‑(Di)alkylethane-1,2-diamineplatinum(II) Compounds as
Precursors for Generating Unsymmetrically Substituted Platinum(IV)
Complexes
Verena Pichler,† Simone Goschl,† Samuel M. Meier,†,‡ Alexander Roller,† Michael A. Jakupec,†,‡
̈
Markus Galanski,*,†,‡ and Bernhard K. Keppler*,†,‡
†Institute of Inorganic Chemistry, University of Vienna, Wahringer Straße 42, A-1090 Vienna, Austria
̈
‡Research Platform “Translational Cancer Therapy Research”, University of Vienna, Wahringer Straße 42, A-1090 Vienna, Austria
̈
S
* Supporting Information
ABSTRACT: Investigations of the influence of bulky groups in the
equatorial ligand sphere of platinum(IV) compounds on the
complexes’ stability and reaction pattern were performed. Four
dihydroxidoplatinum(IV) complexes were reacted with anhydrides,
cinnamoyl chloride, and n-propyl isocyanate and yielded the
symmetric dicarboxylated products or, if steric hindrance was
observed, unsymmetrically substituted monocarboxylated analogues.
With the aim of raising the steric demand, the following ligands were
chosen: N-cyclohexylethane-1,2-diamine, N,N-dimethylethane-1,2-
diamine, N,N-diethylethane-1,2-diamine, and N,N-diisopropyl-
ethane-1,2-diamine. All of the novel complexes were characterized
by electrospray ionization mass spectrometry (ESI-MS), one- and
two-dimensional NMR spectroscopy, elemental analysis, and
reversed-phase HPLC; complexes B3, C3, C6, and D4 were also analyzed by X-ray diffraction. Additionally, the cytotoxicities
of 10 compounds toward the cisplatin-sensitive cell line CH1 and the intrinsically cisplatin-resistant cell lines A549 and SW480
were investigated, and IC50 values down to the nanomolar range were found. To aid in the interpretation of structure−activity
relationships, log kw values as a measure for the lipophilicity were determined for all of the new complexes, and the rates of
reduction of C1, C3, and C4 relative to satraplatin were determined by means of NMR spectroscopy and ESI-MS.
INTRODUCTION
■
Platinum(II) compounds, namely, cisplatin, carboplatin, and
oxaliplatin, belong to an extremely successful group of
chemotherapeutics in worldwide clinical application.1,2
Although these anticancer platinum(II) complexes are widely
used in the treatment of cancer, certain limitations have been
encountered, mainly related to their toxicity, low kinetic
inertness, application in a limited number of cancer types
because of intrinsic drug resistance, and development of
acquired resistance.3−8
The development of platinum(IV) prodrugs is a promising
approach to overcome the mentioned limitations. To date, four
platinum(IV) complexes have entered clinical trials (Figure 1),
but none of them has been approved for clinical use. (OC-6-
Figure 1. Platinum(IV) complexes that have entered clinical trials.
33)-Dichloridodihydroxidobis(isopropylamine)platinum(IV)
Prednisone Against Refractory Cancer (SPARC) phase-III trial
showed no significant improvement in overall survival rates
compared with standard treatment.11 (OC-6-43)-Bis(acetato)-
(adamantylamine)amminedichloridoplatinum(IV) (LA-12) of-
fers a quite similar ligand sphere compared to satraplatin, and
(iproplatin, CHIP, JM9) exhibited high solubility coupled with
low toxicity but was abandoned because of its lack of activity.9
Dose-limiting neurotoxicity led to the abandonment of clinical
trials of (OC-6-22)-tetrachlorido(trans-cyclohexane-1,2-
diamine)platinum(IV) (tetraplatin, ormaplatin).10 (OC-6-43)-
Bis(acetato)amminedichlorido(cyclohexylamine)platinum(IV)
(satraplatin, JM216) is currently the most advanced compound
in different phase-I and -II trials, but the Satraplatin and
Received: April 9, 2013
© XXXX American Chemical Society
A
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clinical trials are currently in progress or have been
finished.12−14 The high kinetic inertness of platinum(IV)
complexes is the reason for a range of advantages compared
with the more labile platinum(II) complexes. They include (i)
an extension of synthetic pathways known from organic
chemistry, (ii) possible oral availability, and (iii) reduced side
effects.15 Interactions with biomolecules take place mainly via
reduction of the platinum(IV) prodrug to platinum(II) species.
These platinum(II) drugs then exhibit the same reactivity as
related platinum(II) compounds (i.e., binding to thiol-
containing molecules is observed within seconds).16,17
The most promising platinum(IV) compounds, satraplatin
and LA-12, share a sterically demanding amine ligand at an
equatorial position. Another effective platinum complex
offering a bulky ligand is picoplatin (ZD0473, JM472,
AMD473), which showed less binding to glutathione or
methionine compared with cisplatin. This behavior is
attributable to the bulky picoline ligand that protects the
platinum center from axial attack, thereby reducing side
reactions with biomolecules in the human body.18 As recently
reported, steric effects may dictate the synthetic access to
platinum(IV) compounds. For example, employing N,N-
dimethylethane-1,2-diamine as a chelating equatorial ligand in
(OC-6-43)-dichlorido(N,N-dimethylethane-1,2-diamine)-
dihydroxidoplatinum(IV) leads to selective monocarboxylation
in the reaction of this complex with succinic anhydride.19
In this work, we examined the influence of increasing the size
of the alkyl group(s) positioned on one of the equatorial
amines on the synthesis, stability, and lipophilicity of
symmetrically (X−Pt−X) and unsymmetrically (X−Pt−Y)
trans-substituted platinum(IV) complexes. It has to be stated
that the unsymmetric trans substitution in all cases generated a
chiral platinum center, and therefore, all of the unsymmetrically
substituted complexes were asymmetric and present as racemic
mixtures. Four platinum(II) precursors A−D based on the
ligands N,N-diisopropylethane-1,2-diamine, N,N-diethylethane-
1,2-diamine, N,N-dimethylethane-1,2-diamine, and N-cyclo-
hexylethane-1,2-diamine, respectively (Figure 2), were oxidized
hexanone and N-Boc-ethane-1,2-diamine, reduction of the
imine and cleavage of the N-Boc protecting group gave amine
S2 in 82% yield. The starting platinum(II) complexes A−D
(Figure 2; also see the Supporting Information) and the
platinum(IV) complexes C1 and C2 (Figure 3) were
synthesized according to standard literature procedures.19,24
All of the platinum(II) precursors were oxidized in aqueous
H2 O2 solution at room temperature, and the
dihydroxidoplatinum(IV) complexes A1−D1 (Figure 3) were
obtained in good yields (55−99%). Compound A1 was
unstable in solution, most probably because of the bulky
isopropyl groups, and therefore, further reactions could not be
performed (Figure S1 in the Supporting Information). The
stable dihydroxido compounds B1, C1, and D1 were
investigated with regard to their reactivity toward succinic
anhydride, acetic anhydride, cinnamoyl chloride, and n-propyl
isocyanate, yielding symmetrically and unsymmetrically sub-
stituted compounds (see the overview in Figure 3).
The reactions of dihydroxido complexes B1 and C1 with a 4-
fold excess of succinic anhydride led to the monocarboxylated
compounds B2 and C2 in excellent yields (Scheme 1). The
diacetato complexes B4, C4, and D4 were observed when acetic
anhydride was used as the solvent (>100 equiv excess)
(Scheme 2). When the amount of acetic anhydride was
reduced to 2 equiv, monoadducts B3 and C3 were isolated in
excellent yields of 75−99%. In case of the satraplatin-related
educt D1, a selective unsymmetric synthesis could not be
performed.
Changing the reactant to cinnamoyl chloride (Scheme 3)
demonstrated more clearly the influence of steric hindrance of
the equatorial ligand. The ethyl groups of N,N-diethylethane-
1,2-diamine blocked one of the axial OH groups most
effectively, as a small excess (2.1 equiv) led to the unsym-
metrically substituted compound B5. A small amount of
symmetric byproduct B6 (<10% yield; see the Supporting
Information) was formed by increasing the excess to 4 equiv,
but no complete turnover could be accomplished. Nevertheless,
the synthesis of a mixed dicarboxylated product was performed
by suspending B5 in excess acetic anhydride, which yielded B9
in excellent yield (84%). Reaction of the less sterically hindered
methyl derivative C1 and even a small excess of cinammoyl
chloride led to the dicarboxylated compound C6 (∼65% yield).
However, when equi- to dimolar amounts of the reagents were
used, the corresponding monocarboxylation product C5 could
be obtained, but further separation with column chromatog-
raphy was necessary to remove minor amounts of the
symmetrically substituted byproduct. Once again, only the
dicarboxylated derivative D6 was found for the satraplatin
analogue D1.
For the most reactive species, n-propyl isocyanate, a 2- or
1.2-fold excess led to the monocarboxylated products B7 and
C7. Increasing the excess of reagent to >100 equiv yielded
dicarboxylated products B8 (see the Supporting Information)
and C8 (Scheme 4). The latter complex was obtained in good
yield, whereas for B8 a yield of only ∼20% was detected.
Furthermore, the compound could not be separated on silica, as
it decomposed under normal-phase chromatographic condi-
tions.
Figure 2. Structures of platinum(II) building blocks A−D. The
structure of JM118 is shown for comparison.
to the trans-hydroxidoplatinum(IV) analogues and further
reacted with succinic or acetic anhydride,20,21 cinnamoyl
chloride,22 or n-propyl isocyanate23 in accord with recently
published procedures. Furthermore, the mono- and disub-
stituted complexes were investigated with respect to their
cytotoxic potency in three human cancer cell lines, namely,
CH1 (ovarian carcinoma), A549 (non-small-cell lung carcino-
ma), and SW480 (colon adenocarcinoma). Additionally, the
rates of reduction of three selected complexes were determined.
RESULTS AND DISCUSSION
In summary, it can be stated that the steric hindrance of the
chelating equatorial ligand increases in the following order: N-
cyclohexylethane-1,2-diamine (series D) < N,N-dimethyl-
ethane-1,2-diamine (series C) < N,N-diethylethane-1,2-diamine
(series B) < N,N-diisopropylethane-1,2-diamine (series A). In
■
Syntheses. All of the chelating diamines were obtained
from commercial suppliers except for N-cyclohexylethane-1,2-
diamine (S2), which was synthesized in two steps (see the
Supporting Information): after imine formation using cyclo-
B
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Figure 3. Schematic overview of the investigated complexes, including their yields. Abbreviations: succ = 3-carboxypropanoato; oac = acetato; cin =
(2E)-3-phenylprop-2-enoato; carb = propylcarbamoato.
Scheme 1. Reactivity of Dihydroxidoplatinum(IV)
Compounds toward Succinic Anhydride
Scheme 3. Reactivity of Dihydroxidoplatinum(IV)
Compounds toward Cinnamoyl Chloride
a
the sterically most demanding cases, unstable compounds were
obtained, in particular A1 and B8. It seems that increasing the
bulkiness at the equatorial position increases the ease with
which monocarboxylated products can be synthesized.
However, this facilitation of the synthesis of unsymmetrically
substituted products is associated with a decrease in compound
stability.
Characterization. All of the novel metal complexes were
characterized by NMR spectroscopy, electrospray ionization
mass spectrometry (ESI-MS), reversed-phase HPLC (RP-
HPLC), and elemental analysis, and complexes B3, C3, C6,
and D4 were also analyzed by X-ray crystallography.
a
Conditions: (I) 2 to 2.1 equiv of cinnamoyl chloride, pyridine,
acetone; (II) 4 equiv of cinnamoyl chloride, pyridine, acetone; (III)
acetic anhydride.
1
The H, 13C, 15N, and 195Pt NMR resonances were in the
expected ranges; 195Pt, 15N, and selected 1H chemical shifts are
listed in Table S1 in the Supporting Information. In the H
NMR spectrum, the NH signals of the equatorially coordinated
N(,N)-(di)alkylethane-1,2-diamine ligands were detected be-
1
Scheme 2. Reactivity of Dihydroxidoplatinum(IV) Compounds toward Acetic Anhydride
C
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Scheme 4. Reactivity of Dihydroxidoplatinum(IV) Compounds toward n-Propyl Isocyanate
Figure 4. ORTEP views of B3, C3, C6, and D4 with atom-labeling schemes. Thermal ellipsoids are drawn at the 50% probability level.
Table 1. Crystal Data and Details of Data Collection for B3, C3, C6, and D4
B3
C3
C6
D4
empirical formula
formula weight
space group
a (Å)
C8H20Cl2N2O3Pt
458.25
C6H16Cl2N2O3Pt
430.20
C22H26Cl2N2O4Pt
648.44
C12H24Cl2N2O4Pt
526.32
triclinic, P1
monoclinic, P21/c
7.8017(5)
12.6113(8)
11.9901(7)
−
triclinic, P1
monoclinic, P21/c
10.3015(3)
22.4208(8)
7.8210(3)
−
̅
̅
7.3751(4)
7.7180(4)
12.7856(9)
101.004(2)
96.950(3)
109.822(2)
658.33(7)
2
7.8654(6)
12.8074(11)
13.3055(12)
65.149(4)
72.991(4)
85.489(4)
1161.51(17)
2
b (Å)
c (Å)
α (deg)
β (deg)
γ (deg)
V (Å3)
98.306(2)
111.494(1)
−
−
1167.33(13)
4
1680.77(10)
4
Z
λ (Å)
0.71073
0.71073
0.71073
0.71073
ρcalcd (g cm−3
)
2.312
2.448
1.854
2.080
crystal size (mm3)
0.10 × 0.04 × 0.03
100(2)
0.15 × 0.04 × 0.02
100(2)
0.10 × 0.10 × 0.03
100(2)
0.15 × 0.15 × 0.10
100(2)
T (K)
μ (mm−1
)
11.057
12.463
6.301
8.681
a
R1
0.0155
0.0232
0.0370
0.0249
b
wR2
0.0340
0.0629
0.0716
0.0538
c
GOF
1.008
1.026
0.963
1.036
a
b
c
2
2
2
R1 = ∑||Fo| − |Fc||/∑|Fo|. wR2 = {∑[w(Fo − Fc2)2]/∑[w(Fo )2]}1/2. GOF = {∑[w(Fo − Fc2)2]/(n − p)}1/2, where n is the number of
reflections and p is the total number of parameters refined.
tween 6.9 and 10.7 ppm, strongly depending on the axial ligand
sphere. In the case of the dihydroxidoplatinum(IV) compounds
A1−C1, the NH signals appeared as broad singlets at ∼7 ppm,
whereas the signals of the dicarboxylated compounds B4, B6,
C4, and C6 were shifted to lower field at ∼9.5 ppm. The
unsymmetrically substituted compounds B2, B3, B5, C2, C3,
and C5 featured a splitting of the NH signals into two broad
singlets as a result of the chiral platinum center, one at ∼7 ppm
and the other at ∼9.5 ppm. Changing the carboxylato group to
carbamate (complexes B7, B8, C7, and C8) led to a downfield
shift of the corresponding signals to 10.0−10.6 ppm. Therefore,
these signals are suitable for differentiating between mono- and
D
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Table 2. Cytotoxicities toward Three Human Cancer Cell Lines
a
IC50 (μM)
IC50 ratio
complex
CH1
A549
SW480
A549/CH1
SW480/CH1
B1
B2
B3
B4
B7
C3
C4
C7
C8
D4
2.5 0.2
9.6 3.2
105 35
239 31
45 12
150 26
5.5 1.3
2.6 0.2
1.7 0.1
42
25
36
42
42
43
75
26
83
18
34
17
8
18
16
7
0.84 0.12
0.72 0.11
0.26 0.06
1.8 0.4
30
30
11
9
3
1
4
7
77 13
65 14
23
6
13
5
0.87 0.10
1.0 0.3
4.4 0.7
5.8 0.7
0.47 0.03
4.2 0.3
115 10
75 13
26
9.9 1.4
16
2
6
0.12 0.03
0.89 0.15
3.3 1.4
4
2
5
b
C1
111 15
145 39
1.3 0.4
35
9
b
C2
8.4 1.0
b
cisplatin
0.16 0.03
3.5 0.3
22
a
Half-maximal inhibitory concentrations in the MTT assay (96 h exposure). Values are means standard deviations obtained from at least three
b
independent experiments. Data taken from ref 19.
bisderivatization. Furthermore, all of the carbamate-containing
complexes showed two signals for the CONH proton in a ratio
of ∼4:1 due to the generation of conformational isomers.23
This effect could also be studied in the 195Pt NMR spectrum. In
the case of D8, four platinum signals and two sets of 1H signals
were observed, as the combination of the chiral center of the N-
cyclohexylethane-1,2-diamine ligand and the conformational
isomers of the carbamate group led to diastereoisomers.
Heating of the sample resulted in the reduction to one set of
signals (Figure S2 in the Supporting Information).
sphere with the general formula [cis,cis,trans-PtIVCl2N2O2]. The
bond distances are close to 2.3 Å for Pt−Cl and 2.0 Å for Pt−N
and Pt−O. These findings are in accordance with similar
structures reported in the literature.26,27 The ethane-1,2-
diamine ligands form a five-membered PtN2C2 chelate cycle
with the Pt(IV) center. The ΘN1−C1−C2−N2 torsion angle, which
serves as a measure of the deviation of the chelate ring from
planarity, is −56.9(2)° in B3, 56(3)° in C3, 51.8(9)° in C6, and
−53.8(3)° in D4.
The steric effects of the N(,N)-(di)alkylethane-1,2-diamine
ligands are also reflected in the X-ray structures. Increasing the
length of the alkyl chain leads to deformation of the square-
planar platinum core. The Cl−Pt−Nalkyl angle increases in
going from N-cyclohexylethane-1,2-diamine [D4, 93.58(8)°]
and N,N-dimethylethane-1,2-diamine [C3, 92.93(16)°; C6,
93.79(15)°] to N,N-diethylethane-1,2-diamine [B3, 95.44(5)°],
whereas on the other side, the Cl−Pt−NH2 angle decreases in
going from D4 [92.11(7)°] to C3 [90.3(2)°] to C6
[89.25(14)°] to B3 [88.60(6)°]. The remaining angles, Cl−
Pt−Cl and NH2−Pt−Nalkyl, are barely influenced. The extent of
the Cl−Pt−Nalkyl angle seems to correlate with the instability of
the platinum(IV) complex in this series.
A common observation in the crystal structures is the
presence of intramolecular hydrogen bonds between the axial
oxygens and the equatorial NH2 of the ligand. In the
symmetrically substituted compound C6, the amine nitrogen
N1 is involved in intramolecular hydrogen bonding to oxygen
atoms O2 and O4 (donor−acceptor contacts of 2.730 and
2.726 Å, respectively), whereas in the case of D4, one hydrogen
bond is located between N1 and O2 (N1···O2, 2.691 Å) and
the second between N2 and O4 (N2···O4, 2.705 Å). In the two
unsymmetrically substituted complexes B3 and C3, only one
hydrogen bond between N1 and O2 is formed. The axial OH
ligand enables further intermolecular hydrogen bonds, which
are found in both cases between coordinated hydroxido
oxygens and N1.
Partition Coefficients. The partition coefficient (kw),
which is the ratio of the concentrations of a drug in lipophilic
and hydrophilic phases, can describe the lipophilicity of a
complex and consequently aid in predicting a part of its
pharmacokinetic behavior in a living organism. In general,
passive diffusion through a membrane bilayer is easier for a
compound with increasing lipophilic character. A clear-cut
All of the measured 195Pt chemical shifts were in the expected
range and in accordance with literature values indicating a
PtIVCl2N2O2 geometry.25,26 Furthermore, the 195Pt NMR
spectra displayed differences between the dihydroxido,
monocarboxylated, and dicarboxylated products. For the
dihydroxido compounds A1, B1, and C1, the 195Pt chemical
shifts were 2412, 2493, and 2764 ppm, respectively.
Consequently, the platinum shift relocation increased with
increasing size of the sterically demanding ligand. In the case of
system B based on the N,N-diethyl chelating ligand, the 195Pt
shift started in the 2578−2587 ppm range indicating
monocarboxylation (B2, B3, and B5), while a shift of 2819−
2615 ppm indicated dicarboxylation (compounds B4 and B6).
Changing the chelating equatorial ligand to N,N-dimethyl-
ethane-1,2-diamine (system C) resulted in an upfield shift of
∼90 ppm for the monohydroxido (C2 and C3, ∼2500 ppm)
and dicarboxylato complexes (B4 and B9, ∼2800 ppm). The
resonances of D4, D6, and D8 were in the range 2742−2812
ppm without an observable influence of the axial ligands.
15N signals were found for the monocarboxylato compounds
between −4.0 and −6.8 ppm and for the dicarboxylato
compounds between −16.6 and −20.5 ppm. Once more,
complexes bearing the chelating ligand including cyclohexyl-
amine did not conform to this behavior, as their 15N shifts
appeared at ∼9.0 ppm.
Crystal Structures. Results of the X-ray diffraction studies
on single crystals of B3, C3, C6, and D4 are shown in Figure 4.
Crystal data, data collection parameters, and structure refine-
ment details are given in Table 1. Selected bond lengths and
angles are listed in Table S2 in the Supporting Information.
Complexes C6/B3 and C3/D4 crystallized in the triclinic space
group P1 and the monoclinic space group P2 /c, respectively.
̅
1
The platinum(IV) centers feature an octahedral coordination
E
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relationship between lipophilicity and drug accumulation was
also reported for platinum anticancer agents.28,29 In recent
times, RP-HPLC measurements have increasingly been used to
determine the lipophilicity of a drug, as the method is highly
reproducible, fast, and independent of concentration. As the
chromatographic system requires an organic modifier (in our
case methanol), extrapolation to 0% methanol is essential to
obtain the value of log kw, which contains information about the
lipophilicity of the compound.27,30,31
Values of log kw for the synthesized compounds are shown in
Table S3 in the Supporting Information. The value for complex
A1 could not be determined because of the instability of the
complex. The investigated set of compounds covers a wide
range of log kw values from 0.05 for the least lipophilic
compound (C3) to 4.42 for the most lipophilic compound
(C6). In general, the lipophilic character increases with the
alkyl-chain length in the equatorial ligand: N,N-dimethylethane-
1,2-diamine (series C) < N,N-diethylethane-1,2-diamine (series
B) < N-cyclohexylethane-1,2-diamine (series D). Furthermore,
compounds with log kw > 2.84 showed insufficient solubility for
cell-culture studies.
cisplatin-resistant cell lines A549 and SW480 to those for the
cisplatin-sensitive cell line CH1, it became obvious that
cytotoxicities of the platinum(IV) complexes were affected to
different extents relative to cisplatin. A549 cells were more
resistant to all of the platinum(IV) complexes (A549/CH1 IC50
ratios = 18−83) than to cisplatin (A549/CH1 IC50 ratio = 8).
Furthermore, A549 cells were more resistant to dicarboxylated
platinum(IV) complexes than to monocarboxylated ones (e.g.,
the A549/CH1 IC50 ratios for C3 and C4 were 43 and 75,
respectively). In contrast, SW480 cells were less resistant to the
dicarboxylated complexes than to compounds with a Pt−OH
group (e.g., SW480/CH1 IC50 ratio = 13 for C3 vs 5 for C4).
The investigated complexes had SW480/CH1 IC50 ratios of 4−
18, indicating that the extent to which SW480 resistance affects
the potency of these complexes is generally less than that for
cisplatin (SW480/CH1 IC50 ratio = 22).
Cytotoxicity versus log kw. Semilogarithmic plots of log
kw versus IC50 (Figure S3 in the Supporting Information)
showed a very rough correlation for the tested complexes. As
the more lipophilic compounds always showed increased
cytotoxicity, we suggest that there is an influence of
lipophilicity, although it is neither strong nor strictly linear.32
Especially compound B1 constitutes an exception. These
results are in good accordance with previously published
results showing that the terminal group of the axial ligand has a
conspicuous influence on the cytotoxicity of platinum(IV)
compounds33 and that correlation of log kw with IC50 is rather
poor.34
Cytotoxicities toward Human Cancer Cell Lines. Ten
compounds with sufficient aqueous solubility were investigated
with respect to their cytotoxicities toward three human cancer
cell lines (CH1, A549, and SW480) by means of a colorimetric
microculture (MTT) assay, and the results were compared with
those for cisplatin and the previously published compounds C1
and C2 (Table 2).19
All of the tested complexes showed a strong capacity to
inhibit growth of the cisplatin-sensitive CH1 cell line, with half-
maximal inhibitory concentration (IC50) values in the range
9.6−0.12 μM. In particular, B7 and C8 showed antiproliferative
activity in nanomolar concentrations and were as potent as
cisplatin. In general, the dicarboxylated complexes were more
active than their respective monocarboxylated counterparts
(e.g., C8 vs C7 and C4 vs C3), presumably because of their
greater lipophilic character. However, the influence of lip-
ophilicity on the cytotoxicity was not always observable,
particularly with respect to increasing the alkyl-chain length
of the N(,N)-(di)alkylethane-1,2-diamine chelating ligand. The
IC50 values for the diacetato compounds B4, C4, and D4 were
found in a small range of 0.72−0.89 μM, although their log kw
values varied broadly from 0.7 to 2.22.
Rates of Reduction. As Wexselblatt and co-workers
recently showed, the rate of reduction of platinum(IV)
complexes is dependent on the axial ligands, at least in the
case of oxaliplatin derivatives.15,35 To investigate whether
similar behavior exists for platinum(IV) complexes containing
an equatorial N2Cl2 coordination sphere, the rates of reduction
of complexes C1, C3, and C4 and the reference compound
satraplatin (Figure 5) were measured by 1H NMR spectroscopy
and ESI-MS.
A similar activity pattern was seen in SW480 cells. Except for
B1, B2, and C3, the tested compounds were equally as potent
as or more potent than cisplatin, with IC50 values as low as 0.47
μM (C8).
Figure 5. Complexes used to investigate the rate of reduction.
The intrinsically cisplatin-resistant A549 cell line seemed to
be indifferent to the mono- and dicarboxylated products, as
compounds of each matched pair (B3 vs B4 and C3 vs C4)
showed similarly moderate cytotoxicities with IC50 values of
∼30 and ∼70 μM, respectively. An exception to this was the
carbamate pair C7 and C8, where the monocarbamate yielded
an IC50 value ∼2.5 times higher than the dicarbamate.
Interestingly, when only the influence of the equatorial chelate
ligand was considered, the cytotoxicity toward this cell line
decreased in the order N-cyclohexylethane-1,2-diamine (series
D) > N,N-diethylethane-1,2-diamine (series B) > N,N-
dimethylethane-1,2-diamine (series C), in accordance with
decreasing lipophilicity. However, as expected, the least
cytotoxic compound, B2, featured a free carboxylic group,
which is in accordance with previous findings.19,26,27 Upon
comparison of the ratios of the IC50 values for the intrinsically
A clear trend in the reduction rates was observed in 1H NMR
experiments depending on the axial ligands (Figure 6). During
these experiments, the metal complex was reacted with ascorbic
acid at a 1:2 molar ratio and a final metal concentration of 0.5
mM. The dihydroxido compound C1 was reduced compara-
tively slowly within a few days. The monohydroxido complex
C3 showed an increased rate of reduction with a half-life of ∼14
h. Intriguingly, the diacetato complex C4 displayed a half-life of
∼30 min and was completely reduced within 4 h. Although
satraplatin features an analogous ligand sphere, it showed a half-
life of ∼6 h for reduction. Varbanov et al.34 showed that a
compound similar to C4 and satraplatin has a half-life of ∼5 h,
which corresponds to the rate of reduction of satraplatin rather
than C4. The ∼10-fold increase in the reduction rate for C4
compared with satraplatin could be a result of diminished
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⧫
■
●
▲
Figure 6. Time courses for the reductions of compounds C1 ( ), C3 ( ), C4 ( ), and satraplatin ( ) incubated with a 2-fold excess of ascorbic
acid in phosphate buffer.
Figure 7. (A) Excerpt of the positive-ion-mode ESI mass spectrum of an incubation solution containing NaAsc and C3 (molar ratio 2:1) after 72 h.
(B) ESI mass spectra showing the time evolution of the incubation solution containing ascorbate and C4 (molar ratio 2:1). The experimental ESI
mass signals include a standard deviation of m/z 0.05. Simulations of isotopic distributions are shown in gray. Abbreviations: en* = N,N-
dimethylethane-1,2-diamine; ac = acetate; asc = ascorbate.
stability due to the bulky methyl groups. Consequently, the
significantly increased reduction rate of C4 suggests that N,N-
dialkylation of the equatorial amine allows fine-tuning of the
reduction rates of diacetatoplatinum(IV) complexes.
The monoacetato complex C3 has a reduction rate between
those of the diacetato and dihydroxido complexes, in agreement
with literature data for platinum(IV) complexes featuring
chlorido ligands.15 In summary, the following trend in the rates
of reduction was observed: C4 > C3 ≈ satraplatin > C1.
ESI-MS experiments confirmed the results of the NMR
measurements, and a similar trend in the rates of reduction of
the platinum(IV) compounds by sodium ascorbate (NaAsc)
was observed. In these experiments, the complexes were
reacted with NaAsc at an identical molar ratio as in the NMR
experiments. The final metal concentration in the reaction
mixture was 50 μM. The compounds were stable in aqueous
solution in the absence of NaAsc for at least 72 h, and
characteristic mass signals were observed, typically correspond-
ing to [M + H]+ or [M + Na]+, where M is C1, C3, C4, or
satraplatin. In addition, ESI-MS allowed the identification of
some of the species formed upon reduction of the platinum-
(IV) complexes.
In the ESI-MS experiments, C1 was not reduced by NaAsc,
and the detected mass signals were identical to those observed
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during stability testing (Figure S4 in the Supporting
Information). On the other hand, C3 was reduced to a
significant extent by NaAsc, and several distinct mass signals
were detected with a characteristic platinum isotopic
distribution (Figure 7A). The mass spectrum recorded
immediately after preparation of the incubation solution
revealed a peak for [(en*)PtCl(OH) + H]+ (en* = N,N-
between. However, to understand the differences in the
chemical and biological behavior in more detail, further
investigations have to be performed.
EXPERIMENTAL SECTION
■
Materials and Methods. All of the solvents and reagents were
obtained from commercial suppliers and were used as received, except
for methanol, which was dried using standard procedures. K2PtCl4 was
obtained from Johnson Matthey (Switzerland). Reverse-osmosis water
was doubly distilled before use. For column chromatography, silica gel
60 (Fluka) was used. The starting platinum(II) compounds (SP-4-3)-
dichlorido(N,N-diisopropylethane-1,2-diamine)platinum(II) (A), (SP-
4-3)-dichlorido(N,N-diethylethane-1,2-diamine)platinum(II) (B),
(SP-4-3)-dichlorido(N,N-dimethylethane-1,2-diamine)platinum(II)
(C), and (SP-4-3)-dichlorido(N-cyclohexylethane-1,2-diamine)-
platinum(II) (D) were synthesized according to literature proce-
dures.24,36 The platinum(IV) complexes C1 and C2 were also
synthesized using previously published procedures.19
dimethylethane-1,2-diamine) at m/z 336.95
0.05 (mtheor =
337.04 Da, 19%) (all intensities are given relative to the most
abundant signal in the mass spectrum). This is indicative of a
rapidly initiated reduction process that is accompanied by the
loss of the axial acetato ligands and hydrolysis of one Pt−Cl
bond. It must be noted that hydrolysis is generally not observed
for kinetically inert platinum(IV) complexes. The latter signal
increased to 83% relative to the parent mass signal [C3 + Na]+
after 6 h. After 72 h, the most prominent Pt-containing mass
signal corresponded to [Pt(asc) + Na]+ (m/z 393.94 0.05,
mtheor = 393.99 Da), indicating even the loss of the chelating
diamine ligand under the conditions of the ESI-MS measure-
ment.
Finally, C4 was quickly reduced by NaAsc (Figure 7B) and
even after 10 min, the most abundant peak in the mass
spectrum corresponded to [(en*)PtCl(OH) + H]+ (m/z
336.94 0.05, mtheor = 337.04, 100%), as similarly observed
with C3. In the same spectrum, the parent mass signal [C4 +
Na]+ was detected at 63% relative abundance. After 72 h,
[(en*)PtCl(OH) + H]+ (100%) and [Pt(asc) + Na]+ (82%)
were detected as thermodynamically stable species, as observed
in the case of C3. The ESI-MS experiments also confirmed that
satraplatin was reduced at a lower rate than C4, although the
two complexes feature similar ligand spheres. However, mass
signals assignable to reduction products were observed only
during the initial 6 h in minor amounts before all of the signals
slowly vanished, indicating decomposition of satraplatin.
Finally, glutathione was not able to reduce any of the
compounds during a period of 72 h.
1H, 13C, 15N, and 195Pt one- and two-dimensional NMR spectra
were recorded with a Bruker Avance III 500 MHz instrument at
500.32 (1H), 125.81 (13C), 50.70 (15N), and 107.55 (195Pt) MHz or
500.10 (1H), 125.75 (13C), 50.68 (15N), and 107.51 (195Pt) MHz in
perdeuterated dimethyl sulfoxide (DMSO-d6) at 298 K. The solvent
residual peaks for 1H and 13C were used as internal references, whereas
195Pt chemical shifts were referenced to external K2PtCl4 and 15N
chemical shifts to external NH4Cl. ESI-MS was carried out with a
Bruker esquire3000 ion trap using MeOH as the solvent. Elemental
analyses were performed by the Microanalytical Laboratory of the
University of Vienna using a PerkinElmer 2400 CHN elemental
analyzer.
Syntheses. General Procedure I: Oxidation of Pt(II) Complexes.
The platinum(II) complex was suspended in H2O, and 30% H2O2 was
added. The reaction mixture was stirred at room temperature (rt).
Subsequently, the solvent was removed under reduced pressure and
the residue was washed with ice-cold ethanol and diethyl ether.
(OC-6-43)-Dichlorido(N,N-diisopropylethane-1,2-diamine)-
dihydroxidoplatinum(IV) (A1). General procedure I was used with
platinum(II) complex A (0.05 g, 0.12 mmol), H2O (15 mL), H2O2
(0.5 mL), and stirring for 4 h. A1 was not stable in solution (Figure S1
in the Supporting Information). Yield: 47 mg (89%). Elemental
analysis for C8H22Cl2N2O2Pt·0.1KCl: calcd C 21.27, H 4.91, N 6.20;
found C 21.09, H 4.54, N 5.84. 1H NMR (DMSO-d6): δ 7.06 (bs, 2H,
NH2); 4.15 (m, 2H, CH); 2.97 (m, 2H, CH2CH2); 2.72 (bs, 2H,
CH2CH2); 1.37 (t, 12H, CH3, J = 7.0 Hz). 195Pt NMR (D2O): δ 2746.
(OC-6-43)-Dichlorido(N,N-diethylethane-1,2-diamine)-
dihydroxidoplatinum(IV) (B1). General procedure I was used with
platinum(II) complex B (0.20 g, 0.52 mmol), H2O (5 mL), H2O2 (1
mL), and stirring for 2.5 h. Yield: 0.18 g (83%). Elemental analysis for
C6H18Cl2N2O2Pt·0.5H2O: calcd C 16.95, H 4.50, N 6.59; found C
CONCLUSION
■
A series of symmetrically and unsymmetrically substituted
platinum(IV) complexes was synthesized in order to investigate
the barrier to synthesis caused by N(,N)-(di)alkylethane-1,2-
diamine chelating ligands. The equatorial dimethylamine unit
hampered the reaction between reagents and axial Pt−OH
groups. This effect was stronger when the methyl substituents
were replaced with ethyl substituents, whereas no effect was
observed in the case of a cyclohexylamine group. However, two
isopropyl substituents led to an unstable compound. The
influence on the reaction pattern was highly replicable, and the
steric hindrance could be overcome by using more reactive
reagents such as acyl halides or isocyanides or an enormous
excess of less reactive anhydrides. The set of compounds was
furthermore investigated with respect to cytotoxicity, lip-
ophilicity, and rate of reduction. The most active compounds
showed promising IC50 values in the low micromolar to
nanomolar concentration range toward three cancer cell lines
(CH1, SW480, and A549), which are comparable to or better
than those of cisplatin. A very rough correlation between
cytotoxicity and lipophilicity was observed, showing only a
minor influence of this parameter. The rate of reduction clearly
changed depending on the axial ligands in the case of an
N2Cl2Pt ground core. The dicarboxylated complex was reduced
extremely rapidly relative to the dihydroxido counterpart; the
half-life of the monocarboxylated analogues was situated in
1
16.61, H 4.29, N 6.34. H NMR (DMSO-d6): δ 6.95 (bs, 2H, NH2),
3.65 (m, 2H, N(CH2CH3)2), 2.76 (bm, 4H, N(CH2CH3)2 + NCH2),
2.64 (bs, 2H, NH2CH2), 1.19 (t, 6H, CH3, J = 7.0 Hz). 13C NMR
(DMSO-d6): δ 63.6 (NCH2), 48.3 (N(CH2CH3)2), 44.6 (NH2CH2),
9.3 (N(CH2CH3)2). 15N NMR (DMSO-d6): δ −1.9. 195Pt NMR
(DMSO-d6): δ 2493.
(OC-6-43)-Dichlorido(N-cyclohexylethane-1,2-diamine)-
dihydroxidoplatinum(IV) (D1). General procedure I was used with
platinum(II) complex D (0.55 g, 1.35 mmol), H2O (15 mL), H2O2
(5.5 mL), and stirring for 7 h. Yield: 0.32 g (55%). Elemental analysis
for C8H20Cl2N2O2Pt: calcd C 21.73, H 4.56, N 6.33; found C 21.39, H
4.17, N 5.97. Insoluble in H2O, N,N-dimethylformamide (DMF),
MeOH, and DMSO.
(OC-6-54)-(3-Carboxypropanoato)dichlorido(N,N-diethylethane-
1,2-diamine)hydroxidoplatinum(IV) (B2). B1 (100 mg; 0.24 mmol)
and succinic anhydride (96 mg; 0.96 mmol) in DMF (2 mL) were
stirred at 40 °C for 18 h. The solvent was removed at 40 °C under
reduced pressure to form a yellow oil. The residue was suspended in
acetone, and the yellow solid was filtered off, washed with acetone and
diethyl ether, and dried in vacuo. The acetone phase was reduced to
H
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half of the volume, and a second fraction was precipitated using diethyl
ether. Yield: 110 mg (89%). Elemental analysis for
C10H22N2O5Cl2Pt·0.5H2O: calcd C 22.87, H 4.41, N 5.33; found C
22.79, H 4.16, N 5.18. 1H NMR (DMSO-d6): δ 12.06 (bs, 1H,
COOH), 9.36 (bs, 1H, NH2), 7.11 (bs, 1H, NH2), 3.65 (m, 1H,
N(CH2CH3)2), 3.44 (m, 1H, N(CH2CH3)2), 3.01−2.87 (bm, 2H,
NCH2CH2N), 2.83 (m, 1H, N(CH2CH3)2), 2.78−2.70 (bm, 2H,
NCH2CH2N), 2.68 (m, 1H, N(CH2CH3)2), 2.49−2.37 (bm, 4H,
PtOOCCH2CH2), 1.66 (bs, 1H, OH), 1.23 (t, 3H, N(CH2CH3)2), J =
7.0 Hz); 1.17 (t, 3H, N(CH2CH3)2, J = 7.0 Hz). 13C NMR (DMSO-
d6): δ 181.2 (PtOOC), 174.6 (COOH), 64.2 (NCH2CH2N), 50.7
(N(CH2CH3)2), 48.7 (N(CH2CH3)2), 44.7 (NCH2CH2N), 32.7
(CH2CH2COOH), 30.4 (CH2CH2COOH), 9.6 (N(CH2CH3)2), 8.7
(N(CH2CH3)2). 15N NMR (DMSO-d6): δ −6.9. 195Pt NMR (DMSO-
d6): δ 2582. ESI-MS (MeOH) m/z: (pos) 516.8 [M + H]+, 538.7 [M
+ Na]+; (neg) 514.6 [M − H]−, 1029.0 [2M − H]−.
(81%). Elemental analysis for C8H18Cl2N2O4Pt: calcd C 20.35, H 3.84,
N 5.93; found C 20.16, H 3.89, N 5.66. 1H NMR (DMSO-d6): δ 9.47
(bs, 2H, NH2), 2.96 (s, 4H, NCH2CH2N), 2.64 (s, 6H, N(CH3)2),
1.95 (s, 6H, COCH3). 13C NMR (DMSO-d6): δ 179.8 (CO), 68.8
(NH2CH2), 50.0 (N(CH3)2), 45.7 (CH3NCH2), 24.4 (COCH3). 15N
NMR (DMSO-d6): δ −18.1. 195Pt NMR (DMSO-d6): δ 2717. ESI-MS
(MeOH) m/z: (pos) 472.8 [M + H]+, 494.8 [M + Na]+, 510.7 [M +
K]+; (neg) 470.3 [M − H]−, 506.6 [M + Cl]−.
(OC-6-43)-Diacetatodichlorido(N-cyclohexylethane-1,2-
diamine)platinum(IV) (D4). General procedure III was used with D1
(125 mg, 0.28 mmol), acetic anhydride (10 mL), and stirring for 10 h.
Yield: 98 mg (66%). Elemental analysis for C12H24Cl2N2O4Pt·0.5H21O:
calcd C 26.92, H 4.71, N 5.23; found C 26.57, H 4.34, N 5.05. H
NMR (DMSO-d6): δ 10.03 (bs, 1H, NH), 9.79 (bs, 1H, NH), 8.13
(bs, 1H, NH), 3.61 (t, 1H, cyclohexyl CH, J = 11 Hz), 2.86−2.63 (m,
4H, CH2CH2), 2.00 (bs, 2H, cyclohexyl CH2), 1.95 (s, 3H, CH3), 1.91
(s, 3H, CH3), 1.75 (bs, 2H, cyclohexyl CH2), 1.67−1.59 (m, 2H,
cyclohexyl CH2), 1.36−1.28 (m, 2H, cyclohexyl CH2), 1.25−1.18 (m,
1H, cyclohexyl CH2), 1.13−1.05 (m, 1H, cyclohexyl CH2). 13C NMR
(DMSO-d6): δ 181.4 (CO), 180.5 (CO), 60.5 (cyclohexyl CH), 51.1
(CH2CH2), 47.2 (CH2CH2), 31.5 (cyclohexyl CH2), 28.0 (cyclohexyl
CH2), 25.6 (cyclohexyl CH2), 25.6 (cyclohexyl CH2), 25.2 (cyclohexyl
CH2), 24.0 (CH3), 23.8 (CH3). 15N NMR (DMSO-d6): δ −9.5 (NH2),
21.5 (NH). 195Pt NMR (DMSO-d6): δ 2768. ESI-MS (MeOH) m/z:
(pos) 429.2 [C8H17N2Cl2Pt + Na]+, 548.2 [M + Na]+; (neg) 526.2 [M
− H]−, 561.0 [M + Cl]−.
General Procedure II. B1 or C1 was suspended in DMF, and acetic
anhydride was added. The solution was stirred at rt and quenched with
a small amount of H2O, and the crude product was precipitated by
addition of diethyl ether. The precipitate was filtered, washed with
diethyl ether, and dried under reduced pressure.
(OC-6-54)-Acetatodichlorido(N,N-diethylethane-1,2-diamine)-
hydroxidoplatinum(IV) (B3). General procedure II was used with B1
(100 mg, 0.24 mmol), DMF (4 mL), acetic anhydride (49 mg, 0.48
mmol), and stirring for 4 h. Yield: 109 mg (99%). Elemental analysis
for C8H20Cl2N2O3Pt·0.8DMF: calcd C 24.17, H 4.99, N 7.59; found C
1
(OC-6-54)-Dichlorido(N,N-diethylethane-1,2-diamine)hydroxido-
[(2E)-3-phenylprop-2-enoato]platinum(IV) (B5). B1 (100 mg, 0.24
mmol) was suspended in dry acetone (8 mL), and pyridine (190 mg,
2.50 mmol) was added. The reaction mixture was heated to 50 °C,
after which cinnamoyl chloride (84 mg, 0.51 mmol) dissolved in dry
acetone (2 mL) was added. After 30 min, the reaction mixture was
quenched with H2O, and acetone was removed under reduced
pressure. The aqueous solution was stored in the refrigerator
overnight. and the formed precipitate was filtered off and washed
with cold ethanol and ether. The crude product was recrystallized in
methanol. Yield: 93 mg (71%). Elemental analysis for
C15H24Cl2N2O3Pt·0.5 H2O: calcd C 32.44, H 4.54, N 5.04; found C
24.25, H 4.78, N 7.54. H NMR (DMSO-d6): δ 9.39 (bs, 1H, NH2),
7.13 (bs, 1H, NH2), 3.65 (m, 1H, N(CH2CH3)2), 3.39 (m, 1H,
N(CH2CH3)2), 3.03−2.94 (bm, 2H, NCH2CH2N), 2.84 (m, 1H,
N(CH2CH3)2), 2.75−2.64 (bm, 3H, NCH2CH2N + N(CH2CH3)2),
1.92 (s, 3H, CH3), 1.65 (s+d, 1H, OH, J = 9.5 Hz), 1.24 (t, 3H,
N(CH2CH3)2, J = 7.0 Hz), 1.17 (t, 3H, N(CH2CH3)2, J = 7.0 Hz). 13
C
NMR (DMSO-d6): δ 180.1 (CO), 64.3 (CH2NR2), 50.9 (N-
(CH2CH3)2), 48.8 (N(CH2CH3)2), 44.6 (CH2NH2), 25.1 (COCH3),
9.6 (N(CH2CH3)2), 8.6 (N(CH2CH3)2). 15N NMR (DMSO-d6): δ
−6.8. 195Pt NMR (DMSO-d6): δ 2587. ESI-MS (MeOH) m/z: (pos)
481.0 [M + Na]+; (neg) 456.9 [M − H]−, 492.0 [M + Cl]−.
(OC-6-54)-Acetatodichlorido(N,N-dimethylethane-1,2-diamine)-
hydroxidoplatinum(IV) (C3). General procedure II was used with C1
(0.10 g, 0.26 mmol), DMF (4 mL), acetic anhydride (53 mg, 0.52
mmol), and stirring for 1 h. The crude product was recrystallized in
ethanol. Yield: 83 mg (75%). Elemental analysis for C6H16Cl2N2O3Pt:
1
32.09, H 4.22, N 4.77. H NMR (DMSO-d6): δ 9.45 (bs, 1H, NH2),
7.62 (m, 2H, phenyl CH), 7.39 (m, 3H, phenyl CH), 7.37 (d, 1H,
PtOOCCHCH, J = 16 Hz), 7.22 (bs, 1H, NH2), 6.49 (d, 1H,
PtOOCCHCH, J = 16 Hz), 3.69 (m, 1H, N(CH2CH3)2), 3.39 (m,
1H, N(CH2CH3)2), 3.05 (m, 2H, NCH2CH2N), 2.91 (m, 1H,
N(CH2CH3)2), 2.71 (m, 3H, NCH2CH2N + N(CH2CH3)2), 1.84 (s,
1H, OH), 1.28 (t, 3H, N(CH2CH3)2, J = 7.0 Hz), 1.19 (t, 3H,
N(CH2CH3)2, J = 7.0 Hz). 13C NMR (DMSO-d6): δ 175.3 (PtOOC),
141.5 (PtCOOCHCH), 135.2 (phenyl C), 130.1 (phenyl CH),
129.3 (2 × phenyl CH), 128.3 (2 × phenyl CH), 124.1
(PtCOOCHCH), 64.4 (CH2CH2), 51.0 (CH2CH3), 48.8
(CH2CH3), 44.8 (CH2CH2), 9.7 (CH3), 8.6 (CH3). 15N NMR
(DMSO-d6): δ −5.9. 195Pt NMR (DMSO-d6): δ 2578. ESI-MS
(MeOH) m/z: (pos) 546.8 [M + H]+, 568.8 [M + Na]+, 584.8 [M +
K]+; (neg) 544.6 [M − H]−, 579.7 [M + Cl]−.
1
calcd C 16.75, H 3.75, N 6.51; found C 16.72, H 3.44, N 6.44. H
NMR (DMSO-d6): δ 9.46 (bs, 1H, NH2), 7.10 (bs, 1H, NH2), 2.89−
2.75 (m, 4H, NCH2CH2N), 2.66 (s+d, 3H, N(CH3)2, J = 12 Hz), 2.59
(s+d, 3H, N(CH3)2, J = 13 Hz), 1.92 (s, 3H, COCH3), 1.49 (s+d, 1H,
OH, J = 9.5 Hz). 13C NMR (DMSO-d6): δ 180.1 (CO), 67.9 (CNR2),
50.3 (N(CH3)2), 48.6 (N(CH3)2), 45.1 (CNH2), 24.9 (COCH3). 15N
NMR (DMSO-d6): δ −6.0. 195Pt NMR (DMSO-d6): δ 2503. ESI-MS
(MeOH) m/z: (pos) 468.9 [M + K]+.
General Procedure III. B1, C1, or D1 was suspended in acetic
anhydride and stirred at rt until a clear solution was formed. The
product was precipitated by slow addition of diethyl ether. The
precipitate was filtered and washed with diethyl ether and dried under
reduced pressure.
(OC-6-54)-Dichlorido(N,N-dimethylethane-1,2-diamine)-
hydroxido[(2E)-3-phenylprop-2-enoato]platinum(IV) (C5). C1 (100
mg, 0.26 mmol) and pyridine (122 mg, 1.55 mmol) were suspended in
dry acetone (10 mL), and cinnamoyl chloride (86 mg, 0.52 mmol) in
acetone (5 mL) was added. The reaction mixture was stirred at rt for 5
h, after which the solvent was removed under reduced pressure. The
residue was dissolved in methanol, and the symmetrically substituted
product was separated from the unsymmetrically substituted one by
column chromatography (4:1 MeOH/EE). Yield: 47 mg (35%).
Elemental analysis for C13H20Cl2N2O3Pt: calcd C 30.13, H 3.89, N
(OC-6-43)-Diacetatodichlorido(N,N-diethylethane-1,2-diamine)-
platinum(IV) (B4). General procedure III was used with B1 (100 mg,
0.24 mmol) and acetic anhydride (2 mL). Yield: 105 mg (95%).
Elemental analysis for C10H22Cl2N2O4Pt: calcd C 24.01, H 4.43, N
1
5.60; found C 23.62, H 4.13, N 5.38. H NMR (DMSO-d6): δ 9.64
(bs, 2H, NH2), 3.56 (m, 2H, N(CH2CH3)2), 2.99 (bm, 4H, CH2CH2),
2.72 (m, 2H, N(CH2CH3)2), 1.95 (s, 6H, COCH3), 1.24 (t, 6H,
N(CH2CH3)2, J = 7.0 Hz). 13C NMR (DMSO-d6): δ 180.1 (CO), 65.8
(CH2N(CH2CH3)2), 50.9 (N(CH2CH3)2), 45.0 (CH2NH2), 24.6
(COCH3), 9.0 (N(CH2CH3)2). 15N NMR (DMSO-d6): δ −20.5. 195Pt
NMR (DMSO-d6): δ 2819. ESI-MS (MeOH) m/z: (pos) 522.7 [M +
Na]+, 538.6 [M + K]+; (neg) 498.4 [M − H]−, 534.4 [M + Cl]−.
(OC-6-43)-Diacetatodichlorido(N,N-dimethylethane-1,2-
diamine)platinum(IV) (C4). General procedure III was used with C1
(100 mg, 0.26 mmol) and acetic anhydride (3 mL). Yield: 98 mg
1
5.40; found C 29.85, H 3.58, N 5.30. H NMR (DMSO-d6): δ 9.51
(bs, 1H, NH2), 7.63 (m, 2H, phenyl CH), 7.39 (m, 4H, 3 × (phenyl
CH + OOCCHCH)), 7.23 (bs, 1H, NH2), 6.54 (d, 1H, OOCCH
CH, J = 15.9 Hz), 2.95−2.80 (m, 4H, NCH2CH2N), 2.71 (bs, 3H,
N(CH3)2), 2.64 (bs, 3H, N(CH3)2), 1.66 (s, 1H, OH). 13C NMR
(DMSO-d6): δ 175.4 (PtOOC), 141.5 (PtCOOCHCH), 135.1
(phenyl C), 130.1 (phenyl CH), 129.3 (2 × phenyl CH), 128.3 (2 ×
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phenyl CH), 124.0 (PtCOOCHCH), 68.0 (CH2N(CH3)2), 50.3
(N(CH3)2), 48.6 (N(CH3)2), 45.3 (H2NCH2). 15N NMR (DMSO-
d6): δ −5.4. 195Pt NMR (DMSO-d6): δ 2493. ESI-MS (MeOH) m/z:
(pos) 518.8 [M + H]+, 540.7 [M + Na]+, 556.7 [M + K]+; (neg) 516.5
[M − H]−, 552.6 [M + Cl]−.
2600 (minor). ESI-MS (MeOH) m/z: (pos) 501.8 [M + H]+, 523.1
[M + Na]+, 539.7 [M + K]+; (neg) 499.5 [M − H]−.
(OC-6-54)-Dichlorido(N,N-dimethylethane-1,2-diamine)-
hydroxidopropylcarbamatoplatinum(IV) (C7). C1 (100 mg, 0.26
mmol) was suspended in DMF (3 mL), and n-propyl isocyanate (26
mg, 0.31 mmol) was added. The suspension was then stirred for 10 h
at rt. The solvent was removed under reduced pressure. The residue
was dissolved in MeOH, and the unsymmetrically substituted product
was separated from the symmetrically substituted one by column
chromatography (5:1 MeOH/EE). Yield: 41 mg (42%). Elemental
analysis for C8H21Cl2N3O3Pt: calcd C 20.30, H 4.47, N 8.88; found C
20.00, H 4.16, N 8.56. 1H NMR (DMSO-d6): δ 10.63 (bs, 1H, NH2),
6.96 (bs, 1H, NH2), 6.38 (major) 5.96 (minor) (bs, 1H, OOCNH),
2.87 (bm, 4H, NCH2CH2N), 2.68 (s+d, 3H, N(CH3)2, J = 12.5 Hz),
2.62 (m, 2H, NHCH2), 2.56 (s+d, 3H, N(CH3)2, J = 12.5 Hz), 1.37
(m, 2H, NHCH2CH2), 0.80 (t, 3H, CH3, J = 7.0 Hz). 13C NMR
(DMSO-d6): δ 165.9 (PtOOC), 67.9 (CH2N(CH3)2), 50.2 (N-
(CH3)2), 48.0 (N(CH3)2), 45.0 (NH2CH2), 43.2 (NHCH2), 23.6
(NHCH2CH2), 11.8 (CH3). 15N NMR (DMSO-d6): δ −4.0 (NH2),
66.3 (NH). 195Pt NMR (DMSO-d6): δ 2530 (major), 2519 (minor).
ESI-MS (MeOH) m/z: (pos) 511.8 [M + K]+; (neg) 472.4 [M − H]−,
507.7 [M + Cl]−.
(OC-6-43)-Dichlorido(N,N-dimethylethane-1,2-diamine)bis[(2E)-
3-phenylprop-2-enoato]platinum(IV) (C6). C1 (50 mg, 0.13 mmol)
and pyridine (102 mg, 1.29 mmol) were suspended in dry acetone (10
mL), and cinnamoyl chloride (107 mg, 0.64 mmol) in acetone (5 mL)
was added. The reaction mixture was refluxed for 2 h and subsequently
cooled to rt. The solvent was removed under reduced pressure, and
the residue was suspended in ethanol (96%). The yellow solid was
filtered off, washed with diethyl ether, and dried in vacuo. Yield: 56 mg
(66%). Elemental analysis for C22H26Cl2N2O4Pt: calcd C 40.75, H
4.04, N 4.32; found C 40.69, H 3.89, N 4.17. 1H NMR (DMSO-d6): δ
9.65 (bs, 2H, NH2), 7.68−7.66 (m, 4H, phenyl CH), 7.44 (d, 2H,
OOCCHCH, J = 16 Hz), 7.42−7.40 (m, 6H, phenyl CH), 6.54 (d,
2H, OOCCHCH, J = 16 Hz), 3.06−3.00 (m, 4H, NCH2CH2N),
2.73 (bs, 6H, N(CH3)2). 13C NMR (DMSO-d6): δ 174.9 (PtOOC),
142.5 (PtCOOCHCH), 134.7 (phenyl C), 130.4 (phenyl CH),
129.4 (2 × phenyl CH), 128.6 (2 × phenyl CH), 122.1 (PtCOOCH
CH), 68.9 (NCH2CH2N), 50.2 (N(CH3)2), 45.9 (NCH2CH2N). 15N
NMR (DMSO-d6): δ −16.6. 195Pt NMR (DMSO-d6): δ 2693. ESI-MS
(MeOH/DMSO) m/z: (pos) 648.9 [M + H]+, 670.9 [M + Na]+,
686.8 [M + K]+.
(OC-6-43)-Dichlorido(N,N-dimethylethane-1,2-diamine)bis-
(propylcarbamato)platinum(IV) (C8). C1 (100 mg, 0.26 mmol) was
suspended in dry acetone (5 mL), and n-propyl isocyanate (87 mg,
1.03 mmol) was added. The solution was stirred at rt for 24 h until the
solid was dissolved completely. The solvent was removed under
reduced pressure, and the residue was suspended in acetone (1 mL).
The product was precipitated by addition of ether, filtered off, and
washed with ether. Yield: 94 mg (65%). Elemental analysis for
C12H28Cl2N4O4Pt: calcd C 25.81, H 5.05, N 10.03; found C 25.90, H
(OC-6-43)-Dichlorido(N-cyclohexylethane-1,2-diamine)bis[(2E)-
3-phenylprop-2-enoato]platinum(IV) (D6). D1 (100 mg, 0.23 mmol)
and pyridine (71 mg, 0.91 mmol) were suspended in dry acetone, and
solid cinnamoyl chloride (0.15 g, 0.91 mmol) was added.
Subsequently, the reaction mixture was stirred at rt until a clear
solution was obtained. A small amount of H2O was added to stop the
reaction, and acetone was removed under reduced pressure. The
solution was kept in the refrigerator overnight, and the formed solid
was filtered off and recrystallized in 8:1 MeOH/H2O. Yield: 72 mg
(45%). Elemental analysis for C26H32Cl2N2O4Pt: calcd C 44.45, H
4.59, N 3.99; found C 44.40, H 4.55, N 3.82. 1H NMR (DMSO-d6): δ
10.21 (bs, 1H, NH2), 9.94 (bs, 1H, NH2), 8.31 (bs, 1H, NH), 7.66 (m,
4H, phenyl CH), 7.49 (d, 1H, CHCH, J = 16.0 Hz), 7.41 (d, 1H,
CHCH, J = 16.0 Hz), 7.40 (m, 6H, phenyl CH), 6.55 (d, 1H, CH
CH, J = 16.0 Hz), 6.49 (d, 1H, CHCH, J = 16.0 Hz), 3.75 (t, 1H,
cyclohexyl CH, J = 11.0 Hz), 2.88−2.72 (m, 4H, CH2CH2), 2.08 (m,
2H, cyclohexyl CH2), 1.70 (m, 3H, cyclohexyl CH2), 1.60 (m, 1H,
cyclohexyl CH2), 1.31 (m, 3H, cyclohexyl CH2), 1.09 (m, 1H,
cyclohexyl CH2). 13C NMR (DMSO-d6): δ 176.5 (CO), 175.7 (CO),
142.9 (PtCOOCHCH), 142.8 (PtCOOCHCH), 134.7 (phenyl
C), 134.6 (phenyl C), 130.5 (phenyl CH), 129.4 (phenyl CH), 129.4
(phenyl CH), 128.6 (phenyl CH), 128.5 (phenyl CH), 121.4
(PtCOOCHCH), 60.8 (cyclohexyl CH), 51.38 (CH2CH2), 47.6
(CH2CH2), 31.7 (cyclohexyl CH2), 28.3 (cyclohexyl CH2), 25.6
1
4.75, N 9.71. H NMR (DMSO-d6): δ 10.29 (bs, 2H, NH2), 6.67 +
6.20 (bs, 2H, OOCNH), 2.89 (m, 4H, NHCH2), 2.89−2.87 (m, 4H,
NCH2CH2N), 2.61 (bs, 6H, N(CH3)2), 1.36 (m, 4H, NHCH2CH2),
0.80 (t, 6H, CH3, J = 7.5 Hz). 13C NMR (DMSO-d6): δ 164.5
(PtOOC), 68.4 (NHCH2), 49.2 (N(CH3)2), 45.3 (NCH2CH2N), 43.2
(NCH2CH2N), 23.3 (NHCH2CH2), 11.8 (CH3). 15N NMR (DMSO-
d6): δ −9.3 (NH2), 67.4 (CONH). 195Pt NMR (DMSO-d6): δ 2720
(major), 2707 (minor). ESI-MS (MeOH) m/z: (pos) 558.9 [M + H]+,
580.8 [M + Na]+.
(OC-6-43)-Dichlorido(N-cyclohexylethane-1,2-diamine)bis-
(propylcarbamato)platinum(IV) (D8). To a stirred suspension of D1
(105 mg, 0.24 mmol) in DMF (1 mL) was added n-propyl isocyanate
(1 mL), and after 3.5 h, the solvent was removed under reduced
pressure. The residue was dissolved in MeOH, and a solid was
precipitated by EtOAc. For complete precipitation, hexane was added
and the suspension was stored overnight at 4 °C. The yellow product
was filtered off, washed with Et2O, and dried in vacuo. Yield: 93 mg
(64%). Elemental analysis for C16H34Cl2N4O4Pt·0.6H2O: calcd C
1
(cyclohexyl CH2), 25.6 (cyclohexyl CH2), 25.22 (cyclohexyl CH2). 15
N
30.83, H 5.69, N 8.99; found C 30.46, H 5.28, N 8.65. H NMR
NMR (DMSO-d6): δ −8.2 (NH2), 22.7 (NH). 195Pt NMR (DMSO-
d6): δ 2743. ESI-MS (MeOH) m/z: (pos) 429.1 [C8H17N2Cl2Pt +
Na]+, 724.2 [M + Na]+; (neg) 699.9 [M − H]−.
(DMSO-d6): δ 11.58 (s, 1H, NH), 11.08 (s, 1H, NH), 7.80 (s, 1H,
NH), 6.76 (major) 6.28 + 6.20 (minor) (s, 2H, CONH), 3.66 (m, 1H,
cyclohexyl CH), 2.92−2.74 (m, 4H, CH2CH2), 2.89 (m, 4H,
NHCH2), 2.09 (m, 2H, cyclohexyl CH2), 1.73 (m, 3H, cyclohexyl
CH2), 1.62 (m, 1H, cyclohexyl CH2), 1.36 (m, 4H, NHCH2CH2), 1.26
(m, 3H, cyclohexyl CH2), 1.10 (m, 1H, cyclohexyl CH2), 0.81 (m, 6H,
CH3). 13C NMR (DMSO-d6): δ 166.1 (CO), 165.7 (CO), 59.9
(cyclohexyl CH), 50.3 (CH2CH2), 46.9 (CH2CH2), 43.1
(CH2CH2CH3), 43.0 (CH2CH2CH3), 31.6 (cyclohexyl CH2), 28.3
(cyclohexyl CH2), 25.7 (cyclohexyl CH2), 25.7 (cyclohexyl CH2), 25.3
(cyclohexyl CH2), 23.4 (CH2CH2CH3), 11.8 (CH2CH2CH3), 11.6
(CH2CH2CH3). 15N NMR (DMSO-d6): δ −3.3 (NH2), 23.5 (NH),
67.3 (CONH). 195Pt NMR (DMSO-d6): δ 2812.2 (major), 2800.7
(minor); diastereomer 2796.3 (major), 2783.9 (minor). ESI-MS
(MeOH) m/z: (pos) 612.0 [M + H]+, 634.0 [M + Na]+.
(OC-6-54)-Dichlorido(N,N-diethylethane-1,2-diamine)hydroxido-
propylcarbamatoplatinum(IV) (B7). B1 (50 mg, 0.12 mmol) was
suspended in dry acetone (5 mL), and n-propyl isocyanate (20 mg,
0.24 mmol) was added. The suspension was then stirred at rt for 3 h.
The solvent was removed under reduced pressure, and the residue was
dissolved in EtOH. Subsequently, the crude product was precipitated
using Et2O, filtered off, and dried in vacuo. Yield: 51 mg (85%).
Elemental analysis for C10H25Cl2N3O3Pt·0.5H2O: calcd C 23.54, H
5.14, N 8.23; found C 23.24, H 4.83, N 7.85. 1H NMR (DMSO-d6): δ
10.58 (bs, 1H, NH2), 6.95 (bs, 1H, NH2), 6.31 (major) 5.96 (minor)
(bs, 1H, NH), 3.64 (m, 1H, CH2), 3.50 (m, 1H, CH2), 2.94−2.50
(bm, 8H, CH2CH2, CH2CH3, CH2CH2CH3), 1.38 (m, 2H, CH2CH3),
1.20 (m, 6H, CH3), 0.82 (t, 3H, CH3, J = 7.5 Hz). 13C NMR (DMSO-
d6): δ 165.79 (CO), 64.45 (NCH2CH2N), 50.29 (CH2CH3), 48.44
(CH2CH3), 44.47 (NCH2CH2N), 43.15 (CH2CH2CH3), 23.58
(CH2CH2CH3), 11.81 (CH2CH2CH3), 9.32 (CH3) 9.12 (CH3). 15N
NMR (DMSO-d6): δ −4.9. 195Pt NMR (DMSO-d6): δ 2615 (major),
(OC-6-54)-Acetatodichlorido(N,N-diethylethane-1,2-diamine)-
[(2E)-3-phenylprop-2-enoato]platinum(IV) (B9). B5 (61 mg, 0.11
mmol) was stirred in 3 mL of acetic anhydride at rt for 24 h.
Subsequently, 1 mL of acetone was added to the reaction mixture, and
the product was precipitated by addition of Et2O. To complete the
J
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precipitation, the suspension was kept in the refrigerator for 48 h. The
solid material was filtered off, washed with Et2O, and dried under
reduced pressure. Yield: 55 mg (84%). Elemental analysis for
C17H26Cl2N2O4Pt·0.3C3H6O: calcd C 35.49, H 4.63, N 4.62; found
C 35.37, H 4.49, N 4.25. 1H NMR (DMSO-d6): δ 9.74 (bs, 1H, NH2),
9.12 (bs, 1H, NH2), 7.65 (m, 2H, phenyl CH), 7.42 (d, 1H,
PtOOCCHCH, J = 16.5 Hz), 7.40 (m, 3H, phenyl CH), 6.49 (d,
1H, PtOOCCHCH, J = 16.5 Hz), 3.56 (m, 2H, N(CH2CH3)2),
3.08−2.99 (bm, 4H, NCH2CH2N), 2.76 (m, 2H, N(CH2CH3)2), 1.98
(s, 3H, CH3), 1.27 (m, 6H, N(CH2CH3)2). 13C NMR (DMSO-d6): δ
179.6 (PtOOC), 174.8 (PtOOC), 142.1 (phenyl C), 134.2 (phenyl
CH), 130.0 (2 × phenyl CH), 128.9 (CHCH), 128.1 (2 × phenyl
CH), 121.8 (CHCH), 65.4 (NCH2CH2N), 50.59 (N(CH2CH3)2),
50.5 (N(CH2CH3)2), 44.7 (NCH2CH2N), 24.2 (CH3), 8.7 (N-
(CH2CH3)2), 8.6 (N(CH2CH3)2). 15N NMR (DMSO-d6): δ −19.0.
195Pt NMR (DMSO-d6): δ 2808. ESI-MS (MeOH) m/z: (pos) 588.5
[M + H]+, 609.7 [M + Na]+, 625.7 [M + K]+; (neg) 586.5 [M − H]−.
Crystallographic Structure Measurements. X-ray diffraction
measurements on C3, B3, D4, and C6 were performed on a Bruker X8
APEX II CCD diffractometer at 100 K. Single crystals were positioned
35 mm from the detector, and 1496, 2844, 631, and 1102 frames were
measured for 30, 20, 5, and 50 s, respectively, over 1° scan width. The
data were processed using the SAINT software package.37 Crystal data,
data collection parameters, and structure refinement details are given
in Table 1. Structures were solved by direct methods and refined by
full-matrix least-squares techniques. Non-H atoms were refined with
anisotropic displacement parameters. H atoms were inserted at
calculated positions and refined with a riding model. Structure
solution was achieved with SHELXS-97 and refinement with
SHELXL-97,38 and graphics were produced with ORTEP-3.39
Cell Lines and Culture Conditions. Human CH1 cells were
kindly provided by Lloyd R. Kelland (CRC Centre for Cancer
Therapeutics, Institute of Cancer Research, Sutton, UK) and SW480
and A549 cells by Brigitte Marian (Institute of Cancer Research,
Department of Medicine I, Medical University of Vienna, Austria).
Cells were grown in 75 cm2 culture flasks (Iwaki/Asahi Technoglass,
Gyouda, Japan) as adherent monolayer cultures in complete culture
medium [minimal essential medium supplemented with 10% heat-
inactivated fetal bovine serum (FBS), 1 mM sodium pyruvate, 4 mM L-
glutamine, and 1% (v/v) nonessential amino acids (from 100× ready-
to-use stock solution), all purchased from Sigma-Aldrich, Vienna,
Austria]. Cultures were maintained at 37 °C in a humidified
atmosphere containing 95% air and 5% CO2.
Cytotoxicity Tests in Cancer Cell Lines. Cytotoxicity was
determined by a colorimetric microculture assay (MTT assay). For
this purpose, CH1, A549, and SW480 cells were harvested from
culture flasks by trypsinization and seeded into 96-well microculture
plates (CytoOne, Starlab) in densities of 1 × 103 (CH1), 2 × 103
(SW480), 3 × 103 (A549) viable cells/well. After a 24 h preincubation,
cells were exposed to dilutions of the test compounds in complete
culture medium (200 μL/well) for 96 h. At the end of the exposure
period, the drug solutions were replaced with RPMI 1640 medium
supplemented with 10% heat-inactivated FBS and 4 mM L-glutamine
(100 μL/well) and MTT solution (MTT reagent in phosphate-
buffered saline; 20 μL/well). After incubation for 4 h, the medium was
removed, and the formazan product formed by viable cells was
dissolved in DMSO (150 μL/well). Optical densities at 550 nm were
measured with a microplate reader (Biotek ELx808) using a reference
wavelength of 690 nm to correct for unspecific absorption. The
quantity of viable cells was expressed in terms of T/C values by
comparison to untreated controls, and IC50 values were calculated
from concentration−effect curves by interpolation. Evaluations were
based on means from at least three independent experiments, each
comprising triplicates for each concentration level.
Reversed-Phase HPLC Measurements.27,30,31 Analytical RP-
HPLC analysis was performed on a Dionex Summit system controlled
by the Dionex Chromeleon 6.60 software. The experimental
conditions were as follows: a silica-based C18 gel as stationary phase
(Agilent Zorbax Eclipse, 4.6 mm × 250 mm); MeOH/15 mM aqueous
formic acid-based mobile phases; UV−vis detection set at 225, 250,
275, and 330 nm; column temperature of 25 °C; flow rate of 1 mL/
min; concentration of 0.25 mM for the investigated complexes; 0.1
mM KI solution as the internal standard; injection volume of 25 μL.
The capacity factor is defined as k′ = (tR − t0)/t0, where tR is the
retention time of the investigated compound and t0 is the retention
time of the internal reference KI (dead-volume marker). Values of tR
were determined for at least three different MeOH/HCOOH ratios.
Extrapolation of the resulting linear dependence of log k′ on the
percentage of MeOH in the solvent afforded the value of log kw: log k′
= log kw − SMeOHφ, where k′ is the capacity factor at the measured
mobile phase composition, kw is the extrapolated capacity factor in
100% H2O, SMeOH is a constant for a given substance and the given
HPLC system, and φ is the volume percentage of the organic modifier
(MeOH) in the mobile phase.
ASSOCIATED CONTENT
■
S
* Supporting Information
Synthesis of ligand S2; characterization of platinum compounds
A, B, B6, and B8; synthesis and characterization of platinum(II)
compound D; time-dependent decomposition of A1 in DMSO-
d6; variable-temperature 1H NMR spectra of D8; plots of log kw
vs IC50; excerpts of mass spectra of C1 and satraplatin after 72
1
h; selected H, 15N, and 195Pt NMR shifts; selected bond
lengths and angles for B3, C3, C6, and D4; log kw values;
elemental analysis results for all of the compounds; and X-ray
crystallographic data (CIF) for B3, C3, C6, and D4. This
material is available free of charge via the Internet at http://
pubs.acs.org.
Reduction Behavior Measurements. The reactivities of
complexes C1, C3, and C4 and the reference compound satraplatin
toward cellular reductants was investigated by NMR spectroscopy and
ESI-MS. For NMR measurements, stock solutions of the compounds
(1 mM) and ascorbic acid (2 mM) in 20 mM phosphate buffer in D2O
(pD 7.51) were prepared. Reaction mixtures were then prepared
(compound:reductant molar ratio = 1:2), and spectra were measured
at ambient temperature over a period of 72 h. The reductions were
monitored by following the decreases in the intensities of the signals at
2.83, 2.12, 2.12, and 2.12 ppm for C1, C3, C4, and satraplatin,
respectively. For ESI-MS measurements, stock solutions of the
compounds (400 μM), NaAsc (200 μM), and glutathione (200 μM)
were prepared. Reaction mixtures were prepared (compound:reduc-
tant molar ratio = 1:2) and incubated for 72 h at 37 °C. Mass spectra
were recorded in the positive- and negative-ion modes after 0, 3, 6, and
72 h. The samples were diluted to 5 μM using 1:1 water/MeOH and
directly infused into an AmaZon SL ESI ion trap (Bruker Daltonics
GmbH, Bremen, Germany). The mass spectra were processed using
ESI Compass 1.3 and DataAnalysis 4.0 software (Bruker Daltonics).
AUTHOR INFORMATION
■
Corresponding Author
*Phone: +43-1-4277-52600. Fax: +43-1-4277-52680. E-mail:
markus.galanski@univie.ac.at (M.G.), bernhard.keppler@
univie.ac.at (B.K.K).
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors are indebted to the FFGAustrian Research
Promotion Agency (811591) and the Austrian Council for
Research and Technology Development (IS526001). Further-
more, we thank Prof. Arion for X-ray structure analysis and
Elisabeth Jirkovsky and Ricarda Bugl for RP-HPLC measure-
ments.
K
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dx.doi.org/10.1021/ic400816g | Inorg. Chem. XXXX, XXX, XXX−XXX