Tetracarboxylatoplatinum(IV) complexes featuring monodentate leaving groups - A rational approach toward exploiting the platinum(IV) prodrug strategy

Article abstract of DOI:10.1016/j.jinorgbio.2015.08.018

A series of novel symmetrically and unsymmetrically coordinated platinum(IV) complexes with monodentate carboxylato ligands was synthesized. The compounds exhibit a general coordination sphere of [Pt(en)(OCOR)₂(OCOR′)(OCOR″], where the carboxylato ligands are represented by acetato and succinic acid monoester ligands. Dicarboxylatoplatinum(II) complexes were synthesized and oxidized symmetrically or unsymmetrically to obtain platinum(IV) complexes, which were subsequently carboxylated with noncyclic anhydrides. The compounds were investigated in detail by elemental analysis, mass spectrometry, infrared and multinuclear (¹H, ¹³C, ¹⁵N, ¹⁹⁵Pt) NMR spectroscopy as well as by X-ray diffraction in some cases. The reduction behavior was followed by NMR spectroscopy, while stability and lipophilicity were examined by analytical reversed phase HPLC measurements. Cytotoxic properties were studied in three human cancer cell lines derived from cisplatin sensitive ovarian teratocarcinoma (CH1/PA-1), cisplatin insensitive colon carcinoma (SW480) and non-small cell lung cancer (A549). Thereby, the most lipophilic (yet water soluble) platinum(IV) complexes showed promising IC₅₀ values in the low micromolar and even nanomolar range, demonstrating the significant advantage of using equatorially coordinated monodentate carboxylato ligands.

Full text of DOI:10.1016/j.jinorgbio.2015.08.018

JIB-09789; No of Pages 13
Journal of Inorganic Biochemistry xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Journal of Inorganic Biochemistry
journal homepage: www.elsevier.com/locate/jinorgbio
Tetracarboxylatoplatinum(IV) complexes featuring monodentate leaving groups — A
rational approach toward exploiting the platinum(IV) prodrug strategy☆
Doris Höfer , Hristo P. Varbanov ⁎, Anton Legin , Michael A. Jakupec a,c, Alexander Roller a,
a
a,b,
a
a,
a,c,
Markus Galanski ⁎, Bernhard K. Keppler
Institute of Inorganic Chemistry, University of Vienna, Waehringer Strasse 42, A-1090 Vienna, Austria
Institut des Sciences et Ingénierie Chimiques, Ecole Polytechnique Fédérale de Lausanne (EPFL), CH-1015, Lausanne, Switzerland
Research Platform “Translational Cancer Therapy Research”, University of Vienna, Waehringer Strasse 42, A-1090 Vienna, Austria
⁎
a
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel symmetrically and unsymmetrically coordinated platinum(IV) complexes with
monodentate carboxylato ligands was synthesized. The compounds exhibit a general coordination sphere
of [Pt(en)(OCOR) (OCOR′)(OCOR″)], where the carboxylato ligands are represented by acetato and
2
Received 31 March 2015
Received in revised form 10 August 2015
Accepted 20 August 2015
Available online xxxx
succinic acid monoester ligands. Dicarboxylatoplatinum(II) complexes were synthesized and oxidized
symmetrically or unsymmetrically to obtain platinum(IV) complexes, which were subsequently carboxylated
with noncyclic anhydrides. The compounds were investigated in detail by elemental analysis, mass spectrome-
Keywords:
Platinum complexes
Synthesis
Characterization
Cytotoxicity
Reduction
1
13 15 195
try, infrared and multinuclear ( H, C, N, Pt) NMR spectroscopy as well as by X-ray diffraction in some cases.
The reduction behavior was followed by NMR spectroscopy, while stability and lipophilicity were examined by
analytical reversed phase HPLC measurements. Cytotoxic properties were studied in three human cancer cell
lines derived from cisplatin sensitive ovarian teratocarcinoma (CH1/PA-1), cisplatin insensitive colon carcinoma
(SW480) and non-small cell lung cancer (A549). Thereby, the most lipophilic (yet water soluble) platinum(IV)
Lipophilicity
complexes showed promising IC50 values in the low micromolar and even nanomolar range, demonstrating
the significant advantage of using equatorially coordinated monodentate carboxylato ligands.
©
2015 Elsevier Inc. All rights reserved.
1
. Introduction
as prodrugs which unfold their cytostatic potency after reduction to
the respective platinum(II) congeners. In consideration of the kinetic in-
ertness, this concept reveals several advantages: (i) expanded synthetic
strategies allow fine-tuning of important pharmacological properties,
(ii) protein interactions and therefore side effects and deactivation can
be decreased, (iii) oral administration is feasible, and (iv) a possibly in-
creased half-life of the prodrug improves accumulation of the platinum
complex at the target site. Nevertheless, the key event in the mecha-
nism of action is the activation (reduction followed by aquation) pri-
marily inside the cancer tissue/cells.
In the 1960s, B. Rosenberg's [1] experiments revealed the antiprolifer-
ative properties of platinum complexes initiating the success story of cis-
platin [cis-diamminedichloridoplatinum(II)] (Fig. 1). Cisplatin, together
with the 2nd and 3rd generation platinum(II) complexes, carboplatin
[
cis-diammine-(1,1-cyclobutanedicarboxylato)platinum(II)] and
oxaliplatin [(trans-1R,2R-cyclohexanediamine)oxalatoplatinum(II)],
still represent an indispensable class of worldwide approved anticancer
agents used in nearly 50% of all tumor chemotherapies [2,3]. Neverthe-
less, severe general toxicity and resistance [4] are relentless impedi-
ments, stimulating enormous efforts in that field of research dedicated
to improve cancer therapy. In this area of bioinorganic chemistry, re-
search was also expanded to other metals with anticancer activity.
Platinum(IV) complexes represent another emerging focus, featur-
ing an altered mode of action in line with the concept of activation by
reduction [5]. Accordingly, kinetically inert platinum(IV) complexes act
In the case of platinum(IV) complexes featuring equatorial
chlorido ligands, the reduction is mainly dependent on the axial li-
gands. Choi et al. [6] reported an increase of the reduction poten-
tial as the electron-withdrawing power of the axial ligands rises
(OH b OCOCH
3 3
b Cl b OCOCF ). The observed trend can be used to ex-
plain the behavior of three platinum(IV) complexes which entered
clinical trials until now (Fig. 1). Tetraplatin was abandoned after phase
I, showing severe neurotoxicity [7] attributed to the immediate reduction
to the reactive platinum(II) species. Iproplatin in contrast was rejected
after phase III clinical trials due to lack of activity [8] caused by insufficient
reduction. A breakthrough was achieved with satraplatin, [(OC-6-43)-
diacetato(ammine)dichlorido(cyclohexylamine)platinum(IV)], the first
platinum complex designed for oral administration. Satraplatin reached
☆
Dedicated to Prof. Giovanni Natile on the occasion of his 70th birthday.
Corresponding author.
⁎
E-mail addresses: hristo.varbanov@univie.ac.at (H.P. Varbanov),
markus.galanski@univie.ac.at (M. Galanski), bernhard.keppler@univie.ac.at
B.K. Keppler).
(
http://dx.doi.org/10.1016/j.jinorgbio.2015.08.018
162-0134/© 2015 Elsevier Inc. All rights reserved.
0
Please cite this article as: D. Höfer, et al., Tetracarboxylatoplatinum(IV) complexes featuring monodentate leaving groups — A rational approach
toward exploiting the platinum(IV) ..., J. Inorg. Biochem. (2015), http://dx.doi.org/10.1016/j.jinorgbio.2015.08.018

2
D. Höfer et al. / Journal of Inorganic Biochemistry xxx (2015) xxx–xxx
Fig. 1. Platinum(II) chemotherapeutics in worldwide use: cisplatin, carboplatin and oxaliplatin; platinum(IV) complexes which entered clinical trials: tetraplatin, iproplatin, and
satraplatin.
phase III clinical trials; unfortunately, no significant improvement in
overall survival rates compared to standard treatment could be observed
In the present work, we report on symmetrically and unsymmetri-
cally coordinated tetracarboxylato platinum(IV) complexes featuring
equatorial monodentate ligands. The coordination sphere was varied
using succinic acid mono esters of different lengths together with
acetato ligands. Unsymmetrically carboxylated complexes were obtain-
ed via oxidation in acetic acid. Subsequent carboxylation was achieved
by using anhydrides of succinic acid mono esters. The compounds
were evaluated with regard to their lipophilicity, stability, reduction be-
havior and antiproliferative activity.
[9,10]. Nevertheless, complexes with axial carboxylato ligands are still in-
teresting as anticancer drug candidates due to their intermediate reduc-
tion potentials and various options for derivatization.
Recent studies revealed that the reduction rate does not always
correlate with the reduction potential of platinum(IV) complexes.
Unusual inertness in presence of ascorbic acid was observed for
tetracarboxylatoplatinum(IV) complexes, prodrugs of carboplatin
[
11] and oxaliplatin [12]. The nitrogen and oxygen donor atoms are
supposed to hinder an inner sphere electron transfer because of their
poor ability to act as electron transfer bridges. Thus, tetracarboxylato
complexes are less likely to be prematurely reduced before arriving at
the target site; hence, they are interesting prodrug candidates [13].
The platinum(IV) prodrug strategy is not only valuable for introduc-
ing tumor specificity but can also be used to overcome barriers which
hinder drug delivery. Lipophilic complexes benefit from a facilitated dif-
fusion through membranes thereby increasing the concentration at the
target site and consequently improving the cytotoxicity [14]. In this con-
text it is worthwhile mentioning that especially succinic ester ligands in
axial position are of high interest due to the possibility of adjusting
physiologically relevant properties (lipophilicity/solubility balance) of
Pt(IV) complexes without compromising their aqueous solubility.
Further optimization can be achieved by fine-tuning the coordina-
tion sphere. Besides the importance of the type of axial ligands, the
equatorial leaving groups significantly affect the reactivity of the re-
spective platinum(II) complexes after reduction of the platinum(IV)
species. Pt(II) compounds featuring a bidentate dicarboxylato ligand
are considerably more inert compared to those analogs exhibiting two
monodentate carboxylato ligands.
2. Experimental
2.1. Material and methods
All solvents and chemicals were obtained from commercial suppliers
and were used as received. K [PtCl ] was purchased from Johnson
2 4
Matthey (Switzerland). Methanol and ethanol were dried according to
standard procedures and water was purified by reversed osmosis and
double distillation before use. Thin layer chromatography was carried
out on silica gel on TLC-PET foils (Fluka) and silica gel 60 (Fluka) was
applied for column chromatography. Reactions involving platinum
complexes were carried out under protection from light and with
glass coated magnetic stirring bars. Succinic acid mono esters (L1–L3)
and the corresponding anhydrides (L4–L6) were synthesized, using
standard procedures (Scheme 1).
(SP-4-2)-(Ethane-1,2-diamine)diiodidoplatinum(II) (1), used as
a precursor for the synthesis of all target species, was prepared by re-
2 4
action of K [PtCl ] with KI and ethane-1,2-diamine, according to
Dhara's method [23]. The respective dicarboxylato platinum(II)
complexes (2–3) were synthesized, as described by Rochon et al.
[24] with slight modifications. (OC-6-22)-Tetraacetato(ethane-1,2-
diamine)platinum(IV) (11) was obtained from (OC-6-22)-(ethane-
1,2-diamine)tetrahydroxidoplatinum(IV) as reported elsewhere
[25]. NMR measurements were recorded on a Bruker Avance III
Additionally, research also concentrated on unsymmetric complexes
with different axial ligands. Song et al. [15] reported the synthesis of
tricarboxylatoplatinum(IV) complexes by nucleophilic substitution of
tetrahydroxido complexes in carboxylic acids. Pichler et al. [9,16,17]
used sterically demanding amine carrier ligands shielding platinum on
one side to yield mono-acetylated complexes. Investigation of the
1
13
15
500 MHz spectrometer at 500.32 ( H), 125.81 ( C), 50.70 ( N)
and 107.55 (1 Pt) MHz in DMF-d
solvent resonances were used as internal reference for H (DMF-d
2.93 ppm (lowfield methyl signal); D O, 4.70 ppm; CDCl , 7.29 ppm)
, 34.6, ppm (lowfield methyl signal)) chemical shifts.
95Pt and N signals were referenced relative to external K
[PtCl
and NH Cl, respectively. Electrospray ionization mass spectra
95
7
2 3
, D O or CDCl at 298 K. The residual
1
oxidation of platinum(II) complexes with H
2
O
2
by NMR measure-
7
,
ments by Dunham et al. [18] revealed that one of the axial ligands
originates from the oxidizing agent while the other one is a solvent
molecule. Thus, unsymmetric complexes were obtained by using sol-
vents different from water [19–21]. Another approach was described
by Zhang et al. [22] in which dihydroxido platinum(IV) precursors were
reacted with activated carboxylic acids to obtain monocarboxylated
platinum(IV) complexes.
2
3
13
and C (DMF-d
7
1
15
2
4
]
4
were measured with a Bruker maXis ESI-QqTOF (electrospray ionization
quadrupole–quadrupole-time-of-flight) spectrometer in the positive and
negative mode using ACN/MeOH 1% H O as solvent (ACN = acetonitrile).
2
Please cite this article as: D. Höfer, et al., Tetracarboxylatoplatinum(IV) complexes featuring monodentate leaving groups — A rational approach
toward exploiting the platinum(IV) ..., J. Inorg. Biochem. (2015), http://dx.doi.org/10.1016/j.jinorgbio.2015.08.018

D. Höfer et al. / Journal of Inorganic Biochemistry xxx (2015) xxx–xxx
3
2
Scheme 1. Synthesis and NMR numbering scheme of succinic acid monoesters and their anhydrides. (i) MeOH (L1), EtOH (L2) or 1-PentOH (L3); (ii) (AcO) O.
1
Elemental analyses were performed at the Microanalytical Laboratory of
the University of Vienna with a PerkinElmer 2400 CHN Series II elemental
analyzer or a Eurovector EA3000 elemental analyzer and are within
4.15, N 5.30. H NMR (D
2
O): δ = 5.22 (bs, 4H, NH
2
), 3.65 (s, 6H, H5′),
13
2.58–2.47 (m, 8H, H3′/H2′), 2.45 (s, 4H, Hen) ppm. C NMR (D
δ = 182.0 (C1′), 176.2 (C4′), 52.2 (C5′), 48.3 (Cen) 30. 6 (C3′), 30.0
(C2′, JPt,C = 34.5 Hz) ppm.
2
O):
3
195
±
0.4% of the calculated values, confirming their ≥95% purity. Infrared
2
Pt NMR (D O): δ = −273 ppm. ESI-MS
+
+
spectra were obtained on a Bruker Vertex 70 FT-IR-spectrometer
(ACN/MeOH 1% H O): (pos) m/z 540.09 [M+Na ] , (neg) 516.10 [M–
2
+
−
with an ATR-unit (attenuated total reflection unit) in the range of
H ] . IR: ν = 3281 bm (νN–H), 3227 bm (νN–H), 2977 w (νC–H),
2954 w (νC–H), 2923 w (νC–H), 1722 s (νas C_O), 1623 s (νas C_O),
1382 s, 1362 s, 1283 m, 1223 s, 1162 s cm 1.
−
1
4
000–400 cm . Intensities of the reported bands are described with
−
s for strong, m for medium and w for weak; broad signals are addition-
ally specified with the letter b in front of these abbreviations.
2.2.2. General procedure II: symmetric oxidation of Pt(II) complexes
2
.2. Synthesis
The platinum(II) complex was suspended in 15% H and the reac-
2 2
O
tion mixture was stirred for 15 min at 40 °C and for further 45 min at
room temperature. The volume of the resulting clear yellow solution
was concentrated under reduced pressure and acetone was added to
precipitate the product. The resulting suspension was cooled to 4 °C
for 1.5 h. The product was collected by filtration, washed with cold
methanol and diethyl ether and dried in vacuo.
2
.2.1. General procedure I: synthesis of Pt(II) complexes
(SP-4-2)-(Ethane-1,2-diamine)diiodidoplatinum(II) 1 was suspended
in an aqueous solution of Ag SO (0.95 eq) and the reaction mixture was
stirred overnight in the dark. To remove precipitated AgI, the suspension
was filtered through a sintered glass funnel with a filter paper disk
2
4
(
MN GF-3). A barium carboxylate solution was prepared by stirring
Ba(OH) ·H O (0.95 eq) and the respective carboxylic acid (2.85 eq) in
2
2
2.2.2.1. (OC-6-33)-Diacetato(ethane-1,2-diamine)dihydroxidoplatinum(IV)
water until a clear solution was formed. The resulting solution was
added slowly to the filtrate and the reaction mixture was stirred for
2 2
(4). General procedure II. 2 (352 mg. 0.94 mmol), 15% H O (6 mL).
1
3
Yield 137 mg (36%). H NMR (D
32.5 Hz), 2.06 (s, 6H, H2a′) ppm. C NMR (D
47.0 (Cen), 22.1 (C2a′) ppm. 195Pt NMR (D
(ACN/MeOH 1%H
2
O): δ = 2.64 (s + d, 4H, Hen, JPt,H
=
13
2
.5 h at room temperature. Precipitated BaSO
4
was removed by filtration
2
O): δ = 181.2 (C1a′),
and the filtrate was evaporated to dryness. The solid was suspended in a
small amount of methanol and was cooled to 4 °C for 3 h. The product
was filtered, washed with diethyl ether and dried in vacuo.
2
O): δ = 3185 ppm. ESI-MS
+
+
2
O): (pos) m/z 430.06 [M+Na ] , (neg) 406.06 [M–
+
−
+ −
H ] , 813.13 [M
2
–H ] . IR: ν = 3412 bw (νPtO–H), 3147 bw (νN–
−
1
H
), 1594 s (νas C_O), 1366 s, 1326 s, 1156 w, 1064 w cm
.
2
.2.1.1. (SP-4-2)-Diacetato(ethane-1,2-diamine)platinum(II) (2). General
procedure I. 1 (800 mg, 1.57 mmol), Ag SO (466 mg, 1.49 mmol) in
O (70 mL), Ba(OH) ·8H O (471 mg, 1.49 mmol) and CH COOH
256 μL, 4.48 mmol) in H O (11 mL). Crystals suitable for X-ray data col-
lection were obtained from diluted MeOH/water solution at 4 °C. Yield
2
4
2.2.2.2. (OC-6-33)-(Ethane-1,2-diamine)(dihydroxido)bis((4-methoxy)-
4-oxobutanoato)platinum(IV) (6). General procedure II. 3 (305 mg.
H
(
2
2
2
3
1
2
0.59 mmol), 15% H
DMF-d
32.3 Hz), 2.62–2.59 (m, 4H, H2′), 2.57–2.54 (m, 4H, H3′) ppm.
NMR (D O/DMF-d ): δ = 179.6 (C1′), 174.0 (C4′), 51.4 (C5′), 47.1
(Cen), 30.9 (C2′, JPt,C = 32.6 Hz), 29.9 (C3′) ppm.
DMF-d ): δ = 3195 ppm. ESI-MS (ACN/MeOH 1%H
2
O
2
(6 mL). Yield 104 mg (32%). H NMR (D
2
O/
3
7
): δ = 3.64 (s, 6H, H5′), 2.76 (s + d, 4H, Hen, J
Pt,H
=
C
1
3
4
1
12 mg (73%). Elemental analysis: C
8.85, H 3.96, N 7.33; found C 18.48, H 3.67, N 7.30. H NMR (D
6
H
14
N
2
O
4
Pt·0.5H
2
O; calcd. C
O):
1
2
2
7
13
3
195
δ = 5.24 (bs, 4H, NH
NMR (D
O): δ = 182.1 (C1a′), 48.1 (Cen), 21.9 (C2a′) ppm. 195Pt
NMR (D O): δ=−286 ppm. ESI-MS (ACN/MeOH 1%H O): (pos)
m/z 396.05 [M+Na ] , (neg) 372.06 [M–H ] . IR: ν = 3215 bm
N–H), 3113 bm (νN–H), 1636 m (νas C_O), 1583 s (νas C_O), 1390 m,
2
), 2.41 (s, 4H, Hen), 1.90 (s, 6H, H2a′) ppm.
C
Pt NMR (D
2
O/
2
7
2
O): (pos) m/z
+
+
+ −
2
2
574.10 [M+Na ] , (neg) 550.10 [M–H ] . IR: ν = 3559 m (νPtO–H),
3281 m (νN–H), 3252 m (νN–H), 2790 w (νC–H), 2749 w (νC–H), 2607 w
+
+
+ −
(
1
ν
(νC–H), 2419 m, 1724 s (νas C_O), 1629 s (νas C_O), 1360 s, 1266 m,
318 m, 1151 w, 1121 w cm−1
.
1236 m, 1168 m cm
−1
.
2
.2.1.2. (SP-4-2)-(Ethane-1,2-diamine)bis((4-methoxy)-4-oxobutanoato)
2.2.3. (OC-6-33)-Triacetato(ethane-1,2-diamine)hydroxidoplatinum(IV)
(5)
36-40% peroxyacetic acid (239 μL, 1.28 mmol, 2 eq) in acetic acid
(4 mL) was added slowly to a suspension of 2 (240 mg, 0.64 mmol) in
acetic acid (4 mL). The obtained clear yellow solution was left stirring
for 48 h at room temperature. The solvent was removed under reduced
platinum(II) (3). General procedure I. 1 (200 mg, 0.39 mmol), Ag
166 mg, 0.37 mmol) in H O (17 mL), Ba(OH) ·8H O (118 mg,
.37 mmol) and 3-(methoxycarbonyl)propanoic acid (L1, 149 mg,
.13 mmol) in H O (6 mL). Yield 170 mg (88%). Elemental analysis:
Pt·0.6H O; calcd. C 27.29, H 4.43, N 5.30; found C 27.06, H
2 4
SO
(
2
2
2
0
1
C
2
H N O
12 22 2 8
2
Please cite this article as: D. Höfer, et al., Tetracarboxylatoplatinum(IV) complexes featuring monodentate leaving groups — A rational approach
toward exploiting the platinum(IV) ..., J. Inorg. Biochem. (2015), http://dx.doi.org/10.1016/j.jinorgbio.2015.08.018

4
D. Höfer et al. / Journal of Inorganic Biochemistry xxx (2015) xxx–xxx
2
δ = 8.49 (bs, 4H, NH JH,H = 6.7 Hz), 2.80 (bs,
), 4.06 (t, 4H, H5, 3
pressure and the brown oily residue was suspended in ethyl acetate in
order to precipitate the product. The resulting suspension was cooled
for 1 h at 4 °C and the product was filtered off, washed with methanol
and diethyl ether and dried in vacuo. Crystals suitable for X-ray data col-
4H, Hen), 2.58–2.55 (m, 4H, H2/H3), 2.54–2.51 (m, 4H, H2/H3), 1.97
(s, 6H, H2a′), 1.65–1.59 (m, 4H, H6), 1.36–1.30 (m, 8H, H7,H8), 0.91
3
13
7
(t, 6H, H9, JH,H = 6.7 Hz) ppm. C NMR (DMF-d ): δ = 180.1 (C1),
lection were obtained in the NMR tube in a mixture of DMF and D
2
O.
), 7.44
), 2.90–2.79 (m, 2H, Hen), 2.73–2.62 (m, 2H, Hen), 1.94 (s,
176.3 (C1a′), 172.6 (C4), 64.2 (C5), 47.9 (Cen). 30.5 (C2/C3), 29.9
(C2/C3), 28.2 (C6), 27.9 (C7), 22.2 (C8), 21.7 (C2a′), 13.5 (C9) ppm.
1
Yield 189 mg (65%). H NMR (DMF-d
bs, 2H, NH
H, H2a′), 1.84 (s, 3H, H2a) ppm. C NMR (DMF-d
7 2
): δ = 8.63 (bs, 2H, NH
1
5
195
(
6
2
N NMR (DMF-d
7
): δ = −21.4 (NH
2
) ppm.
7
Pt NMR (DMF-d ):
13
7
): δ = 179.6
δ = 3495 ppm. ESI-MS (ACN/MeOH 1%H
[M+Na ] , 1517.50 [M +Na ] ; (neg) 746.25 [M–H ] . IR: ν =
2
2
O): (pos) m/z 770.24
3
+ +
+ +
+ −
(
J
C1a), 177.4 (C1a′), 47.4 (Cen), 23.3 (C2a, JPt,C = 27.8 Hz), 22.3 (C2a′,
3
15
Pt,C = 34.5 Hz) ppm. N NMR (DMF-d
7
): δ = −20.0 (NH
2
), −19.9
3190 bm (νN–H), 2958 m (νC–H), 2930 m (νC–H), 2859 w (νC–H), 1730 s
1
95
−1
(NH
2
) ppm.
O): (pos) m/z 472.07 [M+Na ] , (neg) 448.07 [M–H ] . IR:
ν = 3535 w (νPtO–H), 3180 bm (νN–H), 3073 bm (νN–H), 1608 s (νas
7
Pt NMR (DMF-d ): δ = 3284 ppm. ESI-MS (ACN/MeOH
(νas C_O), 1625 m (νas C_O), 1357 s, 1303 s, 1204 s, 1157 s cm
.
+
+
+ −
1
%H
2
2.2.4.4. (OC-6-23)-Triacetato(ethane-1,2-diamine)((4-methoxy)-4-
oxobutanoato)platinum(IV) (8a). General procedure III. 3-
−
1
C_O), 1579 s (νas C_O), 1364 s, 1326 s, 1301 s cm
.
(
Methoxycarbonyl)propanoic anhydride (L4, 193 mg, 0.78 mmol,
2
.2.4. General procedure III: carboxylation with noncyclic anhydrides
The platinum complex together with the corresponding anhydride
1.8 eq), DMF (8 mL), 5 (201 mg, 0.45 mmol), stirring in DMF for
5.5 h at 35–45 °C and for 1 h at rt, column chromatography:
EtOAc/MeOH (6:1–2:1). Yield 145 mg (58%). Elemental analysis:
was stirred in DMF until a clear yellow solution was formed. DMF was
removed under reduced pressure and the crude product was purified
by column chromatography.
13 24 2
C H N O10Pt; calcd. C 27.71, H 4.29, N 4.97; found C 27.68, H
1
4.29, N 4.91. H NMR (DMF-d
H, NH ), 3.65 (s, 3H, H5), 2.80 (bs, 4H, Hen), 2.57–2.54 (m, 2H,
H2), 2.53–2.50 (m, 2H, H3), 1.97 (s, 6H, H2a′), 1.92 (s, 3H, H2a)
ppm. 13C NMR (DMF-d
): δ = 180.0 (C1), 179.0 (C1a), 176.3 (C1a′),
173.1 (C4), 51.1 (C5), 48.0 (Cen), 30.5 (C2, JPt,C = 34.7 Hz), 29.7 (C3),
7 2
): δ = 8.53 (bs, 2H, NH ), 8.47 (bs,
2
2
2
4
.2.4.1. (OC-6-13)-Diacetato(ethane-1,2-diamine)bis((4-methoxy)-
-oxobutanoato)platinum(IV) (7a). General procedure III. 3-
7
3
(
Methoxycarbonyl)propanoic anhydride (L4, 344 mg, 1.40 mmol,
eq), DMF (6 mL), 4 (186 mg, 0.46 mmol), stirring in DMF for 5 h
at 45 °C and 3 h at rt, column chromatography: EtOAc/MeOH
3
15
3
21.9 (C2a), 21.7 (C2a′,
δ = −21.2 (NH
(ACN/MeOH 1% H
2
J
Pt,C = 32.5 Hz) ppm. N NMR (DMF-d
7
):
) ppm. 1 Pt NMR (DMF-d
95
): δ = 3494 ppm. ESI-MS
2
7
+
+
(
6:1–2:1). Yield 165 mg (57%). Elemental analysis: C16
28 2
H N O12P·0.5
O): (pos) m/z 586.10 [M+Na ] , 526.08 [M-
1
+ +
+ −
+ −
H
2
O; calcd. C 29.82, H 4.54, N 4.35; found 29.87, H 4.40, N 4.38. H
3 2
CH COOH+Na ] ; (neg) 562.10 [M–H ] , 1125.21 [M –H ] .
NMR (DMF-d
7
): δ = 8.48 (bs, 4H, NH
2
), 3.65 (s, 6H, H5), 2.80 (bs, 4H,
IR: ν = 3224 bm (νN–H), 2950 w (νC–H), 1735 m (νas C_O),
H
en), 2.58–2.55 (m, 4H, H2), 2.53–2.50 (m, 4H, H3), 1.97 (s, 6H, H2a′)
1641 s (νas C_O), 1622 s (νas C_O), 1361 m, 1293 s, 1178 m cm−1.
1
3
ppm. C NMR (DMF-d
7
): δ = 180.0 (C1), 176.3 (C1a′), 173.0 (C4),
3
5
1.1 (C5), 47.9 (Cen), 30.5 (C2, JPt,C = 38.2 Hz), 29.6 (C3), 21.7 (C2a′,
Pt,C = 32.0 Hz) ppm. N NMR (DMF-d
2.2.4.5. (OC-6-23)-Triacetato(ethane-1,2-diamine)((4-ethoxy)-4-
oxobutanoato)platinum(IV) (8b). General procedure III. 3-(Ethoxy-
carbonyl)propanoic anhydride (L5, 184 mg, 0.67 mmol, 1.6 eq), DMF
(6 mL), 5 (186 mg, 0.41 mmol), stirring in DMF for 2.5 h at 35 °C and
for 6 h at rt, column chromatography: EtOAc/MeOH (10:1–2:1). Crys-
3
15
J
7
): δ = −21.6 (NH
2
) ppm.
O):
3
COOH+Na ] , 538.07
1
95
Pt NMR (DMF-d
pos) m/z 658.12 [M+Na ] , 598.10 [M–CH
7
): δ = 3494 ppm. ESI-MS (ACN/MeOH 1%H
2
+
+
+ +
(
+
+
+ +
[
M–2CH
3
COOH+Na ] , 1293.24 [M
2
+Na ] ; (neg) 634.12 [M–
+
−
H ] . IR: ν = 3230 bm (νN–H), 3048 w (νC–H), 1728 s (νas C_O),
1
2
tals suitable for X-ray data collection were obtained in EtOAc/Et O at
640 s (νas C_O), 1358 s, 1291 s cm−1
.
4 °C. Yield 130 mg (54%). Elemental analysis: C14
calcd. C 28.24, H 4.74, N 4.70; found C 28.24, H 4.45, N 4.80. H NMR
H
N
O
10Pt·H
26
2
2
O;
1
2
.2.4.2. (OC-6-13)-Diacetato(ethane-1,2-diamine)bis((4-ethoxy)-4-
(DMF-d
J
7
): δ = 8.54 (bs, 2H, NH
H,H = 7.1 Hz), 2.80 (bs, 4H, Hen), 2.57–2.54 (m, 2H, H2), 2.52–20.49
(m, 2H, H3), 1.97 (s, 6H, H2a′), 1.92 (s, 3H, H2a), 1.23 (t, 3H, H6,
2 2
), 8.48 (bs, 2H, NH ), 4.11 (q, 2H, H5,
3
oxobutanoato)platinum(IV) (7b). General procedure III. 3-(Ethoxy
carbonyl)propanoic anhydride (L5, 334 mg, 1.22 mmol, 3 eq), DMF
3
13
(
5.5 mL), 4 (165 mg, 0.41 mmol), stirring in DMF for 4 h at 45 °C and
h at rt, column chromatography: EtOAc/MeOH (6:1–4:1). Crystals
suitable for X-ray data collection were obtained after storage in diluted
EtOAc/Et O solution at 4 °C. Yield 165 g (61%). Elemental analysis:
12Pt·0.5H O; calcd. C 32.15, H 4.95, N 4.17; found C 32.15,
H 4.95, N 4.16. H NMR (DMF-d ): δ = 8.49 (bs, 4H, NH ), 4.11 (q, 4H,
H5, 3
H,H = 7.1 Hz), 2.80 (bs, 4H, Hen), 2.57–2.54 (m, 4H, H2), 2.52–
.49 (m, 4H, H3), 1.97 (s, 6H, H2a′), 1.23 (t, 6H, H6,
J
H,H = 7.1 Hz) ppm. C NMR (DMF-d
176.3 (C1a′), 172.6 (C4), 60.1 (C5), 48.0 (Cen), 30.5 (C2, 3
39.2 Hz), 29.9 (C3), 21.9 (C2a), 21.7 (C2a′), 13.8 (C6) ppm.
7
): δ = 180.0 (C1), 179.0 (C1a),
4
Pt,C
J =
1
5
N
95
2
NMR (DMF-d
7
): δ = −21.7 (NH
2
) ppm. 1 Pt NMR (DMF-d
7
):
C
18
H
32
N
2
O
2
2
δ = 3495 ppm. ESI-MS (ACN/MeOH 1%H O): (pos) m/z 600.11
[M+Na ] , 540.09 [M-CH COOH+Na ] , 1177.23 [M +Na ] ;
3 2
(neg) 576.12 [M–H ] . IR: ν = 3260 bm (νN–H), 3194 bw (νN–H),
2987 w (νC–H), 2932 w (νC–H), 1718 m (νas C_O), 1625s (νas C_O),
1358 m, 1284 s, 1246 m, 1215 m, 1169 m, cm−1.
1
+ +
+ +
+ +
7
2
+ −
J
3
2
J
H,H = 7.1 Hz)
1
3
ppm. C NMR (DMF-d
6
1
7
): δ = 180.1 (C1), 176.3 (C1a′), 172.5 (C4),
0.1 (C5), 47.9 (Cen), 30.5 (C2, JPt,C = 39.9 Hz), 29.9 (C3), 21.7 (C2a′),
3
1
5
195
3.8 (C6) ppm. N NMR (DMF-d
7
): δ = −21.5 (NH
2
) ppm.
Pt
2.2.4.6.
(OC-6-23)-Triacetato(ethane-1,2-diamine)((4-pentoxy)-4-
NMR (DMF-d
7
): δ = 3494 ppm. ESI-MS (ACN/MeOH 1% H
2
O):
oxobutanoato)platinum(IV) (8c). General procedure III. 3-(Pentoxy-
carbonyl)propanoic anhydride (L6, 268 mg, 0.75 mmol, 1.6 eq), DMF
(8 mL), 5 (204 mg, 0.45 mmol), stirring in DMF for 4.5 h at 35 °C and
for 4.5 h at rt, column chromatography: EtOAc/MeOH (6:1–4:1). Yield
+
+
+ +
(
pos) m/z 686.15 [M+Na ] , 1349.31 [M +Na ] , 626.13
2
+
+
+ −
[
(
(
M-CH
ν
ν
3
COOH+Na ] ; (neg) 662.15 [M–H ] . IR: ν = 3226 bm
N–H), 3136 bm (νN–H), 2985 w (νC–H), 2924 w (νC–H), 1724 s
as C_O), 1649 s (νas C_O), 1626 s (νas C_O), 1359 m cm−1
.
141 mg (50%). Elemental analysis: C17
3
H
32
N
2
O
10Pt·0.3H
2
O; calcd. C
2.67, H 5.26, N 4.48; found C 32.61, H 5.23, N 4.46. H NMR (DMF-
): δ = 8.54 (bs, 2H, NH ), 8.47 (bs, 2H, NH ), 4.06 (t, 2H, H5,
H,H = 6.7 Hz), 2.80 (bs, 4H, Hen), 2.58–2.55 (m, 4H, H2) 2.53–2.51
1
2
4
.2.4.3. (OC-6-13)-Diacetato(ethane-1,2-diamine)bis((4-pentoxy)-
-oxobutanoato)platinum(IV) (7c). General procedure III. 3-
d
J
7
2
2
3
(
Pentoxycarbonyl)propanoic anhydride (L6, 505 mg, 1.41 mmol, 3 eq),
(m, 4H, H3), 1.97 (s, 6H, H2a′), 1.92 (s, 3H, H2a), 1.65–1.59 (m, 2H,
3
DMF (7 mL), 4 (182 mg, 0.45 mmol), stirring in DMF for 8 h at 35 °C
and for 1 h at rt, column chromatography: EtOAc/MeOH (1:0–10:1).
Yield 157 mg (47%). Elemental analysis: C24
H6), 1.36–1.30 (m, 4H, H7, H8), 0.91 (t, 3H, H9, JH,H = 6.7 Hz) ppm.
1
3
C NMR (DMF-d
7
): δ 180.0 (C1), 179.0 (C1a), 176.3 (C1a′), 172.6
3
44 2
H N O12Pt; calcd. C 38.55,
(C4), 64.2 (C5), 48.0 (Cen), 30.6 (C2, JPt,C = 37.3 Hz), 29.9 (C3), 28.3
(C6), 27.9 (C7) 22.2 (C8), 21.9 (C2a), 21.7 (C2a′), 13.5 (C9) ppm.
1
15
H 5.93, N 3.75; found C 38.66, H 6.26, N 3.96. H NMR (DMF-d
7
):
N
Please cite this article as: D. Höfer, et al., Tetracarboxylatoplatinum(IV) complexes featuring monodentate leaving groups — A rational approach
toward exploiting the platinum(IV) ..., J. Inorg. Biochem. (2015), http://dx.doi.org/10.1016/j.jinorgbio.2015.08.018

D. Höfer et al. / Journal of Inorganic Biochemistry xxx (2015) xxx–xxx
5
NMR (DMF-d
7
): δ = −21.8 (NH
2
), −21.5 (NH
2
) ppm. 195Pt NMR (DMF-
O): (pos) m/z 642.16
succinic acid mono ester was dried in vacuo and was used without fur-
ther purification.
d
7
): δ = 3495 ppm. ESI-MS (ACN/MeOH 1%H
2
+
+
+ +
+ −
[
M+Na ] , 582.14 [M-CH
3
COOH+Na ] ; (neg) 618.16 [M–H ] ,
–H ] . IR: ν = 3230 bm (νN–H), 2954 m (νC–H), 2860 w
C–H), 1740 m (νas C_O), 1622 s (νas C_O), 1361 m, 1293 s,
+
−
1
237.33 [M
2
2.2.5.1. 3-(Methoxycarbonyl)propanoic acid (L1). Methanol (20 mL,
0.5 mol, 10 eq), succinic anhydride (5.007 g, 0.05 mol), 2 h 70 °C. The
colorless highly viscous liquid was cooled in the freezer for 15 min to
(
ν
−
1
1
172 m cm
.
give a white crystalline solid which was dried in vacuo. Yield 6.371 g
1
2
.2.4.7. (OC-6-22)-(Ethane-1,2-diamine)tetrakis((4-methoxy)-4-oxo
(96%), H NMR (CDCl
3
): δ 3.74 (s, 3H, H5), 2.74–2.71 (m, 2H, H2/H3),
butanoato)platinum(IV) (9). General procedure III. (Methoxy
carbonyl)propanoic anhydride (L4, 280 mg, 1.14 mmol, 4.2 eq),
DMF (5 mL), 6 (150 mg, 0.27 mmol), stirring in DMF for 4 h at
2.68–2.65 (m, 2H, H2/H3) ppm.
2.2.5.2. 3-(Ethoxycarbonyl)propanoic acid (L2). Ethanol (8 mL, 0.14 mol,
4
5–50 °C and for 4 h at rt, column chromatography: EtOAc/
3 eq), succinic anhydride (5.027 g, 0.05 mmol), 1 h at 100 °C. Yield
1
MeOH (20:1–6:1). Yield 99 mg (47%). Elemental analysis:
C
3
NH
H
4.945 g (67%) colorless liquid. H NMR (CDCl
3
): δ = 4.19 (q, 2H, H5,
3
22
H
36
N
2
O
16Pt·0.4H
3.25, H 4.33, N 3.42. H NMR (DMF-d
), 3.66 (s, 6H, H5/H5′), 3.65 (s, 6H, H5/H5′), 2.80 (bs, 4H,
en), 2.64–2.61 (m, 4H, H2/H3/H2′/H3′), 2.56–2.51 (m, 12H, H2/
2
O; calcd. C 33.58, H 4.71, N 3.56; found C
JH,H = 7.1 Hz), 2.72–2.70 (m, 2H, H2/H3), 2.66–2.63 (m, 2H, H2/3),
1
3
7
): δ = 8.51 (bs, 4H,
1.28 (t, 3H, H6, JH,H = 7.1 Hz) ppm.
2
2.2.5.3. 3-(Pentoxycarbonyl)propanoic acid (L3). 1-Pentanol (5 mL,
1
3
H3/H2′/H3′) ppm. C NMR (DMF-d
7
): δ = 180.0 (C1/C1′), 177.6
0.05 mmol, 1.5 eq), succinic anhydride (3.122 g, 0.03 mmol), 14 h at
1
(
(
3
C1/C1′), 173.08 (C4/C4′), 173.05 (C4/C4′), 51.1 (C5/C5′), 51.0
C5/C5′), 48.0 (Cen), 30.6 (C2/C3/C2′/C3′), 30.2 (C2/C3/C2′/C3′),
73 °C and 10 h at rt. Yield 5.824 g (99%) colorless liquid. H NMR
3
(CDCl
3
): δ = 4.12 (t, 2H, H5, JH,H = 6.7 Hz), 2.73–2.70 (m, 2H, 2H/3H),
15
0.1 (C2/C3/C2′/C3′), 29.6 (C2/C3/C2′/C3′) ppm. N NMR (DMF-d
7
):
2.67–2.64 (m, 2H, 2H/3H), 1.69–1.63 (m, 2H, H6), 1.41–1.40 (m, 4H,
) ppm. 1 Pt NMR (DMF-d
O): (pos) m/z 802.16 [M+Na ] ; (neg) 778.17 [M–
H ] . IR: ν = 3238 bw (νN–H), 3194 bw (νN–H), 2953 w (νC–H),
95
): δ = 3486 ppm. ESI-MS
7H,8H), 0.93 (t, 3H, H9, JH,H = 7.0 Hz) ppm.
3
δ = −21.4 (NH
ACN/MeOH 1%H
2
7
+
+
(
2
+
−
2.2.6. General procedure V: synthesis of noncyclic anhydrides of succinic
acid mono esters
1
1
727 s (νas C_O), 1630 s (νas C_O), 1343 m, 1305 m, 1237 s, 2306 s,
159 s cm−1.
Acetic anhydride and the corresponding succinic acid mono ester
were heated to 110 °C for 1.5 h under anhydrous conditions. The clear
solution was allowed to cool down to room temperature and acetic
acid and the unreacted excess of acetic anhydride were removed
under reduced pressure while heating to 40 °C.
2
.2.4.8. (OC-6-33)-Diacetato(ethane-1,2-diamine)bis((4-methoxy)-4-
oxobutanoato)platinum(IV) (10). General procedure III. Acetic anhy-
dride (189 mg, 1.85 mmol, 4.7 eq), DMF (8 mL), 6 (217 mg, 0.39
mmol), stirring in DMF for 7 h at 35–40 °C and for 40 min at rt, column
chromatography: EtOAc/MeOH (4:1). Yield 123 mg (49%). Elemental
2.2.6.1. 3-(Methoxycarbonyl)propanoic anhydride (L4). Acetic anhydride
analysis: C16
H 4.59, N 4.43. H NMR (DMF-d
H, H5′), 2.79 (bs, 4H, Hen), 2.63–2.61 (m, 4H, H2′/H3′), 2.55–2.53
H
28
N
2
O
12Pt; calcd. C 30.24, H 4.44, N 4.41; found C 30.32,
(2.9 mL, 30.74 mmol, 4 eq), L1 (1.003 g, 7.59 mmol). Yield 896 mg
1
1
7
): δ = 8.56 (bs, 4H, NH ), 3.65 (s,
2
(96%) colorless liquid. H NMR (CDCl
3
): δ = 3.74 (s, 6H, H5), 2.83 (t,
3
3
6
4H, H2/H3, JH,H = 6.6 Hz), 2.70 (t, 4H, H2/H3, JH,H = 6.6 Hz) ppm.
1
3
(
m, 4H, H2′/H3′), 1.90 (s, 6H, H2a) ppm. C NMR (DMF-d
7
): δ =
1
78.9 (C1a), 177.5 (C1′), 173.0 (C4′), 51.0 (C5′), 48.1 (Cen), 30.2
2.2.6.2. 3-(Ethoxycarbonyl)propanoic anhydride (L5). Acetic anhydride
(12.5 mL, 132.7 mmol, 4 eq), L2 (4.848 g, 33.2 mmol). Yield 4.440 g
3
15
(
(
3
[
(
C2′/C3′), 30.1 (C2′/C3′), 22.0 (C2a, JPt,C = 38.3 Hz) ppm. N NMR
DMF-d
) ppm. 1 Pt NMR (DMF-d
O): (pos) m/z 658.12
M+Na ] ; (neg) 634.1 [M–H ] . IR: ν = 3270 bm (νN–H), 3016 w
C–H), 2934 w (νC–H), 1738 s (νas C_O), 1645 s (νas C_O), 1358 m,
95
): δ =
(98%) colorless liquid. H NMR (CDCl
1
): δ = 4.19 (q, 4H, H5, JH,H
3
=
7
): δ = −21.5 (NH
2
7
3
3
488 ppm. ESI-MS (ACN/MeOH 1%H
2
7.1 Hz), 2.82 (t, 4H, H2/H3,
J
H,H = 6.6 Hz), 2.69 (t, 4H, 2H/3H,
+
+
+ −
3
3
JH,H = 6.5 Hz), 1.29 (t, 6H, H6, JH,H = 7.1 Hz) ppm.
ν
1
265 s, 1224 m, 1163 m cm−1
.
2.2.6.3. 3-(Pentoxycarbonyl)propanoic anhydride (L6). Acetic anhydride
9 mL, 95.83 mmol, 6 eq), L3 (3.004 g, 15.96 mmol). Yield 2.853 g
(
1
3
2
.2.4.9. (OC-6-22)-Tetraacetato(ethane-1,2-diamine)platinum(IV) (11).
(99.7%) colorless liquid. H NMR (CDCl
3
): δ = 4.13 (t, 4H, H5, JH,H
H,H = 6.6 Hz), 2.69 (t, 4H, 2H/3H,
JH,H = 6.6 Hz), 1.69–1.63 (m, 4H, H6), 1.41–1.31 (m, 8H, H7,H8), 0.93
=
3
(OC-6-22)-(Ethane-1,2-diamine)tetrahydroxidoplatinum(IV) (64 mg,
6.8 Hz), 2.82 (t, 4H, H2/H3, J
3
0
.20 mmol) was suspended in acetic anhydride (18 mL) and the reac-
3
tion mixture was left stirring for 10 days in the dark. The solvent was re-
moved under reduced pressure and the solid was suspended in acetone.
The product was collected by filtration and was recrystallized from
(t, 6H, H9, JH,H = 6.9 Hz) ppm.
2.3. Crystallographic structure measurements
acetone/H
calcd. C 24.44, H 4.10, N 5.70; found C 24.33, H 4.14, N 5.61.
NMR (DMF-d ): δ = 8.52 (bs, 4H, NH
H, H2a/H2a′), 1.91 (s, 6H, H2a/H2a′) ppm. C NMR (DMF-d
δ = 178.9 (C1a/C1a′), 176.2 (C1a/C1a′), 48.0 (Cen), 22.0 (C2a/C2a′),
2
O. Yield 52 mg (53%). Elemental analysis: C10
H
20
N
2
O
8
Pt;
H
1
X-ray diffraction measurements of 5, 7b and 8b were performed on a
Bruker D8 VENTURE system at 100 K with MoKα (λ = 0.71073 Å). For 5,
7b and 8b single crystals were positioned at 40, 30 and 40 mm from the
detector and 2680, 806 and 2051 frames were measured each for 12, 10
and 10 s over a 0.4, 0.5 and 0.5° scan width. The data were processed
using SAINT [26]. Crystal data, data collection parameters and structure
refinements details, are given in Table 1.
Absorption corrections were performed by SADABS [27] with multi-
scan absorption correction. Structures were solved by direct and charge
flipping methods and refined by full-matrix least-squares techniques.
Non-H atoms were refined with anisotropic displacement parameters.
H atoms were inserted in calculated positions and refined with riding
and rotating models. Structure solution was achieved with SHELXS
[28] and Olex2 [29] and refinements were carried out with SHELXL
[28], Olex2 [30] and Shelxle [31]. Molecular graphics were implemented
7
2
), 2.80 (bs, 4H, Hen), 1.97 (s,
1
3
6
7
):
1
5
2
1.7 (C2a/C2a′) ppm. N NMR (DMF-d
7
): δ = −21.6 (NH
2
) ppm.
O):
COOH+Na ] ; (neg)
90.08 [M–H ] ,. IR: ν = 3214 bm (νN–H), 3019 w (νC–H), 2930 w (νC–
1
95
Pt NMR (DMF-d
pos) m/z 514.08 [M+Na ] , 454.05 [M-CH
7
): δ = 3496 ppm. ESI-MS (ACN/MeOH 1%H
2
+
+
+ +
(
3
+
−
4
−
1
H
), 1645 s (νas C_O), 1621 s (νas C_O), 1356 m, 1276 s cm
.
2
.2.5. General procedure IV: synthesis of succinic acid mono esters
Succinic anhydride was refluxed in an excess of the respective
alcohol under anhydrous conditions and the remaining alcohol was re-
moved under reduced pressure. Chloroform was added to precipitate
succinic acid which was subsequently removed by filtration. The
Please cite this article as: D. Höfer, et al., Tetracarboxylatoplatinum(IV) complexes featuring monodentate leaving groups — A rational approach
toward exploiting the platinum(IV) ..., J. Inorg. Biochem. (2015), http://dx.doi.org/10.1016/j.jinorgbio.2015.08.018

6
D. Höfer et al. / Journal of Inorganic Biochemistry xxx (2015) xxx–xxx
Table 1
Crystal data, data collection parameters and structure refinement details of 5, 7b and 8b.
5
7b
8b
Empirical formula
Formula weight
Temperature/K
Crystal system
Space group
a [Å]
b [Å]
c [Å]
α [°]
C
11
H
25
N
3
O
8
Pt
C
18
H
32
N
2
O
12Pt
14 32 2
C H N O13Pt
631.50
100 (2)
522.43
100 (2)
663.54
100 (2)
Triclinic
P-1
9.4659 (4)
11.8051 (5)
Monoclinic
P 2 /c
11.4972 (6)
10.1344 (5)
14.1175 (7)
90
Monoclinic
P 2 /c
1
1
10.6172 (6)
25.3437 (17)
8.8791 (6)
90
12.2600 (5)
82.5890 (12)
β [°]
93.0981 (14)
71.0933 (13)
105.3501 (18)
γ [°]
Volume [Å ]
90
69.0017 (13)
1209.89 (9)
90
3
1642.53 (14)
2303.9 (3)
Z
ρ
4
2
4
calc [g · cm−3]
1
2.113
8.587
1.821
5.861
1.821
6.153
μ [mm−
]
F (000)
1016.0
0.168 × 0.037 × 0.024
3.548 to 60.068
656.0
0.131 × 0.124 × 0.106
4.832 to 50.7
1248.0
0.125 × 0.06 × 0.01
4.29 to 50.71
3
Crystal size [mm ]
Θ range for data collection [°]
2
Index ranges
−16 ≤ h ≤ 16, −14 ≤ k ≤ 14, −19 ≤ l ≤ 19
−11 ≤ h ≤ 11, −14 ≤ k ≤ 14, −14 ≤ l ≤ 14
−12 ≤ h ≤ 12, −30 ≤ k ≤ 30, −10 ≤ l ≤ 10
Reflections collected
Independent reflections
58,445
4807
16,581
4405
66,470
4222
[
R
int = 0.0268, Rsigma = 0.0126]
[Rint = 0.0386, Rsigma = 0.0336]
[Rint = 0.0391, Rsigma = 0.0153]
Data/restraints/parameters
Goodness-of-fit on F
4807/0/217
1.111
4405/3/302
1.070
4222/6/300
1.051
2
Final R indexes [I N =2σ (I)]
R
1
= 0.0124
wR = 0.0273
= 0.0154
wR = 0.0280
R
1
= 0.0268
wR = 0.0599
= 0.0327
wR = 0.0621
R
1
= 0.0166
wR = 0.0352
= 0.0208
wR = 0.0365
0.78/−0.52
2
2
2
Final R indexes [all data]
R
1
R
1
R
1
2
2
2
Largest diff. peak/hole [eÅ−3]
0.65/−0.53
1.29/−0.69
2
− F2
2
2
2
2
2
2
2
1/2
R
1
= Σ||F
o
| − |F
c
||/Σ|F
o
|. wR
2
= {Σ[w(F
o
c
) ] / Σ[w(F
o
) ]}i . GOF = {Σ[w(F
o
− F
c
) ] / (n − p)} , where n is the number of reflections and p is the total number of parameters refined.
with Olex2 [29]. X-ray results for 2 are available in the supplementary
data. The structure was not uploaded to the CCDC because of uncer-
tainties in anisotropic behavior at one atom position (C3).
2.5. Stability measurements by RP-HPLC
Stability of the synthesized Pt(IV) complexes was investigated by
means of analytical reversed phase HPLC measurements. HPLC condi-
2
.4. Determination of lipophilicity by RP-HPLC
w
tions were the same as for logk determination. The investigated com-
pounds (0.5 mM) were incubated in water and PBS (phosphate buffered
saline) solution (pH = 7.4) at 37 °C for 24 h. Each sample was run with a
MeOH/0.1% aqueous TFA mobile phase immediately after dissolution,
after 2.5 h, after 5 h and after 24 h of incubation. Gradient eluent condi-
tions were as follows: 5%–40% MeOH in 14 min, 40%–95% MeOH in
9 min. All peaks were integrated relative to the sum of all peaks in the
chromatogram.
The lipophilicity, i.e. the capacity factor k, was determined by analyt-
ical reversed phase HPLC analysis, which were performed on a Dionex
Summit System controlled by Dionex Chromeleon 6.60 software. HPLC
conditions were as follows: silica-based Xbridge C18 (4.6 × 150 mm,
5
μm) column; mobile phase: MeOH/0.1% TFA (2,2,2-trifluoroethanoic
acid) in water (Milli-Q, 18.2 MΩ cm, Milli-Q Advantage A10, Darmstadt,
Germany), column temperature 25 °C, flow rate 1 mL/min, injection
volume 20 μL, UV–vis detection set at 210, 225, 250 and 300 nm;
sample concentration: 0.5 mM in MeOH/0.1%TFA (1:1); concentra-
tion of the external dead volume marker KI: 0.3 mM in MeOH/
2.6. NMR experiments: incubation with ascorbic acid and stability of 2
Reduction of isomers 7a and 10 by ascorbic acid was monitored by
1
0
.1%TFA (1:1). All samples were filtered through a 0.45 μm Nylon fil-
ter (Minisart RC 25, Sartorius AG, Göttingen, Germany) before injec-
tion. Capacity factors k were determined as k = (t − t )/t , where t
and t denote the retention time of the analyte and the retention
time of the external reference KI (dead volume marker), respective-
ly. Retention times t were determined for at least 3 different MeOH/
0.1% TFA) ratios (two independent experiments each) in an
isocratic mode in order to obtain capacity factors k located in the lin-
ear range of −0.5 b logk b 1.5 [32]. Logk values were determined by
extrapolation of logk to 100% aqueous mobile phase according to the
linear Soczewínski–Snyder relationship logk = logk
where k is the capacity factor in the measured mobile phase compo-
sition, k is the extrapolated capacity factor in 100% H O, SMeOH is a
H NMR spectroscopy at 25 °C. The compounds were prepared in
50 mM phosphate buffer (in D
2
O, pD = 7.4) and 1H NMR spectra
R
0
0
R
were recorded over a period of 6 h in order to check the stability of
the complexes. During this time no changes in the NMR spectra could
be detected. Ascorbic acid was added to the NMR sample to yield final
0
1
R
concentrations of 25 mM ascorbic acid and 1 mM complex. H NMR
1
(
spectra were recorded up to a period of 10 days. H NMR experiments
2
e.g. reduction of 7a and 10 as well as aquation of 2 in D O were followed
w
by the change of intensity of the CH signal of the acetato ligands. Inte-
3
gration of the signals was performed relative to the sum of all signals de-
riving from the respective (coordinated and non-coordinated) ligands.
Aquation of 2 was monitored by 1H NMR spectroscopy at 25° in
w
− SMeOHφ
w
2
2
D O. Integration of the well separated signals deriving from the
constant for a substance and a given HPLC system and φ is defined
as the percentage of organic modifier in the mobile phase (in this
diacetato- and monoacetatomonohydroxidoplatinum(II) complexes
and from the free acetato ligands provided a detailed insight into the
case MeOH). Another parameter for describing the lipophilicity φ
was calculated from logk by: φ = −logk /S, where S is the slope
0
aquation process. The ratio of the 3 platinum(II) species [Pt(en)(OAc)
2
],
+
2+
w
0
w
2 2 2
[Pt(en)(OAc)(OH )] and [Pt(en)(OH ) ]
was followed over a period
obtained by plotting logk values as a function of the percentage of
the organic modifier in the mobile phase [33].
of 16 h. The exchange of acetato ligands by chloride was investigated in
aqueous sodium chloride solution at 25 °C over a period of 12 h. Sodium
Please cite this article as: D. Höfer, et al., Tetracarboxylatoplatinum(IV) complexes featuring monodentate leaving groups — A rational approach
toward exploiting the platinum(IV) ..., J. Inorg. Biochem. (2015), http://dx.doi.org/10.1016/j.jinorgbio.2015.08.018

D. Höfer et al. / Journal of Inorganic Biochemistry xxx (2015) xxx–xxx
7
chloride was applied in 10-fold excess. Due to the overlapping signals of
Pt(en)(OAc) ] and [Pt(en)Cl(OAc)], only the extent of released acetato
3. Results and discussion
[
2
ligand could be monitored, which was referenced to the protons of the
ethane-1,2-diamine ligand.
3.1. Synthesis
Dicarboxylatoplatinum(II) complexes 2 and 3 were synthesized by
adapting reported procedures [24] (Scheme 2) with some modifications
featuring yields over 70%. Treatment of compounds 2 and 3 with an
2
.7. Cell lines and culture conditions
CH1 cells (identified via short tandem repeat, STR, profiling as PA-1
2 2
aqueous H O solution (15%) afforded the symmetric dihydroxido
ovarian teratocarcinoma cells by Multiplexion, Heidelberg, Germany;
compare [34]) were obtained from Lloyd R. Kelland (CRC Centre for
Cancer Therapeutics, Institute of Cancer Research, Sutton, UK). A549
complexes 4 and 6 (Scheme 3). As a result of the fast hydrolysis of
dicarboxylatoplatinum(II) complexes (monodendate carboxylato li-
gands) only moderate yields were achieved in these oxidation reactions.
Unsymmetric oxidation was realized with peracetic acid in acetic acid in
order to obtain octahedrally coordinated (OC-6-33)-triacetato(ethane-
1,2-diamine)hydroxidoplatinum(IV) in 66% yield. This approach failed
when applied to 3 due to isomerization, resulting in the formation of a
mixture of products. The carboxylation reactions with noncyclic anhy-
drides of succinic acid mono esters were performed in DMF under anhy-
drous conditions at 35–45 °C [35]. The final products (7a–c, 8a–c, 9 and
10, Schemes 3 and 4) were obtained after purification by column
chromatography as white powders in moderate yields (45–60%). The
respective noncyclic anhydrides were synthesized by mono esterifica-
tion of succinic anhydride with the corresponding alcohols (methanol,
ethanol and 1-pentanol) and subsequent heating (110 °C) in acetic
anhydride. The herein demonstrated methodology for carboxylation
of mono- and dihydroxidoplatinum(IV) complexes represents a
considerable improvement to previous procedures in which carboxyla-
tion with cyclic anhydrides is followed by derivatization with alcohols
[11,35,36]. The utilization of pre-built noncyclic anhydrides of dicarbox-
ylic acid monoesters increases the overall yield and allows saving of one
time-consuming reaction step. Moreover, this method broadens the
spectrum of complexes, which can serve as reactants, since the proce-
dure does not include the synthesis of complexes with free carboxylic
groups, which are difficult to isolate in some cases.
(
non-small cell lung carcinoma) as well as SW480 (colon carcinoma)
cells were kindly provided by Brigitte Marian (Institute of Cancer
Research, Department of Medicine I, Medical University of Vienna,
2
Austria). Cell monolayer adherent cultures were grown in 75 cm cul-
ture flasks (CytoOne, Starlab, UK) in complete medium [i.e., minimal es-
sential medium (MEM) supplemented with 10% heat-inactivated fetal
bovine serum, 1 mM sodium pyruvate, 2 mM L-glutamine, and 1% non-
essential amino acids from 100× ready-to-use stock (all purchased
from Sigma-Aldrich, Austria)]. Cell cultures were incubated at 37 °C in
a moist atmosphere containing 5% CO
.8. Cytotoxicity tests in cancer cell lines
Cytotoxic activity of the compounds was determined by means of
2
in air.
2
the colorimetric microculture MTT assay (MTT = 3-(4,5-dimethyl-2-
thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide). Cells were seeded
into 96-well microculture plates (CytoOne, Starlab, UK) in densities
of 1 × 10 cells (CH1/PA-1), 2 × 103 cells (SW480) and 3 × 103
cells (A549) in a volume of 100 μL per well. Cells were allowed to
settle for 24 h to resume adherent growth prior to drug exposure.
For testing, the drugs were dissolved and serially diluted in complete
medium and then added in aliquots of 100 μL per well. After continuous
exposure for 96 h, drug solutions were replaced with 100 μL of RPMI
medium/MTT mixture [6:1 of RPMI 1640 medium (supplemented
with 10% heat-inactivated fetal bovine serum and 2 mM L-glutamine)/
MTT solution in phosphate-buffered saline (5 mg/mL)]. After incubation
for 4 h, the medium/MTT mixtures were removed, and the formazan
crystals formed by viable cells were dissolved in 150 μL of DMSO
per well. Optical densities at 550 nm were measured with a micro-
plate reader (ELx808 Absorbance Microplate Reader, Bio-Tek, USA),
using a reference wavelength of 690 nm to correct for unspecific
absorption. The quantity of viable cells was expressed as the per-
centage of untreated controls, and 50% inhibitory concentrations
3
3.2. Characterization
All novel platinum complexes were characterized in detail by ele-
mental analysis, electrospray ionization mass spectrometry (ESI-MS),
1
13 15
195
IR and multinuclear ( H, C, N,
Pt) NMR spectroscopy as well as
by X-ray diffraction in the case of 2, 5, 7b and 8b.
3.2.1. Characterization by NMR, IR and ESI-MS
NMR investigations, including one- and two-dimensional H, 13C
1
and 1 Pt as well as 1H, N correlation measurements, allowed in
95
15
195
(
IC50) were calculated from concentration-effect curves by interpo-
depth characterization of the synthesized compounds. Selected
Pt,
1
15
13
lation. Evaluation is based on means from at least three indepen-
dent experiments, each comprising triplicates per concentration
level.
H, N and C chemical shifts are listed in Table 2.
Acquisition of 1 Pt NMR was applied to judge the oxidation state of
95
platinum and to investigate the influence of the ligand sphere and
Scheme 2. Synthesis of dicarboxylatoplatinum(II) complexes 2 and 3 and the tetraacetato complex 11. (i) KI, ethane-1,2-diamine; (ii) Ag
Ba(OCOCH CH COOCH
2 4 2 2 4
SO , Ba(OAc) ; (iii) Ag SO ,
2
2
3 2
) .
Please cite this article as: D. Höfer, et al., Tetracarboxylatoplatinum(IV) complexes featuring monodentate leaving groups — A rational approach
toward exploiting the platinum(IV) ..., J. Inorg. Biochem. (2015), http://dx.doi.org/10.1016/j.jinorgbio.2015.08.018

8
D. Höfer et al. / Journal of Inorganic Biochemistry xxx (2015) xxx–xxx
Scheme 3. Symmetric and unsymmetric oxidation of 2 and 3 and subsequent carboxylation with noncyclic anhydrides of succinic acid monoesters. (i) 15% H
2 2 2 3
O /H O; (ii) CH COOOH/
CH COOH; (iii) 3 eq (ROCOCH CH CO) O/DMF; (iv) 1.5 eq (ROCOCH CH CO) O/DMF; (v) 15% H /H O; (vi) 4.2 eq (CH OCOCH CH CO) O/DMF; (vii) (AcO)
3
2
2
2
2
2
2
2
O
2
2
3
2
2
2
2
O.
geometry on the chemical shift of the central ion. The oxidation from
3284 ppm and for tetracarboxylato complexes 7a-11 195Pt signals were
observed between 3486–3496 ppm. Interestingly, an exchange of acetato
ligands in axial position with longer 4-alkoxy-4-oxobutanoato chains
showed no influence on the resonance of the platinum nucleus while
such an exchange in the equatorial position, resulted in a considerable
difference of the chemical shift (e.g. 10, 3488 ppm; 7a, 3494 ppm).
Pt(II) to Pt(IV) was accompanied by a characteristic downfield shift of
1
95
more than 3000 ppm.
Pt(IV) chemical shifts displayed clear differ-
ences between monohydroxido, dihydroxido, and tetracarboxylated
platinum(IV) compounds. Dihydroxido complexes 4 and 6 were
found at 3185–3195 ppm, the monohydroxido complex 5 resonated at
Scheme 4. NMR numbering scheme for synthesized complexes.
Please cite this article as: D. Höfer, et al., Tetracarboxylatoplatinum(IV) complexes featuring monodentate leaving groups — A rational approach
toward exploiting the platinum(IV) ..., J. Inorg. Biochem. (2015), http://dx.doi.org/10.1016/j.jinorgbio.2015.08.018

D. Höfer et al. / Journal of Inorganic Biochemistry xxx (2015) xxx–xxx
9
Table 2
Selected H,13C, 15N and 195Pt chemical shifts of synthesized compounds in D
1
O (2–4), D
O/DMF-d
(6) and DMF-d
(5, 7–11) in ppm (protons 2a′ and 2a and carbon atoms 1a′, 1a, 2a′, and
2
2
7
7
2a are those of the acetato ligand, dash for equatorial ligands, compare Scheme 3).
Complex
195Pt
1H
13C
15N
NH
2
2a′
2a
1a′
1a
2a′
2a
NH
2
2
3
4
6
5
−286
−273
3185
3195
3284
3494
3494
3495
3494
3495
3495
3486
3488
3496
5.24
5.22
–
–
8.63, 7.44
8.48
8.49
1.90
182.1
21.9
2.06
181.2
22.1
1.94
1.97
1.97
1.97
1.97
1.97
1.97
1.84
177.4
176.3
176.3
176.3
176.3
176.3
176.3
179.6
22.3
21.7
21.7
21.7
21.7
21.7
21.7
23.3
−20.0, −19.9
−21.6
−21.5
−21.4
−21.2
−21.7
−21.8, −21.5
−21.4
−21.5
−21.6
7
7
7
8
8
8
9
1
1
a
b
c
a
b
c
8.49
8.47, 8.53
8.48, 8.54
8.47, 8.54
8.51
8.56
8.52
1.92
1.92
1.92
179.0
179.0
179.0
21.9
21.9
21.9
0
1
1.90
1.91
178.9
178.9
22.0
22.0
1.97
176.2
21.7
1H NMR spectra of symmetrically and unsymmetrically coordinated
complexes showed significant differences. Symmetric tetracarboxylato
complexes 7a–c displayed NH resonances as a broad singlet at around
.49 ppm whereas unsymmetric complexes 8a–8c featured a marked
splitting of this signal giving one singlet at 8.54 and the other one
at 8.47 ppm. Acetato ligands in axial position gave resonances at
resonances detected at 179 ppm (C1a) in complexes 7a–8c and 11. In
C NMR spectra, C2 and C3 could be assigned due to the observed plat-
13
2
inum satellites with a coupling constant of ca. 37 Hz, resulting from cou-
95
15
8
pling with the 1 Pt nucleus. Finally, N chemical shifts of coordinated
ethane-1,2-diamine of complexes 5 and 7–11 were found between
−20 and −21.8 ppm in accord with data of analogous platinum(IV)
complexes found in the literature [37].
1
.92 ppm (H2a) in comparison to equatorial acetato ligands, displaying
a slight downfield shift (H2a′ at 1.97 ppm).
IR spectra of all compounds 2–11 showed broad bands
−
1
An inverse effect was observed for carbonyl atoms of acetato ligands.
Chemical shifts of C_O atoms of equatorially coordinated acetate were
found at 176.3 ppm (C1a′), while axial coordination resulted in carbonyl
[3281–3073 cm ] attributed to N–H stretching vibrations, followed
−
1
by medium to weak C–H bands from 3048–2860 cm in most cases.
Very intensive infrared absorption was observed in the region of
Fig. 2. ORTEP views of 2, 5, 7b and 8b with atom labeling scheme. Thermal ellipsoids are drawn at the 50% probability level.
Please cite this article as: D. Höfer, et al., Tetracarboxylatoplatinum(IV) complexes featuring monodentate leaving groups — A rational approach
toward exploiting the platinum(IV) ..., J. Inorg. Biochem. (2015), http://dx.doi.org/10.1016/j.jinorgbio.2015.08.018

10
D. Höfer et al. / Journal of Inorganic Biochemistry xxx (2015) xxx–xxx
740–1579 cm− resulting from C_O bond stretching. IR spectra of 4–6
1
3.3. Lipophilicity
1
displayed
characteristic
ν(PtO–H)
stretching
vibrations
−
1
[
3559–3412 cm ], which disappeared after carboxylation.
ESI-MS spectra were measured in the positive and negative ion
mode. In all cases, isotopic distribution patterns and m/z values agreed
Lipophilicity represents an important measure to estimate the phar-
macokinetic behavior of metal-based drugs as well as their cytotoxicity
which is also dependent on drug accumulation [11,14]. The latter is
based on a facilitated penetration through membranes by lipophilic
compounds. The lipophilicity of the synthesized Pt(IV) complexes was
determined by RP-HPLC analysis, and is described via the chromato-
+
+
with calculated data. In the positive mode [M+Na ] ions were pre-
dominant in all spectra. Furthermore, loss of CH COOH and
+Na ] ions were observed in some cases. In the negative ion
3
+
+
[
M
2
+
−
mode, all MS spectra exhibited the [M–H ] fragment.
w 0
graphic retention parameters logk and φ . Lipophilicity determination
by RP-HPLC represents a highly reproducible and fast method, which is
independent from concentrations and therefore allows easy determina-
tion of the capacity factor k. The latter describes the ratio of the equilib-
rium concentration of a compound between a stationary phase, which
mimics an apolar organic phase (C18) and a polar phase given by the
aqueous eluent with an organic modifier (MeOH). Extrapolation to
3
.2.2. Crystal structures
X-ray diffraction studies were performed for complexes 2, 5, 7b and
b. The results are shown in Fig. 2. Crystal data, data collection parame-
8
ters and structure refinement details for 5, 7b and 8b are given in
Table 1 and selected bond lengths and angles are listed in Table 3,
respective data for 2 are available in the supplementary data (the
structure was not uploaded to the CCDC because of uncertainties in an-
isotropic behavior at one atom position (C3)). The platinum(II) complex
100% H
2
O results in logk
w
values which ensure comparability and esti-
is defined as the percentage
mation of the respective lipophilicities. φ
0
of organic modifier in the mobile phase, where the logk value is zero
for a given compound (the distribution of a compound is equal in the
stationary and the mobile phase [33]).
2
as well as the unsymmetric complexes 5 and 8b crystallized in the
monoclinic space group P 2 /c while symmetric complex 7b crystallized
1
in the triclinic space group P-1. The platinum(IV) center in 7b and 8b
features a distorted octahedral coordination sphere with equatorial
O1-Pt-O2 angles at around 78.68° (78.27° 7b, 79.28° 8b) and axial O3-
Pt-O4 angles of 176.27° (177.05° 7b, 175.46° 8b). This deformation
can be ascribed to intramolecular hydrogen bonds between amine and
carboxylato ligands. In 7b hydrogen bonds are formed between amines
and equatorial acetato ligands with donor–acceptor contacts ranging
from 2.82 Å (N1 · · · O9) to 2.87 Å (N2 · · · O8). Hydrogen bonds
between amines and axial carboxylato ligands exhibit donor–acceptor
distances between 2.74 Å (N1 · · · O10) and 2.76 Å (N2 · · · O5).
The Pt–O bonds in equatorial position 2.0206 Å (2.012–2.028 Å) are
slightly longer than axial ones at 2.005 Å (2.000–2.009 Å) probably
caused by a difference in trans-influence of the opposing nitrogen and
oxygen atoms. Pt–N bond distances are found at around 2.04 Å. These
observations are consistent with data for similar compounds found in
the literature [25,38]. The chelating ethane-1,2-diamine ligand forms a
The set of investigated compounds comprises a wide range of logk
w
values ranging from 0.44 for the most hydrophilic compound 11 to 4.58
for the most lipophilic compound 7c (Table S3). Nevertheless, the com-
bination of equatorial and axial carboxylate ligands provides good water
solubility allowing cell culture studies even for the most lipophilic com-
pound 7c.
3.4. Cytotoxicity in human cancer cell lines
In vitro cytotoxicity of platinum(II) complexes 2 and 3 and of
platinum(IV) complexes 7a–11 was investigated by means of the MTT
colorimetric assay in three human cancer cell lines (CH1/PA-1, SW480,
A549, Table 4). Hydroxido species 4–6 were not evaluated since purifi-
cation in the required quality could not be achieved.
In general, all novel complexes displayed a better activity in the cis-
platin sensitive cell line CH1/PA-1 compared to SW480 and A549 cancer
cells, which are known to be inherently cis- and carboplatin resistant.
Platinum(II) complexes 2 and 3 show IC50 values between those of cis-
platin and carboplatin, but closer to that of cisplatin. This circumstance
is explainable by the following features: (i) Pt(II) complexes, bearing
monodentate carboxylato leaving groups are subject to faster ligand–
exchange reactions (e.g. aquation, exchange with chloride, etc.), com-
pared to analogs, featuring bidentately coordinated dicarboxylato
ligands (e.g. carboplatin, see next chapter) and therefore behave more
similarly to dichlorido complexes. (ii) The decreased cytotoxicity,
5
-membered ring with the platinum center and gives a distorted
envelope conformation. The deviation from planarity can be described
by the torsion angle ΘN1–C1–C2–N1 which is 52.0(6)° for 2, −55.47(16)°
for 5, –51.6(5)° for 7b and 53.3(3)°for 8b [9].
Table 3
Selected bond lengths and angles of 5, 7b and 8b.
Bond lengths [Å]
5
7b
2.028 (3)
2.022 (3)
2.009 (3)
2.000 (3)
2.042 (4)
2.036 (3)
8b
Pt-O1
Pt-O2
Pt-O3
Pt-O4
Pt-N1
Pt-N2
2.0204 (12)
2.0293 (12)
1.9772 (12)
2.0400 (12)
2.0273 (14)
2.0251 (14)
2.0205 (17)
2.0118 (17)
2.0036 (18)
2.0055 (17)
2.033 (2)
Table 4
Lipophilicity (logk
cancer cell lines.
w
values) and cytotoxicity of investigated complexes in three human
2.036 (2)
a
Complex
logk
w
IC50 [μM]
Angles [°]
5
7b
8b
CH1/PA-1
SW480
A549
O4-Pt-O1
O4-Pt-O2
O4-Pt-O3
O4-Pt-N1
O4-Pt-N2
O1-Pt-O2
O1-Pt-O3
O1-Pt-N2
O2-Pt-O3
O2-Pt-N1
O3-Pt-N1
O3-Pt-N2
N1-Pt-N2
89.00 (5)
89.46 (5)
176.44 (5)
95.69 (5)
92.41 (5)
82.54 (5)
87.66 (5)
95.32 (5)
91.31 (5)
98.26 (5)
87.64 (5)
86.69 (6)
83.71 (6)
89.00 (13)
88.95 (12)
177.07 (12)
93.52 (15)
86.41 (14)
79.09 (12)
89.28 (13)
99.42 (14)
88.39 (12)
99.48 (13)
88.14 (14)
96.21 (14)
82.21 (15)
88.00 (7)
85.40 (7)
175.45 (7)
88.43 (8)
95.07 (8)
78.27 (7)
87.48 (7)
100.38 (8)
93.24 (7)
99.12 (8)
96.07 (8)
86.19 (8)
82.26 (9)
2
3
0.40 ± 0.10
0.41 ± 0.08
6.8 ± 2.0
5.5 ± 0.5
0.059 ± 0.02
11 ± 3
6.4 ± 1.9
2.1 ± 0.5
15 ± 1
24 ± 1
11 ± 3
0.16 ± 0.03
1.4 ± 0.4
14 ± 2
14 ± 1
13 ± 3
20 ± 5
N400
N160
5.8 ± 0.4
N400
274 ± 51
N100
N160
7
7
7
8
8
8
9
1
1
a
b
c
a
b
c
1.45 ± 0.003
2.13 ± 0.013
4.58 ± 0.000
1.07 ± 0.006
1.29 ± 0.016
2.73 ± 0.008
2.57 ± 0.002
1.69 ± 0.004
0.44 ± 0.003
226 ± 53
112 ± 24
2.4 ± 0.5
345 ± 34
161 ± 45
32 ± 11
N160
0
1
N160
N160
327 ± 63
3.5 ± 0.3
85 ± 28
280 ± 82
1.3 ± 0.4
91 ± 10
Cisplatin [17]
Carboplatin [11]
a
Torsion angle [°]
5
7b
8b
50% inhibitory concentrations in CH1/PA-1, A549 and SW480 cells in the MTT assay
96 h exposure). Values are the mean ± standard deviations obtained from at least
three independent experiments.
(
Θ
N1–C1–C2–N1
−55.47 (16)
−51.6 (5)
53.3 (3)
Please cite this article as: D. Höfer, et al., Tetracarboxylatoplatinum(IV) complexes featuring monodentate leaving groups — A rational approach
toward exploiting the platinum(IV) ..., J. Inorg. Biochem. (2015), http://dx.doi.org/10.1016/j.jinorgbio.2015.08.018

D. Höfer et al. / Journal of Inorganic Biochemistry xxx (2015) xxx–xxx
11
compared to cisplatin could be attributed to the different carrier ligands.
It has been shown, that [Pt(en)Cl ] displays slightly higher IC50 values
than [Pt(NH Cl ] in both cisplatin sensitive and resistant cell lines
39]. However, compounds 2 and 3 are equipped with a better water
2
3
)
2
2
[
2
solubility compared to their dichlorido analog [Pt(en)Cl ] and cisplatin,
which offers advantages for further biological studies.
The exchange of one or both axial acetates of 11 with 4-alkoxy-4-
oxobutanoato ligands increases the lipophilicity, as well as the cytotox-
icity of the complexes (7a–c and 8a–c) without compromising the
aqueous solubility. Intriguingly, substituting equatorial acetates with
4
-methoxy-4-oxobutanoate lead to the opposite effect (compare IC50
values for 7a and 9, and 10 and 11 in CH1/PA-1 cells). Moreover, 10
was four times less cytotoxic in CH1/PA-1 cells than its isomer 7a. This
difference cannot be attributed to an alteration in lipophilicity, as both
w 0
isomers expectedly showed similar logk (resp. φ ) values.
In general, a correlation between cytotoxicity and lipophilicity can
be drawn (Fig. 3). Thereby, IC50 values increase in the subseries in the
order: 7c b 7b b 7a and 8c b 8b b 8a, inversely correlating with
2 2
Fig. 4. Aquation of platinum(II) complex 2, [Pt(en)(OAc) ], in D O at 25 °C.
w
their logk values. Symmetric complexes 7a–7c display a higher anti-
proliferative potency compared to their unsymmetrically coordinated
analogs 8a–8c, which is explainable by distinct differences in lipophilic-
ity (compare Table 4). The most lipophilic complex 7c exhibits the low-
est IC50 values (in the nanomolar range in CH1/PA-1 cells) and shows
even a higher cytotoxicity than its platinum(II) counterpart 2. This be-
havior is in accord with previous studies revealing that by increasing
the lipophilicity of diam(m)inebis(carboxylato)dichloridoplatinum(IV)
complexes (cisplatin analogs), compounds with higher cytotoxicity
compared to their platinum(II) analogs can be obtained [36,40,41]. In
contrast, this approach failed in the case of diamminetetracarboxylato
platinum(IV) complexes designed as prodrugs of carboplatin [11]
since the platinum(II) compound generated via reduction is inert due
to a chelating dicarboxylato ligand.
The increased activity of the novel tetracarboxylato complexes in
comparison to the respective carboplatin analogs may be ascribed to
the higher reactivity of the resulting platinum(II) complex featuring
monodentate carboxylato ligands. In order to evaluate this hypothesis,
as well as to find an explanation for the difference in cytotoxicity, espe-
cially of isomers 7a and 10, further investigations regarding the stability
and rate of reduction of Pt(IV) complexes (e.g. 7a and 10) were per-
formed. In addition, the aquation of complex 2 was studied (see next
chapters).
and 32% of acetate was uncoordinated. This represents a clear difference
to carboplatin which shows high stability in aqueous solution and can
be stored for 2 years in ready-to-use infusions [42]. As expected, the ex-
change of acetato ligands by chloride in NaCl solution was also fast
(
Fig. 5). After 12 h 35% of the acetato ligands were uncoordinated.
Since aquation plays an important role in the mode of action of platinum
complexes, this difference will also affect the cytotoxicity. The faster the
activated aqua complexes are formed, the faster a coordination to the
DNA is possible and therefore the chance of drug efflux can be reduced
[4]. Consequently, the enhanced reactivity might be an explanation for
the higher cytotoxicity of tetracarboxylatoplatinum(IV) complexes
bearing monodentate ligands compared to their analogs, featuring
bidentate coordinated dicarboxylates (e.g. carboplatin prodrugs) [11].
3.6. Aqueous stability of novel platinum(IV) complexes under
physiologically relevant conditions
The influence of different carboxylato ligands in equatorial and axial
position of symmetrically and unsymmetrically substituted complexes
on the aqueous stability was investigated in water and PBS solution
using RP HPLC measurements. After incubation at 37 °C for 24 h in
3
2
.5. Stability of platinum(II) complex 2 in D O and in saline solution
1
The stability of 2 was investigated by H NMR spectroscopy in D
and in aqueous sodium chloride solution at ambient temperature
25 °C). Measurements in pure D O revealed considerably fast aquation.
After 12 h less than 50% of the initial complex was found intact (Fig. 4)
2
O
(
2
Fig. 3. Correlation between the cytotoxicity (IC50 values) and lipophilicity (logk
w
values)
2
Fig. 5. Behavior of 2 in sodium chloride solution in D O. Coordinated acetato ligand (left
of investigated platinum(IV) compounds in CH1/PA-1 cancer cells.
signals) and free acetate (right signal).
Please cite this article as: D. Höfer, et al., Tetracarboxylatoplatinum(IV) complexes featuring monodentate leaving groups — A rational approach
toward exploiting the platinum(IV) ..., J. Inorg. Biochem. (2015), http://dx.doi.org/10.1016/j.jinorgbio.2015.08.018

12
D. Höfer et al. / Journal of Inorganic Biochemistry xxx (2015) xxx–xxx
Fig. 6. 1H NMR of isomers 7a (left) and 10 (right) immediately after addition of ascorbic acid (bottom) and after 10 days (top).
water, 90–96% of the analyte was still intact in every sample. In PBS so-
lution (pH = 7.4) 87–92% was unchanged after 24 h. The observed re-
sults are in accordance with the kinetic inertness of Pt(IV) complexes
explaining the significant decrease of the rate of aquation in comparison
to the very reactive platinum(II) counterparts (2, and 3).
exhibited the expected high kinetic inertness; even in the presence of
an excess of ascorbic acid only a very slow reduction could be observed.
With increasing lipophilicity of the axial ligands, platinum(IV) com-
plexes featuring comparable or higher cytotoxicity than their
platinum(II) congeners were obtained. Notably, these compounds are
characterized not only by a high lipophilicity and cytotoxicity, but also
by a high aqueous solubility and stability under physiologically relevant
conditions, even in the presence of bioreductants.
3
.7. Reduction behavior toward ascorbic acid
Consequently, tetracarboxylato complexes with monodentate ligands
are ideal candidates for exploiting the platinum(IV) prodrug strategy. Fur-
ther investigations concerning the reduction behavior and the mode of
action of this novel type of platinum(IV) complexes are necessary.
As mentioned above, the difference observed in cytotoxicity toward
CH1/PA-1 cells between isomers 7a and 10 could not be attributed to
their lipophilicity. In order to find an explanation for this behavior, the
reduction of both complexes was investigated (Fig. 6). Therefore, com-
pounds 7a and 10 were incubated with a 25-fold excess of ascorbic
Abbreviations
2
acid in D O buffered solution at physiological pH. The reduction was
1
followed by H NMR spectroscopy. Both complexes showed a very
slow reduction rate; after 10 days, around 90% of the complexes were
still intact. These findings are consistent with data in the literature for
other tetracarboxylato platinum(IV) complexes [11–13]. Varbanov
et al. [11] reported only 50% reduction after 3 weeks of incubation for
a platinum(IV) analog of carboplatin using the same experimental
conditions. Zhang et al. [12] reported 5% reduction after 12 h for a
tetracarboxylatoplatinum(IV) oxaliplatin analog, and a half-life of
ACN
ATR
ESI-MS
acetonitrile
attenuated total reflection
electrospray ionization mass spectrometry
ESI-QqTOF electrospray ionization quadrupole–quadrupole-time-of-
flight
MEM
MTT
minimal essential medium
3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium
bromide
phosphate buffer saline
2,2,2-trifluoroethanoic acid
2
2
.5 days was found for [Pt(en)(OAc) (ox)] by Chen et al. [13]. In both
PBS
TFA
cases a 10-fold excess of ascorbic acid was applied. Previous investiga-
tions explained the high kinetic inertness of such type of complexes
by the inability of carboxylate and amine ligands to act as good inner
sphere electron transfer ligands.
Acknowledgments
The comparable very slow rate of reduction by ascorbate observed
for novel complexes 7a and 10, and for tetracarboxylato complexes act-
ing as prodrugs for carboplatin cannot explain the observed differences
in cytotoxicity. However, it should be also mentioned, that reduction by
ascorbic acid at physiological pH is only a simplified model, which can-
not fully represent the complicated cell microenvironment. As already
discussed, the higher activity of tetracarboxylato complexes bearing
monodentate leaving groups, compared to those featuring bidentate
carboxylates, is most probably associated with the higher reactivity of
their reduced Pt(II) counterparts.
The support of the FFG — Austrian Research Promotion Agency, the
Austrian Council for Research and Technology Development, the FWF
(Schrödinger fellowship J 3577-B13 to H.V.) and COST CM1105 is grate-
fully acknowledged.
Appendix A. Supplementary data
Crystallographic data have been deposited with the Cambridge
Crystallographic Data Center with numbers CCDC 1056105–1056107.
Copies of data can be obtained, free of charge, on application to CCDC,
1
2 Union Road, Cambridge CB2 1EZ, UK (deposit@ccdc.com.ac.uk).
4
. Conclusion
Supplementary data to this article can be found online at doi:http://
dx.doi.org/10.1016/j.jinorgbio.2015.08.018
A series of tetracarboxylatoplatinum(IV) complexes with
monodentate carboxylato ligands was synthesized and character-
ized by using various analytical methods. Investigation of the aqueous
stability of the complexes showed that respective platinum(II) counter-
parts revealed high reactivity. In contrast, platinum(IV) complexes
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Please cite this article as: D. Höfer, et al., Tetracarboxylatoplatinum(IV) complexes featuring monodentate leaving groups — A rational approach
toward exploiting the platinum(IV) ..., J. Inorg. Biochem. (2015), http://dx.doi.org/10.1016/j.jinorgbio.2015.08.018