Oxidative reactivity and cytotoxic properties of a platinum(II) complex prepared by outer-sphere amide bond coupling

Article abstract of DOI:10.1016/j.poly.2012.07.097

Benzyl amine was coupled to the dangling carboxylic acid groups of the platinum(II) complex [Pt(edda)Cl₂], where edda = ethylenediamine-N, N′-diacetic acid, to give the diamide-tethered complex [Pt(L)Cl ₂] (1), where L = ethylenediamine-N,N′-bis(N-benzylacetamide). Complex 1 was oxidized with both PhICl₂ and Br₂. Oxidation with PhICl₂ cleanly afforded the tetrachloride complex, [Pt(L)Cl₄] (2), whereas oxidation with Br₂ gave rise to several mixed halide complexes of the general formula, [Pt(L)Cl_x- Br_4-x], where x = 1, 2, or 3. Complexes 1 and 2 were fully characterized by ¹H, ¹³C, and ¹⁹⁵Pt NMR spectroscopy, as well as by ESI-MS. These compounds exist as a mixture of diastereomers that arise from the chirality of the two coordinated nitrogen atoms. Crystal structures of 1, 2, and [Pt(L)Cl_xBr_y] (3) are reported. Although refined as the tetrabromide complex [Pt(L)Br ₄], the crystal structure of 3 is a mixture of species with site-occupancy disorder of chloride and bromide ligands. DFT calculations indicate that the two sets of diastereomers of 1 and 2 are effectively thermoneutral, a conclusion that is also supported by the observation of both members of each pair by NMR spectroscopy. The cytotoxicity of 1 and 2 was measured by the MTT assay in HeLa cells and compared to that of cisplatin. Both exhibit IC₅₀ values close to 50 μM and are therefore substantially less toxic than cisplatin, for which the IC₅₀ is 1 μM.

Full text of DOI:10.1016/j.poly.2012.07.097

Polyhedron 58 (2013) 71–78
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Polyhedron
journal homepage: www.elsevier.com/locate/poly
Oxidative reactivity and cytotoxic properties of a platinum(II) complex prepared
by outer-sphere amide bond coupling
⇑
Justin J. Wilson, Stephen J. Lippard
Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Available online 17 August 2012
Benzyl amine was coupled to the dangling carboxylic acid groups of the platinum(II) complex
[Pt(edda)Cl2], where edda = ethylenediamine-N,N0-diacetic acid, to give the diamide-tethered complex
[Pt(L)Cl2] (1), where L = ethylenediamine-N,N0-bis(N-benzylacetamide). Complex 1 was oxidized with
both PhICl2 and Br2. Oxidation with PhICl2 cleanly afforded the tetrachloride complex, [Pt(L)Cl4] (2),
whereas oxidation with Br2 gave rise to several mixed halide complexes of the general formula, [Pt(L)Clx-
Br4-x], where x = 1, 2, or 3. Complexes 1 and 2 were fully characterized by 1H, 13C, and 195Pt NMR spec-
troscopy, as well as by ESI-MS. These compounds exist as a mixture of diastereomers that arise from
the chirality of the two coordinated nitrogen atoms. Crystal structures of 1, 2, and [Pt(L)ClxBry] (3) are
reported. Although refined as the tetrabromide complex [Pt(L)Br4], the crystal structure of 3 is a mixture
of species with site-occupancy disorder of chloride and bromide ligands. DFT calculations indicate that
the two sets of diastereomers of 1 and 2 are effectively thermoneutral, a conclusion that is also supported
by the observation of both members of each pair by NMR spectroscopy. The cytotoxicity of 1 and 2 was
measured by the MTT assay in HeLa cells and compared to that of cisplatin. Both exhibit IC50 values close
This article is dedicated to the memory of
Michelle Millar, a wonderful friend,
colleague, and scientist.
Keywords:
Cisplatin
MTT assay
X-ray crystallography
NMR spectroscopy
Amide coupling
Platinum
to 50 lM and are therefore substantially less toxic than cisplatin, for which the IC50 is 1 lM.
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
alization of this compound via amide bond formation has led to the
synthesis of many new platinum(IV) complexes that were tested for
biological activity [6–11]. This predictable chemistry has also been
used to attach platinum(IV) prodrugs to peptides [12–15] and vari-
ous nano-delivery devices [16–21] for improved anticancer efficacy.
Previously, we reported an analogous amide bond coupling
reaction using the dangling carboxylic acid of the platinum(II)
complex [Pt(edma)Cl2], where edma = ethylenediamine-N-mono-
acetic acid [22]. This chemistry was used to tether a dansyl fluoro-
phore on the ethylenediamine backbone of the platinum(II)
complex. The resulting complex behaved as a fluorescent reporter
for the oxidation state of the platinum center, thus demonstrating
the potential utility of this chemistry for designing complexes with
valuable properties. In the present article we describe amide bond
formation on the platinum(II) complex, [Pt(edda)Cl2] where
edda = ethylenediamine-N,N0-diacetic acid having two free carbox-
ylic acid groups. Oxidation of this new platinum(II) complex by
PhICl2 and Br2 was investigated, together with the cytotoxic prop-
erties of the two chloride complexes in HeLa cells.
Cisplatin is a widely used and effective metal-based chemother-
apeutic agent. The biological properties of this simple coordination
compound were discovered in 1965, leading to subsequent FDA-
approval for the treatment of testicular and bladder cancer in
1978 [1]. Since then, a wide array of platinum complexes have
been synthesized and tested for biological activity with the goal
of finding new platinum-based chemotherapeutics with fewer
toxic side effects and a different spectrum of activity [2]. The two
approved second-generation compounds, carboplatin and oxalipla-
tin, are one result of this research endeavor. Despite the clinical
success of these three compounds, their side effects [3,4] and lack
of efficacy in certain cancer types, primarily due to resistance [5],
drives the search for new platinum-based anticancer agents.
The ability to systematically modify potential platinum antican-
cer agents by predictable and readily controlled chemistry is of va-
lue for the synthesis of new drug candidates having novel
properties. In recent years, this principle has been applied to gener-
ate platinum(IV) prodrugs. For example, the free carboxylic acid
groups of the platinum(IV) compound cis,cis,trans-[Pt(NH3)2Cl2(suc-
cinate)2] can engage in amide bond coupling reactions [6]. Function-
2. Experimental
2.1. General considerations
⇑
All reactions were carried out under normal atmospheric condi-
tions. Solvents were used as received without additional drying or
Corresponding author. Tel.: +1 (617) 253 1892; fax: +1 (617) 258 8150.
E-mail address: lippard@mit.edu (S.J. Lippard).
0277-5387/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.poly.2012.07.097

72
J.J. Wilson, S.J. Lippard / Polyhedron 58 (2013) 71–78
purification. The compounds, [Pt(edda)Cl2] (edda = ethylenedia-
mine-N,N0-diacetic acid) [23] and iodobenzene dichloride [24],
were synthesized as previously described. Benzylamine and car-
bonyldiimidazole (CDI) were purchased from Sigma Aldrich and
used as received.
solution, which was allowed to stir at rt for 1 h. The solution was
filtered and 200 mL of Et2O was added. After 10 min, a fine yellow
solid deposited. This solid was isolated by vacuum filtration,
washed twice with 10 mL of Et2O, and then dried under vacuum.
Yield: 0.108 g (49%). M.p. > 200 °C (gradual browning), 255–
265 °C (dec into black char). 1H NMR (400 MHz, DMF-d7): R,R/
S,S + R,S diasteromers (3:1) d 8.95 + 8.92 (triplets, 2H, NH amide),
7.39–7.28 (overlapping multiplets, 12H, 5H aromatic + NH),
4.50 + 4.45 (doublets, 4H, benzyl CH2), 4.23–3.80 (multiplets, 4H,
CH2 adjacent to amide), 3.60–3.20 (broad multiplets, 4H, CH2 eth-
ylenediamine backbone). 13C{1H} NMR (100 MHz, DMF-d7): R,R/
S,S + R,S diasteromers (3:1) d 166.6 + 166.5, 139.2 + 139.1, 128.7,
127.8, 127.4, 57.4 + 57.2, 55.0 + 54.3, 43.3 + 43.2. 195Pt{1H} NMR
(86 MHz, DMF-d7): R,R/S,S + R,S diasteromers (3:1) d ꢀ370 (minor),
ꢀ378 (major). IR (KBr, cmꢀ1): 3440 m, 3294 m, 3153 w, 3105 w,
2924 w, 2876 w, 1657 s, 1584 w, 1571 m, 1495 vw, 1450 w,
1384 w, 1410 w, 1324 w, 1277 w, 1216 vw, 1068 w, 758 m, 704
m, 508 w. ESI-MS (negative-ion mode): m/z 580.9 ([PtLCl4–4H–
3Cl]ꢀ, Calc. 581.1), 616.9 ([PtLCl4–3H–2Cl]ꢀ, Calc. 617.1), 652.9
([PtLCl4–2H–Cl]ꢀ, Calc. 653.1), 689.0 ([PtLCl4–H]ꢀ, Calc. 689.0),
1381.1 ([2PtLCl4–H]ꢀ, Calc. 1380.9). Anal. Calc. for C20H26Cl4N4O2-
Pt: C, 34.75; H, 3.79; N, 8.10. Found: C, 34.70; H, 3.66; N, 8.20%.
2.2. Physical measurements
NMR spectra were recorded on a Bruker DPX-400 or Varian Mer-
cury spectrometer in the MIT Department of Chemistry Instrumen-
tation Facility at 20 °C. 1H and 13C{1H} NMR spectra were
referenced internally to residual solvent peaks, and chemical shifts
are expressed relative to tetramethylsilane, SiMe4 (d = 0 ppm).
195Pt{1H} NMR spectra were referenced externally to K2PtCl4 in
D2O (d = ꢀ1628 ppm). Electrospray ionization mass spectra (ESI-
MS) were acquired on an Agilent Technologies 1100 series LC-
MSD trap. ESI-MS and NMR spectra of all compounds are given in
the Supplementary data (Figs. S1–S10). Fourier transform infrared
(FTIR) spectra were recorded with a ThermoNicolet Avatar 360
spectrometer running the OMNIC software. Samples were prepared
as KBr disks. Melting points were obtained on a Meltemp apparatus
and are reported uncorrected. X-ray powder diffraction data were
obtained using a Bruker D8 Advance diffractometer equipped with
a CuK
a
radiation source. The sample was placed on a rotating
2.5. Oxidation of 1 with Br2
stage, and data were acquired at every 0.01° at a rate of 0.2 s/step.
To a suspension of 1 (115 mg, 0.185 mmol) in 3 mL of DMF was
2.3. Synthesis of [Pt(L)Cl2] (1)
added Br2 in DMF (0.61 M, 460 lL, 0.28 mmol). The mixture was
left to stir at rt in the absence of light for 0.5 h. The resulting orange
solution was filtered and set up for vapor diffusion with water as
the entering solvent. After 6 days, orange microcrystalline material
deposited. The supernatant was decanted and the remaining solid
was washed with 3 ꢁ 5 mL water, 2 ꢁ 5 mL EtOH, and 2 ꢁ 5 mL
Et2O sequentially, prior to drying in vacuo. This material, as de-
scribed below in Sections 3.3 and 3.4, is composed of a mixture
of platinum(IV) compounds with the general formula [Pt(L)Clx-
Br4-x], where x is a positive integer ꢂ3. Yield: 109 mg. ESI-MS (neg-
ative-ion mode): m/z 581.0 ([PtLCl2Br2–Cl–2Br–4H]ꢀ, Calc. 581.1),
619.0 ([PtLCl2Br2–2Br–3H]ꢀ, Calc. 617.1), 625.0 ([PtLCl2Br2–2Cl–
Br–4H]ꢀ, Calc. 625.1). 660.9 ([PtLCl2Br2–Cl–Br–3H]ꢀ, Calc. 661.0),
698.8 ([PtLCl2Br2–Br–2H]ꢀ, Calc. 697.0 (100%), 699.0 (99.1%)),
706.8 ([PtLCl2Br2–2Cl–3H], Calc. 707.0), 734.8 ([PtLCl3Br–H]ꢀ, Calc.
735.0), 742.8 ([PtLCl2Br2–Cl–2H]ꢀ, Calc. 743.0), 778.8 ([PtLCl2Br2–
H]ꢀ, Calc. 778.9), 822.7 ([PtLClBr3–H]ꢀ, Calc. 822.9), 904.7
([PtLClBr3+Br] or [PtLBr4+Cl], Calc. 904.8).
A solution of CDI (0.465 g, 2.87 mmol) in 50 mL of DMF was
added to a suspension of [Pt(edda)Cl2] (0.619 g, 1.40 mmol) in
16 mL of DMF. The resulting mixture was heated at 60 °C for
10 min, at which point a yellow solution resulted, and then
sparged with N2 for 5 min. Benzylamine (0.307 g, 2.87 mmol) in
40 mL of DMF was added in a dropwise manner to this solution
containing the activated platinum complex. After stirring for
12 h, the solution was concentrated to 15 mL under reduced pres-
sure and elevated temperature (60 °C). The addition of 20 mL of
water afforded the desired compound as an off-white solid, which
was isolated by filtration and washed sequentially with 5 mL of
water, 2 ꢁ 5 mL of ethanol, and 2 ꢁ 5 mL of diethyl ether (Et2O) be-
fore being dried in vacuo. Yield: 0.594 g (68%). M.p. > 280 °C (grad-
ual browning), 302–307 °C (dec into black liquid). 1H NMR
(400 MHz, DMF-d7): R,R/S,S + R,S diasteromers (1:1) d 8.64 (2H,
two overlapping triplets, amide NH), 7.36–7.24 (multiplet, 10H,
aromatic protons), 6.22 + 6.15 (2H, broad singlets, coordinating
NH), 4.48–4.38 (m, 4H, benzyl CH2), 4.31–4.16 (2H, two doublets,
CH adjacent to amide), 3.80–3.62 (two doublet of doublets, 2H,
CH adjacent to amide), 3.21–3.11 (broad multiplet, 2H, CH2 ethy-
lenediamine backbone), 2.72–2.66 (broad multiplet, 2H, CH2 ethy-
lenediamine backbone). 13C{1H} NMR (100 MHz, DMF-d7): R,R/
2.6. Theoretical calculations
DFT calculations were carried out with the ORCA program pack-
age [25]. Geometries were optimized in the gas-phase using the
BP86 functional [26–28]. The def2-TZVP(-f) basis set and the
decontracted def2-TZVP/J auxiliary basis set were used for all
atoms with the zeroth-order regular approximation (ZORA) to ac-
count for relativistic effects [29,30]. Numerical frequency calcula-
tions at the same level of theory revealed the optimized
geometries to be local minima on the potential energy surface
and were used for thermodynamic calculations.
S,S + R,S diasteromers (1:1)
d 168.1 + 168.0, 139.50 + 139.48,
128.7, 127.82, 127.79, 127.3, 55.6 + 54.7, 55.1, 42.91. 195Pt{1H}
NMR (86 MHz, DMF-d7): R,R/S,S + R,S diasteromers (1:1) d ꢀ2347,
ꢀ2362. IR (KBr, cmꢀ1): 3340 m, 3165 m, 3111 m, 2949 w, 1685
m, 1662 s, 1555 m, 1496 w, 1452 w, 1419 m, 1358 w, 1261 m,
1078 w, 1025 w, 986 w, 860 w, 748 w, 695 w, 581 w, 453 w.
ESI-MS (negative-ion mode): m/z 582.9 ([PtLCl2–2H–Cl]ꢀ Calc.
,
583.1), 619.0 ([PtLCl2–H]ꢀ, Calc. 619.1), 1239.1 ([2PtLCl2–H]ꢀ, Calc.
1239.2). Anal. Calc. for C20H26Cl2N4O2Pt: C, 38.72; H, 4.22; N, 9.03.
Found: C, 38.71; H, 4.13; N, 8.96%.
2.7. X-ray crystallographic studies
Single crystals were mounted in Paratone oil on cryoloops and
frozen under a 100 K KRYO-FLEX nitrogen cold stream. In general,
data were collected on a Bruker APEX CCD X-ray diffractometer
2.4. Synthesis of [Pt(L)Cl4] (2)
with graphite-monochromated MoK
a radiation (k = 0.71073 Å)
To a suspension of 1 (200 mg, 0.322 mmol) in 5 mL of DMF, a
solution of PhICl2 (91 mg, 0.33 mmol) in 1 mL of DMF was added
in a dropwise manner. The suspension became a bright yellow
controlled by the APEX2 software package [31]. For 2 and 3, absorp-
tion corrections were applied using SADABS [32]. The structures were
solved using direct methods and refined on F2 with the SHELXTL-97

J.J. Wilson, S.J. Lippard / Polyhedron 58 (2013) 71–78
73
software package [33,34]. Structures were checked for higher sym-
metry using PLATON [35]. All non-hydrogen atoms were located and
refined anisotropically. Hydrogen atoms were placed in idealized
locations and given isotropic thermal parameters equivalent to
either 1.5 (terminal CH3 hydrogen atoms) or 1.2 times the thermal
parameter of the atom to which they were attached. Crystallo-
graphic data collection and refinement parameters are shown in
Table 1. Crystals of 2, obtained by vapor diffusion of water into a
DMF solution, gave good quality data. No problems were encoun-
tered during the solution and refinement of this structure. Specific
refinement details for 1 and 3 are described below.
unit also raises concerns as to whether these two species are sym-
metry-related in a monoclinic space group. Structure solution in
P21/c gave basic atomic connectivity. During refinement, however,
thermal ellipsoids attained unreasonable sizes and many became
non-positive definite upon anisotropic refinement, a problem not
ꢀ
encountered in P1. Furthermore, the merging and refinement sta-
tistics and the standard deviations of the bond distances and an-
gles for the P21/c solution and refinement were substantially
worse than those in the triclinic space group. The two molecules
ꢀ
in the asymmetric unit in the P1 solution exhibit only one obvious
conformational difference. In particular, the tilting of the
five-membered chelate rings is different, as discussed in more
detail in Section 3.4. Searches for higher symmetry space groups
Colorless plates of 1 were obtained by the slow evaporation of a
DMF solution. All crystals screened displayed signs of non-merohe-
dral twinning in the form of split reflections. After full data collec-
tion, the program CELL_NOW [36] was used to look for additional
domains. Two domains were found that accounted for 97% of the
harvested reflections. The second domain was rotated by 6.8°
about the c⁄ axis. The data were integrated over both domains
using SAINT [37]. An absorption correction was applied with the
program TWINABS [38]. The corrected data were then analyzed for
systematic absences and higher metric symmetry with XPREP [39]
to determine the space group. The structure was solved with the
SHELXTL-97 software package and refined using data from both do-
mains. The second domain refined to a scale factor of 8.33%. Two
molecules of 1 are present in the asymmetric unit in the space
ꢀ
with PLATON on the P1 solution were unsuccessful.
X-ray quality crystals of 3 were grown by vapor diffusion of
water into
a DMF solution. The structure solved readily in
P212121. Unexpectedly, all four halide ligands refined best as bro-
mine atoms. From additional characterization data presented in
Sections 3.3 and 3.4, crystals of 3 are believed to be composed a
disordered mixture of [Pt(L)ClxBry] species, with site-occupancy
disorder of chloride and bromide ligands present throughout. De-
spite several attempts, this type of disorder could not be success-
fully modeled. Hence, the Pt–Br distances in this structure should
not be strictly interpreted as such.
ꢀ
group P1. Restraints on the directionality and size of the thermal
2.8. Cell culture and cytotoxicity assays
displacement parameters of the nitrogen and carbon atoms of
the ethylenediamine backbone of one of the molecules in the
asymmetric unit were applied. The largest electron density peak
and hole are 4.78 and ꢀ2.44 eÅ3, located 1.19 Å from Pt2 and
0.78 Å from Pt1, respectively. This large residual density might in
HeLa cells grown as monolayers in Dulbecco’s Modified Eagle
Medium (DMEM) supplemented with 10% fetal bovine serum
(FBS) and 1% penicillin/streptomycin were kept at 37 °C in a
humidified incubator with 5% CO2. Cells were passed every 3–
4 days using stock solutions of trypsin/EDTA to detach the cells.
The cytotoxicities of 1, 2, and cisplatin were measured with the
MTT assay. Cells were seeded in 96 well plates (2000 cells/well)
and incubated for 16-24 h. The medium was then aspirated and re-
placed with 2-fold (for 1 and 2) or 4-fold (for cisplatin) serial
part be due the presence of additional twin domains. It should also
ꢀ
be noted that the space group utilized was P1. Both b and
c are
close to 90°, suggesting that a higher metric symmetry monoclinic
space group might be more appropriate to describe the structure.
Furthermore, the presence of two molecules of 1 in the asymmetric
Table 1
X-ray crystallographic data collection and refinement parameters.
1
2
3
Formula
Formula weight
Space group
a (Å)
b (Å)
c (Å)
C
20H26Cl2N4O2Pt
C20H26Cl4N4O2Pt
691.34
P43212
C20H26Br4N4O2Pt
869.18
P212121
11.1850(5)
12.0391(6)
18.2747(9)
620.44
ꢀ
P1
9.2382(10)
12.9438(14)
18.045(2)
80.2930(17)
88.9920(18)
90.0000(16)
2126.5(4)
4
11.3811(3)
18.4022(11)
a
(°)
b (°)
c
(°)
V (Å3)
2383.63(17)
4
1.926
2460.8(2)
4
2.346
Z
q
calc, (g cmꢀ3
)
1.938
T (°C)
(MoK
h range, (°)
ꢀ173(2)
ꢀ173(2)
ꢀ173(2)
12.222
2.03–29.13
52226
6631
281
100.0
5.42
15.30
l
a
), (mmꢀ1
)
6.874
6.360
1.60–25.06
58892
7479
518
99.4
8.00
12.58
4.93
11.07
2.10–28.71
50603
3092
142
99.9
2.42
4.11
1.92
4.11
Total no. of data
No. of unique data
No. of parameters
Completeness to h (%)
a
R1 (%)
b
wR2 (%)
a
R1 (%) for I > 2
r
5.03
14.95
1.095
b
wR2 (%) for I > 2
r
Goodness-of-fit (GOF)c
Max, min peaks, (eÅꢀ3
Flack parameter
1.024
4.783, ꢀ2.435
1.058
)
0.817, ꢀ0.558
3.799, ꢀ3.319
ꢀ0.012(7)
ꢀ0.045(19)
P
P
a
R1
¼
jjFoj ꢀ jFcjj= jFoj.
ꢀ
ꢁ
ꢀ
ꢁ
2
2
P
P
1=2
wR2 ¼ f ½w F2o ꢀ Fc2 ꢃ= ½w F2o ꢃg
.
b
ꢀ
ꢁ
2
P
1=2
GOF ¼ f ½w F2o ꢀ F2c ꢃ=ðn ꢀ pÞg
where n is the number of data and p is the number of refined parameters.
c

74
J.J. Wilson, S.J. Lippard / Polyhedron 58 (2013) 71–78
dilutions of the desired platinum complex in growth medium.
After a 72 h exposure time, the platinum-containing medium was
removed and replaced with 200 lL of a solution of MTT
(0.8 mg/mL) in serum-free DMEM. The MTT solution was aspirated
after a 4 h incubation period, and the resulting purple formazan
crystals were dissolved with a 3:80 v/v mixture of 35% aqueous
ammonia and DMSO [40]. The absorbance in each well was read
at 550 nm. The absorbance values were normalized to that of the
untreated wells (100% cell viability) and plotted against compound
plementary data). The small, 15 ppm difference between these sig-
nals suggests that the magnetic environments of platinum for the
two species are very similar in solution. Because the coordinating
nitrogen atoms of 1 are chiral, both the enantiomeric R,R/S,S and
meso R,S diastereomers exist. The two distinct, yet chemically sim-
ilar, species observed in solution are assigned as these two
diastereomers.
3.2. Synthesis and characterization of 2
concentration. The 50% growth inhibitory concentration (IC50
)
values were estimated by interpolation of the resulting curves.
The experiment was repeated at least in triplicate, using 6 wells
per concentration level. Reported IC50 values are the averages de-
rived from these independent experiments, and the errors are the
resulting standard deviations. For 1 and 2, the highest concentra-
tion levels utilized 0.2% DMF to solubilize the compounds. A con-
trol experiment revealed that the average cell viability after 72 h
exposure to 0.2% pure DMF is 74 6%. Therefore, the reported
IC50 values of 1 and 2 are expected to be somewhat lower than
those in the absence of the somewhat toxic DMF co-solvent.
The oxidation of 1 with PhICl2 in DMF afforded the diaminotet-
rachloroplatinum(IV) complex, 2, as the only product (Scheme 2).
The IR data of 2 are similar to those of 1, with the major C@O amide
stretching frequency occurring at 1657 cmꢀ1. New Pt–Cl vibra-
tional modes, which typically range between 300 and 400 cmꢀ1
,
could not be observed within the window of the spectrometer used
(4000–400 cmꢀ1). The ESI-MS of 2 in the negative ion mode dis-
plays peaks for the [MꢀH]ꢀ and [2 MꢀH]ꢀ ions, confirming the
presence of the two newly introduced chlorine atoms (Fig. S8, Sup-
plementary data). Three other significant peaks due to the [M–2H–
Cl]ꢀ, [M–3H–2Cl]ꢀ, and [M–4H–3Cl]ꢀ ions were present as well,
corresponding to sequential loss of chloride anions and protons
from the parent fragment.
3. Results and discussion
As for 1, two distinct species are present in solution that we as-
sign to analogous R,R/S,S and R,S diastereomers. For 2 in DMF, how-
ever, the ratio of these two species is approximately 1:3, in
contrast to the 1:1 ratio observed for 1. The NH resonances of
the coordinated amine ligand in the 1H NMR spectrum (Fig. S5,
Supplementary data) are shifted downfield relative to those of 1
and are largely obscured by overlapping resonances of the aro-
matic protons. This overlap prevents observation of 195Pt coupling
to the NH protons, typically observed for platinum(IV) complexes.
The major diastereomer resonates at ꢀ378 ppm in the 195Pt NMR
spectrum, whereas the minor diastereomer resonates at
ꢀ370 ppm (Fig. S7, Supplementary data). The significant downfield
shift (ꢄ2000 ppm) of the 195Pt NMR resonances of 2 relative to
those of 1 is expected on the basis of the greater platinum oxida-
tion state of 2 (+4) in comparison to 1 (+2). The 195Pt NMR chemical
shifts of 2 are similar to those of a related PtIVN2Cl4 complex [22].
3.1. Synthesis and characterization of 1
Previously, we reported that the dangling carboxylic acid of the
platinum(II) complex, [Pt(edma)Cl2] where edma = ethylenedia-
mine-N-monoacetic acid, can engage in coupling reactions with
amines to form stable amide bonds [22]. Inspired by this prior work,
we investigated the amide coupling chemistry of the platinum
complex [Pt(edda)Cl2], where edda is ethylenediamine-N,N0-diace-
tic acid having two terminal carboxylic acid moieties. A suspension
of [Pt(edda)Cl2] in DMF was activated with 2 equiv of 1,10-carbon-
yldiimidazole, resulting in a homogeneous yellow solution. The
addition of benzylamine to the activated complex afforded complex
1, [Pt(L)Cl2] where L = ethylenediamine-N,N0-bis(N-benzylaceta-
mide), as an off-white solid after appropriate workup (Scheme 1).
Formation of the amide bond was verified by IR spectroscopy,
which revealed the disappearance of the free carboxylic acid
stretching frequency near 1720 cmꢀ1 of the starting material and
concomitant formation of a C@O bond stretching frequency at
1662 cmꢀ1. Elemental analysis further verified the bulk composi-
tion of the isolated material. ESI-MS of 1, in the negative ion mode,
gave rise to three major ion peaks (Fig. S4, Supplementary data). In
addition to the commonly observed [MꢀH]ꢀ and [2 MꢀH]ꢀ peaks,
the [M–2H–Cl]ꢀ peak was also detected.
NMR spectroscopy in DMF-d7 revealed the presence of equal
amounts of two species in solution. The 1H NMR spectrum
(Fig. S1, Supplementary data) shows two overlapping triplets cen-
tered at 8.64 ppm, which we assign to the NH proton of the newly
formed amide bond. The NH resonances of the coordinated amines
appear at 6.19 and 6.11 ppm. The 13C NMR spectrum also shows a
doubling of most of the signals expected for 1 (Fig. S2, Supplemen-
tary data). Two peaks in the 195Pt NMR spectrum are observed at
ꢀ2347 and ꢀ2362 ppm in an approximate 1:1 ratio (Fig. S3, Sup-
3.3. Oxidation of 1 with Br2
Treatment of 1 as a suspension in DMF with 1.5 equiv of Br2 led
to the formation of a homogeneous orange solution. Slow diffusion
of water vapor into this solution afforded orange microcrystalline
material. Analysis of the product by ESI-MS revealed an envelope
of molecular ion peaks (Fig. S10, Supplementary data). A peak cor-
responding to the ion of the expected Br2 oxidative addition prod-
uct, [PtLCl2Br2–H]ꢀ, was observed at m/z 778.8 (Calc. 778.9). In
addition, eight other peaks corresponding to the ions, [PtLCl2-n-
Br2-m–nCl–mBr–(1+m+n)H]ꢀ where n and m are integers under
the condition m + n ꢂ3 were detected. These species are analogous
to the [PtLCl4–2H–Cl]ꢀ, [PtLCl4–3H–2Cl]ꢀ, and [PtLCl4–4H–3Cl]ꢀ
ions observed for 2, and arise from different combinations of Cl-
and Br- loss upon ionization. A peak at m/z 734.8 corresponds to
Scheme 1. Synthesis of compound 1.

J.J. Wilson, S.J. Lippard / Polyhedron 58 (2013) 71–78
75
Scheme 2. Synthesis of compound 2.
the ion [PtLCl3Br–H]ꢀ (Calc. 735.0), and a peak at 822.7 is assigned
to the species [PtLClBr3–H]ꢀ (Calc. 822.9). A signal at m/z 904.7
could correspond either to [PtLClBr3+Br]ꢀ or [PtLBr4+Cl]ꢀ (Calc.
for both 904.8). These latter three peaks indicate that partial inter-
molecular halide exchange might occur either in solution or gas-
phase, giving rise to core fragments with the unexpected PtLCl3Br,
PtLClBr3, and PtLBr4 molecular formulae.
cis and trans isomers and diastereomers owing to the chirality of the
coordinating nitrogen atoms. The peaks at ꢀ599 and ꢀ647 ppm are
tentatively assigned to different isomers of the complex, [Pt(L)Cl3-
Br], whereas the peaks between ꢀ1120 and ꢀ1175 ppm are assigned
to isomers of [Pt(L)ClBr3]. Although, to the best of our knowledge,
195Pt NMR data for platinum(IV) complexes with similar N2Cl3Br
and N2ClBr3 coordination spheres have not been reported, the posi-
tions of these peaks are consistent with the ability of softer ligands,
such as bromide, to systematically shift the 195Pt NMR resonance up-
field [44]. In comparing the 195Pt chemical shifts for the N2Cl4 com-
plex 2 and the species observed here, it appears that sequential
substitution of a chloride for a bromide ligand leads to an upfield
shift of 250–300 ppm. Similarly, the 195Pt NMR chemical shift of
the [PtBr6]2ꢀ anion moves upfield by approximately 290 ppm for
each replacement of a chloride for a bromide ligand [45]. The precise
mechanism by which these mixed halide species form remains
uncertain. The presence of a small amount of unreacted platinum(II)
starting material may facilitate halide scrambling via the well
known platinum(II)-catalyzed ligand substitution reactions of plat-
inum(IV) complexes [46].
The 1H NMR spectrum of this material (Fig. S9, Supplementary
data) displays a broad multiplet centered at 8.90 ppm, which is as-
signed to the NH amide resonance. Two distinct triplets, as ob-
served for 1 and 2, could not be clearly resolved from this signal.
With the exception of a sharp doublet at 4.50 ppm assigned to
the benzyl CH2 group, the aliphatic region shows a series of poorly
overlapping multiplets ranging from 4.8 to 3.2 ppm. Therefore, the
1H NMR spectrum is indicative of more than just two species pres-
ent in solution.
Investigation of the solution 195Pt NMR spectrum of this material
confirmed the presence of multiple platinum-containing com-
pounds. At least seven different species are observed in the 195Pt
NMR spectrum. The 195Pt chemical shifts of these compounds range
from ꢀ599 to ꢀ1425 ppm (Fig. 1). These peaks are hypothesized to
arise from a mixture of [Pt(L)ClxBrx-4] compounds with different ra-
tios of bromide and chloride ligands. The peak at ꢀ1425 ppm is
similar to those of the tetrabromide compounds, [Pt(en)Br4]
3.4. Description of crystal structures
Two molecules of 1 crystallize in the asymmetric unit. One of
these molecules is depicted in Fig. 2, and selected bond distances
and angles are collected in Table 2. The other molecule is shown
in the Supplementary data (Fig. S11), along with bond distances
and angles (Table S1). The platinum atom has square-planar coor-
dination, as expected for this ion in the +2 oxidation state. The two
non-symmetry related molecules are meso-R,S diastereomers as
conveyed by their nitrogen centers. A significant difference be-
tween them is the canting of the five-membered chelate ring.
The conformation of non-planar five-membered chelate rings pro-
duces two different chiral orientations of the ligand, referred to as k
and d (Fig. 3) [47]. One of the molecules in the asymmetric unit has
the k chelate ring conformation, whereas the other displays a d
chelate ring conformation. No disorder occurs in the ethylenedia-
mine backbone, confirming that the two molecules are crystallo-
graphically distinct and not symmetry related.
(d = ꢀ1473 ppm)
[41],
[Pt(trans-1,2-diaminocyclohexane)Br4]
(d = ꢀ1525 ppm) [42], and [Pt(cis-1,2-diamoncyclohexane)Br4]
(d = ꢀ1540 ppm) [42], and it is therefore assigned to the species,
[Pt(L)Br4]. The cluster of peaks ranging from ꢀ878 to ꢀ933 ppm fall
in a region expected for platinum(IV) compounds with an N2Cl2Br2
coordination sphere. For example, the compounds cis,cis,trans-
[Pt(NH3)2Cl2Br2], cis,trans-[Pt(trans-1,2-diaminocyclohexane)Cl2-
Br2], and cis,trans-[Pt(cis-1,2-diaminocyclohexane)Cl2Br2] resonate
at ꢀ980, ꢀ976, and ꢀ990 ppm, respectively [42,43]. The observation
of several peaks in this region most likely derives from a mixture of
Complex 2 crystallizes in the chiral space group, P43212 with
half a molecule per asymmetric unit. The platinum center lies on
a crystallographic 2-fold symmetry axis. Complex 2 attains the ex-
pected octahedral coordination geometry for a platinum(IV) ion, as
shown in Fig. 4. The Pt-ligand bond lengths and angles are typical
for this oxidation state and geometry and are summarized in Ta-
ble 3. The 2-fold axis requires that both coordinating nitrogen
atoms attain the same stereochemistry, and for the crystal studied
the complex is the S,S enantiomer. The fact that the Flack parame-
ter refined to a value near zero confirms this choice. Crystals with
the R,R enantiomer presumably comprise half of the sample. The
conformation of the chelate ring is d.
Orange crystals obtained from the oxidation of 1 with Br2 were
analyzed by X-ray diffraction. A structure solution was obtained
using the non-centrosymmetric space group P212121. Despite the
decrease in symmetry from tetragonal to orthorhombic, the unit
cell parameters are similar to those of 2 with a between 11 and
Fig. 1. 195Pt NMR spectrum of the Br2-oxidation products of 1 in DMF-d7. A small
degree of baseline rolling is observed, most likely due to acoustic ringing of the
probe.

76
J.J. Wilson, S.J. Lippard / Polyhedron 58 (2013) 71–78
Table 3
Selected interatomic distances (Å) and angles (degrees) for 2.a
Bonds
Angles
Pt1–N1 2.083(2)
N1–Pt1–N1A 84.28(14) N1–Pt1–Cl2A 92.79(7)
Pt1–Cl1 2.3091(7) N1–Pt1–Cl1
Pt1–Cl2 2.3061(7) N1–Pt1–Cl2
89.69(8)
177.07(7) Cl1–Pt1–Cl1A 177.15(5)
Cl2–Pt1–Cl2A 90.14(4)
Cl1–Pt1–Cl2
90.21(4)
N1–Pt1–Cl1A 88.20(8)
a
Atoms are labeled as indicated in Fig. 4. Numbers in parentheses are the esti-
mated standard deviations of the last significant figures.
and angles are collected in Table 4. The perfect 2-fold symmetry
of 2 is not observed for 3 owing to a difference in the orientation
of a benzyl-amide ligand arm. The complex resolves in the solid-
state as the R,R enantiomer with a k conformation of the five-mem-
bered chelate ring.
Fig. 2. Structure of one of the molecules of 1 in the asymmetric unit. Thermal
ellipsoids are drawn at the 50% probability level.
The structure of [Pt(L)Br4] was somewhat unexpected. By 195Pt
NMR spectroscopy, a number of different species, corresponding to
different ratios of bromide and chloride ligands on the plati-
num(IV) center, were discovered to comprise the bulk microcrys-
talline material isolated from the oxidation of 1 with Br2. We
considered the hypothesis that the X-ray structure solution might
correspond to a homogeneous compound such as the tetrabromide
complex that serendipitously separated from the other species by
fractional crystallization. An X-ray powder pattern of the bulk or-
ange microcrystalline material, however, is in substantial agree-
ment with that of the computed pattern of 3 with only minor
deviations of the intensities at 2h ꢅ 20°. These differences might
reflect the presence of a small number of impurities (Fig. 6). These
impurities might correspond to crystallites of other mixed halide
species in the mixture, as suggested by mass spectrometry and
platinum NMR spectroscopy. In addition, refinement of 3 resulted
in thermal parameters for the bromide ligands greater than that
of the platinum atom and in violation of the Hirshfeld rigid-bond
test [48], consistent with site-position disorder involving lighter
Table 2
Selected interatomic distances (Å) and angles (degrees) for one of the molecules of 1
in the asymmetric unit.a
Bonds
Angels
Pt1–N1
Pt1–N2
Pt1–Cl1
Pt1–Cl2
2.060(8)
2.061(8)
2.310(2)
2.303(2)
N1–Pt1–N2
N1–Pt1–Cl1
N1–Pt1–Cl2
N2–Pt1–Cl1
N2–Pt1–Cl2
Cl1–Pt1–Cl2
84.3(3)
94.1(2)
175.2(2)
178.3(2)
90.9(2)
90.74(9)
a
Atoms are labeled as indicated in Fig. 2. Numbers in parentheses are the esti-
mated standard deviations of the last significant figures.
12 Å and c near 18.3 Å. The structure, solved and refined success-
fully as [Pt(L)Br4] (3), is depicted in Fig. 5. The expected octahedral
coordination geometry for platinum(IV) is attained. Bond lengths
Fig. 3. Depiction of the k and d isomers of 1 found in the asymmetric unit. Hydrogen atoms and phenyl rings are omitted for clarity.
Fig. 4. Structure of 2. Thermal ellipsoids are drawn at the 50% probability level.

J.J. Wilson, S.J. Lippard / Polyhedron 58 (2013) 71–78
77
atoms. The Pt–Br distances determined from the crystal structure
of the related complex, [Pt(en)Br4], are 2.461(2) and 2.488(2) Å
for the axial and equatorial positions, respectively [41]. For 3, the
Pt–Br distances are systematically shorter, ranging from
2.3934(15) to 2.4359(15) Å. These shorter Pt–Br distances are most
likely a consequence of unresolved substitutional disorder of bro-
mide and chloride ligands. Although 3 refined satisfactorily as
[Pt(L)Br4], the powder diffraction, 195Pt NMR spectroscopic and
ESI-MS results all argue in favor of a disordered superposition of
halo species. Accordingly, the Pt–Br distances derived from this
data set should be interpreted with caution.
3.5. DFT calculations
The relative thermodynamic stabilities of the two diastereo-
mers (R,R/S,S and R,S) of 1 and 2 were investigated by DFT calcula-
tions. These diastereomers were optimized in the gas-phase. The
optimized structures are shown in the Supplementary data
(Figs. S12–S15), where their Cartesian coordinates are also depos-
ited (Tables S2–S5). The calculated Gibbs free energy difference at
298 K between the S,S and R,S diastereomers of 1 is 0.32 kcal/mol,
whereas for the diastereomers of 2, the energy difference is
1.09 kcal/mol. For both 1 and 2, the S,S diastereomer is computed
to be the thermodynamically preferred isomer, although these en-
ergy differences are within the error of the DFT calculations. The
small energy differences, which correspond to equilibrium con-
stants of 1.7 and 6.3 for 1 and 2, respectively, are consistent with
the observation of both species in solution by NMR spectroscopy.
For a series of similar platinum(II) and platinum(IV) complexes
bearing related ethylenediamine-N,N0-diester ligands, computed
energy differences between diastereomers are on the order of sev-
eral kcal/mol, thereby explaining the observation of only one of the
isomers in solution by NMR spectroscopy [49–53].
Fig. 6. X-ray powder diffraction pattern of the bulk material isolated from the
oxidation of 1 with Br2 (bottom, black), along with the simulated diffraction powder
from the single crystal structure 3 (top, red). (For interpretation of the references to
colour in this figure legend, the reader is referred to the web version of this article.)
1 and 2 are more than order of magnitude less cytotoxic than cis-
platin, with IC50 values of approximately 50 lM. The fact that both
complexes exhibit identical IC50 values is not unexpected because
cis-diaminetetrachloroplatinum(IV) complexes are rapidly reduced
to their platinum(II) analogs by a variety of biologically relevant
reducing agents [54–56]. Tetraplatin, a related platinum(IV) com-
plex with an N2Cl4 coordination sphere, is reduced within 15 min
in RPMI cell growth medium [57]. Therefore, for 2, reduction most
likely also occurs in the growth medium prior to internalization by
the cells. The low in vitro cytotoxicities of 1 and 2 measured here
suggest that these complexes are poor anticancer drug candidates.
In contrast, several of the aforementioned platinum(II) and
3.6. Cytotoxicity measurements
The antiproliferative effects of 1, 2, and cisplatin as a control
were determined in HeLa cells by the MTT assay. The 50% growth
inhibitory concentration (IC50) values are collected in Table 5. Both
Fig. 5. Structure of 3. Thermal ellipsoids are drawn at the 50% probability level.
Table 4
Selected interatomic distances (Å) and angles (degrees) for 3.a
Bonds
Angles
Pt1–N1
Pt1–N2
2.101(9)
2.103(9)
2.3988(15)
2.3934(15)
2.4197(14)
2.4359(15)
N1–Pt1–N2
N1–Pt1–Br1
N1–Pt1–Br2
N1–Pt1–Br3
N1–Pt1–Br4
N2–Pt1–Br1
83.9(3)
92.6(3)
176.4(3)
88.5(2)
88.8(2)
176.3(2)
N2–Pt1–Br2
N2–Pt1–Br3
N2–Pt1–Br4
Br1–Pt1–Br2
Br1–Pt1–Br3
Br1–Pt1–Br4
92.9(2)
89.2(2)
89.2(2)
90.57(6)
91.92(6)
89.54(6)
Br2–Pt1–Br3
Br2–Pt1–Br4
Br3–Pt1–Br4
89.64(6)
93.02(6)
176.95(6)
Pt1–Br1b
Pt1–Br2b
Pt1–Br3b
Pt1–Br4b
a
Atoms are labeled as indicated in Fig. 5. Numbers in parentheses are the estimated standard deviations of the last significant figures.
These distances are shorter than anticipated owing to partial occupancy of the site by chloride (see text).
b

78
J.J. Wilson, S.J. Lippard / Polyhedron 58 (2013) 71–78
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IC50 values of cisplatin, 1, and 2 in HeLa cells.a
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IC50
1.0 0.8
50
47 11
(
l
M)
Cisplatin
1
2
2
a
Reported errors are standard deviations of at least
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Acknowledgements
This work was supported by Grant CA034992 from the National
Cancer Institute. Spectroscopic instrumentation at the MIT DCIF is
maintained with funding from NIH Grant 1S10RR13886-01. J.J.W.
is the recipient of a David H. Koch Graduate Fellowship. The
authors thank Mr. Carl Brozek and Professor Mircea Dinca˘ for
assistance with and use of a powder X-ray diffractometer, and
Mr. Timothy Johnstone and Dr. Patricia Marqués-Gallego for com-
ments on the manuscript.
Appendix A. Supplementary data
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crystallographic data for 1, 2 and 3. These data can be obtained free
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from the Cambridge Crystallographic Data Centre, 12 Union Road,
Cambridge CB2 1EZ, UK; fax: +44 1223 336 033; or e-mail: depos-
it@ccdc.cam.ac.uk. Supplementary data associated with this article
can be found, in the online version, at http://dx.doi.org/10.1016/
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