Synthesis, characterisation and: In vitro cytotoxicity of mixed ligand Pt(II) oxadiazoline complexes with hexamethylenetetramine and 7-nitro-1,3,5-triazaadamantane

Article abstract of DOI:10.1039/c7dt02406a

trans-Platinum(ii) oxadiazoline complexes with 7-nitro-1,3,5-triazaadamantane (NO₂-TAA) or hexamethylenetetramine (hmta) ligands have been synthesised from trans-[PtCl₂(PhCN)₂] via cycloaddition of nitrones to one of the coordinated nitriles, followed by exchange of the other nitrile by NO₂-TAA or hmta. Stoichiometric control allows for the selective synthesis of mono- and dinuclear complexes where 7-NO₂TAA and hmta act as mono- and bidentate ligands, respectively. Precursors and the target complexes trans-[PtCl₂(hmta)(oxadiazoline)], trans-[PtCl₂(NO₂-TAA)(oxadiazoline)] and trans-[{PtCl₂(oxadiazoline)}₂(hmta)] were characterised by elemental analysis, IR and multinuclear (¹H, ¹³C, ¹⁹⁵Pt) NMR spectroscopy. DFT (B3LYP/6-31G?/LANL08) and AIM calculations suggest a stronger bonding of hmta with the [PtCl₂(oxadiazoline)] fragment, in agreement with the experimentally observed reactivity in the ligand exchange (hmta > 7-NO₂TAA). Replacement of the nitrile by hmta is predicted to be more exothermic than that with 7-NO₂-TAA, although the activation barriers are similar. Protonation of the non-coordinated N atoms is anticipated to weaken the Pt-N bond and lower the activation barrier for ligand exchange. This effect might help activate these compounds in a slightly acidic environment such as some tumour tissues. Ten of the new compounds were tested for their in vitro cytotoxicity in the human cancer cell lines HeLa and A549. Some of the mononuclear complexes are more potent than cisplatin, and their activity is still high in A549 where cisplatin shows little effect. The dinuclear complexes are inactive, presumably due to their lipophilicity and reduced solubility in water.

Full text of DOI:10.1039/c7dt02406a

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DOI: 10.1039/C7DT02406A
All correspondence should be sent to
Dr Gabriele Wagner
Tel
+44 (0) 1244 512427
Department of Natural Sciences
University of Chester
e-mail g.wagner@chester.ac.uk
Thornton Science Park
Pool Lane, Ince, Chester, CH2 4NU,
United Kingdom.
Synthesis, characterisation and in-vitro cytotoxicity of mixed ligand Pt(II)
oxadiazoline complexes with hexamethylenetetramine and 7-nitro-1,3,5-
triazaadamantane.
Stefanie Sieste,a Irina Lifincev,a Nina Steina and Gabriele Wagner,*a,b
a: Institute of Inorganic Chemistry, University of Ulm, Albert-Einstein-Allee 11, D-89081
Ulm, Germany.
b: Department of Natural Sciences, University of Chester, Thornton Science Park, Pool Lane,
Ince, Chester, CH2 4NU, United Kingdom.
TOC Entry
The synthesis, spectroscopic and DFT-computational characterisation of trans-platinum(II)
oxadiazoline complexes with one hexamethylenetetramine or 7-nitro-1,3,5-triazaadamantane
ligand is described. Some of these complexes are more cytotoxic than cisplatin in in vitro tests
with the human cancer cell lines HeLa and A549.
1

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DOI: 10.1039/C7DT02406A
Abstract
Trans-platinum(II) oxadiazoline complexes with 7-nitro-1,3,5-triazaadamantane (NO2-TAA)
or hexamethylenetetramine (hmta) ligands have been synthesised from trans-[PtCl2(PhCN)2]
via cycloaddition of nitrones to one of the coordinated nitriles, followed by exchange of the
other nitrile by NO2-TAA or hmta. Stoichiometric control allows for the selective synthesis of
mono- and dinuclear complexes where 7-NO2TAA and hmta act as mono- and bidentate
ligands, respectively. Precursors and the target complexes trans-[PtCl2(hmta)(oxadiazoline)],
trans-[PtCl2(NO2-TAA)(oxadiazoline)] and trans-[{PtCl2(oxadiazoline)}2(hmta)] were
characterised by elemental analysis, IR and multinuclear (1H, 13C, 195Pt) NMR spectroscopy.
DFT (B3LYP/6-31G*/LANL08) and AIM calculations suggest a stronger bonding of hmta
with the [PtCl2(oxadiazoline)] fragment, in agreement with the experimentally observed
reactivity in the ligand exchange (hmta > 7-NO2TAA). Replacement of the nitrile by hmta is
predicted more exothermic than that with 7-NO2-TAA, although the activation barriers are
similar. Protonation of the non-coordinated N atoms is anticipated to weaken the Pt-N bond
and lower the activation barrier for ligand exchange. This effect might help activate these
compounds in a slightly acidic environment such as some tumour tissues.
Ten of the new compounds were tested for their in vitro cytotoxicity in the human cancer cell
lines HeLa and A549. Some of the mononuclear complexes are more potent than cisplatin,
and their activity is still high in A549 where cisplatin shows little effect. The dinuclear
complexes are inactive, presumably due to their lipophilicity and reduced solubility in water.
Keywords
Platinum complexes, oxadiazoline, hexamethylenetetramine, azaadamantane, cancer therapy
Introduction
Cancer chemotherapy of solid tumours relies heavily on the use of platinum-based drugs,
namely the globally approved cytotoxic Pt(II) compounds cisplatin, carboplatin and
oxaliplatin, together with locally approved derivatives such as lobaplatin, nedaplatin and
heptaplatin.1 Despite their significant therapeutic success there are strong limitations due to
the severe side effects experienced by the patient, and the occurrence of intrinsic or acquired
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resistance. The latter, in the form of cross resistance, drastically restricts theDOthI:e10r.a10p3e9u/Ct7iDcT02406A
options because cancer cells that acquired resistance to one drug will respond poorly to
secondary treatment with other platinum drugs also.
In the search for improved therapeutic methods, much work has gone into the development of
new delivery systems for established drugs, but also into the design of new compounds.1,2
Trans-configured Pt(II) compounds received increasing interest when their in vitro ability to
overcome resistance was recognised. Among the compounds studied there are Pt(II) and
Pt(IV) complexes bearing aliphatic or aromatic amines,3 or higher order nitrogen containing
ligands. Pt(II) iminoether complexes4 have been investigated in much detail with respect to
their mechanism of action. A marked cellular uptake and higher degree of DNA platination,
together with the formation of mainly monofunctional adducts, seems to evoke DNA damage
and intracellular repair mechanisms which are quite different to those caused by cisplatin.
Pt(II) bisamidine complexes5 also show a higher uptake and cellular accumulation than
cisplatin, and this has been attributed to the presence of a phenyl group which increases the
lipophilicity of the complex. Also the trans-configured Pt(II) oxadiazoline complexes, shown
in Scheme 1 and studied in our group,6 are active against a panel of human cancer cells
including cisplatin and carboplatin resistant ones, and the IC50 values are typically in an
acceptable micromolar range, between those of cisplatin and carboplatin. In these compounds,
the substitution pattern can be easily varied, allowing for fine tuning of pharmacologically
relevant parameters such as solubility and transport properties, or even for introduction of
targeting agents that aid the selective uptake in the cancer cells.
Scheme 1. Platinum(II) oxadiazoline complexes with in-vitro cytostatic properties.6
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The synthesis of platinum oxadiazoline complexes follows a straightforward modular scheme
where the ligand is built up in the coordination sphere of the metal by cycloaddition of a
nitrone to a metal coordinated nitrile, as long as the latter is kinetically sufficiently stable.7
Both Pt(IV)8 and Pt(II)9 complexes are easily accessible and can be interconverted into each
other.10,11 The reaction typically occurs with a high degree of chemo- and stereoselectivity, so
that functionalised12 and chiral complexes13 are accessible as well. Mixed ligand complexes
can be made from suitable precursors bearing one nitrile and one other ligand (e.g.
sulfoxide),13 or by mono-cycloaddition to only one of two initially equivalent nitriles14 and
subsequent ligand exchange.11
The latter method lead to complexes bearing a reactive and labile NO2-TAA ligand,15
designed to achieve some selectivity in cellular uptake and enhanced reactivity in tumour cells
with fairly simple means. The non-coordinated nitrogen atoms are expected to partially
protonate in an aqueous medium, to give cationic complexes that are more prone to penetrate
the cell membrane. Moreover, protonation has been shown to weaken the coordination to the
platinum to make the complex more labile, and also the release of formaldehyde from the
ligand is stimulated by protonation. Since tumour tissue is often more acidic than normal
tissue, all these effects should be at work, resulting in a higher activity.
In this work, hexamethylenetetamine (hmta, also known as 1,3,5,7-tetraazaadamantane) is
explored for a similar purpose, and compared with 7-nitro-1,3,5-triazaadamantane. Hmta is
known to coordinate to transition metals in various ways,16 although only very few reports
exist on platinum complexes.17,18 Hmta is used, as hippurate and other salts, against urinary
infections, and the mode of action is assumed to be based on the slow release of
formaldehyde.19 Moreover, hmta is approved in the EU for usage as food preservative (under
the name E239),20 again using the antibiotic activity of formaldehyde released from the
compound under acidic conditions. HMTA has also been reported to enhance the sensitivity
to cisplatin when co-administered.21
4

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Results and Discussion
In this work, we present the synthesis of new Pt(II) oxadiazoline complexes bearing a 7-nitro-
1,3,5-triaza-adamantane ligand (5b, 5e, 5f), the mononuclear hexamethylenetetramine
complexes 7a – 7f and the corresponding dinuclear species 8a – 8f, shown in Scheme 2 and 3.
DFT calculations are used to rationalise the reactivity pattern, and the in vitro cytotoxicity of
selected compounds is assessed.
Synthesis of the Pt(II) complexes
The complexes 5a – 5f and 7a – 7f were synthesised in two steps via the cycloadducts 3a – f,
by reaction of trans-[PtCl2(PhCN)2] (2) with one equivalent of a nitrone 1a – 1f. The mono-
cycloadducts trans-[PtCl2(PhCN)(oxadiazoline)] 3a – 3f were obtained with high selectivity
and in good yields. Their spectroscopic properties correspond closely to those described
previously for related compounds.14,15 When 3a – 3f are reacted with one equivalent of a
tertiary amine such as 7-nitro-1,3,5-triaza-adamantane 4 and hexamethylenetetramine 6, the
benzonitrile ligand is replaced and the mixed ligand oxadiazoline complexes 5a – 5f and 7a –
7f are formed (see Scheme 2). The reaction with 6 is accompanied by the formation of the
dinuclear side products 8a – 8f, in which two PtCl2(oxadiazoline) moieties are coordinated to
one molecule of 6, as shown in Scheme 3. This side reaction, however, can be suppressed
when an excess (1.5 equivalents) of 6 is used. It is worth mentioning that 6, even in a three-
fold excess, does not replace the oxadiazoline ligand from the platinum complex under the
conditions applied, even if the reaction is left for 3 weeks.
The selective synthesis of the dinuclear compounds 8a – 8f was achieved when 3a – 3f and 6
was used in a 2:1 stoichiometry and the reaction time was extended to 2 weeks. The
analogous NO2-TAA complexes can be prepared from 3a – 3f and 4, but the reaction at room
temperature takes 5 weeks to complete. At a higher temperature, formation of the dinuclear
complexes is accompanied by a number of unidentified side products, most likely due to the
decomposition of the azaadamantane framework. This can be concluded from the appearance
of additional signals in the aliphatic range during 1H-NMR monitoring of the reaction.
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Scheme 2. Synthesis of platinum(II) oxadiazoline complexes bearing 7-nitro-1,3,5-triaza-
adamantane15 or hexamethylenetetramine ligands (R = 2-methoxyphenyl (a), 4-methoxy-
phenyl (b), 2,6-dimethoxyphenyl (c), 2,4,6-trimethoxyphenyl (d), 2,3,4-trimethoxyphenyl (e)
and 3,4,5-trimethoxyphenyl (f).
Scheme 3. Synthesis of dinuclear platinum(II) oxadiazoline complexes 8 with the hexa-
methylenetetramine ligand 6 and oxadiazoline ligands (abbreviated as L).
The IR spectra of the mononuclear complexes 7a – f are dominated by the fundamentals of
the C-N vibrations of the hexamethylenetetramine ligand. These bands appear at 1234, 992,
808 and 669 cm-1 in the free hexamethylenetetramine. In the Pt(II) coordinated species they
are split and also experience some shift. Literature data suggest a minor splitting resulting in
closely spaced doublets or triplets when hmta acts as a monodentate ligand,22 whereas
6

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complexes with bidentate bridging hmta ligands show well defined and well separated
bands.23 This, however, seems not to apply as a general rule since compounds 7a – 7f show
clearly separated signals in spite of the monodentate coordination mode. Thus, the resonance
at 1234 cm-1 splits into two bands and experiences a symmetric shift around the band of the
free hmta, whereas those at 992 and 808 cm-1 split and shift, the former to higher, the latter to
lower wavenumbers. A detailed interpretation, however, is complicated by the presence of
signals of the oxadiazoline ligand, among which the C=N and C=C stretch at 1629-1643 and
1593-1610 cm-1 can be clearly assigned, together with the characteristic C-H stretching
vibration of the OMe groups at 2841-2834 cm-1. The dinuclear complexes 8a – 8f show
relatively similar IR spectra, but the signals attributed to the oxadiazoline ligand are
somewhat more intense.
Free hmta is Td symmetric, resulting in the equivalence of all CH2 groups in the NMR.
Therefore, only one singlet is seen in the 1H NMR and also only one signal appears in the 13C
NMR spectrum. When coordination to one nitrogen atom occurs, the local symmetry of the
hmta ligand is C3v, assuming that the rotation around the Pt-N bond is not hindered (and
ignoring the C1 symmetry of the PtCl2(oxadiazoline) moiety). In this case, one would expect
one singlet for the protons Ha and two doublets for the axial and equatorial protons Hb and Hb’
1
13
in the H NMR and two signals in the C NMR, as indicated in Scheme 4. This was indeed
observed in the spectra of compounds 7a – 7f: The proton signals appear in a range of 4.96 to
5.02 ppm (Ha) and at 4.44 and 4.50 ppm (Hb and Hb’), at higher and lower field, respectively,
as compared to the free ligand (4.72 ppm). The signal at 4.50 ppm was attributed to Hb’
because it displays an NOE with Ha in the NOESY spectrum. Consequently, the signal at 4.44
ppm that does not show an NOE with Ha is assigned to Hb. The 13C signals are also more and
less deshielded (79.4 and 73.0 ppm in the complex, as compared to 74.8 ppm in the free
ligand). As a rule, the atoms closer to the Pt coordinated nitrogen appear at lower field,
whereas those further away from the coordination site experience a high field shift. The trans-
configuration of the complexes can be inferred from the absence of NOE signals between the
two organic ligands. Also the 195Pt NMR signal would be expected further downfield in the
corresponding cis-complexes,24 although this effect can be pretty small.14
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Scheme 4. 1H and 13C NMR numbering of the coordinated hexamethylenetetramine ligand in
compounds 7 (top) and 8 (bottom).
When two platinum moieties coordinate to the hmta, the local symmetry is further reduced to
C2v (still assuming free rotation around the Pt-N bonds). This should result in four signals in
1
13
the H NMR and three signals in the C NMR. In the spectra of 8a – 8f, however, further
signal splitting was observed, suggesting the presence of two diastereoisomers with different
configurations at the chiral carbon of the oxadiazoline. Additionally, some of the methoxy
signals in the 1H and 13C spectra of 8c and 8d are split or broadened, which is attributed to the
additional existence of conformers, due to a hindered rotation of the heterocycles around the
Pt-N bonds, when two methoxy substituents are present in ortho-position of the aromatic ring.
These signals should collapse at higher temperature. T-dependent NMR experiments,
however, are complicated by the limited thermal stability of the complexes, and noticeable
decomposition takes place at 60 oC already.
Computational analysis of the ligands and the platinum complexes.
A DFT study and a topological analysis of the charge densities was undertaken for ligands 4
and 6, the representative Pt(II) compounds 3a, 5a, 7a and 8a and their protonated congeners,
with the aim to elucidate the reactivity and the properties of the platinum complexes. The
structures obtained by full geometry optimisation using the B3LYP functional, LANL08 for
Pt and Cl, and 6-31G* for all other atoms compare well with X-ray crystallographic data of
closely related complexes bearing oxadiazoline or azaadamantane ligands,9,11,13,25 and with the
results from other DFT calculations using the same or very similar methods.15,26
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Table 1 shows the bond distances, bond orders and charge densities at the bond critical point
of the Pt-N and Pt-Cl bonds in compounds 5a, 7a, 8a, monoprotonated 5a-H+, 7a-H+,
diprotonated 5a-2H+, 7a-2H+, and triprotonated 7a-3H+. The Pt-Cl bonds show little
response to the nature of the N-ligands, as expected from molecular orbital considerations for
a cis-arrangement where the electronic communication is weak. The trans-positioned N-
ligands, however, clearly communicate with each other, and the Pt-N bond to the oxadiazoline
ligand is weaker when the Pt-N bond to the trans-positioned azaadamantane is stronger (and
vice versa). Judging from the shorter bond length, the higher bond order and the higher charge
density in the bond critical point, the Pt-N bond to the hmta ligand in 7a is stronger than that
to the 7-NO2-TAA ligand in 5a. The azaadamantane cage of free 6 is more electron rich than
that of free 4, as deduced from the higher average AIM charge27 at the amine nitrogens (4: -
0.968; 6: -0.976) and the slightly higher charge density in the cage critical point (4: 0.0984
e/Å; 6: 0.0993 e/Å). The higher negative charge at the N-atoms in 6 suggest stronger σ-donor
properties resulting in the higher tendency to bind to Pt(II), in agreement with the experiment.
The bond between the Pt(II) and the hmta nitrogen is stronger in the mononuclear complex 7a
than in the dinuclear species 8a, suggesting that the coordination of a second Lewis acidic
metal moiety weakens the bond to the first one. A similar effect is observed when the non-
coordinating nitrogen atoms in 7a are protonated to give 7a-H+, 7a-2H+ and 7a-3H+. With
increasing degree of protonation the Pt-N bond weakens and concomitantly the Pt-N(oxa)
bond strengthens. A ligand exchange is thus expected to occur more easily when the hmta
ligand is protonated or a second coordination takes place.
The monoprotonated species 5a-H+ and 7a-H+ also show an interesting charge distribution at
the C-atoms of the azaadamantane ligand. The carbons remote from the coordination site are
electron rich whereas the one that is flanked by the Pt-N and H-N+ moieties is particularly
electron deficient. A nucleophilic attack at this carbon should thus be facilitated, and this has
indeed been observed with 5a where hydrolysis lead to the release of formaldehyde.15 The
same effect can be seen in the dinuclear complex 8a, where the same C-atom is flanked by
two PtCl2(oxadiazoline) moieties. Here, however, a nucleophilic attack appears difficult for
steric reasons and this might explain the relative stability of complexes of this type.
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Table 1. Bond lenghts and electronic properties of the Pt-N and Pt-Cl bonds in compounds 5a, 7a, 8a, monoprotonated 5a-H+, 7a-H+, diprotonated
5a-2H+, 7a-2H+, and triprotonated 7a-3H+.
Bond
Property
5a
5a-H+ 5a-2H+ 7a
8a
7a-H+
2.014
0.399
7a-2H+
1.992
7a-3H+
1.981
bond lenght (Å) 2.035
2.008
0.405
1.989
0.458
2.042
0.348
2.036
0.357
2.035
0.357
Pt-N(oxa)
bond order
0.354
0.454
0.513
ρBCP (e/Å3)
0.7792 0.8489 0.9086 0.7645 0.7854 0.7794 0.8357
0.9009
2.215
0.9461
2.276
bond lenght (Å) 2.135
2.182
0.334
2.234
0.253
2.124
0.431
2.136
0.419
2.136
0.419
2.165
0.345
Pt-N(TAA)
or
bond order
0.420
0.260
0.184
ρBCP (e/Å3)
0.6450 0.5551 0.4768 0.6658 0.6428 0.6419 0.5822
0.4989
2.446
0.4239
2.466
Pt-N(hmta)
Pt-Cl(1)
bond length (Å) 2.447
2.433
0.843
2.443
0.812
2.448
0.816
2.443
0.827
2.443
0.829
2.451
0.808
bond order
0.818
0.811
0.768
ρBCP (e/Å3)
0.4513 0.4670 0.4590 0.4498 0.4556 0.4558 0.4491
0.4564
2.413
0.4415
2.403
bond lenght (Å) 2.435
2.429
0.860
2.413
0.879
2.432
0.873
2.432
0.877
2.432
0.878
2.413
0.902
Pt-Cl(2)
bond order
0.876
0.881
0.891
ρBCP (e/Å3)
0.4668 0.4683 0.4854 0.4653 0.4659 0.4656 0.4856
0.4852
0.4974
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The energy difference ΔE = E(protonated species) – E(unprotonated species) wDaOsI:u10s.e10d39a/Cs7DaT02406A
measure for the protonation energies28 given in Table 2. Comparing the free ligands, it
becomes evident that hmta 6 is more easily protonated than 7-NO2TAA 4, as the reaction 6 +
H+ → 6-H+ is more exothermic than 4 + H+ → 4-H+. This suggests that 4 is less basic than 6,
in agreement with the pKa values of the protonated species 4-H+ and 6-H+ of 3.42 (±0.05)
(see Experimental Part) and 4.89.29 The same trend is seen in the protonation of the platinum
complexes 5a and 7a, where also the NO2TAA complex 5a is less prone to accept H+.
Compared to the free ligands, the platinum complexes are more difficult to protonate, in line
with the electron withdrawing effect the Lewis-acidic Pt(II) center exhibits. A second and
third protonation (where possible) is energetically less favourable than the first protonation,
for free ligands and Pt(II) complexes alike, which also meets our expectations. The
protonation of the complexes 5a and 7a is easier than H+-transfer to the mono-protonated free
ligands, since the binding of the stronger Lewis acid H+ reduces the basicity of the remaining
nitrogens more than the coordination to the weakly Lewis-acidic Pt(II) center.
The ligand exchange reactions of PtCl2(oxa)(PhCN), namely the derivative 3a, were assessed
from the reaction energies ΔE = Ʃ(E(products) – Ʃ(E(reactants). Overall, the exchange of
PhCN by 4 or 6 is exothermic, to a higher degree for hmta 6 than for the 7-NO2TAA ligand 4.
The thermodynamic motivation of the analogous reactions with the mono-protonated ligands
is lower and comes close to thermoneutrality in the case of 4-H+. Thus, protonation of 5a and
possibly also 7a should lead to an equilibrium situation in which 3a co-exists with 5a and 7a.
In the formation of the 2:1 complex 8a, the second coordination is less thermodynamically
motivated than the first one, in agreement with the experimental observations.
The replacement of the oxadiazoline ligand from 3a by reaction with 4 is thermodynamically
disfavoured, and the analogous reaction with 6 is close to thermoneutral. In both cases, the
replacement of the nitrile ligand in 3a is thermodynamically far more favourable, and this
agrees well with the observed selectivity in favour of formation of 5a and 7a.
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Table 2. Reaction energies ΔE and selected activation energies Ea of the protonation and
ligand exchange reactions involving the free ligands 4 and 6 and the platinum complexes 5a,
7a and 8a (kcal/mol).
Protonation reaction
4 + H+ → 4-H+
ΔE
Protonation reaction
6 + H+ → 6-H+
ΔE
-220.6
-113.3
-7.1
-229.0
-120.1
-11.0
-224.5
-127.1
-32.6
ΔE
4-H+ + H+ → 4-2H+
4-2H+ + H+ → 4-3H+
5a + H+ → 5a-H+
6-H+ + H+ → 6-2H+
6-2H+ + H+ → 6-3H+
7a + H+ → 7a-H+
-217.0
-121.3
5a-H+ + H+ → 5a-2H+
7a-H+ + H+ → 7a-2H+
7a-2H+ + H+ → 7a-3H+
Ligand exchange
Ligand exchange
ΔE
Ea
Ea
3a + 4 → 5a + PhCN
-5.13 +15.2 3a + 6 → 7a + PhCN
3a + 6-H+ → 7a-H+ + PhCN
-8.02 +15.3
-3.53
3a + 4-H+ → 5a-H+ + PhCN -1.48
3a + 7a → 8a + PhCN
-4.71
2.74
3a + 6-3H+ → 7a-3H+ + PhCN -30.2
3a + 6 → 10a + oxadiazoline -0.58
3a + 4 → 9a + oxadiazoline
Figure 1. Transition states for the ligand exchange reactions 3a + 4 → 5a + PhCN (left) and
3a + 6 → 7a + PhCN (right).
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Figure 2. Energy profile and characteristic bond distances and angles along the intrinsic
reaction coordinate of the reaction 3a + 6 → 7a + PhCN.
The reaction kinetics for the ligand exchange (3a + 4 or 6 to give 5a or 7a + PhCN) were
assessed from the activation barriers Ea, which are 15.2 kcal/mol and 15.3 kcal/mol,
respectively. Both reactions are thus expected to occur with approximately the same reaction
rate. The observed slower reaction with 7-NO2TAA 4 is probably due to the poor solubility of
this ligand, thus the reaction is hampered by the low availability of the free ligand in solution.
The transition state for ligand exchange (Figure 1) can be best described as a slightly distorted
trigonal bipyramidal structure with the chloro ligands in apical positions at the central Pt atom
and the nitrogen ligands in the trigonal plane. From a mechanistic point of view, addition and
elimination are relatively simultaneous processes, which can be seen from the transition state
geometry and also from the single energy barrier in the energy profile and the changes in
bond distances and angles along the intrinsic reaction coordinate (IRC), shown in Figure 2 for
the reaction 3a + 6 → 7a + PhCN. The Pt-N-C(nitrile) angle starts to bend upon approach of
the azaadamantane ligand, and the Pt-N bond to the nitrile elongates as the Pt-N bond to the
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DOI: 10.1039/C7DT02406A
azaadamantane shortens. The overall process resembles an SNi reaction at a tetrahedral center
where both nucleophile and nucleofuge share the same orbital lobe of the electrophilic center.
The oxadiazoline as a spectator ligand practically does not change any of its parameters along
the IRC, except of a small conformational modification of the phenyl ring. Also the chloro
ligands remain unaffected. Overall, the ligand exchange occurs under retention of the trans-
configuration in the product, in agreement with the experimental observation.
In-vitro cytotoxicity of the Pt(II) complexes
The in vitro cytotoxicity of cisplatin and the new compounds 5a, 7a, 8a, 5b, 7b, 5c, 7c, 7d, 5e
and 7e in the epithelial human cancer cell lines HeLa30 and A54931 was determined by means
of the CellTiter-Glo® luminescent cell viability assay,32 as described in the experimental part.
HeLa cervical cancer cells are known to respond to cisplatin with an IC50 of 1.1 to 1.3 μM,33
whereas the lung cancer cell line A549, with an IC50 of 64 μM, is fairly inert to cisplatin
treatment.34
Figure 3. Cell viability after 24 h of incubation in the presence of 2.5 μM and 10 μM doses of
the platinum compounds cisplatin, 5a, 7a, 8a, 5b, 7b, 5c, 7c, 7d, 5e and 7e. Data are mean
values over three experiments and given relative to untreated cells = 100 %. Cell viabilities
are given relative to a solvent blank = 100 %. Error bars indicate standard deviations.
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DOI: 10.1039/C7DT02406A
Due to the poor solubility of the Pt(II) compounds in aqueous media DMSO had to be used as
a co-solvent. The stability of compound 5a in DMSO over a period of weeks has been
established before.15 Nevertheless, care has been taken to avoid ligand exchange by reducing
the contact to DMSO to a minimum. Thus, the platinum compounds were dissolved in DMSO
and immediately diluted into water to give stock solutions which are 100 μM in platinum and
contain 5 % (v/v) DMSO. These were used immediately without further storage for the
cytotoxicity experiments. Aliquots used to achieve 10 μM or 2.5 μM platinum doses in the
cell suspension introduce 0.5 % (v/v) and 0.125 % (v/v) of DMSO, which should have little
effect on the growth and survival of the cancer cells.35 Any effects on the intracellular ATP
levels in the presence of more than 0.1 % (v/v) DMSO36 are compensated by solvent blank
measurements.
The mononuclear hmta complex 7a, as the most potent compound tested, is significantly more
active than cisplatin and able to reduce the cell viability to 24.3%, using a 2.5 μM dose, where
with cisplatin the cell viability is 47.3%. In contrast to that, the analogous dinuclear complex
8a, bearing the same oxadiazoline ligand as 7a, is totally inactive in the concentration range
tested (2.5 to 10 μM). This might be due to the low polarity of 8a, seen in the high Rf values
in thin layer chromatography. Additionally, since the di-coordinated hmta in 8a will not be
protonated, no cationic species are present in an aqueous medium, resulting in an overall low
solubility and poor transport properties and cellular uptake. The substitution pattern of
compounds 7 has a strong influence on the activity, which decreases in an order 7a > 7c > 7e
> 7b > 7d. It seems that the presence of an OMe group in 4-position of the aromatic ring
attached to the oxadiazoline ligand reduces the in vitro activity of the compound, whereas an
OMe group in 2-position seems to strongly enhance it. The analogous hmta and NO2-TAA
complexes reduce the cell viability to a similar extent (e.g 7b and 5b), suggesting that the
hmta and NO2-TAA ligands do not affect the activity of the compound greatly. NO2-TAA and
hmta may dissociate off at a fairly early stage of delivery and binding, and activity is
determined by the nature of the PtCl2(oxadiazoline) fragment or the hydrolysed form thereof.
An attempted NMR study of the stability of our compounds in the cell culture medium did not
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give conclusive results due to the low concentration (and solubility) of the Pt(II)DcOoI: m10.p10o3u9/nCd7DsT02406A
in the presence of a large amount of culture medium.
Compound 7a, as the only compound, is more active at low concentration (2.5 μM, as
compared to 10 μM), and this effect is reproducible. At the current stage of investigation, we
are not sure whether there is a biochemical reason for this, or whether it is caused by the low
solubility of the compound in an aqueous medium. The 100 μM stock solutions, when left
overnight, show clear signs of precipitation, and the solid, isolated by centrifugation and
analysed by SEM, TEM and EDX, has the correct Pt:Cl:S elemental composition expected for
un-decomposed 7a (that is, Pt:Cl 1:2, no S detetable). Presumably, the 2.5 M solution is
supersaturated and all the platinum compound is bioavailable to the cells, whereas the 10 M
solution precipitates 7a during the cytotoxicity experiment and the cells can only take up the
dissolved compound and not the particulate matter. Clearly, far more detailed studies are
necessary to clarify this effect.
A549 cells show the expected weak response to 2.5 μM cisplatin, and also our new
compounds do not perform any better, except of the TAA complex 5c, which reduces the cell
viability to about 80 %. With the four-fold dose (10 μM), the dinuclear complex 8a, is still
inactive, as in the case of the HeLa cells, but all mononuclear complexes show appreciable
activity, and the structure-activity relation pattern is similar to the one observed for the HeLa
cells at lower concentration. Most of our mononuclear complexes (5a, 5c, 5e, 7a, 7c, 7e) are
in fact more active in A549 than cisplatin, and this could make this class of compounds
interesting for therapeutic applications. Only a few other Pt(II) complexes show a similar
behaviour, as for example a group of cis-Pt(II) complexes with pyrazole derived ligands with
an up to 3-fold potency as compared to cisplatin.37 More often, the activity against A549 is
best of all similar to that of cisplatin, as in the case of cis-Pt(II) amidine complexes.5a
Conclusion
In this work, we described a highly efficient and selective synthesis route to a series of mono-
and dinuclear mixed ligand Pt(II) complexes bearing oxadiazoline and azaadamantane
ligands, and their in-vitro cytotoxicity in two human cancer cell lines (HeLa and A549).
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These complexes were designed to bear one oxadiazoline as an easily modifiable ligand that
allows for fine-adjustment of the pharmacological properties, and one azaadamantane ligand
whose lability can be triggered by protonation in the slightly acidic environment found in
some tumour tissues. This hypothesis has been supported by DFT studies and is in line with
preliminary experimental observations of the chemical reactivity of related NO2TAA
complexes.15 DFT calculations were also used to corroborate the reactivity and selectivity in
the ligand exchange reaction and mechanistic issues, by looking into transition state
geometries, activation barriers and energy profiles of the reaction.
The in-vitro cytotoxicity of ten of the new compounds was tested using the human cancer cell
lines HeLa and A549. Whereas the dinuclear complexes were inactive (most likely due to low
solubility and poor cellular uptake), all mono-nuclear complexes showed a fairly high
activity. This was often higher than that of cisplatin used for comparison, in particular with
the lung cancer cell line A549 which is known to respond poorly to cisplatin. Analogous
NO2TAA and hmta complexes show fairly similar activity, suggesting a dissociation of the
labile ligand at a relatively early stage. For practical reasons (commercial availability, low
cost, faster reactions and easier product purification), hmta as a labile ligand appears slightly
superior, as compared to the NO2TAA. Overall, our in-vitro cytotoxicity results are
promising, but further studies will be necessary to fully assess the potential of these new
compounds with respect to a potential therapeutic use.
Experimental Part
Materials and Instrumentation. Solvents and reagents were obtained from
commercial sources and used as received. Trans-[PtCl2(PhCN)2] 2,11,38 nitrones 1a-1f39 and 7-
NO2TAA 440 were synthesised according to published methods. C, H, N elemental analyses
1
were run on a Vario Micro Cube automatic analyser. Infrared spectra (4000-400 cm ) were
1
recorded on a Bruker Tensor 27 FT-IR using the ATR technique. H, 13C and 195Pt NMR
spectra were acquired on Bruker Avance 500 and Bruker Avance 400 spectrometers at
ambient temperature. 195Pt chemical shifts are given relative to aqueous K2[PtCl4] = 1630
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ppm. All 195Pt signals show half height line widths of 600 – 750 Hz, as a result of unresolved
DOI: 10.1039/C7DT02406A
spin-spin interactions with the quadrupolar 14N nuclei.
Determination of the pKa values of the free ligands 4 and 6.
The pKa values were determined from titrations of 4 and 6 with 0.1 M HCl, according to a
procedure described in the literature.29 Since the pKa values are relatively low, back titration
of the protonated forms 4-H+ and 6-H+ with 0.1 M NaOH was also applied. For this, 0.8 mmol
of compounds 4 or 6 were dissolved in 30 ml of demineralised water and 8 ml of 0.1 M HCl
(1 equivalent) were added. The solution was then titrated with 0.1 M NaOH and the pKa
value was obtained from the titration curve at the half-equivalence point.
4-H+ + H2O → 4 + H3O+ (pKa = 3.42).
6-H+ + H2O → 6 + H3O+ (pKa = 4.87; lit: 4.89,29 4.8641).
Synthesis of the mixed benzonitrile / oxadiazoline complexes.
Trans-PtCl2(PhCN)(oxadiazoline) complexes 3a - 3f were prepared according to the
literature,15 where also the characterisation of compounds 3a, 3c and 3d can be found.15
trans-(Benzonitrile)-dichloro[2,3-dihydro-3-(4-methoxyphenyl)-2-methyl-5-phenyl-1,2,4-
oxadiazole-N4] platinum (3b). Yield 89 %. Elemental analysis calculated for
C23H21Cl2N3O2Pt: C 43.34; H 3.32; N 6.59; found: C 43.12; H 3.22; N 6.68. IR (selected
1
bands), cm : 3046, 3003, 2964 and 2931 (CH), 2837 (CH of OMe), 2289 (CN), 1627
1
m (C=N). H NMR in CDCl3,  (ppm): 3.06 (s, br., 3H, NMe), 3.86 (s, 3H, OMe), 5.95 (s,
br., 1H, N-CH-N), 7.01 (d, 8.9 Hz, 2H) and 7.67 (d, 8.5 Hz, 2H)(aryl-H of N-CH(Ar)-N), 7.52
(t, 7.7 Hz, 2H) and 7.72 (m, 3H)(PhCN-Pt), 7.62 (t, 7.7 Hz, 2H), 7.71 (m, 1H) and 9.01 (d,
13
8.0 Hz, 2H)(aryl-H of PhC=N). C NMR in CDCl3,  (ppm): 45.8 (NMe), 55.3 (OMe), 94.4
(N-CH-N), 114.1 and 130.7 (CH of N-CH(Ar)-N), 130.2 and 160.7 (Cq of N-CH(Ar)-N),
129.3, 133.5 and 134.7 (CH of PhCN-Pt), 109.8 (Cq of PhCN-Pt), 116.5 (CN), 128.6,
130.3 and 134.1 (CH of PhC=N), 122.3 (Cq of PhC=N), 164.3 (C=N). 195Pt NMR in CDCl3, 
(ppm): 2236.
trans-(Benzonitrile)-dichloro[2,3-dihydro-3-(2,3,4-trimethoxyphenyl)-2-methyl-5-
phenyl-1,2,4-oxadiazole-N4] platinum (3e). Yield 90 %. Elemental analysis calculated for
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C25H25Cl2N3O4Pt: C 43.05; H 3.61; N 6.02; found: C 43.13; H 3.48; N 6.17. IR (selected
1
bands), cm : 3060, 2999, 2968 and 2938 (CH), 2836 (CH of OMe), 2289 (CN), 1630
1
m (C=N). H NMR in CDCl3,  (ppm): 3.08 (s, br., 3H, NMe), 3.89, 3.92 and 4.13 (s, 3H
each, OMe), 6.36 (s, br., 1H, N-CH-N), 6.75 (d, 8.8 Hz, 1H) and 7.34 (d, br., 8 Hz, 2H)(aryl-
H of N-CH(Ar)-N), 7.52 (t, 7.9 Hz, 2H) and 7.73 (m, 3H)(PhCN-Pt), 7.63 (t, 7.8 Hz, 2H),
7.69 (m, 1H) and 9.02 (d, 8.0 Hz, 2H)(aryl-H of PhC=N). 13C NMR in CDCl3,  (ppm): 46.8
(NMe), 55.9, 60.8 and 61.4 (3  OMe), 90.4 (N-CH-N), 106.6 and 124.1 (CH of N-CH(Ar)-
N), 121.7, 141.9, 152.3 and 154.9 (Cq of N-CH(Ar)-N), 129.3, 133.5 and 134.8 (CH of
PhCN-Pt), 109.8 (Cq of PhCN-Pt), 116.5 (CN), 128.6, 130.6 and 134.0 (CH of PhC=N),
122.4 (Cq of PhC=N), 161.4 (C=N). 195Pt NMR in CDCl3,  (ppm): 2233.
trans-(Benzonitrile)-dichloro[2,3-dihydro-3-(3,4,5-methoxyphenyl)-2-methyl-5-phenyl-
1,2,4-oxadiazole-N4] platinum (3f). Yield 79 %. Elemental analysis calculated for
C25H25Cl2N3O4Pt: C 43.05; H 3.61; N 6.02; found: C 42.88; H 3.58; N 6.14. IR (selected
1
bands), cm : 3061, 3000, 2965 and 2940 (CH), 2839 (CH of OMe), 2289 (CN), 1625
1
m (C=N). H NMR in CDCl3,  (ppm): 3.06 (s, br., 3H, NMe), 3.90 (s, 3H, OMe), 3.94 (s,
6H, OMe), 5.92 (s, br., 1H, N-CH-N), 6.97 (s, 2H, aryl-H of N-CH(Ar)-N), 7.50 (t, 7.8 Hz,
2H) and 7.69 (m, 3H)(PhCN-Pt), 7.62 (t, 7.9 Hz, 2H), 7.68 (m, 1H) and 9.02 (d, 7.9 Hz,
2H)(aryl-H of PhC=N). 13C NMR in CDCl3,  (ppm): 45.8 (NMe), 56.4 and 60.8 (OMe), 94.7
(N-CH-N), 106.1 (CH of N-CH(Ar)-N), 138.9 and 153.4 (Cq of N-CH(Ar)-N, third Cq not
detected), 129.3, 133.5 and 134.8 (CH of PhCN-Pt), 109.7 (Cq of PhCN-Pt), 116.6 (CN),
128.6, 130.8 and 134.2 (CH of PhC=N), 122.2 (Cq of PhC=N), 163.8 (C=N). 195Pt NMR in
CDCl3,  (ppm): 2226.
Synthesis of the mononuclear mixed triazaadamantane / oxadiazoline complexes.
Trans-PtCl2(7-NO2-TAA)(oxadiazoline) complexes 5a – 5f were prepared by reaction of 3a –
3f with 7-nitro-1,3,5-triazaadamantane 4, according to the literature.15 The compounds 5a, 5c
and 5d have already been described and characterised.15
trans-Dichloro[2,3-dihydro-3-(4-methoxyphenyl)-2-methyl-5-phenyl-1,2,4-oxadiazole-
N4][7-nitro-1,3,5-triazaadamantane-N1]platinum (5b). Yield 88 %. Elemental analysis
calculated for C23H28Cl2N6O4Pt: C 38.45; H 3.93; N 11.70; found: C 38.33; H 4.06; N 11.57.
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1

DOI: 10.1039/C7DT02406A
IR (selected bands), cm : 3055, 2966, 2926 and 2853 (CH), 2839 (CH of OMe), 1636 m
(C=N), 1608 w (C=C), 1540 s (NO2). 1H NMR in CDCl3,  (ppm): 3.02 (s, br., 3H, NMe),
3.87 (s, 3H, OMe), 3.66 (d, 14.2 Hz, 2H) and 3.69 (d, 14.2 Hz, 2H)(TAA Hd and Hd'), 4.17
("s", 2H, TAA Hc), 3.90 (m, 1H) and 4.26 (d, 13.4 Hz, 1H)(TAA Hb and Hb'), 4.52 (dm, 12.9
Hz, 2H) and 4.76 (dm, 12.9 Hz, 2H)(TAA Ha and Ha'), 5.81 (s, br., 1H, N-CH-N), 7.01 (d, 8.4
Hz, 2H) and 7.65 (d, 8.4 Hz, 2H)(aryl-H of N-CH(Ar)-N), 7.59 (t, 7.8 Hz, 2H), 7.70 (t, 7.4
Hz, 1H) and 8.92 (d, 7.9 Hz, 2H)(aryl-H of PhC=N). 13C NMR in CDCl3,  (ppm): 45.7
(NMe), 55.3 (OMe), 58.09 and 58.11 (TAA Cd), 62.5 (TAA Cc), 71.4 (TAA Cb), 78.06 and
78.08 (TAA Ca), 72.8 (TAA C-NO2), 94.3 (N-CH-N), 113.8 and 130.4 (CH of N-CH(Ar)-N),
130.5 and 160.7 (Cq of N-CH(Ar)-N), 128.4, 130.6 and 133.8 (CH of PhC=N), 122.7 (Cq of
PhC=N), 163.5 (C=N). 195Pt NMR in CDCl3,  (ppm): 2170.
trans-Dichloro[2,3-dihydro-3-(2,3,4-trimethoxyphenyl)-2-methyl-5-phenyl-1,2,4-oxadia-
zole-N4][7-nitro-1,3,5-triazaadamantane-N1]platinum (5e). Yield 82%. Elemental
analysis calculated for C25H32Cl2N6O6Pt: C 38.57; H 4.14; N 10.79; found: C 38.20; H 4.07;
1
N 10.51. IR (selected bands), cm : 3053, 2967 and 2878 (CH), 2837 (CH of OMe),
1
1635 m (C=N), 1601 w (C=C), 1538 s (NO2). H NMR in CDCl3,  (ppm): 3.00 (s, br.,
3H, NMe), 3.89 (s, 3H), 3.92 (s, 3H) and 4.12 (s, 3H)(3  OMe), 3.68 (d, 14.3 Hz, 2H) and
3.71 (d, 14.3 Hz, 2H)(TAA Hd and Hd'), 4.24 ("s", 2H, TAA Hc), 3.89 (m, 1H) and 4.27 (dm,
13.5 Hz, 1H)(TAA Hb and Hb'), 4.58 (m, 2H) and 4.81 (d, 13.2 Hz, 2H)(TAA Ha and Ha'),
6.22 (s, br., 1H, N-CH-N), 6.72 (d, 8.5 Hz, 1H) and 7.21 (d, br., 7.0 Hz, 1H)(aryl-H of N-
CH(Ar)-N), 7.61 (t, 7.9 Hz, 2H), 7.71 (t, 7.6 Hz, 1H) and 8.93 (d, 7.3 Hz, 2H)(aryl-H of
PhC=N). 13C NMR in CDCl3,  (ppm): 46.8 (NMe), 55.9, 60.8 and 61.4 (3  OMe), 58.1
(TAA Cd), 62.6 (TAA Cc), 71.4 (TAA Cb), 78.15 and 78.22 (TAA Ca), 72.8 (TAA C-NO2),
90.0 (N-CH-N), 106.8 and 124.1 (CH of N-CH(Ar)-N), 121.9, 141.9, 152.3 and 154.8 (Cq of
N-CH(Ar)-N), 128.4, 130.4 and 133.7 (CH of PhC=N), 122.9 (Cq of PhC=N), 164.7 (C=N).
195Pt NMR in CDCl3,  (ppm): 2172.
trans-Dichloro[2,3-dihydro-3-(3,4,5-trimethoxyphenyl)-2-methyl-5-phenyl-1,2,4-oxadia-
zole-N4][7-nitro-1,3,5-triazaadamantane-N1]platinum (5f). Yield 85 %. Elemental
analysis calculated for C25H32Cl2N6O6Pt: C 38.57; H 4.14; N 10.79; found: C 38.33; H 4.06;
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1
N 10.55. IR (selected bands), cm : 3064, 3001, 2964 and 2942 (CH), 284D0OI: 1(0C.103H9/C7oDfT02406A
OMe), 1629 m (C=N), 1595 w (C=C), 1538 s (NO2). 1H NMR in CDCl3,  (ppm): 3.03 (s,
br., 3H, NMe), 3.91 (s, 3H, OMe), 3.95 (s, 6H, OMe), 3.67 (s, br., 4H, TAA Hd and Hd'), 4.14
(d, 14.3 Hz, 1H) and 4.19 (d, 14.3 Hz, 1H)(TAA Hc and Hc'), 3.89 (m, 1H) and 4.24 (d, 13.3
Hz, 1H)(TAA Hb and Hb'), 4.50 (m, 2H) and 4.72 (dm, 13.3 Hz, 2H)(TAA Ha and Ha'), 5.76
(s, br., 1H, N-CH-N), 6.98 (s, 2H, aryl-H of N-CH(Ar)-N), 7.60 (t, 7.9 Hz, 2H), 7.70 (t, 7.4
Hz, 1H) and 8.94 (d, 7.8 Hz, 2H)(aryl-H of PhC=N). 13C NMR in CDCl3,  (ppm): 45.5
(NMe), 56.4 and 60.9 (OMe), 58.1 (TAA Cd), 62.5 (TAA Cc), 71.4 (TAA Cb), 78.10 and
78.12 (TAA Ca), 72.8 (TAA C-NO2), 94.5 (N-CH-N), 106.6 (CH of N-CH(Ar)-N), 138.9 and
153.2 (Cq of N-CH(Ar)-N, third Cq not detected), 128.4, 130.4 and 133.9 (CH of PhC=N),
122.6 (Cq of PhC=N), 164.3 (C=N). 195Pt NMR in CDCl3,  (ppm): .
Synthesis of the mononuclear mixed hexamethylenetetramine / oxadiazoline complexes.
Trans-PtCl2(PhCN)(oxadiazoline) 3a-3f (0.1 mmol) and hexamethylenetetramine 6 (0.15
mmol) were dissolved in chloroform (1 ml) and stirred at room temperature for 4 days. The
solvent was evaporated and the residual crude products were purified by chromatography on
silica using a CH2Cl2/diethylether gradient of 100:0 to 50:50 as eluent.
trans-Dichloro[2,3-dihydro-3-(2-methoxyphenyl)-2-methyl-5-phenyl-1,2,4-oxadiazole-
N4][hexamethylenetetramine-N1]platinum (7a). Yield 83 %. Elemental analysis
calculated for C22H28Cl2N6O2Pt: C 39.18; H 4.18; N 12.46; found: C 38.95; H 4.27; N 12.75.
1
IR (selected bands), cm : 3063, 2964 and 2888 (CH), 2840 (CH of OMe), 1629 m
(C=N), 1603 w (C=C), 1247, 1225, 1024, 998, 831, 774, 754, 688, 653. 1H NMR in CDCl3,
 (ppm): 3.05 (s, br., 3H, NMe), 3.94 (s, 3H, OMe), 4.45 (d, 12.6 Hz, 3 H) and 4.52 (d, 12.2
Hz, 3 H)(hmta Hb and Hb’), 5.02 (s, 6H, hmta Ha), 6.32 (s, br., 1H, N-CH-N), 6.97 (d, 8.5 Hz,
1H), 7.00 (t, 7.5 Hz, 1H), 7.39 (td, 7.8 Hz, 1.5 Hz, 1H) and 7.50 (d, br., 7.1 Hz, 1H)(aryl-H of
N-CH(Ar)-N), 7.60 (t, 7.6 Hz, 2H), 7.70 (t, 7.5 Hz, 1H) and 8.95 (d, 7.4 Hz, 2H)(aryl-H of
13
PhC=N). C NMR in CDCl3,  (ppm): 47.2 (NMe), 55.7 (OMe), 73.1 (hmta Cb), 79.5 (hmta
Ca), 89.9 (N-CH-N), 110.9, 120.3, 129.3 and 130.7 (CH of N-CH(Ar)-N), 124.3 and 157.5
(Cq of N-CH(Ar)-N), 128.4, 130.4 and 133.6 (CH of PhC=N), 123.0 (Cq of PhC=N), 164.9
(C=N). 195Pt NMR in CDCl3,  (ppm): 2208.
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trans-Dichloro[2,3-dihydro-3-(4-methoxyphenyl)-2-methyl-5-phenyl-1,2,4-oxadiazole-
N4][hexamethylenetetramine-N1]platinum (7b). Yield 78 %. Elemental analysis
calculated for C22H28Cl2N6O2Pt: C 39.18; H 4.18; N 12.46; found: C 39.35; H 4.11; N 12.17.
1
IR (selected bands), cm : 3060, 2963, 2931 and 2885 (CH), 2834 (CH of OMe), 1629 m
(C=N), 1610 w (C=C), 1253, 1228, 1022, 995, 830, 775, 757, 689, 655. 1H NMR in CDCl3,
 (ppm): 3.00 (s, br., 3H, NMe), 3.85 (s, 3H, OMe), 4.44 (d, 12.5 Hz, 3 H) and 4.50 (d, 12.3
Hz, 3 H)(hmta Hb and Hb’), 4.96 (s, 6H, hmta Ha), 5.82 (s, br., 1H, N-CH-N), 6.98 (d, 8.5 Hz,
2H) and 7.64 (d, 8.5 Hz, 2H)(aryl-H of N-CH(Ar)-N), 7.58 (t, 7.8 Hz, 2H), 7.68 (t, 7.3 Hz,
13
1H) and 8.95 (d, 7.9 Hz, 2H)(aryl-H of PhC=N). C NMR in CDCl3,  (ppm): 45.7 (NMe),
55.3 (OMe), 73.0 (hmta Cb), 79.4 (hmta Ca), 94.1 (N-CH-N), 113.7 and 130.4 (CH of N-
CH(Ar)-N), 130.42 and 160.6 (Cq of N-CH(Ar)-N), 128.3, 130.4 and 133.7 (CH of PhC=N),
122.8 (Cq of PhC=N), 163.5 (C=N). 195Pt NMR in CDCl3,  (ppm): 2213.
trans-Dichloro[2,3-dihydro-3-(2,6-dimethoxyphenyl)-2-methyl-5-phenyl-1,2,4-oxadia-
zole-N4][hexamethylenetetramine-N1]platinum (7c). Yield 70 %. Elemental analysis
calculated for C23H30Cl2N6O3Pt: C 39.21; H 4.29; N 11.93; found: C 38.91; H 4.33; N 11.71.
1
IR (selected bands), cm : 3003, 2970, 2931 and 2886 (CH), 2838 (CH of OMe), 1634 m
1
(C=N), 1596 w (C=C), 1253, 1226, 1108, 1022, 995, 830, 776, 761, 689, 656. H NMR in
CDCl3,  (ppm): 3.02 (s, 3H, NMe), 3.64 (s, 3H) and 4.05 (s, 3H)(2  OMe), 4.43 (d, 12.7 Hz,
3 H) and 4.49 (d, 12.7 Hz, 3 H)(hmta Hb and Hb’), 4.96 (s, 6H, hmta Ha), 6.63 (s, 1H, N-CH-
N), 6.55 (d, 8.3 Hz, 1H), 6.66 (d, 8.3 Hz, 1H) and 7.34 (t, 8.3 Hz, 1H)(aryl-H of N-CH(Ar)-
N), 7.58 (t, 7.5 Hz, 2H), 7.66 (m, 1H) and 8.87 (d, 7.7 Hz, 2H)(aryl-H of PhC=N). 13C NMR
in CDCl3,  (ppm): 48.3 (NMe), 55.9 and 56.7 (2  OMe), 73.0 (hmta Cb), 79.3 (hmta Ca),
86.7 (N-CH-N), 103.9, 105.1 and 131.0 (CH of N-CH(Ar)-N), 113.3, 158.5 and 160.6 (Cq of
N-CH(Ar)-N), 128.3, 130.1 and 133.0 (CH of PhC=N), 123.3 (Cq of PhC=N), 163.2 (C=N).
195Pt NMR in CDCl3,  (ppm): 2214.
trans-Dichloro[2,3-dihydro-3-(2,4,6-trimethoxyphenyl)-2-methyl-5-phenyl-1,2,4-oxadia-
zole-N4][hexamethylenetetramine-N1]platinum (7d). Yield 71 %. Elemental analysis
calculated for C24H32Cl2N6O4Pt: C 39.24; H 4.39; N 11.44; found: C 39.37; H 4.43; N 11.60.
1
IR (selected bands), cm : 3008, 2963, 2931 and 2881 (CH), 2836 (CH of OMe), 1606 m
22

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DOI: 10.1039/C7DT02406A
(C=N), 1593 w (C=C), 1452, 1256, 1227, 1121, 1022, 996, 828, 774, 758, 690, 653. H
NMR in CDCl3,  (ppm): 2.99 (s, br., 3H, NMe), 3.62 (s, 3H), 3.82 (s, 3H) and 4.01 (s, 3H)(3
 OMe), 4.43 (d, 12.7 Hz, 3 H) and 4.50 (d, 12.0 Hz, 3 H)(hmta Hb and Hb’), 4.97 (s, 6H,
hmta Ha), 6.51 (s, br., 1H, N-CH-N), 6.09 (d, 2.2 Hz, 1H) and 6.20 (d, 2.2 Hz, 1H)(aryl-H of
N-CH(Ar)-N), 7.56 (t, 7.8 Hz, 2H), 7.65 (t, 7.5 Hz, 1H) and 8.86 (d, 7.9 Hz, 2H)(aryl-H of
PhC=N). 13C NMR in CDCl3,  (ppm): 48.5 (NMe), 55.2, 55.9 and 56.6 (3  OMe), 73.0
(hmta Cb), 79.3 (hmta Ca), 86.9 (N-CH-N), 90.7 and 91.6 (CH of N-CH(Ar)-N), 106.3, 159.2,
161.5 and 162.2 (Cq of N-CH(Ar)-N), 128.3, 130.0 and 133.0 (CH of PhC=N), 123.5 (Cq of
PhC=N), 163.2 (C=N). 195Pt NMR in CDCl3,  (ppm): 2214.
trans-Dichloro[2,3-dihydro-3-(2,3,4-trimethoxyphenyl)-2-methyl-5-phenyl-1,2,4-
oxadiazole-N4][hexamethylenetetramine-N1]platinum (7e). Yield 81%. Elemental
analysis calculated for C24H32Cl2N6O4Pt: C 39.24; H 4.39; N 11.44; found: C 39.36; H 4.32;
1
N 11.74. IR (selected bands), cm : 3059, 2939 and 2879 (CH), 2836 (CH of OMe),
1630 m (C=N), 1600 w (C=C), 1495, 1256, 1227, 1097, 1023, 996, 829, 774, 758, 690,
652. 1H NMR in CDCl3,  (ppm): 3.02 (s, br., 3H, NMe), 3.88 (s, 3H), 3.91 (s, 3H) and 4.12
(s, 3H)(3  OMe), 4.45 (d, 12.8 Hz, 3 H) and 4.52 (d, 12.5 Hz, 3 H)(hmta Hb and Hb’), 5.01 (s,
6H, hmta Ha), 6.24 (s, br., 1H, N-CH-N), 6.70 (d, 8.6 Hz, 1H) and 7.20 (d, br., 8.0 Hz,
1H)(aryl-H of N-CH(Ar)-N), 7.56 (t, 7.8 Hz, 2H), 7.70 (t, 7.3 Hz, 1H) and 8.96 (d, 7.8 Hz,
13
2H)(aryl-H of PhC=N). C NMR in CDCl3,  (ppm): 46.8 (NMe), 55.9, 60.8 and 61.4 (3 
OMe), 73.0 (hmta Cb), 79.5 (hmta Ca), 89.9 (N-CH-N), 106.7 and 124.0 (CH of N-CH(Ar)-
N), 122.0, 141.8, 152.2 and 154.7 (Cq of N-CH(Ar)-N), 128.4, 130.4 and 133.7 (CH of
PhC=N), 123.0 (Cq of PhC=N), 164.4 (C=N). 195Pt NMR in CDCl3,  (ppm): 2217.
trans-Dichloro[2,3-dihydro-3-(3,4,5-trimethoxyphenyl)-2-methyl-5-phenyl-1,2,4-
oxadiazole-N4][hexamethylenetetramine-N1]platinum (7f). Yield 79%. Elemental
analysis calculated for C24H32Cl2N6O4Pt: C 39.24; H 4.39; N 11.44; found: C 38.99; H 4.23;
1
N 11.56. IR (selected bands), cm : 3009, 2995, 2943 and 2887 (CH), 2841 (CH of
OMe), 1643 m (C=N), 1596 w (C=C), 1451, 1331, 1259, 1238, 1226, 1025, 997, 826, 778,
1
690, 654. H NMR in CDCl3,  (ppm): 3.02 (s, br., 3H, NMe), 3.90 (s, 3H, 4-OMe), 3.94 (s,
6H, 3-OMe and 5-OMe), 4.44 (d, 12.9 Hz, 3 H) and 4.49 (d, 12.3 Hz, 3 H)(hmta Hb and Hb’),
23

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4.95 (s, 6H, hmta Ha), 5.78 (s, br., 1H, N-CH-N), 6.98 (s, 2H, aryl-H of N-CH(Ar)-N), 7.58 (t,
13
7.8 Hz, 2H), 7.69 (t, 7.5 Hz, 1H) and 8.96 (d, 7.8 Hz, 2H)(aryl-H of PhC=N). C NMR in
CDCl3,  (ppm): 45.6 (NMe), 56.4 (4-OMe), 60.8 (3-OMe and 5-OMe), 73.0 (hmta Cb), 79.4
(hmta Ca), 94.4 (N-CH-N), 106.5 (CH of N-CH(Ar)-N), 138.8 and 153.3 (Cq of N-CH(Ar)-N,
third Cq not detected), 128.3, 130.4 and 133.8 (CH of PhC=N), 122.7 (Cq of PhC=N), 164.4
(C=N). 195Pt NMR in CDCl3,  (ppm): 2202.
Synthesis of the dinuclear mixed hexamethylenetetramine / oxadiazoline complexes.
Trans-PtCl2(PhCN)(oxadiazoline) 3a-3f (0.22 mmol) and hexamethylenetetramine 6 (0.1
mmol) were dissolved in chloroform (1 ml) and stirred at room temperature for 2 weeks. The
solvent was evaporated and the residual crude products were purified by chromatography on
silica using a CH2Cl2/diethylether gradient of 100:0 to 90:10 as eluent.
[-(hexamethylenetetramine-N1:N3]tetrachlorobis[2,3-dihydro-3-(2-methoxyphenyl)-
2-methyl-5-phenyl-1,2,4-oxadiazole-N4]diplatinum (8a, isomeric mixture). Yield 61 %.
Elemental analysis calculated for C38H44Cl4N8O4Pt2: C 37.76; H 3.67; N 9.27; found: C
1
38.05; H 3.80; N 9.38. IR (selected bands), cm : 3058, 3008, 2968, 2938 and 2914 (CH),
2840 (CH of OMe), 1620 m (C=N), 1605 and 1589 s (C=C), 1247, 1062, 1030, 983,
1
792, 772, 748, 733, 686. H NMR in CDCl3,  (ppm): 3.05 (s, br., 6H, NMe), 3.93 and 3.94
(s, 3H each, OMe), 4.24 (s, 2H, hmta Hc), 4.75 (d, 12.8 Hz, 4H), 4.92 (d, 11.6 Hz, 2H) and
4.93 (d, 11.6 Hz, 2H)(hmta Hb and Hb’), 5.38 (s, 2H, hmta Ha), 6.30 (s, br., 2H, N-CH-N),
6.96 (m, 2H), 7.00 (m, 2H), 7.39 (tm, 7.8 Hz, 2H) and 7.49 (d, br., 6.7 Hz, 2H)(aryl-H of N-
13
CH(Ar)-N), 7.60 (m, 4H), 7.69 (m, 2H) and 8.94 (dm, 7.4 Hz, 4H)(aryl-H of PhC=N). C
NMR in CDCl3,  (ppm): 47.3 (NMe), 55.8 (OMe), 71.3 (hmta Cc), 77.72, 77.78, 77.83 and
77.87 (hmta Cb), 80.8 (hmta Ca), 92.7 (N-CH-N), 110.9, 120.4, 129.1 and 130.7 (CH of N-
CH(Ar)-N), 123.8 and 156.0 (Cq of N-CH(Ar)-N), 128.5, 130.4 and 133.8 (CH of PhC=N),
122.8 (Cq of PhC=N), 164.5 (C=N). 195Pt NMR in CDCl3,  (ppm): -2207.
[-(hexamethylenetetramine-N1:N3]tetrachlorobis[2,3-dihydro-3-(4-methoxyphenyl)-
2-methyl-5-phenyl-1,2,4-oxadiazole-N4]diplatinum (8b, isomeric mixture). Yield 68 %.
Elemental analysis calculated for C38H44Cl4N8O4Pt2: C 37.76; H 3.67; N 9.27; found: C
1
38.01; H 3.33; N 8.94. IR (selected bands), cm : 3063, 2963, 2934 and 2915 (CH), 2837
24

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DOI: 10.1039/C7DT02406A
(CH of OMe), 1631 m (C=N), 1612 s (C=C), 1514, 1348, 1305, 1251, 1176, 1032, 990,
1
909, 850, 774, 731, 689. H NMR in CDCl3,  (ppm): 3.02 (s, br., 6H, NMe), 3.84 (s, 6H,
OMe), 4.22 (s, 2H, hmta Hc), 4.69 (d, 12.7 Hz, 4H), 4.83 (m, 4H, hmta Hb and Hb’), 5.30
(“m”, 2H, hmta Ha), 5.81 (s, br., 2H, N-CH-N), 6.99 (d, 8.8 Hz, 2H), 7.00 (d, 8.8 Hz, 2H) and
7.66 (d, 8.2 Hz, 4H)(aryl-H of N-CH(Ar)-N), 7.59 (t, 7.9 Hz, 4H), 7.66 (t, 7.4 Hz, 2H) and
8.95 (d, 7.7 Hz, 4H)(aryl-H of PhC=N). 13C NMR in CDCl3,  (ppm): 45.7 (NMe), 55.3
(OMe), 71.3 (hmta Cc), 77.45, 77.55, 77.67 and 77.75 (hmta Cb), 80.8 (hmta Ca), 94.1 (N-CH-
N), 113.9 and 130.40 (CH of N-CH(Ar)-N), 130.44 and 160.6 (Cq of N-CH(Ar)-N), 128.5,
130.4 and 133.8 (CH of PhC=N), 122.6 (Cq of PhC=N), 163.5 (C=N). 195Pt NMR in CDCl3, 
(ppm): 2211.
[-(hexamethylenetetramine-N1:N3]tetrachlorobis[2,3-dihydro-3-(2,6-dimethoxy-
phenyl)-2-methyl-5-phenyl-1,2,4-oxadiazole-N4]diplatinum (8c, isomeric mixture).
Yield 60 %. Elemental analysis calculated for C40H48Cl4N8O6Pt2: C 37.86; H 3.81; N 8.83;
1
found: C 38.14; H 3.61; N 8.65. IR (selected bands), cm : 3002, 2966, 2937 and 2911
(CH), 2838 (CH of OMe), 1642 m (C=N), 1596 w (C=C), 1477, 1253, 1108, 1031,
990, 909, 793, 726, 689. 1H NMR in CDCl3,  (ppm): 3.03 (s, 6H, NMe), 3.654 (s, 3H), 3.658
(s, 3H), 4.00 (s, 3H) and 4.05 (s, 3H)(2  OMe), 4.21 (s, 2H, hmta Hc), 4.70 (m, 4H), 4.83 (m,
4H)(hmta Hb and Hb’), 5.27 (“m”, 2H, hmta Ha), 6.61 (s, 2H, N-CH-N), 6.55 (d, 8.4 Hz, 2H),
6.65 (d, 8.4 Hz, 2H) and 7.34 (t, 8.5 Hz, 2H)(aryl-H of N-CH(Ar)-N), 7.59 (t, 7.7 Hz, 4H),
7.67 (t, 7.4 Hz, 2H) and 8.86 (d, 7.5 Hz, 4H)(aryl-H of PhC=N). 13C NMR in CDCl3,  (ppm):
48.4 (NMe), 56.0 and 56.9 (2  OMe), 71.3 (hmta Cc), 77.57, 77.64, 77.67 and 77.68 (hmta
Cb), 80.5 and 80.6 (hmta Ca), 86.8 (N-CH-N), 104.1, 105.0 and 131.0 (CH of N-CH(Ar)-N),
113.2, 158.5 and 160.5 (Cq of N-CH(Ar)-N), 128.5, 130.1 and 133.2 (CH of PhC=N), 123.1
(Cq of PhC=N), 163.2 (C=N). 195Pt NMR in CDCl3,  (ppm): 2214.
[-(hexamethylenetetramine-N1:N3]tetrachlorobis[2,3-dihydro-3-(2,4,6-trimethoxy-
phenyl)-2-methyl-5-phenyl-1,2,4-oxadiazole-N4]diplatinum (8d, isomeric mixture).
Yield 70 %. Elemental analysis calculated for C42H52Cl4N8O8Pt2: C 37.96; H 3.94; N 8.43;
1
found: C 38.11; H 3.81; N 8.09. IR (selected bands), cm : 3001, 2965, 2940, 2915 and 2886
(CH), 2839 (CH of OMe), 1607 m (C=N), 1593 w (C=C), 1451, 1256, 1229, 1205,
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1155, 1121, 1059, 1032, 991, 909, 813, 774, 690. H NMR in CDCl3,  (ppm):D2O.I9: 190.1(0s3,9/bC7rD.,T02406A
6H, NMe), 3.63 (s, 6H), 3.79 (s, 6H), 3.98 (s, 3H) and 4.00 (s, 3H)(3  OMe), 4.23 (“m”, 2H,
hmta Hc), 4.72 (m, 4H), 4.85 (m, 4H)(hmta Hb and Hb’), 5.38 (“m”, 2H, hmta Ha), 6.50 (s,
br., 2H, N-CH-N), 6.09 (d, 2.1 Hz, 2H), 6.21 (d, 2.1 Hz, 1H) and 6.22 (d, 2.1 Hz, 1H)(aryl-H
of N-CH(Ar)-N), 7.58 (t, 7.8 Hz, 4H), 7.65 (t, 7.4 Hz, 2H) and 8.85 (d, 7.7 Hz, 4H)(aryl-H of
PhC=N). 13C NMR in CDCl3,  (ppm): 48.3 (NMe), 55.2, 55.9 and 56.8 (3  OMe), 71.3
(hmta Cc), 77.59, 77.66, 77.68 and 77.95 (hmta Cb), 80.5 and 80.6 (hmta Ca), 86.9 (N-CH-N),
90.74, 90.88, 91.57 and 91.72 (CH of N-CH(Ar)-N), 106.2, 159.3, 161.41, 161.43, 162.28
and 162.34 (Cq of N-CH(Ar)-N), 128.4, 130.0 and 133.1 (CH of PhC=N), 123.3 (Cq of
PhC=N), 163.0 (C=N). 195Pt NMR in CDCl3,  (ppm): 2214.
[-(hexamethylenetetramine-N1:N3]tetrachlorobis[2,3-dihydro-3-(2,3,4-trimethoxy-
phenyl)-2-methyl-5-phenyl-1,2,4-oxadiazole-N4]diplatinum (8e, isomeric mixture).
Yield 66 %. Elemental analysis calculated for C42H52Cl4N8O8Pt2: C 37.96; H 3.94; N 8.43;
1
found: C 38.22; H 4.08; N 8.03. IR (selected bands), cm : 3061, 2998, 2967 and 2943
(CH), 2836 (CH of OMe), 1633 m (C=N), 1601 s (C=C), 1495, 1466, 1384, 1290,
1
1097, 1033, 1012, 992, 909, 795, 730, 690. H NMR in CDCl3,  (ppm): 3.03 (s, br., 6H,
NMe), 3.87 (s, 6H), 3.90 (s, 6H) and 4.11 (s, 6H)(3  OMe), 4.25 (s, br., 2H, hmta Hc), 4.78
(m, 4H), 4.88 (m, 4H)(hmta Hb and Hb’), 5.38 (“m”, 2H, hmta Ha), 6.20 (s, br., 2H, N-CH-
N), 6.70 (d, 8.8 Hz, 2H) and 7.19 (d, br., 8.2 Hz, 2H)(aryl-H of N-CH(Ar)-N), 7.60 (t, 7.5 Hz,
13
4H), 7.68 (t, 7.3 Hz, 2H) and 8.95 (d, 7.5 Hz, 4H)(aryl-H of PhC=N). C NMR in CDCl3, 
(ppm): 46.9 (NMe), 55.9, 60.8 and 61.5 (3  OMe), 71.3 (hmta Cc), 77.56, 77.64, 77.83 and
77.87 (hmta Cb), 81.0 (hmta Ca), 89.9 (N-CH-N), 106.8 and 123.9 (CH of N-CH(Ar)-N),
121.9, 141.8, 152.2 and 154.8 (Cq of N-CH(Ar)-N), 128.5, 130.4 and 133.8 (CH of PhC=N),
122.7 (Cq of PhC=N), 164.5 (C=N). 195Pt NMR in CDCl3,  (ppm): .
[-(hexamethylenetetramine-N1:N3]tetrachlorobis[2,3-dihydro-3-(3,4,5-trimethoxy-
phenyl)-2-methyl-5-phenyl-1,2,4-oxadiazole-N4]diplatinum (8f, isomeric mixture). Yield
67 %. Elemental analysis calculated for C42H52Cl4N8O8Pt2: C 37.96; H 3.94; N 8.43; found: C
1
37.33; H 3.60; N 8.61. IR (selected bands), cm : 3063, 2999, 2965 and 2940 (CH), 2838
(CH of OMe), 1628 m (C=N), 1595 s (C=C), 1451, 1332, 1237, 1124, 1059, 1034, 990,
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DOI: 10.1039/C7DT02406A
730, 689. H NMR in CDCl3,  (ppm): 3.03 (s, br., 6H, NMe), 3.90 (s, 6H, 4-OMe), 3.93 (s,
12H, 2-OMe, 5-OMe), 4.22 (s, 2H, hmta Hc), 4.72 (m, 4H) and 4.80 (m, 4H)(hmta Hb and
Hb’), 5.34 (s, br., 2H, hmta Ha), 5.77 (s, br., 2H, N-CH-N), 6.96 (s, 4H, aryl-H of N-CH(Ar)-
13
N), 7.59 (t, 7.8 Hz, 4H), 7.68 (t, 7.3 Hz, 2H) and 8.96 (m, 4H)(aryl-H of PhC=N). C NMR
in CDCl3,  (ppm): 45.5 (NMe), 56.4 (4-OMe), 60.9 (3-OMe and 5-OMe), 71.2 (hmta Cc),
77.52, 77.57, 77.70 and 77.79 (hmta Cb), 81.0 (hmta Ca), 94.3 (N-CH-N), 106.3 (CH of N-
CH(Ar)-N), 138.9 and 153.2 (Cq of N-CH(Ar)-N, third Cq not detected), 128.5, 130.5 and
134.0 (CH of PhC=N), 122.5 (Cq of PhC=N), 164.3 (C=N). 195Pt NMR in CDCl3,  (ppm):
.
Computational Details
DFT calculations were carried out with the PC GAMESS/Firefly package,42 which is partially
based on the GAMESS(US) source code.43 Results were visualised with MacMOLPlt.44
Molecular geometries were fully optimised using the B3LYP hybrid functional,45 in its
implementation which is based on the VWN1 formula.46 The LANL08 core potential basis
set47 was used for Pt and Cl and the 6-31G* basis set48 for all other atoms. Relative energies
are zero point energy (ZPE) corrected and refer to the energy of the starting materials = 0
kcal/mol. The harmonic vibrational frequencies of all stationary points were computed in
order to characterise them as local minima or transition states. For all transition states, the
vibration associated with the imaginary frequency was examined for being consistent with the
product formation. Intrinsic reaction coordinates (IRC) were traced from the transition states
towards both reactant and product direction along the imaginary mode of vibration using the
algorithm developed by Gonzáles and Schlegel.49 Mayer bond orders50 were calculated as
implemented in PC GAMESS/Firefly. The topological analysis of the charge densities51 was
performed with the software package MORPHY.52
Cytotoxicity Studies
Preparation of the Pt(II) stock solutions. The platinum compounds were dissolved in
DMSO and immediately diluted into water to give solutions which are 100 μM in platinum
and contain 5 % (v/v) DMSO. Aliquots of these solutions used to achieve 10 μM or 2.5 μM
platinum doses in the cell suspension introduce 0.5 % (v/v) and 0.125 % (v/v) of DMSO,
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DOI: 10.1039/C7DT02406A
which should have little effect on the growth and survival of the cancer cells. Any effects on
the intracellular ATP levels in the presence of more than 0.1 % (v/v) DMSO36 are
compensated by solvent blank measurements.
Cell culture. Cell culture reagents were obtained from PAA Laboratories (Cölbe, Germany).
The HeLa and A549 cell lines were obtained from DSMZ (Deutsche Sammlung von
Mikroorganismen und Zellkulturen). Both cell lines were cultured as attached monolayers in
Dulbecco’s Modified Eagle’s Medium (DMEM, high glucose 4.5 g/L) with 10% fetal bovine
serum, 1% MEM non-essential amino acids and 1% Penicillin/Streptomycin supplements.
Cytotoxicity Testing. Cytotoxicity was determined by means of the luminescent cell viability
assay CellTiter-Glo®,53 obtained from Promega, which is based on the luciferase reaction and
measures the ATP content of metabolically active cells (as a measure for the number of living
cells). Cultured cell monolayers were converted into single cell suspension by treatment with
trypsin-EDTA solution, and then seeded into 96-well tissue culture plates at a density of 1 x
105 cells per 100 μl. Cells were allowed to settle under standard culture incubation conditions
for 24 h and then treated with freshly prepared solutions of the platinum compounds, at Pt
concentrations in the cell medium of 2.5 μM and 10 μM, respectively. After 24 h incubation
under standard culture conditions cells were lysed for 10 minutes with the CellTiter-Glo®
reagent solution and the luminescence signal was read using a multiwell plate luminometer.
The quantity of live cells was expressed relative to DMSO treated control cells (“solvent”).
Cell viability data given are mean values over three experiments.
Acknowledgement. The authors wish to thank Johnson Matthey Plc for a loan of
platinum compounds, the University of Chester and the University of Ulm, in particular Prof.
Mika Lindén (Inorganic Chemistry II) for general support of this work, and Prof. Tanja Weil
(Organic Chemistry III) for access to cell lab facilities. We also want to express our sincere
thanks to Dr. Klaus Eisele and Magdalene Zimmermann for their help with the cell viability
studies.
28

Page 29 of 32
Dalton Transactions
View Article Online
DOI: 10.1039/C7DT02406A
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