Cytotoxicity and ROS production of novel Pt(IV)oxaliplatin derivatives with indole propionic acid

Article abstract of DOI:10.1016/j.ica.2019.04.038

The coordination of biologically active moieties to the axial positions of Pt(IV)derivatives of Pt(II)anticancer drugs allows the co-delivery and simultaneous activation of two pro-drugs for combination therapy. Pt(IV)complexes with a redox modulator as an axial ligand can kill cancer cells by a mechanism combining DNA platination and generation of oxidative stress. In this study we evaluated the cytotoxicity of Pt(IV)complexes based on the oxaliplatin scaffold and the pro-oxidant indole-3-propionate in cisplatin-sensitive and cisplatin-resistant ovarian cancer cells. A series of five complexes was synthesized and characterized by ¹H and ¹⁹⁵Pt NMR spectroscopy, IR spectroscopy, mass spectrometry and elemental analysis; trans-[Pt(DACH)(ox)(IPA)(OH)](1), trans-[Pt(DACH)(ox)(IPA)₂](2), trans-[Pt(DACH)(ox)(IPA)(bz)](3), trans-[Pt(DACH)(ox)(IPA)(suc)](4), and trans-[Pt(DACH)(ox)(IPA)(ac)](5)(DACH = 1,2-diaminocyclohexane (1R,2R)-(?), ox = oxalate, IPA = indole-3-propionate, bz = benzoate, suc = succinate and ac = acetate). The complexes were shown to produce cellular reactive oxygen species (ROS)in a time-dependent manner. The most potent ROS producer, complex 1, also elicited the highest cytotoxicity. Complex 1 was shown to form the mono- and bis-adducts [Pt(DACH)(guanosine)(OH)]⁺ and [Pt(DACH)(guanosine)₂]²⁺ in the presence of ascorbic acid, suggesting that on activation the released oxaliplatin will interact with DNA.

Full text of DOI:10.1016/j.ica.2019.04.038

Accepted Manuscript
Cytotoxicity and ROS Production of Novel Pt(IV) Oxaliplatin Derivatives with
Indole Propionic Acid
Dina Tolan, Awatif Rashed Z. Almotairy, Orla Howe, Michael Devereux, Diego
Montagner, Andrea Erxleben
PII:
S0020-1693(19)30274-9
https://doi.org/10.1016/j.ica.2019.04.038
ICA 18883
DOI:
Reference:
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Inorganica Chimica Acta
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Revised Date:
Accepted Date:
27 February 2019
15 April 2019
18 April 2019
Please cite this article as: D. Tolan, A.R.Z. Almotairy, O. Howe, M. Devereux, D. Montagner, A. Erxleben,
Cytotoxicity and ROS Production of Novel Pt(IV) Oxaliplatin Derivatives with Indole Propionic Acid, Inorganica
Chimica Acta (2019), doi: https://doi.org/10.1016/j.ica.2019.04.038
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Cytotoxicity and ROS Production of Novel Pt(IV) Oxaliplatin Derivatives with
Indole Propionic Acid
Dina Tolan, Awatif Rashed Z. Almotairy,
†1
1,2
Orla Howe, Michael Devereux, Diego
2
2
Montagner,* Andrea Erxleben*1
3
1
2
School of Chemistry, National University of Ireland, Galway, Ireland
School of Biological & Health Sciences, Technological University Dublin, City Campus,
Dublin, Ireland
3
Department of Chemistry, Maynooth University, Maynooth, Ireland
These authors contributed equally to this manuscript
present address: Department of Chemistry, Faculty of Science, El-Menoufia University,

†
Shebin El-Kom, Egypt
E-mail: andrea.erxleben@nuigalway.ie; diego.montagner@nuigalway.ie
Abstract
The coordination of biologically active moieties to the axial positions of Pt(IV) derivatives of
Pt(II) anticancer drugs allows the co-delivery and simultaneous activation of two pro-drugs
for combination therapy. Pt(IV) complexes with a redox modulator as an axial ligand can kill
cancer cells by a mechanism combining DNA platination and generation of oxidative stress.
In this study we evaluated the cytotoxicity of Pt(IV) complexes based on the oxaliplatin
scaffold and the pro-oxidant indole-3-propionate in cisplatin-sensitive and cisplatin-resistant
1
ovarian cancer cells. A series of five complexes was synthesized and characterized by H and
195Pt NMR spectroscopy, IR spectroscopy, mass spectrometry and elemental analysis; trans-
[
Pt(DACH)(ox)(IPA)(OH)] (1), trans-[Pt(DACH)(ox)(IPA) ] (2), trans-[Pt(DACH)(ox)(IPA)(bz)] (3),
2
trans-[Pt(DACH)(ox)(IPA)(suc)] (4), and trans-[Pt(DACH)(ox)(IPA)(ac)] (5) (DACH = 1,2-
diaminocyclohexane (1R,2R)-(−), ox = oxalate, IPA = indole-3-propionate, bz = benzoate, suc
=
succinate and ac = acetate). The complexes were shown to produce cellular reactive
oxygen species (ROS) in a time-dependent manner. The most potent ROS producer, complex
1
, also elicited the highest cytotoxicity. Complex 1 was shown to form the mono- and bis-
+
]2+ in the presence of
adducts [Pt(DACH)(guanosine)(OH)] and [Pt(DACH)(guanosine)
2
ascorbic acid, suggesting that on activation the released oxaliplatin will interact with DNA.
Keywords: Oxaliplatin, Pt(IV) prodrugs, Redox Stress, Indole Propionic Acid, Cytotoxicity
1

Introduction
About 50 % of cancer patients who undergo chemotherapy receive one of the three
worldwide approved platinum(II) complexes, cisplatin, carboplatin and oxaliplatin, either
alone or in combination with other anticancer drugs [1]. Co-administration of two or more
chemotherapeutics with independent modes of action is widely used to reduce the risk of
resistance development and to enhance the anticancer efficacy through synergistic effects
[
2]. However, different selectivities and pharmacokinetics of the individual drugs can pose
significant challenges in the application of combination therapy regimens. Oxidation of Pt(II)
to Pt(IV) allows the covalent attachment of a second biologically active entity to the axial
positions of the cisplatin-, carboplatin- or oxaliplatin-scaffold [3-20]. In addition to the
simultaneous delivery of the Pt drug and the co-administered drug, Pt(IV) complexes have
the advantages of a higher stability, lower toxicity, reduced side effects and potential for
oral administration [3,4]. Dual-action Pt(IV) compounds are pro-drugs that require
activation by intracellular reducing agents to release the active Pt(II) species and the axial
ligands (Chart 1) [3,21].
We have recently reported that the coordination of indole carboxylic acids to the axial
positions of the Pt(IV) derivative of cisplatin leads to potent anticancer agents that
overcome cisplatin resistance [22]. Indole carboxylic acids are redox modulators that can act
as pro-oxidants [23-27]. Indole acetic acid, for example, is used in the oxidation therapy of
cancer [23]. We showed that the Pt(IV) indole-3-propionic acid complexes cis,cis,trans-
[
Pt(NH
3
)
2
Cl
2
(IPA)(OH)], cis,cis,trans-[Pt(NH
3
2
) Cl
2
(IPA)
2
], cis,cis,trans-[Pt(NH
3
)
2
2
Cl (IPA)(Bz)]
and cis,cis,trans-[Pt(NH
)
3 2
2
Cl (IPA)(Ac)] (IPA = indole-3-propionate, Bz = benzoate, Ac =
acetate) exert their cytotoxic activity by a dual-action mechanism combining DNA
platination and the generation of reactive oxygen species (ROS). cis,cis,trans-
[
Pt(NH
3
)
2
2
Cl (IPA)(OH)] was found to be up to four times more effective than cisplatin [22].
Oxaliplatin (Chart 1) is a third-generation Pt(II) drug that is approved for the treatment of
metastatic colorectal cancer and is also active against lung and ovarian cancer cell lines
[
28,29]. It contains a more stable leaving group compared to cisplatin and the R,R-
diaminocyclohexane (DACH) chelating ligand [30]. The latter leads to a spectrum of activity
that is different from that of cisplatin and carboplatin. Oxaliplatin also shows a different
resistance profile and is usually better tolerated than cisplatin [31,32]. In continuation of our
previous work we therefore synthesized the oxaliplatin analogues of the cis,cis,trans-
[
Pt(NH
3
)
2
2
Cl (IPA)Y] complexes and investigated their cytotoxicity and ROS generating
capability in cisplatin-sensitive and cisplatin-resistant ovarian cancer cells.
2

Chart 1. The three FDA-approved Pt(II) anticancer drugs, general structure of Pt(IV) pro-
drugs for cisplatin and pro-drug activation by reduction.
Results and Discussion
Chart 2. Structures of complexes 1 - 5.
The five complexes based on oxaliplatin (Chart 2) were synthesised as shown in Scheme 1
and in the Experimental Section in the Supporting Information. Briefly, oxaliplatin was
oxidised to the corresponding di-hydroxido Pt(IV) complex (A) by H
2
O
2
in aqueous solution
(Scheme 1). A was reacted with the activated N-hydroxysuccinimide (NHS) ester of indole-3-
propionic acid (B) in DMSO solution to obtain complex 1. This complex was the precursor to
obtain complexes 3 - 5 that could be isolated by precipitation with diethyl ether from a DMF
solution containing 1 and the corresponding anhydride; benzoic (3), succinic (4) and acetic
(5) anhydride, respectively. 2 was obtained by reaction of A with a large excess of B in
DMSO.
3

The complexes were characterized by 1H and 195Pt-NMR spectroscopy, infra-red
spectroscopy, elemental analysis and mass spectrometry (Supporting Information, Figures
1
S –12S). The purity and the stability of the complexes for 36 h have been evaluated in
HEPES buffer solution by HPLC (Figure 13S). Elemental analyses are in good agreement with
-
-
the expected values for the complexes. In the ESI-MS spectra the [M - H] or [M + Cl] parent
ions of the platinum(IV) complexes were readily observed with the typical isotope pattern of
-1
Pt. The IR spectra displayed characteristic C=O stretching vibrations around 1710 cm and
-
1
1
650 cm for all the complexes while the symmetric and antisymmetric N-H stretching
-1
bands of the amine ligands were found between 3325 and 3400 cm and between 3150 and
-
1
1
3
190 cm . The proton resonances of the coordinated amine ligands in the H NMR spectrum
of 1 appear as multiplet at ca. 7.08 - 8.52-ppm. This multiplet is broadened and downfield
shifted to ca. 8.12 - 8.55 ppm in complexes 2 - 5. The chemical shift values of the amine
protons are well in line with those of previously reported Pt(IV) complexes based on
oxaliplatin [33,34]. The 195Pt NMR spectrum of 1 in DMF (insert D
O) shows a single
2
resonance at 1389 ppm that is shifted downfield to ca. 1590 ppm in the di-carboxylato
complexes 2 - 5. The presence of an additional electron withdrawing carboxylate ligand in 2
-
5 causes a de-shielding effect of about 200 ppm on the platinum signal with respect to the
mono-carboxylated complex 1.
Scheme 1. Syntheses of complexes 1 – 5.
4

The anticancer activities of the complexes were evaluated in two human ovarian carcinoma
cells lines, cisplatin-sensitive A2780 and cisplatin-resistant A2780cis. The cytotoxicity was
determined after incubation for 24 and 48 h using the colorimetric MTT assay and IC50
values are reported in Table 1. For comparison purposes, the cytotoxicities of cisplatin,
oxaliplatin and of the free IPA ligand were assessed under the same experimental
conditions. The complexes are less active than cisplatin, in particular in the sensitive A2780
cell line, whereas they show higher activity than the reference drug oxaliplatin. The IC50
values of 1 - 5 in the A2780 cells are about 3 to 6 times lower at the 48 h timepoint than at
the 24 h timepoint. The decrease in the IC50 value with prolonged exposure time is less
pronounced for cisplatin. That is, the oxaliplatin derivatives take longer to exert their
cytotoxic effects. After 48 h, the activity of 1 is comparable to that of cisplatin (IC50(48 h) =
1
2.6 (1) vs. 10.5 (cisplatin)). The complexes show cross-resistance with cisplatin.
Table 1: IC50 values of the Pt(IV) complexes 1 - 5, cisplatin, oxaliplatin and the free ligand IPA
in A2780 and A2780cisR cells determined by the MTT test.
Complex
IC50 (µM) ± S.D.
A2780
A2780cis
2
4 h
48 h
24 h
66.91 ± 1.8
70.48 ± 1.83
55.26 ± 2.30
48 h
1
2
3
74.40 ± 8.1
53.32 ± 1.8
54.52 ± 1.7
12.62 ± 0.02
15.47 ± 1.72
16.10 ± 1.8
55.68 ± 1.7
46.17 ± 1.61
54.59 ± 2.5
4
5
58.39 ± 8.7
70.10 ± 4.65
16.55 ± 8.5
21.45 ±2.7
132.52 ± 2.7
103.01 ± 6.1
63.69 ± 7.4
47.56 ± 0.68
Cisplatin
20.10 ± 4.65
10.46 ± 2.1
19.80 ± 1.10
> 200
60.80 ± 8.2
110.68 ± 16.8
> 200
29.04 ± 6.7
86.37 ± 2.9
> 200
Oxaliplatin 83.25 ± 5.7
IPA > 200
In the A2780 cell line the most active complex at the 48 h timepoint is the mono-
carboxylated complex 1 with one axial OH group. The bis-substituted complex 2 with two
indole acids in both the axial positions is slightly more active in the resistant cells (48 h
timepoint). The finding that the mono- and the bis-PBA complex exert the strongest
cytotoxic activity is in line with the cytotoxicity data observed for the analogous cisplatin-
based complexes [22].
The generally not exceptional activity of these complexes may be ascribed to the reduction
rate of these complexes under physiological conditions. As mentioned in the introduction,
Pt(IV) complexes are pro-drugs and must be reduced to active Pt(II) agents. The reduction of
5

1
was studied via HPLC in the presence of an excess of ascorbic acid in HEPES buffer at pH 7
and 37 C. As shown in Figure 1, the complex releases the axial IPA ligand and is reduced to
the active oxaliplatin(II) species. However, the reduction kinetics are much slower compared
to the analogous Pt(IV) compound based on cisplatin [22]. After 72 h there is still 19 % of the
starting Pt(IV) complex 1. Under the same conditions, cis,cis,trans-[Pt(NH
3
)
2
2
Cl (IPA)(OH)]
was completely reduced within 24 h [22]. A lower reduction rate corresponds to a lower
cytotoxicity because the active oxaliplatin is not completely released. In general, a low
cytotoxic activity can also be due to a low lipophilicity and poor cellular uptake. However,
we did not further investigate this possibility, as for the analogous cisplatin derivatives no
correlation between lipophilicity, cellular uptake and IC50 value was observed. The slow pro-
drug activation is consistent with the delayed cytotoxicity of 1 whose IC50 value becomes
comparable to that of cisplatin at the 48 h timepoint.
Figure 1. HPLC analysis of the reaction of 1 with ascorbic acid at 37 C and pH 7 at different
times. = ascorbic acid.
*
To further confirm the activation mechanism, 1 was incubated with guanosine (G) in the
presence of sodium ascorbate to simulate the interaction of the platinum drug with the
nucleobases of DNA. The mass spectrum of the reaction mixture (Figure 2) clearly shows the
2
+
+
presence of two peaks, corresponding to the species [Pt(DACH)(G)
2
] (m/z 875.25; {M-H} )
+
and [Pt(DACH)(OH)(G)] (m/z 609.16) which suggests that 1 can bind to the guanosine
residues of DNA upon reduction. (Chart S1). The peak observed at m/z = 742.22 can be
+
assigned to [Pt(DACH)(guanosine)(guanine)-H] resulting from the loss of the sugar entity of
one guanosine ligand. It is worthy of note that the incubation of 1 with guanosine without
the reducing agent ascorbic acid does not give any Pt-guanosine adducts.
6

Figure 2. ESI-MS spectrum of the reaction between 1 and guanosine in presence of sodium
ascorbate.
Indole carboxylic acids are agents that can alter and modify the cellular redox state and the
cisplatin analogues of 1 - 5 induced a significant increase in ROS production. We speculated
that the oxaliplatin-based Pt(IV) complexes with axial indole propionate ligands could also
generate cellular oxidative stress. The generation of ROS in A2780 and A2780cis cells after
incubation with 1 – 5, cisplatin, oxaliplatin and IPA was measured. ROS levels were
monitored over a 2 h period using 2ʹ,7ʹ-dichlorofluorescein diacetate (H DCFDA) as a
2
peroxide-sensitive fluorescent dye and the results are reported in Figure 3 and Figure 14S in
the Supporting Information. All the complexes were able to stimulate the production of ROS
in a time-dependent manner and the increased oxidative stress follows the order 1  4  3

2  5. Complex 1 is the most efficient ROS generator and is significantly more potent than
the free IPA ligand and the reference drugs cisplatin and oxaliplatin. The higher cytotoxic
activity of complex 1 with respect to the other complexes is probably due to a synergistic
effect of the two active species released upon intracellular reduction, oxaliplatin and IPA. As
known, while oxaliplatin will interact with DNA as its cellular target, IPA will increase the
ROS production with these two mechanisms operating simultaneously.
7

Figure 3. Fluorescent detection of ROS using DCFDA in A2780 and A2780cis cells after
exposure to cisplatin (top) and 1 (bottom) over a period of 2 h. H
control. ● 10 min; ● 15 min; ● 30 min; ● 1 h; ● 2 h.
2
2
O was used as a positive
Conclusions
The most active complex of the newly synthesized Pt(IV) derivatives of oxaliplatin,
cis,cis,trans-[Pt(DACH)(ox)(IPA)(OH)] is more potent than oxaliplatin in A2780 ovarian cancer
cells. Overall, compared to their cisplatin-based counterparts reported earlier by us [22], 1 –
5
are only moderately cytotoxic. This is in line with reports on other oxaliplatin/cisplatin pro-
drugs [17] and can be attributed to the relatively slow reduction kinetics of Pt(IV) derivatives
of oxaliplatin. 1 – 5 are more efficient ROS producers in A2780 and A2780cis cells than the
free indole carboxylic acid.
Acknowledgement
A.A. acknowledges a PhD scholarship from Taibah University. D.T. acknowledges the
Egyptian Ministry of Higher Education (MoHE) for funding her research stay at the National
University of Ireland Galway.
8

Appendix A. Supplementary data
Supplementary data to this article can be found online at ...
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1
0

Graphical Abstract
1
1

Highlights



Pt(IV) derivatives of oxaliplatin with a redox modulator in axial position were synthesised.
The complexes produce cellular reactive oxygen species (ROS).
The most potent ROS producer elicits the highest cytotoxicity.
1
2