Synthesis, characterization, and in vitro cytotoxicity of a Kiteplatin-Ibuprofen Pt(IV) prodrug

Article abstract of DOI:10.1016/j.ica.2017.07.019

The Pt(IV) prodrug of kiteplatin, cis-trans-cis-[PtCl₂(RS-Ibuprofen-H)₂(cis-1,4-DACH)], having in the axial positions two molecules of Ibuprofen, has been synthesised, characterized and tested in vitro. The aim was to potentiate the

Full text of DOI:10.1016/j.ica.2017.07.019

Accepted Manuscript
Synthesis, characterization, and in vitro cytotoxicity of a Kiteplatin-Ibuprofen
Pt(IV) prodrug
Alessandra Curci, Nunzio Denora, Rosa Maria Iacobazzi, Nicoletta Ditaranto,
James D. Hoeschele, Nicola Margiotta, Giovanni Natile
PII:
S0020-1693(17)30801-0
http://dx.doi.org/10.1016/j.ica.2017.07.019
ICA 17742
DOI:
Reference:
To appear in:
Inorganica Chimica Acta
Received Date:
Revised Date:
Accepted Date:
23 May 2017
3 July 2017
5 July 2017
Please cite this article as: A. Curci, N. Denora, R.M. Iacobazzi, N. Ditaranto, J.D. Hoeschele, N. Margiotta, G.
Natile, Synthesis, characterization, and in vitro cytotoxicity of a Kiteplatin-Ibuprofen Pt(IV) prodrug, Inorganica
Chimica Acta (2017), doi: http://dx.doi.org/10.1016/j.ica.2017.07.019
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Synthesis, characterization, and in vitro cytotoxicity of a Kiteplatin-Ibuprofen
Pt(IV) prodrug.
1
2
3
1
Alessandra Curci, Nunzio Denora, Rosa Maria Iacobazzi, Nicoletta Ditaranto, James D.
4
1,*
1,*
Hoeschele, Nicola Margiotta, Giovanni Natile.
1
Dipartimento di Chimica, Università degli Studi di Bari Aldo Moro, via E. Orabona 4, 70125 Bari
Italy);
(
2
Dipartimento di Farmacia-Scienze del Farmaco, Università degli Studi di Bari Aldo Moro, via E.
Orabona 4, 70125 Bari (Italy);
3
Istituto Tumori IRCCS Giovanni Paolo II, viale O. Flacco 65, 70124 Bari (Italy).
4
Department of Chemistry, Eastern Michigan University, Ypsilanti, MI, USA 48197.
*
Corresponding Authors
Phone: +39 080 5442759 (N.M.); +39 080 5442774 (G.N.). E-mail: nicola.margiotta@uniba.it
N.M.); giovanni.natile@uniba.it (G.N.).
(
Keywords: cisplatin, Kiteplatin, cis-1,4-DACH, Ibuprofen, Pt(IV) drugs, NSAID, colon cancer.
Abstract
The Pt(IV) prodrug of kiteplatin, cis-trans-cis-[PtCl
2
(RS-Ibuprofen-H) (cis-1,4-DACH)], having in
2
the axial positions two molecules of Ibuprofen, has been synthesised, characterized and tested in
vitro. The aim was to potentiate the cytotoxic effect of kiteplatin with the anti-inflammatory activity
of Ibuprofen. The reduction potential of the conjugate resulted comparable to those of other
reported Pt(IV) carboxylate complexes, ensuring in vivo stability in blood during transport and
intracellular reduction with release of the active species. The cytotoxic activity of the complex
resulted remarkably potentiated reaching nanomolar activity. It is possible that the coordinated
1

Ibuprofen molecules promote the transport and the accumulation of the complex in tumour cells by
increasing its lipophilicity. In addition to the increased uptake, Ibuprofen could also exert an anti-
inflammatory action mediated by inhibition of the COX enzymes which are overexpressed in
tumors. The use of a conjugate with Pt-bound Ibuprofen can ensure similar cellular uptake and
biodistribution for both the anti-inflammatory and the cytotoxic drugs in the exact ratio of 2:1.
Introduction
Cisplatin (Chart 1) has become one of the most widely used chemotherapeutic agents and is mainly
used for the treatment of testicular, ovarian, bladder, and lung cancers, melanoma, lymphomas, and
myelomas.[1],[2] The clinical efficacy demonstrated by the three platinum-based drugs currently
approved by the FDA (cisplatin, carboplatin, and oxaliplatin; Chart 1) has prompted an intense
research aiming to discover new metal-based anticancer drugs with enhanced efficacy, lower
toxicity, and a broader spectrum of activity.[3]
Chart 1. Platinum drugs currently approved by FDA.
Several advantages can be obtained by the use of targeted and/or controlled drug delivery systems
that can improve dramatically the degree and duration of the clinical outcome.[4],[5] Moreover, the
synthesis of prodrugs has been exploited to lessen the limitations of conventional platinum(II)
anticancer drugs.[4] A recent strategy in the development of platinum-based chemotherapeutic
drugs is the assembly in a single complex of two different pharmacologically active
2

agents.[6],[7],[8],[9] This is possible especially in Pt(IV) prodrugs that are known to be rapidly
reduced intracellularly by biological reducing agents, such as glutathione and ascorbic acid, to
release the active ligands and the cytotoxic Pt(II) species.[10],[11]
NSAIDs (Non-Steroidal Anti-Inflammatory Drugs) have been recently used as axial ligands in
Pt(IV) complexes as a consequence of the generally accepted correlation between cancer and
inflammation processes.[12],[13],[14] Indeed, recent work has demonstrated that the inflammation
processes correlated to cancer are responsible for the evolution of the tumor mass with consequent
formation and spreading of metastases. For instance, the occurrence of prostate, colon, and
pancreatic cancer is highly frequent in patients experiencing the inflammation processes prostatitis,
ulcerative colitis, and pancreatitis, respectively. NSAIDs have as major targets the cyclooxygenases
(
COX) enzymes, which are involved in the biosynthesis of prostaglandins and are expressed in two
isoforms, COX-1 and COX-2.[15] COX is also implicated in cisplatin resistance with COX-2 being
overexpressed in many tumors and involved in tumor initiation and progression. For this reason
COX inhibitors and, in particular, COX-2 selective inhibitors are used as cancer-preventive and as
adjuvant agents in chemotherapeutic regimens.[16] As evidenced in clinical and tissue culture
investigations, a problem associated with the combination therapy of antitumor drugs and COX
inhibitors, is the different pharmacokinetic profile and biodistribution of the two drugs administered
separately. To obtain concerted transport into the tumor cells, different groups have pursued the
conjugation of the antiproliferative agent with the anti-inflammatory drug and have prepared
platinum(II) [17] and platinum(IV) [16],[18],[19],[20] prodrugs containing NSAIDs such as aspirin,
diclofenac, indomethacin, and Ibuprofen.
We are currently investigating the cisplatin fourth generation derivative [PtCl
2
(cis-1,4-DACH)]
(
DACH = diaminocyclohexane), also dubbed Kiteplatin (Chart 2), a new candidate platinum
anticancer drug that contains an isomeric form of the diamine ligand present in oxaliplatin (1R,2R-
DACH).[21],[22],[23],[24],[25],[26],[27],[28],[29] Our interest in this compound stems from its
3

activity against cisplatin (2008/C13* ovarian cancer cells) and oxaliplatin (LoVo/LoVo-OXP colon
cancer cells) sensitive/resistant cell lines. The cross-resistance profiles indicate that kiteplatin is as
good as oxaliplatin and significantly better than cisplatin (particularly in the case of colon cancer
cells) toward the sensitive sublines. Furthermore, despite sharing similar lipophilicity with
oxaliplatin and similar propensity for passive diffusion, the accumulation of kiteplatin is not
reduced in oxaliplatin-resistant cells lines. Similarly to cisplatin and oxaliplatin, also Pt(IV)
derivatives of kiteplatin have been prepared (particularly with benzoate or long-chain carboxylates
in axial positions) with the aim of improving the drug uptake and, hence, the pharmacological
activity in cancer cells (Chart 2).[30],[31]
NH2
Cl
Pt
NH2
Cl
[
PtCl (cis-1,4-DACH)]
2
(
kiteplatin)
O
O
O
O
Pt
O
n
n
Cl
Cl
NH2
NH2
Cl
Cl
Pt
O
NH2
NH2
O
O
cis,trans,cis[PtCl (OOC(CH )nCH ) (cis-1,4-DACH)]
2
2
3 2
(n = 0, 2, 4, 8)
cis,trans,cis[PtCl (benzoate) (cis-1,4-DACH)]
2
2
Chart 2. Structures of [PtCl (cis-1,4-DACH)] (kiteplatin) and its Pt(IV) lipophilic derivatives.
2
Noteworthy, the cytotoxic activity of the Pt(IV) kiteplatin-benzoate derivative resulted to be
remarkably higher (ca. 45 times) than that of kiteplatin, reaching nanomolar concentrations against
4

a wide panel of human cancer cells, including cisplatin- and oxaliplatin-resistant colon-cancer
cells.[31]
S-Ibuprofen belongs to the 2-arylpropionic acid group of NSAIDs and is a potent COX-1 and
COX-2 inhibitor while the R isomer is ca. 2-3 orders of magnitude less potent.[32] Although the
mechanism of action is not fully understood,[33] we were attracted by the ability of Ibuprofen to
inhibit tumor initiation and proliferation in prostate and breast cancer and, in particular, in colon
cancer cell lines such as HCA-7, HCT-116, and HCT-15, this latter expressing only COX-1 and
producing nearly no prostaglandin E .
2
With the aim of potentiating the activity of kiteplatin against colon cancer cells, not only by
enhancing the lipophilicity of the platinum compound but also by exerting an additional anti-
inflammatory effect on the tumor cells, in this investigation we have prepared a Pt(IV) prodrug of
kiteplatin having Ibuprofen in the axial positions (Scheme 1). We also hoped that the combination
of an anti-inflammatory and a chemotherapeutic drug would be more effective if the two drugs are
delivered in the form of a single prodrug. Racemic Ibuprofen was extracted by a commercial anti-
inflammatory drug and its acyl chloride was reacted with the dihydroxido Pt(IV)-derivative of
kiteplatin. The Pt(IV) conjugate was fully characterized and its cathodic potential was determined
by cyclic voltammetry. Finally, the new Pt(IV) derivative of kiteplatin was tested in vitro against
HCT-15 and HCT-116 human colon-cancer cell lines.
Experimental
Materials and methods
Commercial reagent grade chemicals and solvents were used as received without further
purification.
1
1
13
H-NMR and [ H- C] HSQC spectra were recorded on a Bruker Avance III 700 MHz instrument.
5

1
13
H and C chemical shifts were referenced using the internal residual peak of the solvent (Acetone-
1
13
195
d
6
: 2.05 ppm for H and 29.92 ppm for C). Pt-NMR spectra were recorded on Bruker Avance
1
95
DPX 300 MHz instrument. Pt NMR spectra were referenced to K
2
PtCl (external standard placed
4
at -1620 ppm with respect to Na [PtCl ]).[34]
2
6
Electrospray ionisation mass spectrometry (ESI-MS) was performed with an electrospray interface
and an ion trap mass spectrometer (1100 Series LC/MSD Trap system Agilent, Palo Alto, CA).
Elemental analyses were carried out with an Eurovector EA 3000 CHN instrument.
Kiteplatin [21] and cis,trans,cis-[PtCl
2
(OH) (cis-1,4-DACH)],[27] were prepared according to
2
already reported procedures and all analytical data were in good agreement with the given
formulation (data not shown).
Extraction of Ibuprofen from BRUFEN® 600mg expired tablets
Ibuprofen ((RS)-2-[4-(2-methylpropyl) phenyl] propanoic acid) was isolated from two commercial
Brufen® 600 mg expired tablets. The tablets were finely ground and suspended in 40 mL of 1M
NaOH. The mixture was stirred at room temperature for few minutes until a white and foamy
suspension was formed. The suspension was extracted with 40 mL of ethyl ether and the aqueous
phase was collected and treated again with 40 mL of Et O for a second extraction. The aqueous
2
phase was separated and filtered through a plug of Celite® to remove the undissolved solids.
Then, the colorless filtrate was acidified with 5M HCl until pH ~1-2 leading to the formation of a
white dispersed solid. The acidic suspension was extracted using 40 mL of ethyl acetate (1:1, v/v
with the aqueous phase) for three times. The organic phase was separated from the aqueous phase,
treated with anhydrous sodium sulfate and then filtered. The filtrate was evaporated to dryness
using a rotary evaporator until the formation of a white solid which was subsequently dried under
1
vacuum. Obtained: 1.11 g (92% yield). H-NMR (acetone-d
6
): 10.66 (s broad, 1H, C(1)OOH ), 7.25
(
d, JH-H = 7.87 Hz, 2H, C(4/5)H), 7.13 (d, JH-H = 7.87 Hz, 2H, C(6/7)H), 3.71 (m, JH-H = 7.10 Hz,
1
H, C(2)H), 2.45 (d, JH-H = 7.16 Hz, 2H, C(8)H
2
), 1.85 (m, JH-H = 6.02 Hz, 1H, C(9)H), 1.42 (d, JH-H
6

1
3
=
7.18 Hz, 3H, C(3)H
29.7 (C(4/5)), 128.0 (C(6/7)), 45.40 (C(8)), 45.18 (C(2)), 30.9 (C(9)), 22.4 (C(10/11)) and 18.9
C(3)) ppm. (See Figure 1 for numbering of atoms)
3
) and 0.88 (d, JH-H = 6.82 Hz, 6H, C(10/11)H
3
) ppm. C NMR (acetone-d ):
6
1
(
Synthesis of cis-trans-cis-[PtCl
2
(RS-Ibuprofen-H)
2
(cis-1,4-DACH)] (RS-Ibuprofen-H = 2-[4-(2-

metylpropyl)phenyl]propanoate; OOC-CH(CH
3
)-C -CH -CH(CH ).
6
H
4
2
3 2
)
The Pt(IV) complex was prepared according to already reported procedures with slight
modifications.[16],[31] Ibuprofen (0.500 g, 2.41 mmol) was solubilized in 2 mL of SOCl and the
2
solution was stirred at room temperature for 18 hours. Then the solution was concentrated by rotary
evaporation until formation of a yellow-brown oil which was diluted with 1.5 mL of acetone. The
acetone solution of Ibuprofen acyl chloride was added to a suspension containing cis,trans,cis-
[
(
PtCl
2
(OH)
2
(cis-1,4-DACH)] (0.050 g, 0.120 mmol) and pyridine (390 μL; 4.828 mmol) in acetone
1.5 mL). The reaction mixture was stirred at 75 °C for 48 h in the dark in a capped round-bottom
pressure flask. After cooling the obtained suspension to room temperature, the white precipitate was
eliminated by filtration. An excess of n-pentane was added to the filtrate to yield a precipitate that
was isolated by filtration of the mother liquor and washed with water and diethyl ether. The
obtained solid was dissolved in acetone and treated with water which caused the precipitation of a
white solid which was separated and dried under vacuum. Obtained 0.032 g (32% yield).
Anal. Calculated for C32
H
48Cl
2
N
2
4
O Pt: C, 48.61; H, 6.12; N, 3.54 %. Found: C, 48.28; H, 6.12; N,
+
m
1
3
.45 %. ESI-MS (MeOH): calc. for C32
H
48Cl
2
N
2
O
4
PtNa, [M+Na] : 813.70. Found: /z 813.26. H-
NMR (acetone-d
6
): 8.41, 8.04, 7.62 and 7.22 (4H, broad, NH ), 7.28 (d, JH-H = 7.47 Hz, 4H,
2
C(4/5)H)
,
7.06 (d, JH-H = 7.47 Hz, 4H, C(6/7)H), 3.68 (m, JH-H = 6.84 Hz, 2H, C(2)H), 3.26 (s, JH-Pt
0.02 Hz, 2H, C(a)H), 2.43 (d, JH-H = 7.18 Hz, 4H, C(8)H ), 1.82 (m, JH-H = 6.54 Hz, 2H, C(9)H),
.56 (br, 4H, C(b/c)H ), 1.47 (br, 4H, C(b/c)H ), 1.37-1.35 (m, 6H, C(3)H ), 0.87 (d, JH-H = 6.65 Hz,
2H, C(10/11)H ) ppm. C NMR (acetone-d
=
8
1
1
2
2
2
3
13
3
6
): 129.4 (C(4/5)), 128.1 (C(6/7)), 50.9 (C(a)), 48.1
7

(
C(2)), 45.5 (C(8)), 31.0 (C(9)), 22.4 (C(10/11)), 20.09 (C(b/c)), 20.09 (C(b/c)), 19.4 (C(3)) ppm.
1
95
Pt NMR (acetone-d ): 1099 ppm. (See Scheme 1 for numbering of atoms).
6
Electrochemical measurements
A CHI 1140B electrochemical analyzer (CH Instruments, Inc.) was used for the cyclic voltammetry
(
CV) measurements performed with a standard three-electrode cell. The working electrode was a
glassy carbon electrode (GC), the reference electrode was KCl-saturated Ag/AgCl, and the auxiliary
electrode was a platinum lamina. The GC working electrode was polished with alumina and then
rinsed with distilled water and dried, to obtain a reproducible surface for all the experiments. The
platinum complex was dissolved in DMSO containing 0.1M [NBu
4
][PF ] as supporting electrolyte,
6
to a final concentration of 1.0 mM. Nitrogen was bubbled through the solution to remove oxygen.
−1
Peak potentials were measured at different scan rates (0.10, 0.15, 0.20, and 0.25 V s ) and reported
vs KCl-saturated Ag/AgCl. The stability of the reference electrode in DMSO was verified
frequently by measuring the potential vs SCE.
Cell viability assay
The human colorectal carcinoma cell lines (no COX-2 expression) HCT-116 and HCT-15 were
cultured in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS),
1
00 U/mL penicillin, 100 μg/mL streptomycin and 2 mM L-glutamine in a 5% CO
atmosphere at 37 °C. All materials for cell culturing were purchased from EuroClone, Italy. The
antitumor activity of the Pt(IV)–Ibuprofen complex, cis-trans-cis-[PtCl (RS-Ibuprofen-H) (cis-1,4-
2
humidified
2
2
DACH)] and of kiteplatin, Ibuprofen, and cisplatin, used as reference compounds, was assessed by
the 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) test performed as
described elsewhere with some modifications.[35] Cells were seeded in 96-well plates with a
density of 5000 cells/well and 24 h post seeding, the medium was removed and replaced with drug-
8

containing medium. In particular, the Pt(IV) conjugate, kiteplatin, and cisplatin were tested in the
concentration range 0.01-100 µM, while Ibuprofen was tested in the range 0.01-800 µM. After 72 h
of incubation, 10 µL of MTT were added to each well and the cells were incubated at 37 °C for
further 2 h. Absorbance at 570 nm was measured using a Perkin Elmer VictorX3 microplate reader
and Prism software (ver. 6d; 2013, GraphPad, USA) was used to calculate the half maximal
inhibitory (IC50) values for each cell line.
Results and Discussion
Extraction of Ibuprofen from BRUFEN® 600mg expired tablets and its characterization
Two Brufen® 600 mg expired tablets were used to extract the sodium salt of Ibuprofen by treatment
with NaOH after removing the lipophilic excipients with an organic phase. The aqueous phase was
acidified to protonate Ibuprofen, which was subsequently extracted twice with ethyl acetate.
1
Ibuprofen was characterized by NMR spectroscopy. The H-NMR spectrum of Ibuprofen in
acetone-d
6
(Figure 1) shows a broad peak falling at 10.66 ppm assigned to the carboxylic proton
(
not shown). The doublets falling at 7.25 and 7.13 ppm, were assigned to the aromatic protons. This
doublet of doublets is typical of a phenyl system disubstituted in para positions (H(4/5) and H(6/7)
in Figure 1). The quartet integrating for one proton at 3.71 ppm and the doublet at 1.42 ppm were
assigned, respectively, to the proton and to the methyl group bound to the chiral carbon (H(2) and
H(3) in Figure 1). The doublet integrating for two protons at 2.45 ppm was assigned to the
methylene protons (H(8) in Figure 1). The multiplet at 1.85 ppm integrating for one proton was
assigned to H(9) while at 0.88 ppm resonates a doublet assigned to the isopropylic methyls
(
H(10/11) in Figure 3).
9

1
Figure 1. H-NMR (700 MHz) of Ibuprofen in acetone-d
6
. The asterisks indicate the residual
solvent peaks.
1
13
The NMR characterization of the carbon atoms was obtained by a [ H- C]-HSQC 2D NMR
experiment also performed in acetone-d (Figure 2). The cross peaks falling at 7.25/128.0 and
.13/129.7 ppm ( H/ C) were attributed to the aromatic C(4/5)H and C(6/7)H. The cross peaks at
6
1
13
7
2
1
13
.45/45.40, 1.85/30.9 and 0.88/22.4 ppm ( H/ C) were assigned to C(8), C(9) and C(10/11),
respectively (see Figure 1 for numbering of atoms). The chiral carbon C(2) resonates at 45.18 ppm
while the methyl carbon C(3) falls at 18.9 ppm (Figure 2).
10

1
13
13
Figure 2. [ H- C] HSQC 2D (176.05 MHz, C) spectrum of Ibuprofen in acetone-d .
6
Synthesis and characterization of cis-trans-cis-[PtCl
2
(RS-Ibuprofen-H) (cis-1,4-DACH)]
2
The Pt(IV) conjugate cis-trans-cis-[PtCl
esterification of the dihydroxido platinum(IV) precursor cis,trans,cis-[PtCl
with the acyl chloride of Ibuprofen, prepared starting from Ibuprofen and SOCl
2
(RS-Ibuprofen-H) (cis-1,4-DACH)] was synthesized by
2
2
(OH)
2
(cis-1,4-DACH)]
2
(Scheme 1). The
reaction was carried out in the presence of pyridine to neutralize the released HCl and in the dark to
avoid the possible spontaneous solvent-assisted reduction of cis,trans,cis-[PtCl
2
(OH) (cis-1,4-
2
DACH)] to kiteplatin.[27,36–38]
11

Scheme 1. Synthesis of the conjugate Pt(IV) complex cis-trans-cis-[PtCl
,4-DACH)].
2
(RS-Ibuprofen-H) (cis-
2
1
12

The ESI-MS spectrum of cis-trans-cis-[PtCl
2
(RS-Ibuprofen-H) (cis-1,4-DACH)] showed the
2
+
presence of a peak at m/z = 813.26 corresponding to [M+Na] as also confirmed by the isotopic
pattern (data not shown).
1
The H NMR spectrum of cis-trans-cis-[PtCl
2
(RS-Ibuprofen-H)
2
(cis-1,4-DACH)] in acetone-d is
6
reported in Figure 3. The doublets falling at 7.28 and 7.06 ppm were assigned to the aromatic
protons of Ibuprofen H(4/5) and H(6/7), respectively. The multiplet at 3.68 ppm was attributed to
the chiral -CH of the coordinated Ibuprofen. This signal appears as a superimposition of two or
more quartets, probably due to the presence of different diasteroisomers in solution (we indeed used
a racemic Ibuprofen). The singlet at 3.68 ppm shows a cross-peak with the signals in the region
1
.35-1.37 ppm in the COSY-spectrum (cross-peak a in Figure 4). These latter signals at 1.35-1.37
ppm confirm the presence of more diasteroisomers in solution and were attributed to the protons of
methyl groups bound to the chiral carbons. The singlet with Pt satellites at 3.26 ppm, assigned to
the methynic protons of coordinated DACH, has a cross-peak with the broad signals at 1.56-1.47
ppm in the 2D COSY spectrum (cross-peak b in Figure 4). The latter two broad signals were
assigned to the methylene protons of cis-1,4-DACH. The multiplet falling at 1.82 ppm was assigned
to the H(9) protons (see Scheme 1 for the numbering of protons). Indeed, the cross-peak d in the 2D
COSY spectrum (Figure 4) correlates the signal of C(9)H with that of the methyl groups
C(10/11)H
3
. The doublet at 2.43 ppm, integrating for two protons, was attributed to C(8)H of
2
Ibuprofen and it was correlated to C(9)H through a cross-peak (cross-peak c in Figure 4). The
doublet integrating for twelve protons at 0.87 ppm was assigned to the isopropyl methyl groups of
1
Ibuprofen. Finally, the H-NMR spectrum shows four very low broad signals at 8.41, 8.04, 7.62 and
7
7
.22 ppm attributed to the aminic protons of coordinated cis-1,4-DACH. In particular, the signal at
1
.22 ppm not visible in the H-NMR spectrum was assigned with the help of the 2D-COSY
experiment (data not shown).
13

1
Figure 3. H NMR (700 MHz) spectrum of cis-trans-cis-[PtCl
in acetone-d . (* indicate the residual solvent peaks, # indicate impurities of the solvent).
2
(RS-Ibuprofen-H) (cis-1,4-DACH)]
2
6
14

Figure 4. Selected region of the 2D COSY (700 MHz) spectrum obtained for cis-trans-cis-
PtCl (RS-Ibuprofen-H) (cis-1,4-DACH)] in acetone-d
[
2
2
6
.
1
13
The NMR characterization of the carbon atoms of the complex was obtained by a [ H- C]-HSQC
2
1
D NMR experiment also performed in acetone-d
6
(Figure 5). The cross peaks falling at 3.26/50.9,
1
13
.56/20.09 and 1.47/20.09 ppm ( H/ C), were attributed to the methynic and methylenic groups of
1
3
DACH, respectively. The aromatic carbons of Ibuprofen resonate at 128.1 and 129.4 ppm. The C
signals at 48.1 and 19.4 ppm were assigned to the chiral carbons C(2) and to the methyl carbons
C(3), respectively.
1
13
13
Figure 5. [ H- C] HSQC 2D (176.05 MHz, C) spectrum of cis-trans-cis-[PtCl
H) (cis-1,4-DACH)] in acetone-d
2
(RS-Ibuprofen-
2
6
.
15

1
95
The Pt-NMR of the Pt(IV) compound is reported in Figure 6. It shows a single peak at 1099 ppm
which is in the range typical for a Pt(IV) atom in a Cl coordination environment.[39,40]
2
O
2
N
2
1
95
Figure 6. Pt-NMR (64.52 MHz) spectrum of cis-trans-cis-[PtCl
DACH)] in acetone-d
2
(RS-Ibuprofen-H) (cis-1,4-
2
6
.
1
13
195
The H, C and Pt chemical shifts are in good agreement with those already reported for the
analogous Pt(IV)-Ibuprofen derivative cis,trans,cis-[PtCl
2
(Ibuprofen)
2
(NH ) ].[19]
3
2
Electrochemical measurements
The reduction potential of Pt(IV) complexes plays a key role in their activity. Therefore, CV
electrochemical measurements of blank and Pt(IV) complex solutions were recorded on a glassy
carbon electrode in DMSO solvent. Contrary to the expectations, the CV plot of the electrolyte
c
solution showed the electro activity of a couple peak with Ep = -0.83 V (vs SCE). Experiments
were then carried out to exclude solvent and electrolyte impurities, along with water traces due to
the well-known hygroscopic nature of the alkyl ammonium salt. Moreover, the scan rate was varied
-
1
c
from 0.10 to 0.25 Vs and it was determined that the ip was directly proportional to the square root
of the scan rate (Figure S1 in Supporting Information), meaning that the electro-activity was due to
a dissolved interfering compound rather than to an adsorbate. It was not possible to fully elucidate
the nature of the interfering compound, most likely it derives from partial degradation of the
ammonium salt to the amine, but its peak was taken into proper account in the analysis of the Pt(IV)
complex voltammogram.
16

Figure 7. Cyclic voltammogram recorded at a glassy carbon electrode on a DMSO solution
containing 0.1M [NBu ][PF ] as supporting electrolyte and 1.0 mM of Pt(IV) complex. Scan rate
4
6
−1
0
.20 V s .
The CV plot (Figure 7) clearly shows that the cathodic peak area is larger than the anodic one, this
can be a consequence of the activity of the analyte being superimposed to that of the electrolyte. By
c
subtracting the contribute of the latter, the Ep of the platinum complex was unequivocally
measured and found equal to -0.93 V (corrected vs SCE) (see also Figure S2 in Supporting
Information). This value is compatible with the reduction potential of similar Pt(IV) complexes [41]
−
and with a chemically irreversible 2e reduction due to the detachment of the two axial ligands upon
reduction of the octahedral Pt(IV) complex to the square-planar Pt(II) species.[27] Moreover, it was
observed that the kinetics of the process is highly affected by the steric hindrance of the axial
ligands. The electrochemical behaviour at low scan rates indicated a slow transfer process to the
electrode causing hysteresis phenomena. For this reason, the experimental scan rate was set
unusually high.
17

c
In conclusion, the CV investigation shows that the Ep measured for the Pt(IV) conjugate with
Ibuprofen is suitable for the intracellular reduction necessary to activate this kind of prodrugs into
the pharmacologically active species.
Cytotoxicity assay
In order to evaluate the antitumor activity of the Pt(IV)–Ibuprofen prodrug, in vitro cytotoxic assays
were performed on two colorectal carcinoma cell lines both characterized by no expression of
COX-2 protein [19,32,42]: HCT-116 and HCT-15. Data from MTT assay after 72 h of incubation
were compared with those obtained for Ibuprofen, kiteplatin, and cisplatin used as reference
compounds. The IC50 values calculated from the dose-response curves reported in Figure 8 are
shown in Table 1. According to already reported data,[32] Ibuprofen is not cytotoxic in the range of
concentrations analyzed, with IC50 values >800 and >708 μM for HCT-15 and HCT-116 cell lines,
respectively. Interestingly, the Pt(IV) conjugate cis-trans-cis-[PtCl
2
(RS-Ibuprofen-H) (cis-1,4-
2
DACH)] is more active than the precursor kiteplatin and the reference compound cisplatin, with
IC50 values of 0.45 and 0.26 µM for HCT-15 and HCT-116, respectively. Specifically, the cis-trans-
cis-[PtCl
2
(RS-Ibuprofen-H) (cis-1,4-DACH)] conjugate is ca. 25 times more effective than its Pt(II)
2
precursor kiteplatin (IC50 of 11 and 7 μM for HCT-115 and HCT-116, respectively) and ca. 40
times more active than cisplatin (IC50 of 11 and 17 μM for HCT-116 and HCT-15 cell lines,
respectively). In a previous work,[31] the Pt(IV) derivative of kiteplatin with the biologically
inactive hydroxido ligands, cis-trans-cis-[PtCl
IC50 value of 10.32 M (MTT test; 72 h) against HCT-15 tumor cell line. This finding indicates that
the higher cytotoxicity found in the present investigation for cis-trans-cis-[PtCl (RS-Ibuprofen-
H) (cis-1,4-DACH)] is likely due to the presence of ibuprofen ligands in the axial positions.
Although the cis-trans-cis-[PtCl (RS-Ibuprofen-H) (cis-1,4-DACH)] complex had very marked
activity, the mechanism responsible for the greater activity of the conjugate with respect to the
2
(OH) (cis-1,4-DACH)], was found to possess an
2
2
2
2
2
18

precursor complex is difficult to identify. It cannot be related to an additional inhibition of COX-2
by Ibuprofen, since both cell lines do not express this enzyme. A possible explanation could be a
better cellular uptake of the conjugate Pt(IV) complex with respect to both kiteplatin and cisplatin.
In fact, the two Ibuprofen axial ligands would confer a greater lipophilicity to the Pt(IV) complex
with consequent facilitate transport through the cell membrane. The axial ibuprofen ligands are
benzoate-like ligands for which in a previous paper was demonstrated that the loss of the axial
ligands is slow (t1/2 of ca. 26 h).[31] Hence, we are quite confident that also the hydrolysis of
ibuprofen is slow. Moreover, also the reduction potential measured by cyclic voltammetry are
compatible with an intracellular reduction of the Pt(IV) complex by biological reductants. However,
we cannot exclude the intervention of some other type of synergistic effect between kiteplatin and
Ibuprofen within the tumor cells and, to clarify this point, other experiments such as cell uptake and
cytotoxicity tests with combinations of kiteplatin and Ibuprofen (administered separately and at a
dose of 1:2) are planned. The resulted combination index will clarify whether there is a synergistic
action between the two compounds.
Table 1. IC50 values [µM] determined in cell viability assays against human colorectal cancer cell
lines HCT-15 and HCT-116 (incubation times of 72 h). Data are mean ± SD (n=3).
Compounds
IC50 (µM)
HCT15
0.45 ± 0.04
11±1
HCT116
0.26 ± 0.03
7 ±1
cis-trans-cis-[PtCl
2
(RS-Ibuprofen-H) (cis-1,4-DACH)]
2
Kiteplatin
Cisplatin
17±2
11±1
Ibuprofen
>800
708 ± 8
19

Figure 8. Cell viability profiles of HCT-15 and HCT-116 cells determined after 72 h of incubation
with cis-trans-cis-[PtCl (RS-Ibuprofen-H) (cis-1,4-DACH)], Kiteplatin, Cisplatin and Ibuprofen.
2
2
Data are mean ± SD (n=3).
Conclusions
In this work we have synthesised, characterized and tested in vitro a Pt(IV) prodrug of kiteplatin,
cis-trans-cis-[PtCl (RS-Ibuprofen-H) (cis-1,4-DACH)], having in the axial positions two molecules
2
2
of Ibuprofen. The complex was designed with the aim of potentiating the cytotoxic effect of
kiteplatin by exploiting the increased intracellular uptake of the compound (due to the high
lipophilicity of the axial ligands) and the potential dual cytotoxic and anti-inflammatory activity.
In order to evaluate the propensity of the Pt(IV) prodrug to undergo intracellular activation to the
corresponding Pt(II) active species by cytoplasmic reducing agents (such as glutathione and
ascorbic acid), the reduction potential of the conjugate was measured electrochemically. The
reduction potential of the conjugate resulted comparable to those of other reported Pt(IV)
carboxylate complexes, ensuring in vivo stability in blood during transport but enabling its
intracellular reduction and consequent activation.
The cytotoxic activity of the complex resulted remarkably potentiated by the presence of the
lipophilic axial ligands reaching nanomolar activity. Thus, the Pt(IV) conjugate showed a markedly
increased cytotoxicity compared to both cisplatin and the Pt(II) precursor kiteplatin. It is possible
20

that the coordinated Ibuprofen molecules promote the transport and the accumulation of the
complex in tumour cells. In addition to the increased uptake, Ibuprofen could also exert an anti-
inflammatory action mediated by inhibition of the enzymes COX-1 and COX-2, which are
overexpressed in tumors. Moreover, the use of a conjugate with Pt-bound Ibuprofen could also
ensure the same cellular uptake and biodistribution for both the anti-inflammatory and the cytotoxic
drugs in the exact ratio of 2:1. Experiments with tumor cells overexpressing COX-1 and/or COX-2
enzymes will tell if an anti-inflammatory action of Ibuprofen can further increase the antitumor
efficacy.
In conclusion, the nanomolar in vitro cytotoxicity of this new Pt(IV)-Ibuprofen prodrug of kiteplatin
makes this compound clinically attractive as antitumor agent for its potential capability to overcome
the side effects of the conventional Pt-based drugs and to act as a dual acting drug. Extension to
other tumor cells and investigation of drug uptake by cells and anti-inflammatory properties are
under way.
Acknowledgments
We acknowledge the University of Bari (Italy), the Italian Ministero dell’Università e della Ricerca,
the Inter-University Consortium for Research on the Chemistry of Metal Ions in Biological Systems
(
C.I.R.C.M.S.B.), and the European Union (COST CM1105: Functional metal complexes that bind
to biomolecules) for support. We are grateful to Prof. Giovanni Lentini (Dipartimento di Farmacia-
Scienze del Farmaco, Università di Bari, Italy) for his precious assistance in the extraction of
Ibuprofen from expired commercial tablets and to Ms. Ilaria Orlando for the preparation and
analyses of the Pt compounds.
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26

Table of Contents (TOC)
27

Highlights
A Pt(IV) prodrug of kiteplatin having in the axial positions two molecules of Ibuprofen has been
synthesised.
The reduction potential of the conjugate ensures in vivo stability in blood during transport and
intracellular reduction with release of the active species.
The cytotoxic activity of the complex results remarkably potentiated reaching nanomolar activity.
28