Platinum(IV) cisplatin derivative trans, cis, cis-bis(heptanoato)amine(cyclohexylamine)dichloridoplatinum(IV) has an enhanced therapeutic index compared to cisplatin for the treatment of non-small cell lung cancer

Article abstract of DOI:10.1016/j.ica.2014.07.043

Lung cancer is the leading cause of cancer-related death in the United States, and non-small cell lung cancer (NSCLC) the most common type. Platinum (Pt) anticancer agents, such as cisplatin, remain a mainstay in the clinic; however, these agents are not tumor-specific and, thus, the patient experiences negative side-effects. We here prepare trans, cis, cis-bis(heptanoato)amine(cyclohexylamine)dichloridoplatinum(IV) and demonstrate that it is greater than 50-fold more toxic toward NSCLC cells than is cisplatin. Furthermore, it has a much improved therapeutic index. This Pt(IV) complex binds to DNA in a manner similar to that of cisplatin, and can be incorporated into mesoporous silica nanoparticles for fine-controlled release and the targeting of tumors.

Full text of DOI:10.1016/j.ica.2014.07.043

Inorganica Chimica Acta xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Inorganica Chimica Acta
journal homepage: www.elsevier.com/locate/ica
Platinum(IV) cisplatin derivative trans, cis,
cis-bis(heptanoato)amine(cyclohexylamine)dichloridoplatinum(IV) has
an enhanced therapeutic index compared to cisplatin for the treatment
of non-small cell lung cancer
Yi Shi a, Bhuvaneswari Koneru a, Warren Redfearn a, Michael L. Miller b, Lara Myers a,
Anthony J. Di Pasqua a,
⇑
a Department of Pharmaceutical Sciences, University of North Texas System College of Pharmacy, University of North Texas Health Science Center, Fort Worth, TX 76107, United States
b Department of Chemistry, Creighton University, Omaha, NE 68178, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Lung cancer is the leading cause of cancer-related death in the United States, and non-small cell lung can-
cer (NSCLC) the most common type. Platinum (Pt) anticancer agents, such as cisplatin, remain a mainstay
in the clinic; however, these agents are not tumor-specific and, thus, the patient experiences negative
side-effects. We here prepare trans, cis, cis-bis(heptanoato)amine(cyclohexylamine)dichloridoplati-
num(IV) and demonstrate that it is greater than 50-fold more toxic toward NSCLC cells than is cisplatin.
Furthermore, it has a much improved therapeutic index. This Pt(IV) complex binds to DNA in a manner
similar to that of cisplatin, and can be incorporated into mesoporous silica nanoparticles for fine-con-
trolled release and the targeting of tumors.
Received 16 July 2014
Accepted 20 July 2014
Available online xxxx
Keywords:
Chemotherapy
Platinum anticancer agent
Non-small cell lung cancer
Therapeutic index
Ó 2014 Published by Elsevier B.V.
DNA binding
Mesoporous silica nanoparticle
1. Introduction
containing sulfur functional groups and mitochondria [13,14].
Pt(IV) complexes have lower ligand exchange rates than Pt(II) com-
Cis-diamminedichloroplatinum(II), cisplatin, shown in Fig. 1,
was the first platinum (Pt)-based drug used as an anti-proliferative
agent and is renowned for its high level and broad spectrum of
anticancer activity [1]. Since Rosenberg discovered the anticancer
activity of cisplatin in the 1960s, a large number of anticancer Pt(II)
drugs have been found [2], but only a few of them are used in clin-
ical therapy [3]. However, side-effects of cisplatin and related Pt(II)
complexes limit their clinical usefulness. Cisplatin along with other
Pt(II) complexes can easily bind with thiol groups on proteins in
plasma, leading to deactivation of cisplatin and severe negative
side effects [2,4]. Furthermore, as cisplatin is highly toxic toward
rapidly dividing healthy cells, severe dose-limiting side-effects
and acquired or intrinsic resistance to cisplatin are frequently
observed; thus, its application is limited [5,6].
plexes and must be ‘‘activated’’ via bio-reduction to their Pt(II)
counterparts before interacting with DNA and other biological tar-
gets; thus, they are considered to be promising prodrugs of biolog-
ically active Pt(II) complexes. The biological reducing agent
ascorbic acid is frequently cited in connection with the reduction
of Pt(IV) complexes to Pt(II) [15–18]. Ascorbic acid is a two electron
reducing agent present in blood plasma with a concentration of
50–150 lM and the cytosol at approximately 1 mM [19,20].
Previously, it was shown that cis, trans, cis-[PtCl2(OH)2(NH3)2]
(oxoplatin) and its carboxylate-modified analog, cis, trans, cis-
[PtCl2(OH)(O2CCH2CH2CO2H)(NH3)2], both Pt(IV) complexes, can
be reduced by ascorbic acid and bind to plasmid pBR322 DNA
[15]. Blatter and co-workers revealed that ascorbic acid-reduced
species of iproplatin and oxoplatin, both Pt(IV) complexes, can
bind to and unwind closed circular PM2 DNA [18]. Furthermore,
the octahedrally coordinated Pt(IV) complexes allow for more flex-
ibility in structure modifications than Pt(II) complexes, including
the potential for axial ligand modification to enhance their ability
to enter tumor cells.
To overcome these drawbacks, a new generation of platinum
agents, Pt(IV) octahedral compounds, have been developed [7–
12]. The binding of cisplatin to nuclear DNA elicits its biologic
effect, although it also interacts with RNA, proteins and enzymes
In this work, the cytotoxicity of a lipophilic Pt(IV) complex,
trans, cis, cis-bis(heptanoato)amine(cyclohexylamine)dichlorido-
platinum(IV), referred to in text as 5 and shown in Fig. 1, with a
⇑
Corresponding author. Tel.: +1 817 735 2144.
E-mail address: Anthony.DiPasqua@unthsc.edu (A.J. Di Pasqua).
http://dx.doi.org/10.1016/j.ica.2014.07.043
0020-1693/Ó 2014 Published by Elsevier B.V.
Please cite this article in press as: Y. Shi et al., Inorg. Chim. Acta (2014), http://dx.doi.org/10.1016/j.ica.2014.07.043

2
Y. Shi et al. / Inorganica Chimica Acta xxx (2014) xxx–xxx
Fig. 1. Percent cell survival versus platinum (Pt) concentration in normal human lung fibroblast cell line, WI-38, and non-small cell lung cancer (NSCLC) cell lines, H596 and
A549. The cells were exposed to cisplatin or complex 5 for 24 h before measurement.
partition coefficient (logPo/w) of 4.10 [21], was synthesized and
tested against WI-38 normal human lung fibroblast cells, and
H596 and A549 human non-small cell lung cancer (NSCLC) cells.
Lung cancer is the leading cause of cancer-related death in the
United States, and non-small cell lung cancer (NSCLC) the most
common type. Pt anticancer agents, such as cisplatin, remain a
mainstay in the clinic for the treatment of this disease [22]. It
was previously shown that 5 is antiproliferative toward A2780
ovarian and HCT116 colon carcinoma cell lines with half maximal
in mesoporous silica MCM-41 nanoparticles [23,24], to better tar-
get tumors. We previously demonstrated that mesoporous silica
MCM-41 nanoparticles accumulate in ovarian and NSCLC tumors
after intraperitoneal and intravenous (i.v.) injection, respectively
[25,26]. Thus, we tested the loading and release of complex 5 from
this type of nanoparticle.
2. Materials and methods
inhibitory concentration (IC50) values of 0.39 0.01 nM and
1.65 0.25 nM, respectively [21]. To further investigate the mech-
anism of action of this Pt(IV) complex, we used pBR322 DNA as a
capture agent for the reduction product of 5 by ascorbic acid,
and investigated the mobility of the platinated DNA forms in an
agarose gel. The lipophilic nature of 5 allows for its encapsulation
2.1. Materials
Cisplatin, tetraethylammonium chloride (Et4NCl), dimethyl-
acetamide (DMA), Dowex 50-W-X8H+ cation exchange resin and
cyclohexylamine were from Sigma–Aldrich (St. Louis, MO);
Please cite this article in press as: Y. Shi et al., Inorg. Chim. Acta (2014), http://dx.doi.org/10.1016/j.ica.2014.07.043

Y. Shi et al. / Inorganica Chimica Acta xxx (2014) xxx–xxx
3
heptanoic anhydride was from Acros Organics (Morris Plains, NJ);
A549, H596 and WI-38 cells were from American Type Culture Col-
lection (Manassas, VA); CellTiter 96Ò AQueous One Solution Cell
Proliferation Assay (MTS) solution was from Promega (Madison,
WI); and pBR322 DNA was from New England Biolabs (Ipswich,
MA).
precipitate was observed, which confirmed a negative test. After
the negative test was obtained, 82 mg of decolorizing carbon was
added to the reaction mixture and stirred for 0.5 h at r.t. and then
centrifuged. The clear supernatant was obtained and 1.92 mL of
HCl (37%) was added. The mixture was left to stand overnight at
r.t. and the pale yellow precipitate was collected via vacuum filtra-
tion and washed with 5 mL of H2O, ethanol and ether, and then
dried in vacuo. Pale yellow product collected: 156.1 mg (63.3%
yield). MS (acetonitrile with NaI) calculated molecular mass of C6-
H16Cl2N2Pt: 382.19 g/mol, observed average molecular mass:
382.25 g/mol. 1H NMR (300 MHz, DMF-d7), d (ppm) = 1.10 (1H,
m), 1.27 (4H, m), 1.58 (1H, m), 1.73 (2H, m), 2.42 (2H, m), 2.93
(1H, m), 4.21 (3H, b), 4.89 (2H, b).
2.2. Synthesis of [PtCl3NH3]ꢀ (1)
All four Pt intermediates were prepared using literature meth-
ods [27], with some modifications. To cisplatin (0.468 g,
1.56 mmol) and tetraethylammonium chloride (Et4NCl, 0.332 g,
1.88 mmol) was added 40 mL of fresh reagent grade DMA. The
solution was stirred while heating at 100 2 °C for 6 h in a
round-bottom flask, while a slow stream of N2 gas was introduced
to the solution by a gas dispersion tube for purging. Fresh DMA was
added to maintain the volume and temperature control, but the
volume of the reaction mixture was allowed to be reduced to
approximately 10 mL by the end of the reaction. After the orange
solution obtained was cooled to room temperature (r.t.), it was
poured into 90 mL of 1/1 v/v hexane/ethyl acetate and was stored
at ꢀ15 °C overnight. The clear solution was decanted and discarded
and an orange oil was obtained when it was warmed to r.t. The
orange oil was dissolved with 8 mL of water and the mixture was
allowed to stand for 30 min at r.t. to allow complete precipitation
of a small amount of unreacted cisplatin. The mixture was then fil-
tered via vacuum filtration; the filtrate contained Et4N[PtCl3NH3].
The filtrate solution was stirred with 2.0 g of rinsed Dowex 50-
W-X8H+ cation exchange resin for 1 h, the resin was filtered via
vacuum filtration, and the volume of the filtrate reduced to
approximately 5 mL at r.t. To the yellow/orange filtrate, 0.6 mL of
saturated KCl solution was added and the mixture stored and left
to evaporate in the refrigerator (ꢁ7 °C) to slowly obtain orange
crystals. Orange crystals collected: 162.9 mg (29.2% yield). Mass
spectrometry (MS) (1:9 H2O:MeOH) calculated molecular mass of
H3Cl3KNPt: 357.57 g/mol, observed average molecular mass:
357.51 g/mol.
2.5. Synthesis of cis, trans, cis-[PtCl2(OH)2achNH3] (4) and then cis,
trans, cis-[PtCl2(OC(O)(CH2)5CH3)2achNH3] (5)
A suspension of complex 3 (0.141 g, 0.37 mmol) in 1.40 mL of
H2O was stirred at r.t. for 3 h. The suspension was heated to
70 °C before 365 lL of H2O2 (30%) was added and stirred for 2 h.
The reaction mixture was then cooled at r.t. and let stand overnight
before it was cooled in an ice bath for 0.5 h. The precipitate was
collected via vacuum filtration. The product was washed with
1.5 mL of H2O, ethanol and then diethyl ether. A very pale yel-
low/off-white powder obtained: 85.7 mg (55.8% yield). Complex
5 was then prepared in a similar manner as [Pt(cis-1,4-dach)-
trans-(acetate)2Cl2] [28,29]. To
a suspension of 4 (75 mg,
0.18 mmol) in 15 mL acetonitrile was added heptanoic anhydride
(0.714 mL, 15-fold). The reaction mixture was refluxed for 15 h
and the clear yellow solution obtained was evaporated to dryness
under reduced pressure. The resulting yellow residue was redis-
solved in approximately 3 mL of acetone and centrifuged. The
supernatant was saved and evaporated to a minimum volume
and kept in the refrigerator (ꢁ7 °C). The pale yellow precipitate
obtained was isolated via centrifugation, resuspended using 1 mL
of diethyl ether, collected via vacuum filtration and washed with
approximately 5 mL of diethyl ether. The product was dried in
vacuo. Off-white powder obtained: 63.1 mg (53.8% yield). MS (ace-
tonitrile with NaI) calculated molecular mass of C20H42N2O4Cl2Pt:
640.54 g/mol, observed average molecular mass: 640.46 g/mol.
1H NMR (300 MHz, DMF-d7), d (ppm) = 0.85 (6H, m), 1.22 (21H,
m), 1.60 (1H, m), 1.72 (2H, m), 2.17 (2H, m), 2.25 (4H, m), 3.00
(1H, m), 7.04 (3H, b), 7.60 (2H, b).
2.3. Synthesis of cis-[PtClIachNH3] (ach is cyclohexylamine) (2)
A solution of NaI (0.324 g, 2.16 mmol) in 0.54 mL of H2O was
added to a stirred solution of complex 1 (0.406 g, 1.27 mmol) in
1.8 mL of H2O in the dark, followed by 149 lL of cyclohexylamine.
The mixture was then stirred at r.t. for 4 h. The yellow precipitate
was collected via vacuum filtration and washed with approxi-
mately 3 mL of water and then approximately 3 mL of ethanol.
The solid was suspended in approximately 3 mL of acetone and
stirred in H2O for 0.5 h, the suspension centrifuged and the light
yellow clear supernatant removed and discarded. This procedure
was repeated three times before the product was dried in vacuo.
Pale yellow powder was collected: 345.6 mg (57.3% yield). MS
(acetonitrile with NaI) calculated molecular mass of C6H16ClIN2Pt:
473.64 g/mol, observed average molecular mass: 473.66 g/mol. 1H
NMR (300 MHz, DMF-d7), d (ppm) = 1.10 (1H, m), 1.31 (4H, m),
1.60 (1H, m), 1.74 (2H, m), 2.43 (2H, m), 2.95 (1H, m), 4.21 (3H,
b), 4.96 (2H, b).
2.6. Cytotoxicity of cisplatin and the complex 5 against A549, H596
and WI-38 cell lines
The studies involving human NSCLC cells, A549 and H596, and
human lung fibroblast cells, WI-38, were carried out under stan-
dard conditions in a humidified, 37 °C, 5% CO2 atmosphere incuba-
tor. The culture medium used for A549 and H596 cells was Roswell
Park Memorial Institute (RPMI) containing 10% fetal calf serum
(FCS), 100
-glutamine. The culture medium used for WI-38 cells was Mini-
mum Essential Medium (MEM) containing 10% FCS, 100 g/mL
lg/mL streptomycin, 100 IU/mL penicillin and 2.0 mM
L
l
streptomycin, 100 IU/mL penicillin and 2.0 mM L-glutamine. Solu-
tions of cisplatin and complex 5 at various concentrations were
prepared. Solid cisplatin was dissolved in 0.9% sodium chloride
and allowed to equilibrate in the dark for 24 h before being diluted
with medium and used to treat cells. Complex 5 was first dissolved
in a small amount of DMSO and then diluted using medium. Two
control groups were included; the first being medium alone and
the second being medium plus cells without treatment of cisplatin
or 5. Ten replicates were done for each concentration and each
2.4. Synthesis of cis-[PtCl2achNH3] (3)
To a stirred suspension of complex 2 (0.307 g, 0.648 mmol) in
3.83 mL of H2O in the dark was added AgNO3 (0.182 g, 1.07 mmol).
The mixture was then stirred at r.t. for 5 h and tested for Ag+: 20
lL
of reaction mixture was taken and centrifuged, and the colorless
supernatant added to a NaCl solution prepared by adding 10 pieces
of NaCl crystal to 1.0 mL of H2O, and no precipitate formed.
Another several pieces of NaCl crystals were added and no
control group. The cells were seeded at 5 ꢂ 104 cells/mL (100
lL/
well) in 96-well plates and allowed to grow for 24 h, after which
Please cite this article in press as: Y. Shi et al., Inorg. Chim. Acta (2014), http://dx.doi.org/10.1016/j.ica.2014.07.043

4
Y. Shi et al. / Inorganica Chimica Acta xxx (2014) xxx–xxx
time the medium was removed and replaced with 100 lL of med-
ium containing Pt complex. After an exposure time of 24 h, the
3. Results
Lipophilic Pt(IV) complex 5 was synthesized and characterized
medium containing the Pt complex was removed and replaced
with 100 lL of fresh medium. To each well, 20 l
L of CellTiter 96Ò
using 1H NMR and mass spectrometry. The toxicities of cisplatin
and 5 toward human NSCLC cells (A549 and H596) and human
lung fibroblast cells (WI-38) were then investigated. Cells were
exposed to cisplatin or complex 5 for 24 h. In Fig. 1, % survival is
plotted against Pt concentration, and the IC50 values listed. Com-
plex 5 is more toxic toward all three cell lines than is cisplatin.
The IC50 values of complex 5 toward H596 and A549 cells are
AQueous One (MTS) solution was added. After 2 h incubation with
the MTS solution, the absorbance was read at 490 nm using a Syn-
ergy™ H1 hybrid multi-mode microplate reader (BioTek). The per-
cent survival of cells treated by each concentration of Pt drug was
calculated using the following equation:
ꢀ
ꢁ
Ad ꢀ Am
Ac ꢀ Am
0.3
lM and 0.2
lM, respectively, while those of cisplatin are
% survival ¼ 100 ꢂ
ð1Þ
16.8
l
M and 68.8
lM, respectively. The IC50 values of complex 5
and cisplatin toward WI-38 cells are 0.8
lM and 30.5 lM,
where Ad is the absorbance of cells treated with Pt drug, Am is the
absorbance of medium alone and Ac is the absorbance of cells with-
out treatment. The percent (%) survival data was fit to an exponen-
tial regression using Microsoft Excel Solver, and IC50 values
determined.
respectively.
The mechanism of action of complex 5 was investigated
through agarose gel electrophoresis. Fig. 2(a) shows the image of
an agarose gel of pBR322 DNA treated with cisplatin in pH 7.4 buf-
fer (Lane 2–8) for 24 h at 37 °C. When r increases, the mobility of
Form I DNA decreases and then, at higher r values, it increases. This
is a typical binding pattern for cisplatin to Form I DNA [30].
Fig. 2(b) shows an image of an agarose gel of pBR322 DNA treated
with 5 (after reduction with ascorbic acid) in pH 7.4 buffer (Lane 4–
7) for 24 h at 37 °C. Plasmid pBR322 DNA was used to capture cis-
platin and the Pt(II) reduction products; DNA binding affects the
mobilities of the DNA in the gel. The pBR322 DNA has both closed
and nicked circular forms in each individual lane; the faster
migrating Form I is the supercoiled closed circular form of DNA
and the slower migrating Form II is the nicked circular form. Pt
complex 5 was first dissolved in a small amount of DMSO and then
diluted using aqueous buffer; the final concentration of DMSO in
each sample was no more than 2.5% (v/v). To examine and exclude
possible effects of the reducing agents and DMSO on the plasmid
DNA, three control groups were run in the gel with complex 5. Lane
1 contains only DNA, lane 2 contains DNA and 2.5% DMSO and lane
3 contains DNA and the reducing agent ascorbic acid. In Fig. 2(b),
with higher r value, the mobility of Form I DNA decreases, reveal-
ing binding of reduced 5 to Form I DNA.
2.7. DNA binding studies
The DNA binding studies were done in a total volume of 20
containing 3.85 M base pairs pBR322 DNA and 10 mM pH 7.4
HEPES buffer (with DMSO when testing 5). The final concentration
of cisplatin and Pt(IV) complex, 5, was in the range of 1.0–6.0
and 20–100 M, respectively. Ascorbic acid was used as the reduc-
lL
l
lM
l
ing agent for 5. The complex was first dissolved in DMSO followed
by the addition of HEPES buffer and ascorbic acid to give an aque-
ous solution containing 250 lM of ascorbic acid and 2.5% DMSO v/
v and was incubated for 72 h at 37 °C in sealed Eppendorf tubes.
Then, pBR322 DNA stock solutions were added. The values of r,
where r = [Pt]/[DNA-bp], in the reaction solutions are given in the
figure captions. The samples were further incubated at 37 °C for
24 h in sealed Eppendorf tubes. After incubation, 2.5
ing buffer containing 0.25% bromophenol blue, 0.25% xylene cyanol
and 40% sucrose was added to each sample. An 8 L volume of each
lL of a load-
l
solution was loaded onto a 1.0% (w/v) agarose gel in 1ꢂ TBE
(0.090 M trisborate and 0.002 M ethylenediaminetetraacetic acid)
buffer, pH 8.0, and electrophoresis was carried out at 70 V for
1.5 h. The gel was then exposed to a 5 ꢂ 10ꢀ5% solution of ethidium
bromide for 30 min, washed (destained) in distilled water for
15 min, and its image captured using a ChemiDoc MP imaging sys-
tem (Bio-Rad). The DNA binding studies with cisplatin were done
in the same manner, but no DMSO or ascorbic acid were used in
sample preparation and only one control group (DNA alone) was
included. The r values are given in the figure caption.
2.8. Incorporation of complex 5 into mesoporous silica (MCM-41)
Mesoporous silica (MCM-41) nanoparticles were prepared as
previously described [26]. The nanoparticles were approximately
400 nm. To a solution containing 1 mg of complex 5 in 200 lL eth-
anol was added 5 mg of MCM-41. The mixture was stirred vigor-
ously at r.t. for 24 h and then centrifuged at 1000g for 10 min.
The clear supernatant was discarded, and the pellet washed twice
with ethanol and once with PBS, and then dried in vacuo. The dried
sample was digested with hydrofluoric acid at 80 °C for 16 h, then
evaporated to dryness, and further digested in 70% nitric acid for
2 h at 70 °C, before being analyzed using a NexION 300D (Perkin
Elmer) inductively coupled plasma-mass spectrometer (ICP-MS).
To study release, 5-containing MCM-41 was added to PBS, pH
7.4, and the mixture vortexed at r.t. for 24 h. After this time, it
was centrifuged at 1000g for 10 min, the supernatant removed
and the pellet dried in vacuo. The dried sample was digested as pre-
viously described and analyzed using ICP-MS.
Fig. 2. Ethidium bromide-stained agarose gels of pBR322 DNA (38.5 lM). The
pBR322 DNA was in the presence of cisplatin (a) or ascorbic acid-reduced complex 5
(b) for 24 h. The fastest migrating band (bottom) is covalently closed circular Form I
DNA, while the slowest migrating band (top) is nicked circular Form II DNA. Lane
and r ([compound]/[DNA-bp]) are: (a) Cisplatin – 1, 0; 2, 0.03; 3, 0.04; 4, 0.05; 5,
0.08; 6, 0.10; 7, 0.13; and 8, 0. (b) Complex
5 – 1, 0; 2, 0 (DMSO); 3, 0
(DMSO + ascorbic acid); 4, 0.52; 5, 1.04; 6, 2.08; and 7, 2.60.
Please cite this article in press as: Y. Shi et al., Inorg. Chim. Acta (2014), http://dx.doi.org/10.1016/j.ica.2014.07.043

Y. Shi et al. / Inorganica Chimica Acta xxx (2014) xxx–xxx
5
Based on ICP-MS results, complex 5 can be loaded into and then
released from mesoporous silica MCM-41 nanoparticles. After 24 h
adsorption at r.t., MCM-41 contains 0.9% w/w of 5. After 24 h in PBS
at r.t., 95.9% of 5 is released from mesoporous silica MCM-41.
Thus, we prepared complex 5, which has the potential to be used
alone or with other therapeutic agents, both radio- and chemother-
apeutic, and administered free or encapsulated in nanocarriers.
This complex has a higher therapeutic index than cisplatin, and
shares its mechanism.
4. Discussion
Acknowledgements
Lung cancer is the leading cause of cancer-related death in the
United States, and approximately 85–90% of lung cancer patients
have NSCLC. NSCLC is extremely difficult to treat, the 5-year sur-
vival rate being 17% [31,32]. Complex 5 was synthesized following
literature procedures [26–28], with some modifications. The syn-
thesis and characterization of 5 and its intermediates are reported
here in detail. It has been reported that complex 5 is highly lipo-
philic, with a logPo/w of 4.10, and its high toxicity toward A2780
ovarian and HCT116 colon carcinoma cell lines has been demon-
strated [21]. We tested and compared the toxicity of complex 5
and cisplatin toward human NSCLC cells, as well as normal human
lung fibroblast cells (Fig. 1). As illustrated in Fig. 1(b)–(c) and (e)–
(f), complex 5 is significantly more toxic toward both NSCLC cell
lines (H596 and A549) than cisplatin. Complex 5 was again more
toxic than cisplatin toward a normal human lung fibroblast cell
line (WI-38); however, the therapeutic indexes (TI) of 5 compared
to cisplatin were much improved. TI is defined as the ratio of the
IC50 of normal cells to that of neoplastic cells [33]. In the case of
H596 relative to WI-38, the TI is 1.82 for cisplatin and 2.63 for 5.
In the case of A549 relative to WI-38, the TI is 0.443 for cisplatin
and 3.79 for 5. Thus, complex 5 could be a candidate for use in
the clinic.
We wish to thank the University of North Texas System College
of Pharmacy for their support of this research, Drs. Laszlo Prokai
and Szabolcs Szarka (University of North Texas Health Science Cen-
ter) for acquiring mass spectra, Dr. Kala Green (Texas Christian
University) for acquiring 1H NMR spectra and Dr. Andras Lacko
and Ms. Nirupama Sabnis (University of North Texas Health Sci-
ence Center) for helpful discussions pertaining to this work.
References
[1] B. Desoize, C. Madoulet, Crit. Rev. Oncol. Hematol. 42 (2002) 317.
[2] R.A. Alderden, M.D. Hall, T.W. Hambley, J. Chem. Educ. 83 (2006) 728.
[3] E. Wong, C.M. Giandomenico, Chem. Rev. 99 (1999) 2451.
[4] A.I. Ivanov, J. Christodoulou, J.A. Parkinson, K.J. Barnham, A. Tucker, J.
Woodrow, P.J. Sadler, J. Biol. Chem. 273 (1998) 14721.
[5] D. Wang, S.J. Lippard, Nat. Rev. Drug Discov. 4 (2005) 307.
[6] B.A. Teicher, Clin. Cancer Res. 14 (2008) 1610.
[7] J.S. Butler, P.J. Sadler, Curr. Opin. Chem. Biol. 17 (2013) 175.
[8] E. Wexselblatt, D. Gibson, J. Inorg. Biochem. 117 (2012) 220.
[9] N.P. Farrell, Curr. Top. Med. Chem. 11 (2011) 2623.
[10] A.M. Montana, C. Batalla, Curr. Med. Chem. 16 (2009) 2235.
[11] W.H. Ang, S. Pilet, R. Scopelliti, F. Bussy, L. Juillerat-Jeanneret, P.J. Dyson, J.
Med. Chem. 48 (2005) 8060.
[12] L.R. Kelland, S.Y. Sharp, C.F. O’Neill, F.I. Raynaud, P.J. Beale, I.R. Judson, J. Inorg.
Biochem. 77 (1999) 111.
Plasmid pBR322 DNA was used in agarose gel electrophoresis to
investigate the mechanism of action of complex 5 in the presence
of the biological relevant reducing agent ascorbic acid (Fig. 2). As
shown in Fig. 2(a), the mobility of Form I DNA changed with
increased r, which indicates that Pt(II) binds to the negatively
supercoiled Form I DNA. The mobility of Form I DNA at first
decreased, indicating Form I DNA became less negatively super-
coiled due to the binding of Pt(II) to DNA. When r was greater than
0.08, Form I DNA became positively supercoiled and its mobility
increased with r. In Fig. 2(b), with greater r value, the mobility of
form I DNA decreases, revealing DNA binding of reduced complex
5 to Form I DNA in a pattern similar to that of cisplatin when r is
less than 0.08.
The lipophilic complex 5 has the potential to be incorporated
into nanocarriers to achieve enhanced tumor accumulation. Preli-
minary studies show that it can be incorporated into mesoporous
silica MCM-41 nanoparticles at approximately 1% w/w, and most
is released in pH 7.4 buffer after 24 h. These 400 nm MCM-41
nanoparticles were previously shown to accumulate in tumors
after i.v. injection into mice [26]. In fact, approximately 4.5% of
the initial dose of radiolabeled MCM-41 per gram of tissue accu-
mulated in tumors after 24 h. Thus, complex 5-loaded MCM-41
nanoparticles could be a promising drug delivery system for the
treatment of NSCLC. Drug loading capacity may be further
improved by modifying the MCM-41 nanoparticles.
[13] H. Baruah, C.G. Barry, U. Bierbach, Curr. Top. Med. Chem. 4 (2004) 1537.
[14] P.D. Lawley, D.H. Phillips, Mutat. Res. 355 (1996) 13.
[15] Y. Shi, S.-A. Liu, D.J. Kerwood, J. Goodisman, J.C. Dabrowiak, J. Inorg. Biochem.
107 (2012) 6.
[16] M. Sinisi, F.P. Intini, G. Natile, Inorg. Chem. 51 (2012) 9694.
[17] E.L. Weaver, R.N. Bose, J. Inorg. Biochem. 95 (2003) 231.
[18] E.E. Blatter, J.F. Vollano, B.S. Krishnan, J.C. Dabrowiak, Biochemistry 23 (1984)
4817.
[19] H. Reiber, M. Ruff, M. Uhr, Clin. Chim. Acta 217 (1993) 163.
[20] P. Washko, D. Rotrosen, M. Levine, Am. J. Clin. Nutr. 54 (1991) 1221S.
[21] P. Gramatica, E. Papa, M. Luini, E. Monti, M.B. Gariboldi, M. Ravera, E. Gabano,
L. Gaviglio, D. Osella, J. Biol. Inorg. Chem. 15 (2010) 1157.
[22] G.J. Peters, A. Avan, M.G. Ruiz, V. Orsini, A. Avan, E. Giovannetti, E.F. Smit,
Anticancer Res. 34 (2014) 435.
[23] D. Tarn, C.E. Ashley, M. Xue, E.C. Carnes, J.I. Zin, C.J. Brinker, Acc. Chem. Res. 46
(2013) 792.
[24] A.J. Di Pasqua, K.K. Sharma, Y.-L. Shi, B.B. Toms, W. Ouellette, J.C. Dabrowiak, T.
Asefa, J. Inorg. Biochem. 7 (2008) 1416.
[25] A.J. Di Pasqua, H. Yuan, Y. Chung, J.-K. Kim, J.E. Huckle, C. Li, M. Sadgrove, T.H.
Tran, M. Jay, X. Lu, J. Nucl. Med. 54 (2013) 111.
[26] A.J. Di Pasqua, M.L. Miller, X. Lu, L. Peng, M. Jay, Inorg. Chim. Acta 393 (2012)
334.
[27] C.M. Giandomenico, M.J. Abrams, B.A. Murrer, J.F. Vollano, M.I. Rheinheimer,
S.B. Wyer, G.E. Bossard, J.D. Higgins III, Inorg. Chem. 34 (1995) 1015.
[28] S. Shamsuddin, C.C. Santillan, J.L. Stark, K.H. Whitmire, Z.H. Siddik, A.R.
Khokhar, J. Inorg. Biochem. 71 (1998) 29.
[29] M.J. Abrams, C.M. Gaindomenico, B.A. Murrer, J.F. Vollano, EP 328274, Aug 16,
1989.
[30] A. Binter, J. Goodisman, J.C. Dabrowiak, J. Inorg. Biochem. 100 (2006) 1219.
[31] M. Ding, J. Yang, Med. Oncol. 31 (2014) 928.
[32] K. Cetin, D.S. Ettinger, Y.-J. Hei, C.D. O’Malley, Clin. Epidemiol. 3 (2011) 139.
[33] P.R. Deepa, S. Vandhana, U. Jayanthi, S. Krishnakumar, Basic Clin. Pharmacol.
Toxicol. 110 (2012) 494.
5. Conclusions
Lung cancer, especially NSCLC, demonstrates high prevalence
and high mortality, and effective treatment options are lacking.
Please cite this article in press as: Y. Shi et al., Inorg. Chim. Acta (2014), http://dx.doi.org/10.1016/j.ica.2014.07.043