Are Pt(IV) Prodrugs That Release Combretastatin A4 True Multi-action Prodrugs?

Article abstract of DOI:10.1021/acs.jmedchem.1c00706

Multi-action Pt(IV) derivatives of cisplatin with combretastatin A4 (CA4) bioactive ligands that are conjugated to Pt(IV) by carbonate are unique because the ligand (IC50 < 10 nM) is dramatically 1000-folds more cytotoxic than cisplatin in vitro. The Pt(IV)-CA4 prodrugs were as cytotoxic as CA4 itself, indicating that the platinum moiety probably plays an insignificant role in triggering cytotoxicity, suggesting that the Pt(IV)-CA4 complexes act as prodrugs for CA4 rather than as true multi-action prodrugs. In vivo tests (Lewis lung carcinoma) show that ctc-[Pt(NH3)2(PhB)(CA4)Cl2] inhibited tumor growth by 93% compared to CA4 (67%), cisplatin (84%), and 1:1:1 cisplatin/CA4/PhB (85%) while displaying <5% body weight loss compared to cisplatin (20%) or CA4 (10%). In this case, and perhaps with other extremely potent bioactive ligands, platinum(IV) acts merely as a self-immolative carrier triggered by reduction in the cancer cell with only a minor contribution to cytotoxicity.

Full text of DOI:10.1021/acs.jmedchem.1c00706

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Article
Are Pt(IV) Prodrugs That Release Combretastatin A4 True Multi-
action Prodrugs?
Claudia Schmidt,# Tomer Babu,# Hana Kostrhunova, Annika Timm, Uttara Basu, Ingo Ott,*
Valentina Gandin,* Viktor Brabec,* and Dan Gibson*
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ABSTRACT: “Multi-action” Pt(IV) derivatives of cisplatin with combretas-
tatin A4 (CA4) bioactive ligands that are conjugated to Pt(IV) by carbonate
are unique because the ligand (IC50 < 10 nM) is dramatically 1000-folds more
cytotoxic than cisplatin in vitro. The Pt(IV)-CA4 prodrugs were as cytotoxic as
CA4 itself, indicating that the platinum moiety probably plays an insignificant
role in triggering cytotoxicity, suggesting that the Pt(IV)-CA4 complexes act as
prodrugs for CA4 rather than as true multi-action prodrugs. In vivo tests
(Lewis lung carcinoma) show that ctc-[Pt(NH3)2(PhB)(CA4)Cl2] inhibited
tumor growth by 93% compared to CA4 (67%), cisplatin (84%), and 1:1:1
cisplatin/CA4/PhB (85%) while displaying <5% body weight loss compared to
cisplatin (20%) or CA4 (10%). In this case, and perhaps with other extremely potent bioactive ligands, platinum(IV) acts merely as a
self-immolative carrier triggered by reduction in the cancer cell with only a minor contribution to cytotoxicity.
acid, 5-fluorouracil, irinotecan, and oxaliplatin) to treat
pancreatic cancer.9 These drugs are administered individually,
making it difficult to control the process since each drug has its
own pharmacokinetic profile and its own toxicity pattern.
In the last 2 decades, octahedral Pt(IV) complexes emerged as
promising candidates for overcoming resistance. The relative
stability of the low-spin d6 octahedral Pt(IV) complexes to
substitution reactions means that they are less reactive toward
biomolecules outside the cells, increasing their chances of
reaching the cancer cells intact. After crossing the cell
membrane, they are activated by reduction, releasing the Pt(II)
drugs in their active form as well as their axial ligands (Scheme
1).10−13 This provides a mechanism for the concurrent release of
a ratiometrically defined combination of bioactive moieties
inside the cancer cell. When two or even more drugs which act
on different cellular targets with different modes of action are
simultaneously released in the cancer cell, we have multi-action
prodrugs.14−16 This approach provides us with the opportunity
to design a larger number of structural alterations compared to
the square-planar Pt(II) complexes and resulted in the synthesis
and evaluation of a large variety of Pt(IV) prodrugs whose
activity is based on the activation-by-reduction concept.
INTRODUCTION
■
Chemotherapy is an important approach for the treatment of
various cancers. Most chemotherapeutic protocols are not
limited to a single drug (monotherapy) but rely on the
combination of several drugs that act by different mechanisms
against different cellular targets in the cancer cells (combination
therapy).1,2 This approach aims to overcome resistance by
ensuring more efficient and complete killing of the cancer cells in
a tumor.
Pt(II) complexes, such as cisplatin and its derivatives
carboplatin or oxaliplatin (Figure 1), have been used worldwide
in the clinic for the treatment of several types of cancers for more
than 40 years.3 These FDA-approved drugs, which exert their
anticancer activity by binding to nuclear DNA, cause severe side
effects that are due in part to their lack of stability under
physiological conditions and lack of selectivity toward cancer
cells. Most patients suffer from ototoxicity and nephrotoxicity,
naming only the most common ones.4,5 However, the major
drawback is the high number of incidences of inherent resistance
as well as resistance to the drugs that tumors develop during
treatment.6 Therefore, in order to overcome resistance, these
platinum drugs are used in the clinic mostly in combination with
other drugs with different potencies, toxicities, and modes of
action to treat a variety of cancers.
Received: April 19, 2021
Some examples of combination therapy with platinum drugs
include the combination of paclitaxel and cisplatin for the
treatment of several cancers;7 folinic acid, 5-fluorouracil, and
oxaliplatin are combined in a three-component protocol
(FOLFOX) for the treatment of colorectal cancer;8 and
FOLFIRINOX is a protocol that combines four drugs (folinic
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Figure 1. FDA-approved Pt(II) drugscisplatin, carboplatin, and oxaliplatin (top). Tubulin inhibitors (bottom).
Scheme 1. Pt(IV) Prodrugs Are Stable Outside the Cell and Are Activated by Reduction in the Cell Releasing the Pt(II) Drug
That Binds to DNA as Well as the Two Axial Ligands
Figure 2. Pt(IV) complexes that were tethered to the axial OH with linkers that are not likely to release the free CA4 following reduction.23−25
Another class of potent anticancer drugs are the antimitotic
agents that disrupt the formation of the mitotic spindle. Drugs of
this family include paclitaxel, docetaxel, colchicine, vincristine,
and vinblastine that are often combined with Pt-based drugs
(Figure 1).17 In this context, we chose to explore the
combination of cisplatin with the bioactive cis-stilbene
combretastatin A4 (CA4) (Figure 1), that is an antimitotic,
antiangiogenic, and antiproliferative molecule first isolated from
an African tree, called Combretum caffrum.18 CA4 is a tubulin
polymerization inhibitor of colchicine type.19,20 Due to the
antiangiogenic properties of the 4-O-phosphate derivative CA-
4P (formally known as fosbretabulin), the compound is
undergoing phase II/III clinical investigations against anaplastic
thyroid cancer in combination with carboplatin and paclitaxel.21
Unfortunately, the three-drug combination did not show any
advantages over the administration of carboplatin and paclitaxel
alone. Considering that the three drugs have all different
pharmacokinetics, combining two bioactive molecules in one
complex might result in a higher activity and a lower overall
toxicity as we have already shown in our latest report on a new
Pt(IV) complex that combined cisplatin and gemcitabine.22
There are several reports describing the Pt(IV) conjugates of
CA4. Because CA4 does not have a carboxylate that can be
directly conjugated to Pt(IV), it had to be modified, and linkers,
which included a carboxylate, were appended to CA4 so that it
could be tethered to the OH of the Pt(IV). The linkers were
conjugated to CA4 by stable ether or ester linkages (Figure
2).23−25 The bonds between the linker and CA4 are fairly stable
B
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Scheme 2. Synthetic Schemes for the Preparation of Compounds
under physiological conditions, suggesting that following
reduction the linker is likely to remain tethered to CA4 and
that the released bioactive agent is the CA4-linker conjugate
rather than free CA4.
molecule to the axial position of Pt(IV) via a carbonate.
Following reduction of the Pt(IV) complex, the carbonate linker
loses the non-toxic CO2, thereby generating the bioactive
molecule in its original unaltered form. These designs
tremendously extend the possibilities for new combinations of
Pt(IV) complexes.
Since even minor structural alterations of the bioactive
molecule can influence the activity of a compound, we decided
to synthesize and study the anticancer properties of a series of
carbonate-linked Pt(IV)-CA4 complexes, which release the
unmodified free CA4 after reduction. Moreover, we wanted to
explore the effect of adding another bioactive ligand to the
second axial position, forming “triple-action” prodrugs16 on the
pharmacological properties of the prodrugs. We chose the
following compounds that have all shown to act synergistically
with cisplatin: the HDAC inhibitors phenylbutyrate (PhB)14
and valproate (Val)26 as well as the PDK inhibitor
dichloroacetate (DCA)27 or octanoate (Oct) that enhances
DNA methylation.28 We investigated the influence of the trans
axial ligands on the stability, rates of reduction, and cytotoxicity
of these complexes and compared a carbonate-linked compound
to its carboxylate-linked counterpart where CA4 was linked to
the metal by a glutaric (glu) linker (Figure 2C). Biological
investigations on the antiproliferative effects on a panel of
different cancer cells were conducted and cellular uptake studies
in MDA-MB-231 breast carcinoma cells were performed. In vivo
studies in Lewis lung carcinoma mice were carried out to assess
their tumor growth inhibition effects and overall toxicity
(indicated by body weight loss) in comparison to the single
application of the reference drugs cisplatin and CA4 and their
co-administration.
Our goal was to prepare multi-action Pt(IV) prodrugs where
CA4 is conjugated to the axial position of Pt(IV) via a carbonate
linkage in order to ensure that following the reduction of Pt(IV),
and loss of CO2, CA4 will be released in the cell in its native
form. The synthetic approach we adopted is similar to the one
we described recently and is depicted in Scheme 2. We activated
the hydroxyl of CA4 by reacting CA4 with N,N′-disuccinimidyl
carbonate (DSC) in the presence of N,N-dimethylpyridine-4-
amine (DMAP) in dichloromethane (DCM) for 3 h. Stirring the
reaction for more than 3 h or adding more equivalents of base
resulted in the formation of a CA4 dimer, where two CA4
molecules are connected through a carbonate linker as the major
product. The activated CA4 was reacted with oxoplatin to obtain
the dual-action prodrugcisPt(CA4)(OH), compound 2, in
good yield (85%). Compound 2 also served as the precursor for
the synthesis of the triple-action compounds 3−7 that were
obtained by carboxylation of the axial OH of 2 with the
appropriate anhydrides (Scheme 2).
While it is intellectually appealing and synthetically
challenging to be able to modify the OH of CA4 in a way that
following activation of the prodrug CA4 will be released in its
free from, several studies demonstrated that the 3′-OH group of
the B-ring is not essential for the interaction of CA4 with tubulin,
and derivatives of CA4 where the OH was modified can retain
good cytotoxicity.29 Thus, for comparison, we also prepared the
complex, cisPt(CA4-glu)(OH), where CA4 was first modified
with glutarate that was then activated with EDC and NHS and
conjugated to the axial OH of Pt(IV), from which the triple-
action derivative cisPt(CA4-glu)(PhB) was prepared (Scheme
2).
RESULTS & DISCUSSION
■
Synthesis and Characterization of the Compounds.
Recently, we reported for the first time on a strategy for tethering
ligands with an OH group to Pt(IV) such that following
reduction of Pt(IV), the original ligands are released.22 We
accomplished this by attaching a hydroxyl group of a bioactive
C
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All complexes were characterized by 1H, 13C, and 195Pt-NMR
as well as by ESI mass spectrometry, and high purity was
confirmed by high-performance liquid chromatography
(HPLC) measurements and elemental analysis. The obtained
data were consistent with the proposed structures (Supporting
Information).
Stability of the Compounds. The stability of compounds
2, 3, 4, and 9 was measured by monitoring the decrease of the
peak of the starting material by reversed-phase HPLC (RP-
HPLC) in ACN/phosphate buffer, in ACN/cell culture
medium, at 37 °C and following the reviewers’ suggestion also
in 1% dimethyl sulfoxide (DMSO)/cell culture medium. The
results are presented in Table 1. The cisPt(CA4-glu)(OH)
glu)(OH) and cisPt(CA4-glu)(PhB) compared to compounds
2, 3, and 4 is probably because carbonate linkers are more
susceptible to hydrolysis than carboxylates.15 The rate of
hydrolysis of the axial CA4 seems to depend on the nature of
the axial ligand trans to the carbonate. When the trans ligand is
also a carbonate (4), the hydrolysis is fast, it is moderate when
the trans ligand is carboxylate (3), and slow when it is OH (2).
Interestingly, the hydrolysis in medium is faster than in buffer for
cisPt(CA4)(PhB), slower for cisPt(CA4)(CA4), and still slow
(>1 day) for cisPt(CA4)(OH) and of course for cisPt(CA4-
glu)(OH) and cisPt(CA4-glu)(PhB). There are some differ-
ences between the half-lives measured in ACN/cell culture
medium and 1% DMSO/cell culture medium particularly for
cisPt(CA4)(OH), but the trend is the same.
Table 1. Reduction and Stability Studies of Pt(IV)
Complexes
Reduction of the Compounds. The rates of reduction of
compounds 2, 3, and 4 were measured by HPLC in a 10-fold
excess of an ascorbic acid solution in phosphate buffer (50 mM,
pH 7.0). As expected, the dual-action compound, 2, was reduced
rapidly due to the efficient electron transfer from ascorbate to
Pt(IV)t1/2 = 9 min. The bis-carbonate complex (4) had a t1/2
of 27 min and compound 3 had a t1/2 of 210 min. The relatively
short half-life of cisPt(CA4)(CA4) could be due to the
combination of hydrolysis, followed by the rapid reduction of
cisPt(CA4)(OH). Because the retention times of CA4-glu and
CA4 were identical, we could not use HPLC to verify whether
the reduction product was CA4 or CA4-glu. Therefore, we
characterized the reduction products of cisPt(CA4)(OH) and
reduction
studies
stability studies
t
1/2 in
t1/2
t1/2
t1/2 in
buffer
(50%;
ACN)
medium
(50%
medium
(1%
buffer
(50%
compound
ACN)
DMSO)
ACN)
(2) cisPt(CA4)(OH)
(3) cisPt(CA4)(PhB)
(4) cisPt(CA4)(CA4)
∼3.5 days
∼10 h
∼1 h
∼3 days
∼4 h
∼3 h
∼1 day
∼3 h
∼2.5 h
9 min
210 min
27 min
110 min
(9)
>21 days
∼12 days ∼4 days
cisPt(CA4-glu)(OH)
(10)
>23 days
∼11 days ∼4.5 days
260 min
cisPt(CA4-glu)(PhB)
1
cisPt(CA4-glu)(OH) by H NMR spectroscopy in DMSO-d6
with a 10-fold excess of ascorbate. As can be seen in Figure 3, the
chemical shifts of free CA4 fall in the range of 6.3−6.9 ppm,
while when bound to Pt(IV), these resonances are shifted
complex is very stable with half-lives >21 and ∼12 days in buffer
and medium, respectively. The higher stability of cisPt(CA4-
Figure 3. Reduction of carbonate vs carboxylate Pt(IV) complexes by ascorbic acid in DMSO-d6 (1H NMR 500 MHz).
D
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Table 2. Antiproliferative Effects of Pt(IV)-CA4 Complexes on a Panel of Different Human Tumor Cells and a Healthy Human
a
Kidney Cell Line
A2780
ovary
A2780cis
ovary
A375
PC9
lung
HT-29
colon
MCF-7
breast
MDA-MB-231 SK-HEP-1
RC-124
kidney
compound
melanoma
breast
liver
unit
cisplatin
2.41 (0.20) 26.46 (3.68) 3.08 (1.54) 9.94 (1.34) 7.99 (1.38)
10.65 (1.04)
8.83 (1.58)
3.55 (0.16)
1.89 (0.28)
3.68 (0.58)
3.51 (0.11)
3.19 (0.33)
1.82 (0.18)
3.12 (0.37)
4.37 (0.16)
4.21 (0.35)
28.00 (2.12) 1.04 (0.14)
μM
1 + cisPt (1:1) 2.40 (0.41) 2.53 (0.31)
1 + cisPt (2:1) 1.04 (0.09) 1.11 (0.04)
2.64 (0.04) 3.33 (0.11) 415.00 (15.56) 3.40 (0.53)
1.07 (0.35) 1.50 (0.14) 192.30 (3.99) 1.67 (0.22)
2.02 (0.81) 4.64 (0.49) 348.21 (14.28) 3.14 (0.32)
3.25 (1.04) 5.82 (1.27) 211.38 (5.62) 2.49 (0.35)
3.52 (0.85) 6.78 (0.52) 101.96 (9.50) 2.77 (0.08)
1.72 (0.78) 4.48 (0.88) 202.79 (1.53) 1.68 (0.23)
4.83 (0.56)
2.49 (0.26)
3.00 (0.29)
4.74 (0.18)
4.02 (0.76)
2.42 (0.10)
4.39 (0.47)
5.62 (0.81)
5.76 (0.87)
10.22 (0.49) nM
4.45 (0.37)
9.31 (1.06)
9.62 (1.90)
nM
nM
nM
1
2
3
4
5
6
7
8
9
2.37 (0.44) 2.80 (0.03)
3.61 (0.78) 4.47 (0.64)
4.01 (0.36) 4.52 (0.60)
2.03 (0.29) 2.46 (0.67)
3.64 (0.18) 3.87 (0.33)
4.18 (0.56) 4.58 (0.85)
5.01 (1.19) 5.61 (1.26)
1.78 (0.12) 1.68 (0.05)
2.03 (0.11) 2.45 (0.43)
11.11 (0.84) nM
5.00 (1.28)
9.73 (0.72)
7.84 (0.08)
nM
nM
nM
2.47 (0.98) 5.89 (0.65) 1100 (370)
3.33 (0.37)
3.94 (0.88) 7.18 (1.76) 139.90 (2.54) 3.66 (0.43)
4.34 (1.58) 9.26 (1.41) 299.73 (7.95) 3.91 (0.18)
1.76 (0.13) 2.29 (0.17)
14.46 (0.71) nM
nM
nM
2.23 (0.66) 3.12 (0.15)
a
Cells were incubated for 72 h at 37 °C. (n = 3).
downfield and fall between 6.4 and 7.2 ppm. Once the Pt(IV) is
reduced, as evidenced by the disappearance of the peak of the
ammine ligands (triplet at 5.9 ppmred asterisk), the aromatic
peaks correspond to those of unbound free CA4. In contrast, the
reduction of cisPt(CA4-glu)(OH), even after 48 h, does not
show any signs of free CA4.
efficacy of prodrugs in vivo. While direct covalent binding of
cisplatin to albumin serves to inactivate the drug by reducing its
bioavailability, the efficacy of Pt(IV) prodrugs can be enhanced
by deliberately targeting (covalently33,34 or non-covalently35)
albumin in vivo, using it to deliver the cytotoxic agent to the
tumor. One such compound, BTP-114, is in clinical trials.36
The binding of cisplatin, cisPt(CA4)OH, cisPt(CA4)(PhB),
and cisPt(CA4-glu)(PhB), which were tested in vivo, to fetal calf
serum (FCS) proteins (10 v/v %) in Dulbecco’s modified
Eagle’s medium (DMEM) was evaluated over 24 h in an ethanol
precipitation assay. The respective complexes were added to the
medium, and aliquots were collected at different time points.
Proteins were precipitated by the addition of ethanol. The
unbound metal content was determined in the supernatant, and
the metal bound to FCS proteins was calculated (%) and the
results are depicted in Figure 4. The values for cisplatin are in
Interestingly, even the two most stable compounds both in
buffer and in medium were reduced rather rapidly, lending
support to the hypothesis that the prodrugs are relatively stable
outside the cells and undergo rapid activation inside the cells.
Antiproliferative Effects on Tumor Cells and Non-
tumorigenic Cells. The antiproliferative effect of the
compounds was evaluated against a panel of eight cancer cell
lines from different organs, both sensitive and resistant to
cisplatin, and one non-tumorigenic human kidney cell line (RC-
124). CA4 is a very potent cytotoxic agent with low nM IC50
values in all the cancer cell lines and is about 1000-fold more
potent than cisplatin. In contrast, the second axial ligands (Val,
Oct, and PhB) are not cytotoxic on their own and have IC50
values in the mM range against various cancer cell lines,14,26,28,30
and the IC50 values for DCA were reported as >100 μM.27,31,32
All the Pt(IV)-CA4 conjugates also exhibited low nM
cytotoxicity (<10 nM) in all cancer cell lines with the exception
of HT-29, where IC50 values varied between 101 and 1100 nM
(Table 2). The IC50 values of the 1:1 and 2:1 mixture of CA4 and
cisplatin reflect merely the IC50 values of CA4 with no apparent
contribution of cisplatin. Interestingly, there is no correlation
between the IC50 values of the compounds and their half-lives in
culture cell medium, nor did we observe any dependence on the
identity of the second bioactive ligand. The lack of correlation
between stability in medium and the cytotoxicity is probably due
to the fact that CA4 itself is so potent, so that even if a Pt(IV)
compound is hydrolyzed or reduced in the medium, and CA4 is
released before it enters the cell, CA4 itself will still exert its
cytotoxic effect with the same efficacy. It is noteworthy that the
nature of the linker between the OH of CA4 and Pt(IV), and
consequently what is released in the cell, can significantly affect
the potency of the prodrug. This was also noticeable in the case
where the carbonate-linked gemcitabine, ctc-[Pt(NH3)2(Gem-
carb)(PhB)Cl2], is significantly more cytotoxic than the
succinate-linked gemcitabine, ctc-[Pt(NH3)2(Gem-suc)(PhB)-
Cl2].22
Figure 4. Platinum binding to FCS proteins in DMEM cell culture
medium over 24 h at 37 °C.
accordance with the values reported in the literature.37 Cisplatin
and cisPt(CA4)(PhB) reach a plateau after 2 h of incubation,
where 62−67% of the compound is bound to the proteins. The
complex cisPt(CA4)(OH) binds slower and to a lower extent to
proteins. After 8 h of incubation only, 45% was found to be
bound to proteins. In contrast, cisPt(CA4-glu)(PhB) binds
rapidly and to a larger extent to FCS proteins in comparison to
Protein Binding of Platinum Complexes. Binding to
serum proteins, particularly albumin, can significantly affect the
E
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Table 3. Cellular Platinum Accumulation and DNA Platination in MDA-MB-231 Cells
a
b
cellular uptake (ng Pt/106 cells)
DNA platination (pg Pt/μg DNA)
c
0.1 μM
1 μM
10 μM
10 μM
oxoplatin
cisplatin
Pt(CA4)(OH)
Pt(CA4-glu)(OH)
Pt(CA4)(PhB)
0.12 0.02
0.8 0.2
1.5 0.4
2.6 0.3
1.1 0.2
3.6 0.8
6
15
18
68
1
3
4
8
7
23
28
1
3
2
0.14 0.01
0.29 0.04
0.5 0.1
5
1
80 10
a
b
Cellular platinum accumulation into MDA-MB-231 cells after 5 h of exposure to 0.1, 1, or 10 μM of the compounds. Platinum associated with
c
DNA in MDA-MB-231 cells after 5 h of exposure to 10 μM of the compounds. Cell viability at the time of harvesting as determined with trypan
blue was not under 89%.
all the other platinum complexes. A plateau is reached after 4 h
with a maximum of 80% platinum bound to FCS.
conjugates than from the cells treated with cisplatin (approx-
imately 3.3−11.7-fold).
We presume that the binding of cisplatin to the proteins is
covalent, while the Pt(IV) compounds are bound to the proteins
non-covalently. In order to see whether lipophilicity affects the
protein binding, we note that based on the retention times on a
C18 column, under identical conditions, the order of
lipophilicity is cis(CA4-glu)(PhB)6.13 min > cis(CA4)-
(PhB) 6.11 min > cis(CA4)(OH) 4.69 min, indicating that a
higher lipophilicity enhances the binding capabilities of the
platinum complexes.
Tubulin Polymerization Inhibition in Cells. CA4 is a
tubulin-binding chemotherapy drug.39 It interferes with the
microtubule dynamics of tubulin acting as a tubulin polymer-
ization inhibitor.40 CA4 is known to bind the β-subunit of
tubulin (colchicine site) and thus prevents cells from producing
microtubules that are essential for cytoskeleton formation.
Whole-cell flow cytometry analysis of microtubules was used
previously to assess the ability of various compounds to alter
tubulin/microtubule dynamics.41,42 The method uses a mild
lysis buffer and stabilization of microtubule network with
glutaraldehyde. We employed this approach to investigate
whether the investigated Pt(IV)-CA4 agents are able to
modulate the tubulin/microtubule dynamics upon entering
the cells (and presumably releasing the combretastatin unit).
MDA-MB-231 cells were treated with a series of concentrations
of CA4, CA4-glu, cisPt(CA4)(OH), and cisPt(CA4-glu)(OH)
for 8 h. The cells were then harvested, fixed, and stained with an
anti-α-tubulin-FITC antibody. It is expected that under these
experimental conditions, compounds that effectively inhibit
tubulin polymerization will show decreased fluorescence
compared to the control. The results (Figure 5) demonstrate a
significant and dose-dependent decrease in FITC fluorescence,
Cell Uptake. Before a metallodrug reaches its pharmaco-
logical target in the cell, it must first accumulate in cells. It has
been demonstrated that a higher accumulation of metallodrugs
generally potentiates antiproliferative activity. Moreover, the
mechanism of action of antitumor metallodrugs is a multi-step
process that includes as the first step cell entry or
accumulation.38 Therefore, the cellular levels of the investigated
compounds were measured after a 5 h exposure of the MDA-
MB-231 cells to the platinum complexes at concentrations 0.1
and 1.0 μM at 37 °C. The relatively high concentration (with
respect to the IC50 values) and short treatment time were chosen
to balance the detection limit and the effort to evaluate cells in a
viable state (cell viability at the moment of harvesting was above
90%). The platinum amount determined with inductively
coupled plasma mass spectrometry (ICP−MS) was related to
the cell counts. The accumulation of platinum from the
investigated Pt(IV)-CA4 conjugates was approximately 1.9−
6.3-fold greater than that from cisplatin, indicating that one
factor responsible for the enhanced antiproliferative activity of
Pt(IV)-CA4 conjugates in comparison with conventional
cisplatin is their increased cellular accumulation (Table 3).
We also determined the accumulation of platinum from the
investigated Pt(IV)-CA4 conjugates when the cellular levels of
platinum from the investigated compounds were measured after
exposure of the MDA-MB-231 cells to the platinum complexes
at the considerably higher concentration (10 μM) when,
however, the cell viability at the moment of harvesting was
only around 89%. The results in Table 3 show that also under
these conditions, the accumulation of platinum from the
investigated Pt(IV)-CA4 conjugates was considerably greater
than that from cisplatin (approximately 2.5−11.3-fold). More-
over, when the cells were treated with the investigated
compounds at their elevated concentration (10 μM), it was
also possible to measure platinum from these compounds
associated with DNA isolated from the treated cells. The level of
DNA platination correlated with the cellular platinum
accumulation in MDA-MB-231 cells, that is, it was considerably
greater in DNA isolated from the cells treated with Pt(IV)-CA4
Figure 5. Microtubule analysis in cells. MDA-MB-231 cells were
treated with indicated concentrations of the investigated compounds
for 8 h, harvested, fixed, and stained with the anti-α-tubulin-FITC
antibody. The fluorescence signal was normalized to the signal of non-
treated control (orange lines: mean SD). Very light gray; light gray
CA4-glu; dark graycisPt(CA4)(OH); and blackcisPt(CA4-glu)-
(OH). The value of fluorescence of untreated control was taken as
100%. The data represent a mean of three independent experiments.
The symbols */**/$ indicate a significant difference from the
untreated control cells with p < 0.05, p < 0.01, and p < 0.001,
respectively, based on a two-tailed Student’s t-test.
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indicating a lower amount of polymerized tubulin mass in the
cells treated with the investigated Pt(IV)-CA4 agents. Thus, the
data support the view that the investigated Pt(IV)-CA4 agents
can act through the inhibition of the tubulin polymerization
similar to free CA4 and CA4-glu, although with slightly lower
efficiency. Moreover, cisPt(CA4-glu)(OH) was more effective
than cisPt(CA4)(OH), which likely reflects the lower cellular
accumulation of cisPt(CA4)(OH) (Table 3). It is noteworthy
that the cisPt(CA4) compounds significantly inhibit cellular
tubulin polymerization at low nM concentrations that are in the
same range as their IC50 values. However, the level of DNA
platination was observed under the conditions when a significant
fraction of cells lost their viability. Thus, these results cannot be
taken as a support for the view that the mechanism of action of
Pt(IV) conjugates involves the processing of platinum−DNA
adducts.
Microtubule Imaging. The microtubule network in MDA-
MB-231 cells non-treated or treated with the tested compounds
was visualized by immunofluorescence. The non-treated cells
and cells treated with oxoplatin display a typical elongated
spindle shape, whereas the treatment with agents containing the
CA4 moiety induced changes in cell morphology, including
condensation of the microtubule material near the cell surface,
loss of membrane protrusions, and microtubule depolymeriza-
tion (Figure 6).
Tubulin Polymerization Inhibition in a Cell-Free
Medium. As cisPt(CA4)(OH) and cisPt(CA4-glu)(OH)
inhibited cellular tubulin polymerization (Figure 5), we
proceeded to find out the effect of Pt(IV)-CA4 compounds
on in vitro tubulin polymerization. Purified tubulin was
incubated in the absence and presence of different concen-
trations of CA4, CA4-glu, cisPt(CA4)(OH), cisPt(CA4-glu)-
(OH), and cisPt(CA4)(PhB), and the effect of the compounds
on the polymerization of tubulin was monitored using the
fluorescence-based tubulin polymerization assay kit (porcine
tubulin; Cytoskeleton, Inc.). Thus, polymerization of porcine
tubulin in the presence or absence of the investigated
compounds was followed by an increase in fluorescence at 37
°C. Figure 7 shows that CA4 inhibited the tubulin assembly in a
concentration-dependent manner with a significant effect visible
at a concentration of CA4 at 1 μM. CA4-glu was considerably
less effective, whereas the efficiency of cisPt(CA4)(OH),
cisPt(CA4-glu)(OH), and cisPt(CA4)(PhB) at 1 μM concen-
tration was almost negligible. These results suggest that the
inhibition of tubulin polymerization inside the cell (Figure 5)
was caused by CA4 released from cisPt-CA4 inside cells,
although the intact cisPt-CA4 complexes might also contribute
at least partially to the effect.
Figure 6. Effects of the investigated compounds on microtubule
organization. MDA-MB-231 cells were incubated with the tested
compounds at the concentrations corresponding to their IC50 values
(MTT, 72 h) for 24 h. Images were obtained with confocal microscopy
of anti-α-tubulin immunofluorescence (red) preparations; DNA was
stained with DAPI (blue). BFbright field. (A) Control non-treated
cells and (B−G) cells treated with individual compounds: (B) CA4,
(C) CA4-glu, (D) cisPt(CA4)(OH), (E) cisPt(CA4-glu)(OH), (F)
cisPt(CA4-glu)(PhB), and (G) oxoplatin. Scale bar represents 25 μm.
Cell Cycle Analysis. We investigated the cell cycle
distribution of MDA-MB-231 cells treated with the investigated
compounds for 24 h. The results are shown in Figure 8. Cisplatin
and oxoplatin arrest the cells in the S phase with cisplatin causing
more pronounced changes. All compounds containing the
combretastatin moiety arrest the cells predominantly in the G2/
M phase, which is typical for compounds which inhibit tubulin
polymerization. The three compounds possessing platinum as
well as combretastatin parts induce G2/M arrest; however, cell
population in the S phase is increased as compared to the two
no-platinum complexes.
than cisplatin. Moreover, all the complexes, with the exception
of cisplatin, have nearly identical IC50 values in the low nM
range, suggesting that CA4 rather than cisplatin is responsible
for the killing of the cancer cells.
These results are markedly different from those reported for
what looks like similar Pt(IV) derivatives of cisplatin where CA4
is conjugated to Pt(IV) via different linkers (Figure 2). For the
prodrug in Figure 2A, the bioactive ligand (CA4 derivative) is
1.25−4.27-fold less potent than cisplatin in four out of five
cancer cell lines, and the complex itself is only 2.58−11.31 times
more potent than cisplatin, suggesting that both moieties
contribute to cell death.23 For the compound in Figure 2B, the
bioactive ligand is 3.72−8.52-fold more potent than cisplatin
and the complex is 11.78−21.83 times more potent than
cisplatin.24 The IC50 value of the bioactive ligand in Figure 2C is
Role of Platinum in Inducing Cell Death. The
cytotoxicity data indicate that the Pt(IV)-CA4 complexes
described here present an interesting example of combining
cisplatin with a bioactive ligand that is 1000-fold more potent
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Figure 7. Tubulin assembly curves. Fluorescence-based tubulin polymerization assay kit was employed. Polymerization was recorded in the absence
(black dots) or presence of the tested compounds at 37 °C.
Therefore, due to this unique case where there is a huge
difference in potencies between the bioactive ligands and
cisplatin, an obvious question is whether the released platinum
(cisplatin) has a significant contribution toward the killing of the
cancer cell. In order to answer this question, we first isolated
DNA from MDA-MB-231 cells treated with Pt(IV)-CA4
compounds under similar conditions to those used for
measuring cellular platinum accumulation in MDA-MB-231
cells treated with cisPt-CA4 compounds (Table 3), that is, a 5 h
exposure of the MDA-MB-231 cells to the platinum complexes
at concentrations 0.1 and 1.0 μM or a 24 h exposure to the
platinum complexes at a concentration of 0.01 μM at 37 °C.
However, under these conditions, no measurable platinum from
cisPt-CA4 compounds was found to be associated with DNA.
These results can be interpreted to mean that DNA in the cells
treated with cisPt-CA4 compounds was not modified by Pt
adducts because the Pt moiety released from Pt-CA4
compounds did not reach the cell nucleus or that it entered
the cell nucleus but did not bind covalently to the DNA or that
its DNA adducts were efficiently repaired. Another eventuality is
that due to the relatively low IC50 values of the investigated
cisPt-CA4 compounds, we could only use in these experiments
relatively low concentrations of the Pt compounds (to warrant
that most of the cells used in this experiment were alive). Thus,
the level of Pt associated with DNA might be so low that it was
below the limit of detection by ICP−MS. Therefore, as we did in
a number of our previous papers,28,43,44 we also used an
additional test that makes it possible to determine whether DNA
is a target of the investigated compounds. This test is based on
the fact that platinum drugs are more cytotoxic in cells deficient
in DNA repair.45 Mutant Chinese hamster ovary CHO-K1 cells
MMC-2 deficient in DNA nucleotide excision repair were more
sensitive (9.7- or 9.3-fold) to killing by cisplatin or oxoplatin
compared with the wild-type cells (Table 4), consistent with the
previously obtained results.45,46 This result confirms that the
Figure 8. Cell cycle distribution of MDA-MB-231 cells following a 24 h
exposure to individual compounds at concentrations corresponding to
their respective IC50 values (MTT, 72 h). The cells were washed, fixed
with 70% ethanol, stained with propidium iodide, and subjected to
FACS analysis. RedG1 phase, blue dashed regionS phase, and
greenG2/M phase. The percentages of MDA-MB-231 cells
corresponding to the individual phases of cell cycle were acquired
with ModFit software and are expressed as mean
experiments).
SD (three
not reported, but the complex has nearly identical IC50 values to
CA4 in four cancer cell lines being 29.2−34.23-fold more potent
than cisplatin. Interestingly, also the oxaliplatin and the
Pt(DACH)Cl2 analogues of this compound have nearly the
same IC50 values as the cisplatin analogues, suggesting that
activity might be due primarily to the bioactive ligand.25 We also
note that in the cell lines that we used, the IC50 of CA4 was in the
low nM range (<10 nM), in agreement with several reports,29
while the values reported for CA4 for the compounds in Figure 2
are in the 100−300 nM range.
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In Vivo Studies. In vitro cytotoxicity studies are unfortu-
nately scarcely effective in predicting the in vivo pharmacody-
namics profile of a putative drug. Therefore, we took a step
forward toward preclinical translational studies by performing
preliminary in vivo anticancer investigations comparing
cisplatin, CA4, cisPt(CA4)(PhB), cisPt(CA4)(OH), and cisPt-
(CA4-glu)(PhB) against the murine Lewis lung carcinoma
(LLC) solid tumor model. Seven days after tumor inoculation,
the tumor-bearing mice were randomized into vehicle control
and treatment groups. Control mice received the vehicle,
whereas treated groups received daily oral doses of Pt(IV)
complexes (20 mg kg−1), daily i.p. of 3 mg kg−1 of cisplatin, and
20 mg kg−1 of CA4, 20 mg kg−1 of PhB, or a combination of
cisplatin, CA4 and PhB (3 mg/kg, ratio 1:1:1). The tumor
growth was evaluated at day 15, and the results are summarized
in Table S1. The changes in body weights were monitored at day
0 and from day 7 onward every 2 days as a sign of systemic
toxicity (Figure 9).
PhB proved to be barely effective in reducing the tumor
volume mass and CA4 induced a 67% reduction of tumor mass
significantly lower compared to cisplatin (84%). The antitumor
effect induced by cisPt(CA4)(OH) was comparable to that
induced by cisplatin (84% inhibition), and, noteworthily,
administration of cisPt(CA4-glu)(PhB) or cisPt(CA4)(PhB)
determined a superior reduction of the tumor mass by 91.5 and
92.6%, respectively. The i.p. treatment with the combination of
cisplatin, CA4, and PhB induced a tumor mass reduction that
was comparable to that of cisplatin alone and significantly lower
than that induced by the triple-acting compounds cisPt(CA4-
glu)(PhB) and cisPt(CA4)(PhB). In addition, the time course
Table 4. Antiproliferative Activity Data [IC50 Mean Values]
Obtained by MTT Test for Chinese Hamster Ovary CHO-K1
Cell Line (Wild Type) and Its Mutant Cell Line MMC-2
a
Deficient in DNA Repair
b
unit
CHO-K1
MMC-2
F
CA4
oxoplatin
cisplatin
cisPt(CA4)(OH)
cisPt(CA4-glu)(OH)
cisPt(CA4)(PhB)
nM
μM
μM
nM
nM
nM
6
40
24
5.3 0.6
5.8 0.7
4.5 0.6
1
2
4
4.9 0.8
4.3 0.5
2.5 0.5
1.4 0.2
1.3 0.3
1.1 0.3
1.2
9.3
9.6
3.8
4.5
4.1
a
The drug treatment period was 72 h. The results are expressed as
mean values SD from three independent experiments, each
b
performed in triplicate. F = factor defined as IC50 (NER-efficient,
CHO-K1)/IC50 (NER-deficient, MMC-2).
important cellular target of the antiproliferative activity of
cisplatin or oxoplatin is nuclear DNA.46 Contrastingly, the
antiproliferative activity of CA4 was almost identical in both cell
lines, indicating that DNA is an unlikely target of the
antiproliferative activity of CA4. CisPt(CA4)(OH), cisPt-
(CA4-glu)(OH), and cisPt(CA4)(PhB) were more active in
MMC-2 cells (3.9-, 4.6-, and 4.2-fold, respectively) than in
CHO-K1 cells; however, the difference in antiproliferative
activities was not as pronounced as in the case of cisplatin and
oxoplatin. This finding suggests that reparable DNA damage by
the platinum moiety released from Pt-CA4 compounds may play
a considerably less decisive role in the mechanism of their
antiproliferative activity in comparison with cisplatin or
oxoplatin.
Figure 9. TopInhibition of tumor growth, days 7−14: animals received daily oral gavage of 20 mg kg−1 of Pt(CA4)(PhB), Pt(CA4)(OH), or
cisPt(CA4-glu)(PhB) and daily i.p. of 3 mg kg−1 of cisplatin and 20 mg kg−1 of CA4. At day 15, animals were sacrificed. BottomBody weight changes
of LLC-bearing C57BL mice treated with the vehicle or the tested compounds. The error bars indicate SD.
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of body weight changes attested that cisplatin and the i.p. drug
combination induced elevated anorexia, whereas all Pt(IV)
derivatives were significantly better tolerated by animals,
inducing only minimal body weight loss (<10%) throughout
the therapeutic experimentation. It is important to notice that
combination treatment with higher doses of cisplatin, CA4, and
PhB were discontinued due to elevated toxicity.
The compound reported in Figure 2A was previously tested in
vivo by Li and co-workers in an HepG-2 tumor xenograft model
and was compared to cisplatin, CA4, and 1:1 cisplatin/CA4.
Similar to what we observed in our study, CA4 that was found to
be most potent in vitro elicited the weakest in vivo anticancer
activity.24 However, in contrast to what we observed with
compounds cisPt(CA4-glu)(PhB) and cisPt(CA4)(PhB), the
complex in Figure 2A displayed inferior tumor inhibition
compared to both cisplatin and the 1:1 mixture. The compound
in Figure 2B was also previously tested against a SK-OV-3
xenograft model by Huang and collaborators, and in this case,
the complex was somewhat more active than CA4 but less active
than cisplatin or a combination of cisplatin and CA4.25
released in the cancer cell probably makes only a limited
contribution to the killing of the cancer cells. The in vitro data
lead us to suggest that these compounds are not true multi-
action cytotoxic agents but rather prodrugs of CA4.
Although the contribution of platinum to the killing of the
cells in vitro is not clear, the conjugation of CA4 to Pt(IV)
significantly enhanced the in vivo tumor inhibition and reduces
the acute toxicity (weight loss) compared to CA4. Notably, CA4
that is the most potent in vitro is the least efficacious in vivo. The
two most efficacious prodrugs, 3 and 8, inhibit tumor growth by
∼92% compared to cisplatin (84%), CA4 (67%), and 1:1
cisplatin/CA4 (85%). This demonstrates again that the in vitro
cytotoxicity does not necessarily translate to in vivo activity.
If indeed, CA4, rather the cisplatin, is primarily responsible for
killing the cancer cells, what is the role of the platinum moiety?
In this case, and perhaps in other cases of Pt(IV) complexes with
extremely potent bioactive ligands, the main role of Pt(IV) is
merely to act as a self-immolative carrier that is activated by
reduction inside the cancer cell and serves to improve the
efficacy of CA4 and reduce its acute toxicity.
It is interesting to note that the “dual-action” compounds with
either an OH, cisPt(CA4)(OH), or Cl axial ligands (Figure 2)
were less efficacious than cisplatin in vivo, while the two “triple-
action” compounds with the PhB ligand were significantly more
active than cisplatin. Although cisPt(CA4)(OH) and cisPt-
(CA4)(PhB) have nearly identical IC50 values in vitro and
despite the fact that cisPt(CA4)(OH) is significantly more
stable than cisPt(CA4)(PhB) in the cell culture medium,
cisPt(CA4)(PhB) is significantly more active in vivo. This might
be because the “dual-action” prodrugs, with the OH or Cl axial
ligands, are reduced significantly faster than the “triple-action”
prodrugs and perhaps they are reduced in vivo prior to reaching
the tumor. Another possible explanation for the differences in
efficacy and toxicity between the “triple-action” compounds,
cisPt(CA4-glu)(PhB) and cisPt(CA4)(PhB), and cisPt(CA4)-
(OH) might be the ability of the former to interact non-
covalently with albumin via PhB, resulting in higher stability and
bioavailability.
While we usually think of Pt(IV) complexes as prodrugs
whose task is to deliver the cytotoxic Pt(II) drugs (sometimes
accompanied by other bioactive moieties) to the cancer cells, we
may need to adjust our viewpoint and should consider these
compounds not as multi-action prodrugs but as prodrugs that
effectively deliver CA4, rather than cisplatin, to the tumor.
EXPERIMENTAL SECTION
■
General Methods. Oxoplatin,46 4-phenylbuturic anhydride,
valproic anhydride, and octanoic anhydride were prepared by following
the established procedures.47 Combretastatin A4 (from Abosyn
Chemical Inc., USA) and DCA anhydride (from Sigma-Aldrich,
Germany) were commercially purchased.
Characterization. All complexes were characterized by H-, 13C-,
1
and 195Pt-NMR and ESI mass spectrometry and the high purity (>95%)
was confirmed by HPLC measurements (see Figures S36−S44) and
elemental analysis. The obtained data were consistent with the
proposed structures.
All NMR data were recorded on a Bruker AVANCE III HD 500
MHz spectrometer. The data were processed using a MestreNova. 1H
and 13C NMR chemical shifts were referenced with the individual
solvent residual peaks of the respective NMR solvents. 195Pt NMR
chemical shifts were reported with respect to the chemical shift of the
standard K2PtCl4 in water at −1624 ppm. Electrospray ionization mass
spectra (ESI-MS) were done using a Thermo Scientific triple
quadrature mass spectrometer (Quantum Access) by the positive
mode ESI. Elemental analyses were performed using a Thermo
Scientific FLASH 2000 element analyzer. All compounds were
characterized by HPLC on a Thermo Scientific UltiMAte 3000
HPLC using one of the following methods.
Purification by Preparative HPLC. Compounds (3)−(9) were
purified on a Thermo Scientific UltiMate 3000 preparative HPLC
equipped with a RP-18 phase column (Phenomenex Luna, length 250
cm × internal diameter 21.2 mm, particle size 10 μM, and pore size 100
A). Method settings: flow rate 15.0 mL/min, wavelength 220 nm (UV
detector), water−ACN gradient (5 min 100% water to equilibrate, in 15
min from 0 to 100% ACN, 10 min 100% ACN), room temperature.
Method A: LC XB-C18 column (Phenomenex Kinetex, length 250 mm,
internal diameter 4.60 mm, particle size 5 μM, and pore size 100A).
Settings: flow rate 1.0 mL/min, wavelength 220 nm (UV detector),
water−ACN gradient (5 min 100% water to equilibrate, in 15 min from
0 to 100% ACN, and 5 min 100% ACN), and room temperature. Used
for compounds (2)−(7). Method B: LC XB-C18 column (Phenom-
enex Kinetex, length 100 mm, internal diameter 4.60 mm, particle size
2.6 μM, and pore size 100A). Settings: flow rate 1.0 mL/min,
wavelength 220 nm (UV detector), water−ACN gradient (1.84 min
CONCLUSIONS
■
The results of these studies provide us with some interesting
insights into Pt(IV) multi-action prodrugs. The prodrugs
described here differ from most other reported Pt(IV) prodrugs
because the free bioactive moiety (CA4) that is released inside
the cancer cell, with low nanomolar IC50 values, is dramatically
(1000 fold) more cytotoxic than the platinum drug (low
micromolar IC50). All six cisPt(CA4) “multi-action” prodrugs,
regardless of the identity of the second bioactive ligand, have low
nanomolar IC50 values against seven out of the eight cancer cell
lines, suggesting that CA4 is the dominant factor in triggering
cell death, raising doubts whether platinum makes any
significant contribution toward cell death. Comparison of the
cytotoxicity data to that of similar compounds that were
previously reported suggests that the nature of the link between
the OH of CA4 and Pt(IV) significantly affects the
pharmacological properties of the prodrugs. Comparing the
IC50 values of the compounds against a pair of cancer cells lines
proficient and deficient in DNA repair (NER), we found that
cisplatin and oxoplatin were about 9-fold more potent against
cells deficient in DNA repair, while the cisPt(CA4) prodrugs
were only about 4-fold more potent. This, in conjunction with
our inability to detect by ICPMS Pt−DNA adducts in the
nucleus, suggests that the small amount of cisplatin that is
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100% water to equilibrate, in 5.84 min from 0 to 100% ACN, 2 min
100% ACN), and room temperature. Used for compounds (8)−(10).
Synthesis of Carbonate Compounds (2)−(7).
3.65 (s, 3H, −OCH3), 3.61 (s, 6H, −OCH3). 13C NMR (126 MHz,
DMSO-d6): δ 157.95, 153.04, 150.94, 141.69, 137.19, 132.47, 129.50,
129.43, 129.10, 126.20, 123.89, 112.86, 106.43, 60.53, 56.14, 56.09.
195Pt NMR (108 MHz, DMSO-d6): δ 1058.53. Elemental analysis for
C19H26Cl2N2O8Pt·*H2O calcd/found: (C 32.86/32.98 H 4.06/3.98 N
4.03/3.83). MS-ESI (positive mode): m/z [M + H]+ 676.93, [M + Na]+
696.87.
(i) 500.0 mg of CA4 (1) (1.581 mmol), 809.5 mg of DSC (3.161
mmol), and 183.4 mg of DMAP (1.502 mmol) were stirred at
room temperature in 20 mL of DCM for 3 h and monitored by
HPLC. Afterward, the solution was washed three times with 20
mL of distilled water and dried by addition of Na2SO4, filtered,
and evaporated to dryness under reduced pressure at room
temperature. The activated CA4 (CA4-DSC) was isolated as a
fluffy white solid, yielding 90.3%. It was used without any further
purification. HPLC purity was > 95%. The product was stored at
−4 °C.
(ii) 300.0 mg of CA4-DSC (0.656 mmol) and 175.3 mg of oxoplatin
(0.525 mmol) were stirred at 35 °C in 25 mL dimethylforma-
mide (DMF) for 16 h. The reaction was monitored by platinum
NMR. After the complete conversion to cisPt(CA4)(OH) (2),
DMF was evaporated under reduced pressure at 35 °C. The
yellow residue was dissolved in 2 mL of MeOH, and 48 mL of
diethyl ether was added to precipitate the pure product. After
centrifugation, diethyl ether was decanted, the product was
washed again twice with 10 mL of diethyl ether, and dried at
room temperature under reduced pressure, yielding 84.8% of
cisPt(CA4)(OH) (2) as a pure pale yellow solid. The product
was stored at −4 °C.
cisPt(CA4)(PhB) (3) 1H NMR (500 MHz, DMSO-d6): δ 7.28 (t, J
= 7.5 Hz, 2H, −CH), 7.19 (m, 3H, −CH), 7.09 (d, J = 8.2 Hz, 2H,
−CH), 7.01 (d, J = 8.2 Hz, 1H, −CH), 6.56 (s, 2H, −CH), 6.47 (m, 2H,
−CH), 3.76 (s, 3H, −OCH3), 3.65 (s, 3H, −OCH3), 3.61 (s, 6H,
−OCH3), 2.60 (t, J = 7.6 Hz, 2H, −CH2), 2.25 (t, J = 7.4 Hz, 2H,
−CH2), 1.75 (p, J = 7.5 Hz, 2H, −CH2). 13C NMR (126 MHz, DMSO-
d6): δ 207.04, 180.80, 157.60, 153.05, 150.79, 142.42, 141.10, 137.20,
132.42, 129.57, 128.95, 128.70, 126.69, 126.17, 123.65, 112.99, 106.41,
60.54, 56.20, 56.09, 35.02, 32.02, 28.75. 195Pt NMR (108 MHz, DMSO-
d6): δ 1233.89. Elemental analysis for C29H36Cl2N2O9Pt calcd/found:
(C 42.34/42.36 H 4.41/4.55 N 3.41/3.11). MS-ESI (positive mode):
m/z [M + H]+ 822.95, [M + NH4]+ 839.74.
cisPt(CA4)(CA4) (4) 1H NMR (500 MHz, DMSO-d6): δ 7.10 (m,
4H −CH), 7.03 (d, J = 8.4 Hz, 2H −CH), 6.56 (s, 4H −CH), 6.47 (q,
4H, −CH), 3.76 (s, 6H −OCH3), 3.65 (s, 6H −OCH3), 3.61 (s, 12H
−OCH3).13C NMR (126 MHz, DMSO-d6): δ 157.38, 153.06, 150.70,
141.00, 137.22, 132.40, 129.65, 129.60, 128.93, 126.75, 123.70, 113.02,
106.43, 60.53, 56.22, 56.09. 195Pt NMR (108 MHz, DMSO-d6): δ
1256.75. Elemental analysis for C38H44Cl2N2O14Pt calcd/found: (C
44.80/44.29 H 4.35/4.40 N 2.75/2.48). MS-ESI (positive mode): m/z
[M + Na]+ 1040.85.
(iii) 80.0 mg of cisPt(CA4)(OH) (2) (0.118 mmol) and either CA4-
DSC or the respective anhydrides (0.473 mmol) were stirred in
2 mL of DMF at room temperature for 24 h up to 5 days. The
reaction was monitored by platinum NMR and HPLC. After the
complete conversion, DMF was evaporated and the pure
products (3)−(7) were obtained as a solid after purification by
preparative HPLC. The products were stored at −4 °C.
1
cisPt(CA4)(DCA) (5) H NMR (500 MHz, DMSO-d6): δ ppm
7.11 (m, 2H−CH), 7.03 (d, J = 8.3 Hz, 1H−CH), 6.53 (s, 2H−CH),
6.47 (q, 2H, −CH), 3.76 (s, 6H −OCH3), 3.65 (s, 6H −OCH3), 3.61
(s, 12H −OCH3). 13C NMR (126 MHz, DMSO-d6): δ ppm 170.87,
157.46, 153.06, 150.71, 140.98, 137.22, 132.40, 129.64, 129.60, 128.92,
126.79, 123.67, 113.02, 106.46, 65.60, 60.54, 56.22, 56.09. 195Pt NMR
(108 MHz, DMSO-d6): δ ppm 1230.24. Elemental analysis for
C21H26Cl4N2O9Pt calcd/found: (C 32.04/32.09 H 3.33/3.30 N
3.56/3.29). MS-ESI (positive mode): m/z [M + H]+ 786.74, [M +
NH4]+, 803.39, [M + Na]+ 808.72.
Synthesis of Carboxylate Compounds (8)−(10).
(iv) 200.0 mg of CA4 (0.634 mmol) was stirred with glutaric
anhydride (0.951 mmol) and TEA (1.903 mmol) in 5 mL of
DCM at room temperature for 2 h. The reaction was monitored
by thin-layer chromatography (2% MeOH in DCM: Rf(CA4)
=
1
0.5, Rf(CA4‑glutaric) = 0.17). After the complete conversion, the
compound was isolated by a separating funnel with DCM and
water (adjusted with hydrochloric acid to pH 2). The organic
phases were combined, dried over Na2SO4, filtered, and
evaporated at room temperature. The desired compound was
obtained as an oil, which was washed with hexane for 1 h.
Hexane was decanted and the white solid residue was washed
twice more with hexane. CA4-glu (8) was suspended in distilled
water and dried by lyophilization overnight and a fluffy white
solid was obtained, yielding 92.6%.
cisPt(CA4)(OCT) (6) H NMR (500 MHz, DMSO-d6): δ ppm
7.08 (m, 2H −CH), 7.01 (d, J = 8.3 Hz, 1H −CH), 6.56 (s, 2H −CH),
6.48 (q, 4H, −CH), 3.76 (s, 6H −OCH3), 3.65 (s, 6H −OCH3), 3.61
(s, 12H −OCH3) 2.24 (t, J = 7.5 Hz, 2H −CH2) 1.46 (m, 2H −CH2),
1.25 (m, 8H −CH2). 0.87 (t, J = 6.8 Hz, 3H −CH3). 13C NMR (126
MHz, DMSO-d6): δ 181.14, 157.59, 153.06, 150.80, 141.11, 137.21,
132.42, 129.58, 128.96, 126.66, 123.68, 112.99, 106.42, 60.53, 56.20,
56.09, 35.67, 31.66, 29.49, 29.02, 25.88, 22.56, 14.45. 195Pt NMR (108
MHz, DMSO-d6): δ ppm 1235.96. Elemental analysis for
C27H40Cl2N2O9Pt calcd/found: (C 40.41/40.76 H 5.02/5.11 N
3.49/3.41). MS-ESI (positive mode): m/z [M + H]+ 802.70, [M +
Na]+ 824.96, [M + K]+ 840.78.
(v) 200 mg of CA4-glu (0.465 mmol) was activated by the addition
of EDC (0.558 mmol) and NHS (0.558 mmol) and stirred in 2
mL of DMF for 4 h at room temperature. The reaction was
monitored by HPLC. After the complete conversion of CA4-glu
to CA4-glu-DSC, DMF was evaporated, and the product was
purified by preparative HPLC, yielding 91.2%.
(vi) 95 mg of CA4-glu-DSC (0.180 mmol) was stirred with oxoplatin
(0.144 mmol) in 15 mL of DMF for 16 h. The reaction was
monitored by 195Pt NMR. After the complete conversion, DMF
was evaporated and the product cisPt(CA4-glu)(OH) (9) was
purified by preparative HPLC and dried by lyophilization. A
pure white solid was obtained with a yield of 33.5%.
(vii) 24 mg of cisPt(CA4-glu)(OH) (0.032 mmol) was dissolved in 3
mL of DMF and phenyl butyric anhydride (0.129 mmol) was
added. The reaction was stirred for 16 h at room temperature.
Afterward, the solvent was removed under reduced pressure.
Methanol was added, and the final complex was purified by
preparative HPLC and dried by lyophilization. A pure white
solid (10) was gained, yielding 61%.
cisPt(CA4)(OH) (2) 1H NMR (500 MHz, DMSO-d6): δ 7.06 (m,
2H, −CH), 6.99 (d, J = 8.4 Hz, 1H, −CH), 6.57 (s, 2H, −CH), 6.47 (q,
J = 12.3 Hz, 2H, −CH), 5.96 (m, 6H, −NH3), 3.75 (s, 3H, −OCH3),
cisPt(CA4)(VAL) (7) 1H NMR (500 MHz, DMSO-d6): δ 7.09 (m,
2H, −CH), 7.02 (d, J = 8.5 Hz, 1H, −CH), 6.57 (s, 2H, −CH), 6.48 (q,
J = 10.5 Hz, 2H, −CH), 3.76 (s, 3H, −OCH3), 3.65 (s, 3H, −OCH3),
3.61 (s, 6H, −OCH3), 2.81 (s, 1H, −CH), 1.38 (m, 8H −CH2), 0.84 (t,
6H, −CH3). 13C NMR (126 MHz, DMSO-d6): δ 184.41, 157.51,
153.04, 150.94, 141.69, 137.21, 132.42, 129.60, 129.59, 128.98, 126.60,
123.76, 112.98, 106.43, 60.53, 56.20, 56.09, 46.59, 42.57, 35.12, 34.60,
25.95, 20.47, 14.63.195Pt NMR (108 MHz, DMSO-d6): δ 1240.19.
Elemental analysis for C27H40Cl2N2O9Pt·EtOH calcd/found: (C
41.04/41.46 H 5.46/5.23 N 3.30/3.39). MS-ESI (positive mode): m/
z [M + H]+ 802.84, [M + Na]+ 824.96.
CA4-glu (8) 1H NMR (500 MHz, DMSO-d6): δ 7.15 (dd, J = 8.4, 2.1
Hz, 1H, −CH), 7.06 (d, J = 8.5, 1H, −CH), 6.99 (d, J = 2.1 Hz, 1H,
−CH), 6.54 (s, 2H, −CH), 6.50 (q, 2H, −CH), 3.74 (s, 3H, −OCH3),
3.64 (s, 3H, −OCH3), 3.60 (s, 6H, −OCH3), 2.56 (t, J = 7.3 Hz, 2H,
−CH2), 2.32 (t, J = 7.4 Hz, 2H, −CH2), 1.81 (p, J = 7.5 Hz, 2H, −CH2).
13C NMR (126 MHz, DMSO-d6): δ 173.99, 170.74, 152.63, 149.99,
138.97, 136.78, 131.97, 129.52, 129.39, 128.27, 127.39, 122.67, 112.65,
105.89, 60.05, 55.85, 55.55, 32.38, 32.32, 20.00. Elemental analysis for
C23H26O8 calcd/found: (C 64.18/63.98 H 6.09/6.20). MS-ESI
K
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(positive mode): m/z [M + H]+ 431.10, [M + NH4]+ 448.11, [M +
Na]+ 453.08, [M + K]+ 469.18.
supplemented with gentamycin (50 mg/L) and fetal bovine serum
superior, standardized (Biochrom GmbH, Berlin) (10% v/v), and were
passaged once a week. RC-124 human kidney cells were maintained in
McCoy’s 5A (modified, with L-glutamine) medium (Life Technolo-
gies), which was supplemented with gentamycin (50 mg/L) and fetal
bovine serum superior, standardized (Biochrom GmbH, Berlin) (10%
v/v), and also passaged once a week. For experiments with RC-124
cells, microtiter plates had been pretreated in the following way: 30 mL
of a sterilized gelatin solution [1.5% (m/V)] was added to each well of
flat-bottom 96-well plates, the plates were covered with their lids,
incubated for 1 h at 37 °C, the excess solution was removed, and the
wells were washed once with phosphate-buffered saline (PBS) (140
mM NaCl, 3 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4; pH 7.4),
and the new cell culture medium was added. HT-29, MCF-7, SK-HEP-
1, and RC-124 cells were obtained from Cell Line Service (CLS,
Eppelheim, Germany) and MDA-MB-231 cells from the Helmholtz
Institute for Infection Research (HZI, Braunschweig, Germany).
A375 human malignant melanoma cells, A2780 human ovarian
carcinoma cells, and A2780cis cisplatin-resistant ovarian carcinoma
cells were cultured in RPMI 1640 (no glutamine) supplemented with
heat-inactivated fetal bovine serum (South American origin) (10% v/
v), gentamycin sulfate solution (1% v/v), and L-glutamine solution 200
nM (1% v/v). PC9 human non-small cell lung carcinoma cells were
maintained in DMEM (high glucose, no glutamine/sodium pyruvate)
supplemented with L-glutamine solution (200 nM) (1% v/v), 10% (v/
v) heat-inactivated fetal bovine serum (South American origin,
European grade), and 1% (v/v) penicillin−streptomycin solution
(containing penicillin G sodium salt: 10,000 units/mL and
streptomycin sulfate: 10 mg/mL) and were passaged twice a week.
Once a week, a solution of cisplatin in RPMI medium (end
concentration: 1 μM) was added to A2780cis cells to maintain the
resistance. All reagents were purchased at Biological Industries. A375,
PC9, A2780, and A2780cis cells were obtained from the American Type
Culture Collection (ATCC, Rockville, MD).
Antiproliferative Assay in Tumorigenic and Non-tumorigenic
Cells. The antiproliferative effects were determined according to a
recently used method with minor modifications.15,48 In short: SK-HEP-
1 cells, HT-29 cells, MDA-MB-231 cells, MCF-7 cells, or RC-124 cells
were transferred into each well of a flat-bottom 96-well microtiter plate
(note: for RC-124, pretreated plates were used, see above) and
incubated at 37 °C/5% CO2 for either 72 h. Stock solutions of the
compounds in cell culture medium (cisplatin as reference) or DMSO
(compounds (1)−(9)) were freshly prepared and diluted with the
respective cell culture medium to graded concentrations (a final
concentration of DMSO: 0.1% v/v). After 72 h of exposure, the cell
biomass was determined by crystal violet (Sigma-Aldrich) staining at a
wavelength of 590 nm.
A357 melanoma cells (4000 cells/well), A2780 ovarian carcinoma
cells (5000 cells/well), A2780cis cisplatin-resistant ovarian carcinoma
cells (7000 cells/well), and PC9 small lung cell carcinoma cells (4000
cells/well) were seeded in flat-bottom 96-well plates and incubated at
37 °C/5% CO2 for 24 h. Stock solutions of the compounds in DMSO
(compounds (1)−(9)) or cell culture medium (cisplatin as reference)
were freshly prepared and diluted with the respective cell culture
medium to graded concentrations (final concentration of DMSO: 0.1%
v/v). After 72 h of exposure, the cell biomass was determined by 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)
(Sigma-Aldrich) staining, and the absorptions were calculated as the
delta of two measurements of the same well at wavelengths 570 and 690
nm.
cisPt(CA4-glu)(OH) (9) 1H NMR (500 MHz, DMSO-d6): δ 7.14
(dd, 1H, −CH), 7.06 (d, 1H, −CH), 6.98 (dd, 1H, −CH), 6.54 (s, 2H,
−CH), 6.50 (q, 2H, −CH), 5.93 (m, 6H, −NH3), 3.75 (s, 3H,
−OCH3), 3.64 (s, 3H, −OCH3), 3.60 (s, 6H, −OCH3), 2.56 (t, 2H,
−CH2), 2.27 (t, 2H, −CH2), 1.77 (p, 2H, −CH2). 13C NMR (126
MHz, DMSO-d6): δ 180.64, 171.58, 153.08, 150.53, 139.53, 137.22,
132.44, 129.96, 129.83, 128.78, 127.75, 123.18, 113.13, 106.37, 60.56,
56.40, 56.04, 35.66, 33.10, 21.58. 195Pt NMR (108 MHz, DMSO-d6): δ
1037.72. Elemental analysis for C23H32Cl2N2O9Pt calcd/found: (C
37.01/37.35 H 4.32/4.02 N 3.75/3.32). MS-ESI (positive mode): m/z
[M + H]+ 746.19, [M + Na]+ 768.10, [M + K]+ 784.06.
cisPt(CA4-glu)(PhB) (10) 1H NMR (500 MHz, DMSO-d6): δ
7.33−7.03 (m, 8H), 6.98 (d, J = 2.2 Hz, 1H), 6.67−6.40 (m, 9H), 3.75
(d, J = 5.7 Hz, 3H), 3.64 (d, J = 1.7 Hz, 3H), 3.61 (d, J = 3.2 Hz, 6H),
2.59 (dt, J = 9.4, J = 7.4 Hz, 4H), 2.35 (t, J = 7.3 Hz, 2H), 2.23 (t, J = 7.3
Hz, 2H), 1.77 (dt, J = 15.0, 7.4 Hz, 4H). 13C NMR (126 MHz, DMSO-
d6): δ 181.10, 180.49, 171.48, 153.08, 150.51, 142.47, 139.50, 137.22,
132.44, 129.97, 129.85, 128.95, 128.77, 128.69, 127.78, 126.15, 123.16,
113.14, 106.37, 60.56, 56.40, 56.04, 48.40, 35.54, 34.91, 32.92, 27.99,
21.35. 195Pt NMR (108 MHz, DMSO-d6): δ 1218.45 Elemental analysis
for C33H42Cl2N2O10Pt calcd/found: (C 44.40/44.18 H 4.74/5.08 N
3.14/3.18). MS-ESI (positive mode): m/z [M + H]+ 892.96, [M + Na]+
914.79, [M + K]+ 731.06.
Stability Studies by RP-HPLC. HPLC studies were performed on a
Thermo Scientific UltiMAte 3000 HPLC. Preparation for ACN/
medium and buffer/medium: 500 μL of a stock solution, containing the
respective compound in acetonitrile (1.2 μM), was prepared and 500
μL of either RPMI cell culture medium (without any supplements) or
phosphate buffer (50 mM, pH 7.0) was added. Preparation for DMSO/
medium: 10 μL of DMSO was added to compounds, and after they
were solubilized, 990 μL of 1640 RPMI were added to achieve a final
concentration of 1% DMSO. After appropriate mixing, the samples
were filtered through a PreTech syringe Nylon filter (13 mm, 0.22 μm),
stored at 37 °C, and injected several times until the decomposition was
completed, and the test complexes were hydrolyzed more than 50%.
Method C: LC XB-C18 column (Phenomenex Kinetex, length 250 mm,
internal diameter 4.60 mm, particle size 5 μM, and pore size 100A).
Settings: flow rate 1.0 mL/min, wavelength 220 nm (UV detector),
water−ACN gradient (2 min 100% water to equilibrate, in 15 min from
0 to 100% ACN, 3 min 100% ACN), and autosampler temperature 37
°C. Used for compounds (2)−(5), (7). Method D: LC XB-C18
column (Phenomenex Kinetex, length 100 mm, internal diameter 4.60
mm, particle size 2.6 μM, and pore size 100A). Settings: flow rate 1.0
mL/min, wavelength 220 nm (UV detector), water−ACN gradient
(1.84 min 100% water to equilibrate, in 5.84 min from 0 to 100% ACN,
2 min 100% ACN), and autosampler temperature 37 °C. Used for
compounds (6) and (8)−(10).
Reduction Studies by RP-HPLC and 1H NMR. RP-HPLC: 500 μL of
a stock solution, containing the respective compound in acetonitrile
(1.2 μM), was prepared and 500 μL of an ascorbic acid solution (12
μM) of phosphate buffer (50 mM, pH 7.0) was added. After
appropriate mixing, the samples were filtered through a PreTech
syringe Nylon filter (13 mm, 0.22 μm), stored at 37 °C, and injected
several times until the reduction was completed, and the test complexes
were fully reduced. Method C: used for compounds (2)−(5), (7).
Method D: used for compounds (6), (8)−(10). 1H NMR: 1 mg of the
respective complexes cisPt(CA4)(OH) and cisPt(CA4-glu)(OH) was
dissolved in 750 μL of DMSO-d6 and 1H NMR were recorded.
Afterward, 10 equiv of ascorbic acid was added to each same sample
tube, mixed carefully to ensure that all reagents are completely
dissolved, and the reduction of each complex was monitored up to 24 h
(every 5 min for cisPt(CA4)(OH) and every hour for cisPt(CA4-
glu)(OH)).
The IC50 values were determined as the concentration that caused
50% inhibition of cell proliferation compared to an untreated control.
The results were calculated as the mean values of three independent
experiments.
Protein Binding Studies with FCS Proteins. Protein binding of the
platinum complexes was studied using a modified precipitation assay
reported earlier by Ma et al.49 FCS was chosen as the representative
protein since it is used in all the cell culture experiments. Cell culture
medium (DMEM) was reconstituted with 10% FCS. The CA4−
platinum complexes were dissolved in DMSO to prepare a stock
Cell Culture. Maintenance: HT-29 human colon carcinoma cells,
MDA-MB-231 human breast cancer cells, MCF-7 human breast
carcinoma cells, and SK-HEP-1 human liver adenocarcinoma cells were
maintained in Dulbecco’s modified Eagle’s medium (4.5 g/L (D)-
glucose, L-glutamine, and pyruvate) from Life Technologies, which was
L
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solution (10 mM), and cisplatin was dissolved in Millipore water and
diluted to the same concentration. Each stock solution was added to the
reconstituted medium such that the final concentration was 50 μM in a
total volume of 5 mL. They were incubated at 37 °C for different time
points (0, 2, 4, 8, and 24 h). Untreated reconstituted media was treated
identically as the other samples and used as the matrix for calibration.
Aliquots (500 μL) of each sample were treated with ice-cold ethanol (1
mL) and stored at −25 °C for 3 h. Thereafter, the samples were
centrifuged (964 g at 4 °C for 30 min). The supernatant was isolated
and stored at −25 °C for the final measurements. An aliquot (500 μL)
of each sample was treated with 10% nitric acid (4.5 mL) and the
samples were further diluted using Millipore water. For calibration, a
standard solution of platinum (1.0 ppm Pt standard solution procured
from Sigma-Aldrich, ICP grade) was added in graded concentrations to
the untreated sample. The platinum in the samples was measured using
ICP optical emission spectroscopy (Agilent Technologies ICP−OES,
5110) equipped with a SPS4 autosampler. The values reported are the
average of three independent measurements.
Cellular Uptake Studies in MDA-MB-231 Cancer Cells. Cellular
accumulations of studied complexes were determined in highly invasive
human breast carcinoma cells MDA-MB-231. In these experiments, 1 ×
106 MDA-MB-231 cells were seeded on 100 mm Petri dishes. After
overnight pre-incubation in a drug-free medium at 37 °C in a 5% CO2
humidified atmosphere, the cells were treated with platinum
compounds and allowed a further 5 h of drug exposure under similar
conditions. After treatment, the cells were extensively washed with PBS
(37 °C), detached by using 0.25% trypsin, washed twice with ice-cold
PBS, and counted. The cell pellets were stored at −70 °C and digested
using a microwave acid (HCl) digestion system (CEM Mars). The
quantity of metal taken up by the cells was determined by ICP−MS
using Agilent 7500 (Agilent, Japan). All experiments were carried out in
triplicate.
The amount of platinum bound to nucleic acids was quantified by ICP−
MS. All measurements were done in triplicate.
To determine the amount of platinum associated with DNA, the cells
were prepared and treated similarly to cellular uptake studies. Following
the treatment, the cells were detached with 0.25% trypsin and
extensively washed, and the pellets were lysed using DNAzol (DNAzol
genomic DNA isolation reagent, MRC) supplemented with RNase A
(100 μg mL−1). DNA was precipitated with ethanol, washed with 70%
ethanol, and resolved in H2O. DNA amount was determined
spectrophotometrically and Pt content was measured with ICP−MS.
The experiment was performed in triplicate.
Antiproliferative Activity of Pt-CA4 Compounds in CHO-K1 and
MMC-2 Cells. Chinese hamster ovary CHO-K1 cell line (wild type) and
its mutant cell line MMC-2 were kindly supplied by Dr. M. Pirsel
(Cancer Research Institute, Slovak Academy of Sciences, Bratislava,
Slovakia). The CHO-K1 and MMC-2 cells were grown in Dulbecco’s
modified Eagle’s medium (DMEM, high glucose 4.5 g L−1, PAA,
Pasching, Austria) supplemented with gentamycin (50 mg mL−1,
Serva) and 10% heat-inactivated fetal bovine serum (PAA, Pasching,
Austria). The cells were cultured in a humidified incubator at 37 °C in a
5% CO2 atmosphere and subcultured two to three times a week with an
appropriate plating density. The cells were seeded in 96-well plates at a
density of 3 × 103 cells/well in 100 μL medium. Following overnight
incubation, the cells were treated with a series of concentrations of the
investigated compounds in a final volume of 200 μL for 72 h. At the end
of the treatment, 20 μL of the tetrazolium compound MTT solution
(Calbiochem, Darmstadt, Germany) (1.25 mg mL−1 in PBS) was
added. After another 2−4 h, the medium was removed, and the
resulting formazan product was dissolved in 100 μL DMSO.
Absorbance was measured at 570 nm (ref. 620 nm) with an absorbance
reader (SUNRISE TECAN, 1086 SCHOELLER). The IC50 values
were determined as the drug concentration corresponding to 50%
inhibition of the control signal.
Analysis of Tubulin Polymerization in MDA-MB-231 Cells. MDA-
MB-231 cells were seeded in 60 mm Petri dishes (3 × 105 cells/well)
and grown overnight. Following the 8 h treatment at the indicated
concentrations, the cells were harvested by trypsinization, washed, and
pelleted. Subsequently, the cells were fixed in 1 mL of microtubule-
stabilizing buffer (80 mM pipes, pH 6.8, 5 mM ethylenediaminetetra-
acetic acid, 1 mM MgCl2, 0.5% Triton X-100, and 0.5% glutaraldehyde)
for 10 min at room temperature. Glutaraldehyde was then quenched
with 0.7 mL of NaBH4 in PBS. The cells were pelleted and resuspended
in antibody dilution solution (PBS, pH 7.4, 2% BSA 0.2% Triton X-100,
0.1% NaN3). After a 3 h incubation with anti-α-tubulin-FITC antibody
(mouse monoclonal; Sigma-Aldrich, Prague; 1:250 dilution), the cells
were analyzed by a FACSVerse flow cytometer (Becton Dickinson,
Germany) and data analyzed using ModFit LT 4.1 software (Verity
Software House).
Tubulin Polymerization Inhibition in a Cell-Free Medium.
Fluorescence-based tubulin polymerization assay kit (porcine tubulin;
Cytoskeleton, Inc.) was used to analyze the influence of the tested
compounds on tubulin polymerization in vitro. The experiment was
performed following the manufacturer’s protocol. Briefly, 2 mg mL−1
tubulin in tubulin glycerol buffer (80 mM pipes; 2 mM MgCl2 0.5 mM
EGTA, pH 6.9; 1 mM GTP; 15% glycerol; and 10 μM fluorescent
reporter) was polymerized in the absence or presence of the tested
compounds (1, 5, and 10 μM) at 37 °C for 40 min. Fluorescence signal
(EX 350 nm/EM 450 nm) was read once per minute using a Varian
Cary Eclipse spectrofluorophotometer.
Quantification of Platinum Bound to DNA Isolated from MDA-
MB-231 Cells. MDA-MB-231 cells were treated with the tested
compounds at a concentration of 0.1 and 1.0 μM for 5 h or 0.01 μM for
24 h at 37 °C. The cell pellets were stored at −70 °C and then lysed in
DNAzol (DNAzol, MRC) supplemented with RNase A (100 μg mL−1).
Genomic DNA was precipitated from the lysate by 100% ethanol,
washed twice with 75% ethanol, and resuspended in 8 mM NaOH. The
DNA content in each sample was determined by UV spectropho-
tometry. To avoid the affection of high DNA concentration on the
detection of platinum in the samples, the DNA samples were digested in
the presence of 30% hydrochloric acid (Suprapur, Merck Millipore).
Microtubule Imaging. MDA-MB-231 cells were grown on cover-
slips in six-well plates and treated with 1 μM compounds (20 μM in the
case of oxoplatin) for 2 h. The cells were washed with PBS, fixed with
4% p-formaldehyde, and processed for immunostaining: permeabiliza-
tion with 0.1% Tween-20 in PBS (PBST), blocking with 10% normal
donkey serum in PBST, incubation with primary antibody (anti-β I
tubulin antibody; Abcam ab179511; 1:500) for 2 h, washed, incubated
with secondary antibody (donkey anti-rabbit IgG Alexa Fluor 647;
Abcam ab150075; 1:200) for 1 h, washed, and mounted with ProLong
Diamond antifade with DAPI. The images were obtained with the laser-
scanning microscope Leica TCS SP8 SMD (Leica Microsystems
BmbH, Wetzlar, Germany).
Cell Cycle Analysis. MDA-MB-231 cells were seeded in six-well
plates (6 × 105 cells/well), and following an overnight incubation, they
were treated with tested compounds at concentrations corresponding
to IC50 values (obtained with the MTT assay after a 72 h treatment).
After a 24 h treatment, the cells were washed, detached with trypsin,
washed twice with PBS, pelleted by centrifugation, and resuspended in
70% ethanol. Following a 1 h fixation, the cells were washed with PBS
(2×) and stained with propidium iodide (50 μg mL−1 in Vindel’s
solution10 mM Tris-Cl (pH = 8.0), 10 mM NaCl, 0.1% Triton X-
100, 100 μg mL−1 RNase A) for 30 min at 37 °C. Cell cycle profiles were
obtained with an FACSVerse flow cytometer (Becton Dickinson,
Germany), and the data were analyzed with the ModFit LT 4.1 (Verity
Software House). The experiment was performed three times.
In Vivo Antitumor Activity in C57BL Lewis Lung Cell Carcinoma
Mice. In vivo anticancer activity toward LLC. All studies involving
animal testing were carried out in accordance with ethical guidelines for
animal research acknowledging the European Directive 2010/63/UE as
to the animal welfare and protection and the related codes of practice
(approval 640/2016-PR). The mice were purchased from Charles
River, housed in steel cages under controlled environmental conditions
(constant temperature, humidity, and 12 h dark/light cycle), and
alimented with commercial standard feed and tap water ad libitum. The
LLC cell line was purchased from ECACC, United Kingdom. The LLC
cell line was maintained in DMEM (Euroclone) supplemented with
10% heat inactivated fetal bovine serum (Euroclone), 10 mM L−1
M
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glutamine, 100 U mL−1 penicillin, and 100 μg/mL streptomycin in a 5%
CO2 air incubator at 37 °C. The LLC was implanted intramuscularly
(i.m.) as a 2 × 106 cell inoculum into the right hind leg of 8 week-old
male C57BL mice (24 3 g body weight). After 7 days from tumor
implantation (visible tumors), mice were randomly divided into eight
groups (five animals per group) and subjected to daily i.p.
administration. Control mice received the vehicle [0.5% DMSO (v/
v) and 99.5% of a saline solution (v/v)], whereas treated groups
received daily doses of animals received daily oral gavage of 20 mg kg−1
of Pt(CA4)(PhB), Pt(CA4)(OH), or cisPt(CA4-glu)(OH), daily i.p.
of 3 mg kg−1 of cisplatin, 20 mg kg−1 of CA4 or PhB, and a combination
of 3 mg kg−1 of cisplatin, CA4, and PhB. At day 15, animals were
sacrificed, the legs were amputated at the proximal end of the femur,
and the inhibition of tumor growth was determined from the difference
in the weights of the tumor-bearing leg and the healthy leg of the
animals, expressed as a percentage referenced to the control animals.
Body weight was measured at day 0 and from day 7 onward every 2 days
and was taken as a parameter for systemic toxicity. All presented values
are the means SD of no less than three measurements.
Author Contributions
#C.S. and T.B. contributed equally to this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
D.G. would like to acknowledge the support of the Israel Science
Foundation (grant 1002/18). This research was also funded by
the ICA-USA board member Bruce Youngman (D.G.). H.K. and
V.B. acknowledge the support of the Czech Science Foundation
(grant 21-27514S). We would like to acknowledge Dr. Gourab
Dey of Prof. Galia Blum’s research group at the Hebrew
University of Jerusalem for helping with MS measurement and
Petra Lippmann from I.O.’s lab at the Technische University of
Braunschweig for supporting us with the crystal violet assay.
REFERENCES
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ASSOCIATED CONTENT
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00706.
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NMR and ESIMS spectra of the compounds as well as the
HPLC chromatograms of all compounds (PDF)
Molecular formula strings of all the tested compounds
(CSV)
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AUTHOR INFORMATION
■
(5) Pabla, N.; Dong, Z. Cisplatin Nephrotoxicity: Mechanisms and
Renoprotective Strategies. Kidney Int. 2008, 73, 994−1007.
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Corresponding Authors
Ingo Ott − Institute of Medicinal and Pharmaceutical
Chemistry, Technische Universität Braunschweig, 38106
Braunschweig, Germany; Email: ingoott@tu-
braunschweig.de
Valentina Gandin − Dipartimento di Scienze del Farmaco,
Universita di Padova, 35131 Padova, Italy;
Email: valentina.gandin@unipd.it
Viktor Brabec − Institute of Biophysics, Czech Academy of
Sciences, 61265 Brno, Czech Republic; Department of
Biophysics, Faculty of Science, Palacky University in Olomouc,
78371 Olomouc, Czech Republic; orcid.org/0000-0002-
8233-1393; Email: brabec@ibp.cz
Dan Gibson − Institute for Drug Research, School of Pharmacy,
The Hebrew University, 91120 Jerusalem, Israel;
orcid.org/0000-0002-1631-4018; Email: dang@
ekmd.huji.ac.il
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Oncology Group Study. J. Clin. Oncol. 1999, 17, 2676.
(8) Yang, Y.-F.; Wang, G.-Y.; He, J.-L.; Wu, F.-P.; Zhang, Y.-N. Overall
Survival of Patients with KRAS Wild-Type Tumor Treated with
FOLFOX/FORFIRI+/-Cetuximab as the First-Line Treatment for
Metastatic Colorectal Cancer: A Meta-Analysis. Medicine 2017, 96,
No. e6335.
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FOLFIRINOX - A New Paradigm in the Treatment of Pancreatic
Cancer. Expert Rev. Anticancer Ther. 2014, 14, 1115−1125.
(10) Wexselblatt, E.; Gibson, D. What Do We Know About the
Reduction of Pt(IV) Pro-Drugs? J. Inorg. Biochem. 2012, 117, 220−229.
(11) Gibson, D. Multi-action Pt(IV) Anticancer Agents; Do We
Understand How They Work? J. Inorg. Biochem. 2019, 191, 77−84.
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(14) Raveendran, R.; Braude, J. P.; Wexselblatt, E.; Novohradsky, V.;
Stuchlikova, O.; Brabec, V.; Gandin, V.; Gibson, D. Pt(IV) Derivatives
of Cisplatin and Oxaliplatin with Phenylbutyrate Axial Ligands are
Potent Cytotoxic Agents that Act by Several Mechanisms of Action.
Chem. Sci. 2016, 7, 2381−2391.
(15) Babu, T.; Sarkar, A.; Karmakar, S.; Schmidt, C.; Gibson, D.
Multiaction Pt(IV) Carbamate Complexes Can Codeliver Pt(II) Drugs
and Amine Containing Bioactive Molecules. Inorg. Chem. 2020, 59,
5182−5193.
(16) Petruzzella, E.; Sirota, R.; Solazzo, I.; Gandin, V.; Gibson, D.
Triple Action Pt(IV) Derivatives of Cisplatin: a New Class of Potent
Authors
Claudia Schmidt − Institute for Drug Research, School of
Pharmacy, The Hebrew University, 91120 Jerusalem, Israel
Tomer Babu − Institute for Drug Research, School of Pharmacy,
The Hebrew University, 91120 Jerusalem, Israel
Hana Kostrhunova − Institute of Biophysics, Czech Academy of
Sciences, 61265 Brno, Czech Republic
Annika Timm − Institute of Medicinal and Pharmaceutical
Chemistry, Technische Universität Braunschweig, 38106
Braunschweig, Germany
Uttara Basu − Institute of Medicinal and Pharmaceutical
Chemistry, Technische Universität Braunschweig, 38106
Braunschweig, Germany
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.1c00706
N
https://doi.org/10.1021/acs.jmedchem.1c00706
J. Med. Chem. XXXX, XXX, XXX−XXX

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