Synthesis and Cytotoxicity of Water-Soluble Dual- And Triple-Action Satraplatin Derivatives: Replacement of Equatorial Chlorides of Satraplatin by Acetates

Article abstract of DOI:10.1021/acs.inorgchem.9b02796

Pt(II) complexes, such as cisplatin and oxaliplatin, are in widespread use as anticancer drugs. Their use is limited by the toxic side effects and the ability of tumors to develop resistance to the drugs. A popular approach to overcome these drawbacks is to use their kinetically inert octahedral Pt(IV) derivatives that act as prodrugs. The most successful Pt(IV) complex in clinical trials to date is satraplatin, cct-[Pt(NH₃)(c-hexylamine)Cl₂(OAc)₂], that upon cellular reduction releases the cytotoxic cis-[Pt(NH₃)(c-hexylamine)Cl₂]. In an attempt to obtain water-soluble and more effective cytotoxic Pt(IV) complexes, we prepared a series of dual- and triple-action satraplatin analogues, where the equatorial chlorido ligands were replaced with acetates and the axial ligands include innocent and bioactive ligands. Replacement of the chlorides with acetates enhanced the water solubility of the compounds and, with one exception, all of the compounds were very stable in buffer. In general, compounds with one or two axial hydroxido ligands were reduced by ascorbate significantly more quickly than compounds with two axial carboxylates. While replacement of the chlorides with acetates in satraplatin led to a reduction in cytotoxicity, the dual- and triple-action analogues with equatorial acetates had low- to sub-micromolar IC₅₀ values in a panel of eight cancer cells. The triple-action compound cct-[Pt(NH₃)(c-hexylamine)(OAc)₂(PhB)(DCA)] was active in all cell lines, causing DNA damage that induced cell cycle inhibition and apoptosis. Its good activity against CT26 cells in vitro translated into good in vivo efficacy against the CT26 allograft, an in vivo model with intrinsic satraplatin resistance. This indicates that multiaction Pt(IV) derivatives of diamine dicarboxylates are interesting anticancer drug candidates.

Full text of DOI:10.1021/acs.inorgchem.9b02796

Article
pubs.acs.org/IC
Cite This: Inorg. Chem. XXXX, XXX, XXX−XXX
Synthesis and Cytotoxicity of Water-Soluble Dual- and Triple-Action
Satraplatin Derivatives: Replacement of Equatorial Chlorides of
Satraplatin by Acetates
Subhendu Karmakar,†,∥ Isabella Poetsch,‡,§,∥ Christian R. Kowol,§ Petra Heffeter,*,‡
and Dan Gibson*,†
†Institute for Drug Research, School of Pharmacy, The Hebrew University, Jerusalem 91120, Israel
‡Institute of Cancer Research and Comprehensive Cancer Center, Department of Medicine I, Medical University of Vienna,
Borschkegasse 8a, 1090 Vienna, Austria
§Institute of Inorganic Chemistry, Faculty of Chemistry, University of Vienna, Vienna, Austria
S
* Supporting Information
ABSTRACT: Pt(II) complexes, such as cisplatin and
oxaliplatin, are in widespread use as anticancer drugs. Their
use is limited by the toxic side effects and the ability of tumors
to develop resistance to the drugs. A popular approach to
overcome these drawbacks is to use their kinetically inert
octahedral Pt(IV) derivatives that act as prodrugs. The most
successful Pt(IV) complex in clinical trials to date is satraplatin,
cct-[Pt(NH3)(c-hexylamine)Cl2(OAc)2], that upon cellular
reduction releases the cytotoxic cis-[Pt(NH3)(c-hexylamine)-
Cl2]. In an attempt to obtain water-soluble and more effective
cytotoxic Pt(IV) complexes, we prepared a series of dual- and
triple-action satraplatin analogues, where the equatorial
chlorido ligands were replaced with acetates and the axial
ligands include innocent and bioactive ligands. Replacement of
the chlorides with acetates enhanced the water solubility of the compounds and, with one exception, all of the compounds were
very stable in buffer. In general, compounds with one or two axial hydroxido ligands were reduced by ascorbate significantly
more quickly than compounds with two axial carboxylates. While replacement of the chlorides with acetates in satraplatin led to
a reduction in cytotoxicity, the dual- and triple-action analogues with equatorial acetates had low- to sub-micromolar IC50 values
in a panel of eight cancer cells. The triple-action compound cct-[Pt(NH3)(c-hexylamine)(OAc)2(PhB)(DCA)] was active in all
cell lines, causing DNA damage that induced cell cycle inhibition and apoptosis. Its good activity against CT26 cells in vitro
translated into good in vivo efficacy against the CT26 allograft, an in vivo model with intrinsic satraplatin resistance. This
indicates that multiaction Pt(IV) derivatives of diamine dicarboxylates are interesting anticancer drug candidates.
mM), resulting in the activation of cisplatin by aquation, where
cis-[Pt(NH3)2Cl2] is converted to the reactive cis-[Pt-
(NH3)2(H2O)Cl]+, which covalently modifies the DNA
(Scheme 1). Thus, cisplatin itself can be considered a prodrug
where a main role of the chlorido ligands is to stabilize the
drug outside the cell and allow its activation inside.
Nowadays one of the most popular approaches to overcome
these drawbacks is to use kinetically inert octahedral Pt(IV)
derivatives of the semilabile square-planar cisplatin.16−18
Pt(IV) complexes are derived from Pt(II) by oxidative
addition, where two ligands are added to the axial positions.
Their kinetic inertness minimizes unwanted extracellular
interactions, but once inside the cancer cell the complex is
INTRODUCTION
■
Platinum (Pt)-based cancer chemotherapy became immensely
popular in the last four decades after its accidental discovery by
Rosenberg et al.1−3 The three FDA-approved Pt(II) drugs
cisplatin, carboplatin, and oxaliplatin (Figure 1) are still
helping cancer patients worldwide in the fight against this
disease.4−7 However, some serious side effects8−10 and
inherent and acquired drug resistances11−14 prompted
inorganic medicinal chemists to design better platinum-based
drugs. Cisplatin, cis-[Pt(NH3)2Cl2], triggers its cytotoxic effect
by losing its chlorido ligands and binding to two adjacent
guanines on the same strand of the nuclear DNA. The drug is
administered intravenously and enters the cancer cell by
passive diffusion and through the human copper transporter
(hCTR).15 Outside the cell, it stays in the dichlorido form due
to the high extracellular chloride concentration (∼100 mM).
Inside the cell, the chloride concentration is lower (4−10
Received: September 19, 2019
© XXXX American Chemical Society
A
DOI: 10.1021/acs.inorgchem.9b02796
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
oacetate (DCA), respectively, as the bioactive ligands. HDAC
inhibitors play a pivotal role in the alteration of epigenetic
mechanisms by changing the structure of chromatin, thereby
controlling gene expression or repression.33−35 Hence, HDAC
inhibitors can act as drugs in epigenetic anticancer therapy.
FDA has already approved the HDAC inhibitors SAHA
(vorinostat),34,36 belinostat,37 and romidepsin38 for the
treatment of T-cell lymphoma and panabiostat39,40 for multiple
myeloma. The inhibition of PDK by DCA leads to activation
of the pyruvate dehydrogenase complex (PDHC), resulting in
a shift of cellular metabolism from glycolysis to glucose
oxidation, which affects the survival of cancer cells.41,42 DCA is
considered an orphan drug, and its anticancer activity is well-
known.43−45
Recently, Hoefer et al. showed that lipophilic Pt(IV)
complexes with four monodentatecarboxylato ligands have
good aqueous solubility and good cytotoxicity.46
Figure 1. Three FDA-approved Pt(II) drugs (cisplatin, carboplatin,
and oxaliplatin) and the three Pt(IV) complexes (iproplatin,
tetraplatin/ormaplatin, and satraplatin) that have entered clinical
trials.
Herein we synthesized dual- and triple-action Pt(IV)
derivatives of cis-[Pt(NH3)(CHA)(OAc)2]. These Pt(IV)
compounds are analogues of satraplatin where the equatorial
chlorides of satraplatin were replaced by acetates and the axial
positions were functionalized with HDAC or PDK inhibitors.
This generates novel complexes, where a single molecule can
deliver multiple drugs having different target sites upon
activation of the prodrug.
reduced, releasing the original Pt(II) drug as well as the two
axial ligands (Scheme 1).
Among the three Pt(IV) drugs (iproplatin, tetraplatin,
satraplatin, Figure 1) that have entered clinical trials,
satraplatin has shown encouraging activity in combination
therapy and as a standalone drug.19−23
Pt(IV) compounds need to be activated inside the cell to
release the Pt(II) drug that itself requires further activation.
Since in this case, the Pt(II) drug is generated inside the cancer
cell, it no longer requires the chlorido ligands for extracellular
stabilization. Therefore, in principle, a Pt(II) drug with any
labile or semilabile leaving group, which is generated inside the
cancer cell, could be active as well. Thus, we decided to explore
the Pt(IV) derivatives of satraplatin where the chlorido ligands
were replaced by acetato groups. On the one hand, acetates
bind to the Pt through the oxygen, making them good leaving
groups, and on the other hand, they can increase the water
solubility of the complexes.
Recently, it has become very popular to conjugate bioactive
ligands to the axial positions of Pt(IV) complexes to attain
“dual action” or “triple action” drugs.24−32 On the basis of our
previous experience, we decided to use histone deacetylase
(HDAC) and/or pyruvate dehydrogenase kinase (PDK)
inhibitors such as 4-phenylbutyrate (PhB) and/or dichlor-
EXPERIMENTAL SECTION
■
Materials and Methods. All of the chemicals and solvents were
procured from reputed commercial sources and used without further
purification.
Reaction mixtures were analyzed by an analytical HPLC system
(Thermo Scientific UltiMate 3000) with a reverse-phase C18 column
(Phenomenex Kinetex, length 250 mm, internal diameter 4.60 mm,
particle size 5 μm, pore size 100 Å). The purity and retention time
(RT) of Pt(IV) complexes reported here were measured with the
aforementioned analytical HPLC system. The samples were dissolved
in either methanol or acetonitrile and eluted with a 0−90% linear
gradient of acetonitrile in water for 30 min under the detection of UV
wavelengths at 220 and 260 nm. Bulk purification of the complexes
was done using a preparative HPLC system (Thermo Scientific
UltiMate 3000) attached with a reverse-phase C18 column
(Phenomenex Luna, Length 250 mm, internal diameter 21.20 mm,
particle size 10 μm, pore size 100 Å) following the same acetonitrile/
water gradient mentioned above. UV detection was monitored at 220
Scheme 1. (Top) Labile Cisplatin Is Stabilized from Aquation Outside the Cell by the High Chloride Concentration and Once
Inside the Cell Is Activated by Aquation That Facilitates DNA Binding and (Bottom) Inert Pt(IV) Is Stable Outside the Cell,
a
and Once Inside, It Is Activated by Reduction That Is Followed by Aquation
a
Pt(IV) complexes do not depend on chloride concentrations for activation; thus, L can be a non-chloride ligand that undergoes aquation.
B
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Article
nm. Collected pure fractions were combined and lyophilized. All of
the NMR data were recorded with a Bruker AVANCE IIIHD 500
MHz spectrometer. All of the data were processed with Bruker
TopSpin 3.5pl7 software, and the chemical shifts were described in
parts per million (ppm). 1H and 13C (proton decoupled) NMR
chemical shifts were referenced with the individual solvent residual
peaks of the respective NMR solvents used. Proton-decoupled 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 measured using a Thermo
Scientific triple-quadrature mass spectrometer (Quantum Access) by
positive mode electrospray ionization. Elemental analyses reported
were performed using a Thermo Scientific FLASH 2000 element
analyzer.
temperature, the reaction mixture was dried completely and purified
by HPLC to give 3 (RT 26.7 min, purity >99%) as a yellow sticky
1
gum. Yield: 50 mg (21.3%). H NMR (500 MHz, MeOD): δ 7.24−
γ
7.11 (m, 10H, CHPhB), 2.71−2.58 (m, 5H, H1 and CH2PhB), 2.40−
α
2.28 (m, 4H, CH2PhB), 2.09−2.03 (m, 8H, H2 and 2 × OCOCH3),
β
1.94−1.82 (m, 4H, CH2PhB), 1.76 (m, 2H, H3), 1.64 (m, 1H, H4),
1.34−1.25 (m, 4H, H2 and H3), 1.19 (m, 1H, H4). 13C NMR (125
MHz, MeOD): δ 183.54 and 183.50 (COPhB), 182.3 and 181.4-
(OCOCH3), 143.4 and 143.2 (CPhB), 129.62 and 129.57 (CHPhB),
129.30 and 129.26 (CHPhB), 126.83 and 126.76 (CHPhB), 56.4 (C1),
36.9 and 36.2 (αCH2PhB), 36.1 and 36.0 (γCH2PhB), 33.3 and 33.2
(C2), 29.2 and 28.9 (βCH2PhB), 26.3 (C4), 26.1 (C3), 23.8 and 22.4
(OCOCH3). 195Pt NMR (107.5 MHz, MeOD): δ 2115.4. ESI-MS:
calculated for [C30H44N2O8Pt + Na]+ 778.26, found 778.06. Anal.
Calcd for C30H44N2O8Pt: C, 47.68; H, 5.87; N, 3.71. Found: C,
47.98; H, 6.19; N, 3.84.
Synthesis. cis-[Pt(NH3)(CHA)(OAc)2]. Silver acetate (369 mg, 2.21
mmol) was added to a solution of cis-[Pt(NH3)(CHA)I2]18 (673 mg,
1.19 mmol; CHA = NH2C6H11) in 12 mL of DMF. After the mixture
was stirred for 14 h at room temperature, precipitated AgI was filtered
off and the filtrate was evaporated to give a brown oil. The oily residue
was dissolved in a minimum volume of dichloromethane and
precipitated with petroleum ether to afford a white solid of the
required cis-diacetato Pt(II) complex. The color of the complex
changes very quickly from white to light brown on exposure to light
and therefore was quickly used for the next reaction. Yield: 430 mg
cct-[Pt(NH3)(CHA)(OAc)2(DCA)2)] (4). To a solution of 1 (160 mg,
0.34 mmol) in DMF (7 mL) was added dichloroacetic anhydride
(320 μL, 2.09 mmol) dropwise, and the mixture was stirred at room
temperature for 14 h. Upon completion, the reaction mixture was
evaporated to dryness and subsequent HPLC purification provided 4
(RT 20.5 min, purity >96%) as a very faint yellow powder. Yield: 50
mg (21.4%). 1H NMR (500 MHz, MeOD): δ 6.27 (s, 1H,
OCOCHCl2), 6.26 (s, 1H, OCOCHCl2), 2.68 (m, 1H, H1), 2.14−
2.05 (m, 8H, H2 and 2 × OCOCH3), 1.77 (m, 2H, H3), 1.66 (m, 1H,
H4), 1.40−1.28 (m, 4H, H2 and H3), 1.20 (m, 1H, H4). 13C NMR
(125 MHz, MeOD): δ 182.3 and 181.0 (OCOCH3), 172.60 and
172.57 (OCOCHCl2), 67.2 and 66.2 (OCOCHCl2), 56.8 (C1), 33.4
and 32.6 (C2), 26.2 (C4), 26.15 and 26.06 (C3), 23.5 and 21.8
(OCOCH3). 195Pt NMR (107.5 MHz, MeOD): δ 2104.5. ESI-MS:
calculated for [C14H24Cl4N2O8Pt + Na]+ 706.98, found 706.71; Anal.
Calcd for C14H24Cl4N2O8Pt: C, 24.54; H, 3.53; N, 4.09. Found: C,
24.86; H, 3.72; N, 4.09.
1
(84%). H NMR (500 MHz, DMF-d7): δ 6.26 (br, 2H, NH2), 4.93
(br, 3H, NH3), 2.49 (m, 1H, H1), 2.32 (m, 2H, H2), 1.75 (s, 3H,
OCOCH3), 1.73 (s, 3H, OCOCH3), 1.69 (m, 2H, H3), 1.56 (m, 1H,
H4), 1.29−1.20 (m, 4H, H2 and H3), 1.09 (m, 1H, H4). 13C NMR
(125 MHz, DMF-d7): δ 180.3 and 179.7 (OCOCH3), 56.5(C1), 34.6
(C2), 26.5 (C4), 25.8(C3), 24.3 and 24.2 (OCOCH3). 195Pt NMR
(107.5 MHz, DMF-d7): δ −1623.4.
cct-[Pt(NH3)(CHA)(OAc)2(OH)2] (1). A 20 mL portion of H2O2
(30% w/v in water) was added to cis-[Pt(NH3)(CHA)(OAc)2] (220
mg, 0.51 mmol) at room temperature. The white solid dissolved
quickly to give a yellow solution. The solution was stirred for another
14 h, after which H2O2 was evaporated to dryness in a stream of
compressed air to give a yellow oil. Diethyl ether was added followed
by sonication and scratching, resulting in a yellow precipitate. Yield:
220 mg (93%). The crude powder was used for the next reactions
without purification. For biological studies, the crude product was
purified by HPLC (RT 10.2 min) to get 1 (purity >96%) as a yellow
crystalline solid. Yield: 63 mg (26.5%). 1H NMR (500 MHz, MeOD):
δ 2.76 (m, 1H, H1), 2.10−2.05 (m, 8H, H2 and 2 × OCOCH3), 1.78
(m, 2H, H3), 1.67 (m, 1H, H4), 1.37−1.19 (m, 5H, H2, H3and H4).
13C NMR (125 MHz, MeOD): δ 182.66 and 182.61 (OCOCH3),
54.5(C1), 34.1 and 33.6(C2), 26.4 (C4), 26.11 and 26.05 (C3), 24.0
and 23.5 (OCOCH3). 195Pt NMR (107.5 MHz, MeOD): δ 1859.8.
ESI-MS: calculated for [C10H24N2O6Pt + H]+ 464.14, found 463.97.
Anal. Calcd for C10H24N2O6Pt·H2O: C, 24.95; H, 5.44; N, 5.82.
found: C, 24.82; H, 5.22, N; 6.15.
cct-[Pt(NH3)(CHA)(OAc)2(PhB)(OH)] (5). cis-[Pt(NH3)(CHA)-
(OAc)2] (230 mg, 0.53 mmol) and 4-phenylbutyric acid (1.7 g,
10.3 mmol) were allowed to dissolve completely in 6 mL of THF, and
to this solution was added H2O2 (150 μL, 1.32 mmol) dropwise. The
yellow reaction mixture was stirred for 14 h at room temperature and
then heated at 45 °C for another 36 h. After that THF was evaporated
and the resulting oil was dissolved in a minimum volume of diethyl
ether to which a large volume of petroleum ether was added. The
whole mixture was refrigerated for 0.5 h and centrifuged to give a
brown oil at the bottom of the centrifuge tube. The brown oil was
preserved, discarding the other liquid on top of it. This procedure was
repeated three times to yield crude 5. Further HPLC purification was
performed to give 5 (RT 17.9 min, purity >98%) as a white powder.
1
Yield: 70 mg (20.7%). H NMR (500 MHz, MeOD): δ 7.26−7.18
(m, 4H, CHPhB), 7.15−7.12 (m, 1H, CHPhB), 2.72−2.60 (m, 3H, H1
and γCH2PhB), 2.35 (t, J = 7.28 Hz, 2H, αCH2PβhB), 2.21−1.97 (m, 8H,
H2 and 2 × OCOCH3), 1.92−1.86 (m, 4H, CH2PhB), 1.77 (m, 2H,
H3), 1.66 (m, 1H, H4), 1.39−1.26 (m, 4H, H2 and H3), 1.21(m, 1H,
H4). 13C NMR (125 MHz, MeOD): δ 184.3 (COPhB), 182.6 and
182.3 (OCOCH3), 143.4 (CPhB), 129.5 (CHPhB), 129.2 (CHPhB),
126.7 (CHPhB), 55.7 (C1), 37.3 (αCH2PhB), 36.1 (γCH2PhB), 33.5 and
33.1 (C2), 29.0 (βCH2PhB), 26.3 (C4), 26.1 and 26.0 (C3), 24.1 and
23.3 (OCOCH3). 195Pt NMR (107.5 MHz, MeOD): δ 1936.6. ESI-
MS: calculated for [C20H34N2O7Pt + H]+ 610.21, found 609.99. Anal.
Calcd for C20H34N2O7Pt: C, 39.41; H, 5.62; N, 4.60. Found: C,
40.02; H, 5.66; N, 4.56.
cct-[Pt(NH3)(CHA)(OAc)2(OAc)2] (2). Excess acetic anhydride was
added to a solution of 1 (250 mg, 0.54 mmol) in DMF (5 mL). The
mixture was heated at 60 °C with stirring for 8 h. After completion of
the reaction (as indicated by 195Pt NMR of the reaction mixture), the
solution was centrifuged and the filtrate was evaporated completely in
a rotary evaporator to give a yellow oil, which was purified by HPLC
to yield 2 (RT 12.1 min, purity >99%) as an off-white solid. Yield: 70
1
mg (23.5%). H NMR (500 MHz, MeOD): δ 2.67 (m, 1H, H1),
2.09−2.03 (m, 14H, 4 × OCOCH3 and H2), 1.77 (m, 2H, H3), 1.66
(m, 1H, H4), 1.35−1.26 (m, 4H, H2 and H3), 1.20 (m, 1H, H4). 13C
NMR (125 MHz, MeOD): δ 182.2 (2 × OCOCH3, Axial), 181.29
and 181.15 (OCOCH3, Equatorial), 56.5 (C1), 33.2 (C2), 26.3 (C4),
26.2 (C3), 23.7 and 23.1 (OCOCH3, Equatorial), 22.3 (2 ×
OCOCH3, Axial). 195Pt NMR (107.5 MHz, MeOD): δ 2116.9. ESI-
MS: calculated for [C14H28N2O8Pt + Na]+ 570.14, found 569.94.
Anal. Calcd for C14H28N2O8Pt: C, 30.71; H, 5.16; N, 5.12. Found: C,
30.63; H, 5.24; N, 5.02.
cct-[Pt(NH3)(CHA)(OAc)2(PhB)(DCA)] (6). Crude 5 (500 mg, 0.82
mmol) in 2 mL of DMF, as received from the previous reaction, was
reacted with dichloroacetic anhydride (480 μL, 3.15 mmol). After
accomplishment of the reaction, the mixture was dried. Purification by
HPLC gave 6 (RT 23.7 min, purity >97%) as a light yellow fluffy
1
powder. Yield: 120 mg (20.2%). H NMR (500 MHz, MeOD): δ
7.26−7.18 (m, 4H, CHPhB), 7.16−7.13 (m, 1H, CHPhB), 6.26−6.25
(d, 1H, CHDCA), 2.72−2.61 (m, 3H, H1 and γCH2PhB), 2.41−2.31 (m,
2H, αCH2PhB), 2.10−2.04 (m, 8H, H2 and 2 × OCOCH3), 1.92−1.83
cct-[Pt(NH3)(CHA)(OAc)2(PhB)2] (3). 4-Phenylbutyric acid anhy-
dride (777 mg, 2.5 mmol) was added to a solution of 1 (145 mg, 0.31
mmol) in 6.5 mL of DMF. After it was stirred for 12 h at room
β
(m, 2H, CH2PhB), 1.75 (m, 2H, H3), 1.63 (m, 1H, H4), 1.34−1.26
(m, 4H, H2 and H3), 1.20 (m, 1H, H4). 13C NMR (125 MHz,
C
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MeOD): δ 183.5 and 183.2 (COPhB), 182.39 and 182.36 (OCOCH3,
Axial), 181.3 and 181.0 (OCOCH3, Equatorial), 172.49 and 172.48
(OCOCHCl2), 143.4 and 143.2 (CPhB), 129.64 and 129.57 (CHPhB),
129.3 and 129.2 (CHPhB), 126.8 and 126.7 (CHPhB), 66.34 and 66.31
(OCOCHCl2), 56.4 and 56.3 (C1), 36.8 and 35.7 (αCH2PhB), 36.09
and 36.06 (γCH2PhB), 33.59 and 33.54 (C2), 32.75 and 32.71 (C2),
29.1 and 28.8 (βCH2PhB), 26.3 and 26.2 (C4), 26.2 and 26.1 (C3),
23.6 and 23.1 (OCOCH3, Equatorial), 21.9 (OCOCH3, Axial). 195Pt
NMR (107.5 MHz, MeOD): δ 2116.1. ESI-MS: calculated for
[C22H34Cl2N2O8Pt+ Na]+ 742.12, found 742.92. Anal. Calcd for
C22H34Cl2N2O8Pt: C, 36.67; H, 4.76; N, 3.89. Found: C, 36.81; H,
4.80; N, 3.98.
Determination of Water Solubility. In order to determine the
water solubility of the synthesized Pt(IV) complexes, the standard
shake flask method was followed. In brief, saturated water solutions of
each complex were prepared by shaking for 24 h in water at room
temperature. After that, each solution was filtered and injected into an
analytical HPLC. The unknown concentrations of complexes in water
were calculated from the standard curve of the respective complexes.
Stability in Phosphate Buffer. The stability of the Pt(IV)
complexes (except complex 3) were studied in 50 mM phosphate
buffer (pH 7.0) at 37 °C. Due to low aqueous solubility, the study of
complex 3 was performed in a 20% mixture of MeCN in 50 mM
phosphate buffer (pH 7.0).
Briefly, the Pt(IV) complexes were incubated at 37 °C in the dark
and injected into analytical HPLC at different time intervals. The
samples were run in a linear gradient of 0−90% acetonitrile in water
over 30 min. The half-lives (t1/2) were determined after linear fitting
of ln(At/A0) vs time (t) by considering a pseudo-first-order rate
equation for the hydrolysis (At = A0e−kt; eq 1), where A0 and At are
the integrated areas of HPLC peaks of the respective complexes at t =
0 and at time t, respectively, and k is the rate constant. Then the t1/2
value was calculated by utilizing the equation t1/2 = 0.693/k.
Rates of Reduction. This study was performed using the same
experimental conditions as those for the stability study in phosphate
buffer (50 mM) with addition of 10 equiv of L-ascorbic acid (pH 7).
The decreasing intensities of the respective peaks of the Pt(IV)
complexes were monitored with analytical HPLC, and t1/2 values were
determined following eq 1 above.
Cell Lines and Culture Conditions. The human and murine
cancer cell lines used in this study are summarized together with the
source and respective growth medium in Table S1. All media were
supplemented with 10% fetal bovine serum. Cultures were regularly
checked for mycoplasma contamination. Cultures were maintained at
37 °C in a humidified atmosphere containing 95% air and 5% CO2.
Cytotoxicity Tests in Cancer Cell Lines. To determine cell
viability, (2−5) × 104 cells/mL (depending on the proliferation speed
of the cell line) were seeded in 96-well plates (100 μL/well) and
allowed to recover for 24 h. Then, cells were exposed to the test drugs
at the indicated concentrations for 72 h. Anticancer activity was
measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) based vitality assay (EZ4U; Biomedica, Vienna,
Austria) following the manufacturer’s recommendations. Cytotoxicity
was calculated using the GraphPad Prism software (using a point-to-
point function) and was expressed as IC50 values calculated from full
dose−response curves (drug concentrations inducing a 50% reduction
of cell number in comparison to untreated control cells cultured in
parallel).
culture plastic may occur especially in case of lipophilic compounds;47
therefore, results were corrected for platinum levels of a blank well
containing no cells.
Time-Lapse Microscopy. Cells in an amount of 4 × 104 cells/
well were seeded in an 8-well μ-slide (ibidi, Martinsried, Germany)
and allowed to recover for 24 h. The cells were treated with the
respective compounds at 5 μM, and the image series was started
directly after treatment in a humidified incubation chamber (37 °C,
5% CO2) on a Livecell Inverted Brightfield Microscope Nikon Eclipse
Ti (20x Super Plan Fluor NA 0.45 Ph1, PCS sCMOS monochrome
camera 4.2MPxl, rolling shutter, Visiview Software).
Morphological Evaluation of CT26 Cells. Briefly, 2 × 105 cells
were seeded in 6-well plates, allowed to recover for 48 h, and treated
for another 24 h. Subsequently, cells were collected and cytospins
prepared using a cytocentrifuge (Cytospin 4, Thermo Scientific,
Waltham, MA, USA) at 400 rpm for 5 min. After fixation with
methanol/acetone (−20 °C, 1/1) cells were stained with 4′,6-
diamidine-2′-phenylindole dihydrochloride (DAPI). For each slide,
200−300 nuclei were scored normal, mitotic, or apoptotic.
Cell Cycle Analysis by Flow Cytometry. CT26 cells were
seeded in 6-well plates at a density of 3 × 105 cells. Cells were treated
at 1 and 2 μM concentrations for 24 h and subsequently trypsinized,
washed with PBS, and fixed in 70% ethanol (1 h at −20 °C). Fixed
cells were centrifuged (8 min, 6000g) and resuspended in 100 μL of
0.9% NaCl, and RNA was digested with 0.2 mg/mL RNase for 30 min
at 37 °C. Nuclei were stained with propidium iodide (PI, 0.01 mg/
mL, Sigma-Aldrich. St. Louis, MO, USA) for 30 min at 4 °C in the
dark. Cells were analyzed by flow cytometry using a BD LSR
FortessaTM instrument, and FlowJo software was used to evaluate the
results.
Immunofluorescence (DNA Damage). Cells in an amount of 4
× 103 CT26 cells were seeded on “PTFE”-printed slides (E63424-06,
Science Services). After 24 h of recovery cells were treated for 19 h
and subsequently fixed in 4% paraformaldehyde (PFA) solution for 10
min at room temperature before washing two times for 1 min in PBS.
Cells were incubated with blocking buffer (5% BSA, 0.2% Triton X, in
PBS) and subsequently incubated with antiphospho-H2AX antibody
(cell signaling (20E3) #9718, 1/200 diluted in PBS with 1% BSA and
0.2% Triton X) for 1 h at room temperature. After two further
washing steps with PBS, cells were incubated (1 h) with the secondary
antibody (antirabbit IgG labeled with FITC, Sigma-Aldrich) and with
phalloidin-tetramethylrhodamine B isothiocyanate (phalloidin
TRITC, P1951, Sigma) 1/500 and 1/1000 diluted in PBS with 1%
BSA and 0.2% Triton X. Cells were counterstained with DAPI before
mounting. Images were obtained using a Zeiss LSM 700 confocal
microscope (Plan-Apochromat 63x/1.4 Oil DIC M27 LSM 700
AxioObserver). Subsequent image handling was carried out using
ImageJ software. The nuclei area from each image was selected and
used to detect the sum of the pixel values in the pH2AX channel
(integrated density). Integrated density was normalized to nuclei area.
From each condition eight images were quantified.
Immunohistochemsitry (DNA Damage). Immunohistochemis-
try was performed according to the standard procedure. Shortly, 4 μm
sections of paraffin-embedded tumor samples were deparaffinized,
blocked for endogenous peroxidase by incubating for 10 min in tris-
buffered saline 0.3% H2O2, and rehydrated. Antigens were retrieved
by boiling the samples in 10 mM citrate buffer (pH 6.0) for 3 min at
1.5 bar. The samples were blocked using Ultra V Block (UltraVision
LP) and subsequently incubated with the primary antibody against
pH2AX (Cell Signaling, #9718, 1/1000). Antibody binding was
detected by HRP polymers (UltraVision LP), and the color was
developed using 3,30-diaminobenzidine. Samples were counterstained
with hematoxylin.
Animal Experiments. All animal experiments were approved by
the local ethics commission and carried out according to the Austrian
and FELASA guidelines for animal care and protection. Eight- to
twelve-week-old female BALB/c mice (weighing ∼20 g) were
purchased from Envigo, Italy. The animals were kept in a pathogen-
free environment, and every procedure was done in a laminar airflow
cabinet.
Total Pt Uptake Levels. CT26 cells (5 × 105/well) were drug-
exposed for 3 h at 37 °C. After two washing steps with PBS, cells were
lysed at room temperature in 500 μL of 67−69% HNO3(83872.270,
VWR) for 1 h. A 400 μL portion of the lysate was added to 7.6 mL of
distilled deionized water, and platinum concentrations were
determined by ICP-MS using an Agilent 7800 ICP-MS instrument.
The ICP-MS Agilent 7800 instrument (Agilent Technologies, Tokyo,
Japan) was equipped with a CETAC ASX-520 autosampler
(Nebraska, USA) and a MicroMist nebulizer at a sample uptake
rate of approximately 0.2 mL/min. The Agilent MassHunter software
package (Workstation Software, version C.01.04, Build 544.17, Patch
3, 2018) was used for data processing. As unspecific binding to cell
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Scheme 2. Synthetic Scheme for the Preparation of Equatorial Acetate Modified Dual- and Triple-Action Satraplatin
Derivatives
Anticancer Activity against CT-26 Cells in Vivo. Murine CT-
26 cells (5 × 105) were injected subcutaneously into the right flank of
female BALB/c mice. Animals were treated with the drug (dissolved
in 5% DMSO) either intraperitoneally or orally at the indicated drug
concentrations at day 4, 6, 8, 11, 13, and 15. Animals were controlled
for distress development every day, and the tumor size was assessed
regularly by caliper measurement. Tumor volume was calculated using
the formula (length × width)2/2.
NMR (DMF-d7) corresponding to the two methyl groups of
the acetates confirm the binding to the platinum (Figure S1B).
These data also correlate with their 13C NMR chemical shifts
at 24.2 and 24.1 ppm, respectively, in DMF-d7, and the
carbonyl 13C chemical shifts were found at 180.3 and 179.7
ppm, respectively (Figure S1C,D). The NH2 and NH3 groups
gave two broad signals at 6.26 and 4.93 ppm, respectively, in
the 1H NMR (Figure S1B). Among the protons of the
cyclohexyl group, the H1 proton (attached to NH2) possesses
the most downfield chemical shift at 2.49 ppm as a one-proton
multiplet. Then the chemical shifts for H2, H3, and H4 came
in a descending order of sequence (Figure S1B). However,
they came in two different sets, in accordance with their axial
and equatorial positions. In 13C NMR the sequence changed as
the chemical shift corresponding to C4 came before that of C3
(Figure S1C).
cis-[Pt(NH3)(CHA)(OAc)2] was oxidized readily to the
Pt(IV) complex 1 with 30% H2O2 (Scheme 2). Apart from a
color change (to yellow), the 195Pt NMR chemical shift at
∼1860 ppm (Figure S2A) confirmed the oxidation to the
Pt(IV) species cct-[Pt(NH3)(CHA)(OAc)2(OH)2]. This
complex is inert and hence was purified by reverse-phase
HPLC using a MeCN/water gradient (Figure S2B) and was
characterized by ESI-MS (Figure S2C) and NMR (Figure
S2D,E); its purity was confirmed by elemental analysis. The
NMR, ESI-MS, and elemental analysis support the formation
of 1.
We acetylated both axial positions of 1 with an excess of
acetic anhydride to produce the Pt(IV) complex 2 (cct-
[Pt(NH3)(CHA)(OAc)2(OAc)2]; Scheme 2). 2 differs from
satraplatin only by replacement of the chlorido ligands by
acetates. The 195Pt NMR spectrum of 2 has a resonance at
2116.9 ppm (Figure S3A), indicating bis-acetylation. Both axial
methyl groups have the same chemical shifts at 2.03 ppm in the
1H NMR (Figure S3C). This is also true in the 13C NMR,
where both of the methyl carbons resonate at 22.2 ppm and
the carbonyls at 182.1 ppm (Figure S3D). Put together, we
conclude that the axial acetates of 2 are equivalent.
RESULTS AND DISCUSSION
Synthesis and Chemical Characterization. The syn-
thesis of the target compounds, dual- and triple-action Pt(IV)
■
Table 1. Chemical Properties of Compounds 1−6: Water
Solubility, Stability in Phosphate Buffer, and Half-Lives in
Ascorbic Acid
water
half-life in
solubility
phosphate buffer
half-life with 10 equiv L-
a
b
c
complex
(mg/mL)
t1/2 (day)
ascorbic acid t1/2 (h)
b
b
1
2
>2
>2 (>3.6
mM)
0.006−0.02
1.26−1.83
0.10−0.12
0.37−0.71
16.5
0.4
b
b
9.6
35.9
c
c
3
4
5
6
18.5
151.9
b
b
0.04
1
b
b
23.9
0.5
b
b
8.2
15.9
c
c
satraplatin 0.4 (0.8 mM)
3.1
0.8
a
b
24 h in water at room temperature 50 mM phosphate buffer pH 7.0
c
at 37 °C. 20% MeCN in 50 mM phosphate buffer pH 7.0 at 37 °C.
derivatives of satraplatin with axial acetato ligands, proceeded
according to Scheme 2. Initially, the precursor, cis-[Pt(NH3)-
(CHA)I2], was synthesized from cis-[Pt(CHA)2I2], following
the procedure of Lippard et al.18 The iodides were replaced
with acetates by reacting cis-[Pt(NH3)(CHA)I2] with 2 equiv
of Ag(OAc) in the polar aprotic solvent DMF. The desired
compound, cis-[Pt(NH3)(CHA)(OAc)2], was obtained as a
white powder in 84% yield. 195Pt NMR of the reaction mixture
indicated a shift of the resonance at −3307.7 ppm (starting cis-
diiododiam(m)ine Pt(II) complex, data not shown) to
−1623.4 ppm (Figure S1A). This chemical shift indicates
that two oxygen atoms are bound to the cis-diam(m)ine Pt(II)
We prepared dual-action Pt(IV) complexes by esterifying
the two axial OH groups of 1 with PhB anhydride or DCA
anhydride to generate complexes 3 and 4, respectively
(Scheme 2). The two PhB ligands in the two axial positions
1
center. Two acetate singlets at 1.75 and 1.73 ppm in the H
E
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a
Table 2. Cytotoxicity Assessed by MTT Assay
IC50 (μM)
drug
HCT-116
A2780
CT-26
B-16
SW480
Capan-1
p31
DU145
CisPt
SatraPt
5.4 0.30
4.0 0.60
45.5 3.34
39.4 3.15
0.4 0.05
1.2 0.20
0.4 0.12
0.3 0.05
1.6 0.57
3.4 2.44
20.6 9.59
24.1 7.74
0.2 0.05
0.6 0.24
0.2 0.04
0.2 0.08
3.9 1.42
5.5 2.19
37.2 7.66
49.2 4.53
1.3 0.07
2.1 0.20
5.3 0.51
1.0 0.02
9.6 3.70
4.0 0.75
>100
7.1 0.40
3.6 0.14
20.7 2.89
43.9 8.39
0.6 0.13
1.2 0.20
0.8 0.38
0.4 0.03
9.3 4.83
6.4 3.64
>100
15.4 8.1
8.0 1.28
96.2 24.08
89.9 26.35
0.6 0.00
1.7 0.16
0.5 0.06
0.4 0.19
1.3 0.89
3.3 0.77
21.8 0.94
27.4 5.51
0.3 0.05
1.1 0.21
0.3 0.02
0.3 0.02
1
2
3
4
5
6
>100
>100
0.5 0.08
1.0 0.11
1.1 0.11
0.3 0.02
0.6 0.32
2.1 1.36
0.3 0.03
0.3 0.12
a
Cells were treated for 72 h with increasing concentrations of test compounds. IC50 values (given as mean SD) indicate the concentration
yielding a 50% reduction in cell viability.
Figure 2. Intracellular platinum accumulation of the investigated
complexes. CT-26 cells were treated with the indicated drugs at 2.5 or
5 μM for 3 h at 37 °C. Intracellular platinum levels were detected by
ICP-MS measurements. Bars depict platinum amount (in ng) per 106
cells as mean SD (n = 3) of one representative experiment.
of 3 are not magnetically equivalent, as is reflected in their
1
chemical shifts in H (Figure S4B) and 13C (Figure S4C,D)
NMR. The HPLC chromatogram has a single peak with a
purity of >99% (Figure S4E). This suggests that there might
not be free rotation around the Pt−PhB bonds due to the
CHA ligand. Similarly, the protons of axial DCA in 4 are
slightly separated, resonating at 6.27 and 6.26 ppm (Figure
S5C). This is supported by 13C NMR (67.2 and 66.2 ppm for
the two DCA CH carbons and 172.6 and 172.5 ppm for DCA
carbonyl carbons; Figure S5D). The lack of free rotation of the
bulky axial ligands (PhB and DCA) is further supported by the
fact that, with the exception of 2 which has small acetato
ligands, the 13C NMR of 3−6 show that each carbon in CHA
has a unique chemical shift. This suggests that there is no free
rotation of the CHA probably due to steric interactions with
the axial ligands.
We tried to prepare the nonsymmetric Pt(IV) complexes by
monocarboxylation of 1 with PhB (complex 5) by reacting 1
with 1.2 equiv of PhB anhydride in DMSO. We monitored the
progress of the reaction by 195Pt NMR. After 20 h of stirring at
room temperature, we observed two peaks at 1957.1 and
2159.1 ppm for unreacted bis-hydroxido (1) and the bis-
carboxylato (3) Pt(IV) complexes, respectively (Figure S6A).
Interestingly, there was no sign of any peak for the
monocarboxylato (5) Pt(IV) at ca. 2000−2100 ppm.
Repeating the reaction in DMF yielded the same result
(Figure S6B).
Figure 3. Morphological changes after drug treatment. (a) Images of
CT-26 cells treated with the new complexes at 5 μM for 24 h. Images
were taken by time-lapse microscopy (every 20 min). The scale bar is
100 μm. (b) CT-26 cells treated with 5 μM of the respective
complexes for 24 h. Nuclei were stained by DAPI, and the
morphology was analyzed. Cells from five representative images
were quantified. Bars depict mean SD (percent of apoptotic cells is
indicated above the respective bar). Significance (**p < 0.01, ****p <
0.0001) in comparison to DMSO control was calculated by one-way
ANOVA using GraphPad Prism software.
oxidized this complex with a slight excess of H2O2 (2.5 equiv)
in the presence of 20 equiv of PhB in THF (Scheme 2). After
the reaction mixture was stirred overnight at room temper-
ature, we observed two large peaks of almost equal intensity in
195Pt NMR at 1947.3 and 2018.8 ppm and a very small signal
at 2097.5 ppm (Figure S6C). The oxidation gave rise to two
major products. The first peak is the usual H2O2 oxidation
product cct-[Pt(NH3)(CHA)(OAc)2(OH)2], and the second
Therefore, we tried to carry out the monocarboxylation of
cct-[Pt(NH3)(CHA)(OAc)2(OH)2] with the help of a method
previously reported by our group.48 In this process, we began
with the Pt(II) complex cis-[Pt(NH3)(CHA)(OAc)2] and
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Figure 4. Changes in cell cycle distribution upon drug treatment in CT26 cells. Cell cycle analysis was done by flow cytometry determining the
DNA content of the ethanol-fixed, PI-stained cells after 24 h of treatment with the indicated drug concentrations. Bars show percentages of cells in
G0/G1, S, and G2/M phases of the cell cycle, calculated by FlowJo software and depicted as mean SD; p values were calculated by multiple t test
analysis (*p < 0.05, **p < 0.01, ***p < 0.001). Individual values are given below.
peak corresponds to 5 with one axial OH (from H2O2) and
one axial PhB. The minor peak results from the biscarbox-
ylation: i.e., 3. The reaction mixture was heated to 40−45 °C
for another 1.5 days, upon which it yielded 5 as a major peak
with 3 as a minor peak (Figure S6D). Here, we see that the
PhB (acid) reacts with cct-[Pt(NH3)(CHA)(OAc)2(OH)2]
directly upon prolonged heating, although in our earlier
report49 the heating of the reaction mixture was strictly
prohibited. Warming the reaction mixture, in this case, yielded
the desired product.
solubilities of satraplatin (0.4 mg/mL, 0.8 mM) and
compounds 2 (>2 mg/mL, 3.6 mM) are compared. We
determined the range of the water solubility of our complexes
1−6, and the data are summarized in Table 1 in mg/mL. With
the exception of 3 and 5, all compounds are more soluble than
satraplatin (0.4 mg/mL).
Stability in Phosphate Buffer. We examined the stability
of our Pt(IV) complexes in 50 mM phosphate buffer at pH 7.0
and 37 °C, and the stability was monitored by HPLC and is
expressed as half-life (t1/2) in Table 1.
Among all the complexes, 5 is the most and 4 is the least
stable (Table 1). We assume that the main cause of instability
is aquationthe replacement of one axial ligand by a
hydroxide. Within less than 1 h (t1/2 ≈ 0.9 h) half of 4 (RT
20.6 min) was hydrolyzed to produce the monohydroxido-
mono-DCA derivative cct-[Pt(NH3)(CHA)(OAc)2(DCA)-
(OH)] (4a, RT 14.8 min; Figure S10A), which was
characterized by ESI-MS, having an MH+ peak at m/z
574.83 (calcd m/z 574.07; Figure S10B). This is in agreement
with what we reported previously.49 After 400 min only 0.5%
of 4 remained intact (Figure S10C). Complex 6 with one DCA
Compound 6 was prepared from 5 by esterification of the
remaining OH group of 5 with DCA anhydride to yield the
triple-action Pt(IV) complex 6 (Scheme 2). HPLC suggests
the existence of two isomers of 6 at RT 23.7 min that cannot
1
be separated (Figure S8A). The H (Figure S8C) and 13C
NMR spectra (Figure S8D−H) were also in accordance with
two isomers.
Water Solubility. One of the goals of replacing the
chlorido ligands with acetato ligands was to try to increase the
aqueous solubility. The enhancement in solubility gained by
just replacing chlorides with acetates is evident when the
G
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Figure 5. Induction of DNA damage by the compound panel. (A) CT26 cells treated with the respective compounds at 2.5 μM for 19 h. Then cells
were fixed with 4% PFA solution. Nuclei were stained with D59API (depicted in gray), actin was stained with TRITC-phalloidin (depicted in
magenta), and DNA damage was visualized by pH2AX staining (depicted in green). The scale bar is 20 μm. (b) Integrated intensity of pH2AX
signals from (a) quantified using ImageJ software. Bars present mean SD (n = 8). The significance in comparison to control samples was
calculated by one-way ANOVA using GraphPad Prism software (*p < 0.05, ***p < 0.001, ****p < 0.0001).
and one PhB attached to the axial position has a long half-life
in buffer (8.2 days). The same is true for the tetraacetato
complex 2 with a half-life of 9.6 days. The species found in
addition to 2 (RT 12.1 min) in the HPLC are the hydrolysis
products of the acetates at RT 11.7 and 9.1 min (Figure
S12A,B). 1, 3, and 5 have half-lives greater than 15 days (Table
1) in buffer and hence their speciations are not discussed here.
It is interesting and important to note that the bis-DCA
compound 4 is significantly less stable than the mono-PhB-
mono-DCA compound 6, attesting to the strong influence of
the electronic properties of the axial ligand on the rate of
hydrolysis of the ligand trans to it.
Rates of Reduction. The design of Pt(IV) prodrugs is
predicated on the assumption that they will remain intact in
the bloodstream and will be reduced primarily inside the
malignant tissue.51,52 It is claimed that GSH, ascorbic acid, and
metalloproteins are among the biological reducing agents
responsible for activation of Pt(IV) prodrugs.50−53 An
important parameter in the mode of action of the Pt(IV)
prodrugs is the rate of reduction. As we cannot measure it
inside the cancer cells, we compared the rates of reduction of
the six new compounds in 50 mM phosphate buffer of pH 7.0
at 37 °C. We expressed their rates of reduction in terms of the
half-lives (t1/2) of the complexes (Table 1).
reductions of 1 (t1/2 = 0.4 h) and 5 (t1/2 = 0.5 h) are attributed
to the facile electron transfer from the ascorbate to the metal
via an inner-sphere interaction of the ascorbate with the axial
hydroxido ligand. Although 4 and 6, like 2 and 3, have only
am(m)ines and carboxylates in their coordination sphere, they
are reduced much more quickly (Table 1). The reason behind
this is the relative instability of the DCA ligand to aquation,
particularly when both axial ligands are DCA (Table 1).51,55
Following replacement of the DCA with hydroxide, the rate of
reduction increases.
Anticancer Activity in Cell Culture. The cytotoxicity of
the six Pt(IV) derivatives of cis-[Pt(NH3)(CHA)(OAc)2] was
measured in eight cancer cell lines of human and murine origin
(Table 2).
Replacing the chlorido ligands of satraplatin by acetates
(compound 2) led to a dramatic reduction in the cytotoxicity.
While the average IC50 values of cisplatin and satraplatin are
∼7 and ∼5 μM, respectively, for 1 and 2 it was (depending on
the tested cell model) between ∼20 and >100 μM. Thus,
compounds 1 and 2 were basically not active in comparison to
cisplatin or satraplatin. Introduction of the bioactive ligands in
the axial positions (3−6) resulted in a significant enhancement
of their potency. The average IC50 values of compounds 3−6
are 0.6, 1.4, 1.1, and 0.4 μM, respectively. It is worth noting
that compounds 3, 5 and 6 have sub-micromolar activities in
all cell lines tested (with the exception of the murine colon
carcinoma CT-26), which is in good agreement with previously
published data on an oxaliplatin derivative of 5.56 Overall, it
seems that the concentration range of the compound’s
cytotoxicity does not depend on the nature of the axial ligand.
The slow rates of reduction observed for compounds 2 (t1/2
= 35.9 h) and 3 (t1/2 = 151.9 h) are in agreement with the
previous reports that Pt(IV) compounds whose coordination
sphere is comprised of am(m)ines and carboxylates are
reduced slowly, presumably due to the slow transfer of
electrons from the ascorbate to the metal.54 The more rapid
H
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Figure 6. In vivo anticancer activity of complex 6. (a) CT-26 cells were injected subcutaneously into the right flank of BALB/c mice. Mice were
treated on day 4, 6, 8, 11, 13, and 15 with 20 mg/kg (p.o.) or 7.2 mg/kg (i.p.) of compound 6. (b) Tumor volumes were calculated as described in
the Experimental Section. Each experimental group contained four animals. Data are means
SEM. (c) Weight of dissected tumors. (d)
Histological evaluation of tumor tissue sections stained by H/E. Blue arrows indicate mitotic cells, and black arrows indicate apoptotic cells: 40×
magnification, scale bar 25 μm, scale bar of bottom corner of enlarged images 50 μm. The lower right corner shows quantification of histological
sections. Three images of each mouse tumor section were analyzed by manual cell counting of mitotic and apoptotic (pyknotic) cells. Bars depict
mean SD (n = 3). Significance was calculated using one-way ANOVA using Graph Pad Prism software. (e) Tumor sections stained for pH2AX:
40× magnification, scale bar 50 μm, scale bar of bottom corner of enlarged images 50 μm.
Consequently, while 4 was slightly less potent than 3, 5, and 6,
the last three essentially have similar cytotoxicities. Still, in
comparison of the dose−response curves, 6 had the best
anticancer activity of all tested drugs in this panel (Figure S14).
Overall, on the basis of the stability and reduction kinetic
data of the compounds (Table 1), the similarity of the IC50
values from 3−6 is rather surprising, which prompted us to
further investigate the biological effects of our test panel.
Cellular Drug Uptake. In order to better understand the
differences in the cytotoxicity of the investigated compounds
(especially the lack of activity in case of 1 and 2), the cellular
drug uptake levels were investigated by ICP-MS in CT-26 cells.
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As shown in Figure 2, distinct differences between the
individual compounds were observed. In line with the very
high IC50 values obtained in the viability assays, the platinum
levels of 1 and 2 were very low (near the limit of detection).
This indicates that these compounds have limited anticancer
activity due to insufficient drug accumulation. This could be
probably explained by a reduced hydrophobicity of these
compounds, as indicated by their retention time on a reversed-
phase C18 column. Compounds 1 and 2 had retention times of
10.1 and 12.1 min, in comparison those of 5 (with one OH) of
17.9 min and 3, 4, and 6 of >20 min. Interestingly, also
compound 5, which was very active in the viability assays, was
characterized by a rather low cellular accumulation (below 0.5
ng of Pt/106 cells and the reference drug satraplatin). This
could probably be explained by the very easy reduction of the
compound, resulting in rapid release of the highly active Pt(II)
species, enabling earlier DNA damage. Finally, compounds 3,
4, and 6 were very efficiently taken up into the cells (in case of
6 up to 10-fold higher than for satraplatin), which is in good
agreement with their anticancer activity in the MTT assays and
indicates also that in the case of these compounds the axial
ligands facilitate the uptake of these drugs.
Cell Death, Cell Cycle Arrest, and DNA-Damage-
Inducing Properties of the New Complexes. Despite their
similar IC50 values, already rough examination using a cell
culture microscope indicated distinct differences in the
morphologies of the treated cells. Consequently, the effects
of the compound panel were followed by live cell microscopy,
which also suggested differences in their underlying mode of
actions (e.g., apoptosis-inducing potential; Figure 3a). To gain
more insights into the mechanisms of our new platinum drugs,
CT26 cells were fixed after 24 h of drug treatment and their
DAPI-stained nuclei evaluated for their apoptotic and mitotic
features (Figure 3b).
This analysis revealed that only treatment with compounds 3
and 6 led to a significant increase of cells with apoptotic
morphology (Figure 3b). Thus, 6 was the most potent
apoptosis inducer of the tested cell panel (16-fold increase in
comparison to control), while 3 led only to an 8-fold increase
in apoptotic cells. No necrotic cells were found in any of the
groups (data not shown). With regard to the mitotic fraction,
already in the control samples only a very low number of cells
with mitotic features was visible (∼0.2%). However, a
complete absence of mitotic cells in samples treated with
compounds 3−6 indicated that all compounds could have an
effect on the cell cycle progression of the cancer cells (data not
shown). Thus, as a next step, we evaluated the cell cycle
distribution in comparison to cisplatin and satraplatin after 24
h of treatment at two concentrations (Figure 4). These
experiments revealed that, in line with our hypothesis, 3−6 (1
and 2 were not included because of their inactivity based on
insufficient drug uptake) affected the cell cycle distribution of
the investigated cancer cells. In more detail, the cells reacted in
most cases (especially in the 1 μM samples) with a 60−80%
increase of the cell population in the G2/M phase (cisplatin
was the most potent compound in this aspect, with a
significant increase of 240% of the G2/M fraction). In
addition, in case of cisplatin, a 3, 4 and 6 shift in the G0/
G1-S ratio was observed, which indicated accumulation of cells
in the S phase as well as G2/M phase after treatment with
these drugs. Whether these differences on cell cycle
distribution between 5 and the other compounds could be
due to the earlier released PhB ligand is speculative and would
need further investigations. Together these data indicate that,
although compounds 3−6 affect the cell cycle progression of
the cancer cells, only 3 and 6 are able to induce apoptosis
under the investigated conditions.
In order to assess whether the observed effects are due to the
more potent formation of DNA adducts, phosphorylation of
histone variant H2AX, a common marker for platinum-induced
DNA damage,57,58 was assessed for all drugs. As shown in
Figure 5, only compounds 3, 4, and 6 together with cisplatin
(which was used as a positive control) were able to
significantly increase the signal in comparison to the control
samples. Out of these drugs, 6 was most active, resulting in an
∼3-fold increase of pH2AX levels in comparison to the control
samples. Overall, this indicates that, after 19 h of treatment, at
least in the case of 4−6, the Pt(IV) complex was reduced to
the active Pt(II) species, which could then bind/interact with
the DNA.
In Vivo Anticancer Activity of Complex 6 against
CT26 Colon Cancer Cells in Vivo. To investigate the
anticancer activity of the most promising compound 6 in vivo,
female BALB/c mice were injected subcutaneously with CT26
cells into the right flank (day 0), resulting in rapid tumor
formation. This model was chosen because it is intrinsically
resistant to satraplatin and is only slightly responsive to
cisplatin treatment.59,60 Treatment with 6 was started on day 4
using 7.2 mg/kg i.p. (equimolar to 3 mg/kg for cisplatin and 5
mg/kg for satraplatin) or 20 mg/kg p.o. (a concentration that
would be equimolar to 8 mg/kg for cisplatin or 13.5 mg/kg for
satraplatin) (Figure 6a). Mice received three therapies per
week for 2 weeks. Therapy was well tolerated with no signs of
toxicity such as weight loss (data not shown). Although during
the first week there were only minor differences in tumor
volume between the treatment groups, on day 7 therapy with 6
resulted in tumor stabilization and subsequently slower tumor
progression in comparison to the solvent-treated animals.
Treatment p.o. via oral gavage had a slightly enhanced
anticancer effect in comparison to i.p. application, resulting
in significantly reduced tumor volumes (in comparison to
control) from day 15 (Figure 6b). On day 18, mice were
sacrificed and tumor weights collected. Both treatment groups
showed a significant reduction in average tumor weight (over
50%) (Figure 6c). Tumors were subsequently histologically
analyzed by H/E staining (for total morphology) and
immunohistologically analyzed for occurrence of pH2AX.
Morphological analysis of the H/E stains for cells with mitotic
and apoptotic features revealed that, while no changes in the
levels of mitotic cells were observed (data not shown), in
samples collected from mice receiving 6 i.p. significantly
enhanced apoptosis levels were detected (Figure 6d). This was
associated with higher levels of H2AX phosphorylation (Figure
6e). Overall, this activity of 6 against CT26 cells in vivo is
promising, as we have recently shown that this model is
intrinsically resistant to satraplatin.60
CONCLUSIONS
■
In the present paper, we chemically and biologically
investigated for the first time the acetato analogues of
platinum(IV) derivatives of cis-[Pt(NH3)(CHA)Cl2] with
bioactive axial ligands. Replacement of the chlorides with
acetates enhanced the water solubility of the compounds. With
the exception of the bis-DCA compound 4 all of the
compounds were very stable in buffer. Overall, the panel of
compounds showed different and partially unexpected
J
DOI: 10.1021/acs.inorgchem.9b02796
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
behaviors. On the basis of our previous experience, we
expected the rather fast reduction of compounds 1, 4, and 5
because of the axial OH ligands of 1 and 5 and the reported
fast hydrolysis of one DCA ligand in the cisplatin analogue of
4. In contrast, the considerably long half-life of 6 (in
comparison to 4) was surprising and indicated that this
compound might be attractive for further biological inves-
tigations. While replacing the chlorides with acetates in
satraplatin led to a reduction in cytotoxicity, the dual- and
triple-action analogues with equatorial acetates had low- to
sub-micromolar IC50 values in a panel of eight cancer cell lines.
We found that compound 6 was most active in cell culture on
the basis of DNA-damage-induced cell cycle inhibition and
apoptosis induction. Due to the strong differences in the
physicochemical properties, the current compound panel was
considered as inappropriate for further biological investigations
regarding the role of DCA and PhB ligands in the observed
biological activity. However, in case of the triple-action
derivative 6, this definitively needs to be done as a next step
in the preclinical evaluation and is a matter of currently
ongoing studies. Fortunately, the good activity of compound 6
against CT26 cells in vitro translated into good in vivo efficacy
against the CT26 allograft, an in vivo model with intrinsic
satraplatin resistance. This indicates that multiaction Pt(IV)
derivatives of diamine dicarboxylates are interesting anticancer
drug candidates.
with flow cytometry. The study was performed in the course of
the COST action CM1105.
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ASSOCIATED CONTENT
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* Supporting Information
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AUTHOR INFORMATION
Corresponding Authors
*P.H.: tel, +43-1-40160-57594; fax, +43-1-40160-957555; e-
mail, petra.heffeter@meduniwien.ac.at.
*D.G.: tel, +97226758702; e-mail, dang@ekmd.huji.ac.il.
ORCID
Subhendu Karmakar: 0000-0002-4840-3015
Christian R. Kowol: 0000-0002-8311-1632
Dan Gibson: 0000-0002-1631-4018
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
D.G. acknowledges the support of the Israel Science
Foundation (grant 1002/18) and the Alex Grass Center for
Drug Design and synthesis. S.K. wants to thank the Planning
and Budgeting Committee (PBC) of the Council for Higher
Education (Israel) and The Hebrew University of Jerusalem
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Hugo Aborghetti as well as Irene Herbacek for their assistance
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