Efficient one-pot synthesis of trans-Pt(ii)(salicylaldimine)(4-picoline)Cl complexes: Effective agents for enhanced expression of p53 tumor suppressor genes

Article abstract of DOI:10.1039/c5dt01098e

A series of trans-Pt(ii)(salicylaldimine)(4-picoline)Cl complexes were synthesized in 78-87% yield using a one-pot procedure from commercially available precursors. The structures of these complexes were characterized by ¹H, ¹⁹F and

Full text of DOI:10.1039/c5dt01098e

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Efficient one-pot synthesis of trans-Pt(II)-
(salicylaldimine)(4-picoline)Cl complexes: effective
agents for enhanced expression of p53 tumor
suppressor genes†
Cite this: DOI: 10.1039/c5dt01098e
Faiz-Ur Rahman,a Amjad Ali,b,c Rong Guo,a Wei-Kun Wang,a Hui Wang,*a
Zhan-Ting Li,a Yuejian Lina and Dan-Wei Zhang*a
A series of trans-Pt(II)(salicylaldimine)(4-picoline)Cl complexes were synthesized in 78–87% yield using a
one-pot procedure from commercially available precursors. The structures of these complexes were
characterized by 1H, 19F and 13C NMR spectroscopy, HRMS (ESI) as well as single crystal X-ray analysis.
Bioactivity investigations including bio-assay, time- and dose-dependent, cell cycle progression study,
caspase 3 and 9 apoptosis marker assay and DNA interaction using pBR322 plasmid DNA by gel electro-
phoresis were performed. The results indicated that the complexes showed promising in vitro cytotoxicity
in MCF-7 and A549 cancer cell lines. Moreover these complexes enhanced the expression of p53 tumor
suppressor gene family members such as p63 and p73.
Received 19th March 2015,
Accepted 23rd April 2015
DOI: 10.1039/c5dt01098e
www.rsc.org/dalton
Pt metal. Therefore the ligand is of great importance in anti-
cancer activities, side effects and also in drug’s resistance.
Introduction
p53 is known as guardian of the genomes and is one of the Presently there are three global (cisplatin, carboplatin, and
key tumor suppressor transcription factor but maintained in a oxaliplatin) and three local (nedaplatin, lobaplatin, and hepta-
low state in cancer cells.1,2 p53 regulates a number of target platin) clinically administered platinum based antitumor
genes, especially those involved in cell proliferation, cell agents (Fig. 1) used for almost all kinds of solid tumor treat-
growth and apoptosis.3–5 Other family members of p53 such ments.23 But the major associated limitations were found to be
as p63 and p736–9 also regulate a diverse network of genes that a number of side effects24 and intrinsic or timely acquired
involve in both cancer inhibition and progression.10–14 Plati- resistance in cancer patients.25,26 Therefore other platinum
num based chemotherapeutic agents including cisplatin are based antitumor agents were developed27 those with different
known to activate p53 expression in multiple cancer cells, e.g. carrier ligand(s) improved cytotoxic effect,28 selectivity,29 stabi-
MCF-7, OVCA-42915 and A549.1,16 Once p53 is activated, it lity, least side effects,20 good delivery30–33 and metal based
enhances different apoptotic pathways leading to the death of drug resistances in cancer patients.15
cancer cells.5
Aforementioned facts increase the interest of the research-
Great progress is being made in exploring the mechanistic ers in novel platinum based anticancer chemistry with strong
action of platinum based antitumor agents.17–22 The primary interest in easy synthesis, stability, favourable therapeutic
target is revealed to be DNA and this platinum–DNA inter-
action is controlled by different carrier ligands attached to the
aDepartment of Chemistry, Fudan University, 220 Handan Road, Shanghai 200433,
China. E-mail: zhangdw@fudan.edu.cn, wanghui@fudan.edu.cn;
Tel: +86 21 65643576
bInstitute of Biomedical Sciences, School of Life Sciences, East China Normal
University, 500 Dongchuan Road, Shanghai 200241, China
cUnit of GIGA Signal Transduction, GIGA-R, University of Liege, Tour GIGA +2 B34,
Avenue de I’hopital, 1, CHU SART-Tilman 4000 Liege, Belgium
†Electronic supplementary information (ESI) available: 1H, 19F, and 13C NMR,
ESI-MS spectra, stability analysis figures and crystallographic data. CCDC
1041822 (C1), 1041823 (C2) and 1041824 (C3). For ESI and crystallographic data
in CIF or other electronic format see DOI: 10.1039/c5dt01098e
Fig. 1 Approved platinum based anticancer drugs.
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indices, lower toxicity, delivery etc. Recently, we have also been
working on anticancer platinum complexes which target acti-
vation of different genomes during cancer cell apoptosis.34
The mono-18,35–38 or bi-functional39 nature of these complexes
is still a matter of further exploration but in either case bio-
activity through targeted genome activation is important,
therefore herein we mainly focused on their synthesis, in vitro
anticancer activities and genome activation effects. In this
study an efficient one-pot procedure has been developed for
the synthesis of functionalized trans-Pt(II)(salicylaldimine)-
(4-picoline)Cl complexes which were studied mechanistically
for their anticancer activities.
Fig. 2 X-ray crystal structures of C1–3.
Table 1 Selected bond lengths (Å), angles (°) and torsion angles (°) of
C1–3
Results and discussion
Entry
Bonds/angles
C1
C2
C3
Synthesis and characterization
1
2
3
4
5
6
7
8
Pt–Cl
Pt–N1
Pt–N2
Pt–O
2.287(3)
1.998(7)
2.033(7)
1.994(7)
93.5(2)
88.5(2)
93.0(3)
84.9(3)
173.4(2)
177.4(3)
8.7(7)
2.291(2)
1.996(5)
2.030(5)
2.001(5)
92.6(2)
89.3(2)
94.1(2)
2.290(4)
1.98(1)
2.01(1)
1.994(9)
92.6(3)
88.8(3)
93.2(4)
85.5(4)
174.2(3)
178.5(4)
12(1)
As shown in Scheme 1, ligands L1–8 were prepared in quanti-
tative yields from the particular salicylaldehyde and the corres-
ponding amine/aniline. Then the reaction mixture of
equivalent quantities of L1–8, K2PtCl4 and NaOAc in MeOH–
DMSO (20 : 1) was reacted for 12 h under reflux. After the
solvent was evaporated at low pressure, an excess amount
(2 drops) of 4-picoline and minimum amount of CH2Cl2 were
added to the mixture in the same pot and was stirred at room
temperature for 18 to 48 h to obtain C1–8 in excellent isolated
yields (78–87%).
∠N1PtCl
∠N2PtCl
∠N1PtO
∠N2PtO
∠OPtCl
∠N1PtN2
∠C1OPtN1
∠C1OPtN2
∠OPtN1C8
∠N2PtN1C8
84.0(2)
9
173.4(1)
177.8(2)
−4.3(5)
176.5(8)
−178.3(5)
−149(5)
10
11a
12a
13a
14a
−172.9(7)
168(6)
131(6)
−167(1)
169.1(9)
−163(14)
All these complexes were characterized by H, 19F and 13C
1
a Dihedral angles.
NMR spectroscopy, as well as HRMS (ESI). Small JPt–H values
(48–52 Hz) indicated that these complexes possess trans-
configuration, in which 4-picoline occupied the trans position
to imine nitrogen.34
crystal data and structure refinement parameters for these
complexes are summarized in the ESI.†
Selected bond lengths, angles and torsion angles of C1–3
are summarized in Table 1. On comparing the data, it was
found that these complexes possess similar crystal structures.
Bond lengths between platinum and other corresponding
elements in these complexes were comparable (entries 1–4).
Around the Pt centre in C1, the angle of ∠N1PtCl was the
biggest, while in C2 and C3, that of ∠N1PtO was the biggest,
and ∠N2PtO was the smallest in all of these complexes (entries
6–8). Pt exhibited a square planar environment because the
angles of ∠OPtCl and ∠N1PtN2 were ca. 180°, respectively
(entries 9 and 10). Small differences were observed in the di-
hedral angles around the O–Pt bond and the N1–Pt bond
(entries 11–14).
Crystallography
Single crystals of C1–3 were obtained by slow evaporation of
their CH2Cl2 solutions. The crystal structures which confirm
the trans geometry of the complexes are shown in Fig. 2. The
Anticancer activities
Cytotoxic effect analyses of complexes C1–8 in MCF-7 and
A549 cancer cell lines were performed. MTT assay results
showed that these complexes have the ability to cause signifi-
cant cell death in both cancer cell lines (Fig. 3). These results
indicate the strong cytotoxic nature of these complexes which
is further explored with the following different cytotoxic
in vitro experiments. Furthermore, we focused only on C2, C5
Scheme 1 One-pot synthesis of complexes C1–8.
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Fig. 3 Cytotoxicity screening of DMSO (negative control), cisplatin
(positive control), and C1–8 in breast MCF-7 and lung A549 cancer
cells, respectively. Cancer cells were incubated with 20 µM cisplatin and
C1–8 for 24 h. Error bars represent the average of three independent
experiments (mean s.d.).
Fig. 5 Activation of tumor suppressor genes such as p53, p63 and p73
by C2, C5 and C7 in MCF-7 cells. 20 µM of each cisplatin and Pt com-
plexes were incubated with MCF-7 cells for 24 h. DMSO was used as a
negative control and cisplatin as a positive control. Error bars represent
the average of three independent experiments (mean s.d.).
and C7 due to their comparatively higher cytotoxic activity.
Their time- and dose-dependent studies showed that C2, C5
and C7 caused significant cell death over 24 h, which was
further increased up to 72 h (Fig. 4A), and that 20 μM quan-
Fig. 6 Protein level analysis by western blot; western blot analysis was
performed for p53, p63 and p73 in cancer cells incubated with 20 µM
cisplatin, C2, C5 and C7 for 24 h. GAPDH serve as a loading control.
tities of these complexes caused significant cell death
(Fig. 4B). Both time- and dose-dependent data were quite com-
parable to those of cisplatin.
To investigate the mechanistic pathways followed by these
complexes for effective cancer cell death,40–42 activation of
tumor suppressor genes such as p53, p63 and p73 by C2, C5
and C7, respectively, was analyzed using real-time PCR
(RT-PCR). Enhanced expression of these tumor suppressor
genes was observed (Fig. 5) and similar results were obtained
for protein levels induced by these genes by western blot
(Fig. 6).
The expression of other selected p53 target genes induced
by the addition of C2, C5 and C7 was also studied. RT-PCR
analysis of p53-repressed target genes showed the abilities of
these complexes to inhibit the expression of p53-repressed
genes (Fig. 7). Moreover, a panel of genes induced by p53 was
selected and the enhancement in these genes’ expressions by
these Pt(II) complexes was also observed (Fig. 8). These results
Fig. 4 (A) Time-dependent study, 20 µM of each cisplatin, C2, C5 and
C7 were incubated with MCF-7 cells for the mentioned time and time-
dependent assays were performed. (B) Dose-dependent studies of cis-
platin, C2, C5 and C7 in MCF-7 cells. Cancer cells were incubated with
the mentioned complexes at given concentration for 24 h and dose-
dependent MTT assays were performed. Error bars represent the
average of three independent experiments (mean s.d.).
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Fig. 7 C2, C5 and C7 enhanced repression of p53 downregulated
genes in MCF-7 cells. 20 µM of each cisplatin, C2, C5 and C7 were incu-
bated with cancer cells for 24 h and p53 target genes were analyzed by
RT-PCR. Error bars represent the average of three independent experi-
ments (mean s.d.).
Fig. 8 Cisplatin C2, C5 and C7 enhanced induction of p53-upregulated
genes in MCF-7 cells. 20 µM of each of each Pt complex was incubated
with MCF-7 cells for 24 h and p53 target genes were analyzed by
RT-PCR. Error bars represent the average of three independent experi-
ments (mean s.d.).
confirmed that C2, C5 and C7 have the potential properties of
activation of p53 and in turn regulate p53-dependent target
gene expressions that result in cancer cells apoptosis.
The cell cycle progression arrest properties were also inves-
tigated and experimental results showed their potency for cell
cycle arrest. It is observed that C2, C5 and C7 can arrest the
DNA resting G0/G1 phase; in contrast these complexes inhibit
the DNA synthesis in the S phase and mitotic G2/M phase
(Fig. 9) leading to inhibition of cancer progression.
Fig. 9 Cell cycle progression studies of cisplatin, C2, C5 and C7 in
MCF-7 cells figure shows G0/G1, S and G2/M phases. (A) Original flow
cytometry data. (B) Representative bar graph of the data of 9A. MCF-7
cells were treated with 20 µM of each mentioned complex for 24 h.
Caspase 3 and 9 are cell death markers, activated during
cell apoptosis. The expression of these markers in response to
the addition of Pt(II) complexes in cancer cells was analyzed.
Both caspase 3 and caspase 9 (Fig. 10) were activated. These
results showed that C2, C5 and C7 enhanced apoptotic path-
ways and thereby caused cancer cell death.
To check the interaction of C2, C5 and C7 with DNA, a gel
electrophoresis study was performed using pBR322 plasmid
DNA. Slow migration of the DNA incubated with cisplatin and
these complexes confirmed their strong interaction with DNA
(Fig. 11) and formed the Pt–DNA adduct. The formation of this
metal DNA adduct inside cancer cells leads to cell death.
In this scenario, the results of synthesis and the discussed
cytotoxic profile of these complexes enabled us to conclude
them in comparison with the other previously reported plati-
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Conclusion
In conclusion eight novel trans-Pt(II)(salicylaldimine)(4-pico-
line)Cl complexes were prepared using an efficient one-pot
1
procedure and structurally characterized using H, 19F and 13C
NMR spectroscopy, HRMS (ESI) as well as single crystal X-ray
analysis. These complexes showed comparative cytotoxicity to
the clinically administered anticancer drug cisplatin. These
newly synthesized platinum complexes probably hold the
advantages of easy and efficient one-step synthesis from com-
mercially available materials, their higher stabilities and com-
parable cytotoxicities to commercially available drugs. Detailed
biological studies suggested that these Pt(II) complexes posses-
sing strong anticancer activities could be used in the develop-
ment of effective anticancer drug candidates.
Experimental
General
All analytical grade solvents and reagents were purchased from
commercial sources and used without further purification
unless otherwise mentioned. K2PtCl4 was purchased from
Sinopharm Chemical Company. pBR322 DNA plasmid was
purchased from Beijing Think-Far Technology Company
Limited. Preparative thin layer chromatography (TLC) was per-
Fig. 10 Activation of caspase 3 and 9 signaling by cisplatin, C2, C5 and
C7, in MCF-7 cells. (A) Original experimental data images. (B) Band inten-
sity plot; 20 µM of each cisplatin and Pt complexes were incubated with
MCF-7 cells for 24 h. Band intensities were analyzed by using myImage
analysis software (Thermo Scientific, Life Technologies). CTL and DMSO
were used as calibrators.
1
formed on precoated silica gel plates (0.4–0.5 mm thick). H,
19F and 13C NMR analyses were conducted using a Bruker
AVANCE III HD 400 MHz spectrophotometer at room tempera-
ture (298 K) in CDCl3 or D2O/DMSO-d6. Positive ion mass
spectra (ESI-MS) were obtained on a Bruker micrOTOF II. X-ray
data were collected on a Bruker APEX DUO diffractometer with
graphite-monochromatized Mo Kα radiation (λ = 0.71073 Å).
All the structures were solved by direct methods of SHELXS-97
and refined by full-matrix least squares techniques using
SHELXL-97 program within WINGX.55,56 Non-hydrogen atoms
of the crystallized compounds were refined with anisotropic
temperature parameters. The hydrogen atoms attached to
carbons were generated geometrically.
Fig. 11 DNA interaction study of cisplatin, C2, C5 and C7 by gel elec-
trophoresis. Cisplatin and the mentioned complexes 20 µM were incu-
bated with pBR322 plasmid DNA at 37 °C for 24 hours and analysed by
gel electrophoresis. DMSO was used as a negative control and cisplatin
as a positive control.
Preparation and characterization
num based cytotoxic agents. The most important fact about
this class of complexes is their easy synthesis from commer-
cially available cheaper raw materials and high stability in
solid as well as in the dissolved state. The other important pro-
perties are their comparative cytotoxic effect and the activation
of tumor suppressor genes (p53). Tumor suppressor gene p53
plays a key role in the cellular responses to genotoxic stress,43
play an important role in cancer cell apoptosis and may also
have a role in DNA repair.44–47 Several other groups showed
the importance of platinum complexes in activation of
p53 genes.48,49 Similarly cisplatin,50,51 oxaliplatin52,53 and car-
boplatin54 are studied for their important p53 gene activation
properties. C2, C5 and C7 caused a programmed cancer cell
death by activating the apoptotic pathways which could be an
important feature of these complexes.
General procedure for the synthesis of complexes C1–
8. A mixture of particular ligands (L1–L8, 0.05 mmol), K2PtCl4
(0.05 mmol), and NaOAc (0.05 mmol) in MeOH–DMSO (20 : 1)
was stirred at reflux temperature. After 12 h (when all ligands
were consumed) the volatiles were evaporated to dryness under
reduced pressure. The yellow gummy material recovered was
dissolved in 6 mL of CH2Cl2, an excess amount of 4-picoline
(2 drops) was added to the mixture and further stirred for
18–48 h (checked by TLC eluted with 1% MeOH–CH2Cl2). After
completion, the reaction mixture was diluted with CH2Cl2 and
washed thoroughly with water to remove all the inorganic
salts. The organic portion was dried with anhydrous sodium
sulphate and vacuum evaporated. The yellow to orange solid
(C1–C8) obtained was dissolved again in a minimum amount
of CH2Cl2 and applied to a short (1.5 × 3 cm) silica column
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eluted with 1% MeOH–CH2Cl2. A pure yellow to orange solid
(C1–C8) was obtained in excellent yield.
Characterization of C1.
Characterization of C4.
1
80% yield, yellow solid. H NMR (400 MHz, CDCl3) δ 8.69 (d,
J = 6.7 Hz, 2H), 7.92 (s, JPt–H = 56 Hz, 1H), 7.48–7.35 (m, 3H),
7.34–7.24 (m, 4H), 7.17 (d, J = 6.1 Hz, 2H), 6.96 (d, J = 8.6 Hz,
1H), 6.66 (t, J = 7.4 Hz, 1H), 2.41 (s, 3H). 13C NMR (100 MHz,
CDCl3) δ 162.7, 159.7, 153.2, 151.7, 150.4, 135.0, 134.3, 128.3,
126.9, 125.7, 124.6, 120.5, 120.3, 116.8, 21.1. HRMS (ESI):
calcd for C19H18ClN2OPt 520.0755: [M + H]+, found: 520.0738.
Characterization of C5.
1
84% yield, orange solid. H NMR (400 MHz, CDCl3) δ 8.69 (d,
J = 6.7 Hz, 2H), 7.60 (s, JPt–H = 48 Hz, 1H), 7.21–7.13 (m, 3H),
7.07 (s, 1H), 6.82 (d, J = 8.6 Hz, 1H), 3.84 (d, J = 6.8 Hz, 2H),
2.59–2.44 (m, 1H), 2.41 (s, 3H), 2.25 (s, 3H), 0.94 (d, J = 6.7 Hz,
6H). 13C NMR (100 MHz, CDCl3) δ 160.6, 158.5, 151.6, 150.2,
135.8, 133.0, 125.6, 125.1, 119.8, 119.3, 72.1, 29.9, 21.1, 20.0,
19.6. HRMS (ESI): calcd for C18H24ClN2OPt 514.1225: [M + H]+,
found: 514.1206.
Characterization of C2.
1
80% yield, orange solid. H NMR (400 MHz, CDCl3) δ 8.68 (d,
J = 5.5 Hz, 2H), 7.91 (s, JPt–H = 52 Hz, 1H), 7.44 (t, J = 7.7 Hz,
1H), 7.32 (d, J = 7.9 Hz, 1H), 7.30–7.21 (m, 2H), 7.17 (d, J = 5.9
Hz, 2H), 7.06 (t, J = 8.5 Hz, 2H), 6.96 (d, J = 8.6 Hz, 1H), 6.67 (t,
J = 7.4 Hz, 1H), 2.42 (s, 3H). 19F NMR (376 MHz, CDCl3) δ
1
80% yield, yellow solid. H NMR (400 MHz, CDCl3) δ 8.70 (d,
J = 6.6 Hz, 2H), 7.86 (s, JPt–H = 52 Hz, 1H), 7.44–7.34 (m, 2H),
7.32–7.26 (m, 3H), 7.25 (d, J = 2.4 Hz, 1H), 7.16 (d, J = 6.1 Hz,
2H), 7.09 (s, 1H), 6.89 (d, J = 8.7 Hz, 1H), 2.41 (s, 3H), 2.26 (s,
3H). 13C NMR (100 MHz, CDCl3) δ 161.0, 159.6, 153.3, 151.6,
150.3, 136.7, 133.2, 128.2, 126.8, 125.8, 125.7, 125.5, 124.6,
120.0, 21.1, 20.0. HRMS (ESI): calcd for C20H20ClN2OPt
534.0912: [M + H]+, found: 534.0897.
−115.77. 13C NMR (100 MHz, CDCl3) δ 162.8, 161.2 (d, JC–F
=
246.1 Hz), 159.9, 151.6, 150.4, 149.3, 135.2, 134.3, 126.1 (d, JC–F
= 8.4 Hz), 125.7, 120.4, 116.8, 115.0 (d, JC–F = 22.8 Hz), 21.1.
HRMS (ESI): calcd for C19H17ClFN2OPt 538.0656: [M + H]+,
found: 538.0655.
Characterization of C6.
Characterization of C3.
1
78% yield, yellow solid. H NMR (400 MHz, CDCl3) δ 8.66 (d,
1
80% yield, orange solid. H NMR (400 MHz, CDCl3) δ 8.69 (d, J = 6.6 Hz, 2H), 7.60 (s, JPt–H = 52 Hz, 1H), 7.17 (d, J = 6.1 Hz,
J = 6.7 Hz, 2H), 7.85 (s, JPt–H = 52 Hz, 1H), 7.27 (d, J = 2.3 Hz, 2H), 7.14–7.07 (m, 1H), 6.96 (dd, J = 8.9, 3.2 Hz, 1H), 6.82 (dd,
1H), 7.26–7.21 (m, 2H), 7.17 (d, J = 6.3 Hz, 2H), 7.11–7.01 (m, J = 9.2, 4.6 Hz, 1H), 3.86 (d, J = 6.8 Hz, 2H), 2.58–2.45 (m, 1H),
3H), 6.88 (d, J = 8.7 Hz, 1H), 2.41 (s, 3H), 2.26 (s, 3H). 19F NMR 2.42 (s, 3H), 0.94 (d, J = 6.7 Hz, 6H). 19F NMR (376 MHz,
(376 MHz, CDCl3) δ −115.93. 13C NMR (100 MHz, CDCl3) CDCl3) δ −130.59. 13C NMR (100 MHz, CDCl3) δ 159.0, 157.7,
δ 161.1 (d, JC–F = 247.4 Hz), 161.1, 159.7, 151.6, 150.4, 149.3, 153.9 (d, JC–F = 234.3 Hz), 152.8, 151.5, 150.4, 125.6, 122.4 (d,
136.9, 133.2, 125.7, 125.6, 126.1 (d, JC–F = 8.4 Hz), 120.1, 119.8, JC–F = 24.1 Hz), 120.7 (d, JC–F = 7.5 Hz), 116.7 (d, JC–F
=
115.0 (d, JC–F = 22.8 Hz), 21.1, 20.0. HRMS (ESI): calcd for 22.2 Hz), 72.3, 29.9, 21.1, 19.5. HRMS (ESI): calcd for
C20H19ClFN2OPt 552.0818: [M + H]+, found: 552.0810.
C17H21ClFN2OPt 518.0969: [M + H]+, found: 518.0961.
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Characterization of C7.
Celle cuture
Cancer cells were incubated in DMEM, supplemented with
10% FBS and maintained at 37 °C and 5% CO2.
Antibodies
The following antibodies were used in western blot experi-
ments: anti-p53 (Santa Cruz), anti-p63 (Abcam), anti-GAPDH
(Santa Cruz), anti-p73 (Santa Cruz). Caspase-3 and caspase-9
(Chemicon), which recognize only the cleaved large subunits
(17 kDa of caspase-3 and 37 kDa of caspase-9), were used in
this study.
1
87% yield, orange solid. H NMR (400 MHz, CDCl3) δ 8.67 (d,
J = 6.7 Hz, 2H), 7.86 (s, JPt–H = 48 Hz, 1H), 7.40 (dd, J = 9.9,
5.5 Hz, 2H), 7.33–7.27 (m, 3H), 7.24–7.14 (m, 3H), 6.99 (dd, J =
8.8, 3.2 Hz, 1H), 6.90 (dd, J = 9.3, 4.6 Hz, 1H), 2.42 (s, 3H). 19F
NMR (376 MHz, CDCl3) δ −129.96. 13C NMR (100 MHz, CDCl3)
δ 159.4, 158.8, 155.2, 152.9, 151.8 (d, JC–F = 156.5 Hz), 151.6,
128.3, 127.1, 125.7, 124.5, 123.5 (d, JC–F = 24.2 Hz), 121.6 (d,
JC–F = 7.5 Hz), 119.2 (d, JC–F = 7.7 Hz), 116.8 (d, JC–F = 22.3 Hz),
21.1. HRMS (ESI): calcd for C19H17ClFN2OPt 538.0656:
[M + H]+, found: 538.0650.
RT-PCR
Total RNA from cells was isolated using TRIZOL (Invitrogen),
following manufacturer’s protocol. Briefly, 0.5–1 µg of total
RNA was reverse transcribed in a total volume of 25 µL, includ-
ing 132 units of Moloney-murine-leukemia virus reverse tran-
scriptase, 26.4 units of RNAase inhibitor, 0.6 µg of (dT) 15
primer, 2 µM dNTPs, and 1× Moloney-murine-leukemia virus
RT buffer provided by Promega (Madison, WI). Aliquots of the
RT products were used for RT-PCR analysis. For qRT-PCR 2 µL
of reverse transcribed cDNA was subjected to RT-PCR using
master mix SYBR-Green (BIORAD) and the Mx3005P-quantitat-
ive RT-PCR system (Stratagene). Each reaction consisted of
SYBR Green (1 : 60 000 final concentration), 40 nM of both
sense and antisense primers, 2 µL of cDNA and H2O to a final
volume of 20 µL.
Characterization of C8.
1
85% yield, orange solid. H NMR (400 MHz, CDCl3) δ 8.65 (d,
J = 6.7 Hz, 2H), 7.85 (s, JPt–H = 48 Hz, 1H), 7.26–7.19 (m, 3H),
7.18 (d, J = 6.0 Hz, 2H), 7.06 (t, J = 8.5 Hz, 2H), 6.98 (dd, J = 8.8,
3.1 Hz, 1H), 6.90 (dd, J = 9.3, 4.5 Hz, 1H), 2.42 (s, 3H). 19F
Preparation of total cell extract and western blot analysis
Cells were washed with phosphate buffered saline and treated
with an extraction buffer (50 mM Tris-HCl, pH 7.4, 1%
Nonidet P-40, 0.25% sodium-deoxycholate, 150 mM NaCl, and
1 mM EDTA) supplemented with 1 mM phenylmethane sulfo-
nyl fluoride, 1 mM sodium-orthovanadate (Na3VO4), 0.1 mM
dithiothreitol, 0.4 µg mL−1 leupeptin/pepstatin). Cell extract
was stored at −20 °C until required. Protein samples were
subject to electrophoresis in 10% SDS polyacrylamide-gel. Sep-
arated proteins were electroblotted to nitrocellulose mem-
branes (Bio-Rad), and the blot was blocked for 1 h at room
temperature with 0.1% PBST blocking buffer with 5% fat-free
dried milk powder. The blot was then incubated with primary
antibodies (1 : 1000 dilutions) at 4 °C overnight, washed with
0.1% TBST 3 times, and incubated with secondary antibodies
(mouse, rabbit) (1 : 5000 dilution) for 1 h. It was washed again
3 times and exposed to an Odyssey LI-COR-scanner.
NMR (376 MHz, CDCl3)
(100 MHz, CDCl3) δ 161.2 (d, JC–F = 246.2 Hz), 159.4, 159.0,
154.1 (d, JC–F = 234.7 Hz), 151.6, 150.6, 149.1, 126.0 (d, JC–F
8.5 Hz), 125.8, 123.7 (d, JC–F = 24.4 Hz), 121.7 (d, JC–F = 7.5 Hz),
119.0 (d, JC–F = 7.9 Hz), 116.7 (d, JC–F = 22.3 Hz), 115.1 (d, JC–F
δ
−115.44, −129.79. 13C NMR
=
=
22.9 Hz), 21.1. HRMS (ESI): calcd for
556.0561: [M + H]+, found: 556.0557.
C19H16ClF2N2OPt
Stability of Pt(II) complexes
The stability of a chemotherapeutic agent is crucial in cytotoxic
studies, so we perfomed stability analysis experiments for C2,
C3 and C5 as model complexes using 1H NMR spectroscopy in
10–15% D2O–DMSO-d6 mixture at room temperature over 0,
24, 48, and 72 h. The comparative spectra are shown in the
ESI† which show that these compounds are quite stable under
the mentioned conditions (Fig. S51–53†).
MTT assay
Cell viability was assessed with a 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium-bromide (MTT) assay in replicates.
Cells were seeded in 96-well plates at 2.5 × 103 cells per well,
Biological study
All biological experiments were performed using our pre- and cultured in DMEM supplemented with 10% FBS. After
viously reported methods with little modifications.34,57,58 Error that, the cells were treated with DMSO (0.5% was the final con-
bars represent the average of the three independent experi- centration of DMSO used in cell culture media), cisplatin or a
ments (mean
controls.
s.d.). Cells with no DMSO were used as particular platinum complex (C1–C8) for the mentioned time
(figures). The drug-containing medium was replaced with
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200 µL of DMEM supplemented with 10% FBS containing
0.5 mg mL−1 MTT reagent and the cells were incubated in a
CO2 incubator at 37 °C for 2 h. The quantity of formazan
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Cell-cycle analysis was carried out to estimate DNA-contents by
flow cytometry as described previously.46 Cells were fixed in ice
cold 70% ethanol, incubated overnight at −20 °C and stained
with propidium-iodide (PI)/Triton X-100 containing RNaseA-
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Gel electrophoresis34
The DNA binding properties of cisplatin C2, C5 and C7 were
investigated by agarose gel electrophoresis. pBR322 plasmid
DNA (50 ng μL−1) was used as the target. Appropriate dilutions
of the tested compounds were made, and the required
volumes of solutions were added to achieve a set of concen-
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24 hours. Afterwards, the agarose gel (up to 1% w/v) contain-
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for 40 min. The obtained bands were imaged using a Mole-
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Acknowledgements
The authors acknowledge the Science and Technology Com-
mission of Shanghai Municipality (13M1400200), Ministry of
Science and Technology of the People’s Republic of China
(2013CB834501), the National Natural Science Foundation of
China (no. 21102017) and China Postdoctoral Association for
providing postdoctoral fellowship.
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