Platinum(II) complexes containing aminophosphonate esters: Synthesis, characterization, cytotoxicity and action mechanism

Article abstract of DOI:10.1016/j.ejmech.2013.04.024

New platinum(II) complexes containing aminophosphonate ester were synthesized and fully characterized, which were found to possess better solubility in both organic solvents and water than cisplatin. These platinum(II) complexes exhibited considerable cyt

Full text of DOI:10.1016/j.ejmech.2013.04.024

European Journal of Medicinal Chemistry 64 (2013) 554e561
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European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Platinum(II) complexes containing aminophosphonate esters:
Synthesis, characterization, cytotoxicity and action mechanism
,
,
b
b
b
Ke-Bin Huang a b, Zhen-Feng Chen b , Yan-Cheng Liu , Zhu-Quan Li , Jian-Hua Wei ,
**
*
Meng Wang b, Xiao-Li Xie b, Hong Liang a
a School of Chemistry of Nankai University, Tianjin 300071, PR China
b Guangxi Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources, School of Chemistry & Chemical Engineering of Guangxi
Normal University, Yucai Road 15, Guilin 541004, Guangxi, PR China
,b,
a r t i c l e i n f o
a b s t r a c t
Article history:
New platinum(II) complexes containing aminophosphonate ester were synthesized and fully charac-
terized, which were found to possess better solubility in both organic solvents and water than cisplatin.
These platinum(II) complexes exhibited considerable cytotoxicity against tumor cells MG-63, SK-OV-3,
HepG2, BEL-7404 and low cytotoxicity to normal human liver cells HL-7702. Their antitumor activities
were achieved through the induction of cell apoptosis and the cell cycle arrest at G1 phase. The elec-
trophoretic mobility studies and CD spectral analysis revealed that the binding mode of complex 6 to
DNA might be different from that of cisplatin.
Received 2 January 2013
Received in revised form
6 April 2013
Accepted 9 April 2013
Available online 17 April 2013
Keywords:
Ó 2013 Elsevier Masson SAS. All rights reserved.
Aminophosphonate ester
Platinum(II) complexes
Synthesis
Crystal structure
Cytotoxicity
Action mechanism
1. Introduction
coordinate platinum and targeted drugs have been designed as an
important strategy to overcome the side effects of cisplatin [7,8].
Despite cisplatin (cis-diamminedichloroplatinum(II), CDDP)-
based chemotherapy is curative for testicular germ cell tumors
(TGCT) and constitutes a component of standard treatment regimes
for ovarian, cervical, bladder, head and neck, small cell and non-
small-cell lung cancers, the development of platinum drugs with
improved antitumor activity continues to be a productive field of
research [1,2]. Currently, the main focus concentrates on designing
cytotoxic agents possessing either oral bioavailability, fewer side
effects, or being able to circumvent intrinsic or acquired CDDP
resistance, which is a major clinical problem [3,4]. To achieve these
goals, chemists employed different strategies for the development
of new platinum anticancer agents with different action mecha-
nisms and broader ranges of antitumor activity [5,6]. Especially, in
the past two decades, new functional ligands have been used to
Because alkaline phosphatase is known to be overexpressed in
the extracellular space of specific tumor cells such as ovarian and
hepatic carcinoma cells [9,10] and the phosphate groups also exhibit
high affinity to calcium ions, introduction of a phosphate group has
been used to design targeted cisplatin analogs [11,12]. Since phos-
phonate esters can be hydrolyzed under biological conditions,
phosphonate esters were used as a strategy to increase solubilityand
enhance transport through cellular membrane [11,12]. Platinum
complexes incorporating functional phosphoric moieties for tar-
geting cancer cells were first reported by Keppler and co-workers in
the early 1990s [13]. More recently, Natile, Bose and Guo also re-
ported platinum phosphonate complexes and found that some of
them had a cytotoxic mechanism different from that of cisplatin
[7,8,14,15]. However, except for Guo’s work, most of published re-
searches attached phosphonate groups to the platinum center as
leaving groups. When the functional moieties are attached to plat-
inum as leaving groups, they may detach during the complicated
physiological process even before reaching the targeted tissues. As a
result, the functional groups of the complexes will be lost. Therefore,
one of the key factors for cancer targeting is to use non-leaving
functional moieties to guide the platinum to specific tissues.
* Corresponding author. Tel./fax: þ86 773 2120958.
** Corresponding author. School of Chemistry of Nankai University, Tianjin
300071, PR China.
E-mail addresses: chenzfubc@yahoo.com, chenzfgxnu@yahoo.com (Z.-F. Chen),
hliang@gxnu.edu.cn (H. Liang).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.04.024

K.-B. Huang et al. / European Journal of Medicinal Chemistry 64 (2013) 554e561
555
Aminophosphonic acids are structural analogs of natural ami-
nocarboxylic acid. The functionalized aminophosphonic acids,
especially, -aminoalkyl-phosphonic derivatives, are a class of
a
important compounds that exhibit intriguing biological activities in
the pharmacological and agrochemical fields [16e19]. We have
designed and synthesized a series of platinum(II) complexes con-
taining aminophosphonate ester groups recently [20]. As the
continuation of this series of researches, herein, we reported six
platinum complexes containing new functional ligands of a-ami-
noalkyl-phosphonate ester derivatives as non-leaving groups. The
anticancer structureeproperty relationship was established for
platinum(II) complexes with various alkyl chain lengths (CH2)m
and three electron-donating and lipophilic methoxy substituents
(R1eR3), in which these amides are known to have the potential to
induce cell apoptosis [21,22].
2. Results and discussion
2.1. Synthesis
The aminophosphonate ester derivatives (LaeLf) were prepared
from pyridinealdehyde, diethylphosphite and various amines via
one-step synthetic route (Scheme 1). The products were charac-
terized by elemental analysis, 1H NMR, 13C NMR and ESI-MS
spectroscopy.
Scheme 2. Synthesis route of the platinum(II) complexes.
Corresponding cis-dichloroplatinum(II) complexes 1e6 were
obtained by reacting cis-Pt(DMSO)2Cl2 with LaeLf in anhydrous
dichloromethane and ethanol (1:1), respectively (Scheme 2).
The synthesized platinum(II) complexes were characterized by
elemental analysis, 1H NMR, 13C NMR and ESI-MS spectroscopy
as well as single crystal X-ray diffraction analysis (for complexes
1, 3, 5, 6).
2.3. In vitro cytotoxic activity
The in vitro cytotoxicities of complexes 1e6 against MG-63,
SK-OV-3, HepG2, BEL-7404 and HL-7702 cell lines were investi-
gated by MTT method and compared with those of cisplatin. As
shown in Table 1, the IC50 values of complexes 1e6 against tumor
cell lines MG-63, SK-OV-3, HepG2 were higher than that of
cisplatin. However, they exhibited lower IC50 (higher cytotoxicity)
toward BEL-7404 than cisplatin. Complex 6 showed the highest
cytotoxicity against BEL-7404 cell line. In addition, complexes 1e6
displayed lower cytotoxicities to normal human liver cells HL-7702
than to the tested tumor cells, and there was about an order of
magnitude of difference. Comparison of the cytotoxicity of com-
plexes 1 and 4, 2 and 5, 3 and 6 against the BEL-7404 tumor cells
indicated that the presence of three methoxy groups at the C3, C4
and C5 of the benzene ring enhanced cytotoxicity. Similar trends
were observed for complexes 1 and 4, 2 and 5, 3 and 6 toward the
other four tested tumor cell lines. Therefore, structures with the
shortest distance of the phenyl ring to the platinum complex and
the presence of the methoxy groups at the C3, C4 and C5 of the
benzene ring exhibited the highest cytotoxicity.
2.2. Structures of platinum(II) complexes
The crystal structures of complexes 1, 3, 5 and 6 indicated that
the platinum centers adopt an approximately square-planar ge-
ometry in which the dihedral angle between the pyridyl ring and
Pt(II) coordination plane is 13.2ꢀ (complex 1), 9.2ꢀ (complex 3),
14.7ꢀ (complex 5) and 7.1ꢀ (complex 6). Selected bond lengths and
angles are given in the captions of Figs. 1 and 2, which are within
the normal range expected for Pt(II) complexes. One notable
feature is that the carbon atom attached to P forms a half chair
conformation with respect to the platinum coordination plane in all
complexes, which is consistent with the steric interaction between
the phosphonate ester moiety and the platinum coordination
plane. The major structural distinctions among the complexes are
the presence or absence of the three electron-donating and lipo-
philic methoxy groups at the C3, C4 and C5 of the benzene ring, and
the different spatial separations of the benzene ring from the
platinum coordination plane. Additionally, complexes 1e6 exhibi-
ted higher solubility in both organic solvents and water compared
with cisplatin.
2.4. Apoptosis study by flow cytometry
Apoptosis is the programmed cell death that controls the
development and homeostasis of multicellular organisms by
elimination of aged, damaged, or mutated cells, which has been
shown to be the key cellular event responsible for the anticancer
activity of most of the anticancer drugs [23]. To determine whether
the observed cell death induced by the complexes was due to
apoptosis, the interaction of BEL-7404 cells with complex 6 was
further investigated using an Annexin V-FITC/propidium iodide
assay (Fig. 3). As phosphatidylserine (PS) exposure usually precedes
loss of plasma membrane integrity in apoptosis, the presence of
annexin Vþ/PIꢁ cells is considered as an indicator of apoptosis.
When treated with complex 6, the population of annexin Vþ/PIꢁ
cells (Q4) was 50.8%, which suggested that apoptotic death was
induced in BEL-7404 cells.
Scheme 1. Synthesis route of aminophosphonate esters.

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K.-B. Huang et al. / European Journal of Medicinal Chemistry 64 (2013) 554e561
ꢀ
ꢀ
Fig. 1. ORTEP drawings of 1 (left) and 3 (right). The hydrogen atoms have been omitted for clarity. Selected bond lengths (A) and angles ( ) for 1: Pt(1)eN(1) 2.0191(71), Pt(1)eN(2)
2.0555(74), Pt(1)eCl(2) 2.3089(24), Pt(1)eCl(1) 2.2830(21); N(1)ePt(1)eN(2) 171.53(24), N(1)ePt(1)eCl(2) 95.69(23), N(1)ePt(1)eCl(1) 171.53(24), N(2)ePt(1)eCl(2) 177.66(21),
N(2)ePt(1)eCl(1) 89.55(21), Cl(2)ePt(1)eCl(1) 92.75(9); for 3: Pt(1)eN(1) 2.0069(84), Pt(1)eN(2) 2.0699 (102), Pt(1)eCl(2) 2.2857 (30), Pt(1)eCl(1) 2.2953 (33); N(1)ePt(1)eN(2)
83.48(34), N(1)ePt(1)eCl(2) 173.43 (29), N(1)ePt(1)eCl(1) 94.10 (27), N(2)ePt(1)eCl(2) 90.55 (25), N(2)ePt(1)eCl(1) 177.54 (22), Cl(2)ePt(1)eCl(1) 91.84 (12).
2.5. Mitochondrial membrane potential detection
genetically regulated, evolutionarily conserved process with
numerous links to many human diseases, most notably cancer [26].
Caspase-3 is able to directly degrade multiple substrates including
structural and regulatory proteins. Thus, therapeutic strategies
designed to stimulate apoptosis by activating Caspase-3 may help
in combating cancer caused by apoptosis deficiency. Some small
molecules have been developed to be selective activators of
Caspase-3. We have therefore investigated whether the Caspase-3
was activated when BEL-7404 cells were exposed to complex 6.
As shown in Fig. 5, cells treated with complex 6 had a significant
increase in the activity of caspase-3 as indicated by a notable shift
in the ratio of green/dark fluorescence versus control.
Mitochondria act as a point of integration for apoptotic signals
originating from both the extrinsic and intrinsic apoptotic pathways.
Mitochondrial dysfunction and the release of apoptogenic factors are
critical events in triggering various apoptotic pathways [24,25]. Loss
of mitochondrial membrane potential is an important indicator of
mitochondrial dysfunction. To delineate the importance of mito-
chondria in complex 6 induced apoptosis, the changes of mito-
chondrial membrane potential were measured by the JC-1 probe,
which was dispersed from aggregated form (red fluorescence) to the
monomeric form (green fluorescence) when mitochondrial mem-
brane potential was lost. As shown in Fig. 4, cells treated with
complex 6 exhibited a significant decrease in mitochondria mem-
brane potential as indicated by a notable shift in the ratio of green/
red fluorescence versus control. These results suggested that mito-
chondria were involved in the initiation of apoptosis.
2.7. Cell cycle analysis
The cell cycle is a set of events that result in cell growth and
division into two daughter cells. The cell cycle is divided into G1, S,
G2, and M stages. To determine whether the suppression of cancer
cell growth by the platinum complexes was caused by cell cycle
arrest, the BEL-7404 cells were treated with complex 6 and cisplatin
respectively at their IC50 concentrations and the cell cycle phases
were assayed via assessing the DNA content of cells stained with
2.6. Caspase-3 activation assay
The Caspase family is well characterized as playing a crucial
role in modulation of programmed cell death (PCD), which is a
ꢀ
ꢀ
Fig. 2. ORTEP drawing of complexes 5 (left) and 6 (right), and the hydrogen atoms have been omitted for clarity. Selected bond lengths (A)angles ( ), for 5: Pt(1)eN(1) 1.9965 (118),
Pt(1)eN(2) 2.0560(90), Pt(1)eCl(1) 2.3045(35), Pt(1)eCl(2) 2.2928(32), N(1)ePt(2)eN(2) 81.93(38), Cl(1)ePt(2)eN(1) 173.60(30), Cl(2)ePt(2)eN(1), 95.19 (30), N(2)ePt(2)eCl(1)
92.21(27), N(2)ePt(2)eCl(2) 176.71(28), Cl(1)ePt(2)eCl(2) 90.75(14); for 6: Pt(1)eN(1) 2.0045(82), Pt(1)eN(2) 2.0571(68), Pt(1)eCl(1) 2.2908(23), Pt(1)eCl(2) 2.2866(26), N(1)e
Pt(1)eN(2) 83.65(32), N(1)ePt(1)eCl(1) 94.77(22), N(1)ePt(1)eCl(2) 174.70(21), Cl(1)ePt(1)eN(2) 178.31(22), Cl(2)ePt(1)eN(2) 91.07(25), Cl(2)ePt(1)eCl(2) 90.51(10).

K.-B. Huang et al. / European Journal of Medicinal Chemistry 64 (2013) 554e561
557
Table 1
IC50 M) values for complexes 1e6 against four human tumor cells and normal
human liver cells.
formation or changes in environment [28,29]. The normal ct-DNA
has a right-handed chiral conformation and exists in B-form in so-
lution. The CD spectrum of DNA is very sensitive to its conforma-
tional changes. It is generally accepted that covalent binding and
intercalative binding can influence the tertiary structure of DNA and
induce changes in the CD spectra of DNA, whereas other non-
covalent binding modes such as electrostatic interaction or groove
binding does not significantly perturb the CD spectra [30].
(
m
Complex
MG-63
SK-OV-3
HepG2
BEL-7404
HL-7702
1
2
3
4
5
6
21.2
18.3
>50
22.3
25.2
21.4
9.5
45.3
22.5
48.2
32.5
>50
18.2
6.4
25.5
>50
42.5
36.2
18.2
25.2
11.5
26.5
32.1
24.8
21.2
17.6
12.5
132.8
123.7
148.5
158.1
98.5
114.9
105.4
86.0
The CD spectrum of ct-DNA shows a positive absorbance peak at
ca. 270 nm and a negative absorbance peak at ca. 245 nm, due to the
Cisplatina
pep base stacking of DNA and the right-hand helicity of B-form
a
Cisplatin was used as positive control.
DNA, respectively [31]. As shown in Fig. S1 (see ESI), the addition of
complex 6 at the concentration of 5 ꢂ 10ꢁ6 M to 2 ꢂ 10ꢁ4 M did not
perturb the positive absorbance of DNA obviously while the
negative absorbance of DNA decreased. The changes in the CD
spectra of the DNA upon addition of cisplatin were different from
that induced by the addition of complex 6, which strongly sug-
gested that complex 6 has different binding mode toward ct-DNA.
The CD result is in agreement with the pUC19 plasmid DNA assay.
propidium iodide as measured by flow cytometry. The flow cyto-
metric data for the BEL-7404 cells treated with complex 6 and
cisplatin are shown in Fig. 6. Treatment of cells with complex 6 for
24 h enhanced cell-cycle arrest at the G1 phase, resulting in pop-
ulation increase in the G1 phase (70.65%) compared with the
control cells (35.36%), and the populations of the S and G2 phase
decreased only to a certain extent compared with the control cells.
We speculated that the decrease of populations in S and G2 phases
was the direct result of the population increase in G1 phase.
However, cisplatin resulted in population increase in the S phase
(48.11% and 35.36% for cisplatin and control, respectively), which is
in agreement with the literature studies regarding cell cycle arrest
by cisplatin [27]. Therefore, the mechanism of the suppression of
tumor cells’ growth by the title platinum(II) complexes might be
different from that of cisplatin.
3. Conclusions
Six new mononuclear platinum complexes 1e6 containing
aminophosphonate esters as the non-leaving group were synthe-
sized and fully characterized. Cytotoxicity assay of complexes 1e6
against MG-63, SK-OV-3, HepG2, BEL-7404 and HL-7702 cells
showed that complex 6 exhibited the highest cytotoxicity against
BEL-7404 cell line, which could be attributed to the structural
features including the shortest separation phenyl ring from the
platinum complex and the presence of methoxy groups at the C3, C4
and C5 of the benzene ring. Complex 6 induced apoptosis in tumor
cells and G1 cell cycle arrest, which is different from cisplatin that
induces S phase cell cycle arrest. The electrophoretic mobility
studies and CD spectral analysis revealed that the binding mode of
complex 6 to DNA might be different from that of cisplatin. Further
studies of this type of new platinum complexes could yield new
leads to improve the potency and selectivity of platinum anticancer
drugs.
2.8. Interaction with pUC19 plasmid DNA
Since these platinum complexes possess the classic cis-dichloro
coordination mode similar to cisplatin, we compared their in-
teractions with ct-DNA with that of cisplatin. As shown by the gel
electrophoresis experiments (Fig. 7), complex 6 induced a small
change in the migration rate of the negatively supercoiled band of
pUC19 plasmid DNA at 20
mM, and steadily decreased the DNA
migration rate with the increase of concentrations. The migration
rate of Form I DNA abruptly decreased when the concentration of
complex 6 reached 60
concentration of complex 6 was further increased to 70
contrast, cisplatin induced no change in migration rate at concen-
trations below 40 M and steadily decreased the DNA migration
rate at concentrations greater than 40 M. These results suggested
m
M, but it then increased notably when the
4. Experimental
mM. In
4.1. Materials and reagents
m
m
All chemicals, unless otherwise noted, were purchased from
Sigma and Alfa Aesar. All materials were used as received without
further purification unless noted specifically. TriseHCleNaCl (TBS)
buffer solution (5 mM Tris, 50 mM NaCl, pH adjusted to 7.35 by
titration with hydrochloric acid using a Sartorius PB-10 pH meter,
Tris ¼ tri(hydroxymethyl)aminomethane) was prepared using
double distilled water. The TBE buffer (1ꢂ) and DNA loading buffer
(6ꢂ) were commercially available. Calf thymus DNA (ct-DNA) was
purchased from Sino-American Biotech Co., Ltd. (Beijing). Ct-DNA
gave a UV absorbance ratio at 260e280 nm of w1.85:1, indicating
that the DNA was effectively free of protein. The DNA concentration
per nucleotide was determined spectrophotometrically by
employing a molar absorption coefficient (6600 Mꢁ1 cmꢁ1) at
that our platinum complexes might interact with DNA via different
mechanism from cisplatin.
2.9. Circular dichroism spectral analysis
Circular dichroism (CD) is a useful technique to assess whether
nucleic acids undergo conformational changes as a result of complex
260 nm. The stock solution of pUC19 plasmid DNA (250
purchased from Takara Biotech Co., Ltd.
mg/mL) was
4.2. Instrumentation
1H NMR, 13C NMR spectra were recorded on a Bruker AV-500
NMR spectrometer with CDCl3 or DMSO-d6 as solvent. Elemental
analyses (C, H, and N) were carried out on a PerkinElmer Series II
CHNS/O 2400 elemental analyzer. ESI-MS spectra for the
Fig. 3. Annexin V/propidium iodide assay of BEL-7404 cells treated by complex 6 (at
IC50 concentration) measured by flow cytometry.

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K.-B. Huang et al. / European Journal of Medicinal Chemistry 64 (2013) 554e561
Fig. 4. Loss of DJm induced by complex 6. Cells were treated with complex 6 for 3 h and examined by JC-1 under fluorescent microscope.
characterization of complexes 1e6 were performed on Thermo-
fisher Scientific Exactive LC-MS Spectrometer.
4.3.1.3. Diethyl anilino(2-pyridinyl)methylphosphonate (Lc). 1H
NMR (500 MHz, CDCl3)
d
8.69 (d, J ¼ 4.6 Hz, 1H), 7.81 (t, J ¼ 7.7 Hz,
1H), 7.63 (d, J ¼ 8.0 Hz, 1H), 7.40e7.35 (m, 1H), 7.05 (dd, J ¼ 11.2,
4.4 Hz, 2H), 6.68e6.59 (m, 3H), 4.02e3.99 (m, 4H), 3.93e3.86 (m,
1H), 1.22 (d, J ¼ 7.0 Hz, 3H), 1.11 (t, J ¼ 7.0 Hz, 3H). 13C NMR
4.3. Synthesis
(125 MHz, CDCl3)
d
154.96 (s), 146.88 (s), 145.83 (d, J ¼ 12.7 Hz),
4.3.1. Synthesis of the ligands
139.56 (s), 129.23 (s), 128.03 (s), 123.59 (d, J ¼ 80.4 Hz), 118.86 (s),
113.87e113.27 (m), 61.82 (s), 61.77 (s), 56.80 (s), 16.27 (s), 16.22 (s).
ESI-MS: m/z 321.14 [Lc þ H]þ
General procedure for the synthesis of aminophosphonate es-
ters ligands [29]. Equimolar amounts (0.2 mol) of pyridinealdehyde,
diethylphosphite and phenyl-amines were refluxed for 1 h at 60 ꢀC.
After the reaction mixture was cooled to room temperature, the oily
residue was purified on a silica gel column (petroleum ether:ethyl
acetate ¼ 1:1). Yellow oil-like product was obtained.
4.3.1.4. Diethyl 2-pyridinyl[(3,4,5-trimethoxyphenethyl)amino]meth-
ylphosphonate (Ld). 1H NMR (500 M Hz, CDCl3)
d 8.59 (s, 1H), 7.67
(d, J ¼ 7.7, 1.6 Hz, 1H), 7.46 (d, J ¼ 7.0 Hz, 1H), 7.28 (m, 1H), 6.44 (d,
J ¼ 5.9 Hz, 2H), 4.21 (dd, J ¼ 11.9, 5.0 Hz, 2H), 4.14e4.05 (m, 2H),
3.84 (d, J ¼ 9.0 Hz, 1H), 3.68e3.54 (m, 9H), 3.15 (s, 2H), 3.01 (s, 2H),
4.3.1.1. Diethyl(phenethylamino)(2-pyridinyl)methylphosphonate
(La). 1H NMR (500 MHz, CDCl3)
d
8.54 (dd, J ¼ 4.8, 1.2 Hz, 1H), 7.62
1.26 (m, 3H), 1.13 (m, 3H). 13C NMR (125 MHz, CDCl3)
d 157.04 (s),
(td, J ¼ 7.7, 1.7 Hz, 1H), 7.37 (dd, J ¼ 8.1, 1.3 Hz, 1H), 7.22 (t, J ¼ 7.4 Hz,
2H), 7.19e7.11 (m, 4H), 4.21 (d, J ¼ 21.3 Hz, 1H), 4.10e4.03 (m, 2H),
4.02e3.95 (m, 1H), 3.89 (tdd, J ¼ 10.3, 7.7, 5.2 Hz, 1H), 2.81e2.72 (m,
4H),1.23 (t, J ¼ 7.1 Hz, 3H),1.14 (t, J ¼ 7.1 Hz, 3H). 13C NMR (125 MHz,
152.13 (s), 149.34 (s), 137.12 (s), 136.48 (s), 134.47 (s), 123.81 (d,
J ¼ 4.1 Hz), 121.72 (s), 105.14 (s), 62.89 (d, J ¼ 7.8 Hz), 62.87 (d,
J ¼ 6.0 Hz), 61.41 (s), 60.17 (s), 60.03 (s), 55.42 (s), 37.21 (s) 16.31 (d,
J ¼ 4.5 Hz), 16.17 (d, J ¼ 4.3 Hz) ESI-MS: m/z 439.18 [Ld þ H]þ.
CDCl3)
d 156.39 (s), 149.19 (s), 139.82 (s), 136.41 (s), 128.74 (d,
J ¼ 9.9 Hz), 128.32 (s), 126.08 (s), 123.42 (d, J ¼ 4.7 Hz), 122.62 (d,
J ¼ 2.5 Hz), 63.35e63.06 (m), 62.79 (d, J ¼ 7.0 Hz), 61.95 (d,
J ¼ 26.9 Hz), 49.98 (d, J ¼ 16.4 Hz), 36.32 (s), 16.37 (d, J ¼ 5.7 Hz),
16.26 (d, J ¼ 5.7 Hz). ESI-MS: m/z 349.17 [La þ H]þ.
4.3.1.5. Diethyl 2-pyridinyl[(3,4,5-trimethoxybenzyl)amino]methyl-
phosphonate (Le). 1H NMR (500 M Hz, CDCl3)
d 8.56 (s, 1H), 7.63 (d,
J ¼ 7.1 Hz, 1H), 7.36 (d, J ¼ 6.3 Hz, 1H), 7.21e7.12 (m, 1H), 6.46 (d,
J ¼ 5.2 Hz, 2H), 4.17 (dd, J ¼ 21.9, 6.0 Hz, 1H), 4.12e4.03 (m, 2H),
4.02e3.95 (m, 1H), 3.89 (dd, J ¼ 9.3, 5.7 Hz, 1H), 3.79e3.71 (m, 10H),
3.51 (d, J ¼ 12.8 Hz, 1H), 1.26e1.21 (m, 3H), 1.13 (q, J ¼ 7.2 Hz, 3H).
4.3.1.2. Diethyl(benzylamino)(2-pyridinyl)methylphosphonate (Lb).
1H NMR (500 MHz, CDCl3)
d
8.60 (dd, J ¼ 4.8, 1.1 Hz, 1H), 7.66 (td,
13C NMR (125 MHz, CDCl3)
d 156.08 (s), 153.12 (s), 149.26 (s), 136.88
J ¼ 7.7, 1.4 Hz, 1H), 7.42 (dd, J ¼ 7.9, 1.0 Hz, 1H), 7.30e7.26 (m, 4H),
7.25e7.18 (m, 2H), 4.20 (d, J ¼ 21.8 Hz,1H), 4.17e4.09 (m, 2H), 4.09e
4.02 (m, 1H), 3.96e3.87 (m, 1H), 3.81 (d, J ¼ 13.3 Hz, 1H), 3.59 (d,
(s), 136.36 (s), 134.97 (s), 123.87 (d, J ¼ 4.7 Hz),122.68 (d, J ¼ 2.3 Hz),
105.14 (s), 63.09 (d, J ¼ 6.8 Hz), 62.77 (d, J ¼ 7.0 Hz), 61.49 (s), 60.77
(s), 60.27 (s), 55.99 (s), 16.42 (d, J ¼ 5.7 Hz), 16.26 (d, J ¼ 5.6 Hz).
ESI-MS: m/z 425.19 [Le þ H]þ.
J ¼ 13.3 Hz, 1H), 1.27 (t, J ¼ 7.1 Hz, 3H), 1.17 (t, J ¼ 7.1 Hz, 3H). 13
C
NMR (125 MHz, CDCl3) d 156.18 (s), 149.32 (s), 139.30 (s), 136.39 (s),
128.40 (s), 128.33 (s), 127.11 (s), 123.82 (d, J ¼ 4.8 Hz), 122.65 (d,
J ¼ 2.5 Hz), 63.19 (d, J ¼ 6.8 Hz), 62.76 (d, J ¼ 6.9 Hz), 52.10 (s), 51.97
(s), 16.42 (d, J ¼ 5.8 Hz), 16.27 (d, J ¼ 5.8 Hz). ESI-MS: m/z 335.13
[Lb þ H]þ.
4.3.1.6. Diethyl 2-pyridinyl(3,4,5-trimethoxyanilino)methylphosphonate
(Lf). 1H NMR (500 MHz, CDCl3)
7.49 (d, J ¼ 7.4 Hz, 1H), 7.18 (s, 1H), 5.21 (d, J ¼ 41.8 Hz, 1H), 4.92 (d,
d
8.56 (s, 1H), 7.63 (d, J ¼ 5.6 Hz, 1H),
Fig. 5. Activation of Caspase-3 induced by complex 6, examined by FITC-DEVD-FMK under a fluorescent microscope.

K.-B. Huang et al. / European Journal of Medicinal Chemistry 64 (2013) 554e561
559
Fig. 6. Cell cycle analysis by flow cytometry of BEL-7404 cells treated with complex 6 and cisplatin at IC50 concentrations.
J ¼ 22.1 Hz, 1H), 4.10 (dd, J ¼ 19.5, 12.8 Hz, 2H), 4.00 (d, J ¼ 2.8 Hz, 1H),
3.86 (s, 1H), 3.68 (dd, J ¼ 11.6, 4.3 Hz, 9H), 1.26 (d, J ¼ 6.2 Hz, 3H), 1.13
(s), 129.11 (s), 128.73 (s), 127.71 (s), 122.82 (s), 121.63 (s), 63.19
(d, J ¼ 5.6 Hz), 62.86 (d, J ¼ 7.3 Hz), 53.14 (s), 50.87 (s), 16.32
(d, J ¼ 4.8 Hz), 16.17 (d, J ¼ 5.1 Hz). ESI-MS: m/z 599.12 [M þ H]þ.
Elemental analysis calculated: C, 34.14; H, 3.74; N, 4.58; Found: C,
34.07; H, 3.70; N, 4.67.
(d, J ¼ 6.2 Hz, 3H). 13C NMR (125 MHz, CDCl3)
d 156.03 (s), 153.76 (s),
149.06 (s), 143.23 (d, J ¼ 13.0 Hz), 136.83 (s), 130.67 (s), 128.82 (s),
122.81 (d, J ¼ 4.4 Hz), 91.69 (s), 63.46 (d, J ¼ 6.9 Hz), 63.26 (d,
J ¼ 7.1 Hz), 60.96 (s), 59.14 (s), 57.94 (s), 55.84 (s), 16.41 (d, J ¼ 5.7 Hz),
16.22 (d, J ¼ 5.7 Hz). ESI-MS: m/z 411.12[Lf þ H]þ.
4.3.2.3. Complex 3. 1H NMR (500 MHz, DMSO-d6)
d 8.55 (d,
J ¼ 4.7 Hz, 1H), 7.80 (t, J ¼ 7.7 Hz, 1H), 7.60 (d, J ¼ 7.9 Hz, 1H), 7.33e
7.28 (m, 1H), 7.03 (t, J ¼ 7.7 Hz, 2H), 6.77 (d, J ¼ 8.2 Hz, 2H), 6.56 (t,
J ¼ 7.2 Hz,1H), 5.11 (d, J ¼ 23.8 Hz,1H), 4.11e4.03 (m, 2H), 3.95 (ddd,
J ¼ 17.3, 10.8, 5.1 Hz, 1H), 3.81 (tt, J ¼ 14.9, 7.2 Hz, 1H), 1.20 (t,
J ¼ 7.1 Hz, 3H), 1.07 (t, J ¼ 7.0 Hz, 3H). 13C NMR (125 MHz, DMSO-d6)
4.3.2. Synthesis of the platinum complexes
General procedure for the synthesis of the platinum complexes:
To a solution of cis-Pt(DMSO)2Cl2 (84 mg, 0.2 mmol) was added Laef
(0.2 mmol) dissolved in 30 mL mixtures of anhydrous dichloro-
methane and ethanol (1:1). The mixture was stirred in the dark at
room temperature for 1 day. The resulting yellow solution was
filtered and crystals or solid powders were obtained by slow
evaporation of filtrate solution.
d
156.96 (s), 149.05 (s), 147.55 (d, J ¼ 13.6 Hz), 137.30 (s), 129.87 (s),
129.11 (d, J ¼ 30.3 Hz), 123.46 (d, J ¼ 3.8 Hz), 123.32 (s), 117.71 (s),
114.00 (s), 63.09 (d, J ¼ 6.8 Hz), 62.86 (d, J ¼ 7.0 Hz), 56.89 (d,
J ¼ 150.1 Hz), 16.74 (d, J ¼ 5.3 Hz), 16.52 (d, J ¼ 5.5 Hz). ESI-MS: m/z
585.13. Elemental analysis calculated: C, 32.89; H, 3.56; N, 4.68;
Found: C, 32.83; H, 3.44; N, 4.79.
4.3.2.1. Complex 1. 1H NMR (500 MHz, DMSO-d6)
d 9.05 (dd,
J ¼ 27.1, 5.8 Hz, 1H), 8.24 (t, J ¼ 7.7 Hz, 1H), 7.66 (dd, J ¼ 18.6, 7.2 Hz,
1H), 7.62e7.57 (m, 1H), 7.28 (d, J ¼ 7.3 Hz, 2H), 7.23 (s, 2H), 7.21
(d, J ¼ 5.4 Hz, 1H), 4.34e4.10 (m, 4H), 3.98e3.85 (m, 1H), 3.10
(d, J ¼ 10.5 Hz, 2H), 3.03 (d, J ¼ 8.2 Hz, 2H), 1.32e1.26 (m, 3H), 1.21
4.3.2.4. Complex 4. 1HNMR (500 MHz, DMSO-d6)
d
9.01 (d, J ¼ 4.8Hz,
1H), 8.21(t, J¼ 3.7Hz,1H), 7.41(d, J¼ 7.9Hz,1H), 7.41(t, J¼ 5.4Hz,1H),
6.72 (s, 2H), 4.29e4.22 (m, 2H), 4.12e4.10 (m, 4H), 4.08 (d, J ¼ 12.5 Hz,
1H), 3.74 (s, 2H), 3.52e3.48 (s, 9H), 1.28 (t, J ¼ 6.7 Hz, 3H), 1.16 (t,
(t, J ¼ 6.9 Hz, 3H). 13C NMR (125 MHz, DMSO-d6)
d 160.70
(d, J ¼ 8.4 Hz), 148.49 (s), 139.76 (s), 137.98 (s), 129.20 (s), 128.98 (s),
J ¼ 7.4 Hz, 3H). 13C NMR (125 MHz, DMSO-d6)
d 160.61 (s), 150.41 (s),
127.00 (s), 125.72 (s), 124.72 (s), 65.30 (d,
J
¼
6.5 Hz),
150.51 (s),148.32 (s),142.14 (s),137.80 (s),128.43 (s),124.65 (s),124.16
(s),111.04 (s),109.49 (s), 65.24 (d, J¼ 4.5 Hz), 63.24(d, J ¼ 7.3 Hz), 60.42
(d, J¼ 11.4Hz), 60.17(s),55.66(d,J¼ 4.7Hz), 55.33(s),41.26(s),16.30e
16.28 (m), 16.43 (d, J ¼ 3.9 Hz). ESI-MS: m/z 703.45 [M þ H]þ.
Elemental analysis calculated: C, 35.64; H, 4.24; N, 4.02; Found: C,
35.86; H, 4.30; N, 3.98.
64.14 (d, J ¼ 6.5 Hz), 58.66 (d, J ¼ 10.7 Hz), 48.81 (s), 34.14 (s), 16.55
(s), 16.50 (s). ESI-MS: m/z 614.42 [M þ H]þ. Elemental analysis
calculated: C, 35.37; H, 4.02; N, 4.65; Found: C, 35.25; H, 3.94;
N, 4.57.
4.3.2.2. Complex 2. 1H NMR (500 MHz, DMSO-d6)
d 8.95 (dd,
J ¼ 21.3, 4.8 Hz, 1H), 8.02 (t, J ¼ 7.4 Hz, 1H), 7.61 (d, J ¼ 18.6 Hz, 1H),
7.40e7.38 (m,1H), 7.13 (t, J ¼ 6.7 Hz, 2H), 6.82 (t, J ¼ 7.2 Hz, 2H), 6.66
(t, J ¼ 5.7 Hz, 1H), 5.31 (d, J ¼ 15.8 Hz, 1H), 4.42e4.31 (m, 4H), 3.53
4.3.2.5. Complex 5. 1H NMR (500 MHz, DMSO-d6)
d 8.78 (d,
J ¼ 5.8 Hz, 1H), 8.01 (t, J ¼ 7.7 Hz, 1H), 7.43 (d, J ¼ 7.7 Hz, 1H), 7.33 (t,
J ¼ 6.4 Hz, 1H), 7.27 (s, 2H), 4.57e4.51 (m, 1H), 4.29e4.22 (m, 2H),
4.18e4.10 (m, 4H), 4.02 (d, J ¼ 12.0 Hz, 1H), 3.78 (s, 3H), 3.77 (s, 6H),
1.25 (t, J ¼ 7.0 Hz, 3H), 1.19 (t, J ¼ 7.0 Hz, 3H). 13C NMR (125 MHz,
(d, J ¼ 11.5 Hz, 2H) 1.24 (t, J ¼ 5.1 Hz, 3H), 1.12 (t, J ¼ 7.0 Hz, 3H). 13
C
NMR (125 MHz, DMSO-d6)
d 158.14 (s), 151.42 (s), 141.33 (s), 137.42
Fig. 7. Electrophoresis in agarose gel of pUC19 plasmid DNA (0.02 mg/mL, 30
the concentration of each platinum complex is indicated in the figure.
m
M base pair) incubated for 4 h at 37 ꢀC with cisplatin, complex 6, respectively. “C” represents control;

560
K.-B. Huang et al. / European Journal of Medicinal Chemistry 64 (2013) 554e561
DMSO-d6)
d
161.64 (d, J ¼ 8.7 Hz), 152.80 (s), 152.54 (s), 147.55 (s),
supplemented with 10% (v/v) fetal bovine serum, 2 mM glutamine,
100 U/mL penicillin, and 100 U/mL streptomycin at 37 ꢀC, in a
highly humidified atmosphere of 95% air/5% CO2. The cytotoxicities
of complexes 1e6 against MG-63, SKOV-3, HepG2, BEL-7404 and
HL-7702 cell lines were examined by the microculture tetrazolium
(MTT) assay [34]. The experiments were carried out using reported
procedure [35]. The growth inhibitory rate of treated cells
was calculated using the data from three replicate tests as
(ODcontrol ꢁ ODtest)/ODcontrol ꢂ 100%. The compounds were incu-
bated with various cell lines for 48 h at five different concentrations
of complexes 1e6 dissolved in fresh media; the range of concen-
trations used is dependent on the complex. The final IC50 values
were calculated by the Bliss method (n ¼ 5). All tests were inde-
pendently repeated at least three times.
142.04 (s), 138.80 (s), 128.83 (s), 125.65 (s), 124.79 (s), 110.01 (s),
109.91 (s), 65.27 (d, J ¼ 6.5 Hz), 64.20 (d, J ¼ 6.9 Hz), 60.58 (d,
J ¼ 10.4 Hz), 60.14 (s), 56.31 (d, J ¼ 4.7 Hz), 56.27 (s), 16.58e16.49
(m), 16.33 (d, J ¼ 5.9 Hz). ESI-MS: m/z 689.22 [M þ H]þ. Elemental
analysis calculated: C, 35.12; H, 4.01; N, 3.98; Found: C, 34.84; H,
4.09; N, 4.06.
4.3.2.6. Complex 6. 1H NMR (500 MHz, DMSO-d6)
d 8.56 (d,
J ¼ 4.7 Hz, 1H), 7.81 (t, J ¼ 7.7 Hz, 1H), 7.62 (t, J ¼ 13.0 Hz, 1H), 7.34e
7.25 (m, 1H), 6.12 (s, 2H), 5.14 (d, J ¼ 23.8 Hz, 1H), 4.08 (p, J ¼ 7.3 Hz,
2H), 3.96 (dt, J ¼ 21.9, 7.2 Hz, 1H), 3.84e3.76 (m, 1H), 3.63 (d,
J ¼ 7.0 Hz, 6H), 3.62 (d, J ¼ 2.9 Hz, 1H), 3.48 (s, 3H), 3.18 (s, 1H), 1.22
(t, J ¼ 7.0 Hz, 3H), 1.08 (t, J ¼ 7.0 Hz, 3H). 13C NMR (125 MHz, DMSO-
d6)
d
157.14 (s), 153.62 (s), 148.97 (s), 144.13 (d, J ¼ 14.6 Hz), 137.34
(s), 129.83 (s), 123.59 (s), 123.32 (s), 92.05 (s), 63.06 (d, J ¼ 6.6 Hz),
62.84 (d, J ¼ 6.8 Hz), 60.52 (s), 56.58 (s), 55.98 (s), 16.77 (d,
J ¼ 5.2 Hz), 16.54 (d, J ¼ 5.5 Hz). ESI-MS: m/z 675.26 [M þ H]þ.
Elemental analysis calculated: C, 33.82; H, 3.62; N, 3.93; Found: C,
33.79; H, 3.88; N, 4.15.
4.5. Apoptosis assays by flow cytometry
The ability of platinum complex 6 to induce apoptosis is eval-
uated in BEL-7404 cell line using Annexin V conjugated with FITC
and propidium iodide (PI) counterstaining by flow cytometry. BEL-
7404 cells of exponential growth were inoculated in 6-well plates
and cultured for 12 h before the platinum compounds were added
to give the indicated final concentrations. After 48-hour incubation,
cells were harvested, washed twice in phosphate-buffered saline
4.3.3. X-ray crystallographic analysis
Suitable crystals of complexes 1, 3, 5, 6 were grown from
dichloromethane and ethanol (1:1). The single crystals were
mounted on glass fibers, and crystal data were collected on a Bruker
Smart Apex II CCD or Rigaku Mercury CCD diffractometer equipped
with graphite monochromated Mo K
room temperature. Absorption correction were applied by using the
multiscan program SADABS [32]. The structures were solved with
direct methods and refined using SHELX-97 programs [33]. The non-
hydrogen atoms were located in successive difference Fourier syn-
thesis. The final refinement was performed by full-matrix least-
squares methods with anisotropic thermal parameters for non-
hydrogen atoms on F2. The hydrogen atoms were added theoreti-
cally and riding on the concerned atoms. The crystallographic data
and refinement details of the structures are summarized Table 2.
(PBS), and resuspended in 100
mL binding buffer (including
140 mmol/L NaCl, 2.5 mmol/L CaCl2 and 10 mmol/L Hepes/NaOH,
ꢀ
pH 7.4) at a concentration of 1 ꢂ 106 cells/mL. Then cells were
a
radiation (
l
¼ 0.71073 A) at
incubated with 5
mL of Annexin V-FITC (in buffer including
10 mmol/L NaCl, 1% bovine serum albumin, 0.02% NaN3 and
50 mmol/L Tris, pH 7.4) and 10 L PI (20 g/mL) for 15 min at room
m
m
temperature in the dark. Cells were kept shielded from light before
being analyzed by flow cytometry using a BectoneDickinson
FACSCalibur.
4.6. Mitochondrial membrane potential detection
Cells incubated with IC50 concentration of complex 6 for 12 h in
poly-HEMA coated 6-wells were collected and resuspended in fresh
medium. After the addition of 0.5 mL JC-1 working solution, the cells
were incubated in a 5% CO2 incubator for 20 min at 37 ꢀC. The staining
solution was removed by centrifugation and cells were washed with
JC-1 staining buffer twice. Cells having low membrane potential were
4.4. Cell lines, culture conditions and cytotoxicity assay (MTT assay)
The cell lines MG-63, SKOV-3, HepG2, BEL-7404 and HL-7702
were obtained from the Shanghai Cell Bank in the Chinese Acad-
emy of Sciences. Cell lines were grown in the RPMI-1640 medium
Table 2
Crystal data and structure refinement details for complexes 1, 3, 5, 6.
Formula
C18H27Cl2N2O4PPt
C16H21Cl2N2O3PPt
C20H29Cl2N2O6PPt
C19H27Cl2N2O6PPt
fw
T/K
632.38
293(2)
586.30
293(2)
690.40
293(2)
676.39
293(2)
Crystal system
Space group
Monoclinic
P21/c
15.4044(5)
8.2538(2)
18.9162(6)
90.00
107.911(4)
90.00
2288.53(12)
4
Orthorhombic
Pbca
5.4808(8)
14.8321(10)
17.5122(8)
90.00
90.00
90.00
4021.0(4)
8
1.937
Triclinic
P-1
Triclinic
P-1
ꢀ
a, A
11.7643(4)
15.8123(10)
16.3386(8)
61.921(6)
72.916(3)
88.301(4)
2541.9(2)
4
9.0916(4)
11.3273(6)
12.2329(4)
104.389(4)
95.129(4),
101.415(4)
1183.32(10)
2
ꢀ
b, A
ꢀ
c, A
ꢀ
ꢀ
ꢀ
a
,
b
,
g
,
3
ꢀ
V, A
Z
Dc, g cmꢁ3
1.835
6.460
1.034
1.804
5.830
1.014
1.893
6.257
1.029
m
, mmꢁ1
7.339
1.018
GOF on F2
Reflns (collected/unique)
10,669/4683
0.0797
0.0593
38,217/4113
0.3935
0.1123
0.2445
20,992/10,383
0.1033
0.0912
9838/4849
0.0998
0.0645
Rint
a
R1 (I > 2
s
(I))
wR2b (all data)
0.1647
0.2741
0.1497
a
R1 ¼ SjjFoj e jFcjj/SjFoj.
b
P
P
wR2 ¼ ½ wðFo2 ꢁ Fc2Þ2= wðFo2Þ2ꢃ1=2
:

K.-B. Huang et al. / European Journal of Medicinal Chemistry 64 (2013) 554e561
561
taken pictures under fluorescent microscope with excitation and
Acknowledgments
emission at 485 and 535 nm, respectively (45ꢂ, Nikon).
This work was supported by the National Natural Science
Foundation of China (No. 21271051), National Basic Research Pro-
gram of China (2012CB723501), IRT1225, and Natural Science
Foundation of Guangxi Province (Nos. 2012GXNSFDA053005,
2012GXNSFDA385001) as well as “BAGUI Scholar” program of
Guangxi province of China.
4.7. Determination of caspase-3 activity
The activation of caspase-3 was examined using a caspase-3
(DEVD-FMK) conjugated to FITC as the fluorescent in situ marker
in living cells (catalog #QIA91, Merck). FITC-DEVD-FMK is cell
permeable, nontoxic, and irreversibly binds to activated caspase-3
in apoptotic cells. After exposure of BEL-7404 cell lines to com-
plex 6 for 8 h, the cultures were washed twice with HBSS
containing 0.1% BSA and then incubated with FITC-DEVD-FMK
Appendix A. Supplementary material
(1 m
L/mL in EBSS) for 1 h in a 37 ꢀC incubator with 5% CO2. The
Supplementary material related to this article can be found at
http://dx.doi.org/10.1016/j.ejmech.2013.04.024.
FITC label in apoptotic cells was examined immediately under
fluorescent microscope with excitation and emission at 485 and
535 nm, respectively (25ꢂ, Nikon).
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4.9. Agarose gel electrophoresis assay [36]
In plasmid DNA unwinding experiments, all compounds were
prepared as 2 ꢂ 10ꢁ3 M stock solutions of DMSO and diluted to 10
and 100
m
M by 1ꢂ TBE buffer. Compounds of various concentra-
tions were mixed with 0.5
TBE buffer so that the same experiment can be repeated twice. All
samples were incubated at 25 ꢀC in dark for 4 h. Then 12
L of each
sample mixed with 2 L DNA loading buffer was electrophoresed at
5 V/cm through 0.8% agarose gel immersed in 1ꢂ TBE buffer solu-
tion for 60 min. Finally, the gel was stained with EB (1.27 M) in
mg DNA and made up to a total 25 mL by
m
m
m
dark for 30 min, followed by visualized on a BIO-RAD imaging
system with a UVevis transilluminator.
4.10. CD absorption spectrometry assay
In the CD absorption spectrometry, the working solution of each
sample was prepared by using 1 ꢂ 10ꢁ4 M DNA and titrating the
complexes into the DNA solution stepwise with the [DNA]/[com-
pound] ratio ranging from 10:0.5 to 10:5. The working solution was
incubated for 10 min after each addition and then its CD spectrum
was recorded at 100 nm/min scan rate. The CD signals of the TBS
were subtracted as the background.
[32] G.M. Sheldrick, SADABS, Version 2.05, University of Go€ttingen, Go€ttingen,
Germany, 2002.
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