Synthesis, cytotoxicity, and apoptosis induction study of antitumor dinuclear platinum(II) complexes

Article abstract of DOI:10.1002/ardp.201200288

Five novel dinuclear platinum(II) complexes with a new chiral ligand, 3-(2-amino-cyclohexylamino)-propionic acid (HP), were designed, prepared and spectrally characterized. The in vitro cytotoxicities of these compounds were evaluated against the HepG-2, MCF-7, A549, and HCT-116 cell lines. The results indicated that all compounds showed cytotoxicity towards the HepG-2 cell line. Particularly, complex X5, which has SO 42- as a bridge, exhibited better cytotoxicity than carboplatin or oxaliplatin against all selected cell lines. Moreover, double dyeing flow cytometric resection indicated that the target compounds inhibited tumor cell growth by inducing apoptosis. Five novel dinuclear platinum(II) complexes with a new chiral ligand, 3-(2-amino- cyclohexylamino)-propionic acid, were designed. The in vitro cytotoxicity results of these compounds against the HepG-2, MCF-7, A549, and HCT-116 cell lines indicated that all compounds showed cytotoxicity towards the HepG-2 cell line. Particularly, complex X5, which has SO 42- as a bridge, exhibited better cytotoxicity than carboplatin or oxaliplatin against all selected cell lines. Copyright

Full text of DOI:10.1002/ardp.201200288

308
Arch. Pharm. Chem. Life Sci. 2013, 346, 308–313
Full Paper
Synthesis, Cytotoxicity, and Apoptosis Induction Study of
Antitumor Dinuclear Platinum(II) Complexes
Gang Xu1,2, Shaohua Gou1,2, and Chuanzhu Gao2,3
1 Jiangsu Province Hi-Tech Key Laboratory for Biomedical Research, Southeast University, Nanjing, China
2 Pharmaceutical Research Center, School of Chemistry and Chemical Engineering, Southeast University,
Nanjing, China
3 School of Life Science and Technology, Kunming University of Science and Technology, Kunming, China
Five novel dinuclear platinum(II) complexes with a new chiral ligand, 3-(2-amino-cyclohexylamino)-
propionic acid (HP), were designed, prepared and spectrally characterized. The in vitro cytotoxicities of
these compounds were evaluated against the HepG-2, MCF-7, A549, and HCT-116 cell lines. The results
indicated that all compounds showed cytotoxicity towards the HepG-2 cell line. Particularly, complex
X5, which has SO42ꢀ as a bridge, exhibited better cytotoxicity than carboplatin or oxaliplatin against
all selected cell lines. Moreover, double dyeing flow cytometric resection indicated that the target
compounds inhibited tumor cell growth by inducing apoptosis.
Keywords: 3-(2-Amino-cyclohexylamino)-propionic acid / Apoptosis induction / Cytotoxicity /
Dinuclear platinum complex
Received: July 13, 2012; Revised: December 24, 2012; Accepted: January 18, 2013
DOI 10.1002/ardp.201200288
Introduction
bridge, is a typical example as a lead compound for its potent
cytotoxicity and different anticancer mechanism from
cisplatin [5–9].
Platinum-based anticancer drugs are widely used for
the treatment of various types of solid tumors. Cisplatin,
carboplatin, and oxaliplatin, the three global drugs, are
used to treat 40–80% of all cancer patients. However,
their application is severely limited by their cumulative
toxicities such as nephrotoxicity, ototoxicity, and peripheral
neuropathy. Additionally, both intrinsic and acquired resist-
ance can develop [1, 2]. These drawbacks have stimulated
the search for other platinum-based anticancer agents
not obeying the classical structure–activity relationship
(SAR) [3].
Initially, Pt(II) ions in multinuclear antitumor platinum
complexes were reported to be linked by amines such as
flexible aliphatic polyamines and aromatic amines with rigid
ring structures [10, 11]. Recently, there have been several
reports on antitumor dinuclear platinum(II) complexes, in
which dicarboxylate and amino acidic anions as well as
halide anions were used as bridges to connect two metal
ions [12–14]. And the related study indicated that the bridges
were found to have an important effect on the cytotoxicity of
the resulting dinuclear complexes.
Multinuclear platinum complexes have been reported
increasingly as an important type of non-classical anticancer
platinum complexes, which could probably overcome the
drug resistance/cross resistance arising from cisplatin and
its analogs [4]. This is partly due to the fact that their
DNA binding mode is different from cisplatin. BBR3464, a
trinuclear platinum complex with 1,6-hexyldiamine as a
In view of an essential role of 1R,2R-diaminocyclohexane
(DACH) in the success of oxaliplatin [15], we have designed a
class of chiral ligands in which carboxylato groups were
introduced to bind one of the nitrogen atoms of 1R,2R-
DACH to generate amino acidic derivatives. In our early work,
two different series of dinuclear platinum(II) complexes of 2-
(((1R,2R)-2-aminocyclohexyl)amino)acetic acid and 2-(((1R,2R)-
2-aminocyclohexyl)amino)propanoic acid, with dicarboxy-
lates or sulfate as bridges were prepared. In vitro evaluations
showed that the dinuclear complexes with succinate (com-
plex S0 and S2) and sulfate (complex S1, Fig. 1) as bridges
exhibited better or comparable activities against HCT-116,
Correspondence: Professor Shaohua Gou, School of Chemistry and
Chemical Engineering, Southeast University, Nanjing 211189, China.
E-mail: sgou@seu.edu.cn
Fax: þ86 25 83272381
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Arch. Pharm. Chem. Life Sci. 2013, 346, 308–313
Antitumor Dinuclear Platinum(II) Complexes
309
derivative directly due to the equivalent reactivity of the
two amino groups in DACH. The ligand was synthesized
via several synthetic steps shown in Scheme 1.
When preparing the dinuclear platinum complexes, we
first synthesized the intermediate [PtPI] by adopting a
similar method described literaturally [17]. And then silver
dicarboxylate/sulfate were used to remove the iodide anions
of [PtPI] to give complexes X1–X5 (Scheme 2).
All dinuclear platinum(II) complexes were characterized by
IR, 1H NMR, ESI-MS spectra, and microanalyses. The elemental
analysis results are in good agreement with the calculated
values. The IR spectra of these complexes are analogous, Pt–N
coordination bonds were confirmed by the examination of
Figure 1. Chemical structures of complexes S0 and S1.
nNH =nNH shifting to lower frequencies compared with their
2
–
free amino groups. On the other hand, the shifts of the C O
–
MCF-7, and HepG-2 cell lines compared with positive controls
[16, 17].
absorption from free carboxylic acids near 1700 cmꢀ1 to a
band near 1541–1647 cmꢀ1 proved that carboxylate anions
were coordinated to Pt(II) ions. The 1H NMR spectra of the
complexes are consistent with their corresponding protons
both in the chemical shifts and the number of hydrogens.
All prepared complexes showed a peak of [MþNa]þ or [MþH]þ
in their positive ESI mass spectra, which are consistent with
the expected molecular formula weights. The mass spectra of
the compounds exhibited four main molecular ion peaks
due to the isotopes of 194Pt (33%), 195Pt (34%), 196Pt (25%),
and 198Pt (7%). It is noted that a new chiral center was
generated at the substituted nitrogen atom of 1R,2R-DACH
after coordination. As stated in our former study including
crystal structure analysis and theoretical calculations [18, 19],
we proposed that the S configuration was preferable in the
nitrogen atom.
Inspired by the result, we introduced another aliphatic
acid, namely propionic acid, to DACH and got a new
chiral ligand, 3-(2-amino-cyclohexylamino)-propionic acid
(abbreviated as HP), to evaluate the relationship between
the substituting carboxylato groups and biological activities
of the resulting compounds. With HP as a carrier group and
different dicarboxylates as bridges, five dinuclear platinum
complexes were prepared and characterized by IR, MS, and
1H NMR spectra. It is noted that all dinuclear platinum com-
plexes showed good aqueous solubility. The results of in vitro
cytotoxicity assays showed that all compounds exhibited affir-
mative activity to selected human cell lines; especially some of
them surpassed that of oxaliplatin against HCT-116. The results
of flow cytometry (FCM) tests showed that they inhibited the
proliferation of tumor cells by inducing cell apoptosis.
The aqueous solubility of the resulting dinuclear com-
pounds was measured, because it is a very important factor
for anticancer platinum drugs. Compared with cisplatin
(1 mg/mL) and oxaliplatin (8 mg/mL), all prepared platinum
compounds have rather good aqueous solubility (ranging
from 18 to 47 mg/mL).
Results and discussion
Mono-Boc protected 1R,2R-DACH was used as the starting
material, since it was hard to get an N-monosubstituted
Scheme 1. Synthetic chart for ligand HP.
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Arch. Pharm. Chem. Life Sci. 2013, 346, 308–313
that of the positive control (carboplatin), which indicated
that sulfate was probably a good choice as linking ligand.
When tested against HepG-2 cells, all complexes showed
antitumor cytotoxicity. Complex X2 that has malonate as a
bridge gave the best performance and its activity was 2.5
times that of carboplatin, while complex X5 also showed very
similar activity to carboplatin.
For HCT-116 cells, complex X5 also exhibited better
activity than oxaliplatin and complex X1 showed very close
activity to the positive control. Interestingly, when HCT-116
was employed, the longer the dicarboxylate bridge is,
the lower the antitumor activity is. Especially, when a
cyclobutyl moiety was introduced to the dicarboxylate
bridge, the antitumor activity significantly decreased, indi-
cating the cyclobutyl fragment had a negative impact on the
activity.
The main difference between complexes S0/S1 and X3/X5 is
the alkyl length of N-substituted carboxylates which leads to
the different size of the PtNO ring resulted from coordina-
tion. The former complex has a N-acetate 1R,2R-DACH ligat-
ing group to generate a five-membered ring, while the latter
has a N-propionate 1R,2R-DACH moiety to generate a six-
membered ring. As we could conclude from the IC50 data,
no dramatic changes were observed between these two series
of complexes. Complexes S1 and X5, bridged by sulfate, had
almost the same activities towards MCF-7, HepG-2, and HCT-
116 cell lines, but the cytotoxicity of X5 against A549 was
much better than its counterpart. Although complexes X3,
S0, and S2 were all connected by succinate, the anticancer
activity of X3 was much decreased compared with S0 and S2
in all the four tested cancer cell lines. It seemed that N-acetate
1R,2R-DACH was a slightly better choice than the N-propio-
nate one as a carrier ligand, and sulfate was a good linker in
these two series of complexes.
Scheme 2. Synthetic route of dinuclear platinum(II) complexes
X1–X5.
The in vitro cytotoxicities of dinuclear platinum compounds
were evaluated by MTT assay using HepG-2 human hepato-
cellular carcinoma cells, MCF-7 human breast cancer cells,
A549 human lung cancer cells and HCT-116 human color-
ectal cancer cells, with carboplatin or oxaliplatin as positive
control. As we can see from Table 1, most complexes showed
cytotoxicity to the selected cell lines.
Complex X5 owning sulfate as a bridge showed good
activity to all the four selected cancer cell lines. When tested
against A549 and MCF-7 cells, complex X5 had much better
activity than other dinuclear compounds in which dicarbox-
ylates served as bridges, and even is twofold more potent than
Table 1. Cytotoxicity of complexes X1–X5 against four tumor cell
lines.
In summary, complex X5 showed much better activity
than other dinuclear compounds, and its activity surpassed
the positive controls against all selected human cell lines.
Complexes X1 and X5, which have shown favorable cyto-
toxicity in MTT assay, were chosen to test their capability to
induce apoptosis of HCT-116 cells. The results are shown in
Fig. 2.
Compound
IC50 [mM]b)
A549
MCF-7
HEPG-2
HCT-116
X1
X2
X3
X4
X5
S0
S1
S2
40
29.4
>50
30
40
>50
>50
34.2
8.1
32
4
43.9
18
9.1
8.2
6.8
5.1
17.8
43.3
>50
In Fig. 2, we can see that the apoptosis rate is very low under
10% in the control (cells without drugs), while complexes X1,
X5, and the positive control inhibited HCT-116 cell prolifer-
ation by inducing apoptosis obviously. It is noted that the
apoptosis rate of complex X5 surpassed oxaliplatin, which is
consistent with the results of the MTT method.
6.4
4.2
>50a)
>50
9.9
7.1
7.9
15.3
Not tested
5.8 [13]
5.1 [13]
4.0
Not tested
4.3
Not tested
11.2
Not tested
>50
9.7
Not tested
Carboplatin
Oxaliplatin
a)
The cytotoxicities of complexes S0 and S1 were tested but not
reported in [13].
Conclusion
b)
All IC50 values (drug concentration giving 50% survival) calcu-
lated based on the Pt content are means ꢁ SD (SD <12% of
We have prepared a new chiral ligand of HP derived from
the mean value) from at least three separate experiments.
1R,2R-DACH. Using HP as a carrier ligand, five novel
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Antitumor Dinuclear Platinum(II) Complexes
311
Figure 2. Induction of apoptosis (HCT-116) of
complexes X1 and X5. Q1, unnatural death;
Q2, late apoptosis and/or necrosis; Q3, normal;
Q4, early apoptosis.
of acetonitrile, and refluxed for 12 h. Pale yellow oil (intermedi-
ate I1) was separated by silica gel column chromatography
(PE(60–90)/EtOAc/methanol ¼ 5:1:1). The intermediate 1 was
then dissolved in 200 mL of methanol and excess HCl/methanol
(5 mol/L) was added, leading to the formation of intermediate I2,
which was finally hydrolyzed under a basic condition to give the
sodium salt of HP, 3.1 g. Yield, 30%. M.W.: 208.0. Found (calcd. for
C9H17N2O2Na), C 51.89 (51.92), H 8.26 (8.17), N 13.55 (13.46). IR
(cmꢀ1): 3330s (br), 2963s, 2926s, 2858s, 1590vs (sh), 1443vs,
1338m, 1300m, 1140m, 1076w, 916m, 879m, 769m, 438m; 1H
NMR (D2O/TSP, ppm): d 1.02–1.24 (m, 4H, 2CH2 of DACH), 1.65–
2.20 (m, 4H, 2CH2 of DACH), 2.22–2.24 (m, 1H, NH2CH of DACH),
2.35–2.46 (m, 2H, CH2CH2COO), 2.69–2.74 (m, 1H, NHCH of
DACH), 2.90–3.35 (m, 2H, NHCH2CH2COO); ESI-MS m/z:
[MþH]þ ¼ 209.0 (100%).
dinuclear platinum(II) complexes were designed and syn-
thesized with dicarboxylates or sulfate as bridges. The
results of in vitro cytotoxicity assays suggested that complex
X5 owning sulfate as a bridge showed better cytotoxicity
against all selected human cell lines than the positive con-
trols. Typical platinum(II) complex indicated that it can
inhibit tumor growth by induction of apoptosis.
Consequently, the prepared dinuclear platinum(II) com-
plexes, especially X5, may be promising leading com-
pounds for further research.
Experimental
Materials and instruments
Intermediate [PtPI]
K2PtCl4 was purchased from Sino-Platinum Co., Ltd. [17, 20];
mono-Boc protected DACH was used as the starting material
and prepared according to the procedure reported [21].
All reagents were of high purity and used without any further
purification. Elemental analysis for C, H, and N was performed
with a Perkin-Elmer 1400C instrument. ESI-MS spectra were
carried out in a Finnigan MAT SSQ 710 (120–1000 am) apparatus
and IR spectra were scanned by a Nicolet IR200 spectropho-
tometer in the range of 4000–400 cmꢀ1 in KBr pellet. 1H NMR
spectra were recorded on a Bruker DRX-500 spectrometer at
500 MHz in D2O using sodium tetramethylsilylpropanoate
(TSP) as an internal reference.
K2PtCl4 (10 mmol) was added to a stirring aqueous solution
(50 mL) of KI (70 mmol). The solution was stirred at 258C
for 30 min under a nitrogen atmosphere to obtain a black
solution of K2PtI4, into which an aqueous solution of
10 mmol sodium salt of HP in 15 mL water was added dropwise
with stirring. After 24 h at 258C in the dark, a yellow precipitate
was filtered, washed sequentially and thoroughly with
water, ethanol and ether, and finally dried in vacuum. Data
for [PtPI]: 2.8 g, yield: 56%, dark yellow solid. Found
(calcd. for C9H17IN2O2Pt), C 21.23 (21.30), H 3.44 (3.35), N 5.37
(5.52), Pt 38.30 (38.47). IR (cmꢀ1): 3432s (br), 3179s, 3106s,
2928m, 2854m, 1594vs, 1443m, 1383s, 1322m, 1255m,
1067m, 1020m, 930w, 884m, 643w, 486m; 1H NMR (D2O/TSP,
ppm): d 1.11–1.66 (m, 6H, 3CH2 of DACH), 2.01–2.78 (m, 6H, 2CH
of DACH and CH2 of DACH and NHCH2CH2COO), 3.27–3.66
(m, 2H, NHCH2CH2COO); ESI-MS m/z: [MþH]þ ¼ 508 (100%),
[MþNa]þ ¼ 530 (50%).
Synthesis
Ligand HP: 3-(2-amino-cyclohexylamino)-propionic acid
Fifty millimolar of mono-Boc protected 1R,2R-DACH, 70 mmol K2CO3
and 50 mmol ethyl 3-bromopropionate were dissolved in 300 mL
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Arch. Pharm. Chem. Life Sci. 2013, 346, 308–313
Complex X1
In vitro cytotoxicity
A suspension of silver oxalate (10 mmol) and [PtPI] (20 mmol) in
200 mL water was stirred at 608C under a nitrogen atmosphere
in the dark for 24 h, the resulting AgI precipitate was filtered off
and washed with water for two times. The filtrate was evaporated
to nearly dryness and to give a white solid, which was washed
with cool water and ethanol for several times, and then dried in
vacuum. Yield 31%. Found (calcd. for C20H34N4O8Pt2), C 28.25
(28.30), H 4.09 (4.01), N 6.53 (6.60), Pt 45.92 (46.01). IR (cmꢀ1):
3479s (br), 3111s, 2937m, 2864m, 1647vs (sh), 1392s, 1322m,
1271m, 1078w, 1030w, 903w, 806m, 572m, 499m; 1H NMR
(D2O/TSP, ppm): d 1.11–1.63 (m, 12H, 6CH2 of 2DACH), 1.92–
2.65 (m, 12H, 2CH2 of 2DACH and 4CH of 2DACH
and 2NHCH2CH2COO), 3.15–3.29 (m, 4H, 2NHCH2CH2OO);
ESI-MS m/z: [MþNa]þ ¼ 871.5 (80%). The synthetic procedures
for complexes X2–X5 were similar to X1.
In vitro cytotoxicity was evaluated against four different human
carcinoma cell lines: HepG-2 human hepatocellular carcinoma
cells, MCF-7 human breast cancer cells, A549 human lung cancer
cells and HCT-116 human colorectal cancer cells. Among them,
MCF-7 and HepG-2 were cultured in RPMI 1640 supplemented
with 10% fetal bovine serum, 100 IU/mL penicillin, and
100 mg/mL streptomycin; HCT-116 was cultured in McCoy’s 5A
(Sigma–Aldrich), which contained 10% fetal bovine serum. Cells
were maintained at 378C in a humidified atmosphere containing
5% CO2. The experimental procedures of MTT assay were described
by Mosmann [22]. Complexes X1–X5, oxaliplatin and carboplatin
were added in final concentrations ranging from 0 to 100 mM.
After 48 h, 10 mL MTT in PBS (5 mg/mL) was added to each well
and the plates were incubated for 3 h at 378C. Remove the liquid
and add DMSO (150 mL) to dissolve the formazan. The OD for each
well was measured on a microplate reader at a wavelength of
490 nm. All cytotoxicity tests were carried out three times paral-
lelly, IC50 values were calculated from curves constructed by
plotting the inhibitory rate of compounds against tumor cells
(%) versus the logarithm of compounds concentrations.
Complex X2
Yield 26%. Found (calcd. for C21H36N4O8Pt2), C 29.31 (29.23),
H 4.26 (4.18), N 6.58 (6.50), Pt 45.10 (45.27). IR (cmꢀ1): 3423s
(br), 3228s, 2937s, 2859s, 1615vs, 1453s, 1355vs, 1253m,
1174m, 1079m, 1031m, 960m, 890m, 722m, 653w, 563m,
499m. 1H NMR (D2O/TSP, ppm): d 1.14–1.61 (m, 12H, 6CH2 of
2DACH), 2.02–2.66 (m, 12H, 2CH2 of 2DACH and 4CH of 2DACH
and 2NHCH2CH2COO), 3.21–3.28 (m, 6H, 2NHCH2CH2COO and
COOCH2COO). ESI-MS m/z: [MþH]þ ¼ 863.5 (30%), [MþK]þ ¼ 901.6
(50%).
Induction of cell apoptosis
Apoptosis of tumor cells induced by complexes X1, X5, or oxa-
liplatin (positive control) was measured by FCM using annexin
V-FITC/PI apoptosis kit according to the instructions. HCT-116
cells were washed in cold phosphate-buffered saline (PBS) and
then centrifuged (5 min, 2000 rpm). Cells were stained with
annexin V-FITC and propidium iodide (PI) in the binding buffer.
After 15 min of incubation at room temperature, the fluor-
escence was tested using a flow cytometer (FACS Calibur, BD
Corp, USA) and the excitation wavelength was 488 nm. Results
were analyzed using Cell Quest Pro software and represented as
percentage of normal and apoptotic cells at various stages. FITC
and PI fluorescences were measured in the FL1 and the FL2
channel, respectively.
Complex X3
Yield 22%. Found (calcd. for C22H38N4O8Pt2), C 29.99 (30.14),
H 4.36 (4.34), N 6.56 (6.39), Pt 44.31 (44.52). IR (cmꢀ1): 3443s
(br), 3232s, 2938s, 2857m, 1616vs (sh), 1387vs, 1261s, 1174m,
1079m, 1030w, 888w, 807m, 640m (br), 547m. 1H NMR (D2O/TSP,
ppm):
d 1.11–1.62 (m, 12H, 6CH2 of 2DACH), 2.02–2.67
(m, 16H, 2CH2 of 2DACH and 4CH of 2DACH and 4H
of COOCH2CH2COO and 2NHCH2CH2COO), 3.09–3.29 (m, 4H,
2NHCH2CH2COO). ESI-MS m/z: [MþH]þ ¼ 877.6 (30%),
[MþNa]þ ¼ 899.5 (30%).
This work is financially supported by the National Natural Science
Foundation of China (Projects 20971022 and 21271041) and the New
Drug Creation Project of the National Science and Technology Major
Foundation of China (Project 2013ZX09402102-001-006) to S.G.
Complex X4
Yield 28%. Found (calcd. for C24H40N4O8Pt2), C 31.79 (31.93),
H 4.50 (4.43), N 6.33 (6.21), Pt 43.09 (43.28). IR (cmꢀ1): 3444vs
(br), 3232vs, 2944s, 2865s, 1615vs, 1374vs, 1253m, 1154m,
1117m, 1079w, 1031w, 890m, 787m, 704m, 654w, 563m,
499m. 1H NMR (D2O/TSP, ppm): d 1.15–1.83 (m, 14H, 6CH2 of
2DACH and 2H of (COO)2C(CH2)2CH2), 1.88–2.86 (m, 16H, 2CH2
of 2DACH and 4CH of 2DACH and 4H of (COO)2C(CH2)2CH2
and 2NHCH2CH2COO), 2.90–3.30 (m, 4H, 2NHCH2CH2COO).
ESI-MS m/z: [MþH]þ ¼ 903.5 (25%), [MþNa]þ ¼ 925.5 (33%).
The authors have declared no conflict of interest.
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