Synthesis, cytotoxicity, and DNA-binding levels of ammine/cyclohexylamine platinum(II) complexes with dicarboxylates

Article abstract of DOI:10.1080/15533170600651462

Four new ammine/cyclohexylamine platinum(II) complexes with dicarboxylates (a-d) have been synthesized and characterized by elemental analysis, conductivity, IR, UV, and ¹HNMR spectra techniques. The cytotoxicity of the complexes was tested by MTT assay. The cell cycle analysis and the levels of total platinum bound to DNA were measured by flow cytometry and ICP-MS. The results show that the cytotoxicity of complexes (a-d) against EJ, HCT-8, BGC-823, HL-60, and MCF-7 cell lines decreases in the sequence: b > a > c > d. Complexes (a-d) have better cytotoxicity against EJ and HL-60 and complex (b) demonstrates cytotoxicity superior to that of the clinically established cisplatin. The complexes (a-d) induced a concentration-dependent accumulation of HL-60 cells in the G₂/M phase of the cell cycle as cisplatin. The levels of total platinum bound to DNA in HL-60 and EJ cells decrease in the sequence: b > cisplatin > a > c > d under the same experimental conditions. Copyright

Full text of DOI:10.1080/15533170600651462

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Synthesis, Cytotoxicity, and DNA‐Binding Levels of
Ammine/Cyclohexylamine Platinum(II) Complexes with
Dicarboxylates
Jinchao Zhang a & Shen Yong a
a Department of Chemistry, College of Chemistry and Environmental Science , Hebei
University , Baoding, P.R. China
Published online: 15 Feb 2007.
To cite this article: Jinchao Zhang & Shen Yong (2006) Synthesis, Cytotoxicity, and DNA‐Binding Levels of Ammine/
Cyclohexylamine Platinum(II) Complexes with Dicarboxylates, Synthesis and Reactivity in Inorganic, Metal-Organic, and Nano-
Metal Chemistry, 36:4, 345-351
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Synthesis and Reactivity in Inorganic, Metal-Organic and Nano-Metal Chemistry, 36:345–351, 2006
Copyright # 2006 Taylor & Francis Group, LLC
ISSN: 0094-5714 print/1532-2440 online
DOI: 10.1080/15533170600651462
Synthesis, Cytotoxicity, and DNA-Binding Levels
of Ammine/Cyclohexylamine Platinum(II)
Complexes with Dicarboxylates
Jinchao Zhang and Shen Yong
Department of Chemistry, College of Chemistry and Environmental Science, Hebei University,
Baoding, P.R. China
1970s, only one (carboplatin) has received worldwide approval
Four new ammine/cyclohexylamine platinum(II) complexes so far. Four drugs (oxaliplatin, nedaplatin, lobaplatin and
with dicarboxylates (a–d) have been synthesized and character-
ized by elemental analysis, conductivity, IR, UV, and 1HNMR
spectra techniques. The cytotoxicity of the complexes was tested
by MTT assay. The cell cycle analysis and the levels of total
platinum bound to DNA were measured by flow cytometry and
SKI2053R) have gained regionally limited approval and
another eight continue to be evaluated in clinical studies. Two
of these (JM-216, ZD0473) have recently entered phase III
studies. Therefore, research work is still worthwhile.[1–5]
ICP-MS. The results show that the cytotoxicity of complexes
(a–d) against EJ, HCT-8, BGC-823, HL-60, and MCF-7 cell
lines decreases in the sequence: b > a > c > d. Complexes (a–d)
have better cytotoxicity against EJ and HL-60 and complex (b)
demonstrates cytotoxicity superior to that of the clinically estab-
lished cisplatin. The complexes (a–d) induced a concentration-
The structure-activity relationships summarized by Cleare
and Hoeschele dominated Pt drug design for over 20 years
and remained valid until relatively recently. This is reflected
in the fact that all Pt compounds that have entered clinical
trials so far adhere to this set of guidelines. However, it has
dependent accumulation of HL-60 cells in the G2/M phase of become quite evident that mere analogues of cisplatin or carbo-
the cell cycle as cisplatin. The levels of total platinum bound to
DNA in HL-60 and EJ cells decrease in the sequence: b >
cisplatin > a > c > d under the same experimental conditions.
platin will probably not offer any substantial clinical advan-
tages over the existing drugs. A number of researchers have
taken a completely different approach to Pt drug design and
have prepared compounds that violate the structure-activity
Keywords ammine/cyclohexylamine platinum(II) complexes,
synthesis, cytotoxicity, cell cycle, DNA binding
relationships but show antitumor activities. Therefore, the
search continues for an improved Pt antitumor agent, motivated
by the desire to design a less toxic, orally active compound that
is non-cross-resistant with cisplatin and carboplatin.[2,4,6,7]
The mixed ammine/amine platinum complexes with chloro
INTRODUCTION
have been reported and demonstrated better activity against cis-
By now, cisplatin is one of the most active chemotherapeutic platin-resistant cells in vitro and more less toxicity than the
agents available for the treatment of a variety of malignancies, parent cisplatin.[8,9] For example, JM-216 has recently entered
especially testicular and ovarian. Despite its success, cisplatin phase III studies. The possible advantage of platinum anticancer
has several disadvantages that include severe toxicity, relatively drugs with decreased reactivity of leaving group is an estab-
narrow range of tumors and resistance to cisplatin. These draw- lished approach which commenced with the clinical success of
backs have been the impetus for the development of an carboplatin. It is reported that the decreased reactivity of carbo-
improved Pt antitumor drug, thousands of Pt compounds have platin reduce the nephrotoxic and neurotoxic side effects of cis-
been synthesized and evaluated as potential antitumor agents. platin. Moreover, decrease in reactivity of leaving groups may
Among the 33 platinum agents that have entered clinical trials also lead to reduced detoxification reactions by intracellular
after the onset of clinical studies with cisplatin in the early thiols. This may increase the efficacy of the drug and help to cir-
cumvent resistance mechanism such as overexpression of gluta-
thione. So, carboxylate platinum complexes seem to be quite
promising than the corresponding chloro analogs. Thus far,
Received 3 November 2005; accepted 8 February 2006.
Address correspondence to Jinchao Zhang, Department of
Chemistry, College of Chemistry and Environmental Science, Hebei
University, Baoding 071002, P.R. China. E-mail: jczhang6970@
yahoo.com.cn
many mixed ammine/amine platinum complexes with chloro
have been reported. But few mixed ammine/amine platinum
complexes with carboxylates were reported.[8–11] Previously
345

346
J. ZHANG AND S. YONG
we reported the synthesis, characterization and antitumor
activity of platinum(II) complexes of mixed ammine/ COO)] (b), [Pt(NH3)(
methylamine with bidentate carboxylates.[12] In the present [Pt(NH3)(
The synthetic procedure for [Pt(NH3)(
NH2)(OOC-CH2-
NH2)(OOC-CH2-CH2-COO)] (c) and
NH2)(OOC-CH55CH-COO)] (d) are similar. The
work, the synthesis, cytotoxicity and DNA-binding levels of synthetic routines of the mixed ammine/cyclohexylamine
new mixed ammine/cyclohexylamine platinum(II) complexes platinum(II) complexes with dicarboxylates are given
with dicarboxylates are reported and discussed.
here (Figure 1).
EXPERIMENTAL
Cytotoxicity Analysis
The complexes were dissolved in phosphate-buffered saline
(PBS) and diluted to the required concentration with culture
medium when used. The cytotoxicity was evaluated by MTT
assay.[15] Briefly, cells were plated in 96-well microassay
culture plates (104 cells per well) and grown overnight at
378C in a 5% CO2 incubator. Compounds were then added to
the wells to achieve final concentrations ranging from 1027
to 1024 M. Control wells were prepared by addition of
culture medium. Wells containing culture medium without
cells were used as blanks. The plates were incubated at 378C
in a 5% CO2 incubator for 48 hr. Upon completion of the incu-
bation, stock MTT dye solution (20 mL, 5 mg/mL) was added
to each well. After 4 hr incubation, 2-propanol (100 mL) was
added to solubilize the MTT formazan. The optical density
of each well was then measured on a microplate spectropho-
tometer at a wavelength of 570 nm. The IC50 value was deter-
mined from plots of % viability against dose of compound
added. Five different human carcinomas were the subjects in
this study: EJ (bladder carcinoma), HCT-8 (colon carcinoma),
BGC-823 (gastric carcinoma), HL-60 (immature granulocyte
leukemia) and MCF-7 (galactophore carcinoma).
Reagents
All reagents and solvents were analytical reagent grade.
MTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl
tetrazolium
bromide), TrisHCl (tris-(hydroxymethyl)-aminomethane, hydro-
chloride), PI (propidium iodide), RNase and Genomic DNA
extraction kit were purchased from Sigma (USA). RPMI 1640
culture medium and FBS (fetal bovine serum) was from
Gibco. Cisplatin was purchased from Qi Lu Pharmaceutical
Factory in China.
Instrumentation and Measurement
Elemental analyses were determined on a EA-1110 elemen-
tal analyzer. The thermal analysis was determined using
RI-GAKU 8150 meter (Ar, 108C min21, Al2O3). Molar con-
.
ductances at room temperature were measured in 1023
M
aqueous solutions using a DSS-11A type conductivity meter.
The IR spectra were recorded in the 400–4000 cm21 range
using KBr pellets and a Perkin-Elmer Model-683 spectro-
photometer. The electronic spectra in H2O were measured on
an UV-3400 Toshniwal spectrophotometer. The 1HNMR
spectra in D2O were recorded on a Brucker AV 400 NMR spec-
trometer. Cell cycle analysis was performed on a BECScan
Flow Cytometer. The levels of total platinum bound to DNA
were measured by PE Elan-5000 ICP-MS (Inductively
Coupled Plasma Mass Spectrometry).
Flow Cytometry Analysis
The cell cycle analysis was performed as described by
Ferlini et al.[16] HL-60 cells were treated with platinum com-
plexes for the indicated times and harvested by centrifugation
at 1200 rpm/min for 5 min at room temperature. Cell pellets
were rinsed with PBS, suspended in a 1:1 (v/v) solution of
PBS and 0.2 M Na2HPO4 2 0.1 M citric acid (pH ¼ 7.5), and
fixed with cold ethanol at 48C for 1 hr. Fixed cells were
washed with PBS and resuspended in a staining solution con-
taining PI (10 mg/mL) and DNase-free RNase (100 mg/mL).
The cell suspensions were incubated at 378C for 1 hr in the
Preparation of Complexes
Precursor
[Pt( NH2)I2]2 (ii) and cis-[Pt(
synthesized according to the literatures.[13,14] [Pt(NH3)
NH2)(OOC)2] (a): cis-[Pt( NH2)(NH3)I2] (0.57 g,
complexes
cis-[Pt(
NH2)2I2]
(i),
NH2)(NH3)I2] (iii) were
(
1 mmol) was mixed with AgNO3 (0.331 g, 1.95 mmol) in
15 mL of water. The mixture was allowed to stir overnight in
the dark. The AgI precipitate was removed by filtration and
1–2 drops of NaCl was added to the filtrate. If a precipitate
(AgCl) appeared immediately, it was filtered out after 10 min
and again 1 drop of NaCl was added to the filtrate. The pro-
cedure was repeated until there was no immediate precipitate
after adding one drop of NaCl solution. When all the silver
ions have been removed, a slight excess of the sodium salt of
the oxalic acid was added to the filtrate. After 6 hr, the
mixture was evaporated to dryness and washed a few times
with a minimum quantity of very cold water. The final
product [Pt(NH3)( NH2)(OOC)2] was dried over P2O5
under vacuum. Yield: 65%.
FIG. 1. Synthetic routines of the mixed ammine/cyclohexylamine
platinum(II) complexes with dicarboxylates.

SYNTHESIS AND CHARACTERIZATION OF NEW PLATINUM COMPLEXES
347
Statistical Analysis
TABLE 1
Physical properties of the complexes
Data were collected from at least three separate exper-
iments. The results are expressed as means + sd. The statisti-
cal differences were analyzed using SPSS0 t-test. p values
less than 0.05 were considered to indicated statistical
differences.
Found (calcd.) (%)
Complex
C
N
H
Pt
V
(i)
22.20
(22.27)
13.09
4.30
(4.33)
2.50
4.01
(4.05)
2.34
—
—
RESULTS AND DISCUSSION
(ii)
(iii)
a
—
—
—
Physical Properties of the Complexes
(13.15)
12.70
(2.56)
4.80
(2.39)
2.70
The physical properties of the complexes are presented in
Table 1. There is good agreement between the calculated and
the found values. Low molar conductances for the complexes
correspond to non-electrolytes.[18]
—
(12.75)
24.12
(4.96)
7.23
(2.85)
3.98
48.96
(48.86)
47.32
5.36
5.68
5.79
6.00
(24.06)
26.31
(7.02)
6.87
(4.04)
4.29
b
(26.15)
28.32
(6.78)
6.58
(4.39)
4.87
(47.20)
45.87
IR Spectra
c
The IR spectra of the complexes (a–d) are similar, the main
bands with tentative assignments are listed in Table 2. The
bands yNH and dNH in the precursor complexes (i–iii) and new
complexes (a–d) shift to lower frequencies than those of free
ammine and cyclohexylamine. New band appears at 444–
483 cm21 and is assigned to Pt-N stretching. Thus it indicates
that they are coordinated with platinum through nitrogen
atoms. The carboxylate group of the complexes (a–d) shows
two bands, an intense antisymmetric carboxylate stretching
(28.10)
28.36
(28.23)
(6.56)
6.69
(6.59)
(4.72)
4.32
(4.27)
(45.65)
45.86
(45.87)
d
dark and analyzed on a flow cytometry. Data were collected by
ModFit LT 2.0 for power software.
y
(as, coo2) and a symmetric stretching y(s,coo2), at about 1650 and
-
1400cm21, respectively. The values of Dy(coo2)(y(as, coo2)
DNA Binding
y
(s, coo2)) of the complexes (a–d) are in the range 248–
The levels of total platinum bound to DNA in HL-60 and EJ
cells were performed as described by Mellish et al.[17] Briefly,
approximately 5 ꢀ 107 HL-60 or EJ cells were seeded in
tissue-culture flasks, then the complexes were added in a con-
centration gradient, each concentration in triplicate, and the
final concentrations were maintained at 10, 25, 50 and
100 mM, respectively. They were incubated at 378C in 5%
CO2 for 4 hr. Cells were then harvested, and DNA was
294 cm21, which is greater than Dy(coo2) of the corresponding
sodium dicarboxylates, so we may suggest that the carboxylate
group is monodentate coordinated through oxygen atoms.[19]
This is further confirmed by the appearance of the peaks of yPt-O
.
Electronic Spectra
No absorption peaks appear for the malonic acid and
extracted according to DNA extraction kit procedure. The succinic acid, one peak appears at 212.0 (n ! pꢁ) and
DNA was dissolved in 300 mL water. The purity and concen- 212.0 nm (p ! pꢁ) in the spectra of the oxalic acid and
tration of DNA was measured by UV spectroscopy. An maleic acid respectively. After formation of the complexes,
aliquot of the remaining sample was sonicated and subjected blue shifts by ca. 18.0 and 7.0 nm in the spectra of the
to platinum analysis by ICP-MS.
complex (a) and (d) respectively, one new peak appears at
TABLE 2
IR data (cm21) of the complexes
Complex
yNH
dNH
y(as, coo2)
y(s, coo2)
Dy(coo2)
yPt-O
yPt-N
(i)
(ii)
(iii)
a
b
c
3195, 3109
3236, 3200
3270, 3200
3260, 3165
3258, 3156
3209, 3125
3268, 3158
1566
1570
1510
1550
1549
1535
1523
444
470
480
470
465
470
483
1679
1669
1647
1656
1385
1401
1399
1405
294
268
248
251
586
569
578
574
d

348
J. ZHANG AND S. YONG
TABLE 3
1HNMR spectra of the ligands and complexes
Compound
Chemical shift (d, ppm)
Cyclohexylamine
Oxalic acid
2.52 (br, 1H, CH (methine)), 0.9–2.0 (m, 10H, CH(alkyl))
—
3.26 (s, 2H, -CH2-)
Malonic acid
Succinic acid
Maleic acid
2.67 (s, 4H, -CH2-CH2-)
6.28 (s, 2H, -CH55CH-)
a
b
c
2.91 (br, 1H, CH (methine)), 1.0–2.2 (m, 10H, CH(alkyl))
2.92 (br, 1H, CH (methine)), 1.1–2.4 (m, 10H, CH(alkyl)), 3.38 (s, 2H, -CH2-)
2.88 (br, 1H, CH (methine)), 1.0–2.4 (m, 10H, CH(alkyl)), 2.82 (s, 4H, -CH2-CH2-)
2.85 (br, 1H, CH (methine)), 1.0–2.3 (m, 10H, CH(alkyl)), 6.35 (s, 2H, -CH55CH-)
d
206.0 nm in the spectra of the complex (b), no absorption peak cytotoxicity against EJ and HL-60 and complex (b) demonstrates
appears for the complex (c).
cytotoxicity superior to that of the clinically established cisplatin.
The mode of action of platinum anticancer drugs is still not
completely understood but it is thought to depend on hydrolysis
reactions where the leaving group is replaced by a water
molecule adding a positive charge on the molecule. The
hydrolysis product is believed to be the active species
reacting mainly with glutathione in the cytoplasm and the
DNA in the nucleus, thus inhibiting replication, transcription
1HNMR
As listed in Table 3, after formation of the complexes, the
protons of the complexes shift to low field compared with
those of free ligands. This is also further confirmed that the
dicarboxylate and cyclohexylamine are coordinated with
platinum through oxygen and nitrogen atoms.
Thermal Stability
Thermal analytical data was listed in Table 4. The thermal
behavior of the complexes (a–d) is similar. They have a
fixed melting point, there is an endothermic peak at
320–3428C coinciding with the melting point. There is a
big endothermic peak (405–8008C), corresponding to
50.25–55.06% weight loss.
Based on the preceding studies, we propose a tentative
coordination structure for the complexes (Figure 2).
FIG. 2. Possible structure of the complexes (a–d).
Cytotoxicity Effect
As listed in Figure 3, the cytotoxicity against EJ, HCT-8,
BGC-823, HL-60, and MCF-7 cell lines decreases in the
sequence: b . a . c . d. Complexes (a–d) have better
TABLE 4
Thermal analytical data of the complexes
Dec. temp.
(8C)
Total
wt. loss
(%)
m.p.
(8C)
Complex
T1
T2
Residue
a
b
c
334
342
328
320
410
405
421
419
800
786
765
776
50.25
51.36
53.47
55.06
Pt
Pt
Pt
Pt
d
FIG. 3. Cytotoxicty of complexes (a–d) against various human carcinomas.

SYNTHESIS AND CHARACTERIZATION OF NEW PLATINUM COMPLEXES
349
and other nuclear functions and arresting cancer cell prolifer-
ation and tumor growth. So the reactivity of leaving groups
is an important factor to affect anticancer activity.[5] In the
present work, for ammine/cyclohexylamine platinum(II) com-
plexes with dicarboxylates, dicarboxylates coordinating with
platinum through oxygen atoms form different chelate cycle,
cycle size affects the reactivity of leaving groups, and further
affect their cytotoxicity.
Cell Cycle Analysis
The effect of the complexes on cell cycle was given in
Table 5. The complexes (a–d) induced a concentration-
dependent accumulation of HL-60 cells in the G2/M phase
of the cell cycle as cisplatin.
DNA Binding
FIG. 4. Levels of total platinum bound to DNA in HL-60 cell after 4-hr
exposure to platinum complexes.
As listed in Figures 4 and 5, the levels of total platinum
bound to DNA in HL-60 and EJ cells decrease in the sequence:
b . cisplatin > a . c . d under the same experimental con-
ditions. It is generally been accepted that platinum-based drugs
exert their cytotoxic effects through the formation of platinum-
DNA adducts. This occurs predominantly at the N7 position of
guanosine. The vast majority of cisplatin-DNA adducts are of
the intrastrand type between adjacent guanines.[20] In general,
the degree of cytotoxicity of cisplatin correlates with the
amount of DNA-platination.[21] However, a study in testicular
cancer germ cell lines found no association between cisplatin
DNA-platination and drug sensitivity and a similar observation
was made in a breast cancer cell line.[22,23] With respect to a
correlation between the cytotoxicity of oxaliplatin and DNA-
platination, the literature is limited. No correlation could be
found between DNA-platination and cytotoxicity. Mellish
et al.[17] reported that no significant correlation was found
between total DNA platination levels and cytotoxicity of the
seven platinum-based drugs in SKOV-3 or in CH1 cell lines.
Roberts et al.[24] reported that significant correlation was found
between total DNA platination levels and cytotoxicity of poly-
nuclear platinum complex in L1210/0 cell line. In our work,
we found that there was also significant correlation between
total DNA platination levels and cytotoxicity of four ammine/
cyclohexylamine platinum complexes with dicarboxylates in
HL-60 and EJ cell lines. The total DNA platination levels
contains some kinds of Pt-DNA adducts formed by platinum
complex such as 1,2-intrastrand adducts between adjacent
guanines or adjacent adenine. This suggests that it is probably
the levelof specific DNA adducts thatis important indetermining
the cytotoxicity of platinum-based drugs.[17] In addition,
TABLE 5
Effect of the platinum complexes on cell cycle in HL-60 cells
Cell cycle (%) (x¯+s
)
Concentration
(mM)
Complex
G1
S
G2/M
Control
Cisplatin
0.0
1.5
3.0
1.5
3.0
1.5
3.0
1.5
3.0
1.5
3.0
31.34 + 1.06ꢁꢁ
25.36 + 1.25
18.98 + 1.02ꢁꢁ
26.38 + 1.06ꢁ
22.65 + 1.65ꢁ
22.78 + 2.03ꢁꢁ
14.65 + 1.23ꢁꢁ
27.56 + 2.13
24.58 + 1.65ꢁꢁ
29.68 + 2.35ꢁꢁ
26.78 + 1.25ꢁꢁ
60.85 + 1.41ꢁꢁ
50.36 + 1.35
45.68 + 2.87ꢁꢁ
51.35 + 4.06ꢁ
48.36 + 5.02ꢁ
47.65 + 4.65ꢁꢁ
43.25 + 3.98ꢁꢁ
53.46 + 4.79
49.68 + 4.75ꢁꢁ
56.98 + 5.98ꢁꢁ
52.36 + 2.65ꢁꢁ
7.80 + 1.18ꢁꢁ
24.28 + 2.35
35.34 + 2.87ꢁꢁꢁ
a
b
c
22.27 + 2.35
28.99 + 2.65ꢁꢁꢁ
29.57 + 2.06ꢁꢁ
42.10 + 3.75ꢁꢁ
18.98 + 0.35
25.74 + 2.03ꢁꢁ
13.34 + 1.32ꢁꢁ
20.86 + 2.98ꢁꢁ
d
ꢁP , 0.05, ꢁꢁP , 0.01 compared with the control group.

350
J. ZHANG AND S. YONG
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FIG. 5. Levels of total platinum bound to DNA in EJ cell after 4-hr exposure
to platinum complexes.
platinum-based drugs might have other important targets apart
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