Synthesis and in vitro antiproliferative activity of platinum(II) complexes with N-monoalkyl 1R,2R-diaminocyclohexane as ligands

Article abstract of DOI:10.1016/j.ejmech.2012.07.026

Nine dichloridoplatinum(II) complexes with N-monoalkyl 1R,2R- diaminocyclohexane as ligands were synthesized and spectrally characterized. Among them, the crystal structure of a typical complex has been determined by X-Ray diffraction. All compounds were evaluated for their in vitro antitumor activity against four human cancer cell lines, which showed selective cytotoxicity comparable to that of positive agents against A549 (human non-small cell lung cancer) cell line. Especially complex 3, cis-[(1R,2R)-N ¹-2-butyl-1,2-diaminocyclohexane-N,N′] dichloroplatinum(II), was much more active in vitro (IC₅₀ = 1.82 μM) than cisplatin against A549. The structure-activity relationship was summarized according to the cytotoxicity and QSAR properties. In addition, flow cytometry and agarose gel electrophoresis experiments were also applied to investigate the mode of action of the representative complexes.

Full text of DOI:10.1016/j.ejmech.2012.07.026

European Journal of Medicinal Chemistry 55 (2012) 297e306
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and in vitro antiproliferative activity of platinum(II) complexes with
N-monoalkyl 1R,2R-diaminocyclohexane as ligands
Yanyan Sun , Fei Liu , Shaohua Gou ,*, Lin Cheng a b, Lei Fang a ,*, Runting Yin a
a
,
b
a
a
,
b
,
,
b
,b
a
Pharmaceutical Research Center, School of Chemistry and Chemical Engineering, Southeast University, Jiangning District, Nanjing 211189, China
Jiangsu Province Hi-Tech Key Laboratory for Bio-medical Research, Southeast University, Nanjing 211189, China
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 11 March 2012
Received in revised form
Nine dichloridoplatinum(II) complexes with N-monoalkyl 1R,2R-diaminocyclohexane as ligands were
synthesized and spectrally characterized. Among them, the crystal structure of a typical complex has
been determined by X-Ray diffraction. All compounds were evaluated for their in vitro antitumor activity
against four human cancer cell lines, which showed selective cytotoxicity comparable to that of positive
12 July 2012
Accepted 17 July 2012
Available online 24 July 2012
1
agents against A549 (human non-small cell lung cancer) cell line. Especially complex 3, cis-[(1R,2R)-N -
0
2
-butyl-1,2-diaminocyclohexane-N,N ] dichloroplatinum(II), was much more active in vitro
(
IC50 ¼ 1.82
mM) than cisplatin against A549. The structureeactivity relationship was summarized
Keywords:
according to the cytotoxicity and QSAR properties. In addition, flow cytometry and agarose gel elec-
trophoresis experiments were also applied to investigate the mode of action of the representative
complexes.
Platinum(II) complexes
N-Monoalkyl 1R,2R-diaminocyclohexane
Cytotoxicity
Apoptosis
DNA cleavage
Ó 2012 Elsevier Masson SAS. All rights reserved.
1. Introduction
With the advantage of oxaliplatin applied in clinic use, a great
number of complexes containing 1R,2R-DACH moiety have been
Despite the success of cisplatin as one of the most active agents
in solid tumor chemotherapy in clinic applications [1,2], many
related platinum-based antitumor agents have been designed and
synthesized according to the structureeactivity relationships (SAR)
summarized by Cleare and Hoeschele in 1973 [3e6] to overcome
the drawbacks of cisplatin including side effects, low water-
solubility, narrow spectrum, and intrinsic or acquired resistance.
By replacing the leaving groups of chloride anions in cisplatin with
synthesized and tested for anticancer activities against various
human solid tumors, and some of them have entered preclinical
and clinical trials [13e16]. For example, miriplatin, (1R,2R-dia-
0
minocyclohexane-N,N )bis(tetradecanoato-O)platinum(II), as the
second DACHeplatinum agent in clinical practice after oxaliplatin,
was approved in the treatment of hepatocellular carcinoma in
Japan in 2009 [17]. Thus, it can be deduced that 1R,2R-DACH is an
effective carrier group in designing DACH-type platinum
derivatives.
1
1
,1-cyclobutanedicarboxylate, carboplatin was launched in the late
980’s, which has been widely used in clinical chemotherapy due to
its reduced side effects compared to its parent compound cisplatin
7]. Oxaliplatin, as the first effective platinum-based agent for the
In addition to DACH, some platinum complexes with DACH
derivatives as carrier ligands have been reported, but these ligands
were mainly involved with ring-substituted DACH derivatives
[18e21] (Fig.1A). As for N-substituted DACH derivatives, onlya small
[
treatment of colorectal cancer, surmounts the cross-resistance with
cisplatin or carboplatin and possesses appropriate water-solubility
and lessened toxicity [8]. The bulky chiral ligand, 1R,2R-dia-
minocyclohexane (1R,2R-DACH), contributes to high cytotoxicity
against cisplatin-resistant cell lines, possibly due to the steric
hindrance effect of the DACHeplatinumeDNA adducts [9e12].
1
2
number of symmetric N ,N -disubstituted DACH derivatives were
used as carrier ligands [22e24] (Fig. 1B). However, the platinum(II)
complexes bearing N-monosubstituted DACH ligands have seldom
been involved, possibly due to the difficulty of obtaining N-mono-
substituted DACH derivatives by controlling the equivalent reac-
tivity of the two amino groups in DACH [25]. Moreover, several non-
1
2
platinum complexes with symmetric N ,N -disubstituted DACH
*
Corresponding authors. Pharmaceutical Research Center, School of Chemistry
ligand have been reported for their structural research including
and Chemical Engineering, Southeast University, Jiangning District, Nanjing 211189,
China. Tel./fax: þ86 25 83272381.
0 0
(R,R )-(N,N )-diisopropylcyclohexyl-1,2-diamine] dichloro ber-
yllium(II) [26], [(R,R )-(N,N )-dibenzylcyclohexyl-1,2-diamine]
[
0
0
E-mail addresses: sgou@seu.edu.cn (S. Gou), fanglei24@googlemail.com
(
L. Fang).
dichloropalladium(II) [27], and Cu(II) [28] complexes.
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.07.026

298
Y. Sun et al. / European Journal of Medicinal Chemistry 55 (2012) 297e306
A
B
R4
R5
2+
H
2
N
R3
R2
O
O
O
O
NH
NH
Cl
Cl
NH
N
N
(ClO4)22-
Pt
Pt
Pt
N
NH
R5
R1
H
2
R4
R
1
2
3
= Me, Et, C(CH
= Me, H
= Me, H
3 3
)
5
R = Me
R
R
4
R = H, OH, Me
1
2
Fig. 1. General formula of reported platinum complexes containing ring-substituted 1R,2R-DACH (A) and symmetric N ,N -disubstituted 1R,2R-DACH (B) derivatives as ligands.
Recently, we have set up a project to study platinum(II)
complexes with N-substituted DACH derivatives as carrier ligands
systematically. By introducing different mono-substituted groups
at one of two nitrogen atoms of 1R,2R-DACH, one of our targets is to
investigate the sterically hindered effect of N-substituted groups in
these complexes on their antitumor activities and summarize the
structureeactivity relationship. In this study, we’d like to report
such a series of dichlorido platinum(II) complexes of the type [cis-
of potassium tetrachloroplatinate and L1eL9 in water (Scheme 1B),
respectively. The synthesized platinum(II) complexes were char-
1
13
acterized by elemental analysis, IR, H NMR, C NMR and ESI-MS
spectra. It is noted that NeH stretching vibrations of complexes
ꢀ
1
1e9 exhibit, in the range 3050e3209 cm , red shifting compared
with the amino group of the corresponding metal-free ligands
(hydrochloride salts). The amino coordination with Pt(II) ions and
PteCl bond formation can be supported by the presence of v (PteN)
ꢀ
1
1
PtLCl
2
], where L represents a number of N-monoalkyl 1R,2R-DACH
and v (PteCl) in 421e499 cm . In the H NMR spectra of complexes
1e9, the broad signals of hydrogen atoms belonging to amino
groups appear in the range of 5.93e6.65 ppm due to the amine
coordination with platinum(II) ions, compared with the metal-free
ligands in the range of 8.70e9.42 ppm. The signals of CeH protons
connected to the amino groups occur in the spectra of
2.40e3.20 ppm as a multiplet, shifting highfield compared with the
corresponding signals (3.22e3.70 ppm) in the free ligands. The
derivatives including linear and cyclic alkyl groups functioned as
steric hindrance.
The in vitro antitumor activities of these compounds against
human non-small cell lung cancer A549, breast carcinoma MCF-7,
colorectal cancer HCT-116 and hepatocellular carcinoma HepG2
cell lines were tested to investigate the effect of the different alkyl
groups at the N1 position of 1R,2R-DACH. In addition, the mecha-
nism of cellular death and the interaction with DNA for typical
compounds have also been discussed in this paper.
13
chemical shifts and carbon numbers of C NMR spectra for
complexes 1e9 were in conformity with the theoretical spectra of
complex structures. All the platinum complexes were characterized
þ
þ
2
. Results and discussion
by ESI-MS, showing the presence of [M þ H] and/or [M ꢀ Cl]
peaks which also display the isotopic distribution of the platinum
element. Elemental analysis confirmed the structures of the target
compounds further.
2.1. Synthesis and characterization
A stereogenic center with either R or S configuration at N1
position can be produced when the N-alkyl substituted 1R,2R-
DACH ligand is coordinated to the platinum(II) ion. In this case, the
diastereoisomeric mixtures may be produced with one alkyl
The preparation of 1R,2R-DACH derivatives (L1eL9) was carried
out starting from 1R,2R-diaminocyclohexane via several synthetic
steps (Scheme 1A). It was difficult to get N-monosubstituted 1R,2R-
DACH derivatives directly due to the equivalent reactivity of the
two amino groups, and therefore N-monoprotected DACH was
firstly synthesized. Usually, amino groups can be protected by
benzyl (Bn), tert-butoxycarbonyl (Boc) [29], benzyloxy-carbonyl
1
substituent (at N position) in the pseudo axial or equatorial posi-
2 2
tion of the PtN Cl square plane. Trying to explicit the structures of
our complexes, we have made a theoretic calculation for complex 1
to observe which configuration, R or S, is energetically preferred by
using HyperChem 7.5 [33]. After building the molecule, we chose
AMBER as the molecular mechanics force field which assigns atom
types and parameters to the molecule. Then amber2 was selected
as the parameter set for the AMBER force field. At last, Polak-Ribiere
was applied as the minimization algorithm to optimize the struc-
ture of the molecule and calculate the minimal energy of the
molecule, which are illustrated in Figure S1 (Supporting
information).
(
Cbz)
[30],
9-fluorenylmethoxycarbonyl
(Fmoc),
4-
methylbenzenesulfonyl (Ts) [31] groups and so on [32]. Among
these protecting groups, Boc was recently reported for N-mono-
protection of trans-1,2-diaminocyclohexane hydrochloride, which
can be easily deprotected with HCl/EtOAc [29]. Hence, in our study
N-mono Boc protected DACH was firstly synthesized and used as
the intermediate. Then the unprotected amino group underwent
Schiff base condensation with the corresponding alkyl/cycloalkyl
aldehyde or ketone, followed by in situ reduction with sodium
borohydride. Subsequently, the Boc group was removed through
hydrolysis in the presence of hydrochloric acid to give the ligands
Upon the calculation result, it is obviously judged that the S
1
configuration at N atom of complex 1 is more thermodynamically
(
L1eL9) in their hydrochloride salts. All the ligands were charac-
stable than the corresponding R configuration resulting from
1
terized by elemental analysis, IR, H NMR, and ESI-MS spectra
which were in conformity with the structure of target compounds.
The specific optical rotations of ligands turned out left-handed
similar to that of the precursor 1R,2R-DACH.
a steric effect of the ligand. Analogous calculation results were also
1
obtained for other complexes. So it is proposed that the N atom in
complexes 1e9 prefers to adopt S configuration.
Furthermore, an X-ray crystallographic analysis of complex 1
showed that two original stereogenic centers of carbon atoms were
maintained as R,R configuration, while the nitrogen atom of
Corresponding
(N-monoalkyl
1R,2R-diaminocyclohexane)
dichloroplatinum(II) complexes 1e9 were obtained by the reaction

Y. Sun et al. / European Journal of Medicinal Chemistry 55 (2012) 297e306
299
NH2 HCl
NH
2
NH2
NH
1R,2R)-DACH
NH2 HCl
NH
10
(i)
(ii)
(iii)
NH
Boc
NH
Boc
2
2
(
11
12
A
R1
R2
H
H
N R
N
3
N R
3
(iv)
(v)
(vi)
2
HCl
NH
Boc
NH
Boc
3
NH2
1
14
L1-L9
R3
H
6
N R
1
NH
Cl
Cl
(vii)
3
(viii)
5
B
L1-L9
Pt
4
NH2
2
N
3
H
4' 3'
2'
2
1
'
1: R3=
2: R3=
3: R3=
4: R3=
5: R3=
7: R3=
8: R3=
4'
5
'
3
'
1' 2'
5'
4
'
∗
∗
6'
3
'
6: R3=
9: R3=
1'
2'
ꢁ
ꢁ
Scheme 1. General synthetic route to ligands L1eL9 (A) and platinum(II) compounds 1e9 (B). (i) HCl, MeOH, 0 C; (ii) H
2
O, (Boc)
2
O, 0 C; (iii) NaOH; (iv) aldehyde or ketone, MeOH;
ꢁ
(
v) NaBH
4
; (vi) HCl/EtOAc, 0 C; (vii) Na
CO
2 3
, H
2
O; (viii) K
2
PtCl
4
, H
2
O, r. t., 5 h.
secondary amine preferred to S configuration and no its corre-
sponding enantiomer was observed in the crystal structure (Fig. 2),
which confirmed the above computational result as expected [34].
It is also noted that complexes 3 and 6 with 2-butyl and 2-pentyl
substituents connecting to one of 1R,2R-DACH nitrogen atoms,
respectively, can introduce a stereogenic center at the tertiary
carbon atom of the substituent (marked in asterisk symbol in
Scheme 1), which may generate a pair of optical enantiomers. Since
we have not obtained the optically pure ligand of N-(2R-butyl or
pentyl)-1R,2R-diaminocyclohexane or N-(2S-butyl or pentyl)-
and N-(2R/2S-pentyl)-1R,2R-diaminocyclohexane dihydrochloride
(L6) were used as racemic compounds in this study to prepare
complexes 3 and 6.
2.2. In vitro cytotoxic activity
The cytotoxicities of platinum complexes 1e9 were tested
in vitro against A549 (human non-small cell lung cancer), MCF-7
human breast carcinoma), HCT-116 (human colorectal cancer)
(
and HepG2 (human hepatocellular carcinoma) cell lines by MTT
assay, respectively. Since the tested complexes showed poor
aqueous solubility, they were all dissolved in DMSO and then
diluted in culture medium so that the effective DMSO concentra-
tion did not exceed 0.5%. The corresponding IC50 values were pre-
sented in Table 1. It was noted that all compounds showed selective
cytotoxicities against A549, which were comparable or superior to
those of cisplatin and oxaliplatin. As for MCF-7 cell line, these
compounds did not exhibit high cytotoxicity except compound 3
compared with cisplatin and oxaliplatin. Besides, HCT-116 and
HepG2 were the least sensitive cell lines to all the platinum
complexes.
1
R,2R-diaminocyclohexane to coordinate with platinum(II) ions,
N-(2R/2S-butyl)-1R,2R-diaminocyclohexane dihydrochloride (L3)
According to the IC50 values for A549 cell line in Table 1, the
1
cytotoxicity of compounds 1e6 with linear alkyl group at the N
position of 1R,2R-DACH showed a convex parabolic change with the
increasing carbon atoms of the alkyl chain of the substituent.
Compounds 2 (R ¼ 1-butyl), 3 (R ¼ 2-butyl), and 4 (R ¼ 2-
methylpropyl) with four carbon atoms of alkyl chain were more
active in vitro (IC50 ¼ 1.82e13.42
m
M) than compounds 1
M), 5 (R ¼ neopentyl, IC50 ¼19.40 M), 6
M), indicating that the different linear
(
R ¼ isopropyl, IC50 ¼14.22
m
m
(R ¼ 2-pentyl, IC50 ¼ 38.66
m
Fig. 2. Molecular structure of complex 1 with atomic numbering. Selected bond
1
ꢁ
four-carbon alkyl substituents as stericallyhinderedgroupsat the N
position of 1R,2R-DACH had moreinfluence on the biological activity
than those bearing three- or five-carbon alkyl chains.
lengths (Å) and bond angles ( ): Pt1eCl1 2.305(3), Pt1eCl2 2.311(3), Pt1eN1 2.037(7),
Pt1eN2 2.064(6), Cl1ePt1eCl2 93.30(8), Cl1ePt1eN1 174.32(17), Cl1ePt1eN2
91.69(18), Cl2ePt1eN1 91.09(17), Cl2ePt1eN2 174.91(18), N1ePt1eN2 84.0(2).

300
Y. Sun et al. / European Journal of Medicinal Chemistry 55 (2012) 297e306
Table 1
Cytotoxicity of compounds 1e9 tested by MTT assay against human tumor cell lines (IC50
, mM).
Compd.
R
IC50
(
m
M)a
A549b
MCF-7c
HCT-116d
HepG2e
1
2
3
4
5
6
7
8
Isopropyl
1-Butyl
2-Butyl
2-Methylpropyl
Neopentyl
2-Pentyl
Cyclobutyl
Cyclopentyl
Cyclohexyl
14.22 ꢂ 1.84
5.50 ꢂ 0.49
1.82 ꢂ 0.11
13.42 ꢂ 2.01
19.40 ꢂ 1.74
38.66 ꢂ 3.48
21.19 ꢂ 1.72
10.26 ꢂ 0.98
16.23 ꢂ 2.15
12.16 ꢂ 1.05
10.02 ꢂ 0.60
46.68 ꢂ 6.10
24.66 ꢂ 1.87
14.45 ꢂ 1.25
38.53 ꢂ 3.18
>100
>100
>100
>100
>100
>100
>100
>100
79.24 ꢂ 6.20
69.48 ꢂ 6.23
5.66 ꢂ 0.55
5.54 ꢂ 0.35
54.50 ꢂ 5.69
82.57 ꢂ 7.65
>100
>100
66.44 ꢂ 5.72
82.22 ꢂ 7.47
>100
>100
>100
>100
>100
54.24 ꢂ 5.25
42.21 ꢂ 5.44
3.92 ꢂ 0.27
6.55 ꢂ 0.78
9
Cisplatin
Oxaliplatin
4.00 ꢂ 0.36
3.57 ꢂ 0.24
a
Mean value ꢂ Standard Deviation from three independent experiments (IC50 is the drug concentration effective in introducing 50% of cell death measured by MTT assay
after 48 h drug exposure). MTT ¼ 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.
b
Human non-small cell lung cancer cell line.
Human breast carcinoma cell line.
Human colorectal cancer cell line.
Human hepatocellular carcinoma cell line.
c
d
e
Compounds 7e9 possessing cycloalkyl substituent at the N1
position of 1R,2R-DACH exhibited comparable cytotoxicity to that
of cisplatin and oxaliplatin against A549. The antitumor activities of
these compounds had no significant change when the cycloalkyl
substituents were modified from four- to six-carbon rings, while
complex 7 was less active in vitro than the others probably due to
the instability of cyclobutane.
important effect on the antitumor activity of the resulting plati-
num(II) complexes. In addition, the low water-solubility of these
complexes owing to the existence of chloride anions limits the
application in practice, which inspires us to develop N-monoalkyl
1R,2R-DACH platinum(II) complexes with hydrophilic anions like
carboxylates as leaving groups by improving the water-solubility of
the related metal complexes in our subsequent research.
From the data in Table 1, for A549 and MCF-7, the order of the
cytotoxic activity of all complexes owning different substituents
was 3 (R ¼ 2-butyl) > 2 (R ¼ 1-butyl) > 4 (R ¼ 2-methylpropyl), 1
2
.3. Inhibition of cell proliferation
(
R ¼ isopropyl), 8 (R ¼ cyclopentyl) > 9 (R ¼ cyclohexyl) > 5
On basis of the result of cytotoxicity above, A549 was the most
(
R ¼ neopentyl) > 7 (R ¼ cyclobutyl) > 6 (R ¼ 2-pentyl), suggesting
sensitive cell line to compounds 1e9. So it was used as the target to
test the inhibition of cell growth by all compounds. For comparison,
cisplatin was also investigated, and the result of cell counting at
different concentrations of drugs was shown in Fig. 3. Cisplatin at low
concentration of 5
Complexes 1e3 had an obvious effect of inhibiting cell growth at the
concentration as low as 5 M within the culture duration of 6 days,
while complexes 5e9 showed inhibition activity at the concentration
of 5e10 Minthesamecellline. Theinhibitioneffectofallcompounds
on the cell proliferation was concentration-dependent.
1
that the complexes with linear alkyl substituents at the N position
of 1R,2R-DACH showed higher cytotoxicity, in general, than those
with cycloalkyl substituents. Among these complexes, complex 3
with 2-butyl substituent as sterically hindered group was the most
effective agent among the synthesized complexes, 5.5e6.7-fold
more potent against A549 and 2.2e3.7-fold less potent against
MCF-7 than positive controls in terms of IC50 values.
mM completely inhibited cell proliferation in A549.
m
For the purpose of delineating the relationship between structure
and cytotoxicity for all compounds, we have applied Hyperchem 7.5
software to make a theoretic calculation about the quantitative
structureeactivity relationship (QSAR) properties including dipole-
moment, Van der Waals surface area, Van der Waals volume, log P,
refractivity, and polarizability. After building the molecule by Hyper-
chem 7.5 as described in Section 2.1, AMBER was chosen as the
molecular mechanics force field and amber2 was selected as the
parameter set. Then Polak-Ribiere was applied as the minimization
algorithm to optimize the structure of the molecule and the QSAR
properties were computed, shown in Table 2. According to the calcu-
lation results, a positive correlation was found between the number of
m
2.4. Apoptosis study by flow cytometry
In order to determine the mechanism of cellular death intro-
duced by the platinum complexes (necrosis or apoptosis), studies of
flowcytometry were undertaken on compounds 2, 3, 8, cisplatin and
oxaliplatin [35]. The tested compounds were incubated with tumor
cells for 24 h at a concentration of 50 mM and the phase diagrams
were shown in Fig. 4. Four areas in the each diagram represent
necrotic cells (Q1, positive for PI and negative for annexin/FITC), live
1
carbon atoms of the substituent at N position and Van der Waals
surface area, Van der Waals volume, refractivity, or polarizability for all
compounds. However, there is no correlation between the properties
and cytotoxicity based on the IC50 values in A549. The order of cyto-
toxicity in A549 was complex 3 > 2 > 8 > 4 >1 >9 > 5 > 7 >6, and the
dipole-moments for all complexes were listed by the order of complex
Table 2
The QSAR properties of all compounds calculated by Hyperchem software.
Compd. Dipole-
Surface
moment area (Å ) (Å )
Volume log P Refractivity Polarizability
2
3
(D)
3
< 2 < 1 < 4 < 6 < 5 < 7 < 8 < 9, suggesting that there is a negative
1
2
3
4
5
6
7
8
9
4.74
4.72
4.40
6.30
9.41
9.16
12.22
20.84
21.56
432.41
483.11
456.00
468.99
471.60
496.73
438.70
460.10
465.75
726.88
800.00
771.75
785.33
810.08
863.15
742.13
782.44
810.06
1.89
2.34
2.36
2.35
2.85
3.15
1.93
2.32
2.72
55.04
59.75
59.57
59.62
64.02
68.77
57.69
62.29
66.89
22.56
24.40
24.40
24.40
26.23
28.07
23.63
25.46
27.29
correlation for all complexes between the cytotoxicity in A549 and
dipole-moment. Besides, it is also noted in Table 2 that the active
compounds (2, 3, 4, and 8) in A549 possess similar values of log P in
a tight range of 2.32e2.36, which demonstrates that a moderate value
of log P is necessary for a promising drug.
As stated above, the number of carbon atoms and the shape of
the N-monosubstituted alkyl group as steric hindrance have an

Y. Sun et al. / European Journal of Medicinal Chemistry 55 (2012) 297e306
301
Cisplatin
Compound 1
1
200000
1400000
Control
Control
1µM
1
5
1
µM
1200000
000000
1000000
µM
5µM
0µM
10µM
25µM
50µM
1
8
00000
00000
00000
00000
0
25µM
5
0µM
8
00000
00000
00000
00000
0
6
4
2
6
4
2
0
1
2
3
4
5
6
0
1
2
3
4
5
6
Time (days)
Time (days)
Compound 2
Compound 3
600000
1400000
1200000
1000000
1
Control
1µM
Control
1µM
1400000
1200000
1000000
5
1
2
5
µM
5
µM
0µM
5µM
0µM
10µM
5µM
50µM
2
8
00000
00000
00000
00000
0
800000
600000
400000
200000
0
6
4
2
0
1
2
3
4
5
6
0
1
2
3
4
5
6
Time (days)
Time (days)
Compound 4
Compound 5
1400000
1200000
1000000
800000
600000
400000
200000
0
1400000
1200000
1000000
Control
1µM
Control
1µM
5
1
2
5
µM
5µM
0µM
5µM
0µM
10µM
25µM
50µM
8
00000
00000
00000
00000
0
6
4
2
0
1
2
2
2
3
4
5
5
5
6
6
6
0
1
2
3
4
5
6
Time (days)
Time (days)
Compound 6
Compound 7
1600000
1400000
1200000
1000000
1400000
Control
1µM
Control
1µM
1
200000
000000
5
µM
5µM
10µM
10µM
25µM
50µM
1
2
5
5µM
0µM
8
00000
00000
00000
00000
0
8
00000
00000
00000
00000
0
6
4
2
6
4
2
0
1
3
4
0
1
2
3
4
5
6
Time (days)
Time (days)
Compound 8
Compound
1400000
1200000
1000000
800000
9
1400000
1200000
1000000
Control
1µM
Control
1µM
5
1
2
5
µM
5µM
0µM
5µM
0µM
10µM
25µM
50µM
8
00000
00000
00000
00000
0
6
4
2
600000
400000
200000
0
0
1
3
4
0
1
2
3
4
5
6
Time (days)
Time (days)
Fig. 3. Inhibition of A549 tumor cell proliferation by synthesized compounds and cisplatin at different concentrations for 6 days of incubation (0e50
m
M).

302
Y. Sun et al. / European Journal of Medicinal Chemistry 55 (2012) 297e306
cells (Q3, negative for annexin and PI), lateapoptosis or necrosis cells
Q2, positive for annexin and PI) and apoptosis cells (Q4, negative for
PI and positive for annexin), respectively.
cytotoxic activity of complex 8 on MCF-7 cell line, this experiment
did not involve complex 8. It was demonstrated that the tested
compounds, in general, induced apoptosis of A549 and MCF-7
tumor cells in line with the cytotoxicity above. The result that
complexes with linear alkyl substituents at the N position showed
higher cytotoxicity than those with cycloalkyl substituents was
(
As for A549 cell line in Fig. 4A, complex 3 showed the highest
population of apoptotic cells (Q4, 46.5%) among the tested
compounds, 1.6-fold higher than cisplatin and 4.7-fold higher than
oxaliplatin. And complex 2 exhibited a slightly lower population of
apoptotic cells (38.0%) than complex 3, but still 1.3-fold higher than
cisplatin and 3.8-fold higher than oxaliplatin at the same concen-
tration. Complex 8 produced an apoptosis rate (13.1%) close to that
of oxaliplatin, lower than that of cisplatin.
1
further testified by flow cytometric assay.
2.5. Cell cycle analysis
It is believed that apoptosis is the immediate cause of cytotox-
icity for the representative compounds, so it is necessary to analyze
the DNA content of cells by flow cytometry to investigate the effect
of complexes on A549 cell-cycle progression and the interaction
with DNA. The result was obtained by flow cytometry after cells
With respect to MCF-7 in Fig. 4B, it was observed that complexes
2
and 3 had a weaker effect on tumor apoptosis than cisplatin with
apoptotic populations of 24.5% and 18.7%, whereas they produced
higher apoptotic populations than oxaliplatin. Considering the low
Fig. 4. Flow cytometric results after the exposure of A549 (A) and MCF-7 (B) cells to the active compounds 2, 3, 8, oxaliplatin and cisplatin (50 mM). Four areas in the diagrams
represent four different cell states: necrotic cells (Q1, positive for PI and negative for annexin/FITC), living cells (Q3, negative for annexin and PI), late apoptotic or necrotic cells (Q2,
positive for annexin and PI) and apoptotic cells (Q4, negative for PI and positive for annexin).

Y. Sun et al. / European Journal of Medicinal Chemistry 55 (2012) 297e306
303
were treated with complexes 2, 3, and 8 for 24 h at the concen-
trations of 10 and 25 M (Fig. 5). At both tested concentrations of
A
C
m
cisplatin, blockade in the S phase (46.13% and 36.39%) was observed
in A549 cells. Complexes 2, 3, and 8 caused accumulation of cells in
the S and G2/M phases relative to negative control tumor cells,
Form II
Form III
Form I
especially at low concentration of 10 mM, indicating that cell-cycle
progression is blocked in the S and G2/M phases by tested
complexes. In particular, induction of apoptosis by complexes was
further confirmed by the appearance of an apoptotic population in
1
1
2
2
3
3
4
4
5
5
1
2
3
4
5
the sub-G1 phase at 10 and 25
mM, which is also characteristic of
B
D
DNA fragmentation produced by complexes. It was notable that
there was no obvious accumulation in sub-G1 for complexes after
Form II
12 h incubation (data not shown). Therefore, we conclude that
Form III
Form I
complexes 2, 3, and 8 inhibit tumor cell proliferation and cause
cytotoxicity by blocking DNA synthesis and arrest cells in the G2/M
phase, and DNA fragmentation is another mode of action for our
complexes.
1
2
3
4
5
Fig. 6. Cleavage of pET22b plasmid DNA and gel electrophoretic mobility pattern in the
presence of various concentrations of tested compounds, (A) cisplatin, (B) compound 2,
2.6. Interaction with pET22b plasmid DNA
(C) compound 3, (D) compound 8. The lanes (from left to right) correspond to
untreated plasmid DNA only (lane 1), then concentrations of 10, 20, 40, and 80 M of
m
It is testified that DNA is one of targets in tumor cells and DNA
tested compounds incubated with DNA (lanes 2e5). Form I, Form II, and Form III
indicate supercoiled, nicked (open circle), and linear forms of pET22b, respectively.
fragmentation is caused by the complexes. Herein, DNA cleavage
produced by representative complexes 2, 3, and 8 was tested by
agarose gel electrophoresis, using pET22b plasmid DNA as the
target. Fig. 6 showed the result of gel electrophoretic mobility of
pET22b plasmid DNA treated with different concentrations of
complexes 2, 3, 8, and cisplatin. In the electrophoretogram, the
untreated plasmid DNA (lane 1), which mainly comprised cova-
lently closed circular (form I, ccc) and a small amount of open
circular (form II, oc) bands, was considered as negative control.
cisplatin and tested compounds, respectively. When pET22b
ꢁ
plasmid DNA was incubated with the compounds at 37 C for 24 h,
it was noted that the mobility of the plasmid DNA had different
changes for the compounds tested compared with negative control.
In Fig. 6A, as for cisplatin, the increase in intensity of the open
circular DNA (form II) and decrease in mobility of the closed circular
DNA (form I) were observed with the increase in concentrations of
the compounds tested, indicating that the supercoil DNA was
relaxed or unwound by cisplatin as a result of covalent binding. It
was also seen that a small amount of linear DNA (form III), whose
electrophoretic mobility was between closed circular and open
Lanes 2e5 stood for concentrations of 10, 20, 40, and 80
mM of
A
5
4
4
3
3
2
2
1
1
0
5
0
5
0
5
0
5
0
5
0
circular forms, appeared at the low concentration (10 mM), whereas
the mobility of form III increased with the increasing of drug
concentration until form III and I overlapped to give rise to a new
band (lane 5).
Control
Cisplatin
Compound 2
Compound 3
Compound 8
With regard to complex 2 (see Fig. 6B), the mobility of supercoil
DNA (form I) had no decrease (compared to that of untreated DNA),
while the increase in intensity of form II and form III was observed
with the increase of complex concentration, resulting from
unwinding of supercoil DNA to partially become open circular and
linear DNA. A similar electrophoresis pattern to that of complex 2
was observed for complex 3 in Fig. 6C, while complex 3 produced
stronger unwinding of supercoil DNA than complex 2. Complex 8
had a weaker cleavage effect on DNA than other complexes just by
producing a little amount of linear DNA at low concentrations
sub-G1
G1
S
G2/M
1
0 µM
B
5
4
4
3
3
2
2
1
1
0
5
0
5
0
5
0
5
0
5
0
(10e20
mM) in Fig. 6D, while it decreased the mobility of supercoil
DNA at high concentrations (40e80
mM).
Control
Cisplatin
Compound 2
Compound 3
Compound 8
In general, both complexes 2 and 3 showed the comparable
cleavage capability and similar DNA binding pattern to that of
cisplatin at low concentration (<10
differently in cleaving plasmid DNA from cisplatin at high
concentration (>40 M). And complex 8, with a cycloalkyl
mM), whereas they behaved
m
1
substituent at N position, showed weaker cleavage ability than
complexes 2 and 3.
sub-G1
G1
S
G2/M
3. Conclusions
2
5 µM
1
In this study, a series of N -alkyl 1R,2R-diaminocyclohexane
Fig. 5. Compounds 2, 3, and 8 introduced cell cycle arrest in A549 cell lines. Cells were
treated in the absence and presence of tested compounds at the concentration of
derivatives were used as ligands to prepare nine novel sterically
hindered platinum(II) complexes with chloride anions as the leaving
group, whose structures have been illustrated by theoretic
10
m
M (A) or 25
mM (B) for 24 h incubation, respectively, and measured by flow
cytometry.

304
Y. Sun et al. / European Journal of Medicinal Chemistry 55 (2012) 297e306
computation together with single crystal X-ray diffraction study on
a typical complex. The biological activities of all metal complexes
were examined against four tumor cell lines. The result of in vitro
antitumor activity indicated that complexes 2 (alkyl group: 1-butyl)
and 3 (alkyl group: 2-butyl) were more active than cisplatin and
oxaliplatin against A549 human non-small cell lung cancer cell line
and exhibited comparable cytotoxicity to cisplatin and oxaliplatin in
MCF-7 (human breast carcinoma cellline). It was noted that the linear
butyl substituents, especially 2-butyl group as steric hindrance,
played a more important role in suppressing tumor cell proliferation
than other alkyl substituents. And this deduction can be further
confirmed from the negative correlation for all complexes between
the cytotoxicity in A549 and dipole-moment. Since the dichlor-
oplatinum(II) complexes are hardly dissolved in water, hydrophilic
anions can be considered to be introduced as leaving groups to
improve the water-solubility of complexes with our ligands.
CH
NH and NH
23.8, 24.3, 27.6 (C4, C5 and CH
3
CH
2
CH
2
), 2.40e2.88(m, 3H, NHCHand NH
2
CH), 5.96e6.62(m, 3H,
13
2 6
) ppm; C NMR (d -DMSO):
d
13.9 (CH ), 19.7 (CH CH ),
3
3
2
3 2 2
CH CH ), 29.0, 31.3 (C3 and C6), 45.4,
þ
60.3, 64.2 (C1, C2 and NHCH
2
) ppm; ESI-MS: m/z (%) [M þ H] ¼ 437.2
þ
(100), [M ꢀ Cl] ¼ 400.1 (70). Anal. calcd. for C10
2 2
H22Cl N Pt: C, 27.53;
H, 5.08; N, 6.42. Found: C, 27.69; H, 5.19; N, 6.30.
1
0
4.1.2.3. cis-[(1R,2R)-N -(2-Butyl)-1,2-diaminocyclohexane-N,N ]
2
0
dichloroplatinum(II) (3). Yellow powder (0.40 g, 92%). ½
a
ꢃ
þ116.3
D
ꢀ
1
(c 0.66 in MeOH). IR (KBr, cm ): 3440(br), 3190(nNeH), 3110(nNeH),
1
2930(
TMS):
DACH and CH
6.15e6.59 (m, 3H, NH and NH
(CH CH ), 18.0 (CHCH
C6 and CH CH ), 58.7, 60.9, 65.9 (C1, C2 and NHCHCH
MS: m/z (%) [M þ H] ¼ 437.2 (65), [M ꢀ Cl] ¼ 400.1 (100).
Anal. calcd. for C10 Pt: C, 27.53; H, 5.08; N, 6.42. Found: C,
27.64; H, 5.15; N, 6.28.
n
CeH), 2860(
1.04e1.21 (m, 6H, CH
CHCH CH ), 2.40e2.85 (m, 3H, NHCH and NH
n
CeH), 487(
n
PteN), 421(
n
PteCl); H NMR (d
6
-DMSO/
d
3
CHCH CH
2
3
), 1.21e2.35 (m, 10H, CH
2
of
3
2
3
2
CH),
13
2
) ppm; C NMR (d
6
-DMSO):
d
11.2
2
3
3
), 20.4, 24.3 (C4 and C5), 27.5, 29.6, 31.9 (C3,
) ppm; ESI-
3
2
3
þ
þ
Flow cytometry assay with representative compounds verified
that complexes 2, 3, and 8 inhibited tumor proliferation by inducing
apoptosis and DNA fragmentation. Cleavage of DNA double helix
produced by complexes was determined from the agarose gel
electrophoresis experiments.
2 2
H22Cl N
1
4.1.2.4. cis-[(1R,2R)-N -(2-Methylpropyl)-1,2-diaminocyclohexane-
0
N,N ]dichloroplatinum(II) (4). Yellow powder (0.38 g, 87%).
2
0
ꢀ1
4
4
4
. Experimental protocols
½
a
ꢃ
þ110.7 (c 0.70 in MeOH). IR (KBr, cm ): 3450(br), 3186(
n
NeH),
D
1
3
117(
NMR (d
CH of DACH and CH(CH
NHCH ), 6.29e6.63 (m, 3H, NH and NH
21.3 (2CH ), 23.8, 24.2 (C4 and C5), 25.6 (CH(CH
and C6), 54.1, 60.2, 65.9 (C1, C2 and NHCH ) ppm; ESI-MS: m/z (%)
[M þ H] ¼ 437.2 (48), [M ꢀ Cl] ¼ 400.1 (100). Anal. calcd. for
Pt: C, 27.53; H, 5.08; N, 6.42. Found: C, 27.42; H, 5.24; N,
n
NeH), 2936(
n
CeH), 2864(
n
CeH), 490(
n
PteN), 452(
n
PteCl);
H
.1. Chemistry
6
-DMSO/TMS):
d
0.93 (m, 6H, CH(CH
3 2
) ), 1.04e2.15 (m, 9H,
2
3
)
2
), 2.48e2.95 (m, 4H, NHCH, NH
2
CH and
6
-DMSO):
13
.1.1. Materials and instruments
2
2
) ppm; C NMR (d
All chemicals and solvents were of analytical reagent grade and
d
3
3 2
)
), 28.1, 31.6 (C3
were used without further purification. Potassium tetra-
chloroplatinate(II) and 1R,2R-diaminocyclohexane were obtained
from a local chemical company. Elemental analyses for C, H and N
were done on a Vario MICRO CHNOS Elemental Analyzer, Ele-
mentar. Infrared spectra were measured on KBr pellets on a Nicolet
2
þ
þ
10 2 2
C H22Cl N
6.23.
ꢀ1 1
1
0
IR200 FT-IR spectrometer in the range of 4000e400 cm . H NMR
4.1.2.5. cis-[(1R,2R)-N -Neopentyl-1,2-diaminocyclohexane-N,N ]
13
20
D
and C NMR spectra were recorded in d
6
-DMSO with a Bruker
dichloroplatinum(II) (5). Yellow powder (0.40 g, 88%). ½
a
ꢃ
þ91.0 (c
ꢀ
1
3
00 MHz spectrometer. Mass spectra were measured on a Bruker
0.85 in MeOH). IR (KBr, cm ): 3192(
n
NeH), 3095(
n
NeH), 2931(
-DMSO/TMS):
of DACH), 2.87e3.06 (m, 4H,
nCeH),
1
Esquire ESI-MS instrument. The specific optical rotations of all
complexes were recorded on a WZZ-2A Automatic Polarimeter.
2859(
(s, 9H, C(CH
NHCH, NH CH and NHCH
NMR (d -DMSO):
C(CH ), 29.5 (3CH
n
CeH), 493(
n
PteN), 433(
n
PteCl); H NMR (d
6
d 1.13
3
)
3
), 1.00e2.26 (m, 8H, CH
2
13
2
2
2
), 6.21e6.55 (m, 3H, NH and NH ) ppm; C
1
4
.1.2. Preparation of cis-[(1R,2R)-N -alkyl-1,2-
6
d
23.3, 24.1 (C4 and C5), 28.2, 30.5, 31.7 (C3, C6 and
), 55.9, 60.1, 66.6 (C1, C2 and NHCH ) ppm; ESI-
0
diaminocyclohexane-N,N ]dichloroplatinum(II) (complexes 1e9)
3
)
3
3
2
þ
To a solution of K
2
PtCl
4
(0.42 g, 1 mmol) in distilled water (10 mL)
was added (1R,2R)-N -alkyl-1,2-diaminocyclohexane dihydrochloride
1 mmol) neutralized by Na CO in distilled water (10 mL). The reaction
MS: m/z (%) [M þ H] ¼ 450.1 (100). Anal. calcd. for C11
H24Cl
2
N
2
Pt: C,
1
29.34; H, 5.37; N, 6.22. Found: C, 29.10; H, 5.34; N, 6.06.
(
2
3
ꢁ
1
0
mixture was then stirred at 25 C for 5 h in the dark under a nitrogen
atmosphere. Yellow precipitate was filtered off, washed withwater, and
then dried in vacuum to yield 85e92%.
4.1.2.6. cis-[(1R,2R)-N -(2-Pentyl)-1,2-diaminocyclohexane-N,N ]
2
0
dichloroplatinum(II) (6). Yellow powder (0.38 g, 85%). ½
a
ꢃ
þ96.5 (c
D
ꢀ
1
0.90 in MeOH). IR (KBr, cm ): 3209(
n
NeH), 3120(
H
n
NeH), 2929(
NMR (d -DMSO/TMS):
), 1.25e2.26 (m, 12H, CH of
), 2.47e2.95 (m, 3H, NHCH and NH CH),
13.9
CH), 23.9, 24.3 (C4 and C5), 29.6,
nCeH),
1
2
861(
1.02e1.20 (m, 6H, CH
CHCH CH
6.25e6.58 (m, 3H, NH and NH
(CH CH ), 19.4, 20.8 (CH CH and CH
31.6, 32.1 (C3, C6 and CH CH CH
NHCHCH
n
CeH), 495(
n
PteN), 445(
CHCH
CH
nPteCl);
6
1
0
4
.1.2.1. cis-[(1R,2R)-N -Isopropyl-1,2-diaminocyclohexane-N,N ]
d
3
CH CH
2 2 3
2
2
0
dichloroplatinum(II) (1). Yellow powder (0.37 g, 89%). ½
a
ꢃ
þ123.8
DACH and CH
3
2
2
3
2
D
ꢀ
1
13
(
2
c 0.85 in MeOH). IR (KBr, cm ): 3475(br), 3194(
n
NeH), 3121(
PteCl); H NMR (d
n
NeH),
2 6
) ppm; C NMR (d -DMSO): d
1
924(
n
CeH), 2867(
n
CeH), 496(
0.95 (m, 6H, CH(CH
n
PteN), 443(
), 1.04e2.16 (m, 8H, CH
CH), 5.95e6.54 (m, 3H, NH and
21.0 (2CH ), 22.5, 24.3 (C4 and
n
6
-DMSO/
2
3
3
2
3
TMS):
2
d
3
)
2
2
of DACH),
3
2
2
), 56.7, 60.9, 65.8 (C1, C2 and
þ
.48e2.95 (m, 3H, NHCH and NH
2
3
) ppm; ESI-MS: m/z (%) [M þ H] ¼ 450.1 (100). Anal. calcd.
NH -DMSO):
2
) ppm; 13C NMR (d
6
d
3
for C11
2 2
H24Cl N Pt: C, 29.34; H, 5.37; N, 6.22. Found: C, 29.19; H, 5.21; N,
C5), 30.1, 32.1 (C3 and C6), 52.5 ((CH
3
)
2
CHNH), 61.4, 65.0 (C1 and
6.18.
þ
þ
C2) ppm; ESI-MS: m/z (%) [M þ H] ¼ 422.2 (100), [M ꢀ Cl] ¼ 386.1
1
0
(
53). Anal. calcd. for C
9
H20Cl
2
N
2
Pt: C, 25.60; H, 4.77; N, 6.63. Found:
4.1.2.7. cis-[(1R,2R)-N -Cyclobutyl-1,2-diaminocyclohexane-N,N ]
2
0
C, 25.35; H, 5.09; N, 6.52.
dichloroplatinum(II) (7). Yellow powder (0.39 g, 91%). ½
a
ꢃ
þ94.3 (c
D
ꢀ1
0
.70 in MeOH). IR (KBr, cm ): 3465(br), 3184(
2934( CeH), 2856( CeH), 485( PteN), 423(
DMSO/TMS): 1.05e2.25 (m, 14H, CH
2.69e3.15 (m, 3H, NHCH and NH
n
NeH), 3050(
PteCl); H NMR (d
n
NeH),
1
0
1
4
.1.2.2. cis-[(1R,2R)-N -(1-Butyl)-1,2-diaminocyclohexane-N,N ]
n
n
n
n
6
-
2
0
dichloroplatinum(II) (2). Yellow powder (0.39 g, 90%). ½
a
ꢃ
þ125.2 (c
d
2
of DACH and cyclobutyl),
CH), 6.08e6.65 (m, 3H, NH and
14.6 (C3 ), 23.8, 24.2 (C4 and C5),
D
ꢀ
1
0
2
.80 in MeOH). IR (KBr, cm ): 3480(br), 3187(
n
NeH), 3117(
n
NeH),
2
1
13
0
935(
n
CeH), 2863(
n
CeH), 490(nPteN), 436(
n
PteCl); H NMR (d
6
-DMSO/
NH
2
) ppm; C NMR (d
6
-DMSO):
d
0
0
TMS):
d
0.98 (t, 3H, CH
2
CH ), 1.12e2.23 (m, 12H, CH
3
2
of DACH and
28.9, 30.3, 31.6, 32.6 (C3, C6, C2 and C4 ), 52.2, 55.3, 62.9 (C1, C2 and

Y. Sun et al. / European Journal of Medicinal Chemistry 55 (2012) 297e306
305
C1 ) ppm; ESI-MS: m/z (%) [M þ H] ¼ 434.1 (62), [M ꢀ Cl]þ ¼ 398.1
0
þ
every 24 h for 6 days using a Neubauer hemocytometer after
trypsinization of cells.
(
100). Anal. calcd. for C10
H20Cl
2
N
2
Pt: C, 27.66; H, 4.64; N, 6.45.
Found: C, 27.79; H, 4.80; N, 6.55.
4
.2.3. Flow cytometry analysis
Annexin V-FITC Apoptosis Detection Kit (BD Pharmingen, USA)
1
0
4
.1.2.8. cis-[(1R,2R)-N -Cyclopentyl-1,2-diaminocyclohexane-N,N ]
20
dichloroplatinum(II) (8). Yellow powder (0.40 g, 89%). ½
a
ꢃ
þ85.7 (c
was used to measure the apoptosis induced by platinum complexes
by means of flow cytometry according to the manufacturer’s
instructions. After apoptosis of selected cell lines (A549 and MCF-7)
D
ꢀ
1
0
2
.65 in MeOH). IR (KBr, cm ): 3430(br), 3178(
n
NeH), 3113(
PteCl); H NMR (d
n
NeH),
1
938(
n
CeH), 2864(
n
CeH), 499(
nPteN), 458(
n
6
-
DMSO/TMS):
d
1.09e2.17 (m, 16H, CH
2
of DACH and cyclopentyl),
by the addition of 50 mM selected compounds and positive controls
2
NH
.58e2.74 (m, 3H, NHCH and NH
2
CH), 6.18e6.57 (m, 3H, NH and
for 24 h, cells were collected by centrifugation. Then cells were
washed with cold water and resuspended in Annexin V-FITC
13
0
2
) ppm; C NMR (d
6
-DMSO):
d
22.7, 23.7, 23.9, 24.4 (C4, C5, C3
0
0
0
6
and C4 ), 29.3, 30.4, 32.1, 32.3 (C3, C6, C2 and C5 ), 58.0, 62.1, 65.3
binding buffer at a concentration of 1 ꢄ 10 cells/mL, followed by
0
þ
(
C1, C2 and C1 ) ppm; ESI-MS: m/z (%) [M ꢀ Cl] ¼ 412.1 (100). Anal.
calcd. for C11 Pt: C, 29.47; H, 4.95; N, 6.25. Found: C, 29.29;
H, 5.07; N, 6.10.
staining with 5
m
L Annexin V-FITC and incubation in the dark at
ꢁ
H22Cl
2
N
2
25 C. Then cells were centrifugated for 5 min and were resus-
pended in Annexin V-FITC binding buffer and propidium iodide
(
10 mL). After 15 min incubation at room temperature, the fluo-
1
0
4
.1.2.9. cis-[(1R,2R)-N -Cyclohexyl-1,2-diaminocyclohexane-N,N ]
rescence was measured using a flow cytometer (BD FACSCalibur,
USA). The results were obtained by using FCSExpress software and
represented as percentage of normal and apoptotic cells.
20
dichloroplatinum(II) (9). Yellow powder (0.42 g, 90%). ½
a
ꢃ
þ94.0 (c
D
ꢀ1
0
2
.85 in MeOH). IR (KBr, cm ): 3440(br), 3187(
n
NeH), 3109(
PteCl); H NMR (d
n
NeH),
1
6
932(
n
CeH), 2856(
n
CeH), 489(
1.02e2.18 (m, 18H, CH
n
PteN), 435(
n
6
-
For cell-cycle study, 1 ꢄ10 A549 cells were seeded in a six-well
ꢁ
DMSO/TMS):
d
2
of DACH and cyclohexyl),
plate and incubated at 37 C overnight. Then cells were incubated
2
NH
.79e3.20 (m, 3H, NHCH and NH
2
CH), 5.93e6.45 (m, 3H, NH and
with the representative complexes for 24 h at 10 and 25 mM. Cells
13
0
2
) ppm; C NMR (d
6
-DMSO):
d
23.9 (C4 ), 24.3, 24.9, 25.3, 25.4
were harvested with trypsin and washed twice with PBS solution.
0
0
0
0
ꢁ
(
C4, C5, C3 and C5 ), 30.1, 30.9, 32.1, 32.6 (C3, C6, C2 and C6 ), 61.1,
Then cells were fixed in cold 70% ethanol and stored at 4 C. On the
0
þ
6
1.6, 65.4 (C1, C2 and C1 ) ppm; ESI-MS: m/z (%) [M þ H] ¼ 462.1
day of analysis, ethanol was removed by centrifugation, and cells
were washed twice with PBS, and then treated with RNase (75 kU/
þ
(
75), [M ꢀ Cl] ¼ 426.1 (100). Anal. calcd. for C12
2 2
H24Cl N Pt: C,
ꢁ
31.17; H, 5.23; N, 6.06. Found: C, 31.31; H, 5.09; N, 6.02.
mL) for 30 min at 37 C. Propidium iodide (PI) was finally added
(50 mg/mL), and samples were processed by a flow cytometer
4
.1.3. X-ray crystallographic analysis and data collection
(FACScan, Becton Dickson, USA). The results were analyzed by
FCSExpress software.
Single crystals of complex 1 from its CH OHeCH Cl solution were
3
2
2
obtained when exposed to air. Crystal data, data collection parameters,
and structure refinement details are given in Table S1 (Supporting
information) CIF file is available as Supporting information.
4.2.4. Gel electrophoresis experiment
The DNA cleavage by complexes 2, 3, 8, and cisplatin was
investigated by agarose gel electrophoresis. pET22b plasmid DNA
(40 ng/mL) was used as the target in this experiment. Appropriate
4
4
.2. Biological studies
dilutions of compounds were made before dissolving them in
DMSO, and different volumes of solutions were added to the tubes
.2.1. Cell culture
to achieve a set of concentrations in the range of 0e80
mM. pET22b
All adherent cell lines including A549 (human non-small cell
DNA (5 L, 200 ng) was added to each tube, and then the mixtures
m
lung cancer), MCF-7 (human breast carcinoma), HepG2 (hepato-
of platinum complexes and pET22b plasmid DNA were incubated at
37 C for 24 h. After that, the agarose gel (made up to 1% w/v)
containing ethidium bromide was prepared using TA buffer (50 mM
ꢁ
cellular carcinoma) and HCT-116 (human colorectal cancer) were
ꢁ
cultured in a humidified, 5% CO
2
atmosphere at 37 C, and main-
tained in monolayer culture in RPMI-1640 medium supplemented
with 10% fetal bovine serum (FBS), 100 g/mL of streptomycin and
00 g/mL of penicillin.
Triseacetate, pH 7.5). The mixtures with loading buffer (1 mL)
m
underwent electrophoresis in agarose gel in TA buffer (50 mM
Triseacetate, pH 7.5) at 100 V for 70 min. The bands were photo-
graphed using a Molecular Imager (Bio-Rad, USA) under UV light.
1
m
4
.2.2. MTT assay and cell growth assay
The IC50 values of all complexes were determined by MTT assay
MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide) [36]. The suspension of 2000 cells/well was plated in
Acknowledgments
(
¼
The authors are grateful to the New Drug Creation Project of the
National Science and Technology Major Foundation of China
9
2
6-well culture plates with culture medium and was incubated for
ꢁ
4 h at 37 C in a 5% CO
2
incubator. The complexes were dissolved
(Project 2010ZX09401-401) and the National Natural Science
in DMSO and diluted to the required concentration with culture
medium (DMSO final concentration < 0.5%). Then the diluted
Foundation of China (Project. 20971022) for financial aids to this
work. Sun would like to thank the Scientific Research Innovation
Project for College Graduates of Jiangsu Province (Project
CX10B_084Z) and Scientific Research Foundation of Graduate
School of Southeast University (Project YBJJ1106). We appreciate
very much Dr. Zhiguo Zhang in Justus-Liebig University for his
assistance in molecular calculation with HyperChem 7.5 software.
solution of complexes was added to the wells, and the cells were
ꢁ
incubated at 37 C in a 5% CO
2
incubator for 48 h. After that, the
L MTT dye solution (5 mg/mL) for 4 h
cultivation. The media with MTT solution were removed with
00 L of DMSO solution. The absorbance of formazane solution
cells were treated with 10
m
1
m
was measured at 540 nm with an automatic microplate ELISA
reader. The IC50 value was determined from the chart of cell
viability (%) against dose of complex added (mM). As for the cell
Appendix A. Supplementary material
proliferation assay, 30,000 cells/well were seeded in 6-well culture
plates. After 6 h for settlement, cells were incubated in different
concentrations of selected drugs and cell numbers were recorded
Supplementary data related to this article can be found online at
http://dx.doi.org/10.1016/j.ejmech.2012.07.026.

3
06
Y. Sun et al. / European Journal of Medicinal Chemistry 55 (2012) 297e306
[19] M. Galanski, A. Yasemi, S. Slaby, M.A. Jakupec, V.B. Arion, M. Rausch,
References
A.A. Nazarov, B.K. Keppler, Eur. J. Med. Chem. 39 (2004) 707e714.
20] M. Galanski, A. Yasemi, M.A. Jakupec, N.G. Keyserlingk, B.K. Keppler, Monatsh.
Chem. 136 (2005) 693e700.
21] S.A. Abramkin, U. Jungwirth, S.M. Valiahdi, C. Dworak, L. Habala, K. Meelich,
W. Berger, M.A. Jakupec, C.G. Hartinger, A.A. Nazarov, M. Galanski,
B.K. Keppler, J. Med. Chem. 53 (2010) 7356e7364.
[
[
[
[
1] B. Lippert (Ed.), Cisplatin: Chemistry and Biochemistry of a Leading Anticancer
Drug, Verlag Helvetica Chimica Acta, Zurich, Switzerland, 2000, p. 563.
2] M.A. Jakupec, M. Galanski, V.B. Arion, C.G. Hartinger, B.K. Keppler, Dalton
Trans. (2008) 183e194.
3] M.A. Fuertes, C. Alonso, J.M. Perez, Chem. Rev. 103 (2003) 645e662.
[
[22] J. Gao, Y. Liu, Y. Zhou, R.A. Zingaro, ChemMedChem 2 (2007) 1723e1729.
[
4] S.W. Johnson, J.P. Stevenson, P.J. O’Dwyer, Cisplatin and its analogues, in:
V.T. DeVita Jr., S. Hellman, S.A. Rosenberg (Eds.), Cancer: Principles and Practice of
Oncology, sixth ed. J.B. Lippincott Company, Philadelphia, PA, 2001, pp. 376e388.
5] M.J. Cleare, J.D. Hoeschele, Platinum Met. Rev. 17 (1973) 3e7.
6] M.J. Cleare, J.D. Hoeschele, Bioinorg. Chem. 2 (1973) 187e192.
7] N.J. Wheate, S. Walker, G.E. Craig, R. Oun, Dalton Trans. 39 (2010) 8113e8127.
8] D. Lebwohl, R. Canetta, Eur. J. Cancer 34 (1998) 1522e1534.
9] J.L. Misset, H. Bleiberg, W. Sutherland, M. Bekradda, E. Cvitkovic, Crit. Rev.
Oncol. Hematol. 35 (2000) 75e93.
[23] J. Gao, Y. Liu, R.A. Zingaro, Chem. Res. Toxicol. 22 (2009) 1705e1712.
[24] D.M. Fisher, P.J. Bednarski, R. Grünert, P. Turner, R.R. Fenton, J.R. Aldrich-
Wright, ChemMedChem 2 (2007) 488e495.
[25] C. Gao, S. Gou, L. Fang, J. Zhao, Bioorg. Med. Chem. Lett. 21 (2011) 1763e1766.
[26] B. Neumuiller, K.Z. Dehnicke, Anorg. Allg. Chem. 633 (2007) 2262e2267.
[27] E. Rafii, B. Dassonneville, A. Heumann, Chem. Comm. (2007) 583e585.
[28] T. Akitsu, Y. Endo, M. Okawara, Y. Kimoto, M. Ohwa, Open Crystallogr. J. 4
(2011) 2e7.
[29] D.W. Lee, H. Ha, W.K. Lee, Synth. Commun. 37 (2007) 737e742.
[30] Y.K. Kim, S.J. Lee, K.H. Ahn, J. Org. Chem. 65 (2000) 7807e7813.
[31] (a) K. Püntener, L. Schwink, P. Knochel, Tetrahedron Lett. 37 (1996)
8165e8168;
[
[
[
[
[
[10] A.M. Di Francesco, A. Ruggiero, R. Riccardi, Cell. Mol. Life Sci. 59 (2002)
1914e1927.
[
[
11] E.Raymond,S.Faivre,J.M.Woynarowski,S.G.Chaney,Semin.Oncol.25(1998)4e12.
12] Z.Z. Zdraveski, J.A. Mello, C.K. Farinelli, J.M. Essigmann, M.G. Marinus, J. Biol.
Chem. 277 (2002) 1255e1260.
(b) W.L. Neumann, G.W. Franklin, K.R. Sample, K.W. Aston, R.H. Weiss,
D.P. Riley, Tetrahedron Lett. 38 (1997) 779e782;
[
[
[
[
[
[
13] C.W. Yu, K.K. Li, S.K. Pang, S.C. Au-Yeung, Y.P. Ho, Bioorg. Med. Chem. Lett. 16
(c) J. Balsells, L. Mejorado, M. Phillips, F. Ortega, G. Aguire, R. Somanathan,
P.J. Walsh, Tetrahedron: Asymmetry 9 (1998) 4135e4142.
[32] (a) T.W. Green, P.G.M. Wuts, Protective Groups in Organic Synthesis, third ed.
Wiley, New York, 1999;
(b) J.M. Mitchell, N.S. Finney, Tetrahedron Lett. 41 (2000) 8431.
[33] HyperChem Professional 7.5, Hypercube, Inc., USA, 2011.
[34] Detailed methods of structure determination are given in the Supporting
information.
(
2006) 1686e1691.
14] X. Liu, H. Shen, H. Zhu, K. Cui, S. Gou, Bioorg. Med. Chem. Lett. 17 (2007)
831e3834.
3
15] C. Rothenburger, M. Galanski, V.B. Arion, H. Görls, W. Weigand, B.K. Keppler,
Eur. J. Inorg. Chem. (2006) 3746e3752.
16] Y. Yu, L. Lou, W. Liu, H. Zhu, Q. Ye, X. Chen, W. Gao, S. Hou, Eur. J. Med. Chem.
43 (2008) 1438e1443.
17] K. Hamamura, T. Kondo, K. Kuroishi, N. Nakamura, R. Suzuki, M. Takeo, K. Ohno,
Y. Takahashi, T. Tanaka, T. Oyaizu, Gastroenterology 140 (2011) S-597.
18] L. Habala, M. Galanski, A. Yasemi, A.A. Nazarov, N.G. Keyserlingk, B.K. Keppler,
Eur. J. Med. Chem. 40 (2005) 1149e1155.
[35] A.M. Montana, F.J. Bernal, J. Lorenzo, C. Farnos, C. Batalla, M.J. Prieto,
V. Moreno, F.X. Aviles, J.M. Mesas, M.T. Alegre, Bioorg. Med. Chem. 16 (2008)
1721e1737.
[36] T.R. Mosmann, J. Immunol. Methods 65 (1983) 55e63.