Antitumor platinum(II) complexes containing platinum-based moieties of present platinum drugs and furoxan groups as nitric oxide donors: Synthesis, DNA interaction, and cytotoxicity

Article abstract of DOI:10.1021/ic301374z

Six novel platinum(II) complexes 1-6 bearing different furoxan moieties as nitric oxide (NO) donors have been designed, synthesized, and characterized by elemental analysis and ¹H NMR, IR, and ESI-MS spectroscopy. The furoxan groups were introduced to the platinum complexes to release NO, which may take synergic action with the platinum-based moieties on the tumor cells. It was found that all compounds exhibited considerable cytotoxicity against human HCT-116 and SGC-7901 cell lines via DNA binding together with NO-releasing features, especially for compound 3. This finding is in accordance with the previous reports that NO hybrids show higher cytotoxicity against colon cancer cell lines compared with their parent compounds.

Full text of DOI:10.1021/ic301374z

Article
pubs.acs.org/IC
Antitumor Platinum(II) Complexes Containing Platinum-Based
Moieties of Present Platinum Drugs and Furoxan Groups as Nitric
Oxide Donors: Synthesis, DNA Interaction, and Cytotoxicity
Jian Zhao,† Shaohua Gou,*,†,‡ Yanyan Sun,†,‡ Lei Fang,†,‡ and Zhimei Wang†,‡
‡
†Pharmaceutical Research Center, School of Chemistry and Chemical Engineering, and Jiangsu Province Hi-Tech Key Laboratory
for Bio-medical Research, Southeast University, Nanjing, China
ABSTRACT: Six novel platinum(II) complexes 1−6 bearing different furoxan moieties as nitric oxide (NO) donors have been
designed, synthesized, and characterized by elemental analysis and 1H NMR, IR, and ESI-MS spectroscopy. The furoxan groups
were introduced to the platinum complexes to release NO, which may take synergic action with the platinum-based moieties on
the tumor cells. It was found that all compounds exhibited considerable cytotoxicity against human HCT-116 and SGC-7901 cell
lines via DNA binding together with NO-releasing features, especially for compound 3. This finding is in accordance with the
previous reports that NO hybrids show higher cytotoxicity against colon cancer cell lines compared with their parent compounds.
reactive molecule that it has a very short half-life in the body
and is impossibly administered in aqueous solution. Besides,
NO is found to be a two-sided sword in the treatment of cancer
because many researches showed that NO plays a dual role as a
proneoplastic and an antineoplastic agent simultaneously.
Although the exact role of NO in oncology remains
controversial, there is a consensus to date that the function
of NO in the treatment of cancer depends on its action
concentration. Basically, the high concentration of NO leads to
inhibition of cell growth and induces apoptotic cell death,
whereas the low concentration promotes cell growth.18,19
Considering such character of NO, researchers are increasingly
interested in NO donors that are stable in vitro and are able to
release a relatively high level of NO in vivo.16
Furoxans (1,2,5-oxadiazole 2-oxides) represent an important
class of NO donors. The biological activities of furoxan
derivatives have been widely studied, including anti-infective
properties, anticancer features, mutagenicity, immunosuppres-
sion, and central muscle relaxant properties.20 Since the
anticancer properties of some benzofuroxans were first reported
by Ghosh and Whitehouse,21−24 many furoxan and benzofur-
oxan derivatives have been tested against human cancer cell
INTRODUCTION
■
The successful application of cisplatin in clinical for cancer
therapies has triggered considerable interest to search for more
effective cisplatin analogues, and as a consequence carboplatin
and oxaliplatin have been successively developed and used in
clinic.1,2 On the basis of the great success of platinum-based
drugs, cisplatin has reached medicinal chemistry textbooks
worldwide.3 However, the cross resistance and severe side
effects of platinum drugs have limited their clinical application
to a great extent. In order to overcome these drawbacks, a
number of drug-design strategies have been employed in the
past years,4−11 and one of the most successful approaches is to
combine platinum-based drugs with some other active
pharmacophore so that a synergic action can be achieved
toward the tumor cells.12−15
Nitric oxide (NO) is one of the most important signaling
molecules involved in many processes of vasodilation, neuro-
transmission, immune system, and apoptosis in human
bodies.16,17 Recently, it has been found that NO has
cytotoxicity against cancer cells, as evidenced by the positive
results of the study concerning the beneficial effects of NO in
oncology.18 The antitumor mechanisms of NO are various,
including (a) inhibiting the proliferation of tumor cells, (b)
causing apoptosis and necrosis of tumor cells, and (c) inducing
the attenuation of angiogenesis.19 However, NO is such a
Received: June 27, 2012
Published: September 7, 2012
© 2012 American Chemical Society
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Scheme 1. Synthetic Route of Complexes 1−6
Figure 1. Schematic representation of the complexes studied in this work.
lines as cytotoxic compounds.25,26 Recently, a few researchers
have demonstrated that NO-donor compounds can enhance
the cytotoxicity of platinum drugs against human cancer
cells27,28 and sensitize drug-resistant human cancer cells to
cisplatin,29 indicating that there is a positive synergy between
NO and platinum-based compounds in the aspect of anticancer.
It is noted that all previous attempts were performed by
administrating NO-donor compounds and platinum drugs as
two individual agents, but there has been no attempt to
combine them into a molecule so far. From this point of view, it
will be interesting and significant to study the hybrid
compounds of platinum-based moieties with NO donors as
anticancer agents.
In this work, we have designed and synthesized a series of
hybrid compounds consisting of both platinum-based moieties
of the present effective platinum drugs and furoxan species for
the first time. It is anticipated that the newly synthesized agents
could bind DNA in the same way as cisplatin and its analogues
and exhibit good cytotoxicity against tumor cells owing to the
anticancer property of NO and its possible synergic action with
platinum-based complexes. At first, we intended to add furoxan
species to the classical carrier ligands [e.g., ammine, (1R,2R)-
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Figure 2. UV−vis spectra of complexes 1−6 in 5% CH3CH2OH/95% H2O recorded at different times over 12 h of incubation at 298 K: (a) complex
1; (b) complex 2; (c) complex 3; (d) complex 4; (e) complex 5; (f) complex 6.
Scheme 2. Hydrolysis Equation for Complex 2
1,2-diaminocyclohexane, (4R,5R)-4,5-bis(aminomethyl)-2-iso-
propyl-1,3-dioxolane]; however, the lack of hydroxyl groups
in these carrier ligands limits the introduction of furoxan
species. Thus, we had to tether the furoxan moieties to the
leaving group ligands containing a hydroxyl group like glycolic
acid and 3-hydroxy-cyclobutane-1,1-dicarboxylic acid. Herein
reported are the synthesis (Scheme 1), characterization, and in
vitro biological properties of a series of novel anticancer
candidates (Figure 1) that combine the platinum moieties of
the present platinum drugs like carboplatin, oxaliplatin, or
heptaplatin with furoxan groups.
The synthesized compounds were spectrally characterized by
elemental analysis and 1H NMR, IR, and electrospray ionization
mass (ESI-MS) spectroscopy. In the ESI-MS spectra, all of the
platinum complexes showed 100% of [M + H]+ or [M + Na]+
peaks, except for 1, which showed 100% of the [M − L1]+
peak. It is noted that complexes 1−6 have three protonated ion
peaks, respectively, because of the existence of the isotopes
1
194Pt (33%), 195Pt (34%), and 196Pt (25%). H NMR spectra
confirmed the existence of furoxan groups, which are
compatible to the chemical structures of the corresponding
complexes.
Stability of Complexes 1−6. Because the stability of
platinum-based compounds is one of the key criteria for being
an anticancer candidate, complexes 1−6 were evaluated by UV
spectral analysis at different times in 5% CH3CH2OH/95%
H2O (Figure 2). No detectable changes of the absorption bands
of complexes 4−6 have been observed within 12 h, which
demonstrates the good stability of these compounds with
bidentate carboxylate under the test conditions. However, the
absorption bands of complex 2 decreased gradually at around
250 nm within the first 7 h and then maintained stability in the
later 5 h, while the absorption bands of complexes 1 and 3
increased gradually at the beginning and then became
unchanged in the later hours, indicating that complexes 1−3
RESULTS AND DISCUSSION
■
Synthesis of Complexes 1−6. Ligands L1 and L2 as well
as platinum(II) complexes 1−6 were prepared following the
procedure shown in Scheme 1. We failed to get L1 by a direct
reaction of glycolic acid with 3,4-bis(phenylsulfonyl)-1,2,5-
oxadizazole oxide, so the carboxylic acid was protected by
esterification and then hydrolyzed under a basic condition with
pH < 10. L2 was obtained in a manner analogous to that of L1.
Because furoxans are stable under acidic conditions and have
high resistance against heat,17 hydrolysis took place under
acidic conditions with tetrabutylammonium bromide as a
phase-transfer catalyst at 100 °C.
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with two monodentate carboxylates were easily hydrolyzed
until was equilibrium reached. It is observed that the UV
spectral changes of the complexes with different carrier ligands
were not the same. A representative equilibrium equation for
complex 2 is shown in Scheme 2.
According to Figure 2, it is noted that the changes of the
absorption bands of complex 3 were very tiny compared to
those of complexes 1 and 2 but somewhat like those of
complexes 4−6 with bidentate carboxylate ligands, demonstrat-
ing that complex 3 was relatively more stable than complexes 1
and 2. The departure of the leaving group from the complex
with two monodentate carboxylates was also detected by the
ESI-MS technique. The typical peak appearing in the ESI-MS
spectrum of complex 1 was attributed to [M − L1]+ species. It
is notable that the departure of the leaving group from the
complex with two monodentate carboxylates was common,
which similarly occurred in the case of cisplatin.30 The
absorption bands at around 228 and 254 nm were assigned
to the furoxan derivatives, which were stable in aqueous
solution. Overall, complexes 4−6 with a bidentate O−O leaving
group were more stable than complexes 1−3, and the
synthesized furoxan derivatives were stable in aqueous solution
as well.
DNA Binding Study. The interaction of platinum
compounds with DNA to form platinum/DNA adducts is the
main mechanism of platinum complexes leading to tumor cell
death and anticancer effects.3,31 With the purpose of assessing
the binding of our synthesized complexes with DNA, agarose
gel electrophoresis was applied to study the interaction of
pET22b plasmid DNA with complexes 1−6 and cisplatin,
respectively. The compounds tested were divided into three
groups, and each group was composed of untreated pET22b
plasmid DNA, cisplatin, and newly synthesized compounds
bearing the same carrier ligands. Each compound was tested at
four different concentrations (10, 50, 100, and 200 μM).
When pET22b plasmid DNA was incubated with complexes
1−6 and cisplatin increased from 10 to 200 μM, it was found
that the intensity of the circular supercoiled DNA (form I)
band decreased, while that of the nicked (form II) bands
increased. This can be attributed to the unwinding of
supercoiled DNA to open circular DNA, demonstrating the
binding between DNA and the platinum complexes (Figure 3).
For complexes 1 and 3, the circular supercoiled DNA
disappeared at a concentration of 200 μM and a small fraction
of linear DNA (form III) appeared, which is indicative of the
strong DNA binding activities of complexes 1 and 3. It was
noticed that the linear DNA was not observed in the case of
complex 2 under the same conditions. As for complexes 4−6,
only a high concentration of complex 6 caused the
disappearance of the circular supercoiled DNA. The DNA
binding abilities of the tested platinum complexes follows the
order 1, 3 > 2, 6, cisplatin > 4, 5, as indicated by the
disappearance of the circular supercoiled DNA at a
concentration of 200 μM. Evidently, the ability of complexes
4−6 with bidentate carboxylate to interact with DNA was lower
than that of complexes 1−3 with two monodentate
carboxylates. This is probably due to the fact that the bidentate
dicarboxylate anion, which forms a six-membered coordination
ring with the metal atom, is more stable and harder to
dissociate from the parent compound, while the corresponding
monodentate anion, which binds the metal atom only with a
single coordination bond, appears to be much easier to
dissociate from the parent compounds. In general, it could be
Figure 3. Gel electrophoretic mobility pattern of pET22b plasmid
DNA when incubated with various concentrations of platinum
complexes. (a) Lanes 1 and 14: control DNA. Lanes 2−5: cisplatin
(10, 50, 100, and 200 μM) + DNA. Lanes 6−9: complex 1 (10, 50,
100, and 200 μM) + DNA. Lanes 10−13: complex 4 (10, 50, 100, and
200 μM) + DNA. (b) Lanes 1 and 14: control DNA. Lanes 2−5:
cisplatin (10, 50, 100, and 200 μM) + DNA; Lanes 6−9: complex 2
(10, 50, 100, and 200 μM) + DNA. Lanes 10−13: complex 5 (10, 50,
100, and 200 μM) + DNA. (c) Lanes 1 and 14: control DNA. Lanes
2−5: cisplatin (10, 50, 100, and 200 μM) + DNA. Lanes 6−9: complex
3 (10, 50, 100, and 200 μM) + DNA. Lanes 10−13: complex 6 (10,
50, 100, and 200 μM) + DNA.
concluded that complexes 1−3 and 6 showed cleavage ability
comparable to that of cisplatin, while the binding ability of
complexes 4 and 5 was a little weak. Notably, the behaviors of
plasmid DNA binding with our complexes and cisplatin are
similar, which is in accordance with our expectation that the
newly synthesized agents could bind DNA in the same manner
as cisplatin.
In Vitro NO-Releasing Study. Considering that the
−
principal oxidation product of NO is NO2 in aqueous
solution, the Griess reaction32 was applied to measure the
quantity of NO released from complexes 1−6. It has been
reported that thiols can induce the release of NO from furoxan
and its derivatives in vitro.17 Therefore, complexes 1−6 were
measured in the presence and absence of L-cysteine to assess
their ability to produce NO under different conditions. The
release of NO from complexes 1−6 was very fast at the
beginning and then gradually tended to remain unchanged at
last in the presence of L-cysteine (Figure 4). The NO-releasing
abilities of complexes generally followed the order 6 > 4, 3, 5, 2
> 1. The final percentage of NO released from complexes 1−6
varied from 27.28% to 44.50%, which was different from some
organic furoxan derivatives that varied widely (0.2−34.3%).33
In contrast, no NO liberated from complexes 1−6 was
observed in the absence of L-cysteine, suggesting that furoxan
moieties are stable under the test conditions, which is in
accordance with the UV spectral results. We know that thiol-
containing molecules, such as GSH and L-cysteine,34,35 could
form stable bonds with platinum atoms and dramatically
decrease the percentage of platinum-based drugs binding to
DNA (only 5−10% of cisplatin binds to nuclear DNA),36,37
which may lead to severe side effects and drug resistance.38,39
Nevertheless, once the newly synthesized compounds were
exposed to the thiol-containing molecules, some of the thiol-
containing nucleophiles would react with the furoxan moieties
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Figure 4. Percentage of NO release from complexes 1−6 in the presence or absence of L-cysteine in vitro.
to release NO, which may partly decrease the binding with
platinum-based moieties. Overall, our compounds exhibited
good NO-releasing properties in the presence of thiols and
were stable under normal conditions in vitro.
In Vitro Cytotoxicity Assay. The in vitro cytotoxicity of
complexes 1−6 and positive agents was evaluated by means of
CCK-8 assay against HCT-116 (human colorectal cancer cell
line) and SGC-7901 (human gastric cancer cell line).
Carboplatin and oxaliplatin, whose structures are similar to
those of our synthesized complexes, were used as positive
controls. According to the IC50 values (Table 1) for HCT-116,
HCT-116 colon cancer cell line; i.e., compound 4 bearing a
NO-releasing moiety showed 2-fold higher cytotocixity than its
parent drug (carboplatin), suggesting that the furoxan species
probably made a contribution to the cytotoxic activity on HCT-
116.
According to the IC50 values, the relative orders of the
cytotoxicity of the newly synthesized compounds against HCT-
116 and SGC-7901 cell lines are 3 > 2 > 1 > 6 > 4 > 5 and 3 >
5 > 6 > 2 > 1 > 4, respectively. It is notable that complexes 1−3
with two monodentate carboxylates showed cytotoxicity
superior to that of their corresponding compounds with
bidentate dicarboxylates, which are in line with the gel
electrophoresis results, demonstrating that the interaction of
platinum-based moieties with DNA played the leading role in
the anticancer effect. In a comparison of the same leaving
ligands, the anticancer activities of different carrier ligands
followed the order (4R,5R)-4,5-bis(aminomethyl)-2-isopropyl-
1,3-dioxolane > (1R,2R)-1,2-diaminocyclohexane > ammine.
Complex 3 is the most effective agent among the synthesized
complexes, which has cytotoxicity similar to that of oxaliplatin
against both HCT-116 and SGC-7901 cell lines. In general,
complexes 1−6 showed considerable anticancer activity against
the HCT-116 cell line, and most of them exhibited cytotoxic
efficacy better than that of carboplatin and close to that of
oxaliplatin.
Table 1. In Vitro Cytotoxicity (IC50, μM) of Complexes 1−6,
Carboplatin, and Oxaliplatin against Human Cancer Cell
a
Lines
IC50 (μM)
b
c
compound
HCT-116
SGC-7901
carboplatin
oxaliplatin
complex 1
complex 2
complex 3
complex 4
complex 5
complex 6
273.05 12.3
57.04 5.61
64.06 7.81
57.21 4.32
39.43 2.34
111.91 7.89
142.15 12.12
87.06 8.07
58.11 4.31
17.35 1.23
217.93 10.13
94.23 5.14
34.64 2.13
248.07 21.14
59.10 4.59
70.83 2.91
a
b
Each value is the mean of three independent experiments. Human
CONCLUSION
c
■
colorectal cancer cell. Human gastric cancer cell.
Furoxan moieties were used for the first time as NO donors in
the form of carboxylate to combine with platinum-based
moieties of the present platinum drugs to prepare six novel
platinum-based compounds. As expected, the DNA binding
properties of the platinum-based compounds, analogous to
those of cisplatin, were proven by agarose gel electrophoresis.
In consideration of the complexity of the cellular system, the
synergic action of NO-donor and platinum-based moieties was
not studied in this paper. However, the newly synthesized
compounds exhibited good stability and NO-releasing proper-
ties and showed considerable anticancer activity against HCT-
116 and SGC-7901 cells in vitro. Besides, the interaction of
complexes with DNA, which formed platinum/DNA adducts,
played the leading role in the anticancer effects of complexes
1−6, while the furoxan species were important for the cytotoxic
activity on the HCT-116 colon cancer cell line. Thus, these
compounds are promising anticancer agents and deliver a new
view for the design of anticancer drugs beyond those currently
in use.
complexes 1−6 showed cytotoxicity comparable to that of
oxaliplatin, which is the first platinum-based complex effective
in the treatment of colorectal cancer. As for SGC-7901, the
synthesized compounds showed cytotoxicity comparable to that
of carboplatin and less cytotoxicity than oxaliplatin. Obviously,
complexes 1−6 were more sensitive to the colon cancer cell
line than the gastric cancer cell line. It has been reported that
NO hybrids, which seemed safer, were from 1.7- to 1083-fold
more potent than their corresponding parent compounds
against HT-29 and HCT-15 colon cancer cell lines in vitro.19,40
Besides, NO aspirin promoted the anticancer activity compared
with their parent compounds against the HT-29 colon cancer
cell line by stimulating phophorylation of MAP kinases P38 and
JNK in vitro.41 Moreover, NO-donating aspirin showed a
strong inhibitory effect against intestinal carcinogenesis in Min
(APCMin/+) mice in vivo.42 On the basis of the previous
reports,40−42 it is rational to conjecture that furoxan species
may more or less play a role in the cytotoxic efficacy against the
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water and 30 mL of THF was added to the filtrate and stirred for 24 h
at room temperature. After that, the mixture was cooled to room
temperature and concentrated to 5 mL, affording the precipitate. The
precipitate was filtered and washed with cold distilled water.
Complex 1. cis-[Pt(NH3)2I2] was used as the starting material.
Yield: 0.50 g (60.9%), pale-yellow solid. Elem anal. Calcd for
C20H20N6O14S2Pt: C, 29.02; H, 2.44; N, 10.15; S, 7.75. Found C,
28.88; H, 2.29; N, 10.40; S, 7.89. IR (KBr, cm−1): 3444 (m), 1708 (w),
1623 (s), 1458 (w), 1350 (w), 1164 (m), 834 (w), 730 (w), 687 (w),
597 (s), 553 (w). 1H NMR (DMSO-d6 + D2O): δ 8.08−8.05 (m, 2H,
Ph), 7.97−7.88 (m, 1H, Ph), 7.83−7.74 (m, 2H, Ph), 4.99 (s, 1H,
CH2), 4.94 (s, 1H, CH2), 4.58 (s, 2H, CH2). ESI-MS: m/z 528 (100%;
[M − L1]+).
EXPERIMENTAL SECTION
■
Materials and Instrumentation. All reagents and solvents were
of analytical purity and were used without any further purification.
Potassium tetrachloroplatinate(II) was purchased from a local
chemical company. 1H NMR spectra were produced on a Bruker
500 MHz spectrometer. ESI-MS spectra were measured on a Bruker
instrument. IR spectra were obtained on a Nicolet IR 200
spectrophotometer. All UV absorption spectra were recorded on a
Shimadzu UV-1700 UV−vis spectrophotometer.
Synthesis and Characterization. Synthesis of Ethyl Glycolate.
Glycolic acid (7.6 g, 0.1 mol) and concentrated sulfuric acid (5 mL)
were added at room temperature to a mixture solution of absolute
ethyl alcohol (17.4 mL, 0.3 mol) and benzene (50 mL) in a 250 mL
flask equipped with an oil−water separator. The mixture was heated
under reflux for 12 h and then evaporated under vacuum to remove
the rest of ethanol and benzene; 50 mL of water was added and
extracted with ethyl acetate (3 × 30 mL). The extract was washed with
water (3 × 30 mL) and dried with anhydrous sodium sulfate. The
solvent was evaporated off under vacuum. Ethyl glycolate was
obtained, which was used without further purification for the synthesis
of a. Yield: 7.8 g (75%). IR (KBr, cm−1): 3398 (m), 2984 (m), 1741
(s), 1437 (w), 1223 (s), 1098 (s), 1021 (w).
Complex 2. cis-{Pt[(1R,2R)-1,2-diaminocyclohexane]Cl2} was used
as the starting material.45 Yield: 0.61 g (67.1%), white crystalline solid.
Elem anal. Calcd for C26H28N6O14S2Pt: C, 34.40; H, 3.11; N, 9.26; S,
7.06. Found: C, 34.12; H, 3.34; N, 9.45; S, 6.95. IR (KBr, cm−1): 3443
(w), 3216 (w), 3101 (w), 2941 (w), 1616 (s), 1550 (s), 1453 (m),
1
1295 (w), 1164 (m), 1021 (w), 730 (w), 597 (s), 555 (w). H NMR
(DMSO-d6): δ 8.07−8.02 (m, 2H, Ph), 7.95−7.85 (m, 1H, Ph), 7.80−
7.70 (m, 2H, Ph), 6.28−5.81 (m, 4H, NH2), 4.93 (s, 2H, CH2), 4.51
(s, 2H, CH2), 2.49−1.01 (m, 10H, CH2 of DACH). ESI-MS: m/z 930
(100%; [M + Na]+).
Synthesis of L1. Ethyl glycolate (0.62 g, 6 mmol) and 3,4-
bis(phenylsulfonyl)furoxan43 (0.73 g, 2 mmol) were dissolved in 40
mL of tetrahydrofuran (THF) at room temperature. 25% NaOH (0.72
mL, 6 mmol) was added dropwise at 25 °C within 5 min to a stirred
solution. The mixture was stirred for 2 h at 25 °C, and 40 mL of water
was added. THF was removed under vacuum at 30 °C, and white
deposits (a) were obtained by filtration. The intermediate a was
dissolved in a mixture solution of 25 mL of water and 25 mL of THF.
A pH of 10 was maintained by the addition of a 1 M NaOH aqueous
solution, and the reaction was terminated when the pH was stable. The
white solid was filtered off, and the pH of the filtrate was adjusted to 4
with a 1 M HCl solution. THF was removed under vacuum at 30 °C,
affording white precipitate of L1. Yield: 0.37 g (62%), white solid.
Elem anal. Calcd for C10H8N2O7S: C, 40.00; H, 2.69; N, 9.33; S, 10.68.
Complex 3. cis-{Pt[(4R,5R)-4,5-bis(aminomethyl)-2-isopropyl-1,3-
dioxolane] I2} was used as starting material.46 Yield: 0.65 g (66.9%).
Pale yellow solid. Elemental analysis calculated for C28H32N6O16S2Pt
(%): C, 34.75; H, 3.33; N, 8.68; S, 6.63. Found C, 34.49; H, 3.56; N,
1
8.95; S, 6.34. IR (KBr)/cm−1: 3442 (m), 1710 (m), 1624 (s), 1551
(s), 1459 (m), 1354 (w), 1252 (w), 1163 (s), 1086 (w), 731 (m), 599
(s); H NMR (d6-DMSO+D2O): δ 8.06−8.02 (m, 2H, Ph), 7.95−7.91
(m, 1H, Ph), 7.78−7.73 (m, 2H, Ph), 4.94 (s, 2H, CH2), 4.84−4.83 (d,
J(H,H)=4.5 Hz, 1H, CH), 4.43 (s, 2H, CH2), 4.25 (s, 1H, CH), 4.22
(s, 1H, CH), 3.15 (m, 1H, CH2NH2), 2.99 (d, 1H, CH2NH2), 2.65
(m, 2H, CH2NH2), 1.86−1.79 (m, 1H, CHMe2), 0.92 ppm (d, J(H,H)
=7 Hz, 6H, C(CH3)2); ESI-MS: m/z [M+Na]+ = 990 (100%).
Complex 4. cis-[Pt(NH3)2I2] was used as the starting material.
Yield: 0.36 g (58.9%), pale-yellow solid. Elem anal. Calcd for
C14H16N4O9SPt: C, 27.50; H, 2.64; N, 9.16; S, 5.24. Found: C,
27.89; H, 2.76; N, 8.89; S, 5.10. IR (KBr, cm−1): 3445 (w), 1615 (s),
1542 (s), 1451 (w), 1359 (s), 1167 (m), 742 (w), 684 (w), 599 (s),
556 (w). 1H NMR (DMSO-d6 + D2O): δ 8.00−7.99 (d, J(H,H) = 7.5
Hz, 2H, Ph), 7.89−7.86 (t, J(H,H) = 7.5 Hz, 1H, Ph), 7.75−7.72 (t,
J(H,H) = 7.5 Hz, 2H, Ph), 4.93−4.90 (t, J(H,H) = 7.0 Hz, 1H, CH of
cyclobutyl), 3.38−3.34 (m, 2H, CH2 of cyclobutyl), 2.79−2.71 (m,
2H, CH2 of cyclobutyl). ESI-MS: m/z 612 (10%; [M + H]+), 634
(100%; [M + Na]+).
1
Found: C, 39.68; H, 2.78; N, 9.22; S, 10.89. H NMR (DMSO-d6): δ
13.51 (m, 1H, OH), 8.05−8.03 (m, 2H, Ph), 7.92−7.89 (m, 1H, Ph),
7.77−7.74 (m, 2H, Ph), 5.07 (s, 2H, CH2). ESI-MS:m/z 299 (100%;
[M − H]−).
Synthesis of L2. Diethyl 3-hydroxycyclobutane-1,1-dicarboxylate44
(1.30 g, 6 mmol) and 3,4-bis(phenylsulfonyl)furoxan (0.73 g, 2 mmol)
were dissolved in 40 mL of THF at room temperature. 25% NaOH
(0.72 mL, 6 mmol) was added dropwise at 25 °C within 5 min to a
stirred solution. The mixture was stirred for 2 h at 25 °C, and 40 mL of
water was added. The solvent was removed under vacuum at 30 °C,
and white deposits (b) were obtained by filtration. The intermediate b
was suspended in 100 mL of water, a small amount of tetra-n-
butylammonium bromide was added as a phase-transfer catalyst, and
then 5 mL of concentrated hydrochloric acid was added to the
solution. The mixture was heated under reflux for 12 h and extracted
with ethyl acetate (3 × 50 mL). The extract was washed with water (3
× 30 mL) and separated by column chromatography (PE:EtOAc =
2:1). Yield: 0.25 g (32%), white solid. Elem anal. Calcd for
C14H12N2O9S: C, 43.75; H, 3.15; N, 7.29; S, 8.34. Found: C, 43.43;
H, 3.23; N, 7.46; S, 8.71. IR (KBr, cm−1): 2923 (m), 1712 (s), 1618
(s), 1552 (m), 1450 (m), 1416 (m), 1357 (w), 1292 (w), 1163 (s),
Complex 5. cis-{Pt[(1R,2R)-1,2-diaminocyclohexane]Cl2} was used
as the starting material. Yield: 0.44 g (64.2%), white crystalline solid.
Elem anal. Calcd for C20H24N4O9SPt: C, 34.73; H, 3.50; N, 8.10; S,
4.64. Found: C, 34.56; H, 3.37; N, 8.41; S, 4.54. IR (KBr, cm−1): 3448
(m), 3200 (w), 3095 (w), 2946 (w), 1620 (s), 1544 (m), 1450 (w),
1
1358 (m), 1168 (m), 900 (w), 732 (w), 596 (s), 554 (w). H NMR
(DMSO-d6): δ 8.04−8.01 (m, 2H, Ph), 7.93−7.88 (m, 1H, Ph), 7.78−
7.73 (m, 2H, Ph), 5.92 (m, 2H, NH2), 5.23−5.20 (m, 2H, NH2),
4.95−4.89 (m, 1H, CH of cyclobutyl), 3.40−3.38 (m, 2H, CH2 of
cyclobutyl), 2.83−2.73 (m, 2H, CH2 of cyclobutyl), 2.04−0.85 (m,
10H, CH2 of DACH). ESI-MS: m/z 692 (30%; [M + H]+), 714
(100%; [M + Na]+).
1
999 (w), 736 (w), 599 (s), 552 (m). H NMR (DMSO-d6): δ 13.12
Complex 6. cis-{Pt[(4R,5R)-4,5-bis(aminomethyl)-2-isopropyl-1,3-
dioxolane]I2} was used as the starting material. Yield: 0.49 g (65.9%),
yellow solid. Elem anal. Calcd for C22H28N4O11SPt: C, 35.16; H, 3.75;
N, 7.45; S, 4.27. Found: C, 34.87; H, 3.89; N, 7.68; S, 4.04. 1 IR (KBr,
cm−1): 3443 (m), 2971 (w), 1721 (w), 1619 (s), 1545 (m), 1451 (w),
(m, 2H, OH), 8.04−8.02 (m, 2H, Ph), 7.91−7.88 (m, 1H, Ph), 7.76−
7.73 (m, 2H, Ph), 5.14−5.11 (m, 1H, CH of cyclobutyl), 2.94−2.88
(m, 2H, CH2 of cyclobutyl), 2.66−2.62 (m, 2H, CH2 of cyclobutyl).
ESI-MS: m/z 407 (100%; [M + Na]+).
Standard Procedure for the Preparation of Complexes 1−6. A
mixture of cis-[Pt(Am)I2] or cis-[Pt(Am)Cl2] (1 mmol) [Am = 2-
ammine, (1R,2R)-1,2-diaminocyclohexane, and (4R,5R)-4,5-bis-
(aminomethyl)-2-isopropyl-1,3-dioxolane] and silver nitrate (2
mmol) in distilled water was stirred in the dark for 24 h at 40 °C.
The reaction mixture was filtered under vacuum, and then L1 (2
mmol) or L2 (1 mmol) mixed with NaOH (2 mmol) in 20 mL of
1
1358 (m), 1165 (m), 1091 (w), 734 (w), 597 (s), 556 (w). H NMR
(DMSO-d6 + D2O): δ 8.03−8.02 (d, J(H,H) = 7.5 Hz, 2H, Ph), 7.92−
7.89 (t, J(H,H) = 7.5 Hz, 1H, Ph), 7.77−7.74 (t, J(H,H) = 7.5 Hz, 2H,
Ph), 4.94−4.90 (m, 1H, CH of cyclobutyl), 4.85 (m, 1H, CH), 4.39 (s,
1H, CH), 4.26 (s, 1H, CH), 3.22−3.19 (m, 2H, CH2 of cyclobutyl),
3.12 (m, 1H, CH2NH2), 3.00 (d, 1H, CH2NH2), 2.80 (m, 2H, CH2 of
cyclobutyl), 2.67 (m, 2H, CH2NH2), 2.19−2.17 (m, 1H, CHMe2),
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Inorganic Chemistry
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0.87−0.86 (d, 6H, C(CH3)2). ESI-MS: m/z 752 (30%, [M + H]+),
(5) Yu, Y.; Domianello, S.; Legin, A. A.; Jakupec, M. A.; Arion, V. B.;
Kukushkin, V. Y.; Galanski, M.; Keppler, B. K. Inorg. Chem. 2011, 50,
10673−10681.
774 (100%; [M + Na]+).
Stability Studies. The absorption spectra of complexes 1−6 in the
UV−vis region were recorded on a Shimadzu UV-1700 UV−vis
spectrophotometer. The electronic spectra of complexes 1−6
dissolved in 5% CH3CH2OH/95% H2O were obtained at different
times over 12 h at room temperature.
Interaction with Plasmid DNA. The pET22b plasmid DNA was
used as the target to treat with complexes 1−6 and cisplatin to
investigate the cleavage of DNA by agarose gel electrophoresis. The
complexes were dissolved and diluted to different desired concen-
trations with distilled water, then 5 μL of pET22b plasmid DNA (0.5
μg/mL) was added to the solution, and the resulting solution was
incubated in a water bath at 37 °C in the dark for 24 h for DNA
unwinding. After that, the mixtures with a loading buffer (1 μL) were
loaded onto the 1.0% agarose gel stained with ethidium bromide, and
electrophoresis was carried out under a TA buffer (50 mM Tris-
acetate, pH 7.5) for 60 min at 100 V. The gels were photographed by a
Molecular Imager (Bio-Rad, USA) under UV light.
NO Release in Vitro. The standard curve of nitrite concentration
against absorbance was measured as described before.47 Complexes 1−
6 were diluted to 12.5 μM with either (i) a phosphate-buffered
solution (PBS) at pH 7.4 or (ii) PBS containing 5 mM L-cysteine at
pH 7.4, and then Griess reagent was added to the solution to react
with the nitrite at different times. The data were acquired by
measuring the absorbance at 540 nm with a UV spectrophotometer.
Cytotoxicity Studies. The cytotoxicity of all of the complexes
against HCT-116 and SGC-7901 cells was determined by means of the
colorimetric assay CCK-8 (Beyotime, Beijing, China). The cells were
plated at 5000 cells per well in 96-well culture plates with a culture
medium and incubated for 24 h at 37 °C in a water atmosphere (5%
CO2). The compounds with the desired concentrations were obtained
by dissolution in DMSO and dilution with a culture medium (DMSO
final concentration < 0.4%). Then the diluted solution of complexes
was treated with the cells for 48 h at 37 °C in a 5% CO2 incubator.
After that, 10 μL of a freshly diluted CCK-8 solution (5 mg/mL in
PBS) was added to each well and the plate was incubated for 4 h. The
cell survival was evaluated by measuring the absorbance at 540 nm
with an automatic microplate ELISA reader. IC50 values were
calculated from the chart of the cell viability (%) against the
compound concentration (μM). All experiments were carried out in
triplicate.
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AUTHOR INFORMATION
■
Corresponding Author
*E-mail: sgou@seu.edu.cn.
Notes
(28) Yasuda, H.; Nakayama, K.; Watanabe, M.; Suzuki, S.; Fuji, H.;
Okinaga, S.; Kanda, A.; Zayasu, K.; Sasaki, T.; Asada, M.; Suzuki, T.;
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The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors are grateful to the National Natural Science
Foundation of China (Project. 20971022) and the New Drug
Creation Project of the National Science and Technology
Major Foundation of China (Project 2010ZX09401-401) for
financial aid to this work.
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