Study on Antitumor Platinum(II) Complexes of Chiral Diamines with

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Journal of Medicinal Chemistry

Study on Antitumor Platinum(II) Complexes of Chiral Diamines with Dicyclic Species as Steric Hindrance Fengfan Liu,†, ‡ Shaohua Gou, †, ‡* Feihong Chen, †, ‡ Lei Fang, †, ‡* Jian Zhao† †

Pharmaceutical Research Center and School of Chemistry and Chemical Engineering,

Southeast University, Nanjing 211189, China ‡

Jiangsu Province Hi-Tech Key Laboratory for Bio-medical Research, Southeast University,

Nanjing 211189, China Supporting Information ABSTRACT:

A

series

of

platinum(II)

complexes,

characteristic

of

chiral

trans-bicyclo[2.2.2]octane-7,8-diamine as ligand possessing dicyclic steric hindrance, were designed and synthesized. Biological evaluation showed that almost all complexes had cytotoxic activity against the tested cancer cell lines, among which most of chiral (R,R)-enantiomeres had stronger cytotoxicity than their (S,S)-counterparts, and 2a, [trans-bicyclo[2.2.2]octane-7R,8R-diamine](oxalato-O,O’)platinum(II),

is

the

most

effective agent. Significantly, its counterpart, 2b, was much sensitive to cisplatin resistant SGC7901/CDDP cancer cell line at a higher degree than 2a. Docking study and agarose gel electrophoresis revealed that the interaction of 2a with DNA was similar to that of oxaliplatin. Western blot analysis demonstrated that 2a could induce a better effect than cisplatin on mitochondrial-dependent apoptosis pathway. Kinetic study indicated that the dicyclic ligand can accelerate the reaction rate of the complex.

ACS Paragon Plus Environment


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INTRODUCTION Since cisplatin was firstly approved by FDA in 1978, platinum complexes have become a class of important anticancer drugs.1,2,3 Up to date, many platinum complexes have been widely employed in cancer chemtherapy, which has brought about a multibillion-dollar industry.4-11 In spite of their success, the clinical application of platinum drugs also suffers from several drawbacks including serious toxicity, instinct and acquired drug resistance.12-15 Therefore, much effort has been made to overcome the side effects and improve the antitumor activity of platinum-based drugs.16-19 Oxaliplatin, one of the best sale anticancer drugs, has become the standard first line therapy in case of metastatic colorectal cancer, which can exhibit activity against cisplatin and carboplatin resistant cancers.20 It is noted that oxaliplatin possesses a cycloalkyl framework, 1R,2R-diaminocyclohexane (abbreviated as 1R,2R-DACH), which acts as a carrier ligand providing steric hindrance when interacting with DNA. The unique and potent antitumor activity of oxaliplatin is believed to be attributed to a [Pt(1R,2R-DACH)]2+ unit that plays a pivotal role in increasing cell uptake and inhibiting DNA mismatch repair.21,22 Although numerous platinum complexes of trans-1,2-DACH analogs have recently been studied to explore the sterically hindered platinum complexes,23,24,25 only a few of platinum complexes of carrier ligands with dicyclic framework as steric hindrance were involved. So far, there are limited examples of such ligands involved in two types of (i) 1,2-diamine like 1,2-camphordiamine

(Figures

1A

and

1B),26

and

(ii)

1,4-diamine

like

1,2-bis(aminomethyl)carbobicyclic derivatives (Figures 1C and 1D).27,28 It is obvious that the latter type ligand could offer less steric hindrance than the former one when they


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combine with platinum(II) ions, since the complexes of 1,4-diamine could leave more space when binding to DNA. In addition, picoplatin is a sterically hindered platinum(II) complex which is significant for its ability to overcome cisplatin resistance due to its unique non-planar structure between pyridine ring and the coordination square plane around the metal atom.29 Thus, it is an effective way to overcome the platinum drug cross-resistance between cisplatin and its analogs by introducing sterically hindered moieties in the structure of platinum complexes. Recently, chiral trans-bicyclo[2.2.2]octane-7,8-diamine (abbreviated as L) has been reported for asymmetric organocatalysis.30,31 Such a 1,2-diamine, which has a bridging alkyl group to connect a diaminocyclohexane moiety similar to 1,2-DACH, is liable to function as a carrier ligand via coordination with a platinum atom to form antitumor complexes. Particularly, the steric configuration of the 1,2-diamine upon the dicyclic skeleton might influence the interaction of the resulting platinum complex with DNA, because the hindrance of the dicycloalkyl moiety could prevent the active platinum(II) complex from detoxification reactions by biological nucleophiles, which are generally believed to play a role in mechanisms underlying tumor resistance to platinum compounds.24,25,32 Besides, dicarboxylates like oxalate, malonate and 1,1-cyclobutanedicarboxylate, applied as leaving groups in place of chloride anions (Figure 2), can have a significant effect on the stability, pharmacokinetic and bioavailability of the platinum complexes as well.25,28 Herein

we

report

four

pairs

of

chiral

platinum(II)

complexes

of

trans-bicyclo[2.2.2]octane-7R,8R-diamine (abbreviated as LR) /trans-bicyclo[2.2.2]octane7S,8S-diamine (abbreviated as LS) and their biological evaluation against three human cancer cell lines together with the assay of the typical compounds against cisplatin resistant cancer cell lines. In addition, chemical and biological properties of these compounds have



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been studied to explicit their preliminary mechanism of action.

Figure 1. Known platinum complexes of carrier ligands with dicyclic framework.

Figure 2. Chemical structures of oxaliplatin, picoplatin and the target complexes.

RESULTS AND DISCUSSION Synthesis of Chiral Ligands and Their Complexes. The ligand L was prepared by methods as described in literature reports.30,31 The racemeric compound was resolved by its diastereoenantiomeric salt with the corresponding optical dibenzoyl tartaric acid. Although LS was reported previously, LR was obtained for the first time by resolution of L with (2R, 3R)-dibenzoyl tartaric acid. Optical enantiomer purity of both LR and LS were measured


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(Supporting Information, Figure S1), which are almost equal to the values as reported previously.30

Scheme 1. Synthetic route and chemical structures of the target complexes.

Reaction of LR with K2PtCl4 in water led to the formation of yellow complex 1a. The chloride anions were removed from complex 1a with related silver dicarboxylates resulting in the production of white complexes 2a-4a, respectively. The corresponding complexes 1b and 2b-4b of LS were prepared by the same procedure. The synthetic process is showed in Scheme 1. All compounds were spectrally characterized by IR, 1H NMR and 13C NMR spectra as well as ESI-MS spectroscopy. In the IR spectra of the platinum complexes, N-H stretching vibrations located in range 3110-3274 cm-1 (double peaks), which showed the obvious red shifting in contrast to the amino group of the free ligand in range 3384-3403 cm-1, due to the



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amino group coordinating with Pt(II) ions. The νas (C-O) vibration of complexes appeared in 1643-1660 cm-1 which was characteristic of coordinated carboxylate ligands, while the νs (C-O) vibration exhibited in 1361-1392 cm-1. In the 1H NMR spectra，the proton signals of the L moiety in the complexes overlapped in the range of 1.14-2.35 ppm, while those attributed to C-H groups connecting to the amino groups occurred between 2.26 and 2.35 ppm. The CH-N signal of the complexes presented the split by the coupling to the Pt-bound NH and the coupling to Pt195, which were different from those of L appearing a single peak in 2.48 ppm. Two amine protons of complexes 2a-4a/2b-4b appeared in range 5.13-5.69 ppm due to amine coordination with platinum, which were different from those of L in 1.47-1.48 ppm. Apart from the above in common, the characteristic proton signals of cyclobutyl and methylene groups in complexes 3a-4b, were also observed in their corresponding 1H NMR spectra.

13

C NMR peaks of all complexes were compatible to the

proposed chemical structures. The ESI-MS showed [M+H]+ or [M+Na]+ peaks which were in agreement with the proposed molecular formula of the metal complexes. CD spectra of complexes 1a and 1b in powders were measured for confirmation of their related optical configurations, in which two compounds clearly gave the opposite cotton effect (Supporting Information, Figure S2). In addition, the optical rotation degrees of the rest complexes proved that the corresponding pair compounds (2a/2b, 3a/3b and 4a/4b) are optical enantiomers as expected.


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In Vitro Cytotoxicity Assay. The cytotoxicity of complexes 1a-4a/1b-4b were firstly evaluated in vitro by means of MTT assay against human HCT-116, HepG-2 and A549 cancer cell lines with cisplatin, carboplatin and oxaliplatin as positive controls. The corresponding IC50 values are given in Table 1. The result showed that all complexes had rather potent cytotoxicity against HCT-116, HepG-2 and A549 cell lines, and the IC50 values were within the range of 4.45-41.19 µM, 7.86-36.84 µM and 7.72-66.72 µM, respectively. It is noted that complex 2a is the most effective agent among the synthesized compounds. Its cytotoxic activity against HCT-116 cell line is comparable to that of oxaliplatin but a bit higher than that of cisplatin (IC50 = 4.45 µM vs IC50 = 6.50 µM), while its cytotoxicity against A549 (IC50 = 7.72 µM) is in magnitude at the same order as cisplatin (IC50 = 3.86 µM) but a little superior to that of oxaliplatin. Meanwhile, 2a has nearly the same cytotoxicity as oxaliplatin against HepG-2 cell line (IC50 = 10.74 µM vs IC50 = 12.80 µM), while showing less cytotoxicity than that of cisplatin (IC50 = 3.65). However, the cytotoxic activity of 2b, the enantiomer of 2a, is 2.41-fold, 1.95-fold and 4.23-fold less potent than 2a against HCT-116, HepG-2 and A549, respectively. As compared with carboplatin, almost all of the synthesized complexes showed superior cytotoxicity against all the tested cell lines to carboplatin.



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Table 1. In vitro cytotoxicity of complexes 1a-4b, cisplatin, carboplatin and oxaliplatin.

IC50 (µM)a

a

Complex

HCT-116b

HepG-2c

A549d

cisplatin

6.50±0.47

3.65±0.31

3.86±0.28

carboplatin

44.53±4.64

39.08±2.95

55.00±4.76

oxaliplatin

3.28±0.34

12.80±1.28

8.95±0.78

1a

9.69±0.91

7.86±0.61

46.43±3.86

1b

17.62±1.26

23.11±1.87

43.20±4.13

2a

4.45±0.33

10.74±1.05

7.72±0.64

2b

10.73±1.16

20.89±2.03

32.67±2.92

3a

6.88±0.54

20.28±1.46

39.93±3.76

3b

19.64±1.76

36.84±2.87

26.42±2.29

4a

38.16±2.91

24.48±2.17

58.09±4.94

4b

41.19±3.84

27.44±2.36

66.72±5.81

IC50 is the drug concentration effective in inhibiting 50% of the cell growth measured by

the MTT assay after 72 h drug exposure. Each value is the mean of three independent experiments. b HCT-116: human colorectal cancer cell line. c HepG-2: human hepatocellular carcinoma cell line. d A549: human non-small cell lung cancer cell line. By analyzing the structure-activity relationship, it was noted that the introduction of dicyclic framework on the cyclohexane ring could improve the in vitro antitumor activity of the resulting Pt(II) complexes. It was also noticed that the trans-(R,R) congener, except


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individual cases, showed a little greater potency than its corresponding trans-(S,S) enantiomer. This phenomenon must be related to the stereo configuration of the complex, but we have not got a clear idea about it so far. Since a similar feature was also observed in oxaliplatin and its S,S-configured counterpart,33 and our a-class complexes could be regarded as oxaliplatin analogs, it might be understandable that a-class complexes usually had higher biological activity than b-class complexes. However, the leaving ligand also had an important influence on the antitumor activity of the Pt(II) complex in addition to the stereo configuration. According to the above biological result, complex 2a together with its counterpart 2b was selected for further evaluation against normal noncancerous cell line and cisplatin resistant cancer cell lines by the MTT assay. Since both normal SGC7901 and cisplatin resistant SGC7901/CDDP human gastric cancer cell lines were presently available in our laboratory, they were used for contrast with cisplatin and oxaliplatin as positive controls. The results, listed in Table 2, indicated that 2a had potent cytotoxicity against SGC7901 cancer cell line comparable to that of cisplatin or oxaliplatin as expected, and 2b was only half as potent as that of 2a. However, in contrast to the data of 2a and 2b against the cisplatin resistant SGC7901/CDDP cancer cell line, it was found that 2b had remarkably potent cytotoxic activity, not only 1.67-fold more potent than 2a, but 1.12-fold and 2.54-fold stronger than cisplatin and oxaliplatin, respectively. It was of much significance to note that 2b has a small resistant factor (RF) of 0.85, while the RF value of 2a is 2.44. Such a RF difference between 2a and 2b is obviously due to their spatial configurations of these two enantiomers, which means that the trans-(S,S) enantiomer is much biologically



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sensitive to the SGC7901/CDDP cancer cell line. The introduction of dicyclic framework on 1,2-DACH can increase the antitumor activity of the resulting Pt(II) complex due to its steric hindrance as anticipated, but the spatial configuration of the ligand plays an important role on the ability of the corresponding Pt(II) complex to overcome cisplatin resistance. Moreover, oxaliplatin had notably less ability to overcome cisplatin resistant SGC7901/CDDP cancer cells upon the corresponding RF data of cisplatin (3.01) and oxaliplatin (4.70). The reason is also postulated as the spatial configuration of the carrier ligand of 1R,2R-DACH in oxaliplatin. Table 2. Cytotoxicity of Complexes 2a and 2b towards SGC7901, SGC7901/CDDP and HUVEC cell lines. IC50 (µM) a

a

Complex

SGC7901b

SGC7901/CDDPc

Resistant Factor d

HUVECe

cisplatin

4.23±0.34

12.75±1.03

3.01

25.28±2.34

oxaliplatin

6.18±0.55

29.04±2.05

4.70

31.77±2.98

2a

7.96±0.76

19.39±2.01

2.44

44.34±3.78

2b

13.88±1.34

11.43±2.09

0.85

51.26±4.67

IC50 is the drug concentration effective in inhibiting 50% of the cell growth measured by

the MTT assay after 72 h drug exposure. Each value is the mean of three independent experiments.

b

SGC7901:

human

gastric

cancer

cell

line.

c

SGC7901/CDDP:

cisplatin-resistant human gastric cancer cell line. d RF (resistant factor) is defined as IC50 in SGC7901CDDP/ IC50 in SGC7901. e HUVEC: human umbilical vein endothelial cell line.


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Except above, both 2a and 2b as typical complexes were measured against human umbilical vein endothelial cell (HUVEC). The results showed that the IC50 values of 2a and 2b against HUVEC were 44.34 and 51.26 µM, respectively, higher than the IC50 values for the SGC7901 cancer cell lines. Interestingly, 2a and 2b is less toxic than cisplatin and oxaliplatin towards HUVEC cell line, despite 2a and 2b shows strong cyctotoxity against normal cancer lines (Tables 1 and 2). This result is indicative of a selective toxicity of the complexes for the cancer cells over the normal cell. Cellular Uptake. As the most active complex in the cytotoxicity assay, 2a was then chosen for the cellular uptake test. For the investigation of the configuration-dependent cellular uptake, 2b, the enantiomer of 2a, was also selected. The extent of cellular uptake was measured by treating SGC7901 and SGC7901/CDDP cells with 20 µM of 2a, 2b and oxaliplatin for 12 h. The concentration of platinum in the tumor cells was evaluated by the technique of ICP-MS. As shown in Table 3, the content of 2a taken up by the SGC7901 cells was 1.62 times higher than that of 2b. Interestingly, in the case of SGC7901/CDDP cells, the uptake of 2b by the SGC7901/CDDP cells was significantly higher than 2a. Upon the results from the cytotoxicity and cellular uptake tests, it seems that there might be a positive correlation between these two tests, namely, the enhanced cellular uptake can result in the increase of the cytotoxicity. However, platinum accumulation of our complexes in the tested cells was not always high compatible to its lipophilicity when compared with oxaliplatin. This indicated that there is not a clear correlation between cellular uptake and lipophilicity of the platinum compounds, which has been reported previously.34,35 In spite of the increased amount of platinum content in uptake of 2b in SGC7901/CDDP cells, its ability to overcome cisplatin resistance was not merely dominated by its lipophilicity.36 It is known



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that platinum drug accumulation is probably due to a combination of passive, active and facilitated transport mechanisms.37 Although 2a and 2b are complexes of enantiomeric neutral ligands with the same hydrophobic character, they have different accumulations both in cisplatin-active cells and cisplatin-resistant cells, indicating that the uptake of 2a and 2b might be via a carrier-mediated way. Table 3. Cellular uptake of 2a and 2b in SGC7901 and SGC7901/CDDP cells after 12h of incubation.a

Pt content (nanograms/106 cells)

a

Complex

SGC7901

SGC7901/CDDP

oxaliplatin

75.6 ± 4.5

68.4 ± 3.4

2a

40.8 ± 2.4

63.6 ± 2.8

2b

25.2 ± 1.7

78.0 ± 4.1

Intracellular accumulation of oxaliplatin, 2a and 2b (20 µM) in SGC7901 and

SGC7901/CDDP cells after 12h. Each value shown in the table is in nanograms of platinum per 106 cells. Results are expressed as the mean ± SD for three independent experiments. It is noted that both 2a and 2b showed almost the same intracellular platinum levels as oxaliplatin, but they did not correlate with the cytotoxicity against SGC7901/CDDP cells (Table 2). The uncorrelation between the intracellular platinum levels and the cytotoxicity of complexes may be owing to the fact that the intracellular platinum level is, although important, not the only factor deciding the cytotoxicity of the complexes. Many of other factors, such as the amount of the complexes that finally act on DNA, the inactivation of the


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complexes due to the bounding to GSH, also have influences on the cytotoxicity. Actually, similar results have been already reported before.38,39 Apoptosis Study with Complex 2a. According to the result of cytotoxicity assay, the apoptotic analysis of 2a against HepG-2 and HCT-116 was carried out by an Annexin V-FITC/PI assay, respectively. The tested complexes were incubated with HepG-2 and HCT-116 cells for 24h at a concentration of 50 µM. Q1-Q4 represent four different cell states: necrotic cells, late apoptotic or necrotic cells, living cells, apoptotic cells, respectively (in Figures 3 and 4). As showed in Figure 3, the effect on apoptotic rate of the HepG-2 cell line for 2a is slightly higher than oxaliplatin after 24h incubation. Compared with the IC50 values of 2a (IC50 = 10.74 µM) and oxaliplatin (IC50 = 12.80 µM), the results indicated that the cytotoxicity and apoptosis rate had a positive correlation in HepG-2 tumor cells. As for HCT-116 cell line, 2a showed an apoptosis rate close to that of oxaliplatin. As shown in Figure 4, after treated with 2a (50 µM) for 24 h, the early apoptotic rate increased (from 1.88% to 11.12%) and the late apoptotic rate also increased strikingly (from 3.49% to 30.62%). Oxaliplatin showed a lower population of apoptotic cells than complex 2a. In contrast to the apoptotic rate of the HepG-2 cell line, HCT-116 cell line was relatively sensitive to 2a that own a dicyclic ligand analogous to 1R,2R-DACH in oxaliplatin. Overall, the tested complexes could cause cancer cell death based on an apoptotic pathway.



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Figure 3. Flow cytometric analysis of the distribution of HepG-2 cells treated with complex 2a and oxaliplatin at 50 µM.

Figure 4. Flow cytometric analysis of the distribution of HCT-116 cells treated with complex 2a and oxaliplatin at 50 µM.

Western Blot Analysis. To further understand the mechanism of action of the newly synthesized complexes, three mitochondrial related apoptotic proteins of Bax, Bcl-2 and Caspase-3 were tested in HepG-2 and HCT-116 cells treated with cisplatin, 1a and 2a by the western blot analysis (Figure 5). The result showed that cisplatin, 1a and 2a could increase the ratio of Bax/Bcl-2 proteins both in HepG-2 cells and HCT-116 cells. Moreover, it was noted that both 1a and 2a could effectively decrease the downstream expression of prototype

apoptotic

executioner

Caspase-3

and

increase


the

expression

of

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Cleaved-caspase-3, all of which could trigger apoptosis of tumor cells. It was demonstrated through the results of the expression of apoptosis-regulated proteins that both 1a and 2a could induce a better activation function than cisplatin on mitochondrial-dependent apoptosis pathway since they contain LR as a carrier ligand with steric hindrance. As a highly complex and tightly regulated process, the apoptosis in cells involved many different signaling pathways.40 Among this, the platinum-induced apoptosis was to some extent caused by the mitochondrial-dependent apoptosis pathway.

Figure 5. HepG-2 and HCT-116 cells treated with 50 µM of cisplatin, 1a and 2a for 12 hours were examined for the expression of apoptosis-regulated proteins using western blot analysis. Equal loading was testified by the detection of β-actin. The results were obtained from three independent experiments.

Interaction with pET22b Plasmid DNA. It is well known that the primary target of platinum-based drugs within DNA is the N7 atom of guanine. Thus, in order to study the interaction of the newly synthesized complex with DNA, docking study of 2a into duplex



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DNA (PDB code 3LPV) was performed using MOE software. The result indicates that the [Pt(LR)]2+ fragment is bound to DNA in the major groove owing to the resistance of the dicyclic framework, generating a spatial bulk/unpolar region (Figure 6). Meanwhile, it is noted that 2a can effectively bind to guanines of the DNA via a covalent bond between Pt and the amine groups of guanines. Compared with the former researches,24,33 the interaction mode of 2a has a similar behavior to that of oxaliplatin which contains 1R,2R-DACH as a carrier ligand (Supporting Information, Figure S3). Therefore, it seems to be reasonable to deduce that the dicyclic framework containing 1,2-DACH moiety in our complex can provide certain spatial hindrance and prevents the part of active unit, [Pt(LR)]2+, from the coordination site of DNA.

Figure 6. Docking study of 2a intrastrand cross-link in a DNA dodecamer duplex.

The DNA unwinding ability of complex 2a was tested by agarose gel electrophoresis, in which cisplatin and oxaliplatin were used as positive controls. The gel electrophoretic mobility pattern of plasmid DNA treated with different concentrations of cisplatin, oxaliplatin and complex 2a is shown in Figure 7. The untreated plasmid DNA was used as


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negative control, which mainly consisted of covalently closed circular (Form I) and a small amount of open circle form (Form II) that presented two bands in gel. All platinum compounds were incubated with pET22b plasmid DNA at 37oC for 24h. It can be seen that lanes 1-8 correspond to the DNA distort pattern produced by the tested compounds with concentrations gradient of 0, 10, 20, 40, 80, 160, 320 and 640 µM, respectively. Different concentration of the compounds led to the different change of agarose gel relative electrophoretic mobility (REM) of DNA molecules. With the increased of concentration of cisplatin from 10 to 640 µM, open circular form DNA obviously increased. This can be attributed to the covalently closed circular DNA to open circle form, demonstrating the binding between the Pt(II) and DNA. However, as for complex 2a incubation with plasmid DNA on the same condition, the increase in intensity of the Form II band was different from that of cisplatin, but similar to that of oxaliplatin, hinting the structural similarity between 2a and oxaliplatin. The different effects of 2a on the mobility of plasmid DNA compared to cisplatin can very likely result from different DNA binding ability of these two complexes. By analyzing the results of REM of DNA molecules and the structure of the tested complexes, we can reasonably conclude that the interaction of complex 2a with DNA in a different way from cisplatin is partly owing to the introduction of dicyclic groups like oxaliplatin.



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Figure 7. Gel electrophoretic mobility pattern of pET22b plasmid DNA incubated at 37oC with various concentrations of tested compounds for 24h. The lanes correspond to untreated plasmid DNA (lane 1), concentrations of 10, 20, 40, 80, 160, 320, 640µM of cisplatin(a), oxaliplatin(b) and complex 2a(c) (lanes 2–8), respectively, incubated with DNA.

Kinetics Study. Kinetic property is important for the pharmacological profile of the platinum complexes. 41, 42 In comparison of complex 2a with oxaliplatin, both compounds bearing oxalate as a leaving ligand showed considerable cytotoxicity in vitro. Thus, it is meaningful to study and compare their kinetic reactivity, which can be helpful to learn more about the chemical and biological property of the newly synthesized compounds. In our study, L-methionine (abbreviated as L-Met), a small amino acid molecule, was especially chosen as a nucleophile and used to react with 2a, because such a reaction can be easily monitored by UV-Vis spectroscopy. The reaction was carried out at 37 °C under pseudo-first-order conditions by using at least a 10-fold excess of nucleophile. Hepes buffer was used to control the pH at 7.4. The UV-Vis spectra were used to record the reaction of L-Met with complex 2a and oxaliplatin, respectively (Supporting Information, Figure S4). According to the previous reports,43,44,45 the reaction should occur in two steps.


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However, the kinetic traces at 260 nm for complex 2a and oxaliplatin gave the best reproducible data when they were fitted with single-exponential functions (Supporting Information, Figure S5), which is probably due to the fact that the absorbance changes of the second step were too small to be observed. To our knowledge, the observed reaction was assigned to the ring open reaction, and a linear dependence of kobs on the nucleophile concentration was found, and the results obeyed eq 1 (kobs = k1 + k2[L-Met]) (Figure 8). The value of k2 for complex 2a was a little higher than that of oxaliplatin under the test condition, indicating that the ring open reaction rate of 2a with L-met was faster than that of oxaliplatin. The reaction of oxaliplatin and L-Met has ever been studied with liquid chromatography previously.46 According to the literature report, the degradation rate constant of oxaliplatin was 0.092 M-1S-1 (5.5 M-1 min-1), which agrees reasonably well with our first step reaction rate constant (0.088 M-1S-1).

Figure 8. Plots of kobs vs L-Met concentration for complex 2a and oxaliplatin. T = 310 K, pH = 7.4.

The above result showed that the kinetic reactivity of 2a was slightly improved as



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compared with oxaliplatin due to the application of LR instead of 1R,2R-DACH as a carrier ligand. It may be reasonably deduced that such a kinetic reactivity of 2a could affect the departure rate of the leaving group, oxalate, from the active Pt(II) species.

CONCLUSION. A series of chiral platinum(II) complexes were designed and prepared by applying optical L as a carrier ligand with dicyclic steric hindrance. Biological assay indicated that almost all compounds had cytotoxic activity against the first tested three cancer cell lines. It is noted that most of (R,R)-enantiomeres have stronger cytotoxicity than their (S,S)-counterparts. Particularly, complex 2a has superior or comparable cytotoxic activity to those of cisplatin and oxaliplatin against HCT-116, HepG-2 and A549 cancer cell lines. Further in vitro assay turned out that 2a had potent cytotoxicity against SGC7901 cancer cell line comparable to that of cisplatin or oxaliplatin as expected. However, 2b had remarkably potent cytotoxic activity against the cisplatin resistant SGC7901/CDDP cancer cell line in contrast to 2a and oxaliplatin with a small RF of 0.85. The RF difference between 2a and 2b resulted from their configurations, which means that the trans-(S,S) enantiomer is much biologically sensitive to the SGC7901/CDDP cancer cell line. Interestingly, 2a shows stronger cyctotoxity than 2b against normal cancer cell lines, but it is less cytotoxic than cisplatin and oxaliplatin towards HUVEC cell line. Due to the structural similarity to oxaliplatin, the tested complexes could cause cancer cell death based on an apoptotic pathway. Western blot study indicated that the induced apoptosis by the typical complex was to some extent caused by the mitochondrial-dependent apoptosis pathway. Interaction of the platinum complex with DNA showed that the dicyclic framework containing 1,2-DACH moiety in the complex can provide certain spatial


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hindrance, which is similar to oxaliplatin. Furthermore, kinetic study on complex 2a demonstrated that the application of LR instead of 1R,2R-DACH as a carrier ligand can accelerate the reaction rate of 2a. Overall, the introduction of dicyclic framework on 1,2-DACH, which has brought about increased lipophilicity, slightly improved kinetic reactivity and structurally different DNA adducts, can increase the antitumor activity of the resulting Pt(II) complex and partly overcome cisplatin resistance due to its steric hindrance. EXPERIMENTAL SECTION Materials and Instruments. All chemicals and solvents were of analytical reagent grade and were used without further purification. Potassium tetrachloroplatinate(II) was obtained from a local chemical company (Lingfeng Chemical Ltd.). Human cancer cell lines including SGC7901/CDDP were obtained from Nanjing KeyGEN BioTECH company. Elemental analyses for C, H, and N were done on a Vario MICRO CHNOS Elemental Analyzer (Elementar). The specific optical rotations of all complexes were recorded on a WZZ-2A Automatic Polarimeter. Infrared spectra were recorded on KBr pellets on a Nicolet IR200 FT-IR spectrometer in the range of 4000-400 cm-1. 1H NMR and

13

C NMR spectra

were recorded in CDCl3 and d6-DMSO with a Bruker 300 or 500 MHz spectrometer. Mass spectra were measured on an Agilent 6224 TOF LC/MS instrument. UV/Vis spectra and kinetic traces were recorded on Shimadzu UV1700 equipped with a thermostatic controlled cell holder. CD spectra of the powders were measured on Jasco J-810 Spectropolarimeter. Platinum contents were measured on an Optima 5300DV ICP-MS instrument. Ligand LS: trans-bicyclo[2.2.2]octane-7S,8S-diamine. LS was prepared by following the



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procedure described by Moteki et al.30 White powder, yield: 1.41g (26%). [α]D30 = -68o (c = 1.46, MeOH; 99.26% ee (Supporting Information, Figure S1)); IR(KBr, cm-1): 3403(νN-H, m)，3385(νN-H, m), 2926m, 2856m, 1574 m, 1465m, 1350m, 959w, 815w. 1H NMR(500 MHz, CDCl3): δ1.30-1.36(m, 2H, CH2CHCH2), 1.39(s, 2H, CH2CHCH2), 1.48(s, 4H, CHNH2), 1.50-1.55(m, 4H, CH2CHCH2), 1.65-1.71(m, 2H, CH2CHCH), 2.48(s, 2H, CHNH2). ESI-MS: m/z [M+H]+ = 141.

Ligand LR: trans-bicyclo[2.2.2]octane-7R,8R-diamine. The procedure for preparing LR was similar to that of LS, using (2R, 3R)-dibenzoyl tartaric acid as chiral selectors. White powder, yield: 1.36g (25%). [α]D30=67o(c=1.44, MeOH; 98.02% ee) (Supporting Information, Figure S1)); IR (KBr, cm-1): 3403(νN-H, m)，3384(νN-H, m), 2924m, 2854m, 1574m, 1463m, 1348m, 957 w, 815w. 1H NMR (500 MHz, CDCl3): δ1.30-1.35(m, 2H, CH2CHCH2), 1.38(s, 2H, CH2CHCH2), 1.47(s, 4H, CHNH2), 1.49-1.54(m, 4H, CH2CHCH2), 1.64-1.71(m, 2H, CH2CHCH), 2.48(s, 2H, CHNH2). ESI-MS: m/z [M+H]+ = 141. Preparation of Complexes 1a and 1b. Under nitrogen and protection from light, an aqueous solution (25 mL) of K2PtCl4 (2.07 g, 5 mmol) was added to LR (0.70 g, 5 mmol) in water (5 mL). The reaction mixture was then stirred at room temperature for 10h and yellow solids deposited. The product was filtered off, washed with ethanol and distilled water, and then dried in vacuum (yield 89%). Complex 1b was obtained by following the same procedure.

Complex 1a: yellow powder, yield: 2.43g (89%). Elem anal. Calcd for C8H16Cl2N2Pt: C,


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23.66; H, 3.98; N, 6.90. Found: C, 23.41; H, 4.25; N, 7.28. IR (KBr, cm-1): 3279(νN-H, m), 3194(νN-H, m), 2939m, 2869m, 1569m, 477w, 411w; 1H NMR(300 MHz, d6-DMSO): δ 1.19-1.77(m, 8H, CH2 of LR), 1.80-1.91(m, 2H, CH of LR), 2.53(d, 2H, J = 5.2 Hz, 2×NH2CH), 5.80-6.51(m, 4H, 2×CHNH2); 13C NMR(d6-DMSO/TMS, ppm): δ19.15, 19.40, 25.99, 26.03, 31.24, 63.10, 64.07.

Complex 1b: yellow powder, yield: 2.45g (90%). Elem anal. Calcd for C8H16Cl2N2Pt: C, 23.66; H, 3.98; N, 6.90. Found: C, 23.72; H, 3.83; N, 7.16. IR (KBr, cm-1): 3278(νN-H, m), 3193(νN-H, m), 2939m, 2868m, 1569m, 480w, 412w; 1H NMR(300 MHz, d6-DMSO): δ 1.23-1.50(m, 6H, CH2 of LS), 1.72-1.80(m, 4H, CH of LS), 2.52(d, 2H, J = 5.2 Hz, 2×NH2CH), 5.82-6.22(m, 4H, 2×CHNH2); 13C NMR(d6-DMSO/TMS, ppm): δ19.13, 19.38, 25.98, 26.03, 31.25, 31.27, 63.20, 64.08.

General Synthesis of Complexes 2a-4b. A mixture of complex 1a or 1b (1 mmol) and silver dicarboxylate (1 mmol) in distilled water (80 mL) was heated to 50°C and stirred for 24h in the dark under nitrogen and protection from light. After the mixture was cooled to the room temperature, precipitated white solid (AgCl) was removed by filtration. The filtrate was concentrated to 10 mL by a rotatory evaporator at 45°C and then kept cool at 5°C for 6h. White crystals were filtered off, washed with a small quantity of chilled water, and then dried at 30°C in vacuum. Greater than 95% purity of complexes 2a-4b was confirmed by elemental analysis. Complex 2a: white powder, yield: 0.30g (71%). [α]D30=140.0o (c = 0.20, DMF:H2O = 1:1). Elem anal. Calcd for C10H16N2O4Pt: C, 28.37; H, 3.81; N, 6.62. Found: C, 28.63; H,



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3.57; N, 6.83. IR (KBr, cm-1): 3274(νN-H, m), 3131(νN-H, m), 2943m, 2872m, 1704s, 1660s, 1589m, 1372s, 1153w, 807w; 1H NMR(300 MHz, d6-DMSO): δ1.17-1.65(m, 8H, CH2 of LR), 1.69-1.72(m, 2H, CH of LR), 2.26(d, 2H, J = 9.0 Hz, 2×NH2CH), 5.31-5.69(m, 4H, 2×CHNH2); 13C NMR(d6-DMSO/TMS, ppm): δ19.22, 25.99, 31.17, 64.46, 165.87; ESI-MS: m/z [M+Na]+ = 446 (100%). Complex 2b: white powder, yield: 0.33 (72%).[α]D30=-137.4o (c = 0.20, DMF : H2O = 1:1) Elem anal. Calcd for C10H16N2O4Pt: C, 28.37; H, 3.81; N, 6.62. Found: C, 28.58; H, 3.63; N, 6.76. IR (KBr, cm-1): 3271(νN-H, m), 3129(νN-H, m), 2948m, 2891m, 1709S, 1658S, 1593m, 1375S, 1147w, 813w; 1H NMR(300 MHz, d6-DMSO): δ 1.21-1.45(m, 6H, CH2 of LS), 1.63-1.70(m, 4H, CH2 of LS), 2.27(d, 2H, J = 9.0 Hz, 2×NHCH2), 5.30-5.69(m, 4H, 2×CHNH2); 13C NMR(d6-DMSO/TMS, ppm): δ19.19, 25.96, 31.14, 64.44, 165.83; ESI-MS: m/z[M+Na]+= 446 (100%). Complex 3a: white powder, yield: 0.29g (68%). [α]D30=102.7o (c = 0.22, DMF : H2O = 1:1). Elem anal. Calcd for C11H18N2O4Pt: C, 30.21; H, 4.15; N, 6.41. Found: C, 30.46; H, 3.88; N, 6.63. IR (KBr, cm-1): 3219(νN-H, m), 3110(νN-H, m), 2946m, 2870m, 1648S, 1392m, 1369S, 968w, 749w; 1H NMR(300 MHz, d6-DMSO): δ1.14-1.47(m, 8H, CH2 of LR), 1.66-1.73(m, 2H, CH of LR), 2.32(d, 2H, J = 9.0 Hz, 2×CHNH2), 3.20(s, 2H, CH2(COO)2), 5.24-5.52(m, 4H, 2×CHNH2)；13C NMR(d6-DMSO/TMS, ppm): δ19.19, 25.96, 31.12, 50.22, 64.62, 174.00; ESI-MS: m/z [M+H]+= 438(100%). Complex 3b: white powder，yield：0.30g (69%). [α]D30=-103.8o (c = 0.22, DMF:H2O = 1:1). Elem anal. Calcd for C11H18N2O4Pt: C, 30.21; H, 4.15; N, 6.41. Found: C, 30.55; H,


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3.91; N, 6.59. IR (KBr, cm-1): 3218(νN-H, m), 3114(νN-H, m), 2947m, 2872m, 1647S, 1395m, 1367S, 971w, 750w; 1H NMR(300 MHz, d6-DMSO): δ1.16-1.44(m, 6H, CH2 of LS), 1.60-1.70(m, 4H, CH2 of LS), 2.35(d, 2H, J = 9.0 Hz, 2×NHCH2), 3.21(s, 2H, CH2(COO)2), 5.24-5.51(m, 4H, 2×CHNH2); 13C NMR(d6-DMSO/TMS, ppm): δ19.17, 25.93, 31.10, 50.16, 64.60, 173.98; ESI-MS: m/z[M+H]+= 438(100%). Complex 4a: white powder, yield: 0.32g (67%).[α]D30=122.8o (c = 0.25, DMF:H2O = 1:1). Elem anal. Calcd for C14H22N2O4Pt: C, 35.22; H, 4.64; N, 5.87. Found: C, 34.97; H, 4.43; N, 6.13. IR (KBr, cm-1): 3209(νN-H, m), 3117(νN-H, m), 2921m, 2868m, 1643S, 1605s, 1361S, 905w; 1H NMR(300 MHz, d6-DMSO): δ 1.18-1.68m(m, 12H, CH and CH2 of LR and CH2 of cyclobutane), 2.28(d, 2H, J = 9.0 Hz, 2×NHCH2), 2.68(t, 4H, J = 6.0 Hz, 2×CH2 of cyclobutane), 5.13-5.47(m, 4H, 2×CHNH2);

13

C NMR(d6-DMSO/TMS, ppm): δ15.01,

19.23, 25.99, 30.31, 31.19, 55.52, 64.73, 177.36; ESI-MS: m/z [M+H]+ = 478 (100%). Complex 4b: white powder, yield: 0.31g (66%). [α]D30=-119.6o (c = 0.25, DMF:H2O = 1:1)Elem anal. Calcd for C14H22N2O4Pt: C, 35.22; H, 4.64; N, 5.87. Found: C, 35.01; H, 4.91; N, 5.72. IR (KBr, cm-1): 3208(νN-H, m), 3116(νN-H, m), 2923m, 2866m, 1644S, 1606s, 1362S, 906w; 1H NMR(300 MHz, d6-DMSO): δ 1.19-1.69(m, 12H, CH and CH2 of LS and CH2 of cyclobutane), 2.27(d, 2H, J = 9.0 Hz, 2×NHCH2), 2.71(t, 4H, J = 6.0 Hz, CH2 of cyclobutane), 5.15-5.47(m, 4H, 2×CHNH2);

13

C NMR(d6-DMSO/TMS, ppm): δ14.90,

19.21, 25.97, 30.29, 31.17, 55.51, 64.71, 177.33. ESI-MS: m/z[M+H]+ = 478 (100%).

Cell Culture. All adherent cell lines including human colorectal carcinoma cell line (HCT-116), hepatocellular carcinoma cell line (HepG-2), non-small cell lung cancer cell



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line (A549), gastric cancer cell line (SGC7901), cisplatin-resistant gastric cancer cell line (SGC7901/CDDP) and human umbilical vein endothelial cell line (HUVEC),

were

cultured in a humidified, 5% CO2 atmosphere at 37°C, and maintained in monolayer culture in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), 100 mg/mL of streptomycin and 100 mg/mL of penicillin.

Cytotoxicity Analysis (IC50). MTT assay: the cells were seeded in 96-well tissue cultured plates at a density of 5000 cells/well. After overnight incubation, the cells were treated with the diluted solution of complexes, which were obtained by dissolving in DMF and diluting with culture medium (DMF final concentration < 0.4%). After 72 h of incubation at 37oC, 10 µL of a freshly diluted 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) solution (5 mg/mL) were added to each well and the plate was incubated at 37 °C in a humidified 5% CO2 atmosphere for 4 h. At the end of the incubation period the medium was removed and the formazan product was dissolved in 150 µL of DMSO. The cell viability was evaluated by measurement of the absorbance at 490 nm, using an Absorbance Reader (Bio-Rad). IC50 values (compound concentration that produces 50% of cell growth inhibition) were calculated from curves constructed by plotting cell survival inhibitory rate (%) versus drug concentration logarithm. All experiments were made in quintuplication. The reading values were converted to the percentage of control (% cell survival). Cytotoxic effects were expressed as IC50 values. Cellular uptake test. SGC-7901 and SGC-7901/CDDP cells were seeded in 6-well plates. After the cells reached about 80% confluence, 20 µM of oxaliplatin, 2a or 2b was added, respectively. After 12 h incubation, cells were collected and washed 3 times with ice-cold


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PBS, then centrifuged at 1000×g for 10 min and resuspended in 1 mL PBS. A volume of 100 µL was taken out to determine the cell density. The rest of the cells was spun down and digested at 65oC in 200 µL 65% HNO3 for 10 h. The Pt level in cells was measured by ICP-MS. Cell Apoptosis Study by Flow Cytometry. Apoptosis induced by platinum complexes was tested by flow cytometry using an Annexin V–FITC Apoptosis Detection Kit (Roche, USA) according to the manufacturer’s instructions. After induced apoptosis of selected cell lines (HepG-2 and HCT-116) by the addition of 50 µM selected compounds and oxaliplatin (50 µM) (positive controls) for 24 h, cells were collected by centrifugation (5 min, 25 oC, 2000 rpm). The cells were then washed twice with cold water and resuspended in Annexin V–FITC binding buffer at a concentration of 1×106 cells mL-1. Cells were stained with 5 mL Annexin V–FITC and incubated in the dark at 25 oC for 10 min. The cell suspension was centrifuged for 5 min (25oC, 2000 rpm) and cells were resuspended in Annexin V–FITC binding buffer. Propidium iodide (10 mL) was added, and the tubes were placed on ice, away from light. The fluorescence was measured using a flow cytometer (FACScan, Becton Dickson, USA). The results were analyzed by FCSExpress software and were expressed as the percentage of normal and apoptotic cells at various stages. Western Blot Analysis. HepG-2 and HCT-116 cells (5 × 105 cells) were seeded in their culture flasks and incubated until 90% confluent. The cells were treated with platinum complexes at concentration of 50 µM for 12 h. The dead cells and media were aspirated and the rest were washed with ice cold PBS (phosphate buffered saline). A solution of 150µL of lysis buffer (100 mM Tris-Cl, pH 6.8, 4%(m/v) sodium dodecylsulfonate, 20% (v/v) glycerol, 200 mM β-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride, and 1 g/mL aprotinin) was added to each one. The cells were scrapped and the lysate was collected. The cell lysate was homogenized by passing through a syringe with a needle for 15 to 20 times



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on ice and allowed to sit on ice for 15 min, centrifuged at 4 °C and 13,200 RPM for 15 min. The supernatant was harvested. The protein concentration was quantified by BCA (bicinchoninic acid) protein assay (Peirce, Rockford, IL) and adjusted to equal concentration. The samples (20µg/lane) were separated by SDS-PAGE using an 8–16% Tris-Glycine gel (Invitrogen) and followed by transferring proteins onto a nitrocellulose membrane. The membrane was blocked with 5% nonfat dry milk in TBST (Tris buffered saline plus 0.1% Tween 20) for 1 h and then incubated with primary antibody (1:2000 for β-actin, Bax, Bcl-2, Caspase-3, Santa Cruz, USA) at 4 °C overnight. After washing with PBST for three times, the membranes were incubated for 1 h with an IRDye™ 800 conjugated secondary antibody diluted 1:20000 in PBST, and the labeled proteins were detected with an Odyssey Scanning System (Li-COR., Lincoln, Nebraska, USA). Gel Electrophoresis Study. According to the reported method,25 interaction of platinum(II) complexes (cisplatin, oxaliplatin and complex 2a) with pET22b plasmid DNA was tested by agarosegel electrophoresis. Cisplatin and oxaliplatin were used as positive control，and pET22b plasmid DNA of 50 ng/µL was used as the target in the experiment. Appropriate dilutions of the tested compounds were made, and the required volumes of solutions were added to achieve a set of concentrations in the range of 0-640 µM; pET22b DNA (5 µL) was added to each tube, and the mixtures of platinum complexes and pET22b plasmid DNA were then incubated at 37°C for 24h. Afterward, the agarosegel (made up to 1% w/v) was prepared by TA buffer (50 mM Tris-acetate, pH 7.5). The mixtures with loading buffer (1 µL) underwent electrophoresis in agarose gel in TA buffer at 90V for 80min. Bands were imaged using a Molecular Imager (Tanon, China) under UV light. Kinetics Study. The kinetic reactions were studied by observing the change in absorbance


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at suitable wavelengths under pseudo-first-order conditions (at least 10-flod excess of L-Met over the platinum(II) complexes). A suitable wavelength for kinetic trace was determined by recording spectra of the reaction mixture over the wavelength range 200-500 nm, and 260 nm was selected to observe for all the kinetics measurement. The reactions were studied at 37 ± 0.1oC and pH 7.4 (Hepes, 0.25 M) in the presence of 0.1 M NaClO4.

Supporting Information. HPLC purity of LR and LS; CD spectra of 1a and 1b in powders; Crystal structure of an oxaliplatin 1,2-d(GpG) intrastrand cross-link in a DNA Dodecamer duplex. Time-dependent UV-vis spectra recorded for the reaction of platinum complexes with L-Met at different times. Absorbance-time trace recorded for the reaction of complex 2a with L-Met. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. *E-mail:[email protected] ACKNOWLEDGMENTS The authors appreciate the reviewers so much for their significant and helpful suggestions and comments on our manuscript. We are grateful to the National Natural Science Foundation of China (Project 21272041), the Fundamental Research Funds for the Central Universities (Project 2242013K30011), and the New Drug Creation Project of the National Science and Technology Major Foundation of China (Project 2013ZX09402102-001-006)



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for financial aids to this work. We would like to thank the State Key Laboratory of Natural Medicines at China Pharmaceutical University for use of MOE software.

ANCILLARY INFORMATION DACH

diaminocyclohexane

Form I

covalently closed circular DNA

Form II

open circlar DNA

L

trans-bicyclo[2.2.2]octane-7,8-diamine

LR

trans-bicyclo[2.2.2]octane-7R,8R-diamine

LS

trans-bicyclo[2.2.2]octane-7S,8S-diamine

REM

relative electrophoretic mobility

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Study on Antitumor Platinum(II) Complexes of Chiral Diamines with

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