Design, Synthesis, and Biological Features of Platinum(II) Complexes

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Design, Synthesis, and Biological Features of Platinum(II) Complexes with Rigid Steric Hindrance Zhimei Wang,†,‡ Haiyan Yu,†,‡ Shaohua Gou,*,†,‡ Feihong Chen,†,‡ and Lei Fang*,†,‡ †

Pharmaceutical Research Center and School of Chemistry and Chemical Engineering and ‡Jiangsu Province Hi-Tech Key Laboratory for Biomedical Research, Southeast University, Nanjing 211189, China S Supporting Information *

ABSTRACT: A series of platinum(II) complexes, with Nmonosubstituted 1R,2R-diaminocyclohexane bearing methoxysubstituted benzyl groups as carrier ligands, were designed and synthesized. The newly prepared compounds, with chloride anions as leaving groups, were found to be very active against the tested cancer cell lines, including a cisplatin-resistant cell line. Despite their efficacy against tumor cells, they also showed low toxicity to a human normal liver cell line. Among them, complex 1 had superior cytotoxic activity against A549, HCT116, MCF-7, SGC7901, and SGC7901/CDDP cancer cell lines. The DNA binding assay is of further special interest, as an unusual monofunctional binding mode was found, due to the introduction of a rigid substituted aromatic ring in the 1R,2R-diaminocyclohexane framework as steric hindrance. The linkage of complex 1 with DNA was stable and insensitive to nucleophilic attack. Moreover, studies including cellular uptake, gel electrophoresis, apoptosis and cell cycle, and Western blot analysis have provided insight into the high potency of this compound.

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can be repaired by enzymes.8 So it is possible to promote antitumor activity of the platinum drugs by enhancing formation of stable bifunctional adducts with DNA. From the viewpoint of steric hindrance, the nature of the amine carrier ligand has a strong influence on the stability of the platinum complexes.4b−d Platinum(II) complexes bearing only one labile ligand and forming one bond to the DNA nucleobase are defined as monofunctional complexes, which show significant cellular response different from the classic bifunctional, charge-neutral platinum-based drugs.4a,9 Recently, monofunctional platinum(II) complexes have attracted much attention, due to their unique antitumor features. To improve the potency and cellular uptake of monofunctional platinum(II) complexes, various bulky groups have been introduced to the ligands. Structural studies suggested that ligands containing bulky species, like aromatic rings, are crucial for the anticancer activity of monofunctional platinum(II) complexes,9 due to their ability to intercalate into DNA base pairs. Structural modification of monofunctional platinum(II) complexes, with bulky steric groups, would give rise to greater efficacy than cisplatin and oxaliplatin in established human cancer cell lines. Thus, it can be concluded that the steric hindrance group has a strong influence on the activity of both monofunctional and bifunctional platinum complexes, which might be a key factor to reduce resistance compared to cisplatin. So far, a number of

INTRODUCTION Since platinum(II) has a high affinity for sulfur, after administrating a platinum-based drug in the human body, there is a strong possibility for nucleophilic attack by S-donor biomolecules, which are present in large quantities in the form of peptides, proteins, and enzymes.1 Thus, the toxic effects are mostly caused by the binding of platinum complexes with Scontaining biomolecules.2 One of the important issues in platinum drug design is to maintain the activity of the platinum complex before it reaches the DNA target. The drug delivery systems might be able to specifically target cancer cells and release the active platinum complexes in a controlled fashion.3 But at present, the pharmacological use of new therapeutics is often limited by a safe and effective drug-delivery system. Except in drug delivery based systems, introduction of a steric hindrance group to a ligand has become a useful strategy for the convenient and efficient synthesis of platinum compounds that may avoid the inactivity of platinum drugs.4 It is believed that the interaction of platinum anticancer drugs with DNA is by either a reversible or an irreversible mechanism, and the reversible binding mode is sensitive to nucleophilic attack.5 According to the binding mode with DNA, platinum(II) compounds can be divided into two classes: bifunctional and monofunctional. Bifunctional platinum(II) complexes, such as cisplatin, exert antitumor activities through intrastrand and/or interstrand linkages with DNA.6 Mechanistic studies suggest that, due to the small size of the ammine, the linkages of cisplatin and its analogues with DNA are very sensitive to nucleophilic attack,7 and the interstrand linkages © XXXX American Chemical Society

Received: February 12, 2016

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DOI: 10.1021/acs.inorgchem.6b00361 Inorg. Chem. XXXX, XXX, XXX−XXX

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Inorganic Chemistry Scheme 1. Preparation of the Ligand

proposed. The specific optical rotations of the ligands were lefthanded, as that of 1R,2R-DACH. A routine reaction, between K2PtCl4 and the corresponding ligand in water, generated complexes 1−3, respectively. Removing chloride anions from complexes 1−3 with the corresponding silver dicarboxylate resulted in the production of complexes 4−15 (Scheme 2). All complexes were spectrally characterized by 1H and 13C NMR spectroscopy, as well as ESIMS spectroscopy.

different-sized alkyl moieties have been successfully rendered for steric hindrance in the ligands derived from trans-1,2diaminocyclohexane (DACH) in our group.10 The related studies indicated that a few representative platinum(II) complexes with these alkyl groups have good antitumor activity and possess cellular responses different from those of cisplatin and oxaliplatin.11 However, these linear or cyclic alkyl groups attached to the DACH framework are flexible, relative to the coordination plane around the metal atom, which may lead to the formation of stereoisomers as well. So the substitution of a flexible group for a rigid one seems necessary. In this paper, a benzyl group with a methoxy substituent has been selectively introduced to the ligand containing the DACH skeleton as rigid steric resistance (Scheme 1). The benzyl group, as a good πconjugated unit, is insusceptible to biological nucleophiles with its rigid skeleton, and the appending methoxy group on the phenyl ring is a popular substituent in drugs with significant biological features. In this way, three new ligands (Figure 1)

Scheme 2. Synthetic Steps To Prepare Platinum(II) Complexes 1−15

In the 1H NMR spectra, the proton signals of the cyclohexyl moiety were found in the range of 0.77−2.20 ppm, and signals of C−H groups connecting to the amino groups occurred between 2.11 and 2.90 ppm. The chemical shift of the methylene group (NHCH2Ar) was between 3.12 and 4.31 ppm, and signals at 6.41−8.21 ppm were due to the phenyl ring. Compared with the corresponding ligand, the chemical shifts of alkyl species on DACH of the complexes moved to a higher field. The chemical shift of the methyl group was between 3.63 and 3.83, upfield shifted in comparison to that of the corresponding free ligand (3.81−3.92 ppm). Also, the signals of the methylene group (NHCH2Ar) moved upfield, compared with the corresponding ligand (4.14−4.71 ppm). 13C NMR peaks of all complexes were compatible to the proposed chemical structures. In the ESI-MS spectra, 100% [M − Cl]+ was detected in complexes 1−3 and 100% [M − H]− was discovered in complexes 4−15, which were composed of a few isotopic peaks owing to the presence of platinum isotopes, suggesting the bonding between Pt(II) ions and the ligands. The specific optical rotations of complexes were left-handed. As reported formerly,11a−d the S configuration at the N1 atom of the complex with a pending benzyl group was more

Figure 1. Chemical structures of ligands.

have been obtained, which were used to prepare a series of platinum(II) complexes with chloride anions or dicarboxylates as leaving groups (Figure 2). The resulting platinum(II) complexes have been biologically assayed against a few human cancer cell lines, including a cisplatin-resistant cancer cell line. Furthermore, chemical and biological properties of typical compounds have been investigated to explore the mechanism of action, in addition to the function of steric resistance resulting from the ligand.

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RESULTS AND DISCUSSION Synthesis and Characterization. The ligands (L1−L3), prepared by following the procedure shown in Scheme 1, were characterized by microanalysis, ESI-MS, and 1H and 13C NMR spectra, which were consistent with the chemical structure

Figure 2. Chemical structures of the synthesized platinum(II) complexes. B

DOI: 10.1021/acs.inorgchem.6b00361 Inorg. Chem. XXXX, XXX, XXX−XXX

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Inorganic Chemistry thermodynamically stable than the corresponding R configuration, resulting from a steric effect of the ligand according to molecular calculation, which has also been proven by the crystallographic data of analogous metal complexes.11a,e In Vitro Cytotoxicity. The cytotoxic activity of complexes 1−15 against three cancer cell lines was first evaluated in vitro by means of an MTT assay with cisplatin and oxaliplatin as references. The corresponding IC50 values are shown in Table 1. From the cytotoxicity profiles, complex 1 was the most

oxaliplatin, complex 2 was more active against A549 (11.71 μM) and MCF-7 (9.15 μM), and less active against HCT-116 (9.46 μM). Complex 3 had a higher cytotoxic activity than oxaliplatin against the A549 cell line, while its cytotoxicity against MCF-7 cells was the same order of magnitude as that of oxaliplatin. Therefore, the synthesized complexes, with chloride anions as leaving groups, had strong anticancer activity, comparable or even superior to those of cisplatin and oxaliplatin against A549, MCF-7, and HCT-116 cell lines. However, complexes 4−15, with the leaving groups of chloride anions replaced by dicarboxylates, showed moderate or low cytotoxicity compared to their precursors, except that complex 8 had nearly the same cytotoxicity as oxaliplatin against the MCF-7 cell line. On the basis of the results above, the preferred complexes 1− 3 were selected for further research on LO2, SGC7901, and SGC7901/CDDP cell lines. The results are presented in Table 2. It is noted that, compared with cisplatin and oxaliplatin, complexes 1 and 2 were less toxic toward human normal cell line, but they showed stronger cytotoxicity against SGC7901 and SGC7901/CDDP, which indicated a selective toxicity of the complexes for the cancer cells over the normal cell. Meanwhile, complex 3 was more toxic toward the LO2 cell line than oxaliplatin, but less toxic than cisplatin. Its cytotoxic activity on SGC7901 was almost as potent as that of oxaliplatin, but it was less potent than cisplatin. Moreover, complexes 1−3 had the potential to overcome cisplatin resistance, because their resistance factors were in the range of 0.59−0.91 (RF < 2 can be regarded as non-cross-resistant).12 By analyzing the structure−activity relationship, it was learned that the leaving ligand had an important influence on antitumor activity. Compared with compounds with chloride anions as leaving groups, most of the other complexes with dicaroxylates did not show potent antitumor activity, which might be due to the fact that the departure rate of dicaroxylates chelating the metal atom was slower than that of the monodentate chloride anions. It was also noticed that the substituent (methoxy group) on the ortho- and meta-positions had significantly better cytotoxic activity than the para one. It has been extensively demonstrated that the nature of the amine carrier ligands has a strong influence on the activity of platinum complexes.13 The interaction of cisplatin with biological molecules has been found to be one of the key factors for its serious toxicity. The high stability of the DACH− platinum fragment in oxaliplatin leads to a significantly different reaction behavior from that of cisplatin. In this study, by attaching the methoxy-substituted benzyl group to the 1,2DACH framework, the resulting platinum(II) complexes 1−3

Table 1. In Vitro Cytotoxicity of Complexes 1−15 against Human Tumor Cell Lines IC50 (μM)a complex

A549b

HCT-116c

MCF-7d

cisplatin oxaliplatin 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

4.20 ± 0.35 17.17 ± 1.08 2.17 ± 0.12**,## 11.71 ± 0.94## 14.83 ± 1.38 >200 >200 37.06 ± 2.98 41.70 ± 3.12 35.97 ± 2.87 42.75 ± 3.27 >200 108.42 ± 9.21 >200 64.35 ± 4.65 >200 136.33 ± 12.11

8.71 ± 0.76 5.36 ± 0.32 6.02 ± 0.58 9.46 ± 0.64 36.15 ± 2.87 101.20 ± 9.39 >200 42.21 ± 3.70 118.44 ± 11.29 21.53 ± 1.68 >200 >200 >200 >200 40.60 ± 3.67 93.25 ± 7.23 >200

3.46 ± 0.26 10.05 ± 0.89 0.80 ± 0.04**,## 9.15 ± 0.78 16.19 ± 1.02 74.24 ± 5.98 89.22 ± 6.89 32.04 ± 2.67 47.47 ± 3.39 16.05 ± 1.03 >200 >200 >200 121.86 ± 10.22 39.25 ± 2.63 >200 42.59 ± 3.69

a

IC50 is the drug concentration effective at inhibiting 50% of the cell growth measured by the MTT assay after 72 h of drug exposure expressed as the mean value ± standard deviation of three independent experiments (*P < 0.05, **P < 0.01, compared with cisplatin; #P < 0.05, ##P < 0.01, compared with oxaliplatin). bA549 is human non-small-cell lung cancer cell line. cHCT-116 is human colorectal cancer cell line. dMCF-7 is human breast carcinoma cell line.

effective agent among the synthesized compounds. Its IC50 value for the MCF-7 cell line was determined to be 0.80 μM, which is up to 4-fold superior to cisplatin (3.46 μM) and 12fold superior to oxaliplatin (10.05 μM). Its IC50 value for the A549 cell line was 2.17 μM, which is up to 2-fold superior to cisplatin (4.20 μM) and 8-fold superior to oxaliplatin (17.17 μM), while its cytotoxicity against the HCT-116 cell line (6.02 μM) was nearly the same as that of oxaliplatin (5.36 μM), but a bit higher than that of cisplatin (8.71 μM). Compared with

Table 2. In Vitro Cytotoxicity of Complexes toward LO2, SGC7901, and SGC7901/CDDP Cell Lines IC50(μM)a complex

LO2b

SGC7901c

cisplatin oxaliplatin 1 2 3

7.47 ± 0.51 16.95 ± 1.23 18.31 ± 1.04**,## 23.10 ± 2.05**,## 14.75 ± 0.89*

2.14 ± 0.17 14.30 ± 1.05 3.21 ± 0.28## 8.77 ± 0.36## 17.77 ± 1.14

SGC7901/CDDPd

RFe

12.61 ± 1.20 9.87 ± 0.87 2.93 ± 0.42**, 8.51 ± 0.59# 10.48 ± 1.01

5.89 0.69 0.91 0.97 0.59

#

a IC50 is the drug concentration effective in inhibiting 50% of the cell growth measured by the MTT assay after 72 h of drug exposure expressed as the mean value ± standard deviation of three independent experiments (*P < 0.05, **P < 0.01, compared with cisplatin; #P < 0.05, ##P < 0.01, compared with oxaliplatin). bLO2 is normal human liver cell line. cSGC7901 is human gastric cancer cell line. dSGC7901/CDDP is human gastric cancer cisplatin resistant cell line. eResistance factor (RF) is IC50 resistant/IC50 sensitive.

C

DOI: 10.1021/acs.inorgchem.6b00361 Inorg. Chem. XXXX, XXX, XXX−XXX

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Inorganic Chemistry

from the significant DNA bending at 1,2-intrastrand cross-links that occurs following treatment with bifunctional platinum agents.6a This might be the reason that the rate of migration for DNA treated with complex 1 was slower than that for cisplatin, when interacting with pET22b plasmid DNA. As mentioned above, the critical structural features of monofunctional platinum complexes, like aromatic rings, were used as an intercalator to DNA. Except the intercalator, the linker chain was selected to favor a DNA binding mechanism in which the two components act in a functionally interdependent manner.16 Therefore, the linker was designed to be flexible enough to allow strainless dual binding. However, the benzyl group of complex 1, a rigid one without a flexible linker, could not intercalate into DNA base pairs, but it provided a limited spacing between platinum and nucleophiles. Moreover, hydrogen bond interactions might be involved in the binding with DNA via O atom from the methoxy substituent with H atoms from NH and/or OH moieties of DNA. Thus, it is proposed that the substituent position on the phenyl ring can provide the appropriate steric hindrance and hydrogen bond effect, which may explain why the complex with a substituent on the orthoor meta-position had better cytotoxic activity than the parasubstituted one. To learn more about the chemical and biological properties of the new kind of monofunctional platinum complexes, the reaction of complex 1 with GMP in the presence of GSH was carried out. A biological molecule such as GSH is present in concentrations of 0.5−10 mM in most cells, and it does interact with the platinum compound.17 High levels of intracellular glutathione, which was in cisplatin-resistant cells, have been found to be correlated with low platinum activity.18 So GSH was selected as a S-donor nucleophile to investigate the stability of the adduct with DNA. The reaction of complex 1 with GMP in the presence of GSH was monitored by HPLC and LC−MS, and typical diagrams are shown in Figure S5 (SI). Besides the major product I, the reaction in the presence of GSH gave rise to product II, as detected by HPLC. Positive ESI-MS analysis of II (Figure S6, SI) showed a doubly charged cationic peak at m/z 763.20175, corresponding to the molecular formula of [C48H73N12O16PSPt2]2+, which can be ascribed to the [Pt(L1)]2+ fragment binding with one GMP and one GSH. This assignment was also confirmed by theoretic molecular weights and isotopic distribution patterns. The DNA−Pt−protein complexes, like adduct II, have been reported in the reaction of cisplatin with peptide−nucleotide hybrids, but no data are available on their biological significance.9c Since the purpose of this reaction was to investigate the stability of the adduct with DNA, a concentration change of the products was achieved (Figure 4). As shown in Figure 4a, the binding of complex 1 to GMP was very rapid within the first day, and then the reaction rate slowed down gradually. The phenomenon was almost the same in the reaction of complex 1 with both GMP and GSH (Figure 4b), except the concentration of adduct I reached a plateau after 4 days. Moreover, adduct I appeared to be invariable in the presence of GSH, for its area remained constant between 4 and 8 days, which indicated the stability of DNA binding. Also, the area of adduct II remained unchanged after a short decrease between 3 and 8 days. In addition, except the cobinding situation with GMP, GSH’s combination with complex 1 only was not found,19 even when the macro range gradient elution of solvent A was set from 95% to 0% within 100 min, indicating that the

are found to be more active toward the cisplatin-resistant SGC7901 cell line than cisplatin but less toxic toward normal human liver cells. This can be significantly attributed to the steric hindrance effect caused by the methoxy-substituted benzyl group, which might reduce the platinum’s reactivity toward biological molecules and bring about the lower toxicity of the complexes toward human normal cell lines but higher activity against the cisplatin-resistant cancer cell line. DNA Binding Studies. Upon the remarkable antitumor features, complex 1 was selected for further investigation. Since DNA is the putative target for platinum anticancer drugs, the interaction with DNA has been widely studied for platinum complexes. Therefore, the DNA binding ability of complex 1 was first investigated by gel electrophoresis, in which cisplatin and oxaliplatin were used as positive controls. The DNA chain can generally be divided into closed circular DNA or supercoil DNA, linear DNA, and open circular DNA. As shown in Figure 3, it mainly contains closed circular DNA and open circular

Figure 3. Gel electrophoretic pattern of pET22b plasmid DNA incubated with various concentrations of tested platinum(II) compounds for 24 h. The lanes correspond to untreated plasmid DNA (lane 0), concentrations of 10, 40, 160, and 640 μM of cisplatin (lanes 1−4), oxaliplatin (lanes 5−8), and complex 1 (lanes 9−12) respectively incubated with DNA. Forms I and II represented closed circular DNA and open circular DNA, respectively.

DNA, which are defined as form I and form II, respectively. It can be seen that with the increased concentration of platinum complexes from 10 to 640 μM, the rate of migration for closed circular DNA (form I) and open circular DNA (form II) all decreased. The significant differences between complex 1 and controls were the band density and the migration distance. As mentioned before, bifunctional platinum(II) complexes mainly exert antitumor activities through intrastrand cross-links with DNA. Complex 1, owning two labile chloride anions, might form a bifunctional adduct with DNA. Guanosine 5′monophosphate (GMP), including N-donor atoms, is a DNA model molecule,14 so it was used to characterize the binding mode of the synthesized complex, which was detected by using HPLC−UV and LC−MS techniques. As seen in Figure S2 of the Supporting Information (SI), the reaction of complex 1 with GMP gave rise to one main product (I), as detected by HPLC. According to the positive ESI-MS analysis of I (Figure S3, SI), a single-charged cationic peak at m/z 791.18738, corresponding to the molecular formula of [C24H36N7O10PPt]+, can be assigned to complex 1 combined with one GMP accompanied by loss of two chlorine atoms. This assignment was also confirmed by theoretic molecular weights and isotopic distribution patterns. It has been reported that cisplatin, oxaliplatin, and their analogues could form the bifunctional adduct with the N7 atoms of guanine bases.15 Although the bifunctional adduct of complex 1 was detected by LC−MS (Figure S4, SI), its concentration was too low to be detected by HPLC (UV detector). Hence, by introducing the benzyl group as steric hindrance, a new kind of monofunctional platinum complex was found. Studies on the monofunctional compound revealed that little distortion of the DNA double helix is induced upon platination,4a,9a but our result is quite different D

DOI: 10.1021/acs.inorgchem.6b00361 Inorg. Chem. XXXX, XXX, XXX−XXX

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Inorganic Chemistry

Solubility and Stability. The aqueous solubility of complex 1 is approximately 1 mg/mL at 25 °C, which is the same as that of cisplatin. The stability of complex 1 in water was studied by HPLC with cisplatin as reference [Figure S7 (SI) and Figure 5]. It is noted that both complex 1 and cisplatin

Figure 5. Time-dependent stability of complex 1 (1 mg/mL) in water (pH 6.0), DMF (pH 6.0), and the buffer (pH 6.2) and cisplatin (1 mg/mL) in water (pH 6.0) determined by HPLC profiles.

Figure 4. Time-dependent distribution of each species on HPLC profiles. (a) Adduct I of complex 1 (0.25 mM) with GMP (0.50 mM) in deionized water. (b) Adducts I and II of complex 1 (0.25 mM) with GMP (0.25 mM) in the presence of GSH (0.25 mM) in deionized water.

gradually hydrolyzed after being dissolved in water; however, only 32.2% of complex 1 hydrolyzed compared with 37.7% of cisplatin after 24 h. As time extended to 48 h, cisplatin turned out to be much more seriously decomposed than complex 1. These results indicated that complex 1 is more stable than cisplatin in aqueous solution. The effect of complex 1 in DMF and the buffer used for biochemical experiments was also investigated. As shown in Figure 5, 33.6% of complex 1 hydrolyzed in DMF and 33.2% in the buffer after 24 h, which are almost equivalent to the value in water, demonstrating that a solvent like DMF and the buffer had hardly significant effects on the stability of the compound. Cell Cycle Analysis. Since the monofunctional platinum complex 1 showed significant antitumor features different from those of its analogue oxaliplatin, the biological analysis was further investigated. The cell cycle, a cycle stage that renews cells from preparation to division to two cells, mainly includes G1, S, G2, and M phases. Measurement of the cell cycle can determine the amount of DNA over varying periods. The cell cycle with complex 1 was carried out on the MCF-7 cell line at 50 μM for 24 h, with cisplatin and oxaliplatin as positive controls. The result can be seen in Figure 6. Figure 6a shows the cell cycle results of three platinum drugs, and Figure 6b is a bar graph showing the DNA counts after treatment with different platinum drugs. In the cell cycle diagram, from left to right was G1, S, and G2 phase; in the control, 60.91% was in G1 phase, 23.08% was in S phase, and 3.59% was in G2 phase. When incubated with cisplatin, the cell cycle arrest of S and G2 phases increased obviously (from 23.08% to 29.02% and from 3.09% to 7.89%) and additional phases reduced correspondingly. When incubated with oxaliplatin, cell cycle arrest occurred mainly in the G1 phase (from 60.91% to 76.63%). Notably, the cell cycle arrest of complex 1 primarily occurred in the G2 phase (from 3.09% to 5.53%) and in the S phase (from 23.08% to 23.40%). Consequently, the cell cycle arrest of cisplatin and complex 1 against MCF-7 cell line occurred in the G2 phase, while for oxaliplatin it occurred in the G1 phase. This indicated that the mechanism of cell cycle arrest induced by complex 1 was clearly different from that of its parent molecule oxaliplatin. The different inhibition characters of complex 1 and oxaliplatin may arise from their distinct DNA binding modes, which further confirmed our former results.

platinum complex, with the good π-conjugated unit benzyl group and the appending donor methoxy group, was insusceptible to nucleophiles. Taken together, these results showed that the linkage of complex 1 with DNA was stable and insensitive to nucleophilic attack. Cellular Uptake Properties. Except the inactivation of the complexes due to their binding to GSH, the cellular uptake of complexes is one of the important factors affecting cytotoxicity. As the most active complex in the cytotoxicity assay, complex 1 was chosen to examine the cellular uptake on MCF-7 cell, while cisplatin and oxaliplatin were used as positive controls. The result can be seen in Table 3. It was obvious that cellular uptake Table 3. Cellular Uptake of Complex 1 on MCF-7 Cells after 12 h of Incubationa complex

Pt content (ng/106 cells) in MCF-7 cells

cisplatin oxaliplatin 1

148.6 ± 14 118.6 ± 23 106.6 ± 19**,#

a

Intracellular accumulation of cisplatin, oxaliplatin, and complex 1 in MCF-7 cells (per 106 cells) after 12 h at a concentration of 50 μM. Results are expressed as the mean ± SD for three independent experiments (*P < 0.05, **P < 0.01, compared with cisplatin; #P < 0.05, ##P < 0.01, compared with oxaliplatin).

of these three complexes was on the same order of magnitude. Cisplatin had the highest concentration of uptake at 148.6 ng/ 106 cells, oxaliplatin was at 118.6 ng/106 cells, and complex 1 had the lowest uptake at 106.6 ng/106 cells. Given the results from the cytotoxicity and cellular uptake tests, complex 1 was proven to be the most efficient one against the MCF-7 cell line, with the lowest cellular uptake and highest cytotoxic activity. The lack of correlation between the intracellular levels and the cytotoxicity of complex 1 might be owed to the fact that the intracellular platinum level was not the key factor dominating the cytotoxicity, but the amount of the complexes finally acting on DNA was.20 As the DNA binding studies showed, complex 1 had a low reactivity toward biological nucleophiles but a stable binding behavior with DNA. E

DOI: 10.1021/acs.inorgchem.6b00361 Inorg. Chem. XXXX, XXX, XXX−XXX

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Figure 7. (a) Apoptosis analysis of MCF-7 cells incubated with cisplatin, oxaliplatin, and complex 1 at 50 μM for 24 h, compared with the control group cells. (b) Histogram of apoptosis rate. Figure 6. (a) Effect on the cell cycle of MCF-7 cells treated with cisplatin (G1, 49.27%; S, 29.02%; G2, 7.89%), oxaliplatin (G1, 76.63%; S, 7.96%; G2, 1.9%), and complex 1 (G1, 54.75%; S, 23.40%; G2, 5.53%) at 50 μM for 24 h, compared with the control group cells (G1, 60.91%; S, 23.08%; G2, 3.59%). (b) The percentages of MCF-7 cells in the different phases of the cell cycle.

Apoptosis Studies. To observe the changes in cell death, cell apoptosis investigation of complex 1 was carried out at 50 μM for 24 h on the MCF-7 cell line, with cisplatin and oxaliplatin as positive controls. In Figure 7a, necrosis, late apoptosis, viable cells, and early apoptosis were repressed in quadrants of Q1, Q2, Q3, and Q4, respectively. Figure 7b showed the apoptosis rate of each platinum drug in the histogram. As shown in Figure 7a, cisplatin achieved an apoptosis rate of 73.33% (17.81% early apoptosis and 55.52% late apoptosis), and oxaliplatin attained an apoptosis rate of 15.55% (5.18% early apoptosis and 10.37% late apoptosis), while complex 1 achieved 28.08% apoptosis rate (16.53% early apoptosis and 11.55% late apoptosis). Comparing complex 1 with the negative control, the early death increased from 3.34% to 16.33% and late death grew from 2.96% to 11.55%. The apoptosis rate of complex 1 was significantly greater than that of oxaliplatin, under the same condition either in early apoptosis or late apoptosis. These results indicated that complex 1 triggered cancer cell death via an apoptotic pathway. To further investigate the mechanism of apoptosis induced by complex 1, Bax, Bcl-2, and procaspase-3 proteins were investigated in MCF-7 cells by Western blot analysis; cisplatin and oxaliplatin were used as positive controls. As shown in Figure 8, a conspicuous increase of Bax expression was detected in cells after treatment with cisplatin, oxaliplatin, and complex 1, which could trigger the apoptosis. Meanwhile, an obvious decrease of Bcl-2 and procaspase-3 expression was observed in

Figure 8. Expression of apoptosis-regulated proteins examined by Western blot analysis after treatment of MCF-7 cells with cisplatin, oxaliplatin, and complex 1 at a concentration of 50 μM. Equal loading was testified by the detection of β-actin. The results were obtained from three independent experiments.

cells, especially those incubated with cisplatin and complex 1. All of the data indicated that complex 1 could induce cell apoptosis by an intrinsic mitochondrial pathway.

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CONCLUSION In this study, a series of novel platinum(II) complexes with Nmonosubstituted 1R,2R-diaminocyclohexane bearing methoxysubstituted benzyl groups as carrier ligands was designed and prepared. Biological assays indicated that complex 1 had potent cytotoxic activity against A549, HCT-116, MCF-7, and SGC7901 cancer cell lines and was less toxic to human normal liver LO2 cell line, compared with cisplatin and oxaliplatin. Meanwhile, another two complexes, 2 and 3, with chloride anions as leaving groups, also showed strong anticancer activity comparable or even superior to those of cisplatin and oxaliplatin, whereas other complexes with dicarboxylates, except individual cases, exhibited moderate or low cytotoxicity. Moreover, the cytotoxicity of complexes 1−3 against a cisplatin-resistant cell (SGC7901/CDDP) indicated that they F

DOI: 10.1021/acs.inorgchem.6b00361 Inorg. Chem. XXXX, XXX, XXX−XXX

Article

Inorganic Chemistry

123.7, 123.9, 134.1, 134.6, 160.3. ESI-HRMS: calcd for [M + H]+ 235.1805, found 235.1803. Anal. Calcd (%) for C14H24Cl2N2O: C 54.73, H 7.87, N 9.12. Found: C 54.60, H 7.91, N 9.01. 1 L2·2HCl. Yield: 73%. [α]20 D = −30.7 (c = 0.33 in H2O). H NMR (500 MHz, D2O): δ = 1.32−1.39 (m, 2H; CH2 of DACH), 1.39−1.62 (m, 2H; CH2 of DACH), 1.81−1.88 (m, 2H; CH2 of DACH), 2.15− 2.18 (d, 3J(H,H) = 15 Hz,1H; CH2 of DACH), 2.39−2.42 (d, 3J(H,H) = 15 Hz, 1H; CH2 of DACH), 3.41−3.53 (m, 2H; CH of DACH), 3.81 (s, 3H; CH3), 4.14−4.41 (dd, 2H; CH2), 7.04−7.43 (m, 4H; ArH). 13C NMR (500 MHz, D2O): δ = 21.8 (C4), 22.0 (C5), 25.6 (C6), 28.9 (C3), 48.2 (C7), 50.8 (C2), 55.0 (C1), 58.0 (CH3), 117.0, 125.3, 134.1, 134.5, 134.6, 160.3. ESI-HRMS: calcd for [M + H]+ 235.1805, found 235.1809. Anal. Calcd (%) for C14H24Cl2N2O: C 54.73, H 7.87, N 9.12. Found: C 54.55, H 7.95, N 9.04. 1 L3·2HCl. Yield: 77%. [α]20 D = −51.0 (c = 0.41 in H2O). H NMR (500 MHz, D2O): δ = 1.39−1.41 (m, 2H; CH2 of DACH), 1.54−1.62 (m, 2H; CH2 of DACH), 1.80−1.88 (m, 2H; CH2 of DACH), 2.16− 2.19 (d, 3J(H,H) = 15 Hz,1H; CH2 of DACH), 2.41−2.44 (d, 3J(H,H) = 15 Hz, 1H; CH2 of DACH), 3.45−3.50 (m, 2H; CH of DACH), 3.84 (s, 3H; CH3), 4.16−4.44 (dd, 2H; CH2), 7.04−7.46 (m, 4H; ArH). 13C NMR (75 MHz, D2O): δ = 24.9 (C4), 25.0 (C5), 28.7 (C6), 31.9 (C3), 51.0 (C7), 53.9 (C2), 58.1 (C1), 60.7 (CH3), 114.8, 121.8, 130.0, 131.4, 158.7. ESI-HRMS: calcd for [M + H]+ 235.1805, found 235.1806. Anal. Calcd (%) for C14H24Cl2N2O: C 54.73, H 7.87, N 9.12. Found: C 54.67, H 7.99, N 8.98. Standard Procedure for the Preparation of Complexes 1−3. K2PtCl4 (2.08 g, 5.00 mmol) and a ligand (1.17 g, 5.00 mmol) which was neutralized with Na2CO3 was mixed in aqueous solution (80 mL) and kept stirring at 40 °C for 18 h shaded from light. During the process, a yellowish precipitate formed and was filtered out. The solid was washed with water repeatedly and then rinsed with a small amount of ethanol. After being dried in a vacuum, a yellowish powder was obtained. Complex 1, [Pt(L1)Cl2]. Yield: 88%. [α]20 D = 154.0 (c = 0.20, in DMF). 1H NMR (500 MHz, DMSO-d6): δ = 0.90−0.99 (m, 2H; CH2 of DACH), 1.29−1.36 (m, 2H; CH2 of DACH), 1.52−1.59 (m, 2H; CH2 of DACH), 1.79−1.81 (m, 1H; CH2 of DACH), 2.01−2.04 (m, 1H; CH2 of DACH), 2.15−2.43 (m, 2H; CH of DACH), 3.82 (s, 3H; CH3), 3.78−4.02 (m, 2H; CH2), 6.98−8.21 (m, 4H; ArH). 13C NMR (500 MHz, DMSO-d6): δ = 24.05 (C4), 24.09 (C5), 28.50 (C6), 31.71 (C3), 44.03 (C7), 55.40 (C2), 61.04 (C1), 65.14 (CH3), 110.98, 120.59, 122.52, 129.82, 131.93, 157.91. ESI-HRMS: calcd for [M − Cl]+ 464.1063, found 464.1077. Anal. Calcd (%) for C14H22Cl2N2OPt: C 33.61, H 4.43, N 5.60. Found: C 33.77, H 4.39, N 5.54. Complex 2, [Pt(L2)Cl2]. Yield: 82%. [α]20 D = 124.3 (c = 0.17, in DMF). 1H NMR (500 MHz, DMSO-d6): δ = 0.86−0.95 (m, 2H; CH2 of DACH), 1.21−1.28 (m, 2H; CH2 of DACH), 1.45−1.53 (m, 2H; CH2 of DACH), 1.70−1.73 (m, 1H; CH2 of DACH), 1.90−1.93 (m, 1H; CH2 of DACH), 2.19−2.35 (m, 2H; CH of DACH), 3.83 (s, 3H; CH3), 3.62−3.91 (m, 2H; CH2), 6.92−8.01 (m, 4H; ArH). 13C NMR (500 MHz, DMSO-d6): δ = 24.35 (C4), 24.60 (C5), 27.82 (C6), 31.66 (C3), 48.73 (C7), 55.30 (C2), 61.83 (C1), 65.70 (CH3), 113.60, 123.18, 129.41, 136.18, 149.52, 159.31; ESI-HRMS: calcd for [M + H] + 235.1805, found 464.1081. Anal. Calcd (%) for C14H 22Cl2N2OPt: C 33.61, H 4.43, N 5.60. Found: C 33.64, H 4.51, N 5.47. Complex 3, [Pt(L3)Cl2]. Yield: 80%. [α]20 D = 139.2 (c = 0.19, in DMF). 1H NMR (500 MHz, DMSO-d6): δ = 0.77−0.86 (m, 2H; CH2 of DACH), 1.10−1.17 (m, 2H; CH2 of DACH), 1.38−1.46 (m, 2H; CH2 of DACH), 1.70−1.73 (m, 1H; CH2 of DACH), 1.90−1.93 (m, 1H; CH2 of DACH); 2.11−2.29 (m, 2H; CH of DACH), 3.78 (s, 3H; CH3), 3.75−3.84 (m, 2H; CH2), 6.95−8.01 (m, 4H; ArH). 13C NMR (500 MHz, DMSO-d6): δ = 23.79 (C4), 24.12 (C5), 27.72 (C6), 31.47 (C3), 48.12 (C7), 55.06 (C2), 60.27 (C1), 63.70 (CH3), 113.89, 125.77, 126.32, 131.84, 137.40, 159.08. ESI-HRMS: calcd for [M − Cl]+ 464.1063, found 464.1080. Anal. Calcd (%) for C14H22Cl2N2OPt: C 33.61, H 4.43, N 5.60. Found: C 33.50, H 4.54, N 5.49. Standard Procedure for the Preparation of Complexes 4−6. The synthetic method includes the following two steps. The corresponding complex (0.50 g, 1.00 mmol) 1−3 was first suspended in water (100 mL), and then sliver nitrate (0.34 g, 2.00 mmol) was

all had the potential to overcome cisplatin resistance. The DNA binding assay demonstrated that complex 1, with a benzyl moiety containing a methoxyl group at ortho position as steric hindrance, had the ability to bind DNA monofunctionally, which is significantly different from classical bifunctional crosslinking platinum(II) complexes. In particular, the monofunctional DNA conjugation of complex 1 is not via the traditional metalloinsertion but via the covalent binding between platinum and the N7-guanine site. Moreover, HPLC and LC−MS studies showed that complex 1 was insusceptible to nucleophiles, and the linkage of complex 1 with DNA was stable and insensitive to nucleophilic attack as well. The irreversible binding affinities for DNA could make up for its lower cellular uptake, as compared with those of cisplatin and oxaliplatin. A cell cycle study on the MCF-7 cell line indicated that the mechanism of cell cycle arrest induced by complex 1 was different from that of oxaliplatin. Complex 1 could trigger cancer cell death via an apoptotic pathway. Furthermore, Western blot analysis indicated that the apoptosis induced by complex 1 was to some extent caused by an intrinsic mitochondrial pathway. All these features, including the satisfactory activity toward the tested cancer cell lines, especially the cisplatin-resistant line, the binding mode versus other oxaliplatin derivatives, and cellular uptake and apoptosis properties, bear out that the introduction of the rigid steric hindrance is very useful to promote the anticancer activity of the resulting platinum complexes and potentially to overcome cisplatin drug resistance.

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EXPERIMENTAL SECTION

Materials and Instruments. The reagents and materials required in the experiment were chemically pure without further purification. Potassium tetrachloroplatinate(II) was purchased from a local chemical company (Lingfeng Chemical Ltd.). The cells used to styudy biological activity were obtained from KeyGEN BioTECH Co. The specific optical rotations were measured on a WZZ-2A automatic polarimeter. 1H and 13C NMR spectra were measured in DMSO-d6 with a Bruker 300 or 500 MHz spectrometer. Mass spectra were measured by an Agilent 6224 ESI/TOF MS instrument. Elemental analyses of C, H, and N used a Vario MICRO CHNOS elemental analyzer (Elementar). HPLC was performed on an Agilent 1260 system. LC−MS were performed on an Agilent 1260-6224 system. Standard Procedure for the Preparation of Ligands. The method of synthesized ligands included three steps (Scheme 1). To a solution of Boc-protected cyclohenxanediamine (8.56 g, 40 mmol) in 150 mL of methanol was added o-methoxybenzaldehyde, mmethoxybenzaldehyde, or p-methoxybenzaldehyde (9.92 g, 80 mmol), and the mixture was kept stirring at room temperature for 2 h. Then, NaBH4 (4.54 g, 120 mmol) was added under ice water bath condition. When the reaction was completed, 10 mL of water was added to quench the reaction. The solvent was removed under reduced pressure, and a white solid was obtained and then extracted by adding organic phase. Next, the solvent was removed to get an oily substance. For long-term preservation, the hydrochloride salt of the ligand was prepared as follows: the oil was first dissolved with 75 mL of anhydrous diethyl ether. Then, 150 mL of 2 mol/L AcOEt/HCl was slowly added at ice water bath temperature, and a white solid deposited. At last, to get a white powder, the solid was collected by rapid filtration and dried in a vacuum desiccator. 1 L1·2HCl. Yield: 75%. [α]20 D = −41.2 (c = 0.49 in H2O). H NMR (500 MHz, D2O): δ = 1.35−1.40 (m, 2H; CH2 of DACH), 1.42−1.65 (m, 2H; CH2 of DACH), 1.80−1.87 (m, 2H; CH2 of DACH), 2.17− 2.20 (d, 3J(H,H) = 15 Hz,1H; CH2 of DACH), 2.39−2.42 (d, 3J(H,H) = 15 Hz, 1H; CH2 of DACH), 3.39−3.44 (m, 2H; CH of DACH), 3.92 (s, 3H; CH3), 4.25−4.71 (dd, 2H; CH2), 7.05−7.53 (m, 4H; ArH). 13C NMR (75 MHz, D2O): δ = 24.8 (C4), 24.9 (C5), 28.7 (s; C6), 31.8 (C3), 47.8 (C7), 53.5 (C2), 58.1 (C1), 60.8 (CH3), 114.0, G

DOI: 10.1021/acs.inorgchem.6b00361 Inorg. Chem. XXXX, XXX, XXX−XXX

Article

Inorganic Chemistry added. After the mixture was kept stirring at 38 °C for 24 h with shading from light, it was filtered. Potassium oxalate (ox, 0.14 g, 1.00 mmol) in water (15 mL) was added to the above filtrate. The reaction proceeded in the dark at room temperature with a large amount of solid precipitated. After 12 h, the solid no longer significantly increased, and the solid was filtered out, washed with water repeatedly, and dried in a vacuum to give a pale yellow powder. Complex 4, [Pt(L1)(ox)]. Yield: 66%. 1H NMR (500 MHz, DMSOd6): δ = 0.89−1.09 (m, 2H; CH2 of DACH), 1.11−1.23 (m, 2H; CH2 of DACH), 1.40−1.54 (m, 2H; CH2 of DACH), 1.80−1.86 (m, 2H; CH2 of DACH), 2.18−2.27 (m, 2H; CH of DACH), 3.67 (s, 3H; CH3), 3.42−3.75 (m, 2H; CH2), 6.93−7.43 (m, 4H; ArH). 13C NMR (500 MHz, DMSO-d6): δ = 24.02 (C4), 24.62 (C5), 28.80 (C6), 32.07 (C3), 46.35 (C7), 55.67 (C2), 63.06 (C1), 66.53 (CH3), 111.15, 121.10, 123.08, 130.47, 133.66, 158.47, 166.04 (C1″), 166.98 (C3″). ESI-HRMS: calcd for [M − H]− 516.1104, found 516.1100. Anal. Calcd (%) for C16H 22N2O5Pt: C 37.14, H 4.29, N 5.41. Found: C 37.21, H 4.23, N 5.29. Complex 5, [Pt(L2)(ox)]. Yield: 59%. 1H NMR (500 MHz, DMSOd6): δ = 1.03−1.14 (m, 2H; CH2 of DACH), 1.28−1.36 (m, 2H; CH2 of DACH), 1.56−1.64 (m, 2H; CH2 of DACH), 1.83−1.86 (m, 1H; CH2 of DACH), 2.07−2.10 (m, 1H; CH2 of DACH), 2.66−2.85 (m, 2H; CH of DACH), 3.67 (s, 3H; CH3), 3.42−3.75 (m, 2H; CH2), 6.42−7.40 (m, 4H; ArH). 13C NMR (500 MHz, DMSO-d6): δ = 24.28 (C4), 24.68 (C5), 28.10 (C6), 32.06 (C3), 50.91 (C7), 55.37 (C2), 61.59 (C1), 65.38 (CH3), 114.38, 117.05, 123.40, 129.83, 135.85, 159.75, 165.91 (C1″), 166.87 (C3″). ESI-HRMS: calcd for [M − H]− 516.1104, found 516.1101. Anal. Calcd (%) for C16H22N2O5Pt: C 37.14, H 4.29, N 5.41. Found: C 37.20, H 4.24, N 5.39. Complex 6, [Pt(L3)(ox)]. Yield: 56%. 1H NMR (500 MHz, DMSOd6): δ = 1.04−1.13 (m, 2H; CH2 of DACH), 1.30−1.38 (m, 2H; CH2 of DACH), 1.58−1.66 (m, 2H; CH2 of DACH), 1.80−1.83 (m, 1H; CH2 of DACH), 2.06−2.09 (m, 1H; CH2 of DACH), 2.63−2.81 (m, 2H; CH of DACH), 3.65 (s, 3H; CH3), 3.61−3.76 (m, 2H; CH2), 6.41−7.53 (m, 4H; ArH). 13C NMR (500 MHz, DMSO-d6): δ = 24.23 (C4), 24.67 (C5), 27.96 (C6), 32.06 (C3), 50.04 (C7), 55.54 (C2), 61.64 (C1), 64.70 (CH3), 109.62, 114.27, 122.57, 126.20, 132.80, 159.73, 165.91 (C1″), 166.93 (C3″). ESI-HRMS: calcd for [M − H]− 516.1104, found 516.1110. Anal. Calcd (%) for C16H22N2O5Pt: C 37.14, H 4.29, N 5.41. Found: C 37.06, H 4.35, N 5.33. Standard Procedure for the Preparation of Complexes 7−9. The synthetic procedure was similar to that of complexes 4−6, except sodium malonate (mal) was used to take the place of potassium oxalate. Complex 7, [Pt(L1)(mal)]. Yield: 65%. 1H NMR (500 MHz, DMSOd6): δ = 1.04−1.13 (m, 2H; CH2 of DACH), 1.30−1.39 (m, 2H; CH2 of DACH), 1.58−1.64 (m, 2H; CH2 of DACH), 1.80−1.83 (m, 1H; CH2 of DACH), 2.17−2.20 (m, 1H; CH2 of DACH), 2.60−2.79 (m, 2H; CH of DACH), 2.67 (s, 2H; CH2 of (COO)2CH2), 3.69 (s, 3H; CH3), 3.72−3.85 (m, 2H; CH2 of NHCH2Ph), 4.27−4.31 (m, 1H; NH), 4.82−5.42 (dd, 2H; NH2), 6.68−7.51 (m, 4H; ArH). 13C NMR (500 MHz, DMSO-d6): δ = 23.99 (C4), 24.48 (C5), 28.96 (C6), 34.69 (C3), 46.27 (C7), 55.55 (C2″), 56.14 (C2), 62.04 (C1), 67.98 (CH3), 107.72, 126.86, 127.35, 138.73, 140.20, 153.67, 172.00 (C1″ and C3″). ESI-HRMS: calcd for [M − H]− 530.1260, found 530.1263. Anal. Calcd (%) for C17H 24N2O5Pt: C 38.42, H 4.55, N 5.27. Found: C 38.27, H 4.68, N 5.20. Complex 8, [Pt(L2)(mal)]. Yield: 65%. 1H NMR (500 MHz, DMSOd6): δ = 1.03−1.12 (m, 2H; CH2 of DACH), 1.29−1.39 (m, 2H; CH2 of DACH), 1.58−1.62 (m, 2H; CH2 of DACH), 1.78−1.82 (m, 1H; CH2 of DACH), 2.10−2.14 (m, 1H; CH2 of DACH), 2.55−2.82 (m, 2H; CH of DACH), 2.68 (s, 2H; CH2 of (COO) 2CH2), 3.66 (s, 3H; CH3), 4.01−4.15 (m, 2H; CH2 of NHCH2Ph), 6.47−7.40 (m, 4H; ArH). 13C NMR (500 MHz, DMSO-d6): δ = 23.63 (C4), 24.41 (C5), 28.94 (C6), 33.91 (C3), 47.91 (C7), 55.15 (C2), 58.34 (C2″), 61.54 (C1), 67.64 (CH3), 113.07, 114.34, 123.97, 126.02, 154.36, 159.88, 171.83 (C1″ and C3″); ESI-HRMS: calcd for [M − H]− 530.1260, found 530.1276. Anal. Calcd (%) for C17H24N2O5Pt: C 38.42, H 4.55, N 5.27. Found: C 38.31, H 4.63, N 5.18.

Complex 9, [Pt(L3)(mal)]. Yield: 50%. 1H NMR (500 MHz, DMSOd6): δ = 1.05−1.15 (m, 2H; CH2 of DACH), 1.31−1.41 (m, 2H; CH2 of DACH), 1.60−1.63 (m, 2H; CH2 of DACH), 1.76−1.80 (m, 1H; CH2 of DACH), 2.13−2.17 (m, 1H; CH2 of DACH), 2.50−2.90 (m, 2H; CH of DACH), 2.69 (s, 2H; CH2 of (COO)2CH2), 3.65 (s, 3H; CH3), 3.96−4.10 (m, 2H; CH2 of NHCH2Ph), 6.61−7.13 (m, 4H; ArH). 13C NMR (500 MHz, DMSO-d6): δ = 23.55 (C4), 24.73 (C5), 29.18 (C6), 34.89 (C3), 46.43 (C7), 55.18 (C2), 57.09 (C2″), 61.81 (C1), 67.72 (CH3), 111.15, 121.10, 123.08, 130.47, 133.66, 158.47, 166.04 (C1″), 166.98 (C3″); ESI-HRMS: calcd for [M − H]− 530.1260, found 530.1277. Anal. Calcd (%) for C17H24N2O5Pt: C 38.42, H 4.55, N 5.27. Found: C 38.50, H 4.41, N 5.18. Standard Procedure for the Preparation of Complexes 10− 12. The synthesis was analogous to that of complexes 4−6, except silver 1,1-cyclobutanedicarboxylate (cbda) was used to replace silver nitrate and potassium oxalate. Complex 10, [Pt(L1)(cbda)]. Yield: 70%. 1H NMR (500 MHz, DMSO-d6): δ = 1.05−1.40 (m, 4H; CH2 of DACH), 1.60−1.91 (m, 4H; CH2 of DACH), 1.93−2.07 (m, 2H; CH2 of cbda), 2.17−2.28 (m, 2H; CH2 of cbda), 2.63−2.81 (m, 2H; CH of DACH), 3.72 (s, 3H; CH3), 3.67−3.81 (m, 2H; CH2 of NHCH2Ph), 6.62−7.55 (m, 4H; ArH). 13C NMR (500 MHz, DMSO-d6): δ = 23.76 (C4), 24.68 (C5), 28.50 (C6), 29.37 (C4″ and C6″), 34.89 (C3), 46.46 (C5″), 49.31 (C7), 55.47 (C2), 56.13 (C1), 62.13 (C2″), 68.01 (CH3), 107.64, 126.87, 127.37, 138.52, 140.20, 153.69, 177.72 (C1″ and C3″). ESIHRMS: calcd for [M − H]− 570.1573, found 570.1570. Anal. Calcd (%) for C20H 28N2O5Pt: C 42.03, H 4.94, N 4.90. Found: C 42.14, H 5.01, N 4.83. Complex 11, [Pt(L2)(cbda)]. Yield: 55%. 1H NMR (500 MHz, DMSO-d6): δ = 1.13−1.27 (m, 4H; CH2 of DACH), 1.65−1.95 (m, 4H; CH2 of DACH), 1.95−2.00 (m, 2H; CH2 of cbda), 2.17−2.27 (m, 2H; CH2 of cbda), 2.86 (m, 2H; CH of DACH), 3.70 (s, 3H; CH3), 4.02−4.11 (m, 2H; CH2 of NHCH2Ph), 6.45−7.39 (m, 4H; ArH). 13C NMR (500 MHz, DMSO-d6): δ = 22.98 (C4), 23.96 (C5), 28.34 (C6), 28.55 (C4″), 28.72 (C6″), 33.29 (C3), 48.28 (C5″ and C7), 54.60 (C2), 57.90 (s; C1), 60.61 (C2″), 67.16 (CH3), 107.71, 110.95, 112.42, 125.22, 153.52, 156.84, 176.50 (C1″), 176.87 (C3″). ESIHRMS: calcd for [M − H]− 570.1573, found 570.1581. Anal. Calcd (%) for C20H28N2O5Pt: C 42.03, H 4.94, N 4.90. Found: C 42.11, H 5.02, N 4.79. Complex 12, [Pt(L3)(cbda)]. Yield: 55%. 1H NMR (500 MHz, DMSO-d6): δ = 1.05−1.41 (m, 4H; CH2 of DACH), 1.62−1.92 (m, 4H; CH2 of DACH), 1.96−2.13 (m, 4H; CH2 of cbda), 2.17−2.26 (m, 2H; CH2 of cbda), 2.73 (m, 2H; CH of DACH), 3.65 (s, 3H; CH3), 3.92−4.11 (m, 2H; CH2 of NHCH2Ph), 6.59−7.52 (m, 4H; ArH). 13C NMR (500 MHz, DMSO-d6): δ = 23.52 (C4), 24.39 (C5), 28.19 (C6), 28.91 (C4″), 29.02 (C6″), 34.33 (C3), 48.98 (C7), 54.80 (C2), 56.47 (C1), 61.57 (C2″), 67.47 (CH3), 109.61, 119.17, 122.35, 138.21, 145.20, 156.49, 177.26 (C1″), 177.36 (C3″). ESI-HRMS: calcd for [M − H]− 570.1573, found 570.1583. Anal. Calcd (%) for C20H28N2O5Pt: C 42.03, H 4.94, N 4.90. Found: C 42.15, H 5.00, N 4.82. Standard Procedure for the Preparation of Complexes 13− 15. The preparation was similar to that of complexes 4−6, except sodium 3-hydroxy-1,1-cyclobutane dicarboxylate (hcbda) was used to take the place of potassium oxalate. Complex 13, [Pt(L1)(hcbda)]. Yield: 58%. 1H NMR (500 MHz, DMSO-d6): δ = 1.05−1.39 (m, 4H; CH2 of DACH), 1.65−1.81 (m, 4H; CH2 of DACH), 1.91−2.07 (m, 2H; CH2 of hcbda), 2.20−2.38 (m, 2H; CH2 of hcbda), 2.59−2.61 (m, 2H; CH of DACH), 3.72 (s, 3H; CH3), 3.57−3.85 (m, 2H; CH2 of NHCH2Ph), 4.22−4.31 (m, 1H; OH), 6.62−7.52 (m, 4H; ArH). 13C NMR (500 MHz, DMSOd6): δ = 23.70 (C4), 23.93 (C5), 28.66 (C6), 31.55 (C4″ and C6″), 31.66 (C3), 45.72 (C7), 46.87 (C2″), 55.58 (C2 and C1), 61.05 (CH3), 65.97 (C5″), 111.00, 120.46, 122.32, 129.88, 131.20, 158.04, 172.89 (C1″ and C3″). ESI-HRMS: calcd for [M − H]− 586.1522, found 586.1524. Anal. Calcd (%) for C20H 28N2O6Pt: C 40.89, H 4.80, N 4.77. Found: C 40.80, H 4.92, N 4.66. Complex 14, [Pt(L2)(hcbda)]. Yield: 43%. 1H NMR (500 MHz, DMSO-d6): δ = 1.03−1.43 (m, 4H; CH2 of DACH), 1.62−1.98 (m, 4H; CH2 of DACH), 2.11−2.27 (m, 2H; CH2 of hcbda), 2.20−2.39 H

DOI: 10.1021/acs.inorgchem.6b00361 Inorg. Chem. XXXX, XXX, XXX−XXX

Article

Inorganic Chemistry (m, 2H; CH2 of hcbda), 2.84 (m, 2H; CH of DACH), 3.67 (s, 3H; CH3), 4.04−4.31 (m, 2H; CH2 of NHCH2Ph), 4.24−4.31 (m, 1H; OH), 6.47−7.39 (m, 4H; ArH). 13C NMR (500 MHz, DMSO-d6): δ = 23.76 (C4), 24.49 (C5), 28.96 (C6), 34.41 (C4″ and C6″), 46.71 (C3 and C7), 46.73 (C2″), 55.29 (C2 and C1), 62.16 (CH3), 68.00 (C5″), 108.42, 111.76, 126.98, 128.29, 134.93, 157.63, 178.01 (C1″ and C3″). ESI-HRMS: calcd for [M − H]− 586.1522, found 586.1530. Anal. Calcd (%) for C20H28N2O6Pt: C 40.89, H 4.80, N 4.77. Found: C 40.91, H 4.91, N 4.74. Complex 15, [Pt(L3)(hcbda)]. Yield: 78%. 1H NMR (500 MHz, DMSO-d6): δ = 1.16−1.37 (m, 4H; CH2 of DACH), 1.65−1.82 (m, 4H; CH2 of DACH), 1.91−2.07 (m, 2H; CH2 of hcbda), 2.22−2.39 (m, 2H; CH2 of HCBD), 2.75 (m, 2H; CH of DACH), 3.65 (s, 3H; CH3), 3.97−4.10 (m, 2H; CH2 of NHCH2Ph), 4.21−4.26 (m, 1H; OH), 6.59−7.54 (m, 4H; ArH). 13C NMR (500 MHz, DMSO-d6): δ = 23.28 (C4), 24.21 (C5), 28.74 (C6), 41.20 (C4″ and C6″), 41.80 (C3), 44.33 (C7), 47.28 (C2″), 54.66 (C2), 56.33 (C1), 61.56 (CH3), 67.31(C5″), 109.50, 119.75, 122.23, 138.02, 145.04, 156.52, 176.95 (C1″), 177.44 (C3″). ESI-HRMS: calcd for [M − H]− 586.1522, found 586.1532. Anal. Calcd (%) for C20H28N2O6Pt: C 40.89, H 4.80, N 4.77. Found: C 40.87, H 4.90, N, 4.68. Cell Culture. A549 (human non-small-cell lung cancer cell line), HCT-116 (human colorectal cancer cell line), MCF-7 (human breast carcinoma cell line), SGC7901 (human gastric cancer cell line), and SGC7901/DDP (human gastric cancer cisplatin resistant cell line) as well as LO2 (normal human liver cell line) were maintained in a humidified atmosphere of 5% CO2 and 95% air at 37 °C. Certain cells were cultured in RPMI-1640 medium with 10% fetal bovine serum (FBS), and others were cultured in Dulbecco’s modified Eagle medium. All media were also supplemented with streptomycin (100 μg/mL) and ampicillin sodium (100 μg/mL). Cytotoxicity Analysis (IC50). The in vitro activity of the synthesized platinum(II) complexes was determined by the MTT method. The cultured cells with better vitality were transferred to a 96well plate such that the density of the cells was 5000 per well, and they were incubated overnight. Then, the compounds were dissolved by DMF and diluted with medium to various concentrations (the final concentration of DMF was less than 0.4%). After being incubated at 37 °C for 72 h, cells were stained with 3-(4,5-dimethyl-2-thiazolyl)2,5-diphenyl-2H-tetrazolium bromide (MTT) (5 mg/mL) for another 4 h, and then dissolved with 150 μL of DMSO. The UV absorption intensity was detected with an ELISA reader at 490 nm. The IC50 values were calculated by SPSS software after three parallel experiments. Statistical analyses were performed using an unpaired, two-tailed Student’s t test. All comparisons are made relative to controls, and the significance of the difference is indicated as *P < 0.05 and **P < 0.01, compared with cisplatin, and #P < 0.05 and ##P < 0.01, compared with oxaliplatin. Gel Electrophoresis Study. Complex 1 was analyzed by gel electrophoresis with cisplatin and oxaliplatin as positive controls, PET22b plasmid DNA was chosen as the target. The main steps were as follows: first, the platinum drugs were diluted to a concentration from 0 to 640 μM (ultrasonic vibration was necessary for high concentration solutions) and incubated with 5 μL of PET22b plasmid DNA at 37 °C water bath for 24 h. Second, 5 μL of drug−DNA mixtures containing loading buffer were added to agarose gel (ultrapure grade, Bio-Rad, made up to 1% w/v), and the gel was run in TA buffer (45 mM Tris-acetate, pH 7.5) for 60 min in 100 V. Last, but not least, the experiment data were obtained by a Molecular Imager (Gel Doc XR Imaging System, Bio-Rad). Reaction of Complex 1 with GMP. The reactions were prepared by mixing aliquots of 0.5 mM complex 1 and 1 mM GMP. No buffer was used to prevent the increased activation of the complexes due to coordination or interfering signals in the observed peak areas. The pH value was 6.0. The final solution (complex 1 at 0.25 mM, GMP at 0.50 mM) was kept at 37 °C in the dark. The sample was taken for HPLC analysis or LC−MS analysis without purification. Reaction of Complex 1 with GMP in the Presence of GSH. The reactions were prepared by mixing aliquots of 0.5 mM complex 1, 1 mM GMP, and 1 mM GSH. No buffer was used to prevent the

increased activation of the complexes due to coordination or interfering signals in the observed peak areas. The pH value was 6.0. The final solution (complex 1 at 0.25 mM, GMP at 0.25 mM, and GSH at 0.25 mM) was kept at 37 °C in the dark. The sample was taken for HPLC analysis or LC−MS analysis without purification. HPLC Studies. High-performance liquid chromatography (HPLC) was performed on an Agilent 1260 system equipped with a Phoenix C18 column (250 × 4.6 mm, 5 μm). HPLC profiles were recorded with UV detection at 210 nm, with mobile phase A, water containing 15 mM ammonium acetate (CH3CO2NH4), and mobile phase B, methanol. For analytical assays, the flow rate was 0.8 mL/min. The gradient of solvent A for complex 1 was as follows: 80% from 0 to 5 min, from 80% to 60% within 70 min, and reset to 80% from 75 to 80 min. LC−MS Studies. High-performance liquid chromatography and electrospray ionization time-of-flight mass spectrometry (HPLC ESI/ TOF MS) was performed on an Agilent 1260-6224 system. The LC analysis method was the same as the HPLC−UV one, and a splitting ration to MS was 1/5. Positive-ion electrospray ionization mass spectra were obtained at a capillary temperature of 350 °C with spray voltage 4000 V. The mass accuracy of all measurements was within 0.0001 m/ z unit. Cellular Uptake Test. MCF-7 cells with good activity were seeded in 6-well plates in 5% CO2 at 37 °C. After the cell density reached 80%, complex 1, cisplain, or oxaliplatin was added to each well at a concentration of 20 μM, and the plates were incubated for 12 h. Then, cells were collected and washed three times with ice-cold PBS, followed by centrifugation for 10 min and resuspension in PBS (1 mL). Then, 100 μL of suspension was taken out for measuring the cell density. The remaining cells were digested by HNO3 (200 μL, 65%) at 65 °C for 10 min. The results were measured by ICP-MS after three parallel experiments. Statistical analyses were performed using an unpaired, two-tailed Student’s t test. All comparisons are made relative to controls, and the significance of the difference is indicated as *P < 0.05 and **P < 0.01, compared with cisplatin, and #P < 0.05 and ##P < 0.01, compared with oxaliplatin. Solubility and Stability. The stability of the platinum agents was monitored by HPLC on an Agilent 1260 system, equipped with a Phoenix C18 column (250 × 4.6 mm, 5 μm). HPLC profiles were recorded with UV detection at 200 nm. The mobile phase for complex 1 in water was CH3CN/H2O (60:40), for complex 1 in DMF the buffer was CH3CN/H2O (40:60), and for cisplatin in water the buffer was CH3CN/H2O (10:90). The flow rate was 1 mL/min. To test the stability in water, complex 1 and cisplatin were diluted to 10 mg with 10 mL of purified water and incubated at 37 °C in the dark. To test the stability in DMF or the buffer, 10 mg of complex 1 was diluted with 1 mL of DMF or 1 mL of Dulbecco’s modified Eagle medium and 9 mL purified water and incubated at 37 °C in the dark. The sample was taken for HPLC analysis without purification. Cell Cycle Measurement. MCF-7 cells with good vitality were transferred into 6-well plates, with a density of 10 000 per well, and cultured overnight at 37 °C. Then, the tested platinum complexes were incubated with cells for 24 h. All adherent and floating cells were collected and washed twice with PBS. Then, the cells were fixed with 70% ethanol at 4 °C for 24 h. After that, fixed cells were washed with PBS. After being centrifuged, cells were stained with 50 μg/mL propidium iodide solution containing 100 μg/mL RNase for 0.5 h at 37 °C. The sample (at least 1 × 104 cells) was measured by flow cytometry (FAC Scan, Becton Dickenson) using Cell Quest software and recording propidium iodide (PI) in the FL2 channel. Cell Apoptosis Study by Flow Cytometry. MCF-7 cell line was used in the apoptosis experiment. Cisplatin and oxaliplatin were positive controls at a concentration of 50 μM. Specific operations were as follows: MCF-7 cells with good activity (5 × 105 cells per plate) were transferred to 6-well plates and cultured overnight in 5% CO2 at 37 °C. Platinum drugs were added, which were diluted to a concentration of 50 μM. After 24 h, the cells were digested with trypsin and washed twice with cold PBS. Then, cells were collected by centrifugation (2000 rpm, 5 min). After that, cells were resuspended in binding buffer (10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl2, pH I

DOI: 10.1021/acs.inorgchem.6b00361 Inorg. Chem. XXXX, XXX, XXX−XXX

Article

Inorganic Chemistry

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7.4) and incubated with annexin V-FITC (100 ng/mL) and then with propidium iodide (2 μg/mL) for 15 min in the dark at room temperature. At last, the fluorescence of cells was detected by an annexin V-FITC apoptosis detection kit (Roche) according to the manufacturer’s protocol, and cells were analyzed by a computer station running CellQuest software. Western Blot Analysis. The active preferred MCF-7 cells were seeded until the cell density reached 80%. Then 50 μM platinum complexes were added, and the cells were cultured for 12 h at 37 °C. Proteins were extracted by lysis buffer. The concentration of protein was measured by the BCA (bicinchoninic acid) assay with a Varioskan multimode microplate spectrophotometer (Thermo, Waltham, MA). Then equal amounts of protein (20 mg/lane) were separated by 10% sodium dodecyl sulfate−polyacrylamide gel electrophoresis (SDS− PAGE). After that, protein samples were transferred onto polyvinylidene difluoride (PVDF) Immobilon-P membrane (Bio-Rad) with a transblot apparatus (Bio-Rad). The blots, blocked with 5% nonfat milk in TBST (Tris-buffered saline plus 0.1% Tween 20) for 1 h, were incubated with primary antibodies diluted in PBST [1:2000 for β-actin, 1:500 for Bax (BD Pharmagin), and 1:500 for Bcl-2 (Cell Signal)] overnight at 4 °C. After that, the membrane was washed with PBST three times and incubated with IRDye 800 conjugated secondary antibody for 1 h at 37 °C. Detection was performed by an Odyssey scanning system (Li-COR, Lincoln, Nebraska).

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.inorgchem.6b00361. Chemical structures of GMP, GSH, and GS; ESI-MS spectroscopy for monofunctional and bifunctional adducts; and HPLC detection of the reaction of complex 1and GMP in the absence or presence of GSH (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*S.G. e-mail: [email protected]. *L.F. e-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are grateful to the National Natural Science Foundation of China (Project 21272041), the New Drug Creation Project of the National Science and Technology Major Foundation of China (Project 2013ZX09402102-001-006), and the Fundamental Research Funds for the Central Universities (Project 2242013K30011) for financial aid to this work. We also want to express our gratitude to the Priority Academic Program Development of Jiangsu Higher Education Institutions for the construction of fundamental facilities.

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REFERENCES

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DOI: 10.1021/acs.inorgchem.6b00361 Inorg. Chem. XXXX, XXX, XXX−XXX