A Potential Bone-Targeting Hypotoxic Platinum(II) Complex with an

Publication Date (Web): March 7, 2018. Copyright © 2018 American Chemical Society ... Top of Page; Abstract; Introduction; Results and Discussion; Co...
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Article Cite This: Inorg. Chem. XXXX, XXX, XXX−XXX

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A Potential Bone-Targeting Hypotoxic Platinum(II) Complex with an Unusual Cytostatic Mechanism toward Osteosarcoma Cells Zhenqin Zhang,†,‡ Zhenzhu Zhu,§ Cheng Luo,† Chengcheng Zhu,† Changli Zhang,† Zijian Guo,*,† and Xiaoyong Wang*,§ †

State Key Laboratory of Coordination Chemistry, School of Chemistry and Chemical Engineering, and §State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing 210023, People’s Republic of China ‡ School of Pharmacy, Nanjing Medical University, Nanjing 211166, People’s Republic of China S Supporting Information *

ABSTRACT: Osteosarcoma (OS) is the most common primary pediatric bone tumor lethal to children and adolescents. Chemotherapeutic agents such as cisplatin are not effective for OS because of their poor accessibility to this cancer and severe systemic toxicity. In this study, a lipophilic platinum(II) complex bearing a bisphosphonate bonetargeting moiety, cis-[PtL(NH3)2Cl]NO3 {BPP; L = tetraethyl [2-(pyridin-2-yl)ethane-1,1-diyl]bisphosphonate}, was prepared and characterized by NMR, electrospray ionization mass spectrometry, and single-crystal X-ray crystallography. The cytotoxicity of BPP toward OS cell lines U2OS and MG63 was tested by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. BPP exhibits moderate inhibition against U2OS cells through a mechanism involving both DNA binding and a mevalonate pathway. The acute toxicity of BPP to mice is 7-fold lower than that of cisplatin. The relative low systemic toxicity may result from the steric hindrance of the ligand, which blocks BPP approaching the bases of DNA. The results suggest that incorporating bisphosphonates into a platinum complex not only enhances its bone-targeting property but also minimizes its reactivity toward DNA and thereby lowers the systematic toxicity of the complex. The diminished cytotoxicity of BPP could be compensated for by increasing the therapeutic dose with marginal harm. This strategy provides a new possibility for overcoming the ineffectiveness and systemic toxicity of platinum drugs in the treatment of OS.

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INTRODUCTION Osteosarcoma (OS) is responsible for considerable deaths of children and adolescents worldwide.1 The 5-year survival rate reaches merely 20% for metastatic and recurrent OS patients, which lags far behind the survival rate of 58−82% for childhood cancers.2 Because of the lack of knowledge about the driving oncogenes and peculiar tumor sites, primarily in the knee joint and appendicular skeleton, therapy for OS is extremely difficult.3 Standard treatments for OS include surgery and neoadjuvant and adjuvant chemotherapy;4 among them, platinum-based anticancer agents such as cisplatin are a mainstay in the clinic.5 Despite great therapeutic success, systemic toxicities, such as neurotoxicity, ototoxicity, and nephrotoxicity, are major barriers for achieving ideal efficacy of platinum drugs,6 which intensifies the need for developing new platinum anticancer drugs with minimized systemic toxicity. Drug targeting is among the most promising approaches to reducing the systemic toxicity, which could potentially enhance the efficacy of cancer therapy.7 As highly effective inhibitors of bone resorption, bisphosphonates (BPs) can bind preferentially to bone mineral and alleviate osteolytic metastases8,9 and, therefore, are the most common bone-targeting agents.10 Extensive interest has © XXXX American Chemical Society

emerged on the development of Pt-BP complexes; some have proven to be more effective than BPs as a single agent in preventing tumor growth.11 Recently, we found that dinuclear platinum complexes with BP targeting groups demonstrate potential selectivity for the OS cell line U2OS; moreover, BP modification can reduce the acute toxicity of these complexes to some extent.12 However, their mechanism of action is similar to that of typical platinum drugs, and therefore the toxicity of platinum drugs was inherited. To further optimize our previous design, we adopted another strategy that maintains the bone-targeting property but minimizes the reactivity of the platinum center to DNA. Herein we report the structure of cis-[PtL(NH3)2Cl]NO3 {BPP; L = tetraethyl [2-(pyridin-2-yl)ethane-1,1-diyl]bisphosphonate} and its antitumor potential toward OS cells. The molecular mechanism of BPP was compared with our previous dinuclear BP-Pt complexes. We found that, besides DNA binding, farnesol (FOH) was involved in the apoptotic pathway of U2OS cells induced by BPP. It is gratifying that the Received: January 11, 2018

A

DOI: 10.1021/acs.inorgchem.7b03261 Inorg. Chem. XXXX, XXX, XXX−XXX

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Inorganic Chemistry

authenticated by the crystal structure of BPP, with P2 being closer to the py ring. The selected bond lengths and angles are listed in the caption of Figure 1 and are in the normal range for platinum(II) complexes. The dihedral angle between the py and platinum(II) planes is 73.11°. As a result, the extrusion effect of the py ring leads both N1−Pt1−N2 and N1−Pt1−Cl1 to deviate from 90°. Interestingly, the angle of C5−N1−Pt1 (125.7°) is larger than that of C1−N1−Pt1 (117.0°), implying that the BP moiety exerts some repulsion toward the platinum(II) plane or some steric hindrance exists above the platinum(II) plane. Actually, one of the BP tetraesters is situated immediately above the platinum(II) plane so that the distance between Cl1 and C14 is only 5.716 Å, which may make it difficult for BPP to approach the bases of DNA. The crystal parameters and structure refinement details are summarized in Table 1.

in vivo acute toxicity of BPP is remarkably lower than that of dinuclear BP-Pt complexes and cisplatin.

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RESULTS AND DISCUSSION Synthesis and Characterization. BPP was obtained following the procedure shown in Scheme 1. The cationic

Scheme 1. Synthetic Route to BPPa

Table 1. Crystallographic Data and Structure Refinement for BPP a

DMSO = dimethyl sulfoxide; DMF = N,N-dimethylformamide.

chemical formula fw cryst syst space group a (Å) b (Å) c (Å) h k l Z F(000) Dcalc (g cm−3) V (Å3) Nref, Npar R1, wR2 GOF on F2

platinum(II) complex cis-[Pt(NH3)2(py)Cl]+ (py = pyridyl) has shown significant anticancer activity in murine tumor models and the potential for overcoming drug resistance.13 The steric hindrance of py is supposed to slow down the reaction of BPP with glutathione (GSH) and circumvent the GSHmediated drug resistance. Thus, we believe that incorporation of the BP moiety with cis-[Pt(NH3)2(py)Cl]+ is a rational design for our goal. The ligand and BPP were characterized by 1 H, 13C, 31P NMR spectroscopy and electrospray ionization mass spectrometry (ESI-MS; Figures S1−S10). BPP was also characterized by 195 Pt NMR (Figure S11) and X-ray crystallography. The purity of BPP was determined to be >98% by high-performance liquid chromatography (HPLC; Figure S12). X-ray crystallography analysis revealed that BPP crystallized in the orthorhombic space group Pbca. The platinum center adopts an approximate square-planar geometry (Figure 1). The magnetic nonequivalence of the two phosphorus atoms is

C15H33ClF6N3O6P3Pt 788.89 orthorhombic Pbca 18.9342(14) 12.9262(9) 23.6419(17) −22 to +22 −15 to +8 −28 to +28 8 3088 1.811 5786.3(7) 21087, 5087 0.0808, 0.1731 1.122

Lipophilicity and Cellular Uptake. The lipophilicity parameter (log P) is a key factor that influences the drug to pass through the cellular membrane and determines the mode of administration.14 Therefore, we tested the log P of BPP using the shake-flask method in a 1-octanol/phosphate buffer system (pH 7.4).15 BPP is regularly distributed between water and noctanol (Figure S13). The log P value is calculated to be −1.7 ± 0.1 (Table S1), showing that BPP is more lipophilic than cisplatin (log P = −2.3).16 Thus, BPP is expected to pass through the lipid bilayer of the cell membrane more easily than cisplatin. However, the actual cellular uptake of BPP in U2OS cells is 1.813 ± 0.067 μg of platinum/106 cells, as determined by inductively coupled plasma mass spectrometry (ICP-MS) after incubation at 37 °C for 24 h, less than that of cisplatin (3.566 ± 0.408 μg of platinum/106 cells; Figure 2). The results indicate that BPP cannot enter U2OS cells as efficiently as cisplatin. The contradiction might be attributed to the positive charge on BPP, which makes it difficult to approach the cell membrane. Nevertheless, other factors may also affect the cellular uptake,17 and the exact reason for the phenomenon is unknown. The cellular uptake of BPP in human normal LO2 hepatic cells (1.248 ± 0.167 μg of platinum/106 cells) is even lower than that of in U2OS cells. This reduced cellular uptake might lead to a reduction of the general toxicity of the complex.

Figure 1. Crystal structure of BPP. Hydrogen atoms and nitrate ion are omitted for clarity. Selected bond length (Å) and angles (deg): N1−Pt1 2.010(5), N2−Pt1 2.032(5), N3−Pt1 2.015(5), Cl1−Pt1 2.284(2); N1−Pt1−Cl1 90.82(15), N1−Pt1−N2 91.0(2), N3−Pt1− Cl1 90.13(17), N3−Pt1−N2 88.1(2), C1−N1−Pt1 117.0(4), C5− N1−Pt1 125.7(4), N1−Pt1−N3 179.0(2), N2−Pt1−Cl1 177.76(16). B

DOI: 10.1021/acs.inorgchem.7b03261 Inorg. Chem. XXXX, XXX, XXX−XXX

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Figure 2. Cellular uptake of BPP and cisplatin (10 μM) in U2OS and LO2 cells (106) after incubation at 37 °C for 24 h. Data are presented as the mean ± standard deviation of three independent experiments.

Antiproliferative Activity. The cytotoxicity of BPP toward OS cell lines U2OS and MG-63 was tested by 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, with cisplatin as a positive reference. The corresponding half-maximal inhibitory concentration (IC50) values in Table 2

Figure 3. Impact of BPP (5.0 μM) on the cell cycle of U2OS cells after incubation for 48 h and PI staining, analyzed by flow cytometry. The data are mean percentages of the cells at the G0/G1, S, and G2/M phases.

Table 2. IC50 Values (μM) of BPP at 48 h cell

BPP

ligand

cisplatin

U2OS MG-63 LO2

16.03 ± 3.11 51.61 ± 3.08 13.71 ± 3.03

>100 >100 not tested

8.60 ± 2.40 8.66 ± 0.50 1.67 ± 0.76

V-FITC staining combined with propidium iodide (PI) to examine whether BPP could induce U2OS cells to go into apoptosis. The population of the early (Annexin V+/P−) and late (Annexin V+/PI+) apoptotic cells reached 47.6% after the cells were incubated with BPP (20.0 μM) for 48 h (Figure 4), indicating that BPP can induce apoptosis of U2OS cells. In comparison with cisplatin, BPP is less potent (0.42-fold) in inducing apoptosis.

reveal that BPP is cytotoxic against the U2OS cells but is less potent than cisplatin. MG-63 cells are insensitive to BPP. The ligand is not cytotoxic against both cancer cell lines, indicating that the combination of the platinum moiety with BP is crucial for the anticancer efficacy of BPP, which not only provides a cytotoxic metal center but also produces a lipophilic complex.18 The toxicity of BPP to normal cells was tested on the LO2 cells. In comparison with cisplatin, BPP is 8.2-fold less toxic toward LO2, indicating mild harm to normal cells. The concentrationdependent cytotoxic profiles of BPP against U2OS, MG-63, and LO2 cell lines are shown in Figure S14. Arrest of the Cell Cycle. The effect of BPP on the cell cycle of U2OS cells was investigated by flow cytometry. As shown in Figure 3, after exposure to BPP for 48 h, the cells arrested at the G0/G1 phase barely changed; the cells at the S phase decreased moderately from 42.7% to 32.7%, and those at the G2/M phase increased from 18.7% to 35.3%. The cells were evenly arrested at the three phases, which is different from the effect of cisplatin. The lower toxicity of BPP to the LO2 cell line was also verified by the cell cycle progression. As expected, after exposure to BPP for 48 h, the cells arrested at the S phase decreased from 56.2% to 46.5%, and the cells at the G2/M phase increased moderately from 1.5% to 10.3%. By contrast, the cells arrested by cisplatin at the S phase decreased from 56.2% to 33.5%, and the cells at the G2/M phase increased dramatically from 1.5% to 54.6% (Figure S15). The difference in the cell cycle distribution of U2OS and LO2 cells after BPP treatment may result from the distinct metabolic mode between cancer and normal cells. Apoptosis. Apoptosis is a programmed cell death in multicellular organisms, which plays a crucial role in maintaining homeostasis. Phosphatidylserine of the cytoplasmic membrane was thus measured by flow cytometry using Annexin

Figure 4. Flow cytometry analysis of U2OS cells after incubation with BPP (20.0 μM) for 48 h and subsequent staining with Annexin V and PI.

Interaction with DNA. In a previous study, we found that dinuclear BP-Pt complexes derived from cisplatin and oxaliplatin can react with DNA.12 The interactions between BPP and circulating-tumor DNA (CT-DNA) or supercoiled pUC19 DNA were also investigated by circular dichroism (CD) spectroscopy or agarose gel electrophoresis. In similar conditions, however, BPP only induced slight changes in the conformation of CT-DNA (Figure S16A). In agarose gel electrophoresis, systematic mild changes in the mobility and intensity of supercoiled pUC19 DNA were observed (Figure C

DOI: 10.1021/acs.inorgchem.7b03261 Inorg. Chem. XXXX, XXX, XXX−XXX

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Inorganic Chemistry S16B). The results suggest that BPP could weakly bind to DNA, even in the medium with a relatively low level of chloride (Figure S16C). To further verify the interaction between BPP and DNA, we analyzed the platination of DNA using ICP-MS. As shown in Figure 5, the percentage of DNA-bound platinum

Scheme 2. Proposed Mechanism for Intervening the Mevalonate Pathway by BPP

Figure 5. Percentage of bound platinum after BPP and cisplatin were incubated with pUC19 plasmid DNA in a buffer (10.0 mM Tris−HCl, 10 mM NaCl, pH 7.4) at 37 °C for 24 h. The data represent the average of two independent experiments ± standard deviation.

observed with the addition of FOH (Figure S18). Specifically, the IC50 value is 16.03 ± 3.11 μM for BPP treatment alone, whereas those for the simultaneous treatment of BPP plus 10.0 and 20.0 μM FOH are 23.70 ± 5.38 and 36.09 ± 7.59 μM, respectively. FOH alone is not cytotoxic toward U2OS cells. The result again suggests that BPP is involved in the mevalonate pathway. Interestingly, our results coincide with the recent observations reported by Lin et al.,25 where they demonstrated that a platinum complex derived from N-BPs exerted anticancer effects by inhibiting the mevalonate pathway. However, the molecular mechanism of the complex still remains unclear; in particular, the action of the platinum center is unexplored. Because the addition of extra FOH could make up the loss of cellular FOH or decrease the concentration of BPP, more dying cells were rescued. Moreover, the positively charged BPP might be capable of mimicking the isoprenoid pyrophosphate carbocation transition state involved in the FPP biosynthesis.26 In short, apart from DNA damage, BPP also intervenes with the mevalonate pathway to exert the anticancer effect. Acute Toxicity. Finally, we tested the median lethal dose (LD50) of BPP to evaluate its acute toxicity. The mortality and changes in the body weight were recorded for 14 days after BPP was intravenously injected into mice. The survival rates and changes in body weight of the mice after treatment with different doses of BPP are shown in Figure 6. No death was observed up to 50.0 mg kg−1 dose of BPP. On the basis of the data, the LD50 value of BPP was calculated to be 61.16 mg kg−1, 7-fold higher than that of cisplatin (LD50 = 8.6 mg kg−1),27 indicating that the toxicity of BPP is quite low. In comparison with our previous dinuclear BP-Pt complexes (LD50 4.32−5.10 mg kg−1),12 BPP has a much lower acute toxicity. The relatively low systemic toxicity may result from the steric hindrance of the ligand and low cellular uptake of BPP. In fact, 31P and 1H NMR and ESI-MS spectra (Figures S19 and S20) show that the reaction of BPP with GSH is much slower than that of cisplatin in similar conditions (30 min to 2.5 h).28 As we mentioned above, the application of platinum drugs is largely restricted by serious systemic toxicities. Here we demonstrate that the toxicity could be reduced by tethering the platinum moiety to a BP targeting group, which has not been reported so far.

for BPP (40%). Taken together, BPP can moderately bind to DNA compared with cisplatin. The inconspicuous changes in the CD spectrum and gel electrophoresis may be due to the fact that BPP has only one labile leaving ligand (Cl−) and could only produce a monofunctional Pt-DNA adduct. Such an adduct would not change the base pair twist angle at the binding site very much and thereby just causes small changes in the CD spectrum and the mobility of supercoiled DNA. Another reason may be due to the steric hindrance of the BP groups, which block BPP from approaching the bases of DNA (see Figure 1). The results completely meet our design anticipation mentioned above, that is, minimizing the reactivity of the platinum center to DNA. Involvement of BPP in the Mevalonate Pathway. The mevalonate pathway for cholesterol biosynthesis and protein prenylation has been related to various aspects of tumor development and progression.19 Nitrogen-containing BPs (NBPs) could inhibit farnesyl pyrophosphate (FPP) synthase, a key enzyme of the mevalonate pathway, and further alter protein prenylation, which are crucial for the growth and survival of cancer cells.8 N-BPs have shown dramatic anticancer effects in vitro and in some in vivo models of cancer mediated by the inhibition of FPP synthase.20,21 As an important intermediate in the mevalonate pathway,22 FOH and FPP can transform to each other.23 To ascertain whether BPP is involved in the mevalonate pathway, we studied the reaction of BPP with FOH using ESI-MS. The esterification of FOH with BPP was verified by two species observed at m/z 1021.83 and 1347.92, respectively (Figure S17). Thus, BPP could deplete FOH in U2OS cells. According to the established mevalonate pathway, the depletion of FOH could decrease the level of FPP and thereby result in the downregulation of Ras prenylation (Scheme 2). As a result, the cell survival signaling pathway is impaired, leading to the inhibition of cell proliferation.24 The proliferation of U2OS cells in the presence of BPP and different concentrations of FOH were then investigated by MTT assay. Partial growth rescue from the inhibition of BPP is D

DOI: 10.1021/acs.inorgchem.7b03261 Inorg. Chem. XXXX, XXX, XXX−XXX

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prepared in pure ethanol and diluted in a cell culture medium to the desired concentration immediately before use. The final concentration of ethanol in culture media was below 0.1% (v/v). Tetraethyl methylenediphosphonate was synthesized according to the previous literature.29 Electrospray ionization mass spectrometry (ESI-MS) spectra were recorded using an LCQ fleet ESI-MS spectrometer (Thermo Scientific). The 1H, 13C, 31P, and 195Pt NMR spectra were acquired on a Bruker DRX-500 spectrometer at 298 K. The circular dichroism (CD) experiments were performed on a Jasco J-810 spectropolarimeter (Japan Spectroscopic, Japan) in a 1-cm-path-length cylindrical quartz cell at room temperature. The images of agarose gel electrophoresis were obtained by using a Bio-Rad Gel-Doc XR imaging system, and quantification analysis was performed with Quantity One software. The ICP-MS data were obtained on an ELAN9000 inductively coupled plasma mass spectrometer (PerkinElmer Inc., U.S.A.). Synthesis and Characterization. Synthesis of 2-(2Pyridinylethylidene)bis(phosphonic acid) Tetraethyl Ester (L). It was prepared by a published method with some modifications.30 2(Chloromethyl)pyridine hydrochloride (26.46 g, 0.16 mol) was dissolved in water (35 mL), and the solution was made alkaline by adding sodium hydroxide (6.72 g, 0.16 mol) in water (70 mL). The mixture was extracted with chloroform, the extract was dried over anhydrous MgSO4, and the solvent was removed in vacuo. The residue was purified by vacuum distillation to give 2-(chloromethyl)pyridine as a pale-red oil. In a separate bottle, tetraethyl methylenediphosphonate (23.04 g, 80 mmol) in dry DMSO (20 mL) was added to a mixture of 60% NaH (4.189 g, 96 mmol) in DMSO (150 mL) at 273 K under nitrogen. The reaction mixture was stirred at 273 K for 30 min and then at room temperature for 2 h. 2-(Chloromethyl)pyridine (8.77 g, 72 mmol) was added dropwise to the stirring reaction mixture. The reaction was allowed to stir for an additional 72 h at room temperature and then quenched by the addition of saturated aqueous ammonium chloride. The reaction mixture was extracted with methylene chloride, and the organic extracts were combined and concentrated under reduced pressure. The mixture was redissolved in excess toluene, washed with water and brine, dried over Na2SO4, and concentrated under vacuum. The product is purified by flash chromatography with isopropyl alcohol in methylene chloride in silica gel. 1H NMR (500 MHz, CDCl3): δ 8.53 (d, J = 4.35 Hz, 1H), 7.57 (t, J = 7.60 Hz, 1H), 7.21 (d, J = 7.73 Hz, 1H), 7.11 (t, J = 7.3 Hz, 1H), 4.18−4.03 (m, 8H), 3.55 (tt, J = 23.42 and 6.14 Hz, 1H), 3.36 (td, J = 16.13 and 6.40 Hz, 2H), 1.24 (dt, J = 11.55 and 7.02 Hz, 12H). 13C NMR (125 MHz, CDCl3): δ 157.84, 148.61, 135.81, 123.31, 121.13, 62.16 (dd, J = 30.36 and 3.72 Hz), 34.85 (t, J = 132.85 Hz), 33.01, 15.88 (d, J = 3.19 Hz). 31 P NMR (202 MHz, CDCl3): δ 23.39. ESI-MS (m/z, positive mode). Found (calcd): [L + H]+, 380.33 (380.14); [L + Na]+, 402.42 (402.32); [2L + Na]+, 781.25 (781.26). Synthesis of BPP. Cisplatin (150 mg, 0.5 mmol) and AgNO3 (85 mg, 0.5 mmol) were stirred in anhydrous DMF (8.5 mL) in the dark at 325 K for 24 h. The resulting AgCl precipitate was removed by filtration, and L (160 mg, 0.425 mmol) was added to the filtrate. After stirring for an additional 24 h at 325 K, CH2Cl2 (100 mL) was added to the reaction mixture, and the solution was stirred for 10 min. The white precipitate was removed by filtration, and the filtrate was evaporated under reduced pressure. The resulting oil was dissolved in CHCl3 (4 mL) and added to diethyl ether (100 mL). The white precipitate was collected and washed by diethyl ether. Yellow crystals suitable for single-crystal X-ray diffraction analysis were obtained by slow evaporation of the complex and NaPF6 solution. A yellow precipitate of BPP was formed in 72% yield. 1H NMR (500 MHz, CDCl3): δ 9.08 (d, J = 5.10 Hz, 1H), 7.77 (t, J = 7.49 Hz, 1H), 7.49 (d, J = 8.04 Hz, 1H), 7.34 (t, J = 6.33 Hz, 1H), 4.64 (brs, 3H), 4.33 (brs, 3H), 4.26 (t, J = 7.18 Hz, 2H), 4.17−4.11 (m, 8H), 3.51 (t, J = 7.45 Hz, 1H), 1.42−1.29 (m, 12H). 13C NMR (125 MHz, CDCl3): δ 160.89, 154.01, 138.31, 128.00, 124.47, 63.73, 62.86 (d, J = 5.93 Hz), 34.58, 16.31 (dd, J = 11.78 and 5.59 Hz). 31P NMR (202 MHz, CDCl3): δ 21.71, 21.06. 195Pt NMR (107 MHz, CDCl3): δ −2289.49. ESI-MS (m/z, positive mode). Found (calcd): [PtL(NH3)2Cl − H2O

Figure 6. Survival rates (A) and changes in the body weight (B) of mice treated intravenously with different doses of BPP for 14 days. The change in the body weight was calculated as (body weight/body weight at day 1) × 100%. Control values were collected from mice treated with PBS.

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CONCLUSIONS In summary, a BP-modified platinum(II) complex displays effective inhibition against OS cells. The BP targeting group not only improves the selectivity of the platinum complex toward sarcoma cells but also reduces the systemic toxicity. Although the cytotoxicity of BPP is moderate in comparison with cisplatin, the imperfectness could be compensated for by raising the therapeutic dose because of its low toxicity. The cytostatic action of BPP is related to both DNA binding and the mevalonate pathway, which provides new insight into the antitumor mechanism of platinum complexes.

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EXPERIMENTAL SECTION

Materials and Methods. Cisplatin, NaPF6, diethyl phosphonate, 2-(chloromethyl)pyridine hydrochloride, L-glutathione (GSH), and 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma. Farnesol (FOH) was purchased from J&K Chemical Inc. Supercoiled pUC19 plasmid DNA was purchased from TaKaRa Biotechnology (Dalian). All chemicals were used as received without further purification unless otherwise stated. The human OS cell lines U2OS and MG-63 and the human hepatic cell line LO2 originated from the Cancer Institute and Hospital, Chinese Academy of Medical Sciences. Male ICR mice, aged 6 weeks and weighing 20−22 g, were purchased from the Model Animal Research Centre of Nanjing University. Anhydrous CH2Cl2, N,N-dimethylformamide (DMF), and dimethyl sulfoxide (DMSO) were prepared by refluxing or stirring in the presence of calcium hydride for 72 h. A stock solution of FOH was E

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K in the dark. The cells were analyzed using a BD LSR Fortessa flow cytometry. Detection of Apoptosis. U2OS cells were exposed to 20.0 μM BPP or cisplatin. The cells were kept at 310 K under 5% CO2 for 48 h and harvested for apoptotic assay. The cells were resuspended in a staining solution consisting of 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid/NaOH (pH 7.4), 140 mM NaCl, and 2.5 mM CaCl2. Annexin V-FITC (5 μL, 1 μg mL−1) and PI (5 μL, 1 μg mL−1) were added to the suspension and incubated at room temperature in the dark for 15 min before analysis by flow cytometry. At least 10000 events were acquired for each sample. Studies on DNA Interaction. The CT-DNA stock solution was prepared as we described previously.34 CD spectra were recorded at room temperature in the wavelength range of 220−320 nm after CTDNA (0.1 mM) was incubated with BPP at different molar ratios in buffer (5 mM Tris−HCl, 50 mM NaCl, pH 7.4) at 310 K for 24 h in the dark. Three scans were performed for each spectrum, and the buffer background was subtracted. Different concentrations of BPP were incubated with pUC19 plasmid DNA (200 ng) at 310 K for 24 h. Samples were examined by electrophoretic mobility shift assay through 1% agarose gel with a TAE buffer (50 mM Tris acetate and 1 mM ethylenediaminetetraacetic acid) for 2 h at 100 V. The resultant gels were stained with 0.5 μg mL−1 ethidium bromide in a TAE buffer and visualized under UV light. The results of electrophoresis were obtained by using a Bio-Rad Gel-Doc XR imaging system. DNA Platination. After electrophoresis, DNA in the resultant gels was extracted by a gel extraction kit. Briefly, the nicked, linear, and supercoiled DNA under UV light were sliced using a wide, sharp scalpel, incubated with an equal volume of binding buffer (XP2) at 50−60 °C for 7 min, and shaken every 2−3 min. DNA was collected in a HitBind DNA Mini Column by centrifuging at a maximum speed (≥13000g) for 1 min at room temperature. After the addition of 300 μL of binding buffer (XP2) and centrifugation, the DNA was collected in a clean 1.5 mL microcentrifuge tube by maximum-speed centrifugation. The samples were treated with 0.2 mL of hot (∼90 °C) concentrated nitric acid for 2 h, and the total platinum content was determined by ICP-MS. DNA platination percentage = (platinum in nicked and linear DNA)/(platinum in nicked and linear DNA) + (platinum in supercoiled DNA). Two independent experiments were performed in triplicate, and the values are the means ± standard deviation. Reactions with FOH. BPP (6.3 mg, 0.0098 mmol) was incubated with FOH (2.17 mg, 0.0098 mmol) in PBS (1.0 mL) at 310 K, and the reaction solution was monitored by ESI-MS at 48 h. Acute Toxicity. Mice were housed in cages in a ventilated room at 20 ± 2 °C and 60 ± 10% relative humidity with a 12 h light/dark cycle. Water and commercial laboratory complete food for mice were available ad libitum. They were acclimated to this environment for 5 days before treatment. Determination of the median lethal dose (LD50) was carried out in accordance with the method provided by the OECD guideline 423. Prior to dosing, food but not water was withheld for 4 h. Stock solutions of BPP were homogeneously suspended in PBS prior to injection. Different concentrations of the complexes were intravenously administered to each group of four mice. The group that received an identical anount of PBS served as a control. After injection, the changes in the body weight were recorded over a period of 14 days. The survival rate of the mice was calculated as (number of live mice/total number of mice tested) × 100%. The LD50 value of BPP was calculated according to the OECD guideline 425. Animal assays were performed in compliance with the local ethnics committee. Reactions with GSH. BPP (3.61 mg, 0.0056 mmol) was dissolved in D2O (0.5 mL) in an NMR tube. GSH (2.45 mg, 0.0132 mmol) was added to the NMR tube, and the obtained solution was monitored by 31 P and 1H NMR and ESI-MS at different time intervals while the samples were kept at 310 K.

+ H] + (C 15 H 36 N 3 O 7 P 2 Pt), 627.25 (627.16); [PtL(NH 3 ) 2 Cl] + (C15H33N3O6P2ClPt), 643.25 (643.12); {2[PtL(NH3)2Cl] + Cl}+ (C30H66N6O12P4Cl3Pt2), 1321.92 (1321.21). HPLC Study. The sample was centrifuged, and the supernatant was subjected to analysis at 220 nm via a Beckman Coulter HPLC instrument equipped with a C18 reverse-phase column (eluent: 50/50 H2O/CH3OH). X-ray Crystallography. X-ray crystallography data were collected on a Bruker SMART APEX CCD area-detector diffractometer operating in the φ−ω scan mode with graphite-monochromated Mo Kα radiation (λ = 0.71073 Å) at 298 K. Empirical absorption corrections were carried out using a multiscan program. The SMART software was used for data acquisition and the SAINT software for data extraction.31 The structure of BPP was solved by direct methods and refined on F2 by full matrix least-squares methods using the SHELXTL program.32 All non-hydrogen atoms were refined anisotropically. Hydrogen atoms were included in calculated positions and refined with isotropic thermal parameters. Lipophilicity Determination. The lipophilicity was measured in a 1-octanol/buffer system using the shake-flask method.15 Solutions of BPP (50, 100, 150, and 200 μM) were prepared in the phosphate buffer (10 mM, pH 7.4) presaturated with 1-octanol. Equal volumes (2.0 mL) of the solution and 1-octanol presaturated with the phosphate buffer were mixed and placed in a thermostatic (25.0 ± 0.1 °C) air-bath orbital shaker at 200 rpm for 4 h. The samples were separated into two phases after centrifugation at 2500 rpm for 15 min. The concentration of the solute in the aqueous phase was determined by spectrophotometry (λmax = 267 nm). According to the law of mass conservation, the drug concentration of the corresponding 1-octanol phase and the lipo−hydro partition coefficient Po/w (Po/w = Co/Cw = Ao/Aw, where A stands for absorbance) were calculated. Platinum Cellular Uptake. U2OS or LO2 cells (15 × 104) were seeded in two 6-well plates, allowed to grow for 18 h, and later treated with BPP and cisplatin (10.0 μM), respectively, for 24 h. After incubation, the medium was aspirated, and all of the wells were washed with phosphate-buffered saline (PBS) three times. The cells were trypsinized, and live cells were counted by the Trypan Blue method. Later on, cells were treated with 0.5 mL of hot (≈90 °C) concentrated nitric acid for 2 h. The samples were analyzed by ICP-MS to determine the total platinum content per well. The amount of platinum was calculated by subtracting the average amount of platinum found in the blank wells from the average amount of platinum found in the cell-containing wells and normalizing to the average number of cells per well. Anticancer Activity Assays. The cytotoxicity of BPP was tested against the human OS cell lines (U2OS and MG-63) and the human hepatic cell line (LO2) by MTT assay.33 Briefly, the U2OS, MG-63, and LO2 cells were inoculated in 96-well plates and incubated in different media. U2OS and LO2 were cultured in Dulbecco’s modified Eagle’s medium with 10% heat-inactivated fetal bovine serum. MG-63 cells were cultured in a minimum essential medium with 10% heatinactivated fetal bovine serum. These cells were cultured at 37 °C in an atmosphere of 5% CO2 and 95% air and 100% relative humidity overnight. Different concentrations of BPP or BPP plus FOH (10.0 or 20.0 μM) in a PBS buffer solution (pH 7.4) were added to the culture medium to reach specific concentrations. After the cells were incubated for an additional 48 h, MTT (20 μL, 5 mg mL−1 in PBS) was added to each well. The culture plates were centrifuged, and the medium was removed after another 4 h of incubation. DMSO (200 μL) was added to each well, and the absorbance of dissolved formazan was measured at 570 nm using an ELISA plate reader. The mean of three independent results was taken as the inhibition rate (%) or IC50. Cell Cycle Analysis. U2OS or LO2 cells were exposed to 5.0 μM BPP or cisplatin at 310 K under 5% CO2 for 48 h. Trypsin was added to the culture medium,and adherent cells were collected after mild centrifugation (1000 rpm, 179g force). The cells were washed with PBS, fixed in an ice-cold ethanol (70%) for 6 h, pelleted by centrifugation, washed with PBS, incubated with RNase A for 30 min at 310 K, and stained with PI (1 mg mL−1) for another 30 min at 273 F

DOI: 10.1021/acs.inorgchem.7b03261 Inorg. Chem. XXXX, XXX, XXX−XXX

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(7) Wang, X. Y.; Guo, Z. J. Targeting and delivery of platinum-based anticancer drugs. Chem. Soc. Rev. 2013, 42, 202−224. (8) Rogers, M. J.; Crockett, J. C.; Coxon, F. P.; Mönkkönen, J. Biochemical and molecular mechanisms of action of bisphosphonates. Bone 2011, 49, 34−41. (9) Fournier, P. G. J.; Daubiné, F.; Lundy, M. W.; Rogers, M. J.; Ebetino, F. H.; Clézardin, P. Lowering bone mineral affinity of bisphosphonates as a therapeutic strategy to optimize skeletal tumor growth inhibition in vivo. Cancer Res. 2008, 68, 8945−8953. (10) Cole, L. E.; Vargo-Gogola, T.; Roeder, R. K. Targeted delivery to bone and mineral deposits using bisphosphonate ligands. Adv. Drug Delivery Rev. 2016, 99, 12−27. (11) (a) Bose, R. N.; Maurmann, L.; Mishur, R. J.; Yasui, L.; Gupta, S.; Grayburn, W. S.; Hofstetter, H.; Salley, T. Non-DNA-binding platinum anticancer agents: Cytotoxic activities of platinum-phosphato complexes towards human ovarian cancer cells. Proc. Natl. Acad. Sci. U. S. A. 2008, 105, 18314−18319. (b) Qiu, L.; Lv, G. C.; Cao, Y.; Chen, L. P.; Yang, H.; Luo, S. N.; Zou, M. F.; Lin, J. G. Synthesis and biological evaluation of novel platinum complexes of imidazolylcontaining bisphosphonates as potential anticancer agents. JBIC, J. Biol. Inorg. Chem. 2015, 20, 1263−1275. (c) Galanski, M.; Slaby, S.; Jakupec, M. A.; Keppler, B. K. Synthesis, characterization, and in vitro antitumor activity of osteotropic diam(m)ineplatinum(II) complexes bearing a N,N-bis(phosphonomethyl)glycine ligand. J. Med. Chem. 2003, 46, 4946−4951. (d) Margiotta, N.; Capitelli, F.; Ostuni, R.; Natile, G. A new dinuclear platinum complex with a nitrogencontaining germinal bisphosphonate as potential anticancer compound specifically targeted to bone tissues. J. Inorg. Biochem. 2008, 102, 2078−2086. (e) Margiotta, N.; Ostuni, R.; Gandin, V.; Marzano, C.; Piccinonna, S.; Natile, G. Synthesis, characterization, and cytotoxicity of dinuclear platinum-bisphosphonate complexes to be used as prodrugs in the local treatment of bone tumors. Dalton Trans. 2009, 48, 10904−10913. (f) Tušek-Božić, L.; Juribašić, M.; Scarcia, V.; Furlani, A. Platinum(II) complexes of 8-quinolylmethylphosphonates: Synthesis, characterization and antitumor activity. Polyhedron 2010, 29, 2527−2536. (g) Margiotta, N.; Ostuni, R.; Piccinonna, S.; Natile, G.; Zanellato, I.; Boidi, C. D.; Bonarrigo, I.; Osella, D. Platinumbisphosphonate complexes have proven to be inactive chemotherapeutics targeted for malignant mesothelioma because of inappropriate hydrolysis. J. Inorg. Biochem. 2011, 105, 548−557. (12) Zhang, Z. Q.; Wang, X. Y.; Luo, C.; Zhu, C. C.; Wang, K.; Zhang, C. L.; Guo, Z. J. Dinuclear platinum(II) complexes with bonetargeting groups as potential anti-osteosarcoma agents. Chem. - Asian J. 2017, 12, 1659−1667. (13) (a) Lovejoy, K. S.; Todd, R. C.; Zhang, S.; McCormick, M. S.; D’Aquino, J. A.; Reardon, J. T.; Sancar, A.; Giacomini, K. M.; Lippard, S. J. cis-Diammine(pyridine)chloroplatinum(II), a monofunctional platinum(II) antitumor agent: Uptake, structure, function, and prospects. Proc. Natl. Acad. Sci. U. S. A. 2008, 105, 8902−8907. (b) Wang, D.; Zhu, G.; Huang, X.; Lippard, S. J. X-ray structure and mechanism of RNA polymerase II stalled at an antineoplastic monofunctional platinum-DNA adduct. Proc. Natl. Acad. Sci. U. S. A. 2010, 107, 9584−9589. (14) Manallack, D. T.; Prankerd, R. J.; Yuriev, E.; Oprea, T. I.; Chalmers, D. K. The significance of acid/base properties in drug discovery. Chem. Soc. Rev. 2013, 42, 485−496. (15) Reithofer, M. R.; Bytzek, A. K.; Valiahdi, S. M.; Kowol, C. R.; Groessl, M.; Hartinger, C. G.; Jakupec, M. A.; Galanski, M.; Keppler, B. K. Tuning of lipophilicity and cytotoxic potency by structural variation of anticancer platinum(IV) complexes. J. Inorg. Biochem. 2011, 105, 46−51. (16) Oldfield, S. P.; Hall, M. D.; Platts, J. A. Calculation of lipophilicity of a large, diverse dataset of anticancer platinum complexes and the relation to cellular uptake. J. Med. Chem. 2007, 50, 5227−5237. (17) (a) Puckett, C. A.; Ernst, R. J.; Barton, J. K. Exploring the cellular accumulation of metal complexes. Dalton Trans. 2010, 39, 1159−1170. (b) Poynton, F. E.; Bright, S. A.; Blasco, S.; Williams, D. V.; Kelly, J. M.; Gunnlaugsson, T. The development of ruthenium(II)

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.inorgchem.7b03261. Supplementary tables and figures concerning NMR and ESI-MS spectra of the ligand, BPP, and GSH, UV spectra of BPP, crystallographic data, cellular uptake, cytotoxic profile, cell cycle analysis, and CD spectra (PDF) Accession Codes

CCDC 1491820 contains the supplementary crystallographic data for this paper. These data can be obtained free of charge via www.ccdc.cam.ac.uk/data_request/cif, or by emailing data_ [email protected], or by contacting The Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033.

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AUTHOR INFORMATION

Corresponding Authors

*Tel: +862589684549. Fax: +862583314502. E-mail: zguo@ nju.edu.cn (Z.G.). *Tel: +862589684549. Fax: +862583314502. E-mail: boxwxy@ nju.edu.cn (X.W.). ORCID

Zijian Guo: 0000-0003-4986-9308 Xiaoyong Wang: 0000-0002-8338-9773 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the National Natural Science Foundation of China (Grants 31570809, 91213305, and 31700714), National Basic Research Program of China (Grants 2015CB856300 and 2011CB935800), Natural Science Foundation of Jiangsu Province (Grant BK20150054), and Scientific Research Funds of Nanjing Medical University (Grant KY109NJMUZD15024).

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REFERENCES

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DOI: 10.1021/acs.inorgchem.7b03261 Inorg. Chem. XXXX, XXX, XXX−XXX