2.1. Materials, Measurements, and Data Analysis

Cisplatin, (3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), 2-(chloromethyl)pyridine hydrochloride, human serum albumin (HSA), and L-glutathione (GSH) were purchased from sigma. Supercoiled pUC19 plasmid DNA and 6X loading buffer (0.05% bromophenol blue, 0.035% xylene cyanol FF, 36% glycerol, and 20 mM EDTA) were purchased from Takara Biotechnology (Dalian) Co., Ltd. Calf thymus DNA (CT-DNA), Tris and ethidium bromide (EB) were purchased from Sunshine Bio. Co., Ltd (Nanjing, China). All chemicals were used as received without further purification unless otherwise stated. The human osteosarcoma cell lines U2OS and MG-63, the human breast carcinoma cell line MCF-7 and the human cervical cancer cell line HeLa were originated from the Cancer Institute & Hospital, Chinese Academy of Medical Sciences.

Anhydrous N, N-dimethylformamide (DMF), and dimethylsulfoxide (DMSO) were prepared by refluxing or stirring the commercial reagent in the presence of calcium hydride for 72 hours. 2-[2-pyridinylethylidene]bis[phosphonic acid] tetraethyl ester was synthesized according to the previous literature [26].

The ¹H, ¹³C, ³¹P, and ¹⁹⁵Pt NMR spectra were acquired on a Bruker DRX-500 spectrometer. Electrospray ionization mass spectrometry (ESI-MS) spectra were recorded using the LCQ fleet ESI-MS spectrometer (Thermo Scientific), and the isotopic distribution pattern for the complex was simulated using the ISOPRO 3.0 program. Elemental analyses were carried out with Elementar Vario ELIII. FT-IR spectra were recorded on a Tensor-27 FT-IR spectrometer (Bruker, Germany) with a resolution of 2 cm⁻¹ and a spectral range of 4000–400 cm⁻¹. The high-performance liquid chromatography (HPLC) sample was centrifuged, and the supernatant was subjected to analysis at 220 nm via an SPD-20AV Shimadzu HPLC instrument equipped with a C18 reverse-phase column (eluent: 50/50 H₂O/CH₃OH). 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 UV-vis spectra were determined on a Shimadzu UV 3600 (UV-vis-near-IR) spectrophotometer. 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 inductively coupled plasma mass spectrometry (ICP-MS) data were obtained on ELAN9000 ICP-MS (PerkinElmer Inc., U. S. A.). The optical density (OD) of formazan was tested on ThermoScientific Varioskan Flash.

All cell experiments were conducted in triplicate. Statistical analyses were performed with the SPSS software. Data were expressed as the mean ± standard deviation.

2.2. Synthesis of Tetraethyl 2,2-Bis(2-Pyridinylmethyl) Methylidene-1,1-Bisphosphonate (Ligand)

2-Chloromethyl pyridine hydrochloride (5.292 g, 32 mmol) was dissolved in water (7 mL), and the solution was made alkaline by adding sodium hydroxide (1.344 g, 32 mmol) to water (14 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, 2-[2-pyridinylethylidene]bis(phosphonic acid) tetraethyl ester (9.31 g, 24.56 mmol) in 20 mL dry DMSO was added to a mixture of 60% N (1.08 g, 29.47 mmol) in DMSO 20 mL at 0°C under nitrogen. The reaction mixture was stirred at 0°C for 30 min then at room temperature for 2 h. 2-Chloromethylpyridine (2.82 g, 20 mmol) was then added dropwise to the stirring reaction mixture. The reaction was allowed to stir for an additional 96 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 H₂O and brine, dried over Na₂SO₄, and concentrated under a vacuum. The yellow product is purified by flash chromatography with isopropyl alcohol in methylene chloride in silica gel. ¹H NMR (500 MHz, CDCl₃): δ 8.60 (s, 2 H), 7.77 (d, J = 7.74 Hz, 2 H), 7.58 (t, J = 7.42 Hz, 2 H), 7.15 (s, 2 H), 4.02–3.97 (m, 8 H), 3.64 (dd, J = 12.15, 2.58 Hz, 4 H), and 1.12 (td, J = 7.05, 2.03 Hz, 12 H). ¹³C NMR (125 MHz, CDCl₃): δ 158.14, 148.37, 135.39, 126.55, 121.43, 62.46, 50.55, 36.71, and 16.16. ³¹P NMR (202 MHz, CDCl₃): δ 23.84. IR (KBr) cm⁻¹: 3431, 2977, 2930, 2869, 1624, 1584, 1470, 1437, 1390, 1356, 1250, 1022, 968, 874, 807, 686, 613, and 513. ESI-MS (m/z, positive mode). Found (calcd): [Ma + H]⁺, 471.33 (471.44), [2M + Na]⁺, and 963.17 (963.88).

2.3. Synthesis of DBPP

Cisplatin (75 mg, 0.25 mmol) and AgNO₃ (42.5 mg, 0.25 mmol) were stirred in anhydrous DMF (4.0 mL) in the dark at 40°C for 24 h. The resulting AgCl precipitate was removed by filtration and Ligand (94.27 mg, 0.2 mmol) was added to the filtrate. After stirring for an additional 24 h at 40°C, AgNO₃ (34.0 mg, 0.20 mmol) was added to the solution, and the solution was stirred for another 24 h at 40°C. The resulting AgCl precipitate was removed by filtration, and CH₂Cl₂ (100 mL) was added to the reaction mixture. 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). A yellow precipitate of DBPP was formed at a 58% yield. ¹H NMR (500 MHz, CDCl₃): δ 9.44 (s, 2 H), 7.68 (t, J = 7.85 Hz, 2 H), 7.58 (d, J = 7.75 Hz, 2 H), 7.45 (t, J = 5.04 Hz, 2 H), 5.05 (s, 4 H),4.42 (m, 6 H), 4.11 (t, J = 13.56 Hz, 2 H), 3.67 (s, 4 H), 1.87 (s, 6 H), and 1.50 (t, J = 7.01 Hz, 6 H); ¹³C NMR (125 MHz, CDCl₃): δ 161.02, 152.68, 138.44, 129.96, 125.19, 64.77, 42.26, 16.55, and 15.70; ³¹P NMR (202 MHz, CDCl₃): δ 22.88, 20.55; ¹⁹⁵Pt NMR (107 MHz, CDCl₃): δ−2287.651. IR (KBr) cm⁻¹: 3433, 3225, 2990, 1611, 1484, 1443, 1376, 1356, 1223, 1049, 968, 780, 653, 607, and 533. ESI-MS (m/z, positive mode). [C₂₁H₃₈N₄O₆P₂Pt]⁺ found (calcd.): 698.33 (699.19); [C₂₁H₃₈N₄O₆P₂Pt]²⁺ found (calcd.): 349.92 (349.59). Calculated for C₂₁H₃₈N₆O₁₂P₂Pt: C, 30.62; H, 4.65; N, 10.20. Found: C, 30.68; H, 4.42; N, 10.13%. The purity of DBPP was determined to be >98% by HPLC. The retention time was 5.07 minute.

2.4. Computational Modeling

Electronic properties of the chemical structures of the ligand and DBPP were calculated using density functional theory (DFT). In particular, the hybrid functional B3LYP, electron basis sets 6-311G (d, p), and an effective core potential LanL2DZ basis set as implemented in the Gaussian09 suite of programs were used for full geometry optimization. In all calculations, the integral equation form (IEFPCM) of the polarization continuum model was used to simulate the solvent (water) environment. The frequencies of all geometries are calculated at the same level. HOMO (highest occupied molecular orbital) and LUMO (lowest unoccupied molecular orbital) were calculated for the optimized geometries.

2.5. Anticancer Activity Assays

The in vitro MTT assay was carried out according to a previously reported procedure [28]. Briefly, the U2OS (osteosarcoma), MG-63 (osteosarcoma), MCF-7 (breast carcinoma), and HeLa (cervical cancer) cells were inoculated in 96-well plates and incubated in different media. U2OS was maintained in DMEM with 10% fetal bovine serum. MG-63, MCF-7, and HeLa cells were maintained in RPMI 1640 medium with 10% fetal bovine serum. These cells were cultured at 37°C in an atmosphere of 5% CO₂ and 95% air and 100% relative humidity overnight. After plating, different concentrations of DBPP in PBS were added to the above culture medium. After the cells were incubated for another 48 h, MTT (20 μL, 5 mg mL⁻¹ in PBS) was added to each well. After another 4 h of incubation, the culture plates were centrifuged, and the medium was removed. Then DMSO (200 μL) was added to each well, and the absorbance of dissolved formazan was measured at 570 nm by an ELISA plate reader. The inhibition rate (%) or IC₅₀ was the mean of three independent results.

2.6. Lipophilicity Determination

Lipophilicity determination was carried out by using the 1-octanol/buffer system of the shake-flask method [29]. Briefly, solutions of DBPP (50, 100, 150, and 200 μM) were prepared in the phosphate buffer (10 mM, pH 7.4) which was presaturated with 1-octanol. 2.0 mL of the solution and equal volumes of 1-octanol were mixed and placed in a thermostatic (25.0 ± 0.1°C) air-bath orbital shaker at 200 rpm for 4 h. After centrifugation at 2500 rpm for 15 min, the samples were separated into two phases. According to the law of mass conservation, the concentration of the solute in the aqueous phase was determined by spectrophotometry (λ_max = 267 nm). The drug concentration of the corresponding 1-octanol phase and the lipo-hydro partition coefficient P_o/w(C_o/Cw = A_o/A_w), where A stands for absorbance) were calculated.

2.7. Platinum Cellular Uptake

U2OS cells (15 × 10⁴) were seeded in 6-well plates, which were allowed to grow for 18 h, then treated with DBPP (50.0 μM and 100.0 μM) for 24 h, 48 h, and 72 h, respectively. After incubation, the medium was removed 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. After that, cells were suspended in 500 μL PBS and digested with 0.5 mL of hot (≈95°C) concentrated nitric acid for 2 h, followed by the addition of H₂O₂ (50 μL) and HCl (100 μL). The obtained homogenized solution was diluted by water and 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 it to the average number of cells per well.

2.8. Studies on DNA Interaction

The DNA binding experiments were performed in Tris-HCl/NaCl buffer (5 mM Tris-HCl, 50 mM NaCl, pH 7.4) using different concentrations of DBPP. After CT-DNA was incubated with different ratios of DBPP at 37°C for 24 h in the dark, CD spectra were recorded in the wavelength range of 220–320 nm. Each CD spectrum was recorded after averaging over at least 3 accumulations, and the buffer background was subtracted.

The impact on the secondary structure of plasmid DNA was determined by agarose gel electrophoresis. pUC19 plasmid DNA (200 ng) was treated with different concentrations of DPP at 37°C for 24 h. All the samples were finally loaded onto a 1% agarose gel containing EB (0.5 μg·mL⁻¹). Electrophoresis was carried out for 2 h at 100 V in a TAE buffer (50 mM Tris-acetate and 1 mM ethylenediaminetetraacetic acid). The resulting bands were visualized under UV light. The resulting photographs were obtained by using a Bio-Rad Gel-Doc XR imaging system.

2.9. Interaction with HSA

Reactions of DBPP with HSA were carried out in PBS (10 mM, pH 7.4). Reactions were monitored by UV-Vis and absorption spectra were obtained after HSA (3.0 μM) with different concentrations of DBPP in PBS at 37°C for 24 h in the dark.

2.10. Solubility Test

Solubility testing was carried out using distilled water and chloroform as test media. Each test was repeated 3 times. One milliliter of each test medium was put in a glass tube and stirred with a constant speed of 80 rpm for 10 min after the start of the test. Then, the samples were incubated at 37°C for 30 min and centrifuged for 10 min at 10,000 rpm. The solvent of each sample was removed, and the dry sample was redissolved in 1.0 mL of distilled water. The platinum concentration of the samples was determined by ICP-MS. The dissolution values were calculated and expressed in the form of Mol/L (M).

2.11. Stability of DBPP

DBPP (ca. 0.0056 mmol) was dissolved in a solution of deuterated aqueous and placed into an NMR tube. GSH (2.45 mg, ca. 0.0132 mmol) was added to the same NMR tube. Then, the tube was kept at 37°C, and the obtained solution was monitored by ¹H NMR and ³¹P NMR at different time intervals.

3.1. Characterization

Preparation of DBPP was conducted according to the procedure shown in Scheme 1. Briefly, the ligand was straightforwardly obtained from nucleophilic substitution reaction of 2-(chloromethyl)pyridine hydrochloride and 2-[2-pyridinylethylidene]bis(phosphonic acid) tetraethyl ester. Then, the ligand was reacted with cationic cis-[Pt(NH₃)₂Cl(DMF)]⁺, after which AgNO₃ was added to afford DBPP in high yield. Since there are two pyridyl rings in DBPP, it is assumed that DBPP is more difficult to react with GSH. In addition, the coordination of the ligand with platinum (II) affords an eight-membered ring. To the best of our knowledge, it is the first example reported till now.

The ligand and the resulting platinum complex (DBPP) have been characterized by using NMR, ESI-MS, and IR spectroscopy. Compared to the free ligand, the aromatic proton signals in the ¹H NMR data of DBPP significantly shift to a lower field, indicating that the pyridine nitrogen has been coordinated to the Pt^II center. Additionally, ³¹P NMR data of the free ligand provided a singlet at 23.84 ppm, whereas two new signals at 22.88 and 20.55 ppm were observed for the DBPP indicating a change in the P atoms’ signals as a result of coordination with platinum. Moreover, the ¹⁹⁵Pt NMR signal from DBPP falling at −2287.651 ppm further confirmed that the Pt^II complex was successfully developed. The IR spectrum of the ligand and DBPP complex showed a POO-stretching band in the 1000–1250 region [30]. The ESI-MS spectrum of DBPP showed two main peaks at m/z 349.92 and 698.33, which could be ascribed to the positively charged species [C₂₁H₃₈N₄O₆P₂Pt]²⁺ and [C₂₁H₃₈N₄O₆P₂Pt]⁺, respectively.

3.2. DFT Calculation

We carried out the DFT calculation using a collection of Gaussian 09 programs in order to theoretically comprehend the coordination of DBPP [31–33]. Figure 1 demonstrates how the central Pt²⁺ coordinates with two N atoms from NH₃ and two N atoms from the pyridine units, the latter of which almost stand in a straight line in opposite directions. Figure 1 also displays the spatial distributions of the HOMO and LUMO of the ligand and DBPP. The ligand’s electron density, whether HOMO or LUMO, is evenly distributed, primarily on the pyridine unit. In contrast, the majority of the electrons in the HOMO orbital are distributed on the N-Pt binding site in the DBPP upon binding with platinum ions. Additionally, DBPP’s energy gap (E) between HOMO and LUMO is 3.28 eV, lower than the ligand’s (5.98 eV), indicating that DBPP is more stable than the free ligand.

3.3. Antiproliferative Activity

The cytotoxic potency of DBPP was evaluated. As shown in Table 1, DBPP did not show significant activity against MG-63, MCF-7, and HeLa cell lines at a concentration of 50 and 100 μM after 24 h incubation. However, DBPP exhibits a significant inhibitory effect on U2OS cells’ ability to proliferate, indicating a higher selectivity than cisplatin. The high systemic toxicity of cisplatin is thought to be associated with nonselectivity [34]. By redirecting drugs at the specific site of injury or disease is the most promising strategy to compel the associated drug resistance and may be helpful in lowering systematic toxicities [35]. The difference in selectivity between DBPP and cisplatin may be due to the presence of a bisphosphonate moiety in DBPP as opposed to cisplatin.

At the concentration of 50 and 100 μM, the relative cellular viability is 85.8% ± 1.6% and 55.7% ± 3.0%, respectively (Table 1). With the extension of incubation time from 24 h to 48 h, the relative cellular viability reduced to 65.6% ± 3.8% and 48.2% ± 0.9%, respectively. When the incubation time was extended to 72 h, the relative cellular viability reduced to 58.3% ± 4.8% and 30.7% ± 1.6%, respectively (Table 2). The 50% inhibition appeared at concentrations up to about 60 μM. In contrast, cisplatin was more cytotoxic. For example, the relative cellular viability of cisplatin toward MG-63 at 10.0 μM concentration for 24 h is 59.0% ± 1.0%.

Analysis of the structure-activity relationship revealed that the leaving group has an important influence on the cytotoxicity. The result of cytotoxicity indicated that DBPP which has no leaving group in the structure, shows moderate cytotoxicity against U2OS cells but is quite less potent than cisplatin and BPP, the latter of which have at least one leaving the group in the structure [26].

3.4. Lipophilicity and Cellular Uptake

The lipophilicity parameter (log  P) is a crucial factor in membrane transport [36, 37]. Here, the log  P value of DBPP was determined as water-octanol partition coefficients using the shake-flask method. The log  P is calculated to be −1.1 ± 0.1 (Figure 2), showing that DBPP is more lipophilic than BPP (log  P = −1.7) [26] and cisplatin (log  P = −2.3) [38].

The U2OS cellular uptake of DBPP was determined by ICP-MS. When U2OS cells were treated with 10.0 μM cisplatin for 24 h, the concentration of cellular was 2.6 ± 0.2 μg/10⁶ cells. However, treating U2OS cells with 50.0 μM of DBPP for 24 h only resulted in 0.6 ± 0.5 μg/10⁶ cells concentration of cellular platinum (Table 3), which was quite lower than that of cisplatin. Prolonging the incubation time to 48 h or 72 h could not bring about a significant increase in cell uptake. After exposure to 100.0 μM of DBPP for 48 and 72 h, the concentration of cellular platinum rose to 3.7 ± 0.5 and 5.0 ± 0.3 μg/10⁶ cells, respectively. In addition, the results indicate that the Pt uptake is correlated with the cytotoxicity.

3.5. Interaction with DNA

Since DNA is the primary target of Pt (II)-based antitumor complexes, to get more insight into the antitumor activity of DBPP, the ability of DBPP to bind calf thymus DNA (CT-DNA) and unwind supercoiled pUC19 DNA was investigated. Figure 3(a) displays the circular dichroism CD spectra of CT-DNA in the absence or presence of varying amounts of DBPP, where the negative band at 245 nm and the positive one at 275 nm represent the characteristics of B-DNA. With the rising of the [DBPP]/[DNA] ratio from 0 to 0.8, the intensity of the negative bands decreases and the positive bands increases. Moreover, the maximum wavelength of the negative bands has a tendency to redshift. Such a changing tendency indicates a conformational conversion from B-DNA to A-DNA. The changes of the negative and positive bands are quite similar to that of DNA modified by cisplatin and this suggests that DBPP can induce a similar formation of intrastrand cross-links with cis-[PtCl₂(Py)₂] [39]. Figure 3(b) shows the DNA unwinding property of DBPP by native agarose gel electrophoresis using pUC19 DNA. Upon incubation with DBPP, retardation of supercoiled DNA was observed, indicating untwisting by DNA binding. Additionally, the separation between supercoiled and relaxed DNA decreases as the molar ratio (r_i) increases. The coalescence point r_i (c) for complete removal of supercoiled DNA is 1.8, which is higher than that of cisplatin (r_i (c) = 0.076) [40]. The results suggest that DBPP cannot perturb the tertiary structure of supercoiled DNA as efficiently as cisplatin, which is consistent with their cytotoxicity.

3.6. Interaction with HSA

HSA plays a crucial role in the transport of drugs because of its remarkable binding properties and the highest abundance in blood plasma [41]. UV-Vis spectrophotometry was used as a simple method to explore the interaction of DBPP with HSA. The effect of DBPP on the UV-Vis absorption of HSA is shown in Figure 4. With the addition of DBPP, the absorption intensity of HSA was enhanced with an obvious blue shift of about 12 nm (from 278 to 266 nm) indicating that the microenvironment of tryptophan (Trp), tyrosine (Tyr), and phenylalanine (Phe) residues were altered and they were extended into the aqueous milieu [42]. These observations confirm that DBPP can destroy the tertiary structure of HSA and form a novel complex with HSA.

3.7. Stability of DBPP

Having the U2OS selectively inhibited platinum complex in hand, we next turned our attention to investigating the reactivity between DBPP and Glutathione (GSH). GSH is a soluble tripeptide whose intracellular concentrations are between 0.5 to 10 mM. As a nucleophilic molecule, GSH could chelate with platinum (II) to form a Pt (GS)₂ conjugate, which is then exported out of the cells. It would decrease the platinum (II)-mediated DNA damage and weaken the therapeutic efficacy. The interactions between GSH and Pt (II) complexes are therefore considered to play a critical role in mechanisms that are relevant to the inactivation of platinum complexes, their resistance, and side effects [43–45].

Figure 5 demonstrated that DBPP has good solubility in chloroform. Since solubility affects the bioavailability and individual variability of drugs [46], we carried out the solubility test to explore the details of the solubility of DBPP. The solubility values of DBPP in distilled water and chloroform were 0.13 and 0.084 M, respectively. Therefore, the reaction of DBPP with GSH in D₂O was studied at millimolar concentrations at 37°C by ³¹P NMR and ¹H NMR.

As shown in Figure 6(a), the peaks of DBPP are located at 24.88 and 20.09 ppm. After even 168 hours incubation, no obvious changes were observed, indicating that DBPP is quite inert to GSH. Similar results were observed in Figure 6(b). Overall, these results demonstrate that DBPP can hardly chelate with GSH. This is a promising result since GSH-Pt conjugates cannot form, suggesting that DBPP could avoid the GSH-induced side effects. It is worth mentioning that due to the high inertness of GSH, the cytotoxicity deficiency of DBPP may be compensated by raising the therapeutic dose.