English

• 0.   Introduction

  Platinum-based drugs are classic anti-cancer chemotherapeutics and are still widely used as first - line therapy to treat solid neoplasms, mainly because of their broad - spectrum anticancer activities, their distinct therapeutic effects, and their preferable economy^[1-2]. Currently, approximately 50% of all chemotherapy regimens including platinum - based drugs are utilized as the main therapy or co-therapy for clinical cancer treatment^[3]. Commonly used platinum - based drugs include cisplatin, carboplatin, oxaliplatin, nedaplatin, lobaplatin, and heptaplatin. All of these drugs are square-planar platinum(Ⅱ) complexes with two leaving groups and two non -leaving ligands coordinated to the Pt(Ⅱ) center^[1]. Because of their structural similarity, platinum - based drugs share many general characteristics. Hence, there are indeed existing some common problems when referring to the advantages and disadvantages of platinum - based drugs. And this clinical dilemma is a serious impediment to the development of new platinum-based drugs with novel antitumor mechanisms. More unfavorably, platinum-based drugs generally demonstrate several serious side effects, including nephrotoxicity, myelosuppression, neurotoxicity, and ototoxicity, which dramatically limit their clinical use^[4-7]. Another major obstacle to developing new platinum compounds with novel antitumor mechanisms is drug resistance^[8-9]. It is therefore of vital importance to optimize the chemical properties, bioactivities and metabolic pathways, and mechanism of actions of novel platinum-based compounds.

  Among the strategies used to overcome these adversities, one of the most common approaches is to design and exploit monofunctional platinum complexes which contain only one leaving group (in contrast with traditional platinum drugs) ^[10-14]. Under this principle, many monofunctional Pt complexes have been thoroughly synthesized, and their anticancer activities and mechanisms of action have been investigated^[15-20]. In comparison with classical bifunctional drugs such as cisplatin, which forms bifunctional DNA adducts, monofunctional Pt complexes interact with DNA in a different manner, forming only Pt-G monoadducts^[21-23]. Furthermore, most monofunctional Pt complexes undergo hydrolysis at a slower rate (than classical bifunctional Pt drugs), and they are less reactive to thiol molecules (e. g., glutathione) in vivo, mainly due to their comparative chemical inertness and stronger hydrophobicity^[24]. Conclusively, monofunctional Pt(Ⅱ) complexes bind only one strand of DNA and might target cancer without causing auditory side effects, thus having more potential development and application significance than bifunctional drugs, including cisplatin.

  Considering efforts to improve the anticancer activities of platinum - based agents, the selection of non-leaving ligands is especially important when designing and preparing monofunctional platinum complexes. Groups containing nitrogen (N) donors are usually employed as leaving groups due to the thermodynamic stability of the Pt—N bonds. Thus, many monofunctional platinum complexes adopt a tridentate Pt-NNN system. For instance, Liang′s group synthesized two monofunctional Pt(Ⅱ) complexes containing jatrorrhizine derivative ligands ([Pt(Jat1)Cl]Cl and [Pt(Jat2) Cl]Cl). Both of these compounds displayed remarkable antitumor activity on HeLa cells (with IC₅₀ values of 15.01 and 1.00 nmol·L^-1, respectively), and this antitumor activity was achieved by targeting p53 and telom-erase^[25]. Guo and co - workers developed a triphe-nylphosphonium-modified terpyridine platinum(Ⅱ) com-plex (TTP). TTP exhibited excellent cytotoxicity against cisplatin - insensitive human ovarian cancer cells and preferential inhibition to mitochondrial TrxR^[26].

  In the present study, a novel binuclear monofunctional Pt complex with a Pt-NNN coordination mode, [Pt₂(BPA-TPA)Cl₂]Cl₂ (Pt₂-BPA-TPA, BPA - TPA=2, 6 - bis((bis(pyridin - 2 - ylmethyl)amino)methyl)pyridine), was synthesized (Scheme 1). Pt₂-BPA-TPA was then characterized by NMR and high-resolution mass spectroscopy (HRMS). X - ray single crystal diffraction was also used to determine the structure of Pt₂-BPA-TPA. Thereafter, the in vitro DNA cleavage activity of Pt₂-BPA-TPA and its toxicity in the A549 cancer cell line were determined. Besides, we provided evidence for a credible molecular mechanism underlying its antitumor activities. Together, our results prove that Pt₂-BPA-TPA is a promising candidate for novel anticancer therapy.

  Scheme 1

  

  Scheme 1.  Synthetic routes of ligand BPA-TPA and complex Pt₂-BPA-TPA

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  1.   Experimental

  1.1   Materials and physical measurements

  All reagents were commercially obtained and, unless otherwise stated, were used without further purification. 2, 6 - Pyridinedimethanol was purchased from Nanjing Chemlin Co., Ltd. Bis-(2-pyridylmethyl) amine (DPA) and K₂PtCl₄ were obtained from Adamas Reagent Co., Ltd. CCK-8 (cell counting kit-8) was purchased from Dojindo Molecular Technologies Inc., Tokyo, Japan.

  NMR spectra were measured on an AVANCE Ⅲ HD 400 MHz spectrometer. Electrospray ionization (ESI) mass spectra were obtained using an LCQ Fleet ThermoFisher mass spectrometer. Fluorescence spectra and UV - Vis absorption spectrum were determined on a Hitachi F - 4700 fluorescence spectrometer and VARIAN CARY 50 Conc UV - visible spectrophotometer, respectively. The crystal structure of the complex was determined on a Rigaku XtaLAB PRO Ⅱ singlecrystal diffractometer.

  1.2   Synthesis of complex Pt₂-BPA-TPA

  2,6-Bis(bromomethyl)pyridine was synthesized by previously reported methods^[27]. BPA - TPA was prepared according to the reference^[28]. A 2, 6 - bis(bromomethyl)pyridine (1.27 g, 4.8 mmol) solution, excess K₂CO₃ (1.59 g, 11.5 mmol), and DPA (1.92 g, 9.6 mmol) were added to CH₃CN under N₂ atmosphere and stirred at 30 ℃ for 2 h. After completion of the reaction, the mixture was filtered and the solvent was removed. The resulting yellow oil was extracted four times using boiling petroleum ether (b. p. 60 - 90 ℃) under vigorous stirring. BPA-TPA was then precipitated from the gradually cooled petroleum solution as white crystals. The purified product (2.41 g) was obtained with a yield of 56%. ¹H NMR (400 MHz, CDCl₃): δ 8.51 (d, J=4.8 Hz, 4H), 7.65 - 7.57 (m, 9H), 7.43 (d, J=7.7 Hz, 2H), 7.14 - 7.11 (m, 4H), 3.91 (s, 8H), 3.89 (s, 4H). 13C NMR (100 MHz, CDCl₃): δ 159.32, 158.61, 149.05, 136.87, 136.45, 123.00, 122.00, 121.17, 60.20. HRMS in CH₃OH (m/z): Calcd. [M+H]⁺ 502.272; Found: 502.322.

  K₂PtCl₄ (410 mg, 1 mmol) was first dissolved in 10 mL deionized water and then mixed with 10 mL methanol containing BPA-TPA (248 mg, 0.5 mmol) in a roundbottom flask. The resulting mixture was stirred in the dark for 48 h at room temperature. A beige precipitate was collected by centrifugation, and this was carefully washed with dichloromethane, ethanol, acetone, and anhydrous ether. The purified product of Pt₂-BPA -TPA (52 mg) was obtained with a yield of 21%. ¹H NMR (400 MHz, DMSO-d₆): δ 8.60 (d, J=5.0 Hz, 4H), 8.20-8.16 (m, 4H), 7.74 (d, J=7.9 Hz, 4H), 7.59 (s, 3H), 7.55 (t, J=6.4 Hz, 4H), 5.40 (d, J=15.7 Hz, 4H), 5.06 (d, J=15.8 Hz, 4H), 4.26 (s, 4H). HRMS in CH₃CN (m/z): Calcd. [M-Cl]⁺ 998.095 7; Found: 998.096 4. Calcd. [M-2Cl]²⁺ 481.065 2; Found: 481.063 7. Calcd. [M-2Cl+6H₂O]²⁺ 534.095 8; Found: 534.092 3.

  1.3   Single-crystal structure determination

  The crystal structure of Pt₂-BPA-TPA was determined by the single - crystal X - ray diffraction method. Whitish block single crystals were obtained upon vapor diffusion of ethyl acetate into a solution of the complex in DMSO. Data for Pt₂-BPA-TPA was collected on a hybrid pixel array detector (HPAD) at 293 K using graphite monochromatic Cu Kα radiation (λ =0.154 184 nm) in the multi - scan mode. The structure was solved by direct methods using the SHELXS program of the SHELXTL package and refined using full-matrix leastsquares methods with SHELXL (semi -empirical absorption corrections were applied using the SADABS program). SQUEEZE was adopted to reduce the effect originating from disordered solvent molecules. All hydrogen atoms were placed in a theoretical position. Crystal data and details of data collection and refinement for Pt₂-BPA-TPA are summarized in Table 1.

  Table 1

  

  Table 1.  Crystal parameters and structure refinements for Pt₂-BPA-TPA

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  Parameter	Pt2-BPA-TPA
  Chemical formula	C32H37Cl4N7O2Pt2
  Formula weight	1 083.66
  Temperature / K	293
  Crystal size / mm	0.06×0.05×0.04
  Crystal system	Monoclinic
  Space group	P21/c
  a / nm	1.259 36(3)
  b / nm	1.486 16(3)
  c / nm	2.187 45(5)
  β/(°)	105.058(2)
  V / nm3	3.953 48(16)
  Z	4
  Dc / (g·cm-3)	1.821
  μ / mm-1	15.832
  F(000)	2 072
  θ range for data collection / (°)	2.068 0-67.317 0
  Index ranges (h, k, l)	-15 ≤ h ≤ 14,
  	-11 ≤ k≤ 17,
  	-26 ≤ l ≤ 26
  Nref, Npar	6 885, 425
  Completeness / %	98.68
  Final R indices [I > 2σ(I)]	R1=0.091 7, wR2=0.246 3
  R indices (all data)	R1=0.097 7, wR2=0.251 1
  Goodness-of-fit on F2	1.028

  CCDC: 2115372.

  1.4   DNA cleavage studies

  DNA cleavage efficiency was assessed by agarose gel electrophoresis employing supercoiled pBR322 plasmid DNA. Briefly, supercoiled pBR322 plasmid DNA (200 ng) in 1×TAE buffer (Tris-HCl-EDTA) was treated with Pt₂-BPA -TPA or BPA -TPA (5 -20 μmol· L^-1) in a final volume of 20 μL. All samples were incubated at 37 ℃ for 1.5 h. After treatment, loading buffer (3 μL) was added, and agarose gel electrophoresis was performed on a 1% gel (containing 1.0 μg·mL^-1 neutral red) at 100 V for 1 h in diluted 1×TAE buffer. The gel was then visualized by photographing intercalated neutral red fluorescence under a UV illuminator. The cleavage efficiency was measured by determining the ability of the compound to excise supercoiled pBR322 DNA (SC) to its nicked circular (NC) and linear (L) forms.

  1.5   CCK-8 assay

  The cytotoxicity of Pt₂-BPA-TPA and cisplatin to human lung cancer A549 cells was measured using the CCK-8 assay. CCK -8 contains Dojindo′s highly watersoluble tetrazolium salt, 2-(2-methoxy-4-nitrophenyl)-3-(4- nitrophenyl)-5 -(2, 4-disulfophenyl)-2H-tetrazolium sodium salt (WST - 8), which produces a water - soluble formazan dye upon reduction in the presence of an electron mediator. A549 cells were seeded in 96 - well plates at a density of 4×103 cells per well and then incubated overnight. The adherent cells were then treated with different concentrations of drugs. After two days, 10 μL of the CCK -8 solution was added to each well of the plate, and the plate was incubated for 3 h in the incubator (37 ℃, φ_CO2=5%). Finally, the OD ₄₅₀ value (optical density at 450 nm) was measured in a microplate reader.

  1.6   Western blot analysis

  A549 cells (or HepG2 cells) were homogenized in lysis buffer (#9803, CST, USA), and total protein was isolated by centrifugation at 12 000 r·min^-1 for 15 min at 4 ℃. Total protein concentration in the lysates was quantified using a bicinchoninic acid (BCA) protein assay kit (A53225, Thermo Fisher Scientific, USA) according to the manufacturer′s instructions. The samples were then subjected to western blot analysis using specific antibodies against α-tubulin (GB11001, ServiceBio, Wuhan, China), cleaved caspase-3 (9661S, CST, USA), and p21 (A1483, ABclonal, Wuhan, China) as described previously^[29].

  1.7   Statistical analysis

  The data were analyzed by SPSS 19.0 software and presented as means±SD from at least three independent experiments. All the statistical results were analyzed according to the Student′s t-test and Graphpad Prism 9.0 (GraphPad Software Inc., La Jolla, USA) was used to compare group means. A value of p < 0.05 represented statistically significant.

  2.   Results and discussion

  2.1   NMR and HRMS characterization of Pt₂-BPA-TPA

  The structural characteristics of ligand BPA -TPA and complex Pt₂-BPA -TPA were elucidated by NMR and HRMS. The ¹H NMR spectra of Pt₂-BPA-TPA and BPA-TPA in DMSO-d ₆ are shown in Fig. 1. In comparison with the spectrum of BPA- TPA, proton signals in Pt₂-BPA-TPA were shifted significantly to the lower field, suggesting that Pt(Ⅱ) was coordinated to the ligand, and thus providing evidence of the formation of a binuclear Pt(Ⅱ) complex in Pt₂-BPA-TPA. The coordination of Pt increased steric hindrance in the two pyridyl groups, eliciting magnetic nonequivalence in the nearby methylene protons. Consequently, the H7 singlet at 3.79 was split into two doublets at 5.4 and 5.1. The existence of Pt₂-BPA-TPA in the CH₃CN solution was also studied with HRMS. The m/z peaks for [(Pt₂-BPA-TPA)-Cl]⁺, [(Pt₂-BPA-TPA)-2Cl]²⁺, and [(Pt₂-BPA-TPA)-2Cl+6H₂O]²⁺ were observed at 998.096 4, 481.063 7, and 534.092 3, respectively. The results indicate that complex Pt₂-BPA-TPA is stable in CH₃CN.

  Figure 1

  

  Figure 1.  ¹H NMR spectra of complex Pt₂-BPA-TPA and the corresponding ligand BPA-TPA

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  2.2   Crystal structure

  As shown in Fig. 2, binuclear Pt complex Pt₂-BPA-TPA crystallizes in a monoclinic system with space group P2₁/c (centrosymmetric space group). The two Pt ions are symmetrically located in the center of the two pyridine ring arms which originate from DPA. Each Pt(Ⅱ) ion adopts an approximate planar quadrilateral geometry associated with the [PtN₃Cl] arrangement, which is comprised of two pyridyl nitrogen atoms (for Pt1, N1 and N3; for Pt2, N4 and N6), a tertiary amine nitrogen atom (for Pt1, N2; for Pt2, N5), and a chloride atom (for Pt1, Cl1; for Pt2, Cl2). Using Pt1 as an example, the two diagonal positions are analogously occupied by N1 and N3 to yield Pt1—N1 (0.202 7(14) nm) and Pt1—N3 (0.203 9(14) nm). The dihedral angle between Cl1—Pt1—N3 and N1—Pt1—N2 is 2.166°. This dihedral angle deviates from the ideal value of 0°, implying a distorted square - planar configuration of [PtN₃Cl]. Selected bond lengths and bond angles are listed in Table 2.

  Figure 2

  

  Figure 2.  Crystal structure of Pt₂-BPA-TPA with 50% thermal ellipsoids

  Hydrogen atoms are omitted for clarity

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  Table 2

  

  Table 2.  Selected bond lengths (nm) and angles (°) for Pt₂-BPA-TPA

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  Pt1—Cl1	0.230 4(4)	Pt1—N1	0.202 7(14)	Pt1—N2	0.203 1(15)
  Pt1—N3	0.203 9(14)	Pt2—Cl2	0.229 2(5)	Pt2—N4	0.202 0(17)
  Pt2—N5	0.200	9(19)	Pt2—N6	0.204	0(14)
  Cl1—Pt1—N1	95.7(5)	Cl1—Pt1—N2	179.5(4)	Cl1—Pt1—N3	97.1(4)
  N1—Pt1—N2	83.9(6)	N1—Pt1—N3	167.0(6)	N2—Pt1—N3	83.3(6)
  Cl2—Pt2—N4	95.7(6)	Cl2—P2—N6	97.6(5)	Cl2—Pt2—N5	178.2(4)
  N4—Pt2—N6	166.2(7)	N4—Pt2—N5	83.3(7)	N5—Pt2—N6	83.2(6)

  2.3   DNA cleavage activity

  The main target of platinum anticancer agents is generally believed to be nuclear DNA. The mechanism of action of Pt(Ⅱ) complexes involves the formation of Pt- DNA adducts that induce DNA damage by interfering with DNA transcription and replication, ultimately leading to cell death. To investigate the DNA cleavage activity of complex Pt₂-BPA-TPA, we performed agarose gel electrophoresis experiments using pBR322 plasmid DNA (1 μL, 0.2 μg) in Tris - HCl/NaCl (50 mmol·L^-1, pH=7.2) buffer. A significant difference in the migration rates of the three types of DNA could be observed through gel electrophoresis. The migration rates can be expected to differ as follows: supercoiled form (Form Ⅰ, SC) > linear (Form Ⅲ, L) > nicked circular (Form Ⅱ, NC)^[30]. Fig. 3 shows the results of gel electrophoretic separation of pBR322 plasmid DNA treated with increasing concentrations of Pt₂-BPA-TPA, and with the free ligand BPA - TPA. Both Pt₂ - BPA-TPA and BPA-TPA cleaved Form Ⅰ DNA into Form Ⅱ DNA and Form Ⅲ DNA in a concentrationdependent manner. At the lowest concentration tested (5 μmol·L^-1), supercoiled Form Ⅰ DNA was still observed, implying the complex′s weak nuclease activity at such a faint dose. Surprisingly, at 10 μmol·L^-1 concentration, Pt₂ - BPA-TPA and BPA-TPA exhibited efficient cleavage activity; accordingly, Form Ⅰ was relaxed to generate Form Ⅱ and Ⅲ, suggesting that double - stranded DNA was thoroughly cleaved. Under the same concentration conditions, the DNA cleavage activity of the binuclear Pt(Ⅱ) complex was not significantly higher than that of the ligand. We hypothesize that the introduction of two Pt(Ⅱ) ions does not significantly change the spatial structure of the ligand.

  Figure 3

  

  Figure 3.  Agarose gel electrophoresis diagrams for cleavage of pBR322 plasmid DNA induced by complex Pt₂-BPA-TPA and ligand BPA-TPA

  Lane 1: Control; Lane 2: DNA+BPA-TPA (5 μmol·L^-1); Lane 3: DNA+Pt₂-BPA-TPA (5 μmol·L^-1); Lane 4: DNA+BPA-TPA (10 μmol·L^-1); Lane 5: DNA+Pt₂-BPA-TPA (10 μmol·L^-1); Lane 6: DNA+BPA-TPA (20 μmol·L^-1); Lane 7: DNA+Pt₂-BPA-TPA (20 μmol·L^-1)

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  2.4   In vitro cytotoxicity

  The binuclear Pt(Ⅱ) complex Pt₂-BPA-TPA was evaluated for its toxicity against human lung cancer A549 cells using the CCK -8 assay. Cisplatin was used as a positive control. The results are shown in Fig. 4. The IC₅₀ value of Pt₂-BPA-TPA was approximately 25 μmol·L^-1, which was significantly lower than that of cisplatin (45 μmol·L^-1). Therefore, Pt₂-BPA-TPA demonstrated increased anticancer activity on A549 cells (compared to cisplatin). As a class of non-classical platinum(Ⅱ) complexes, monofunctional platinum(Ⅱ) complexes have been widely studied to overcome the resistance and side effects of classical bifunctional platinum(Ⅱ) complexes. For instance, Guo and coworkers reported a series of mononuclear monofunctional platinum(Ⅱ) complexes with excellent anticancer activity and further investigated the anticancer mechanism^{[18, 31-32]}. Mao and coworkers developed a mononuclear monofunctional platinum(Ⅱ) complex based on lonidamine which possesses prominent cytotoxic activity against triple - negative breast cancer MDA - MB - 231 cells^[11]. Nevertheless, it is interesting that the binuclear monofunctional platinum(Ⅱ) complex Pt₂ - BPA-TPA probably exhibits a new mechanism, even if its antican-cer activity is modest in our present work.

  Figure 4

  

  Figure 4.  Dose-dependent curves showing the cytotoxicity ofcisplatin and complex Pt₂-BPA-TPA in A549 cells

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  2.5   Expression of apoptosis-related proteins

  Since complex Pt₂-BPA-TPA exhibited increased anticancer activity (compared to cisplatin), we further explored its anticancer mechanism in greater detail. p21 protein and caspase family proteins are both known to be key regulators of the apoptotic pathway. If p21 is produced in excess, the affected cell proceeds to apoptosis^[33]. Caspase-3 is an important" executioner" of this apoptotic process^{[18, 34]}. To gain an insight into the molecular mechanism of cell death triggered by Pt₂-BPA-TPA, we used western blot assays to detect p21 and caspase - 3 protein levels in HepG2, A549 cells, and rat myocardial (H9c2) cells. All three cell types were treated with Pt₂-BPA-TPA at concentrations of 10 or 20 μmol·L^-1. For comparison, these cells were also treated with cisplatin at 1 and 5 μmol· L^-1. As shown in Fig. 5, p21 and cleaved - caspase - 3 protein levels in HepG2 and A549 cells were markedly increased by Pt₂-BPA-TPA treatment. In A549 cells, a six-fold increase in the p21 protein level was observed, and this was significantly higher than that observed in HepG2 cells. No significant increase in either p21 protein or cleaved-caspase-3 protein levels was observed in normal H9c2 cells. These data provide solid evidence that Pt₂-BPA-TPA induces critical nuclear DNA damage, subsequently inducing an upregulation in the protein levels of important downstream apoptosis-related proteins, including p21 and cleavedcaspase-3.

  Figure 5

  

  Figure 5.  Expression of p21 and cleaved-caspase-3 (C-casp 3) in HepG2, A549, and H9c2 cells after incubation with cisplatin (Cis-Pt) and Pt₂-BPA-TPA for 24 h, where α-tubulin used as an internal control (CON)

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  3.   Conclusions

  In summary, we have successfully prepared a new binuclear monofunctional Pt(Ⅱ) complex, Pt₂-BPA-TPA, based on the Pt - NNN coordination pattern and employing polypyridyl as the non -leaving group. Single-crystal X-ray diffraction analysis reveals that Pt₂ - BPA-TPA belongs to the monoclinic system with space group P 2₁/c. The cleavage activity of Pt₂-BPA-TPA towards supercoiled pBR322 plasmid DNA was also studied by agarose gel electrophoresis, demonstrating that Pt₂-BPA-TPA could effectively cleave Form Ⅰ DNA to Form Ⅱ DNA and Form Ⅲ DNA. CCK-8 analysis revealed that Pt ₂-BPA-TPA could inhibit the growth of A549 cells and that Pt ₂-BPA-TPA demonstrated higher cytotoxicity than cisplatin after 48 h incubation. Moreover, western blot analyses provided evidence that the anticancer mechanism of Pt₂-BPA-TPA involves the upregulation of protein levels of important downstream apoptosis -related proteins, including p21 and cleaved - caspase - 3. Generally, Pt₂-BPA-TPA, a refinedly synthesized binuclear monofunctional Pt complex, is a promising candidate for novel anticancer therapy.

  
  Acknowledgments: This project was supported by the Educational Commission of Hubei Province of China (Grant No. D20212802), the Natural Science Foundation of Hubei Province (Grant No. 2021CFB439), and the Scientific Projects of Health Commission of Hubei Province (Grant No.WJ2019Q022). Declaration of competing interest: All authors of this paper declare that they have no competing interests.
  
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  Scheme 1  Synthetic routes of ligand BPA-TPA and complex Pt₂-BPA-TPA

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  Figure 1  ¹H NMR spectra of complex Pt₂-BPA-TPA and the corresponding ligand BPA-TPA

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  Figure 2  Crystal structure of Pt₂-BPA-TPA with 50% thermal ellipsoids

  Hydrogen atoms are omitted for clarity

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  Figure 3  Agarose gel electrophoresis diagrams for cleavage of pBR322 plasmid DNA induced by complex Pt₂-BPA-TPA and ligand BPA-TPA

  Lane 1: Control; Lane 2: DNA+BPA-TPA (5 μmol·L^-1); Lane 3: DNA+Pt₂-BPA-TPA (5 μmol·L^-1); Lane 4: DNA+BPA-TPA (10 μmol·L^-1); Lane 5: DNA+Pt₂-BPA-TPA (10 μmol·L^-1); Lane 6: DNA+BPA-TPA (20 μmol·L^-1); Lane 7: DNA+Pt₂-BPA-TPA (20 μmol·L^-1)

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  Figure 4  Dose-dependent curves showing the cytotoxicity ofcisplatin and complex Pt₂-BPA-TPA in A549 cells

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  Figure 5  Expression of p21 and cleaved-caspase-3 (C-casp 3) in HepG2, A549, and H9c2 cells after incubation with cisplatin (Cis-Pt) and Pt₂-BPA-TPA for 24 h, where α-tubulin used as an internal control (CON)

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  Table 1.  Crystal parameters and structure refinements for Pt₂-BPA-TPA

  Parameter	Pt2-BPA-TPA
  Chemical formula	C32H37Cl4N7O2Pt2
  Formula weight	1 083.66
  Temperature / K	293
  Crystal size / mm	0.06×0.05×0.04
  Crystal system	Monoclinic
  Space group	P21/c
  a / nm	1.259 36(3)
  b / nm	1.486 16(3)
  c / nm	2.187 45(5)
  β/(°)	105.058(2)
  V / nm3	3.953 48(16)
  Z	4
  Dc / (g·cm-3)	1.821
  μ / mm-1	15.832
  F(000)	2 072
  θ range for data collection / (°)	2.068 0-67.317 0
  Index ranges (h, k, l)	-15 ≤ h ≤ 14,
  	-11 ≤ k≤ 17,
  	-26 ≤ l ≤ 26
  Nref, Npar	6 885, 425
  Completeness / %	98.68
  Final R indices [I > 2σ(I)]	R1=0.091 7, wR2=0.246 3
  R indices (all data)	R1=0.097 7, wR2=0.251 1
  Goodness-of-fit on F2	1.028

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  Table 2.  Selected bond lengths (nm) and angles (°) for Pt₂-BPA-TPA

  Pt1—Cl1	0.230 4(4)	Pt1—N1	0.202 7(14)	Pt1—N2	0.203 1(15)
  Pt1—N3	0.203 9(14)	Pt2—Cl2	0.229 2(5)	Pt2—N4	0.202 0(17)
  Pt2—N5	0.200	9(19)	Pt2—N6	0.204	0(14)
  Cl1—Pt1—N1	95.7(5)	Cl1—Pt1—N2	179.5(4)	Cl1—Pt1—N3	97.1(4)
  N1—Pt1—N2	83.9(6)	N1—Pt1—N3	167.0(6)	N2—Pt1—N3	83.3(6)
  Cl2—Pt2—N4	95.7(6)	Cl2—P2—N6	97.6(5)	Cl2—Pt2—N5	178.2(4)
  N4—Pt2—N6	166.2(7)	N4—Pt2—N5	83.3(7)	N5—Pt2—N6	83.2(6)

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