English

• Cancer seriously endangers people′s life and safety. In 2018, the World Health Organization reported that cancer was the second leading cause of death, killing about 9.6 million people worldwide^[1]. Chemotherapy is a major method widely applied in current clinical cancer treatment^[2-3]. Cisplatin is one of the most effective chemotherapeutic drugs approved by the Food and Drug Administration^[4]. However, cisplatin is limited in clinical use due to its severe side effects^[5-7]. Therefore, a range of metal complexes have been designed and synthesized for the treatment of cancer^[8-11], of which ruthenium complexes are the most promising to be the next generation of anticancer agents^[12-13].

  Many Ru complexes, such as KP -1019 and NAMI-A, have been widely studied due to their excellent clinical efficacy^[14-15]. Ru complexes have been applied in various fields of medicinal chemistry against tumors with different biological targets^[16-17]. In addition, aroylhydrazones possess excellent biological and pharmaceutical activities, including anticancer, anti - inflammatory, and anti - tubercular activities^[18]. More importantly, previous studies have been showed that anticancer activity of metal - base complexes derived from aroylhydrazones were higher than that of the ligands alone^[19-20]. Hence, the design of aroylhydrazone complexes chelated with ruthenium ion for anticancer treatment has an important prospect.

  The primary target of metal - based agents to kill tumors is DNA, which causes amount of DNA lesions, thus preventing transcription and replication^[21-22]. Considering the above factors, we designed and synthesized a Ru(Ⅱ) complex derived from aroylhydrazones (Scheme 1). Subsequently, the structure of Ru(Ⅱ) complex was measured via X-ray crystallography and solved. Finally, the anticancer activity of Ru(Ⅱ) complex and its effect on DNA were investigated in vitro.

  Scheme 1

  

  Scheme 1.  Synthetic route for [Ru(L)(DMSO)₂Cl₂] (1)

  下载: 全尺寸图片 幻灯片

  1.   Experimental

  1.1   Materials and general methods

  The chemical reagents and solvent used were analytically pure and purchased from Sigma or Innochem Company. Tumor cell lines were supplied by the Shanghai institute for biochemistry and cell biology.

  1.2   Synthesis of [Ru(L)(DMSO)₂Cl₂] (1)

  A mixture of 0.1 mmol 4 - hydroxybenzhydrazide and 0.1 mmol 3-methyl-2-thiophenecarboxaldehyde were dissolved in 20 mL methanol and refluxed. Then the ligand L was obtained. Subsequently, 0.1 mmol RuCl₃ and 50 μL DMSO (dimethyl sulfoxide) were added and stirred for another 2 h (Scheme 1). The resulting solution was evaporated at room temperature, and the yellow -brown crystals crystallized after about 7 d. The purity of complex 1 (>95%) was detected by HPLC (Fig. S1). Anal. Calcd. for C₁₇H₂₆Cl₂N₂O₅RuS₃(%): C, 33.66; H, 4.32; N, 4.62. Found(%): C, 33.72; H, 4.35; N, 4.58.

  1.3   X⁃ray crystallography

  A single crystal (0.18 mm×0.19 mm×0.22 mm) of 1 was picked out and then measured by a Bruker SMART Apex Ⅱ CCD diffractometer with graphite - monochromated Mo Kα radiation (λ =0.071 073 nm). The diffraction data were collected. The structure was solved by direct methods and refined with the XL refinement package using least squares minimization by olex 2.0 software^[23]. All non - hydrogen atoms were anisotropically refined. All hydrogen atoms were fixed in calculated positions and refined isotropically. The data is listed in Table 1, and selected bond lengths and angles are listed in Table 2.

  Table 1

  

  Table 1.  Crystal data and structure refinement for complex 1

  下载: 导出CSV

  Empirical formula	C17H24Cl2N2O4RuS3·H2O		Dc/(g·cm-3)	1.673
  Formula weight	606.55		μ/mm-1	1.163
  Crystal system	Orthorhombic		F(000)	2 464
  Space group	Pbca		Reflection collected	12 030
  a/nm	1.702 43(6)		Independent reflection	4 241(Rint=0.033 8, Rσ=0.042 4)
  b/nm	1.595 86(7)		Data, restraint, parameter	4 241, 0, 276
  c/nm	1.773 00(15)		Goodness-of-fit on F2	1.149
  Volume/nm3	4.817 0(5)		Final R indexes[I≥2σ(I)]	R1=0.065 7, wR2=0.139 7
  Z	8		Final R indexes (all data)	R1=0.098 5, wR2=0.163 4

  Table 2

  

  Table 2.  Selected bond lengths (nm) and angles (°) of complex 1

  下载: 导出CSV

  Ru1-S1	0.223 1(3)	Ru1-Cl2	0.238 4(3)	Ru1-N1	0.209 2(8)
  Ru1-Cl1	0.240 0(3)	Ru1-O1	0.209 3(7)	Ru1-S2	0.225 2(3)
  S1-Ru1-Cl1	91.87(12)	O1-Ru1-Cl1	88.4(2)	N1-Ru1-Cl2	84.8(3)
  S1-Ru1-Cl2	92.49(13)	O1-Ru1-Cl2	86.7(2)	N1-Ru1-O1	77.6(3)
  S1-Ru1-S2	93.21(10)	O1-Ru1-S2	90.9(2)	N1-Ru1-S2	168.5(2)
  Cl2-Ru1-Cl1	171.35(12)	N1-Ru1-S1	98.2(2)	S2-Ru1-Cl1	93.46(10)
  O1-Ru1-S1	175.8(2)	N1-Ru1-Cl1	87.2(3)	S2-Ru1-Cl2	93.75(12)

  CCDC: 2080249.

  1.4   MTT assays

  The human balder cell lines T24 were cultured in 96-well plate and incubated with the selected concentrations of complex 1 for 48 h. Then the MTT (3-(4, 5-dimethylthiazol-2-yl)- 2, 5-diphenyltetrazolium bromide) solution was added to each well and incubated for 4 h. The medium was replaced with 100 μL DMSO. The absorbance was measured at an excitation wavelength of 570 or 630 nm with an enzyme-labelling instrument.

  1.5   DNA cleavage studies

  The super-coiled plasmid pBR322 DNA was incu-bated with complex 1 for 4 h. Subsequently, agarose gel electrophoresis was employed to isolate pBR322 DNA in the reaction solution. These results were captured by camera for analyzing the ability of complex 1 to cleave DNA.

  1.6   Western⁃blotting

  T24 cells were co - incubated with Ru(Ⅱ) complex for 24 h, and then the cells were harvested and lysed to obtain the proteins. The concentrations of proteins were measured using BCA assay. The proteins were isolated by SDS-polyacrylamide gel, transferred to a PVDF (polyvinylidene fluoride) membrane and blocked with fat-free milk. Then, the membrane was incubated with primary antibodies and secondary antibodies respectively.

  1.7   DNA metalation

  Two millions T24 cells were placed into 10 cm dishes, and incubated with complex 1 for 24 h. T24 cells were collected by centrifugation and washed once time. Subsequently, DNA was extracted using a PureLink Genomic DNA Mini Kit (Invitrogen). DNA purity was detected by absorbance measurements at 260 and 280 nm. The DNA solution was added 1 mL of concentrated HNO₃ and 0.5 mL of 30% H₂O₂. Finally, these samples were detected by ICP-MS.

  1.8   Cellular uptake

  T24 cells were seeded into 10 cm dishes, and then incubated with complex 1. After 24 h, the cells were collected and washed. Subsequently, the cellular components were isolated and extracted according to the instructions of the mitochondrial isolation kit and the nuclear isolation kit, respectively. The samples were detected by ICP-MS.

  1.9   Docking

  The binding mode of complex 1 in DNA (PDB ID: 1D64) was investigated using AutoDock 4.0. Target receptor (DNA) and complex 1 were prepared via docking protocol and saved into‘PDBQT ’format. And then the‘autogrid’and‘autodock’were performed, respectively. Energy-scoring function was used to ensure the best Ru(Ⅱ) complex-DNA pose.

  1.10   Intracellular reactive oxygen species (ROS) analysis

  T24 cells were cultured into 6 - well plates, and treated with complex 1 for 24 h. After incubation, T24 cells were collected and stained in the dark with 2′, 7′- dichlorodihydro - fluorescein diacetate (H₂DCF - DA) for 30 min. Intracellular ROS generation was detected by flow cytometry.

  1.11   Comet assay

  T24 cells were incubated with complex 1 for 24 h, and the cells were harvested. 1×10⁴ cells were embedded in 80 μL low -gelling-temperature agarose and rapidly pipetted onto a pre-coated microscope slide. These slides were electrophoresed for 20 min, and then stained with PI for 30 min in dark. Finally, the slides were graphed using a laser confocal microscope.

  2.   Results and discussion

  2.1   Structure of 1

  X-ray diffraction analysis reveals that 1 crystalliz-es in the orthorhombic space group Pbca. As shown in Fig. 1, the structure of complex 1 is composed of a central Ru(Ⅱ)ion, two DMSO molecules, two chloride ions, and a Schiff base ligand L. The central Ru(Ⅱ) forms a six - coordinated environment, which is composed of one N atom (Ru1—N1 0.209 2 nm) and one O atom (Ru1—O1 0.209 3 nm) of HL, two S atoms (Ru1—S1 0.223 1 nm, Ru1—S2 0.225 2 nm) of DMSO and two Cl atoms (Ru1—Cl1 0.240 0 nm, Ru1—Cl2 0.238 4 nm).

  Figure 1

  

  Figure 1.  ORTEP diagram of complex 1 showing 30% probability level ellipsoids

  One crystalline water molecule is not shown

  下载: 全尺寸图片 幻灯片

  2.2   Anticancer activity of 1

  The cytotoxicity of 1 against T24 cells was assessed using the MTT assay. The IC₅₀ values of HL, RuCl₃ and DMSO were greater than 50 μmol·L^-1. These results indicate that the compounds do not exhibit significant antitumor activity. While the synthesized complex 1 from the ligand and RuCl₃ exhibited remarkably activity (IC₅₀=15.28 μmol·L^-1), as shown in Fig. 2.

  Figure 2

  

  Figure 2.  Cytotoxicity of 1 at different concentrations

  下载: 全尺寸图片 幻灯片

  2.3   Cellular accumulation

  The cellular uptake of 1 by T24 cells was measured using ICP-MS. As shown in Fig. 3, the contents of ruthenium in cells, nuclei and mitochondria after treatment of 1 were 1.23, 0.28 and 0.11 nmol, respectively. These results suggest that 1 can efficiently enter the cell and accumulate in the nucleus.

  Figure 3

  

  Figure 3.  ICP-MS analysis of total Ru contents in cytoplasm, mitochondria, and in nucleus of T24 cells (1×10⁶ cells) treated with 1 for 24 h

  下载: 全尺寸图片 幻灯片

  2.4   Anti-tumour mechanism of 1

  2.4.1   DNA cleavage

  DNA is the primary intracellular target of many PT/Ru drugs in the treatment of tumors^[21-22]. Hence, the DNA-cleaving activity of 1 was investigated by an agarose gel electrophoresis assay using pBR322 DNA. The control group cannot cleave the PBR322 DNA, whereas 1 can efficiently relax the supercoiled form (Form Ⅰ) of pBR322 DNA into open circular form (Form Ⅱ) in a dose - dependent manner. These results strongly suggest that complex 1 has a strong effect on DNA (Fig. 4).

  Figure 4

  

  Figure 4.  Agarose gel electrophoresis patterns for cleavage of pBR322 DNA by 1

  下载: 全尺寸图片 幻灯片

  2.4.2   Docking studies

  To better understand the mode between B - DNA and 1, molecular docking was carried out for studying the interactions. Complex 1 fitted well into the curved profile of the B-DNA in the minor groove located in the A-T rich region (Fig. 5). The energy minimization structure indicates that 1 is stabilized by hydrophobic contact, van der Waals forces and B - DNA functional groups.

  Figure 5

  

  Figure 5.  Molecular docked model of 1 with B-DNA

  下载: 全尺寸图片 幻灯片

  2.4.3   DNA damage studies

  To further confirm the ability of 1 acting on DNA in tumor cells, the genomic DNA was extracted from T24 cells after treated with 1. The Ru content in the genomic DNA was at highly level, and the significantly elevated level of Ru content was in a dose - dependent fashion (Fig. 6).

  Figure 6

  

  Figure 6.  Amount of Ru in genomic DNA (1 μg) of T24 cells after treatment of 1

  下载: 全尺寸图片 幻灯片

  T24 cells were induced apparent DNA damage co- incubated with complex 1, which was characterized by the tail DNA. The number and length of tail DNA were in positive correlation with the degree of DNA damage. The 1 - treated group produced more and longer tail DNA in a dose-dependent manner than the control group (Fig. 7).

  Figure 7

  

  Figure 7.  Comet assay for analyzing DNA damage of T24 cells after treatment of 1

  Tail DNA was marked with white arrow

  下载: 全尺寸图片 幻灯片

  Western blotting assays showed that the expression level of γH2AX (a well - known marker for DNA double-strand breaks) in the control group was kept at lower levels, while that in the treatment group significantly increased (Fig. 8). Taken together, these results strongly indicate that direct interaction with DNA is a possible mechanism for complex 1 to play a role in killing tumor cells.

  Figure 8

  

  Figure 8.  Expression level of γH2AX treated with 1

  下载: 全尺寸图片 幻灯片

  2.4.4   ROS generation

  Generally, ROS is, at least in part, responsible for the cleavage of DNA^[24-26]. Therefore, the amount of intracellular ROS after treatment of 1 was investigated by flow cytometry using the DCFH-DA. T24 cells treated with 1 led to a shift in the fluorescence peak to the right compared to the un - treatment group, which indicate that 1 significantly increases intracellular ROS levels in a dose-dependent fashion (Fig. 9).

  Figure 9

  

  Figure 9.  Analysis of ROS levels in T24 cells treated with 1 for 24 h

  下载: 全尺寸图片 幻灯片

  3.   Conclusions

  A Ru(Ⅱ) complex, [Ru(L)(DMSO)₂Cl₂] (1), was synthesized and its anticancer activity was investigated. Complex 1 exhibited effective activities against T24 cells, whereas the ligands had no significant cytotoxicity. Complex 1 is possible to kill tumor cells through the following mechanisms: 1 can bind to DNA bases, form DNA adducts, induce genomic DNA damage, and may block transcription and replication. Additionally, 1 can produce amount of ROS, causing oxidative loss of T24 cells, which leads to tumor cell death. In summary, complex 1 has potential as an anti-cancer chemotherapeutic agent. Our studies may be useful in guiding the development for designing new metal - base antitumor agents.

  
  Supporting information is available at http://www.wjhxxb.cn
  
• 1. [1]

     El-Kordy E A. J. Microsc. Ultrastruct. , 2019, 7: 153-164 doi: 10.4103/JMAU.JMAU_21_19

  2. [2]

     Mukaya E H, Mbianda X Y. Mini-Rev. Med. Chem. , 2020, 20: 726-738 doi: 10.2174/1389557519666191107142926

  3. [3]

     Schweigert M, Dubecz A, Stein H J. Nat. Rev. Gastroenterol. Hepatol. , 2013, 10: 230-244 doi: 10.1038/nrgastro.2012.236

  4. [4]

     Rancoule C, Guy J B, Vallard A, Ben Mrad M, Rehailia A, Magné N. Bull. Cancer, 2017, 104: 167-176 doi: 10.1016/j.bulcan.2016.11.011

  5. [5]

     Rosenberg B, Van Camp L, Krigas T I. Nature, 1965, 205: 698-699 doi: 10.1038/205698a0

  6. [6]

     Rosenberg B, Van Camp L, Grimley E B, Thomson A J. J. Biol. Chem. , 1967, 242: 1347-1352 doi: 10.1016/S0021-9258(18)96186-7

  7. [7]

     Notaro A, Frei A, Rubbiani R, Jakubaszek M, Basu U, Koch S, Mari C, Dotou M, Blacque O, Gouyon J, Bedioui F, Rotthowe N, Winter R F, Goud B, Ferrari S, Tharaud M, Řezáčová M, Humajová J, Tomšík P, Gasser G. J. Med. Chem. , 2020, 63: 5568-5584 doi: 10.1021/acs.jmedchem.0c00431

  8. [8]

     Misirlic-Dencic S, Poljarevic J, Isakovic A M, Sabo T, Markovic I, Trajkovic V. Curr. Med. Chem. , 2020, 27: 380-410 doi: 10.2174/0929867325666181031114306

  9. [9]

     Sun Y, Heidary D K, Zhang Z, Richards C I, Glazer E C. Mol. Pharm. , 2018, 15: 3404-3416 doi: 10.1021/acs.molpharmaceut.8b00407

  10. [10]

      Mudavath R, Vuradi R K, Bathini U, Narsimha N, Kunche S, Sunitha S N T, Ch S D. Nucleosides Nucleotides Nucleic Acids, 2019, 38: 874-900 doi: 10.1080/15257770.2019.1618470

  11. [11]

      Leskovac A, Petrovic S, Lazarevic-Pasti T, Krstic M, Vasic V. J. Biol. Inorg. Chem. , 2018, 23: 689-704 doi: 10.1007/s00775-018-1560-x

  12. [12]

      Biancalana L, Pampaloni G, Marchetti F. Chimia (Aarau), 2017, 71: 573-579 doi: 10.2533/chimia.2017.573

  13. [13]

      Alessio E, Messori L. Molecules, 2019, 24: 1995-2014 doi: 10.3390/molecules24101995

  14. [14]

      Trondl R, Heffeter P, Kowol C R, Jakupec M A, Berger W, Keppler B K. Chem. Sci. , 2014, 5: 2925-2932 doi: 10.1039/C3SC53243G

  15. [15]

      Nardon C, Brustolin L, Fregona D. Future Med. Chem. , 2016, 8: 211-226 doi: 10.4155/fmc.15.175

  16. [16]

      Notaro A, Gasser G. Chem. Soc. Rev. , 2017, 46: 7317-7337 doi: 10.1039/C7CS00356K

  17. [17]

      Hartinger C G, Jakupec M A, Zorbas-Seifried S, Groessl M, Egger A, Berger W, Zorbas H, Dyson P J, Keppler B K. Chem. Biodivers. , 2008, 5: 2140-2155 doi: 10.1002/cbdv.200890195

  18. [18]

      Samanta B, Chakraborty J, Shit S, Batten S R, Jensen P, Masuda J D, Mitra S. Inorg. Chim. Acta, 2007, 360: 2471-2484 doi: 10.1016/j.ica.2006.12.019

  19. [19]

      Deng J G, Gou Y, Chen W. Fu X, Deng H. Bioorg. Med. Chem. , 2016, 24: 2190-2198 doi: 10.1016/j.bmc.2016.03.033

  20. [20]

      Monfared H H, Vahedpour M, Yeganeh M M, Ghorbanloo M, Mayer P, Janiak C. Dalton. Trans. , 2011, 40: 1286-1294 doi: 10.1039/c0dt00371a

  21. [21]

      Rocha C R, Silva M M, Quinet A, Cabral-Neto J B, Menck C. Clinics (Sao Paulo), 2018, 73: 478-488

  22. [22]

      Rodríguez-Corrales J Á, Wang J, Winkel B S J, Brewer K J. ChemBioChem, 2018, 19: 2216-2224 doi: 10.1002/cbic.201800369

  23. [23]

      Dolomanov O V, Bourhis L J, Gildea R J, Howard J A K, Puschmann H. J. Appl. Crystallogr. , 2009, 42: 339-341 doi: 10.1107/S0021889808042726

  24. [24]

      Celik H, Arinç E. J. Pharm. Pharm. Sci. , 2008, 11: 68-82

  25. [25]

      Pracharova J, Radosova M T, Dvorak T E, Thangappan R, Arivanandhan M. Dalton. Trans. , 2016, 45: 13179-13186 doi: 10.1039/C6DT01467D

  26. [26]

      Wani T H, Chakrabarty A, Shibata N, Yamazaki H, Guengerich F P, Chowdhury G. Chem. Res. Toxicol. , 2017, 30: 1622-1628 doi: 10.1021/acs.chemrestox.7b00127

• 

  Scheme 1  Synthetic route for [Ru(L)(DMSO)₂Cl₂] (1)

  下载: 全尺寸图片 幻灯片
  

  Figure 1  ORTEP diagram of complex 1 showing 30% probability level ellipsoids

  One crystalline water molecule is not shown

  下载: 全尺寸图片 幻灯片
  

  Figure 2  Cytotoxicity of 1 at different concentrations

  下载: 全尺寸图片 幻灯片
  

  Figure 3  ICP-MS analysis of total Ru contents in cytoplasm, mitochondria, and in nucleus of T24 cells (1×10⁶ cells) treated with 1 for 24 h

  下载: 全尺寸图片 幻灯片
  

  Figure 4  Agarose gel electrophoresis patterns for cleavage of pBR322 DNA by 1

  下载: 全尺寸图片 幻灯片
  

  Figure 5  Molecular docked model of 1 with B-DNA

  下载: 全尺寸图片 幻灯片
  

  Figure 6  Amount of Ru in genomic DNA (1 μg) of T24 cells after treatment of 1

  下载: 全尺寸图片 幻灯片
  

  Figure 7  Comet assay for analyzing DNA damage of T24 cells after treatment of 1

  Tail DNA was marked with white arrow

  下载: 全尺寸图片 幻灯片
  

  Figure 8  Expression level of γH2AX treated with 1

  下载: 全尺寸图片 幻灯片
  

  Figure 9  Analysis of ROS levels in T24 cells treated with 1 for 24 h

  下载: 全尺寸图片 幻灯片

  Table 1.  Crystal data and structure refinement for complex 1

  Empirical formula	C17H24Cl2N2O4RuS3·H2O		Dc/(g·cm-3)	1.673
  Formula weight	606.55		μ/mm-1	1.163
  Crystal system	Orthorhombic		F(000)	2 464
  Space group	Pbca		Reflection collected	12 030
  a/nm	1.702 43(6)		Independent reflection	4 241(Rint=0.033 8, Rσ=0.042 4)
  b/nm	1.595 86(7)		Data, restraint, parameter	4 241, 0, 276
  c/nm	1.773 00(15)		Goodness-of-fit on F2	1.149
  Volume/nm3	4.817 0(5)		Final R indexes[I≥2σ(I)]	R1=0.065 7, wR2=0.139 7
  Z	8		Final R indexes (all data)	R1=0.098 5, wR2=0.163 4

  下载: 导出CSV

  Table 2.  Selected bond lengths (nm) and angles (°) of complex 1

  Ru1-S1	0.223 1(3)	Ru1-Cl2	0.238 4(3)	Ru1-N1	0.209 2(8)
  Ru1-Cl1	0.240 0(3)	Ru1-O1	0.209 3(7)	Ru1-S2	0.225 2(3)
  S1-Ru1-Cl1	91.87(12)	O1-Ru1-Cl1	88.4(2)	N1-Ru1-Cl2	84.8(3)
  S1-Ru1-Cl2	92.49(13)	O1-Ru1-Cl2	86.7(2)	N1-Ru1-O1	77.6(3)
  S1-Ru1-S2	93.21(10)	O1-Ru1-S2	90.9(2)	N1-Ru1-S2	168.5(2)
  Cl2-Ru1-Cl1	171.35(12)	N1-Ru1-S1	98.2(2)	S2-Ru1-Cl1	93.46(10)
  O1-Ru1-S1	175.8(2)	N1-Ru1-Cl1	87.2(3)	S2-Ru1-Cl2	93.75(12)

  下载: 导出CSV
•