English

• 0.   Introduction

  Cancer, also known as a malignant tumor, is a kind of major disease that poses a serious threat to public health^[1-2]. The treatment methods for cancer generally include surgery, chemotherapy, and radiotherapy, together with targeted therapy and immunotherapy emerged in recent years^[3-4]. Among these, chemotherapy has always played an important role in the clinical treatment of cancer. The development of new anticancer drugs with higher activity and different mechanisms of action is of great significance for improving the efficiency of cancer treatment.

  Since the discovery of cisplatin (CDDP) by Barnett Rosenberg et al. in 1965 for its strong inhibitory effect on tumor cells and the final FDA approval in 1979, altogether three generations of platinum anticancer drugs have been developed and successfully used in clinical. This also marked the inception of the research field of medicinal inorganic chemistry^[5-8]. However, the toxic side effects and resistance issues heavily limit their extensive applications. Consequently, the creation of non-platinum anticancer complexes that operate via mechanisms distinct from those of platinum-based drugs has garnered extensive interest among researchers in the field of chemistry and pharmacology. This area has emerged as a focal point in the realm of medicinal chemistry research in recent years^[9-10]. Copper is one of the essential metal elements in organisms, and also a necessary trace element in the metabolism of both normal cells and tumor cells. It was well‑studied that an increased level of copper in serum and tissue could propel angiogenesis, tumor growth, and metastasis^[11-12]. Considering that these three processes are indispensable steps in cancer progression, copper-targeted anticancer therapy is feasible and some promising copper complexes as drug candidates have been designed and investigated^{[11, 13]}.

  On the other hand, anthracyclines are a kind of effective anticancer agent distinguished by their unique four-membered ring structure and sugar moieties connected via glycosidic bonds. Doxorubicin and daunorubicin exemplify this class of drugs, renowned for their established effectiveness against a range of malignancies^[14-15]. They continue to be indispensable in the treatment of both solid tumors and hematological cancers. However, their administration is frequently associated with severe adverse effects, such as cardiotoxicity, and they may induce drug resistance due to their anthraquinone framework, which can diminish their therapeutic advantages^[16-17]. Therefore, it is necessary to structurally improve anthracyclines by modifying their anthraquinone nucleus structure. In our previous studies, the anthrahydrazone structure was adopted instead to prevent lipid peroxidation by the quinone group of anthracyclines, so that the potential cardiotoxicity could be avoided^[18]. In recent years, some meaningful metal complexes of the new anthrahydrazone ligands have been synthesized and investigated for their anticancer activities^[19-21]. It was also found that the 9-/10-side chain of anthrahydrazone, as an important pharmacophore, could modulate the anticancer efficacy based on the structure-activity relationship (SAR).

  Therefore, we report here the first pair of anthrahydrazone bearing pyridine as a side chain on either 9- or 9-/10-position, which are respectively named as 9-pyridine anthrahydrazone (9-PAH) and 9, 10-pyridine anthrahydrazone (9, 10-PAH), as well as their corresponding copper complexes, [Cu(L1)Cl₂]Cl (1) and {[Cu₄(μ₂-Cl)₃Cl₄(9, 10-PAH)₂(DMSO)₂]Cl₂}_n (2), where the new ligand L1 is formed by coupling two 9-PAH ligands in the coordination reaction (Scheme 1). Their in vitro anticancer activities were screened and the cell cycle arrest, cell apoptosis, and DNA binding properties were further examined and discussed. The work presented here is promising to modulate the anticancer activities of the anthrahydrazone and its metal complexes.

  Scheme 1

  

  Scheme 1.  Synthetic route of the two anthrahydrazone ligands, 9-PAH and 9, 10-PAH, as well as the new ligand L1 coupled by two 9-PAH

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  1.   Experimental

  1.1   Materials and instruments

  All chemicals were commercially available and were used without further purification unless specifically noted otherwise. 9-Anthraldehyde and CuCl₂·2H₂O were sourced from Aladdin Biochemical Technology Co., Ltd (Shanghai). 2-Hydrazinopyridine was obtained from Shanghai Bide Pharmatech Co., Ltd. Other chemical reagents were procured from Xi-long Chemical Co., Ltd. (Shantou). MTT (3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyltetrazolium bromide), RNase A, and PI (propidium iodide) were acquired from Sigma‑Aldrich. Annexin V‑FITC (fluorescein isothiocyanate)/PI was sourced from BD Biosciences.

  NMR spectra were acquired using a Bruker AV-400 spectrometer, with TMS and solvent signals serving as internal standards. High-resolution electrospray ionization-mass spectroscopy (HR ESI-MS) was measured on an Exactive liquid chromatography-MS mass spectrometer. Infrared spectra (IR) were recorded on a PerkinElmer FT-IR Spectrometer. Elemental analyses were conducted on a PerkinElmer Series Ⅱ CHNS/O 2400 elemental analyzer. Fluorescence spectra were recorded by an RF-5301 PC fluorescence spectrophotometer.

  1.2   Synthesis and characterization of 9-PAH and 9, 10-PAH

  The synthetic method of the 9-PAH ligand could be referenced to what we have previously reported^[19-20]. A suspension of 9-anthraldehyde (0.412 g, 2 mmol) and 2-hydrazinopyridine (0.218 g, 2 mmol) in 10 mL of anhydrous methanol was refluxed at 65 ℃ for 6 h. The completion of the reaction, indicated by the formation of an orange-yellow precipitate, could be monitored by using thin-layer chromatography (TLC). The precipitate was filtered and recrystallized using chloroform or dichloromethane to yield the orange crystallite, which was the target ligand, 9-PAH (Yield: 70%). In contrast to the synthesis of 9-PAH, 9, 10-PAH was synthesized using 9, 10‑anthracenedicarboxaldehyde (0.234 g, 1 mmol) in place of 9‑anthraldehyde, and employed twice the amount of 2‑hydrazpyridine (0.261 g, 2.4 mmol) in methanol solution. The mixed solution was stirred for reaction at 65 ℃ for 4 h, then was filtered after cooled to room temperature. The target ligand, 9, 10-PAH, was then achieved in an orange crystalline solid (Yield: 85%).

  Characterization of 9-PAH(C₂₀H₁₅N₃): HRMS(m/z): 298.133 4 [9-PAH+H]⁺. Elemental analysis Calcd. for C₂₀H₁₅N₃(%): C 80.78, H 5.08, N 14.13; Found(%): C 80.85, H 5.13, N 14.03. ¹H NMR (400 MHz, DMSO-d₆): δ 11.15 (s, 1H), 9.30 (s, 1H), 8.74 (d, J=8.6 Hz, 2H), 8.60 (s, 1H), 8.19 (dd, J=5.0, 1.8 Hz, 1H), 8.11 (d, J=8.2 Hz, 2H), 7.70-7.53 (m, 5H), 7.25 (d, J=8.4 Hz, 1H), 6.82-6.79 (m, 1H). ¹³C NMR (101 MHz, DMSO-d₆): δ 157.0, 148.0, 138.2, 137.7, 131.1, 129.2, 129.0, 128.2, 126.8, 126.2, 125.4, 124.9, 115.2, 106.1 (Fig.S1, S2, S5, S9, Supporting information).

  Characterization of 9, 10-PAH(C₂₆H₂₀N₆): HRMS(m/z): 417.181 6 [9, 10-PAH+H]⁺. Elemental analysis Calcd. for C₂₆H₂₀N₆(%): C 74.98, H 4.84, N 20.18; Found(%): C 75.09, H 4.79, N 20.12. ¹H NMR (400 MHz, DMSO-d₆): δ 11.19 (s, 2H), 9.24 (s, 2H), 8.72 (d, J=3.2 Hz, 4H), 8.20-8.18 (m, 2H), 7.66 (d, J=5.2 Hz, 6H), 7.21 (d, J=8.4 Hz, 2H), 6.84-6.80 (m, 2H). ¹³C NMR (125 MHz, DMSO-d₆): δ 157.0, 148.0, 138.2, 137.6, 129.1, 127.8, 126.4, 125.5, 115.3, 106.2. (Fig.S3, S4, S6, S10).

  1.3   Synthesis and characterization of complexes 1 and 2

  9-PAH (0.1 mmol, 0.030 0 g) and CuCl₂·2H₂O (0.2 mmol, 0.034 0 g) were dissolved in a mixed solution consisting of 30 mL of ethanol and 6 mL of chloroform. The mixture was allowed to react in reflux at 65 ℃ for 10 h. Subsequently, the reaction was stopped and the mixture was filtered after sufficient cooling down. Furthermore, the target metal complex 1 was obtained by the slow evaporation of the filtrate at room temperature for 3 d, from which the brown block single crystals could be also harvested. Yield: 75%. HRMS (m/z): 688.117 6 for [M-2Cl-H]⁺; 591.227 4 for [M-Cu-3Cl]⁺ (Fig.S7 and S11).

  9, 10-PAH (0.009 6 mmol, 0.004 0 g) and CuCl₂·2H₂O (0.028 8 mmol, 0.004 9 g) were dissolved in the mixed solution with 0.5 mL CH₃CN and 0.14 mL DMSO. Then the reaction mixture was heated to 60 ℃ allowing it to react for 3 d. After cooling to room temperature, the brown crystals of complex 2 were formed. Yield: 55%. HRMS (m/z): 718.878 2 for [2Cu+9, 10-PAH+5Cl]^-; 682.901 2 for [2Cu+9, 10-PAH+4Cl-H]^- (Fig.S8 and S12).

  1.4   X-ray crystallography

  The data collection of single crystals of the complexes was carried out using Bruker or Rigaku Oxford CCD diffractometer, which was equipped with a graphite monochromator and Mo Kα radiation (λ=0.071 07 nm) at room temperature. The structure was determined through direct methods and refined utilizing the OLEX2 and SHELXL-97 software programs. The non-hydrogen atoms were identified via successive difference Fourier synthesis. The ultimate refinement was achieved by employing full-matrix least-squares methods, with anisotropic thermal parameters for non‑ hydrogen atoms on F². The hydrogen atoms were theoretically positioned and placed in a riding configuration on the corresponding atoms. The crystallographic data and refinement specifics of the structural analyses were comprehensively summarized in Table 1 and 2. The packing diagram and the 2D topological network of the polymer structure of complex 2 are shown in Fig.S13-S15.

  Table 1

  

  Table 1.  Crystallographic data and structure refinement parameters for complexes 1 and 2

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  Parameter	1	2
  Empirical formula	C40H27Cl3CuN6	C28H26Cl4.5Cu2N6OS
  Formula weight	761.57	781.21
  Temperature/K	293(2)	296.15
  Crystal system	Triclinic	Triclinic
  Space group	P1	P1
  a/nm	1.214 63(4)	1.270 63(9)
  b/nm	1.278 47(4)	1.276 25(10)
  c/nm	1.288 89(4)	1.314 81(10)
  α/(°)	67.991(3)	106.594 0(10)
  β/(°)	76.950(3)	107.573 0(10)
  γ/(°)	75.834(3)	91.960 0(10)
  Volume/nm3	1.779 06(10)	1.931 0(3)
  Z	2	2
  Dc/(g·cm-3)	1.422	1.345
  μ/mm-1	0.878	1.495
  F(000)	778.0	791.0
  2θ range/(°)	6.6-52.74	3.36-53.34
  Reflection collected	44 173	24 138
  Index ranges	-15 ≤ h ≤ 15, -15 ≤ k ≤ 15, -16 ≤ l ≤ 16	-15 ≤ h ≤ 16, -16 ≤ k ≤ 16, -16 ≤ l ≤ 16
  Independent reflection	7 259 (Rint=0.037 3)	8 077 (Rint=0.039 5)
  Data, restraint, number of parameters	7 259, 0, 469	8 077, 3, 390
  Goodness-of-fit on F2	1.649	1.077
  Final R indexes [I≥2σ(I)]	R1=0.054 5, wR2=0.157 3	R1=0.046 9, wR2=0.131 0
  Final R indexes (all data)	R1=0.061 8, wR2=0.161 7	R1=0.063 8, wR2=0.139 1
  Largest diff. peak and hole/(e·nm-3)	2 400, -1 370	1 230, -1 120

  Table 2

  

  Table 2.  Selected bond lengths (nm) and angles (°) for complexes 1 and 2

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  1
  Cu1—Cl2	0.222 58(9)	Cu1—N4	0.198 5(2)	Cu1—N2	0.209 2(3)
  Cu1—Cl1	0.247 42(11)	Cu1—N6	0.196 2(3)
  Cl2—Cu1—Cl1	104.95(4)	N6—Cu1—Cl2	98.36(8)	N2—Cu1—Cl2	98.75(7)
  N4—Cu1—Cl2	160.51(8)	N6—Cu1—Cl1	109.67(9)	N2—Cu1—Cl1	89.77(8)
  N4—Cu1—Cl1	94.11(8)	N6—Cu1—N4	78.58(11)
  N4—Cu1—N2	76.94(10)	N6—Cu1—N2	149.65(11)
  2
  Cu2—Cl3	0.227 89(9)	Cu2—N6	0.202 0(3)	Cu1—O1	0.195 6(2)
  Cu2—Cl4	0.224 28(10)	Cu1—Cl2	0.260 06(5)	Cu1—N1	0.205 6(3)
  Cu2—N4	0.204 6(3)	Cu1—Cl1	0.224 46(10)	Cu1—N3	0.199 9(3)
  Cl4—Cu2—Cl3	94.18(4)	N4—Cu2—Cl3	173.95(8)	N1—Cu1—Cl2	93.45(8)
  N6—Cu2—Cl3	94.88(8)	Cl1—Cu1—Cl2	109.96(4)	N3—Cu1—N1	79.49(11)
  N6—Cu2—Cl4	157.40(9)	O1—Cu1—Cl2	93.89(8)
  N6—Cu2—N4	79.06(10)	O1—Cu1—N1	87.28(10)

  1.5   Biological activity assay

  1.5.1   Cell culture

  The human cancer cell lines, including T‑24, Hep-G2, MGC-803, HeLa-229, SK-OV-3, and NCI-H460, as well as the human normal liver cell line HL-7702, were all obtained from the Shanghai Cell Bank of the Chinese Academy of Sciences. They were cultivated in DMEM or Gibco Medium with 10% fetal bovine serum, 100 U·mL^-1 of penicillin, and 100 μg·mL^-1 of streptomycin. The condition for cell incubation was maintained at 37 ℃, with a 5% CO₂/95% air (volume fraction) of humidified atmosphere.

  1.5.2   Cytotoxicity analysis by MTT assay

  The tested cells were transplanted into 96-well plates and incubated for 24 h at 37 ℃ with a 5% CO₂/95% air of atmosphere. Then the cells were incubated and treated with 20 μL of each compound at varying concentrations (1.25, 2.5, 5, 10, and 20 μmol·L^-1), along with the control. 10 μL of MTT solution (5 mg·mL^-1) was added after 48 h-incubation, and continued incubation for 4-6 h. Subsequently, the formed formazan crystals were solubilized in 100 μL of DMSO. The absorbance values were recorded to determine IC₅₀ values using the Bliss method. The assay was replicated three times.

  1.5.3   Analysis for cell cycle

  The selected cancer cells (SK-OV-3 and HeLa-229) were cultured at 37 ℃ for 48 h in 6-well plates after the treatment with the gradient concentrations (ca. 0.5×, 1.0×, 2.0× of IC₅₀ values) of complex 2, and the cell culture medium was set for negative control. After the incubation, the cells were collected, washed, and suspended in the pre-cooled PBS (phosphate-buffered saline). Then the collected cells were fixed in ice-cold ethanol overnight at -20 ℃ before being thoroughly rinsed with pre-cooled PBS and subsequently stained by RNase A and PI solution for analysis.

  1.5.4   Analysis for cell apoptosis

  SK-OV-3 and HeLa-229 cells were cultured in 6-well plates under controlled conditions of 37 ℃, following which they were treated with different concentrations of complex 2. After a 24-hour co-incubation period with 2, the cells were labeled using the AO/EB kit, adhering strictly to the prescribed instructions for 5-10 min. Ultimately, the cells were observed and imaged using an inverted fluorescent microscope.

  1.6   Spectroscopic analysis for DNA binding

  Complex 2 was diluted with DMSO to achieve a stock solution at 2 mmol·L^-1 concentration, while the DNA reservoir concentration was maintained at 2 mmol·L^-1. For DNA competitive binding studies by fluorescent spectral analysis, a mixed solution with a c_GR/c_DNA ratio of 1∶10 was refrigerated overnight to ensure complete DNA dissolution. Subsequently, the GR-DNA mixed solution was added to the sample cuvette, along with 3 mL of Tris buffer. Using a pipette, equal volumes of the 2 mmol·L^-1 compound stock solution were incrementally added into the sample cuvette. As the concentration ratio of 2 to GR-DNA increased, the fluorescence spectrum was monitored. The quenching constant (K_sv) for 2, representing its quenching ability, was derived through the linear fitting of I₀/I vs c_Q, utilizing the Stern‑Volmer equation: I₀/I=1+K_svc_Q, where I₀ and I signify the emission peak intensities of the GR-DNA system in the absence and presence of 2 as the quencher, respectively, and c_Q denotes the concentration of the quencher.

  2.   Results and discussion

  2.1   Synthesis

  As depicted in Scheme 1, two anthrahydrazone ligands, 9-PAH and 9, 10-PAH, were synthesized in a similar synthetic route, utilizing 9-anthraldehyde or 9, 10-anthracenedicarboxaldehyde together with 2-hydrazinopyridine as the starting materials^[20]. Subsequently, two corresponding copper(Ⅱ) complexes were respectively synthesized via the solution method within EtOH/CHCl₃ or CH₃CN/DMSO. It is noteworthy to observe that during the synthesis of complex 1, two 9-PAH ligands underwent a coupling reaction, leading to the formation of a new ligand, designated as L1 (C₄₀H₂₈N₆). This newly formed ligand L1 subsequently deprotonated and coordinated with Cu(Ⅱ), resulting in the actual chemical formulation of 1, which is [Cu(L1)Cl₂]Cl. The structures of the two copper complexes were characterized by HRMS, elemental analysis (C, H, N), and single-crystal X-ray diffraction analysis. Furthermore, the stability of each compound in an aqueous solution was evaluated using UV-Vis spectral analysis, which suggested that each metal complex retained its coordination state in solution for 48 h without newly emerged absorption peaks (Fig.S16-S19).

  2.2   Crystal structure analysis of complexes 1 and 2

  The X-ray diffraction analysis indicated that the crystal structures of complexes 1 and 2 both belong to the triclinic crystal system with space group P1. Details of the structural refinement parameters and the crystallographic data were summarized in Table 1 and 2.

  It should be noted that the two 9-PAH ligands have been connected into one ligand (L1) by the internal cyclization reaction between the C atom from the imine —C=N group and the pyridyl N atom of one 9-PAH ligand^[22-24], and further by the inter-coupling between its N atom from the amine —NH group and the C atom of the imine —C=N group of another 9-PAH ligand. Meanwhile, it can be observed that there exists a free Cl^-, acting as a counterion, which suggests that the new coupled ligand L1 formed to be an overall +1 cation when coordinating with Cu(Ⅱ). Given that the N atom (N3) of the pyridine and the N atom (N1) of the imine —C=N group are both in the quaternized form, considering their characteristic C=N bond, the amine —NH group (N2) coordinated with Cu(Ⅱ) should be deprotonated. For complex 1, the Cu(Ⅱ) center adopts a five-coordinated square pyramidal configuration, as shown in Fig. 1A. The central Cu(Ⅱ) is coordinated by three different N atoms (N2, N4, N6) and two Cl atoms, respectively. The three N atoms are respectively from the amine —NH group (N2), as well as the imine —C=N group (N4) and the pyridine (N6). One Cl atom coordinates to Cu(Ⅱ) to form the basis of the pyramid, together with the three N atoms. While the other Cl atom is located at the top of the pyramid. From the perspective of the bond distances and angles, in 1, the Cu—Cl distances [0.222 58(9) and 0.247 42(11) nm] are longer than the Cu—N distances [0.198 5(2), 0.196 2(3), and 0.209 2(3) nm]. The two chelated angles formed by the tridentated 9-PAH (after coupling) to Cu(Ⅱ) (N2—Cu1—N4) and (N4—Cu1—N6) were determined to be 76.94(10)° and 78.58(11)°, respectively.

  Figure 1

  

  Figure 1.  Crystal structures of two copper complexes 1 (A) and 2 (B)

  The solvent molecules are omitted for clarity.

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  Meanwhile, the two five-coordinated Cu2 atoms symmetrically bridge the adjacent structural units via one coordinated Cl atom, respectively, thus forming a 1D chain-like polymeric structure. Additionally, each structural unit contains two symmetric free Cl^- serving as counterions.

  The crystal structure of complex 2 is shown in Fig. 1B. Comparatively, 2 constitutes a polymeric structure, wherein each structural unit encompasses two centrosymmetric binuclear copper complexes of 9, 10-PAH. The four Cu ions all adopt a similar five-coordinated square pyramidal geometry. Specifically, each 9, 10-PAH ligand, via its two side chains of pyridinyl anthrahydrazone, forms a five‑coordinated binuclear structure with two Cu atoms (Cu1 and Cu2). For Cu1, it is coordinated respectively by two Cl atoms (Cl1, Cl2), one DMSO solvent molecule, and one 9, 10-PAH ligand via the chelated N1 atom of the imine —C=N group and the pyridyl N3 atom, which form the basis of the pyramid together with the O atom of DMSO and the Cl1 atom, while Cl2 occupies the top of the pyramid. The coordinated Cl2, located at the symmetry center of the structural unit, further coordinates with Cu1A to bridge the two binuclear copper structures (Fig.S13). Meanwhile, 9, 10-PAH coordinates to Cu2 with the N4 atom of the imine —C=N group and the pyridyl N6 atom on the other side chain, together with two Cl atoms (Cl3, Cl4). While one Cl3A atom from the adjacent structural unit further coordinates with Cu2 to form a similar five-coordinated square pyramidal configuration. Therefore, the two five-coordinated Cu2 atoms symmetrically connect the adjacent structural units via one coordinated Cl atoms, respectively, thus forming a 1D chain-like polymeric structure, as depicted in Fig.S14. Additionally, each structural unit contains two symmetric free Cl^- serving as counterions (Fig.S15). Such coordination structure could be also supported by the empirical molecular formula for the crystallographic data of 2, C₂₈H₂₆Cl_4.5Cu₂N₆OS.

  2.3   In vitro cytotoxicity

  The in vitro cytotoxic effects of all the compounds were evaluated by MTT assay towards the T-24, Hep-G2, MGC-803, HeLa-229, SK-OV-3, NCI-H460 cancer cell lines as well as a normal liver cell line, HL-7702. The cisplatin and CuCl₂·2H₂O were utilized as positive controls. As presented in Table 3, neither the ligands nor CuCl₂·2H₂O demonstrated growth inhibition activities against the tested cancer cell lines, with all IC₅₀ values exceeding 20 μmol·L^-1 except for SK-OV-3. In contrast, complexes 1 and 2 exhibited significant and broad-spectrum growth inhibition activities, with IC₅₀ values ranging from 8 to 16 μmol·L^-1. Notably, they showed cytotoxic selectivity towards different cell lines. The SK-OV-3 and T-24 cells were more sensitive to 1, with IC₅₀ values of (7.06±0.79) μmol·L^-1 and (8.23±0.30) μmol·L^-1, respectively, whereas 2 was most effective against HeLa-229 and SK-OV-3 cells, with IC₅₀ values of (5.92±0.32) μmol·L^-1 and (6.48±0.39) μmol·L^-1, respectively. The inhibitory activities of both complexes against these mentioned cancer cell lines were comparable to or even twice those of cisplatin, indicating a notable synergistic effect under the coordination of the ligands with Cu(Ⅱ). Furthermore, both complexes also exhibited cytotoxicity toward normal liver cells with IC₅₀ values tested to be (15.10±0.76) μmol·L^-1 and (8.09±0.22) μmol·L^-1, suggesting that they are both potential cytotoxic compounds.

  Table 3

  

  Table 3.  IC₅₀ values for the tested compounds towards various cancer cell lines and the normal liver cell line  μmol·L^-1

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  Compound	T-24	HepG-2	MGC-803	HeLa-229	SK-OV-3	NCI-H460	HL-7702
  9-PAH	> 20	> 20	> 20	> 20	> 20	> 20	> 20
  1	8.23±0.30	12.68±1.35	10.65±0.48	15.76±1.36	7.06±0.79	12.22±1.52	15.10±0.76
  9, 10-PAH	> 20	> 20	> 20	> 20	> 20	> 20	> 20
  2	13.07±0.03	15.63±0.92	8.67±0.68	5.92±0.32	6.48±0.39	15.52±0.94	8.09±0.22
  CuCl2·2H2O	> 20	> 20	> 20	> 20	18.71±0.83	> 20	> 20
  Cisplatin[20]	14.79±2.74	18.55±0.78	17.79±2.50	18.55±0.78	6.84±0.43	18.29±1.02	12.67±1.27

  2.4   Cell cycle analysis

  Cell cycle arrest represents a pivotal mechanism underpinning the anticancer efficacy of a myriad of small-molecule drugs. To elucidate the anticancer mechanism of complex 2, we utilized flow cytometry to investigate the cell cycle arrest induced by 2 over 48 h treatment, focusing on the two most sensitive cell lines, SK-OV-3 and HeLa-229, as depicted in Fig. 2. A marked elevation in the proportion of S phase in HeLa-229 cells was observed following treatment with 3, 6, and 12 μmol·L^-1 of 2, with the populations increasing from 18.35% to 24.33%, 26.38%, and 31.68%, respectively. These results suggested that 2 induces cell cycle arrest in the S phase of HeLa-229 cells effectively, potentially inhibiting cellular proliferation by disrupting DNA synthesis during this critical phase of the cell cycle. Conversely, the distribution of cell cycle phases exhibited negligible variations across the concentration range of 3.3-13.1 μmol·L^-1 in SK-OV-3 cells, suggesting that cell cycle arrest might not constitute the primary mechanism underlying the effects exerted by 2 on these cellular entities. Upon comparing the cycle arrest results of these selected cell lines, discernible disparities emerged in the manner through which complex 2 exerts its activity against diverse cancer cell types.

  Figure 2

  

  Figure 2.  Populations of the cell cycle of SK-OV-3 and HeLa-229 cells under the treatment of complex 2 for 48 h in different concentrations

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  2.5   Cell apoptosis

  Apoptosis is also acknowledged as a pivotal mechanism by which small-molecule drugs inhibit cellular proliferation. To morphologically assess the potential apoptosis induced by complex 2 in cancer cells, we employed the AO/EB double staining assay, utilizing a fluorescence-inverted microscope for observation. The resultant morphological alterations induced by 2 after a 24-hour incubation in both HeLa-229 and SK-OV-3 cells are illustrated in Fig. 3. It can be seen that the addition of 2 precipitated a progressive transformation in cell morphology, evidenced by a transition towards a more diminutive and spherical form. Simultaneously, the manifestation of orange or orange-red fluorescence in cells became notably apparent. These results were particularly illustrated in SK-OV-3 cells exposed to the highest concentration of 13 μmol·L^-1, wherein the intensity of the orange-red fluorescence was markedly enhanced. These observations suggested that the cells are undergoing a process of apoptosis.

  Figure 3

  

  Figure 3.  Cell apoptosis indicated by AO/EB staining in the HeLa-229 and SK-OV-3 cells treated with complex 2

  Images were acquired under fluorescence microscopy.

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  2.6   GR-competitive binding

  GelRed (GR) represents a new class of DNA probes that exemplify the intercalation mechanism within DNA. It possesses the capacity to form soluble complexes with calf thymus DNA (ct-DNA). Given its reduced toxicity in comparison to the traditional DNA intercalator (ethidium bromide, EtBr), GR is increasingly recognized as an environmentally benign substitute for EtBr. DNA serves as a pivotal pharmacological target for various metallo-chemotherapeutic agents, thereby emphasizing the significance of investigating the interactions between DNA and these metal complexes. Several distinct modes of interaction can occur between the metal complexes and DNA, including intercalation between adjacent base pairs within the DNA helix and association with the grooves of the DNA backbone. The intercalative potential of complex 2 was assessed through the competitive experiment compared with GR. When intercalated between DNA base pairs, GR exhibits intense fluorescence emission at approximately 600 nm. However, this fluorescence is quenched when GR is displaced by a competing intercalator.

  As illustrated in Fig. 4, the dashed line represents the fluorescence of the GR‑DNA system. In the absence of complex 2, the system exhibited a prominent fluorescence peak at 600 nm with an intensity of I₀=623.75. In contrast, the solid lines depict the changes in the fluorescence intensity of the GR-DNA mixed with the added complex 2 at different concentrations. Notably, when the ratio of c_GR∶c_DNA∶c₂ reached 1∶10∶6, the fluorescence intensity decreased to I₆=286.29, with a 54.25% reduction. Upon increasing the ratio to 1∶10∶10, the fluorescence intensity reduced to the minimum value (I_min=176.44), with a significant reduction of 71.71%. This observation highlights the potent competitive interaction between the complex and GR, thereby affirming the ability of 2 to effectively intercalate within DNA base pairs. Consequently, a substantial quantity of GR is displaced, leading to a gradual decrease in fluorescence intensity and a marked augmentation of the quenching effect. Through the linear fitting analysis by the Stern-Volmer equation, the quenching constant (K_sv) of 2 on the GR-DNA system was evaluated to be 1.343×10³ L·mol^-1, with the R² value of 0.981 4. Additionally, the quenching effects on the fluorescence intensity were found to be a non-linear pattern, suggesting the possibility of both dynamic and static quenching mechanisms between 2 and DNA. As a result, the underlying mechanism responsible for the quenching of GR fluorescence by complex 2 might involve a dual mode of action.

  Figure 4

  

  Figure 4.  Fluorescence spectroscopic analysis for the DNA binding of complex 2 under competition with the GR-DNA system

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  3.   Conclusions

  In this work, two anthrahydrazone ligands bearing pyridyl side-chain on the 9- or 9, 10-position of the anthracene were designed and characterized, which respectively afforded the corresponding copper complex by coordination with Cu(Ⅱ). Both complexes 1 and 2 exhibited significant cytotoxicity towards the selected cancer cells, in which 2 showed a certain superiority. In the presence of the copper center, it was found that 2 exhibits anticancer activity by arresting the cell cycle and inducing cell apoptosis in the most sensitive cancer cells, in which the copper center should play the key role. Viewed from some previously reported metal complexes of anthrahydrazones, the metal centers are also proved to be crucial for modulating the anticancer activity of anthradydrazone ligands. Much convincing evidence has also suggested that the coordinated Cu(Ⅱ)/Cu(Ⅰ) could potentiate the anticancer activities of various bioactive ligands. Nevertheless, more persuasive conclusions need to be drawn based on extensive ligand/complex designing, anticancer screening, and comparative action mechanism analyses in terms of structure-activity relationship.

  
  Acknowledgments: This work was financially supported by the National Natural Science Foundation of China (Grant No.22167005) and the Research Startup Funding for High-level Talents & Scientific Research Initiation Projects of Yunnan Open University (Grants No.060123070, 061024023, 060123076). Conflicts of interest: There are no conflicts to declare.
  Supporting information is available at http://www.wjhxxb.cn
  
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• 

  Scheme 1  Synthetic route of the two anthrahydrazone ligands, 9-PAH and 9, 10-PAH, as well as the new ligand L1 coupled by two 9-PAH

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  Figure 1  Crystal structures of two copper complexes 1 (A) and 2 (B)

  The solvent molecules are omitted for clarity.

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  Figure 2  Populations of the cell cycle of SK-OV-3 and HeLa-229 cells under the treatment of complex 2 for 48 h in different concentrations

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  Figure 3  Cell apoptosis indicated by AO/EB staining in the HeLa-229 and SK-OV-3 cells treated with complex 2

  Images were acquired under fluorescence microscopy.

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  Figure 4  Fluorescence spectroscopic analysis for the DNA binding of complex 2 under competition with the GR-DNA system

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  Table 1.  Crystallographic data and structure refinement parameters for complexes 1 and 2

  Parameter	1	2
  Empirical formula	C40H27Cl3CuN6	C28H26Cl4.5Cu2N6OS
  Formula weight	761.57	781.21
  Temperature/K	293(2)	296.15
  Crystal system	Triclinic	Triclinic
  Space group	P1	P1
  a/nm	1.214 63(4)	1.270 63(9)
  b/nm	1.278 47(4)	1.276 25(10)
  c/nm	1.288 89(4)	1.314 81(10)
  α/(°)	67.991(3)	106.594 0(10)
  β/(°)	76.950(3)	107.573 0(10)
  γ/(°)	75.834(3)	91.960 0(10)
  Volume/nm3	1.779 06(10)	1.931 0(3)
  Z	2	2
  Dc/(g·cm-3)	1.422	1.345
  μ/mm-1	0.878	1.495
  F(000)	778.0	791.0
  2θ range/(°)	6.6-52.74	3.36-53.34
  Reflection collected	44 173	24 138
  Index ranges	-15 ≤ h ≤ 15, -15 ≤ k ≤ 15, -16 ≤ l ≤ 16	-15 ≤ h ≤ 16, -16 ≤ k ≤ 16, -16 ≤ l ≤ 16
  Independent reflection	7 259 (Rint=0.037 3)	8 077 (Rint=0.039 5)
  Data, restraint, number of parameters	7 259, 0, 469	8 077, 3, 390
  Goodness-of-fit on F2	1.649	1.077
  Final R indexes [I≥2σ(I)]	R1=0.054 5, wR2=0.157 3	R1=0.046 9, wR2=0.131 0
  Final R indexes (all data)	R1=0.061 8, wR2=0.161 7	R1=0.063 8, wR2=0.139 1
  Largest diff. peak and hole/(e·nm-3)	2 400, -1 370	1 230, -1 120

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  Table 2.  Selected bond lengths (nm) and angles (°) for complexes 1 and 2

  1
  Cu1—Cl2	0.222 58(9)	Cu1—N4	0.198 5(2)	Cu1—N2	0.209 2(3)
  Cu1—Cl1	0.247 42(11)	Cu1—N6	0.196 2(3)
  Cl2—Cu1—Cl1	104.95(4)	N6—Cu1—Cl2	98.36(8)	N2—Cu1—Cl2	98.75(7)
  N4—Cu1—Cl2	160.51(8)	N6—Cu1—Cl1	109.67(9)	N2—Cu1—Cl1	89.77(8)
  N4—Cu1—Cl1	94.11(8)	N6—Cu1—N4	78.58(11)
  N4—Cu1—N2	76.94(10)	N6—Cu1—N2	149.65(11)
  2
  Cu2—Cl3	0.227 89(9)	Cu2—N6	0.202 0(3)	Cu1—O1	0.195 6(2)
  Cu2—Cl4	0.224 28(10)	Cu1—Cl2	0.260 06(5)	Cu1—N1	0.205 6(3)
  Cu2—N4	0.204 6(3)	Cu1—Cl1	0.224 46(10)	Cu1—N3	0.199 9(3)
  Cl4—Cu2—Cl3	94.18(4)	N4—Cu2—Cl3	173.95(8)	N1—Cu1—Cl2	93.45(8)
  N6—Cu2—Cl3	94.88(8)	Cl1—Cu1—Cl2	109.96(4)	N3—Cu1—N1	79.49(11)
  N6—Cu2—Cl4	157.40(9)	O1—Cu1—Cl2	93.89(8)
  N6—Cu2—N4	79.06(10)	O1—Cu1—N1	87.28(10)

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  Table 3.  IC₅₀ values for the tested compounds towards various cancer cell lines and the normal liver cell line  μmol·L^-1

  Compound	T-24	HepG-2	MGC-803	HeLa-229	SK-OV-3	NCI-H460	HL-7702
  9-PAH	> 20	> 20	> 20	> 20	> 20	> 20	> 20
  1	8.23±0.30	12.68±1.35	10.65±0.48	15.76±1.36	7.06±0.79	12.22±1.52	15.10±0.76
  9, 10-PAH	> 20	> 20	> 20	> 20	> 20	> 20	> 20
  2	13.07±0.03	15.63±0.92	8.67±0.68	5.92±0.32	6.48±0.39	15.52±0.94	8.09±0.22
  CuCl2·2H2O	> 20	> 20	> 20	> 20	18.71±0.83	> 20	> 20
  Cisplatin[20]	14.79±2.74	18.55±0.78	17.79±2.50	18.55±0.78	6.84±0.43	18.29±1.02	12.67±1.27

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