Synthesis and Anti-Cancer Investigations of Novel Copper(II) Complexes Based on Adenine

Synthesis and Anti-Cancer Investigations of Novel Copper(II) Complexes Based on Adenine | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Synthesis and Anti-Cancer Investigations of Novel Copper(II) Complexes Based on Adenine Xiaoyan Zhai, Hussein Hanibah, Nor Zakiah Nor Hashim, Juzheng Zhang, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3997929/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 01 Jun, 2024 Read the published version in Journal of Molecular Structure → Version 1 posted You are reading this latest preprint version Abstract Platinum-based chemotherapeutics have played a critical role in oncology for decades. However, their broader utility is hindered by the advent of severe side effects and the emergence of drug resistance. The pursuit of alternative agents, particularly non-platinum (non-Pt) metal complexes, has gained momentum in current research. Designing efficacious non-Pt metal agents that target DNA poses a complex challenge. In this study, we present the strategic design, synthesis, and thorough characterization of two innovative copper(II) complexes leveraging adenine as a ligand, a potential avenue to overcome these challenges. Our investigation demonstrates the superior cytotoxicity of these copper(II) complexes compared to the benchmark cisplatin, with complex C2 exhibiting the most promising anticancer activity, showcasing an impressive IC 50 value of 4.51 µM in MGC-803 cells. Mechanistic insights underscore that complex C2 executes its cytotoxic effects by instigating DNA damage, orchestrating cell cycle arrest at the G2 phase, perturbing mitochondrial membrane potential, inducing ROS production, and ultimately triggering apoptotic pathways. These findings significantly emphasize the potential of designing novel adenine-based anticancer metal complexes targeting DNA, portraying a compelling trajectory for advancing anticancer drug development. Adenine Copper(II) Complexes DNA Targeting Reactive oxygen species Anticancer Cytotoxicity Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 1 Introduction Platinum-based chemotherapeutics have played a pivotal role in treating solid tumors for over five decades by effectively inducing DNA damageKomeda 2011 ; Xiaoyong and Zijian 2012 ; McQuitty 2014 ; Kenny and Marmion 2019 ; Ghosh 2019 ; Xian et al. 2021 ; Zhang et al. 2022 ; Su et al. 2022 ). However, the advancement of platinum drugs is hindered by prevalent drug resistance, significant side effects, low water solubility, and target ambiguity. Notably, drug resistance and severe limitations in the development of platinum metal-based drugs pose critical challenges (Kim et al. 2020 ; Pan et al. 2021 ; Lu et al. 2022b ; Van Nyen et al. 2022 ). It is imperative to explore alternative metal-based complexes to circumvent the resistance associated with platinum drugs. Recognizing the success of cisplatin, researchers have embarked on extensive investigations into other metal-based complexes. Recent studies have underscored the potential of non-platinum based complexes, exhibiting enhanced potency, reduced toxicity, and higher target specificity, emerging as promising candidates for novel chemotherapeutic agents (Ott and Gust 2007 ; Milacic et al. 2008 ; Erxleben 2019 ; Ma et al. 2019 ; Imberti et al. 2020 ; Malik et al. 2021 ; Gourdon et al. 2022 ; Ferraro et al. 2022 ; Islam et al. 2022 ; Lu et al. 2022a ; Sinicropi et al. 2022 ; Devi et al. 2023 ). Within this realm, copper (Cu) complexes have garnered significant attention for their potential applications as chemotherapeutic agents. (Cvek et al. 2008 ; Paterson and Donnelly 2011 ; Zhang et al. 2017 ; Mo et al. 2018 ; Spengler et al. 2018 ; Ohui et al. 2019 ; Yu et al. 2019 ; MacHado et al. 2020 ; Gou et al. 2021 ; da Silva et al. 2022 ; Shen et al. 2022 ; Khalil et al. 2022 ). Copper, being an essential element for sustaining life, actively participates in vital metabolic pathways within cells as cofactors. Moreover, Cu ions possess the capacity to generate reactive oxygen species (ROS) through metal oxidation and reduction processes. Reactive oxygen free radicals can engage in numerous reactions with DNA, proteins, and lipids, leading to mutations and cellular damage (Moloney and Cotter 2018 ; Kaarniranta et al. 2019 ; Srinivas et al. 2019 ; Yan et al. 2019 ; Yuan et al. 2020 ; Jiang et al. 2022 ; Tsvetkov et al. 2022 ; Tang et al. 2022 ; Cobine and Brady 2022 ). Over the last two decades, several studies have demonstrated the cytotoxicity of Cu complexes, even at nanomolar levels. Hence, Cu complexes exhibit substantial potential to supersede cisplatin as the next generation of metal-based anticancer drugs (Cvek et al. 2008 ; Paterson and Donnelly 2011 ; Zhang et al. 2017 ; Mo et al. 2018 ; Spengler et al. 2018 ; Ohui et al. 2019 ; Yu et al. 2019 ; MacHado et al. 2020 ; Gou et al. 2021 ; da Silva et al. 2022 ; Shen et al. 2022 ; Khalil et al. 2022 ). Nevertheless, these Cu complexes have yet to demonstrate explicit DNA targeting properties, potentially resulting in off-target effects upon administration. Adenine, also known as vitamin B4, holds a crucial role as a fundamental compound aiding in DNA and RNA synthesis, constituting a vital component of human genetic material (Heyn and Esteller 2015 ; Parashar et al. 2018 ; Khodadadi et al. 2021 ; Yu et al. 2021 ; Varma et al. 2022 ). To develop a copper complex with precise targeting and excellent DNA affinity, we propose a novel approach using adenine as the foundation. DNA is comprised of nitrogenous bases, deoxyribose, and phosphate. In DNA synthesis, cells utilize amino acids or free nitrogenous bases, such as adenine, for salvage synthesis, a less energy-intensive and simpler process than de novo synthesis. The design rationale of the study employs a unique approach by utilizing adenine as the lead compound to synthesize a series of copper complexes that employ tridentate coordination donors (OH, N, N) (Fig. 1 ). This approach aims to enhance their targeting ability and affinity for DNA, optimize the salvage synthesis pathway to enable copper (Cu) complexes to effectively interact with DNA, increase reactive oxygen species (ROS) levels, induce DNA damage, and ultimately impede cancer cell proliferation (Guo et al. 2010 ; Tabrizi and Abyar 2019 ; Fan et al. 2020 ; Battaglia et al. 2020 ; Hayes et al. 2020 ). The objective of the study is to overcome the significant challenges posed by platinum-based chemotherapeutics, including drug resistance, notable side effects, low water solubility, and target ambiguity. Non-platinum metal complexes have shown promise as potential chemotherapeutic agents, with enhanced potency, reduced toxicity, and heightened target specificity. These characteristics make them a suitable alternative to platinum-based chemotherapeutics as novel anticancer drugs. This work focuses on copper complexes with tridentate adenine Schiff base ligand modifications. Two adenine-based copper complexes (C1-C2) were synthesized and evaluated for their activity across various cancer cell lines. The potential of these Cu complexes to induce DNA damage was investigated, and the mechanism by which these Cu complexes inhibit cancer cells in vitro was elucidated. The transition towards non-platinum metal complexes is a compelling option, and this research contributes to the growing body of work aimed at exploring these alternatives for the development of more effective and safer chemotherapeutic agents. 2 Experimental sections 2.1 Materials 6-Hydrazinopurine, copper(II) chloride (CuCl 2 ), and a salicylaldehyde derivative were sourced from Aladdin, Shanghai, China. MTT, JC-1, 2′,7-dichlorodihydrofluorescein diacetate (DCFH2-DA), and AO/EB were procured from Sigma-Aldrich. All biological antibodies were acquired from Abcam. Commercial sources supplied all other solvents and reagents, which were used without further purification. The 1 H and 13 C NMR analyses were conducted at room temperature (298 K) in DMSO-d6 using a Bruker AVANCE III HD 400 MHz spectrophotometer. X-ray crystallographic data were collected using a Rigaku HyPix diffractometer. 2.2 Synthesis of copper(II) complexes The ligands based on adenine were synthesized through a reaction involving 6-Hydrazinyl-9H-purine and various aldehydes. Specifically, 6-Hydrazinyl-9H-purine (3 mmol) and the corresponding aldehyde (3 mmol) were dissolved in 10 mL of ethanol and stirred for a duration of 6 hours. The resulting product was filtered to obtain a light yellow solid. The chemical structures of these ligands were meticulously characterized using High-Resolution Mass Spectrometry (HRMS), as well as 1 H and 13 C Nuclear Magnetic Resonance (NMR) spectroscopy. Subsequently, the Cu(II) complexes were synthesized by reacting the ligands (0.10 mmol) with CuCl 2 (0.10 mmol) in a solvent mixture of methanol (1.5 mL) and dichloromethane (1.5 mL). The reaction was conducted under solvothermal conditions at 65°C for a period of 24 hours. Crystals suitable for X-ray diffraction analysis were carefully collected to further elucidate the molecular structure and bonding arrangements within the complexes. L1 : 1 H NMR (500 MHz, DMSO- d 6 ) δ 13.25 (s, 1H), 11.84 (s, 1H), 11.53 (s, 1H), 8.34 (d, J = 8.1 Hz, 3H), 7.34 (s, 1H), 7.25 (t, J = 7.6 Hz, 1H), 6.93–6.88 (m, 2H). 13 C NMR (126 MHz, DMSO- d 6 ) δ 157.32, 152.41, 145.33, 141.38, 130.86, 119.56, 119.22, 118.77, 117.26. HRMS:m/z 255.0916 [M + H] L2 : 1 H NMR (500 MHz, DMSO- d 6 ) δ 13.20 (s, 1H), 11.82 (s, 1H), 11.32 (s, 1H), 8.31 (t, J = 19.8 Hz, 3H), 7.12 (s, 1H), 7.05 (d, J = 8.1 Hz, 1H), 6.81 (d, J = 8.2 Hz, 1H), 2.24 (s, 3H). 13 C NMR (126 MHz, DMSO- d 6 ) δ 155.07, 152.37, 145.40, 141.31, 131.47, 130.75, 127.97, 118.77, 117.03, 20.31. HRMS:m/z 269.1073 [M + H] 2.3 Bioactivity Cell cultures were maintained in Dulbecco's Modified Eagle Medium (DMEM, Gibco) supplemented with 10% (v/v) fetal bovine serum (FBS, Gibco), 100 U/mL penicillin (Sigma), 100 U/mL streptomycin (Sigma), and 2 mM glutamine (Sigma) at 37°C in a humidified atmosphere containing 95% air and 5% CO2. Cu(II) complexes were dissolved in dimethylformamide (DMF). To assess the in vitro cytotoxicity of the Cu(II) complexes against both cancer and normal cell lines, a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT, Sigma) assay was conducted. Cells were exposed to Cu(II) complexes and free ligands at five different concentrations for 48 hours. The growth inhibitory rates were measured using an enzyme labeling instrument at a double wavelength of 570/630 nm. The growth inhibitory rates were computed using the formula: (OD control - OD test) / OD control × 100%. 2.4 Cu(II) complex C2 induced mitochondrial damage MGC-803 cells were cultured and incubated with Cu(II) complex C1/C2 at a concentration of 4.51 µM for 6 hours. The incubation was carried out in poly-HEMA coated 6-well plates to prevent cell adhesion. Subsequently, the cells were harvested and resuspended in a fresh culture medium. To assess mitochondrial membrane potential, 0.5 mL of JC-1 and DAPI working solution was added to the cell suspension. The cells were then incubated at 37°C for 25 minutes. Following incubation, the staining solution was carefully removed through centrifugation. The cells were rinsed three times with JC-1 staining buffer to ensure proper removal of excess staining solution. The stained cells were promptly subjected to analysis using confocal microscopy to evaluate the changes in mitochondrial membrane potential induced by the treatment with Cu(II) complex C2. 2.5 Enhancement of Cu(II) complex C2 at intracellular ROS levels MGC-803 cells were seeded at a density of 1 × 105 cells/well on poly-L-lysine-coated coverslips within 6-well plates using heat-deactivated complete RPMI. The cells were subsequently treated with Cu(II) complex C1/C2 at a concentration of 4.51 µM for a duration of 6 hours. Following the treatment, the cells were washed thrice with phosphate-buffered saline (PBS) and then exposed to 25 µM H2DCFDA (Sigma, excitation/emission: 488 nm/515 nm) for 20 minutes at 37°C. After the incubation period, excess H2DCFDA was removed, and the cells were rinsed thrice with PBS. The coverslips carrying the treated cells were then washed with ultrapure water, mounted on slides, and promptly subjected to analysis utilizing a fluorescence microscope. This methodology enabled the assessment of intracellular reactive oxygen species (ROS) levels induced by Cu(II) complex C1/C2 treatment. 2.6 Expression of γH2AX in MGC-803 cells induced by Cu(II) complex C2 MGC-803 cells were seeded on 6-well plates at a density of 1×10 5 cells per well. After incubation for 12 hours, the cells were treated with different concentrations of Cu(II) complex C1/C2 (4.51 µM) for 24 hours, and then the cells were incubated with rabbit γH2AX polyclonal antibody (diluted with 1% BSA) at 4°C overnight. The cells were then incubated with Alexa Fluor 488-conjugated secondary antibody for 2 hours. γH2AX expression was evaluated by confocal microscopy. 2.7 Comet assay Following treatment with Cu(II) complex C1/C2 (4.51 µM) for 24 hours, cells were adjusted to a concentration of 10 6 -10 7 cells/mL after centrifugation. A 0.5% normal melting point agarose (NMA) solution was preheated at 56℃, and 80 µL was gently applied onto a preheated glass slide. A clean cover glass was quickly placed over the agarose, and the slide was allowed to solidify at 4℃ for 10 minutes. Subsequently, 10 µL of PBS containing 1000 cells and 75 µL of 0.5% low melting point agarose (LMA) were mixed at 37℃. The cover glass was gently removed, and the LMA-cell mixture was applied on top of the first solidified agarose layer. A clean cover glass was placed over the mixture and allowed to solidify at 4℃ for 10 minutes. Finally, 85 µL of 0.5% LMA was preheated at 37℃ and applied to the solidified LMA layer. A cover glass was placed on top to allow solidification. The cover glass was removed, and the slide was immersed in freshly prepared cell lysate for at least 1 hour. Post cell lysis, the slide was rinsed twice with PBS to remove excess salt. The slide was placed in a horizontal electrophoresis tank and submerged in a newly prepared alkaline electrophoresis buffer. Alkaline hydrolysis was performed for 20 minutes to allow DNA unwinding under alkaline conditions, forming single-stranded DNA and facilitating DNA breakage and migration during electrophoresis. 2.8 Cell cycle arrest by Cu(II) complex C2 MGC-803 cells were seeded at a density of 1×10 5 cells in 6-well plates and incubated at 37°C for 12 hours to allow cell attachment and growth. Following incubation, the cells were treated with Cu(II) complex C1/C2 at a concentration of 4.51 µM for 48 hours. Subsequently, the cancer cells were collected in separate 1.5 mL EP tubes, and 500 µL of 70% ice-cold ethanol was added. The cells were then incubated at -20°C overnight. Following incubation, centrifugation at 1500 rpm for 5 minutes was performed to discard the ethanol. Next, the cells were treated with a staining solution of 1 mg/mL propidium iodide (PI) and 10% RnaseA and incubated in the dark for 0.5 hours. 2.9 AO/EB assay Glass cover slides were pre-placed in a 6-well culture plate, and cell suspension was inoculated for 12 hours. Subsequently, cells were treated with Cu(II) complex C1/C2 at a concentration of 4.51 µM for an additional 24 hours. Following the incubation, the reagent kit's instructions were followed to prepare a mixed working solution of acridine orange (AO) and ethidium bromide (EB) totaling 600 µL. This solution was carefully added to the cells in the six-well plate, and the cells were incubated in the dark for 25 minutes. After the incubation period, the cells were washed twice with phosphate-buffered saline (PBS). The cover glass was then placed on the slide, and the samples were immediately observed under a fluorescence microscope to assess the effects of Cu(II) complex C1/C2 treatment on cellular morphology and viability. 2.10 Induced apoptosis by Cu(II) complex C2. Flow cytometry was employed to assess the apoptosis-inducing potential of C2 in MGC-803 cells. Initially, MGC-803 cells were seeded in 6-well plates and incubated for 12 hours. Subsequently, the cells were treated with Cu(II) complex C1/C2 (4.51 µM) for 24 hours. Following the treatment, the cells were harvested, washed three times with phosphate-buffered saline (PBS), and resuspended in 120 µL of binding buffer to achieve a final 1 × 10 6 cells/mL concentration. The cell suspension was treated with 5 µL of V-FITC (5 µg/mL) and incubated for 25 minutes at 37°C in a light-protected environment. Following the incubation, 10 µL of propidium iodide was added to the cell suspension and incubated for 20 minutes at 25°C under light protection. Apoptosis was analyzed using flow cytometry, providing valuable insights into the apoptotic effects induced by Cu(II) complex C2 on MGC-803 cells. 3 Results and discussion 3.1 Design and structure of ligands L1-L2 and Cu complexes C1-C2 The Cu complexes synthesized in this study are rooted in two fundamental considerations. Firstly, Cu complexes hold substantial promise to supersede cisplatin, representing a progressive leap in the realm of metal-based anticancer drugs (Cvek et al. 2008 ; Paterson and Donnelly 2011 ; Zhang et al. 2017 ; Mo et al. 2018 ; Spengler et al. 2018 ; Ohui et al. 2019 ; Yu et al. 2019 ; MacHado et al. 2020 ; Gou et al. 2021 ; da Silva et al. 2022 ; Shen et al. 2022 ; Khalil et al. 2022 ). Secondly, leveraging adenine, a pivotal free nitrogenous base in DNA salvage synthesis pathways, as the foundational compound for synthesizing Cu complexes with a high affinity for DNA. Figure 1 succinctly illustrates the synthetic approach, elucidating the straightforward preparation of Cu complexes C1-C2 through the reaction of 6-Hydrazinopurine with distinct aldehydes. The chemical structures of ligands L1-L2 were meticulously characterized using 1 H NMR and 13 C NMR spectroscopy. Furthermore, the structures of Cu(II) complexes C1-C2 were elucidated through X-ray crystallography. These Cu(II) complexes were synthesized by reacting CuCl 2 with varying 6-Hydrazinopurine Schiff-base ligands in a mixture of CH 3 OH and CH 2 Cl 2 at 65°C, as depicted in Fig. 1 . The crystal structures of the two complexes were thoroughly analyzed via single-crystal diffraction. The relevant data and analysis for C1-C2 are summarized in Table 1 and Table 2 . Table 1 Crystal data for Cu(II) complexes. C1 C2 formula C 12 H 9 Cu 1 N 6 O 1 Cl1 C 13 H 11 Cu 1 N 6 O 1 Cl1 formula weight 350.9922 365.0079 space group P2/c P2/n a, Ẳ 11.0708(8) 14.7343(6) b, Ẳ 6.8388(5) 6.7905(3) c, Ẳ 20.0281(13) 15.6089(6) α, deg 90 90 β, deg 92.438(6) 96.142(4) γ, deg 90 90 V, Ẳ3 1514.97(18) 1552.76(11) Temperature 100.01(10) 100 K Z 7 4 Final R indexes [I > = 2σ (I)] R 1 = 0.1791, wR 2 = 0.4541 R 1 = 0.0402, wR 2 = 0.0936 Final R indexes [all data] R 1 = 0.1919, wR 2 = 0.4620 R 1 = 0.0571, wR 2 = 0.1063 Table 2 Selected bond lengths (Ẳ) and angles (deg) for Cu(II) complexes. C1 C2 Cu1-O1 1.918(8) 1.901(2) Cu1-N4 1.988(9) 1.991(3) Cu1-N6 1.970(9) 1.952(3) Cu1-Cl1 2.243(3) 2.2276(9) O1-Cu1-N4 172.2(4) 172.51(10) O1-Cu1-N6 91.7(4) 91.90(10) O1-Cu1-Cl1 90.9(3) 91.31(7) N4-Cu1-Cl1 96.1(3) 96.03(8) N6-Cu1-N4 81.5(4) 80.74(11) N6-Cu1-Cl1 176.3(3) 176.75(8) 3.2 Structure-activity relationships of L1-L2 and C1-C2 The cytotoxicity assessment of ligands L1-L2 and their respective Cu complexes C1-C2 was conducted using the MTT method, with cisplatin as a control to gauge the activity of the Cu complexes. As shown in Table 3 , the cytotoxic activity against cancer cell lines was notably higher for Cu complexes C1-C2 in comparison to ligands L1-L2. This observation highlights a substantial increase in cytotoxicity due to Cu coordination. Moreover, complex C2 demonstrated superior cytotoxicity when compared to both complex C1 and cisplatin. This finding suggests that the introduction of a methyl group, replacing one of the hydrogen atoms at the C-5 position in salicylaldehyde, significantly enhances the cytotoxicity of the Cu complex. Notably, complex C2 exhibited the highest cytotoxicity with MGC-803 cells (4.51 ± 0.26 µM). The Cu complexes demonstrated an approximately twofold increase in cytotoxicity against MGC-803 cells compared to the ligands alone. However, it is essential to address the noteworthy increase in cytotoxicity levels observed in normal cells (HL7702) by these complexes. Despite the substantial cytotoxic activity of complex C2 against MGC-803 cells in vitro relative to C1, the elevated cytotoxicity towards normal cells necessitates further investigation. Ongoing research focuses on delving into the mechanism underlying the inhibitory effect of C2 on cancer cell growth. Table 3 IC 50 (µM) Values of ligands L1-L2 and its Cu complexes C1-C2 against different cancer cells and HL7702 cells for 48 h. compounds MGC-803 SK-OV-3 A549 T24 HL7702 L1 9.47 ± 1.02 9.75 ± 1.86 11.81 ± 1.30 8.44 ± 1.04 26.94 ± 2.55 L2 8.03 ± 0.69 9.51 ± 1.04 11.67 ± 1.27 6.81 ± 0.52 28.61 ± 2.73 C1 5.14 ± 0.44 7.33 ± 0.78 7.17 ± 0.85 6.69 ± 0.33 13.85 ± 1.28 C2 4.51 ± 0.26 6.87 ± 0.36 6.51 ± 0.39 5.24 ± 0.57 11.67 ± 1.03 cisplatin 16.83 ± 1.52 21.35 ± 1.99 19.48 ± 2.64 17.88 ± 1.91 19.41 ± 1.84 CuCl 2 > 50 > 50 > 50 > 50 > 50 3.3 Anti-cancer mechanisms of Cu(II) complexes 3.3.1 Induce mitochondrial damage by Cu(II) complex C2 Mitochondria represent significant targets for metal complexes, and various pathways of cancer cell death are closely associated with mitochondrial function (Erxleben 2019 ; Olelewe and Awuah 2023 ; Scalcon et al. 2023 ). In this study, we investigated the impact of the Cu(II) complex C2 on mitochondrial membrane potential (△Ψm) in MGC-803 cells, a key indicator of mitochondrial health. The staining of MGC-803 cells with JC-1, followed by examination using confocal microscopy, allowed us to evaluate changes in mitochondrial membrane potential. In normal cells, the aggregated form of JC-1 emits a red fluorescence, reflecting an intact mitochondrial membrane potential. Conversely, in cells with mitochondrial dysfunction, JC-1 remains in its monomeric form, exhibiting green fluorescence, often associated with apoptotic cells. Remarkably, our findings demonstrated the increase in green (monomeric) fluorescence when MGC-803 cells were treated with C2, as illustrated in Fig. 2 . These alterations in mitochondrial membrane potential strongly suggest that C2 induces damage to the mitochondria. Such damage to the mitochondrial membrane potential can trigger cascades of events leading to altered cellular processes and ultimately cell death. The observed impact on mitochondrial function further highlights the potential of C2 as an effective agent in combating cancer by disrupting vital cellular pathways. Further investigations are warranted to elucidate the precise mechanisms and downstream effects of mitochondrial damage induced by C2. 3.3.2 Promote the generation of ROS by Cu(II) complex C2 Mitochondria, as the principal subcellular organelles responsible for generating reactive oxygen species (ROS), play a critical role in cellular oxidative processes (Yang et al. 2016 ). When mitochondria experience dysfunction, there is often an upsurge in ROS production, triggering various apoptotic signaling pathways. Therefore, in this study, we sought to investigate the impact of C2 on ROS generation within the MGC-803 cell line. We utilized 2',7'-dichlorofluorescein diacetate (DCFH-DA) and fluorescence microscopy to measure intracellular ROS levels. Figure 3 illustrates the outcomes of this analysis. In the control group, a weak fluorescence image was observed, suggesting relatively low ROS levels. Conversely, following incubation with Cu(II) complex C2, MGC-803 cells exhibited green fluorescence, indicating a substantial increase in ROS levels. The augmentation of ROS levels following C2 treatment signifies the induction of oxidative stress within the cells. Elevated ROS levels can trigger a multitude of cellular responses, including oxidative damage to biomolecules, initiation of apoptotic cascades, and overall perturbation of cellular homeostasis. The robust increase in ROS generation resulting from C2 treatment underlines its potential as a potent agent for inducing oxidative stress-mediated cytotoxicity in cancer cells. Further research is imperative to comprehend the precise mechanisms by which C2 modulates ROS and its subsequent effects on cellular fate. 3.3.3 Induce DNA damage by Cu(II) complex C2 Given adenine's crucial role as a free nitrogenous base in DNA synthesis, its integration into the Cu(II) complex C2 structure enhances the complex's targeting ability and affinity for DNA. This integration suggests a potential mechanism for the observed cytotoxicity and anticancer activity. Excessive levels of reactive oxygen species (ROS) generated due to the action of Cu(II) complex C2 can inflict damage not only on DNA but also induce cell cycle arrest, amplifying the potential anticancer effects.(Perillo et al. 2020 ) DNA damage can manifest in various forms, including base modification, DNA single-strand breaks (SSBs), DNA intra- and inter-strand cross-links, and the most severe form, DNA double-strand breaks (DSBs). Among these, DSBs are considered highly detrimental to the DNA structure. To confirm the capability of Cu(II) complex C2 in inducing DNA damage, we conducted immunofluorescence analysis targeting histone H2AX phosphorylation foci (γH2AX), a recognized marker for DNA DSBs, in MGC-803 cells. The phosphorylation of serine 139 on H2AX, forming γH2AX, is a characteristic response to DNA DSBs, providing a clear biomarker reflecting the extent of DNA damage (Rogakou et al. 1998 ). As depicted in Fig. 4 , upon treatment of MGC-803 cells with 4.51 µM C1/C2 for 4 hours, a substantial upregulation in the fluorescence intensity of γH2AX was observed. This observation strongly suggests that adenine's incorporation into Cu(II) complex C2 significantly enhances its targeting ability and affinity for DNA, ultimately resulting in notable DNA damage. The induced DNA damage can be regarded as a crucial factor contributing to the heightened cytotoxicity and potential anticancer properties exhibited by Cu(II) complex C2. Further investigations will delve into unraveling the intricate mechanisms underpinning this DNA-targeted therapeutic approach. To further validate the DNA-damaging effects of Cu(II) complex C2, we employed the comet assay, a robust tool for detecting and quantifying DNA single- and double-strand breaks in cells. This assay allows for a precise assessment of the extent of DNA damage induced by the compound under investigation. As illustrated in Fig. 5 , the comet assay results clearly demonstrate that C2 elicited longer-length tails compared to the control and C1 in MGC-803 cells, underscoring the severe DNA damage induced by C2. In addition, to determine whether C2 interacted with DNA, we performed the molecular docking studies using the promising lead compound C2 to the DNA molecule. (Malik et al. 2021 ) The results of molecular docking showed that there are potential binding sites of C2 in DNA (Fig. 6 A-C). In addition, the kinetics of C2 binding to DNA was studied by measuring the amount of Cu bound to precipitated ct-DNA (Fig. 6 D). The compelling evidence presented in this study collectively affirms that copper complexes engineered based on the adenine framework possess a potent capability to instigate substantial DNA damage. The capacity to induce significant DNA damage marks a pivotal attribute in the potential application of these complexes as targeted agents for cancer treatment. Further exploration of the mechanistic intricacies underlying the DNA-damaging properties of copper complexes and their potential implications in cancer therapy is warranted for a comprehensive understanding and effective clinical translation. 3.3.4 Induce cell cycle blocks by Cu(II) complex C2 Following the confirmation of DNA damage induction by copper complex C2 through comet assay and γH2AX staining, our focus shifted to investigating whether C2 could disrupt the cell cycle. In Fig. 7 , the data obtained after treating the cells with complex C1/C2 at 4.51 µM for 48 hours revealed a noteworthy increase in cells residing in the G2 phase of the cell cycle. Specifically, cells in the G2 phase increased to 18.86% compared to the control (6.24%), providing strong evidence that complex C2 effectively arrests the MGC-803 cell cycle at the G2 phase. This observed cell cycle arrest is a crucial finding, as it suggests a potential mechanism through which copper complex C2 exerts its cytotoxic effects on cancer cells. Cell cycle regulation is fundamental in cell proliferation and maintaining genomic stability. Disruption of this process can impair cell division and lead to cell death. Therefore, the ability of copper complex C2 to induce cell cycle arrest, particularly at the G2 phase, underscores its potential as a potent anti-cancer agent by interfering with vital cellular processes. Further research is warranted to elucidate the precise molecular pathways underlying this observed cell cycle arrest and its implications for cancer treatment. 3.3.5 Mediate apoptosis by Cu(II) complex C2 Acridine orange (AO)/ethyl bromide (EB) staining is a well-established technique utilized to detect cell apoptosis. In this method, AO is capable of penetrating living cells and binding to DNA, emitting a green fluorescence. Conversely, EB can only penetrate cells that are no longer viable, binding to DNA and emitting an orange-red fluorescence (Liu et al. 2015 ). Apoptotic cells typically exhibit intensified red fluorescence and manifest distinct characteristics, such as circular rosary-like structures, pyknotic nuclei, or blocky formations. As depicted in Fig. 8 , the AO/EB staining results unequivocally demonstrate that C2 significantly induces apoptosis in MGC-803 cells. This observation further strengthens the evidence of C2's potent anticancer properties, as induction of apoptosis is a pivotal mechanism for inhibiting cancer cell growth and promoting cell death. The ability of C2 to trigger apoptosis underscores its potential as a targeted therapeutic agent in cancer treatment. Further investigations are warranted to decipher the precise apoptotic pathways modulated by C2, providing invaluable insights for its prospective clinical application. In addition to the techniques employed earlier, annexin V and propidium iodide (PI) staining coupled with flow cytometry analyses were utilized to further evaluate Cu(II) complex C2's capacity to induce apoptosis in MGC-803 cells. The results, illustrated in Fig. 9 , unequivocally demonstrate that treatment with complex C1/C2 at a concentration of 4.51 µM for 24 hours triggered apoptosis in the MGC-803 cells. Specifically, the percentage of cells undergoing early apoptosis escalated from 2.79% in the control group to a maximum of 24.66% in the C2 group. Concurrently, the percentages of cells undergoing late apoptosis increased from 0.83% in the control group to a maximum of 17.33%. These compelling results collectively underscore the potential of complex C2 as a potent apoptotic inducer. Apoptosis, a highly regulated cell death mechanism, plays a fundamental role in maintaining tissue homeostasis and eliminating damaged or malignant cells. The ability of C2 to significantly induce apoptosis further solidifies its potential as an efficacious anticancer agent. Understanding the precise molecular pathways and mechanisms through which C2 orchestrates apoptosis is crucial for harnessing its full therapeutic potential and advancing its application in cancer treatment. Further investigations are warranted to elucidate the intricacies of C2-induced apoptosis, paving the way for potential apoptotic inducers. 4 Conclusions In summary, our study successfully designed, synthesized, and characterized two novel Cu(II) complexes based on adenine. These complexes demonstrated potent anticancer activity against the MGC-803 cell line, surpassing the cytotoxicity of cisplatin. Notably, enhancing the structure of salicylaldehyde at the C-5 position with a methyl group significantly amplified the cytotoxic effects. Particularly, Cu(II) complex C2 emerged as a formidable chemotherapeutic agent, exerting its efficacy through multiple mechanisms, including heightened ROS levels, induction of DNA damage, disruption of mitochondrial function, and initiation of apoptosis. These compelling findings underscore the potential of Cu(II) complex based on adenine as a promising anticancer agent that warrants in-depth exploration and validation for its potential clinical application. Comparatively, this work builds on the established role of platinum-based chemotherapeutics while addressing their limitations, particularly severe side effects and drug resistance. The presented copper(II) complexes offer a promising alternative and pave the way for further research and development in the field of non-platinum metal-based anticancer drugs. The superior cytotoxicity of these adenine-based copper(II) complexes, especially complex C2, demonstrates their potential to outperform cisplatin, marking a significant stride in anticancer drug discovery. Additionally, the mechanistic insights into the cytotoxic effects of C2 underscore the potential of adenine-based anticancer metal complexes that specifically target DNA, presenting an exciting avenue for advancing the development of effective anticancer agents. Declarations Contributions of Authors Xiaoyan Zhai : Visualization, Formal analysis, Methodology, Software, Data Curation, Writing - Original Draft. Hussein Hanibah : Supervision, Conceptualization, Resources, Validation, Project administration. Nor Zakiah Nor Hashim : Supervision, Writing- Reviewing and Editing. Juzheng Zhang : Supervision, Resources, Funding acquisition. Xianli Ma: Project administration, Validation. Lilan Wei: Investigation, Software. Xiaoqun Zhou: Conceptualization, Resources . Acknowledgements This work was supported by the Guangxi Natural Science Foundation for the study (No. 2023GXNSFBA026313), China's National Natural Science Foundation (No. 82204208). Ethical Approval The authors declare that they have no known competing personal relationships that could have appeared to influence the work reported in this paper. Funding The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Xiaoyan Zhai reports financial support was provided by Guangxi Natural Science Foundation. Xiaoyan Zhai reports financial support was provided by China’s National Natural Science Foundation. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Availability of data and materials Data available on request due to privacy/ethical restrictions. References Battaglia AM, Chirillo R, Aversa I, et al (2020) Ferroptosis and Cancer: Mitochondria Meet the “Iron Maiden” Cell Death. 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Supplementary Files floatimage1.jpeg Table of contents graphic Cite Share Download PDF Status: Published Journal Publication published 01 Jun, 2024 Read the published version in Journal of Molecular Structure → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3997929","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":276221959,"identity":"94f0f6c7-177b-42a1-bf63-ee86bff700e2","order_by":0,"name":"Xiaoyan Zhai","email":"","orcid":"","institution":"Universiti Teknologi MARA","correspondingAuthor":false,"prefix":"","firstName":"Xiaoyan","middleName":"","lastName":"Zhai","suffix":""},{"id":276221960,"identity":"0522226e-5801-484a-b2de-8062871a1588","order_by":1,"name":"Hussein Hanibah","email":"","orcid":"","institution":"Universiti Teknologi MARA","correspondingAuthor":false,"prefix":"","firstName":"Hussein","middleName":"","lastName":"Hanibah","suffix":""},{"id":276221961,"identity":"d155242f-467d-4af4-b8fb-03d4d20018c7","order_by":2,"name":"Nor Zakiah Nor Hashim","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA30lEQVRIiWNgGAWjYNCCAxIMBuwNDAcgHKK18BxgOHCABC0MDAYSCVDVhLSYs/c+/vDjjIW8ueQbw8Mfcxjk+G4ksG7mwaPFsue4gWHPDQnDnbNzDA4c3MZgLHkjge02Pi0GN9IYEng+SDBuuA3RkriBGC0H/3yQsN9w8wxYSz0xWhibeW5IAA3nAWtJMCCo5cwxZmaZMxLJG86kFRw4u03CcOaZh2035+DTcryN+eObY3W2G44f3vyhcpuNPN/x5GM33uDRgg4kgJixgQmfw7ADxh8kaxkFo2AUjIJhDABx81uVsQ+xngAAAABJRU5ErkJggg==","orcid":"","institution":"Universiti Teknologi MARA","correspondingAuthor":true,"prefix":"","firstName":"Nor","middleName":"Zakiah Nor","lastName":"Hashim","suffix":""},{"id":276221962,"identity":"746895d5-8010-429d-a061-32df410761c1","order_by":3,"name":"Juzheng Zhang","email":"","orcid":"","institution":"Guilin Medical University","correspondingAuthor":false,"prefix":"","firstName":"Juzheng","middleName":"","lastName":"Zhang","suffix":""},{"id":276221963,"identity":"988ef820-e635-4b23-a730-26fa85531b30","order_by":4,"name":"Xianli Ma","email":"","orcid":"","institution":"Guangxi University","correspondingAuthor":false,"prefix":"","firstName":"Xianli","middleName":"","lastName":"Ma","suffix":""},{"id":276221964,"identity":"338689fc-c75a-4d38-864c-971b65e5432d","order_by":5,"name":"Lilan Wei","email":"","orcid":"","institution":"Guilin Medical University","correspondingAuthor":false,"prefix":"","firstName":"Lilan","middleName":"","lastName":"Wei","suffix":""},{"id":276221965,"identity":"f80f7d93-9da9-41aa-8181-b6c79cd7c212","order_by":6,"name":"Xiaoqun Zhou","email":"","orcid":"","institution":"Guilin Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xiaoqun","middleName":"","lastName":"Zhou","suffix":""}],"badges":[],"createdAt":"2024-02-28 22:37:11","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3997929/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3997929/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1016/j.molstruc.2024.138836","type":"published","date":"2024-06-01T12:46:14+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":52108798,"identity":"4d0fd825-c147-4e94-b02f-974d59f28a67","added_by":"auto","created_at":"2024-03-06 20:27:30","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":151439,"visible":true,"origin":"","legend":"\u003cp\u003eSynthesis of adenine Schiff-base ligand L1-L2 and corresponding Cu(II) complexes C1-C2.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-3997929/v1/481c61e40401891c22422d3b.png"},{"id":52108484,"identity":"59676343-975a-4ee2-a488-4dd8d7d4681c","added_by":"auto","created_at":"2024-03-06 20:19:30","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":243768,"visible":true,"origin":"","legend":"\u003cp\u003eDecreased mitochondrial membrane potential after treatment with the C1/C2. MGC-803 cancer cells were incubated with 4.51 μM C1/C2 for 6 h and treated with JC-1, and observed by laser confocal microscopy.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-3997929/v1/2b62441e66218096f1bbd2f6.png"},{"id":52108483,"identity":"07d44b32-7b45-474f-aefc-b20de41f2cd6","added_by":"auto","created_at":"2024-03-06 20:19:30","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":90675,"visible":true,"origin":"","legend":"\u003cp\u003eThe intracellular reactive oxygen species (ROS) are determined by fluorescence microscopy after treatment with the C1/C2. MGC-803 cancer cells were incubated with 4.51 μM C1/C2 for 6 h, treated with DCFH-DA, and observed by fluorescence microscopy.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-3997929/v1/2a7c2ce205e82465eb90dc4b.png"},{"id":52108486,"identity":"97fc1874-6491-4deb-8501-d8bfcde628c1","added_by":"auto","created_at":"2024-03-06 20:19:30","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":246104,"visible":true,"origin":"","legend":"\u003cp\u003eDNA damaged by C1/C2, γH2AX as a marker for DNA double-strand breaks. MGC-803 cancer cells were incubated with 4.51 μM C1/C2 for 24 h, immunofluorescence staining of γH2AX was determined by laser confocal microscopy.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-3997929/v1/cdccc05fd14b18ab35555291.png"},{"id":52108800,"identity":"ed358bc5-bb40-428a-8ca9-6f7955a84313","added_by":"auto","created_at":"2024-03-06 20:27:30","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":79780,"visible":true,"origin":"","legend":"\u003cp\u003eImages from comet assay. MGC-803 cells were treated with 4.51 μM C1/C2 for 24 h.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-3997929/v1/c2a90531e2b754d9429b744c.png"},{"id":52108490,"identity":"393a92ec-3454-4d1e-b78b-fd1eeb6f7459","added_by":"auto","created_at":"2024-03-06 20:19:31","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":249642,"visible":true,"origin":"","legend":"\u003cp\u003e(A-C) Docking studies of C2 with DNA. (D) Percentage of unbound Cu at various time points after exposure to ct-DNA.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-3997929/v1/f7c62140ada09a23ff8ee359.png"},{"id":52108488,"identity":"c956963e-749d-4617-ac0d-544680a2cb2f","added_by":"auto","created_at":"2024-03-06 20:19:30","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":104254,"visible":true,"origin":"","legend":"\u003cp\u003eCell cycle arrest after being treated with 4.51 μM C1/C2 for 48 h in MGC-803 cells was tested by flow cytometry.\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-3997929/v1/931fff706dbc72b3254e68dc.png"},{"id":52108491,"identity":"eaa5b74d-703f-4c0e-bc72-f566ab693402","added_by":"auto","created_at":"2024-03-06 20:19:31","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":355839,"visible":true,"origin":"","legend":"\u003cp\u003eAO/EB fluorescence staining was performed on MGC-803 cells following treatment with 4.51 μM C1/C2 for 24 hours. The resulting AO/EB images were analyzed using a fluorescence microscope.\u003c/p\u003e","description":"","filename":"floatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-3997929/v1/27240feed4ac263295d16f0c.png"},{"id":52108492,"identity":"b176de93-a92c-49c1-88d7-32ff8585dc0e","added_by":"auto","created_at":"2024-03-06 20:19:31","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":132288,"visible":true,"origin":"","legend":"\u003cp\u003eApoptosis was observed in MGC-803 cells after treatment with 4.51 μM C1/C2 for 24 hours, compared to the control cells. The apoptotic cell distributions induced by C1/C2 were determined using PI and FITC-annexin V staining via flow cytometry.\u003c/p\u003e","description":"","filename":"floatimage10.png","url":"https://assets-eu.researchsquare.com/files/rs-3997929/v1/41844105a957a76c7b8b6bc4.png"},{"id":57918854,"identity":"dcdea988-5ce5-46ed-b880-3ba41e0e6fa5","added_by":"auto","created_at":"2024-06-07 12:46:24","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2375105,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3997929/v1/a2f1f3e7-60b8-4a88-82a8-5bca3f6028d8.pdf"},{"id":52108487,"identity":"6cc186f7-022b-4ca8-b930-2cb998e43408","added_by":"auto","created_at":"2024-03-06 20:19:30","extension":"jpeg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":466625,"visible":true,"origin":"","legend":"\u003cp\u003eTable of contents graphic\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3997929/v1/34916972d1333f823e163806.jpeg"}],"financialInterests":"No competing interests reported.","formattedTitle":"Synthesis and Anti-Cancer Investigations of Novel Copper(II) Complexes Based on Adenine","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003ePlatinum-based chemotherapeutics have played a pivotal role in treating solid tumors for over five decades by effectively inducing DNA damageKomeda \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Xiaoyong and Zijian \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; McQuitty \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Kenny and Marmion \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Ghosh \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Xian et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Zhang et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Su et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). However, the advancement of platinum drugs is hindered by prevalent drug resistance, significant side effects, low water solubility, and target ambiguity. Notably, drug resistance and severe limitations in the development of platinum metal-based drugs pose critical challenges (Kim et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Pan et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Lu et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2022b\u003c/span\u003e; Van Nyen et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). It is imperative to explore alternative metal-based complexes to circumvent the resistance associated with platinum drugs. Recognizing the success of cisplatin, researchers have embarked on extensive investigations into other metal-based complexes.\u003c/p\u003e \u003cp\u003eRecent studies have underscored the potential of non-platinum based complexes, exhibiting enhanced potency, reduced toxicity, and higher target specificity, emerging as promising candidates for novel chemotherapeutic agents (Ott and Gust \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Milacic et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Erxleben \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Ma et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Imberti et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Malik et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Gourdon et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Ferraro et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Islam et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Lu et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2022a\u003c/span\u003e; Sinicropi et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Devi et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Within this realm, copper (Cu) complexes have garnered significant attention for their potential applications as chemotherapeutic agents. (Cvek et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Paterson and Donnelly \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Zhang et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Mo et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Spengler et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Ohui et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Yu et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; MacHado et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Gou et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; da Silva et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Shen et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Khalil et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Copper, being an essential element for sustaining life, actively participates in vital metabolic pathways within cells as cofactors. Moreover, Cu ions possess the capacity to generate reactive oxygen species (ROS) through metal oxidation and reduction processes. Reactive oxygen free radicals can engage in numerous reactions with DNA, proteins, and lipids, leading to mutations and cellular damage (Moloney and Cotter \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Kaarniranta et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Srinivas et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Yan et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Yuan et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Jiang et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Tsvetkov et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Tang et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Cobine and Brady \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Over the last two decades, several studies have demonstrated the cytotoxicity of Cu complexes, even at nanomolar levels. Hence, Cu complexes exhibit substantial potential to supersede cisplatin as the next generation of metal-based anticancer drugs (Cvek et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Paterson and Donnelly \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Zhang et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Mo et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Spengler et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Ohui et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Yu et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; MacHado et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Gou et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; da Silva et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Shen et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Khalil et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Nevertheless, these Cu complexes have yet to demonstrate explicit DNA targeting properties, potentially resulting in off-target effects upon administration.\u003c/p\u003e \u003cp\u003eAdenine, also known as vitamin B4, holds a crucial role as a fundamental compound aiding in DNA and RNA synthesis, constituting a vital component of human genetic material (Heyn and Esteller \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Parashar et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Khodadadi et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Yu et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Varma et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). To develop a copper complex with precise targeting and excellent DNA affinity, we propose a novel approach using adenine as the foundation. DNA is comprised of nitrogenous bases, deoxyribose, and phosphate. In DNA synthesis, cells utilize amino acids or free nitrogenous bases, such as adenine, for salvage synthesis, a less energy-intensive and simpler process than de novo synthesis. The design rationale of the study employs a unique approach by utilizing adenine as the lead compound to synthesize a series of copper complexes that employ tridentate coordination donors (OH, N, N) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). This approach aims to enhance their targeting ability and affinity for DNA, optimize the salvage synthesis pathway to enable copper (Cu) complexes to effectively interact with DNA, increase reactive oxygen species (ROS) levels, induce DNA damage, and ultimately impede cancer cell proliferation (Guo et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Tabrizi and Abyar \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Fan et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Battaglia et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Hayes et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The objective of the study is to overcome the significant challenges posed by platinum-based chemotherapeutics, including drug resistance, notable side effects, low water solubility, and target ambiguity. Non-platinum metal complexes have shown promise as potential chemotherapeutic agents, with enhanced potency, reduced toxicity, and heightened target specificity. These characteristics make them a suitable alternative to platinum-based chemotherapeutics as novel anticancer drugs.\u003c/p\u003e \u003cp\u003eThis work focuses on copper complexes with tridentate adenine Schiff base ligand modifications. Two adenine-based copper complexes (C1-C2) were synthesized and evaluated for their activity across various cancer cell lines. The potential of these Cu complexes to induce DNA damage was investigated, and the mechanism by which these Cu complexes inhibit cancer cells in vitro was elucidated. The transition towards non-platinum metal complexes is a compelling option, and this research contributes to the growing body of work aimed at exploring these alternatives for the development of more effective and safer chemotherapeutic agents.\u003c/p\u003e"},{"header":"2 Experimental sections","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Materials\u003c/h2\u003e \u003cp\u003e6-Hydrazinopurine, copper(II) chloride (CuCl\u003csub\u003e2\u003c/sub\u003e), and a salicylaldehyde derivative were sourced from Aladdin, Shanghai, China. MTT, JC-1, 2\u0026prime;,7-dichlorodihydrofluorescein diacetate (DCFH2-DA), and AO/EB were procured from Sigma-Aldrich. All biological antibodies were acquired from Abcam. Commercial sources supplied all other solvents and reagents, which were used without further purification. The \u003csup\u003e1\u003c/sup\u003eH and \u003csup\u003e13\u003c/sup\u003eC NMR analyses were conducted at room temperature (298 K) in DMSO-d6 using a Bruker AVANCE III HD 400 MHz spectrophotometer. X-ray crystallographic data were collected using a Rigaku HyPix diffractometer.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003e2.2 Synthesis of copper(II) complexes\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eThe ligands based on adenine were synthesized through a reaction involving 6-Hydrazinyl-9H-purine and various aldehydes. Specifically, 6-Hydrazinyl-9H-purine (3 mmol) and the corresponding aldehyde (3 mmol) were dissolved in 10 mL of ethanol and stirred for a duration of 6 hours. The resulting product was filtered to obtain a light yellow solid. The chemical structures of these ligands were meticulously characterized using High-Resolution Mass Spectrometry (HRMS), as well as \u003csup\u003e1\u003c/sup\u003eH and \u003csup\u003e13\u003c/sup\u003eC Nuclear Magnetic Resonance (NMR) spectroscopy.\u003c/p\u003e \u003cp\u003eSubsequently, the Cu(II) complexes were synthesized by reacting the ligands (0.10 mmol) with CuCl\u003csub\u003e2\u003c/sub\u003e (0.10 mmol) in a solvent mixture of methanol (1.5 mL) and dichloromethane (1.5 mL). The reaction was conducted under solvothermal conditions at 65\u0026deg;C for a period of 24 hours. Crystals suitable for X-ray diffraction analysis were carefully collected to further elucidate the molecular structure and bonding arrangements within the complexes.\u003c/p\u003e \u003cp\u003e \u003cb\u003eL1\u003c/b\u003e: \u003csup\u003e1\u003c/sup\u003eH NMR (500 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) δ 13.25 (s, 1H), 11.84 (s, 1H), 11.53 (s, 1H), 8.34 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.1 Hz, 3H), 7.34 (s, 1H), 7.25 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.6 Hz, 1H), 6.93\u0026ndash;6.88 (m, 2H). \u003csup\u003e13\u003c/sup\u003eC NMR (126 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) δ 157.32, 152.41, 145.33, 141.38, 130.86, 119.56, 119.22, 118.77, 117.26. HRMS:m/z 255.0916 [M\u0026thinsp;+\u0026thinsp;H]\u003c/p\u003e \u003cp\u003e \u003cb\u003eL2\u003c/b\u003e: \u003csup\u003e1\u003c/sup\u003eH NMR (500 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) δ 13.20 (s, 1H), 11.82 (s, 1H), 11.32 (s, 1H), 8.31 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;19.8 Hz, 3H), 7.12 (s, 1H), 7.05 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.1 Hz, 1H), 6.81 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.2 Hz, 1H), 2.24 (s, 3H). \u003csup\u003e13\u003c/sup\u003eC NMR (126 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) δ 155.07, 152.37, 145.40, 141.31, 131.47, 130.75, 127.97, 118.77, 117.03, 20.31. HRMS:m/z 269.1073 [M\u0026thinsp;+\u0026thinsp;H]\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Bioactivity\u003c/h2\u003e \u003cp\u003eCell cultures were maintained in Dulbecco's Modified Eagle Medium (DMEM, Gibco) supplemented with 10% (v/v) fetal bovine serum (FBS, Gibco), 100 U/mL penicillin (Sigma), 100 U/mL streptomycin (Sigma), and 2 mM glutamine (Sigma) at 37\u0026deg;C in a humidified atmosphere containing 95% air and 5% CO2. Cu(II) complexes were dissolved in dimethylformamide (DMF).\u003c/p\u003e \u003cp\u003eTo assess the in vitro cytotoxicity of the Cu(II) complexes against both cancer and normal cell lines, a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT, Sigma) assay was conducted. Cells were exposed to Cu(II) complexes and free ligands at five different concentrations for 48 hours. The growth inhibitory rates were measured using an enzyme labeling instrument at a double wavelength of 570/630 nm. The growth inhibitory rates were computed using the formula: (OD control - OD test) / OD control \u0026times; 100%.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Cu(II) complex C2 induced mitochondrial damage\u003c/h2\u003e \u003cp\u003eMGC-803 cells were cultured and incubated with Cu(II) complex C1/C2 at a concentration of 4.51 \u0026micro;M for 6 hours. The incubation was carried out in poly-HEMA coated 6-well plates to prevent cell adhesion. Subsequently, the cells were harvested and resuspended in a fresh culture medium.\u003c/p\u003e \u003cp\u003eTo assess mitochondrial membrane potential, 0.5 mL of JC-1 and DAPI working solution was added to the cell suspension. The cells were then incubated at 37\u0026deg;C for 25 minutes. Following incubation, the staining solution was carefully removed through centrifugation. The cells were rinsed three times with JC-1 staining buffer to ensure proper removal of excess staining solution.\u003c/p\u003e \u003cp\u003eThe stained cells were promptly subjected to analysis using confocal microscopy to evaluate the changes in mitochondrial membrane potential induced by the treatment with Cu(II) complex C2.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Enhancement of Cu(II) complex C2 at intracellular ROS levels\u003c/h2\u003e \u003cp\u003eMGC-803 cells were seeded at a density of 1 \u0026times; 105 cells/well on poly-L-lysine-coated coverslips within 6-well plates using heat-deactivated complete RPMI. The cells were subsequently treated with Cu(II) complex C1/C2 at a concentration of 4.51 \u0026micro;M for a duration of 6 hours.\u003c/p\u003e \u003cp\u003eFollowing the treatment, the cells were washed thrice with phosphate-buffered saline (PBS) and then exposed to 25 \u0026micro;M H2DCFDA (Sigma, excitation/emission: 488 nm/515 nm) for 20 minutes at 37\u0026deg;C. After the incubation period, excess H2DCFDA was removed, and the cells were rinsed thrice with PBS.\u003c/p\u003e \u003cp\u003eThe coverslips carrying the treated cells were then washed with ultrapure water, mounted on slides, and promptly subjected to analysis utilizing a fluorescence microscope. This methodology enabled the assessment of intracellular reactive oxygen species (ROS) levels induced by Cu(II) complex C1/C2 treatment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Expression of γH2AX in MGC-803 cells induced by Cu(II) complex C2\u003c/h2\u003e \u003cp\u003eMGC-803 cells were seeded on 6-well plates at a density of 1\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells per well. After incubation for 12 hours, the cells were treated with different concentrations of Cu(II) complex C1/C2 (4.51 \u0026micro;M) for 24 hours, and then the cells were incubated with rabbit γH2AX polyclonal antibody (diluted with 1% BSA) at 4\u0026deg;C overnight. The cells were then incubated with Alexa Fluor 488-conjugated secondary antibody for 2 hours. γH2AX expression was evaluated by confocal microscopy.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Comet assay\u003c/h2\u003e \u003cp\u003eFollowing treatment with Cu(II) complex C1/C2 (4.51 \u0026micro;M) for 24 hours, cells were adjusted to a concentration of 10\u003csup\u003e6\u003c/sup\u003e-10\u003csup\u003e7\u003c/sup\u003e cells/mL after centrifugation. A 0.5% normal melting point agarose (NMA) solution was preheated at 56℃, and 80 \u0026micro;L was gently applied onto a preheated glass slide. A clean cover glass was quickly placed over the agarose, and the slide was allowed to solidify at 4℃ for 10 minutes.\u003c/p\u003e \u003cp\u003eSubsequently, 10 \u0026micro;L of PBS containing 1000 cells and 75 \u0026micro;L of 0.5% low melting point agarose (LMA) were mixed at 37℃. The cover glass was gently removed, and the LMA-cell mixture was applied on top of the first solidified agarose layer. A clean cover glass was placed over the mixture and allowed to solidify at 4℃ for 10 minutes.\u003c/p\u003e \u003cp\u003eFinally, 85 \u0026micro;L of 0.5% LMA was preheated at 37℃ and applied to the solidified LMA layer. A cover glass was placed on top to allow solidification. The cover glass was removed, and the slide was immersed in freshly prepared cell lysate for at least 1 hour.\u003c/p\u003e \u003cp\u003ePost cell lysis, the slide was rinsed twice with PBS to remove excess salt. The slide was placed in a horizontal electrophoresis tank and submerged in a newly prepared alkaline electrophoresis buffer. Alkaline hydrolysis was performed for 20 minutes to allow DNA unwinding under alkaline conditions, forming single-stranded DNA and facilitating DNA breakage and migration during electrophoresis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Cell cycle arrest by Cu(II) complex C2\u003c/h2\u003e \u003cp\u003eMGC-803 cells were seeded at a density of 1\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells in 6-well plates and incubated at 37\u0026deg;C for 12 hours to allow cell attachment and growth. Following incubation, the cells were treated with Cu(II) complex C1/C2 at a concentration of 4.51 \u0026micro;M for 48 hours.\u003c/p\u003e \u003cp\u003eSubsequently, the cancer cells were collected in separate 1.5 mL EP tubes, and 500 \u0026micro;L of 70% ice-cold ethanol was added. The cells were then incubated at -20\u0026deg;C overnight. Following incubation, centrifugation at 1500 rpm for 5 minutes was performed to discard the ethanol. Next, the cells were treated with a staining solution of 1 mg/mL propidium iodide (PI) and 10% RnaseA and incubated in the dark for 0.5 hours.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 AO/EB assay\u003c/h2\u003e \u003cp\u003eGlass cover slides were pre-placed in a 6-well culture plate, and cell suspension was inoculated for 12 hours. Subsequently, cells were treated with Cu(II) complex C1/C2 at a concentration of 4.51 \u0026micro;M for an additional 24 hours.\u003c/p\u003e \u003cp\u003eFollowing the incubation, the reagent kit's instructions were followed to prepare a mixed working solution of acridine orange (AO) and ethidium bromide (EB) totaling 600 \u0026micro;L. This solution was carefully added to the cells in the six-well plate, and the cells were incubated in the dark for 25 minutes.\u003c/p\u003e \u003cp\u003eAfter the incubation period, the cells were washed twice with phosphate-buffered saline (PBS). The cover glass was then placed on the slide, and the samples were immediately observed under a fluorescence microscope to assess the effects of Cu(II) complex C1/C2 treatment on cellular morphology and viability.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10 Induced apoptosis by Cu(II) complex C2.\u003c/h2\u003e \u003cp\u003eFlow cytometry was employed to assess the apoptosis-inducing potential of C2 in MGC-803 cells. Initially, MGC-803 cells were seeded in 6-well plates and incubated for 12 hours. Subsequently, the cells were treated with Cu(II) complex C1/C2 (4.51 \u0026micro;M) for 24 hours.\u003c/p\u003e \u003cp\u003eFollowing the treatment, the cells were harvested, washed three times with phosphate-buffered saline (PBS), and resuspended in 120 \u0026micro;L of binding buffer to achieve a final 1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells/mL concentration. The cell suspension was treated with 5 \u0026micro;L of V-FITC (5 \u0026micro;g/mL) and incubated for 25 minutes at 37\u0026deg;C in a light-protected environment.\u003c/p\u003e \u003cp\u003eFollowing the incubation, 10 \u0026micro;L of propidium iodide was added to the cell suspension and incubated for 20 minutes at 25\u0026deg;C under light protection. Apoptosis was analyzed using flow cytometry, providing valuable insights into the apoptotic effects induced by Cu(II) complex C2 on MGC-803 cells.\u003c/p\u003e \u003c/div\u003e"},{"header":"3 Results and discussion","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Design and structure of ligands L1-L2 and Cu complexes C1-C2\u003c/h2\u003e \u003cp\u003eThe Cu complexes synthesized in this study are rooted in two fundamental considerations. Firstly, Cu complexes hold substantial promise to supersede cisplatin, representing a progressive leap in the realm of metal-based anticancer drugs (Cvek et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Paterson and Donnelly \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Zhang et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Mo et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Spengler et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Ohui et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Yu et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; MacHado et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Gou et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; da Silva et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Shen et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Khalil et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Secondly, leveraging adenine, a pivotal free nitrogenous base in DNA salvage synthesis pathways, as the foundational compound for synthesizing Cu complexes with a high affinity for DNA. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e succinctly illustrates the synthetic approach, elucidating the straightforward preparation of Cu complexes C1-C2 through the reaction of 6-Hydrazinopurine with distinct aldehydes.\u003c/p\u003e \u003cp\u003eThe chemical structures of ligands L1-L2 were meticulously characterized using \u003csup\u003e1\u003c/sup\u003eH NMR and \u003csup\u003e13\u003c/sup\u003eC NMR spectroscopy. Furthermore, the structures of Cu(II) complexes C1-C2 were elucidated through X-ray crystallography. These Cu(II) complexes were synthesized by reacting CuCl\u003csub\u003e2\u003c/sub\u003e with varying 6-Hydrazinopurine Schiff-base ligands in a mixture of CH\u003csub\u003e3\u003c/sub\u003eOH and CH\u003csub\u003e2\u003c/sub\u003eCl\u003csub\u003e2\u003c/sub\u003e at 65\u0026deg;C, as depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The crystal structures of the two complexes were thoroughly analyzed via single-crystal diffraction. The relevant data and analysis for C1-C2 are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCrystal data for Cu(II) complexes.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eC1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eC2\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eformula\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eC\u003csub\u003e12\u003c/sub\u003eH\u003csub\u003e9\u003c/sub\u003eCu\u003csub\u003e1\u003c/sub\u003eN\u003csub\u003e6\u003c/sub\u003eO\u003csub\u003e1\u003c/sub\u003eCl1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eC\u003csub\u003e13\u003c/sub\u003eH\u003csub\u003e11\u003c/sub\u003eCu\u003csub\u003e1\u003c/sub\u003eN\u003csub\u003e6\u003c/sub\u003eO\u003csub\u003e1\u003c/sub\u003eCl1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eformula\u003c/p\u003e \u003cp\u003eweight\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e350.9922\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e365.0079\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003espace\u003c/p\u003e \u003cp\u003egroup\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eP2/c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eP2/n\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ea, Ẳ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e11.0708(8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e14.7343(6)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eb, Ẳ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6.8388(5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.7905(3)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ec, Ẳ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e20.0281(13)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15.6089(6)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eα, deg\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e90\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eβ, deg\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e92.438(6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e96.142(4)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eγ, deg\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e90\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eV, Ẳ3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1514.97(18)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1552.76(11)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTemperature\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e100.01(10)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e100 K\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eZ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFinal R indexes [I\u0026thinsp;\u0026gt;\u0026thinsp;=\u0026thinsp;2σ (I)]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eR\u003csub\u003e1\u003c/sub\u003e\u0026nbsp;= 0.1791, wR\u003csub\u003e2\u003c/sub\u003e\u0026nbsp;= 0.4541\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eR\u003csub\u003e1\u003c/sub\u003e\u0026nbsp;= 0.0402, wR\u003csub\u003e2\u003c/sub\u003e\u0026nbsp;= 0.0936\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFinal R indexes [all data]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eR\u003csub\u003e1\u003c/sub\u003e\u0026nbsp;= 0.1919, wR\u003csub\u003e2\u003c/sub\u003e\u0026nbsp;= 0.4620\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eR\u003csub\u003e1\u003c/sub\u003e\u0026nbsp;= 0.0571, wR\u003csub\u003e2\u003c/sub\u003e\u0026nbsp;= 0.1063\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSelected bond lengths (Ẳ) and angles (deg) for Cu(II) complexes.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eC1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eC2\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCu1-O1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.918(8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.901(2)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCu1-N4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.988(9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.991(3)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCu1-N6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.970(9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.952(3)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCu1-Cl1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2.243(3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.2276(9)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eO1-Cu1-N4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e172.2(4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e172.51(10)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eO1-Cu1-N6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e91.7(4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e91.90(10)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eO1-Cu1-Cl1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e90.9(3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e91.31(7)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eN4-Cu1-Cl1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e96.1(3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e96.03(8)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eN6-Cu1-N4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e81.5(4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e80.74(11)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eN6-Cu1-Cl1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e176.3(3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e176.75(8)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Structure-activity relationships of L1-L2 and C1-C2\u003c/h2\u003e \u003cp\u003eThe cytotoxicity assessment of ligands L1-L2 and their respective Cu complexes C1-C2 was conducted using the MTT method, with cisplatin as a control to gauge the activity of the Cu complexes. As shown in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, the cytotoxic activity against cancer cell lines was notably higher for Cu complexes C1-C2 in comparison to ligands L1-L2. This observation highlights a substantial increase in cytotoxicity due to Cu coordination.\u003c/p\u003e \u003cp\u003eMoreover, complex C2 demonstrated superior cytotoxicity when compared to both complex C1 and cisplatin. This finding suggests that the introduction of a methyl group, replacing one of the hydrogen atoms at the C-5 position in salicylaldehyde, significantly enhances the cytotoxicity of the Cu complex. Notably, complex C2 exhibited the highest cytotoxicity with MGC-803 cells (4.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26 \u0026micro;M). The Cu complexes demonstrated an approximately twofold increase in cytotoxicity against MGC-803 cells compared to the ligands alone.\u003c/p\u003e \u003cp\u003eHowever, it is essential to address the noteworthy increase in cytotoxicity levels observed in normal cells (HL7702) by these complexes. Despite the substantial cytotoxic activity of complex C2 against MGC-803 cells in vitro relative to C1, the elevated cytotoxicity towards normal cells necessitates further investigation. Ongoing research focuses on delving into the mechanism underlying the inhibitory effect of C2 on cancer cell growth.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eIC\u003csub\u003e50\u003c/sub\u003e (\u0026micro;M) Values of ligands L1-L2 and its Cu complexes C1-C2 against different cancer cells and HL7702 cells for 48 h.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003ecompounds\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMGC-803\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSK-OV-3\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eA549\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eT24\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eHL7702\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eL1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e9.47\u0026thinsp;\u0026plusmn;\u0026thinsp;1.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e9.75\u0026thinsp;\u0026plusmn;\u0026thinsp;1.86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e11.81\u0026thinsp;\u0026plusmn;\u0026thinsp;1.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8.44\u0026thinsp;\u0026plusmn;\u0026thinsp;1.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e26.94\u0026thinsp;\u0026plusmn;\u0026thinsp;2.55\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eL2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e9.51\u0026thinsp;\u0026plusmn;\u0026thinsp;1.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e11.67\u0026thinsp;\u0026plusmn;\u0026thinsp;1.27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6.81\u0026thinsp;\u0026plusmn;\u0026thinsp;0.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e28.61\u0026thinsp;\u0026plusmn;\u0026thinsp;2.73\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6.69\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e13.85\u0026thinsp;\u0026plusmn;\u0026thinsp;1.28\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.87\u0026thinsp;\u0026plusmn;\u0026thinsp;0.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e11.67\u0026thinsp;\u0026plusmn;\u0026thinsp;1.03\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ecisplatin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e16.83\u0026thinsp;\u0026plusmn;\u0026thinsp;1.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e21.35\u0026thinsp;\u0026plusmn;\u0026thinsp;1.99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e19.48\u0026thinsp;\u0026plusmn;\u0026thinsp;2.64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e17.88\u0026thinsp;\u0026plusmn;\u0026thinsp;1.91\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e19.41\u0026thinsp;\u0026plusmn;\u0026thinsp;1.84\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCuCl\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;50\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Anti-cancer mechanisms of Cu(II) complexes\u003c/h2\u003e \u003cdiv id=\"Sec17\" class=\"Section3\"\u003e \u003ch2\u003e3.3.1 Induce mitochondrial damage by Cu(II) complex C2\u003c/h2\u003e \u003cp\u003eMitochondria represent significant targets for metal complexes, and various pathways of cancer cell death are closely associated with mitochondrial function (Erxleben \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Olelewe and Awuah \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Scalcon et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In this study, we investigated the impact of the Cu(II) complex C2 on mitochondrial membrane potential (△Ψm) in MGC-803 cells, a key indicator of mitochondrial health. The staining of MGC-803 cells with JC-1, followed by examination using confocal microscopy, allowed us to evaluate changes in mitochondrial membrane potential.\u003c/p\u003e \u003cp\u003eIn normal cells, the aggregated form of JC-1 emits a red fluorescence, reflecting an intact mitochondrial membrane potential. Conversely, in cells with mitochondrial dysfunction, JC-1 remains in its monomeric form, exhibiting green fluorescence, often associated with apoptotic cells. Remarkably, our findings demonstrated the increase in green (monomeric) fluorescence when MGC-803 cells were treated with C2, as illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eThese alterations in mitochondrial membrane potential strongly suggest that C2 induces damage to the mitochondria. Such damage to the mitochondrial membrane potential can trigger cascades of events leading to altered cellular processes and ultimately cell death. The observed impact on mitochondrial function further highlights the potential of C2 as an effective agent in combating cancer by disrupting vital cellular pathways. Further investigations are warranted to elucidate the precise mechanisms and downstream effects of mitochondrial damage induced by C2.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section3\"\u003e \u003ch2\u003e3.3.2 Promote the generation of ROS by Cu(II) complex C2\u003c/h2\u003e \u003cp\u003eMitochondria, as the principal subcellular organelles responsible for generating reactive oxygen species (ROS), play a critical role in cellular oxidative processes (Yang et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). When mitochondria experience dysfunction, there is often an upsurge in ROS production, triggering various apoptotic signaling pathways. Therefore, in this study, we sought to investigate the impact of C2 on ROS generation within the MGC-803 cell line.\u003c/p\u003e \u003cp\u003eWe utilized 2',7'-dichlorofluorescein diacetate (DCFH-DA) and fluorescence microscopy to measure intracellular ROS levels. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e illustrates the outcomes of this analysis. In the control group, a weak fluorescence image was observed, suggesting relatively low ROS levels. Conversely, following incubation with Cu(II) complex C2, MGC-803 cells exhibited green fluorescence, indicating a substantial increase in ROS levels.\u003c/p\u003e \u003cp\u003eThe augmentation of ROS levels following C2 treatment signifies the induction of oxidative stress within the cells. Elevated ROS levels can trigger a multitude of cellular responses, including oxidative damage to biomolecules, initiation of apoptotic cascades, and overall perturbation of cellular homeostasis. The robust increase in ROS generation resulting from C2 treatment underlines its potential as a potent agent for inducing oxidative stress-mediated cytotoxicity in cancer cells. Further research is imperative to comprehend the precise mechanisms by which C2 modulates ROS and its subsequent effects on cellular fate.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section3\"\u003e \u003ch2\u003e3.3.3 Induce DNA damage by Cu(II) complex C2\u003c/h2\u003e \u003cp\u003eGiven adenine's crucial role as a free nitrogenous base in DNA synthesis, its integration into the Cu(II) complex C2 structure enhances the complex's targeting ability and affinity for DNA. This integration suggests a potential mechanism for the observed cytotoxicity and anticancer activity. Excessive levels of reactive oxygen species (ROS) generated due to the action of Cu(II) complex C2 can inflict damage not only on DNA but also induce cell cycle arrest, amplifying the potential anticancer effects.(Perillo et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) DNA damage can manifest in various forms, including base modification, DNA single-strand breaks (SSBs), DNA intra- and inter-strand cross-links, and the most severe form, DNA double-strand breaks (DSBs). Among these, DSBs are considered highly detrimental to the DNA structure. To confirm the capability of Cu(II) complex C2 in inducing DNA damage, we conducted immunofluorescence analysis targeting histone H2AX phosphorylation foci (γH2AX), a recognized marker for DNA DSBs, in MGC-803 cells. The phosphorylation of serine 139 on H2AX, forming γH2AX, is a characteristic response to DNA DSBs, providing a clear biomarker reflecting the extent of DNA damage (Rogakou et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). As depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, upon treatment of MGC-803 cells with 4.51 \u0026micro;M C1/C2 for 4 hours, a substantial upregulation in the fluorescence intensity of γH2AX was observed. This observation strongly suggests that adenine's incorporation into Cu(II) complex C2 significantly enhances its targeting ability and affinity for DNA, ultimately resulting in notable DNA damage. The induced DNA damage can be regarded as a crucial factor contributing to the heightened cytotoxicity and potential anticancer properties exhibited by Cu(II) complex C2. Further investigations will delve into unraveling the intricate mechanisms underpinning this DNA-targeted therapeutic approach.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further validate the DNA-damaging effects of Cu(II) complex C2, we employed the comet assay, a robust tool for detecting and quantifying DNA single- and double-strand breaks in cells. This assay allows for a precise assessment of the extent of DNA damage induced by the compound under investigation. As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, the comet assay results clearly demonstrate that C2 elicited longer-length tails compared to the control and C1 in MGC-803 cells, underscoring the severe DNA damage induced by C2.\u003c/p\u003e \u003cp\u003eIn addition, to determine whether C2 interacted with DNA, we performed the molecular docking studies using the promising lead compound C2 to the DNA molecule. (Malik et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) The results of molecular docking showed that there are potential binding sites of C2 in DNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-C). In addition, the kinetics of C2 binding to DNA was studied by measuring the amount of Cu bound to precipitated ct-DNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003eThe compelling evidence presented in this study collectively affirms that copper complexes engineered based on the adenine framework possess a potent capability to instigate substantial DNA damage. The capacity to induce significant DNA damage marks a pivotal attribute in the potential application of these complexes as targeted agents for cancer treatment. Further exploration of the mechanistic intricacies underlying the DNA-damaging properties of copper complexes and their potential implications in cancer therapy is warranted for a comprehensive understanding and effective clinical translation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section3\"\u003e \u003ch2\u003e3.3.4 Induce cell cycle blocks by Cu(II) complex C2\u003c/h2\u003e \u003cp\u003eFollowing the confirmation of DNA damage induction by copper complex C2 through comet assay and γH2AX staining, our focus shifted to investigating whether C2 could disrupt the cell cycle. In Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e, the data obtained after treating the cells with complex C1/C2 at 4.51 \u0026micro;M for 48 hours revealed a noteworthy increase in cells residing in the G2 phase of the cell cycle. Specifically, cells in the G2 phase increased to 18.86% compared to the control (6.24%), providing strong evidence that complex C2 effectively arrests the MGC-803 cell cycle at the G2 phase.\u003c/p\u003e \u003cp\u003eThis observed cell cycle arrest is a crucial finding, as it suggests a potential mechanism through which copper complex C2 exerts its cytotoxic effects on cancer cells. Cell cycle regulation is fundamental in cell proliferation and maintaining genomic stability. Disruption of this process can impair cell division and lead to cell death. Therefore, the ability of copper complex C2 to induce cell cycle arrest, particularly at the G2 phase, underscores its potential as a potent anti-cancer agent by interfering with vital cellular processes. Further research is warranted to elucidate the precise molecular pathways underlying this observed cell cycle arrest and its implications for cancer treatment.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section3\"\u003e \u003ch2\u003e3.3.5 Mediate apoptosis by Cu(II) complex C2\u003c/h2\u003e \u003cp\u003eAcridine orange (AO)/ethyl bromide (EB) staining is a well-established technique utilized to detect cell apoptosis. In this method, AO is capable of penetrating living cells and binding to DNA, emitting a green fluorescence. Conversely, EB can only penetrate cells that are no longer viable, binding to DNA and emitting an orange-red fluorescence (Liu et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Apoptotic cells typically exhibit intensified red fluorescence and manifest distinct characteristics, such as circular rosary-like structures, pyknotic nuclei, or blocky formations.\u003c/p\u003e \u003cp\u003eAs depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e, the AO/EB staining results unequivocally demonstrate that C2 significantly induces apoptosis in MGC-803 cells. This observation further strengthens the evidence of C2's potent anticancer properties, as induction of apoptosis is a pivotal mechanism for inhibiting cancer cell growth and promoting cell death. The ability of C2 to trigger apoptosis underscores its potential as a targeted therapeutic agent in cancer treatment. Further investigations are warranted to decipher the precise apoptotic pathways modulated by C2, providing invaluable insights for its prospective clinical application.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn addition to the techniques employed earlier, annexin V and propidium iodide (PI) staining coupled with flow cytometry analyses were utilized to further evaluate Cu(II) complex C2's capacity to induce apoptosis in MGC-803 cells. The results, illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e, unequivocally demonstrate that treatment with complex C1/C2 at a concentration of 4.51 \u0026micro;M for 24 hours triggered apoptosis in the MGC-803 cells.\u003c/p\u003e \u003cp\u003eSpecifically, the percentage of cells undergoing early apoptosis escalated from 2.79% in the control group to a maximum of 24.66% in the C2 group. Concurrently, the percentages of cells undergoing late apoptosis increased from 0.83% in the control group to a maximum of 17.33%. These compelling results collectively underscore the potential of complex C2 as a potent apoptotic inducer.\u003c/p\u003e \u003cp\u003eApoptosis, a highly regulated cell death mechanism, plays a fundamental role in maintaining tissue homeostasis and eliminating damaged or malignant cells. The ability of C2 to significantly induce apoptosis further solidifies its potential as an efficacious anticancer agent. Understanding the precise molecular pathways and mechanisms through which C2 orchestrates apoptosis is crucial for harnessing its full therapeutic potential and advancing its application in cancer treatment. Further investigations are warranted to elucidate the intricacies of C2-induced apoptosis, paving the way for potential apoptotic inducers.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"4 Conclusions","content":"\u003cp\u003eIn summary, our study successfully designed, synthesized, and characterized two novel Cu(II) complexes based on adenine. These complexes demonstrated potent anticancer activity against the MGC-803 cell line, surpassing the cytotoxicity of cisplatin. Notably, enhancing the structure of salicylaldehyde at the C-5 position with a methyl group significantly amplified the cytotoxic effects. Particularly, Cu(II) complex C2 emerged as a formidable chemotherapeutic agent, exerting its efficacy through multiple mechanisms, including heightened ROS levels, induction of DNA damage, disruption of mitochondrial function, and initiation of apoptosis. These compelling findings underscore the potential of Cu(II) complex based on adenine as a promising anticancer agent that warrants in-depth exploration and validation for its potential clinical application. Comparatively, this work builds on the established role of platinum-based chemotherapeutics while addressing their limitations, particularly severe side effects and drug resistance. The presented copper(II) complexes offer a promising alternative and pave the way for further research and development in the field of non-platinum metal-based anticancer drugs. The superior cytotoxicity of these adenine-based copper(II) complexes, especially complex C2, demonstrates their potential to outperform cisplatin, marking a significant stride in anticancer drug discovery. Additionally, the mechanistic insights into the cytotoxic effects of C2 underscore the potential of adenine-based anticancer metal complexes that specifically target DNA, presenting an exciting avenue for advancing the development of effective anticancer agents.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eContributions of Authors\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eXiaoyan Zhai\u003c/strong\u003e\u003cstrong\u003e:\u003c/strong\u003e Visualization, Formal analysis, Methodology, Software, Data Curation, Writing - Original Draft. \u003cstrong\u003eHussein Hanibah\u003c/strong\u003e\u003cstrong\u003e:\u0026nbsp;\u003c/strong\u003eSupervision, Conceptualization, Resources, Validation, Project administration. \u003cstrong\u003eNor Zakiah Nor Hashim\u003c/strong\u003e\u003cstrong\u003e:\u003c/strong\u003e Supervision, Writing- Reviewing and Editing. \u003cstrong\u003eJuzheng Zhang\u003c/strong\u003e\u003cstrong\u003e:\u003c/strong\u003e Supervision, Resources, Funding acquisition. \u003cstrong\u003eXianli Ma:\u003c/strong\u003e Project administration, Validation. \u003cstrong\u003eLilan Wei:\u0026nbsp;\u003c/strong\u003eInvestigation, Software. \u0026nbsp;\u003cstrong\u003eXiaoqun Zhou:\u0026nbsp;\u003c/strong\u003eConceptualization, Resources\u003cstrong\u003e.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Acknowledgements\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Guangxi Natural Science Foundation for the study (No. 2023GXNSFBA026313), China\u0026apos;s National Natural Science Foundation (No. 82204208).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Ethical Approval\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Funding\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Xiaoyan Zhai reports financial support was provided by Guangxi Natural Science Foundation. Xiaoyan Zhai reports financial support was provided by China\u0026rsquo;s National Natural Science Foundation. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Availability of data and materials\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eData available on request due to privacy/ethical restrictions.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBattaglia AM, Chirillo R, Aversa I, et al (2020) Ferroptosis and Cancer: Mitochondria Meet the \u0026ldquo;Iron Maiden\u0026rdquo; Cell Death. Cells 9:1\u0026ndash;26. https://doi.org/10.3390/CELLS9061505\u003c/li\u003e\n\u003cli\u003eCobine PA, Brady DC (2022) Cuproptosis: Cellular and molecular mechanisms underlying copper-induced cell death. 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Mol Med Rep 15:3\u0026ndash;11. https://doi.org/10.3892/MMR.2016.6022\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Adenine, Copper(II) Complexes, DNA Targeting, Reactive oxygen species, Anticancer, Cytotoxicity","lastPublishedDoi":"10.21203/rs.3.rs-3997929/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3997929/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePlatinum-based chemotherapeutics have played a critical role in oncology for decades. However, their broader utility is hindered by the advent of severe side effects and the emergence of drug resistance. The pursuit of alternative agents, particularly non-platinum (non-Pt) metal complexes, has gained momentum in current research. Designing efficacious non-Pt metal agents that target DNA poses a complex challenge. In this study, we present the strategic design, synthesis, and thorough characterization of two innovative copper(II) complexes leveraging adenine as a ligand, a potential avenue to overcome these challenges. Our investigation demonstrates the superior cytotoxicity of these copper(II) complexes compared to the benchmark cisplatin, with complex C2 exhibiting the most promising anticancer activity, showcasing an impressive IC\u003csub\u003e50\u003c/sub\u003e value of 4.51 \u0026micro;M in MGC-803 cells. Mechanistic insights underscore that complex C2 executes its cytotoxic effects by instigating DNA damage, orchestrating cell cycle arrest at the G2 phase, perturbing mitochondrial membrane potential, inducing ROS production, and ultimately triggering apoptotic pathways. These findings significantly emphasize the potential of designing novel adenine-based anticancer metal complexes targeting DNA, portraying a compelling trajectory for advancing anticancer drug development.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e","manuscriptTitle":"Synthesis and Anti-Cancer Investigations of Novel Copper(II) Complexes Based on Adenine","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-06 20:19:25","doi":"10.21203/rs.3.rs-3997929/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a48d9f2f-b8e9-4eec-98ae-7f2084a9d8df","owner":[],"postedDate":"March 6th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-06-07T12:46:14+00:00","versionOfRecord":{"articleIdentity":"rs-3997929","link":"https://doi.org/10.1016/j.molstruc.2024.138836","journal":{"identity":"journal-of-molecular-structure","isVorOnly":true,"title":"Journal of Molecular Structure"},"publishedOn":"2024-06-01 12:46:14","publishedOnDateReadable":"June 1st, 2024"},"versionCreatedAt":"2024-03-06 20:19:25","video":"","vorDoi":"10.1016/j.molstruc.2024.138836","vorDoiUrl":"https://doi.org/10.1016/j.molstruc.2024.138836","workflowStages":[]},"version":"v1","identity":"rs-3997929","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3997929","identity":"rs-3997929","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}