Investigation of Antimicrobial, Antioxidant, and Anticancer Properties of Substituted Phenethylamine-Based Imine and Metal Complexes

Investigation of Antimicrobial, Antioxidant, and Anticancer Properties of Substituted Phenethylamine-Based Imine and Metal Complexes | 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 Investigation of Antimicrobial, Antioxidant, and Anticancer Properties of Substituted Phenethylamine-Based Imine and Metal Complexes Taha Yasin BAYRAM, Merve YILDIRIM, Elif AKSAKAL, Bunyamin OZGERİS, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5767315/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Imine is a bioactive molecule formed by the reaction of primary amine with aldehyde or ketone. Imines can form stable complexes with metals due to a C = N group in their structures. These complexes have antibacterial, antifungal, anticancer, and antioxidant properties. Based on the literature data, this study synthesized substituted phenethylamine-based imine compounds copper (Cu) and zinc (Zn) metal complexes. The synthesized imine-metal complexes' antimicrobial, anticancer, and antioxidant activities were evaluated. The antimicrobial activity of the metal complexes was tested against pathogens using the disk diffusion method. No antimicrobial activity was observed for the metal complexes. The anticancer activity of the metal complexes was investigated on lung cancer cell line (A549) and healthy dermal fibroblast cell line (HDF) using WST-8 and SRB assay methods. The results revealed dose-dependent anticancer activity of the metal complexes in the A549 cell line, with IC50 values ​​ranging from 43.65 to 99.36 µg/mL. Additionally, dose-dependent cytotoxic effects of the compounds were observed in HDF cells. The responses of the compounds to free radicals and oxidative stress were evaluated using ABTS and CUPRAC methods. However, no antioxidant activity was detected for the metal complexes. Based on these analyses, it is predicted that imine-metal complexes may be potential candidates as anticancer agents. Imine Copper Zinc metal complex antimicrobial activity anticancer activity antioxidant activity Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Introduction In 1864, the German scientist Hugo Schiff coined the name "imine" or "Schiff base." [ 1 ]. The reversible acid-catalyzed reaction of a primary amine and an aldehyde or ketone forms imine. Imine compounds are potent ligands in chemical processes due to their great productivity and ease of synthesis [ 2 ] and are extensively employed in industrial, biological, and medical applications [ 3 ]. Imıne also has various biological effects such as anticancer [ 4 ], antibacterial [ 5 ], antifungal [ 6 ], and antioxidant [ 7 ]. Imine compounds have steric and electronegativity characteristics due to electron-donating atoms in their structure. These characteristics allow stable metal complexes to form widely used in analytical chemistry, agrochemicals, pharmaceuticals, and medical chemistry. [ 8 , 9 ]. The C = N group in imines' backbone structure enables them to combine with metal ions to generate complexes resulting in various biological characteristics. As a result, research on the synthesis, characterization, and biological activity of imine-metal complexes has become increasingly important in recent years [ 10 ]. It is known in the literature that the imine metal complex has strong antioxidant properties. Imine-metal complexes are one type of these that exhibit antioxidant activity by giving free radicals protons or electrons. [ 11 ]. Accordingly, much research focuses on imine-metal complexes' production and antioxidant properties. For instance, Jafari et al. (2017) synthesized and characterized imine-metal complexes and examined their antioxidant activities, reporting that they exhibited antioxidant activity [ 12 ]. In a different investigation, imines and four distinct metal complexes were used to create novel compounds, which were then analyzed using analytical and spectroscopic techniques. The antioxidant activity of these compounds was assessed using four different methods, and it was shown that the imine-metal complexes exhibited more potent antioxidant activity than their imine ligands [ 13 ]. The properties of the metals contained in the compounds determine the antioxidant activity of imine-metal complexes [ 14 ]. Today, resistance to commercially available antibiotics is on the rise, while it is predicted that resistance to metal-based antibiotics is less likely to develop [ 15 ]. Observations in hospitals where pathogenic organisms do not grow on metal-based surfaces increase optimism about this possibility [ 16 ]. Metals and metal complexes are widely used in various fields, particularly industrial applications. However, using metals as antimicrobial agents dates back thousands of years [ 17 ]. Additionally, the United Nations' Sustainable Development Goals highlight the potential of metal-based antimicrobials as promising agents in combating infectious diseases. The literature also reports antimicrobial activity exhibited by compounds formed with metal complexes. Among these, imine-metal complexes have been shown to possess significant antimicrobial activity [ 18 – 20 ]. This study synthesizes an imine compound based on 4-methoxyphenethylamine [ 21 ] and its novel Cu (II) and Zn (II) metal complexes. These metal complexes' antimicrobial, antioxidant, and anticancer potentials were investigated. This study aims to synthesize substituted phenethylamine-based imine and metal complexes to develop new bioactive substances. Experimental Section Materials and Methods The following materials were used for chemical synthesis: Benzaldehyde (Sigma Aldrich), 4-Methyl Phenethylamine (Acros Organics), 4-Methoxy Phenethylamine (J&K Scientific), Methylene Chloride (Sigma Aldrich), Molecular Sieve 4(Å) (Sigma Aldrich), n-Hexane (TEKKİM), Triethylamine (Sigma Aldrich), Chloroform D1 (Sigma Aldrich), Ethanol (Sigma Aldrich), Copper (II) Chloride Dihydrate (Sigma Aldrich), and Zinc Chloride (Sigma Aldrich). For antioxidant activity studies, the following were used: 2,2’-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) (Fluka), potassium persulfate (K 2 S 2 O 8 ) (Carlo Erba), ascorbic acid, neocuproine (Isolab), and ammonium acetate (Sigma Aldrich). For antimicrobial activity studies, the following were used: Luria Bertani Agar (LBA) (Miller MERCK), Potato Dextrose Agar (PDA) (Oxoid), Dimethyl Sulfoxide (DMSO) (Merck), Mueller Hinton Agar (MHA) (Oxoid), and Mueller Hinton Broth (MHB) (Biolife). For anticancer activity studies, the following were procured and used: Dulbecco's Modified Eagle Medium (DMEM) (Biowest), RPMI-1640 (Ecotech), Fetal Bovine Serum (FBS) (Gibco), L-Glutamine (WISENT INC), PenStrep (Gibco), Phosphate Buffered Saline (PBS) (Gibco), Hydrogen Peroxide (Sigma Aldrich), Trypsin/EDTA (Gibco), and CVDK-8 (Ecotech). Instruments This study utilized IR spectroscopy and X-ray diffraction (XRD) devices to characterize chemical structures. The IR spectra were analyzed using an IRTracer-100 FTIR spectrophotometer with parameters ranging from 4000 to 400 cm⁻¹. Powder X-ray diffraction (XRD) analysis was conducted using an EMPYREAN system with a Cu X-ray source and a wavelength of λ = 1.5406 Å. Methods Imin Synthesis Based on ( E)-N-Benzylidene-2-(4-methoxyphenethyl)ethanamine (3) Benzaldehyde (1) (0.500 g, 1 eq.) and 4-Methoxyphenethylamine (2) (1.06 g, 1 eq.) were placed in a 100 mL beaker, and 1.0 g of molecular sieve (4Å) was added. The mixture was stirred with a glass rod until warming occurred. Methylene chloride (20 mL) was added to the mixture and filtered using filter paper [ 22 ]. Methylene chloride was removed using an evaporator. The product was crystallized using hexane-methylene chloride to yield yellow crystals of the imine (3) with an 89.52% yield (1.01 g) [ 21 ]. General Method for Synthesis of Imin-Metal Complexes (4–5) Metal salts (Copper (II), Chloride Dihydrate, and Zinc Chloride) (1 eq.) were dissolved in ethanol (15 mL), and 1.0 g of molecular sieve (4Å) was added. The mixture was allowed to stand at room temperature for 30–40 minutes. In a 250 mL two-neck round-bottom flask, imine ligand (2 eq.) was dissolved in ethanol (15 mL). Nitrogen (N 2 ) gas was used to create an inert atmosphere. Once inert conditions were achieved, the metal solutions dried with molecular sieves were added dropwise, followed by triethylamine (2 eq., 0.72 mL). The mixture was refluxed overnight at 100°C. After completion, the reaction mixture was transferred to a sterile centrifuge tube and centrifuged at 9000 rpm for 15 minutes. The supernatant was discarded, and the solid particles were washed with ethanol (10 mL) and centrifuged again at 9000 rpm for 15 minutes. The particles were dried at room temperature. The imin-copper metal complex (4) was obtained as a brown solid with a yield of 69% (0.389 g). The imin-zinc metal complex (5) was obtained as a brown solid with a yield of 88% (0.497 g) [ 23 ]. Antibacterial Tests Disc Diffusion Test One of the most commonly used methods for antibacterial testing, the disc diffusion test was employed. Bacterial inoculum was prepared according to 0.5 McFarland standard and spread on petri dishes. Metal complexes (4–5) prepared at a concentration of 200 µM were impregnated into ten µL disc papers and placed on the Petri dishes. After 24 hours of incubation at 37°C, the zone diameters were measured to determine antibacterial sensitivity. DMSO was used as a negative control [ 24 ]. Antioxidant Activity Studies ABTS Radical Scavenging Activity : This method is based on the discoloration of the blue-green ABTS•+ radical solution in the presence of antioxidants, measured spectrophotometrically. A solution of 7 mM ABTS and 2.45 mM potassium persulfate was mixed in a 1:1 ratio and incubated in the dark at room temperature for 12–16 hours. After incubation, the mixture was diluted with ethanol to absorb 0.7 ± 0.02 at 734 nm. To 1 mL of this ABTS solution, 10 µL of metal complex solutions prepared at different concentrations were added. After 6 minutes of incubation at room temperature, absorbance at 734 nm was measured. Ascorbic acid was used as a standard. Each experiment was repeated thrice, and the results averaged [ 25 ]. Cupric Reducing Antioxidant Capacity (CUPRAC) The reducing capacity of the synthesized compounds was determined using the Cu 2+ reducing power method, as reported by [ 26 ]. CuCl 2 solution (0.25 mL, 0.01 M), ethanolic neocuproine solution (0.25 mL, 7.5×10⁻³ M), and ammonium acetate buffer solution (0.25 mL, 1 M) were added to different concentrations (6.25–200 µg/mL) of metal complexes. The total volume was adjusted to 2 mL with dH 2 O, mixed for 30 minutes, and absorbance was measured at 450 nm using a spectrophotometer. Anticancer and Cytotoxicity Studies Anticancer analysis was performed on the A549 lung cancer cell line, while cytotoxicity was tested on healthy human dermal fibroblast (HDF) cells. A549 cells were cultured in RPMI-1640 medium supplemented with 10% Fetal Bovine Serum, 1% Penicillin/Streptomycin, and 1% L-glutamine at 37°C in a 5% CO 2 incubator. HDF cells were cultured similarly but in a DMEM medium. Cells were passaged every few days based on density. When the cells in T25 flasks reached 70–80% confluency, they were detached using trypsin, and cell counts were performed using a Neubauer chamber under an inverted microscope. Synthesized imin-metal complexes were dissolved in DMSO and prepared at 200, 100, 50, 25, 12.5, and 6.25 µg/mL concentrations. These were applied to the cells and incubated for 48 hours. All experiments were conducted in triplicate. The control group was medium only, with methotrexate and 1% DMSO as positive controls. WST-8 Assay for Cell Proliferation The WST-8 cell proliferation assay was performed to determine the compounds' cytotoxic and anticancer activities and optimal doses. The commercial “Cell Viability Detection Kit-8 (CVDK-8)” was used following the kit protocol. After incubating cells treated with imin-metal complexes for 48 hours, 50 µL of WST-8 solution was added to each well under sterile and dark conditions. The cultures were incubated at 37°C in 5% CO 2 for 1–4 hours. The absorbance of the formazan crystals formed was measured at 450 nm, and cell viability percentages for each dose were calculated [ 27 ]. SRB Assay for Cell Proliferation The sulforhodamine B (SRB) assay was performed to analyze the effects of imin-metal complexes on total protein levels in A549 and HDF cells. A549 and HDF cells at 70–80% confluency were seeded into 96-well plates and incubated at 37°C in 5% CO 2 for 24 hours. After incubation, IC50 concentrations of the imin-metal complexes were applied in triplicate and incubated for 48 hours. After incubation, the media was removed, and 100 µL of 10% cold trichloroacetic acid was added to each well and incubated at + 4°C for 1 hour to fix the cellular proteins. After fixation, the trichloroacetic acid was removed, and wells were washed with deionized water. SRB dye (50 µL) was added to each well and incubated in the dark at room temperature for 30 minutes. The dye was removed, wells were washed with 1% acetic acid and air-dried, and 200 µL of 10 mM tris base was added. The plate was shaken at 150 rpm for 15 minutes, and absorbance was measured at 564 nm. Total protein amounts in the cells were calculated. The control group was medium only, with methotrexate and 1% DMSO as positive controls [ 28 ]. Statistical Analyses Data obtained from the studies were evaluated using GraphPad Prism 10.0 Software (GraphPad Software, La Jolla, CA). Unpaired t-tests were performed to calculate p-values, with p < 0.05 considered significant. Results and Discussion Synthesis Design Imin-metal complexes, which are biologically active compounds, have been shown to possess antibacterial, antifungal [ 16 ], antioxidant [ 29 ], and anticancer activities [ 23 ], attracting significant interest in the fields of bioorganic and bioinorganic chemistry. In imin-metal complex reactions, the process typically begins with imine synthesis, followed by adding metal salts to yield mono-, bi-, tri-, or tetra-dentate complexes [ 30 ]. In this study, the imine intermediate (3) was synthesized by reacting benzaldehyde and 4-methoxy phenethylamine, the structure of which is known in the literature, using the synthesis method described above [ 21 ]. Subsequently, the imine was reacted with Copper (II) Chloride Dihydrate and Zinc Chloride metal salts to produce the corresponding imin-metal complexes (4–5) with yields ranging between 68–89%. The synthesis design for the target compounds is illustrated in Fig. 1 ., while the structures of the synthesized products are presented in Fig. 2 . Structural Characterization The structure of the 4-methoxy phenethylamine-based imine (3) synthesized in this study is known in the literature [ 31 ]. However, the structures of the imine-copper and imine-zinc metal complexes are novel and have yet to be previously reported. The synthesized imin-metal complexes (4–5) were characterized using FTIR, mass spectrometry, and XRD analyses. All spectral data confirmed the structures of the synthesized compounds. FTIR Analysis FTIR data revealed the characteristic CH = N signal of the metal complex at 1645 cm⁻¹, and the Metal-N bond showed transmittance values in the range of 530–551 cm⁻¹ (Fig. 3 – 5 ). These results are consistent with similar structures in the literature. When imines are used as ligands, the electronegative properties of the metals are known to cause shifts in the characteristic peaks of the ligand [ 32 ]. For the synthesized metal complexes, the CH = N peak characteristic of the imine shifted from 1645 cm⁻¹ to 1595 cm⁻¹ and 1610 cm⁻¹ after binding to copper and zinc, respectively. Additionally, signals in the 500–600 cm⁻¹ range, corresponding to metal-nitrogen bonds, were observed in the FTIR data, consistent with previously reported values in the literature [ 33 ]. These findings demonstrate that the FTIR analysis of the synthesized metal complexes aligns well with literature-reported structures of similar compounds. Mass Spectroscopy Analysis Mass spectroscopy analysis of the imine-metal complexes revealed that the molecular ion peaks for the imine-copper (4) and imine-zinc metal complexes (5) were observed at 542 m/z (S1-S2). The predicted molecular masses of the complexes, calculated using ChemBioDraw software, were also 542 m/z. This agreement between the experimental and theoretical values confirms the structures of the synthesized imine-metal complexes. The mass spectrometry analysis also provided detailed information about the composition of the imine-metal complexes, further supporting that the metal ions are bound to the imine structure. This evidence validates the successful synthesis and structural integrity of the complexes. XRD Analysis The XRD patterns provided clear evidence of the formation of crystalline structures in the synthesized imine-metal complexes (Fig. 6 .). The XRD graph of the imine-zinc complex (5) displayed distinct and sharp peaks, indicating the successful coordination of imine groups with zinc ions to form a well-ordered crystalline structure. These sharp peaks are characteristic of high crystallinity and confirm the structural regularity of the complex. In contrast, the XRD graph of the imine-copper complex (4) showed a different peak distribution, with lower intensity and broader peaks than the zinc complex. This suggests the copper complex has a less regular crystalline structure or exhibits amorphous characteristics. While some peak positions overlapped with those of the zinc complex, the overall pattern was less defined, reflecting a different or less ordered phase structure. These results highlight the significant variation in crystallinity and structural organization between the complexes, influenced by the specific metal ion used. The lattice method was used to index the main peaks of the X-ray diffraction pattern [ 34 ]. The parameters were calculated according to the Scherrer equation [ 35 ]. Table 1 presents the crystal size, hkl indices, and 2-theta maxima. Table 1 The XRD peaks and crystal thickness were calculated. Compound 2-theta maximum hkl indices crystal size Imıne (3) Ligand 11.00724 100 16.39094856 Compound (4) Copper 14.13604 100 0.947813954 Compound (5) Zinc 34.10437 220 61.6247131 Coordination Chemistry Insights The formation of imine-metal complexes occurs through the coordination of the imine group, typically the azomethine (-C = N-) moiety, with metal ions. This coordination is driven by the interaction between the imine group's electron-donating properties and the metal ions' electron-accepting nature [ 36 ]. The observed differences in crystallinity and structural order can be attributed to variations in the electronic and geometric properties of the zinc and copper ions. These factors influence the coordination environment and packing arrangement in the crystal lattice. The XRD analysis validates the successful formation of imine-metal complexes and demonstrates the structural diversity resulting from interactions with different metal ions. This underscores the versatility of imine groups as ligands in coordination chemistry, enabling the formation of complexes with varying crystalline properties [ 37 ]. Antimicrobial Activity Results The antimicrobial activity of the synthesized imine-copper and imine-zinc metal complexes was evaluated against a range of pathogens, including Gram-negative bacteria : A. baumannii and E. coli Gram-positive bacteria : Methicillin-resistant Staphylococcus aureus (MRSA) and Enterococcus faecalis Yeasts : Candida albicans and Candida dubliniensis . Disc diffusion tests were conducted successfully to determine the antimicrobial efficacy (Table 2 ). No zone of inhibition was observed around the discs impregnated with the complexes, indicating that the synthesized compounds lack antimicrobial activity against the tested pathogens. The disc diffusion data in Table 2 confirms the absence of antimicrobial activity. Despite the promising structural properties of imine-metal complexes, the synthesized compounds did not demonstrate significant antimicrobial potential in this study. Further modifications to the structure or testing against additional strains may provide insights into their potential biological activity in other contexts. Table 2 Disc diffusion results of imine-metal complexes Microbial Strains Imine-Metal Complex Compound (4) Compound (5) A. baumannii ATCC BAA- 1605 - - MRSA ATCC 43300 - - E. coli ATCC BAA-2523 - - E. faecalis ATCC 49452 - - C. albicans ATCC 10231 - - C. tropicalis KÜEN 1025 - - Imine-metal complexes, particularly those involving copper and zinc, have been widely studied for their antimicrobial potential. While some studies highlight promising activity, others indicate limited or no efficacy, highly dependent on the ligand structure and metal coordination. Studies have reported that imine-copper metal complexes exhibit significant antimicrobial activity against Gram-positive and Gram-negative bacteria and fungi due to their structural properties [ 38 ]. However, antimicrobial efficacy can vary significantly based on the structural features of the complex. Some imine-copper complexes demonstrate no activity, indicating that not all synthesized complexes are effective [ 39 ]. Imine-zinc complexes have garnered attention for their potential antimicrobial activity, often showing enhanced efficacy compared to their free ligands due to synergistic effects [ 40 ]. Nonetheless, not all imine-zinc complexes exhibit antimicrobial properties, highlighting the variability in activity based on ligand structure and metal coordination [ 41 ]. In the current study, synthesized imine-copper (4) and imine-zinc (5) complexes exhibited no antimicrobial activity against tested bacterial strains, including A. baumannii , E. coli , MRSA, E. faecalis , and fungal strains like C. albicans and C. dubliniensis . This result aligns with reports that not all imine-metal complexes possess antimicrobial properties and underscores the structural specificity of their activity. While specific imine-metal complexes have demonstrated antifungal properties [ 42 ], others have shown no such activity [ 6 ]. The absence of antifungal activity in the complexes synthesized here aligns with studies indicating that antifungal efficacy depends heavily on structural and coordination features [ 43 ]. The findings suggest that while imine-metal complexes have potential antimicrobial applications, their activity is not universal and depends on specific structural and coordination factors. This variability highlights the need for further exploration and optimization to improve their efficacy against resistant pathogens. Antioxidant Activity The study investigated the antioxidant activities of imine-copper (4) and imine-zinc metal complexes using the ABTS and CUPRAC methods. As a result of these studies, it was determined that the metal complexes exhibited no antioxidant activity compared to the positive controls, ascorbic acid and trolox (Fig. 7 – 8 ). In the literature, the antioxidant activity of imine metal complexes, particularly those containing transition metals, is reported to be significant due to their potential therapeutic applications. Studies have shown that these complexes exhibit enhanced antioxidant properties compared to their free ligands [ 39 ]. Additionally, other studies have reported that metal complexes demonstrate dose-dependent antioxidant activities, often surpassing the activities of free ligands. However, they are generally less effective than standard antioxidants such as ascorbic acid [ 44 ]. The literature also comprehensively investigates imine copper-zinc metal complexes' antioxidant and non-antioxidant activities, revealing a complex interaction between their chemical structures and biological effects. These complexes, especially those containing copper(II) and zinc(II), exhibit significant antioxidant properties, frequently outperforming their free ligand counterparts but generally falling short of the effectiveness of standard antioxidants like ascorbic acid. Copper (II) and zinc (II) complexes have demonstrated noteworthy antioxidant activities in studies that show dose-dependent effects in scavenging free radicals such as DPPH and hydroxyl radicals [ 44 ]. However, in the current study, metal complexes (4–5) showed no antioxidant activity compared to ascorbic acid and trolox. Effects of Imine-Metal Complexes on Cell Viability: Analysis of WST-8 and SRB Assays This study investigated the effects of imine-metal complexes on A549 and HDF cell lines using WST-8 and SRB assays. The WST-8 assay revealed that the imine-metal complexes exhibited dose-dependent anticancer activity in the A549 cell line, with an IC50 value calculated to be 43.65–99.36 µg/ml (Fig. 9 ). Additionally, the imine-metal complexes demonstrated dose-dependent cytotoxic effects in the HDF cells (Fig. 10 ). The SRB assay detects cellular total proteins based on cell viability. The cellular total proteins at the IC50 concentration of the imine-metal complexes were examined for the SRB assay. As a result of this test, the total protein content in the A549 cells treated with the imine-copper metal complex was calculated to be 28%, and for the HDF cells, it was 25%. For the A549 cells treated with the imine-zinc metal complex, the total protein content was calculated to be 39%, while for the HDF cells, it was 46% (Fig. 11 .). The anticancer activity of imine metal complexes has garnered significant attention due to their unique properties and mechanisms of action. Recent studies emphasize that various imine metal complexes exhibit vigorous anticancer activity against cancer cell lines [ 45 ]. The investigation of imine metal complexes, particularly in the context of A549 lung cancer cells, highlights their potential for anticancer applications [ 46 ]. Research has shown that the imine-copper metal complex can effectively target cancer cells and often outperforms traditional agents such as cisplatin [ 47 ]. Copper complexes, especially those complexed with imine ligands, can induce cell death through a novel mechanism called cuproptosis, which occurs via copper [ 48 ]. The synthesis of imine-copper metal complexes has demonstrated promising results in targeting A549 lung cancer cells. In a study by Gul et al., 2020, it was reported that the imine-copper complex exhibited anticancer activity by influencing autophagy and apoptosis in the A549 cell line. The analysis conducted in this study revealed dose-dependent anticancer activity of the imine-copper complex in the A549 cancer cell line, consistent with the literature [ 49 ]. However, the study also observed dose-dependent cytotoxic effects of the synthesized imine-copper complexes in the HDF healthy cell line. It is known in the literature that imine-copper complexes enhance cytotoxicity through their ability to bind to DNA. Diimine-copper complexes have shown significant cytotoxic effects against various cancer cell lines due to their strong DNA-binding capabilities [ 50 ]. The imine-copper complex synthesized in this study is characterized by a diimine structure in its chemical composition. The anticancer activity of imine and zinc metal complexes has attracted considerable attention due to their potential efficacy and reduced side effects compared to traditional treatments. Studies suggest these complexes can interact with DNA and exhibit cytotoxic effects against various cancer cell lines. Zinc (II) diimine complexes have demonstrated promising anticancer properties by increasing their ability to interact with DNA, enhancing their cytotoxicity against cancer cells [ 51 ]. Specifically, complexes formed with cobalt zinc and imine ligands have shown significant DNA-binding affinity, which is crucial for their anticancer activities [ 52 ]. Zinc complexes have been found to perform better than traditional platinum-based drugs in selectivity and efficacy, indicating their potential as safer alternatives [ 51 ]. The analysis conducted in this study revealed dose-dependent anticancer activity of the imine-zinc metal complex in the A549 cancer cell line, consistent with the literature. However, the HDF healthy cell line also observed dose-dependent cytotoxic effects. Declarations Acknowledgments: This study was supported by TUBITAK-2209-A University Students Research Projects Support Program (2022/02). We want to thank TUBITAK for their financial support and research conditions. Author Contributions : TYB, MY, and EA performed the analyses and wrote the first draft of the manuscript. AG and BO supervised the study and interpreted the results. All authors contributed to the revision of the manuscript and have read and approved the submitted version. Data availability statements: All data generated or analyzed during this study are included in this published article Conflicts of Interest: The authors declare no conflicts of interest References Hrichi H, Elkanzi N, Bakr R. 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An Explicative Review on the Current Advancement in Schiff Base-Metal Complexes as Anticancer Agents Evolved in the Past Decade: Medicinal Chemistry Aspects. Med Chem. 2023;19(10):960–85. doi: 10.2174/1573406419666230707105221 . Yang Y, Guo L, Tian Z, Gong Y, Zheng H, Zhang S, et al. Novel and Versatile Imine-N-Heterocyclic Carbene Half-Sandwich Iridium(III) Complexes as Lysosome-Targeted Anticancer Agents. Inorg Chem. 2018;57(17):11087–98. doi: 10.1021/acs.inorgchem.8b01656 . Gomes RN, Silva ML, Gomes KS, Lago JHG, Cerchiaro G. Synthesis, characterization, and cytotoxic effects of new copper complexes using Schiff-base derivatives from natural sources. J Inorg Biochem. 2024;250:112401. doi: 10.1016/j.jinorgbio.2023.112401 . Laurent R, Maraval V, Bernardes-Génisson V, Caminade A-M. Dendritic Pyridine–Imine Copper Complexes as Metallo-Drugs. Molecules. 2024 doi: 10.3390/molecules29081800 . Gul NS, Khan TM, Chen M, Huang KB, Hou C, Choudhary MI, et al. New copper complexes inducing bimodal death through apoptosis and autophagy in A549 cancer cells. J Inorg Biochem. 2020;213:111260. doi: 10.1016/j.jinorgbio.2020.111260 . Alvarez N, Rocha A, Collazo V, Ellena J, Costa-Filho AJ, Batista AA, et al. Development of Copper Complexes with Diimines and Dipicolinate as Anticancer Cytotoxic Agents. Pharmaceutics. 2023;15(5). doi: 10.3390/pharmaceutics15051345 . Babgi BA, Domyati D, Abdellattif MH, Hussien MA. Evaluation of the Anticancer and DNA-Binding Characteristics of Dichloro(diimine)zinc(II) Complexes. Chemistry. 2021 doi: 10.3390/chemistry3040086 . Mukherjee D, Reja S, Sarkar K, Fayaz TKS, Kumar P, Kejriwal A, et al. In vitro cytotoxicity activity of copper complexes of imine and amine ligands: A combined experimental and computational study. Inorganic Chemistry Communications. 2022;146:110190. doi: https://doi.org/10.1016/j.inoche.2022.110190 . Additional Declarations No competing interests reported. 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2","display":"","copyAsset":false,"role":"figure","size":28245,"visible":true,"origin":"","legend":"\u003cp\u003eSynthesized imine-metal complex\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5767315/v1/5a91a70e809cf5c139f04b82.jpg"},{"id":73277909,"identity":"a4d403ba-0085-4413-9aa5-0fb032c93267","added_by":"auto","created_at":"2025-01-08 12:05:41","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":37910,"visible":true,"origin":"","legend":"\u003cp\u003eFTIR result of 4- Methoxy Phenethylamine based imine (3)\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5767315/v1/e1c1743a387a4b3f0a14649c.jpg"},{"id":73278964,"identity":"d85bd9a7-d874-4b4b-9b43-66ec7a884472","added_by":"auto","created_at":"2025-01-08 12:13:41","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":26877,"visible":true,"origin":"","legend":"\u003cp\u003eFTIR result of imin-copper metal complex (4)\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5767315/v1/73c0eb2027b0e2c66f6e875c.jpg"},{"id":73277483,"identity":"a9cf7a56-be34-49aa-b32f-ba2766167316","added_by":"auto","created_at":"2025-01-08 11:57:43","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":37151,"visible":true,"origin":"","legend":"\u003cp\u003eFTIR result of imine-Zinc metal complex (5)\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5767315/v1/7a3029b277a337ba1f957923.jpg"},{"id":73277915,"identity":"cb49d783-007c-4f2c-8d83-5d10585420be","added_by":"auto","created_at":"2025-01-08 12:05:41","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":48732,"visible":true,"origin":"","legend":"\u003cp\u003eXRD result of Imine-Metal Complexes\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5767315/v1/f0fee266d19dc90c6f9dd1f4.jpg"},{"id":73278966,"identity":"c2b848e7-40f9-44d7-849a-a25347975fdd","added_by":"auto","created_at":"2025-01-08 12:13:41","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":51354,"visible":true,"origin":"","legend":"\u003cp\u003eABTS analysis results of imine-metal complexes\u003c/p\u003e","description":"","filename":"7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5767315/v1/4ac10e854ae0f90b36dc4ce1.jpg"},{"id":73278965,"identity":"8435fb99-919e-40a2-b811-25d875e10537","added_by":"auto","created_at":"2025-01-08 12:13:41","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":34807,"visible":true,"origin":"","legend":"\u003cp\u003eCUPRAC analysis results of imine-metal complexes\u003c/p\u003e","description":"","filename":"8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5767315/v1/4221902060035ae0e6a31b65.jpg"},{"id":73277440,"identity":"e197dfd0-adad-40f9-8f66-5ecd05e93667","added_by":"auto","created_at":"2025-01-08 11:57:41","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":43959,"visible":true,"origin":"","legend":"\u003cp\u003ePercent cell viability result of A549 imine metal complexes\u003c/p\u003e","description":"","filename":"9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5767315/v1/4cabd5ae4a56ea3e0e152a0e.jpg"},{"id":73277442,"identity":"99909bc7-ace3-4088-bcbf-b4ed659f187a","added_by":"auto","created_at":"2025-01-08 11:57:41","extension":"jpg","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":43411,"visible":true,"origin":"","legend":"\u003cp\u003ePercent cell viability result of HDF imine metal complexes\u003c/p\u003e","description":"","filename":"10.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5767315/v1/838d570ab5977d1770dd5265.jpg"},{"id":73277919,"identity":"b1e0f2f3-c535-4a54-8edf-17793cd08dad","added_by":"auto","created_at":"2025-01-08 12:05:42","extension":"jpg","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":42955,"visible":true,"origin":"","legend":"\u003cp\u003ePercentage of total protein of A549 and HDF imine metal complex\u003c/p\u003e","description":"","filename":"11.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5767315/v1/7dce52f29209b13f6cd85674.jpg"},{"id":74345053,"identity":"df3cc1b4-71a6-414c-8603-07dd856feb90","added_by":"auto","created_at":"2025-01-21 09:39:08","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1382763,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5767315/v1/23fa95c8-068f-4128-84b5-31dd6d2c2cfd.pdf"},{"id":73277448,"identity":"b0ecdf15-562e-4a07-90ea-bca343c3a2ca","added_by":"auto","created_at":"2025-01-08 11:57:41","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":173127,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterialTYB.docx","url":"https://assets-eu.researchsquare.com/files/rs-5767315/v1/0b3a7e5997ad4dd81866a486.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Investigation of Antimicrobial, Antioxidant, and Anticancer Properties of Substituted Phenethylamine-Based Imine and Metal Complexes","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIn 1864, the German scientist Hugo Schiff coined the name \"imine\" or \"Schiff base.\" [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The reversible acid-catalyzed reaction of a primary amine and an aldehyde or ketone forms imine. Imine compounds are potent ligands in chemical processes due to their great productivity and ease of synthesis [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e] and are extensively employed in industrial, biological, and medical applications [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Imıne also has various biological effects such as anticancer [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], antibacterial [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], antifungal [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], and antioxidant [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eImine compounds have steric and electronegativity characteristics due to electron-donating atoms in their structure. These characteristics allow stable metal complexes to form widely used in analytical chemistry, agrochemicals, pharmaceuticals, and medical chemistry. [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The C\u0026thinsp;=\u0026thinsp;N group in imines' backbone structure enables them to combine with metal ions to generate complexes resulting in various biological characteristics. As a result, research on the synthesis, characterization, and biological activity of imine-metal complexes has become increasingly important in recent years [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIt is known in the literature that the imine metal complex has strong antioxidant properties. Imine-metal complexes are one type of these that exhibit antioxidant activity by giving free radicals protons or electrons. [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Accordingly, much research focuses on imine-metal complexes' production and antioxidant properties. For instance, Jafari et al. (2017) synthesized and characterized imine-metal complexes and examined their antioxidant activities, reporting that they exhibited antioxidant activity [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. In a different investigation, imines and four distinct metal complexes were used to create novel compounds, which were then analyzed using analytical and spectroscopic techniques. The antioxidant activity of these compounds was assessed using four different methods, and it was shown that the imine-metal complexes exhibited more potent antioxidant activity than their imine ligands [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. The properties of the metals contained in the compounds determine the antioxidant activity of imine-metal complexes [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eToday, resistance to commercially available antibiotics is on the rise, while it is predicted that resistance to metal-based antibiotics is less likely to develop [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Observations in hospitals where pathogenic organisms do not grow on metal-based surfaces increase optimism about this possibility [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Metals and metal complexes are widely used in various fields, particularly industrial applications. However, using metals as antimicrobial agents dates back thousands of years [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Additionally, the United Nations' Sustainable Development Goals highlight the potential of metal-based antimicrobials as promising agents in combating infectious diseases. The literature also reports antimicrobial activity exhibited by compounds formed with metal complexes. Among these, imine-metal complexes have been shown to possess significant antimicrobial activity [\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThis study synthesizes an imine compound based on 4-methoxyphenethylamine [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] and its novel Cu (II) and Zn (II) metal complexes. These metal complexes' antimicrobial, antioxidant, and anticancer potentials were investigated. This study aims to synthesize substituted phenethylamine-based imine and metal complexes to develop new bioactive substances.\u003c/p\u003e"},{"header":"Experimental Section","content":"\n\u003ch3\u003eMaterials and Methods\u003c/h3\u003e\n\u003cp\u003eThe following materials were used for chemical synthesis: Benzaldehyde (Sigma Aldrich), 4-Methyl Phenethylamine (Acros Organics), 4-Methoxy Phenethylamine (J\u0026amp;K Scientific), Methylene Chloride (Sigma Aldrich), Molecular Sieve 4(\u0026Aring;) (Sigma Aldrich), n-Hexane (TEKKİM), Triethylamine (Sigma Aldrich), Chloroform D1 (Sigma Aldrich), Ethanol (Sigma Aldrich), Copper (II) Chloride Dihydrate (Sigma Aldrich), and Zinc Chloride (Sigma Aldrich). For antioxidant activity studies, the following were used: 2,2\u0026rsquo;-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) (Fluka), potassium persulfate (K\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e8\u003c/sub\u003e) (Carlo Erba), ascorbic acid, neocuproine (Isolab), and ammonium acetate (Sigma Aldrich). For antimicrobial activity studies, the following were used: Luria Bertani Agar (LBA) (Miller MERCK), Potato Dextrose Agar (PDA) (Oxoid), Dimethyl Sulfoxide (DMSO) (Merck), Mueller Hinton Agar (MHA) (Oxoid), and Mueller Hinton Broth (MHB) (Biolife). For anticancer activity studies, the following were procured and used: Dulbecco's Modified Eagle Medium (DMEM) (Biowest), RPMI-1640 (Ecotech), Fetal Bovine Serum (FBS) (Gibco), L-Glutamine (WISENT INC), PenStrep (Gibco), Phosphate Buffered Saline (PBS) (Gibco), Hydrogen Peroxide (Sigma Aldrich), Trypsin/EDTA (Gibco), and CVDK-8 (Ecotech).\u003c/p\u003e\n\u003ch3\u003eInstruments\u003c/h3\u003e\n\u003cp\u003eThis study utilized IR spectroscopy and X-ray diffraction (XRD) devices to characterize chemical structures. The IR spectra were analyzed using an IRTracer-100 FTIR spectrophotometer with parameters ranging from 4000 to 400 cm⁻\u0026sup1;. Powder X-ray diffraction (XRD) analysis was conducted using an EMPYREAN system with a Cu X-ray source and a wavelength of λ\u0026thinsp;=\u0026thinsp;1.5406 \u0026Aring;.\u003c/p\u003e\n\u003ch3\u003eMethods\u003c/h3\u003e\n\u003cp\u003e \u003cb\u003eImin Synthesis Based on (\u003c/b\u003e \u003cb\u003eE)-N-Benzylidene-2-(4-methoxyphenethyl)ethanamine\u003c/b\u003e \u003cb\u003e(3)\u003c/b\u003e\u003c/p\u003e \u003cp\u003eBenzaldehyde (1) (0.500 g, 1 eq.) and 4-Methoxyphenethylamine (2) (1.06 g, 1 eq.) were placed in a 100 mL beaker, and 1.0 g of molecular sieve (4\u0026Aring;) was added. The mixture was stirred with a glass rod until warming occurred. Methylene chloride (20 mL) was added to the mixture and filtered using filter paper [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Methylene chloride was removed using an evaporator. The product was crystallized using hexane-methylene chloride to yield yellow crystals of the imine (3) with an 89.52% yield (1.01 g) [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eGeneral Method for Synthesis of Imin-Metal Complexes (4–5)\u003c/h3\u003e\n\u003cp\u003eMetal salts (Copper (II), Chloride Dihydrate, and Zinc Chloride) (1 eq.) were dissolved in ethanol (15 mL), and 1.0 g of molecular sieve (4\u0026Aring;) was added. The mixture was allowed to stand at room temperature for 30\u0026ndash;40 minutes. In a 250 mL two-neck round-bottom flask, imine ligand (2 eq.) was dissolved in ethanol (15 mL). Nitrogen (N\u003csub\u003e2\u003c/sub\u003e) gas was used to create an inert atmosphere. Once inert conditions were achieved, the metal solutions dried with molecular sieves were added dropwise, followed by triethylamine (2 eq., 0.72 mL). The mixture was refluxed overnight at 100\u0026deg;C. After completion, the reaction mixture was transferred to a sterile centrifuge tube and centrifuged at 9000 rpm for 15 minutes. The supernatant was discarded, and the solid particles were washed with ethanol (10 mL) and centrifuged again at 9000 rpm for 15 minutes. The particles were dried at room temperature. The imin-copper metal complex \u003cb\u003e(4)\u003c/b\u003e was obtained as a brown solid with a yield of 69% (0.389 g). The imin-zinc metal complex \u003cb\u003e(5)\u003c/b\u003e was obtained as a brown solid with a yield of 88% (0.497 g) [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eAntibacterial Tests\u003c/h3\u003e\n\u003cp\u003e \u003cstrong\u003eDisc Diffusion Test\u003c/strong\u003e \u003cp\u003eOne of the most commonly used methods for antibacterial testing, the disc diffusion test was employed. Bacterial inoculum was prepared according to 0.5 McFarland standard and spread on petri dishes. Metal complexes (4\u0026ndash;5) prepared at a concentration of 200 \u0026micro;M were impregnated into ten \u0026micro;L disc papers and placed on the Petri dishes. After 24 hours of incubation at 37\u0026deg;C, the zone diameters were measured to determine antibacterial sensitivity. DMSO was used as a negative control [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eAntioxidant Activity Studies\u003c/h2\u003e \u003cp\u003e \u003cb\u003eABTS Radical Scavenging Activity\u003c/b\u003e: This method is based on the discoloration of the blue-green ABTS\u0026bull;+ radical solution in the presence of antioxidants, measured spectrophotometrically. A solution of 7 mM ABTS and 2.45 mM potassium persulfate was mixed in a 1:1 ratio and incubated in the dark at room temperature for 12\u0026ndash;16 hours. After incubation, the mixture was diluted with ethanol to absorb 0.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 at 734 nm. To 1 mL of this ABTS solution, 10 \u0026micro;L of metal complex solutions prepared at different concentrations were added. After 6 minutes of incubation at room temperature, absorbance at 734 nm was measured. Ascorbic acid was used as a standard. Each experiment was repeated thrice, and the results averaged [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eCupric Reducing Antioxidant Capacity (CUPRAC)\u003c/strong\u003e \u003cp\u003eThe reducing capacity of the synthesized compounds was determined using the Cu\u003csup\u003e2+\u003c/sup\u003e reducing power method, as reported by [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. CuCl\u003csub\u003e2\u003c/sub\u003e solution (0.25 mL, 0.01 M), ethanolic neocuproine solution (0.25 mL, 7.5\u0026times;10⁻\u0026sup3; M), and ammonium acetate buffer solution (0.25 mL, 1 M) were added to different concentrations (6.25\u0026ndash;200 \u0026micro;g/mL) of metal complexes. The total volume was adjusted to 2 mL with dH\u003csub\u003e2\u003c/sub\u003eO, mixed for 30 minutes, and absorbance was measured at 450 nm using a spectrophotometer.\u003c/p\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAnticancer and Cytotoxicity Studies\u003c/h3\u003e\n\u003cp\u003eAnticancer analysis was performed on the A549 lung cancer cell line, while cytotoxicity was tested on healthy human dermal fibroblast (HDF) cells. A549 cells were cultured in RPMI-1640 medium supplemented with 10% Fetal Bovine Serum, 1% Penicillin/Streptomycin, and 1% L-glutamine at 37\u0026deg;C in a 5% CO\u003csub\u003e2\u003c/sub\u003e incubator. HDF cells were cultured similarly but in a DMEM medium. Cells were passaged every few days based on density. When the cells in T25 flasks reached 70\u0026ndash;80% confluency, they were detached using trypsin, and cell counts were performed using a Neubauer chamber under an inverted microscope. Synthesized imin-metal complexes were dissolved in DMSO and prepared at 200, 100, 50, 25, 12.5, and 6.25 \u0026micro;g/mL concentrations. These were applied to the cells and incubated for 48 hours. All experiments were conducted in triplicate. The control group was medium only, with methotrexate and 1% DMSO as positive controls.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eWST-8 Assay for Cell Proliferation\u003c/strong\u003e \u003cp\u003eThe WST-8 cell proliferation assay was performed to determine the compounds' cytotoxic and anticancer activities and optimal doses. The commercial \u0026ldquo;Cell Viability Detection Kit-8 (CVDK-8)\u0026rdquo; was used following the kit protocol. After incubating cells treated with imin-metal complexes for 48 hours, 50 \u0026micro;L of WST-8 solution was added to each well under sterile and dark conditions. The cultures were incubated at 37\u0026deg;C in 5% CO\u003csub\u003e2\u003c/sub\u003e for 1\u0026ndash;4 hours. The absorbance of the formazan crystals formed was measured at 450 nm, and cell viability percentages for each dose were calculated [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eSRB Assay for Cell Proliferation\u003c/strong\u003e \u003cp\u003eThe sulforhodamine B (SRB) assay was performed to analyze the effects of imin-metal complexes on total protein levels in A549 and HDF cells. A549 and HDF cells at 70\u0026ndash;80% confluency were seeded into 96-well plates and incubated at 37\u0026deg;C in 5% CO\u003csub\u003e2\u003c/sub\u003e for 24 hours. After incubation, IC50 concentrations of the imin-metal complexes were applied in triplicate and incubated for 48 hours. After incubation, the media was removed, and 100 \u0026micro;L of 10% cold trichloroacetic acid was added to each well and incubated at +\u0026thinsp;4\u0026deg;C for 1 hour to fix the cellular proteins. After fixation, the trichloroacetic acid was removed, and wells were washed with deionized water. SRB dye (50 \u0026micro;L) was added to each well and incubated in the dark at room temperature for 30 minutes. The dye was removed, wells were washed with 1% acetic acid and air-dried, and 200 \u0026micro;L of 10 mM tris base was added. The plate was shaken at 150 rpm for 15 minutes, and absorbance was measured at 564 nm. Total protein amounts in the cells were calculated. The control group was medium only, with methotrexate and 1% DMSO as positive controls [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003c/p\u003e\n\u003ch3\u003eStatistical Analyses\u003c/h3\u003e\n\u003cp\u003eData obtained from the studies were evaluated using GraphPad Prism 10.0 Software (GraphPad Software, La Jolla, CA). Unpaired t-tests were performed to calculate p-values, with p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 considered significant.\u003c/p\u003e"},{"header":"Results and Discussion","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eSynthesis Design\u003c/h2\u003e \u003cp\u003eImin-metal complexes, which are biologically active compounds, have been shown to possess antibacterial, antifungal [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], antioxidant [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], and anticancer activities [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], attracting significant interest in the fields of bioorganic and bioinorganic chemistry. In imin-metal complex reactions, the process typically begins with imine synthesis, followed by adding metal salts to yield mono-, bi-, tri-, or tetra-dentate complexes [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this study, the imine intermediate (3) was synthesized by reacting benzaldehyde and 4-methoxy phenethylamine, the structure of which is known in the literature, using the synthesis method described above [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Subsequently, the imine was reacted with Copper (II) Chloride Dihydrate and Zinc Chloride metal salts to produce the corresponding imin-metal complexes (4\u0026ndash;5) with yields ranging between 68\u0026ndash;89%. The synthesis design for the target compounds is illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e., while the structures of the synthesized products are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eStructural Characterization\u003c/h2\u003e \u003cp\u003eThe structure of the 4-methoxy phenethylamine-based imine (3) synthesized in this study is known in the literature [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. However, the structures of the imine-copper and imine-zinc metal complexes are novel and have yet to be previously reported. The synthesized imin-metal complexes (4\u0026ndash;5) were characterized using FTIR, mass spectrometry, and XRD analyses. All spectral data confirmed the structures of the synthesized compounds.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eFTIR Analysis\u003c/h2\u003e \u003cp\u003eFTIR data revealed the characteristic CH\u0026thinsp;=\u0026thinsp;N signal of the metal complex at 1645 cm⁻\u0026sup1;, and the Metal-N bond showed transmittance values in the range of 530\u0026ndash;551 cm⁻\u0026sup1; (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). These results are consistent with similar structures in the literature. When imines are used as ligands, the electronegative properties of the metals are known to cause shifts in the characteristic peaks of the ligand [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFor the synthesized metal complexes, the CH\u0026thinsp;=\u0026thinsp;N peak characteristic of the imine shifted from 1645 cm⁻\u0026sup1; to 1595 cm⁻\u0026sup1; and 1610 cm⁻\u0026sup1; after binding to copper and zinc, respectively. Additionally, signals in the 500\u0026ndash;600 cm⁻\u0026sup1; range, corresponding to metal-nitrogen bonds, were observed in the FTIR data, consistent with previously reported values in the literature [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. These findings demonstrate that the FTIR analysis of the synthesized metal complexes aligns well with literature-reported structures of similar compounds.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eMass Spectroscopy Analysis\u003c/h2\u003e \u003cp\u003eMass spectroscopy analysis of the imine-metal complexes revealed that the molecular ion peaks for the imine-copper (4) and imine-zinc metal complexes (5) were observed at \u003cb\u003e542 m/z\u003c/b\u003e (S1-S2). The predicted molecular masses of the complexes, calculated using ChemBioDraw software, were also 542 m/z. This agreement between the experimental and theoretical values confirms the structures of the synthesized imine-metal complexes.\u003c/p\u003e \u003cp\u003eThe mass spectrometry analysis also provided detailed information about the composition of the imine-metal complexes, further supporting that the metal ions are bound to the imine structure. This evidence validates the successful synthesis and structural integrity of the complexes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eXRD Analysis\u003c/h2\u003e \u003cp\u003eThe XRD patterns provided clear evidence of the formation of crystalline structures in the synthesized imine-metal complexes (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e.). The XRD graph of the imine-zinc complex (5) displayed distinct and sharp peaks, indicating the successful coordination of imine groups with zinc ions to form a well-ordered crystalline structure. These sharp peaks are characteristic of high crystallinity and confirm the structural regularity of the complex. In contrast, the XRD graph of the imine-copper complex (4) showed a different peak distribution, with lower intensity and broader peaks than the zinc complex. This suggests the copper complex has a less regular crystalline structure or exhibits amorphous characteristics. While some peak positions overlapped with those of the zinc complex, the overall pattern was less defined, reflecting a different or less ordered phase structure. These results highlight the significant variation in crystallinity and structural organization between the complexes, influenced by the specific metal ion used.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe lattice method was used to index the main peaks of the X-ray diffraction pattern [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. The parameters were calculated according to the Scherrer equation [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e presents the crystal size, hkl indices, and 2-theta maxima.\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\u003eThe XRD peaks and crystal thickness were calculated.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\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 \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCompound\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2-theta maximum\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ehkl indices\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ecrystal size\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eImıne (3) Ligand\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e11.00724\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e16.39094856\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCompound (4) Copper\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e14.13604\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.947813954\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCompound (5) Zinc\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e34.10437\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e220\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e61.6247131\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=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eCoordination Chemistry Insights\u003c/h2\u003e \u003cp\u003eThe formation of imine-metal complexes occurs through the coordination of the imine group, typically the azomethine (-C\u0026thinsp;=\u0026thinsp;N-) moiety, with metal ions. This coordination is driven by the interaction between the imine group's electron-donating properties and the metal ions' electron-accepting nature [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. The observed differences in crystallinity and structural order can be attributed to variations in the electronic and geometric properties of the zinc and copper ions. These factors influence the coordination environment and packing arrangement in the crystal lattice. The XRD analysis validates the successful formation of imine-metal complexes and demonstrates the structural diversity resulting from interactions with different metal ions. This underscores the versatility of imine groups as ligands in coordination chemistry, enabling the formation of complexes with varying crystalline properties [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eAntimicrobial Activity Results\u003c/h2\u003e \u003cp\u003eThe antimicrobial activity of the synthesized imine-copper and imine-zinc metal complexes was evaluated against a range of pathogens, including \u003cb\u003eGram-negative bacteria\u003c/b\u003e: \u003cem\u003eA. baumannii\u003c/em\u003e and \u003cem\u003eE. coli\u003c/em\u003e \u003cb\u003eGram-positive bacteria\u003c/b\u003e: Methicillin-resistant \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (MRSA) and \u003cem\u003eEnterococcus faecalis\u003c/em\u003e \u003cb\u003eYeasts\u003c/b\u003e: \u003cem\u003eCandida albicans\u003c/em\u003e and \u003cem\u003eCandida dubliniensis\u003c/em\u003e. Disc diffusion tests were conducted successfully to determine the antimicrobial efficacy (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). No zone of inhibition was observed around the discs impregnated with the complexes, indicating that the synthesized compounds lack antimicrobial activity against the tested pathogens. The disc diffusion data in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e confirms the absence of antimicrobial activity. Despite the promising structural properties of imine-metal complexes, the synthesized compounds did not demonstrate significant antimicrobial potential in this study. Further modifications to the structure or testing against additional strains may provide insights into their potential biological activity in other contexts.\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\u003eDisc diffusion results of imine-metal 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\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eMicrobial Strains\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eImine-Metal Complex\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCompound (4)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCompound (5)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eA. baumannii ATCC BAA- 1605\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMRSA\u003c/b\u003e \u003cb\u003eATCC 43300\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eE. coli ATCC BAA-2523\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eE. faecalis ATCC 49452\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eC. albicans ATCC 10231\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eC. tropicalis K\u0026Uuml;EN 1025\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\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\u003eImine-metal complexes, particularly those involving copper and zinc, have been widely studied for their antimicrobial potential. While some studies highlight promising activity, others indicate limited or no efficacy, highly dependent on the ligand structure and metal coordination. Studies have reported that imine-copper metal complexes exhibit significant antimicrobial activity against Gram-positive and Gram-negative bacteria and fungi due to their structural properties [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. However, antimicrobial efficacy can vary significantly based on the structural features of the complex. Some imine-copper complexes demonstrate no activity, indicating that not all synthesized complexes are effective [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Imine-zinc complexes have garnered attention for their potential antimicrobial activity, often showing enhanced efficacy compared to their free ligands due to synergistic effects [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Nonetheless, not all imine-zinc complexes exhibit antimicrobial properties, highlighting the variability in activity based on ligand structure and metal coordination [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. In the current study, synthesized imine-copper (4) and imine-zinc (5) complexes exhibited no antimicrobial activity against tested bacterial strains, including \u003cem\u003eA. baumannii\u003c/em\u003e, \u003cem\u003eE. coli\u003c/em\u003e, MRSA, \u003cem\u003eE. faecalis\u003c/em\u003e, and fungal strains like \u003cem\u003eC. albicans\u003c/em\u003e and \u003cem\u003eC. dubliniensis\u003c/em\u003e. This result aligns with reports that not all imine-metal complexes possess antimicrobial properties and underscores the structural specificity of their activity. While specific imine-metal complexes have demonstrated antifungal properties [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e], others have shown no such activity [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The absence of antifungal activity in the complexes synthesized here aligns with studies indicating that antifungal efficacy depends heavily on structural and coordination features [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. The findings suggest that while imine-metal complexes have potential antimicrobial applications, their activity is not universal and depends on specific structural and coordination factors. This variability highlights the need for further exploration and optimization to improve their efficacy against resistant pathogens.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eAntioxidant Activity\u003c/h2\u003e \u003cp\u003eThe study investigated the antioxidant activities of imine-copper (4) and imine-zinc metal complexes using the ABTS and CUPRAC methods. As a result of these studies, it was determined that the metal complexes exhibited no antioxidant activity compared to the positive controls, ascorbic acid and trolox (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn the literature, the antioxidant activity of imine metal complexes, particularly those containing transition metals, is reported to be significant due to their potential therapeutic applications. Studies have shown that these complexes exhibit enhanced antioxidant properties compared to their free ligands [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Additionally, other studies have reported that metal complexes demonstrate dose-dependent antioxidant activities, often surpassing the activities of free ligands. However, they are generally less effective than standard antioxidants such as ascorbic acid [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. The literature also comprehensively investigates imine copper-zinc metal complexes' antioxidant and non-antioxidant activities, revealing a complex interaction between their chemical structures and biological effects. These complexes, especially those containing copper(II) and zinc(II), exhibit significant antioxidant properties, frequently outperforming their free ligand counterparts but generally falling short of the effectiveness of standard antioxidants like ascorbic acid. Copper (II) and zinc (II) complexes have demonstrated noteworthy antioxidant activities in studies that show dose-dependent effects in scavenging free radicals such as DPPH and hydroxyl radicals [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. However, in the current study, metal complexes (4\u0026ndash;5) showed no antioxidant activity compared to ascorbic acid and trolox.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eEffects of Imine-Metal Complexes on Cell Viability: Analysis of WST-8 and SRB Assays\u003c/h2\u003e \u003cp\u003eThis study investigated the effects of imine-metal complexes on A549 and HDF cell lines using WST-8 and SRB assays. The WST-8 assay revealed that the imine-metal complexes exhibited dose-dependent anticancer activity in the A549 cell line, with an IC50 value calculated to be 43.65\u0026ndash;99.36 \u0026micro;g/ml (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e). Additionally, the imine-metal complexes demonstrated dose-dependent cytotoxic effects in the HDF cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe SRB assay detects cellular total proteins based on cell viability. The cellular total proteins at the IC50 concentration of the imine-metal complexes were examined for the SRB assay. As a result of this test, the total protein content in the A549 cells treated with the imine-copper metal complex was calculated to be 28%, and for the HDF cells, it was 25%. For the A549 cells treated with the imine-zinc metal complex, the total protein content was calculated to be 39%, while for the HDF cells, it was 46% (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003e.).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe anticancer activity of imine metal complexes has garnered significant attention due to their unique properties and mechanisms of action. Recent studies emphasize that various imine metal complexes exhibit vigorous anticancer activity against cancer cell lines [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. The investigation of imine metal complexes, particularly in the context of A549 lung cancer cells, highlights their potential for anticancer applications [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Research has shown that the imine-copper metal complex can effectively target cancer cells and often outperforms traditional agents such as cisplatin [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Copper complexes, especially those complexed with imine ligands, can induce cell death through a novel mechanism called cuproptosis, which occurs via copper [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. The synthesis of imine-copper metal complexes has demonstrated promising results in targeting A549 lung cancer cells. In a study by Gul et al., 2020, it was reported that the imine-copper complex exhibited anticancer activity by influencing autophagy and apoptosis in the A549 cell line. The analysis conducted in this study revealed dose-dependent anticancer activity of the imine-copper complex in the A549 cancer cell line, consistent with the literature [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. However, the study also observed dose-dependent cytotoxic effects of the synthesized imine-copper complexes in the HDF healthy cell line. It is known in the literature that imine-copper complexes enhance cytotoxicity through their ability to bind to DNA. Diimine-copper complexes have shown significant cytotoxic effects against various cancer cell lines due to their strong DNA-binding capabilities [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. The imine-copper complex synthesized in this study is characterized by a diimine structure in its chemical composition.\u003c/p\u003e \u003cp\u003eThe anticancer activity of imine and zinc metal complexes has attracted considerable attention due to their potential efficacy and reduced side effects compared to traditional treatments. Studies suggest these complexes can interact with DNA and exhibit cytotoxic effects against various cancer cell lines. Zinc (II) diimine complexes have demonstrated promising anticancer properties by increasing their ability to interact with DNA, enhancing their cytotoxicity against cancer cells [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Specifically, complexes formed with cobalt zinc and imine ligands have shown significant DNA-binding affinity, which is crucial for their anticancer activities [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Zinc complexes have been found to perform better than traditional platinum-based drugs in selectivity and efficacy, indicating their potential as safer alternatives [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. The analysis conducted in this study revealed dose-dependent anticancer activity of the imine-zinc metal complex in the A549 cancer cell line, consistent with the literature. However, the HDF healthy cell line also observed dose-dependent cytotoxic effects.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments:\u003c/strong\u003e This study was supported by TUBITAK-2209-A University Students Research Projects Support Program (2022/02). We want to thank TUBITAK for their financial support and research conditions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e: TYB, MY, and EA performed the analyses and wrote the first draft of the manuscript. AG and BO supervised the study and interpreted the results. All authors contributed to the revision of the manuscript and have read and approved the submitted version.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statements:\u003c/strong\u003e All data generated or analyzed during this study are included in this published article\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest: \u003c/strong\u003eThe authors declare no conflicts of interest\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eHrichi H, Elkanzi N, Bakr R. Novel Β-lactams and Thiazolidinone Derivatives from 1,4-dihydroquinoxaline Schiff\u0026rsquo;s Base: Synthesis, Antimicrobial Activity and Molecular Docking Studies. 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Inorganic Chemistry Communications. 2022;146:110190. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.inoche.2022.110190\u003c/span\u003e\u003cspan address=\"10.1016/j.inoche.2022.110190\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"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":"Imine, Copper, Zinc, metal complex, antimicrobial activity, anticancer activity, antioxidant activity","lastPublishedDoi":"10.21203/rs.3.rs-5767315/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5767315/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eImine is a bioactive molecule formed by the reaction of primary amine with aldehyde or ketone. Imines can form stable complexes with metals due to a C\u0026thinsp;=\u0026thinsp;N group in their structures. These complexes have antibacterial, antifungal, anticancer, and antioxidant properties. Based on the literature data, this study synthesized substituted phenethylamine-based imine compounds copper (Cu) and zinc (Zn) metal complexes. The synthesized imine-metal complexes' antimicrobial, anticancer, and antioxidant activities were evaluated. The antimicrobial activity of the metal complexes was tested against pathogens using the disk diffusion method. No antimicrobial activity was observed for the metal complexes. The anticancer activity of the metal complexes was investigated on lung cancer cell line (A549) and healthy dermal fibroblast cell line (HDF) using WST-8 and SRB assay methods. The results revealed dose-dependent anticancer activity of the metal complexes in the A549 cell line, with IC50 values ​​ranging from 43.65 to 99.36 \u0026micro;g/mL. Additionally, dose-dependent cytotoxic effects of the compounds were observed in HDF cells. The responses of the compounds to free radicals and oxidative stress were evaluated using ABTS and CUPRAC methods. However, no antioxidant activity was detected for the metal complexes. Based on these analyses, it is predicted that imine-metal complexes may be potential candidates as anticancer agents.\u003c/p\u003e","manuscriptTitle":"Investigation of Antimicrobial, Antioxidant, and Anticancer Properties of Substituted Phenethylamine-Based Imine and Metal Complexes","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-01-08 11:57:35","doi":"10.21203/rs.3.rs-5767315/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":"e22a77b9-5371-4e2a-8459-d18b38dc0719","owner":[],"postedDate":"January 8th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-01-21T09:38:33+00:00","versionOfRecord":[],"versionCreatedAt":"2025-01-08 11:57:35","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5767315","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5767315","identity":"rs-5767315","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}