Metal complex compounds containing conjugated double six-membered rings, and preparation method and application thereof

Abstract

This invention belongs to the field of antibacterial material preparation technology, and discloses a class of metal coordination compounds containing conjugated double six-membered rings, their preparation methods, and applications. By coordinating transition metal ions with dicyandiamide, a series of coordination compounds with conjugated double six-membered ring structures are constructed. Controllable preparation from small molecules to polymers is achieved by introducing different linkers. Specifically, using N,N-dimethylformamide as a solvent, and dicyandiamide, metal chlorides, and linkers as raw materials, the reaction is carried out at 70°C in a specific ratio for 24 hours. After filtration, washing, and drying, small molecule materials and polymer materials are obtained, respectively. This preparation method is simple, uses widely available raw materials, and all materials exhibit high surface positive potentials and high antibacterial properties against *Escherichia coli* and *Staphylococcus aureus*.

Description

Technical Field

[0001] This invention belongs to the field of antibacterial materials, specifically relating to a class of metal coordination compounds containing conjugated double six-membered rings, their preparation methods, and applications. Background Technology

[0002] Microorganisms are widely distributed in Earth's ecosystems, permeating all aspects of human production and daily life, and are closely related to human health. Common clinical infections are often induced by strains of Gram-positive and Gram-negative bacteria, which can lead to various serious diseases. Antibiotics were once a key means of combating bacterial infections, but due to their irrational and excessive use, multidrug-resistant strains have emerged and spread rapidly. Furthermore, the long development cycle and high cost of new antibiotics have severely impacted the traditional antibiotic treatment system. Therefore, it is urgent to explore effective strategies to combat multidrug-resistant strains and prevent the spread of "superbugs." The research and application of antimicrobial materials has become an important direction for solving these problems. Therefore, there is an urgent need to develop novel antimicrobial materials, especially those that are less likely to induce bacterial resistance.

[0003] In response to the increasingly serious challenge of bacterial resistance, researchers have been exploring novel antibacterial strategies in recent years, with significant progress made, particularly in the fields of cationic polymers, metal complexes, and single-atom nanozymes. For example, Yu et al. reviewed cationic antimicrobial polymers (CAPs) such as quaternary ammonium salts, guanidine salts, and quaternary phosphonium salts, which utilize the electrostatic interaction between their positive charge and bacterial cell membranes to achieve highly efficient bactericidal activity, and have a lower risk of resistance compared to traditional antibiotics (Coordination Chemistry Reviews 447 (2021) 214128). Gong et al. constructed a supramolecular structure assembled from the cell-penetrating peptide octaarginine (R8) and the anionic surfactant SDS. The layered aggregates showed a bactericidal rate of 99.9% against Staphylococcus aureus and Escherichia coli at a concentration of 25 μg / mL, and significantly promoted wound healing in a mouse infection model (Advanced Materials 2025, 202411388). Zhang et al. prepared PSACNZs-N2-C material by controlling the Co-N coordination number in cobalt single-atom nanozymes, which achieved a bactericidal rate of 99.7% against Staphylococcus aureus at a concentration of 400 μg / mL (Acta Biomaterialia 164 (2023) 563–576). Zhao et al. developed guanidine-functionalized polyamino acid PArg 20Through a phage-like mechanism of "adsorption-penetration-destruction," it rapidly kills Candida albicans within 10 minutes, with a MIC value as low as 2-4 μg / mL, and significantly reduces fungal load in mouse corneal and systemic infection models (ACSNano 2025, 19(49), 41605-41622). Zigale et al. further designed Co-NC single-atom nanozymes with oxidase activity, which achieved a >99% kill rate against tetracycline-resistant bacteria by generating reactive oxygen species without added H2O2, and effectively promoted wound healing (ACS Appl. Mater. Interfaces 2026, 18, 3669-3683). Although the antibacterial effects of nanomaterials have been continuously improved through the continuous updating of various antibacterial strategies, the bactericidal effect of existing technologies is still unsatisfactory. Therefore, it is particularly important to find materials with low minimum antibacterial concentrations and antibacterial strategies with excellent bactericidal effects under short light exposure times.

[0004] The guanidino functional group, due to its unique properties, provides insights for designing novel antibacterial materials. Studies have shown that the positively charged guanidino group can form specific bidentate hydrogen bonds with negatively charged phosphate groups in the phospholipid layer of bacterial cell membranes, thereby achieving efficient targeted binding and physical membrane disruption, and exhibiting lower cytotoxicity compared to quaternary ammonium salts. Metal coordination compounds, due to their designable structures, diverse geometries, and rich physicochemical properties, show broad application prospects in catalysis, biomedicine, and other fields. By coordinating organic ligands with targeted membrane disruption capabilities with antibacterial metal ions, it is hoped that novel materials with both targeted recognition and multi-mechanism synergistic antibacterial activity can be constructed. However, how to design and synthesize metal coordination compounds with well-defined structures, good stability, and excellent antibacterial properties, and deeply understand the intrinsic relationship between their structure and antibacterial activity, remains a current research hotspot and challenge. Summary of the Invention

[0005] In response to the need to develop antibacterial materials with better performance in the context of existing technologies, this invention provides a class of metal coordination compounds containing conjugated double six-membered rings, their preparation methods, and applications. The preparation method is simple, uses widely available raw materials, and exhibits highly efficient antibacterial properties against Escherichia coli and Staphylococcus aureus.

[0006] To address the problems in the existing technology, the technical solution adopted by this invention is as follows:

[0007] A method for preparing a class of metal coordination compounds containing conjugated double six-membered rings includes the following steps:

[0008] S1, Dicyandiamide is dissolved in sufficient N,N-dimethylformamide to form a dicyandiamide solution;

[0009] S2, add the transition metal chloride to sufficient N,N-dimethylformamide, sonicate to form a transition metal ion solution, then add it dropwise to the dicyandiamide solution, add the linker under vigorous stirring, stir continuously at 70°C, add the reaction solution dropwise to a large amount of continuously stirred deionized water to precipitate the product, filter, and wash three times each with water and acetone.

[0010] S3, after the cleaned solid was dried overnight in a vacuum oven at 50°C, the metal coordination compound was collected.

[0011] The transition metal chloride is CuCl2, CoCl2, or ZnCl2; the linker is selected from n-hexanol or 1,6-hexanediol.

[0012] Preferably, when the linker is n-hexanol, the molar ratio of dicyandiamide, transition metal ions and n-hexanol is 2:1:2, and the resulting metal coordination compound is a small molecule material, denoted as M-DCHe.

[0013] Preferably, when the linker is 1,6-hexanediol, the molar ratio of dicyandiamide, transition metal ions and 1,6-hexanediol is 2:1:1, and the resulting metal coordination compound is a polymer material, denoted as M-DCHDO.

[0014] The metal coordination compound containing conjugated double six-membered rings prepared by any of the above preparation methods.

[0015] Preferably, the metal coordination compound containing conjugated double six-membered rings has a conjugated double six-membered ring core structural unit formed by the coordination of dicyandiamide and transition metal ions, with a Zeta potential > +30 mV, which can efficiently target and bind to negatively charged bacterial membranes and is stably dispersed in water. Moreover, the metal coordination compound containing conjugated double six-membered rings has a high surface positive charge.

[0016] The above-mentioned metal coordination compounds containing conjugated double six-membered rings are used in the preparation of antibacterial materials.

[0017] Preferably, the antibacterial activity includes antibacterial activity against one or a mixture of two of Escherichia coli or Staphylococcus aureus.

[0018] Preferably, under light-free conditions, the metal coordination compound exhibits excellent antibacterial properties against both Escherichia coli and Staphylococcus aureus, with a minimum inhibitory concentration in the range of 0.3-5 μg / mL.

[0019] Specifically, when the metal coordination compound containing a conjugated double six-membered ring is the polymer material Co-DCHDO, it exhibits excellent activity under light-free conditions, with a minimum inhibitory concentration (MIC) of 0.3 μg / mL. Under visible light irradiation at 490-500 nm, the antibacterial effect is significantly enhanced, with a MIC of 0.2 μg / mL.

[0020] An antibacterial material comprising the aforementioned metal coordination compound containing conjugated double six-membered rings.

[0021] Beneficial effects:

[0022] Improving biocompatibility and addressing the poor antibacterial performance of low-concentration materials have always been pain points in the field of antibacterial materials. This invention relates to a class of metal coordination compounds containing conjugated double six-membered rings, their preparation method, and applications. These metal coordination compounds possess advantages such as good chemical stability, ease of preparation, and environmental friendliness, effectively achieving highly efficient antibacterial activity against *Escherichia coli* and *Staphylococcus aureus*. Specific advantages are as follows:

[0023] The raw materials used in this invention are inexpensive, low-cost, and readily available. The one-pot reaction synthesis method makes preparation convenient and simple to operate. Controllable preparation from small molecules to polymers can be achieved by adjusting the linkers.

[0024] This invention relates to a class of metal coordination compounds containing conjugated double six-membered rings. The core structural unit of the conjugated double six-membered ring is formed by the coordination of dicyandiamide and transition metal ions. The zeta potential is > +30 mV, and all of them have high surface positive charge. They can efficiently target and bind to negatively charged bacterial membranes and are stably dispersed in water. They are novel metal coordination planar chelate coordination compound materials with high bactericidal activity.

[0025] This invention discloses a class of metal coordination compounds containing conjugated double six-membered rings that exhibit excellent antibacterial properties against *Escherichia coli* and *Staphylococcus aureus* under light-free conditions, with minimum inhibitory concentrations (MICs) ranging from 0.3 to 5 μg / mL. Among these, the cobalt complexes show particularly outstanding antibacterial activity; Co-DCHe achieves a 100% bactericidal rate against *Staphylococcus aureus* at a concentration of 2 μg / mL. Notably, Co-DCHDO not only demonstrates excellent activity under light-free conditions with a MIC of 0.3 μg / mL, but its antibacterial effect is significantly enhanced under visible light irradiation at 490-500 nm, reaching a MIC of 0.2 μg / mL. Furthermore, it exhibits good biocompatibility. Attached Figure Description

[0026] Figure 1 The images show the infrared spectra of three small molecule materials and their ligands.

[0027] Figure 2 The figures show the proton NMR spectra of three small molecule materials: (a) Cu-DCHe, (b) Co-DCHe, and (c) Zn-DCHe.

[0028] Figure 3 This is the electrospray ionization mass spectrum of Cu-DCHe.

[0029] Figure 4 Matrix-assisted laser desorption / ionization time-of-flight mass spectra of Co-DCHe.

[0030] Figure 5 This is the electrospray ionization mass spectrum of Zn-DCHe.

[0031] Figure 6 The X-ray photoelectron spectrum of Cu-DCHe includes the full spectrum of Cu-DCHe as well as the Cu 2p, C 1s, N 1s, and O 1s spectra.

[0032] Figure 7 The X-ray photoelectron spectrum of Co-DCHe includes the full spectrum of Co-DCHe as well as the spectra of Co 2p, C 1s, N 1s, and O 1s.

[0033] Figure 8 The X-ray photoelectron spectrum of Zn-DCHe includes the full spectrum of Zn-DCHe as well as the Zn 2p, C 1s, N 1s, and O 1s spectra.

[0034] Figure 9 The diagram shows the zeta potentials of three small molecule materials and their ligands.

[0035] Figure 10 The images show the infrared spectra of three polymer materials and their ligands.

[0036] Figure 11 The image shows the 1H NMR spectrum of the copper coordination polymer Cu-DCHDO.

[0037] Figure 12 This is a gel permeation chromatogram of Cu-DCHDO.

[0038] Figure 13 The X-ray absorption fine structure spectra of Cu-DCHDO are shown below, where (a) is the K-edge absorption spectrum of Cu, (b) is the R-space spectrum of Cu, and (c) is the wavelet transform k-side spectrum of Cu foil, Cu-DCHDO, Cu₂O, and CuO. 3 Weighted extended X-ray absorption fine structure spectrum.

[0039] Figure 14 The X-ray photoelectron spectrum of Cu-DCHDO includes the full spectrum of Cu-DCHDO as well as the Cu 2p, C 1s, N 1s, and O 1s spectra.

[0040] Figure 15 The images show scanning electron microscope (SEM) images of Cu-DCHDO and the corresponding energy dispersive spectral (EDS) elemental distribution maps.

[0041] Figure 16 The diagram shows the zeta potentials of three polymer materials and their ligands.

[0042] Figure 17 The solid-state UV diffuse reflectance spectrum of the Co coordination polymer Co-DCHDO;

[0043] Figure 18 The effects of three small molecule materials (Cu-DCHe, Co-DCHe, and Zn-DCHe) at different concentrations (0, 1, 2, 3, and 4 μg / mL) on 1×10⁻⁶ ppm were investigated under light-free conditions. 6 Antibacterial properties of Escherichia coli at CFU / mL.

[0044] Figure 19 The effects of three small molecule materials (Cu-DCHe, Co-DCHe, and Zn-DCHe) at different concentrations (0, 1, 2, 3, and 4 μg / mL) on 1×10⁻⁶ ppm were investigated under light-free conditions. 6 Bar chart showing the bactericidal rate of CFU / mL Escherichia coli.

[0045] Figure 20 The effects of three small molecule materials (Cu-DCHe, Co-DCHe, and Zn-DCHe) at different concentrations (0, 1, 2, 3, and 4 μg / mL) on 1×10⁻⁶ ppm were investigated under light-free conditions. 6 Antibacterial properties of CFU / mL Staphylococcus aureus.

[0046] Figure 21 The effects of three small molecule materials (Cu-DCHe, Co-DCHe, and Zn-DCHe) at different concentrations (0, 1, 2, 3, and 4 μg / mL) on 1×10⁻⁶ ppm were investigated under light-free conditions. 6 Bar chart showing the bactericidal rate of CFU / mL Staphylococcus aureus.

[0047] Figure 22 The effects of three polymer materials (Cu-DCHDO, Co-DCHDO, and Zn-DCHDO) at different concentrations (0, 0.5, 1, and 1.5 μg / mL) on 1×10⁻⁶ μg / mL were investigated under light-free conditions. 6 Antibacterial properties of Escherichia coli at CFU / mL.

[0048] Figure 23 The effects of three polymer materials (Cu-DCHDO, Co-DCHDO, and Zn-DCHDO) at different concentrations (0, 0.5, 1, and 1.5 μg / mL) on 1×10⁻⁶ μg / mL were investigated under light-free conditions. 6 Bar chart showing the bactericidal rate of CFU / mL Escherichia coli.

[0049] Figure 24Three polymer materials (Cu-DCHDO, Co-DCHDO, and Zn-DCHDO) at different concentrations (0, 0.5, 1, and 1.5 μg / mL) were compared with the commercial antibacterial agent polyhexamethylene biguanide hydrochloride (PHMB) under light-free conditions to inhibit the growth of 1×10⁻⁶ micrograms of bacteria. 6 Antibacterial properties of CFU / mL Staphylococcus aureus.

[0050] Figure 25 Three polymer materials (Cu-DCHDO, Co-DCHDO, and Zn-DCHDO) at different concentrations (0, 0.5, 1, and 1.5 μg / mL) were compared with the commercial antibacterial agent polyhexamethylene biguanide hydrochloride (PHMB) under light-free conditions to inhibit the growth of 1×10⁻⁶ micrograms of bacteria. 6 Bar chart showing the bactericidal rate of CFU / mL Staphylococcus aureus.

[0051] Figure 26 Different concentrations (0, 0.1, 0.2, 0.3, 0.4, 0.5 μg / mL) of the polymer Co-DCHDO and the commercial antibacterial agent polyhexamethylene biguanide hydrochloride (PHMB) were compared under visible light irradiation at 490-500 nm to investigate the effect of 1×10⁻⁶ μg / mL on the antibacterial activity of 1×10⁻⁶ μg / mL. 6 Antibacterial properties of CFU / mL Staphylococcus aureus.

[0052] Figure 27 Different concentrations (0, 0.1, 0.2, 0.3, 0.4, 0.5 μg / mL) of the polymer Co-DCHDO and the commercial antibacterial agent polyhexamethylene biguanide hydrochloride (PHMB) were compared under visible light irradiation at 490-500 nm to investigate the effect of 1×10⁻⁶ μg / mL on the antibacterial activity of 1×10⁻⁶ μg / mL. 6 Bar chart showing the bactericidal rate of CFU / mL Staphylococcus aureus.

[0053] Figure 28 CCK-8 cytotoxicity assays of M-DCHe at concentrations of (a) 5 µg / mL and (b) 25 µg / mL.

[0054] Figure 29 CCK-8 cytotoxicity assays of M-DCHDO at concentrations of (a) 2 µg / mL and (b) 10 µg / mL. Detailed Implementation

[0055] The present invention reacts a metal salt, dicyandiamide and a linker (n-hexanol or 1,6-hexanediol) in N,N-dimethylformamide to obtain the six metal coordination compounds containing conjugated double six-membered rings.

[0056] The raw materials used in this invention are all commercially available products.

[0057] Example 1: Synthesis of Cu-DCHe

[0058] First, 20 mmol of dicyandiamide was dissolved in 20 mL of N,N-dimethylformamide to form a dicyandiamide solution. Then, 10 mmol of CuCl2 was added to sufficient N,N-dimethylformamide, and the solution was ultrasonically homogenized to form CuCl2. 2+ Solution. Then, Cu 2+ The solution was added dropwise to a dicyandiamide solution, followed by the addition of 20 mmol of n-hexanol under vigorous stirring at 70°C. The reaction mixture was then added dropwise to a large volume of continuously stirred deionized water to precipitate the product. The precipitate was filtered, washed three times each with water and acetone, and then dried overnight in a vacuum oven at 50°C. The final dried product was named Cu-DCHe.

[0059] Example 2 Synthesis of Co-DCHe

[0060] First, 20 mmol of dicyandiamide was dissolved in 20 mL of N,N-dimethylformamide to form a dicyandiamide solution. Then, 10 mmol of CoCl2 was added to sufficient N,N-dimethylformamide, and the solution was sonicated to form CoCl2. 2+ Solution. Then, Co 2+ The solution was added dropwise to a dicyandiamide solution, followed by the addition of 20 mmol of n-hexanol under vigorous stirring at 70°C. The reaction mixture was then added dropwise to a large volume of continuously stirred deionized water to precipitate the product. The precipitate was filtered, washed three times each with water and acetone, and then dried overnight in a vacuum oven at 50°C. The final dried product was named Co-DCHe.

[0061] Example 3 Synthesis of Zn-DCHe

[0062] First, 20 mmol of dicyandiamide was dissolved in 20 mL of N,N-dimethylformamide to form a dicyandiamide solution. Then, 10 mmol of ZnCl2 was added to sufficient N,N-dimethylformamide, and the solution was sonicated to form ZnCl2. 2+ Solution. Then, add Zn 2+ The solution was added dropwise to a dicyandiamide solution, followed by the addition of 20 mmol of n-hexanol under vigorous stirring at 70°C. The reaction mixture was then added dropwise to a large volume of continuously stirred deionized water to precipitate the product. The precipitate was filtered, washed three times each with water and acetone, and then dried overnight in a vacuum oven at 50°C. The final dried product was named Zn-DCHe.

[0063] Example 4 Synthesis of Cu-DCHDO

[0064] First, 20 mmol of dicyandiamide was dissolved in 20 mL of N,N-dimethylformamide to form a dicyandiamide solution. Then, 10 mmol of CuCl2 was added to sufficient N,N-dimethylformamide, and the solution was ultrasonically homogenized to form CuCl2. 2+ Solution. Then, Cu 2+ The solution was added dropwise to a dicyandiamide solution, followed by the addition of 10 mmol of 1,6-hexanediol under vigorous stirring at 70°C. The reaction mixture was then added dropwise to a large volume of continuously stirred deionized water to precipitate the product. The precipitate was filtered, washed three times each with water and acetone, and then dried overnight in a vacuum oven at 50°C. The final dried product was named Cu-DCHDO.

[0065] Example 5 Synthesis of Co-DCHDO

[0066] First, 20 mmol of dicyandiamide was dissolved in 20 mL of N,N-dimethylformamide to form a dicyandiamide solution. Then, 10 mmol of CoCl2 was added to sufficient N,N-dimethylformamide, and the solution was sonicated to form CoCl2. 2+ Solution. Then, Co 2+ The solution was added dropwise to a dicyandiamide solution, followed by the addition of 10 mmol of 1,6-hexanediol under vigorous stirring at 70°C. The reaction mixture was then added dropwise to a large volume of continuously stirred deionized water to precipitate the product. The precipitate was filtered, washed three times each with water and acetone, and then dried overnight in a vacuum oven at 50°C. The dried product was then collected. The final product was named Co-DCHDO.

[0067] Example 6 Synthesis of Zn-DCHDO

[0068] First, 20 mmol of dicyandiamide was dissolved in 20 mL of N,N-dimethylformamide to form a dicyandiamide solution. Then, 10 mmol of ZnCl2 was added to sufficient N,N-dimethylformamide, and the solution was sonicated to form ZnCl2. 2+ Solution. Then, add Zn 2+ The solution was added dropwise to a dicyandiamide solution, followed by the addition of 10 mmol of 1,6-hexanediol under vigorous stirring at 70°C. The reaction mixture was then added dropwise to a large volume of continuously stirred deionized water to precipitate the product. The precipitate was filtered, washed three times each with water and acetone, and then dried overnight in a vacuum oven at 50°C. The final dried product was named Zn-DCHDO.

[0069] Figure 1The infrared spectra of the three small molecule materials and their ligands clearly show that all three materials exhibit characteristic vibrational peaks of -NH-, -NH2 functional groups and CH bonds. The characteristic cyano (C≡N) peak unique to the ligand dicyandiamide completely disappears in the spectra of the three small molecule materials, while the characteristic vibrational peak of the COC bond appears. This phenomenon confirms that a nucleophilic addition reaction occurred between n-hexanol and the ligand. In addition, the characteristic band of the imino group (C=NH) shifts to the high-frequency direction after the coordination reaction, indicating that the N atom in the ligand participates in the coordination process.

[0070] Figure 2 The images show the 1H NMR spectra of three small molecule materials. Characteristic peaks corresponding to the -NH2, -NH-, and CO-CH functional groups appeared in the spectra of all three materials, and the integral area ratio of each characteristic peak was 2:1:2. Characteristic peaks corresponding to alkyl chains could also be observed. These characteristics are in perfect agreement with the geometric configuration of the materials, further confirming the correctness of the configuration.

[0071] Figure 3 , Figure 4 and Figure 5 The mass spectra of three small molecule materials, Cu-DCHe, Co-DCHe, and Zn-DCHe, show high-intensity characteristic signal peaks. The mass-to-charge ratio of each characteristic peak is consistent with the theoretical mass-to-charge ratio of the three small molecule materials, confirming the successful generation of the target materials.

[0072] Figure 6 , Figure 7 and Figure 8 The X-ray photoelectron spectra of three small molecule materials, Cu-DCHe, Co-DCHe, and Zn-DCHe, are shown. Spectral analysis reveals the elemental composition, oxidation state of the metal ions, and coordination environment of the atoms in these three materials, comprehensively verifying the accuracy of their molecular structures.

[0073] Figure 9 The Zeta potential diagrams of the three small molecule materials and their ligands show that all three materials exhibit a high surface positive charge (>+30 mV). This characteristic enables them to efficiently target and bind to the negatively charged bacterial cell membrane, providing a structural basis for their antibacterial properties.

[0074] Based on the combined results of infrared spectroscopy, proton nuclear magnetic resonance spectroscopy, mass spectrometry, and X-ray photoelectron spectroscopy, it can be fully demonstrated that the three chelating coordination small molecule materials have been successfully prepared.

[0075] Figure 10 The infrared spectra of three polymer materials and their ligands are shown. The characteristic cyano (C≡N) peak unique to the dicyandiamide ligand in the polymer materials completely disappears, while the peak at ~1675 cm⁻¹ disappears.-1 The presence of a C=NH conjugate absorption peak, which is slightly shifted compared to the ligand dicyandiamide, confirms the successful formation of the same core coordination unit as the small molecule in the polymer.

[0076] Figure 11 The image shows the 1H NMR spectrum of Cu-DCHDO, a copper-coordinated polymer among the three polymer materials. It can be seen that the spectrum of Cu-DCHDO shows characteristic peaks consistent with those of the aforementioned small molecule materials, corresponding to the -NH2, -NH- and CO-CH functional groups, respectively. The integral area ratio of each characteristic peak is 2:1:2. At the same time, characteristic peaks corresponding to alkyl chains can be observed, confirming that Cu-DCHDO has a similar geometric configuration to the small molecule materials.

[0077] The gel permeation chromatography results of Cu-DCHDO are as follows: Figure 12 As shown in the figure, analysis reveals that the number-average molecular weight Mn of Cu-DCHDO is 2.5 × 10⁻⁶. 3 The polydispersity index (PDI) was 1.364 g / mol, indicating that Cu-DCHDO had been successfully polymerized and the polymer had good dispersibility.

[0078] Figure 13 The fine structure X-ray absorption spectrum of Cu-DCHDO; Figure 14 The X-ray photoelectron spectrum of Cu-DCHDO; combined with Figures 13-14 The information indicates that Cu in Cu-DCHDO exhibits a mixed valence state, where Cu... 2+ With Cu + The ratio was 10:1; subsequently, the coordination of Cu in Cu-DCHDO was further analyzed by extended X-ray absorption fine structure spectroscopy, and the atomic-level dispersion state of the metal was preliminarily verified. In the Cu element R-space spectrum of Cu-DCHDO, the main peak of Cu at 1.51 Å is significantly different from the Cu-Cu peak at 2.23 Å ​​in Cu foil, which further rules out the presence of Cu clusters or nanoparticles in Cu-DCHDO. Although this characteristic peak is closer to the Cu-O coordination at 1.56 Å in CuO, according to the R-space characteristic peak information of Cu-N coordination in the reported literature, the R-space characteristic peak positions of Cu-N bond and Cu-O coordination are almost coincident. Furthermore, since dicyandiamide itself does not contain O element in its structure, this characteristic peak should be attributed to Cu-N coordination bond. Moreover, through wavelet transform k... 3 Weighted extended X-ray absorption fine structure spectroscopy further confirmed the coordination of Cu atoms.

[0079] Figure 15The image shows a scanning electron microscope (SEM) image of Cu-DCHDO. It can be clearly observed that Cu-DCHDO exhibits an irregular blocky morphology, and the energy-dispersive X-ray spectroscopy mapping image of the SEM shows a uniform distribution of C, N, Cl, and Cu elements, proving the uniformity of Cu-DCHDO preparation.

[0080] Figure 16 The diagram shows the zeta potentials of three polymer materials and their ligands. The surface potentials of all three polymer materials are greater than +40 mV, and they all have higher surface positive charge than smaller molecular materials. Among them, Cu-DCHDO has a surface potential as high as +59.5 mV, indicating that it has a very strong ability to target and bind to negatively charged bacterial cell membranes.

[0081] Figure 17 The image shows the solid-state UV diffuse reflectance spectrum of Co-DCHDO. It can be seen that Co-DCHDO exhibits a characteristic absorption peak in the wavelength range of 490~510nm. This characteristic indicates that Co-DCHDO has photoresponsive potential, providing a theoretical basis for its subsequent photocatalytic or photoantibacterial performance research.

[0082] Based on the same synthesis method and similar characterization data, it can be reasonably inferred that Co-DCHDO and Zn-DCHDO have the same coordination core structure and similar polymer backbone structure as Cu-DCHDO.

[0083] Example 7: Six metal coordination compound materials containing conjugated double six-membered rings with 1×10⁻⁶ ohms 6 Antibacterial test of Escherichia coli and Staphylococcus aureus at CFU / mL concentration

[0084] The *Escherichia coli* and *Staphylococcus aureus* used in this invention are from Wenzhou Weiqiong Microbial Technology Co., Ltd., with *Escherichia coli* catalog number HC-16007 and strain number CMCC(B)44102; and *Staphylococcus aureus* catalog number HC-16005 and strain number CMCC(B)26003.

[0085] This invention uses a concentration of 1×10⁻⁶ in 1 mL. 6 Different concentrations of small molecules (Cu-DCHe, Co-DCHe, Zn-DCHe) or polymers (Cu-DCHDO, Co-DCHDO, Zn-DCHDO) were added to CFU / mL *E. coli* / *Staphylococcus aureus* bacterial cultures, followed by co-culturing at 37°C in air atmosphere for 24 hours. For photocatalytic antibacterial experiments, a 490-500 nm LED light source (50 mW / cm²) was used during co-culturing. 2 Irradiate for 30 minutes.

[0086] Both the bacterial diluent and the material dispersion solution were sterile PBS solutions; the co-culture time with the bacteria was 24 hours.

[0087] The formula for calculating the sterilization rate is as follows:

[0088]

[0089] Among them, the colony count refers to the number of bacterial colonies on the TSA medium after the culture is completed.

[0090] (1) Take 0.1 mL of resuscitated Escherichia coli or Staphylococcus aureus bacterial solution and measure the transmittance (OD) at a wavelength of 600 nm. 600 =1), resulting in a bacterial concentration of 1×10⁻⁶. 9 CFU / mL, take a certain amount of bacterial suspension and mix it with sterile PBS solution to reduce the bacterial concentration to 1×10⁻⁶. 8 CFU / mL.

[0091] (2) Weigh the antibacterial material and add it to sterile PBS solution to prepare a suspension with a concentration of 1 mg / mL.

[0092] (3) Select sterile 24-well plates and perform triple replicates for each material concentration to eliminate errors. For example, for a material concentration of 1 μg / mL, first add 0.8 mL of PBS solution to each well, then add 0.1 mL of the material suspension, and finally add 0.1 mL of diluted bacterial solution. Ensure that the total liquid volume in each well is 1 mL. At this point, the number of bacteria in each well is 1 × 10⁻⁶. 6 CFU / mL. For photocatalytic antibacterial experiments, a 490-500 nm LED light source (50 mW / cm²) was used during co-culture. 2 Irradiate for 30 minutes. After spotting, place the well plate in a 37°C incubator with air atmosphere to co-culture the bacteria and antibacterial material for 24 hours. The control group for each concentration is the same concentration of bacterial solution without the addition of this antibacterial material.

[0093] (4) Remove the well plate after co-culture. Dilute the solution concentration in the well plate to 1 / 10 of the original concentration, take 10 μL of the diluted liquid, drop it onto TSA medium, and spread it evenly with a spreader. Then invert the spread culture dish and place it in a 37°C incubator under air atmosphere, and wait for the bacteria to grow for 24 hours.

[0094] (5) Take out the petri dish, take pictures of the growth of the colonies, count the number of colonies, and calculate the sterilization rate.

[0095] Figure 18The effects of three small molecule materials (Cu-DCHe, Co-DCHe, and Zn-DCHe) at different concentrations (0, 1, 2, 3, and 4 μg / mL) on 1×10⁻⁶ ppm were investigated under light-free conditions. 6 The antibacterial properties of CFU / mL Escherichia coli were evaluated, with the control group consisting of an equal volume of sterile PBS solution without the added material. Figure 19 The effects of three small molecule materials (Cu-DCHe, Co-DCHe, and Zn-DCHe) at different concentrations (0, 1, 2, 3, and 4 μg / mL) on 1×10⁻⁶ ppm were investigated under light-free conditions. 6 Bar chart showing the bactericidal rate of Escherichia coli at CFU / mL; Figure 20 The effects of three small molecule materials (Cu-DCHe, Co-DCHe, and Zn-DCHe) at different concentrations (0, 1, 2, 3, and 4 μg / mL) on 1×10⁻⁶ ppm were investigated under light-free conditions. 6 The antibacterial properties of CFU / mL Staphylococcus aureus were evaluated, with the control group consisting of an equal volume of sterile PBS solution without the added material. Figure 21 The effects of three small molecule materials (Cu-DCHe, Co-DCHe, and Zn-DCHe) at different concentrations (0, 1, 2, 3, and 4 μg / mL) on 1×10⁻⁶ ppm were investigated under light-free conditions. 6 Bar chart showing the bactericidal rate of CFU / mL Staphylococcus aureus.

[0096] Figure 18 The study demonstrated the efficacy of three small-molecule antibacterial materials (Cu-DCHe, Co-DCHe, and Zn-DCHe) at different concentrations (0, 1, 2, 3, and 4 μg / mL) against 1×10⁻⁶ micrograms of bacteria under light-free conditions. 6 Plate plot showing the antibacterial properties of Escherichia coli at a concentration of CFU / mL. Figure 19 The bar charts showing the corresponding bactericidal rates reveal that all three small molecule materials exhibited concentration-dependent antibacterial activity; that is, as the concentration increased, the number of colonies on the plate decreased significantly, and the bactericidal rate gradually increased. Zn-DCHe showed the best antibacterial activity, with a bactericidal rate approaching 100% at a concentration of 4 μg / mL and a minimum inhibitory concentration (MIC) of 3 μg / mL. Cu-DCHe and Co-DCHe also achieved bactericidal rates exceeding 90% at 4 μg / mL, with MICs of 5 μg / mL for both. Overall, Zn-DCHe showed better inhibitory effects against *Escherichia coli* than Cu-DCHe and Co-DCHe. Figure 20 , Figure 21 Three small molecule materials with the same concentration gradient under no light exposure were compared to 1×10⁻⁶. 6Data on the antibacterial properties and bactericidal rates of Staphylococcus aureus at CFU / mL concentrations showed that, compared to Escherichia coli, the three small molecules exhibited more significant inhibitory effects on Staphylococcus aureus. Cu-DCHe and Zn-DCHe achieved near-complete sterilization at a concentration of 3 μg / mL, with a sterilization rate of 100% and a MIC of 3 μg / mL. Co-DCHe achieved a sterilization rate of 100% at 2 μg / mL, with a MIC of 2 μg / mL. This indicates that the three small molecule materials have superior antibacterial activity against Gram-positive bacteria (Staphylococcus aureus) compared to Gram-negative bacteria (Escherichia coli).

[0097] The antibacterial effects of three polymer materials (Cu-DCHDO, Co-DCHDO, and Zn-DCHDO) against Escherichia coli and Staphylococcus aureus were further investigated, and the results are as follows: Figure 22-27 As shown. Specifically:

[0098] Figure 22 The effects of three polymer materials (Cu-DCHDO, Co-DCHDO, and Zn-DCHDO) at different concentrations (0, 0.5, 1, and 1.5 μg / mL) on 1×10⁻⁶ μg / mL were investigated under light-free conditions. 6 The antibacterial properties of CFU / mL Escherichia coli were evaluated, with the control group consisting of an equal volume of sterile PBS solution without the added material. Figure 23 The effects of three polymer materials (Cu-DCHDO, Co-DCHDO, and Zn-DCHDO) at different concentrations (0, 0.5, 1, and 1.5 μg / mL) on 1×10⁻⁶ μg / mL were investigated under light-free conditions. 6 Bar chart showing the bactericidal rate of Escherichia coli at CFU / mL; Figure 24 Three polymer materials (Cu-DCHDO, Co-DCHDO, and Zn-DCHDO) at different concentrations (0, 0.5, 1, and 1.5 μg / mL) were compared with the commercial antibacterial agent polyhexamethylene biguanide hydrochloride (PHMB) under light-free conditions to inhibit the growth of 1×10⁻⁶ micrograms of bacteria. 6 The antibacterial properties of CFU / mL Staphylococcus aureus were evaluated, with the control group consisting of an equal volume of sterile PBS solution without the added material. Figure 25 Three polymer materials (Cu-DCHDO, Co-DCHDO, and Zn-DCHDO) at different concentrations (0, 0.5, 1, and 1.5 μg / mL) were compared with the commercial antibacterial agent polyhexamethylene biguanide hydrochloride (PHMB) under light-free conditions to inhibit the growth of 1×10⁻⁶ micrograms of bacteria. 6 Bar chart showing the bactericidal rate of CFU / mL Staphylococcus aureus; Figure 26Different concentrations (0, 0.1, 0.2, 0.3, 0.4, 0.5 μg / mL) of the polymer Co-DCHDO and the commercial antibacterial agent polyhexamethylene biguanide hydrochloride (PHMB) were compared under visible light irradiation at 490-500 nm to investigate the effect of 1×10⁻⁶ μg / mL on the antibacterial activity of 1×10⁻⁶ μg / mL. 6 The antibacterial properties of CFU / mL Staphylococcus aureus were evaluated, with the control group consisting of an equal volume of sterile PBS solution without the added material. Figure 27 Different concentrations (0, 0.1, 0.2, 0.3, 0.4, 0.5 μg / mL) of the polymer Co-DCHDO and the commercial antibacterial agent polyhexamethylene biguanide hydrochloride (PHMB) were compared under visible light irradiation at 490-500 nm to investigate the effect of 1×10⁻⁶ μg / mL on the antibacterial activity of 1×10⁻⁶ μg / mL. 6 Bar chart showing the bactericidal rate of CFU / mL Staphylococcus aureus.

[0099] Figure 22 , Figure 23 The antibacterial properties and bactericidal rates of these materials against Escherichia coli at different concentrations (0, 0.5, 1, 1.5 μg / mL) and under no-light conditions were presented. Compared with the aforementioned small molecule materials, the polymer materials exhibited superior antibacterial activity. Among them, Zn-DCHDO achieved a bactericidal rate of 100% at 1.5 μg / mL with a MIC of 1.5 μg / mL, Co-DCHDO achieved a bactericidal rate of 96% at 1.5 μg / mL with a MIC of 1.5 μg / mL, and Cu-DCHDO achieved a bactericidal rate of approximately 65% ​​at 1.5 μg / mL with a MIC of 2 μg / mL. Figure 24 , Figure 25 The antibacterial properties of three polymer materials and the commercial antibacterial agent polyhexamethylene biguanide hydrochloride (PHMB) against Staphylococcus aureus were compared under no-light conditions. Notably, Co-DCHDO showed the most significant bactericidal effect against Staphylococcus aureus, achieving 100% sterilization at a concentration of 0.5 μg / mL, with a minimum inhibitory concentration (MIC) below 0.5 μg / mL. Its antibacterial efficacy is comparable to that of the commercial antibacterial agent PHMB. Based on... Figure 17 The solid-state UV diffuse reflectance spectroscopy results of Co-DCHDO show that Co-DCHDO has a characteristic absorption peak in the 490–510 nm range, indicating that it may have photocatalytic antibacterial potential. Therefore, further research was conducted using a 490–500 nm LED visible light source (50 mW / cm²). 2 Photocatalytic antibacterial experiment under irradiation ( Figure 26 , Figure 27Experimental results showed that light irradiation significantly enhanced the antibacterial effect of Co-DCHDO. At a concentration of 0.2 μg / mL, the bactericidal rate of the light-irradiated group reached 100%, and compared with the unirradiated group (MIC of 0.3 μg / mL), the MIC of the light-irradiated group was further reduced to 0.2 μg / mL, consistent with the minimum inhibitory concentration of polyhexamethylene biguanide (PHMB). This result confirms the highly efficient antibacterial activity of Co-DCHDO against Staphylococcus aureus, possessing antibacterial efficacy comparable to the commercial antibacterial agent PHMB. Overall, the antibacterial effect of polymer materials is superior to that of smaller molecular weight materials, with a significantly higher bactericidal rate at the same concentration, demonstrating the significant advantage of polymer materials in antibacterial efficacy.

[0100] Example 8: Biosafety Testing of Six Metal Coordination Compounds Containing Conjugated Bisix-Membered Rings

[0101] In addition to highly effective and broad-spectrum antibacterial properties, antibacterial materials must also possess good biocompatibility to ensure their effectiveness in practical biological applications. To evaluate the cytotoxicity of M-DCHe and M-DCHDO to somatic cells, primary mouse bone marrow mesenchymal stem cells (BMSCs) were used as experimental subjects, and changes in cell count were measured using a cell counting kit-8 (CCK-8) and an enzyme-linked immunosorbent assay (ELISA) reader. In the experiment, based on previously determined minimum in vitro antibacterial concentrations, concentrations of 5 μg / mL and 25 μg / mL were selected for the small molecule M-DCHe, and concentrations of 2 μg / mL and 10 μg / mL were selected for the polymeric M-DCHDO. Subsequently, after co-culturing with cells for 24 hours and 48 hours, neither M-DCHe nor M-DCHDO had any effect on cell growth, and their cytotoxicity was negligible.

[0102] Specifically, in this embodiment, 6-week-old male rats were selected, and bone marrow mesenchymal stem cells (BMSCs) were isolated from their femurs. The BMSCs were cultured in α-MEM medium (Gibco, USA) containing 1% penicillin-streptomycin and 10% fetal bovine serum (FBS) (complete medium). Passage 0 BMSCs were passaged to passage 2 for subsequent experiments. The cells were cultured at 2 × 10⁶ cells per well. 3Second-generation BMSCs were seeded in 96-well plates at a density of [number] cells per well and cultured in complete medium for 12 h. After 12 h, BMSCs were treated with small molecule material (M-DCHe) at concentrations of 5 μg / mL and 25 μg / mL, and with polymer (M-DCHDO) at concentrations of 2 μg / mL and 10 μg / mL, respectively. Wells without added materials served as the control group. The biocompatibility of the small molecule material M-DCHe and the polymer material M-DCHDO was assessed using a CCK-8 assay kit (Solarbio, China) at predetermined time points (24 h and 48 h). The test results are shown below. Figure 28 and Figure 29 As shown in the figure, after co-culturing with mouse primary bone marrow mesenchymal stem cells (BMSCs) for 24 and 48 hours, the cell viability of each material group was expressed as the optical density OD value at a wavelength of 450 nm (OD). 450 (This is indicated by the symbol). Compared with the control group, the OD of the M-DCHe and M-DCHDO treatment groups was significantly higher. 450 The values ​​showed no significant difference, indicating that none of them had a significant impact on cell growth, and their toxicity to cells was negligible.

[0103] In summary, this invention achieves highly efficient antibacterial activity against *Escherichia coli* and *Staphylococcus aureus* by preparing a class of metal coordination compounds (including small molecules and polymers) containing conjugated double six-membered rings. All compounds possess high surface positive charges, enabling them to exert potent bactericidal effects even at low concentrations. Co-DCHDO, in particular, exhibits antibacterial efficacy comparable to commercially available PHMB under visible light irradiation, with good biocompatibility. The materials of this invention solve the problem of poor antibacterial efficacy at low concentrations, providing a strong candidate for the development of novel antibacterial agents.

[0104] This invention provides a metal coordination compound material containing conjugated double six-membered rings that exhibits highly efficient antibacterial properties against Escherichia coli and Staphylococcus aureus, solving the problem of poor antibacterial effects against Escherichia coli and Staphylococcus aureus at low material concentrations.

Claims

1. A method for preparing a class of metal coordination compounds containing conjugated double six-membered rings, characterized in that, Includes the following steps, S1, Dicyandiamide is dissolved in sufficient N,N-dimethylformamide to form a dicyandiamide solution; S2, add the transition metal chloride to sufficient N,N-dimethylformamide, sonicate to form a transition metal ion solution, then add it dropwise to the dicyandiamide solution, add the linker under vigorous stirring, stir continuously at 70°C, add the reaction solution dropwise to a large amount of continuously stirred deionized water to precipitate the product, filter, and wash three times each with water and acetone. S3, after the cleaned solid was dried overnight in a vacuum oven at 50°C, the metal coordination compound was collected. The transition metal chloride is CuCl2, CoCl2, or ZnCl2; the linker is selected from n-hexanol or 1,6-hexanediol. When the linker is n-hexanol, the molar ratio of dicyandiamide, transition metal ions and n-hexanol is 2:1:2, and the resulting metal coordination compound is a small molecule material, denoted as M-DCHe. When the linker is 1,6-hexanediol, the molar ratio of dicyandiamide, transition metal ions and 1,6-hexanediol is 2:1:

1. The resulting metal coordination compound is a polymer material, denoted as M-DCHDO.

2. A metal coordination compound containing a conjugated double six-membered ring prepared by the preparation method according to any one of claims 1.

3. The metal coordination compound containing conjugated double six-membered rings according to claim 2, characterized in that, The metal coordination compound containing a conjugated double six-membered ring comprises a core structural unit of a conjugated double six-membered ring formed by the coordination of dicyandiamide and transition metal ions, with a Zeta potential > +30 mV. It can efficiently target and bind to negatively charged bacterial membranes, disperse stably in water, and all of the metal coordination compounds containing the conjugated double six-membered ring have high surface positive charge.

4. The application of the metal coordination compound containing conjugated double six-membered rings as described in claim 2 in the preparation of antibacterial materials.

5. The application according to claim 4, characterized in that, The antibacterial activity includes antibacterial activity against one or a combination of two of Escherichia coli or Staphylococcus aureus.

6. The application according to claim 5, characterized in that, Under light-free conditions, the metal coordination compound exhibits excellent antibacterial properties against both Escherichia coli and Staphylococcus aureus, with a minimum inhibitory concentration in the range of 0.3-5 μg / mL.

7. An antibacterial material, characterized in that, The metal coordination compound containing the conjugated double six-membered ring as described in claim 2.

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