Small-molecule antibacterial compounds with dual targeting of DNA and membranes and uses thereof

Abstract

The application discloses a small-molecule antibacterial compound with DNA and membrane double targeting, which has the following general structure: The series of molecules have the application potential as a novel double-targeting antibacterial agent with high efficiency and environmental protection due to the small molecular weight, good biocompatibility and easy production and the like.

Description

Technical Field

[0001] This invention relates to antibacterial compounds, and more particularly to a small molecule antibacterial compound with dual DNA and membrane targeting, its preparation method, and its application. Background Technology

[0002] The development of novel antimicrobial molecules is a crucial means of addressing the global antibiotic resistance crisis. Currently, most antibiotics used clinically work by binding to key bacterial proteins through a single-target approach to inhibit bacterial growth. This "single drug, single target" model is insufficient to address the evolution of antibiotic resistance caused by bacterial gene mutations, which is the root cause of the widespread prevalence of antibiotic resistance in clinical practice.

[0003] Compared to proteins, cell membranes and DNA, also important bacterial biomolecules, possess multi-target site structures, making them potential candidate targets for multi-target antibacterial drugs. Furthermore, cell membranes and DNA share certain structural similarities: both exhibit high overall negative charge and a degree of local hydrophobicity. Therefore, it is hoped that a single hydrophobic, positively charged molecule can simultaneously target multiple binding sites on both the bacterial cell membrane and DNA, thereby inhibiting the evolution of bacterial resistance through this "dual multi-target" mechanism.

[0004] In the study of simultaneous targeting of bacterial membrane and DNA, Liu Runhui's team reported a poly(2-oxazoline) in 2020. This poly(2-oxazoline) enters bacteria through membrane disruption and strongly interacts with DNA to generate reactive oxygen species (ROS), exhibiting good bactericidal activity against Staphylococcus aureus, including MRSA. This study demonstrates that poly(2-oxazoline), as a novel functional peptide mimic with multiple biological functions, has various application potentials. In 2021, the inventors' team reported a method to address the drug resistance crisis: introducing a dual-selective mechanism of action into an amidine-rich polymer. This polymer can simultaneously disrupt the bacterial membrane and bind to bacterial DNA, thus exhibiting a high therapeutic index against bacterial types resistant to multiple drugs (including colistin), such as ESKAPE strains and clinical isolates. These works fully demonstrate the potential of bacterial cell membranes and DNA as dual-target antibacterial agents, achieving certain results in alleviating bacterial resistance to antibiotics and improving biocompatibility, further advancing the research of novel dual-target antibacterial drugs. However, these research subjects are mainly high molecular weight oligomers, and they all have problems such as high toxicity and poor pharmacokinetic properties.

[0005] Compared to macromolecular drugs, small molecule drugs have certain advantages in terms of toxicity, pharmacokinetics, and pharmacokinetics. Although research on small molecule drugs that target bacterial cell membranes and DNA individually is progressing steadily, small molecule compounds that achieve dual targeting of these two targets have not yet been successfully developed. Summary of the Invention

[0006] To address the aforementioned problems, this invention discloses a small molecule antibacterial compound with dual DNA and membrane targeting, its preparation method, and its applications.

[0007] A small molecule antibacterial compound with dual DNA and membrane targeting has the following general structural formula:

[0008]

[0009] This compound is synthesized by liquid-phase synthesis, in which a diacetyl linker and an azide-containing cationic molecule undergo a 2+3 cyclization addition reaction in the presence of anhydrous copper sulfate and sodium ascorbate to synthesize the antibacterial molecule.

[0010] It can be used as a DNA inhibitor and has promising applications in the treatment of bacterial-mediated diseases (such as infections and antibiotic prophylaxis).

[0011] This invention discloses the design and application of an antibacterial molecule that targets both bacterial DNA and the cell membrane. Based on the high-throughput synthesis characteristics of copper-catalyzed click chemistry, a series of antibacterial molecules were designed. DNA and bacterial cell membranes share certain structural similarities, both exhibiting high overall negative charge and a degree of local hydrophobicity. By utilizing the hydrophobic and positively charged molecular structure of the compound, simultaneous targeting of both the bacterial cell membrane and DNA can be achieved.

[0012] The novel antibacterial molecules designed in this invention possess symmetrical structural features. Through copper-catalyzed click chemistry, they achieve modular combinations of hydrophobic and positively charged groups, enabling high-throughput synthesis. Because click chemistry offers high quantification and the catalysts and solvents do not interfere with subsequent bacterial and cellular activity assays, the molecular library synthesized using click chemistry can be directly applied to subsequent biological property screening without purification. In addition to screening for antibacterial activity, hemolytic toxicity, and cytotoxicity, this invention also investigated the in vivo toxicity of selected compounds in mice, further ensuring the biosafety of the selected compounds. Due to their small molecular weight, good biocompatibility, and ease of synthesis, this series of molecules has the potential to be used as a highly efficient and environmentally friendly novel dual-targeting antibacterial agent.

[0013] The beneficial effects of this invention are as follows: The antibacterial molecules of this invention exhibit excellent antibacterial activity. Molecular lipid interference experiments and scanning electron microscopy experiments have demonstrated that these molecules have good binding ability to bacterial cell membranes; DNA gel electrophoresis experiments have demonstrated their DNA binding ability. Preferred molecules exhibit low hemolytic toxicity, low mammalian cytotoxicity, and low acute toxicity in mice (compared to pentamidine, chlorhexidine, and polymyxin), and show good therapeutic effects in mouse epidermal wound models and tail vein infection blood flow models. Due to their small molecular weight, good biocompatibility, and ease of synthesis and production, this series of molecules has the potential to be used as a novel, highly efficient, and environmentally friendly dual-targeting antibacterial agent. Attached Figure Description

[0014] Figure 1 Synthetic steps and structure of dual-targeted antibacterial molecules

[0015] Figure 2 The NMR spectrum of XIX-57;

[0016] Figure 3 The NMR spectrum of XIX-54;

[0017] Figure 4 The NMR spectrum of I-54;

[0018] Figure 5 The NMR spectrum of XIV-57;

[0019] Figure 6 The NMR spectrum of XIV-54;

[0020] Figure 7 The NMR spectrum of XV-54;

[0021] Figure 8 The NMR spectrum of I-46;

[0022] Figure 9 The NMR spectrum of XI-54;

[0023] Figure 10 The NMR spectrum of I I-1;

[0024] Figure 11 Cytotoxicity test for compound XIX-57

[0025] Figure 12 Schematic diagram of mouse epidermal wound infection with antibacterial molecule XIX-57

[0026] Figure 13 MIC fold change of different compounds after the addition of PG / PE

[0027] Figure 14 SEM imaging of XIX-57

[0028] Figure 15 XIX-57 combined with its single component and other control compounds in DNA gel experiments. Detailed Implementation

[0029] The technical solution of the present invention will be specifically described below through specific embodiments and in conjunction with the accompanying drawings. Unless otherwise specified, the components or devices in the following embodiments are all general standard parts or components known to those skilled in the art, and their structures and principles can be learned by those skilled in the art through technical manuals or conventional experimental methods.

[0030] The first objective of this invention is to provide an antimicrobial molecule with dual DNA and membrane targeting capabilities, having the following general structural formula:

[0031]

[0032] Wherein the R1 group is any one of the following formulas.

[0033]

[0034] The R2 group is a group with an lipocationic structure, selected from any of the following formulas.

[0035]

[0036] The second objective of this invention is to provide a method for preparing the dual-targeting molecule, which employs a liquid-phase synthesis method. In this method, a linker (di-alkynyl linker) and an azide-containing cationic molecule undergo a 2+3 cyclization addition reaction in the presence of anhydrous copper sulfate and sodium ascorbate to synthesize the dual-targeting molecule. The synthetic route is as follows.

[0037]

[0038] A third objective of this invention is to provide the application of the aforementioned dual-targeting molecule as an antibacterial agent. This molecule utilizes its molecular structure to interact with bacterial membranes and DNA, achieving a significant inhibitory effect on bacterial growth.

[0039] Example 1: High-throughput synthesis of dual-targeting molecules.

[0040] High-throughput synthesis method: Using the appropriate solvents, 100 mM stock solutions of alkynyl compounds (DMSO), azide compounds (DMSO), sodium ascorbate (water), and copper sulfate (water) were prepared respectively. Then, 10, 20, 10, and 10 μL were added sequentially to 96-well plates, respectively. Following this, 50 μL of DMSO was added for dilution, and the plates were incubated overnight in a microplate shaker at 1000 rpm and 25°C to obtain a 10 mM stock solution. Click-screen libraries for all compounds in the examples were prepared according to the above method.

[0041] Example 2: Large-scale synthesis of the dual-targeting molecule XIX-57

[0042] Large-scale synthetic method: In the molecular synthesis process, the alkynyl parent compound and the cationic group of azide are mixed in a 1:2 molar ratio using a copper-catalyzed click chemistry method, and the alkynyl and azide are added in the presence of anhydrous copper sulfate and sodium ascorbate. The specific preparation process of XIX-57 is as follows:

[0043]

[0044] Step 1:

[0045] Synthesis of 2-azidoethylamine

[0046] NaN3 (23.1 mmol), 2-chloroethylamine hydrochloride (76.9 mmol), and purified water (80 mL) were added sequentially to a round-bottom flask (250 mL), and the mixture was reacted at 85 °C for 15 h. After the reaction was complete, the reaction solution was cooled to 10 °C, and KOH (11 mmol) was added and stirred for 1.5 h. The reaction solution was then poured into a separating funnel and extracted with diethyl ether (5 × 100 mL). The organic phase was dried over anhydrous sodium sulfate, and the diethyl ether was removed by concentration under reduced pressure to give product 4, a light brown liquid (yield = 87%).

[0047] Step 2:

[0048] Synthesis of 1-(2-azidoethyl)guanidine

[0049] 2-Azide ethylamine (31.1 mmol), S-ethylisothiourea hydrobromide (27 mmol), and triethylamine (27 mmol) were mixed, and then acetonitrile (25 mL) and deionized water (1.2 mL) were added. The mixture was reacted at room temperature for 16 h. After the reaction was complete, the solvent was evaporated to obtain a viscous, transparent, oily liquid. The azide compound corresponding to 57 was separated by silica gel column chromatography (eluting solvent: ethanol:ethyl acetate = 5:1).

[0050] Step 3:

[0051] Synthesis of diacetylene linkers

[0052] Bisphenol (1 mmol), potassium carbonate (4.4 mmol), acetone (50 mL), and bromopropyne (2.2 mmol) were added sequentially to a round-bottom flask and reacted overnight at 80 °C. After the reaction was complete, the solvent was evaporated and 60 mL of ethyl acetate was added. The organic phase was washed with 30 mL of 5% K₂CO₃ aqueous solution and 30 mL of deionized water, respectively, and dried with anhydrous sodium sulfate. Finally, the organic phase was collected and the crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 5:1 as eluent) to obtain the diyne linker corresponding to XIX (yield = 85%).

[0053] Step 4:

[0054] The product from step 2 (10 mmol), the product from step 3 (10 mmol), and CuBr (0.5 mmol) were sequentially added to a round-bottom flask containing 30 mL of DMF, and reacted overnight at room temperature. After the reaction was complete, the insoluble matter was filtered off, and the reaction solution was poured into 200 mL of anhydrous diethyl ether. The mixture was allowed to stand overnight, and a precipitate was formed to give the final product XIX-57 (yield = 20%). Its NMR spectrum is shown below. Figure 2 As shown.

[0055] Example 3: Large-scale synthesis of the dual-targeting molecule XIX-54

[0056] The synthesis route for XIX-54 is as follows:

[0057]

[0058] Step 1:

[0059] Synthesis of 3-azidopropylamine

[0060] NaN3 (23.1 mmol) and 3-chloropropylamine (76.9 mmol) were added to a round-bottom flask containing 80 mL of water and reacted at 85 °C for 15 h. After the reaction was complete, the reaction solution was cooled to 10 °C, and KOH (11 mmol) was added and stirred for 1.5 h. The reaction solution was then placed in a separating funnel and extracted with diethyl ether (5 × 100 mL). The organic phase was dried over anhydrous sodium sulfate, and the diethyl ether was removed by concentration under reduced pressure to obtain 3-azidopropylamine, which was a light brown liquid (yield = 87%).

[0061] Step 2:

[0062] Synthesis of 1-(3-azidopropyl)guanidine

[0063] 3-Azidepropylamine (31.1 mol), S-ethylisothiourea hydrobromide (27 mol), triethylamine (27 mol), and acetonitrile (24 mL) were added to a round-bottom flask. Deionized water (1.2 mL) was added to the solution, and the mixture was stirred at 25 °C for 16 h. After the reaction was complete, the solvent was removed by rotary evaporation, and the azide compound corresponding to the viscous, transparent, oily liquid 54 was obtained by silica gel column chromatography (eluent: ethanol:ethyl acetate = 5:1).

[0064] Step 3:

[0065] Synthesis of diacetylene linkers

[0066] Bisphenol (1 mmol), potassium carbonate (4.4 mmol), acetone (50 mL), and bromopropyne (2.2 mmol) were sequentially added to a round-bottom flask containing acetone (50 mL) and reacted overnight at 80 °C. After the reaction was complete, the solvent was evaporated and 60 mL of ethyl acetate was added. The organic phase was washed with 5% K₂CO₃ aqueous solution (30 mL) and deionized water (30 mL), respectively, and dried with anhydrous sodium sulfate. Finally, the organic phase was collected and the crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 5:1 as eluent) to obtain the diyne linker corresponding to XIX (yield = 85%).

[0067] Step 4:

[0068] The product from step 2 (10 mmol), the product from step 3 (10 mmol), and CuBr (0.5 mmol) were sequentially added to a round-bottom flask containing 30 mL of DMF, and reacted overnight at room temperature. After the reaction was complete, the insoluble matter was filtered off, and the reaction solution was poured into 200 mL of anhydrous diethyl ether. The mixture was allowed to stand overnight, and a precipitate was formed to give the final product XIX-54 (yield = 20%). Its NMR spectrum is shown below. Figure 3 As shown.

[0069] Example 4: Large-scale synthesis of dual-targeting molecule I-54

[0070] The synthetic route for I-54 is as follows:

[0071]

[0072] Step 1:

[0073] Synthesis of 3-azidopropylamine

[0074] NaN3 (23.1 mmol), 3-chloropropylamine (76.9 mmol), and purified water (80 mL) were added sequentially to a round-bottom flask, and the mixture was reacted at 85 °C for 15 h. After the reaction was complete, the reaction solution was cooled to 10 °C, and KOH (11 mmol) was added and stirred for 1.5 h. The reaction solution was then placed in a separating funnel and extracted with diethyl ether (5 × 100 mL). The organic phase was dried over anhydrous sodium sulfate, and the diethyl ether was removed by concentration under reduced pressure to obtain the product, which was a light brown liquid (yield = 87%).

[0075] Step 2:

[0076] Synthesis of 1-(3-azidopropyl)guanidine

[0077] Azide propylamine (31.1 mol), S-ethylisothiourea hydrobromide (27 mol), triethylamine (27 mol), and acetonitrile (24 mL) were added to a round-bottom flask. Deionized water (1.2 mL) was added to the solution, and the mixture was stirred at 25 °C for 16 h. After the reaction was complete, the solvent was removed by rotary evaporation, and the azide compound corresponding to the viscous, transparent, oily liquid 54 was obtained by silica gel column chromatography (eluent: ethanol:ethyl acetate = 5:1).

[0078] Step 3:

[0079] Synthesis of diacetylene linkers

[0080] Bisphenol (1 mmol), potassium carbonate (4.4 mmol), and bromopropyne (2.2 mmol) were sequentially added to a round-bottom flask containing 50 mL of acetone and reacted overnight at 80 °C. After the reaction was complete, the solvent was evaporated and 60 mL of ethyl acetate was added. The organic phase was washed with 30 mL of 5% K₂CO₃ aqueous solution and 30 mL of deionized water, respectively, and dried with anhydrous sodium sulfate. Finally, the organic phase was collected and the crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 5:1 as eluent) to obtain the diyne linker corresponding to I (yield = 85%).

[0081] Step 4:

[0082] The product from step 2 (10 mmol), the product from step 3 (10 mmol), and CuBr (0.5 mmol) were sequentially added to a round-bottom flask containing 30 mL of DMF, and reacted overnight at room temperature. After the reaction was complete, the insoluble matter was filtered off, and the reaction solution was poured into 200 mL of anhydrous diethyl ether. The mixture was allowed to stand overnight, and a precipitate was formed to give the final product I-54 (yield = 20%), whose NMR spectrum is shown below. Figure 4 As shown.

[0083] Example 5: Large-scale synthesis of the dual-targeting molecule XIV-57

[0084] Synthetic route of XIV-57:

[0085]

[0086] Step 1:

[0087] Synthesis of 2-azidoethylamine

[0088] NaN3 (23.1 mmol), 2-chloroethylamine (76.9 mmol), and purified water (80 mL) were added sequentially to a round-bottom flask, and the mixture was reacted at 85 °C for 15 h. After the reaction was complete, the reaction solution was cooled to 10 °C, and KOH (11 mmol) was added and stirred for 1.5 h. The reaction solution was then poured into a separating funnel and extracted with diethyl ether (5 × 100 mL). The organic phase was dried over anhydrous sodium sulfate, and the diethyl ether was removed by concentration under reduced pressure to obtain the product, which was a light brown liquid (yield = 87%).

[0089] Step 2:

[0090] Synthesis of 1-(2-azidoethyl)guanidine

[0091] 2-Azide ethylamine (31.1 mmol), S-ethylisothiourea hydrobromide (27 mmol), and triethylamine (27 mmol) were mixed, and then acetonitrile (25 mL) and deionized water (1.2 mL) were added. The mixture was reacted at room temperature for 16 h. After the reaction was complete, the solvent was evaporated to obtain a viscous, transparent, oily liquid. The azide compound corresponding to 57 was separated by silica gel column chromatography (eluting solvent: ethanol:ethyl acetate = 5:1).

[0092] Step 3:

[0093] Synthesis of diacetylene linkers

[0094] Bisphenol (1 mmol), potassium carbonate (4.4 mmol), and bromopropyne (2.2 mmol) were added sequentially to a round-bottom flask containing acetone (50 mL) and reacted overnight at 80 °C. After the reaction was complete, the solvent was evaporated and 60 mL of ethyl acetate was added. The organic phase was washed with 5% K₂CO₃ aqueous solution (30 mL) and deionized water (30 mL), respectively, and dried with anhydrous sodium sulfate. Finally, the organic phase was collected and the crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 5:1 as eluent) to obtain the diyne linker corresponding to XIV (yield = 85%).

[0095] Step 4:

[0096] The product from step 2 (10 mmol), the product from step 3 (10 mmol), and CuBr (0.5 mmol) were sequentially added to a round-bottom flask containing 30 mL of DMF, and reacted overnight at room temperature. After the reaction was complete, the insoluble matter was filtered off, and the reaction solution was poured into 200 mL of anhydrous diethyl ether. The mixture was allowed to stand overnight, and a precipitate was formed to give the final product XIV-57 (yield = 20%). Its NMR spectrum is shown below. Figure 5 As shown

[0097] Example 6: Mass synthesis of the dual-targeting molecule XIV-54

[0098] The synthetic route for XIV-54 is as follows:

[0099]

[0100] Step 1:

[0101] NaN3 (23.1 mmol), 3-chloropropylamine (76.9 mmol), and purified water (80 mL) were added sequentially to a round-bottom flask (250 mL), and the mixture was reacted at 85 °C for 15 h. After the reaction was complete, the reaction solution was cooled to 10 °C, and KOH (11 mmol) was added and stirred for 1.5 h. The reaction solution was then placed in a separating funnel and extracted with diethyl ether (5 × 100 mL). The organic phase was dried over anhydrous sodium sulfate, and the diethyl ether was removed by concentration under reduced pressure to obtain the product, which was a light brown liquid (yield = 87%).

[0102] Step 2:

[0103] Synthesis of 1-(3-azidopropyl)guanidine

[0104] Azide propylamine (31.1 mol), S-ethylisothiourea hydrobromide (27 mol), triethylamine (27 mol), and acetonitrile (24 mL) were added to a round-bottom flask. Deionized water (1.2 mL) was added to the solution, and the mixture was stirred at 25 °C for 16 h. After the reaction was complete, the solvent was removed by rotary evaporation, and the azide compound corresponding to the viscous, transparent, oily liquid 54 was obtained by silica gel column chromatography (eluent: ethanol:ethyl acetate = 5:1).

[0105] Step 3:

[0106] Synthesis of diacetylene linkers

[0107] Bisphenol (1 mmol), potassium carbonate (4.4 mmol), acetone (50 mL), and bromopropyne (2.2 mmol) were added sequentially to a round-bottom flask and reacted overnight at 80 °C. After the reaction was complete, the solvent was evaporated and 60 mL of ethyl acetate was added. The organic phase was washed with 30 mL of 5% K₂CO₃ aqueous solution and 30 mL of deionized water, respectively, and dried with anhydrous sodium sulfate. Finally, the organic phase was collected and the crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 5:1 as eluent) to obtain the diyne linker corresponding to XIV (yield = 85%).

[0108] Step 4:

[0109] The product from step 2 (10 mmol), the product from step 3 (10 mmol), and CuBr (70 mg, 0.5 mmol) were sequentially added to a round-bottom flask containing 30 mL of DMF, and reacted overnight at room temperature. After the reaction was complete, the insoluble matter was filtered off, and the reaction solution was poured into 200 mL of anhydrous diethyl ether. The mixture was allowed to stand overnight, and a precipitate was formed to give the final product XIV-54 (yield = 20%), whose NMR spectrum is shown below. Figure 6 As shown.

[0110] Example 7: Large-scale synthesis of the dual-targeting molecule XV-54

[0111] The synthesis route of XV-54 is as follows:

[0112]

[0113] Step 1:

[0114] NaN3 (23.1 mmol), 3-chloropropylamine (76.9 mmol), and water (80 mL) were added sequentially to a round-bottom flask, and the mixture was reacted at 85 °C for 15 h. After the reaction was complete, the reaction solution was cooled to 10 °C, and KOH (11 mmol) was added and stirred for 1.5 h. The reaction solution was then placed in a separating funnel and extracted with diethyl ether (5 × 100 mL). The organic phase was dried in anhydrous sodium sulfate, and the diethyl ether was removed by concentration under reduced pressure to obtain the product, which was a light brown liquid (yield = 87%).

[0115] Step 2:

[0116] Synthesis of 1-(2-azidoethyl)guanidine

[0117] 3-Azidepropylamine (31.1 mmol), S-ethylisothiourea hydrobromide (27 mmol), and triethylamine (27 mmol) were mixed, and then acetonitrile (25 mL) and deionized water (1.2 mL) were added. The mixture was reacted at room temperature for 16 h. After the reaction was complete, the solvent was removed by rotary evaporation to obtain a viscous, transparent, oily liquid. The azide compound corresponding to 57 was obtained by silica gel column chromatography (eluting solvent: ethanol:ethyl acetate = 5:1).

[0118] Step 3:

[0119] Synthesis of diacetylene linkers

[0120] Bisphenol (290 mg, 1 mmol), potassium carbonate (607.2 mg, 4.4 mmol), acetone (50 mL), and bromopropyne (254.7 mg, 2.2 mmol) were added sequentially to a round-bottom flask and reacted overnight at 80 °C. After the reaction was complete, the solvent was evaporated and 60 mL of ethyl acetate was added. The organic phase was washed with 30 mL of 5% K₂CO₃ aqueous solution and 30 mL of deionized water, respectively, and dried over anhydrous sodium sulfate. Finally, the organic phase was collected and purified by silica gel column chromatography (petroleum ether / ethyl acetate = 5:1 as eluent) to obtain the diyne linker corresponding to XV (yield = 85%).

[0121] Step 4:

[0122] The product from step 2 (10 mmol), the product from step 3 (10 mmol), and CuBr (0.5 mmol) were sequentially added to a round-bottom flask containing 30 mL of DMF, and reacted overnight at room temperature. After the reaction was complete, the insoluble matter was filtered off, and the reaction solution was poured into 200 mL of anhydrous diethyl ether. The mixture was allowed to stand overnight, and a precipitate was formed to give the final product XV-54 (yield = 20%), whose NMR spectrum is shown below. Figure 7 As shown.

[0123] Example 8: Large-scale synthesis of dual-targeting molecule I-46

[0124] The synthetic route for I-46 is as follows:

[0125]

[0126] Step 1:

[0127] Synthesis of 1-azido-3-chloropropane

[0128] NaN3 (12.7 mmol) and 1-bromo-3-chloropropane (12.7 mmol) were added to a solution containing 20 mL of DMF and reacted at room temperature for 18 h. After the reaction was complete, an equal volume of deionized water was added, and the reaction mixture was extracted three times with diethyl ether (30 mL). The organic phase was then washed three times with saturated brine. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain 1-azido-3-chloropropane in 83% yield.

[0129] Step 2:

[0130] Synthesis of 46

[0131] 1-Azide-3-chloropropane (12.7 mmol), 1-benzylimidazole (12.7 mmol), and acetonitrile (10 mL) were added sequentially to a round-bottom flask and stirred under reflux at 85 °C for two days. The mixture was then concentrated under vacuum, the residue was dissolved in methanol, and the insoluble matter was filtered out. A large amount of diethyl ether was then added to precipitate the solid. This process was repeated 2-3 times to obtain the azide compound corresponding to 46.

[0132] Step 3:

[0133] Synthesis of diacetylene linkers

[0134] Bisphenol (1 mmol), potassium carbonate (4.4 mmol), acetone (50 mL), and bromopropyne (2.2 mmol) were added sequentially to a round-bottom flask and reacted overnight at 80 °C. After the reaction was complete, the solvent was evaporated and 60 mL of ethyl acetate was added. The organic phase was washed with 30 mL of 5% K₂CO₃ aqueous solution and 30 mL of deionized water, respectively, and dried with anhydrous sodium sulfate. Finally, the organic phase was collected and the crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 5:1 as eluent) to give the diyne linker corresponding to I (yield = 85%).

[0135] Step 4:

[0136] The product from step 2 (10 mmol), the product from step 3 (10 mmol), and CuBr (0.5 mmol) were sequentially added to a round-bottom flask containing 30 mL of DMF, and reacted overnight at room temperature. After the reaction was complete, the insoluble matter was filtered off, and the reaction solution was poured into 200 mL of anhydrous diethyl ether. The mixture was allowed to stand overnight, and a precipitate was formed to give the final product I-46 (yield = 20%), whose NMR spectrum is shown below. Figure 8 As shown.

[0137] Example 9: Mass synthesis of the dual-targeting molecule XI-54

[0138] Synthesis route of XI-54:

[0139]

[0140] Step 1:

[0141] NaN3 (23.1 mmol), 3-chloropropylamine (76.9 mmol), and purified water (80 mL) were added sequentially to a round-bottom flask, and the mixture was reacted at 85 °C for 15 h. After the reaction was complete, the reaction solution was cooled to 10 °C, and KOH (11 mmol) was added and stirred for 1.5 h. The reaction solution was then placed in a separating funnel and extracted with diethyl ether (5 × 100 mL). The organic phase was dried over anhydrous sodium sulfate, and the diethyl ether was removed by concentration under reduced pressure to obtain the product, which was a light brown liquid (yield = 87%).

[0142] Step 2:

[0143] Synthesis of 1-(3-azidopropyl)guanidine

[0144] Azide propylamine (31.1 mol), S-ethylisothiourea hydrobromide (27 mol), triethylamine (27 mol), and acetonitrile (24 mL) were added to a round-bottom flask. Deionized water (1.2 mL) was added to the solution, and the mixture was stirred at 25 °C for 16 h. After the reaction was complete, the solvent was removed by rotary evaporation, and the azide compound corresponding to the viscous, transparent, oily liquid 54 was obtained by silica gel column chromatography (eluent: ethanol:ethyl acetate = 5:1).

[0145] Step 3:

[0146] Synthesis of diacetylene linkers

[0147] Bisphenol (1 mmol), potassium carbonate (4.4 mmol), acetone (50 mL), and bromopropyne (2.2 mmol) were added sequentially to a round-bottom flask and reacted overnight at 80 °C. After the reaction was complete, the solvent was evaporated and 60 mL of ethyl acetate was added. The organic phase was washed with 30 mL of 5% K₂CO₃ aqueous solution and 30 mL of deionized water, respectively, and dried over anhydrous sodium sulfate. Finally, the organic phase was collected and the crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 5:1 as eluent) to obtain the diyne linker corresponding to XI (yield = 85%).

[0148] Step 4:

[0149] The product from step 2 (10 mmol), the product from step 3 (10 mmol), and CuBr (0.5 mmol) were sequentially added to a round-bottom flask containing 30 mL of DMF, and reacted overnight at room temperature. After the reaction was complete, the insoluble matter was filtered off, and the reaction solution was poured into 200 mL of anhydrous diethyl ether. The mixture was allowed to stand overnight, and a precipitate was formed to give the final product XI-54 (yield = 20%), whose NMR spectrum is shown below. Figure 9 As shown.

[0150] Example 10: Mass synthesis of dual-targeting molecule II-1

[0151] The synthetic route for I I-1 is as follows:

[0152]

[0153] Step 1:

[0154] Synthesis of 1-azido-3-chloropropane

[0155] 12.7 mmol of NaN3 and 12.7 mmol of 1-bromo-3-chloropropane were added to a round-bottom flask containing 20 mL of DMF and stirred at room temperature for 18 h. After the reaction was complete, an equal volume of deionized water was added, and the reaction mixture was extracted three times with diethyl ether (30 mL). The organic phase was then washed three times with saturated brine. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain 1-azido-3-chloropropane in 83% yield.

[0156] Step 2:

[0157] Synthesis of 1

[0158] 1-Azide-3-chloropropane (12.7 mmol), triphenylphosphine (12.7 mmol), and acetonitrile (10 mL) were added sequentially to a round-bottom flask and stirred under reflux at 85 °C for two days. After the reaction was complete, the mixture was concentrated under vacuum, and the residue was dissolved with a small amount of DCM. The insoluble matter was filtered off, and a large amount of diethyl ether was added to the filtrate to precipitate the solid. This process was repeated 2-3 times to obtain the azide compound corresponding to 1.

[0159] Step 3:

[0160] Synthesis of diacetylene linkers

[0161] Bisphenol (1 mmol), potassium carbonate (4.4 mmol), acetone (50 mL), and bromopropyne (2.2 mmol) were added sequentially to a round-bottom flask and reacted overnight at 80 °C. After the reaction was complete, the solvent was evaporated and 60 mL of ethyl acetate was added. The organic phase was washed with 30 mL of 5% K₂CO₃ aqueous solution and 30 mL of deionized water, respectively, and dried over anhydrous sodium sulfate. Finally, the organic phase was collected and the crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 5:1 as eluent) to obtain the diyne linker corresponding to II (yield = 85%).

[0162] Step 4:

[0163] The product from step 2 (10 mol), the product from step 3 (10 mmol), and CuBr (0.5 mmol) were sequentially added to a round-bottom flask containing 30 mL of DMF, and reacted overnight at room temperature. After the reaction was complete, the insoluble matter was filtered off, and the reaction solution was poured into 200 mL of anhydrous diethyl ether. The mixture was allowed to stand overnight, and a precipitate was formed to give the final product, I I-1 (yield = 20%), whose NMR spectrum is shown below. Figure 10 As shown.

[0164] Example 11: Tests of the antibacterial and hemolytic activities of the compound

[0165] The compounds were dissolved separately in DMSO, and their antibacterial activity against Staphylococcus aureus was tested using the standard broth dilution method. The results of their antibacterial and hemolytic activities are shown in the table below.

[0166]

[0167]

[0168]

[0169] Of the compounds tested, 259 compounds had MIC values ​​below 128 μM against Staphylococcus aureus, with 139 of these compounds having MIC values ​​below 16 μM. This indicates that these compounds possess good antibacterial properties. The cationic group significantly influences the antibacterial activity of the compounds; quaternary phosphonium salts almost all exhibited strong antibacterial activity, with MIC values ​​as low as 2 μM. Imidazole salts, quaternary ammonium salts, and guanidines also showed good antibacterial activity. Free amines exhibited poorer cationic activity, with the lowest MIC value reaching 16 μM. All compounds showed no hemolytic toxicity to blood cells at a concentration of 128 μM and demonstrated good biocompatibility.

[0170] Example 12: Cytotoxicity test of XIX-57, an antibacterial molecule with highly effective antibacterial properties

[0171] XIX-57 was serially diluted with sterile PBS in sterile 96-well plates and then added to cell-containing culture medium. 20 μL of the compound solution was added to each well, resulting in final concentrations of (256, 128, 64, 32, 8, 4, 2 μg / mL). After static incubation for 24 h, the supernatant culture medium was aspirated and discarded. 100 μL of PBS was added to each well, and the cells were washed three times. 0.5 mg / mL MTT solution was added under light-protected conditions, and the cells were incubated for 1.5 h. The MTT solution was then removed, and 100 μL of dimethyl sulfoxide was added to each well. The cells were then incubated at 37°C for 5 min. The absorbance at 595 nm was read using a microplate reader, and the data was processed using GraphPad. The results are shown below.Figure 11 As shown, when the compound concentration was 256 μg / mL, the cell viability of HEK-293T remained above 80%, indicating that compound XIX-57 had low cytotoxicity to HEK-293T and good biosafety.

[0172] Example 13: Mouse activity assay of the antibacterial molecule XIX-57 with highly effective antibacterial effect

[0173] The antibacterial effect of compound XIX-57 in a mouse wound model was evaluated, and the experimental results are as follows: Figure 12 As shown. Adult male ICR mice (6-8 weeks old, 25g each) were anesthetized by intraperitoneal injection of chloral hydrate (50mg / kg), and an open excision wound (3-4cm) was created on the back of the mouse. 2 ), inoculate the excised wound area with a Staphylococcus aureus suspension (10). 8 CFU (carbohydrate, hydroxychloroquine, and hydroxychloroquine) was used to confirm infection. Mice were divided into three groups (PBS, XIX-57, and vancomycin), with four mice in each group. All treatment groups received a dose of 30 mg / kg of drug, and ultrapure water was used as the solvent. Treatment began 24 hours after infection, with the drug applied to the wound area and repeated for 7 days, during which the bacterial load on the wound surface was measured. The mice's condition was observed and recorded daily, including activity level, wound area, and body weight. On the first day after drug treatment, the bacterial load in the XIX-57 treatment group was reduced by at least two orders of magnitude compared to the PBS group, and slightly better than the vancomycin treatment group. This experiment demonstrates that XIX-57 is comparable to vancomycin in vivo in mice, exhibiting good antibacterial activity.

[0174] Example 14: Lipid interference experiment of the compound

[0175] The compound was dissolved in DMSO, and the antibacterial activity against Staphylococcus aureus was tested using the standard broth dilution method. The above-mentioned Staphylococcus aureus-containing bacterial suspension was prepared to a concentration of 5 × 10⁻⁶. 5 A working solution of CFU / mL was prepared, with either PE or PG added (to a final concentration of 128 μg / mL), and the antibacterial activity of the compound under different lipid interference conditions was determined. For example... Figure 13As shown, in the presence of PG, the MICs of XIX-1, XIX-57, XIX-54, XIX-57, and XIX-54 increased by 32, 18, 32, 60, and 18 times, respectively, indicating that these compounds have strong PG binding ability. In particular, the change in MIC of XIX-57 is comparable to that of chlorhexidine and YB10. Chlorhexidine is a broad-spectrum bactericide with strong membrane targeting; YB10 is a novel oligomer with a strong ability to disrupt bacterial cell membranes. This experiment demonstrates that the binding of compounds to PG is crucial to their antibacterial activity. Similarly, by comparing the changes in MIC before and after the addition of PE, the change in MIC of XIX-57 was still the most significant, with a change of 8 times.

[0176] Example 15: Test of membrane perturbation of bacteria by compound XIX (SEM observation)

[0177] Scanning electron microscopy (SEM) imaging of bacterial surface morphology allows for the study of the effects of drugs on bacterial cell membranes. First, single colonies of *Staphylococcus aureus* were picked and cultured overnight on CAMHB medium at 37°C and 220 rpm. Next, the bacteria were aliquoted into new shake tubes, 3 mL per tube. Then, XIX-57 was added to the shake tubes at final concentrations of 4×MIC, 2×MIC, MIC, and 1 / 2×MIC, with shake tubes without XIX-57 serving as a negative control. These shake tubes were then incubated at 37°C and 220 rpm for 4 hours. Afterward, 1 mL of bacterial culture from each shake tube was transferred to a new centrifuge tube and centrifuged at 3200 rpm for 3-5 minutes, removing the supernatant. Then, 1 mL of 2% glutaraldehyde solution was added to the centrifuge tube to fix the bacteria for 2 hours. After fixation, the sample was centrifuged at 3200 rpm for 3-5 minutes. After removing the supernatant, the bacteria were dehydrated sequentially using 30%, 50%, 70%, and 90% ethanol aqueous solutions and anhydrous ethanol solutions, followed by freeze-drying. The bacterial morphology was observed and recorded after gold sputtering. Figure 14 As shown, under non-growth conditions, the Staphylococcus aureus in the control group had normal morphology and intact, plump cells. The Staphylococcus aureus treated with XIX-57 showed numerous bacterial fragments, cell deformation, and surface depressions in some bacteria. Under growth conditions, treatment with 16 μg / mL of the drug resulted in severe cell damage in most bacteria. XIX-57 demonstrated a very significant ability to disrupt bacterial cell membranes under both growth and non-growth conditions.

[0178] Example 16: Agarose gel electrophoresis experiment to determine the binding ability of compounds to DNA

[0179] The binding affinity of compounds to DNA was determined by agarose gel electrophoresis. Under alkaline conditions (pH 8.0 of the buffer), DNA carries a negative charge and migrates towards the positive electrode through the gel medium in an electric field. Different DNA fragments exhibit different migration rates in the electric field due to their varying molecular weights and conformations. Ethidium bromide (EB) can intercalate between base pairs in DNA molecules, forming fluorescent complexes. After UV irradiation, different bands can be separated, achieving molecular weight identification. When a compound binds to DNA to form a complex, its molecular weight and charge change, ultimately hindering the migration of the DNA complex on the gel. Pre-extracted plasmid pCo ld-ctx-m-15 (20 ng / μL) was mixed with different concentrations of XIX-57 and incubated at 37°C for 30 min. After incubation, the mixed solutions of different concentrations were rapidly mixed with Loading Buffer and added to the drug loading wells on the agarose gel. The gel was then run on a 1% agarose gel containing Gerid (2 μg / mL) for 30 min. DNA band shifts can be observed and photographed under ultraviolet light. For example... Figure 15 As shown, compared to XIX and 57 alone which do not bind to DNA, XIX-57 can strongly bind to DNA to prevent it from migrating on the gel, demonstrating the multidentate effect, which indicates that XIX-57 has a strong DNA binding ability.

[0180] Many specific details have been set forth in the foregoing description to provide a thorough understanding of the present invention. However, the above description is merely a preferred embodiment of the present invention, and the present invention can be implemented in many other ways different from those described herein. Therefore, the present invention is not limited to the specific embodiments disclosed above. Furthermore, any person skilled in the art can make many possible variations and modifications to the technical solutions of the present invention, or modify them into equivalent embodiments, using the methods and techniques disclosed above, without departing from the scope of the present invention. Any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention, without departing from the content of the present invention, shall still fall within the protection scope of the present invention.

Claims

1. A small molecule antibacterial compound with dual DNA and membrane targeting, characterized in that, The structural formula of the antibacterial compound is: or 。 2. A method for preparing the compound of claim 1, characterized in that, The specific preparation processes are as follows: ； 。 3. An antibacterial agent, characterized in that, Includes any of the compounds described in claim 1.

4. A DNA inhibitor, characterized in that, Includes any of the compounds described in claim 1.

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