9-aminoacridines with multiple electron withdrawing substituents, processes for their preparation and uses thereof

Abstract

The application belongs to the technical field of pharmaceutical chemistry, and discloses a kind of aminoacridine compounds with multi-electron-withdrawing substituent group with broad-spectrum antibacterial activity and can kill bacteria quickly and a preparation method and purposes thereof.The compounds are prepared by Ullmann coupling reaction, ring closure reaction and aminolysis reaction, and the structures of the compounds are shown in formula I-III.Bacteriostatic activity experiment research shows that the compounds have clear bacteriostatic activity on various coccobacilli and bacilli, the minimum inhibitory concentration (MIC) of the compounds on staphylococcus aureus and staphylococcus epidermidis reaches 0.25 μg / mL, and the minimum inhibitory concentration MIC of the compounds on staphylococcus capitis can reach 0.5 μg / mL.The compounds can be used for antibacterial drug development, and have the advantages of high antibacterial activity, broad antibacterial spectrum and fast bactericidal effect.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical chemistry and discloses a 9-aminoacridine compound with multiple electron-withdrawing substituents that has antibacterial activity, as well as its preparation method and uses. Background Technology

[0002] Penicillin was used in the 1940s to treat diseases caused by infections such as diphtheria, anthrax, streptococcus, and pneumococcus. The discovery of penicillin opened up the history of human use of antibiotics and became one of the greatest discoveries in 20th-century medicine.

[0003] In clinical use, penicillin has revealed many shortcomings, such as instability to acid, narrow antibacterial spectrum, inability to be taken orally, inability to be administered orally, and easy development of drug resistance. Subsequently, penicillin was structurally modified, resulting in a series of modified penicillin compounds (Formula IV). These modified compounds have shown better clinical application value. For example, enzyme-resistant penicillin oxacillin sodium can be used for penicillin G-resistant Staphylococcus aureus infections, acid-resistant penicillin phenacetin can be taken orally, and broad-spectrum penicillin amoxicillin has expanded the antibacterial spectrum.

[0004]

[0005] Structural modifications to penicillin have yielded a series of antibiotics with improved efficacy. Many similar examples exist, such as the modification of natural cephalosporin C, which resulted in five generations of cephalosporins: the first-generation cephalosporin cefadroxil, the second-generation cephalosporin cefuroxime, the third-generation cephalosporin cefotaxime, the fourth-generation cephalosporin cefazolin, and the fifth-generation cephalosporin cefepime (formula V). These cephalosporins exhibit changes in their antibacterial activity. The first, second, and third-generation cephalosporins show a gradual decrease in antibacterial activity against Gram-positive cocci, but a gradual increase in antibacterial activity against Gram-negative bacilli. Fourth-generation cephalosporins are primarily suitable for infections caused by multidrug-resistant Gram-negative bacteria, while fifth-generation cephalosporins are primarily suitable for infections caused by multidrug-resistant Gram-positive bacteria.

[0006]

[0007] In summary, structural modification of active compounds to obtain a series of drugs with the same parent ring structure is an important method in new drug research.

[0008] Antibiotics are prone to developing resistance during use, and bacterial resistance poses a serious threat to human health and economic development worldwide. In 2019 alone, approximately 5 million people died globally due to drug-resistant bacteria (①Christopher JLM, et al. Global burden of bacterial antimicrobial resistance in 2019: a systematic analysis, Lancet, 2022, 399: 629.). In 2023, the World Health Organization (WHO) released the Global Antimicrobial Resistance and Use Surveillance System (GLASS) report: 2022 (https: / / www.who.int / publications / i / item / 9789240062702). This report analyzed antimicrobial resistance trends since 2017, focusing on monitoring bloodstream infections, gastrointestinal infections, gonorrhea, and urinary tract infections, and the resistance of eight bacterial pathogens associated with these diseases (Acinetobacter spp., Escherichia coli, Klebsiella pneumoniae, Salmonella spp., Staphylococcus aureus, Streptococcus pneumoniae, Shigella spp., and Neisseria gonorrhoeae). The report found that among bloodstream infection pathogens reported in 76 countries and regions, the infection rates of "superbugs" resistant to third-generation cephalosporins, Escherichia coli, and methicillin-resistant Staphylococcus aureus were 42% and 35%, respectively. This demonstrates that bacterial resistance is becoming an increasingly serious problem.

[0009] Antibiotic resistance mechanisms have also received widespread attention from scientists, but the exact mechanisms of bacterial resistance still have many unknowns (③Lewis K, et al. Sophisticated natural products as antibiotics. Nature, 2024, 632, 39; ④Alexander JAN, et al. Structural basis of broad-spectrum β-lactam resistance in Staphylococcus aureus. Nature, 2023, 613, 375; ⑤Lázár V, et al. Antibiotic combinations reduce Staphylococcus aureus clearance. Nature, 2022, 610, 540).

[0010] In June 2024, the World Health Organization (WHO) released its latest report on antimicrobial agents (including antibiotics) in global clinical and preclinical development. The report noted that although the number of antimicrobial agents in clinical development increased from 80 in 2021 to 97 in 2023, there remains an urgent need for new innovative drugs to treat serious infections and replace those that have become ineffective due to widespread use (⑥ https: / / www.who.int / news / item / 14-06-2024-who-releases-report-on-state-of-development-of-antibacterials). In conclusion, antibiotic resistance is a growing global problem, and the discovery of antibiotics with novel structural features will provide new options for overcoming antibiotic resistance. Summary of the Invention

[0011] 9-Aminoacridine, ethacridine lactate, and acridine yellow hydrochloride (Formula VI) are all acridine compounds with antibacterial activity. (⑦ Pengfei She, et al. Repurposing 9-aminoacridine as an adjuvant enhances the antimicrobial effects of rifampin against multidrug-resistant klebsiella pneumoniae. Microbiol Spectr. 2023, 11(3):1; ⑧ Yamamoto, et al. Effect of 6,9-diamino-2-ethoxyacridine on bacteriophages and animal viruses, Antimicrobial Agents and) Chemotherapy (1961-70), 1968, 668; ⑨ Zhang Chuanshi, et al. Treatment of 54 cases of infectious pleuropneumonia in calves with intravenous injection of acridinium hydrochloride. Heilongjiang Animal Husbandry and Veterinary Medicine, 2011, (04): 76). Among them, ethacridine lactate is widely used for wound cleaning and disinfection, and acridinium hydrochloride is widely used as a veterinary drug for the prevention and treatment of intestinal infections in cattle, sheep, pigs and other animals.

[0012]

[0013] As can be seen from Formula VI, 9-aminoacridine, ethacridine lactate, and acridine yellow hydrochloride are all acridine compounds. For ease of description, we will label the three aromatic rings contained in acridine as ring A, ring B, and ring C, respectively (Formula VII). The common structural feature of these three acridine compounds is that the substituents on the acridine parent ring are all electron-donating amino or ethoxy substituents. Further literature review also found that the reported acridine compounds with antibacterial activity are all compounds containing electron-donating substituents.

[0014] In the design, synthesis and structure-activity relationship study of antitumor drugs, the applicant unexpectedly discovered that acridine compound 1 (Formula VII), as a synthetic intermediate, has significant antibacterial activity. Its structural feature is that both ring A and ring C contain electron-withdrawing substituents. Its minimum inhibitory concentration (MIC) against Staphylococcus aureus and Escherichia coli can reach 2 μg / mL (the MICs of amoxicillin against Staphylococcus aureus and Escherichia coli are 2 μg / mL and 4 μg / mL, respectively).

[0015]

[0016] Compound 1, discovered by the applicant, has an acridine parent ring and differs significantly from known acridine compounds with antibacterial activity, such as 9-aminoacridine, ethacridine lactate, and acridine yellow hydrochloride, in the following two ways:

[0017] (1) Structural differences: 9-aminoacridine, ethacridine lactate, and acridine yellow hydrochloride have only electron-donating substituents on the acridine ring, while compound 1 has electron-withdrawing substituents on both the A and C rings of acridine.

[0018] (2) Difference in activity: Using the micro-broth dilution method, the applicant evaluated the antibacterial activity of 9-aminoacridine and its salts, ethacridine lactate, acridine yellow hydrochloride, and compound 1. The results are shown in Table 1. As can be seen from the table below, compound 1, which has multiple electron-withdrawing substituents, has a significantly improved antibacterial effect compared to known acridine compounds.

[0019] Antibacterial activity of acridine compounds

[0020]

[0021] Based on the above findings, the applicant conducted extensive literature review and systematically studied the design, synthesis, and antibacterial activity evaluation of aminoacridine compounds containing electron-withdrawing substituents. The newly designed compound retained the structural characteristics of compound 1, namely, that both the A and C rings of 9-aminoacridine contain electron-withdrawing substituents. Further synthesis and antibacterial activity evaluation yielded beneficial research results.

[0022] Therefore, the present invention aims to provide a class of 9-aminoacridine compounds with multiple electron-withdrawing substituents, which have significant antibacterial effects.

[0023] The 9-aminoacridine compounds with multiple electron-withdrawing substituents described in this invention have the structural formulas shown in Formulas I to III:

[0024]

[0025] In Formula I, R1, R2, R3, and R4 are hydrogen atoms or electron-withdrawing substituents, and at least one of R1-R4 is an electron-withdrawing substituent.

[0026] In Formula II, R1, R2, R3, and R4 are hydrogen atoms or electron-withdrawing substituents, and at least one of R1-R4 is an electron-withdrawing substituent.

[0027] In Formula III, R1, R2, R3, and R4 are hydrogen atoms or electron-withdrawing substituents, and at least one of R1-R4 is an electron-withdrawing substituent.

[0028] It may also be other pharmaceutically acceptable salts, or solvates, or hydrates, or prodrugs, or metabolites thereof.

[0029] The present invention also provides a method for preparing the aforementioned compound, comprising the following steps:

[0030] (1) Using aniline with different electron-withdrawing substituents as raw materials, 4-nitro-2-chlorobenzoic acid, 5-nitro-2-chlorobenzoic acid and 5-fluoro-2-bromobenzoic acid were reacted with Ullmann coupling reaction to obtain intermediates 1a-11a;

[0031]

[0032] (2) Intermediates 1a to 11a undergo cyclization reactions with phosphorus oxychloride to obtain intermediates 1b to 11b;

[0033]

[0034] (3) Intermediates 1b-11b undergo ammonolysis reactions with phenol and ammonium carbonate, respectively, to obtain target compounds 1-11;

[0035]

[0036] The applicant of this invention conducted research on 9-aminoacridine compounds with multiple electron-withdrawing substituents, obtaining compounds 1-11 as shown in Formulas I to III above, and their structures were confirmed. The content of each compound was greater than 99.0% as determined by high-performance liquid chromatography. Based on this, the activity of the obtained compounds was evaluated. This invention also provides methods and results for evaluating the activity of the aforementioned compounds, including the following:

[0037] In vitro antibacterial activity was evaluated using the micro-broth dilution method: The aforementioned compounds of this invention were prepared into an antibacterial compound solution with a concentration of 256 μg / mL. After adding MH broth medium to 96-well plates, the antibacterial compound solution was diluted twofold to 10 different concentration gradients: 128, 64, 32, 16, 8, 4, 2, 1, 0.5, and 0.25 μg / mL. *Escherichia coli* (Gram-negative bacteria) and *Staphylococcus aureus* (Gram-positive bacteria) were used as test bacteria, and vancomycin, meropenem, tigecycline, and amoxicillin were used as positive control drugs. The inoculated 96-well plates were placed in an incubator at 37°C for 16 hours. Bacterial growth was observed, and the first well that did not become cloudy was taken as the minimum inhibitory concentration (MIC) of the compound. The antibacterial properties of some compounds of this invention in 96-well plates are as follows: Figure 1 As shown.

[0038] The 9-aminoacridine compounds with multiple electron-withdrawing substituents described in this invention exhibit excellent activity against a variety of cocci, indicating that these compounds possess superior antibacterial activity. In particular, compounds 1, 3, and 8 have MIC values ​​between 0.25 and 1 μg / mL against Staphylococcus aureus, superior to the antibacterial activity of clinically used drugs amoxicillin and tigecycline; compounds 2 and 3 have MIC values ​​of 1 μg / mL against Enterococcus faecalis, showing superior antibacterial activity compared to the positive control drug vancomycin; compounds 3 and 4 have MIC values ​​of 1 μg / mL and 2 μg / mL against Staphylococcus albus, respectively, similar to the antibacterial activity of the positive control drug levofloxacin. Compounds 3 and 4 have MIC values ​​of 0.5 μg / mL against Staphylococcus capitella, similar to the antibacterial activity of the positive control drug levofloxacin; the 9-aminoacridine compounds with multiple electron-withdrawing substituents described in this invention also exhibit significant antibacterial activity against a variety of bacilli. The MIC values ​​of compounds 1 and 3 against Shigella were 0.5 μg / mL and 1 μg / mL, respectively, similar to those of the positive control drug levofloxacin. The MIC value of compound 3 against Enterobacter sakazakii was 2 μg / mL, superior to the positive control drug ampicillin.

[0039] The present invention has the following beneficial effects:

[0040] This invention discloses 9-aminoacridine compounds with multiple electron-withdrawing substituents possessing antibacterial activity, their preparation methods, and uses. These compounds exhibit excellent antibacterial activity in antibacterial activity tests, with a minimum inhibitory concentration (MIC) reaching 0.25 μg / mL. The compounds prepared by this invention can be used for antibacterial drug development, possessing advantages such as high antibacterial activity, broad antibacterial spectrum, and rapid bactericidal effect, providing a new option for developing antibacterial drugs.

[0041] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention. Attached Figure Description

[0042] Figure 1 This is a diagram showing the antibacterial activity of some of the compounds of this invention in a 96-well plate;

[0043] Figure 2 Microscopic images of the antibacterial effects of compound 3 and control drugs of the present invention on Staphylococcus aureus (a is compound 3, b is the negative control without the compound, c is ethacridine lactate, and d is vancomycin).

[0044] Figure 3 This is a line graph showing the bactericidal kinetics of compound 3 of the present invention against Staphylococcus aureus.

[0045] Figure 4 This is a line graph showing the bactericidal kinetics of compound 3 of the present invention against Escherichia coli.

[0046] Figure 5 This is the ultraviolet absorption spectrum of the interaction between compound 3 of the present invention and DNA. Detailed Implementation

[0047] The following detailed description of specific embodiments, presented in the form of experimental examples, further illustrates the above-described content of the present invention. However, this should not be construed as limiting the scope of the subject matter of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention.

[0048] The chemical reagents used in the specific embodiments of the present invention are obtained by purchasing commercially available products.

[0049] The instruments used for detection and analysis in this invention are as follows:

[0050] Nuclear magnetic resonance imaging (MRI) scanner, specifically the Bruker Advance Digital 400 MRI scanner from Switzerland.

[0051] High-resolution mass spectrometry, Bruker SolanX 70FT-MS (Switzerland); Agilent 6540TOF.

[0052] Sterilizer, Bingshan Songyang Biotechnology (Dalian) Co., Ltd., MVS-83 Vertical Pressure Steam Sterilizer.

[0053] 37℃ incubator, Shanghai Yiheng DHP-9602 (vertical) constant temperature incubator.

[0054] Shaker, Shanghai Jinghong Experimental Equipment Co., Ltd. THZ-412 benchtop constant temperature shaker.

[0055] UV-2600 Spectrophotometer, Shimadzu Analytical Technology R&D Co., Ltd.

[0056] Experiments 1-3 are synthesis experiments of compounds, and Experiments 4-8 are activity evaluation experiments of compounds.

[0057] Experimental Example 1 uses the preparation of compound 1 as an example to prepare a series of compounds.

[0058] In a three-necked flask (250 mL), 10 g of 2-chloro-5-nitrobenzoic acid (0.05 mol, 1 eq) was dissolved in 40 mL of N,N-dimethylformamide. Then, 6.67 g of 4-fluoroaniline (0.06 mol, 1.2 eq), 13.82 g of potassium carbonate (0.1 mol, 2.0 eq), 8.30 g of potassium iodide (0.05 mol, 1 eq), and 0.32 g of copper powder (0.005 mol, 0.1 eq) were added. The mixture was heated under reflux for 10 h, and the reaction progress was monitored by TLC (petroleum ether: ethyl acetate = 1:1). After the reaction was complete, the system was cooled to room temperature, and excess copper powder and potassium carbonate were removed by filtration. The pH of the filtrate was adjusted to 4 with 2 mol / L hydrochloric acid, and the residue was dried to obtain a yellow solid, designated as intermediate 1a, for use in the next reaction step.

[0059] In a three-necked flask (250 mL), intermediate 1a (10.5 g, 0.038 mol, 1 eq) prepared above was dissolved in toluene (15 mL). Phosphorus oxychloride (5.81 g, 0.038 mol, 1 eq) was added under ice bath conditions, and the mixture was heated under reflux for 2 h. The reaction progress was monitored by TLC (petroleum ether: ethyl acetate = 3:1). After the reaction was completed, the system was cooled to room temperature, and toluene and phosphorus oxychloride were evaporated to dryness. Crushed ice and ammonia were added, and the mixture was stirred at room temperature for 30 min. 50 mL of DCM was added for extraction, followed by washing with saturated brine, drying with anhydrous sodium sulfate, and evaporating the organic phase using a rotary evaporator. The organic phase was washed with diethyl ether to obtain a yellow solid 1b.

[0060] 1b (2.73 g, 1 mmol, 1 eq) and ammonium carbonate (1.4 g, 1.2 mmol, 1.2 eq) were reacted with phenol (5 g, 2 mmol, 2 eq) in 120 °C for 4 h. The reaction progress was monitored by TLC (petroleum ether: ethyl acetate = 1:1). After the reaction was completed, the system was cooled to room temperature, and 10 mL of 10% sodium hydroxide aqueous solution was added to remove excess phenol. The mixture was then filtered to obtain a red solid compound 1.

[0061]

[0062]

[0063] Experimental Example 2 uses the preparation of compound 3 as an example to prepare a series of two compounds.

[0064] In a three-necked flask (250 mL), 10 g of 2-chloro-4-nitrobenzoic acid (0.05 mol, 1 eq) was dissolved in 40 mL of N,N-dimethylformamide. Then, 6.67 g of 4-chloroaniline (0.06 mol, 1.2 eq), 13.82 g of potassium carbonate (0.1 mol, 2.0 eq), 8.30 g of potassium iodide (0.05 mol, 1 eq), and 0.32 g of copper powder (0.005 mol, 0.1 eq) were added. The mixture was heated under reflux for 10 h, and the reaction progress was monitored by TLC (petroleum ether:ethyl acetate = 1:1). After the reaction was complete, the system was cooled to room temperature, and excess copper powder and potassium carbonate were removed by filtration. The pH of the filtrate was adjusted to 4 with 2 mol / L hydrochloric acid, and the residue was dried by filtration to obtain a yellow solid, designated as intermediate 3a, for use in the next reaction.

[0065] In a three-necked flask (250 mL), the intermediate 3a (10.5 g, 0.038 mol, 1 eq) prepared above was dissolved in toluene (15 mL). Phosphorus oxychloride (5.81 g, 0.038 mol, 1 eq) was added under ice bath conditions, and the mixture was heated to reflux for 2 h. The reaction progress was monitored by TLC (petroleum ether: ethyl acetate = 3:1). After the reaction was completed, the system was cooled to room temperature, and the toluene and phosphorus oxychloride were evaporated to dryness. Crushed ice and ammonia were added, and the mixture was stirred at room temperature for 30 min. 50 mL of DCM was added for extraction, followed by washing with saturated brine, drying with anhydrous sodium sulfate, and evaporating the organic phase using a rotary evaporator. The organic phase was washed with diethyl ether to obtain a yellow solid 3b.

[0066] 3b (2.73 g, 1 mmol, 1 eq) and ammonium carbonate (1.4 g, 1.2 mmol, 1.2 eq) were reacted with phenol (5 g, 2 mmol, 2 eq) in 120 °C for 4 h. The reaction progress was monitored by TLC (petroleum ether: ethyl acetate = 1:1). After the reaction was completed, the system was cooled to room temperature, and 10 mL of 10% sodium hydroxide aqueous solution was added to remove excess phenol. The mixture was then filtered to obtain a red solid compound 3.

[0067] Following the preparation method of Experimental Example 2, compounds 4 to 7 were obtained:

[0068]

[0069]

[0070] Experimental Example 3 uses the preparation of compound 8 as an example to prepare a series of three compounds.

[0071] In a three-necked flask (250 mL), 10 g of 2-bromo-5-fluorobenzoic acid (0.05 mol, 1 eq) was dissolved in 40 mL of N,N-dimethylformamide. Then, 6.67 g of 3-fluoroaniline (0.06 mol, 1.2 eq), 13.82 g of potassium carbonate (0.1 mol, 2.0 eq), 8.30 g of potassium iodide (0.05 mol, 1 eq), and 0.32 g of copper powder (0.005 mol, 0.1 eq) were added. The mixture was heated under reflux for 10 h, and the reaction progress was monitored by TLC (petroleum ether:ethyl acetate = 1:1). After the reaction was complete, the system was cooled to room temperature, and excess copper powder and potassium carbonate were removed by filtration. The pH of the filtrate was adjusted to 4 with 2 mol / L hydrochloric acid, and the residue was dried by filtration to obtain a yellow solid, designated as intermediate 8a, for use in the next reaction step.

[0072] In a three-necked flask (250 mL), the intermediate 8a (10.5 g, 0.038 mol, 1 eq) prepared above was dissolved in toluene (15 mL). Phosphorus oxychloride (5.81 g, 0.038 mol, 1 eq) was added under ice bath conditions, and the mixture was heated to reflux for 2 h. The reaction progress was monitored by TLC (petroleum ether: ethyl acetate = 3:1). After the reaction was completed, the system was cooled to room temperature, and the toluene and phosphorus oxychloride were evaporated to dryness. Crushed ice and ammonia were added, and the mixture was stirred at room temperature for 30 min. 50 mL of DCM was added for extraction, followed by washing with saturated brine, drying with anhydrous sodium sulfate, and evaporating the organic phase using a rotary evaporator. The organic phase was washed with diethyl ether to obtain a yellow solid 8b.

[0073] 8b (2.73 g, 1 mmol, 1 eq) and ammonium carbonate (1.4 g, 1.2 mmol, 1.2 eq) were reacted with phenol (5 g, 2 mmol, 2 eq) in 120 °C for 4 h. The reaction progress was monitored by TLC (petroleum ether: ethyl acetate = 1:1). After the reaction was completed, the system was cooled to room temperature, and 10 mL of 10% sodium hydroxide aqueous solution was added to remove excess phenol. The mixture was then filtered to obtain a red solid compound 8.

[0074] Following the preparation method of Example 3, compounds 9–11 were obtained:

[0075]

[0076]

[0077] The beneficial effects of the compounds of the present invention are illustrated below using experimental examples 4-8.

[0078] Example 4 Evaluation of antibacterial activity against Escherichia coli and Staphylococcus aureus

[0079] The activity of the compounds synthesized in this invention against *Escherichia coli* and *Staphylococcus aureus*, both facultative anaerobes, was evaluated using a micro-broth dilution method. The culture medium used was MHB broth. The specific operating steps are as follows:

[0080] (1) Prepare an antibacterial compound solution with a concentration of 25600 μg / mL and store it in a refrigerator at 4℃ for later use.

[0081] (2) For the test bacteria Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus), single colonies of 0.5–1 mm were picked up with an inoculation loop and inoculated into MHB broth medium. The medium was incubated on a shaker at 37°C and 220 rpm for 4 hours, and then diluted to a bacterial count of approximately 1 × 10⁻⁶. 5 Prepare a bacterial culture at CFU / mL for later use.

[0082] (3) Take 10 μL of the antibacterial compound solution with a concentration of 25600 μg / mL into a 1.5 mL sterile centrifuge tube, and add 990 μL of sterile ultrapure water to dilute it to a compound solution with a concentration of 256 μg / mL.

[0083] (4) Take a 96-well plate and add 100 μL of MHB broth to each well. Then add 100 μL of the compound solution to the first well. Dilute stepwise using the two-fold dilution method to obtain the antibacterial compound solution. The concentrations of the first to tenth wells are 128, 64, 32, 16, 8, 4, 2, 1, 0.5, and 0.25 μg / mL, respectively. Finally, add 100 μL of the prepared bacterial suspension to make the total volume of each well 200 μL. The eleventh well contains 200 μL of MHB broth, and the twelfth well contains 100 μL of MHB broth and 100 μL of bacterial suspension as a blank control. Place the inoculated 96-well plate in a 37°C incubator and observe the bacterial growth for 16 hours. Vancomycin, meropenem, tigecycline, and amoxicillin are used as positive controls. Perform at least three parallel tests for each compound. The MIC value of the compound is the first well that does not become cloudy (indicating no bacterial growth) as observed by the naked eye.

[0084] Table 1. MIC results of the compounds of this invention against Escherichia coli and Staphylococcus aureus.

[0085]

[0086] As shown in Table 1, compounds 1 through 11 of the present invention all exhibit a certain degree of antibacterial activity, which can be summarized as follows:

[0087] (1) Compound 3 has a MIC of 0.25 μg / mL against Escherichia coli, which is superior to the antibacterial activity of the positive control drugs tigecycline and amoxicillin;

[0088] (2) Compound 3 has a MIC of 0.25 μg / mL against Staphylococcus aureus, which is superior to the antibacterial activity of the positive control drugs vancomycin, tigecycline and amoxicillin.

[0089] (3) Other compounds have an anti-Staphylococcus aureus MIC of 1-4 μg / mL, and have good antibacterial activity.

[0090] Example 5: Evaluation of antibacterial activity against Enterococcus faecalis and various Staphylococcus species.

[0091] The activity of the compounds synthesized in this invention against Enterococcus faecalis, various Staphylococcus aureus, and various Bacillus was evaluated using a micro-broth dilution method. Since the Enterococcus faecalis, various Staphylococcus aureus, and various Bacillus used can grow rapidly under aerobic conditions, cationic-regulated MHB broth (CAMHB broth) was used as the culture medium. The specific operating steps are as follows:

[0092] (1) Prepare an antibacterial compound solution with a concentration of 25600 μg / mL and store it in a refrigerator at 4℃ for later use.

[0093] (2) For the test bacteria, *Enterococcus faecalis*, *S. capitis*, *S. epidermidis*, *S. lentus*, *Shigella flexneri*, *Enterobacter cloacae*, and *Enterobacter sakazakii* were picked up with an inoculation loop, and single colonies of 0.5–1 mm were inoculated into CAMHB broth medium and incubated on a shaker at 37°C and 220 rpm for 4 hours. The culture was then diluted to approximately 1 × 10⁻⁶ bacteria. 5 Prepare a bacterial culture at CFU / mL for later use.

[0094] (3) Take 10 μL of the antibacterial compound solution with a concentration of 25600 μg / mL into a 1.5 mL sterile centrifuge tube, and add 990 μL of sterile ultrapure water to dilute it to a compound solution with a concentration of 256 μg / mL.

[0095] (4) Take a 96-well plate and add 100 μL of CAMHB broth to each well. Then add 100 μL of the compound solution to the first well. Dilute stepwise using the two-fold dilution method to obtain the antibacterial compound solution. The concentrations of the first to tenth wells are 128, 64, 32, 16, 8, 4, 2, 1, 0.5, and 0.25 μg / mL, respectively. Finally, add 100 μL of the prepared bacterial suspension to make the total volume of each well 200 μL. The eleventh well contains 200 μL of CAMHB broth, and the twelfth well contains 100 μL of CAMHB broth and 100 μL of bacterial suspension as a blank control. Place the inoculated 96-well plate in a 37°C incubator and observe the bacterial growth for 16 hours. Vancomycin, erythromycin, tigecycline, and chlorhexidine are used as positive controls. Perform at least three parallel tests for each compound. The MIC value of the compound is the first well that does not become cloudy (indicating no bacterial growth) as observed by the naked eye.

[0096] Table 2. MIC results of the compounds of this invention against Enterococcus faecalis.

[0097]

[0098]

[0099] As shown in Table 2, the compounds of this invention exhibit certain anti-Enterococcus faecalis activity, which can be summarized as follows:

[0100] The compounds of this invention have MIC values ​​of 1–16 μg / mL against Enterococcus faecalis. Among them, compounds 2 and 3 have an MIC value of 1 μg / mL against Enterococcus faecalis, exhibiting significant antibacterial activity superior to the positive control drug vancomycin.

[0101] Table 3. MIC results of the compounds of this invention against Staphylococcus aureus.

[0102]

[0103] As shown in Table 3, the compounds of this invention exhibit strong anti-staphylococcal activity, which can be summarized as follows:

[0104] (1) The MIC values ​​of the compounds of the present invention against Staphylococcus aureus are 1 to 16 μg / mL. Among them, the MIC values ​​of compounds 3 and 4 against Staphylococcus aureus are 1 μg / mL and 2 μg / mL, respectively, which are similar to the activity of the positive control levofloxacin.

[0105] (2) The MIC values ​​of the compounds of the present invention against Staphylococcus aureus are 0.5–16 μg / mL. Among them, the MIC values ​​of compounds 3 and 4 against Staphylococcus aureus are 0.5 μg / mL, which are similar to the activity of the positive control drug levofloxacin.

[0106] (3) The MIC values ​​of the compounds of the present invention against Staphylococcus epidermidis are 0.5–16 μg / mL. Among them, the MIC values ​​of compounds 3 and 4 against Staphylococcus epidermidis are 0.5 μg / mL, which is the same as the activity of the positive control levofloxacin; the MIC values ​​of compounds 1 and 2 against Staphylococcus epidermidis are 4 μg / mL, which show good antibacterial activity.

[0107] Table 4. MIC results of the compounds of this invention against bacilli.

[0108]

[0109] As shown in Table 4, the compounds of this invention exhibit strong antibacterial activity, which can be summarized as follows:

[0110] (1) The MIC values ​​of the compounds of the present invention against Shigella are 0.25 to 16 μg / mL, and the MIC value of compound 1 against Shigella is 0.25 μg / mL, which is comparable to the activity of the positive control drug levofloxacin.

[0111] (2) The MIC values ​​of the compounds of the present invention against Enterobacter cloacae are 0.25-32 μg / mL, and the MIC value of compound 3 is 0.25 μg / mL, which is comparable to the activity of the positive control drug levofloxacin.

[0112] (3) The MIC values ​​of the compounds of the present invention against Enterobacter sakazakii are 2-32 μg / mL, and the MIC value of compound 3 is 2 μg / mL, which is superior to the positive control drug ampicillin.

[0113] As shown in Tables 1-4, these compounds exhibit good antibacterial activity against Enterococcus faecalis, various Staphylococcus species, and various bacilli.

[0114] Microscopic images of the antibacterial activity of compound 3 against Staphylococcus aureus in Experiment Example 6.

[0115] The Staphylococcus aureus activity of the compounds synthesized in this invention was evaluated using the micro-broth dilution method. The Staphylococcus aureus used can grow rapidly under aerobic conditions; therefore, cationic-regulated MHB broth (CAMHB broth) was used as the culture medium. The specific operating steps are as follows:

[0116] (1) Prepare an antibacterial compound solution with a concentration of 25600 μg / mL and store it in a refrigerator at 4℃ for later use.

[0117] (2) For the test bacteria Staphylococcus aureus, pick up single colonies of 0.5-1 mm with an inoculation loop and inoculate them into CAMHB broth medium. Incubate on a shaker at 37°C and 220 rpm for 4 hours, then dilute to approximately 1×10⁻⁶ bacteria. 5 Prepare a bacterial culture at CFU / mL for later use.

[0118] (3) Take 10 μL of the antibacterial compound solution with a concentration of 25600 μg / mL into a 1.5 mL sterile centrifuge tube, and add 990 μL of sterile ultrapure water to dilute it to a compound solution with a concentration of 256 μg / mL.

[0119] (4) Take a 96-well plate and add 100 μL of CAMHB broth to each well. Then add 100 μL of the compound solution to the first well. Dilute stepwise using the two-fold dilution method to obtain the antibacterial compound solution. The concentrations of the first to tenth wells are 128, 64, 32, 16, 8, 4, 2, 1, 0.5, and 0.25 μg / mL, respectively. Finally, add 100 μL of the prepared bacterial suspension to make the total volume of each well 200 μL. The eleventh well contains 200 μL of CAMHB broth, and the twelfth well contains 100 μL of CAMHB broth and 100 μL of bacterial suspension as a blank control. Place the inoculated 96-well plate in a 37°C incubator and observe the bacterial growth for 16 hours. Vancomycin is used as a positive control. Each compound is tested in at least three parallel experiments. The MIC value of the compound is the first well that does not become cloudy (indicating no bacterial growth) as observed by the naked eye. Take 10 μL from the well containing the compound's MIC value, drop it onto a glass slide, and stain it with crystal violet. Observe under a 20x microscope. The following results are shown in the appendix. Figure 2 .

[0120] As we can see from the images, compound 3 has a better bactericidal effect against Staphylococcus aureus than ethacridine lactate and vancomycin.

[0121] Experiment Example 7: Bactericidal Kinetics Experiment

[0122] (1) Using an inoculation loop, pick single colonies of Staphylococcus aureus and Escherichia coli, each measuring 0.5–1 mm, and inoculate them into MHB broth medium. Incubate on a shaker at 37°C and 220 rpm for 16–18 h, then dilute with MHB broth medium at a concentration of 1 × 10⁻⁶. 4 The sample was divided into 7 EP tubes and cultured for 2 hours. Then, 4×MIC compound and ethacridine lactate (control drug) were added and cultured for another 2 hours.

[0123] (2) At each specified time point of 0, 1, 2, 3, 4, and 6 h after adding the compound, 100 μL of bacterial culture was aspirated and centrifuged at 3500 rpm for 2 min. The supernatant was discarded, the culture was resuspended in PBS, and then diluted 10-fold. 100 μL of the diluted bacterial culture was evenly spread on MHA solid agar plates and incubated in a constant temperature incubator at 37 °C.

[0124] (3) After incubation for 16 hours, calculate the total number of bacteria on the agar plate. Then, calculate the number of colonies per milliliter based on the dilution factor.

[0125] The broken line was obtained by calculating the number of colonies per milliliter. Figure 3 (Staphylococcus aureus) and Figure 4 (E. coli). Through... Figure 3 It can be seen that for compound 3, when Staphylococcus aureus was administered at 4×MIC, all bacteria were killed after 3 hours of treatment, while some bacteria remained unkilled after 6 hours for the control drug ethacridine lactate. This demonstrates that compound 3 has a much faster rate of killing Staphylococcus aureus than ethacridine lactate. Figure 4 It can be seen that for compound 3, when E. coli was administered 4×MIC, the bacteria were completely killed after 3 hours of treatment, while the control drug ethacridine lactate still had some bacteria remaining after 6 hours. This shows that compound 3 kills E. coli much faster than ethacridine lactate.

[0126] Experiment 8: DNA Binding Experiment

[0127] Compound 3 was prepared to a concentration of 10. -5 The test solution for M was prepared by adding ctDNA to a concentration of 2.5 × 10⁻⁶. -5 The mother liquor of M. A certain volume of 10 -5 The M compound solution was placed in a cuvette, and ctDNA stock solution was continuously added dropwise to increase the ctDNA concentration in the cuvette from 1 × 10⁻⁶. -5 M increases gradually to 10 × 10 -5 M, measuring the ultraviolet absorption spectrum of DNA. For example... Figure 5 As shown in the formula. The binding constant (Kb) of compound 4 with calf thymus DNA was calculated to be 2.0 × 10⁻⁶. 4 .

Claims

1,9-Aminoacridine compounds, characterized in that, Selected from one of the following compounds: 。 2. The use of the 9-aminoacridine compound according to claim 1 in the preparation of antibacterial drugs, characterized in that, As an active ingredient, it or its hydrochloride, lactate or quaternary ammonium salt is prepared into an antibacterial drug.

3. The use of the 9-aminoacridine compound according to claim 2 in the preparation of antibacterial drugs, characterized in that, It or its hydrochloride, lactate or quaternary ammonium salt can be prepared into broad-spectrum antibacterial drugs.

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