9-amino acridine compound with multiple electron-withdrawing substituents as well as preparation method and application of 9-amino acridine compound

Abstract

The invention belongs to the technical field of medical chemistry, and discloses an aminoacridine compound with multiple electron withdrawing substituents, which has broad-spectrum antibacterial activity and can quickly sterilize, and a preparation method and application thereof. The compound is prepared through Ullmann coupling reaction, ring closing reaction and ammonolysis reaction, and the structure of the compound is shown as a formula I-a formula III. Bacteriostatic activity experiment research shows that the compound has definite bacteriostatic activity to various cocci and bacilli, the minimum inhibitory concentration (MIC) to staphylococcus aureus and staphylococcus epidermidis reaches 0.25 mu g / mL, and the minimum inhibitory concentration (MIC) to staphylococcus capitis can reach 0.5 mu g / mL. The compound can be used for developing antibacterial drugs, and has the advantages of high antibacterial activity, wide antibacterial spectrum, rapid sterilization and the like. And # imgabs0 #.

Description

Technical Field

[0001] The invention belongs to the field of pharmaceutical chemistry and discloses a 9-aminoacridine compound containing multiple electron-withdrawing substituents and having antibacterial effect, a preparation method and application thereof. Background Art

[0002] Penicillin was used in the 1940s to treat diseases caused by infections such as diphtheria, anthrax, streptococci and pneumococci. The discovery of penicillin opened up the history of human application of antibiotics and became one of the greatest discoveries in the medical field in the 20th century.

[0003] During clinical use, penicillin revealed many shortcomings, such as instability to acid, narrow antibacterial spectrum, inability to be taken orally, only injection, and easy development of drug resistance. Subsequently, penicillin was structurally modified, and a series of penicillin structural modifications (Formula IV) were obtained. These modifications showed better clinical application value, such as enzyme-resistant penicillin oxacillin sodium can be used for penicillin G-resistant Staphylococcus aureus infections, acid-resistant penicillin pheneticillin can be taken orally, and broad-spectrum penicillin amoxicillin expands the antibacterial spectrum.

[0004]

[0005] The structural modification of penicillin has obtained a series of antibiotics with better application effects. There are many similar examples. For example, the modification of natural cephalosporin C has successively obtained a total of 5 generations of cephalosporin drugs, such as the first-generation cephalosporin drug cefuroxil, the second-generation cephalosporin drug cefuroxime, the third-generation cephalosporin drug ceftriaxone, the fourth-generation cephalosporin drug cefazolin and the fifth-generation cephalosporin ceftobiprole (Formula V). These cephalosporins have undergone changes in antibacterial effects. The antibacterial activity of the first, second and third generation cephalosporins against Gram-positive cocci has gradually weakened, but the antibacterial activity against Gram-negative bacilli has gradually increased. The fourth generation cephalosporins are mainly suitable for infections with multidrug-resistant Gram-negative bacteria, and the fifth generation cephalosporins are mainly suitable for infections with multidrug-resistant Gram-positive bacteria.

[0006]

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

[0008] Antibiotics are prone to develop drug resistance during use, and bacterial resistance poses a serious threat to human health and economic development worldwide. In 2019 alone, about 5 million people died worldwide from 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 released the Global Antimicrobial Resistance and Use Surveillance System Report (②WHO. Global antimicrobial resistance and use surveillance system (GLASS) report: 2022. https: / / www.who.int / publications / i / item / 9789240062702), which analyzed the trend of antimicrobial resistance since 2017, focusing on monitoring bloodstream infections, gastrointestinal infections, gonorrhea and urinary tract infections, and the drug resistance of 8 bacterial pathogens related to the above diseases (Acinetobacter, Escherichia coli, Klebsiella pneumoniae, Salmonella, Staphylococcus aureus, Streptococcus pneumoniae, Shigella, Neisseria gonorrhoeae), and found that the bloodstream infection pathogens reported in 76 countries and regions, "super bacteria" third-generation cephalosporin-resistant Escherichia coli and methicillin-resistant Staphylococcus aureus infection rates were 42% and 35% respectively. It can be seen that the problem of bacterial resistance is becoming more and more serious.

[0009] The mechanism of antibiotic resistance has also received widespread attention from scientific and technological personnel, but there are still many unknowns about the exact mechanism of bacterial resistance (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 released its latest report on antimicrobial drugs (including antibiotics) in clinical and preclinical development worldwide. The report pointed out that although the number of antimicrobial drugs in clinical development increased from 80 in 2021 to 97 in 2023, new innovative drugs are still urgently needed to treat serious infections and replace those that have become ineffective due to widespread use (⑥https: / / www.who.int / news / item / 14-06-2024-who-releases-report-on-state-of-development-of-antibacterials). In short, antibiotic resistance is an increasingly serious global problem, and the discovery of antibiotics with new structural features will provide new options for overcoming antibiotic resistance. Summary of the invention

[0011] 9-aminoacridine, ethacridine lactate, and acridinium hydrochloride (Formula VI) are all acridine compounds with antibacterial effects (⑦Pengfei She, et al. Repurposing 9-aminoacridine as an adjuvant enhances the antimicrobial effects of rifampin against multidrug-resistant klebsiellapneumonia. Microbiol Spectr. 2023, 11(3): 1; ⑧Yamamoto, 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. Intravenous injection of acriflavine hydrochloride to treat 54 cases of contagious pleuropneumonia in calves. Heilongjiang Animal Husbandry and Veterinary Medicine, 2011, (04): 76). Among them, ethacridine lactate is widely used for wound debridement and disinfection, and acriflavine hydrochloride is widely used as a veterinary drug to prevent and treat intestinal infections in cattle, sheep, pigs and other animals.

[0012]

[0013] As can be seen from Formula VI, 9-aminoacridine, ethacridine lactate, and acridinium hydrochloride are all acridine compounds. For the convenience of description, we mark the three aromatic rings contained in acridine as ring A, ring B, and ring C in turn (Formula VII). The common structural feature of the above three acridine compounds is that the substituents on the acridine mother ring are all electron-donating amino or ethoxy substituents. Further literature research also found that the structural features of the reported acridine compounds with antibacterial effects are all compounds containing electron-donating substituents.

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

[0015]

[0016] The compound 1 discovered by the applicant has an acridine mother ring and has the following two significant differences compared with the known acridine compounds with antibacterial effects, such as 9-aminoacridine, ethacridine lactate, and acriflavine hydrochloride:

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

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

[0019] Antibacterial activity of acridine compounds

[0020]

[0021] Based on the above findings, the applicant conducted a systematic study on the design, synthesis and antibacterial activity evaluation of aminoacridine compounds containing electron-withdrawing substituents after extensive literature research. The newly designed compound retains the structural characteristics of compound 1, that is, the A ring and C ring of 9-aminoacridine both contain electron-withdrawing substituents. After further synthesis and antibacterial activity evaluation, beneficial research results were obtained.

[0022] To this end, 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 of the present invention have the structural formulas shown in Formula I to Formula 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 thereof, or solvates thereof, or hydrates thereof, or prodrugs thereof, 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 starting materials, they reacted with 4-nitro-2-chlorobenzoic acid, 5-nitro-2-chlorobenzoic acid and 5-fluoro-2-bromobenzoic acid to produce intermediates 1a-11a;

[0031]

[0032] (2) Intermediates 1a-11a undergo ring-closure reaction with phosphorus oxychloride to obtain intermediates 1b-11b;

[0033]

[0034] (3) Intermediate 1b-11b is subjected to ammonolysis reaction with phenol and ammonium carbonate to obtain target compound 1-11;

[0035]

[0036] The applicant of the present invention has conducted research on 9-aminoacridine compounds with multiple electron-withdrawing substituents, obtained compounds 1-11 shown in the above formula I to formula III, and confirmed their structures. The contents were all greater than 99.0% by high-performance liquid chromatography analysis. On this basis, the obtained compounds were evaluated for activity. The present invention also provides the activity evaluation method and results of the aforementioned compounds, including the following contents:

[0037] The in vitro antibacterial activity was evaluated by the microbroth dilution method: the aforementioned compounds of the present invention were prepared into an antibacterial compound solution with a concentration of 256 μg / mL. After adding MH broth culture medium to a 96-well plate, the antibacterial compound solution was diluted into 10 different concentration gradients of 128, 64, 32, 16, 8, 4, 2, 1, 0.5, and 0.25 μg / mL by the two-fold dilution method. Escherichia coli (Gram-negative bacteria) and Staphylococcus aureus (Gram-positive bacteria) were used as the test bacteria, and vancomycin, meropenem, tigecycline, and amoxicillin were used as positive control drugs. The inoculated 96-well plate was placed in a 37°C incubator for culture. After 16 hours, the growth of the bacterial solution was observed. The first well plate that did not become turbid by naked eye observation was the minimum inhibitory concentration (MIC) of the compound. The antibacterial effects of some compounds of the present invention in 96-well plates are shown in the figure. Figure 1 shown.

[0038] The 9-aminoacridine compounds with multiple electron-withdrawing substituents of the present invention show good activity against a variety of cocci, indicating that the compounds have excellent antibacterial activity. In particular, the MIC values ​​of compound 1, compound 3 and compound 8 against Staphylococcus aureus are between 0.25 and 1 μg / mL, which is better than the antibacterial activity of clinical drugs amoxicillin and tigecycline; the MIC values ​​of compound 2 and compound 3 against Enterococcus faecalis are 1 μg / mL, and the antibacterial effect is better than the positive control drug vancomycin; the MIC values ​​of compound 3 and compound 4 against Staphylococcus albus are 1 μg / mL and 2 μg / mL, respectively, which are similar to the antibacterial activity of the positive control drug levofloxacin. The MIC values ​​of compound 3 and compound 4 against Staphylococcus capitis are 0.5 μg / mL, which is similar to the antibacterial activity of the positive control drug levofloxacin; the 9-aminoacridine compounds with multiple electron-withdrawing substituents of the present invention show significant antibacterial activity against a variety of bacilli. The MIC values ​​of compound 1 and compound 3 against Shigella dysenteriae were 0.5 μg / mL and 1 μg / mL, respectively, which were similar to the positive drug levofloxacin. The MIC value of compound 3 against Enterobacter sakazakii was 2 μg / mL, which was better than the positive control drug ampicillin.

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

[0040] The present invention discloses a 9-aminoacridine compound with multiple electron-withdrawing substituents having antibacterial activity, a preparation method and a use thereof. The compound exhibits excellent antibacterial effect in an antibacterial activity test, and the minimum inhibitory concentration MIC reaches 0.25 μg / mL. The compound prepared by the present invention can be used for the development of antibacterial drugs, has the advantages of high antibacterial activity, broad antibacterial spectrum, and rapid sterilization, and provides a new option for the development of drugs with antibacterial activity.

[0041] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 The antibacterial effects of some compounds of the present invention in 96-well plates are shown;

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

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

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

[0046] Figure 5 It is the ultraviolet absorption spectrum diagram of the interaction between compound 3 of the present invention and DNA. DETAILED DESCRIPTION

[0047] The following is a further detailed description of the above contents of the present invention through specific implementation examples in the form of experimental examples, but this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to 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 the detection and analysis of the present invention are as follows:

[0050] Nuclear magnetic resonance instrument: Advance Digital 400 nuclear magnetic resonance instrument produced by Bruker, Switzerland.

[0051] High-resolution mass spectrometry, solanX 70FT-MS from Bruker, Switzerland; Agilent 6540TOF.

[0052] Sterilizer, MVS-83 vertical pressure steam sterilizer produced by Bingshan Songyang Biotechnology (Dalian) Co., Ltd.

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

[0054] Shaker, THZ-412 desktop constant temperature oscillator from Shanghai Jinghong Experimental Equipment Co., Ltd.

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

[0056] Experimental Examples 1 to 3 are experiments on the synthesis of compounds, and Experimental Examples 4 to 8 are experiments on the activity evaluation of compounds.

[0057] Experimental Example 1 Preparation of Series 1 Compounds Using Compound 1 as an Example

[0058] In a three-necked flask (250 mL), 2-chloro-5-nitrobenzoic acid (10 g, 0.05 mol, 1 eq) was dissolved in N, N-dimethylformamide (40 mL), and then 4-fluoroaniline (6.67 g, 0.06 mol, 1.2 eq), potassium carbonate (13.82 g, 0.1 mol, 2.0 eq), potassium iodide (8.30 g, 0.05 mol, 1 eq), copper powder (0.32 g, 0.005 mol, 0.1 eq) were added and heated to reflux for 10 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 the excess copper powder and potassium carbonate were removed by suction filtration. The filtrate was adjusted to a pH of 4 with 2 mol / L hydrochloric acid, and then filtered and dried to obtain a yellow solid, which was called intermediate 1a, for the next reaction.

[0059] In a three-necked flask (250 mL), the 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, heated to reflux for 2 h, and 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, toluene and phosphorus oxychloride were dried, crushed ice and ammonia were added, and stirred at room temperature for 30 min, 50 mL of DCM was added for extraction, washed with saturated brine, dried over anhydrous sodium sulfate, and the organic phase was dried with a rotary evaporator, and washed with 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) as solvent at 120 °C for 4 h. The reaction progress was monitored by TLC (petroleum ether: ethyl acetate = 1:1). After the reaction, the system was cooled to room temperature, 10 mL of 10% aqueous sodium hydroxide solution was added to remove excess phenol, and red solid compound 1 was obtained by filtration.

[0061]

[0062]

[0063] Experimental Example 2 Preparation of Series 2 Compounds Using Compound 3 as an Example

[0064] In a three-necked flask (250 mL), 2-chloro-4-nitrobenzoic acid (10 g, 0.05 mol, 1 eq) was dissolved in N, N-dimethylformamide (40 mL), and then 4-chloroaniline (6.67 g, 0.06 mol, 1.2 eq), potassium carbonate (13.82 g, 0.1 mol, 2.0 eq), potassium iodide (8.30 g, 0.05 mol, 1 eq), copper powder (0.32 g, 0.005 mol, 0.1 eq) were added and heated to reflux for 10 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 the excess copper powder and potassium carbonate were removed by suction filtration. The filtrate was adjusted to pH 4 with 2 mol / L hydrochloric acid, and then filtered and dried to obtain a yellow solid, which was called intermediate 3a, for the next reaction.

[0065] In a three-necked flask (250 mL), the intermediate 3a prepared above (10.5 g, 0.038 mol, 1 eq) was dissolved in toluene (15 mL), phosphorus oxychloride (5.81 g, 0.038 mol, 1 eq) was added under ice bath conditions, heated to reflux for 2 h, and 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, toluene and phosphorus oxychloride were dried, crushed ice and ammonia were added, stirred at room temperature for 30 min, 50 mL of DCM was added for extraction, washed with saturated brine, dried over anhydrous sodium sulfate, and the organic phase was dried with a rotary evaporator, and washed with 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) as solvent at 120 °C for 4 h. The reaction progress was monitored by TLC (petroleum ether: ethyl acetate = 1:1). After the reaction, the system was cooled to room temperature, 10 mL of 10% aqueous sodium hydroxide solution was added to remove excess phenol, and red solid compound 3 was obtained by filtration.

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

[0068]

[0069]

[0070] Experimental Example 3 Preparation of Series 3 Compounds Using Compound 8 as an Example

[0071] In a three-necked flask (250 mL), 2-bromo-5-fluorobenzoic acid (10 g, 0.05 mol, 1 eq) was dissolved in N, N-dimethylformamide (40 mL), and then 3-fluoroaniline (6.67 g, 0.06 mol, 1.2 eq), potassium carbonate (13.82 g, 0.1 mol, 2.0 eq), potassium iodide (8.30 g, 0.05 mol, 1 eq), copper powder (0.32 g, 0.005 mol, 0.1 eq) were added and heated under reflux for 10 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 the excess copper powder and potassium carbonate were removed by suction filtration. The filtrate was adjusted to pH 4 with 2 mol / L hydrochloric acid, and then filtered and dried to obtain a yellow solid, which was called intermediate 8a, for the next reaction.

[0072] In a three-necked flask (250 mL), the intermediate 8a prepared above (10.5 g, 0.038 mol, 1 eq) was dissolved in toluene (15 mL), phosphorus oxychloride (5.81 g, 0.038 mol, 1 eq) was added under ice bath conditions, heated to reflux for 2 h, and the reaction progress was monitored by TLC (petroleum ether: ethyl acetate = 3: 1). After the reaction, the system was cooled to room temperature, toluene and phosphorus oxychloride were dried, crushed ice and ammonia were added, stirred at room temperature for 30 min, 50 mL of DCM was added for extraction, washed with saturated brine, dried over anhydrous sodium sulfate, the organic phase was dried with a rotary evaporator, and washed with 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) as solvent at 120 °C for 4 h. The reaction progress was monitored by TLC (petroleum ether: ethyl acetate = 1:1). After the reaction, the system was cooled to room temperature, 10 mL of 10% aqueous sodium hydroxide solution was added to remove excess phenol, and red solid compound 8 was obtained by filtration.

[0074] According to the preparation method of Experimental Example 3, compounds 9 to 11 were obtained:

[0075]

[0076]

[0077] The following experimental examples 4 to 8 are used to illustrate the beneficial effects of the compounds of the present invention.

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

[0079] The microbroth dilution method was used to evaluate the activity of the compound synthesized in the present invention against Escherichia coli and Staphylococcus aureus, both of which are facultative anaerobic bacteria, and the culture medium was MHB broth. The specific operation steps are as follows:

[0080] (1) The test compound was prepared into an antibacterial compound solution with a concentration of 25600 μg / mL and stored in a refrigerator at 4°C for later use.

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

[0082] (3) Take 10 μL of the antibacterial compound solution with a concentration of 25600 μg / mL in 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, add 100 μL of MHB broth medium to each well, then add 100 μL of compound solution to the first well, and dilute stepwise by two-fold dilution method to obtain 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 solution to make the total volume of each well 200 μL. The eleventh well is 200 μL of MHB broth medium, and the twelfth well is added with 100 μL of MHB broth medium and 100 μL of bacterial solution as a blank control. The inoculated 96-well plate is placed in a 37°C incubator for incubation, and the growth of the bacterial solution is observed for 16 hours. At the same time, vancomycin, meropenem, tigecycline and amoxicillin are used as positive control drugs. Each compound is tested in parallel at least three times, and the first well plate that does not become turbid (indicating no bacterial growth) observed by naked eye is the MIC value of the compound.

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

[0085]

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

[0087] (1) The MIC of compound 3 against Escherichia coli reached 0.25 μg / mL, which was superior to the antibacterial activity of positive control drugs tigecycline and amoxicillin;

[0088] (2) The MIC of compound 3 against Staphylococcus aureus reached 0.25 μg / mL, which was superior to the antibacterial activity of the positive control drugs vancomycin, tigecycline, and amoxicillin;

[0089] (3) The MIC of other compounds against Staphylococcus aureus was 1-4 μg / mL, which showed good antibacterial effect.

[0090] Experimental Example 5 Evaluation of antibacterial activity against Enterococcus faecalis and various Staphylococci

[0091] The microbroth dilution method was used to evaluate the activity of the compound synthesized by the present invention on Enterococcus faecalis, various Staphylococci, and various Bacilli. The Enterococcus faecalis, various Staphylococci, and various Bacilli used can grow rapidly under aerobic conditions. Therefore, the culture medium uses cation-adjusted MHB broth (CAMHB broth). The following are the specific steps:

[0092] (1) The test compound was prepared into an antibacterial compound solution with a concentration of 25600 μg / mL and stored in a refrigerator at 4°C for later use.

[0093] (2) For the test bacteria Enterococcus faecalis, Staphylococcus capitis, Staphylococcus epidermidis, Staphylococcus lentus, Shigella flexneri, Enterobacter cloacae, and Enterobacter sakazakii, a single colony of 0.5 to 1 mm was picked up with an inoculation loop and inoculated into CAMHB broth. The culture was incubated on a shaker at 37°C and 220 rpm for 4 h, and then diluted to a bacterial count of about 1 × 10 5 CFU / mL of bacterial solution for future use.

[0094] (3) Take 10 μL of the antibacterial compound solution with a concentration of 25600 μg / mL in 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, add 100 μL of CAMHB broth medium to each well, then add 100 μL of compound solution to the first well, and dilute it step by step by two-fold dilution method to obtain 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 solution to make the total volume of each well 200 μL. The eleventh well is 200 μL of CAMHB broth medium, and the twelfth well is added with 100 μL of CAMHB broth medium and 100 μL of bacterial solution as a blank control. The inoculated 96-well plate is placed in a 37°C incubator for incubation, and the growth of the bacterial solution is observed for 16 hours. At the same time, vancomycin, erythromycin, tigecycline and chlorhexidine are used as positive control drugs. Each compound is tested in parallel at least three times, and the first well plate that does not become turbid (indicating no bacterial growth) observed by naked eye is the MIC value of the compound.

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

[0097]

[0098]

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

[0100] The MIC value of the compounds of the present invention to Enterococcus faecalis is 1-16 μg / mL. Among them, the MIC value of Compound 2 and Compound 3 to Enterococcus faecalis is 1 μg / mL, showing a significant antibacterial effect, which is better than the positive control drug vancomycin.

[0101] Table 3 MIC results of the compounds of the present invention against Staphylococcus

[0102]

[0103] As can be seen from Table 3, the compounds of the present 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 albus are 1 to 16 μg / mL. Among them, the MIC values ​​of compound 3 and compound 4 against Staphylococcus albus 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 capitis are 0.5-16 μg / mL. Among them, the MIC values ​​of compounds 3 and 4 against Staphylococcus capitis 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 to 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 positive control levofloxacin; the MIC values ​​of compounds 1 and 2 against Staphylococcus epidermidis are 4 μg / mL, showing good antibacterial effects.

[0107] Table 4 MIC results of the compounds of the present invention against Bacillus

[0108]

[0109] As can be seen from Table 4, the compounds of the present 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 dysenteriae are 0.25-16 μg / mL, and the MIC value of compound 1 against Shigella dysenteriae is 0.25 μg / mL, which is equivalent to the activity of the positive drug levofloxacin.

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

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

[0113] According to Tables 1 to 4, this class of compounds showed good antibacterial activity against Enterococcus faecalis, various Staphylococci and various Bacilli.

[0114] Experimental Example 6 Microscope image of the antibacterial activity of compound 3 against Staphylococcus aureus

[0115] The microbroth dilution method was used to evaluate the activity of the compound synthesized by the present invention against Staphylococcus aureus. The Staphylococcus aureus used can grow rapidly under aerobic conditions. Therefore, the culture medium used was cation-adjusted MHB broth (CAMHB broth). The specific operation steps are as follows:

[0116] (1) The test compound was prepared into an antibacterial compound solution with a concentration of 25600 μg / mL and stored in a refrigerator at 4°C for later use.

[0117] (2) Use an inoculation loop to pick up a single colony of 0.5-1 mm for the test bacteria Staphylococcus aureus and inoculate it into CAMHB broth medium. Incubate it on a shaker at 37°C and 220 rpm for 4 h, then dilute it to a concentration of about 1×10 5 CFU / mL of bacterial solution for future use.

[0118] (3) Take 10 μL of the antibacterial compound solution with a concentration of 25600 μg / mL in 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, add 100 μL of CAMHB broth medium to each well, then add 100 μL of compound solution to the first well, and dilute stepwise by two-fold dilution method to obtain 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 solution to make the total volume of each well 200 μL. The eleventh well is 200 μL of CAMHB broth medium, and the twelfth well is added with 100 μL of CAMHB broth medium and 100 μL of bacterial solution as a blank control. The inoculated 96-well plate is placed in a 37°C incubator for incubation, and the growth of the bacterial solution is observed for 16 hours. At the same time, vancomycin is used as a positive control drug. Each compound is tested in parallel at least three times, and the first well plate that does not become turbid (indicating no bacterial growth) observed by naked eye is the MIC value of the compound. Take out 10 μL from the well with the compound MIC value, drop it on the slide, and stain it with crystal violet. Observe it under a 20x microscope and get the following results (see attached) Figure 2 .

[0120] From the pictures we can see that compound 3 has better bactericidal effect on Staphylococcus aureus than ethacridine lactate and vancomycin.

[0121] Experimental Example 7 Bactericidal kinetics experiment

[0122] (1) Use an inoculation loop to pick up single colonies of Staphylococcus aureus and Escherichia coli of 0.5-1 mm in size and inoculate them into MHB broth. Incubate on a shaker at 37°C and 220 rpm for 16-18 h. Then dilute 1×10 4 times and dispensed into 7 ep tubes respectively. After culturing for 2 h, 4×MIC compound and ethazacrilin lactate (control drug) were added and the culture was continued.

[0123] (2) Take 100 μL of bacterial solution at each designated time point of 0, 1, 2, 3, 4, and 6 h after adding the compound, centrifuge at 3500 rpm for 2 min. Pour off the supernatant, resuspend in PBS, and dilute it 10 times. Take 100 μL of the diluted bacterial solution and evenly spread it on the MHA solid agar plate and incubate it in a constant temperature incubator at 37°C.

[0124] (3) After incubation for 16 hours, count 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 is obtained by calculating the number of colonies per milliliter Figure 3 (Staphylococcus aureus) and Figure 4 (Escherichia coli). Figure 3 It can be seen that for compound 3, when Staphylococcus aureus was given 4×MIC, all the bacteria were killed after 3 hours, while some bacteria were not killed after 6 hours of the control drug ethacridine lactate. This shows that the killing speed of compound 3 against Staphylococcus aureus is much stronger than that of ethacridine lactate. Figure 4 It can be seen that for compound 3, when Escherichia coli was administered 4×MIC, all the bacteria were killed after 3 hours of action, while after 6 hours of the control drug ethacridine lactate, some bacteria were still not killed. This shows that the killing speed of compound 3 for Escherichia coli is much faster than that of ethacridine lactate.

[0126] Experimental Example 8 DNA Binding Experiment

[0127] Compound 3 was prepared at a concentration of 10 -5 M of the test solution, and the ctDNA was prepared to 2.5×10 -5 M mother liquor. -5 The M compound solution was placed in a cuvette, and the ctDNA mother solution was continuously added to it, so that the ctDNA concentration in the cuvette increased from 1×10 -5 M increases gradually to 10×10 -5 M, measure the UV absorption spectrum of DNA. Figure 5 As shown. Through the formula The binding constant Kb of compound 4 and calf thymus DNA was calculated to be 2.0×10 4 .

Claims

1. 9-aminoacridine compound, characterized in that: It has the structural formula shown in Formula I, Formula II and Formula III:

2. The 9-aminoacridine compound according to claim 1, characterized in that: The compound is selected from the following compounds:

3. A method for preparing the 9-aminoacridine compound according to claim 1, characterized in that: This is accomplished by the following steps: (1) Using aniline with different electron-withdrawing substituents as raw materials, they were subjected to Ullmann coupling reaction with 4-nitro-2-chlorobenzoic acid, 5-nitro-2-chlorobenzoic acid and 5-fluoro-2-bromobenzoic acid to obtain intermediates 1a-11a: (2) Intermediates 1a-11a undergo ring-closure reaction with phosphorus oxychloride to obtain intermediates 1b-11b: (3) Intermediate 1b-11b undergoes aminolysis reaction with phenol and ammonium carbonate to obtain target compound 1-11:

4. The pharmaceutical use of the 9-aminoacridine compound according to claim 1 or 2, characterized in that: As an active ingredient, it or its hydrochloride, lactate or quaternary ammonium salt is prepared into an antibacterial drug.

5. The pharmaceutical use of the 9-aminoacridine compound according to claim 4, characterized in that: The compound or its hydrochloride, lactate or quaternary ammonium salt is prepared into a broad-spectrum antibacterial drug.

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