Beta-diketone amide compound and application thereof in preparation of gram bacterium infection resisting medicine

Abstract

The invention provides a beta-diketoamide compound and application thereof in preparation of a gram infection resistant drug, the structural formula of the beta-diketoamide compound is as shown in formula (1), in the formula (1), R1 is H or halogen; r2 is nitro, alkyl, halogen or methoxyl. The beta-diketoamide compound disclosed by the invention comprises gram-positive bacteria such as staphylococcus aureus, enterococcus faecalis, enterococcus faecium and staphylococcus epidermidis and other clinical isolates, and shows antibacterial activity; after being combined with polymyxin, the compound has synergistic bacteriostatic activity on gram-negative bacteria such as acinetobacter baumannii, klebsiella pneumoniae, escherichia coli and pseudomonas aeruginosa, and can remove biofilms formed by staphylococcus aureus and enterococcus faecalis.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, in particular to beta-diketoamide compounds and applications thereof in preparing medicaments for resisting Gram infection. Background Art

[0002] Antibiotic resistance (AMR) is a core challenge facing global public health. The World Health Organization predicts that by 2050, AMR could cause 10 million deaths annually. ESKAPE pathogens, represented by Enterococcus faecalis, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Escherichia coli, are the main pathogens of hospital-acquired infections. These bacteria utilize multidrug resistance mechanisms to evade existing antimicrobial treatments. According to the National Bacterial Resistance Surveillance Network, Escherichia coli and Klebsiella pneumoniae are still the main isolates in infectious disease departments, highlighting the serious threat posed by drug-resistant bacteria to clinical treatment.

[0003] The formation of bacterial biofilms is one of the key mechanisms leading to increased drug resistance. Biofilms are complex three-dimensional structures formed by bacteria adhering to biological or non-biological surfaces and secreting an extracellular polymer matrix to wrap themselves. This unique structure not only provides a physical barrier for bacteria, reducing their sensitivity to antibiotics by 10-1000 times, but also effectively evades attacks from the host immune system. Data from the National Institutes of Health (NIH) show that at least 80% of human bacterial infections are associated with biofilms, including chronic and difficult-to-treat infections such as surgical site infections, osteomyelitis, and diabetic ulcers. The persistent inflammation and tissue damage caused by biofilms significantly increase the difficulty of treatment and patient mortality.

[0004] Existing antimicrobial strategies face multiple challenges in combating biofilm infections: traditional antibiotics have difficulty penetrating the biofilm matrix and are prone to inducing drug-resistant mutations; host immune cells are limited in their function within the biofilm microenvironment, making them unable to effectively eliminate bacteria. Therefore, developing novel antimicrobial agents that can target biofilm structures, interfere with bacterial adhesion and aggregation mechanisms, or disrupt the extracellular matrix has become crucial for overcoming current therapeutic bottlenecks. Summary of the Invention

[0005] To address the above technical issues, the present invention discloses β-diketoamide compounds and their use in the preparation of anti-Gram infection drugs. The β-diketoamide compounds exhibit antibacterial activity against clinical isolates of Gram-positive bacteria, including Staphylococcus aureus, Enterococcus faecalis, Enterococcus faecium, and Staphylococcus epidermidis. In particular, when combined with the clinical antibiotic polymyxin, they exhibit synergistic antibacterial activity against Gram-negative bacteria, such as Klebsiella pneumoniae and Escherichia coli, and can reverse polymyxin resistance. This provides a reference for the development of antibacterial drugs based on β-diketoamide compounds and for combination therapy against Gram-negative bacteria.

[0006] To this end, the technical solution adopted in the present invention is:

[0007] β-diketoamide compounds, whose structural formula is shown in formula (1):

[0008]

[0009] Among them, R 1 is H or halogen; R 2 is nitro, alkyl, halogen or methoxy.

[0010] The technical solution of the present invention discloses a new compound, such as the above-mentioned β-diketone amide compound, which has the effect of inhibiting the growth of Gram-positive bacteria and biofilm formation. The β-diketone amide compound combined with polymyxin has a synergistic inhibitory effect on the growth of Gram-negative bacteria.

[0011] As a further improvement of the present invention, the β-diketoamide compound is KL-A4, KL-A7, KL-A10, KL-A13, KL-A22 or KL-A23, and the structural formulas of KL-A4, KL-A7, KL-A10, KL-A13, KL-A22 and KL-A23 are shown in the following formulas (2) to (7):

[0012]

[0013] The above formulas (2) to (7) are named KL-A4, KL-A7, KL-A10, KL-A13, KL-A22 and KL-A23, respectively.

[0014] The present invention discloses the use of the above-mentioned β-diketoamide compounds in preparing drugs for resisting Gram-bacterial infection. The Gram-bacteria include Gram-positive bacteria and Gram-negative bacteria. The Gram-positive bacteria are at least one of Staphylococcus aureus, Enterococcus faecalis, Enterococcus faecium, Staphylococcus epidermidis, or Streptococcus pneumoniae; and the Gram-negative bacteria are at least one of Acinetobacter baumannii, Klebsiella pneumoniae, Escherichia coli, and Pseudomonas aeruginosa. The β-diketoamide compounds are used to prepare drugs for resisting Gram-positive bacterial infection; and the β-diketoamide compounds are used in combination with polymyxin to prepare drugs for resisting Gram-negative bacterial infection.

[0015] As a further improvement of the present invention, the drug comprises polymyxin and the β-diketone amide compound. The β-diketone amide compound and polymyxin have a synergistic effect of inhibiting the growth of Gram-negative bacteria.

[0016] As a further improvement of the present invention, the concentration of the β-diketoamide compound in the treatment system is not less than 0.20 μM.

[0017] As a further improvement of the present invention, the medicine is an injection, tablet, pill, capsule, suspension, granule, spray or emulsion.

[0018] The present invention also discloses the use of the above-mentioned β-diketoamide compound in preparing a coating for inhibiting Gram-positive bacteria. The coating is used on the surface of medical devices. The Gram-positive bacteria include Gram-positive bacteria and Gram-negative bacteria. The Gram-positive bacteria are at least one of Staphylococcus aureus, Enterococcus faecalis, Enterococcus faecium, Staphylococcus epidermidis, or Streptococcus pneumoniae. The Gram-negative bacteria are at least one of Acinetobacter baumannii, Klebsiella pneumoniae, Escherichia coli, and Pseudomonas aeruginosa. The β-diketoamide compound is used to prepare a coating for inhibiting Gram-positive bacteria. The β-diketoamide compound is combined with polymyxin to prepare a coating for inhibiting Gram-negative bacteria.

[0019] The invention discloses a coating for inhibiting Gram bacteria, which comprises the beta-diketoamide compound.

[0020] The present invention also discloses the use of the above-mentioned β-diketoamide compounds in preparing a disinfectant for inhibiting Gram-positive bacteria, wherein the Gram-positive bacteria include Gram-positive bacteria and Gram-negative bacteria, wherein the Gram-positive bacteria are at least one of Staphylococcus aureus, Enterococcus faecalis, Enterococcus faecium, Staphylococcus epidermidis, or Streptococcus pneumoniae, and the Gram-negative bacteria are at least one of Acinetobacter baumannii, Klebsiella pneumoniae, Escherichia coli, and Pseudomonas aeruginosa; the β-diketoamide compounds are used to prepare a disinfectant for inhibiting Gram-positive bacteria; and the β-diketoamide compounds are combined with polymyxin to prepare a disinfectant for inhibiting Gram-negative bacteria.

[0021] The invention discloses a Gram-inhibiting disinfectant, which comprises the beta-diketoamide compound.

[0022] The present invention also discloses a drug for resisting Gram-positive bacterial infection, comprising the above-mentioned β-diketoamide compound, wherein the Gram-positive bacteria is at least one of Staphylococcus aureus, Enterococcus faecalis, Enterococcus faecium, Staphylococcus epidermidis or Streptococcus pneumoniae.

[0023] The present invention also discloses a drug for resisting Gram-negative bacterial infection, comprising the above-mentioned β-diketoamide compound and polymyxin, wherein the Gram-negative bacteria is at least one of Acinetobacter baumannii, Klebsiella pneumoniae, Escherichia coli, and Pseudomonas aeruginosa.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] The technical solution of the present invention discloses a new β-diketoamide compound (including KL-A4, KL-A7, KL-A10, KL-A13, KL-A22 and KL-A23) and its medical use. The β-diketoamide compound (including KL-A4, KL-A7, KL-A10, KL-A13, KL-A22 and KL-A23) exhibits antibacterial activity against clinical isolates of Gram-positive bacteria including Staphylococcus aureus, Enterococcus faecalis, Enterococcus faecium, Staphylococcus epidermidis, and exhibits synergistic antibacterial activity against Gram-negative bacteria such as Acinetobacter baumannii, Klebsiella pneumoniae, Escherichia coli and Pseudomonas aeruginosa after combination with polymyxin. It can also eliminate the biofilms formed by Staphylococcus aureus and Enterococcus faecalis. In particular, KL-A10 showed the best antibacterial effect and exhibited minimal toxicity against mammalian cells, including normal human lung epithelial cells BEAS-2B and human hepatic stellate cells LX-2, at the MIC concentration, making it suitable for clinical use. This suggests that β-diketoamide compounds (including KL-A4, KL-A7, KL-A10, KL-A13, KL-A22, and KL-A23) have potential application in the clinical treatment of bacterial infections. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 3. The growth curves of KL-A10 of the embodiment of the present invention against Gram-positive bacteria (MSSA, MRSA, S.epidermidsSE1457, E.faecalis OG1RF); wherein (a) is for MSSA SA113, (b) is for MRSA YUSA145, (c) is for S.epidermids SE1457, and (d) is for E.faecalis OG1RF.

[0027] Figure 2 The data show the development of drug resistance to MRSAYUSA145 at 1 / 2×MIC for 32 consecutive days in the examples of the present invention, with linezolid (LZD) serving as a positive control.

[0028] Figure 3 The confocal laser scanning microscope observations of the effects of KL-A10, VAN, and KL-2 on the removal of mature biofilm of Staphylococcus aureus SA113 in an embodiment of the present invention are shown; wherein, (a) is a blank sample, (b) is 25 μM KL-2, (c) is 25 μM KL-2, (d) is 25 μM vancomycin, (e) is 25 μM KL-A10, and (f) is 50 μM KL-A10.

[0029] Figure 4The present invention is a checkerboard method analysis of the synergistic antibacterial activity of KL-A10 and polymyxin B against polymyxin-resistant strains of Gram-negative bacteria (Escherichia coli resistant strains and Klebsiella pneumoniae); wherein, (a) and (b) are respectively the checkerboard method analysis of the synergistic antibacterial activity results of KL-A10 and polymyxin B combination against Escherichia coli resistant strains MG1655 (MCR-1) and MG1655-PB, (c) and (d) are respectively the checkerboard method analysis of the synergistic antibacterial activity results of KL-A10 and polymyxin B combination against Klebsiella pneumoniae (e) and (f) are the results of the synergistic antibacterial activity of KL-2 combined with polymyxin B against Escherichia coli resistant strains MG1655 (MCR-1) and MG1655-PB, respectively, analyzed by the checkerboard assay. (g) and (h) are the results of the synergistic antibacterial activity of KL-2 combined with polymyxin B against Klebsiella pneumoniae resistant strains K2044 (MCR-1) and K2044-PB, respectively, analyzed by the checkerboard assay.

[0030] Figure 5 The present invention is an embodiment of the present invention, wherein (a) is a survival curve of the MRS A USA 300 infection model of the greater wax moth; wherein, (a) is a survival curve of 106 CFU of Staphylococcus aureus USA 300, treated with 25 μM and 50 μM KL-A10, and 6.25 μM VAN and 25 μM KL-2; (b) to (f) are representative images of the toxicity assay of the greater wax moth treated with drugs after 48 hours of treatment, wherein dark brown represents death; (b) is a blank sample, (c) PBS treatment group is used as a negative control, (d) is 25 μM KL-2, (e) is 6.25 μM vancomycin treatment as a positive control, and (f) is 25 μM KL-A10.

[0031] Figure 6 The lung bacterial load of MRSAUSA300 after treatment with KL-A10 of the present invention, KL-2 as a control, and normal saline (six mice in each group). *, p<0.05; **, p<0.01. DETAILED DESCRIPTION

[0032] The preferred embodiments of the present invention are described in further detail below.

[0033] β-diketoamide compounds, the structural formula of which is shown in formula (1); wherein, R 1 is H or a halogen group; R 2 is a substituent such as nitro, alkyl, halogen and methoxy.

[0034]

[0035] Specifically, the β-diketoamide molecular derivatives involved in this embodiment are shown in Table 1.

[0036] Table 1

[0037]

[0038]

[0039] The synthetic routes of the β-diketone amide compounds (KL-A1 to KL-A23) in Table 1 are: (1) Synthesis of substituted β-ketoesters (Formulas 1a-1d):

[0040]

[0041] The specific steps are as follows: 1 mmol of substituted acetophenone and 2 mmol of diethyl oxalate were dissolved in 5 mL of THF. Sodium hydride (4 mmol) was slowly added in portions with stirring. The reaction was allowed to react at 40°C for 3 h, and the reaction progress was monitored by thin-layer chromatography (TLC). After completion of the reaction, the mixture was quenched, the pH was adjusted to neutral with 1N HCl, and the mixture was extracted with ethyl acetate (3 × 10 mL). The organic phases were combined, dried over anhydrous Na2SO4, concentrated under reduced pressure, and recrystallized (dichloromethane / petroleum ether) to obtain products 1a-1d (yields: 0.1757-0.2230 g, 79.8-90.3%).

[0042] (2) Synthesis of substituted β-keto acids (Formula 2a-2d):

[0043]

[0044] The specific steps are as follows: Products 1a-1d (1 mmol) were dissolved in 6 mL of anhydrous methanol, followed by the addition of a 1N aqueous solution of NaOH, and the reaction was heated under reflux for 4 h. The reaction progress was monitored by TLC. After completion of the reaction, the solvent was removed, the pH was adjusted to 2-3 with 1N HCl, and the mixture was filtered and dried to obtain solid products 2a-2d. (Product yield: 0.1324-0.2005 g, 68.8-88.5%)

[0045] (3) Synthesis of aromatic β-diketone amide compounds (KL-A2 to KL-A12, KL-A14 to KL-A23). The synthetic route is:

[0046]

[0047] The specific steps are as follows: 2a-2d (1 mmol) was dissolved in 5 mL of anhydrous THF. Under room temperature stirring, a THF solution of substituted aniline (1.2 mmol) and EEDQ (1.1 mmol) was slowly added dropwise. The reaction was allowed to react at room temperature for 20 h. The reaction progress was monitored by TLC. After the reaction, 10% aqueous citric acid solution and saturated sodium bicarbonate solution were added for washing. The product was extracted with ethyl acetate (3×10 mL), dried over anhydrous Na2SO4, concentrated under reduced pressure, and recrystallized (dichloromethane / petroleum ether) to obtain the product. The product was filtered and dried, or purified by column chromatography (EA:PE = 1:120 to 1:20) to obtain products KL-A2 to KL-A12, KL-A14 to KLA23. (Product yield: 0.0353-0.1739 g, 10.5-55.2%).

[0048] (4) Synthesis of aromatic β-diketone amide compounds (KL-A1, KL-A13), the synthetic route is:

[0049]

[0050] The specific steps are as follows: 2a-2d (1 mmol), substituted aniline (1 mmol), and sodium acetate (1 mmol) are dissolved in 5 mL of acetic acid and reacted at 100°C for 3 hours. The reaction progress is monitored by TLC. After the reaction is completed, the temperature is cooled and the reaction solution is poured into water. A brown solid precipitates and is then purified by column chromatography (EA:PE = 1:15) to obtain products KL-A1 and KL-A13. (Product yield: 0.1186-0.1696 g, 34.2-54.3%)

[0051]

[0052] As shown in the above synthetic route, the first step is to synthesize different substituted β-ketoesters 1a-1d by reacting substituted acetophenone and diethyl oxalate under alkaline conditions of sodium hydride; the β-ketoesters 1a-1d undergo hydrolysis in alkaline conditions of sodium hydroxide to obtain β-keto acids 2a-2d; finally, amide condensation of 2a-2d with substituted aniline in the presence of EEDQ or sodium acetate ultimately yields β-diketoamide compounds KL-A1 to KL-A23.

[0053] Following the above route, β-diketoamide compounds KL-A1 to KL-A23 were synthesized. The product structures and characterization data are as follows:

[0054] (Z)-2-hydroxy-N-(4-nitrophenyl)-4-oxo-4-phenylbut-2-enamide(KL-A1)

[0055]

[0056] Brown solid:yield 0.1696g,54.3%;m.p.185.2-187.6℃; 1 H NMR(400MHz,CDCl3)δ9.30(s,1H),8.29(d,J=9.1Hz,2H),8.04(d,J=7.3Hz,2H),7.89(d,J=9.2Hz,2H),7.64(t,J=6.8Hz,1H),7.53(t,J=7.7Hz,2H),7.31(s,1H); 13 C NMR(100MHz,CDCl3)δ185.7,179.3,159.5,144.3,142.3,134.1,133.1,129.1,127.8,125.3,119.6,93.7;HRMS(ESI)m / z calcd for C 16 H 12 N2O5 + ([M+H] + )313.0746,found 313.0745.

[0057] (Z)-N-(2,4-dichlorophenyl)-2-hydroxy-4-oxo-4-phenylbut-2-enamide(KL-A2)

[0058]

[0059] Yellow solid:yield 0.1223 g,36.4%;m.p.255.4-257.6℃; 1 H NMR(400 MHz,DMSO)δ12.03(s,1H),8.02(d,J=7.6 Hz,2H),7.75(s,1H),7.63(t,J=7.3 Hz,1H),7.54(t,J=7.5 Hz,2H),7.43(d,J=8.7 Hz,1H),7.14(d,J=8.7 Hz,1H),6.65(s,1H); 13 C NMR(100 MHz,CDCl3)δ189.8,162.0,151.6,137.0,134.2,133.8,132.3,131.0,128.9,128.5,127.0,94.3;HRMS(ESI)m / z calcd for C 16 H 11 Cl2NO3 + ([M+H] +)336.0116,found 336.0117.

[0060] (Z)-N-(4-fluorophenyl)-2-hydroxy-4-oxo-4-phenylbut-2-enamide(KL-A3)

[0061]

[0062] Yellow solid:yield 0.0622 g,21.8%;m.p.163.4-165.3℃; 1 H NMR(400 MHz,DMSO)δ12.03(s,1H),8.02(d,J=7.6 Hz,2H),7.75(s,1H),7.63(t,J=7.3 Hz,1H),7.54(t,J=7.5 Hz,2H),7.43(d,J=8.7 Hz,1H),7.14(d,J=8.7 Hz,1H),6.65(s,1H); 13 C NMR(100 MHz,CDCl3)δ189.8,162.0,151.6,137.0,134.2,133.8,132.3,131.0,128.9,128.5,127.0,94.3;HRMS(ESI)m / z calcd for C 16 H 12 FNO3 + ([M+H] + )286.0801,found 286.0802.

[0063] (Z)-4-(4-chlorophenyl)-2-hydroxy-4-oxo-N-(p-tolyl)but-2-enamide(KL-A4)

[0064]

[0065] Yellow solid:yield 0.1743 g,55.2%;m.p.214.1-216.3℃; 1 H NMR(400 MHz,DMSO)δ10.61(s,1H),8.10(d,J=8.5 Hz,2H),7.72(d,J=8.3 Hz,2H),7.66(d,J=8.4Hz,2H),7.22(s,1H),7.18(d,J=8.3 Hz,2H),2.28(s,3H); 13C NMR(100 MHz,CDCl3)δ

[0066] 183.9,180.6,158.7,140.0,135.1,134.0,132.0,129.8,129.3,129.0,93.8,21.0;HRMS(ESI)

[0067] m / z calcd for C 17 H 14 ClNO3 + ([M+H] + )316.0735,found 316.0736.

[0068] (Z)-4-(4-chlorophenyl)-2-hydroxy-N-(4-methoxyphenyl)-4-oxobut-2-enamide

[0069] (KL-A5)

[0070]

[0071] Yellow solid:yield 0.2525 g,76.1%;m.p.234.2-236.1℃; 1 H NMR(400 MHz, DMSO) δ10.60(s,1H),8.10(d,J=8.5Hz,2H),7.75(d,J=8.9Hz,2H),7.65(d,J=8.5 Hz,2H),7.22(s,1H),6.95(d,J=9.0 Hz,2H),3.75(s,3H); 13 C NMR(100 MHz,CDCl3)δ

[0072] 183.9,180.6,158.6,157.0,140.0,132.0,129.8,129.3,129.0,121.4,114.4,93.8,55.5;HRMS(ESI)m / z calcd for C 17 H 14 ClNO4 + ([M+H] + )332.0611,found 332.0612.

[0073] (Z)-4-(4-chlorophenyl)-2-hydroxy-4-oxo-N-(o-tolyl)but-2-enamide(KL-A6)

[0074]

[0075] Yellow solid:yield 0.1190 g,37.7%;m.p.210.5–212.6℃; 1 H NMR(400 MHz,DMSO)δ10.19(s,1H),8.10(d,J=6.2 Hz,2H),7.65(d,J=8.1 Hz,2H),7.43(d,J=8.3Hz,1H),7.31–7.27(m,1H),7.22(d,J=12.3 Hz,3H),2.23(s,3H); 13 C NMR(100MHz,CDCl3)δ184.1,180.5,158.8,140.1,134.6,132.0,130.7,129.3,129.0,128.3,127.1,125.6,121.5,93.8,17.6;HRMS(ESI)m / z calcd for C 17 H 14 ClNO3 + ([M+H] + )316.0662,found316.0663.

[0076] (Z)-4-(4-chlorophenyl)-N-(4-ethylphenyl)-2-hydroxy-4-oxobut-2-enamide(KL-A7)

[0077]

[0078] Yellow solid:yield 0.0745 g,22.6%;m.p.195.6-197.2℃; 1 H NMR(400 MHz,DMSO)δ10.61(s,1H),8.10(d,J=8.7 Hz,2H),7.73(d,J=8.4 Hz,2H),7.65(d,J=8.7Hz,2H),7.22(d,J=1.4 Hz,2H),7.20(s,1H),2.58(d,J=7.6 Hz,2H),1.17(t,J=7.6 Hz,3H); 13 C NMR(100 MHz,CDCl3)δ183.9,180.6,158.7,141.5,134.2,132.0,129.3,129.0,128.6,119.9,93.8,28.4,15.6;HRMS(ESI)m / z calcd for C 18 H 16 ClNO3+ ([M+H] + )330.0892,found 330.0891.

[0079] methyl(Z)-4-(4-(4-chlorophenyl)-2-hydroxy-4-oxobut-2-enamido)benzoate

[0080] (KL-A8)

[0081]

[0082] Yellow solid:yield 0.0619 g,17.2%;m.p.283.4-285.5℃; 1 H NMR(400 MHz, DMSO) δ11.01(s,1H),8.11(d,J=7.8Hz,2H),7.97(dd,J=13.6,4.8Hz,4H),7.66(d,J =7.8Hz,2H),7.24(s,1H),3.84(s,3H); 13 C NMR(100 MHz,CDCl3)δ184.1,179.9,166.4,159.1,140.5,140.3,131.7,131.1,129.4,129.0,126.7,119.2,93.7,52.2;HRMS(ESI)m / zcalcdfor C 18 H 14 ClNO5 + ([M+H] + )360.0561,found 360.0563.

[0083] (Z)-N-(4-(tert-butyl)phenyl)-4-(4-chlorophenyl)-2-hydroxy-4-oxobut-2-enamide(KL-A9)

[0084]

[0085] Yellow solid:yield 0.0517 g,15.4%;m.p.265.6-267.4℃; 1 H NMR(400 MHz,DMSO)δ10.64(s,1H),8.11(d,J=7.8 Hz,2H),7.74(d,J=7.9 Hz,2H),7.65(d,J=7.8Hz,2H),7.39(d,J=7.9 Hz,2H),7.23(s,1H),1.27(s,9H); 13C NMR(100 MHz,CDCl3)δ

[0086] 183.9,180.6,158.7,148.4,140.1,133.9,132.0,129.3,129.0,126.1,119.6,93.8,34.5,31.4;HRMS(ESI)m / z calcd for C 20 H 20 ClNO3 + ([M+H] + )358.1205,found358.1205.

[0087] (Z)-N-(4-bromophenyl)-4-(4-chlorophenyl)-2-hydroxy-4-oxobut-2-enamide

[0088] (KL-A10)

[0089]

[0090] Yellow solid:yield 0.1490 g,39.2%;m.p.292.6-294.5℃; 1 H NMR(400 MHz,DMSO)δ10.83(s,1H),8.10(d,J=8.4 Hz,2H),7.82(d,J=8.7 Hz,2H),7.65(d,J=8.5Hz,2H),7.57(d,J=8.8 Hz,2H),7.22(s,1H); 13 C NMR(100 MHz,CDCl3)δ184.0,180.1,158.9,140.2,135.6,132.3,131.8,129.4,129.0,121.4,118.1,93.7;HRMS(ESI)m / z calcdforC 16 H 11 BrClNO3Na + ([M+Na] + )401.9503,found 401.9503.

[0091] (Z)-N,4-bis(4-chlorophenyl)-2-hydroxy-4-oxobut-2-enamide(KL-A11)

[0092]

[0093] Yellow solid:yield 0.0750 g,22.3%;m.p.282.1-284.5℃; 1 H NMR(400 MHz,DMSO)δ10.83(s,1H),8.10(d,J=8.5Hz,2H),7.88(d,J=8.8Hz,2H),7.66(d,J=8.5Hz,2H),7.44(d,J=8.8 Hz,2H),7.22(s,1H); 13 C NMR(100 MHz,CDCl3)δ184.1,180.1,158.9,140.2,135.2,131.8,130.4,129.3,129.1,121.1,93.7;HRMS(ESI)m / z calcdforC 16 H 11 Cl2NO3Na + ([M+Na] + )358.0008,found 358.0009.

[0094] (Z)-4-(4-chlorophenyl)-N-(4-fluorophenyl)-2-hydroxy-4-oxobut-2-enamide(KL-A12)

[0095]

[0096] Yellow solid:yield 0.0801 g,25.0%;m.p.265.2-267.3℃; 1 H NMR(400 MHz,DMSO)δ10.79(s,1H),8.13(d,J=8.7 Hz,2H),7.88(dd,J=9.2,5.0 Hz,2H),7.68(d,J=8.7 Hz,2H),7.23(d,J=9.3 Hz,3H); 13 C NMR(100 MHz,CDCl3)δ184.0,180.3,158.7,140.2,132.7,131.9,129.3,129.0,121.6,116.1,115.9,93.8;HRMS(ESI)m / z calcdforC 16 H 11 ClFNO3 + ([M+H] + )320.0411,found 320.0412.

[0097] (Z)-4-(4-chlorophenyl)-2-hydroxy-N-(4-nitrophenyl)-4-oxobut-2-enamide(KL-A13)

[0098]

[0099] Yellow solid:yield 0.1186 g,34.2%;m.p.212.3-214.6℃; 1 H NMR(400 MHz,DMSO)δ11.24(s,1H),8.29(d,J=9.3 Hz,2H),8.12(dd,J=8.9,4.8 Hz,4H),7.66(d,J=8.7 Hz,2H),7.25(s,1H); 13 C NMR(100 MHz,CDCl3)δ184.3,179.5,159.3,142.2,140.5,129.4,129.1,125.3,119.6,93.7;HRMS(ESI)m / z calcd for C 16 H 11 ClN2O5Na + ([M+Na] + )369.0249,found 369.0247.

[0100] (Z)-4-(4-chlorophenyl)-N-(2,4-dichlorophenyl)-2-hydroxy-4-oxobut-2-enamide(KL-A14)

[0101]

[0102] White solid:yield 0.0689 g,18.6%;m.p.257.6-259.4℃; 1 H NMR(400 MHz,DMSO)δ10.31(s,1H),8.13(d,J=8.6 Hz,2H),7.87(d,J=8.7 Hz,1H),7.80(d,J=2.3 Hz,1H),7.67(d,J=8.6 Hz,2H),7.55–7.52(m,1H),7.24(s,1H); 13C NMR(100 MHz,CDCl3)δ184.4,179.3,159.0,140.3,132.3,131.8,130.2,129.4,129.1,128.1,124.2,121.8,93.7;HRMS(ESI)m / z calcd for C 16 H 10 Cl3NO3 + ([M+H] + )369.9726,found 369.9728.

[0103] (Z)-4-(4-fluorophenyl)-2-hydroxy-4-oxo-N-(p-tolyl)but-2-enamide(KL-A15)

[0104]

[0105] Yellow solid:yield 0.1496 g,50.0%;m.p.120.3-122.5℃; 1 H NMR(400 MHz,DMSO)δ10.59(s,1H),8.19(dd,J=8.9,5.5 Hz,2H),7.72(d,J=8.4 Hz,2H),7.43(t,J=8.8 Hz,2H),7.21(s,1H),7.18(d,J=8.3 Hz,2H),2.29(s,3H); 13 C NMR(100 MHz,CDCl3)δ184.5,179.7,167.4,158.8,135.1,134.1,130.3,129.8,119.8,116.3,116.1,93.7,21.0;HRMS(ESI)m / z calcd for C 17 H 14 FNO3 + ([M+H] + )300.0958,found 300.0959.

[0106] (Z)-N-(4-ethylphenyl)-4-(4-fluorophenyl)-2-hydroxy-4-oxobut-2-enamide(KL-A16)

[0107]

[0108] Yellow solid:yield 0.0577 g,18.4%;m.p.188.2-190.4℃; 1 H NMR(400 MHz,DMSO)δ10.23(s,1H),8.19(s,2H),7.45–7.38(m,3H),7.30(s,2H),7.20(s,2H),2.62–2.57(m,2H),1.12(d,J=5.7 Hz,3H); 13 C NMR(100 MHz,DMSO)δ179.8,175.0,154.2,129.3,125.6,124.1,122.2,121.1,117.1,111.6,111.4,19.6,9.2;HRMS(ESI)m / z calcdforC 18 H 16 FNO3 + ([M+H] + )314.1114,found 314.1113.

[0109] (Z)-4-(4-fluorophenyl)-2-hydroxy-N-(4-methoxyphenyl)-4-oxobut-2-enamide(KL-A17)

[0110]

[0111] Yellow solid:yield 0.1340 g,44.4%;m.p.194.1-196.3℃; 1 H NMR(400 MHz,DMSO)δ10.58(s,1H),8.18(dd,J=8.9,5.5 Hz,2H),7.76(d,J=9.1 Hz,2H),7.43(s,2H),7.21(s,1H),6.96–6.93(m,2H),3.75(s,3H); 13 C NMR(100 MHz,CDCl3)δ184.5,179.7,158.7,157.1,130.3,129.8,121.4,116.3,116.1,114.4,93.7,55.5;HRMS(ESI)m / z calcdforC 17 H 14 FNO4 + ([M+H] + )316.0907,found 316.0908.

[0112] (Z)-N-(4-bromophenyl)-4-(4-fluorophenyl)-2-hydroxy-4-oxobut-2-enamide(KL-A18)

[0113]

[0114] White solid:yield 0.0699 g,19.2%;m.p.275.3-277.6℃; 1 H NMR(400 MHz,DMSO)δ10.82(s,1H),8.19(dd,J=9.0,5.4 Hz,2H),7.82(d,J=8.9 Hz,2H),7.57(d,J=8.9 Hz,2H),7.42(d,J=8.9 Hz,2H),7.22(s,1H); 13 C NMR(100 MHz,CDCl3)δ184.6,179.3,159.0,135.7,132.3,130.4,121.4,118.1,116.4,116.2,93.6;HRMS(ESI)m / z calcdforC 16 H 11 BrFNO3Na + ([M+Na] + )385.9799,found 385.9800.

[0115] (Z)-N-(4-chlorophenyl)-4-(4-fluorophenyl)-2-hydroxy-4-oxobut-2-enamide(KL-A19)

[0116]

[0117] White solid:yield 0.0671 g,21.0%;m.p.224.5-227.8℃; 1 H NMR(400 MHz,DMSO)δ10.85(s,1H),8.21(dd,J=8.8,5.5 Hz,2H),7.90(d,J=8.9 Hz,2H),7.47(s,2H),7.43(d,J=8.7 Hz,2H),7.24(s,1H); 13C NMR(100 MHz,CDCl3)δ184.59,179.3,159.0,135.2,130.4,129.4,121.1,116.4,116.2,93.6;HRMS(ESI)m / z calcd for C 16 H 11 ClFNO3Na + ([M+Na] + )342.0304,found 342.0304.

[0118] (Z)-N,4-bis(4-fluorophenyl)-2-hydroxy-4-oxobut-2-enamide(KL-A20)

[0119]

[0120] Yellow solid:yield 0.30799 g,26.3%;m.p.218.5-220.4℃; 1 H NMR(400 MHz,DMSO)δ10.79(s,1H),8.20(t,J=6.1Hz,2H),7.90–7.86(m,2H),7.44(t,J=8.1Hz,2H),7.24(s,3H); 13 C NMR(100 MHz,CDCl3)δ184.6,179.4,167.5,164.9,158.9,132.7,130.3,129.8,121.6,116.4,116.1,115.9,93.7;HRMS(ESI)m / z calcd for C 16 H 11 F2NO3 +

[0121] ([M+H] + )304.0707,found 304.0709.

[0122] (Z)-N-(2-chloro-4-fluorophenyl)-4-(4-fluorophenyl)-2-hydroxy-4-oxobut-2-enamide(KL-A21)

[0123]

[0124] White solid:yield 0.0353 g,10.5%;m.p.311.2-313.5℃;1 H NMR(400 MHz,DMSO)δ10.32(s,1H),8.19(dd,J=8.8,5.4 Hz,2H),7.76(dd,J=8.9,5.8 Hz,1H),7.61(dd,J=8.6,2.9 Hz,1H),7.42(t,J=8.8 Hz,2H),7.32(td,J=8.6,2.9 Hz,1H),7.21(s,1H); 13 CNMR(100 MHz,CDCl3)δ184.7,178.7,159.0,130.4,122.3,116.9,116.6,116.4,116.2,115.0,114.7,93.7;HRMS(ESI)m / z calcd for C 16 H 10 ClF2NO3Na + ([M+Na] + )360.0210,found 360.0210.

[0125] (Z)-4-(4-bromophenyl)-N-(4-chlorophenyl)-2-hydroxy-4-oxobut-2-enamide(KL-A22)

[0126]

[0127] Yellow solid:yield 0.0799 g,21.0%;m.p.247.3-249.7℃; 1 H NMR(400 MHz,DMSO)δ10.85(s,1H),8.02(d,J=8.3 Hz,2H),7.88(d,J=7.5 Hz,2H),7.80(d,J=7.4Hz,2H),7.44(d,J=8.2 Hz,2H),7.22(s,1H); 13 C NMR(100 MHz,CDCl3)δ184.1,180.3,158.9,135.1,132.3,130.4,129.3,129.1,121.1,93.7;HRMS(ESI)m / z calcd forC 16 H 11 BrClNO3 + ([M+H] + )378.9611,found 378.9613.

[0128] (Z)-N,4-bis(4-bromophenyl)-2-hydroxy-4-oxobut-2-enamide(KL-A23)

[0129]

[0130] Yellow solid: yield 0.0825g, 19.4%; mp253.6-255.2℃; 1 H NMR (400MHz, DMSO) δ10.85 (s, 1H), 8.02 (d, J = 7.4Hz, 2H), 7.82 (d, J = 9.0Hz, 4H), 7.57 (d, J = 8.8Hz, 2H), 7.22 (s, 1H); 13 C NMR(100MHz, CDCl3)δ184.1,180.2,158.9,135.6,132.3,129.0,121.4,118.1,93.7; HRMS(ESI)m / z calcd for C 16 H 11 Br2NO3Na + ([M+Na] + )447.8978,found447.8978.

[0131] For the above compounds, the antibacterial and antimicrobial properties were tested in the following examples.

[0132] Example 1

[0133] Determination of antibacterial activity of β-diketoamide compounds

[0134] (1) Strain source

[0135] 80 Gram-positive bacteria (comprising 20 MRSA, 20 methicillin-sensitive Staphylococcus aureus (MSSA), 20 Enterococcus faecalis and 20 Staphylococcus epidermidis) and 8 Gram-negative bacteria (comprising 2 strains each of Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae and Escherichia coli) clinical strains used in the present embodiment were collected from different inpatients in the hospital or from the experimental strains preserved in this laboratory. All clinical strains were identified by Phoenix 100 automatic microbial identification system, and matrix-assisted laser desorption ionization / time-of-flight mass spectrometry (MALDI-TOF-MS) was adopted to re-identify all strains after the secondary culture. Quality control strain Staphylococcus aureus ATCC29213 was purchased from ATCC strain library.

[0136] (2) Main instruments and reagents

[0137] Micropipette, Phoenix-100 fully automated bacterial identification / drug susceptibility system, fully automated mass spectrometry detection system IVD MALDI Biotyper (Bruker, Germany), fully automated growth curve analyzer, CAMHB culture medium, TSB culture medium, FV3000 laser confocal microscope. Custom compound library, CCK-8 kit, polymyxin B, crystal violet, LIVE / DEADBacLight TM Fluorescent dye, glucose, 96-well cell culture plates.

[0138] In this example, a 96-well plate was used to conduct an experiment on the effect of β-diketonamide compounds on the growth of Gram-positive bacteria (S. epidermids SE1457, MRSAYUSA145, MSSA SA113, E. faecalis OG1RF, E. faecium EMF64). The specific steps were as follows: the minimum inhibitory concentration of β-diketonamide compounds was determined by broth microdilution method, and the β-diketonamide compounds were serially diluted in a 96-well plate with cationic Mueller Hinton broth medium (CAMHB). The volume of each well was 100 μL, and the highest concentration tested was 100 μM. After culturing different strains overnight, the turbidity of the bacterial suspension was adjusted to 0.5 McFarland turbidity, and the bacterial suspension was diluted with CAMHB broth medium at a ratio of 1:200 and inoculated into a 96-well plate to make a final concentration of approximately 5×10 5 CFU / mL. Bacterial suspensions were incubated with varying concentrations of compound at 37°C for 18 hours. Wells containing only bacterial suspension without compound served as bacterial growth controls (positive controls); wells containing only culture medium served as negative controls. The experiment was repeated three times. The MIC was defined as the lowest drug concentration at which bacterial growth was not visible to the naked eye.

[0139] The culture (100 μL) in the wells with the compound MIC or above in the 96-well plate was transferred to the CAMHB agar plate and incubated at 37°C for 24 h. No colonies were visible on the plate (colonies ≤ 4), which was the minimum bactericidal concentration (MBC).

[0140] In this example, the antibacterial activity of β-diketonamide compounds against Gram-positive bacteria (S. epidermids SE1457, MRSAYUSA145, MSSA SA113, E. faecalis OG1RF, and E. faecium EMF64) was evaluated using the broth microdilution method. The minimum bactericidal concentrations (MBCs) of the β-diketonamide compounds were also determined. Vancomycin (VAN) was used as a positive control. The MIC and MBC results are shown in Table 2.

[0141] Table 2 Antibacterial and bactericidal activities of β-diketoamide compounds against Gram-positive bacteria

[0142]

[0143] MIC a Indicates the minimum inhibitory concentration of the target compound against S.epidermids SE1457, MRSAYUSA145, MSSA SA113, E.faecalis OG1RF, and E.faecium EMF64. MBC b It represents the minimum bactericidal concentration of the target compound against S. epidermids SE1457, MSSASA113 and E. faecalis OG1RF.

[0144] The results in Table 2 show that some β-diketoamide compounds have good antibacterial activity against five Gram-positive bacteria. Among them, KL-A4, KL-A7, KL-A10, KL-A13, KL-A22 and KL-A23 had comparable antibacterial activities against MRSAYUSA145, with MICs ranging from 3.125 to 6.25 μM; KL-A4, KL-A6, KL-A7, KL-A9, KL-A10, KL-A11, KL-A18, KL-A19, KL-A22 and KL-A23 had better antibacterial activities against S.epidermids SE1457, with MICs ranging from 1.56 to 6.25 μM; KL-A4, KL-A6, KL-A7, KL-A9, KL-A10, KL-A11, KL-A18, KL-A19, KL-A22 and KL-A23 had better antibacterial activities against MSSA SA113 had good antibacterial activity with MICs of 3.125 μM; KL-A1, KL-A4, KL-A6, KL-A7, KL-A10, KL-A11, KL-A12, KL-A13, KL-A15, KL-A16, KL-A18, KL-A19, KL-A21, KL-A22, and KL-A23 had good antibacterial activity against E. faecalis OG1RF with MICs ranging from 1.56 to 3.125 μM; EMF64 exhibited good antibacterial activity, with MICs ranging from 1.56 to 3.125 μM.

[0145] The MIC test results of β-diketoamide compounds against Gram-negative bacteria Klebsiella pneumoniae (K.peneumoniae K2044) and Escherichia coli (E.coliATCC25922) showed that all compounds had no inhibitory effect on Klebsiella pneumoniae (K.peneumoniae K2044) and Escherichia coli (E.coli ATCC25922), and the MICs values ​​were all greater than 100μM.

[0146] In summary, most derivatives exhibited strong antibacterial activity against Gram-positive bacteria. Compounds KL-A4, KL-A7, KL-A10, KL-A13, KL-A22, and KL-A23 exhibited the best antibacterial activity against Gram-positive bacteria, with MICs ranging from 1.56 to 6.25 μM. Compound KL-A10 exhibited the best bactericidal activity against Staphylococcus aureus, with MBCs ranging from 25 to 50 μM. The activity of KL-A10 was evaluated below.

[0147] Example 2

[0148] In this example, a 96-well plate was used to test the antibacterial activity MIC of β-diketonamide compounds against Gram-negative bacteria (K. peneumoniae K2044, E. coli ATCC25922). The specific steps were as follows: the minimum inhibitory concentration of β-diketonamide compounds was determined by broth microdilution method. The β-diketonamide compounds were serially diluted in a 96-well plate with cationic Mueller Hinton broth (CAMHB). The volume of each well was 100 μL, and the highest concentration tested was 100 μM. After overnight culture of different strains, the turbidity of the bacterial suspension was adjusted to 0.5 McFarland turbidity, and the bacterial suspension was diluted with CAMHB broth at a ratio of 1:200 and inoculated into a 96-well plate to a final concentration of approximately 5×10 5 CFU / mL. Bacterial suspensions were incubated with varying concentrations of compound at 37°C for 18 hours. Wells containing bacterial suspension without compound served as bacterial growth controls (positive controls); wells containing culture medium alone served as negative controls. The MIC was defined as the lowest drug concentration at which bacterial precipitation was not visible to the naked eye.

[0149] Table 3 shows the MIC results of β-diketoamide compounds against K. pneumoniae K2044 and E. coli ATCC25922. β-diketoamide compounds had no inhibitory effect on either K. pneumoniae K2044 or E. coli ATCC25922, with MICs greater than 100 μM. This indicates that β-diketoamide compounds have no significant inhibitory effect on Gram-negative bacteria.

[0150] Table 3 Antibacterial activity of β-diketoamide compounds against Gram-negative bacteria

[0151]

[0152] MIC a It represents the minimum inhibitory concentration of the target compound against K.pneumoniaeK2044 and E.coliATCC25922.

[0153] Example 3

[0154] Cytotoxicity of β-diketoamides against human hepatic stellate cells (LX-2) and lung epithelial cells (BEAS-2B): Human hepatic stellate cells (LX-2) and lung epithelial cells (BEAS-2B) were seeded in 96-well plates containing 1% heat-inactivated fetal bovine serum, 1% penicillin-streptomycin, and 1% DMEM. The cells were incubated at 37°C in a 5% CO2 atmosphere for 24 hours (approximately 2000-4000 cells per well). Subsequently, β-diketoamides at varying concentrations (0.39-100 μM) were added and incubated in a constant-temperature incubator for 24 hours. After 24 hours of incubation, 10 μL of CCK-8 was added to each well using a CCK-8 assay kit and incubated for 1-2 hours. The absorbance at 450 nm was measured using a high-content microplate reader, and cell viability was calculated according to the following formula. The blank control was made up of only culture medium, and the control group was made up of no drug. Each group was repeated 6 times. The concentration of the compound that inhibited 50% cell growth (IC 50 ).

[0155]

[0156] Most β-diketoneamide compounds have low toxicity to human hepatic stellate cells (LX-2) and lung epithelial cells (BEAS-2B), among which KL-A1, KL-A8, KL-A9, KL-A14, KL-A16, KL-A17, KL-A21, KL-A22, and KL-A23 have the highest IC values ​​for LX-2 cells. 50 The IC values ​​of KL-A2, KL-A3, KL-A7, KL-A10, KL-A11, KL-A13, KL-A14, KL-A17, and KL-A22 against BEAS-2B cells were >100 μM. 50 Values ​​>100 μM.

[0157] Table 4 Cytotoxicity of β-diketoamide compounds

[0158]

[0159] IC 50 d is the concentration of compound that inhibits the growth of LX-2 and BEAS-2B cells by 50%, the highest concentration tested was 100 μM.

[0160] Example 4

[0161] In this example, the minimum inhibitory concentration (MIC) of KL-A10 against 80 clinically isolated Gram-positive strains (including 20 MSSA, 20 MRSA, 20 Enterococcus faecalis, and 20 Staphylococcus epidermidis) was determined by microbroth dilution. The specific steps were as follows: an overnight culture solution was taken and the turbidity was adjusted to 0.5 McFarland (the bacterial count was approximately 1.0-1.5 × 10 8 cfu / mL). Dilute the bacterial solution 1:100 with CAMHB medium and add to a 96-well plate, with 12 wells per row. Set up 10 gradient wells for KL-A10 (200, 100, 50, 25, 12.5, 6.25, 3.125, 1.56, 0.78, and 0.39 μM). Add 200 μL of this bacterial solution to the 11th well as a positive control, and add 200 μL of CAMHB medium to the 12th well as a negative control. Incubate at 37°C for 18 hours and observe the results. The MIC value is calculated based on the drug concentration at which no bacterial precipitation is visible to the naked eye. Follow the above steps, using KL-2 as a control sample (CAS number 900308-51-2).

[0162] In this example, the statistical results of the MIC values ​​of KL-A10 against Gram-positive bacteria such as Staphylococcus aureus, Staphylococcus epidermidis, and Enterococcus faecalis are shown in Table 5. It can be seen that KL-A10 has a relatively broad spectrum of antibacterial activity against a variety of Gram-positive bacteria, and the MIC values ​​are mainly distributed between 0.78 μM and 6.25 μM, among which the inhibitory effect on Enterococcus faecalis is the best, with a MIC of 0. 50 As shown in Table 5, the MIC of KL-A10 against Staphylococcus aureus (including MRSA and MSSA), Staphylococcus epidermidis, and Enterococcus faecalis is 1.56 μM. 50 Both were lower than the control drug KL-2, indicating that the antibacterial effect of KL-A10 was better than that of KL-2.

[0163] Table 5 Minimum inhibitory concentration distribution of KL-A10 and control samples against Staphylococcus aureus, Staphylococcus epidermidis and Enterococcus faecalis

[0164]

[0165] MSSA: Methicillin-sensitive Staphylococcus aureus; MRSA: Methicillin-resistant Staphylococcus aureus. S.epidermids: Staphylococcus epidermidis; E.faecalis: Enterococcus faecalis. MIC 50MIC is the lowest drug concentration that can inhibit 50% of the number of strains. 90 It is the lowest drug concentration that can inhibit 90% of the number of strains.

[0166] Example 5

[0167] Experiment on the effect of KL-A10 on the growth curve of Gram-positive bacteria.

[0168] In order to verify whether KL-A10 can inhibit the growth of Gram-positive bacteria, we used different sub-inhibitory concentrations of KL-A10 to treat Staphylococcus epidermidis (SE1457), Staphylococcus aureus (SA113), MRSA (YUSA145) and Enterococcus faecalis (OG1RF), and measured their absorbance at 600 nm (OD) at different time points. 600 ) value, the specific steps are as follows: take the overnight culture liquid and dilute it 1000 times with tryptone soy broth (TSB) medium and add it to 96-well plate, add different concentrations (1 / 8×, 1 / 4×, 1 / 2× and 1×MIC) of KL-A10 and place it in an automatic growth curve analyzer, and measure the OD every 1 hour. 600 The absorbance value was used to detect the content of planktonic bacteria in the culture supernatant. The incubation temperature was 37°C and the growth curve was drawn.

[0169] Growth curve analysis Figure 1 As shown in the figure, even at a concentration of 1 / 8×MIC, KL-A10 can restrict the growth of all Gram-positive bacteria. As the concentration increases, bacterial growth will also lag accordingly. At a concentration of 1×MIC, the growth of Gram-positive bacteria is completely inhibited. These results preliminarily indicate that KL-A10 has good growth inhibition activity against Gram-positive bacteria and has the potential to be developed as a drug against Gram-positive bacterial infections.

[0170] Example 6

[0171] When bacteria are continuously exposed to subinhibitory concentrations of antibiotics, the risk of developing resistance increases significantly, posing a significant challenge to healthcare. Therefore, we investigated the development of resistance in Staphylococcus aureus after exposure to KL-A10. The following steps were used: Methicillin-resistant Staphylococcus aureus (MRSA) YUSA145 was induced to resist KL-A10. Linezolid (LZD) served as an antibiotic control. Overnight cultures of MRSA YUSA145 were diluted 1:100 into medium containing KL-A10 and linezolid at concentrations of 1 / 2 × the MIC, respectively, and incubated at 37°C for 24 hours. After 24 hours, the next passage was performed, for a total of 32 passages. The MIC values ​​of the drugs against MRSA YUSA145 were measured every four passages.

[0172] The results are as follows Figure 2 As shown, Staphylococcus aureus YUSA145 rapidly developed resistance to the positive control antibiotic linezolid after eight passages, with the MIC value increasing fourfold. By the 30th passage, the MIC had increased 16-fold compared to the parent strain. In contrast, the MIC of KL-A10 remained stable during passage, ultimately increasing only twofold. These results suggest that KL-A10 is less likely to induce bacterial resistance than linezolid.

[0173] Example 7

[0174] Microbial pathogens enhance their survival in hosts and induce chronic infections by forming biofilms. Therefore, we investigated the ability of KL-A10 to eliminate mature biofilms. The procedure was as follows: Overnight cultures of Staphylococcus aureus SA113 were inoculated into 6-well plates at a ratio of 1:200 with fresh TSBG medium (containing 1% glucose) for 24 hours to allow mature biofilms to form. After 24 hours of incubation, the supernatant was removed and KL-A10 was added at concentrations of 25 μM and 50 μM. Vancomycin at 25 μM served as a positive control, while a drug-free, DMSO-only plate served as a negative control. KL-2 at 25 μM and 50 μM served as other controls. The plates were incubated at 37°C for 24 hours. Following incubation, the plates were washed twice with PBS and stained with 1 μM SYTO9 and 1 μM propidium iodide (PI) for 15 minutes in the dark. The samples were then prepared for confocal microscopy. SYTO9, a penetrating nucleic acid dye, can label both live and dead bacteria, emitting green fluorescence. PI, on the other hand, can only penetrate dead bacteria with damaged membranes, where it binds to DNA, producing red fluorescence and competitively inhibiting SYTO9's staining efficiency. Confocal laser scanning microscopy was then used to observe the thickness of the biofilm removed.

[0175] The results are as follows Figure 3 As shown, KL-A10 at a concentration of 25 μM significantly reduced the thickness of mature biofilms, with the effect being even more pronounced at a concentration of 50 μM. This indicates that KL-A10 is effective in removing mature S. aureus biofilms. The control, KL-2 at a concentration of 25 μM, had little effect on the thickness of mature biofilms. While KL-2 at a concentration of 50 μM did reduce biofilm thickness, the effect was less pronounced than that of KL-A10.

[0176] Example 8

[0177] In this example, the synergistic activity of KL-A10 and polymyxin B against resistant strains of Gram-negative bacteria Escherichia coli and Klebsiella pneumoniae was evaluated using a checkerboard assay: the drugs were serially diluted two-fold in a 96-well plate in CAMHB to a final volume of 100 μl. The polymyxin-resistant strains tested were Escherichia coli MG1655 (MCR-1), with a polymyxin MIC of 4 μg / mL; MG1655-PB, with a polymyxin MIC of 128 μg / mL; Klebsiella pneumoniae K2044-PB, with a polymyxin MIC of 64 μg / mL; and Klebsiella pneumoniae K2044 (MCR-1), with a polymyxin MIC of 256 μg / mL. After overnight culture of different strains, the turbidity of the bacterial suspension was adjusted to 0.5 McFarland turbidity, and the bacterial suspension was diluted with CAMHB broth at a ratio of 1:200 and inoculated into a 96-well plate to make the final concentration of about 5×10 5 CFU / mL. Wells containing only bacterial suspension without compound served as bacterial growth controls (positive controls); wells containing only culture medium served as negative controls, and KL-2 was used as a control. Bacterial suspensions were incubated with varying concentrations of compound at 37°C for 18 hours. The MIC was defined as the lowest drug concentration at which bacterial growth was not visible to the naked eye. OD600 was also measured, and bacterial growth was analyzed by calculating the ratio of each well to the untreated control well.

[0178] The results are as follows Figure 4 As shown, KL-A10 and polymyxin B exhibited synergistic antibacterial activity against polymyxin-resistant strains of Gram-negative bacteria (Escherichia coli and Klebsiella pneumoniae), enhancing the antibacterial activity of polymyxin B against polymyxin-resistant strains: 50 μM KL-A10 reduced the MIC of MG1655 (MCR-1) against polymyxin B from 4 μg / mL to 0.5 μg / mL. 100 μM KL-A10 reduced the MIC of MG1655-PB against polymyxin B from 128 μg / mL to 1 μg / mL. 12.5 μM KL-A10 reduced the MIC of K2044 (MCR-1) against polymyxin B from 256 μg / mL to 2 μg / mL. KL-A10 at 12.5 μM can reduce the MIC of K2044-PB against polymyxin B from 64 μg / mL to 2 μg / mL, indicating that KL-A10 has the potential to be used as an antibiotic adjuvant in combination with polymyxin B to treat polymyxin-resistant bacteria.

[0179] Although the control sample KL-2 also showed enhanced antibacterial activity against polymyxins, its activity was lower than that of KL-A10. 50μM KL-A10 reduced the MIC of MG1655 (MCR-1) against polymyxins from 4μg / mL to 1μg / mL. 100μM KL-A10 reduced the MIC of MG1655-PB against polymyxins from 128μg / mL to 2μg / mL. 12.5μM KL-A10 reduced the MIC of K2044-PB (MCR-1) against polymyxins from 256μg / mL to 8μg / mL. 12.5μM KL-A10 reduced the MIC of K2044-PB against polymyxins from 64μg / mL to 16μg / mL.

[0180] Example 9

[0181] The wax moth infection assay has been widely used as an effective in vivo model for testing antimicrobial drugs and their toxicity. To evaluate the therapeutic efficacy of KL-A10 against G. mellonella, we employed MRSAUSA300 for infection. The steps were as follows: First, an infection dose of 106 colonies of MRSAUSA300 was injected near the right hind limb of the G. mellonella. One hour after infection, the moths were treated with 25 μM and 50 μM KL-A10, respectively, with PBS serving as a control. Following treatment, the G. mellonella survival was observed for 48 hours at 37°C in the dark, and the larval status of the G. mellonella was photographed and recorded.

[0182] The results are as follows Figure 5 As shown, after infection with USA300, all G. mellonella larvae in the PBS-treated group died. Compared to the PBS-treated control group, KL-A10 at a concentration of 25 μM increased G. mellonella survival. The therapeutic effect of 25 μM KL-A10 was comparable to that of vancomycin (6.25 μM), with both achieving a 40% survival rate. In contrast, the survival rate of G. mellonella larvae treated with the control, KL-2, was 10% after 48 hours. These results demonstrate that KL-A10 exhibits superior antibacterial efficacy in vivo.

[0183] Example 10

[0184] The anti-infection effect of KL-A10 in the mouse lung infection model: 8-week-old mice were selected and infected with MRSAUSA300 in the lungs by intranasal drops (108 CFU) to evaluate the therapeutic effect of KL-A10 in vivo. Each mouse was given 30 μL of bacteria, with 15 μL dropped into each nostril for lung infection. 2 hours after infection, the mice were randomly divided into 4 groups (6 per group) and injected with 200 μL of 0.9% NaCl, KL-A10 (10 mg / kg), KL-A10 (20 mg / kg), KL-2 (10 mg / kg), and KL-2 (20 mg / kg) for treatment. 12 hours after infection, the second drug injection was performed. 12 hours after the last administration, the mice were euthanized. Lung tissue was collected, weighed, and then homogenized with 0.9% NaCl. Finally, serial dilutions were performed and plated onto TSA plates for counting, followed by overnight incubation at 37°C. The number of colonies on the plates was counted, from which the bacterial load per unit mass of lung tissue was calculated. In addition, lung sections from mice in the different dosing groups were obtained for pathological analysis.

[0185] like Figure 6 The bacterial loads shown in the results show that treatment with 10 mg / kg KL-A10 reduced the bacterial load by approximately 0.5 log, while treatment with 20 mg / kg KL-A10 reduced the lung bacterial load by approximately 0.8 log, demonstrating superior efficacy to the positive control linezolid at the same dose. Similarly, KL-A10 was more effective than the control KL-2 at the same dose. Treatment with 10 mg / kg KL-2 did not significantly reduce the bacterial load, while treatment with 10 mg / kg KL-2 reduced the bacterial load by approximately 0.5 log. This demonstrates the potential of KL-A10 to combat bacterial infections in vivo.

[0186] In the above examples, 23 β-diketoamide derivatives were synthesized. By measuring their minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and biological activities such as cytotoxicity and hemolytic activity, a preliminary structure-activity relationship between the structure and biological activity of these compounds was summarized.

[0187] The results of biological activity assays showed that (1) some β-diketone amide derivatives had good antibacterial effects against Gram-positive bacteria (S. epidermidis SE1457, MRSAYUSA145, MSSA SA113, E. faecalis OG1RF, E. faecaium EMF64), among which compound KL-A10 had the best antibacterial activity against five Gram-positive bacteria, with MICs ranging from 1.56 to 3.125 μM, and KL-A10 had the best bactericidal activity, with an MBC of 25 to 50 μM; however, these β-diketone amide derivatives did not show antibacterial activity against Gram-negative bacteria K. pneumoniae K2044 and E. coli ATCC25922; the KL-A10 molecule had low proliferative toxicity against human hepatic stellate cells (LX-2) and lung epithelial cells (BEAS-2B). In summary, β-diketoamide derivatives, especially KL-A10, have the potential to be developed into new antibacterial agents.

[0188] The in vitro and in vivo antibacterial efficacy of KL-A10 was further evaluated. A comparison of the distribution of minimum inhibitory concentrations (MICs) of KL-A10 against MRSA, MSSA, S. epidermids, and E. faecalis revealed that the MICs of KL-A10 against these four Gram-positive bacteria ranged from 0.78 to 6.25 μM, with an MIC50 of 3.125 μM. The effect of KL-A10 on the planktonic growth of Gram-positive bacteria was determined, demonstrating a concentration-dependent inhibitory effect. MRSAYUSA145 was cultured at subinhibitory concentrations of KL-A10 for resistance testing. Compared with the antibiotic linezolid, KL-A10 was less likely to induce bacterial resistance. Laser confocal microscopy was used to observe the clearance of mature MSSA biofilms by KL-A10, demonstrating a clearance effect at a concentration of 25 μM. Biofilm thickness was significantly reduced compared to the control group. In addition, KL-A10 can enhance the antibacterial activity of polymyxins against Gram-negative bacteria. In vivo experiments have shown that KL-A10 can increase the survival rate of greater wax moth at a concentration of 25 μM, and in a mouse lung infection experiment, KL-A10 has an anti-infective therapeutic effect on mice.

[0189] The above is a further detailed description of the present invention in conjunction with specific preferred embodiments, and the specific implementation of the present invention should not be considered to be limited to these descriptions. For those skilled in the art to which the present invention belongs, several simple deductions or substitutions can be made without departing from the concept of the present invention, and all of these should be considered to fall within the scope of protection of the present invention.

Claims

1. β-diketoamide compounds, characterized in that Its structural formula is as follows (1): Among them, R 1 is H or halogen; R 2 is nitro, alkyl, halogen or methoxy.

2. The β-diketoamide compound according to claim 1, characterized in that It is KL-A4, KL-A7, KL-A10, KL-A13, KL-A22 or KL-A23, and the structural formulas of KL-A4, KL-A7, KL-A10, KL-A13, KL-A22 and KL-A23 are shown in the following formulas (2) to (7):

3. Use of the β-diketoamide compound according to claim 1 or 2 for preparing an anti-Gram infection drug, characterized in that: The Gram-positive bacteria include Gram-positive bacteria and Gram-negative bacteria, the Gram-positive bacteria are at least one of Staphylococcus aureus, Enterococcus faecalis, Enterococcus faecium, Staphylococcus epidermidis or Streptococcus pneumoniae, and the Gram-negative bacteria are at least one of Acinetobacter baumannii, Klebsiella pneumoniae, Escherichia coli, and Pseudomonas aeruginosa; the β-diketoamide compound is used to prepare a drug for resisting Gram-positive bacterial infection; the β-diketoamide compound is combined with polymyxin to prepare a drug for resisting Gram-negative bacterial infection.

4. Use of the β-diketoamide compound according to claim 3 for preparing an anti-Gram infection drug, characterized in that: The concentration of the β-diketoamide compound in the treatment system is not less than 0.20 μM.

5. Use of the β-diketoamide compound according to claim 3 for preparing an anti-Gram infection drug, characterized in that: The medicine is in the form of an injection, tablet, pill, capsule, suspension, granule, spray or emulsion.

6. Use of the β-diketoamide compound according to claim 1 or 2 for preparing a coating for inhibiting Gram bacteria, characterized in that: The coating is used on the surface of medical devices, the Gram-positive bacteria include Gram-positive bacteria and Gram-negative bacteria, the Gram-positive bacteria are at least one of Staphylococcus aureus, Enterococcus faecalis, Enterococcus faecium, Staphylococcus epidermidis or Streptococcus pneumoniae, and the Gram-negative bacteria are at least one of Acinetobacter baumannii, Klebsiella pneumoniae, Escherichia coli, and Pseudomonas aeruginosa; the β-diketoamide compound is used to prepare a coating that inhibits Gram-positive bacteria; the β-diketoamide compound is combined with polymyxin to prepare a coating that inhibits Gram-negative bacteria.

7. Use of the β-diketoamide compound according to claim 1 or 2 for preparing a Gram-inhibiting disinfectant, characterized in that: The Gram-positive bacteria include Gram-positive bacteria and Gram-negative bacteria, the Gram-positive bacteria are at least one of Staphylococcus aureus, Enterococcus faecalis, Enterococcus faecium, Staphylococcus epidermidis or Streptococcus pneumoniae, and the Gram-negative bacteria are at least one of Acinetobacter baumannii, Klebsiella pneumoniae, Escherichia coli, and Pseudomonas aeruginosa; the β-diketoamide compound is used to prepare a disinfectant for inhibiting Gram-positive bacteria; the β-diketoamide compound is combined with polymyxin to prepare a disinfectant for inhibiting Gram-negative bacteria.

8. A drug for resisting Gram-positive bacterial infection, characterized in that: The β-diketoamide compound according to any one of claims 1 to 2, wherein the Gram-positive bacteria is at least one of Staphylococcus aureus, Enterococcus faecalis, Enterococcus faecium, Staphylococcus epidermidis or Streptococcus pneumoniae.

9. A drug for resisting Gram-negative bacterial infection, characterized in that: The method comprises the β-diketoamide compound according to any one of claims 1 to 2 and polymyxin, wherein the Gram-negative bacteria is at least one of Acinetobacter baumannii, Klebsiella pneumoniae, Escherichia coli, and Pseudomonas aeruginosa.

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