Synthesis of a new class of schiff base gold(III) compounds and their antibacterial applications

Abstract

The application discloses synthesis of a novel Schiff base gold (III) compound and antibacterial application thereof, and belongs to the technical field of medicine preparation; gold sodium aurothiomalate has significant antibacterial activity, but the Au-S bond thereof is easily broken by intracellular reducing thiol, and the gold sodium aurothiomalate is metabolized before reaching a target point to produce toxic side effects; based on the gold sodium aurothiomalate, developing a gold compound with stronger stability is one of strategies for overcoming drug-resistant bacteria; the Schiff base itself has antibacterial activity, and after being combined with gold (III) ions, the Schiff base can stabilize gold atoms and improve the activity of the gold atoms; in vitro antibacterial activity shows that Au6 and Au13 have significant antibacterial advantages on carbapenem and polymyxin-resistant Klebsiella pneumoniae, the minimum inhibitory concentration (MIC) is 10 micromoles, and the antibacterial activity is at least 16 times higher than that of the gold sodium aurothiomalate; in vivo antibacterial activity experiments prove that Au6 and Au13 can reduce mouse skin infection and promote wound healing, and can prolong the survival time of abdominal infection mice, and have important practical application value.

Description

Technical Field

[0001] This invention relates to the technical field of drug preparation, specifically to the synthesis of a novel class of Schiff gold (III) compounds and their antibacterial applications; more specifically, to the application of Schiff gold (III) compounds in the resistance to carbapenem-resistant and polymyxin-resistant Gram-negative bacteria. Background Technology

[0002] The threat posed by multidrug-resistant bacteria has become a major challenge in the health field, especially the emergence of multidrug-resistant Gram-negative bacteria; among them, carbapenem-resistant Enterobacteriaceae (CRE) have attracted much attention. CRE refers to Enterobacteriaceae that develop resistance to any carbapenem antibiotic (such as imipenem, ertapenem, or domperidone). Their resistance develops through multiple mechanisms, including the production of carbapenemases, such as class A enzymes (e.g., KPC), class B enzymes (e.g., NDM), and class D enzymes (e.g., OXA-48), excessive production of AmpC enzymes, and the production of... The presence of broad-spectrum β-lactamase (ESBL), loss of outer membrane porin, and high expression of efflux pumps in CREs is particularly concerning. The acquisition of the polymyxin resistance gene MCR-1 by CREs is especially worrying, as polymyxins are considered the last line of defense against multidrug-resistant Gram-negative bacterial infections. The proliferation of ESBL-producing CREs, coupled with the acquisition of the polymyxin resistance gene MCR-1, could lead to untreatable bacterial infections. Therefore, there is an urgent need to develop antimicrobial drugs targeting Gram-negative bacteria that are simultaneously resistant to carbapenems and polymyxins.

[0003] Aurnofen is a drug used to treat rheumatoid arthritis. Studies have shown that aurnofen exerts its antibacterial effect by inhibiting the activity of thioreductase (TrxR). Aurnofen is also a dual inhibitor of metallo-β-lactamases, which can effectively restore the sensitivity of carbapenem- and polymyxin-resistant bacteria to antibiotics. However, aurnofen has the disadvantage of poor stability. Schiff bases are a class of ligands with antibacterial potential that can effectively stabilize the gold center and improve its antibacterial activity. Therefore, developing novel Schiff base gold (III) antibacterial agents is a promising therapeutic strategy.

[0004] There are currently no reports on the activity of the above compounds against carbapenem-resistant and polymyxin-resistant Gram-negative bacteria. Summary of the Invention

[0005] To address the aforementioned problems, this invention aims to overcome the defect that aurinophene is easily metabolized by biomolecules containing thiol groups, leading to the significant loss of coordinating ligands before interaction with the target enzyme. This invention synthesizes novel Schiff base gold (III) compounds. Schiff bases are a class of high-performance, simple-to-synthesize ligands with antibacterial potential, effectively stabilizing metal ions of various valence states and enhancing their activity. Furthermore, gold (III) has a richer range of coordination modes than gold (I). Therefore, two novel Schiff base gold (III) compounds, Au6 and Au13, were synthesized. These compounds exhibit good antibacterial activity against carbapenem-resistant and polymyxin-resistant Klebsiella pneumoniae, with a minimum inhibitory concentration (MIC) of 10 μM. In animal studies, they significantly improved infections caused by carbapenem-resistant and polymyxin-resistant Klebsiella pneumoniae, promoted wound healing, and prolonged the survival time of mice with severe peritoneal infections.

[0006] The technical solution of this invention is: a novel Schiff gold (III) compound, comprising compounds Au6 and Au13, the chemical structural formula of which is shown below:

[0007]

[0008] Furthermore, a method for synthesizing the aforementioned novel Schiff base gold (III) compounds.

[0009] Furthermore, a pharmaceutical composition comprising a therapeutically effective amount of one or more novel Schiff base gold (III) compounds as described above and a pharmaceutically acceptable carrier.

[0010] Furthermore, the application of the aforementioned novel Schiff base gold (III) compounds in resistance to carbapenem-resistant and polymyxin-resistant Gram-negative bacteria.

[0011] Specifically, the study focused on the preparation of two novel Schiff base gold (III) compounds, Au6 and Au13, which can effectively inhibit carbapenem-resistant and polymyxin-resistant Klebsiella pneumoniae, and their in vitro and in vivo inhibitory effects on these bacteria. Using Schiff bases with antibacterial potential and excellent performance as ligands, and combining them with gold (III) compounds with more diverse coordination modes, novel Schiff base gold (III) compounds, Au6 and Au13, with stronger stability than auronophen, were synthesized. In vitro studies showed that compounds Au6 and Au13 exhibited superior inhibitory effects against carbapenem-resistant and polymyxin-resistant Klebsiella pneumoniae compared to auronophen. In vivo experiments demonstrated that compounds Au6 and Au13 could effectively treat skin infections caused by carbapenem-resistant and polymyxin-resistant Klebsiella pneumoniae and prolong the survival time of mice with severe peritonitis.

[0012] The beneficial effects of this invention are as follows: Compounds Au6 and Au13 in this invention have significant inhibitory effects on carbapenem-resistant and polymyxin-resistant Klebsiella pneumoniae, with a MIC of 10 μM; in animal experiments, compounds Au6 and Au13 can significantly control skin infections caused by carbapenem-resistant and polymyxin-resistant Klebsiella pneumoniae, promote wound healing, and effectively prolong the survival time of mice with severe peritoneal infections; Schiff base gold (III) compounds have the advantages of inexpensive and readily available raw materials, simple synthesis, outstanding stability, few side effects, and significant antibacterial effects, and have great development potential. Attached Figure Description

[0013] Figure 1 These are the 1H and 1C spectra of the Schiff base gold (III) compound Au1 in the embodiments of the present invention; (a) is the 1H spectrum, and (b) is the 1C spectrum.

[0014] Figure 2 These are the 1H and 1C spectra of the Schiff base gold (III) compound Au2 in the embodiments of the present invention; (a) is the 1H spectrum, and (b) is the 1C spectrum.

[0015] Figure 3 These are the 1H and 1C spectra of the Schiff base gold (III) compound Au3 in the embodiments of the present invention; (a) is the 1H spectrum, and (b) is the 1C spectrum.

[0016] Figure 4 These are the proton and carbon spectra of the Schiff base gold (III) compound Au4 in the embodiments of the present invention; (a) is the proton spectrum, and (b) is the carbon spectrum.

[0017] Figure 5 These are the proton and carbon spectra of the Schiff base gold (III) compound Au5 in the embodiments of the present invention; (a) is the proton spectrum, and (b) is the carbon spectrum.

[0018] Figure 6 These are the proton and carbon spectra of the Schiff base gold (III) compound Au6 in the embodiments of the present invention; (a) is the proton spectrum, and (b) is the carbon spectrum.

[0019] Figure 7 These are the proton and carbon spectra of the Schiff base gold (III) compound Au7 in the embodiments of the present invention; (a) is the proton spectrum, and (b) is the carbon spectrum.

[0020] Figure 8 These are the proton and carbon spectra of the Schiff base gold (III) compound Au8 in the embodiments of the present invention; (a) is the proton spectrum, and (b) is the carbon spectrum.

[0021] Figure 9 These are the proton and carbon spectra of the Schiff base gold (III) compound Au9 in the embodiments of the present invention; (a) is the proton spectrum, and (b) is the carbon spectrum.

[0022] Figure 10 These are the 1H and 1C spectra of the Schiff base gold (III) compound Au10 in the embodiments of the present invention; (a) is the 1H spectrum, and (b) is the 1C spectrum.

[0023] Figure 11 These are the 1H and 1C spectra of the Schiff base gold (III) compound Au11 in the embodiments of the present invention; (a) is the 1H spectrum, and (b) is the 1C spectrum.

[0024] Figure 12 These are the 1H and 1C spectra of the Schiff base gold (III) compound Au12 in the embodiments of the present invention; (a) is the 1H spectrum, and (b) is the 1C spectrum.

[0025] Figure 13 These are the 1H and 1C spectra of the Schiff base gold (III) compound Au13 in the embodiments of the present invention; (a) is the 1H spectrum, and (b) is the 1C spectrum.

[0026] Figure 14 This invention relates to the analysis of the in vitro TrxR activity and action sites of Schiff base gold (III) compounds Au6 and Au13 in the embodiments of the present invention;

[0027] Figure 15 This is a diagram showing the in vitro GR activity and action site analysis of Schiff base gold (III) compounds Au6 and Au13 in the embodiments of the present invention;

[0028] Figure 16 This is a structural diagram showing the healing of wounds in mice infected with carbapenem- and polymyxin-resistant Klebsiella pneumoniae using Schiff base gold (III) compounds Au6 and Au13 in the embodiments of the present invention from 1 to 14 days.

[0029] Figure 17 This is a graph showing the effect of Schiff base gold (III) compounds Au6 and Au13 on wound size and body weight in mice with wound infection, as described in this embodiment of the invention.

[0030] Figure 18 This is a diagram illustrating the effects of Schiff base gold (III) compounds Au6 and Au13 on the pathology and immunohistochemistry of infected wounds in mice, as described in this invention.

[0031] Figure 19 This is a graph showing the effect of Schiff base gold (III) compounds Au6 and Au13 on immunofluorescence experiments of infected mouse wound tissue in the embodiments of the present invention;

[0032] Figure 20 This is a schematic diagram illustrating the effect of Schiff base gold (III) compounds Au6 and Au13 on the survival time of mice infected with lethal doses of carbapenem- and polymyxin-resistant Klebsiella pneumoniae in embodiments of the present invention. Detailed Implementation

[0033] To more clearly illustrate the technical solution of the present invention, the technical solution of the present invention will be further described in detail below:

[0034] Carbapenem- and polymyxin-resistant Klebsiella pneumoniae refers to bacteria that are resistant to carbapenems or polymyxins when tested with carbapenems or polymyxins according to CLSI methods (disk method or dilution method) and the results are judged as drug-resistant according to CLSI-M100 standards. MIC refers to the minimum inhibitory concentration, which is an indicator of the antibacterial activity of an antimicrobial drug. It refers to the lowest drug concentration that can inhibit the growth of pathogens in the culture medium after 18 to 24 hours of in vitro bacterial culture.

[0035] Example 1 illustrates the synthesis of Schiff gold (III) compounds Au6 and Au13 using the synthesis of these compounds as an example. The structural formulas of Au6 and Au13 are listed in detail, and the structures of other Schiff gold (III) compounds are briefly described.

[0036] 1. Structural form:

[0037]

[0038] By conjugating Schiff base ligands with antibacterial potential with gold(III) while retaining the cyclohexanediamine and NNOO coordination mode of oxaliplatin, the Schiff base gold(III) compounds Au1-Au13 were obtained through chemical synthesis. Their structures were then determined using 1H and 1C NMR spectroscopy (e.g.,...). Figure 1-13 (As shown).

[0039] Taking the synthesis methods of Au6 and Au13 as examples, the synthesis methods of Schiff base gold (III) compounds Au1-Au13 are described in detail: 7-methoxy-2-naphthol is used as the starting material, and 7-methoxy-2-hydroxynaphthol is obtained by substituting the aldehyde group at the ortho position of the hydroxyl group under the action of magnesium chloride and paraformaldehyde; using this as the starting material, the asymmetric Schiff base ligand is synthesized by a stepwise method, and finally the Schiff base gold (III) compound is synthesized.

[0040] The synthetic route is shown below:

[0041]

[0042] Specifically, the synthesis method of compound Au6 is as follows: (1): Accurately weigh 7-methoxy-2-naphthol (4.00 g, 22.96 mmol), dissolve it in 50 mL of acetonitrile, then add magnesium chloride (4.37 g, 45.92 mmol), adjust the pH to approximately 10 with triethylamine, and finally add paraformaldehyde (7.58 g, 252.60 mmol). Reflux at 98 °C for 17 h. Quench the reaction with water. After the reaction solution cools to room temperature, adjust the pH to approximately 2 with concentrated hydrochloric acid, extract with ethyl acetate, and repeat 3 times. An organic phase was added, anhydrous sodium sulfate was added to remove water, and silica gel column chromatography (petroleum ether: ethyl acetate = 100:3) was used to obtain the product 2-hydroxy-7-methoxy-1-naphthaldehyde; (2): 2-hydroxy-7-methoxy-1-naphthaldehyde (150.00 mg, 0.74 mmol) was accurately weighed, 10 mL of ethanol was added, and it was heated to completely dissolve it. Then, it was added dropwise to an ethanol (1 mL) solution containing cis-cyclohexanediamine (84.70 mg, 0.74 mmol) under ice bath conditions, and stirred in an ice bath for 2 h. Then, 1 mL of ethanol solution containing o-vanillin (112.90 mg, 0.74 mmol) was added dropwise. After stirring at room temperature, a bright yellow precipitate formed after 12 h. The precipitate was filtered and washed with a small amount of ethanol to obtain the crude product. The crude product was then dissolved in an appropriate amount of ethanol, heated under reflux, cooled at -20 °C for 6 h, filtered, washed with a small amount of ethanol, and dried to obtain ligand L6. (3): 2 mL of dichloromethane solution containing L6 (65.20 mg, 0.15 mmol) was added dropwise to 1 mL of solution containing NaAuCl4·2H2O (60.0 mg, 0.74 mmol). In an ethanol solution containing 0 mg (0.15 mmol), the mixture was stirred until homogeneous. Ammonium hexafluorophosphate (143.40 mg, 0.90 mmol) was added. The mixture was then stirred at room temperature for 24 h under argon protection, protected from light. After the reaction was complete, an orange-red solid precipitated. The solid was filtered, washed with a small amount of dichloromethane and ethanol, and the filter cake was dissolved in an appropriate amount of acetonitrile. The ammonium hexafluorophosphate was removed by filtration, and the filtrate was recovered and dried under evaporation to obtain the crude product. A small amount of dichloromethane and methanol was added, and the mixture was stirred at 45 °C for 10 min. This process was repeated three times, and the mixture was washed with a small amount of dichloromethane and methanol and dried under evaporation to obtain Au6.

[0043] Synthesis of compound Au13: (4): Accurately weigh 2,4-dihydroxybenzaldehyde (1.00 g, 7.24 mmol) and 1-(2-chloroethyl)piperidine hydrochloride (1.33 g, 7.24 mmol), add 50 mL of acetone to dissolve completely, then add sodium bicarbonate (1.22 g, 14.48 mmol), reflux for 3 days; after the reaction is complete, filter, and use silica gel column chromatography (ethyl acetate) to obtain the intermediate 2-hydroxy-4-(2- (piperidin-1-yl)ethoxy)benzaldehyde; (5): Accurately weigh 2-hydroxy-7-methoxy-1-naphthaldehyde (200.00 mg, 0.99 mmol), add 10 mL of ethanol, heat to dissolve completely, and then dropwise into an ethanol (1 mL) solution containing cis-cyclohexanediamine (112.90 mg, 0.99 mmol) under ice bath conditions. Stir in an ice bath for 2 h, and then dropwise add 2-hydroxy-4-(2-(piperidin-1-yl)ethoxy)benzaldehyde. A solution of 1 mL of ethanol containing 246.60 mg (0.99 mmol) was stirred at room temperature. After 12 h, the solution turned pale yellow and clear. L13 was obtained by silica gel column chromatography (dichloromethane:methanol = 100:3); (6): 2 mL of dichloromethane solution containing L13 (66.60 mg, 0.13 mmol) was added dropwise to 1 mL of ethanol solution containing NaAuCl4·2H2O (50.00 mg, 0.13 mmol), stirred evenly, and then added... Ammonium hexafluorophosphate (102.40 mg, 0.65 mmol) was added, and the mixture was stirred at room temperature for 24 h under argon protection and in the dark. After the reaction was completed, an orange-red solid precipitated. The solid was filtered, washed with a small amount of dichloromethane and ethanol, and the filter cake was dissolved in an appropriate amount of acetonitrile. The ammonium hexafluorophosphate was removed by filtration, and the filtrate was recovered and dried under evaporation to obtain the crude product. Methanol was then added, and the mixture was stirred at 55 °C for 10 min. Toluene was then added, and the mixture was allowed to stand at -20 °C for more than 6 h. The mixture was then filtered, washed with a small amount of dichloromethane and methanol, and dried under evaporation to obtain Au13.

[0044] In addition, the synthetic route of Au1-13 is described in the examples as follows:

[0045]

[0046]

[0047] Among them, (a)MgCl2,(CH2O) n ,TEA,CH3CN,reflux,17h; (b)(1)EtOH,0℃,2h,(2)RT,12h;(c)NaAuCl4·2H2O,NH4PF6,DCM,EtOH,Ar,RT,24h.

[0048] Au1 compound data: orange-red solid, yield 12.4%, purity >95%. 1H NMR (500MHz, DMSO-d6) δ9.51(s,1H,N=CH),8.80(s,1H,N=CH),8.14(d,J=9.1Hz,1H,ArH),7.99(d,J=7.9 Hz,1H,ArH),7.90(d,J=8.7Hz,1H,ArH),7.79-7.73(m,2H,ArH),7.28(d,J=10.0Hz,2H,ArH),7.16(dd,J= 8.8,2.2Hz,1H,ArH),7.03(t,J=7.4Hz,1H,ArH),4.71(s,1H,N-CH),4.54(dd,J=10.4,5.2Hz,1H,N-CH), 3.99(s,3H,OCH3),2.83(d,J=15.7Hz,1H),2.21-1.95(m,3H),1.78(d,J=12.5Hz,1H),1.59-1.31(m,3H). 13 C NMR (126MHz, DMSO-d6) δ162.24,160.13,159.82,159.79,154.19,139.31,138.94,136.52,135.15,130.96,122. 74,120.11,118.95,117.98,117.34,114.67,109.42,103.72,72.74,71.13,55.78,28.91,23.17,22.65,18.24. ESI-MS(+)[m / z]:597.15[M-PF6] + .

[0049] Au2 compound data: orange-red solid, yield 19.5%, purity >95%. 1H NMR(500MHz,DMSO-d6)δ9.49(d,1H,N=CH),8.78(d,J=1.8Hz,1H,N=CH),8.16-8.07(m,2H,ArH),7.89(dd,J=13.3, 8.8Hz,1H,ArH),7.77(s,1H,ArH),7.26(d,J=9.0Hz,1H,ArH),7.19-7.12(m,2H,ArH),6.97(td,J=8.3,2.5Hz,1H, ArH), 4.69 (d, 1H, J = 4.4Hz, N-CH), 4.53 (dt, J = 10.1, 4.7Hz, 1H, N-CH), 3.98 (d, J = 9.1Hz, 3H, OCH3), 2.80 (d, J = 15. 8Hz,1H),2.19-2.08(m,1H),2.01(d,J=12.9Hz,1H),1.90-1.75(m,2H),1.58-1.44(m,2H),1.37(t,J=12.9Hz,1H). 13 CNMR(126MHz,DMSO-d6)δ162.17,161.76,160.15,159.19,154.20,139.40,135.11,130.95,122.73,117.62,117 .17,114.67,109.35,108.21,107.96,107.78,104.25,103.78,73.05,71.02,55.77,28.89,25.42,22.65,18.16. ESI-MS(+)[m / z]:615.17[M-PF6] + .

[0050] Au3 compound data: orange-red solid, yield 17.4%, purity >95%. 1H NMR (500MHz, DMSO-d6) δ9.41(s,1H,N=CH),8.75(s,1H,N=CH),8.16(d,J=9.1Hz,1H,ArH),7.90(dd,J=2 0.0,8.8Hz,1H,ArH),7.80-7.70(m,2H,ArH),7.29(d,J=9.1Hz,1H,ArH),7.21-7.12(m,2H,ArH),6.90(d d,J=10.4,7.9Hz,1H,ArH),4.70(d,J=4.8Hz,1H,N-CH),4.61-4.55(m,1H,N-CH),3.98(d,J=7.8Hz,3H, OCH3),2.65-2.62(m,1H),2.38-2.34(m,1H),2.13-1.98(m,2H),1.71(s,1H),1.53(s,2H),1.23(s,1H). 13 C NMR (126MHz, DMSO-d6) δ161.62,160.63,154.42,153.72,153.61,139.97,139.06,135.58,131.49,123.26,11 7.62, 115.17 (d, J = 12.7Hz), 109.91, 108.71, 104.35, 75.32, 73.16, 72.37, 56.22, 28.97, 23.54, 22.04, 20.60. ESI-MS(+)[m / z]:615.09[M-PF6] + .

[0051] Au4 compound data: orange-red solid, yield 18.1%, purity >95%. 1H NMR (500MHz, DMSO-d6) δ9.46(s,1H,N=CH),8.65(s,1H,N=CH),8.10(dd,J=9.1,2.5Hz,1H,ArH),7.85(dd,J=8.8,2.6Hz,1H,Ar H),7.78(dd,J=8.1,2.6Hz,1H,ArH),7.71(t,J=2.5Hz,1H,ArH),7.22(dd,J=9.1,2.5Hz,1H,ArH),7.13(dt,J=2.3,8.9Hz,1H, ArH),7.06(s,1H,ArH),6.80(d,J=8.1Hz,1H,ArH),4.68(s,1H,N-CH),4.55-4.48(m,1H,N-CH),3.97(s,3H,OCH3),2.79(d,J= 15.6Hz, 1H), 2.34 (d, J = 2.5Hz, 3H, CH3), 2.18-1.98 (m, 3H), 1.78 (d, J = 12.0Hz, 1H), 1.58-1.45 (m, 2H), 1.36 (t, J = 12.9Hz, 1H). 13 C NMR (126MHz, DMSO-d6) δ162.34,160.22,159.87,159.14,154.20,150.89,139.41,136.23,135.24,131.06,122.83, 120.76,117.81,117.73,117.44,114.76,109.50,103.78,72.97,71.00,55.87,29.09,23.41,22.66,21.85,18.41. ESI-MS(+)[m / z]:611.15[M-PF6] + .

[0052] Au5 compound data: brownish-red solid, yield 14.2%, purity >95%. 1H NMR (500MHz, DMSO-d6) δ9.52(s,1H,N=CH),8.72(s,1H,N=CH),8.13(d,J=9.1Hz,1H,ArH),7.89(d,J=8 .8Hz,1H,ArH),7.76(t,J=2.9Hz,2H,ArH),7.58(dd,J=8.6,2.3Hz,1H,ArH),7.26(d,J=9.1Hz,1H,ArH) ,7.22-7.13(m,2H,ArH),4.70(s,1H,N-CH),4.56-4.50(m,1H,N-CH),3.99(s,3H,OCH3),2.81(d,J=15. 7Hz, 1H), 2.30 (s, 3H, CH3), 2.18-1.97 (m, 3H), 1.78 (d, J = 11.8Hz, 1H), 1.58-1.43 (m, 2H), 1.37 (t, 1H). 13 C NMR(126MHz,DMSO-d6)δ162.25,160.11,159.61,158.13,154.14,140.28,139.26,135.37,135.14,130.94,127.60, 122.72,119.65,117.71,117.36,114.63,109.39,103.71,72.67,71.03,55.78,28.91,23.18,22.62,19.45,18.21. ESI-MS(+)[m / z]:611.13[M-PF6] + .

[0053] Au6 compound data: orange-red solid, yield 13.3%, purity >95%. 1H NMR (500MHz, DMSO-d6) δ9.52(s,1H,N=CH),8.76(s,1H,N=CH),8.15(d,J=9.2Hz,1H,ArH),7.89(d,J=8.8Hz,1H,ArH),7.76(d ,J=2.4Hz,1H,ArH),7.54(d,J=8.1Hz,1H,ArH),7.39(d,J=7.7Hz,1H,ArH),7.29(d,J=9.0Hz,1H,ArH),7.16(dd,J=8.9,2.4H z,1H,ArH),6.94(t,J=7.9Hz,1H,ArH),4.72(s,1H,N-CH),4.54(dt,J=10.5,4.9Hz,1H,N-CH),4.00(s,3H,OCH3),3.89(s,3H ,OCH3),2.84(d,J=15.6Hz,1H),2.20-1.97(m,3H),1.83-1.74(m,1H),1.51(dt,J=25.8,13.5Hz,2H),1.36(t,J=13.0Hz,1H). 13 C NMR(126MHz,DMSO-d6)δ162.32,160.13,159.88,154.13,150.72,148.80,139.36,135.14,130.95,127.05,122.72, 120.08,118.78,118.49,117.37,114.65,109.38,103.74,72.76,71.14,56.03,55.78,28.97,23.23,22.62,18.29. ESI-MS(+)[m / z]:627.13[M-PF6] + .

[0054] Au7 compound data: orange-red solid, yield 17.3%, purity >95%. 1H NMR (500MHz, DMSO-d6) δ9.50(s,1H,N=CH),8.58(s,1H,N=CH),8.13(d,J=9.2Hz,1H,ArH),7.87(dd,J=15.1,8.9H z,2H,ArH),7.75(d,J=2.3Hz,1H,ArH),7.22(d,J=9.1Hz,1H,ArH),7.15(dd,J=8.7,2.2Hz,1H,ArH),6.82(d,J=2. 4Hz,1H,ArH),6.65(dd,J=8.9,2.4Hz,1H,ArH),4.64(d,1H,N-CH),4.51(dt,J=10.3,4.9Hz,1H,N-CH),3.99(s,3H ,OCH3),3.89(s,3H,OCH3),2.79(d,J=16.7Hz,1H),2.21-1.95(m,2H),1.78(d,J=12.5Hz,1H),1.56-1.18(m,4H). 13 C NMR(126MHz,DMSO-d6)δ168.48,162.33,162.13,160.12,157.82,154.14,139.28,137.68,135.18,130.96,122.71, 117.40,114.63,113.73,109.39,109.13,103.70,100.27,73.09,70.39,56.11,55.77,28.86,23.17,22.68,18.28. ESI-MS(+)[m / z]:627.14[M-PF6] + .

[0055] Au8 compound data: brownish-red solid, yield 11.6%, purity >95%. 1H NMR (500MHz, DMSO-d6) δ9.53(d,J=5.6Hz,1H,N=CH),8.74(d,J=5.4Hz,1H,N=CH),8.12(t,J=7.4Hz,1H,ArH),7.88( t,J=7.3Hz,1H,ArH),7.76(d,J=6.0Hz,1H,ArH),7.52(d,J=6.3Hz,1H,ArH),7.42(q,J=5.5,4.3Hz,1H,ArH),7.27- 7.19(m,2H,ArH),7.17-7.12(m,1H,ArH),4.70(s,1H,N-CH),4.58-4.46(m,1H,N-CH),3.98(d,J=5.7Hz,3H,OCH3), 3.78(d,J=5.8Hz,3H,OCH3),2.80(d,J=14.6Hz,1H),2.17-1.96(m,5H),1.79(d,J=13.4Hz,1H),1.59-1.28(m,1H). 13 CNMR(126MHz,DMSO-d6)δ162.22,160.13,159.61,155.08,154.17,151.36,139.28,135.15,130.95,128.52,122.74 ,119.09,118.85,117.35,115.86,114.67,109.38,103.69,72.65,71.15,55.80,55.73,29.10,23.10,22.80,18.16. ESI-MS(+)[m / z]:627.13[M-PF6] + .

[0056] Au9 compound data: orange-red solid, yield 17.6%, purity >95%. 1H NMR (500MHz, DMSO-d6) δ9.40(s,1H,N=CH),8.70(s,1H,N=CH),8.11(d,J=9.1Hz,1H,ArH),7.86(d,J=8.8Hz,1H,ArH),7.70( s,1H,ArH),7.61(t,J=8.3Hz,1H,ArH),7.24(d,J=9.1Hz,1H,ArH),7.14(dd,J=8.8,2.2Hz,1H,ArH),6.84(d,J=8.6Hz,1H,A rH),6.55(d,J=8.1Hz,1H,ArH),4.68(q,J=4.5Hz,1H,N-CH),4.56(dt,J=9.9,4.8Hz,1H,N-CH),3.98(s,3H,OCH3),3.92(s, 3H,OCH3),2.61(d,J=15.9Hz,1H),2.31(s,1H),2.14-1.98(m,2H),1.72(s,1H),1.61-1.46(m,2H),1.37(t,J=12.0Hz,1H). 13 C NMR(126MHz,DMSO-d6)δ162.24,160.83,160.76,160.12,153.91,153.25,139.35,139.02,135.14,130.99,122.74, 117.38,114.62,110.63,110.19,109.45,103.75,100.64,72.55,71.56,56.93,55.77,27.50,24.22,21.87,18.79. ESI-MS(+)[m / z]:627.18[M-PF6] + .

[0057] Au10 compound data: orange-red solid, yield 13.7%, purity >95%. 1H NMR (500MHz, DMSO-d6) δ9.54(s,1H,N=CH),8.79(s,1H,N=CH),8.14(d,J=9.2Hz,1H,ArH),8.00(d,J=2.6Hz,1H,ArH),7.89( d,J=8.8Hz,1H,ArH),7.84(dd,J=8.9,2.6Hz,1H,ArH),7.78(d,J=2.4Hz,1H,ArH),7.26(d,J=9.0Hz,1H,ArH),7.22(d,J=8.9 Hz,1H,ArH),7.16(dd,J=8.8,2.3Hz,1H,ArH),4.70(s,1H,N-CH),4.52(dt,J=10.6,5.0Hz,1H,N-CH),3.99(s,3H,OCH3),2.8 4(d,J=15.4Hz,1H),2.15-2.05(m,2H),2.00(d,J=10.7Hz,1H),1.80(d,J=12.9Hz,1H),1.59-1.35(m,3H),1.32(s,9H,CH3). 13 C NMR(126MHz,DMSO-d6)δ162.27,160.13,160.00,158.13,154.22,141.14,139.26,137.04,135.18,132.14,130.96,122 .74,119.44,117.55,117.41,114.67,109.41,103.71,72.72,71.03,55.80,33.87,31.01,29.06,23.01,22.81,18.17. ESI-MS(+)[m / z]:653.18[M-PF6] + .

[0058] Au11 compound data: orange-red solid, yield 11.1%, purity >95%. 1 H NMR (500MHz,

[0059] DMSO-d6)δ9.57(s,1H,N=CH),8.77(d,1H,N=CH),8.14(d,J=9.4Hz,1H,ArH),7.93-7.85

[0060] (m,2H,ArH),7.79(s,1H,ArH),7.72(s,1H,ArH),7.17(t,J=8.5Hz,2H,ArH),4.69(s,1H,N-CH),4.52(s,1H,N-CH),3.99(d,J=2 .7Hz,3H,OCH3),2.84(d,J=16.4Hz,3H),2.11-1.96(m,4H),1.79(d,1H),1.51(d,J=2.5Hz,9H,CH3),1.33(d,J=2.8Hz,9H,CH3). 13 CNMR(126MHz,DMSO-d6)δ162.35,160.46,160.12,157.15,154.21,140.18,139.18,137.70,135.25,133.26,130.95,130.58,1 22.70,119.94,117.69,114.63,109.51,103.66,72.42,71.00,55.78,35.58,33.99,31.07,29.59,29.12,22.99,22.78,18.26. ESI-MS(+)[m / z]:709.26[M-PF6] + .

[0061] Au12 compound data: brownish-red solid, yield 13.7%, purity >95%. 1 H NMR (500MHz, DMSO-d6) δ9.53(s,1H,N=CH),8.82(s,1H,N=CH),8.15(d,J=9.1Hz,1H,ArH),8.07(d,J=8.3Hz,1H,ArH),7.89(dd,J =17.0,8.2Hz,3H,ArH),7.78(s,1H,ArH),7.60(s,1H,ArH),7.52(dt,J=15.0,7.2Hz,3H,ArH),7.41(d,J=8.5Hz,1H,ArH),7.26(d ,J=9.0Hz,1H,ArH),7.17(dd,J=8.4,1.7Hz,1H,ArH),4.73(s,1H,N-CH),4.55(dt,J=10.5,4.9Hz,1H,N-CH),3.99(s,3H,OCH3), 2.85(d,J=15.0Hz,1H),2.21-2.12(m,2H),2.06-1.99(m,1H),1.80(d,J=12.6Hz,1H),1.59-1.47(m,2H),1.39(t,J=12.6Hz,1H). 13C NMR (126MHz, DMSO-d6) δ162.27,160.17,160.10,159.28,154.21,150.16,139.39,138.19,137.04,135.20,131.01,129.30,129. 25,127.23,122.78,119.24,117.66,117.34,115.14,114.72,109.46,103.75,72.93,71.05,55.80,29.02,23.18,22.69,18.28. ESI-MS(+)[m / z]:673.14[M-PF6] + .

[0062] Au13 compound data: orange-red solid, yield 17.5%, purity >95%. 1 H NMR (500MHz, DMSO-)

[0063] d6)δ9.47(s,1H,N=CH),8.59(s,1H,N=CH),8.13(d,J=10.0Hz,1H,ArH),7.89(dd,J=5.0

[0064] Hz,2H,ArH),7.75(s,1H,ArH),7.18(dd,J=10.0Hz,2H,ArH),6.93(s,1H,ArH),6.71(d,J=10.0Hz,1H,ArH),4.65(s,1H,N-CH),4.54-4.49(m,1H, N-CH), 4.47 (t, J = 5.0Hz, 2H), 3.98 (s, J = 6.1Hz, 5H, OCH3, OCH2), 2.77 (d, J = 15.6Hz, 1H), 2.18-1.95 (m, 4H), 1.85-1.65 (m, 6H), 1.57-1.28 (m, 7H). 13 C NMR(126MHz,DMSO-d6)δ162.29,162.08,160.16,158.05,154.19,139.33,137.80,135.20,131.00,122.73,117.33,114.67,1 14.26,109.42,109.38,103.74,101.12,73.09,70.51,62.99,55.79,54.56,52.87,28.96,23.20,22.64,22.53,21.07,18.30. ESI-MS(+)[m / z]:724.20[M-PF6] + .

[0065] 2. Drugs and reagents: Aurinofen and other gold compounds were dissolved in DMF; imipenem and polymyxin B were purchased from a certain technology company; Klebsiella pneumoniae ATCC BAA-1705 (carbapenem- and polymyxin-resistant), Pseudomonas aeruginosa ATCC BAA-2108, Escherichia coli ATCC BAA-2140, and Staphylococcus aureus ATCC 700699 were purchased from a certain biological standard resource center; in addition, four bacterial strains were isolated from the hospital, including carbapenem- and polymyxin-resistant Klebsiella pneumoniae (CRKP) (NCBI: WH2023-10), carbapenem-resistant Acinetobacter baumannii (CRAB) (NCBI: JAKLSN000000000), carbapenem-resistant Pseudomonas aeruginosa, and carbapenem-resistant Escherichia coli; yeast extract, trypsin, and sodium chloride were all products of a certain biotechnology company.

[0066] Example 2: Screening of in vitro antibacterial activity of Schiff base gold (III) compounds Au6 and Au13:

[0067] In accordance with the Clinical and Laboratory Standards in Incubation (CLSI) guidelines, the antibacterial efficacy of Schiff base gold (III) compounds was determined using the broth microdilution method; the final concentration of the compound was 40 μM; after incubation at 37°C for 24 h, bacterial growth was measured in 96-well plates using an ELISA reader; MIC was defined as the lowest drug concentration that inhibits the growth of pathogens in the culture medium after 18 to 24 h of in vitro bacterial culture.

[0068] Table 1. Screening of Schiff base gold (III) compounds Au1-Au13 against multidrug-resistant bacteria (MIC)

[0069]

[0070]

[0071] Example 3: Analysis of the inhibitory activity and action sites of Schiff gold (III) compounds Au6 and Au13 on TrxR enzyme in the lung:

[0072] Gold complexes significantly inhibited TrxR, and this inhibition reduced the cellular antioxidant capacity, leading to an increase in reactive oxygen species (ROS). These effects are often closely related to its antibacterial activity. A plasmid containing the TrxR gene was constructed and transformed into *E. coli* to express the *Klebsiella pneumoniae* TrxR protein in vitro. The inhibitory activities of compounds Au6 and Au13 on TrxR and their target sites were determined. First, the enzyme was purified by nickel affinity chromatography and gel filtration chromatography. The collected fractions were analyzed by reducing SDS-PAGE, showing the presence of CRKP TrxR (with a molecular weight of 38 kDa) in the gel. Figure 14 A). Next, the in vitro TrxR enzyme activity was determined by DTNB reduction activity. This method utilizes the reduction of TrxR to generate a colored 2-nitro-5-thiobenzoate (λmax: 412 nm); the results showed that Auranofin (IC) 50 =30.9μM), Au6(IC) 50 =19.9μM) and Au13(IC 50 =18.3 μM) both showed inhibitory activity against TrxR ( Figure 14 B); Subsequently, the irreversible inhibitory effect of Au6 and Au13 on recombinant TrxR was verified by evaluating the activities of desalted and non-desalted enzymes. Figure 14 C); To further investigate the covalent binding sites between Au6, Au13, and TrxR, TrxR was pre-reduced with NADPH, resulting in the reduction of the disulfide bonds of redox activity. The TrxR enzyme was then co-incubated with Au6 and Au13 to assess its activity. The results showed that NADPH-reduced TrxR was sensitive to treatment with Au6 and Au13, suggesting that these complexes may bind to redox active structures. Figure 14 D); To further verify this hypothesis, GSH was pre-incubated with Au6 and Au13 respectively, and then TrxR was co-incubated with Au6 and Au13 conjugates of GSH to measure TrxR activity; the results showed that pre-incubation of GSH with Au6 and Au13 eliminated its inhibitory effect on recombinant TrxR (D); Figure 14 E); These findings suggest that Au6 and Au13 possess the ability to modify with thiohydrogen groups, thereby achieving irreversible inhibition of bacterial TrxR activity by targeting redox active structures.

[0073] Example 4: Analysis of the inhibitory activity and action sites of Schiff base gold (III) compounds Au6 and Au13 on pulmonary GR enzyme:

[0074] Based on the above results, it was found that Auranofin and its structurally modified complexes (Au6, Au13) were not effective inhibitors of TrxR; the glutathione system and the Trx system are two synergistic disulfide reductase systems that perform antioxidant functions and play key roles in various cellular activities; importantly, the GSH-GR system present in Gram-negative bacteria provides strong support for the Trx system; therefore, further investigation was conducted to determine whether the gold complexes (Auranofin, Au6, and Au13) inhibited glutathione reductase (GR) activity; firstly, the Klebsiella pneumoniae GR protein was recombinantly expressed in Escherichia coli, and the enzyme was subsequently purified by nickel affinity chromatography and gel filtration chromatography; the collected fractions were analyzed by reducing SDS-PAGE, showing the presence of CRKP GR (with a molecular weight of 45 kDa) in the gel. Figure 15 A) GR catalyzes the reduction of GSSG to regenerate GSH, while simultaneously oxidizing NADPH to generate NADP. + The dehydrogenation rate of NADPH can be determined by measuring the rate of absorbance reduction at 340 nm, thereby calculating GR activity. The inhibitory effect of GR is estimated by observing the rate of NADPH reduction in the presence of Auranofin, Au6, or Au13; results show that Au6 and Au13 can rapidly inhibit GR in a dose-dependent manner, with Au6 having an IC50 value of [missing value]. 50 The value is 11.62 μM, and the IC of Au13 is... 50 The value was 5.39 μM; in contrast, at a concentration of 40 μM, Auranofin did not show any inhibitory effect on GR. Figure 15 B); Subsequently, the irreversible inhibitory effect of Au6 and Au13 on recombinant GR was further confirmed by measuring the activities of desalted and non-desalted enzymes (B). Figure 15 C); To further investigate the covalent binding sites between Au6, Au13, and GR, GR was pre-reduced with NADPH, leading to the reduction of redox-active disulfide bonds. The activity was assessed by co-incubating the enzyme with Au6 and Au13. The results showed that NADPH-reduced GR was sensitive to treatment with Au6 and Au13, suggesting that these complexes may bind to redox-active structures. Figure 15 D); To further verify this hypothesis, GSH was pre-incubated with Au6 and Au13 respectively, and then GR was co-incubated with GSH-conjugated Au6 and Au13 to measure GR activity; the study found that pre-incubation of GSH with Au6 and Au13 eliminated its inhibitory effect on recombinant GR ( Figure 15E); These results indicate that Au6 and Au13 have strong sulfhydryl modification effects and irreversibly inhibit GR activity by targeting redox active structures; in contrast, Auranofin did not show inhibitory activity against GR, which may be the main reason for its poor antibacterial activity against CRKP; in summary, the results suggest that Au6 and Au13 mainly induce intracellular redox imbalance in bacteria by inhibiting GR activity, thereby leading to bacterial death.

[0075] Example 5: In vivo anti-infective evaluation of Schiff base gold (III) compounds Au6 and Au13:

[0076] Based on the determination of minimum inhibitory concentration (MIC), compounds Au6 and Au13 both exhibited good antibacterial activity against carbapenem- and polymyxin-resistant Klebsiella pneumoniae, with activity at least 16 times higher than that of aurinofen. Therefore, the systematic evaluation of the in vivo anti-infective activity of compounds Au6 and Au13 in mice was conducted using the following methods:

[0077] 22-25g BALB / c mice were acclimatized for one week. A 1.2*1.2cm incision was made in the skin on the back of the mice using surgical scissors, and 10 mg of methylphenidate was injected into the wound surface. 9 A wound infection model was established using 100 μL of CFU / mL bacterial suspension (carbapenem- and polymyxin-resistant Klebsiella pneumoniae). Mice were randomly divided into 6 groups (3 mice per group). 24 hours after infection, the following treatments were administered: control group received saline solution applied to the wound; H2O2 group (positive control) received 10 mM hydrogen peroxide solution applied to the wound; AF group (positive control) received 5 mM aurinol solution applied to the wound; polymyxin B group (positive control) received 5 mM polymyxin B solution applied to the wound; Au6 group received 5 mM Au6 solution applied to the wound; and Au13 group received 5 mM Au13 solution applied to the wound. Wound infection status was continuously observed in mice for 14 days. Figure 16 Record the wound ( Figure 17 A) Mouse weight ( Figure 17 B) Changes were observed, and pathological staining, immunohistochemical experiments, and inflammatory cytokine analysis were performed on the wound tissue.

[0078] Compared with the control group, H2O2 group, auron-methyl group, and polymyxin B group, compounds Au6 and Au13 showed significant antibacterial effects, effectively promoting wound healing and significantly improving wound area recovery; changes in mouse body weight confirmed that compounds Au6 and Au13 had no toxicity during treatment; hematoxylin and eosin (H&E) staining and immunohistochemical analysis (IL-6 and TNF-α) showed no toxicity. Figure 18 The results showed that, compared with other groups, the skin tissue inflammation in the Au6 and Au13 treatment groups was significantly reduced, and the expression of inflammatory factors was also significantly decreased; in addition, the results were further confirmed by tissue immunofluorescence experiments. Figure 19 This study confirmed that, compared with the control group, the infiltration of inflammatory cells (macrophages and T cells) in the Au6 and Au13 treatment groups was significantly reduced, resulting in a reduction of local tissue inflammation. In summary, in vivo data showed that the in vivo antibacterial activity of compounds Au6 and Au13 was superior to that of aurinol and polymyxin B, and could significantly reduce local tissue infection and promote wound healing.

[0079] Example 6: Effects of Schiff gold (III) compounds Au6 and Au13 on the survival rate of CRKP-infected mice:

[0080] Female BALB / c mice (18-20g) were acclimatized for one week; they were then randomly divided into a control group, a polymyxin B group, an Au6 group, and an Au13 group (n=6); each mouse was then intraperitoneally injected with 200μL of CRKP suspension (10 9 CFU / mL); 1 hour after infection, mice were treated with PBS, polymyxin B, Au6, and Au13, respectively; survival rates were observed and recorded within 48 hours. Figure 20 All experiments were conducted in three biological replicates. In contrast, three mice in both the Au6 and Au13 treatment groups survived for more than 24 hours, with one mouse in the Au6 treatment group surviving for more than 48 hours and two mice in the Au13 treatment group surviving for more than 48 hours. The results indicate that both Au6 and Au13 can prolong the survival time of mice with severe peritonitis and demonstrate a therapeutic advantage against multidrug-resistant Gram-negative bacteria.

Claims

1. A novel class of Schiff base gold(III) compounds, characterized in that, The compounds are Au6 and Au13, and their chemical structural formulas are shown below: 、 。 2. The method for synthesizing a novel Schiff base gold(III) compound according to claim 1, characterized in that, The synthetic route is shown below: 。 3. A pharmaceutical composition comprising a therapeutically effective amount of a novel Schiff base gold (III) compound as described in claim 1 and a pharmaceutically acceptable carrier.

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