Synthesis and antibacterial application of novel Schiff base gold (III) compound

Abstract

The invention discloses synthesis and antibacterial application of a novel Schiff base gold (III) compound, and belongs to the technical field of medicine preparation. Golden nofen has remarkable antibacterial activity, but an Au-S bond of the gold nofen is easily damaged by reducing thiol in cells and is metabolized before reaching a target spot to generate toxic and side effects, and development of a gold compound with higher stability based on the gold nofen is one of strategies for overcoming drug-resistant bacteria; schiff base has antibacterial activity and can stabilize gold atoms and improve the activity of the gold atoms after being combined with gold (III) ions; in-vitro antibacterial activity shows that Au6 and Au13 have remarkable antibacterial advantages on carbapenems and polymyxin-resistant klebsiella pneumoniae, the minimum inhibitory concentration (MIC) is 10 mu M, and the antibacterial activity of Au6 and Au13 is improved by at least 16 times compared with that of Jinnofen; in-vivo antibacterial activity experiments prove that Au6 and Au13 can reduce mouse skin infection, promote wound healing and prolong the survival time of mice with abdominal infection, and have important practical application value.

Description

Technical Field

[0001] The present invention relates to the technical field of drug preparation, and in particular, to the synthesis of a novel Schiff base gold (III) compound and its antibacterial application; in particular, to the application of the Schiff base gold (III) compound in resisting carbapenem-resistant and polymyxin-resistant Gram-negative bacteria. Background Art

[0002] The hazards of multidrug-resistant bacteria have 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 are resistant to any carbapenem antibiotics (such as imipenem, ertapenem or doripenem); their resistance has multiple mechanisms, including the production of carbapenemases, such as class A enzymes (such as KPC), class B enzymes (such as NDM) and class D enzymes (such as OXA-48), overproduction of AmpC enzymes, production of The acquisition of the polymyxin resistance gene MCR-1 by CRE is particularly worrying because polymyxins are considered the last line of defense for the treatment of multidrug-resistant Gram-negative bacterial infections. The massive reproduction of ESBLs-producing CRE, coupled with the acquisition of the polymyxin resistance gene MCR-1 by these bacteria, may lead to untreatable bacterial infections. Therefore, there is an urgent need to develop antimicrobial drugs for Gram-negative bacteria that are resistant to both carbapenems and polymyxins.

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

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

[0005] In view of the above problems, in order to solve the defect that auranofin is easily metabolized by biological macromolecules containing thiol groups, resulting in a large amount of loss of the coordinated ligand before reacting with the target enzyme, the present invention synthesized a new Schiff base gold (III) compound; Schiff base is a class of ligands with excellent performance, simple synthesis and antibacterial potential, which can effectively stabilize metal ions of various valence states and improve their activity; in addition, gold (III) has a richer coordination mode than gold (I); therefore, two new Schiff base gold (III) compounds Au6 and Au13 were synthesized, which have good antibacterial activity against carbapenem-resistant and polymyxin-resistant Klebsiella pneumoniae, with a minimum inhibitory concentration of 10 μM, and can significantly improve the infection caused by carbapenem-resistant and polymyxin-resistant Klebsiella pneumoniae at the animal level, promote wound healing, and prolong the survival time of mice with severe abdominal infection.

[0006] The technical solution of the present invention is: a new type of Schiff base gold (III) compound described in the present invention, the Schiff base gold (III) compound includes compound Au6 and compound Au13, and its chemical structure is shown as follows:

[0007]

[0008] Furthermore, a method for synthesizing the novel Schiff base gold (III) compound is provided.

[0009] Furthermore, a pharmaceutical composition contains 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 novel Schiff base gold (III) compound is used to fight carbapenem-resistant and polymyxin-resistant Gram-negative bacteria.

[0011] Specifically, two new Schiff base gold (III) compounds Au6 and Au13 that can effectively inhibit carbapenem- and polymyxin-resistant Klebsiella pneumoniae were prepared, and their inhibitory effects on carbapenem- and polymyxin-resistant Klebsiella pneumoniae in vivo and in vitro were studied; Schiff bases with antibacterial potential and excellent performance were used as ligands in combination with gold (III) with richer coordination patterns to synthesize new Schiff base gold (III) compounds Au6 and Au13 that are more stable than auranofin; in vitro studies showed that compounds Au6 and Au13 have better inhibitory effects on carbapenem- and polymyxin-resistant Klebsiella pneumoniae than auranofin; in vivo experiments proved that compounds Au6 and Au13 can effectively treat skin infections caused by carbapenem- and polymyxin-resistant Klebsiella pneumoniae, and prolong the survival time of mice with severe abdominal infections.

[0012] The beneficial effects of the present invention are as follows: the compounds Au6 and Au13 in the present invention have significant inhibitory effects on carbapenem-resistant and polymyxin-resistant Klebsiella pneumoniae, with an MIC of 10 μM; in animal experiments, the 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 abdominal infections; the Schiff base gold (III) compound has the advantages of cheap and readily available raw materials, simple synthesis, outstanding stability, few side effects and significant antibacterial effects, and has great development potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 1 are hydrogen and carbon spectra of the Schiff base gold (III) compound Au1 in the embodiment of the present invention; (a) is the hydrogen spectrum, and (b) is the carbon spectrum;

[0014] Figure 2 1 are hydrogen and carbon spectra of the Schiff base gold (III) compound Au2 in the embodiment of the present invention; (a) is the hydrogen spectrum, and (b) is the carbon spectrum;

[0015] Figure 3 1 are hydrogen and carbon spectra of the Schiff base gold (III) compound Au3 in the embodiment of the present invention; (a) is the hydrogen spectrum, and (b) is the carbon spectrum;

[0016] Figure 4 1 are hydrogen and carbon spectra of the Schiff base gold (III) compound Au4 in the embodiment of the present invention; (a) is the hydrogen spectrum, and (b) is the carbon spectrum;

[0017] Figure 5 1 are hydrogen and carbon spectra of the Schiff base gold (III) compound Au5 in the embodiment of the present invention; (a) is the hydrogen spectrum, and (b) is the carbon spectrum;

[0018] Figure 6 1 are hydrogen and carbon spectra of the Schiff base gold (III) compound Au6 in the embodiment of the present invention; (a) is the hydrogen spectrum, and (b) is the carbon spectrum;

[0019] Figure 7 1 are hydrogen and carbon spectra of the Schiff base gold (III) compound Au7 in the embodiment of the present invention; (a) is the hydrogen spectrum, and (b) is the carbon spectrum;

[0020] Figure 8 1 are hydrogen and carbon spectra of the Schiff base gold (III) compound Au8 in the embodiment of the present invention; (a) is the hydrogen spectrum, and (b) is the carbon spectrum;

[0021] Fig. 9 1 are hydrogen and carbon spectra of the Schiff base gold (III) compound Au9 in the embodiment of the present invention; (a) is the hydrogen spectrum, and (b) is the carbon spectrum;

[0022] Fig.10 1 and 2 are hydrogen and carbon spectra of the Schiff base gold (III) compound Au10 in the embodiment of the present invention; (a) is the hydrogen spectrum, and (b) is the carbon spectrum;

[0023] Fig.11 1 and 2 are hydrogen and carbon spectra of the Schiff base gold (III) compound Au11 in the embodiment of the present invention; (a) is the hydrogen spectrum, and (b) is the carbon spectrum;

[0024] Fig.12 1 and 2 are hydrogen and carbon spectra of the Schiff base gold (III) compound Au12 in the embodiment of the present invention; (a) is the hydrogen spectrum, and (b) is the carbon spectrum;

[0025] Fig.13 1 and 2 are hydrogen and carbon spectra of the Schiff base gold (III) compound Au13 in the embodiment of the present invention; (a) is the hydrogen spectrum, and (b) is the carbon spectrum;

[0026] Fig.14 The present invention is an analysis of the in vitro TrxR activity and action site of the Schiff base gold (III) compounds Au6 and Au13 in the embodiments of the present invention;

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

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

[0029] Fig.17 This is an analysis chart of the effects of Schiff base gold (III) compounds Au6 and Au13 on wound size and weight of mice infected with wounds in the embodiment of the present invention;

[0030] Fig.18 This is an analysis diagram of the effects of Schiff base gold (III) compounds Au6 and Au13 on the pathology and immunohistochemistry of infected wounds in mice in the embodiments of the present invention;

[0031] Fig.19 This is an analysis diagram of the effects of Schiff base gold (III) compounds Au6 and Au13 on immunofluorescence experiments of infected wound tissues in mice according to an embodiment of the present invention;

[0032] Fig. 20 It is a schematic diagram of 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 the embodiments of the present invention. DETAILED DESCRIPTION

[0033] In order to more clearly illustrate the technical solution of the present invention, the technical solution of the present invention is further described in detail below:

[0034] Carbapenem-resistant or polymyxin-resistant Klebsiella pneumoniae means that: according to the CLSI method (strip method or dilution method), Klebsiella pneumoniae is tested with carbapenems or polymyxin antibiotics, and the results are judged as drug-resistant according to the CLSI-M100 standard; MIC refers to the minimum inhibitory concentration, which is an indicator to measure the antibacterial activity of antibiotics. 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 Taking the synthesis method of Schiff base gold (III) compounds Au6 and Au13 as an example, the synthesis of Schiff base gold (III) compounds is introduced; the structural formulas of Au6 and Au13 are listed in detail, and the structures of other Schiff base gold (III) compounds are briefly listed:

[0036] 1. Structural formula:

[0037]

[0038] The Schiff base ligand with antibacterial potential was combined with gold (III), and the cyclohexanediamine and NNOO coordination modes of oxaliplatin were retained. Schiff base gold (III) compounds Au1-Au13 were obtained by chemical synthesis. The structures of the compounds were determined by hydrogen and carbon nuclear magnetic resonance spectra (e.g. Figure 1-13 shown).

[0039] Taking the synthesis methods of Au6 and Au13 as examples, the synthesis methods of Schiff base gold (III) compounds Au1-Au13 are introduced in detail: 7-methoxy-2-naphthol is used as the starting material, and the aldehyde group is substituted at the ortho position of the hydroxyl group under the action of magnesium chloride and polyformaldehyde to obtain 7-methoxy-2-hydroxynaphthaldehyde; using this as the raw material, asymmetric Schiff base ligands are synthesized by a step-by-step method, and finally Schiff base gold (III) compounds are synthesized.

[0040] Its synthetic route is as follows:

[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 value to 10 with triethylamine, and finally add paraformaldehyde (7.58 g, 252.60 mmol), reflux at 98°C; after 17 hours, add water to quench, after the reaction solution is cooled to room temperature, adjust the pH value to 2 with concentrated hydrochloric acid, extract with ethyl acetate, repeat 3 times, and the resultant is The organic phase was dehydrated by adding anhydrous sodium sulfate, and the product 2-hydroxy-7-methoxy-1-naphthaldehyde was obtained by silica gel column chromatography (petroleum ether: ethyl acetate = 100:3); (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, and then it was dropped into an ethanol (1 mL) solution containing cis-cyclohexanediamine (84.70 mg, 0.74 mmol) under ice bath conditions, and stirred for 2 h in an ice bath, and then Then, an ethanol (1 mL) solution containing o-vanillin (112.90 mg, 0.74 mmol) was added dropwise, and the mixture was stirred at room temperature. After 12 h, a bright yellow precipitate was precipitated, which was filtered off and washed with a small amount of ethanol to obtain a crude product. The crude product was then dissolved in an appropriate amount of ethanol, heated to reflux, cooled at -20°C for 6 h, filtered off, 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 NaAuCl4·2H2O (60.0 0mg, 0.15mmol) in ethanol solution, stir evenly, add ammonium hexafluorophosphate (143.40mg, 0.90mmol), protect with argon, avoid light, and stir at room temperature for 24h; after the reaction, an orange-red solid is precipitated, filter it, wash it with a small amount of dichloromethane and ethanol, dissolve the filter cake in an appropriate amount of acetonitrile, filter it to remove the ammonium hexafluorophosphate, recover the filtrate and spin dry it to obtain a crude product; add a small amount of dichloromethane and methanol, stir at 45°C for 10min, repeat 3 times, wash it with a small amount of dichloromethane and methanol, and dry it to obtain Au6;

[0043] Synthesis method 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 completely dissolve them, then add sodium bicarbonate (1.22 g, 14.48 mmol), and reflux for 3 days; after the reaction is completed, filter and use silica gel column chromatography (ethyl acetate) to obtain the intermediate 2-hydroxy-4-(2- (5): Accurately weigh 2-hydroxy-7-methoxy-1-naphthaldehyde (200.00 mg, 0.99 mmol), add 10 mL of ethanol, heat to completely dissolve it, then drop it 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 drop 2-hydroxy-4-(2-(piperidin-1-yl)ethoxy)benzaldehyde (246.60 mg, 0.99 mmol) in ethanol (1 mL), stirred at room temperature, after 12 h, the solution turned light yellow and clear, and 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 dropped into 1 mL of ethanol solution containing NaAuCl4·2H2O (50.00 mg, 0.13 mmol), stirred evenly, and added Add ammonium hexafluorophosphate (102.40 mg, 0.65 mmol), protect with argon, avoid light, and stir at room temperature for 24 hours; after the reaction, an orange-red solid precipitates, filter it, wash it with a small amount of dichloromethane and ethanol, dissolve the filter cake in an appropriate amount of acetonitrile, filter it to remove the ammonium hexafluorophosphate, recover the filtrate and spin dry it to obtain a crude product; then add methanol, stir at 55°C for 10 minutes, then add toluene, let it stand at -20°C for more than 6 hours, filter it, wash it with a small amount of dichloromethane and methanol, and dry it to obtain Au13.

[0044] In addition, the synthetic route of Au1-13 is described in the embodiment 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: brown-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: brown-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: brown-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 (500 MHz, 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: Auranofin and other gold compounds were dissolved in DMF; imipenem and polymyxin B were purchased from a certain technology company; Klebsiella pneumoniae ATCC BAA-1705 (resistant to carbapenems and polymyxins), 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-resistant 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, tryptone, 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] The antimicrobial effects of Schiff base gold (III) compounds were determined by broth microdilution in accordance with the Clinical and Laboratory Standards Institute (CLSI) guidelines; the final concentration of the compound was 40 μM; the plates were incubated at 37°C for 24 h, and bacterial growth was measured in 96-well plates using an ELISA reader; the MIC was defined as the lowest drug concentration that inhibited the growth of pathogens in culture medium after 18 to 24 h of in vitro bacterial culture.

[0068] Table 1. Screening of the activity 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 site of Schiff base gold (III) compounds Au6 and Au13 on TrxR enzyme:

[0072] Gold complexes have a significant inhibitory effect on TrxR. Inhibition of TrxR will reduce the antioxidant capacity of cells and lead to an increase in reactive oxygen species (ROS). These effects are often closely related to its antibacterial activity. By constructing a plasmid with the TrxR gene and transforming it into Escherichia coli, the TrxR protein of Klebsiella pneumoniae was expressed in vitro, and the inhibitory activity and target of compounds Au6 and Au13 on TrxR 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 38kDa in the gel. Fig.14 A). Then, the in vitro TrxR activity was determined by DTNB reduction activity, which utilizes the reduction of TrxR to generate colored 2-nitro-5-thiobenzoate (λmax: 412nm); the results showed that Auranofin (IC 50 =30.9μM), Au6(IC 50 =19.9 μM) and Au13 (IC 50 =18.3 μM) showed inhibitory activity against TrxR ( Fig.14 B); Subsequently, the irreversible inhibition of recombinant TrxR by Au6 and Au13 was verified by evaluating the activities of desalted and non-desalted enzymes ( Fig.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 redox-active disulfide bonds, and then the TrxR enzyme was 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 ( Fig.14 D); To further verify this hypothesis, GSH was pre-incubated with Au6 and Au13, respectively, and then TrxR was co-incubated with GSH-conjugated Au6 and Au13 to measure TrxR activity; the results showed that pre-incubation of GSH with Au6 and Au13 eliminated their inhibitory effects on recombinant TrxR ( Fig.14 E); These findings suggest that Au6 and Au13 have the ability to modify sulfhydryl groups, thereby achieving irreversible inhibition of bacterial TrxR activity by targeting redox-active structures.

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

[0074] According to the above results, it was found that Auranofin and the 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 a key role in a variety of cellular activities; importantly, the GSH-GR system present in Gram-negative bacteria provides strong support for the Trx system; therefore, it was further studied whether the gold complexes (Auranofin, Au6 and Au13) have an inhibitory effect on glutathione reductase (GR) activity; first, the Klebsiella pneumoniae GR protein was recombinantly expressed in Escherichia coli, and then 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 GR with a molecular weight of 45 kDa in the gel ( Fig.15 A); GR catalyzes the reduction of GSSG to regenerate GSH and simultaneously oxidizes NADPH to generate NADP + ; The dehydrogenation rate of NADPH can be determined by measuring the rate of decrease in absorbance at 340 nm, thereby calculating GR activity. The inhibitory effect of GR was estimated by observing the rate of NADPH reduction in the presence of Auranofin, Au6, or Au13; The results showed that Au6 and Au13 could rapidly inhibit GR in a dose-dependent manner, and the IC of Au6 was 50 The value is 11.62 μM, and the IC of Au13 50 The value was 5.39 μM; in contrast, at a concentration of 40 μM, Auranofin showed no inhibitory effect on GR ( Fig.15 B); Subsequently, the irreversible inhibitory effect of Au6 and Au13 on recombinant GR was further confirmed by measuring the activity of desalted and non-desalted enzymes ( Fig.15 C); To further investigate the covalent binding sites between Au6, Au13 and GR, GR was pre-reduced with NADPH, resulting in the reduction of redox-active disulfide bonds, and the enzymes were incubated with Au6 and Au13 to assess their activity; 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 ( Fig.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 their inhibitory effects on recombinant GR ( Fig.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 showed no inhibitory activity against GR, which may be the main reason for its poor antibacterial activity against CRKP; Taken together, the results suggest that Au6 and Au13 induce redox imbalance in bacteria mainly by inhibiting GR activity, leading to bacterial death.

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

[0076] Through the determination of minimum inhibitory concentration, compounds Au6 and Au13 both showed good antibacterial activity against carbapenem-resistant and polymyxin-resistant Klebsiella pneumoniae, and their activity was at least 16 times higher than that of auranofin; therefore, a systematic evaluation of the in vivo anti-infection activity of compounds Au6 and Au13 at the mouse animal level was carried out, and the specific method was as follows:

[0077] 22-25g BALB / c mice were fed adaptively for 1 week, and surgical scissors were used to cut a 1.2*1.2cm wound on the back skin of the mice. 10 9 Wound infection model was established with 100 μL of bacterial solution (carbapenem-resistant and polymyxin-resistant Klebsiella pneumoniae) and randomly divided into 6 groups, with 3 mice in each group. After 24 h of infection, the control group was given normal saline for external application to the wound; the H2O2 group (positive control) was given 10 mM hydrogen peroxide for external application to the wound; the AF group (positive control) was given 5 mM auranofin for external application to the wound; the polymyxin B group (positive control) was given 5 mM polymyxin B for external application to the wound; the Au6 group was given 5 mM Au6 for external application to the wound; the Au13 group was given 5 mM Au13 for external application to the wound. The wound infection of mice was observed continuously for 14 days ( Fig.16 ), record wounds ( Fig.17 A) Mouse weight ( Fig.17 B) changes, and conduct pathological staining, immunohistochemistry experiments, inflammatory cytokine analysis, etc. on the wound tissue.

[0078] Compared with the control group, H2O2 group, auranofin group and polymyxin B group, compounds Au6 and Au13 had significant antibacterial effects and could effectively promote wound healing. The recovery of wound area was significantly improved. The 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) ( Fig.18 ) 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 reduced; in addition, tissue immunofluorescence experiments ( Fig.19 ) 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 reduced local tissue inflammation; In summary, the in vivo data showed that the in vivo antibacterial activity of compounds Au6 and Au13 was superior to that of the auranofin group and polymyxin B, and could significantly reduce local tissue infection and promote wound healing.

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

[0080] Female BALB / c mice (18-20 g) were adaptively raised for one week and randomly divided into a control group, a polymyxin B group, an Au6 group, and an Au13 group (n=6). Then, each mouse was intraperitoneally injected with 200 μL of CRKP suspension (10 9 CFU / mL); 1 hour after infection, mice were treated with PBS, polymyxin B, Au6 and Au13 respectively; the survival rate within 48 hours was observed and recorded ( Fig. 20 ); All experiments were performed in three biological replicates; in contrast, three mice in the Au6 and Au13 treatment groups survived for more than 24 h, one mouse in the Au6 treatment group survived for more than 48 h, and two mice in the Au13 treatment group survived for more than 48 h; The results showed that both Au6 and Au13 could prolong the survival time of mice with severe peritoneal infection and showed therapeutic advantages against multidrug-resistant Gram-negative bacteria.

Claims

1. A novel Schiff base gold (III) compound, characterized in that: The compounds include compounds Au6 and Au13.

2. A novel Schiff base gold (III) compound according to claim 1, characterized in that: The Schiff base ligand was combined with gold (III), the cyclohexanediamine and NNOO coordination modes of oxaliplatin were retained, and Schiff base gold (III) compounds Au1-Au13 were obtained by chemical synthesis method.

3. A novel Schiff base gold (III) compound according to claim 1, characterized in that: The chemical structural formulas of the compounds Au6 and Au13 are shown below:

4. A method for synthesizing a novel Schiff base gold (III) compound according to claim 1.

5. The method for synthesizing a novel Schiff base gold (III) compound according to claim 4, characterized in that: Its synthetic route is as follows:

6. A pharmaceutical composition comprising a therapeutically effective amount of one or more novel Schiff base gold (III) compounds as claimed in any one of claims 1 to 5 and a pharmaceutically acceptable carrier.

7. Use of a novel Schiff base gold (III) compound according to any one of claims 1 to 5 in combating carbapenem-resistant and polymyxin-resistant Gram-negative bacteria.

8. The use according to claim 7, characterized in that In vitro, the compounds Au6 and Au13 had inhibitory effects on carbapenem- and polymyxin-resistant Klebsiella pneumoniae.

9. The use according to claim 7, characterized in that: In vivo, the compounds Au6 and Au13 treated skin infections caused by carbapenem- and polymyxin-resistant Klebsiella pneumoniae.

10. The use according to claim 7, characterized in that: In vivo, the compounds Au6 and Au13 prolonged the survival of severe intra-abdominal infections caused by carbapenem- and polymyxin-resistant Klebsiella pneumoniae.

Images