4-(pyridin-4-yl)-1H-imidazole-2-amine derivatives and compositions targeting key protein machinery for outer membrane assembly of Gram-negative bacteria

Abstract

Disclosed herein are 4-(pyridin-4-yl)-1H-imidazole-2-amine derivatives and compositions that target key protein machinery in the outer membrane assembly of Gram-negative bacteria, belonging to the field of chemical and pharmaceutical technology. The present invention provides a small molecule compound represented by formula (I): #imgabs0#. This small molecule compound can serve as a novel target for the inner membrane lipoprotein localization machinery (LolCDE) of Gram-negative bacteria and exhibits antibacterial activity, providing a new approach for specific antibacterial treatment of Gram-negative bacteria.

Description

Technical Field

[0001] The present invention belongs to the field of chemical medicine technology, and specifically relates to 4-(pyridin-4-yl)-1H-imidazole-2-amine derivatives and compositions that target key protein machinery assembled in the outer membrane of Gram-negative bacteria. Background Art

[0002] Gram-negative bacteria, such as Klebsiella pneumoniae, are a medical nightmare. These pathogens are highly adaptable, virulent, and rapidly develop resistance. Only a handful of antibiotics can effectively combat them, yet these antibiotics also destroy beneficial gut bacteria. Furthermore, infections caused by multidrug-resistant bacteria have become a major global health crisis. Bacteria continuously evolve and acquire resistance through various mechanisms, such as mutation, horizontal gene transfer, and biofilm formation. These pathogens are increasingly undermining the effectiveness of existing antibiotics, necessitating the development of new antibiotics. Gram-negative bacteria include Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae, and Salmonella typhimurium, as well as carbapenem-resistant Acinetobacter baumannii, third-generation cephalosporin-resistant Enterobacteriaceae, and carbapenem-resistant Enterobacteriaceae. Gram-negative bacteria possess an innate asymmetric inner and outer membrane system that selectively allows for the permeability of nutrients and shields them from antibiotics.

[0003] The "Lol system" is a class of proteins that only exist in Gram-negative bacteria and is an effective way to bypass the defense of Gram-negative bacteria. In recent years, a series of small molecule compounds (Lolamicin, Abaucin) and peptides (Zosurabalpin, Darobactin) targeting key protein machines in the inner and outer membranes of Gram have been discovered.

[0004] (Lolamicin), (Abaucin)

[0005] These substances exhibit excellent antibacterial activity and safety, and can broadly kill Gram-negative bacteria resistant to multiple antibiotics without disrupting the intestinal flora. These research results further demonstrate the effectiveness of the key protein machinery in the inner and outer membranes of Gram-negative bacteria as a new antibiotic target. However, small molecule compounds that target the key protein machinery in the inner and outer membranes of Gram-negative bacteria are currently very limited, and there is an urgent need to provide more targeted small molecule compounds with better antibacterial activity. Summary of the Invention

[0006] Technical issues:

[0007] The present invention is designed to provide some new small molecule compounds that target the Gram-negative bacteria inner membrane lipoprotein localization machinery (LolCDE) and have antibacterial activity, providing a new approach for Gram-negative bacteria-specific antibacterial treatment.

[0008] Technical solution:

[0009] In one aspect, the present invention provides a class of 4-(pyridin-4-yl)-1-(4-pyridin-1-yl)-1-(4-pyridin-2-yl)-2-(4-pyridin-3-yl)-1-(4-pyridin-4-yl)-1-(4-pyridin-3 ...4-yl)-1-(4- H -Imidazole-2-amine derivatives, or pharmaceutically acceptable salts thereof, whose structure is shown in formula (I):

[0010] ,

[0011] Wherein, n is the number of substituents R1 on the pyridine ring, and is 0, 1, 2, or 3, and each R1 is independently selected from a C1-C4 alkyl group;

[0012] R2 is independently selected from substituted or unsubstituted C6-C 10 Aryl or C5-C 10 Heteroaryl, wherein the substituted group is selected from cyano (CN), halogen (F, Cl, Br, I).

[0013] In one embodiment of the present invention, the C1-C4 alkyl group is specifically: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl.

[0014] In one embodiment of the present invention, C6-C 10 The aryl group may specifically be phenyl.

[0015] In one embodiment of the present invention, C5-C 10 The heteroaryl group can be specifically selected .

[0016] In one embodiment of the present invention, R2 is specifically selected from: .

[0017] In one embodiment of the present invention, the 4-(pyridin-4-yl)-1H-imidazole-2-amine derivatives can be specifically selected from:

[0018] .

[0019] In one embodiment of the present invention, the pharmaceutically acceptable salt comprises an inorganic salt or an organic salt; wherein the inorganic salt is selected from hydrochloride, hydrobromide, hydroiodide, perchlorate, sulfate, bisulfate, nitrate, phosphate, acid phosphate; and the organic salt is selected from formate, acetate, trifluoroacetate, propionate, pyruvate, glycolate, oxalate, malonate, succinate, glutarate, fumarate, maleate, lactate, malate, citrate, tartrate, methanesulfonate, ethanesulfonate, benzenesulfonate, salicylate, p-toluenesulfonate, and ascorbate.

[0020] On the other hand, the present invention also provides a pharmaceutical composition comprising the above-mentioned 4-(pyridin-4-yl)-1H-imidazole-2-amine derivative or a pharmaceutically acceptable salt thereof, and a pharmaceutical excipient.

[0021] In one embodiment of the present invention, the pharmaceutical excipients include any one or more of the following: solvents, propellants, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integrities, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoaming agents, thickeners, inclusion agents, humectants, flocculants and deflocculating agents, filter aids, and release retardants.

[0022] In one embodiment of the present invention, the pharmaceutical composition further comprises a pharmaceutical carrier, and the pharmaceutical carrier is selected from microcapsules, microspheres, nanoparticles and liposomes.

[0023] In one embodiment of the present invention, the dosage forms of the pharmaceutical composition include: injection, lyophilized powder for injection, suspension, implant, embolic agent, capsule, tablet, pill and oral solution.

[0024] In another aspect, the present invention also provides the use of the above-mentioned 4-(pyridin-4-yl)-1H-imidazole-2-amine derivatives or pharmaceutically acceptable salts thereof in the preparation of Gram-negative bacteria-specific antibiotic drugs.

[0025] Beneficial effects:

[0026] The present invention 4- (pyridin-4-yl) -1 H -Imidazole-2-amine derivatives have significant antibacterial activity, with the minimum inhibitory concentration as low as 0.0078 μg / mL.

[0027] Furthermore, the 4-(pyridin-4-yl)-1H-imidazole-2-amine derivatives of the present invention can have a significant affinity with the target protein LolCDE, with an affinity of K d It can reach 496-831 μmol / L.

[0028] Based on the above effects, the present invention provides a new approach for specific antibacterial treatment of Gram-negative bacteria. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The synthetic route of compounds 1-7.

[0030] Figure 2 This is the molecular docking diagram of compound 5 and lolCDE.

[0031] Figure 3 This is the SDS-PAGE gel image of the target protein LolCDE purification.

[0032] Figure 4 A comparison chart of the affinity determination results between the target protein LolCDE and compounds 1-7. DETAILED DESCRIPTION

[0033] The technical solution of the invention is described in detail below with reference to the accompanying drawings:

[0034] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

[0035] Example 1

[0036] Preparation of compound 1:

[0037]

[0038] Synthesis of Intermediate 1: 3-(2-bromoacetyl)phenol (1 mmol) and 2.5 equivalents of 2-aminopyrimidine (2.5 mmol) were added to a round-bottom flask, followed by 10 mL of ethanol as solvent. The mixture was heated at 80°C for 3 hours. The reaction was monitored using thin-layer chromatography on silica gel plates. After completion of the reaction, the mixture was cooled to room temperature, filtered, washed with petroleum ether, and dried under reduced pressure to obtain Intermediate 1, which was used directly in the next reaction without further purification.

[0039] Synthesis of Intermediate 2: 1 mmol of Intermediate 1 was placed in a round-bottom flask. An appropriate amount (10 mL) of acetonitrile was added as solvent, along with 1.5 equivalents of Boc2O and 1.5 equivalents of DMAP. The mixture was allowed to react at room temperature for 2 hours. The reaction was monitored using thin-layer chromatography on silica gel plates. After completion, the reaction solution was filtered, washed with petroleum ether, and dried under reduced pressure to yield Intermediate 2, which was used directly in the next step.

[0040] Synthesis of Intermediate 3: 1 mmol of Intermediate 2 and 1.7 equivalents of 4-bromopyridine (1.7 mmol) were added to a two-necked flask. 5 equivalents of potassium carbonate (5 mmol) and N,N-dimethylacetamide (20 mL) were added as the solvent. The mixture was purged with N₂ for 10 minutes. Under N₂, 0.1 equivalents of Pd(OAc)₂ and 0.1 equivalents of PCy₃·HBF₄ were added. The reaction mixture was purged with N₂ for 5 minutes and then heated and stirred at 125°C for 7 hours. The reaction was monitored by thin-layer chromatography on silica gel. After completion, the reaction mixture was diluted with an appropriate amount of water and extracted five times with ethyl acetate. The organic layers were combined and washed five times with water. The organic layers were collected, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography (dichloromethane:methanol = 15:1) to obtain Intermediate 3.

[0041] Synthesis of Intermediate 4: 1 mmol of Intermediate 3 was added to a round-bottom flask and dissolved in 5 mL of dichloromethane. 5 mL of hydrochloric acid and dioxane solution was added and stirred at room temperature for 3 hours. After completion of the reaction, the solvent was removed by distillation under reduced pressure to obtain Intermediate 4, which was used directly in the next reaction.

[0042] Synthesis of intermediate 5: Intermediate 4 (1 mmol) was reacted with 1 equivalent of 7-(chloromethyl)imidazo[1,2- a Pyridine (1 mmol) was added to a round-bottom flask, along with cesium carbonate (3 mmol) and N,N-dimethylformamide (10 mL) as the solvent. The mixture was heated at 80°C for 3 hours. The reaction was monitored using thin-layer chromatography on silica gel plates. After the reaction was completed and cooled to room temperature, an appropriate amount of water was added to the system. Extraction was performed with ethyl acetate, followed by washing with water five times and saturated brine once. The organic phase was collected, dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography (dichloromethane:methanol = 10:1) to obtain intermediate 5.

[0043] Synthesis of Compound 1: 1 mmol of intermediate 5 was placed in a round-bottom flask and dissolved in 20 mL of ethanol. 5 equivalents of hydrazine hydrate (5 mmol) was added and allowed to react at 80°C for 5 hours. The reaction was monitored using thin-layer chromatography on silica gel plates. After completion of the reaction, the solvent was removed by distillation under reduced pressure and the product was purified by column chromatography (dichloromethane:methanol = 10:1) to yield Compound 1.

[0044] NMR data of compound 1:

[0045] 1 H NMR (400 MHz, DMSO- d6 ) δ 8.54 (d, J= 7.0 Hz, 1H), 8.40 - 8.30 (m,2H), 7.94 (s, 1H), 7.62 - 7.53 (m, 2H), 7.39 - 7.25 (m, 3H), 7.10 (t, J = 2.0Hz, 1H), 7.05 - 6.96 (m, 2H), 6.91 (dd, J = 7.0, 1.7 Hz, 1H), 5.61 (s, 2H), 5.15 (s, 2H). 13 C NMR (101 MHz, DMSO- d6 ) δ 158.14, 150.88, 149.41 (2C), 144.27,141.04, 134.38, 134.03, 133.40, 129.77, 127.00, 120.56, 120.09 (2C), 114.54(2C), 114.18, 113.91, 113.23, 111.58 (2C), 68.19.

[0046] According to the same preparation method as compound 1, the synthetic route is as follows Figure 1 As shown, 4-bromopyridine is replaced by other pyridine raw materials to obtain compound 2-4 accordingly.

[0047]

[0048] According to the same preparation method as compound 1, the synthetic route is as follows Figure 1 As shown, 4-bromopyridine was replaced by 4-bromo-2-methylpyridine as the starting material, and 7-(chloromethyl)imidazo[1,2- a ] Pyridine was replaced by 4-fluorobenzyl chloride, m-cyanobenzyl chloride, and 6-(chloromethyl)quinoline raw materials, respectively, to obtain compounds 5-7 accordingly.

[0049]

[0050] Molecular docking of compound 5 and lolCDE:

[0051] The structure of the small molecule compound was drawn by KingDraw and converted using Chem3D. The small molecule was pre-processed using Autodock Tools and saved as a PDBQT file. The crystal structure of lolCDE (https: / / www.rcsb.org / structure / 7ARK) was retrieved from the PDB database (https: / / www.rcsb.org / ) and pre-processed using Autodock Tools to remove redundant molecules and water molecules, merge polar hydrogen atoms, and save as a PDBQT file. Finally, Autodock Vina was used to dock the small molecule to the active site near the lipoprotein. The molecular docking of compound 5 with the target protein LolCDE is shown in Figure 2. Figure 2 shown.

[0052] Example 2: Minimum inhibitory concentration determination

[0053] In this example, the two-fold dilution method was used to determine the minimum inhibitory concentrations of small molecule compounds (compounds 1-7 and control compounds A and B).

[0054] Carbapenem-resistant Escherichia coli, Klebsiella pneumoniae, carbapenem-resistant Klebsiella pneumoniae, Pseudomonas aeruginosa, carbapenem-resistant Pseudomonas aeruginosa, and Stenotrophomonas maltophilia were all provided by West China Hospital of Sichuan University.

[0055] Preparation of test drug solutions: All small molecule compounds were dissolved in DMSO. For the determination of the minimum inhibitory concentration (MIC), small molecule compounds were dissolved in MH medium to concentrations of 256 μg / mL, 128 μg / mL, 64 μg / mL, 32 μg / mL, 16 μg / mL, 8 μg / mL, 4 μg / mL, 2 μg / mL, 1 μg / mL, and 0.5 μg / mL.

[0056] First, prepare a sterile 96-well plate. Add 200 μL of the test drug solution to the first well of each row, and 100 μL of 6% DMSO to the remaining wells. Subsequently, transfer 100 μL of the test drug solution from the first well to the second well and mix thoroughly. Continue this dilution to the 11th well, ensuring compound concentrations range from 0.0078 μg / mL to 256 μg / mL. No drug is added to the 12th well. Finally, add 100 μL of the test bacterial solution (Escherichia coli MG1655 ΔTolC, Escherichia coli ATCC25922, carbapenem-resistant E. coli, Klebsiella pneumoniae, carbapenem-resistant K. pneumoniae, Pseudomonas aeruginosa, carbapenem-resistant Pseudomonas aeruginosa, and Stenotrophomonas maltophilia) at an OD value of 0.001 to each well. Incubate the 96-well plate at 37°C for 20 hours. After the incubation period, 20 μL of 1% TTC (2,3,5-triphenyltetrazolium chloride) was added to each well and the cells were incubated at 37°C for 10 minutes to allow for color development. The final assay results are shown in Table 1-2.

[0057] Table 1 Minimum inhibitory concentration of each compound against Escherichia coli ATCC 25922

[0058]

[0059] The structure of control A is .

[0060] The structure of control B is .

[0061] The results in Table 1 above show that compounds 1-7 have antibacterial activity against Escherichia coli ATCC25922 strain, among which compounds 5, 6 and 7 have the best antibacterial effect, and the minimum inhibitory concentration can be lower than 0.0078 μg / mL.

[0062] Table 2 Minimum inhibitory concentrations of compounds 2, 3, 5, 6, and 7 against other or carbapenem-resistant Gram-negative bacteria

[0063]

[0064] The results in Table 2 above show that compounds 2, 3, 5, 6 and 7 have significant antibacterial effects on extended-spectrum β-lactamase Escherichia coli, Klebsiella pneumoniae and carbapenem-resistant Klebsiella pneumoniae.

[0065] Example 3: In vitro expression and purification of target protein LolCDE

[0066] (1) Transform the pTRC99a-LolCDE plasmid into competent E. coli C43. After 12 h, pick a single clone in 100 mL LB (containing 100 μg / mL Ampicillin) and culture at 37°C and 220 rpm.

[0067] (2) After 9 hours, inoculate 6 L of LB and culture for expansion at 37°C, 220 rpm, or induce overnight at 20°C, 180 rpm for 15-20 hours.

[0068] (3) After centrifugation at 4,000 rpm for 15 min, the bacterial pellet was collected and resuspended in 120 mL of lysis buffer containing 50 mM Hepes, pH 7.8, 300 mM NaCl, and 10% (v / v) glycerol, supplemented with PMSF to a final concentration of 1 mM;

[0069] (4) After high-pressure crushing at 800 bar, the membrane pellet was collected by centrifugation at 140,000 × g for 50 min at 4°C, and the membrane pellet was resuspended in 50 mL of lysis buffer supplemented with 1% DDM and dissolved at room temperature for 30 min;

[0070] (5) After centrifugation at 140,000 × g for 25 min at 4°C, the supernatant was bound to a 3 mL Ni-NTA column (MCE) at room temperature for 40 min;

[0071] (6) Wash the cells with 70 mL of lysis buffer supplemented with 30 mM imidazole and 0.03% (wt / vol) DDM (Anatrace), and elute the target protein with 20 mL of lysis buffer containing 250 mM imidazole and 0.03% (wt / vol) DDM.

[0072] (7) Finally, the eluted target protein was further desalted in a buffer solution (50 mM Hepes, 150 mM NaCl, and 0.03% DDM, pH 7.8). SDS-PAGE was used to verify the expression of the target protein.

[0073] The SDS-PAGE results of the target protein LolCDE expression and purification are as follows Figure 3 shown.

[0074] Example 4: Biomembrane Interference Technology of Small Molecule Compounds and Target Protein LolCDE

[0075] (1) Add 200 μL of buffer (50 mM Hepes, 150 mM NaCl, 0.03% DDM) to the pre-wet plate and pre-wet the sensor (Ni-NTA) for 10 min.

[0076] (2) Protein loading: define the sensor position and the positions of the buffer and protein, with a protein loading of 12 nm;

[0077] (3) Determine affinity. Define the sensor positions and the positions of the small molecule for gradient dilution. Add 200 μL of 15% DMSO buffer (50 mM Hepes, 150 mM NaCl, 0.03% DDM) to the first four wells. Add 200 μL of the small molecule compound sample sequentially to the next eight wells according to the concentration gradient. Programming: Custom 180 s, Association and Disassociation times are both 80 s, and Baseline 120 s.

[0078] The results are as follows Figure 4 and shown in Table 3.

[0079] Table 3

[0080]

[0081] The affinity mentioned in the table refers to the affinity of the compound to the target. The smaller the value, the stronger the affinity. The specific calculation formula is as follows: K d =K off / K on

[0082] The dissociation rate constant k off It reflects the stability of the complex, the percentage of the complex that dissociates per second, that is, 1% of the complex dissociates per second, the unit is s -1 ; Binding rate constant k on Represents the binding rate of the complex, the number of complexes produced per second at a substance concentration of 1M, in M -1 s -1 Affinity K d It reflects the strength of the interaction binding ability, and the unit is M.

[0083] The results showed that compounds 3, 5, and 7 had good affinities with the target protein LolCDE, with affinities of 496 μmol / L, 683 μmol / L, and 831 μmol / L, respectively.

[0084] In summary, it can be seen that compounds 3, 5, and 7 of the present invention not only have excellent antibacterial effects, but also have significant affinity effects with the target protein LolCDE.

[0085] The embodiments provided above are not intended to limit the scope of the present invention, nor are the steps described to limit their execution order. Any obvious improvements to the present invention made by those skilled in the art in combination with existing common knowledge shall fall within the scope of protection defined by the claims of the present invention.

Claims

1. A class of 4-(pyridin-4-yl)-1-(4-pyridin-1-yl)-1-(4-pyridin-2-yl)-2-nitropropene derivatives targeting key protein machinery of Gram inner and outer membranes H -Imidazole-2-amine derivatives or pharmaceutically acceptable salts thereof, characterized in that, Specific selection: 。 2. A class of 4-(pyridin-4-yl)-1-(4-pyridin-2-yl)-1-(4-pyridin-3-yl)-2-(4-pyridin-4 ... H -Imidazole-2-amine derivatives or pharmaceutically acceptable salts thereof, characterized in that, The pharmaceutically acceptable salts include inorganic salts or organic salts; wherein the inorganic salts are selected from: hydrochloride, hydrobromide, hydroiodide, perchlorate, sulfate, bisulfate, nitrate, phosphate, acid phosphate; the organic salts are selected from: formate, acetate, trifluoroacetate, propionate, pyruvate, glycolate, oxalate, malonate, succinate, glutarate, fumarate, maleate, lactate, malate, citrate, tartrate, methanesulfonate, ethanesulfonate, benzenesulfonate, salicylate, p-toluenesulfonate, ascorbate.

3. A pharmaceutical composition, characterized in that It contains a class of 4-(pyridin-4-yl)-1-(pyridin-2-yl)-1-(pyridin-3-yl)-2-(pyridin-4-yl)-1-(pyridin-3-yl)-2-(pyridin-3-yl)-1 ... H -imidazole-2-amine derivatives or pharmaceutically acceptable salts thereof, and pharmaceutical excipients.

4. The pharmaceutical composition according to claim 3, characterized in that The pharmaceutical excipients are selected from any one or more of the following: solvents, propellants, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integrators, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoaming agents, thickeners, inclusion agents, humectants, flocculants and deflocculating agents, filter aids, and release retardants.

5. The pharmaceutical composition according to claim 3, characterized in that The pharmaceutical composition comprises a pharmaceutical carrier, and the pharmaceutical carrier is selected from microcapsules, microspheres, nanoparticles and liposomes.

6. The pharmaceutical composition according to claim 3, characterized in that The dosage form of the pharmaceutical composition is selected from the group consisting of injection, freeze-dried powder for injection, suspension, implant, embolic agent, capsule, tablet, pill and oral solution.

7. A class of 4-(pyridin-4-yl)-1-(4-pyridin-2-yl)-1-(4-pyridin-3-yl)-2-(4-pyridin-4-yl)-1-(4-pyridin-3-yl)-2-(4-pyridin-3-yl)-1-(4-pyridin-3-yl)-1-(4-pyridin-4-yl)-1-(4-pyridin-3 ... H - Use of imidazole-2-amine derivatives or pharmaceutically acceptable salts thereof in the preparation of Gram-negative bacteria-specific antibiotic drugs.

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