4-(pyridine-4-yl)-1H-imidazole-2-amine derivatives and compositions targeting Gram-negative bacterium outer membrane assembly key protein machines

Abstract

The invention discloses 4-(pyridine-4-yl)-1H-imidazole-2-amine derivatives and compositions of a machine for assembling key proteins on an outer membrane of targeted gram-negative bacteria, and belongs to the technical field of chemical medicines. The invention provides a small molecule compound as shown in a formula (I): # imgabs0 #, the small molecule compound can be used as a brand new targeting gram-negative bacterium inner membrane lipoprotein positioning machine (LolCDE) and has bacteriostatic activity, and a new way is provided for specific antibacterial treatment of gram-negative bacteria.

Description

Technical Field

[0001] The present invention belongs to the technical field of chemical medicine, and particularly relates to 4-(pyridin-4-yl)-1H-imidazole-2-amine derivatives and compositions targeting key protein machines for the outer membrane assembly of Gram-negative bacteria. Background Art

[0002] Gram-negative bacteria, such as Klebsiella pneumoniae, are a nightmare in medicine. These pathogenic bacteria have strong adaptability, toxicity, and the ability to rapidly develop drug resistance. Only a very small number of antibiotics can eliminate them, yet these antibiotics also damage beneficial gut bacteria in the human body. In addition, infectious diseases caused by multi-drug resistant bacteria have become an important global health crisis. Bacteria continuously evolve and acquire drug resistance through various mechanisms (such as gene mutation, horizontal gene transfer, and biofilm formation). These pathogens are increasingly weakening the effectiveness of existing antibiotics, and there is an urgent need to develop new antibiotics. Gram-negative bacteria include Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae, Salmonella typhimurium, carbapenem-resistant Acinetobacter baumannii, third-generation cephalosporin-resistant Enterobacter, and carbapenem-resistant Enterobacter, etc. Gram-negative bacteria have an innate asymmetric inner and outer membrane system, which can selectively permeate nutrients and shield antibiotic drugs.

[0003] The "Lol system" is a class of proteins that only exist in Gram-negative bacteria and is also an effective way to bypass the defense of Gram-negative bacteria. In recent years, a series of small molecule compounds (Lolamicin, Abaucin) and polypeptides (Zosurabalpin, Darobactin) targeting key protein machines in the inner and outer membranes of Gram-negative bacteria have been successively discovered, (Lolamicin), (Abaucin) These substances exhibit good antibacterial activity and safety, can broadly kill Gram-negative bacteria resistant to a variety of antibiotics without destroying the gut flora. These research results further demonstrate the effectiveness of key protein machines in the inner and outer membranes of Gram-negative bacteria as new antibiotic targets. However, there are still very limited small molecule compounds targeting key protein machines in the inner and outer membranes of Gram-negative bacteria, and there is an urgent need to provide more small molecule compounds with better targeting and antibacterial activity. Summary of the Invention

[0004] Technical Problem: The present invention is designed to provide some novel small molecule compounds targeting the inner membrane lipoprotein localization machine (LolCDE) of Gram-negative bacteria and having antibacterial activity, providing a new approach for the specific antibacterial treatment of Gram-negative bacteria.

[0005] Technical solution: On the one hand, the present invention provides a class of 4-(pyridin-4-yl)-1H-imidazole-2-amine derivatives, or pharmaceutically acceptable salts thereof, having a structure as shown in formula (I): H , In the formula, n is the number of substituents R1 on the pyridine ring, taking values of 0, 1, 2, 3, and each R1 is independently selected from C1-C4 alkyl groups; R2 is independently selected from substituted or unsubstituted C6-C 10 aryl or C5-C 10 heteroaryl, and the substituted groups are selected from cyano (CN), halogen (F, Cl, Br, I).

[0006] In one embodiment of the present invention, the C1-C4 alkyl group is specifically: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl.

[0007] In one embodiment of the present invention, the C6-C 10 aryl is specifically phenyl.

[0008] In one embodiment of the present invention, the C5-C 10 heteroaryl is specifically selected from .

[0009] In one embodiment of the present invention, R2 is specifically selected from: .

[0010] In one embodiment of the present invention, the 4-(pyridin-4-yl)-1H-imidazole-2-amine derivative is specifically selected from: .

[0011] In one embodiment of the present invention, the pharmaceutically acceptable salts include inorganic salts or organic salts; among them, 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.

[0012] On the other hand, the present invention also provides a pharmaceutical composition containing the above-mentioned 4-(pyridin-4-yl)-1H-imidazole-2-amine derivative or its pharmaceutically acceptable salt, and pharmaceutical excipients. ​

[0013] In one embodiment of the present invention, the pharmaceutical excipients include any one or more of the following: solvents, propellants, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, antiadhesives, chelating agents, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoaming agents, thickening agents, clathrates, humectants, flocculants and deflocculants, filter aids, release retardants.

[0014] In one embodiment of the present invention, the pharmaceutical composition further includes a pharmaceutical carrier, and the pharmaceutical carrier is selected from microcapsules, microspheres, nanoparticles and liposomes.

[0015] In one embodiment of the present invention, the dosage forms of the pharmaceutical composition include: injection solutions, freeze-dried powder injections for injection, suspensions, implants, embolization agents, capsules, tablets, pills and oral liquids.

[0016] On the other hand, the present invention also provides the use of the above-mentioned 4-(pyridin-4-yl)-1H-imidazol-2-amine derivatives or pharmaceutically acceptable salts thereof in the preparation of Gram-negative bacteria-specific antibiotic drugs.

[0017] Advantageous effects: The 4-(pyridin-4-yl)-1 H -imidazol-2-amine derivatives of the present invention have significant antibacterial activity, and the minimum inhibitory concentration can be as low as 0.0078 μg / mL.

[0018] Furthermore, the 4-(pyridin-4-yl)-1H-imidazol-2-amine derivatives of the present invention can have a significant affinity for the target protein LolCDE, and the affinity K d can reach 496 - 831 μmol / L.

[0019] Based on the above effects, the present invention provides a new approach for Gram-negative bacteria-specific antibacterial treatment. Description of the drawings

[0020] Figure 1 is the synthetic route diagram of compounds 1 - 7.

[0021] Figure 2 is the molecular docking diagram of compound 5 and lolCDE.

[0022] Figure 3 is the SDS-PAGE gel diagram of the purification of the target protein LolCDE.

[0023] Figure 4 is the comparison diagram of the affinity determination results of the target protein LolCDE and compounds 1 - 7. Detailed implementation mode

[0024] The technical solution of the invention will be described in detail below with reference to the accompanying drawings: Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various modifications and decorations without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

[0025] Example 1 Preparation of Compound 1:

[0026] 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, 10 mL of ethanol was added as a solvent, and the mixture was heated at 80 °C for 3 hours. The reaction was monitored by thin-layer chromatography silica gel plate. After the reaction was completed and cooled to room temperature, the reaction solution was filtered, washed with petroleum ether, and dried under reduced pressure to obtain Intermediate 1, which was directly used in the next step without any purification.

[0027] 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 a solvent, 1.5 equivalents of Boc2O and 1.5 equivalents of DMAP were added, and the reaction was carried out at room temperature for 2 hours. The reaction was monitored by thin-layer chromatography silica gel plate. After the reaction was completed, the reaction solution was filtered, washed with petroleum ether, and dried under reduced pressure to obtain Intermediate 2, which was directly used in the next step.

[0028] 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) was added, N,N-dimethylacetamide (20 mL) was added as a solvent, purged with N2 for 10 minutes, and 0.1 equivalent of Pd(OAc)2 and 0.1 equivalent of PCy3·HBF4 were added under a N2 atmosphere. The reaction mixture was purged with N2 for 5 minutes and further heated and stirred at 125 °C for 7 hours. The reaction was monitored by thin-layer chromatography silica gel plate. After the reaction was completed, the reaction solution was diluted with an appropriate amount of water, extracted 5 times with ethyl acetate, the organic layers were combined, washed 5 times with water, the organic layer was collected, dried over anhydrous sodium sulfate, and concentrated under reduced pressure, and purified by column chromatography (dichloromethane:methanol = 15:1) to obtain Intermediate 3.

[0029] Synthesis of Intermediate 4: 1 mmol of Intermediate 3 was added to a round-bottom flask, dissolved in an appropriate amount (5 mL) of dichloromethane, 5 mL of hydrochloric acid dioxane solution was added, and the mixture was stirred at room temperature for 3 hours. After the reaction was completed, the solvent was removed by distillation under reduced pressure to obtain Intermediate 4, which was directly used in the next step.

[0030] Synthesis of Intermediate 5: Add Intermediate 4 (1 mmol) and 1 equivalent of 7-(chloromethyl)imidazo[1,2- a pyridine (1 mmol) into a round-bottom flask, add cesium carbonate (3 mmol), add N,N-dimethylformamide (10 mL) as the solvent, and heat at 80 °C for 3 hours. Detect the reaction using a thin-layer chromatography silica gel plate. After the reaction is completed, wait for it to cool to room temperature, add an appropriate amount of water to the system, extract with ethyl acetate, wash with water 5 times, wash with saturated brine 1 time, collect the organic phase, dry with anhydrous sodium sulfate, concentrate, and purify by column chromatography (dichloromethane:methanol = 10:1) to obtain Intermediate 5.

[0031] Synthesis of Compound 1: Take 1 mmol of Intermediate 5 and place it in a round-bottom flask, add 20 mL of ethanol to dissolve it, add 5 equivalents of hydrazine hydrate (5 mmol), and react at 80 °C for 5 hours. Detect the reaction using a thin-layer chromatography silica gel plate. After the reaction is completed, distill off the solvent under reduced pressure, and purify by column chromatography (dichloromethane:methanol = 10:1) to obtain Compound 1.

[0032] 1H NMR data of Compound 1: 1 H NMR (400 MHz, DMSO- d6 ) δ 8.54 (d, J 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 J = 2.0 Hz, 1H), 7.05 - 6.96 (m, 2H), 6.91 (dd, J 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. According to the same preparation method as Compound 1, the synthetic route is as Figure 1As shown, 4-bromopyridine was replaced with other pyridine raw materials, and compounds 2-4 were correspondingly obtained.

[0033]

[0034] According to the same preparation method as compound 1, the synthetic route is as Figure 1 shown. 4-bromopyridine was replaced with 4-bromo-2-methylpyridine raw material, and 7-(chloromethyl)imidazo[1,2- a pyridine was replaced with 4-fluorobenzyl chloride, 3-chlorobenzyl cyanide, and 6-(chloromethyl)quinoline raw materials respectively, and compounds 5-7 were correspondingly obtained.

[0035]

[0036] Molecular docking of compound 5 with lolCDE: The structure of the small molecule compound was drawn by KingDraw and converted using Chem3D. The small molecule was preprocessed 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 preprocessed using Autodock Tools to remove excess molecules and water molecules, merge polar hydrogen atoms, and save as a PDBQT file. Finally, the small molecule was docked to the active site near the lipoprotein using Autodock Vina. The molecular docking of compound 5 with the target protein LolCDE is as Figure 2 shown.

[0037] Example 2: Determination of minimum inhibitory concentration In this example, the minimum inhibitory concentration of small molecule compounds (compounds 1-7 and control compounds A, B) was determined by the two-fold dilution method.

[0038] 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.

[0039] Preparation of the drug solution to be tested: All small molecule compounds were dissolved in DMSO. When determining the minimum inhibitory concentration of the drug, the small molecule compounds were dissolved in MH medium to 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.

[0040] First, prepare a sterile 96-well plate. Add 200 μL of the drug solution to be tested into the first well of each row, and add 100 μL of 6% DMSO into the remaining wells. Subsequently, take 100 μL of the drug solution to be tested from the first well and transfer it to the second well, and mix well. Dilute the drug solution to the 11th well in this way successively, so that the compound concentration is in the range of 0.0078 μg / mL - 256 μg / mL; and 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 Escherichia coli, Klebsiella pneumoniae, carbapenem-resistant Klebsiella pneumoniae, Pseudomonas aeruginosa, carbapenem-resistant Pseudomonas aeruginosa, and Stenotrophomonas maltophilia) with an OD value of 0.001 into each well, and place the 96-well plate in an incubator at 37°C for 20 hours. After the incubation, add 20 μL of 1% TTC (2,3,5-triphenyltetrazolium chloride) into each well, and let it stand at 37°C for 10 minutes for the color reaction. The final measurement results are shown in Table 1-2.

[0041] Table 1 Determination results of the minimum inhibitory concentration of each compound against Escherichia coli ATCC 25922

[0042] The structure of Control A is 。

[0043] The structure of Control B is 。

[0044] The results in Table 1 above show that Compounds 1-7 have antibacterial activity against the Escherichia coli ATCC25922 strain, among which Compounds 5, 6, and 7 have the best antibacterial effects, and the minimum inhibitory concentration can be lower than 0.0078 μg / mL.

[0045] Table 2 Determination results of the minimum inhibitory concentration of Compounds 2, 3, 5, 6, and 7 against other or carbapenem-resistant Gram-negative bacteria

[0046] The results in Table 2 above show that Compounds 2, 3, 5, 6, and 7 have significant antibacterial effects against extended-spectrum β-lactamase Escherichia coli, Klebsiella pneumoniae, and carbapenem-resistant Klebsiella pneumoniae.

[0047] Example 3: In vitro expression and purification of the target protein LolCDE (1) Transfer the PTRC99a-LolCDE plasmid into Escherichia coli competent cell C43. After 12 h, pick a single colony into 100 mL LB (containing 100 μg / mL Ampicillin), and culture it at 37°C with 220 rpm; (2) After 9 h, add 6 L of LB, and expand the culture at 37 °C and 220 rpm, and induce overnight at 20 °C and 180 rpm for 15 - 20 h; (3) After centrifugation at 4,000 rpm for 15 min, collect the cell precipitate, and then resuspend it in 120 mL of lysis buffer containing 50 mM Hepes, pH 7.8, 300 mM NaCl and 10% (v / v) glycerol, and supplement with PMSF at a final concentration of 1 mM; (4) After high-pressure crushing at 800 bar, centrifuge at 4 °C and 140,000×g for 50 min to collect the membrane precipitate. Resuspend the membrane precipitate in 50 mL of lysis buffer supplemented with 1% DDM, and dissolve the membrane at room temperature for 30 min; (5) After centrifugation at 4 °C and 140,000×g for 25 min, bind the supernatant to 3 mL of Ni-NTA column (MCE) at room temperature for 40 min; (6) Wash the impurities 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.

[0048] (7) Finally, further desalt the eluted target protein in buffer (50 mM Hepes, 150 mM NaCl and 0.03% DDM, pH 7.8). Verify the expression of the target protein by SDS-PAGE.

[0049] The SDS-PAGE results of the expression and purification of the target protein LolCDE are as Figure 3 shown.

[0050] Example 4: Biomembrane Interference Technology of Small Molecule Compounds and Target Protein LolCDE (1) Add 200 μL of buffer (50 mM Hepes, 150 mM NaCl, 0.03% DDM) to the pre-wetted plate to pre-wet the sensor (Ni-NTA) for 10 min; (2) Load the protein, define the position of the sensor and the positions of the buffer and the protein, and load the protein by 12 nm; (3)Measure the affinity, define the position of the sensor and the position of the small molecule with gradient dilution. Add 200 μL of buffer containing 15% DMSO (50 mM Hepes, 150 mM NaCl, 0.03% DDM) to the first four wells, and then add 200 μL of small molecule compound samples to the next eight wells in sequence according to the concentration gradient. Program design: Custom 180 s, with the time of Association and Disassociation both being 80 s, and Baseline 120 s.

[0051] The results are as Figure 4 shown in

[0052] Table 3

[0053] The meaning of the affinity involved in the table is the magnitude of the affinity of the compound for the target. The smaller the value, the stronger the affinity. The specific calculation formula is as follows: K d =K off / K on where the dissociation rate constant k off reflects the stability of the complex. The percentage of the complex dissociated per second, that is, 1% of the complex dissociates per second, and the unit is s -1 ; the association rate constant k on represents the association rate of the complex. At a substance concentration of 1 M, the number of complexes generated per second, and the unit is M -1 s -1 . The affinity K d reflects the strength of the interaction binding ability, and the unit is M.

[0054] The results show that compounds 3, 5, and 7 have good affinities for the target protein LolCDE, and the affinities are 496 μmol / L, 683 μmol / L, and 831 μmol / L respectively.

[0055] In summary, it can be seen that compounds 3, 5, and 7 of the present invention can not only have excellent antibacterial effects, but also have significant affinity effects with the target protein LolCDE.

[0056] The above-provided embodiments are not intended to limit the scope covered by the present invention, nor are the described steps intended to limit their execution order. Obvious improvements made by those skilled in the art to the present invention in combination with the existing common general knowledge also fall within the protection scope defined by the claims of the present invention.

Claims

1. A class of 4-(pyridin-4-yl)-1-imidazole-2-amine derivatives targeting key protein machines in the inner and outer membranes of Gram-negative bacteria, or pharmaceutically acceptable salts thereof, characterized in that, H - Imidazole-2-amine derivatives, or pharmaceutically acceptable salts thereof, characterized in that, Its structure is shown in formula (I): ， In the formula, n is the number of substituents R1 on the pyridine ring, taking values of 0, 1, 2, or 3, and each R1 is independently selected from C1-C4 alkyl groups; R2 is independently selected from substituted or unsubstituted C6-C 10 aryl or C5-C 10 heteroaryl, wherein the substituted groups are selected from cyano and halogen.

2. A class of 4-(pyridin-4-yl)-1-imidazole-2-amine derivatives or pharmaceutically acceptable salts thereof targeting key protein machines in the inner and outer membranes of Gram-negative bacteria according to claim 1, characterized in that, H - imidazole-2-amine derivatives, or pharmaceutically acceptable salts thereof, characterized in that, C6-C 10 The aryl group is a phenyl group, C5-C 10 The heteroaryl group is or 。 3. A class of 4-(pyridin-4-yl)-1-imidazole-2-amine derivatives or pharmaceutically acceptable salts thereof targeting key protein machines in the inner and outer membranes of Gram-negative bacteria according to claim 1, characterized in that, H R2 is specifically selected from: . ​ 4. A class of 4-(pyridin-4-yl)-1-imidazole-2-amine derivatives targeting key protein machines in the inner and outer membranes of Gram-negative bacteria, or a pharmaceutically acceptable salt thereof, characterized in that, H The said 4-(pyridin-4-yl)-1 H -imidazole-2-amine derivatives are specifically selected from: ​ 。 5. A class of 4-(pyridin-4-yl)-1-imidazole-2-amine derivatives or pharmaceutically acceptable salts thereof targeting key protein machines in Gram-positive and Gram-negative outer membranes, according to any one of claims 1-4, characterized in that, H The pharmaceutically acceptable salts include inorganic salts or organic salts; among them, the inorganic salts are selected from hydrochloride, hydrobromide, hydroiodide, perchlorate, sulfate, bisulfate, nitrate, phosphate, and acid phosphate; the organic salts are selected from: formate, acetate, trifluoroacetate, propionate, pyruvate, glycolate, oxalate, malonate, succinate, glutarate, fumarate, maleate, lactate, malate, citrate, tartrate, mesylate, esylate, benzenesulfonate, salicylate, p-toluenesulfonate, and ascorbate. ​ 6. A pharmaceutical composition, characterized in that, It contains a class of 4-(pyridin-4-yl)-1-imidazole-2-amine derivatives targeting key protein machines of Gram-positive and Gram-negative outer membranes as described in any one of claims 1-5, or a pharmaceutically acceptable salt thereof, and pharmaceutical excipients. H -imidazole-2-amine derivatives, or a pharmaceutically acceptable salt thereof, and pharmaceutical excipients.

7. The pharmaceutical composition according to claim 6, characterized in that, The pharmaceutical excipients are selected from any one or more of the following: solvents, propellants, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, antiadhesives, chelating agents, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoaming agents, thickeners, clathrates, humectants, flocculants and deflocculants, filter aids, and release retardants.

8. The pharmaceutical composition according to claim 6, characterized in that, The pharmaceutical composition contains a pharmaceutical carrier, and the pharmaceutical carrier is selected from microcapsules, microspheres, nanoparticles, and liposomes.

9. The pharmaceutical composition according to claim 6, characterized in that, The dosage form of the pharmaceutical composition is selected from: injection solutions, freeze-dried powder injections for injection, suspensions, implants, embolization agents, capsules, tablets, pills, and oral liquids.

10. Use of a 4-(pyridin-4-yl)-1-imidazol-2-amine derivative or a pharmaceutically acceptable salt thereof according to any one of claims 1-5 for preparing a Gram-negative bacteria-specific antibiotic drug, which targets key protein machinery of the Gram-negative bacteria outer and inner membranes H -imidazol-2-amine derivatives, or pharmaceutically acceptable salts thereof, in the preparation of Gram-negative bacteria-specific antibiotic drugs.

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