4-(1H-pyrazol-4-yl)pyridine derivatives targeting Gram-negative bacterial lipoprotein transport pathway, compositions and uses thereof

Abstract

The present invention discloses 4-(1H-pyrazol-4-yl)pyridine derivatives, compositions and uses thereof targeting the lipoprotein transport pathway of Gram-negative bacteria, and belongs to the field of chemical medicine technology. The present invention provides 4-(1H-pyrazol-4-yl)-2,6-dimethylpyridine and 4-(1H-pyrazol-4-yl)pyridine derivatives with the following structures:, the compounds can target the key protein machinery of the inner and outer membranes of Gram, and have significant antibacterial activity; at the same time, they can target the key protein machinery of the inner and outer membranes of Gram, and have good affinity with the target protein LolCDE, providing a new approach for specific antibacterial treatment of Gram-negative bacteria.

Description

Technical Field

[0001] The present invention belongs to the technical field of chemical medicine, and specifically relates to 4-(1H-pyrazol-4-yl)pyridine derivatives targeting the lipoprotein transport pathway of Gram-negative bacteria, compositions and uses thereof. Background Art

[0002] Gram-negative bacteria, such as Klebsiella pneumoniae, are a medical nightmare. These pathogens are highly adaptable, toxic, and have the ability to rapidly develop resistance. Only a few antibiotics can kill them, but these antibiotics also destroy beneficial intestinal bacteria. In addition, infectious diseases caused by multidrug-resistant bacteria have become a major global health crisis. Bacteria continue to evolve and acquire 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 and Salmonella typhimurium, carbapenem-resistant Acinetobacter baumannii, third-generation cephalosporin-resistant Enterobacteriaceae, and carbapenem-resistant Enterobacteriaceae. Gram-negative bacteria have an innate asymmetric inner and outer membrane system that can selectively pass nutrients and shield antibiotics.

[0003] "LolCDE" is a class of proteins that only exist in Gram-negative bacteria and are extremely important for bacterial survival. If the function of this protein is inhibited, the bacteria will not survive. 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 show good antibacterial activity and safety, and can kill a wide range of Gram-negative bacteria that are resistant to multiple antibiotics without destroying the intestinal flora. These research results further demonstrate the effectiveness of the key protein machinery of the inner and outer membranes of Gram-negative bacteria as a new antibiotic target.

[0006] However, small molecule compounds targeting the key protein machinery of the inner and outer membranes of Gram are still very limited, and there is an urgent need to provide more small molecule compounds that can effectively inhibit bacteria and have better targeting. Summary of the invention

[0007] Technical issues:

[0008] 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.

[0009] Technical solution:

[0010] In one aspect, the present invention provides a class of 4-(1H-pyrazol-4-yl)pyridine derivatives targeting key protein machinery of Gram inner and outer membranes, or a pharmaceutically acceptable salt thereof, the structure of which is shown below:

[0011] ,

[0012] In the formula, X and Y are independently selected from CR2 and N;

[0013] R1 is H, C 1-3 Alkyl, R2 is H, C 1-3 alkyl.

[0014] In one embodiment of the present invention, the 4-(1H-pyrazol-4-yl)-2,6-dimethylpyridine and 4-(1H-pyrazol-4-yl)pyridine derivatives are specifically selected from:

[0015] .

[0016] 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, and acid phosphate; 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.

[0017] On the other hand, the present invention also provides a pharmaceutical composition, which contains the above-mentioned 4-(1H-pyrazol-4-yl)-2,6-dimethylpyridine and 4-(1H-pyrazol-4-yl)pyridine derivatives or pharmaceutically acceptable salts thereof, and pharmaceutical excipients.

[0018] 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, defoamers, thickeners, inclusion agents, humectants, flocculants and deflocculating agents, filter aids, and release retardants.

[0019] 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.

[0020] 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.

[0021] In another aspect, the present invention also provides use of the above-mentioned 4-(1H-pyrazol-4-yl)-2,6-dimethylpyridine derivatives or pharmaceutically acceptable salts thereof in the preparation of Gram-negative bacteria-specific antibiotic drugs.

[0022] Beneficial effects:

[0023] The 4-(1H-pyrazol-4-yl)pyridine derivatives of the present invention also have significant antibacterial activity, and the minimum inhibitory concentration is below 512 μg / mL, and the lowest can be as low as 1 μg / mL.

[0024] Furthermore, the 4-(1H-pyrazol-4-yl)pyridine derivatives of the present invention can have affinity with the target protein LolCDE, thereby effectively targeting the key protein machinery of the inner and outer membranes of Gram-negative bacteria.

[0025] The present invention combines the above two aspects to provide a new approach for specific antibacterial treatment of Gram-negative bacteria. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is the synthetic route of compound 1.

[0027] Figure 2 This is the structure of compound 1-6.

[0028] Figure 3 This is the molecular docking diagram of compound 1 and lolCDE.

[0029] Figure 4 This is the SDS-PAGE gel image of the target protein LolCDE purification.

[0030] Figure 5This is the result of affinity determination between the target protein LolCDE and small molecule compounds. DETAILED DESCRIPTION

[0031] The technical solution of the invention is described in detail below with reference to the accompanying drawings:

[0032] Although the present invention has been disclosed as above in the form of a preferred embodiment, 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.

[0033] The following examples involve 6 compounds (Compounds 1-6), the corresponding chemical structures are as follows Figure 2 As shown, and in accordance with Figure 1 Prepared by the synthetic route shown.

[0034] Example 1 Preparation of Compounds

[0035] Preparation of compound 1:

[0036]

[0037] Synthesis of intermediate 1: Add methyl 3-hydroxybenzoate (1 mmol) and 1 equivalent of 1-(chloromethyl)-4-fluorobenzene (1 mmol) to a round-bottom flask, add cesium carbonate (2 mmol), add N, N-dimethylformamide (5 mL) as a solvent, and heat at 80°C for 3 hours. Detect the reaction with a thin layer chromatography silica gel plate. After the reaction is completed, 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 once, collect the organic phase, dry with anhydrous sodium sulfate, and concentrate to obtain the crude product of intermediate 1, which is directly used in the next step reaction.

[0038] Synthesis of intermediate 2: 2.2 mmol LiHMDS (1 mmol / L dissolved in THF) was added to a double-necked flask, 5 mL THF was added as solvent, N2 was protected, 1.1 mmol 2,4,6-trimethylpyridine was added at 0°C, and the reaction was allowed to react for 1 hour at 0°C. Then 1 mmol of intermediate 1 was added under N2 protection, and the reaction was moved to room temperature and stirred for 3 hours. The reaction was detected by thin layer chromatography silica gel plate. After the reaction was completed, 1 mL 6N HCl was slowly added to the system to quench, and then the pH was adjusted to 8 with excess saturated sodium bicarbonate solution, extracted with ethyl acetate (3×20 mL), and the organic phase was collected, dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography (petroleum ether: ethyl acetate = 3:1) to obtain intermediate 2.

[0039] Synthesis of compound 1: 1 mmol of intermediate 2 was placed in a round-bottom flask, 3 mL of DMF-DMA was added, and the mixture was stirred at 105°C for 1 hour. The solvent was then distilled off under reduced pressure, 6.5 mmol of hydrazine hydrate and 5 mL of ethanol were added, and the mixture was reacted at 80°C for 1 hour. After the reaction was completed, the solvent was distilled off under reduced pressure, and the mixture was purified by column chromatography (dichloromethane: methanol = 20:1) to obtain compound 1.

[0040] 1 H NMR (400 MHz, Chloroform- d ) δ 8.16 (s, 1H), 7.52 (m, J = 8.0, 3.5,0.8 Hz, 2H), δ 7.47 (s, 1H), 7.41 (ddd, J = 7.6, 2.2, 1.3 Hz, 1H), 7.35 – 7.29(m, 2H), 7.26 – 7.18 (m, 2H), 7.08 (s, 2H), 6.93 (ddd, J = 7.9, 2.2, 1.3 Hz,1H), 4.96 (t, J = 0.8 Hz, 2H), 2.51 (s, 6H). 13 C NMR (101 MHz, Chloroform- d ) δ163.56, 161.59, 158.57, 158.11 (2C), 138.73, 137.35, 134.09, 133.52, 130.28,130.27, 130.13, 130.06, 123.78, 121.26, 117.71, 116.14, 115.46, 115.28,113.44, 69.56, 24.38 (2C).

[0041] According to the same preparation method as compound 1, 3-hydroxybenzoic acid methyl ester is replaced by other methyl formate raw materials, and 2,4,6-trimethylpyridine is replaced by 4-methylpyridine, and compound 2-6 is obtained accordingly. The corresponding structural information is shown below:

[0042]

[0043] Molecular docking of compound 1 and lolCDE:

[0044] 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 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 1 with the target protein LolCDE is shown below. Figure 3 shown.

[0045] Example 2: Minimum inhibitory concentration determination

[0046] In this example, the two-fold dilution method was used to determine the minimum inhibitory concentration of small molecule compounds (compounds 1-6).

[0047] 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.

[0048] Preparation of drug solutions to be tested: All small molecule compounds were dissolved in DMSO. When determining the minimum inhibitory concentration of drugs, small molecule compounds were dissolved in MH medium to 512 μg / mL, 256 μg / mL, 128 μg / mL, 64 μg / mL, 32 μg / mL, 16 μg / mL, 8 μg / mL, 4 μg / mL, 2 μg / mL and 1 μg / mL.

[0049] First, prepare a sterile 96-well plate. Add 200 μL of the drug solution to be tested to the first well of each row, and add 100 μL of 6% DMSO to the remaining wells. Subsequently, transfer 100 μL of the drug solution to be tested from the first well to the second well and mix well. Dilute the drug solution to the 11th well in this way, while no drug is added to the 12th well. Finally, add 100 μL of the test bacteria 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 to each well, and place the 96-well plate at 37°C for 20 hours. After the incubation, 20 μL of 1% TTC (2,3,5-triphenyltetrazolium chloride) was added to each well and incubated at 37°C for 10 minutes for color development. The final assay results are shown in Table 1.

[0050] Table 1 Minimum inhibitory concentration of compounds 1-6 against Escherichia coli MG1655 ΔTolC

[0051]

[0052] The results in Table 1 above show that compounds 1, 2, 3, 4, 5, and 6 have antibacterial activity against Escherichia coli MG1655 ΔTolC and Escherichia coli ATCC25922 strains, among which compounds 1, 3, 4, and 5 have better antibacterial effects, and the minimum inhibitory concentrations are all as low as 512 μg / mL or less, among which compound 1 is the best, which can be as low as 1 μg / mL.

[0053] Table 2 Minimum inhibitory concentration of compound 1 against other or carbapenem-resistant Gram-negative bacteria

[0054]

[0055] The results in Table 2 above show that compound 1 has excellent antibacterial effects on a variety of Gram-negative bacteria.

[0056] Example 3: In vitro expression and purification of target protein LolCDE

[0057] (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.

[0058] (2) After 9 hours, inoculate 6 L of LB and expand the culture at 37°C 220 rpm, and induce overnight at 20°C 180 rpm for 15-20 hours;

[0059] (3) After centrifugation at 4,000 rpm for 15 min, the bacterial pellet was collected and then 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;

[0060] (4) After high-pressure crushing at 800 bar, the membrane precipitate was collected by centrifugation at 140,000 × g for 50 min at 4°C, and the membrane precipitate was resuspended in 50 mL of lysis buffer supplemented with 1% DDM and dissolved at room temperature for 30 min;

[0061] (5) After centrifugation at 140,000 × g for 25 min at 4°C, the supernatant was combined with a 3 mL Ni-NTA column (MCE) at room temperature for 40 min;

[0062] (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.

[0063] (7) Finally, the eluted target protein was further desalted in a buffer (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.

[0064] The SDS-PAGE results of the expression and purification of the target protein LolCDE are shown in Figure 4 shown.

[0065] Example 4: Biomembrane interference technology of small molecule compounds and target protein LolCDE

[0066] (1) Add 200 μL 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.

[0067] (2) Loading protein: define the sensor position and the position of the buffer and protein, with protein loading of 12 nm;

[0068] (3) Determine affinity, define the sensor position and the position of the gradient dilution of small molecules, add 200 μL of 15% DMSO buffer (50 mM Hepes, 150 mM NaCl, 0.03% DDM) to the first four wells, and add 200 μL of small molecule compound samples in the next 8 wells according to the concentration gradient. Program design: Custom 180 s, Association and Disassociation time are both 80 s, Baseline 120 s.

[0069] The above affinity test results are shown in Table 3 and Figure 5 shown.

[0070] Table 3 Affinity test results of various small molecule compounds for the target protein LolCDE

[0071]

[0072] Among them, the structure of Lolamicin is .

[0073] The affinities involved in the table (K d ) means 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

[0074] The dissociation rate constant k is off It reflects the stability of the complex, the percentage of the complex dissociated 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 concentration of 1M substance, in M -1 s -1 . Affinity K d It reflects the strength of the interaction binding ability, and the unit is M.

[0075] The results show that compared with Lolamicin, compounds 1-4 and compound 6 have better affinity with the target protein LolCDE.

[0076] In summary, it can be seen that compounds 1, 3, and 4 of the present invention can not only have good antibacterial effects, but also have excellent affinity effects with the target protein LolCDE.

[0077] The embodiments provided above are not intended to limit the scope of the present invention, and the steps described are not intended to limit the execution order thereof. Those skilled in the art may make obvious improvements to the present invention in combination with existing common knowledge, which also fall within the scope of protection defined by the claims of the present invention.

Claims

1. A 4-(1H-pyrazol-4-yl)pyridine derivative, or a pharmaceutically acceptable salt thereof, whose structure is shown below: ,or .

2. The 4-(1H-pyrazol-4-yl)pyridine derivative or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The pharmaceutically acceptable salt is an inorganic salt or an organic salt; wherein the inorganic salt is selected from hydrochloride, hydrobromide, hydroiodide, perchlorate, sulfate, bisulfate, nitrate, and phosphate; and the organic salt is selected from formates, acetates, trifluoroacetates, propionates, pyruvates, glycolates, oxalates, malonates, succinates, glutarates, fumarates, maleates, lactates, malates, citrates, tartrates, methanesulfonates, ethanesulfonates, benzenesulfonates, salicylates, p-toluenesulfonates, and ascorbates.

3. The 4-(1H-pyrazol-4-yl)pyridine derivative or a pharmaceutically acceptable salt thereof according to claim 2, characterized in that: The inorganic salt is acid phosphate.

4. A pharmaceutical composition, characterized in that Contains the 4-(1H-pyrazol-4-yl)pyridine derivative according to any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof, and a pharmaceutical excipient.

5. The pharmaceutical composition according to claim 4, characterized in that The pharmaceutical excipients are selected from any one or more of the following: solvents, propellants, emulsifiers, colorants, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, anti-adhesive agents, integrities, penetration enhancers, pH regulators, buffers, plasticizers, foaming agents, defoamers, thickeners, humectants, filter aids, and release retardants.

6. The pharmaceutical composition according to claim 4, characterized in that Pharmaceutical excipients are coating materials.

7. The pharmaceutical composition according to claim 4, characterized in that The pharmaceutical excipient is a flocculant or a deflocculating agent.

8. The pharmaceutical composition according to claim 4, characterized in that The medicinal excipient is a fragrance.

9. The pharmaceutical composition according to claim 4, characterized in that The pharmaceutical excipient is an inclusion compound.

10. The pharmaceutical composition according to claim 4, characterized in that The pharmaceutical excipient is an adhesive.

11. The pharmaceutical composition according to claim 4, characterized in that The pharmaceutical excipient is a surfactant.

12. The pharmaceutical composition according to claim 4, characterized in that The dosage form of the pharmaceutical composition is selected from the group consisting of injection, lyophilized powder for injection, suspension, implant, embolic agent, capsule, tablet, pill and oral solution.

13. Use of the 4-(1H-pyrazol-4-yl)pyridine derivative according to any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof, in the preparation of a Gram-negative bacteria-specific antibiotic.

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