Ebbuselenium derivative, pharmaceutical composition and application of ebbuselenium derivative

Abstract

The invention discloses an ebbuselenium derivative, a pharmaceutical composition and application of the ebbuselenium derivative. The ebbeselenium derivative provided by the invention has the characteristic of inhibiting PPK activity, reduces the polyphosphoric acid (polyP) level of bacteria, reduces the formation of biological membranes, reduces the athletic ability of the bacteria, reduces the generation of virulence factors, can also enhance the sensitivity of the bacteria to antibiotics, and can be used for preparing a medicine for preventing and treating the diseases, such as the diseases, the pests and the like. Therefore, the compound is used for preventing and treating bacterial infection diseases.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical chemistry, and in particular to an ebselen derivative, a pharmaceutical composition and application thereof. Background Art

[0002] Pseudomonas aeruginosa is a Gram-negative bacterium and a conditional pathogen. It is listed by the World Health Organization as an urgent priority pathogen in urgent need of antibiotics. The pathogen is widely present in nature and easily infects patients with impaired immune systems such as cystic fibrosis, burns, immunodeficiency, chronic obstructive pulmonary disease, cancer and severe respiratory infections, which can cause very serious or even fatal tissue damage and inflammation. Pseudomonas aeruginosa can grow and multiply in large numbers on moist surfaces, so it is widely present on medical equipment and is the most common type of hospital-acquired infection and drug-resistant pathogens. Epidemiological studies have shown that nearly 700,000 people die each year from antibiotic-resistant bacterial infections, of which 7.1% of deaths are caused by Pseudomonas aeruginosa. Pseudomonas aeruginosa shows inherent and acquired resistance to many antibiotics, making its treatment particularly challenging.

[0003] Pseudomonas aeruginosa is known for its remarkable adaptability, using multiple means to survive within the host, such as flagellar motility, biofilm formation, and release of virulence factors. In the early stages of infection, P. aeruginosa navigates to the host surface using polar flagella and adheres through the action of adhesins, initiating the colonization process. Subsequently, these bacteria can form biofilms—complex bacterial communities surrounded by an extracellular matrix—which are often associated with persistent P. aeruginosa infections and resistance to antibiotics. Free-floating (planktonic) and biofilm-associated P. aeruginosa produce a range of virulence factors, among which pyocyanin and pyoverdine are particularly prominent. Pyoverdine is the major siderophore of P. aeruginosa, which functions to bind and transport iron into bacterial cells and is responsible for the green color of P. aeruginosa cultures. Pyoverdine, on the other hand, is a blue redox-active phenazine that primarily exerts its toxicity through interaction with molecular oxygen, leading to the production of superoxide anions, which can damage host tissues. The production of pyrocyanin has been shown to be associated with increased biofilm formation, which in turn has been shown to enhance the production of pyocyanin, an interaction that reflects the interconnectedness of the various pathogenic features exhibited by Pseudomonas aeruginosa. Targeting these pathogenic features during Pseudomonas aeruginosa infection is currently the most effective strategy to combat Pseudomonas aeruginosa infection, as such strategies inhibit the pathogenicity of Pseudomonas aeruginosa without causing direct bacterial death, which avoids the development of bacterial resistance; in addition, many antibiotics are not specific to pathogens and may have adverse effects on commensal microbial communities. This may lead to reinfection or increased susceptibility to other infectious pathogens. However, therapeutic strategies that target pathogenic features can avoid these deleterious consequences.

[0004] Polyphosphate (polyP) is a linear polymer composed of many phosphate groups connected by phosphate bonds. It has multiple biological functions in bacteria, including energy storage, metabolic regulation, stress response, biofilm formation and expression of pathogenicity factors. In Pseudomonas aeruginosa, polyphosphate affects pathogenicity in various ways, especially plays an important role in biofilm formation, and is also involved in the expression of pathogenicity factors such as pyrochloroquine and pyrocyanine. In addition, polyphosphate enhances bacterial resistance to antibiotics and enhances bacterial survival under antibiotic pressure. In other pathogenic bacteria, polyphosphate is also closely related to pathogenicity and antibiotic sensitivity. Therefore, targeting the homeostasis of polyphosphate has become a potential anti-pathogenic strategy for Pseudomonas aeruginosa and other pathogenic bacteria.

[0005] Polyphosphate is synthesized by polyphosphate kinases (PPKs), which are classified into two distinct types: PPK1 and PPK2. PPK1 catalyzes the synthesis of polyphosphate primarily by transferring a γ-phosphate group from ATP, whereas the PPK2 enzyme prefers to consume polyphosphate to phosphorylate nucleosides. However, PPK2 can also synthesize polyphosphate, albeit with relatively weak activity. PPK1 has been identified as a key factor in the pathogenicity of Pseudomonas aeruginosa, and studies have shown that deletion of the ppk1 gene impairs bacterial quorum sensing, motility, biofilm formation, and overall pathogenicity in animal models. Recently, PPK2 has also been shown to be an important target for disrupting polyphosphate-mediated pathogenicity in Pseudomonas aeruginosa. Overall, the roles of PPK1 and PPK2 in the pathogenicity and survival of a variety of other clinically important pathogens have also been recognized. Due to their pathogenicity in bacteria and the lack of homologous enzymes in mammals, PPKs have been considered as potential targets for novel antimicrobial drug discovery.

[0006] Therefore, it is necessary to develop a new compound for inhibiting polyphosphate kinase. Summary of the invention

[0007] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the first aspect of the present invention provides an ebselen derivative that can effectively inhibit PPKs activity, reduce the polyphosphate level of bacteria, reduce the formation of biofilms, reduce the motility of bacteria, reduce the production of virulence factors, and enhance the sensitivity of bacteria to antibiotics.

[0008] The second aspect of the present invention also provides a pharmaceutical composition.

[0009] The third aspect of the present invention also provides a use of an ebselen derivative in the preparation of a polyphosphate kinase inhibitor drug.

[0010] The fourth aspect of the present invention also provides a use of an ebselen derivative in a drug for treating and / or preventing bacterial infection.

[0011] According to the first aspect of the present invention, an ebselen derivative having a structure described by formula (I) or formula (II), or a pharmaceutically acceptable salt thereof is provided:

[0012]

[0013] Wherein, R1 is selected from H, C 1～6 alkyl or halogen;

[0014] R2 is selected from pyridyl, adamantyl, substituted or unsubstituted phenyl, wherein the substituted group includes furanyl, thienyl, C 1～6substituted or unsubstituted C 3-12 The saturated heterocyclic group, 1～3 The alkyl and C 1～3 Alkoxy substituted benzyl or C 2～6 Ester-substituted C 1～6 The alkyl group;

[0015] and when R1 is selected from H, R2 is not phenyl;

[0016] R3 is selected from phenyl substituted with hydroxyl;

[0017] R4 is selected from C 1～6 an alkyl-substituted phenyl group;

[0018] R5 is selected from H or C 1～6 The alkyl group;

[0019] R6 is selected from C 1～7 alkyl or halogenated alkyl substituted benzyl; or R6 and the adjacent N atom form a 6-membered heterocyclic ring;

[0020] R7 is selected from tert-butyloxycarbonyl.

[0021] The ebselen derivative according to the embodiment of the present invention has at least the following beneficial effects:

[0022] The ebselen derivatives provided by the present invention have the characteristics of inhibiting PPK activity, reducing the polyphosphate (polyP) level of bacteria, reducing the formation of biofilms, reducing the motility of bacteria, reducing the production of virulence factors, and enhancing the sensitivity of bacteria to antibiotics, thereby being used for preventing and treating bacterial infection diseases.

[0023] According to a preferred embodiment of the present invention, R1 is selected from H or halogen.

[0024] According to a preferred embodiment of the present invention, R2 is selected from pyridyl, adamantyl, substituted or unsubstituted phenyl, wherein the substituted group includes furanyl, thienyl, C 1～3 Alkoxy, hydroxyl, substituted or unsubstituted C containing 1 to 3 heteroatoms selected from N and O 3-8 The unsaturated heterocyclic group, 1～3 The alkyl and C 1～3 Alkoxy substituted benzyl or C 2～6 The ester-substituted C 1～3 of alkyl.

[0025] According to a preferred embodiment of the present invention, the ebselen derivative is selected from the following structural formula:

[0026]

[0027] According to a second aspect of the present invention, a pharmaceutical composition is provided, comprising the ebselen derivative described in the first aspect of the present invention; and a pharmaceutically acceptable excipient.

[0028] According to a preferred embodiment of the present invention, the pharmaceutical composition further comprises an antibiotic.

[0029] According to a preferred embodiment of the present invention, the antibiotic is selected from at least one of tetracycline, amoxicillin, cefixime, vancomycin and norfloxacin.

[0030] Use of ebselen or an ebselen derivative according to the third aspect of the present invention or the pharmaceutical composition according to the second aspect of the present invention in the preparation of a polyphosphate kinase inhibitor;

[0031] The ebselen derivative has a structure described by formula (I) or formula (II):

[0032]

[0033] Wherein, R1 is selected from H, C 1～6 Alkoxy, C 1～6 alkyl or halogen;

[0034] R2 is selected from pyridyl, adamantyl, substituted or unsubstituted phenyl, wherein the substituted groups include hydroxyl, furanyl, thienyl, C 1～6 substituted or unsubstituted C 3-12 The unsaturated heterocyclic group, 1～3 The alkyl and C 1～3 Alkoxy substituted benzyl or C 2～6 The ester-substituted C 1～6 and when R1 is selected from H, R2 is not phenyl;

[0035] R3 is selected from phenyl substituted with hydroxyl;

[0036] R4 is selected from C 1～6 an alkyl-substituted phenyl group;

[0037] R5 is selected from H or C 1～6 The alkyl group;

[0038] R6 is selected from C 1～7 alkyl or halogenated alkyl substituted benzyl; or R6 and the adjacent N atom form a 6-membered heterocyclic ring;

[0039] R7 is selected from tert-butyloxycarbonyl.

[0040] According to a preferred embodiment of the present invention, the polyphosphate kinase includes PPK1 and PPK2.

[0041] According to a preferred embodiment of the present invention, the ebselen derivative has a structure described by formula (I) or formula (II):

[0042]

[0043] Wherein, R1 is selected from H, C 1～6 alkyl or halogen;

[0044] R2 is selected from pyridyl, adamantyl, substituted or unsubstituted phenyl, wherein the substituted group includes furanyl, thienyl, C 1～6 substituted or unsubstituted C 3-12 The saturated heterocyclic group, 1～3 The alkyl and C 1～3 Alkoxy substituted benzyl or C 2～6 The ester-substituted C 1～6 The alkyl group;

[0045] and when R1 is selected from H, R2 is not phenyl;

[0046] R3 is selected from phenyl substituted with hydroxyl;

[0047] R4 is selected from C 1～6 an alkyl-substituted phenyl group;

[0048] R5 is selected from H or C 1～6 The alkyl group;

[0049] R6 is selected from C 1～7 alkyl or halogenated alkyl substituted benzyl; or R6 and the adjacent N atom form a 6-membered heterocyclic ring;

[0050] R7 is selected from tert-butyloxycarbonyl.

[0051] Use of ebselen or an ebselen derivative according to the fourth aspect of the present invention or the pharmaceutical composition according to the second aspect of the present invention in the preparation of a drug for treating and / or preventing bacterial infection caused by polyphosphate kinase;

[0052] The ebselen derivative has a structure described by formula (I) or formula (II):

[0053]

[0054] Wherein, R1 is selected from H, C 1～6 Alkoxy, C 1～6alkyl or halogen;

[0055] R2 is selected from pyridyl, adamantyl, substituted or unsubstituted phenyl, wherein the substituted groups include hydroxyl, furanyl, thienyl, C 1～6 substituted or unsubstituted C 3-12 The unsaturated heterocyclic group, 1～3 The alkyl and C 1～3 Alkoxy substituted benzyl or C 2～6 The ester-substituted C 1～6 and when R1 is selected from H, R2 is not phenyl;

[0056] R3 is selected from phenyl substituted with hydroxyl;

[0057] R4 is selected from C 1～6 an alkyl-substituted phenyl group;

[0058] R5 is selected from H or C 1～6 The alkyl group;

[0059] R6 is selected from C 1～7 alkyl or halogenated alkyl substituted benzyl; or R6 and the adjacent N atom form a 6-membered heterocyclic ring;

[0060] R7 is selected from tert-butyloxycarbonyl.

[0061] In the present invention, the ebselen derivative can effectively inhibit the activity of PPK enzyme, and has a good effect on infections caused by bacteria that rely on PPK enzyme to regulate the production of polyphosphate and exert toxic effects.

[0062] According to a preferred embodiment of the present invention, the bacteria include Pseudomonas aeruginosa, Klebsiella pneumoniae, Salmonella, Bacillus subtilis, Acinetobacter baumannii, Enterobacter cloacae or Enterococcus.

[0063] Definitions and general terms

[0064] “C 1-6 "alkyl" means an alkyl group having 1 to 6 carbon atoms, including C 1-6 Straight chain alkyl, C 1-6 The branched alkyl and C 3-6 The cycloalkyl group may be, for example, a straight-chain alkyl group having 1, 2, 3, 4, 5 or 6 carbon atoms, a branched-chain alkyl group having 1, 2, 3, 4, 5 or 6 carbon atoms, or a cycloalkyl group having 3, 4, 5 or 6 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, n-hexyl, cyclopropyl, methylcyclopropyl, ethylcyclopropyl, cyclopentyl, methylcyclopentyl, cyclohexyl, etc.1-7 Alkyl", "C 1-3 The "alkyl group" has a similar explanation, except that the number of carbon atoms is different.

[0065] “C 1-6 "alkoxy" means an alkoxy group having 1 to 6 carbon atoms, including C 1-6 Straight chain alkoxy, C 1-6 Branched alkoxy and C 2-6 The cycloalkoxy group may be, for example, a straight chain alkoxy group having 1, 2, 3, 4, 5 or 6 carbon atoms, a branched chain alkoxy group having 1, 2, 3, 4, 5 or 6 carbon atoms, or a cycloalkoxy group having 2, 3, 4, 5 or 6 carbon atoms, for example, a methoxy group, an ethoxy group, a n-propoxy group, an isopropoxy group, etc. 1-3 The "alkoxy group" has a similar explanation, except that the number of carbon atoms is different.

[0066] "a substituted or unsubstituted C containing 1 to 3 heteroatoms selected from N, O and S 3-12 The term "saturated heterocyclic group" refers to a heterocyclic group having 3 to 12 carbon atoms, wherein the ring atoms in the heterocyclic ring contain heteroatoms, the number of heteroatoms is 1 to 3, and the heteroatoms are at least one selected from N, O and S, the heterocyclic ring is a saturated group, and optionally at least one H in the group is replaced by C 1～6 substituted by an alkyl group.

[0067] “C 2～6 The ester group of The ester group, wherein C 2～6 The ester group refers to an ester group having 2 to 6 carbon atoms in total, and representative examples include methyl formate, ethyl formate, ethyl acetate, methyl acetate and the like.

[0068] The pharmaceutically acceptable adjuvants of the present invention include any solvent, solid excipient, diluent, adhesive, disintegrant, or other liquid excipient, dispersant, flavoring agent or suspending agent, surfactant, isotonic agent, thickener, emulsifier, preservative, solid adhesive, glidant or lubricant, etc., suitable for the specific target dosage form. As described in the following documents: In Remington: The Science and Practice of Pharmacy, 21st edition, 2005, ed. DB Troy, Lippincott Williams & Wilkins, Philadelphia, and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and JC Boylan, 1988-1999, Marcel Dekker, New York, the contents of the documents herein are combined to show that different adjuvants can be applied to the preparation of pharmaceutically acceptable compositions and their known preparation methods. Except insofar as any conventional excipients are incompatible with the compounds of the present invention, for example by producing any undesirable biological effect or interacting in a deleterious manner with any other component of the pharmaceutically acceptable composition, their use is contemplated by the present invention.

[0069] Substances that can be used as pharmaceutically acceptable excipients include, but are not limited to, ion exchangers; aluminum; aluminum stearate; lecithin; serum proteins, such as human serum albumin; buffer substances such as phosphates; glycine; sorbic acid; potassium sorbate; partial glyceride mixtures of saturated vegetable fatty acids; water; salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts; colloidal silicon; magnesium trisilicate; polyvinyl pyrrolidone; polyacrylates; waxes; polyethylene-polyoxypropylene-blocking polymers; lanolin; sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as carboxymethyl cellulose sodium cellulose, ethylcellulose and cellulose acetate; gum powder; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycol compounds such as propylene glycol and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethanol; phosphate buffer solution; and other non-toxic suitable lubricants such as sodium lauryl sulfate and magnesium stearate; colorants; release agents; coatings; sweeteners; flavorings; fragrances; preservatives and antioxidants.

[0070] The pharmaceutically acceptable salts of the present invention include salts formed with inorganic acids, organic acids, alkali metals, alkaline earth metals and basic amino acids. Including, but not limited to: hydrochloride, sulfate, bisulfate, nitrate, hydrobromide, hydroiodide, carbonate, bicarbonate, sulfite, bisulfite, pyrosulfate, monohydrogen phosphate, dihydrogen phosphate, perchlorate, persulfate, hemisulfate, bisulfate, thiocyanate, phosphate, pyrophosphate, metaphosphate; suitable organic acid salts include, but not limited to: formate, acetate, propionate, butyrate, benzoate, malonate, succinate, pyruvate, methanesulfonate, ethanesulfonate, propanesulfonate, citrate, 4-nitrobenzoate, benzenesulfonate, p-toluenesulfonate, malate, propiolate, 2-butyrate, 2-hydroxy-ethanesulfonate, Vinyl acetate, tartrate, L-tartrate, fumarate, isethionate, maleate, lactate, lactobionate, pamoate, salicylate, galactarate, glucoheptonate, mandelate, 1,2-ethanedisulfonate, 2-naphthalenesulfonate, oxalate, trifluoroacetate, trifluoromethanesulfonate, adipate, suberate, sebacate, butyne-1,4-dioate, hexyne-1,6-dioate, glycolate, alginate, ascorbate, isoascorbate, aspartate, L-aspartate, glutamate, L-glutamate, 2-phenoxybenzoate, 2-(4-hydroxybenzoyl)benzoate, acetoacetate, 2-hydroxy Ethylsulfonate, benzenesulfonate, borate, chlorobenzoate, camphorate, itaconate, camphorsulfonate, L-camphorsulfonate, methylbenzoate, dinitrobenzoate, sulfamate, lactobionate, galacturonate, cyclopentylpropionate, dodecyl sulfate, acrylate, cyclopentanepropionate, glycerophosphate, methoxybenzoate, digluconate, gluconate, heptanoate, hexanoate, 2-hydroxy-ethanesulfonate, trimethylacetate, glucuronate, laurate, phthalate, phenylacetate, lauryl sulfate, 2-acetoxybenzoate, nicotinate, cinnamate, oleate, palmitate, pamoate, pectinate, phthalate, Glutarate, hydroxymaleate, hydroxybenzoate, phenylacetate, 3-hydroxy-2-naphthoate, 3-phenylpropionate, isobutyrate, pivalate, picrate, stearate, 2,2-dichloroacetate, acylated amino acid salts, alginate, 4-acetamidobenzenesulfonate, decanoate, cholate, octanoate, nonanoate, cyclamate, phthalate, cysteine ​​hydrochloride, sorbate, pamoate, mucate, glycinate hydrochloride, naphthalene disulfonate, xylene sulfonate, cystine dihydrochloride, undecanoate, polyvinyl sulfonate, sulfosalicylate, phenylbutyrate, 4-hydroxybutyrate, polyvinyl sulfate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, and valerate.

[0071] Other features and advantages of the present invention will be set forth in the description which follows, and in part will be apparent from the description, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0072] The above and / or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0073] Figure 1 The IC values ​​of ebselen of Example 1 of the present invention for PPK1 and PPK2 are 50 Inhibition curve and enzymatic rate curve of PPK1 in the presence of ebselen. The experiment was repeated three times independently (n=3), *P<0.05, **P<0.01, ***P<0.001.

[0074] Figure 2 It is a CD binding analysis diagram between PPK1 and PPK2 and ebselen of Example 1 of the present invention.

[0075] Figure 3 This is a graph showing the inhibition of PolyP production by Pseudomonas aeruginosa by ebselen in Example 1 of the present invention.

[0076] Figure 4 This is a graph showing the inhibition of biofilm formation of Pseudomonas aeruginosa by ebselen in Example 1 of the present invention.

[0077] Figure 5 This is a graph showing the inhibition of the motility of Pseudomonas aeruginosa by ebselen in Example 1 of the present invention.

[0078] Figure 6 This is a graph showing the inhibition of toxin production by Pseudomonas aeruginosa by ebselen in Example 1 of the present invention.

[0079] Figure 7 This is a graph showing the effect of the combined use of ebselen and antibiotics in Example 1 of the present invention on the growth of Pseudomonas aeruginosa. DETAILED DESCRIPTION

[0080] The following are specific embodiments of the present invention, and the technical solutions of the present invention are further described in conjunction with the embodiments, but the present invention is not limited to these embodiments.

[0081] Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0082] Example 1

[0083] This example provides an ebselen (Ebselen; EB-1), the reaction equation and preparation method thereof are as follows:

[0084]

[0085] S1. Weigh o-iodobenzoic acid (CAS No.: 88-67-5, compound 1, 1.00 g, 4.03 mmol), EDCI (1.11 g, 5.76 mmol) and DMAP (47 mg, 0.38 mmol) in a 250 mL round-bottom flask, add CH2Cl2 (30 mL), slowly add aniline (CAS No.: 62-53-3; 357 mg, 3.84 mmol) at 0°C; then move to room temperature (about 25°C) and stir. After reacting for about 3 hours, monitor by TLC until the reaction of the raw materials is complete. Add 80 mL of water to quench the reaction, then add CH2Cl2 to extract three times (40 mL × 3), then wash three times with saturated NaHCO3 solution (40 mL × 3) and HCl solution (1M, 40 mL × 3) and combine the organic phases, dry the organic layer with anhydrous Na2SO4, and concentrate under reduced pressure to remove CH2Cl2 to obtain a crude product. The crude product does not need to be purified and is directly used in the next step.

[0086] S2, add CuI (177mg, 0.93mmol), o-phenanthroline (167mg, 0.93mmol), selenium powder (293mg, 3.71mmol) and K2CO3 (640mg, 4.64mmol) to the crude product (1.00g, 3.09mmol) obtained in step S1 in a 250mL round-bottom flask, add DMF (20mL), heat to 120℃ under nitrogen protection and stir for 6h, monitor by TLC until the raw material reaction is complete. Filter, add 200mL water to the filtrate, then add CH2Cl2 to extract three times (50mL×3), then wash three times with saturated brine (150mL×3) and combine the organic phases, dry the organic layer with anhydrous Na2SO4, concentrate under reduced pressure to remove CH2Cl2, purify by flash column chromatography (pure CH2Cl2), and obtain 340mg of light yellow solid compound EB-01, with a yield of 40%.

[0087] NMR data of compound EB-01: 1 H-NMR (600MHz, DMSO-d6) δ8.10(d,J=8.0Hz,1H),7.92(d,J=8.6Hz,1H),7.69(t,J=8.3Hz ,1H),7.65(d,J=7.5Hz,2H),7.51–7.47(m,1H),7.47–7.44(m,2H),7.27(t,J=7.4Hz,1H).

[0088] Example 2

[0089] This embodiment provides an ebselen derivative EB-2, and its preparation method is the same as that of Example 1, except that: in step S1, o-iodobenzoic acid is replaced by 2-iodo-5-methoxybenzoic acid (CAS No.: 54413-93-3).

[0090] In step S2, compound EB-2 is obtained (yield 40%).

[0091]

[0092] Compound EB-02: yellow solid; 1 H NMR(400MHz,DMSO-d6)δ7.96(d,J=8.8Hz,1H),7.64(d,J=7.5Hz,2H),7.49–7.42(m,2H) ,7.41(d,J=2.7Hz,1H),7.33(dd,J=8.8,2.8Hz,1H),7.27(t,J=7.4Hz,1H),3.86(s,3H).

[0093] Example 3

[0094] This embodiment provides an ebselen derivative EB-3, and its preparation method is the same as that of Example 1, except that: in step S1, o-iodobenzoic acid is replaced by 5-fluoro-2-iodobenzoic acid (CAS No.: 52548-63-7).

[0095] In step S2, compound EB-3 was obtained (yield: 22%).

[0096]

[0097] Compound EB-3: green solid; 1H NMR (400 MHz, DMSO-d6) δ 8.12 (dd, J = 8.8, 4.9 Hz, 1H), 7.68–7.56 (m, 4H), 7.46 (t, J = 7.9 Hz, 2H), 7.28 (t, J = 7.4 Hz, 1H).

[0098] Example 4

[0099] This embodiment provides an ebselen derivative EB-4, and its preparation method is the same as that of Example 1, except that: in step S1, aniline is replaced by p-aminoanisole (CAS No.: 104-94-9).

[0100] In step S2, compound 2b was obtained (yield 35%).

[0101]

[0102] S3. Add 2b (380 mg, 1.26 mmol) to a 100 mL round-bottom flask, add 10 mL of ultra-dry CH2Cl2, move to a -78 ° C cold trap and stir for 10 minutes, add BBr3 (520 mg, 2.52 mmol) dropwise under nitrogen protection at -78 ° C, and add dropwise in 30 minutes. Then slowly warm the reaction system to 0 ° C and stir for about two hours, and monitor by TLC until the raw material reacts completely. Add 15 mL of saturated NaHCO3 solution to the reaction system to quench the reaction, filter, and collect the filter residue. The filter residue is purified by flash column chromatography (CH2Cl2: MeOH = 50: 1) to obtain 310 mg of yellow solid EB-4 with a yield of 85%.

[0103]

[0104] Compound EB-04: 1 H NMR (400MHz, DMSO-d6) δ9.61 (s, 1H), 8.08 (d, J = 8.0Hz, 1H), 7.87 (d, J = 7.3Hz, 1H), 7.6 6(t,J=7.1Hz,1H),7.46(t,J=7.4Hz,1H),7.35(d,J=8.7Hz,2H),6.82(d,J=8.7Hz,2H).

[0105] Example 5

[0106] This embodiment provides an ebselen derivative EB-6, and its preparation method is as follows:

[0107] S1. Add EB-04 (87 mg, 0.30 mmol) to a 50 mL round-bottom flask, add 2 mL DMF, then add ethyl bromoacetate (CAS No.: 105-36-2; 80 mg, 0.48 mmol) and K2CO3 (64 mg, 0.46 mmol), stir the reaction system at room temperature for about two hours, and monitor by TLC until the reaction of the raw materials is complete. Add 20 mL of water to the reaction system, then add CH2Cl2 to extract three times (15 mL×3), then wash three times with saturated brine (40 mL×3) and combine the organic phases, dry the organic layer with anhydrous Na2SO4, concentrate under reduced pressure to remove CH2Cl2, and purify by flash column chromatography (pure CH2Cl2) to obtain 95 mg of white solid EB-6 with a yield of 84%.

[0108]

[0109] Compound EB-06: 1H NMR (600MHz, DMSO-d6) δ8.08(d,J=8.0Hz,1H),7.92–7.87(m,1H),7.67(td,J=8.3,7.8,1.4Hz,1H),7.51(d,J=9. 0Hz, 2H), 7.48 (t, J = 7.0Hz, 1H), 7.01 (d, J = 9.0Hz, 2H), 4.82 (s, 2H), 4.19 (q, J = 7.1Hz, 2H), 1.23 (t, J = 7.1Hz, 3H).

[0110] Example 6

[0111] This embodiment provides an ebselen derivative EB-5, and its preparation method is as follows:

[0112] S1. Add EB-06 (65 mg, 0.17 mmol) and 10 mL CH2Cl2 to a 50 mL round-bottom flask, followed by triethylamine (87 mg, 0.85 mmol). Slowly add lithium hydroxide (41 mg, 1.7 mmol) to the reaction system under an ice bath, react at 0°C for about two hours, monitor by TLC until the reaction of the raw materials is complete, dropwise add concentrated hydrochloric acid to adjust the pH value of the reaction system to about 3, filter, collect the filter residue, and purify by flash column chromatography (CH2Cl2: MeOH = 10: 1) to obtain 50 mg of white solid EB-5 with a yield of 83%.

[0113]

[0114] Compound EB-5: white solid; 1 H NMR (400MHz, DMSO-d6) δ9.46(s,1H),8.07(d,J=8.0Hz,1H),7.88(d,J=8.6Hz,1H),7.67(ddd,J=8.2,7.3,1.4Hz,1H),7.55–7.43(m,5H),1.49(s,9H).

[0115] Example 7

[0116] This embodiment provides an ebselen derivative EB-7, and its preparation method is the same as that of Example 1, except that: in step S1, aniline is replaced by N-BOC-p-phenylenediamine (CAS No.: 71026-66-9).

[0117] In step S2, compound EB-7 was obtained (yield: 26%).

[0118]

[0119] Compound EB-7: white solid; 1H NMR (400MHz, DMSO-d6) δ9.46(s,1H),8.07(d,J=8.0Hz,1H),7.88(d,J=8.6Hz,1H),7.67(ddd,J=8.2,7.3,1.4Hz,1H),7.55–7.43(m,5H),1.49(s,9H).

[0120] Example 8

[0121] This embodiment provides an ebselen derivative EB-8, and its preparation method is the same as that of Example 1, except that: in step S1, aniline is replaced by N-BOC-m-phenylenediamine (CAS No.: 68621-88-5).

[0122] In step S2, compound EB-8 was obtained (yield: 29%).

[0123]

[0124] Compound EB-8: white solid; 1 H NMR(400MHz,DMSO-d6)δ9.50(s,1H),8.07(d,J=8.0Hz,1H),7.90(d,J=7.7Hz,1H),7.8 4(s,1H),7.68(t,J=7.6Hz,1H),7.48(t,J=7.4Hz,1H),7.37–7.28(m,3H),1.49(s,9H).

[0125] Example 9

[0126] This embodiment provides an ebselen derivative EB-9, and its preparation method is as follows:

[0127] S1. Weigh 3-(N-tert-butyloxycarbonylamino)benzoic acid (CAS No.: 111331-82-9, compound 1, 2.00 g, 8.44 mmol), EDCI (1.94 g, 10.12 mmol) and DMAP (103 mg, 0.84 mmol) in a 250 mL round-bottom flask, add CH2Cl2 (30 mL), and slowly add morpholine (CAS No.: 110-91-8; 808 mg, 9.29 mmol) at 0°C; then move to room temperature (about 25°C) and stir. After reacting for about 3 hours, monitor by TLC until the reaction of the raw materials is complete. Add 80 mL of water to quench the reaction, then add CH2Cl2 to extract three times (40 mL×3), then wash three times with saturated NaHCO3 solution (40 mL×3) and saturated NH4Cl solution (40 mL×3) respectively and combine the organic phases. The organic layer is then dried over anhydrous Na2SO4 and concentrated under reduced pressure to remove CH2Cl2 to obtain a crude product. The crude product does not need to be purified and is directly used in the next step.

[0128]

[0129] S2. Add CH2Cl2 (30 mL) to the crude product compound 2 (2.1 g, 6.86 mmol) obtained in S1, slowly add trifluoroacetic acid (7.82 g, 68.6 mmol) dropwise and react at room temperature for about two hours. Monitor by TLC until the reaction of the raw material is complete. Directly spin dry, add CH2Cl2 (30 mL), EDCI (1.79 g, 9.34 mmol), DMAP (76 mg, 0.62 mmol) and o-iodobenzoic acid (CAS No.: 88-67-5, 1.85 g, 7.46 mmol) to the reaction system. After reacting at room temperature for about 3 hours, monitor by TLC until the reaction of the raw material is complete. Add 80 mL of water to quench the reaction, then add CH2Cl2 to extract three times (40 mL×3), then wash three times with saturated NaHCO3 solution (40 mL×3) and saturated NH4Cl solution (40 mL×3) respectively and combine the organic phases. The organic layer is then dried over anhydrous Na2SO4 and concentrated under reduced pressure to remove CH2Cl2 to obtain a crude product. The crude product does not need to be purified and is directly used in the next step.

[0130]

[0131] S3, add potassium tert-butoxide (1.01g, 9.02mmol) and selenium powder (573mg, 7.25mmol) to the crude product compound 3 (1.58g, 3.62mmol) obtained in step S2 in a 250mL round-bottom flask, slowly add DMF (20mL) in an ice bath and stir, heat to 120°C under nitrogen protection and stir for 6h, monitor by TLC until the raw material reaction is complete. Filter, add 200mL water to the filtrate, then add CH2Cl2 to extract three times (50mL×3), then wash three times with saturated brine (150mL×3) and combine the organic phases, dry the organic layer with anhydrous Na2SO4, concentrate under reduced pressure to remove CH2Cl2, and purify by flash column chromatography (CH2Cl2:MeOH=100:1) to obtain 378mg of white solid compound EB-09, with a yield of 27%.

[0132]

[0133] Compound EB-09: white solid; 1H NMR(400MHz, DMSO-d6)δ8.10(d,J=8.0Hz,1H),7.92(d,J=8.7Hz,1H),7.77–7.74(m,1H),7.73–7.6 7(m,2H),7.56–7.51(m,1H),7.49(t,J=7.5Hz,1H),7.30(dt,J=7.6,1.2Hz,1H),3.74–3.36(m,8H).

[0134] Example 10

[0135] This embodiment provides an ebselen derivative EB-10; its preparation method is basically the same as that of Example 8, except that: in step S1, morpholine is replaced by cyclohexylmethylamine (CAS No.: 3218-02-8).

[0136] In step S2, compound EB-10 was obtained (yield: 23%).

[0137]

[0138] Compound EB-10: white solid; 1 H NMR (400MHz, DMSO-d6) δ8.21(d,J=8.0Hz,1H),8.04(t,J=1.8Hz,1H),7.91(d,J =7.7Hz,1H),7.80(d,J=8.0Hz,1H),7.72(d,J=7.8Hz,1H),7.71–7.65(m,1H),7 .52(t,J=8.0Hz,1H),7.48(t,J=7.0Hz,1H),3.12(t,J=6.4Hz,2H),1.70(t,J=1 2.4Hz, 4H), 1.64–1.48 (m, 2H), 1.27–1.07 (m, 3H), 0.92 (q, J = 10.6, 9.5Hz, 2H).

[0139] Embodiment 11

[0140] This embodiment provides an ebselen derivative EB-11; its preparation method is basically the same as that of Example 8, except that: in step S1, morpholine is replaced by 4-(trifluoromethyl)benzylamine (CAS No.: 3300-51-4).

[0141] In step S2, compound EB-11 was obtained (yield: 29%).

[0142]

[0143] Compound EB-11: white solid; 1H NMR (400MHz, DMSO-d6) δ9.27(t,J=5.9Hz,1H),8.16–8.11(m,2H),7.92(d,J=6.9Hz,1H),7.86–7. 79(m,2H),7.70(d,J=8.1Hz,3H),7.61–7.53(m,3H),7.49(t,J=7.0Hz,1H),4.58(d,J=5.8Hz,2H).

[0144] Example 12

[0145] This embodiment provides an ebselen derivative EB-12; its preparation method is basically the same as that of Example 1, except that: aniline in step S1 is replaced by 2-aminopyridine (CAS No.: 504-29-0).

[0146] In step S2, compound EB-12 was obtained (yield: 15%).

[0147]

[0148] Compound EB-12: yellow solid; 1 H NMR(400MHz,DMSO-d6)δ8.79(d,J=7.3Hz,1H),8.31(d,J=9.3Hz,1H),7.90(t ,J=8.4Hz,1H),7.76–7.68(m,2H),7.56–7.47(m,2H),7.06(t,J=7.5Hz,1H).

[0149] Embodiment 13

[0150] This embodiment is an ebselen derivative EB-13, and its preparation method is as follows:

[0151] S1. Weigh 4-isopropyl salicylaldehyde (CAS No.: 536-32-3, compound 1, 0.95 g, 5.79 mmol), iodomethane (2.46 g, 17.3 mmol) and potassium carbonate (2.4 g, 17.4 mmol) in a 250 mL round-bottom flask, add DMF (15 mL), react at room temperature for about two hours, monitor by TLC until the reaction of the raw materials is complete, add 80 mL of water to the reaction system, then add CH2Cl2 to extract three times (50 mL×3), then wash three times with saturated brine (150 mL×3) and combine the organic phases, dry the organic layer over anhydrous Na2SO4, and concentrate under reduced pressure to remove CH2Cl2 to obtain a crude product, which does not need to be purified and is directly used in the next step.

[0152]

[0153] S2. To the crude product compound 2 (1.54 g, 8.55 mmol) obtained in S1, tert-butyl carbamate (3.00 g, 25.6 mmol) and triethylsilane (2.98 g, 25.6 mmol) were added, followed by acetonitrile (25 mL). Trifluoroacetic acid (1.95 g, 17.1 mmol) was slowly added dropwise at room temperature. The reaction was carried out for about one and a half hours at room temperature. TLC was used to monitor the complete reaction of the raw materials. Excess trifluoroacetic acid (9.75 g, 85.5 mmol) was added. The reaction was continued for another half an hour at room temperature. TLC was used to monitor the complete reaction. The mixture was directly spin-dried. CH2Cl2 (30 mL), EDCI (2.22 g, 11.62 mmol), DMAP (95 mg, 0.78 mmol) and o-iodobenzoic acid (CAS No.: 88-67-5, 2.02 g, 8.15 mmol) were added to the reaction system. The reaction was carried out for about 3 hours at room temperature. TLC was used to monitor the complete reaction. Add 80 mL of water to quench the reaction, then add CH2Cl2 to extract three times (40 mL×3), then wash three times with saturated NaHCO3 solution (40 mL×3) and HCl solution (1M, 40 mL×3) respectively and combine the organic phases. The organic layer is then dried over anhydrous Na2SO4 and concentrated under reduced pressure to remove CH2Cl2 to obtain a crude product. The crude product does not need to be purified and is directly used in the next step.

[0154]

[0155] S3, add CuI (281mg, 1.47mmol), o-phenanthroline (266mg, 1.47mmol), selenium powder (466mg, 5.90mmol) and K2CO3 (1.02g, 7.39mmol) to the crude product compound 3 (2.01g, 4.91mmol) obtained in step S2 in a 250mL round-bottom flask, add DMF (20mL), heat to 120℃ under nitrogen protection and stir for 6h, monitor by TLC until the raw material reaction is complete. Filter, add 200mL water to the filtrate, then add CH2Cl2 to extract three times (50mL×3), then wash three times with saturated brine (150mL×3) and combine the organic phases, dry the organic layer with anhydrous Na2SO4, concentrate under reduced pressure to remove CH2Cl2, purify by flash column chromatography (pure CH2Cl2), and obtain 750mg of light yellow solid compound EB-13, with a yield of 39%.

[0156]

[0157] Compound EB-13: white solid; 1H NMR (400MHz, DMSO-d6) δ7.98(d,J=8.0Hz,1H),7.82(d,J=8.5Hz,1H),7.58(t,J=8.3Hz,1H),7.41(t,J=7.9Hz,1H),7.11(d,J=7.7H z,1H),6.91(d,J=1.2Hz,1H),6.79(dd,J=7.7,1.3Hz,1H),4.80(s,2H),3.86(s,3H),2.87(p,J=6.9Hz,1H),1.20(d,J=6.9Hz,6H).

[0158] Embodiment 14

[0159] This embodiment provides an ebselen derivative EB-14; its preparation method is basically the same as that of Example 1, except that in step S1, aniline is replaced by furfurylamine (CAS No.: 617-89-0).

[0160] In step S2, compound EB-14 was obtained (yield: 21%).

[0161]

[0162] Compound EB-14: yellow solid; 1 H NMR (400MHz, DMSO-d6) δ8.00(d,J=8.0Hz,1H),7.84(d,J=6.9Hz,1H),7.64(dd,J=1.7,1.0Hz,1H),7.60(t,J=8.3Hz,1H),6.50–6.40(m,2H),4.92(s,2H).

[0163] Embodiment 15

[0164] This embodiment provides an ebselen derivative EB-15; its preparation method is basically the same as that of Example 1, except that: in step S1, aniline is replaced by 2-thiophenemethylamine (CAS No.: 27757-85-3).

[0165] In step S2, compound EB-15 was obtained (yield: 18%).

[0166]

[0167] Compound EB-15: purple solid; 1H NMR (600MHz, DMSO-d6) δ8.01(d,J=8.0Hz,1H),7.84(d,J=7.7Hz,1H),7.60(t,J=8.2Hz,1H),7.46(d, J=6.2Hz,1H),7.42(t,J=7.4Hz,1H),7.14(d,J=3.1Hz,1H),6.99(dd,J=5.0,3.5Hz,1H),5.08(s,2H).

[0168] Example 16

[0169] This embodiment provides an ebselen derivative EB-16; its preparation method is basically the same as that of Example 1, except that: in step S1, aniline is replaced by 2-methoxyethylamine (CAS No.: 109-85-3).

[0170] In step S2, compound EB-16 was obtained (yield: 23%).

[0171]

[0172] Compound EB-16: light red solid; 1 H NMR(400MHz,DMSO-d6)δ8.03(d,J=8.0Hz,1H),7.81(d,J=7.0Hz,1H),7.62–7.56(m,1 H),7.40(t,J=7.0Hz,1H),3.90(t,J=5.2Hz,2H),3.56(t,J=5.2Hz,2H),3.32(s,3H).

[0173] Embodiment 17

[0174] This embodiment provides an ebselen derivative EB-17, and its preparation method is as follows:

[0175] S1. Add EB-16 (60 mg, 2.34 mmol) to a 100 mL round-bottom flask, add 10 mL of ultra-dry CH2Cl2, move to a -78 ° C cold trap and stir for 10 minutes, add BBr3 (963 mg, 4.70 mmol) dropwise under nitrogen protection at -78 ° C, and add dropwise in 30 minutes. Then slowly warm the reaction system to 0 ° C and stir for about two hours, and monitor by TLC until the raw material reacts completely. Add 15 mL of saturated NaHCO3 solution to the reaction system to quench the reaction, filter, and collect the residue. The residue is purified by flash column chromatography (CH2Cl2: MeOH = 20: 1) to obtain 480 mg of yellow solid EB-17 with a yield of 84%.

[0176]

[0177] Compound EB-17: yellow solid; 1 H NMR (400MHz, DMSO-d6) δ8.02(d,J=8.0Hz,1H),7.81(d,J=6.9Hz,1H),7.59(t,J=6.9Hz,1H) ,7.40(t,J=7.0Hz,1H),5.10(t,J=5.1Hz,1H),3.80(t,J=5.4Hz,2H),3.62(q,J=5.3Hz,2H).

[0178] Embodiment 18

[0179] This embodiment provides an ebselen derivative EB-18; its preparation method is basically the same as that of Example 1, except that: in step S1, aniline is replaced by 1-(2-aminoethyl)piperidine (CAS No.: 27578-60-5).

[0180] In step S2, compound EB-18 was obtained (yield: 18%).

[0181]

[0182] Compound EB-18: pale yellow solid; 1 H NMR (500MHz, DMSO-d6) δ8.00(d,J=7.9Hz,1H),7.80(d,J=7.7Hz,1H),7.56(t,J=8.1Hz,1H),7. 37(t,J=7.8Hz,1H),3.86–3.82(m,2H),2.55–2.46(m,6H),1.66(p,J=5.5Hz,4H),1.46(s,2H).

[0183] Embodiment 19

[0184] This embodiment provides an ebselen derivative EB-19; its preparation method is basically the same as that of Example 1, except that: in step S1, aniline is replaced by 1-(2-aminoethyl)morpholine (CAS No.: 2038-03-1).

[0185] In step S2, compound EB-19 was obtained (yield: 11%).

[0186]

[0187] Compound EB-19: yellow solid; 1H NMR (600MHz, DMSO-d6) δ8.00(d,J=7.9Hz,1H),7.81(d,J=8.3Hz,1H),7.57(t,J=8.2Hz, 1H),7.38(t,J=7.9Hz,1H),3.88–3.84(m,2H),3.71(t,J=4.6Hz,4H),2.59–2.51(m,6H).

[0188] Embodiment 20

[0189] This embodiment provides an ebselen derivative EB-20; its preparation method is basically the same as that of Example 1, except that: in step S1, aniline is replaced by 1-(2-aminoethyl)piperazine (CAS No.: 934-98-5).

[0190] In step S2, compound EB-20 was obtained (yield: 16%).

[0191]

[0192] Compound EB-20: reddish brown solid; 1 H NMR(500MHz,DMSO-d6)δ8.02(d,J=8.0Hz,1H),7.80(d,J=7.7Hz,1H),7.57(t,J=7.5 Hz,1H),7.37(t,J=7.4Hz,1H),3.89–3.80(m,2H),2.59–2.44(m,10H),2.22(s,3H).

[0193] Embodiment 21

[0194] This embodiment provides an ebselen derivative EB-21; its preparation method is basically the same as that of Example 1, except that: in step S1, aniline is replaced by glycine tert-butyl ester (CAS No.: 6456-74-2).

[0195] In step S2, compound EB-21 was obtained (yield: 25%).

[0196]

[0197] Compound EB-21: white solid; 1 H NMR (500MHz, DMSO-d6) δ8.05(d,J=8.0Hz,1H),7.82(d,J=7.6Hz,1H),7.62(t,J=7.5Hz,1H),7.42(t,J=7.4Hz,1H),4.40(s,2H),1.42(s,9H).

[0198] Embodiment 22

[0199] This embodiment provides an ebselen derivative EB-22; its preparation method is basically the same as that of Example 1, except that: in step S1, aniline is replaced by 2-aminoadamantane (CAS No.: 13074-39-0).

[0200] In step S2, compound EB-22 was obtained (yield: 31%).

[0201]

[0202] Compound EB-22: white solid; 1 H NMR (500MHz, DMSO-d6) δ8.01(d,J=7.9Hz,1H),7.72(d,J=7.6Hz,1H),7.57(t,J=7. 4Hz, 1H), 7.38 (t, J = 7.3Hz, 1H), 2.32 (s, 6H), 2.11 (s, 3H), 1.69 (q, J = 12.2Hz, 6H).

[0203] Embodiment 23

[0204] This embodiment provides an ebselen derivative EB-23; the preparation method thereof is as follows:

[0205] S1. Add EB-02 (30 mg, 0.10 mmol) and 2 mL CH2Cl2 to a 50 mL round-bottom flask, and then add p-toluene thiophenol (CAS No.: 106-45-6; 28 mg, 0.23 mmol). Stir the reaction system at room temperature for about two hours, monitor by TLC until the reaction of the raw materials is complete, concentrate under reduced pressure to remove CH2Cl2, and purify by flash column chromatography (pure CH2Cl2) to obtain 26 mg of white solid EB-23, with a yield of 61%.

[0206]

[0207] Compound EB-23: white solid; 1 H NMR (400MHz, DMSO-d6) δ10.49(s,1H),9.47(s,1H),8.18(d,J=50.5Hz,2H),7.65–7.55(m,1H),7.54– 7.46(m,2H),7.43(m,1H),7.40–7.30(m,2H),7.15–7.02(m,2H),6.79(d,J=6.4Hz,2H),2.23(s,3H).

[0208] Performance Testing

[0209] 1. PPK1 protein expression and purification:

[0210] Plasmid transformation: 1 μL of expression plasmid was added to 100 μL of E. coli.BL21 competent cells (thawed on ice), ice bathed for 30 mins, and then placed in a 42°C water bath for heat shock for 90 s. After heat shock, it was quickly transferred to ice and ice bathed for 2 mins. Then, 1 mL of LB liquid culture medium was added on the clean bench and activated at 37°C, 200 rpm for 1 h. After centrifugation at 6000 rpm for 1 min, the bacteria were collected, the supernatant was aspirated (leaving 200 μL), the bacteria were resuspended by blowing with a pipette tip, and the bacterial liquid was evenly applied to LB solid culture medium containing 100 mg / L kanamycin, and then cultured at 37°C for 12-16 h.

[0211] Protein expression and extraction: After the monoclonal colonies were grown, 6 monoclonal colonies were picked up with a small pipette tip and placed in 3 mL of LB medium containing kanamycin, and incubated at 37°C, 180 rpm for 16 h; 1 mL of bacterial solution was added to 500 mL of TB medium containing kanamycin, and incubated at 37°C, 180 rpm for 3 to 4 hours until OD 600 When the value is between 0.6 and 0.8, add 0.3-0.5 mM IPTG and incubate at 18°C, 180 rpm for 16 hours overnight; centrifuge the bacterial solution at 4000 rpm, 4°C for 30 minutes; remove the supernatant, resuspend the cell pellet in 25 mL Buffer, and incubate on ice for 45 minutes; heat shock the cells at 37°C for 10 minutes; centrifuge the bacterial solution at 18000 rpm for 30 minutes, remove the supernatant; resuspend the cell pellet in 25 mL Buffer, add 3 mL 1 M Na2CO3 solution and 1 M KCl solid to the lysate; stir at 4°C for 1 hour, and remove cell debris by centrifugation at 18000 rpm for 30 minutes.

[0212] Protein purification: dilute the supernatant with an equal volume of distilled water, and mix the liquid with the Ni column equilibrated with Buffer, and incubate the Ni column beads and the mixed solution at 4°C for 45 minutes; use 15mL of imidazole gradient elution with different concentrations (10mM, 50mM×2, 75mM, 100mM, 200mM, 300mM); collect samples of each elution fraction, and electrophoresed at 300V, 400mA for 45mins, and identify the fractions by SDS-Page development; collect the elution fractions containing the target protein, and dialyze with 1L of Buffer overnight; separate the elution fractions by FPLC, and store the protein in liquid nitrogen at -80°C.

[0213] 2. PPK2 protein expression and purification:

[0214] Transform the plasmid into E.coli.BL21 competent cells; after monoclonal colonies grow, pick up 6 monoclonal colonies with a small pipette tip and place them in 3mL LB medium containing kanamycin, incubate at 37℃, 180rpm for 16h; take 1mL of bacterial solution and add it to 500mL TB medium containing kanamycin, incubate at 37℃, 180rpm for 3 to 4 hours until OD 600 When the value is between 0.6 and 0.8, add 0.5 mM IPTG and incubate at 18°C, 180 rpm for 16 h overnight; centrifuge the bacterial solution at 4000 rpm, 4°C for 30 mins; remove the supernatant and resuspend the cell pellet in 25 mL of Buffer; add PMSF and lysozyme to the lysate, let it stand for 30 mins, then ultrasonicate for 25 mins to lyse the bacteria and release the protein; centrifuge the mixture at 18000 rpm for 30 mins, take the supernatant and remove the cell debris.

[0215] 3. In vitro PPK inhibitor activity test:

[0216] Experiments were performed using equimolar amounts of PPK1 or PPK2 enzyme (275 nM) to ensure that comparisons between different enzymes were valid. For the polyphosphate synthesis activity of PPK1 and PPK2B, specific reaction buffers and conditions were used. In kinetic experiments, the concentration of ATP was varied to assess its effect on enzyme activity. The reaction mixture was incubated at 37°C for 30 min, and then a 50 μl sample was removed and added to a solution containing acid fuchsin to stop the reaction and quantify polyphosphate. The amount of polyphosphate was recorded using a specific absorbance ratio (A630 / 530 nm) and calibrated using a standard curve. The data curves were fitted using GraphPad Prism software to determine the IC 50 value.

[0217] The experimental results are shown in Table 1. It can be seen from Table 1 that the ebselen and its derivatives prepared by the scheme of the present invention have an inhibitory effect on the PPK enzyme of Pseudomonas aeruginosa.

[0218] Table 1

[0219]

[0220]

[0221] IC values ​​of EB-1 for PPK1 and PPK2 50 Inhibition curve diagram; and PPK1 enzymatic rate curve in the presence of ebselen Figure 1 ,in, Figure 1 a is the IC of ebselen (EB-1) against PPK1 and PPK2 50Inhibition curve; IC of ebselen against PPK1 50 The value was 36.74±1.79; IC 50 The value is 11.35±1.12; Figure 1 b is the Michaelis-Menten enzymatic reaction kinetic curve of ibselen to PPK1; the results show that the Michaelis constant Km of ibselen to substrate ATP in the presence of PPK1 remains basically unchanged, while the maximum reaction rate constant Ki is reduced, indicating that ibselen may inhibit PPK1 enzyme activity in a non-competitive inhibition manner.

[0222] 4. Inhibition experiment of Pseudomonas aeruginosa PolyP production:

[0223] Culture bacteria in 5mL LB medium containing inhibitors or DMSO at 37℃, 180rpm overnight. Centrifuge the cultured bacterial solution at 3900rpm for 10mins to precipitate the cells; resuspend the cell pellet in 1mL MOPS medium and add the same concentration of inhibitors or DMSO as the initial culture; incubate at 37℃, 180rpm for 2h. Centrifuge the pellet again, resuspend it in 250μL GITC lysis buffer, and then heat shock at 95℃ for 5mins; add 15μL of 10% SDS and 0.5mL of 95% ethanol to the tube in turn, mix well; transfer to a silica gel column, centrifuge at 10000rpm for 1min, and elute with 50μL distilled water.

[0224] The experimental results are as follows Figure 3 As shown, Figure 3 a is the concentration-dependent inhibition of ebselen on the production of PolyP by Pseudomonas aeruginosa; Figure 3 b is the inhibition of PolyP production by 100 μM ebselen on WT, PPK1 knockout, PPK2 knockout, and PPK1 and PPK2 double knockout Pseudomonas aeruginosa. The results showed that the production of PolyP in Pseudomonas aeruginosa decreased with the increase of ebselen concentration, indicating that ebselen can inhibit the production of PolyP in Pseudomonas aeruginosa.

[0225] 5. Biofilm formation experiment:

[0226] A small amount of Pseudomonas aeruginosa seed culture was picked up and put into 3mL LB medium, and incubated overnight at 37°C and 180rpm for 16 hours; 30μL of bacterial solution was taken and added into 3mL M63 medium, and incubated overnight at 37°C and 180rpm for 16 hours; PA strain was inoculated into a 96-well tissue culture plate, 100μL of bacterial solution was added to each well, and the plate was incubated at 37°C for 16 hours to allow the biofilm to form slowly; after the biofilm was formed, the plate was washed twice with sterile water to remove unattached bacteria and culture medium residues; the biofilm was stained with 0.1% crystal violet solution; after staining, the plate was washed twice with sterile water again to remove unbound dye; the stained biofilm ring was dissolved with 30% acetic acid for subsequent absorbance measurement, and the absorbance of the dissolved dye was recorded at a wavelength of 570nm, and the absorbance was used to reflect the formation of the biofilm.

[0227] The experimental results are as follows Figure 4 As shown, Figure 4 A is the inhibition of biofilm formation by ebselen on wild-type, PPK1 knockout, PPK2 knockout, and PPK1 and PPK2 double knockout types of Pseudomonas aeruginosa; Figure 4 B shows the time gradient treatment of ebselen for WT and PPK1 and PPK2 double knockout Pseudomonas aeruginosa; Figure 4 C is the biofilm formation of Pseudomonas aeruginosa treated with ebselen according to the concentration gradient for 16 hours. The results show that ebselen inhibits the biofilm formation of Pseudomonas aeruginosa in a concentration-dependent manner.

[0228] 6. Bacterial motility and invasiveness test

[0229] A semi-solid culture medium was prepared by mixing 0.3% Bacto agar and 2.5% LB culture medium, and 100 mM inhibitor or an equal volume of DMSO was added to the agar as a control; before pouring the plate, the agar solution containing the compound or DMSO was poured into the culture dish, and a pipette tip dipped in an overnight LB culture was used to perform a point inoculation in the center of the culture plate, and the inoculated culture dish was placed in a sealed container and incubated at 30°C for 18 hours; after the incubation, the bacterial movement diameter in the culture dish was measured, and the movement diameter of the bacteria on the agar surface can reflect its movement ability.

[0230] The experimental results are as follows Figure 5 As shown, Figure 5 A is the inhibition of motility of wild-type Pseudomonas aeruginosa, PPK1 knockout type, PPK2 knockout type, and PPK1 and PPK2 double knockout Pseudomonas aeruginosa by ebselen; Figure 5 B is the measurement of the movement radius of wild-type and PPK1 and PPK2 double knockout Pseudomonas aeruginosa after ebselen treatment; Figure 5C is the growth curve of Pseudomonas aeruginosa under the treatment of ebselen or DMSO. The results show that ebselen can significantly reduce the movement radius of Pseudomonas aeruginosa, indicating that ebselen can reduce the movement and invasiveness of Pseudomonas aeruginosa.

[0231] 7. Pseudomonas aeruginosa toxin quantitative experiment:

[0232] Pseudomonas aeruginosa was cultured in 50 mL of 2.5% LB medium at 37°C and 180 rpm for 18 hours. The culture medium was centrifuged at 18,000 rpm for 30 mins, the precipitate was removed and the supernatant was retained. The supernatant after centrifugation was filtered through a filter membrane; the absorbance of the supernatant was measured at 403 nm using a spectrophotometer to quantify Pyoverdine. Pyocyanin was first extracted with 3 mL of chloroform and then treated with 1 mL of hydrochloric acid to obtain a red product. The absorbance of the red product was measured at 520 nm to quantify Pyocyanin. In order to eliminate any possible color interference, the cell-free blank control reading was subtracted from the absorbance reading of each sample.

[0233] The experimental results are as follows Figure 6 As shown, Figure 6 A is the effect of ebselen on the production of Pyoverdine by Pseudomonas aeruginosa; Figure 6 B is the effect of ebselen on the production of Pyocyanin by Pseudomonas aeruginosa. The results show that ebselen can reduce the production of Pyoverdine and Pyocyanin toxins by Agrobacterium aeruginosa.

[0234] 8. Pseudomonas aeruginosa antibiotic resistance experiment:

[0235] Pick a small amount of Pseudomonas aeruginosa seed stock into 3 mL LB medium and culture it at 37°C, 180 rpm overnight. Dilute the overnight culture of Pseudomonas aeruginosa strain into LB medium at a ratio of 1:100. When the OD of the culture reaches 600 When the p-value reached about 0.6, 100 μM ebselen and 7.2 μM antibiotics were added to the culture medium, and the bacteria were cultured on a shaker at 180 rpm at 37°C; after 3, 6, 9, or 12 hours of incubation, the bacteria were collected. The culture medium was centrifuged at 12,000 rpm for 2 mins, the supernatant was discarded, and then washed with phosphate-buffered saline (PBS) to remove non-bacterial components in the culture medium, and the number of residual bacteria was determined by plate counting.

[0236] The experimental results are as follows Figure 7 As shown, Figure 7 A is the effect of combined use of ibucillin and tetracycline on the growth of Pseudomonas aeruginosa; Figure 7 B is the significance analysis of the time-killing curve; Figure 7C is the time-kill curve of colony growth. The results show that ebselen can enhance the inhibitory effect of tetracycline antibiotics on the growth of Pseudomonas aeruginosa.

[0237] 9. CD binding experiment:

[0238] The sample protein is completely dissolved in the solvent to form a uniform and transparent solution. The nitrogen flow rate is controlled to reduce light scattering and oxidation of the solution. The buffer and solvent should be checked separately before preparing the solution to ensure that they have no absorption interference within the measurement wavelength range. The CD signal of the buffer without the sample is measured to facilitate the subsequent subtraction of the background signal from the CD signal of the sample. The CD signal of the sample is measured in the range of about 190-250nm, and the background signal is subtracted from the CD signal of the sample to obtain a net CD spectrum.

[0239] The experimental results are as follows Figure 2 As shown, the results showed that the CD values ​​of ebselen and Pseudomonas aeruginosa PPK1 and PPK2B changed significantly before and after binding, indicating that ebselen can strongly bind to both PPK1 and PPK2B.

[0240] The above is a detailed description of the embodiments of the present invention, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge scope of ordinary technicians in the relevant technical field without departing from the purpose of the present invention.

Claims

1. An ebselen derivative having a structure described in formula (I) or formula (II), or a pharmaceutically acceptable salt thereof: in, R1 is selected from H, C 1～6 alkyl or halogen; R2 is selected from pyridyl, adamantyl, substituted or unsubstituted phenyl, wherein the substituted group includes furanyl, thienyl, C 1～6 substituted or unsubstituted C 3-12 The saturated heterocyclic group, 1～3 The alkyl and C 1～3 Alkoxy substituted benzyl or C 2～6 Ester-substituted C 1～6 The alkyl group; and when R1 is selected from H, R2 is not phenyl; R3 is selected from phenyl substituted with hydroxyl; R4 is selected from C 1～6 an alkyl-substituted phenyl group; R5 is selected from H or C 1～6 The alkyl group; R6 is selected from C 1～7 alkyl or halogenated alkyl substituted benzyl; or R6 and the adjacent N atom form a 6-membered heterocyclic ring; R7 is selected from tert-butyloxycarbonyl.

2. The ebselen derivative according to claim 1, characterized in that R1 is selected from H or halogen.

3. The ebselen derivative according to claim 1 or 2, characterized in that R2 is selected from pyridyl, adamantyl, substituted or unsubstituted phenyl, wherein the substituted group includes furanyl, thienyl, C 1～3 Alkoxy, hydroxyl, substituted or unsubstituted C containing 1 to 3 heteroatoms selected from N and O 3-8 The unsaturated heterocyclic group, 1～3 The alkyl and C 1～3 Alkoxy substituted benzyl or C 2～6 The ester-substituted C 1～3 of alkyl.

4. The ebselen derivative according to claim 1 or 2, characterized in that Selected from the following structural formulas:

5. A pharmaceutical composition, characterized in that The invention comprises the ebselen derivative according to any one of claims 1 to 4; and a pharmaceutically acceptable excipient.

6. The pharmaceutical composition according to claim 5, characterized in that The pharmaceutical composition also includes an antibiotic.

7. The pharmaceutical composition according to claim 6, characterized in that The antibiotic is selected from at least one of tetracycline, amoxicillin, cefixime, vancomycin and norfloxacin.

8. Use of ebselen or an ebselen derivative or the pharmaceutical composition according to claims 5 to 7 in the preparation of a polyphosphate kinase inhibitor; The ebselen derivative has a structure described by formula (I) or formula (II): in, R1 is selected from H, C 1～6 Alkoxy, C 1～6 alkyl or halogen; R2 is selected from pyridyl, adamantyl, substituted or unsubstituted phenyl, wherein the substituted groups include hydroxyl, furanyl, thienyl, C 1～6 substituted or unsubstituted C 3-12 The saturated heterocyclic group, 1～3 The alkyl and C 1～3 Alkoxy substituted benzyl or C 2～6 Ester-substituted C 1～6 and when R1 is selected from H, R2 is not phenyl; R3 is selected from phenyl substituted with hydroxyl; R4 is selected from C 1～6 Alkyl-substituted phenyl; R5 is selected from H or C 1～6 The alkyl group; R6 is selected from C 1～7 alkyl or halogenated alkyl substituted benzyl; or R6 and the adjacent N atom form a 6-membered heterocyclic ring; R7 is selected from tert-butyloxycarbonyl.

9. Use of ebselen or an ebselen derivative or the pharmaceutical composition according to claims 5 to 7 in the preparation of a drug for treating and / or preventing bacterial infection caused by polyphosphate kinase; The ebselen derivative has a structure described by formula (I) or formula (II): in, R1 is selected from H, C 1～6 Alkoxy, C 1～6 alkyl or halogen; R2 is selected from pyridyl, adamantyl, substituted or unsubstituted phenyl, wherein the substituted groups include hydroxyl, furanyl, thienyl, C 1～6 substituted or unsubstituted C 3-12 The saturated heterocyclic group, 1～3 The alkyl and C 1～3 Alkoxy substituted benzyl or C 2～6 The ester-substituted C 1～6 and when R1 is selected from H, R2 is not phenyl; R3 is selected from phenyl substituted with hydroxyl; R4 is selected from C 1～6 Alkyl-substituted phenyl; R5 is selected from H or C 1～6 The alkyl group; R6 is selected from C 1～7 alkyl or halogenated alkyl substituted benzyl; or R6 and the adjacent N atom form a 6-membered heterocyclic ring; R7 is selected from tert-butyloxycarbonyl.

10. The use according to claim 9, characterized in that: The bacteria include Pseudomonas aeruginosa, Klebsiella pneumoniae, Salmonella, Bacillus subtilis, Acinetobacter baumannii, Enterobacter cloacae or Enterococcus.

Images