An ebselen derivative, pharmaceutical composition and use thereof

Abstract

The application discloses an ebselen derivative, a pharmaceutical composition and application thereof. The ebselen derivative has a structure as shown in formula (I) or formula (II). The ebselen derivative provided by the application has the characteristics of inhibiting PPK activity, reducing the polyP level of bacteria, reducing the formation of a biofilm, reducing the movement ability of bacteria, reducing the generation of a virulence factor, and enhancing the sensitivity of bacteria to antibiotics, and thus can be used for preventing and treating bacterial infectious diseases.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of pharmaceutical chemistry, and particularly relates to an ebselen derivative, a pharmaceutical composition and application thereof. BACKGROUND

[0002] Pseudomonas aeruginosa is a gram-negative bacterium and a conditional pathogen. It is listed as an urgent priority pathogen for antibiotics by the World Health Organization. The pathogen widely exists in nature and is easy to infect patients with impaired immune systems such as cystic fibrosis, burns, immunodeficiency, chronic obstructive pulmonary disease, cancer and severe respiratory tract infection, which can cause very serious and even fatal tissue damage and inflammation. Pseudomonas aeruginosa can grow and reproduce in large quantities on wet surfaces, so it widely exists on medical equipment and is the most common one among hospital-acquired infections and drug-resistant pathogens. Epidemiological studies show that nearly 700,000 people die from antibiotic-resistant bacterial infections every year, 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] P. aeruginosa is known for its remarkable adaptability, using a variety of ways to survive within the host, such as through flagella motility, biofilm formation, and release of pathogenic factors. At the early stage of infection, P. aeruginosa uses polar flagella to navigate towards the host surface and adhere through the action of adhesins, initiating the process of colonization. Subsequently, these bacteria can form biofilms - complex bacterial communities encased in extracellular matrix - often associated with persistent P. aeruginosa infection and resistance to antibiotics. Both free-floating (planktonic) and biofilm-associated P. aeruginosa produce a range of pathogenic factors, among which pyocyanin and pyoverdine are particularly notable. Pyoverdine, the major siderophore of P. aeruginosa, functions to bind and transport iron into the bacterial cell and is also responsible for the green color of P. aeruginosa culture broth. On the other hand, pyoverdine is a blue-colored redox-active phenazine, whose toxicity is primarily through interaction with molecular oxygen, leading to the production of superoxide anion, which can damage host tissues. Pyoverdine production has been shown to be associated with increased biofilm formation, and in turn, biofilm formation has been shown to enhance pyocyanin production, an interaction that embodies the interconnection of various pathogenic features exhibited by P. aeruginosa. Targeting these pathogenic features during the course of P. aeruginosa infection is currently the most effective strategy to combat P. aeruginosa infection, as this strategy suppresses the pathogenicity of P. aeruginosa without causing direct death of the bacteria, which avoids the development of bacterial resistance; in addition, many antibiotics are not specific to pathogens and thus can have adverse effects on symbiotic microbial communities. This can lead to reinfection or increased susceptibility to other infectious pathogens. However, treatment strategies that target pathogenic features can avoid these harmful consequences.

[0004] Polyphosphate (polyP) is a linear polymer of many phosphate groups linked by phosphoanhydride bonds, which has multiple biological functions in bacteria, including energy storage, metabolic regulation, stress response, biofilm formation, and pathogenic factor expression. In P. aeruginosa, polyphosphate influences pathogenicity in various ways, particularly playing an important role in biofilm formation and also being involved in the expression of pathogenic factors such as pyoverdine and pyocyanin. In addition, polyphosphate enhances bacterial resistance to antibiotics, enhancing the survivability of bacteria under antibiotic pressure. In other pathogenic bacteria, polyphosphate is also closely related to pathogenic mechanisms and antibiotic sensitivity. Therefore, targeting the homeostasis of polyphosphate has become a potential anti-pathogenic strategy for P. aeruginosa and other pathogenic bacteria.

[0005] Polyphosphates are synthesized by polyphosphate kinases (PPKs), which are divided into two distinct classes: PPK1 and PPK2. PPK1s primarily catalyze the synthesis of polyphosphates by transferring a γ-phosphate group from ATP, whereas PPK2 enzymes preferentially consume polyphosphates to phosphorylate nucleosides. However, PPK2s can also synthesize polyphosphates, albeit with relatively weak activity. PPK1 has been identified as a key factor in P. aeruginosa pathogenicity, and studies have shown that the deletion of 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 of P. aeruginosa. In general, the roles of PPK1 and PPK2 in the pathogenicity and survival of other various 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 the discovery of new antibacterial drugs.

[0006] Therefore, it is necessary to develop a new compound for inhibiting polyphosphate kinase. SUMMARY

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

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

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

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

[0011] The ebselen derivative provided by the first aspect of the present application has the structure of formula (I) or formula (II), or a pharmaceutically acceptable salt thereof:

[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 substituted or unsubstituted phenyl, wherein the substituted group includes 1～6Alkyl, hydroxyl, or substituted or unsubstituted C atoms containing 1-3 heteroatoms selected from N, O, and S. 3-12 saturated heterocyclic groups, C 1～3 alkyl and C 1～3 alkoxy-substituted benzyl or C 2～6 ester-substituted C 1～6 Alkyl groups;

[0015] Furthermore, when R1 is selected from H, R2 is not a phenyl group;

[0016] R3 is selected from hydroxylated phenyl groups;

[0017] R4 is selected from C 1～6 Alkyl-substituted phenyl;

[0018] R5 is selected from H or C. 1～6 Alkyl groups;

[0019] R6 is selected from C 1～7 alkyl or haloalkane-substituted benzyl groups; or R6 forming a 6-membered heterocycle with an adjacent N atom;

[0020] R7 is selected from tert-butyloxycarbonyl.

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

[0022] The ibuselenline derivative provided by this invention has the characteristic of inhibiting PPK activity, reducing the level of bacterial polyphosphate (polyP), reducing biofilm formation, reducing bacterial motility, reducing the production of virulence factors, and also enhancing the sensitivity of bacteria to antibiotics, thereby being used for the prevention and treatment of bacterial infectious 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 groups, wherein the substituted groups include Furanyl, thiophene, C 1～3 Alkyl, hydroxyl, or substituted or unsubstituted C atoms containing 1-3 heteroatoms selected from N or O. 3-8 Unsaturated heterocyclic groups, C 1～3 alkyl and C 1～3 alkoxy-substituted benzyl or C 2～6 ester-substituted C 1～3 Alkyl groups.

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

[0026]

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

[0028] According to a preferred embodiment of the present invention, the pharmaceutical composition further includes 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] The use of an ibuselenoid or an ibuselenoid derivative, or a pharmaceutical composition according to the second aspect of the present invention, in the preparation of a polyphosphate kinase inhibitor;

[0031] The ibuselenline derivative has the structure described in formula (I) or formula (II):

[0032]

[0033] Among them, R1 is selected from H and C. 1～6 alkoxy, C 1～6 Alkyl or halogen;

[0034] R2 is selected from pyridyl, adamantyl, substituted or unsubstituted phenyl groups, wherein the substituted groups include hydroxyl, Furanyl, thiophene, C 1～6 Alkyl, hydroxyl, or substituted or unsubstituted C atoms containing 1-3 heteroatoms selected from N, O, and S. 3-12 Unsaturated heterocyclic groups, C 1～3 alkyl and C 1～3 alkoxy-substituted benzyl or C 2～6 ester-substituted C 1～6 Alkyl groups; and when R1 is selected from H, R2 is not a phenyl group;

[0035] R3 is selected from hydroxylated phenyl groups;

[0036] R4 is selected from C 1～6 Alkyl-substituted phenyl;

[0037] R5 is selected from H or C. 1～6 Alkyl groups;

[0038] R6 is selected from C 1～7 alkyl or haloalkane-substituted benzyl groups; or R6 forming a 6-membered heterocycle with an adjacent N atom;

[0039] R7 is selected from tert-butyloxycarbonyl.

[0040] According to a preferred embodiment of the present application, the polyphosphatase kinase comprises PPK1 and PPK2.

[0041] According to a preferred embodiment of the present application, the ebselen derivative has a structure as described in 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 comprises furyl, thienyl, C 1～6 alkoxy, hydroxyl, substituted or unsubstituted C 3-12 saturated heterocyclic group containing 1-3 heteroatoms selected from N, O and S, benzyl substituted with C 1～3 alkyl and C 1～3 alkoxy, or C 2～6 ester group substituted C 1～6 alkyl;

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

[0046] R3 is selected from hydroxyl substituted phenyl;

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

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

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

[0050] R7 is selected from tert-butyloxycarbonyl.

[0051] According to a fourth aspect of the present application, there is provided use of ebselen or an ebselen derivative or the pharmaceutical composition according to the second aspect of the present application in the manufacture of a medicament for the treatment and / or prevention of bacterial infection caused by polyphosphate kinase.

[0052] The ebselen derivative has a structure as described in Formula (I) or Formula (II):

[0053]

[0054] wherein R1 is selected from H, C 1～6 alkoxy, C 1～6alkyl or halo;

[0055] R2 is selected from pyridyl, adamantyl, substituted or unsubstituted phenyl, wherein the substituted group includes hydroxy, furyl, thienyl, C 1～6 alkoxy, hydroxy, substituted or unsubstituted C 3-12 unsaturated heterocyclyl, benzyl substituted with C 1～3 alkyl and C 1～3 alkoxy; and when R1 is selected from H, R2 is not phenyl; 2～6 ester; and when R1 is selected from H, R2 is not phenyl; 1～6 alkyl; and when R1 is selected from H, R2 is not phenyl;

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

[0057] R4 is selected from phenyl substituted with C 1～6 alkyl;

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

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

[0060] R7 is selected from tert-butyloxycarbonyl.

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

[0062] According to a preferred embodiment of the present application, the bacteria include P. aeruginosa, K. pneumoniae, Salmonella, B. subtilis, A. baumannii, E. cloacae or Enterococcus.

[0063] Definitions and general terms

[0064] "C 1-6 alkyl" means an alkyl group having a total number of carbon atoms of 1-6, including linear C 1-6 alkyl, branched C 1-6 alkyl and cyclic C 3-6 alkyl, for example, can be a linear alkyl group having a total number of carbon atoms of 1, 2, 3, 4, 5 or 6, a branched alkyl group having a total number of carbon atoms of 1, 2, 3, 4, 5 or 6, or a cyclic alkyl group having a total number of carbon atoms of 3, 4, 5 or 6, for example, can be a methyl group, an ethyl group, an n-propyl group, an iso-propyl group, an n-butyl group, an iso-butyl group, a tert-butyl group, an n-pentyl group, an iso-pentyl group, an n-hexyl group, a cyclopropyl group, a methylcyclopropyl group, an ethylcyclopropyl group, a cyclopentyl group, a methylcyclopentyl group, a cyclohexyl group, etc.1-7 Alkyl group, C 1-3 "alkyl" has a similar interpretation, except that the number of carbon atoms is different.

[0065] “C 1-6 "alkoxy group" refers to an alkoxy group with a total number of 1-6 carbon atoms, including C64 and C64. 1-6 straight-chain alkoxy, C 1-6 Branched alkoxy groups and C 2-6 The cycloalkoxy group can be, for example, a straight-chain alkoxy group with a total of 1, 2, 3, 4, 5, or 6 carbon atoms; a branched-chain alkoxy group with a total of 1, 2, 3, 4, 5, or 6 carbon atoms; or a cycloalkoxy group with a total of 2, 3, 4, 5, or 6 carbon atoms, such as methoxy, ethoxy, n-propoxy, isopropoxy, etc. Regarding "C 1-3 The "alkoxy group" has a similar explanation, except that the number of carbon atoms is different.

[0066] "C containing 1-3 heteroatoms selected from N, O, and S, either substituted or unsubstituted" 3-12 "Saturated heterocyclic group" refers to a heterocyclic group with a total number of carbon atoms of 3-12, wherein the cyclic atoms in the heterocycle contain heteroatoms, and the number of heteroatoms is 1-3, and the heteroatoms are selected from at least one of N, O and S, the heterocycle is a saturated group, and optionally at least one H in the group is converted to carbon by C. 1～6 Alkyl groups are substituted.

[0067] “C 2～6 The ester group represents the structural formula as follows: The ester group, wherein C 2～6 The ester group is represented by an ester group with a total number of carbon atoms of 2 to 6. Representative examples include methyl formate, ethyl formate, ethyl acetate, methyl acetate, etc.

[0068] Pharmaceutically acceptable excipients of the present application include any solvent, solid excipient, diluent, binding agent, disintegrant, or other liquid excipient, dispersing agent, flavoring agent or suspending agent, surface active agent, isotonic agent, thickening agent, emulsifying agent, preservative, solid binder, glidant or lubricant, and the like, suitable for the particular target dosage form. As described in the following references: In Remington: The Science and Practice of Pharmacy, 21st edition, 2005, ed. D. B. Troy, Lippincott Williams & Wilkins, Philadelphia, and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York, the contents of which are incorporated herein, indicate the different excipients that can be applied to the formulation of pharmaceutically acceptable compositions and their known methods of preparation. The use of any conventional excipient is contemplated to be within the scope of the present application, except to the extent that any conventional excipient is incompatible with the compounds of the present application, for example, produces any adverse biological effect or interacts in a deleterious manner with any other component(s) of the pharmaceutically acceptable composition.

[0069] Substances which can serve as pharmaceutically-acceptable excipients include, but are not limited to, ion exchangers, alumina, 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 silica, magnesium trisilicate, polyvinyl pyrrolidone, polyacrylates, waxes, polyethylene-polyoxypropylene- block polymers, wool fat, sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; powdered tragacanth; 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; glycols such as propylene glycol and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer solutions; and other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate; coloring agents; releasing agents; coating agents; sweetening agents; flavoring agents; perfuming agents; preservatives; and antioxidants.

[0070] Pharmaceutically acceptable salts of the present application include salts with inorganic acids, organic acids, alkali metals, alkaline earth metals and basic amino acids. These include, but are not limited to: hydrochlorides, sulfates, bisulfates, nitrates, hydrobromides, hydroiodides, carbonates, bicarbonates, sulfites, bisulfites, pyrosulfates, monohydrogen phosphates, dihydrogen phosphates, perchlorates, persulfates, hemisulfates, bisulfates, thiocyanates, phosphates, pyrophosphates, metaphosphates; suitable organic acid salts include, but are not limited to: formates, acetates, propionates, butyrates, benzoates, malonates, succinates, pyruvates, mesylates, esylates, propylsulfonates, citrates, 4-nitrobenzoates, phenylsulfonates, p-toluenesulfonates, malates, propiolates, 2-butynoates, 2-hydroxy-ethanesulfonates, vinylacetates, tartrates, L-tartrates, fumarates, isethionates, maleates, lactates, lactobionates, pamoates, salicylates, galactarates, glucoheptanoates, mandelates, 1,2-ethanedisulfonates, 2-naphthalenesulfonates, oxalates, trifluoroacetates, trifluoromethanesulfonates, adipates, suberates, sebacates, butyn-1,4-dioates, hexyn-1,6-dioates, glycolates, alginates, ascorbates, isoascorbates, aspartates, L-aspartates, glutarates, L-glutarates, 2-phenoxybenzoates, 2-(4-hydroxybenzoyl)benzoates, acetoacetates, 2-hydroxyethanesulfonates, benzenesulfonates, borates, chlorobenzoates, camphorates, itaconates, camphorsulfonates, levocamphorsulfonates, methylbenzoates, dinitrobenzoates, sulfamates, lactobionates, galacturonates, cyclopentanepropionates, dodecylsulfates, acrylates, cyclopentanepropionates, glycerophosphates, methoxybenzoates, digluconates, gluconates, heptanoates, hexanoates, 2-hydroxy-ethanesulfonates, trimethylacetates, glucuronates, laurates, phthalates, phenylacetates, lauryl sulfates, 2-acetoxybenzoates, nicotinates, cinnamates, oleates, palmitates, pamoates, pectates, phthalates, glutarates, hydroxymaleates, hydroxybenzoates, phenylacetates, 3-hydroxy-2-naphthoates, 3-phenylpropionates, isobutyrates, neopentanoates, picrates, stearates, 2,2-dichloroacetates, acylated amino acid salts, alginates, 4-acetamidobenzoates, cucurbitates, cholate, octanoates, nonanoates, cyclamic acids, pthalates, hemisulfates, sorbates, pamoates, mucates, glycine hydrochlorides, naphthalene disulfonates, xylene sulfonates, cysteine hydrochlorides, undecanoates, polyvinylsulfonates, sulfosalicylates, phenylbutyrates, 4-hydroxybutyrates, polyvinylsulfates, naphthalene-1-sulfonates, naphthalene-2-sulfonates, and valerates.

[0071] Other features and advantages of the invention will be set forth in the description which follows, and will be apparent in part from the description, or may be learned by practicing the invention. Attached Figure Description

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

[0073] Figure 1 The IC50 of ebuselenline in Example 1 of this invention for PPK1 and PPK2 is... 50 Inhibition curve and enzymatic rate curve of PPK1 in the presence of ebuselenline. Three independent replicate experiments (n=3), *P<0.05, **P<0.01, ***P<0.001.

[0074] Figure 2 This is a CD binding analysis diagram between PPK1, PPK2, and ebuselenline from Example 1 of this invention.

[0075] Figure 3 This is a graph showing the inhibition of PolyP generation by ebuselenine in Example 1 of the present invention.

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

[0077] Figure 5 This is a graph showing the inhibitory effect of ebuselenine (from Example 1 of this invention) on the motility of Pseudomonas aeruginosa.

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

[0079] Figure 7 This is a graph showing the effect of the combined use of ebuselenol and antibiotics on the growth of Pseudomonas aeruginosa in Example 1 of this invention. Detailed Implementation

[0080] The following are specific embodiments of the present invention, and the technical solutions of the present invention will be 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 this invention are all conventional reagents, methods and equipment in this technical field.

[0082] Example 1

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

[0084]

[0085] S1, 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) were taken in a 250 mL round bottom flask, CH2Cl2(30 mL) was added, aniline (CAS No: 62-53-3; 357 mg, 3.84 mmol) was added slowly at 0 °C, then the reaction mixture was allowed to stir at room temperature (about 25 °C) for about 3 h, after completion of the reaction, as monitored by TLC. The reaction was quenched by adding 80 mL of water, then extracted with CH2Cl2three times (40 mL x 3), washed with saturated NaHCO3solution (40 mL x 3) and HCl solution (1 M, 40 mL x 3) three times each and the organic layer was combined, dried over anhydrous Na2SO4, concentrated under reduced pressure to remove CH2Cl2, to get the crude product, which was used as such for the next step without further purification.

[0086] S2, to the crude product (1.00 g, 3.09 mmol) obtained from step S1, CuI (177 mg, 0.93 mmol), o-phenanthroline (167 mg, 0.93 mmol), selenium powder (293 mg, 3.71 mmol) and K2CO3(640 mg, 4.64 mmol) were taken in a 250 mL round bottom flask, DMF (20 mL) was added, the reaction mixture was stirred at 120 °C for 6 h under nitrogen atmosphere, after completion of the reaction, as monitored by TLC. The reaction mixture was filtered, the filtrate was added with 200 mL of water, then extracted with CH2Cl2three times (50 mL x 3) and washed with saturated brine solution three times (150 mL x 3) and the organic layer was combined, dried over anhydrous Na2SO4, concentrated under reduced pressure to remove CH2Cl2, purified by flash column chromatography (pure CH2Cl2) to get 340 mg of compound EB-01 as a light yellow solid in 40% yield.

[0087] NMR data of compound EB-01: 1 H-NMR (600 MHz, DMSO-d6) δ 8.10 (d, J = 8.0 Hz, 1H), 7.92 (d, J = 8.6 Hz, 1H), 7.69 (t, J = 8.3 Hz, 1H), 7.65 (d, J = 7.5 Hz, 2H), 7.51 - 7.47 (m, 1H), 7.47 - 7.44 (m, 2H), 7.27 (t, J = 7.4 Hz, 1H).

[0088] Example 2

[0089] This example provides a ebselen derivative EB-2, the preparation method of which 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] Compound EB-2 is prepared in step S2 (yield: 40%).

[0091]

[0092] Compound EB-02: yellow solid; 1 H NMR (400 MHz, DMSO-d6) δ 7.96 (d, J = 8.8 Hz, 1H), 7.64 (d, J = 7.5 Hz, 2H), 7.49 - 7.42 (m, 2H), 7.41 (d, J = 2.7 Hz, 1H), 7.33 (dd, J = 8.8, 2.8 Hz, 1H), 7.27 (t, J = 7.4 Hz, 1H), 3.86 (s, 3H).

[0093] Example 3

[0094] This example provides a ebselen derivative EB-3, the preparation method of which 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] Compound EB-3 is prepared in step S2 (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 example provides a ebselen derivative EB-4, the preparation method of which 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] Compound 2b is prepared in step S2 (yield: 35%).

[0101]

[0102] S3, Into a 100 mL round-bottom flask was added 2b (380 mg, 1.26 mmol), 10 mL of super dry CH2Cl2, and moved into a -78 °C cold bath for stirring for 10 min. BBr3(520 mg, 2.52 mmol) was added dropwise under nitrogen protection at -78 °C. The dropwise addition was completed in 30 min. The reaction system was then slowly warmed to 0 °C and stirred for about two hours. TLC monitoring showed that the reaction was complete. 15 mL of saturated NaHCO3solution was added to quench the reaction, filtered, and the filtrate was collected. The filtrate was 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 (400 MHz, DMSO-d6) δ 9.61 (s, 1H), 8.08 (d, J = 8.0 Hz, 1H), 7.87 (d, J = 7.3 Hz, 1H), 7.66 (t, J = 7.1 Hz, 1H), 7.46 (t, J = 7.4 Hz, 1H), 7.35 (d, J = 8.7 Hz, 2H), 6.82 (d, J = 8.7 Hz, 2H).

[0105] Example 5

[0106] This example provides a ebselen derivative EB-6, and a preparation method thereof as follows:

[0107] S1, Into a 50 mL round-bottom flask was added EB-04 (87 mg, 0.30 mmol), 2 mL of DMF, followed by the addition of ethyl bromoacetate (CAS No: 105-36-2; 80 mg, 0.48 mmol) and K2CO3(64 mg, 0.46 mmol). The reaction system was stirred at room temperature for about two hours. TLC monitoring showed that the reaction was complete. 20 mL of water was added to the reaction system, followed by the addition of CH2Cl2for extraction three times (15 mL x 3), and then washed with saturated brine three times (40 mL x 3) and combined organic phase. The organic layer was dried with anhydrous Na2SO4, concentrated under reduced pressure to remove CH2Cl2, and purified 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 (400 MHz, DMSO-d6) δ 8.08 (d, J = 8.0 Hz, 1H), 7.92 - 7.87 (m, 1H), 7.67 (td, J = 8.3, 7.8, 1.4 Hz, 1H), 7.51 (d, J = 9.0 Hz, 2H), 7.48 (t, J = 7.0 Hz, 1H), 7.01 (d, J = 9.0 Hz, 2H), 4.82 (s, 2H), 4.19 (q, J = 7.1 Hz, 2H), 1.23 (t, J = 7.1 Hz, 3H).

[0110] Example 6

[0111] This example provides a ebselen derivative EB-5, the preparation method of which is as follows:

[0112] S1, 50 mL round-bottom flask was added EB-06 (65 mg, 0.17 mmol), 10 mL CH2Cl2, followed by the addition of triethylamine (87 mg, 0.85 mmol). Lithium hydroxide (41 mg, 1.7 mmol) was slowly added to the reaction system under ice bath, and the reaction was carried out at 0°C for about two hours. TLC monitoring was performed until the raw material was completely reacted. The pH value of the reaction system was adjusted to about 3 by adding concentrated hydrochloric acid, and then filtered to collect the filter residue. Purification was performed 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 (400 MHz, DMSO-d6) δ 9.46 (s, 1H), 8.07 (d, J = 8.0 Hz, 1H), 7.88 (d, J = 8.6 Hz, 1H), 7.67 (ddd, J = 8.2, 7.3, 1.4 Hz, 1H), 7.55 - 7.43 (m, 5H), 1.49 (s, 9H).

[0115] Example 7

[0116] This example provides a ebselen derivative EB-7, the preparation method of which 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] Compound EB-7 (yield 26%) was prepared in step S2.

[0118]

[0119] Compound EB-7: white solid; 1H NMR (400 MHz, DMSO-d6) δ 9.46 (s, 1H), 8.07 (d, J = 8.0 Hz, 1H), 7.88 (d, J = 8.6 Hz, 1H), 7.67 (ddd, J = 8.2, 7.3, 1.4 Hz, 1H), 7.55 - 7.43 (m, 5H), 1.49 (s, 9H).

[0120] Example 8

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

[0122] The compound EB-8 is prepared in step S2 (yield 29%).

[0123]

[0124] Compound EB-8: white solid; 1 H NMR (400 MHz, DMSO-d6) δ 9.46 (s, 1H), 8.07 (d, J = 8.0 Hz, 1H), 7.88 (d, J = 8.6 Hz, 1H), 7.67 (ddd, J = 8.2, 7.3, 1.4 Hz, 1H), 7.55 - 7.43 (m, 5H), 1.49 (s, 9H).

[0125] Example 9

[0126] This example provides a ebselen derivative EB-9, the preparation method of which is as follows:

[0127] S1, 3-(N-tert-butoxycarbonylamino)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) were weighed into a 250 mL round-bottom flask, CH2Cl2(30 mL) was added, and morpholine (CAS No.: 110-91-8; 808 mg, 9.29 mmol) was slowly added at 0 °C; then the reaction was stirred at room temperature (about 25 °C), and TLC monitoring showed that the reaction was complete after about 3 h. The reaction was quenched by adding 80 mL of water, extracted with CH2Cl2three times (40 mL x 3), washed with saturated NaHCO3solution (40 mL x 3) and saturated NH4Cl solution (40 mL x 3) three times each, and the organic phases were combined and dried over anhydrous Na2SO4. CH2Cl2was removed by reduced pressure concentration to obtain the crude product, which was used directly in the next step without purification.

[0128]

[0129] S2, To the crude product compound 2 (2.1 g, 6.86 mmol) prepared in S1, CH2Cl2(30 mL) was added, and trifluoroacetic acid (7.82 g, 68.6 mmol) was slowly added dropwise. The reaction was allowed to proceed at room temperature for about two hours, and TLC was used to monitor the completion of the reaction. CH2Cl2(30 mL) was added to the reaction system, and 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) were added. The reaction was allowed to proceed at room temperature for about 3 h, and TLC was used to monitor the completion of the reaction. 80 mL of water was added to quench the reaction, and CH2Cl2was used to extract three times (40 mL x 3). The organic phase was washed three times with saturated NaHCO3solution (40 mL x 3) and saturated NH4Cl solution (40 mL x 3), and then combined. The organic layer was dried with anhydrous Na2SO4, and CH2Cl2was removed by concentration under reduced pressure to obtain the crude product. The crude product was used directly in the next step without purification.

[0130]

[0131] S3, To the crude product compound 3 (1.58 g, 3.62 mmol) prepared in S2, potassium tert-butoxide (1.01 g, 9.02 mmol) and selenium powder (573 mg, 7.25 mmol) were added in a 250 mL round-bottom flask. DMF (20 mL) was slowly added dropwise under ice bath stirring, and the temperature was raised to 120°C. The reaction was allowed to proceed for 6 h, and TLC was used to monitor the completion of the reaction. The filtrate was filtered, and 200 mL of water was added to the filtrate. CH2Cl2was used to extract three times (50 mL x 3), and the organic phase was washed three times with saturated brine (150 mL x 3) and then combined. The organic layer was dried with anhydrous Na2SO4, and CH2Cl2was removed by concentration under reduced pressure. The product was purified by flash column chromatography (CH2Cl2:MeOH = 100:1) to obtain 378 mg of white solid compound EB-09, with a yield of 27%.

[0132]

[0133] Compound EB-09: white solid; 1H NMR (400 MHz, DMSO-d6) δ 8.10 (d, J = 8.0 Hz, 1H), 7.92 (d, J = 8.7 Hz, 1H), 7.77 - 7.74 (m, 1H), 7.73 - 7.67 (m, 2H), 7.56 - 7.51 (m, 1H), 7.49 (t, J = 7.5 Hz, 1H), 7.30 (dt, J = 7.6, 1.2 Hz, 1H), 3.74 - 3.36 (m, 8H).

[0134] Example 10

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

[0136] The compound EB-10 is prepared in step S2 (the yield is 23%).

[0137]

[0138] Compound EB-10: white solid; 1 H NMR (400 MHz, DMSO-d6) δ 8.10 (d, J = 8.0 Hz, 1H), 7.92 (d, J = 8.7 Hz, 1H), 7.77 - 7.74 (m, 1H), 7.73 - 7.67 (m, 2H), 7.56 - 7.51 (m, 1H), 7.49 (t, J = 7.5 Hz, 1H), 7.30 (dt, J = 7.6, 1.2 Hz, 1H), 3.74 - 3.36 (m, 8H).

[0139] Example 11

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

[0141] The compound EB-11 is prepared in step S2 (the yield is 29%).

[0142]

[0143] Compound EB-11: white solid; 1H NMR (400 MHz, DMSO-d6) δ 9.27 (t, J = 5.9 Hz, 1H), 8.16 - 8.11 (m, 2H), 7.92 (d, J = 6.9 Hz, 1H), 7.86 - 7.79 (m, 2H), 7.70 (d, J = 8.1 Hz, 3H), 7.61 - 7.53 (m, 3H), 7.49 (t, J = 7.0 Hz, 1H), 4.58 (d, J = 5.8 Hz, 2H).

[0144] Example 12

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

[0146] The compound EB-12 is prepared in step S2 (yield is 15%).

[0147]

[0148] Compound EB-12: yellow solid; 1 H NMR (400 MHz, DMSO-d6) δ 8.79 (d, J = 7.3 Hz, 1H), 8.31 (d, J = 9.3 Hz, 1H), 7.90 (t, J = 8.4 Hz, 1H), 7.76 - 7.68 (m, 2H), 7.56 - 7.47 (m, 2H), 7.06 (t, J = 7.5 Hz, 1H).

[0149] Example 13

[0150] This example is a ebselen derivative EB-13, its preparation method is as follows:

[0151] S1, 4-isopropyl salicylaldehyde (CAS No.: 536-32-3, compound 1, 0.95 g, 5.79 mmol), methyl iodide (2.46 g, 17.3 mmol) and potassium carbonate (2.4 g, 17.4 mmol) are weighed into a 250 mL round-bottom flask, DMF (15 mL) is added, and the reaction is carried out at room temperature for about two hours. TLC monitoring is carried out until the raw material is completely reacted. 80 mL of water is added to the reaction system, followed by extraction with CH2Cl2three times (50 mL x 3), washing with saturated brine three times (150 mL x 3), and then combining the organic phases. The organic layer is dried over anhydrous Na2SO4, and CH2Cl2is removed by concentration under reduced pressure to obtain the crude product. The crude product is used directly in the next step without purification.

[0152]

[0153] S2, To the crude product compound 2 (1.54 g, 8.55 mmol) prepared in step S1 was added tert-butyl carbamate (3.00 g, 25.6 mmol) and triethylsilane (2.98 g, 25.6 mmol), followed by acetonitrile (25 mL), and trifluoroacetic acid (1.95 g, 17.1 mmol) was slowly added dropwise at room temperature. The reaction was allowed to proceed at room temperature for about one and a half hours, and TLC monitoring was performed until the starting material was completely reacted. Excess trifluoroacetic acid (9.75 g, 85.5 mmol) was added, and the reaction was allowed to proceed at room temperature for another half hour, and TLC monitoring was performed until the reaction was complete. The reaction system was directly dried by spinning, CH2Cl2(30 mL) was added to the reaction system, 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, and the reaction was allowed to proceed at room temperature for about 3 hours, and TLC monitoring was performed until the reaction was complete. The reaction was quenched by adding 80 mL of water, and then CH2Cl2was extracted three times (40 mL x 3). The organic phase was washed three times with saturated NaHCO3solution (40 mL x 3) and HCl solution (1 M, 40 mL x 3), and then combined. The organic layer was dried with anhydrous Na2SO4, and CH2Cl2was removed by concentration under reduced pressure to obtain a crude product. The crude product was used directly in the next step without purification.

[0154]

[0155] S3, To the crude product compound 3 (2.01 g, 4.91 mmol) prepared in step S2 was added CuI (281 mg, 1.47 mmol), o-phenanthroline (266 mg, 1.47 mmol), selenium powder (466 mg, 5.90 mmol), and K2CO3(1.02 g, 7.39 mmol) in a 250 mL round-bottom flask, and DMF (20 mL) was added. After stirring at 120°C for 6 hours under nitrogen protection, TLC monitoring was performed until the starting material was completely reacted. Filtration was performed, 200 mL of water was added to the filtrate, and then CH2Cl2was extracted three times (50 mL x 3). The organic phase was washed three times with saturated brine (150 mL x 3) and then combined. The organic layer was dried with anhydrous Na2SO4, and CH2Cl2was removed by concentration under reduced pressure. Purification was performed by flash column chromatography (pure CH2Cl2) to obtain 750 mg of yellowish solid compound EB-13 at a yield of 39%.

[0156]

[0157] Compound EB-13: white solid; 1H NMR (400 MHz, DMSO-d6) δ 7.98 (d, J = 8.0 Hz, 1H), 7.82 (d, J = 8.5 Hz, 1H), 7.58 (t, J = 8.3 Hz, 1H), 7.41 (t, J = 7.9 Hz, 1H), 7.11 (d, J = 7.7 Hz, 1H), 6.91 (d, J = 1.2 Hz, 1H), 6.79 (dd, J = 7.7, 1.3 Hz, 1H), 4.80 (s, 2H), 3.86 (s, 3H), 2.87 (p, J = 6.9 Hz, 1H), 1.20 (d, J = 6.9 Hz, 6H).

[0158] Example 14

[0159] This example provides a ebselen derivative EB-14; its preparation method is substantially the same as that of Example 1, the difference is that: in step S1 aniline is replaced with furfurylamine (CAS No.: 617-89-0).

[0160] Compound EB-14 is prepared in step S2 (yield is 21%).

[0161]

[0162] Compound EB-14: yellow solid; 1 H NMR (400 MHz, DMSO-d6) δ 7.98 (d, J = 8.0 Hz, 1H), 7.82 (d, J = 8.5 Hz, 1H), 7.58 (t, J = 8.3 Hz, 1H), 7.41 (t, J = 7.9 Hz, 1H), 7.11 (d, J = 7.7 Hz, 1H), 6.91 (d, J = 1.2 Hz, 1H), 6.79 (dd, J = 7.7, 1.3 Hz, 1H), 4.80 (s, 2H), 3.86 (s, 3H), 2.87 (p, J = 6.9 Hz, 1H), 1.20 (d, J = 6.9 Hz, 6H).

[0163] Example 15

[0164] This example provides a ebselen derivative EB-15; its preparation method is substantially the same as that of Example 1, the difference is that: in step S1 aniline is replaced with 2-thiophenemethylamine (CAS No.: 27757-85-3).

[0165] Compound EB-15 is prepared in step S2 (yield is 18%).

[0166]

[0167] Compound EB-15: purple solid; 1H NMR (400 MHz, DMSO-d6) δ 8.03 (d, J = 8.0 Hz, 1H), 7.81 (d, J = 7.0 Hz, 1H), 7.62 - 7.56 (m, 1H), 7.40 (t, J = 7.0 Hz, 1H), 3.90 (t, J = 5.2 Hz, 2H), 3.56 (t, J = 5.2 Hz, 2H), 3.32 (s, 3H).

[0168] Example 16

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

[0170] Compound EB-16 is prepared in step S2 (yield is 23%).

[0171]

[0172] Compound EB-16: light red solid; 1 H NMR (400 MHz, DMSO-d6) δ 8.03 (d, J = 8.0 Hz, 1H), 7.81 (d, J = 7.0 Hz, 1H), 7.62 - 7.56 (m, 1H), 7.40 (t, J = 7.0 Hz, 1H), 3.90 (t, J = 5.2 Hz, 2H), 3.56 (t, J = 5.2 Hz, 2H), 3.32 (s, 3H).

[0173] Example 17

[0174] This example provides a ebselen derivative EB-17, its preparation method is as follows:

[0175] S1, 100 mL round bottom flask was added EB-16 (60 mg, 2.34 mmol), 10 mL of super dry CH2Cl2, moved into-78 ℃ cold trap stirring for 10 minutes, under the protection of nitrogen-78 ℃ dropwise added BBr3 (963 mg, 4.70 mmol), 30 minutes dropwise. Then the reaction system was slowly warmed to 0 ℃ stirring for about two hours, TLC monitoring to the complete reaction of raw materials. To the reaction system was added 15 mL of saturated NaHCO3 solution to quench the reaction, filtration, collection of filter residue. The filter residue was purified by flash column chromatography (CH2Cl2:MeOH = 20:1), to obtain 480 mg of yellow solid EB-17, yield was 84%.

[0176]

[0177] Compound EB-17: yellow solid; 1 H NMR (400 MHz, DMSO-d6) δ 8.02 (d, J = 8.0 Hz, 1H), 7.81 (d, J = 6.9 Hz, 1H), 7.59 (t, J = 6.9 Hz, 1H), 7.40 (t, J = 7.0 Hz, 1H), 5.10 (t, J = 5.1 Hz, 1H), 3.80 (t, J = 5.4 Hz, 2H), 3.62 (q, J = 5.3 Hz, 2H).

[0178] Example 18

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

[0180] Compound EB-18 is prepared in step S2 (the yield is 18%).

[0181]

[0182] Compound EB-18: yellow solid; 1 H NMR (500 MHz, DMSO-d6) δ 8.00 (d, J = 7.9 Hz, 1H), 7.80 (d, J = 7.7 Hz, 1H), 7.56 (t, J = 8.1 Hz, 1H), 7.37 (t, J = 7.8 Hz, 1H), 3.86 - 3.82 (m, 2H), 2.55 - 2.46 (m, 6H), 1.66 (p, J = 5.5 Hz, 4H), 1.46 (s, 2H).

[0183] Example 19

[0184] This example provides a ebselen derivative EB-19; its preparation method is substantially the same as that of Example 1, the difference is that: in step S1, aniline is replaced by 1-(2-aminoethyl)piperidine (CAS No.: 27578-60-5).

[0185] Compound EB-19 is prepared in step S2 (the yield is 11%).

[0186]

[0187] Compound EB-19: yellow solid; 1H NMR (600 MHz, DMSO-d6) δ 8.00 (d, J = 7.9 Hz, 1H), 7.81 (d, J = 8.3 Hz, 1H), 7.57 (t, J = 8.2 Hz, 1H), 7.38 (t, J = 7.9 Hz, 1H), 3.88 - 3.84 (m, 2H), 3.71 (t, J = 4.6 Hz, 4H), 2.59 - 2.51 (m, 6H).

[0188] Example 20

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

[0190] Compound EB-20 is prepared in step S2 (the yield is 16%).

[0191]

[0192] Compound EB-20: red-brown solid; 1 H NMR (500 MHz, DMSO-d6) δ 8.02 (d, J = 8.0 Hz, 1H), 7.80 (d, J = 7.7 Hz, 1H), 7.57 (t, J = 7.5 Hz, 1H), 7.37 (t, J = 7.4 Hz, 1H), 3.89 - 3.80 (m, 2H), 2.59 - 2.44 (m, 10H), 2.22 (s, 3H).

[0193] Example 21

[0194] This example provides a ebselen derivative EB-21; its preparation method is substantially the same as that of Example 1, the difference is that: in step S1, aniline is replaced with tert-butyl glycinate (CAS No.: 6456-74-2).

[0195] Compound EB-21 is prepared in step S2 (the yield is 25%).

[0196]

[0197] Compound EB-21: white solid; 1 H NMR (500 MHz, DMSO-d6) δ 8.05 (d, J = 8.0 Hz, 1H), 7.82 (d, J = 7.6 Hz, 1H), 7.62 (t, J = 7.5 Hz, 1H), 7.42 (t, J = 7.4 Hz, 1H), 4.40 (s, 2H), 1.42 (s, 9H).

[0198] Example 22

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

[0200] Compound EB-22 is prepared in step S2 (yield is 31%).

[0201]

[0202] Compound EB-22: white solid; 1 H NMR (400 MHz, DMSO-d6) δ 10.49 (s, 1H), 9.47 (s, 1H), 8.18 (d, J = 50.5 Hz, 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 = 6.4 Hz, 2H), 2.23 (s, 3H).

[0203] Example 23

[0204] This example provides a ebselen derivative EB-23; its preparation method is as follows:

[0205] S1, 50 mL round-bottom flask was added EB-02 (30 mg, 0.10 mmol), 2 mL CH2Cl2, then p-toluenethiol (CAS No.: 106-45-6; 28 mg, 0.23 mmol) was added. The reaction system was stirred at room temperature for about two hours, TLC monitoring until the raw material was completely reacted, CH2Cl2was removed by reduced pressure concentration, and 26 mg of white solid EB-23 was obtained by flash column chromatography (pure CH2Cl2), the yield was 61%.

[0206]

[0207] Compound EB-23: white solid; 1 H NMR (400 MHz, DMSO-d6) δ 10.49 (s, 1H), 9.47 (s, 1H), 8.18 (d, J = 50.5 Hz, 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 = 6.4 Hz, 2H), 2.23 (s, 3H).

[0208] Performance test

[0209] 1. PPK1 protein expression and purification:

[0210] Plasmid transformation: Take 1 μL expression plasmid into 100 μL E. coli. BL21 competent cells (melted on ice), ice bath 30 mins, then put into 42°C water bath for 90 s, after heat shock, quickly transfer to ice, ice bath 2 mins, then add 1 mL LB liquid medium on the clean bench, 37°C, 200 rpm, activate 1 h; collect the bacteria after centrifugation at 6000 rpm for 1 min, suck off the supernatant (leave 200 μL), resuspend the bacteria with a gun head, evenly smear the bacterial liquid on the LB solid medium containing 100 mg / L kanamycin, and then culture at 37°C for 12-16 h.

[0211] Protein expression and extraction: After growing a single colony, use a small gun head to pick 6 single colonies and place them in 3 mL LB medium containing kanamycin, 37°C, 180 rpm for 16 h; take 1 mL bacterial liquid and add it to 500 mL TB medium containing kanamycin, 37°C, 180 rpm for 3 to 4 hours until OD 600 value is between 0.6-0.8, at this time, add 0.3-0.5 mM IPTG, and incubate at 18°C, 180 rpm for 16 h overnight; centrifuge the bacterial liquid at 4000 rpm, 4°C for 30 mins; remove the supernatant, resuspend the cell pellet in 25 mL Buffer, and incubate on ice for 45 mins; heat shock the cells at 37°C for 10 mins; centrifuge the bacterial liquid at 18000 rpm for 30 mins, remove the supernatant; resuspend the cell pellet in 25 mL Buffer, add 3 mL 1M Na2CO3 solution and 1M KCl solid to the lysate; stir at 4°C for 1 h, then remove the cell debris by centrifugation at 18000 rpm for 30 mins.

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

[0213] 2, PPK2 protein expression and purification:

[0214] The plasmid was transformed into E. coli BL21 competent cells; after growing single colonies, 6 single colonies were picked with a small gun head and placed in 3 mL LB medium containing kanamycin, incubated at 37°C, 180 rpm for 16 h; 1 mL of bacterial solution was added to 500 mL TB medium containing kanamycin, incubated at 37°C, 180 rpm for 3 to 4 hours until OD 600 was between 0.6-0.8, at which time 0.5 mM IPTG was added, and incubated at 18°C, 180 rpm for 16 h overnight; the bacterial solution was centrifuged at 4000 rpm, 4°C for 30 mins; the supernatant was removed, and the cell pellet was resuspended in 25 mL Buffer; PMSF and lysozyme were added to the lysate, and after standing for 30 mins, ultrasonic treatment was performed for 25 mins to lyse the bacteria and release the protein; the mixture was centrifuged at 18000 rpm for 30 mins, and the supernatant was taken and the cell debris was removed.

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

[0216] Experiments were performed using equimolar amounts of PPK1 or PPK2 enzymes (275 nM) to ensure that comparisons between different enzymes were valid, and for PPK1 and PPK2B polyphosphate synthesis activity, specific reaction buffers and conditions were used, and 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 minutes, and then 50 μl of sample was taken and added to an acidic fuchsin solution to stop the reaction and quantify the polyphosphate; a specific absorption ratio (A630 / 530 nm) was used to record the amount of polyphosphate, and a standard curve was used for calibration, and data curves were fitted using GraphPad Prism software to determine IC 50 values.

[0217] The experimental results are shown in Table 1, and from Table 1 it can be seen that the ebselen and its derivatives prepared according to the scheme of the present application have inhibitory activity on the PPK enzyme of P. aeruginosa.

[0218] Table 1

[0219]

[0220]

[0221] IC 50 inhibition curves of ebselen (EB-1) on PPK1 and PPK2; and the enzymatic rate curve of PPK1 in the presence of ebselen are shown in Figure 1 wherein, Figure 1 a is the IC 50Inhibition curve; IC50 of ebuselenline against PPK1 50 The value is 36.74±1.79; IC for PPK2 50 The value is 11.35 ± 1.12; Figure 1 b is the Michaelis-Menten enzymatic reaction kinetic curve of ibuprofen to PPK1; the results show that the Michaelis constant Km of ibuprofen to substrate ATP remains basically unchanged in the presence of PPK1, while the maximum reaction rate constant Ki is reduced, indicating that ibuprofen may inhibit PPK1 enzyme activity in a non-competitive manner.

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

[0223] Bacteria were cultured overnight at 37°C and 180 rpm in 5 mL LB medium containing inhibitors or DMSO. The cultured bacterial suspension was centrifuged at 3900 rpm for 10 min to pellet the cells; the cell pellet was resuspended in 1 mL MOPS medium, and the same concentration of inhibitor or DMSO as the initial culture was added; the mixture was incubated at 37°C and 180 rpm for 2 h. The pellet was centrifuged again, resuspended in 250 μL GITC lysis buffer, and then heat-shocked at 95°C for 5 min; 15 μL of 10% SDS and 0.5 mL of 95% ethanol were added sequentially to the tube and mixed thoroughly; the mixture was transferred to a silica gel column, centrifuged at 10000 rpm for 1 min, and eluted with 50 μL of distilled water.

[0224] Experimental results are as follows Figure 3 As shown, where, Figure 3 a represents the concentration-dependent inhibition of PolyP production by ebuselenol in Pseudomonas aeruginosa; Figure 3 b represents the inhibition of PolyP production in WT-type, PPK1 knockout-type, PPK2 knockout-type, and PPK1 / PPK2 double-knockout Pseudomonas aeruginosa by 100 μM ebuselenine. The results indicate that PolyP production in Pseudomonas aeruginosa decreases with increasing ebuselenine concentration, suggesting that ebuselenine can inhibit PolyP production in Pseudomonas aeruginosa.

[0225] 5. Biofilm formation experiment:

[0226] A small amount of P. aeruginosa stock bacteria was taken into 3 mL of LB medium, incubated at 37°C, 180 rpm for 16 hours; 30 μL of bacterial solution was taken into 3 mL of M63 medium, incubated at 37°C, 180 rpm for 16 hours; P. aeruginosa strain was inoculated in a 96-well tissue culture plate, 100 μL of bacterial solution was added to each well, and 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 residual medium; the biofilm was stained with 0.1% crystal violet solution; after staining, the plate was washed twice with sterile water to remove unbound stain; the stained biofilm ring was dissolved with 30% acetic acid for subsequent absorbance measurement, and the absorbance of the dissolved stain was recorded at 570 nm wavelength, and the absorbance reflected the formation of the biofilm.

[0227] The experimental results are shown in Table 1. Figure 4 Figure 4 A is the inhibition of biofilm formation of ebselen on P. aeruginosa wild type, PPK1 knockout type, PPK2 knockout type, and PPK1 and PPK2 double knockout type; Figure 4 B is the measurement of the movement radius of wild type and PPK1 and PPK2 double knockout P. aeruginosa treated with ebselen; Figure 4 C is the biofilm formation of P. aeruginosa treated with ebselen at a concentration gradient for 16 hours. The results show that ebselen inhibits the formation of P. aeruginosa biofilm in a concentration-dependent manner.

[0228] 6. Bacterial motility invasion experiment

[0229] A semi-solid medium was prepared using 0.3% Bacto agar and 2.5% LB medium, and 100 mM of the 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 a culture dish, and a pipette tip dipped in overnight LB culture was used to inoculate the center of the medium plate, and the inoculated culture dish was placed in a sealed container and incubated at 30°C for 18 hours; after incubation, the diameter of bacterial movement in the culture dish was measured, and the diameter of bacterial movement on the agar surface could reflect the motility of the bacteria.

[0230] The experimental results are shown in Table 1. Figure 5 Figure 5 A is the inhibition of biofilm formation of ebselen on P. aeruginosa wild type, PPK1 knockout type, PPK2 knockout type, and PPK1 and PPK2 double knockout type; Figure 5 B is the measurement of the movement radius of wild type and PPK1 and PPK2 double knockout P. aeruginosa treated with ebselen; Figure 5 ​​C is the growth curve of P. aeruginosa under the treatment of ebselen or DMSO. The results show that ebselen can significantly reduce the motility radius of P. aeruginosa, indicating that ebselen can reduce the motility and invasiveness of P. aeruginosa.

[0231] 7. Quantitative experiment of P. aeruginosa toxin:

[0232] P. aeruginosa was cultured in 50 mL of 2.5% LB medium at 37°C and 180 rpm for 18 hours, the medium was centrifuged at 18000 rpm for 30 mins, the precipitate was removed and the supernatant was reserved, the centrifuged supernatant was filtered through a filter membrane; the absorbance of the supernatant was measured at 403 nm using a spectrophotometer to quantify Pyoverdine, 3 mL of chloroform was used to extract Pyocyanin, then 1 mL of hydrochloric acid was used to treat it 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 absorbance reading of the blank control without cells was subtracted from the absorbance reading of each sample.

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

[0234] 8. Antibiotic resistance experiment of P. aeruginosa:

[0235] A small amount of P. aeruginosa stock bacteria was picked into 3 mL of LB medium and cultured at 37°C and 180 rpm overnight, the overnight culture of the P. aeruginosa strain was diluted into LB medium at a ratio of 1:100, when the OD 600 of the culture reached about 0.6, 100 μM of ebselen and 7.2 μM of antibiotic were added to the medium, and the bacteria were cultured at 37°C at a speed of 180 rpm on a shaker; after 3 hours, 6 hours, 9 hours or 12 hours of incubation, the bacteria were collected. Centrifugation was performed at 12000 rpm for 2 mins, the supernatant was discarded, then phosphate buffered saline (PBS) was used for washing to remove non-bacterial components in the medium, and plate counting method was used to determine the number of residual bacteria.

[0236] The experimental results are shown in Figure 7 , wherein Figure 7 A is the effect of ebselen combined with tetracycline on the growth of P. aeruginosa; Figure 7 B is the significance analysis of the time-kill curve; Figure 7C is the time-kill curve of the colony growth. The results show that ebselen can enhance the inhibitory effect of tetracycline antibiotic on the growth of P. aeruginosa.

[0237] 9. CD binding experiment:

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

[0239] The experimental results are shown in Table 1. Figure 2 The results show that the CD values of ebselen and P. aeruginosa PPK1 and PPK2B before and after binding change significantly, indicating that ebselen and PPK1 and PPK2B can produce strong binding.

[0240] The above is described in detail in combination with the embodiments of the present application, but the present application is not limited to the above embodiments, and various changes can be made within the knowledge range possessed by those skilled in the art without departing from the purpose of the present application.

Claims

1. Use of ebselen or an ebselen derivative or a pharmaceutical composition in the manufacture of a polyphosphatase inhibitor, wherein the pharmaceutical composition comprises the ebselen derivative; and pharmaceutically acceptable excipients; the ebselen derivative has a structure according to Formula (I) or Formula (II): ###0001### (I) (II) and when R1 is H, R2 is not phenyl; R3 is selected from the group consisting of hydroxy-substituted phenyl; R4 is selected from the group consisting of H, halogen, and C1-C6 alkyl; R5 is selected from the group consisting of H, halogen, and C1-C6 alkyl; R6 is selected from the group consisting of H, halogen, and C1-C6 alkyl; and R7 is selected from the group consisting of H, halogen, and C1-C6 alkyl; and R8 is selected from the group consisting of H, halogen, and C1-C6 alkyl; and when R1 is H, R2 is not phenyl; R3 is selected from the group consisting of hydroxy-substituted phenyl; R4 is selected from the group consisting of H, halogen, and C1-C6 alkyl; R5 is selected from the group consisting of H, halogen, and C1-C6 alkyl; R6 is selected from the group consisting of H, halogen, and C1-C6 alkyl; and R7 is selected from the group consisting of H, halogen, and C1-C6 alkyl; and R8 is selected from the group consisting of H, halogen, and C1-C6 alkyl; and when R1 is H, R2 is not phenyl; R3 is selected from the group consisting of hydroxy-substituted phenyl; R4 is selected from the group consisting of H, halogen, and C1-C6 alkyl; R5 is selected from the group consisting of H, halogen, and C1-C6 alkyl; R6 is selected from the group consisting of H, halogen, and C1-C6 alkyl; and R7 is selected from the group consisting of H, halogen, and C1-C6 alkyl; and R8 is selected from the group consisting of H, halogen, ​ ​ ； wherein R1is selected from H, C 1~6 alkoxy, C 1~6 alkyl or halogen; R2is selected from adamantyl, substituted or unsubstituted phenyl, wherein the substituted group is selected from hydroxy, ; alkyl of C 1~6 ; hydroxy, substituted or unsubstituted C 3-12 ; saturated heterocyclyl of C 1~3 ; alkyl of C 1~3 ; alkoxy of C 2~6 ; ester of C 1~6 ; alkyl of C The substituents are alkyl groups substituted with C 1~6 O. ​ ​ R4is selected from C 1~6 alkyl substituted phenyl; R5is selected from H or C 1~6 alkyl; ​ 2. Use according to claim 1, characterized in that, ​ 3. Use according to claim 1 or 2, characterized in that, R2is selected from adamantyl, substituted or unsubstituted phenyl, wherein the substituted group is selected from ; substituted or unsubstituted C 1~3 ; substituted or unsubstituted C 3-8 ; substituted or unsubstituted C 1~3 ; substituted or unsubstituted C 1~3 ; substituted or unsubstituted C 2~6 ; substituted or unsubstituted C 1~3 ; substituted or unsubstituted C 4. Use according to claim 1 or 2, characterized in that, ​ 。 5. The use according to claim 1, characterized in that, ​ 6. Use according to claim 5, characterized in that, ​ 7. Use of an ebselen derivative for the manufacture of a polyphosphatase inhibitor, characterized in that, ​ 、 。

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