Active oxygen radical responsive polymyxin prodrug compounds and uses thereof

Abstract

The present application provides a polymyxin prodrug compound with the structure shown in formula (I) or a stereoisomer or a pharmaceutically acceptable salt thereof. The polymyxin prodrug compound of the present application can effectively reduce the biological toxicity of polymyxin, selectively activate in bacterial infected tissues, achieve bacterial killing in infected sites, improve the bioavailability of polymyxin, achieve sustainable development and utilization of classic antibiotics, expand the scope of future clinical application of polymyxin, and has a broad clinical application prospect.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and in particular to a polymyxin prodrug compound responsive to active oxygen free radicals and applications thereof. Background Art

[0002] Drug-resistant bacterial infections continue to threaten human health and life. Approximately 700,000 people die worldwide each year from antibiotic resistance. If the problem remains unchecked, the death toll from antibiotic resistance could reach 10 million by 2050. This means that global deaths from drug-resistant infections will surpass those from cancer. In 2020, the World Health Organization stated that declining private investment and insufficient innovation in the development of new antibiotics are undermining efforts to combat drug-resistant infections. The 60 antimicrobial drugs currently under development, including 50 antibiotics and 10 biologics, have shown little effectiveness compared to existing treatments, and few new drugs are being developed for the most difficult-to-treat Gram-negative resistant bacteria.

[0003] Gram-negative bacteria, such as Klebsiella pneumoniae and Escherichia coli, can cause serious and often fatal infections, posing a health and life-threatening threat to people with weakened or underdeveloped immune systems, such as newborns, as well as the elderly, those undergoing surgery, and those undergoing cancer treatment. According to the European Medicines Agency (EMEA), approximately two-thirds of deaths caused by antibiotic-resistant bacteria in Europe are due to Gram-negative infections. Gram-negative bacteria also cause 45-70% of ventilator-associated pneumonia (VAP) cases, 20-30% of catheter-related bloodstream infections, and other infections associated with intensive care units, such as surgical site or urinary tract infections (UTIs). According to the World Health Organization, the three most deadly Gram-negative resistant bacteria are Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacteriaceae.

[0004] Polymyxins first appeared in clinical practice as antimicrobial drugs in the 1970s, with the main products including polymyxin B and polymyxin E. They are composed of a cationic seven-membered peptide ring and a ternary peptide with a fatty acid chain. They kill bacteria by disrupting the bacterial membrane, allowing the bacterial contents to leak out, a process known as a "self-uptake mechanism." Clinically, polymyxins typically require high doses and long-term treatment to achieve optimal therapeutic effects. This treatment often has potential toxicity to the human kidneys and nervous system, limiting their use in clinical treatment. However, in the 21st century, due to the increasing prevalence of bacterial resistance, polymyxins have been re-introduced into clinical use and are considered the last line of defense against multidrug-resistant Gram-negative bacteria. However, the side effects induced by polymyxins remain a challenge.

[0005] Currently, two polymyxin prodrugs are in clinical use: polymyxin sulfate for oral and topical use, and polymyxin sodium methanesulfonate (CMS) for injection. CMS modifies the five amino groups on the Dab residue in polymyxin, creating a less toxic, inactive prodrug. This prodrug is then converted to the active polymyxin in vivo and exerts its antibacterial activity. Because CMS is less toxic than polymyxin sulfate, it can be used parenterally. However, the clinical application of CMS still faces several challenges. For example, approximately 60% of CMS is excreted in the urine within the first 24 hours of administration, leaving only a small amount hydrolyzed to the active polymyxin, resulting in low drug bioavailability. Furthermore, many researchers have attempted to develop new polymyxin prodrugs. Zhu et al. modified the hydroxyl groups of two threonine amino acids in polymyxin E with acetate-terminated polyethylene glycol methyl ether. Experimental results indicate that this prodrug effectively reduces the nephrotoxicity of polymyxin E. However, due to the slow conversion of the prodrug to the active ingredient in vivo, the antibacterial activity is reduced, necessitating a higher dosage. Summary of the Invention

[0006] Based on this, the present invention provides a new class of polymyxin prodrug compounds, which exist in an inactive form in normal tissues, cause very little damage to normal tissues, and have few toxic side effects. When stimulated by reactive oxygen free radicals (ROS) at the site of bacterial infection, they release active polymyxin components, thereby effectively killing bacteria.

[0007] The present invention includes the following technical solutions.

[0008] A polymyxin prodrug compound having a structure represented by formula (I) or a stereoisomer thereof or a pharmaceutically acceptable salt thereof:

[0009]

[0010] wherein R1 is selected from:

[0011] R2 and R3 are each independently selected from C1-C8 alkyl;

[0012] R is selected from the group consisting of a boronic acid group and a substituted or unsubstituted boronic ester group.

[0013] In some embodiments, R is selected from the group consisting of: a borate ester group obtained by reacting a boric acid group with a sugar containing an adjacent dihydroxy structure; or

[0014] In some embodiments, the sugar containing an vicinal dihydroxy structure is a monosaccharide, a disaccharide or a trisaccharide.

[0015] In some embodiments, the polymyxin prodrug compound has a structure shown in the following formula (II):

[0016]

[0017] wherein R1 is selected from:

[0018] R2 and R3 are each independently selected from C1-C8 alkyl;

[0019] Each R4 and R5 are independently selected from: H, C1-C6 alkyl, one or more R6-substituted C1-C6 alkyl, or R4, R5 and the -OBO- connected thereto form the following structure:

[0020]

[0021] Each R6 is independently selected from: H, hydroxyl, carboxyl, carbonyl, aldehyde, C1-C6 alkoxy;

[0022] Each R7, R8, R9, R 10 Each independently selected from: H, C1-C8 alkyl, C1-C8 alkoxy, one or more R 12 Substituted C1-C8 alkyl, one or more R 12 Substituted C1-C8 alkoxy, carboxyl, aldehyde, or R7 and R 10 is hydrogen, R8, R9 and the carbon atom to which they are connected together form one or more R 13 substituted 5-6 membered oxygen-containing heterocyclic group;

[0023] Each R 12 Each independently selected from: H, hydroxyl, carboxyl, carbonyl, aldehyde, C1-C6 alkoxy, hydroxy-substituted C1-C6 alkyl, one or more R 15 substituted 5-6 membered oxygen-containing heterocycloalkoxy group;

[0024] Each R 13 Each independently selected from: H, hydroxyl, one or more R 16 Substituted C1-C6 alkyl, one or more R 15 Substituted 5-6 membered oxygen-containing heterocycloalkoxy, one or more R 17 Substituted C1-C8 alkoxy;

[0025] Each R 15 Each independently selected from: H, hydroxyl, hydroxyl-substituted C1-C6 alkyl, one or more R 18 substituted 5-6 membered oxygen-containing heterocycloalkoxy group;

[0026] Each R 16Each independently selected from: H, hydroxyl, one or more R 15 substituted 5-6 membered oxygen-containing heterocycloalkoxy group;

[0027] Each R 17 Each independently selected from: H, hydroxyl, hydroxyl-substituted C1-C6 alkyl, one or more R 19 substituted 5-6 membered oxygen-containing heterocyclic group;

[0028] Each R 18 Each independently selected from: H, hydroxyl, hydroxyl-substituted C1-C6 alkyl;

[0029] Each R 19 Each independently selected from: H, hydroxyl, hydroxyl-substituted C1-C6 alkyl, one or more R 18 Substituted 5-6 membered oxygen-containing heterocycloalkoxy group.

[0030] In some embodiments, R2 is selected from C3-C4 alkyl; R3 is selected from C2-C5 alkyl.

[0031] In some embodiments, R4 and R5 are both H, or R4, R5 and the -OBO- attached thereto form the following structure:

[0032]

[0033] R7, R8, R9 and R 10 Each independently selected from: H, C1-C4 alkyl, C1-C4 alkoxy, one or more R 12 Substituted C1-C4 alkyl, one or more R 12 Substituted C1-C4 alkoxy, carboxyl, aldehyde, or R7 and R 10 is hydrogen, R8, R9 and the carbon atom to which they are connected together form one or more R 13 substituted 6-membered oxygen-containing heterocyclic group.

[0034] In some embodiments, each R 12 Each independently selected from: H, hydroxyl, carboxyl, carbonyl, aldehyde, C1-C3 alkoxy, hydroxy-substituted C1-C3 alkyl, one or more R 15 Substituted 6-membered oxygen-containing heterocycloalkoxy; wherein each R 15 Each is independently selected from the group consisting of: H, hydroxy, and C1-C3 alkyl substituted with hydroxy.

[0035] In some embodiments, each R 13 Each independently selected from: H, hydroxyl, one or more R 16 Substituted C1-C3 alkyl, one or more R 15Substituted 6-membered oxygen-containing heterocycloalkoxy, one or more R 17 Substituted C1-C3 alkoxy; wherein each R 15 are independently selected from: H, hydroxy, hydroxy-substituted C1-C3 alkyl, one or more R 18 Substituted 5-6 membered oxygen-containing heterocycloalkoxy; each R 16 Each independently selected from: H, hydroxyl, one or more R 15 Substituted 6-membered oxygen-containing heterocycloalkoxy; each R 17 are independently selected from: H, hydroxy, hydroxy-substituted C1-C3 alkyl, one or more R 19 Substituted 5-6 membered oxygen-containing heterocyclic group; each R 18 Each R is independently selected from: H, hydroxyl, hydroxyl-substituted C1-C3 alkyl; 19 are independently selected from: H, hydroxy, hydroxy-substituted C1-C3 alkyl, one or more R 18 Substituted 5-6 membered oxygen-containing heterocycloalkoxy group.

[0036] In some embodiments, R4 and R5 are both H, or R4, R5 and the -OBO- attached thereto form the following structure:

[0037]

[0038] In some embodiments, the polymyxin prodrug compound is selected from the following compounds:

[0039]

[0040]

[0041]

[0042]

[0043]

[0044] Among them, R1 is R2 is R3 is selected from or,

[0045] R1 is R2 is R3 is or,

[0046] R1 is R2 is R3 is selected from or,

[0047] R1 is R2 is R3 is selected from or,

[0048] R1 is R2 is R3 is selected from or,

[0049] R1 is R2 is R3 is selected from

[0050] The present invention also provides the use of the above-mentioned polymyxin prodrug compound, including the following technical solutions.

[0051] Use of the above-mentioned polymyxin prodrug compound or its stereoisomer or pharmaceutically acceptable salt in the preparation of a drug for treating bacterial infection.

[0052] In some embodiments, the bacteria are Gram-negative bacteria.

[0053] In some embodiments, the bacteria are Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Salmonella typhimurium, or Enterobacteriaceae.

[0054] In some embodiments, the bacteria are Klebsiella pneumoniae, Enterobacter cloacae, Salmonella typhimurium, Escherichia coli and Pseudomonas aeruginosa.

[0055] The present invention also provides an antibacterial drug.

[0056] The specific technical solutions are as follows:

[0057] A drug for treating bacterial infection is prepared from active ingredients and pharmaceutically acceptable excipients. The active ingredient includes the above-mentioned polymyxin prodrug compound or its stereoisomer or a pharmaceutically acceptable salt thereof.

[0058] Based on the above technical solution, the present invention has the following beneficial effects:

[0059] The present invention utilizes the difference in ROS concentration between bacterial infection sites and normal tissue sites to structurally modify clinically approved polymyxins B and E. By modifying them with phenylboronic acid or phenylboronic acid, ROS-responsive polymyxin prodrug compounds are obtained. The prodrug compounds exist in an inactive form in normal tissues and are inactive polymyxin prodrugs under blood circulation conditions, causing very little damage to normal tissues and exhibiting low cytotoxicity. At the bacterial infection site, ROS-sensitive chemical bonds modified with phenylboronic acid or phenylboronic acid esters are broken by the action of ROS, converting the inactive polymyxin prodrug into a polymyxin with high antibacterial activity. This allows highly selective killing of bacteria (especially drug-resistant Gram-negative bacteria) at the infection site, significantly improving the antibacterial effect while significantly reducing the toxic side effects of the polymyxin. The polymyxin prodrug compound of the present invention can effectively reduce the biological toxicity of polymyxin, selectively activate in bacterially infected tissues, kill bacteria at the infected site, improve the bioavailability of polymyxin, realize the sustainable development and utilization of classic antibiotics, expand the scope of future clinical application of polymyxin, and has broad clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 The invention provides a synthetic route for phenylboronic acid-modified polymyxins, phenylboronic acid-modified polymyxins and sugar-modified polymyxin prodrugs.

[0061] Figure 2 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolane)-benzyl nitrophenyl carbonate (ABE) 1 H NMR characterization spectrum.

[0062] Figure 3 Phenylboronic acid ester modified polymyxin E 1 H NMR characterization spectrum.

[0063] Figure 4 Phenylboronic acid-modified polymyxin E 1 H NMR characterization spectrum.

[0064] Figure 5 Phenylboronic acid ester modified polymyxin B 1 H NMR characterization spectrum.

[0065] Figure 6 Phenylboronic acid-modified polymyxin B 1 H NMR characterization spectrum.

[0066] Figure 7 Mass spectra of polymyxin E modified with phenylboronic acid ester (a) and after ROS response (b).

[0067] Figure 8 Xylose-modified polymyxin prodrug1 H NMR characterization spectrum.

[0068] Figure 9 Arabinose-modified polymyxin prodrug 1 H NMR characterization spectrum.

[0069] Figure 10 Fructose-modified polymyxin prodrug 1 H NMR characterization spectrum.

[0070] Figure 11 Glucose-modified polymyxin prodrug 1 H NMR characterization spectrum.

[0071] Figure 12 Galactose-modified polymyxin prodrug 1 H NMR characterization spectrum.

[0072] Figure 13 Mannose-modified polymyxin prodrug 1 H NMR characterization spectrum.

[0073] Figure 14 Lactose-modified polymyxin prodrug 1 H NMR characterization spectrum.

[0074] Figure 15 Maltotriose modified polymyxin prodrug 1 H NMR characterization spectrum.

[0075] Figure 16 Raffinose modified polymyxin prodrug 1 H NMR characterization spectrum.

[0076] Figure 17 This is the hemolytic activity result of polymyxin E modified with phenylboronic acid ester.

[0077] Figure 18 This is the ROS sensitivity result of phenylboronic acid ester-modified polymyxin E in response to different concentrations of H2O2 for different times (5min, 15min, 30min).

[0078] Figure 19 This is the antibacterial kinetic results of polymyxin E modified with phenylboronic acid ester.

[0079] Figure 20 This is the antibacterial kinetics result of polymyxin B modified with phenylboronic acid ester.

[0080] Figure 21 This is a graph showing the bactericidal kinetics of polymyxin E modified with phenylboronic acid ester.

[0081] Figure 22 This is the MTT result of the cytotoxicity of polymyxin E and polymyxin B modified with phenylboronic acid ester on HK-2 cells.

[0082] Figure 23 Maximum tolerated dose of sugar-modified ROS-responsive polymyxin prodrugs.

[0083] Figure 24 The therapeutic effect of polymyxin E modified with phenylboronic acid ester on pneumonia.

[0084] Figure 25 The therapeutic effect of lactose-modified polymyxin E on pneumonia. DETAILED DESCRIPTION

[0085] The experimental methods in the following examples of the present invention, where specific conditions are not specified, are generally carried out under conventional conditions or conditions recommended by the manufacturers. The various commonly used chemical reagents used in the examples are all commercially available products.

[0086] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0087] The terms "comprise," "comprising," and "having," and any variations thereof, are intended to cover non-exclusive inclusions. For example, a process, method, apparatus, product, or device comprising a series of steps is not limited to the listed steps or modules but may optionally include steps not listed, or other steps inherent to the process, method, product, or device.

[0088] In this application, "plurality" refers to two or more. "And / or" describes the relationship between related objects, indicating that three possible relationships exist. For example, "A and / or B" can mean: A exists alone, A and B exist simultaneously, or B exists alone. The character " / " generally indicates that the related objects are in an "or" relationship.

[0089] In the compounds of the present invention, when any variable (such as R 4 、R 5If a substituent (e.g., ) occurs more than once in any component, its definition on each occurrence is independent of its definition at every other occurrence. Likewise, combinations of substituents and variables are permissible so long as such combinations result in a stable compound. A line drawn from a substituent into a ring system indicates that the indicated bond may be attached to any substitutable ring atom. If the ring system is polycyclic, this means that such a bond may be attached only to any suitable carbon atom in an adjacent ring. It will be understood that one of ordinary skill in the art can select substituents and substitution patterns in the compounds of the present invention to provide chemically stable compounds that can be readily synthesized from readily available starting materials using techniques in the art and the methods set forth below. If a substituent is itself substituted with more than one group, it will be understood that these groups may be on the same carbon atom or on different carbon atoms, so long as the structure is stable. The phrase "optionally substituted with one or more substituents" is considered equivalent to the phrase "optionally substituted with at least one substituent," and in such cases, preferred embodiments will have 0-3 substituents.

[0090] As used herein, the term "alkyl" is intended to include both branched and straight-chain saturated aliphatic hydrocarbon groups having a specified number of carbon atoms. For example, the definition of "C1-C6 alkyl" includes groups having 1, 2, 3, 4, 5, or 6 carbon atoms in a straight or branched chain. For example, "C1-C6 alkyl" specifically includes methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, pentyl, and hexyl.

[0091] The term "alkoxy" refers to a group having an -O-alkyl structure, such as -OCH3, -OCH2CH3, -OCH2CH2CH3, -O-CH2CH(CH3)2, -OCH2CH2CH2CH3, -O-CH(CH3)2, etc.

[0092] The term "oxygen-containing heterocyclic group" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic substituent, wherein one or more ring atoms are O atoms and the remaining ring atoms are carbon atoms, for example, tetrahydropyranyl.

[0093] The term "oxyheterocycloalkoxy" refers to a group in which a carbon atom of an oxygen-containing heterocyclic ring is linked to -O-, for example:

[0094] The drug for treating bacterial infection of the present invention can be used in non-human mammals or humans.

[0095] The pharmaceutically acceptable excipients used in the drug for treating bacterial infection of the present invention refer to: one or more compatible solid or liquid fillers or gel substances, which are suitable for human use and must have sufficient purity and sufficiently low toxicity.

[0096] "Compatibility" herein means that the components of the composition can be blended with the active ingredient of the present invention (polymyxin prodrug compound or its stereoisomer or pharmaceutically acceptable salt) and with each other without significantly reducing the efficacy of the active ingredient.

[0097] The pharmaceutically acceptable excipients used in the drug for treating bacterial infection of the present invention include, but are not limited to, one or more of the following materials: at least one of solvents, excipients, fillers, extenders, binders, humectants, disintegrants, dissolution buffers, absorption accelerators, adsorbents, diluents, solubilizers, emulsifiers, lubricants, wetting agents, suspending agents, flavoring agents and fragrances.

[0098] Examples of pharmaceutically acceptable excipients include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers (such as ), wetting agents (such as sodium lauryl sulfate), colorants, flavorings, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0099] There is no particular limitation on the administration of the active ingredient or pharmaceutical composition of the present invention. Representative administration routes include (but are not limited to): oral, rectal, parenteral (intravenous, intramuscular or subcutaneous) and the like.

[0100] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules.

[0101] In these solid dosage forms, the active ingredient is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with:

[0102] (a) fillers or extenders, for example, starch, lactose, sucrose, glucose, mannitol and silicic acid;

[0103] (b) binders, for example, hydroxymethylcellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and gum arabic;

[0104] (c) humectants, for example, glycerin;

[0105] (d) disintegrants, for example, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate;

[0106] (e) a buffering solvent, for example, paraffin;

[0107] (f) absorption accelerators, for example, quaternary ammonium compounds;

[0108] (g) wetting agents, for example, cetyl alcohol and glyceryl monostearate;

[0109] (h) adsorbents, for example, kaolin;

[0110] (i) Lubricants, for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, or mixtures thereof. In capsules, tablets and pills, the dosage form may also contain a buffering agent.

[0111] The solid dosage forms can also be prepared using coatings and shells, such as enteric coatings and other materials known in the art. They can contain opacifying agents, and the release of the active ingredient in such compositions can be delayed in a certain portion of the digestive tract. Examples of embedding components that can be used are polymeric substances and waxes.

[0112] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, or tinctures. In addition to the active ingredient, the liquid dosage form may contain inert diluents commonly used in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropyl alcohol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butylene glycol, dimethylformamide, and oils, particularly cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil, and sesame oil, or mixtures thereof. In addition to these inert diluents, the composition may also contain adjuvants, such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents, and fragrances.

[0113] In addition to the active ingredients, suspensions may contain suspending agents such as, for example, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances.

[0114] Compositions for parenteral injection may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.

[0115] One example of a ROS-responsive polymyxin prodrug compound of the present invention can be synthesized according to the following method: p-nitrophenyl chloroformate and 4-(hydroxymethyl)phenylboronic acid pinacol ester are reacted as raw materials with triethylamine as an acidifying agent to produce 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolane)-benzylnitrophenyl carbonate (ABE). ABE reacts with polymyxin E and polymyxin B, respectively, to produce phenylboronate-modified ROS-responsive polymyxin E and polymyxin B prodrugs. The phenylboronate-modified polymyxin E and polymyxin B prodrugs are hydrolyzed with concentrated hydrochloric acid to produce phenylboronic acid-modified ROS-responsive polymyxin E and polymyxin B prodrugs. The phenylboronic acid-modified ROS-responsive polymyxin E and polymyxin B prodrugs are then reacted with a compound containing an ortho-dihydroxy group to produce sugar-modified polymyxin E and polymyxin B prodrugs with improved water solubility.

[0116] In some embodiments, the method for synthesizing a phenylboronic acid-modified polymyxin E or polymyxin B prodrug comprises the following steps:

[0117] (1) p-Nitrophenyl chloroformate and 4-(hydroxymethyl)phenylboronic acid pinacol ester are dissolved in anhydrous tetrahydrofuran, and anhydrous triethylamine is added as an acidifying agent. After the reaction is completed, the mixture is extracted with ethyl acetate, washed with saturated sodium bicarbonate and dilute hydrochloric acid in sequence, dried with anhydrous magnesium sulfate, filtered, rotary evaporated, passed through a silica gel column, and rotary evaporated to prepare 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborane)-benzyl nitrophenyl carbonate (ABE).

[0118] (2) Dissolve polymyxin E or polymyxin B in saturated sodium bicarbonate, add ABE dissolved in dimethylformamide, and after the reaction is completed, remove the solvent by distillation under reduced pressure, dissolve in dichloromethane, centrifuge, collect the supernatant, remove the solvent, precipitate in ether, and dialyze to obtain phenylboronic acid ester-modified polymyxin E or phenylboronic acid ester-modified polymyxin B.

[0119] (3) Dissolving the phenylboronic acid ester-modified polymyxin prodrug in dimethylformamide, adding concentrated hydrochloric acid dropwise, and dialyzing after the reaction to obtain phenylboronic acid-modified polymyxin E or phenylboronic acid-modified polymyxin B.

[0120] In some embodiments, a method for preparing a sugar-modified ROS-responsive polymyxin E and polymyxin B prodrug compound obtained by reacting a sugar containing an ortho-dihydroxy structure with phenylboronic acid comprises the following steps: reacting a phenylboronic acid-modified polymyxin E or polymyxin B prodrug with a sugar containing an ortho-dihydroxy structure in a 0.1 M NaOH solution, and dialyzing against a 0.01 M NaOH solution after the reaction to obtain a phenylboronic acid-modified polymyxin E or a related derivative of phenylboronic acid-modified polymyxin B with improved water solubility.

[0121] The above-described preparation method can prepare a series of ROS-responsive polymyxin prodrug compounds. In the present invention, the ROS-responsive polymyxin E prodrug modified with phenylboronic acid ester is represented as ABE-Colistin; the ROS-responsive polymyxin B prodrug modified with phenylboronic acid ester is represented as ABE-Polymyxin B; the ROS-responsive polymyxin E prodrug modified with phenylboronic acid ester is represented as ABA-Colistin; the ROS-responsive polymyxin B prodrug modified with phenylboronic acid ester is represented as ABA-Polymyxin B; the sugar-modified ROS-responsive polymyxin E prodrug obtained by the reaction of a sugar containing an ortho-dihydroxy structure with phenylboronic acid is expressed as X-ABA-Colistin, wherein X represents the abbreviation of different sugars, for example, the ROS-responsive polymyxin E prodrug obtained by the reaction with lactose is expressed as Lac-ABA-Colistin, the ROS-responsive polymyxin E prodrug obtained by the reaction with fructose is expressed as Fru-ABA-Colistin, and the ROS-responsive polymyxin E prodrug obtained by the reaction with galactose is expressed as Gal-ABA-Colistin; the sugar-modified ROS-responsive polymyxin B prodrug obtained by the reaction of a sugar containing an ortho-dihydroxy structure with phenylboronic acid is expressed as X-ABA-Polymyxin B, wherein X represents the abbreviation of different sugars.

[0122] The polymyxins and polymyxins used in the following examples are commercially available products. Polymyxins and polymyxins are known to have various substructures, as follows:

[0123]

[0124]

[0125]

[0126] The following are specific examples.

[0127] Example 1: Preparation of Phenylboronic Acid-Modified ROS-Responsive ABA-Colistin

[0128] 1) Preparation of 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolane)-benzyl nitrophenyl carbonate (ABE)

[0129] Weigh 0.47 g of p-nitrophenyl chloroformate and 0.5 g of 4-(hydroxymethyl)phenylboronic acid pinacol ester into a 100 mL round-bottom flask, dissolve in 20 mL of anhydrous tetrahydrofuran (THF), add 600 μL of anhydrous triethylamine under stirring, and stir at room temperature for 1 hour; add 30 mL of ethyl acetate, transfer to a 125 mL separatory funnel, let it stand, add 250 mL of saturated NaHCO3 solution three times to wash, discard the lower aqueous layer, and add 250 mL of ethanol three times. The product was washed with HCl (1M), the lower aqueous layer was discarded, the upper liquid was poured into a 100 mL beaker, an appropriate amount of anhydrous MgSO4 was added, and the mixture was stirred overnight to dry. Then, the product was filtered into a 500 mL round-bottom flask, 2 mL of silica gel was added to the filtered solution, and the product was rotary evaporated, dry-loaded, and purified by column chromatography. The eluent was ethyl acetate and n-hexane (V:V = 1:20), and 0.52 g of ABE was obtained with a yield of 61.2%. 1 H NMR characterization spectrum Figure 2 shown.

[0130] 2) Preparation of Phenylboronic ABE-Colistin

[0131] 100 mg of polymyxin E sulfate (purchased from Shanghai Yuanye Biotechnology Co., Ltd.; CAS: 1264-72-8, product number: S17057; its polymyxin E is a mixture of two components, polymyxin E1 and polymyxin E2) was weighed. 4 mL of saturated sodium bicarbonate was added to a 25 mL round-bottom flask and stirred to dissolve. ABE solution (200 mg of ABE dissolved in 1 mL of dimethylformamide (DMF)) was added to the round-bottom flask and stirred for 24 hours. The solvent was removed by distillation under reduced pressure at 50°C, and the solution was dissolved with 10 mL of dichloromethane. The solution was centrifuged to remove the precipitate, and the solution was concentrated. After precipitation with ether, the solution was dissolved with 5 mL of dimethyl sulfoxide (DMSO), dialyzed with water, and lyophilized to obtain 103 mg of phenylboronic acid ester-modified polymyxin E (denoted as ABE-Colistin) with a yield of 48.4%. 1 HNMR characterization spectrum Figure 3 Its mass spectrum is shown as Figure 7 As shown in Figure a.

[0132] 3) Preparation of phenylboronic acid-modified ABA-Colistin

[0133] Weigh 50 mg of ABE-Colistin into a 25 mL round-bottom flask, add 4 mL of DMSO, stir to dissolve, add 0.3 mL of concentrated hydrochloric acid (37 wt%), stir to react for 6 h, dialyze with water, and lyophilize to obtain 35 mg of phenylboronic acid-modified polymyxin E (denoted as ABA-Colistin), with a yield of 84.1%. 1 H NMR characterization spectrum Figure 4

[0134] Example 2: Preparation of phenylboronic acid-modified ROS-responsive ABA- Polymyxin B

[0135] 1) Preparation of 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan)-benzyl nitrophenyl carbonate (ABE)

[0136] The same as Example 1.

[0137] 2) Preparation of phenylboronate-modified ABE-Polymyxin B

[0138] The method is the same as Example 1, and 105 mg of phenylboronate-modified polymyxin B (denoted as ABE-Polymyxin B) is prepared from polymyxin B sulfate (purchased from Dalian Melun Biotechnology Co., Ltd.; CAS: 1405-20-5, product number: MB1188; its polymyxin B is a mixture containing polymyxin B1, polymyxin B2, polymyxin B3, and Ile-polymyxin B1 four structures) with a yield of 50.4%, and its structure is as shown in the following figure: 1 The H NMR characterization spectrum is as shown in the following figure: Figure 5

[0139] 3) Preparation of phenylboronic acid-modified ABA-Polymyxin B

[0140] The method is the same as Example 1, and 34 mg of phenylboronic acid-modified polymyxin B (denoted as ABA-Polymyxin B) is prepared with a yield of 80.9%, and its structure is as shown in the following figure: 1 The H NMR characterization spectrum is as shown in the following figure: Figure 6

[0141] Example 3: Preparation of sugar-modified ROS-responsive polymyxin E prodrug

[0142] 80 mg (0.039 mmol) of ABA-Colistin is weighed in a round-bottom flask, 4 mL of 0.1M NaOH solution is added, stirred and dissolved, 664.8 mg (1.012 mmol) of lactose is added, stirred for 24 h, dialyzed with 0.01M NaOH solution, and freeze-dried to prepare 88 mg of lactose-modified ROS-responsive polymyxin E prodrug with a yield of 62.8%.

[0143] Other sugar-modified polymyxin E prodrugs are prepared according to the method of this example, and the feeding ratio is shown in the following Table 1:

[0144] Table 1

[0145] ​​​

[0146]

[0147] Xylose-modified polymyxin E prodrug 1 H NMR characterization spectrum Figure 8 As shown; Arabinose modified polymyxin E prodrug 1 H NMR characterization spectrum Figure 9 As shown; fructose modified polymyxin E prodrug 1 H NMR characterization spectrum Figure 10 As shown; Glucose modified polymyxin E prodrug 1 H NMR characterization spectrum Figure 11 As shown; Galactose modified polymyxin E prodrug 1 H NMR characterization spectrum Figure 12 As shown; Mannose modified polymyxin E prodrug 1 HNMR characterization spectrum Figure 13 As shown; lactose modified polymyxin E prodrug 1 H NMR characterization spectrum Figure 14 As shown; maltotriose modified polymyxin E prodrug 1 H NMR characterization spectrum Figure 15 As shown; Raffinose modified polymyxin E prodrug 1 H NMR characterization spectrum Figure 16 shown.

[0148] Example 4:

[0149] Antimicrobial activity, hemolytic toxicity, and cytotoxicity of ROS-responsive polymyxin prodrugs ABE-Colistin and ABE-Polymyxin B.

[0150] 1) Hemolysis test:

[0151] Take an appropriate amount of whole sheep blood in a centrifuge tube, dilute it with 1×PBS to prepare a 4% (v / v) sheep blood solution; use PBS to prepare a series of drug solutions of different concentrations, add them to the EP tube at 100μL / tube, and then add an equal volume of 4% sheep blood; PBS and an equal volume of 4% sheep blood are mixed as a negative control group; 0.1% Triton and an equal volume of 4% sheep blood are mixed as a positive control group; after the samples are mixed, they are placed at 37°C for incubation for 60 minutes. Subsequently, the samples are placed at 4°C for centrifugation (1000rpm / 5min); after centrifugation, 100μL of the supernatant is placed in a 96-well plate, and the absorbance is measured at 576nm to calculate the hemolysis rate. The calculation formula is as follows: Hemolytic activity = (OD experimental group - OD negative group) ÷ (OD positive group - OD negative group) × 100%. As Figure 17The hemolytic activity of colistin and ABE-colistin was compared, indicating that polymyxin E modified with phenylboronic acid ester has lower hemolytic activity and can be better used in vivo.

[0152] 2) Inhibitory kinetics:

[0153] Weigh the ABE-Colistin material powder, add it to a 1.5mL Eppendorf tube, then add anhydrous DMSO and vortex to completely dissolve it, to prepare ABE-Colistin with a series of concentrations; weigh the Colisitin powder, add it to a 1.5mL Eppendorf tube, then add sterile water and vortex to completely dissolve it, to prepare Colistin with a series of concentrations; incubate the ABE-Colistin with different concentrations of H2O2 (0, 1, 2mmol) for different times (5min, 15min, 30min).

[0154] Escherichia coli (ATCC35218) was collected by centrifugation, washed three times with PBS, and diluted to an appropriate concentration using M9 medium. A series of drug solutions (0 mM H2O2 + ABE-Colistin group, 1 mM H2O2 + ABE-Colistin group, 2 mM H2O2 + ABE-Colistin group, and Colistin group) were then added to make the final bacterial concentration 1 × 10 6 CFU / mL. The samples were incubated at 37°C and taken out after 24 hours. The absorbance at 600nm was measured to calculate the bacterial inhibition efficiency. The calculation formula is: Inhibition rate = (100-(OD value of the treatment group-OD value of the blank background group) ÷ (OD value of the blank control group-OD value of the blank background group)) × 100%. The results are as follows Figure 18 As shown, at 5 minutes, ABE-Colistin incubated with 1mM H2O2 could not completely inhibit bacterial growth, while ABE-Colistin incubated with 2mM H2O2 could completely inhibit bacterial growth. Previous experimental data show that the MIC concentration of Colisitin is approximately 0.25μg / mL; however, if the incubation time is extended, ABE-Colistin can also completely inhibit bacterial growth at a concentration of 0.25μg / mL after incubation under 1mM H2O2 conditions; after incubation for 15 minutes and 30 minutes, the curve trends are almost the same, indicating that ABE-Colistin modified with phenylboronic acid ester is more sensitive to ROS and responds faster, and can effectively inhibit bacterial growth in a ROS environment.

[0155] ABE-Colistin with a series of concentrations was incubated with 1 mM H2O2 for 30 min. Escherichia coli (ATCC35218), Pseudomonas aeruginosa (ATCC27853), Klebsiella pneumoniae (ATCC700603), Enterobacter cloacae (ATCC700323), Salmonella enterica serovar Typhimurium (ATCC14028), and Escherichia coli (ATCC25922) were collected by centrifugation, washed three times with PBS, and diluted to an appropriate concentration using LB medium. A series of drug solutions (0 mM H2O2 + ABE-Colistin group, 1 mM H2O2 + ABE-Colistin group, and Colistin group) were then added to achieve a final bacterial concentration of 1 × 10 6 CFU / mL. The samples were incubated at 37°C and taken out after 24 hours. The absorbance at 600nm was measured to calculate the bacterial inhibition efficiency. The calculation formula is: Inhibition rate = (100-(OD value of the treatment group-OD value of the blank background group) ÷ (OD value of the blank control group-OD value of the blank background group)) × 100%. The results are as follows Figure 19 As shown in the results, ABE-Colistin incubated with 1 mM H2O2 can completely inhibit bacterial growth in the concentration range of 0.25 μg / mL to 32 μg / mL, while ABE-Colistin not treated with H2O2 has no effect on inhibiting bacterial growth, indicating that phenylboronic acid ester-modified ABE-Colistin can not only sensitively respond to ROS, but also selectively and effectively inhibit the growth of multiple Gram-negative bacteria in a ROS environment.

[0156] ABE-Polymyxin B with a series of concentrations was incubated with 1 mmol H2O2 for 30 min. Escherichia coli (ATCC35218) and Pseudomonas aeruginosa (ATCC27853) were collected by centrifugation, washed three times with PBS, and diluted to the appropriate concentration using LB medium. A series of drug solutions (0 mM H2O2 + ABE-Colistin group, 1 mM H2O2 + ABE-Colistin group, Colistin group) were then added to achieve a final bacterial concentration of 1 × 10 6 CFU / mL. The samples were incubated at 37°C and taken out after 24 hours. The absorbance at 600nm was measured to calculate the bacterial inhibition efficiency. The calculation formula is: Inhibition rate = (100-(OD value of the treatment group-OD value of the blank background group) ÷ (OD value of the blank control group-OD value of the blank background group)) × 100%. The results are as follows Figure 20As shown in the results, ABE-Polymyxin B incubated with 1 mM H2O2 can completely inhibit bacterial growth in the concentration range of 0.25 μg / mL to 2 μg / mL, while ABE-Colistin without H2O2 treatment has no effect on inhibiting bacterial growth, indicating that ABE-Polymyxin B modified with phenylboronic acid ester can not only sensitively respond to ROS, but also can selectively and effectively inhibit bacterial growth in a ROS environment.

[0157] 3) Bactericidal kinetics:

[0158] A series of ABE-Colistin concentrations were incubated with 1 mmol H2O2 for 30 min. Escherichia coli (ATCC35218) and Pseudomonas aeruginosa (ATCC27853) were collected by centrifugation, washed three times with PBS, and diluted to the appropriate concentration using M9 medium. A series of drug solutions (0 mM H2O2 + ABE-Colistin group, 1 mM H2O2 + ABE-Colistin group, and Colistin group) were then added to achieve a final bacterial concentration of 1 × 10 6 CFU / mL. The sample was incubated at 37°C for 0 hours: the bacterial solution of the untreated group was diluted 100 times and 1000 times, and 20 μL of the bacterial solution was taken at each dilution factor and spread on the agar plate; after incubation at 37°C for 2 hours, the bacteria were diluted 100 times and 1000 times with LB in a 96-well plate on ice, and 20 μL of the bacterial solution was taken at each dilution factor and spread on the agar plate. The agar plate was incubated at 37°C for 12 hours, and then the colonies were counted to calculate the bacterial survival rate. The calculation formula is: Bacterial survival rate = the number of colonies in the ABE-Colistin treatment group ÷ the number of colonies in the blank control group × 100%. The experimental results are as follows. Figure 21 As shown, it can be seen that the ROS-responsive ABE-Colistin modified with phenylboronic acid ester can be activated in the ROS environment and kill bacteria.

[0159] 4) Cytotoxicity assay:

[0160] HK-2 cells were used for the experiment, and 100 μL / well (about 1 x 10 4), placed in a 37°C, CO2 cell culture incubator for 24 hours; added different concentrations of ABE-Colistin solution, Colistin solution, ABE-Polymyxin B solution and Polymyxin B solution respectively; then placed in a 37°C, CO2 cell culture incubator for 24 hours, added 10 μL of MTT solution (5 mg / mL) to each well, incubated at 37°C for 4 hours to reduce MTT to formazan; aspirated the supernatant, added 200 μL of DMSO to each well to dissolve the formazan, and shook on a shaker for 30 minutes, protecting from light throughout the process; the absorbance of each well was detected by an enzyme reader at a wavelength of 490 nm to calculate the cell viability. The calculation formula is: Cell survival rate % = (OD value of drug-added cells-blank OD value) ÷ (OD value of control cells-blank OD value) × 100%. Note: No cells were added to the blank group, and other operations were the same as the experimental group. The experimental results are as follows Figure 22 As shown, the activity of HK-2 cells in the ABE-Colistin and ABE-Polymyxin B groups was basically unaffected by increasing drug concentrations, and phenylboronic acid ester-modified ABE-Colistin and ABE-Polymyxin B had no toxic effect on HK-2 cells.

[0161] Example 5:

[0162] Antibacterial activity of sugar-modified ROS-responsive polymyxin prodrugs.

[0163] Weigh the sugar-modified polymyxin prodrug (X-ABA-Colistin) powder, add it to a 1.5 mL Eppendorf tube, then add sterile water and vortex it to completely dissolve it, to prepare a series of X-ABA-Colistin concentrations; weigh the ABA-Colistin powder, add it to a 1.5 mL Eppendorf tube, then add sterile water and vortex it to completely dissolve it, to prepare a series of ABA-Colistin concentrations; weigh the ABE-Colistin powder, add it to a 1.5 mL Eppendorf tube, then add sterile water and vortex it to completely dissolve it, to prepare a series of ABE-Colistin concentrations; weigh the Colisitin powder, add it to a 1.5 mL Eppendorf tube, then add sterile water and vortex it to completely dissolve it, to prepare a series of Colistin concentrations; incubate the X-ABA-Colistin series with 1 mM H2O2 for 6 hours.

[0164] Escherichia coli (ATCC35218) and Pseudomonas aeruginosa (ATCC27853) were collected by centrifugation, washed three times with PBS, and diluted to an appropriate concentration using LB medium. A series of drug solutions were then added (0 mM H2O2 + X-ABA-Colistin group, 1 mM H2O2 + X-ABA-Colistin group, 0 mM H2O2 + ABA-Colistin group, 1 mM H2O2 + ABA-Colistin group, 0 mM H2O2 + ABE-Colistin group, 1 mM H2O2 + ABE-Colistin group, Colistin group) to make the final bacterial concentration of 1 × 10 6 CFU / mL. The samples were incubated at 37°C and removed after 24 hours. The absorbance at 600 nm was measured to determine the minimum inhibitory concentration (MIC) (i.e., the lowest concentration that completely inhibited bacterial growth). The results are shown in Table 2. All sugar-modified prodrugs exhibited superior antibacterial activity against E. coli than against P. aeruginosa. Xylose- and raffinose-modified polymyxin prodrugs exhibited superior antibacterial activity compared to other sugar-modified prodrugs. Both prodrugs had MICs of 0.5 μg / mL (calculated based on the polymyxin E content) against E. coli. The MIC for the raffinose-modified polymyxin prodrug against P. aeruginosa was 1 μg / mL, while the MIC for the xylose-modified polymyxin prodrug against P. aeruginosa was 2 μg / mL. The remaining sugar-modified polymyxin prodrugs had MICs of 1-2 μg / mL against E. coli and 2-4 μg / mL against P. aeruginosa. The MIC values ​​of the active ingredient polymyxin against Escherichia coli and Pseudomonas aeruginosa were both 0.25 μg / mL. The above data show that the modified polymyxin prodrug can not only sensitively respond to ROS, but also selectively and effectively inhibit bacterial growth in a ROS environment.

[0165] Table 2 Antibacterial activity of sugar-modified polymyxin prodrugs against Gram-negative bacteria after incubation with H2O2

[0166]

[0167] Example 6:

[0168] Maximum tolerated dose of polymyxin prodrugs in response to ROS.

[0169] The MTD of ICR mice for polymyxin prodrugs with different sugar modifications was detected. Six-week-old female ICR mice were used to test the MTD. The specific test method is as follows: a mouse is injected with a ROS-responsive polymyxin prodrug injection at a dose of 50 mg / kg (the concentration of polymyxin E) through the tail vein. After observing for 30 minutes, if there is no obvious change in the activity of the mouse, another mouse is given a dose of 100 mg / kg and observed for 30 minutes. If there is no obvious change in the activity of the mouse, the same concentration of drug is injected into two mice every 12 hours and continued to be observed. When none of the five mice died 24 hours after the injection of the drug, it is determined that the mouse's MTD for the drug is ≥100 mg / kg. Considering that the actual dosage concentration will not exceed 100 mg / kg, the drug concentration will no longer be increased. If a mouse dies during the MTD test, the existing dosage is reduced by 10 mg / kg and the above steps are repeated until the MTD value is measured. The results are as follows. Figure 23 As shown, the MTDs of the five sugar-modified polymyxin prodrugs, Ara-ABA-Colistin, Fru-ABA-Colistin, Gal-ABA-Colistin, Lac-ABA-Colistin, and Mal-ABA-Colistin, are all greater than 100 mg / kg (calculated based on the content of polymyxin E); the MTDs of Glu-ABA-Colistin and Man-ABA-Colistin are 90 mg / kg; the MTD of ABE-Colistin is 75 mg / kg, and the MTDs of Xy-ABA-Colistin and Raf-ABA-Colistin are 20 mg / kg and 15 mg / kg, respectively. Although the MTD values ​​of the polymyxin prodrugs modified with xylose and raffinose are not high, they are still 3-4 times higher than the MTD value of polymyxin (5 mg / kg), indicating that the sugar-modified polymyxin prodrugs prepared by the present invention can significantly reduce the in vivo toxicity of polymyxin.

[0170] Example 7:

[0171] Therapeutic effect of ABE-Colistin on pneumonia in mice infected with Escherichia coli ATCC35218

[0172] ICR female mice aged 6-7 weeks and weighing 25-30 g were used. Escherichia coli (ATCC35128) in the logarithmic phase were collected and washed three times with PBS to obtain a PBS bacterial suspension. The absorbance at 600 nm was measured to determine the bacterial concentration. Finally, the bacterial suspension was diluted to a concentration of 2 × 10 8CFU / mL. Mice were anesthetized by intraperitoneal injection of sterile 1% sodium pentobarbital solution. After checking that the mouse was in a deep anesthetized state, it was fixed on the operating table. The tongue of the mouse was pulled outward with sterile surgical forceps to fully expose the throat space. The auxiliary tube was inserted into the trachea along the throat, and the catheter was inserted into the auxiliary tube. 50 μL of the concentration of 2×10 8 A suspension of Escherichia coli (ATCC35128) containing 100 CFU / mL of bacteria was placed in the mouse cage after recovery and continued to be housed normally under the original feeding conditions. The infection state lasted for 12 hours. Colistin (3 mg / kg) injection solution was prepared using 5% sterile glucose solution. ABE-Colistin (3 mg / kg), ABE-Colistin (15 mg / kg), and ABE-Colistin (75 mg / kg) were first prepared with DMSO to form a stock solution, then diluted with 5% sterile glucose solution to prepare the injection solution (the volume of DMSO should not exceed 5% of the total injection volume). Treatment was administered via tail vein injection. A blank control group was injected with an equal volume of sterile PBS solution as a control. After 12 hours of treatment, the mice were sacrificed, and the intact lungs were removed. The lungs were repeatedly rinsed in sterile PBS to remove blood, then dried with sterile absorbent paper and placed in a homogenization tube. After the net lung weight is recorded, sterile water is added to each homogenate tube to make the total volume about 0.8 mL, and all homogenate tubes are kept on ice. Lung tissue homogenate is obtained using a high-speed tissue homogenizer. The tissue homogenate is placed on ice and gradiently diluted with sterile PBS. After dilution 10 times, 100 times, and 1000 times, 10 μL of each sample at each dilution factor, including the original solution, is added dropwise to the agar plate and coated. The agar plate is placed in a 37°C incubator overnight and counted. The bacterial survival rate of the tissue (CFU / g) = the number of bacteria in each sample / the tissue weight of the sample, and the mean±SD value is taken for statistical plotting. The results are shown in the figure. Figure 24 As shown, the in vivo therapeutic effect of ABE-Colistin (3 mg / mL Colistin) was similar to that of the Colistin (3 mg / mL) group. As the dose of ABE-Colistin increased, the bacterial load per gram of lung tissue in mice decreased by more than one order of magnitude, and no mice died. This result shows that ABE-Colistin can achieve superior therapeutic effects compared to Colistin as the dose increases while maintaining safety in vivo.

[0173] Example 8:

[0174] The therapeutic effect of Lac-ABA-Colistin on pneumonia-induced mice infected with Escherichia coli ATCC35218.

[0175] The infection method is the same as that in Example 7. The administration method is: Colistin (3 mg / kg) and Lac-ABA-Colistin (3 mg / kg Colistin) are diluted with 5% sterile glucose solution to form an injection solution, and the drug is administered through the tail vein injection. The blank control group is injected with an equal volume of 5% sterile glucose solution as a control, and the drugs are administered 12 hours and 18 hours after infection, respectively. After 24 hours of infection, the mice are killed, the intact lungs are taken out, and they are repeatedly rinsed in sterile PBS to remove the blood. The water is then wiped dry with sterile absorbent paper, and the lungs are placed in a homogenate tube with sterile water, and stored on ice. A high-speed tissue homogenate is used to obtain lung tissue homogenate. The tissue homogenate is placed on ice and graded diluted with sterile PBS. After dilution of 10 times, 100 times, and 1000 times, 10 μL of each sample at each dilution multiple, including the original solution, is added dropwise to the agar plate for coating. The agar plate is placed in a 37°C incubator overnight and counted. The results are shown in the figure below. Figure 25 As shown, the number of bacteria in the infected lungs of the Lac-ABA-Colistin group was reduced by 10-fold compared to the 5% glucose group, indicating that Lac-ABA-Colistin has a certain therapeutic effect on pneumonia. In addition, the number of bacteria in the infected lungs of the Lac-ABA-Colistin group and the Colistin group was comparable, indicating that the lactose-modified polymyxin prodrug has similar in vivo antibacterial activity as polymyxin.

[0176] The technical features of the above-described embodiments can be combined arbitrarily. To make the description concise, not all possible combinations of the technical features in the following embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0177] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be determined by the appended claims.

Claims

1. A polymyxin prodrug compound having the structure represented by formula (I) or a pharmaceutically acceptable salt thereof: in, R1 is selected from: R2 and R3 are each independently selected from C1-C8 alkyl; R is selected from the group consisting of: a borate ester group obtained by reacting a boric acid group with a sugar containing an adjacent dihydroxy structure, or The sugar containing an adjacent dihydroxy structure is a monosaccharide, a disaccharide or a trisaccharide.

2. A polymyxin prodrug compound or a pharmaceutically acceptable salt thereof, characterized in that: The polymyxin prodrug compound has a structure shown in the following formula (II): wherein R1 is selected from: R2 and R3 are each independently selected from C1-C8 alkyl; Each R4 and R5 are independently selected from: H, C1-C6 alkyl, or R4, R5 and the -OBO- connected thereto form the following structure: Each R7, R8, R9, R 10 Each independently selected from: H, C1-C8 alkyl, one or more R 12 Substituted C1-C8 alkyl, or R7 and R 10 is hydrogen, R8, R9 and the carbon atom to which they are connected together form one or more R 13 substituted 5-6 membered oxygen-containing heterocyclic group; Each R 12 are independently selected from: hydroxyl, carboxyl, carbonyl, aldehyde; Each R 13 Each independently selected from: H, hydroxyl, one or more R 16 Substituted C1-C6 alkyl, one or more R 15 Substituted 5-6 membered oxygen-containing heterocycloalkoxy, one or more R 17 Substituted C1-C8 alkoxy; Each R 15 Each independently selected from: H, hydroxyl, hydroxyl-substituted C1-C6 alkyl, one or more R 18 substituted 5-6 membered oxygen-containing heterocycloalkoxy group; Each R 16 Each independently selected from: H, hydroxyl, one or more R 15 substituted 5-6 membered oxygen-containing heterocycloalkoxy group; Each R 17 Each independently selected from: H, hydroxyl, hydroxyl-substituted C1-C6 alkyl, one or more R 19 substituted 5-6 membered oxygen-containing heterocyclic group; Each R 18 Each independently selected from: H, hydroxyl, hydroxyl-substituted C1-C6 alkyl; Each R 19 Each independently selected from: H, hydroxyl, hydroxyl-substituted C1-C6 alkyl, one or more R 18 Substituted 5-6 membered oxygen-containing heterocycloalkoxy group.

3. The polymyxin prodrug compound or a pharmaceutically acceptable salt thereof according to claim 1 or 2, characterized in that: R2 is selected from C3-C4 alkyl; R3 is selected from C2-C5 alkyl.

4. The polymyxin prodrug compound or a pharmaceutically acceptable salt thereof according to claim 2, characterized in that: R4 and R5 are both H, or R4, R5 and the -OBO- connected thereto form the following structure: R7, R8, R9 and R 10 Each independently selected from: H, C1-C4 alkyl, one or more R 12 Substituted C1-C4 alkyl, or R7 and R 10 is hydrogen, R8, R9 and the carbon atom to which they are connected together form one or more R 13 substituted 6-membered oxygen-containing heterocyclic group.

5. The polymyxin prodrug compound or a pharmaceutically acceptable salt thereof according to claim 4, characterized in that: Each R 13 Each independently selected from: H, hydroxyl, one or more R 16 Substituted C1-C3 alkyl, one or more R 15 Substituted 6-membered oxygen-containing heterocycloalkoxy, one or more R 17 Substituted C1-C3 alkoxy; wherein each R 15 are independently selected from: H, hydroxy, hydroxy-substituted C1-C3 alkyl, one or more R 18 Substituted 5-6 membered oxygen-containing heterocycloalkoxy; each R 16 Each independently selected from: H, hydroxyl, one or more R 15 Substituted 6-membered oxygen-containing heterocycloalkoxy; each R 17 are independently selected from: H, hydroxy, hydroxy-substituted C1-C3 alkyl, one or more R 19 Substituted 5-6 membered oxygen-containing heterocyclic group; each R 18 Each R is independently selected from: H, hydroxyl, hydroxyl-substituted C1-C3 alkyl; 19 are independently selected from: H, hydroxy, hydroxy-substituted C1-C3 alkyl, one or more R 18 Substituted 5-6 membered oxygen-containing heterocycloalkoxy group.

6. The polymyxin prodrug compound or a pharmaceutically acceptable salt thereof according to claim 2, characterized in that: R4 and R5 are both H, or R4, R5 and the -OBO- connected thereto form the following structure:

7. The polymyxin prodrug compound or a pharmaceutically acceptable salt thereof according to claim 1 or 2, characterized in that: The polymyxin prodrug compound is selected from the following compounds: Among them, R1 is R2 is R3 is selected from or, R1 is R2 is R3 is or, R1 is R2 is R3 is selected from or, R1 is R2 is R3 is selected from or, R1 is R2 is R3 is selected from or, R1 is R2 is R3 is selected from 8. Use of the polymyxin prodrug compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 7 in the preparation of an antibacterial drug; the bacteria are Gram-negative bacteria.

9. The use according to claim 8, characterized in that The bacteria are Acinetobacter baumannii, Pseudomonas aeruginosa, Salmonella typhimurium, and Enterobacteriaceae.

10. The use according to claim 9, characterized in that The bacteria are Klebsiella pneumoniae, Enterobacter cloacae, Salmonella typhimurium, Escherichia coli and Pseudomonas aeruginosa.

11. A drug for treating bacterial infection, characterized in that: The invention is prepared from active ingredients and pharmaceutically acceptable excipients, wherein the active ingredient comprises the polymyxin prodrug compound according to any one of claims 1 to 7 or a pharmaceutically acceptable salt thereof.

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