CN115490757A - Active oxygen free radical responsive polymyxin prodrug compound and its application - Google Patents

Abstract

The invention provides a polymyxin prodrug compound with a structure shown in a formula (I) or a stereoisomer or a pharmaceutically acceptable salt thereof. The polymyxin prodrug compound can effectively reduce the biological toxicity of polymyxin, is selectively activated in a bacterial infection tissue, kills bacteria at an infected part, improves the bioavailability of the polymyxin, realizes the sustainable development and utilization of classical antibiotics, expands the clinical application range of the polymyxin in the future, and has wide clinical application prospect.

Description

Active oxygen free radical responsive polymyxin prodrug compound and its application

technical field

The invention relates to the technical field of medicines, in particular to a polymyxin prodrug compound responsive to active oxygen free radicals and an application thereof.

Background technique

Antibiotic-resistant bacterial infections continue to threaten human life and health. About 700,000 people worldwide die from antibiotic resistance every year. Antibiotic resistance could kill 10 million people by 2050 if the problem goes unchecked. This means that drug-resistant infections will kill more people globally than cancer. Falling private investment and insufficient innovation in the development of new antibiotics are undermining human efforts to fight drug-resistant infections, the World Health Organization said in 2020. The 60 antimicrobial drugs currently in development, including 50 antibiotics and 10 biologics, have shown little success compared with existing treatments, and few new drugs are being developed for the most difficult-to-treat Gram-negative, drug-resistant bacteria .

Gram-negative bacteria, such as Klebsiella pneumoniae and E. coli, can cause serious and often fatal infections in people with weakened or underdeveloped immune systems, such as newborns, as well as the elderly, those undergoing surgery, and cancer Treating people poses a health and life threat. According to the Medicines Agency (EMEA), around two-thirds of deaths caused by antibiotic-resistant bacteria in Europe are caused by Gram-negative bacterial infections. Gram-negative bacteria also cause 45-70% of ventilator-associated pneumonia (VAP) cases, 20-30% of catheter-associated bloodstream infections, and other infections associated with the ICU, such as surgical site or urinary tract infections (UTIs). According to the report of the World Health Organization, the three most deadly Gram-negative drug-resistant bacteria are Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacteriaceae.

Polymyxin appeared clinically as an antimicrobial drug in the 1970s, and its main products include polymyxin B and polymyxin E. They are composed of a cationic seven-membered peptide ring and a three-membered peptide with a fatty acid chain. By destroying the bacterial membrane, the contents of the bacteria are leaked, which is called the "self-uptake mechanism", thereby achieving the purpose of killing bacteria. The clinical use of polymyxin usually requires high doses and long-term treatment to achieve the best therapeutic effect, and this treatment often has potential toxicity to the human kidney and nervous system, making polymyxin in clinical treatment more effective. Use is restricted. However, in the 21st century, due to the emerging problem of bacterial drug resistance, polymyxin has been used clinically again and is considered to be the last line of defense against multi-drug resistant Gram-negative bacteria. However, side effects induced by polymyxins are still a problem.

There are currently two colistin prodrugs in clinical use: colistin sulfate for oral and topical use and colistin sodium methanesulfonate (CMS) for injectable use. CMS becomes a less toxic inactive prodrug by modifying the five amino groups on the Dab residue in polymyxin, which is converted into the active ingredient polymyxin in vivo and exerts an antibacterial effect. Since CMS is less toxic than polymyxin sulfate, it can be used as a parenteral drug. However, there are still some problems in the clinical application of CMS, such as: about 60% of CMS will be excreted through urine within 24 hours before administration, only a small part of CMS can be hydrolyzed into active polymyxin, and the drug bioavailability Low. In addition, many researchers also tried to develop new polymyxin prodrugs. Zhu et al. used acetic acid-capped polyethylene glycol methyl ether to modify the two threonine hydroxyl groups in polymyxin E. Experiments have shown that this prodrug can effectively reduce the nephrotoxicity of polymyxin, but due to the slow conversion rate of the prodrug into the active ingredient in the body, the antibacterial activity decreases accordingly, and the dosage needs to be increased.

Contents of the invention

Based on this, the present invention provides a new class of polymyxin prodrug compounds, which exist in an inactive form in normal tissues, have very little damage to normal tissues, have little toxic and side effects, and are activated at bacterial infection sites. The stimulation of oxygen free radicals (ROS) releases active polymyxin components, thereby effectively killing bacteria.

The present invention includes the following technical solutions.

A polymyxin prodrug compound having a structure shown in formula (I) or a stereoisomer thereof or a pharmaceutically acceptable salt thereof:

Wherein, R ₁ is selected from:

R ₂ and R ₃ are independently selected from C ₁ -C ₈ alkyl groups;

R is selected from: boronic acid group, substituted or unsubstituted boronic acid ester group.

或者

In some of these embodiments, R is selected from: a borate ester group obtained by reacting a boronic acid group with a sugar containing an adjacent dihydroxy structure,

or

In some of these embodiments, the sugar containing an ortho-dihydroxy structure is a monosaccharide, a disaccharide or a trisaccharide.

In some of these embodiments, the polymyxin prodrug compound has the structure shown in the following formula (II):

Wherein, R ₁ is selected from:

R ₂ and R ₃ are independently selected from C ₁ -C ₈ alkyl groups;

Each R ₄ , R ₅ is independently selected from: H, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkyl substituted by one or more R ₆ , or R ₄ , R ₅ and -OBO connected thereto - together form the following structure:

Each R ₆ is independently selected from: H, hydroxyl, carboxyl, carbonyl, aldehyde, C ₁ -C ₆ alkoxy;

Each R ₇ , R ₈ , R ₉ , and R ₁₀ are independently selected from: H, C ₁ -C ₈ alkyl, C ₁ -C ₈ alkoxy, C ₁ -C ₈ substituted by one or more R ₁₂ Alkyl, one or more R ₁₂ substituted C ₁ -C ₈ alkoxy, carboxyl, aldehyde, or R ₇ and R ₁₀ are hydrogen, R ₈ , R ₉ and the carbon atoms connected to them together form one or more A 5-6 membered oxygen-containing heterocyclic group substituted by R ₁₃ ;

Each R ₁₂ is independently selected from: H, hydroxyl, carboxyl, carbonyl, aldehyde, C ₁ -C ₆ alkoxy, hydroxyl substituted C ₁ -C ₆ alkyl, one or more R ₁₅ substituted 5- 6-membered oxygen-containing heterocycloalkoxy;

Each R ₁₃ is independently selected from: H, hydroxyl, one or more R ₁₆ substituted C ₁ -C ₆ alkyl, one or more R ₁₅ substituted 5-6 membered oxygen-containing heterocycloalkoxy groups, one or multiple R ₁₇ substituted C ₁ -C ₈ alkoxy groups;

Each R ₁₅ is independently selected from: H, hydroxyl, hydroxyl substituted C ₁ -C ₆ alkyl, one or more R ₁₈ substituted 5-6 membered oxygen-containing heterocycloalkoxy;

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

Each R ₁₇ is independently selected from: H, hydroxyl, hydroxyl substituted C ₁ -C ₆ alkyl, one or more R ₁₉ substituted 5-6 membered oxygen-containing heterocyclic groups;

Each R ₁₈ is independently selected from: H, hydroxyl, hydroxyl substituted C ₁ -C ₆ alkyl;

Each R ₁₉ is independently selected from: H, hydroxyl, C ₁ -C ₆ alkyl substituted by hydroxyl, 5-6 membered oxygen-containing heterocycloalkoxy substituted by one or more R ₁₈ .

In some of these embodiments, R ₂ is selected from C ₃ -C ₄ alkyl; R ₃ is selected from C ₂ -C ₅ alkyl.

In some of these embodiments, both R ₄ and R ₅ are H, or R ₄ , R ₅ and -OBO- connected to them together form the following structure:

R ₇ , R ₈ , R ₉ and R ₁₀ are independently selected from: H, C ₁ -C ₄ alkyl, C ₁ -C ₄ alkoxy, C ₁ -C ₄ alkane substituted by one or more R ₁₂ group, one or more C ₁ -C ₄ alkoxy groups substituted by R ₁₂ , carboxyl, aldehyde, or R ₇ and R ₁₀ are hydrogen, R ₈ , R ₉ and the carbon atoms connected to them together form one or more A 6-membered oxygen-containing heterocyclic group substituted by R ₁₃ .

In some of these embodiments, each R ₁₂ is independently selected from: H, hydroxyl, carboxyl, carbonyl, aldehyde, C ₁ -C ₃ alkoxy, hydroxyl substituted C ₁ -C ₃ alkyl, one or more A 6-membered oxygen-containing heterocycloalkoxy group substituted by R ₁₅ ; wherein, each R ₁₅ is independently selected from: H, hydroxyl, and C ₁ -C ₃ alkyl substituted by hydroxyl.

In some of these embodiments, each R ₁₃ is independently selected from: H, hydroxyl, C ₁ -C ₃ alkyl substituted by one or more R ₁₆ , 6-membered oxygen-containing heterocyclic ring substituted by one or more R ₁₅ Alkoxy, C ₁ -C ₃ alkoxy substituted by one or more R ₁₇ ; wherein, each R ₁₅ is independently selected from: H, hydroxyl, C ₁ -C ₃ alkyl substituted by hydroxyl, one or more Each R ₁₈ substituted 5-6 membered oxygen-containing heterocycloalkoxy; each R ₁₆ is independently selected from: H, hydroxyl, one or more R ₁₅ substituted 6-membered oxygen-containing heterocycloalkoxy; each R ₁₇ are each independently selected from: H, hydroxyl, hydroxyl-substituted C ₁ -C ₃ alkyl, one or more R ₁₉ substituted 5-6 membered oxygen-containing heterocyclic groups; each R ₁₈ is independently selected from: H , hydroxyl, hydroxyl substituted C ₁ -C ₃ alkyl; each R ₁₉ is independently selected from: H, hydroxyl, hydroxyl substituted C ₁ -C ₃ alkyl, one or more R ₁₈ substituted 5-6 members Oxygen heterocycloalkoxy.

In some of these embodiments, both R ₄ and R ₅ are H, or R ₄ , R ₅ and -OBO- connected to them together form the following structure:

In some of these embodiments, the polymyxin prodrug compound is selected from the following compounds:

R₂为

R₃选自

或者，where _R1 is

_R2 is

_{R3 is} selected from

or,

R₂为

R₃为

或者， _R1 is

_R2 is

_R3 is

or,

R₂为

R₃选自

或者， _R1 is

_R2 is

_{R3 is} selected from

or,

R₂为

R₃选自

或者， _R1 is

_R2 is

_{R3 is} selected from

or,

R₂为

R₃选自

或者， _R1 is

_R2 is

_{R3 is} selected from

or,

R₂为

R₃选自

_R1 is

_R2 is

_{R3 is} selected from

The present invention also provides the application of the above-mentioned polymyxin prodrug compound, including the following technical solutions.

Application of the above-mentioned polymyxin prodrug compound or its stereoisomer or pharmaceutically acceptable salt thereof in the preparation of a medicament for treating bacterial infection.

In some of these embodiments, the bacteria are Gram-negative bacteria.

In some of these embodiments, the bacteria are Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Salmonella typhimurium, Enterobacteriaceae bacteria.

In some of these embodiments, the bacteria are Klebsiella pneumoniae, Enterobacter cloacae, Salmonella typhimurium, Escherichia coli and Pseudomonas aeruginosa.

The invention also provides an antibacterial drug.

The specific technical scheme is as follows:

A medicament for treating bacterial infection, which is prepared from active ingredients and pharmaceutically acceptable auxiliary materials. The active ingredients include the above-mentioned polymyxin prodrug compound or its stereoisomer or its pharmaceutically acceptable salt.

Based on the above technical scheme, the present invention has the following beneficial effects:

The present invention utilizes the concentration difference of ROS between the bacterial infection site and the normal tissue site to modify the structure of clinically approved polymyxin B and E, and obtain ROS-responsive polymyxin by modifying phenylboronic acid ester or phenylboronic acid Prodrug compound, the prodrug compound exists in an inactive form in normal tissues, and it is an inactive polymyxin prodrug under blood circulation conditions, which has very low damage to normal tissues and low cytotoxicity; The role of ROS, the ROS-sensitive chemical bond modified by phenylboronic acid or phenylboronic acid ester breaks the chain, and the inactive polymyxin prodrug is transformed into polymyxin with high antibacterial activity, which can kill the infection site with high selectivity. Bacteria (especially drug-resistant Gram-negative bacteria), significantly improve the antibacterial effect while significantly reducing the toxic and side effects of polymyxin. The polymyxin prodrug compound of the present invention can effectively reduce the biological toxicity of polymyxin, selectively activate in bacterial infection tissue, realize the killing of bacteria at the infection site, improve the bioavailability of polymyxin, and achieve the classic The sustainable development and utilization of antibiotics has expanded the scope of clinical application of polymyxin in the future, and has broad clinical application prospects.

Description of drawings

Fig. 1 is a synthesis route of phenylboronate-modified polymyxin, phenylboronic acid-modified polymyxin and sugar-modified polymyxin prodrug.

Fig. 2 is the ¹ H NMR characterization spectrum of 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborin)-benzylnitrophenyl carbonate (ABE).

Fig. 3 is a ¹ H NMR characterization spectrum of phenylboronate-modified polymyxin E.

Fig. 4 is a ¹ H NMR characterization spectrum of polymyxin E modified with phenylboronic acid.

Fig. 5 is a ¹ H NMR characterization pattern of polymyxin B modified with phenylboronate.

Fig. 6 is a ¹ H NMR characterization spectrum of polymyxin B modified with phenylboronic acid.

Figure 7 is the mass spectrum of phenylboronate-modified polymyxin E (a) and its ROS response (b).

Fig. 8 is a ¹ H NMR characterization spectrum of xylose-modified polymyxin prodrug.

Fig. 9 is a ¹ H NMR characterization spectrum of the arabinose-modified polymyxin prodrug.

Fig. 10 is the ¹ H NMR characterization spectrum of the fructose-modified polymyxin prodrug.

Fig. 11 is the ¹ H NMR characterization spectrum of the glucose-modified polymyxin prodrug.

Fig. 12 is a ¹ H NMR characterization spectrum of galactose-modified polymyxin prodrug.

Fig. 13 is the ¹ H NMR characterization spectrum of the mannose-modified polymyxin prodrug.

Fig. 14 is the ¹ H NMR characterization spectrum of the lactose-modified polymyxin prodrug.

Fig. 15 is a ¹ H NMR characterization spectrum of maltotriose-modified polymyxin prodrug.

Fig. 16 is the ¹ H NMR characterization spectrum of the raffinose-modified polymyxin prodrug.

Figure 17 is a graph showing the results of hemolytic activity of phenylboronate-modified polymyxin E.

Fig. 18 is a diagram showing the ROS response sensitivity results of phenylboronate-modified polymyxin E in response to different concentrations of H ₂ O ₂ for different times (5 min, 15 min, 30 min).

Fig. 19 is a graph showing the results of bacteriostatic kinetics of polymyxin E modified with phenylboronate.

Fig. 20 is a graph showing the results of bacteriostatic kinetics of polymyxin B modified by phenylboronate.

Figure 21 is a diagram showing the results of bactericidal kinetics of phenylboronate-modified polymyxin E.

Figure 22 is a graph showing the cytotoxic MTT results of phenylboronate-modified polymyxin E and polymyxin B on HK-2 cells.

Figure 23 is the maximum tolerated dose of sugar-modified ROS-responsive polymyxin prodrugs.

Figure 24 shows the therapeutic effect of phenylboronate-modified polymyxin E on pneumonia.

Figure 25 shows the therapeutic effect of lactose-modified polymyxin E on pneumonia.

detailed description

The experimental methods without specific conditions indicated in the following examples of the present invention are generally in accordance with conventional conditions, or in accordance with the conditions suggested by the manufacturer. Various commonly used chemical reagents used in the examples are all commercially available products.

Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by one of ordinary skill in the technical field of the present invention. The terms used in the description of the present invention are only for the purpose of describing specific embodiments, and are not used to limit the present invention.

The terms "including" and "having" and any variations thereof in the present invention are intended to cover a non-exclusive inclusion. For example, a process, method, device, product or equipment that includes a series of steps is not limited to the listed steps or modules, but optionally also includes steps that are not listed, or optionally also includes for these processes, Other steps inherent in a method, product, or apparatus.

The "plurality" mentioned in the present invention means two or more. "And/or" describes the association relationship of associated objects, indicating that there may be three types of relationships, for example, A and/or B may indicate: A exists alone, A and B exist simultaneously, and B exists independently. The character "/" generally indicates that the contextual objects are an "or" relationship.

^In compounds described herein, when any variable (eg, R4, R5, etc. ⁾ occurs more than once in any component, its definition at each occurrence is independent of its definition at each other occurrence. Also, combinations of substituents and variables are permissible only if such combinations render the compounds stable. 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 it means that such bonds are only to any suitable carbon atoms of adjacent rings. It is understood that one of ordinary skill in the art can select substituents and substitution patterns on the compounds of the present invention to provide compounds that are chemically stable and can be readily synthesized from readily available starting materials by skill in the art and by methods set forth below. If a substituent is itself substituted with more than one group, it is understood that these groups may be on the same carbon atom or on different carbon atoms, so long as the structure is stabilized. The phrase "optionally substituted with one or more substituents" is considered equivalent to the phrase "optionally substituted with at least one substituent" and in this case preferred embodiments will have 0-3 substituents.

The term "alkyl" as used herein is meant to include both branched and straight chain saturated aliphatic hydrocarbon groups having the specified number of carbon atoms. For example, the definition of "C ₁ -C ₆ " in "C ₁ -C ₆ alkyl" includes groups having 1, 2, 3, 4, 5 or 6 carbon atoms arranged in a linear or branched chain. For example, "C ₁ -C ₆ alkyl" specifically includes methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, pentyl, hexyl.

The term "alkoxy" refers to a group with an -O-alkyl structure, such as -OCH ₃ , -OCH ₂ CH ₃ , -OCH ₂ CH ₂ CH ₃ , -O-CH ₂ CH(CH ₃ ) ₂ , - OCH ₂ CH ₂ CH ₂ CH ₃ , -O-CH(CH ₃ ) ₂ and the like.

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

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

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

The pharmaceutically acceptable adjuvant used in the medicine for treating bacterial infection of the present invention refers to: one or more compatible solid or liquid fillers or gel substances, which are suitable for human use and must have sufficient purity and low toxicity.

"Compatibility" here refers to the ability of each component in the composition to be compatible with the active ingredient of the present invention (polymyxin prodrug compound or its stereoisomer or pharmaceutically acceptable salt thereof) and between them Blending with each other without significantly reducing the efficacy of the active ingredients.

The pharmaceutically acceptable adjuvant used in the medicament for treating bacterial infection of the present invention includes but not limited to one or more of the following materials: solvent, excipient, filler, compatibilizer, binder, humectant , disintegrating agent, retarding agent, absorption accelerator, adsorbent, diluent, solubilizer, emulsifier, lubricant, wetting agent, suspending agent, flavoring agent and perfume at least one.

)、润湿剂(如十二烷基硫酸钠)、着色剂、调味剂、稳定剂、抗氧化剂、防腐剂、无热原水等。Examples of pharmaceutically acceptable excipients include cellulose and its derivatives (such as sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid , magnesium stearate), calcium sulfate, vegetable oil (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifiers (such as

), wetting agent (such as sodium lauryl sulfate), coloring agent, flavoring agent, stabilizer, antioxidant, preservative, pyrogen-free water, etc.

The administration method of the active ingredient or pharmaceutical composition of the present invention is not particularly limited, and representative administration methods include (but not limited to): oral, rectal, parenteral (intravenous, intramuscular or subcutaneous) and the like.

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

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

(a) fillers or extenders such as starch, lactose, sucrose, glucose, mannitol and silicic acid;

(b) binders such as hydroxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and acacia;

(c) humectants, for example, glycerin;

(d) disintegrants such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate;

(e) slow solvent, for example, paraffin;

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

(g) humectants, for example, cetyl alcohol and glyceryl monostearate;

(h) Adsorbents, for example, kaolin;

(i) Lubricants such as 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 buffering agents.

The solid dosage form can also be prepared with coatings and shell materials, such as enteric coatings and other materials known in the art. They may contain opacifying agents and the release of the active ingredient from such compositions may be in a certain part of the alimentary canal in a delayed manner. Examples of usable embedding components are polymeric substances and waxy substances.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active ingredient, liquid dosage forms may contain inert diluents conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1 , 3-butanediol, dimethylformamide and oils, especially cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or mixtures of these substances, etc. Besides such inert diluents, the compositions can also contain adjuvants, such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

Suspensions, in addition to the active ingredient, may contain suspending agents, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar, mixtures of these substances, and the like.

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 vehicles include water, ethanol, polyols, and suitable mixtures thereof.

One of the examples of the ROS-responsive polymyxin prodrug compound of the present invention can be synthesized according to the following method: with p-nitrophenyl chloroformate and 4-(hydroxymethyl) phenylboronic acid pinacol ester as raw materials , using triethylamine as an acidic agent, react to obtain 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborin)-benzylnitrophenyl carbonate (ABE). ABE was reacted with polymyxin E and polymyxin B to obtain phenylboronate-modified ROS-responsive polymyxin E and polymyxin B prodrugs. Phenylboronic acid ester-modified polymyxin E and polymyxin B prodrugs were hydrolyzed with concentrated hydrochloric acid to obtain phenylboronic acid-modified ROS-responsive polymyxin E and polymyxin B prodrugs. Phenylboronic acid-modified ROS-responsive polymyxins E and B were reacted with compounds containing ortho-dihydroxy groups to obtain sugar-modified polymyxins E and B prodrugs with improved water solubility.

In some of these embodiments, the synthetic method of polymyxin E or polymyxin B prodrug modified by phenylboronic acid comprises the following steps:

(1) Dissolve p-nitrophenyl chloroformate and 4-(hydroxymethyl) phenylboronic acid pinacol ester with anhydrous tetrahydrofuran, add anhydrous triethylamine as an ancidic agent, after the reaction finishes, use ethyl acetate ester extraction, and successively washed with saturated sodium bicarbonate and dilute hydrochloric acid, dried with anhydrous magnesium sulfate, suction filtered, rotary evaporated, passed through a silica gel column, and rotary evaporated to prepare 4-(4,4,5,5-tetramethyl -1,3,2-dioxaborolane)-benzylnitrophenyl carbonate (ABE).

(2) Dissolve polymyxin E or polymyxin B with saturated sodium bicarbonate, add ABE dissolved in dimethylformamide, after the reaction is over, remove the solvent by distillation under reduced pressure, dissolve in dichloromethane, and centrifuge , take the supernatant, remove the solvent, precipitate in ether, and dialyze to obtain phenylboronate-modified polymyxin E or phenylboronate-modified polymyxin B.

(3) Dissolve the polymyxin prodrug modified by phenylboronic acid ester with dimethylformamide, add concentrated hydrochloric acid dropwise, and dialyze after the reaction to obtain polymyxin E modified by phenylboronic acid or polymyxa modified by phenylboronic acid Prime B.

In some of these embodiments, a method for preparing a sugar-modified ROS-responsive polymyxin E and polymyxin B prodrug compound based on the reaction of a sugar containing an ortho-dihydroxy structure and phenylboronic acid comprises the following steps : The polymyxin E or polymyxin B prodrug modified by phenylboronic acid reacts with the sugar containing the ortho-dihydroxy structure in 0.1M NaOH solution, and dialyzes with 0.01M NaOH solution after the reaction to obtain water-soluble Improved phenylboronic acid-modified polymyxin E or related derivatives of phenylboronic acid-modified polymyxin B.

A series of ROS-responsive polymyxin prodrug compounds can be prepared by the preparation method described above. In the present invention, the ROS-responsive polymyxin B prodrug of phenylboronate modification is expressed as ABE-Colistin; the ROS-responsive polymyxin B prodrug of phenylboronate modification is expressed as ABE-Polymyxin B; The boronic acid-modified ROS-responsive polymyxin B prodrug is represented as ABA-Colistin; the phenylboronate-modified ROS-responsive polymyxin B prodrug is represented as ABA-Polymyxin B; The sugar-modified ROS-responsive polymyxin E prodrug obtained by reacting sugar with phenylboronic acid is expressed as X-ABA-Colistin, where X represents the abbreviation of different sugars, such as the ROS-responsive polymyxin E obtained by reacting lactose The prodrug is represented as Lac-ABA-Colistin, the ROS-responsive polymyxin E prodrug obtained by reacting with fructose is represented as Fru-ABA-Colistin, and the ROS-responsive polymyxin E prodrug obtained by reacting with galactose is represented It is Gal-ABA-Colistin; the sugar-modified ROS-responsive polymyxin B prodrug based on the reaction of a sugar containing an ortho-dihydroxy structure and phenylboronic acid is expressed as X-ABA-Polymyxin B, where X represents the different sugar abbreviation.

Both polymyxin E and polymyxin B used in the following examples are commercially available products. The known substructures of polymyxin E and polymyxin B are as follows:

The following are specific examples.

Example 1: Preparation of phenylboronic acid-modified ROS-responsive ABA-Colistin

1) Preparation of 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborane)-benzylnitrophenyl carbonate (ABE)

Weigh 0.47g of p-nitrophenyl chloroformate and 0.5g of 4-(hydroxymethyl) phenylboronic acid pinacol ester in a 100mL round bottom flask, dissolve with 20mL of anhydrous tetrahydrofuran (THF), and dissolve in Add 600 μL of anhydrous triethylamine under stirring conditions, stir and react at room temperature for 1 h; add 30 mL of ethyl acetate, transfer to a 125 mL separatory funnel, let stand, add 250 mL of saturated NaHCO ₃ solution three times for washing, discard The lower aqueous layer was washed by adding 250mL HCl (1M) three times, discarding the lower aqueous layer, pouring the upper liquid into a 100mL beaker, adding an appropriate amount of anhydrous MgSO ₄ , stirring overnight to dry, and then suction-filtering it into a 500mL round bottom In the flask, add 2mL of silica gel to the solution obtained by suction filtration, rotary evaporation, dry loading, column chromatography purification, eluent is ethyl acetate and n-hexane (V:V=1:20), to obtain 0.52g ABE , the yield is 61.2%, and its ¹ H NMR characterization spectrum is shown in FIG. 2 .

2) Preparation of ABE-Colistin modified by phenylboronate

Weigh 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 combination of polymyxin E ₁ and Polymyxin E ₂ mixture of two components). In a 25mL round-bottom flask, add 4mL saturated sodium bicarbonate, stir to dissolve, add ABE solution (200mgABE dissolved in 1mL dimethylformamide (DMF)) into the round-bottom flask, stir for 24h, at 50°C The solvent was distilled off under reduced pressure, dissolved with 10 mL of dichloromethane, centrifuged to remove the precipitate, concentrated solution, precipitated with ether, dissolved with 5 mL of dimethyl sulfoxide (DMSO), dialyzed with water successively, and freeze-dried to obtain 103 mg of phenylboron The yield of ester-modified polymyxin E (referred to as ABE-Colistin) was 48.4%. Its ¹ HNMR characterization spectrum is shown in FIG. 3 , and its mass spectrum is shown in figure a in FIG. 7 .

3) Preparation of ABA-Colistin modified with phenylboronic acid

Weigh 50 mg of ABE-Colistin in 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 for 6 h, dialyze with water, freeze-dry to obtain 35 mg of phenylboronic acid-modified polymyxa The yield of element E (referred to as ABA-Colistin) was 84.1%, and its ¹ H NMR characterization spectrum is shown in FIG. 4 .

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

1) Preparation of 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborane)-benzylnitrophenyl carbonate (ABE)

With embodiment 1.

2) Preparation of phenylboronate-modified ABE-Polymyxin B

The method is the same as in Example 1, using polymyxin B sulfate (purchased from Dalian Meilun Biotechnology Co., Ltd.; CAS: 1405-20-5, product number: MB1188; its polymyxin B contains polymyxin B ₁ , polymyxin B ₂ , polymyxin B ₃ , and mixture of four structures of Ile-polymyxin B ₁ ) were prepared to obtain 105 mg of phenylboronate-modified polymyxin B (denoted as ABE- Polymyxin B), the yield is 50.4%, and its ¹ H NMR characterization spectrum is shown in FIG. 5 .

3) Preparation of ABA-Polymyxin B modified by phenylboronic acid

The method was the same as in Example 1, and 34 mg of phenylboronic acid-modified polymyxin B (referred to as ABA-Polymyxin B) was prepared with a yield of 80.9%. The ¹ H NMR characterization pattern is shown in FIG. 6 .

Example 3: Preparation of sugar-modified ROS-responsive polymyxin E prodrugs

Weigh 80mg (0.039mmol) of ABA-Colistin into a round bottom flask, add 4mL of 0.1M NaOH solution, stir to dissolve, add 664.8mg (1.012mmol) of lactose, stir for 24h, dialyze with 0.01M NaOH solution, freeze-dry, 88 mg of lactose-modified ROS-responsive polymyxin E prodrug was prepared with a yield of 62.8%.

According to the method of this embodiment, other sugar-modified polymyxin E prodrugs were prepared, and the feeding ratio is shown in the following table 1:

Table 1

The ¹ H NMR characterization spectrum of the xylose-modified polymyxin E prodrug is shown in Figure 8; the ¹ H NMR characterization spectrum of the arabinose-modified polymyxin E prodrug is shown in Figure 9; the fructose-modified polymyxin E prodrug The ¹ H NMR characterization spectrum of colistin E prodrug is shown in Figure 10; the ¹ H NMR characterization spectrum of glucose-modified polymyxin E prodrug is shown in Figure 11; the galactose-modified polymyxin E prodrug The ¹ H NMR characterization spectrum of the drug is shown in Figure 12; the ¹ H NMR characterization spectrum of the mannose-modified polymyxin E prodrug is shown in Figure 13; the ¹ H NMR characterization of the lactose-modified polymyxin E prodrug The spectrum is shown in Figure 14; the ¹ H NMR characterization spectrum of the maltotriose-modified polymyxin E prodrug is shown in Figure 15; the ¹ H NMR characterization spectrum of the raffinose-modified polymyxin E prodrug is shown in Figure 16 shows.

Example 4:

Antibacterial activity, hemolytic toxicity and cytotoxicity of ROS-responsive polymyxin prodrugs ABE-Colistin and ABE-Polymyxin B.

1) Hemolysis test:

Take an appropriate amount of sheep whole blood in a centrifuge tube, dilute it with 1×PBS, and prepare a 4% (v/v) sheep blood solution; use PBS to prepare a series of drug solutions, add 100 μL/tube to the EP tube, and then add Equal volume of 4% sheep blood; PBS and equal volume of 4% sheep blood were mixed as a negative control group; 0.1% Triton and equal volume of 4% sheep blood were mixed as a positive control group; samples were mixed and placed in Incubate at 37°C for 60min. Subsequently, the sample was placed at 4° C. for centrifugation (1000 rpm/5 min); after centrifugation, 100 μL of the supernatant was taken into a 96-well plate, and the absorbance was measured at 576 nm 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%. Figure 17 shows the hemolytic activity of Colistin and ABE-Colistin, indicating that polymyxin E modified with phenylboronate has lower hemolytic activity and can be better used in vivo.

2) Antibacterial kinetics:

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

Escherichia coli (ATCC35218) was collected by centrifugation, washed 3 times with PBS, the bacterial solution was diluted to an appropriate concentration with M9 medium, and a series of drug solutions were added (0mM H ₂ O ₂ +ABE-Colistin group, 1mM H ₂ O ₂ + ABE-Colistin group, 2mM H ₂ O ₂ +ABE-Colistin group, Colistin group), so that the final concentration of bacteria was 1×10 ⁶ CFU/mL. The samples were incubated at 37°C, and the samples were taken out after 24 hours, the absorbance value at 600nm was measured, and the bacterial inhibition efficiency was calculated. The calculation formula is: inhibition rate=(100-(OD value of treatment group-OD value of blank background group)÷(OD value of blank control group-OD value of blank background group))×100%. The results are shown in Figure 18. At 5 minutes, ABE-Colistin incubated with 1mM H ₂ O ₂ could not completely inhibit the growth of bacteria, but ABE-Colistin incubated with 2mM H ₂ O ₂ could completely inhibit the growth of bacteria. It was known from the previous experimental data that Colisitin The MIC concentration of ABE-Colistin is about 0.25μg/mL; however, after prolonging the incubation time, ABE-Colistin can completely inhibit the growth of bacteria at a concentration of 0.25μg/mL after incubation under the condition of 1mM H ₂ O ₂ ; after incubation for 15 minutes and 30 minutes, the curve trend It is almost consistent, indicating that the phenylboronate-modified ABE-Colistin has higher sensitivity and faster response to ROS, and can effectively inhibit bacterial growth in the ROS environment.

Incubate serial concentrations of ABE-Colistin with 1mM H ₂ O ₂ for 30 minutes; centrifuge to collect E. Salmonella (ATCC14028), Escherichia coli (ATCC25922), washed 3 times with PBS, diluted the bacterial solution to an appropriate concentration with LB medium, and then added a series of drug solutions (0mM H ₂ O ₂ +ABE-Colistin group, 1mM H ₂ O ₂ + ABE-Colistin group, Colistin group), so that the final concentration of bacteria was 1×10 ⁶ CFU/mL. The samples were incubated at 37°C, and the samples were taken out after 24 hours, the absorbance value at 600nm was measured, and the bacterial inhibition efficiency was calculated. Calculation formula: antibacterial rate=(100-(OD value of treatment group-OD value of blank background group)÷(OD value of blank control group-OD value of blank background group))×100%. The results are shown in Figure 19, ABE-Colistin incubated with 1mM H ₂ O ₂ can completely inhibit bacterial growth in the concentration range of 0.25 μg/mL to 32 μg/mL, while ABE-Colistin without H ₂ O ₂ treatment has no The effect of inhibiting bacterial growth indicated that phenylboronate-modified ABE-Colistin could not only sensitively respond to ROS, but also selectively effectively inhibit the growth of a variety of Gram-negative bacteria in the ROS environment.

Incubate serial concentrations of ABE-Polymyxin B with 1 mmol H ₂ O ₂ for 30 minutes; collect Escherichia coli (ATCC35218) and Pseudomonas aeruginosa (ATCC27853) by centrifugation, wash with PBS three times, dilute the bacterial solution to an appropriate concentration with LB medium, and then A series of drug solutions (0mM H ₂ O ₂ +ABE-Colistin group, 1mM H ₂ O ₂ +ABE-Colistin group, Colistin group) were added to make the final concentration of bacteria 1×10 ⁶ CFU/mL. The samples were incubated at 37°C, and the samples were taken out after 24 hours, the absorbance value at 600nm was measured, and the bacterial inhibition efficiency was calculated. Calculation formula: antibacterial rate=(100-(OD value of treatment group-OD value of blank background group)÷(OD value of blank control group-OD value of blank background group))×100%. The results are shown in Figure 20, ABE-Polymyxin B incubated with 1mM H ₂ O ₂ can completely inhibit bacterial growth in the concentration range of 0.25 μg/mL-2 μg/mL, while ABE-Colistin without H ₂ O ₂ treatment does not Any effect of inhibiting bacterial growth indicated that phenylboronate-modified ABE-Polymyxin B could not only sensitively respond to ROS, but also selectively effectively inhibit bacterial growth under ROS environment.

3) Sterilization kinetics:

Incubate serial concentrations of ABE-Colistin with 1 mmol H ₂ O ₂ for 30 min, respectively. Escherichia coli (ATCC35218) and Pseudomonas aeruginosa (ATCC27853) were collected by centrifugation, washed three times with PBS, diluted with M9 medium to an appropriate concentration, and then added a series of drug solutions (0mM H ₂ O ₂ +ABE-Colistin group , 1 mM H ₂ O ₂ +ABE-Colistin group, Colistin group), so that the final concentration of bacteria was 1×10 ⁶ CFU/mL. Incubate the sample at 37°C for 0 hours: Dilute the bacterial solution of the non-medicated group 100 times and 1000 times, and spread 20 μL of the bacterial solution on the agar plate for each dilution; after incubation at 37°C for 2 hours, the 96-well plate was Dilute the bacteria 100 times and 1000 times with LB on ice, and spread 20 μL of the bacterial solution on the agar plate for each dilution multiple, place the agar plate at 37 ° C for 12 hours, count the colonies, and 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 shown in Figure 21. It can be seen that the phenylboronate-modified ROS-responsive ABE-Colistin can be activated in the ROS environment and kill bacteria.

4) Cytotoxicity test:

HK-2 cells were selected for the experiment, 100 μL/well (about 1x 10 ⁴ ) of cells were added to a 96-well plate, and cultured in a CO ₂ cell incubator at 37°C for 24 hours; different concentrations of ABE-Colistin solution, Colistin solution, ABE-Polymyxin B solution, and Polymyxin B solution; then placed in a 37°C, CO ₂ cell culture incubator and incubated for 24 hours, added 10 μL of MTT solution (5 mg/mL) to each well, and incubated at 37°C for 4 hours to make MTT Reduce to formazan; suck out the supernatant, add 200 μL DMSO to each well to dissolve the formazan, shake well on the shaker for 30 minutes, and avoid light during the whole process; detect the absorbance value of each well at a wavelength of 490nm with a microplate reader, and calculate the cell viability . The calculation formula is: cell viability%=(OD value of drug-added cells-blank OD value)÷(control cell OD value-blank OD value)×100%. Note: Cells were not added to the blank group, and other operations were the same as the experimental group. The experimental results are shown in Figure 22. The activity of HK-2 cells in the ABE-Colistin and ABE-Polymyxin B groups was basically not affected with the increase of the drug concentration. -2 cells have no toxic effect.

Example 5:

Antibacterial activity of sugar-modified ROS-responsive polymyxin prodrugs.

Weigh the sugar-modified polymyxin prodrug (X-ABA-Colistin) powder, add it to a 1.5mL Ep tube, add sterile water and vortex to dissolve it completely, and prepare X-ABA-Colistin with a series concentration ;Weigh the ABA-Colistin powder, add it to a 1.5mL Ep tube, add sterile water and vortex to dissolve it completely, and prepare ABA-Colistin with a series concentration; weigh the ABE-Colistin powder, add it to a 1.5mL Ep tube Add sterile water and vortex to dissolve completely to prepare ABE-Colistin with a series concentration; weigh Colisitin powder, add it to a 1.5mL Ep tube, add sterile water to vortex to dissolve completely, and prepare A series of concentrations of Colistin; a series of concentrations of X-ABA-Colistin were incubated with 1mM H ₂ O ₂ for 6h.

Escherichia coli (ATCC35218) and Pseudomonas aeruginosa (ATCC27853) were collected by centrifugation, washed three times with PBS, diluted with LB medium to an appropriate concentration, and then added a series of drug solutions (0mM H ₂ O ₂ +X-ABA- Colistin group, 1mM H ₂ O ₂ +X-ABA-Colistin group, 0mM H ₂ O ₂ +ABA-Colistin group, 1mM H ₂ O ₂ +ABA-Colistin group, 0mM H ₂ O ₂ +ABE-Colistin group, 1mM H ₂ O ₂ +ABE-Colistin group, Colistin group), so that the final concentration of bacteria was 1×10 ⁶ CFU/mL. The sample was incubated at 37° C., and the sample was taken out after 24 hours, and the absorbance value at 600 nm was measured to obtain the minimum inhibitory concentration (ie, the minimum concentration that can completely inhibit the growth of bacteria). The results are shown in Table 2. It can be seen from the table that the antibacterial activity of all sugar-modified prodrugs to Escherichia coli is better than that to Pseudomonas aeruginosa. Compared with other sugar-modified prodrugs, the polymyxin prodrugs modified by xylose and raffinose showed better antibacterial properties, and the MIC values of the two prodrugs against E. coli were both 0.5 μg/mL Colistin E content calculation), the MIC value of the polymyxin prodrug of raffinose modification to Pseudomonas aeruginosa is 1g/mL, the polymyxin prodrug of xylose modification is to Pseudomonas aeruginosa The MIC value of 2μg/mL. The MIC values of the remaining sugar-modified polymyxin prodrugs to Escherichia coli were 1-2 μg/mL, and the MIC values to Pseudomonas aeruginosa were 2-4 μg/mL. The MIC values of the active ingredient polymyxin against Escherichia coli and Pseudomonas aeruginosa are both 0.25 μg/mL. The above data indicated that the modified polymyxin prodrug could not only sensitively respond to ROS, but also selectively effectively inhibit the growth of bacteria in ROS environment.

Table 2 Antibacterial activity of sugar-modified polymyxin prodrugs against Gram-negative bacteria after incubation with H ₂ O ₂

Embodiment 6:

Maximum tolerated dose of ROS-responsive polymyxin prodrugs.

Examination of the MTD of different sugar-modified polymyxin prodrugs in ICR mice. Six-week-old female ICR mice were used to test MTD, and the specific test method was as follows: a mouse was injected with ROS-responsive polymyxin before the tail vein at a dose of 50 mg/kg (the concentration of polymyxin E). After 30 minutes of observation, if there is no significant change in the activity of the mouse, then give another mouse a dose of 100 mg/kg and observe for 30 minutes. The mice were injected with the same concentration of the drug and continued to observe. When none of the five mice died 24 hours after the injection of the drug, it was determined that the MTD of the mouse was greater than or equal to 100 mg/kg. Considering that the actual administration concentration will not exceed 100mg/kg, the drug concentration will not be increased any further. If the mice die during the MTD test, reduce the current dose by 10 mg/kg and repeat the above steps until the MTD value is measured. The results are shown in Figure 23, wherein Ara-ABA-Colistin, Fru-ABA-Colistin, Gal-ABA-Colistin, Lac-ABA-Colistin, Mal-ABA-Colistin these five sugar modified polymyxin prodrugs The MTDs are all greater than 100mg/kg (calculated based on the content of polymyxin E); the MTDs of Glu-ABA-Colistin and Man-ABA-Colistin are 90mg/kg; the MTDs of ABE-Colistin are 75mg/kg, The MTDs of Colistin and Raf-ABA-Colistin are 20mg/kg and 15mg/kg, respectively. Although the MTD value of the polymyxin prodrug modified by xylose and raffinose is not high, it is still 3-4 times higher than the MTD value (5mg/kg) of polymyxin, which shows that the sugar modification prepared by the present invention The polymyxin prodrug can significantly reduce the in vivo toxicity of polymyxin.

Embodiment 7:

Therapeutic effect of ABE-Colistin on pneumonia mice infected with Escherichia coli ATCC35218

ICR female mice aged 6-7 weeks and weighing 25-30 g were used. Collect the escherichia coli (ATCC35128) that is in the logarithmic phase, wash 3 times with PBS, after obtaining the PBS bacterial suspension, measure the absorbance value at 600nm to determine the bacterial concentration, and finally dilute and prepare the required bacterial suspension concentration of the animal infection model as 2×10 ⁸ CFU/mL. Mice were anesthetized by intraperitoneal injection of sterile 1% pentobarbital sodium solution. After checking that the mouse is in a state of deep anesthesia, fix it on the operating table, pull the tongue of the mouse outward with sterile surgical forceps, fully expose the throat space, insert the auxiliary tube into the trachea along the throat, and then Insert the catheter into the auxiliary tube. Inject 50 μL of Escherichia coli (ATCC35128) bacterial suspension with a concentration of 2×10 ⁸ CFU/mL into the lungs of each mouse, wait for it to wake up, transfer it to the mouse cage, and continue to raise normally under the original feeding conditions, and the infection state lasts for 12 hours . Colistin (3mg/kg) injection solution is prepared with 5% sterile glucose solution, ABE-Colistin (3mg/kg Colistin), ABE-Colistin (15mg/kg Colistin), ABE-Colistin (75mg/kg Colistin) is first prepared with DMSO into mother liquor, then diluted with 5% sterile glucose solution into injection solution (the volume of DMSO should not exceed 5% of the total injection volume), and treated by tail vein injection, and the blank control group was injected with an equal volume of sterile PBS solution as a contrast . After 12 hours of treatment, the mice were killed, and the complete lungs were taken out, rinsed repeatedly in sterile PBS to remove blood, then dried with sterile absorbent paper, and put into a homogenization tube. After the net lung weight was recorded, sterile water was added to each homogenate tube to make a total volume of about 0.8 mL, and all homogenate tubes were stored on ice. A homogenate of lung tissue was obtained using a high-speed tissue homogenizer. Put the tissue homogenate on ice, and use sterile PBS to carry out serial dilution. After diluting 10 times, 100 times, and 1000 times, take 10 μL of each sample for each dilution factor including the original solution and drop it on the agar plate to coat the plate. The agar plates were counted overnight in an incubator at 37°C. 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 was used for statistical mapping. The results are shown in Figure 24, the in vivo therapeutic effect of ABE-Colistin (3mg/mL Colistin) was similar to that of Colistin (3mg/mL) group, and as the dose of ABE-Colistin increased, the amount of bacteria per gram of mouse lung tissue decreased by 1 more than an order of magnitude, and the mice did not die. The results show that ABE-Colistin can obtain better therapeutic effect than Colistin with the increase of dose while ensuring safety in vivo.

Embodiment 8:

Therapeutic effect of Lac-ABA-Colistin on pneumonia mice infected with Escherichia coli ATCC35218.

The infection mode is the same as in Example 7. The administration method is: Colistin (3mg/kg), Lac-ABA-Colistin (3mg/kg Colistin) are diluted with 5% sterile glucose solution to form an injection solution, administered by tail vein injection, and the blank control group is injected with an equal volume of As a control, 5% sterile glucose solution was administered at 12 hours and 18 hours after infection respectively. At 24 hours after infection, the mice were sacrificed, and the complete lungs were taken out, rinsed repeatedly in sterile PBS to remove blood, and then treated with sterile Bacteria-absorbing paper to dry the water, put into a homogenization tube with sterile water, and store on ice. A homogenate of lung tissue was obtained using a high-speed tissue homogenizer. Put the tissue homogenate on ice, and use sterile PBS to carry out serial dilution. After diluting 10 times, 100 times, and 1000 times, take 10 μL of each sample for each dilution factor including the stock solution and drop it on the agar plate to coat the plate. , and the agar plates were counted overnight in a 37°C incubator. The results are shown in Figure 25, the number of bacteria in the infected lungs with Lac-ABA-Colistin was reduced by 10 times compared to the 5% glucose group, showing 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 was comparable to that of the Colistin group, indicating that the lactose-modified polymyxin prodrug and polymyxin have similar antibacterial activity in vivo.

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

The above-mentioned embodiments only express several implementation modes of the present invention, and the description thereof is relatively specific and detailed, but should not be construed as limiting the patent scope of the present invention. It should be pointed out that those skilled in the art can make several modifications and improvements without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent for the present invention should be based on the appended claims.

Claims (15)

1. A polymyxin prodrug compound having a structure shown in formula (I) or its stereoisomer or a pharmaceutically acceptable salt thereof:

Wherein, R ₁ is selected from:

R ₂ and R ₃ are independently selected from C ₁ -C ₈ alkyl groups; R is selected from: boronic acid group, substituted or unsubstituted boronic acid ester group. 2. The polymyxin prodrug compound or its stereoisomer or its pharmaceutically acceptable salt according to claim 1, characterized in that R is selected from the group consisting of boronic acid groups and those containing adjacent dihydroxyl structures The boronate group obtained by the reaction of sugar,

or

3. The polymyxin prodrug compound or its stereoisomer or its pharmaceutically acceptable salt according to claim 1, is characterized in that, the described sugar containing adjacent dihydroxy structure is monosaccharide, dihydroxy sugar or trisaccharides. 4. The polymyxin prodrug compound or its stereoisomer or pharmaceutically acceptable salt thereof according to claim 1, wherein the polymyxin prodrug compound has the following formula (II) Structure shown:

Wherein, R ₁ is selected from:

R ₂ and R ₃ are independently selected from C ₁ -C ₈ alkyl groups; Each R ₄ , R ₅ is independently selected from: H, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkyl substituted by one or more R ₆ , or R ₄ , R ₅ and -OBO connected thereto - together form the following structure:

Each R ₆ is independently selected from: H, hydroxyl, carboxyl, carbonyl, aldehyde, C ₁ -C ₆ alkoxy; Each R ₇ , R ₈ , R ₉ , and R ₁₀ are independently selected from: H, C ₁ -C ₈ alkyl, C ₁ -C ₈ alkoxy, C ₁ -C ₈ substituted by one or more R ₁₂ Alkyl, one or more R ₁₂ substituted C ₁ -C ₈ alkoxy, carboxyl, aldehyde, or R ₇ and R ₁₀ are hydrogen, R ₈ , R ₉ and the carbon atoms connected to them together form one or more A 5-6 membered oxygen-containing heterocyclic group substituted by R ₁₃ ; Each R ₁₂ is independently selected from: H, hydroxyl, carboxyl, carbonyl, aldehyde, C ₁ -C ₆ alkoxy, hydroxyl substituted C ₁ -C ₆ alkyl, one or more R ₁₅ substituted 5- 6-membered oxygen-containing heterocycloalkoxy; Each R ₁₃ is independently selected from: H, hydroxyl, one or more R ₁₆ substituted C ₁ -C ₆ alkyl, one or more R ₁₅ substituted 5-6 membered oxygen-containing heterocycloalkoxy groups, one or multiple R ₁₇ substituted C ₁ -C ₈ alkoxy groups; Each R ₁₅ is independently selected from: H, hydroxyl, hydroxyl substituted C ₁ -C ₆ alkyl, one or more R ₁₈ substituted 5-6 membered oxygen-containing heterocycloalkoxy; Each R ₁₆ is independently selected from: H, hydroxyl, one or more R ₁₅ substituted 5-6 membered oxygen-containing heterocycloalkoxy; Each R ₁₇ is independently selected from: H, hydroxyl, hydroxyl substituted C ₁ -C ₆ alkyl, one or more R ₁₉ substituted 5-6 membered oxygen-containing heterocyclic groups; Each R ₁₈ is independently selected from: H, hydroxyl, hydroxyl substituted C ₁ -C ₆ alkyl; Each R ₁₉ is independently selected from: H, hydroxyl, C ₁ -C ₆ alkyl substituted by hydroxyl, 5-6 membered oxygen-containing heterocycloalkoxy substituted by one or more R ₁₈ . 5. The polymyxin prodrug compound or its stereoisomer or pharmaceutically acceptable salt thereof according to any one of claims _1-4 , wherein R is selected from C ₃ -C ₄ alkane group; R ₃ is selected from C ₂ -C ₅ alkyl. 6. The polymyxin prodrug compound or its stereoisomer or pharmaceutically acceptable salt thereof according to claim 4, characterized in that R ₄ and R ₅ are both H, or R ₄ , R ₅ Together with its connected -OBO-, it forms the following structure:

R ₇ , R ₈ , R ₉ and R ₁₀ are independently selected from: H, C ₁ -C ₄ alkyl, C ₁ -C ₄ alkoxy, C ₁ -C ₄ alkane substituted by one or more R ₁₂ group, one or more C ₁ -C ₄ alkoxy groups substituted by R ₁₂ , carboxyl, aldehyde, or R ₇ and R ₁₀ are hydrogen, R ₈ , R ₉ and the carbon atoms connected to them together form one or more A 6-membered oxygen-containing heterocyclic group substituted by R ₁₃ . 7. The polymyxin prodrug compound or its stereoisomer or its pharmaceutically acceptable salt according to claim 6, is characterized in that, each R ₁₂ are independently selected from: H, hydroxyl, carboxyl, Carbonyl, aldehyde, C ₁ -C ₃ alkoxy, C ₁ -C ₃ alkyl substituted by hydroxy, 6-membered oxygen-containing heterocycloalkoxy substituted by one or more R ₁₅ ; wherein, each R ₁₅ is independently is selected from: H, hydroxy, C ₁ -C ₃ alkyl substituted by hydroxy. 8. The polymyxin prodrug compound or its stereoisomer or its pharmaceutically acceptable salt according to claim 6, is characterized in that, each R ₁₃ is independently selected from: H, hydroxyl, one or Multiple R ₁₆ substituted C ₁ -C ₃ alkyl groups, one or more R ₁₅ substituted 6-membered oxygen-containing heterocycloalkoxy groups, one or more R ₁₇ substituted C ₁ -C ₃ alkoxy groups; where , each R ₁₅ is independently selected from: H, hydroxyl, hydroxyl substituted C ₁ -C ₃ alkyl, one or more R ₁₈ substituted 5-6 membered oxygen-containing heterocycloalkoxy; each R ₁₆ is independently are selected from: H, hydroxyl, 6-membered oxygen-containing heterocycloalkoxy substituted by one or more R ₁₅ ; each R ₁₇ is independently selected from: H, hydroxyl, C ₁ -C ₃ alkyl substituted by hydroxyl, One or more R ₁₉ substituted 5-6 membered oxygen-containing heterocyclic groups; each R ₁₈ is independently selected from: H, hydroxyl, hydroxyl substituted C ₁ -C ₃ alkyl; each R ₁₉ is independently selected from : H, hydroxyl, C ₁ -C ₃ alkyl substituted by hydroxy, 5-6 membered oxygen-containing heterocycloalkoxy substituted by one or more R ₁₈ . 9. The polymyxin prodrug compound or its stereoisomer or pharmaceutically acceptable salt thereof according to claim 4, characterized in that R ₄ and R ₅ are both H, or R ₄ , R ₅ Together with its connected -OBO-, it forms the following structure:

10. The polymyxin prodrug compound or its stereoisomer or pharmaceutically acceptable salt thereof according to any one of claims 1-4, characterized in that the polymyxin prodrug compound is selected from from the following compounds:

where _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

11. Use of the polymyxin prodrug compound or its stereoisomer or its pharmaceutically acceptable salt according to any one of claims 1-10 in the preparation of a medicament for treating bacterial infection. 12. The application according to claim 11, characterized in that the bacteria are Gram-negative bacteria. 13. The application according to claim 12, characterized in that the bacteria are Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Salmonella typhimurium, Enterobacteriaceae bacteria. 14. The application according to claim 13, characterized in that the bacteria are Klebsiella pneumoniae, Enterobacter cloacae, Salmonella typhimurium, Escherichia coli and Pseudomonas aeruginosa. 15. A medicine for the treatment of bacterial infections, characterized in that it is prepared from active ingredients and pharmaceutically acceptable adjuvants, the active ingredients comprising the polymyxin prodrug compound according to any one of claims 1-10 or a stereoisomer thereof or a pharmaceutically acceptable salt thereof.

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