WO2022262823A1 - 活性氧自由基响应的多粘菌素前药化合物及其应用 - Google Patents

活性氧自由基响应的多粘菌素前药化合物及其应用 Download PDF

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WO2022262823A1
WO2022262823A1 PCT/CN2022/099235 CN2022099235W WO2022262823A1 WO 2022262823 A1 WO2022262823 A1 WO 2022262823A1 CN 2022099235 W CN2022099235 W CN 2022099235W WO 2022262823 A1 WO2022262823 A1 WO 2022262823A1
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substituted
polymyxin
hydroxyl
alkyl
independently selected
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PCT/CN2022/099235
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English (en)
French (fr)
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熊梦华
鲍燕
邓映雪
黄芮
黄松音
王均
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华南理工大学
中山大学孙逸仙纪念医院
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Publication of WO2022262823A1 publication Critical patent/WO2022262823A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/12—Cyclic peptides, e.g. bacitracins; Polymyxins; Gramicidins S, C; Tyrocidins A, B or C
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/50—Cyclic peptides containing at least one abnormal peptide link
    • C07K7/54—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring
    • C07K7/60—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring the cyclisation occurring through the 4-amino group of 2,4-diamino-butanoic acid
    • C07K7/62—Polymyxins; Related peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • 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.
  • 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
  • EMEA Medicines Agency
  • VAP ventilator-associated pneumonia
  • UMI urinary tract infections
  • 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.
  • polymyxin has been used clinically again and is considered to be the last line of defense against multi-drug resistant Gram-negative bacteria.
  • side effects induced by polymyxins are still a problem.
  • colistin prodrugs 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.
  • 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.
  • ROS oxygen free radicals
  • the present invention includes the following technical solutions.
  • 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.
  • R is selected from: a borate ester group obtained by reacting a boronic acid group with a sugar containing an adjacent dihydroxy structure, or
  • the sugar containing an ortho-dihydroxy structure is a monosaccharide, a disaccharide or a trisaccharide.
  • the polymyxin prodrug compound has the structure shown in the following formula (II):
  • 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;
  • 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 .
  • R 2 is selected from C 3 -C 4 alkyl; R 3 is selected from C 2 -C 5 alkyl.
  • 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 .
  • 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.
  • 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 substituted by one
  • both R 4 and R 5 are H, or R 4 , R 5 and -OBO- connected to them together form the following structure:
  • the polymyxin prodrug compound is selected from the following compounds:
  • 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
  • the present invention also provides the application of the above-mentioned polymyxin prodrug compound, including the following technical solutions.
  • the bacteria are Gram-negative bacteria.
  • the bacteria are Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Salmonella typhimurium, Enterobacteriaceae bacteria.
  • the bacteria are Klebsiella pneumoniae, Enterobacter cloacae, Salmonella typhimurium, Escherichia coli and Pseudomonas aeruginosa.
  • the invention also provides an antibacterial drug.
  • 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.
  • 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
  • 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.
  • 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 by 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.
  • 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.
  • any variable eg, R4, R5, etc.
  • its definition at each occurrence is independent of its definition at each other occurrence.
  • 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.
  • substituents are 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.
  • alkyl as used herein is meant to include both branched and straight chain saturated aliphatic hydrocarbon groups having the specified number of carbon atoms.
  • 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.
  • “C 1 -C 6 alkyl” specifically includes methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, pentyl, hexyl.
  • 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.
  • 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.
  • oxygen-containing heterocyclic group 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.
  • 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 Tween ), wetting agent (such as sodium lauryl sulfate), coloring agent, flavoring agent, stabilizer, antioxidant, preservative, pyrogen-free water, etc.
  • cellulose and its derivatives such as sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, etc.
  • gelatin such as talc
  • solid lubricants such as stearic acid , magnesium stearate
  • 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.
  • the active ingredient is admixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with:
  • fillers or extenders such as starch, lactose, sucrose, glucose, mannitol and silicic acid;
  • binders such as hydroxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and acacia;
  • humectants for example, glycerin
  • disintegrants such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate;
  • absorption accelerators for example, quaternary ammonium compounds
  • humectants for example, cetyl alcohol and glyceryl monostearate
  • Adsorbents for example, kaolin
  • Lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, or mixtures thereof.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the synthetic method of polymyxin E or polymyxin B prodrug modified by phenylboronic acid comprises the following steps:
  • 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.
  • 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 expressed as ABA-Colistin;
  • the phenylboronate-modified ROS-responsive polymyxin B prodrug is expressed 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
  • 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:
  • 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).
  • ABE solution 200mg ABE dissolved in 1mL dimethylformamide (DMF)
  • DMF dimethylformamide
  • 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 benzene
  • the borate-modified polymyxin E (referred to as ABE-Colistin) has a yield of 48.4%. Its 1 H NMR characterization spectrum is shown in FIG. 3 , and its mass spectrum is shown in figure a in FIG. 7 .
  • 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 .
  • 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
  • Example 2 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 .
  • 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 spectrum of the lactose-modified polymyxin E prodrug
  • the characterization spectrum is shown in Figure 14; the 1 H NMR characterization spectrum of the maltotriose-modified polymyxin E prodrug
  • 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.
  • 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.
  • 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.
  • ABE-Colistin The MIC concentration of ABE-Colistin is about 0.25 ⁇ g/mL; however, after prolonging the incubation time, ABE-Colistin can completely inhibit the growth of bacteria at a concentration of 0.25 ⁇ g/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.
  • 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%.
  • ABE-Colistin incubated with 1mM H 2 O 2 can completely inhibit bacterial growth in the concentration range of 0.25 ⁇ g/mL to 32 ⁇ g/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.
  • 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 1 mM H 2 O 2 can completely inhibit bacterial growth in the concentration range of 0.25 ⁇ g/mL to 2 ⁇ g/mL, while ABE-Colistin B without H 2 O 2 treatment There was no effect of inhibiting bacterial growth, indicating that phenylboronate-modified ABE-Polymyxin B could not only sensitively respond to ROS, but also selectively effectively inhibit bacterial growth under ROS environment.
  • HK-2 cells were selected for the experiment, 100 ⁇ L/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 ⁇ L 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 ⁇ L 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 .
  • cell viability% (OD value of drug-added cells-blank OD value) ⁇ (control cell OD value-blank OD value) ⁇ 100%.
  • 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.
  • Escherichia coli ATCC352178 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.
  • 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 values of the remaining sugar-modified polymyxin prodrugs to Escherichia coli were 1-2 ⁇ g/mL
  • the MIC values to Pseudomonas aeruginosa were 2-4 ⁇ g/mL.
  • the MIC values of the active ingredient polymyxin against Escherichia coli and Pseudomonas aeruginosa are both 0.25 ⁇ g/mL.
  • Embodiment 6 is a diagrammatic representation of Embodiment 6
  • 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.
  • 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 is a diagrammatic representation of Embodiment 7:
  • mice 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.
  • 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) first with DMSO
  • ABE-Colistin 3mg/kg Colistin
  • ABE-Colistin 15mg/kg Colistin
  • ABE-Colistin 75mg/kg Colistin
  • DMSO dilute into injection solution with 5% sterile glucose solution (the volume of DMSO cannot exceed 5% of the total injection volume), and treat by tail vein injection, and the blank control group injects an equal volume of sterile PBS solution as control.
  • 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.
  • 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 ⁇ L 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.
  • CFU/g the number of bacteria in each sample/the tissue weight of the sample
  • 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 is a diagrammatic representation of Embodiment 8
  • 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.
  • 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 it into a homogenate tube with sterile water, and store it on ice.
  • Lung tissue homogenate was obtained using a high-speed tissue homogenizer. Put the tissue homogenate on ice, and use sterile PBS for serial dilution, after dilution 10 times, 100 times, and 1000 times, take 10 ⁇ L 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 an incubator at 37°C.
  • 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.
  • 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.

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Abstract

一种具有式(I)所示结构的多粘菌素前药化合物或其立体异构体或其药学上可接受的盐。多粘菌素前药化合物可以有效降低多粘菌素的生物毒性，选择性在细菌感染组织激活，实现对感染部位细菌的杀伤，提高多粘菌素的生物利用度，实现了经典抗生素的可持续开发利用，扩大了未来多粘菌素在临床应用的范围，具有广阔的临床应用前景。

Description

活性氧自由基响应的多粘菌素前药化合物及其应用 技术领域
本发明涉及药物技术领域，具体涉及一种活性氧自由基响应的多粘菌素前药化合物及其应用。
背景技术
耐药性细菌感染不断威胁着人类的生命健康安全，全球每年因抗生素耐药性死亡的人数大约为70万人。如果该问题不加以遏制，到2050年因抗生素耐药性死亡的人数将达到1000万。这意味着，全球因耐药性感染而死亡的人数将超过癌症。世界卫生组织2020年表示，私人投资的下降和新抗生素开发的创新不足，正在削弱人类与耐药菌感染作斗争的努力。目前正在研发的60种抗菌药物，包括50种抗生素和10种生物制剂，与现有的治疗方法相比收效甚微，并且针对最难治的革兰氏阴性菌耐药细菌研发的新药很少。
革兰氏阴性细菌，例如克雷伯氏肺炎菌和大肠杆菌，可引起严重且往往致命的感染，对免疫系统较弱或尚未完全发育的人，如新生儿，以及老年人、接受手术和癌症治疗的人，构成健康和生命威胁。根据药品管理局(EMEA)，在欧洲，大约三分之二由耐抗生素细菌引起的死亡是由革兰氏阴性菌感染造成的。革兰氏阴性细菌还导致45-70％的呼吸机相关性肺炎(VAP)病例，20-30％的导管相关性血流感染，以及与重症监护室相关的其他感染，如手术部位或尿路感染(UTIs)。根据世界卫生组织的报告，目前最为致命的三种革兰氏阴性耐药菌为鲍曼不动杆菌(Acinetobacter baumannii)、绿脓杆菌(Pseudomonas aeruginosa)和肠杆菌科细菌。
多粘菌素在20世纪70年代作为抗微生物药出现在临床，主要产品包括多粘菌素B和多粘菌素E。它们是由阳离子七元肽环和一个带有脂肪酸链的三元肽构成，通过破坏细菌膜使得细菌的内容物泄露，被称为“自摄取机制”，从而达到杀伤细菌的目的。在临床上多粘菌素的使用通常需要通过高剂量以及长期治疗才能达到最佳治疗效果，而这种治疗方式往往对人肾脏和神经系统产生潜在毒性，使得多粘菌素在临床治疗中的使用受到限制。但在21世纪，由于其细菌耐药性问题不断凸显，多粘菌素重新在临床上使用，被认为是针对多重耐药革兰氏阴性细菌的最后一道防线。然而，多粘菌素所诱导的副作用依旧是现在所面临的难题。
目前有两种多粘菌素前药应用于临床：口服和局部使用的多粘菌素硫酸盐及注射使用的多粘菌素甲磺酸钠(CMS)。CMS通过修饰多粘菌素中Dab残基上的五个氨基从而变成一种毒性较小的无活性前药，并在体内转化为活性成分多粘菌素并发挥抗菌作用。由于CMS的 毒性比多粘菌素硫酸盐小，所以可以作为非肠道用药。但是CMS的临床应用仍存在一些问题，例如：CMS在给药前24小时内约60％的CMS会通过尿液排出体外，只有少部分的CMS可以水解为活性多粘菌素，药物生物利用率低。除此之外，许多研究人员也尝试开发新的多粘菌素前药，Zhu等人利用醋酸封端的聚乙二醇甲基醚修饰多粘菌素E中的两个苏氨酸的羟基。实验表明这种前药可有效降低多粘菌素的肾毒性，但是由于前药在体内转化为活性成分的速率慢，抗菌活性随之降低，需要增大给药剂量。
发明内容
基于此，本发明提供了一类新的多粘菌素前药化合物，该前药在正常组织以无活性的形式存在，对正常组织的伤害非常小，毒副作用小，在细菌感染部位受到活性氧自由基(ROS)的刺激，释放出活性的多粘菌素成分，从而有效杀伤细菌。
本发明包括如下技术方案。
具有式(I)所示结构的多粘菌素前药化合物或其立体异构体或其药学上可接受的盐：
Figure PCTCN2022099235-appb-000001
其中，R 1选自：
Figure PCTCN2022099235-appb-000002
R 2、R 3分别独立地选自C 1-C 8烷基；
R选自：硼酸基、取代或者未取代的硼酸酯基。
在其中一些实施例中，R选自：由硼酸基与含有邻位二羟基结构的糖反应得到的硼酸酯基、
Figure PCTCN2022099235-appb-000003
或者
Figure PCTCN2022099235-appb-000004
在其中一些实施例中，所述含有邻位二羟基结构的糖为单糖、双糖或者三糖。
在其中一些实施例中，所述的多粘菌素前药化合物具有如下式(II)所示结构：
Figure PCTCN2022099235-appb-000005
其中，R 1选自：
Figure PCTCN2022099235-appb-000006
R 2、R 3分别独立地选自C 1-C 8烷基；
各R 4、R 5分别独立地选自：H、C 1-C 6烷基、一个或多个R 6取代的C 1-C 6烷基、或者R 4、R 5和与其相连的-O-B-O-一起形成如下结构：
Figure PCTCN2022099235-appb-000007
各R 6分别独立地选自：H、羟基、羧基、羰基、醛基、C 1-C 6烷氧基；
各R 7、R 8、R 9、R 10分别独立地选自：H、C 1-C 8烷基、C 1-C 8烷氧基、一个或多个R 12取代的C 1-C 8烷基、一个或多个R 12取代的C 1-C 8烷氧基、羧基、醛基，或者R 7和R 10为氢，R 8、R 9和与其相连的碳原子一起形成一个或多个R 13取代的5-6元含氧杂环基；
各R 12分别独立地选自：H、羟基、羧基、羰基、醛基、C 1-C 6烷氧基、羟基取代的C 1-C 6烷基、一个或多个R 15取代的5-6元含氧杂环烷氧基；
各R 13分别独立地选自：H、羟基、一个或多个R 16取代的C 1-C 6烷基、一个或多个R 15取代的5-6元含氧杂环烷氧基、一个或多个R 17取代的C 1-C 8烷氧基；
各R 15分别独立地选自：H、羟基、羟基取代的C 1-C 6烷基、一个或多个R 18取代的5-6元含氧杂环烷氧基；
各R 16分别独立地选自：H、羟基、一个或多个R 15取代的5-6元含氧杂环烷氧基；
各R 17分别独立地选自：H、羟基、羟基取代的C 1-C 6烷基、一个或多个R 19取代的5-6 元含氧杂环基；
各R 18分别独立地选自：H、羟基、羟基取代的C 1-C 6烷基；
各R 19分别独立地选自：H、羟基、羟基取代的C 1-C 6烷基、一个或多个R 18取代的5-6元含氧杂环烷氧基。
在其中一些实施例中，R 2选自C 3-C 4烷基；R 3选自C 2-C 5烷基。
在其中一些实施例中，R 4和R 5均为H，或者R 4、R 5和与其相连的-O-B-O-一起形成如下结构：
Figure PCTCN2022099235-appb-000008
R 7、R 8、R 9和R 10分别独立地选自：H、C 1-C 4烷基、C 1-C 4烷氧基、一个或多个R 12取代的C 1-C 4烷基、一个或多个R 12取代的C 1-C 4烷氧基、羧基、醛基，或者R 7和R 10为氢，R 8、R 9和与其相连的碳原子一起形成一个或多个R 13取代的6元含氧杂环基。
在其中一些实施例中，各R 12分别独立地选自：H、羟基、羧基、羰基、醛基、C 1-C 3烷氧基、羟基取代的C 1-C 3烷基、一个或多个R 15取代的6元含氧杂环烷氧基；其中，各R 15分别独立地选自：H、羟基、羟基取代的C 1-C 3烷基。
在其中一些实施例中，各R 13分别独立地选自：H、羟基、一个或多个R 16取代的C 1-C 3烷基、一个或多个R 15取代的6元含氧杂环烷氧基、一个或多个R 17取代的C 1-C 3烷氧基；其中，各R 15分别独立地选自：H、羟基、羟基取代的C 1-C 3烷基、一个或多个R 18取代的5-6元含氧杂环烷氧基；各R 16分别独立地选自：H、羟基、一个或多个R 15取代的6元含氧杂环烷氧基；各R 17分别独立地选自：H、羟基、羟基取代的C 1-C 3烷基、一个或多个R 19取代的5-6元含氧杂环基；各R 18分别独立地选自：H、羟基、羟基取代的C 1-C 3烷基；各R 19分别独立地选自：H、羟基、羟基取代的C 1-C 3烷基、一个或多个R 18取代的5-6元含氧杂环烷氧基。
在其中一些实施例中，R 4和R 5均为H，或者R 4、R 5和与其相连的-O-B-O-一起形成如下结构：
Figure PCTCN2022099235-appb-000009
在其中一些实施例中，所述多粘菌素前药化合物选自如下化合物：
Figure PCTCN2022099235-appb-000010
Figure PCTCN2022099235-appb-000011
Figure PCTCN2022099235-appb-000012
Figure PCTCN2022099235-appb-000013
Figure PCTCN2022099235-appb-000014
其中，R 1为
Figure PCTCN2022099235-appb-000015
R 2为
Figure PCTCN2022099235-appb-000016
R 3选自
Figure PCTCN2022099235-appb-000017
或者，
R 1为
Figure PCTCN2022099235-appb-000018
R 2为
Figure PCTCN2022099235-appb-000019
R 3为
Figure PCTCN2022099235-appb-000020
或者，
R 1为
Figure PCTCN2022099235-appb-000021
R 2为
Figure PCTCN2022099235-appb-000022
R 3选自
Figure PCTCN2022099235-appb-000023
或者，
R 1为
Figure PCTCN2022099235-appb-000024
R 2为
Figure PCTCN2022099235-appb-000025
R 3选自
Figure PCTCN2022099235-appb-000026
或者，
R 1为
Figure PCTCN2022099235-appb-000027
R 2为
Figure PCTCN2022099235-appb-000028
R 3选自
Figure PCTCN2022099235-appb-000029
或者，
R 1为
Figure PCTCN2022099235-appb-000030
R 2为
Figure PCTCN2022099235-appb-000031
R 3选自
Figure PCTCN2022099235-appb-000032
本发明还提供了上述多粘菌素前药化合物的应用，包括如下技术方案。
上述的多粘菌素前药化合物或其立体异构体或其药学上可接受的盐在制备治疗细菌感染的药物中的应用。
在其中一些实施例中，所述细菌为革兰氏阴性细菌。
在其中一些实施例中，所述细菌为克雷伯氏肺炎菌、鲍曼不动杆菌、绿脓杆菌、鼠伤寒沙门氏菌、肠杆菌科细菌。
在其中一些实施例中，所述细菌为肺炎克雷伯菌、阴沟肠杆菌，鼠伤寒沙门氏菌、大肠杆菌和绿脓杆菌。
本发明还提供了一种抗菌药。
具体技术方案如下：
一种治疗细菌感染的药物，由活性成分和药学上可接受的辅料制备得到，所述活性成分包括上述的多粘菌素前药化合物或其立体异构体或其药学上可接受的盐。
基于上述技术方案，本发明具有以下有益效果：
本发明利用细菌感染部位与正常组织部位ROS的浓度差异，对于临床批准的多粘菌素B和E进行结构修饰，通过对其修饰苯硼酸酯或苯硼酸，获得ROS响应的多粘菌素前药化合物，该前药化合物在正常组织以无活性形式存在，在血液循环条件下是无活性的多粘菌素前药，对正常组织的伤害非常低，细胞毒性小；在细菌感染部位受ROS的作用，苯硼酸或苯硼酸酯修饰的ROS敏感化学键断链，无活性的多粘菌素前药转变为高抗菌活性的多粘菌素，从而可以高选择性的杀死感染部位的细菌(尤其是耐药的革兰氏阴性菌)，显著提高抗菌效果的同时显著降低多粘菌素的毒副作用。本发明的多粘菌素前药化合物可以有效降低多粘菌素的生物毒性，选择性在细菌感染组织激活，实现对感染部位细菌的杀伤，提高多粘菌素的生物利用度，实现了经典抗生素的可持续开发利用，扩大了未来多粘菌素在临床应用的范围，具有广阔的临床应用前景。
附图说明
图1为苯硼酸酯修饰的多粘菌素、苯硼酸修饰的多粘菌素以及糖修饰的多粘菌素前药的合成路线。
图2为4-(4,4,5,5-四甲基-1,3,2-二恶硼烷)-苄基硝苯基碳酸酯(ABE)的 1H NMR表征图谱。
图3为苯硼酸酯修饰的多粘菌素E的 1H NMR表征图谱。
图4为苯硼酸修饰的多粘菌素E的 1H NMR表征图谱。
图5为苯硼酸酯修饰的多粘菌素B的 1H NMR表征图谱。
图6为苯硼酸修饰的多粘菌素B的 1H NMR表征图谱。
图7为苯硼酸酯修饰的多粘菌素E(a)以及其经ROS响应后(b)的质谱。
图8为木糖修饰的多粘菌素前药的 1H NMR表征图谱。
图9为阿拉伯糖修饰的多粘菌素前药的 1H NMR表征图谱。
图10为果糖修饰多粘菌素前药的 1H NMR表征图谱。
图11为葡萄糖修饰多粘菌素前药的 1H NMR表征图谱。
图12为半乳糖修饰多粘菌素前药的 1H NMR表征图谱。
图13为甘露糖修饰多粘菌素前药的 1H NMR表征图谱。
图14为乳糖修饰多粘菌素前药的 1H NMR表征图谱。
图15为麦芽三糖修饰多粘菌素前药的 1H NMR表征图谱。
图16为棉子糖修饰多粘菌素前药的 1H NMR表征图谱。
图17为苯硼酸酯修饰的多粘菌素E的溶血活性结果图。
图18为苯硼酸酯修饰的多粘菌素E对不同浓度的H 2O 2响应不同时间(5min，15min，30min)的ROS响应敏感性结果图。
图19为苯硼酸酯修饰的多粘菌素E的抑菌动力学结果图。
图20为苯硼酸酯修饰的多粘菌素B的抑菌动力学结果图。
图21为苯硼酸酯修饰的多粘菌素E的杀菌动力学结果图。
图22为苯硼酸酯修饰的多粘菌素E与多粘菌素B对HK-2细胞的细胞毒性MTT结果图。
图23为糖修饰的ROS响应的多粘菌素前药的最大耐受剂量。
图24为苯硼酸酯修饰的多粘菌素E对肺炎的治疗效果。
图25为乳糖修饰的多粘菌素E对肺炎的治疗效果。
具体实施方式
本发明下列实施例中未注明具体条件的实验方法，通常按照常规条件，或按照制造厂商所建议的条件。实施例中所用到的各种常用化学试剂，均为市售产品。
除非另有定义，本发明所使用的所有的技术和科学术语与属于本发明的技术领域的技术人员通常理解的含义相同。本发明的说明书中所使用的术语只是为了描述具体的实施例的目的，不用于限制本发明。
本发明的术语“包括”和“具有”以及它们任何变形，意图在于覆盖不排他的包含。例如包含了一系列步骤的过程、方法、装置、产品或设备没有限定于已列出的步骤或模块，而是可选地还包括没有列出的步骤，或可选地还包括对于这些过程、方法、产品或设备固有的其它步骤。
在本发明中提及的“多个”是指两个或两个以上。“和/或”，描述关联对象的关联关系，表示可以存在三种关系，例如，A和/或B，可以表示：单独存在A，同时存在A和B，单独存在B这三种情况。字符“/”一般表示前后关联对象是一种“或”的关系。
本发明所述化合物中，当任何变量(例如R 4、R 5等)在任何组分中出现超过一次，则其每次出现的定义独立于其它每次出现的定义。同样，允许取代基及变量的组合，只要这种 组合使化合物稳定。自取代基划入环系统的线表示所指的键可连接到任何能取代的环原子上。如果环系统为多环，其意味着这种键仅连接到邻近环的任何适当的碳原子上。要理解本领域普通技术人员可选择本发明化合物的取代基及取代型式而提供化学上稳定的并可通过本领域技术和下列提出的方法自可容易获得的原料容易合成的化合物。如果取代基自身被超过一个基团取代，应理解这些基团可在相同碳原子上或不同碳原子上，只要使结构稳定。短语“任选被一个或多个取代基取代”被认为与短语“任选被至少一个取代基取代”相当且在此情况下优选的实施方案将具有0-3个取代基。
本文所用术语“烷基”意指包括具有特定碳原子数目的支链的和直链的饱和脂肪烃基。例如，“C 1-C 6烷基”中“C 1-C 6”的定义包括以直链或支链排列的具有1、2、3、4、5或6个碳原子的基团。例如，“C 1-C 6烷基”具体包括甲基、乙基、正丙基、异丙基、正丁基、叔丁基、异丁基、戊基、己基。
术语“烷氧基”指具有-O-烷基结构的基团，如-OCH 3、-OCH 2CH 3、-OCH 2CH 2CH 3、-O-CH 2CH(CH 3) 2、-OCH 2CH 2CH 2CH 3、-O-CH(CH 3) 2等。
术语“含氧杂环基”为饱和或部分不饱和的单环或多环环状取代基，其中一个或多个环原子为O原子，其余环原子为碳，例如：四氢吡喃基。
术语“含氧杂环烷氧基”指含氧杂环基的环碳原子与-O-连接的基团，例如：
Figure PCTCN2022099235-appb-000033
本发明的用于治疗细菌感染的药物可以用于非人哺乳动物或者人。
本发明的用于治疗细菌感染的药物中所用的药学上可接受的辅料指的是：一种或多种相容性固体或液体填料或凝胶物质，它们适合于人使用，而且必须有足够的纯度和足够低的毒性。
“相容性”在此指的是组合物中各组分能和本发明的活性成分(多粘菌素前药化合物或其立体异构体或其药学上可接受的盐)以及它们之间相互掺和，而不明显降低活性成分的药效。
本发明的用于治疗细菌感染的药物所用的药学上可接受的辅料包括但不限于如下材料中的一种或多种：溶剂、赋形剂、填料、增容剂、粘合剂、保湿剂、崩解剂、缓溶剂、吸收加速剂、吸附剂、稀释剂、增溶剂、乳化剂、润滑剂、润湿剂、悬浮剂、矫味剂和香料中的至少一种。
药学上可以接受的辅料部分例子有纤维素及其衍生物(如羧甲基纤维素钠、乙基纤维素钠、纤维素乙酸酯等)、明胶、滑石、固体润滑剂(如硬脂酸、硬脂酸镁)、硫酸钙、植物油(如豆油、芝麻油、花生油、橄榄油等)、多元醇(如丙二醇、甘油、甘露醇、山梨醇等)、乳化剂(如吐温
Figure PCTCN2022099235-appb-000034
)、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。
本发明的活性成分或药物组合物的施用方式没有特别限制，代表性的施用方式包括(但并不限于)：口服、直肠、肠胃外(静脉内、肌肉内或皮下)等。
用于口服给药的固体剂型包括胶囊剂、片剂、丸剂、散剂和颗粒剂。
在这些固体剂型中，活性成分与至少一种常规惰性赋形剂(或载体)混合，如柠檬酸钠或磷酸二钙，或与下述成分混合：
(a)填料或增容剂，例如，淀粉、乳糖、蔗糖、葡萄糖、甘露醇和硅酸；
(b)粘合剂，例如，羟甲基纤维素、藻酸盐、明胶、聚乙烯基吡咯烷酮、蔗糖和阿拉伯胶；
(c)保湿剂，例如，甘油；
(d)崩解剂，例如，琼脂、碳酸钙、马铃薯淀粉或木薯淀粉、藻酸、某些复合硅酸盐、和碳酸钠；
(e)缓溶剂，例如，石蜡；
(f)吸收加速剂，例如，季胺化合物；
(g)润湿剂，例如，鲸蜡醇和单硬脂酸甘油酯；
(h)吸附剂，例如，高岭土；
(i)润滑剂，例如，滑石、硬脂酸钙、硬脂酸镁、固体聚乙二醇、十二烷基硫酸钠，或其混合物。胶囊剂、片剂和丸剂中，剂型也可包含缓冲剂。
所述的固体剂型还可采用包衣和壳材制备，如肠衣和其它本领域公知的材料。它们可包含不透明剂，并且，这种组合物中活性成分的释放可以延迟的方式在消化道内的某一部分中释放。可采用的包埋组分的实例是聚合物质和蜡类物质。
用于口服给药的液体剂型包括药学上可接受的乳液、溶液、悬浮液、糖浆或酊剂。除了活性成分外，液体剂型可包含本领域中常规采用的惰性稀释剂，如水或其它溶剂，增溶剂和乳化剂，例知，乙醇、异丙醇、碳酸乙酯、乙酸乙酯、丙二醇、1,3-丁二醇、二甲基甲酰胺 以及油，特别是棉籽油、花生油、玉米胚油、橄榄油、蓖麻油和芝麻油或这些物质的混合物等。除了这些惰性稀释剂外，组合物也可包含助剂，如润湿剂、乳化剂和悬浮剂、甜味剂、矫味剂和香料。
除了活性成分外，悬浮液可包含悬浮剂，例如，乙氧基化异十八烷醇、聚氧乙烯山梨醇和脱水山梨醇酯、微晶纤维素、甲醇铝和琼脂或这些物质的混合物等。
用于肠胃外注射的组合物可包含生理上可接受的无菌含水或无水溶液、分散液、悬浮液或乳液，和用于重新溶解成无菌的可注射溶液或分散液的无菌粉末。适宜的含水和非水载体、稀释剂、溶剂或赋形剂包括水、乙醇、多元醇及其适宜的混合物。
本发明的ROS响应的多粘菌素前药化合物的案例之一可以按照下述方法合成：以对硝基苯基氯甲酸酯和4-(羟甲基)苯硼酸频哪醇酯为原料，以三乙胺为敷酸剂，反应得到4-(4,4,5,5-四甲基-1,3,2-二恶硼烷)-苄基硝苯基碳酸酯(ABE)。ABE分别与多粘菌素E及多粘菌素B反应，得到苯硼酸酯修饰的ROS响应的多粘菌素E和多粘菌素B前药。苯硼酸酯修饰的多粘菌素E和多粘菌素B前药用浓盐酸水解后得到苯硼酸修饰的ROS响应的多粘菌素E和多粘菌素B前药。苯硼酸修饰的ROS响应的多粘菌素E和多粘菌素B与含有邻位二羟基结构的化合物反应得到水溶性改善的糖修饰的多粘菌素E和多粘菌素B前药。
在其中一些实施方案中，苯硼酸修饰的多粘菌素E或多粘菌素B前药的合成方法包括以下步骤：
(1)用无水四氢呋喃溶解对硝基苯基氯甲酸酯和4-(羟甲基)苯硼酸频哪醇酯，加入无水三乙胺作为敷酸剂，反应结束后，用乙酸乙酯萃取，并依次用饱和碳酸氢钠和稀盐酸洗涤，用无水硫酸镁干燥，抽滤，旋蒸，过硅胶柱，旋蒸，制备得到4-(4,4,5,5-四甲基-1,3,2-二恶硼烷)-苄基硝苯基碳酸酯(ABE)。
(2)用饱和碳酸氢钠溶解多粘菌素E或多粘菌素B，加入用二甲基甲酰胺溶解的ABE，反应结束后，减压蒸馏除去溶剂，溶于二氯甲烷中，离心，取上清，抽除溶剂，在乙醚中沉淀，透析得到苯硼酸酯修饰的多粘菌素E或苯硼酸酯修饰的多粘菌素B。
(3)用二甲基甲酰胺溶解苯硼酸酯修饰的多粘菌素前药，滴加浓盐酸，反应结束后透析得到苯硼酸修饰的多粘菌素E或苯硼酸修饰的多粘菌素B。
在其中一些实施方案中，一种基于含有邻位二羟基结构的糖与苯硼酸反应得到的糖修饰的ROS响应的多粘菌素E和多粘菌素B前药化合物的制备方法包括以下步骤：苯硼酸修饰 的多粘菌素E或多粘菌素B前药与含有邻位二羟基结构的糖在0.1M NaOH溶液中反应，反应结束后用0.01M NaOH溶液透析，即可得到水溶性改善的苯硼酸修饰的多粘菌素E或苯硼酸修饰的多粘菌素B的相关衍生物。
由以上所述的制备方法可以制得一系列ROS响应的多粘菌素前药化合物。本发明中，苯硼酸酯修饰的ROS响应的多粘菌素E前药表示为ABE-Colistin；苯硼酸酯修饰的ROS响应的多粘菌素B前药表示为ABE-Polymyxin B；苯硼酸修饰的ROS响应的多粘菌素E前药表示为ABA-Colistin；苯硼酸酯修饰的ROS响应的多粘菌素B前药表示为ABA-Polymyxin B；基于含有邻位二羟基结构的糖与苯硼酸反应得到的糖修饰的ROS响应的多粘菌素E前药表示为X-ABA-Colistin，其中X代表不同糖的缩写，例如与乳糖反应得到的ROS响应的多粘菌素E前药表示为Lac-ABA-Colistin、与果糖反应得到的ROS响应的多粘菌素E前药表示为Fru-ABA-Colistin、与半乳糖反应得到的ROS响应的多粘菌素E前药表示为Gal-ABA-Colistin；基于含有邻位二羟基结构的糖与苯硼酸反应得到的糖修饰的ROS响应的多粘菌素B前药表示为X-ABA-Polymyxin B，其中X代表不同糖的缩写。
以下实施例中所用的多粘菌素E和多粘菌素B均为市售产品。已知的多粘菌素E和多粘菌素B有多种亚结构，具体如下：
Figure PCTCN2022099235-appb-000035
Figure PCTCN2022099235-appb-000036
Figure PCTCN2022099235-appb-000037
以下为具体实施例。
实施例1：制备苯硼酸修饰的ROS响应的ABA-Colistin
1)4-(4,4,5,5-四甲基-1,3,2-二恶硼烷)-苄基硝苯基碳酸酯(ABE)的制备
称量0.47g对硝基苯基氯甲酸酯和0.5g 4-(羟甲基)苯硼酸频哪醇酯于一个100mL的圆底烧瓶中，用20mL的无水四氢呋喃(THF)溶解，在搅拌的条件下加入600μL的无水三乙胺，在室温下搅拌反应1h；加入30mL乙酸乙酯，转移到125mL的分液漏斗中，静置，分三次加入250mL饱和NaHCO 3溶液洗涤，弃去下层水层，分三次加入250mL HCl(1M)洗涤，弃去下层水层，将上层液体倒于100mL烧杯中，加入适量无水MgSO 4，搅拌过夜干燥，然后将其抽滤至500mL的圆底烧瓶中，往抽滤所得的溶液中加入2mL硅胶，旋蒸，干法上样，柱层析纯化，洗脱剂为乙酸乙酯和正己烷(V:V＝1:20)，得到0.52g ABE，收率61.2％，其 1H NMR表征图谱如图2所示。
2)苯硼酸酯修饰的ABE-Colistin的制备
称量100mg的多粘菌素E硫酸盐(购自上海源叶生物科技有限公司；CAS：1264-72-8，产品编号：S17057；其多粘菌素E是含有多粘菌素E 1和多粘菌素E 2两种成分的混合物)。于25mL圆底烧瓶中，加入4mL饱和碳酸氢钠，搅拌溶解，将ABE溶液(200mg ABE溶于1mL二甲基甲酰胺(DMF))，加入到圆底烧瓶中，搅拌反应24h，在50℃下减压蒸馏除去溶剂，用10mL二氯甲烷溶解，离心，除掉沉淀，浓缩溶液，用乙醚沉淀后，用5mL二甲亚砜(DMSO)溶解，并依次用水透析，冻干，得103mg苯硼酸酯修饰的多粘菌素E(记为ABE-Colistin)，收率48.4％，其 1H NMR表征图谱如图3所示，其质谱如图7中的a图所示。
3)苯硼酸修饰的ABA-Colistin的制备
称量50mg的ABE-Colistin于25mL圆底烧瓶中，加入4mL DMSO，搅拌溶解，加入0.3mL浓盐酸(37wt％)，搅拌反应6h，用水透析，冻干，得35mg苯硼酸修饰的多粘菌素E(记为ABA-Colistin)，收率84.1％，其 1H NMR表征图谱如图4所示。
实施例2：制备苯硼酸修饰的ROS响应的ABA-Polymyxin B
1)4-(4,4,5,5-四甲基-1,3,2-二恶硼烷)-苄基硝苯基碳酸酯(ABE)的制备
同实施例1。
2)苯硼酸酯修饰的ABE-Polymyxin B的制备
方法同实施例1，利用多粘菌素B硫酸盐(购自大连美仑生物技术有限公司；CAS：1405-20-5，产品编号：MB1188；其多粘菌素B是含有多粘菌素B 1、多粘菌素B 2、多粘菌素B 3、Ile-多粘菌素B 1四种结构的混合物)制备得到105mg苯硼酸酯修饰的多粘菌素B(记为ABE-Polymyxin B)，收率50.4％，其 1H NMR表征图谱如图5所示。
3)苯硼酸修饰的ABA-Polymyxin B的制备
方法同实施例1，制备得到34mg苯硼酸修饰的多粘菌素B(记为ABA-Polymyxin B)，收率80.9％，其 1H NMR表征图谱如图6所示。
实施例3：糖修饰的ROS响应的多粘菌素E前药的制备
称量80mg(0.039mmol)的ABA-Colistin于圆底烧瓶中，加入4mL 0.1M NaOH溶液，搅拌溶解，加入664.8mg(1.012mmol)乳糖，搅拌反应24h,用0.01M NaOH溶液透析，冻干，制备得到88mg乳糖修饰的ROS响应的多粘菌素E前药，收率62.8％。
按本实施例的方法制备了其他糖修饰的多粘菌素E前药，其投料比如下表1所示：
表1
Figure PCTCN2022099235-appb-000038
Figure PCTCN2022099235-appb-000039
木糖修饰的多粘菌素E前药的 1H NMR表征图谱如图8所示；阿拉伯糖修饰的多粘菌素E前药的 1H NMR表征图谱如图9所示；果糖修饰的多粘菌素E前药的 1H NMR表征图谱如图10所示；葡萄糖修饰的多粘菌素E前药的 1H NMR表征图谱如图11所示；半乳糖修饰的多粘菌素E前药的 1H NMR表征图谱如图12所示；甘露糖修饰的多粘菌素E前药的 1H NMR表征图谱如图13所示；乳糖修饰的多粘菌素E前药的 1H NMR表征图谱如图14所示；麦芽三糖修饰的多粘菌素E前药的 1H NMR表征图谱如图15所示；棉子糖修饰的多粘菌素E前药的 1H NMR表征图谱如图16所示。
实施例4：
ROS响应的多粘菌素前药ABE-Colistin以及ABE-Polymyxin B的抗菌活性、溶血毒性和细胞毒性。
1)溶血实验：
取适量羊全血于离心管中，用1×PBS稀释，配制4％(v/v)羊血溶液；使用PBS配制一系列浓度的药物溶液，按100μL/管加入到EP管中，再加入等体积的4％羊血；其中，PBS和等体积的4％羊血混合，作为阴性对照组；0.1％Triton和等体积的4％羊血混合，为阳性对照组；样品混匀后置于37℃中孵育60min。随后，将样品置于4℃进行离心(1000rpm/5min)；离心后，取100μL的上清到96孔板中，并于576nm处测定吸光度，计算溶血率。计算公式如下：溶血活性＝(OD实验组-OD阴性组)÷(OD阳性组-OD阴性组)×100％。如图17为Colistin，和ABE-Colistin的溶血活性，说明苯硼酸酯修饰后的多粘菌素E具有较低的溶血活性，可以更好的在体内应用。
2)抑菌动力学：
称取ABE-Colistin材料粉末，加入1.5mL的Ep管中，再加入无水DMSO涡旋使其完全溶解，配制成系列浓度的ABE-Colistin；称取Colisitin粉末，加入1.5mL的Ep管中，再 加入无菌水涡旋使其完全溶解，配制成系列浓度的Colistin；将系列浓度的ABE-Colistin分别与不同浓度的H 2O 2(0,1,2mmol)孵育不同时间(5min、15min、30min)。
离心收集大肠杆菌(ATCC35218)，PBS清洗3次，使用M9培养基稀释菌液至合适浓度，再加入一系列浓度的药物溶液(0mM H 2O 2+ABE-Colistin组，1mM H 2O 2+ABE-Colistin组，2mM H 2O 2+ABE-Colistin组，Colistin组)，使得细菌终浓度为1×10 6CFU/mL。将样品置于37℃中孵育，在24小时后取出样品，测定在600nm处的吸光度值，计算抑制细菌效率。计算公式为：抑菌率＝(100-(处理组的OD值-空白背景组的OD值)÷(空白对照组的OD值-空白背景组的OD值))×100％。结果如图18所示，5分钟时，1mM H 2O 2孵育的ABE-Colistin无法完全抑制细菌生长，2mM H 2O 2孵育的ABE-Colistin可以完全抑制细菌生长，由以前实验数据得知Colisitin的MIC浓度大约在0.25μg/mL；但是延长孵育时间，ABE-Colistin在1mM H 2O 2条件下孵育后也能够在0.25μg/mL浓度完全抑制细菌生长；孵育15分钟和30分钟，曲线趋势几乎一致，说明苯硼酸酯修饰的ABE-Colistin对ROS的敏感性较高且响应速度较快，在ROS环境下可以有效抑制细菌生长。
将系列浓度的ABE-Colistin分别与1mM H 2O 2孵育30min；离心收集大肠杆菌(ATCC35218)，绿脓杆菌(ATCC27853)，肺炎克雷伯菌(ATCC700603)，阴沟肠杆菌(ATCC700323)，鼠伤沙门氏菌(ATCC14028)，大肠杆菌(ATCC25922)，PBS清洗3次，使用LB培养基稀释菌液至合适浓度，再加入一系列浓度的药物溶液(0mM H 2O 2+ABE-Colistin组，1mM H 2O 2+ABE-Colistin组，Colistin组)，使得细菌终浓度为1×10 6CFU/mL。将样品置于37℃中孵育，在24小时后取出样品，测定在600nm处的吸光度值，计算抑制细菌效率。计算公式：抑菌率＝(100-(处理组的OD值-空白背景组的OD值)÷(空白对照组的OD值-空白背景组的OD值))×100％。结果如图19所示，1mM H 2O 2孵育的ABE-Colistin在0.25μg/mL～32μg/mL的浓度范围内能够完全抑制细菌生长，而没有经过H 2O 2处理的ABE-Colistin没有任何抑制细菌生长的作用，说明苯硼酸酯修饰的ABE-Colistin不仅可以敏感地响应ROS，并且可以选择性地在ROS环境下有效抑制多种革兰氏阴性菌的生长。
将系列浓度的ABE-Polymyxin B分别与1mmol H 2O 2孵育30min；离心收集大肠杆菌(ATCC35218)，绿脓杆菌(ATCC27853)，PBS清洗3次，使用LB培养基稀释菌液至合适浓度，再加入一系列浓度的药物溶液(0mM H 2O 2+ABE-Colistin组，1mM H 2O 2+ABE-Colistin组，Colistin组)，使得细菌终浓度为1×10 6CFU/mL。将样品置于37℃中孵育，在24小时后取出样品，测定在600nm处的吸光度值，计算抑制细菌效率。计算公式：抑菌率＝(100-(处理组的OD值-空白背景组的OD值)÷(空白对照组的OD值-空白背景组的OD值))×100％。结果如图20所示，1mM H 2O 2孵育的ABE-Polymyxin B在0.25μg/mL～2 μg/mL的浓度范围内能够完全抑制细菌生长，而没有经过H 2O 2处理的ABE-Colistin没有任何抑制细菌生长的作用，说明苯硼酸酯修饰的ABE-Polymyxin B不仅可以敏感地响应ROS，并且可以选择性地在ROS环境下有效抑制细菌的生长。
3)杀菌动力学：
将系列浓度的ABE-Colistin分别与1mmol H 2O 2孵育30min。离心收集大肠杆菌(ATCC35218)和绿脓杆菌(ATCC27853)，PBS清洗3次，使用M9培养基稀释菌液至合适浓度，再加入一系列浓度的药物溶液(0mM H 2O 2+ABE-Colistin组，1mM H 2O 2+ABE-Colistin组，Colistin组)，使得细菌终浓度为1×10 6CFU/mL。将样品置于37℃中孵育0小时：将未加药组菌液稀释100倍，1000倍，各稀释倍数取20μL菌液在琼脂板上涂布；37℃孵育2小时后，96孔板在冰上用LB稀释细菌100倍，1000倍，各稀释倍数取20μL菌液在琼脂板上涂布，将琼脂板置于37℃中培养12小时后，进行菌落计数，计算细菌存活率。计算公式为：细菌存活率＝ABE-Colistin处理组的菌落数÷空白对照组的菌落数×100％。实验结果如图21所示，可见苯硼酸酯修饰的ROS响应的ABE-Colistin可以在ROS环境下激活，并且杀死细菌。
4)细胞毒性实验：
选用HK-2细胞进行实验，在96孔板中加入细胞100μL/孔(约1x 10 4)，置于37℃，CO 2细胞培养箱中培养24小时；分别加入不同浓度的ABE-Colistin溶液、Colistin溶液、ABE-Polymyxin B溶液以及Polymyxin B溶液；再置于37℃，CO 2细胞培养箱中孵育24小时，每孔加入10μL的MTT溶液(5mg/mL)，37℃孵育4小时，使MTT还原成甲臜；吸出上清液，每孔加入200μL DMSO使甲臜溶解，在摇床上摇匀30分钟，全程避光；酶标仪在490nm波长处检测每孔的吸光值，进行细胞活力计算。计算公式为：细胞存活率％＝(加药细胞OD值-空白OD值)÷(对照细胞OD值-空白OD值)×100％。注：空白组不加入细胞，其他操作同实验组。实验结果如图22所示，ABE-Colistin和ABE-Polymyxin B组的HK-2细胞活性随着药物浓度的提高基本不受影响，苯硼酸酯修饰的ABE-Colistin和ABE-Polymyxin B对HK-2细胞没有毒性作用。
实施例5：
糖修饰的ROS响应的多粘菌素前药的抗菌活性。
称取糖修饰的多粘菌素前药(X-ABA-Colistin)粉末，加入1.5mL的Ep管中，再加入无菌水涡旋使其完全溶解，配制成系列浓度的X-ABA-Colistin；称取ABA-Colistin粉末，加入1.5mL的Ep管中，再加入无菌水涡旋使其完全溶解，配制成系列浓度的ABA-Colistin；称取ABE-Colistin粉末，加入1.5mL的Ep管中，再加入无菌水涡旋使其完全溶解，配制成 系列浓度的ABE-Colistin；称取Colisitin粉末，加入1.5mL的Ep管中，再加入无菌水涡旋使其完全溶解，配制成系列浓度的Colistin；将系列浓度的X-ABA-Colistin分别与1mM H 2O 2孵育6h。
离心收集大肠杆菌(ATCC35218)，绿脓杆菌(ATCC27853)，PBS清洗3次，使用LB培养基稀释菌液至合适浓度，再加入一系列浓度的药物溶液(0mM H 2O 2+X-ABA-Colistin组，1mM H 2O 2+X-ABA-Colistin组，0mM H 2O 2+ABA-Colistin组，1mM H 2O 2+ABA-Colistin组，0mM H 2O 2+ABE-Colistin组，1mM H 2O 2+ABE-Colistin组，Colistin组)，使得细菌终浓度为1×10 6CFU/mL。将样品置于37℃中孵育，在24小时后取出样品，测定在600nm处的吸光度值，得到最低抑菌浓度(即能够完全抑制细菌生长的最低浓度)。结果如表2所示，从表中可以看到所有糖修饰的前药对大肠杆菌的抑菌活性均好于对铜绿假单胞菌的抑菌活性。木糖和棉子糖修饰的多粘菌素前药相对于其他糖修饰的前药显示出更好的抑菌性能，两种前药对大肠杆菌的MIC值均为0.5μg/mL(以多粘菌素E的含量计算)，棉子糖修饰的多粘菌素前药对铜绿假单胞菌的MIC值为1g/mL，木糖修饰的多粘菌素前药对铜绿假单胞菌的MIC值为2μg/mL。其余糖修饰的多粘菌素前药对大肠杆菌的MIC值为1-2μg/mL，对铜绿假单胞菌的MIC值为2-4μg/mL。而活性成分多粘菌素对大肠杆菌和绿脓假单胞菌的MIC值均为0.25μg/mL。上述数据表明修饰后的多粘菌素前药不仅可以敏感地响应ROS，并且可以选择性地在ROS环境下有效抑制细菌的生长。
表2糖修饰的多粘菌素前药与H 2O 2孵育后对革兰氏阴性菌的抑菌活性
Figure PCTCN2022099235-appb-000040
实施例6：
ROS响应的多粘菌素前药的最大耐受剂量。
检测ICR小鼠对不同糖修饰的多粘菌素前药的MTD。测试MTD使用的是六周龄的雌性ICR小鼠，具体测试方法如下：向一只小鼠以50mg/kg(多粘菌素E的浓度)的剂量尾静脉注射ROS响应的多粘菌素前药注射液，观察30min后若小鼠的活动情况无明显变化则向另一只小鼠以100mg/kg剂量给药并观察30min，若小鼠的活动情况无明显变化，每间隔12h，向两只小鼠注射相同浓度的药物并继续观察，待五只小鼠在注射药物24h后均未出现死亡时，则判定小鼠对该药物的MTD≥100mg/kg。因考虑到实际给药浓度不会超过100mg/kg，故不再继续增大药物浓度。若在测试MTD期间出现小鼠死亡情况，则在现有给药剂量的基础上减少10mg/kg并重复上述步骤，直至测出MTD值。结果如图23所示，其中Ara-ABA-Colistin、Fru-ABA-Colistin、Gal-ABA-Colistin、Lac-ABA-Colistin、Mal-ABA-Colistin这五种糖修饰的多粘菌素前药的MTD均大于100mg/kg(以多粘菌素E的含量计算)；Glu-ABA-Colistin、Man-ABA-Colistin的MTD为90mg/kg；ABE-Colistin的MTD为75mg/kg，Xy-ABA-Colistin和Raf-ABA-Colistin的MTD分别为20mg/kg和15mg/kg。尽管木糖和棉子糖修饰的多粘菌素前药的MTD值不高，但仍比多粘菌素的MTD值(5mg/kg)高3-4倍，这表明本发明制备的糖修饰的多粘菌素前药可显著降低多粘菌素的体内毒性。
实施例7：
ABE-Colistin对大肠杆菌ATCC35218感染的肺炎小鼠的治疗效果
使用6-7周龄、体重为25-30g的ICR雌性小鼠。收集处于对数期的大肠杆菌(ATCC35128)，用PBS清洗3次，得到PBS细菌悬浮液后，测定600nm处的吸光值确定细菌浓度，最后稀释配制成动物感染模型所需的细菌悬浮液浓度为2×10 8CFU/mL。对小鼠通过腹腔注射无菌1％戊巴比妥钠溶液进行麻醉。检查小鼠已经处于深层麻醉状态后，将其固定于操作台上，用无菌手术镊将小鼠舌头向外牵扯，充分暴露出咽喉部空间，将辅助管顺着喉部插入气管内，再将导管插入辅助管内。每只小鼠肺部注入50μL，浓度为2×10 8CFU/mL的大肠杆菌(ATCC35128)细菌悬浮液，等待苏醒后转置鼠笼中，继续以原饲养条件正常饲养，感染状态持续12小时。Colistin(3mg/kg)的注射溶液用5％无菌葡萄糖溶液配制，ABE-Colistin(3mg/kg Colistin)，ABE-Colistin(15mg/kg Colistin)，ABE-Colistin(75mg/kg Colistin)先用DMSO配制成母液，再用5％无菌葡萄糖溶液稀释成注射溶液(DMSO的体积不可超过总注射体积的5％)，通过尾静脉注射给药治疗，空白对照组注射等体积的无菌PBS溶液作为对照。治疗12小时，将小鼠处死，取出完整的肺部，在无菌的PBS中反复漂洗，除去血液，再用无菌吸水纸拭干水分，装入匀浆管中。净肺部重量记录完 毕后，每个匀浆管中加入无菌水，使得总体积约为0.8mL，所有匀浆管置于冰上保存。利用高速组织均质器得到肺组织匀浆。组织匀浆置于冰上，用无菌PBS进行梯度稀释，稀释10倍，100倍，1000倍后，包括原液在内的每个稀释倍数取每个样品10μL滴加至琼脂板上涂板，琼脂板于37℃恒温箱中过夜后计数。组织的细菌存活率(CFU/g)＝每个样品的细菌数量/该样品的组织重量，取mean±SD值统计作图。结果如图24所示，ABE-Colistin(3mg/mL Colistin)的体内治疗效果与Colistin(3mg/mL)组相近，随着ABE-Colistin的剂量增大，小鼠每克肺组织细菌量降低1个数量级以上，并且小鼠未出现死亡情况。该结果表明ABE-Colistin在体内保证安全的情况下随着剂量增大可以获得比Colistin更优异的治疗效果。
实施例8：
Lac-ABA-Colistin对大肠杆菌ATCC35218感染的肺炎小鼠的治疗效果。
感染方式与实施例7相同。给药方式为：Colistin(3mg/kg)、Lac-ABA-Colistin(3mg/kg Colistin)用5％无菌葡萄糖溶液稀释成注射溶液，通过尾静脉注射给药治疗，空白对照组注射等体积的5％无菌葡萄糖溶液作为对照，分别于感染12小时和18小时给药，感染24小时，将小鼠处死，取出完整的肺部，在无菌的PBS中反复漂洗，除去血液，再用无菌吸水纸拭干水分，装入加入无菌水的匀浆管中，置于冰上保存。利用高速组织均质器得到肺组织匀浆。将组织匀浆置于冰上，用无菌PBS进行梯度稀释，稀释10倍，100倍，1000倍后，包括原液在内的每个稀释倍数取每个样品10μL滴加至琼脂板上涂板，琼脂板于37℃恒温箱中过夜后计数。结果如图25所示，Lac-ABA-Colistin的感染肺部中细菌的数量相对于5％葡萄糖组的减少了10倍，显示出Lac-ABA-Colistin对肺炎具有一定的治疗效果。另外，Lac-ABA-Colistin组与Colistin组的感染肺部中细菌的数量相当，表明乳糖修饰的多粘菌素前药与多粘菌素具有相近的体内抗菌活性。
以上所述实施例的各技术特征可以进行任意的组合，为使描述简洁，未对以下实施例中的各个技术特征所有可能的组合都进行描述，然而，只要这些技术特征的组合不存在矛盾，都应当认为是本说明书记载的范围。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对本发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。

Claims (15)

  1. 具有式(I)所示结构的多粘菌素前药化合物或其立体异构体或其药学上可接受的盐：
    Figure PCTCN2022099235-appb-100001
    其中，R 1选自：
    Figure PCTCN2022099235-appb-100002
    R 2、R 3分别独立地选自C 1-C 8烷基；
    R选自：硼酸基、取代或者未取代的硼酸酯基。
  2. 根据权利要求1所述的多粘菌素前药化合物或其立体异构体或其药学上可接受的盐，其特征在于，R选自：由硼酸基与含有邻位二羟基结构的糖反应得到的硼酸酯基、
    Figure PCTCN2022099235-appb-100003
    或者
    Figure PCTCN2022099235-appb-100004
  3. 根据权利要求1所述的多粘菌素前药化合物或其立体异构体或其药学上可接受的盐，其特征在于，所述含有邻位二羟基结构的糖为单糖、双糖或者三糖。
  4. 根据权利要求1所述的多粘菌素前药化合物或其立体异构体或其药学上可接受的盐，其特征在于，所述多粘菌素前药化合物具有如下式(II)所示结构：
    Figure PCTCN2022099235-appb-100005
    其中，R 1选自：
    Figure PCTCN2022099235-appb-100006
    R 2、R 3分别独立地选自C 1-C 8烷基；
    各R 4、R 5分别独立地选自：H、C 1-C 6烷基、一个或多个R 6取代的C 1-C 6烷基、或者R 4、R 5和与其相连的-O-B-O-一起形成如下结构：
    Figure PCTCN2022099235-appb-100007
    各R 6分别独立地选自：H、羟基、羧基、羰基、醛基、C 1-C 6烷氧基；
    各R 7、R 8、R 9、R 10分别独立地选自：H、C 1-C 8烷基、C 1-C 8烷氧基、一个或多个R 12取代的C 1-C 8烷基、一个或多个R 12取代的C 1-C 8烷氧基、羧基、醛基，或者R 7和R 10为氢，R 8、R 9和与其相连的碳原子一起形成一个或多个R 13取代的5-6元含氧杂环基；
    各R 12分别独立地选自：H、羟基、羧基、羰基、醛基、C 1-C 6烷氧基、羟基取代的C 1-C 6烷基、一个或多个R 15取代的5-6元含氧杂环烷氧基；
    各R 13分别独立地选自：H、羟基、一个或多个R 16取代的C 1-C 6烷基、一个或多个R 15取代的5-6元含氧杂环烷氧基、一个或多个R 17取代的C 1-C 8烷氧基；
    各R 15分别独立地选自：H、羟基、羟基取代的C 1-C 6烷基、一个或多个R 18取代的5-6元含氧杂环烷氧基；
    各R 16分别独立地选自：H、羟基、一个或多个R 15取代的5-6元含氧杂环烷氧基；
    各R 17分别独立地选自：H、羟基、羟基取代的C 1-C 6烷基、一个或多个R 19取代的5-6元含氧杂环基；
    各R 18分别独立地选自：H、羟基、羟基取代的C 1-C 6烷基；
    各R 19分别独立地选自：H、羟基、羟基取代的C 1-C 6烷基、一个或多个R 18取代的5-6元含氧杂环烷氧基。
  5. 根据权利要求1-4任一项所述的多粘菌素前药化合物或其立体异构体或其药学上可接受的盐，其特征在于，R 2选自C 3-C 4烷基；R 3选自C 2-C 5烷基。
  6. 根据权利要求4所述的多粘菌素前药化合物或其立体异构体或其药学上可接受的盐，其特征在于，R 4和R 5均为H，或者R 4、R 5和与其相连的-O-B-O-一起形成如下结构：
    Figure PCTCN2022099235-appb-100008
    R 7、R 8、R 9和R 10分别独立地选自：H、C 1-C 4烷基、C 1-C 4烷氧基、一个或多个R 12取代 的C 1-C 4烷基、一个或多个R 12取代的C 1-C 4烷氧基、羧基、醛基，或者R 7和R 10为氢，R 8、R 9和与其相连的碳原子一起形成一个或多个R 13取代的6元含氧杂环基。
  7. 根据权利要求6所述的多粘菌素前药化合物或其立体异构体或其药学上可接受的盐，其特征在于，各R 12分别独立地选自：H、羟基、羧基、羰基、醛基、C 1-C 3烷氧基、羟基取代的C 1-C 3烷基、一个或多个R 15取代的6元含氧杂环烷氧基；其中，各R 15分别独立地选自：H、羟基、羟基取代的C 1-C 3烷基。
  8. 根据权利要求6所述的多粘菌素前药化合物或其立体异构体或其药学上可接受的盐，其特征在于，各R 13分别独立地选自：H、羟基、一个或多个R 16取代的C 1-C 3烷基、一个或多个R 15取代的6元含氧杂环烷氧基、一个或多个R 17取代的C 1-C 3烷氧基；其中，各R 15分别独立地选自：H、羟基、羟基取代的C 1-C 3烷基、一个或多个R 18取代的5-6元含氧杂环烷氧基；各R 16分别独立地选自：H、羟基、一个或多个R 15取代的6元含氧杂环烷氧基；各R 17分别独立地选自：H、羟基、羟基取代的C 1-C 3烷基、一个或多个R 19取代的5-6元含氧杂环基；各R 18分别独立地选自：H、羟基、羟基取代的C 1-C 3烷基；各R 19分别独立地选自：H、羟基、羟基取代的C 1-C 3烷基、一个或多个R 18取代的5-6元含氧杂环烷氧基。
  9. 根据权利要求4所述的多粘菌素前药化合物或其立体异构体或其药学上可接受的盐，其特征在于，R 4和R 5均为H，或者R 4、R 5和与其相连的-O-B-O-一起形成如下结构：
    Figure PCTCN2022099235-appb-100009
  10. 根据权利要求1-4任一项所述的多粘菌素前药化合物或其立体异构体或其药学上可接受的盐，其特征在于，所述多粘菌素前药化合物选自如下化合物：
    Figure PCTCN2022099235-appb-100010
    Figure PCTCN2022099235-appb-100011
    Figure PCTCN2022099235-appb-100012
    Figure PCTCN2022099235-appb-100013
    其中，R 1为
    Figure PCTCN2022099235-appb-100014
    R 2为
    Figure PCTCN2022099235-appb-100015
    R 3选自
    Figure PCTCN2022099235-appb-100016
    或者，
    R 1为
    Figure PCTCN2022099235-appb-100017
    R 2为
    Figure PCTCN2022099235-appb-100018
    R 3为
    Figure PCTCN2022099235-appb-100019
    或者，
    R 1为
    Figure PCTCN2022099235-appb-100020
    R 2为
    Figure PCTCN2022099235-appb-100021
    R 3选自
    Figure PCTCN2022099235-appb-100022
    或者，
    R 1为
    Figure PCTCN2022099235-appb-100023
    R 2为
    Figure PCTCN2022099235-appb-100024
    R 3选自
    Figure PCTCN2022099235-appb-100025
    或者，
    R 1为
    Figure PCTCN2022099235-appb-100026
    R 2为
    Figure PCTCN2022099235-appb-100027
    R 3选自
    Figure PCTCN2022099235-appb-100028
    或者，
    R 1为
    Figure PCTCN2022099235-appb-100029
    R 2为
    Figure PCTCN2022099235-appb-100030
    R 3选自
    Figure PCTCN2022099235-appb-100031
  11. 权利要求1-10任一项所述的多粘菌素前药化合物或其立体异构体或其药学上可接 受的盐在制备治疗细菌感染的药物中的应用。
  12. 根据权利要求11所述的应用，其特征在于，所述细菌为革兰氏阴性细菌。
  13. 根据权利要求12所述的应用，其特征在于，所述细菌为克雷伯氏肺炎菌、鲍曼不动杆菌、绿脓杆菌、鼠伤寒沙门氏菌、肠杆菌科细菌。
  14. 根据权利要求13所述的应用，其特征在于，所述细菌为肺炎克雷伯菌、阴沟肠杆菌、鼠伤寒沙门氏菌、大肠杆菌和绿脓杆菌。
  15. 一种治疗细菌感染的药物，其特征在于，由活性成分和药学上可接受的辅料制备得到，所述活性成分包括权利要求1-10任一项所述的多粘菌素前药化合物或其立体异构体或其药学上可接受的盐。
PCT/CN2022/099235 2021-06-18 2022-06-16 活性氧自由基响应的多粘菌素前药化合物及其应用 WO2022262823A1 (zh)

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CN103946233A (zh) * 2011-11-18 2014-07-23 诺瓦克塔生物系统有限公司 多黏菌素衍生物
WO2018108154A1 (zh) * 2016-12-16 2018-06-21 中国医学科学院医药生物技术研究所 多粘菌素衍生物及其制备方法和应用
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