WO2023138211A1 - 星型β-抗菌糖肽、其制备方法及应用 - Google Patents

星型β-抗菌糖肽、其制备方法及应用 Download PDF

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WO2023138211A1
WO2023138211A1 PCT/CN2022/134136 CN2022134136W WO2023138211A1 WO 2023138211 A1 WO2023138211 A1 WO 2023138211A1 CN 2022134136 W CN2022134136 W CN 2022134136W WO 2023138211 A1 WO2023138211 A1 WO 2023138211A1
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substituted
unsubstituted
antibacterial
star
general formula
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PCT/CN2022/134136
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English (en)
French (fr)
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司张勇
朱潇
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苏州万维生命科学技术有限公司
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Priority claimed from CN202210076643.9A external-priority patent/CN116515102A/zh
Priority claimed from CN202211186482.5A external-priority patent/CN117815249A/zh
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Publication of WO2023138211A1 publication Critical patent/WO2023138211A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7016—Disaccharides, e.g. lactose, lactulose
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G69/00—Macromolecular compounds obtained by reactions forming a carboxylic amide link in the main chain of the macromolecule
    • C08G69/02—Polyamides derived from amino-carboxylic acids or from polyamines and polycarboxylic acids
    • C08G69/08—Polyamides derived from amino-carboxylic acids or from polyamines and polycarboxylic acids derived from amino-carboxylic acids
    • C08G69/14—Lactams
    • C08G69/22—Beta-lactams

Definitions

  • the application specifically relates to a star-shaped ⁇ -antibacterial glycopeptide, its preparation method and its application in medicine, belonging to the field of medicine.
  • Gram-negative bacteria have a low-penetrability outer membrane and an efficient efflux pump system.
  • the low-penetrability outer membrane can prevent drug molecules from entering the bacterial internal target, and its efficient efflux pump system can discharge the antibiotics that have entered the bacterial internal target to the outside of the bacterial body.
  • These two processes greatly limit the concentration accumulation of antibiotics at their drug targets, causing the antibiotics to lose their effectiveness.
  • Gram-negative bacteria resistant to carbapenems have been listed by the World Health Organization as a group of bacteria that urgently need to find new antibiotics or new treatments.
  • beta peptides Compared with natural alpha peptides, beta peptides have an extra methylene group on the backbone. This endows beta peptides with greater molecular chain flexibility, allowing the formation of different types of secondary structures to exhibit an amphiphilic conformation, which is one of the key elements contributing to the antimicrobial activity of antimicrobial peptides.
  • Another outstanding feature of beta peptides is their resistance to cleavage by proteases, resulting in increased stability in vivo. These properties make beta peptides more attractive as candidate antibacterial agents. However, the antibacterial properties and biocompatibility of the existing beta polypeptides still need to be further improved.
  • IFDs invasive fungal infections
  • US CDC U.S. Centers for Disease Control and Prevention
  • AMR Emergency Antimicrobial Resistance
  • azoles including imidazoles such as clotrimazole, ketoconazole, etc., and triazoles such as fluconazole, itraconazole, etc.
  • CYP51 cytochrome P450-dependent enzyme-14 ⁇ -lanolin alcohol demethylase
  • Increased permeability eventually inhibits fungal growth and replication (Clinical infectious diseases: an official publication of the Infectious Diseases Society of America, 62(3), 362-368); polyenes (amphotericin B, nystatin, etc.) can combine with ergosterol on the fungal cytoplasmic membrane, resulting in membrane decomposition or increased membrane permeability, causing Cell contents overflow and die (Actapharmaceutica Sinica.B, 11(8), 2585-2604); flucytosines (such as 5-fluorocytosine) can inhibit thymidine synthase and affect DNA and protein synthesis (Nature communications, 12(1), 3418); and echinocandins (such as caspofungin, micafungin, etc.), can inhibit the main component of fungal cell wall 1 , the synthesis of 3- ⁇ -D-glucan, etc.
  • flucytosines such as 5-fluorocytosine
  • echinocandins such as caspofungin, micafungin
  • the main purpose of this application is to provide a star-shaped ⁇ -antibacterial glycopeptide, its preparation method and its use in the preparation of drugs for preventing or treating fungal infections, so as to overcome the shortcomings of the prior art.
  • Core is a structural unit with a cyclic group
  • R 1 , R 2 , R 3 , R 4 Each independently selected from H, halogen, substituted or unsubstituted C1-C20 alkyl, substituted or unsubstituted C1-C20 alkoxy, substituted or unsubstituted C2-C20 alkenyl, substituted or unsubstituted C2-C20 alkynyl, substituted or unsubstituted C3-C20 alicyclic group, substituted or unsubstituted C6-C15 aryl, substituted or unsubstituted C7-C30 alkyl-aryl, substituted or unsubstituted C3-C20 hetero Ring, substituted or unsubstituted C4-C30 alkyl-heterocycle, substituted or unsubstituted C5-C15 heteroaryl, C1-C20 hydroxyalkyl, cyano, amino, guanidino, nitro or hydroxyl;
  • n 0-6, m is 3-100, and x and y are 1-50.
  • the second aspect of the present application provides a star-shaped ⁇ -antibacterial glycopeptide, the outer layer of which is a helical glycopeptide block, and the inner layer is a positively charged polydimethylaminobeta lactam block.
  • the structure of the star-shaped ⁇ -antibacterial glycopeptide is shown in general formula (I).
  • a third aspect of the present application provides a method for preparing a compound represented by general formula (I), the method comprising:
  • the precursor compound comprising Core is polymerized with the compound shown in formula (II) and the compound shown in formula (III) to obtain the polymer shown in formula (IV);
  • the fourth aspect of the present application provides a pharmaceutical composition, comprising a compound represented by general formula (I) or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.
  • the fifth aspect of the present application provides the use of the compound represented by the general formula (I) or a pharmaceutically acceptable salt thereof or the pharmaceutical composition as an antibacterial agent.
  • the sixth aspect of the present application provides the use of the compound represented by general formula (I) or a pharmaceutically acceptable salt thereof or the pharmaceutical composition in the preparation of a medicament for treating or preventing bacterial infection or a disease caused by the bacterial infection.
  • the seventh aspect of the present application provides the use of the compound represented by the general formula (I) or a pharmaceutically acceptable salt thereof or the pharmaceutical composition as an efflux pump inhibitor and/or a bacterial membrane permeabilizing agent.
  • the eighth aspect of the present application provides a method for treating or preventing a bacterial infection or a disease caused by the bacterial infection in a subject, the method comprising administering an effective amount of the compound represented by general formula (I) or the pharmaceutical composition to the subject.
  • the bacteria are multidrug resistant.
  • the method further comprises administering to the subject an effective amount of another antibacterial agent.
  • antibacterial agents include, but are not limited to, but not limited to ammonia pyrine, chlorocelalin, phenazylin and chiraxin, cephalosporins such as cefchlo, ceptopyol, cephalipinone, cephalosporin, cephalus, cephalosporte, cephalosporte, and cefulin; Large cycloids, ninosone, tetracycline and amino glycoside.
  • said other antibacterial agents can also be selected from rifampicin, ciprofloxacin, levofloxacin, piperacillin, cotrimoxazole, gentamicin, lobumycin, erythromycin ethylsuccinate, erythromycin, clarithromycin, novobiocin, spiramycin, acetylspiramycin, chloramphenicol, trimethoprim, sulfamethoxazole, carbenicillin, polymyxin B, colistin, amikacin, kanamycin, neomycin, netilmicin, streptomycin, tobramycin, paromomycin, Geldanamycin, herbimycin, clocarbaef, doripenem, cilastatin, cefadroxil, cephalothin, cephalexin, cefamandole, cefprozil, cefuroxime, cefixime, cefdinir, cefditor
  • the ninth aspect of the present application provides the use of the compound represented by general formula (I) or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition, or the star-shaped ⁇ -antibacterial glycopeptide in the preparation of a drug for preventing or treating fungal infection.
  • the tenth aspect of the present application provides the use of the compound represented by the general formula (I) or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition, or the star-shaped ⁇ -antibacterial glycopeptide in the preparation of an antifungal drug synergist.
  • the eleventh aspect of the present application provides the use of the compound represented by the general formula (I) or its pharmaceutically acceptable salt or the pharmaceutical composition, or the star-shaped ⁇ -antibacterial glycopeptide in the preparation of products for the prevention, diagnosis, detection, protection, treatment or research of pathogenic fungi and their directly related diseases.
  • the twelfth aspect of the present application provides a method for treating or preventing a fungal infection or a disease caused by the fungal infection in a subject, the method comprising administering an effective amount of the star ⁇ -antibacterial glycopeptide or the pharmaceutical composition to the subject.
  • the fungi include fungi of the genus Aspergillus, Candida, etc., especially fungi with drug resistance, but are not limited thereto.
  • the method further comprises co-administering the star ⁇ -antibacterial glycopeptide and an antifungal agent to the subject.
  • the antifungal drugs include azoles, polyenes, flucytosines, echinocandins, etc., but are not limited thereto.
  • the compound represented by the general formula (I) provided by this application is a non-natural ⁇ antibacterial glycopeptide. Its chemical structure is a block star polymer. A hydration isolation layer further reduces protein adsorption, especially to better reduce the cytotoxicity caused by positive charges.
  • the compound or its salt can be used in combination with different antibiotics to restore and enhance the bactericidal effect of the antibiotics.
  • star-shaped ⁇ -antibacterial glycopeptide provided by this application when used in combination with commonly used antifungal drugs, it can effectively overcome the resistance of drug-resistant fungi to antifungal drugs, and can guarantee and improve the killing effect on drug-resistant fungi while greatly reducing the amount of anti-fungal drugs used. It has excellent preventive or therapeutic effects on fungal infections, especially drug-resistant fungal infections and diseases caused by them.
  • Fig. 1 shows a kind of star type ⁇ -antibacterial glycopeptide 1H NMR spectrum in embodiment 1.
  • FIG. 2 shows a gel permeation chromatogram of a star ⁇ -antibacterial glycopeptide in Example 1 before deprotection.
  • FIG. 3 shows the cytotoxicity of a star ⁇ -antibacterial glycopeptide in Example 1 before deprotection.
  • Figure 4 shows that the star-shaped ⁇ -antibacterial glycopeptide enhances the bactericidal effect of rifampicin on carbapenem-resistant Acinetobacter baumannii by the checkerboard broth microdilution assay in Example 1.
  • FIG. 5 shows that the star-shaped ⁇ -antibacterial glycopeptide enhances the bactericidal effect of roxithromycin on carbapenem-resistant Acinetobacter baumannii by the checkerboard broth microdilution assay in Example 1.
  • FIG. 6 shows that the star-shaped ⁇ -antibacterial glycopeptide enhances the bactericidal effect of clarithromycin on carbapenem-resistant Acinetobacter baumannii as determined by checkerboard broth microdilution in Example 1.
  • FIG. 7 shows that the star-shaped ⁇ -antibacterial glycopeptide enhances the bactericidal effect of erythromycin against carbapenem-resistant Acinetobacter baumannii by the checkerboard broth microdilution assay in Example 1.
  • Figure 8 shows that the star-shaped ⁇ -antibacterial glycopeptide enhances the bactericidal effect of novobiocin on carbapenem-resistant Acinetobacter baumannii by the checkerboard broth microdilution assay in Example 1.
  • FIG. 9 shows that the star-shaped ⁇ -antibacterial glycopeptide enhances the bactericidal effect of spiramycin against carbapenem-resistant Acinetobacter baumannii by the checkerboard broth microdilution assay in Example 1.
  • FIG. 10 shows that the star-shaped ⁇ -antibacterial glycopeptide enhances the bactericidal effect of acetylspiramycin against carbapenem-resistant Acinetobacter baumannii by the checkerboard broth microdilution assay in Example 1.
  • Figure 11 shows that the combination of star ⁇ -antibacterial glycopeptide and rifampicin has excellent bactericidal effect on carbapenem-resistant Acinetobacter baumannii through the time-killing curve measurement in Example 1.
  • Figure 12 shows that the combination of star ⁇ -antibacterial glycopeptide and novobiocin has excellent bactericidal effect on carbapenem-resistant Acinetobacter baumannii through the time-killing curve measurement in Example 1.
  • FIG. 13 shows that the combination of star ⁇ -antibacterial glycopeptide and clarithromycin has excellent bactericidal effect on carbapenem-resistant Acinetobacter baumannii through the time-killing curve measurement in Example 1.
  • Figure 14 shows that the star ⁇ -antibacterial glycopeptide enhances the bactericidal effect of novobiocin on Acinetobacter vulgaris baumannii (ATCC19606) by the checkerboard broth microdilution assay in Example 1.
  • Figure 15 shows that the star-shaped ⁇ -antibacterial glycopeptide enhances the bactericidal effect of erythromycin on Acinetobacter vulgaris baumannii (ATCC19606) by the checkerboard broth microdilution assay in Example 1.
  • Figure 16 shows that the star-shaped ⁇ -antibacterial glycopeptide enhances the bactericidal effect of clarithromycin on Acinetobacter vulgaris baumannii (ATCC19606) by the checkerboard broth microdilution assay in Example 1.
  • Figure 17 shows that the star-shaped ⁇ -antibacterial glycopeptide enhances the bactericidal effect of roxithromycin on Acinetobacter vulgaris baumannii (ATCC19606) by the checkerboard broth microdilution assay in Example 1.
  • Figure 18 shows that the star ⁇ -antibacterial glycopeptide enhances the bactericidal effect of acetylspiramycin on Acinetobacter vulgaris baumannii (ATCC19606) by the checkerboard broth microdilution assay in Example 1.
  • Figure 19 shows that the star-shaped ⁇ -antibacterial glycopeptide enhances the bactericidal effect of erythromycin ethylsuccinate on Acinetobacter vulgaris (ATCC19606) by the checkerboard broth microdilution assay in Example 1.
  • Figure 20 shows that the star-shaped ⁇ -antibacterial glycopeptide enhances the bactericidal effect of rifampicin on Acinetobacter vulgaris baumannii (ATCC19606) by the checkerboard broth microdilution assay in Example 1.
  • Figure 21 shows that the star ⁇ -antibacterial glycopeptide enhances the bactericidal effect of nalidixic acid against Acinetobacter vulgaris baumannii (ATCC 19606) by the checkerboard broth microdilution assay in Example 1.
  • Fig. 22 shows the bactericidal curve of the combination of star ⁇ -antibacterial glycopeptide and itraconazole against drug-resistant Aspergillus fumigatus in Example 1.
  • Fig. 23 shows the bactericidal curve of the combination of star ⁇ -antibacterial glycopeptide and itraconazole against drug-resistant Candida albicans in Example 1.
  • Core is a structural unit with a cyclic group
  • R 1 , R 2 , R 3 , R 4 Each independently selected from H, halogen, substituted or unsubstituted C1-C20 alkyl, substituted or unsubstituted C1-C20 alkoxy, substituted or unsubstituted C2-C20 alkenyl, substituted or unsubstituted C2-C20 alkynyl, substituted or unsubstituted C3-C20 alicyclic group, substituted or unsubstituted C6-C15 aryl, substituted or unsubstituted C7-C30 alkyl-aryl, substituted or unsubstituted C3-C20 hetero ring, substituted or unsubstituted C4-C30 alkyl-heterocycle, substituted or unsubstituted C5-C15 heteroaryl, C1-C20 hydroxyalkyl, cyano, amino, guanidino, nitro or hydroxyl;
  • n 0-6, m is 3-100, and x and y are 1-50.
  • the structural units with cyclic groups include substituted or unsubstituted 3-12 membered cycloalkyl groups, substituted or unsubstituted 3-12 membered heterocyclic groups, substituted or unsubstituted 6-10 membered aryl groups or substituted or unsubstituted 5-10 membered heteroaryl groups, organic macromolecular residues with cyclic groups (such as cyclodextrin molecular residues) or inorganic compound molecular residues with cyclic groups (such as cage polysilsesquioxane molecular residues).
  • the structural unit having a cyclic group is selected from substituted or unsubstituted phenyl groups.
  • the compound is a compound represented by general formula (I-1):
  • the aforementioned alkyl group can be straight or branched, and can be optionally substituted, for example, can be selected from methyl, ethyl, 1-propyl or n-propyl, 2-propyl or isopropyl, 1-butyl or n-butyl, 2-methyl-1-propyl or isobutyl, 1-methylpropyl or sec-butyl, 1,1-dimethylethyl or tert-butyl, 1-pentyl, 2-pentyl, 3-pentyl, 2-methyl-2-butyl, 3- Methyl-2-butyl, 3-methyl-1-butyl, 2-methyl-1-butyl, 1-hexyl, 2-hexyl, 3-hexyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 3-methyl-3-pentyl, 2-methyl-3-pentyl, 2,3-dimethyl-2-butyl and 3,3
  • the aforementioned alkoxy group may be linear or branched, for example, may be selected from but not limited to methoxy, ethoxy, propoxy, butoxy and the like.
  • the aforementioned alkenyl groups may be linear or branched, and may be optionally substituted, ie, substituted or unsubstituted.
  • the aforementioned alkenyl group may be selected from, but not limited to, ethenyl, propenyl, butenyl, 1,4-butadienyl, pentenyl, hexenyl, 4-methylhex-1-enyl, 4-ethyl-2-methylhex-1-enyl and the like.
  • the aforementioned alkynyl groups may be straight or branched, and may be optionally substituted.
  • the aforementioned alkynyl group may be selected from, but not limited to, ethynyl, propynyl, butynyl and the like.
  • the aforesaid alicyclic group includes cycloalkyl and cycloalkenyl, for example, can be selected from but not limited to cyclopropane, cyclobutane, cyclopentane, cyclopentene, cyclopentadiene, cyclohexane, cyclohexene, 1,3-cyclohexadiene, 1,4-cyclohexadiene, cycloheptane and cycloheptene and the like.
  • the aforementioned aryl group can be a 6-membered carbocyclic aromatic ring such as phenyl, or a 7-12-membered bicyclic ring such as naphthalene, indane, and 1,2,3,4-tetrahydroquinoline, etc., and is not limited thereto.
  • the aforementioned heterocyclic ring is an aliphatic spirocyclic ring comprising at least one heteroatom selected from N, O and S, which can be selected from but not limited to 1-pyrrolidinyl, 2-pyrrolidinyl, 2,4-imidazolidinyl, 2,3-pyrazolidinyl, 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-piperidinyl, 2,5-piperazinyl, pyranyl, 2-morpholinyl, oxiranyl, aziridinyl, azetidinyl, tetrahydropyridine Pyridyl, thiomorpholinyl, thienyl, piperazinyl, homopiperazinyl, homopiperidinyl, azepanyl, oxepanyl, thienyl, 1,4-oxepanyl, 1,4-dioxepane, 1,4-oxepane
  • the aforementioned alkyl heterocycle refers to a chemical substituent comprising an alkyl group coupled to a heterocycle or a substituted heterocycle.
  • the aforementioned heteroaryl refers to an aromatic heterocyclic ring, which may be formed by five, six, seven, eight, nine or more atoms.
  • Heteroaryl groups can be optionally substituted. For example, it can be selected from but not limited to aromatic C5-C15 heterocyclic groups containing one oxygen or sulfur atom or up to four nitrogen atoms, or a combination of one oxygen or sulfur atom and up to two nitrogen atoms, and their substituted and benzo and pyridofused derivatives.
  • R 1 , R 2 , and R 3 are each independently selected from C1-C6 alkyl, hydroxyl or amino groups.
  • R3 can also be selected from amine, hydroxyl, and the like.
  • R 4 , R 5 and R 6 are hydrogen atoms.
  • the compound is a compound represented by the following formula:
  • Some embodiments of the present application also provide a star-shaped ⁇ -antibacterial glycopeptide, the outer layer of which is a helical glycopeptide block, and the inner layer is a positively charged polydimethylaminobeta lactam block.
  • the structure of the star-shaped ⁇ -antibacterial glycopeptide is shown in general formula (I).
  • the source of the glycopeptide block includes monosaccharide glucose, galactose, mannose, altrose, glucosamine, galactosamine, mannose amino or disaccharide maltose, but not limited thereto.
  • the star-shaped ⁇ -antibacterial glycopeptide provided in the examples of the present application is a type of star-shaped block polymer.
  • the outer layer of the helical glycopeptide can be better exposed to the outer layer of the compound to bind to the sugar receptor on the surface of bacteria to prevent infection; form a layer of hydration to prevent protein adsorption; block the combination of the cationic block in the inner layer and mammalian cells, and further improve biocompatibility.
  • Some embodiments of the present application also provide a method for preparing the compound represented by general formula (I), the method comprising:
  • the precursor compound comprising Core is polymerized with the compound shown in formula (II) and the compound shown in formula (III) to obtain the polymer shown in formula (IV);
  • the compound with a protecting group represented by formula (II) and the compound with a protecting group represented by formula (III) can be synthesized according to methods known in the art.
  • glucose-derived beta lactam AS(Bn)
  • AS(Bn) glucose-derived beta lactam
  • Cbz carboxybenzyl-protected beta lactam
  • DM(Cbz) carboxybenzyl-protected beta lactam with amino group can be synthesized according to literature (Angew.Chem.Int.Ed.2020doi.org/10.1002/anie.201914304).
  • protecting group is well known in the art, and generally refers to a chemical group that reacts with and combines with a functional group in a molecule to prevent the functional group from participating in the subsequent reaction of the molecule, but the group can be removed subsequently to regenerate the unprotected functional group.
  • the protecting group includes but not limited to benzyl, carboxybenzyl (Cbz), tert-butoxycarbonyl (Boc), etc., preferably carboxybenzyl.
  • step (a) includes: preparing the protected polymer in the presence of lithium bis(trimethylsilyl)amide and 4-tert-butylbenzoyl chloride.
  • reaction temperature used in step (a) may be 0°C to 66°C, preferably at room temperature.
  • step (b) includes: deprotecting the protected polymer under conditions of sodium and liquid ammonia.
  • reaction temperature used in step (b) may be -80°C to -50°C, preferably -78°C to -55°C.
  • Some embodiments of the present application also provide a pharmaceutical composition, comprising the compound represented by the general formula (I) or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.
  • Said salt can be any salt, organic or inorganic addition salt, especially any pharmaceutically acceptable organic or inorganic addition salt commonly used in pharmacy, for example the compound shown in general formula (I) and inorganic acid such as hydrochloric acid, hydrobromic acid, hydroiodic acid or with organic acid such as formic acid, acetic acid, acetoacetic acid, trifluoroacetic acid, trifluoromethanesulfonic acid, p-toluenesulfonic acid, methanesulfonic acid reaction formation salt.
  • inorganic acid such as hydrochloric acid, hydrobromic acid, hydroiodic acid or with organic acid such as formic acid, acetic acid, acetoacetic acid, trifluoroacetic acid, trifluoromethanesulfonic acid, p-toluenesulfonic acid, methanesulfonic acid reaction formation salt.
  • the pharmaceutical composition further comprises other antibacterial agents.
  • the other antibacterial agents include but are not limited to ampicillin, cloxacillin, oxacillin and piperacillin, cephalosporins such as cefaclor, cefamandole, cefazolin, cefoperazone, cefotaxime, cefoxitin, ceftazidime, ceftriaxone and cephalosporins; carbapenems include imipenem and meropenem; Glycosides.
  • the other antibacterial agents can also be selected from rifampicin, ciprofloxacin, levofloxacin, piperacillin, cotrimoxazole, gentamicin, lobumycin, erythromycin ethylsuccinate, erythromycin, clarithromycin, novobiocin, spiramycin, acetylspiramycin, chloramphenicol, trimethoprim, sulfamethoxazole, carbenicillin, polymyxin B, colistin, amikacin, kanamycin, neomycin, netilmicin, streptomycin, tobramycin, paromomycin, Geer Demycin, herbimycin, clocarbacephalosporin, doripenem, cilastatin, cefadroxil, cephalothin, cephalexin, cefamandole, cefprozil, cefuroxime, cefixime, cefdinir, cef
  • the other antibacterial agents include but not limited to antibiotics against Gram-positive bacteria, and the antibacterial range of the antibacterial agents includes Gram-negative bacteria. Further, using the compound represented by the general formula (I) or a pharmaceutically acceptable salt thereof can expand the antibacterial range of antibiotics against Gram-positive bacteria, making it also effective against Gram-negative bacteria.
  • the aforementioned pharmaceutically acceptable carrier includes pharmaceutically acceptable materials, compositions, excipients, etc., such as liquid or solid fillers, diluents, excipients or solvent encapsulation materials, specifically including but not limited to the following substances: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; tragacanth gum powder; malt; ; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols such as propylene glycol; polyols such as glycerin, sorbitol, mannitol and polyethylene glycol; esters such as ethyl oleate and ethyl laurate;
  • pharmaceutically inert, inorganic or organic excipients may be used for the preparation of the pharmaceutical compositions.
  • lactose, talc, stearic acid and its salts, fats, waxes, solid or liquid polyols, natural oils and hardened oils and the like can be used as pharmaceutically acceptable carriers.
  • water, alcohol, glycerin, polyols and their appropriate mixtures, and vegetable oils can be used as pharmaceutically acceptable carriers.
  • the preparation can be sterilized in various ways, including filtering through a bacteria-retaining filter, or adding a sterilizing agent in the form of a sterile solid composition.
  • Some embodiments of the present application also provide the use of the compound represented by the general formula (I) or a pharmaceutically acceptable salt thereof or the pharmaceutical composition as an antibacterial agent.
  • antibacterial agent refers to any substance or combination that can: (i) inhibit, reduce or prevent the growth of bacteria; (ii) have the ability to inhibit or reduce the ability of bacteria to produce infection in a subject; (iii) have the ability to inhibit or reduce the ability of bacteria to reproduce or maintain infectivity in the environment.
  • Some embodiments of the present application also provide the use of the compound represented by the general formula (I) or a pharmaceutically acceptable salt thereof or the pharmaceutical composition in the preparation of a medicament for treating or preventing bacterial infection or a disease caused by the bacterial infection.
  • the bacteria include at least one of multi-drug resistant bacteria and sensitive bacteria.
  • the bacterium has multi-drug resistance, that is, the bacterium is resistant to multiple drugs with different chemical structures and/or has drug resistance to drugs targeting different targets.
  • the bacteria include Gram-negative bacteria (such as Escherichia coli, Pseudomonas aeruginosa, Helicobacter pylori, Klebsiella pneumoniae, etc.), Gram-positive bacteria, etc., especially carbapenem-resistant Gram-negative bacteria, such as carbapenem-resistant Acinetobacter baumannii.
  • Gram-negative bacteria such as Escherichia coli, Pseudomonas aeruginosa, Helicobacter pylori, Klebsiella pneumoniae, etc.
  • carbapenem-resistant Gram-negative bacteria such as carbapenem-resistant Acinetobacter baumannii.
  • the drug can be prepared in various forms, and can be used in various ways such as oral administration, injection, external application, embolization, and aerosol.
  • Formulations suitable for oral administration may be tablets, troches, lozenges, aqueous or oil suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs.
  • the medicine may also contain sweeteners, flavoring agents, coloring agents, preservatives, and antioxidants.
  • the preparations suitable for injection can be in the form of sterile injectable aqueous solutions or oil suspensions, and the solvents used therein include but are not limited to water, Ringer's solution, sodium chloride solution, glucose solution and the like.
  • Formulations suitable as suppositories are solid at ordinary temperatures but liquid in the rectum where the drug carrier will melt in the rectum to release the drug without being irritating to the rectum.
  • Some embodiments of the present application also provide the use of the compound represented by the general formula (I) or a pharmaceutically acceptable salt thereof or the pharmaceutical composition as an efflux pump inhibitor and/or a bacterial membrane permeabilizing agent.
  • the definition of the efflux pump inhibitor and/or bacterial membrane permeabilizing agent is well known in the art.
  • the cell efflux pump refers to a protein assembly that exports substrate molecules from the cytoplasm or periplasm of cells in an energy-dependent manner.
  • the bacterial membrane permeabilizing agent refers to any compound that can reduce the integrity of the bacterial cell plasma membrane or destroy the bacterial cell plasma membrane.
  • Some embodiments of the present application also provide a method for treating or preventing a bacterial infection or a disease caused by the bacterial infection in a subject, the method comprising administering an effective amount of the compound represented by general formula (I) or the pharmaceutical composition to the subject.
  • the bacteria are multidrug resistant.
  • the method further comprises administering to the subject an effective amount of another antibacterial agent.
  • the other antibacterial agents are as described above, and will not be repeated here.
  • those skilled in the art can determine the optimal dosage ratio of the compound represented by the general formula (I) and the other antibacterial agents in a manner known in the art.
  • the compound represented by the general formula (I) can be administered alone or in combination with one or more other antibacterial agents (simultaneously, sequentially or separately) in an effective amount to a subject in need.
  • a subject may be any human or non-human animal, preferably a mammal, more preferably a human.
  • the therapeutically effective dose of the compound represented by the general formula (I) and/or other antibacterial agents when used alone may be lower than the standard dose.
  • the therapeutically effective dose can be 1%-99%, 1%-90%, 1%-80%, 1%-70%, 1%-60%, 1%-50%, 1%-40%, 1%-30%, 1%-20%, 5%-20%, 1%-10%, 0.1%-1%, 0.01%-1%, 0.001%-1%, etc. of the standard dose.
  • Some embodiments of the present application also provide the use of the compound represented by general formula (I) or a pharmaceutically acceptable salt thereof or the star-shaped ⁇ -antibacterial glycopeptide (also referred to as "antibacterial polypeptide") in the preparation of drugs for preventing or treating fungal infections.
  • the star-shaped ⁇ -antibacterial glycopeptide includes at least one of the compound represented by the general formula (I) and the stereoisomer, tautomer, N-oxide, hydrate, solvate or pharmaceutically acceptable salt of the compound;
  • Core is a structural unit with a cyclic group
  • R 1 , R 2 , R 3 , R 4 Each independently selected from H, halogen, substituted or unsubstituted C1-C20 alkyl, substituted or unsubstituted C1-C20 alkoxy, substituted or unsubstituted C2-C20 alkenyl, substituted or unsubstituted C2-C20 alkynyl, substituted or unsubstituted C3-C20 alicyclic group, substituted or unsubstituted C6-C15 aryl, substituted or unsubstituted C7-C30 alkyl-aryl, substituted or unsubstituted C3-C20 hetero ring, substituted or unsubstituted C4-C30 alkyl-heterocycle, substituted or unsubstituted C5-C15 heteroaryl, C1-C20 hydroxyalkyl, cyano, amino, guanidino, nitro or hydroxyl;
  • n 0-6, m is 3-100, and x and y are 1-50.
  • the use includes: combining the star ⁇ -antibacterial glycopeptide with antifungal drugs to prevent or treat fungal infections or diseases caused by fungal infections.
  • the drug can be prepared in various forms, and can be used in various ways such as oral administration, injection, external application, embolization, and aerosol.
  • the formulations suitable for oral administration may be tablets, troches, lozenges, water or oil suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs, ointments, etc.
  • the medicine may also contain sweeteners, flavoring agents, coloring agents, preservatives, antioxidants and the like.
  • Some embodiments of the present application also provide the use of the star-shaped ⁇ -antibacterial glycopeptide in the preparation of antifungal drug synergists.
  • Some embodiments of the present application also provide an antifungal pharmaceutical composition, comprising the star-shaped ⁇ -antibacterial glycopeptide and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition also includes an antifungal drug.
  • the fungi may be drug-sensitive fungi, drug-neutral fungi or drug-resistant fungi, especially drug-resistant fungi.
  • the fungus may be a fungus of the order Saccharomycetales, Onygenales or Hyphomycetales. More specifically, the fungi can be drug-sensitive fungi of Saccharomycetales, drug-sensitive fungi of Onygenales, drug-sensitive fungi of Hyphomycetales, drug-neutral fungi of Saccharomycetales, drug-neutral fungi of Onygenales, drug-neutral fungi of Hyphomycetales, yeast Drug-resistant fungi of Saccharomycetales, drug-resistant fungi of Onygenales, drug-resistant fungi of Hyphomycetales, etc.
  • the fungi may be fungi of the family Cryptococcaceae, Arthrodermataceae, Hyphomycet and the like. More specifically, the fungus can be a drug-sensitive fungus of the family Cryptococcae (Cryptacoccaceae), a drug-sensitive fungus of the Pachydermaceae (Arthrodermataceae), a drug-sensitive fungus of the Hyphomycet family, a drug-neutral fungus of the Cryptococcaceae (Cryptacoccaceae), a drug-neutral fungus of the Arthrodermaceae (Arthrodermataceae), or a drug-sensitive fungus of the Hyphomycet family.
  • the fungus can be a drug-sensitive fungus of the family Cryptococcae (Cryptacoccaceae), a drug-sensitive fungus of the Pachydermaceae (Arthrodermataceae), a drug-sensitive fungus of the Hyphomycet family
  • Moderate fungi drug-resistant fungi of the family Cryptococcaceae (Cryptacoccaceae), drug-resistant fungi of the family Arthrodermataceae, drug-resistant fungi of the family Hyphomycet, etc.
  • the fungi may be fungi of the genus Aspergillus, Candida, Candida, Microsporum, Epidermophyton, etc. More specifically, the fungi can be drug-sensitive fungi of the genus Candida, drug-sensitive fungi of the genus Microsporum, drug-sensitive fungi of the genus Epidermophyton, drug-neutral fungi of the genus Candida, drug-neutral fungi of the genus Microsporum, drug-neutral fungi of the genus Epidermophyton, and drug-neutral fungi of the genus Candida. Drug-resistant fungi of the genus Microsporum, drug-resistant fungi of the genus Epidermophyton, etc.
  • the fungus may be Candida albicans, Microsporum gypseum, Epidermophyton floccosum and the like. More specifically, the fungus can be drug-sensitive Candida albicans, drug-sensitive Microsporum gypseum, drug-sensitive Epidermophyton floccosum, drug-neutral Candida albicans, drug-neutral Microsporum gypseum, drug-neutral Isotropic Epidermophyton floccosum, drug-resistant Candida albicans, drug-resistant Microsporum gypseum, drug-resistant Epidermophyton floccosum, Aspergillus fumigatus, Candida auris ) etc.
  • the pathogenic fungi include dermatophytes, deep infection fungi and the like.
  • the dermatophytes may include: Trichophyton, such as Trichophyton rubrum, Trichophyton mentagrophyton, Trichophyton violaceum; Epidermophyton, such as Epidermophyton flocculum; Microsporum, such as Microsporum audouin, Microsporum canis, Microsporum plasteriformis; Malassezia sp.;
  • the deep infection fungi may include: Candida; Cryptococcus neoformans; Pneumocystis; Aspergillus; Histoplasma; Fusarium; Penicillium; Mucor;
  • the antifungal drugs include azoles (such as imidazoles such as clotrimazole, ketoconazole, miconazole, econazole, etc. and triazoles such as fluconazole, itraconazole, voriconazole, posaconazole, etc.), polyenes (such as amphotericin B, nystatin, etc.), allylamines (such as terbinafine, etc.), flucytosines (such as 5-fluorocytosine, etc.), echinocandins (such as caspofungin, micafungin, etc. ) antifungal drugs, etc., and are not limited thereto, for example, may also include griseofulvin, compound sulfamethoxazole, pentamidine isethionate, polymyxin, etc.
  • azoles such as imidazoles such as clotrimazole, ketoconazole, miconazole, econazole, etc. and tri
  • the dosage of the antifungal drug is 0-2048 ⁇ g/mL, and the dosage of the antibacterial polypeptide is 0-512 ⁇ g/mL.
  • Some embodiments of the present application also provide a method for treating or preventing a fungal infection or a disease caused by the fungal infection in a subject, the method comprising administering an effective amount of the star ⁇ -antibacterial glycopeptide or the pharmaceutical composition to the subject.
  • the fungus is resistant.
  • the method further comprises administering to the subject an effective amount of another antifungal drug.
  • the antifungal drugs are as described above and will not be repeated here.
  • those skilled in the art can determine the optimal dosage ratio of the star-shaped ⁇ -antibacterial glycopeptide and the antifungal drug in a manner known in the art.
  • the star-shaped ⁇ -antibacterial glycopeptide for example, when the compound represented by the general formula (I) is used in combination with commonly used antifungal drugs, there is a synergistic effect, which can not only effectively overcome the resistance of drug-resistant fungi to commonly used antifungal drugs, but also significantly reduce the usage of commonly used antifungal drugs, for example, the therapeutically effective dose of commonly used antifungal drugs can be significantly lower than its standard dose, such as 1% to 99%, 1% to 90%, 1% to 80%, 1% to 70%, 1% to 60%, 1% to 1% of the standard dose.
  • the therapeutically effective dose of itraconazole may be 1/512 of its standard dose.
  • the star-shaped ⁇ -antibacterial glycopeptide of the present application can also overcome the resistance of bacteria to commonly used antibiotics, and can effectively kill multi-drug resistant bacteria by using it in combination with commonly used antibiotics, thereby preventing or improving bacterial infections or diseases caused by them (see CN2022100766439).
  • the star-shaped ⁇ -antibacterial glycopeptide of the present application can also overcome the resistance of bacteria to commonly used antibiotics, and can effectively kill multi-drug resistant bacteria by using it in combination with commonly used antibiotics, thereby preventing or improving bacterial infections or diseases caused by them (see CN2022100766439).
  • some antibiotics that have the effect of killing fungi by using them in combination with the antibacterial polypeptide, they can also kill drug-resistant fungi and drug-resistant bacteria at the same time, reduce the amount of antibiotics and antifungal drugs administered, and reduce the burden of drugs on the patient's liver and kidney system. This is very useful for the treatment of some diseases caused by co-infection of bacteria and fungi.
  • Example 1 The structural formula of a star-shaped ⁇ -antibacterial glycopeptide (also named antibacterial polypeptide 1) provided in this example is as follows:
  • a method for synthesizing the star-shaped ⁇ -antibacterial glycopeptide comprises the steps:
  • Glucose-derived beta lactam (AS(Bn)) was synthesized according to literature (J.Am.Chem.Soc.134, 16255-16264 (2012)). According to the literature (Angew.Chem.Int.Ed.2020doi.org/10.1002/anie.201914304), a carboxybenzyl (Cbz)-protected beta lactam (DM(Cbz)) with an amine group was synthesized.
  • the whole polymerization process was carried out in an inert gas-protected glove box.
  • 0.4MDM(Cbz) and AS(Bn) 0.02M 1,3,5-benzenetricarboxylic acid chloride and 0.15M lithium bis(trimethylsilyl)amide (LiHMDS) were prepared in anhydrous tetrahydrofuran.
  • a solution of 1ml 1,3,5-benzenetricarbonyl chloride, 900 ⁇ l DM(Cbz) was added to a 10ml oven-dried round bottom flask and mixed. Stir at 30°C for 3 min to make the solution evenly mixed.
  • 1 ml of LiHMDS solution was injected into the reaction flask to initiate polymerization.
  • the star ⁇ -antibacterial glycopeptide was successfully synthesized by anionic ring-opening polymerization, which is a block star ⁇ -polypeptide.
  • the outer layer of the block star-shaped ⁇ -polypeptide is a helical glycopeptide block, which can improve biocompatibility, prevent protein adsorption and prevent infection
  • the inner layer is a positively charged polydimethylaminobeta lactam block, which can increase the permeability of the bacterial outer membrane and destroy the bacterial molecular efflux pump.
  • the molecular structure of the polypeptide is confirmed by H NMR spectroscopy.
  • the integral ratio of the outer glycopeptide block and the inner positively charged polydimethylaminobeta lactam block is 1:2, which is consistent with the monomer feed ratio.
  • the molecular weight of this polypeptide is 26 by the degree of polymerization of the polymkeric substance measured by gel permeation chromatography (GPC), close to the monomer/initiator feed ratio of 27: 1, and has relatively narrow molecular weight distribution
  • Carbapenem-resistant Acinetobacter baumannii is the first bacterium on the world's most drug-resistant and most threatening human health "super bacteria" list published by the World Health Organization (WHO).
  • Table 1 is the drug resistance table of carbapenem-resistant Acinetobacter baumannii (DR-AB). It can be seen that it has developed resistance to almost all antibiotics commonly used in clinical practice.
  • the activity of the star-shaped ⁇ -antibacterial glycopeptide is tested by using this kind of bacteria, and the test methods include:
  • a deep-well plate is used to longitudinally arrange a 2-fold gradient concentration solution of the sample (starting from 4 ⁇ 256 ⁇ g/ml, and the last well is MHB).
  • Embodiment 2 The structural formula of a star-shaped ⁇ -antibacterial glycopeptide (also named antibacterial polypeptide 2) provided in this embodiment is as follows:
  • the structure of Core is the same as the Core in the star ⁇ -antibacterial glycopeptide in Example 1.
  • the 1H NMR characterization spectrum of the star-shaped ⁇ -antibacterial glycopeptide includes: ⁇ 5.66 (1H, br), 4.02-4.39 (3H), 3.65 (2H, br), 3.37-2.96 (9H), 1.37 (6H, br).
  • the synthesis method of this star-shaped ⁇ -antibacterial glycopeptide can refer to Example 1, but the beta lactam monomer used is a monomethyl-substituted beta lactam, and its synthesis method refers to (J.Am.Chem.Soc.2007,129,15474-15476), other steps and raw materials are basically the same as in Example 1.
  • Embodiment 3 The structural formula of a star-shaped ⁇ -antibacterial glycopeptide (also named antibacterial polypeptide 3) provided in this embodiment is as follows:
  • the structure of Core is the same as the Core in the star ⁇ -antibacterial glycopeptide in Example 1.
  • the 1H NMR characterization spectrum of the star-shaped ⁇ -antibacterial glycopeptide includes: ⁇ 5.66 (1H, br), 4.02 (2H), 3.65-2.75 (13H).
  • the synthesis method of the star-shaped ⁇ -antibacterial glycopeptide can refer to Example 1, but the beta lactam monomer used is a beta lactam without methyl substitution, and its synthesis method refers to (J.Am.Chem.Soc.2014, 136, 4333-4342), other steps and raw materials are basically the same as in Example 1.
  • Embodiment 4 The structural formula of a star-shaped ⁇ -antibacterial glycopeptide (also named antibacterial polypeptide 4) provided in this embodiment is as follows:
  • the structure of Core is the same as the Core in the star ⁇ -antibacterial glycopeptide in Example 1.
  • the 1H NMR characterization spectrum of the star-shaped ⁇ -antibacterial glycopeptide includes: ⁇ 5.66 (1H, br), 4.53 (2H, br), 4.02 (1H, br), 3.65 (2H, br), 3.37-2.58 (8H).
  • the synthesis method of the star-shaped ⁇ -antibacterial glycopeptide can refer to Example 1, but the beta lactam monomer used is a single base-free substitution, and the methylene amino group protected by Cbz is changed from the alpha position to the beta lactam at the beta position.
  • the synthesis method refers to (J.Am.Chem.Soc.2013, 135, 5270-5273), and other steps and raw materials are basically the same as in Example 1.
  • Embodiment 5 The structural formula of a star-shaped ⁇ -antibacterial glycopeptide (also named antibacterial polypeptide 5) provided in this embodiment is as follows:
  • the structure of Core is the same as the Core in the star ⁇ -antibacterial glycopeptide in Example 1.
  • the 1H NMR characterization spectrum of the star-shaped ⁇ -antibacterial glycopeptide includes: ⁇ 5.66 (1H, br), 4.32 (3H), 4.02 (2H), 3.65 (2H, br), 3.37-3.0 (5H), 2.50 (4H, br), 1.8-1.3 (12H).
  • the synthesis method of the star-shaped ⁇ -antibacterial glycopeptide can refer to Example 1, but the beta lactam monomer used is a beta lactam with no methyl substitution and a Cbz-protected methylene amino carbon chain length increased to 4.
  • the synthesis method refers to (Nat. Commun. 2019, 10, 1-14), and other steps and raw materials are basically the same as in Example 1.
  • Embodiment 6 The structural formula of a star-shaped ⁇ -antibacterial glycopeptide (also named antibacterial polypeptide 6) provided in this embodiment is as follows:
  • the structure of Core is the same as the Core in the star ⁇ -antibacterial glycopeptide in Example 1.
  • the 1H NMR characterization spectrum of the star-shaped ⁇ -antibacterial glycopeptide includes: ⁇ 5.66 (1H, br), 4.60-4.14 (1H), 4.12-3.52 (4H, br, m), 3.20-2.58 (7H, br, m), 1.37 (12H, br, m).
  • the synthesis method of the star-shaped ⁇ -antibacterial glycopeptide can refer to Example 1, but the beta lactam monomer used is changed from glucose to altrolactam monomer.
  • the synthesis method is referred to (J.Am.Chem.Soc.2017, 139, 14217-14223), and other steps and raw materials are basically the same as in Example 1.
  • Embodiment 7 The structural formula of a star-shaped ⁇ -antibacterial glycopeptide (also named antibacterial polypeptide 7) provided in this embodiment is as follows:
  • the structure of Core is the same as the Core in the star ⁇ -antibacterial glycopeptide in Example 1.
  • the 1H NMR characterization spectrum of the star-shaped ⁇ -antibacterial glycopeptide includes: ⁇ 5.82 (1H, br), 4.37-4.14 (1H), 4.12-3.52 (4H, br, m), 3.85-2.58 (11H, br, m), 1.37 (12H, br, m).
  • the synthesis method of the star-shaped ⁇ -antibacterial glycopeptide can refer to Example 1, but the beta lactam monomer used therein is changed from glucose to galactinolactam monomer.
  • the synthesis method is referred to (J.Am.Chem.Soc.2016, 138, 6532-6540), and other steps and raw materials are basically the same as in Example 1.
  • Embodiment 8 The structural formula of a star-shaped ⁇ -antibacterial glycopeptide (also named antibacterial polypeptide 8) provided in this embodiment is as follows:
  • Core is 1,2,3,4-cyclobutane tetracarbonyl.
  • the 1H NMR characterization spectrum of the star-shaped ⁇ -antibacterial glycopeptide includes: ⁇ 5.66 (1H, br), 4.04 (1H), 3.77-3.53 (2H, br), 3.48-2.69 (9H, br, m), 3.20-2.58, 1.37 (12H, br).
  • the synthesis method of the star-shaped ⁇ -antibacterial glycopeptide can refer to Example 1, the difference is that in step (2), 1,2,3,4-cyclobutanetetracarbonyl chloride is used to replace 1,3,5-benzenetricarboxylic acid chloride, and other steps and raw materials are basically the same as in Example 1.
  • Embodiment 9 The structural formula of a star-shaped ⁇ -antibacterial glycopeptide (also named antibacterial polypeptide 9) provided in this embodiment is as follows:
  • Core is 1,2,3,4,5,6-cyclohexane hexacarbonyl.
  • the 1H NMR characterization spectrum of the star-shaped ⁇ -antibacterial glycopeptide includes: ⁇ 5.66 (1H, br), 4.04 (1H), 3.77-3.53 (2H, br), 3.48-2.69 (9H, br, m), 3.20-2.58, 1.37 (12H, br).
  • the synthesis method of the star-shaped ⁇ -antibacterial glycopeptide can refer to Example 1, the difference is that in step (2), 1,2,3,4,5,6-cyclohexanehexayl chloride is used to replace 1,3,5-benzenetricarboxylic acid chloride, and other steps and raw materials are basically the same as in Example 1.
  • Example 1 the activity of the star-shaped ⁇ -antibacterial glycopeptides provided in Examples 2-9 was tested by using carbapenem-resistant Acinetobacter baumannii in this example. The results showed that these star-shaped ⁇ -antibacterial glycopeptides were used in combination with various antibiotic adjuvants, which can also increase the bactericidal range of antibiotics, enhance the bactericidal effect of antibiotics, reduce the possibility of bacterial drug resistance, and the effect is similar to that of the antibacterial glycopeptides in Example 1.
  • Ampicillin/Sulbactam > 32 drug resistance
  • Levofloxacin > 8 drug resistance
  • Piperacillin > 128 drug resistance
  • Cotrimoxazole > 320 drug resistance
  • Gentamicin > 16 drug resistance
  • Ceftazidime > 64 drug resistance
  • Ceftriaxone > 64 drug resistance
  • Cefepime > 64 drug resistance
  • Piperacillin/Tazobactam > 128 drug resistance
  • C.albicans Candida albicans, number ATCC 64124; resistant to a variety of azoles and echinocandins
  • A. fumigatus Aspergillus fumigatus, number ATCC MYA-3627; itraconazole resistance
  • RPMI-1640 medium and potato dextrose agar (PDA) medium were used for the test.
  • the formulations of the two media are shown in Tables 2 and 3 below.
  • RPMI-1640 medium mixed buffer Accurately weigh 34.53g MOPS powder and dissolve it in 1L RPMI-1640 liquid medium. After dissolving, use 1M NaOH solution to adjust the pH value to 7.
  • PDA medium Accurately weigh 35g of PDB medium powder and 17.5g of agar powder and dissolve in 1L of ultrapure water. After fully dissolving, autoclave at 121°C for 20 minutes in a high-pressure steam sterilizer, and store in a refrigerator at 4°C for later use.
  • Test method Use the method in CLSI-M27-A2 (Measurement of Antifungal Drug Susceptibility by Broth Dilution Method) of the American Clinical Laboratory Standardization Association to make appropriate modifications for the relevant test of Candida albicans (P.Wayne, CLSI document M27-A2, 2002); use the industry standard of the People's Republic of China-Anti-filamentous fungal drug susceptibility test broth dilution method (WS/T411-2013) to carry out the relevant test of Aspergillus fumigatus (WS/T 41) after suitability modification 1-2013, Broth Dilution Method for Drug Susceptibility Testing against Filamentous Fungi [S]).
  • MIC minimum inhibitory concentration
  • FIC fractional inhibitory concentration
  • time-kill curve After selecting the sample with synergistic effect-antifungal drug combination to be tested, dilute the cultured fungus to the concentration of the bacterial solution required by the micro-dilution method, one part for normal culture (without adding antibacterial drugs), and one part for the drug combination at a selected concentration, and culture them simultaneously in a 96-well plate. 0-10 7 gradient dilution, and take 10 ⁇ L to the PDA plate, after 36-48 hours of culture for colony counting.
  • Table 4 Synergistic effect of antifungal drugs by antimicrobial peptide 1 against itraconazole-resistant Aspergillus fumigatus at 24 hours
  • MIC in Table 4 is the minimum inhibitory concentration.
  • Antibacterial peptide 1 Antibacterial peptide synthesized in the laboratory, test concentration: 1/4MIC.
  • MIC* Concentration of a single component when the combination has an antibacterial effect. Enhancement factor: MIC/MIC*.
  • FIC index partial inhibitory concentration index. Mechanism of action: 1.
  • Inhibit the synthesis of ergosterol change the permeability of the cell membrane to cause antibacterial effect
  • 1* Inhibit the synthesis of ergosterol, change the permeability of the cell membrane to cause antibacterial effect, and cause squalene to accumulate in the lipid droplets in the cytoplasm, destroy the cell membrane and cause bactericidal effect
  • 3, Inhibit thymidine synthase affect DNA and protein synthesis to inhibit fungal growth;
  • Inhibit Aspergillus and Candida cells ⁇ (1,3)-D-glycoside synthase an important component of wall synthesis, leads to antibacterial and bactericidal effects, the same below.
  • Table 6 Synergistic effect of antibacterial polypeptide 1 on antifungal drugs against drug-resistant Candida albicans at 48h
  • antimicrobial polypeptide 1 also had a relatively obvious synergistic effect when used in combination with a variety of common antifungal drugs, and the synergistic inhibitory effect of azole antifungal drugs was slightly better than that of nystatin.
  • the MIC* value of the antifungal drug can be reduced to 1/4-1/512 of the MIC, which proves that antibacterial polypeptide 1 can have a good synergistic effect on antifungal drugs.
  • FIG. 22 shows the bactericidal curve of itraconazole combined with antibacterial polypeptide 1 against drug-resistant Aspergillus fumigatus.
  • the strain used is itraconazole-resistant Aspergillus fumigatus, numbered ATCC MYA-3627; Aspergillus fumigatus control: RPMI1640+ bacterial liquid.
  • Drug combination concentration the component concentration at the lowest FIC index. It can be seen that the antibacterial effect of Aspergillus fumigatus under the combined action of two drugs with synergistic effect.
  • the drug-added group mainly has antibacterial effect, and the number of fungi in the well decreases slowly with the increase of time until the fungal concentration is about 300 CFU/mL.
  • FIG. 23 shows the bactericidal curve of itraconazole combined with antibacterial polypeptide 1 against drug-resistant Candida albicans.
  • Strains used Candida albicans, resistant to echinocandins and azoles, numbered ATCC 64124; Candida albicans control: RPMI1640+ bacteria solution.
  • Drug combination concentration the component concentration at the lowest FIC index. It can be seen that Candida albicans also has a similar trend, that is, the control group enters the logarithmic phase at about 6 hours, and the overall trend is the standard curve; the drug-dosed group has a relatively obvious inhibitory effect under the synergistic effect of the two drugs. After the number of fungi in the wells decreased by an order of magnitude in the first 3 hours, the fungal concentration remained stable at about 300 CFU/mL in the subsequent 45 hours.
  • antimicrobial polypeptide 1 was combined with 12 commonly used antifungal drugs in vitro to determine its synergistic effect on two drug-resistant fungi.
  • the results show that the antibacterial polypeptide 1 can effectively overcome the resistance of fungi to antifungal drugs and greatly reduce the amount of antifungal drugs used.
  • the experimental results show that when antibacterial peptides are used in combination with antifungal drugs, their respective antibacterial mechanisms play a role of mutual promotion, so that drug-resistant fungi are more effectively inhibited.
  • antibacterial polypeptide 1 has good synergistic effect when used in combination with various antifungal drugs.
  • Test Example 3 This test example tests the performance of antibacterial polypeptide 2 to antibacterial polypeptide 9, as follows:
  • C.albicans Candida albicans, No. ATCC 64124
  • A.fumigatus Aspergillus fumigatus, No. ATCC MYA-3627
  • Trichophyton rubrum Red Trichophyton, No. ATCC MYA-4438
  • Epidermophyton floccosum Epidermophyton flocculus, No.
  • Microsporum gy pseum Microsporum gypseum (Microsporum gypsum, number ATCC 14683), Filobasidiella bacillispora (Cryptococcus neoformans, number ATCC32609), Histoplasmosis capsulati (capsular histoplasma, number ATCC12700), Fusarium moniliforme (Fusarium moniliforme, number ATCC 10052), Rhizopus oryzae (rice Rhizopus, number ATCC96382), Penicillium marneffei (Penicillium marneffei, number ATCC24100).
  • antibacterial polypeptide 2 to antibacterial polypeptide 9 amphotericin B, 5-fluorocytosine, fluconazole, ketoconazole, itraconazole, voriconazole, miconazole nitrate, micafungin (purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., Beijing Solebao Technology Co., Ltd., etc.).
  • Example 7 For the aforementioned strains, culture was carried out with reference to Example 1 and the corresponding culture medium and culture methods well known in the art, and then the method basically the same as that of Example 1 was used for testing. The corresponding test results are shown in Table 7 below.
  • Table 7 The inhibitory effect of antibacterial polypeptide 2 ⁇ antibacterial polypeptide 9 and different antifungal drugs on different fungi at 48h
  • antibacterial polypeptide 2 to antibacterial polypeptide 9 when used in combination with commonly used antifungal drugs, they can also effectively overcome the resistance of drug-resistant fungi to commonly used antifungal drugs, and the effect is similar to that of the antibacterial polypeptide of Test Example 1.
  • the applicant also prepared other antibacterial polypeptides and their derivatives mentioned in the specification of this application by referring to the preparation methods of the aforementioned antibacterial polypeptide 1 to antibacterial polypeptide 9, such as pharmaceutically acceptable salts (including hydrochloride, hydrobromide, acetate, etc.), and also tested their performance.
  • pharmaceutically acceptable salts including hydrochloride, hydrobromide, acetate, etc.

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Abstract

本申请公开了一种星型β-抗菌糖肽、其制备方法及应用。所述星型β-抗菌糖肽具有通式(I)所示结构：该通式(I)中各取代基与说明书中的定义相同。所述星型β-抗菌糖肽或其可药用的盐具有良好的生物相容性和抗菌性能，以及低的细胞毒性，并可以与不同抗生素联合使用，从而恢复和增强抗生素的杀菌效果。并且，所述星型β-抗菌糖肽与常用抗真菌药物联合使用时，可以有效克服耐药真菌对抗真菌药物的耐药性，对耐药真菌进行有效的抑制和灭杀，在耐药真菌感染或由其引起的疾病的预防或治疗方法中有广阔应用前景。

Description

星型β-抗菌糖肽、其制备方法及应用
本申请基于并要求于2022年1月21日递交的申请号为202210076643.9、发明名称为“星型β-抗菌糖肽、其制备方法及应用”的中国专利申请以及2022年9月27日递交的申请号为202211186482.5、发明名称为“抗菌多肽在制备预防或治疗真菌感染的药物中的用途”的中国专利申请的优先权。
技术领域
本申请具体涉及一种星型β-抗菌糖肽、其制备方法及在医药上的应用，属于医药领域。
背景技术
抗生素的发现和应用对于现代医学的发展起到了极大地推动作用。然而在过去几十年的抗生素治疗中，细菌不可避免的要选择逃生突变来产生对抗生素的耐药性。碳青霉烯类抗生素是目前难治的革兰氏阴性菌感染的最后一种有效抗生素。然而碳青霉烯类药物在医院的大量使用促进了耐碳青霉烯革兰氏阴性菌的快速出现和传播。同时，尽管人们做出了各种努力去开发新型抗生素，但是新的抗生素的发现几乎陷入了停滞状态，在过去五十年都没有一种新的类型的可以杀死革兰氏阴性菌的抗生素被发现。
在诸多原因中，最主要的挑战在于革兰氏阴性菌具有低穿透性外膜和高效外排泵系统，其中低穿透性的外膜可以阻碍药物分子进入到细菌内部靶点，同时其高效的外排泵系统可以把进到细菌内部靶点的抗生素排到细菌体外。这两个过程极大地限制了抗生素在其药物靶点的浓度积累，从而导致抗生素失去效果。现在耐碳青霉烯类的革兰氏阴性菌已经被世界卫生组织列为急需寻找新的抗生素或者新的治疗方法的一类细菌。
天然抗菌肽的发现极大地激发了人们对这类抗菌药物的广泛研究，以期将其转化为临床应用。因为它们具有快速广谱抗菌效力和耐药频率低的优点。尽管最初的实验结果令人兴奋，有超过三千多种抗菌肽被发现，但是天然抗菌肽从研究发现到转化为临床应用的成功案例有限，目前为止只有五种天然抗菌肽被批准用于临床应用，包括乳链菌肽、短杆菌肽、多粘菌素、达托霉素和蜂毒肽。在各种原因中，主要缺点在于它们的细胞毒性、在体内不稳定，容易被蛋白吸附和高生产成本。
与天然的阿尔法多肽相比，贝塔多肽在主链上多了一个额外的亚甲基。这赋予贝塔多肽更大的分子链的灵活性，允许形成不同类型的二级结构以表现出两亲构象，这是有助于抗菌 肽抗菌活性的关键要素之一。贝塔多肽的另一个突出特点是它们对蛋白酶裂解的抵抗力，从而提高了体内稳定性。这些特性使贝塔多肽作为候选抗菌剂更具有吸引力。但现有的贝塔多肽的抗菌性能、生物相容性等仍有待进一步提升。
近年来，侵袭性真菌感染(IFDs)已经对公共健康造成了巨大威胁。许多机会性致病真菌存在我们周围的生活环境中，并可以产生大量的空气传播孢子。人类每天暴露于生物气溶胶等各种环境真菌病原体中。虽然大多数环境真菌在健康个体中不会引起明显的病理生理事件，但那些健康或免疫力受损的人容易感染一系列疾病，包括浅表性、过敏性、慢性和危及生命的感染病征。其中，曲霉属(如烟曲霉Aspergillus fumigatus)、念珠菌属(如耳念珠菌Candida auris)等真菌展现出较强的内在抗性及获得性耐药性。这两种病原菌已被列入美国疾病控制与预防中心(US CDC)于2019年发布的紧急抗菌素耐药性(AMR)威胁清单。寻找应对耐药性真菌感染的治疗方法刻不容缓。
常用抗真菌药物主要可分为以下几种：唑类(包括咪唑类如克霉唑、酮康唑等及三唑类氟康唑、伊曲康唑等)，可通过抑制细胞色素P450依赖酶——14α-羊毛脂醇脱甲基酶(CYP51)阻止真菌细胞膜主要成分麦角甾醇的合成，麦角甾醇的缺失伴随14α-甲基化甾醇的蓄积会使真菌细胞膜的流动性发生改变，导致膜相关性酶的活性降低，通透性增加，最终使真菌的生长和复制受到抑制(Clinical infectious diseases：an official publication of the Infectious Diseases Society of America，62(3)，362-368)；多烯类(两性霉素B、制霉菌素等)，可与真菌胞质膜上的麦角甾醇结合，致使膜分解或增加膜通透性、造成细胞内容物外溢而死亡(Actapharmaceutica Sinica.B，11(8)，2585-2604)；氟胞嘧啶类(如5-氟胞嘧啶)，可抑制胸腺嘧啶核苷合成酶，影响DNA及蛋白质合成(Nature communications，12(1)，3418)；以及棘白菌素类(如卡泊芬净、米卡芬净等)，可抑制真菌细胞壁的主要成分1，3-β-D-葡聚糖的合成等(Clinical microbiology and infection：the official publication of the European Society of Clinical Microbiology and Infectious Diseases，25(7)，792-798)。但事实上，随着真菌耐药性(和耐受性)的不断出现，导致药物治疗耐药真菌感染的过程举步维艰。
对于耐药真菌感染的治疗，一个明显的解决方案是新型药物的研发，但由于较高的时间及资金成本对大多数公司来说意味着较高的风险，所以这并不是一个可以迅速解决问题的方法(Clinical microbiology and infection：the official publication of the European Society of Clinical Microbiology and Infectious Diseases，20Suppl6，11-18)。在这种形势下，联合用药的协同作用可以作为一种可以在短时间内验证增强疗效并加以推广的方案，以对抗耐药真菌的感染。然而现有的联合用药效果并不显著。例如Hanane Yousfi等对多粘菌素与抗真菌药物的体外联合用药试验结果显示，对于白假丝酵母菌，多粘菌素仅能将抗真菌药物的有效作用浓度减少 到单独使用时的1/2(两性霉素B)、1/16(伊曲康唑)以及无减少(氟康唑)，而这需要使用1-64μg/mL的多粘菌素，这会带来较高的血药浓度。在这种情况下，患者的肾脏负担必须加以考虑(In vitro polymyxin activity against clinical multidrug-resistant fungi.Antimicrobial resistance and infection control，8，66.)。所以迫切需要一种有较低毒性且能对常用抗真菌药物产生协同增效作用的物质，以丰富抗真菌感染的治疗方案。
发明内容
本申请的主要目的在于提供一种星型β-抗菌糖肽、其制备方法及在制备预防或治疗真菌感染的药物中的用途，以克服现有技术的不足。
为实现前述发明目的，本申请采用的技术方案包括：
本申请的第一个方面提供了一种通式(I)所示的化合物：
Figure PCTCN2022134136-appb-000001
其中，Core为具有环状基团的结构单元；
R 1、R 2、R 3、R 4各自独立的选自H、卤素、取代或未取代的C1-C20烷基、取代或未取代的C1-C20烷氧基、取代或未取代的C2-C20烯基、取代或未取代的C2-C20炔基、取代或未取代的C3-C20脂环基、取代或未取代的C6-C15芳基、取代或未取代的C7-C30烷基-芳基、取代或未取代的C3-C20杂环、取代或未取代的C4-C30烷基-杂环、取代或未取代的C5-C15杂芳基、C1-C20羟烷基、氰基、氨基、胍基、硝基或者羟基；
n为0～6，m为3～100，x、y为1～50。
本申请的第二个方面提供了一种星型β-抗菌糖肽，其外层为螺旋结构的糖肽嵌段，内层为带正电荷的聚二甲基氨基贝塔内酰胺嵌段。该星型β-抗菌糖肽的结构如通式(I)所示。
本申请的第三个方面提供了一种制备通式(I)所示化合物的方法，所述方法包括：
(i)依据反应式a，使包含Core的前驱体化合物与式(II)所示的化合物、式(III)所示的化合物发生聚合反应，获得式(IV)所示的聚合物；
Figure PCTCN2022134136-appb-000002
(ii)依据反应式b，对式(IV)所示的聚合物进行脱保护
Figure PCTCN2022134136-appb-000003
本申请的第四个方面提供了一种药物组合物，包含通式(I)所示的化合物或其可药用盐以及药学上可接受的载体。
本申请的第五个方面提供了通式(I)所示的化合物或其可药用盐或者所述药物组合物作为抗菌剂的用途。
本申请的第六个方面提供了通式(I)所示的化合物或其可药用盐或者所述药物组合物在制备治疗或预防细菌感染或由所述细菌感染引起的疾病的药物中的用途。
本申请的第七个方面提供了通式(I)所示的化合物或其可药用盐或者所述药物组合物作为细胞外排泵抑制剂和/或细菌膜透化剂的用途。
本申请的第八个方面提供了在受试者中治疗或预防细菌感染或由所述细菌感染引起的疾病的方法，该方法包括向受试者施用有效量的通式(I)所示的化合物或者所述药物组合物。
在一些实施方式中，所述细菌具有多重耐药性。
在一些实施例中，所述的方法还包括向受试者施用有效量的其它抗菌剂。所述其它抗菌剂包括但不限于氨苄西林、氯唑西林、苯唑西林和哌拉西林、头孢菌素例如头孢克洛、头孢孟多、头孢唑林、头孢哌酮、头孢噻肟、头孢西丁、头孢他啶、头孢曲松和头孢菌素；碳青霉烯类包括例如亚胺培南和美罗培南；和糖肽类、大环内酯类、喹诺酮类、四环素类和氨基糖苷类。此外所述其它抗菌剂还可以选自利福平、环丙沙星、左氧氟沙星、哌拉西林、复方新诺明、庆大霉素、洛布霉素、琥乙红霉素、红霉素、克拉霉素、新生霉素、螺旋霉素、乙 酰螺旋霉素、氯霉素、甲氧苄啶、磺胺甲恶唑、羧苄青霉素、多粘菌素B、粘菌素、阿米卡星、卡那霉素、新霉素、奈替米星、链霉素、妥布霉素、巴龙霉素，格尔德霉素、除莠霉素，氯碳头孢，多利培南、西司他丁、头孢羟氨苄、头孢噻吩、头孢氨苄、头孢孟多、头孢丙烯、头孢呋辛、头孢克肟、头孢地尼、头孢托仑、头孢泊肟、头孢布烯、头孢唑肟、头孢吡肟、替考拉宁、万古霉素、罗红霉素、醋竹桃霉素、秦利霉素、大观霉素、阿莫西林、羧苄西林、双氯西林、氟氯西林、美洛西林、甲氧西林、萘夫西林、青霉素、替卡西林、杆菌肽、依诺沙星、加替沙星、莫西沙星、诺氟沙星、曲伐沙星、磺胺米隆、偶氮磺胺、磺胺醋酰、磺胺甲二唑、磺胺、柳氮磺吡啶、磺胺异噁唑、甲氧苄啶磺胺甲噁唑、地美环素、多西环素、米诺环素、土霉素、四环素、胂凡纳明、克林霉素、林可霉素、乙胺丁醇、磷霉素、夫西地酸、呋喃唑酮、异烟肼、利奈唑胺、双唑秦栓、莫匹罗星、呋喃妥因、吡嗪酰胺、奎奴普丁/达福普汀、异福酰胺、替硝唑中的任意一种或多种的组合。
本申请的第九个方面提供了通式(I)所示的化合物或其可药用盐或者所述药物组合物，或者星型β-抗菌糖肽在制备预防或治疗真菌感染的药物中的用途。
本申请的第十个方面提供了所述通式(I)所示的化合物或其可药用盐或者所述药物组合物，或者星型β-抗菌糖肽在制备抗真菌药物增效剂中的用途。
本申请的第十一个方面提供了所述通式(I)所示的化合物或其可药用盐或者所述药物组合物，或者星型β-抗菌糖肽在制备用于预防、诊断、检测、保护、治疗或研究病原真菌及其直接相关疾病的产品中的用途。
本申请的第十二个方面提供了在受试者中治疗或预防真菌感染或由所述真菌感染引起的疾病的方法，该方法包括向受试者施用有效量的所述星型β-抗菌糖肽或者所述药物组合物。
在一些实施方式中，所述真菌包括曲霉属、念珠菌属真菌等，特别是具有耐药性的真菌，且不限于此。
在一些实施例中，所述的方法还包括向受试者联合施用所述星型β-抗菌糖肽及抗真菌药物。所述抗真菌药物包括唑类、多烯类、氟胞嘧啶类、棘白菌素类抗真菌药物等，且不限于此。
本申请提供的通式(I)所示的化合物是一种非天然β抗菌糖肽，其化学结构呈现为嵌段星型聚合物，其外层为螺旋结构的糖肽嵌段，内层为带正电荷的聚二甲基氨基贝塔内酰胺嵌段，通过这种星型结构(star structure)，不仅可以起到增加细菌外膜通透性和破坏细菌分子外排泵的作用，而且可以利用外层的糖肽嵌段模拟细胞表面多糖分子，进一步抑制细菌感染，同时还可以通过形成一层水化隔离层，进一步降低蛋白吸附，尤其是还可以更好的降低由于正电荷所导致的细胞毒性。该化合物或其盐等可以与不同抗生素联合使用，从而恢复和增强抗 生素的杀菌效果。
并且，本申请提供的星型β-抗菌糖肽与常用抗真菌药物联合使用时，能有效克服耐药真菌对抗真菌药物的耐药性，可以在大幅降低抗真菌药物使用量的情况下，保障及提升对耐药真菌的灭杀效果，对真菌感染，尤其是耐药真菌感染及由其引起的疾病具有优异的预防或治疗效果。
附图说明
为了更清楚地说明本申请实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本申请中记载的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1示出了实施例1中一种星型β-抗菌糖肽1H NMR图谱。
图2示出了实施例1中一种去保护之前的星型β-抗菌糖肽的凝胶渗透色谱图。
图3示出了实施例1中一种去保护之前的星型β-抗菌糖肽的细胞毒性。
图4示出了实施例1中通过棋盘肉汤微量稀释测定显示星型β-抗菌糖肽增强利福平对于耐碳青霉烯类鲍曼不动杆菌的杀菌作用。
图5示出了实施例1中通过棋盘肉汤微量稀释测定显示星型β-抗菌糖肽增强罗红霉素对于耐碳青霉烯类鲍曼不动杆菌的杀菌作用。
图6示出了实施例1中通过棋盘肉汤微量稀释测定显星型β-抗菌糖肽增强克拉霉素对于耐碳青霉烯类鲍曼不动杆菌的杀菌作用。
图7示出了实施例1中通过棋盘肉汤微量稀释测定显示星型β-抗菌糖肽增强红霉素对于耐碳青霉烯类鲍曼不动杆菌的杀菌作用。
图8示出了实施例1中通过棋盘肉汤微量稀释测定显示星型β-抗菌糖肽增强新生霉素对于耐碳青霉烯类鲍曼不动杆菌的杀菌作用。
图9示出了实施例1中通过棋盘肉汤微量稀释测定显示星型β-抗菌糖肽增强螺旋霉素对于耐碳青霉烯类鲍曼不动杆菌的杀菌作用。
图10示出了实施例1中通过棋盘肉汤微量稀释测定显示星型β-抗菌糖肽增强乙酰螺旋霉素对于耐碳青霉烯类鲍曼不动杆菌的杀菌作用。
图11示出了实施例1中通过时间杀菌曲线测定显示星型β-抗菌糖肽和利福平联合用药对于耐碳青霉烯类鲍曼不动杆菌的具有优秀杀菌作用。
图12示出了实施例1中通过时间杀菌曲线测定显示星型β-抗菌糖肽和新生霉素联合用药对于耐碳青霉烯类鲍曼不动杆菌的具有优秀杀菌作用。
图13示出了实施例1中通过时间杀菌曲线测定显示星型β-抗菌糖肽和克拉霉素联合用药对于耐碳青霉烯类鲍曼不动杆菌的具有优秀杀菌作用。
图14示出了实施例1中通过棋盘肉汤微量稀释测定显示星型β-抗菌糖肽增强新生霉素对于普通鲍曼不动杆菌(ATCC19606)的杀菌作用。
图15示出了实施例1中通过棋盘肉汤微量稀释测定显示星型β-抗菌糖肽增强红霉素对于普通鲍曼不动杆菌(ATCC19606)的杀菌作用。
图16示出了实施例1中通过棋盘肉汤微量稀释测定显示星型β-抗菌糖肽增强克拉霉素对于普通鲍曼不动杆菌(ATCC19606)的杀菌作用。
图17示出了实施例1中通过棋盘肉汤微量稀释测定显示星型β-抗菌糖肽增强罗红霉素对于普通鲍曼不动杆菌(ATCC19606)的杀菌作用。
图18示出了实施例1中通过棋盘肉汤微量稀释测定显示星型β-抗菌糖肽增强乙酰螺旋霉素对于普通鲍曼不动杆菌(ATCC19606)的杀菌作用。
图19示出了实施例1中通过棋盘肉汤微量稀释测定显示星型β-抗菌糖肽增强琥乙红霉素对于普通鲍曼不动杆菌(ATCC19606)的杀菌作用。
图20示出了实施例1中通过棋盘肉汤微量稀释测定显示星型β-抗菌糖肽增强利福平对于普通鲍曼不动杆菌(ATCC19606)的杀菌作用。
图21示出了实施例1中通过棋盘肉汤微量稀释测定显示星型β-抗菌糖肽增强萘啶酮酸对于普通鲍曼不动杆菌(ATCC19606)的杀菌作用。
图22示出了实施例1中将星型β-抗菌糖肽与伊曲康唑联用对耐药烟曲霉的杀菌曲线图。
图23示出了实施例1中将星型β-抗菌糖肽与伊曲康唑联用对耐药白色念珠菌的杀菌曲线图。
具体实施方式
鉴于现有技术的缺陷，本案发明人经长期研究和大量实践，得以提出本申请的技术方案，下面将对本申请的技术方案进行清楚、完整地描述，显然，所描述的实施例是本申请一部分实施例，而不是全部的实施例。基于本申请中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本申请保护的范围。另外需要说明的是，除非另有定义，本文使用的所有技术和科学术语与本领域普通技术人员通常理解的含义相同。
本申请的一些实施例提供了一种通式(I)所示的化合物：
Figure PCTCN2022134136-appb-000004
或者所述化合物的立体异构体、互变异构体、N-氧化物、水合物、溶剂化物或盐，或它们的混合物，特别是所述化合物的可药用盐；
其中，Core为具有环状基团的结构单元；
R 1、R 2、R 3、R 4各自独立的选自H、卤素、取代或未取代的C1-C20烷基、取代或未取代的C1-C20烷氧基、取代或未取代的C2-C20烯基、取代或未取代的C2-C20炔基、取代或未取代的C3-C20脂环基、取代或未取代的C6-C15芳基、取代或未取代的C7-C30烷基-芳基、取代或未取代的C3-C20杂环、取代或未取代的C4-C30烷基-杂环、取代或未取代的C5-C15杂芳基、C1-C20羟烷基、氰基、氨基、胍基、硝基或者羟基；
n为0～6，m为3～100，x、y为1～50。
在一些实施方式中，所述具有环状基团的结构单元包括取代或未取代的3～12元环烷基、取代或未取代的3～12元杂环基、取代或未取代的6～10元芳基或者取代或未取代的5～10元杂芳基、具有环状基团的有机大分子残基(例如环糊精分子残基)或者具有环状基团的无机化合物分子残基(例如笼型聚倍半硅氧烷分子残基)。
进一步的，所述具有环状基团的结构单元选自取代或未取代的苯基。
在一些较为具体的实施方案中，所述化合物为通式(I-1)所示的化合物：
Figure PCTCN2022134136-appb-000005
其中，R 5、R 6选自H、卤素、取代或未取代的C1-C20烷基、取代或未取代的C1-C20烷氧基、取代或未取代的C2-C20烯基、取代或未取代的C2-C20炔基、取代或未取代的C3-C20脂环基、取代或未取代的C6-C15芳基、取代或未取代的C7-C30烷基-芳基、取代或未取代的C3-C20杂环、取代或未取代的C4-C30烷基-杂环、取代或未取代的C5-C15杂芳基、C1-C20羟烷基、氰基、氨基、胍基、硝基或者羟基。
在一些实施方式中，前述的烷基可以是直链或支链的，并可以任选地被取代，例如可以选自甲基、乙基、1-丙基或正丙基、2-丙基或异丙基、1-丁基或正丁基、2-甲基-1-丙基或异丁基、1-甲基丙基或仲丁基、1，1-二甲基乙基或叔丁基、1-戊基、2-戊基、3-戊基、2-甲基-2-丁基、3-甲基-2-丁基、3-甲基-1-丁基、2-甲基-1-丁基，1-己基，2-己基，3-己基，2-甲基-2-戊基，3-甲基-2-戊基，4-甲基-2-戊基，3-甲基-3-戊基，2-甲基-3-戊基、2，3-二甲基-2-丁基和3，3-二甲基-2-丁基等，且不限于此。
在一些实施方式中，前述烷氧基可以是直链或支链的，例如可以选自但不限于甲氧基、乙氧基、丙氧基、丁氧基等。
在一些实施方式中，前述烯基可以是直链或支链的，并且可以被任选地被取代，即被取代或未被取代。例如，前述烯基可以选自但不限于乙烯基、丙烯基、丁烯基、1，4-丁二烯基、戊烯基、己烯基、4-甲基己-1-烯基、4-乙基-2-甲基己-1-烯基等。
在一些实施方式中，前述炔基可以是直链或支链的，并且可以被任选地被取代。例如，前述炔基可以选自但不限于乙炔基、丙炔基、丁炔基等。
在一些实施方式中，前述脂环族包括环烷基和环烯基，例如可以选自但不限于环丙烷、环丁烷、环戊烷、环戊烯、环戊二烯、环己烷、环己烯、1，3-环己二烯、1，4-环己二烯、环庚烷和环庚烯等。
在一些实施方式中，前述芳基可以是苯基等6元碳环芳环，也可以是萘、茚满和1，2，3，4-四氢喹啉等7-12元双环等，且不限于此。
在一些实施方式中，前述杂环是包含至少一个选自N、O和S的杂原子的脂族螺环，其可以选自但不限于1-吡咯烷基、2-吡咯烷基、2，4-咪唑烷基、2，3-吡唑烷基、1-哌啶基、2-哌啶基、3-哌啶基、4-哌啶基、2，5-哌嗪基、吡喃基、2-吗啉基、环氧乙烷基、氮丙啶基、氮杂环丁烷基、四氢吡啶基，硫代吗啉基，噻吩基，哌嗪基，高哌嗪基，高哌啶基，氮杂环庚烷基，氧杂环庚烷基、噻吩基、1，4-氧杂杂环烷基、1，4-二氧杂环庚烷、1，4-氧杂杂环庚烷、4-氧杂环庚烷，4-氧杂环庚烷，1-氧杂环庚烷、四氢呋喃基，四氢噻吩基、四氢噻吩基、2-吡喃基、四氢噻吩基3-吡咯啉基、二氢吲哚基、2H-吡喃基、4H-吡喃基、1，4-二氧杂环己烷基、1，3-二氧戊环基、吡唑啉基、吡唑立啶基、二噻吩基、二噻吩基、吡唑烷基咪唑啉基、嘧啶壬基、1，1-二氧代-硫代二氢吡啶基、3-氮杂双环[3.1.0]己基、3-氮杂双环[4.1.0]庚基等，且不限于此。
在一些实施方式中，前述烷基杂环是指包含与杂环或取代的杂环偶联的烷基的化学取代基。
在一些实施方式中，前述杂芳基是指芳族杂环，其可以由五个、六个、七个、八个、九个或更多原子形成。杂芳基可以任选地被取代。例如可以选自但不限于包含一个氧或硫原子或最多四个氮原子，或一个氧或硫原子和最多两个氮原子的组合的芳族C5-C15杂环基团，以及它们的取代以及苯并和吡啶并稠合衍生物。
较为优选的，R 1、R 2、R 3各自独立的选自C1-C6烷基、羟基或者胺基。
在一些情况下，R 3也可选自胺基、羟基等。
较为优选的，R 4、R 5、R 6选用氢原子。
在一个较为具体的实施案例中，所述化合物为下式所示的化合物：
Figure PCTCN2022134136-appb-000006
本申请的一些实施例还提供了一种星型β-抗菌糖肽，其外层为螺旋结构的糖肽嵌段，内层为带正电荷的聚二甲基氨基贝塔内酰胺嵌段。该星型β-抗菌糖肽的结构如通式(I)所示。
其中，所述糖肽嵌段的来源包括单糖葡萄糖、半乳糖、甘露糖、阿卓糖、胺基葡萄糖、胺基半乳糖、胺基甘露糖或双糖麦芽糖，但不限于此。
本申请实施例所提供的星型β-抗菌糖肽是一类星型嵌段聚合物，其外层螺旋糖肽可以更好的暴露在化合物外层，起到结合细菌表面糖受体，预防感染；形成一层水化层，防止蛋白吸附；阻挡内层的阳离子嵌段和哺乳动物细胞的结合，进一步提高生物相容性。
本申请的一些实施例还提供了一种制备通式(I)所示化合物的方法，所述方法包括：
(i)依据反应式a，使包含Core的前驱体化合物与式(II)所示的化合物、式(III)所示的化合物发生聚合反应，获得式(IV)所示的聚合物；
Figure PCTCN2022134136-appb-000007
(ii)依据反应式b，对式(IV)所示的聚合物进行脱保护
Figure PCTCN2022134136-appb-000008
其中，式(II)所示的带保护基团的化合物、式(III)所示的带保护基团的化合物可以按照本领域已知的方式合成。例如，可以根据文献(J.Am.Chem.Soc.134，16255-16264(2012))合成葡萄糖衍生的贝塔内酰胺(AS(Bn))。以及，可以根据文献(Angew.Chem.Int.Ed.2020doi.org/10.1002/anie.201914304)合成羧基苄基(Cbz)保护的带胺基的贝塔内酰胺(DM(Cbz))。
其中，所述“保护基团”的定义是本领域习知的，一般是指与分子中的官能团反应并结合以防止该官能团参与分子的后续反应的化学基团，但该基团随后可以被去除从而再生未保护的官能团。在本申请中，所述保护基团包括但不限于苄基、羧基苄基(Cbz)、叔丁氧基羰基(Boc)等，优选为羧基苄基。
进一步的，步骤(a)包括：在双(三甲基甲硅烷基)氨基锂和4-叔丁基苯甲酰氯存在下制备所述保护聚合物。
进一步的，步骤(a)中使用的反应温度可以为0℃～66℃，优选为室温反应。
进一步的，步骤(b)包括：在钠和液氨条件下使所述保护聚合物脱保护。
进一步的，步骤(b)中使用的反应温度可以为-80℃～-50℃，优选为-78℃～-55℃。
本申请的一些实施例还提供了一种药物组合物，包含所述通式(I)所示的化合物或其可药用盐以及药学上可接受的载体。
在本说明书中，所述可药用盐的定义是本领域习知的，即：在适当的药用评估范围内，适用于与人体组织器官和低等动物的组织器官接触的那些盐，没有过度的毒性、刺激、过敏反应或类似的反应。所述盐可以是任何盐，有机或无机加成盐，特别是药学上常用的任何药学上可接受的有机或无机加成盐，例如通式(I)所示化合物与无机酸如盐酸、氢溴酸、氢碘酸或与有机酸如甲酸、乙酸、乙酰乙酸、三氟乙酸、三氟甲磺酸、对甲苯磺酸、甲磺酸反应形成的盐。
在一些较为优选的实施方式中，所述药物组合物还包含其它抗菌剂。
进一步的，所述其它抗菌剂包括但不限于氨苄西林、氯唑西林、苯唑西林和哌拉西林、头孢菌素例如头孢克洛、头孢孟多、头孢唑林、头孢哌酮、头孢噻肟、头孢西丁、头孢他啶、 头孢曲松和头孢菌素；碳青霉烯类包括例如亚胺培南和美罗培南；和糖肽类、大环内酯类、喹诺酮类、四环素类和氨基糖苷类。此外所述其它抗菌剂还可以选自利福平、环丙沙星、左氧氟沙星、哌拉西林、复方新诺明、庆大霉素、洛布霉素、琥乙红霉素、红霉素、克拉霉素、新生霉素、螺旋霉素、乙酰螺旋霉素、氯霉素、甲氧苄啶、磺胺甲恶唑、羧苄青霉素、多粘菌素B、粘菌素、阿米卡星、卡那霉素、新霉素、奈替米星、链霉素、妥布霉素、巴龙霉素，格尔德霉素、除莠霉素，氯碳头孢，多利培南、西司他丁、头孢羟氨苄、头孢噻吩、头孢氨苄、头孢孟多、头孢丙烯、头孢呋辛、头孢克肟、头孢地尼、头孢托仑、头孢泊肟、头孢布烯、头孢唑肟、头孢吡肟、替考拉宁、万古霉素、罗红霉素、醋竹桃霉素、秦利霉素、大观霉素、阿莫西林、羧苄西林、双氯西林、氟氯西林、美洛西林、甲氧西林、萘夫西林、青霉素、替卡西林、杆菌肽、依诺沙星、加替沙星、莫西沙星、诺氟沙星、曲伐沙星、磺胺米隆、偶氮磺胺、磺胺醋酰、磺胺甲二唑、磺胺、柳氮磺吡啶、磺胺异噁唑、甲氧苄啶磺胺甲噁唑、地美环素、多西环素、米诺环素、土霉素、四环素、胂凡纳明、克林霉素、林可霉素、乙胺丁醇、磷霉素、夫西地酸、呋喃唑酮、异烟肼、利奈唑胺、双唑秦栓、莫匹罗星、呋喃妥因、吡嗪酰胺、奎奴普丁/达福普汀、异福酰胺、替硝唑中的任意一种或多种的组合。
其中，所述其它抗菌剂包括但不限于抗革兰氏阳性菌抗生素，而所述抗菌剂的抗菌范围包括革兰氏阴性菌。进一步的，利用所述通式(I)所示的化合物或其可药用盐，可以扩展抗革兰氏阳性菌抗生素的抗菌范围，使其对于革兰氏阴性菌也有效果。
在本说明书中，前述药学上可接受的载体包括药学上可接受的材料、组合物、赋形剂等，例如液体或固体填充剂、稀释剂、赋形剂或溶剂包封材料，具体包括但不限于如下物质：糖类，例如乳糖、葡萄糖和蔗糖；淀粉，如玉米淀粉和马铃薯淀粉；纤维素及其衍生物，如羧甲基纤维素钠、乙基纤维素和醋酸纤维素；黄蓍胶粉；麦芽；明胶；滑石；赋形剂，如可可脂和栓剂蜡；油类，如花生油、棉籽油、红花油、芝麻油、橄榄油、玉米油和豆油；二醇类，如丙二醇；多元醇，如甘油、山梨糖醇、甘露糖醇和聚乙二醇；油酸乙酯、月桂酸乙酯等酯类；琼脂；缓冲剂，如氢氧化镁和氢氧化铝；海藻酸；无菌蒸馏水；乙醇；pH缓冲溶液；聚酯、聚碳酸酯或聚酐；等等。
在一些实施方式中，为了制备所述药物组合物，可以使用药学惰性的无机或有机赋形剂。例如，为了制备丸剂、粉剂、明胶胶囊或栓剂等，可以使用乳糖、滑石粉、硬脂酸及其盐、脂肪、蜡、固体或液体多元醇、天然油和硬化油等作为药学上可接受的载体。为了制备注射液、口服液、喷剂、气溶胶制剂、粉剂等，可以使用水、醇、甘油、多元醇及其合适的混合物以及植物油等作为药学上可接受的载体。进一步的，在制得前述制剂后，可以通过多种方式对制剂进行灭菌，包括通过细菌截留过滤器过滤，或者通过加入无菌固体组合物形式的灭 菌剂。
本申请的一些实施例还提供了所述通式(I)所示的化合物或其可药用盐或者所述药物组合物作为抗菌剂的用途。
在本说明书中，所述“抗菌剂”是指能够：(i)抑制、减少或防止细菌生长的任何物质或其组合；(ii)具备抑制或降低细菌在受试者中产生感染的能力；(iii)具备抑制或降低细菌在环境中繁殖或保持传染性的能力。
本申请的一些实施例还提供了所述通式(I)所示的化合物或其可药用盐或者所述药物组合物在制备治疗或预防细菌感染或由所述细菌感染引起的疾病的药物中的用途。
其中，所述细菌包括具有多重耐药性细菌、敏感细菌中的至少一种。
进一步的，所述细菌具有多重耐药性，即，细菌对不同化学结构的多种药物具有耐药性和/或对针对不同靶标的药物具有耐药性。
在本说明书中，所述细菌包括革兰氏阴性菌(例如大肠杆菌、铜绿假单胞菌、幽门螺杆菌、肺炎克雷伯菌等)、革兰氏阳性菌等，尤其是耐碳青霉烯革兰氏阴性菌，例如耐碳青霉烯鲍曼不动杆菌。
在本说明书中，所述药物可以是多种形式的制剂，并可以通过口服、注射、外敷、栓塞、气雾等多种方式使用。适于口服的制剂形式可以是片剂、糖锭剂、锭剂、水或油混悬液、可分散粉末或颗粒、乳液、硬或软胶囊，或糖浆剂或酏剂等。并且，所述药物还可以含有甜味剂、矫味剂、着色剂、防腐剂、抗氧化剂。适于注射的制剂形式可以是无菌注射水溶液形式或油混悬液形式，其中采用的溶剂包括但不限于水、林格氏液、氯化钠溶液、葡萄糖溶液等。适于作为栓剂的制剂形式在普通温度下为固体但在直肠中为液体，其中的药物载体可以在直肠中会溶化而释放药物且对直肠无刺激性。
本申请的一些实施例还提供了所述通式(I)所示的化合物或其可药用盐或者所述药物组合物作为细胞外排泵抑制剂和/或细菌膜透化剂的用途。
其中，所述细胞外排泵抑制剂和/或细菌膜透化剂的定义是本领域熟知的。具体的，所述细胞外排泵是指以能量依赖性方式从细胞的细胞质或周质输出底物分子的蛋白质组装体。所述细菌膜透化剂是指能够降低细菌细胞质膜的完整性或破坏细菌细胞质膜的任何化合物。
本申请的一些实施例还提供了在受试者中治疗或预防细菌感染或由所述细菌感染引起的疾病的方法，该方法包括向受试者施用有效量的通式(I)所示的化合物或者所述药物组合物。
在一些实施方式中，所述细菌具有多重耐药性。
在一些实施方式中，所述的方法还包括向受试者施用有效量的其它抗菌剂。所述其它抗菌剂如前所述，此处不再赘述。并且，本领域技术人员可以按照本领域习知的方式确定通式(I) 所示化合物与所述其它抗菌剂的最佳用量比例。
需要说明的是，通式(I)所示化合物可以以有效量单独或与一种或多种其他抗菌剂组合(同时、顺序或分开)施用于有此需要的受试者。
在本说明书中，受试者可以是任何人类或非人类动物，优选哺乳动物，更优选人类。
由于通式(I)所示化合物与所述其它抗菌剂在联合使用时存在协同效应，所以通式(I)所示化合物和/或其他抗菌剂单独使用时的治疗有效剂量可以低于其标准剂量。例如，治疗有效剂量可为标准剂量的1％～99％、1％～90％、1％～80％、1％～70％、1％～60％、1％～50％、1％～40％、1％～30％、1％～20％、5％～20％、1％～10％、0.1％～1％、0.01％～1％、0.001％～1％等。
本申请的一些实施例还提供了通式(I)所示的化合物或其可药用盐或者所述星型β-抗菌糖肽(亦可称为“抗菌多肽”)在制备预防或治疗真菌感染的药物中的用途。
所述星型β-抗菌糖肽包括通式(I)所示的化合物以及所述化合物的立体异构体、互变异构体、N-氧化物、水合物、溶剂化物或可药用盐中的至少一种；
Figure PCTCN2022134136-appb-000009
其中，Core为具有环状基团的结构单元；
R 1、R 2、R 3、R 4各自独立的选自H、卤素、取代或未取代的C1-C20烷基、取代或未取代的C1-C20烷氧基、取代或未取代的C2-C20烯基、取代或未取代的C2-C20炔基、取代或未取代的C3-C20脂环基、取代或未取代的C6-C15芳基、取代或未取代的C7-C30烷基-芳基、取代或未取代的C3-C20杂环、取代或未取代的C4-C30烷基-杂环、取代或未取代的C5-C15杂芳基、C1-C20羟烷基、氰基、氨基、胍基、硝基或者羟基；
n为0～6，m为3～100，x、y为1～50。
在一些实施方式中，所述的用途包括：将所述星型β-抗菌糖肽与抗真菌药物联用，以预防或治疗真菌感染或真菌感染引起的疾病。
在本申请中，所述药物可以是多种形式的制剂，并可以通过口服、注射、外敷、栓塞、气雾等多种方式使用。适于口服的制剂形式可以是片剂、糖锭剂、锭剂、水或油混悬液、可分散粉末或颗粒、乳液、硬或软胶囊，或糖浆剂或酏剂、膏剂等。并且，所述药物还可以含有甜味剂、矫味剂、着色剂、防腐剂、抗氧化剂等。
本申请的一些实施例还提供了所述星型β-抗菌糖肽在制备抗真菌药物增效剂中的用途。
本申请的一些实施例还提供了一种抗真菌药物组合物，包含所述星型β-抗菌糖肽以及药学上可接受的载体。所述药物组合物还包含抗真菌药物。
在本申请中，所述真菌可以为药物敏感性真菌、药物中等性真菌或耐药性真菌，特别是耐药性真菌。
例如，所述真菌可以为酵母目(Saccharomycetales)、爪甲团囊菌目(Onygenales)或丝孢目(Hyphomycetales)真菌。更具体的，所述真菌可以为酵母目(Saccharomycetales)的药物敏感性真菌、爪甲团囊菌目(Onygenales)的药物敏感性真菌、丝孢目(Hyphomycetales)的药物敏感性真菌、酵母目(Saccharomycetales)的药物中等性真菌、爪甲团囊菌目(Onygenales)的药物中等性真菌、丝孢目(Hyphomycetales)的药物中等性真菌、酵母目(Saccharomycetales)的耐药性真菌、爪甲团囊菌目(Onygenales)的耐药性真菌、丝孢目(Hyphomycetales)的耐药性真菌等。
例如，所述真菌可以为隐球酵母科(Cryptacoccaceae)、裸囊菌科(Arthrodermataceae)和丝孢科(Hyphomycet)真菌等。更具体的，所述真菌可以为隐球酵母科(Cryptacoccaceae)的药物敏感性真菌、裸囊菌科(Arthrodermataceae)的药物敏感性真菌、丝孢科(Hyphomycet)的药物敏感性真菌、隐球酵母科(Cryptacoccaceae)的药物中等性真菌、裸囊菌科(Arthrodermataceae)的药物中等性真菌、丝孢科(Hyphomycet)的药物中等性真菌、隐球酵母科(Cryptacoccaceae)的耐药性真菌、裸囊菌科(Arthrodermataceae)的耐药性真菌和丝孢科(Hyphomycet)的耐药性真菌等。
例如，所述真菌可以为曲霉属、念珠菌属、假丝酵母属(Candida)、小孢子菌属(Microsporum)、表皮癣菌属(Epidermophyton)真菌等。更具体的，所述真菌可以为假丝酵母属(Candida)的药物敏感性真菌、小孢子菌属(Microsporum)的药物敏感性真菌、表皮癣菌属(Epidermophyton)的药物敏感性真菌、假丝酵母属(Candida)的药物中等性真菌、小孢子菌属(Microsporum)的药物中等性真菌、表皮癣菌属(Epidermophyton)的药物中等性真菌、假丝酵母属(Candida)的耐药性真菌、小孢子菌属(Microsporum)的耐药性真菌、表皮癣菌属(Epidermophyton)的耐药性真菌等。
例如，所述真菌可以为白色念珠菌(Candida albicans)、石膏样小孢子菌(Microsporum gypseum)和絮状表皮癣菌(Epidermophyton floccosum)真菌等。更具体的，所述真菌可以为药物敏感性的白色念珠菌(Candida albicans)、药物敏感性的石膏样小孢子菌(Microsporum gypseum)、药物敏感性的絮状表皮癣菌(Epidermophyton floccosum)、药物中等性的白色念珠菌(Candida albicans)、药物中等性的石膏样小孢子菌(Microsporum gypseum)、药物中等性的絮状表皮癣菌(Epidermophyton floccosum)、耐药性的白色念珠菌(Candida albicans)、耐药性的石膏样小孢子菌(Microsporum gypseum)、耐药性的絮状表皮癣菌(Epidermophyton floccosum)、 烟曲霉(Aspergillus fumigatus)、耳念珠菌(Candida auris)等。
较为典型的，所述致病真菌包括皮肤癣菌、深部感染真菌等。所述皮肤癣菌可以包括：毛癣菌属(Trichophyton)，例如红色毛癣菌、须癣毛癣菌、紫色毛癣菌；表皮癣菌属(Epidermophyton)，例如絮状表皮癣菌；小孢子癣菌属(Microsporum)，例如奥杜盎小孢子菌、犬小孢子菌、石膏样小孢子菌；马拉色菌属；等等。所述深部感染真菌可以包括：假丝酵母菌；新生隐球菌；肺孢子菌属；曲霉菌属；组织胞浆菌属；镰刀菌属；青霉菌属；毛霉菌属；等等。
在本申请中，所述抗真菌药物包括唑类(如咪唑类如克霉唑、酮康唑、咪康唑、益康唑等及三唑类氟康唑、伊曲康唑、伏立康唑、泊沙康唑等)、多烯类(如两性霉素B、制霉菌素等)、烯丙胺类(如特比奈芬等)、氟胞嘧啶类(如5-氟胞嘧啶等)、棘白菌素类(如卡泊芬净、米卡芬净等)抗真菌药物等，且不限于此，例如还可以包括灰黄霉素、复方磺胺甲恶唑、羟乙磺酸戊烷脒、多黏菌素等。
在一个实施例中，所述抗真菌药物的用量为0-2048μg/mL，所述抗菌多肽的用量为0-512μg/mL。
本申请的一些实施例还提供了在受试者中治疗或预防真菌感染或由所述真菌感染引起的疾病的方法，该方法包括向受试者施用有效量的所述星型β-抗菌糖肽或者所述药物组合物。
在一些实施方式中，所述真菌具有耐药性。
在一些实施方式中，所述的方法还包括向受试者施用有效量的其它抗真菌药物。所述抗真菌药物如前所述，此处不再赘述。并且，本领域技术人员可以按照本领域习知的方式确定所述星型β-抗菌糖肽与所述抗真菌药物的最佳用量比例。
所述星型β-抗菌糖肽，例如通式(I)所示化合物与常用抗真菌药物联合使用时，存在协同效应，不仅可以有效克服耐药真菌对常用抗真菌药物的耐药性，且还可以显著降低常用抗真菌药物的使用量，例如可以使常用抗真菌药物的治疗有效剂量明显低于其标准剂量，例如为标准剂量的1％～99％、1％～90％、1％～80％、1％～70％、1％～60％、1％～50％、1％～40％、1％～30％、1％～20％、5％～20％、1％～10％、0.1％～1％、0.01％～1％、0.001％～1％等。示例性的，在一些情况下，将所述星型β-抗菌糖肽与伊曲康唑等联合使用时，伊曲康唑的治疗有效剂量可以为其标准剂量的1/512。
另外，本申请人在此前的研究中还发现，本申请的星型β-抗菌糖肽还可以克服细菌对常用抗生素的耐药性，通过与常用抗生素联合使用，可以有效杀死多重耐药的细菌，由此可以预防或质量细菌感染或由此引起的疾病(参阅CN2022100766439)。如此，对于一些具有灭杀真菌的功效的抗生素来说，通过将其与所述抗菌多肽联用，还可同时灭杀耐药真菌和耐药 细菌，且降低抗生素及抗真菌药物的施用量，减少药物对患者肝肾系统等造成的负担，这对于一些由细菌和真菌共同感染引起的疾病的治疗是非常有用的。
下面结合若干优选实施例及附图对本申请的技术方案做进一步详细说明，本实施例在以发明技术方案为前提下进行实施，给出了详细的实施方式和具体的操作过程，但本申请的保护范围不限于下述的实施例。
实施例1本实施例提供的一种星型β-抗菌糖肽(亦可被命名为抗菌多肽1)的结构式如下：
Figure PCTCN2022134136-appb-000010
一种合成该星型β-抗菌糖肽的方法包括如下步骤：
(1)贝塔内酰胺单体制备
根据文献(J.Am.Chem.Soc.134，16255-16264(2012))合成葡萄糖衍生的贝塔内酰胺(AS(Bn))。根据文献(Angew.Chem.Int.Ed.2020doi.org/10.1002/anie.201914304)合成羧基苄基(Cbz)保护的带胺基的贝塔内酰胺(DM(Cbz))。
(2)聚合的一般程序
整个聚合过程在惰性气体保护的手套箱中进行。首先，在无水四氢呋喃中配制0.4MDM(Cbz)和AS(Bn)，0.02M 1，3，5-苯三甲酰氯和0.15M双(三甲基甲硅烷基)酰胺锂(LiHMDS)。然后，将1ml 1，3，5-苯三甲酰氯、900μl DM(Cbz)的溶液加入10ml烘箱干燥的圆底烧瓶中混合。在30℃下搅拌3min使溶液混合均匀。随后将1ml LiHMDS溶液注入反应烧瓶中以引发聚合。在室温下反应一段时间后，通过TLC确认DM(Cbz)单体完全消耗后，将450μl AS(Bn)溶液添加到反应混合物继续反应直至单体全部消耗，之后用几滴甲醇淬灭反应。通过在二氯甲烷中重复溶解(3个循环)和在己烷中沉淀来纯化聚合物。
(3)聚合物脱保护
将140mg保护聚合物和54mg叔丁醇钾(KOt-Bu)溶解在5.0ml无水THF中。在-78℃和氮气下，将聚合物溶液滴加到快速搅拌的钠(120mg)的液氨(～15ml)溶液中。钠无水甲苯和己烷中洗涤并在加入前切成小块。反应在-78℃下反应4h，然后逐滴加入饱和氯化铵进行淬灭反应直至蓝色消失。剩余的氨在室温过夜蒸发。过滤所得溶液用去离子水洗涤，并用1000Da的截止透析袋透析2天，每2～3小时换一次水。最后，通过冷冻干燥获得白色固体样品。
本实施例通过阴离子开环聚合成功合成了该星型β-抗菌糖肽，其为一种嵌段星型β-多肽。该嵌段星型β-多肽外层为螺旋结构的糖肽嵌段，可以起到提高生物相容性，预防蛋白吸附以及预防感染的作用，内层为带正电荷的聚二甲基氨基贝塔内酰胺嵌段，起到增加细菌外膜通透性和破坏细菌分子外排泵的作用。参阅图1所示，该多肽的分子结构由核磁共振氢谱证实，外层糖肽嵌段和内层带正电荷的聚二甲基氨基贝塔内酰胺嵌段的积分比例为1∶2，和单体投料比一致。参阅图2，该多肽的分子量通过凝胶渗透色谱(GPC)测量的聚合物的聚合度为26，接近于27∶1的单体/引发剂进料比，并且具有相对较窄的分子量分布
Figure PCTCN2022134136-appb-000011
耐碳青霉烯类的鲍曼不动杆菌是在世界卫生组织(WHO)发表的世界上最具耐药性、最能威胁人类健康的“超级细菌”列表上第一位的细菌。表1是耐碳青霉烯鲍曼不动杆菌(DR-AB)耐药性表格，可以看出它对现在临床上常用的抗生素几乎都产生了耐药性。
本实施例中利用该种细菌对该星型β-抗菌糖肽的活性进行了测试，测试方法包括：
1.最低抑菌浓度(MIC)值检测
1)活化：从LB平板上划取一个单菌落加入至含有5ml MHB培养基的离心管中，置于摇床，37℃，175rpm，振荡培养过夜。
2)复培：吸取50μl活化菌液加至含有5ml MHB培养基的离心管中，振荡培养3-4h。
3)配置抗生素溶液(10mg/ml)。
4)在96孔板的B排-F排每个孔加入50μL MHB，再分别吸取50μL MHB加至B1、C1、D1、E1、F1孔。
5)从B1、C1、D1、E1、F1孔中分别吸取10.24μL MHB并丢弃。
6)分别吸取10.24μL抗生素溶液，加至B1、C1、D1、E1、F1孔中，并混合均匀。
7)从第一列(B2-F1)孔中吸取50μL混合液至第二列孔位(B2-F2)，并吹打混匀。依次重复直至最后一列孔位(B12-F12)。
8)从最后一列孔位(B12-F12)吸取50μL混合液并丢弃。
9)将复培的细菌从摇床取出。
10)稀释的菌悬液，并使用酶标仪测定OD值直至将菌悬液稀释为OD≈0.2(此时菌液约 10 9CFU/ml)。然后将菌悬液再稀释1000倍即可得到10 6CFU/ml菌悬液。
11)分别吸取50μL，106CFU/ml菌悬液至前5排孔位(B2-F12)。
12)吸取菌悬液和MHB将第6排孔位(G2-G12)的正负对照补齐(6个正对照如G2-G6：100μL MHB；6个负对照如G7-G12：50μL MHB+50μL菌悬液)。
13)将待测96孔板振荡3-4min，封口后放入生化培养箱37℃培养16～18h。
14)从培养箱中取出96孔板，目测浑浊度以确定MIC值，再使用酶标仪测量595nm OD值并记录，导出数据并根据折点解释进行判断。
2.棋盘稀释法测量联合用药效果
1)活化、复培菌种并配置抗生素溶液(与MIC实验步骤1)-3)相同)。
2)使用深孔板横向配置抗生素的2倍梯度浓度溶液(从16×MIC起始，最后一孔为MHB)。
3)使用深孔板纵向配置样品的2倍梯度浓度溶液(从4×256μg/ml起始，最后一孔为MHB)。
4)使用多道移液器从深孔板中分别吸取25μL抗生素梯度浓度溶液与样品梯度浓度溶液，分别按行、列加满整个96孔板。
5)每个孔加入50μL，106CFU/ml菌悬液(配置方法与MIC实验步骤10)相同)。
6)封口、振荡、培养及读数(与MIC实验步骤13)-14)相同)。
经以上活性测试发现，通过将本实施例的星型β-抗菌糖肽与多种抗生素辅助剂联合使用，可以实现的效果包括但不限于：
(1)增加抗生素的杀菌范围，可以把只对革兰氏阳性菌有效的抗生素(如利福平)也变得对革兰氏阴性菌有效果，参阅图3；
(2)极大地增强抗生素的杀菌效果，降低多种抗生素的最低杀菌浓度达到16-256倍，参阅图4-图9；
(3)降低细菌耐药性产生的可能性，因为有两个分子同时作用于不同位置，细菌很难同时做出针对性变异。
实施例1中星型β-抗菌糖肽增强其它抗菌素对于耐碳青霉烯类鲍曼不动杆菌的杀菌作用的一些实验结果还可参阅图4-图13，对于普通鲍曼不动杆菌的杀菌作用参阅图14-图21。
实施例2：本实施例提供的一种星型β-抗菌糖肽(亦可被命名为抗菌多肽2)的结构式如下：
Figure PCTCN2022134136-appb-000012
其中，Core的结构与实施例1星型β-抗菌糖肽中的Core相同。相较于实施例1，该星型β-抗菌糖肽的1H NMR表征图谱包含：δ5.66(1H，br)，4.02-4.39(3H)，3.65(2H，br)，3.37-2.96(9H)，1.37(6H，br)。该星型β-抗菌糖肽的合成方法可以参考实施例1，但所采用的贝塔内酰胺单体为单甲基取代的贝塔内酰胺，其合成方法参照(J.Am.Chem.Soc.2007，129，15474-15476)，其他步骤和原料与实施例1基本相同。
实施例3：本实施例提供的一种星型β-抗菌糖肽(亦可被命名为抗菌多肽3)的结构式如下：
Figure PCTCN2022134136-appb-000013
其中，Core的结构与实施例1星型β-抗菌糖肽中的Core相同。相较于实施例1，该星型β-抗菌糖肽的1H NMR表征图谱包含：δ5.66(1H，br)，4.02(2H)，3.65-2.75(13H)。该星型β-抗菌糖肽的合成方法可以参考实施例1，但所采用的贝塔内酰胺单体为无甲基取代的贝塔内酰胺，其合成方法参照(J.Am.Chem.Soc.2014，136，4333-4342)，其他步骤和原料与实施例1基本相同。
实施例4：本实施例提供的一种星型β-抗菌糖肽(亦可被命名为抗菌多肽4)的结构式如下：
Figure PCTCN2022134136-appb-000014
其中，Core的结构与实施例1星型β-抗菌糖肽中的Core相同。相较于实施例1，该星型β-抗菌糖肽的1H NMR表征图谱包含：δ5.66(1H，br)，4.53(2H，br)4.02(1H，br)，3.65(2H，br)，3.37-2.58(8H)。该星型β-抗菌糖肽的合成方法可以参考实施例1，但所采用的贝塔内酰胺单体为单无基取代，同时Cbz保护的亚甲基胺基从α位变到β位的贝塔内酰，其合成方法参照(J.Am.Chem.Soc.2013，135，5270-5273)，其他步骤和原料与实施例1基本相同。
实施例5：本实施例提供的一种星型β-抗菌糖肽(亦可被命名为抗菌多肽5)的结构式如下：
Figure PCTCN2022134136-appb-000015
其中，Core的结构与实施例1星型β-抗菌糖肽中的Core相同。相较于实施例1，该星型β-抗菌糖肽的1H NMR表征图谱包含：δ5.66(1H，br)，4.32(3H)，4.02(2H)，3.65(2H，br)，3.37-3.0(5H).2.50(4H，br)，1.8-1.3(12H)。该星型β-抗菌糖肽的合成方法可以参考实施例1，但所采用的贝塔内酰胺单体为无甲基取代同时Cbz保护的亚甲基胺基碳链长度增加到4的贝塔内酰胺，其合成方法参照(Nat.Commun.2019，10，1-14)，其他步骤和原料与实施例1基本相同。
实施例6：本实施例提供的一种星型β-抗菌糖肽(亦可被命名为抗菌多肽6)的结构式如下：
Figure PCTCN2022134136-appb-000016
其中，Core的结构与实施例1星型β-抗菌糖肽中的Core相同。相较于实施例1，该星型β-抗菌糖肽的1H NMR表征图谱包含：δ5.66(1H，br)，4.60-4.14(1H)，4.12-3.52(4H，br，m)，3.20-2.58(7H，br，m)，1.37(12H，br，m)。该星型β-抗菌糖肽的合成方法可以参考实施例1，但所采用的贝塔内酰胺单体从葡萄糖变为阿卓糖内酰胺单体，其合成方法参照(J.Am.Chem.Soc.2017，139，14217-14223)，其他步骤和原料与实施例1基本相同。
实施例7：本实施例提供的一种星型β-抗菌糖肽(亦可被命名为抗菌多肽7)的结构式如下：
Figure PCTCN2022134136-appb-000017
其中，Core的结构与实施例1星型β-抗菌糖肽中的Core相同。相较于实施例1，该星型β-抗菌糖肽的1H NMR表征图谱包含：δ5.82(1H，br)，4.37-4.14(1H)，4.12-3.52(4H，br，m)，3.85-2.58(11H，br，m)，1.37(12H，br，m)。该星型β-抗菌糖肽的合成方法可以参考实施例1， 但其中采用的贝塔内酰胺单体从葡萄糖变为半乳糖内酰胺单体，其合成方法参照(J.Am.Chem.Soc.2016，138，6532-6540)，其他步骤和原料与实施例1基本相同。
实施例8：本实施例提供的一种星型β-抗菌糖肽(亦可被命名为抗菌多肽8)的结构式如下：
Figure PCTCN2022134136-appb-000018
其中，Core为1，2，3，4-环丁烷四碳基。相较于实施例1，该星型β-抗菌糖肽的1HNMR表征图谱包含：δ5.66(1H，br)，4.04(1H)，3.77-3.53(2H，br)，3.48-2.69(9H，br，m)，3.20-2.58，1.37(12H，br)。该星型β-抗菌糖肽的合成方法可以参考实施例1，区别在于：步骤(2)中是采用1，2，3，4-环丁烷四酰氯替换了1，3，5-苯三甲酰氯，其他步骤和原料与实施例1基本相同。
实施例9：本实施例提供的一种星型β-抗菌糖肽(亦可被命名为抗菌多肽9)的结构式如下：
Figure PCTCN2022134136-appb-000019
其中，Core为1，2，3，4，5，6-环己烷六碳基。相较于实施例1，该星型β-抗菌糖肽的1H NMR表征图谱包含：δ5.66(1H，br)，4.04(1H)，3.77-3.53(2H，br)，3.48-2.69(9H，br，m)，3.20-2.58，1.37(12H，br)。该星型β-抗菌糖肽的合成方法可以参考实施例1，区别在于：步骤(2)中是采用1，2，3，4，5，6-环己烷六酰氯替换了1，3，5-苯三甲酰氯，其他步骤和原料与实施例1基本相同。
测试例1
参照实施例1的方法，利用本实施例中利用耐碳青霉烯鲍曼不动杆菌对实施例2-实施例9提供的星型β-抗菌糖肽的活性进行测试，结果显示，这些星型β-抗菌糖肽与多种抗生素辅助剂联合使用，其也能增加抗生素的杀菌范围，增强抗生素的杀菌效果，降低细菌耐药性产生的可能性，且效果与实施例1的抗菌糖肽接近。
此外，本申请人还参照前述实施例1-9，制备了本申请说明书中述及的其它星型β-抗菌糖肽，并也对其抗菌性能等进行了测试，结果显示，这些星型β-抗菌糖肽与多种抗生素辅助剂联合使用，在增加抗生素的杀菌范围、增强抗生素的杀菌效果、降低细菌耐药性产生的可能 性等方面的效果均远优于现有的糖源性阳离子嵌段聚(β-肽)、β-肽糖共聚物等。
表1耐碳青霉烯鲍曼不动杆菌(CR-AB)耐药性
抗生素 MIC(μg/ml) 耐药性
环丙沙星 ＞＝4 耐药
氨苄西林\舒巴坦 ＞＝32 耐药
左氧氟沙星 ＞＝8 耐药
哌拉西林 ＞＝128 耐药
复方新诺明 ＞＝320 耐药
庆大霉素 ＞＝16 耐药
头孢哌酮\舒巴坦 8 耐药
头孢他啶 ＞＝64 耐药
头孢曲松 ＞＝64 耐药
头孢吡肟 ＞＝64 耐药
哌拉西林\他唑巴坦 ＞＝128 耐药
美罗培南 ＞＝16 耐药
洛布霉素 ＞＝16 耐药
亚胺培南 ＞＝16 耐药
测试例2
本测试例对抗菌多肽1的性能进行测试，具体如下：
1.菌种：使用两株耐药真菌：C.albicans(白色念珠菌，编号ATCC 64124；对多种唑类及棘白菌素类药物耐药)及A.fumigatus(烟曲霉，编号ATCC MYA-3627；伊曲康唑耐药)进行测试。
2.药物：除制霉菌素选用自北京索莱宝科技有限公司及实验室自合成的抗菌多肽1外，其余抗真菌药物均来自于上海阿拉丁生化科技股份有限公司(制霉菌素、5-氟胞嘧啶、氟康唑、酮康唑、伊曲康唑、伏立康唑、硝酸咪康唑、硝酸益康唑、特比萘芬、泊沙康唑、克霉唑、卡泊芬净)。
3.培养基：使用RPMI-1640培养基和马铃薯葡萄糖琼脂(PDA)培养基进行试验，具体材料包括：RPMI-1640液体培养基(来自于上海秦坦科技股份有限公司，商品编号C8016-500mL)；3-吗啉丙磺酸(来自于苏州亚科科技股份有限公司，编号M0001，缩写为MOPS，下同)；马铃薯葡萄糖肉汤(PDB)培养基粉末(来自于北京索莱宝科技有限公司，商品编号HB0129-2)；琼脂(上海碧云天生物技术有限公司，商品编号：#ST004D)。两种培养基的配方见下表2、3。
表2：RPMI-1640培养基成分
成分 含量(g/L)
D-葡萄糖 2.00
L-谷氨酰胺 0.30
碳酸氢钠 2.00
MOPS 34.53
表3：PDA培养基成分
成分 含量(g/L)
马铃薯浸粉 5.00
蛋白胨 10.00
葡萄糖 15.00
氯化钠 5.00
琼脂 17.50
根据试验需要，配制如下两种培养基：
RPMI-1640培养基混合缓冲剂：准确称量34.53gMOPS粉末溶解于1LRPMI-1640液体培养基，溶解后使用1M NaOH溶液调节pH值至7。
PDA培养基：准确称量35gPDB培养基粉末及17.5g琼脂粉溶解于1L超纯水。充分溶解后于高压蒸汽灭菌器中121℃高压灭菌20min，保存在4℃冰箱冷藏备用。
4.测试方法：使用美国临床实验室标准化协会CLSI-M27-A2(肉汤稀释法测定抗真菌药物敏感性)中的方法进行适当修改以进行白色念珠菌的相关测试(P.Wayne，CLSI document M27-A2，2002)；使用中华人民共和国行业标准-抗丝状真菌药物敏感性试验肉汤稀释法(WS/T411-2013)进行适宜性修改后进行烟曲霉的相关测试(WS/T 411-2013，抗丝状真菌药物敏感性试验肉汤稀释法[S])。
最低抑菌浓度(MIC)测定：使用微量肉汤稀释法进行测定，测试浓度为512-0.25μg/mL的2倍梯度浓度，每种菌对每种药物均测试5次重复及正负对照。
最低分级抑菌浓度(FIC)测定：对MIC＞0.5的抗真菌药物与抗菌多肽1(如下亦称样品)进行联合用药试验，使用微量肉汤棋盘法进行测定，在96孔板上分别测定样品与抗真菌药物的单用及两药联用时的抗菌作用效果，并计算部分抑菌浓度指数(FIC index)值。其FIC值≤0.5为协同，0.5-2为无关或相加，＞2为拮抗。FIC值计算方法如下所示，其中MIC*为联用时有抑菌效果的单一组分浓度。
Figure PCTCN2022134136-appb-000020
时间杀菌曲线(Time-Kill curve)测定：选定待测的有协同作用的样品-抗真菌药物组合后，将培养的真菌稀释至微量稀释法所需的菌液浓度，一份正常培养(无抗菌药物添加)，一份加入选定浓度的药物组合，于96孔板中同时培养，并于11个培养时间点(0、0.5、1、2、3、4、5、6、7、24、48h)时分别取出一组孔内菌液进行10 0-10 7的梯度稀释，并取10μL至PDA平板上，培养36-48h后进行菌落计数。
测试结果分析：
由于抗菌多肽1与各抗真菌药物联用组合的最低分级抑菌浓度不尽相同，为方便结果对比，统计了浓度为1/4MIC的抗菌多肽1与常见抗真菌药物联用时对抗耐药真菌的协同增效作用。
参阅表4、表5，根据试验结果可知，1/4MIC的抗菌多肽1与抗真菌药物在对烟曲霉作用时，前24h的协同效果最好，对常用抗真菌药物基本都有协同作用，可将其作用效果增强4-256倍；24h后协同作用虽有一定程度的衰减，但至48h时则仍有伊曲康唑、特比奈芬、5-氟胞嘧啶(5-FC)具有协同作用，增效作用为1-8倍。在所有联用组合中，抗菌多肽1对伊曲康唑的协同增效作用最高，所有FIC index值均小于2，即无拮抗作用产生。
表4：24h时抗菌多肽1在抗耐伊曲康唑的烟曲霉时对抗真菌药物的协同增效作用
Figure PCTCN2022134136-appb-000021
注：表4中MIC为最小抑菌浓度。抗菌多肽1：实验室合成样品抗菌多肽，测试浓度：1/4MIC。MIC*：联用有抗菌效果时单一组分的浓度。增强倍数：MIC/MIC*。FIC index：部分抑菌浓度指数。作用机理：1，抑制麦角甾醇合成，改变细胞膜通透性造成抑菌作用；1*：抑制麦角甾醇合成，改变细胞膜通透性导致抑菌作用，且造成角鲨烯堆积于胞浆中的脂滴，破坏细胞 膜导致杀菌作用；2，结合麦角甾醇，改变膜通透性导致真菌死亡；3，抑制胸腺嘧啶核苷合成酶，影响DNA及蛋白质合成以抑制真菌生长；4，抑制曲霉菌属和念珠菌属细胞壁合成的重要成份β(1，3)-D-糖苷合成酶导致抑菌及杀菌作用，下同。
表5：48h时抗菌多肽1在抗耐伊曲康唑的烟曲霉时对抗真菌药物的协同增效作用
Figure PCTCN2022134136-appb-000022
表6：48h时抗菌多肽1在抗耐药白色念珠菌时对抗真菌药物的协同增效作用
Figure PCTCN2022134136-appb-000023
如表6所示，在对白色念珠菌的试验中，抗菌多肽1与多种常见抗真菌药物联用时同样有较为明显的协同效果，其中唑类抗真菌药的协同抑制效果略好于制霉菌素。在具有协同作 用的药物组合中，药物作用48h时，抗真菌药物的MIC*值可降至MIC的1/4-1/512，证明抗菌多肽1可对抗真菌药物起良好的增效作用。
请参阅图22示出了伊曲康唑与抗菌多肽1联用对耐药烟曲霉的杀菌曲线。其中使用的菌株为耐伊曲康唑烟曲霉，编号ATCC MYA-3627；烟曲霉对照：RPMI1640+菌液。药物联用浓度：最低FIC index时组分浓度。可以看到，烟曲霉在有协同效果的两药联用作用下的抑菌效果。无联用药物作用时，显示为正常真菌时间生长曲线的趋势，于7h后进入对数期；加药物组主要是抑菌作用，孔内真菌数量随时间的增加而缓慢减少直至真菌浓度为300CFU/mL左右。
请参阅图23示出了伊曲康唑与抗菌多肽1联用对耐药的白色念珠菌的杀菌曲线。使用菌株：白色念珠菌，棘白菌素类及唑类耐药，编号ATCC 64124；白色念珠菌对照：RPMI1640+菌液。药物联用浓度：最低FIC index时组分浓度。可以看到，白色念珠菌也有类似的趋势，即对照组在6h左右进入对数期，整体呈标准曲线趋势；加药组在两种药物的协同作用下起着较为明显的抑制作用，约前3h将孔内真菌数量下降了一个数量级后，真菌浓度在后续45h一直稳定在300CFU/mL左右。
本测试例通过将抗菌多肽1与12种常用抗真菌药物进行体外联用，以测定其对两种耐药真菌的协同增效作用。结果表明，该抗菌多肽1可以有效克服真菌对抗真菌药物的耐药性，极大降低抗真菌药物的使用量。实验结果说明抗菌多肽与抗真菌药物联用时，其各自的抗菌机理起到相互促进的作用，使耐药真菌更加有效地被抑制。总体来说，抗菌多肽1在与多种抗真菌药物联用时均有良好的协同增效作用。
测试例3本测试例对抗菌多肽2～抗菌多肽9的性能进行测试，具体如下：
1.菌种：C.albicans(白色念珠菌，编号ATCC 64124)、A.fumigatus(烟曲霉，编号ATCC MYA-3627)、Trichophyton rubrum(红色毛癣菌，编号ATCC MYA-4438)、Epidermophyton floccosum(絮状表皮癣菌，编号ATCC52066)、Microsporum gypseum(石膏样小孢子菌，编号ATCC 14683)、Filobasidiella bacillispora(新生隐球菌，编号ATCC32609)、Histoplasmosis capsulati(荚膜组织胞浆菌，编号ATCC12700)、Fusarium moniliforme(串珠镰刀菌，编号ATCC 10052)、Rhizopus oryzae(米根霉，编号ATCC96382)、Penicillium marneffei(马尔尼菲青霉菌，编号ATCC24100)。
2.药物：抗菌多肽2～抗菌多肽9、两性霉素B、5-氟胞嘧啶、氟康唑、酮康唑、伊曲康唑、伏立康唑、硝酸咪康唑、米卡芬净(购自上海阿拉丁生化科技股份有限公司、北京索莱宝科技有限公司等)。
针对前述的各菌种，参照实施例1及本领域熟知的相应培养基和培养方法进行培养，之后采用与实施例1基本相同的方法进行测试。相应测试结果如下表7所示。
表7：48h时抗菌多肽2～抗菌多肽9与不同抗真菌药物对不同真菌的抑制效果
Figure PCTCN2022134136-appb-000024
Figure PCTCN2022134136-appb-000025
Figure PCTCN2022134136-appb-000026
Figure PCTCN2022134136-appb-000027
Figure PCTCN2022134136-appb-000028
Figure PCTCN2022134136-appb-000029
Figure PCTCN2022134136-appb-000030
Figure PCTCN2022134136-appb-000031
Figure PCTCN2022134136-appb-000032
Figure PCTCN2022134136-appb-000033
Figure PCTCN2022134136-appb-000034
Figure PCTCN2022134136-appb-000035
Figure PCTCN2022134136-appb-000036
Figure PCTCN2022134136-appb-000037
Figure PCTCN2022134136-appb-000038
可以看到，抗菌多肽2～抗菌多肽9抗菌多肽与常用抗真菌药物联合使用时，其也能有效克服耐药真菌对常用抗真菌药物的耐药性，且效果与测试例1的抗菌多肽接近。
此外，本申请人还参照前述抗菌多肽1～抗菌多肽9的制备方法，制备了本申请说明书中述及的其它抗菌多肽及其衍生物等，例如可药用的盐(包括盐酸盐、氢溴酸盐、乙酸盐等)，并也对其性能等进行了测试，结果显示，这些抗菌多肽及其衍生物与多种抗真菌药物联合使用，在降低真菌耐药性、增强抗真菌药物对耐药真菌的灭杀作用等方面均有优异表现。
应当理解，本申请的技术方案不限于上述具体实施案例的限制，凡是在不脱离本申请宗旨和权利要求所保护的范围情况下，根据本申请的技术方案做出的技术变形，均落于本申请的保护范围之内。

Claims (31)

  1. 一种通式(I)所示的化合物：
    Figure PCTCN2022134136-appb-100001
    其中，Core为具有环状基团的结构单元；
    R 1、R 2、R 3、R 4各自独立的选自H、卤素、取代或未取代的C1-C20烷基、取代或未取代的C1-C20烷氧基、取代或未取代的C2-C20烯基、取代或未取代的C2-C20炔基、取代或未取代的C3-C20脂环基、取代或未取代的C6-C15芳基、取代或未取代的C7-C30烷基-芳基、取代或未取代的C3-C20杂环、取代或未取代的C4-C30烷基-杂环、取代或未取代的C5-C15杂芳基、C1-C20羟烷基、氰基、氨基、胍基、硝基或者羟基；
    n为0～6，m为3～100，x、y为1～50。
  2. 如权利要求1所述的通式(I)所示的化合物，其特征在于：所述具有环状基团的结构单元包括取代或未取代的3～12元环烷基、取代或未取代的3～12元杂环基、取代或未取代的6～10元芳基或者取代或未取代的5～10元杂芳基、具有环状基团的有机大分子残基或者具有环状基团的无机化合物分子残基。
  3. 如权利要求2所述的通式(I)所示的化合物，其特征在于：所述具有环状基团的有机大分子残基包括环糊精分子残基。
  4. 如权利要求2所述的通式(I)所示的化合物，其特征在于：所述具有环状基团的无机化合物分子残基包括笼型聚倍半硅氧烷分子残基。
  5. 如权利要求2所述的通式(I)所示的化合物，其特征在于：所述具有环状基团的结构单元选自取代或未取代的苯基。
  6. 如权利要求5所述的通式(I)所示的化合物，其特征在于，所述化合物为通式(I-1)所示的化合物：
    Figure PCTCN2022134136-appb-100002
    R 5、R 6选自H、卤素、取代或未取代的C1-C20烷基、取代或未取代的C1-C20烷氧基、取代或未取代的C2-C20烯基、取代或未取代的C2-C20炔基、取代或未取代的C3-C20脂环基、取代或未取代的C6-C15芳基、取代或未取代的C7-C30烷基-芳基、取代或未取代的C3-C20杂环、取代或未取代的C4-C30烷基-杂环、取代或未取代的C5-C15杂芳基、C1-C20羟烷基、氰基、氨基、胍基、硝基或者羟基。
  7. 如权利要求6所述的通式(I)所示的化合物，其特征在于：R 1、R 2、R 3各自独立的选自C1-C6烷基、羟基或胺基。
  8. 如权利要求6所述的通式(I)所示的化合物，其特征在于：R 4、R 5、R 6为氢原子。
  9. 如权利要求6所述的通式(I)所示的化合物，其特征在于，所述化合物为下式所示的化合物：
    Figure PCTCN2022134136-appb-100003
  10. 如权利要求1-9中任一项所述通式(I)所示的化合物的立体异构体、互变异构体、N-氧化物、水合物、溶剂化物或可药用盐。
  11. 一种星型β-抗菌糖肽，其特征在于，所述星型β-抗菌糖肽的外层为螺旋结构的糖肽嵌段，内层为带正电荷的聚二甲基氨基贝塔内酰胺嵌段。
  12. 如权利要求11所述的星型β-抗菌糖肽，其特征在于，所述糖肽嵌段的来源包括单糖葡萄糖、半乳糖、甘露糖、阿卓糖、胺基葡萄糖、胺基半乳糖、胺基甘露糖或双糖麦芽糖。
  13. 如权利要求11或12所述的星型β-抗菌糖肽，其特征在于，所述星型β-抗菌糖肽的结构如通式(I)所示：
    Figure PCTCN2022134136-appb-100004
    其中，Core为具有环状基团的结构单元；
    R 1、R 2、R 3、R 4各自独立的选自H、卤素、取代或未取代的C1-C20烷基、取代或未取代的C1-C20烷氧基、取代或未取代的C2-C20烯基、取代或未取代的C2-C20炔基、取代或未取代的C3-C20脂环基、取代或未取代的C6-C15芳基、取代或未取代的C7-C30烷基-芳基、取代或未取代的C3-C20杂环、取代或未取代的C4-C30烷基-杂环、取代或未取代的C5-C15杂芳基、C1-C20羟烷基、氰基、氨基、胍基、硝基或者羟基；
    n为0～6，m为3～100，x、y为1～50。
  14. 一种制备权利要求1所述通式(I)所示化合物的方法，其特征在于，所述方法包括：
    (i)依据反应式a，使包含Core的前驱体化合物与式(II)所示的化合物、式(III)所示的化合物发生聚合反应，获得式(IV)所示的聚合物；
    Figure PCTCN2022134136-appb-100005
    (ii)依据反应式b，对式(IV)所示的聚合物进行脱保护
    Figure PCTCN2022134136-appb-100006
  15. 如权利要求14所述的方法，其特征在于，步骤(a)包括：在双(三甲基甲硅烷基)氨基锂存在下制备所述保护聚合物；
    和/或，步骤(b)包括：在钠和液氨条件下使所述保护聚合物脱保护。
  16. 一种药物组合物，其特征在于，包含：
    权利要求1-9中任一项所述通式(I)所示的化合物或其可药用盐，
    或者，权利要求11-13中任一项所述星型β-抗菌糖肽；以及
    药学上可接受的载体。
  17. 如权利要求16所述的药物组合物，其特征在于：所述药物组合物还包含其它抗菌剂。
  18. 如权利要求17所述的药物组合物，其特征在于：所述其它抗菌剂包括氨苄西林、氯唑西林、苯唑西林、哌拉西林、头孢菌素、碳青霉烯类、糖肽类、大环内酯类、喹诺酮类、四环素类、氨基糖苷类、利福平、环丙沙星、左氧氟沙星、哌拉西林、复方新诺明、庆大霉素、洛布霉素、琥乙红霉素、红霉素、克拉霉素、新生霉素、螺旋霉素、乙酰螺旋霉素、氯 霉素、甲氧苄啶、磺胺甲恶唑、羧苄青霉素、多粘菌素B、粘菌素、阿米卡星、卡那霉素、新霉素、奈替米星、链霉素、妥布霉素、巴龙霉素，格尔德霉素、除莠霉素、氯碳头孢、多利培南、西司他丁、头孢羟氨苄、头孢噻吩、头孢氨苄、头孢孟多、头孢丙烯、头孢呋辛、头孢克肟、头孢地尼、头孢托仑、头孢泊肟、头孢布烯、头孢唑肟、头孢吡肟、替考拉宁、万古霉素、罗红霉素、醋竹桃霉素、泰利霉素、大观霉素、阿莫西林、羧苄西林、双氯西林、氟氯西林、美洛西林、甲氧西林、萘夫西林、青霉素、替卡西林、杆菌肽、依诺沙星、加替沙星、莫西沙星、诺氟沙星、曲伐沙星、磺胺米隆、偶氮磺胺、磺胺醋酰、磺胺甲二唑、磺胺、柳氮磺吡啶、磺胺异噁唑、甲氧苄啶磺胺甲噁唑、地美环素、多西环素、米诺环素、土霉素、四环素、胂凡纳明、克林霉素、林可霉素、乙胺丁醇、磷霉素、夫西地酸、呋喃唑酮、异烟肼、利奈唑胺、双唑泰栓、莫匹罗星、呋喃妥因、吡嗪酰胺、奎奴普丁/达福普汀、异福酰胺、替硝唑中的任意一种或多种的组合；所述头孢菌素包括头孢克洛、头孢孟多、头孢唑林、头孢哌酮、头孢噻肟、头孢西丁、头孢他啶、头孢曲松中的任意一种或多种的组合；所述碳青霉烯类包括亚胺培南和美罗培南中的任意一种或多种的组合。
  19. 权利要求1-9中任一项所述通式(I)所示的化合物或其可药用盐或者权利要求16-18中任一项所述药物组合物作为抗菌剂的用途。
  20. 根据权利要求19所述的用途，其特征在于：所述药物组合物所包含的其它抗菌剂包括抗革兰氏阳性菌抗生素，而所述抗菌剂的抗菌范围包括革兰氏阴性菌。
  21. 权利要求1-9中任一项所述通式(I)所示的化合物或其可药用盐、权利要求11-13中任一项所述星型β-抗菌糖肽或者权利要求16-18中任一项所述药物组合物在制备治疗或预防细菌感染或由所述细菌感染引起的疾病的药物中的用途。
  22. 如权利要求21所述的用途，其特征在于：所述细菌包括具有多重耐药性细菌、敏感细菌中的至少一种。
  23. 权利要求1-9任一项所述通式(I)所示的化合物或其可药用盐、权利要求11-13中任一项所述星型β-抗菌糖肽或者权利要求16-18中任一项所述药物组合物作为细胞外排泵抑制剂和/或细菌膜透化剂的用途。
  24. 权利要求1-9中任一项所述通式(I)所示的化合物或其可药用盐或者权利要求11-13中任一项所述星型β-抗菌糖肽在制备预防或治疗真菌感染的药物中的用途。
  25. 如权利要求24所述的用途，其特征在于，所述的用途包括：将所述抗菌多肽与抗真菌药物联用，以预防或治疗真菌感染或真菌感染引起的疾病。
  26. 如权利要求24-25中任一项所述的用途，其特征在于：所述真菌包括药物敏感性真菌、药物中等性真菌或耐药性真菌。
  27. 如权利要求26所述的用途，其特征在于：所述真菌为耐药性真菌。
  28. 如权利要求25所述的用途，其特征在于：所述抗真菌药物包括唑类、多烯类、氟胞嘧啶类或棘白菌素类抗真菌药物。
  29. 权利要求1-9中任一项所述通式(I)所示的化合物或其可药用盐或者权利要求11-13中任一项所述星型β-抗菌糖肽在制备抗真菌药物增效剂中的用途。
  30. 如权利要求29所述的用途，其特征在于：所述抗真菌药物包括唑类、多烯类、氟胞嘧啶类或棘白菌素类抗真菌药物。
  31. 一种抗真菌药物组合物，其特征在于包括：
    权利要求1-9中任一项所述通式(I)所示的化合物或其可药用盐，
    或者，权利要求11-13中任一项所述星型β-抗菌糖肽；以及
    药学上可接受的载体；
    抗真菌药物，所述抗真菌药物包括唑类、多烯类、氟胞嘧啶类或棘白菌素类抗真菌药物。
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CN107073020A (zh) * 2014-08-22 2017-08-18 贝尔维特格生物医学研究所（Idibell） 预防和/或治疗感染的方法和试剂
CN110452374A (zh) * 2019-08-30 2019-11-15 苏州大学 具有高效基因递送能力的三维球形α螺旋阳离子聚多肽及其制备方法与应用
WO2021087021A1 (en) * 2019-10-29 2021-05-06 Wisconsin Alumni Research Foundation Peptide-nanoparticle conjugates
CN115087465A (zh) * 2019-10-29 2022-09-20 威斯康星校友研究基金会 肽-纳米颗粒缀合物

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