WO2020119773A1 - 两性霉素b肽衍生物 - Google Patents

两性霉素b肽衍生物 Download PDF

Info

Publication number
WO2020119773A1
WO2020119773A1 PCT/CN2019/124970 CN2019124970W WO2020119773A1 WO 2020119773 A1 WO2020119773 A1 WO 2020119773A1 CN 2019124970 W CN2019124970 W CN 2019124970W WO 2020119773 A1 WO2020119773 A1 WO 2020119773A1
Authority
WO
WIPO (PCT)
Prior art keywords
gly
aeeac
pharmaceutically acceptable
acceptable salt
compound according
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/CN2019/124970
Other languages
English (en)
French (fr)
Inventor
东圆珍
冯军
张喜全
张金华
赵文杰
朱冰
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shanghai Duomirui Biotechnology Ltd
Shanghai Institute of Pharmaceutical Industry
Chia Tai Tianqing Pharmaceutical Group Co Ltd
Original Assignee
Shanghai Duomirui Biotechnology Ltd
Shanghai Institute of Pharmaceutical Industry
Chia Tai Tianqing Pharmaceutical Group Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Shanghai Duomirui Biotechnology Ltd, Shanghai Institute of Pharmaceutical Industry, Chia Tai Tianqing Pharmaceutical Group Co Ltd filed Critical Shanghai Duomirui Biotechnology Ltd
Priority to CN201980079769.8A priority Critical patent/CN113166188B/zh
Publication of WO2020119773A1 publication Critical patent/WO2020119773A1/zh
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Images

Classifications

    • 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
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/10—Antimycotics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H17/00—Compounds containing heterocyclic radicals directly attached to hetero atoms of saccharide radicals
    • C07H17/04—Heterocyclic radicals containing only oxygen as ring hetero atoms
    • C07H17/08—Hetero rings containing eight or more ring members, e.g. erythromycins
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • the invention belongs to the field of medicine, and relates to amphotericin B peptide derivatives, preparation methods and applications thereof, and specifically relates to a series of amphotericin B peptide derivatives with high solubility, low toxicity and good antibacterial activity And its synthetic preparation and application.
  • Amphotericin B is a polyene broad-spectrum antifungal drug, suitable for the treatment of the following fungal infection diseases: Candidiasis, Cryptococcosis, Blastomycosis ), coccidioidomycosis, mucormycosis caused by Mucor, sporothrichosis caused by Sporothrix, and most of Aspergillus ) Caused by aspergillosis (aspergillosis) and so on.
  • amphotericin B Since the amphotericin B was isolated from metabolites of Streptomyces in 1955, this compound has attracted much attention.
  • amphotericin B is the gold standard for clinical treatment of deep fungal infections and systemic systemic infections, and it is the only effective therapeutic drug for certain fatal systemic fungal infections; on the other hand, at therapeutic doses, Amphotericin B has serious toxic and side effects, such as hemolytic toxicity, nephrotoxicity, neurotoxicity, etc., and amphotericin B has very poor water solubility. After oral administration of this product, it is less and unstable from the gastrointestinal tract. The application of amphotericin B is greatly restricted.
  • amphotericin B liposomes are made of vesicles formed by phospholipid bilayer membranes wrapped with drug molecules and targeted
  • the new drug with drug delivery function is better tolerated than ordinary preparations. On the one hand, it can be distributed more in the liver, spleen, and lungs, while in other organs, especially in kidney tissue, the concentration is lower.
  • the cholesterol component in liposomes can reduce the binding of drugs to cholesterol in human cells and enhance the binding of ergosterol to fungal cells, with relatively few side effects on the kidneys and the like.
  • amphotericin B liposome preparations also have the following disadvantages: First, the antibacterial activity of liposome preparations is inferior to amphotericin B, and the therapeutic dose needs to be increased. Second, the cost of liposome preparations is higher. The price is relatively expensive. 3. The instability of the liposome itself. 4. The liposome preparation has not fundamentally eliminated the nephrotoxicity and side effects of amphotericin B.
  • amphotericin B used as the lead compound, a solid phase and liquid phase combined experimental method ,Peptide reaction of amphotericin B to synthesize a series of amphotericin B peptide derivatives, including H-(Gly)n-OH or hydrophilicity formed by combining AEEAc and Gly (n is an integer, the range is 2-10) Series of amphotericin B peptide derivatives, while retaining their antibacterial activity, improve their water solubility, and even further reduce their hemolytic toxicity, nephrotoxicity and other toxic side effects.
  • the present invention relates to amphotericin B peptide derivatives and their synthetic preparation methods and applications, in particular to improving the solubility of amphotericin B and reducing the toxicity of amphotericin B, while retaining the antibacterial activity of amphotericin B Amphotericin B peptide derivative and its synthetic preparation method and application.
  • amphotericin B peptide derivative specifically refers to the synthesis of R 2 -(Gly)n-OH or AEEAc combined with Gly by using R 2 -AEEAc-OH and R 2- Gly-OH as raw materials and adopting the solid phase synthesis method
  • the resulting series of compounds are then coupled with amphotericin B through an amide bond, and finally the amino protecting group is removed, and then purified to obtain the target compound.
  • the invention relates to compounds of general formula [I] or derivatives thereof:
  • R 1 is a hydrophilic polymer part
  • R 2 is an amino protecting group such as Fmoc, Boc or H
  • R 3 is H or C 1-4 hydrocarbon group or phenyl
  • R 4 is OH or H
  • the hydrophilic polymer part may be a H-(Gly)n-OH polymer part containing H-Gly-OH monomer, where n takes an integer of 2-20, preferably n takes 2 -15 integer, optimally, n is 5
  • the hydrophilic polymer part may also be a polypeptide part composed of Gly and AEEAc through a peptide bond, the length of the polypeptide part is 2-20 peptides, preferably 2- 15 peptides, optimally, 5 peptides.
  • the present invention relates to the synthetic preparation of amphotericin B peptide derivatives.
  • the present invention relates to the derivative having improved anti-fungal activity while improving solubility and even further reducing toxicity.
  • the present invention relates to water-soluble polypeptide compounds, including but not limited to H-(Gly)n-OH or a polypeptide composed of Gly and AEEAc through a peptide bond.
  • amphotericin B derivative referred to in the present invention is connected by an amide bond.
  • the amphotericin B derivative mentioned in the present invention comprises: a water-soluble polypeptide part, which is connected to the sugar amine structure on the amphotericin B through a stable amide bond, so this type of compound It can be referred to as amphotericin B polypeptide derivative (DTY-AMB) for short.
  • the water-soluble polypeptide refers to a polypeptide in which H-(Gly)n-OH or AEEAc is combined with Gly
  • the water-soluble polypeptide part refers to a part formed by removing the -OH and -H from the terminal amino acid in the corresponding polypeptide .
  • DTY-AMB also contains a class of derivatives where the carboxyl group on C 16 is esterified, such as hydrocarbyl esters.
  • T is a polymer containing Gly or Gly derivatives, or a combination polypeptide composed of AEEAc and Gly and derivatives thereof.
  • R 1 is selected from -(Gly)n-, -(AEEAc)n-Gly-, -(AEEAc-Gly)n-AEEAc-, -AEEAc-Gly-, -(AEEAc-Gly)n -, -(Gly-AEEAc)n-Gly-, -(AEEAc)n-Gly-(AEEAc)m-, -(AEEAc)n-(Gly)m- and -(Gly)n-AEEAc-(Gly) m-, where n and m are each independently an integer of 2-9.
  • R 1 is selected from -(AEEAc)n-Gly-, -(AEEAc-Gly)n-AEEAc-, -(AEEAc-Gly)n-, -(Gly-AEEAc)n-Gly- and -(AEEAc ) n-Gly-(AEEAc)m-, wherein n and m are each independently an integer of 2-9 and at the same time satisfy the number of AEEAc in R 1 is greater than or equal to 3.
  • R 2 is H.
  • R 3 is H.
  • R 2 is an amino protecting group such as Fmoc, Boc or H
  • R 3 is H or C 1-4 hydrocarbon group or phenyl
  • R 4 is OH or H
  • the hydrophilic polypeptide part may be T (T is composed of Gly or a combination of AEEA and Gly), where the length of T is 2-10 peptides, preferably 3-7 peptides, and most preferably 5 peptides.
  • the hydrophilic polypeptide part of DTY-AMB has different lengths, and may contain 2-20 monomers, preferably, 2-10 monomers, and optimally, 5 monomers, In the present invention, DTY-AMB may contain 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 monomers .
  • the monomer may be Gly, or Gly and AEEAc.
  • the hydrophilic polypeptide part of DTY-AMB is T, and T has different lengths, and may contain 2-10 monomers, preferably, 3-7 monomers, and optimally, 5 Monomers, in some specific embodiments, DTY-AMB may contain 2, 3, 4, 5, 6, 7, 8, 9, 10 monomers.
  • the monomer may be Gly, or Gly and AEEAc.
  • the C 16 position of DTY-AMB contains a free carboxyl group or a hydrocarbyl ester of carboxyl group, including methyl ester, ethyl ester, propyl ester, butyl ester, phenyl ester and the like.
  • the DTY-AMB containing the hydrophilic polypeptide part T has good bacteriostatic activity, and at the same time, it is not easily hydrolyzed by enzymes because it is connected by an amide bond.
  • the invention relates to compounds of formula [II] or derivatives thereof, wherein: R 3 is H; R 2 -T- is selected from:
  • the present invention relates to compounds of formula [II] or derivatives thereof, wherein R 3 is H; R 2 -T- is H-AEEAc-XYZ-AEEAc-; X is AEEAc or Gly, and Y is AEEAc Or Gly, and Z is AEEAc or Gly.
  • the invention relates to compounds of formula [III] or derivatives thereof:
  • the invention relates to compounds of formula [IV] or derivatives thereof:
  • the compound of the present invention is a compound of the following formula or a derivative thereof, wherein the peptide modification site may be an amino group in AMB, that is, an amino group of a glycosamino group:
  • the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising the above compound or a pharmaceutically acceptable salt thereof.
  • the present invention relates to a method for preventing and/or treating fungal infections in a subject in need thereof, wherein the subject is administered a therapeutically effective amount of the above compound or a pharmaceutically acceptable salt thereof.
  • the fungal infection is selected from fungal infections such as Cryptococcus, Blastomyces dermatitis, Candida albicans, Candida krusei, and Candida parapsilosis, Coccidioides immitis, Mucor, Sporothrix schenckii, and Aspergillus fumigatus.
  • the subject has Cryptococcosis, Blastomycosis, Candidiasis, Coccidioidomycosis, mucormycosis caused by Mucor , Sporrotrichosis caused by Sporothrix, aspergillosis caused by most Aspergillus.
  • the present invention relates to the use of the above compounds or pharmaceutically acceptable salts thereof for the preparation of medicaments, such as antifungal drugs.
  • the fungus is selected from, for example, Cryptococcus, dermatitis, Candida albicans, Candida krusei, Candida parapsilosis, Coccidioides sphaeroides, Mucor spp., S. sclerotiorum and Aspergillus fumigatus.
  • the above-mentioned compound of the present invention or a pharmaceutically acceptable salt thereof has considerable antifungal activity, and has improved solubility, and may even further have reduced toxicity.
  • the present invention relates to the above compounds or pharmaceutically acceptable salts thereof for preventing and/or treating fungal infections.
  • the fungal infection is selected from the group of fungal infections such as Cryptococcus, Blastodermatitis, Candida albicans, Candida krusei, Candida parapsilosis, Coccidioides, Mucor, Mycet Schenck or Aspergillus fumigatus.
  • the above compound or a pharmaceutically acceptable salt thereof is used to treat diseases caused by fungal infections selected from the group consisting of cryptococcosis, blastomycosis, candidiasis, coccidioidomycosis, and mucormycosis caused by mucormycosis , Spore hyphae caused by spore hyphae, aspergillosis caused by most aspergillus.
  • diseases caused by fungal infections selected from the group consisting of cryptococcosis, blastomycosis, candidiasis, coccidioidomycosis, and mucormycosis caused by mucormycosis , Spore hyphae caused by spore hyphae, aspergillosis caused by most aspergillus.
  • the above-mentioned compound of the present invention or a pharmaceutically acceptable salt thereof has considerable antifungal activity, and has improved solubility, and may even further have reduced toxicity.
  • the present invention relates to a method for preparing the above compound or a pharmaceutically acceptable salt thereof.
  • the method includes: (1) solid-phase synthesis of a polypeptide on a resin, cleaving the resulting polypeptide product with a weak acid, and filtering, Rotate steam, then add the first organic solvent to dissolve, spin steam, precipitate with the second organic solvent, and dry to obtain the polypeptide containing the amino protecting group; (2) Activate the polypeptide containing the amino protecting group, and then in anhydrous The reaction with amphotericin B in the solvent is carried out in the presence of a catalyst amount of base; and optionally, (3) the amino protecting group of the polypeptide portion containing the amino protecting group is removed.
  • the weak acid described in step (1) includes but is not limited to trifluoroethanol, and the weak acid can be formulated with dichloromethane at 1:4 (V/V) as a weak acid solution.
  • the first organic solvent in step (1) may be selected from DCM and THF.
  • the second organic solvent in step (1) may be selected from diethyl ether, isopropyl ether, and methyl tert-butyl ether.
  • the anhydrous solvent described in step (2) is selected from DMF and DMSO.
  • the base described in step (2) includes but is not limited to N,N-diisopropylethylamine (DIEA).
  • DIEA N,N-diisopropylethylamine
  • a removal agent selected from the following group is used to remove the amino protecting group: piperidine (PIP) solution, preferably 10 wt% to 40 wt% PIP in DMF solution, more preferably 20 wt% to 25 wt% PIP Of DMF solution.
  • PIP piperidine
  • the invention relates to a compound selected from the following or a pharmaceutically acceptable salt thereof:
  • HT- is any one selected from the following:
  • the main pathogen of deep fungal infections is still Candida albicans, and the phenomenon of drug resistance is also the most prominent. Therefore, the prevention and treatment of deep infections of Candida albicans is also the focus of the research field of antifungal infections.
  • the water-soluble polymer part T DTY-AMB has a good antibacterial effect on Candida albicans.
  • DTY-AMB can be in the form of a pharmaceutically acceptable salt.
  • DTY-AMB can be used as an effective pharmaceutical ingredient for oral preparations; it can also be used as an effective pharmaceutical ingredient for injections, such as intravenous injection, subcutaneous injection, intramuscular injection, etc.; and can also be used as an effective pharmaceutical ingredient for topical medications.
  • DTY-AMB can be made into an effective dosage unit of medicine through the existing medical technology.
  • the effective dosage unit can be in the form of oral, tablet, capsule or liquid.
  • the pharmaceutical ingredient can be made into a preparation containing water, wherein the water content is not less than 50%.
  • Oral preparations can be in the form of liquids, suspensions, powders, tablets, and capsules; tablets containing various excipients (such as calcium carbonate, calcium phosphate, etc.) can also be made into disintegrating preparations.
  • excipients such as calcium carbonate, calcium phosphate, etc.
  • the pharmaceutical ingredients can be released in a controlled manner, including slow release or rapid release, and the controlled release dosage of the relevant pharmaceutical ingredients can be achieved by known pharmaceutical techniques.
  • the pharmaceutical composition may contain 0.1%-99.9% DTY-AMB (DTY-AMB is calculated by weight), and the optimal content is 1%-70%.
  • Figure 3 is the general route of DTY-AMB chemical synthesis.
  • the chemical synthesis of DTY-AMB includes the first, second and third steps below.
  • the first step includes solid phase synthesis of polypeptide R 2 -T and activation of the series of polypeptides.
  • the present invention provides a method for synthesizing the series of polypeptides. In some aspects, the method includes: synthesizing the series of polypeptides. In some schemes, solid-phase synthesis technology can be used to prepare the series of polypeptides, including:
  • step (1) The product of step (1) is cleaved with a weak acid, filtered, and rotary evaporation, then the appropriate amount of organic solvent is added to dissolve the peptide, rotary evaporation is repeated, repeated 2-3 times, and finally the organic solvent is precipitated and dried to obtain the target polypeptide .
  • the step (1) includes the following steps:
  • the amino protecting group includes but is not limited to tert-butoxycarbonyl (Boc), benzyloxycarbonyl (Z) or 9-fluorenyl-methylcarbonyl (Fmoc), preferably 9-fluorenyl-methylcarbonyl (Fmoc), etc.
  • the protective group includes but is not limited to this, and a reasonable choice can be made according to specific circumstances.
  • an appropriate amount of R 2 -AEEAc-OH or R 2 -Gly-OH is weighed and an appropriate amount of high steric hindered base is taken, added with DCM, for example, 10 mL of DCM to dissolve, and then put into the reactor.
  • the high steric hindrance reagents include but are not limited to N,N-diisopropylethylamine (DIEA).
  • an amino protecting group removing agent for example 1mL 20% PIP/DMF
  • the solvent used in the liquid phase environment of the step (a) is selected from dimethylformamide (DMF), dichloromethane (DCM), N-methylpyrrolidone (NMP), preferably DCM and DMF.
  • DMF dimethylformamide
  • DCM dichloromethane
  • NMP N-methylpyrrolidone
  • the removal agent of the amino protecting group needs to be added with a removal agent of the amino protection group.
  • the removal agent of the amino protection group is a piperidine (PIP) solution with a concentration of 10%-40% (PIP/DMF).
  • PIP piperidine
  • the removal time is 20-50 min; the preferred concentration is 20%-25% (PIP/DMF) and the removal time is 25-35 min.
  • the coupling of the amino acid in the step (a) requires the addition of a coupling reagent.
  • the coupling reagent is composed of a carbodiimide type reagent or a benzotriazolium salt type reagent and 1-hydroxybenzotriazole (HOBt).
  • the carbodiimide type reagents include but are not limited to dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC) or N-diaminopropyl-N-ethylcarbodiimide (EDC).
  • DCC dicyclohexylcarbodiimide
  • DIC diisopropylcarbodiimide
  • EDC N-diaminopropyl-N-ethylcarbodiimide
  • the benzotriazonium salt type reagents include but are not limited to 2-(1H-benzotriazo L-1-yl)-1,1,3,3-tetramethylurea tetrafluoroborate (TBTU), O-benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate (HBTU), hexafluorophosphate benzotriazole-1-oxytris(dimethylamino) Phosphorus (BOP) or hexafluorophosphate benzotriazol-1-yl-oxytripyrrolidinyl phosphorus (PyBOP).
  • 2-(1H-benzotriazo L-1-yl)-1,1,3,3-tetramethylurea tetrafluoroborate TBTU
  • HBTU O-benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate
  • BOP hexaflu
  • the coupling reagent is preferably diisopropylcarbodiimide (DIC) and 1-hydroxybenzotriazole (HOBt), or 2-(1H-benzotriazo L-1-yl)-1,1 , 3,3-tetramethylurea tetrafluoroborate (TBTU) and 1-hydroxybenzotriazole (HOBt), further preferably DIC (diisopropylcarbodiimide) and 1-hydroxybenzotriazole (HOBt).
  • DIC diisopropylcarbodiimide
  • HOBt 2-(1H-benzotriazo L-1-yl)-1,1 , 3,3-tetramethylurea tetrafluoroborate
  • TBTU 3,3-tetramethylurea tetrafluoroborate
  • TBTU 3,3-tetramethylurea tetrafluoroborate
  • TBTU 3,3-tetramethylurea tetrafluoroborate
  • HBt 1-hydroxybenz
  • the "monitoring" in the step (a) uses a ninhydrin detection method to monitor the condensation reaction of the polypeptide.
  • the sequential coupling of amino acids in the step (a) refers to linking amino acids one by one from the C-terminus to the N-terminus according to the amino acid sequence of the polypeptide.
  • the weak acid described in step (2) includes but is not limited to trifluoroethanol, and the weak acid can be formulated with dichloromethane at 1:4 (V/V) as a weak acid solution.
  • the other part of the reaction in the first step is the activation of a polypeptide containing an amino protecting group, wherein the polypeptide T is a 2-10 peptide, preferably a 3-7 peptide, and most preferably a 5 peptide.
  • the activation of the polypeptide carboxyl group includes: activated ester method, symmetric anhydride method, azide method, etc., preferably a milder activated ester method; used activation reagent: carbodiimide type reagent or benzotriazonium salt type reagent It is composed of 1-hydroxybenzotriazole (HOBt) or succinimide (HOSU).
  • the carbodiimide type reagents include but are not limited to dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC) or N-diaminopropyl-N-ethylcarbodiimide (EDC).
  • DCC dicyclohexylcarbodiimide
  • DIC diisopropylcarbodiimide
  • EDC N-diaminopropyl-N-ethylcarbodiimide
  • the benzotriazonium salt type reagents include but are not limited to 2-(1H-benzotriazo L-1-yl)-1,1,3,3-tetramethylurea tetrafluoroborate ( TBTU), O-benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate (HBTU), hexafluorophosphate benzotriazole-1-oxytris(dimethylamino) Phosphorus (BOP) or hexafluorophosphate benzotriazol-1-yl-oxytripyrrolidinyl phosphorus (PyBOP), preferably diisopropylcarbodiimide (DIC) and hydroxysuccinimide (HOSU);
  • the solvent in the liquid phase system used for activation is preferably an organic solvent, including DMF, DMSO, DCM, THF, etc., preferably THF (tetrahydrofuran).
  • the amino protecting group includes but is not limited to tert-butoxycarbonyl (Boc), benzyloxycarbonyl (Z) or 9-fluorenyl-methylcarbonyl (Fmoc), preferably 9-fluorenyl-methylcarbonyl (Fmoc);
  • the activation of the ester is at 30°C for 1-2 hours.
  • the addition of a catalyst amount of base is beneficial to complete activation.
  • the base reagents include but are not limited to N,N-diisopropylethylamine (DIEA); after rotary evaporation, Compound 2 can be obtained by removing the organic solvent.
  • DIEA N,N-diisopropylethylamine
  • compound 2 is reacted with amphotericin B in an anhydrous solvent such as DMF and DMSO at room temperature for 1-2 hours.
  • the reaction requires protection from light.
  • a catalyst amount of base is beneficial to complete the reaction.
  • the base reagents include but are not limited to N,N-diisopropylethylamine (DIEA); compound 4 and compound 4 can be obtained
  • DIEA N,N-diisopropylethylamine
  • compound 4 and compound 4 can be obtained
  • Preparative purification can be performed by semi-preparative RP-HPLC.
  • the third step is to perform a deamino protecting group on the polypeptide containing an amino protecting group.
  • the amino protecting group includes but is not limited to tert-butoxycarbonyl (Boc), benzyloxycarbonyl (Z) or 9-fluorenyl-methylcarbonyl (Fmoc), preferably 9-fluorenyl-methylcarbonyl (Fmoc); removal of the amino protecting group requires the addition of an amino protecting group removing agent.
  • the amino protecting group removing agent is piperidine (PIP) solution with a concentration of 10% -40% (PIP/DMF), the removal time is 20-50min; the preferred concentration is 20%-25% (PIP/DMF), the removal time is 25-35min.
  • 5-deoxyamphophilic B can also be used as a starting material instead of amphotericin B, 5-deoxyamphophilic B, that is, the group OH at the R 4 position of amphotericin B becomes H; related 5- Deoxyamphophilic B can be synthesized.
  • the preparation method of the amphotericin B peptide derivative provided by the present invention may further include a purification step.
  • the purification method used includes but is not limited to reverse phase chromatography or ion exchange chromatography, preferably reverse phase chromatography.
  • the in vitro antibacterial activity of the amphotericin B peptide derivative of the present invention can be identified by measuring its minimum inhibitory concentration (MIC).
  • MIC minimum inhibitory concentration
  • NCCLS National Committee for Clinical Laboratory Standardization
  • Amphotericin B was used as a positive control.
  • the in vitro activity measurement shows that the amphotericin B peptide derivative provided by the present invention has good activity against Candida albicans.
  • ethyl is "optionally” substituted with halogen, meaning that ethyl can be unsubstituted (CH 2 CH 3 ), mono-substituted (such as CH 2 CH 2 F), poly-substituted (such as CHFCH 2 F, CH 2 CHF 2 etc.) or completely substituted (CF 2 CF 3 ).
  • halogen meaning that ethyl can be unsubstituted (CH 2 CH 3 ), mono-substituted (such as CH 2 CH 2 F), poly-substituted (such as CHFCH 2 F, CH 2 CHF 2 etc.) or completely substituted (CF 2 CF 3 ).
  • C mn means that there are mn carbon atoms in this part.
  • carbon 3-10 cycloalkyl means that the cycloalkyl has 3-10 carbon atoms.
  • Carbon 0-6 alkylene means that the alkylene has 0-6 carbon atoms, and when the alkylene has 0 carbon atoms, the group is a bond.
  • C 1-6 means that the group may have 1 carbon atom, 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms, or 6 carbon atoms.
  • any variable (such as R) appears more than once in the composition or structure of a compound, its definition in each case is independent. So, for example, if a group is replaced by 2 Rs, then each R has an independent option.
  • halo or halogen refers to fluorine, chlorine, bromine and iodine.
  • acyl refers to a -CO- group.
  • hydroxyl refers to the -OH group.
  • amino refers to the -NH 2 group.
  • alkyl refers to a hydrocarbon group of the formula C n H 2n +.
  • the alkyl group may be linear or branched.
  • hydrocarbon 1-6 alkyl refers to a monovalent straight or branched chain aliphatic group containing 1 to 6 carbon atoms (eg, methyl, ethyl, n-propyl, isopropyl, n-butyl, Isobutyl, sec-butyl, tert-butyl, n-pentyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl, neopentyl, 3,3-dimethylpropyl , Hexyl, 2-methylpentyl, etc.).
  • the alkyl portion (ie, alkyl) of alkoxy, alkylamino, dialkylamino, alkylsulfonyl, and alkylthio groups has the same definition as above.
  • alkoxy refers to -O-alkyl.
  • alkylthio refers to -S-alkyl.
  • alkylamino refers to -NH (alkyl).
  • cycloalkyl refers to a carbocyclic ring that is fully saturated and can exist as a single ring, a condensed ring, or a spiro ring. Unless otherwise indicated, the carbocyclic ring is usually a 3 to 10 membered ring, preferably a 3 to 8 membered ring.
  • Non-limiting examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, norbornyl (bicyclo[2.2.1]heptyl), bicyclo[2.2.2]octyl, adamant Alkyl and so on.
  • aryl refers to an all-carbon monocyclic or fused polycyclic aromatic ring group having a conjugated ⁇ electron system.
  • the aryl group may have 6-20 carbon atoms, 6-14 carbon atoms, or 6-12 carbon atoms.
  • Non-limiting examples of aryl groups include, but are not limited to, phenyl, naphthyl, anthracenyl, and the like.
  • heteroaryl refers to a monocyclic or fused polycyclic ring system, which contains at least one ring atom selected from N, O, S, and the remaining ring atoms are C, and has at least one aromatic ring.
  • Preferred heteroaryl groups have a single 4 to 8 membered ring, especially 5 to 8 membered ring, or have multiple fused rings containing 6 to 14, especially 6 to 10 ring atoms.
  • heteroaryl groups include, but are not limited to, pyrrolyl, furyl, thienyl, imidazolyl, oxazolyl, pyrazolyl, pyridyl, pyrimidinyl, pyrazinyl, quinolinyl, isoquinolinyl , Tetrazolyl, triazolyl, triazinyl, benzofuranyl, benzothienyl, indolyl, isoindolyl, etc.
  • substituted means that any one or more hydrogen atoms on a specific atom are replaced with a substituent, as long as the valence state of the specific atom is normal and the compound after substitution is stable.
  • pharmaceutically acceptable refers to those compounds, materials, compositions and/or dosage forms, which are within the scope of reliable medical judgment and are suitable for use in contact with human and animal tissues without excessive The toxicity, irritation, allergic reactions or other problems or complications are commensurate with a reasonable benefit/risk ratio.
  • metal salts for example, metal salts, ammonium salts, salts with organic bases, salts with inorganic acids, salts with organic acids, salts with basic or acidic amino acids, and the like can be mentioned .
  • metal salts include, but are not limited to, alkali metal salts, such as sodium salt, potassium salt, etc.; alkaline earth metal salts, such as calcium salt, magnesium salt, barium salt, etc.; aluminum salt, etc.
  • Non-limiting examples of salts with organic bases include, but are not limited to, trimethylamine, triethylamine, pyridine, picoline, 2,6-lutidine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, Salt formed by dicyclohexylamine, etc.
  • Non-limiting examples of salts with inorganic acids include, but are not limited to, salts with hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid, and the like.
  • Non-limiting examples of salts with organic acids include, but are not limited to, with formic acid, acetic acid, trifluoroacetic acid, fumaric acid, oxalic acid, malic acid, maleic acid, tartaric acid, citric acid, succinic acid, methanesulfonic acid, benzene Salts formed by sulfonic acid, p-toluenesulfonic acid, etc.
  • Non-limiting examples of salts with basic amino acids include, but are not limited to, salts with arginine, lysine, ornithine, and the like.
  • Non-limiting examples of salts with acidic amino acids include, but are not limited to, salts with aspartic acid, glutamic acid, and the like.
  • pharmaceutical ingredient refers to one or more compounds of the present application or salts thereof and excipients, diluents, or carriers commonly used in the art for delivering biologically active compounds to organisms (eg, humans) Of the preparation.
  • the purpose of the pharmaceutical composition is to facilitate the administration of the compound of the present application to an organism.
  • pharmaceutically acceptable excipients, diluents, or carriers refers to those excipients, diluents, or carriers that do not have a significant stimulating effect on the organism and do not impair the biological activity and performance of the active compound.
  • Suitable carriers, diluents and excipients are well known to those skilled in the art and include such materials as carbohydrates, waxes, water-soluble and/or water-swellable polymers, hydrophilic or hydrophobic materials, gelatin, oil, Solvent, water and other raw materials.
  • polypeptide refers to a compound formed by connecting more than two amino acids through peptide bonds, such as but not limited to 2 peptide, 3 peptide, 4 peptide, 5 peptide, 6 peptide, 7 peptide, 8 peptide, 9 peptide, 10 Peptide, 15 peptide, 20 peptide.
  • AEEAc refers to 2-(2-(2-aminoethoxy)ethoxy)acetic acid.
  • Gly refers to glycine.
  • the present application also includes compounds of the present application that are the same as those described herein, but one or more atoms are replaced by an isotope labeled with an atom having an atomic weight or mass number different from the atomic weight or mass number usually found in nature.
  • isotopes that can be incorporated into the compounds of the present application include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine, and chlorine, such as 2 H, 3 H, 11 C, 13 C, 14 C, 13 N, 15 N, 15 O, 17 O, 18 O, 31 P, 32 P, 35 S, 18 F, 123 I, 125 I, 36 Cl, etc.
  • Certain isotopically-labeled compounds of the present application can be used in the analysis of compound and/or substrate tissue distribution. Tritiated (ie 3 H) and carbon-14 (ie 14 C) isotopes are especially preferred because of their ease of preparation and detection. In addition, substitution with heavier isotopes (such as deuterium (ie, 2 H)) can provide certain therapeutic advantages resulting from higher metabolic stability (eg, increased in vivo half-life or reduced dosage requirements), and therefore in certain situations The following may be preferred. Positron emission isotopes such as 15 O, 13 N, 11 C, and 18 F can be used in positron emission tomography (PET) studies to determine substrate occupancy.
  • PET positron emission tomography
  • the isotopically labeled compounds of the present application can generally be prepared by isotopically labeled reagents instead of unisotopically labeled reagents by the following procedures similar to those disclosed in the schemes and/or examples below.
  • the compounds of the present application can be prepared by various synthetic methods well known to those skilled in the art, including the specific embodiments listed below, the embodiments formed by the combination with other chemical synthetic methods, and equivalents well known to those skilled in the art Alternatively, preferred embodiments include but are not limited to the examples of the present application.
  • Figure 1 shows the chemical structure of amphotericin B.
  • FIG. 2 shows the overall chemical structure of DTY-AMB containing T.
  • This structure contains a sugar amine structure with an amino group in the sugar amine.
  • the hydrophilic polypeptide part T (T is composed of Gly-OH or AEEAc-OH and Gly-OH (Combined) can be coupled to amphotericin B through a stable amide bond, which is not easily hydrolyzed by enzymes and is more stable in the body.
  • the solubility of amphotericin B is improved, on the other hand, the amphotericin B is reduced. Toxicity of mycin B.
  • Figure 3 depicts the general route of DTY-AMB chemical synthesis.
  • Figure 4 is a chemical structure diagram of the target product DMR078.
  • Figure 5 is the absorption spectrum of DMR078 and AMB;
  • Figure 5A shows the absorption spectrum of DMR078 and AMB (12.7 ⁇ g/mL) in methanol;
  • Figure 5B shows the DMR078 and AMB (in PBS buffer) 12.7 ⁇ g/mL);
  • FIG. 5C shows the absorption spectrum of DMR078 (62.4 ⁇ g/mL) in PBS buffer.
  • Amino acids are: Fmoc-AEEAc-OH and Fmoc-Gly-OH
  • Synthetic reagents HATU, DMF, DCM, DIEA, piperidine.
  • CS-BIO type peptide synthesizer Waters600 semi-preparative high-performance liquid chromatograph, Beckman centrifuge, Buchi rotary evaporator.
  • the crude product was purified by semi-preparative RP-HPLC under the following conditions.
  • the collected product was analyzed by Agilent 1260 HPLC under the following conditions.
  • the target components with a purity greater than 90% are collected, rotary evaporated, and freeze-dried.
  • Amino acid sequence 2-10 peptide containing Gly-OH, or AEEAc-OH and Gly-OH
  • amphotericin B peptide derivative of HT-AMB the synthesis method refers to compound DMR078, namely 1Fmoc-T (T is Gly, or a combination of AEEAc and Gly hydrophilic peptide, peptide Length range is 2-10); 2 Preparation of Fmoc-T-Osu; 3 Fmoc-T-Osu modifies the amino group of amphotericin B, piperidine removes Fmoc, and finally obtains the crude product of amphotericin B peptide derivative of HT-AMB.
  • the crude product was purified by RP-HPLC; according to the general formula [II], Table 3 lists the abbreviations and relative molecular masses of the compounds of this example:
  • amphotericin B and amphotericin B peptide derivative prepared by the same method as in Example 1.
  • their solubility was measured using double distilled water. The results are shown in Table 4.
  • H-G-A-G-A-G-AMB ⁇ 0.5 H-A-G-A-G-A-AMB(DMR078) >60 H-A-A-G-A-AMB(DMR079) >60 H-A-G-A-G-AMB >60 H-G-A-G-A-G-AMB >60 H-G-A-G-A-G-AMB >70 H-(AEEAc) 9 -Gly-AMB >100
  • A refers to AEEAc
  • G refers to Gly
  • AMB is almost insoluble in water; when the number of AEEAc is less than 3, Gly is increased, and the solubility is not improved. If all are Gly, H-(Gly) 5 -AMB is dissolved in water and left at room temperature for half an hour , Will form micelles; when the number of AEEAc is more than 3, the amount of Gly increases and decreases, AMB peptide derivatives have higher solubility, DMR078 significantly improves water solubility; however, with the hydrophilic peptide After prolonged, the solubility of amphotericin B peptide derivatives increases, but the antibacterial activity can be significantly reduced, or even no antibacterial activity.
  • Unstable three parts AMB parent structure are C 13 ketal structure and a half, seven and a conjugated double bond structure of C 19-bit ⁇ - glycosidic bond, so the Chinese Pharmacopoeia amphotericin B using a predetermined dark, refrigerated Storage.
  • the amphotericin B peptide derivative prepared by the invention is modified on the structure of amphotericin B, so similar degradation pathways also exist.
  • the minimum inhibitory concentration (MIC) of each antibacterial drug was determined according to the micro-broth dilution method recommended by the National Committee for Clinical Laboratory Standardization (NCCLS).
  • the bacterial culture medium was Mueller-Hinton (MH) broth medium and Candida albicans medium Hyclone modified RPMI-1640 medium was used.
  • the fungal growth medium is modified Martin medium, and the specific preparation method is as follows: 1L medium contains 2% glucose, 0.2% yeast extract powder, 0.5% fish meal peptone, 0.05% magnesium sulfate, 0.1% dipotassium hydrogen phosphate, according to the above ratio Weigh each substance in a corresponding amount, dissolve it in a certain amount of purified water, adjust the volume to 1 L, adjust the pH to 7.2, add 2% agar, and sterilize at 121°C for 30 min at high temperature.
  • the fungal MIC test uses Hyclone modified RPMI-1640 medium.
  • the specific preparation method is as follows: Weigh 18.00g of glucose dissolved in a certain amount of purified water, dilute to 500mL, and sterilize at 115°C for 15min. In a sterile environment, add 500 mL of RPMI-1640 medium to the sterilized glucose solution, store it at 4°C, and store it until use.
  • Hyclone modified RPMI-1640 liquid The turbidity of the culture medium is adjusted to 0.5 McLay standard, which is equivalent to containing 1 ⁇ 10 6 ⁇ 5 ⁇ 10 6 CFU (colony-forming unit) per milliliter. Take this bacterial suspension with Hyclone modified RPMI-1640 liquid medium After 1:20 dilution, a further 1:50 dilution is used as the inoculum. The bacterial suspension concentration is equivalent to 1 ⁇ 10 3 ⁇ 5 ⁇ 10 3 CFU/ml.
  • the drug concentrations in wells 1-10 were 32 ⁇ g/mL, 16 ⁇ g/mL, 8 ⁇ g/mL, 4 ⁇ g/mL, 2 ⁇ g/mL, 1 ⁇ g/mL, 0.5 ⁇ g/mL, 0.25 ⁇ g/mL, 0.125 ⁇ g/mL, 0.0625 ⁇ g /mL, hole 11 is a blank control without antibacterial drugs and inoculants, and hole 12 is a negative control without antibacterial drugs.
  • the 96-well plate inoculated with fungi was placed in an air incubator at 28°C and incubated for 40-50 hours.
  • the minimum drug concentration without fungal growth is the minimum inhibitory concentration (MIC) of the sample.
  • the MIC measurement results of each antibacterial drug are shown in Table 5.
  • A refers to AEEAc
  • G refers to Gly
  • PBS buffer solution take 100mL 0.2mol/L Na 2 HPO 4 ⁇ 12H 2 O mother liquor, and adjust its pH value to 7.4 with 0.2mol/L NaH 2 PO 4 ⁇ 2H 2 O mother liquor Measure 50 mL of the above buffer and dilute it with purified water 20 times, then weigh 9.00 g of NaCl and add it.
  • pH8.0 PBS buffer solution take 100mL 0.2mol/L Na 2 HPO 4 ⁇ 12H 2 O mother liquor, and adjust its pH value to 8.0 with 0.2mol/L NaH 2 PO4 ⁇ 2H 2 O mother liquor Take 50mL of the above buffer solution and dilute it with purified water 20 times, then weigh and add 9.00g NaCl to it.
  • the drug concentration of antimicrobial drugs in holes 1-10 is 640 ⁇ g/mL, 320 ⁇ g/mL, 160 ⁇ g/mL, 80 ⁇ g/mL, 40 ⁇ g/mL, 20 ⁇ g/mL, 10 ⁇ g/mL, 5 ⁇ g/mL, 2.5 ⁇ g/mL, 1.25 ⁇ g/mL
  • the drug concentration of the amphotericin B of the control drug is 12.8 ⁇ g/mL, 6.4 ⁇ g/mL, 3.2 ⁇ g/mL, 1.6 ⁇ g/mL, 0.8 ⁇ g/mL, 0.4 ⁇ g/mL, 0.2 ⁇ g/mL, 0.1 ⁇ g/mL, 0.05 ⁇ g/mL, 0.025 ⁇ g/mL
  • the 11th well is the negative control of red blood cell suspension + PBS
  • the 12th well is the positive control of red blood cell suspension + double distilled water. Finally, it was placed in a 37°C incubator for 1 hour.
  • A refers to AEEAc
  • G refers to Gly
  • Example 7 Spectral properties and self-aggregation characteristics of AMB and DMR078
  • Disodium hydrogen phosphate Na 2 HPO 4 ⁇ 12H 2 O: 3.58g
  • FIG. 5A shows the spectrum of DMR078 and AMB (12.7 ⁇ g/mL) in methanol
  • Figure 5B shows the spectrum of DMR078 and AMB (12.7 ⁇ g/mL) in PBS buffer
  • Figure 5C shows The spectrum of DMR078 (62.4 ⁇ g/mL) in PBS buffer is shown.
  • Example 8 In vitro HEK293T cytotoxicity assay
  • HEK293T cells NaCl, KCl, Na 2 HPO 4 ⁇ 12H 2 O, KH 2 PO 4 , MTT, DMEM
  • Disodium hydrogen phosphate Na 2 HPO 4 ⁇ 12H 2 O: 3.58g
  • Collect the logarithmic phase cells adjust the concentration of the cell suspension, add 100 ⁇ L to each well, plate the density of the cells to be tested to 0.5 ⁇ 10 4 cells/well (the edge wells are filled with sterile PBS), 5% CO 2 , and incubate at 37°C overnight. Cover the bottom of the cell monolayer with a well (96-well flat bottom plate), discard the supernatant, and then add the drug with a gradient concentration, a total of 8 concentration gradients, 100 ⁇ L per well, set a double well, 5% CO 2 , 37 Incubate for 24-48 hours at °C, and observe under an inverted microscope.
  • compositions and/or methods disclosed and claimed herein can be prepared and implemented without undue experimentation.
  • compositions and methods of the present invention have been described according to preferred embodiments, it will be apparent to those skilled in the art that without departing from the concept, spirit and scope of the present invention, the compositions and/or methods described herein can be Or the method and the steps of the method or the order of the steps are changed.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Medicinal Chemistry (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Epidemiology (AREA)
  • Molecular Biology (AREA)
  • Engineering & Computer Science (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Communicable Diseases (AREA)
  • Oncology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • Genetics & Genomics (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

属于医药领域，具体涉及两性霉素B肽衍生物及其制备方法。所述两性霉素B肽衍生物对耐药细菌、真菌具有广谱、高效的杀菌效果。

Description

两性霉素B肽衍生物 技术领域
本发明属于医药领域，涉及到两性霉素B肽衍生物及其制备方法与应用，具体而言涉及到一系列具有较高溶解性、低毒性、较好抗菌活性的两性霉素B肽衍生物及其合成制备与应用。
背景技术
两性霉素B(Amphotericin B，AMB)是一种多烯类广谱抗真菌药，适用于下列真菌感染疾病的治疗：念珠菌病(Candidiasis)、隐球菌病(Cryptococcosis)、芽生菌病(Blastomycosis)、球孢子菌病(Coccidioidomycosis)，由毛霉菌(Mucor)所致的毛霉菌病(mucormycosis)，由孢子丝菌(Sporothrix)引起的孢子丝菌病(Sporotrichosis)，由大部分曲霉菌(Aspergillus)所致的曲霉菌病(aspergillosis)等。
自1955年从链霉菌属代谢物中分离得到两性霉素B以来，该化合物就备受瞩目。一方面，两性霉素B是临床上治疗深部真菌感染及全身系统性感染的金标准，并且对于某些致命性全身真菌感染，它是唯一有效的治疗药物；另一方面，在治疗剂量时，两性霉素B有比较严重的毒副作用，例如溶血毒性、肾毒性、神经系统毒性等，而且两性霉素B水溶解性极差，口服该品后自胃肠道吸收少且不稳定，因此临床上两性霉素B的应用受到了极大的限制。
虽然研究表明，作为药物载体的脂质体可以显著降低两性霉素B的毒副作用，两性霉素B脂质体是利用磷脂双分子层膜形成的囊泡包裹药物分子而制成的具有靶向给药功能的新型药物，较普通制剂具有更好的耐受性，一方面它可以较多地分布在肝、脾、肺，而在其它脏器尤其在肾组织内的浓度较低，另一方面，脂质体中的胆固醇成分可降低药物与人体细胞中胆固醇的结合而增强对真菌细胞麦角固醇的结合，对肾脏等的副作用相对较小。但是两性霉素B脂质体类制剂也存在如下的缺点：一、脂质体类制剂的抗菌活性差于两性霉素B，需要加大治疗剂量，二、脂质体类制剂成本较高，价格比较昂贵，三、脂质体本身的不稳定性，四、脂质体类制剂并没有从根本上消除两性霉素B的肾毒性等毒副作用。
鉴于两性霉素B所述的特点及两性霉素B化学结构改造的历史，结合发明人掌握的固相合成多肽技术：以两性霉素B为先导化合物，采用固相和液相联合的实验方法，对两性霉素B进行接肽反应，合成一系列两性霉素B肽衍生物，包括H-(Gly)n-OH或由AEEAc与Gly组合而成的亲水性(n为整数，范围为2-10)系列的两性霉素B肽衍生物，在保留其抗菌活性的同时，提高其水溶性，甚至进一步降低其溶血毒性、肾毒性等毒副作用。
发明内容
一方面，本发明涉及两性霉素B肽衍生物及其合成制备方法与应用，具体而言涉及到改善两性霉素B溶解性，降低两性霉素B毒性，但同时保留两性霉素B抗菌活性的两性霉素B肽衍生物及其合成制备方法与应用。该两性霉素B肽衍生物特指：以R 2-AEEAc-OH及R 2-Gly-OH为原料，采用固相合成的方式，合成R 2-(Gly)n-OH或AEEAc与Gly组合而成的系列化合物，然后通过酰胺键与两性霉素B进行偶联，最后脱除氨基保护基，再经过纯化即可得到目标化合物。
一方面，本发明涉及通式为[I]的化合物或其衍生物：
Figure PCTCN2019124970-appb-000001
其中，R 1为亲水性多聚物部分；R 2为Fmoc、Boc等氨基保护基或H；R 3为H或C 1-4烃基或苯基；R 4为OH或H；可供选择地，亲水性多聚物部分可以为含H-Gly-OH单体的H-(Gly)n-OH多聚物的部分，在这里n取2-20整数，较优地，n取2-15整数，最优地，n为5；亲水性多聚物部分也可为由Gly以及AEEAc通过肽键构成的多肽部分，多肽部分的长度为2-20肽，较优地为2-15肽，最优地，为5肽。
在一些方案中，本发明涉及到两性霉素B肽衍生物的合成制备方法。
在一些方案中，本发明涉及到该衍生物在保留抗真菌活性的同时，改善了溶解性，甚至进一步降低了毒性。
在一些方案中，本发明涉及到水溶性多肽化合物，包括但不限于H-(Gly)n-OH或Gly与AEEAc通过肽键构成的多肽。
在一些方案中，本发明所指的两性霉素B衍生物是通过酰胺键连接的。
在一些方案中，本发明所提到的两性霉素B衍生物包含：水溶性的多肽部分，该多肽部分通过一个稳定的酰胺键与两性霉素B上的糖胺结构相连，因此该类化合物可以简称为两性霉素B多肽衍生物(DTY-AMB)。在本发明中，水溶性多肽指代H-(Gly)n-OH或AEEAc与Gly组合的多肽，水溶性多肽部分是指相应的多肽中的末端氨基酸脱去-OH和-H之后形成的部分。
DTY-AMB也包含C 16上羧基被酯化的一类衍生物，比如烃基类酯。T是含有Gly或Gly衍生物、或由AEEAc及Gly构成的组合多肽及其衍生物的多聚物。
在一些优选方案中，R 1选自-(Gly)n-、-(AEEAc)n-Gly-、-(AEEAc-Gly)n-AEEAc-、-AEEAc-Gly-、-(AEEAc-Gly)n-、-(Gly-AEEAc)n-Gly-、-(AEEAc)n-Gly-(AEEAc)m-、-(AEEAc)n-(Gly)m-和-(Gly)n-AEEAc-(Gly)m-，其中，n和m各自独立地为2-9的整数。优选地，R 1选自-(AEEAc)n-Gly-、-(AEEAc-Gly)n-AEEAc-、-(AEEAc-Gly)n-、-(Gly-AEEAc)n-Gly-和-(AEEAc)n-Gly-(AEEAc)m-，其中，n和m各自独立地为2-9的整数且同时满足R 1中的AEEAc的数量大于等于3。
在一些优选方案中，R 2为H。
在一些优选方案中，R 3为H。
一方面，本发明中DTY-AMB的结构为[II]:
Figure PCTCN2019124970-appb-000002
R 2为Fmoc、Boc等氨基保护基或者H；R 3为H或C 1-4烃基或苯基；R 4为OH或H；可供选择地，亲水性多肽部分可以为T(T由Gly或AEEA及Gly组合而成)，在这里T的长度范围为2-10肽，较优地，为3-7肽，最优地，为5肽。
在一些方案中，DTY-AMB中亲水性多肽部分有不同的长度，可能含有2-20个单体，较优地，含有2-10个单体，最优地，含有5个单体，在本发明中，DTY-AMB可以含有2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20个单体。该单体可能为Gly、或Gly及AEEAc。
在一些方案中，DTY-AMB中亲水性多肽部分是T，T有不同的长度，可能含有2-10个单体，较优地，含有3-7个单体，最优地，含有5个单体，在一些具体实施方案中，DTY-AMB可以含有2、3、4、5、6、7、8、9、10个单体。该单体可能为Gly、或Gly及AEEAc。
在一些方案中，DTY-AMB的C 16位上含有一个游离羧基或者羧基的烃基酯，包括甲酯、乙酯、丙酯、丁酯、苯酯等。
本发明中，含有亲水性多肽部分T的DTY-AMB具有较好的抑菌活性，同时由于是通过酰胺键连接的而不易被酶水解。
另一方面，本发明涉及式[II]的化合物或其衍生物，其中：R 3为H；R 2-T-选自：
H-AEEAc-Gly-，
H-Gly-Gly-，
H-AEEAc-AEEAc-Gly-，
H-(Gly) 5-，
H-AEEAc-AEEAc-Gly-Gly-Gly-，
H-Gly-Gly-AEEAc-Gly-Gly-，
H-Gly-AEEAc-Gly-AEEAc-Gly-，
H-AEEAc-Gly-AEEAc-Gly-AEEAc-，
H-AEEAc-AEEAc-Gly-AEEAc-AEEAc-，
H-AEEAc-Gly-AEEAc-Gly-AEEAc-Gly-，
H-Gly-AEEAc-Gly-AEEAc-Gly-AEEAc-，
H-Gly-AEEAc-Gly-AEEAc-Gly-AEEAc-Gly-，或
H-(AEEAc) 9-Gly-。
在一些方案中，本发明涉及式[II]的化合物或其衍生物，其中，R 3为H；R 2-T-为H-AEEAc-X-Y-Z-AEEAc-；X为AEEAc或Gly，Y为AEEAc或Gly，且Z为AEEAc或Gly。
在一些方案中，本发明涉及式[III]的化合物或其衍生物：
Figure PCTCN2019124970-appb-000003
在一些方案中，本发明涉及式[IV]的化合物或其衍生物：
Figure PCTCN2019124970-appb-000004
在一些方案中，本发明的化合物是下式的化合物或其衍生物，其中肽修饰位点可以为AMB中的氨基，即糖胺基的氨基：
H-AEEAc-Gly-AMB，
H-Gly-Gly-AMB，
H-AEEAc-AEEAc-Gly-AMB，
H-(Gly) 5-AMB，
H-AEEAc-AEEAc-Gly-Gly-Gly-AMB，
H-Gly-Gly-AEEAc-Gly-Gly-AMB，
H-Gly-AEEAc-Gly-AEEAc-Gly-AMB，
H-AEEAc-Gly-AEEAc-Gly-AEEAc-AMB(式[III]，DMR078)，
H-AEEAc-AEEAc-Gly-AEEAc-AEEAc-AMB(式[IV]，DMR079)，
H-AEEAc-Gly-AEEAc-Gly-AEEAc-Gly-AMB，
H-Gly-AEEAc-Gly-AEEAc-Gly-AEEAc-AMB，
H-Gly-AEEAc-Gly-AEEAc-Gly-AEEAc-Gly-AMB，或
H-(AEEAc) 9-Gly-AMB。
在一些方案中，本发明涉及包含上述化合物或其药用盐的药物组合物。
在一些方案中，本发明涉及对有需要的受试者预防和/或治疗真菌感染的方法，其中，向所述受试者给予治疗有效量的上述化合物或其药用盐。在优选的方案中，所述真菌感染选自如下真菌的感染：例如隐球菌(Cryptococcus)、皮炎芽生菌(Blastomyces dermatitis)、白色念珠菌(Candida albicans)、克柔念珠菌(Candida krusei)、近平滑念珠菌(Candida parapsilosis)、粗球孢子菌(Coccidioides immitis)、毛霉菌(Mucor)、申克孢子丝菌(Sporothrix schenckii)和烟曲菌(Aspergillus fumigatus)。例如，所述受试者患有隐球菌病(Cryptococcosis)、芽生菌病(Blastomycosis)、念珠菌病(Candidiasis)、球孢子菌病(Coccidioidomycosis)，由毛霉菌所致的毛霉菌病(mucormycosis)，由孢子丝菌(Sporothrix)引起的孢子丝菌病(Sporotrichosis)，由大部分曲霉菌(Aspergillus)所致的曲霉菌病(aspergillosis)。
在一些方案中，本发明涉及上述化合物或其药用盐用于制备药物、例如抗真菌药物的用途。优选地，所述真菌选自例如隐球菌、皮炎芽生菌、白色念珠菌、克柔念珠菌、近平滑念珠菌、粗球孢子菌、毛霉菌、申克孢子丝菌和烟曲菌。优选地，与两性霉素B相比，本发明的上述化合物或其药用盐具有相当的抗真菌活性，并具有改善的溶解性，甚至可进一步具有降低的毒性。
在一些方案中，本发明涉及用于预防和/或治疗真菌感染的上述化合物或其药用盐。优选地，所述真菌感染选自如下真菌的感染：例如隐球菌、皮炎芽生菌、白色念珠菌、克柔念珠菌、近平滑念珠菌、粗球孢子菌、毛霉菌、申克孢子丝菌或烟曲菌。进一步优选地，上述化合物或其药用盐用于治疗真菌感染引起的选自如下的疾病：隐球菌病、芽生菌病、念珠菌病、球孢子菌病，由毛霉菌所致的毛霉菌病，由孢子丝菌引起的孢子丝菌病，由大部分曲霉菌所致的曲霉菌病。优选地，与两性霉素B相比，本发明的上述化合物 或其药用盐具有相当的抗真菌活性，并具有改善的溶解性，甚至可进一步具有降低的毒性。
在一些方案中，本发明涉及制备上述化合物或其药用盐的方法，优选地，所述方法包括：(1)在树脂上固相合成多肽，将得到的多肽产物用弱酸进行裂解，过滤，旋蒸，然后加入第一有机溶剂溶解，旋蒸，用第二有机溶剂沉淀，干燥，得到含有氨基保护基的多肽；(2)将所述含有氨基保护基的多肽进行活化，然后在无水溶剂中与两性霉素B在催化剂量的碱存在下进行反应；以及任选地，(3)脱除含有氨基保护基的多肽部分的氨基保护基。
步骤(1)所述的弱酸包括但不限于三氟乙醇，并且该弱酸可与二氯甲烷按1:4(V/V)进行配制作为弱酸溶液。
步骤(1)所述的第一有机溶剂可选自DCM和THF。
步骤(1)所述的第二有机溶剂可选自乙醚、异丙醚、甲基叔丁基醚。
步骤(2)所述的无水溶剂选自DMF和DMSO。
步骤(2)所述的碱包括但不限于N,N-二异丙基乙胺(DIEA)。
在步骤(3)中，采用选自如下的脱除剂来脱除氨基保护基：哌啶(PIP)溶液，优选10wt％-40wt％的PIP的DMF溶液，更优选20wt％-25wt％的PIP的DMF溶液。
在一些方案中，本发明涉及选自如下的化合物或其药用盐：
Figure PCTCN2019124970-appb-000005
其中，R 3为H；H-T-为选自如下中的任一种：
Figure PCTCN2019124970-appb-000006
(H-AEEAc-Gly-)，
Figure PCTCN2019124970-appb-000007
(H-Gly-Gly-)，
Figure PCTCN2019124970-appb-000008
(H-AEEAc-AEEAc-Gly-)，
Figure PCTCN2019124970-appb-000009
(H-(Gly) 5-)，
Figure PCTCN2019124970-appb-000010
(H-AEEAc-AEEAc-Gly-Gly-Gly-)，
Figure PCTCN2019124970-appb-000011
(H-Gly-Gly-AEEAc-Gly-Gly-)，
Figure PCTCN2019124970-appb-000012
(H-Gly-AEEAc-Gly-AEEAc-Gly-)，
Figure PCTCN2019124970-appb-000013
(H-AEEAc-Gly-AEEAc-Gly-AEEAc-)，
Figure PCTCN2019124970-appb-000014
(H-AEEAc-AEEAc-Gly-AEEAc-AEEAc-)，
Figure PCTCN2019124970-appb-000015
(H-AEEAc-Gly-AEEAc-Gly-AEEAc-Gly-)，
Figure PCTCN2019124970-appb-000016
(H-Gly-AEEAc-Gly-AEEAc-Gly-AEEAc-)，
Figure PCTCN2019124970-appb-000017
(H-Gly-AEEAc-Gly-AEEAc-Gly-AEEAc-Gly-)，或
Figure PCTCN2019124970-appb-000018
(H-(AEEAc) 9-Gly-)。
目前深部真菌感染的主要病原菌仍然是白色念珠菌，耐药现象也最为突出，因此白色念珠菌深部感染的防治也是抗真菌感染研究领域的重点，本发明中，含有水溶性多聚物部分T的DTY-AMB对白色念珠菌具有良好的抑菌效果。
DTY-AMB可以是药学上可接受的盐的形式。
DTY-AMB可以作为口服制剂的有效药物成分；也可以充当注射用药的有效药物成分，如静脉注射、皮下注射、肌肉注射等；也可以作为局部用药的有效药物成分。
DTY-AMB可以通过现有的医药学技术制成药物有效剂量单位，该有效剂量单位的形式可以为口服、药片、胶囊或液体等剂型。
药物成分可制成含水的制剂，其中水分不低于50％。
口服制剂可以为液体、悬浮液、粉末、药片及胶囊等形式；其中包含各种不同赋形剂(比如：碳酸钙、磷酸钙等)的药片也可以做成崩解制剂。
药物成分可以通过可控的方式进行释放，包括缓释或快速释放，相关药物成分的可控释放剂量可以通过已知的医药学技术进行实现。
药物成分可以包含有0.1％-99.9％的DTY-AMB(DTY-AMB按重量计算)，较优含量为1％-70％。
图3为DTY-AMB化学合成的总路线图，DTY-AMB的化学合成包括下文的第一步、第二步及第三步。
第一步包括固相合成多肽R 2-T及该系列多肽的活化，本发明提供了一种该系列多肽的合成方法。在一些方案中，所述方法包括：合成该系列多肽。在一些方案中，可采用固相合成技术制备该系列多肽，包括：
(1)在树脂上固相合成多肽；
(2)将步骤(1)的产物用弱酸进行裂解，过滤，旋蒸，然后加入适量的有机溶剂溶解多肽，再次旋蒸，重复2-3次，最后用有机溶剂沉淀，干燥，得到目标多肽。
任选地，所述步骤(1)包括如下步骤：
(a)浸泡树脂—投料(首个氨基酸及高位阻碱)—测定树脂取代值—脱除氨基保护基—洗涤—监测—偶联氨基酸—监测—洗涤—脱除氨基保护基—顺序偶联剩余氨基酸—直到最后一个氨基酸，而后进行洗涤不进行脱除氨基保护基；其中，氨基保护基是指为保护参与缩合反应的氨基而引入的化学基团。所述的氨基保护基包括但不限于叔丁氧羰基(Boc)、苄氧羰基(Z)或9-芴基-甲基羰基(Fmoc)，优选9-芴基-甲基羰基(Fmoc)等，所述保护基包括但不限于此，可根据具体情况进行合理选择。
所述步骤(a)的投料，即称取适量的R 2-AEEAc-OH或R 2-Gly-OH及吸取适量的高位阻碱，加入DCM、例如10mL DCM溶解后，投入到反应器中。
所述高位阻碱试剂包括但不限于N,N-二异丙基乙胺(DIEA)。
所述步骤(a)的测定树脂取代值，即分别取适量的偶联上首个氨基酸的树脂于两个EP管中，干燥称重(W)，分别加入氨基保护基的脱除剂(例如1mL 20％PIP/DMF)进行反应(例如反应30min)以脱除氨基保护基，取该脱保护液(例如100μL)，加入有机溶剂(例如10mL DMF；稀释倍数为S，例如101倍)，测定301nm的吸光值A(在本文中，也称为“A 301”)，取代值(SD)公式为：SD＝A 301*S/(7800*W)，W 的单位为g。
所述步骤(a)的液相环境所用溶剂选自：二甲基甲酰胺(DMF)、二氯甲烷(DCM)、N-甲基吡咯烷酮(NMP)，优选DCM及DMF。
所述步骤(a)中脱除氨基保护基需要加入氨基保护基的脱除剂，氨基保护基的脱除剂选用哌啶(PIP)溶液，浓度10％-40％(PIP/DMF)，脱除时间为20-50min；优选浓度为20％-25％(PIP/DMF)，脱除时间25-35min。
所述步骤(a)中氨基酸的偶联需要加入偶联试剂，偶联试剂由碳二亚胺型试剂或者苯并三氮唑鎓盐型试剂和1-羟基苯并三唑(HOBt)组成。
所述碳二亚胺型试剂包括但不限于二环己基碳二亚胺(DCC)、二异丙基碳二亚胺(DIC)或N-二氨基丙基-N-乙基碳二亚胺(EDC)。
所述苯并三氮唑鎓盐型试剂包括但不限于2-(1H-苯并三偶氮L-1-基)-1,1,3,3-四甲基脲四氟硼酸酯(TBTU)、O-苯并三唑-N,N,N',N'-四甲基脲六氟磷酸盐(HBTU)、六氟磷酸苯并三唑-1-氧基三(二甲氨基)磷(BOP)或六氟磷酸苯并三唑-1-基-氧基三吡咯烷基磷(PyBOP)。
所述偶联试剂优选二异丙基碳二亚胺(DIC)和1-羟基苯并三唑(HOBt)、或2-(1H-苯并三偶氮L-1-基)-1,1,3,3-四甲基脲四氟硼酸酯(TBTU)和1-羟基苯并三唑(HOBt)，进一步优选DIC(二异丙基碳二亚胺)和1-羟基苯并三唑(HOBt)。
所述步骤(a)中的“监测”采用茚三酮检测法监测多肽的缩合反应。
所述步骤(a)中的顺序偶联氨基酸是指根据多肽氨基酸序列从C端向N端逐个连接氨基酸。
步骤(2)所述的弱酸包括但不限于三氟乙醇，并且该弱酸可与二氯甲烷按1:4(V/V)进行配制作为弱酸溶液。
第一步反应中的另一部分为含有氨基保护基的多肽的活化，其中多肽T为2-10肽，较优地，为3-7肽，最优地，为5肽。多肽羧基的活化包括：活化酯法、对称酸酐法、叠氮化法等，优选较为温和的活化酯法；所用的活化试剂：由碳二亚胺型试剂或者苯并三氮唑鎓盐型试剂和1-羟基苯并三唑(HOBt)或琥珀酰亚胺(HOSU)组成。所述碳二亚胺型试剂包括但不限于二环己基碳二亚胺(DCC)、二异丙基碳二亚胺(DIC)或N-二氨基丙基-N-乙基碳二亚胺(EDC)。所述苯并三氮唑鎓盐型试剂包括但不限于2-(1H-苯并三偶氮L-1-基)-1,1,3,3-四甲基脲四氟硼酸酯(TBTU)、O-苯并三唑-N,N,N',N'-四甲基脲六氟磷酸盐(HBTU)、六氟磷酸苯并三唑-1-氧基三(二甲氨基)磷(BOP)或六氟磷酸苯并三唑-1-基-氧基三吡咯烷基磷(PyBOP)，优选二异丙基碳二亚胺(DIC)和羟基琥珀酰亚胺(HOSU)；活化时所用的液相体系中的溶剂优选有机溶剂，包括DMF、DMSO、DCM、THF等，优选THF(四氢呋喃)。所述的氨基保护基包括但不限于叔丁氧羰基(Boc)、苄氧羰基(Z)或9-芴基-甲基羰基(Fmoc)，优选9-芴基-甲基羰基(Fmoc)；酯的活化在30℃下，1-2小时，加入催化剂量的碱，有利于活化完全，所述碱试剂包括但不限于N,N-二异丙基乙胺(DIEA)；经过旋蒸，除去有机溶剂即可得到化合物2。
第二步，化合物2在无水溶剂中，如DMF、DMSO，室温下与两性霉素B进行1-2小时的反应。反应要求避光，较优地，加入催化剂量的碱，有利于反应完全，所述碱试剂包括但不限于N,N-二异丙基乙胺(DIEA)；即可得到化合物4，化合物4可以通过半制备型RP-HPLC进行制备纯化。
第三步，对含有氨基保护基的多肽进行脱氨基保护基，所述的氨基保护基包括但不限于叔丁氧羰基(Boc)、苄氧羰基(Z)或9-芴基-甲基羰基(Fmoc)，优选9-芴基-甲基羰基(Fmoc)；脱除氨基保护基需要加入氨基保护基的脱除剂，氨基保护基的脱除剂选用哌啶(PIP)溶液，浓度10％-40％(PIP/DMF)，脱除时间为20-50min；优选浓度为20％-25％(PIP/DMF)，脱除时间25-35min。通过纯化，即可得到化合物5(即，DTY-AMB)。
另外，5-脱氧两性霉素B也可以代替两性霉素B来充当起始原料，5-脱氧两性霉素B即两性霉素B的R 4位置的基团OH变为H；相关的5-脱氧两性霉素B可以通过合成得到。
特别有益的是为满足医药用途的质量要求，本发明所提供的两性霉素B肽衍生物的制备方法还可以进一步包括纯化步骤。所采用的纯化方法包括但不限于反相色谱法或离子交换色谱法，优选反相色谱法。
本发明所涉及两性霉素B肽衍生物的体外抗菌活性可以通过测定其最低抑菌浓度(MIC)来鉴定。美国临床实验室标准化委员会(NCCLS)推荐采用微量肉汤稀释法来测定各抗菌药物的最低抑菌浓度(MIC)，培养基采用改良型RPMI-1640培养基。以两性霉素B作为阳性对照。体外活性测定表明，本发明提供的两性霉素B肽衍生物具有较好的抗白色念珠菌活性。
定义
除非另有说明，本申请中所用的下列术语具有下列含义。一个特定的术语在没有特别定义的情况下不应该被认为是不确定的或不清楚的，而应该按照本领域普通的含义去理解。当本文中出现商品名时，意在指代其对应的商品或其活性成分。
术语“任选”或“任选地”是指随后描述的事件或情况可以发生或不发生，该描述包括发生所述事件或情况和不发生所述事件或情况。例如，乙基“任选”被卤素取代，指乙基可以是未被取代的(CH 2CH 3)、单取代的(如CH 2CH 2F)、多取代的(如CHFCH 2F、CH 2CHF 2等)或完全被取代的(CF 2CF 3)。本领域技术人员可理解，对于包含一个或多个取代基的任何基团，不会引入任何在空间上不可能存在和/或不能合成的取代或取代模式。
本文所用的C m-n指该部分中具有m-n个碳原子。例如，“碳 3-10环烷基”指该环烷基具有3-10个碳原子。“碳 0-6亚烷基”指该亚烷基具有0-6个碳原子，当亚烷基具有0个碳原子时，该基团为键。
本文中的数字范围，是指给定范围中的各个整数。例如“C 1-6”是指该基团可具有1个碳原子、2个碳原子、3个碳原子、4个碳原子、5个碳原子或6个碳原子。
当任何变量(例如R)在化合物的组成或结构中出现一次以上时，其在每一种情况下的定义都是独立的。因此，例如，如果一个基团被2个R所取代，则每个R都有独立的选项。
术语“卤”或“卤素”是指氟、氯、溴和碘。
术语“酰基”指-CO-基团。
术语“羧基”指-COOH基团。
术语“羟基”指-OH基团。
术语“氨基”指-NH 2基团。
术语“烷基”是指通式为C nH 2n+1的烃基。该烷基可以是直链或支链的。例如，术语“烃 1-6烷基”指含有1至6个碳原子的单价直链或支链脂肪族基团(例如甲基、乙基、正丙基、异丙基、正丁基、异丁基、仲丁基、叔丁基、正戊基、1-甲基丁基、2-甲基丁基、3-甲基丁基、新戊基、3,3-二甲基丙基、己基、2-甲基戊基等)。类似地，烷氧基、烷基氨基、二烷基氨基、烷基磺酰基和烷硫基的烷基部分(即烷基)具有上述相同定义。
术语“烷氧基”指-O-烷基。术语“烷硫基”指-S-烷基。术语“烷基氨基”指-NH(烷基)。
术语“环烷基”指完全饱和的并且可以以呈单环、稠环或螺环存在的碳环。除非另有指示，该碳环通常为3至10元环，优选为3至8元环。环烷基非限制性实例包括但不限于环丙基、环丁基、环戊基、环己基、降冰片基(双环[2.2.1]庚基)、双环[2.2.2]辛基、金刚烷基等。
术语“芳基”是指具有共轭的π电子体系的全碳单环或稠合多环的芳香环基团。例如，芳基可以具有6-20个碳原子、6-14个碳原子或6-12个碳原子。芳基的非限制性实例包括但不限于苯基、萘基和蒽基等。
术语“杂芳基”是指单环或稠合多环体系，其中含有至少一个选自N、O、S的环原子，其余环原子为C，并且具有至少一个芳香环。优选的杂芳基具有单个4至8元环、尤其是5至8元环，或具有包含6至14个、尤其是6至10个环原子的多个稠合环。杂芳基的非限制性实例包括但不限于吡咯基、呋喃基、噻吩基、咪唑基、噁唑基、吡唑基、吡啶基、嘧啶基、吡嗪基、喹啉基、异喹啉基、四唑基、三唑基、三嗪基、苯并呋喃基、苯并噻吩基、吲哚基、异吲哚基等。
术语“被取代”是指特定原子上的任意一个或多个氢原子被取代基取代，只要特定原子的价态是正常的并且取代后的化合物是稳定的。当取代基为氧代(即＝O)时，意味着两个氢原子被取代，氧代不会发生在芳香基上。
术语“药学上可接受的”是针对那些化合物、材料、组合物和/或剂型而言，它们在可靠的医学判断的范围之内，适用于与人类和动物的组织接触使用，而没有过多的毒性、刺激性、过敏性反应或其它问题或并发症，与合理的利益/风险比相称。
作为药学上可接受的盐，例如，可以提及金属盐、铵盐、与有机碱形成的盐、与无机酸形成的盐、与有机酸形成的盐、与碱性或者酸性氨基酸形成的盐等。金属盐的非限制性实例包括但不限于碱金属的盐，例如钠盐、钾盐等；碱土金属的盐，例如钙盐、镁盐、钡盐等；铝盐等。与有机碱形成的盐的非限制性实例包括但不限于与三甲胺、三乙胺、吡啶、甲基吡啶、2,6-二甲基吡啶、乙醇胺、二乙醇胺、三乙醇胺、环己胺、二环己基胺等形成的盐。与无机酸形成的盐的非限制性实例包括但不限于与盐酸、氢溴酸、硝酸、硫酸、磷酸等形成的盐。与有机酸形成的盐的非限制性实例包括但不限于与甲酸、乙酸、三氟乙酸、富马酸、草酸、苹果酸、马来酸、酒石酸、柠檬酸、琥珀酸、甲磺酸、苯磺酸、对甲基苯磺酸等形成的盐。与碱性氨基酸形成的盐的非限制性实例包括但不限于与精氨酸、赖氨酸、鸟氨酸等形成的盐。与酸性氨基酸 形成的盐的非限制性实例包括但不限于与天冬氨酸、谷氨酸等形成的盐。
术语“药物成分”是指一种或多种本申请的化合物或其盐与在本领域中通常接受的用于将生物活性化合物输送至有机体(例如人)的赋形剂、稀释剂、或载体的制剂。药物组合物的目的是有利于对有机体给予本申请的化合物。
术语“药学上可接受的赋形剂、稀释剂、或载体”是指对有机体无明显刺激作用，而且不会损害该活性化合物的生物活性及性能的那些赋形剂、稀释剂、或载体。合适的载体、稀释剂及赋形剂是本领域技术人员熟知的，并且包括诸如碳水化合物、蜡、水溶性和/或水可膨胀的聚合物、亲水性或疏水性材料、明胶、油、溶剂、水等原料。
术语“多肽”是指由2个以上的氨基酸通过肽键连接而形成的化合物，例如但不限于2肽、3肽、4肽、5肽、6肽、7肽、8肽、9肽、10肽、15肽、20肽。
在本文中，除非另有说明，否则术语“包含、包括和含有(comprise、comprises和comprising)”或等同物为开放式表述，意味着除所列出的要素、组分和步骤外，还可涵盖其它未指明的要素、组分和步骤。
术语“AEEAc”是指2-(2-(2-氨基乙氧基)乙氧基)乙酸。术语“Gly”是指甘氨酸。
本申请还包括与本文中记载的那些相同的，但一个或多个原子被原子量或质量数不同于自然中通常发现的原子量或质量数的原子置换的同位素标记的本申请化合物。可结合到本申请化合物的同位素的实例包括氢、碳、氮、氧、磷、硫、氟、碘和氯的同位素，诸如分别为 2H、 3H、 11C、 13C、 14C、 13N、 15N、 15O、 17O、 18O、 31P、 32P、 35S、 18F、 123I、 125I和 36Cl等。
某些同位素标记的本申请化合物(例如用 3H及 14C标记的那些)可用于化合物和/或底物组织分布分析中。氚化(即 3H)和碳-14(即 14C)同位素由于它们易于制备和检测是尤其优选的。此外，用较重同位素(诸如氘(即 2H))取代可以提供某些由更高的代谢稳定性产生的治疗优点(例如增加的体内半衰期或降低的剂量需求)，并且因此在某些情形下可能是优选的。正电子发射同位素，诸如 15O、 13N、 11C和 18F可用于正电子发射断层扫描(PET)研究以测定底物占有率。通常可以通过与公开于下文的方案和/或实施例中的那些类似的下列程序，通过同位素标记试剂取代未经同位素标记的试剂来制备同位素标记的本申请化合物。
本申请的化合物可以通过本领域技术人员所熟知的多种合成方法来制备，包括下面列举的具体实施方式、其与其它化学合成方法的结合所形成的实施方式以及本领域技术人员所熟知的等同替换方式，优选的实施方式包括但不限于本申请的实施例。
本申请具体实施方式的化学反应是在合适的溶剂中完成的，所述的溶剂须适合于本申请的化学变化及其所需的试剂和物料。为了获得本申请的化合物，有时需要本领域技术人员在已有实施方式的基础上对合成步骤或者反应流程进行修改或选择。
为了描述和公开的目的，以引用的方式将所有的专利、专利申请和其它已确定的出版物在此明确地并入本文。这些出版物仅因为它们的公开早于本申请的申请日而提供。所有关于这些文件的日期的声明或这些文件的内容的表述是基于申请者可得的信息，并且不构成任何关于这些文件的日期或这些文件的内容的正确性的承认。而且，在任何国家，在本中对这些出版物的任何引用并不构成关于该出版物成为本领域的公知常识的一部分的认可。
以下实施例仅代表本发明阐述的一个方面，不是本发明主题的局限。
附图说明
图1为两性霉素B的化学结构。
图2为包含T的DTY-AMB整体化学结构，此结构中包含了一个糖胺结构，糖胺中有一个氨基，亲水性多肽部分T(T由Gly-OH或AEEAc-OH及Gly-OH组合而成)可以通过稳定的酰胺键与两性霉素B偶联，该酰胺键不容易被酶水解，在体内较稳定，一方面改善了两性霉素B的溶解性，另一方面降低了两性霉素B的毒性。
图3描述了DTY-AMB化学合成总路线图。
图4为目标产物DMR078的化学结构图。
图5为DMR078及AMB的吸收光谱图；其中图5A示出了处于甲醇中的DMR078及AMB(12.7μg/mL)的吸收光谱图；图5B示出了处于PBS缓冲液中的DMR078及AMB(12.7μg/mL)的吸收光谱图；图5C示出了处于PBS缓冲液中的DMR078(62.4μg/mL)的吸收光谱图。
具体实施方式
实施例1：DMR078的制备与纯化
亲水多肽结构：H-AEEAc-Gly-AEEAc-Gly-AEEAc-OH
DMR078结构：
Figure PCTCN2019124970-appb-000019
(1)材料及试剂
2-CTC树脂，取代值0.945mmol/g。
氨基酸为：Fmoc-AEEAc-OH及Fmoc-Gly-OH
合成试剂：HATU、DMF、DCM、DIEA、哌啶。
(2)仪器
CS-BIO型多肽合成仪、Waters600半制备型高效液相色谱仪、Beckman离心机、Buchi旋蒸仪。
(3)操作步骤(以1g树脂为例)
a.固相化学合成多肽
称取2-CTC树脂1.00g，置于多肽合成仪反应器中，加入10mL DCM，浸泡1h，称取2-3倍量的Fmoc-AEEAc-OH及吸取4-6倍量的DIEA加入10mL DCM溶解后，投入反应器中，进行反应，反应温度为室温(25℃及其以上，否则延长反应时间)，反应二小时，即第一个氨基酸偶联到了树脂上，然后DCM洗涤树脂6次，测定此时树脂的取代值(SD)；随后加入20％PIP/DMF溶液10mL，混合30min脱除氨基保护基，DMF洗涤树脂6次，偶联第二个氨基酸，称取三倍量的Fmoc-Gly-OH、HATU及吸取六倍量的DIEA，加入10mL DMF溶解后，进行反应，反应温度为室温，以茚三酮反应监测反应进程，监测无色则为反应完成，用DMF洗涤树脂6次。而后，即可按照上述偶联第二个氨基酸的方法继续进行下一个氨基酸的偶联反应，如此循环，直至全部氨基酸偶联完成。
b.裂解及沉淀
多肽合成结束后，称湿重。按照5mL裂解试剂/1g树脂的比例加入裂解试剂，试剂配比为TFE:DCM＝1:4(V:V)，室温搅拌反应1小时，过滤至25mL的旋蒸瓶中，40℃将液体蒸干，然后向旋蒸瓶中加入10mL DCM，再次将液体旋蒸至干，反复进行2-3次，最后加入3mL DCM，溶解切割的多肽，并将溶液转移至50mL离心管中，加入40mL乙醚，放置-20℃冰箱中20min，离心，真空干燥，称重粗肽。
c.液相反应制备两性霉素B肽衍生物
称取干燥后的粗肽0.1mmol、HOSU 0.095mmol、DIC 15μL，并加入5mL THF(25℃，反应2小时)，旋蒸除去THF；加入5mL DMSO溶解反应产物，加入0.095mmol两性霉素B以及7μL DIEA室温下反应1～2小时，加入20％PIP/DMF溶液4mL，反应10～20min，即得到粗产物。
按如下条件，用半制备型RP-HPLC，对粗产物进行纯化。
纯化
色谱柱：Nano Micro C18制备柱(10mm×250mm，10μm)
流速：5mL/min
检测波长：409nm
流动相：A相：1％HAC/水
B相：1％HAC/乙腈
梯度洗脱程序如表1。
表1梯度洗脱程序
Figure PCTCN2019124970-appb-000020
按如下条件，用安捷伦1260HPLC，对收集产物进行分析。
色谱柱：YMC-pack ODS-AQ C18分析柱(4.6mm×250mm，5μm)
流速：1mL/min
检测波长：215nm、383nm及405nm
流动相：A相：0.05％TFA/水
B相：0.05％TFA/乙腈
梯度洗脱程序如表2。
表2梯度洗脱程序
Figure PCTCN2019124970-appb-000021
收集纯度大于90％的目标组分，旋蒸，冷冻干燥。经ESI-MS进行分子量确证，M/Z＝1473(M+H) +与理论分子量相符。
实施例2：含有T的其它两性霉素B肽衍生物的制备与纯化
氨基酸序列：为含有Gly-OH、或AEEAc-OH及Gly-OH的2-10肽
产物结构：
Figure PCTCN2019124970-appb-000022
合成H-T-AMB的两性霉素B肽衍生物，合成方法参照化合物DMR078，即①采用固相合成法合成Fmoc-T(T为Gly、或AEEAc及Gly组合而成的亲水性多肽，肽的长度范围为2-10)；②制备Fmoc-T-Osu；③Fmoc-T-Osu修饰两性霉素B的氨基，哌啶脱除Fmoc，最终得到H-T-AMB的两性霉素B肽衍生物粗品。采用RP-HPLC对粗产物进行纯化；根据化合物通式[II]，表3列举了该实施例的化合物的简称及相对分子质量：
表3化合物简称及相对分子质量
名称 相对分子质量
H-(Gly-Gly)-AMB 1038
H-(AEEAc) 9-Gly-AMB 2287
实施例3：两性霉素B肽衍生物溶解性的测定
两性霉素B及两性霉素B肽衍生物(采用与实施例1相同的方法制备)溶解性的测定，本发明中，采用双蒸水对它们的溶解性进行了测定，结果如表4。
表4溶解性测定结果
化合物 溶解性(mg/mL)
AMB <0.001
H-A-G-AMB <0.5
H-G-G-AMB <0.5
H-A-A-G-AMB <0.5
H-(Gly) 5-AMB <0.5
H-A-A-G-G-G-AMB <0.5
H-G-G-A-G-G-AMB <0.5
H-G-A-G-A-G-AMB <0.5
H-A-G-A-G-A-AMB(DMR078) >60
H-A-A-G-A-A-AMB(DMR079) >60
H-A-G-A-G-A-G-AMB >60
H-G-A-G-A-G-A-AMB >60
H-G-A-G-A-G-A-G-AMB >70
H-(AEEAc) 9-Gly-AMB >100
注：A指代AEEAc；G指代Gly
结果显示，AMB几乎不溶于水；当AEEAc个数少于3时，增加Gly，溶解度没有改善，如若全部为Gly时，H-(Gly) 5-AMB，其溶解于水后，室温放置半小时，会形成胶束；当AEEAc的个数多于3时，Gly的量增加与减少，AMB肽衍生物均有较高的溶解度，DMR078明显改善了水溶解性；然而，随着亲水性多肽的延长，两性霉素B肽衍生物的溶解度增大，抑菌活性却可明显下降，甚至无抑菌活性。
实施例4：两性霉素B肽衍生物的稳定性
AMB母体结构的三个不稳定部位分别为C 13位半缩酮结构、七个共轭双键结构及C 19位的β-糖苷键，故中国药典中规定两性霉素B采用避光、冷藏的方式进行贮藏。本发明制备的两性霉素B肽衍生物是在两性霉素B结构上进行了修饰，故也存在相似的降解途径。①在甲醇溶液中(室温12h)，两性霉素B肽衍生物C 13位半缩酮结构会被甲基化；②在低pH(如TFA)下，容易产生m/z 801的杂质，推测其C 19的β-糖苷键断裂；③在避光室温条件下，其水溶液共轭七烯键很容易氧化，而低温(-20℃)避光条件下，化合物共轭七烯键只有轻微的氧化，故制备的两性霉素B肽衍生物也采取避光、冷藏的方式进行贮藏；同时试验表明两性霉素B与亲水性多肽之间的酰胺键非常稳定，在4℃、25℃下，以水、PBS为溶剂，样品在放置4h、12h、24h、48h后，采用RP-HPLC(215nm、383nm、405nm)未检测到断裂的亲水性多肽。
实施例5：体外抗菌活性测定
根据美国临床实验室标准化委员会(NCCLS)推荐的微量肉汤稀释法测定各抗菌药物的最低抑菌浓度(MIC)，细菌培养基采用Mueller-Hinton(MH)肉汤培养基，白色念球菌培养基采用Hyclone改良型RPMI-1640培养基。
具体步骤为：
(1)抗菌药物贮存液制备：
精确配制浓度为320μg/mL的AMB肽衍生物和阳性对照品两性霉素B的各贮存液，置于-20℃环境中避光保存备用。
(2)培养基的配制：
真菌生长培养基采用改良马丁培养基，具体配制方法如下：1L培养基中含有2％葡萄糖、0.2％酵母浸出粉、0.5％鱼粉蛋白胨、0.05％硫酸镁、0.1％磷酸氢二钾，按上述比例称取相应量的各物质，溶解于一定量纯净水之后，定容至1L，调pH至7.2，加入2％琼脂，121℃高温灭菌30min。
真菌MIC测试采用Hyclone改良型RPMI-1640培养基，具体配制方法如下：称取18.00g葡萄糖溶于一定量纯净水中，定容到500mL，115℃高温灭菌15min。在无菌环境中，向已灭菌的葡萄糖溶液中加入500mL RPMI-1640培养基，混合后4℃贮存备用。
(3)接种物的制备：
挑取在平板上生长48h的真菌单菌落，转接到改良型马丁培养基的斜面培养基上，28℃孵育24h，增菌后处于对数生长期的菌液用Hyclone改良型RPMI-1640液体培养基调整其浊度达到0.5麦氏标准，相当于每毫升含1×10 6～5×10 6CFU(colony-forming unit)，取此菌悬液用Hyclone改良型RPMI-1640液体培养基进行1:20稀释后，再进行1:50稀释作为待接种物，菌悬液浓度相当于1×10 3～5×10 3CFU/ml。
(4)稀释抗菌药物的制备及菌液接种：
取一块96孔板，在第1孔中加入160μL RPMI-1640液体培养基，第2-12孔中各加入100μL RPMI-1640液体培养基，然后向第1孔加入抗菌药物原液(320μg/mL)40μL，混匀，接着吸取100μL至第2孔，混匀后再从第2孔中吸取100μL至第3孔，如此连续倍比稀释至第10孔，并从第10孔中吸取100μL弃去，然后向第1-10孔及第12孔中加入上述制备好的接种物各100μL，使各孔的最终菌液浓度约为2.5×10 3CFU/ml。第1-10孔药物浓度分别为32μg/mL、16μg/mL、8μg/mL、4μg/mL、2μg/mL、1μg/mL、0.5μg/mL、0.25μg/mL、0.125μg/mL、0.0625μg/mL，第11孔为不含抗菌药物及接种物的空白对照，第12孔为不含抗菌药物的阴性对照。
(5)孵育
接种真菌的96孔板置于28℃空气孵箱中孵育40～50h。
(6)结果
以肉眼观察，无真菌生长的最低药物浓度即为该样品的最低抑菌浓度(MIC)。各抗菌药物的MIC测定结果如表5所示。
表5 MIC测定结果
化合物 MIC(μmol/L)
AMB 1.08
H-A-G-AMB 1.78
H-G-G-AMB 1.93
H-A-A-G-AMB 1.69
H-(Gly) 5-AMB 1.65
H-A-A-G-G-G-AMB 1.44
H-G-G-A-G-G-AMB 1.54
H-G-A-G-A-G-AMB 1.44
H-A-G-A-G-A-AMB(DMR078) 1.36-2.72
H-A-A-G-A-A-AMB(DMR079) 2.56
H-A-G-A-G-A-G-AMB 2.61
H-G-A-G-A-G-A-AMB 5.22-10.46
H-G-A-G-A-G-A-G-AMB 5.03-10.08
注：A指代AEEAc；G指代Gly
实施例6：体外溶血活性测定
1.材料及试剂
无菌脱纤维羊血，NaH 2PO 4·2H 2O，Na 2HPO 4·12H 2O，NaCl
2.仪器设备
Allegra X-22R型低温高速离心机，电子天平，微量移液器，SHP-150型生化培养箱，96well板，酶标仪3.实验方法
(1)PBS(磷酸缓冲盐生理盐水)缓冲液的配制
0.2mol/L NaH 2PO 4·2H 2O母液配制：准确称取NaH 2PO 4·2H 2O 3.12g，加纯净水搅拌溶解后，定容至100mL。
0.2mol/L Na 2HPO 4·12H 2O母液配制：准确称取Na 2HPO 4·12H 2O 14.32g，加纯净水搅拌溶解后，定容至200mL。
0.01mol/L pH7.4的PBS缓冲液：取100mL 0.2mol/L Na 2HPO 4·12H 2O母液，用0.2mol/L NaH 2PO 4·2H 2O母液将其pH值调至7.4，量取50mL上述缓冲液用纯净水稀释20倍，再称取9.00g NaCl加入其中。
0.01mol/L pH8.0的PBS缓冲液：取100mL 0.2mol/L Na 2HPO 4·12H 2O母液，用0.2mol/L NaH 2PO4·2H 2O母液将其pH值调至8.0，量取50mL上述缓冲液用纯净水稀释20倍，再称取9.00g NaCl加入其中。
(2)受试物的制备
准确称量待检测的两性霉素B肽衍生物样品，用pH 8.0的PBS缓冲液配制成浓度为2.56mg/mL的待测液(由于衍生物样品中有分离纯化时残留的HAC，用pH 8.0的PBS缓冲液配制可以使得最终溶液pH达到7.3-7.4，另外溶解度较差的衍生物不再测定溶血毒性)，-20℃冷藏备用。准确称取两性霉素B，首先用DMSO配置成5.12mg/mL，然后用pH 7.4的PBS缓冲液稀释成51.2μg/mL的待测液。
(3)羊血红细胞的处理
取无菌脱纤维羊血数毫升，加入约10倍量的pH 7.4的PBS缓冲液，摇匀，1500r/min离心15分钟，除去上清液，沉淀的红细胞再用pH 7.4的PBS缓冲液按照上述方法洗涤2-3次，至上清液不显红色为止。将所得红细胞用pH 7.4的PBS缓冲液配成2％的混悬液，供试验用。
(4)溶血毒性测定步骤
取一块96孔板，在第1-11孔中加入100μL pH 7.4的PBS缓冲液，在12孔加入100μL双蒸水，然后向第1孔加入抗菌药物原液(2560μg/mL)或两性霉素B原液(51.2μg/mL)100μL，混匀，接着吸取100μL至第2孔，混匀后再从第2孔中吸取100μL至第3孔，如此连续倍比稀释至第10孔，并从第10孔中吸取100μL弃去，然后向第1-10孔及第11-12孔中加入上述制备好的接种物各100μL 2％的红细胞混悬液，使每孔最终红细胞浓 度约为1×10 8细胞/孔。抗菌药物的第1-10孔药物浓度分别为640μg/mL、320μg/mL、160μg/mL、80μg/mL、40μg/mL、20μg/mL、10μg/mL、5μg/mL、2.5μg/mL、1.25μg/mL，对照药物两性霉素B的第1-10孔药物浓度分别为12.8μg/mL、6.4μg/mL、3.2μg/mL、1.6μg/mL、0.8μg/mL、0.4μg/mL、0.2μg/mL、0.1μg/mL、0.05μg/mL、0.025μg/mL，第11孔为红细胞混悬液+PBS的阴性对照，第12孔为红细胞混悬液+双蒸水的阳性对照。最后置于37℃的恒温箱中温育1h。
(5)孵育
温育1h后，取出96well板，600r/min离心15分钟，取上清用酶标仪测定540nm下的OD值。按照下列公式计算溶血率，其中阴性对照为PBS，阳性对照为双蒸水。溶血率＝(试品OD-阴性OD)/(阳性OD-阴性OD)
(6)结果
两性霉素B肽衍生物的体外溶血毒性结果见表6。
表6体外溶血毒性结果
化合物 体外溶血毒性(μg/mL)
AMB 6.4
H-A-G-A-G-A-AMB(DMR078) >640
H-A-A-G-A-A-AMB(DMR079) >640
H-A-G-A-G-A-G-AMB >640
H-G-A-G-A-G-A-AMB >640
H-G-A-G-A-G-A-G-AMB >640
注：A指代AEEAc；G指代Gly
实施例7：AMB及DMR078光谱性质和自聚集特征
1.材料及试剂
NaCl，KCl，Na 2HPO 4·12H 2O，KH 2PO 4，DMSO，CH 3OH。
2.仪器设备
电子天平，恒温水浴槽，UV1800紫外可见分光光度计
3.实验方法
(1)PBS(磷酸缓冲盐生理盐水)缓冲液的配制
PBS，pH 7.4，1L
磷酸二氢钾(KH 2PO4)：0.27g
磷酸氢二钠(Na 2HPO 4·12H 2O)：3.58g
氯化钠(NaCl)：8.00g
氯化钾(KCl)：0.20g
加水约800mL充分搅拌溶解，然后加入盐酸调pH至7.4，定容到1L
(2)受试物的制备
准确称量待检测的DMR078，分别用DMSO、pH 7.4的PBS缓冲液配制成浓度为1.28和2.56mg/mL的待测液，-20℃冷藏备用。准确称取AMB，用DMSO配置成1.28mg/mL，-20℃冷藏备用。
(3)测定步骤
首先，将20μL等份的每种DMSO溶液加入到2mL甲醇或2mL PBS缓冲盐中(消除DMSO的影响)，另外分别取20μL、30μL、40μL及50μL的DMR078 2.56mg/mL溶液加入到2mL PBS缓冲盐中；其次，上述溶液在30℃温育30分钟；最后，记录每个样品300-430nm范围内UV-Vis光谱数据。
4)结果
AMB及DMR078(H-A-G-A-G-A-AMB)的光谱图结果见图5。图5A示出了处于甲醇中的DMR078及AMB(12.7μg/mL)的光谱图；图5B示出了处于PBS缓冲液中的DMR078及AMB(12.7μg/mL)的光谱图；图5C示出了处于PBS缓冲液中的DMR078(62.4μg/mL)的光谱图。
文献报道AMB在PBS缓冲液中存在多个形式，即单体形式、可溶性自聚集以及不可溶的自聚集。而吸光度A348/A409的比率已被用作AMB自聚集(s)的衡量指标；由上图可知，AMB以及DMR078在甲醇溶液中不会产生自聚集(图5A)；在相同的浓度(12.7μg/mL)下，AMB在409nm处的吸收和光谱形状显示：AMB具有明显的自聚集现象，然而，衍生物DMR078单体形式的比例很高，不存在自聚集现象(图5B)； 在更高的浓度(62.4μg/mL)下，在409nm处的吸收和光谱形状显示：衍生物DMR078单体形式的比例仍很高，略微存在自聚集现象(图5C)。
实施例8：体外HEK293T细胞毒性测定
1.材料及试剂
HEK293T细胞，NaCl，KCl，Na 2HPO 4·12H 2O，KH 2PO 4，MTT，DMEM
2.仪器设备
电子天平，微量移液器，多功能酶标仪，A2型生物安全柜，微孔板恒温振荡器，CO2培养箱，离心机，倒置显微镜，恒温水浴槽
3.实验方法
(1)PBS(磷酸缓冲盐生理盐水)缓冲液的配制
PBS，pH 7.4，1L
磷酸二氢钾(KH 2PO 4)：0.27g
磷酸氢二钠(Na 2HPO 4·12H 2O)：3.58g
氯化钠(NaCl)：8.00g
氯化钾(KCl)：0.20g
加水约800mL充分搅拌溶解，然后加入盐酸调pH至7.4，定容到1L
(2)受试物的制备
准确称量待检测的DMR078，用无菌pH 7.4的PBS缓冲液配制成浓度为3.00mg/mL的待测液(使用无菌注射器及0.22μm滤头，在超净工作台中过滤)，-20℃冷藏备用。准确称取两性霉素B，用DMSO配置成10.24mg/mL，然后用pH 7.4的PBS缓冲液稀释成1.024mg/mL的待测液(用无菌pH 7.4的PBS缓冲液在超净工作台中稀释)。
(3)细胞肾毒性测定步骤及孵育
收集对数期细胞，调整细胞悬液浓度，每孔加入100μL，铺板使待测细胞调密度至0.5×10 4细胞/孔(边缘孔用无菌PBS填充)，5％CO 2，37℃孵育过夜。至细胞单层铺满孔底(96孔平底板)，弃去上清液，再加入梯度浓度的药物，共8个浓度梯度，每孔100μL，设1个复孔，5％CO 2，37℃孵育24-48小时，倒置显微镜下观察。弃去上清液，再每孔加入10μL MTT溶液(5mg/mL，即0.5％MTT)，继续培养4-6h。吸去孔内培养液，每孔加入100μL二甲基亚砜，置微孔振荡器上低速振荡10min，使结晶物充分溶解。在酶联免疫检测仪OD 580nm处测量各孔的吸光值。同时设置调零孔(培养基、MTT、二甲基亚砜)，阳性对照孔(细胞、相同浓度的药物溶解介质、培养基、MTT、二甲基亚砜)，1％二甲基亚砜对照孔(细胞、培养基、MTT、二甲基亚砜)。
(4)结果
AMB及DMR078的体外肾毒性结果见表7。
表7体外肾毒性结果
化合物 EC50(μg/mL)
AMB 25.6
DMR078 1500
由EC50的数据可得：与AMB相比，化合物DMR078的体外肾细胞毒性明显降低。
根据本发明所公开的内容，在此所公开并要求保护的所有组合物和/或方法均无需进行过多实验即可进行制备和实施。虽然根据优选实施方案对本发明的组合物和方法进行了描述，但对本领域技术人员而言，在不背离本发明的概念、精神和范围的情况下，可对在此所述的组合物和/或方法以及所述方法的步骤或步骤的顺序进行改变。
本文所引用的所有文献的公开内容通过引用结合于此，引用程度为，他们提供示例性的、程序上和其他的细节补充本文所述内容。

Claims (20)

  1. 式[I]所示的化合物或其药用盐：
    Figure PCTCN2019124970-appb-100001
    其中，R 1选自亲水性多聚物部分；
    R 2选自氨基保护基或H；
    R 3选自H或C 1-4烃基或苯基；
    R 4为OH或H；
    可选择地，所述亲水性多聚物部分可以为H-(Gly)n-OH多聚物的部分，其中n取1-20的整数，较优地，n取2-15的整数，最优地，n为5；
    可选择地，所述亲水性多聚物部分可以为由Gly与AEEAc通过肽键构成的多肽部分，多肽部分的长度为2-20肽，较优地为2-15肽，最优地，为5肽；
    可选择地，所述氨基保护基为Fmoc或Boc。
  2. 根据权利要求1所述的化合物或其药用盐，所述亲水性多聚物部分是H-(Gly)n-OH多聚物的部分，其中n取2-20的整数，较优地，n取2-15的整数，最优地，n为5。
  3. 根据权利要求1所述的化合物或其药用盐，所述亲水性多聚物部分是由Gly与AEEAc通过肽键构成的5肽部分。
  4. 根据权利要求1所述的化合物或其药用盐，所述R 1选自-(Gly)n-、-(AEEAc)n-Gly-、-(AEEAc-Gly)n-AEEAc-、-AEEAc-Gly-、-(AEEAc-Gly)n-、-(Gly-AEEAc)n-Gly-、-(AEEAc)n-Gly-(AEEAc)m-、-(AEEAc)n-(Gly)m-和-(Gly)n-AEEAc-(Gly)m-，其中，n和m各自独立地为2-9的整数。
  5. 根据权利要求1或4所述的化合物或其药用盐，所述R 1选自-(AEEAc)n-Gly-、-(AEEAc-Gly)n-AEEAc-、-(AEEAc-Gly)n-、-(Gly-AEEAc)n-Gly-和-(AEEAc)n-Gly-(AEEAc)m-，其中，n和m各自独立地为2-9的整数且同时满足R 1中的AEEAc的数量大于等于3。
  6. 根据权利要求1-5任一项所述的化合物或其药用盐，其中，R 2为H。
  7. 根据权利要求1-6任一项所述的化合物或其药用盐，其中，R 3为H。
  8. 根据权利要求1-7任一项所述的化合物或其药用盐，所述化合物具有式[II]：
    Figure PCTCN2019124970-appb-100002
    其中，R 2为Fmoc、Boc或者H；
    R 3为H或C 1-4烃基或苯基；
    R 4为OH或H；
    可选择地，T是含有Gly或其衍生物、或者由AEEAc及Gly构成的组合多肽及其衍生物的多聚物部分，在这里T的长度范围为2-10肽，较优地，为3-7肽，最优地，为5肽。
  9. 根据权利要求1-8任一项所述的化合物或其药用盐，其中，R 3选自H、甲基、乙基、丙基、1-甲基乙基、丁基、1-甲基丙基、2-甲基丙基、1,1-二甲基乙基或苯基。
  10. 根据权利要求8或9所述的化合物或其药用盐，其中，R 3为H；R 2-T-选自：
    H-AEEAc-Gly-，
    H-Gly-Gly-，
    H-AEEAc-AEEAc-Gly-，
    H-(Gly) 5-，
    H-AEEAc-AEEAc-Gly-Gly-Gly-，
    H-Gly-Gly-AEEAc-Gly-Gly-，
    H-Gly-AEEAc-Gly-AEEAc-Gly-，
    H-AEEAc-Gly-AEEAc-Gly-AEEAc-，
    H-AEEAc-AEEAc-Gly-AEEAc-AEEAc-，
    H-AEEAc-Gly-AEEAc-Gly-AEEAc-Gly-，
    H-Gly-AEEAc-Gly-AEEAc-Gly-AEEAc-，
    H-Gly-AEEAc-Gly-AEEAc-Gly-AEEAc-Gly-，
    或H-(AEEAc) 9-Gly-。
  11. 根据权利要求8-10任一项所述的化合物或其药用盐，其中，R 3为H；R 2-T-为H-AEEAc-X-Y-Z-AEEAc-；X为AEEAc或Gly，Y为AEEAc或Gly，且Z为AEEAc或Gly。
  12. 根据权利要求1-11任一项所述的化合物或其药用盐，所述化合物具有式[V]：
    Figure PCTCN2019124970-appb-100003
    其中，R 3为H；H-T-为选自如下中的任一种：
    Figure PCTCN2019124970-appb-100004
    Figure PCTCN2019124970-appb-100005
  13. 根据权利要求1-12任一项所述的化合物或其药用盐，其中，所述化合物选自：
    Figure PCTCN2019124970-appb-100006
  14. 制备权利要求1-13任一项所述的化合物或其药用盐的方法，包括：(1)在树脂上固相合成多肽，将得到的多肽产物用弱酸进行裂解，过滤，旋蒸，然后加入第一有机溶剂溶解，旋蒸，用第二有机溶剂沉淀，干燥，得到含有氨基保护基的多肽；(2)将所述含有氨基保护基的多肽进行活化，然后在无水溶剂中与两性霉素B在催化剂量的碱存在下进行反应；以及任选地，(3)脱除含有氨基保护基的多肽部分的氨基保护基。
  15. 一种药物组合物，其中，所述组合物包含权利要求1-13任一项所述的化合物或其药用盐的药物组合物。
  16. 权利要求1-13任一项所述的化合物或其药用盐用于制备药物、优选抗真菌药物的用途。
  17. 权利要求16所述的用途，与两性霉素B相比，所述化合物或其药用盐具有相当的抗真菌活性，并具有改善的溶解性。
  18. 权利要求16或17所述的用途，所述真菌选自例如隐球菌、皮炎芽生菌、白色念珠菌、克柔念珠菌、近平滑念珠菌、粗球孢子菌、毛霉菌、申克孢子丝菌和烟曲菌。
  19. 一种对有需要的受试者预防和/或治疗真菌感染的方法，其中，向所述受试者给予治疗有效量的权利要求1-13任一项所述的化合物或其药用盐。
  20. 权利要求19所述的方法，其中，所述真菌感染选自如下真菌的感染：隐球菌(Cryptococcus)、皮炎芽生菌(Blastomyces dermatitis)、白色念珠菌(Candida albicans)、克柔念珠菌(Candida krusei)、近平滑念珠菌(Candida parapsilosis)、粗球孢子菌(Coccidioides immitis)、毛霉菌(Mucor)、申克孢子丝菌(Sporothrix schenckii)和烟曲菌(Aspergillus fumigatus)。
PCT/CN2019/124970 2018-12-13 2019-12-13 两性霉素b肽衍生物 Ceased WO2020119773A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201980079769.8A CN113166188B (zh) 2018-12-13 2019-12-13 两性霉素b肽衍生物

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201811521926.X 2018-12-13
CN201811521926 2018-12-13

Publications (1)

Publication Number Publication Date
WO2020119773A1 true WO2020119773A1 (zh) 2020-06-18

Family

ID=71076256

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2019/124970 Ceased WO2020119773A1 (zh) 2018-12-13 2019-12-13 两性霉素b肽衍生物

Country Status (2)

Country Link
CN (1) CN113166188B (zh)
WO (1) WO2020119773A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114133428A (zh) * 2021-12-09 2022-03-04 华东理工大学 两性霉素b亲水性多肽衍生物及其应用

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003000738A2 (de) * 2001-06-21 2003-01-03 Fresenius Kabi Deutschland Gmbh Wasserlösliches, einen aminozucker aufweisendes antibiotikum in form eines polysaccharidkonjugats
CN107375939A (zh) * 2017-07-19 2017-11-24 中国药科大学 用于治疗真菌感染的两性霉素b多肽类水凝胶载药体系
CN108714151A (zh) * 2018-06-15 2018-10-30 中国科学院长春应用化学研究所 一种两性霉素b抗真菌纳米药物及其制备方法

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4272525A (en) * 1978-10-23 1981-06-09 Schering Corporation Derivatives of polyene macrolide antibiotics containing an amino sugar moiety, process for the preparation thereof, and pharmaceutical compositions containing them
ZA795508B (en) * 1978-10-23 1981-05-27 Schering Corp Derivatives of polyene macrolide antibiotics containing an amino sugar moiety, process for the preparation thereof, and pharmaceutical compositions containing them
WO2001051061A1 (en) * 2000-01-14 2001-07-19 Intrabiotics Pharmaceuticals, Inc. Derivatives of polyene macrolides and preparation and use thereof
KR20150027217A (ko) * 2012-06-15 2015-03-11 블러트 에스.에이. 항진균성 항생 암포테리신 b의 n-치환된 제2 세대 유도체, 및 이의 제조 및 응용 방법
US9750817B2 (en) * 2015-08-13 2017-09-05 Ramot At Tel-Aviv University Ltd. Amphotericin B derivatives

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003000738A2 (de) * 2001-06-21 2003-01-03 Fresenius Kabi Deutschland Gmbh Wasserlösliches, einen aminozucker aufweisendes antibiotikum in form eines polysaccharidkonjugats
CN107375939A (zh) * 2017-07-19 2017-11-24 中国药科大学 用于治疗真菌感染的两性霉素b多肽类水凝胶载药体系
CN108714151A (zh) * 2018-06-15 2018-10-30 中国科学院长春应用化学研究所 一种两性霉素b抗真菌纳米药物及其制备方法

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114133428A (zh) * 2021-12-09 2022-03-04 华东理工大学 两性霉素b亲水性多肽衍生物及其应用
CN114133428B (zh) * 2021-12-09 2023-10-27 华东理工大学 两性霉素b亲水性多肽衍生物及其应用

Also Published As

Publication number Publication date
CN113166188A (zh) 2021-07-23
CN113166188B (zh) 2023-06-20

Similar Documents

Publication Publication Date Title
CN103550781B (zh) 树状大分子自组装药物载体及其制备方法和应用
BRPI0721259A2 (pt) composto, composiÇço farmacÊutica, uso de composto, e, processo para a fabricaÇço de composto
CN106334194B (zh) 缀合物及其制备方法和用途
CN108164584A (zh) Vap多肽及其在制备靶向诊疗肿瘤药物中的应用
CN114790226A (zh) 一种抗菌肽及其制备方法
WO2017152756A1 (zh) cRGD-厄洛替尼缀合物及其制备方法
CN113121627A (zh) 两性霉素b甲酯肽衍生物及其制备方法
EP4410816A1 (en) Peptide
CN113166188B (zh) 两性霉素b肽衍生物
CN104311641B (zh) 一种抗术后疤痕的可降解多支化糖肽水凝胶及其制备方法
CN111423493B (zh) 一种棕榈酸化抗酶解抗菌肽及其制备方法和应用
CN110862458B (zh) 具有酸激活抗肿瘤活性的组合肽及其临床应用
CN111247162B (zh) 多黏菌素类似物及其制备方法
CN118384289A (zh) 一种肿瘤靶向肽及多肽偶联药物的制备方法及应用
CN110669087B (zh) 两性霉素b肽衍生物及其制备方法
CN110917139A (zh) 多分枝生物素修饰的乳腺癌靶向脂质体的制备和应用
CN117510611A (zh) 一种抗菌肽及其制备方法和应用
JP4195089B2 (ja) 亜酸化物単位から製造される巨大環式化合物
CN115785222A (zh) 一类酸激活抗肿瘤肽及其应用
CA3230326A1 (en) Peptides with antimicrobial activities
KR101732124B1 (ko) 신규한 피부투과성 펩타이드 및 이것의 용도
CN109897089A (zh) 一种整合素配体vs多肽及其在制备肿瘤靶向诊治递药系统中的应用
JP4817335B2 (ja) 新規抗菌性ペプチド
CN112300250B (zh) 阿尼芬净类似物及其制备方法
JP3472821B2 (ja) 新規ペプチド、及び当該ペプチドを用いた組成物

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 19894551

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 19894551

Country of ref document: EP

Kind code of ref document: A1