WO2019085926A1 - Analogue de polymyxine et son procédé de préparation - Google Patents

Analogue de polymyxine et son procédé de préparation Download PDF

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Publication number
WO2019085926A1
WO2019085926A1 PCT/CN2018/112889 CN2018112889W WO2019085926A1 WO 2019085926 A1 WO2019085926 A1 WO 2019085926A1 CN 2018112889 W CN2018112889 W CN 2018112889W WO 2019085926 A1 WO2019085926 A1 WO 2019085926A1
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Prior art keywords
dab
leu
deletion
ala
polypeptide
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PCT/CN2018/112889
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English (en)
Chinese (zh)
Inventor
冯军
张喜全
路建光
东圆珍
张友
唐汉卿
徐宏江
Original Assignee
上海医药工业研究院
正大天晴药业集团股份有限公司
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Application filed by 上海医药工业研究院, 正大天晴药业集团股份有限公司 filed Critical 上海医药工业研究院
Priority to CN201880070045.2A priority Critical patent/CN111247162B/zh
Publication of WO2019085926A1 publication Critical patent/WO2019085926A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/12—Cyclic peptides, e.g. bacitracins; Polymyxins; Gramicidins S, C; Tyrocidins A, B or C
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/50—Cyclic peptides containing at least one abnormal peptide link
    • C07K7/54—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring
    • C07K7/60—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring the cyclisation occurring through the 4-amino group of 2,4-diamino-butanoic acid
    • C07K7/62—Polymyxins; Related peptides
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention belongs to the field of medicine, relates to an antibacterial peptide and a preparation method and application thereof, and particularly relates to a group of polymyxin analogs having high antibacterial activity and a synthetic preparation and application thereof.
  • Polymyxin is a class of cyclic peptide antibiotics produced by Bacillus polymyxa discovered in 1947. It consists of various components, including polymyxin A, polymyxin B, polymyxin C, and more. Colistin D, polymyxin E, usually have a narrow antibacterial spectrum and are only resistant to Gram-negative bacteria. It is commonly used clinically to treat infections caused by Gram-negative bacteria. However, due to its severe nephrotoxicity and neurotoxicity, it is gradually replaced by other antibiotics (aminoglycosides, etc.) with low toxicity and good antibacterial effect. Until the emergence of multi-drug resistant bacteria in the past 20 years, polymyxin has once again entered the field of vision as a clinical therapeutic drug.
  • polymyxin products currently on the market are mainly polymyxin B sulfate, polymyxin E sulfate and sodium polymyxin E methanesulfonate.
  • the antibacterial activity of polymyxin B and polymyxin E is slightly different. In general, the activity of polymyxin B is stronger than that of polymyxin E (Chen Guanrong, the clinical application of polymyxin and the response to super bacteria [J]. Medical Herald, 2011, 30 (2): 135-140).
  • Natural polymyxin is generally composed of a fatty acyl chain and a cyclic heptapeptide linked by a linear tripeptide, wherein the 4-position amino acid L-Dab ( ⁇ , ⁇ -diaminobutyric acid) is condensed with the 10-position amino acid L-Thr to form a heptapeptide. ring.
  • L-Dab ⁇ , ⁇ -diaminobutyric acid
  • the structures of polymyxin B and polymyxin E are very similar, with only differences in the 6 amino acids. Among them, the 6-position amino acid of polymyxin B is D-Phe, and the 6-position amino acid of polymyxin E is D-Leu (Cui, et.al. Research Development of Polymyxins [J]. World Notes on Antibiotics, 2015 , 36(5): 205-210).
  • the invention relates to a polypeptide and a preparation method and application thereof, in particular to a group of polymyxin analogs having high antibacterial activity, and synthetic preparation and application thereof.
  • the application relates to polypeptides and pharmaceutically acceptable salts thereof, which polypeptides have at least 70% sequence identity to the sequences of Formula I.
  • the polypeptide has the following sequence: R1-Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-Xaa7-Xaa8-Xaa9-Xaa10-Xaa11-Xaa12 (Formula I), wherein R1 is: Fat a straight or branched C 6 -C 20 acyl group or deletion; Xaa1 is: Leu, Ala, AEEAc, Phe, IPhe, BrPhe, or 3FPhe, or a deletion; Xaa2 is: Leu, Ala, Arg, or AEEAc , or deletion; Xaa3 is: Dab, Ala, Leu, or Orn, or deletion; Xaa4 is: Thr, Ala, Dab, or Lys;
  • the polypeptide is a cyclic peptide.
  • the polypeptide is formed by the condensation of Xaa6 with a side chain group of Xaa12 to form an amide linkage, and optionally, the pendant group comprises an amino group and/or a carboxyl group.
  • the polypeptide is obtained by dehydration of an amino group with a carboxyl group to form an amide bond.
  • the R 1 is selected from the group consisting of heptanoyl, methylheptanoyl, octanoyl, methyloctanoyl, decanoyl, methyl decanoyl, decanoyl, methyl decanoyl, lauroyl, myristoyl, Palmitoyl, 17-carboxylic acid-heptadecanoyl or a deletion, preferably n-octanoyl, decanoyl, lauroyl, palmitoyl, 17-carboxylic acid-heptadecanoyl or a deletion, more preferably n-octanoyl or a deletion
  • Xaa1 is preferably Leu or deleted
  • Xaa2 is preferably Leu
  • Xaa3 is preferably Dab
  • Xaa3 is preferably Dab
  • Xaa4 is preferably Thr
  • Xaa4 is preferably Dab
  • polypeptide has the formula:
  • R1 is: an aliphatic straight-chain or branched C 6 -C 20 acyl group or a deletion
  • Xaa1 is: Leu, Ala, AEEAc, Phe, IPhe, BrPhe, 3FPhe or a deletion
  • Xaa2 is: Leu, Ala, Arg, AEEAc or deletion
  • Xaa3 is: Dab, Ala, Leu, Orn or deletion
  • Xaa4 is: Thr, Ala, Dab or Lys
  • Xaa5 is: Dab, Lys, Orn, Ala or deletion
  • Xaa6 is: Lys or Dab
  • Xaa7 is: Dab, Ala, Lys, Orn or deletion
  • Xaa8 is: D-Leu, D-Phe, Lys or D-Ala
  • Xaa9 is: Leu or Ala
  • Xaa10 is: Dab, Leu, Orn, Ala, D-
  • the R1 is selected from the group consisting of heptanoyl, methylheptanoyl, octanoyl, methyloctanoyl, decanoyl, methyldecanoyl, decanoyl, methyldecanoyl, lauroyl, myristoyl , palmitoyl, 17-carboxylic acid-heptadecanoyl or a deletion, preferably n-octanoyl, decanoyl, lauroyl, palmitoyl, 17-carboxylic acid-heptadecanoyl or a deletion, more preferably n-octanoyl or Deletion;
  • Xaa1 is preferably Leu or deleted, Xaa2 is preferably Leu, Xaa3 is preferably Dab, Xaa3 is preferably Dab, Xaa4 is preferably Thr, Xaa5 is preferably Dab,
  • polypeptide has the formula:
  • R1 is: an aliphatic straight-chain or branched C 6 -C 20 acyl group or a deletion
  • Xaa1 is: Leu, Ala, AEEAc, Phe, IPhe, BrPhe, 3FPhe or a deletion
  • Xaa2 is: Leu, Ala, Arg, AEEAc or deletion
  • Xaa3 is: Dab, Ala, Leu, Orn or deletion
  • Xaa4 is: Thr, Ala, Dab, Lys
  • Xaa5 is: Dab, Lys, Orn, Ala or deletion
  • Xaa7 is: Dab, Ala, Lys, Orn or deletion
  • Xaa8 is: D-Leu, D-Phe, Lys, D-Ala
  • Xaa9 is: Leu, Ala
  • Xaa10 is: Dab, Leu, Orn, Ala, D-Leu or deletion
  • Xaa11 is: Da
  • polypeptide has the formula:
  • R1 is: an aliphatic straight-chain or branched C 6 -C 20 acyl group or a deletion
  • Xaa1 is: Leu, Ala, AEEAc, Phe, IPhe, BrPhe, 3FPhe or a deletion
  • Xaa2 is: Leu, Ala, Arg, AEEAc or deletion
  • Xaa3 is: Dab, Ala, Leu, Orn or deletion
  • Xaa4 is: Thr, Ala, Dab or Lys
  • Xaa5 is: Dab, Lys, Orn, Ala or deletion
  • Xaa7 is: Dab, Ala, Lys, Orn or deletion
  • Xaa8 is: D-Leu, D-Phe, Lys or D-Ala
  • Xaa9 is: Leu or Ala
  • Xaa10 is: Dab, Leu, Orn, Ala, D-Leu or deletion
  • Xa11 is: Dab
  • the R1 is selected from the group consisting of heptanoyl, methylheptanoyl, octanoyl, methyloctanoyl, decanoyl, methyldecanoyl, decanoyl, methyldecanoyl, lauroyl, myristoyl , palmitoyl, 17-carboxylic acid-heptadecanoyl or a deletion, preferably n-octanoyl, decanoyl, lauroyl, palmitoyl, 17-carboxylic acid-heptadecanoyl or a deletion, more preferably n-octanoyl or Deletion;
  • Xaa1 is preferably Leu or deleted, Xaa2 is preferably Leu, Xaa3 is preferably Dab, Xaa4 is preferably Dab, Xaa4 is preferably Thr, Xaa5 is preferably Dab,
  • the polypeptide is a peptide or cyclic peptide consisting of the sequence represented by R1-Leu-Dab-Thr-Dab-Xaa6-Dab-Xaa8-Leu-Dab-Dab-Glu, wherein R1 is preferably n-octanoyl , decanoyl, lauroyl, palmitoyl, 17-carboxyheptadecanoyl or a deletion, more preferably n-octanoyl or a deletion; Xaa6 is preferably Lys or Dab, and Xaa8 is preferably D-Phe or D-Leu.
  • the polypeptide is a peptide or cyclic peptide consisting of the sequence represented by R1-Dab-Thr-Dab-Xaa6-Dab-Xaa8-Leu-Dab-Dab-Glu, wherein R1 is preferably n-octane Acyl, decanoyl, lauroyl, palmitoyl, 17-carboxylic acid-heptadecanoyl or a deletion, more preferably n-octanoyl or a deletion; Xaa6 is preferably Lys or Dab, and Xaa8 is preferably D-Phe or D-Leu.
  • the polypeptide comprises the sequence H 2 N-Leu-Dab-Thr-Dab-Lys-Dab-D-Phe-Leu-Dab-Dab-Glu, or the sequence H 2 N-Dab- A peptide or cyclic peptide of Thr-Dab-Lys-Dab-D-Phe-Leu-Dab-Dab-Glu.
  • the polypeptide of the invention comprises at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95 with the above polypeptide. %, 96%, 97%, 98%, 99% sequence identity amino acid sequence.
  • the polypeptide of the invention has an amino acid sequence of at least 1, 2, 3, 4, 5, 6 amino acid additions, substitutions, and/or deletions as compared to the polypeptide described above.
  • the polypeptide of the invention has at least 1, 2, 3, 4, 5, 6 amino acid substitutions compared to the above polypeptide, wherein the substitution can be a conservative substitution, And/or approximate replacement.
  • the polypeptide of the invention is a truncated amino acid of six, seven, eight, nine, ten, eleven amino acid lengths of the above polypeptide.
  • polypeptides provided herein are a low toxicity polypeptide.
  • the application also provides the use of the aforementioned polypeptide or a pharmaceutically acceptable salt thereof.
  • the polypeptides of the present invention can be used for bacteriostatic, antibacterial or antibacterial substances.
  • the antimicrobial substance is an antimicrobial or antimicrobial agent.
  • the bacterium is a bacterium or a fungus. It is particularly surprising that the polypeptides of the invention can be used against anti-negative or anti-resistant bacteria, or for the preparation of anti-negative or anti-resistant bacteria.
  • the bacterium, fungus or drug resistant bacterium is Acinetobacter baumanii, drug resistant Acinetobacter baumanii, Pseudomonas aeruginosa, resistant One or more of Pseudomonas aeruginosa, Bacillus subtilis, Staphylococcus aureus, E.
  • coli and/or candida Albicans, More preferably, it is Pseudomonas aeruginosa, a drug-resistant Acinetobacter baumannii, and a drug-resistant Pseudomonas aeruginosa.
  • the invention relates to an antimicrobial substance.
  • the antimicrobial substance is an antimicrobial or antimicrobial agent.
  • the antibacterial substance or the microbial agent of the present invention includes the polypeptide of the present invention, and one or more of a pharmaceutically acceptable salt and/or a carrier and an excipient.
  • the present application is directed to a pharmaceutical composition
  • a pharmaceutical composition comprising a polypeptide of the present application, or a pharmaceutically acceptable salt thereof.
  • the pharmaceutical compositions of the present application further comprise a pharmaceutically acceptable excipient, diluent or carrier.
  • the present application relates to the use of the polypeptide or a pharmaceutically acceptable salt thereof for the manufacture of a medicament and/or a pharmaceutical composition for preventing or treating an infectious disease.
  • the present application relates to a method of treating a disease in a mammal comprising administering to a mammal in need of such treatment, preferably a human, a therapeutically effective amount of a polypeptide of the invention, or a pharmaceutically acceptable salt thereof, or a pharmaceutical combination thereof Things.
  • the above combination therapies can be administered in the following manner: (a) a single pharmaceutical composition comprising a polypeptide of the present application, at least one additional therapeutic agent described herein, and a pharmaceutically acceptable excipient, diluent or carrier; Or (b) two separate pharmaceutical compositions comprising (i) a first composition comprising a polypeptide of the present application and a pharmaceutically acceptable excipient, diluent or carrier, and (ii) comprising at least one An additional therapeutic agent and a second composition of a pharmaceutically acceptable excipient, diluent, or carrier.
  • the pharmaceutical compositions can be administered simultaneously or sequentially and in any order.
  • the invention relates to a set of polypeptides or pharmaceutically acceptable salts thereof, the amino acid sequences of which are as follows:
  • polypeptide is a cyclic peptide having the amino acid sequence shown below:
  • R1 is: an aliphatic straight or branched C 6 -C 20 acyl group or a deletion
  • Xaa1 is: Leu, Ala, AEEAc, Phe, IPhe, BrPhe, 3FPhe or deletion;
  • Xaa2 is: Leu, Ala, Arg, AEEAc or deletion;
  • Xaa3 is: Dab, Ala, Leu, Orn or missing;
  • Xaa4 is: Thr, Ala, Dab, Lys;
  • Xaa5 is: Dab, Lys, Orn, Ala or missing;
  • Xaa6 is: Lys, Glu, Dab;
  • Xaa7 is: Dab, Ala, Lys, Orn or missing;
  • Xaa8 is: D-leu, D-Phe, Lys, D-Ala;
  • Xaa9 is: Leu, Ala;
  • Xaa10 is: Dab, Leu, Orn, Ala, D-Leu or deletion;
  • Xaa11 is: Dab, Leu, Orn, Ala, D-Leu or deletion;
  • Xaa12 is: Glu, Lys, Asp;
  • the R1 is selected from the group consisting of heptanoyl, methylheptanoyl, octanoyl, methyloctanoyl, decanoyl, methyldecanoyl, decanoyl, methyldecanoyl, lauroyl, meat Myristoyl, palmitoyl, 17-carboxylic acid-heptadecanoyl or a deletion
  • Xaa1 is preferably Leu or deleted
  • Xaa2 is preferably Leu
  • Xaa3 is preferably Dab
  • Xaa4 is preferably Thr
  • Xaa5 is preferably Dab
  • Xaa6 is preferably Lys or Dab
  • Xaa7 is preferably Dab
  • Xaa8 is preferably D-Phe or D-Leu
  • Xaa9 is preferably Leu
  • Xaa10 is preferably Dab
  • Xaa11 is preferably
  • amino acid residues of the polypeptides involved in the present invention include natural amino acids and unnatural amino acids, wherein the three-letter codes corresponding to the natural amino acids involved are shown in Table 1, the unnatural amino acids involved and the corresponding letter codes and The structure is shown in Table 2.
  • the amino acid according to the present invention is an L-form amino acid by default unless it is specifically limited.
  • the polypeptide of the present invention is a cyclic peptide which is obtained by dehydration of an amino group (or a carboxyl group) of a Xaa6 and Xaa12 side chain with a carboxyl group (or an amino group) to form an amide bond.
  • the peptides of the present invention may have a C-terminus either in the form of a carboxyl group or in the form of an amide, preferably in the form of an amide.
  • a cyclic peptide (ZAMP18) is disclosed, the amino acid sequence of which is:
  • a further preferred embodiment of the invention discloses a cyclic peptide with a fatty chain (ZAMP29) having an amino acid sequence of:
  • a group of cyclic peptides CTAMP-41, CTAMP-42, CTAMP-43, CTAMP-44, CTAMP-45, CTAMP-46, CTAMP-47, ZAMP-3, ZAMP-4 are disclosed.
  • the polypeptide of the invention may be a peptide having a linear primary structure, and in another aspect, the polypeptide of the invention may be a cyclic peptide.
  • the polypeptide of the invention comprises at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92 of the peptide or cyclic peptide shown in any of the above sequences. %, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity amino acid sequence.
  • the polypeptide of the invention comprises a addition, substitution and/or deletion of at least 1, 2, 3, 4, 5, 6 amino acids with a polypeptide as set forth in any of the above sequences. Amino acid sequence.
  • the polypeptide of the invention comprises a substitution of at least 1, 2, 3, 4, 5, 6 amino acids with a polypeptide as set forth in any of the above sequences, wherein the substitution may be Conservative substitutions, and/or approximate replacements.
  • the polypeptide of the invention is a continuous truncation of six, seven, eight, nine, ten, eleven amino acid lengths of the polypeptide shown in any of the above sequences.
  • the invention also provides a process for the preparation of the above peptide or cyclic peptide.
  • the method comprises: synthesizing the polypeptide.
  • the above polypeptides can be prepared using solid phase synthesis techniques, including:
  • step (1) The product of the step (1) is cleaved with a strong acid; a side chain protecting group scavenger is added, filtered, and the polypeptide is precipitated with 5-20 volumes of an organic solvent, centrifuged, and the organic solvent is repeatedly washed and precipitated, and dried to obtain a crude peptide.
  • step (1) comprises the following steps:
  • the amino protecting group refers to a chemical group introduced to protect the amino group involved in the condensation reaction.
  • the amino protecting group includes, but is not limited to, t-butoxycarbonyl (Boc), benzyloxycarbonyl (Z) or 9-fluorenyl-methoxycarbonyl (Fmoc), preferably 9-fluorenyl-methoxycarbonyl (Fmoc).
  • Arg can be protected with pentamethylbenzofuran-5-sulfonyl (Pbf); Dab, Orn can be tert-butoxycarbonyl ( Boc) protection; Thr can be protected with tert-butyl (tBu).
  • Fmoc-L-Dab(Boc)-OH has the structure shown below:
  • the protecting group includes, but is not limited to, a reasonable selection according to a specific situation.
  • the solvent used in the liquid phase environment of the step (a) is selected from the group consisting of dimethylformamide (DMF), dichloromethane (DCM), N-methylpyrrolidone (NMP), preferably DMF.
  • DMF dimethylformamide
  • DCM dichloromethane
  • NMP N-methylpyrrolidone
  • the removal of the amino protecting group in the step (a) requires the removal of the amino protecting group, and the removal agent of the amino protecting group is selected from piperidine (PIP) solution at a concentration of 10-40% (PIP/DMF).
  • PIP piperidine
  • the 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 consisting of a carbodiimide type reagent or a benzotriazole salt type reagent and 1-hydroxybenzotriazole (HOBt). .
  • the carbodiimide type reagent includes, but is not limited to, dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC) or N-diaminopropyl-N-ethylcarbodiimide. (EDC).
  • DCC dicyclohexylcarbodiimide
  • DIC diisopropylcarbodiimide
  • EDC N-diaminopropyl-N-ethylcarbodiimide.
  • the benzotriazolium salt type reagent includes, but is not limited to, 2-(1H-benzotriazo L-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU), O-benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate (HBTU), benzotriazole-1-oxotris(dimethylamino) hexafluorophosphate Phosphorus (BOP) or benzotriazol-1-yl-oxytripyrrolidinylphosphonium hexafluorophosphate (PyBOP).
  • TBTU 2-(1H-benzotriazo L-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate
  • HBTU O-benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate
  • BOP
  • the coupling reagent is preferably diisopropylcarbodiimide (DIC) and 1-hydroxybenzotriazole (HOBt), or 2-(1H-benzotriazo L-1-yl)-1,1. , 3,3-tetramethyluronium tetrafluoroborate (TBTU) and 1-hydroxybenzotriazole (HOBt), further preferably DIC (diisopropylcarbodiimide) and 1-hydroxybenzotriazole (HOBt).
  • the "monitoring” in step (a) uses a ninhydrin assay to monitor the condensation reaction of the polypeptide.
  • the sequential linking of amino acids in the step (a) means that the amino acids are linked one by one from the C-terminus to the N-terminus according to the amino acid sequence of the polypeptide.
  • the cyclized amino acid in the step (b) is Xaa6 and Xaa12, and a ring is formed by forming an amide bond between a side chain amino group and a carboxyl group.
  • step (b) an orthogonal protection strategy is employed for the ring formation, the amino protecting group on the side chain of the ring-forming amino acid is allyloxycarbonyl (Alloc), and the carboxyl protecting group is allyl (OAll).
  • step (b) requires the addition of a remover, and the remover used is tetrakis(triphenylphosphine)palladium ((Pd(PPh 3 ) 4 )).
  • step (b) requires the addition of a scavenger, and the scavenger may be selected from H 3 N ⁇ BH 3 , Me 2 NH ⁇ BH 3 or PhSiH 3 , preferably PhSiH 3 .
  • the cyclization reaction in the step (b) requires the addition of a coupling reagent consisting of a carbodiimide type reagent or a tertiary amine type and a benzotriazole salt type reagent or a pyridine triazolium salt type.
  • carbodiimide type reagent includes, but is not limited to, dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC) or N-diaminopropyl-N-ethylcarbamate Imine (EDC).
  • DCC dicyclohexylcarbodiimide
  • DIC diisopropylcarbodiimide
  • EDC N-diaminopropyl-N-ethylcarbamate Imine
  • the tertiary amine type reagents include, but are not limited to, N,N-diisopropylethylamine (DIEA).
  • the benzotriazolium salt type reagent includes, but is not limited to, 2-(1H-benzotriazo L-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU), O-benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate (HBTU), benzotriazole-1-oxotris(dimethylamino) hexafluorophosphate Phosphorus (BOP) or benzotriazol-1-yl-oxytripyrrolidinylphosphonium hexafluorophosphate (PyBOP).
  • TBTU 2-(1H-benzotriazo L-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate
  • HBTU O-benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate
  • BOP
  • the pyridine triazolium salt type includes, but is not limited to, (3H-1,2,3-triazolo[4,5-b]pyridin-3-yloxy)tri-1-pyrrolidinyl hexafluorophosphate (PyAOP), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), 7-azabenzotriazole-1 - methoxytris(dimethylamino)phosphine hexafluorophosphate (AOP).
  • 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) or N,N-diisopropylethylamine (DIEA) and 2-(7-azo Benzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU).
  • DIC diisopropylcarbodiimide
  • HOBt 1-hydroxybenzotriazole
  • TBTU 3,3-tetramethylurea tetrafluoroborate
  • DIEA N,N-diisopropylethylamine
  • DIEA N,N-diisopropylethylamine
  • HATU 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate
  • the step (1) further comprises: (c) the resin peptide obtained in the step (b) is linked to a fatty acid chain, which may be a mono-, di- or other polybasic fatty acid chain.
  • the coupling agent in the step (c) is diisopropylcarbodiimide (DIC) and 1-hydroxybenzotriazole (HOBt), and the reaction solvent is DMF.
  • the side chain protecting group scavenger according to the step (2) includes, but not limited to, thioanisole, triisopropylsilane, phenol, water, 1,2-ethanedithiol, m-cresol or any two or two of the above
  • the above combination is prepared and prepared by trifluoroacetic acid or hydrofluoric acid at 5-20% (V/V).
  • the step (1) is carried out by using Wang resin; if the C-terminus of the polypeptide of the present invention is in the form of an amide, the step (1) is synthesized by using Rink Amide MBHA resin. .
  • polypeptide preparation method provided by the present invention may further comprise a purification step in order to meet the quality requirements for medical use.
  • Purification methods employed include, but are not limited to, reverse phase chromatography or ion exchange chromatography, preferably reverse phase chromatography.
  • the in vitro antibacterial activity of the polypeptides of the invention can be identified by determining its minimum inhibitory concentration (MIC).
  • MIC minimum inhibitory concentration
  • NCCLS American Clinical Laboratory Standardization Committee
  • MH Mueller-Hinton
  • RPMI-1640 modified RPMI-1640. base.
  • Amphotericin B, polymyxin E sulfate and vancomycin hydrochloride were used as positive controls.
  • antimicrobial peptides provided by the present invention have higher antibacterial activity, particularly anti-negative bacteria (such as Pseudomonas aeruginosa) or anti-resistant bacteria (such as drug-resistant Acinetobacter baumannii, drug-resistant patina)
  • anti-negative bacteria such as Pseudomonas aeruginosa
  • anti-resistant bacteria such as drug-resistant Acinetobacter baumannii, drug-resistant patina
  • aliphatic straight-chain or branched C 6 -C 20 acyl group means a substituent containing a carbonyl group moiety and a non-carbonyl group moiety, and the non-carbonyl group includes an aliphatic straight chain or a branch.
  • Chain groups include, but are not limited to, alkyl groups, cycloalkyl groups, alkene groups, and alkynyl groups.
  • the invention includes, but is not limited to, acyl groups found in known polymyxin compounds, including heptanoyl, methylheptanoyl (including (S)-6-methylheptanoyl), octanoyl, methyl octyl Acyl (including (S)-6-methyloctanoyl, (S)-7-methyloctanoyl), decanoyl, methyldecanoyl (including (S)-6-methyldecanoyl, (S)- 7-methyldecanoyl and (S)-8-methyldecanoyl) and decanoyl.
  • acyl groups found in known polymyxin compounds including heptanoyl, methylheptanoyl (including (S)-6-methylheptanoyl), octanoyl, methyl octyl Acyl (including (S)-6-methyloctanoyl, (S)-7-methyloc
  • lauroyl, palmitoyl, myristoyl and 17-carboxylic acid-heptadecanoyl are also included.
  • the "lauroyl”, “palmitoyl” and “myristoyl” refer to a hydroxy group in lauric acid, palmitic acid, and myristic acid substituted.
  • the 17-carboxylic acid-heptadecanoyl group generally means:
  • fatty acid chain refers to a substituent comprising an aliphatic straight or branched chain moiety and one, two or more carboxyl group moieties including, but not limited to, an alkyl group, a ring An alkyl group, an alkene group, an alkynyl group or the like, the one, two or more carboxyl group moieties are meant to comprise a monocarboxyl group, a dicarboxyl group or more, and the term “monobasic fatty acid chain” means a substituent comprising an aliphatic straight or branched chain moiety and a carboxyl group moiety, the term “dibasic fatty acid chain” means a substituent comprising an aliphatic straight or branched chain moiety and two carboxyl group moieties, the term “ “polybasic fatty acid chain” means a substituent comprising an aliphatic straight or branched chain moiety and a plurality of carboxyl group moieties
  • substituted means that any one or more hydrogen atoms on a particular atom are replaced by a substituent as long as the valence of the particular atom is normal and the substituted compound is stable.
  • an ethyl group “optionally” substituted with halo refers to an ethyl group may be unsubstituted (CH 2 CH 3), monosubstituted (e.g., CH 2 CH 2 F), polysubstituted (e.g. CHFCH 2 F, CH 2 CHF 2, etc.) or completely substituted (CF 2 CF 3 ). It will be understood by those skilled in the art that for any group containing one or more substituents, no substitution or substitution pattern that is sterically impossible to exist and/or which cannot be synthesized is introduced.
  • C mn means having mn carbon atoms in this moiety.
  • carbon 3-10 cycloalkyl means that the cycloalkyl has 3 to 10 carbon atoms.
  • Carbon 0-6 alkylene means that the alkylene group has 0 to 6 carbon atoms, and when the alkylene group has 0 carbon atoms, the group is a bond.
  • C1-6 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 eg, R
  • its definition in each case is independent.
  • each R has an independent option.
  • halo or halogen refers to fluoro, chloro, bromo and iodo.
  • acyl refers to a -CO- group.
  • hydroxy refers to an -OH group.
  • cyano refers to a -CN group.
  • mercapto refers to a -SH group.
  • amino means -NH 2 group.
  • nitro refers to a -NO 2 group.
  • alkyl refers to a hydrocarbon group of the formula C n H 2n +.
  • the alkyl group can be straight or branched.
  • hydrocarbon 1 - 6 alkyl refers to a monovalent straight or branched aliphatic group containing from 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.).
  • alkyl moiety i.e., alkyl
  • an alkoxy group an alkylamino group, a dialkylamino group, an alkylsulfonyl group, and an alkylthio group
  • alkyl i.e., alkyl
  • alkoxy refers to -O-alkyl
  • alkylthio refers to -S-alkyl.
  • alkylamino refers to -NH(alkyl).
  • dialkylamino refers to -N(alkyl) 2 .
  • alkenyl refers to a straight or branched unsaturated aliphatic hydrocarbon group having at least one double bond consisting of a carbon atom and a hydrogen atom.
  • alkenyl groups include, but are not limited to, ethenyl, 1-propenyl, 2-propenyl, 1-butenyl, isobutenyl, 1,3-butadienyl, and the like.
  • alkynyl means a straight or branched unsaturated aliphatic hydrocarbon group having at least one triple bond composed of a carbon atom and a hydrogen atom.
  • alkynyl groups include, but are not limited to, ethynyl (-C a carbon atom), 1-propynyl (-C group (atoms, and 3), 2-propynyl (-CH 2 -CH (original) And 1,3-butadiynyl (-C alkynyl (sub and hydrogen) and the like.
  • cycloalkyl refers to a carbocyclic ring that is fully saturated and can exist as a single ring, fused ring or spiro ring. Unless otherwise indicated, the carbocyclic ring is typically a 3 to 10 membered ring, preferably a 3 to 8 membered ring.
  • Non-limiting examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, norbornyl (bicyclo[2.2.1]heptyl), bicyclo[2.2.2]octyl, diamond Alkyl and the like.
  • aryl refers to an all-carbon monocyclic or fused polycyclic aromatic ring group having a conjugated ⁇ -electron system.
  • an aryl group can 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 containing at least one ring atom selected from N, O, S, the remaining ring atoms being C, and having at least one aromatic ring.
  • Preferred heteroaryl groups have a single 4 to 8 membered ring, especially a 5 to 8 membered ring, or a plurality of fused rings containing from 6 to 14, especially from 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, fluorenyl, isodecyl and the like.
  • treating means administering a compound or formulation described herein to prevent, ameliorate or eliminate a disease or one or more symptoms associated with the disease, and includes:
  • terapéuticaally effective amount means (i) treating or preventing a particular disease, condition or disorder, (ii) alleviating, ameliorating or eliminating one or more symptoms of a particular disease, condition or disorder, or (iii) preventing or delaying The amount of a compound of the present application in which one or more symptoms of a particular disease, condition, or disorder are described herein.
  • the amount of a compound of the present application which constitutes a “therapeutically effective amount” will vary depending on the compound, the condition and severity thereof, the mode of administration, and the age of the mammal to be treated, but can be routinely determined by those skilled in the art It is determined by its own knowledge and the present disclosure.
  • pharmaceutically acceptable is for those compounds, materials, compositions and/or dosage forms that are within the scope of sound medical judgment and are suitable for use in contact with human and animal tissues without Many toxic, irritating, allergic reactions or other problems or complications are commensurate with a reasonable benefit/risk ratio.
  • a metal salt, an ammonium salt, a salt with an organic base, a salt with an inorganic acid, a salt with an organic acid, a salt with a basic or acidic amino acid, or the like can be mentioned.
  • metal salts include, but are not limited to, salts of alkali metals such as sodium salts, potassium salts, and the like; salts of alkaline earth metals such as calcium salts, magnesium salts, barium salts, and the like; aluminum salts and the like.
  • Non-limiting examples of salts formed with organic bases include, but are not limited to, with trimethylamine, triethylamine, pyridine, picoline, 2,6-lutidine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, A salt formed by dicyclohexylamine or the like.
  • Non-limiting examples of salts formed with inorganic acids include, but are not limited to, salts formed with hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid, and the like.
  • Non-limiting examples of salts formed 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. a salt formed of a sulfonic acid, p-toluenesulfonic acid or the like.
  • Non-limiting examples of salts formed with basic amino acids include, but are not limited to, salts formed with arginine, lysine, ornithine, and the like.
  • Non-limiting examples of salts formed with acidic amino acids include, but are not limited to, salts formed with aspartic acid, glutamic acid, and the like.
  • pharmaceutical composition refers to one or more compounds of the present application, or salts thereof, and excipients, diluents, or agents generally accepted in the art for delivery of a biologically active compound to an organism (eg, a human), or Formulation of the carrier.
  • the purpose of the pharmaceutical composition is to facilitate administration of the compounds of the present application to an organism.
  • pharmaceutically acceptable excipient, diluent, or carrier refers to those excipients, diluents, or carriers that do not significantly irritate the organism and which do not impair the biological activity and properties of the active compound.
  • Suitable carriers, diluents and excipients are well known to those skilled in the art and include, for example, carbohydrates, waxes, water soluble and/or water swellable polymers, hydrophilic or hydrophobic materials, gelatin, oil, Raw materials such as solvents and water.
  • the present application also includes isotopically labeled compounds of the present application which are identical to those described herein, but wherein one or more atoms are replaced by an atom having a different atomic weight or mass than the atomic or mass number normally found in nature.
  • isotopes that may 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 respectively.
  • isotopically-labeled compounds of the present application can be used in compound and/or substrate tissue distribution assays.
  • Deuterated (i.e., 3 H) and carbon-14 (i.e., 14 C) isotopes are especially preferred for their ease of preparation and detectability.
  • 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 thus in some cases The following may be preferred.
  • Positron emitting 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.
  • Isotopically labeled compounds of the present application can generally be prepared by substituting an isotopically labeled reagent for an unisotopically labeled reagent by procedures similar to those disclosed in the schemes and/or examples disclosed below.
  • the compounds of the present application can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, combinations thereof with other chemical synthesis methods, and those well known to those skilled in the art. Equivalent alternatives, preferred embodiments include, but are not limited to, embodiments of the present application.
  • the invention includes, but is not limited to, the following:
  • polypeptide or a pharmaceutically acceptable salt thereof which polypeptide has at least 70% sequence identity to the sequence of Formula I:
  • R1 is: an aliphatic straight-chain or branched C 6 -C 20 acyl group or a deletion
  • Xaa1 is: Leu, Ala, AEEAc, Phe, IPhe, BrPhe, 3FPhe or deletion;
  • Xaa2 is: Leu, Ala, Arg, AEEAc or deletion;
  • Xaa3 is: Dab, Ala, Leu, Orn or missing;
  • Xaa4 is: Thr, Ala, Dab or Lys
  • Xaa5 is: Dab, Lys, Orn, Ala or missing;
  • Xaa6 is: Lys, Glu or Dab;
  • Xaa7 is: Dab, Ala, Lys, Orn or missing;
  • Xaa8 is: D-Leu, D-Phe, Lys or D-Ala;
  • Xaa9 is: Leu or Ala
  • Xaa10 is: Dab, Leu, Orn, Ala, D-Leu or deletion;
  • Xaa11 is: Dab, Leu, Orn, Ala, D-Leu or deletion;
  • Xaa12 is: Glu, Lys or Asp.
  • polypeptide of claim 1 or a pharmaceutically acceptable salt thereof, wherein the polypeptide is a cyclic peptide.
  • polypeptide according to any one of items 1 to 2, wherein the polypeptide is obtained by dehydration of Xaa6 and a side chain group of Xaa12 to form an amide bond, optionally, the side chain group.
  • the group contains an amino group and/or a carboxyl group.
  • R1 is selected from the group consisting of heptanoyl, methylheptanoyl, octanoyl, methyloctanoyl, decanoyl, methyl decanoyl, hydrazine Acyl, methyl decanoyl, lauroyl, myristoyl, palmitoyl, 17-carboxylic acid-heptadecanoyl or a deletion, preferably n-octanoyl, decanoyl, lauroyl, palmitoyl, 17-carboxylic acid-ten Heptanoyl or deletion, more preferably n-octanoyl or deletion;
  • Xaa1 is preferably Leu or deleted, Xaa2 is preferably Leu, Xaa3 is preferably Dab, Xaa4 is preferably Thr, Xaa5 is preferably Dab, Xaa1 is preferably Leu or deleted, Xaa
  • R1 is: an aliphatic straight-chain or branched C 6 -C 20 acyl group or a deletion
  • Xaa1 is: Leu, Ala, AEEAc, Phe, IPhe, BrPhe, 3FPhe or deletion;
  • Xaa2 is: Leu, Ala, Arg, AEEAc or deletion;
  • Xaa3 is: Dab, Ala, Leu, Orn or missing;
  • Xaa4 is: Thr, Ala, Dab or Lys
  • Xaa5 is: Dab, Lys, Orn, Ala or missing;
  • Xaa6 is: Lys or Dab
  • Xaa7 is: Dab, Ala, Lys, Orn or missing;
  • Xaa8 is: D-Leu, D-Phe, Lys or D-Ala;
  • Xaa9 is: Leu or Ala
  • Xaa10 is: Dab, Leu, Orn, Ala, D-Leu or deletion;
  • Xaa11 is: Dab, Leu, Orn, Ala, D-Leu or deletion.
  • R1 is selected from the group consisting of heptanoyl, methylheptanoyl, octanoyl, methyloctanoyl, decanoyl, methyldecanoyl, decanoyl, methyl Decanoyl, lauroyl, myristoyl, palmitoyl, 17-carboxylic acid-heptadecanoyl or a deletion, preferably n-octanoyl, decanoyl, lauroyl, palmitoyl, 17-carboxylic acid-heptadecanoyl or Deletion, more preferably n-octanoyl or deletion;
  • Xaa1 is preferably Leu or deletion
  • Xaa2 is preferably Leu
  • Xaa3 is preferably Dab
  • Xaa4 is preferably Dab
  • Xaa4 is preferably Thr
  • Xaa5 is preferably Da
  • R1 is: an aliphatic straight-chain or branched C 6 -C 20 acyl group or a deletion
  • Xaa1 is: Leu, Ala, AEEAc, Phe, IPhe, BrPhe, 3FPhe or deletion;
  • Xaa2 is: Leu, Ala, Arg, AEEAc or deletion;
  • Xaa3 is: Dab, Ala, Leu, Orn or missing;
  • Xaa4 is: Thr, Ala, Dab or Lys
  • Xaa5 is: Dab, Lys, Orn, Ala or missing;
  • Xaa7 is: Dab, Ala, Lys, Orn or missing;
  • Xaa8 is: D-Leu, D-Phe, Lys or D-Ala;
  • Xaa9 is: Leu or Ala
  • Xaa10 is: Dab, Leu, Orn, Ala, D-Leu or deletion;
  • Xaa11 is: Dab, Leu, Orn, Ala, D-Leu or deletion;
  • Xaa12 is: Glu or Asp.
  • R1 is selected from the group consisting of heptanoyl, methylheptanoyl, octanoyl, methyloctanoyl, decanoyl, methyldecanoyl, decanoyl, methylhydrazine Acyl, lauroyl, myristoyl, palmitoyl, 17-carboxylic acid-heptadecanoyl or a deletion, preferably n-octanoyl, decanoyl, lauroyl, palmitoyl, 17-carboxylic acid-heptadecanoyl or a deletion More preferably, it is n-octanoyl or a deletion; Xaa1 is preferably Leu or a deletion, Xaa2 is preferably Leu, Xaa3 is preferably Dab, Xaa3 is preferably Dab, Xaa1
  • R1 is: an aliphatic straight-chain or branched C 6 -C 20 acyl group or a deletion
  • Xaa1 is: Leu, Ala, AEEAc, Phe, IPhe, BrPhe, 3FPhe or deletion;
  • Xaa2 is: Leu, Ala, Arg, AEEAc or deletion;
  • Xaa3 is: Dab, Ala, Leu, Orn or missing;
  • Xaa4 is: Thr, Ala, Dab or Lys
  • Xaa5 is: Dab, Lys, Orn, Ala or missing;
  • Xaa7 is: Dab, Ala, Lys, Orn or missing;
  • Xaa8 is: D-Leu, D-Phe, Lys or D-Ala;
  • Xaa9 is: Leu or Ala
  • Xaa10 is: Dab, Leu, Orn, Ala, D-Leu or deletion;
  • Xaa11 is: Dab, Leu, Orn, Ala, D-Leu or deletion.
  • polypeptide according to any one of items 1 to 4, wherein the polypeptide is represented by R1-Leu-Dab-Thr-Dab-Xaa6-Dab-Xaa8-Leu-Dab-Dab-Glu a sequence consisting of a peptide or a cyclic peptide, wherein R1 is preferably n-octanoyl, decanoyl, lauroyl, palmitoyl, 17-carboxylic acid-heptadecanoyl or a deletion, more preferably n-octanoyl or a deletion; Xaa6 is preferably Lys Or Dab, Xaa8 is preferably D-Phe or D-Leu.
  • polypeptide according to item 11 or a pharmaceutically acceptable salt thereof, which is a peptide consisting of H 2 N-Leu-Dab-Thr-Dab-Lys-Dab-D-Phe-Leu-Dab-Dab-Glu Or a cyclic peptide.
  • polypeptide of claim 12 or a pharmaceutically acceptable salt thereof, the amino acid sequence of the polypeptide is as follows:
  • polypeptide according to any one of items 1 to 4, wherein the polypeptide is a sequence represented by R1-Dab-Thr-Dab-Xaa6-Dab-Xaa8-Leu-Dab-Dab-Glu A peptide or cyclic peptide consisting of wherein R1 is preferably n-octanoyl, decanoyl, lauroyl, palmitoyl, 17-carboxylic acid-heptadecanoyl or a deletion, more preferably n-octanoyl or a deletion; Xaa6 is preferably Lys or Dab Xaa8 is preferably D-Phe or D-Leu.
  • polypeptide according to item 14 or a pharmaceutically acceptable salt thereof, which is a peptide or a ring consisting of n-octanoyl-Dab-Thr-Dab-Lys-Dab-D-Phe-Leu-Dab-Dab-Glu Peptide.
  • polypeptide of claim 15 or a pharmaceutically acceptable salt thereof, the amino acid sequence of the polypeptide being as follows:
  • polypeptide of clause 1-3 or a pharmaceutically acceptable salt thereof, the amino acid sequence of the polypeptide having at least 70% sequence identity to one of the following sequences:
  • step (1) The product of the step (1) is cleaved with a strong acid; a side chain protecting group scavenger is added, filtered, and the polypeptide is precipitated with 5-20 volumes of an organic solvent, centrifuged, washed with an organic solvent, and dried to obtain a crude peptide.
  • step (1) is performed in a liquid phase environment, and specifically comprises:
  • step (a) the solvent used in the liquid phase environment of step (a) is selected from the group consisting of: dimethylformamide (DMF), dichloromethane (DCM), N-methylpyrrolidone (NMP), Preference is given to DMF;
  • the amino protecting group is selected from the group consisting of tert-butoxycarbonyl (Boc), benzyloxycarbonyl (Z) or 9-fluorenyl-methoxycarbonyl (Fmoc), preferably 9-fluorenyl-methoxycarbonyl (Fmoc).
  • a coupling reagent consisting of a carbodiimide type reagent or a benzotriazine.
  • Imidazolium salt type reagent and 1-hydroxybenzotriazole HBt
  • DIC diisopropylcarbodiimide
  • HBt 1-hydroxybenzotriazole
  • TBTU 2-(1H-benzene And trisazo-l-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate
  • HBt 1-hydroxybenzotriazole
  • DIC diisopropylcarbodiimide
  • 1-hydroxybenzotriazole 2-(1H-benzene And trisazo-l-1-yl)-1,1,3,3-tetramethyluronium t
  • step (b) is carried out by orthogonal protection strategy, and the amino protecting group on the side chain of the ring-forming amino acid is allyloxycarbonyl (Alloc).
  • the carboxy protecting group is an allyl group (OAll).
  • step (b) comprises deamination protection, and the removal of Alloc and OAll requires the addition of a degreasing agent, which is tetrakis(triphenylphosphine)palladium (Pd(PPh) 3 ) 4 ).
  • step (b) further requires the addition of a scavenger selected from the group consisting of H 3 N ⁇ BH 3 , Me 2 NH ⁇ BH 3 or PhSiH 3 , preferably PhSiH 3 .
  • a coupling reagent consisting of a carbodiimide type reagent or a tertiary amine type and a benzotriene a razoloxyl salt type reagent or a pyridine triazolium salt type; preferably diisopropylcarbodiimide (DIC) and 1-hydroxybenzotriazole (HOBt), or 2-(1H-benzotriazo) L-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) and 1-hydroxybenzotriazole (HOBt) or N,N-diisopropylethylamine ( DIEA) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU); further preferably N,N-diis
  • step (1) further comprises: (c) linking the resin peptide obtained in the step (b) to a fatty acid chain or a polybasic fatty acid chain.
  • step (c) is carried out in the presence of a coupling agent in a DMF solvent, the coupling agent being diisopropylcarbodiimide (DIC) and 1-hydroxyl Benzotriazole (HOBt).
  • DIC diisopropylcarbodiimide
  • HOBt 1-hydroxyl Benzotriazole
  • bacterium or fungus is Acinetobacter baumanii, drug resistant Acinetobacter baumanii, Pseudomonas aeruginosa, Drug resistant Pseudomonas aeruginosa, Bacillus subtilis, Staphylococcus aureus, E. coli, and/or candida Albicans One or several.
  • amino acids are: Fmoc-Glu(OAll)-OH, Fmoc-Dab(Boc)-OH, Fmoc-D-Phe-OH, Fmoc-Leu-OH, Fmoc-D-Leu-OH, Fmoc-Lys(Alloc)- OH, Fmoc-Thr(tBu)-OH
  • Synthetic reagents HOBt, DIC, DMF, DCM, piperidine, tetrakis(triphenylphosphine)palladium, phenylsilane, sodium diethyldithiocarbamate.
  • PSI300 peptide synthesis instrument Waters600 semi-preparative high performance liquid chromatography, centrifuge.
  • the resin was dried under vacuum and weighed.
  • the peptide was precipitated by adding 10 volumes of ice diethyl ether to the lysate, centrifuged, and the supernatant was discarded, and the precipitate was repeatedly washed with ice diethyl ether for 4 to 5 times, dried in vacuo, and the crude peptide was weighed.
  • the crude peptide was purified by semi-preparative RP-HPLC.
  • the target component with a purity greater than 95% was collected, steamed under low pressure, and lyophilized.
  • amino acids are: Fmoc-Glu(OAll)-OH, Fmoc-Dab(Boc)-OH, Fmoc-D-Phe-OH, Fmoc-Leu-OH, Fmoc-D-Leu-OH, Fmoc-Lys(Alloc)- OH, Fmoc-Thr(tBu)-OH
  • Synthetic reagents HOBt, DIC, DMF, DCM, piperidine, tetrakis(triphenylphosphine)palladium, phenylsilane, sodium diethyldithiocarbamate, n-octanoic acid.
  • PSI300 peptide synthesis instrument Waters600 semi-preparative high performance liquid chromatography, centrifuge.
  • the resin was dried under vacuum and weighed.
  • the peptide was precipitated by adding 10 volumes of ice diethyl ether to the lysate, centrifuged, and the supernatant was discarded, and the precipitate was repeatedly washed with ice diethyl ether for 4 to 5 times, dried in vacuo, and the crude peptide was weighed.
  • the crude peptide was purified by semi-preparative RP-HPLC.
  • the target component with a purity greater than 95% was collected, steamed under low pressure, and lyophilized.
  • Molecular weight confirmation by ESI-MS, m/z 606.25 (M+2H) 2+ is consistent with the theoretical molecular weight of 1210.34.
  • amino acids are: Fmoc-Glu(OAll)-OH, Fmoc-Asp(OAll)-OH, Fmoc-Dab(Boc)-OH, Fmoc-Leu-OH, Fmoc-D-Leu-OH, Fmoc-D-Phe- OH, Fmoc-Lys(Alloc)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Orn(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Phe(4- CF 3 )-OH, Fmoc-Phe(4-Br)-OH, Fmoc-Phe(4-I)-OH, Fmoc-Ala-OH, Fmoc-D-Ala-OH, Fmoc-AEEA-OH,
  • Reagents HOBt, DIC, DMF, piperidine, n-octanoic acid, citric acid, lauric acid, palmitic acid, t-butyl monodecanoate.
  • PSI300 peptide synthesizer Waters600 semi-preparative high performance liquid chromatography, Q-Tof MicroYA019 mass spectrometer, magnetic stirrer.
  • the polypeptide in Table 7 was prepared and purified in a manner similar to the procedure of ac in Example 1, and if it was a fatty chain polypeptide, it was prepared according to the procedure ac in Example 2, and the fraction having a purity greater than 95% was collected, and steamed, Freeze-dried.
  • the measured values of ESI-MS are shown in Table 7.
  • the minimum inhibitory concentration (MIC) of each antimicrobial peptide was determined according to the micro-broth dilution method recommended by the American Committee for Clinical Laboratory Standards (NCCLS).
  • the bacterial culture medium was Mueller-Hinton (MH) broth medium, Candida albicans medium.
  • Hyclone modified RPMI-1640 medium was used.
  • the above antimicrobial peptide at a concentration of 320 ⁇ g/ml and 80 ⁇ g/ml positive control amphotericin B, polymyxin E sulfate and vancomycin hydrochloride were precisely prepared.
  • the prepared stock solutions were stored in an environment of -20 ° C for use.
  • the inoculating loop uses the inoculating loop to pick 3 to 5 colonies of similar morphology to be inoculated, inoculate them in 4 to 5 ml of MH broth, and incubate at 35 ° C for 12 to 16 h.
  • the bacterial liquid was corrected to a MH turbidity standard with MH broth, and contained about 1 to 2 ⁇ 10 8 CFU/ml.
  • the above bacterial suspension was diluted 1:1000 with MH broth and set aside.
  • the drug concentrations in the first 1-10 well 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, and 0.0625 ⁇ g, respectively.
  • the 11th hole is a blank control without antibacterial drugs and inoculum
  • the 12th hole is a negative control without antibacterial drugs.
  • the 96-well plates inoculated with bacteria were incubated in a normal air incubator at 35 ° C for 16-20 h, and the 96-well plates inoculated with fungi were incubated in a 28 ° C air incubator for 40-50 h.
  • the lowest drug concentration without bacterial growth was observed by the naked eye as the minimum inhibitory concentration (MIC) of the sample.
  • MIC minimum inhibitory concentration
  • LD50 Median Lethal Dose refers to the exposure dose of harmful substances or radiation that can kill half of the test subjects. It is a common parameter for describing the toxicity of harmful substances or radiation. LD50 is usually characterized by the ratio of the mass of the hazardous substance to the weight of the test organism and can be used to compare the relative toxicity of different substances.
  • mice male, weighing 20-22 g, were from Shanghai Xipuer-Beikai Experimental Animal Co., Ltd. The mice were housed in an animal laboratory at 20 ⁇ 2 ° C for 12 hours, and the experimental animals were given free access to water and food. Randomly grouped by weight.
  • intravenous administration was carried out at a dose of 10 ml/kg.
  • mice were used for pre-experiment, and the lethal dose range of 0% and 100% mortality of the test substance was measured, and then the formal test was conducted.
  • the number of formal test groups was 5 dose groups, and the number of animals in each group was 10.
  • One mouse was administered, and the test sample was administered intravenously at 5 mg/kg (administered volume 0.2 ml). The injection is completed in about 20s. If the animal does not die, another mouse is taken and a higher dose is given. If the animal dies or is in a state of sudden death, take another one and give a lower dose. The dose of animal survival was supplemented to 3, and the survival state of the animals was observed. Find 0% and 100% death doses.
  • the inter-group dose ratio was set, and the number of groups was 5, and the number of animals in each group was 10.
  • the immediate response of the animals after administration was observed and recorded. Body weight changes were monitored and the status of the animals after administration was recorded. If an animal dies, it should be recorded, and the experiment is terminated 7 days after administration, and all animals are sacrificed. Based on the experimental results, the LD50 was determined.
  • compositions and methods of the present invention have been described in accordance with the preferred embodiments, those skilled in the art may, without departing from the concept, the spirit and the scope of the present invention, The compositions and/or methods described herein, as well as the order of the steps or steps of the methods, are varied.

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Abstract

La présente invention se rapporte au domaine des médicaments, et concerne un analogue de polymyxine présentant une activité antibactérienne élevée, un procédé de préparation correspondant, et une application associée. L'analogue de polymyxine a un effet bactéricide à large spectre et à haut rendement sur des bactéries et des champignons résistants aux médicaments.
PCT/CN2018/112889 2017-10-31 2018-10-31 Analogue de polymyxine et son procédé de préparation WO2019085926A1 (fr)

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CN110669107B (zh) * 2018-07-02 2024-04-02 上海医药工业研究院 抗菌肽及其制备方法
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WO2012051663A1 (fr) * 2010-10-21 2012-04-26 Monash University Composés antimicrobiens

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WO2002055543A2 (fr) * 2001-01-16 2002-07-18 Univ Ramot Composes, compositions pharmaceutiques et procedes pour le traitement de bacteriemie et/ou septicemie
US7935503B2 (en) * 2007-07-20 2011-05-03 University Of Georgia Research Foundation, Inc. Methods for producing and using polymyxins
FI20085469A0 (fi) * 2008-02-08 2008-05-16 Northern Antibiotics Oy Polymyksiinijohdannaiset, joissa on lyhyt rasvahappohäntä, ja niiden käyttöjä
US9096649B2 (en) * 2008-02-08 2015-08-04 Northern Antibiotics Ltd. Polymyxin derivatives and uses thereof
WO2009139635A1 (fr) * 2008-05-15 2009-11-19 Prosensa Technologies B.V. Dérivés de polymyxine et leurs utilisations
CN101851270A (zh) * 2009-04-03 2010-10-06 梁浩 一种多粘菌素衍生物及其制备方法
EP2679302A1 (fr) * 2012-06-28 2014-01-01 Zentrum für biomedizinische Technologie der Donau- Universität Krems Agent de sorption sélectif pour le nettoyage sanguin extracorporel
RU2730012C2 (ru) * 2013-05-22 2020-08-14 Сперо Терапьютикс, Инк. Производные полимиксина и их применение в комбинированной терапии совместно с различными антибиотиками
EP3173421A1 (fr) * 2015-11-30 2017-05-31 Universitat de Barcelona Composés peptidiques utiles en tant qu'agents antibactériens

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