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WO2019223644A1 - 多肽及其药物组合物和用途 - Google Patents

多肽及其药物组合物和用途 Download PDF

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Publication number
WO2019223644A1
WO2019223644A1 PCT/CN2019/087595 CN2019087595W WO2019223644A1 WO 2019223644 A1 WO2019223644 A1 WO 2019223644A1 CN 2019087595 W CN2019087595 W CN 2019087595W WO 2019223644 A1 WO2019223644 A1 WO 2019223644A1
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ieeiqkk
compound
ieeiskk
ieeiykk
influenza virus
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PCT/CN2019/087595
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English (en)
French (fr)
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王潮
钟武
曹瑞源
刘克良
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中国人民解放军军事科学院军事医学研究院
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Publication of WO2019223644A1 publication Critical patent/WO2019223644A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; 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
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present application relates to the field of biomedicine, and in particular, to polypeptides and pharmaceutical compositions and uses thereof.
  • Influenza Virus belongs to the Orthomyxoviridae family of influenza viruses (Influenza Virus) and is a single-stranded negative-strand RNA virus. According to the differences between Nucleoprotein (NP) and Membrane Protei (MP), influenza viruses can be divided into three subtypes: A, B, and C (ie, A, B, and C). Influenza A Virus (IAVs) can infect humans, various birds, and other mammals. Due to the susceptibility to mutations in its antigenicity, it can cause seasonal influenza, and it has repeatedly caused worldwide pandemics. The health hazard is enormous. According to the latest estimates from the U.S. Centers for Disease Control and Prevention, the World Health Organization, and global health partners, as of December 2017, there are 6 to 1.2 billion seasonal flu patients each year, causing about 3 to 5 million serious illnesses, many of which Up to 650,000 people have died of respiratory diseases caused by seasonal flu.
  • IAVs Influenza A Virus
  • influenza virus infection-neuraminidase inhibitors such as oseltamivir and zanamivir
  • M2 ion channel blockers such as amantadine.
  • Neuraminidase inhibitors selectively inhibit the activity of neuraminidase, prevent the virus from being released from infected cells to nearby uninfected cells, and have an inhibitory effect on influenza A and B viruses
  • M2 ion channel blockers prevent the virus from unshelling Releases genetic material RNA into the host cytoplasm, making the virus unable to replicate and is only effective against influenza A viruses.
  • H1N1 and H3N2 influenza virus strains are resistant to amantadines, oseltamivir has also induced the generation of H1N1 virus resistant strains.
  • the development of new anti-influenza virus drugs targeting new targets to overcome the virus The drug resistance has great practical significance.
  • the surface spikes of influenza virus are composed of Hemagglutinin (HA) and Neuraminidase (NA).
  • Hemagglutinin HA mediates the process of virus adsorption and entry into host cells during the life cycle of the influenza virus.
  • Current research suggests that after the virus approaches the host cell, the HA1 subunit of HA will first bind to the ⁇ -2,3 or ⁇ -2,6 sialic acid receptor on the glycoprotein / glycolipid end of the host cell membrane, and the virus enters through endocytosis The host cell is enclosed in an endosome.
  • the loop domain of HA2 undergoes a "loop-to-helix transition" conformational transition, causing it to be NHR Repeat) region forms a long coiled triple helix core, and finally a CHR (C-terminal Heptads Repeat) region containing a short alpha helix and a long alpha helix is folded back in a "V" shape and acts on a trimer hydrophobic groove formed by NHR A six-helix spiral (Six Helix Bundle, 6HB) is formed, which completes the process of membrane fusion.
  • NHR C-terminal Heptads Repeat
  • fusion inhibitors designed based on the 6HB formation mechanism have been reported in a variety of type I enveloped viruses, among them, T20 peptides also serve as the first clinically marketed fusion inhibitors in the treatment of AIDS.
  • T20 peptides also serve as the first clinically marketed fusion inhibitors in the treatment of AIDS.
  • fusion inhibitors against influenza viruses that can directly inhibit the HA-mediated membrane fusion process from peptides derived from the HA2CHR region, which may be related to the complex fusion mechanism of the influenza virus itself.
  • influenza virus fusion needs to undergo endocytosis first, triggering the fusion process under low pH conditions in the cell inclusion body.
  • the artificially designed alpha helix peptides that can target the IAVs and NHR regions were conjugated with lipid molecules with membrane anchoring functions. Lipopeptide can effectively inhibit influenza A H1N1 and H3N2 influenza viruses.
  • the present application provides a compound of formula (I) or a compound having at least 80% identity therewith, a stereoisomer or a pharmaceutically acceptable salt thereof,
  • X a represents a hydrophobic amino acid, and each X a is the same or different;
  • X d represents a hydrophobic amino acid, and each X d is the same or different;
  • X e is selected from the following amino acids: Ser, Asn, Gln, Glu, Asp, Lys, Arg, His, Tyr, Trp, Met, and Cys, each X e being the same or different;
  • E is Glu
  • R 1 represents cholesterol, steroids, sphingosine, and fatty acids (such as C 6-24 fatty acids);
  • L 1 is selected from Gly, ⁇ -alanine ( ⁇ -Ala), ⁇ -aminobutyric acid (GABA), 6-aminohexanoic acid (6-Aca), and NH 2- (CH 2 CH 2 -O) n- CH 2 CH 2 -COOH, where n is an integer selected from 1-25;
  • n is selected from 2, 3, 4, 5, 6, 7, 8, 9, and 10.
  • X a is an amino acid residue that can have a hydrophobic interaction with the transmembrane subunit NHR region of a type I enveloped virus fusion protein.
  • X a is selected from Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, and Met, each X a being the same or different.
  • X a is selected from Ala, Val, Leu, Ile, Phe, and Tyr, each X a being the same or different.
  • X a is Ile.
  • X d is an amino acid residue that can have a hydrophobic interaction with the transmembrane subunit NHR region of a type I enveloped virus fusion protein.
  • X d is selected from Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp and Met, each X d being the same or different.
  • X d is selected from Ala, Val, Leu, Ile, Phe, and Tyr, each X d being the same or different.
  • X d is Ile.
  • X e is an amino acid residue that can interact polarly with the NMR region of a transmembrane subunit of a type I enveloped virus fusion protein.
  • X e is selected from Ser, Gln, Glu, Lys, His, Tyr, and Trp, each X e being the same or different.
  • X e is Gln, Ser or Tyr.
  • X e is Gln.
  • R 1 is selected from C 6-24 saturated fatty acids.
  • R 1 is selected from caprylic acid, capric acid, lauric acid, myristic acid, and palmitic acid.
  • L 1 is selected from Gly, ⁇ -alanine ( ⁇ -Ala), 6-aminocaproic acid (6-Aca), and NH 2- (CH 2 CH 2 -O) 7- CH 2 CH 2 -COOH.
  • L 1 is selected from Gly, ⁇ -alanine ( ⁇ -Ala), and 6-aminocaproic acid (6-Aca).
  • L 1 is ⁇ -alanine ( ⁇ -Ala).
  • m is selected from 3, 4, 5, 6, 7, and 8.
  • m is selected from 4, 5, and 6.
  • n is 5.
  • X a is Ile;
  • Xd is Ile;
  • X e is Gln, Ser or Tyr;
  • R 1 is palmitic acid;
  • L 1 is Gly, ⁇ -alanine ( ⁇ -Ala), 6 -Aminohexanoic acid (6-Aca) or NH 2- (CH 2 CH 2 -O) 7 -CH 2 CH 2 -COOH;
  • m is 5.
  • the compound is selected from:
  • a is ⁇ -Ala
  • z is 6-Aca
  • p is NH 2- (CH 2 CH 2 -O) 7 -CH 2 CH 2 -COOH
  • G is Gly
  • C 8 is caprylic acid
  • C 10 is capric acid
  • C 12 is lauric acid
  • C 14 is myristic acid
  • C 16 is palmitic acid.
  • the compound of the invention has at least 90% identity with the compound of formula (I), preferably at least 91% identity, at least 92% identity, at least 93% identity, at least 94% Identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity.
  • the present application provides a pharmaceutical composition
  • a pharmaceutical composition comprising a compound of formula (I) or a compound having at least 80% identity therewith, a stereoisomer or a pharmaceutically acceptable salt thereof, and a One or more pharmaceutically acceptable carriers or excipients.
  • the pharmaceutical composition of the present application can be made into tablets, sustained-release tablets, controlled-release tablets, dragees, hard or soft capsules, aqueous or oily suspensions, emulsions, dispersible powders or granules , Syrups or elixirs, drip pills, pellets, or oral solutions.
  • Pharmaceutical compositions for oral use may also contain, for example, one or more colorants, sweeteners, flavoring agents, and / or preservatives.
  • Suitable excipients for tablets include, inert diluents such as lactose, sodium carbonate, calcium phosphate or calcium carbonate; disintegrants such as corn starch and alginic acid; binders such as starch; lubricants such as magnesium stearate , Stearic acid or talc; preservatives such as ethyl or propyl paraben; and antioxidants such as ascorbic acid and the like.
  • the tablets may be uncoated or they may be coated to alter their disintegrating effect and subsequent absorption of the active ingredient in the gastrointestinal tract or to improve their stability and / or appearance, which can be used in any case Conventional coating agents and methods well known in the art.
  • Suitable excipients for hard capsules include inert solid diluents such as calcium carbonate, calcium phosphate or kaolin, and the like.
  • Suitable excipients for soft capsules include water or oils such as peanut oil, liquid paraffin or olive oil, and the like.
  • Aqueous suspensions generally contain the active ingredient in the form of micronized powder and one or more dispersants, wetting agents or suspending agents, such as sodium carboxymethyl cellulose, methyl cellulose, hydroxypropyl methyl ester Cellulose, sodium alginate, polyvinyl-pyrrolidone, tragacanth and gum arabic; etc .; dispersants or wetting agents, such as condensates of lecithin or alkenyl oxide with fatty acids (such as polyoxyethylene stearate) ), Or the condensation product of ethylene oxide with a long-chain fatty alcohol, such as ethylene cetyl heptyl alcohol, or the condensation product of ethylene oxide with a partial ester derived from a fatty acid and hexitol, such as polyoxygenation Ethylene sorbitol monooleate, or the condensation product of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, such as polyethylene sorbitan monoo
  • Aqueous suspensions may also contain one or more preservatives (e.g., ethyl or propyl parabens), antioxidants (e.g., ascorbic acid), colorants, flavoring agents, and / or sweeteners (e.g., Sucrose, saccharin and aspartame).
  • preservatives e.g., ethyl or propyl parabens
  • antioxidants e.g., ascorbic acid
  • colorants e.g., ascorbic acid
  • flavoring agents e.g., ascorbic acid
  • sweeteners e.g., Sucrose, saccharin and aspartame
  • Oily suspensions may be formulated by suspending the active ingredient in a vegetable oil (such as peanut oil, olive oil, sesame oil or coconut oil) or a mineral oil (such as liquid paraffin). Oily suspensions may also contain thickening agents such as beeswax, solid paraffin or cetyl alcohol. Sweeteners and flavoring agents as described above may be added to enhance the mouthfeel of the oral formulation.
  • the pharmaceutical composition can be preserved by the addition of an antioxidant such as ascorbic acid.
  • the pharmaceutical composition of the present application may also take the form of an oil-in-water emulsion.
  • the oily phase may be a vegetable oil, such as olive oil or peanut oil, or a mineral oil, such as liquid paraffin or a mixture thereof.
  • Suitable emulsifiers may be, for example, natural gums such as acacia or astragalus gum, natural phospholipids such as soybean lecithin, and esters or partial esters derived from fatty acids and hexitol anhydrides (such as sorbitan monooleate), And a condensation product of the partial ester and ethylene oxide, such as polyethylene oxide sorbitan monooleate.
  • Emulsions may also contain sweeteners, flavoring agents, preservatives, and the like.
  • Syrups and elixirs may be formulated with sweeteners (such as glycerol, propylene glycol, sorbitol, aspartame, or sucrose), and may also contain a demulcent, a preservative, a flavoring agent, and / or a colorant.
  • sweeteners such as glycerol, propylene glycol, sorbitol, aspartame, or sucrose
  • the pharmaceutical composition When administered parenterally (e.g., intravenously, subcutaneously or intramuscularly), the pharmaceutical composition can be prepared as a sterile aqueous or oily solution, sterile powder, liposome, emulsion, microemulsion, Nanoemulsions or microcapsules.
  • the pharmaceutical composition may also be in the form of a sterile aqueous or oily suspension for injection, which may be formulated according to known methods using one or more suitable dispersing, wetting agents and / or suspending agents, these The reagents are as described above.
  • the sterile injectable preparation may also be a sterile aqueous or oleaginous suspension for injection in a diluent or solvent, which is non-toxic and gastrointestinal acceptable, such as a solution in 1,3-butanediol .
  • the amount of active ingredient which is combined with one or more excipients to produce a single dosage form can be determined depending on the host treated and the particular route of administration.
  • formulations for oral administration to humans generally contain, for example, 0.5 mg to 2 g of active ingredient and appropriate and conventional amounts of excipients (approximately 5-98% of the total weight of the composition).
  • a unit preparation generally contains about 1 mg to 500 mg of the active ingredient.
  • the dosage of the pharmaceutical composition for therapeutic or preventive purposes should be adjusted according to the nature and severity of the disorder, the age and sex of the animal or patient, and the route of administration.
  • the pharmaceutical composition When used for treatment or prevention, it is generally administered in a daily dose in the range of, for example, 1 mg to 100 mg / kg of body weight, and divided doses may be administered if necessary.
  • lower dosages are used for parenteral administration, for example, intravenous administration generally uses dosages in the range of, for example, 1 mg to 10 mg / kg of body weight.
  • the present application provides a compound of formula (I) or a compound having at least 80% identity, a stereoisomer, or a pharmaceutically acceptable salt or pharmaceutical composition thereof for the preparation of an influenza virus and Use of a target cell membrane fusion drug.
  • influenza virus is an influenza A virus.
  • influenza virus is selected from H1N1 and H3N2.
  • the target cells are cell lines or cells from a subject.
  • the present application provides a compound of formula (I), or a compound having at least 80% identity, a stereoisomer or a pharmaceutically acceptable salt thereof, that inhibits the fusion of an influenza virus with a target cell membrane in vitro. use.
  • influenza virus is an influenza A virus.
  • influenza virus is selected from H1N1 and H3N2.
  • the target cells are cell lines or cells from a subject.
  • the present application provides a compound of formula (I) or a compound having at least 80% identity, a stereoisomer or a pharmaceutically acceptable salt or pharmaceutical composition thereof in the preparation of a prophylactic or therapeutic agent and Use of medicine or anti-flu virus medicine for diseases related to influenza virus infection.
  • influenza virus is an influenza A virus.
  • influenza virus is selected from H1N1 and H3N2.
  • the disease associated with influenza virus infection is selected from the group consisting of H1N1 influenza and H3N2 influenza.
  • the application provides a compound of formula (I) or a compound having at least 80% identity therewith, a stereoisomer or a pharmaceutically acceptable salt or pharmaceutical composition thereof for inhibiting influenza virus Fusion with target cell membrane.
  • influenza virus is an influenza A virus.
  • influenza virus is selected from H1N1 and H3N2.
  • the target cells are cell lines or cells from a subject.
  • the compound, or a compound having at least 80% identity therewith, a stereoisomer or a pharmaceutically acceptable salt or pharmaceutical composition thereof is used in an in vivo method.
  • the present application provides a compound of formula (I) as described above or a compound having at least 80% identity therewith, a stereoisomer or a pharmaceutically acceptable salt or pharmaceutical composition thereof for use in prevention or treatment Diseases related to influenza virus infection or anti-influenza virus.
  • influenza virus is an influenza A virus.
  • influenza virus is selected from H1N1 and H3N2.
  • the disease associated with influenza virus infection is selected from the group consisting of H1N1 influenza and H3N2 influenza.
  • the present application provides a method for inhibiting fusion of an influenza virus with a target cell membrane, which comprises administering to a cell an effective amount of a compound of formula (I) as described above or a compound having at least 80% identity with the same, a steric difference Or a pharmaceutically acceptable salt or pharmaceutical composition.
  • influenza virus is an influenza A virus.
  • influenza virus is selected from H1N1 and H3N2.
  • the target cells are cell lines or cells from a subject.
  • the method is performed in vivo.
  • the method is performed in vitro.
  • the application provides an antiviral method comprising administering to a subject in need thereof an effective amount of a compound of formula (I), or a compound having at least 80% identity therewith, Isomers or pharmaceutically acceptable salts or pharmaceutical compositions.
  • influenza virus is an influenza A virus.
  • influenza virus is selected from H1N1 and H3N2.
  • the application provides a method of preventing or treating a disease associated with an influenza virus infection, comprising administering to a subject in need thereof an effective amount of a compound of formula (I) % Identity compounds, stereoisomers or pharmaceutically acceptable salts or pharmaceutical compositions thereof.
  • influenza virus is an influenza A virus.
  • influenza virus is selected from H1N1 and H3N2.
  • the disease associated with influenza virus infection is selected from the group consisting of H1N1 influenza and H3N2 influenza.
  • fatty acid refers to an aliphatic carbon chain containing one carboxyl group at one end. According to the degree of carbon chain saturation, it can be divided into saturated fatty acids, monounsaturated fatty acids and polyunsaturated fatty acids. Among them, the saturated fatty acids have the structural formula of C x H 2x + 1 COOH. According to the length of the carbon chain, it can be divided into short-chain fatty acids (the number of carbon atoms on the carbon chain is less than 6), medium-chain fatty acids (the number of carbon atoms on the carbon chain is 6-12), and long-chain fatty acids (the carbon on the carbon chain) The number of atoms is greater than 12).
  • hydrophobic amino acid mainly includes tyrosine, tryptophan, phenylalanine, valine, leucine, isoleucine, proline, methionine and Alanine.
  • the term "identity" is used to refer to a sequence match between two polypeptides or between two nucleic acids.
  • a position in two compared sequences is occupied by the same base or amino acid monomer subunit (e.g., a position in each of the two DNA molecules is occupied by adenine, or two Each position of the polypeptide is occupied by lysine)
  • the molecules are identical at that position.
  • the "percent identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of compared positions x 100. For example, if 6 of the 10 positions of two sequences match, the two sequences are 60% identical.
  • the DNA sequences CTGACT and CAGGTT share 50% identity (3 positions out of a total of 6 positions match).
  • comparisons are made when two sequences are aligned to produce maximum identity.
  • Such alignment can be achieved by using, for example, the method of Needleman et al. (1970) J. Mol. Biol. 48: 443-453, which can be conveniently performed by a computer program such as the Align program (DNAstar, Inc).
  • the algorithm of E.Meyers and W.Miller Comput.Appl. Biosci., 4: 11-17 (1988)
  • the PAM120 weight residue table is used.
  • the term "subject" refers to an animal, particularly a mammal, preferably a human.
  • the term "effective amount" refers to an amount sufficient to obtain or at least partially obtain a desired effect.
  • a prophylactically effective amount refers to an amount sufficient to prevent, prevent, or delay the onset of a disease
  • a therapeutically effective amount refers to an amount sufficient to cure or at least partially prevent a disease and its complications in a patient already suffering from the disease. It is well within the ability of those skilled in the art to determine such an effective amount.
  • the amount effective for therapeutic use will depend on the severity of the disease to be treated, the overall state of the patient's own immune system, the general condition of the patient such as age, weight and sex, the manner in which the drug is administered, and other treatments administered concurrently and many more.
  • Figure 1 shows how NBD-IIQ16 enters MDCK cells and its distribution in inclusion bodies as observed by a laser confocal microscope.
  • IAVs Type A (type A) influenza virus
  • MALDI-TOF-MS Matrix-assisted laser desorption ionization time-of-flight mass spectrometry
  • NHR N-terminal heptad repeat
  • NMP N-Methyl pyrrrolidone
  • TFA trifluoroacetic acid
  • Trp Tryptophan
  • the Rink amide resin used as a solid-phase synthesis carrier in the examples is a product of Tianjin Nankai Synthesis Co., Ltd.
  • HBTU, HOBt, DIEA and Fmoc protected natural amino acids or D-type unnatural amino acids are the products of Shanghai Gill Biochemical Co., Ltd. and Beijing Okinas Technology Co., Ltd.
  • NMP N-methylpyrrolidone
  • TFA trifluoroacetic acid
  • DMF and DCM are products of Sinopharm Chemical Reagent Co., Ltd.
  • Chromatographically pure acetonitrile is a Fisher product.
  • Other reagents are domestically produced analytical products unless otherwise specified.
  • Peptide synthesis was performed using standard Fmoc solid-phase methods. With Rink Amide resin, the peptide chain is extended from the C-terminus to the N-terminus.
  • the condensing agent is HBTU / HOBt / DIEA.
  • the deprotecting agent was a piperidine / DMF solution.
  • the lysate was trifluoroacetic acid (TFA).
  • the crude peptide was dissolved in water and lyophilized. It was separated and purified by medium pressure liquid chromatography or high pressure liquid chromatography (HPLC), and the pure peptide content was greater than 90%.
  • Matrix-assisted laser analytical time-of-flight mass spectrometry MALDI-TOF-MS determines the molecular weight of peptide sequences.
  • the peptide sequence was synthesized using a CEM microwave peptide synthesizer.
  • Blocking reagent 20% v / v acetic anhydride in DMF.
  • Rink Amide resin 0.23 g (0.1 mmol) of Rink Amide resin was weighed and placed in the reactor of CEM microwave peptide synthesizer, and then protected amino acids, condensation reagents, activated bases, deprotection reagents, and blocking reagents were configured according to the above concentrations. Automatic peptide synthesizer for synthesis. After completion, the peptide resin was transferred to a peptide solid-phase synthesis reactor, washed twice with DMF, anhydrous methanol, and DCM, respectively, and dried under vacuum at room temperature to obtain 1.25 g of the peptide resin.
  • Peptide resin [containing Lys (Dde) special amino acid)] swell resin with a little DCM for 20 min, and drained.
  • Dde protection group removal reagent 2ml of hydrazine hydrate is dissolved in 40ml of DMF in a volume ratio of 20: 1. The deprotection reagent was added to the resin, stirred at room temperature for 3 minutes, dried, and repeated 4 times to remove the reaction.
  • Peptide resin lysis Weigh 1.31g of peptide resin synthesized by a microwave synthesizer, put it into a 250ml eggplant-shaped bottle, and ice bath. A lysate was prepared by adding 10 ml of 1 g of peptide resin. The TFA needs to be cooled in the ice bath for 30 minutes or stored in the refrigerator before use; add the prepared lysate to the peptide resin under the ice bath condition, and slowly stir the electromagnetically, the resin turns orange-red, and react for 30 minutes in the ice bath condition, and then Remove the ice bath and continue to stir the reaction for 150 minutes. The reaction is complete. Add 200 ml of ice ether cooled at 4 ° C under vigorous stirring to precipitate a white precipitate.
  • Crude peptides are purified by medium pressure or high pressure chromatography.
  • the chromatographic column was a C8 column, and the eluents were acetonitrile, water, and a small amount of TFA.
  • the column was equilibrated with 200 ml of 20% acetonitrile / water / 0.1% TFA solution in advance.
  • a ⁇ -Ala; z: 6-Aca; p: NH 2- (CH 2 CH 2 -O) 7 -CH 2 CH 2 -COOH; G: Gly; C 8 : caprylic acid, C 10 : capric acid , C 12 : lauric acid, C 14 : myristic acid, C 16 : palmitic acid.
  • the peptides were respectively incubated with the target peptide N66 derived from the H3N2HA region (residues 40-105 of the N3 region of the H3N2 fusion protein). Incubation method: The peptide was mixed with N66 equimolarly and incubated at 30 ° C for 30 min. Sodium acetate buffer (pH 5.0) The mixture was diluted to a final concentration of 10 ⁇ M for testing.
  • a MOS-450 circular dichroic analyzer was used for spectral scanning of the peptide solution, and the parameters were set: the scanning wavelength was 180-280nm, the scanning optical path was 1mm, the scanning unit was 1nm, and the scanning repetition number of each sample was 3 times.
  • Polypeptides with a typical ⁇ -helical structure appear on the CD spectrum as negative peaks at 208 nm and 222 nm and positive peaks at 195 nm, while ⁇ -helicality is based on a molar ellipticity of -33,000 (deg ⁇ cm 2 at 222 nm).
  • Dmol -1 is 100% alpha helicity to calculate the percentage of helix content of the polypeptide and complex to be tested.
  • the stability of the polypeptide described herein to interact with the target N66 and form a conjugate is determined by a CD temperature scan.
  • the specific method is as follows: transfer the above-mentioned polypeptide for measuring the CD signal into the sample cell (can also be reconfigured), set the CD instrument program to temperature scanning, the detection wavelength is 220nm, and the scanning range is 20-98 ° C. Obtain the change curve of CD signal with temperature, and calculate the Tm value according to the curve. The results of circular dichroism are shown in Table 2.
  • Peptide samples were diluted at a ratio of 0.2% BSA in DMEM and incubated with 100TCID 50 / mL of influenza virus A / Puerto Rico / 8/34 (H1N1) or A / Aichi / 2/68 (H3N2) for 30 minutes. After 30 min, the virus and peptide mixture was added to the cells to adsorb the virus. After 1 hour, the virus was adsorbed, the virus mixture was removed, and 3 ml of a 1% agarose-containing maintenance solution (1 ⁇ g / ml TPCK-trypsin) was added to cover the cells.
  • a normal MDCK cell negative control group, a virus infection positive control group and an antiviral drug oseltamivir control group were set up. After incubation at 37 ° C for 5 hours with 5% CO 2 , the morphological changes of the cells were observed daily under an inverted microscope.
  • the antiviral activity was measured by the MTT method: the cell plate after 48 h of culture was removed, the medium was removed, and 100 ⁇ l of a medium containing 0.5 mg / ml MTT was added, and cultured at 37 ° C and 5% CO 2 in the dark for 4 h; then the medium was discarded Add 150 ⁇ l of DMSO to dissolve and shake for 10 min, determine the OD value at 570 nm, calculate the IC 50 of the compound using Prism 5.01, and repeat the test three times. The results of the activity test are shown in Table 3.
  • the cells selected in this experiment were the target cells MDCK cells in the influenza virus infection model.
  • the peptides were labeled with the green fluorescent label NBD, and the situation of NBD-IIQ16 entering MDCK cells and the distribution of inclusion bodies were directly observed by a laser confocal microscope. After incubating the peptide with MDCK cells, they were thoroughly washed with PBS, and the cells were fixed after LysoTracher-red labeling. Peptides that emit green fluorescence can enter cells and are widely distributed in the red area of inclusion bodies labeled with LysoTracher (green fluorescence and red fluorescence are superimposed to form yellow fluorescence). The ability to penetrate the membrane and enter inclusion bodies may be an important reason why lipopeptides inhibit the fusion of influenza virus membranes.
  • the N-terminus of IIQ16 was covalently conjugated to the fluorescent molecule NBD and named NBD-IIQ16.
  • Experiment MDCK cells prior to administration 24h 10 4 cells were seeded in small NEST 15mm dish and incubated at 37 °C 5% CO 2 incubator. After 24 hours, the culture solution was aspirated, and 1 mL of NBD-IIQ16 DMEM culture solution was added, and incubated at 37 ° C for 2 hours. After that, the culture solution was aspirated, washed three times with PBS, and then 1 ml of 50 nM LysoTracker Red / DMEM solution was added, followed by incubation at 37 ° C for 30 minutes.
  • LysoTracker Red has an excitation wavelength of 577 nm. The results are shown in Figure 1.

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Abstract

提供多肽及其药物组合物和用途，具体地，提供式(I)所示化合物或与其具有至少80％同一性的化合物、其立体异构体或药学上可接受的盐，其药物组合物及其在制备抗流感病毒感染或预防或治疗与流感病毒感染相关的疾病的药物中的用途，Ac-[X aEEX dX eKK] m-L 1-K(R 1)-NH 2 (I)。

Description

多肽及其药物组合物和用途
本申请是以CN申请号为201810499379.3，申请日为2018年5月23日的申请为基础，并主张其优先权，该CN申请的公开内容在此作为整体援引加入本申请中。
技术领域
本申请涉及生物医药领域，具体涉及多肽及其药物组合物和用途。
背景技术
流行性感冒病毒(Influenza Virus)属正粘病毒科(Orthomyxoviridae)流感病毒属(Influenza Virus)，是单股负链RNA病毒。根据核蛋白(Nucleoprotein，NP)和膜蛋白(Membrane protei，MP)的不同，流感病毒可分为A、B、C(即甲、乙、丙)三种亚型。A型流感病毒(Influenza A Virus，IAVs)可感染人类、各种禽类和其他多种哺乳动物，由于其抗原性易发生变异，会引起季节性流感，并多次引起世界性大流行，对人类健康危害极大。根据美国疾病控制和预防中心、世界卫生组织和全球卫生合作伙伴的最新估计，截止2017年12月，每年季节性流感患者高达6-12亿，造成约300万至500万例严重疾病，其中多达65万人死于由季节性流感引起的呼吸道疾病。
目前临床用于治疗流感病毒感染的药物主要有两类-神经氨酸酶抑制剂(如奥司他韦、扎那米韦)和M2离子通道阻滞剂(如金刚烷胺)。神经氨酸酶抑制剂选择性抑制神经氨酸酶的活性，阻止病毒由感染细胞释放至临近未感染细胞，对A、B型流感病毒均有抑制作用；M2离子通道阻滞剂阻止病毒脱壳释放遗传物质RNA到宿主细胞质中，使病毒无法进行复制，只对A型流感病毒有效。由于当前的H1N1和H3N2型流感病毒株对金刚烷胺类药物耐药，奥司他韦也诱导了H1N1病毒耐药毒株的产生，针对新靶点的新型抗流感病毒药物的研发以克服病毒的耐药性，具有重大的现实意义。
流感病毒表面棘突由血凝素(Hemagglutinin，HA)和神经氨酸酶(Neuraminidase，NA)组成。血凝素HA在流感病毒生命周期中介导了病毒吸附和进入宿主细胞的过程。目前研究认为，病毒靠近宿主细胞后，HA的HA1亚基会首先与宿主细胞膜糖蛋白/糖脂末端的α-2,3或α-2,6唾液酸受体结合，病毒通过内吞作用进入宿主细胞并被包裹在内涵体中。在内涵体的酸性条件下(pH～5.5)，原本在中性pH条件下为紧闭“夹子” 状结构的HA1区域构象发生重排，并逐步与HA2区域解离。HA2随即发生构象重排，隐藏在蛋白中的融合肽暴露出来并插到内涵体膜上，随后HA2的环形域发生“loop-to-helix transition”构象转变，使其上NHR(N-terminal Heptads Repeat)区域形成较长的卷曲三螺旋内核，最后包含一个短α螺旋和一个长α螺旋的CHR(C-terminal Heptads Repeat)区域通过“V”字形折回并作用于NHR构成的三聚体疏水槽形成“发卡”样稳定的六股螺旋(Six helix bundle,6HB)，完成膜融合过程。
虽然基于6HB形成机制而设计的融合抑制剂在多种I型包膜病毒中均有报道，其中，T20多肽还作为第一种临床上市的融合抑制剂在治疗AIDS中发挥作用。但针对流感病毒的融合抑制剂，尚无来源于HA2CHR区域的多肽能够直接抑制HA介导的膜融合过程的报道，这可能与流感病毒自身具有复杂的融合机制有关。不同于HIV-1病毒直接在宿主细胞膜表面进行融合，流感病毒融合需要首先经历内吞，在细胞内含体中的低pH条件下触发融合过程。
发明内容
基于IAVs的病毒–宿主细胞膜融合机制和六股α螺旋束(6HB)的本质特征，将可以靶向IAVs NHR区域的人工设计α螺旋肽与具有膜锚定功能的脂质分子共缀，所设计的脂肽可以有效抑制甲型H1N1和H3N2流感病毒。
因此，在一个方面本申请提供式(I)所示化合物或与其具有至少80％同一性的化合物、其立体异构体或药学上可接受的盐，
Ac-[X aEEX dX eKK] m-L 1-K(R 1)-NH 2
(I)
其中，X a表示疏水性氨基酸，各X a相同或不同；
X d表示疏水性氨基酸，各X d相同或不同；
X e选自下述氨基酸：Ser、Asn、Gln、Glu、Asp、Lys、Arg、His、Tyr、Trp、Met和Cys，各X e相同或不同；
E为Glu；
K为Lys；
R 1表示胆固醇、类固醇、鞘氨醇和脂肪酸(例如C 6-24脂肪酸)；
L 1选自Gly、β-丙氨酸(β-Ala)、γ-氨基丁酸(GABA)、6-氨基己酸(6-Aca)和NH 2-(CH 2CH 2-O) n-CH 2CH 2-COOH，其中，n为选自1-25的整数；
m选自2、3、4、5、6、7、8、9和10。
在某些优选的实施方案中，X a为可与I型包膜病毒融合蛋白跨膜亚基NHR区域发生疏水相互作用的氨基酸残基。
在某些优选的实施方案中，X a选自Ala、Val、Leu、Ile、Pro、Phe、Tyr、Trp和Met，各X a相同或不同。
在某些优选的实施方案中，X a选自Ala、Val、Leu、Ile、Phe和Tyr，各X a相同或不同。
在某些优选的实施方案中，X a为Ile。
在某些优选的实施方案中，X d为可与I型包膜病毒融合蛋白跨膜亚基NHR区域发生疏水相互作用的氨基酸残基。
在某些优选的实施方案中，X d选自Ala、Val、Leu、Ile、Pro、Phe、Tyr、Trp和Met，各X d相同或不同。
在某些优选的实施方案中，X d选自Ala、Val、Leu、Ile、Phe和Tyr，各X d相同或不同。
在某些优选的实施方案中，X d为Ile。
在某些优选的实施方案中，X e为可与I型包膜病毒融合蛋白跨膜亚基NHR区域发生极性相互作用的氨基酸残基。
在某些优选的实施方案中，X e选自Ser、Gln、Glu、Lys、His、Tyr和Trp，各X e相同或不同。
在某些优选的实施方案中，X e为Gln、Ser或Tyr。
在某些优选的实施方案中，X e为Gln。
在某些优选的实施方案中，R 1选自C 6-24饱和脂肪酸。
在某些优选的实施方案中，R 1选自辛酸、癸酸、月桂酸、肉豆蔻酸和棕榈酸。
在某些优选的实施方案中，L 1选自Gly、β-丙氨酸(β-Ala)、6-氨基己酸(6-Aca)和NH 2-(CH 2CH 2-O) n-CH 2CH 2-COOH，其中，n＝1、2、3、4、5、7、9、11或23。
在某些优选的实施方案中，L 1选自Gly、β-丙氨酸(β-Ala)、6-氨基己酸(6-Aca)和NH 2-(CH 2CH 2-O) 7-CH 2CH 2-COOH。
在某些优选的实施方案中，L 1选自Gly、β-丙氨酸(β-Ala)和6-氨基己酸(6-Aca)。
在某些优选的实施方案中，L 1为β-丙氨酸(β-Ala)。
在某些优选的实施方案中，m选自3、4、5、6、7和8。
在某些优选的实施方案中，m选自4、5和6。
在某些优选的实施方案中，m为5。
在某些优选的实施方案中，X a为Ile；Xd为Ile；X e为Gln、Ser或Tyr；R 1为棕榈酸；L 1为Gly、β-丙氨酸(β-Ala)、6-氨基己酸(6-Aca)或NH 2-(CH 2CH 2-O) 7-CH 2CH 2-COOH；m为5。
在某些优选的实施方案中，所述化合物选自：
IEEIQKK IEEIQKK IEEIQKK IEEIQKK IEEIQKK-a-K(C 8)，
IEEIQKK IEEIQKK IEEIQKK IEEIQKK IEEIQKK-a-K(C 10)，
IEEIQKK IEEIQKK IEEIQKK IEEIQKK IEEIQKK-a-K(C 12)，
IEEIQKK IEEIQKK IEEIQKK IEEIQKK IEEIQKK-a-K(C 14)，
IEEIQKK IEEIQKK IEEIQKK IEEIQKK IEEIQKK-a-K(C 16)，
IEEIQKK IEEIQKK IEEIQKK IEEIQKK IEEIQKK-G-K(C 16)，
IEEIQKK IEEIQKK IEEIQKK IEEIQKK IEEIQKK-z-K(C 16)，
IEEIQKK IEEIQKK IEEIQKK IEEIQKK IEEIQKK-p-K(C 16)，
IEEISKK IEEISKK IEEISKK IEEISKK IEEISKK-a-K(C 16)，
IEEISKK IEEISKK IEEISKK IEEISKK IEEISKK-G-K(C 16)，
IEEISKK IEEISKK IEEISKK IEEISKK IEEISKK-z-K(C 16)，
IEEISKK IEEISKK IEEISKK IEEISKK IEEISKK-p-K(C 16)，
IEEIYKK IEEIYKK IEEIYKK IEEIYKK IEEIYKK-a-K(C 16)，
IEEIYKK IEEIYKK IEEIYKK IEEIYKK IEEIYKK-G-K(C 16)，
IEEIYKK IEEIYKK IEEIYKK IEEIYKK IEEIYKK-z-K(C 16)，
IEEIYKK IEEIYKK IEEIYKK IEEIYKK IEEIYKK-p-K(C 16)，
IEEIQKK IEEISKK IEEISKK IEEISKK IEEIQKK-a-K(C 16)，和
IEEIQKK IEEIYKK IEEIYKK IEEIYKK IEEIQKK-a-K(C 16)；
其中，a为β-Ala，z为6-Aca，p为NH 2-(CH 2CH 2-O) 7-CH 2CH 2-COOH，G为Gly，C 8为辛酸，C 10为癸酸，C 12为月桂酸，C 14为肉豆蔻酸，C 16为棕榈酸。
在本申请的某些优选实施方案中，本发明的化合物与式(I)化合物具有至少90％同一性，优选至少91％同一性、至少92％同一性、至少93％同一性、至少94％同一性、至少95％同一性、至少96％同一性、至少97％同一性、至少98％同一性、或至少99％同一性。
在另一个方面，本申请提供一种药物组合物，其含有前文所述式(I)化合物或与其具有至少80％同一性的化合物、其立体异构体或药学上可接受的盐，以及一种或多种药学上可接受的载体或赋形剂。
当口服给药时，本申请的药物组合物可制成片剂、缓释片、控释片、锭剂、硬或软胶囊、水性或油混悬剂、乳剂、可分散的散剂或颗粒剂、糖浆剂或酏剂、滴丸、微丸或口服溶液。用于口服使用的药物组合物还可以含有例如一种或多种着色剂、甜味剂、矫味剂和/或防腐剂。
用于片剂的适宜的赋形剂包括，惰性稀释剂例如乳糖、碳酸钠、磷酸钙或碳酸钙；崩解剂例如玉米淀粉和藻酸；粘合剂例如淀粉；润滑剂例如硬脂酸镁、硬脂酸或滑石粉；防腐剂例如对羟基苯甲酸乙酯或丙酯；和抗氧剂，例如抗坏血酸等。片剂可以是未包衣的，也可以采用包衣以改变其崩解作用以及活性成分在胃肠道内的后续吸收作用，或改进其稳定性和/或外观，在任意情况中，均可使用该领域熟知的常规包衣剂和方法。
用于硬胶囊的适宜的赋形剂包括，惰性固体稀释剂例如碳酸钙、磷酸钙或高岭土等。用于软胶囊的适宜的赋形剂包括水或油例如花生油、液体石蜡或橄榄油等。
水性混悬剂一般含有微粉形式的活性成分和一种或多种分散剂、湿润剂或助悬剂，所述助悬剂例如为羧甲基纤维素钠、甲基纤维素、羟丙基甲基纤维素、藻酸钠、聚乙烯-吡咯烷酮、西黄蓍胶和阿拉伯胶等；分散剂或湿润剂，例如卵磷脂或烯基氧化物与脂肪酸的缩合物(例如聚氧乙烯硬脂酸酯)，或环氧乙烷与长链脂肪醇的缩合产物，例如十七氧化亚乙基鲸蜡醇，或环氧乙烷与衍生自脂肪酸与己糖醇的偏酯的缩合产物，例如聚氧化乙烯山梨糖醇一油酸酯，或环氧乙烷与衍生自脂肪酸和己糖醇酐的偏酯的缩合产物，例如聚乙烯脱水山梨糖醇一油酸酯。水性混悬剂还可含有一种或多种防腐剂(例如对羟基苯甲酸乙酯或丙酯)、抗氧剂(例如抗坏血酸)、着色剂、矫味剂、和/或甜味剂(例如蔗糖，糖精和天冬酰苯丙氨酸甲酯)等。
可通过将活性成分悬浮在植物油(例如花生油、橄榄油、芝麻油或椰子油)或矿物油(例如液体石蜡)中来配制油性混悬剂。油性混悬剂也可含有增稠剂例如蜂蜡、固体石蜡或鲸蜡醇。可以加入如上所述的甜味剂和矫味剂以提升口服制剂的口感。所述药物组合物可以通过加入抗氧剂例如抗坏血酸来防腐。
本申请的药物组合物也可以采用水包油的乳剂的形式。油相可以是植物油，例如橄榄油或花生油，或者矿物油，例如液体石蜡或者它们的混合物。适当的乳化剂可以 是例如，天然树胶例如阿拉伯胶或西黄芪胶，天然磷脂例如大豆卵磷脂，和衍生自脂肪酸和己糖醇酐的酯或偏酯(例如脱水山梨糖醇一油酸酯)，以及所述偏酯与环氧乙烷的缩合产物，例如聚氧化乙烯脱水山梨糖醇一油酸酯。乳剂也可含有甜味剂、矫味剂和防腐剂等。
糖浆剂和酏剂可与甜味剂(例如甘油、丙二醇、山梨糖醇、阿司帕坦或蔗糖)配制，也可含有缓和剂、防腐剂、矫味剂和/或着色剂等。
当非肠道给药(例如经静脉内、皮下或肌肉内给药)时，所述药物组合物可制成灭菌的水性或油性溶液、无菌粉末、脂质体、乳剂、微乳剂、纳米乳剂或微囊。
所述药物组合物还可以是注射用无菌水性或油性混悬剂的形式，其可以按照已知方法利用一种或多种适宜的分散剂、湿润剂和/或助悬剂来配制，这些试剂如上所述。无菌注射制剂也可以是在稀释剂或溶剂中的注射用无菌水性或油性混悬剂，所述稀释剂或溶剂无毒且肠胃可接受，例如在1，3-丁二醇中的溶液。
有关制剂的其他信息可参考Comprehensive Medicinal Chemistry的第5卷，25.2章(Corwin Hanschl；Chairman of Editorial Board)，PergamonPress1990。
可以根据被治疗宿主和具体给药途径的不同，来确定与一种或多种赋形剂混合以制备单一剂量形式的活性成分的量。例如，用于对人口服给药的制剂一般含有例如0.5mg-2g的活性成分以及适当的和常规量的赋形剂(约占组合物总重的5-98％)。单位制剂中一般约含有1mg-500mg的活性成分。有关给药途径和给药方案的进一步信息可参考Comprehensive Medicinal Chemistry的第5卷，25.3章(Corwin Hanschl；Chairman of Editorial Board)，Pergamon Press 1990。
供治疗或预防目的的药物组合物的给药量，应根据病症的性质和严重性、动物或患者的年龄和性别和给药途径等进行调整。
在基于治疗或预防使用所述药物组合物时，一般是以日剂量在例如1mg-100mg/kg体重的范围内给药，如有需要可以分剂量给药。通常，以肠胃外途径给药时采用较低剂量，例如经静脉内给药时，一般采用例如1mg-10mg/kg体重范围内的剂量。
在另一个方面，本申请提供本前文所述式(I)化合物或与其具有至少80％同一性的化合物、其立体异构体或药学上可接受的盐或药物组合物在制备抑制流感病毒与靶细胞膜融合的药物中的用途。
在某些优选的实施方案中，所述流感病毒为甲型流感病毒。
在某些优选的实施方案中，所述流感病毒选自H1N1和H3N2。
在某些优选的实施方案中，所述靶细胞为细胞系或来自受试者的细胞。
在另一个方面，本申请提供前文所述式(I)化合物或与其具有至少80％同一性的化合物、其立体异构体或药学上可接受的盐在体外抑制流感病毒与靶细胞膜融合中的用途。
在某些优选的实施方案中，所述流感病毒为甲型流感病毒。
在某些优选的实施方案中，所述流感病毒选自H1N1和H3N2。
在某些优选的实施方案中，所述靶细胞为细胞系或来自受试者的细胞。
在另一个方面，本申请提供前文所述式(I)化合物或与其具有至少80％同一性的化合物、其立体异构体或药学上可接受的盐或药物组合物在在制备预防或治疗与流感病毒感染相关的疾病的药物或抗流感病毒药物中的用途。
在某些优选的实施方案中，所述流感病毒为甲型流感病毒。
在某些优选的实施方案中，所述流感病毒选自H1N1和H3N2。
在某些优选的实施方案中，所述与流感病毒感染相关的疾病选自甲型H1N1流感和甲型H3N2流感。
在另一个方面，本申请提供前文所述式(I)化合物或与其具有至少80％同一性的化合物、其立体异构体或药学上可接受的盐或药物组合物，其用于抑制流感病毒与靶细胞膜融合。
在某些优选的实施方案中，所述流感病毒为甲型流感病毒。
在某些优选的实施方案中，所述流感病毒选自H1N1和H3N2。
在某些优选的实施方案中，所述靶细胞为细胞系或来自受试者的细胞。
在某些优选的实施方案中，所述化合物或与其具有至少80％同一性的化合物、其立体异构体或药学上可接受的盐或药物组合物用于体内方法中。
在另一个方面，本申请提供前文所述式(I)化合物或与其具有至少80％同一性的化合物、其立体异构体或药学上可接受的盐或药物组合物，其用于预防或治疗与流感病毒感染相关的疾病或抗流感病毒。
在某些优选的实施方案中，所述流感病毒为甲型流感病毒。
在某些优选的实施方案中，所述流感病毒选自H1N1和H3N2。
在某些优选的实施方案中，所述与流感病毒感染相关的疾病选自甲型H1N1流感和甲型H3N2流感。
在另一个方面，本申请提供一种抑制流感病毒与靶细胞膜融合的方法，其包括向细胞施用有效量的前文所述式(I)化合物或与其具有至少80％同一性的化合物、其立体异构体或药学上可接受的盐或药物组合物。
在某些优选的实施方案中，所述流感病毒为甲型流感病毒。
在某些优选的实施方案中，所述流感病毒选自H1N1和H3N2。
在某些优选的实施方案中，所述靶细胞为细胞系或来自受试者的细胞。
在某些优选的实施方案中，所述方法在体内进行。
在某些优选的实施方案中，所述方法在体外进行。
在另一个方面，本申请提供一种抗病毒的方法，其包括向有此需要的受试者施用有效量的前文所述式(I)化合物或与其具有至少80％同一性的化合物、其立体异构体或药学上可接受的盐或药物组合物。
在某些优选的实施方案中，所述流感病毒为甲型流感病毒。
在某些优选的实施方案中，所述流感病毒选自H1N1和H3N2。
在另一个方面，本申请提供一种预防或治疗与流感病毒感染相关的疾病的方法，其包括向有此需要的受试者施用有效量的前文所述式(I)化合物或与其具有至少80％同一性的化合物、其立体异构体或药学上可接受的盐或药物组合物。
在某些优选的实施方案中，所述流感病毒为甲型流感病毒。
在某些优选的实施方案中，所述流感病毒选自H1N1和H3N2。
在某些优选的实施方案中，所述与流感病毒感染相关的疾病选自甲型H1N1流感和甲型H3N2流感。
在本申请中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的细胞培养、分子遗传学、核酸化学、免 疫学实验室操作步骤均为相应领域内广泛使用的常规步骤。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。
如本文中所使用的，术语“脂肪酸”是指一端含有一个羧基的脂肪族碳链。根据碳链的饱和度可分为饱和脂肪酸、单不饱和脂肪酸和多不饱和脂肪酸，其中，饱和脂肪酸具有C xH 2x+1COOH的结构通式。根据碳链的长度又可分为短链脂肪酸(碳链上的碳原子数小于6)、中链脂肪酸(碳链上的碳原子数为6-12)和长链脂肪酸(碳链上的碳原子数大于12)。
如本文中所使用的，术语“疏水性氨基酸”主要包括酪氨酸、色氨酸、苯丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、甲硫氨酸和丙氨酸。
如本文中所使用的，术语“同一性”用于指两个多肽之间或两个核酸之间序列的匹配情况。当两个进行比较的序列中的某个位置都被相同的碱基或氨基酸单体亚单元占据时(例如，两个DNA分子的每一个中的某个位置都被腺嘌呤占据，或两个多肽的每一个中的某个位置都被赖氨酸占据)，那么各分子在该位置上是同一的。两个序列之间的“百分数同一性”是由这两个序列共有的匹配位置数目除以进行比较的位置数目×100的函数。例如，如果两个序列的10个位置中有6个匹配，那么这两个序列具有60％的同一性。例如，DNA序列CTGACT和CAGGTT共有50％的同一性(总共6个位置中有3个位置匹配)。通常，在将两个序列比对以产生最大同一性时进行比较。这样的比对可通过使用，例如，可通过计算机程序例如Align程序(DNAstar,Inc)方便地进行的Needleman等人(1970)J.Mol.Biol.48：443-453的方法来实现。还可使用已整合入ALIGN程序(版本2.0)的E.Meyers和W.Miller(Comput.Appl Biosci.，4:11-17(1988))的算法，使用PAM120权重残基表(weight residue table)、12的缺口长度罚分和4的缺口罚分来测定两个氨基酸序列之间的百分数同一性。此外，可使用已整合入GCG软件包(可在www.gcg.com上获得)的GAP程序中的Needleman和Wunsch(J MoI Biol.48:444-453(1970))算法，使用Blossum 62矩阵或PAM250矩阵以及16、14、12、10、8、6或4的缺口权重(gap weight)和1、2、3、4、5或6的长度权重来测定两个氨基酸序列之间的百分数同一性。
如本文中所使用的，术语“受试者”是指动物，特别是哺乳动物，优选人。
如本文中所使用的，术语“有效量”是指，足以获得或至少部分获得期望的效果的量。例如，预防有效量是指，足以预防，阻止，或延迟疾病的发生的量；治疗有效量是指，足以治愈或至少部分阻止已患有疾病的患者的疾病和其并发症的量。测定这样 的有效量完全在本领域技术人员的能力范围之内。例如，对于治疗用途有效的量将取决于待治疗的疾病的严重度，患者自己的免疫系统的总体状态，患者的一般情况例如年龄、体重和性别，药物的施用方式，以及同时施用的其他治疗等等。
附图说明
图1显示了激光共聚焦显微镜观察的NBD-IIQ16进入MDCK细胞的情况及在内含体中的分布。
具体实施方式
下面将结合实施例对本发明的实施方案进行详细描述，但是本领域技术人员将会理解，下列实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
在本申请中使用的缩写具有下面的含义：
Ala(Alanine,A)：丙氨酸
Arg(Arginine,R)：精氨酸
Asn(Asparagine,N)：天冬酰胺
Asp(Aspartic acid,D)：天冬氨酸
CHR(C-terminal heptad repeat)：C端重复序列
Cys(Cysteine,C)：半胱氨酸
DCM(Dichloromethane)：二氯甲烷
DMF(N,N-Dimethyl formamide)：二甲基甲酰胺
Fmoc(Fluorenylmethoxycarbonyl)：芴甲氧羰基
Gln(Glutamine,Q)：谷氨酰胺
Glu(Glutamic acid,E)：谷氨酸
Gly(Glycine,G)：甘氨酸
HBTU：2-(1H-1-羟基苯并三唑)-1,1,3,3-四甲基脲六氟磷酸
His(Histidine,H)：组氨酸
HOBt(1-Hydroxylbenzotriazole anhydrous)：1-羟基苯并三氮唑
HPLC(High performance liquid chromatography)：高效液相色谱
IAVs：A型(甲型)流行性感冒病毒
Ile(Isoleucine,I)：异亮氨酸
Leu(Leucine,L)：亮氨酸
Lys(Lysine,K)：赖氨酸
MALDI-TOF-MS：基质辅助激光解吸电离飞行时间质谱
Met(Methionine,M)：甲硫氨酸
NHR(N-terminal heptad repeat)：N端重复序列
NMP(N-Methyl pyrrolidone)：N-甲基吡咯烷酮
Phe(Phenylalanine,F)：苯丙氨酸
Pro(Proline,P)：脯氨酸
Ser(Serine,S)：丝氨酸
TFA(trifluoroacetic acid)：三氟乙酸
Thr(Threonine,T)：苏氨酸
Trp(Tryptophan,W)：色氨酸
Tyr(Tyrosine,Y)：酪氨酸
Val(Valine,V)：缬氨酸
实施例所用固相合成载体Rink酰胺树脂为天津南开合成责任有限公司产品。HBTU、HOBt、DIEA以及Fmoc保护的天然氨基酸或D型的非天然氨基酸为上海吉尔生化公司以及北京欧凯纳斯科技有限公司产品。N-甲基吡咯烷酮(NMP)、三氟乙酸(TFA)为北京百灵威科技有限公司产品。DMF、DCM为国药集团化学试剂有限公司产品。色谱纯乙腈为Fisher公司产品。其他试剂如无说明均为国产分析纯产品。
实施例1：化合物的制备
多肽合成采用标准的Fmoc固相方法。选用Rink Amide树脂，肽链由C端向N端延长。缩合剂为HBTU/HOBt/DIEA。脱保护剂为哌啶/DMF溶液。裂解液为三氟乙酸(TFA)，粗肽水溶解后冻干保存。用中压液相色谱法或高压液相色谱法(HPLC)分离纯化，纯肽含量大于90％。基质辅助激光解析飞行时间质谱(MALDI-TOF-MS) 确定肽序列分子量。
利用CEM微波多肽合成仪合成肽序列。
合成条件如下：
保护氨基酸：0.2M保护氨基酸的DMF溶液，
缩合试剂：0.45M HBTU/HOBt的DMF溶液，
活化碱：2M DIEA的NMP溶液，
脱保护试剂：20％v/v哌啶的DMF溶液，
封闭试剂：20％v/v乙酸酐的DMF溶液。
称取Rink Amide树脂0.23g(0.1mmol)置于CEM微波多肽合成仪反应器中，然后将保护氨基酸，缩合试剂，活化碱，脱保护试剂，封闭试剂按上述浓度配置好后，用CEM微波全自动多肽合成仪进行合成。完成后肽树脂转移至多肽固相合成反应器中，分别用DMF、无水甲醇、DCM各洗涤两遍，室温真空干燥，得肽树脂1.25g。
肽树脂[含有Lys(Dde)特殊氨基酸)]加少许DCM溶胀树脂20min，抽干。配制脱除Dde保护基试剂：2ml水合肼溶于40ml DMF中，体积比20:1。将脱保护试剂加入树脂中，室温搅拌3min，抽干，重复4次，脱除反应毕。称取3equiv的饱和脂肪酸(辛酸、癸酸、月桂酸、肉蔻酸或棕榈酸)、0.11g HBTU(3equiv)和40mg HOBt(3equiv)溶于6ml DMF，加入反应器，再加入320μl DIEA(6equiv)，室温搅拌反应1h。反应毕，抽干反应液，DMF、无水甲醇、DCM交替洗涤树脂两次，乙醚再洗去残留饱和脂肪酸，真空干燥，得肽树脂1.31g。
裂解液(体积百分比)：三氟乙酸：间甲酚：苯甲硫醚：水＝8.5:0.5:0.5:0.5。
肽树脂的裂解：称取微波合成仪合成好的肽树脂1.31g，放入250ml茄形瓶中，冰浴。按1克肽树脂加入10ml的量配制裂解液。TFA需预先冰浴降温30min或者预先存放于冰箱中使用；将配好的裂解液加入到冰浴条件下的肽树脂中，慢速电磁搅拌，树脂变橙红色，冰浴条件下反应30min，然后撤掉冰浴室温下继续搅拌反应150min，反应完成，剧烈搅拌下加入预先在4℃冷却的冰乙醚200ml，析出白色沉淀，继续搅拌30min后静置30min；用G4砂芯漏斗滤出析出物，用冰乙醚反复洗涤滤饼3遍，晾干。加入双蒸水30ml，乙腈10ml使固体充分溶解，抽滤，滤液冻干得粗肽1.04g。
粗肽的纯化：粗肽用中压或高压色谱进行纯化。色谱柱为C8柱，洗脱剂为乙腈，水及少量TFA。具体操作步骤：称取粗肽1.00g，加水20ml，乙腈5ml使固体溶解，离心10min(3000转/分钟)取上清液，上清液经0.23μm无菌滤膜过滤后上样。色谱 柱预先用20％乙腈/水/0.1％TFA溶液200ml平衡。上样后继续用20％乙腈/水/0.1％TFA溶液200ml冲洗，高效液相检测洗脱液成分。根据液相检测结果逐渐升高乙腈含量，直至所纯化的多肽主峰被洗脱出来。合并洗脱液，旋转蒸发除去全部乙腈溶剂，冻干纯肽，HPLC检测含量大于90％，MALDI-TOF确证分子量。各多肽序列如表1所示：
表1 多肽序列信息
编号 多肽序列 分子量
1 IEEIQKK   IEEIQKK   IEEIQKK   IEEIQKK 4730.5
2 IEEIQKK   IEEIQKK   IEEIQKK   IEEIQKK 4758.7
3 IEEIQKK   IEEIQKK   IEEIQKK   IEEIQKK 4786.1
4 IEEIQKK   IEEIQKK   IEEIQKK   IEEIQKK 4814.2
5 IEEIQKK   IEEIQKK   IEEIQKK   IEEIQKK 4842.7
6 IEEIQKK   IEEIQKK   IEEIQKK   IEEIQKK 4827.6
7 IEEIQKK   IEEIQKK   IEEIQKK   IEEIQKK 4884.8
8 IEEIQKK   IEEIQKK   IEEIQKK   IEEIQKK 5214.3
9 IEEISKK   IEEISKK   IEEISKK   IEEISKK 4639.0
10 IEEISKK   IEEISKK   IEEISKK   IEEISKK 4621.8
11 IEEISKK   IEEISKK   IEEISKK   IEEISKK 4679.2
12 IEEISKK   IEEISKK   IEEISKK   IEEISKK 5009.1
13 IEEIYKK   IEEIYKK   IEEIYKK   IEEIYKK 5019.2
14 IEEIYKK   IEEIYKK   IEEIYKK   IEEIYKK 5000.2
15 IEEIYKK   IEEIYKK   IEEIYKK   IEEIYKK 5061.8
16 IEEIYKK   IEEIYKK   IEEIYKK   IEEIYKK 5388.7
17 IEEIQKK   IEEISKK   IEEISKK   IEEISKK 4718.6
18 IEEIQKK   IEEIYKK   IEEIYKK   IEEIYKK 4948.5
其中，a:β-Ala；z:6-Aca；p:NH 2-(CH 2CH 2-O) 7-CH 2CH 2-COOH；G：Gly；C 8：辛酸，C 10：癸酸，C 12：月桂酸，C 14：肉豆蔻酸，C 16：棕榈酸。
实施例2：多肽的圆二色谱分析
将多肽分别与衍生于H3N2HA区域的靶标肽N66(H3N2融合蛋白NHR区域40-105残基)孵育，孵育方法：将多肽与N66等摩尔混合后30℃孵育30min，醋酸钠缓冲液(pH 5.0)稀释混合物至终浓度10μM进行测试。
使用MOS-450圆二色谱仪对多肽溶液进行波谱扫描，设置参数：扫描波长180-280nm，扫描光程1mm，扫描单位1nm，每个样品扫描重复次数3次。具备典型α螺旋结构的多肽在CD谱图上表现为在208nm和222nm处的负峰和195nm处的正峰，α螺旋度则是以在222nm处的摩尔椭圆率为-33000(deg·cm 2·dmol -1)为100％α螺 旋度来计算待测多肽及复合物的螺旋含量百分比。接下来，通过CD温度扫描测定本申请所描述多肽与靶标N66相互作用并形成结合物的稳定性。具体方法如下：将上述用于测定CD信号的多肽转入样品池(也可以重新配置)，将CD仪器程序设定为温度扫描，检测波长220nm，扫描范围20-98℃，进行程序温度扫描后得到CD信号随温度变化曲线，根据曲线计算Tm值。圆二色谱结果见表2。
表2 圆二色谱测定多肽的α螺旋含量
Figure PCTCN2019087595-appb-000001
圆二色谱分析表明，在PBS(pH＝5.0)中，多肽可以与靶标N66相互作用形成典型的α螺旋结构。抗病毒活性较好的多肽与靶标N66形成复合物的螺旋含量较高且形成复合物的稳定性也相对较高。这些结果表明多肽的抗病毒活性与其靶标形成异源 6HB的螺旋度和热稳定性有关。
实施例3：化合物抑制IAVs致细胞病变实验(IC 50)
以10 4细胞/每孔的数量将MDCK细胞(购自美国菌种保存中心ATCC)接种到96孔培养板上，细胞培养液使用含10％胎牛血清(FBS)的DMEM培养液，37℃5％CO 2培养24h。次日镜下观察，待细胞长至80％-90％时弃去培养液，用PBS(pH 7.4)洗2次除去残留的含血清培养基。使用含0.2％BSA的DMEM培养液倍比稀释多肽样品，预先与100TCID 50/mL的流感病毒A/Puerto Rico/8/34(H1N1)或A/Aichi/2/68(H3N2)共孵30min，30min后病毒与多肽混合液加入细胞中吸附病毒。1h后，吸附完病毒，移去病毒混合液，加入3ml含1％琼脂糖的维持液(1μg/ml TPCK-胰酶)覆盖细胞。同时设立正常MDCK细胞阴性对照组、病毒感染阳性对照组和抗病毒阳性药奥司他韦对照组。37℃5％CO 2倒置培养48h后，同时每日在倒置显微镜下观察细胞形态变化情况。用MTT法进行抗病毒活性测定：取出48h培养后的细胞板，移去培养基，加入100μl含0.5mg/ml MTT的培养基，37℃5％CO 2避光培养4h；之后弃去培养基，加入150μl的DMSO溶解振荡10min，测定在570nm处的OD值，使用Prism5.01计算化合物的IC 50，重复试验三次。活性测试结果如表3所示。
表3 多肽抗流感活性测试
Figure PCTCN2019087595-appb-000002
Figure PCTCN2019087595-appb-000003
由表3活性结果可知，所有多肽均可有效抑制甲型流感毒株的复制，其中化合物3、4、5、8、9、10、13、14、15抑制甲型H1N1流感病毒株活性达到低μM水平与阳性对照Oseltamivir acid(化合物19)相当；化合物2、5、6、7、9、11、12、13、16、17抑制甲型H3N2流感病毒活性与阳性对照Oseltamivir acid相当。
实施例4：激光共聚焦显微镜成像实验研究多肽进入细胞内含体
该实验中选用的细胞是流感病毒侵染模型中的靶细胞MDCK细胞，多肽用绿色荧光标签NBD标记，通过激光共聚焦显微镜直接观察NBD-IIQ16进入MDCK细胞的情况及在内含体的分布。多肽与MDCK细胞孵育后由PBS充分清洗，经LysoTracher-red标记后固定细胞。发射绿色荧光的多肽能够进入细胞并广泛分布于经LysoTracher标记的内含体红色区域(绿色荧光与红色荧光叠加后形成黄色荧光)。能够透膜并进入内含体的功能可能是脂肽抑制流感病毒膜融合的重要原因。
将IIQ16(化合物5)的N端共价缀合荧光分子NBD，并命名为NBD-IIQ16。实验采用MDCK细胞，给药前24h在NEST 15mm小皿中接种10 4个细胞，在37℃ 5％CO 2培养箱中培养。24h后吸出培养液，加入1mL NBD-IIQ16的DMEM培养液，37℃孵育2h。之后吸出培养液，用PBS清洗3次，再加入1ml 50nM的LysoTracker Red/DMEM溶液，37℃培养30min。吸出培养液，依次用2mg/ml肝素钠/PBS溶液洗3次，PBS洗3次，使用3％多聚甲醛固定样本，在Carl Zeiss LSM510meta激光共聚焦显微镜下观察。LysoTracker Red激发波长为577nm。结果如图1所示。
尽管本发明的具体实施方式已经得到了详细的阐述，本领域技术人员将会理解。根据已经公开的所有教导，可以对那些细节进行各种修改和替换，这些改变均在本发明的保护范围之内。本发明的全部范围有所附权利要求及其任何等同物给出。

Claims (16)

  1. 式(I)所示化合物或与其具有至少80％同一性的化合物、其立体异构体或药学上可接受的盐，
    Ac-[X aEEX dX eKK] m-L 1-K(R 1)-NH 2
    (I)
    其中，X a表示疏水性氨基酸，各X a相同或不同；
    X d表示疏水性氨基酸，各X d相同或不同；
    X e选自下述氨基酸：Ser、Asn、Gln、Glu、Asp、Lys、Arg、His、Tyr、Trp、Met和Cys，各X e相同或不同；
    E为Glu；
    K为Lys；
    R 1选自胆固醇、类固醇、鞘氨醇和脂肪酸；
    L 1选自Gly、β-丙氨酸(β-Ala)、γ-氨基丁酸(GABA)、6-氨基己酸(6-Aca)和NH 2-(CH 2CH 2-O) n-CH 2CH 2-COOH，其中，n为选自1-25的整数；
    m选自2、3、4、5、6、7、8、9和10。
  2. 权利要求1的化合物或与其具有至少80％同一性的化合物、其立体异构体或药学上可接受的盐，其中X a选自Ala、Val、Leu、Ile、Pro、Phe、Tyr、Trp和Met，各X a相同或不同；
    优选地，X a为Ile。
  3. 权利要求1或2的化合物或与其具有至少80％同一性的化合物、其立体异构体或药学上可接受的盐，其中X d选自Ala、Val、Leu、Ile、Pro、Phe、Tyr、Trp和Met，各X d相同或不同；
    优选地，X d为Ile。
  4. 权利要求1-3任一项的化合物或与其具有至少80％同一性的化合物、其立体异构体或药学上可接受的盐，其中X e选自Ser、Gln和Lys，各X e相同或不同；
    优选地，X e为Gln、Ser或Tyr；
    优选地，X e为Gln。
  5. 权利要求1-4任一项的化合物或与其具有至少80％同一性的化合物、其立体异构体或药学上可接受的盐，其中R 1选自C 6-24饱和脂肪酸；
    优选地，R 1选自辛酸、癸酸、月桂酸、肉豆蔻酸和棕榈酸；
    优选地，R 1为棕榈酸。
  6. 权利要求1-5任一项的化合物或与其具有至少80％同一性的化合物、其立体异构体或药学上可接受的盐，其中L 1选自Gly、β-Ala、6-Aca和NH 2-(CH 2CH 2-O) n-CH 2CH 2-COOH，其中，n＝1、2、3、4、5、7、9、11或23；
    优选地，L 1选自Gly、β-丙氨酸(β-Ala)、6-氨基己酸(6-Aca)和NH 2-(CH 2CH 2-O) 7-CH 2CH 2-COOH；
    优选地，L 1选自Gly、β-丙氨酸(β-Ala)和6-氨基己酸(6-Aca)。
  7. 权利要求1-6任一项的化合物或与其具有至少80％同一性的化合物、其立体异构体或药学上可接受的盐，其中所述化合物选自：
    IEEIQKK IEEIQKK IEEIQKK IEEIQKK IEEIQKK-a-K(C 8)，
    IEEIQKK IEEIQKK IEEIQKK IEEIQKK IEEIQKK-a-K(C 10)，
    IEEIQKK IEEIQKK IEEIQKK IEEIQKK IEEIQKK-a-K(C 12)，
    IEEIQKK IEEIQKK IEEIQKK IEEIQKK IEEIQKK-a-K(C 14)，
    IEEIQKK IEEIQKK IEEIQKK IEEIQKK IEEIQKK-a-K(C 16)，
    IEEIQKK IEEIQKK IEEIQKK IEEIQKK IEEIQKK-G-K(C 16)，
    IEEIQKK IEEIQKK IEEIQKK IEEIQKK IEEIQKK-z-K(C 16)，
    IEEIQKK IEEIQKK IEEIQKK IEEIQKK IEEIQKK-p-K(C 16)，
    IEEISKK IEEISKK IEEISKK IEEISKK IEEISKK-a-K(C 16)，
    IEEISKK IEEISKK IEEISKK IEEISKK IEEISKK-G-K(C 16)，
    IEEISKK IEEISKK IEEISKK IEEISKK IEEISKK-z-K(C 16)，
    IEEISKK IEEISKK IEEISKK IEEISKK IEEISKK-p-K(C 16)，
    IEEIYKK IEEIYKK IEEIYKK IEEIYKK IEEIYKK-a-K(C 16)，
    IEEIYKK IEEIYKK IEEIYKK IEEIYKK IEEIYKK-G-K(C 16)，
    IEEIYKK IEEIYKK IEEIYKK IEEIYKK IEEIYKK-z-K(C 16)，
    IEEIYKK IEEIYKK IEEIYKK IEEIYKK IEEIYKK-p-K(C 16)，
    IEEIQKK IEEISKK IEEISKK IEEISKK IEEIQKK-a-K(C 16)，和
    IEEIQKK IEEIYKK IEEIYKK IEEIYKK IEEIQKK-a-K(C 16)；
    其中，a为β-Ala，z为6-Aca，p为NH 2-(CH 2CH 2-O) 7-CH 2CH 2-COOH，G为Gly，C 8为辛酸，C 10为癸酸，C 12为月桂酸，C 14为肉豆蔻酸，C 16为棕榈酸。
  8. 药物组合物，其含有权利要求1-7任一项的化合物或与其具有至少80％同一性的化合物、其立体异构体或药学上可接受的盐，以及一种或多种药学上可接受的载体或赋形剂。
  9. 权利要求1-7任一项的化合物或与其具有至少80％同一性的化合物、其立体异构体或药学上可接受的盐或权利要求8的药物组合物在制备抑制流感病毒与靶细胞膜融合的药物中的用途；优选地，所述流感病毒为甲型流感病毒；优选地，所述流感病毒选自H1N1和H3N2。
  10. 权利要求1-7任一项的化合物或与其具有至少80％同一性的化合物、其立体异构体或药学上可接受的盐或权利要求8的药物组合物在制备预防或治疗与流感病毒感染相关的疾病的药物或抗流感病毒药物中的用途；优选地，所述流感病毒为甲型流感病毒；优选地，所述流感病毒选自H1N1和H3N2；优选地，所述与流感病毒感染相关的疾病选自甲型H1N1流感和甲型H3N2流感。
  11. 权利要求1-7任一项的化合物或与其具有至少80％同一性的化合物、其立体异构体或药学上可接受的盐在体外抑制流感病毒与靶细胞膜融合中的用途；优选地，所述流感病毒为甲型流感病毒；优选地，所述流感病毒选自H1N1和H3N2；优选地，所述靶细胞为细胞系或来自受试者的细胞。
  12. 权利要求1-7任一项的化合物或与其具有至少80％同一性的化合物、其立体异构体或药学上可接受的盐或权利要求8的药物组合物，其用于抑制流感病毒与靶细胞膜融合；优选地，所述流感病毒为甲型流感病毒；优选地，所述流感病毒选自H1N1 和H3N2；优选地，所述靶细胞为细胞系或来自受试者的细胞；优选地，所述化合物或与其具有至少80％同一性的化合物、其立体异构体或药学上可接受的盐或药物组合物用于体内方法中。
  13. 权利要求1-7任一项的化合物或与其具有至少80％同一性的化合物、其立体异构体或药学上可接受的盐或权利要求8的药物组合物，其用于预防或治疗与流感病毒感染相关的疾病或抗流感病毒；优选地，所述流感病毒为甲型流感病毒；优选地，所述流感病毒选自H1N1和H3N2；优选地，所述与流感病毒感染相关的疾病选自甲型H1N1流感和甲型H3N2流感。
  14. 一种抑制流感病毒与靶细胞膜融合的方法，其包括向细胞施用有效量的权利要求1-7任一项的化合物或与其具有至少80％同一性的化合物、其立体异构体或药学上可接受的盐或权利要求8的药物组合物；优选地，所述流感病毒为甲型流感病毒；优选地，所述流感病毒选自H1N1和H3N2；优选地，所述靶细胞为细胞系或来自受试者的细胞；优选地，所述方法在体内进行；优选地，所述方法在体外进行。
  15. 一种抗病毒的方法，其包括向有此需要的受试者施用有效量的权利要求1-7任一项的化合物或与其具有至少80％同一性的化合物、其立体异构体或药学上可接受的盐或权利要求8的药物组合物；优选地，所述流感病毒为甲型流感病毒；优选地，所述流感病毒选自H1N1和H3N2。
  16. 一种预防或治疗与流感病毒感染相关的疾病的方法，其包括向有此需要的受试者施用有效量的权利要求1-7任一项的化合物或与其具有至少80％同一性的化合物、其立体异构体或药学上可接受的盐或权利要求8的药物组合物；优选地，所述流感病毒为甲型流感病毒；优选地，所述流感病毒选自H1N1和H3N2；优选地，所述与流感病毒感染相关的疾病选自甲型H1N1流感和甲型H3N2流感。
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