WO2019223644A1 - Polypeptide, and pharmaceutical composition and use thereof - Google Patents

Abstract

Provided are a polypeptide, and a pharmaceutical composition and use thereof. In particular, provided are a compound represented by formula (I) or a compound having at least 80% of identity thereto, a stereoisomer or pharmaceutically-acceptable salt of the compound, a pharmaceutical composition of the compound, and use of the compound in the preparation of drugs against influenza virus infections or drugs for preventing or treating diseases associated with the influenza virus infections, Ac-[X_aEEX_dX_eKK_]m-L₁-K(R₁)-NH₂ (I).

Description

Polypeptide and pharmaceutical composition and use thereof

This application is based on an application with a CN application number of 201810499379.3 and an application date of May 23, 2018, and claims its priority. The disclosure of this CN application is hereby incorporated by reference in its entirety.

Technical field

The present application relates to the field of biomedicine, and in particular, to polypeptides and pharmaceutical compositions and uses thereof.

Background technique

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.

At present, there are two main types of drugs used clinically to treat influenza virus infection-neuraminidase inhibitors (such as oseltamivir and zanamivir) and 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. As the current 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 α-2,3 or α-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. Under the acidic conditions of the endosome (pH ~ 5.5), the conformation of the HA1 region, which was originally a tightly-clamped "clip" -like structure at neutral pH, rearranged and gradually dissociated from the HA2 region. HA2 then undergoes a conformational rearrangement. The fusion peptide hidden in the protein is exposed and inserted into the endosome membrane. Subsequently, 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.

Although 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. However, there are no reports of 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. Unlike the HIV-1 virus, which fuses directly on the surface of the host cell membrane, influenza virus fusion needs to undergo endocytosis first, triggering the fusion process under low pH conditions in the cell inclusion body.

Summary of the Invention

Based on the virus-host cell membrane fusion mechanism of IAVs and the essential characteristics of six alpha helix bundles (6HB), 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.

Accordingly, in one aspect 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,

Ac- [X _a EEX _d X _e KK] _m -L ₁ -K (R ₁ ) -NH ₂

(I)

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;

K is Lys;

R ₁ represents cholesterol, steroids, sphingosine, and fatty acids (such as C _6-24 fatty acids);

L _{1 is} selected from Gly, β-alanine (β-Ala), γ-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;

m is selected from 2, 3, 4, 5, 6, 7, 8, 9, and 10.

In certain preferred embodiments, 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.

In certain preferred embodiments, X _{a is} selected from Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, and Met, each X _{a being the} same or different.

In certain preferred embodiments, X _{a is} selected from Ala, Val, Leu, Ile, Phe, and Tyr, each X _{a being the} same or different.

In certain preferred embodiments, X _a is Ile.

In certain preferred embodiments, 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.

In certain preferred embodiments, X _{d is} selected from Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp and Met, each X _{d being the} same or different.

In certain preferred embodiments, X _{d is} selected from Ala, Val, Leu, Ile, Phe, and Tyr, each X _{d being the} same or different.

In certain preferred embodiments, X _d is Ile.

In certain preferred embodiments, 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.

In certain preferred embodiments, X _{e is} selected from Ser, Gln, Glu, Lys, His, Tyr, and Trp, each X _{e being the} same or different.

In certain preferred embodiments, X _e is Gln, Ser or Tyr.

In certain preferred embodiments, X _e is Gln.

In certain preferred embodiments, R _{1 is} selected from C _6-24 saturated fatty acids.

In certain preferred embodiments, R _{1 is} selected from caprylic acid, capric acid, lauric acid, myristic acid, and palmitic acid.

In certain preferred embodiments, L _{1 is} selected from Gly, β-alanine (β-Ala), 6-aminocaproic acid (6-Aca), and NH _2- (CH ₂ CH ₂ -O) _n- CH ₂ CH ₂ -COOH, where n = 1, ₂ , 3, 4, 5, 7, 9, 11, or 23.

In certain preferred embodiments, L _{1 is} selected from Gly, β-alanine (β-Ala), 6-aminocaproic acid (6-Aca), and NH _2- (CH ₂ CH ₂ -O) _7- CH ₂ CH ₂ -COOH.

In certain preferred embodiments, L _{1 is} selected from Gly, β-alanine (β-Ala), and 6-aminocaproic acid (6-Aca).

In certain preferred embodiments, L ₁ is β-alanine (β-Ala).

In certain preferred embodiments, m is selected from 3, 4, 5, 6, 7, and 8.

In certain preferred embodiments, m is selected from 4, 5, and 6.

In certain preferred embodiments, m is 5.

In certain preferred embodiments, X _a is Ile; Xd is Ile; X _e is Gln, Ser or Tyr; R ₁ is palmitic acid; L ₁ is Gly, β-alanine (β-Ala), 6 -Aminohexanoic acid (6-Aca) or NH _2- (CH ₂ CH ₂ -O) ₇ -CH ₂ CH ₂ -COOH; m is 5.

In certain preferred embodiments, the compound is selected from:

IEEIQKK IEEIQKK IEEIQKK IEEIQKK IEEIQKK-aK (C ₈ ),

IEEIQKK IEEIQKK IEEIQKK IEEIQKK IEEIQKK-aK (C ₁₀ ),

IEEIQKK IEEIQKK IEEIQKK IEEIQKK IEEIQKK-aK (C ₁₂ ),

IEEIQKK IEEIQKK IEEIQKK IEEIQKK IEEIQKK-aK (C ₁₄ ),

IEEIQKK IEEIQKK IEEIQKK IEEIQKK IEEIQKK-aK (C ₁₆ ),

IEEIQKK IEEIQKK IEEIQKK IEEIQKK IEEIQKK-GK (C ₁₆ ),

IEEIQKK IEEIQKK IEEIQKK IEEIQKK IEEIQKK-zK (C ₁₆ ),

IEEIQKK IEEIQKK IEEIQKK IEEIQKK IEEIQKK-pK (C ₁₆ ),

IEEISKK IEEISKK IEEISKK IEEISKK IEEISKK-aK (C ₁₆ ),

IEEISKK IEEISKK IEEISKK IEEISKK IEEISKK-GK (C ₁₆ ),

IEEISKK IEEISKK IEEISKK IEEISKK IEEISKK-zK (C ₁₆ ),

IEEISKK IEEISKK IEEISKK IEEISKK IEEISKK-pK (C ₁₆ ),

IEEIYKK IEEIYKK IEEIYKK IEEIYKK IEEIYKK-aK (C ₁₆ ),

IEEIYKK IEEIYKK IEEIYKK IEEIYKK IEEIYKK-GK (C ₁₆ ),

IEEIYKK IEEIYKK IEEIYKK IEEIYKK IEEIYKK-zK (C ₁₆ ),

IEEIYKK IEEIYKK IEEIYKK IEEIYKK IEEIYKK-pK (C ₁₆ ),

IEEIQKK IEEISKK IEEISKK IEEISKK IEEIQKK-aK (C ₁₆ ), and

IEEIQKK IEEIYKK IEEIYKK IEEIYKK IEEIQKK-aK (C ₁₆ );

Where a is β-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, and C ₁₀ is capric acid , C ₁₂ is lauric acid, C ₁₄ is myristic acid, and C ₁₆ is palmitic acid.

In certain preferred embodiments of the present application, 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.

In another aspect, the present application provides 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.

When administered orally, 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 monooleate. 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).

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.

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 .

For additional information on formulations, please refer to Comprehensive Medicine, Volume 5, Chapter 25.2 (Corwin Hanschl; Chair of Editorial Board), Pergamon Press 1990.

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. For example, 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. For further information on the route of administration and schedule, please refer to Volume 5 of the Comprehensive Medical Chemistry Chapter 25.3 (Corwin Hanschl; Chair of Editorial Board), Pergamon Press 1990.

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.

When the pharmaceutical composition is 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. Generally, 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.

In another aspect, 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.

In certain preferred embodiments, the influenza virus is an influenza A virus.

In certain preferred embodiments, the influenza virus is selected from H1N1 and H3N2.

In certain preferred embodiments, the target cells are cell lines or cells from a subject.

In another aspect, 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.

In certain preferred embodiments, the influenza virus is an influenza A virus.

In certain preferred embodiments, the influenza virus is selected from H1N1 and H3N2.

In certain preferred embodiments, the target cells are cell lines or cells from a subject.

In another aspect, 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.

In certain preferred embodiments, the influenza virus is an influenza A virus.

In certain preferred embodiments, the influenza virus is selected from H1N1 and H3N2.

In certain preferred embodiments, the disease associated with influenza virus infection is selected from the group consisting of H1N1 influenza and H3N2 influenza.

In another aspect, 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.

In certain preferred embodiments, the influenza virus is an influenza A virus.

In certain preferred embodiments, the influenza virus is selected from H1N1 and H3N2.

In certain preferred embodiments, the target cells are cell lines or cells from a subject.

In certain preferred embodiments, 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.

In another aspect, 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.

In certain preferred embodiments, the influenza virus is an influenza A virus.

In certain preferred embodiments, the influenza virus is selected from H1N1 and H3N2.

In certain preferred embodiments, the disease associated with influenza virus infection is selected from the group consisting of H1N1 influenza and H3N2 influenza.

In another aspect, 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.

In certain preferred embodiments, the influenza virus is an influenza A virus.

In certain preferred embodiments, the influenza virus is selected from H1N1 and H3N2.

In certain preferred embodiments, the target cells are cell lines or cells from a subject.

In certain preferred embodiments, the method is performed in vivo.

In certain preferred embodiments, the method is performed in vitro.

In another aspect, 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.

In certain preferred embodiments, the influenza virus is an influenza A virus.

In certain preferred embodiments, the influenza virus is selected from H1N1 and H3N2.

In another aspect, 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.

In certain preferred embodiments, the influenza virus is an influenza A virus.

In certain preferred embodiments, the influenza virus is selected from H1N1 and H3N2.

In certain preferred embodiments, the disease associated with influenza virus infection is selected from the group consisting of H1N1 influenza and H3N2 influenza.

In this application, unless otherwise stated, scientific and technical terms used herein have the meaning commonly understood by those skilled in the art. In addition, the cell culture, molecular genetics, nucleic acid chemistry, and immunology laboratory procedures used in this article are all routine procedures widely used in the corresponding field. Meanwhile, in order to better understand the present invention, definitions and explanations of related terms are provided below.

As used herein, the term "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).

As used herein, the term "hydrophobic amino acid" mainly includes tyrosine, tryptophan, phenylalanine, valine, leucine, isoleucine, proline, methionine and Alanine.

As used herein, the term "identity" is used to refer to a sequence match between two polypeptides or between two nucleic acids. When 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), then 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. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 positions out of a total of 6 positions match). Generally, 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)), which has been integrated into the ALIGN program (version 2.0), can also be used, and the PAM120 weight residue table is used. , A gap length penalty of 12, and a gap penalty of 4 to determine the percent identity between two amino acid sequences. In addition, the Needleman and Wunsch (J MoI Biol. 48: 444-453 (1970)) algorithm integrated into the GAP program of the GCG software package (available at www.gcg.com) can be used, using the Blossom 62 matrix or PAM250 matrix with gap weights of 16, 14, 12, 10, 8, 6, or 4 and length weights of 1, 2, 3, 4, 5, or 6 to determine the percent identity between two amino acid sequences .

As used herein, the term "subject" refers to an animal, particularly a mammal, preferably a human.

As used herein, the term "effective amount" refers to an amount sufficient to obtain or at least partially obtain a desired effect. For example, 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. For example, 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.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows how NBD-IIQ16 enters MDCK cells and its distribution in inclusion bodies as observed by a laser confocal microscope.

Detailed ways

The embodiments of the present invention will be described in detail below with reference to examples, but those skilled in the art will understand that the following examples are only used to illustrate the present invention, and should not be considered as limiting the scope of the present invention. If the specific conditions are not indicated in the examples, the conventional conditions or the conditions recommended by the manufacturer are used. If the reagents or instruments used are not specified by the manufacturer, they are all conventional products that are commercially available.

The abbreviations used in this application have the following meanings:

Ala (Alanine, A): Alanine

Arg (Arginine, R): arginine

Asn (Asparagine, N): Asparagine

Asp (Aspartic acid, D): Aspartic acid

CHR (C-terminal heptad repeat): C-terminal repeat

Cys (Cysteine, C): Cysteine

DCM (Dichloromethane): methylene chloride

DMF (N, N-Dimethyl formamide): dimethylformamide

Fmoc (Fluorenylmethoxycarbonyl):

Gln (Glutamine, Q): Glutamine

Glu (Glutamic acid, E): glutamic acid

Gly (Glycine, G): Glycine

HBTU: 2- (1H-1-hydroxybenzotriazole) -1,1,3,3-tetramethylurea hexafluorophosphate

His (Histidine, H): Histidine

HOBt (1-Hydroxylbenzotriazole anhydrous): 1-hydroxybenzotriazole

HPLC (High Performance Liquid Chromatography): High Performance Liquid Chromatography

IAVs: Type A (type A) influenza virus

Ile (Isoleucine, I): Isoleucine

Leu (Leucine, L): Leucine

Lys (Lysine, K): Lysine

MALDI-TOF-MS: Matrix-assisted laser desorption ionization time-of-flight mass spectrometry

Met (Methionine, M): Methionine

NHR (N-terminal heptad repeat): N-terminal repeat

NMP (N-Methyl pyrrrolidone): N-methylpyrrolidone

Phe (Phenylalanine, F): Phenylalanine

Pro (Proline, P): Proline

Ser (Serine, S): Serine

TFA (trifluoroacetic acid): trifluoroacetic acid

Thr (Threonine, T): Threonine

Trp (Tryptophan, W): Tryptophan

Tyr (Tyrosine, Y): Tyrosine

Val (Valine, V): Valine

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. N-methylpyrrolidone (NMP) and trifluoroacetic acid (TFA) are products of Beijing Bailingwei Technology Co., Ltd. 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.

Example 1: Preparation of compounds

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.

The synthesis conditions are as follows:

Protected amino acids: 0.2M DMF solution of protected amino acids,

Condensation reagent: 0.45M HBTU / HOBt DMF solution,

Activated base: 2M DIEA NMP solution,

Deprotection reagent: 20% v / v piperidine in DMF,

Blocking reagent: 20% v / v acetic anhydride in DMF.

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. Preparation of 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. Weigh 3equiv saturated fatty acids (octanoic acid, capric acid, lauric acid, myristic acid or palmitic acid), 0.11g HBTU (3equiv) and 40mg HOBt (3equiv) in 6ml DMF, add to the reactor, and add 320 μl DIEA (6equiv ), The reaction was stirred at room temperature for 1 h. After the reaction, the reaction solution was drained, and the resin was washed twice with DMF, anhydrous methanol, and DCM. The ether was washed with residual saturated fatty acids, and dried under vacuum to obtain 1.31 g of peptide resin.

Lysate (% by volume): trifluoroacetic acid: m-cresol: anisole: water = 8.5: 0.5: 0.5: 0.5.

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. Continue stirring for 30 minutes and let stand for 30 minutes. Filter the precipitate with a G4 sand funnel. The filter cake was repeatedly washed 3 times with iced ether and air-dried. 30 ml of double distilled water and 10 ml of acetonitrile were added to fully dissolve the solid, suction filtration was performed, and the filtrate was lyophilized to obtain 1.04 g of a crude peptide.

Purification of crude peptides: 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. Specific operation steps: Weigh 1.00 g of crude peptide, add 20 ml of water and 5 ml of acetonitrile to dissolve the solid, centrifuge for 10 min (3000 rpm) to take the supernatant, and apply the supernatant after filtering through a 0.23 μm sterile filter. The column was equilibrated with 200 ml of 20% acetonitrile / water / 0.1% TFA solution in advance. After the sample was loaded, it was rinsed with 200ml of 20% acetonitrile / water / 0.1% TFA solution, and the composition of the eluent was detected by high performance liquid phase. According to the results of liquid phase detection, the acetonitrile content was gradually increased until the main peak of the purified peptide was eluted. The eluents were combined, all acetonitrile solvents were removed by rotary evaporation, and the pure peptide was lyophilized. The content of HPLC was greater than 90%, and the molecular weight was confirmed by MALDI-TOF. The sequence of each polypeptide is shown in Table 1:

Table 1.Peptide sequence information

Numbering	Peptide sequence	Molecular weight
1	IEEIQKK IEEIQKK IEEIQKK	4730.5
2	IEEIQKK IEEIQKK IEEIQKK	4758.7
3	IEEIQKK IEEIQKK IEEIQKK	4786.1
4	IEEIQKK IEEIQKK IEEIQKK	4814.2
5	IEEIQKK IEEIQKK IEEIQKK	4842.7
6	IEEIQKK IEEIQKK IEEIQKK	4827.6
7	IEEIQKK IEEIQKK IEEIQKK	4884.8
8	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	5019.2
14	IEEIYKK IEEIYKK IEEIYKK	5000.2
15	IEEIYKK IEEIYKK IEEIYKK	5061.8
16	IEEIYKK IEEIYKK IEEIYKK	5388.7
17	IEEIQKK IEEISKK IEEISKK IEEISKK	4718.6
18	IEEIQKK IEEIYKK IEEIYKK IEEIYKK	4948.5

Among them, a: β-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.

Example 2: Circular dichroism analysis of peptides

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 μM 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 α-helical structure appear on the CD spectrum as negative peaks at 208 nm and 222 nm and positive peaks at 195 nm, while α-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. Next, 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.

Table 2 Cyclic dichromatographic determination of the α-helix content of peptides

Circular dichroism analysis showed that in PBS (pH = 5.0), the peptide can interact with the target N66 to form a typical α-helical structure. Polypeptides with good antiviral activity and the target N66 form a complex with a higher helix content and the stability of the complex formation is relatively high. These results indicate that the antiviral activity of a polypeptide is related to the helicity and thermostability of its target to form heterologous 6HB.

Example 3: Experiment of Compound Inhibiting IAVs-induced Cytopathic Disease (IC ₅₀ )

To ¹⁰⁴ cells / per well number of MDCK cells (available from the American Type Culture Collection ATCC) were seeded into 96-well culture plates, cell culture medium, DMEM culture medium containing 10% fetal bovine serum (FBS) is, 37 [deg.] C Incubate at 5% CO _{2 for} 24 h. Observe under the microscope the next day. When the cells reach 80% -90%, discard the culture medium and wash twice with PBS (pH 7.4) to remove the residual serum-containing medium. 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 μg / ml TPCK-trypsin) was added to cover the cells. At the same time, 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 μl 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 μl 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.

Table 3 Peptide anti-influenza activity test

From the activity results in Table 3, it can be known that all polypeptides can effectively inhibit the replication of influenza A strains, among which compounds 3, 4, 5, 8, 9, 10, 13, 14, and 15 inhibit the activity of influenza A H1N1 influenza virus strains to reach a low level. The μM level was comparable to that of the positive control Oseltamivir acid (Compound 19); compounds 2, 5, 6, 7, 9, 11, 12, 13, 16, 17 inhibited influenza A H3N2 influenza virus activity comparable to the positive control Oseltamivir acid.

Example 4: Laser Confocal Microscopy Imaging Experimental Study of Peptide Entry into Cell Inclusion Body

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 (compound 5) 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 ℃ 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. Aspirate the culture solution, wash it 3 times with 2mg / ml sodium heparin / PBS solution, wash 3 times with PBS, fix the sample with 3% paraformaldehyde, and observe under Carl Zeiss LSM510meta laser confocal microscope. LysoTracker Red has an excitation wavelength of 577 nm. The results are shown in Figure 1.

Although specific embodiments of the present invention have been described in detail, those skilled in the art will understand. According to all the teachings that have been disclosed, various modifications and substitutions can be made to those details, and these changes are all within the protection scope of the present invention. The full scope of the invention is given by the appended claims and any equivalents thereof.

Claims (16)

1. A compound represented by formula (I) or a compound having at least 80% identity therewith, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof,

   Ac- [X _a EEX _d X _e KK] _m -L ₁ -K (R ₁ ) -NH ₂

   (I)

   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;

   K is Lys;

   R _{1 is} selected from cholesterol, steroids, sphingosine and fatty acids;

   L _{1 is} selected from Gly, β-alanine (β-Ala), γ-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;

   m is selected from 2, 3, 4, 5, 6, 7, 8, 9, and 10.
2. The compound of claim 1, or a compound having at least 80% identity thereto, _a stereoisomer or a pharmaceutically acceptable salt thereof, wherein X _{a is} selected from the group consisting of Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, and Met, each X _{a is the} same or different;

   Preferably, X _a is Ile.
3. The compound of claim 1 or 2 or a compound having at least 80% identity, a stereoisomer or a pharmaceutically acceptable salt thereof, wherein X _{d is} selected from Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp and Met, each X _{d is the} same or different;

   Preferably, X _d is Ile.
4. The compound according to any one of claims 1 to 3, or a compound having at least 80% identity therewith, a stereoisomer or a pharmaceutically acceptable salt thereof, wherein X _{e is} selected from the group consisting of Ser, Gln, and Lys, each X _{e being the} same or different;

   Preferably, X _e is Gln, Ser or Tyr;

   Preferably, X _e is Gln.
5. A compound according to any one of claims 1 to 4 or a compound having at least 80% identity therewith, a stereoisomer thereof or a pharmaceutically acceptable salt thereof, wherein R _{1 is} selected from C _6-24 saturated fatty acids;

   Preferably, R _{1 is} selected from caprylic acid, capric acid, lauric acid, myristic acid and palmitic acid;

   Preferably, R ₁ is palmitic acid.
6. The compound according to any one of claims 1 to 5 or a compound having at least 80% identity therewith, a stereoisomer thereof or a pharmaceutically acceptable salt thereof, wherein L _{1 is} selected from Gly, β-Ala, 6-Aca and NH _2- (CH ₂ CH ₂ -O) _n -CH ₂ CH ₂ -COOH, where n = 1, ₂ , 3, 4, 5, 7, 9, 11 or 23;

   Preferably, L _{1 is} selected from Gly, β-alanine (β-Ala), 6-aminohexanoic acid (6-Aca), and NH _2- (CH ₂ CH ₂ -O) ₇ -CH ₂ CH ₂ -COOH ;

   Preferably, L _{1 is} selected from Gly, β-alanine (β-Ala) and 6-aminohexanoic acid (6-Aca).
7. A compound according to any one of claims 1-6, or a compound having at least 80% identity therewith, a stereoisomer or a pharmaceutically acceptable salt thereof, wherein the compound is selected from:

   IEEIQKK IEEIQKK IEEIQKK IEEIQKK IEEIQKK-aK (C ₈ ),

   IEEIQKK IEEIQKK IEEIQKK IEEIQKK IEEIQKK-aK (C ₁₀ ),

   IEEIQKK IEEIQKK IEEIQKK IEEIQKK IEEIQKK-aK (C ₁₂ ),

   IEEIQKK IEEIQKK IEEIQKK IEEIQKK IEEIQKK-aK (C ₁₄ ),

   IEEIQKK IEEIQKK IEEIQKK IEEIQKK IEEIQKK-aK (C ₁₆ ),

   IEEIQKK IEEIQKK IEEIQKK IEEIQKK IEEIQKK-GK (C ₁₆ ),

   IEEIQKK IEEIQKK IEEIQKK IEEIQKK IEEIQKK-zK (C ₁₆ ),

   IEEIQKK IEEIQKK IEEIQKK IEEIQKK IEEIQKK-pK (C ₁₆ ),

   IEEISKK IEEISKK IEEISKK IEEISKK IEEISKK-aK (C ₁₆ ),

   IEEISKK IEEISKK IEEISKK IEEISKK IEEISKK-GK (C ₁₆ ),

   IEEISKK IEEISKK IEEISKK IEEISKK IEEISKK-zK (C ₁₆ ),

   IEEISKK IEEISKK IEEISKK IEEISKK IEEISKK-pK (C ₁₆ ),

   IEEIYKK IEEIYKK IEEIYKK IEEIYKK IEEIYKK-aK (C ₁₆ ),

   IEEIYKK IEEIYKK IEEIYKK IEEIYKK IEEIYKK-GK (C ₁₆ ),

   IEEIYKK IEEIYKK IEEIYKK IEEIYKK IEEIYKK-zK (C ₁₆ ),

   IEEIYKK IEEIYKK IEEIYKK IEEIYKK IEEIYKK-pK (C ₁₆ ),

   IEEIQKK IEEISKK IEEISKK IEEISKK IEEIQKK-aK (C ₁₆ ), and

   IEEIQKK IEEIYKK IEEIYKK IEEIYKK IEEIQKK-aK (C ₁₆ );

   Where a is β-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, and C ₁₀ is capric acid , C ₁₂ is lauric acid, C ₁₄ is myristic acid, and C ₁₆ is palmitic acid.
8. A pharmaceutical composition comprising a compound according to any one of claims 1 to 7 or a compound having at least 80% identity therewith, a stereoisomer or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable Carrier or excipient.
9. A compound according to any one of claims 1 to 7 or a compound having at least 80% identity therewith, a stereoisomer or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 8 in the preparation of an influenza virus to inhibit fusion of a target cell membrane Use in a medicament; preferably, the influenza virus is an influenza A virus; preferably, the influenza virus is selected from the group consisting of H1N1 and H3N2.
10. A compound according to any one of claims 1 to 7 or a compound having at least 80% identity therewith, a stereoisomer or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 8 in the preparation for the prevention or treatment of influenza virus infection Use of a drug for a related disease or an anti-influenza virus medicine; preferably, the influenza virus is an influenza A virus; preferably, the influenza virus is selected from the group consisting of H1N1 and H3N2; preferably, the influenza virus is associated with an influenza virus infection The disease is selected from influenza A H1N1 and influenza A H3N2.
11. Use of a compound according to any one of claims 1 to 7 or a compound having at least 80% identity therewith, a stereoisomer or a pharmaceutically acceptable salt thereof for inhibiting the fusion of an influenza virus with a target cell membrane in vitro; preferably, all The influenza virus is an influenza A virus; preferably, the influenza virus is selected from H1N1 and H3N2; preferably, the target cell is a cell line or a cell from a subject.
12. A compound according to any one of claims 1 to 7 or a compound having at least 80% identity therewith, a stereoisomer or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 8 for inhibiting influenza virus and a target Cell membrane fusion; preferably, the influenza virus is influenza A virus; preferably, the influenza virus is selected from H1N1 and H3N2; preferably, the target cell is a cell line or a cell from a subject; preferably, The compound, or a compound having at least 80% identity therewith, a stereoisomer thereof, or a pharmaceutically acceptable salt or pharmaceutical composition is used in an in vivo method.
13. A compound according to any one of claims 1 to 7 or a compound having at least 80% identity therewith, a stereoisomer or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 8 for the prevention or treatment of influenza Diseases related to viral infection or anti-influenza virus; preferably, the influenza virus is influenza A virus; preferably, the influenza virus is selected from H1N1 and H3N2; preferably, the disease related to influenza virus infection is selected from H1N1 and H3N2 influenza.
14. A method for inhibiting fusion of an influenza virus with a target cell membrane, comprising administering to a cell an effective amount of a compound according to any one of claims 1 to 7 or a compound having at least 80% identity therewith, a stereoisomer or a pharmaceutically acceptable The accepted salt or the pharmaceutical composition of claim 8; preferably, the influenza virus is influenza A virus; preferably, the influenza virus is selected from H1N1 and H3N2; preferably, the target cell is a cell line or derived from Cells of the subject; preferably, the method is performed in vivo; preferably, the method is performed in vitro.
15. An antiviral method comprising administering to a subject in need thereof an effective amount of a compound according to any one of claims 1-7 or a compound having at least 80% identity therewith, a stereoisomer or a pharmaceutically acceptable An acceptable salt or the pharmaceutical composition of claim 8; preferably, the influenza virus is influenza A virus; preferably, the influenza virus is selected from the group consisting of H1N1 and H3N2.
16. A method for 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 according to any one of claims 1 to 7 or a compound having at least 80% identity therewith, A stereoisomer or a pharmaceutically acceptable salt thereof or the pharmaceutical composition of claim 8; preferably, the influenza virus is influenza A virus; preferably, the influenza virus is selected from the group consisting of H1N1 and H3N2; preferably, The diseases associated with influenza virus infection are selected from the group consisting of H1N1 influenza and H3N2 influenza.

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