Methods for optimizing viral membrane fusion inhibitors and broad-spectrum anti-coronavirus lipopeptides and their applications

Abstract

The present disclosure relates to a method for optimizing a viral membrane fusion inhibitor, and a broad-spectrum anti-coronavirus lipopeptide and its application. The present disclosure provides a compound or a pharma- ceutically acceptable salt or derivative thereof, the compound being represented by formula (I) or formula (II), X1 being an amino-terminal protecting group, X2 being a polypeptide, the amino acid sequence being (EAAAK)n or A[(EAAAK)n]A, X3 being lysine or cysteine ​​or 2,3-diaminopropionic acid or ornithine or 2,4-diaminobutanoic acid or 2,7-diaminoheptanoic acid, X4 being a lipophilic compound functional group modified to X3, or a lipophilic compound functional group modified to K of X2, and X5 being a carboxy-terminal protecting group. The compound of the present disclosure is stable in nature and is used in the preparation of a pharmaceutical composition for preventing and treating diseases caused by coronavirus, and is a highly effective and broad-spectrum novel coronavirus membrane fusion inhibitor, the pharmaceutical composition being used for the prevention and treatment of diseases caused by coronavirus. JPEG2024531265000007.jpg45141

Description

[Technical Field]

[0001] The present disclosure relates to methods for optimizing viral membrane fusion inhibitors and broad-spectrum anti-coronavirus lipopeptides and their applications. [Background technology]

[0002] Membrane fusion is a crucial biological phenomenon, culminating in physiological processes such as zygote formation and intracellular vesicle trafficking. Many viruses that pose serious threats to human health, such as HIV, influenza, hepatitis, Ebola, Zika, SARS, Middle East Respiratory Syndrome (MERS), and the novel coronavirus (SARS-CoV-2), currently wreaking havoc on human populations, also infect host cells via membrane fusion. Viral membrane fusion is mediated by fusion proteins present on the surface of virus particles, such as the gp41 subunit of the HIV envelope protein and the S2 subunit of the coronavirus spike S protein. Fusion proteins typically contain key functional domains, such as the fusion peptide (FP), heptapeptide repeat domain 1 (HR1), heptapeptide repeat domain 2 (HR2), and transmembrane region (TM). During viral membrane fusion, the fusion protein undergoes a dramatic conformational change, first exposing the FP and inserting into the target cell membrane. Then, HR1 forms a trimer helix, and HR2 reverse-folds into the groove formed by the HR1 trimer to form a typical 6-helix bundle (6-HB) structure, drawing the viral and cellular membranes together and initiating fusion, allowing viral genetic material to pass through the fusion pore and enter the target cell. Studies have shown that polypeptides derived from many viral HR1 and HR2 regions act as viral membrane fusion inhibitors by competitively binding to the pre-fusion fusion protein and inhibiting the formation of the 6-HB structure. Currently, the HIV treatment T20 (enfuvir peptide) is the only viral membrane fusion inhibitor approved for clinical use by the FDA in the United States, and the development of antiviral drugs targeting this target is gaining increasing attention. In recent years, lipopeptides, which utilize modifications of lipid compounds (e.g., fatty acids and cholesterol) to improve the half-life and antiviral activity of polypeptides, have attracted attention in the development of viral membrane fusion inhibitors (Non-Patent Document 1).

[0003] Coronaviruses (CoVs) are enveloped, single-stranded, positive-sense RNA viruses classified into four genera: α, β, γ, and δ. Currently known human CoVs include HCoV-229E and HCoV-NL63 of the α genus and HCoV-OC43, CoV-HKU1, SARS-CoV, MERS-CoV, and SARS-CoV-2 of the β genus. HCoV-229E, HCoV-NL63, HCoV-OC43, and CoV-HKU1 are common pathogens that typically cause only common cold symptoms and account for approximately 10%–30% of upper respiratory tract infections in adults, but can still cause severe or fatal illness in children, the elderly, and immunocompromised patients. On the other hand, SARS-CoV, MERS-CoV, and SARS-CoV-2 are highly pathogenic pathogens that cause severe lung disease with a high mortality rate. SARS-CoV-2 shares 79.5% and 96% sequence identity with SARS-CoV and the bat coronavirus SL-CoV-RaTG13, respectively, and uses the same cellular receptor (ACE2). SARS-CoV-2 has a higher transmission capacity than SARS-CoV. By the end of January 2022, approximately 360 million confirmed cases of COVID-19 had been reported worldwide, of which over 5.6 million patients had died (www.who.int). As SARS-CoV-2 continues to spread, variants of particular concern (VOCs), including alpha, beta, gamma, delta, and omicron, continue to emerge. As a result, vaccines and drugs often become less effective or even ineffective. Therefore, the development of efficient, broad-spectrum coronavirus inhibitors is needed.

[0004] Thus, there is a need in the art for the development of efficient and broad-spectrum coronavirus inhibitors that can inhibit many different types of coronaviruses, including those with different mutations. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Xue J, Chong H, Zhu Y, Zhang J, Tong L, Lu J, Chen T, Cong Z, Wei Q, He Y. 2022. Efficient treatment and pre-exposure prophylaxis in rhesus macaques by an HIV fusion-inhibitory lipopeptide. Cell 185: 131-144 e18. [Non-Patent Document 2] Zhu Y, Yu D, Hu Y, Wu T, Chong H, He Y. 2021. SARS-CoV-2-derived fusion inhibitor lipopeptides exhibit highly potent and broad-spectrum activity against divergent human coronaviruses. Signal Transduct Target Ther 6: 294. [Non-Patent Document 3] Yu D, Zhu Y, Yan H, Wu T, Chong H, He Y. 2021. Pan-coronavirus fusion inhibitors possess potent inhibitory activity against HIV-1, HIV-2, and simian immunodeficiency virus. Emerg Microbes Infect 10: 810-821. [Non-Patent Document 4] [ PubMed ] Yu D, Zhu Y, Jiao T, Wu T, Xiao X, Qin B, Chong H, Lei X, Ren L, Cui S, Wang J, He Y.2021. Structure-based design and characterization of novel fusion-inhibitory lipopeptides against SARS-CoV-2 and emerging variants. Emerg Microbes Infect 10:1227–1240.

Direct Environment 5

Outdoor Configuration6

Direct Environment 7

[0006] The scientific research team disclosed herein has been engaged in the research and development of viral membrane fusion inhibitors and has succeeded in designing broad-spectrum lipopeptide-based coronavirus membrane fusion inhibitors that have relatively strong inhibitory activity against SARS-CoV-2 and its mutant strains (Non-Patent Documents 2-5).

[0007] The design of lipopeptide-based viral membrane fusion inhibitors typically requires the incorporation of a linker, which acts as a connecting arm, between the polypeptide sequence and the lipid moiety (e.g., fatty acid or cholesterol). While the binding site of the lipid moiety is expected to be the viral or cellular membrane, the binding site of the lipid moiety and the polypeptide is different because the polypeptide accumulates in the target region. Polypeptides tend to have strong structural rigidity and form stable secondary structures. Therefore, a flexible linker is often used to connect the polypeptide and lipid moiety, allowing the polypeptide and lipid moieties to form appropriate conformations to bind to their respective binding sites, while simultaneously fully fulfilling their respective roles and avoiding mutual interference due to steric hindrance. Common flexible linkers include combinations of glycine (G) and serine (S), such as (GGGGS)n and (GSGSG)n. The distance between the structural domains can be expanded or contracted by changing the size of n. Another common flexible linker is the low-molecular-weight polyethylene glycol (PEG)n, where n is often in the range of 2 to 24. Lipopeptides used as coronavirus membrane fusion inhibitors reported in the literature also employ flexible linkers. For example, IPB02V1 to IPB02V5 are all PEG8 (Non-Patent Document 2), IPB24 to IPB27 are PEG4, PEG5, and PEG8, respectively (Non-Patent Document 4), EKL1C is GSG (Non-Patent Document 6), EK1C4 is a tandem linkage of GSGSG and PEG4 (Non-Patent Document 7), and [SARSHRC-PEG4]2-chol is PEG4 (Non-Patent Document 8). A common rigid linker used in preparing fusion proteins is the (EAAAK)n sequence, which can form an α-helix. This sequence has internal hydrogen bonds and a tightly linked peptide backbone, making it rigid and stable.Another type of rigid linker has a Pro-rich sequence (XP)n, where X can be any amino acid, preferably alanine, lysine, or glutamic acid. The (XP)n sequence does not have a helical structure, but the proline in the sequence increases the rigidity of the backbone and is effective in separating structural domains.

[0008] There is no precedent for the use of a rigid linker in currently prepared lipopeptide-based viral membrane fusion inhibitors. In this disclosure, we have inventively used the EAAAK sequence as a rigid linker to prepare broad-spectrum lipopeptide coronavirus membrane fusion inhibitors, imparting a pronounced helical structure to the polypeptide and significantly improving the antiviral activity and stability of the inhibitor.

[0009] The present disclosure uniquely provides methods for optimizing viral membrane fusion inhibitors and broad-spectrum anti-coronavirus lipopeptides and their responses.

[0010] In the present disclosure, it has been discovered for the first time that compounds IPB29 and IPB30, particularly IPB29, having the rigid linker EAAAK and formula I have at least 70-fold increased inhibitory activity against COVID-19 compared to compounds without such a linker, and approximately 8-fold increased inhibitory activity compared to compounds with flexible linkers such as PEG or GSGSG.

[0011] The inventors also found that the increased activity of lipopeptide IPB29 is due to the addition of the rigid linker EAAAK, which significantly increases the helical content of the lipopeptide and simultaneously increases its stability. Furthermore, this lipopeptide IPB29 can inhibit many different types of coronaviruses, including but not limited to SARS-CoV-2 and its various mutant strains, SARS-CoV, MERS-CoV, HCoV-229E, HCoV-OC43, and HCoV-NL63.

[0012] Specifically, in this disclosure, novel compounds characterized by significantly improved α-helical structure, stability, and antiviral activity were prepared by using a rigid linker EAAAK sequence with an α-helical structure as the linking arm between the polypeptide sequence and the lipid compound. [Means for solving the problem]

[0013] In order to achieve the above object, the present disclosure provides a compound represented by the following formula (I) or (II), or a pharmaceutically acceptable salt thereof, or a derivative thereof: [ka]

[0014] In formula (I) and formula (II), X1 is an amino-terminal protecting group, In formula (I) and formula (II), X2 is a polypeptide, and the amino acid sequence is (EAAAK)n or A[(EAAAK)n]A, where n is a natural number of 5 or less and represents the number of repeats of the EAAAK sequence; In formula (I), X3 is lysine or cysteine ​​or 2,3-diaminopropionic acid (Dap) or ornithine (Orn) or 2,4-diaminobutanoic acid (Dab) or 2,7-diaminoheptanoic acid (Dah); In formula (I), X4 is a lipophilic compound functional group modified to X3, In formula (II), X4 is a lipophilic compound functional group modified by K of X2; In formula (I) and formula (II), X5 is a carboxy-terminal protecting group.

[0015] Illustratively, X1 is an acetyl group (Ac), an amino group (NH2), a maleoyl group, a succinyl group, a tert-butoxycarbonyl group, or a benzyloxy group, or any other hydrophobic group or polymeric carrier group.

[0016] Illustratively, X5 is an amino group (NH2), a carboxyl group, a hydroxyl group, an amide group, or a tert-butoxycarbonyl group, or any other hydrophobic group or polymeric carrier group.

[0017] Exemplary lipophilic compounds include cholesterol succinate monoester, 2-cholesteryl acetic acid, 2-cholesteryl propionic acid, 3-cholesteryl propionic acid, 2-cholesteryl butyric acid, 2-cholesteryl isobutyric acid, 3-cholesteryl butyric acid, 3-cholesteryl isobutyric acid, 4-cholesteryl butyric acid, 2-cholesteryl valeric acid, 2-cholesteryl isovaleric acid, 3-cholesteryl valeric acid, 5-cholesteryl valeric acid, 2-cholesteryl hexanoic acid, 6-cholesteryl hexanoic acid, 2-cholesteryl heptanoic acid, 7-cholesteryl heptanoic acid, 2-cholesteryl octanoic acid, 8-cholesteryl octanoic acid, cholesteryl bromoacetate, fatty acids having 8 to 20 carbon atoms (e.g., octadecanoic acid), dihydro(neuro)sphingosine, vitamin E, and other lipid compounds.

[0018] Illustratively, the lipophilic compound is stearyl chloride.

[0019] Exemplary of the compound is lipopeptide IPB29, which is a compound represented by formula (I), in which X1 is Ac, X2 is EAAAK, X3 is lysine, the lipophilic compound is cholesterol succinate monoester, and X5 is NH2.

[0020] Exemplary of the compound is lipopeptide IPB30, which is a compound represented by formula (I), in which X1 is Ac, X2 is EAAAK, X3 is lysine, the lipophilic compound is stearyl chloride, and X5 is NH2.

[0021] Among them, EAAAK refers to an embodiment in which n in (EAAAK)n is equal to 1.

[0022] The present disclosure further provides multimers of the following (a1) or (a2) or (a3), among which: (a1) A multimer formed of any of the compounds described above, (a2) A multimer formed from any of the pharmaceutically acceptable salts described above, (a3) A multimer formed from any of the above-described derivatives.

[0023] The present disclosure further provides the following (b1), (b2), (b3), or (b4) applications of any of the compounds described above, or pharmaceutically acceptable salts thereof, or derivatives thereof, wherein: (b1) Application in the preparation of coronavirus membrane fusion inhibitors, (b2) Application in the preparation of a drug for preventing and / or treating a disease caused by a coronavirus, (b3) Application as a coronavirus membrane fusion inhibitor, (b4) Applications for preventing and / or treating diseases caused by coronaviruses.

[0024] The present disclosure further provides a product comprising any of the compounds described above, or a pharmaceutically acceptable salt thereof, or a derivative thereof, wherein the product performs the following function (c1) or (c2): (c1) Function as a coronavirus membrane fusion inhibitor, (c2) The function of preventing and / or treating diseases caused by coronaviruses.

[0025] The present disclosure further covers the use of a linker polypeptide in the preparation of a product that enhances the antiviral activity and / or stability of a viral membrane fusion inhibitor, wherein the amino acid sequence of the linker polypeptide is (EAAAK)n or A[(EAAAK)n]A, where n is a natural number equal to or less than 5.

[0026] The present disclosure further provides a method for enhancing the antiviral activity and / or stability of a viral membrane fusion inhibitor, the method comprising linking a linker polypeptide to the viral membrane fusion inhibitor, wherein the amino acid sequence of the linker polypeptide is (EAAAK)n or A[(EAAAK)n]A, where n is a natural number of 5 or less.

[0027] The present disclosure further provides a method for preparing a modified viral membrane fusion inhibitor, comprising the following steps (d1) or (d2): (d1) A lipopeptide is obtained by linking an unmodified viral membrane fusion inhibitor to an X3 (X4) group using a linker polypeptide having an amino acid sequence of (EAAAK)n or A[(EAAAK)n]A as a linking arm, wherein X4 in the X3 (X4) group is modified to X3, X3 is lysine or cysteine ​​or 2,3-diaminopropionic acid (Dap) or ornithine (Orn) or 2,4-diaminobutyric acid (Dab) or 2,7-diaminoheptanoic acid (Dah), X4 is a lipophilic compound functional group, and n is a natural number of 5 or less; (d2) Using a linker polypeptide having an amino acid sequence of (EAAAK)n or A[(EAAAK)n]A as a linking arm, the unmodified viral membrane fusion inhibitor is linked to X4 to obtain a lipopeptide, wherein X4 is a lipophilic compound functional group and is modified to K of the linker polypeptide, and n is a natural number of 5 or less; The lipopeptides are so-called modified viral membrane fusion inhibitors.

[0028] The present disclosure further provides a modified viral membrane fusion inhibitor of the following (e1) or (e2), wherein: (e1) A lipopeptide is obtained by linking an unmodified viral membrane fusion inhibitor to an X3 (X4) group using a linker polypeptide having an amino acid sequence of (EAAAK)n or A[(EAAAK)n]A as a linking arm, wherein X4 in the X3 (X4) group is modified to X3, X3 is lysine or cysteine ​​or 2,3-diaminopropionic acid (Dap) or ornithine (Orn) or 2,4-diaminobutyric acid (Dab) or 2,7-diaminoheptanoic acid (Dah), X4 is a lipophilic compound functional group, and n is a natural number of 5 or less; (e2) A linker polypeptide having an amino acid sequence of (EAAAK)n or A[(EAAAK)n]A is used as a linking arm to link the unmodified viral membrane fusion inhibitor to X4 to obtain a lipopeptide, wherein X4 is a lipophilic compound functional group and is modified to K of the linker polypeptide, and n is a natural number of 5 or less; The lipopeptides are so-called modified viral membrane fusion inhibitors.

[0029] The present disclosure further covers pharmaceutical compounds that are any of the compounds described above or pharmaceutically acceptable salts or derivatives thereof, or multimers described above.

[0030] The pharmaceutical compound is used for the following purposes (f1), (f2), (f3), (f4), (f5), or (f6), among which: (f1) Use as a coronavirus membrane fusion inhibitor, (f2) Use for preventing and / or treating diseases caused by coronaviruses, (f3) Use for anti-coronavirus purposes, (f4) Use for inhibiting coronavirus cell fusion, (f5) Use for inhibiting coronavirus from entering cells, (f6) Use for inhibiting the replication of coronaviruses.

[0031] The present disclosure further provides a method for treating and / or preventing coronavirus infection in an animal, said method comprising administering said pharmaceutical compound to the subject animal, thereby inhibiting coronavirus from infecting the animal.

[0032] X2 in any of the above is a rigid linker.

[0033] The linker polypeptide according to any of the above is a rigid linker polypeptide.

[0034] X2 in any of the above is a rigid linker having an α-helical structure.

[0035] The linker polypeptide according to any of the above is a rigid linker polypeptide having an α-helical structure.

[0036] In any of the above, n may be 1, 2, 3, 4 or 5.

[0037] X2 described in any of the above can function as a linking arm and significantly increase the α-helical structure, thereby improving the stability and antiviral activity of the compound, or a pharmaceutically acceptable salt thereof, or a derivative thereof.

[0038] Any of the above-described linker polypeptides functions as a linking arm and significantly increases the α-helical structure, thereby improving the stability and antiviral activity of the viral membrane fusion inhibitor.

[0039] When X3 is lysine, the lipid compound is linked via the amino group of its side chain.

[0040] When X3 is cysteine, the lipid compound is linked via the sulfhydryl group of its side chain.

[0041] When X3 is lysine, the preferred lipophilic compound is cholesterol succinic acid monoester, which is linked to the side chain of lysine by an amidation reaction.

[0042] When X4 is cysteine, the preferred lipophilic compound is cholesteryl bromoacetate.

[0043] In formula (I) and formula (II), the abbreviations for amino acids have the meanings well known in the art, such as S for serine, V for valine, N for asparagine, I for isoleucine, Q for glutamine, K for lysine, E for glutamic acid, D for aspartic acid, R for arginine, L for leucine, A for alanine, G for glycine, Y for tyrosine, and C for cysteine.

[0044] The amino acid may be an L-amino acid.

[0045] To improve the bioavailability, stability, and / or antiviral activity of a polypeptide, one or more (e.g., 2 to 5, 2 to 4, or 2 to 3) amino acids in the polypeptide may be replaced with amino acids having a D-form conformation, artificially modified amino acids, rare amino acids occurring in nature, etc.

[0046] D-amino acids refer to amino acids that correspond to the L-amino acids that make up proteins.

[0047] Artificially modified amino acids refer to common L-amino acids that make up proteins and have been modified by methylation, phosphorylation, etc.

[0048] Rare amino acids found in nature include uncommon proteinogenic and non-proteinogenic amino acids, such as 5-hydroxylysine, methylhistidine, γ-aminobutyric acid, and homoserine.

[0049] The present disclosure provides novel methods for designing lipopeptides as viral membrane fusion inhibitors, and lipopeptide inhibitors prepared using the methods or strategies have significantly increased helical structural characteristics and significantly increased antiviral activity. Pharmaceutically acceptable salts, solvates, chelates, or non-covalent complexes formed by the compounds, drug precursors based on the compounds, or mixtures of any of the above forms are also part of the present disclosure.

[0050] The present disclosure provides methods for preventing and treating diseases caused by coronaviruses, comprising compounds of the present disclosure.

[0051] The present disclosure further provides a pharmaceutical composition for preventing and treating diseases caused by coronavirus, comprising a compound according to the present disclosure. Preferably, the pharmaceutical composition is used for preventing and treating diseases caused by coronavirus.

[0052] The coronaviruses referred to in the present disclosure include various coronaviruses embodied in the Examples, but are not particularly limited thereto in the present disclosure, such as SARS-CoV-2 (wild SARS-CoV-2 strains and their various mutant strains; the entire genome sequence of the wild SARS-CoV-2 can be found in GenBank: MN908947.3, and the mutant strains can be alpha, beta, gamma, delta, and omicron strains), SARS-CoV, MERS-CoV, and other coronaviruses (e.g., HCoV-229E, HCoV-OC43, and HCoV-NL63), including bat-derived coronaviruses and pangolin-derived coronaviruses.

[0053] The compounds include different optical isomers, racemates and / or mixtures thereof. In the above cases, single enantiomers or diastereoisomers, such as optically active isomers, can be obtained by asymmetric synthesis or racemic resolution. Resolution of racemates can be achieved by various methods, such as conventional recrystallization using a resolving agent or chromatographic methods.

[0054] The compounds include different cis and / or trans isomers of double bonds.

[0055] A derivative of a compound may be a solvate of the compound, a complex of the compound, a chelate of the compound, or a non-covalent conjugate of the compound. A derivative of a compound may also be a drug precursor based on the compound (e.g., an ester or amide derivative of the compound).

[0056] In preparing the medicament, mixtures of any form of the above compounds may be used.

[0057] The prepared drug may contain a mixture of any of the above compounds in any form.

[0058] The modified viral membrane fusion inhibitors prepared using the methods of the present disclosure have significantly increased helical structural characteristics, resulting in significantly increased antiviral activity and / or stability.

[0059] Further aspects of the present disclosure are described in detail below or are partially embodied in the examples of the present disclosure. Hereinafter, unless otherwise specified, the amounts of different components and reaction conditions used herein may be interpreted as "approximately" or "about" in all cases. Therefore, unless otherwise specified, the numerical parameters recited below and in the claims are approximate parameters, and different numerical parameters may be obtained under different experimental conditions due to differences in standard error.

[0060] In the present specification, if there is any discrepancy or doubt between the chemical structure and the chemical name of a compound, the compound will be precisely defined according to the chemical structure. The compounds described herein may contain one or more chiral centers and / or structures such as double bonds, and stereoisomers, including double bond isomers (e.g., geometric isomers), optical enantiomers, or diastereomers, may also exist. Therefore, any chemical structure within the scope of the present specification, whether or not it contains such a structure in part or in its entirety, includes all possible enantiomers and diastereomers of the compound, as well as any single stereoisomer (e.g., simple geometric isomer, simple enantiomer, or simple diastereomer) and any mixture of these isomers. These racemates and stereoisomeric mixtures can also be further resolved into their component enantiomers or stereoisomers using chiral separation techniques or chiral molecule synthesis methods, as understood by those skilled in the art.

[0061] In practical application, the drug of the present disclosure can be administered directly to a patient for the purpose of treating and / or preventing coronavirus infection, or can be administered in combination with an appropriate carrier or excipient. Carrier materials include water-soluble carrier materials (e.g., polyethylene glycol, polyvinylpyrrolidone, organic acids, etc.), poorly soluble carrier materials (e.g., ethyl cellulose, cholesteryl stearate, etc.), and enteric carrier materials (e.g., cellulose phthalate acetate, carboxymethyl ethyl cellulose, etc.), but these are not particularly limited in the present disclosure. Water-soluble carrier materials are preferred. These materials can be used to prepare various dosage forms, including, but not limited to, tablets, capsules, lozenges, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal formulations, oral tablets, suppositories, and lyophilized powders. These may also be conventional formulations, sustained-release formulations, controlled-release formulations, or various microparticle delivery systems. Various carriers well known in the art can be used to prepare tablets into unit dosage forms. Examples of the carrier include diluents and absorbents such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, clay, microcrystalline cellulose, and aluminum silicate; wetting agents and adhesives such as water, glycerin, polyethylene glycol, ethanol, propanol, starch slurry, dextrin, syrup, honey, glucose solution, acacia mucilage, gelatin glue, sodium carboxymethylcellulose, shellac, methylcellulose, potassium phosphate, and polyvinylpyrrolidone; and drying agents such as cellulose acetate, cellulose acetate, cellulose acetate syrup ... Disintegrants such as dried starch, alginate, agar powder, brown seaweed starch, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene, sorbitol fatty acid esters, sodium dodecyl sulfonate, methylcellulose, and ethylcellulose; disintegration inhibitors such as sucrose, glyceride tristearate, cocoa butter, and hydrogenated oils; absorption promoters such as quaternary ammonium salts and sodium dodecyl sulfate; and lubricants such as talc, silica, corn starch, stearates, boric acid, liquid paraffin, and polyethylene glycol.Tablets can also be prepared as coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or double-layer and multi-layer tablets. Various carriers known in the art can be used to prepare unit-dosage pills. Examples of suitable carriers include diluents and absorbents such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oils, polyvinylpyrrolidone, Gelucire, kaolin, and talc; gum arabic, tragacanth, gelatin, ethanol, honey, liquid sugar, adhesives such as rice paste and flour paste; and disintegrants such as agar powder, dry starch, alginates, sodium dodecylsulfonate, methylcellulose, and ethylcellulose. Various carriers known in the art can be used to prepare unit-dosage suppositories. Examples of suitable carriers include polyethylene glycol, lecithin, cocoa butter, higher alcohols, esters of higher alcohols, gelatin, and semi-synthetic glycerides. To prepare the unit dosage form into an injectable preparation such as a solution, emulsion, lyophilized powder, or suspension, any diluent commonly used in the art, such as water, ethanol, polyethylene glycol, 1,3-propanediol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, or polyoxyethylene sorbitol fatty acid ester, can be used. To prepare an isotonic injection, an appropriate amount of sodium chloride, glucose, or glycerin can be added to the injection, and conventional cosolvents, buffers, pH adjusters, and the like can also be added. Depending on actual needs, colorants, preservatives, flavorings, flavoring agents, sweeteners, or other materials can also be added to the pharmaceutical preparation. The above dosage forms can be used for injections, including subcutaneous, intravenous, intramuscular, and intraperitoneal injections; intraluminal administration, such as rectal and vaginal; respiratory administration, such as nasal; and mucosal administration. Among the above administration routes, injection, atomized inhalation, nasal spray, or nasal instillation are preferred.

[0062] The dosage of the drug of the present disclosure depends on many factors, such as the nature and severity of the disease to be prevented or treated, the sex, age, weight and individual response of the patient or animal, the specific active ingredient used, the route and frequency of administration, etc. The above-mentioned dose may be administered in a single dosage form or in several divided doses, for example, two, three or four divided doses.

[0063] The drugs disclosed herein can be used directly alone for the treatment and prevention of coronavirus infections, or in combination with one or more other antiviral drugs to improve overall therapeutic efficacy. These antiviral agents include, but are not limited to, neutralizing antibodies, protease inhibitors, RNA-dependent RNA polymerase (RdRp) inhibitors, and viral entry inhibitors. The neutralizing antibodies may be one or more selected from amvalvimab (BRII-196), romursevimab (BRII-198), casirivimab, idevimab, sotrovimab, bamlanivimab, etc., and the protease inhibitors may be paxlovir, darunavir, lopinavir / ritonavir, etc. ), the RdRp inhibitor may be one or more selected from Molnupiravir, Favipiravir, Remdesivir, Sofosbuvir, and the like, and the viral entry inhibitor may be one or more selected from Arbidol, hydroxychloroquine, and the like.

[0064] For a particular patient, the specific therapeutically effective dose level will be determined based on a variety of factors, including the disorder being treated and the severity of the disorder, the activity of the specific active ingredient used, the specific composition used, the patient's age, weight, general health, sex, and diet, the timing of administration, route of administration and excretion rate of the specific active ingredient used, duration of treatment, drugs used in combination or simultaneously with the specific active ingredient used, and similar factors well known in the medical field. It is common practice in the art to start the dose of the active ingredient, for example, at a dose of the active ingredient at or below the level required to achieve the desired therapeutic effect, and gradually increase the dose until the desired effect is achieved. [Effects of the Invention]

[0065] The inventors of the present disclosure have discovered for the first time that compounds IPB29 and IPB30 of formula (I) having a rigid linker EAAAK, particularly IPB29, have at least 70-fold improved inhibitory activity against the novel coronavirus compared to compounds without the linker, and approximately 8-fold improved inhibitory activity compared to compounds with a flexible linker (e.g., PEG or GSGSG).

[0066] At the same time, the inventors of the present disclosure found that the increased activity of lipopeptide IPB29 is due to the rigid linker EAAAK, which significantly increases the helical content of the lipopeptide and simultaneously improves its stability. Furthermore, such lipopeptide IPB29 can inhibit many different types of novel coronaviruses.

[0067] The compounds disclosed herein are stable in nature and are highly effective and broad-spectrum novel coronavirus membrane fusion inhibitors that can be used to prepare pharmaceutical compositions for the prevention and treatment of coronavirus-induced diseases. The pharmaceutical compositions are used for the prevention and treatment of coronavirus-induced diseases. [Brief explanation of the drawings]

[0068] [Figure 1]The α-helical content (left panel) and thermal stability (right panel) of lipopeptides as viral membrane fusion inhibitors are shown. [Figure 2] The α-helical content (left panel) and thermal stability (right panel) of lipopeptide-target sequence polypeptide complexes as viral membrane fusion inhibitors are shown. [Figure 3] The inhibitory activity of lipopeptides against SARS-CoV-2 infection of 293T / ACE2 cells (left panel) or Huh-7 cells (right panel). [Figure 4] Figure 1 shows the inhibitory activity of lipopeptides against infection of 293T / ACE2 cells with various SARS-CoV-2 mutant strains. [Figure 5] Figure 1 shows the inhibitory activity of lipopeptides against infection of Huh-7 cells with various SARS-CoV-2 mutant strains. [Figure 6] 1 shows the inhibitory activity of lipopeptides against other coronaviruses. [Figure 7] FIG. 1 shows in vitro cytotoxicity testing of lipopeptides. [Figure 8] FIG. 1 shows stability analysis of lipopeptides IBP24 and IPB29. [Figure 9] The inhibitory effect of a novel lipopeptide on cell membrane fusion mediated by the S protein of SARS-CoV-2 is shown. The target cells in the left panel are 293T / ACE2 cells, and those in the right panel are Huh-7 cells. DETAILED DESCRIPTION OF THE INVENTION

[0069] The scientific research team disclosed herein has been engaged in the long-term research and development of viral membrane fusion inhibitors, and as a result, has designed a lipopeptide-based broad-spectrum coronavirus membrane fusion inhibitor that has relatively strong inhibitory activity against SARS-CoV-2 and its mutant strains (Non-Patent Documents 2-5).

[0070] The design of lipopeptide-based viral membrane fusion inhibitors typically requires the incorporation of a linker between the polypeptide sequence and the lipid moiety (e.g., fatty acid or cholesterol) as a connecting arm. The expected binding site of the lipid moiety is the viral or cellular membrane, while the polypeptide aggregates at the target region, so the binding site of the lipid moiety and the polypeptide are different. Because polypeptides tend to form stable secondary structures and have strong structural rigidity, a flexible linker is usually used to connect the polypeptide and lipid moiety, allowing the polypeptide and lipid moiety to form appropriate conformations and bind to their respective binding sites. At the same time, they fully fulfill their respective roles while avoiding interactions due to steric hindrance. Common flexible linkers include combinations of glycine (G) and serine (S), such as (GGGGS)n or (GSGSG)n, where the size of n can be varied to increase or decrease the distance between structural domains. Another common flexible linker is the low-molecular-weight polyethylene glycol (PEG)n, where n is often in the range of 2 to 24. Lipopeptides that have been reported in the literature as coronavirus membrane fusion inhibitors also use flexible linkers. For example, IBP02V1 to IBP02V5 all use PEG8 (Non-Patent Document 2), IPB24 to IPB27 use PEG4, PEG5, PEG6, and PEG8, respectively (Non-Patent Document 4), EKL1C uses GSG (Non-Patent Document 6), and EK1C4 uses GSGSG and PEG4 linked in series (Non-Patent Document 7). HRCFor example, PEG4 is used in [-PEG4]2-chol (Non-Patent Document 8). A typical rigid linker used in preparing fusion proteins has an (EAAAK)n sequence that can form an α-helix, and is a rigid and stable sequence due to internal hydrogen bonds and a tightly connected peptide chain backbone. Another type of rigid linker has a Pro-rich sequence (XP)n, where X is any amino acid, preferably alanine, lysine, or glutamic acid. Although the (XP)n sequence does not have a helical structure, the proline increases the rigidity of the backbone, allowing for effective separation of structural domains.

[0071] The present disclosure will be described in more detail below with reference to specific embodiments. However, these examples are intended only to illustrate the present disclosure and do not limit the scope of the present disclosure. The examples provided below can serve as guidelines for further improvements by those skilled in the art and do not limit the present disclosure in any manner. Those skilled in the art can achieve the technical solutions of the present disclosure by appropriately modifying relevant parameters after reading the contents of this specification. It should be noted that all similar substitutions and modifications are obvious to those skilled in the art, and all such substitutions and modifications are included within the scope of the present disclosure. In addition, the methods of the present disclosure have been described using preferred examples. Those skilled in the art can implement and apply the technical solutions of the present disclosure by modifying the compounds and preparation methods described herein or by making appropriate modifications and combinations without departing from the content, spirit, and scope of the present disclosure.

[0072] Unless otherwise specified, the experimental methods in the following examples are all conventional methods and are carried out in accordance with the techniques or conditions described in the literature or in accordance with the product specifications. Unless otherwise specified, the materials and reagents used in the following examples are all commercially available.

[0073] Example 1: Preparation of Lipopeptides The lipopeptides IPB29, IPB30, IPB20, IPB24, and IPB28 were prepared. Both IPB29 and IPB30 have the EAAAK (SEQ ID NO: 4) rigid linker. The amino acid sequences of IPB29 and IPB30 are shown in SEQ ID NO: 1. SEQ ID NO: 1: SVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKEAAAKK.

[0074] Among these, the lipopeptides IPB20 (which does not have a linker as a linking arm), IPB24 (which has the flexible linker PEG4 as a linking arm, obtained from Fmoc-NH-PEG4-CH2CH2COOH as a synthetic raw material), and IPB28 (which has the flexible linker GSGSG (SEQ ID NO: 5) as a linking arm) were all control substances.

[0075] Lipopeptide IPB29 and lipopeptide IPB30 both satisfy the following general formula: [ka]

[0076] In lipopeptide IPB29, X1 is an amino-terminal protecting group Ac, X2 is an EAAAK rigid linker, X3 is a lysine residue, X4 is a cholesterol succinate monoester group modified to X3, and X5 is a carboxy-terminal protecting group NH2. In lipopeptide IPB30, X1 is an amino-terminal protecting group Ac, X2 is an EAAAK rigid linker, X3 is a lysine residue, X4 is a stearyl chloride group modified to X3, and X5 is a carboxy-terminal protecting group NH2.

[0077] The sequence structures of the five lipopeptides are shown in Table 1. In the five lipopeptides, Ac represents an acetyl group, NH represents an amino group, and EAAAK represents a short peptide with the amino acid sequence EAAAK. The structural differences among the five lipopeptides are shown in Table 2.

[0078] [Table 1]

[0079] [Table 2]

[0080] 1) Chemical reagents required for preparation Chemical reagents, such as various Fmoc amino acids, N,N'-diisopropylcarbodiimide (DIC), 1-hydroxybenzotriazole (HOBt), N,N-dimethylformamide (DMF), piperidine (PIPE), ninhydrin, acetic anhydride (AcO), N,N'-diisopropylethylamine (DIEA), hydrazine hydrate, cholesterol succinate monoester, stearyl chloride, trifluoroacetic acid (TFA), ethanedithiol (EDT), thioanisole (TA), triisopropylsilane (TIPS), phenol, and N-fluorenylmethyloxycarbonyl-tetrapolyethyleneglycol-carboxylic acid (Fmoc-NH-PEG-CHCHCOOH), were all purchased from major chemical suppliers without further purification before use.

[0081] Examples of amino acids with protecting groups (hereinafter also referred to as "protected amino acids") used in polypeptide synthesis include Fmoc-Lys(Dde)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Gly-OH, Fmoc-Ala-OH, Fmoc-Ser(tBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Ile-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Val-OH, and Fmoc-Arg(Pbf)-OH. The definitions of the abbreviations are as well known: Fmoc is 9-fluorenylmethoxycarbonyl, Dde is 1-(4,4-dimethyl-2,6-dioxocyclohexenyl)ethyl, Boc is tert-butoxycarbonyl, tBu is tert-butyl, OtBu is tert-butoxy, Trt is trityl, and Pbf is (2,3-dihydro-2,2,4,6,7-pentamethylbenzofuran-5-yl)sulfonyl.

[0082] 2) Synthesis of peptide resin The peptide resin was prepared by sequentially coupling protected amino acids corresponding to the amino acid sequence of the polypeptide to Rink Amide MBHA resin as a carrier resin through Fmoc deprotection and coupling reaction.

[0083] 2-1) Linking the first protected amino acid of the main chain 0.3 mmol of the first protected amino acid, Fmoc-Lys(Dde)-OH, and 0.3 mmol of HOBt were dissolved in an appropriate amount of DMF, and then 0.3 mmol of DIC was slowly added to the DMF solution of the protected amino acid while shaking. The reaction was allowed to proceed with shaking at room temperature for 5 minutes to obtain an activated protected amino acid solution, which was then ready for later use.

[0084] 0.1 mmol of Rink Amide MBHA resin (0.35 mmol / g*0.3 g) was taken and deprotected with 25% PIPE / DMF solution (volume ratio) for 20 minutes (twice), washed, and filtered to obtain the Fmoc-deprotected resin.

[0085] The activated first protected amino acid solution was added to the resin from which Fmoc had been removed, and a coupling reaction was carried out for 60 minutes. After filtration and washing, a resin containing the first protected amino acid Fmoc-Lys(Dde) was obtained.

[0086] 2-2) Linking of other protected amino acids to the main chain The other protected amino acids corresponding to the polypeptide were sequentially linked in the same manner as for linking the first protected amino acid of the main chain, yielding a resin containing the amino acids of the main chain. Finally, the N-terminus was blocked by acetylation using 0.3 mmol of AcO and 6 mmol of DIEA, completing the synthesis of the main chain. After each step of the reaction, the reaction was controlled by the Kaiser test. If the condensation reaction of an individual amino acid was incomplete, the condensation reaction was repeated until the desired peptide was obtained.

[0087] 2-3) Linking of side chains (a) The Dde protecting group on the C-terminal lysine side chain was removed by treating the resin with as little hydrazine hydrate / DMF solution (volume ratio) as possible (10 min, twice), followed by filtration and washing to obtain the Dde-free resin.

[0088] (a) Cholesterol modification of polypeptides was performed as follows. 0.3 mmol of cholesterol succinate monoester and 0.3 mmol of HOBt were dissolved in an appropriate amount of DMF. 0.3 mmol of DIC was then slowly added to the solution containing cholesterol succinate monoester and HOBt, and the mixture was shaken at room temperature for 5 minutes. The resulting solution containing cholesteryl succinate monoester, HOBt, and DIC was added to the Dde-free resin obtained in step (a), and the coupling reaction was carried out for 60 minutes. The resulting peptide resin was then filtered, washed, and dried.

[0089] (C) Stearylation of the polypeptide was carried out as follows: 0.3 mmol of stearyl chloride and 0.6 mmol of DIEA were dissolved in an appropriate amount of DMF, slowly added to the Dde-free resin obtained in step (A), and the reaction was carried out with shaking at room temperature for 60 minutes. The peptide resin was then obtained by filtration, washing, and drying.

[0090] 3) Preparation of crude product The peptide resin was mixed with the cleavage reagent (15 mL / g resin) and mixed uniformly. The mixture was then shaken at 30°C for 3 hours to cleave the target polypeptide from the resin and remove the side-chain protecting groups. The filtrate from the reaction mixture was collected, and the resin was washed three times with a small amount of TFA / DCM. The combined filtrate was precipitated with anhydrous ether and centrifuged. The filter cake was washed twice with cold anhydrous ether and dried under reduced pressure to obtain a gray-white powder, which was the crude lipopeptide.

[0091] The cleavage reagent consisted of trifluoroacetic acid: 1,2-ethanedithiol: thioanisole: phenol: H2O: triisopropylsilane in a volume ratio of 68.5:10:10:5:3.5:1.

[0092] 4) Preparation of pure product The crude lipopeptide was dissolved in water / acetonitrile, stirred, and centrifuged to remove insoluble material for further use. Purification was performed using reverse-phase high-performance liquid chromatography (RPLC). An Agela C18 chromatography column (10 μm, 100 Å, 50 × 250 mm) was used, with mobile phases A (0.05% TFA and 2% acetonitrile in water) and B (90% acetonitrile / water). The flow rate was 25 mL / min, and the UV detection wavelength was 220 nm. The crude solution was loaded onto the column and gradient elution was performed. The corresponding purified components were collected and directly lyophilized to remove the solvent, yielding the purified trifluoroacetate salt of the fluffy polypeptide.

[0093] The purified polypeptide trifluoroacetate salts were redissolved in water and acetonitrile, and a large amount of anion exchange resin (acetate form) was added. The mixture was stirred for 3 hours. After filtration and rinsing the ion exchange resin with a water / acetonitrile mixture, the combined filtrates were lyophilized to obtain the purified polypeptide acetate salts (i.e., the lipopeptides shown in Table 1).

[0094] The chemical structures of the lipopeptides shown in Table 1 were identified by MALDI-TOF mass spectrometry, and their purity was measured using analytical high-performance liquid chromatography (Agela C18-4.6 × 250 mm, flow rate 1 mL / min). The purity of all synthesized lipopeptides exceeded 95%.

[0095] Example 2: Structural characteristics of lipopeptides and analysis of their interaction with target sequences The secondary structure (α-helix) and thermal stability of the test lipopeptide, as well as the interaction between the test lipopeptide and the target sequence mimetic polypeptide, were measured using circular dichroism (CD) technology, with reference to papers published by the present inventor (Non-Patent Documents 4 and 5). The target sequence mimetic polypeptide N52 is derived from the HR1 sequence of the S2 subunit of the SARS-CoV-2 spike protein, and the sequence structure of N52 is as follows: Ac-FNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQ-NH2 (the amino acid sequence contained in N52 is shown in SEQ ID NO: 6)

[0096] The test lipopeptides were lipopeptide IPB29, lipopeptide IPB30, lipopeptide IPB20, lipopeptide IPB24, or lipopeptide IPB28 prepared in Example 1, and the test conjugate was a mixture of the test lipopeptide and N52.

[0097] 1) A test lipopeptide (or test complex) was prepared into a 10 μM solution (in the case of a test complex, 10 μM means that the concentrations of both the test lipopeptide and N52 were 10 μM) using phosphate buffer (PBS, pH 7.2), and the solution was left in a water bath at 37°C for 30 minutes.

[0098] 2) The solution obtained in step 1 was transferred to the corresponding cuvette, and the molar ellipticity [θ]λ of the solution was scanned in the wavelength range of 195-270 nm using a Jasco spectropolarimeter (model number J-815). A typical α-helical structure may have maximum negative peaks at 208 nm and 222 nm. To correct the spectral values, a PBS blank was subtracted, and the peak value of -33,000 degrees cm² dmol² was used as the reference for 100% α-helical content. The percentage of α-helical content was calculated from the molar ellipticity of the solution at a wavelength of 222 nm.

[0099] 3) The solution obtained in step 1 was placed in a thermal stability detection cuvette, and the CD temperature control module was adjusted to scan the temperature-dependent change in the solution [θ]222 from 20 to 98°C at a rate of 2°C / min. The melting graph was smoothed, and the midpoint temperature (Tm) of the thermal dissociation transition was calculated using Origin software, reflecting the degree of helical thermal stability.

[0100] The CD results for the test lipopeptides are shown in Figure 1. The α-helical content of IPB20, without either a flexible or rigid linker, was 51%, while the α-helical contents of IPB24 and IPB28, with flexible linkers, were 19% and 20%, respectively. This indicates that the introduction of a flexible linker increases the rotational freedom of the entire lipopeptide, increasing the overall molecular entropy of the lipopeptide and stabilizing the secondary structure of the polypeptide. The α-helical contents of IPB29 and IPB30, with rigid linkers, were 74% and 62%, respectively. This indicates that the introduction of the EAAAK sequence significantly increases the helical structure of the lipopeptide. This is likely due to the EK salt bridge in the EAAAK linker conjugating with the salt bridge in the α-helical secondary structure of the polypeptide, stabilizing its secondary structure. At the same time, A is more likely to form an α-helical structure than G. Lipopeptides with a relatively high α-helical content can effectively reduce the binding entropy of the polypeptide moiety to the target, increasing the binding constant and improving activity. Furthermore, a relatively stable secondary structure facilitates protease hydrolysis within the polypeptide antibody, improving its stability in vivo and contributing to its antiviral activity.

[0101] The CD results for the test complexes are shown in Figure 2. Each lipopeptide interacted with the target sequence mimetic polypeptide to form complexes with typical α-helical structures. The α-helical contents of the IPB20-N52, IPB24-N52, and IPB28-N52 complexes were 66%, 48%, and 37%, respectively, while the α-helical contents of the IPB29-N52 and IPB30-N52 complexes were 68% and 53%, respectively (Figure 2, left panel). The Tm values ​​of the IPB20-N52, IPB24-N52, and IPB28-N52 complexes were 90°C, 90°C, and 89°C, respectively, while the Tm values ​​of the IPB29-N52 and IPB30-N52 complexes were 86°C and 78°C, respectively (Figure 2, right panel). This indicates that lipopeptides incorporating an EAAAK linker can enhance their ability to bind to cell membranes or viral membranes while maintaining specific and stable binding to targets.

[0102] Example 3: Inhibitory effects of lipopeptides on the novel coronavirus SARS-CoV-2 and its mutants The test cells were 293T / ACE2 cells or Huh-7 cells. 293T cells were a product of the American Type Culture Collection (ATCC, catalog number CRL-3216), Huh-7 cells were a product of the National Laboratory Cell Resources Sharing Service Platform, and 293T / ACE2 cells were described in Non-Patent Document 2.

[0103] 1) Inhibitory effect of lipopeptides on the novel coronavirus SARS-CoV-2 The test lipopeptides were lipopeptide IPB29, lipopeptide IPB30, lipopeptide IPB20, lipopeptide IPB24, or lipopeptide IPB28, as prepared in Example 1.

[0104] 1-1) Preparation of SARS-CoV-2 pseudovirus The plasmid expressing the S protein of SARS-CoV-2 was designated pCoV2-S and is described in the "Single-cycle infection assay" section of the Materials and Methods section of Non-Patent Document 5 under "A plasmid expressing the S protein of SARS-CoV-2." The HIV backbone plasmid, pNL4-3.luc.RE, was provided by the National Institutes of Health AIDS Reagent and Reference Program (Cat. No. 3418).

[0105] 293T cells were co-transfected with pCoV2-S and pNL4-3.luc.RE at a 1:1 ratio and cultured in a 37°C, 5% CO2 cell culture incubator for 48 hours. The supernatant containing the SARS-CoV-2 pseudovirus was then collected, filtered, and stored at -80°C for future use.

[0106] The prepared SARS-CoV-2 pseudovirus is the "SARS-CoV-2 pseudovirus (SARS-CoV-2PV, hereinafter also referred to as "SARS-CoV-2WT")" described in Non-Patent Document 2.

[0107] 1-2) Inhibitory effect of lipopeptides on SARS-CoV-2 (a) Test lipopeptides were dissolved in deionized water, and the concentrations were measured. The test lipopeptides were then diluted to the starting concentration with DMEM medium and diluted three-fold in a 96-well cell culture plate, with each well containing 50 μL of lipopeptide solution. Nine dilutions were prepared, with three parallel wells for each dilution, and a control well containing 50 μL of DMEM medium was also prepared.

[0108] (B) After step (A) is completed, 50 μL of the pseudovirus prepared in step (A) is added per well (virus amount: 500 TCID 50 ) was added and incubated at room temperature for 30 minutes.

[0109] (c) Test cells cultured in advance at a concentration of 10 × 10 4The cell suspension was adjusted to 15 μg / mL, and DEAE-dextran was added to the cell suspension to a concentration of 15 μg / mL. The cell suspension was then added to the 96-well plate (100 μL / well) obtained in step (a) and cultured in a 37°C, 5% CO2 cell incubator for 48 hours.

[0110] (d) After step (c) is completed, the supernatant is discarded, and 30 μL of cell lysis solution is added to each well. After lysis at room temperature for 15 minutes, luciferase substrate (Promega) is added, and the relative fluorescence units (RLU) are measured using a microplate chemiluminescence detector. An inhibition rate graph and the 50% inhibitory concentration (IC50) of the drug are plotted. 50 ) was calculated and the figure was created.

[0111] The results are shown in Figure 3. The IC values ​​of IPB20, IPB24, IPB28, IPB29, and IPB30 inhibiting SARS-CoV-2 infection in 293T / ACE2 cells. 50 The IC values ​​for IPB20, IPB24, IPB28, IBP29 and IPB30 to inhibit SARS-CoV-2 infection in Huh-7 cells were 63.85 nM, 5.51 nM, 6.91 nM, 0.57 nM and 4.19 nM, respectively. 50The values ​​were 39 nM, 2.43 nM, 2.88 nM, 0.53 nM, and 2.77 nM, respectively. IPB29 and IPB20 differ in that IPB29 contains the rigid linker EAAAK, while IPB20 lacks a rigid or flexible linker. IPB29 showed approximately 112-fold improved antiviral activity in 293T / ACE2 cells and approximately 74-fold improved antiviral activity in Huh-7 cells compared to IPB20. IPB29 and IPB24 differ in that IPB29 contains the rigid linker EAAAK, while IPB24 contains the flexible linker PEG4. IPB29 showed approximately 10-fold improved antiviral activity in 293T / ACE2 cells and approximately 5-fold improved antiviral activity in Huh-7 cells compared to IPB24. IPB29 and IPB28 differ in that IPB29 contains the rigid linker EAAAK, while IPB28 contains the flexible linker GSGSG. Compared to IPB28, IPB29 exhibited approximately 12-fold improved antiviral activity in 293T / ACE2 cells and approximately 5-fold improved antiviral activity in Huh-7 cells. Experimental results demonstrated that the EAAAK linker significantly improved the inhibitory activity of lipopeptides as inhibitors, as demonstrated by the inhibitory activity of IPB29 and IPB30 against SARS-CoV-2.

[0112] 2) Inhibitory effect of lipopeptides on mutant forms of the novel coronavirus SARS-CoV-2 The test lipopeptides were lipopeptide IPB29, lipopeptide IPB30, lipopeptide IPB24, or lipopeptide IPB28, as prepared in Example 1.

[0113] 2-1) Creation of pseudoviruses of mutant SARS-CoV-2 Pseudoviruses of mutant SARS-CoV-2 novel coronaviruses were prepared, specifically as shown in Figure 4. See step 1-1) for the method, except that the plasmid expressing the SARS-CoV-2 S protein was replaced with a plasmid expressing the S protein of a SARS-CoV-2 mutant (a single-point mutation or a typical epidemic strain). The pseudovirus of the SARS-CoV-2 D614G mutant prepared is the so-called "D614G PV" described in Non-Patent Document 2.

[0114] 2-2) Inhibitory effect of lipopeptides on SARS-CoV-2 mutants The inhibitory effect was measured in the same manner as in step 1-2), and the measurement results when 293T / ACE2 cells were used as test cells are shown in Figure 4. The IC values ​​of IPB24, IPB28, IPB29, and IPB30, which inhibit the Delta strain in 293T / ACE2 cells, were 50 The IC values ​​were 4.94 nM, 6.30 nM, 0.79 nM and 3.57 nM for IPB24, IPB28 and IPB30, respectively. 50 The IC values ​​of IPB24, IPB28, IPB29, and IPB30 were approximately 6-fold, 8-fold, and 4-fold higher than those of IPB29, respectively. 50 The IC values ​​were 4.51 nM, 4.51 nM, 0.47 nM and 1.78 nM for IPB24, IPB28 and IPB30, respectively. 50 The values ​​were approximately 10-fold, 10-fold, and 4-fold higher than those of IPB29, respectively.

[0115] The results for Huh-7 cells are shown in Figure 5. The IC values ​​of IPB24, IPB28, IPB29, and IPB30 inhibiting Delta strain infection in Huh-7 cells were 50 The IC values ​​were 3.46 nM, 4.34 nM, 0.56 nM and 2.17 nM for IPB24, IPB28 and IPB30, respectively. 50The IC values ​​of IPB24, IPB28, IPB29, and IPB30 were approximately 6-, 8-, and 4-fold higher than those of IPB29, respectively. 50 The IC values ​​were 2.56 nM, 2.46 nM, 0.46 nM and 1.46 nM for IPB24, IPB28 and IPB30, respectively. 50 The values ​​were approximately 6-fold, 5-fold, and 3-fold higher than those of IPB29, respectively.

[0116] These results demonstrate that novel lipopeptides (e.g., IPB29 and IPB30) exhibit good inhibitory activity against various SARS-CoV-2 mutants (mutant strains) as viral membrane fusion inhibitors.

[0117] Example 4: Inhibitory effects of lipopeptides on other coronaviruses The test lipopeptides were lipopeptide IPB29, lipopeptide IPB30, lipopeptide IPB24, or lipopeptide IPB28 prepared in Example 1, and the test cells were 293T / ACE2 cells or Huh-7 cells.

[0118] 4-1) Preparation of other coronavirus pseudoviruses Various other coronavirus pseudoviruses were prepared, including bat-derived coronavirus (bat RaTG13), pangolin-derived coronavirus (PCoV-GD or PCoV-GX), SARS-CoV, MERS-CoV, HCoV-NL63, and HCoV-229E.

[0119] See step 1-1) of Example 3 for the method, except that the plasmid expressing the SARS-CoV-2 S protein was replaced with a plasmid expressing the S protein of another coronavirus. The prepared SARS-CoV pseudovirus was the "SARS-CoV PV" described in Non-Patent Document 2, the prepared MERS-CoV pseudovirus was the "MERS-CoV PV" described in Non-Patent Document 2, the prepared HCoV-NL63 pseudovirus was the "HCoV-NL63 PV" described in Non-Patent Document 2, and the prepared HCoV-229E pseudovirus was the "HCoV-229E PV" described in Non-Patent Document 2.

[0120] 4-2) Inhibitory effects of lipopeptides on other coronaviruses The inhibitory activity was measured in the same manner as in step 1-2) of Example 3, and the results are shown in Figure 6. The results demonstrated that the novel lipopeptides can effectively inhibit infection with the above seven types of viruses. In 293T / ACE2 cells, the IC values ​​of IPB24, IPB28, and IPB30, which inhibit infection with the bat RaTG13 strain, PCoV-GD strain, and PCoV-GX strain, were 50 The IC values ​​of IPB24, IPB28, and IPB30 inhibited infection of SARS-CoV, MERS-CoV, HCoV-NL63, and HCoV-229E strains in Huh-7 cells. 50 The maximum inhibitory activity was approximately 11-fold, 7-fold, 9-fold, and 7-fold that of IPB29, respectively. These results indicate that the lipopeptides IPB29 and IPB30, especially IPB29, exhibit strong inhibitory effects against other coronaviruses, and that IPB29 still exhibits the strongest inhibitory activity against SARS-CoV, PCoV-GD, and PCoV-GX, which are closely related to SARS-CoV-2, compared to other lipopeptides.

[0121] Example 5: Analysis of in vitro cytotoxicity and therapeutic index of lipopeptides The test lipopeptides were lipopeptide IPB29, lipopeptide IPB30, lipopeptide IPB24, or lipopeptide IPB28 prepared in Example 1, and the test cells were 293T / ACE2 cells or Huh-7 cells.

[0122] The in vitro cytotoxicity test of the test lipopeptides was performed using a CCK-8 cell proliferation / toxicity test kit (Abbkine, product number KTC011001) according to the following procedure: 1) In a 96-well cell culture plate, the test lipopeptides were diluted 3-fold, and 100 μL of lipopeptide solution was added to each well, resulting in nine dilutions with three parallel wells per dilution. Additionally, a control well was added with DMEM medium (100 μL per well). 2) 10 × 10 4 The test cell suspension (100 μL / mL) was added to the 96-well cell culture plate obtained in step 1) at a volume of 100 μL / well and cultured for 48 hours under conditions of 37°C and 5% CO2. 3) After step 2) was completed, 20 μL of CCK-8 solution was added to each well, and the plate was incubated in an incubator for another 2 hours, after which the absorbance at 450 nm (OD450) was measured using a microplate reader. An inhibition rate graph was created using GraphPad Prism software, and the median cytotoxic concentration (CCT) of the drug was calculated. 50 ) was calculated.

[0123] As shown in Figure 7, the CC of four lipopeptides, IBP24, IBP28, IBP29, and IBP30, on 293T / ACE2 cells was significantly increased. 50 were 14.36 μM, 12.28 μM, 23.94 μM, and 45.46 μM, respectively, and CC 50 The values ​​were 15.02 μM, 15.97 μM, 22.75 μM, and 44.43 μM, respectively. This indicates that IPB29 and IPB30, especially IPB30 modified with stearyl chloride, have relatively low cytotoxicity.

[0124] CC 50 / I C 50Analysis (IC 50 The data obtained in Example 3 confirmed that all four polypeptides had very high selective therapeutic indices (TI). For example, IPB24, IPB28, IPB29, and IPB30 exhibited high TI values ​​of approximately 3184, 2723, 50936, and 25539, respectively, for inhibitory activity against infection of 293T / ACE2 cells with Omicron mutant strains. The TI values ​​of IPB29 were approximately 16-fold, 19-fold, and 2-fold higher than those of IPB24, IPB28, and IPB30, respectively. Furthermore, IPB24, IPB28, IPB29, and IPB30 exhibited high TI values ​​of approximately 5867, 6492, 49457, and 30432, respectively, for inhibitory activity against infection of Huh-7 cells with Omicron mutant strains. The TI values ​​of IPB29 were approximately 8-fold, 8-fold, and 1.6-fold higher than those of IPB24, IPB28, and IPB30, respectively. These results indicate that IPB29 and IPB30 have a higher therapeutic index and higher druggability than IPB24 and IPB28.

[0125] Example 6: Stability studies of lipopeptide inhibitors In this example, the inventors compared the stability of representative lipopeptides IPB24 and IPB29 from various angles, including protease digestion, liver microsome digestion, incubation with human serum, and prolonged storage at 37°C. Using the lipopeptides IPB29 and IPB24 prepared in Example 1 as test lipopeptides, the antiviral activity of the lipopeptides was measured in the same manner as in Step 1 of Example 3 (test cells: 293T / ACE2 cells).

[0126] 1) Protease digestion The test proteases used were proteinase K, trypsin, and α-chymotrypsin, which were manufactured by Sigma-Aldrich under product numbers P2308, T4799, and C4129, respectively.

[0127] The test lipopeptide and the test protease were mixed to final concentrations of 2 mg / mL and 0.1 mg / mL, respectively, and incubated at 37°C for 0, 30, 60, 120, or 180 minutes, after which the antiviral activity of the lipopeptide was measured.

[0128] 2) Digestion of liver microsomes The human liver microsome (mixture) reagent for the Phase I metabolic stability test kit was purchased from Beijing Huizhi Taikang Pharmaceutical Technology Co., Ltd. (product number 0111A1.03). The experimental method was performed according to the manufacturer's instructions. First, 10 μL of solution A, 2 μL of solution B, and 28 μL of 0.1 M PBS buffer were mixed uniformly and pre-incubated at 37°C for 5 minutes. After pre-incubation, the mixture was dispensed into 40 μL tubes and heated in a 37°C water bath to prepare the pre-incubation solution for further use. 154 μL of 0.1 M PBS buffer, 5 μL of liver microsomes, and 1 μL of 4 mM test lipopeptide solution were mixed, followed by addition of 40 μL of pre-incubation solution. The mixture was then immediately placed in a 37°C water bath for incubation and time measurement. After different incubation times, 200 μL of pre-chilled acetonitrile was added to the incubation system to terminate the reaction, and the antiviral activity of the lipopeptide was measured.

[0129] 3) Human serum stability experiment 20% human serum was mixed with the test lipopeptide at a final concentration of 150 μM and incubated at 37° C. for 0, 5, 30, 60, 120, or 180 minutes, after which the antiviral activity of the lipopeptide was measured.

[0130] 4) Temperature stability experiment Aqueous solutions of test lipopeptides at a concentration of 300 μM were left at 37° C. for different periods of time, and the changes in their antiviral activity were then measured.

[0131] 5) Analysis of results The experimental results are shown in Figure 8. The antiviral activity of IPB24 and IPB29 treated with proteinase K, trypsin, or α-chymotrypsin showed no significant change compared to the untreated lipopeptides (i.e., the treatment group with 0 incubation time), suggesting that single enzyme digestion has limited effect on the stability of lipopeptides. On the other hand, the inhibitory activity of IPB24 against SARS-CoV-2 infection of 293T / ACE2 cells was clearly reduced by treatment with human liver microsomes for 72 and 96 hours, and its IC 50 Although the levels increased approximately 8-fold and 14-fold, respectively, the effect on the antiviral activity of IPB29 was limited. Liver microsomes contain most phase I enzymes, and the microsomal mixed-function oxidase system, primarily composed of CYP450, is the most important. Further investigation is needed into the components that affect the activity of IPB24.

[0132] IPB24 and IPB29 were more sensitive to treatment with 20% human serum. The antiviral activity of IPB24 was reduced approximately 24-fold after 5 min of incubation, and approximately 32-, 36-, 37-, and 42-fold after 30, 60, 120, and 180 min of incubation, respectively. On the other hand, IPB29 was significantly more resistant to human serum, with approximately 4-, 10-, 12-, 13-, and 13-fold reductions in its antiviral activity after 5 min of incubation and 180 min of incubation, respectively.

[0133] The antiviral activity of IPB24 incubated at 37°C for 3, 7, 14, 21, and 28 days, respectively, gradually decreased over time, especially by more than fivefold after 28 days. In contrast, the antiviral activity of IPB29 remained unchanged or was much smaller under the same incubation conditions. Further studies are needed to determine the temperature stability of lipopeptides over longer periods.

[0134] In conclusion, IPB29 exhibited significantly improved stability compared with IPB24, demonstrating a clear advantage in stability, further supporting the superior druggability of the IPB29 helical lipopeptide with the EAAAK sequence.

[0135] Example 7: Inhibitory effect of novel lipopeptides on SARS-CoV-2 S protein-mediated cell-cell membrane fusion The lipopeptides IPB29, IPB30, and IPB20 prepared in Example 1 were used as test lipopeptides, and the test cells were 293T / ACE2 cells and Huh-7 cells.

[0136] To further evaluate the anti-SARS-CoV-2 activity of the novel lipopeptide inhibitors, a cell-cell fusion inhibition experiment based on the DSP system was conducted in this disclosure. For specific methods, please refer to the "Cell-cell fusion assay" sections of Non-Patent Documents 4 and 5. The procedure was as follows:

[0137] 1) 293T effector cell suspension (1.5 x 10 4 cells / 100 μL / well) into a 96-well plate, and simultaneously add 293T / ACE2 or Huh-7 target cell suspension (1.5 × 10 5 The cells were cultured at 37°C and 5% CO2 at 10 cm cell culture dish.

[0138] 2) After 16 hours of incubation, the pCoV2-S plasmid and pDSP 1-7 The plasmids were co-transfected into 293T effector cells, and simultaneously pDSP 8-11 The plasmids were introduced into 293T / ACE2 or Huh-7 target cells, and the cells were continued to be cultured.

[0139] 3) After 24 hours, the polypeptides were diluted three-fold in a 96-well plate, resulting in nine dilutions in three parallel wells for each concentration. The diluted polypeptides were added to the effector cells and incubated for 1 hour in a 37°C, 5% CO2 cell culture incubator.

[0140] 4) Preheat the DMEM complete medium, add EnduRen Live Cell Substrate (Promega) at a ratio of 1:4000, and resuspend the centrifuged 293T / ACE2 or Huh-7 target cells in the resulting medium to a cell concentration of 3 × 10 5 The solution was adjusted to 1 mL / mL and incubated at 37°C, 5% CO2 for 30 minutes.

[0141] 5) 293T / ACE2 or Huh7 target cells were added to 293T effector cells at a volume of 100 μL / well, and the mixture was centrifuged at 400 g for 1 minute to ensure sufficient contact between the effector and target cells. The mixed cells were then cultured for 2 hours.

[0142] 6) Luciferase activity (RLU) was read using a microplate photometer, and the inhibition rate and IC 50 was calculated.

[0143] As shown in Figure 9, when the target cells were 293T / ACE2 (left panel), the IC values ​​of IPB20, IPB29, and IPB30, which inhibited cell-cell membrane fusion mediated by the S protein of SARS-CoV-2, were significantly higher than those of IPB20, IPB29, and IPB30. 50 The IC values ​​of IPB20, IPB29, and IPB30 for inhibiting SARS-CoV-2 S protein-mediated cell-cell membrane fusion were 3.65 nM, 0.2 nM, and 0.44 nM, respectively, when the target cells were Huh-7 (right panel). 50 The ATP concentrations were 4.01 nM, 0.31 nM, and 0.45 nM, respectively. These experimental results confirmed that the new membrane fusion inhibitors IPB29 and IPB30 exhibit relatively strong inhibitory activity against SARS-CoV-2 S protein-mediated cell-cell fusion.

[0144] The present disclosure has been described in detail above. Those skilled in the art can practice the present disclosure in a relatively wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the present disclosure and without undue experimentation. Although specific examples are shown in the present disclosure, it should be understood that further improvements can be made to the present disclosure. That is, in accordance with the principles of the present disclosure, the present application is intended to include any modification, use, or improvement of the present disclosure, including modifications made by conventional techniques that are outside the scope of the present disclosure and are well known in the art. Application of some essential features may be made within the scope of the following appended claims. (Addendum) The present disclosure includes the following aspects. Item 1: A compound represented by the following formula (I) or formula (II), or a pharmaceutically acceptable salt or derivative thereof, [ka] In formula (I) and formula (II), X 1 is an amino-terminal protecting group, In formula (I) and formula (II), X 2 is a polypeptide having an amino acid sequence of (EAAAK)n or A[(EAAAK)n]A, where n is a natural number of 5 or less and represents the number of repeats of the EAAAK sequence; In formula (I), X 3 is lysine or cysteine ​​or 2,3-diaminopropionic acid (Dap) or ornithine (Orn) or 2,4-diaminobutanoic acid (Dab) or 2,7-diaminoheptanoic acid (Dah), In formula (I), X 4 is X 3 a lipophilic compound functional group modified with In formula (II), X 4 is X 2 K is a lipophilic compound modified with a functional group, In formula (I) and formula (II), X 5 is a carboxy-terminal protecting group, or a pharmaceutically acceptable salt or derivative thereof. Item 2: The compound is a compound represented by formula (I), Among them, X 1 is an acetyl group (Ac), and X 2 is EAAAK and X 3 is lysine, the lipophilic compound is cholesterol succinate monoester, and X 5 NH 2 2. The compound according to item 1, wherein: Item 3: The compound is a compound represented by formula (I), Among them, X 1 is an acetyl group (Ac), and X 2 is EAAAK and X 3 is lysine, the lipophilic compound is stearyl chloride, and X 5 NH 2 2. The compound according to item 1, wherein: Item 4: A multimer of any one of the following (a1) to (a3): (a1) A multimer formed of the compound according to any one of items 1 to 3, (a2) A multimer formed from the pharmaceutically acceptable salt according to any one of items 1 to 3, (a3) A multimer formed from the derivative according to any one of items 1 to 3. Item 5: Any of the following applications (b1) to (b4): (b1) Application in the preparation of coronavirus membrane fusion inhibitors, (b2) Application in the preparation of a drug for preventing and / or treating a disease caused by a coronavirus, (b3) Application as a coronavirus membrane fusion inhibitor, (b4) The compound according to any one of items 1 to 3, or a pharmaceutically acceptable salt thereof, or a compound according to any one of items 1 to 3, which is used for preventing and / or treating a disease caused by coronavirus. Applications of derivatives. Item 6: A product comprising the compound according to any one of Items 1 to 3, or a pharmaceutically acceptable salt thereof, or a derivative thereof, The product performs the following function (c1) or (c2): (c1) Function as a coronavirus membrane fusion inhibitor, (c2) A product whose function is to prevent and / or treat diseases caused by coronaviruses. Item 7: Use of a linker polypeptide in the preparation of a product that enhances the antiviral activity and / or stability of a viral membrane fusion inhibitor, The amino acid sequence of the linker polypeptide is (EAAAK)n or A[(EAAAK)n]A, where n is a natural number of 5 or less. Item 8: A method for enhancing the antiviral activity and / or stability of a viral membrane fusion inhibitor, comprising: The method includes a step of linking a linker polypeptide to a viral membrane fusion inhibitor, wherein the amino acid sequence of the linker polypeptide is (EAAAK)n or A[(EAAAK)n]A, where n is a natural number of 5 or less. Item 9: A method for preparing a modified viral membrane fusion inhibitor, comprising the following step (d1) or step (d2): (d1) Using a linker polypeptide having the amino acid sequence (EAAAK)n or A[(EAAAK)n]A as a linking arm, the unmodified viral membrane fusion inhibitor and X 3 (X 4 ) groups to obtain a lipopeptide, and X 3 (X 4 ) group, X 4 is X 3 modified to X 3 is lysine or cysteine ​​or 2,3-diaminopropionic acid (Dap) or ornithine (Orn) or 2,4-diaminobutyric acid (Dab) or 2,7-diaminoheptanoic acid (Dah), and X 4 is a lipophilic compound functional group, n is a natural number of 5 or less, (d2) Using a linker polypeptide having the amino acid sequence (EAAAK)n or A[(EAAAK)n]A as a linking arm, the unmodified viral membrane fusion inhibitor and X 4 Lipopeptides are obtained by linking groups, among which X 4 is a lipophilic compound functional group, which is modified to K of the linker polypeptide, and n is a natural number of 5 or less; A preparation method, wherein the lipopeptide is a modified viral membrane fusion inhibitor. Item 10: A modified viral membrane fusion inhibitor according to the following (e1) or (e2): (e1) Using a linker polypeptide having the amino acid sequence (EAAAK)n or A[(EAAAK)n]A as a linking arm, the unmodified viral membrane fusion inhibitor and X 3 (X 4 ) groups to obtain a lipopeptide, and X 3 (X 4 ) group, X 4 is X 3 modified to X 3 is lysine or cysteine ​​or 2,3-diaminopropionic acid (Dap) or ornithine (Orn) or 2,4-diaminobutyric acid (Dab) or 2,7-diaminoheptanoic acid (Dah), and X 4 is a lipophilic compound functional group, n is a natural number of 5 or less, (e2) Using a linker polypeptide having the amino acid sequence (EAAAK)n or A[(EAAAK)n]A as a linking arm, the unmodified viral membrane fusion inhibitor and X 4 Lipopeptides are obtained by linking groups, among which X 4 is a lipophilic compound functional group, which is modified to K of the linker polypeptide, and n is a natural number of 5 or less; The lipopeptide is a modified viral membrane fusion inhibitor.

Claims

1. A compound represented by the following formula (I) or a pharmaceutically acceptable salt thereof: 【Chemical 1】 In formula (I), X １ is an acetyl group (Ac), In formula (I), X ２ is a polypeptide having the amino acid sequence EAAAK, and in formula (I), X ３ is lysine, In formula (I), X ４ is X ３ a lipophilic compound group modified as above, wherein the lipophilic compound is cholesterol succinic acid monoester or stearyl chloride; A compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein X 5 is NH 2.

2. The compound is a compound represented by formula (I), wherein X １ is an acetyl group (Ac), and X ２ is EAAAK, and X ３ is lysine, the lipophilic compound is cholesterol succinate monoester, and X ５ is NH ２ 2. The compound of claim 1, wherein:

3. The compound is a compound represented by formula (I), wherein X １ is an acetyl group (Ac), and X ２ is EAAAK, and X ３ is lysine, the lipophilic compound is stearyl chloride, and X ５ is NH ２ 2. The compound of claim 1, wherein:

4. A multimer of (a1) or (a2) below: (a1) a multimer formed of the compound according to any one of claims 1 to 3; (a2) A multimer formed with the pharmaceutically acceptable salt according to any one of claims 1 to 3.

5. The compound according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof for use in the following (b1) or (b2): (b1) Preparation of coronavirus membrane fusion inhibitors; (b2) Preparation of a medicament for preventing and / or treating a disease caused by a coronavirus.

6. A product comprising the compound according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof, and performing the following function (c1) or (c2): (c1) Function as a coronavirus membrane fusion inhibitor; (c2) The ability to prevent and / or treat diseases caused by coronaviruses.

7. 1. Use of a linker polypeptide in the preparation of a product that enhances the antiviral activity and / or stability of a viral membrane fusion inhibitor, comprising: The viral membrane fusion inhibitor comprises the compound according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof; The amino acid sequence of the linker polypeptide is EAAAK.

8. A method for enhancing the antiviral activity and / or stability of a viral membrane fusion inhibitor, comprising: The method includes the step of linking a linker polypeptide X 2 as a linking arm to the unmodified viral membrane fusion inhibitor, and further linking an X 3 (X 4 ) group to obtain a lipopeptide, wherein the lipopeptide is a compound of the following formula (I): 【Chemistry 2】 In formula (I), X 1 is an acetyl group (Ac); In formula (I), the linker polypeptide X2 has the amino acid sequence of EAAAK; in formula (I), in the X3 (X4) group, X4 is modified on X3, X3 is lysine, and X4 is a lipophilic compound group, and the lipophilic compound is cholesterol succinic acid monoester or stearyl chloride; The method wherein, in formula (I), X 5 is NH 2 .

9. The linker polypeptide X2 consisting of the amino acid sequence of EAAAK is used as a linking arm to bind the unmodified viral membrane fusion inhibitor and X2. ３ (X ４ ) group to obtain a lipopeptide, The lipopeptide is a compound of formula (I): 【Chemistry 3】 In formula (I), X 1 is an acetyl group (Ac); In formula (I), in the X 3 (X 4 ) group, X 4 is modified on X 3 , X 3 is lysine, X 4 is a lipophilic compound group, and the lipophilic compound is cholesterol succinic acid monoester or stearyl chloride; In formula (I), X 5 is NH 2 ; A method for preparing a modified viral membrane fusion inhibitor, wherein the lipopeptide is a modified viral membrane fusion inhibitor.

Images