Polypeptide-small molecule conjugate for inhibiting novel coronavirus

Abstract

The invention provides a conjugate of a polypeptide and a small organic molecule, a preparation method thereof, a pharmaceutical composition, a composition and application. The polypeptide is obtained by covalent coupling of a small organic molecule transmembrane serine protease inhibitor and a polypeptide with antiviral activity. The polypeptide-small molecule conjugate has the effect of resisting the novel coronavirus.

Description

Technical Field

[0001] The present disclosure belongs to the field of biomedicine and relates to a polypeptide-small molecule conjugate for inhibiting viruses. Background Art

[0002] SARS-CoV-2 has strong pathogenicity and rapid transmission ability. The emergence of its variants such as Delta and Omicron has enhanced its infectivity and pathogenicity and reduced its sensitivity to existing vaccines and drugs. The process by which SARS-CoV-2 enters host cells through the membrane fusion pathway mainly consists of the following steps. First, the receptor-binding domain (RBD) of the spike protein on the virus surface recognizes and binds to the host cell surface receptor angiotensin-converting enzyme 2 (ACE2), causing the virus to attach to the host cell surface. After the RBD receptor binds to ACE2, the S2' site of the S protein is exposed, and then the host protease type II transmembrane serine protease (TTSPs) cleaves and activates this site to initiate membrane fusion. Among them, transmembrane serine protease 2 (TMPRSS2) is considered to be the most important hydrolase. The above process further induces a series of conformational changes in the S2 subunit, transitioning from a metastable state to a lower energy state. Theoretically, by using a transmembrane serine protease 2 inhibitor, the co-expression of the ACE2 receptor on the host cell surface can be inhibited, further inhibiting the entry of SARS-CoV-2 into host cells.

[0003] Currently, there is still no specific drug for SARS-CoV-2. Therefore, in the foreseeable future, the COVID-19 pandemic is likely to remain a global health threat, and it is still of great significance to develop highly efficient and specific therapeutic and preventive drugs. Summary of the Invention

[0004] The present disclosure provides a polypeptide-small molecule conjugate, which has a highly efficient and specific inhibitory effect on the novel coronavirus (SARS-CoV-2).

[0005] In a first aspect of the present disclosure, there is provided a polypeptide-small molecule conjugate or its prodrug, tautomer, optical isomer, geometric isomer, solvate or its pharmaceutically acceptable salt, wherein the polypeptide-small molecule conjugate comprises an organic small molecule transmembrane serine protease inhibitor and a polypeptide moiety with antiviral activity.

[0006] The second aspect of the present disclosure provides a method for preparing the polypeptide-small molecule conjugate described herein, which includes linking the organic small molecule transmembrane serine protease inhibitor described herein with a polypeptide moiety having antiviral activity. In some preferred embodiments, the method for preparing the polypeptide-small molecule conjugate described herein includes covalently coupling the organic small molecule transmembrane serine protease inhibitor described herein with a polypeptide moiety having antiviral activity through a linker.

[0007] The third aspect of the present disclosure provides a pharmaceutical composition, which includes the polypeptide-small molecule conjugate described herein, and one or more pharmaceutically acceptable excipients or carriers.

[0008] The fourth aspect of the present disclosure provides a composition, which includes the polypeptide-small molecule conjugate described herein and other antiviral drugs.

[0009] The fifth aspect of the present disclosure provides the use of the polypeptide-small molecule conjugate, pharmaceutical composition or composition described herein in the preparation of a drug for preventing and / or treating viral infections.

[0010] The sixth aspect of the present disclosure provides a method for inhibiting viral infection in vitro for non-therapeutic purposes, by adding a therapeutically effective amount of the polypeptide-small molecule conjugate, pharmaceutical composition or composition described herein to a test sample.

[0011] The seventh aspect of the present disclosure provides a method for treating or preventing viral infection, which includes administering to a subject a prophylactically or therapeutically effective amount of the polypeptide-small molecule conjugate, pharmaceutical composition or composition described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 Shows the mass spectrum of the small molecule Camostat-1 in Example 1.

[0013] Figure 2 Shows the mass spectrum of the small molecule Camostat-2 in Example 1.

[0014] Figure 3 Shows the mass spectrum of the small molecule Camostat-3 in Example 1.

[0015] Figure 4 Shows the mass spectrum of the small molecule Camostat-A in Example 1.

[0016] Figure 5 Shows the mass spectrum of the small molecule Camostat-B in Example 1.

[0017] Figure 6 Shows the 1H NMR spectrum of the small molecule Camostat-1 in Example 1.

[0018] Figure 7 The carbon-13 nuclear magnetic resonance spectrum of the small molecule Camostat-1 in Example 1 is shown.

[0019] Figure 8 The proton nuclear magnetic resonance spectrum of the small molecule Camostat-2 in Example 1 is shown.

[0020] Figure 9 The carbon-13 nuclear magnetic resonance spectrum of the small molecule Camostat-2 in Example 1 is shown.

[0021] Figure 10 The proton nuclear magnetic resonance spectrum of the small molecule Camostat-3 in Example 1 is shown.

[0022] Figure 11 The carbon-13 nuclear magnetic resonance spectrum of the small molecule Camostat-3 in Example 1 is shown.

[0023] Figure 12 The proton nuclear magnetic resonance spectrum of the small molecule Camostat-A in Example 1 is shown.

[0024] Figure 13 The carbon-13 nuclear magnetic resonance spectrum of the small molecule Camostat-A in Example 1 is shown.

[0025] Figure 14 The proton nuclear magnetic resonance spectrum of the small molecule Camostat-B in Example 1 is shown.

[0026] Figure 15 The carbon-13 nuclear magnetic resonance spectrum of the small molecule Camostat-B in Example 1 is shown.

[0027] Figure 16 The high performance liquid chromatography (HPLC) graph of the compound IPB19N1-CA in Example 2 (purity 97.9%) is shown.

[0028] Figure 17 The mass spectrum of the compound IPB19N1-CA in Example 2 is shown.

[0029] Figure 18 The high performance liquid chromatography (HPLC) graph of the compound IPB19N1-CB in Example 3 (purity 98.7%) is shown.

[0030] Figure 19 The mass spectrum of the compound IPB19N1-CB in Example 3 is shown.

[0031] Figure 20 The high performance liquid chromatography (HPLC) graph of the compound IPB19N2-CA in Example 4 (purity 99%) is shown.

[0032] Figure 21Shows the mass spectrum of compound IPB19N2-CA in Example 4.

[0033] Figure 22 Shows the high performance liquid chromatography (HPLC) diagram (purity 98%) of compound IPB19C2-CB in Example 5.

[0034] Figure 23 Shows the mass spectrum of compound IPB19C2-CB in Example 5.

[0035] Figure 24 Shows the schematic diagram of the binding of the pharmacodynamic active peptide and the small molecule pharmacophore. Detailed implementation manners

[0036] The following specific embodiments illustrate the implementation manners of the present application. Those skilled in the art can easily understand other advantages and effects of the present application from the content disclosed in this specification.

[0037] Unless otherwise specified, all numbers representing content, concentration, ratio, mass, volume, time, temperature, thickness, technical effects, etc. used in this specification and claims should be understood to be modified by the term "about" or "substantially" in any case. Therefore, unless there is a contrary indication, the numerical parameters listed in the following specification and appended claims are approximate values. For those skilled in the art, they can vary according to the desired properties and effects sought through this disclosure and should be interpreted according to the number of significant digits and the conventional rounding method or the manner understood by those skilled in the art for each numerical parameter.

[0038] Although the numerical ranges and parameters of the broad scope of the present disclosure are approximate values, the values set forth in the specific embodiments are provided as precisely as possible. However, any numerical value will inherently contain certain errors that are necessarily caused by the standard deviations found in their corresponding test measurements. Each numerical range given in this specification will include each narrower numerical range falling within that broader numerical range as if these narrower numerical ranges were all expressly written herein.

[0039] When used herein, the expression "A and / or B" includes three cases: (1) A; (2) B; and (3) A and B. The expression "A, B and / or C" includes seven cases: (1) A; (2) B; (3) C; (4) A and B; (5) A and C; (6) B and C; and (7) A, B and C. The meanings of similar expressions can be deduced by analogy.

[0040] Polypeptide-small molecule conjugate

[0041] The present application provides a polypeptide-small molecule conjugate or a prodrug, tautomer, optical isomer, geometric isomer, solvate or pharmaceutically acceptable salt thereof. The polypeptide-small molecule conjugate comprises an organic small molecule transmembrane serine protease inhibitor and a polypeptide moiety having antiviral activity.

[0042] In some embodiments, in the polypeptide-small molecule conjugate provided herein, the organic small molecule transmembrane serine protease inhibitor and the polypeptide moiety having antiviral activity are covalently linked through a linker. In some preferred embodiments, the linker provided herein comprises at least one of a linker and lysine.

[0043] In some embodiments, the polypeptide-small molecule conjugate provided herein comprises a structure represented by Formula I, Formula II or Formula III:

[0044] α-P-Z-K(X)-β, α-X-Z-P-β, α-P–K(Z–X)-β,

[0045] Formula I Formula II Formula III

[0046] Wherein, α is selected from amino, acetyl, maleoyl, succinyl, tert-butoxycarbonyl, benzyloxycarbonyl or dansyl; β is selected from amide, carboxyl or carboxyl derivative; K represents lysine; P represents a polypeptide moiety having antiviral activity; Z represents a linker; X represents an organic small molecule transmembrane serine protease inhibitor; "-" indicates a covalent connection relationship between two molecules; the fragment X or Z-X in "()" indicates that it is connected to K through the side chain of K.

[0047] In some embodiments, the polypeptide moiety provided herein is derived from the HR2 domain of the S2 subunit of the SARS-CoV-2 spike protein.

[0048] In some embodiments, the polypeptide provided herein is selected from any one of the following (a)-(d): (a) SVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIK (SEQ ID NO:1); (b) ISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL (SEQ ID NO:2); (c) a functional variant of SVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIK containing 0-7 conservative amino acid substitutions; (d) a functional variant of ISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL containing 0-7 conservative amino acid substitutions. The polypeptide provided herein can bind to HR1 of the S2 subunit of the SARS-CoV-2 spike protein, rendering the interaction between HR1 and HR2 ineffective and unable to promote viral membrane-host cell membrane fusion, so that the virus cannot enter the host cell and release its genetic material, further inhibiting the infection of the virus to the human body.

[0049] In some embodiments, the linker provided herein is a flexible linker. In some preferred embodiments, the linker provided herein has the following structure: (PEN) n or (PEG) n -(CH2) m , where n is selected from any integer from 1 to 35, m is selected from any integer from 1 to 10, and "-" indicates a covalent connection relationship between two molecules. In some preferred embodiments, n is 4, 12, or 24. In some preferred embodiments, m is 2. PEG herein is polyethylene glycol.

[0050] In some embodiments, the organic small molecule transmembrane serine protease inhibitor provided herein comprises at least one of the organic small molecule transmembrane serine protease 2 inhibitors. In a preferred embodiment, the organic small molecule transmembrane serine protease 2 inhibitor provided herein is selected from: camostat, camostat mesylate, VD4162, phenylmethylsulfonyl fluoride, 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride, and benzamidine hydrochloride.

[0051] In some embodiments, the organic small molecule transmembrane serine protease 2 inhibitor provided herein comprises at least one of Compound I and Compound II.

[0052] Compound I has the structure of

[0053]

[0054] Compound II has the structure of

[0055]

[0056] In some embodiments, the organic small molecule transmembrane serine protease inhibitor provided herein is linked to the polypeptide moiety provided herein by a click reaction, such as an azide-alkyne cycloaddition reaction.

[0057] As described herein, "click reaction", also known as click chemistry, link chemistry or rapid assembly combinatorial chemistry, is a synthetic concept introduced by chemist K B Sharpless in 2001. The main idea is to rapidly and reliably complete the chemical synthesis of various molecules through the splicing of small units. It especially emphasizes the development of new combinatorial chemistry methods based on the formation of carbon-heteroatom bonds (C-X-C), and to simply and efficiently obtain molecular diversity by means of these reactions (click reactions). Common click reactions include: cycloaddition reactions, nucleophilic ring-opening reactions, carbonyl chemistry other than aldol reactions, and addition reactions of carbon-carbon multiple bonds.

[0058] In some embodiments, the organic small molecule transmembrane serine protease inhibitor provided herein contains an alkynyl group, and the lysine in the polypeptide-small molecule conjugate contains an azide moiety, and the alkynyl group is conjugated to the azide moiety by a click reaction. In some preferred embodiments, the organic small molecule transmembrane serine protease inhibitor provided herein contains an alkynyl group, and the lysine in the polypeptide-small molecule conjugate contains an azide moiety, and the alkynyl group is linked to the azide moiety by an azide-alkyne cycloaddition reaction. In some embodiments, the organic small molecule transmembrane serine protease inhibitor provided herein contains an azide moiety, and the lysine in the polypeptide-small molecule conjugate contains an alkynyl group, and the alkynyl group is conjugated to the azide moiety by a click reaction. In some preferred embodiments, the organic small molecule transmembrane serine protease inhibitor provided herein contains an azide moiety, and the lysine in the polypeptide-small molecule conjugate contains an alkynyl group, and the alkynyl group is linked to the azide moiety by an azide-alkyne cycloaddition reaction.

[0059] In some embodiments, the azide moiety provided herein contains any structure containing an azide group and capable of covalently linking to the polypeptide linker moiety, such as azidoacetic acid, azidobenzene, biotin-azide, epoxide-azide, aldehyde-azide, alkyne-azide, azidoacrylate, azidopropionic acid, 2-azidopropionic acid, azidobutyric acid, azido-polylysine, etc. In some preferred embodiments, the azide moiety provided herein contains azidoacetic acid, azidopropionic acid, 2-azidopropionic acid or azidobutyric acid.

[0060] In some embodiments, the click reaction is an azide-alkyne cycloaddition reaction. The azide-alkyne cycloaddition reaction refers to the cycloaddition reaction between azide and alkyne catalyzed by Cu(I). This reaction was developed by chemists such as Morten Meldal and K.B. Sharpless, and it is characterized by high efficiency, selectivity, and ease of operation. In the reaction, first, the copper(I) catalyst (such as Cu(I) bromide or Cu(I) sulfate) is activated, and usually a ligand (such as dimethylamine (DMA) or triphenylphosphine (TPP)) is required to improve the selectivity and efficiency of the reaction. Then, a copper-azide complex is formed, that is, the azide forms a complex with the activated copper(I) catalyst. Through the action of the copper(I) catalyst on the alkyne, the electrophilicity of the triple-bond carbon atoms of the alkyne is enhanced. The nitrogen atom of the azide attacks the carbon atom of the alkyne as a nucleophile to form an intermediate, that is, a [3+2] ring. Finally, the intermediate undergoes rearrangement to ultimately form a stable 1,2,3-triazole ring. The reaction can proceed rapidly under mild conditions, which is suitable for coupling the polypeptide part of the polypeptide-small molecule conjugate and the organic small molecule transmembrane serine protease inhibitor. It can specifically label target molecules in a complex biological system without generating additional side reaction substances. During the reaction process, the formation of the triazole ring provides a platform for further chemical modification, and various functional groups can be introduced.

[0061] In some embodiments, the polypeptide-small molecule conjugate structures provided herein include one or more of the following:

[0062] Compound I-PEG4-SVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIK-CONH2;

[0063] Compound II-PEG4-SVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIK-CONH2;

[0064] Compound I-PEG 12 -SVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIK-CONH2;

[0065] Compound II-PEG 12 -SVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIK-CONH2;

[0066] Compound I-PEG 24 -SVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIK-CONH2;

[0067] Compound II-PEG 24 -SVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIK-CONH2;

[0068] Ac-SVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIK-PEG4-K(Compound I)-CONH2; Ac-SVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIK-PEG4-K(Compound II)-CONH2; Ac-SVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIK-PEG 12 -K(Compound I)-CONH2; Ac-SVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIK-PEG 12 -K(Compound II)-CONH2; Ac-SVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIK-PEG 24 -K(Compound I)-CONH2; Ac-SVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIK-PEG 24 -K(Compound II)-CONH2,

[0069] wherein "-" represents a covalent connection relationship between two molecules; Compound I or II within "()" is connected to K through the side chain of K,

[0070] Compound I has the structure of

[0071]

[0072] Compound II has the structure of

[0073]

[0074] In this article, the term "polypeptide" refers to a compound formed by dehydration condensation of three or more amino acids linked together by peptide bonds. Polypeptides can be prepared by chemical synthesis or obtained by protein hydrolysis. The types of amino acids in polypeptides can include, but are not limited to: natural or non-natural amino acids such as L-amino acids, D-amino acids, α-amino acids or β-amino acids, etc.

[0075] In this article, the term "transmembrane serine protease" (TMPRSSs), also known as type II transmembrane serine proteases (TTSPs), is a family of proteins located on the cell membrane with a conserved serine protease domain. More than twenty members have been found in mammals. The basic structures of TMPRSSs are similar. The protease domain at the C-terminus is extracellular, the N-terminus is intracellular, and they also have a single transmembrane domain. The difference lies in the backbone region. TMPRSSs have a variety of important physiological functions, and abnormal functions can cause various diseases such as deafness, cancer, anemia, and hypertension.

[0076] In this article, by connecting the polypeptide and the small molecule in the polypeptide-small molecule conjugate, dual-functional and dual-targeting effects can be achieved, successfully targeting the key target sites of two consecutive links in the process of the virus entering the host cell through the membrane fusion pathway. On the basis of maintaining the activity of the first target ligand, the activity of the second target ligand is obtained, and the combined effects of the two target ligands result in enhanced therapeutic effects, or even synergistic effects when they cooperate with each other.

[0077] In this article, the term "derived" means that atoms or groups in a certain original substance (or compound) are treated by at least one of the processes including substitution, removal, condensation, addition, coupling, and conjugation reactions to form a more complex or simpler product with a chemical bond change compared to the original substance.

[0078] In this article, SARS-CoV-2 refers to severe acute respiratory syndrome coronavirus 2, which has genomic variations compared with previously known coronaviruses and is also known as the "novel coronavirus", and it is a positive-sense RNA virus. SARS-CoV-2 is a member of the Coronaviridae family of viruses, which are large, enveloped, single-stranded RNA viruses with a diameter between 65 and 125 nanometers. Under an electron microscope, the virus particles appear roughly spherical and have finger-like extensions (glycoproteins) on their surface, which are usually called spikes.

[0079] In this text, the term "HR2 region" is a key domain in the S2 subunit of the spike (S) protein of the SARS-CoV-2 virus. The S protein is an important protein that mediates the binding of the virus to the host cell receptor and membrane fusion. It consists of two subunits, S1 and S2. The S1 subunit is mainly responsible for recognizing and binding to the receptor (such as ACE2) on the surface of the host cell, while the S2 subunit participates in the fusion process of the virus with the host cell membrane. In the S2 subunit, the HR2 region (heptapeptide repeat 2) and the HR1 region (heptapeptide repeat 1) together participate in the formation of the six-helix bundle (6-HB) structure, which is a key step in the virus membrane fusion process. During the process of the virus infecting the host cell, the S2 subunit of the S protein undergoes conformational changes, and the interaction between HR1 and HR2 is a key factor promoting the fusion of the virus membrane with the host cell membrane. This fusion process is crucial for the virus to enter the host cell and release its genetic material. The polypeptide part of the polypeptide-small molecule conjugate of the present disclosure is derived from the HR2 domain of the S2 subunit of the SARS-CoV-2 spike protein, and this structure can be mimicked as the HR2 domain of the S2 subunit of the SARS-CoV-2 spike protein, so as to achieve binding to the HR1 of SARS-CoV-2, resulting in the failure of the interaction between HR1 and HR2, being unable to promote the fusion of the virus membrane with the host cell membrane, causing the virus to be unable to enter the host cell and release its genetic material, and further inhibiting the infection of the virus to the human body.

[0080] In this text, amino acids are represented using standard three-letter or single-letter coding rules. Specifically, alanine - Ala (A), cysteine - Cys (C), aspartic acid - Asp (D), glutamic acid - Glu (E), phenylalanine - Phe (F), glycine - Gly (G), histidine - His (H), isoleucine - Ile (I), lysine - Lys (K), leucine - Leu (L), methionine - Met (M), asparagine - Asn (N), proline - Pro (P), glutamine - Gln (Q), arginine - Arg (R), serine - Ser (S), threonine - Thr (T), valine - Val (V), tryptophan - Trp (W), tyrosine - Tyr (Y).

[0081] In this text, VD4162 is a macrocyclic inhibitor of serine protease, and its structure is shown as follows:

[0082]

[0083] In some embodiments, the polypeptide-small molecule conjugate structure provided in this text is composed of an organic small molecule transmembrane serine protease 2 inhibitor and an HR2 domain derivative capable of binding to the HR1 domain of the S2 subunit of the SARS-CoV-2 spike protein through polyethylene glycol PEG4, polyethylene glycol PEG12 or polyethylene glycol PEG 24 It is composed of a connection. In some structures, a fragment including Compound I or Compound II is connected to a lysine residue. In this case, the fragment can exist in the form of a side-chain fragment. For example, the polypeptide-small molecule conjugate part includes a fragment of K(Compound I)-CONH2, wherein Compound I within "()" is covalently connected to the adjacent lysine through the side chain of lysine.

[0084] During the process of SARS-CoV-2 entering host cells through the membrane fusion pathway, the receptor-binding domain (RBD) of the spike protein on the virus surface recognizes and binds to the receptor angiotensin-converting enzyme 2 (ACE2) on the host cell surface, enabling the virus to attach to the host cell surface. After the RBD receptor binds to ACE2, the S2' site of the S protein is exposed. Subsequently, host protease type II transmembrane serine protease (TTSPs) cleaves and activates this site to initiate membrane fusion. After membrane fusion, a series of conformational changes occur in the S2 subunit, the fusion peptide FP is exposed and inserted into the target cell membrane, three heptad repeat sequence 1 (HR1) molecules interact to form an elongated trimer, and three heptad repeat sequence 2 molecules (HR2) attach in an antiparallel manner to the hydrophobic groove formed between HR1s, forming a stable six-helix bundle (6-HB) structure. This structure pulls the virus envelope closer to the host cell membrane to cause membrane fusion. Subsequently, the virus genome is released through the fusion pore into the host cell to complete the invasion. This process is continuous. Theoretically, inhibiting any link in this process can prevent the virus from invading host cells. The polypeptide-small molecule conjugate of the present disclosure can target the key sites in two consecutive steps of the process of entering host cells, namely the protease type II transmembrane serine protease (TTSPs) in the first step, which can cleave and activate the S2' site of the S protein and initiate membrane fusion, and the HR1 region in the second step, preventing the formation of a stable 6-HB structure by SARS-CoV-2. Among them, the organic small molecule transmembrane serine protease 2 inhibitor in the polypeptide-small molecule conjugate can inhibit the cleavage and activation of the S2' site of the S protein, and the polypeptide part can bind to the HR1 region to prevent the generation of the 6-HB structure. In this way, the activity of the ligand of the HR1 region can be obtained on the basis of maintaining the activity of the ligand of transmembrane serine protease 2. It has been verified that the cooperation between the two ligands achieves a significantly enhanced therapeutic effect, even reaching a synergistic effect, thereby better preventing SARS-CoV-2 from entering host cells through the membrane fusion pathway, and further preventing and / or treating early infections and transmissions of COVID-19.

[0085] In addition, while retaining the advantages of multi-drug combination or multi-component composite preparations, the polypeptide-small molecule conjugates of the present disclosure can also reduce the interactions between drugs and their associated toxic and side effects, have uniform pharmacokinetic properties, do not have the problems of dosage and ratio in combination drug use, can reduce the dosage and improve the therapeutic effect.

[0086] As used herein, the term "pharmaceutically acceptable salt" includes acid addition salts and base addition salts. Suitable acid addition salts are formed from acids that form non-toxic salts. Examples thereof include, but are not limited to: acetate, adipate, aspartate, benzoate, benzenesulfonate, bicarbonate / carbonate, bisulfate / sulfate, borate, camphorsulfonate, citrate, cyclohexanesulfamate, ethanedisulfonate, formate, fumarate, glucoheptonate, gluconate, glucuronate, hexafluorophosphate, 2-(4-hydroxybenzyl)benzoate, hydrochloride / chloride, hydrobromide / bromide, hydroiodide / iodide, 2-hydroxyethanesulfonate, lactate, malate, maleate, malonate, mesylate, methylsulfate, naphthoate, 2-naphthalenesulfonate, nicotinate, nitrate, oxalate, palmitate, phosphate / hydrogenphosphate / dihydrogenphosphate, pyroglutamate, glucuronate, stearate, salicylate, tannate, tartrate, tosylate, and trifluoroacetate. Suitable base addition salts are formed from bases that form non-toxic salts. Examples thereof include, but are not limited to: aluminum, arginine, calcium, choline, diethylamine, diethanolamine, glycine, lysine, magnesium, meglumine, ethanolamine, potassium, sodium, tromethamine, and zinc salts. Half salts of acids and bases can also be formed, such as half sulfate and half calcium salts. For a review of suitable salts, see Handbook of Pharmaceutical Salts: Properties, Selection and Use by Stahl and Wermuth (Wiley-VCH, 2002).

[0087] Preparation method

[0088] The present disclosure provides a method for preparing the polypeptide-small molecule conjugates described herein, which includes covalently linking the organic small molecule transmembrane serine protease inhibitor described herein with a polypeptide having antiviral activity. In some preferred embodiments, the method for preparing the polypeptide-small molecule conjugates described herein provided by the present disclosure includes covalently coupling the organic small molecule transmembrane serine protease inhibitor with the polypeptide having antiviral activity through a linker.

[0089] In some embodiments, the organic small molecule transmembrane serine protease inhibitor and the polypeptide having antiviral activity can be linked through a click reaction, such as an azide-alkyne cycloaddition reaction.

[0090] In some embodiments, the peptides described herein can be synthesized by sequentially bonding amino acids using solid-phase peptide synthesis techniques. Solid-phase peptide synthesis techniques are well-known and can be practiced proficiently by those skilled in the art. In this method, the synthesis of the peptides in the compounds of the present disclosure can be carried out by sequentially binding the desired amino acid residues one by one to the growing peptide chain according to the general principles of solid-phase methods. These methods are disclosed in a number of references, including Merrifield, R.B., Solid phase synthesis (Nobel lecture). Angew Chem 24:799-810 (1985); and Barany et al., The Peptides, Analysis, Synthesis and Biology, Volume 2, Gross, E. and Meienhofer, J. editors, Academic Press 1-284 (1980). In the synthesis of peptides, the reactive side-chain groups of each amino acid residue can be protected with suitable protecting groups to prevent chemical reactions at that site before the protecting groups are removed. For example, when reacting at the carboxyl group of an amino acid or fragment, the α-amino group is protected with a protecting group (e.g., 9-fluorenylmethoxycarbonyl (Fmoc), tert-butoxycarbonyl (Boc)), and then the α-amino protecting group is selectively removed to allow subsequent reactions at that site. In the preparation, orthogonal protecting groups can be used as appropriate to prepare polypeptides containing side chains, for example, Fmoc-Ser(tBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Arg(pbf)-OH. During the process, any suitable reagent can be used to cleave the compound from the solid phase, for example, a combination of trifluoroacetic acid (TFA), triisopropylsilane (TIS), and water.

[0091] For the collection and purification of the peptide product, those skilled in the art can adopt any known method, for example, precipitating the final product by adding cold ether and collecting it by filtration, and purifying it using, for example, high-pressure chromatography.

[0092] Although the synthesis is mainly described with reference to solid-phase peptide synthesis methods, it should be understood that other synthesis methods can also be used to prepare the compounds of the present invention.

[0093] Pharmaceutical composition, composition

[0094] The present disclosure provides a pharmaceutical composition comprising the polypeptide-small molecule conjugate described herein, and one or more pharmaceutically acceptable excipients. The present disclosure provides a pharmaceutical composition comprising the polypeptide-small molecule conjugate described herein, and one or more pharmaceutically acceptable carriers or excipients.

[0095] The present disclosure provides a composition comprising the polypeptide-small molecule conjugate described herein and other antiviral drugs. The composition achieves a better inhibitory effect on viruses and / or improves the problem of viral drug resistance through the combination of multiple drugs.

[0096] The pharmaceutical compositions and compositions of the present disclosure can be formulated in any manner known in the art, including but not limited to solid, semi-solid or liquid system dosage forms such as tablets, capsules, caplets, suspensions, powders, lyophilized preparations, suppositories, eye drops, skin patches, orally soluble preparations, sprays, aerosols, etc.

[0097] The pharmaceutical compositions and compositions of the present disclosure can be immediate release and / or modified release formulations, including delayed release, sustained release, pulsed release, controlled release, targeted release and programmed release formulations.

[0098] As used herein, "pharmaceutically acceptable excipients" refer to components that are non-toxic to the subject other than the active ingredient in the pharmaceutical composition. Pharmaceutically acceptable excipients include but are not limited to excipients (such as diluents, carriers, etc.) and additives (such as stabilizers, preservatives, solubilizers, buffers, etc.). Excipients may include polyvinylpyrrolidone, gelatin, hydroxypropyl cellulose (HPC), gum arabic, polyethylene glycol, mannitol, sodium chloride and sodium citrate. For injectable preparations or other liquid dosage forms for administration, preferably water containing at least one or more buffering components may be used, and stabilizers, preservatives and solubilizers may also be employed. For solid dosage forms for administration, any one of a variety of thickeners, fillers, extenders and carrier additives may be used, such as starch, sugar, cellulose derivatives, fatty acids, etc. For topical dosage forms for administration, any one of a variety of creams, ointments, gels, lotions, etc. may be used. For most pharmaceutical preparations, the inactive ingredients may constitute a larger part of the preparation by weight or volume. For pharmaceutical preparations, it also encompasses the use of any one of a variety of metered release, sustained release or extended release formulations and additives such that the dosage can be formulated to deliver the compounds of the present disclosure over a period of time.

[0099] The term "pharmaceutically acceptable carrier" includes pharmaceutically acceptable materials, compositions, or vehicles such as liquid or solid fillers, diluents, excipients, solvents, or encapsulating materials involved in carrying or transporting the peptides of the present disclosure within a subject or carrying or transporting the peptides of the present disclosure to a subject such that they can perform their intended functions. Each salt or carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject. Some examples of materials that can be used as pharmaceutically acceptable carriers include: sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethyl cellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; diols such as propylene glycol; polyols such as glycerol, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethanol; phosphate buffer solutions; diluents; granulating agents; lubricants; binders; disintegrants; wetting agents; emulsifiers; coloring agents; demolding agents; coating agents; sweetening agents; flavoring agents; perfuming agents; preservatives; antioxidants; plasticizers; gelling agents; thickening agents; hardening agents; setting agents; suspending agents; surfactants; humectants; carriers; stabilizers; and other non-toxic compatible substances used in pharmaceutical formulations, or any combination thereof.

[0100] The polypeptide-small molecule conjugates, pharmaceutical compositions, and compositions of the present disclosure can be administered by means such as topical administration, systemic administration, intralesional administration, mucosal administration, intrabuccal administration, oral administration, transdermal administration, inhalation administration, intranasal administration, central nervous system administration, transpulmonary administration, intravesical administration, urethral administration, vaginal administration, intravenous, subcutaneous, intramuscular, intraperitoneal injection, etc. The excipients in the pharmaceutical compositions and compositions of the present disclosure are adapted to their administration routes.

[0101] In some embodiments, the polypeptide-small molecule conjugates, pharmaceutical compositions, and compositions of the present disclosure can be delivered orally, for example, by tablets or capsules. The polypeptide-small molecule conjugates, pharmaceutical compositions, and compositions can be packaged in enteric protectants, preferably such that the compound is not released until the tablet or capsule is transported to the stomach and optionally further transported to a portion of the small intestine.

[0102] In some embodiments, the polypeptide-small molecule conjugates, pharmaceutical compositions, and compositions of the present disclosure can be administered by injection. Pharmaceutical forms suitable for injection include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that it can be administered by syringe. The form must be stable under the conditions of preparation and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, or liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils. Therapeutic administration can also be achieved through sustained-release injection formulations, such as formulations that allow subcutaneous injection, including: nanospheres / microspheres, liposomes, emulsions, gels, insoluble salts, or suspensions.

[0103] In some embodiments, the polypeptide-small molecule conjugates, pharmaceutical compositions, and compositions of the present disclosure can be administered intranasally. The polypeptide-small molecule conjugates, pharmaceutical compositions, and compositions can be in the form of an aqueous solution, such as a solution including saline, citrate, or other common excipients or preservatives. They can also be in the form of dry formulations or powders.

[0104] Use

[0105] The present disclosure provides the use of the polypeptide-small molecule conjugates, pharmaceutical compositions, or compositions provided herein in the preparation of drugs for preventing and / or treating viral infections. In some embodiments, the viral infection is a coronavirus infection. In some preferred embodiments, the viral infection is a novel severe acute respiratory syndrome coronavirus infection.

[0106] The organic small molecule transmembrane serine protease inhibitor and the polypeptide portion in the polypeptide-small molecule conjugate contribute to the key target sites of two consecutive links in the process of the virus entering host cells through the membrane fusion pathway. On the basis of maintaining the activity of the first target ligand, the activity of the second target ligand is obtained, and the two target ligands cooperate to achieve enhanced efficacy, and even play a synergistic effect.

[0107] The present disclosure provides a method for treating or preventing viral infections, including administering to a subject a prophylactically or therapeutically effective amount of the polypeptide-small molecule conjugates, pharmaceutical compositions, or compositions described herein.

[0108] The present disclosure provides a method for in vitro inhibiting viral infections for non-therapeutic purposes, by adding an effective amount of the polypeptide-small molecule conjugates, pharmaceutical compositions, or compositions described herein to a test sample to inhibit the test sample from being infected by the virus.

[0109] As used herein, the term "subject" includes animals, such as vertebrates, preferably mammals, such as dogs, cats, pigs, cows, sheep, horses, rodents (e.g., mice, rats or guinea pigs) or primates (e.g., gorillas, chimpanzees and humans).

[0110] As used herein, the term "treatment" refers to alleviating or ameliorating a disease or disorder (i.e., slowing or arresting the development of the disease or at least one clinical symptom); or alleviating or ameliorating at least one physical parameter or biomarker associated with the disease or disorder.

[0111] As used herein, an "effective amount" is an amount sufficient to elicit a desired therapeutic, prophylactic, inhibitory or other effect by any of the above-described means or by any other means known in the art, an amount that results in a benefit or achieves an effect as compared to a corresponding subject not receiving such amount. The amount is low enough within the scope of reasonable medical judgment to avoid serious side effects. The effective amount of the polypeptide-small molecule conjugate, pharmaceutical composition or composition herein will vary with factors such as the selected polypeptide-small molecule conjugate, pharmaceutical composition or composition; the route of administration; the severity of the disease being treated; the age, body size, weight and physical condition of the patient being treated; the medical history of the patient being treated; the duration of treatment; the nature of concurrent treatments; the desired therapeutic effect, etc., but can still be determined in a conventional manner by those skilled in the art.

[0112] The various embodiments and preferences disclosed above can be combined with each other (as long as they are not inherently contradictory to each other), and the various embodiments thus formed are regarded as part of the disclosure of this application.

[0113] The technical solutions of the present disclosure will be described more clearly and explicitly below by way of examples. It should be understood that these examples are only for illustrative purposes and are by no means intended to limit the protection scope of the present disclosure.

[0114] Examples

[0115] Hereinafter, the examples of the present application will be described. The examples described below are exemplary and are only used to explain the present application and should not be construed as a limitation of the present application. For those not specified in the examples regarding specific techniques or conditions, the techniques or conditions described in the literature in the art or according to the product specification are followed. For reagents or instruments not specified as to the manufacturer, they are all conventional products that can be obtained commercially.

[0116] The Chinese explanations of the abbreviations or English full names used in this application are shown in Table 1 below:

[0117] Table 1. Comparison of Chinese explanations of abbreviations or English full names used

[0118]

[0119] The resin raw material used in the examples is Rink Amide resin, and the substitution constant is 0.53 mmol / g.

[0120] Example 1: Synthesis of Compounds I (ie Camostat-A) and II (ie Camostat-B) The synthesis routes of small molecule compounds Camostat-A and Camostat-B are as follows:

[0121]

[0122] Reagents and reaction conditions: (a) triethylamine, acetonitrile, reflux at 0°C to 90°C; (b) DCC, pyridine, 0°C to room temperature; (c) TFA, DCM; (d) HATU, DIEA, 2-Propynylamine, room temperature; (e) HATU, DIEA, 2-Propynylamine, room temperature.

[0123] (1) Synthesis of 2-(tert-butoxy)-2-oxoethyl-2-(4-hydroxyphenyl)acetate (Camostat-1)

[0124] Weigh 20 grams (131.44 mmol) of p-hydroxyphenylacetic acid and 30.6 grams (157.73 mmol) of tert-butyl bromoacetate into a reaction bottle, add 320 milliliters of acetonitrile and stir to dissolve, add 21.9 milliliters (157.73 mmol) of triethylamine dropwise while stirring under ice bath conditions. After the addition is complete, heat to reflux at 85°C for 8 hours. After the reaction is completed, cool the reaction solution to room temperature, adjust the pH to about 7 with 1 mol hydrochloric acid solution, extract the reaction solution three times with ethyl acetate (EA), combine the organic phases, wash the organic phases three times with saturated sodium chloride water, dry over anhydrous sodium sulfate, and concentrate under reduced pressure. Purify by column chromatography (PE:EA=5:1) to obtain 23.74 grams of white solid with a yield of 67.83%. Figure 1 , Figure 6 and Figure 7 As shown, the relevant data are as follows: 1H NMR (600 MHz, DMSO-d6), δ9.31 (s, 1H), 7.07 (d, J = 8.5 Hz, 2H), 6.70 (d, J = 8.4 Hz, 2H), 4.52 (s, 2H), 3.60 (s, 2H), 1.38 (s, 9H). 13C NMR (151 MHz, DMSO-d6), δ171.13, 166.72, 156.31, 130.36, 123.97, 115.13, 81.52, 61.22, 39.02, 27.62. ESI-MS, m / z: for C 14 H 18The O5 was calculated to be 266.12, and 289.11 (M+Na)+ was found.

[0125] (2) Synthesis of 4-{2-[2-(tert-Butoxy)-2-oxoethoxy]-2-oxoethyl}phenyl 4-guanidinobenzoate (Camostat-2)

[0126] Under nitrogen protection, 13.36 g (61.95 mmol) of 4-guanidinobenzoic acid hydrochloride was added to the reaction flask, dissolved in 650 mL of anhydrous pyridine. 13.94 g (67.58 mmol) of N,N'-dicyclohexylcarbodiimide (DCC) was added under ice bath conditions, and the mixture was stirred for 30 minutes. Then the ice bath was removed, and stirring was continued at room temperature for 1 h. 15 g (56.32 mmol) of Camostat-1 was added and the reaction was stirred for 4 h. The reaction solution was filtered, and the precipitate was washed several times with a small amount of anhydrous pyridine. The filtrate was collected, concentrated under reduced pressure, and a small amount of acetone was added to remove the residual pyridine solvent. It was diluted with anhydrous ether, filtered again, the filtrate was collected, and the ether was allowed to evaporate. The crude product was purified by column chromatography (DCM:MeOH = 5:1) to obtain 7.72 g of a pale yellow solid with a yield of 32.1%. As Figure 2 、 Figure 8 and Figure 9 shown, the relevant data are as follows: 1H NMR (600 MHz, DMSO-d6), δ 8.16 (d, J = 8.7 Hz, 2H), 7.94 (s, 4H), 7.41 (dd, J = 28.8, 8.6 Hz, 4H), 7.24 (d, J = 8.5 Hz, 2H), 4.57 (s, 2H), 3.82 (s, 2H), 1.40 (s, 9H). 13C NMR (151 MHz, DMSO-d6), δ 171.15, 167.12, 164.47, 156.20, 149.97, 141.70, 132.23, 131.91, 131.09, 125.79, 123.10, 122.24, 82.08, 61.85, 49.05, 28.11. ESI-MS, m / z: For C 22 H 25 N3O6 calculated to be 427.17, found 428.18 (M+H)+.

[0127] (3) Synthesis of 2-{{2-{4-[(4-Guanidinobenzoyl)oxy]phenyl}acetoxy}}acetic acid (Camostat-3)

[0128] Weigh 7.2 g (16.85 mmol) of Camostat-2 into a reaction flask, dissolve it by stirring with 200 mL of dichloromethane (DCM), then add 100 mL of trifluoroacetic acid (TFA) continuously. The solution becomes clear and is stirred at room temperature for 2.5 h. The reaction solution is concentrated under reduced pressure, and TFA is removed by freeze-drying. The crude product is purified by column chromatography (DCM:MeOH = 3:1) to obtain 9.5 g of a white solid. The yield is 91.2%. As Figure 3 , Figure 10 and Figure 11 shown, the relevant data are as follows: 1H NMR (600 MHz, DMSO-d6) δ 13.09 (s, 1H), 8.18–8.14 (m, 2H), 7.88 (s, 4H), 7.46–7.37 (m, 4H), 7.26–7.21 (m, 2H), 4.60 (s, 2H), 3.83 (s, 2H). 13C NMR (151 MHz, Methanol-d4), δ 171.33, 169.99, 164.32, 156.33, 150.01, 140.45, 131.89, 131.51, 130.36, 127.44, 123.54, 121.32, 60.64, 39.22. ESI-MS, m / z: For C 18 H 17 N3O6 calculated as 371.11, found 372.12 (M + H)+.

[0129] (4) Synthesis of 4-{2-oxo-2-[2-oxo-2-(prop-2-yn-1-ylamino)ethoxy]ethyl}phenyl 4-guanidinobenzoate (Camostat-A, i.e., Compound I)

[0130] Weigh 3 g (8.08 mmol) of Camostat-3 into a reaction flask, dissolve it by stirring with 60 mL of N,N-dimethylformamide (DMF), then add 3.38 g (8.89 mmol) of N,N-diisopropylethylamine (DIEA) and 1.15 g (8.89 mmol) of 2-(7-azabenzotriazol)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU). After stirring at room temperature for 2 h, add 0.49 g of propargylamine and stir at room temperature for 18 h. The reaction solution is diluted with 60 mL of H2O, extracted three times with EA, the organic phases are combined, washed three times with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated. Purification by column chromatography (DCM:MeOH = 5:1) gives 1.93 g of a pale yellow oil, and the yield is 58.4%. As Figure 4 , Figure 12 and Figure 13As shown below, the relevant data are as follows: 1H NMR (600 MHz, DMSO-d6) δ 8.52 (t, J = 5.4 Hz, 1H), 8.16 (d, J = 8.7 Hz, 2H), 7.69 (s, 4H), 7.41 (dd, J = 18.8, 8.6 Hz, 4H), 7.23 (d, J = 8.5 Hz, 2H), 4.53 (s, 2H), 3.90 (dd, J = 5.5, 2.5 Hz, 2H), 3.84 (s, 2H), 3.14 (t, J = 2.5 Hz, 1H). 13C NMR (151 MHz, Methanol-d4) δ 171.07, 168.25, 164.33, 156.31, 150.05, 140.41, 131.86, 131.53, 130.38, 123.58, 121.38, 78.85, 70.93, 62.26, 39.15, 27.84. ESI-MS, m / z: For C 21 H 20 N4O5 calculated as 408.14, found 409.16 (M + H)+.

[0131] (5) Synthesis of 4-{{2-{2-[methyl(prop-2-yn-1-yl)amino]-2-oxoethoxy}-2-oxoethyl}}phenyl 4-guanidinobenzoate (Camostat-B, i.e., Compound II)

[0132] Weigh 5 g (13.47 mmol) of Camostat-3 and dissolve it in 80 mL of DMF. Add 1.92 g (8.89 mmol) of DIEA and 5.64 g (8.89 mmol) of HATU. After stirring at room temperature for 2 h, add 1.02 g of propargylamine and stir at room temperature for 18 h. Dilute the reaction solution with 80 mL of H2O, extract it three times with EA, combine the organic phases, wash the organic phases three times with saturated sodium chloride solution, dry over anhydrous sodium sulfate, and concentrate. Purify by column chromatography (DCM:MeOH = 5:1) to obtain 3.06 g of a yellow oil, with a yield of 55.8%. As Figure 5 、 Figure 14 and Figure 15As shown below, the relevant data are as follows: 1H NMR (600 MHz, Acetone-d6) δ 8.26 (d, J = 8.5 Hz, 2H), 7.64 (d, J = 8.5 Hz, 2H), 7.56 (s, 2H), 7.46 (dd, J = 11.1, 8.6 Hz, 2H), 7.24 (t, J = 8.6 Hz, 2H), 4.97 - 4.66 (m, 2H), 4.53 - 4.15 (m, 2H), 3.91 - 3.78 (m, 2H), 3.78 - 3.65 (m, 1H), 3.11 - 2.93 (m, 3H), 2.76 (d, J = 27.2 Hz, 1H). 13C NMR (151 MHz, Methanol-d4) δ 171.39, 167.41, 164.31, 156.33, 150.01, 140.44, 131.94, 131.51, 130.42, 123.54, 121.31, 77.51, 72.18, 61.33, 39.23, 35.88, 32.08. ESI-MS, m / z: For C 22 H 22 N4O5 calculated as 422.16, found 423.17 (M + H)+.

[0133] Example 2: Synthesis of IPB19N1-CA

[0134] All polypeptides were completed by the standard Fmoc-protected solid-phase peptide synthesis method. The peptide chain was elongated from the carboxyl terminus (C-terminus) to the amino terminus (N-terminus). The first step of solid-phase peptide synthesis was to covalently bind the first amino acid at the C-terminus of the target peptide chain to the reaction site of the insoluble polymer resin, then remove the amino protection of the first amino acid, and carry out an amide condensation reaction with the carboxyl group of the next activated amino acid. The steps of "washing the peptide resin - removing amino protection - washing the peptide resin - condensation" were repeated until the peptide chain connection was completed.

[0135] In this study, Rink-Amide resin was used as the solid-phase carrier. The amount of polypeptide synthesized in a single reaction was 0.5 mmol (the resin loading was 0.53 mmol / g, and the resin usage was 0.94 g). The resin was washed successively with DMF for 2 times, methanol for 2 times, and DCM for 2 times, each time for 2 minutes. HOBT / HBTU / DIEA (3 eq / 3 eq / 3 eq, reaction for 2 h) or HOBT / DIC (3 eq / 6 eq, reaction for 4 h) was selected as the condensation system; DMF was used as the solvent, and 20% piperidine (piperidine / DMF, v / v = 1 / 4) was used as the Fmoc deprotection reagent; acetic anhydride / DIEA (v / v = 1 / 1) was used as the acetylation reagent; 5% hydrazine hydrate (hydrazine hydrate / DMF, v / v = 1 / 19) was used as the deprotection reagent for the Dde protecting group; ninhydrin solution (0.5 g dissolved in 5 mL of methanol solution) / anhydrous pyridine (v / v = 1 / 1) was used as the detection reagent for reaction monitoring. The metal bath was heated to about 100 °C and heated for 5 minutes. When there were exposed amino groups, the resin was blue or dark brown, and when there were no exposed amino groups, the resin was colorless and transparent.

[0136] The specific operation is as follows: First, the weighed resin was placed in a reactor, and the resin was swollen with 20 mL of a DCM and DMF mixture (v / v = 7 / 3) for 1 hour, then drained. The resin was washed successively with DMF for 2 times, MeOH for 2 times, and DCM for 2 times, each time for 2 minutes. Then, 20% piperidine was used to remove the Fmoc protection of the amino group on the resin, exposing the reaction site. First, 20 mL of the deprotection reagent was added and reacted for 5 minutes, then drained. Then, 20 mL of the deprotection reagent was added again and reacted for 25 minutes, then drained. The resin was washed, and the completion of deprotection was detected with the ninhydrin / pyridine reagent. Next, the first amino acid of the target peptide was coupled. Three equivalents of the amino acid and the condensing agent were weighed and dissolved for activation, and then added to the reactor, and stirred at room temperature for 4 h. For the polypeptide with an azide modification at the C-terminus, the first amino acid introduced onto the resin was lysine (Lys) with a Dde-protected side-chain amino group. After the reaction was stopped, the reaction solution was drained, the resin was washed, the completion of the reaction was detected with the ninhydrin / pyridine reagent, then deprotected with piperidine, washed, and the next amino acid was coupled. After each amino acid was coupled, the steps of washing the peptide resin, deprotecting, washing, and then condensing the next amino acid were repeated until the peptide sequence was synthesized.

[0137] Azide modification at the N-terminus of the peptide sequence can be achieved by directly condensing azidoacetic acid with deprotected PEG4 through carboxyl and amino groups. After peptide synthesis, the peptide chain needs to be cleaved from the resin. Preparation of the cleavage solution: TFA / anisole / m-cresol / water / ethanedithiol are prepared in a volume ratio of 8.25 / 0.5 / 0.5 / 0.5 / 0.25. Generally, 10 mL of cleavage solution is prepared for 0.1 mmol of peptide resin. The operation is as follows: After peptide synthesis, dry the peptide resin with ether. Weigh 0.1 mmol of peptide resin each time and transfer it to a 500 mL eggplant flask. Add 10 mL of cleavage solution under ice bath conditions, stir for 30 minutes, then remove the ice bath, and continue to stir at room temperature for 4 h. Then add 400 mL of ice-cold anhydrous ether and stir vigorously for 30 minutes. Observe the precipitation of white precipitate. Filter off the reaction solution with a G4 funnel and wash the filter cake repeatedly with a small amount of anhydrous ether. Collect the filtrate and freeze-dry it to obtain the crude peptide. Purify it by HPLC preparative to obtain a pure peptide with a purity > 90%.

[0138] Couple the azide-modified polypeptide inhibitor and the small molecule inhibitor with a terminal alkyne modification using a copper(I)-catalyzed azide-alkyne cycloaddition reaction (CuAAC). The catalytic system used is copper sulfate pentahydrate (CuSO4·5H2O) and the reducing agent sodium L-ascorbate, with a mixed solution of tert-butanol and dd H2O (double-distilled water) as the solvent. Weigh 10 mg of the azide-modified polypeptide PEG4-SVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIK-CONH2 with a purity greater than 90% and 2.4-fold equivalents of the small molecule compound I (Camostat-A) with a terminal alkyne modification each time. After dissolving them separately with 3 mL of tert-butanol / dd H2O solution (v / v = 1 / 1), mix the above two solutions. Weigh 10-fold equivalents of CuSO4·5H2O and 50-fold equivalents of sodium L-ascorbate, dissolve and mix them with 4 mL of dd H2O to obtain an orange-yellow solution. Add this solution to the above mixture, vortex at room temperature for 5 minutes, and then monitor the reaction by analytical RP-HPLC. Purify it by preparative RP-HPLC to obtain a pure peptide with a purity > 95%, as Figure 16 shown. Put the purified pure peptide into a mass spectrometer for analysis to obtain the mass spectrometry results as Figure 17 shown.

[0139] Example 3: Synthesis of Compound IPB19N1-CB

[0140] All polypeptides were synthesized by the standard Fmoc-protected solid-phase peptide synthesis method. The peptide chain was extended from the carboxyl terminus (C-terminus) to the amino terminus (N-terminus). The first step of solid-phase peptide synthesis was to covalently bind the first amino acid at the C-terminus of the target peptide chain to the reaction site of the insoluble polymer resin, then remove the amino protection of the first amino acid, and carry out an amide condensation reaction with the carboxyl group of the next activated amino acid. The steps of "washing the peptide resin - removing the amino protection - washing the peptide resin - condensation" were repeated until the peptide chain connection was completed.

[0141] In this study, Rink-Amide resin was used as the solid-phase carrier. The amount of polypeptide synthesized per batch was 0.5 mmol (the resin loading was 0.53 mmol / g, and the resin usage was 0.94 g). The resin was washed successively with DMF for 2 times, methanol for 2 times, and DCM for 2 times, 2 minutes each time. HOBT / HBTU / DIE (3 eq / 3 eq / 3 eq, reaction for 2 h) or HOBT / DIC (3 eq / 6 eq, reaction for 4 h) was selected as the condensation system; DMF was used as the solvent, and 20% piperidine (piperidine / DMF, v / v = 1 / 4) was used as the Fmoc deprotection reagent; acetic anhydride / DIEA (v / v = 1 / 1) was used as the acetylation reagent; 5% hydrazine hydrate (hydrazine hydrate / DMF, v / v = 1 / 19) was used as the deprotection reagent for the Dde protecting group; ninhydrin solution (0.5 g dissolved in 5 mL of methanol solution) / anhydrous pyridine (v / v = 1 / 1) was used as the detection reagent for reaction monitoring. The metal bath was heated to about 100 °C and heated for 5 minutes. When there were exposed amino groups, the resin was blue or dark brown, and when there were no exposed amino groups, the resin was colorless and transparent.

[0142] The specific operations are as follows: First, put the weighed resin into the reactor, swell the resin with 20 mL of a DCM and DMF mixture (v / v = 7 / 3) for 1 hour, then drain it, and wash the resin successively with DMF twice, MeOH twice, and DCM twice, each time for 2 minutes. Then, use 20% piperidine to remove the Fmoc protection of the amino group on the resin, exposing the reaction site. First, add 20 mL of the deprotection reagent and react for 5 minutes, then drain it. Then add another 20 mL of the deprotection reagent and react for 25 minutes, drain it again, wash the resin, and detect the completion of deprotection with ninhydrin / pyridine reagent. Next, connect the first amino acid of the target peptide. Weigh three equivalents of the amino acid and the condensing agent, dissolve and activate them, then add them to the reactor, and stir at room temperature for 4 h. For the polypeptide with an azide modification at the C-terminus, the first amino acid introduced onto the resin is lysine (Lys) with a Dde-protected side-chain amino group. After stopping the reaction, drain the reaction solution, wash the resin, detect the completion of the reaction with ninhydrin / pyridine reagent, then deprotect with piperidine, wash, and connect the next amino acid. After connecting each amino acid, the steps of washing the peptide resin, deprotecting, washing, and then condensing the next amino acid are required until the peptide sequence synthesis is completed.

[0143] For the azide modification at the N-terminus of the peptide sequence, it can be directly completed by condensing azidoacetic acid and deprotected PEG4 for the carboxylic acid and amino groups. After the polypeptide synthesis is completed, the peptide chain needs to be cleaved from the resin. Preparation of the cleavage solution: TFA / anisole / m-cresol / water / ethanedithiol are prepared in a volume ratio of 8.25 / 0.5 / 0.5 / 0.5 / 0.25. Generally, 10 mL of the cleavage solution needs to be prepared for 0.1 mmol of peptide resin. The operation is as follows: After the polypeptide synthesis is completed, dry the peptide resin with ether. Each time, weigh 0.1 mmol of the peptide resin and transfer it to a 500 mL eggplant-shaped flask. Under ice bath conditions, add 10 mL of the cleavage solution, stir for 30 minutes, then remove the ice bath, continue to stir at room temperature for 4 h, add 400 mL of ice-cold anhydrous ether, and stir vigorously for 30 minutes. Observe the precipitation of white precipitate. Filter off the reaction solution with a G4 funnel, and wash the filter cake repeatedly with a small amount of anhydrous ether. Collect the filtrate and freeze-dry it to obtain the crude polypeptide. Purify it by HPLC preparation to obtain a pure peptide with a purity > 90%.

[0144] The azide-modified polypeptide inhibitor and the small molecule inhibitor modified with a terminal alkyne group were coupled using a copper-catalyzed azide-alkyne cycloaddition reaction (CuAAC). The catalytic system used was copper sulfate pentahydrate (CuSO4·5H2O) and the reducing agent sodium L-ascorbate, with a mixed solution of tert-butanol and dd H2O (double-distilled water) as the solvent. Each time, 10 mg of the azide-modified polypeptide PEG4-SVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIK-CONH2 with a purity greater than 90% and 2.4 equivalents of the small molecule compound II (Camostat-B) modified with a terminal alkyne group were weighed. After being fully dissolved in 3 mL of tert-butanol / dd H2O solution (v / v = 1 / 1) respectively, the above two solutions were mixed. 10 equivalents of CuSO4·5H2O and 50 equivalents of sodium L-ascorbate were weighed and dissolved and mixed evenly in 4 mL of dd H2O to obtain an orange-yellow solution. This solution was added to the above mixture. After vortexing at room temperature for 5 minutes, the reaction was monitored by analytical RP-HPLC. The pure peptide with a purity > 95% was purified by preparative RP-HPLC, as Figure 18 shown. The purified pure peptide was put into a mass spectrometer for analysis, and the mass spectrometry results were obtained as Figure 19 shown.

[0145] Example 4: Synthesis of Compound IPB19N2-CA

[0146] All polypeptides were completed using the standard Fmoc-protected solid-phase peptide synthesis method. The peptide chain was extended from the carboxyl terminus (C-terminus) to the amino terminus (N-terminus). The first step of solid-phase peptide synthesis was to covalently bind the first amino acid at the C-terminus of the target peptide chain to the reaction site of the insoluble polymer resin, and then remove the amino protection of the first amino acid and carry out an amide condensation reaction with the carboxyl group of the next activated amino acid. The steps of "washing the peptide resin - removing amino protection - washing the peptide resin - condensation" were repeated until the peptide chain connection was completed.

[0147] In this study, Rink-Amide resin was used as the solid-phase carrier. The amount of polypeptide synthesized in a single reaction was 0.5 mmol (the resin loading was 0.53 mmol / g, and the resin usage was 0.94 g). The resin was washed successively with DMF for 2 times, methanol for 2 times, and DCM for 2 times, each time for 2 minutes. HOBT / HBTU / DIEA (3 eq / 3 eq / 3 eq, reaction for 2 h) or HOBT / DIC (3 eq / 6 eq, reaction for 4 h) was selected as the condensation system; DMF was used as the solvent, and 20% piperidine (piperidine / DMF, v / v = 1 / 4) was used as the Fmoc deprotection reagent; acetic anhydride / DIEA (v / v = 1 / 1) was used as the acetylation reagent; 5% hydrazine hydrate (hydrazine hydrate / DMF, v / v = 1 / 19) was used as the deprotection reagent for the Dde protecting group; ninhydrin solution (0.5 g dissolved in 5 mL of methanol solution) / anhydrous pyridine (v / v = 1 / 1) was used as the detection reagent for reaction monitoring. The metal bath was heated to about 100 °C and heated for 5 minutes. When there were exposed amino groups, the resin was blue or dark brown, and when there were no exposed amino groups, the resin was colorless and transparent.

[0148] The specific operation is as follows: First, the weighed resin was placed in a reactor and swollen with a 20 mL mixture of DCM and DMF (v / v = 7 / 3) for 1 hour, then dried by suction. The resin was washed successively with DMF for 2 times, MeOH for 2 times, and DCM for 2 times, each time for 2 minutes. Then, 20% piperidine was used to remove the Fmoc protection of the amino group on the resin, exposing the reaction site. First, 20 mL of the deprotection reagent was added and reacted for 5 minutes, then dried by suction. Then, 20 mL of the deprotection reagent was added again and reacted for 25 minutes, then dried by suction. The resin was washed, and the completion of deprotection was detected with the ninhydrin / pyridine reagent. Next, the first amino acid of the target peptide was coupled. Three equivalents of the amino acid and the condensing agent were weighed, dissolved and activated, and then added to the reactor, and stirred at room temperature for 4 h. For the polypeptide with an azide modification at the C-terminus, the first amino acid introduced onto the resin was lysine (Lys) with a Dde-protected side-chain amino group. After the reaction was stopped, the reaction solution was dried by suction, the resin was washed, the completion of the reaction was detected with the ninhydrin / pyridine reagent, then deprotected with piperidine, washed, and the next amino acid was coupled. After each amino acid was coupled, it was necessary to go through the steps of washing the peptide resin, deprotection, washing, and then condensing the next amino acid until the peptide sequence synthesis was completed.

[0149] For the azide modification at the N-terminus of the peptide sequence, azidoacetic acid can be directly coupled with the deprotected PEG 12The condensation of carboxylic acid and amino group can be completed. After the polypeptide synthesis is completed, the peptide chain needs to be cleaved from the resin. Preparation of the cleavage solution: TFA / anisole / m-cresol / water / ethanedithiol are prepared at a volume ratio of 8.25 / 0.5 / 0.5 / 0.5 / 0.25. Generally, 10 mL of cleavage solution needs to be prepared for 0.1 mmol of peptide resin. The operation is as follows: After the polypeptide synthesis is completed, the peptide resin is dried with ether. Each time, 0.1 mmol of peptide resin is weighed and transferred to a 500 mL eggplant flask. 10 mL of cleavage solution is added under ice bath conditions, and after stirring for 30 minutes, the ice bath is removed, and stirring is continued at room temperature for 4 h. Then, 400 mL of ice-cold anhydrous ether is added and stirred vigorously for 30 minutes, and it is observed that a white precipitate precipitates. The reaction solution is removed by suction filtration with a G4 funnel, and the filter cake is washed repeatedly with a small amount of anhydrous ether. The filtrate is collected and freeze-dried to obtain the crude polypeptide. The pure peptide with a purity >90% is obtained by preparative HPLC purification.

[0150] The azide-modified polypeptide inhibitor and the small molecule inhibitor modified with a terminal alkyne group are coupled using a copper(I)-catalyzed terminal azide-alkyne cycloaddition reaction (CuAAC). The catalytic system used is copper sulfate pentahydrate (CuSO4·5H2O) and the reducing agent sodium L-ascorbate, and a mixed solution of tert-butanol and dd H2O (double-distilled water) is used as the solvent. Each time, 10 mg of the azide-modified polypeptide PEG 12 -SVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIK-CONH2 with a purity greater than 90% and 2.4-fold equivalents of the small molecule compound I (Camostat-A) modified with a terminal alkyne group are separately dissolved in 3 mL of tert-butanol / dd H2O solution (v / v = 1 / 1) sufficiently. Then, the above two solutions are mixed. 10-fold equivalents of CuSO4·5H2O and 50-fold equivalents of sodium L-ascorbate are weighed and dissolved in 4 mL of dd H2O and mixed evenly to obtain an orange-yellow solution. This solution is added to the above mixture. After vortexing at room temperature for 5 minutes, the reaction is monitored by analytical RP-HPLC. The pure peptide with a purity >95% is obtained by preparative RP-HPLC purification, as Figure 20 shown. The purified pure peptide is put into a mass spectrometer for analysis to obtain the mass spectrometry results as Figure 21 shown.

[0151] Example 5: Synthesis of Compound IPB19C2-CB

[0152] All polypeptides were synthesized using the standard Fmoc-protected solid-phase peptide synthesis method. The peptide chain was elongated from the carboxyl terminus (C-terminus) to the amino terminus (N-terminus). The first step of solid-phase peptide synthesis was to covalently bind the first amino acid at the C-terminus of the target peptide chain to the reaction site of the insoluble polymer resin, then remove the amino protection of the first amino acid, and carry out an amide condensation reaction with the carboxyl group of the next activated amino acid. The steps of "washing the peptide resin - removing amino protection - washing the peptide resin - condensation" were repeated until the peptide chain was connected.

[0153] In this study, Rink-Amide resin was used as the solid-phase carrier. The amount of polypeptide synthesized per batch was 0.5 mmol (the resin loading was 0.53 mmol / g, and the resin usage was 0.94 g). The resin was washed successively with DMF for 2 times, methanol for 2 times, and DCM for 2 times, each time for 2 minutes. HOBT / HBTU / DIEA (3 eq / 3 eq / 3 eq, reaction for 2 h) or HOBT / DIC (3 eq / 6 eq, reaction for 4 h) was selected as the condensation system; DMF was used as the solvent, and 20% piperidine (piperidine / DMF, v / v = 1 / 4) was used as the Fmoc deprotection reagent; acetic anhydride / DIE (v / v = 1 / 1) was used as the acetylation reagent; 5% hydrazine hydrate (hydrazine hydrate / DMF, v / v = 1 / 19) was used as the deprotection reagent for the Dde protecting group; ninhydrin solution (0.5 g dissolved in 5 mL of methanol solution) / anhydrous pyridine (v / v = 1 / 1) was used as the detection reagent for reaction monitoring. The metal bath was heated to about 100 °C and heated for 5 minutes. When there were exposed amino groups, the resin was blue or dark brown, and when there were no exposed amino groups, the resin was colorless and transparent.

[0154] The specific operations are as follows: First, put the weighed resin into the reactor, swell the resin with 20 mL of a mixed solution of DCM and DMF (v / v = 7 / 3) for 1 hour, and then drain it. Wash the resin successively with DMF twice, MeOH twice, and DCM twice, each time for 2 minutes. Then, use 20% piperidine to remove the Fmoc protection of the amino group on the resin, exposing the reaction site. First, add 20 mL of the deprotection reagent and react for 5 minutes, then drain it. Add another 20 mL of the deprotection reagent and react for 25 minutes, then drain it. Wash the resin, and detect the completion of deprotection with ninhydrin / pyridine reagent. Next, connect the first amino acid of the target peptide. Weigh three equivalents of the amino acid and the condensing agent, dissolve and activate them, and then add them to the reactor. Stir and react at room temperature for 4 h. For the polypeptide with an azide modification at the C-terminus, the first amino acid introduced onto the resin is lysine (Lys) with a Dde-protected side-chain amino group. After stopping the reaction, drain the reaction solution, wash the resin, detect the completion of the reaction with ninhydrin / pyridine reagent, then carry out deprotection with piperidine, wash it, and connect the next amino acid. After connecting each amino acid, it is necessary to go through the steps of washing the peptide resin, deprotecting, washing, and then condensing the next amino acid until the peptide sequence synthesis is completed.

[0155] For the azide modification at the N-terminus of the peptide sequence, the condensation of carboxylic acid and amino group can be directly completed by reacting azidoacetic acid with the deprotected PEG. 12 After the polypeptide synthesis is completed, the peptide chain needs to be cleaved from the resin. Preparation of the cleavage solution: Prepare TFA / anisole / m-cresol / water / ethanedithiol in a volume ratio of 8.25 / 0.5 / 0.5 / 0.5 / 0.25. Generally, 10 mL of the cleavage solution is prepared for 0.1 mmol of peptide resin. The operation is as follows: After the polypeptide synthesis is completed, dry the peptide resin with ether. Each time, weigh 0.1 mmol of the peptide resin and transfer it to a 500 mL eggplant flask. Add 10 mL of the cleavage solution under ice bath conditions, stir for 30 minutes, then remove the ice bath, and continue to stir at room temperature for 4 h. Then, add 400 mL of ice-cold anhydrous ether and stir vigorously for 30 minutes. Observe the precipitation of white precipitate. Filter the reaction solution with a G4 funnel, and wash the filter cake repeatedly with a small amount of anhydrous ether. Collect the filtrate and freeze-dry it to obtain the crude polypeptide. Purify it by HPLC preparation to obtain a pure peptide with a purity > 90%.

[0156] Use the copper-catalyzed azide-alkyne cycloaddition reaction (CuAAC) to couple the polypeptide inhibitor with an azide modification and the small molecule inhibitor with a terminal alkyne modification. The catalytic system used is copper sulfate pentahydrate (CuSO4·5H2O) and the reducing agent sodium L-ascorbate, and a mixed solution of tert-butanol and dd H2O (double-distilled water) is used as the solvent. Each time, weigh 10 mg of the polypeptide Ac-SVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIK-PEG with an azide modification and a purity greater than 90%. 12-K-CONH2 and 2.4 equivalents of the small molecule compound II modified with terminal alkynyl group (Camostat-B) were each fully dissolved in 3 mL of tert-butanol / ddH2O solution (v / v = 1 / 1), and then the above two solutions were mixed. 10 equivalents of CuSO4·5H2O and 50 equivalents of sodium L-ascorbate were weighed and dissolved in 4 mL of ddH2O and mixed well to obtain an orange-yellow solution. This solution was added to the above mixture, vortexed at room temperature for 5 minutes, and the reaction was monitored by analytical RP-HPLC. The pure peptide with a purity > 95% was purified by preparative RP-HPLC, as shown in Figure 22 as follows. The purified pure peptide was placed in a mass spectrometer for analysis, and the mass spectrometry results were obtained as shown in Figure 23 as follows.

[0157] Example 6: Anti-SARS-CoV-2 pseudovirus infection experiment

[0158] To detect the inhibitory activity of the polypeptide against the infection of coronavirus PsV (pseudovirus, the preparation method refers to Yang P, Yang Y, Wu Y, Huang C, Ding Y, Wang X, Wang S. An optimized and robust SARS-CoV-2 pseudovirus system for viral entry research. J Virol Methods. 2021, 295:114221.), the target cells (Caco-2 cells, American Type Culture Collection) were placed in a 96-well plate at a density of 1×10 4 cells per well one day before infection. PsV was mixed with an equal volume of the polypeptide, polypeptide-small molecule conjugate or small molecule, and serially diluted with PBS at 37 °C for 30 minutes. This mixture was transferred to Huh-7 cells. The medium was changed after 12 hours and the cells were cultured for another 48 hours. Then, the cells were washed with PBS, lysed with a lysis reagent (Promega), and the relative light units (RLU) were detected by using a luciferase assay kit. The results are shown in Table 2. The preparation method of the polypeptide-small molecule conjugate in Table 2 refers to Examples 2-5.

[0159] Table 2. Anti-viral infection results

[0160]

[0161]

[0162] According to the above steps and material information, a series of polypeptide-small molecule conjugates are obtained by conjugating two alkynyl-modified forms of camostat to the N-terminus and C-terminus of the peptide sequence of peptide IPB19 derived from the HR2 region of the SARS-CoV-2 spike protein S2 subunit. The conjugation steps are referred to Figure 24 . The polypeptide-small molecule conjugates in Table 2 showed significant inhibitory activity in Caco-2 (TMPRSS2+) cells. The inhibitory activity was increased by 4 to 18.5 times compared to its parental peptide IPB19 and by 7.7 to 35 times compared to its parental small molecule inhibitor camostat. Among them, the four conjugates IPB19N1-CA, IPB19N1-CB, IPB19N2-CA and IPB19C2-CB had the best inhibitory activity, and the IC 50 values were 0.169 ± 0.093 μM, 0.164 ± 0.071 μM, 0.131 ± 0.068 μM and 0.163 ± 0.102 μM, respectively. The results showed that the designed polypeptide-small molecule derivatives or conjugates had significantly improved inhibitory activity compared to the corresponding polypeptides or small molecules alone, revealing a strong synergistic effect between the two target ligands in the conjugate.

Claims

1. A polypeptide-small molecule conjugate or a prodrug, tautomer, optical isomer, geometric isomer, solvate or pharmaceutically acceptable salt thereof, wherein the polypeptide-small molecule conjugate comprises an organic small molecule transmembrane serine protease inhibitor and a polypeptide moiety having antiviral activity.

2. The polypeptide-small molecule conjugate according to claim 1, wherein the organic small molecule transmembrane serine protease inhibitor and the polypeptide moiety having antiviral activity are covalently coupled via a linker, preferably, the linker comprises at least one of a linker and lysine.

3. The polypeptide-small molecule conjugate according to claim 1 or 2, which comprises a structure represented by formula I, formula II or formula III: α-P-Z-K(X)-β, α-X-Z-P-β, α-P–K(Z–X)-β, Formula I Formula II Formula III Among them, α is selected from amino, acetyl, maleoyl, succinyl, tert-butoxycarbonyl, benzyloxycarbonyl or dansyl; β is selected from amido, carboxyl or carboxyl derivative; K represents lysine; P represents a polypeptide moiety having antiviral activity; Z represents a linker; X represents an organic small molecule transmembrane serine protease inhibitor; "-" indicates a covalent connection relationship between two molecules; the fragment X or Z-X in "()" indicates that it is connected to K through the side chain of K.

4. The polypeptide-small molecule conjugate according to any one of claims 1-3, wherein the polypeptide moiety is derived from the HR2 domain of the S2 subunit of the SARS-CoV-2 spike protein.

5. The polypeptide-small molecule conjugate according to claim 4, wherein the polypeptide moiety is selected from any one of the following (a)-(d): (a) SVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIK; (b) ISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL; (c) A functional variant of SVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIK containing 0-7 conservative amino acid substitutions; (d) A functional variant of ISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL containing 0-7 conservative amino acid substitutions.

6. The polypeptide-small molecule conjugate according to any one of claims 1-5, wherein the linker is a flexible linker. Preferably, the linker has the following structure: (PEG) n or (PEG) n -(CH2) m , where n is any integer selected from 1 to 35, m is any integer selected from 1 to 10, and "-" indicates a covalent connection relationship between two molecules.

7. The polypeptide-small molecule conjugate according to any one of claims 1-6, wherein the organic small molecule transmembrane serine protease inhibitor comprises at least one of organic small molecule transmembrane serine protease 2 inhibitors, preferably, the organic small molecule transmembrane serine protease 2 inhibitor is selected from: camostat, camostat mesylate, VD4162, phenylmethylsulfonyl fluoride, 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride and benzamidine hydrochloride.

8. The polypeptide-small molecule conjugate according to any one of claims 1-6, wherein the organic small molecule transmembrane serine protease 2 inhibition comprises at least one of Compound I and Compound II, and Compound I has the structure of Compound II has the structure of 9. The polypeptide-small molecule conjugate according to any one of claims 1-8, wherein the organic small molecule transmembrane serine protease inhibitor is linked to the polypeptide moiety by a click reaction.

10. The polypeptide-small molecule conjugate according to claim 9, wherein the organic small molecule transmembrane serine protease inhibitor comprises an alkynyl group, and the lysine in the polypeptide-small molecule conjugate comprises an azide moiety, and the alkynyl group is conjugated to the azide moiety by a click reaction; or the organic small molecule transmembrane serine protease inhibitor comprises an azide moiety, and the lysine in the polypeptide-small molecule conjugate comprises an alkynyl group, and the alkynyl group is conjugated to the azide moiety by a click reaction.

11. The polypeptide-small molecule conjugate according to claim 10, wherein the azide moiety is selected from any one of azidoacetic acid, azidobenzene, biotin-azide, epoxide-azide, aldehyde-azide, alkyne-azide, azide-acrylate, azidopropionic acid, 2-azidopropionic acid, azidobutyric acid, and azido-polylysine.

12. The polypeptide-small molecule conjugate according to any one of claims 1 to 11, wherein the structure of the polypeptide-small molecule conjugate comprises one or more of the following: Compound I-PEG4-SVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIK-CONH2; Compound II-PEG4-SVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIK-CONH2; Compound I-PEG 12 -SVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIK-CONH2; Compound II-PEG 12 -SVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIK-CONH2; Compound I-PEG 24 -SVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIK-CONH2; Compound II-PEG 24 -SVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIK-CONH2; Ac-SVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIK-PEG4-K(Compound I)-CONH2; Ac-SVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIK-PEG4-K(Compound II)-CONH2; Ac-SVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIK-PEG 12 -K (Compound I)-CONH2; Ac-SVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIK-PEG 12 -K (Compound II)-CONH2; Ac-SVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIK-PEG 24 -K (Compound I)-CONH2; Ac-SVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIK-PEG 24 -K (Compound II)-CONH2; wherein "-" indicates a covalent connection relationship between two molecules; the compound in "()" is linked to K through the side chain of K. Compound I has the structure of Compound II has the structure of 13. The method for preparing the polypeptide-small molecule conjugate according to any one of claims 1 to 12, comprising covalently linking the organic small molecule transmembrane serine protease inhibitor to the polypeptide with antiviral activity. Preferably, it comprises covalently coupling the organic small molecule transmembrane serine protease inhibitor to the polypeptide moiety with antiviral activity through a linker.

14. A pharmaceutical composition comprising the polypeptide-small molecule conjugate according to any one of claims 1 to 12, and one or more pharmaceutically acceptable excipients or carriers.

15. A composition comprising the polypeptide-small molecule conjugate according to any one of claims 1 to 12, and other antiviral drugs.

16. Use of the polypeptide-small molecule conjugate according to any one of claims 1 to 12, the pharmaceutical composition according to claim 14, or the composition according to claim 15 in the preparation of a drug for preventing and / or treating viral infections.

17. The application according to claim 16, wherein the viral infection is a coronavirus infection, preferably a novel severe acute respiratory syndrome coronavirus infection.

18. A method for in vitro inhibiting viral infection for non-therapeutic purposes, by adding an effective amount of the polypeptide-small molecule conjugate according to any one of claims 1 to 12, the pharmaceutical composition according to claim 14, or the composition according to claim 15 to a test sample.

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