WO2022156620A1 - Broad-spectrum coronavirus membrane fusion inhibitor and pharmaceutical use thereof - Google Patents

Abstract

The present invention relates to the field of medicine synthesis. Disclosed is a broad-spectrum coronavirus membrane fusion inhibitor. The broad-spectrum coronavirus membrane fusion inhibitor disclosed by the present invention can be used for preparing a pharmaceutical composition for preventing and treating diseases caused by coronavirus. Further disclosed is use of the pharmaceutical composition for preventing and treating diseases caused by coronavirus.

Description

A kind of broad-spectrum coronavirus membrane fusion inhibitor and medicinal use thereof

This application claims the priority of the Chinese patent application filed on January 19, 2021 with the application number 202110070935.7 and the invention titled "A broad-spectrum coronavirus membrane fusion inhibitor and its medicinal use", the entire contents of which are Incorporated herein by reference.

technical field

The invention relates to the field of medicine, in particular to a broad-spectrum coronavirus membrane fusion inhibitor and its medicinal use.

Background technique

Coronavirus (CoV) is a single-stranded positive-stranded RNA virus with an envelope, which is divided into four genera: α, β, γ and δ. Among them, α and β CoV only infect mammals, while γ and δ CoV mainly infect birds. . Seven species of CoV are currently known to infect humans (HCoV), including HCoV-229E and HCoV-NL63 of the alpha genus, HCoV-OC43, CoV-HKU1, SARS-CoV, MERS-CoV of the beta genus and the recently emerged 2019 novel coronavirus SARS -CoV-2. The first 4 HCoVs are common global epidemic pathogens that usually cause only common cold symptoms, accounting for about 10% to 30% of upper respiratory tract infections in adults, but can still cause severe or even severe cases in children, the elderly and immunocompromised patients. deadly diseases; SARS-CoV, MERS-CoV and SARS-CoV-2 are highly pathogenic pathogens that can cause severe lung disease with high fatality rates.

The scientific research team of the present invention has been committed to the research and development of virus membrane fusion inhibitor drugs, and has developed a series of polypeptide membrane fusion inhibitors with strong inhibitory activity against SARS-CoV-2, and they can also effectively inhibit SARS-CoV-2 For details, see Chinese patent CN202010181328.3. Since the purpose of the invention provides lead compounds for the further development of highly pathogenic HCoV therapeutic drugs, the purpose of the present invention is to further develop a broad-spectrum, highly active anti-coronavirus virus membrane on the basis of the above-mentioned patent. fusion inhibitors to meet clinical needs.

SUMMARY OF THE INVENTION

In view of this, the present invention provides a novel coronavirus membrane fusion inhibitor and use thereof.

In order to achieve the above object, the present invention first provides a compound whose structure is shown in formula I or a pharmaceutically acceptable salt, solvate, chelate, non-covalent complex, and a prodrug based on the compound. one or more of:

Ac-Ser-Val-Val-Asn-Ile-Gln-Lys-Glu-Ile-Asp-Arg-Leu-Asn-Glu-Val-Ala-Lys-Asn-Leu-Asn-Glu-Ser-Leu-Ile- Asp-Leu-Gln-Glu-Leu-Gly-Lys-Tyr-Glu-Gln-Tyr-Ile-AA1-AA2(R)-AA3

Formula I

AA1 in formula I includes (( _PEGn1 ( _CH2 ) _n2CO ) _n3 ) _n4- ,

in:

n1 includes an integer from 1 to 30;

n2 includes an integer from 1 to 5;

n3 includes an integer from 1 to 5;

n4 includes an integer from 1 to 5.

AA2 in formula I is selected from Lys, Dap, Orn, Dab or Dah;

AA in _formula I is selected from NH or OH;

R in formula I is selected from cholesteryl 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, or 8-cholesteryl octanoic acid.

In the second aspect, the present invention also provides one or more of the compounds or their pharmaceutically acceptable salts, solvates, chelates, non-covalent complexes, and prodrugs based on the compounds Application in the preparation of broad-spectrum coronavirus membrane fusion inhibitor.

In a third aspect, the present invention also provides a broad-spectrum coronavirus membrane fusion inhibitor, including the compound or a pharmaceutically acceptable salt, solvate, chelate, non-covalent complex, and medicines based on the compound One or more of the precursors, and acceptable excipients or auxiliaries.

In a fourth aspect, the present invention also provides one or more of the compounds or their pharmaceutically acceptable salts, solvates, chelates, non-covalent complexes, and prodrugs based on the compounds, And/or the application of the broad-spectrum coronavirus membrane fusion inhibitor in the preparation of a pharmaceutical composition for preventing and/or treating diseases caused by coronavirus.

In some specific embodiments of the present invention, the coronavirus includes, but is not limited to, one or more of SARS-CoV, MERS-CoV, 2019-nCoV, or common HCoV.

In some specific embodiments of the present invention, the common HCoV includes, but is not limited to, one or more of HCoV-229E, HCoV-OC43 or HCoV-NL63.

In the fifth aspect, the present invention also provides a medicine, including one of the compound or a pharmaceutically acceptable salt, solvate, chelate, non-covalent complex, prodrug based on the compound, or Multiple, and/or the broad-spectrum coronavirus membrane fusion inhibitor, and pharmaceutically acceptable excipients.

In a sixth aspect, the present invention also provides a drug combination, including the drug and any other active ingredients. In the present invention, "any other active ingredient" is not limited, any active ingredient can be combined with the compound provided by the present invention or its pharmaceutically acceptable salt, solvate, chelate, non-covalent complex, based on the compound One or more of the prodrugs on the basis, and/or the combined use of the broad-spectrum coronavirus membrane fusion inhibitor, are all within the protection scope of the present invention.

In a seventh aspect, the present invention also provides one or more of the compounds or their pharmaceutically acceptable salts, solvates, chelates, non-covalent complexes, and prodrugs based on the compounds, The application of the broad-spectrum coronavirus membrane fusion inhibitor, the medicine, and/or the medicine combination in the prevention and/or treatment of diseases caused by coronavirus.

In an eighth aspect, the present invention also provides a method for preventing and/or treating diseases caused by coronavirus, administering the compound or a pharmaceutically acceptable salt, solvate, chelate, non-covalent complex, based on the compound Based on one or more of the prodrugs, the broad-spectrum coronavirus membrane fusion inhibitor, the drug, and/or the drug combination.

More contents involved in the present invention are described in detail below, or some of them can also be realized in the embodiments of the present invention.

Unless otherwise indicated, the amounts used herein to indicate the amounts of the various components and reaction conditions can be interpreted as "approximately" or "approximately" in any case. Correspondingly, unless otherwise specified, the numerical parameters quoted in the following and in the claims are approximate parameters, and different numerical parameters may be obtained due to differences in standard errors under respective experimental conditions.

In this paper, when there is disagreement or doubt between the chemical structural formula and the chemical name of a compound, the chemical structural formula is used to define the compound exactly. The compounds described herein may contain one or more chiral centers, and/or double bonds and the like, and may also exist as stereoisomers, including double bond isomers (such as geometric isomers), optically active enantiomers or diastereomers. Accordingly, any chemical structure within the scope of the description herein, whether in part or in the whole structure containing similar structures above, includes all possible enantiomers and diastereomers of the compound, including Any single stereoisomer (eg, pure geometric isomer, pure enantiomer, or pure diastereomer) and any mixture of these isomers are included. These racemic and stereoisomeric mixtures can also be further resolved into their constituent enantiomers or stereoisomers by those skilled in the art using continuous separation techniques or chiral molecular synthesis methods body.

Compounds of formula I include, but are not limited to, optical isomers, racemates and/or other mixtures of these compounds. In the above case, single enantiomer or diastereomer, such as optical isomer, can be obtained by asymmetric synthesis method or racemate separation method. Resolution of the racemates can be accomplished by various methods, such as conventional recrystallization with a resolution-promoting agent, or by chromatography. In addition, compounds of formula I also include cis and/or trans isomers with double bonds.

The compounds of the present invention include, but are not limited to, the compounds of formula I and all of their various pharmaceutically usable forms. The different pharmaceutically acceptable forms of these compounds include various pharmaceutically acceptable salts, solvates, complexes, chelates, non-covalent complexes, prodrugs based on the aforementioned substances and the aforementioned forms of these compounds. any mixture.

The compound shown in the formula I provided by the present invention has stable properties, is an efficient and broad-spectrum novel coronavirus membrane fusion inhibitor, and is used to prepare a pharmaceutical composition for preventing and treating diseases caused by coronavirus. for the prevention and treatment of diseases caused by the coronavirus.

Description of drawings

Figure 1 α-helix content of the complex between the novel inhibitor and the target sequence polypeptide;

Figure 2. The thermal stability Tm value of the complex of the novel inhibitor and the target sequence polypeptide.

Detailed ways

The invention discloses a broad-spectrum coronavirus membrane fusion inhibitor and its medicinal use, and those skilled in the art can learn from the content of this article and appropriately improve relevant parameters to achieve. It should be particularly pointed out that all similar substitutions and modifications are obvious to those skilled in the art, and they are deemed to be included in the present invention. The method of the present invention has been described by the preferred embodiments, and it is obvious that relevant persons can make changes or appropriate changes and combinations of the compounds and preparation methods described herein without departing from the content, spirit and scope of the present invention, to achieve and Apply the technology of the present invention.

The corresponding Chinese names of the English abbreviations involved in the present invention are shown in the following table:

The preparation method of the polypeptide includes: preparing a peptide resin by a solid-phase polypeptide synthesis method, and then subjecting the peptide resin to acidolysis to obtain a crude product, and finally purifying the crude product to obtain a pure product; wherein the step of preparing the peptide resin by the solid-phase polypeptide synthesis method is to prepare the peptide resin by the solid-phase polypeptide synthesis method. The corresponding protected amino acids or fragments in the following sequences are sequentially connected by solid-phase coupling synthesis to prepare peptide resins:

In the above preparation method, the dosage of the Fmoc-protected amino acid or the protected amino acid fragment is 1.2-6 times of the total moles of the resin charged; preferably 2.5-3.5 times.

In the above preparation method, the substitution value of the carrier resin is 0.2-1.0 mmol/g resin, and the preferred substitution value is 0.3-0.5 mmol/g resin.

As a preferred solution of the present invention, the solid-phase coupling synthesis method is as follows: the protected amino acid-resin obtained in the previous step is subjected to a coupling reaction with the next protected amino acid after removing the Fmoc protecting group. The deprotection time for de-Fmoc protection is 10-60 minutes, preferably 15-25 minutes. The coupling reaction time is 60-300 minutes, preferably 100-140 minutes.

The coupling reaction needs to add a condensation reagent, and the condensation reagent is selected from DIC (N,N-diisopropylcarbodiimide), N,N-dicyclohexylcarbodiimide, benzotriazole hexafluorophosphate -1-yl-oxytripyrrolidinophosphorus, 2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethylurea hexafluorophosphate , benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate or O-benzotriazole-N,N,N',N'-tetramethylurea tetrafluoro One of boronate esters; preferably N,N-diisopropylcarbodiimide. The molar amount of the condensation reagent is 1.2-6 times, preferably 2.5-3.5 times, the total moles of amino groups in the amino resin.

The coupling reaction needs to add an activating reagent, and the activating reagent is selected from 1-hydroxybenzotriazole or N-hydroxy-7-azabenzotriazole, preferably 1-hydroxybenzotriazole. The dosage of the activating agent is 1.2-6 times, preferably 2.5-3.5 times, the total moles of amino groups in the amino resin.

As a preferred solution of the present invention, the reagent for removing Fmoc protection is a PIP/DMF (piperidine/N,N-dimethylformamide) mixed solution, and the mixed solution contains piperidine in an amount of 10-30% (V ). The dosage of the de-Fmoc protecting reagent is 5-15 mL per gram of amino resin, preferably 8-12 mL per gram of amino resin.

Preferably, the peptide resin is subjected to acid hydrolysis while removing the resin and the side chain protecting group to obtain a crude product:

Further preferably, the acid hydrolyzing agent used during the acidolysis of the peptide resin is a mixed solvent of trifluoroacetic acid (TFA), 1,2-ethanedithiol (EDT) and water, and the volume ratio of the mixed solvent is: TFA It is 80-95%, EDT is 1-10%, and the balance is water.

More preferably, the volume ratio of the mixed solvent is: TFA is 89-91%, EDT is 4-6%, and the balance is water. The optimal volume ratio of the mixed solvent is: TFA is 90%, EDT is 5%, and the balance is water.

The dosage of the acid hydrolyzing agent is 4-15 mL of acid hydrolyzing agent per gram of peptide resin; preferably, 7-10 mL of acid hydrolyzing agent is required per gram of peptide resin.

The cleavage time using the acid hydrolyzing agent is 1 to 6 hours at room temperature, preferably 3 to 4 hours.

Further, the crude product is purified by high performance liquid chromatography and freeze-dried to obtain pure product, and the concrete method is:

Take the crude product, add water and stir, adjust the pH value to dissolve completely, the solution is filtered with a 0.45 μm mixed microporous membrane, and purified for subsequent use;

High performance liquid chromatography was used for purification. The chromatographic packing for purification was 10 μm reversed-phase C18, the mobile phase system was 0.1% TFA/water solution-0.1% TFA/acetonitrile solution, and the flow rate of the 77mm*250mm column was 90mL/min. Gradient system elution, cyclic injection purification, take the crude product solution and load it into the chromatographic column, start the mobile phase elution, collect the main peak and evaporate the acetonitrile to obtain the purified intermediate concentrate;

Take the purified intermediate concentrate and filter it with a 0.45 μm membrane filter for later use;

High performance liquid chromatography was used for salt exchange, the mobile phase system was 1% acetic acid/water solution-acetonitrile, the chromatographic packing for purification was 10μm reversed-phase C18, and the flow rate of the 77mm*250mm column was 90mL/min (according to different specifications. chromatographic column, adjust the corresponding flow rate); adopt gradient elution, cyclic sample loading method, load the sample into the chromatographic column, start the mobile phase elution, collect the spectrum, observe the change of absorbance, collect the main peak of salt change and use the analytical liquid phase to detect Purity, combined with the main peak solution of exchanging salt, concentrated under reduced pressure to obtain pure acetic acid aqueous solution, and obtained pure product after freeze-drying.

The raw materials and reagents used in a broad-spectrum coronavirus membrane fusion inhibitor provided by the present invention and its pharmaceutical use can be purchased from the market.

Below in conjunction with embodiment, the present invention is further elaborated:

Example 1 Synthesis of Polypeptide

1. Synthesis of peptide resin

Using Rink Amide BHHA resin as the carrier resin, through de-Fmoc protection and coupling reaction, the peptide resin is prepared by coupling with the corresponding protected amino acid sequence of the polypeptide amino acid sequence in turn.

(1) Access to the first protected amino acid in the main chain

Take 0.03mol of the first protected amino acid and 0.03mol of HOBt, dissolve with an appropriate amount of DMF; take another 0.03mol of DIC, slowly add it to the DMF solution of the protected amino acid under stirring, and stir and react at room temperature for 30 minutes to obtain the activated protection Amino acid solution, ready for use.

Take 0.01 mol of Rink amide MBHA resin (substitution value is about 0.4 mmol/g), use 20% PIP/DMF solution to deprotect for 25 minutes, wash and filter to obtain the resin without Fmoc.

The activated first protected amino acid solution is added to the resin from which Fmoc has been removed, and the coupling reaction is carried out for 60-300 minutes, filtered and washed to obtain a resin containing one protected amino acid.

(2) Access to other protected amino acids in the main chain

Using the same method as the above-mentioned access to the first protected amino acid of the main chain, the other corresponding protected amino acids mentioned above are connected in turn,

A resin containing main chain amino acids is obtained.

(3) Access to the side chain

Take 0.03mol cholesteryl succinate monoester and 0.03mol HOBt, dissolve with an appropriate amount of DMF; take another 0.03mol DIC, slowly add it to the protected amino acid DMF solution under stirring, and stir and react at room temperature for 30 minutes.

Take 2.5mmol of tetrakistriphenylphosphine palladium and 25mmol of phenylsilane, dissolve with an appropriate amount of dichloromethane, enter into the resin containing the main chain amino acid, stir and deprotect for 4 hours, filter and wash, and obtain the resin de-Allocated for subsequent use.

The activated cholesteryl succinate monoester solution is added to the resin from which Alloc has been removed, the coupling reaction is performed for 60-300 minutes, and the peptide resin is obtained by filtering, washing and drying.

2. Preparation of crude product

Take the above peptide resin, add a cleavage reagent (cleavage reagent 10mL/g resin) with a volume ratio of TFA:water:EDT=95:5:5, stir well, stir at room temperature for 3 hours, filter the reaction mixture with a sand core funnel, collect The filtrate and resin were washed 3 times with a small amount of TFA, the filtrate was combined, concentrated under reduced pressure, precipitated with anhydrous ether, washed with anhydrous ether for 3 times, and the crude product was obtained as an off-white powder.

3. Preparation of pure product

Take the above crude product, add water and stir to dissolve, the solution is filtered with a 0.45 μm mixed microporous membrane, and purified for use. High performance liquid chromatography was used for purification. The chromatographic packing for purification was 10 μm reversed-phase C18, the mobile phase system was 0.1% TFA/water solution-0.1% TFA/acetonitrile solution, and the flow rate of the 30mm*250mm column was 20mL/min. Gradient system elution, cyclic injection purification, take the crude product solution and load it into the chromatographic column, start the mobile phase elution, collect the main peak and evaporate the acetonitrile to obtain the purified intermediate concentrate;

The concentrated solution of the purified intermediate was filtered with a 0.45 μm filter membrane for use, and the salt was exchanged by high performance liquid chromatography. The mobile phase system was 1% acetic acid/water solution-acetonitrile, and the chromatographic packing for purification was 10 μm reversed-phase C18, 30mm*250mm The flow rate of the chromatographic column is 20mL/min (the corresponding flow rate can be adjusted according to the chromatographic column of different specifications); the gradient elution, cyclic sample loading method is adopted, the sample is loaded into the chromatographic column, the mobile phase elution is started, the spectrum is collected, and the observation Changes in absorbance, collect the main peak of changing salt and check the purity with analytical liquid phase, combine the main peak solution of changing salt, concentrate under reduced pressure to obtain pure acetic acid aqueous solution, and freeze-dry to obtain pure product.

The following lipopeptide compounds were synthesized using the methods described above:

Table 1

At the same time, the control compounds for research were synthesized:

Table 2

Example 2 Inhibitory effect on viral infection

1. Experimental materials and methods

For the plasmid expressing the S protein of SARS-CoV-2 virus (defined as pCoV2-S), see the paper published by the inventor (Zhu et al.Cross-reactive neutralization of SARS-CoV-2 by serum antibodies from recovered SARS1 patients and immunized animals.Sci Adv.2020;6(45):eabc9999 and Zhu et al.Design of Potent Membrane Fusion Inhibitors Against SARS-CoV-2, an Emerging Coronavirus with High Fusogenic Activity.J Virol.2020;94(14):00653 -20). The HIV backbone plasmid pNL4-3.luc.RE was provided by the National Institutes of Health AIDS Reagents and References Project (Cat. No. 3418); 293T cells were purchased from the American Type Culture Collection (ATCC, Cat. No. CRL-3216); The target cell Huh-7 was purchased from the National Experimental Cell Resource Sharing Service Platform (No. 3111C0001CCC000679); the target cell 293T/ACE2 was prepared and preserved by our laboratory (Cao et al. Potent and persistent antibody responses against the receptor-binding domain of SARS-CoV) spike protein in recovered patients. Virology Journal, 2010, 7:299). The basic steps of antiviral experiments based on S protein pseudovirus are as follows:

(1) Preparation of pseudovirus: 293T cells were co-transfected with pCoV2-S plasmid and pNL4-3.luc.RE at a ratio of 1:1, cultured in a 37°C, 5% _CO2 cell incubator for 48 hours, and the cells containing pseudovirus were collected. The virus supernatant was filtered and stored at -80°C for later use.

(2) Use deionized water or dimethyl sulfoxide (DMSO) to dissolve the polypeptide to be tested and determine the concentration, then dilute the polypeptide to the initial concentration with DMEM medium, and perform 3-fold dilution in the wells of 96 cell culture plates , the final volume of the peptide solution was 50 μL/well, and 3 duplicate wells and 9 dilution gradients were set. DMEM medium without peptide was used as a control.

(3) 50 μL of pseudovirus per well was added to the wells of the drug dilution plate, and then incubated at room temperature for 30 minutes.

(4) Adjust the concentration of pre-cultured 293T/ACE2 cells or Huh-7 cells to 10×10 ⁴ /mL suspension, and add DEAE-dextran to a final concentration of 15 μg/mL, and then add the cells to 96 containing virus In the well plate, 100 μL/well. Incubate for 48 hours in a 37°C, 5% _CO2 cell incubator.

(5) After discarding the supernatant, add 30 μL/well of cell lysate, lyse at room temperature for 15 minutes, add luciferase substrate (Promega), measure relative fluorescence units (RLU) with a microplate luminometer, and calculate Inhibition rate curves and drug median inhibitory concentrations ( _IC50 ) were prepared.

2. Experimental results and analysis

The experimental results show that the new membrane fusion inhibitors IBP24~IPB27 can inhibit the activity of SARS-CoV-2 pseudovirus (PV) infecting 293T/ACE2 or Huh-7 target cells. The average IC50 values of the three experiments are shown in the table below. It can be seen that the new membrane fusion The inhibitor was 12-25 times more active than IPB20.

Table 3 Inhibitory activity against SRAS-CoV-2 cell infection

Example 3 Inhibitory effect on viral cell membrane fusion

1. Experimental materials and methods

In order to further evaluate the anti-SARS-CoV-2 activity of the polypeptide inhibitor, the present invention carried out a DSP-based cell-cell fusion inhibition experiment (methods in Zhu et al. Design of Potent Membrane Fusion Inhibitors Against SARS-CoV-2, an Emerging Coronavirus with High Fusogenic Activity.J Virol.2020;94(14):00653-20), the specific steps are as follows:

(1) Spread the 293T effector cell suspension (1.5×10 ⁴ cells/100 μL/well) in a 96-well plate, and at the same time spread the 293T/ACE2 target cell suspension (15×10 ⁴ cells/mL) on a 10-cm Cell culture dishes were cultured at 37°C and 5% CO ₂ .

(2) After 16 hours of culture, the pCoV2-S plasmid and the pDSP1-7 plasmid were co-transfected into 293T effector cells, while the pDSP8-11 plasmid was transfected into 293T/ACE2 target cells, and then the cells were cultured.

(3) After 24 hours, the polypeptide was serially diluted 3 times in a 96-well plate, and 3 duplicate wells and 9 dilution gradients were set. Diluted polypeptides were added to effector cells and incubated for 1 hour at 37°C, 5% _CO2 cell incubator.

(4) DMEM complete medium was preheated and EnduRen live cell substrate (Promega) was added at a ratio of 1:4000, and then used to resuspend the 293T/ACE2 target cells collected by centrifugation, and the cell concentration was adjusted to 30×10 ⁴ / mL, incubate for 30 min at 37°C, 5% CO ₂ .

(5) 293T/ACE2 target cells were added to 293T effector cells at 100 μL/well, then centrifuged at 400×g for 3 minutes so that the effector cells and target cells were fully contacted, and then the mixed cells were cultured for 2 hours.

(6) Read the luciferase activity (RLU) in a microplate luminometer, and calculate the inhibition rate and IC ₅₀ value.

2. Experimental results and analysis

The experimental results are shown below, indicating that the inhibitory activity of novel membrane fusion inhibitors IBP24-IPB27 on the ability of SARS-CoV-2 S protein to mediate cell-cell fusion is 4-5 times higher than that of IPB20.

Table 4 Inhibitory activity on SRAS-CoV-2 cell fusion

Example 4 Inhibitory effect on other coronaviruses

The preparation and antiviral experimental methods of pseudoviruses (PV) such as SARS-CoV and MERS-CoV are the same as those described in Example 2.

The experimental results show that the novel membrane fusion inhibitor has a good inhibitory effect on SARS-CoV, MERS-CoV, HCoV-NL63 and HCoV-229E human coronavirus at the same time, and the inhibitory activity is 1.5-5 times higher than that of IPB20.

Table 5 Broad-spectrum inhibitory activity against other human coronaviruses

Example 5 Interaction analysis of novel inhibitors and target sequences

Circular dichroism (CD) technique was used to determine the interaction between the inhibitor of the present invention and the target sequence mimetic polypeptide, including the secondary structure (α-helix) and thermal stability of the formed complex. The target sequence mimic peptide used in the experiment is derived from the HR1 sequence of the S2 subunit of the SARS-CoV-2 spike protein, as follows (as shown in SEQ ID No. 7):

Ac-Phe-Asn-Gly-Ile-Gly-Val-Thr-Gln-Asn-Val-Leu-Tyr-Glu-Asn-Gln-Lys-Leu-Ile-Ala-Asn-Gln-Phe-Asn-Ser- Ala-Ile-Gly-Lys-Ile-Gln-Asp-Ser-Leu-Ser-Ser-Thr-Ala-Ser-Ala-Leu-Gly-Lys-Leu-Gln-Asp-Val-Val-Asn-Gln- Asn-Ala-Gln-NH ₂

1. Experimental materials and methods

The inhibitor and the target sequence mimic polypeptide were dissolved in phosphate buffered saline (PBS) at pH 7.2 and mixed, the final concentration of each polypeptide was 10 μM, placed in a water bath at 37°C for 30 minutes, and then the polypeptide solution was moved to the corresponding ratio. In the color dish, use the Jasco spectropolarimeter (model J-815) to scan the change of the molar ellipticity [θ]λ of the peptide solution in the wavelength range of 195-270nm. The typical α-helical structure can show the largest negative peaks at 208nm and 222nm. , subtract the PBS blank control to correct the spectral value, in the calculation process, the peak value is -33000degree.cm2.dmol-1 as the standard of 100% α-helix content, and the polypeptide α-helix is calculated according to the molar ellipticity of the polypeptide solution at 222nm percentage of content. Then, the polypeptide solution was added to the corresponding cuvette for thermal stability detection, and the CD temperature control module was adjusted to scan the variation of [θ]222 of the polypeptide solution with temperature at 20-98°C at a speed of 2°C per minute. The melting curve was smoothed, and the midpoint temperature (Tm) of the thermal dissociation transition was calculated by Origin software to reflect the thermal stability of the spiral.

2. Experimental results and analysis

The experimental results show that: each lipopeptide inhibitor can interact with the target sequence mimic polypeptide to form a complex, which has a typical α-helix structure (see Figure 1). What is more prominent is that the complex has a very high thermal stability Tm value (see Figure 2), indicating that the inhibitor is relatively stable in binding to the target.

A kind of broad-spectrum coronavirus membrane fusion inhibitor provided by the present invention and its medicinal use have been introduced in detail above. The principles and implementations of the present invention are described herein by using specific examples, and the descriptions of the above embodiments are only used to help understand the method and the core idea of the present invention. It should be pointed out that for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can also be made to the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

Claims (10)

1. A compound whose structure is shown in formula I or one or more of its pharmaceutically acceptable salts, solvates, chelates, non-covalent complexes, and prodrugs based on the compound: Ac- Ser-Val-Val-Asn-Ile-Gln-Lys-Glu-Ile-Asp-Arg-Leu-Asn-Glu-Val-Ala-Lys-Asn-Leu-Asn-Glu-Ser-Leu-Ile-Asp- Leu-Gln-Glu-Leu-Gly-Lys-Tyr-Glu-Gln-Tyr-Ile-AA1-AA2(R)-AA3

   Formula I

   AA1 in formula I includes (( _PEGn1 ( _CH2 ) _n2CO ) _n3 ) _n4- ,

   in:

   n1 includes an integer from 1 to 30;

   n2 includes an integer from 1 to 5;

   n3 includes an integer from 1 to 5;

   n4 includes an integer from 1 to 5;

   AA2 in formula I is selected from Lys, Dap, Orn, Dab or Dah;

   AA in _formula I is selected from NH or OH;

   R in formula I is selected from cholesteryl 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, or 8-cholesteryl octanoic acid.
2. One or more of the compound of claim 1 or a pharmaceutically acceptable salt, solvate, chelate, non-covalent complex, and prodrug based on the compound are used in the preparation of broad-spectrum coronary Applications of Viral Membrane Fusion Inhibitors.
3. Broad-spectrum coronavirus membrane fusion inhibitor, is characterized in that, comprises the compound as claimed in claim 1 or its pharmaceutically acceptable salt, solvate, chelate, non-covalent complex, medicine based on this compound basis One or more of the precursors, and acceptable excipients or auxiliaries.
4. The compound of claim 1 or one or more of a pharmaceutically acceptable salt, solvate, chelate, non-covalent complex, prodrug based on the compound, and/or as The application of the broad-spectrum coronavirus membrane fusion inhibitor of claim 3 in the preparation of a pharmaceutical composition for preventing and/or treating diseases caused by coronavirus.
5. The application of claim 4, wherein the coronavirus includes but is not limited to one or more of SARS-CoV, MERS-CoV, 2019-nCoV or common HCoV.
6. The application of claim 5, wherein the common HCoV includes but is not limited to one or more of HCoV-229E, HCoV-OC43 or HCoV-NL63.
7. A medicine, characterized in that it comprises the compound of claim 1 or one of a pharmaceutically acceptable salt, solvate, chelate, non-covalent complex, a prodrug based on the compound, or Multiple, and/or broad-spectrum coronavirus membrane fusion inhibitors as claimed in claim 3, and pharmaceutically acceptable adjuvants.
8. The drug combination is characterized in that it comprises the drug as claimed in claim 7 and any other active ingredients.
9. The compound of claim 1 or one or more of the pharmaceutically acceptable salts, solvates, chelates, non-covalent complexes, and prodrugs based on the compound, as claimed in claim 3 Described broad-spectrum coronavirus membrane fusion inhibitor, the medicine as claimed in claim 7, and/or the application of medicine combination as claimed in claim 8 in preventing and/or treating the disease caused by coronavirus.
10. A method for preventing and/or treating diseases caused by coronavirus, characterized in that, administering the compound according to claim 1 or a pharmaceutically acceptable salt, solvate, chelate, non-covalent complex, based on the compound One or more in the prodrugs above, the broad-spectrum coronavirus membrane fusion inhibitor as claimed in claim 3, the medicine as claimed in claim 7, and/or the drug combination as claimed in claim 8 .

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