WO2021164677A1 - Inhibitor capable of resisting fusion of respiratory syncytial virus - Google Patents

Abstract

Provided is an inhibitor capable of resisting the fusion of respiratory syncytial virus (RSV) and falls within the field of pharmaceutical synthesis. The inhibitor capable of resisting the fusion of respiratory syncytial virus provided by the present invention can be used for treating diseases, such as respiratory syncytial virus pneumonia.

Description

A Kind of Anti-Syncytial Virus Membrane Fusion Inhibitor

This application claims the priority of a Chinese patent application filed with the Chinese Patent Office on February 21, 2020, the application number is 202010108065.3, and the invention title is "an anti-syncytial virus membrane fusion inhibitor", the entire content of which is incorporated by reference In this application.

Technical field

The invention relates to an anti-syncytial virus membrane fusion inhibitor and its use.

Background technique

Human Respiratory Syncytial Virus (RSV) is widely distributed all over the world and is an important viral pathogen causing lower respiratory tract infections. RSV infection can lead to high hospitalization rates and mortality in infants, young children, the elderly, and immunodeficiency populations worldwide. About 70% of infants are infected with RSV within one year of birth. The vast majority of children will be infected within two years after birth. One third of infants and young children who die from acute lower respiratory infections are caused by RSV infection. The World Health Organization (WHO) report believes that about 64 million children worldwide are infected with RSV every year, and nearly 3.5 million children are admitted to hospital because of severe RSV infection, which is 16 times that of influenza. Among them, about 200,000 children under the age of 5 are infected each year. Death due to infection with the RSV virus is a major factor in the hospitalization and death of children worldwide. In the United States, 20% to 25% of infant pneumonia and 50% to 75% of bronchiolitis are caused by RSV. A study data from South Korea showed that the 20-day all-cause mortality rate in patients over 18 years of age with RSV infection was higher than that of influenza (18.4% vs. 6.7%); compared with the seasonal influenza group, the risk of death caused by RSV infection was significantly higher high. In Beijing, 48% of viral pneumonia and 58% of bronchiolitis are caused by RSV (1980-1984); in Guangzhou, 31.4% of pneumonia and bronchiolitis in children are caused by RSV (1973-1986). WHO data also shows that there are nearly 30 million elderly patients infected with RSV worldwide each year, and at least 2 million are severely ill hospitalized patients. Statistics show that RSV infection accounts for 20% of deaths among people over 65. The results of a latest epidemiological survey show that in the UK alone, there are about 487,247 people requiring medical treatment, 17,799 hospitalizations, and 8,482 deaths per RSV epidemic season in adults over 18 years of age. Among them, 65-year-old patients accounted for about the above. 36% of medical treatments, 79% of hospitalizations, and 93% of deaths. China currently has more than 240 million people over the age of 60, all of whom are at high risk of RSV infection. The burden on families and society is huge. Since there is no effective vaccine to prevent RSV infection and effective drugs for treatment of RSV infection in the world, it has brought a great burden to the health care system of all countries in the world.

As early as 1996, the American company Trimeris designed a group of anti-RSV polypeptide membrane fusion inhibitors based on the HRB sequence of RSV. They have strong anti-RSV activity in cell models. The most prominent one is T118, which contains 35 amino acid residues. Subsequent research has developed some peptide RSV fusion inhibitors, but it is difficult to significantly improve the anti-RSV activity, especially relatively short peptides, which often have significantly reduced activity. Currently, there is no peptide RSV fusion inhibitor. Enter the report of clinical trials.

Summary of the invention

The invention provides a new anti-syncytial virus membrane fusion inhibitor and its use.

To achieve the above objective, the present invention first provides a compound represented by structure I, a pharmaceutically acceptable salt, solvate, chelate or non-covalent complex formed by the compound, and a drug based on the compound Precursor, or any mixture of the above forms.

Ac-AA1-Glu-AA3-Val-Asn-Lys-Lys-Ile-Glu-AA10-Ser-Leu-Lys-AA14-Ile-Glu-AA17-Ser-Asp-Lys-AA21-Leu-Glu-AA24- Val-Asn-Lys-AA28-AA29(R1)-AA30(R2)-AA31

Structure I

AA1 is Ile, or Leu;

AA3 is Gln, or Glu;

AA10 is Gln or Glu;

AA14 is Phe, or Lys;

AA17 is Lys, or Glu;

AA21 is Leu, or Lys;

AA24 is Asn, or Glu;

AA28 is Gly or Lys;

AA29 is Lys, or Dap, or Orn, or Dab, or Dah;

AA30 means Cys, or does not exist;

When AA30 is Cys, R1 is H;

When AA30 is not present, R2 is not present;

AA31 is NH ₂ or OH.

R1 in structure I is H, or cholesterol succinate monoester, or 2-cholesterol acetic acid, or 2-cholesterol propionic acid, or 2-cholesterol butanoic acid, or 2-cholesterol isobutyrate, or It is 2-cholesterol valeric acid, or 2-cholesterol isovaleric acid, or 2-cholesterol hexanoic acid, HO ₂ C(CH ₂ ) _n1 CO-(γGlu) _n2 -(PEG _n3 (CH2) _n4 CO) _n5- , Or CH ₃ (CH ₂ ) _n1 CO-(γGlu) _n2 -, or absent;

Wherein: n1 is an integer from 10 to 20;

n2 is an integer from 1 to 5;

n3 is an integer from 1 to 30;

n4 is an integer from 1 to 5;

n5 is an integer from 1 to 5.

R2 in structure I is cholesterol acetate, or cholesterol propionate, or cholesterol butyrate, or cholesterol isobutyrate, or cholesterol valerate, or cholesterol isovalerate, or hexyl Cholesterol ester, or non-existent.

The present invention also provides a pharmaceutical composition comprising the compound according to the present invention, and the pharmaceutical composition provided with the compound of the present invention is used for preparing a medicine for the treatment of diseases.

Preferably, the pharmaceutical composition is used in the preparation of a medicine for treating syncytial virus pneumonia.

More content involved in the present invention is described in detail below, or some can also be experienced in the embodiments of the present invention.

Unless otherwise indicated, the quantities and reaction conditions of different components used in this text can be interpreted as "approximately" or "approximately" in any case. Correspondingly, unless explicitly specified, the digital parameters quoted in the following and in the claims are approximate parameters. Due to the difference in standard errors under respective experimental conditions, different digital parameters may be obtained.

In this article, when the chemical structural formula and chemical name of a compound are in disagreement or doubt, 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 similar structures. There may also be stereoisomers, including double bond isomers (such as geometric isomers), optical rotation Enantiomers or diastereomers. Correspondingly, any chemical structure within the scope described herein, whether part or the entire structure contains the above-mentioned similar structure, includes all possible enantiomers and diastereomers of the compound, including A simple stereoisomer (such as a simple geometric isomer, a simple enantiomer or a simple diastereomer) and any mixture of these isomers. These mixtures of racemic isomers and stereoisomers can be further resolved into their constituent enantiomers or stereoisomers by those skilled in the art using continuous separation techniques or chiral molecule synthesis methods. body.

The compounds of structural formula I include, but are not limited to, optical isomers, racemates and/or other mixtures of these compounds. In the above case, a single enantiomer or diastereomer, such as an optical isomer, can be obtained by asymmetric synthesis or racemate resolution. The resolution of racemates can be achieved by different methods, such as conventional recrystallization with reagents that assist resolution, or chromatographic methods. In addition, the compounds of structural formula I also contain cis and/or trans isomers with double bonds.

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

The above-mentioned prodrug includes the ester or amide derivative of the compound represented by structural formula I contained in the compound.

The compound shown in structure I provided by the present invention has stable properties, is a new type of anti-syncytial virus membrane fusion inhibitor, and can be used for the treatment of syncytial virus pneumonia.

Description of the drawings

In order to explain the embodiments of the present invention or the technical solutions in the prior art more clearly, the following will briefly introduce the drawings that need to be used in the description of the embodiments or the prior art.

Figure 1 shows the inhibitory activity on RSV-EGFP infected target cells;

Figure 2 shows the inhibitory activity on RSV-Luc;

Figure 3 shows the effect on the weight change of RSV-infected mice;

Figure 4 shows the in vivo imaging detection of RSV-infected mice;

Figure 5 shows the statistical analysis of the fluorescence signal of the nasal cavity of RSV-infected mice;

Figure 6 shows the statistical analysis of fluorescence signals in the lungs of RSV-infected mice;

Figure 7 shows the quantitative analysis of RSV virus levels in the lungs of RSV-infected mice by RT-qPCR;

Figure 8 shows the quantitative analysis of RSV replication ability in mouse lung tissue by enzyme-linked immunospot method.

Detailed ways

The invention discloses an anti-syncytial virus membrane fusion inhibitor and its use. Those skilled in the art can learn from the content of this article and appropriately improve the relevant parameters to achieve it. In particular, it should be pointed out that all similar replacements and modifications are obvious to those skilled in the art, and they are all deemed to be included in the present invention. The method of the present invention has been described through preferred embodiments, and the relevant personnel can obviously modify or appropriately change and combine 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 Chinese names corresponding to the English abbreviations involved in the present invention are shown in the following table:

Table 1

Example 1 Preparation of Compound 1

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

The preparation method includes: preparing the peptide resin by solid-phase peptide synthesis, then acid hydrolyzing the peptide resin to obtain the crude product, and finally the crude product is purified to obtain the pure product; wherein the step of preparing the peptide resin by the solid-phase peptide synthesis method is to solidify the peptide resin on the carrier resin. The phase coupling synthesis method sequentially connects the corresponding protected amino acids or fragments in the following sequences to prepare peptide resins:

In the above preparation method, the amount of the Fmoc-protected amino acid or protected amino acid fragment is 1.2-6 times 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: the protected amino acid-resin obtained in the previous step reaction removes the Fmoc protective group and then couples with the next protected amino acid. The deprotection time for 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 requires the addition of a condensation reagent, which is selected from DIC (N,N-diisopropylcarbodiimide), N,N-dicyclohexylcarbodiimide, and benzotriazole hexafluorophosphate -1-yl-oxytripyrrolidinyl phosphorus, 2-(7-aza-1H-benzotriazol-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 boric acid esters; preferably N,N-diisopropylcarbodiimide. The molar amount of the condensation reagent is 1.2-6 times the total moles of amino groups in the amino resin, preferably 2.5-3.5 times.

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 amount of the activating reagent is 1.2-6 times the total moles of amino groups in the amino resin, preferably 2.5-3.5 times.

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 10-30% piperidine (V ). The amount of the de-Fmoc protection reagent is 5-15 mL per gram of amino resin, preferably 8-12 mL per gram of amino resin.

Preferably, the peptide resin undergoes acid hydrolysis to simultaneously remove the resin and side chain protecting groups to obtain a crude product:

More preferably, the acid hydrolyzing agent used in the acid hydrolysis 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 as follows: TFA is 89% to 91%, EDT is 4% to 6%, and the balance is water. Optimally, the 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 an 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 the pure product. The specific method is as follows:

Take the crude product, add water and stir, adjust the pH value to completely dissolve, filter the solution with a 0.45μm mixed microporous membrane, and purify it for use;

Purification was carried out by high performance liquid chromatography. The chromatographic packing used for purification was 10μm reversed-phase C18, the mobile phase system was 0.1% TFA/water solution-0.1% TFA/acetonitrile solution, and the flow rate of 77mm*250mm chromatographic column was 90mL/min. Gradient system elution, cyclic injection purification, take the crude solution and load it on 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 0.45μm filter membrane for later use;

High performance liquid chromatography was used for salt exchange, the mobile phase system was 1% acetic acid/water-acetonitrile, the chromatographic packing used for purification was 10μm reversed-phase C18, the flow rate of 77mm*250mm column was 90mL/min (according to different specifications Chromatographic column, adjust the corresponding flow rate); Adopt gradient elution, cyclic loading method, load the sample on 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 to detect For purity, combine the salt-changing main peak solutions, concentrate under reduced pressure to obtain pure aqueous acetic acid solution, and obtain pure product after freeze-drying.

1. Synthesis of peptide resin

Using Rink Amide BHHA resin as the carrier resin, through de-Fmoc protection and coupling reactions, sequentially coupled with the protected amino acids shown in the table below to prepare peptide resins. The protected amino acids corresponding to the protected amino acids used in this example are as follows:

Table 2

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

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

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 Fmoc-free resin.

The activated first protected amino acid solution is added to the Fmoc-free resin, the coupling reaction is 60-300 minutes, and the resin is filtered and washed to obtain a resin containing 1 protected amino acid.

(2) Access to the 2nd to 30th protected amino acids of the main chain

Using the same method as described above for accessing the first protected amino acid of the main chain, sequentially accessing the corresponding second to 30th protected amino acids above to obtain a peptide resin.

2. Preparation of crude product

Take the above peptide resin, add the lysis reagent (lysis reagent 10mL/g resin) with a volume ratio of TFA:water:EDT=95:5:5, stir evenly, stir at room temperature for 3 hours, and filter the reaction mixture using a sand core funnel to collect The filtrate and the resin were washed 3 times with a small amount of TFA. The filtrate was combined and concentrated under reduced pressure. Anhydrous ether was added for precipitation, and the precipitate was washed with anhydrous ether for 3 times, and the off-white powder was drained to obtain a crude product.

3. Preparation of pure product

Take the above-mentioned crude product, add water and stir to dissolve, and filter the solution with a 0.45μm mixed microporous membrane for purification for use. Purification was carried out by high performance liquid chromatography. The chromatographic packing used for purification was 10μm 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 solution and load it on the chromatographic column, start the mobile phase elution, collect the main peak and evaporate the acetonitrile to obtain the purified intermediate concentrate;

The purified intermediate concentrate was filtered with a 0.45μm filter membrane for use, and the salt was replaced by high performance liquid chromatography. The mobile phase system was 1% acetic acid/water solution-acetonitrile, and the purification chromatographic packing was 10μm 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 different specifications of the chromatographic column); the gradient elution is adopted, and the cyclic loading method is adopted. For the change of absorbance, collect the main peak of salt exchange and check the purity with the analytical liquid phase. Combine the main peak of salt exchange solution and concentrate under reduced pressure to obtain a pure aqueous acetic acid solution, which is freeze-dried to obtain 7.6 g of pure product with a purity of 97.5%. The total yield is 22.1%. The molecular weight is 3442.2 (100% M+H).

Example 2 Preparation of Compound 2

Ac-Ile-Glu-Glu-Val-Asn-Lys-Lys-Ile-Glu-Glu-Ser-Leu-Lys-Lys-Ile-Glu-Glu-Ser-Asp-Lys-Lys-Leu-Glu-Glu- Val-Asn-Lys-Lys-Lys-NH2

The preparation method is the same as in Example 1. The protected amino acids used are as follows:

table 3

8.6 g of pure product was obtained, the purity was 97.9%, and the total yield was 24.4%. The molecular weight is 3527.1 (100% M+H).

Example 3 Preparation of Compound 3

Ac-Ile-Glu-Gln-Val-Asn-Lys-Lys-Ile-Glu-Gln-Ser-Leu-Lys-Phe-Ile-Glu-Lys-Ser-Asp-Lys-Leu-Leu-Glu-Asn- Val-Asn-Lys-Gly-Lys-Cys (cholesterol acetate)-NH ₂

1. Synthesis of peptide resin

The method is the same as in Example 1. The protected amino acids used are as follows:

Table 4

2. Preparation of crude product

Take the above peptide resin, add the lysis reagent (lysis reagent 10mL/g resin) with a volume ratio of TFA:water:EDT=95:5:5, stir evenly, stir at room temperature for 3 hours, and filter the reaction mixture using a sand core funnel to collect The filtrate and resin were washed 3 times with a small amount of TFA. The filtrate was combined and concentrated under reduced pressure. Anhydrous ether was added to precipitate, and then the precipitate was washed with anhydrous ether for 3 times. The precipitate was drained to obtain an off-white powder, which was dissolved in pure DMSO and added Equimolar trifluoroacetic acid solution of cholesteryl bromoacetate is added with pure diisopropylethylamine to adjust to alkaline, and the reaction is followed by RP-HPLC, and the crude solution is obtained after the reaction is completed.

3. Preparation of pure product

The method is the same as in Example 1.

7.2 g of pure product was obtained, the purity was 98.6%, and the total yield was 18.1%. The molecular weight is 3971.8 (100% M+H).

Example 4 Preparation of Compound 4

Ac-Leu-Glu-Gln-Val-Asn-Lys-Lys-Ile-Glu-Gln-Ser-Leu-Lys-Phe-Ile-Glu-Lys-Ser-Asp-Lys-Leu-Leu-Glu-Asn- Val-Asn-Lys-Gly-Lys-Cys (cholesterol acetate)-NH ₂

The preparation method is the same as in Example 3. The protected amino acids used are as follows:

table 5

The pure product was 6.5 g, the purity was 96.9%, and the total yield was 16.4%. The molecular weight is 3971.6 (100% M+H).

Example 5 Preparation of Compound 5

Ac-Ile-Glu-Gln-Val-Asn-Lys-Lys-Ile-Glu-Gln-Ser-Leu-Lys-Phe-Ile-Glu-Lys-Ser-Asp-Lys-Leu-Leu-Glu-Asn- Val-Asn-Lys-Gly-Lys (cholesterol succinate monoester)-NH ₂

1. Synthesis of peptide resin

Using Rink Amide BHHA resin as the carrier resin, through the removal of Fmoc protection and coupling reaction, the peptide resin is prepared by coupling with the protected amino acids shown in the following table in sequence. The protected amino acids corresponding to the protected amino acids used in this example are as follows:

Table 6

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

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

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 Fmoc-free resin.

The activated first protected amino acid solution is added to the Fmoc-free resin, the coupling reaction is 60-300 minutes, and the resin is filtered and washed to obtain a resin containing 1 protected amino acid.

(2) Access to the 2nd to 30th protected amino acids of the main chain

Using the same method as described above for accessing the first protected amino acid of the main chain, sequentially accessing the corresponding second to 30th protected amino acids above to obtain a resin containing the main chain amino acid.

(3) Lys (Alloc) side chain deprotection

Take 2.5mmol of tetrakistriphenylphosphine palladium and 25mmol of phenylsilane, dissolve with appropriate amount of dichloromethane, deprotect for 4 hours, filter and wash to obtain the Alloc-free resin for later use.

(4) Access side chain modification

Using the same method as described above for accessing the first protected amino acid of the main chain, accessing the modification corresponding to the side chain to obtain a peptide resin.

2. Preparation of crude product

The method is the same as in Example 1.

3. Preparation of pure product

The method is the same as in Example 1.

7.7 g of pure product was obtained, the purity was 96.5%, and the total yield was 19.7%. The molecular weight is 3910.8 (100% M+H).

Example 6 Preparation of Compound 6

Ac-Ile-Glu-Gln-Val-Asn-Lys-Lys-Ile-Glu-Gln-Ser-Leu-Lys-Phe-Ile-Glu-Lys-Ser-Asp-Lys-Leu-Leu-Glu-Asn- Val-Asn-Lys-Gly-Lys(γGlu-Pal)-NH ₂

The preparation method is the same as in Example 5. The protected amino acids used are as follows:

Table 7

7.1 g of the pure product was obtained, the purity was 97.6%, and the total yield was 18.6%. The molecular weight is 3809.6 (100% M+H).

Example 7 Preparation of Compound 7

Ac-Ile-Glu-Gln-Val-Asn-Lys-Lys-Ile-Glu-Gln-Ser-Leu-Lys-Phe-Ile-Glu-Lys-Ser-Asp-Lys-Leu-Leu-Glu-Asn- Val-Asn-Lys-Gly-Lys(AEEA-AEEA-γGlu-18 alkanedioic acid)-NH ₂

The preparation method is the same as in Example 5. The protected amino acids used are as follows:

Table 8

The pure product was 6.6 g, the purity was 97.2%, and the total yield was 15.9%. The molecular weight is 4157.8 (100% M+H).

Example 8 Determination of antiviral activity in vitro

Experimental method 1:

The experimental method based on green fluorescent protein reporter gene labeling RSV virus (RSV-EGFP) is carried out in reference 3.

Plate HEp-2 cells at a seeding density of 2×10 ⁴ cells/well. After culturing for 24 hours, the peptides were serially diluted by 3 times and mixed with 3000PFU of RSV-EGFP respectively. After incubating for 5 minutes at 37°C under 5% CO2, the above mixture was added to a 96-well plate containing HEp-2 cells. Continue to incubate at 37°C for 48 hours. Uninfected HEp-2 cells were used as cell negative controls, and virus-infected wells without drug treatment were used as positive controls. A multifunctional microplate reader was used to detect the fluorescence intensity at an excitation wavelength of 479nm and an emission wavelength of 517nm, and GraphPad was used to calculate the relative inhibition rate of virus infection and IC50 value. The experimental results are shown in the following table and Figure 1.

Table 9 Inhibitory activity on RSV-EGFP infected target cells

Group	Code	IC50 value
Control peptide	T118	14.3μM
Compound 1	SV29	5.3μM
Compound 2	SV29EK	12.7μM
Compound 3	SV29-Chol	0.05μM
Compound 4	SV29L-Chol	0.09μM

Experimental method 2:

The antiviral activity of the new RSV fusion inhibitor was further evaluated using RSV virus (RSV-luc) based on the luciferase reporter gene marker.

The peptide drugs were diluted in a 3-fold gradient in a 96-well plate, with 3 replicate wells for each peptide, 9 dilution gradients, and a final volume of 50μL/well. Then, 50μL (100TCID ₅₀ )RSV was added to the 96-well plate containing the peptide drugs. -luc virus solution, incubate at room temperature for 1h. Prepare a Hep-2 cell suspension with a concentration of 10×10 ⁴ /mL with DMEM medium, mix well and add to the above 96-well plate, 100 μL/well. After culturing in a 37℃5% CO2 cell incubator for 48 hours, discard the supernatant, pat dry on a clean absorbent paper, add 30μL/well of cell lysate, lyse for 15min, use Bright-Glo Luciferase Assay reagent ( Promega) measures the relative fluorescence unit (RLU) of each well. Finally, Graphpad software was used to process the obtained data to calculate the IC ₅₀ value of each peptide drug. The experimental results are shown in the following table and Figure 2.

Table 10 Inhibitory activity on RSV-Luc

Group	Code	IC50 value
Compound 1	SV29	2.4μM
Compound 2	SV29EK	2.3μM
Compound 3	SV29-Chol	0.01μM

Example 9 Determination of antiviral activity in vivo

The antiviral activity was determined using a mouse animal model.

1. Experimental method

8-week-old SFP female BALB/c mice (purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.) were used for pharmaceutical evaluation. The experiment set up PBS treatment control group, RSV infection control group, SV29 treatment group and SV29-Chol treatment Group. Each group of 4-6. Under anesthesia with avertin (250mg/Kg), 50μl of a polypeptide PBS solution with a concentration of 50μM was first administered by nasal drops, 15 minutes later, RSV-Luc virus (5 x 10 ⁴ PFU) infection was administered via the nasal route. The changes in the weight of the mice were tested every day. The Lumina II Small Animal Live Imaging System (Lumina II Small Animal Live Imaging System) was used to detect the virus infection, and the mouse live imaging was performed 10 minutes after the injection of 50 μl of fluorescein substrate D-Luciferin (7.5 mg/ml; PBS). Five days after infection, the mice were euthanized, the lung tissues were weighed, milled, and total RNA was extracted to quantitatively detect RSV infection in lung tissues using RT-qPCR method. The PCR primers used are designed and synthesized in reference 4.

Enzyme-linked immunospot method to detect the amount of virus in mouse lung tissue: HEp-2 cells were inoculated in a 96-well plate, 2×104 cells/well; mouse lung tissue was weighed and ground (0.1g lung tissue/0.1ml PBS(0.1 ％BSA)), centrifuge at 10,000×g at 4℃ for 5min to separate the supernatant; serially dilute and add to the above 96-well plate, 3 replicates/dilution, negative control well cells only add maintenance solution, and incubate at 37℃ for 1h Then discard the culture medium, add 1% methylcellulose, 100μl/well; continue to culture at 37°C for 3 days, fix and block, add goat anti-human RSV polyclonal antibody (1:500 dilution), HRP-labeled rabbit anti-goat antibody ( 1: 5 000 dilution), TMB color development, count the number of virus spots under an inverted microscope.

2. Experimental results

(1) Effect on mouse body weight

Compared with the PBS-treated control mice, the body weight of the mice began to decrease 1 day after RSV infection, and the most decreased 2 days after infection. SV29 and SV29-Chol were administered intranasally according to the above method, and the results did not significantly affect the body weight of the mice. The result is shown in Figure 3.

(2) RSV-infected mice live imaging detection

As shown in Figure 4 to Figure 6, the results of the detection using the small animal in vivo imaging system showed that there was no fluorescence signal in the PBS treatment group, and the RSV infected persons could see obvious fluorescence signals on day 1-5, which were distributed in the nasal cavity and the mouse. Lungs. After SV29 and SV29-Chol treatment, RSV was significantly reduced in the nasal cavity from the untreated RSV control group 1 to 4 days after treatment, especially during the peak period of virus replication on the second day. The results showed that the RSV signal in the SV29 treatment group decreased, but the decrease in the SV29-Chol treatment group was not significant.

(3) Quantitative analysis of lung viruses in RSV-infected mice

As shown in Figures 7-8, the results of quantitative detection of RSV replication levels in the lungs of mice in each experimental group using quantitative PCR methods show that both SV29 and SV29-Chol treatments can significantly reduce the amount of RSV virus in the lungs of mice . Relatively speaking, the effect of SV29 is better than that of SV29-Chol. At the same time, the enzyme-linked immunospot method was used to analyze the replication level of RSV virus in the lungs of mice. The results showed that both SV29 and SV29-Chol can reduce the amount of RSV virus in the lungs to a very low level.

The solution provided by the present invention has been described in detail above. This article uses specific examples to illustrate the principle and implementation of the present invention. The description of the above examples is only used to help understand the method and 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 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 (5)

1. A compound represented by structure I:

   Ac-AA1-Glu-AA3-Val-Asn-Lys-Lys-Ile-Glu-AA10-Ser-Leu-Lys-AA14-Ile-Glu-AA17-Ser-Asp-Lys-AA21-Leu-Glu-AA24- Val-Asn-Lys-AA28-AA29(R1)-AA30(R2)-AA31

   Structure I

   AA1 is Ile, or Leu;

   AA3 is Gln, or Glu;

   AA10 is Gln or Glu;

   AA14 is Phe, or Lys;

   AA17 is Lys, or Glu;

   AA21 is Leu, or Lys;

   AA24 is Asn, or Glu;

   AA28 is Gly or Lys;

   AA29 is Lys, or Dap, or Orn, or Dab, or Dah;

   AA30 means Cys, or does not exist;

   When AA30 is Cys, R1 is H;

   When AA30 is not present, R2 is not present;

   AA31 is NH ₂ or OH;

   R1 in structure I is H, or cholesterol succinate monoester, or 2-cholesterol acetic acid, or 2-cholesterol propionic acid, or 2-cholesterol butanoic acid, or 2-cholesterol isobutyrate, or It is 2-cholesterol valeric acid, or 2-cholesterol isovaleric acid, or 2-cholesterol hexanoic acid, HO ₂ C(CH ₂ ) _n1 CO-(γGlu) _n2 -(PEG _n3 (CH2) _n4 CO) _n5- , Or CH ₃ (CH ₂ ) _n1 CO-(γGlu) _n2 -, or absent;

   Wherein: n1 is an integer from 10 to 20;

   n2 is an integer from 1 to 5;

   n3 is an integer from 1 to 30;

   n4 is an integer from 1 to 5;

   n5 is an integer from 1 to 5;

   R2 in structure I is cholesterol acetate, or cholesterol propionate, or cholesterol butyrate, or cholesterol isobutyrate, or cholesterol valerate, or cholesterol isovalerate, or hexyl Cholesterol ester, or non-existent.
2. The compound according to claim 1, comprising a pharmaceutically acceptable salt, solvate, chelate or non-covalent complex of the compound, a prodrug based on the compound, or any mixture of the above forms .
3. The compound according to claim 1 and claim 2 is used to prepare a pharmaceutical composition for the treatment of diseases.
4. The pharmaceutical composition according to claim 3, which is used in the preparation of a medicine for treating syncytial virus pneumonia.
5. The compound of structure I according to claim 1, comprising a method of using the compound for the treatment of syncytial virus pneumonia.

Images