CN111303245B

CN111303245B - Anti-syncytial virus membrane fusion inhibitor

Info

Publication number: CN111303245B
Application number: CN202010108065.3A
Authority: CN
Inventors: 周述靓; 王鹏; 邓岚
Original assignee: Chengdu Aoda Biotechnology Co ltd
Current assignee: Chengdu Aoda Biotechnology Co ltd
Priority date: 2020-02-21
Filing date: 2020-02-21
Publication date: 2023-06-27
Anticipated expiration: 2040-02-21
Also published as: CN111303245A; WO2021164677A1

Abstract

The invention relates to the field of medicine synthesis, and discloses an anti-syncytial virus (RSV) membrane fusion inhibitor. The anti-syncytial virus membrane fusion inhibitor is used for preparing a pharmaceutical composition for treating diseases, and the pharmaceutical composition is used for preparing medicines for treating syncytial virus pneumonia.

Description

Anti-syncytial virus membrane fusion inhibitor

Technical Field

The invention relates to an anti-syncytial virus membrane fusion inhibitor and application thereof.

Background

Human Respiratory Syncytial Virus (RSV) is a major viral pathogen causing lower respiratory tract infections, widely distributed throughout the world. RSV infection can lead to high hospitalization and mortality in infants, elderly and immunocompromised individuals worldwide. About 70% of infants in one year of birth are infected with RSV, most children are infected within two years after birth, and 1/3 of infants who die from acute lower respiratory tract infections are caused by RSV infection. World Health Organization (WHO) reports that about 6400 thousands of children are worldwide infected with RSV each year, and nearly 350 tens of thousands of children are admitted as being heavier than influenza, and that about 20 tens of thousands of children under the year of age die from infection with RSV virus each year, is a major factor in 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. One study data from korea showed that: total cause mortality in 20 days of patients infected with RSV over 18 years old is higher than influenza (18.4% vs. 6.7%); the risk of mortality due to RSV infection is significantly higher compared to seasonal influenza groups. In Beijing, 48% of viral pneumonia and 58% of bronchiolitis are caused by RSV (1980-1984); in Guangzhou, 31.4% of pediatric pneumonia and bronchiolitis are caused by RSV (1973-1986). WHO data also shows that worldwide, elderly patients with annual RSV infection are nearly 3000 tens of thousands, at least 200 tens of thousands of severe hospitalized patients. Statistics show that RSV infection accounts for 20% of cases of death in elderly over 65 years old. A recent epidemiological investigation showed that 487,247 persons need medical treatment, 17,799 hospitalization and 8,482 deaths were saved in each RSV epidemic season in adults over 18 years old in the united kingdom alone, with 65 years old accounting for about 36% of the medical treatments, 79% of the hospitalizations and 93% of the deaths, respectively. The current population over 60 years old in China is over 2.4 hundred million, and belongs to the high-risk group infected by RSV, and the burden faced by families and society is huge. Since there is no effective vaccine for preventing RSV infection and no effective drug for treating RSV infection worldwide, a great burden is imposed on the health care system of countries around the world.

As early as 1996, the American company Trimeris designed a group of anti-RSV polypeptide membrane fusion inhibitors according to the HRB sequence of RSV, and the anti-RSV polypeptide membrane fusion inhibitors have stronger anti-RSV activity in a cell model, wherein the most prominent is T118 containing 35 amino acid residues, and later researches successively develop some polypeptide RSV fusion inhibitors, but the anti-RSV activity is hardly improved obviously, especially relatively short polypeptides, often have obviously reduced activity, and no report that the polypeptide RSV fusion inhibitors enter clinical experiments is yet seen.

Disclosure of Invention

The invention provides a novel syncytial virus resistant membrane fusion inhibitor and application thereof.

To achieve the above object, the present invention provides a compound of the formula I, a pharmaceutically acceptable salt, solvate, chelate or non-covalent complex thereof, a prodrug based on the compound, or a mixture of any 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

Structural formula I

AA1 in the structure I is Ile or Leu;

AA3 in structure I is Gln, or Glu;

AA10 in structure I is gin, or Glu;

AA14 in structure I is Phe, or Lys;

AA17 in structure I is Lys, or Glu;

AA21 in structure I is Leu, or Lys;

AA24 in structure I is Asn, or Glu;

AA28 in structure I is Gly, or Lys;

AA29 in structure I is Lys, or is Dap, or is Orn, or is Dab, or is Dah;

AA30 in structure I is Cys, or absent;

when AA30 in the structure I is Cys, R1 is H;

when AA30 in structure I is absent, R2 is absent;

AA31 in structure I is NH ₂ Or OH.

R1 in structure I is succinic acid cholesterol monoester, or is 2-cholesterol acetic acid, or is 2-cholesterol propionic acid, or is 2-cholesterol butyric acid, or is 2-cholesterol isobutyric acid, or is 2-cholesterol valeric acid, or is 2-cholesterol isovaleric acid, or is 2-cholesterol caproic acid, HO ₂ C(CH ₂ ) _n1 CO-(γGlu) _n2 -(PEG _n3 (CH2) _n4 CO) _n5 -, or is CH ₃ (CH ₂ ) _n1 CO-(γGlu) _n2 -, or is 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 cholesterol caproate, or is absent.

The invention also provides a pharmaceutical composition comprising the compound according to the invention, and the use of the pharmaceutical composition of the compound for preparing a medicament for treating a disease.

Preferably, the pharmaceutical composition is used for preparing medicines for treating the syncytial virus pneumonia.

Further details of the invention are set forth in the accompanying drawings and the description below, or may be learned by practice of the invention.

Unless otherwise indicated, the amounts of the various components, reaction conditions, and the like, are used herein and are to be construed in any sense as "generally", "about". Accordingly, unless explicitly indicated otherwise, the numerical parameters set forth in the following claims are approximations that may vary depending upon the standard deviation employed under the particular circumstances.

Herein, when the chemical structural formula and chemical name of a compound are divergent or ambiguous, the compound is defined exactly by the chemical structural formula. The compounds described herein may contain one or more chiral centers, and/or double bonds and the like, and stereoisomers, including isomers of double bonds (such as geometric isomers), optical enantiomers or diastereomers, may also be present. Accordingly, any chemical structure within the scope of the description herein, whether partial or whole containing such structures, includes all possible enantiomers and diastereomers of the compound, including any single stereoisomer (e.g., a single geometric isomer, a single enantiomer, or a single diastereomer), and mixtures of any of these isomers. These racemic isomers and mixtures of stereoisomers may also be resolved further into their constituent enantiomers or stereoisomers by methods known to those skilled in the art using continuous separation techniques or chiral molecule synthesis.

The compounds of formula I include, but are not limited to, optical isomers, racemates and/or other mixtures of these compounds. In the above cases, single enantiomers or diastereomers, such as optical isomers, may be obtained by asymmetric synthesis or resolution of racemates. Resolution of the racemate can be accomplished in various ways, such as recrystallization with conventional resolution-aiding reagents, or by chromatographic methods. In addition, the compounds of the 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 of formula I and all of their various pharmaceutically acceptable forms. Pharmaceutically useful different forms of these compounds include various pharmaceutically acceptable salts, solvates, complexes, chelates, non-covalent complexes, prodrugs based on the above, and mixtures of any of these forms.

The prodrugs include ester or amide derivatives of the compounds of formula I contained within the compounds.

The compound shown in the structure I provided by the invention has stable properties, is a novel syncytial virus membrane fusion inhibitor, and can be used for treating syncytial virus pneumonia.

Detailed Description

The invention discloses an anti-syncytial virus membrane fusion inhibitor and application thereof, and a person skilled in the art can properly improve related parameters by referring to the content of the text. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the process of the present invention has been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that variations and modifications can be made in the compounds and methods of preparation described herein, or in appropriate combinations, without departing from the spirit and scope of the invention.

The Chinese names corresponding to the English abbreviations in the invention are shown in the following table:

english abbreviations	Chinese name	English abbreviations	Chinese name
				Fmoc	9-fluorenylmethoxycarbonyl	OtBu	Tert-butoxy radical
tBu	Tert-butyl group	Boc	Boc acid tert-butyl ester
				Trt	Trityl radical	Leu	Leucine (leucine)
Ala	Alanine (Ala)	Lys	Lysine
				Asn	Asparagine derivatives	Phe	Phenylalanine (Phe)
Asp	Aspartic acid	Ser	Serine (serine)
				Cys	Cysteine (S)	Val	Valine (valine)
Gln	Glutamine	Dab						2, 4-diaminobutyric acid
				Glu	Glutamic acid	Dah			2, 7-diaminoheptanoic acid
Gly	Glycine (Gly)	Dap	2, 3-diaminopropionic acid
				Ile	Isoleucine (Ile)	Orn	Ornithine

Drawings

1. FIG. 1 inhibitory Activity against RSV-EGFP infection of target cells

2. FIG. 2 inhibitory Activity against RSV-Luc

3. FIG. 3 effect on weight change in RSV infected mice

4. FIG. 4 in vivo imaging detection of animals infected with RSV in mice

5. FIG. 5 statistical analysis of fluorescence signals at nasal sites of RSV infected mice

6. FIG. 6 statistical analysis of pulmonary fluorescence signals from RSV infected mice

7. FIG. 7RT-qPCR quantitative analysis of viral levels of RSV in the lungs of RSV infected mice

8. FIG. 8 quantitative analysis of mouse Lung tissue RSV replication ability by ELISA spots

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 comprises the following steps: preparing peptide resin by adopting a solid-phase polypeptide synthesis method, acidolysis is carried out on the peptide resin to obtain a crude product, and finally, the crude product is purified to obtain a pure product; wherein the step of preparing peptide resin by solid-phase polypeptide synthesis method comprises the steps of sequentially accessing corresponding protected amino acid or fragment in the following sequence on carrier resin by solid-phase coupling synthesis method to prepare peptide resin:

in the preparation method, the dosage of the Fmoc-protected amino acid or the protected amino acid fragment is 1.2-6 times of the total mole number of the resin; preferably 2.5 to 3.5 times.

In the 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 scheme of the invention, the solid phase coupling synthesis method is as follows: the protected amino acid-resin obtained in the previous step is subjected to Fmoc protecting group removal and then is subjected to coupling reaction with the next protected amino acid. The deprotection time for Fmoc deprotection is 10 to 60 minutes, preferably 15 to 25 minutes. The coupling reaction time is 60 to 300 minutes, preferably 100 to 140 minutes.

The coupling reaction needs to add a condensation reagent, wherein the condensation reagent is selected from DIC (N, N-diisopropyl carbodiimide), N, N-dicyclohexylcarbodiimide, benzotriazol-1-yl-oxy-tripyrrolidinylphosphine hexafluorophosphate, 2- (7-aza-1H-benzotriazol-1-yl) -1, 3-tetramethylurea hexafluorophosphate, benzotriazol-N, N, N ', N' -tetramethylurea hexafluorophosphate or O-benzotriazol-N, N, N ', N' -tetramethylurea tetrafluoroborate; n, N-diisopropylcarbodiimide is preferred. The molar amount of the condensing agent is 1.2 to 6 times, preferably 2.5 to 3.5 times, the total molar amount of the 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, and is preferably 1-hydroxybenzotriazole. The amount of the activating agent to be used is 1.2 to 6 times, preferably 2.5 to 3.5 times, the total mole number of the amino groups in the amino resin.

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

Preferably, the peptide resin is subjected to acidolysis and simultaneously the resin and side chain protecting group are removed to obtain a crude product:

further preferably, the acidolysis agent used in 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 as follows: 80-95% of TFA, 1-10% of EDT and the balance of water.

Still more preferably, the volume ratio of the mixed solvent is: 89-91% TFA, 4-6% EDT and the balance water. Optimally, the volume ratio of the mixed solvent is as follows: TFA 90%, EDT 5%, balance water.

The dosage of the acidolysis agent is 4-15 mL of acidolysis agent required by each gram of peptide resin; preferably, 7 to 10mL of acidolysis agent is required per gram of peptide resin.

The time for cleavage with acidolysis agent is 1 to 6 hours, preferably 3 to 4 hours, at room temperature.

Further, the crude product is purified by high performance liquid chromatography and freeze-dried to obtain a pure product, and the specific method comprises the following steps:

taking a crude product, adding water, stirring, adjusting the pH value to be completely dissolved, filtering the solution by using a 0.45 mu m mixed microporous filter membrane, and purifying for later use;

purifying by high performance liquid chromatography, wherein the chromatographic packing for purification is reverse phase C18 with the size of 10 μm, the mobile phase system is 0.1% TFA/water solution-0.1% TFA/acetonitrile solution, the flow rate of a chromatographic column with the size of 77mm and 250mm is 90mL/min, eluting by a gradient system, circularly sampling and purifying, sampling the crude product solution into the chromatographic column, starting mobile phase eluting, collecting main peaks, evaporating acetonitrile, and obtaining purified intermediate concentrated solution;

collecting purified intermediate concentrate, and filtering with 0.45 μm filter membrane;

changing salt by high performance liquid chromatography, wherein the mobile phase system is 1% acetic acid/water solution-acetonitrile, the chromatographic column flow rate of 10 μm reversed phase C18 with 77mm x 250mm chromatographic packing for purification is 90mL/min (corresponding flow rate can be adjusted according to chromatographic columns of different specifications); adopting a gradient elution and cyclic loading method, loading in a chromatographic column, starting mobile phase elution, collecting a spectrum, observing the change of absorbance, collecting a salt-exchange main peak, analyzing the liquid phase to detect the purity, combining the salt-exchange main peak solutions, concentrating under reduced pressure to obtain a pure acetic acid aqueous solution, and freeze-drying to obtain a pure product.

1. Synthesis of peptide resins

The Rink Amide BHHA resin is used as carrier resin, and is coupled with the protected amino acid shown in the following table in sequence through Fmoc protection removal and coupling reaction to prepare the peptide resin. The protected amino acids corresponding to the protected amino acids used in this example are shown below:

(1) Access to backbone 1 st protected amino acid

Taking 0.03mol of 1 st protected amino acid and 0.03mol of HOBt, and dissolving the 1 st protected amino acid and the HOBt with a proper amount of DMF; and (3) adding 0.03mol of DIC into the protected amino acid DMF solution slowly under stirring, and stirring and reacting for 30 minutes in a room temperature environment to obtain an activated protected amino acid solution for later use.

0.01mol of Rink amide MBHA resin (substitution value about 0.4 mmol/g) was taken and deprotected with 20% PIP/DMF solution for 25 min, washed and filtered to give Fmoc-removed resin.

And adding the activated 1 st protected amino acid solution into Fmoc-removed resin, performing coupling reaction for 60-300 minutes, and filtering and washing to obtain the resin containing 1 protected amino acid.

(2) Accessing the 2 nd to 30 th protected amino acid of the main chain

The same method of accessing the 1 st protected amino acid of the main chain is adopted, and the corresponding 2 nd to 30 th protected amino acids are sequentially accessed to obtain the peptide resin.

2. Preparation of crude product

Adding a cracking reagent (10 mL/g resin) with a volume ratio of TFA to water to EDT=95 to 5 into the peptide resin, uniformly stirring, stirring at room temperature for reaction for 3 hours, filtering a reaction mixture by using a sand core funnel, collecting filtrate, washing the resin with a small amount of TFA for 3 times, combining the filtrates, concentrating under reduced pressure, adding anhydrous diethyl ether for precipitation, washing the precipitation with anhydrous diethyl ether for 3 times, and pumping to obtain white-like powder which is a crude product.

3. Preparation of pure product

Taking the crude product, adding water, stirring and dissolving, filtering the solution by using a 0.45 mu m mixed microporous filter membrane, and purifying for later use. Purifying by high performance liquid chromatography, wherein the chromatographic packing for purification is reverse phase C18 with the size of 10 μm, the mobile phase system is 0.1% TFA/water solution-0.1% TFA/acetonitrile solution, the flow rate of a chromatographic column with the size of 30mm or 250mm is 20mL/min, eluting by a gradient system, circularly sampling and purifying, sampling the crude product solution into the chromatographic column, starting mobile phase eluting, collecting main peaks, evaporating acetonitrile, and obtaining purified intermediate concentrated solution;

filtering the purified intermediate concentrate with 0.45 μm filter membrane for use, changing salt by high performance liquid chromatography, wherein the mobile phase system is 1% acetic acid/water solution-acetonitrile, the chromatographic column flow rate of purification column is 20mL/min (corresponding flow rate can be adjusted according to chromatographic columns of different specifications) with reversed phase C18 of 10 μm and 30mm x 250 mm; adopting gradient elution, circulating sample loading method, loading in chromatographic column, starting mobile phase elution, collecting spectrum, observing change of absorbance, collecting salt-changing main peak, analyzing liquid phase to detect purity, mixing salt-changing main peak solutions, concentrating under reduced pressure to obtain pure acetic acid aqueous solution, freeze drying to obtain pure product 7.6g, purity of 97.5%, and total yield of 22.1%. The molecular weight was 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 was carried out in the same way as in example 1, using the protected amino acids as indicated in the following table:

peptide sequence n =	Protecting amino acids
		1	Fmoc-Lys(Boc)
2	Fmoc-Lys(Boc)
		3	Fmoc-Lys(Boc)
4	Fmoc-Asn(Trt)
		5	Fmoc-Val
6	Fmoc-Glu(OtBu)
		7	Fmoc-Glu(OtBu)
8	Fmoc-Leu
		9	Fmoc-Lys(Boc)
10	Fmoc-Lys(Boc)
		11	Fmoc-Asp(OtBu)
12	Fmoc-Ser(tBu)
		13	Fmoc-Glu(OtBu)
14	Fmoc-Glu(OtBu)
		15	Fmoc-Ile
16	Fmoc-Lys(Boc)
		17	Fmoc-Lys(Boc)
18	Fmoc-Leu
		19	Fmoc-Ser(tBu)
20	Fmoc-Glu(OtBu)
		21	Fmoc-Glu(OtBu)
22	Fmoc-Ile
		23	Fmoc-Lys(Boc)
24	Fmoc-Lys(Boc)
		25	Fmoc-Asn(Trt)
26	Fmoc-Val
		27	Fmoc-Glu(OtBu)
28	Fmoc-Glu(OtBu)
		29	Fmoc-Ile
30	Ac ₂ O

8.6g of pure product is obtained, the purity is 97.9%, and the total yield is 24.4%. The molecular weight was 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 (cholesteryl acetate) -NH ₂

1. Synthesis of peptide resins

The procedure is as in example 1, using the protected amino acids as shown in the following table:

peptide sequence n =	Protecting amino acids
		1	Fmoc-Cys(Trt)
2	Fmoc-Lys(Boc)
		3	Fmoc-Gly
4	Fmoc-Lys(Boc)
		5	Fmoc-Asn(Trt)
6	Fmoc-Val
		7	Fmoc-Asn(Trt)
8	Fmoc-Glu(OtBu)
		9	Fmoc-Leu
10	Fmoc-Leu
		11	Fmoc-Lys(Boc)
12	Fmoc-Asp(OtBu)
		13	Fmoc-Ser(tBu)
14	Fmoc-Lys(Boc)
		15	Fmoc-Glu(OtBu)
16	Fmoc-Ile
		17	Fmoc-Phe
18	Fmoc-Lys(Boc)
		19	Fmoc-Leu
20	Fmoc-Ser(tBu)
		21	Fmoc-Gln(Trt)
22	Fmoc-Glu(OtBu)
		23	Fmoc-Ile
24	Fmoc-Lys(Boc)
		25	Fmoc-Lys(Boc)
26	Fmoc-Asn(Trt)
		27	Fmoc-Val
28	Fmoc-Gln(Trt)
		29	Fmoc-Glu(OtBu)
30	Fmoc-Ile
		31	Ac ₂ O

2. Preparation of crude product

Adding a cracking reagent (10 mL/g resin) with a volume ratio of TFA to water to EDT=95 to 5 into the peptide resin, uniformly stirring, stirring at room temperature for reaction for 3 hours, filtering a reaction mixture by using a sand core funnel, collecting filtrate, washing the resin with a small amount of TFA for 3 times, combining the filtrates, concentrating under reduced pressure, adding anhydrous diethyl ether for precipitation, washing the precipitate with the anhydrous diethyl ether for 3 times, pumping to obtain white-like powder, dissolving the white-like powder into pure DMSO, adding a trifluoroacetic acid solution of equimolar bromoacetic acid cholesterol ester, adding pure diisopropylethylamine for regulating to be alkaline, tracking reaction by using RP-HPLC, and obtaining a crude product solution after the reaction is finished.

3. Preparation of pure product

The procedure is as in example 1,

7.2g of pure product is obtained, the purity is 98.6%, and the total yield is 18.1%. The molecular weight was 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 (cholesteryl acetate) -NH ₂

The preparation was carried out in the same way as in example 3, using the protected amino acids as indicated in the following table:

6.5g of pure product is obtained, the purity is 96.9%, and the total yield is 16.4%. The molecular weight was 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 (succinic acid cholesterol monoester) -NH ₂

1. Synthesis of peptide resins

(1) Access to backbone 1 st protected amino acid

(2) Accessing the 2 nd to 30 th protected amino acid of the main chain

The same method of accessing the 1 st protected amino acid of the main chain is adopted, and the corresponding 2 nd to 30 th protected amino acids are sequentially accessed to obtain the resin containing the main chain amino acid.

(3) Lys (Alloc) side chain deprotection

2.5mmol of tetraphenylphosphine palladium and 25mmol of phenylsilane are taken, dissolved with a proper amount of dichloromethane, deprotected for 4 hours, filtered and washed to obtain dealloc resin for later use.

(4) Access side chain modification

The peptide resin is obtained by accessing the modification corresponding to the side chain by the same method of accessing the 1 st protected amino acid of the main chain.

2. Preparation of crude product

The procedure is as in example 1,

3. preparation of pure product

The procedure is as in example 1,

7.7g of pure product is obtained, the purity is 96.5%, and the total yield is 19.7%. The molecular weight was 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 was carried out in the same way as in example 5, using the protected amino acids as indicated in the following table:

7.1g of pure product is obtained, the purity is 97.6%, and the total yield is 18.6%. The molecular weight was 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-gamma Glu-18 alkanedioic acid) -NH ₂

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

Example 8 determination of in vitro antiviral Activity

Experimental method 1:

experimental method based on Green fluorescent protein reporter labeled RSV virus (RSV-EGFP) reference 3 was performed.

HEp-2 cells were plated at a seeding density of 2X10 ⁴ Cells/wells. After 24 hours of incubation, the polypeptides were serially diluted 3-fold, mixed with 3000PFU RSV-EGFP, and incubated at 37℃for 5 minutes under 5% CO2, and the above mixture was added to 96-well plates containing HEp-2 cells, followed by incubation at 37℃for 48 hours. Uninfected HEp-2 cells were used as cell negative control and uninfected virus-infected wells were used as positive control. The fluorescence intensity was measured with a multifunctional microplate reader at an excitation wavelength of 479nm and an emission wavelength of 517nm, and the relative inhibition rate of viral infection and the IC50 value were calculated with a GraphPad, and the experimental results are shown in the following Table and FIG. 1.

Inhibitory Activity against RSV-EGFP infection of target cells

Group of	Code	IC50 value
			Control polypeptides	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 novel RSV fusion inhibitor was further evaluated using a luciferase reporter-based labeled RSV virus (RSV-luc).

The polypeptide drugs were subjected to 3-fold gradient dilution in 96-well plates, 3 multiplex wells per polypeptide, 9 dilution gradients, final volume of 50. Mu.L/well, followed by addition of 50. Mu.L (100 TCID ₅₀ ) RSV-luc virus solution was incubated at room temperature for 1h. The concentration of the culture medium of DMEM is 10 multiplied by 10 ⁴ Per mL of Hep-2 cell suspension, after homogenization, 100. Mu.L/well was added to the 96-well plate described above. After 48 hours of incubation in a 37℃5% CO2 cell incubator, the supernatant was discarded, gently patted dry on clean absorbent paper, 30. Mu.L/well of cell lysate was added, and after 15 minutes of lysis, the relative fluorescence units (RLU) per well was determined using Bright-Glo Luciferase Assay reagent (Promega). Finally, the obtained data are subjected to Graphpad softwareProcessing to calculate IC for each polypeptide drug ₅₀ Values, experimental results are shown in the following table and fig. 2.

Inhibitory Activity against RSV-Luc

Group of	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

Antiviral activity assays were performed using a mouse animal model.

1. Experimental method

Pharmaceutical evaluations were performed using 8 week old SFP female BALB/c mice (purchased from Experimental animal technologies Inc., studies, beijing) and were tested in PBS-treated control, RSV-infected control, SV 29-treated and SV 29-Chol-treated groups. 4-6 per group. Under the anesthesia of avermectin (250 mg/Kg), 50. Mu.l of a 50. Mu.M polypeptide PBS solution was first administered by nasal drip, and 15 minutes later, RSV-Luc virus (5X 10) ⁴ PFU) infection. Mice were tested daily for weight change. The virus infection was detected using a small animal live imaging system (Lumina II Small Animal Live Imaging System), and live imaging of mice was performed 10 minutes after injection of 50. Mu.l of the Luciferin substrate D-Luciferin (7.5 mg/ml; PBS). Mice were euthanized 5 days after infection, lung tissues were weighed and milled, total RNA was extracted and the lung tissue RSV infection was quantitatively detected by RT-qPCR. PCR primer reference 4 was used for the design synthesis.

ELISA spot method for detecting lung tissue virus amount of mice: HEp-2 cells were seeded in 96-well plates, 2X10 4 cells/well; mouse lung tissue was weighed, ground (0.1 g lung tissue/0.1 ml PBS (0.1% BSA)), and the supernatant was isolated by centrifugation at 10 000 Xg for 5min at 4 ℃; serial dilutions were added to the 96-well plate, 3 multiplex wells/dilution, only the maintenance solution was added to the cells in the negative control wells, incubation was performed at 37℃for 1h, the culture solution was discarded, and 1% methylcellulose was added, 100. Mu.l/well; after further incubation for 3d at 37℃the goat anti-human RSV polyclonal antibody (1:500 dilution), HRP-labeled rabbit anti-goat antibody (1:5,000 dilution) and TMB developed were added in sequence and the number of plaques counted under an inverted microscope.

2. Experimental results

(1) Effects on mouse body weight

Mice body weight began to drop 1 day after RSV infection, most frequently at 2 days after infection, compared to PBS-treated control mice. SV29 and SV29-Chol treatments were given nasally as described above, with no significant effect on mouse body weight.

The results are shown in FIG. 3.

(2) In vivo imaging detection of RSV infected mice

The results of detection by using the living animal imaging system are shown in fig. 4-6, and the results show that the PBS treatment group does not have fluorescence signals, and the RSV infected person can see obvious fluorescence signals on days 1-5 and are distributed in the nasal cavity and lung of the mouse. RSV was significantly reduced at nasal sites 1 to 4 days after SV29 and SV29-Chol treatment compared to untreated RSV control, especially at the peak of viral replication on day 2. The results demonstrate a decrease in RSV signal in the SV29 treated group, but no decrease in the SV29-Chol treated group was apparent.

(3) Quantitative analysis of RSV infected mice pulmonary viruses

The quantitative PCR method is used for quantitatively detecting the pulmonary RSV replication level of mice in each experimental group, and the results are shown in figures 7-8, and the results show that. Both SV29 and SV29-Chol treatment significantly reduced the amount of RSV virus in the lungs of mice. In contrast, the effect of SV29 is superior to that of SV 29-Chol. Meanwhile, the replication level of the mouse lung RSV is analyzed by an ELISA spot method, and the results show that both SV29 and SV29-Chol can reduce the virus amount of the lung RSV to a very low level.

Claims

1. A compound of structure I, characterized by:

the structure I is as follows:

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 ；

or alternatively, the first and second heat exchangers may be,

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-NH ₂ ；

or alternatively, the first and second heat exchangers may be,

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 (cholesteryl acetate) -NH ₂ ；

Or alternatively, the first and second heat exchangers may be,

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 (cholesteryl acetate) -NH ₂ 。

2. A pharmaceutically acceptable salt or solvate comprising the compound of claim 1.

3. A pharmaceutical composition comprising a compound of claim 1.

4. Use of the pharmaceutical composition of claim 3 in the preparation of a medicament for the treatment of syncytial virus pneumonia.