CN111303245A - Anti-syncytial virus membrane fusion inhibitor - Google Patents

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 a medicament 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 widely distributed throughout the world and is an important viral pathogen causing lower respiratory tract infections. RSV infection can lead to high rates of hospitalization and mortality among infants, elderly, and immunodeficient people worldwide. Approximately 70% of infants are infected with RSV within one year of birth, most children are infected within two years after birth, and 1/3 of infants dying from acute lower respiratory tract infection are caused by RSV infection. The World Health Organization (WHO) reports that approximately 6400 million children are infected with RSV worldwide each year, nearly 350 million children are admitted to the hospital because of a heavy RSV infection, 16 times as much as influenza, and children under about 20 million years ofage 5 die each year because of RSV infection are a major cause of hospitalization and death of children worldwide. In the united states, 20% to 25% of infantile pneumonia and 50% to 75% of bronchiolitis are caused by RSV. One study data from korea shows: the overall 20-day-old mortality in RSV infected patients over 18 years of age was higher than influenza (18.4% vs. 6.7%); the risk of mortality due to RSV infection was significantly higher compared to the seasonal influenza group. 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 nearly 3000, at least 200, severe hospitalizations are given to elderly patients with RSV infection worldwide each year. Statistics show that the RSV infection accounts for 20% of the death cases of the elderly over 65 years old. One recent epidemiological survey showed that 487,247 saved medical treatment needs, 17,799 hospitalizations and 8,482 deaths occurred per RSV epidemic season in adults over 18 years of age in the uk, with 65-year-old patients accounting for approximately 36% of medical treatment, 79% of hospitalizations and 93% of deaths, respectively. China currently has over 2.4 billion population over 60 years old, belongs to high risk group infected by RSV, and has huge burden for families and society. Because no effective vaccine for preventing RSV infection and no effective medicament for treating RSV infection exist all over the world, great burden is brought to health care systems of all countries all over the world.

In 1996, an anti-RSV polypeptide membrane fusion inhibitor group is designed by American company Trimeris according to the HRB sequence of RSV, the anti-RSV polypeptide membrane fusion inhibitor group has strong anti-RSV activity in a cell model, wherein T118 containing 35 amino acid residues is most prominent, and a plurality of polypeptide RSV fusion inhibitors are successively developed in later research, but the anti-RSV activity is difficult to be obviously improved, particularly relatively short polypeptides have obviously reduced activity, and no report that the polypeptide RSV fusion inhibitor enters clinical tests is found at present.

Disclosure of Invention

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

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

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

AA1 is Ile, or Leu;

AA3 is Gln, or Glu;

AA10 is Gln, or Glu;

AA14 is Phe, or Lys;

AA17 is Lys, or is Glu;

AA21 is Leu, or Lys;

AA24 is Asn, or Glu;

AA28 is Gly, or is Lys;

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

AA30 is Cys, or is absent;

when AA30 is Cys, R1 is H;

when AA30 is absent, R2 is absent;

AA31 is NH₂Or OH.

R1 in structure I is H, or succinic acid cholesterol monoester, or 2-cholesterol acetic acid, or 2-cholesterol propionic acid, or 2-cholesterol butyric acid, or 2-cholesterol isobutyric acid, or 2-cholesterol valeric acid, or 2-cholesterol isovaleric acid, or 2-cholesterol hexanoic acid, HO, or cholesterol ester₂C(CH₂)_n1CO-(γGlu)_n2-(PEG_n3(CH2)_n4CO)_n5-, or is CH₃(CH₂)_n1CO-(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 cholesteryl acetate, or is cholesteryl propionate, or is cholesteryl butyrate, or is cholesteryl isobutyrate, or is cholesteryl valerate, or is cholesteryl isovalerate, or is cholesteryl hexanoate, or is absent.

The invention also provides pharmaceutical compositions comprising a compound according to the invention and the use of a pharmaceutical composition comprising a compound of the invention for the preparation of a medicament for the treatment of a disease.

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

Further details of the invention are set forth below, or some may be appreciated in embodiments of the invention.

Unless otherwise indicated, the amounts of the various ingredients, reaction conditions, and the like used herein are to be construed in any case to mean "about". Accordingly, unless expressly stated otherwise, all numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the standard deviation found in the respective experimental conditions.

Herein, when a chemical structural formula and a chemical name of a compound are ambiguous or ambiguous, the compound is exactly defined 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 (e.g., geometric isomers), optical enantiomers, or diastereomers, may also be present. Accordingly, any chemical structure within the scope of the description, whether partial or complete, including similar structures as described above, includes all possible enantiomers and diastereomers of the compound, including any stereoisomer alone (e.g., pure geometric isomers, pure enantiomers, or pure diastereomers), as well as any mixture of such stereoisomers. Mixtures of these racemates and stereoisomers may also be further resolved into the enantiomers or stereoisomers of their constituent members by those skilled in the art using non-stop separation techniques or methods of chiral molecular 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 case, a single enantiomer or diastereomer, such as an optical isomer, can be obtained by asymmetric synthesis or racemate resolution. Resolution of the racemates can be accomplished by various methods, such as conventional recrystallization from resolution-assisting reagents, or by chromatographic methods. In addition, the 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 pharmaceutically acceptable different forms. The pharmaceutically acceptable different forms of these compounds include various pharmaceutically acceptable salts, solvates, complexes, chelates, non-covalent complexes, prodrugs based on the above and any mixtures of these forms.

The prodrug comprises an ester or amide derivative of the compound shown as the structural formula I contained in the compound.

The compound shown in the structure I provided by the invention has stable property, is a novel anti-syncytial virus membrane fusion inhibitor, and can be used for treating the 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 appropriately improve related parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the process of the present invention has been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications of the compounds and processes described herein, as well as other changes and combinations of the foregoing, may be made to implement and use the techniques of the present invention without departing from the spirit and scope of the invention.

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

english abbreviation	Name of Chinese	English abbreviation	Name of Chinese
				Fmoc	9-fluorenylmethoxycarbonyl group	OtBu	Tert-butoxy radical
tBu	Tert-butyl radical	Boc	Boc-acyl
				Trt	Trityl radical	Leu	Leucine
Ala	Alanine	Lys	Lysine
				Asn	Asparagine	Phe	Phenylalanine
Asp	Aspartic acid	Ser	Serine
				Cys	Cysteine	Val	Valine
Gln	Glutamine	Dab						2, 4-diaminobutyric acid
				Glu	Glutamic acid	Dah			2, 7-Diaminoheptanoic acid
Gly	Glycine	Dap						2, 3-diaminopropionic acid
				Ile	Isoleucine	Orn	Ornithine

Drawings

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

FIG. 2 inhibitory Activity on RSV-Luc

FIG. 3 Effect on weight Change in RSV infected mice

FIG. 4 in vivo animal imaging assay for RSV infected mice

FIG. 5 statistical analysis of fluorescence signals of nasal cavity part of RSV-infected mouse

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

FIG. 7RT-qPCR method for quantitative analysis of RSV levels in lung of RSV infected mice

FIG. 8 quantitative analysis of mouse lung tissue RSV replication ability by ELISA speckle method

EXAMPLE 1 preparation ofCompound 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-NH₂

The preparation method comprises the following steps: preparing peptide resin by adopting a solid-phase polypeptide synthesis method, carrying out acidolysis on the peptide resin to obtain a crude product, and finally purifying the crude product to obtain a pure product; the step of preparing the peptide resin by the solid-phase polypeptide synthesis method is to sequentially insert corresponding protective amino acids or fragments in the following sequences on a carrier resin by the solid-phase coupling synthesis method to prepare the 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 charged 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 preferable substitution value is 0.3-0.5 mmol/g resin.

In a preferred embodiment of the present invention, the solid-phase coupling synthesis method comprises: and (3) after the Fmoc protecting group of the protected amino acid-resin obtained in the previous step is removed, carrying out coupling reaction with the next protected amino acid. The deprotection time for removing Fmoc protection is 10-60 minutes, and preferably 15-25 minutes. The coupling reaction time is 60-300 minutes, and preferably 100-140 minutes.

The coupling reaction needs to add a condensation reagent, and the condensation reagent is selected from one of DIC (N, N-diisopropyl carbodiimide), N, N-dicyclohexylcarbodiimide, benzotriazole-1-yl-oxy tripyrrolidinophosphonium hexafluorophosphate, 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 tetrafluoroborate; n, N-diisopropylcarbodiimide is preferred. The molar consumption of the condensation reagent is 1.2-6 times of the total molar number of amino groups in the amino resin, and preferably 2.5-3.5 times.

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

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

Preferably, the peptide resin is subjected to acidolysis while removing the resin and side chain protecting groups to obtain a crude product:

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

More preferably, the volume ratio of the mixed solvent is: 89-91% of TFA, 4-6% of EDT and the balance of 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 per gram of the peptide resin; preferably, 7-10 mL of acidolysis agent is required per gram of peptide resin.

The time for cracking by using the acidolysis agent is 1-6 hours, preferably 3-4 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:

adding water into the crude product, stirring, adjusting pH value to completely dissolve, filtering the solution with 0.45 μm mixed microporous membrane, and purifying;

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

filtering the purified intermediate concentrated solution with 0.45 μm filter membrane for use;

performing salt exchange by high performance liquid chromatography, wherein the mobile phase system is 1% acetic acid/water solution-acetonitrile, the chromatographic packing for purification is reversed phase C18 with 10 μm, and the flow rate of 77mm × 250mm chromatographic column is 90mL/min (corresponding flow rate can be adjusted according to chromatographic columns with different specifications); loading the sample into a chromatographic column by adopting a gradient elution and circulating sample loading method, starting mobile phase elution, collecting a map, observing the change of the absorbance, collecting a main salt exchange peak, detecting the purity by using an analysis liquid phase, combining main salt exchange 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

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

(1) 1 st protected amino acid inserted into main chain

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

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

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

(2) 2-30 th protected amino acid connected to main chain

And sequentially inoculating the corresponding 2 nd to 30 th protected amino acids by the same method for inoculating the 1 st protected amino acid in the main chain to obtain the peptide resin.

2. Preparation of crude product

Adding a cleavage reagent (10 mL of cleavage reagent/g of resin) with the volume ratio of TFA, water and EDT (95: 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 ether for precipitation, washing the precipitate with anhydrous ether for 3 times, and drying to obtain white-like powder, namely a crude product.

3. Preparation of the pure product

Dissolving the crude product in water under stirring, filtering the solution with 0.45 μm mixed microporous membrane, and purifying. Purifying by high performance liquid chromatography, wherein the chromatographic packing for purification is 10 μm reversed phase C18, the mobile phase system is 0.1% TFA/water solution-0.1% TFA/acetonitrile solution, the flow rate of a30 mm by 250mm chromatographic column is 20mL/min, eluting by a gradient system, circularly sampling for purification, sampling the crude product solution in the chromatographic column, starting the mobile phase for elution, collecting the main peak, and evaporating acetonitrile to obtain a purified intermediate concentrated solution;

filtering the purified intermediate concentrated solution with 0.45 μm filter membrane for use, and performing salt exchange by high performance liquid chromatography with 1% acetic acid/water solution-acetonitrile as mobile phase system, 10 μm reversed phase C18 as purification chromatographic filler, and 20mL/min of 30 mm/250 mm chromatographic column flow rate (corresponding flow rate can be adjusted according to chromatographic columns of different specifications); adopting gradient elution and circulation sample loading method, loading sample in chromatographic column, starting mobile phase elution, collecting atlas, observing change of absorbance, collecting main peak of salt exchange and analyzing liquid phase to detect purity, combining main peak solutions of salt exchange, concentrating under reduced pressure to obtain pure acetic acid water solution, and 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 ofCompound 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 procedure is as in example 1, using the protected amino acids as in the following table:

the peptide sequence n ═	Protectedamino 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	Ac2O

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

EXAMPLE 3 preparation ofCompound 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) -NH₂

1. Synthesis of peptide resins

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

the peptide sequence n ═	Protectedamino 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	Ac2O

2. Preparation of crude product

Taking the peptide resin, adding a cracking reagent (10 mL of the cracking reagent/gram of the resin) with the volume ratio of TFA: water: EDT (95: 5), 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 for 3 times by using a small amount of TFA, merging the filtrate, concentrating under reduced pressure, adding anhydrous ether for precipitation, washing the precipitate for 3 times by using the anhydrous ether, drying to obtain white-like powder, dissolving the white-like powder in pure DMSO, adding equal mol of trifluoroacetic acid solution of cholesteryl bromoacetate, adding pure diisopropylethylamine for adjusting to be alkaline, carrying out RP-HPLC tracking reaction, and obtaining a crude product solution after the reaction is finished.

3. Preparation of the pure product

The procedure was as in example 1,

7.2g of pure product is obtained, the purity is 98.6 percent, and the total yield is 18.1 percent. 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) -NH₂

The procedure is as in example 3, using the protected amino acids as in the following table:

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

EXAMPLE 5 preparation ofCompound 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) -NH₂

1. Synthesis of peptide resins

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

(1) 1 st protected amino acid inserted into main chain

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

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

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

(2) 2-30 th protected amino acid connected to main chain

And sequentially inoculating the corresponding 2 nd to 30 th protected amino acids by the same method for inoculating the 1 st protected amino acid of the main chain to obtain the resin containing the main chain amino acid.

(3) Lys (alloc) side chain deprotection

Taking 2.5mmol of tetratriphenylphosphine palladium and 25mmol of phenylsilane, dissolving with a proper amount of dichloromethane, deprotecting for 4 hours, filtering and washing to obtain a resin without Alloc for later use.

(4) Side chain modification by grafting

And (3) adopting the same method for accessing the 1 st protected amino acid of the main chain, and accessing a modifier corresponding to the side chain to obtain the peptide resin.

2. Preparation of crude product

The procedure was as in example 1,

3. preparation of the pure product

The procedure was as in example 1,

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

EXAMPLE 6 preparation ofCompound 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)-NH₂

The procedure is as in example 5, using the protected amino acids as in the following table:

7.1g of pure product is obtained, the purity is 97.6 percent, and the total yield is 18.6 percent. 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) -NH₂

The procedure is as in example 5, using the protected amino acids as in the following table:

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

EXAMPLE 8 in vitro determination of antiviral Activity

Experimental method 1:

experimental methods based on the Green fluorescent protein reporter Gene-labeled RSV virus (RSV-EGFP) were performed inreference 3.

HEp-2 cells were plated at a density of 2X10⁴Cells/well. After 24 hours of incubation, the polypeptides were serially diluted 3-fold, mixed with 3000PFU of RSV-EGFP, and incubated at 37 ℃ for 5 minutes in 5% CO2, and the mixture was added to a 96-well plate containing HEp-2 cells and incubated at 37 ℃ for an additional 48 hours. Uninfected HEp-2 cells were used as a negative control for cells, and virus-infected wells that were not treated with drug were used as a positive control. The fluorescence intensity is detected by adopting a multifunctional enzyme-labeling instrument when the excitation wavelength is 479nm and the emission wavelength is 517nm, the relative inhibition rate of virus infection and the IC50 value are calculated by GraphPad, and the experimental results are shown in the following table and figure 1.

Inhibitory Activity against RSV-EGFP infection of target cells

Group of	Code	IC50 value
			Control polypeptide	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 inhibitors was further evaluated using RSV virus based on a luciferase reporter marker (RSV-luc).

Polypeptide drugs were diluted in a 3-fold gradient in 96-well plates with 3 multiple wells per polypeptide, 9 dilution gradients, and a final volume of 50 μ L/well, followed by addition of 50 μ L (100 TCID) to the 96-well plates containing the polypeptide drugs₅₀) RSV-luc virus solution, incubated for 1h at room temperature. Prepared with DMEM medium at a concentration of 10 × 10⁴The resulting suspension of Hep-2 cells was mixed well and added to the above 96-well plate at 100. mu.L/well. After culturing in a 5% CO2 cell culture chamber at 37 ℃ for 48 hours, the supernatant was discarded, gently patted dry on a clean absorbent paper, 30. mu.L/well of cell lysate was added, and the relative fluorescence units (RLU) per well were measured using Bright-Glo Luciferase Assay reagent (Promega) after 15min of lysis. Finally, Graphpad software is used for processing the obtained data and calculating the IC of each polypeptide drug₅₀The values, experimental results are shown in the following table and fig. 2.

Inhibitory Activity on 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 mouse animal models.

1. Experimental methods

The pharmaceutical evaluation was carried out using 8-week-old SFP female BALB/c mice (purchased from Beijing Wintotongliwa laboratory animal technology Co., Ltd.) which were treated with PBS, RSV infection, SV29 and SV 29-Chol. 4-6 of them are in each group. Under Avermectin (250mg/Kg) anesthesia, 50. mu.l of 50. mu.M polypeptide PBS solution was first administered by nasal drip, and RSV-Luc virus (5X 10) was administered by nasal route 15 minutes later⁴PFU) infection. Mice were tested daily for weight change. Virus infection was detected using a mouse in vivo Imaging System (Lumina II Small Animal Live Imaging System) and mice were imaged invivo 10 minutes after injection of 50. mu.l of D-Luciferin substrate (7.5 mg/ml; PBS). Mice were euthanized 5 days after infection, lung tissue weighed and groundAnd grinding, extracting total RNA, and quantitatively detecting the RSV infection condition of the lung tissue by adopting an RT-qPCR method. The PCR primers used reference 4 were designed for synthesis.

Detecting the lung tissue virus amount of the mouse by an enzyme-linked immunosorbent assay: HEp-2 cells were seeded in 96-well plates at2X 104 cells/well; mouse lung tissue was weighed, ground (0.1g lung tissue/0.1 ml PBS (0.1% BSA)), centrifuged at 10000 Xg for 5min at 4 ℃ to separate the supernatant; sequentially diluting in a series of ways, adding into the 96-well plate, adding 3 multiple wells/dilution, adding only maintenance solution into negative control well cells, incubating at 37 ℃ for 1h, then discarding culture solution, adding 1% methylcellulose, and adding 100 μ l/well; culturing at 37 deg.C for 3 days, fixing, sealing, sequentially adding goat anti-human RSV polyclonal antibody (1: 500 dilution), HRP-labeled rabbit anti-goat antibody (1: 5000 dilution), TMB developing, and counting virus spots under inverted microscope.

2. Results of the experiment

(1) Effect on mouse body weight

Mice began to loseweight 1 day after RSV infection and declined the most 2 days after infection compared to PBS-treated control mice. No significant effect on body weight was observed in mice treated with SV29 and SV29-Chol by nasal drip as described above.

The results are shown in FIG. 3.

(2) RSV infected mouse in vivo imaging detection

The results of detection by using a small animal living body imaging system are shown in figures 4-6, and the results show that no fluorescence signal is seen in a PBS (phosphate buffer solution) treatment group, and obvious fluorescence signals can be seen in 1-5 days of RSV (respiratory syncytial virus) infected persons and are distributed in the nasal cavity and the lung of a mouse. RSV was significantly reduced in thenasal cavity site 1 to 4 days after SV29 and SV29-Chol treatment compared to untreated RSV control group, particularly at the peak of virus replication atday 2. The results indicated a decrease in RSV signal in the SV29 treated group, but not in the SV29-Chol treated group.

(3) Quantitative analysis of RSV-infected mouse pulmonary virus

The results of quantitative detection of lung RSV replication levels of mice of each experimental group by the quantitative PCR method are shown in FIGS. 7-8, and the results show that. Both SV29 and SV29-Chol treatments significantly reduced RSV viral load in the lungs of mice. In contrast, SV29 is superior to SV 29-Chol. Meanwhile, the RSV virus replication level in the lung of a mouse is analyzed by adopting an enzyme-linked immunospot assay, and the result shows that SV29 and SV29-Chol can reduce the RSV virus amount in the lung to a very low level.

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

AA1 is Ile, or Leu;

AA3 is Gln, or Glu;

AA10 is Gln, or Glu;

AA14 is Phe, or Lys;

AA17 is Lys, or is Glu;

AA21 is Leu, or Lys;

AA24 is Asn, or Glu;

AA28 is Gly, or is Lys;

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

AA30 is Cys, or is absent;

when AA30 is Cys, R1 is H;

when AA30 is absent, R2 is absent;

AA31 is NH₂Or OH.

R1 in structure I is H, or succinic acid cholesterol monoester, or 2-cholesterol acetic acid, or 2-cholesterol propionic acid, or 2-cholesterol butyric acid, or 2-cholesterol isobutyric acid, or 2-cholesterol valeric acid, or 2-cholesterol isovaleric acid, or 2-cholesterol hexanoic acid, HO, or cholesterol ester₂C(CH₂)_n1CO-(γGlu)_n2-(PEG_n3(CH2)_n4CO)_n5-, or is CH₃(CH₂)_n1CO-(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 cholesteryl acetate, or is cholesteryl propionate, or is cholesteryl butyrate, or is cholesteryl isobutyrate, or is cholesteryl valerate, or is cholesteryl isovalerate, or is cholesteryl hexanoate, or is absent.

2. A 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 a mixture of any of the foregoing.

3. A compound according to claim 1 and claim 2 for the preparation of a pharmaceutical composition for the treatment of a disease.

4. The pharmaceutical composition of claim 3, for use in the manufacture of a medicament for the treatment of syncytial virus pneumonia.

5. A compound of structure I according to claim 1, comprising the compound for use in a method of treatment of syncytial virus pneumonia.

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