CN116162136A

CN116162136A - Anti-syncytial virus membrane fusion inhibitor

Info

Publication number: CN116162136A
Application number: CN202111403215.4A
Authority: CN
Inventors: 于海宁; 周述靓; 邓岚; 潘文强
Original assignee: Chengdu Aoda Biotechnology Co ltd
Current assignee: Chengdu Aoda Biotechnology Co ltd
Priority date: 2021-11-24
Filing date: 2021-11-24
Publication date: 2023-05-26
Also published as: WO2023093708A1

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.

The research and development of the virus membrane fusion inhibitor drugs are continuously carried out by the scientific research team, a series of polypeptide membrane fusion inhibitors with strong inhibition activity on syncytial virus (RSV) are developed, and the polypeptide membrane fusion inhibitors can also effectively inhibit the infection of the RSV, and see Chinese patent CN202010108065.3 in detail.

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-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-(AA1)n-AA2(R)-AA3

Structural formula I

AA1 in structure I is Lys, or is Dap, or is Dab, or is Orn, or is Dah, or is Dao, or is Asp, or is Glu, or is Ada, or is Apm, or is Asu;

AA2 in structure I is Lys, or is Dap, or is Dab, or is Orn, or is Dah, or is Dao, or is

Asp [ NH (CH) ₂ ) _m NH]Or Glu [ NH (CH) ₂ ) _m NH]Or Ada [ NH (CH) ₂ ) _m NH]Or Apm [ NH (CH) ₂ ) _m NH]Or is Asu [ NH (CH) ₂ ) _m NH]；

Wherein: m is an integer from 2 to 10;

AA3 in structure I is NH ₂ Or is OH;

n in structure I is an integer from 1 to 10;

r in the structure I is 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 caproic acid.

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	Pbf	(2, 3-dihydro-2, 4,6, 7-pentamethylbenzofuran-5-yl) sulfonyl
Ala	Alanine (Ala)	Leu	Leucine (leucine)
				Arg	Arginine (Arg)	Lys	Lysine
Asn	Asparagine derivatives	Phe	Phenylalanine (Phe)
				Asp	Aspartic acid	Pro	Proline (proline)
Cys	Cysteine (S)	Ser	Serine (serine)
				Gln	Glutamine	Thr	Threonine (Thr)
Glu	Glutamic acid	Trp	Tryptophan
				Gly	Glycine (Gly)	Tyr	Tyrosine
His	Histidine	Val	Valine (valine)
				Ile	Isoleucine (Ile)	Dah	2, 7-diaminoheptanoic acid
Aib	Amino isobutyric acid	Dao	2, 8-diamino octanoic acid
				5-Ava	5-Aminopentanoic acid	Ada	2-aminocaproic acid
Dap	2, 3-diaminopropionic acid	Apm	2-amino heptanoic acid
				Dab	2, 4-diaminobutyric acid	Asu	2-Aminooctanoic acid
Orn	Ornithine

Example 1 preparation of Compounds

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 polypeptide sequence on carrier resin by solid phase coupling synthesis method, and preparing 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

And (3) using Rink Amide BHHA resin as carrier resin, and coupling with protected amino acid corresponding to the polypeptide sequence sequentially through Fmoc protection removal and coupling reaction to prepare the peptide resin.

(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) Access backbone protected amino acids

The same method of accessing the 1 st protected amino acid of the main chain is adopted, and the corresponding protected amino acids in the polypeptide sequence are sequentially accessed to obtain the peptide resin.

(3) Side chain access

Taking 0.02mol of succinic acid cholesterol monoester and 0.02mol of HOBt, and dissolving the cholesterol monoester and the HOBt with a proper amount of DMF; another 0.02mol DIC was added slowly with stirring to the protected amino acid DMF solution and reacted for 30 minutes with stirring at room temperature.

2.5mmol of tetraphenylphosphine palladium and 25mmol of phenylsilane are taken and dissolved by a proper amount of dichloromethane, and enter into resin containing main chain amino acid, and the resin is stirred for deprotection for 4 hours, filtered and washed to obtain dealloc resin for standby.

Adding activated succinic acid cholesterol monoester solution into dealloc resin, coupling for 60-300 min, filtering, washing and drying to obtain 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 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 the pure peptide.

The following lipopeptides were synthesized using the above procedure:

example 2 determination of solubility

The measurement results were as follows

Example 3 determination of in vitro antiviral Activity

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 processed by utilizing Graphpad software, and the IC of each polypeptide drug is calculated ₅₀ Values, experimental results are shown in the following table.

Inhibitory Activity against RSV-Luc

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Claims

1. A compound of structure I:

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-(AA1)n-AA2(R)-AA3

structural formula I

AA2 in structure I is Lys, or is Dap, or is Dab, or is Orn, or is Dah, or is Dao, or is Asp [ NH (CH) ₂ ) _m NH]Or Glu [ NH (CH) ₂ ) _m NH]Or Ada [ NH (CH) ₂ ) _m NH]Or Apm [ NH (CH) ₂ ) _m NH]Or is Asu [ NH (CH) ₂ ) _m NH]；

Wherein: m is an integer from 2 to 10;

AA3 in structure I is NH ₂ Or is OH;

n in structure I is an integer from 1 to 10;

2. A compound according to claim 1, comprising a pharmaceutically acceptable salt, solvate, chelate or non-covalent complex thereof, based on a prodrug of the compound, or a mixture of any of the foregoing forms.

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

4. The pharmaceutical composition according to 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 pneumonitis.