CN114957403B

CN114957403B - Broad-spectrum coronavirus membrane fusion inhibitor and pharmaceutical application thereof

Info

Publication number: CN114957403B
Application number: CN202110206577.8A
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: 2021-02-24
Filing date: 2021-02-24
Publication date: 2023-09-26
Anticipated expiration: 2041-02-24
Also published as: CN114957403A

Abstract

The invention relates to the field of medicine synthesis, and discloses a broad-spectrum coronavirus membrane fusion inhibitor. The broad-spectrum coronavirus membrane fusion inhibitor is used for preparing a pharmaceutical composition for preventing and treating diseases caused by coronaviruses, and the pharmaceutical composition is used for preventing and treating the diseases caused by the coronaviruses.

Description

Broad-spectrum coronavirus membrane fusion inhibitor and pharmaceutical application thereof

Technical Field

The invention relates to a broad-spectrum coronavirus membrane fusion inhibitor and a pharmaceutical application thereof.

Background

Coronaviruses (covs) are enveloped single-stranded positive-strand RNA viruses that are divided into four genera α, β, γ and δ, where the genera α and β CoV only infect mammals and the genera γ and δ CoV primarily infect birds. Currently 7 types of CoVs are known to infect humans (HCoVs), including HCoV-229E and HCoV-NL63 of the alpha genus, HCoV-OC43 of the beta genus, coV-HKU1, SARS-CoV, MERS-CoV, and the newly emerging 2019 new coronavirus SARS-CoV-2. The first 4 hcovs are common global epidemic pathogens, usually cause only common cold symptoms, accounting for about 10% to 30% of upper respiratory tract infections in adults, but can still cause serious and even fatal diseases for children, the elderly and immunocompromised patients; SARS-CoV, MERS-CoV and SARS-CoV-2 are highly pathogenic pathogens, which can cause serious pulmonary diseases with high mortality.

The research and development of virus membrane fusion inhibitor medicine is continuously carried out by the scientific research team, a series of polypeptide membrane fusion inhibitors with strong inhibition activity on SARS-CoV-2 are developed, and the polypeptide membrane fusion inhibitors can also effectively inhibit SARS-CoV infection, and see Chinese patent CN202010181328.3 in detail. Since the invention provides lead compounds for further developing highly pathogenic HCoV therapeutic drugs, the invention aims to further develop a coronavirus virus membrane fusion inhibitor with broad spectrum and high activity based on the above patent so as to meet the clinical demands.

Disclosure of Invention

The invention provides a novel coronavirus 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.

AA1 in Structure I is (AA 4) _m Or is absent;

wherein:

m is an integer from 1 to 10;

AA4 is Gly, or Ser;

AA2 in structure I is 2, 5-dioxaborolidin-1-yl- (CH) ₂ ) _n COOH

Wherein:

n is an integer from 1 to 10.

AA3 in structure I is ((PEG) _n1 (CH ₂ ) _n2 CO) _n3 ) _n4 -；

Wherein:

n1 is an integer from 1 to 30;

n2 is an integer from 1 to 5;

n3 is an integer from 1 to 5;

n4 is an integer from 1 to 5.

X2 in the structure I is 1, X1 is 0, and X4 is 0;

in structure I, X2 is 2, X1 is 1, X3 is 0, and X4 is 1.

The invention provides methods for preventing and treating diseases caused by coronaviruses comprising the compounds of the invention.

The invention also provides a pharmaceutical composition comprising the compound for preventing and treating diseases caused by coronaviruses.

Preferably, the pharmaceutical composition is used for preventing and treating diseases caused by coronaviruses.

Coronaviruses according to the present invention are coronaviruses including but not limited to SARS-CoV, MERS-CoV, 2019-nCoV and ordinary HCoV (e.g. 229E, OC43 and NL 63).

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 compound shown in the structure I has stable properties, is a novel high-efficiency and broad-spectrum coronavirus membrane fusion inhibitor, and is used for preparing a pharmaceutical composition for preventing and treating diseases caused by coronaviruses.

Detailed Description

The invention discloses a broad-spectrum coronavirus membrane fusion inhibitor and pharmaceutical application thereof, and a person skilled in the art can properly improve related parameters by referring to the content of the present disclosure. 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:

EXAMPLE 1 Synthesis of Polypeptides

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 corresponding protecting amino acid of the polypeptide amino acid sequence in sequence 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 to other protected amino acids of the backbone

The same method of accessing the 1 st protected amino acid of the main chain is adopted, other corresponding protected amino acids are sequentially accessed,

obtaining the resin containing main chain amino acid.

(3) Side chain access

Taking 0.03mol of succinic acid cholesterol monoester and 0.03mol of HOBt, and dissolving the cholesterol monoester and the HOBt with a proper amount of DMF; another 0.03mol 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 Linear peptide

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 linear peptide crude product.

3. Preparation of crude product

Taking a linear peptide crude product and maleimide cholesterol derivative (the molar ratio of the crude product to maleimide group is 1:1), dissolving the crude product and the maleimide cholesterol derivative in 30% acetonitrile/water solution, regulating the pH value to 6.0-7.0 by using 1% NaOH solution, and regulating the pH value to 4.0 by using 1% trifluoroacetic acid solution after reacting for 2 hours to obtain a crude product solution.

4. Preparation of pure product

Filtering the crude product solution with a 0.45 μm mixed microporous filter membrane, and purifying for 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, cyclic loading, loading in chromatographic column, starting mobile phase elution, collecting spectrum, observing absorbance change, collecting salt-exchange main peak, analyzing liquid phase to detect purity, mixing salt-exchange main peak solutions, concentrating under reduced pressure to obtain pure acetic acid aqueous solution, and freeze drying to obtain pure product.

The following lipopeptides were synthesized using the above procedure:

the structure of the control compound is:

example 2 inhibition of viral infection

1. Experimental materials and methods

Plasmids expressing the S protein of SARS-CoV-2 virus (defined as pCoV 2-S), see published papers by the inventors (Zhu et al cross-reactive neutralization of SARS-CoV-2by serum antibodies from recovered SARS1 patients and immunized animals.Sci Adv.2020;6 (45): eabc9999 and Zhu et al design of Potent Membrane Fusion Inhibitors Against SARS-CoV-2,an Emerging Coronavirus with High Fusogenic Activity.J Virol.2020;94 (14): 00653-20). HIV backbone plasmid pNL4-3.Luc. RE was supplied by the national institutes of health AIDS reagent and reference project (catalog number 3418); 293T cells were purchased from American type culture Collection (ATCC, catalog number CRL-3216); target cell Huh-7 was purchased from the national laboratory cell resource sharing service platform (number 3111C0001CCC 000679); target cells 293T/ACE2 were prepared and stored by the present laboratory (Cao et al, potential and persistent antibody responses against the receptor-binding domain of SARS-CoV spike protein in recovered components, virology Journal,2010, 7:299). The basic steps of an antiviral experiment based on S protein pseudovirus are as follows:

(1) Preparation of pseudoviruses: the pCoV2-S plasmid and pNL4-3.Luc. RE were co-transfected into 293T cells at 1:1 at 37℃with 5% CO ₂ Culturing in cell incubator for 48 hr, collecting supernatant containing pseudovirus, filtering, and storing at-80deg.C.

(2) The polypeptide to be detected is dissolved by deionized water or dimethyl sulfoxide (DMSO) and the concentration is measured, then the polypeptide is diluted to the initial concentration by a DMEM culture medium, 3 times of dilution is carried out in 96 cell culture plate holes, the final volume of the polypeptide solution is 50 mu L/hole, and 3 compound holes and 9 dilution gradients are arranged. DMEM medium without polypeptide was used as a control.

(3) Pseudoviruses were added to the drug dilution plate wells at 50 μl per well and then incubated for 30 minutes at room temperature.

(4) The concentration of 293T/ACE2 cells or Huh-7 cells cultured in advance was adjusted to 10X 10 ⁴ Per mL of suspension, and DEAE-dextran was added at a final concentration of 15. Mu.g/mL, and then the cells were added to a 96-well plate containing virus at 100. Mu.L/well. At 37℃with 5% CO ₂ The cells were cultured in a cell incubator for 48 hours.

(5) After removing the supernatant, 30. Mu.L/well of the cell lysate was added, and after 15 minutes of lysis at room temperature, a luciferase substrate (Promega Co.) was added, the relative fluorescence units (RLU) were measured by a microplate photometer, and the inhibition rate curve and the half-maximal Inhibition Concentration (IC) of the drug were calculated ₅₀ )。

2. Experimental results and analysis

The experimental results show that: the average IC50 values of three experiments of the novel membrane fusion inhibitors IBP 24-IPB 27 on inhibiting the activity of SARS-CoV-2 Pseudovirus (PV) infection 293T/ACE2 or Huh-7 target cells are shown in the following table, and the novel membrane fusion inhibitor has the activity improved by more than 13 times compared with the IPB 20.

EXAMPLE 3 inhibition of viral cell membrane fusion

1. Experimental materials and methods

To further evaluate the anti-SARS-CoV-2 activity of polypeptide inhibitors, the present invention conducted a DSP-based cell-cell fusion inhibition assay (method of Zhu et al design of Potent Membrane Fusion Inhibitors Against SARS-CoV-2,an Emerging Coronavirus with High Fusogenic Activity.J Virol.2020;94 (14): 00653-20) as follows:

(1) 293T effector cell suspensions (1.5X10) ⁴ mu.L/well) was plated in 96-well plates while 293T/ACE2 target cell suspensions (15X 10) ⁴ Individual/mL) was spread on 10-cm cell culture dishes and placed at 37℃and 5% CO ₂ Culturing under the condition.

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

(3) After 24 hours, polypeptides were subjected to 3-fold gradient dilutions in 96-well plates, 3 multiplex wells and 9 dilution gradients were set. Adding diluted polypeptide to effector cells at 37deg.C, 5% CO ₂ Incubate in cell incubator for 1 hour.

(4) DMEM complete medium was pre-warmed and mixed at 1:4000 ratio Enduren viable cell substrate (Promega Co.) was added and then used to resuspend the centrifuged 293T/ACE2 target cells to adjust the cell concentration to 30X 10 ⁴ /mL, at 37℃with 5% CO ₂ Incubate for 30 minutes under conditions.

(5) 293T/ACE2 target cells were added to 293T effector cells at 100. Mu.L/well, and then centrifuged at 400Xg for 3 minutes for sufficient contact between effector cells and target cells, followed by culturing the mixed cells for 2 hours.

(6) Luciferase activity (RLU) was read in microplate luminometer and inhibition and IC were calculated ₅₀ Values.

2. Experimental results and analysis

The experimental results are shown below, which show that the inhibition activity of the novel membrane fusion inhibitor IBP 24-IPB 27 on SARS-CoV-2S protein mediated cell-cell fusion capability is improved by more than 3 times compared with the IPB20 activity.

Claims

1. A coronavirus membrane fusion inhibitor having the structural formula:

compound 1

Ac-Ser-Val-Val-Asn-Ile-Gln-Lys-Glu-Ile-Asp-Arg-Leu-Asn-

Glu-Val-Ala-Lys-Asn-Leu-Asn-Glu-Ser-Leu-lle-Asp-Leu-Gln-

Glu-Leu-Gly-Lys-Tyr-Glu-Gln-Tyr-Ile-Gly-Ser-Gly-Ser-Gly-

Cys[2,5-dioxopyrrolidin-1-yl-(CH ₂ ) ₂ CO-PEG ₄ CH ₂ COO-cholesterol]-NH ₂ ；

Compound 2

2. A pharmaceutically acceptable salt of the coronavirus membrane fusion inhibitor of claim 1.

3. A pharmaceutical composition comprising the coronavirus membrane fusion inhibitor of claim 1.

4. Use of the coronavirus membrane fusion inhibitor of claim 1 in the manufacture of a medicament for the prevention and treatment of diseases caused by coronaviruses, said coronaviruses being SARS-CoV-2.