CN115068587A - Glycopeptide compound with antiviral activity and application thereof - Google Patents

Abstract

The invention discloses a glycopeptide compound with antiviral activity and application thereof in preparing antiviral drugs. The glycopeptide compound has a structure shown in a general formula (I), wherein R ₁ Selected from N-decylaminoethyl, N-p-chlorobiphenylmethylaminoethyl, 4-trifluoromethylbenzyl, 4' -chlorobiphenylmethyl, or docosahexenoyl; r ₂ Selected from hydroxyl, hydroxylamine, 4-fluorobenzylamine, 4-cyanobenzylamine or 4-nitrophenylethylamine. The glycopeptide compound has the activity of resisting the new coronavirus, and has great potential value for preparing a medicament for treating coronavirus infection, particularly the new coronavirus infection.

Description

Glycopeptide compound with antiviral activity and application thereof

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to a glycopeptide compound with antiviral activity and application thereof.

Background

Glycopeptide antibiotics are traditional antibacterial drugs, and research proves that: glycopeptide drugs also have antiviral activity, and reference is made to reports such as: journal of Medicinal Chemistry,2005,48(11):3885-3890, Journal of Biological Chemistry,2016,291(17):9218-9232, and patent CN104958755A, etc. Recent studies have shown that: teicoplanin, one of the representatives of glycopeptide drugs, has the effect of preventing new coronaviruses from invading cells (see the report of the bioRxiv paper "Teicoplanin patent blocks of 2019-nCoV" (doi: https:// doi. org/10.1101/2020.02.05.935387) of the platform preprinted in the biological field). Therefore, the glycopeptide antibiotic has potential value as a new coronary pneumonia medicament.

In the existing report, the patent CN101928331A granted by the invention in china discloses a glycopeptide compound with a completely new structure, as shown in compound (ii), which is structurally characterized in that the amino acid residues at the 4-position and the 6-position of the peptide skeleton have L-vancosamine. The studies have confirmed that the derivatives of compound (ii) have higher antibacterial activity and safety compared to the glycopeptide drugs already on the market, and these studies are described in: bioorg Med Chem Lett,2011,21: 6732-. The glycopeptide compound is subjected to series derivatization, and the in vitro anti-new coronavirus activity is researched, so that a new idea is provided for the development of anti-new coronavirus medicines.

Disclosure of Invention

In view of this, the invention uses the compound (II) disclosed in Chinese patent CN101928331A as raw material, and derivatizes the compound to obtain a group of improved glycopeptide compounds. Tests prove that the glycopeptide compound has the activity of resisting the new coronavirus; further tests show that the activity of part of glycopeptide compounds of the invention against new coronavirus is obviously superior to that of vancomycin and teicoplanin, and the glycopeptide compounds can be used for preparing medicines for treating coronavirus infection, particularly treating new coronary pneumonia.

Therefore, the first objective of the present invention is to provide an application of glycopeptide compound in the preparation of antiviral drugs, wherein the glycopeptide compound has a structure as shown in the following general formula (I):

wherein:

R ₁ selected from N-decylaminoethyl, N-p-chlorobiphenylmethylaminoethyl, 4-trifluoromethylbenzyl, 4' -chlorobiphenylmethyl, or docosahexenoyl;

R ₂ selected from hydroxyl, hydroxylamine, 4-fluorobenzylamine, 4-cyanobenzylamine or 4-nitrophenylethylamine.

In the present invention, the glycopeptide compound is preferably: the R is ₁ Is N-decylaminoethyl or 4' -chlorobiphenylmethyl, and said R ₂ Is a hydroxylamine group, a 4-fluorobenzylamine group, a 4-cyanobenzylamine group or a 4-nitrophenylethylamino group.

Further preferably, said R ₁ Is 4' -chlorobiphenylmethyl, and, said R ₂ Is 4-fluorobenzylamine group or 4-cyanobenzylamine group.

Further, the virus is a novel coronavirus.

Further, the antiviral drug dosage forms include injections, oral preparations, inhalants, and external preparations.

When used for injection, the injection can be prepared by intravenous injection, intravenous drip, subcutaneous injection, intramuscular injection and intraperitoneal injection; for oral administration, it can be made into solid preparation such as tablet or capsule; for inhalation therapy, it can be made into aerosol, spray and powder spray; for external use, it can be made into ointment or loaded on pharmaceutically acceptable carrier.

Furthermore, the glycopeptide compound in the pharmaceutical dosage form is 0.1-99.9% by weight.

Preferably, the glycopeptide compound in the pharmaceutical dosage form is 0.5-99.5% by weight.

In the invention, the glycopeptide compound in the antiviral drug is in a salt form.

Further, the salt form of the glycopeptide compound comprises: hydrochloride, hydrobromide, sulphate, nitrate, phosphate, formate, acetate, propionate, oxalate, malonate, succinate, fumarate, maleate, lactate, malate, tartrate, citrate, picrate, methanesulphonate, aspartate, and glutamate.

Another object of the present invention is to provide a glycopeptide compound having antiviral activity, wherein the glycopeptide compound has a structure represented by the following general formula (I):

wherein:

R ₁ selected from N-decylaminoethyl, N-p-chlorobiphenylmethylaminoethyl, 4-trifluoromethylbenzyl, 4' -chlorobiphenylmethyl, or docosahexenoyl;

R ₂ selected from hydroxyl, hydroxylamine, 4-fluorobenzylamine, 4-cyanobenzylamine or 4-nitrophenylethylamine.

After the test, the test paper is tested,the glycopeptide compound has the activity of resisting new coronavirus; the anti-new coronavirus activity of part of glycopeptide compounds is obviously superior to that of vancomycin and teicoplanin, and R in the structural formula of the glycopeptide compounds ₁ Is 4' -chlorobiphenylmethyl, said R ₂ When the compound is 4-fluorobenzylamine or 4-cyanobenzylamine, the compound has better selective inhibition effect on new coronavirus, and has great potential value for preparing medicaments for treating coronavirus infection, particularly new coronavirus infection.

Drawings

FIG. 1 shows the results of activity test of Compounds 3, 4, 5, 6 and 9 and vancomycin and teicoplanin inhibiting SARS-CoV-2 pseudovirus and VSV-pseudotype virus new coronavirus, respectively, in example 5;

FIG. 2 shows the results of activity tests of Compounds 3, 4, 5, 6 and 9 and vancomycin and teicoplanin, respectively, inhibiting SARS-CoV-2 pseudovirus attachment in example 6;

FIG. 3 shows the results of activity tests of compounds 3, 4, 5, 6 and 9 and vancomycin and teicoplanin in example 7 for inhibiting the binding of RBD domain of Spike protein to ACE2 protein;

FIG. 4 shows the results of the activity test of compounds 3, 4, 5, 6 and 9 in example 8 in binding to the RBD domain of Spike protein.

Detailed Description

The glycopeptide compounds of the present invention may be prepared by methods described in the above published literature reports, and it should be understood that the examples detailed below are for illustration only and are not intended to limit the scope of the present invention.

In the present invention, the method for obtaining the raw material compound (ii) is described in chinese patent CN101928331A, and other raw materials, apparatuses, and the like in the present invention are commercially available.

In the present invention, unless otherwise stated, all room temperatures refer to temperatures of 20 ℃ to 30 ℃; the proportions of the eluents are all volume percentages; the yield is the molar yield. The following abbreviations have the following meanings, undefined abbreviations have their commonly accepted meanings:

DIEA N, N-diisopropylethylamine

DMSO dimethyl sulfoxide

ESI electrospray ionization mass spectrometry

1H-NMR hydrogen nuclear magnetic resonance spectrum

2019-nCoV 2019 novel coronavirus

PICB 2-methylpyridine-N-borane

PyBOP 1H-benzotriazol-1-yl-oxytripyrrolidinyl hexafluorophosphate

TFA trifluoroacetic acid

Example 1 preparation of Compounds

1. Dispersing compound (II) (2.0g, 1.3mmol) in 20mL DMSO-methanol (1:1, v/v) at room temperature, then adding DIEA (0.4mL, 2.6mmol) and 4' -chlorobiphenyl-4-carbaldehyde (0.4g, 1.7mmol), heating to 65 ℃ and stirring for 2h, standing and cooling to room temperature; adding PICB (0.3g, 2.6mmol), stirring at room temperature for 2h, adding TFA (0.4mL, 5.2mmol), stirring at room temperature for 1h, pouring the reaction solution into 150mL ethyl acetate, separating out insoluble substances, filtering, washing the filter cake with a small amount of ethyl acetate, and removing the solvent; the resulting solid was dissolved in 10mL of DMSO, DIEA (0.7mL, 3.9mmol) and 4-nitrophenylethylamine hydrochloride (0.53g, 2.6mmol) were added in that order, stirred well, then charged into PyBOP (1.4g, 2.6mmol), stirred at room temperature for 3h, the reaction mixture was poured into 150mL of acetone, the insoluble material was precipitated by stirring, filtered, the filter cake was washed with a small amount of acetone, and the solvent was removed.

2. The solid obtained above was purified using reverse phase polymeric filler Uni PSA25-300, eluted with a solution of 0.04% TFA in acetonitrile-water (acetonitrile to water ratio 1:4 by volume) and the eluate was concentrated to dryness to give 0.81g of a white solid.

The chromatographic purity of the obtained white solid was determined to be 98.0% with a yield of 32.0%.

And (3) respectively using electrospray ionization mass spectrum and nuclear magnetic resonance hydrogen spectrum to characterize the product, and confirming the structure: ¹ H- NMR(600MHz,DMSO-d ₆ +D ₂ O)δ(ppm):8.50-8.05(7H),8.00-7.32(10H),7.20-7.05(7H), 6.53-6.46(3H),6.03-5.68(3H),5.50-5.02(3H),4.90-4.87(1H),4.84-4.52(6H),3.97-3.74 (4H),3.55-3.17(5H),3.10-2.76(7H),2.70-2.30(4H),2.25-2.03(1H),2.05-1.13(12H), 0.84-0.75(6H)；C ₉₄ H ₁₀₅ Cl ₃ N ₁₂ O ₂₇ calculated molecular weight: 1938.63, found: 1939.6(M + H).

The preparation methods of the remaining glycopeptide compounds referred to in the examples are the same as the preparation method of compound 9. R of glycopeptide compound of the present invention ₁ And R ₂ The structural formula, the molecular formula and the ESI measured value of the compound are shown in Table 1, and the calculated molecular weight and the ESI measured value of each glycopeptide compound obtained by the preparation are also substantially consistent.

TABLE 1 Structure of respective glycopeptide compounds

Example 2 salt formation example 1

Adding 5mL of saturated hydrogen chloride methanol solution into 100mg of the compound 6, stirring at room temperature for 10min, evaporating under reduced pressure, adding 30mL of acetone, stirring, filtering, and removing the solvent to obtain 100mg of a trihydrochloride white solid of the compound 6, analyzing Cl elements: theoretical 10.58%, found 10.62%.

Example 3 salt formation example 2

1.0g of Compound 9 was dissolved in 10mL of water at room temperature, 43. mu.L of commercial hydrochloric acid (HCl one-fold stoichiometric) was added thereto, stirred at room temperature for 10min, and lyophilized to give 1.0g of Compound 9 as a monohydrochloride white solid, elemental Cl analysis: theoretical 7.17%, found 7.18%.

In addition, in the salt forming process, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, methanesulfonic acid, aspartic acid or glutamic acid are respectively used for replacing hydrogen chloride in the above embodiments, and then the corresponding salt is obtained.

Example 4 formulation example (injection)

It should be noted that the present embodiment is for illustration only, and is not intended to limit the scope of the present invention. The term "active ingredient" means each compound, solvate, tautomer, optical isomer, prodrug, pharmaceutically acceptable salt and the like in the present invention.

In the pharmaceutical dosage form, the content of the glycopeptide compound as an active ingredient is 0.1-99.9% by weight, and more preferably, the content of the glycopeptide compound in the pharmaceutical dosage form is 0.5-99.5% by weight.

Specifically, an injection can be prepared as follows:

active ingredient 100mg

Isotonic saline 1000mL

Solutions of the above ingredients are typically administered intravenously to a patient at a rate of 1 mL/min.

In addition, the general dosage of the pharmaceutical preparation applied to a patient in need of treatment can be referred to the existing dosage of vancomycin and teicoplanin, for example, an adult can be 0.1-2.0 g/day, and the dosage can be changed according to the age, the disease condition and the like of the patient.

Example 5 assay for inhibition of Neocoronaviruses by glycopeptide Compounds

The experimental principle is as follows: in this example, the effect of the novel glycopeptide compound against the new coronavirus was detected at 293T/ACE2 cell level, and the specific and highly inhibitory compound was selected by testing the inhibition of the novel glycopeptide compound against the pseudovirus carrying the Spike protein gene of the new coronavirus and the Spike protein replication-deficient VSV-pseudotype virus of the new coronavirus, and comparing the inhibition differences. The 293T/ACE2 cell line is infected by pseudovirus based on lentivirus to evaluate the effect of the novel glycopeptide compound on the new coronavirus.

The detection method comprises the following steps: this example uses the luciferase assay system (Promega) to measure luciferase activity to calculate the survival rate of the virus.

The testing steps are as follows:

1. the new coronavirus pseudovirus was obtained by cotransfection of 293T cells with psPAX2, plenti-luc-/GFP and the virus envelope protein expression plasmid pCAGGS-nCoV-Sopt.

2. 293T/ACE2 cells at 10 ⁴ The cells/well were plated in 96-well plates and cultured for 12 h. Diluting compounds with different concentrations in a new coronavirus pseudovirus mixed culture medium with the same volume, and adding the diluted compounds into a pre-inoculated 293T/ACE2 for infection; after 12 hours, the medium was refreshed and cultured for another 48 hours.

3. Equal volumes of DMSO were added to media containing different concentrations of the compounds to achieve a final DMSO concentration of 1% (v/v).

4. Luciferase activity was measured using the luciferase assay system (Promega).

The virus survival (%) was calculated as follows:

viral inhibition (%) ═ 1-viral survival (%)

The experimental procedure was performed in a total of 3 replicates.

The virus inhibition test of various novel glycopeptide compounds shows that under the condition of 10 mu M concentration, a plurality of glycopeptide compounds can effectively block viruses from entering cells to realize the infection of the cells, and have the activity of resisting new coronavirus, and the results are shown in table 2.

TABLE 2 inhibition ratio of glycopeptide compounds against viruses

As can be seen from the results in table 2, all of the 10 glycopeptide compounds in example 1 showed significant inhibitory effects against the new coronaviruses, and the antiviral activities thereof were significantly higher than those of the existing teicoplanin, vancomycin and the compound of formula (II); among them, compounds 3, 4, 5, 6 and 9 had better anti-neocoronaviral activity.

FIG. 1 shows the results of the activity test of the compounds 3, 4, 5, 6 and 9 and vancomycin and teicoplanin in this example against SARS-CoV-2 pseudovirus and VSV-pseudotype virus neocoronavirus respectively, and the EC50 range of the compound of this example against neocoronavirus is 0.8-2.0 μm, wherein the compounds 5 and 6, namely R, is 0.8 μm ₁ Is 4' -chlorobiphenylmethyl, and R ₂ When the compound is 4-fluorobenzylamine or 4-cyanobenzylamine, the compound has the best selective inhibition effect on new coronavirus, and has great potential value for preparing medicines for treating coronavirus infection, particularly new coronavirus infection, so that the compound has important development value and popularization significance.

Example 6 inhibition of the attachment of SARS-CoV-2 pseudovirus by Compounds

Studies have shown that the entry process of the virus is the primary step in achieving its spread. SARS-CoV-2 first attaches to the specific host cell receptor ACE2 via Spike protein (Spike), fuses with the host cell and invades the host cell. Therefore, to further demonstrate the inhibitory effect of each compound on the novel coronavirus, this example was conducted to test the inhibitory activity of each compound on the adhesion of SARS-CoV-2 pseudovirus, and the test principle, detection method, test procedure, and the like were similar to those of example 5, except that: the pseudovirus was premixed with compounds of different concentrations to infect 293T/ACE2 cells, adsorbed on ice for 1 hour and then replaced with fresh medium for further culture for 48 hours to determine luciferase activity.

FIG. 2 shows the activity test results of compounds 3, 4, 5, 6 and 9 and vancomycin and teicoplanin in example 1 for inhibiting the attachment of SARS-CoV-2 pseudovirus, respectively, and according to the results shown in FIG. 2, it can be seen that compounds 3, 4, 5, 6 and 9 all have high inhibitory activity, the compound of this example has an EC50 action range of 4-20 μ M, and the inhibitory activity of each compound is significantly higher than that of teicoplanin and vancomycin. The experimental result shows that the glycopeptide compound in the embodiment 1 can achieve the purpose of blocking the virus from invading host cells by inhibiting the attachment of the virus.

Example 7 Compounds inhibit the binding of the RBD domain of the Spike protein to the ACE2 protein

This example further demonstrates the inhibitory effect of each compound on the binding of the RBD region of the Spike protein to the ACE2 protein, since binding of the RBD region to ACE2 is the primary step in the attachment of the SARS-CoV-2 virus.

The experimental principle is as follows: the novel coronavirus (SARS-CoV-2) Spike (Spike, S) glycoprotein has two subunits: the S1 subunit and the S2 subunit, S1 subunit have ACE2 receptor binding site RBD region. This example uses homogeneous time-resolved fluorescence (HTRF) to detect the direct effect of each compound on the binding of the RBD domain of Spike protein to ACE2 protein. In the experiment, energy transfer occurs when the RBD region of the Spike protein is close enough to the ACE2 protein, and the energy donor is Europium lanthanide (Europium) in rare earth elements, the structure is stable, the d2 is an energy acceptor, and the energy acceptor is coupled with a corresponding antibody. Thus, when a compound or antibody blocks its interaction, the signal ratio decreases.

The detection method comprises the following steps: in the present case, the effect of the compound on the Binding of ACE2/spike (RBD) was evaluated by using ACE2/spike (WT) Binding Assay Kits of the CISBIO company.

The experimental steps are as follows:

1. respectively preparing 25nM Tag1-SARS-CoV-2Spike working solution, 75nM Tag2-ACE2 working solution, 1 times concentration anti-Tag1-Eu ³⁺ Working solution, 1-time concentration anti-Tag2-d2 working solution.

2. Preparing a 96-well shallow white plate, and adding a compound and the solution into each well: 2 μ L of compound, 4 μ L of Tag1-SARS-CoV-2Spike and 4 μ L of anti-Tag1-Eu ³⁺ Incubate at room temperature for 15 min.

3. Mixing anti-Tag1-Eu ³⁺ Premix with anti-Tag2-d2 in equal volume, add 10 μ L of premix per well, seal and incubate at room temperature for 3 hours.

4. Read on Victor plate reader.

The calculation method is as follows:

FIG. 3 shows the results of activity tests of compounds 3, 4, 5, 6 and 9 and vancomycin and teicoplanin in example 1 for inhibiting the binding of RBD domain of Spike protein to ACE2 protein; the results shown in FIG. 3 show that the RBD regions of the compounds 3, 4, 5, 6 and 9 and the ACE2 protein have significantly higher binding activity than vancomycin and teicoplanin, and the action EC50 range is 10-60 μ M. Experimental results show that the glycopeptide compound in the embodiment 1 can directly inhibit the combination of the RBD region of the Spike protein and the ACE2 protein, and further inhibit the new coronavirus.

Example 8 Compounds capable of binding to the RBD domain of Spike protein

The experimental principle is as follows: the intermolecular interaction technology can directly measure the equilibrium dissociation constant and the like among molecules, and plays an important role in the research of virus invasion mechanisms, the development of antibodies, the screening of micromolecular drugs and the research and development of vaccines. Surface Plasmon Resonance (SPR) technology is widely used with its outstanding accuracy, stability and high reproducibility. In the invention, based on the fact that each compound can directly inhibit the combination of an RBD region and ACE2, the competitive combination relationship among the compound, ACE2 and RBD is presumed. In this example, the association dissociation reaction of each compound with the RBD region was measured by SPR technique.

The experimental steps are as follows:

1. preparing a buffer solution: protein-coupled buffer solution 1.05 × HBS-EP +; interaction buffer solution: 1.0 × HBS-EP + (pH7.4), 5% DMSO.

2. Pre-enrichment: protein coupling conditions were investigated.

3. Protein coupling: RBD protein coupling conditions are that the concentration is about 25 mug/ml, the system is a sodium acetate solution with pH5.0, the chip activation time is 420s, and the blocking time is 420 s.

4. Protein-compound interaction assay:

(1) solvent calibration solutions were prepared with reference to table 3 below:

TABLE 3 solvent correction solution preparation Table

(2) The following series of concentrations were prepared according to compound solubility and chip surface testing, and the measurement conditions are shown in tables 4 and 5 below:

TABLE 4 measurement conditions of the Compounds

TABLE 5 Compound solution preparation Table

FIG. 4 shows the results of the activity test of the compounds 3, 4, 5, 6 and 9 of example 1, respectively, binding to the RBD domain of Spike protein; from the results shown in FIG. 4, it can be seen that the compounds of example 1, Nos. 3, 4, 5, 6 and 9, which have KD values of 2.56. mu.M, 2.77. mu.M, 9.27. mu.M, 4.35. mu.M and 12.19. mu.M, respectively, are capable of binding to the RED region of the Spike protein and have superior binding activity. Therefore, the glycopeptide compound disclosed by the invention can be combined with an RBD (receptor binding domain) to further seal the interaction between ACE2 and the RBD, so that the virus invasion can be inhibited.

The embodiments of the present invention have been described in detail, but the embodiments are merely examples, and the present invention is not limited to the embodiments described above. Any equivalent modifications and substitutions to those skilled in the art are also within the scope of the present invention.

Claims (10)

1. The application of the glycopeptide compound in preparing antiviral drugs is characterized in that the glycopeptide compound has a structure shown in the following general formula (I):

wherein:

R ₁ selected from N-decylaminoethyl, N-p-chlorobiphenylmethylaminoethyl, 4-trifluoromethylbenzyl, 4' -chlorobiphenylmethyl, or docosahexenoyl;

R ₂ selected from the group consisting of a hydroxyl group, a hydroxylamine group, a 4-fluorobenzylamine group, a 4-cyanobenzylamine group, and a 4-nitrophenylethylamine group.

2. Use according to claim 1, wherein R is ₁ Is N-decylaminoethyl or 4' -chlorobiphenylmethyl, and said R ₂ Is a hydroxylamine group, a 4-fluorobenzylamine group, a 4-cyanobenzylamine group or a 4-nitrophenylethylamino group.

3. Use according to claim 2, wherein R is ₁ Is 4' -chlorobiphenylmethyl, and said is 4-fluorobenzylamino or 4-cyanobenzylamino.

4. The use according to claim 1, wherein the virus is a novel coronavirus.

5. The use as claimed in claim 1, wherein the dosage form of the antiviral drug comprises injection, oral preparation, inhalant, and external preparation.

6. The use of claim 5, wherein the glycopeptide compound is present in the pharmaceutical dosage form in an amount of 0.1% to 99.9% by weight.

7. The use of claim 6, wherein the glycopeptide compound is present in the pharmaceutical dosage form in an amount of 0.5% to 99.5% by weight.

8. The use of claim 1, wherein the glycopeptide compound is in the form of a salt.

9. The use of claim 8, wherein the salt form of the glycopeptide compound comprises: hydrochloride, hydrobromide, sulfate, nitrate, phosphate, formate, acetate, propionate, oxalate, malonate, succinate, fumarate, maleate, lactate, malate, tartrate, citrate, picrate, methanesulfonate, aspartate, and glutamate.

10. A glycopeptide compound having antiviral activity, wherein the glycopeptide compound has a structure represented by the following general formula (I):

wherein:

R ₁ selected from N-decylaminoethyl, N-p-chlorobiphenylmethylaminoethyl, 4-trifluoromethylbenzyl, 4' -chlorobiphenylmethyl, or docosahexenoyl;

R ₂ selected from hydroxyl, hydroxylamine, 4-fluorobenzylamine, 4-cyanobenzylamine or 4-nitrophenylethylamine.

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