CN112939981A

CN112939981A - Nucleoside compound, pharmaceutically acceptable salt, composition and application thereof

Info

Publication number: CN112939981A
Application number: CN202110066452.XA
Authority: CN
Inventors: 不公告发明人
Original assignee: Jiaxing Jinpaite Biotechnology Co ltd
Current assignee: Jiaxing Jinpaite Biotechnology Co ltd
Priority date: 2021-01-19
Filing date: 2021-01-19
Publication date: 2021-06-11
Anticipated expiration: 2041-01-19
Also published as: CN112939981B

Abstract

The invention relates to a nucleoside compound, pharmaceutically acceptable salts, a composition and application thereof, wherein the nucleoside compound has a structure shown in a formula (I):

Description

Nucleoside compound, pharmaceutically acceptable salt, composition and application thereof

Technical Field

The invention relates to the technical field of medicinal chemistry, in particular to a nucleoside compound, a pharmaceutically acceptable salt, a composition and application thereof.

Background

Feline calicivirus is a single-stranded RNA virus without an envelope and with a spherical capsid, is highly contagious, can cause moderate and self-limited acute oral problems and upper respiratory diseases, and is one of the most common pathogenies of feline infectious upper respiratory diseases. At present, no targeted medicine is available for the cat stomatitis problem caused by the feline calicivirus, hormones or antibiotics are generally adopted for the supportive therapy, the cat stomatitis is easy to relapse, and the cat stomatitis is treated by a full-mouth tooth extraction operation in severe cases, so that great pain is brought to the cat. Therefore, a series of effective anti-calicivirus compounds are urgently needed.

Disclosure of Invention

Based on the above, there is a need for nucleoside compounds, pharmaceutically acceptable salts, compositions and uses thereof. The nucleoside compound has a better effect of resisting feline calicivirus, so the nucleoside compound has the potential of preparing medicaments for treating or preventing related diseases caused by the feline calicivirus.

A nucleoside compound or a pharmaceutically acceptable salt thereof having the structure of formula (I):

wherein:

R₁is-H, halogen, -CN, -C_1-6Alkane, C_2-6Olefins or C_2-6An alkyne;

R₃is-H or-CN;

R₄is-H or-CN.

In one embodiment, R₁is-H, -F, -Br, -I or-CN; and/or

R₄is-H or-CN.

In one embodiment, R₁、R₃And R₄At least one of which is-CN.

In one embodiment, the nucleoside compound is selected from any one of the following compounds:

a composition comprising at least one of the above nucleoside compounds.

In one embodiment, the composition further comprises a therapeutic agent.

In one embodiment, the therapeutic agent is one or more of a corticosteroid, a protease inhibitor, and an anti-inflammatory signal transduction modulator.

In one embodiment, the therapeutic agent is a compound of formula (II):

the use of the above nucleoside compound or a pharmaceutically acceptable salt thereof, or the above composition for the manufacture of a medicament for the treatment or prevention of feline calicivirus infection, feline infectious abdoviral infection, or a disease caused by coronavirus infection.

In one embodiment, the disease caused by feline calicivirus is feline stomatitis.

In one embodiment, the disease caused by feline infectious abdominal virus infection is feline infectious peritonitis.

In one embodiment, the coronavirus is human coronavirus (HCoV)229E or a novel coronavirus SARS-CoV-2.

Advantageous effects

The nucleoside compound has better effect of resisting feline calicivirus, so the nucleoside compound has the potential of preparing medicaments for treating or preventing diseases (such as feline stomatitis) caused by the feline calicivirus. .

Detailed Description

In order that the invention may be more fully understood, a more particular description of the invention will now be rendered by reference to specific embodiments thereof that are illustrated in the appended drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

Interpretation of terms

"alkyl" refers to a saturated aliphatic hydrocarbon group, including straight and branched chain groups. C_1-6Alkyl refers to an alkyl group containing 1 to 6 carbon atoms. Non-limiting examples include: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1-dimethylpropyl, 1, 2-dimethylpropyl, 2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1, 2-trimethylpropyl, 1-dimethylbutyl, 1, 2-dimethylbutyl, 2-dimethylbutyl, 1, 3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2, 3-dimethylbutyl. C_1-4Alkyl refers to an alkyl group containing 1 to 4 carbon atoms. In one embodiment, C_1-4The alkyl group is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl or sec-butyl. Alkyl groups may be substituted or unsubstituted, and when substituted, the substituent may be substituted at any available point of attachment.

"alkenyl" refers to hydrocarbons containing a normal, secondary, tertiary, or ring carbon atom having at least one site of unsaturation, i.e., a carbon-carbon sp2 double bond. Phrases containing the term, e.g., "C2_-6The alkenyl group "means an alkenyl group having 2 to 6 carbon atoms. Suitable examples include, but are not limited to: vinyl (-CH ═ CH2), allyl (-CH2CH ═ CH2), cyclopentenyl (-C5H7) and 5-hexenyl (-CH2CH ═ CH 2).

"alkynyl" refers to a hydrocarbon containing a normal, secondary, tertiary, or ring carbon atom having at least one site of unsaturation, i.e., a carbon-carbon sp triple bond. Phrases containing this term, such as "C2-₆The alkynyl group means an alkynyl group having 2 to 6 carbon atoms. Suitable examples include, but are not limited to: ethynyl (-C.ident.CH) and propargyl (-CH 2C.ident.CH).

In the description of the present invention, "a plurality" means two or more unless specifically defined otherwise.

The compounds of the present invention may exist in unsolvated forms as well as solvated forms containing pharmaceutically acceptable solvents such as water, ethanol, and the like, i.e., both solvated and unsolvated forms.

In the present invention, a certain substitutable site may be substituted with one or more substituents, and when a plurality of substituents are present at the substitutable site, the plurality of substituents may be the same as or different from each other.

In the present invention, a pharmaceutical may comprise, in addition to one or more of the compounds or compositions described herein, a mixture of a physiologically/pharmaceutically acceptable salt or prodrug and other chemical components, as well as other components. Such as physiologically/pharmaceutically acceptable carriers and excipients. Among the excipients, may be one or more buffers, stabilizers, anti-adherents, surfactants, wetting agents, lubricants, emulsifiers, binders, suspending agents, disintegrants, fillers, adsorbents, preservatives for coatings (enteric or sustained release), antioxidants, opacifying agents, glidants, processing aids, colorants, sweeteners, fragrances, flavoring agents, and other known additives.

"pharmaceutically acceptable salt" or "pharmaceutically acceptable salt" refers to an organic or inorganic salt of a pharmaceutically acceptable compound.

When the compound is acidic or includes sufficiently acidic bioisosteres, the appropriate "pharmaceutically acceptable salt" refers to a salt prepared from a pharmaceutically acceptable non-toxic base including inorganic and organic bases. The salts are derived from inorganic bases containing aluminum, ammonium, calcium, copper, iron, lithium, magnesium, manganese, potassium, sodium, octyl, and the like. Particular embodiments include ammonium, calcium, magnesium, potassium, and sodium salts. Salts are derived from pharmaceutically acceptable organic non-toxic bases including salts of primary, secondary and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins such as arginine, betaine, caffeine, choline, N, N.sup.1-dibenzylethylenediamine, ethylenediamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, reduced glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, meglumine, morpholine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine, and the like.

When the compound is basic or includes sufficiently basic bioisosteres, salts can be prepared from pharmaceutically acceptable non-toxic acids, including inorganic and organic acids. Such acids include acetic, benzenesulfonic, benzoic, camphorsulfonic, citric, ethanesulfonic, fumaric, gluconic, hydrobromic, hydrochloric, isethionic, lactic, maleic, malic, mandelic, methanesulfonic, mucic, nitric, pamoic, pantothenic, phosphoric, sulfuric, succinic, tartaric, p-toluenesulfonic acid and the like. Particular embodiments include citric acid, hydrobromic acid, hydrochloric acid, phosphoric acid, sulfuric acid, maleic acid, tartaric acid. Other exemplary salts include, but are not limited to, sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, sulfate, phosphate, acid phosphate, isonicotinic acid, lactic acid, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, fumarate, maleate, gentisate, gluconate, glucuronate, gluconate, formate, benzoate, glutamate, methylsulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate (e.g., 1, 1' -methylene-bis- (2-hydroxy-3-naphthoate)).

In addition, the pharmaceutical preparation containing the compound may be tablets, capsules, oral liquids, pills, granules, powders, ointments, patches, suppositories, buccal tablets, eye drops, eye ointments, ear drops, sprays, aerosols, inhalants, injections, and the like.

The term "therapeutically effective amount" refers to the amount of an effective compound or pharmaceutical agent that is the minimum amount necessary to ameliorate, cure or treat one or more symptoms of a disease or disorder.

In addition, the compounds and compositions of the present invention may be administered alone or in combination with other agents. For combination therapy with more than one active agent, when the active agents are in separate dosage formulations, the active agents may be administered separately or in combination. In addition, administration of one agent may be performed before, simultaneously with, or after administration of another agent. When administered in combination with other agents, the "effective amount" of the second agent will depend on the type of drug used.

The present invention encompasses all crystalline and amorphous forms of the compound or a pharmaceutically acceptable salt thereof. In some embodiments, the compound or pharmaceutically acceptable salt thereof also represents an unlabeled form as well as an isotopically labeled form of the compound. Isotopically labeled compounds have the structure given herein, in which one or more atoms are replaced by an atom having a selected atomic mass or mass number. Examples of isotopes that can be incorporated into compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, and chlorine, such as, but not limited to, 2H (deuterium, D), 3H (tritium), 11C, 13C, 14C, 15N, 18F, 31P, 32P, 35S, 36Cl, and 125I. The invention also includes compounds in which 1 to n of the hydrogens attached to the carbon atoms are replaced with deuterium, where n is the number of hydrogens in the molecule. The compounds exhibit increased metabolic resistance and, therefore, can be used to increase the half-life of the compounds when administered to a mammal. Deuterium labeled or substituted compounds of the present invention may have improved DMPK (drug metabolism and pharmacokinetics) properties with respect to distribution, metabolism and excretion (ADME). Substitution with heavier isotopes such as deuterium may afford certain therapeutic advantages resulting from greater metabolic stability, for example increased in vivo half-life, reduced dosage requirements and/or improved therapeutic index. In the compounds of the present invention, any atom not specifically designated as a specific isotope is intended to represent any stable isotope of that atom. Unless otherwise stated, when a position is specifically designated as H or hydrogen, that position is understood to have hydrogen in its natural abundance isotopic composition.

Route of administration

One or more compounds of the invention are administered by any route appropriate to the organism being treated (e.g., cat). Suitable routes include oral, rectal, nasal, pulmonary, topical (including buccal and sublingual), and parenteral (including subcutaneous, intramuscular), and the like.

The compounds or pharmaceutical compositions of the present invention may also be included in a kit.

Detailed explanation

One embodiment of the present invention provides a nucleoside compound having a structure represented by formula (I):

wherein:

R₁is-H, halogen, -CN, -C_1-6Alkane, C_2-6Olefins or C_2-6An alkyne;

R₃is-H or-CN;

R₄is-H or-CN.

In one embodiment, in the structure of formula (I), R₁is-H, halogen, -CN, methyl, ethyl, propyl, isopropyl, ethenyl, propenyl, ethynyl or propynyl.

In one embodiment, in the structure of formula (I), R₁is-H, -F, -Br, -I or-CN.

In one embodiment, in the structure of formula (I), R₄is-H or-CN.

In one embodiment, in the structure of formula (I), R₃is-H or-CN.

In one embodiment, in the structure of formula (I), R₁、R₃And R₄At least one of which is-CN.

an embodiment of the present invention also provides a composition comprising the above nucleoside compound.

In one embodiment, the composition comprises two or more of the nucleoside compounds.

In one embodiment, the composition comprises at least one compound of the following structure:

in one embodiment, the composition comprises at least one nucleoside compound. Further, the above composition further comprises a therapeutic agent.

In one embodiment, the therapeutic agent is a compound of formula (II):

the compound with the structure shown in the formula (I) and the compound shown in the formula (II) are combined to form a composition, and the experimental result shows that the components have obvious synergistic effect and have obvious inhibition effect in experiments of resisting feline calicivirus, feline infectious abdominal virus infection and coronavirus infection. The compositions of the present invention are shown to have potential for the treatment of diseases caused by feline calicivirus, coronavirus infection.

In one embodiment, the composition is composed of a nucleoside compound represented by formula (I) and a compound represented by formula (II).

An embodiment of the present invention provides a use of the above nucleoside compound or a pharmaceutically acceptable salt thereof, or the above composition for the preparation of a medicament for treating or preventing an antiviral.

An embodiment of the present invention provides a use of the above nucleoside compound or a pharmaceutically acceptable salt thereof, or the above composition for the preparation of a medicament for treating or preventing a disease caused by feline calicivirus.

An embodiment of the present invention provides a method for treating or preventing diseases caused by feline calicivirus, comprising the step of administering an effective amount of the above nucleoside compound or a pharmaceutically acceptable salt thereof, or the above composition to a subject to be treated.

An embodiment of the present invention provides a use of the above nucleoside compound or a pharmaceutically acceptable salt thereof, or the above composition for the preparation of a medicament for treating or preventing a disease caused by feline infectious abdominovirus infection.

In one embodiment, the disease caused by feline infectious peritonitis is feline infectious peritonitis.

An embodiment of the present invention provides a method for treating or preventing a disease caused by feline infectious disease virus infection, comprising the step of administering an effective amount of the above nucleoside compound or a pharmaceutically acceptable salt thereof, or the above composition to a subject to be treated.

An embodiment of the present invention provides a use of the above nucleoside compound or a pharmaceutically acceptable salt thereof, or the above composition for the preparation of a medicament for treating or preventing a disease caused by coronavirus infection.

In one embodiment, the coronavirus is human coronavirus (HCoV) 229E.

In one embodiment, the coronavirus is a feline coronavirus.

In one embodiment, the coronavirus is a novel coronavirus SARS-CoV-2.

An embodiment of the present invention provides a method for treating or preventing a disease caused by coronavirus infection, comprising the step of administering an effective amount of the above nucleoside compound or a pharmaceutically acceptable salt thereof, or the above composition to a subject to be treated.

Further, the subject to be treated is a human or a veterinary; further, the subject to be treated is a human or a cat.

An embodiment of the present invention provides a medicament comprising 1) the above nucleoside compound or a pharmaceutically acceptable salt thereof, or the above composition; 2) pharmaceutically acceptable adjuvants.

An embodiment of the present invention provides a feed comprising the above nucleoside compound or a pharmaceutically acceptable salt thereof, or the above composition.

The present invention will be described below with reference to specific examples.

The compounds in the following examples are each:

example 1

To a mixture of compound a (0.20g) and DMF (2mL) was added N-bromosuccinimide (NBS) (122mg), and the mixture was stirred at 10-20 ℃ for 1 hour. LCMS monitoring indicated the formation of target Ms and the reaction was prepared for purification. Compound a-1(0.20g, 78.5% yield, 99.7% purity) was obtained as a white solid.¹HNMR:(400MHz,DMSO-d6)：δ7.98(s,H),7.06(s,1H),6.25(s,1H),5.20(s,1H),4.93(m,1H),4.53(dd,1H),4.05(m,1H),3.93(m,1H),3.64-3.67(m,1H),3.48-3.51(m,1H).

To a mixture of Compound a-1(0.50g) and DMF (2mL) was added Zn (CN)₂(0.67g,5.71mmol, 362. mu.L, 5.99eq), CuI (18.2mg, 95.3. mu. mol,0.10eq) and Pd (PPh)₃)₄(441mg, 381. mu. mol,0.40eq), under nitrogen, and the reaction was stirred at 140 ℃ for 2 hours. LCMS showed generation of target Ms. The reaction was filtered, and 10mL of water was added to the filtrate, followed by extraction with dichloromethane (20mL × 2). After liquid separation, organic phases are combined and concentrated to be dried to obtain a crude product of the compound a-2, and the crude product is sent to preparation and purification to obtain 0.18g of the compound a-2 with the HPLC purity of 97.9%.¹HNMR:(400MHz,DMSO-d6)：δ8.21(s,1H),7.48(s,1H),6.29(d,1H),5.20(s,1H),4.93(m,1H),4.53(dd,1H),4.05(m,1H),3.93(m,1H),3.65(m,1H),3.53(m,1H),2.61(m,2H).

To a mixture of compound a-2(0.15g) and pyridine (10mL) was added N, N-dimethylformamide dimethyl acetal (0.12g), and the reaction mixture was stirred at 10-20 ℃ for 2.5 hours. The reaction was monitored by thin layer silica gel chromatography and a new spot was observed (EA: MeOH 9:1, R)_f0.4), the reaction solution was concentrated under reduced pressure and purified to obtain compound 1(0.11g, 97.2% purity) as a white solid.¹HNMR:(400MHz,D₂O)：δ8.82(s,1H),8.02(s,1H),7.21(d,1H),4.91(d,1H),4.26-4.29(m,1H),4.24-4.27(m,1H),3.87-3.89(m,1H),3.75-3.76(m,1H),3.25-3.27(m,6H).

To a mixture of compound a (2.00g) and pyridine (56mL) was added N, N-dimethylformamide dimethyl acetal (1.23g), and the reaction mixture was stirred at 10-20 ℃ for 1.5 hours. Monitoring the reaction by thin-layer silica gel chromatography: shows a new point formation (EA: MeOH ═ 10:1, R)_f0.30). The reaction solution was concentrated under reduced pressure and purified to obtain compound 2(2.11g,5.73mmol, 83.4% yield, 94.0% purity) as a white solid.¹HNMR:(400MHz,D₂O)：δ8.79(s,1H),8.00(s,1H),7.03-7.04(d,1H),6.92-6.94(d,1H),4.89-4.90(d,1H),4.28-4.30(m,1H),4.22-4.25(m,1H),3.85-3.88(m,1H),3.73-3.75(m,1H),3.24-3.26(m,6H).

To a mixture of compound b (2.00g) and pyridine (60mL) was added N, N-dimethylformamide dimethyl acetal (1.25g), and the reaction mixture was stirred at 10-20 ℃ for 2.5 hours. Monitoring the reaction by thin-layer silica gel chromatography: shows a new point formation (EA: MeOH ═ 10:1, R)_f0.42). The reaction solution was concentrated under reduced pressure and purified to obtain compound 3(1.85g, 96.0% purity) as a white solid.¹H NMR(400MHz,CD₃OD)δ8.65(s,1H),8.06(d,1H),6.77(d,1H),6.66(d,1H),5.49(d,1H),4.46(t,1H),4.36(d,1H),3.85(d,1H),3.80(d,1H)，3.26-3.28(m,6H).

Activity assay

1. Anti-feline calicivirus (FCV-Kaos) activity assay:

1.1 recovery of F81 cells (Cat kidney cells, purchased from ATCC):

1) opening a constant-temperature water bath kettle in advance, and setting the temperature to be 38 ℃;

2) taking out the cell freezing tube from the liquid nitrogen, putting the cell freezing tube into a water bath kettle for 2-3 min, and completely melting the cell liquid;

3) centrifuging the cell freezing tube for 1000r/min and 10 min;

4) discarding the upper layer liquid of the cell freezing tube in the biological safety cabinet, and taking care not to suck the cells;

5) adding 1mL of 10% DMEM medium along the wall of the cryopreservation tube, slightly adding the DMEM medium, and removing the liquid after cleaning, wherein the cells are not blown up;

6) adding 1mL of 10% DMEM medium into the freezing tube, and gently mixing uniformly;

7) adding the liquid into a T25 cell bottle, supplementing 5-6 mL of 10% DMEM medium, uniformly mixing, placing at 37 ℃ with 5% CO₂Culturing in a constant temperature incubator, and observing the cell state every day.

1.2, subculture of F81 cells:

1) DMEM medium containing 8% newborn calf serum at 37 deg.C and 5% CO₂Culturing F81 cells in a constant temperature incubator;

2) when the cells grow to a monolayer, pouring out the original culture solution in the cell bottle, sucking 2-3 mL of serum-free DMEM by using a Pasteur tube, adding the serum-free DMEM into the cell bottle, washing for three times, and pouring out the culture solution;

3) adding 2-3 mL of 0.05% pancreatin for digestion;

4) standing at 37 deg.C for 5% CO₂Maintaining at constant temperature for 1min until digestion is completed;

5) adding a proper volume of 8% DMEM medium into the culture bottle, and gently and uniformly blowing;

6) averagely dividing the liquid in the cell bottle into two new cell culture bottles, and supplementing each liquid to 5-6 mL;

7) the cell vial was placed at 37 ℃ in 5% CO₂Culturing in a constant temperature incubator, and observing the cell state at intervals.

1.3 titration of viral infectivity (TCID)₅₀Measurement of (2)

Feline FCV-Kaos virus (commercially available from ATCC american type culture collection) was serially diluted 10-fold in different concentrations with maintenance medium, and 3 wells were vertically repeated, sequentially inoculated into 96-well plates of host cells grown in monolayers, and cell controls were set. 37 ℃ and 5% CO₂Culturing in a virus incubator, observing day by day under an inverted microscope, continuously observing for 96 hours,adding 1% neutral red 50 μ L, dyeing at 37 deg.C for 1h, discarding the dye solution, washing the excess dye with washing solution, adding 100 μ L of decolorizing solution, decolorizing at room temperature for 10min, and measuring OD value with microplate reader at 540nm wavelength. Calculating the half infection concentration (TCID) of the virus liquid according to Reed-Muench formula₅₀):

Cell viability ═ (OD value-blank OD value)/(normal cell OD value-blank OD value for each group);

(ii) the rate of cell morbidity is 1-cell survival;

cell distance ═ (above 50% lesion rate-50%)/(-above 50% lesion rate-below 50% lesion rate);

TCID₅₀anti lg (greater than 50% CPE percent virus dilution + beta);

TCID of FCV-Kaos Virus in F81 cells₅₀

The results are shown in Table 2:

TABLE 196 hours Virus TCID₅₀Measurement of

1.4 antiviral test method of Compound

The cell maintenance solution for compound test sample is serially diluted by two times of the original concentration for 10 dilutions, 3 multiple wells are set, 50 μ L of the cell maintenance solution is added into 96-well wells transversely inoculated into F81 cat kidney cells grown into a monolayer, and 100 times of TCID is added into each well₅₀The feline calicivirus FCV-Kaos 50 mu L is provided with a virus control group, a cell control group and a drug toxicity group, and 3 multiple wells. 37 ℃ and 5% CO₂Culturing in incubator, observing cytopathic effect day by day, staining with 1% neutral red when virus contrast has more than 90% pathological changes, and measuring OD value with microplate reader at 540nm wavelength. The half effective concentration of the drug (EC50) was calculated using the Reed-Muench method:

EC50 ═ Antilog (value-distance of dilution of virus above 50% percent CPE) ] × C

(Note: C is the final concentration of the drug in the first well)

2. Detection of anti-feline-transmitted-abdominal-virus activity

2.1, a measuring method:

protection assay for feline infectious peritonitis virus cells feline kidney (CRFK) cells and FIPV-79-1146 (feline infectious abdominovirus) were used. Colloquially, the virus and cells are mixed and incubated for 7 days in the presence of the test compound. The virus was pre-titrated to allow control wells to undergo 85% to 95% apoptosis due to virus replication. Since the test compound inhibits viral replication, an antiviral effect is observed in the presence of the test compound. Each assay plate contained cell control wells (cells only), virus control wells (cells and virus), compound toxicity control wells (cells and compound only), compound colorimetric control wells (compound only), and experimental wells (compound, cells and virus). Determination of EC of cells by MTS (MTS (3- (4, 5-dimethylthiazol-2-yl) -5- (3-carboxymethoxyphenyl) -2- (4-sulfophenyl) -2H-tetrazolium) cell proliferation kit₅₀(concentration of 50% cell survival) and cytotoxic CC₅₀(concentration that resulted in 50% cell death).

2.2 cell preparation

Cat kidney (CRFK) cells (commercially available from the American Type Culture Collection (ATCC)) were grown in DMEM medium (2.0mM L-glutamine, 100 units/ml penicillin and 100. mu.g/ml streptomycin) supplemented with 10% Fetal Bovine Serum (FBS). Using standard cell culture techniques at 1: the cells were subcultured twice a week at a split ratio of 10. Total cell number and percent viability were determined using a hemocytometer and trypan blue exclusion. For cells to be used in the assay, cell viability must be greater than 95%. 1X10 the day before the measurement⁴Concentration of individual cells/well cells were seeded in 96-well tissue culture plates.

2.3 preparation of viruses

The virus used for the assay was feline infectious abdominal virus FIPV-79-1146 (commercially available from the American Type Culture Collection (ATCC)) grown in CRFK cells, producing the virus and serving as a stock virus pool. For each assay, a pre-titrated aliquot of the virus was removed from-70 ℃ conditions and thawed to room temperature in a biosafety cabinet. The virus was then suspended and diluted into tissue culture medium such that the amount of virus added to each well was an amount determined to produce between 85% and 95% cell kill 4-5 days after infection.

2.4 cell staining

After 5 days of infection, cell viability was determined and compound toxicity quantified using MTS cell proliferation kit staining. Cell viability and cytotoxicity of the compounds were determined by quantifying the soluble formazan product produced by MTS metabolism by the mitochondrial enzyme that metabolises active cells. 20-25. mu.L of MTS reagent was added to each well, followed by 5% CO at 37 ℃₂Microtiter plates were incubated for 4-6 hours under conditions and cell viability was then determined. The plates were read spectrophotometrically at 490/650nm using a SpectraMax Plus plate reader.

3. Detection of anti-SARS-CoV-2 activity of novel coronavirus

Cytopathic effect (CPE) experiment, which detects the activity of the compound against human novel coronavirus SARS-CoV-2 in vitro and simultaneously detects the toxicity of the compound to green monkey kidney cell Vero-E6.

3.1 preparation of Compounds

Test compounds were formulated as 20mM stock solutions using DMSO solutions, and compounds were tested at 8 concentrations, diluted in 3-fold gradients, in duplicate wells.

3.2 Experimental protocol

Vero-E6 cells in good growth state were subjected to digestion and passaging, and the cells were seeded into 96-well test plates at a density of 10,000 cells per well with cell growth media and cultured overnight in a 5% CO2 incubator at 37 ℃. The following day, compound diluted in multiples (8 concentration points, duplicate wells) was added, followed by virus at 200TCID per well₅₀Cells were added. Cell controls (cells, no compound treatment or viral infection), virus controls (cells infected with virus, no compound treatment) and broth controls (broth only) were set. The final concentration of DMSO in the culture medium was 0.5%. The cells were cultured in a 5% CO2 incubator at 35 ℃ for 3 days. Cell viability was measured using the cell viability assay kit CellTiter Glo (Promega). The cytotoxicity test and the antiviral test are the same, but no virus infection exists. Antiviral Activity and cytotoxic component of CompoundsThe inhibition rate (%) of the compound against the virus-induced cytopathic effect and the survival rate (%) of MRC5 cells were expressed separately at different concentrations. The calculation formula is as follows:

inhibition (%) ═ test well readings-mean of virus controls)/(mean of cell controls-mean of virus controls) × 100

Cell viability (%) — x 100 (test well reading-broth control average)/(cell control average-broth control average)

Inhibition and cell viability rates of compounds were analyzed by nonlinear fit using GraphPad Prism (version 5) and half effective (EC50) and half cytotoxic (CC50) concentrations of compounds were calculated.

4. Detection of anti-human coronavirus (HCoV)229E Activity

Cytopathic effect (CPE) assay, compounds were tested for in vitro anti-human coronavirus (HCoV)229E activity, as well as for toxicity to MRC5 cells.

4.1 preparation of Compounds

Test compounds were formulated as 20mM stock solutions using DMSO solutions, control compound Remdesivir, compound will test 8 concentrations, 3-fold gradient dilution, duplicate wells.

4.2 cells and viruses

MRC5 cells and HCoV 229E strain were purchased from ATCC. Cells were cultured in EMEM (Sigma) medium supplemented with 10% fetal bovine serum (Hyclone), 1% double antibody (Hyclone), 1% L-glutamine (Gibco) and 1% non-essential amino acids (Gibco). EMEM (Sigma) culture medium supplemented with 5% fetal bovine serum (Hyclone), 1% double antibody (Hyclone), 1% L-glutamine (Gibco) and 1% non-essential amino acid (Gibco) was used as experimental culture medium.

4.3 protocol

MRC5 cells were seeded at a density of 20,000 cells per well into 96 well test plates and cultured overnight in a 5% CO2, 37 ℃ incubator. The next day, compound diluted in multiples (8 concentration points, duplicate wells) was added, followed by virus addition to cells at 200TCID50 per well. Cell controls (cells, no compound treatment or viral infection), virus controls (cells infected with virus, no compound treatment) and broth controls (broth only) were set. The final concentration of DMSO in the culture medium was 0.5%. The cells were cultured in a 5% CO2 incubator at 35 ℃ for 3 days. Cell viability was measured using the cell viability assay kit CellTiter Glo (Promega). The cytotoxicity test and the antiviral test are the same, but no virus infection exists. The antiviral activity and cytotoxicity of the compounds were represented by the inhibition rate (%) of the compound against the virus-induced cytopathic effect and the survival rate (%) of MRC5 cells at different concentrations, respectively. The calculation formula is as follows:

Inhibition and cell viability of the compounds were analyzed by nonlinear fit using GraphPad Prism (version 5) and the median Effective Concentration (EC) of the compound was calculated₅₀) And half the Cytotoxic Concentration (CC)₅₀) The value is obtained.

Test article comparison compound:

TABLE 2

(a: less than 100nM, b: 100nM to 1000nM, c: 1000nM to 10. mu.M, d: more than 10. mu.M, FCV represents the result of detection of anti-feline calicivirus activity, FIPV represents the result of detection of anti-feline-transmissible-abdoviral activity, SARS-CoV-2 represents the result of detection of anti-novel coronavirus SARS-CoV-2 activity, HCoV 229E represents the result of detection of anti-human coronavirus (HCoV)229E activity.)

As can be seen from the table above, the compound provided by the invention has stronger inhibitory action on feline calicivirus, feline infectious abdovirus, human coronavirus and feline coronavirus infection, and has the potential of preparing anti-virus medicaments.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims

1. A nucleoside compound or a pharmaceutically acceptable salt thereof, characterized by having a structure represented by formula (I):

wherein:

R₁is-H, halogen, -CN, -C_1-6Alkane, -C_2-6Olefins or-C_2-6An alkyne;

R₃is-H or-CN;

R₄is-H or-CN.

2. The nucleoside compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein R is₁is-H, -F, -Br, -I or-CN; and/or

R₄is-H or-CN.

3. The nucleoside compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein R is₁、R₃And R₄At least one of which is-CN.

4. The nucleoside compound or a pharmaceutically acceptable salt thereof according to claim 1, which is selected from any one of the following compounds:

5. a composition comprising at least one nucleoside compound according to any one of claims 1 to 4.

6. The composition of claim 5, wherein the composition further comprises a therapeutic agent.

7. The composition of claim 6, wherein the therapeutic agent is one or more of a corticosteroid, a protease inhibitor, and an anti-inflammatory signal transduction modulator.

8. The composition of claim 6, wherein the therapeutic agent is a compound of formula (II):

9. use of a nucleoside compound according to any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof, or a composition according to any one of claims 5 to 8 in the manufacture of a medicament for the treatment or prophylaxis of a disease caused by feline calicivirus infection, feline-borne enterovirus infection, or coronavirus infection.

10. Use according to claim 9, characterized in that the disease caused by feline calicivirus is feline stomatitis;

the disease caused by feline infectious peritonitis is feline infectious peritonitis;

the coronavirus is human coronavirus or feline coronavirus.