CN114681472A

CN114681472A - Application of glucosamine and derivatives thereof as antiviral drugs

Info

Publication number: CN114681472A
Application number: CN202210316621.5A
Authority: CN
Inventors: 段小涛; 张学敏; 齐琦
Original assignee: Academy of Military Medical Sciences AMMS of PLA
Current assignee: Academy of Military Medical Sciences AMMS of PLA
Priority date: 2019-03-13
Filing date: 2019-03-13
Publication date: 2022-07-01
Anticipated expiration: 2039-03-13
Also published as: CN109771432B; CN114732822A; CN114681472B; CN114732823B; CN114732823A; CN114732822B; CN109771432A

Abstract

Application of glucosamine and derivatives thereof as antiviral drugs is provided. The invention provides an application of a glucosamine compound shown in the following general formula I and pharmaceutically acceptable salts or solvates thereof in preparing antiviral drugs. The compound has good antiviral effect, especially has very obvious broad spectrum of antiviral effect, and can be used for preparing antiviral medicaments.

Description

Application of glucosamine and derivatives thereof as antiviral drugs

The application is a divisional application of Chinese patent application' application number: 201910192721X, application date: 2019, 3 and 13).

Technical Field

The invention belongs to the field of medicines, and particularly relates to application of glucosamine and derivatives thereof in preparation of antiviral medicines and a preparation method thereof.

Background

N-acetyl-D-glucosamine is the minimum unit of chitin component and is also the basic unit of many important polysaccharides in biological cells. It is an amino monosaccharide generated by substituting OH group on 2-position in glucose molecule with acetylamino group. Chemically, it can be easily synthesized by acetylating the amino group at the 2-position in glucosamine molecule, and naturally occurring N-acetyl-D-glucosamine can be obtained by hydrolyzing the outer shell of crustaceans (such as crab and shrimp) by biotechnology and then refining. The substance has many important physiological functions in the organism. The main application effects are as follows:

1. anti-cancer, anti-tumor and immunomodulatory effects;

2. effects of promoting bone loss healing and quality osteoarthritis; and

3. improving skin water retention, relieving pachylosis, and inhibiting fine wrinkles.

Although N-acetyl-glucosamine has various physiological effects, the application of N-acetyl-glucosamine in the field of antivirus is not reported, the inventor of the invention finds that N-acetyl-glucosamine and derivatives thereof have obvious inhibitory effect on various viruses in the process of experiments, and the safety of N-acetyl-glucosamine is verified in various aspects, so that the N-acetyl-glucosamine has wide prospect as the development of antiviral drugs.

Disclosure of Invention

In one aspect of the invention, there is provided a use of a glucosamine compound represented by the general formula I, a pharmaceutically acceptable salt or solvate thereof in the preparation of an antiviral drug:

wherein R and R' are each independently selected from the group consisting of hydrogen, substituted or unsubstituted C1-C8 alkyl, substituted or unsubstituted C1-C8 alkylcarbonyl, substituted or unsubstituted C6-C15 aryl, substituted or unsubstituted C7-C15 aralkyl, substituted or unsubstituted C6-C15 arylcarbonyl, substituted or unsubstituted C6-C15 arylsulfonyl, substituted or unsubstituted C7-C15 aryloxycarbonyl, and substituted or unsubstituted saturated or unsaturated 5-or 6-membered heterocyclylaminocarbonyl containing 1 to 3 heteroatoms selected from the group consisting of N, O and S;

among the various substituents of R and R', the term "substituted" means that the substituent further contains 1 to 3 substituents selected from the group consisting of C1-C4 alkyl, C1-C4 alkoxy, C2-C4 formate and halogen.

R1, R2, R3 and R4 are each independently hydrogen, substituted or unsubstituted C1-C8 alkyl, substituted or unsubstituted C3-C8 cycloalkyl, substituted or unsubstituted C1-C8 alkylcarbonyl, substituted or unsubstituted amino, substituted or unsubstituted C5 to C12 aryl, substituted or unsubstituted C6 to C13 aralkyl, substituted or unsubstituted saturated or unsaturated 5-or 6-membered heterocyclic group containing 1 to 3 heteroatoms selected from N, O and S, phosphate, dipotassium phosphate, monopotassium phosphite, disodium phosphate and monosodium phosphite.

The halogen is selected from fluorine, chlorine, bromine or iodine.

Preferably, said R and R' are each independently selected from the group consisting of hydrogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 alkylcarbonyl, substituted or unsubstituted C6-C10 aryl, substituted or unsubstituted C7-C10 aralkyl, substituted or unsubstituted C6-C10 arylcarbonyl, substituted or unsubstituted C6-C10 arylsulfonyl, substituted or unsubstituted C7-C10 aryloxycarbonyl, and substituted or unsubstituted saturated or unsaturated 6-membered heterocyclylaminocarbonyl containing 1 to 3 heteroatoms selected from the group consisting of N, O and S.

Further preferably, said R and R' are each independently selected from hydrogen, substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted phenyl C1-C4 alkylcarbonyl, substituted or unsubstituted phenyl C1-C4 alkyl, substituted or unsubstituted phenylcarbonyl, substituted or unsubstituted phenylsulfonyl, substituted or unsubstituted benzylcarbonyl, and substituted or unsubstituted saturated or unsaturated 6-membered heterocyclylaminocarbonyl containing 1 to 3N heteroatoms.

Preferably, in the above various substituents for R and R', the term "substituted" means that the substituent further contains 1 or 2 substituents selected from the group consisting of methyl, ethyl, propyl, methoxy, ethoxy, propoxy, carbomethoxy, fluorine and chlorine.

Further preferably, R and R' are each independently selected from hydrogen, methyl, ethyl, propyl, butyl, methylcarbonyl, ethylcarbonyl, propylcarbonyl, butylcarbonyl, monochloromethylcarbonyl, monochloroethylcarbonyl, monochloropropylcarbonyl, monochlorobutylcarbonyl, dichloromethylcarbonyl, dichloroethylcarbonyl, dichloropropylcarbonyl, dichlorobutylcarbonyl, monofluoromethylcarbonyl, fluoroethylcarbonyl, fluoropropylcarbonyl, monofluorobutylcarbonyl, difluoromethylcarbonyl, difluoroethylcarbonyl, difluoropropylcarbonyl, difluorobutylcarbonyl, phenyl, benzyl, phenethyl, phenylpropyl, phenylbutyl, methoxy-substituted benzyl, methoxy-substituted phenethyl, methoxy-substituted phenylpropyl, methoxy-substituted phenylbutyl, ethoxy-substituted benzyl, ethoxy-substituted phenethyl, Ethoxy-substituted phenylpropyl, ethoxy-substituted phenylbutyl, carbomethoxy-substituted phenyl, pyridylaminocarbonyl, phenylcarbonyl, methylphenylcarbonyl, ethylphenylcarbonyl, propylphenylcarbonyl, methylphenylsulfonyl, ethylphenylsulfonyl, propylphenylsulfonyl, and benzylcarbonyl.

Preferably, the R1, R2, R3 and R4 are each independently hydrogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 alkylcarbonyl, substituted or unsubstituted amino, substituted or unsubstituted C6 to C10 aryl, substituted or unsubstituted C6 to C10 aralkyl, phosphate, dipotassium phosphate, monopotassium phosphite, disodium phosphate and monosodium phosphite.

Further preferably, R1, R2, R3 and R4 are each independently hydrogen, methyl, ethyl, propyl, butyl, methylcarbonyl, ethylcarbonyl, propylcarbonyl, butylcarbonyl, monochloromethylcarbonyl, monochloroethylcarbonyl, monochloropropylcarbonyl, monochlorobutylcarbonyl, dichloromethylcarbonyl, dichloroethylcarbonyl, dichloropropylcarbonyl, dichlorobutylcarbonyl, fluoromethylcarbonyl, fluoroethylcarbonyl, fluoropropylcarbonyl, monofluorobutylcarbonyl, difluoromethylcarbonyl, difluoroethylcarbonyl, difluoropropylcarbonyl, difluorobutylcarbonyl, phenyl, benzyl, phenethyl, phenylpropyl, phenylbutyl, methoxy-substituted benzyl, methoxy-substituted phenethyl, methoxy-substituted phenylpropyl, methoxy-substituted phenylbutyl, ethoxy-substituted benzyl, ethoxy-substituted phenethyl, methyl-, ethoxy-substituted phenylcarbonyl, dichlorobutylcarbonyl, phenyl, benzyl, fluoromethylcarbonyl, dichloromethylcarbonyl, dichloroethylcarbonyl, dichlorobutylcarbonyl, benzyl, methoxy-substituted benzyl, methoxy-carbonyl, methoxy-substituted benzyl, ethoxy-carbonyl, benzyl, substituted with a group, an ester, a substituted with a group, a salt, a, Ethoxy-substituted phenylpropyl, ethoxy-substituted phenylbutyl, dipotassium phosphate, and monopotassium phosphite.

Preferably, the compound of formula I according to the invention is selected from the following compounds:

in another aspect of the present invention, there is provided an antiviral pharmaceutical composition comprising a compound represented by formula I, a pharmaceutically acceptable salt or solvate thereof, as an active ingredient, and a pharmaceutically acceptable carrier.

The antiviral pharmaceutical composition according to the present invention can be used for the treatment and prevention of viral diseases caused by RNA viruses that activate the mitochondrial antiviral signal protein (MAVS) pathway selected from flaviviruses, enteroviruses, influenza viruses, coronaviruses, filoviruses, arenaviruses, togaviruses, paramyxoviruses, rhabdoviruses, african swine fever viruses, and the like;

wherein the flavivirus is selected from the group consisting of dengue virus, Zika virus, Japanese encephalitis virus, yellow fever virus, West Nile virus, and Kyasanan encephalitis virus; the enterovirus is selected from EV71, EV68, Coxsackie virus A6, Coxsackie virus B3, Coxsackie virus A16 and poliovirus; the influenza virus is selected from H1N1(WSN, CA06, PR8), H2N3, H5N1, H7N9, drug-resistant influenza A virus, oseltamivir-resistant influenza virus and dengue virus; the coronavirus is selected from SARS and MERS; the bunyavirus is selected from viruses such as fever with thrombocytopenia syndrome virus (SFTSV), rift valley fever virus, hantavirus and the like; the filovirus is selected from the group consisting of ebola virus and marburg virus; the arenavirus is a lassa fever virus; the togavirus is selected from eastern equine encephalitis virus, western equine encephalitis virus, venezuelan equine encephalitis virus and the like: the paramyxovirus is selected from henipan virus; the rhabdovirus is selected from vesicular stomatitis virus and rabies virus.

According to another aspect of the present invention, the glucosamine compound represented by the general formula I, the pharmaceutically acceptable salt or the solvate thereof according to the present invention is used in combination with at least one drug selected from oseltamivir, peramivir, zanamivir, sofosbuvir and ribavirin for preparing an antiviral drug.

The virus pharmaceutical composition according to the present invention may be formulated in various preparation forms including, but not limited to, capsules, tablets, injections, suppositories, infusion solutions, liniments, emulsions and the like.

Advantageous effects

The compound has good antiviral effect, particularly has very obvious broad spectrum of antiviral effect, and can be used for preparing antiviral medicaments.

Drawings

FIG. 1 is a graph showing the comparison of the expression levels of interferon beta (IFN-. beta.) and interferon inducible protein Ifit1 for Compound 3 in example 1;

FIG. 2 is a graph testing the level of activation of phosphorylated IRF3(pIRF3) against Compound 3 in example 2;

FIG. 3 is a graph testing the level of activation of phosphorylated IRF3(pIRF3) against different dosing amounts of Compound 3 in example 2;

FIG. 4 is a graph showing the comparison of the lung tissue expression levels of IFN-. beta.and Ifit1 mRNA in the mice of the test example 3 and the control group;

FIG. 5 is a graph showing a comparison of the viral load in lung tissues of mice in the test example 3 in the administration group and the control group;

FIG. 6 is a graph showing the degree of inflammatory cell infiltration in lung tissues of the test example 3 and the control group;

FIG. 7 is a graph showing the comparison of the survival rate of IAV-infected mice in the test example 4 in the administration group with that in the control group;

FIG. 8 is a graph showing the survival rate of IAV-H274Y-infected mice in the test example 4 in the administration group and the control group;

FIG. 9 is a graph showing the comparison of the survival rates of two groups of mice infected with VSV in the test example 5 between the administered group and the control group;

FIG. 10 is a graph showing the comparison of the survival rates of two groups of mice infected with SA14 in the test example 5 between the administration group and the control group;

FIG. 11 is a graph showing the comparison of the survival rates of mice infected with SFTAV between the administration group and the control group in test example 5;

FIG. 12 is a graph showing the comparison of the survival rates of two groups of mice infected with CA6 between the administration group and the control group in test example 5;

FIG. 13 is a graph showing the comparison of the survival rates of mice infected with SARS in the test example 5 between the administered group and the control group;

FIG. 14 is a graph comparing the survival rates of mice infected with Ebola in the two groups tested for the administration group and the control group in example 5;

FIG. 15 is a graph showing the comparison of the survival rates of mice infected with Lassa fever virus in the test example 5 in the two groups;

FIG. 16 is a graph showing the comparison of the survival rates of two groups of mice infected with EEE between the administration group and the control group in test example 5;

FIG. 17 is a graph showing the comparison of the survival rates of two groups of mice infected with RhV in the test example 5 between the administration group and the control group;

FIG. 18 is a graph showing the survival rate of mice in the two groups infected with Henepanib virus in the test example 5;

FIG. 19 is a graph showing the comparison of the survival rates of mice infected with ASFV between the administration group and the control group in test example 5;

FIG. 20 is a graph showing the comparison of the survival rates of two groups of mice infected with HCV in the test example 5 between the administered group and the control group.

Detailed Description

Hereinafter, the present invention will be described in detail. Before the description is made, it should be understood that the terms used in the present specification and the appended claims should not be construed as limited to general and dictionary meanings, but interpreted based on the meanings and concepts corresponding to technical aspects of the present invention on the basis of the principle that the inventor is allowed to define terms appropriately for the best explanation. Accordingly, the description proposed herein is just a preferable example for the purpose of illustrations only, not intended to limit the scope of the invention, so it should be understood that other equivalents and modifications could be made thereto without departing from the spirit and scope of the invention.

The inventor of the invention finds that the glucosamine compound shown in the general formula I, and the pharmaceutically acceptable salt or solvate thereof can effectively inhibit the occurrence of viral diseases.

In addition, according to the application of the compound shown in the general formula I in preparing antiviral drugs, the invention develops a novel pharmaceutical composition which contains the compound shown in the general formula I as an active ingredient, pharmaceutically acceptable salt or solvate thereof and a pharmaceutically acceptable carrier.

The pharmaceutically acceptable salt is a conventional non-toxic salt formed by reacting the compound with the general formula (I) and an inorganic acid or an organic acid. For example, the conventional non-toxic salts can be prepared by reacting the compound of formula (I) with inorganic acids including hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, sulfamic acid, phosphoric acid and the like, or organic acids including citric acid, tartaric acid, lactic acid, pyruvic acid, acetic acid, benzenesulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, naphthalenesulfonic acid, ethanesulfonic acid, naphthalenedisulfonic acid, maleic acid, malic acid, malonic acid, fumaric acid, succinic acid, propionic acid, oxalic acid, trifluoroacetic acid, stearic acid, pamoic acid, hydroxymaleic acid, phenylacetic acid, benzoic acid, salicylic acid, glutamic acid, ascorbic acid, p-aminobenzenesulfonic acid, 2-acetoxybenzoic acid, isethionic acid and the like; or sodium salt, potassium salt, calcium salt, aluminum salt or ammonium salt formed by the compound of the general formula (I) and propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, aspartic acid or glutamic acid after forming ester and then forming inorganic base; or methylamine salt, ethylamine salt or ethanolamine salt formed by the compound of the general formula (I) and organic base; or the compound of the general formula (I) forms ester with lysine, arginine and ornithine and then forms corresponding inorganic acid salt with hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid and phosphoric acid or forms corresponding organic acid salt with formic acid, acetic acid, picric acid, methanesulfonic acid and ethanesulfonic acid.

The term "pharmaceutically acceptable carrier" refers to any formulation or carrier medium capable of delivering an effective amount of an active agent of the present invention, without interfering with the biological activity of the active agent, and without toxic side effects to the host or patient, and representative carriers include water, oils, vegetables and minerals, cream bases, lotion bases, ointment bases, and the like. These include suspending agents, viscosity enhancers, skin penetration enhancers, and the like. Their preparation is known to those skilled in the cosmetic or topical pharmaceutical field. For additional information on the carrier, reference may be made to Remington: the Science and Practice of Pharmacy,21st Ed., Lippincott, Williams & Wilkins (2005), The contents of which are incorporated herein by reference.

The term "effective amount" or "therapeutically effective amount" with respect to a drug or pharmacologically active agent refers to a sufficient amount of the drug or agent that is non-toxic but achieves the desired effect. For oral dosage forms of the invention, an "effective amount" of one active agent in a composition is the amount required to achieve the desired effect when combined with another active agent in the composition. The determination of an effective amount varies from person to person, depending on the age and general condition of the recipient and also on the particular active substance, and an appropriate effective amount in an individual case can be determined by a person skilled in the art according to routine tests.

Various dosage forms of the pharmaceutical composition of the present invention can be prepared according to conventional preparation methods in the pharmaceutical field. The unit dose of the preparation formula comprises 0.05-200mg of the compound with the general formula (I), and preferably the unit dose of the preparation formula comprises 0.1-100 mg of the compound with the general formula (I).

The compounds and pharmaceutical compositions of the present invention may be administered to mammals, including humans and animals, clinically, by oral, nasal, dermal, pulmonary, or gastrointestinal routes of administration. Most preferably oral. The optimal daily dosage is 0.01-200mg/kg body weight, and can be administered in one time or 0.01-100mg/kg body weight in several times. Regardless of the method of administration, the optimal dosage for an individual will depend on the particular treatment. Usually starting with a small dose and gradually increasing the dose until the most suitable dose is found.

The following examples are given by way of illustration of embodiments of the invention and are not to be construed as limiting the invention, and it will be understood by those skilled in the art that modifications may be made without departing from the spirit and scope of the invention. Unless otherwise specified, reagents and equipment used in the following examples are commercially available products.

Experimental example 1

Commercially available cas 90-77-7

Experimental example 2

Commercially available cas of 10034-20-5

Experimental example 3

Commercially available cas of 10036-64-3

Experimental example 4

Synthetic references for compounds Journal of Biotechnology,115(2), 157-166; 2005

¹H NMR(400MHz,D₂O):0.95(3H,m),2.16(2H,m),3.29(1H,dd,),3.62(1H,dd),3.58 (1H,dd),3.61–3.67(2H,m),3.72(1H,dd),4.99(1H,d,J＝3.5Hz,H-1).ESI-MS:236.5 [M+H]⁺

Experimental example 5

¹H NMR(400MHz,D₂O):0.95(3H,m),1.32(2H,m),2.16(2H,m),3.29(1H,dd,), 3.62(1H,dd),3.58(1H,dd),3.61–3.67(2H,m),3.72(1H,dd),4.99(1H,d,J＝3.5Hz,H-1). ESI-MS:250.1[M+H]⁺

Experimental example 6

Cas available commercially 7784-54-5

Experimental example 7

Reference Organic & Biomolecular Chemistry,12(45), 9180-9183; 2014 is prepared.

¹H NMR(DMSO-d₆400MHz) δ 8.55(3H, s, NH3),7.48-7.45(2H, m, ArCH),7.42-7.27 (16H, m, ArCH),7.16-7.14(2H, m ArCH),4.89(1H, d, J ═ 11.0Hz, PhCHH),4.86(1H, d, J ═ 8.5Hz, H-1),4.84(1H, d, J ═ 12.0Hz, PhCHH),4.81(1H, d, J ═ 11.0Hz, PhCHH),4.70(1H, d, J ═ 11.5Hz, PhCHH),4.65(1H, d, J ═ 11.0Hz, PhCHH),4.58(1H, d, J ═ 11.5, PhCHH), 4.56H (1H, d, J ═ 11.5Hz, PhCHH), 4.5H, 4.65(1H, d, J ═ 11.0Hz, PhCHH),4.58(1H, d, J ═ 11.5, H, 5H, 4.5H, 3H, 5H, 3H, 5Hz, and 3H, 4.5H, 3H, 4.5H, 4H, 4.5 Hz, 4.5H, 4H, 1H, 4H, 1H, and H, 4H, 1H, 4H, 1H, 4H, 1H, 4H, 1H, 4H, 1H, 4H, 1H, 4H, 1H&H-5&H-6a&H-6b),3.05(1H, dd, J ═ 10.5 and 8.5Hz, H-2); 1.84(3H, s, Me) ESI-MS 604.3[ M + Na ]]⁺

Experimental example 8

Reference Carbohydrate Research,340(11),1876 and 1884; 2005.

¹H NMR(CD₃OD，400MHz)δ:5.32(1H,d),4.57(1H,d),3.72(1H,m),3.30(1H,m),2.96 (2H,dq),2.90(2H,m),2.79(1H,dd),2.39(1H,dd),1.22(3H,t),1.15(3H,t).MS:m/z 208.2 (M+H)⁺

Experimental example 9

Reference Carbohydrate Research,340(11),1876 and 1884; 2005.

¹H NMR(CD₃OD，400MHz)δ:5.40(1H,d),4.70(1H,d),3.84(1H,dd),3.45(1H,dd), 3.37(1H,t),3.01(2H,m),2.99(1H,dd),2.98(2H,m),2.56(1H,dd),1.69(2H,m),1.00(3H, t),0.98(3H,t)。MS:m/z 222.2(M+H)+,244.1(M+Na)⁺.

Experimental example 10

1.80g (10mmol) of D- (-) -fructose was added to a 50ml three-necked flask, freshly distilled benzylamine was added at 0 ℃ (3-8 equiv.) and the reaction was warmed to room temperature, then heated to 40 ℃ and reacted for 20h. The crude product was purified by column separation to give a white solid in 1.05 g 41.18% yield.¹H NMR (DMSO-d6，400MHz)δ:α-anomer 5.02(H-1dd),6.21(C1-OH,d),2.69(H-2,dd),1.97(NH,br), 3.82,3.70(CH₂,d),7.17-7.40(Ph m),3.66(H-3,m),4.50(C3-OH,d),3.32(H-4,m),4.67(C4- OH,d),3.51(H-5,m),3.47(H-6x,m),3.62(H-6y,m),4.36(C6-OH,t)；β-anomer 4.95(H-1dd), 6.15(C1-OH,d),2.89(H-2,dd),2.22(NH,br),3.79,3.67(CH₂,d),7.17-7.40(Ph m),4.10 (H-3,m),4.50(C3-OH,d),3.67(H-4,m),4.76(C4-OH,br),3.77(H-5,m),3.33(H-6x,m),3.57 (H-6y,m),4.35(C6-OH,t).MS:m/z 256.2(M+H)⁺

Experimental example 11

The synthesis method is referred to example 10.¹H NMR(DMSO-d6，400MHz)δ:α-anomer 5.05(H-1dd), 6.23(C1-OH,d),2.73(H-2,dd),2.01(NH,br),3.82,3.72(CH₂,d),7.19-7.40(Ph，m), 3.65(H-3,m),4.51(C3-OH,d),3.33(H-4,m),4.68(C4-OH,d),3.53(H-5,m),3.49(H-6x,m), 3.61(H-6y,m),4.33(C6-OH,t)；β-anomer 4.93(H-1dd),6.16(C1-OH,d),2.89(H-2,dd),2.25 (NH,br),3.77,3.65(CH₂,d),7.17-7.40(Ph，m),4.11(H-3,m),4.51(C3-OH,d),3.66(H-4,m), 4.77(C4-OH,br),3.78(H-5,m),3.35(H-6x,m),3.58(H-6y,m),4.35(C6-OH,t),3.85(Ome, s).MS:m/z 300.2(M+H)⁺

Experimental example 12

References Tetrahedron Letters,43(15), 2705-; 2002.

¹H NMR(DMSO-d6，400MHz)δ:5.02(H-1dd),6.22(C1-OH,d),2.69(H-2,dd), 1.97(NH,br),3.82,3.70(CH₂,d),7.23-7.38(Ph,m),3.66(H-3,m),4.50(C3-OH,d),3.32(H-4, m),4.67(C4-OH,d),3.51(H-5,m),3.47(H-6x,m),3.62(H-6y,m),4.36(C6-OH,t)；3.50 (OCH₃,s),MS:m/z 284.3(M+H)⁺

Experimental example 13

Reference Organic & Biomolecular Chemistry,10(35), 7103-; 2012 is prepared.

¹H NMR(400MHz,D₂O)δ4.48(d,J＝8.5Hz,1H,H-1),3.79(dd,J＝12.6Hz,4.3Hz, 1H,H-6a),3.61(dd,J＝12.4Hz,6.4Hz,1H,H-6b),3.52(dd,J＝10.6,8.4Hz,1H,H-3),3.45 (s,3H,OCH₃),3.39–3.28(m,2H,H-5,H-4),2.85(dd,J＝10.6Hz,8.5Hz,1H,H-2),2.52(s, 3H,NCH3).MS:m/z 206.1(M+H)⁺

Experimental example 14

Reference Tetrahedron,46(16), 5533-42; 1990 by the general formula.

¹HNMR(400MHz,D₂O) 4.93(IH, d.J ═ 4.0Hz, I-H), 4.3-3.6(5H, m,3,4,5,6 and 6' -H), 3.42(3H, s, -OMe),2.76(iH, dd, Jp10.0,4.0Hz,2-H) and 2.51(6H, s, NMe)₂)。MS:m/z 222.1 (M+H)⁺

Experimental example 15

References Tetrahedron,74(1), 19-27; 2018.

¹H NMR(400MHz,CD₃OD)δ5.55(d,J＝3.4Hz,1H),4.05(dd,J＝10.9,8.6Hz,1H), 3.80-3.76(m,2H,),3.74(dd,J＝12.8,7.4Hz),3.45(dd,J＝9.8,8.4Hz,1H),3.27(dd,J＝10.8, 3.4Hz,1H),3.03(s,6H).MS:m/z 208.1(M+H)⁺

Experimental example 16

Reference Journal of Carbohydrate Chemistry,32(7), 411-; 2013.

¹H NMR(400MHz,D₂O)δ7.71-7.36(m,5H),5.02(s,0.81H),4.92-4.81(m,0.35H), 4.13(t,J＝9.7Hz,0.85H),4.00-3.65(m,4.15H),3.58(t,J＝9.6Hz,1.04H),3.53-3.31(m, 0.77H).MS:m/z 256.1(M+H)⁺

Experimental example 17

Reference Journal of Carbohydrate Chemistry,32(7), 411-; 2013.

¹H NMR(400MHz,D₂O)δ7.45(t,J＝9.4Hz,2H),7.15(d,J＝8.7Hz,2H),5.01(s, 0.83H),4.08(t,J＝9.8Hz,0.84H),3.96-3.58(m,7.45H),3.51(t,J＝9.5Hz,1.1H),3.35(s, 1.8H),MS:m/z 286.1(M+H)⁺

Experimental example 18

Reference Journal of Carbohydrate Chemistry,32(7), 411-423; 2013.

¹H NMR(400MHz,D₂O)δ7.40(t,J＝8.4Hz,1H),6.91(d,J＝8.4Hz,1H),6.81(t,J＝ 8.4Hz,1H),6.68(t,J＝8.4Hz,1H),5.03(s,0.83H),4.06(t,J＝9.2Hz,0.84H),3.95-3.54(m, 7.45H),3.53(t,J＝9.4Hz,1.1H),3.38(s,1.8H),MS:m/z 286.1(M+H)⁺

Experimental example 19

Reference Journal of Carbohydrate Chemistry,32(7), 411-; 2013.

¹H NMR(400MHz,D₂O)δ7.42(t,J＝8.2Hz,1H),7.39(d,J＝8.2Hz,1H),7.08(d,J＝8.2Hz,1H),7.03(s,1H),5.05(s,0.83H),4.03(t,J＝9.2Hz,0.84H),3.954-3.55(m,7.45H), 3.54(t,J＝9.4Hz,1.1H),3.39(s,1.8H),MS:m/z 314.1(M+H)⁺

Experimental example 20

220mg (0.74mmoL) of triphosgene are dissolved in 10mL of anhydrous tetrahydrofuran at room temperature, and 5mL of a tetrahydrofuran solution containing 0.89g (2mmoL) of 1,3,4, 6-tetra-O-acetyl-a-D-glucosamine and 0.38mL (2.2mmoL) of diisopropylethylamine are slowly added dropwise over a period of about 5 minutes under nitrogen. After stirring the reaction for a further 30 minutes, 15mL of a tetrahydrofuran solution containing 0.18g (2mmoL) of 4-aminopyridine and 0.38mL (2.2mmoL) of diisopropylethylamine were added. After the reaction mixture was stirred for 5 hours, the stirring was stopped, the organic solvent was distilled off under reduced pressure, diluted with 50mL of ethyl acetate and diluted with 20mL of 10% NaHCO₃Washing for 3 times, adding anhydrous MgSO₄Dry overnight. Distilling under reduced pressure the next day to remove organic solvent to obtain yellow extract. And (4) performing column chromatography separation (silica gel 300-400 meshes, and ethyl acetate-methanol 20:1 as a mobile phase) to obtain a white solid. Crystallization from methanol gave 0.81g of white crystals in 82% yield.

¹H NMR(DMSO-d₆，400MHz)δ8.90(s，1H，NH)，8.30(d，2H，J＝6Hz，PyH)， 7.34(d，2H，J＝6Hz，PyH)，6.53(d，J＝9.2Hz，1H，NH)，6.02(d，J＝3.2Hz，1H， H-1)，5.18(t，J＝9.60Hz，1H，H-3)，5.05(t，J＝10Hz，1H，H-4)，4.20-3.99(m，4H， H-2，H-5，H-6a，6b)，2.20，2.02，2.00，1.95(12H，4Ac)：8.90(s，1H，NH)，8.30 (d，2H，J＝6Hz，PyH)，7.34(d，2H，J＝6Hz，PyH)，6.53(d，J＝9.2Hz，1H，NH)， 6.02(d，J＝3.2Hz，1H，H-1)，5.18(t，J＝9.60Hz，1H，H-3)，5.05(t，J＝10Hz，1H， H-4)，4.20-3.99(m，4H，H-2，H-5，H-6a，6b)，2.20，2.02，2.00，1.95(12H，4Ac) ESI-MS:300[M+1]⁺

Experimental example 21

The references Huaxue Yanjiu Yu Yingyong,20(3), 290-; 2008, obtaining; yield: 96.0 percent.

¹HNMR(DMSO-d₆，400MHz)δ:7.95～7.54(m,5H,Ar),7.45～7.42(m,1H,J_1,2＝7.6Hz, H-1,β-),6.52(d,J_NH,24.8Hz,1H, NH), 5.04-4.57 (m,4H,4OH), 3.81-3.72 (m,1H, H-4), 3.73-3.71 (m,1H, H-3), 3.66-3.63 (m,2H, H-6 and H-6'), 3.51-3.42 (m,1H, H-5), 3.20-3.16 (m,1H, H-2). ESI-MS 284[ M +1 ]]⁺

Experimental example 22

¹HNMR(DMSO-d₆，400MHz)δ:7.92～7.54(m,4H,Ar),7.45～7.42(m,1H,J_1,2＝7.6Hz, H-1,β-),6.52(d,J_NH,24.8Hz,1H, NH), 5.04-4.57 (m,4H,4OH), 3.81-3.72 (m,1H, H-4), 3.73-3.71 (m,1H, H-3), 3.66-3.63 (m,2H, H-6 and H-6'), 3.51-3.42 (m,1H, H-5), 3.20-3.16 (m,1H, H-2), 2.35(s, 3H). ESI-MS:298[ M +1 ]]⁺

Experimental example 23

References Advanced Synthesis & Catalysis,356(14-15), 3199-3213; 2014 is prepared.

¹H NMR(400MHz,D₂O)δ5.33(H-1,dd)3.21(H-2,m),3.80(H-3,dd),3.71(H-4,dd), 3.86(H-5,dt),3.43(H-6,m),2.61(PH),1.84(3H,CH₃).ESI-MS:301.1[M+1]⁺

Experimental example 24

References Advanced Synthesis & Catalysis,356(14-15), 3199-3213; 2014.

¹H NMR(400MHz,D₂O)δ5.38(H-1,dd)3.20(H-2,m)4 3.80(H-3,dd)3.72(H-4, dd),3.88(H-5,dt)3.45(H-6,m)2.66(PH).ESI-MS:260[M+1]⁺

Experimental example 25

Reference documents: khimiya Prirodnykh Soedinenii, (6), 787-90; 1987.

¹H NMR(400MHz,CD₃OD) δ 4.46(1H, d, J ═ 8.5Hz, H-1),3.67-3.65(2H, m, H-6a, and H-6b),3.66(3H, s, CH3),3.56(3H, s, CH3),3.55(3H, s, CH3),3.44(1H, ddd, J ═ 3.5,6.0, and 9.5Hz, H-5),3.42(3H, s, CH3),3.39(1H, dd, J ═ 8.5, and 10.5Hz, H-3),3.29(1H, dd, J ═ 8.5, and 9.5Hz, H-4),2.85(1H, dd, J ═ 8.5, and 10.5Hz, H2); ESI-MS 300.2[ M + Na ]]⁺

Experimental example 26

Reference documents: organic & Biomolecular Chemistry,12(45), 9180-9183; 2014.

¹H NMR(400MHz,CD₃OD) δ 4.46(1H, d, J ═ 8.5Hz, H-1),3.67-3.65(2H, m, H-6a, and H-6b),3.66(3H, s, CH3),3.56(3H, s, CH3),3.55(3H, s, CH3),3.44(1H, ddd, J ═ 3.5,6.0, and 9.5Hz, H-5),3.42(3H, s, CH3),3.39(1H, dd, J ═ 8.5, and 10.5Hz, H-3),3.29(1H, dd, J ═ 8.5, and 9.5Hz, H-4),2.85(1H, dd, J ═ 8.5, and 10.5Hz, H2); ESI-MS 258.1[ M + Na ]]⁺

Experimental example 27

Reference documents: journal of Biological Chemistry,289(46), 32056-; 2014 is prepared.

1H-NMR:(400MHz,CDCl3)δ[ppm]2.03(s,3H,-N-CO-CH₃),2.06(s,6H,2× -O-CO-CH₃),3.27(s,3H,-O-CH₃),3.34(s,3H,-O-CH₃),3.69(dd,3JH,H＝9.8,4.9Hz,1H, H-3),3.83(ddd,3JH,H＝10.1,5.7,2.3Hz,1H,H-5),4.04(dd,2JH,H＝12.2,3JH,H＝2.4Hz, 1H,H-6a),4.21(dd,2JH,H＝12.2,3JH,H＝5.7Hz,1H,H-6b),4.52(ddd,3JH,H＝7.6,4.9,1.5 Hz,1H,H-2),4.75(d,3JH,H＝1.4Hz,1H,H-1),4.92(dd,3JH,H＝10.0,10.0Hz,1H,H-4), 5.82(d,3JH,H＝7.7Hz,1H,NH).ESI-MS:[M+Na]⁺356.1

Experimental example 28

Reference documents: organic Letters,19(5), 1040-; 2017.

¹HNMR(DMSO-d₆，400MHz)δ7.74(d,2H),7.70(s,1H),7.41(d,2H),5.62(d,1H), 3.54-3.79(m,7H),2.87(s,1H),2.35(s,3H).ESI-MS:334.1[M+1]⁺

Experimental example 29

D-glucosamine hydrochloride (1.0g, 0.0046mol) was dissolved in 40mL of methanol and 3 times the amount of sodium methoxide solution, and continuously stirred at room temperature for 0.5-1 h. Phenoxyacetyl chloride (1.28g, 0.007mol) was slowly added dropwise to the free aminosugar solution using a dropping funnel, a white precipitate was observed to form during the addition, and the mixture was stirred at room temperature for 4 hours after the addition. The reaction solution was adjusted to pH 2-3 with 12mol/L hydrochloric acid solution, and the resulting white precipitate was filtered under normal pressure to obtain a white solid, which was washed with glacial methanol (2mL × 3), glacial ethyl ether (2mL × 3), and ice water (2mL × 3), and dried with infrared rays. 1.35g of a white powdery solid was obtained, yield: 68 percent of; a white solid.

¹H NMR(400MHz,DMSO)δ7.59(d,J＝8.3Hz,1H,NH),7.29(d,J＝5.3Hz,2H,ArH), 6.98–6.96(m,3H,ArH),6.57(t,J＝4.9Hz,1H,OH),5.22(d,J＝3.6Hz,1H,1-H),4.99(dd, J＝8.0,4.1Hz,2H,OH),4.51(d,J＝0.8Hz,2H,OCH₂CO),4.43(s,1H,OH),3.67(d,J＝1.7 Hz,1H,2-H),3.60(d,J＝4.9Hz,2H,3-H,5-H),3.50-3.44(m,2H,6-H,6’-H),3.16(d,J＝9.1 Hz,1H,4-H)；ESI-MS:300[M+1]⁺。

Test example 1: evaluation of antiviral Effect of Compound 3 (N-acetyl-glucosamine) at cellular level

Primary macrophages (BMDMs) of mice were differentiated and cultured in vitro, differentiated to the seventh day, and were pretreated with compound 3 at a final cell concentration of 20mM for 3 hours (administration group), and PBS-treated group was used as a control group (control group). Then, cells are infected by influenza virus (IAV) with the infection number of 1MOI, cell samples are collected after infection for 3 hours and 6 hours, RNA is extracted, and the difference of expression levels of interferon beta (IFN-beta) and interferon inducible protein Ifit1 is detected by a real-time fluorescence quantitative PCR method. As shown in fig. 1, the expression levels of IFN- β (left) and Ifit1 (right) mRNA in the cells of the administered group were significantly increased after IAV infection compared to the control group, which demonstrates that compound 3 can enhance the expression level of interferon pathway induced by viral infection at the cellular level, thereby exerting antiviral effect.

Test example 2

H274Y mutation is carried out on NA gene of IAV by using a point mutation kit, and a mutant strain recovery virus is obtained by using plasmid system packaging based on a virus reverse genetics technology. And after sequencing verification, amplifying the recovered virus by using MDCK cells, and collecting the virus, namely the drug-resistant strain mutant strain (IAV-H274Y) of the influenza virus. Research shows that the IAV-H274Y mutant strain has obvious drug resistance to the antiviral drug oseltamivir sold on the market, and experiments prove that the compound 3 has certain antiviral activity to influenza virus drug-resistant strains. BMDM was differentiated and cultured in vitro, and until the seventh day of differentiation, compound 3 was administered to cells at a final concentration of 20mM or 40mM for 3 hours of pretreatment (administration group), and PBS-treated group was used as a control (control group). Cells were then infected with IAV-H274Y at a multiplicity of infection of 1MOI, and cell samples were collected 3 hours and 6 hours after infection for testing. On the one hand, total protein of cells was extracted and the level of activation of phosphorylated IRF3(pIRF3) was detected by Western blotting (Western blot) (fig. 2), and on the other hand, RNA was extracted and the difference in the expression level of interferon beta (IFN- β) mRNA was detected by real-time fluorescent quantitative PCR (fig. 3). The results show that the pIRF3 activation level in 20mM and 40mM cells of the administration group is obviously enhanced compared with that in the control group after IAV-H274Y is infected (figure 2), and the expression level of IFN-beta mRNA in the cells of the administration group is also obviously enhanced compared with that in the control group (figure 3), which proves that the compound 3 has certain effect of resisting drug-resistant influenza virus infection.

Experimental data for compounds 1 through 29 against drug resistant influenza virus infection against the mutation of the resistant strain of influenza virus (IAV-H274Y) are listed in table 1 below.

Test example 3: evaluation of antiviral Activity of Compound 3 at animal adult level

C57 mice of identical sex, weekly 40801and body weight were selected and divided equally into two groups, one group of mice fed with compound 3-containing mouse food (25mg/kg) (administration group) and the other group of mice fed with normal mouse food without compound 3 as a control (control group). After 3 days of feeding, mice were infected with influenza virus IAV (1X 10)⁵PFU/only). After 24 hours of infection, the mice were sacrificed by removing the neck, dissected, and the lung tissues of the infected mice were taken out for lysis, and the interferon activation level and virus replication condition in the lung tissues were examined. The results showed that the lung tissues of mice in the administration group had significantly increased expression levels of IFN-. beta.and Ifit1 mRNA compared with the control group (FIG. 4). Meanwhile, a plaque experiment shows that the virus load in the lung tissue of a mouse in an administration group is remarkably reduced compared with that of a control group (figure 5), the inflammatory cell infiltration degree in the lung tissue is also relieved (figure 6), and the lung tissue sample after infection is detected through the experiment to prove that the compound 3 has certain antiviral activity at the level of an animal adult.

Test example 4:

to further verify the antiviral effect of compound 3 in vivo, C57 mice of identical sex, week 40801and body weight were selected and divided equally into two groups, one group of mice fed compound 3-containing mouse chow (25mg/kg) (dosing group) and the other group of mice fed compound 3-free normal mouse chow as control (control group). After 3 days of feeding, mice were given nasal drops of influenza virus IAV (1 x 10)⁵PFU/mouse), the survival condition of two groups of mice is poorAnd (3) distinguishing. The results show that feeding compound 3 can significantly increase the survival rate of IAV infected mice (fig. 7).

Test example 5: evaluation of the broad Spectrum of antiviral Effect of Compound 3

In experiments using different species of RNA viruses, such as Vesicular Stomatitis Virus (VSV) of the genus vesiculovirus of the family Rhabdoviridae, Coxsackie type 6 Virus (Coxsachievirus A6, CA6) of the genus Enterovirus of the family MicroRNAviridae, Severe fever associated thrombocytopenia syndrome Virus (SFTSV) of the genus phlebovirus of the family Bunyaviridae, Japanese encephalitis Virus SA14 strain (Japanese encephalitis Virus, JEV, SA14) of the genus phlebovirus of the family Coronaviridae, Ebola Virus (Ebola Virus) of the genus Ebola Virus of the family Filoviridae, Lassa Virus (Lassa fevervirus) of the genus Sauroviridae, Orthovirus of the genus Orthovirus of the family Swallowing, Orthovirus (African rhinovirus), Coxsackie Virus (African Virus of the genus Africaceae, African Virus of the genus African Virus of the family Rhabdoviridae (African Virus of the family Rhabdoviridae, Africa Virus RhV) of the genus Africa Virus of the family Rhabdoviridae, ASFV), sex, peri \40801, and weight consistent C57 and ICR mice were selected, and the mice were divided equally into two groups, one group of mice was fed with the grain of the compound 3-containing mice (25mg/kg) (administration group), and the other group of mice was fed with the grain of the normal mice without the compound 3 as a control (control group). 3 days after feeding, mice were infected with IAV-H274Y virus (FIG. 8), VSV virus (FIG. 9), SA14 virus (FIG. 10), SFTSV virus (FIG. 11), CA6 virus (FIG. 12), SARS virus (FIG. 13), Ebola virus (FIG. 14), Lassa fever virus (FIG. 15), EEE virus (FIG. 16), RhV virus (FIG. 17), Henripa virus (FIG. 18), ASFV virus (FIG. 19), and HCV virus (FIG. 20), respectively. The results show that the survival rate of mice after the infection with the above-mentioned RNA virus can be significantly improved by feeding compound 3.

The broad spectrum experimental data of compounds 1 to 29 against the antiviral effects of Vesicular Stomatitis Virus (VSV) of the Vesicular Virus genus of the rhabdoviridae family, coxsackievirus type 6 Virus (coxsackievirus a6, CA6) of the enterovirus genus of the microrna family, Severe fever with thrombocytopenia syndrome Virus (SFTSV) of the phlebovirus genus of the bunyaviridae family, and Japanese encephalitis Virus SA14 strain (Japanese encephalitis Virus, JEV, SA14) of the flavivirus genus of the flaviviridae family are listed in table 1 below.

Claims

1. The application of the glucosamine compound shown in the general formula I, and the pharmaceutically acceptable salt or solvate thereof in preparing antiviral drugs:

among the various substituents of R and R', the term "substituted" means that the substituent further contains 1 to 3 substituents selected from the group consisting of C1-C4 alkyl, C1-C4 alkoxy, C2-C4 formate and halogen;

r1, R2, R3 and R4 are each independently hydrogen, substituted or unsubstituted C1-C8 alkyl, substituted or unsubstituted C3-C8 cycloalkyl, substituted or unsubstituted C1-C8 alkylcarbonyl, substituted or unsubstituted amino, substituted or unsubstituted C5 to C12 aryl, substituted or unsubstituted C6 to C13 aralkyl, substituted or unsubstituted saturated or unsaturated 5-or 6-membered heterocyclic group containing 1 to 3 heteroatoms selected from N, O and S, phosphate, dipotassium phosphate, monopotassium phosphite, disodium phosphate and monosodium phosphite;

the halogen is selected from fluorine, chlorine, bromine or iodine.

2. The use according to claim 1 for the preparation of an antiviral medicament, wherein the compound of formula I according to the invention is a compound of formula 3:

3. the use according to claim 1 for the preparation of an antiviral medicament for use in combination with at least one medicament selected from oseltamivir, peramivir, zanamivir, sofosbuvir and ribavirin.

4. Use according to claim 1 in the preparation of an antiviral medicament, said virus being an influenza virus.

5. The use according to claim 4 in the manufacture of an antiviral medicament, said influenza virus being selected from the group consisting of H1N1(WSN, CA06, PR8), H2N3, H5N1, H7N9, drug-resistant influenza A virus, oseltamivir-resistant influenza virus.