阅读说明：本技术 抗rna病毒药物喹啉衍生物及其应用 (Quinoline derivative of anti-RNA virus medicine and application thereof ) 是由 李洪林 刁妍妍 赵振江 李诗良 朱丽丽 于 2020-03-30 设计创作，主要内容包括：本发明公开了式I所示化合物,或其药学上可接受的盐在治疗RNA病毒感染的药物中的应用。这些药物具备广谱且优异的抗RNA病毒活性,并且对正常细胞的毒性较低。(The invention discloses an application of a compound shown in a formula I or a pharmaceutically acceptable salt thereof in a medicine for treating RNA virus infection. These drugs have a broad spectrum and excellent anti-RNA virus activity and are less toxic to normal cells.)

1. Use of a compound of formula I, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for combating RNA viral infection:

in the formula, R₁Selected from: H. halogen, substituted or unsubstituted C1-6 alkyl, substituted or unsubstituted C1-6 alkoxy, nitro, hydroxy, cyano, amino;

m is an integer selected from 1 to 4;

ring A is a 5-7 membered aromatic ring or a 5-7 membered heteroaromatic ring containing 1-3 heteroatoms selected from N, O or S;

R₂selected from: H. substituted or unsubstituted aryl (preferably phenyl), substituted or unsubstituted C1-8 alkyl, substituted or unsubstituted C1-6 acyl, substituted or unsubstituted C1-6 alkoxy, halogen, nitro, hydroxy, cyano, amino; or, two R₂Can combine to form a 5-7 membered heterocyclic ring containing 1-3 heteroatoms selected from N, O or S (preferably O);

n is an integer selected from 1 to 5;

R₄selected from: H. hydroxy, cyano, substituted or unsubstituted C1-6 alkyl, substituted or unsubstituted C1-6 alkoxy.

2. The use according to claim 1, wherein R is₁Selected from: F. br, substituted or unsubstituted C1-3 alkyl;

m is 1 or 2;

ring A is phenyl;

R₂selected from: substituted or unsubstituted phenyl, substituted or unsubstituted C1-4 alkyl, halogen;

n is 1 or 2;

R₄selected from: H. substituted or unsubstituted C1-3 alkyl.

3. The use according to claim 1, wherein the compound of formula I is the following compound:

4. use according to claim 3, wherein the compound of formula I is the following compound:

5. the use according to claim 4, wherein the compound of formula I is the following compound:

6. the use of any one of claims 1 to 5, wherein the RNA virus includes, but is not limited to: coronaviruses such as Severe acute respiratory syndrome coronavirus (SARS-CoV), Merscov and 2019 New coronavirus (2019-nCoV), Ebola virus, bunyavirus, avian influenza H9N2, H1N1, H7N9, arenavirus, rabies virus, hepatitis C HCV, hepatitis B HBV, human immunodeficiency virus HIV-1, herpes simplex virus HSV-1, HSV-2 and the like.

7. A pharmaceutical composition comprising a compound of formula I as claimed in any one of claims 1 to 6, or a pharmaceutically acceptable salt thereof, in combination with other antiviral agents.

8. The pharmaceutical composition of claim 7, wherein the compound of formula I is the following compound:

9. the pharmaceutical composition of claim 8, wherein the compound of formula I is the following compound:

10. the pharmaceutical composition of any one of claims 7-9, wherein the other antiviral drugs include, but are not limited to: one or more of lopinavir, ritonavir, ribavirin, ridciclovir, oseltamivir, tamiflu, raney rice, virperamivir, abidol and chloroquine (chloroquine phosphate); one or more of lopinavir, ritonavir, ribavirin, rituxivir, and chloroquine (chloroquine phosphate) are preferred.

Technical Field

The present invention relates to the field of pharmaceutical chemistry; in particular, the invention relates to application of buquinar in preparing a medicament for treating RNA virus infection and treating virus infection.

Background

Diseases caused by acute viral infections are an important threat to public health and safety, and these viruses include not only the well-known influenza viruses but also some new outbreaks of new viruses such as coronaviruses.

Diseases caused by viral infection are important threats to public health safety, and these viruses include not only the well-known influenza viruses, Ebola viruses, bunyaviruses, avian influenza viruses H9N2, H1N1, H7N9, arenaviruses, rabies viruses, HCV, HBV, HIV-1, HSV-2, etc., but also coronaviruses such as Severe acute respiratory syndrome coronavirus (SARS-CoV), Zhongdong respiratory syndrome coronavirus (MERS-CoV), and 2019 novel coronavirus (2019-nCoV).

Coronaviruses (Coronaviruses) are a large family of viruses widely found in nature, susceptible to humans and a variety of animals, and are named for their virion surface resembling coronaries. Coronaviruses belong to the family coronaviridae. Based on systematic analysis of viral nucleic acid sequences, coronavirus was classified into four major groups, α, β, γ and a new putative genus in the ninth report of the international committee for viral classification. Among the 7 kinds of beta-type coronavirus which can infect humans, human coronavirus 229E (HCoV-229E), human coronavirus NL63(HCoV-NL63), human coronavirus OC43(HCoV-OC43), hong Kong type I human coronavirus (HCoV-HKU1), severe acute respiratory syndrome coronavirus (SARS-CoV), middle east respiratory syndrome coronavirus (MERS-CoV) and 2019 novel coronavirus (2019-nCoV), respectively. Among them, 2019-nCoV brings serious harm to human health.

Diseases caused by acute viral infections share some common features: 1) short course of disease (1-2 weeks), rapid development (rapid development within a few days after onset); 2) severe illness and even death can easily occur in high risk group; 3) the spread of people is easy to cause; 4) rapid viral replication often leads to an excessive inflammatory response.

Currently, antiviral drugs are based on functional proteins targeting viruses, i.e., targeted drugs need to be developed for each virus. Although the antiviral drug can achieve high specificity and selectivity, the long-term use of the antiviral drug in large quantities often causes drug resistance, and the development cost is high, the time is long, and the predictability is lacked.

Viruses, as organisms of parasitic life, must be propagated depending on the resources of the host cell. Therefore, there is a great need in the art for small molecule drugs designed to target host cells on which viruses live in order to obtain broad spectrum antiviral drugs.

Disclosure of Invention

The purpose of the present invention is to provide a drug having broad-spectrum and excellent antiviral activity; these drugs have low toxicity to normal cells; thereby laying a material foundation for developing a new generation of antiviral drugs.

In a first aspect, the present invention provides the use of a compound of formula I, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for combating RNA viral infection:

in the formula, R₁Selected from: H. halogen, substituted or unsubstituted C1-6 alkyl, substituted or unsubstituted C1-6 alkoxy, nitro, hydroxy, cyano, amino;

m is an integer selected from 1 to 4;

ring A is a 5-7 membered aromatic ring or a 5-7 membered heteroaromatic ring containing 1-3 heteroatoms selected from N, O or S;

R₂selected from: H. substituted or notSubstituted aryl (preferably phenyl), substituted or unsubstituted C1-8 alkyl, substituted or unsubstituted C1-6 acyl, substituted or unsubstituted C1-6 alkoxy, halogen, nitro, hydroxy, cyano, amino; or, two R₂Can combine to form a 5-7 membered heterocyclic ring containing 1-3 heteroatoms selected from N, O or S (preferably O);

n is an integer selected from 1 to 5;

R₄selected from: H. hydroxy, cyano, substituted or unsubstituted C1-6 alkyl, substituted or unsubstituted C1-6 alkoxy.

In a preferred embodiment, R₁Selected from: H. halogen, substituted or unsubstituted C1-3 alkyl;

m is 1 or 2;

ring A is phenyl or a 5-membered heteroaromatic ring containing 1-3 heteroatoms selected from O or S;

R₂selected from: H. substituted or unsubstituted phenyl, substituted or unsubstituted C1-8 alkyl, substituted or unsubstituted C1-3 acyl, substituted or unsubstituted C1-3 alkoxy, halogen; or, two R₂Can be combined to form a 5-membered ring containing 1-3O atoms;

n is an integer selected from 1 to 3;

R₄selected from: H. substituted or unsubstituted C1-3 alkyl. .

In a specific embodiment, R₁Selected from: F. br, substituted or unsubstituted C1-3 alkyl;

m is 1 or 2;

ring A is phenyl;

R₂selected from: substituted or unsubstituted phenyl, substituted or unsubstituted C1-4 alkyl, halogen;

n is 1 or 2;

R₄selected from: H. substituted or unsubstituted C1-3 alkyl.

In a specific embodiment, the compound of formula I is the following:

in a specific embodiment, the compound of formula I is the following:

in a specific embodiment, the compound of formula I is the following:

in particular embodiments, the RNA viruses include, but are not limited to: coronaviruses such as Severe acute respiratory syndrome coronavirus (SARS-CoV), Merscov and 2019 New coronavirus (2019-nCoV), Ebola virus, bunyavirus, avian influenza H9N2, H1N1, H7N9, arenavirus, rabies virus, hepatitis C HCV, hepatitis B HBV, human immunodeficiency virus HIV-1, herpes simplex virus HSV-1, HSV-2 and the like.

In preferred embodiments, the RNA viruses include, but are not limited to: severe acute respiratory syndrome coronavirus (SARS-CoV), middle east respiratory syndrome coronavirus (MERS-CoV) and 2019 novel coronavirus (2019-nCoV), Ebola virus, fever-induced thrombocytopenia syndrome virus (sftsv), avian influenza virus H9N 2.

In a second aspect, the present invention provides a pharmaceutical composition comprising a compound of formula I as described in the first aspect, or a pharmaceutically acceptable salt thereof, and an additional antiviral agent.

In a specific embodiment, the compound of formula I is the following compound:

in a specific embodiment, the compound of formula I is the following:

in a preferred embodiment, the pharmaceutical composition is for use against RNA viral infection.

In preferred embodiments, the RNA viruses include, but are not limited to: coronaviruses such as Severe acute respiratory syndrome coronavirus (SARS-CoV), Merscov and 2019 New coronavirus (2019-nCoV), Ebola virus, bunyavirus, avian influenza H9N2, H1N1, H7N9, arenavirus, rabies virus, hepatitis C HCV, hepatitis B HBV, human immunodeficiency virus HIV-1, herpes simplex virus HSV-1, HSV-2 and the like.

In preferred embodiments, the RNA viruses include, but are not limited to: severe acute respiratory syndrome coronavirus (SARS-CoV), middle east respiratory syndrome coronavirus (MERS-CoV) and 2019 novel coronavirus (2019-nCoV), Ebola virus, fever-induced thrombocytopenia syndrome virus (sftsv), avian influenza virus H9N 2.

In particular embodiments, the other antiviral drugs include, but are not limited to: one or more of lopinavir, ritonavir, ribavirin, ridciclovir, oseltamivir, tamiflu, raney rice, virperamivir, abidol and chloroquine (chloroquine phosphate); one or more of lopinavir, ritonavir, ribavirin, rituxivir, and chloroquine (chloroquine phosphate) are preferred.

In a third aspect, the present invention provides a method of treating an RNA viral infection, the method comprising administering to a subject in need of treatment for a viral infection a therapeutically effective amount of a compound of formula I according to the first aspect or a pharmaceutically acceptable salt thereof or a pharmaceutical composition according to the second aspect.

In preferred embodiments, the RNA viruses include, but are not limited to: coronaviruses such as Severe acute respiratory syndrome coronavirus (SARS-CoV), Merscov and 2019 New coronavirus (2019-nCoV), Ebola virus, bunyavirus, avian influenza H9N2, H1N1, H7N9, arenavirus, rabies virus, hepatitis C HCV, hepatitis B HBV, human immunodeficiency virus HIV-1, herpes simplex virus HSV-1, HSV-2 and the like.

In preferred embodiments, the RNA viruses include, but are not limited to: severe acute respiratory syndrome coronavirus (SARS-CoV), middle east respiratory syndrome coronavirus (MERS-CoV) and 2019 novel coronavirus (2019-nCoV), Ebola virus, fever-induced thrombocytopenia syndrome virus (sftsv), avian influenza virus H9N 2.

In a fourth aspect, the present invention provides a pharmaceutical composition for treating RNA viral infection comprising a therapeutically effective amount of a compound of formula I according to the first aspect or a pharmaceutically acceptable salt thereof.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Detailed Description

The inventor finds that the compound has broad-spectrum and excellent antiviral activity, particularly has remarkable inhibitory activity on newly-appeared 2019 new coronavirus (2019-nCoV), Ebola virus, bunyavirus, avian influenza virus H9N2 and the like, and has low toxicity. The present invention has been completed based on this finding.

Definition of

The 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. For clarity, some terms used herein are defined as follows.

As used herein, "alkyl" refers to a saturated branched or straight chain hydrocarbon group. Specifically, the term "alkyl" as used herein refers to a saturated branched or straight chain hydrocarbon group having 1 to 10 carbon atoms, preferably 2 to 8 carbon atoms, 1 to 6 carbon atoms, 1 to 4 carbon atoms, 1 to 3 carbon atoms, and the like. Specific examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, heptyl, and the like. In this context, alkyl groups may be substituted by 1 or more substituents, for example by halogen or haloalkyl. For example, the alkyl group may be an alkyl group substituted with 1 to 4 fluorine atoms, such as a trifluoromethyl group, or the alkyl group may be an alkyl group substituted with a fluoroalkyl group.

As used herein, "alkoxy" refers to a group represented by RO-, i.e., a group further bonded to the remainder of the molecule through an oxygen atom, wherein R is an alkyl group as described above. Specifically, the term "alkoxy" as used herein refers to alkoxy groups having 1 to 10 carbon atoms, preferably 2 to 8 carbon atoms, 1 to 6 carbon atoms, 1 to 4 carbon atoms, 1 to 3 carbon atoms, and the like. Specific examples of alkyl groups include, but are not limited to, methoxy, ethoxy, propoxy, butoxy, and the like. Herein, an alkoxy group may be substituted with 1 or more substituents, for example, with halogen or haloalkyl. For example, the alkoxy group may be an alkoxy group substituted with 1 to 4 fluorine atoms, such as a trifluoromethoxy group, or the alkyl group may be an alkoxy group substituted with a fluoroalkyl group.

As used herein, "amino" refers to a group of formula "NRxRy", wherein Rx and Ry may be independently selected from H or C1-C3 alkyl or C1-C3 haloalkyl. In a specific embodiment, "amino" as used herein refers to NH 2.

Herein, "halogen" refers to fluorine, chlorine, bromine and iodine. In a preferred embodiment, halogen is chlorine or fluorine; more preferably fluorine.

As used herein, "aryl" or "aromatic ring" have the same meaning and refer to any functional group or substituent derived from a simple aromatic ring. In a specific embodiment, aryl as described herein is phenyl.

Herein, "heteroaryl" or "heteroaromatic ring" have the same meaning, which refers to an aryl group having one or more heteroatoms. The heteroatom may be N, O or an S atom. In particular embodiments, heteroaryl groups described herein are thienyl or furyl.

As used herein, acyl refers to the group designated "R-C (O) -", wherein R is selected from H or substituted or unsubstituted lower alkyl, e.g., C1-3 alkyl.

Compounds of the invention

As used herein, the terms "compound of the invention" and "compound of formula I" have the same meaning and are used interchangeably.

In the invention, the inventor finds that the compound medicine has broad-spectrum and excellent inhibitory activity on coronavirus, particularly new 2019 new coronavirus (2019-nCoV), Ebola virus, bunyavirus, avian influenza virus H9N2 and the like. In a specific embodiment, the compound of the invention is a compound of formula I or a pharmaceutically acceptable salt thereof:

wherein A ring, R₁-R₄M and n are as described above.

In particular, the inventors found that buquina has very significant inhibitory activity against coronaviruses, in particular 2019 neocoronaviruses (2019-nCoV), ebola viruses, bunyaviruses and avian influenza viruses H9N 2.

Buquinar (Brequinar, 96187-53-0) was initially of interest due to its therapeutic effects on a variety of human solid tumors, but has been clinically discontinued due to its more severe toxic side effects. The compound can also be used as a potent immunosuppressant and widely used for treating systemic lupus erythematosus and colitis to inhibit organ transplant rejection.

In addition, one skilled in the art can understand, based on the common general knowledge in the art and the contents of the present invention, that the compound of the present invention can form a salt or an ester due to the carboxyl group contained therein, and further can form a prodrug.

Virus

The RNA viruses described herein (RNA viruses) are a type of biological virus whose genetic material consists of ribonucleic acids (RNA ribonuclear acids), which are usually single-stranded (ssRNA) and also double-stranded (dsRNA).

The term "coronavirus (Coronaviruses)" as used herein is a single-stranded positive-strand RNA virus belonging to the order Nidovirales (Nidovirales) Coronaviridae (Coronaviridae) orthocoronaviridae (orthocoronaviridae). The virus can infect various species such as human, bat, pig, mouse, cow, horse, goat, monkey, etc. There are known 7 kinds of human-infecting coronavirus (HCoV), including middle east respiratory syndrome-associated coronavirus (MERSR-CoV) and severe acute respiratory syndrome-associated coronavirus (SARSr-CoV).

The most recent coronavirus is a novel coronavirus of the genus betagenus, which is named 2019-nCoV by WHO and is the 7 th coronavirus capable of infecting human. At present, no effective vaccine and therapeutic drug aiming at coronavirus exist, and virus diffusion is mainly controlled through precautionary measures, epidemic situation is closely monitored, and suspected cases are isolated and observed. At present, no specific treatment method for coronavirus exists, and symptomatic support treatment is mainly adopted.

The present invention further provides, on the basis of the above compounds, a method for the treatment of viruses, in particular RNA viruses, including but not limited to: pharmaceutical compositions of infections with coronaviruses, such as severe acute respiratory syndrome coronavirus (SARS-CoV), middle east respiratory syndrome coronavirus (MERCoV) and 2019 New coronavirus (2019-nCoV), Ebola virus, bunyavirus, avian influenza H9N2, H1N1, H7N9, arenavirus, rabies virus, hepatitis C HCV, hepatitis B HBV, human immunodeficiency virus HIV-1, herpes simplex virus HSV-1, HSV-2, and the like, comprising a therapeutically effective amount of a compound of the invention, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient. In a preferred embodiment, the compounds or pharmaceutical compositions of the invention may be used for the treatment of infections caused by severe acute respiratory syndrome coronavirus (SARS-CoV), middle east respiratory syndrome coronavirus (MERS-CoV) and 2019 novel coronavirus (2019-nCoV), ebola virus, fever-induced thrombocytopenia syndrome virus (sftsv), avian influenza virus H9N 2.

Examples of pharmaceutically acceptable salts of the compounds of the present invention include, but are not limited to, inorganic and organic acid salts, such as hydrochloride, hydrobromide, sulfate, citrate, lactate, tartrate, maleate, fumarate, mandelate and oxalate salts; and inorganic and organic base salts formed with bases such as sodium hydroxy, TRIS (hydroxymethyl) aminomethane (TRIS, tromethamine) and N-methylglucamine.

Although the requirements vary from person to person, the skilled person can determine the optimal dosage of each active ingredient in the pharmaceutical composition of the invention. Typically, the compounds of the present invention, or pharmaceutically acceptable salts thereof, are administered orally to a mammal daily in an amount of from about 0.0025 to 50 mg/kg body weight. But preferably about 0.01 to 10 mg per kg is administered orally. For example, a unit oral dosage may include from about 0.01 to 50 mg, preferably from about 0.1 to 10 mg, of a compound of the present invention. A unit dose may be administered one or more times daily in one or more tablets, each tablet containing from about 0.1 to 50 mg, conveniently from about 0.25 to 10 mg, of a compound of the invention or a solvate thereof.

The pharmaceutical compositions of the present invention may be formulated in a form suitable for various routes of administration, including but not limited to, administration by parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, buccal, intrathecal, intracranial, nasal or topical routes for the treatment of tumors and other diseases. The amount administered is an amount effective to ameliorate or eliminate one or more symptoms. For the treatment of a particular disease, an effective amount is an amount sufficient to ameliorate or in some way reduce the symptoms associated with the disease. Such amounts may be administered as a single dose or may be administered according to an effective treatment regimen. The amount administered may be sufficient to cure the disease, but is generally administered to ameliorate the symptoms of the disease. Repeated administration is generally required to achieve the desired improvement in symptoms. The dosage of the drug will depend on the age, health and weight of the patient, the type of concurrent treatment, the frequency of treatment, and the desired therapeutic benefit.

The pharmaceutical preparation of the present invention can be administered to any mammals as long as they can obtain the therapeutic effects of the compound of the present invention. Of these mammals, the most important is human. The compounds of the present invention or pharmaceutical compositions thereof are useful for treating ulcerative colitis.

The pharmaceutical preparations of the present invention can be manufactured in a known manner. For example, by conventional mixing, granulating, dragee-making, dissolving, or lyophilizing processes. In the manufacture of oral formulations, solid excipients and active compounds may be combined, optionally grinding the mixture. If desired or necessary after addition of suitable amounts of auxiliaries, the granulate mixture is processed to give tablets or dragee cores.

Suitable adjuvants are, in particular, fillers, for example sugars such as lactose or sucrose, mannitol or sorbitol; cellulose preparations or calcium phosphates, such as tricalcium phosphate or calcium hydrogen phosphate; and binders, such as starch pastes, including corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, or polyvinylpyrrolidone. If desired, disintegrating agents such as the starches mentioned above, as well as carboxymethyl starch, cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate may be added. Adjuvants are, in particular, flow regulators and lubricants, for example silica, talc, stearates, such as calcium magnesium stearate, stearic acid or polyethylene glycol. If desired, a suitable coating resistant to gastric juices can be provided to the tablet core. For this purpose, concentrated saccharide solutions can be used. Such solutions may contain gum arabic, talc, polyvinyl pyrrolidone, polyethylene glycol and/or titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. For the preparation of coatings resistant to gastric juices, suitable cellulose solutions can be used, for example cellulose acetate phthalate or hydroxypropylmethyl cellulose phthalate. Dyes or pigments may be added to the coating of the tablet or lozenge core. For example, for identifying or for characterizing combinations of active ingredient doses.

The method of administration of the pharmaceutical composition includes, but is not limited to, various methods of administration known in the art, and can be determined according to the actual condition of the patient. These methods include, but are not limited to, parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, buccal, intrathecal, intracranial, nasal, or topical routes of administration.

In addition to the compounds of the present invention, the pharmaceutical compositions of the present invention may further comprise other antiviral agents which may be selected from one or more of lopinavir, ritonavir, ribavirin, ridciclovir, oseltamivir, tamiflu, lanimivir, peramivir and chloroquine (chloroquine phosphate); one or more of lopinavir, ritonavir, ribavirin, rituxivir, and chloroquine (chloroquine phosphate) are preferred.

THE ADVANTAGES OF THE PRESENT INVENTION

1. The invention discovers a series of medicines with broad spectrum and excellent antiviral activity for the first time, and particularly has obvious inhibitory activity on the newly appeared 2019 neocoronavirus (2019-nCoV), Ebola virus, bunyavirus and avian influenza H9N 2;

2. these drugs have low toxicity to normal cells;

3. the medicaments lay a material foundation for researching and developing a new generation of antiviral medicaments, thereby having important academic value and practical significance.

The technical solution of the present invention is further described below with reference to specific embodiments, but the following examples are not intended to limit the present invention, and all of the various application methods adopted according to the principles and technical means of the present invention belong to the scope of the present invention.

The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers. Unless otherwise indicated, percentages and parts are by weight.

Example 1 evaluation of inhibitory Activity of the Compounds of the present invention against 2019 Neocrown Virus (2019-nCoV), Ebola Virus, avian influenza Virus A/guangZhou/99(H9N2) and cytotoxicity thereof

The material and the method are as follows: the buciquina is commercially available, the purity is more than 98%, and the derivatives of the buciquina series are purchased from a compound library.

The detection method and the result are as follows:

1. pharmacodynamic experiment for detecting 2019-resistant novel coronavirus (2019-nCoV) by using a fluorescent quantitative PCR method:

reference (Wu Zhong, et al, remdevir and chloroquinone infection of the recombinant engineered novel coronavirus (2019-nCoV) in vitro, Cell Research (2020), 1-3, Zhou, p., et al, a pneumonia outbreak infected with a new coronavirus of probable origin. nature 2020), on Vero E6 cells (ATCC-1586) infected with an infection amount MOI of 2019BetaCoV/Wuhan/WIV 04/2019) of 0.03, while co-culturing was performed with the addition of drugs of different dilution concentrations, the drugs were diluted with DMSO and the DMSO dilution was used as a control. The infection liquid is DMEM + 0.2% BSA, after 48 hours of infection, cell supernatant is collected, virus RNA in the supernatant is extracted through a virus RNA extraction kit, and the copy number of the virus RNA in the cell supernatant is detected through real-time quantitative reverse transcription PCR (qRT-PCR) (Qiagen), so that the virus replication efficiency is reflected. Data processing was performed by Graphpad prism software to determine the semi-Inhibitory Concentration (IC) of the compound against the virus₅₀)。

2. Drug effect experiment of Bright-Glo detection of anti-Ebola virus replicon

Ebola virus replication subsystem (EBOV-NP, EBOV-VP35, EBOV-VP30, EBOV-MG, EBOV-L)

As can be known from the name list of pathogenic microorganisms infected among people, the hazard degree of the Ebola viruses is classified as the first type, the Ebola viruses are required to be operated and carried out in a laboratory with a BSL-4 biological safety level, and in order to reduce the biological safety risk, an Ebola virus Replication subsystem is selected for carrying out an antiviral efficacy test, the System can completely reflect the Replication efficiency of the Ebola viruses, and the System is a common System for Screening the anti-Ebola viruses (Jasenosky L D, Neumann G, Kawaoka Y. minigene-Based Reporter System capable for High-Throughput Screening and/or transport Screening of from Compounds to inhibitors of Ebola viruses and chemical therapy.2010.54(7): 3007). The ebola virus Replication System consists of mini-genome expression plasmid MG for recombinant expression of T7 promoter and luciferase gene and 4 auxiliary plasmids for respectively expressing L, NP, VP35 and VP30 proteins, when the replicon System is transfected into cells for Replication, T7 RNA polymerase induces T7 promoter to start, and further drives luciferase gene expression (Jasenoskk L D, Neumann G, Kawaoka Y.minigene-Based Reporter System Suitable for High-Throughput Screening of compound Able to Inhibit viral Replication and/or transport. analytical reagents & chemother. 2010.54(7): 3007). The replication efficiency of the replicon is positively correlated with the expression quantity of the luciferase gene, so that the replication efficiency of the replicon can be measured by the expression quantity of the luciferase gene in a cell system treated by the drug in drug evaluation, and the inhibition degree of the drug on the Ebola replicon can be evaluated. Bright-Glo reagent (Promega) was used to measure the amount of luciferase expressed. A number of 2X 104 plates of BSR T7/5 cells (Generation of bone resonance synthetic Virus (Brsv) from Cdna: Brsv Ns2 Is Not expression for Virus Replication in Tissue Culture, and the Human Rsv lead enzyme as a Functional Brsv Genome journal of virology 1999.73(1): 251-259, stably transfected to express the T7 RNA polymerase gene, in a cell Culture medium MEM + 10% FBS + 1% L-Glutamine + 2% MAA + 1% P/S in a 96 well plate with a white bottom, were used when the cells were 80% full. The ebola virus five plasmid replication system was prepared as follows:

transfection: the plasmids of the above-mentioned system were all dissolved in 25. mu.lIn the L Opti-MEM serum-free culture medium, marking as an A tube; mu.L of Lipo 2000 was dissolved in 25. mu.L of Opti-MEM serum free medium and recorded as tube B. A, B tubes were mixed together and recorded as tube C, and left at room temperature for 20 min. mu.L/well was added to the treated 96-well plate using a line gun (negative control used a system without EBOV-L plasmid). Shaking at 800rpm for 2 hr, 3-fold dilution of the compound using Opti-MEM serum-free medium, 8 gradient, 3 multiple wells, 50. mu.L per well in shaken white-bottomed 96-well plates, placing at 37 ℃ in 5% CO₂After incubation in an incubator for 24 hours, 50. mu.L of Bright-Glo reagent was added to each well, shaken in the dark for 3min, mixed well, and then allowed to stand for 10 min. Reading in a microplate reader, recording Luminescence (Luminescence) value, and performing data processing by Graphpad prism software to obtain half inhibitory concentration IC of the compound to virus₅₀。

3. Cell Titer-Glo test for drug effect of anti-avian influenza virus capable of infecting human

The experiment is based on a Cell Titer-Glo reagent luminescence detection method, and the inhibitory activity of the compound on avian influenza virus A/guangZhou/99(H9N2) capable of infecting human is detected. MDCK cells with the number of 2 x 104 are plated in a 96-well white-bottom cell culture plate, after the cells grow into a monolayer, the original culture solution is discarded, 50 mu L of 20TCID 50H 9N2 avian influenza virus suspension and 50 mu L of drug solution diluted in multiple proportion are added, each concentration is at least 3 multiple wells, and the virus infection solution is DMEM + 0.2% BSA +25mM HEPES +1 mu g/mL TPCK. Meanwhile, a normal cell control group and a virus control group are set. The 96-well cell culture plate was placed in a 5% CO2 incubator at 37 ℃ and observed every day under a microscope for viral-induced CPE. The virus control group was removed from the incubator when 75% -100% CPE appeared. Add 25. mu.l per wellShaking the reagent for 3min in dark place, and standing for 10 min. The plate is placed in a microplate reader for reading, and the value of Luminescence (Luminescence) is recorded. Data processing was performed by Graphpad prism software to determine the half inhibitory concentration IC of the compound against the virus₅₀。

4. CellTiter-Glo test for drug toxicity (CC)₅₀)

Adenosine Triphosphate (ATP) participates in a plurality of enzymatic reactions in organisms, is an index of living cell metabolism, can detect the survival condition of cells by detecting the ATP content in the cells, and the CellTiter-Glo living cell detection adopts luciferase as a detection object, wherein the luciferase needs the participation of ATP in the luminescence process, and only the respiration action and other life activity processes of metabolic activity cells can generate ATP. Adding equal volume of CellTiter-Glo reagent into the cell culture medium, measuring the cold light value, wherein the light signal is in direct proportion to the ATP amount in the system, and the ATP is in positive correlation with the number of living cells, thereby determining the survival condition of the cells. Plating 2 × 104 cells in a 96-well white-bottom plate, removing the original culture solution after the cells grow into a monolayer, diluting the compound with infection solution (DMEM + 0.2% BSA +25mM HEPES +1 μ g/mL TPCK) to different concentrations, and adding the diluted compound into the 96-well plate; 100 μ l per well, 5 duplicate wells per concentration, with only 0.1mL of infected cells as a normal control; placing at 37 ℃ and 5% CO₂And taking the incubator out of the incubator after 72 hours, cooling the incubator to room temperature, adding 25 mu l of CellTiter-Glo reagent into each well, shaking the incubator for 3min in a dark place, uniformly mixing the reagents, standing the mixture for 10min, placing the mixture in a microplate reader for reading, and recording the value of Luminescence (Luminescence). The semi-toxic concentration (CC) of the compound to the cells was determined by data processing using Graphpad prism software₅₀) And a nontoxic limit concentration (MNCC); percent cell viability ═ test/control wells Luminescence values x 100%.

TABLE 1 evaluation results of Compound Activity

TABLE 2 antiviral efficacy and safety of buquina (Compound 1)

Discussion of the related Art

The results show that some buquina series compounds have good inhibitory activity on 2019 neocoronaviruses (2019-nCoV), Ebola viruses and avian influenza viruses A/guangZhou/99(H9N2), and the buquina has good safety and good application prospects. Particularly for 2019 new coronavirus, the application research of the buquina needs to be accelerated.

EXAMPLE 2 evaluation of antiviral Activity of the combination of the Compound of the present invention with other antiviral drugs

The present inventors have further tested the combination of buquinavir with other antiviral drugs of the prior art, including one or more of lopinavir, ritonavir, ribavirin, ridciclovir, oseltamivir, tamiflu, lanimivir, peramivir, and chloroquine (chloroquine phosphate).

The results show that the combination of the buquinar and the antiviral drugs can generate better treatment effect; wherein the combination with lopinavir, ritonavir, ribavirin, rituxivir and chloroquine (chloroquine phosphate) has relatively better treatment effect.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.