CN108125955A - A kind of broad-spectrum antiviral inhibitor - Google Patents
A kind of broad-spectrum antiviral inhibitor Download PDFInfo
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- CN108125955A CN108125955A CN201810143531.4A CN201810143531A CN108125955A CN 108125955 A CN108125955 A CN 108125955A CN 201810143531 A CN201810143531 A CN 201810143531A CN 108125955 A CN108125955 A CN 108125955A
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- 0 COC(C#CC1)=C(CN(*)*)C=C1Nc1c(ccc(*)c2)c2ncc1 Chemical compound COC(C#CC1)=C(CN(*)*)C=C1Nc1c(ccc(*)c2)c2ncc1 0.000 description 1
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/47—Quinolines; Isoquinolines
- A61K31/4706—4-Aminoquinolines; 8-Aminoquinolines, e.g. chloroquine, primaquine
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Abstract
The invention belongs to pharmaceutical technology fields, are related to a kind of broad-spectrum antiviral inhibitor.Particularly, the purposes for preventing and/or treating virus infection or the disease caused by virus infects is used for the present invention relates to the compound with formula (I) structure.The compound that the application further relates to have formula (I) structure is used to prepare prevention and/or treats the purposes of the drug of virus infection or the disease caused by virus infects.
Description
Technical field
The invention belongs to pharmaceutical technology fields.Particularly, the present invention relates to the compounds with formula (I) structure for preventing
And/or the purposes of the infection for the treatment of virus or the disease caused by virus infects.The application further relates to the change with formula (I) structure
Close the purposes that object is used to prepare prevention and/or the drug of the infection for the treatment of virus or the disease caused by virus infects.
Background technology
The popular Health and Living to the mankind of viral infectious brings tremendous influence.At present, virus infection is anti-
It controls and depends on vaccine inoculation and/or antiviral treatment.However, vaccine is because of its higher price, and needs low-temperature storage,
It is made to be difficult to be widely used；Also, it can not also obtain real breakthrough currently for the vaccine research of many viruses.Antiviral agent
Object has been increasingly becoming treatment viral infectious mainstream, wherein most antiviral drugs are by inhibiting virus genomic
Transcription carrys out viral interference duplication.In general, these Drug inhibitions participate in viral genome transcription specific protein, such as polymerase or
Transcriptase；Host cell is depended on however, as virus to carry out viral genome duplication, therefore these drugs are often
Generate undesirable toxicity.Further, since the high degree of specificity of said medicine, the micromutation in viral genome is produced through being generally sufficient to
The raw Strain resistant to the drug.Meanwhile said medicine is usually only capable of inhibiting active virus replication, it can not be thorough
Eliminate the virus hidden or hidden in host.
In short, in view of currently available vaccine and antiviral drugs are all limited to some defects, it is clinical in for many
It is infected caused by virus all without effective prevention vaccine and therapy, therefore, finds new broad-spectrum antiviral medicament
It is extremely urgent.
Invention content
In the present invention, unless otherwise stated, Science and Technology noun used herein has art technology
The normally understood meaning of personnel institute.Also, the operating procedures such as cell culture used herein, biochemistry, cell biology
It is widely used conventional steps in corresponding field.Meanwhile for a better understanding of the present invention, relational language is provided below
Definition and explanation.
As used herein, term " Ebola virus (Ebola virus, EBOV) " refers to, filamentous virus section
(Filoviridae) a kind of minus-stranded rna virus, to cause Ebola hemorrhagic fever (EBHF, also known as ebola disease viral disease
(EVD)) pathogen.
As used herein, term " Marburg virus (Marburg virus, MARV) " refers to, filamentous virus section
(Filoviridae) a kind of minus-stranded rna virus, to cause the cause of disease of marburg hemorrhagic fever (also known as marburg disease)
Body.
As used herein, term " rabies viruses (Rabies virus, RV) " refers to, Rhabdoviridae
(Rhabdoviridae) a kind of minus-stranded rna virus of Lyssavirus (Lyssavirus), to cause rabic cause of disease
Body.
As used herein, the aliphatic group of term " alkyl " including saturation, including straight chained alkyl (such as first
Base, ethyl, propyl, butyl, amyl, hexyl, heptyl, octyl group, nonyl, decyl etc.), branched alkyl (such as isopropyl, tertiary fourth
Base, isobutyl group etc.), cycloalkyl (such as cyclopropyl, cyclopenta, cyclohexyl, suberyl, cyclooctyl etc.), alkyl-substituted cycloalkanes
Base group or the alkyl group of cycloalkyl substitution.In the present invention, " C is statedx-CyAlkyl " refers to the carbon with particular range
The alkyl group of atom.For example, statement " C1-C6Straight chained alkyl " refers to the straight chained alkyl for including 1-6 carbon atom, specific real
Example includes but not limited to methyl, ethyl, propyl, normal-butyl, n-pentyl or n-hexyl；For example, statement " C1-C6Branched alkyl " is
Refer to the branched alkyl for including 1-6 carbon atom, specific example includes but not limited to isopropyl, tertiary butyl or isobutyl group；For example,
State " C3-C6Cycloalkyl " refers to the cycloalkyl for including 3-6 carbon atom, and specific example includes but not limited to cyclopropyl, ring fourth
Base, cyclopenta or cyclohexyl.
As used herein, term " pharmaceutically acceptable salt " refers to, is deposited in compound provided by the present invention
Basic functionality (such as secondary amine group or tertiary amine group) and the salt of appropriate inorganic or organic anion (acid) formation,
And include but not limited to, inorganic acid salt, such as hydrofluoride, hydrochloride, hydrobromate, hydriodate, nitrate, perchloric acid
Salt, sulfate, phosphate etc.；Rudimentary alkyl sulfonate, such as mesylate, fluoroform sulphonate, esilate；Aryl sulfonic acid
Salt, such as benzene sulfonate, P-TOLUENE SULFO ACID 99's salt；Acylate, such as acetate, malate, fumarate, succinate, lemon
Hydrochlorate, tartrate, oxalates, maleate etc.；Amino-acid salt, as glycinate, trimethylglycine salt, arginine salt,
Ornithine salt, glutamate, aspartate etc..
As used herein, term " pharmaceutically acceptable ester " refers to, is deposited in compound provided by the present invention
The esters that are formed with appropriate acid (for example, carboxylic acid or oxygen-containing inorganic acid) of-OH.Suitable ester group includes but not limited to, first
Acid esters, acetic acid esters, propionic ester, butyrate, acrylate, ethyl succinate, hard fatty acid ester or palmitate.
As used herein, term " effective quantity " refers to, it is sufficient to obtain or at least partly obtain desired effect
Amount.For example, " prevention condition effective amount " refers to, it is sufficient to prevent, prevents or postpone disease (for example, virus is infected or felt by virus
Dye caused by disease) generation amount；" treatment condition effective amount " refers to, it is sufficient to cure or at least partly prevent to have suffered from disease
The disease of patient and the amount of its complication of disease (for example, virus infection or disease caused by virus infects).It measures in this way
Effective quantity completely within the limit of power of those skilled in the art.For example, therapeutical uses, which will effectively be measured, to be depended on
The severity of disease to be treated, the overall status of the immune system of patient oneself, the ordinary circumstance of patient such as age, weight
And gender, the method for application of drug and the other treatment that is administered simultaneously etc..
As used herein, term " subject " includes but not limited to various animals, such as mammal, such as ox
Section animal, equid, caprid, porcine animals, canid, felid, rabbit section animal, rodent (for example,
Mouse or rat), non-human primate (for example, macaque or machin) or people.In some embodiments, the subject
(such as people) virus infection, alternatively, the risk with virus infection.
Present inventor has found that the 4-aminoquinoline compounds with specific structure can significantly inhibit virus for the first time
Infection, have apparent antiviral activity.And before the application, the antiviral activity of this kind of compound did not appeared in the newspapers.
Therefore, in one aspect, the present invention provides logical formula (I) compound represented or its pharmaceutically acceptable salt or
Ester is for the prevention in subject and/or treats the purposes of virus infection or the disease caused by virus infection or is used for
Prepare the purposes for the drug for preventing and/or treating virus infection or the disease caused by virus infects in subject；
Wherein,
R1It independently is hydrogen, halogen or C1-C6Alkyl；
R2、R3It is each independently C1-C6Alkyl or C3-C6Cycloalkyl.
In certain preferred aspects, R1Selected from hydrogen, halogen and C1-C3Alkyl.
In certain preferred aspects, R1Selected from hydrogen and halogen (such as-F ,-Cl ,-Br or-I).
In certain preferred aspects, R1For halogen (such as-F ,-Cl ,-Br or-I).
In certain preferred aspects, R1For-Cl.
In certain preferred aspects, R2、R3It is each independently C1-C4Linear or branched alkyl group or C3-C4Cycloalkanes
Base.
In certain preferred aspects, R2、R3It is each independently methyl, ethyl, n-propyl, isopropyl, ring third
Base, normal-butyl, sec-butyl, tertiary butyl, isobutyl group or cyclobutyl.
In certain preferred aspects, R2With R3It is identical.
In certain preferred aspects, R2、R3For ethyl.
In certain preferred aspects, the compound has chemical formula as follows.
In certain preferred aspects, the virus is selected from Ebola virus (EBOV), Marburg virus (MARV)
With rabies viruses (RV).
In certain preferred aspects, the disease caused by virus infects is selected from Ebola hemorrhagic fever, horse
That fort Hemorrhagic fever and rabies.
In certain preferred aspects, the pharmaceutically acceptable salt is inorganic acid salt or acylate.At certain
In a little preferred embodiments, the pharmaceutically acceptable salt is acylate, such as citrate, fumarate, Malaysia
Hydrochlorate or tartrate.In certain preferred aspects, the pharmaceutically acceptable salt is inorganic acid salt, such as hydrochloric acid
Salt, sulfate, phosphate, mesylate or benzene sulfonate.It is described pharmaceutically acceptable in certain exemplary implementations
Salt is hydrochloride.
In certain preferred aspects, the drug includes the chemical combination as described above of prevention or therapeutically effective amount
Object.
In certain preferred aspects, the drug can be any form known to medical domain.It is for example, described
Drug can be tablet, pill, suspension, emulsion, solution, gelling agent, capsule, pulvis, powder, granule, elixir, ingot
The forms such as agent, suppository, injection (including parenteral solution, injection sterile powder and concentrated solution for injection), inhalant, spray.
In certain preferred aspects, the drug is tablet, pill, capsule, granule, powder, solution, suspension, note
Penetrate liquid or injection sterile powder.
In certain preferred aspects, the drug optionally further includes the other pharmacy with antiviral activity
Activating agent.
In certain preferred aspects, the other forms of pharmacologically active agents is to can be used for treating rabies virus infection
And/or rabic drug, such as rabies vacciness, rabies antiserum or anti-rabies immune globulin are (for example, resist mad dog
Patient's immunoglobulin).
In certain preferred aspects, the other forms of pharmacologically active agents is to can be used for treatment Ebola virus infection
And/or the drug of Ebola hemorrhagic fever.
In certain preferred aspects, the other forms of pharmacologically active agents is to can be used for treatment Marburg virus infection
And/or the drug of Marburg virus Hemorrhagic fever.
It is known in the art, it can be used for anti-Ebola virus or Marburg virus through what FDA ratified currently without any one
Vaccine or drug, treatment means are mainly the supportive treatment taken according to patients clinical situation, such as application contains electricity
The liquid of solution matter carries out implementing venous transfusion or oral rehydration.Therefore, in the present invention, statement " can be used for treating Ebola
Virus infection and/or the drug of Ebola hemorrhagic fever " " can be used for the infection for the treatment of Marburg virus and/or marburg hemorrhagic fever
Drug " mean that those resist the virus activity and/or vaccines for being envisaged for the virus in clinical experimental stage with potential
Or drug.
In certain preferred aspects, the drug includes the compound as described above of unit dose, such as wraps
The compound of any amount containing 1-2000mg, such as 10-1500mg, such as 20-1000mg, such as 50-500mg, such as
50-100mg.In the present invention, the dosage of the compound can be according to the state of an illness weight of patient or subject, age, body
The factors such as weight, gender, administering mode and the course for the treatment of are adjusted.
In the present invention, the drug can be applied by any suitable method known in the art, such as oral,
It is parenteral, rectum, transpulmonary or the modes such as administer locally to.When for taking orally, oral preparation, such as mouth can be made in the drug
Oral solid preparation, such as tablet, capsule, pill, granule；Or, oral liquid, such as oral solution, oral mixed suspension
Agent, syrup etc..When oral preparation is made, the drug can also include suitable filler, adhesive, disintegrant, profit
Lubrication prescription etc.；Particularly, in order to improve the wettability of hydrophobic compound and improve its dissolubility, the drug preferably includes parent
Waterborne polymeric, such as hydroxypropyl cellulose (HPC), hydroxypropyl methyl cellulose (HPMC) or carboxymethyl cellulose (CMC) etc..
When for parenteral, injection can be made in the drug, dense molten including parenteral solution, injection sterile powder and injection
Liquid.When injection is made, the conventional method in existing pharmaceutical field may be used to be produced in the drug.It is noted when preparing
When penetrating agent, the drug can contain pharmaceutically acceptable carrier such as sterile water, ringer's solution and isotonic sodium chlorrde solution,
Also suitable additives such as antioxidant, buffer and bacteriostatic agent can be added according to the property of drug.When for rectally
When, suppository etc. can be made in the drug.During for transpulmonary administration, inhalant or spray etc. can be made in the drug.
In certain preferred aspects, the subject is mammal, such as non-human primate, such as
People.
On the other hand, the present invention provides a kind of prevention and/or the infection for the treatment of virus or caused by virus infects
Disease method, including to subject in need apply a effective amount of logical formula (I) compound represented or its pharmacy
Upper acceptable salt or ester；
Wherein,
R1It independently is hydrogen, halogen or C1-C6Alkyl；
R2、R3It is each independently C1-C6Alkyl or C3-C6Cycloalkyl.
In certain preferred aspects, R1Selected from hydrogen, halogen and C1-C3Alkyl.
In certain preferred aspects, R1Selected from hydrogen and halogen (such as-F ,-Cl ,-Br or-I).
In certain preferred aspects, R1For halogen (such as-F ,-Cl ,-Br or-I).
In certain preferred aspects, R1For-Cl.
In certain preferred aspects, R2、R3It is each independently C1-C4Linear or branched alkyl group or C3-C4Cycloalkanes
Base.
In certain preferred aspects, R2、R3It is each independently methyl, ethyl, n-propyl, isopropyl, ring third
Base, normal-butyl, sec-butyl, tertiary butyl, isobutyl group or cyclobutyl.
In certain preferred aspects, R2With R3It is identical.
In certain preferred aspects, R2、R3For ethyl.
In certain preferred aspects, the compound has structure shown below formula.
In certain preferred aspects, the virus is selected from Ebola virus (EBOV), Marburg virus (MARV)
With rabies viruses (RV).
In certain preferred aspects, the disease caused by virus infects is selected from Ebola hemorrhagic fever, horse
That fort Hemorrhagic fever and rabies.
In certain preferred aspects, the pharmaceutically acceptable salt is inorganic acid salt or acylate.At certain
In a little preferred embodiments, the pharmaceutically acceptable salt is acylate, such as citrate, fumarate, Malaysia
Hydrochlorate or tartrate.In certain preferred aspects, the pharmaceutically acceptable salt is inorganic acid salt, such as hydrochloric acid
Salt, sulfate, phosphate, mesylate or benzene sulfonate.It is described pharmaceutically acceptable in certain exemplary implementations
Salt is hydrochloride.
In certain preferred aspects, can using compound as described above as pharmaceutical composition carry out prepare and
Using.In certain preferred aspects, described pharmaceutical composition can include prevention or therapeutically effective amount it is as described above
Compound.In certain preferred aspects, described pharmaceutical composition can be any form known to medical domain.Example
Such as, described pharmaceutical composition can be tablet, pill, suspension, emulsion, solution, gelling agent, capsule, pulvis, granule,
Elixir, pastille, suppository, injection (including parenteral solution, injection sterile powder and concentrated solution for injection), inhalant, spray
Etc. forms.In certain preferred aspects, described pharmaceutical composition be tablet, it is pill, capsule, granule, powder, molten
Liquid, suspension, parenteral solution or injection sterile powder.
In the present invention, described pharmaceutical composition can be applied by any suitable method known in the art, example
It is such as oral, parenteral, rectum, transpulmonary or administer locally to mode.When for taking orally, mouth can be made in described pharmaceutical composition
Formulation, such as oral solid formulation, such as tablet, capsule, pill, granule；Or, oral liquid, it is such as oral molten
Liquor, oral suspensions, syrup etc..When oral preparation is made, described pharmaceutical composition can also include suitable filling
Agent, adhesive, disintegrant, lubricant etc.；Particularly, in order to improve the wettability of hydrophobic compound and improve its dissolubility,
Described pharmaceutical composition preferably includes hydrophilic polymer, such as hydroxypropyl cellulose (HPC), hydroxypropyl methyl cellulose
(HPMC) or carboxymethyl cellulose (CMC) etc..When for parenteral, injection can be made in described pharmaceutical composition, including
Parenteral solution, injection sterile powder and concentrated solution for injection.When injection is made, described pharmaceutical composition may be used existing
Conventional method in pharmaceutical field is produced.When preparing injection, described pharmaceutical composition, which can contain, pharmaceutically may be used
The carrier of receiving such as sterile water, ringer's solution and isotonic sodium chlorrde solution can also add in suitable attached according to the property of drug
Add agent such as antioxidant, buffer and bacteriostatic agent.When for rectally, suppository can be made in described pharmaceutical composition
Deng.During for transpulmonary administration, inhalant or spray etc. can be made in described pharmaceutical composition.
In certain preferred aspects, the method is optionally further included using other with antiviral activity
Forms of pharmacologically active agents.This other forms of pharmacologically active agents can be applied prior to, concurrently with, or after application compound as described above.
In certain preferred aspects, can as above institute be applied with any amount of 1~2000mg/kg subject's weight
The compound stated, such as with 1~1500mg/kg body weight/days, 1~1000mg/kg body weight/days, 5~1000mg/kg body weight/days,
5~500mg/kg body weight/days, 5~200mg/kg body weight/days, 5~100mg/kg body weight/days or 10~100mg/kg body weight/days
Amount apply compound as described above.In certain preferred aspects, can with 4 times a day, 3 times a day, daily 2
It is secondary, one time a day, every 1 time on the two or mode 1 times a week apply compound as described above, optionally take the circumstances into consideration weekly or monthly
Repeat dosage regimen as described above.In the present invention, the dosage of the compound can be according to the disease of patient or subject
The factors such as feelings weight, age, weight, gender, administering mode and the course for the treatment of are adjusted.
In certain preferred aspects, the subject is mammal, such as non-human primate, such as
People.
Advantageous effect of the invention
Due to lacking effective vaccine/drug and treatment means, at present human infection Ebola virus, Marburg virus,
The death rate after the virus such as rabies viruses is high.Present inventor is found that one kind has by largely studying and screening
The compound of broad anti-viral activity.The compound can significantly interfere with a variety of viruses (for example, Ebola virus, Marburg disease
Poison or rabies viruses) to the course of infection of cell, and significant protective effect has been shown in animal model, have apparent
Antiviral activity.Thus, which can be used for prevention and/or the infection for the treatment of virus or the disease caused by virus infects,
This discovery is of great significance for the prevention of the viral infectious such as Ebola virus, Marburg virus, rabies viruses.
Embodiment of the present invention is described in detail below in conjunction with drawings and examples, but people in the art
Member will be understood that drawings below and embodiment are merely to illustrate the present invention rather than the restriction to the scope of the present invention.With reference to the accompanying drawings
With the following detailed description of preferred embodiment, various purposes of the invention and advantageous aspect are to those skilled in the art
It will be apparent.
Description of the drawings
Fig. 1 shows in embodiment 2 that RV-04 divides RV pseudovirus, EBOV pseudovirus and MARV pseudovirus inhibiting rates
Analyse curve.Wherein, abscissa represents the activity (μM) of RV-04, and ordinate represents inhibiting rate (%).The results show that RV-04
A variety of pseudovirus can be significantly inhibited.
Fig. 2 shown in embodiment 3, the analysis curve of the cytotoxicity of RV-04.Wherein, abscissa represents the work of RV-04
With concentration (μM), ordinate represents inhibiting rate (%).The results show that RV-04 is without apparent cytotoxicity.
Fig. 3 is shown in embodiment 4, using fluorescence microscope RV-04 to point of the neutralization activity of mad dog live virus
Analyse result.The results show that RV-04 has apparent mad dog live virus neutralization activity.
Fig. 4 shows in embodiment 4 that RV-04 is to the analysis curve of mad dog live virus inhibiting rate.Wherein, abscissa table
Show the activity (μM) of RV-04, ordinate represents inhibiting rate (%).The results show that RV-04 can significantly inhibit mad dog live virus
Infection to cell.
Fig. 5 shown in embodiment 5, the analysis of antiviral activities of the RV-04 in Ebola virus infects In vivo model
As a result.The results show that RV-04 can significantly inhibit infection of the Ebola virus to mouse.
Fig. 6 shown in embodiment 5, the analysis of antiviral activities of the RV-04 in Marburg virus infects In vivo model
As a result.The results show that RV-04 can significantly inhibit infection of the Marburg virus to mouse.
Fig. 7-Fig. 9 is respectively illustrated in embodiment 6, by surface plasma resonance technology to RV-04 and RV, EBOV and
MARV virus membrane antigens combine the testing result of activity.The results show that RV-04 is respectively provided with a variety of virus membrane antigens certain knot
Close activity.
Specific embodiment
It is intended to illustrate the present invention the embodiment of (rather than limiting the invention) to describe the present invention referring now to following.
Unless specifically stated otherwise, otherwise basically according to known in the art and normal described in various bibliography
Rule method carries out the experiment and method described in embodiment.The person that is not specified actual conditions in embodiment, according to normal condition or system
The condition for making quotient's suggestion carries out.Reagents or instruments used without specified manufacturer, being can be by the routine of acquisition purchased in market
Product.Those skilled in the art know that embodiment describes the present invention by way of example, and are not intended to limit the required guarantor of the present invention
The range of shield.Entire disclosure case mentioned in this article and other references are merged into herein with its full text by reference.
In following embodiments of the present invention, RV-04 refers to Camoquin (national standard, by Chinese food medicine
Product examine is determined research institute's standard substance and is provided), with chemical formula as follows：
The structure of 1. pseudovirus of embodiment
The 1.1 antiviral drugs screening principles based on pseudovirus
Envelope protein in virus is responsible for identifying the receptor of target cell surface so as to the process for starting absorption and penetrating, thus
Invade cell.In addition, a kind of virus of phenotype mixing phenomena prompting occurred when two kinds of viruses infect a kind of cell simultaneously
Cyst membrane can be integrated into the particle surface of another different virus.Based on above 2 points, in the mistake of research Virus entry cell
Journey and its tissue tropism and receptor etc. produce a kind of new technology --- pseudovirus technology.
Current pseudovirus then refers to that a kind of retrovirus can integrate the cyst membrane sugar of another variety classes virus
Albumen, so as to the cyst membrane with exogenous virus formed, and genome remains retrovirus genomic characterization itself
Virus.Pseudovirion is that the virus protein carrying provided by incasing cells is wrapped in the viral vectors recombinant of foreign gene,
There was only the ability of single infection to target cell, be replication defect type, new virion cannot be generated after infection.Though this virus
Cell can be so invaded, but due to being replication defect type after cell is invaded, and the structural proteins for being assembled into virus are outer
What source provided, it is impossible to the self-replacation of virus is carried out, so very safe.Due to pseudovirus biting property of parent and course of infection with
Euvirus is identical, therefore the early process that can be infected with simulated virus, and reporter gene (fluorescein is carried in pseudovirus
Enzyme), various detection and analysises can be fast, easily carried out, the pseudovirus of this laboratory structure can not only be used for studying disease
The relationship of poison and host cell, the receptor of clonal virus, it is often more important that can be used for screening antiviral drugs, evaluation vaccine is exempted from
Effect of epidemic disease etc..
The present invention is using the plasmid of viral glycoprotein and skeleton plasmid cotransfection 293T cells is expressed, so as to obtain cape horn fever
Poison.With the drug effect after this pseudovirus and doubling dilution for a period of time after, pseudovirus sensitive cells is added in, in 37 DEG C of 5%CO2
After continuing culture in incubator 48 hours, luminous value is detected.If drug prevents viruses adsorption, into cell, sample well
Luminous value can decay or disappear；If drug, without antiviral activity, sample well luminous value is suitable with virus control luminous value.
The preparation of 1.2 pseudovirus
A) preparation of rabies viruses (RV) pseudovirus
Using transfection reagent Lipofectamine 2000, by envelope plasmid pCAG.CVSpCMV-CVS and skeleton plasmid
PSG3 Δs env.cmv.Fluc is (referring to Nie J, et al.Sci Rep.2017Feb 20；7:42769.) according to mass ratio 3:1
Ratio cotransfection 293T cells, after transfection 4-6h or so replace culture medium, be subsequently placed in 37 DEG C, 5%CO2It is cultivated in incubator
48h or so.The cell culture medium supernatant after transfection is collected, centrifugation removes cell fragment, and 0.45 μm of filter filtering is sub-packed in
In 1.5mL centrifuge tubes, saved backup in -80 DEG C of refrigerators.
B) preparation of Ebola virus (EBOV) pseudovirus
Using transfection reagent Lipofectamine 3000, by envelope plasmid pcDNA3.1-EBOV-ZAIRE-GP-8A (ginsengs
See Liu, Q.et al.Sci Rep.2017Mar 30；7:45552.) and skeleton plasmid pSG3 Δs env.cmvFluc is (referring to Nie
J,et al.Sci Rep.2017Feb20；7:42769.) according to mass ratio 1:2 ratio cotransfection 293T cells, 4- after transfection
6h or so replaces culture medium, is subsequently placed in 37 DEG C, 5%CO248h or so is cultivated in incubator.Collect the cell culture medium after transfection
Supernatant, centrifugation remove cell fragment, and 0.45 μm of filter filtering is sub-packed in 1.5mL centrifuge tubes, is preserved in -80 DEG C of refrigerators standby
With.
C) preparation of Marburg virus (MARV) pseudovirus
Using transfection reagent Lipofectamine 3000, by envelope plasmid pcDNA3.1-MARV-GP (referring to Zhang,
L.et al.Hum Vaccin Immunother.2017Aug3；13(8):1811-1817.) and skeleton plasmid pSG3 Δs
Env.cmv.Fluc is (referring to Nie J, et al.Sci Rep.2017Feb 20；7:42769.) according to mass ratio 1:2 ratio
Cotransfection 293T cells, 4-6h or so replaces culture medium after transfection, is subsequently placed in 37 DEG C, 5%CO248h or so is cultivated in incubator.
The cell culture medium supernatant after transfection is collected, centrifugation removes cell fragment, and 0.45 μm of filter filtering is sub-packed in 1.5mL centrifuge tubes
In, it is saved backup in -80 DEG C of refrigerators.
D) preparation of vesicular stomatitis virus (VSV) pseudovirus
Using transfection reagent Lipofectamine 2000, by envelope plasmid pHEF-VSVG (by US National health research
Institute (NIH) aids reagent projects provide) and skeleton plasmid pSG3 Δs env.cmv.Fluc (referring to Nie J, et al.Sci
Rep.2017Feb 20；7:42769.) mass ratio 1:2 ratio cotransfection 293T cells, 4-6h or so replaces culture after transfection
Base is subsequently placed in 37 DEG C, 5%CO248h or so is cultivated in incubator.The cell culture medium supernatant after transfection is collected, is centrifuged, is removed
Cell fragment, 0.45 μm of filter filtering, is sub-packed in 1.5mL centrifuge tubes, is saved backup in -80 DEG C of refrigerators.
1.3 pseudovirus titer determinations
1.3.1RV, the titration of EBOV, MARV, VSV pseudovirus
A) pseudovirus frozen will be dispensed from -80 DEG C of refrigerators taking-up room-temperature water baths defrostings, it is dilute that 5 times of series are done in 96 orifice plates
It releases, 11 gradients, 4 multiple holes, 100 μ L of final volume, last is classified as cell controls.
B) preprepared cell 293T cells (converging rate up to more than 80%) in incubator are taken out, with T75 culture bottles
For, the culture medium abandoned in bottle is inhaled, adds in 1 × PBS of 5ml cleaning cells, after 1 × PBS is abandoned in suction, 2.5ml is added in and has diluted 5 times
0.25% pancreas enzyme -EDTA, it is made to submerge cell dissociation 45 seconds, suction abandons pancreatin, is placed in cell incubator and stands 4 minutes, treat
Cell detachment, adds in 10ml DMEM culture mediums and pancreatin, and after blowing and beating mixing, cell count will be thin with DMEM complete mediums
Born of the same parents are diluted to 5 × 105A/ml.The 100 μ L of addition per hole, i.e., 5 × 10496 orifice plates are put into incubator, culture 48h (RV, EBOV by/hole
Cultivate 48h) or 72h (MARV cultivates 72h).
C) 96 orifice plates are taken out after 48h from cell incubator, is inhaled from sample well and abandons 100 μ L supernatants, add in 100 μ L balances
To the Bright-Glo Luciferase Assay Reagents of room temperature, it is protected from light 2min.
D) after reaction, cell is made fully to split pressure-vaccum 3-5 times repeatedly of the liquid in reacting hole with multichannel pipettor
Solution, is sucked out 150 μ L liquid from every hole, is added in corresponding 96 hole chemiluminescence detection plates, is placed in chemiluminescence detector, uses
BrighGlo programs read luminous value.
1.3.2 the calculating of TCID50
To be higher than the 3 of the luminous value of cell control well times for cut off values, pseudovirus is calculated with Reed-Muench methods
TCID50 is as follows shown：
1. calculate each viral dilution positive number of perforations (a) and negative number of perforations (b)；
2. calculate positive and negative hole cumulative number：Positive hole cumulative number from bottom to top add up (c), negative hole cumulative number by
It is upper to add up (d) downwards；
3. calculate the percentage in positive hole：Ratio=(c)/((c)+(d)) × 100；
4. calculate distance proportion：Distance proportion=(percent positive -50 for being more than 50%)/(big number is in 50% positive
Percentage-be less than 50% percent positive)；
5. logarithm+distance proportion × dilute of the highest extension rate of the percent positive of the logarithm of TCID50=more than 50%
Release the logarithm of coefficient.
Evaluations of the embodiment 2.RV-04 to the inhibitory activity of a variety of pseudovirus
In the present embodiment, RV pseudovirus, EBOV pseudovirus and the MARV pseudovirus prepared by using embodiment 1
To evaluate RV-04 to above-mentioned 3 kinds of viral inhibitory activity.
2.1 experimental procedure
Since the pseudovirus skeleton plasmid pSG3 Δs env.cmvFluc that the present invention uses is from HIV, in order to exclude
Drug inhibits the possibility of pseudovirus by preventing the process of reverse-transcription of skeleton HIV, and the present invention is utilizing pseudovirus primary dcreening operation medicine
While object, secondary screening is carried out to exclude false positive using pseudovirus is fitted into comprising vesicular stomatitis virus (VSV) memebrane protein.Meter
Pseudovirus is fitted into VSV memebrane proteins for calculation drug and target viral memebrane protein is fitted into pseudovirus (that is, RV pseudovirus, EBOV pseudovirus
Or MARV pseudovirus) 50% inhibition concentration (IC50) ratio, when the value be more than 3 when, it is possible to determine that exclude the drug and pass through
The process of reverse-transcription of skeleton HIV is prevented to inhibit the possibility of pseudovirus.Its specific experiment step is as follows：
2.1.1RV-04 to RV pseudovirus, EBOV pseudovirus, MARV pseudovirus inhibitory activity evaluation
A) RV-04 to 30mmol/L is dissolved using DMSO.
B) RV-04 is serially diluted in 96 orifice plates from 150 times of dilution startings, continuous 3 times of progress, 8 dilutions, 2
Multiple holes, final volume are 100 μ L, and first row and secondary series are respectively cell controls (only plus culture medium and cell) and virus control
(only plus viral and cell).
C) RV pseudovirus, EBOV pseudovirus, MARV pseudovirus, VSV pseudovirus are diluted to corresponding TCID50/ml respectively
50 μ L are added in per hole, make the RV pseudovirus in the hole containing 200TCID50/ respectively in final volume, the EBOV pseudovirus in 400TCID50/ holes,
The MARV pseudovirus in 100-800TCID50/ holes, the VSV pseudovirus in 200TCID50/ holes are that 96 orifice plates are put into 37 DEG C, 5%CO2
Cultivate 1h.
D) when incubation time to half an hour, take out preprepared 293T cells in incubator (converge rate up to 80% with
On), pancreas enzyme treated cell is same as above.Cell is diluted to 5 × 10 with DMEM complete mediums5A/ml.
E) when incubation time is small to 1, add 100 μ L 5 × 10 per hole into 96 orifice plates5The cell of a/ml makes every hole thin
Born of the same parents are 5 × 104It is a, 96 orifice plates are put into incubator, 37 DEG C, 5%CO2Cultivate 48h (RV, EBOV pseudovirus) or 72h (MARV cape horn fevers
Poison).
F) 96 orifice plates are taken out from cell incubator after 48h or 72h, is inhaled and abandoned from each loading hole with multichannel pipettor
150 μ L supernatants, then add in 100 μ L Bright-Glo Luciferase Assay Reagents, and room temperature is protected from light 2min.
G) after reaction, cell is made fully to split pressure-vaccum 3-5 times repeatedly of the liquid in reacting hole with multichannel pipettor
Solution, is sucked out 150 μ L liquid from every hole, is added in corresponding 96 hole chemiluminescence detection plates, is placed in chemiluminescence detector, uses
BrighGlo programs read luminous value.
2.1.2 the computational methods of inhibitory activity
A) inhibiting rate of calculating drug, inhibiting rate=[(luminous intensity mean value-cell control well of sample sets shines by force 1-
Spend mean value)/(virus control wells luminous intensity mean value-cell control well luminous intensity mean value)] × 100%；And with drug concentration
For abscissa, inhibiting rate draws amount effect curve for ordinate.
B) according to the inhibiting rate curve of drug, the ID50 and IC50 of drug are calculated using following equation.
Drug ID50=lg (1/ drug is less than the dilution of 50% fluorescent value ratio)+(50- drugs are less than 50% fluorescent value
The fluorescent value percentage in ratio hole)/(higher than the fluorescent value percentage in 50% fluorescent value ratio hole-it is less than 50% fluorescent value ratio
The fluorescent value percentage in hole) × lg (drug dilution multiple).
Drug IC50 (μm ol/L)=30000/ drug ID50.
2.2 experimental result
RV-04 is to the inhibiting rate curve of RV pseudovirus, EBOV pseudovirus and MARV pseudovirus as shown in Figure 1, wherein RV-04
IC50 to rabies viruses pseudovirus (RV) is 4.2 μM, and the IC50 to Ebola virus pseudovirus (EBOV) is 1.8 μM, to horse
The IC50 of your fort virus-virus pseudovirus (MARV) is 1.4 μM, and the IC50 to VSV pseudovirus is 20.4 μM；RV-04 is to VSV vacations
The IC50 ratios of virus and target pseudovirus are noticeably greater than 3.The above results show that RV-04 is false to mad dog, Ebola, Marburg
Virus has significant inhibiting effect, and it interferes pseudovirus pair by inhibiting the combination of virus membrane antigen and target cell
The infection of cell.
The Cytotoxic evaluation of embodiment 3.RV-04
3.1 experimental procedure
In the present embodiment, it is quantitative determined by ATP to evaluate influence of the RV-04 drugs to cell activity, detection is former
Reason is as follows：
ATP is an index of living cells metabolism, is usedLuminescence method cell viability detects
Kit detects after drug and cytosis number of viable cells, cell cracking in culture by being quantitative determined to ATP
It is directly proportional to existing ATP amounts with the luminous signal of generation, and ATP amounts are directly directly proportional to the cell quantity in culture.It is logical
Influence of the drug to ATP luminous signals in cell is crossed, calculates 50% cytotoxic concentration (CC50) of drug.Calculate drug
50% cytotoxic concentration (CC50) and drug to the ratio (that is, therapeutic index SI=CC50/IC50) of the IC50 of target viral,
When the therapeutic index is more than 3, positive drug of the drug to inhibit virus is judged, that is, show that the drug has significant inhibit
Virus activity, and without apparent cytotoxicity.Its specific experiment step is as follows：
3.1.1RV-04 the Cytotoxic evaluation on 293T cells
A) RV-04 is serially diluted in 96 orifice plates from 150 times of dilution startings, continuous 3 times of progress, 8 dilutions, 2
Multiple holes, final volume are 100 μ L, and first row and secondary series are respectively negative control (only adding culture medium, be not added with cell) and cell pair
According to (only plus culture medium and cell).
B) cell controls and sample well add in 50 μ L culture mediums per hole, 96 orifice plates are put into 37 DEG C, 5%CO2Cultivate 1h.
C) preprepared 293T cells (converging rate up to more than 80%) in incubator are taken out, pancreas enzyme treated cell is same
On.Cell is diluted to 5 × 10 with DMEM complete mediums5A/ml.100 μ L complete mediums, remaining hole are added in first row
100 μ L are added in per hole and have been diluted to 5 × 105The 293T cells of a/ml, it is 5 × 10 to make every hole cell4It is a, 96 orifice plates are put into and are incubated
Case, 37 DEG C, 5%CO2Cultivate 48h.
D) 96 orifice plates are taken out after 48h from cell incubator, is inhaled from every hole with multichannel pipettor and abandons 150 μ L supernatants, so
After add in 100 μ LDetection reagent, room temperature are protected from light 10min.
E) after reaction, cell is made fully to split pressure-vaccum 3-5 times repeatedly of the liquid in reacting hole with multichannel pipettor
Solution, 150 μ L liquid are sucked out from every hole, is added in corresponding 96 hole chemiluminescence detection plate of black, is placed in chemiluminescence detector
In, read luminous value with CellTiter-Glo programs.
3.2 experimental result
The results are shown in Figure 2, and wherein RV-04 is 87.6 μM to the CC50 of cell, shows RV-04 below 87.6 μM to thin
Born of the same parents do not have toxicity；Also, described in the CC50 values of RV-04 and embodiment 23 kinds of pseudovirus (RV pseudovirus, EBOV pseudovirus and
MARV pseudovirus) the ratio (i.e. therapeutic index SI=CC50/IC50) of IC50 be noticeably greater than 3.The above results further demonstrate that,
RV-04 has significant inhibition rabies viruses, Ebola virus, Marburg disease cytotoxic activity, and without apparent cytotoxicity.
Evaluations of the embodiment 4.RV-04 to the neutralization activity of mad dog live virus
In the present embodiment, using mad dog live virus to evaluate neutralization activity of the RV-04 drugs to rabies viruses.
It is operated with reference to FFIT, continuous 3 times of dilutions is carried out after the RV-04 of 30mM is diluted 150 times, totally 8 dilutions,
Room temperature is by RV-04 and 50 CVS plants of μ L rabies viruses (building library for National Institute for Food and Drugs Control to preserve) (6.25 × 105It is glimmering
Light titre/ml) it is incubated 1 hour, CVS plants of rabies viruses and PBS and DMSO are incubated 1 hour by PBS and DMSO control groups respectively；It
Afterwards, each hole adds in bsr cell and (builds library for National Institute for Food and Drugs Control to preserve) combined inoculation, then continues in room temperature thin
Born of the same parents cultivate r for 24 hours.Then carry out direct fluorescent staining.After cell culture, cells and supernatant is discarded, PBS is cleaned 1 time, with
Virus remaining in cell conditioned medium is eliminated as much as, acetone is fixed, Fluorescent Staining Observation intracellular viral infections situation.
The results are shown in Figure 3 for microscopy, wherein, PBS and DMSO control groups in fluorescence strong positive, illustrate virus infection BSR
Cell, and be proliferated；And the fluorescence intensity of RV-04 medicine groups is substantially reduced trend, shows that it has apparent neutralize
The effect that CVS plants of rabies viruses.The fluorescent staining in 1-4 holes of RV-04 groups is very weak, illustrates that virus is neutralized by drug, loses
Cell infection ability；And microscopy observation find in 1-4 holes drug on cell without influence.Existed by the way that RV-04 is calculated
It is at room temperature 14.8 μM (Fig. 4) to the IC50 of CVS plants of rabies viruses.
The above results show that RV-04 has potential neutralization activity to mad dog live virus.
The evaluation of embodiment 5.RV-04 interior resisting virus activity
In the present embodiment, using through EBOV virus or MARV virus attacks after BALB/c mouse as model, investigated RV-04
Interior resisting virus activity.
Antiviral activity evaluation of the 5.1 RV-04 drugs in EBOV pseudovirus infection models
The sterile water dissolution of RV-04 drugs, used pseudovirus are pHIV-ZGP-Fluc pseudovirus (i.e. by embodiment 1
The Ebola's pseudovirus prepared).4-5 week old female BAl BIc/c mouse 20 is bought and is studied in Chinese food drug assay
Institute divides 2 groups, every group 10.Experimental group：, for D0, to attack poison on the day of attacking poison a few days ago (Day-2), attack malicious the previous day (Day-
1) 2 hours (- H2) carries out gastric infusion (dosage is 1.8mg/200 μ L) respectively before, attacking poison, and H0 attacks poison, attacks toxic agent amount and is
60AID50 2.67×107TCID50；Control group：With the experimental group sterile water of gavage same volume in the same way simultaneously,
H0 attacks poison, and it is identical with experimental group to attack toxic agent amount.The 4th day after poison is attacked, give every mouse peritoneal and inject 100 μ L amobarbitals
Sodium (240mg/kg) and 100 μ L animal luminous substrates, reaction carry out inspection light, observation animal hair in living imaging instrument after ten minutes
Light level.
The results are shown in Figure 5, and compared with the control group, the light levels of RV-04 administration groups are significantly weaker (Fig. 5 A), RV-04
The total light flux (Total flux) of administration group has dropped 67.24% (Fig. 5 B) compared with the control group.The above results show small
In in mouse body, RV-04 can significantly inhibit the infection of Ebola virus, have significant interior resisting virus activity.
Antiviral activity evaluation of the 5.2 RV-04 drugs in MARV pseudovirus infection models
The sterile water dissolution of RV-04 drugs, used pseudovirus are pHIV-MARV-GP-Fluc pseudovirus (i.e. by reality
Apply the MARV pseudovirus that example 1 prepares).4-5 week old female BAl BIc/c mouse 12, buys and is ground in Chinese food drug assay
Study carefully institute, divide 2 groups, every 6.Experimental group：, for D0, to attack poison on the day of attacking poison a few days ago (Day-2), attack malicious the previous day (Day-1),
Attack and carry out gastric infusion (dosage is 1.8mg/200 μ L) before poison 2 hours (- H2) respectively, H0 attacks poison, attack toxic agent amount for 1.27 ×
107TCID50；Control group：The sterile water of gavage same volume, H0 attack poison in the same way simultaneously with experimental group, attack toxic agent amount
It is identical with experimental group.The 4th day after poison is attacked, give every mouse peritoneal inject 100 μ L yellow Jackets (240mg/kg) and
100 μ L animal luminous substrates, reaction carry out inspection light in living imaging instrument after ten minutes, observe animal light levels.
The results are shown in Figure 6, and compared with the control group, the light levels of RV-04 administration groups are significantly weaker (Fig. 6 A), RV-04
The total light flux (Total flux) of administration group has dropped 84% (Fig. 6 B) compared with the control group.The above results show in mouse
In in vivo, RV-04 can significantly inhibit the infection of Marburg virus, have significant interior resisting virus activity.
Embodiment 6.RV-04 is combined the evaluation of activity with a variety of virus membrane antigens
In the present embodiment, RV-04 is analyzed to a variety of virus membrane antigens by surface plasma resonance (SPR) technology
The combination activity of (EBOV, MARV, RV).
By EBOV and MARV virus membrane antigens extracellular fragment (gp-EBOLA, gp-MARV) (by Gao Fu institutes of Institute of Micro-biology of the Chinese Academy of Sciences
Scholar seminar present), RV CTN Strain (by National Institute for Food and Drugs Control prepare preserve) be separately fixed at CM5 chips
As stationary phase on three channels, by mobile phase of the RV-04 of different diluted concentrations (gradient dilution is proceeded by from 100 μM, is divided
Wei not be 100 μM, 50 μM, 25 μM, 12.5 μM, 6.25 μM, 3.125 μM, 1.56 μM).Buffer is PBS+0.05%Tween20+
5%DMSO is measured on 25 DEG C, Biacor eS200.
As a result respectively as Figure 7-9, wherein Fig. 7 A-9A are the SPR sensorgram of the RV-04 of different diluted concentrations；Fig. 7 B-
9B is change curve of the steady-state response value relative to different RV-04 concentration, and wherein vertical line represents equilibrium dissociation constant (KD) value,
RV-04 and the K that 3 kinds of virus membrane antigens (RV, EBOV, MARV) combine are obtained by BiacoreS200 measureDValue is respectively 32 μ
Mol/L, 32 μm of ol/L, 22 μm of ol/L illustrate that RV-04 and three kinds of viral albumen have certain combination activity.
Although the specific embodiment of the present invention has obtained detailed description, it will be appreciated by those skilled in the art that：Root
According to all introductions announced, details can be carry out various modifications and be changed, and these change in the guarantor of the present invention
Within the scope of shield.The whole of the present invention is divided into be provided by appended claims and its any equivalent.
Claims (10)
1. logical formula (I) compound represented or its pharmaceutically acceptable salt or ester be used to prepare in subject prevention and/or
The purposes of the drug of the infection for the treatment of virus or the disease caused by virus infects；
Wherein,
R1It independently is hydrogen, halogen or C1-C6Alkyl；
R2、R3It is each independently C1-C6Alkyl or C3-C6Cycloalkyl.
2. the purposes of claim 1, wherein, R1Selected from hydrogen, halogen and C1-C3Alkyl；
Preferably, R1Selected from hydrogen and halogen (such as-F ,-Cl ,-Br or-I)；
Preferably, R1For halogen (such as-F ,-Cl ,-Br or-I)；
Preferably, R1For-Cl.
3. the purposes of claims 1 or 2, wherein, R2、R3It is each independently C1-C4Linear or branched alkyl group or C3-C4Cycloalkanes
Base；
Preferably, R2、R3It is each independently methyl, ethyl, n-propyl, isopropyl, cyclopropyl, normal-butyl, sec-butyl, tertiary fourth
Base, isobutyl group or cyclobutyl；
Preferably, R2With R3It is identical；
Preferably, R2、R3For ethyl.
4. the purposes of any one of claim 1-3, wherein, the compound has chemical formula as follows.
5. the purposes of any one of claim 1-4, wherein, the virus is selected from Ebola virus, Marburg virus and rabies
Poison.
6. the purposes of any one of claim 1-5, wherein, the disease caused by virus infects is selected from Ebola's bleeding
Heat, marburg hemorrhagic fever and rabies.
7. the purposes of any one of claim 1-6, wherein, the pharmaceutically acceptable salt is hydrochloride, sulfate or phosphoric acid
Salt；
Preferably, the pharmaceutically acceptable salt is hydrochloride.
8. the purposes of any one of claim 1-7, wherein, the drug further includes the other pharmacy with antiviral activity and lives
Property agent；
Preferably, the other forms of pharmacologically active agents is to can be used for treatment rabies virus infection and/or rabic drug, such as
Rabies vacciness, rabies antiserum or anti-rabies immune globulin (for example, rabies human immunoglobulin(HIg))；
Preferably, the other forms of pharmacologically active agents is available for the infection for the treatment of Ebola virus and/or Ebola hemorrhagic fever
Drug；
Preferably, the other forms of pharmacologically active agents is to can be used for the infection for the treatment of Marburg virus and/or Marburg virus bleeding
The drug of heat.
9. the purposes of any one of claim 1-8, wherein, the drug is tablet, pill, capsule, granule, powder, molten
Liquid, suspension, parenteral solution or injection sterile powder.
10. the purposes of any one of claim 1-9, wherein, the subject is mammal, such as non-human primate,
Such as people.
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| US20160113920A1 (en) * | 2014-10-24 | 2016-04-28 | Keck Graduate Institute Of Applied Life Sciences | Compositions and methods for inhibiting bacterial and viral pathogens |
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| PETER B. MADRID ET AL.: ""A systematic screen of FDA-approved drugs for inhibitors of biological threat agents"", 《PLOS ONE》 * |
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| CN111747944B (en) * | 2020-07-13 | 2022-09-30 | 复旦大学 | Broad-spectrum anti-enveloped virus compound, composition and application thereof |
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