阅读说明：本技术 247种化合物及其组合物在抗新型冠状病毒感染中的应用 (247 compounds and their use in combating infection by new coronaviruses ) 是由 李佳 赵强 蒋华良 苏明波 张文茹 程曦 臧奕 郑明月 蒋映艳 于 2020-06-22 设计创作，主要内容包括：本发明涉及247种化合物有效成分在抗冠状病毒中的应用。具体而言,本发明涉及表1所示的247种化合物或其组合物作为抑制新型冠状病毒PL～(pro)蛋白活性的抑制剂；和/或(b)治疗和/或预防、缓解由新型冠状病毒感染引起的相关疾病的药物中的用途。(The invention relates to application of 247 effective components of compounds in resisting coronavirus. In particular, the invention relates to 247 compounds shown in table 1 or compositions thereof as inhibitors of novel coronavirus PL pro An inhibitor of protein activity; and/or (b) the treatment and/or prevention, alleviation of related diseases caused by novel coronavirus infections.)

1. Use of an active ingredient or a formulation containing said active ingredient, wherein said active ingredient is: any one compound or combination thereof or pharmaceutically acceptable salt thereof, or combination thereof, selected from the 247 compounds of table 1;

and, the preparation of the active ingredient or a pharmaceutical composition containing the active ingredient is used for preparing (a) a formulation for inhibiting coronavirus PL^proInhibitors of protein active ingredients; and/or (b) a medicament for the treatment and/or prevention, amelioration of a related disease caused by a coronavirus infection.

2. The use of claim 1, wherein the coronavirus is a human-infecting coronavirus selected from the group consisting of: SARS-CoV (Severe acute respiratory syndrome coronavirus), 2019 novel coronavirus (2019-nCoV or SARS-CoV-2), MERS-CoV (Middle East respiratory syndrome coronavirus), or common cold-causing coronavirus; the common cold-causing coronavirus is preferably Human coronavirus OC43(Human coronavirus OC43), Human coronavirus 229E (Human coronavirus 229E), Human coronavirus NL63(Human coronavirus NL63), and Human coronavirus HKUL (Human coronavirus HKUL).

3. The use of claim 1, wherein the disease associated with coronavirus is selected from the group consisting of: cold symptoms caused by human coronavirus, high risk symptom infection, respiratory infection, pneumonia and its complications, novel coronavirus pneumonia caused by SARS-CoV-2 (Corona Virus Disease 2019, COVID-19), or their combination.

4. The use according to claim 1, wherein the active ingredient is selected from the group of pharmaceutically active ingredients consisting of: CD0945-G005, CD0946-B009, CD3226-D010, CD3531-C011 or pharmaceutical compositions thereof.

5. The use of claim 1, wherein said composition or medicament comprises: oral and non-oral formulations.

6. A pharmaceutical composition, comprising:

(a1) a first active ingredient selected from the group consisting of: 247 compounds of table 1 or a pharmaceutically acceptable salt or composition thereof;

(a2) optionally a second active ingredient which is an antiviral drug selected from the group consisting of: interferons, RNA-dependent RNA polymerase inhibitors (e.g., Remdesivir (Rudexilevir or GS-5734), Favipiravir (favipiravir), Galidesivir, GS-441524); lopinavir (Lopinavir), Ritonavir (Ritonavir), Nelfinavir (Nelfinavir); chloroquine (Chloroquine, Sigma-C6628), hydroxychloroquine (hydroxychloroquine), cyclosporine (cyclosporine), colimycin (carromycin), baicalin (baicain), baicalein (baicalein), Naphthoquine (napthoquine), Ciclesonide (Ciclesonide), Ribavirin (Ribavirin), Penciclovir (Penciclovir), Leflunomide (Leflunomide), Teriflunomide (Teriflunomide), naftifimide (temulomide), nafamostat (nafamostat), nitate (nitazoxanide), Darunavir (Darunavir), Arbidol (Arbidol), Camostat (Camostat), Niclosamide (nicamide), bartinib (barnitinib), rurunavir (raltinib), Saquinavir (Saquinavir), sinavir (quinavir), or a pharmaceutically acceptable salt thereof;

and/or said second active ingredient is selected from the group consisting of: zinc (Zinc), Fingolimod (Fingolimod), vitamin c (vitamin c), Olmesartan Medoxomil (Olmesartan Medoxomil), valsartan (valsartan), Losartan (Losartan), Thalidomide (thiaidomide), glycyrrhizic acid (glycyrrhetic acid), Artemisinin (Artemisinin), dihydroartemisinin (dihydroartesunine), Artesunate (Artesunate), Artemisone (Artemisone), Azithromycin (Azithromycin), Escin (Escin), Naproxen (Naproxen), or combinations thereof;

and (b) a pharmaceutically acceptable carrier.

7. The pharmaceutical composition of claim 6, wherein the pharmaceutical composition is for inhibiting coronavirus PL^proAnd (3) protein activity.

8. Use of a pharmaceutical composition according to claim 6 for the preparation of a medicament for (a) inhibiting coronavirus; and/or (b) a medicament for the treatment and/or prevention, amelioration of a related disease caused by a coronavirus infection.

9. Novel coronavirus PL inhibition^proA method of producing a protein, comprising the steps of:

mixing a first active ingredient or a preparation containing the first active ingredient with PL of coronavirus (SARS-CoV-2)^proProtein contact, inhibition of said coronavirus PL^proThe activity of the protein; or

Contacting a first active ingredient or a formulation comprising said first active ingredient with a coronavirus (SARS-CoV-2) to inhibit said coronavirus PL^proActivity of the protein, thereby inhibiting replication of the coronavirus;

wherein the first active ingredient is selected from the group consisting of the compounds of table 1 or pharmaceutically acceptable salts thereof or combinations thereof.

10. The method of claim 9, wherein the first active ingredient is selected from the group consisting of: CD0945-G005, CD0946-B009, CD3226-D010, CD3531-C011, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.

Technical Field

The invention relates to the field of medicines, in particular to application of 247 effective components of compounds in resisting virus infection, especially application in resisting coronavirus infection.

Background

In acute infectious diseases, most of the infectious diseases are viral infectious diseases, the incidence rate of the viral infectious diseases is high, and the death rate is high. Because the detection and diagnosis means are limited, the outbreak of new epidemic caused by new viruses often has the characteristics of paroxysmal, random, unpredictable and the like, once the outbreak occurs, if no effective prevention and treatment means exists, the large-scale epidemic is very easy to cause, and the health and life safety of people is seriously threatened.

Coronaviruses (Coronaviruses) are single-stranded positive-strand RNA viruses belonging to the order Nidovirales (Nidovirales) Coronaviridae (Coronaviridae) orthocoronaviridae (orthocoronaviridae) and can infect various species such as humans, bats, pigs, mice, cows, horses, goats, monkeys, etc. There are 6 known human-infecting coronaviruses (HCoV), including middle east respiratory syndrome-associated coronavirus (MERSR-CoV) and severe acute respiratory syndrome-associated coronavirus (SARSr-CoV).

The novel coronavirus 2019-nCoV (SARS-CoV-2) is the 7 th coronavirus infecting human, and can cause severe pneumonia. The 2019-nCoV virus transmission path is not completely mastered, is known to be transmitted by droplets and contact, and has the possibility of infection of people and medical staff, certain community transmission risk and virus variation. There is currently no specific preventive or therapeutic approach for diseases caused by the novel coronavirus.

2019-nCoV coronavirus belongs to coronavirus of coronavirus family, and is single-chain positive virus with envelopeA sense RNA virus. Similar to other known coronaviruses, the 2019-nCoV coronaviruses also complete the propagation of the offspring viruses through the processes of adsorption, penetration, uncoating, biosynthesis, assembly and release of the offspring viruses and the like. 2019-nCoV coronavirus infects host cells, the spike glycoprotein initiated on the surface of a virus envelope is combined with a receptor on the surface of the host cells, then membrane fusion occurs, the virus enters the host cells, the virus genetic material single-chain positive-sense RNA is released under the action of organelles such as cell lysosome, and the like, and the polyprotein is generated by translation under the action of protein synthesis elements such as mitochondria, ribosome and the like of the host cells and necessary raw materials, and then two essential cysteine proteases of the 2019-nCoV coronavirus are obtained: papain (PL)^pro) And 3C-like protease (3C-like protease,3 CL)^pro) Cleavage at specific sites processes the polyprotein precursors, producing a number of non-structural proteins that are important to the viral life cycle. Under the action of these nonstructural proteins, the viral RNA replicates the daughter viral nucleic acid material and translates to a large amount of the desired structural proteins, completing assembly and release of the daughter virus. Any link or key enzyme of the life cycle of 2019-nCoV coronavirus infected cells can be used as a research target of antiviral drugs, such as cysteine protease PL for hydrolyzing and cutting polyprotein precursors^proAnd 3CL^proRNA polymerase responsible for completing replication of progeny viral genetic material, and the like.

At present, no specific vaccine or antiviral drug exists for severe pneumonia diseases caused by SARS-CoV-2 coronavirus. These infectious diseases seriously affect the life health of people, and the research and development of antiviral drugs with good effect is urgent. Aiming at SARS-CoV-2 coronavirus, a low-toxicity and high-efficiency antiviral drug is developed to meet the clinical requirements of SARS-CoV-2 coronavirus infected patients at home and abroad, and has great social significance.

In view of the above, there is an urgent need in the art to develop inhibitors against SARS-CoV-2 coronavirus for the treatment of pneumonia caused by infection with the novel coronavirus.

Disclosure of Invention

The object of the present invention is to provideEffective inhibition of novel coronavirus PL^proA medicinal active component of the protein and a new application thereof in diseases such as pneumonia caused by novel coronavirus infection.

In a first aspect of the present invention, there is provided a use of an active ingredient or a formulation containing the active ingredient, wherein the active ingredient is any one compound or combination thereof selected from the 247 compounds in table 1 (i.e. any one compound or combination thereof numbered 1 to 247 in table 1), or a pharmaceutically acceptable salt or composition thereof:

and, the preparation of the active ingredient or a pharmaceutical composition containing the active ingredient is used for preparing (a) a formulation for inhibiting coronavirus PL^proInhibitors of protein active ingredients; and/or (b) a medicament for the treatment and/or prevention, amelioration of a related disease caused by a coronavirus infection.

In another preferred embodiment, the preparation of the active ingredient or a pharmaceutical composition containing the active ingredient is used for preparing (a) inhibiting novel coronavirus (SARS-CoV-2) PL^proInhibitors of protein active ingredients; and/or (b) a medicament for the treatment and/or prevention, amelioration of the associated diseases caused by the novel coronavirus infection.

In another preferred embodiment, the coronavirus is a human-infecting coronavirus selected from the group consisting of: SARS-CoV (Severe acute respiratory syndrome coronavirus), 2019 novel coronavirus (2019-nCoV or SARS-CoV-2), MERS-CoV (Middle East respiratory syndrome coronavirus), or common cold-causing coronavirus; the common cold-causing coronavirus is preferably Human coronavirus OC43(Human coronavirus OC43), Human coronavirus 229E (Human coronavirus 229E), Human coronavirus NL63(Human coronavirus NL63), and Human coronavirus HKUL (Human coronavirus HKUL).

In another preferred embodiment, the coronavirus-induced related disease is selected from the group consisting of: cold symptoms caused by human coronavirus, high risk symptom infection, respiratory infection, pneumonia and its complications, novel coronavirus pneumonia caused by SARS-CoV-2 (Corona Virus Disease 2019, COVID-19), or their combination.

In another preferred example, the related disease caused by the 2019 novel coronavirus infection is selected from the group consisting of: respiratory infections, pneumonia and its complications, or combinations thereof.

In another preferred embodiment, the active ingredient is selected from the group consisting of: CD0945-G005, CD0946-B009, CD3226-D010, CD3531-C011 or pharmaceutical compositions thereof.

In another preferred embodiment, the pharmaceutical composition containing the active ingredient may also contain other antiviral agents.

In another preferred embodiment, the medicament further comprises an additional component selected from the group consisting of: anti-lung injury drugs, anti-inflammatory drugs or drugs with immunomodulatory effects.

In another preferred embodiment, the medicament further comprises an additional component selected from the group consisting of: zinc (Zinc), Fingolimod (Fingolimod), vitamin c (vitamin c), Olmesartan Medoxomil (Olmesartan Medoxomil), valsartan (valsartan), Losartan (Losartan), Thalidomide (thiaidomide), glycyrrhizic acid (glycyrrhetic acid), Artemisinin (Artemisinin), dihydroartemisinin (dihydroartemesinin), Artesunate (Artesunate), Artemisone (Artemisone), Azithromycin (Azithromycin), Escin (Escin), Naproxen (Naproxen), or combinations thereof.

In another preferred embodiment, the composition or medicament comprises: oral and non-oral formulations.

In another preferred embodiment, the formulation comprises: powder, granule, capsule, injection, tincture, oral liquid, tablet, buccal tablet, or dripping pill.

In a second aspect of the present invention, there is provided a pharmaceutical composition comprising:

(a1) a first active ingredient selected from the group consisting of: 247 compounds of table 1 or a pharmaceutically acceptable salt or composition thereof;

(a2) optionally a second active ingredient which is an antiviral drug selected from the group consisting of: interferons, RNA-dependent RNA polymerase inhibitors (e.g., Remdesivir (Rudexilevir or GS-5734), Favipiravir (favipiravir), Galidesivir, GS-441524); lopinavir (Lopinavir), Ritonavir (Ritonavir), Nelfinavir (Nelfinavir); chloroquine (Chloroquine, Sigma-C6628), hydroxychloroquine (hydroxychloroquine), cyclosporine (cyclosporine), colimycin (carromycin), baicalin (baicain), baicalein (baicalein), Naphthoquine (napthoquine), Ciclesonide (Ciclesonide), Ribavirin (Ribavirin), Penciclovir (Penciclovir), Leflunomide (Leflunomide), Teriflunomide (Teriflunomide), naftifimide (temulomide), nafamostat (nafamostat), nitate (nitazoxanide), Darunavir (Darunavir), Arbidol (Arbidol), Camostat (Camostat), Niclosamide (nicamide), bartinib (barnitinib), rurunavir (raltinib), Saquinavir (Saquinavir), sinavir (quinavir), or a pharmaceutically acceptable salt thereof;

and/or said second active ingredient is selected from the group consisting of: zinc (Zinc), Fingolimod (Fingolimod), vitamin c (vitamin c), Olmesartan Medoxomil (Olmesartan Medoxomil), valsartan (valsartan), Losartan (Losartan), Thalidomide (Thalidomide), glycyrrhizic acid (glycyrrhizic acid), Artemisinin (Artemisinin), dihydroartemisinin (dihydroartemisinin), Artesunate (Artesunate), Artemisone (artemosone), Azithromycin (Azithromycin), Escin (Escin), Naproxen (Naproxen), or combinations thereof;

and (b) a pharmaceutically acceptable carrier.

In another preferred embodiment, the second active ingredient is selected from the group consisting of: (Y1) RNA replicase inhibitors (e.g., Remdesivir (redevir or GS-5734)); (Y2) Lopinavir (Lopinavir); (Y3) Ritonavir (Ritonavir); (Y4) favipiravir; (Y5) Chloroquine (Chloroquine, Sigma-C6628), hydroxychloroquine (hydroxychloroquine), or a pharmaceutically acceptable salt thereof (e.g., Chloroquine phosphate), (Y6) Nelfinavir (Nelfinavir); (Y7) any combination of the above Y1 to Y6.

In another preferred embodiment, the pharmaceutical composition is used for inhibiting coronavirus PL^proAnd (3) protein activity.

At another placeIn a preferred embodiment, the pharmaceutical composition is used for inhibiting 2019 novel coronavirus (2019-nCoV)/(SARS-CoV-2) PL^proAnd (3) protein activity.

In a third aspect of the invention, there is provided the use of a pharmaceutical composition according to the second aspect for the preparation of a medicament for (a) inhibiting a coronavirus; and/or (b) a medicament for the treatment and/or prevention, amelioration of a related disease caused by a coronavirus infection.

In another preferred example, the inhibitor is used for preparing (a) inhibiting 2019 novel coronavirus (SARS-CoV-2)/(2019-nCoV) PL^proAn inhibitor of protein activity; and/or (b) a medicament for treating and/or preventing, and/or alleviating related diseases caused by 2019 novel coronavirus (SARS-CoV-2) infection.

In a fourth aspect of the present invention, there is provided a method for inhibiting a novel coronavirus PL^proA method of producing a protein comprising the steps of:

mixing a first active ingredient or a preparation containing the first active ingredient with PL of coronavirus (SARS-CoV-2)^proProtein contact, inhibition of said coronavirus PL^proThe activity of the protein; or

Contacting a first active ingredient or a formulation comprising said first active ingredient with a coronavirus (SARS-CoV-2) to inhibit said coronavirus PL^proActivity of the protein, thereby inhibiting replication of the coronavirus;

wherein the first active ingredient is selected from the group consisting of the compounds of table 1 or pharmaceutically acceptable salts thereof or combinations thereof.

In another preferred embodiment, the first active ingredient is selected from the group consisting of: CD0945-G005, CD0946-B009, CD3226-D010, CD3531-C011, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.

In another preferred embodiment, the first active ingredient is a pharmaceutical active ingredient of the following group:

in another preferred embodiment, the method is an in vitro method.

In another preferred embodiment, the method is non-therapeutic and non-diagnostic.

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.

Drawings

None.

Detailed Description

The present inventors have conducted extensive and intensive studies and, as a result of extensive screening, have unexpectedly developed 247 pharmaceutically active ingredients effective in inhibiting novel coronavirus PL for the first time^proA protein. Experiments have shown that the active ingredients (represented by CD0945-G005, CD0946-B009, CD3226-D010, and CD 3531-C011) of the present invention can efficiently inhibit the novel coronavirus PL^proThe activity of the protein. The present invention has been completed based on this finding.

Specifically, the present invention discloses pharmaceutically active ingredients selected from the group consisting of: use of CD0945-G005 against novel coronaviruses. The pharmaceutical active ingredient of the present invention has excellent inhibitory activity against novel coronavirus PL^proThe protein activity can inhibit the replication of the novel coronavirus, and has good clinical application prospect.

Term(s) for

As used herein, "active ingredient of the present invention", "inhibiting novel coronavirus PL of the present invention^proActive ingredients of protein "," 247 compounds "," 247 active ingredients ", and" active ingredients of the present invention "are used interchangeably and mean having excellent inhibitory activity against coronavirus PL^proAny active ingredient for protein activity, including any one of the 247 compounds selected from table 1 and combinations thereof.

As used herein, "formulation of the present invention" refers to a formulation containing an active ingredient of the present invention.

As used herein, the term "comprising" or variations thereof, such as "comprises" or "comprising," etc., are understood to imply the inclusion of stated elements or components but not the exclusion of any other elements or components.

As used herein, the terms "novel coronavirus", "2019-nCov" or "SARS-CoV-2" are used interchangeably, the 2019 novel coronavirus being the 7 th coronavirus known to infect humans and causing new coronary pneumonia (COVID-19), one of the serious infectious diseases threatening global human health.

Coronavirus (coronavirus)

Coronaviruses (CoV) belong to the family of the Nidovirales (Nidovirales) Coronaviridae (Coronaviridae), a enveloped positive-strand RNA virus, a subfamily of which contains four genera, alpha, beta, delta and gamma.

Among the currently known human-infecting coronaviruses, HCoV-229E and HCoV-NL63 belong to the genus alpha, and HCoV-OC43, SARS-CoV, HCoV-HKU1, MERS-CoV and SARS-CoV-2 are all the genus beta. SARS-CoV-2 is also known as 2019-nCov.

Highly pathogenic coronavirus "SARS-CoV" and "middle east respiratory syndrome" MERS-CoV both belong to the genus beta coronavirus. The novel coronavirus (SARS-CoV-2) has about 80% similarity to SARS-CoV and 40% similarity to MERS-CoV, and also belongs to the genus beta coronavirus.

The genome of the virus is a single-strand positive-strand RNA, is one of RNA viruses with the largest genome, and codes of the RNA viruses comprise replicase and PL^proProteins, envelope proteins, nucleocapsid proteins, and the like. In the initial stage of viral replication, the genome is translated into two peptide chains of up to several thousand amino acids, the precursor Polyprotein (Polyprotein), which is subsequently cleaved by proteases to produce nonstructural proteins (e.g., RNA polymerase and helicase) and structural proteins (e.g., PL)^proProtein) and accessory proteins.

Active Compounds and active ingredients of the invention

In the present invention, effective inhibition of coronavirus PL is provided^proActive ingredient of protein activity. The active ingredient is selected from the compounds of table 1 shown below, or pharmaceutically acceptable salts thereof, or combinations thereof.

TABLE 1247 Compounds

Experiments show that the active ingredients of the invention can effectively inhibit PL of novel coronavirus^proProtein activity, thereby preventing, treating and/or ameliorating diseases associated with the novel coronavirus.

As used herein, "the active ingredient of the present invention", "the active compound of the present invention inhibiting coronavirus replication" are used interchangeably and refer to a compound having an excellent activity of inhibiting coronavirus replication.

It is to be understood that the active ingredients of the present invention include any one of the compounds of table 1 and combinations thereof.

Or a pharmaceutically acceptable salt, enantiomer, diastereomer or racemate thereof, or a prodrug thereof. It is to be understood that the active ingredients of the present invention also include crystalline forms of the active compounds of the present invention, amorphous compounds, and deuterated compounds, among others.

The "pharmaceutically acceptable salts" are conventional non-toxic salts formed by reacting the active compounds of the present invention with inorganic or organic acids. For example, conventional non-toxic salts can be prepared by reacting the active compounds of the present invention with inorganic acids including hydrochloric, hydrobromic, sulfuric, nitric, sulfamic, phosphoric, and the like, or organic acids including citric, tartaric, lactic, pyruvic, acetic, benzenesulfonic, p-toluenesulfonic, methanesulfonic, naphthalenesulfonic, ethanesulfonic, naphthalenedisulfonic, maleic, malic, malonic, fumaric, succinic, propionic, oxalic, trifluoroacetic, stearic, pamoic, hydroxymaleic, phenylacetic, benzoic, salicylic, glutamic, ascorbic, p-aminobenzenesulfonic, 2-acetoxybenzoic, isethionic, and the like; or sodium, zinc, potassium, calcium, aluminum or ammonium salts of the active compounds of the invention formed with 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 in an ester and then with an inorganic base; or the corresponding inorganic acid salt formed by the active compound of the invention and lysine, arginine and ornithine and then hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid or phosphoric acid, or the corresponding organic acid salt formed by the active compound of the invention and formic acid, acetic acid, picric acid, methanesulfonic acid or ethanesulfonic acid; or a sodium salt, a zinc salt, a potassium salt, a calcium salt, an aluminum salt or an ammonium salt of a carboxyl group/phenolic hydroxyl group in the molecule of the active compound of the present invention and an inorganic base.

Furthermore, the active ingredients of the invention are particularly suitable for use in combination with other anti-coronavirus drugs. Representative other anti-coronavirus drugs include (but are not limited to): interferons, RNA-dependent RNA polymerase inhibitors (e.g., Remdesivir (Rudexilevir or GS-5734), Favipiravir (favipiravir), Galidesvir, GS-441524); 3CL protease inhibitors (e.g., GC-376), Lopinavir (Lopinavir), Ritonavir (Ritonavir), Nelfinavir (Nelfinavir); chloroquine (Chloroquine, Sigma-C6628), hydroxychloroquine, cyclosporine (cyclosporine), clarithromycin (carrlomycin), baicalin (baicain), baicalein (baicalein), Naphthoquine (napheroquine), Ciclesonide (Ciclesonide), Ribavirin (Ribavirin), Penciclovir (Penciclovir), Leflunomide (Leflunomide), Teriflunomide (Teriflunomide), nafamostat (nafamostat), nitazoxanide (nitazoxanide), Darunavir (Darunavir), Arbidol (Arbidol), Camostat (Camostat), Niclosamide (nicamide), bartinib (baricitinib), cricotinib (rulitinib), Dasatinib (Dasatinib), saquinar (quinacrid), quinavir (bevir), or a pharmaceutically acceptable salt thereof. The interferon comprises one or more of interferon alpha-2 a, interferon alpha-2 b, interferon alpha-n 1, interferon alpha-n 3, interferon beta-1 a and interferon beta-1 b.

In addition, since SARS-CoV-2 infection can cause acute lung injury, inflammatory response and even cytokine storm, the active ingredients of the present invention are also particularly suitable for use in combination with drugs having an effect of ameliorating acute lung injury, an anti-inflammatory effect or an immune modulating effect. Representative drugs include, but are not limited to, Zinc (Zinc), Fingolimod (Fingolimod), vitamin c (vitamin c), Olmesartan Medoxomil (olmestanan Medoxomil), valsartan (valsartan), Losartan (Losartan), Thalidomide (Thalidomide), glycyrrhizic acid (glycyrrhetic acid), Artemisinin (artemizin), dihydroartemisinin (dihydroartesunine), Artesunate (Artesunate), Artemisone (Artemisone), Azithromycin (Azithromycin), Escin (Escin), Naproxen (Naproxen).

Preferably, the active ingredient of the invention is combined with artemisinin drugs (one or more of artemisinin, dihydroartemisinin, artesunate and arteannuin). A large number of researches show that the artemisinin drugs have multiple anti-inflammatory and immunoregulatory mechanisms, realize anti-inflammatory and immunoregulatory functions by inhibiting T cell proliferation and activation, inhibiting B cell activation and antibody generation, increasing regulatory T cells and reducing release of inflammatory cytokines, and are expected to relieve immune injury symptoms caused by SARS-CoV-2 infection.

Preferably, the active ingredient of the invention is combined with artemisinin drugs (one or more of artemisinin, dihydroartemisinin, artesunate and arteannuin) and azithromycin.

The active ingredient of the invention can inhibit the infection activity of novel coronavirus such as SARS-CoV-2. Therefore, when the active ingredient of the present invention is therapeutically administered or administered, the infection with the novel coronavirus of 2019 (SARS-CoV-2) can be inhibited, thereby achieving an antiviral effect.

Pharmaceutical composition and application

The invention also provides application of the active compound for inhibiting the replication of the coronavirus, or one or more mixtures of pharmaceutically acceptable salts or prodrugs thereof as an active ingredient in preparing a medicament for treating and/or preventing and relieving diseases related to respiratory tract infection, pneumonia and the like caused by infection of the coronavirus such as 2019 novel coronavirus.

The pharmaceutical composition provided by the present invention preferably contains 0.001-99 wt% of active ingredient, preferably 0.1-90 wt% or 1-50 wt% of active compound of the present invention as active ingredient, the rest being pharmaceutically acceptable carrier, diluent or solution or salt solution.

If necessary, one or more pharmaceutically acceptable carriers can be added into the medicine. The carrier comprises diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption enhancers, surfactants, adsorption carriers, lubricants and the like which are conventional in the pharmaceutical field.

The compounds and pharmaceutical compositions provided herein may be in a variety of forms such as tablets, capsules, powders, syrups, solutions, suspensions and aerosols, and the like, and may be presented in a suitable solid or liquid carrier or diluent and in a suitable sterile vehicle for injection or instillation.

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 formulation generally comprises 0.05 to 400mg of active compound according to the invention, preferably 1 to 500mg of active compound according to the invention.

The compounds and pharmaceutical compositions of the present invention may be administered to mammals in clinical use, including humans and animals, by oral, nasal, dermal, pulmonary or gastrointestinal routes of administration. Most preferably oral. Most preferably, the daily dose is 0.01-400mg/kg body weight, and is administered once or in portions of 0.01-200mg/kg body weight. 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 drug or inhibitor of the present invention can be administered by a variety of different means, e.g., by injection, spray, nasal drop, eye drop, osmotic, absorption, physical or chemical mediated methods, into the body such as muscle, intradermal, subcutaneous, intravenous, mucosal tissue; or mixed with other materials or encapsulated and introduced into body.

Typically, the active ingredient of the invention or a pharmaceutical composition containing it is administered in unit dosage form, either enterally or parenterally, such as orally, intravenously, intramuscularly, subcutaneously, nasally, oromucosally, ocularly, pulmonary and respiratory, dermally, vaginally, rectally, and the like.

The dosage form for administration may be a liquid dosage form, a solid dosage form, or a semi-solid dosage form. The liquid dosage forms can be solution (including true solution and colloidal solution), emulsion (including o/w type, w/o type and multiple emulsion), suspension, injection (including water injection, powder injection and infusion), eye drop, nose drop, lotion, liniment, etc.; the solid dosage form can be tablet (including common tablet, enteric coated tablet, buccal tablet, dispersible tablet, chewable tablet, effervescent tablet, orally disintegrating tablet), capsule (including hard capsule, soft capsule, and enteric coated capsule), granule, powder, pellet, dripping pill, suppository, pellicle, patch, aerosol (powder), spray, etc.; semisolid dosage forms can be ointments, gels, pastes, and the like.

The active ingredients of the invention can be prepared into common preparations, sustained release preparations, controlled release preparations, targeting preparations and various particle delivery systems.

In order to formulate the active ingredients of the present invention into tablets, a wide variety of excipients known in the art can be used, including diluents, binders, wetting agents, disintegrants, lubricants, glidants. The diluent can be starch, dextrin, sucrose, glucose, lactose, mannitol, sorbitol, xylitol, microcrystalline cellulose, calcium sulfate, calcium hydrogen phosphate, calcium carbonate, etc.; the wetting agent can be water, ethanol, isopropanol, etc.; the adhesive can be starch slurry, dextrin, syrup, Mel, glucose solution, microcrystalline cellulose, acacia slurry, gelatin slurry, sodium carboxymethylcellulose, methylcellulose, hydroxypropyl methylcellulose, ethyl cellulose, acrylic resin, carbomer, polyvinylpyrrolidone, polyethylene glycol, etc.; the disintegrant can be dry starch, microcrystalline cellulose, low-substituted hydroxypropyl cellulose, crosslinked polyvinylpyrrolidone, crosslinked sodium carboxymethylcellulose, sodium carboxymethyl starch, sodium bicarbonate and citric acid, polyoxyethylene sorbitol fatty acid ester, sodium dodecyl sulfate, etc.; the lubricant and glidant may be talc, silica, stearate, tartaric acid, liquid paraffin, polyethylene glycol, etc.

The tablets may be further formulated into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or double-layer and multi-layer tablets.

To prepare the administration units into capsules, the active ingredients of the invention can be mixed with diluents, glidants and the mixture placed directly into hard or soft capsules. Or mixing the effective components with diluent, binder, and disintegrating agent, making into granule or pellet, and placing into hard capsule or soft capsule. The diluents, binders, wetting agents, disintegrants, glidants used to prepare the tablets of the invention may also be used to prepare the capsules of the invention.

In order to prepare the active ingredients of the invention into injection, water, ethanol, isopropanol, propylene glycol or the mixture of the water, the ethanol, the isopropanol and the propylene glycol can be used as a solvent, and a proper amount of solubilizer, cosolvent, pH regulator and osmotic pressure regulator which are commonly used in the field can be added. The solubilizer or cosolvent can be poloxamer, lecithin, hydroxypropyl-beta-cyclodextrin, etc.; the pH regulator can be phosphate, acetate, hydrochloric acid, sodium hydroxide, etc.; the osmotic pressure regulator can be sodium chloride, mannitol, glucose, phosphate, acetate, etc. For example, mannitol and glucose can be added as proppant for preparing lyophilized powder for injection.

In addition, colorants, preservatives, flavors, or other additives may also be added to the pharmaceutical preparation, if desired.

The active ingredient or composition of the present invention can be taken alone or in combination with other therapeutic or symptomatic agents.

When the active ingredient of the present invention acts synergistically with other therapeutic agents, its dosage should be adjusted according to the actual circumstances.

The main advantages of the invention include:

(a) the active compound of the invention can effectively inhibit SARS-CoV-2 (novel coronavirus) and has obvious antiviral effect. Wherein WNN3369-C011 inhibits the IC of SARS-CoV₅₀A value of about 0.18. mu.M; IC of NCRW0370-F002 for inhibiting SARS-CoV-2₅₀The value was only 0.28. mu.M; IC of RUS0093-G006 for inhibiting SARS-CoV-2₅₀A value of about 0.32. mu.M; IC for WNN3768-D008 inhibiting SARS-CoV-2₅₀A value of about 0.5. mu.M; IC of PC0003-B003 for inhibiting SARS-CoV-2₅₀The value was about 0.51. mu.M.

(b) The active compound of the invention has low toxic and side effects and good drug property. The active ingredients of the invention are provided with good medical prospect in the field of anti-neocoronary pneumonia. Wherein, when representing compound CD0945-G005 inhibits the activity of SARS-CoV by 94.26%, the cytotoxicity is only 6.03%.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are only for illustrating the present invention and are not intended to limit the scope of the present invention. The experimental procedures, without specific conditions being noted in the following examples, are generally performed according to conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are percentages and parts by weight.

Example 1 anti-SARS-CoV-2 Virus surface PL^proprotein-Activity assay

The experimental apparatus used in this example: microplate reader envision tm (PerkinElmer, USA), experimental materials: recombinant SARS-CoV 2PL^proFull-length protein using Escherichia coliExpressing and purifying the expression system to obtain; the polypeptide substrate Z-RLRGG-AMC was synthesized by Gill Biochemical company.

The test principle is as follows: based on SARS-CoV-2PL^proThe protein is a basic characteristic of proteolytic enzyme, and a screening system for detecting the activity of SARS-CoV-2PLpro protein by a fluorescence method is established. SARS-CoV-2PL^proThe protein can specifically recognize and cut the diglycine polypeptide, the activity detection can adopt fluorescent polypeptide as a substrate, and the activity of the proteolytic enzyme is reflected by detecting the generation of a fluorescent signal.

Specifically, SARS-CoV 2PL^proIs a proteolytic enzyme, which can specifically recognize and cut the diglycine polypeptide, and the activity detection can adopt fluorescent polypeptide Z-RLRGG-AMC as a substrate when SARS-CoV 2PL^proWhen protease activity is exerted, the polypeptide chain is cleaved to release fluorescent AMC, so that AMC can release 460nm emitted light under 355nm excitation light, and SARS-CoV 2PL is reacted by detecting generation of 460nm fluorescence signal^proActivity of (2).

The test method comprises the following steps: the total volume of the test is 50 mu L, and the specific reaction system is as follows: 20mM Tris pH8.0, 0.01% Tween20,0.5mM DTT,40nM PL^pro50 μ M substrate polypeptide and different concentration gradients of compounds, while a DMSO only solvent control was set up.

The samples were dissolved in DMSO and stored at low temperature, and the concentration of DMSO in the final system was controlled within a range that did not affect the detection activity. The samples are tested for activity under a single concentration of the primary screen, e.g., 10. mu.g/mL. For samples that exhibit activity under certain conditions, e.g., an Inhibition% Inhibition greater than 50, the activity dose dependence, i.e., IC, is tested₅₀Values, obtained by non-linear fitting of sample concentrations by sample activity, were calculated as Graphpad Prism 5 using software, as model for fitting was sigmoidal dose-response (variable slope), and for most inhibitor screening models, the bottom and top of the fitted curve were set at 0 and 100.

The experimental results are as follows: the activity of 247 compounds for inhibiting SARS-Cov-2 was determined as shown in Table 2.

Table 2: 247 Compounds for inhibiting SARS-Cov-2PL^proActivity of proteins

EXAMPLE 2 anti-SARS-CoV-2 Virus cytotoxicity assay

The test principle is as follows: the cell infected with SARS-CoV-2 virus is used as virus host cell, and the test sample has SARS-CoV-2 copying blocking activity, which can reflect the antiviral activity of SARS-CoV-2 infection key target. The detection index is the activity level of a reporter gene on the pseudovirus genome.

The test method comprises the following steps:

inoculating virus-infected cells to 96-well culture plate one day in advance, setting active plate and cytotoxic plate respectively, placing at 37 deg.C and 5% CO₂And (5) culturing. The activity assay plate and the cytotoxicity assay plate are added with samples with different dilution concentrations and SARS-CoV-2 pseudovirus suspension according to the same sample adding mode, and a virus control, a cell control and a sample control are set. After further 3 days of culture, the cytotoxic plates were assayed for cell viability using the MTT method. And (3) sucking the culture solution by using an active plate, adding 100 mu L of cell lysate into each hole, performing shake lysis for 5 minutes, adding 100 mu L of Luciferase reaction detection solution into each hole, performing shake incubation for 5 minutes, and determining the chemiluminescence value.

The evaluation method comprises the following steps:

cytotoxicity (MTT method): by comparing the OD values of the virus control, cell control and sample control, the viability of the cells was calculated and further the cytotoxic effect of the samples was calculated.

The experimental results are as follows:

the data for determining the anti-SARS-CoV-2 virus activity and cytotoxicity of 247 compounds are shown in Table 3:

TABLE 3247 anti-SARS-CoV-2 virus activity and cytotoxicity of compounds

As can be seen from Table 3, the compounds of the present invention not only effectively inhibit SARS-CoV-2 virus activity, but also ensure low cytotoxicity. In particular, the representative compound CD0945-G005 exhibited 94.26% antiviral activity, and the cytotoxicity was only 6.03%. Therefore, the preferable compound of the invention can be widely applied to clinic and has good medicinal prospect.

Discussion of the related Art

1. Coronavirus is an RNA virus whose proliferation depends on the regeneration of maternal RNA by RNA-dependent RNA polymerase (RdRp), where SARS-Cov-2 is the cause of new coronary pneumonia, SARS-Cov-2PL^proThe protein is one of key proteins of which 2019-nCoV key protein RdRp becomes an active form, and SARS-Cov-2PL^proHas been considered as one of the drug targets for treating coronary pneumonia. The compounds in Table 2 of the present invention showed very excellent SARS-Cov-2PL^proInhibiting the activity. The compounds of the invention are directed against SARS-Cov-2PL^proIC of₅₀The value can be as low as 0.18 +/-0.03 mu M, which indicates that the compound has the capability of inhibiting the proliferation of SARS-Cov-2, thereby achieving the purpose of treating the new coronary pneumonia.

2. The coronavirus is taken as a virus subspecies, has relatively similar pathogenesis and whole life cycle, and mainly comprises SARS-CoV (Severe acute respiratory syndrome coronavirus), 2019 novel coronavirus (2019-nCoV or SARS-CoV-2), MERS-CoV (Middle East respiratory syndrome coronavirus) or common cold coronavirus; the common cold-causing coronavirus is preferably Human coronavirus OC43(Human coronavirus OC43), Human coronavirus 229E (Human coronavirus 229E), Human coronavirus NL63(Human coronavirus NL63), Human coronavirus HKUl (Human coronavirus HKUl) and the like, different coronaviruses have papain-like PLpro with high homology, so that the novel compound discovered by SARS-CoV-2PLpro possibly has the function of inhibiting other coronaviruses, and therefore the compound also provides a novel solution for other coronaviruses.

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.