WO2024027844A1 - Pharmaceutical composition and use thereof - Google Patents

Abstract

The present invention provides a pharmaceutical composition and use thereof. Specifically, the present invention provides a pharmaceutical composition for preventing and/or treating coronavirus infection and pharmaceutical use thereof. The pharmaceutical composition comprises a) an effective amount of a compound represented by formula (I) or a pharmaceutically acceptable salt thereof; and b) a therapeutically effective amount of a 3CL protease inhibitor. The pharmaceutical composition of the present invention can be used as a coronavirus inhibitor, has a significant synergistic effect, is used for treating and/or preventing and relieving diseases caused by coronaviruses, and has the advantage of having good efficacy, being highly safe, reducing the resistance of viruses, reducing toxic and side effects of drugs, and the like.

Description

A pharmaceutical composition and its use

This application claims priority from the Chinese patent application with application number 202210941259.0 filed on August 5, 2022, the entire content of which is incorporated herein by reference in its entirety.

Technical field

The invention belongs to the field of pharmacy and relates to a pharmaceutical composition for preventing and/or treating coronavirus infection and its use.

Background technique

Coronavirus is a type of enveloped positive-stranded single-stranded RNA virus. Since records began, various parts of the world have been hit by the coronavirus. Novel coronavirus (SARS-CoV-2) is a highly contagious coronavirus. On March 11, 2020, the World Health Organization (WHO) announced that SARS-CoV-2 infection causes new coronavirus pneumonia (COVID-19). Entering a global pandemic, as of July 28, 2022, the COVID-19 epidemic has lasted for more than two years. Since the outbreak of SARS-CoV-2, many mutated virus strains have emerged, many of which are more infectious and pathogenic than the original virus strains. SARS-CoV-2 will continue to evolve over time, and mutant strains with greater infectiousness, pathogenicity, or immune evasion capabilities may emerge in the future. Therefore, the development of specific drugs against SARS-CoV-2 is an important issue that needs to be solved urgently internationally, and it is also a necessity for future strategic reserves.

Currently, combination drugs in the antiviral field are being accepted by more and more doctors and patients. The combined use of antiviral drugs with different targets or different mechanisms of action can not only improve the therapeutic effect of the disease, but also reduce viral drug resistance and reduce drug side effects.

VV116 is an orally available nucleoside analog triisobutyrate prodrug against SARS-CoV-2. This compound can be rapidly metabolized into the parent nucleoside in the body and converted into its triphosphate active form within the cell. The VV116 nucleoside triphosphate form can compete with the endogenous natural nucleoside triphosphate to bind to the active center of viral RNA-dependent RNA polymerase, thereby inhibiting the transcription and replication process of the virus and exerting an antiviral effect. VV116 has been approved for the treatment of COVID-19 in Uzbekistan, and multiple clinical trials are being actively advanced. The structural formula of VV116 is as follows:

Nirmatrelvir (PF-07321332) is an orally available 3CL protease (3C-likeprotease) inhibitor that can effectively inhibit the activity of 3CL protease in a variety of coronaviruses, thus exerting antiviral effects. 3CL protease is a cysteine protease whose main function is to cleave coronavirus polyprotein to produce various functional proteins, including RNA-dependent RNA polymerase, helicase, single-stranded RNA binding protein, exo-ribose Nucleases, endoribonucleases, 2′-O-ribose methyltransferases, etc. These functional proteins all play an important role in virus replication. Nirmatrelvir is the active ingredient in the anti-coronavirus drug Paxlovid. Another ingredient in Paxlovid is ritonavir, which is mainly used to slow down the metabolism of nirmatrelvir. Paxlovid has received Emergency Use Authorization (EUA) for the treatment of COVID-19 in the United States, United Kingdom, European Union and Canada. The structural formula of Nirmatrelvir is as follows:

Molnupiravir (EIDD2801) is the first oral antiviral drug approved for the treatment of patients with new coronavirus pneumonia. Its main plasma metabolite β-D-N4-hydroxycytidine (NHC) is a cytosine nucleoside derivative. The compound shows significant inhibitory effects on the replication of various viruses (influenza virus, hepatitis C virus, SARS, MERS, SARS-CoV-2, etc.) and is a broad-spectrum antiviral nucleoside analogue. NHC will be converted into its triphosphate active form in cells. After this active form is incorporated into the viral RNA chain by the viral RNA-dependent RNA polymerase, it will not cause the termination of the nucleic acid chain elongation, but will make the virus lethal. mutations, thereby exerting antiviral effects. Molnupiravir has been approved in the UK to treat COVID-19 and has received Emergency Use Authorization (EUA) from the US Food and Drug Administration (FDA). The structural formula of Molnupiravir is as follows:

Contents of the invention

The inventor of the present invention unexpectedly discovered that the combined use of pyrrolotriazine base nucleoside analogues (compounds represented by formula (I)) and 3CL protease inhibitors can play a synergistic anti-coronavirus effect, and it has a mutagenic effect. There is no synergistic effect in the combined use of nucleoside analogs NHC based on the mechanism. Therefore, the present invention mainly involves the following aspects.

A first aspect of the invention relates to a pharmaceutical composition containing:

a) A therapeutically effective amount of a compound represented by formula (I) or a pharmaceutically acceptable salt thereof; and

b) A therapeutically effective amount of 3CL protease inhibitor;

In the formula,

R ₁ and R ₂ are each independently selected from hydrogen, C _1-20 alkanoyl group, C _3-10 cycloalkylformyl group, and amino C _1-20 alkanoyl group;

Or R ₁ and R ₂ are connected to each other to form

R ₃ is selected from hydrogen, C _1-20 alkanoyl group, C _3-10 cycloalkylformyl group, and amino C _1-20 alkanoyl group;

R ₄ is selected from hydrogen, deuterium, and halogen;

X is selected from -CH ₂ -, -CD ₂ -.

In some embodiments, R ₁ and R ₂ are each independently selected from hydrogen, C _1-10 alkanoyl, C _3-7 cycloalkylformyl, and amino C _1-10 alkanoyl;

Or R ₁ and R ₂ are connected to each other to form

R ₃ is selected from hydrogen, C _1-20 alkanoyl group, C _3-7 cycloalkylformyl group, and amino C _1-10 alkanoyl group;

R ₄ is selected from hydrogen, deuterium, and halogen;

X is selected from -CH ₂ -, -CD ₂ -.

In some embodiments, R ₁ and R ₂ are each independently selected from hydrogen, formyl, acetyl, propionyl, butyryl, isobutyryl, valeryl, pivaloyl, hexanoyl, 2-ethylbutyryl and 3,3-dimethylbutanoyl, cyclopropanoyl, cyclobutanoyl, cyclopentanoyl, cyclohexaneformyl, cycloheptanoyl, α-aminoisovaleryl, or R ₁ and R ₂ mutually formed by connection

Preferably, R ₁ and R ₂ are each independently selected from hydrogen, isobutyryl, cyclopropanoyl, or R ₁ and R ₂ are connected to each other to form

In some embodiments, R _is selected from hydrogen, formyl, acetyl, propionyl, butyryl, isobutyryl, valeryl, pivaloyl, hexanoyl, 2-ethylbutyryl, 2-methylpentanoyl Acyl, 2-propylpentanoyl, 3,3-dimethylbutanoyl, dodecanoyl, myristanoyl, hexadecanoyl, octadecanoyl, eicosanoyl, cyclopropanoyl, cyclopropanoyl Butyroyl, cyclopentanoyl, cyclohexylformyl, cycloheptanoyl, aminoacetyl, α-aminopropionyl, α-aminoisovaleryl;

Preferably, R ₃ is selected from hydrogen, propionyl, isobutyryl, pivaloyl, 2-ethylbutyryl, 2-methylvaleryl, 2-propylvaleryl, tetradecanoyl, hexadecanoyl , octadecanoyl, cyclopropanoyl, cyclopentanoyl, cyclohexanoyl, α-aminoisovaleryl.

In some embodiments, _R4 is selected from hydrogen, deuterium, fluorine, chlorine and iodine, preferably hydrogen, deuterium, fluorine.

In some embodiments, the compound represented by formula (I) or a pharmaceutically acceptable salt thereof is any compound having the following structure:

In some embodiments, the compound represented by formula (I) is selected from compounds 11, 14, 16, 20, 27, 30, 32, 37, 41, 68 and 69. Preferably, the compound of formula (I) is Compound 69.

In some embodiments, the 3CL protease inhibitor is selected from one or more of the following: nirmatrelvir, S-217622, EDP-235, GC-376, PBI-0451, FB2001, VV993 and SIM0417; preferably, the The 3CL protease inhibitor is nirmatrelvir.

In some embodiments, the pharmaceutical composition further comprises:

c) Synergists; and

d) Optional pharmaceutically acceptable carrier or excipient.

In some embodiments, the potentiator is ritonavir.

In some embodiments, the mass ratio of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof to the 3CL protease inhibitor is 0.01-99.99:1, preferably 0.1-20:1, and further preferably 0.5-4:1, more preferably 0.5:1, 1:1, 1.5:1, 2:1, 2:3, 4:3.

In some embodiments, in the case where the pharmaceutical composition contains a synergist, in the pharmaceutical composition, the compound represented by formula (I) or a pharmaceutically acceptable salt thereof and 3CL protease inhibitor The mass ratio of the agent and the synergist is 1:0.01-99.99:0.01-99.99, preferably 1:0.1-20:0.1-20, and more preferably 1:0.2-5:0.2-5.

In some embodiments, the compound represented by formula (I) or a pharmaceutically acceptable salt thereof and the 3CL protease inhibitor are in the same preparation unit, or the compound represented by formula (I) or a pharmaceutically acceptable salt thereof Acceptable salts and 3CL protease inhibitors are available in different strength formulation units.

In some embodiments, the compound represented by formula (I) or a pharmaceutically acceptable salt thereof and the 3CL protease inhibitor are administered simultaneously, separately, or sequentially.

The second aspect of the present invention relates to the use of the pharmaceutical composition according to the first aspect of the present invention in the preparation of a medicament, wherein the medicament is (a) an inhibitor for inhibiting coronavirus replication; or (b) for the treatment of and /or drugs to prevent or alleviate diseases caused by coronavirus infection.

In some embodiments, the coronavirus is one or more selected from the following:

(1) Coronaviruses that infect humans: such as severe acute respiratory syndrome coronavirus SARS-CoV (Severe acute respiratory syndrome coronavirus, SARS-CoV), 2019 new coronavirus (2019-nCoV or SARS-CoV-2), Middle East respiratory syndrome coronavirus Syndrome coronavirus MERS-CoV (Middle East respiratory syndrome coronavirus, MERS-CoV), human coronavirus OC43 (Human coronavirus OC43), human coronavirus 229E (Human coronavirus 229E), human coronavirus NL63 (Human coronavirus NL63), human Coronavirus HKUl(Human coronavirus HKUl);

(2) Coronaviruses that infect animals: such as porcine epidemic diarrhea virus (PEDV) and feline infectious peritonitis virus (FIFV).

In some embodiments, the disease caused by coronavirus infection is one or more selected from the following:

(D1) Common colds, high-risk symptomatic infections, respiratory infections, pneumonia and their complications caused by human coronavirus infection;

(D2) Porcine epidemic diarrhea caused by porcine epidemic diarrhea virus;

(D3) Feline infectious peritonitis caused by feline coronavirus;

Preferably, the disease caused by coronavirus infection is a disease caused by SARS-CoV-2 infection, especially one or more selected from the following: respiratory tract infection caused by SARS-CoV-2 infection, pneumonia and its complications;

Preferably, the complications include cardiac arrhythmia, myocarditis, respiratory failure, liver function impairment and renal function impairment.

Beneficial effects:

The in vitro cell experiment of the present invention shows that the combination of VV116 (compound 69) and nirmatrelvir can inhibit the proliferation of SARS-CoV-2 and HCoV-OC43 in cells, and the effect is significantly higher than that of the drug alone, and good treatment can be achieved at low doses. The effect is significant synergy.

The efficacy experiment of the in vivo suckling mouse model of the present invention shows that the combination of VV116 and nirmatrelvir can effectively inhibit the load of HCoV-OC43 in the brain and lungs, and the effect is higher than that of the drug alone, and has an obvious synergistic effect.

Therefore, the combination of VV116 and nirmatrelvir can be used as a coronavirus inhibitor to treat diseases caused by coronavirus infection. It has the advantages of good efficacy, high safety, reduced viral drug resistance, and reduced drug side effects.

Description of the drawings

Figure 1 is the in vitro inhibitory activity test results and synergistic calculation results of the combination of VV116 and nirmatrelvir at different concentrations against the SARS-CoV-2 delta variant strain in Example 1.

Figure 2 is the in vitro inhibitory activity test results and synergistic calculation results of the combination of VV116 and nirmatrelvir at different concentrations on HCoV-OC43 in Example 2.

Figure 3 is the in vitro inhibitory activity test results and superposition effect calculation results of the combination of VV116 and NHC at different concentrations against the SARS-CoV-2 delta variant strain in Example 3.

Figure 4 is the results of the in vivo inhibitory activity test of VV116, nirmatrelvir and ritonavir on the HCoV-OC43 infected suckling mouse model in Example 4 alone or in combination.

Figure 5 is the in vivo inhibitory activity test results of VV116, nirmatrelvir and ritonavir alone or in combination in Example 5 on the K18-hACE2 mouse model infected with the SARS-CoV-2 delta variant strain.

Detailed ways

Terminology:

In the present invention, unless otherwise expressly stated, the terms used in the present invention have the meanings defined below. Terms not expressly defined herein have general meanings generally understood by those skilled in the art.

A dash ("-") not between two letters or symbols indicates the attachment site of the substituent. For example, "-CH ₂ -", "-CD ₂ -" means that the group is attached to the rest of the molecule through a carbon atom. However, "-" may be omitted when the attachment point of the substituent is obvious to a person skilled in the art, such as for substituents such as halogen.

When the group has a wavy line , the wavy line indicates where the group is attached to the rest of the molecule.

As used herein, "alkanoyl" refers to a fully saturated linear or branched monovalent hydrocarbon group linked by an acyl group. Alkanoyl groups preferably contain 1-20 carbon atoms, more preferably 1-16 carbon atoms, 1-10 carbon atoms, 1-8 carbon atoms, 1-6 carbon atoms, 1-4 carbon atoms, 1 -3 or 1-2 carbon atoms. The number before the alkanoyl group indicates the number of carbon atoms. For example, "C _1-20 alkanoyl" means an alkanoyl group having 1 to 20 carbon atoms, and so on. Representative examples of alkanoyl include, but are not limited to, formyl, acetyl, propionyl, butyryl, isobutyryl, 2-ethylbutyryl, 3,3-dimethylbutyryl, valeryl, isovaleryl, Pivaloyl, 2-methylpentanoyl, 2-propylpentanoyl, 2,2-dimethylpentanoyl, 2,3-dimethylpentanoyl, hexanoyl, 3-methylhexanoyl, heptanoyl , octanoyl, nonanoyl, decanoyl, dodecanoyl, myristanoyl, hexadecanoyl, octadecanoyl, eicosanoyl, etc.

As used herein, "cycloalkylformyl" refers to a saturated non-aromatic carbocyclic ring linked by a formyl group, including mono-, bi- or tricyclic rings, preferably monocyclic rings. Preferably it has 3-10 carbon atoms, more preferably 3-8 ring carbon atoms, such as 3-7, 3-6 ring carbon atoms. "C _3-10 cycloalkylformyl" is intended to include C ₃ , C ₄ , C ₅ , C ₆ , C ₇ and C ₈ cycloalkyl groups attached to formyl, and so on. Representative examples of cycloalkylformyl include, but are not limited to, cyclopropylformyl, cyclopentanoyl, cyclohexylformyl, cycloheptylformyl, cyclooctanoyl.

As used herein, "aminoalkanoyl" refers to an alkanoyl group, as defined herein, in which one or more hydrogen atoms are substituted by an amino group. Representative examples of aminoalkanoyl include, but are not limited to, aminoacetyl, α-aminopropionyl, α-aminoisovaleryl, and the like.

As used herein, "pharmaceutically acceptable salts" refer to salts that retain the biological effects and properties of the compounds of the invention and which are not biologically or otherwise undesirable. Non-limiting examples of such salts include non-toxic, inorganic or organic base or acid addition salts of the compounds of the invention. In many cases, the compounds of the invention are capable of forming acid and/or base salts due to the presence of amino and/or carboxyl groups or groups similar thereto. Pharmaceutically acceptable acid addition salts can be formed with inorganic acids and organic acids. Inorganic acids from which salts can be derived include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like, with hydrobromic acid being preferred. Organic acids from which salts may be derived include, for example, acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, Mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, etc. Pharmaceutically acceptable base addition salts can be formed with inorganic and organic bases. Inorganic bases from which salts can be derived include, for example, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum and the like; ammonium, potassium, sodium, calcium and magnesium salts are particularly preferred. Organic bases from which salts may be derived include, for example, primary, secondary and tertiary amines, substituted amines (including naturally occurring substituted amines), cyclic amines, basic ion exchange resins and the like, in particular such as isopropylamine, Trimethylamine, diethylamine, triethylamine, tripropylamine and ethanolamine. Pharmaceutically acceptable salts of the present invention can be synthesized from the parent compound (basic or acidic moiety) by conventional chemical methods. In general, the salts can be prepared by reacting the free acid form of the compound with a stoichiometric amount of an appropriate base (eg hydroxide, carbonate, bicarbonate, etc. of Na, Ca, Mg or K ) reaction or reacting the free base form of the compound with a stoichiometric amount of an appropriate acid. This type of reaction is usually carried out in water or organic solvents or a mixture of the two. In general, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile are preferred where feasible. Other suitable salts can be found in Remington's Pharmaceutical Sciences, 20th ed., Mack Publishing Company, Easton, Pa., (1985), which is incorporated by reference.

As used herein, "pharmaceutically acceptable excipients" include any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, Absorption delaying agents, salts, preservatives, drugs, drug stabilizers, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, said similar substances and combinations thereof, are well known to those of ordinary skill in the art (see, e.g., Remington's Pharmaceutical Sciences, 18th ed., Mack Printing Company, 1990, pp. 1289-1329, incorporated herein as refer to). Unless any conventional carrier is incompatible with the active ingredient, it is contemplated for use in the therapeutic or pharmaceutical compositions.

As used herein, a "therapeutically effective amount" of a compound of the present invention refers to an amount of a compound of the present invention that elicits a biological or medical response in an individual or ameliorates symptoms, slows or delays the progression of a disease, prevents a disease, or the like. The "therapeutically effective amount" may be determined by the participating physician or veterinary practitioner and will vary with the compound, the disease state treated, the severity of the disease treated, the age and related health conditions of the individual, the route and form of administration, the attending physician It varies depending on factors such as the judgment of the physician or veterinary practitioner.

As used herein, "individual" refers to an animal. Preferably, the animal is a mammal. Individuals also refer to, for example, primates (eg, humans), cattle, sheep, goats, horses, dogs, cats, rabbits, rats, mice, fish, birds, and the like. In a preferred embodiment, the individual is a human.

As used herein, "inhibition" refers to the alleviation or inhibition of a particular condition, symptom or condition or disease, or a significant reduction in the baseline activity of a biological activity or process.

As used herein, the term "treating" any disease or condition in one embodiment means ameliorating the disease or condition (ie, preventing or slowing the progression of the disease or at least one clinical symptom thereof). In another embodiment, "treating" or "treating" refers to improving at least one physical parameter, which may not be noticeable to the patient. In another embodiment, "treating" or "treating" refers to modulating a disease or disorder physically (e.g., stabilizing perceived symptoms) or physiologically (e.g., stabilizing parameters of the body) or both.

As used herein, "prevention" refers to the administration of one or more pharmaceutical substances, in particular a compound of the invention and/or a pharmaceutically acceptable salt thereof, to an individual with a predisposition to the disease in order to prevent the individual from suffering from the disease. .

After extensive and in-depth research and experiments, the inventor unexpectedly discovered that the combined use of a pyrrolotriazine base nucleoside analog (an effective amount of a compound represented by formula (I)) and a 3CL protease inhibitor can achieve Synergistic anti-coronavirus effect. Experiments have shown that the pharmaceutical composition of the present invention has significant anti-coronavirus activity and is expected to be used in the treatment of 2019 new coronavirus (SARS-CoV-2), human coronavirus OC43 (Human coronavirus OC43) and other coronavirus infections. has obvious advantages, and the present invention is completed on this basis.

Specifically, the present invention discloses the use of a type of pharmaceutical composition in anti-coronavirus, which can be used to prepare drugs for treating diseases, conditions or indications caused by coronavirus infection. The viruses are severe acute respiratory syndrome coronavirus SARS-CoV (Severe acute respiratory syndrome coronavirus, SARS-CoV), 2019 new coronavirus (SARS-CoV-2), Middle East respiratory syndrome coronavirus MERS-CoV (Middle East respiratory syndrome coronavirus, MERS-CoV), human coronavirus OC43 (Human coronavirus OC43), human coronavirus 229E (Human coronavirus 229E), human coronavirus NL63 (Human coronavirus NL63), human coronavirus HKUl (Human coronavirus HKUl), pig Epidemic diarrhea virus (PEDV) or feline infectious peritonitis virus (FIFV).

Specifically, the present invention provides the use of a type of pharmaceutical composition in the preparation of virus inhibitors, for example, in the preparation of treatment and/or prevention, alleviation of severe acute respiratory syndrome coronavirus SARS-CoV (Severe acute respiratory syndrome coronavirus, SARS -CoV), 2019 novel coronavirus (SARS-CoV-2), Middle East respiratory syndrome coronavirus MERS-CoV (Middle East respiratory syndrome coronavirus, MERS-CoV), human coronavirus OC43 (Human coronavirus OC43), human coronavirus Diseases caused by infection with 229E (Human coronavirus 229E), human coronavirus NL63 (Human coronavirus NL63), human coronavirus HKUl (Human coronavirus HKUl), porcine epidemic diarrhea virus (PEDV) and/or feline infectious peritonitis virus (FIFV) uses in medicines. The type of pharmaceutical composition described in this article can significantly inhibit the proliferation of coronaviruses such as the 2019 novel coronavirus and HCoV-OC43 in cells both in vitro and in vivo, and the effect is significantly higher than that of the drug alone, and Low doses can achieve good inhibitory effects, have significant synergistic effects and have good clinical application prospects.

The present invention will be described in detail below with reference to examples. It should be noted that the embodiments of the present invention are limited to illustrating the present invention and have no limiting effect. The relevant test methods and other various experimental operations involved in the examples are all conventional techniques in the field. If there are no special instructions in the text, those of ordinary skill in the field can refer to various commonly used reference books before the filing date of the present invention. , scientific and technological literature or relevant instructions, manuals, etc. to be implemented.

Materials and reagents

VV116 and EIDD2801 were provided by Suzhou Wangshan Wangshui Biopharmaceutical Co., Ltd. with a purity greater than 98%; Nirmatrelvir was purchased from Nanjing Zhengji Pharmaceutical Research Co., Ltd. with a purity greater than 98%; Ritonavir was purchased from Taizhou Kerui Biotechnology Co., Ltd. with a purity greater than 98 %. Balb/c suckling mice aged 5-6 days were purchased from Beijing Vitong Lihua Experimental Animal Technology Co., Ltd. K18-hACE2 mice (male, 7-8 weeks) were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. (Jiangsu, China) ).

The new coronavirus was obtained through application from the National Virus Resource Bank, and the anti-new coronavirus in vivo and in vitro activity tests were carried out in a biosafety level three laboratory. Animal experiments complied with the standards for the use and care of experimental animals and were approved by the Life Sciences Research Ethical Review Committee of the Wuhan Institute of Virology, Chinese Academy of Sciences. The antiviral in vivo activity test was conducted in a biosafety level 3 laboratory.

Example 1:

experiment method:

1) Evenly spread African monkey kidney cells (Vero E6) in a 48-well plate, with 50,000-60,000 cells per well, culture them in DMEM+10% FBS culture medium, and place them in a 37°C cell culture incubator for 12 hours;

2) Discard the cell supernatant, replace the culture medium with DMEM+2% FBS containing the drug or drug combination, and incubate in a 37°C cell culture incubator for 1 hour. Drug concentration combination design plan: VV116 and nirmatrelvir each design 7 concentrations, the concentrations are 2.0μM, 0.67μM, 0.22μM, 0.074μM, 0.024μM, 0.008μM, 0μM respectively. The concentration of each drug is related to the concentration of the other drug. Make combinations. The specific drug concentration combinations are shown in Figure 1;

3) Add the new coronavirus SARS-CoV-2 delta variant strain, MOI=0.01 per well, mix gently, and incubate in a 37°C cell culture incubator for 24 hours;

4) Collect the cell supernatant, extract the viral RNA in the supernatant, and use qRT-PCR to detect the viral copy number.

test results:

The results are shown in Figure 1. Quantitative assessment of the virus inhibition rate by measuring viral copy number using real-time quantitative polymerase chain reaction (qRT-PCR) can reflect the inhibitory effect of VV116 and nirmatrelvir on SARS-CoV-2. As can be seen from the figure, VV116 and nirmatrelvir have obvious inhibitory effects on SARS-CoV-2 in a dose-dependent manner. The effect of combined medication is significantly higher than that of individual medications. VV116 in the concentration range of 0-0.67μM is the same as 0-0.074μM. The combination of nirmatrelvir in the concentration range has a significant synergistic effect. In particular, the combination of 0.024 μM VV116 and 0.008 μM and 0.024 μM nirmatrelvir (the molar ratio of VV116 to nirmatrelvir is 1-3:1) has the strongest synergistic effect.

Example 2:

experiment method:

1) Evenly spread human malignant embryonic rhabdomyomas cells (RD) in a 48-well plate, with 60,000-80,000 cells per well, culture them in DMEM+10% FBS culture medium, and place them in a 37°C cell culture incubator for 12 hours. ;

2) Discard the cell supernatant, replace the culture medium with DMEM+2% FBS containing the drug or drug combination, and incubate in a 37°C cell culture incubator for 1 hour. Drug concentration combination design plan: VV116 and nirmatrelvir are designed with 6 concentrations each. The concentrations of VV116 are 5.0μM, 2.5μM, 2.0μM, 1.5μM, 1.0μM, 0.0μM, and the concentrations of nirmatrelvir are 0.30μM, 0.25μM, 0.20μM, 0.15μM, 0.10μM, 0.0μM, the concentration of each drug is combined with the concentration of another drug. The specific drug concentration combination is shown in Figure 2.

3) Add human coronavirus HCoV-OC43, MOI=0.1 per well, mix gently, and incubate in a 37°C cell culture incubator for 48 hours;

4) Collect the cell supernatant, extract the viral RNA in the supernatant, and use qRT-PCR to detect the viral copy number.

test results:

The results are shown in Figure 2. Quantitative evaluation of the virus inhibition rate by measuring viral copy number using real-time quantitative polymerase chain reaction (qRT-PCR) can reflect the inhibitory effect of VV116 and nirmatrelvir on HCoV-OC43. As can be seen from the figure, VV116 and nirmatrelvir have an obvious inhibitory effect on HCoV-OC43 in a dose-dependent manner. The effect of combined medication is significantly higher than that of individual medications. VV116 in the 0-2.5 μM concentration range is the same as 0-0.15 μM concentration range. The combination of nirmatrelvir has significant synergistic effect.

Example 3:

experiment method:

1) Evenly spread African monkey kidney cells (Vero E6) in a 48-well plate, with 50,000-60,000 cells per well, culture them in DMEM+10% FBS culture medium, and place them in a 37°C cell culture incubator for 12 hours;

2) Discard the cell supernatant, replace the culture medium with DMEM+2% FBS containing the drug or drug combination, and incubate in a 37°C cell culture incubator for 1 hour. Drug concentration combination design plan: Set the VV116 concentration as 2.0μM, 1.0μM, 0.5μM, 0.2μM, 0.1μM, 0.0μM, and set the NHC concentration as 10.0μM, 5.0μM, 2.5μM, 1.2μM, 0.6μM, 0.3μM , 0.0μM, the concentration of each drug is combined with the concentration of another drug, the specific drug concentration combinations are shown in Table 3;

3) Add the new coronavirus SARS-CoV-2 delta variant strain, MOI=0.01 per well, mix gently, and incubate in a 37°C cell culture incubator for 24 hours;

4) Collect the cell supernatant, extract the viral RNA in the supernatant, and use qRT-PCR to detect the viral copy number.

test results:

The results are shown in Figure 3. Quantitative assessment of the virus inhibition rate by measuring viral copy number using real-time quantitative polymerase chain reaction (qRT-PCR) can reflect the inhibitory effect of VV116 and NHC on SARS-CoV-2. It can be seen from the figure that VV116 and NHC have an obvious inhibitory effect on SARS-CoV-2 in a dose-dependent manner. The combination of drugs has a certain additive effect and has no synergistic effect.

Example 4:

Experimental method: 5-6 day old Balb/c suckling mice were divided into six groups, seven in each group, and infected with HCoV-OC43 virus through intranasal instillation. (1×104TCID50/mouse), and administered intragastrically 1 hour later (day 0). The dosage regimen was that each group of Balb/c suckling mice were administered EIDD2801 250mpk, VV116 50mpk, nirmatrelvir 50mpk+ritonavir 50mpk, and VV116 50mpk+ respectively. nirmatrelvir 50mpk+ritonavir 50mpk, vehicle control group (Vehicle group, the solvent is 40% PEG400+10% HS 15+50% ultrapure water) and blank control group (Mock group). After that, except for the blank control group, animals in each group were treated every day Administration was given once by gavage, and the behavioral changes and weight changes of the suckling mice were observed. On the 5th day, anatomy was performed, and the brain and lungs of the mice were taken for qPCR to detect the number of virus copies in the tissues (viral copies; mpk=mg/kg). .

test results:

The results are shown in Figure 4. In the HCoV-OC43-infected suckling mouse model, quantitative real-time polymerase chain reaction (qRT-PCR) measurement of viral copy number in the brain and lungs of suckling mice to quantitatively evaluate the virus inhibition rate can reflect VV116, nirmatrelvir and Inhibitory effect of ritonavir on HCoV-OC43. It can be seen from the figure that compared with the vehicle control group, VV116 alone can reduce the viral load in the brain and lungs of suckling mice by 4log10 and 3log10 respectively; using nirmatrelvir and ritonavir alone can reduce the load by 6log10 and 3log10 respectively; The combination of VV116, nirmatrelvir and ritonavir can reduce the viral load in the brain and lungs of suckling mice by 6log10 and 4log10 respectively; the above results show that the combination of VV116, nirmatrelvir and ritonavir has a significant inhibitory effect on HCoV-OC43 in suckling mice. The effect of combined medication is significantly higher than that of individual medication, with obvious synergistic effect (Note: PF-07321332 is nirmatrelvir, Rito is ritonavir).

Example 5:

This example is an in vivo activity test against SARS-CoV-2 delta mutant strains in mice.

Experimental method: Male K18-hACE2 mice aged 7-8 weeks were randomly divided into 5 groups, with 9 mice in each group. After anesthetizing the mice with isoflurane, they were intranasally infected with the SARS-CoV-2 delta variant strain (50 μl 1 × 10 ³ PFU/ml), administered intragastrically 2 hours later (day 0), each group of K18-hACE2 mice were administered VV116 100mpk, VV116 50mpk, nirmatrelvir 100mpk+ritonavir 50mpk, VV116 50mpk+nirmatrelvir 100mpk+ritonavir 50mpk, In the vehicle control group (5% DMSO+5% Solutol HS-15+5% PEG400+85% Saline), animals in each group were administered intragastrically twice a day. On day 2, some mice were dissected (5 per group), and on day 4, the remaining mice in each group were treated the same. Viral RNA in lung tissue was extracted using RNeasy Mini Kit (Qiagen kit), reverse transcribed according to the instructions of the reverse transcription kit (PrimeScript Reverse Transcriptase, Takara), and then measured by real-time quantitative polymerase chain reaction (qRT-PCR). Viral copy number in mouse lungs, calculated by standard plasmid concentration. The tissue grinding fluid was serially diluted and incubated with Vero E6 cells, and then cultured in carboxymethyl cellulose medium for 4-5 hours. The number of plaques was obtained by crystal violet cell staining, and the virus titer in the mouse lung tissue was calculated ( PFU stands for plaque forming unit).

Experimental results:

The results are shown in Figure 5. In the mouse model of SARS-CoV-2 delta variant infection, the viral copy number in the lungs of mice was determined by quantitative real-time polymerase chain reaction (qRT-PCR) and the virus droplets in the lung tissue of mice were determined by plaque assay. Degree to quantitatively evaluate the virus inhibition rate can reflect VV116, nirmatrelvir+ritonavir, The inhibitory effect of VV116+nirmatrelvir+ritonavir on SARS-CoV-2 delta variant strains. As can be seen from the figure, on days 2 and 4, compared with the vehicle control group, VV116 (100mpk or 50mpk) alone or nirmatrelvir+ritonavir (100mpk+50mpk, NMT group) increased the viral load in the lungs of mice. The amount decreased by 1.0-2.0log10; the combination of VV116, nirmatrelvir and ritonavir (50mpk+100mpk+50mpk, Combo group) reduced the viral load in the lungs of mice to a higher degree than that of the single medication group, on the 2nd and 4th days , the decrease range reaches 3.0-4.0log10. It can be seen from the decrease in virus titers in the lungs of mice that on day 4, VV116 (50mpk) alone has a stronger antiviral effect than nirmatrelvir+ritonavir (100mpk+50mpk) alone; high-dose VV116 (100mpk) and drugs The combination (VV116 50mpk+nirmatrelvir 100mpk+ritonavir 50mpk) can reduce the virus titer below the detection limit on both the 2nd and 4th day. The above results show that the combination of VV116, nirmatrelvir and ritonavir has a significant inhibitory effect on the SARS-CoV-2 delta variant in mice, and the effect of the combination is significantly higher than that of the drug alone.

Claims (10)

1. A pharmaceutical composition containing:

   a) A therapeutically effective amount of a compound represented by the following formula (I) or a pharmaceutically acceptable salt thereof; and

   b) A therapeutically effective amount of 3CL protease inhibitor;
   

   In formula (I),

   R ₁ and R ₂ are each independently selected from hydrogen, C _1-20 alkanoyl group, C _3-10 cycloalkylformyl group, and amino C _1-20 alkanoyl group;

   Or R ₁ and R ₂ are connected to each other to form

   R ₃ is selected from hydrogen, C _1-20 alkanoyl group, C _3-10 cycloalkylformyl group, and amino C _1-20 alkanoyl group;

   R ₄ is selected from hydrogen, deuterium, and halogen;

   X is selected from -CH ₂ -, -CD ₂ -.
2. The pharmaceutical composition according to claim 1, characterized in that, in formula (I),

   R ₁ and R ₂ are each independently selected from hydrogen, C _1-10 alkanoyl group, C _3-7 cycloalkylformyl group, and amino C _1-10 alkanoyl group;

   Or R ₁ and R ₂ are connected to each other to form

   R ₃ is selected from hydrogen, C _1-10 alkanoyl group, C _3-7 cycloalkylformyl group, and amino C _1-10 alkanoyl group;

   R ₄ is selected from hydrogen, deuterium, and halogen;

   X is selected from -CH ₂ -, -CD ₂ -.
3. The pharmaceutical composition according to claim 1 or 2, characterized in that, in formula (I),

   R ₁ and R ₂ are each independently selected from hydrogen, formyl, acetyl, propionyl, butyryl, isobutyryl, valeryl, pivaloyl, hexanoyl, 2-ethylbutyryl and 3,3-di Methylbutyryl, cyclopropylformyl, cyclobutanoyl, cyclopentanoyl, cyclohexaneformyl, cycloheptylformyl, α-aminoisovaleryl, or R1 and R2 connected to each other to form

   Preferably, R ₁ and R ₂ are each independently selected from hydrogen, isobutyryl, cyclopropanoyl, or R ₁ and R ₂ are connected to each other to form

   R ₃ is selected from hydrogen, formyl, acetyl, propionyl, butyryl, isobutyryl, valeryl, pivaloyl, hexanoyl, 2-ethylbutyryl, 2-methylvaleryl, 2-propyl Valeryl, 3,3-dimethylbutanoyl, dodecanoyl, myristanoyl, hexadecanoyl, octadecanoyl, eicosanoyl, cyclopropanoyl, cyclobutanoyl, cyclopentyl Formyl, cyclohexaneformyl, cycloheptylformyl, aminoacetyl, α-aminopropionyl, α-aminoisovaleryl;

   Preferably, R ₃ is selected from hydrogen, propionyl, isobutyryl, pivaloyl, 2-ethylbutyryl, 2-methylvaleryl, 2-propylvaleryl, tetradecanoyl, hexadecanoyl , octadecanoyl, cyclopropylformyl, cyclopentanoyl, cyclohexane Formyl, α-aminoisovaleryl;

   R ₄ is selected from hydrogen, deuterium, fluorine, chlorine and iodine, preferably hydrogen, deuterium and fluorine;

   X is selected from -CH ₂ -, -CD ₂ -.
4. The pharmaceutical composition according to any one of claims 1 to 3, characterized in that the compound represented by formula (I) or a pharmaceutically acceptable salt thereof is any compound having the following structure:
   
   
5. The pharmaceutical composition according to any one of claims 1 to 4, characterized in that the compound represented by formula (I) is selected from compounds 11, 14, 16, 20, 27, 30, 32, 37, 41, 68 and 69; preferably, the compound of formula (I) is compound 69.
6. The pharmaceutical composition according to any one of claims 1 to 5, wherein the compound of formula (I) is compound 69, and the 3CL protease inhibitor is selected from one or more of the following: nirmatrelvir, S -217622, EDP-235, GC-376, PBI-0451, FB2001, VV993 and SIM0417; preferably, the 3CL protease inhibitor is nirmatrelvir.
7. The pharmaceutical composition according to any one of claims 1 to 6, characterized in that the pharmaceutical composition further contains:

   c) Synergists; and

   d) Optional pharmaceutically acceptable carrier or excipient;

   Preferably, the synergist is ritonavir.
8. The pharmaceutical composition according to any one of claims 1 to 7, characterized in that the mass ratio of the compound represented by formula (I) or its pharmaceutically acceptable salt to the 3CL protease inhibitor is 0.01-99.99 ∶1, preferably 0.1-20:1, further preferably 0.5-4:1, even more preferably 0.5:1, 1:1, 1.5:1, 2:1, 2:3, 4:3;

   In the case where the pharmaceutical composition contains a synergist, in the pharmaceutical composition, the compound represented by formula (I) or a pharmaceutically acceptable salt thereof, a 3CL protease inhibitor, and a synergist are The mass ratio is 1:0.01-99.99:0.01-99.99, preferably 1:0.1-20:0.1-20, and further preferably 1:0.2-5:0.2-5.
9. The use of the pharmaceutical composition according to any one of claims 1 to 8 in the preparation of medicines, wherein the medicine is (a) a coronavirus replication inhibitor; or (b) for treatment and/or prevention, relief Medicines for illnesses caused by coronavirus infections.
10. The use according to claim 9, characterized in that,

    The coronavirus is one or more selected from the following:

    (1) Coronaviruses that infect humans: such as severe acute respiratory syndrome coronavirus SARS-CoV (Severe acute respiratory syndrome coronavirus, SARS-CoV), 2019 new coronavirus (2019-nCoV or SARS-CoV-2), Middle East respiratory syndrome coronavirus Syndrome coronavirus MERS-CoV (Middle East respiratory syndrome coronavirus, MERS-CoV), human coronavirus OC43 (Human coronavirus OC43), human coronavirus 229E (Human coronavirus 229E), human coronavirus NL63 (Human coronavirus NL63), human Coronavirus HKU1(Human coronavirus HKU1);

    (2) Coronaviruses that infect animals: such as porcine epidemic diarrhea virus (PEDV) and feline infectious peritonitis virus (FIFV);

    The disease caused by coronavirus infection is one or more selected from the following:

    (D1) Common colds, high-risk symptomatic infections, respiratory infections, pneumonia and their complications caused by human coronavirus infection;

    (D2) Porcine epidemic diarrhea caused by porcine epidemic diarrhea virus;

    (D3) Feline infectious peritonitis caused by feline coronavirus;

    Preferably, the disease caused by coronavirus infection is a disease caused by SARS-CoV-2 infection, especially one or more selected from the following: respiratory tract infection caused by SARS-CoV-2 infection, pneumonia and its complications;

    Preferably, the complications include cardiac arrhythmia, myocarditis, respiratory failure, liver function impairment and renal function impairment.