Novel use of imidazo[1,2-a]pyrazine compound

Abstract

Disclosed is a use of an imidazo[1,2-a]pyrazine compound having a structure represented by formula (I) or a pharmaceutically acceptable salt or a stereoisomer thereof in preparation of a coronavirus PLpro inhibitor. In the present invention, it is found that a derivative compound using imidazo[1,2-a]pyrazine as a parent nucleus can effectively inhibit the activity of SARS-CoV-2 PLpro, and the structure of the compound is distinctly different from that of any known coronavirus PLpro inhibitor; the compound is a novel small molecule inhibitor capable of inhibiting the activity of coronavirus PLpro, and has the advantages of good druggability and low cytotoxicity; the preferred compound has no evident cytotoxicity to BEAS-2B cells within the tested concentration range (up to 200 μM), and is expected to become a broad-spectrum anti-coronavirus drug.

Description

New applications of imidazo[1,2-a]pyrazine compounds Technical Field

[0001] The present invention belongs to the field of medicine, and in particular relates to a new application of an imidazo[1,2-a]pyrazine compound in medicine. Background Art

[0002] Coronaviruses are a large class of viruses that are widely present in nature. Currently, 7 types of coronaviruses are known to infect humans and are classified as human coronaviruses. Human coronaviruses include HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV (which causes severe acute respiratory syndrome SARS) and MERS-CoV (which causes Middle East respiratory syndrome MERS), as well as the newly discovered 2019 novel coronavirus (2019-nCoV or SARS-CoV-2, referred to as the new coronavirus). The new coronavirus is extremely contagious and pathogenic, leading to the global pandemic of coronavirus disease 2019 (COVID-19, previously known as COVID-19 and later as COVID-19 infection). In less than 20 years, coronaviruses have caused multiple global public health crises such as SARS, MERS, and COVID-19, posing a serious threat to human health. In order to respond to the current COVID-19 pandemic and other coronavirus infections, it is necessary to develop broad-spectrum anti-coronavirus drugs.

[0003] After different coronaviruses infect the host, they all go through the same life cycle: the viral genes first translate and express two polyproteins, pp1a and pp1ab. These two polyproteins need to be cut into independent non-structural proteins (Nsp1 to Nsp16) by two coronavirus proteases: papain-like protease (PLpro) and 3C-like protease (3CLpro) before the virus can replicate. PLpro is responsible for cutting and releasing Nsp1 to Nsp3. PLpro can also catalyze the removal of ubiquitin and interferon-stimulated gene 15 (ISG15) attached to host proteins, evading the host's antiviral innate immune response. The PLpro of the new coronavirus has a high homology and the same function as the PLpro of various human coronaviruses, but has a low similarity with the human protease sequence. Given the important role of PLpro in viral replication and its high conservation among coronaviruses, it is an ideal target for the development of anti-coronavirus drugs. Small molecule inhibitors developed for the PLpro of the new coronavirus can also inhibit the PLpro of other coronaviruses.

[0004] Most of the reported small-molecule inhibitors of PLpro against the novel coronavirus are derived from previously reported PLpro inhibitors against SARS-CoV or MERS-CoV, or are derived through modification of these structures. Examples include GRL0617 and its derivatives (Rac5c, Rac3j, Rac3k), reserpine, levothyroxine, proanthocyanidins, and disulfiram. Small-molecule inhibitors with novel structures directly screened against PLpro against the novel coronavirus will play an important role in the treatment of coronavirus infections, especially those caused by the novel coronavirus.

[0005] Summary of the Invention

[0006] Based on this, the present invention discovered a class of imidazo[1,2-a]pyrazine compounds, which have good inhibitory activity against coronavirus PLpro and can be used to prepare anti-coronavirus drugs.

[0007] The present invention includes the following technical solutions.

[0008] Use of an imidazo[1,2-a]pyrazine compound having a structure represented by formula (I) or a pharmaceutically acceptable salt or a stereoisomer thereof in the preparation of a coronavirus PLpro inhibitor,

[0009] Wherein, n is selected from: 1, 2, 3, 4, 5;

[0010] Each R is independently selected from the group consisting of hydrogen, C1-C8 alkyl, C1-C8 alkoxy, C1-C8 alkylthio, 3-8 membered heterocyclyl, halogen, hydroxy, nitro, -N(R1)2, -C(=O)OR2, -C(=O)N(R1)2, -N(R1)-C(=O)R2;

[0011] Each R1 is independently selected from: hydrogen, C1-C8 alkyl;

[0012] Each R2 is independently selected from: C1-C8 alkyl;

[0013] A is selected from: one or more R' substituted or unsubstituted C6-C 10 Aryl, one or more R'-substituted or unsubstituted C5-C8 cycloalkyl groups;

[0014] Each R' is independently selected from the group consisting of hydrogen, halogen, -C(=O)N(R3)2, -C(=O)R4, C1-C8 alkyl, C1-C8 alkoxy, C1-C8 alkylthio, halogen-substituted C1-C8 alkyl, 3-8 membered heterocyclic group, hydroxyl, and nitro; or two adjacent R's together with the carbon atom to which they are attached form a 5-8 membered heterocyclic ring;

[0015] Each R3 is independently selected from the group consisting of hydrogen, C1-C8 alkyl, C1-C8 alkyl substituted with a 3-8 membered heterocyclyl, C1-C8 alkyl substituted with a 5-8 membered heteroaryl, C3-C8 cycloalkyl, and C1-C8 alkyl substituted with -N(R1)2;

[0016] R4 is selected from the group consisting of: hydroxy, C1-C8 alkyl, C1-C8 alkoxy, and 3-8 membered heterocyclic group.

[0017] In some embodiments, each R is independently selected from the group consisting of hydrogen, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 alkylthio, 5-6 membered heterocyclyl, halogen, hydroxy, nitro, -N(R1)2, -C(=O)OR2, -C(=O)N(R1)2, -N(R1)-C(=O)R2;

[0018] Each R1 is independently selected from: hydrogen, C1-C3 alkyl;

[0019] Each R2 is independently selected from: C1-C3 alkyl.

[0020] In some embodiments, each R is independently selected from: hydrogen, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 alkylthio, morpholinyl, chlorine, hydroxyl, -N(R1)2, -C(=O)OR2;

[0021] Each R1 is a C1-C3 alkyl group;

[0022] R2 is selected from: C1-C3 alkyl.

[0023] In some embodiments, each R is independently selected from the group consisting of hydrogen, methyl, methoxy, methylthio, morpholino, chlorine, bromine, fluorine, dimethylamino, hydroxyl, methoxycarbonyl, methylcarbamoyl, ethylcarbamoyl, acetamido, nitro, and amino.

[0024] In some embodiments, A is selected from: one or more R'-substituted or unsubstituted phenyl, cyclopentyl, cyclohexyl.

[0025] In some embodiments, each R' is independently selected from: hydrogen, halogen, -C(=O)N(R3)2, -C(=O)R4, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 alkylthio, halogen-substituted C1-C3 alkyl, 5-6 membered heterocyclic group, hydroxyl, nitro; or two adjacent R's together with the carbon atom to which they are attached form a 5-6 membered heterocyclic ring;

[0026] Each R3 is independently selected from the group consisting of hydrogen, C1-C3 alkyl, C1-C3 alkyl substituted with a 5-6 membered heterocyclyl, C1-C3 alkyl substituted with a 5-6 membered heteroaryl, C5-C6 cycloalkyl, and C1-C3 alkyl substituted with -N(R1)2;

[0027] Each R1 is independently selected from: hydrogen, C1-C3 alkyl;

[0028] R4 is selected from the group consisting of: hydroxyl, C1-C3 alkyl, C1-C3 alkoxy, and 5-6 membered heterocyclic group.

[0029] In some embodiments, each R' is independently selected from: hydrogen, chlorine, -C(=O)NHR3, -C(=O)R4, methyl, C1-C3 alkoxy; or two adjacent R's and the carbon atom to which they are attached together form a 5-membered oxygen-containing heterocyclic ring;

[0030] R3 is selected from the group consisting of: hydrogen, C1-C3 alkyl, morpholino-substituted C1-C3 alkyl, furyl-substituted C1-C3 alkyl, tetrahydrofuranyl-substituted C1-C3 alkyl, C5-C6 cycloalkyl, dimethylamino-substituted C1-C3 alkyl;

[0031] R4 is selected from the group consisting of: hydroxy, methoxy, and 5-6 membered heterocyclic group.

[0032] In some embodiments, each R' is independently selected from: hydrogen, -C(=O)NHR3, -C(=O)R4, methyloxy, methyl, isopropyl, chlorine, ethyl, trifluoromethyl; or two adjacent R's and the carbon atom to which they are attached together form a 5-membered oxygen-containing heterocyclic ring;

[0033] R3 is selected from the group consisting of hydrogen, morpholinyl-substituted ethyl, ethyl, furanyl-substituted methyl, methyl, cyclohexyl, isopropyl, tetrahydrofuranyl-substituted methyl, and dimethylamino-substituted ethyl;

[0034] R4 is selected from the group consisting of hydroxy, methoxy, ethoxy, morpholinyl, and piperidinyl.

[0035] In some embodiments, the imidazo[1,2-a]pyrazine compound has a structure shown in the following formula (II):

[0036] Wherein, R5 and R6 are independently selected from the aforementioned R; R7 and R8 are independently selected from the aforementioned R'.

[0037] In some embodiments, R5 is selected from the group consisting of: hydrogen, C1-C3 alkoxy, chlorine;

[0038] R6 is selected from: hydrogen, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 alkylthio, morpholinyl, chlorine, hydroxyl, -N(R1)2, -C(=O)OR2; each R1 is C1-C3 alkyl; R2 is selected from: C1-C3 alkyl.

[0039] In some embodiments, R5 is selected from the group consisting of: hydrogen, methoxy, chlorine;

[0040] R6 is selected from the group consisting of hydrogen, methyl, methoxy, methylthio, morpholino, chlorine, dimethylamino, hydroxyl, and methoxycarbonyl.

[0041] In some embodiments, R7 is selected from the group consisting of hydrogen, -C(=O)NHR3, -C(=O)R4, C1-C3 alkoxy; R3 in R7 is selected from the group consisting of hydrogen, C1-C3 alkyl, morpholinyl-substituted C1-C3 alkyl, furyl-substituted C1-C3 alkyl, tetrahydrofuranyl-substituted C1-C3 alkyl, C5-C6 cycloalkyl; R4 in R7 is selected from the group consisting of hydroxy, methoxy, and 5-6 membered heterocyclyl;

[0042] R8 is selected from the group consisting of hydrogen, chlorine, -C(=O)NHR3, -C(=O)R4, and C1-C3 alkoxy; R3 in R8 is selected from the group consisting of hydrogen, C1-C3 alkyl, C1-C3 alkyl substituted with morpholinyl, C1-C3 alkyl substituted with furanyl, C5-C6 cycloalkyl, and C1-C3 alkyl substituted with dimethylamino; R4 in R8 is selected from the group consisting of hydroxyl and 5-6 membered heterocyclyl;

[0043] Alternatively, R7 and R8 together with the carbon atom to which they are connected form a 5-membered oxygen-containing heterocyclic ring.

[0044] In some embodiments, R7 is selected from the group consisting of hydrogen, -C(=O)NHR3, -C(=O)R4, methyloxy; R3 in R7 is selected from the group consisting of hydrogen, ethyl substituted by morpholinyl, ethyl, methyl substituted by furanyl, methyl, cyclohexyl, isopropyl, methyl substituted by tetrahydrofuranyl; R4 in R7 is selected from the group consisting of hydroxy, methoxy, morpholinyl, piperidinyl;

[0045] R8 is selected from the group consisting of hydrogen, chlorine, -C(=O)NHR3, -C(=O)R4, and methyloxy; R3 in R8 is selected from the group consisting of hydrogen, morpholinyl-substituted ethyl, ethyl, furanyl-substituted methyl, methyl, cyclohexyl, isopropyl, and dimethylamino-substituted ethyl; R4 in R8 is selected from the group consisting of hydroxy, morpholinyl, and piperidinyl;

[0046] Alternatively, R7 and R8 together with the carbon atom to which they are connected form a 5-membered oxygen-containing heterocyclic ring.

[0047] In some embodiments, R5 and R6 are independently selected from methoxy and hydroxy.

[0048] In some embodiments, R7 and R8 are both methoxy.

[0049] In some embodiments, at least one of R5 and R6 is hydrogen; one of R7 and R8 is hydrogen and the other is not hydrogen.

[0050] The present invention also provides the use of the imidazo[1,2-a]pyrazine compound or a pharmaceutically acceptable salt or a stereoisomer thereof in the preparation of an anti-coronavirus drug.

[0051] The present invention also provides the use of the imidazo[1,2-a]pyrazine compound or a pharmaceutically acceptable salt or a stereoisomer thereof in the preparation of a drug for preventing and / or treating diseases caused by coronavirus.

[0052] In some embodiments, the coronavirus is SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU, preferably SARS-COV-2 (new coronavirus).

[0053] The present invention also provides an anti-coronavirus drug, which is prepared from an active ingredient and a pharmaceutically acceptable excipient. The active ingredient includes the imidazo[1,2-a]pyrazine compound or a pharmaceutically acceptable salt or a stereoisomer thereof.

[0054] The present invention also provides a method for preventing and / or treating diseases caused by coronavirus, comprising the following technical solutions.

[0055] A method for preventing and / or treating diseases caused by coronavirus, comprising: administering a safe and effective amount of the imidazo[1,2-a]pyrazine compound or a pharmaceutically acceptable salt or stereoisomer thereof; or administering a safe and effective amount of the anti-coronavirus drug.

[0056] In some embodiments, the coronavirus is SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU, preferably SARS-COV-2 (new coronavirus).

[0057] The present invention discovered that a class of derivative compounds with imidazo[1,2-a]pyrazine as the parent nucleus can effectively inhibit the activity of the new coronavirus PLpro. The structure of this class of compounds is significantly different from any known coronavirus PLpro inhibitors. It is a new class of small molecule inhibitors that can inhibit the activity of coronavirus PLpro and has the advantages of good drugability and low cytotoxicity. The preferred compounds have no obvious cytotoxicity to BEAS-2B cells within the tested concentration range (up to 200μM). Therefore, the imidazo[1,2-a]pyrazine compounds discovered by the present invention have the effect of resisting coronavirus infection, especially the new coronavirus infection, and are expected to become broad-spectrum anti-coronavirus drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 shows the drugability analysis results of compound P19453.

[0059] FIG2 shows the drugability analysis results of compound P22445.

[0060] FIG3 shows the drugability analysis results of compound P22475.

[0061] FIG4 shows the cytotoxicity results of compounds P19453, P22445, and P22475. DETAILED DESCRIPTION

[0062] The technical solutions of the present invention are further illustrated by specific examples below. Those skilled in the art should understand that the examples are only for helping to understand the present invention and should not be regarded as specific limitations of the present invention.

[0063] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0064] The terms "comprise," "comprising," and "having," and any variations thereof, are intended to cover non-exclusive inclusions. For example, a process, method, apparatus, product, or device comprising a series of steps is not limited to the listed steps or modules but may optionally include steps not listed, or other steps inherent to the process, method, product, or device.

[0065] In this application, "plurality" refers to two or more. "And / or" describes the relationship between related objects, indicating that three possible relationships exist. For example, "A and / or B" can mean: A exists alone, A and B exist simultaneously, or B exists alone. The character " / " generally indicates that the related objects are in an "or" relationship.

[0066] In the compounds of the present invention, when any variable (such as R 1If a substituent (e.g., ) occurs more than once in any component, its definition at each occurrence is independent of its definition at every other occurrence. Likewise, combinations of substituents and variables are permissible so long as such combinations result in a stable compound. A line drawn from a substituent into the ring system indicates that the indicated bond may be attached to any substitutable ring atom. If the ring system is polycyclic, this means that such bonds may be attached only to any suitable carbon atom in an adjacent ring. It will be understood that one of ordinary skill in the art can select substituents and substitution patterns in the compounds of the present invention to provide compounds that are chemically stable and readily synthesized from readily available starting materials using techniques in the art and the methods set forth below. If a substituent is itself substituted with more than one group, it will be understood that these groups may be on the same carbon atom or on different carbon atoms so long as the structure is stable.

[0067] As used herein, the term "alkyl" is intended to include both branched and straight-chain saturated aliphatic hydrocarbon groups having a specified number of carbon atoms. For example, the definition of "C1-C6 alkyl" includes groups having 1, 2, 3, 4, 5, or 6 carbon atoms in a straight or branched arrangement. For example, "C1-C6 alkyl" specifically includes methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, pentyl, and hexyl.

[0068] The term "alkoxy" used in the present invention refers to a group having an -O-alkyl structure, such as -OCH3, -OCH2CH3, -OCH2CH2CH3, -O-CH2CH(CH3)2, -OCH2CH2CH2CH3, -O-CH(CH3)2, etc.

[0069] The term "alkylthio" used in the present invention refers to a group having an -S-alkyl structure, such as -SCH3, -SCH2CH3, -SCH2CH2CH3, -SCH2CH(CH3)2, -SCH2CH2CH2CH3, -SCH(CH3)2, etc.

[0070] The term "heterocyclic group" as used in the present invention is a saturated or partially unsaturated monocyclic or polycyclic cyclic substituent (including monocyclic, spirocyclic, fused cyclic, bridged ring, etc.), wherein one or more ring atoms are selected from N, O or S(O)m (wherein m is an integer from 0 to 2) as a heteroatom, and the remaining ring atoms are carbon. An oxygen-containing heterocyclic group means that at least one ring atom is O.

[0071] As used herein, the term "heteroaryl" refers to an aromatic ring containing one or more heteroatoms selected from O, N, or S. The aromatic ring may be monocyclic, bicyclic, or polycyclic, and includes, but is not limited to, quinolinyl, pyrazolyl, pyrrolyl, thienyl, furanyl, pyridyl, pyrimidinyl, pyrazinyl, triazolyl, imidazolyl, oxazolyl, isoxazolyl, and pyridazinyl. "Heteroaryl" is also understood to include any nitrogen-containing heteroaryl N-oxide derivative. Attachment of the heteroaryl group may be through a carbon atom or a heteroatom.

[0072] As will be understood by those skilled in the art, "halo" or "halo" as used herein refers to chlorine, fluorine, bromine and iodine.

[0073] The anti-coronavirus drug provided by the present invention comprises an active ingredient within a safe and effective amount (i.e., the imidazo[1,2-a]pyrazine compound or a pharmaceutically acceptable salt or stereoisomer thereof described in the present invention), and a pharmaceutically acceptable excipient. In the method for preventing and / or treating diseases caused by coronavirus provided by the present invention, the active ingredient (i.e., the imidazo[1,2-a]pyrazine compound or a pharmaceutically acceptable salt or stereoisomer thereof described in the present invention) administered to the patient is also within a safe and effective amount. When administering the drug, a safe and effective amount of the compound of the present invention is administered to a mammal (such as a human) in need of treatment, wherein the dosage at the time of administration is a pharmaceutically effective dosage. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health status, which are all within the skill range of a skilled physician.

[0074] Here, "safe and effective amount" means an amount of the active ingredient sufficient to significantly improve the condition without causing serious side effects. "Pharmaceutically acceptable excipients" refer to one or more compatible solid or liquid fillers or gels suitable for human use, of sufficient purity, and with sufficiently low toxicity. "Compatibility" here refers to the ability of the components of the composition to blend with the active ingredient of the present invention, and with each other, without significantly reducing the efficacy of the active ingredient.

[0075] Examples of pharmaceutically acceptable excipients include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers (such as ), wetting agents (such as sodium lauryl sulfate), colorants, flavorings, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0076] There is no particular limitation on the administration of the active ingredient or pharmaceutical composition of the present invention. Representative administration methods include (but are not limited to): oral, intratumoral, rectal, parenteral (intravenous, intramuscular or subcutaneous) and the like.

[0077] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules.

[0078] In these solid dosage forms, the active ingredient is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with:

[0079] (a) fillers or extenders, for example, starch, lactose, sucrose, glucose, mannitol and silicic acid;

[0080] (b) binders, for example, hydroxymethylcellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and gum arabic;

[0081] (c) humectants, for example, glycerin;

[0082] (d) disintegrants, for example, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate;

[0083] (e) a buffering solvent, for example, paraffin;

[0084] (f) absorption accelerators, for example, quaternary ammonium compounds;

[0085] (g) wetting agents, for example, cetyl alcohol and glyceryl monostearate;

[0086] (h) adsorbents, for example, kaolin;

[0087] (i) Lubricants, for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, or mixtures thereof. In capsules, tablets and pills, the dosage form may also contain a buffering agent.

[0088] The solid dosage forms can also be prepared using coatings and shells, such as enteric coatings and other materials known in the art. They can contain opacifying agents, and the release of the active ingredient in such compositions can be delayed in a certain portion of the digestive tract. Examples of embedding components that can be used are polymeric substances and waxes.

[0089] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, or tinctures. In addition to the active ingredient, the liquid dosage form may contain inert diluents commonly used in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropyl alcohol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butylene glycol, dimethylformamide, and oils, particularly cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil, and sesame oil, or mixtures thereof. In addition to these inert diluents, the composition may also contain adjuvants, such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents, and fragrances.

[0090] In addition to the active ingredients, suspensions may contain suspending agents such as, for example, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances.

[0091] Compositions for parenteral injection may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.

[0092] The compounds of the present invention may be administered alone or in combination with other drugs known to treat or improve similar conditions. When administered in combination, the original drug's mode of administration and dosage remain unchanged, while the compound of formula (I) of the present invention is taken simultaneously or subsequently. When the compound of formula (I) is taken simultaneously with one or more other drugs, it is preferred to use a pharmaceutical composition containing one or more known drugs and the compound of formula (I). Drug combinations also include taking the compound of formula (I) and one or more other known drugs during overlapping time periods. When the compound of formula (I) is used in combination with one or more other drugs, the dosage of the compound of formula (I) or the known drug may be lower than when they are taken alone.

[0093] The following are specific examples. All reagents, materials and raw materials in the following examples can be obtained from commercial channels.

[0094] Example 1: Testing of the inhibitory activity of imidazo[1,2-a]pyrazine compounds on the PLpro activity of the new coronavirus

[0095] An in vitro enzyme activity experiment was used to test the inhibitory effect of imidazo[1,2-a]pyrazine compounds on the activity of PLpro of the new coronavirus. PLpro can hydrolyze the substrate RLRGG-AMC to generate free AMC. The fluorescence emission value of free AMC at 380nm wavelength and 460nm wavelength was detected by a microplate reader to calculate the activity of PLpro. After the addition of small molecule inhibitors, the activity of PLpro was inhibited and the fluorescence emission value of AMC decreased. After adding different small molecule compounds of fixed concentration (50μM) to the reaction system, the changes in PLpro activity were reflected by the readings of the microplate reader, and the inhibition rate of PLpro activity was calculated. The specific steps are as follows:

[0096] 1.1 Reaction buffer composition: 10 mM DTT, 20 mM Tris, 150 mM NaCl, 2% DMSO. The substrate RLRGG-AMC was purchased from Beyotime and dissolved in DMSO to a 10 mM stock solution. SARS-CoV-2 PLpro was purchased from Sino Biological and dissolved in nuclease-free water to a 125 ng / μL stock solution.

[0097] 1.2 Prepare substrate: Add 2 μL of substrate stock solution to 198 μL of reaction buffer and mix well to obtain a 100 μM substrate working solution.

[0098] 1.3 Prepare PLpro: Add 10 μL of PLpro stock solution to 90 μL of reaction buffer and mix thoroughly to obtain 12.5 ng / μL PLpro working solution.

[0099] 1.4 For the experimental group, 10 μL of PLpro working solution and 2 μL of the imidazo[1,2-a]pyrazine compounds listed in Table 1 (all purchased from Shanghai Taoshu) (final concentration 50 μM) were mixed. For the control group, no compound was added and 10 μL of PLpro working solution and 2 μL of compound solvent were mixed. The cells were then incubated at 37°C for 10 minutes.

[0100] 1.5 Quickly add 10 μL of the substrate working solution prepared in 1.2 to the system in 1.4. Use a microplate reader to incubate at 37°C for 15 minutes, with an interval of 30 seconds between each cycle. The excitation wavelength is set to 380 nm and the emission wavelength is set to 460 nm.

[0101] 1.6 Calculation of Inhibition Rate: Use time (min) as the X-axis and fluorescence (RFU) as the Y-axis. Fit the linear region and calculate RFU / min as the enzyme activity. Calculate the compound's inhibition rate against PLpro based on the difference in enzyme activity between the experimental and control groups. The calculation formula is as follows: Inhibition Rate = (1 - Enzyme Activity in Experimental Group / Enzyme Activity in Control Group) × 100%.

[0102] The results of the in vitro enzyme activity test are shown in Table 1. The results showed that all 168 compounds based on the imidazo[1,2-a]pyrazine core had varying degrees of inhibitory effect on PLpro, of which 21 compounds had an inhibition rate of greater than 45% on SARS-CoV-2 PLpro at 50 μM and were used for subsequent IC50 determination.

[0103] Table 1 Inhibitory effect of imidazo[1,2-a]pyrazine compounds on coronavirus PLpro activity at a concentration of 50 μM

[0104] Example 2: IC50 determination of imidazo[1,2-a]pyrazine compounds for inhibiting the activity of PLpro of the new coronavirus

[0105] The same experimental principle as in Example 1 was used. Different concentrations of imidazo[1,2-a]pyrazine compounds were added to the reaction system. The changes in PLpro activity were reflected by the readings of the microplate reader, and the IC50 of the small molecule compound's inhibition of PLpro was calculated. The specific steps were as follows:

[0106] 2.1 Reaction buffer composition: 10 mM DTT, 20 mM Tris, 150 mM NaCl, 2% DMSO. The substrate RLRGG-AMC was purchased from Beyotime and dissolved in DMSO to a 10 mM stock solution. SARS-CoV-2 PLpro was purchased from Sino Biological and dissolved in nuclease-free water to a 125 ng / μL stock solution.

[0107] 2.2 Prepare substrate: Add 2 μL of substrate stock solution to 198 μL of reaction buffer and mix well to obtain a 100 μM substrate working solution.

[0108] 2.3 Preparation of PLpro: Add 10 μL of PLpro stock solution to 90 μL of reaction buffer and mix thoroughly to obtain 12.5 ng / μL of PLpro working solution.

[0109] 2.4 For the experimental groups, 10 μL of PLpro working solution and 2 μL of imidazo[1,2-a]pyrazine compounds (final concentrations: 100 μM, 50 μM, 25 μM, 12.5 μM, 6.25 μM, 3.125 μM, 1.5625 μM, 0.7812 μM, 0.3906 μM, 0.1953 μM, 0.0976 μM, 0.0488 μM, and 0.0244 μM) were mixed. For the control group, no compound was added and 10 μL of PLpro working solution and 2 μL of compound solvent were mixed. The cells were then incubated at 37°C for 10 minutes.

[0110] 2.5 Quickly add 10 μL of the substrate working solution prepared in 2.2 to the system in 2.4. Use a microplate reader to incubate at 37°C and read continuously for 15 minutes, with an interval of 30 seconds between each cycle. The excitation wavelength is set to 380 nm and the emission wavelength is set to 460 nm.

[0111] 2.6 Calculation of inhibition rate: With time (min) as the X-axis and fluorescence value (RFU) as the Y-axis, fit the linear region and obtain RFU / min as the enzyme activity. Then, calculate the inhibition rate of the test compound at different concentrations on SARS-COV2 PLpro based on the enzyme activity of the experimental and control groups. The calculation formula is as follows: Inhibition rate = (1-enzyme activity of the experimental group / enzyme activity of the control group) × 100%

[0112] 2.7 Calculation of IC50: Calculate the IC50 of each derivative using GraphPad Prism software, with X being the drug concentration and Y being the enzyme inhibition rate.

[0113] The inhibition results of 21 imidazo[1,2-a]pyrazine compounds on PLpro activity are shown in Table 2. Among them, the three compounds with the best inhibitory effects, P19453, P22445, and P22475, had IC50 values ​​of 6.45 μM, 13.94 μM, and 16.57 μM, respectively.

[0114] Table 2 IC50 results of imidazo[1,2-a]pyrazine compounds inhibiting the activity of coronavirus PLpro

[0115] Example 3: Swiss ADME evaluation of the drugability of compounds P19453, P22445, and P22475

[0116] The structures of compounds P19453, P22445, and P22475 were mapped in SwissADME (http: / / www.swissadme.ch / ), and their ADME properties were exported for druggability assessment. SwissADME's evaluation results showed that all three compounds had good druggability, as shown in Figures 1-3.

[0117] Example 4: Detection of cytotoxicity of compounds P19453, P22445, and P22475

[0118] The cytotoxicity of P19453, P22445, and P22475 was tested using a CCK-8 assay. The CCK-8 kit was purchased from Shanghai Yisheng. The specific steps are as follows:

[0119] 4.1 Plating: Human bronchial epithelial cells (BEAS-2B cells) in the logarithmic growth phase were seeded in 96-well plates (100 μl / well). Three replicates were made for each group, and the number of cells per well was set at 5×10 3 The cells were placed in a 37°C 5% CO2 incubator and cultured for 12 h.

[0120] 4.2 Cells were treated with various concentrations of P19453, P22445, and P22475. All experimental groups were treated with DMSO. The concentrations were 100 μM, 50 μM, 25 μM, 12.5 μM, 6.25 μM, and 3.125 μM, with a volume of 2 μL. The control group received 2 μL of DMSO. Cells were then incubated at 37°C in a 5% CO2 incubator.

[0121] After 24 hours, wash the cells twice with PBS and add 100 μL of premixed CCK8 solution (culture medium:CCK8 working solution = 9:1) to each well, avoiding the introduction of air bubbles. Continue incubating at 37°C in a 5% CO2 incubator for 2 hours.

[0122] 4.4 Use a microplate reader to measure the absorbance D(λ) of each well at a wavelength of 450 nm. Analyze the D(λ) values ​​of the zero adjustment group, control group, and experimental group to determine the cell viability. Cell viability reflects the cytotoxicity of the drug. The groups are as follows:

[0123] Experimental group (culture medium containing cells, CCK-8, and test drugs)

[0124] Control group (culture medium containing cells, CCK-8, but without the drug to be tested)

[0125] Zero adjustment group (culture medium without cells and test substances, containing CCK-8)

[0126] The CCK8 results are shown in FIG4 , which show that the three compounds had no obvious cytotoxicity to BEAS-2B cells within the tested concentration range (up to 200 μM).

[0127] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0128] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. Use of an imidazo[1,2-a]pyrazine compound having a structure represented by formula (I) or a pharmaceutically acceptable salt thereof or a stereoisomer thereof in the preparation of a coronavirus PLpro inhibitor, in, n is selected from: 1, 2, 3, 4, 5; Each R is independently selected from the group consisting of hydrogen, C1-C8 alkyl, C1-C8 alkoxy, C1-C8 alkylthio, 3-8 membered heterocyclyl, halogen, hydroxy, nitro, -N(R1)2, -C(=O)OR2, -C(=O)N(R1)2, -N(R1)-C(=O)R2; Each R1 is independently selected from: hydrogen, C1-C8 alkyl; Each R2 is independently selected from: C1-C8 alkyl; A is selected from: one or more R' substituted or unsubstituted C6-C 10 Aryl, one or more R'-substituted or unsubstituted C5-C8 cycloalkyl; Each R' is independently selected from: hydrogen, halogen, -C(=O)N(R3)2, -C(=O)R4, C1-C8 alkyl, C1-C8 alkoxy, C1-C8 alkylthio, halogen-substituted C1-C8 alkyl, 3-8 membered heterocyclic group, hydroxyl, nitro; or two adjacent R's and the carbon atoms to which they are connected together form a 5-8 membered heterocyclic ring; Each R3 is independently selected from the group consisting of hydrogen, C1-C8 alkyl, C1-C8 alkyl substituted with 3-8 membered heterocyclyl, C1-C8 alkyl substituted with 5-8 membered heteroaryl, C3-C8 cycloalkyl, and C1-C8 alkyl substituted with -N(R1)2; R4 is selected from the group consisting of: hydroxyl, C1-C8 alkyl, C1-C8 alkoxy, and 3-8 membered heterocyclic group.

2. The use according to claim 1, characterized in that: Each R is independently selected from the group consisting of hydrogen, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 alkylthio, 5-6 membered heterocyclyl, halogen, hydroxyl, nitro, -N(R1)2, -C(=O)OR2, -C(=O)N(R1)2, -N(R1)-C(=O)R2; Each R1 is independently selected from: hydrogen, C1-C3 alkyl; Each R2 is independently selected from: C1-C3 alkyl.

3. The use according to claim 2, characterized in that: Each R is independently selected from the group consisting of hydrogen, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 alkylthio, morpholinyl, chlorine, hydroxyl, -N(R1)2, -C(=O)OR2; Each R1 is a C1-C3 alkyl group; R2 is selected from: C1-C3 alkyl.

4. The use according to claim 2, characterized in that: Each R is independently selected from the group consisting of hydrogen, methyl, methoxy, methylthio, morpholino, chlorine, bromine, fluorine, dimethylamino, hydroxyl, methoxycarbonyl, methylcarbamoyl, ethylcarbamoyl, acetamido, nitro, and amino.

5. The use according to any one of claims 1 to 4, characterized in that: A is selected from: one or more R'-substituted or unsubstituted phenyl, cyclopentyl, cyclohexyl.

6. The use according to any one of claims 1 to 4, characterized in that: Each R' is independently selected from the group consisting of hydrogen, halogen, -C(=O)N(R3)2, -C(=O)R4, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 alkylthio, halogen-substituted C1-C3 alkyl, 5-6 membered heterocyclic group, hydroxyl, nitro; or two adjacent R's and the carbon atoms to which they are connected together form a 5-6 membered heterocyclic ring; Each R3 is independently selected from the group consisting of hydrogen, C1-C3 alkyl, C1-C3 alkyl substituted with a 5-6-membered heterocyclyl, C1-C3 alkyl substituted with a 5-6-membered heteroaryl, C5-C6 cycloalkyl, and C1-C3 alkyl substituted with -N(R1)2; Each R1 is independently selected from: hydrogen, C1-C3 alkyl; R4 is selected from the group consisting of: hydroxyl, C1-C3 alkyl, C1-C3 alkoxy, and 5-6 membered heterocyclic group.

7. The use according to claim 6, characterized in that: Each R' is independently selected from: hydrogen, chlorine, -C(=O)NHR3, -C(=O)R4, methyl, C1-C3 alkoxy; or two adjacent R's and the carbon atom to which they are connected together form a 5-membered oxygen-containing heterocyclic ring; R3 is selected from the group consisting of: hydrogen, C1-C3 alkyl, morpholinyl-substituted C1-C3 alkyl, furyl-substituted C1-C3 alkyl, tetrahydrofuranyl-substituted C1-C3 alkyl, C5-C6 cycloalkyl, dimethylamino-substituted C1-C3 alkyl; R4 is selected from: hydroxy, methoxy, and 5-6 membered heterocyclic group.

8. The use according to claim 6, characterized in that: Each R' is independently selected from: hydrogen, -C(=O)NHR3, -C(=O)R4, methyloxy, methyl, isopropyl, chlorine, ethyl, trifluoromethyl; or two adjacent R's and the carbon atom to which they are connected together form a 5-membered oxygen-containing heterocyclic ring; R3 is selected from the group consisting of hydrogen, morpholinyl substituted ethyl, ethyl, furanyl substituted methyl, methyl, cyclohexyl, isopropyl, tetrahydrofuranyl substituted methyl, dimethylamino substituted ethyl; R4 is selected from the group consisting of hydroxy, methoxy, ethoxy, morpholinyl, and piperidinyl.

9. The use according to claim 1, characterized in that: The imidazo[1,2-a]pyrazine compound has a structure shown in the following formula (II): Wherein, R5 and R6 are independently selected from R described in any one of claims 1 to 8; R7 and R8 are independently selected from R' described in any one of claims 1 to 8.

10. The use according to claim 9, characterized in that: R5 is selected from: hydrogen, C1-C3 alkoxy, chlorine; R6 is selected from: hydrogen, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 alkylthio, morpholinyl, chlorine, hydroxyl, -N(R1)2, -C(=O)OR2; each R1 is C1-C3 alkyl; R2 is selected from: C1-C3 alkyl.

11. The use according to claim 10, characterized in that: R5 is selected from: hydrogen, methoxy, chlorine; R6 is selected from the group consisting of hydrogen, methyl, methoxy, methylthio, morpholinyl, chlorine, dimethylamino, hydroxyl, and methoxycarbonyl.

12. The use according to claim 9, characterized in that: R7 is selected from the group consisting of hydrogen, -C(=O)NHR3, -C(=O)R4, C1-C3 alkoxy; R3 in R7 is selected from the group consisting of hydrogen, C1-C3 alkyl, C1-C3 alkyl substituted with morpholinyl, C1-C3 alkyl substituted with furanyl, C1-C3 alkyl substituted with tetrahydrofuranyl, C5-C6 cycloalkyl; R4 in R7 is selected from the group consisting of hydroxyl, methoxy, 5-6 membered heterocyclic group; R8 is selected from the group consisting of hydrogen, chlorine, -C(=O)NHR3, -C(=O)R4, C1-C3 alkoxy; R3 in R8 is selected from the group consisting of hydrogen, C1-C3 alkyl, C1-C3 alkyl substituted with morpholinyl, C1-C3 alkyl substituted with furanyl, C5-C6 cycloalkyl, C1-C3 alkyl substituted with dimethylamino; R4 in R8 is selected from the group consisting of hydroxyl, 5-6 membered heterocyclic group; Alternatively, R7 and R8 together with the carbon atom to which they are connected form a 5-membered oxygen-containing heterocyclic ring.

13. The use according to claim 12, characterized in that: R7 is selected from the group consisting of hydrogen, -C(=O)NHR3, -C(=O)R4, methyloxy; R3 in R7 is selected from the group consisting of hydrogen, ethyl substituted by morpholinyl, ethyl, methyl substituted by furanyl, methyl, cyclohexyl, isopropyl, methyl substituted by tetrahydrofuranyl; R4 in R7 is selected from the group consisting of hydroxy, methoxy, morpholinyl, piperidinyl; R8 is selected from the group consisting of hydrogen, chlorine, -C(=O)NHR3, -C(=O)R4, methyloxy; R3 in R8 is selected from the group consisting of hydrogen, ethyl substituted by morpholinyl, ethyl, methyl substituted by furanyl, methyl, cyclohexyl, isopropyl, ethyl substituted by dimethylamino; R4 in R8 is selected from the group consisting of hydroxyl, morpholinyl, piperidinyl; Or R7 and R8 together with the carbon atom to which they are connected form a 5-membered oxygen-containing heterocyclic ring.

14. The use according to any one of claims 9 to 13, characterized in that: R5 and R6 are independently selected from: methoxy and hydroxy.

15. The use according to any one of claims 9 to 13, characterized in that: R7 and R8 are both methoxy.

16. The use according to any one of claims 9 to 13, characterized in that: At least one of R5 and R6 is hydrogen; one of R7 and R8 is hydrogen and the other is not hydrogen.

17. The use according to claim 1, characterized in that: The imidazo[1,2-a]pyrazine compound is selected from the following compounds:

18. Use of the imidazo[1,2-a]pyrazine compound or a pharmaceutically acceptable salt or a stereoisomer thereof as claimed in any one of claims 1 to 17 in the preparation of an anti-coronavirus drug.

19. Use of the imidazo[1,2-a]pyrazine compound or a pharmaceutically acceptable salt or a stereoisomer thereof as claimed in any one of claims 1 to 17 in the preparation of a medicament for preventing and / or treating a disease caused by a coronavirus.

20. The use according to any one of claims 18 to 19, characterized in that: The coronaviruses are SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-229E, HCoV-OC43, HCoV-NL63, and HCoV-HKU.

21. The use according to claim 20, characterized in that The coronavirus is SARS-CoV-2.

22. An anti-coronavirus drug, characterized in that: The invention is prepared from active ingredients and pharmaceutically acceptable excipients, wherein the active ingredients include the imidazo[1,2-a]pyrazine compound described in any one of claims 1 to 17 or a pharmaceutically acceptable salt thereof or a stereoisomer thereof.

23. A method for preventing and / or treating a disease caused by a coronavirus, characterized in that: The method comprises: administering an effective amount of the imidazo[1,2-a]pyrazine compound described in any one of claims 1 to 17 or a pharmaceutically acceptable salt thereof or a stereoisomer thereof; or administering an effective amount of the anti-coronavirus drug described in claim 22.

24. The method according to claim 23, characterized in that The coronaviruses are SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-229E, HCoV-OC43, HCoV-NL63, and HCoV-HKU.

25. The method according to claim 24, characterized in that The coronavirus is SARS-CoV-2.

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