Substituted oxadiazole-aniline compound and application thereof

Abstract

The invention belongs to the technical field of medicines, and discloses a substituted oxadiazole-aniline compound, a preparation method thereof, a pharmaceutical composition taking the compound as an active component, and application of the substituted oxadiazole-aniline compound and the pharmaceutical composition in treatment and / or prevention of infectious diseases caused by coronavirus. Specifically, the invention relates to a compound shown as a formula (I) and an isomer thereof, or a pharmaceutically acceptable salt thereof, and a pharmaceutical composition containing the compound disclosed by the invention, and R1, R2, R3, R4 and n are described in the specification. The invention aims to prepare a new compound with anti-coronavirus activity, and the new compound can be used as a potential new drug for treatment or prophylactic treatment of infectious diseases caused by viruses, especially pneumonia diseases caused by coronaviruses. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the field of medical technology. In particular, it relates to substituted oxadiazole-aniline compounds represented by general formula (I), preparation methods thereof, pharmaceutical compositions with the compounds as active ingredients, and their use in treating and / or preventing infectious diseases caused by coronavirus (SARS-CoV), especially novel coronavirus (SARS-CoV-2). Background Art

[0002] Viral infections continue to threaten human life and health. Currently, there is still a lack of effective antiviral drugs for most viruses. The non-structural proteins involved in viral replication (replicases such as proteases, polymerases and helicases) are still highly conserved. Inhibitors targeting highly conserved viral replicases are more suitable for the development of broad-spectrum antiviral drugs.

[0003] Based on the structure of coronavirus, it is of great significance to find targeted, highly effective and low-toxic drugs to treat the virus. SARS-CoV-2 encodes 16 non-structural proteins. As the protein structures are analyzed one by one, drug development technologies based on the druggable target structure of coronavirus have begun to be widely adopted. Representative of these is the successful launch of Paxlovid, an inhibitor targeting Mpro. The papain-like protease (PLpro) encoded by SARS-CoV-2 is a catalytic domain of the NSP3 protein. Together with Mpro, it is responsible for the maturation and cleavage of viral precursor proteins and is an essential protein for viral replication in cells. In addition, PLpro also mediates the deubiquitination and de-ISGization of antiviral proteins, thereby participating in the regulation of host antiviral innate immunity. The multiple functions of PLpro in the viral life cycle make it an important research and development target for anti-new coronavirus drugs. GRL0617 is a non-covalent binding inhibitor discovered after screening for SARS-CoV PLpro. It has high inhibitory activity and target enzyme affinity. The three-dimensional structure of PLpro has been resolved, and the crystal structure of its complex with GRL0617 has also been reported. These research results have laid the foundation for the research and development of PLpro inhibitors. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a substituted oxadiazole-aniline compound with novel structure and PLpro activity against coronavirus SARS-CoV / SARS-CoV-2. The present invention finds that the substituted oxadiazole-aniline compound has strong PLpro inhibitory activity and anti-coronavirus SARS-CoV-2 effect, and has low cytotoxicity, and can be used for infectious diseases caused by viruses, especially the treatment or preventive treatment of diseases caused by the new coronavirus. The present invention is completed based on the above findings. SUMMARY OF THE INVENTION

[0006] To this end, the present invention provides, in a first aspect, a compound represented by general formula (I) and an isomer thereof, or a pharmaceutically acceptable salt thereof,

[0007]

[0008] in,

[0009] R 1 is hydrogen, methyl, ethyl, propyl, isopropyl, hydroxyl;

[0010] R 2 is a substituted or unsubstituted phenyl group, a substituted or unsubstituted biphenyl group, a substituted or unsubstituted diphenyl ether group, or a substituted or unsubstituted carbazolyl group;

[0011] R 3 independently selected from hydrogen, F, Cl, Br, trifluoromethyl, trifluoromethoxy, C 1 -C 3 Alkyl, C 1 -C 3 Alkoxy, n represents 1, 2, 3 or 4 substituents;

[0012] R 4 is substituted or unsubstituted phenyl, substituted or unsubstituted thienyl;

[0013] The R 2 or R 4 The substituents in the can be selected from the following groups: F, Cl, Br, hydroxyl, amino, nitro, cyano, monofluoromethyl, difluoromethyl, trifluoromethyl, trifluoromethoxy, thienyl, vinyl, ethynyl, hydroxymethyl, carboxyl, sulfonic acid, boric acid, methylthio, C 1 -C 4 Alkyl, C 1 -C 4 Alkoxy.

[0014] In a preferred embodiment, the compound has the structural formula as shown in (II):

[0015]

[0016] in,

[0017] R 1 is hydrogen, methyl, hydroxyl;

[0018] R 2 is a substituted or unsubstituted phenyl group, a substituted or unsubstituted biphenyl group, a substituted or unsubstituted diphenyl ether group, or a substituted or unsubstituted carbazolyl group;

[0019] R 3independently selected from hydrogen, F, Cl, Br, trifluoromethyl, trifluoromethoxy, C 1 -C 3 Alkyl, C 1 -C 3 Alkoxy, n represents 1, 2, 3 or 4 substituents;

[0020] R 5 is hydrogen, cyano, carboxyl, trifluoromethyl, methoxy, morpholinyl, alkynyl, acetylamino, acetyl, hydroxyl, hydroxymethyl;

[0021] The R 2 The substituents in the group may be selected from the following groups: F, Cl, Br, hydroxyl, amino, nitro, cyano, monofluoromethyl, difluoromethyl, trifluoromethyl, trifluoromethoxy, hydroxymethyl, C 1 -C 4 Alkyl, C 1 -C 4 Alkoxy.

[0022] In another preferred embodiment, the compound has the structural formula as shown in (III):

[0023]

[0024] in,

[0025] R 1 is hydrogen, methyl, hydroxyl;

[0026] R 2 is a substituted or unsubstituted phenyl group, a substituted or unsubstituted biphenyl group, a substituted or unsubstituted diphenyl ether group, or a substituted or unsubstituted carbazolyl group;

[0027] R 3 independently selected from hydrogen, F, Cl, Br, trifluoromethyl, trifluoromethoxy, C 1 -C 3 Alkyl, C 1 -C 3 Alkoxy, n represents 1, 2, 3 or 4 substituents;

[0028] R 5 is hydrogen, carboxyl, cyano, boric acid;

[0029] The R 2 The substituents in the group may be selected from the following groups: F, Cl, Br, hydroxyl, amino, nitro, cyano, monofluoromethyl, difluoromethyl, trifluoromethyl, trifluoromethoxy, thienyl, hydroxymethyl, C 1 -C 4 Alkyl, C 1 -C 4 Alkoxy.

[0030] The pharmaceutically acceptable salts described in the present invention are salts formed by the compounds of the present invention and an acid selected from the group consisting of hydrochloric acid, p-toluenesulfonic acid, tartaric acid, maleic acid, lactic acid, methanesulfonic acid, sulfuric acid, phosphoric acid, citric acid, acetic acid or trifluoroacetic acid, preferably hydrochloric acid, p-toluenesulfonic acid or trifluoroacetic acid.

[0031] The compound according to any one of the first aspects of the present invention is the target compound of the present invention prepared in the examples (represented by structural formula or described by systematic nomenclature) and its isomers, and pharmaceutically acceptable salts thereof.

[0032] According to any one of the compounds of the first aspect of the present invention, it is a compound selected from the following:

[0033]

[0034]

[0035]

[0036] The second aspect of the present invention provides a method for preparing any one of the compounds of the first aspect of the present invention, comprising the following steps:

[0037]

[0038] Compound A is mixed with compound B in a suitable solvent (e.g., dichloromethane, tetrahydrofuran, acetonitrile, 1,4-dioxane, preferably 1,4-dioxane) under the action of reagents CDMT and NMM in the air or inert gas (Ar or N 2 ) under protection, placed at -10°C-50°C for reaction for 1-24 hours, preferably at room temperature for reaction for 2-3 hours, then heated to reflux, reacted for 1-24 hours, preferably 6-12 hours, to obtain a compound represented by formula C;

[0039] The compound represented by formula C is placed in a suitable solvent (such as methanol, ethanol, methanol / water, ethanol / water, preferably methanol / water) in the presence of ammonium chloride and metal powder (zinc powder, iron powder, preferably zinc powder) at -10°C to 60°C for 1 to 24 hours, preferably at room temperature for 2 to 4 hours, to obtain a compound represented by formula D;

[0040] The compound represented by formula D is mixed with compound R in a suitable solvent (such as dichloromethane, tetrahydrofuran, acetonitrile, 1,2-dichloroethane, preferably 1,2-dichloroethane). 4 CHO is reacted with a reducing agent (e.g. sodium borohydride, sodium cyanoborohydride, sodium triacetoxyborohydride, preferably sodium triacetoxyborohydride) under acidic conditions (e.g. formic acid, acetic acid, preferably acetic acid) at -10°C to 50°C for 1 to 24 hours, preferably at room temperature for 6 to 12 hours, to obtain a compound represented by formula (I).

[0041] The third aspect of the present invention provides use of the compound and its isomers, or pharmaceutically acceptable salts thereof, as described in any one of the first aspects of the present invention in the preparation of papain-like protease (PLpro) inhibitors.

[0042] The fourth aspect of the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of any compound and its isomers according to the first aspect of the present invention, or a pharmaceutically acceptable salt thereof, and optionally one or more pharmaceutically acceptable excipients.

[0043] The fifth aspect of the present invention provides the use of the compound and its isomers, or pharmaceutically acceptable salts thereof, described in any one of the first aspects of the present invention, or the pharmaceutical composition described in any one of the fourth aspects of the present invention in the preparation of drugs for treating and / or preventing infectious diseases caused by coronavirus.

[0044] The foregoing description only summarizes certain aspects of the present invention, but is not limited to these aspects. These aspects and other aspects will be described in more detail and complete below. DETAILED DESCRIPTION OF THE INVENTION

[0046] Various aspects and features of the present invention are further described below.

[0047] The various terms and phrases used in the present invention have the general meanings known to those skilled in the art. Even so, the present invention still hopes to provide a more detailed description and explanation of these terms and phrases. If the terms and phrases mentioned are inconsistent with the known meanings, the meanings expressed in the present invention shall prevail. The following are the definitions of various terms used in the present invention, which apply to the terms used in the entire specification of this application, unless otherwise specified in specific circumstances.

[0048] In general, the term "substituted or unsubstituted" means that one or more hydrogen atoms in a given structure are replaced by a specified substituent. Unless otherwise indicated, an optional substituent group can be substituted at each substitutable position of the group. When a given structure always has more than one position that can be substituted by one or more substituents selected from a specific group, the substituents can be substituted the same or differently at each position.

[0049] C i -C j = represents a moiety having an integer number "i" (inclusive) to an integer number "j" (inclusive) of carbon atoms. Thus, for example, C 1 -C 3 The alkyl group refers to an alkyl group having 1 to 3 (inclusive) carbon atoms.

[0050] As used herein, the term "alkyl" refers to an alkyl group having a specified number of carbon atoms, which is a straight chain or branched chain alkyl group, and which may include its subgroups, for example, referring to "C 1 -C 3 When "alkyl" is used, it may also include C 1 -C 2 Alkyl represents a sub-range of groups, as well as specific groups such as methyl, ethyl, n-propyl, isopropyl.

[0051] As used herein, the terms "alkoxy" and "alkylamino" are conventional expressions and refer to an alkyl group attached to the remainder of the molecule through an oxygen atom or an amine group, respectively, wherein the alkyl group is as described herein.

[0052] As used herein, the term "haloalkyl" means that the hydrogen on the alkyl group is replaced by one or more halogen atoms, such examples include, but are not limited to, monofluoromethyl, monofluoromethoxy and the like.

[0053] As used herein, the term "ring" refers to a substituted or unsubstituted cycloalkyl group. The so-called ring includes fused rings. The number of atoms in the ring is usually defined as the number of ring members, for example, "C 3 -C 6 "Ring" means a ring of 3 to 6 atoms.

[0054] As used herein, the terms "halogen," "halo," and the like refer to fluorine (F), chlorine (Cl), or bromine (Br).

[0055] In the present invention, "room temperature" refers to a temperature from 10°C to 40°C. In some embodiments, "room temperature" refers to a temperature from 20°C to 30°C; in other embodiments, room temperature refers to 25°C.

[0056] As used herein, the term "effective amount" refers to the amount of a drug that can achieve the desired treatment of a disease or condition described herein in a subject.

[0057] As used herein, the term "pharmaceutically acceptable" when describing, for example, a "pharmaceutically acceptable salt" means that the salt is not only physiologically acceptable to a subject, but also refers to a synthetic substance having pharmaceutical use value.

[0058] As described herein, the term "pharmaceutical composition", which may also refer to a "composition", can be used to achieve the treatment of the diseases or disorders described herein in a subject, especially a mammal.

[0059] "Treatment" for illness includes:

[0060] (1) preventing the disease, that is, preventing the clinical symptoms of the disease from occurring in a mammal that is exposed to or susceptible to the disease but does not experience or show symptoms of the disease,

[0061] (2) inhibiting the disease, i.e., arresting or reducing the progression of the disease or its clinical symptoms,

[0062] (3) Alleviate the disease, that is, cause recovery of the disease or its clinical symptoms.

[0063] "Therapeutically effective amount" refers to the amount of a compound that is sufficient to achieve treatment of a disease when administered to a mammal for the treatment of the disease. The therapeutically effective amount will vary depending on the compound, the disease to be treated and its severity, and the age, weight, sex, and other factors of the mammal. The therapeutically effective amount may also refer to any amount of the compound that is sufficient to achieve the desired beneficial effect, including preventing, inhibiting, or alleviating the disease as described in (1)-(3) above. For example, the amount of the compound may be between 0.1-250 mg / kg, or preferably, 0.5-100 mg / kg, or more preferably, 1-50 mg / kg, or even more preferably, 2-20 mg / kg. Preferably, the amount of the compound is administered to the mammal twice a day. More preferably, the amount of the compound is administered to the mammal once a day.

[0064] As described herein, the term "disease and / or condition" refers to a physical state of the subject, which is related to the disease and / or condition described in the present invention. For example, the disease and / or condition described in the present invention refers to a tuberculosis infectious disease.

[0065] As described herein, the term "subject" may refer to a patient or other animal, particularly a mammal, such as a human, monkey, etc., that receives the compound of formula I or its pharmaceutical composition for treating the disease or condition described in the present invention.

[0066] Another aspect of the present invention also relates to a pharmaceutical composition having the compound of the present invention as an active ingredient. The pharmaceutical composition can be prepared according to methods known in the art. The compound of the present invention can be combined with one or more pharmaceutically acceptable solid or liquid excipients and / or adjuvants to prepare any dosage form suitable for human or animal use.

[0067] The compounds of the present invention or pharmaceutical compositions containing the same can be administered in unit dosage form, and the administration route can be enteral or parenteral, such as oral, intravenous, intramuscular, subcutaneous, nasal, oral mucosa, eyes, lungs and respiratory tract, skin, vagina, rectum, etc.

[0068] The dosage form can be a liquid dosage form, a solid dosage form or a semisolid dosage form. Liquid dosage forms can be solutions (including true solutions and colloidal solutions), emulsions (including o / w types, w / o types and multiple emulsions), suspensions, injections (including water injections, powder injections and infusions), eye drops, nasal drops, lotions and liniments, etc.; solid dosage forms can be tablets (including ordinary tablets, enteric-coated tablets, lozenges, dispersible tablets, chewable tablets, effervescent tablets, orally disintegrating tablets), capsules (including hard capsules, soft capsules, enteric-coated capsules), granules, powders, micropills, dropping pills, suppositories, films, patches, aerosols (powders), sprays, etc.; semisolid dosage forms can be ointments, gels, pastes, etc.

[0069] The compound of the present invention can be made into common preparations, sustained-release preparations, controlled-release preparations, targeted preparations and various microparticle drug delivery systems.

[0070] In order to prepare the compound of the present invention into tablets, various excipients known in the art can be widely used, including diluents, binders, wetting agents, disintegrants, lubricants, and solubilizers. The diluent may be starch, dextrin, sucrose, glucose, lactose, mannitol, sorbitol, xylitol, microcrystalline cellulose, calcium sulfate, calcium hydrogen phosphate, calcium carbonate, etc.; the wetting agent may be water, ethanol, isopropanol, etc.; the binder may be starch slurry, dextrin, syrup, honey, glucose solution, microcrystalline cellulose, acacia slurry, gelatin slurry, sodium carboxymethyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, ethyl cellulose, acrylic resin, carbomer, polyvinyl pyrrolidone, polyethylene glycol, etc.; the disintegrant may be dry starch, microcrystalline cellulose, low-substituted hydroxypropyl cellulose, cross-linked polyvinyl pyrrolidone, cross-linked sodium carboxymethyl cellulose, sodium carboxymethyl starch, sodium bicarbonate and citric acid, polyoxyethylene sorbitan fatty acid ester, sodium dodecyl sulfate, etc.; the lubricant and solubilizing agent may be talc, silicon dioxide, stearate, tartaric acid, liquid paraffin, polyethylene glycol, etc.

[0071] The tablets can be further made into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or double-layer tablets and multi-layer tablets.

[0072] In order to prepare the dosing unit into a capsule, the active ingredient compound of the present invention can be mixed with a diluent and a cosolvent, and the mixture can be directly placed in a hard capsule or a soft capsule. The active ingredient compound of the present invention can also be first prepared into granules or pellets with a diluent, a binder, and a disintegrant, and then placed in a hard capsule or a soft capsule. The diluents, binders, wetting agents, disintegrants, and cosolvents used to prepare tablets of the compound of the present invention can also be used to prepare capsules of the compound of the present invention.

[0073] To prepare the compound of the present invention into an injection, water, ethanol, isopropanol, propylene glycol or a mixture thereof can be used as a solvent and an appropriate amount of a solubilizer, cosolvent, pH adjuster, and osmotic pressure regulator commonly used in the art can be added. The solubilizer or cosolvent can be poloxamer, lecithin, hydroxypropyl-β-cyclodextrin, etc.; the pH adjuster can be phosphate, acetate, hydrochloric acid, sodium hydroxide, etc.; the osmotic pressure regulator can be sodium chloride, mannitol, glucose, phosphate, acetate, etc. If a lyophilized powder injection is prepared, mannitol, glucose, etc. can also be added as a support agent.

[0074] Furthermore, if necessary, colorants, preservatives, perfumes, flavoring agents or other additives may be added to the pharmaceutical preparations.

[0075] In order to achieve the purpose of medication and enhance the therapeutic effect, the drug or pharmaceutical composition of the present invention can be administered by any known administration method.

[0076] The compound or composition of the present invention can be taken alone or in combination with other therapeutic drugs or symptomatic drugs. When the compound of the present invention has a synergistic effect with other therapeutic drugs, its dosage should be adjusted according to the actual situation.

[0077] Beneficial technical effects

[0078] The compounds of the present invention have strong PLpro inhibitory activity, and the inhibitory effects (IC 50 ) was less than 30 μM, and the PLpro inhibitory activity IC 50 <10μM. The antiviral activity of the two compounds was better than that of the PLpro inhibitor GRL0617, showing a strong anti-coronavirus effect. In addition, these compounds had low cytotoxicity to Vero E6 cells (IC 50 >100 μM), indicating that the compounds have good safety. The present invention provides a class of novel compounds with novel structures, strong in vitro PLpro inhibitory activity and low cytotoxicity, which can be used in drugs for inhibiting PLpro activity, especially as therapeutic agents for diseases related to coronavirus infection. DETAILED DESCRIPTION

[0079] The present invention can be described in detail by the following examples, but it is not intended to limit the present invention in any way. The present invention has been described in detail herein, and specific embodiments thereof are disclosed therein, and it is apparent to those skilled in the art that various changes and modifications can be made to the specific embodiments of the present invention without departing from the spirit and scope of the present invention.

[0080] For all of the following examples, standard manipulations and purification methods known to those skilled in the art may be used. The structures of the compounds were determined by H NMR (1 H NMR).

[0081] Preparation Examples Section

[0082] The structures of the compounds were determined by H NMR spectroscopy ( 1 H NMR). The H NMR shift (δ) is given in parts per million (ppm). The coupling constant (J) is given in Hertz (Hz). The NMR spectrum is measured using a 400 Hz, 500 Hz or 700 Hz NMR instrument. Deuterated dimethyl sulfoxide (DMSO-d 6 ) or deuterated chloroform (CDCl 3 ) was used as solvent and tetramethylsilane (TMS) was used as internal standard.

[0083] Example 1

[0084] N-(3-Trifluoromethyl-benzyl)-2-methyl-5-(((5-(5-(1-(2-fluoro-[1,1′-biphenyl]-4-yl)ethyl)-1,2,4-oxadiazol-3-yl)aniline

[0085]

[0086] Synthesis route:

[0087]

[0088] Experimental steps:

[0089] Step 1: Preparation of 5-(1-(2-fluoro-[1,1′-biphenyl]-4-yl)ethyl)-3-(4-methyl-3-nitrophenyl)-1,2,4-oxadiazole (C-1)

[0090] In a 100 mL reaction bottle, 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT, 5250 mg, 30.0 mmol) and N-methylmorpholine (NMM, 9090 mg, 90.0 mmol) were dissolved in 1,4-dioxane (150 mL), stirred at room temperature for 15 minutes, 2-(2-fluoro-[1,1′-biphenyl]-4-yl)propionic acid (A-1, 7320 mg, 30.0 mmol) was added, stirred at room temperature for 60 minutes, (Z)-N′-hydroxy-4-methyl-5-nitrobenzenecarboxamidine (B-1, 5265 mg, 30.0 mmol) was added, stirred at room temperature for 2 hours, heated to reflux for 6 hours. Concentrated under reduced pressure, separated by silica gel column chromatography (ethyl acetate / petroleum ether=5 / 95), intermediate C-1 was obtained as a white solid of 7.39 g, with a yield of 61%.

[0091] 1H NMR (400 MHz, DMSO-d 6 )δ8.51(s,1H),8.22(d,J=8.0Hz,1H),7.71(d,J=8.1Hz,1H),7.54(d,J=7.8Hz,3H),7.47(t,J=7.6Hz,2 H),7.44–7.37(m,2H),7.33(d,J=8.0Hz,1H),4.80(q,J=7.2Hz,1H),2.60(s,3H),1.78(d,J=6.8Hz,3H).

[0092] Step 2: Preparation of 5-(5-(1-(2-fluoro-[1,1′-biphenyl]-4-yl)ethyl)-1,2,4-oxadiazol-3-yl)-2-methylaniline (D-1)

[0093] In a 25 mL reaction bottle, the intermediate 5-(1-(2-fluoro-[1,1′-biphenyl]-4-yl)ethyl)-3-(4-methyl-3-nitrophenyl)-1,2,4-oxadiazole (C-1, 404 mg, 1.0 mmol) was dissolved in 5 ml methanol and 5 ml dichloromethane, saturated aqueous ammonium chloride solution (5 mL) was added, stirred evenly, zinc powder (1308 mg, 20.0 mmol) was added, and stirred at room temperature for 3 hours. Filter, concentrate under reduced pressure, and separate by silica gel column chromatography (methanol / dichloromethane = 3 / 97) to obtain intermediate D-1 as an off-white solid 345 mg, with a yield of 93%.

[0094] 1 H NMR (400 MHz, CDCl 3 )δ7.53(d,J=9.2Hz,2H),7.46–7.34(m,4H),7.39–7.34(m,2H),7.23–7.15(m,3H),4.49(q,J=7.2Hz,1H),2.24(s,3H),1.84(d,J=7.2Hz,3H).

[0095] Step 3: Preparation of N-(3-trifluoromethyl-benzyl)-2-methyl-5-(((5-(5-(1-(2-fluoro-[1,1′-biphenyl]-4-yl)ethyl)-1,2,4-oxadiazol-3-yl)aniline (Compound 1)

[0096] In a 25 mL reaction bottle, the intermediate 5-(5-(1-(2-fluoro-[1,1′-biphenyl]-4-yl)ethyl)-1,2,4-oxadiazol-3-yl)-2-methylaniline (D-1, 80 mg, 0.21 mmol), 3-trifluoromethylbenzaldehyde (73 mg, 0.42 mmol), glacial acetic acid (75 mg, 1.26 mmol) were dissolved in 1,2-dichloroethane (8 mL), stirred at room temperature for 3 hours, and then sodium triacetoxyborohydride (267 mg, 1.26 mmol) was slowly added. Concentrated under reduced pressure, separated by silica gel column chromatography (methanol / dichloromethane=10 / 90), compound I-1 (compound 1), 53 mg of off-white solid, yield 47%.

[0097] 1 H NMR (400 MHz, CDCl 3 )δ7.68(s,1H),7.60(d,J=7.5Hz,1H),7.57–7.48(m,3H),7.48–7.40(m,4H),7.40–7.33(m,2H),7.3 0(s,1H),7.23–7.15(m,3H),4.51(s,2H),4.47(q,J=7.2Hz,1H),2.23(s,3H),1.82(d,J=4.8Hz,3H).

[0098] Example 2

[0099] N-(3-Methoxy-benzyl)-2-methyl-5-(((5-(5-(1-(2-fluoro-[1,1′-biphenyl]-4-yl)ethyl)-1,2,4-oxadiazol-3-yl)aniline

[0100]

[0101] Using 3-methoxybenzaldehyde (57 mg, 0.42 mmol) as raw material, similar operation steps as the third step in Example 1 were used to react with intermediate D-1 to obtain compound I-2 (compound 2), 58 mg of off-white solid, with a yield of 56%.

[0102] 1 H NMR (400 MHz, CDCl 3)δ7.55–7.50(m,2H),7.47–7.40(m,4H),7.40–7.33(m,2H),7.31–7.26(m,1H),7.24–7.13(m,3H),7.03–6.95(m,2H), 6.84(d,J=8.3Hz,1H),4.48(q,J=7.2Hz,1H),4.42(s,2H),3.80(d,J=1.6Hz,3H),2.20(s,3H),1.83(d,J=7.3Hz,3H).

[0103] Example 3

[0104] N-(4-Morpholine-benzyl)-2-methyl-5-(((5-(5-(1-(2-fluoro-[1,1′-biphenyl]-4-yl)ethyl)-1,2,4-oxadiazol-3-yl)aniline

[0105]

[0106] Using 4-(4-morpholinyl)benzaldehyde (80 mg, 0.42 mmol) as raw material, similar operation steps as the third step in Example 1 were used to react with intermediate D-1 to obtain compound I-3 (compound 3), 66 mg of off-white solid, with a yield of 64%.

[0107] 1 H NMR (400 MHz, CDCl 3 )δ7.56–7.50(m,2H),7.48–7.39(m,4H),7.39–7.30(m,4H),7.25–7.20(m,2H),7.20–7.13(m,1H),6.93( s,2H),4.49(q,J=7.7Hz,1H),4.35(s,2H),3.87(s,5H),3.17(s,4H),2.18(s,3H),1.84(d,J=7.1Hz,3H).

[0108] Example 4

[0109] N-(4-Ethylene-benzyl)-2-methyl-5-(((5-(5-(1-(2-fluoro-[1,1′-biphenyl]-4-yl)ethyl)-1,2,4-oxadiazol-3-yl)aniline

[0110]

[0111] Using 4-ethynylbenzaldehyde (55 mg, 0.42 mmol) as raw material, similar operation steps as the third step in Example 1 were used to react with intermediate D-1 to obtain compound I-4 (compound 4), 49 mg of off-white solid, with a yield of 48%.

[0112] 1 H NMR (400 MHz, CDCl 3 )δ7.56–7.46(m,4H),7.46–7.40(m,4H),7.40–7.32(m,3H),7.29(s,1H),7.25–7 .14(m,3H),4.51–4.43(m,3H),3.05(s,1H),2.21(s,3H),1.82(d,J=7.1Hz,3H).

[0113] Example 5

[0114] N-(4-Acetylamino-benzyl)-2-methyl-5-(((5-(5-(1-(2-fluoro-[1,1′-biphenyl]-4-yl)ethyl)-1,2,4-oxadiazol-3-yl)aniline

[0115]

[0116] Using 4-acetaminobenzaldehyde (69 mg, 0.42 mmol) as raw material, similar operation steps as the third step in Example 1 were adopted to react with intermediate D-1 to obtain compound I-5 (compound 5), 47 mg of off-white solid, with a yield of 43%.

[0117] 1 H NMR (400 MHz, CDCl 3 )δ7.56–7.50(m,2H),7.50–7.45(m,2H),7.45–7.39(m,3H),7.39–7.31(m,4H),7.23–7.14( m,4H),4.48(q,J=7.2Hz,1H),4.39(s,2H),2.19(s,3H),2.16(s,3H),1.83(d,J=7.2Hz,3H).

[0118] Example 6

[0119] N-(4-Acetyl-benzyl)-2-methyl-5-(((5-(5-(1-(2-fluoro-[1,1′-biphenyl]-4-yl)ethyl)-1,2,4-oxadiazol-3-yl)aniline

[0120]

[0121] Using 4-acetylbenzaldehyde (62 mg, 0.42 mmol) as raw material, similar operation steps as the third step in Example 1 were used to react with intermediate D-1 to obtain compound I-6 (compound 6), 55 mg of off-white solid, with a yield of 52%.

[0122] 1 H NMR (400 MHz, CDCl 3 )δ7.94(d,J=7.9Hz,2H),7.54–7.47(m,4H),7.46–7.39(m,4H),7.39–7.33(m,1H),7.27(s,1H),7.2 2–7.15(m,3H),4.52(s,2H),4.46(q,J=7.2Hz,1H),2.58(s,3H),2.23(s,3H),1.81(d,J=7.2Hz,3H).

[0123] Example 7

[0124] N-(3-Hydroxy-benzyl)-2-methyl-5-(((5-(5-(1-(2-fluoro-[1,1′-biphenyl]-4-yl)ethyl)-1,2,4-oxadiazol-3-yl)aniline

[0125]

[0126] Using m-hydroxybenzaldehyde (51 mg, 0.42 mmol) as raw material, similar operation steps as the third step in Example 1 were used to react with intermediate D-1 to obtain compound I-7 (compound 7), 50 mg of off-white solid, with a yield of 50%.

[0127] 1 H NMR (400 MHz, CDCl 3 )δ7.53–7.47(m,2H),7.45–7.38(m,4H),7.38–7.33(m,1H),7.30(s,1H),7.23–7.10(m,4H),6.90(d,J=7.6Hz,1 H),6.83(s,1H),6.73(d,J=8.1Hz,1H),4.47(q,J=7.2Hz,1H),4.26(s,2H),2.15(s,3H),1.82(d,J=7.2Hz,3H).

[0128] Example 8

[0129] N-(3,5-Dihydroxy-benzyl)-2-methyl-5-(((5-(5-(1-(2-fluoro-[1,1′-biphenyl]-4-yl)ethyl)-1,2,4-oxadiazol-3-yl)aniline

[0130]

[0131] Using 3,5-dihydroxybenzaldehyde (58 mg, 0.42 mmol) as raw material, similar operation steps as the third step in Example 1 were used to react with intermediate D-1 to obtain compound I-8 (compound 8), 44 mg of off-white solid, with a yield of 42%.

[0132] 1 H NMR (400 MHz, CDCl 3 )δ7.48–7.41(m,2H),7.40–7.34(m,2H),7.34–7.27(m,3H),7.16–7.07(m,3H),6.99(d,J=7.8Hz,1H ),6.28(s,2H),6.21(s,1H),4.39(q,J=7.3Hz,1H),3.95(s,2H),1.98(s,3H),1.74(d,J=7.2Hz,3H).

[0133] Example 9

[0134] N-(4-Hydroxymethyl-benzyl)-2-methyl-5-(((5-(5-(1-(2-fluoro-[1,1′-biphenyl]-4-yl)ethyl)-1,2,4-oxadiazol-3-yl)aniline

[0135]

[0136] Using p-hydroxymethylbenzaldehyde (57 mg, 0.42 mmol) as raw material, similar operation steps as the third step in Example 1 were used to react with intermediate D-1 to obtain compound I-9 (compound 9), 52 mg of off-white solid, with a yield of 50%.

[0137] 1 H NMR (400 MHz, CDCl 3 )δ7.57–7.50(m,2H),7.49–7.43(m,3H),7.43–7.39(m,4H),7.39–7.32(m,3H),7.25–7.15( m,3H),4.69(s,2H),4.48(q,J=7.3Hz,1H),4.45(s,2H),2.22(s,3H),1.84(d,J=6.7Hz,3H).

[0138] Example 10

[0139] N-(3-Carboxy-benzyl)-4-methyl-3-(5-benzyl-1,2,4-oxadiazol-3-yl)aniline

[0140]

[0141] Synthetic route:

[0142]

[0143] Experimental steps:

[0144] Step 1: Preparation of 5-benzyl-3-(4-methyl-3-nitrophenyl)-1,2,4-oxadiazole (C-2)

[0145] In a 100 mL reaction bottle, 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT, 5250 mg, 30.0 mmol) and N-methylmorpholine (NMM, 9090 mg, 90.0 mmol) were dissolved in 1,4-dioxane (150 mL), stirred at room temperature for 15 minutes, 2-phenylacetic acid (A-10, 4085 mg, 30 mmol) was added, stirred at room temperature for 60 minutes, (Z)-N′-hydroxy-2-methyl-5-nitrobenzenecarboxamidine (5265 mg, 30.0 mmol) was added, stirred at room temperature for 2 hours, heated to reflux for 6 hours. Concentrated under reduced pressure, separated by silica gel column chromatography (ethyl acetate / petroleum ether=5 / 95), intermediate C-2 was obtained as a white solid (6.33 g) with a yield of 71%.

[0146] Step 2: Preparation of 5-(5-benzyl-1,2,4-oxadiazol-3-yl)-2-methylaniline (D-2)

[0147] In a 25 mL reaction bottle, the intermediate 5-benzyl-3-(4-methyl-3-nitrophenyl)-1,2,4-oxadiazole (C-2, 295 mg, 1.0 mmol) was dissolved in 5 ml methanol and 5 ml dichloromethane, saturated aqueous ammonium chloride solution (5 mL) was added, stirred evenly, zinc powder (1308 mg, 20.0 mmol) was added, and stirred at room temperature for 3 hours. Filter, concentrate under reduced pressure, and separate by silica gel column chromatography (methanol / dichloromethane = 3 / 97) to obtain intermediate D-2 as an off-white solid 211 mg, with a yield of 80%.

[0148] 1 H NMR (400 MHz, CDCl 3 )δ7.40–7.36(m,3H),7.36–7.35(m,1H),7.35–7.33(m,1H),7.32–7.29(m,1H) ,7.08(d,J=8.1Hz,1H),6.72(dd,J=8.1,2.6Hz,1H),4.29(s,2H),2.49(s,3H).

[0149] Step 3: Preparation of N-(3-carboxy-benzyl)-4-methyl-3-(5-benzyl-1,2,4-oxadiazol-3-yl)aniline (Compound 10)

[0150] In a 25 mL reaction bottle, the intermediate 5-(5-benzyl-1,2,4-oxadiazole-3-yl)-2-methylaniline (D-2, 56 mg, 0.21 mmol), 3-carboxybenzaldehyde (63 mg, 0.42 mmol), glacial acetic acid (75 mg, 1.26 mmol) were dissolved in 1,2-dichloroethane (8 mL), stirred at room temperature for 3 hours, and then sodium triacetoxyborohydride (267 mg, 1.26 mmol) was slowly added. Concentrated under reduced pressure, separated by silica gel column chromatography (methanol / dichloromethane = 10 / 90), compound I-10 (compound 10), 55 mg of off-white solid, yield 66%.

[0151] 1 H NMR (400 MHz, CDCl 3 )δ8.11(s,1H),8.01(d,J=7.8Hz,1H),7.61(d,J=7.6Hz,1H),7.43(t,J=7.7Hz,1H),7.39–7.35(m,3H),7.35–7.34(m ,1H),7.33–7.29(m,2H),7.09(d,J=8.2Hz,1H),6.64(dd,J=8.2,2.6Hz,1H),4.42(s,2H),4.28(s,2H),2.48(s,3H).

[0152] Embodiment 11

[0153] 5-(((3-(5-Benzyl-1,2,4-oxadiazol-3-yl)-5-(trifluoromethyl)-phenyl)amino)methyl)thiophene-2-carboxylic acid

[0154]

[0155] Synthetic route:

[0156]

[0157] Experimental steps:

[0158] Step 1: Preparation of 5-benzyl-3-(3-nitro-5-(trifluoromethyl)phenyl)-1,2,4-oxadiazole (C-3)

[0159] In a 100 mL reaction bottle, 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT, 5250 mg, 30.0 mmol) and N-methylmorpholine (NMM, 9090 mg, 90.0 mmol) were dissolved in 1,4-dioxane (150 mL), stirred at room temperature for 15 minutes, 2-phenylacetic acid (A-2, 4085 mg, 30 mmol) was added, stirred at room temperature for 60 minutes, (Z)-N′-hydroxy-3-nitro-5-(trifluoromethyl)benzamidamide (B-3, 7474 mg, 30 mmol) was added, stirred at room temperature for 2 hours, heated to reflux for 6 hours. Concentrated under reduced pressure, separated by silica gel column chromatography (ethyl acetate / petroleum ether = 5 / 95), intermediate C-3 was obtained as a white solid 7.22 g, yield 70%.

[0160] 1 H NMR (400 MHz, CDCl 3 )δ9.11(s,1H),8.67(s,1H),8.60(s,1H),7.41–7.37(m,4H),7.36–7.30(m,1H),4.34(s,2H).

[0161] Step 2: Preparation of 3-trifluoromethyl-5-(5-benzyl-1,2,4-oxadiazol-3-yl)aniline (D-3)

[0162] In a 25 mL reaction bottle, the intermediate 5-benzyl-3-(3-nitro-5-(trifluoromethyl)phenyl)-1,2,4-oxadiazole (C-3, 349 mg, 1.0 mmol) was dissolved in 5 ml methanol and 5 ml dichloromethane, saturated aqueous ammonium chloride solution (5 mL) was added, stirred evenly, zinc powder (1308 mg, 20.0 mmol) was added, and stirred at room temperature for 3 hours. Filter, concentrate under reduced pressure, and separate by silica gel column chromatography (methanol / dichloromethane = 3 / 97) to obtain intermediate D-3 as an off-white solid 270 mg, with a yield of 85%.

[0163] Step 3: Preparation of 5-(((3-(5-benzyl-1,2,4-oxadiazol-3-yl)-5-(trifluoromethyl)-phenyl)amino)methyl)thiophene-2-carboxylic acid (Compound 11)

[0164] In a 25 mL reaction bottle, the intermediate 3-trifluoromethyl-5-(5-benzyl-1,2,4-oxadiazole-3-yl)aniline (D-3, 67 mg, 0.21 mmol), 5-formyl-2-thiophenecarboxylic acid (66 mg, 0.42 mmol), glacial acetic acid (75 mg, 1.26 mmol) were dissolved in 1,2-dichloroethane (8 mL), stirred at room temperature for 3 hours, and then sodium triacetoxyborohydride (267 mg, 1.26 mmol) was slowly added. Concentrated under reduced pressure, separated by silica gel column chromatography (methanol / dichloromethane = 10 / 90), compound I-11 (compound 11), 61 mg of off-white solid, yield 63%.

[0165] 1 H NMR (400 MHz, DMSO-d 6 )δ7.58(d,J=3.8Hz,1H),7.50(s,1H),7.41–7.35(m,5H),7.35–7.25(m,2H ),7.12(d,J=3.7Hz,1H),7.10(s,1H),4.64(d,J=5.9Hz,2H),4.44(s,2H).

[0166] Example 12

[0167] 5-(((3-(5-(1-phenylethyl)-1,2,4-oxadiazol-3-yl)-5-(trifluoromethyl)phenyl)amino)methyl)thiophene-2-carboxylic acid

[0168]

[0169] Synthetic route:

[0170]

[0171] Experimental steps:

[0172] Step 1: Preparation of 3-(3-nitro-5-(trifluoromethyl)phenyl)-5-(1-phenylethyl)-1,2,4-oxadiazole (C-4)

[0173] In a 100 mL reaction bottle, 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT, 5250 mg, 30.0 mmol) and N-methylmorpholine (NMM, 9090 mg, 90.0 mmol) were dissolved in 1,4-dioxane (150 mL), stirred at room temperature for 15 minutes, 2-phenylpropionic acid (A-3, 4500 mg, 30.0 mmol) was added, stirred at room temperature for 60 minutes, (Z)-N′-hydroxy-3-nitro-5-(trifluoromethyl)benzamidamide (B-3, 7474 mg, 30.0 mmol) was added, stirred at room temperature for 2 hours, heated to reflux for 6 hours. Concentrated under reduced pressure, separated by silica gel column chromatography (ethyl acetate / petroleum ether=5 / 95), intermediate C-4 was obtained as a white solid (7.11 g, yield 65%).

[0174] 1 H NMR (400 MHz, CDCl 3 )δ9.13(d,J=1.8Hz,1H),8.68(s,1H),8.60(s,1H),7.40–7.35(m,4H),7.35–7.28(m,1H),4.51(q,J=7.2Hz,1H),1.86(d,J=7.3Hz,3H).

[0175] Step 2: Preparation of 3-trifluoromethyl-5-(5-(1-phenylethyl)-1,2,4-oxadiazol-3-yl)aniline (D-4)

[0176] In a 25 mL reaction bottle, the intermediate 3-(3-nitro-5-(trifluoromethyl)phenyl)-5-(1-phenylethyl)-1,2,4-oxadiazole (C-4, 363 mg, 1.0 mmol) was dissolved in 5 ml methanol and 5 ml dichloromethane, saturated aqueous ammonium chloride solution (5 mL) was added, stirred evenly, zinc powder (1308 mg, 20.0 mmol) was added, and stirred at room temperature for 3 hours. Filter, concentrate under reduced pressure, and separate by silica gel column chromatography (methanol / dichloromethane = 3 / 97) to obtain intermediate D-4 as an off-white solid 271 mg, with a yield of 81%.

[0177] 1 H NMR (400 MHz, CDCl 3 )δ7.73(s,1H),7.55(s,1H),7.38–7.32(m,4H),7.31–7.27(m,1H),7.01(s,1H),4.46(q,J=7.3Hz,1H),1.81(d,J=7.3Hz,3H).

[0178] Step 3: Preparation of 5-(((3-(5-(1-phenylethyl)-1,2,4-oxadiazol-3-yl)-5-(trifluoromethyl)phenyl)amino)methyl)thiophene-2-carboxylic acid (Compound 12)

[0179] In a 25 mL reaction bottle, the intermediate 3-trifluoromethyl-5-(5-benzyl-1,2,4-oxadiazole-3-yl)aniline (D-4, 70 mg, 0.21 mmol), 5-formyl-2-thiophenecarboxylic acid (66 mg, 0.42 mmol), glacial acetic acid (75 mg, 1.26 mmol) were dissolved in 1,2-dichloroethane (8 mL), stirred at room temperature for 3 hours, and then sodium triacetoxyborohydride (267 mg, 1.26 mmol) was slowly added. Concentrated under reduced pressure, separated by silica gel column chromatography (methanol / dichloromethane = 10 / 90), compound I-12 (compound 12), 66 mg of off-white solid, yield 66%.

[0180] 1 H NMR (400 MHz, DMSO-d 6 )δ7.59(d,J=3.7Hz,1H),7.53(s,1H),7.42–7.34(m,5H),7.34–7.25(m,2H),7 .13(d,J=3.7Hz,1H),7.10(brs,1H),4.70–4.61(m,3H),1.72(d,J=7.2Hz,3H).

[0181] Embodiment 13

[0182] 5-(((3-(5-(Hydroxy(phenyl)ethyl)-1,2,4-oxadiazol-3-yl)-5-(trifluoromethyl)phenyl)amino)methyl)thiophene-2-carboxylic acid

[0183]

[0184] Synthetic route:

[0185]

[0186] Experimental steps:

[0187] Step 1: Preparation of (3-(3-nitro-5-(trifluoromethyl)phenyl)-1,2,4-oxadiazol-5-yl)(phenyl)methanol (C-5)

[0188] In a 100 mL reaction bottle, 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT, 5250 mg, 30.0 mmol) and N-methylmorpholine (NMM, 9090 mg, 90.0 mmol) were dissolved in 1,4-dioxane (150 mL), stirred at room temperature for 15 minutes, 2-hydroxy-2-phenylacetic acid (A-4, 4564 mg, 30.0 mmol) was added, stirred at room temperature for 60 minutes, (Z)-N′-hydroxy-3-nitro-5-(trifluoromethyl)benzamidamide (B-3, 7474 mg, 30.0 mmol) was added, stirred at room temperature for 2 hours, heated to reflux for 6 hours. Concentrated under reduced pressure, separated by silica gel column chromatography (ethyl acetate / petroleum ether = 5 / 95), intermediate C-5 was obtained as a white solid (7.00 g, yield 64%).

[0189] 1 H NMR (400 MHz, CDCl 3 )δ9.14(s,1H),8.70(s,1H),8.63(s,1H),7.59–7.52(m,2H),7.49–7.37(m,3H),6.15(s,1H).

[0190] Step 2: Preparation of 3-trifluoromethyl-5-(5-(hydroxy(phenyl)methyl)-1,2,4-oxadiazol-3-yl)aniline (D-5)

[0191] In a 25 mL reaction bottle, the intermediate (3-(3-nitro-5-(trifluoromethyl)phenyl)-1,2,4-oxadiazol-5-yl)(phenyl)methanol (C-5, 365 mg, 1.0 mmol) was dissolved in 5 ml methanol and 5 ml dichloromethane, saturated aqueous ammonium chloride solution (5 mL) was added, stirred evenly, zinc powder (1308 mg, 20.0 mmol) was added, and stirred at room temperature for 3 hours. Filter, concentrate under reduced pressure, and separate by silica gel column chromatography (methanol / dichloromethane = 3 / 97) to obtain intermediate D-5 as an off-white solid 297 mg, with a yield of 89%.

[0192] 1 H NMR (400 MHz, CDCl 3 )δ7.72(s,1H),7.56–7.49(m,3H),7.46–7.36(m,3H),7.01(s,1H),6.09(s,1H).

[0193] Step 3: Preparation of 5-(((3-(5-(hydroxy(phenyl)ethyl)-1,2,4-oxadiazol-3-yl)-5-(trifluoromethyl)phenyl)amino)methyl)thiophene-2-carboxylic acid (Compound 13)

[0194] In a 25 mL reaction bottle, the intermediate 3-trifluoromethyl-5-(5-(hydroxy(phenyl)methyl)-1,2,4-oxadiazol-3-yl)aniline (D-5, 70 mg, 0.21 mmol), 5-formyl-2-thiophenecarboxylic acid (66 mg, 0.42 mmol), and glacial acetic acid (75 mg, 1.26 mmol) were dissolved in 1,2-dichloroethane (8 mL). After stirring at room temperature for 3 hours, sodium triacetoxyborohydride (267 mg, 1.26 mmol) was slowly added. The mixture was concentrated under reduced pressure and separated by silica gel column chromatography (methanol / dichloromethane = 10 / 90) to obtain compound I-13 (compound 13), 52 mg of off-white solid, and a yield of 52%.

[0195] 1 H NMR (400 MHz, DMSO-d 6 )δ7.60(d,J=3.7Hz,1H),7.54–7.48(m,3H),7.45–7.32(m,4H),7.29(t,J=6.1Hz,1H),7.13(d, J=3.7Hz,1H),7.10(s,1H),6.88(d,J=5.1Hz,1H),6.14(d,J=4.7Hz,1H),4.64(d,J=5.8Hz,2H).

[0196] Embodiment 14

[0197] 5-(((5-(5-(Hydroxy(phenyl)ethyl)-1,2,4-oxadiazol-3-yl)-2-methyl-phenyl)amino)methyl)thiophene-2-carboxylic acid

[0198]

[0199] Synthetic route:

[0200]

[0201] Experimental steps:

[0202] Step 1: Preparation of (3-(4-methyl-3-nitrophenyl)-1,2,4-oxadiazol-5-yl)(phenyl)methanol (C-6)

[0203] In a 100 mL reaction bottle, 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT, 5250 mg, 30.0 mmol) and N-methylmorpholine (NMM, 9090 mg, 90.0 mmol) were dissolved in 1,4-dioxane (150 mL), stirred at room temperature for 15 minutes, 2-hydroxy-2-phenylacetic acid (A-5, 4564 mg, 30.0 mmol) was added, stirred at room temperature for 60 minutes, (Z)-N′-hydroxy-4-methyl-3-nitrobenzamidamide (B-1, 5585 mg, 30.0 mmol) was added, stirred at room temperature for 2 hours, heated to reflux for 6 hours. Concentrated under reduced pressure, separated by silica gel column chromatography (ethyl acetate / petroleum ether = 5 / 95), intermediate C-6 was obtained as a white solid (6.67 g, yield 71%).

[0204] 1 H NMR (400 MHz, CDCl 3 )δ8.18(s,1H),7.63(d,J=7.8Hz,1H),7.44–7.36(m,2H),7.35–7.28(m,3H),7.14(d,J=7.7Hz,1H),6.04(s,1H),2.14(s,3H).

[0205] Step 2: Preparation of (3-(3-amino-4-methylphenyl)-1,2,4-oxadiazol-5-yl)(phenyl)methanol (D-6)

[0206] In a 25 mL reaction bottle, the intermediate (3-(4-methyl-3-nitrophenyl)-1,2,4-oxadiazol-5-yl)(phenyl)methanol (C-6, 311 mg, 1.0 mmol) was dissolved in 5 ml methanol and 5 ml dichloromethane, saturated aqueous ammonium chloride solution (5 mL) was added, stirred evenly, zinc powder (1308 mg, 20.0 mmol) was added, and stirred at room temperature for 3 hours. Filter, concentrate under reduced pressure, and separate by silica gel column chromatography (methanol / dichloromethane = 3 / 97) to obtain intermediate D-6 as an off-white solid 252 mg, with a yield of 90%.

[0207] 1 H NMR (400 MHz, DMSO-d 6 )δ7.51(d,J=7.2Hz,2H),7.44–7.37(m,2H),7.37–7.32(m,1H),7.11–7.03(m,2H),6.81(d,J=5.0Hz,1H),6.09(d,J=5.0Hz,1H),2.10(s,3H).

[0208] Step 3: Preparation of 5-(((5-(5-(hydroxy(phenyl)ethyl)-1,2,4-oxadiazol-3-yl)-2-methyl-phenyl)amino)methyl)thiophene-2-carboxylic acid (Compound 14)

[0209] In a 25 mL reaction bottle, the intermediate (3-(3-amino-4-methylphenyl)-1,2,4-oxadiazol-5-yl)(phenyl)methanol (D-6, 59 mg, 0.21 mmol), 5-formyl-2-thiophenecarboxylic acid (66 mg, 0.42 mmol), glacial acetic acid (75 mg, 1.26 mmol) were dissolved in 1,2-dichloroethane (8 mL), stirred at room temperature for 3 hours, and then sodium triacetoxyborohydride (267 mg, 1.26 mmol) was slowly added. Concentrated under reduced pressure, separated by silica gel column chromatography (methanol / dichloromethane = 10 / 90), compound I-14 (compound 14), 55 mg of off-white solid, yield 62%.

[0210] 1 H NMR (400 MHz, DMSO-d 6 )δ7.57(d,J=3.7Hz,1H),7.50(d,J=7.6Hz,2H),7.40(t,J=7.4Hz,2H),7.37–7.31(m,1H),7.17(s,2H),7.12–7.0 6(m,2H),6.80(d,J=5.0Hz,1H),6.13(t,J=6.2Hz,1H),6.08(d,J=4.0Hz,1H),4.60(d,J=5.6Hz,2H),2.20(s,3H).

[0211] Embodiment 15

[0212] (5-(((5-(5-(1-(4-isobutylphenyl)ethyl)-1,2,4-oxadiazol-3-yl)-2-methylphenyl)amino)methyl)thiophen-2-yl)boronic acid

[0213]

[0214] Synthesis route:

[0215]

[0216] Experimental steps:

[0217] Step 1: Preparation of 5-(1-(4-isobutylphenyl)ethyl)-3-(4-methyl-3-nitrophenyl)-1,2,4-oxadiazole (C-7)

[0218] In a 100 mL reaction bottle, 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT, 700 mg, 4.0 mmol) and N-methylmorpholine (NMM, 1212 mg, 12.0 mmol) were dissolved in 1,4-dioxane (30 mL), stirred at room temperature for 15 minutes, 2-(4-isobutylphenyl)propionic acid (A-6, 824 mg, 4.0 mmol) was added, stirred at room temperature for 60 minutes, (Z)-N′-hydroxy-2-methyl-5-nitrobenzenecarboxamidine (B-1, 702 mg, 3.6 mmol) was added, stirred at room temperature for 2 hours, heated to reflux for 6 hours. Concentrated under reduced pressure, separated by silica gel column chromatography (ethyl acetate / petroleum ether = 5 / 95), intermediate C-7 was obtained as a white solid 837 mg, yield 57%.

[0219] 1 H NMR (400 MHz, CDCl 3 )δ8.82(s,1H),8.33(dd,J=8.4,1.6Hz,1H),7.59(d,J=8.0Hz,1H),7.41(d,J=8.0Hz,2H),7.28(s,1H),4.59(q,J =7.2Hz,1H),2.79(s,3H),2.59(d,J=7.2Hz,2H),2.02–1.97(m,1H),1.95(d,J=7.2Hz,3H),1.03(d,J=6.8Hz,6H).

[0220] Step 2: Preparation of 5-(5-(1-(4-isobutylphenyl)ethyl)-1,2,4-oxadiazol-3-yl)-2-methylaniline (D-7)

[0221] In a 25 mL reaction bottle, the intermediate 5-(1-(4-isobutylphenyl)ethyl)-3-(4-methyl-3-nitrophenyl)-1,2,4-oxadiazole (C-7, 365 mg, 1.0 mmol) was dissolved in 5 ml methanol and 5 ml dichloromethane, saturated aqueous ammonium chloride solution (5 mL) was added, stirred evenly, zinc powder (1308 mg, 20.0 mmol) was added, and stirred at room temperature for 3 hours. Filter, concentrate under reduced pressure, and separate by silica gel column chromatography (methanol / dichloromethane = 3 / 97) to obtain intermediate D-7 as an off-white solid 247 mg, with a yield of 73%.

[0222] 1 H NMR (400 MHz, CDCl 3)δ7.44(d,J=8.0Hz,1H),7.42(s,1H),7.28(s,1H),7.13(t,J=8.8Hz,3H),4.43(q,J=7.2Hz,1H),2 .45(d,J=6.4Hz,2H),2.24(s,3H),1.88–1.83(m,1H),1.80(d,J=7.2Hz,3H),0.90(d,J=6.4Hz,6H).

[0223] Step 3: Preparation of (5-(((5-(5-(1-(4-isobutylphenyl)ethyl)-1,2,4-oxadiazol-3-yl)-2-methylphenyl)amino)methyl)thiophen-2-yl)boronic acid (Compound 15)

[0224] In a 25 mL reaction bottle, the intermediate 5-(5-(1-(4-isobutylphenyl)ethyl)-1,2,4-oxadiazol-3-yl)-2-methylaniline (D-7, 201 mg, 0.6 mmol), 5-formyl-2-thiopheneboronic acid (187 mg, 1.2 mmol), and glacial acetic acid (216 mg, 3.6 mmol) were dissolved in 1,2-dichloroethane (20 mL). After stirring at room temperature for 3 hours, sodium triacetoxyborohydride (760 mg, 3.6 mmol) was slowly added. The mixture was concentrated under reduced pressure and separated by silica gel column chromatography (methanol / dichloromethane = 10 / 90) to obtain compound I-15 (compound 15), 83 mg of yellow solid, with a yield of 29%.

[0225] 1 H NMR (400 MHz, CDCl 3 )δ7.46–7.43(m,2H),7.28(s,1H),7.24(s,1H),7.17–7.08(m,4H),6.98(s,1H),4.63(s,2H),4.43(q,J=7.2 Hz,1H),2.45(d,J=7.2Hz,2H),2.20(s,3H),1.86–1.84(m,1H),1.79(d,J=7.6Hz,3H),0.89(d,J=6.8Hz,6H).

[0226] Example 16

[0227] 5-(((5-(5-(1-(4-isobutylphenyl)ethyl)-1,2,4-oxadiazol-3-yl)-2-methylphenyl)amino)methyl)thiophene-2-carboxylic acid

[0228]

[0229] Using 5-formyl-2-thiophenecarboxylic acid (115 mg, 1.0 mmol) as raw material, similar operation steps as the third step in Example 15 were used to react with intermediate D-7 to obtain compound I-16 (Compound 16), 123 mg of off-white solid, with a yield of 52%.

[0230] 1 H NMR (400 MHz, DMSO-d 6 )δ12.9(brs,1H),7.58(d,J=3.6Hz,1H),7.25(d,J=7.6Hz,2H),7.20–7.17(m,2H),7.14–7.11(m,4H),6.13(brs,1H),4.61(d,J= 5.2Hz,2H),4.55(q,J=7.2Hz,1H),2.41(d,J=6.8Hz,2H),2.21(s,3H),1.79(m,1H),1.67(d,J=7.2Hz,3H),0.84(d,J=6.4Hz,6H).

[0231] Embodiment 17

[0232] 5-(((5-(5-(1-(2-fluoro-[1,1′-biphenyl]-4-yl)ethyl)-1,2,4-oxadiazol-3-yl)-2-methylphenyl)amino)methyl)thiophene-2-carbonitrile

[0233]

[0234] Using 5-cyanothiophene-2-carboxaldehyde (58 mg, 0.42 mmol) as raw material, similar operation steps as the third step in Example 1 were used to react with intermediate D-1 to obtain compound I-17 (compound 17), 55 mg of off-white solid, with a yield of 53%.

[0235] 1 H NMR (400 MHz, DMSO-d 6 )δ7.82(d,J=4.0Hz,1H),7.54(d,J=7.6Hz,3H),7.51–7.46(m,2H),7.42–7.36(m,2H),7.29(d,J=8.0Hz,1H),7.24–7.1 7(m,3H),7.09(s,1H),6.22(brs,1H),4.71(q,J=7.2Hz,1H),4.67(d,J=6.0Hz,2H),2.21(s,3H),1.73(d,J=7.2Hz,3H).

[0236] Embodiment 18

[0237] 5-(((5-(5-(1-(2-fluoro-[1,1′-biphenyl]-4-yl)ethyl)-1,2,4-oxadiazol-3-yl)-2-methylphenyl)amino)methyl)thiophene-2-carboxylic acid

[0238]

[0239] Using 5-formyl-2-thiophenecarboxylic acid (49 mg, 0.42 mmol) as raw material, similar operation steps as the third step in Example 1 were adopted to react with intermediate D-1 to obtain compound I-18 (compound 18), 41 mg of off-white solid, with a yield of 38%.

[0240] 1 H NMR (400 MHz, DMSO-d 6 )δ12.89(brs,1H),7.55-7.53(m,4H),7.48(t,J=6.8Hz,2H),7.42–7.36(m,2H),7.29(d,J=8.0Hz,1H),7.21–7.15(m ,2H),7.11(s,2H),6.14(brs,1H),4.70(t,J=7.2Hz,1H),4.61(d,J=5.6Hz,2H),2.21(s,3H),1.73(d,J=7.2Hz,3H).

[0241] Embodiment 19

[0242] 5-(((5-(5-(1-(3-phenoxyphenyl)ethyl)-1,2,4-oxadiazol-3-yl)-2-methylphenyl)amino)methyl)thiophene-2-carboxylic acid

[0243]

[0244] Synthesis route:

[0245]

[0246] Experimental steps:

[0247] Step 1: Preparation of 3-(4-methyl-3-nitrophenyl)-5-(1-(3-phenoxyphenyl)ethyl)-1,2,4-oxadiazole (C-8)

[0248] In a 100 mL reaction bottle, 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT, 350 mg, 2.0 mmol) and N-methylmorpholine (NMM, 606 mg, 6.0 mmol) were dissolved in 1,4-dioxane (20 mL), stirred at room temperature for 15 minutes, 2-(3-phenoxyphenyl)propionic acid (A-7, 484 mg, 2.0 mmol) was added, stirred at room temperature for 60 minutes, (Z)-N′-hydroxy-2-methyl-5-nitrobenzenecarboxamidine (B-1, 351 mg, 1.8 mmol) was added, stirred at room temperature for 2 hours, heated to reflux for 6 hours. Concentrated under reduced pressure, separated by silica gel column chromatography (ethyl acetate / petroleum ether = 5 / 95), intermediate C-8 was obtained as a white solid 577 mg, yield 72%.

[0249] 1 H NMR (400 MHz, DMSO-d 6 )δ8.47(s,1H),8.18(d,J=7.6Hz,1H),7.70(d,J=8.0Hz,1H),7.39(t,J=9.2Hz,3H),7.14(d,J=7.2Hz,2H),7.09(s, 1H),7.02(d,J=8.0Hz,2H),6.90(dd,J=8.2,2.4Hz,1H),4.71(q,J=7.2Hz,1H),2.60(s,3H),1.72(d,J=7.2Hz,3H).

[0250] Step 2: Preparation of 2-methyl-5-(5-(1-(3-phenoxyphenyl)ethyl)-1,2,4-oxadiazol-3-yl)aniline (D-8)

[0251] In a 25 mL reaction bottle, the intermediate 3-(4-methyl-3-nitrophenyl)-5-(1-(3-phenoxyphenyl)ethyl)-1,2,4-oxadiazole (C-8, 300 mg, 0.74 mmol) was dissolved in 5 ml methanol and 5 ml dichloromethane, saturated aqueous ammonium chloride solution (5 mL) was added, stirred evenly, zinc powder (967 mg, 14.8 mmol) was added, and stirred at room temperature for 3 hours. Filter, concentrate under reduced pressure, and separate by silica gel column chromatography (methanol / dichloromethane = 5 / 95) to obtain intermediate D-8, 230 mg of oily liquid, yield 84%.

[0252] 1 H NMR (400 MHz, DMSO-d 6)δ7.43(s,1H),7.37–7.35(m,2H),7.30–7.26(m,5H),7.19–7.18(m,2H),7.12–7.0 5(m,2H),5.17(brs,2H),4.60(q,J=4.0Hz,1H),2.11(s,3H),1.68(d,J=4.0Hz,3H).

[0253] Step 3: Preparation of 5-(((2-methyl-5-(5-(1-(3-phenoxyphenyl)ethyl)-1,2,4-oxadiazol-3-yl)phenyl)amino)methyl)thiophene-2-carboxylic acid (Compound 19)

[0254] In a 25 mL reaction bottle, the intermediate 2-methyl-5-(5-(1-(3-phenoxyphenyl)ethyl)-1,2,4-oxadiazol-3-yl)aniline (D-8, 115 mg, 0.3 mmol), 5-formyl-2-thiopheneboronic acid (71 mg, 0.6 mmol), and glacial acetic acid (111 mg, 1.86 mmol) were dissolved in 1,2-dichloroethane (12 mL). After stirring at room temperature for 3 hours, sodium triacetoxyborohydride (394 mg, 1.86 mmol) was slowly added. Concentrated under reduced pressure, separated by silica gel column chromatography (methanol / dichloromethane = 10 / 90), compound I-19 (compound 19) was obtained as a white solid 17 mg, yield 10%.

[0255] 1 H NMR (400 MHz, DMSO-d 6 )δ12.89(brs,1H),7.57(d,J=4.0Hz,1H),7.40-7.34(m,3H),7.17–7.14(m,3H),7.12–7.09(m,3H),7.05– 7.01(m,3H),6.89(d,J=8.4Hz,1H),6.13(brs,1H),4.64-4.59(m,3H),2.21(s,3H),1.67(d,J=7.2Hz,3H).

[0256] Embodiment 20

[0257] 5-(((5-(5-(1-(3-phenoxyphenyl)ethyl)-1,2,4-oxadiazol-3-yl)-2-methylphenyl)amino)methyl)thiophene-2-cyano

[0258]

[0259] Using 5-cyanothiophene-2-carboxaldehyde (170 mg, 1.24 mmol) as raw material, similar operation steps as the third step in Example 19 were adopted to react with intermediate D-8 to obtain compound I-20 (compound 20), liquid 130 mg, yield 43%.

[0260] 1 H NMR (400 MHz, DMSO-d 6 )δ7.82(s,1H),7.37(d,J=7.2Hz,3H),7.23(s,1H),7.18–7.15(m,3H),7.12–7.01(m,5H),6. 89(d,J=8.4Hz,1H),4.66(s,2H),4.62(q,J=7.2Hz,1H),2.21(s,3H),1.67(d,J=7.2Hz,3H).

[0261] Embodiment 21

[0262] 5-(((5-(5-(1-(6-chloro-9H-carbazol-2-yl)ethyl)-1,2,4-oxadiazol-3-yl)-2-methylphenyl)amino)methyl)thiophene-2-carboxylic acid

[0263]

[0264] Synthesis route:

[0265]

[0266] Experimental steps:

[0267] Step 1: Preparation of 5-(1-(6-chloro-9H-carbazole-2-yl))ethyl)-3-(4-methyl-3-nitrophenyl)-1,2,4-oxadiazole (C-9)

[0268] In a 100 mL reaction bottle, 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT, 700 mg, 4.0 mmol) and N-methylmorpholine (NMM, 1212 mg, 12.0 mmol) were dissolved in 1,4-dioxane (30 mL), stirred at room temperature for 15 minutes, 2-(6-chloro-9H-carbazole-2-yl)propionic acid (A-8, 1092 mg, 4.0 mmol) was added, stirred at room temperature for 60 minutes, (Z)-N′-hydroxy-2-methyl-5-nitrobenzenecarboxamidine (B-1, 702 mg, 3.6 mmol) was added, stirred at room temperature for 2 hours, heated to reflux for 6 hours. Concentrated under reduced pressure, separated by silica gel column chromatography (ethyl acetate / petroleum ether = 5 / 95), intermediate C-9 was obtained as a yellow solid 970 mg, yield 56%.

[0269] 1 H NMR (400 MHz, DMSO-d 6 )δ11.42(s,1H),8.51(d,J=2.0Hz,1H),8.26–8.18(m,2H),8.14(d,J=8.0Hz,1H),7.70(d,J=8.0Hz,1H),7.50–7.48(m,2 H),7.37(dd,J=8.8,2.0Hz,1H),7.19(dd,J=8.0,1.6Hz,1H),4.86(q,J=7.2Hz,1H),2.60(s,3H),1.83(d,J=7.2Hz,3H).

[0270] Step 2: Preparation of 5-(1-(6-chloro-9H-carbazole-2-yl)ethyl)-3-(4-methyl-3-nitrophenyl)-1,2,4-oxadiazole (D-9)

[0271] In a 25 mL reaction bottle, the intermediate 5-(1-(6-chloro-9H-carbazole-2-yl))ethyl)-3-(4-methyl-3-nitrophenyl)-1,2,4-oxadiazole (C-9, 304 mg, 0.7 mmol) was dissolved in 5 ml methanol and 5 ml dichloromethane, saturated aqueous ammonium chloride solution (5 mL) was added, stirred evenly, zinc powder (15 mg, 14.0 mmol) was added, and stirred at room temperature for 3 hours. Filter, concentrate under reduced pressure, and separate by silica gel column chromatography (methanol / dichloromethane = 5 / 95) to obtain intermediate D-9 as a white solid 205 mg, with a yield of 73%.

[0272] 1 H NMR (400 MHz, DMSO-d 6 )δ11.42(s,1H),8.19(s,1H),8.14(d,J=8.0Hz,1H),7.50–7.47(m,2H),7.37(dd,J=8.8,2.4Hz,1H),7.31(s,1H), 7.18(d,J=8.4Hz,1H),7.13–7.06(m,2H),5.17(s,2H),4.79(q,J=7.2Hz,1H),2.10(s,3H),1.80(d,J=7.2Hz,3H).

[0273] Step 3: Preparation of 5-(((5-(5-(1-(6-chloro-9H-carbazole-2-yl)ethyl)-1,2,4-oxadiazole-3-yl)-2-methylphenyl)amino)methyl)thiophene-2-carboxylic acid (Compound 21)

[0274] In a 25 mL reaction bottle, the intermediate 5-(((1-(6-chloro-9H-carbazole-2-yl))ethyl)-1,2,4-oxadiazolyl)-2-methylaniline (D-9, 58 mg, 0.14 mmol), 5-formyl-2-thiophenecarboxylic acid (32 mg, 0.28 mmol), glacial acetic acid (50 mg, 0.84 mmol) were dissolved in 1,2-dichloroethane (6 mL), stirred at room temperature for 3 hours, and then sodium triacetoxyborohydride (178 mg, 0.84 mmol) was slowly added. Concentrated under reduced pressure, separated by silica gel column chromatography (methanol / dichloromethane = 10 / 90), compound I-21 (compound 21), 43 mg of white solid, yield 56%.

[0275] 1 H NMR (400 MHz, DMSO-d 6 )δ11.62(s,1H),8.18(s,1H),8.12(d,J=8.0Hz,1H),7.49(d,J=8.4Hz,1H),7.45(s,2H),7.36(d,J=8.8Hz,1H),7.20–7 .16(m,4H),7.04(s,1H),6.09(s,1H),4.76(q,J=7.2Hz,1H),4.57(d,J=5.6Hz,2H),2.20(s,3H),1.77(d,J=7.2Hz,3H).

[0276] Embodiment 22

[0277] 5-(((5-(5-(1-([1,1′-biphenyl]-3-yl)ethyl)-1,2,4-oxadiazol-3-yl)-2-methylphenyl)amino)methyl)thiophene-2-carboxylic acid

[0278]

[0279] Synthetic route:

[0280]

[0281] Experimental steps:

[0282] Step 1: Preparation of 5-(1-([1,1′-biphenyl]-3-yl)ethyl)-3-(4-methyl-3-nitrophenyl)-1,2,4-oxadiazole (C-10)

[0283] In a 100 mL reaction bottle, 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT, 350 mg, 2.0 mmol) and N-methylmorpholine (NMM, 606 mg, 6.0 mmol) were dissolved in 1,4-dioxane (20 mL), stirred at room temperature for 15 minutes, 2-([1,1′-biphenyl]-3-yl)propionic acid (A-9, 470 mg, 2.0 mmol) was added, stirred at room temperature for 60 minutes, (Z)-N′-hydroxy-4-methyl-3-nitrobenzamidamide (B-1, 351 mg, 1.8 mmol) was added, stirred at room temperature for 2 hours, heated to reflux for 6 hours. Concentrated under reduced pressure, separated by silica gel column chromatography (ethyl acetate / petroleum ether = 5 / 95), intermediate C-10 was obtained as a white solid 550 mg, yield 71%.

[0284] Step 2: Preparation of 5-(5-(1-([1,1′-biphenyl]-3-yl)ethyl)-1,2,4-oxadiazol-3-yl)-2-methylaniline (D-10)

[0285] In a 25 mL reaction bottle, the intermediate 5-(1-([1,1′-biphenyl]-3-yl)ethyl)-3-(4-methyl-3-nitrophenyl)-1,2,4-oxadiazole (C-10, 550 mg, 1.4 mmol) was dissolved in 4 ml methanol and 4 ml dichloromethane, saturated aqueous ammonium chloride solution (7 mL) was added, stirred evenly, zinc powder (1800 mg, 28.0 mmol) was added, and stirred at room temperature for 3 hours. Filter, concentrate under reduced pressure, and separate by silica gel column chromatography (methanol / dichloromethane = 3 / 97) to obtain intermediate D-10 as an off-white solid 396 mg, with a yield of 73%.

[0286] 1 H NMR (400 MHz, CDCl 3 )δ7.58(d,J=6.4Hz,3H),7.54–7.48(m,1H),7.46–7.40(m,5H),7.37–7.33(m,2H) ,7.14(d,J=7.7Hz,1H),4.52(q,J=7.2Hz,1H),2.22(s,3H),1.86(d,J=7.2Hz,3H).

[0287] Step 3: Preparation of 5-(((5-(5-(1-([1,1′-biphenyl]-3-yl)ethyl)-1,2,4-oxadiazol-3-yl)-2-methylphenyl)amino)methyl)thiophene-2-carboxylic acid (Compound 22)

[0288] In a 25 mL reaction bottle, the intermediate 5-(5-(1-([1,1′-biphenyl]-3-yl)ethyl)-1,2,4-oxadiazol-3-yl)-2-methylaniline (D-10, 70 mg, 0.19 mmol), 5-formyl-2-thiophenecarboxylic acid (59 mg, 0.38 mmol), and glacial acetic acid (68 mg, 1.14 mmol) were dissolved in 1,2-dichloroethane (7 mL). After stirring at room temperature for 3 hours, sodium triacetoxyborohydride (242 mg, 1.14 mmol) was slowly added. The mixture was concentrated under reduced pressure and separated by silica gel column chromatography (methanol / dichloromethane = 10 / 90) to obtain compound I-22 (compound 22), 25 mg of off-white solid, and a yield of 26%.

[0289] 1 H NMR (400 MHz, DMSO-d 6 )δ12.91(brs,1H),7.66–7.55(m,5H),7.47(s,3H),7.39–7.33(m,2H),7.17(d,J=6.9Hz,2H),7.10– 7.08(m,2H),6.12(brs,1H),4.70(q,J=7.2Hz,1H),4.60(s,2H),2.20(s,3H),1.75(d,J=7.2Hz,3H).

[0290] Embodiment 23

[0291] 5-(((5-(5-(1-(4′-ethoxy-[1,1′-biphenyl]-3-yl)ethyl)-1,2,4-oxadiazol-3-yl)-2-methylphenyl)amino)methyl)thiophene-2-carboxylic acid

[0292]

[0293] Synthesis route:

[0294]

[0295] Experimental steps:

[0296] Step 1: Preparation of 5-(1-(4′-ethoxy-[1,1′-biphenyl]-3-yl)ethyl)-3-(4-methyl-3-nitrophenyl)-1,2,4-oxadiazole (C-11)

[0297] In a 100 mL reaction bottle, 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT, 595 mg, 3.4 mmol) and N-methylmorpholine (NMM, 1010 mg, 10.0 mmol) were dissolved in 1,4-dioxane (25 mL), stirred at room temperature for 15 minutes, 2-(4′-ethoxy-[1,1′-biphenyl]-3-yl)propionic acid (A-10, 710 mg, 3.4 mmol) was added, stirred at room temperature for 60 minutes, (Z)-N′-hydroxy-2-methyl-5-nitrobenzenecarboxamidine (B-1, 585 mg, 3.0 mmol) was added, stirred at room temperature for 2 hours, heated to reflux for 6 hours. Concentrated under reduced pressure, separated by silica gel column chromatography (ethyl acetate / petroleum ether=5 / 95), intermediate C-11 was obtained as a white solid (1017 mg), with a yield of 75%.

[0298] 1 H NMR (400 MHz, CDCl 3 )δ7.58–7.48(m,3H),7.47–7.37(m,4H),7.30(d,J=7.7Hz,1H),7.14(d,J=7.7Hz,1H),6.96(d,J=8.9Hz,2H ), 4.50 (q, J = 7.6Hz, 1H), 4.08 (q, J = 6.8Hz, 2H), 2.22 (s, 3H), 1.85 (d, J = 7.6Hz, 3H), 1.45 (t, J = 6.8Hz, 3H).

[0299] Step 2: Preparation of 5-(5-(1-(4′-ethoxy-[1,1′-biphenyl]-3-yl)ethyl)-1,2,4-oxadiazol-3-yl)-2-methylaniline (D-11)

[0300] In a 25 mL reaction bottle, the intermediate 5-(1-(4′-ethoxy-[1,1′-biphenyl]-3-yl)ethyl)-3-(4-methyl-3-nitrophenyl)-1,2,4-oxadiazole (C-11, 377 mg, 1.0 mmol) was dissolved in 5 ml methanol and 5 ml dichloromethane, saturated aqueous ammonium chloride solution (5 mL) was added, stirred evenly, zinc powder (1300 mg, 20.0 mmol) was added, and stirred at room temperature for 3 hours. Filter, concentrate under reduced pressure, and separate by silica gel column chromatography (methanol / dichloromethane = 5 / 95) to obtain intermediate D-11 as a white solid (275 mg) with a yield of 79%.

[0301] 1H NMR (400MHz, DMSO) δ7.61–7.52(m,4H),7.44–7.39(m,1H),7.31–7.27(m,2H),7.12–7.05(m,2H),7.02–6.99(m,2H) ,5.17(s,2H),4.69(t,J=7.6Hz,1H),4.05(t,J=7.2Hz,2H),2.10(s,3H),1.76(d,J=7.2Hz,3H),1.33(t,J=7.6,3H).

[0302] Step 3: Preparation of 5-(((5-(5-(1-(4′-ethoxy-[1,1′-biphenyl]-3-yl)ethyl)-1,2,4-oxadiazol-3-yl)-2-methylphenyl)amino)methyl)thiophene-2-carboxylic acid (Compound 23)

[0303] In a 25 mL reaction bottle, the intermediate 5-(5-(1-(4′-ethoxy-[1,1′-biphenyl]-3-yl)ethyl)-1,2,4-oxadiazol-3-yl)-2-methylaniline (D-11, 400 mg, 1.0 mmol), 5-formyl-2-thiophenecarboxylic acid (312 mg, 2.0 mmol), and glacial acetic acid (360 mg, 6.0 mmol) were dissolved in 1,2-dichloroethane (20 mL). After stirring at room temperature for 3 hours, sodium triacetoxyborohydride (1272 mg, 6.0 mmol) was slowly added. Concentrated under reduced pressure, separated by silica gel column chromatography (methanol / dichloromethane = 10 / 90), compound I-23 (compound 23) was obtained as a light yellow solid 210 mg, with a yield of 39%.

[0304] 1 H NMR (400 MHz, DMSO-d 6 )δ7.61–7.59(m,2H),7.56–7.52(m,3H),7.41(t,J=7.6Hz,1H),7.27(d,J=2.0Hz,1H),7.20–7.15(m,2H),7.06–7.08(m,2H),7.0(d,J=8.4H z,2H),6.12(brs,1H),4.66(q,J=7.2Hz,1H),4.60(s,2H),4.06(q,J=6.8Hz,2H),2.20(s,3H),1.74(d,J=7.2Hz,3H),1.34(t,J=6.8Hz,3H).

[0305] Embodiment 24

[0306] 5-(((2-methyl-5-(5-(3-(thiophen-2-yl)benzyl)-1,2,4-oxadiazol-3-yl)phenyl)amino)methyl)thiophene-2-carboxylic acid

[0307]

[0308] Synthesis route:

[0309]

[0310] Step 1: Preparation of 3-(4-methyl-3-nitrophenyl)-5-(3-(thiophen-2-yl)benzyl)-1,2,4-oxadiazole (C-12)

[0311] In a 100 mL reaction bottle, 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT, 119 mg, 0.68 mmol) and N-methylmorpholine (NMM, 206 mg, 2.04 mmol) were dissolved in 1,4-dioxane (15 mL), stirred at room temperature for 15 minutes, 2-(3-(thiophen-2-yl)phenyl)acetic acid (A-12, 149 mg, 0.68 mmol) was added, stirred at room temperature for 60 minutes, (Z)-N′-hydroxy-2-methyl-5-nitrobenzenecarboxamidine (B-1, 118 mg, 0.61 mmol) was added, stirred at room temperature for 2 hours, and heated to reflux for 6 hours. Concentrated under reduced pressure, separated by silica gel column chromatography (ethyl acetate / petroleum ether = 5 / 95), intermediate C-12 was obtained as a yellow liquid (215 mg), with a yield of 84%.

[0312] 1 H NMR (400 MHz, CDCl 3 )δ8.67(s,1H),8.18(d,J=8.0Hz,1H),7.62(s,1H),7.57(d,J=8.0Hz,1H),7.46(d,J=8.0Hz ,1H),7.39(t,J=7.6Hz,1H),7.34–7.29(m,3H),7.09–7.08(m,1H)4.34(s,2H),2.66(s,3H).

[0313] Step 2: Preparation of 2-methyl-5-(5-(3-(thiophen-2-yl)benzyl)-1,2,4-oxadiazol-3-yl)aniline (D-12)

[0314] In a 25 mL reaction bottle, the intermediate 3-(4-methyl-3-nitrophenyl)-5-(3-(thiophen-2-yl)benzyl)-1,2,4-oxadiazole (C-12, 377 mg, 1.0 mmol) was dissolved in 5 ml methanol and 5 ml dichloromethane, saturated aqueous ammonium chloride solution (5 mL) was added, stirred evenly, zinc powder (1300 mg, 20.0 mmol) was added, and stirred at room temperature for 3 hours. Filter, concentrate under reduced pressure, and separate by silica gel column chromatography (methanol / dichloromethane = 5 / 95) to obtain intermediate D-12 as a white solid 275 mg, with a yield of 79%.

[0315] 1 H NMR (400 MHz, CDCl 3 )δ7.62(s,1H),7.55(d,J=8.0Hz,1H),7.43–7.35(m,3H),7.32–7.26(m,3H ),7.14(d,J=7.6Hz,1H),7.08(t,J=4.4Hz,1H),4.31(s,2H),2.21(s,3H).

[0316] Step 3: Preparation of 5-(((2-methyl-5-(5-(3-(thiophen-2-yl)benzyl)-1,2,4-oxadiazol-3-yl)phenyl)amino)methyl)thiophene-2-carboxylic acid (Compound 24)

[0317] In a 25 mL reaction bottle, the intermediate 2-methyl-5-(5-(3-(thiophen-2-yl)benzyl)-1,2,4-oxadiazol-3-yl)aniline (D-12, 70 mg, 0.2 mmol), 5-formyl-2-thiophenecarboxylic acid (46 mg, 0.4 mmol), glacial acetic acid (72 mg, 1.2 mmol) were dissolved in 1,2-dichloroethane (7 mL), stirred at room temperature for 3 hours, and then sodium triacetoxyborohydride (253 mg, 1.2 mmol) was slowly added. Concentrated under reduced pressure, separated by silica gel column chromatography (methanol / dichloromethane = 10 / 90), compound I-24 (compound 24), 25 mg of white solid, yield 26%.

[0318] 1 H NMR (400 MHz, DMSO-d 6)δ12.90(brs,1H),7.69(s,1H),7.60(d,J=8.0Hz,1H),7.57–7.52(m,3H),7.41(t,J=7.6Hz,1H),7.30(d,J=8.0 Hz,1H),7.16–7.14(m,3H),7.09–7.07(m,2H),6.12(brs,1H),4.60(d,J=6.0Hz,2H),4.44(s,2H),2.20(s,3H).

[0319] Embodiment 25

[0320] 5-(((2-methyl-5-(5-(1-(3-(thiophen-2-yl)phenyl)ethyl)-1,2,4-oxadiazol-3-yl)phenyl)amino)methyl)thiophene-2-carboxylic acid

[0321]

[0322] Synthetic route:

[0323]

[0324] Experimental steps:

[0325] Step 1: Preparation of 3-(4-methyl-3-nitrophenyl)-5-(1-(3-(thiophen-2-yl)phenyl)ethyl)-1,2,4-oxadiazole (C-13)

[0326] In a 100 mL reaction bottle, 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT, 376 mg, 2.1 mmol) and N-methylmorpholine (NMM, 636 mg, 6.3 mmol) were dissolved in 1,4-dioxane (30 mL), stirred at room temperature for 15 minutes, 2-(3-(thiophen-2-yl)phenyl)propionic acid (A-12, 490 mg, 2.1 mmol) was added, stirred at room temperature for 60 minutes, (Z)-N′-hydroxy-4-methyl-3-nitrobenzamidamide (B-1, 370 mg, 1.89 mmol) was added, stirred at room temperature for 2 hours, and heated to reflux for 6 hours. Concentrated under reduced pressure, separated by silica gel column chromatography (ethyl acetate / petroleum ether=5 / 95), intermediate C-13 was obtained as a white solid (589 mg, yield 65%).

[0327] 1 H NMR (400 MHz, CDCl 3)δ8.65(s,1H),8.16(d,J=7.6Hz,1H),7.57(s,1H),7.50(d,J=7.6Hz,1H),7.42(d,J=8.0Hz,1H),7.33(t,J= 7.6Hz,1H),7.30–7.21(m,3H),7.04-7.03(m,1H),4.47(q,J=7.2Hz,1H),2.62(s,3H),1.82(d,J=7.2Hz,3H).

[0328] Step 2: Preparation of 2-methyl-5-(5-(1-(3-(thiophen-2-yl)phenyl)ethyl)-1,2,4-oxadiazol-3-yl)aniline (D-13)

[0329] In a 25 mL reaction bottle, the intermediate 3-(4-methyl-3-nitrophenyl)-5-(1-(3-(thiophen-2-yl)phenyl)ethyl)-1,2,4-oxadiazole (C-13, 440 mg, 1.1 mmol) was dissolved in 3 ml of methanol and 3 ml of dichloromethane, and a saturated aqueous ammonium chloride solution (6 mL) was added, stirred evenly, and zinc powder (1350 mg, 22.5 mmol) was added, and stirred at room temperature for 3 hours. Filter, concentrate under reduced pressure, and separate by silica gel column chromatography (methanol / dichloromethane = 3 / 97) to obtain intermediate D-13 as an off-white solid 371 mg, with a yield of 93%.

[0330] 1 H NMR (400 MHz, CDCl 3 )δ7.60(s,1H),7.53(d,J=8.0Hz,1H),7.45–7.41(m,2H),7.35(t,J=7.6Hz,1H),7.32–7.23(m,4H),7. 14(d,J=7.6Hz,1H),7.07(t,J=4.4Hz,1H),4.48(q,J=7.2Hz,1H),2.22(s,3H),1.84(d,J=7.2Hz,3H).

[0331] Step 3: Preparation of 5-(((2-methyl-5-(5-(1-(3-(thiophen-2-yl)phenyl)ethyl)-1,2,4-oxadiazol-3-yl)phenyl)amino)methyl)thiophene-2-carboxylic acid (Compound 25)

[0332] In a 25 mL reaction bottle, the intermediate 2-methyl-5-(5-(1-(3-(thiophen-2-yl)phenyl)ethyl)-1,2,4-oxadiazol-3-yl)aniline (D-13, 100 mg, 0.27 mmol), 5-formyl-2-thiophenecarboxylic acid (63 mg, 0.55 mmol), and glacial acetic acid (100 mg, 1.66 mmol) were dissolved in 1,2-dichloroethane (8 mL). After stirring at room temperature for 3 hours, sodium triacetoxyborohydride (351 mg, 1.66 mmol) was slowly added. Concentrated under reduced pressure, separated by silica gel column chromatography (methanol / dichloromethane = 10 / 90), compound I-25 (compound 25) was obtained as an off-white solid 14 mg, yield 10%.

[0333] 1 H NMR (400 MHz, DMSO-d 6 )δ12.89(brs,1H),7.67(s,1H),7.59–7.53(m,4H),7.41(t,J=7.7Hz,1H),7.28(d,J=7.7Hz,1H),7.21–7.14(m,3H), 7.11–7.09(m,2H),6.13(brs,1H),4.67(q,J=7.2Hz,1H),4.61(d,J=5.7Hz,2H),2.21(s,3H),1.73(d,J=7.2Hz,3H).

[0334] Biological activity test

[0335] 1. In vitro protease inhibition activity test

[0336] Determination method: Fluorescence method was used to determine the half maximal inhibitory concentration (IC) of the compound against SARS-CoV-2 PLpro protease. 50 ).

[0337] Experimental principle: The characteristic substrate of PLpro protease is labeled with fluorescence quenching. If the substrate is digested, it will emit characteristic fluorescence, and if it is not digested, it will not emit fluorescence. The digestion rate of the substrate by the protease is detected by measuring the fluorescence value at different inhibitor concentrations, and the half-inhibitory concentration value (IC50) of the compound against PLpro is obtained according to the digestion reaction rate at different inhibitor concentrations. 50 ).

[0338] Experimental methods:

[0339] (1) Use DMSO to dilute the compound in series.

[0340] (2) Add reaction buffer, test compounds of different concentrations and PLpro protease in a 96-well transparent-bottom black plate (Greiner, 655096) according to the reaction system.

[0341] (3) Place the reaction plate in a 37°C incubator for 8 to 10 minutes to allow the enzymatic reaction to proceed, then add the fluorescent substrate.

[0342] (4) Use an enzyme-labeled instrument to detect fluorescence intensity in real time.

[0343] (5) The data obtained from the test were fitted using Origin and Graphpad to obtain the IC 50 Numeric value.

[0344] Experimental results:

[0345] Table 1. Results of the inhibitory effects of the compounds of the present invention on PLpro

[0346]

[0347] From the data in Table 1, it can be seen that the compounds of the present invention have good PLpro inhibitory effects, among which 8 compounds have PLpro inhibitory activity IC 50 <10 μM.

[0348] 2. Cytotoxicity assay

[0349] Determination method: MTT method Experimental principle: Cell activity is determined by the reduction of oxidized 3-(4,5-dimethylthiazo-2-yl)-2,5-diphenyl tetrazolium bromide (trade name: Thiazole Blue) / MTT [3-(4,5-dimethylthiazo-2-yl)-2,5-diphenyl tetrazolium bromide] to an insoluble blue formazan compound through mitochondrial dehydrogenase (such as succinate dehydrogenase), which is then dissolved in DMSO and then colored for determination. The conversion amount is positively correlated with the number of living cells.

[0350] Experimental methods:

[0351] (1) Digest Vero E6 cells that have been cultured to the logarithmic growth phase with 0.25% trypsin for 2 to 3 minutes, discard the digestion solution, add an appropriate amount of culture medium, mix well, take 20 μL, count under a microscope using a hemocytometer, and prepare a cell suspension of appropriate concentration for later use. At the same time, use PBS (phosphate buffered solution) to prepare

[0352] 5g / L MTT solution was filtered and sterilized for later use.

[0353] (2) The test drug was dissolved in DMSO and diluted 50-fold with culture medium to make the highest test concentration. The test drug was then serially diluted with culture medium at a ratio of 1:2 on a 96-well plate. Eight concentrations were set for each compound, with the highest concentration being 200 μM. Four parallel wells were set for each concentration, with 100 μL / well.

[0354] (3) The prepared cell suspension was inoculated into a 96-well plate at 100 μL / well with a cell concentration of 4×10 5 cell / mL. At the same time, set up cell control wells without drug and culture medium blank control wells.

[0355] (4) After 48 hours of culture, add 10 μL / well of MTT and continue culturing for 4 hours. Remove the culture plate, carefully discard the culture medium in the wells, add 100 μL of DMSO to each well, shake until the formazan particles are completely dissolved, and measure the optical density (OD) at a wavelength of 570 nm using an enzyme-linked immunosorbent assay. 570 ).

[0356] (5) Data processing: Cell survival percentage (%) = [(cell control OD 570 Value - OD of drug-added group 570 value) / (cell control OD 570 Value - Blank OD 570 The dose-response curve was fitted using Origin software to calculate the concentration of each compound at which 50% of the cells survived (CC 50 ).

[0357] Experimental results:

[0358] Table 2. Cytotoxicity results of the compounds of the present invention

[0359]

[0360] It can be seen from the data in Table 2 that the compounds of the present invention have low cytotoxicity and exhibit high safety.

[0361] 3. Antiviral activity detection

[0362] Determination method: Fluorescence quantitative PCR method to detect the total amount of RNA in the supernatant of cell culture medium Experimental principle: During PCR amplification, a specific fluorescent probe is added at the same time as a pair of primers. The probe is an oligonucleotide with a reporter fluorescent group and a quencher fluorescent group labeled at both ends. When the probe is intact, the fluorescent signal emitted by the reporter group is absorbed by the quencher group; at the beginning, the probe binds to any single strand of DNA. During PCR amplification, the 5'-3' exonuclease activity of the Taq enzyme will enzymatically degrade the probe, separating the reporter fluorescent group and the quencher fluorescent group, so that the fluorescent monitoring system can receive the fluorescent signal, that is, for each DNA chain amplified, a fluorescent molecule is formed, achieving complete synchronization between the accumulation of the fluorescent signal and the formation of the PCR product.

[0363] Experimental methods:

[0364] (1) Vero E6 cells that have been cultured to the logarithmic growth phase are digested with 0.25% trypsin for 2 to 3 minutes, the digestion solution is discarded, an appropriate amount of culture medium is added, mixed, and 20 μL is taken out for counting under a microscope using a hemacytometer to prepare a cell suspension of appropriate concentration for later use.

[0365] (2) The test drug was dissolved in DMSO and diluted 50-fold with culture medium to make the highest test concentration. The test drug was then serially diluted with culture medium at a ratio of 1:2 on a 96-well plate. Eight concentrations were set for each compound, with the highest concentration being 200 μM. Four parallel wells were set for each concentration, with 100 μL / well.

[0366] (3) The prepared cell suspension was inoculated into a 96-well plate at 100 μL / well with a cell concentration of 4×10 5 cell / mL.

[0367] (4) After incubating cells with different dilutions of drug working solution (200ul) for 1h, 100TCID 50 The coronavirus was incubated for 2 h.

[0368] (5) Remove the cell culture medium from the 96-well culture plate and add different concentrations of compound dilutions, 200 μL per well, and 3 replicates per concentration. At the same time, set up drug-free cell control wells, culture medium blank control wells, solvent control wells, and virus control wells.

[0369] (6) After culturing for 48 hours, the culture supernatant was added to AVL lysis buffer and repeatedly pipetted for 15 seconds to ensure sufficient lysis. RNA was then extracted from the cell culture supernatant according to the RNA extraction kit procedure.

[0370] (7) After the extracted RNA, fluorescent probe and polymerase are evenly mixed, the RNA in the supernatant is quantitatively detected using the QPCR method.

[0371] (8) Data processing: Cell antiviral activity = [(Ct value of drug-treated group - Ct value of virus control group / (Ct value of cell control group - Ct value of virus control group)] × 100%. GraphPad software was used to fit the dose-response curve and calculate the concentration of each compound at which the antiviral activity reached 50% (EC 50 ).

[0372] Experimental results:

[0373] Table 3. Antiviral test results of compounds

[0374]

[0375] It can be seen from the data in Table 3 that the antiviral activity of compounds 18 and 19 of the present invention is better than that of GRL0617 with a similar mechanism of action, and exhibits a stronger anti-coronavirus effect.

Claims

1. A compound represented by formula (I) and its isomers, or a pharmaceutically acceptable salt thereof: in, R1 is hydrogen, methyl, ethyl, propyl, isopropyl, or hydroxyl; R2 is a substituted or unsubstituted phenyl group, a substituted or unsubstituted biphenyl group, a substituted or unsubstituted diphenyl ether group, or a substituted or unsubstituted carbazole group; R3 is independently selected from hydrogen, F, Cl, Br, trifluoromethyl, trifluoromethoxy, C1-C3 alkyl, C1-C3 alkoxy, and n represents 1, 2, 3 or 4 substituents; R4 is a substituted or unsubstituted phenyl group, a substituted or unsubstituted thienyl group; The substituents in R2 or R4 may be selected from the following groups: F, Cl, Br, hydroxyl, amino, nitro, cyano, monofluoromethyl, difluoromethyl, trifluoromethyl, trifluoromethoxy, thienyl, vinyl, ethynyl, hydroxymethyl, carboxyl, sulfonic acid, boric acid, methylthio, C1-C4 alkyl, C1-C4 alkoxy.

2. The compound according to claim 1 and its isomers, or pharmaceutically acceptable salts thereof, wherein the compound is represented by general formula (II): in, R1 is hydrogen, methyl, or hydroxyl; R2 is a substituted or unsubstituted phenyl group, a substituted or unsubstituted biphenyl group, a substituted or unsubstituted diphenyl ether group, or a substituted or unsubstituted carbazole group; R3 is independently selected from hydrogen, F, Cl, Br, trifluoromethyl, trifluoromethoxy, C1-C3 alkyl, C1-C3 alkoxy, and n represents 1, 2, 3 or 4 substituents; R5 is hydrogen, cyano, carboxyl, trifluoromethyl, methoxy, morpholinyl, ethynyl, acetylamino, acetyl, hydroxyl, hydroxymethyl; The substituents in R2 may be selected from the following groups: F, Cl, Br, hydroxyl, amino, nitro, cyano, monofluoromethyl, difluoromethyl, trifluoromethyl, trifluoromethoxy, hydroxymethyl, C1-C4 alkyl, C1-C4 alkoxy.

3. The compound according to claim 1 and its isomers, or pharmaceutically acceptable salts thereof, wherein the compound is represented by the general formula (III): in, R1 is hydrogen, methyl, or hydroxyl; R2 is a substituted or unsubstituted phenyl group, a substituted or unsubstituted biphenyl group, a substituted or unsubstituted diphenyl ether group, or a substituted or unsubstituted carbazole group; R3 is independently selected from hydrogen, F, Cl, Br, trifluoromethyl, trifluoromethoxy, C1-C3 alkyl, C1-C3 alkoxy, and n represents 1, 2, 3 or 4 substituents; R5 is hydrogen, carboxyl, cyano, or boric acid; The substituents in R2 may be selected from the following groups: F, Cl, Br, hydroxyl, amino, nitro, cyano, monofluoromethyl, difluoromethyl, trifluoromethyl, trifluoromethoxy, thienyl, hydroxymethyl, C1-C4 alkyl, C1-C4 alkoxy.

4. The compound according to any one of claims 1 to 3 and its isomer, or a pharmaceutically acceptable salt thereof, which is selected from the following compounds:

5. A method for preparing a compound according to any one of claims 1 to 3, comprising the following steps: Compound A and compound B are condensed and cyclized to obtain compound C, which is then reduced to obtain compound D. Compound D and an aldehyde substituted by R4 are reduced by amination to obtain a compound represented by formula (I); Wherein, R1, R2, R3, R4, and n are defined as any one of claims 1-3.

6. Use of the compound according to any one of claims 1 to 4 and its isomers, or pharmaceutically acceptable salts thereof, in the preparation of papain-like protease (PLpro) inhibitors.

7. A pharmaceutical composition, characterized in that The composition comprises a therapeutically and / or preventively effective amount of the compound and its isomers according to any one of claims 1 to 4, or a pharmaceutically acceptable salt thereof, and optionally one or more pharmaceutically acceptable excipients.

8. Use of the compound according to any one of claims 1 to 4 and its isomers, or a pharmaceutically acceptable salt thereof, or the composition according to claim 7 in the preparation of a medicament for treating and / or preventing an infectious disease caused by a coronavirus.

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