Substituted oxadiazole-anilines and uses thereof

Abstract

The application belongs to the technical field of medicine, and discloses a substituted oxadiazole-aniline compound, a preparation method thereof, a pharmaceutical composition taking the compound as an active ingredient, and application of the compound in treatment and / or prevention of infectious diseases caused by a coronavirus. Specifically, the application relates to a compound shown in formula (I) and isomers thereof, or a pharmaceutically acceptable salt thereof and a pharmaceutical composition containing the compound, wherein R1, R2, R3, R4 and n are as described in the specification. The application aims to prepare a new compound with anti-coronavirus activity, which can be used as a potential new drug for treatment or preventive treatment of infectious diseases caused by a virus, in particular, pneumonia diseases caused by a coronavirus.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medicine. In particular, it relates to substituted oxadiazole-aniline compounds represented by general formula (I), a preparation method thereof, a pharmaceutical composition taking the compound as an active ingredient, and the application thereof in the treatment and / or prevention of infectious diseases caused by coronavirus (SARS-CoV), especially the novel coronavirus (SARS-CoV-2). BACKGROUND

[0002] Viral infection has been a constant threat to human life and health. At present, there is still a lack of effective antiviral drugs for most viruses. Non-structural proteins (replicases such as proteases, polymerases and helicases) involved in viral replication are still highly conserved. Inhibitors targeting highly conserved viral replicases are more suitable for the development of broad-spectrum antiviral drugs.

[0003] According to the structure of coronavirus, it is of great significance to find a targeted, efficient and low-toxic drug for treating the virus. SARS-CoV-2 encodes 16 non-structural proteins. With the protein structure being analyzed one by one, the drug development technology based on the structure of the druggable target of coronavirus has been widely adopted, among which the successful listing of the inhibitor Paxlovid targeting Mpro is representative. Papain-like protease (PLpro) encoded by SARS-CoV-2 is a catalytic domain of NSP3 protein, which is responsible for the maturation cleavage of viral precursor protein together with Mpro, and is an essential protein for viral replication in cells. In addition, PLpro also mediates the deubiquitination and deISGylation 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 target for anti-SARS-CoV-2 drugs. GRL0617 is a non-covalent binding inhibitor discovered after screening against SARS-CoV PLpro, which has high inhibitory activity and target enzyme affinity. At present, 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 lay a foundation for the development of PLpro inhibitors. SUMMARY

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

[0006] To this end, the present application provides a compound represented by the general formula (I) and isomers thereof, or a pharmaceutically acceptable salt thereof,

[0007]

[0008] wherein,

[0009] R1 is hydrogen, methyl, ethyl, propyl, isopropyl, hydroxyl;

[0010] R2 is substituted or unsubstituted phenyl, substituted or unsubstituted biphenyl, substituted or unsubstituted diphenyl ether, substituted or unsubstituted carbazolyl;

[0011] 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;

[0012] R4 is substituted or unsubstituted phenyl, substituted or unsubstituted thienyl;

[0013] The substituents in R2 or R4 can be optionally selected from the following groups: F, Cl, Br, hydroxyl, amino, nitro, cyano, monofluoromethyl, difluoromethyl, trifluoromethyl, trifluoromethoxy, thienyl, vinyl, ethynyl, hydroxymethyl, carboxyl, sulfonic acid, boronic acid, methylthio, C1-C4 alkyl, C1-C4 alkoxy.

[0014] In a preferred embodiment, the compound is represented by the general formula (II):

[0015]

[0016] wherein,

[0017] R1 is hydrogen, methyl, hydroxyl;

[0018] R2 is substituted or unsubstituted phenyl, substituted or unsubstituted biphenyl, substituted or unsubstituted diphenyl ether, substituted or unsubstituted carbazolyl;

[0019] 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;

[0020] R5 is hydrogen, cyano, carboxyl, trifluoromethyl, methoxy, morpholinyl, alkynyl, acetylamino, acetyl, hydroxyl, hydroxymethyl;

[0021] The substituents in R2may be optionally selected from the group consisting of F, Cl, Br, hydroxy, amino, nitro, cyano, monofluoromethyl, difluoromethyl, trifluoromethyl, trifluoromethoxy, thienyl, hydroxymethyl, C1-C4alkyl, C1-C4alkoxy.

[0022] In another preferred embodiment, the compound has the structure of formula (III):

[0023]

[0024] wherein,

[0025] R1is hydrogen, methyl, hydroxy;

[0026] R2is substituted or unsubstituted phenyl, substituted or unsubstituted biphenyl, substituted or unsubstituted diphenyl ether, substituted or unsubstituted carbazolyl;

[0027] R3is independently selected from hydrogen, F, Cl, Br, trifluoromethyl, trifluoromethoxy, C1-C3alkyl, C1-C3alkoxy, and n represents 1, 2, 3 or 4 substituents;

[0028] R5is hydrogen, carboxyl, cyano, boronic acid group;

[0029] The substituents in R2may be optionally selected from the group consisting of F, Cl, Br, hydroxy, amino, nitro, cyano, monofluoromethyl, difluoromethyl, trifluoromethyl, trifluoromethoxy, thienyl, hydroxymethyl, C1-C4alkyl, C1-C4alkoxy.

[0030] The pharmaceutically acceptable salt in the present application is a salt of the compound of the present application with 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 and trifluoroacetic acid. Preferably, it is hydrochloric acid, p-toluenesulfonic acid or trifluoroacetic acid.

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

[0032] The compound according to any one of the first aspect of the present application is a compound selected from the group consisting of:

[0033]

[0034]

[0035]

[0036] The second aspect of the present application provides a method for preparing the compound according to any one of the first aspect of the present application, which comprises the following steps:

[0037]

[0038] Compound A is reacted with compound B in a suitable solvent (e.g. dichloromethane, tetrahydrofuran, acetonitrile, 1,4-dioxane, preferably 1,4-dioxane) in the presence of reagent C DMT and NMM under air or inert gas (Ar or N2) protection at -10°C-50°C for 1-24 hours, preferably at room temperature for 2-3 hours, followed by heating to reflux for 1-24 hours, preferably for 6-12 hours, to obtain a compound of formula C;

[0039] The compound of formula C is reacted with ammonium chloride and metal powder (zinc powder, iron powder, preferably zinc powder) in a suitable solvent (e.g. methanol, ethanol, methanol / water, ethanol / water, preferably methanol / water) at -10°C-60°C for 1-24 hours, preferably at room temperature for 2-4 hours, to obtain a compound of formula D;

[0040] The compound of formula D is reacted with compound R4CHO in a suitable solvent (e.g. dichloromethane, tetrahydrofuran, acetonitrile, 1,2-dichloroethane, preferably 1,2-dichloroethane) under acidic conditions (e.g. formic acid, acetic acid, preferably acetic acid) and a reducing agent (e.g. sodium borohydride, sodium cyanoborohydride, sodium triacetoxyborohydride, preferably sodium triacetoxyborohydride) at -10°C-50°C for 1-24 hours, preferably at room temperature for 6-12 hours, to obtain a compound of formula (I).

[0041] The third aspect of the present application provides use of the compound of any one of the first aspect of the present application and isomers thereof, or a pharmaceutically acceptable salt thereof, in the preparation of a papain-like protease (PLpro) inhibitor.

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

[0043] The fifth aspect of the present application provides use of the compound of any one of the first aspect of the present application and isomers thereof, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of any one of the fourth aspect of the present application in the preparation of a drug for treating and / or preventing an infectious disease caused by a coronavirus.

[0044] The foregoing outlines certain aspects of the present application, but is not limited to such aspects. These aspects and other aspects will become more fully apparent from the following description. DETAILED DESCRIPTION

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

[0047] Various terms and phrases used herein have their ordinary meaning within the technical field and art, and the present application can use these terms and phrases consistent with their ordinary meanings unless otherwise expressly set forth herein. However, the present application still seeks to define and explain these terms and phrases in further detail and in a manner that is consistent with the present application, and in the event of a conflict between the ordinary meaning and the meaning expressed herein, the meaning expressed herein shall control. The following are definitions of various terms used herein, which are applicable to the terms as used throughout this specification, unless otherwise indicated.

[0048] Generally, the term "substituted or unsubstituted" means that one or more hydrogen atoms of the given structure are replaced with a particular substituent. Unless otherwise indicated, an optional substituent group can be substituted at any available position on the group. When the given structure can be substituted at more than one position with one or more substituents selected from a particular group, the substituents can be the same or different at each position.

[0049] C i -C j represents a moiety having an integer "i" (inclusive) to an integer "j" (inclusive) number of carbon atoms. Thus, for example, C1-C3 alkyl refers to an alkyl group having from 1 to 3 (inclusive) carbon atoms.

[0050] As used herein, the term "alkyl" refers to an alkyl group having the indicated number of carbon atoms, which is straight-chained or branched, and which can include subgroups thereof, e.g., where "C1-C3 alkyl" is mentioned, it can also include groups represented by the sub-range of C1-C2 alkyl, as well as specific groups such as methyl, ethyl, n-propyl, isopropyl.

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

[0052] As used herein, the term "haloalkyl" refers to an alkyl group in which the hydrogens have been replaced by one or more halogen atoms, examples of which include, but are not limited to, monofluoromethyl, monofluoromethoxy, and the like.

[0053] As used herein, the term "cyclo" refers to a substituted or unsubstituted cycloalkyl group. By cyclo is meant a fused ring. The number of atoms in the ring is generally defined as the number of members of the ring, e.g., "C3-C6 cyclo" refers to a ring arranged around 3-6 atoms.

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

[0055] As used herein, "room temperature" means a temperature from 10 °C to 40 °C. In some embodiments, "room temperature" means a temperature from 20 °C to 30 °C; in other embodiments, room temperature means 25 °C.

[0056] As used herein, the term "effective amount" means an amount of a drug that is effective in achieving the desired treatment of a disease or condition described herein in a subject.

[0057] As used herein, the term "pharmaceutically acceptable" such as in the description of "pharmaceutically acceptable salts" means that the salts are physiologically acceptable as well as commensurate with a reasonable benefit / risk ratio pertinent to use in pharmaceuticals.

[0058] As used herein, the term "pharmaceutical composition", which can also be referred to as "composition", is intended to encompass a product comprising specified ingredients in predetermined amounts.

[0059] "Treating" or "treatment" of a disease includes:

[0060] (1) preventing the disease, i.e., causing the clinical symptoms of the disease not to develop in a mammal that is exposed to or predisposed to the disease but does not yet experience or display symptoms of the disease,

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

[0062] (3) relieving the disease, i.e., causing the regression of the disease or its clinical symptoms.

[0063] "Therapeutically effective amount" means the amount of a compound that, when administered to a mammal for treatment of a disease, is sufficient to effect treatment for that disease. The therapeutically effective amount will vary depending on the compound, the disease to be treated, and its severity, and the age, the body weight, the sex of the mammal, and the like. A therapeutically effective amount can also mean any amount of a compound that is sufficient to achieve a desired beneficial effect, including preventing the disease, inhibiting the disease, or relieving the disease, as described in (1)-(3) above. For example, the amount of a compound can 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 used herein, the term "disease and / or condition" means a physical state of the subject that is associated with a disease and / or condition described herein. For example, the disease and / or condition described herein refers to a Mycobacterium tuberculosis infectious disease.

[0065] As used herein, the term "subject" can refer to a patient or other animal, particularly a mammal, such as a human, monkey, etc., who receives a compound of Formula I or a pharmaceutical composition thereof of the present application for treatment of a disease or disorder described herein.

[0066] In another aspect, the present application also relates to a pharmaceutical composition comprising a compound of the present application as an active ingredient. The pharmaceutical composition can be prepared according to methods known in the art. The compound of the present application or a pharmaceutical composition comprising it can be administered in unit dosage form, and can be administered enterally or parenterally, such as orally, intravenously, intramuscularly, subcutaneously, nasally, buccally, ocularly, pulmonarily, dermally, vaginally, rectally, etc.

[0067] The compound of the present application or a pharmaceutical composition comprising it can be administered in unit dosage form, and can be administered enterally or parenterally, such as orally, intravenously, intramuscularly, subcutaneously, nasally, buccally, ocularly, pulmonarily, dermally, vaginally, rectally, etc.

[0068] The dosage form can be a liquid dosage form, a solid dosage form or a semi-solid dosage form. The liquid dosage form can be a solution (including true solution and colloidal solution), an emulsion (including o / w type, w / o type and multiple emulsion), a suspension, an injection (including water injection, powder injection and infusion), eye drops, nose drops, lotion and liniment, etc.; the solid dosage form can be a tablet (including ordinary tablet, enteric tablet, buccal tablet, dispersible tablet, chewable tablet, effervescent tablet, oral disintegrating tablet), a capsule (including hard capsule, soft capsule, enteric capsule), a granule, a powder, a pellet, a drop, a suppository, a film, a patch, an aerosol (powder) mist, a spray, etc.; the semi-solid dosage form can be an ointment, a gel, a paste, etc.

[0069] The compound of the present application can be prepared into a common preparation, a sustained-release preparation, a controlled-release preparation, a targeted preparation and various microparticle drug delivery systems.

[0070] To make tablets of the compounds of the present application, various excipients known in the art can be widely used, including diluents, binders, wetting agents, disintegrants, lubricants, solubilizers. The diluents can be starch, dextrin, sucrose, glucose, lactose, mannitol, sorbitol, xylitol, microcrystalline cellulose, calcium sulfate, calcium hydrogen phosphate, calcium carbonate, etc.; the wetting agents can be water, ethanol, isopropyl alcohol, etc.; the binders can be starch paste, dextrin, sugar syrup, honey, glucose solution, microcrystalline cellulose, acacia paste, gelatin paste, sodium carboxymethyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, ethyl cellulose, acrylic acid resin, carbomer, polyvinyl pyrrolidone, polyethylene glycol, etc.; the disintegrants can 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 sorbitol fatty acid ester, sodium dodecylsulfate, etc.; the lubricants and solubilizers can be talc, silicon dioxide, stearate, tartaric acid, liquid paraffin, polyethylene glycol, etc.

[0071] The tablets can also 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] To make capsules of the administration units, the effective ingredient, the compounds of the present application, can be mixed with diluents and solubilizers, and the mixture can be directly put into hard capsules or soft capsules. The effective ingredient, the compounds of the present application, can also be first mixed with diluents, binders and disintegrants to make granules or pellets, and then put into hard capsules or soft capsules. The various diluents, binders, wetting agents, disintegrants and solubilizers used for making tablets of the compounds of the present application can also be used for making capsules of the compounds of the present application.

[0073] To make injections of the compounds of the present application, water, ethanol, isopropyl alcohol, propylene glycol or their mixtures can be used as solvents, and appropriate amounts of solubilizers, solubilizers, pH adjustors and osmotic pressure adjustors commonly used in the art can be added. The solubilizers or solubilizers can be poloxamer, lecithin, hydroxypropyl-β-cyclodextrin, etc.; the pH adjustors can be phosphate, acetate, hydrochloric acid, sodium hydroxide, etc.; the osmotic pressure adjustors can be sodium chloride, mannitol, glucose, phosphate, acetate, etc. If lyophilized powder injections are prepared, mannitol, glucose, etc. can also be added as supporting agents.

[0074] In addition, if necessary, coloring agents, preservatives, flavors, flavoring agents or other additives can also be added to the pharmaceutical preparations.

[0075] To achieve the purpose of medication and enhance the therapeutic effect, the drugs or pharmaceutical compositions of the present application can be administered by any known administration method.

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

[0077] Beneficial technical effects

[0078] The compounds in this invention exhibit strong PLpro inhibitory activity, with 11 compounds showing inhibitory effects (IC50, 100 mg / L). 50 The PLpro inhibitory activity of 8 compounds was less than 30 μM, with an IC50 value. 50 <10 μM. The two compounds exhibited superior antiviral activity compared to the PLpro inhibitor GRL0617, demonstrating strong anti-coronavirus activity. Furthermore, these compounds showed low cytotoxicity to Vero E6 cells (IC50). 50 The concentrations (>100 μM) indicate that these compounds have good safety profiles. This invention provides a novel class of compounds with strong in vitro PLpro inhibitory activity and low cytotoxicity, which can be used in drugs that inhibit PLpro activity, particularly as therapeutic agents for conditions related to coronavirus infection. Detailed Implementation

[0079] The present invention will be described in detail through the following embodiments, but this does not imply any adverse limitation on the invention. The present invention has been described in detail herein, and specific embodiments thereof have also been disclosed. It will be 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 thereof.

[0080] For all the following embodiments, standard operating and purification methods known to those skilled in the art were used. The structure of the compound was determined by proton nuclear magnetic resonance spectroscopy (NMR). 1 It was determined by H NMR.

[0081] Preparation examples section

[0082] The structure of the compound was determined by nuclear magnetic resonance (NMR) hydrogen spectrum (1H NMR). 1 The NMR spectrum was determined by 1H NMR. The 1H NMR shift (δ) is given in parts per million (ppm). The coupling constant (J) is in Hertz (Hz). The NMR spectra were measured using a 400 Hz, 500 Hz, or 700 Hz NMR spectra, with deuterated dimethyl sulfoxide (DMSO-d6) or deuterated chloroform (CDCl3) as solvents and tetramethylsilane (TMS) as an 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)anilino)methyl)phenoxy)acetamide

[0085]

[0086] Synthesis route:

[0087]

[0088] Experimental procedure:

[0089] First step: 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 flask, 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT, 5250 mg, 30.0 mmol), 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)propanoic acid (A-1, 7320 mg, 30.0 mmol) was added, stirred at room temperature for 60 minutes, (Z)-N'-hydroxy-4-methyl-5-nitrobenzimidamide (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) to give intermediate C-1, white solid 7.39 g, yield 61%.

[0091] 1 H NMR (400 MHz, DMSO-d6) δ 8.51 (s, 1H), 8.22 (d, J = 8.0 Hz, 1H), 7.71 (d, J = 8.1 Hz, 1H), 7.54 (d, J = 7.8 Hz, 3H), 7.47 (t, J = 7.6 Hz, 2H), 7.44 - 7.37 (m, 2H), 7.33 (d, J = 8.0 Hz, 1H), 4.80 (q, J = 7.2 Hz, 1H), 2.60 (s, 3H), 1.78 (d, J = 6.8 Hz, 3H).

[0092] Second step: 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 vial, 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, then slowly added sodium triacetoxyborohydride (267 mg, 1.26 mmol). Concentrated under reduced pressure, separated by silica gel column chromatography (methanol / dichloromethane = 10 / 90) to obtain compound I-1 (compound 1), white solid 53 mg, yield 47%.

[0094] 1 H NMR (400 MHz, CDC13) δ 7.53 (d, J = 9.2 Hz, 2H), 7.46 - 7.34 (m, 4H), 7.39 - 7.34 (m, 2H), 7.23 - 7.15 (m, 3H), 4.49 (q, J = 7.2 Hz, 1H), 2.24 (s, 3H), 1.84 (d, J = 7.2 Hz, 3H).

[0095] Third step: 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)phenyl)amino)methyl)benzamide (compound 1)

[0096] In a 25 mL reaction vial, 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, then slowly added sodium triacetoxyborohydride (267 mg, 1.26 mmol). Concentrated under reduced pressure, separated by silica gel column chromatography (methanol / dichloromethane = 10 / 90) to obtain compound I-1 (compound 1), white solid 53 mg, yield 47%.

[0097] 1 H NMR (400 MHz, CDC13) δ 7.53 (d, J = 9.2 Hz, 2H), 7.46 - 7.34 (m, 4H), 7.39 - 7.34 (m, 2H), 7.23 - 7.15 (m, 3H), 4.49 (q, J = 7.2 Hz, 1H), 2.24 (s, 3H), 1.84 (d, J = 7.2 Hz, 3H).

[0098] Example 2

[0099] N-(3-Fluoro-benzyl)-2-methyl-5-(((5-(5-(1-(2-fluoro-[1,1'-biphenyl]-4-yl)ethyl)-1,2,4-oxadiazol-3-yl)phenyl)amino)methyl)benzamide

[0100]

[0101] Using 3-methoxybenzaldehyde (57 mg, 0.42 mmol) as the starting material, a similar procedure to the third step in Example 1 was used to react with intermediate D-1 to give compound I-2 (compound 2) as a white-like solid 58 mg in 56% yield.

[0102] 1 H NMR (400 MHz, CDC13) δ 7.55 - 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 = 8.3 Hz, 1H), 4.48 (q, J = 7.2 Hz, 1H), 4.42 (s, 2H), 3.80 (d, J = 1.6 Hz, 3H), 2.20 (s, 3H), 1.83 (d, J = 7.3 Hz, 3H).

[0103] Example 3

[0104] N-(3-Fluoro-benzyl)-2-methyl-5-(((5-(5-(1-(2-fluoro-[1,1'-biphenyl]-4-yl)ethyl)-1,2,4-oxadiazol-3-yl)phenyl)amino)methyl)benzamide

[0105]

[0106] Using 4-(4-morpholinyl)benzaldehyde (80 mg, 0.42 mmol) as the starting material, a similar procedure to the third step in Example 1 was used to react with intermediate D-1 to give compound I-3 (compound 3) as a white-like solid 66 mg in 64% yield.

[0107] 1H NMR (400 MHz, CDC13) δ 7.56 - 7.50 (m, 2 H), 7.48 - 7.39 (m, 4 H), 7.39 - 7.30 (m, 4 H), 7.25 - 7.20 (m, 2 H), 7.20 - 7.13 (m, 1 H), 6.93 (s, 2 H), 4.49 (q, J = 7.7 Hz, 1 H), 4.35 (s, 2 H), 3.87 (s, 5 H), 3.17 (s, 4 H), 2.18 (s, 3 H), 1.84 (d, J = 7.1 Hz, 3 H).

[0108] Example 4

[0109] N-(4-ethynyl-benzyl)-2-methyl-5-(((5-(5-(1-(2-fluoro-[1,1'-biphenyl]-4-yl)ethyl)-1,2,4-oxadiazol-3-yl)phenyl)amino)methyl)benzamide

[0110]

[0111] Using 4-ethynylbenzaldehyde (55 mg, 0.42 mmol) as the starting material, a similar procedure to the third step in Example 1 was used to react with Intermediate D-1 to give compound I-4 (compound 4) as a white solid, 49 mg, 48% yield.

[0112] 1 H NMR (400 MHz, CDC13) δ 7.56 - 7.46 (m, 4 H), 7.46 - 7.40 (m, 4 H), 7.40 - 7.32 (m, 3 H), 7.29 (s, 1 H), 7.25 - 7.14 (m, 3 H), 4.51 - 4.43 (m, 3 H), 3.05 (s, 1 H), 2.21 (s, 3 H), 1.82 (d, J = 7.1 Hz, 3 H).

[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)phenyl)amino)methyl)benzamide

[0115]

[0116] Using p-acetylamino benzaldehyde (69 mg, 0.42 mmol) as the starting material, a similar procedure to the third step in Example 1 was used to react with Intermediate D-1 to give compound I-5 (compound 5) as a white solid, 47 mg, 43% yield.

[0117] 1H NMR (400 MHz, CDC13) δ 7.56 - 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 = 7.2 Hz, 1H), 4.39 (s, 2H), 2.19 (s, 3H), 2.16 (s, 3H), 1.83 (d, J = 7.2 Hz, 3H).

[0118] Example 6

[0119] N-(4- Acetyl-benzyl)-2-methyl-5-(((5-(5-(l-(2-fluoro-[l,r-biphenyl]-4-yl)ethyl)- 1,2,4-oxadiazol-3-yl)amino)methyl)phenyl)methanesulfonamide

[0120]

[0121] Using 4-acetylbenzaldehyde (62 mg, 0.42 mmol) as the starting material, a similar procedure to the third step in Example 1 was used to react with Intermediate D-1 to give compound I-6 (Compound 6) as a white solid 55 mg, 52% yield.

[0122] 1 H NMR (400 MHz, CDC13) δ 7.94 (d, J = 7.9 Hz, 2H), 7.54 - 7.47 (m, 4H), 7.46 - 7.39 (m, 4H), 7.39 - 7.33 (m, 1H), 7.27 (s, 1H), 7.22 - 7.15 (m, 3H), 4.52 (s, 2H), 4.46 (q, J = 7.2 Hz, 1H), 2.58 (s, 3H), 2.23 (s, 3H), 1.81 (d, J = 7.2 Hz, 3H).

[0123] Example 7

[0124] N-(3-Hydroxy-benzyl)-2-methyl-5-(((5-(5-(l-(2-fluoro-[l,r-biphenyl]-4-yl)ethyl)- 1,2,4-oxadiazol-3-yl)amino)methyl)phenyl)methanesulfonamide

[0125]

[0126] Using 3-hydroxybenzaldehyde (51 mg, 0.42 mmol) as the starting material, a similar procedure to the third step in Example 1 was used to react with Intermediate D-1 to give compound I-7 (Compound 7) as a white solid 50 mg, 50% yield.

[0127] 1H NMR (400 MHz, CDC13) δ 7.53 - 7.47 (m, 2 H), 7.45 - 7.38 (m, 4 H), 7.38 - 7.33 (m, 1 H), 7.30 (s, 1 H), 7.23 - 7.10 (m, 4 H), 6.90 (d, J = 7.6 Hz, 1 H), 6.83 (s, 1 H), 6.73 (d, J = 8.1 Hz, 1 H), 4.47 (q, J = 7.2 Hz, 1 H), 4.26 (s, 2 H), 2.15 (s, 3 H), 1.82 (d, J = 7.2 Hz, 3 H).

[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)phenyl)amino)methyl)benzamide

[0130]

[0131] Using 3,5-dihydroxybenzaldehyde (58 mg, 0.42 mmol) as the starting material, a similar procedure to the third step in Example 1 was used to react with Intermediate D-1 to give compound I-8 (compound 8) as a white solid 44 mg in 42% yield.

[0132] 1 H NMR (400 MHz, CDC13) δ 7.53 - 7.47 (m, 2 H), 7.45 - 7.38 (m, 4 H), 7.38 - 7.33 (m, 1 H), 7.30 (s, 1 H), 7.23 - 7.10 (m, 4 H), 6.90 (d, J = 7.6 Hz, 1 H), 6.83 (s, 1 H), 6.73 (d, J = 8.1 Hz, 1 H), 4.47 (q, J = 7.2 Hz, 1 H), 4.26 (s, 2 H), 2.15 (s, 3 H), 1.82 (d, J = 7.2 Hz, 3 H).

[0133] Example 9

[0134] 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)phenyl)amino)methyl)benzamide

[0135]

[0136] Using similar procedure as in Example 1, Step 3, starting from 4- methoxybenzaldehyde (57 mg, 0.42 mmol), and reacting with intermediate D-1, compound I-9 (compound 9) was obtained as a white solid 52 mg, yield 50%.

[0137] 1 H NMR (400 MHz, CDC13) δ 7.57 - 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 = 7.3 Hz, 1H), 4.45 (s, 2H), 2.22 (s, 3H), 1.84 (d, J = 6.7 Hz, 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 procedure:

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

[0145] In a 100 mL reaction flask, 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT, 5250 mg, 30.0 mmol), 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-nitrobenzimidamide (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) to obtain intermediate C-2, white solid 6.33 g, yield 71%.

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

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

[0148] 1 H NMR (400 MHz, CDC13) δ 7.40 - 7.36 (m, 3 H), 7.36 - 7.35 (m, 1 H), 7.35 - 7.33 (m, 1 H), 7.32 - 7.29 (m, 1 H), 7.08 (d, J = 8.1 Hz, 1 H), 6.72 (dd, J = 8.1, 2.6 Hz, 1 H), 4.29 (s, 2 H), 2.49 (s, 3 H).

[0149] Third Step: 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 vial, intermediate 5-(5-benzyl-l,2,4-oxadiazol-3-yl)-2- methylbenzeneamine (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), after stirring at room temperature for 3 hours, sodium triacetoxyborohydride (267 mg, 1.26 mmol) was slowly added. Concentration under reduced pressure, separation by silica gel column chromatography (methanol / dichloromethane = 10 / 90) to obtain compound I-10 (Compound 10), white solid 55 mg, yield 66%.

[0151] 1 H NMR (400 MHz, CDC13) δ 7.40 - 7.36 (m, 3 H), 7.36 - 7.35 (m, 1 H), 7.35 - 7.33 (m, 1 H), 7.32 - 7.29 (m, 1 H), 7.08 (d, J = 8.1 Hz, 1 H), 6.72 (dd, J = 8.1, 2.6 Hz, 1 H), 4.29 (s, 2 H), 2.49 (s, 3 H).

[0152] Example 11

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

[0154]

[0155] Synthetic route:

[0156]

[0157] Experimental procedure:

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

[0159] In a 100 mL reaction flask, 2-chloro-4,6-dimethoxy-l,3,5-triazine (CDMT, 5250 mg, 30.0 mmol), 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)benzimidamide (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) to obtain intermediate C-3, white solid 7.22 g, yield 70%.

[0160] 1 H NMR (400 MHz, CDC13) δ 9.11 (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] Second step: Preparation of 3-trifluoromethyl-5-(5-benzyl-l,2,4-oxadiazol-3-yl)aniline (D-3)

[0162] In a 25 mL reaction vial, 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 of methanol and 5 mL of dichloromethane, saturated aqueous ammonium chloride solution (5 mL) was added, stirred uniformly, zinc powder (1308 mg, 20.0 mmol) was added, stirred at room temperature for 3 hours. Filtration, concentration under reduced pressure, separation by silica gel column chromatography (methanol / dichloromethane = 3 / 97), to obtain intermediate D-3, white solid 270 mg, yield 85%.

[0163] Third step: 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 vial, intermediate 3-trifluoromethyl-5-(5-benzyl-1,2,4- oxadiazol-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), after stirring at room temperature for 3 hours, sodium triacetoxyborohydride (267 mg, 1.26 mmol) was slowly added. Concentration under reduced pressure, separation by silica gel column chromatography (methanol / dichloromethane = 10 / 90), to obtain compound I-11 (compound 11), white solid 61 mg, yield 63%.

[0165] 1 H NMR (400 MHz, DMSO-d6) δ 7.58 (d, J = 3.8 Hz, 1H), 7.50 (s, 1H), 7.41 - 7.35 (m, 5H), 7.35 - 7.25 (m, 2H), 7.12 (d, J = 3.7 Hz, 1H), 7.10 (s, 1H), 4.64 (d, J = 5.9 Hz, 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 procedure:

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

[0173] In a 100 mL reaction flask, 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT, 5250 mg, 30.0 mmol), N-methylmorpholine (NMM, 9090 mg, 90.0 mmol) were dissolved in 1,4-dioxane (150 mL), stirred at room temperature for 15 minutes, 2-phenylpropanoic acid (A-3, 4500 mg, 30.0 mmol) was added, stirred at room temperature for 60 minutes, (Z)-N'-hydroxy-3-nitro-5-(trifluoromethyl)benzimidamide (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) to obtain intermediate C-4, white solid 7.11 g, yield 65%.

[0174] 1 H NMR (400 MHz, CDCl3) δ 9.13 (d, J = 1.8 Hz, 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 = 7.2 Hz, 1H), 1.86 (d, J = 7.3 Hz, 3H).

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

[0176] In a 25 mL reaction flask, 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 of methanol and 5 ml of dichloromethane, saturated aqueous ammonium chloride solution (5 mL) was added, stirred uniformly, zinc powder (1308 mg, 20.0 mmol) was added, stirred at room temperature for 3 hours. Filtered, concentrated under reduced pressure, separated by silica gel column chromatography (methanol / dichloromethane = 3 / 97) to obtain intermediate D-4, white solid 271 mg, yield 81%.

[0177] 1 H NMR (400 MHz, CDCl3) δ 7.73 (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 = 7.3 Hz, 1H), 1.81 (d, J = 7.3 Hz, 3H).

[0178] Third Step: 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 flask, intermediate 3-trifluoromethyl-5-(5-benzyl-1,2,4- oxadiazol-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, then sodium triacetoxyborohydride (267 mg, 1.26 mmol) was added slowly. Concentrated under reduced pressure, separated by silica gel column chromatography (methanol / dichloromethane = 10 / 90) to obtain compound I-12 (Compound 12), white solid 66 mg, yield 66%.

[0180] 1 H NMR (400 MHz, DMSO-d6) δ 7.59 (d, J = 3.7 Hz, 1H), 7.53 (s, 1H), 7.42 - 7.34 (m, 5H), 7.34 - 7.25 (m, 2H), 7.13 (d, J = 3.7 Hz, 1H), 7.10 (brs, 1H), 4.70 - 4.61 (m, 3H), 1.72 (d, J = 7.2 Hz, 3H).

[0181] Example 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 Procedure:

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

[0188] In a 100 mL reaction flask, 2-chloro-4,6-dimethoxy-l,3,5-triazine (CDMT, 5250 mg, 30.0 mmol) was 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)benzimidamide (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) to obtain intermediate C-5, white solid 7.00 g, yield 64%.

[0189] 1 H NMR (400 MHz, CDC13) δ 9.14 (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] Second step: Preparation of 3-trifluoromethyl-5-(5-(hydroxy(phenyl)methyl)-l,2,4-oxadiazol-3-yl)aniline (D-5)

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

[0192] 1 H NMR (400 MHz, CDC13) δ 9.14 (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).

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

[0194] In a 25 mL reaction vial, 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), glacial acetic acid (75 mg, 1.26 mmol) were dissolved in 1,2-dichloroethane (8 mL) and stirred at room temperature for 3 hours, then sodium triacetoxyborohydride (267 mg, 1.26 mmol) was added slowly. Concentrated under reduced pressure, separated by silica gel column chromatography (methanol / dichloromethane = 10 / 90) to obtain compound I-13 (compound 13), off-white solid 52 mg, yield 52%.

[0195] 1 H NMR (400 MHz, DMSO-d6) δ 7.60 (d, J = 3.7 Hz, 1H), 7.54 - 7.48 (m, 3H), 7.45 - 7.32 (m, 4H), 7.29 (t, J = 6.1 Hz, 1H), 7.13 (d, J = 3.7 Hz, 1H), 7.10 (s, 1H), 6.88 (d, J = 5.1 Hz, 1H), 6.14 (d, J = 4.7 Hz, 1H), 4.64 (d, J = 5.8 Hz, 2H).

[0196] Example 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 procedure:

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

[0203] In a 100 mL reaction flask, 2-chloro-4,6-dimethoxy-l,3,5-triazine (CDMT, 5250 mg, 30.0 mmol) was 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-nitrobenzimidamide (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) to give intermediate C-6, white solid 6.67 g, yield 71%.

[0204] 1 H NMR (400 MHz, CDC13) δ 8.18 (s, 1H), 7.63 (d, J = 7.8 Hz, 1H), 7.44 - 7.36 (m, 2H), 7.35 - 7.28 (m, 3H), 7.14 (d, J = 7.7 Hz, 1H), 6.04 (s, 1H), 2.14 (s, 3H).

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

[0206] In a 25 mL reaction flask, intermediate (3-(4-methyl-3-nitrophenyl)-l,2,4-oxadiazol-5-yl)(phenyl)methanol (C-6, 311 mg, 1.0 mmol) was dissolved in 5 ml of methanol and 5 ml of dichloromethane, saturated aqueous ammonium chloride solution (5 mL) was added, stirred uniformly, zinc powder (1308 mg, 20.0 mmol) was added, stirred at room temperature for 3 hours. Filtered, concentrated under reduced pressure, separated by silica gel column chromatography (methanol / dichloromethane = 3 / 97) to give intermediate D-6, white solid 252 mg, yield 90%.

[0207] 1 H NMR (400 MHz, DMSO-d6) δ 7.51 (d, J = 7.2 Hz, 2H), 7.44 - 7.37 (m, 2H), 7.37 - 7.32 (m, 1H), 7.11 - 7.03 (m, 2H), 6.81 (d, J = 5.0 Hz, 1H), 6.09 (d, J = 5.0 Hz, 1H), 2.10 (s, 3H).

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

[0209] In a 25 mL reaction flask, 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), and stirred at room temperature for 3 hours, then sodium triacetoxyborohydride (267 mg, 1.26 mmol) was added slowly. Concentrated under reduced pressure, separated by silica gel column chromatography (methanol / dichloromethane = 10 / 90) to obtain compound I-14 (Compound 14), white solid 55 mg, yield 62%.

[0210] 1 H NMR (400 MHz, DMSO-d6) δ 7.57 (d, J = 3.7 Hz, 1H), 7.50 (d, J = 7.6 Hz, 2H), 7.40 (t, J = 7.4 Hz, 2H), 7.37 - 7.31 (m, 1H), 7.17 (s, 2H), 7.12 - 7.06 (m, 2H), 6.80 (d, J = 5.0 Hz, 1H), 6.13 (t, J = 6.2 Hz, 1H), 6.08 (d, J = 4.0 Hz, 1H), 4.60 (d, J = 5.6 Hz, 2H), 2.20 (s, 3H).

[0211] Example 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] Synthetic route:

[0215]

[0216] Experimental procedure:

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

[0218] In a 100 mL reaction flask, 2-chloro-4,6-dimethoxy-l,3,5-triazine (CDMT, 700 mg, 4.0 mmol) was dissolved in 1,4-dioxane (30 mL) and stirred at room temperature for 15 minutes. Then, 2-(4-isobutylphenyl)propanoic acid (A-6, 824 mg, 4.0 mmol) was added and stirred at room temperature for 60 minutes. Then, (Z)-N'-hydroxy-2-methyl-5-nitrobenzimidamide (B-1, 702 mg, 3.6 mmol) was added and stirred at room temperature for 2 hours. Then, the reaction mixture was heated to reflux for 6 hours. After completion of the reaction, the solvent was removed under reduced pressure. The residue was separated by silica gel column chromatography (ethyl acetate / petroleum ether = 5 / 95) to obtain intermediate C-7 as a white solid (837 mg, 57% yield).

[0219] 1 H NMR (400 MHz, CDC13) δ 8.82 (s, 1H), 8.33 (dd, J = 8.4, 1.6 Hz, 1H), 7.59 (d, J = 8.0 Hz, 1H), 7.41 (d, J = 8.0 Hz, 2H), 7.28 (s, 1H), 4.59 (q, J = 7.2 Hz, 1H), 2.79 (s, 3H), 2.59 (d, J = 7.2 Hz, 2H), 2.02 - 1.97 (m, 1H), 1.95 (d, J = 7.2 Hz, 3H), 1.03 (d, J = 6.8 Hz, 6H).

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

[0221] In a 25 mL reaction flask, intermediate 5-(l-(4-isobutylphenyl)ethyl)-3-(4-methyl-3- nitrophenyl)-l,2,4-oxadiazole (C-7, 365 mg, 1.0 mmol) was dissolved in 5 mL of methanol and 5 mL of dichloromethane. Then, saturated aqueous ammonium chloride solution (5 mL) was added and stirred well. Then, zinc powder (1308 mg, 20.0 mmol) was added and stirred at room temperature for 3 hours. After completion of the reaction, the reaction mixture was filtered and concentrated under reduced pressure. The residue was separated by silica gel column chromatography (methanol / dichloromethane = 3 / 97) to obtain intermediate D-7 as a white solid (247 mg, 73% yield).

[0222] 1H NMR (400 MHz, CDC13) δ 7.44 (d, J = 8.0 Hz, 1H), 7.42 (s, 1H), 7.28 (s, 1H), 7.13 (t, J = 8.8 Hz, 3H), 4.43 (q, J = 7.2 Hz, 1H), 2.45 (d, J = 6.4 Hz, 2H), 2.24 (s, 3H), 1.88 - 1.83 (m, 1H), 1.80 (d, J = 7.2 Hz, 3H), 0.90 (d, J = 6.4 Hz, 6H).

[0223] Third Step: Preparation of (5-(((5-(5-(1-(4-isobutylphenyl)ethyl)-1,2,4-oxadiazol-3-yl)-2-methylphenyl)amino)methyl)thiophene-2-carboxylic acid

[0224] In a 25 mL reaction bottle, 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), glacial acetic acid (216 mg, 3.6 mmol) were dissolved in 1,2-dichloroethane (20 mL), stirred at room temperature for 3 hours, then slowly added sodium triacetoxyborohydride (760 mg, 3.6 mmol). Concentrated under reduced pressure, separated by silica gel column chromatography (methanol / dichloromethane = 10 / 90) to obtain compound I-15 (compound 15), yellow solid 83 mg, yield 29%.

[0225] 1 H NMR (400 MHz, CDC13) δ 7.46 - 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 = 7.2 Hz, 2H), 2.20 (s, 3H), 1.86 - 1.84 (m, 1H), 1.79 (d, J = 7.6 Hz, 3H), 0.89 (d, J = 6.8 Hz, 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 the starting material, a similar procedure to the third step in Example 15 was used to react with Intermediate D-7 to give compound I-16 (Compound 16), white solid-like 123 mg, yield 52%.

[0230] 1 H NMR (400 MHz, DMSO-d6) δ 12.9 (br s, 1H), 7.58 (d, J = 3.6 Hz, 1H), 7.25 (d, J = 7.6 Hz, 2H), 7.20 - 7.17 (m, 2H), 7.14 - 7.11 (m, 4H), 6.13 (br s, 1H), 4.61 (d, J = 5.2 Hz, 2H), 4.55 (q, J = 7.2 Hz, 1H), 2.41 (d, J = 6.8 Hz, 2H), 2.21 (s, 3H), 1.79 (m, 1H), 1.67 (d, J = 7.2 Hz, 3H), 0.84 (d, J = 6.4 Hz, 6H).

[0231] Example 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 the starting material, a similar procedure to the third step in Example 1 was used to react with Intermediate D-1 to give compound I-17 (Compound 17), white solid-like 55 mg, yield 53%.

[0235] 1 H NMR (400 MHz, DMSO-d6) δ 7.82 (d, J = 4.0 Hz, 1H), 7.54 (d, J = 7.6 Hz, 3H), 7.51 - 7.46 (m, 2H), 7.42 - 7.36 (m, 2H), 7.29 (d, J = 8.0 Hz, 1H), 7.24 - 7.17 (m, 3H), 7.09 (s, 1H), 6.22 (br s, 1H), 4.71 (q, J = 7.2 Hz, 1H), 4.67 (d, J = 6.0 Hz, 2H), 2.21 (s, 3H), 1.73 (d, J = 7.2 Hz, 3H).

[0236] Example 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 the starting material, a similar procedure as in Example 1, Step 3 was used to react with Intermediate D-1 to give compound I-18 (Compound 18) as a off-white solid 41 mg in 38% yield.

[0240] 1 H NMR (400 MHz, DMSO-d6) δ 12.89 (br s, 1H), 7.55 - 7.53 (m, 4H), 7.48 (t, J = 6.8 Hz, 2H), 7.42 - 7.36 (m, 2H), 7.29 (d, J = 8.0 Hz, 1H), 7.21 - 7.15 (m, 2H), 7.11 (s, 2H), 6.14 (br s, 1H), 4.70 (t, J = 7.2 Hz, 1H), 4.61 (d, J = 5.6 Hz, 2H), 2.21 (s, 3H), 1.73 (d, J = 7.2 Hz, 3H).

[0241] Example 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] Synthetic route:

[0245]

[0246] Experimental procedure:

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

[0248] In a 100 mL reaction flask, 2-chloro-4,6-dimethoxy-l,3,5-triazine (CDMT, 350 mg, 2.0 mmol), 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)propanoic acid (A-7, 484 mg, 2.0 mmol) was added, stirred at room temperature for 60 minutes, (Z)-N'-hydroxy-2-methyl-5-nitrobenzimidamide (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) to obtain intermediate C-8, white solid 577 mg, yield 72%.

[0249] 1 H NMR (400 MHz, DMSO-d6) δ 8.47 (s, 1H), 8.18 (d, J = 7.6 Hz, 1H), 7.70 (d, J = 8.0 Hz, 1H), 7.39 (t, J = 9.2 Hz, 3H), 7.14 (d, J = 7.2 Hz, 2H), 7.09 (s, 1H), 7.02 (d, J = 8.0 Hz, 2H), 6.90 (dd, J = 8.2, 2.4 Hz, 1H), 4.71 (q, J = 7.2 Hz, 1H), 2.60 (s, 3H), 1.72 (d, J = 7.2 Hz, 3H).

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

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

[0252] 1H NMR (400 MHz, DMSO-d6) δ 7.43 (s, 1H), 7.37 - 7.35 (m, 2H), 7.30 - 7.26 (m, 5H), 7.19 - 7.18 (m, 2H), 7.12 - 7.05 (m, 2H), 5.17 (brs, 2H), 4.60 (q, J = 4.0 Hz, 1H), 2.11 (s, 3H), 1.68 (d, J = 4.0 Hz, 3H).

[0253] Third Step: 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, 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), glacial acetic acid (111 mg, 1.86 mmol) were dissolved in 1,2-dichloroethane (12 mL), stirred at room temperature for 3 hours, then slowly added sodium triacetoxyborohydride (394 mg, 1.86 mmol). Concentrated under reduced pressure, separated by silica gel column chromatography (methanol / dichloromethane = 10 / 90) to obtain Compound I-19 (Compound 19), white solid 17 mg, yield 10%.

[0255] 1 H NMR (400 MHz, DMSO-d6) δ 7.43 (s, 1H), 7.37 - 7.35 (m, 2H), 7.30 - 7.26 (m, 5H), 7.19 - 7.18 (m, 2H), 7.12 - 7.05 (m, 2H), 5.17 (brs, 2H), 4.60 (q, J = 4.0 Hz, 1H), 2.11 (s, 3H), 1.68 (d, J = 4.0 Hz, 3H).

[0256] Example 20

[0257] 5-(((5-(5-(1-(3-Phenoxyphenyl)ethyl)-1,2,4-oxadiazol-3-yl)-2-methylphenyl)amino)methyl)thiophene-2-carboxylic acid

[0258]

[0259] Using similar procedure as in Step 3 of Example 19, 5-cyanothiophene-2- carboxaldehyde (170 mg, 1.24 mmol) was reacted with Intermediate D-8 to give compound I-20 (Compound 20) as a liquid 130 mg, 43% yield.

[0260] 1 H NMR (400 MHz, DMSO-d6) δ 7.82 (s, 1H), 7.37 (d, J = 7.2 Hz, 3H), 7.23 (s, 1H), 7.18 - 7.15 (m, 3H), 7.12 - 7.01 (m, 5H), 6.89 (d, J = 8.4 Hz, 1H), 4.66 (s, 2H), 4.62 (q, J = 7.2 Hz, 1H), 2.21 (s, 3H), 1.67 (d, J = 7.2 Hz, 3H).

[0261] Example 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] Synthetic route:

[0265]

[0266] Experimental procedure:

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

[0268] In a 100 mL reaction flask, 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT, 700 mg, 4.0 mmol), 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-carbazol-2-yl)propanoic acid (A-8, 1092 mg, 4.0 mmol) was added, stirred at room temperature for 60 minutes, (Z)-N'-hydroxy-2-methyl-5-nitrobenzimidamide (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) to give intermediate C-9, yellow solid 970 mg, yield 56%.

[0269] 1 H NMR (400 MHz, DMSO-d6) δ 11.42 (s, 1H), 8.51 (d, J = 2.0 Hz, 1H), 8.26 - 8.18 (m, 2H), 8.14 (d, J = 8.0 Hz, 1H), 7.70 (d, J = 8.0 Hz, 1H), 7.50 - 7.48 (m, 2H), 7.37 (dd, J = 8.8, 2.0 Hz, 1H), 7.19 (dd, J = 8.0, 1.6 Hz, 1H), 4.86 (q, J = 7.2 Hz, 1H), 2.60 (s, 3H), 1.83 (d, J = 7.2 Hz, 3H).

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

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

[0272] 1 H NMR (400 MHz, DMSO-d6) δ 11.42 (s, 1H), 8.51 (d, J = 2.0 Hz, 1H), 8.26 - 8.18 (m, 2H), 8.14 (d, J = 8.0 Hz, 1H), 7.70 (d, J = 8.0 Hz, 1H), 7.50 - 7.48 (m, 2H), 7.37 (dd, J = 8.8, 2.0 Hz, 1H), 7.19 (dd, J = 8.0, 1.6 Hz, 1H), 4.86 (q, J = 7.2 Hz, 1H), 2.60 (s, 3H), 1.83 (d, J = 7.2 Hz, 3H).

[0273] Third Step: Preparation of 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 (Compound 21)

[0274] In a 25 mL reaction vial, intermediate 5-(((1-(6-chloro-9H-carbazol-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) and stirred at room temperature for 3 hours. Then, sodium triacetoxyborohydride (178 mg, 0.84 mmol) was added slowly. After concentration under reduced pressure, the compound was separated by silica gel column chromatography (methanol / dichloromethane = 10 / 90) to give compound I-21 (compound 21) as a white solid 43 mg, yield 56%.

[0275] 1 H NMR (400 MHz, DMSO-d6) δ 11.62 (s, 1H), 8.18 (s, 1H), 8.12 (d, J = 8.0 Hz, 1H), 7.49 (d, J = 8.4 Hz, 1H), 7.45 (s, 2H), 7.36 (d, J = 8.8 Hz, 1H), 7.20 - 7.16 (m, 4H), 7.04 (s, 1H), 6.09 (s, 1H), 4.76 (q, J = 7.2 Hz, 1H), 4.57 (d, J = 5.6 Hz, 2H), 2.20 (s, 3H), 1.77 (d, J = 7.2 Hz, 3H).

[0276] Example 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 procedure:

[0282] First step: 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 flask, 2-chloro-4,6-dimethoxy-l,3,5-triazine (CDMT, 350 mg, 2.0 mmol), N-methylmorpholine (NMM, 606 mg, 6.0 mmol) were dissolved in 1,4-dioxane (20 mL), stirred at room temperature for 15 min, 2-([l,l'-biphenyl]-3-yl)propanoic acid (A-9, 470 mg, 2.0 mmol) was added, stirred at room temperature for 60 min, (Z)-N'-hydroxy-4-methyl-3-nitrobenzimidamide (B-1, 351 mg, 1.8 mmol) was added, stirred at room temperature for 2 h, heated to reflux for 6 h. Concentrated under reduced pressure, separated by silica gel column chromatography (ethyl acetate / petroleum ether = 5 / 95) to give intermediate C-10, white solid 550 mg, yield 71%.

[0284] Second step: Preparation of 5-(5-(l-([l,l'-biphenyl]-3-yl)ethyl)-l,2,4-oxadiazol-3-yl)-2-methylaniline (D-10)

[0285] In a 25 mL reaction flask, intermediate 5-(l-([l,l'-biphenyl]-3-yl)ethyl)-3-(4-methyl-3-nitrophenyl)-l,2,4-oxadiazole (C-10, 550 mg, 1.4 mmol) was dissolved in 4 ml of methanol and 4 ml of dichloromethane, saturated aqueous ammonium chloride solution (7 mL) was added, stirred uniformly, zinc powder (1800 mg, 28.0 mmol) was added, stirred at room temperature for 3 h. Filtered, concentrated under reduced pressure, separated by silica gel column chromatography (methanol / dichloromethane = 3 / 97) to give intermediate D-10, white solid 396 mg, yield 73%.

[0286] 1 H NMR (400 MHz, CDC13) δ 7.58 (d, J = 6.4 Hz, 3H), 7.54 - 7.48 (m, 1H), 7.46 - 7.40 (m, 5H), 7.37 - 7.33 (m, 2H), 7.14 (d, J = 7.7 Hz, 1H), 4.52 (q, J = 7.2 Hz, 1H), 2.22 (s, 3H), 1.86 (d, J = 7.2 Hz, 3H).

[0287] Third step: Preparation of 5-(((5-(5-(l-([l,l'-biphenyl]-3-yl)ethyl)-l,2,4-oxadiazol-3-yl)-2-methylphenyl)amino)methyl)thiophene-2-carboxylic acid (Compound 22)

[0288] In a 25 mL reaction vial, 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), glacial acetic acid (68 mg, 1.14 mmol) were dissolved in 1,2-dichloroethane (7 mL) and stirred at room temperature for 3 hours. Then, sodium triacetoxyborohydride (242 mg, 1.14 mmol) was added slowly. After concentration under reduced pressure, the compound was separated by silica gel column chromatography (methanol / dichloromethane = 10 / 90) to give compound I-22 (compound 22) as a white solid, 25 mg, 26% yield.

[0289] 1 H NMR (400 MHz, DMSO-d6) δ 12.91 (br s, 1H), 7.66 - 7.55 (m, 5H), 7.47 (s, 3H), 7.39 - 7.33 (m, 2H), 7.17 (d, J = 6.9 Hz, 2H), 7.10 - 7.08 (m, 2H), 6.12 (br s, 1H), 4.70 (q, J = 7.2 Hz, 1H), 4.60 (s, 2H), 2.20 (s, 3H), 1.75 (d, J = 7.2 Hz, 3H).

[0290] Example 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] Synthetic route:

[0294]

[0295] Experimental procedure:

[0296] First step: 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 flask, 2-chloro-4,6-dimethoxy-l,3,5-triazine (CDMT, 595 mg, 3.4 mmol), 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-[l,l'-biphenyl]-3-yl)propanoic acid (A-10, 710 mg, 3.4 mmol) was added, stirred at room temperature for 60 minutes, (Z)-N'-hydroxy-2-methyl-5-nitrobenzimidamide (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) to obtain intermediate C-11, white solid 1017 mg, yield 75%.

[0298] 1 H NMR (400 MHz, CDC13) δ 7.58 - 7.48 (m, 3H), 7.47 - 7.37 (m, 4H), 7.30 (d, J = 7.7 Hz, 1H), 7.14 (d, J = 7.7 Hz, 1H), 6.96 (d, J = 8.9 Hz, 2H), 4.50 (q, J = 7.6 Hz, 1H), 4.08 (q, J = 6.8 Hz, 2H), 2.22 (s, 3H), 1.85 (d, J = 7.6 Hz, 3H), 1.45 (t, J = 6.8 Hz, 3H).

[0299] Second step: Preparation of 5-(5-(l-(4'-ethoxy-[l,l'-biphenyl]-3-yl)ethyl)-l,2,4- oxadiazol-3-yl)-2-methylaniline (D-11)

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

[0301] 1H NMR (400 MHz, DMSO) δ 7.61 - 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 = 7.6 Hz, 1H), 4.05 (t, J = 7.2 Hz, 2H), 2.10 (s, 3H), 1.76 (d, J = 7.2 Hz, 3H), 1.33 (t, J = 7.6, 3H).

[0302] Third Step: 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 flask, 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), glacial acetic acid (360 mg, 6.0 mmol) were dissolved in 1,2-dichloroethane (20 mL), stirred at room temperature for 3 hours, then slowly added sodium triacetoxyborohydride (1272 mg, 6.0 mmol). Concentrated under reduced pressure, separated by silica gel column chromatography (methanol / dichloromethane = 10 / 90) to obtain compound I-23 (Compound 23), yellowish solid 210 mg, yield 39%.

[0304] 1 H NMR (400 MHz, DMSO-d6) δ 7.61 - 7.59 (m, 2H), 7.56 - 7.52 (m, 3H), 7.41 (t, J = 7.6 Hz, 1H), 7.27 (d, J = 2.0 Hz, 1H), 7.20 - 7.15 (m, 2H), 7.06 - 7.08 (m, 2H), 7.0 (d, J = 8.4 Hz, 2H), 6.12 (br s, 1H), 4.66 (q, J = 7.2 Hz, 1H), 4.60 (s, 2H), 4.06 (q, J = 6.8 Hz, 2H), 2.20 (s, 3H), 1.74 (d, J = 7.2 Hz, 3H), 1.34 (t, J = 6.8 Hz, 3H).

[0305] Example 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] Synthetic route:

[0309]

[0310] First step: 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 flask, 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT, 119 mg, 0.68 mmol), N-methylmorpholine (NMM, 206 mg, 2.04 mmol) were dissolved in 1,4-dioxane (15 mL) and 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-nitrobenzimidamide (B-1, 118 mg, 0.61 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) to obtain intermediate C-12, 215 mg of yellow liquid, yield 84%.

[0312] 1 H NMR (400 MHz, CDC13) δ 8.67 (s, 1H), 8.18 (d, J = 8.0 Hz, 1H), 7.62 (s, 1H), 7.57 (d, J = 8.0 Hz, 1H), 7.46 (d, J = 8.0 Hz, 1H), 7.39 (t, J = 7.6 Hz, 1H), 7.34 - 7.29 (m, 3H), 7.09 - 7.08 (m, 1H) 4.34 (s, 2H), 2.66 (s, 3H).

[0313] Second step: 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 vial, 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 well, zinc powder (1300 mg, 20.0 mmol) was added, stirred at room temperature for 3 hours. Filtered, concentrated under reduced pressure, separated by silica gel column chromatography (methanol / dichloromethane = 5 / 95) to give intermediate D-12, white solid 275 mg, yield 79%.

[0315] 1 H NMR (400 MHz, CDCI3) d 7.62 (s, 1 H), 7.55 (d, J = 8.0 Hz, 1 H), 7.43 - 7.35 (m, 3H), 7.32 - 7.26 (m, 3H), 7.14 (d, J = 7.6 Hz, 1 H), 7.08 (t, J = 4.4 Hz, 1 H), 4.31 (s, 2H), 2.21 (s, 3H).

[0316] Third step: 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 vial, 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, then sodium triacetoxyborohydride (253 mg, 1.2 mmol) was added slowly. Concentrated under reduced pressure, separated by silica gel column chromatography (methanol / dichloromethane = 10 / 90) to give Compound I-24 (Compound 24), white solid 25 mg, yield 26%.

[0318] 1 H NMR (400 MHz, CDCI3) d 7.62 (s, 1 H), 7.55 (d, J = 8.0 Hz, 1 H), 7.43 - 7.35 (m, 3H), 7.32 - 7.26 (m, 3H), 7.14 (d, J = 7.6 Hz, 1 H), 7.08 (t, J = 4.4 Hz, 1 H), 4.31 (s, 2H), 2.21 (s, 3H).

[0319] Example 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 procedure:

[0325] First step: 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 flask, 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT, 376 mg, 2.1 mmol), 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)propanoic acid (A-12, 490 mg, 2.1 mmol) was added, stirred at room temperature for 60 minutes, (Z)-N'-hydroxy-4-methyl-3-nitrobenzimidamide (B-1, 370 mg, 1.89 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) to obtain intermediate C-13, white solid 589 mg, yield 65%.

[0327] 1 H NMR (400 MHz, CDCl3) δ 8.65 (s, 1H), 8.16 (d, J = 7.6 Hz, 1H), 7.57 (s, 1H), 7.50 (d, J = 7.6 Hz, 1H), 7.42 (d, J = 8.0 Hz, 1H), 7.33 (t, J = 7.6 Hz, 1H), 7.30-7.21 (m, 3H), 7.04-7.03 (m, 1H), 4.47 (q, J = 7.2 Hz, 1H), 2.62 (s, 3H), 1.82 (d, J = 7.2 Hz, 3H).

[0328] Second step: 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 vial, 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, saturated aqueous ammonium chloride solution (6 mL) was added, stirred uniformly, zinc powder (1350 mg, 22.5 mmol) was added, stirred at room temperature for 3 hours. Filtration, concentration under reduced pressure, separation by silica gel column chromatography (methanol / dichloromethane = 3 / 97) to obtain intermediate D-13, white solid 371 mg, yield 93%.

[0330] 1 H NMR (400 MHz, CDC13) δ 7.60 (s, 1H), 7.53 (d, J = 8.0 Hz, 1H), 7.45 - 7.41 (m, 2H), 7.35 (t, J = 7.6 Hz, 1H), 7.32 - 7.23 (m, 4H), 7.14 (d, J = 7.6 Hz, 1H), 7.07 (t, J = 4.4 Hz, 1H), 4.48 (q, J = 7.2 Hz, 1H), 2.22 (s, 3H), 1.84 (d, J = 7.2 Hz, 3H).

[0331] Third Step: 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 vial, 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), glacial acetic acid (100 mg, 1.66 mmol) were dissolved in 1,2-dichloroethane (8 mL), stirred at room temperature for 3 hours, then sodium triacetoxyborohydride (351 mg, 1.66 mmol) was slowly added. Concentration under reduced pressure, separation by silica gel column chromatography (methanol / dichloromethane = 10 / 90) to obtain compound I-25 (Compound 25), white solid 14 mg, yield 10%.

[0333] 1H NMR (400 MHz, DMSO-d6) δ 12.89 (br s, 1H), 7.67 (s, 1H), 7.59 - 7.53 (m, 4H), 7.41 (t, J = 7.7 Hz, 1H), 7.28 (d, J = 7.7 Hz, 1H), 7.21 - 7.14 (m, 3H), 7.11 - 7.09 (m, 2H), 6.13 (br s, 1H), 4.67 (q, J = 7.2 Hz, 1H), 4.61 (d, J = 5.7 Hz, 2H), 2.21 (s, 3H), 1.73 (d, J = 7.2 Hz, 3H).

[0334] Biological activity test

[0335] 1. In vitro protease inhibitory activity test

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

[0337] Principle of experiment: The characteristic substrate of PLpro protease is labeled by fluorescence quenching. If the substrate is digested, it will emit characteristic fluorescence, and if it is not digested, it will not emit fluorescence. By detecting the fluorescence value under the action of different concentrations of inhibitors, the speed of the protease in digesting the substrate is detected, and then the half inhibitory concentration value (IC 50 ) of the compound against PLpro is obtained according to the speed of the digestion reaction under different concentrations of inhibitors.

[0338] Method of experiment:

[0339] (1) The compound is diluted by a factor of 2 using DMSO.

[0340] (2) The reaction system is added in order in a 96-well transparent bottom black plate (Greiner, 655096), including reaction buffer, different concentrations of the compound to be tested, and PLpro protease.

[0341] (3) The reaction plate is placed in a 37°C incubator for enzymatic reaction for 8-10 minutes, and then the fluorescence substrate is added.

[0342] (4) Real-time detection of fluorescence intensity is performed using an enzyme marker.

[0343] (5) The data obtained by detection are fitted using Origin and Graphpad to obtain the IC 50 value.

[0344] Results of experiment:

[0345] Table 1. Results of the inhibitory effect of the compounds in the embodiments of the present invention on PLpro

[0346]

[0347] As shown in Table 1, the compounds of this invention exhibit good PLpro inhibitory activity, with eight compounds showing an IC50 inhibitory activity of PLpro. 50 <10μM.

[0348] 2. Cytotoxicity detection

[0349] Assay Method: MTT Assay Principle: Cell viability is determined by the reduction of oxidized 3-(4,5-dimethylthiazo-2-yl)-2,5-diphenyltetrazolium bromide (trade name: thiazolyl) / MTT [3-(4,5-dimethylthiazo-2-yl)-2,5-diphenyltetrazolium bromide] to a poorly soluble blue formazan compound by mitochondrial dehydrogenases (such as succinate dehydrogenase). The formazan compound is dissolved in DMSO and then reacted with the formazan to determine its color. The amount of formazan is positively correlated with the number of viable cells.

[0350] Experimental methods:

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

[0352] Filter and sterilize a 5 g / L MTT solution for later use.

[0353] (2) Dissolve the test drug in DMSO, dilute it 50 times with culture medium to prepare the highest concentration of the test drug, and then use culture medium to perform serial dilution at 1:2 on a 96-well plate. Set 8 concentrations for each compound, with the highest concentration being 200 μM. Set 4 parallel wells for each concentration, 100 μL / well.

[0354] (3) The prepared cell suspension was seeded into 96-well plates at a density of 100 μL / well, with a cell concentration of 4 × 10⁻⁶ cells / well. 5 Cells / mL. Control wells containing no drug and blank control wells containing culture medium were also included.

[0355] (4) After 48 hours of culture, add MTT 10 μL / well, continue to culture for 4 hours. Take out the culture plate, carefully discard the culture medium in the well, add DMSO 100 μL per well, shake until the formazan particles are completely dissolved, and then use an enzyme-linked immunoassay instrument to measure the optical density value (OD) at 570 nm wavelength. 570

[0356] (5) Data processing: cell survival percentage (%) = [(cell control OD 570 value - drug addition group OD 570 value) / (cell control OD 570 value - blank OD 570 value)] x 100%. Use Origin software to perform dose-response curve fitting to calculate the concentration of various compounds at 50% cell survival (CC 50 ).

[0357] Experimental results:

[0358] Table 2, cytotoxicity results of the compounds of the present application

[0359]

[0360] From the data in Table 2, it can be seen that the cytotoxicity of the compounds of the present application is low, and high safety is shown.

[0361] 3. Anti-viral activity detection

[0362] Detection method: fluorescence quantitative PCR method is used to detect the total amount of RNA in the cell culture medium supernatant. Experimental principle: when PCR amplification is performed, a specific fluorescent probe is added at the same time as a pair of primers. The probe is an oligonucleotide, and the two ends are labeled with a reporter fluorescent group and a quencher fluorescent group, respectively. When the probe is complete, the fluorescent signal emitted by the reporter group is absorbed by the quencher group; at the beginning, the probe is combined on any single strand of DNA, and during PCR amplification, the 5' to 3' exonuclease activity of Taq enzyme degrades the probe, so that the reporter fluorescent group and the quencher fluorescent group are separated, so that the fluorescence monitoring system can receive the fluorescence signal, that is, for every DNA strand amplified, a fluorescent molecule is formed, realizing the complete synchronization of fluorescence signal accumulation and PCR product formation.

[0363] Experimental method:

[0364] (1) The Vero E6 cells that have been cultured to the logarithmic growth phase are digested with 0.25% trypsin for 2-3 min, the digestion solution is aspirated and discarded, an appropriate amount of culture solution is added, mixed, 20 μL is taken and counted under a microscope using a blood cell counter, and a suitable concentration of cell suspension is prepared for standby.

[0365] ​(2) The test drug is dissolved in DMSO, diluted 50 times with the culture medium to prepare the highest concentration of the test drug, and then serially diluted by 1:2 on the 96-well plate with the culture medium, 8 concentrations for each compound, the highest concentration is 200 uM, 4 parallel holes for each concentration, 100 uL / hole.

[0366] (3) The prepared cell suspension is inoculated in the 96-well plate, 100 uL / hole, and the cell concentration is 4x10 5 cell / mL.

[0367] (4) After the drug working solution (200 ul) of different dilutions is incubated with the cells for 1 hour, 100 TCID 50 of the coronavirus is added and incubated for 2 hours.

[0368] (5) The cell culture solution in the 96-well plate is removed, and different concentrations of compound diluents are added, 200 uL / hole, 3 parallel holes for each concentration. At the same time, the cell control hole without drug, the culture medium blank control hole, the solvent control hole and the virus control hole are set.

[0369] (6) After 48 hours of culture, the culture supernatant is taken and added into the AVL lysis solution, and repeatedly blown and sucked for 15S to ensure sufficient lysis, and then the RNA extraction in the cell culture supernatant is carried out according to the steps of the RNA extraction kit.

[0370] (7) After the extracted RNA, fluorescent probe and polymerase are uniformly mixed, the QPCR method is used for quantitative detection of the RNA in the supernatant.

[0371] (8) Data processing: cell antiviral activity = [(drug group Ct value-virus control group Ct value) / (cell control group Ct value-virus control group Ct value)]x100%. The dose-response relationship curve fitting is carried out by using the GraphPad software, and the concentration (EC 50 ) of each compound reaching 50% of the antiviral activity is calculated.

[0372] Experimental results:

[0373] Table 3, antiviral detection results of the compounds

[0374]

[0375] As can be seen from the data in Table 3, the antiviral activities of the compounds 18 and 19 of the present application are better than that of the GRL0617 with the same action mechanism, and the strong anti-coronavirus effect is shown.

Claims

1. A compound of formula (I) and pharmaceutically acceptable salts thereof: wherein, R1 is hydrogen, methyl, ethyl, propyl, isopropyl; R2 is substituted or unsubstituted phenyl, substituted or unsubstituted biphenyl, substituted or unsubstituted diphenyl ether, substituted or unsubstituted carbazolyl; R3 is independently selected from hydrogen, F, Cl, Br, trifluoromethyl, trifluoromethoxy, C1-C3 alkyl, C1-C3 alkoxy, n represents 1, 2, 3 or 4 substituents; R4 is substituted or unsubstituted phenyl, substituted or unsubstituted thienyl; the substituents in R4 can be optionally selected from F, Cl, Br, monofluoromethyl, difluoromethyl, trifluoromethyl, trifluoromethoxy, thienyl, carboxyl, C1-C4 alkyl, C1-C4 alkoxy; the substituents in R2 can be optionally selected from F, Cl, Br, monofluoromethyl, difluoromethyl, trifluoromethyl, trifluoromethoxy, thienyl, C1-C4 alkyl, C1-C4 alkoxy.

2. The compound of claim 1 and pharmaceutically acceptable salts thereof, said compound is represented by general formula (II), wherein, R1 is hydrogen, methyl; R2 is substituted or unsubstituted phenyl, substituted or unsubstituted biphenyl, substituted or unsubstituted diphenyl ether, substituted or unsubstituted carbazolyl; R3 is independently selected from hydrogen, F, Cl, Br, trifluoromethyl, trifluoromethoxy, C1-C3 alkyl, C1-C3 alkoxy, n represents 1, 2, 3 or 4 substituents; R5 is hydrogen, carboxyl, trifluoromethyl, methoxy; the substituents in R2 can be optionally selected from F, Cl, Br, monofluoromethyl, difluoromethyl, trifluoromethyl, trifluoromethoxy, C1-C4 alkyl, C1-C4 alkoxy.

3. The compound of claim 1 and pharmaceutically acceptable salts thereof, said compound is represented by general formula (III), wherein, R1 is hydrogen, methyl; R2 is substituted or unsubstituted phenyl, substituted or unsubstituted biphenyl, substituted or unsubstituted diphenyl ether, substituted or unsubstituted carbazolyl; R3 is independently selected from hydrogen, F, Cl, Br, trifluoromethyl, trifluoromethoxy, C1-C3 alkyl, C1-C3 alkoxy, n represents 1, 2, 3 or 4 substituents; R5 is hydrogen, carboxyl; the substituents in R2 can be optionally selected from F, Cl, Br, monofluoromethyl, difluoromethyl, trifluoromethyl, trifluoromethoxy, thienyl, C1-C4 alkyl, C1-C4 alkoxy.

4. The compound of any one of claims 1 to 3 and pharmaceutically acceptable salts thereof, which is selected from the following compounds:

5. A method for preparing the compound of any one of claims 1 to 3, comprising the following steps: condensation of compound A with compound B, ring closure to obtain compound C, reduction to obtain compound D, amination reduction of compound D with R4-substituted aldehyde to obtain the compound of formula (I); wherein, R1, R2, R3, R4, n are defined as in any one of claims 1 to 3. ​ ​ ​ ​ wherein, ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 6. Use of a compound according to any one of claims 1 to 4, and pharmaceutically acceptable salts thereof, for the manufacture of a papain-like protease (PLpro) inhibitor.

7. A pharmaceutical composition, characterized by, The composition comprises a therapeutically and / or prophylactically effective amount of a compound according to any one of claims 1 to 4, and pharmaceutically acceptable salts thereof, and optionally one or more pharmaceutically acceptable excipients.

8. Use of a compound according to any one of claims 1 to 4, and pharmaceutically acceptable salts thereof, or a composition according to claim 7, for the manufacture of a medicament for the treatment and / or prevention of an infectious disease caused by a coronavirus.

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