CN119320367A - Substituted oxadiazole-aniline compound and application thereof - Google Patents

Abstract

The present invention belongs to the field of medical technology, and discloses substituted oxadiazole-aniline compounds, preparation methods thereof, pharmaceutical compositions with the compounds as active ingredients, and their use in treating and/or preventing infectious diseases caused by coronaviruses. Specifically, the present invention relates to compounds represented by formula (I) and isomers thereof, or pharmaceutically acceptable salts thereof, and pharmaceutical compositions comprising the compounds of the present invention, wherein R ₁ , R ₂ , R ₃ , R ₄ and n are as described in the specification. The present invention aims to prepare new compounds with anti-coronavirus activity, which, as potential new drugs, can be used for the treatment or preventive treatment of infectious diseases caused by viruses, especially pneumonia diseases caused by coronaviruses.

Description

Substituted oxadiazole-aniline compound and application thereof

Technical Field

The invention belongs to the technical field of medicines. In particular to a substituted oxadiazole-aniline compound shown in a general formula (I), a preparation method thereof, a pharmaceutical composition taking the compound as an active ingredient and application thereof in treating and/or preventing infectious diseases caused by coronavirus (SARS-CoV), in particular to novel coronavirus (SARS-CoV-2).

Background

Viral infections continue to threaten human life and health, and currently there is a lack of effective antiviral drugs for most viruses. The nonstructural proteins involved in viral replication (proteases, polymerases and helicases, etc. replicases) are still highly conserved. Inhibitors against highly conserved viral replicases are more suitable for broad-spectrum antiviral drug development.

According to the coronavirus structure, the targeted, efficient and low-toxicity medicine for treating the virus is found, and has important significance. SARS-CoV-2 encodes 16 nonstructural proteins, and as the protein structure is resolved one by one, drug development techniques based on coronavirus druggable target structures have begun to be widely adopted, with representative successful marketing of the Mpro-targeted inhibitor Paxlovid. SARS-CoV-2 encoded papain (PLpro) is a catalytic domain of NSP3 protein, and is responsible for maturation cleavage of viral precursor proteins, which are essential proteins for replication of the virus in cells, in conjunction with Mpro. In addition PLpro also mediates de-ubiquitination and de-ISG of anti-viral proteins, thereby participating in modulating host antiviral innate immunity. PLpro multiple functions in the viral life cycle make it an important development target for anti-novel coronavirus drugs. GRL0617 is a non-covalent binding inhibitor which is found after screening aiming at SARS-CoV PLpro, has higher inhibition activity and target enzyme affinity, the three-dimensional structure of PLpro is analyzed at present, the compound crystal structure of the GRL0617 is also reported, and the research results lay a foundation for the research and development of PLpro inhibitors.

Disclosure of Invention

The invention aims to provide a substituted oxadiazole-aniline compound which has a novel structure and PLpro activity against coronavirus SARS-CoV/SARS-CoV-2. The invention discovers that the substituted oxadiazole-aniline compound has strong PLpro inhibition activity and anti-coronavirus SARS-CoV-2 effect, has low cytotoxicity, can be used for treating or preventing infectious diseases caused by viruses, particularly diseases caused by novel coronaviruses, and has been completed based on the discovery.

Summary of The Invention

To this end, a first aspect of the present invention provides a compound of formula (I) and isomers thereof, or pharmaceutically acceptable salts thereof,

Wherein,

R ₁ is hydrogen, methyl, ethyl, propyl, isopropyl, hydroxy;

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

R ₃ is independently selected from hydrogen, F, cl, br, trifluoromethyl, trifluoromethoxy, C ₁-C₃ alkyl, C ₁-C₃ alkoxy, n represents 1,2, 3 or 4 substituents;

R ₄ is substituted or unsubstituted phenyl, substituted or unsubstituted thienyl;

The substituents in R ₂ or R ₄ may be selected from F, cl, br, hydroxy, amino, nitro, cyano, monofluoromethyl, difluoromethyl, trifluoromethyl, trifluoromethoxy, thienyl, vinyl, ethynyl, hydroxymethyl, carboxy, sulfonate, borate, methylthio, C ₁-C₄ alkyl, C ₁-C₄ alkoxy.

In a preferred embodiment, the compound has the structural formula (II):

Wherein,

R ₁ is hydrogen, methyl or hydroxy;

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

R ₃ is independently selected from hydrogen, F, cl, br, trifluoromethyl, trifluoromethoxy, C ₁-C₃ alkyl, C ₁-C₃ alkoxy, n represents 1,2, 3 or 4 substituents;

R ₅ is hydrogen, cyano, carboxyl, trifluoromethyl, methoxy, morpholinyl, alkynyl, acetamido, acetyl, hydroxy, hydroxymethyl;

the substituents in R ₂ may be any selected from F, cl, br, hydroxy, amino, nitro, cyano, monofluoromethyl, difluoromethyl, trifluoromethyl, trifluoromethoxy, hydroxymethyl, C ₁-C₄ alkyl, C ₁-C₄ alkoxy.

In another preferred embodiment, the compound has the structural formula (III):

Wherein,

R ₁ is hydrogen, methyl or hydroxy;

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

R ₃ is independently selected from hydrogen, F, cl, br, trifluoromethyl, trifluoromethoxy, C ₁-C₃ alkyl, C ₁-C₃ alkoxy, n represents 1,2, 3 or 4 substituents;

R ₅ is hydrogen, carboxyl, cyano or boric acid group;

The substituent in R ₂ may be any one selected from F, cl, br, hydroxy, amino, nitro, cyano, monofluoromethyl, difluoromethyl, trifluoromethyl, trifluoromethoxy, thienyl, hydroxymethyl, C ₁-C₄ alkyl, C ₁-C₄ alkoxy.

The pharmaceutically acceptable salts of the compounds of the present invention are those formed 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 or trifluoroacetic acid. Hydrochloric acid, p-toluenesulfonic acid or trifluoroacetic acid are preferred.

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

A compound according to any one of the first aspects of the invention which is a compound selected from the group consisting of:

in a second aspect the present invention provides a process for the preparation of a compound according to any one of the first aspects of the invention comprising the steps of:

Compound a is reacted 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 under the protection of air or inert gas (Ar or N ₂) at-10 ℃ to 50 ℃ for 1 to 24 hours, preferably room temperature for 2 to 3 hours, followed by heating to reflux for 1 to 24 hours, preferably 6 to 12 hours, to give a compound represented by formula C;

The compound shown in the formula C is reacted for 1 to 24 hours at-10 ℃ to 60 ℃ under the action of ammonium chloride and metal powder (zinc powder, iron powder, preferably zinc powder) in a proper solvent (such as methanol, ethanol, methanol/water, ethanol/water, preferably methanol/water), wherein the room temperature is preferred for 2 to 4 hours to obtain the compound shown in the formula D;

The compound of formula D is reacted with the compound R ₄ CHO 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) with a reducing agent (e.g., sodium borohydride, sodium cyanoborohydride, sodium triacetoxyborohydride, preferably sodium triacetoxyborohydride) at-10℃to 50℃for 1 to 24 hours, wherein room temperature is preferred for 6 to 12 hours to give the compound of formula (I).

In a third aspect, the invention provides the use of a compound according to any one of the first aspects of the invention, or an isomer thereof, or a pharmaceutically acceptable salt thereof, in the preparation of a papain (PLpro) inhibitor.

In a fourth aspect, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of a compound according to any one of the first aspect of the present invention, or an isomer thereof, or a pharmaceutically acceptable salt thereof, and optionally one or more pharmaceutically acceptable excipients.

In a fifth aspect, the present invention provides the use of a compound according to any one of the first aspects of the present invention, or an isomer thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to any one of the fourth aspects of the present invention, in the manufacture of a medicament for the treatment and/or prophylaxis of infectious diseases caused by coronaviruses.

The foregoing merely outlines certain aspects of the invention and is not limited in this regard. These and other aspects are described more fully below.

Detailed Description

Various aspects and features of the invention are described further below.

Various terms and phrases used herein have the ordinary and customary meaning as understood by those skilled in the art, and even though they are still intended to be more fully described and explained herein, the terms and phrases used herein are to be understood and to have a meaning inconsistent with the ordinary and customary meaning as set forth herein. The following are definitions of various terms used in the present application, which are applicable to terms used throughout the specification of the present application unless otherwise specified in the specific context.

In general, the term "substituted or unsubstituted" means that one or more hydrogen atoms in a given structure are replaced by a specific substituent. An optional substituent group may be substituted at each substitutable position of the group unless otherwise indicated. When a given structure is always substituted in more than one position with one or more substituents selected from the group consisting of specific groups, then the substituents may be the same or different at each position.

C _i-C_j represents a moiety having an integer of "i" (including i) to an integer of "j" (including j) carbon atoms. Thus, for example, a C ₁-C₃ alkyl group refers to an alkyl group having 1 to 3 (including 1 and 3) carbon atoms.

As used herein, the term "alkyl" refers to an alkyl group having the indicated number of carbon atoms, which is a straight or branched chain alkyl group, and which may include sub-groups thereof, for example where reference is made to "C ₁-C₃ alkyl" it may also include sub-ranges of groups represented by C ₁-C₂ alkyl groups, as well as specific groups such as methyl, ethyl, n-propyl, isopropyl.

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

As described herein, the term "haloalkyl" means that a hydrogen on an alkyl group is substituted with one or more halogen atoms, examples of which include, but are not limited to, monofluoromethyl, monofluoromethoxy, and the like.

As described herein, the term "ring" means a substituted or unsubstituted cycloalkyl group. The so-called rings include fused rings. The number of atoms on a ring is generally defined as the number of ring elements, e.g. "C ₃-C₆ ring" means 3-6 atoms arranged around a ring.

As described herein, the terms "halogen", "halo", and the like, represent fluorine (F), chlorine (Cl), or bromine (Br).

"Room temperature" in the present invention means a temperature of from 10 ℃ to 40 ℃. In some embodiments, "room temperature" refers to temperatures from 20 ℃ to 30 ℃, and in other embodiments, room temperature refers to 25 ℃.

As used herein, the term "effective amount" refers to the amount of drug that achieves the desired treatment of a disease or disorder described herein in a subject.

As used herein, the term "pharmaceutically acceptable" means, for example, when describing a "pharmaceutically acceptable salt," that salt is not only physiologically acceptable to the subject, but also refers to a synthetic substance that is pharmaceutically useful.

As used herein, the term "pharmaceutical composition," which may also refer to "compositions," may be used to effect treatment of a disease or disorder described herein in a subject, particularly a mammal.

"Treatment" of a disease includes:

(1) Preventing the disease, i.e., preventing a mammal exposed to or susceptible to the disease but not experiencing or exhibiting symptoms of the disease from developing clinical symptoms of the disease,

(2) Inhibiting the disease, i.e., preventing or reducing the progression of the disease or its clinical symptoms,

(3) Alleviating the disease, i.e., causing the recovery of the disease or its clinical symptoms.

"Therapeutically effective amount" refers to the amount of a compound that is sufficient to effect treatment of a disease when administered to a mammal for the purpose of treating 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, etc. of the mammal. A therapeutically effective amount may also refer to any amount of a compound that is sufficient to achieve the desired benefit, including prevention of a disease, inhibition of a disease, or alleviation of a disease as described in (1) - (3) above. For example, the amount of the compound may be in the range of 0.1 to 250mg/kg, or preferably, 0.5 to 100mg/kg, or more preferably, 1 to 50mg/kg, or even more preferably, 2 to 20mg/kg. Preferably, the amount of the compound is administered to the mammal twice daily. More preferably, the amount of the compound is administered to the mammal once daily.

As used herein, the term "disease and/or disorder" refers to a physical state of the subject that is associated with the disease and/or disorder of the present invention. For example, the diseases and/or conditions described herein refer to Bacillus tuberculosis infectious diseases.

As used herein, the term "subject" may refer to a patient or other animal, particularly a mammal, e.g., human, monkey, etc., receiving a compound of formula I of the invention or a pharmaceutical composition thereof for treatment of a disease or condition described herein.

In a further aspect the invention relates to pharmaceutical compositions comprising the compounds of the invention as active ingredient. The pharmaceutical compositions may be prepared according to methods well known in the art. Any dosage form suitable for human or animal use may be made by combining the compounds of the invention with one or more pharmaceutically acceptable solid or liquid excipients and/or adjuvants.

The compound of the present invention or a pharmaceutical composition containing the same may be administered in unit dosage form by the enteral or parenteral route such as oral, intravenous, intramuscular, subcutaneous, nasal, oral mucosal, ocular, pulmonary and respiratory routes, skin, vagina, rectum and the like.

The dosage form may be a liquid, solid or semi-solid dosage form. The liquid dosage forms can be solutions (including true solutions and colloidal solutions), emulsions (including o/w type, w/o type and multiple emulsions), suspensions, injections (including water injection, powder injection and transfusion), eye drops, nasal drops, lotions, liniments and the like, the solid dosage forms can be tablets (including common tablets, enteric-coated tablets, buccal tablets, dispersible tablets, chewable tablets, effervescent tablets, orally disintegrating tablets), capsules (including hard capsules, soft capsules and enteric-coated capsules), granules, powders, pellets, dripping pills, suppositories, films, patches, aerosol (powder) mists, sprays and the like, and the semisolid dosage forms can be ointments, gels, pastes and the like.

The compound of the invention can be prepared into common preparations, slow release preparations, controlled release preparations, targeted preparations and various microparticle administration systems.

For the preparation of the compounds of the present invention into tablets, various excipients known in the art may be widely used, including diluents, binders, wetting agents, disintegrants, lubricants, cosolvents. The diluent can be starch, dextrin, sucrose, glucose, lactose, mannitol, sorbitol, xylitol, microcrystalline cellulose, calcium sulfate, calcium hydrophosphate, calcium carbonate and the like, the wetting agent can be water, ethanol, isopropanol and the like, the binder can 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, polyvinylpyrrolidone, polyethylene glycol and the like, the disintegrating agent can be dry starch, microcrystalline cellulose, low-substituted hydroxypropyl cellulose, crosslinked polyvinylpyrrolidone, crosslinked sodium carboxymethyl cellulose, sodium carboxymethyl starch, sodium bicarbonate, citric acid, polyoxyethylene sorbitol fatty acid ester, sodium dodecyl sulfonate and the like, and the lubricant and the cosolvent can be talcum powder, silicon dioxide, stearate, tartaric acid, liquid paraffin, polyethylene glycol and the like.

The tablets may be further formulated into coated tablets, such as sugar coated tablets, film coated tablets, enteric coated tablets, or bilayer and multilayer tablets.

In order to prepare the administration unit into a capsule, the compound of the present invention as an active ingredient may be mixed with a diluent and a cosolvent, and the mixture may be directly placed in a hard capsule or a soft capsule. The active ingredient of the compound can be prepared into particles or pellets by mixing with a diluent, an adhesive and a disintegrating agent, and then placed into hard capsules or soft capsules. The various diluents, binders, wetting agents, disintegrants and co-solvents used to prepare tablets of the compounds of the invention may also be used to prepare capsules of the compounds of the invention.

For the preparation of the compound of the present invention into injection, water, ethanol, isopropanol, propylene glycol or their mixture may be used as solvent, and appropriate amount of solubilizer, cosolvent, pH regulator and osmotic pressure regulator commonly used in the art may be added. The solubilizer or cosolvent can be poloxamer, lecithin, hydroxypropyl-beta-cyclodextrin, etc., the pH regulator can be phosphate, acetate, hydrochloric acid, sodium hydroxide, etc., and the osmotic pressure regulator can be sodium chloride, mannitol, glucose, phosphate, acetate, etc. For example, mannitol, glucose, etc. can be added as propping agent for preparing lyophilized powder for injection.

In addition, colorants, preservatives, fragrances, flavoring agents, or other additives may also be added to the pharmaceutical formulation, if desired.

For the purpose of administration, the drug or the pharmaceutical composition of the present invention can be administered by any known administration method to enhance the therapeutic effect.

The compounds or compositions of the present invention may be administered alone or in combination with other therapeutic or symptomatic agents. When the compound of the present invention has a synergistic effect with other therapeutic agents, its dosage should be adjusted according to the actual circumstances.

Beneficial technical effects

The compound has stronger PLpro inhibition activity, the inhibition effect (IC ₅₀) of 11 compounds is less than 30 mu M, and the inhibition activity IC ₅₀ of PLpro of 8 compounds is less than 10 mu M.2 compounds have better antiviral activity than PLpro inhibitor GRL0617 and show stronger anti-coronavirus effect. In addition, the compounds have low cytotoxicity (IC ₅₀ >100 mu M) to Vero E6, and the compounds are shown to have better safety. The invention provides a novel compound with novel structure, strong in vitro PLpro inhibition activity and low cytotoxicity, which can be used for inhibiting the application of PLpro in medicaments, in particular to the application of the novel compound as a therapeutic agent for diseases related to coronavirus infection.

Detailed Description

The invention will now be described in more detail by way of the following examples, which are not meant to be limiting in any way. The present invention has been described in detail herein, and specific embodiments thereof are also 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 invention without departing from the spirit and scope of the invention.

For all of the following examples, standard procedures and purification methods known to those skilled in the art may be used. The structure of the compound was determined by nuclear magnetic resonance hydrogen spectroscopy (¹ H NMR).

Preparation example section

The structure of the compound was determined by nuclear magnetic resonance hydrogen spectroscopy (¹ H NMR). The nuclear magnetic resonance hydrogen spectral shift (δ) is given in parts per million (ppm). The coupling constant (J) is in hertz (Hz). Nuclear magnetic resonance spectroscopy was performed using 400Hz, 500Hz, or 700Hz nmr, with deuterated dimethyl sulfoxide (DMSO-d ₆) or deuterated chloroform (CDCl ₃) as solvents, and Tetramethylsilane (TMS) as an internal standard.

Example 1

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)

The synthetic route is as follows:

The experimental steps are as follows:

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)

2-Chloro-4, 6-dimethoxy-1, 3, 5-triazine (CDMT, 5250mg,30.0 mmol) and N-methylmorpholine (NMM, 90.90 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, 7320mg,30.0 mmol) was added, stirred at room temperature for 60 minutes, and (Z) -N' -hydroxy-4-methyl-5-nitrobenzamidine (B-1, 5265mg,30.0 mmol) was added, stirred at room temperature for 2 hours, and heated to reflux for 6 hours. Concentrated under reduced pressure, and separated by silica gel column chromatography (ethyl acetate/petroleum ether=5/95) to give intermediate C-1 as a white solid (7.39 g, yield 61%).

¹H NMR(400MHz,DMSO-d₆)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,2H),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).

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)

In a 25mL reaction flask, intermediate 5- (1- (2-fluoro- [1,1' -biphenyl ] -4-yl) ethyl) -3- (4-methyl-3-nitrophenyl) -1,2, 4-oxadiazole (C-1, 404mg,1.0 mmol) was dissolved in 5mL methanol and 5mL dichloromethane, saturated aqueous ammonium chloride solution (5 mL) was added, stirred well, zinc powder (268 mg,20.0 mmol) was added, and stirred at room temperature for 3 hours. Filtration, concentration under reduced pressure, separation by silica gel column chromatography (methanol/dichloromethane=3/97) gave intermediate D-1 as an off-white solid 345mg in 93% yield.

¹H NMR(400MHz,CDCl₃)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).

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) aniline (Compound 1)

In a 25mL reaction flask, intermediate 5- (5- (1- (2-fluoro- [1,1' -biphenyl ] -4-yl) ethyl) -1,2, 4-oxadiazol-3-yl) -2-methylaniline (D-1, 80mg,0.21 mmol), 3-trifluoromethylbenzaldehyde (73 mg,0.42 mmol) and glacial acetic acid (75 mg,1.26 mmol) were dissolved in 1, 2-dichloroethane (8 mL) and sodium triacetoxyborohydride (267 mg,1.26 mmol) was slowly added after stirring at room temperature for 3 hours. Concentrating under reduced pressure, and separating with silica gel column chromatography (methanol/dichloromethane=10/90) to obtain compound I-1 (compound 1), and the yield is 47% as off-white solid 53 mg.

¹H NMR(400MHz,CDCl₃)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),7.30(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).

Example 2

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)

Using 3-methoxybenzaldehyde (57 mg,0.42 mmol) as a starting material and following a similar procedure to the third step in example 1, it was reacted with intermediate D-1 to give compound I-2 (compound 2) as an off-white solid 58mg in 56% yield.

¹H NMR(400MHz,CDCl₃)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).

Example 3

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

Using 4- (4-morpholinyl) benzaldehyde (80 mg,0.42 mmol) as a starting material and following a similar procedure to the third step in example 1, reaction with intermediate D-1 gave compound I-3 (compound 3) as an off-white solid 66mg in 64% yield.

¹H NMR(400MHz,CDCl₃)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).

Example 4

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

Using 4-ethynylbenzaldehyde (55 mg,0.42 mmol) as a starting material and following a similar procedure to the third step in example 1, reaction with intermediate D-1 gave compound I-4 (compound 4) as an off-white solid 49mg in 48% yield.

¹H NMR(400MHz,CDCl₃)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).

Example 5

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

Using acetamidobenzaldehyde (69 mg,0.42 mmol) as a starting material and following a similar procedure to the third step in example 1, reaction with intermediate D-1 gave compound I-5 (compound 5), as an off-white solid, 47mg in 43% yield.

¹H NMR(400MHz,CDCl₃)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).

Example 6

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)

Using 4-acetophenone (62 mg,0.42 mmol) as a starting material and following a similar procedure to the third step in example 1, reaction with intermediate D-1 gave compound I-6 (compound 6), 55mg as an off-white solid in 52% yield.

¹H NMR(400MHz,CDCl₃)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),7.22–7.15(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).

Example 7

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)

Using m-hydroxybenzaldehyde (51 mg,0.42 mmol) as a starting material and following the similar procedure in the third step of example 1, it was reacted with intermediate D-1 to give compound I-7 (compound 7), as an off-white solid, 50mg, in 50% yield.

¹H NMR(400MHz,CDCl₃)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,1H),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).

Example 8

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)

Using 3, 5-dihydroxybenzaldehyde (58 mg,0.42 mmol) as a starting material and following a similar procedure to the third step in example 1, it was reacted with intermediate D-1 to give compound I-8 (compound 8) as an off-white solid 44mg in 42% yield.

¹H NMR(400MHz,CDCl₃)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).

Example 9

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)

Using p-hydroxymethylbenzaldehyde (57 mg,0.42 mmol) as a starting material and following a similar procedure to the third step in example 1, it was reacted with intermediate D-1 to give compound I-9 (compound 9) as an off-white solid, 52mg, yield 50%.

¹H NMR(400MHz,CDCl₃)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).

Example 10

N- (3-carboxy-benzyl) -4-methyl-3- (5-benzyl-1, 2, 4-oxadiazol-3-yl) aniline

The synthetic route is as follows:

The experimental steps are as follows:

First step preparation of 5-benzyl-3- (4-methyl-3-nitrophenyl) -1,2, 4-oxadiazole (C-2)

2-Chloro-4, 6-dimethoxy-1, 3, 5-triazine (CDMT, 5250mg,30.0 mmol) and N-methylmorpholine (NMM, 90.90 mg,90.0 mmol) were dissolved in 1, 4-dioxane (150 mL) in a 100mL reaction flask, stirred at room temperature for 15 minutes, 2-phenylacetic acid (A-10, 4085mg,30 mmol) was added, stirred at room temperature for 60 minutes, and (Z) -N' -hydroxy-2-methyl-5-nitrobenzamidine (5265 mg,30.0 mmol) was added, stirred at room temperature for 2 hours and heated to reflux for 6 hours. Concentrated under reduced pressure, and separated by silica gel column chromatography (ethyl acetate/petroleum ether=5/95) to give intermediate C-2 as a white solid (6.33 g, yield 71%).

Second step preparation of 5- (5-benzyl-1, 2, 4-oxadiazol-3-yl) -2-methylaniline (D-2)

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

¹H NMR(400MHz,CDCl₃)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).

Third step preparation of N- (3-carboxy-benzyl) -4-methyl-3- (5-benzyl-1, 2, 4-oxadiazol-3-yl) aniline (Compound 10)

In a 25mL reaction flask, intermediate 5- (5-benzyl-1, 2, 4-oxadiazol-3-yl) -2-methylaniline (D-2, 56mg,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) and after stirring at room temperature for 3 hours, sodium triacetoxyborohydride (267 mg,1.26 mmol) was slowly added. Concentrating under reduced pressure, and separating with silica gel column chromatography (methanol/dichloromethane=10/90) to obtain compound I-10 (compound 10), off-white solid 55mg, yield 66%.

¹H NMR(400MHz,CDCl₃)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).

Example 11

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

The synthetic route is as follows:

The experimental steps are as follows:

first step preparation of 5-benzyl-3- (3-nitro-5- (trifluoromethyl) phenyl) -1,2, 4-oxadiazole (C-3)

2-Chloro-4, 6-dimethoxy-1, 3, 5-triazine (CDMT, 5250mg,30.0 mmol), N-methylmorpholine (NMM, 90.90 mg,90.0 mmol) were dissolved in 1, 4-dioxane (150 mL), stirred at room temperature for 15 min, 2-phenylacetic acid (A-2, 4085mg,30 mmol) was added, stirred at room temperature for 60 min, (Z) -N' -hydroxy-3-nitro-5- (trifluoromethyl) benzamide (B-3, 7474mg,30 mmol) was added, stirred at room temperature for 2h, and heated to reflux for 6 h. Concentrated under reduced pressure, and separated by silica gel column chromatography (ethyl acetate/petroleum ether=5/95) to give intermediate C-3 as a white solid (7.22 g, yield 70%).

¹H NMR(400MHz,CDCl₃)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).

Second step preparation of 3-trifluoromethyl-5- (5-benzyl-1, 2, 4-oxadiazol-3-yl) aniline (D-3)

In a 25mL reaction flask, intermediate 5-benzyl-3- (3-nitro-5- (trifluoromethyl) phenyl) -1,2, 4-oxadiazole (C-3, 349mg,1.0 mmol) was dissolved in 5mL methanol and 5mL dichloromethane, saturated aqueous ammonium chloride solution (5 mL) was added, stirred well, zinc powder (428 mg,20.0 mmol) was added, and stirred at room temperature for 3 hours. Filtration, concentration under reduced pressure, separation by silica gel column chromatography (methanol/dichloromethane=3/97) to give intermediate D-3 as an off-white solid 270mg in 85% yield.

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)

In a 25mL reaction flask, intermediate 3-trifluoromethyl-5- (5-benzyl-1, 2, 4-oxadiazol-3-yl) aniline (D-3, 67mg,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 after stirring at room temperature for 3 hours, sodium triacetoxyborohydride (267 mg,1.26 mmol) was slowly added. Concentrating under reduced pressure, and separating with silica gel column chromatography (methanol/dichloromethane=10/90) to obtain compound I-11 (compound 11), off-white solid 61mg, yield 63%.

¹H NMR(400MHz,DMSO-d₆)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).

Example 12

5- (((3- (5- (1-Phenylethyl) -1,2, 4-oxadiazol-3-yl) -5- (trifluoromethyl) phenyl) amino) methyl) thiophene-2-carboxylic acid

The synthetic route is as follows:

The experimental steps are as follows:

First step preparation of 3- (3-nitro-5- (trifluoromethyl) phenyl) -5- (1-phenethyl) -1,2, 4-oxadiazole (C-4)

2-Chloro-4, 6-dimethoxy-1, 3, 5-triazine (CDMT, 5250mg,30.0 mmol), N-methylmorpholine (NMM, 90.90 mg,90.0 mmol) were dissolved in1, 4-dioxane (150 mL), stirred at room temperature for 15 min, 2-phenylpropionic acid (A-3, 4500mg,30.0 mmol) was added, stirred at room temperature for 60 min, and (Z) -N' -hydroxy-3-nitro-5- (trifluoromethyl) benzamide (B-3, 7474mg,30.0 mmol) was added, stirred at room temperature for 2h, and heated to reflux for 6 h. Concentrated under reduced pressure, and separated by silica gel column chromatography (ethyl acetate/petroleum ether=5/95) to give intermediate C-4 as a white solid (7.11 g, yield 65%).

¹H NMR(400MHz,CDCl₃)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).

Second step preparation of 3-trifluoromethyl-5- (5- (1-phenylethyl) -1,2, 4-oxadiazol-3-yl) aniline (D-4)

In a 25mL reaction flask, intermediate 3- (3-nitro-5- (trifluoromethyl) phenyl) -5- (1-phenethyl) -1,2, 4-oxadiazole (C-4,803 mg,1.0 mmol) was dissolved in 5mL methanol and 5mL dichloromethane, saturated aqueous ammonium chloride solution (5 mL) was added, stirred well, zinc powder (428 mg,20.0 mmol) was added, and stirred at room temperature for 3 hours. Filtration, concentration under reduced pressure, separation by silica gel column chromatography (methanol/dichloromethane=3/97) gave intermediate D-4 as an off-white solid 271mg in 81% yield.

¹H NMR(400MHz,CDCl₃)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).

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)

In a 25mL reaction flask, intermediate 3-trifluoromethyl-5- (5-benzyl-1, 2, 4-oxadiazol-3-yl) aniline (D-4, 70mg,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 sodium triacetoxyborohydride (267 mg,1.26 mmol) was slowly added. Concentrating under reduced pressure, and separating with silica gel column chromatography (methanol/dichloromethane=10/90) to obtain compound I-12 (compound 12), off-white solid 66mg, yield 66%.

¹H NMR(400MHz,DMSO-d₆)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).

Example 13

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

The synthetic route is as follows:

The experimental steps are as follows:

first step preparation of (3- (3-nitro-5- (trifluoromethyl) phenyl) -1,2, 4-oxadiazol-5-yl) (phenyl) methanol (C-5)

2-Chloro-4, 6-dimethoxy-1, 3, 5-triazine (CDMT, 5250mg,30.0 mmol), N-methylmorpholine (NMM, 90.90 mg,90.0 mmol) were dissolved in 1, 4-dioxane (150 mL), stirred at room temperature for 15 min, 2-hydroxy-2-phenylacetic acid (A-4, 4564mg,30.0 mmol) was added, stirred at room temperature for 60min, (Z) -N' -hydroxy-3-nitro-5- (trifluoromethyl) benzamide (B-3, 7474mg,30.0 mmol) was added, stirred at room temperature for 2h, and heated to reflux for 6 h. Concentrated under reduced pressure, and separated by silica gel column chromatography (ethyl acetate/petroleum ether=5/95) to give intermediate C-5 as a white solid (7.00 g) in 64% yield.

¹H NMR(400MHz,CDCl₃)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).

Second step preparation of 3-trifluoromethyl-5- (5- (hydroxy (phenyl) methyl) -1,2, 4-oxadiazol-3-yl) aniline (D-5)

In a 25mL reaction flask, intermediate (3- (3-nitro-5- (trifluoromethyl) phenyl) -1,2, 4-oxadiazol-5-yl) (phenyl) methanol (C-5, 365mg,1.0 mmol) was dissolved in 5mL methanol and 5mL dichloromethane, saturated aqueous ammonium chloride solution (5 mL) was added, stirred well, zinc powder (428 mg,20.0 mmol) was added, and stirred at room temperature for 3 hours. Filtration, concentration under reduced pressure, separation by silica gel column chromatography (methanol/dichloromethane=3/97) gave intermediate D-5 as an off-white solid 297mg in 89% yield.

¹H NMR(400MHz,CDCl₃)δ7.72(s,1H),7.56–7.49(m,3H),7.46–7.36(m,3H),7.01(s,1H),6.09(s,1H).

Third step 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)

In a 25mL reaction flask, intermediate 3-trifluoromethyl-5- (5- (hydroxy (phenyl) methyl) -1,2, 4-oxadiazol-3-yl) aniline (D-5, 70mg,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 sodium triacetoxyborohydride (267 mg,1.26 mmol) was slowly added after stirring at room temperature for 3 hours. Concentrating under reduced pressure, and separating with silica gel column chromatography (methanol/dichloromethane=10/90) to obtain compound I-13 (compound 13), off-white solid 52mg, yield 52%.

¹H NMR(400MHz,DMSO-d₆)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).

Example 14

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

The synthetic route is as follows:

The experimental steps are as follows:

first step preparation of (3- (4-methyl-3-nitrophenyl) -1,2, 4-oxadiazol-5-yl) (phenyl) methanol (C-6)

2-Chloro-4, 6-dimethoxy-1, 3, 5-triazine (CDMT, 5250mg,30.0 mmol), N-methylmorpholine (NMM, 90.90 mg,90.0 mmol) were dissolved in1, 4-dioxane (150 mL), stirred at room temperature for 15 min, 2-hydroxy-2-phenylacetic acid (A-5, 4564mg,30.0 mmol) was added, stirred at room temperature for 60 min, and (Z) -N' -hydroxy-4-methyl-3-nitrobenzamidine amide (B-1, 5585mg,30.0 mmol) was added, stirred at room temperature for 2h, and heated to reflux for 6 h. Concentrated under reduced pressure, and separated by silica gel column chromatography (ethyl acetate/petroleum ether=5/95) to give intermediate C-6 as a white solid (6.67 g, yield 71%).

¹H NMR(400MHz,CDCl₃)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).

Second step preparation of (3- (3-amino-4-methylphenyl) -1,2, 4-oxadiazol-5-yl) (phenyl) methanol (D-6)

In a 25mL reaction flask, intermediate (3- (4-methyl-3-nitrophenyl) -1,2, 4-oxadiazol-5-yl) (phenyl) methanol (C-6, 311mg,1.0 mmol) was dissolved in 5mL methanol and 5mL dichloromethane, saturated aqueous ammonium chloride solution (5 mL) was added, stirred well, zinc powder (428 mg,20.0 mmol) was added, and stirred at room temperature for 3 hours. Filtration, concentration under reduced pressure, separation by silica gel column chromatography (methanol/dichloromethane=3/97) to give intermediate D-6 as an off-white solid 252mg in 90% yield.

¹H NMR(400MHz,DMSO-d₆)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).

Third step 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)

In a 25mL reaction flask, intermediate (3- (3-amino-4-methylphenyl) -1,2, 4-oxadiazol-5-yl) (phenyl) methanol (D-6, 59mg,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 after stirring at room temperature for 3 hours, sodium triacetoxyborohydride (267 mg,1.26 mmol) was slowly added. Concentrating under reduced pressure, and separating with silica gel column chromatography (methanol/dichloromethane=10/90) to obtain compound I-14 (compound 14), off-white solid 55mg, yield 62%.

¹H NMR(400MHz,DMSO-d₆)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),7.12–7.06(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).

Example 15

(5- (((5- (5- (1- (4-Isobutylphenyl) ethyl) -1,2, 4-oxadiazol-3-yl) -2-methylphenyl) amino) methyl) thiophen-2-yl) boronic acid

The synthetic route is as follows:

The experimental steps are as follows:

first step preparation of 5- (1- (4-isobutylphenyl) ethyl) -3- (4-methyl-3-nitrophenyl) -1,2, 4-oxadiazole (C-7)

2-Chloro-4, 6-dimethoxy-1, 3, 5-triazine (CDMT, 700mg,4.0 mmol), N-methylmorpholine (NMM, 1212mg,12.0 mmol) are dissolved in 1, 4-dioxane (30 mL), stirred at room temperature for 15 min, 2- (4-isobutylphenyl) propionic acid (A-6, 284 mg,4.0 mmol) is added, stirred at room temperature for 60 min, and (Z) -N' -hydroxy-2-methyl-5-nitrobenzamidine (B-1, 702mg,3.6 mmol) is added, stirred at room temperature for 2h, and heated to reflux for 6h. Concentrated under reduced pressure, and separated by silica gel column chromatography (ethyl acetate/petroleum ether=5/95) to give intermediate C-7 as a white solid 837mg in 57% yield.

¹H NMR(400MHz,CDCl₃)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).

Second step preparation of 5- (5- (1- (4-isobutylphenyl) ethyl) -1,2, 4-oxadiazol-3-yl) -2-methylaniline (D-7)

In a 25mL reaction flask, intermediate 5- (1- (4-isobutylphenyl) ethyl) -3- (4-methyl-3-nitrophenyl) -1,2, 4-oxadiazole (C-7, 365mg,1.0 mmol) was dissolved in 5mL of methanol and 5mL of dichloromethane, saturated aqueous ammonium chloride solution (5 mL) was added, stirred well, zinc powder (428 mg,20.0 mmol) was added, and stirred at room temperature for 3 hours. Filtration, concentration under reduced pressure, separation by silica gel column chromatography (methanol/dichloromethane=3/97) gave intermediate D-7 as an off-white solid 247mg in 73% yield.

¹H NMR(400MHz,CDCl₃)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).

Third step (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)

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

¹H NMR(400MHz,CDCl₃)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＝7.2Hz,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).

Example 16

5- (((5- (5- (1- (4-Isobutylphenyl) ethyl) -1,2, 4-oxadiazol-3-yl) -2-methylphenyl) amino) methyl) thiophene-2-carboxylic acid

Using 5-formyl-2-thiophenecarboxylic acid (115 mg,1.0 mmol) as a starting material, and the similar procedure as in the third step of example 15, was reacted with intermediate D-7 to give compound I-16 (compound 16), an off-white solid 123mg, yield 52%.

¹H NMR(400MHz,DMSO-d₆)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).

Example 17

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

Using 5-cyanothiophene-2-carbaldehyde (58 mg,0.42 mmol) as a starting material and the similar procedure as in the third step of example 1, reacted with intermediate D-1 to give compound I-17 (compound 17), an off-white solid, 55mg, yield 53%.

¹H NMR(400MHz,DMSO-d₆)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),7.24–7.17(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).

Example 18

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

Using 5-formyl-2-thiophenecarboxylic acid (49 mg,0.42 mmol) as a starting material and the similar procedure as in the third step of example 1, the reaction was carried out with intermediate D-1 to give compound I-18 (compound 18), an off-white solid, 41mg, yield 38%.

¹H NMR(400MHz,DMSO-d₆)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).

Example 19

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

The synthetic route is as follows:

The experimental steps are as follows:

first step preparation of 3- (4-methyl-3-nitrophenyl) -5- (1- (3-phenoxyphenyl) ethyl) -1,2, 4-oxadiazole (C-8)

2-Chloro-4, 6-dimethoxy-1, 3, 5-triazine (CDMT, 350mg,2.0 mmol), N-methylmorpholine (NMM, 606mg,6.0 mmol) were dissolved in 1, 4-dioxane (20 mL), stirred at room temperature for 15min, 2- (3-phenoxyphenyl) propionic acid (A-7, 284 mg,2.0 mmol) was added, stirred at room temperature for 60 min, and (Z) -N' -hydroxy-2-methyl-5-nitrobenzamidine (B-1, 351mg,1.8 mmol) was added, stirred at room temperature for 2h, and heated to reflux for 6 h. Concentrated under reduced pressure, and separated by silica gel column chromatography (ethyl acetate/petroleum ether=5/95) to give intermediate C-8 as a white solid 577mg in 72% yield.

¹H NMR(400MHz,DMSO-d₆)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).

Second step preparation of 2-methyl-5- (5- (1- (3-phenoxyphenyl) ethyl) -1,2, 4-oxadiazol-3-yl) aniline (D-8)

In a 25mL reaction flask, intermediate 3- (4-methyl-3-nitrophenyl) -5- (1- (3-phenoxyphenyl) ethyl) -1,2, 4-oxadiazole (C-8, 300mg,0.74 mmol) was dissolved in 5mL methanol and 5mL dichloromethane, saturated aqueous ammonium chloride solution (5 mL) was added, stirred well, zinc powder (967 mg,14.8 mmol) was added, and stirred at room temperature for 3 hours. Filtration, concentration under reduced pressure, separation by silica gel column chromatography (methanol/dichloromethane=5/95) gave intermediate D-8 as an oily liquid 230mg in 84% yield.

¹H NMR(400MHz,DMSO-d₆)δ7.43(s,1H),7.37–7.35(m,2H),7.30–7.26(m,5H),7.19–7.18(m,2H),7.12–7.05(m,2H),5.17(brs,2H),4.60(q,J＝4.0Hz,1H),2.11(s,3H),1.68(d,J＝4.0Hz,3H).

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)

In a 25mL reaction flask, intermediate 2-methyl-5- (5- (1- (3-phenoxyphenyl) ethyl) -1,2, 4-oxadiazol-3-yl) aniline (D-8, 115mg,0.3 mmol), 5-aldehyde-2-thiopheneboronic acid (71 mg,0.6 mmol), glacial acetic acid (111 mg,1.86 mmol) were dissolved in 1, 2-dichloroethane (12 mL) and sodium triacetoxyborohydride (390 mg,1.86 mmol) was slowly added after stirring at room temperature for 3 hours. Concentrating under reduced pressure, and separating with silica gel column chromatography (methanol/dichloromethane=10/90) to obtain compound I-19 (compound 19), 17mg of white solid, and yield 10%.

¹H NMR(400MHz,DMSO-d₆)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).

Example 20

5- (((5- (5- (1- (3-Phenoxyphenyl) ethyl) -1,2, 4-oxadiazol-3-yl) -2-methylphenyl) amino) methyl) thiophene-2-cyano-group

Using 5-cyanothiophene-2-carbaldehyde (170 mg,1.24 mmol) as a starting material and the similar procedure of the third step in example 19, reacted with intermediate D-8 to give compound I-20 (compound 20), liquid 130mg, yield 43%.

¹H NMR(400MHz,DMSO-d₆)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).

Example 21

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

The synthetic route is as follows:

The experimental steps are as follows:

First step preparation of 5- (1- (6-chloro-9H-carbazol-2-yl)) ethyl) -3- (4-methyl-3-nitrophenyl) -1,2, 4-oxadiazole (C-9)

2-Chloro-4, 6-dimethoxy-1, 3, 5-triazine (CDMT, 700mg,4.0 mmol), N-methylmorpholine (NMM, 1212mg,12.0 mmol) are dissolved in 1, 4-dioxane (30 mL), stirred at room temperature for 15min, 2- (6-chloro-9H-carbazol-2-yl) propionic acid (A-8, 1092mg,4.0 mmol) is added, stirred at room temperature for 60 min, and (Z) -N' -hydroxy-2-methyl-5-nitrobenzamidine (B-1, 702mg,3.6 mmol) is added, stirred at room temperature for 2H, and heated to reflux for 6H. Concentrated under reduced pressure, and separated by silica gel column chromatography (ethyl acetate/petroleum ether=5/95) to give intermediate C-9 as a yellow solid 970mg in 56% yield.

¹H NMR(400MHz,DMSO-d₆)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,2H),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).

Second step preparation of 5- (1- (6-chloro-9H-carbazol-2-yl) ethyl) -3- (4-methyl-3-nitrophenyl) -1,2, 4-oxadiazole (D-9)

In a 25mL reaction flask, intermediate 5- (1- (6-chloro-9H-carbazol-2-yl)) ethyl) -3- (4-methyl-3-nitrophenyl) -1,2, 4-oxadiazole (C-9, 304mg,0.7 mmol) was dissolved in 5mL methanol and 5mL dichloromethane, saturated aqueous ammonium chloride solution (5 mL) was added, stirred well, zinc powder (15 mg,14.0 mmol) was added, and stirred at room temperature for 3 hours. Filtration, concentration under reduced pressure, silica gel column chromatography (methanol/dichloromethane=5/95) separation, intermediate D-9, white solid 205mg, yield 73%.

¹H NMR(400MHz,DMSO-d₆)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).

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)

In a25 mL reaction flask, intermediate 5- (((1- (6-chloro-9H-carbazol-2-yl)) ethyl) -1,2, 4-oxadiazolyl) -2-methylaniline (D-9, 58mg,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 sodium triacetoxyborohydride (178 mg,0.84 mmol) was slowly added after stirring at room temperature for 3 hours. Concentrating under reduced pressure, and separating with silica gel column chromatography (methanol/dichloromethane=10/90) to obtain compound I-21 (compound 21), wherein the yield is 56% and the yield is 43 mg.

¹H NMR(400MHz,DMSO-d₆)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).

Example 22

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

The synthetic route is as follows:

The experimental steps are as follows:

First step preparation of 5- (1- ([ 1,1' -biphenyl ] -3-yl) ethyl) -3- (4-methyl-3-nitrophenyl) -1,2, 4-oxadiazole (C-10)

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

Second step preparation of 5- (5- (1- ([ 1,1' -biphenyl ] -3-yl) ethyl) -1,2, 4-oxadiazol-3-yl) -2-methylaniline (D-10)

In a 25mL reaction flask, intermediate 5- (1- ([ 1,1' -biphenyl ] -3-yl) ethyl) -3- (4-methyl-3-nitrophenyl) -1,2, 4-oxadiazole (C-10, 550mg,1.4 mmol) was dissolved in 4mL methanol and 4mL dichloromethane, saturated aqueous ammonium chloride solution (7 mL) was added, stirred well, zinc powder (180 mg,28.0 mmol) was added, and stirred at room temperature for 3 hours. Filtration, concentration under reduced pressure, separation by silica gel column chromatography (methanol/dichloromethane=3/97) gave intermediate D-10 as an off-white solid 396mg in 73% yield.

¹H NMR(400MHz,CDCl₃)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).

Third step 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)

In a 25mL reaction flask, intermediate 5- (5- (1- ([ 1,1' -biphenyl ] -3-yl) ethyl) -1,2, 4-oxadiazol-3-yl) -2-methylaniline (D-10, 70mg,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 sodium triacetoxyborohydride (242 mg,1.14 mmol) was slowly added after stirring at room temperature for 3 hours. Concentrating under reduced pressure, and separating with silica gel column chromatography (methanol/dichloromethane=10/90) to obtain compound I-22 (compound 22), off-white solid 25mg, yield 26%.

¹H NMR(400MHz,DMSO-d₆)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).

Example 23

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

The synthetic route is as follows:

The experimental steps are as follows:

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)

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

¹H NMR(400MHz,CDCl₃)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).

Second step preparation of 5- (5- (1- (4 '-ethoxy- [1,1' -biphenyl ] -3-yl) ethyl) -1,2, 4-oxadiazol-3-yl) -2-methylaniline (D-11)

In a 25mL reaction flask, intermediate 5- (1- (4 '-ethoxy- [1,1' -biphenyl ] -3-yl) ethyl) -3- (4-methyl-3-nitrophenyl) -1,2, 4-oxadiazole (C-11, 377mg,1.0 mmol) was dissolved in 5mL methanol and 5mL dichloromethane, saturated aqueous ammonium chloride solution (5 mL) was added, stirred well, zinc powder (1300 mg,20.0 mmol) was added, and stirred at room temperature for 3 hours. Filtration, concentration under reduced pressure, separation by silica gel column chromatography (methanol/dichloromethane=5/95) gave intermediate D-11 as a white solid 275mg in 79% yield.

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).

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)

In a 25mL reaction flask, intermediate 5- (5- (1- (4 '-ethoxy- [1,1' -biphenyl ] -3-yl) ethyl) -1,2, 4-oxadiazol-3-yl) -2-methylaniline (D-11, 400mg,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) and sodium triacetoxyborohydride (127 mg,6.0 mmol) was slowly added after stirring at room temperature for 3 hours. Concentrating under reduced pressure, and separating with silica gel column chromatography (methanol/dichloromethane=10/90) to obtain compound I-23 (compound 23), 210mg of pale yellow solid, and yield 39%.

¹H NMR(400MHz,DMSO-d₆)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＝8.4Hz,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).

Example 24

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

The synthetic route is as follows:

First step preparation of 3- (4-methyl-3-nitrophenyl) -5- (3- (thiophen-2-yl) benzyl) -1,2, 4-oxadiazole (C-12)

2-Chloro-4, 6-dimethoxy-1, 3, 5-triazine (CDMT, 119mg,0.68 mmol) and N-methylmorpholine (NMM, 206mg,2.04 mmol) were dissolved in 1, 4-dioxane (15 mL), stirred at room temperature for 15 min, 2- (3- (thiophen-2-yl) phenyl) acetic acid (A-12, 149mg,0.68 mmol) was added, stirred at room temperature for 60min, and (Z) -N' -hydroxy-2-methyl-5-nitrobenzamidine (B-1, 118mg,0.61 mmol) was added, stirred at room temperature for 2 h, and heated to reflux for 6 h. Concentrated under reduced pressure, and separated by silica gel column chromatography (ethyl acetate/petroleum ether=5/95) to give intermediate C-12 as a yellow liquid, 215mg, in 84% yield.

¹H NMR(400MHz,CDCl₃)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).

Second step preparation of 2-methyl-5- (5- (3- (thiophen-2-yl) benzyl) -1,2, 4-oxadiazol-3-yl) aniline (D-12)

In a 25mL reaction flask, 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 5mL methanol and 5mL dichloromethane, saturated aqueous ammonium chloride solution (5 mL) was added, stirred well, zinc powder (1300 mg,20.0 mmol) was added, and stirred at room temperature for 3 hours. Filtration, concentration under reduced pressure, separation by silica gel column chromatography (methanol/dichloromethane=5/95) gave intermediate D-12 as a white solid 275mg in 79% yield.

¹H NMR(400MHz,CDCl₃)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).

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)

In a 25mL reaction flask, intermediate 2-methyl-5- (5- (3- (thiophen-2-yl) benzyl) -1,2, 4-oxadiazol-3-yl) aniline (D-12, 70mg,0.2 mmol), 5-formyl-2-thiophenecarboxylic acid (46 mg,0.4 mmol), glacial acetic acid (72 mg,1.2 mmol) were dissolved in1, 2-dichloroethane (7 mL), and after stirring at room temperature for 3 hours, sodium triacetoxyborohydride (255 mg,1.2 mmol) was slowly added. Concentrating under reduced pressure, and separating with silica gel column chromatography (methanol/dichloromethane=10/90) to obtain compound I-24 (compound 24), white solid 25mg, yield 26%.

¹H NMR(400MHz,DMSO-d₆)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＝8.0Hz,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).

Example 25

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

The synthetic route is as follows:

The experimental steps are as follows:

First step preparation of 3- (4-methyl-3-nitrophenyl) -5- (1- (3- (thiophen-2-yl) phenyl) ethyl) -1,2, 4-oxadiazole (C-13)

2-Chloro-4, 6-dimethoxy-1, 3, 5-triazine (CDMT, 376mg,2.1 mmol), N-methylmorpholine (NMM, 636mg,6.3 mmol) are dissolved in 1, 4-dioxane (30 mL), stirred at room temperature for 15 min, 2- (3- (thiophen-2-yl) phenyl) propionic acid (A-12, 490mg,2.1 mmol) is added, stirred at room temperature for 60 min, and (Z) -N' -hydroxy-4-methyl-3-nitrobenzamidine amide (B-1, 370mg,1.89 mmol) is added, stirred at room temperature for 2h, and heated to reflux for 6 h. Concentrated under reduced pressure, and separated by silica gel column chromatography (ethyl acetate/petroleum ether=5/95) to give intermediate C-13 as a white solid 589mg in 65% yield.

¹H NMR(400MHz,CDCl₃)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).

Second step preparation of 2-methyl-5- (5- (1- (3- (thiophen-2-yl) phenyl) ethyl) -1,2, 4-oxadiazol-3-yl) aniline (D-13)

In a 25mL reaction flask, intermediate 3- (4-methyl-3-nitrophenyl) -5- (1- (3- (thiophen-2-yl) phenyl) ethyl) -1,2, 4-oxadiazole (C-13, 440mg,1.1 mmol) was dissolved in 3mL methanol and 3mL dichloromethane, saturated aqueous ammonium chloride (6 mL) was added, stirred well, zinc powder (1350 mg,22.5 mmol) was added, and stirred at room temperature for 3 hours. Filtration, concentration under reduced pressure, separation by silica gel column chromatography (methanol/dichloromethane=3/97) gave intermediate D-13 as an off-white solid 371mg in 93% yield.

¹H NMR(400MHz,CDCl₃)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).

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)

In a 25mL reaction flask, the intermediate 2-methyl-5- (5- (1- (3- (thiophen-2-yl) phenyl) ethyl) -1,2, 4-oxadiazol-3-yl) aniline (D-13, 100mg,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), stirred at room temperature for 3 hours, and then sodium triacetoxyborohydride (351 mg,1.66 mmol) was slowly added. Concentrating under reduced pressure, and separating with silica gel column chromatography (methanol/dichloromethane=10/90) to obtain compound I-25 (compound 25), off-white solid 14mg, yield 10%.

¹H NMR(400MHz,DMSO-d₆)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).

Biological Activity test

1. In vitro protease inhibition Activity assay

Assay methods fluorescence assay of compounds for protease half-inhibitory concentration against SARS-CoV-2PLpro (half maximal inhibitory concentration, IC ₅₀).

Experimental principle: fluorescence quenching a substrate characteristic of PLpro protease is labeled, characteristic fluorescence is emitted if the substrate is digested, and if not digested, fluorescence is not emitted, and the digestion substrate speed of protease is detected by detecting the fluorescence values under the action concentration of different inhibitors, so that the half inhibition concentration value (IC ₅₀) of the compound against PLpro is obtained according to the digestion reaction speeds under the different inhibitor concentrations.

The experimental method comprises the following steps:

(1) Compounds were diluted in a double ratio of concentration using DMSO.

(2) Reaction buffer, different concentrations of the compound to be tested and PLpro protease are added into a 96-well transparent bottom blackboard (Greiner, 655096) in sequence according to the reaction system.

(3) And placing the reaction plate into a 37 ℃ incubator for enzymatic reaction for 8-10 minutes, and then adding the fluorogenic substrate.

(4) And (5) detecting the fluorescence intensity in real time by adopting an enzyme-labeled instrument.

(5) The data obtained by detection are subjected to data fitting by adopting Origin and Graphpad to obtain the IC ₅₀ value.

Experimental results:

TABLE 1 results of inhibition of PLpro by the inventive example compounds

From the data in table 1, the compounds of the present invention have good PLpro inhibition, wherein 8 compounds PLpro have inhibitory activity IC ₅₀ <10 μm.

2. Cytotoxicity detection

The experimental principle of MTT method is that the activity of cells is determined by reducing 3- (4, 5-dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide (trade name: thiazole blue)/MTT [3- (4, 5-dimethylthiazo-2-yl) -2,5-diphenyl tetrazolium bromide ] in an oxidation state into insoluble blue formazan (formazan) compound by intramitochondrial dehydrogenase (such as succinic dehydrogenase) and developing after DMSO dissolution, and the conversion amount is positively related to the number of living cells.

The experimental method comprises the following steps:

(1) And (3) digesting the Vero E6 cells cultured to the logarithmic growth phase with 0.25% pancreatin for 2-3 min, absorbing and discarding the digestive juice, adding a proper amount of culture solution, uniformly mixing, taking 20 mu L, counting under a microscope with a hemocytometer, and preparing cell suspension with proper concentration for later use. At the same time formulate with PBS (phosphate buffered solution)

5G/L MTT solution, filtering and sterilizing for standby.

(2) Test drugs were dissolved in DMSO and diluted 50-fold with medium to give the highest concentration tested, and serial dilutions were then made in 96-well plates 1:2 with medium at 8 concentrations per compound, up to 200. Mu.M, 4 parallel wells per concentration, and 100. Mu.L/well.

(3) The prepared cell suspension was inoculated into 96-well plates at a cell concentration of 4X 10 ⁵ cells/mL at 100. Mu.L/well. And simultaneously setting a cell control hole without medicine and a culture medium blank control hole.

(4) After 48 hours of incubation, 10. Mu.L/well MTT was added and incubation was continued for 4 hours. The plates were removed, the medium was carefully discarded in wells, 100 μl of DMSO was added to each well, and after shaking until the formazan particles were completely dissolved, their optical density values (OD ₅₇₀) were measured with an enzyme-linked immunosorbent assay at 570nm wavelength.

(5) Data treatment, percent of cell survival (%) = [ (cell control OD ₅₇₀ value-dosing group OD ₅₇₀ value)/(cell control OD ₅₇₀ value-blank OD ₅₇₀ value) ]x100%. Dose-response curve fitting was performed with Origin software to calculate the concentration of each compound at 50% cell survival (CC ₅₀).

Experimental results:

TABLE 2 cytotoxicity results of the compounds of the invention

As can be seen from the data in Table 2, the compounds of the present invention have low cytotoxicity and exhibit high safety.

3. Antiviral activity assay

The experimental principle of detecting the total amount of RNA in the cell culture medium supernatant by a fluorescence quantitative PCR method is that a specific fluorescent probe is added while a pair of primers is added during PCR amplification, the probe is an oligonucleotide, and a report fluorescent group and a quenching fluorescent group are respectively marked at two ends of the probe. When the probe is complete, the fluorescent signal emitted by the reporter group is absorbed by the quenching group, and when the probe is combined on any single strand of DNA, during PCR amplification, the 5 '-3' -end exonuclease activity of Taq enzyme cleaves and degrades the probe, so that the reporter fluorescent group and the quenching fluorescent group are separated, and a fluorescent monitoring system can receive the fluorescent signal, namely, one fluorescent molecule is formed every time one DNA strand is amplified, and the accumulation of the fluorescent signal and the formation of PCR products are completely synchronous.

The experimental method comprises the following steps:

(1) And (3) digesting the Vero E6 cells cultured to the logarithmic growth phase with 0.25% pancreatin for 2-3 min, absorbing and discarding the digestive juice, adding a proper amount of culture solution, uniformly mixing, taking 20 mu L, counting under a microscope with a hemocytometer, and preparing cell suspension with proper concentration for later use.

(2) Test drugs were dissolved in DMSO and diluted 50-fold with medium to give the highest concentration tested, and serial dilutions were then made in 96-well plates 1:2 with medium at 8 concentrations per compound, up to 200. Mu.M, 4 parallel wells per concentration, and 100. Mu.L/well.

(3) The prepared cell suspension was inoculated into 96-well plates at a cell concentration of 4X 10 ⁵ cells/mL at 100. Mu.L/well.

(4) After incubation of different dilutions of the drug working fluid (200 ul) with the cells for 1h, coronavirus was added to 100TCID ₅₀ and incubated for 2h.

(5) Cell culture medium was removed from 96-well plates, and compound dilutions were added at different concentrations, 200 μl per well, 3 multiplex wells per concentration. Cell control wells without drug, medium blank control wells, vehicle control wells and virus control wells were also provided.

(6) After 48 hours of culture, the culture supernatant is added into AVL lysate, repeated blowing and sucking for 15S ensures sufficient lysis, and then RNA extraction in the cell culture supernatant is carried out according to the steps of RNA extraction kit.

(7) And (3) uniformly mixing the extracted RNA, the fluorescent probe and the polymerase, and quantitatively detecting the RNA in the supernatant by adopting a QPCR method.

(8) Data processing = [ (drug-loaded group Ct value-virus control group Ct value/(cell control group Ct value-virus control group Ct value) ]. Times.100%. The concentration of each compound at 50% antiviral activity (EC ₅₀) was calculated by dose-response curve fitting using GraphPad software.

Experimental results:

TABLE 3 antiviral detection results of Compounds

As can be seen from the data in Table 3, the antiviral activity of the compounds 18 and 19 of the present invention is superior to GRL0617, which has a similar mechanism of action, and shows a stronger anti-coronavirus effect.

Claims (8)

1. A compound of formula (I) and isomers thereof, or pharmaceutically acceptable salts thereof:

Wherein,

R ₁ is hydrogen, methyl, ethyl, propyl, isopropyl, hydroxy;

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

R ₃ is independently selected from hydrogen, F, cl, br, trifluoromethyl, trifluoromethoxy, C ₁-C₃ alkyl, C ₁-C₃ alkoxy, n represents 1,2, 3 or 4 substituents;

R ₄ is substituted or unsubstituted phenyl, substituted or unsubstituted thienyl;

The substituents in R ₂ or R ₄ may be selected from F, cl, br, hydroxy, amino, nitro, cyano, monofluoromethyl, difluoromethyl, trifluoromethyl, trifluoromethoxy, thienyl, vinyl, ethynyl, hydroxymethyl, carboxy, sulfonate, borate, methylthio, C ₁-C₄ alkyl, C ₁-C₄ alkoxy.

2. The compound according to claim 1, which is represented by the general formula (II),

Wherein R ₁ is hydrogen, methyl or hydroxyl;

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

R ₃ is independently selected from hydrogen, F, cl, br, trifluoromethyl, trifluoromethoxy, C ₁-C₃ alkyl, C ₁-C₃ alkoxy, n represents 1,2, 3 or 4 substituents;

R ₅ is hydrogen, cyano, carboxy, trifluoromethyl, methoxy, morpholinyl, ethynyl, acetamido, acetyl, hydroxy, hydroxymethyl;

the substituents in R ₂ may be any selected from F, cl, br, hydroxy, amino, nitro, cyano, monofluoromethyl, difluoromethyl, trifluoromethyl, trifluoromethoxy, hydroxymethyl, C ₁-C₄ alkyl, C ₁-C₄ alkoxy.

3. The compound according to claim 1, which is represented by the general formula (III),

Wherein,

R ₁ is hydrogen, methyl or hydroxy;

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

R ₃ is independently selected from hydrogen, F, cl, br, trifluoromethyl, trifluoromethoxy, C ₁-C₃ alkyl, C ₁-C₃ alkoxy, n represents 1,2, 3 or 4 substituents;

R ₅ is hydrogen, carboxyl, cyano or boric acid group;

The substituent in R ₂ may be any one selected from F, cl, br, hydroxy, amino, nitro, cyano, monofluoromethyl, difluoromethyl, trifluoromethyl, trifluoromethoxy, thienyl, hydroxymethyl, C ₁-C₄ alkyl, C ₁-C₄ alkoxy.

4. A compound according to any one of claims 1 to 3, selected from the following compounds, and isomers thereof, or pharmaceutically acceptable salts thereof:

5. A process for preparing a compound according to any one of claims 1 to 3, comprising the steps of:

Condensing the compound A and the compound B, closing a ring to obtain a compound C, then carrying out a reduction reaction to obtain a compound D, and carrying out amination reduction on the compound D and aldehyde substituted by R ₄ to obtain a compound shown in the formula (I);

Wherein R ₁、R₂、R₃、R₄, n are as defined in any one of claims 1 to 3.

6. Use of a compound according to any one of claims 1 to 4, or an isomer thereof, or a pharmaceutically acceptable salt thereof, for the preparation of a papain (PLpro) inhibitor.

7. A pharmaceutical composition comprising a therapeutically and/or prophylactically effective amount of a compound according to any one of claims 1 to 4, or an isomer thereof, or a pharmaceutically acceptable salt thereof, and optionally one or more pharmaceutically acceptable excipients.

8. Use of a compound according to any one of claims 1 to 4, or an isomer thereof, or a pharmaceutically acceptable salt thereof, or a composition according to claim 7, for the preparation of a medicament for the treatment and/or prevention of infectious diseases caused by coronaviruses.