3, 5-disubstituted-1, 2, 4-triazole compound as well as preparation method and application thereof

Abstract

The invention discloses a 3, 5-disubstituted-1, 2, 4-triazole compound with a novel structure as well as a preparation method and application thereof, the method relates to two synthesis routes, the route I is that substituted o-amino thiophenol and aldehyde ester substituted five-membered or six-membered heteroaromatic ring react to generate key ester; the route II is that halogen substituted aromatic heterocycle and ester substituted five-membered or six-membered aromatic heterocycle boric acid or boric acid ester are subjected to Suzuki coupling reaction under alkaline conditions to generate key ester. According to the invention, five-membered or six-membered heteroaromatic esters containing different heteroaromatic ring substitutions are generated in two routes, and then the esters are subjected to a series of hydrazinolysis, ring closure, nucleophilic substitution and other reactions to obtain the target product 3, 5-disubstituted-1, 2, 4-triazole compound. The invention further discloses application of the 3, 5-disubstituted-1, 2, 4-triazole compound in preparation of antiviral drugs, and the 3, 5-disubstituted-1, 2, 4-triazole compound has a certain prospect in the field of discovery of new small-molecule drugs for inhibiting coronavirus.

Description

Technical Field

[0001] The invention belongs to the technical field of pharmaceutical chemistry and relates to a 3,5-disubstituted-1,2,4-triazole compound and a preparation method and application thereof. Background Art

[0002] Middle East Respiratory Syndrome Coronavirus (MERS-CoV) is a new zoonotic viral pathogen discovered in the Middle East in 2012. The viral infectious disease caused by this coronavirus infection is called Middle East Respiratory Syndrome (MERS) (N. Engl. J. Med. 2012, 367, 1814-1820). As of May 31, 2019, at least 27 countries around the world have reported cases of MERS-CoV infection, with a mortality rate of up to 35%. MERS-CoV is transmitted from animals to humans and then spreads from person to person. After infection with the virus, it can cause severe respiratory diseases, with symptoms including fever, cough and shortness of breath, accompanied by a high rate of acute renal failure or death.

[0003] MERS-CoV is an enveloped single-stranded RNA virus belonging to the beta coronavirus C lineage, which mainly enters cells through the spike protein (S protein) (Viruses 2019, 11, 59; Lancet. 2004, 363, 938-947). The S protein includes an S1 subunit containing a receptor binding domain (RBD), and an S2 subunit containing a fusion peptide (FP), a long heptad repeat 1 domain (HR1), and a short heptad repeat 2 domain (HR2) (J. Virol. 2003, 77, 8801-8811; J. Biol. Chem. 2004, 279, 49414-49419). MERS-CoV binds to the dipeptidyl peptidase-4 (DPP4) receptor on the surface of the host cell through the RBD in the S protein. Subsequently, S2 changes its conformation and inserts its FP into the plasma membrane or endosomal membrane. HR2 combines with HR1 to form a six-helix bundle (6-HB) fusion core, allowing the virus and cell membrane to bind tightly and fuse, thereby entering the host cell (Science 2020, 367, 1260-1263; Nature 2013, 495, 251-254; Cell Res. 2013, 23, 986-993.). From the perspective of pathogenic mechanism, the recognition and binding of MERS-CoV S protein with host cell receptors is particularly important for the virus to invade cells. Therefore, blocking the recognition and binding of viral S protein and host cell receptors is an important target for the development of anti-MERS-CoV drugs.

[0004] So far, drugs for treating MERS-CoV are mainly divided into the following categories: (1) clinically approved drugs, such as the combination of ribavirin and interferon; (2) protease inhibitors; (3) antiviral peptides; and (4) nucleic acid drugs.

[0005] Lopinavir, ribavirin, etc. have inhibitory activity against SARS-CoV and MERS-CoV (Antimicrob.AgentsChemother.2014,58,4875-4884; J.Clin.Virol.2004,31,69-75; Sci.Rep.2013,3,1686; Antivir.Res.2005,66,159-163). Remdesivir can effectively inhibit the replication of SARS-CoV, MERS-CoV and SARS-CoV-2, EC 50 The values ​​were 0.069μM, 0.074μM and 0.77μM respectively (CellRes.2020,30,269-271;Mbio 2018,9,e00221-18). Chloroquine can inhibit the entry and replication of SARS-CoV, MERS-CoV and SARS-CoV-2, and the EC 50 The values ​​were 6.54μM, 6.28μM and 1.13μM respectively (Antimicrob.Agents Chemother.2014,58,4875-4884; Cell Res.2020,30,269-271; Antimicrob.Agents Chemother.2014,58,4885-4893). Antipsychotic drugs such as chlorpromazine, triflupromazine and fluphenazine also showed the effect of inhibiting the replication of SARS-CoV, MERS-CoV and SARS-CoV-2. EC 50 The value is 4.03-21.4 μM (Antimicrob. Agents Chemother. 2014, 58, 4885-4893; J. Virol. 2020, 94, e01218-20).

[0006] At present, no specific drugs or vaccines have been approved for the prevention or treatment of MERS-CoV infection, and many basic and clinical studies on anti-MERS-CoV drugs are underway. Among the inhibitors of MERS-CoV, antiviral peptides have been studied the most. HR2P, discovered in 2014, can effectively inhibit MERS-CoV replication by preventing S protein-mediated cell-to-cell fusion by interacting with the HR1 domain (Nat. Commun. 2014, 5, 3067). In 2016, a short peptide P9, a broad-spectrum inhibitor of respiratory viruses, was reported. When the administration concentration was higher than 25 μg / ml, its inhibition rate against SARS-CoV and MERS-CoV was as high as more than 95% (Sci. Rep. 2016, 6, 22008). In 2018, a research group designed and synthesized a series of hydrocarbon-staple peptides that inhibit MERS-CoV pseudovirus infection and S protein-mediated cell-to-cell fusion (J. Med. Chem. 2018, 61, 2018-2026). Among them, P21S10 is the most effective fusion inhibitor. In 2019, the pan-coronavirus fusion inhibitory activity peptide EK1 targeting the HR1 domain was screened and improved, and its pan-coronavirus fusion inhibitory activity IC 50 =0.19-0.62μM (Int. J. Antimicrob. Agents 2018, 52, 730-732).

[0007] Although some progress has been made in the study of peptide inhibitors of S protein, the research on small molecule compounds with pan-coronavirus inhibitory activity against S protein is still limited. In 2018, it was reported that three natural products with good inhibitory ability against the entry of MERS-CoV S protein pseudovirus were screened from natural products: dihydrotanshinone, E-64-C and E-64-D (Sci. Adv. 2019, 5, eaav4580). In addition, K22, SSAA09E2, luteolin and Quercetin are all reported small molecule inhibitors of S protein, but there is a problem of poor inhibitory activity (J. Virol. 2013, 87, 8017-8028; PLoS Pathog. 2014, 10, e1004166; Science 2019, 363, eaar6221; J. Virol. 2004, 78, 11334-11339). A patent authorized in 2023 reported a 4-(benzothiazol-2-yl)-N-substituted aniline compound with pan-coronavirus inhibitory activity. This type of small molecule compound has an inhibitory activity against MERS-CoV pseudovirus up to the nanomolar level and also has certain inhibitory activity against SARS-CoV-2. However, there are problems such as the compounds being more toxic and the relatively unsatisfactory inhibitory activity of compounds with weaker toxicity against live viruses (CN202010374044.6).

[0008] Although there are many types of MERS-CoV inhibitors in the basic or clinical research stage, these drugs still have limited inhibitory effects and toxic side effects on MERS-CoV. It is urgent to develop new high-efficiency, low-toxic small molecule inhibitors to deal with viral diseases that may break out at any time. Summary of the invention

[0009] In order to solve the deficiencies in the prior art, the present invention provides a 3,5-disubstituted-1,2,4-triazole compound and a preparation method and application thereof.

[0010] In the present invention, the 3,5-disubstituted-1,2,4-triazole compound has the following general structural formula (I):

[0011]

[0012] In formula (I), L is any of the following five-membered or six-membered aromatic heterocyclic structures:

[0013]

[0014] In formula (I), R 1 Any of the following aromatic heterocyclic structures:

[0015]

[0016] Where R 4 is hydrogen, halogen, C1-C6 alkyl, C1-C6 alkoxy, hydroxy, amino, alkylamino;

[0017] In formula (I), R 2 is hydrogen, C1-C6 alkyl, C3-C8 cycloalkyl, benzyl;

[0018] In formula (I), R 3 is a C1-C6 alkyl group, (n=1, 2, 3), (n=1, 2, 3),

[0019] Preferably, R 1 Any of the following aromatic heterocyclic structures:

[0020]

[0021] Among them, R 4 It is hydrogen, halogen, methyl, methoxy, hydroxyl, and amino.

[0022] Preferably, R 2 It is hydrogen, methyl, ethyl, isopropyl, n-propyl, n-butyl, cyclopropyl, cyclopentyl, cyclohexyl or benzyl.

[0023] Preferably, R 3 Methyl, ethyl, (n=1, 2, 3), (n=1),

[0024] Specifically, when R in formula (I) 1 for When the 3,5-disubstituted-1,2,4-triazole compound has a structural formula as shown in formula (II):

[0025]

[0026] Among them, L, R 2 , R 3 , R 4 The same as defined in formula (I).

[0027] Specifically, when L in formula (II) is a benzene ring and a 2,5-disubstituted thiophene ring, respectively, the structural formulas of the 3,5-disubstituted 1,2,4-triazole compounds are shown in formulas (III-1) and (III-2), respectively:

[0028]

[0029] Among them, R 2 , R 3 , R 4 The same as defined in formula (I).

[0030] The present invention also provides a method for preparing a 3,5-disubstituted-1,2,4-triazole compound (as shown in formula (I) or (II)), which comprises the following two routes:

[0031] Route 1:

[0032] (1) in a first solvent, a substituted o-aminothiophenol and a five-membered or six-membered aromatic heterocycle substituted with an aldehyde or ester group react to generate an intermediate IV-1a;

[0033] (2) In a third solvent, the intermediate IV-1a obtained in step (1) undergoes ester hydrazinolysis reaction with hydrazine hydrate to generate intermediate IV-2a;

[0034] (3) in a fourth solvent, reacting the intermediate IV-2a obtained in step (2) with isothiocyanates substituted with different alkyl groups to generate an intermediate IV-3a;

[0035] (4) In a fifth solvent, the intermediate IV-3a obtained in step (3) undergoes a ring-closing reaction in an alkaline aqueous solution to generate an intermediate IV-4a;

[0036] (5) In a sixth solvent, in the presence of a base, the intermediate IV-4a obtained in step (4) undergoes a nucleophilic substitution reaction with a halide to obtain the target product, i.e., a compound represented by formula (II);

[0037] The reaction process of route 1 is shown in reaction formula (a):

[0038]

[0039] Among them, L, R 2 , R 3 , R 4 The definition is the same as that of formula (I); X is Cl, Br, I;

[0040] Route 2:

[0041] (1) In a second solvent, in the presence of a base and a palladium catalyst, a halogen-substituted aromatic heterocycle and an ester-substituted five-membered or six-membered aromatic heterocycle boronic acid or boronic ester undergo a palladium-catalyzed Suzuki coupling reaction under alkaline conditions to produce an intermediate IV-1b;

[0042] (2) In a third solvent, the intermediate IV-1b obtained in step (1) undergoes ester hydrazinolysis reaction with hydrazine hydrate to generate an intermediate IV-2b;

[0043] (3) in a fourth solvent, reacting the intermediate IV-2b obtained in step (2) with isothiocyanates substituted with different alkyl groups to generate an intermediate IV-3b;

[0044] (4) In a fifth solvent, the intermediate IV-3b obtained in step (3) undergoes a ring-closing reaction in an alkaline aqueous solution to generate an intermediate IV-4b;

[0045] (5) In a sixth solvent, in the presence of a base, the intermediate IV-4b obtained in step (4) undergoes a nucleophilic substitution reaction with a halide to obtain the target product, i.e., the compound represented by formula (I);

[0046] The reaction process of route 2 is shown in reaction formula (b):

[0047]

[0048] Among them, L, R 1 , R 2 , R 3 The definition is the same as that of formula (I); X is Cl, Br, I; and R is hydroxy or alkoxy.

[0049] In step (1), the first solvent is one or more of dimethyl sulfoxide, N,N-dimethylformamide, toluene, etc.; preferably, it is dimethyl sulfoxide.

[0050] In step (1), the molar ratio of the substituted o-aminothiophenol to the five-membered or six-membered aromatic heterocycle substituted with aldehyde or ester groups is 1:1 to 1.05:1; preferably, 1:1.

[0051] In step (1), the reaction temperature for the reaction between the substituted o-aminothiophenol and the five-membered or six-membered aromatic heterocycle substituted with the aldehyde or ester group is 120° C. to 140° C.; preferably, 130° C.

[0052] In step (1), the reaction time of the substituted o-aminothiophenol and the five-membered or six-membered aromatic heterocycle substituted with the aldehyde or ester group is 4 to 6 hours; preferably, 6 hours.

[0053] In step (1), the second solvent is one or more of methanol, ethanol, 1,4-dioxane, toluene, water, etc.; preferably, it is a mixed solvent of toluene, ethanol and water.

[0054] In step (1), the molar ratio of the halogen (chlorine, bromine, iodine) substituted aromatic heterocycle to the ester substituted five-membered or six-membered aromatic heterocycle boronic acid or boronic ester is 1:1.5 to 1:2; preferably, 1:1.5.

[0055] In step (1), the palladium catalyst is one or more of tetrakistriphenylphosphine palladium, bistriphenylphosphine palladium dichloride (II), 1,1'-bis(diphenylphosphino)ferrocene palladium dichloride (II) dichloromethane complex, etc.; preferably, it is tetrakistriphenylphosphine palladium.

[0056] In step (1), the molar ratio of the halogen (chlorine, bromine, iodine) substituted aromatic heterocycle to the palladium catalyst is 1:0.05 to 1:0.10; preferably, it is 1:0.05.

[0057] In step (1), the base is one or more of potassium carbonate, sodium carbonate, etc.; preferably, it is potassium carbonate.

[0058] In step (1), the molar ratio of the base to the halogen (chlorine, bromine, iodine) substituted aromatic heterocycle is 3:1 to 5:1; preferably, it is 3:1.

[0059] In step (1), the reaction temperature of the Suzuki coupling reaction is 80°C to 100°C; preferably, 85°C.

[0060] In step (1), the reaction time of the Suzuki coupling reaction is 5 to 12 hours; preferably, 8 hours.

[0061] In step (2), the third solvent is one or more of ethanol, methanol, isopropanol, etc.; preferably, it is ethanol.

[0062] In step (2), the molar ratio of the intermediate IV-1a or IV-1b to 85% hydrazine hydrate is 1:5 to 1:10; preferably, 1:10.

[0063] In step (2), the reaction temperature is 80°C to 90°C; preferably, 85°C.

[0064] In step (2), the reaction time is 4 to 8 hours; preferably, 8 hours.

[0065] In step (3), the fourth solvent is one or more of ethanol, acetonitrile, methanol, etc.; preferably, it is ethanol.

[0066] In step (3), the molar ratio of the intermediate IV-2a or IV-2b to the isothiocyanates substituted with different alkyl groups is 1:1.1 to 1:1.5; preferably, 1:1.2.

[0067] In step (3), the reaction temperature is 80°C to 90°C; preferably, 85°C.

[0068] In step (3), the reaction time is 6 to 12 hours; preferably, 12 hours.

[0069] In step (4), the fifth solvent is one or more of sodium hydroxide aqueous solution, sodium bicarbonate aqueous solution, potassium carbonate aqueous solution, etc.; preferably, it is sodium hydroxide aqueous solution.

[0070] In step (4), the reaction temperature is 95°C to 105°C; preferably, 100°C.

[0071] In step (4), the reaction time is 4 to 6 hours; preferably, 4 hours.

[0072] In step (5), the sixth solvent is one or more of N,N-dimethylformamide, acetone, dichloromethane, etc.; preferably, it is N,N-dimethylformamide.

[0073] In step (5), the base is one or more of potassium carbonate, potassium tert-butoxide, sodium tert-butoxide, etc.; preferably, it is potassium tert-butoxide.

[0074] In step (5), the molar ratio of the base to the intermediate IV-4a or IV-4b is 2:1 to 5:1; preferably, 3:1.

[0075] In step (5), the molar ratio of the intermediate IV-4a or IV-4b to different halides is 1:1.05 to 1:1.2;

[0076] Preferably, it is 1:1.2.

[0077] In step (5), the reaction temperature is 15°C to 35°C, preferably 25°C.

[0078] In step (5), the reaction time is 4 to 6 hours; preferably, 5 hours.

[0079] In a specific embodiment, the preparation method of the 3,5-disubstituted-1,2,4-triazole compound (as shown in formula (I)) comprises the following steps:

[0080] (1) Substituted o-aminothiophenol as a raw material and a five-membered or six-membered aromatic heterocycle substituted with an aldehyde or ester group are reacted in a molar ratio of 1:1 in dimethyl sulfoxide at 130° C. for 4 to 6 hours to obtain intermediate IV-1a (route 1);

[0081] Alternatively, a halogen (chlorine, bromine, iodine) substituted aromatic heterocycle as a raw material and an ester substituted five-membered or six-membered aromatic heterocycle boronic acid or boronic ester in a molar ratio of 1:1.5 are used to carry out a Suzuki coupling reaction catalyzed by tetrakistriphenylphosphine palladium (5 mol%) in a mixed solvent (toluene: ethanol: water = 2:1:1) at 85°C under alkaline conditions for 5 to 12 hours to generate intermediate IV-1b (route 2);

[0082] (2) Intermediate IV-1a or IV-1b is reacted with 85% hydrazine hydrate in ethanol to undergo ester hydrazinolysis reaction, wherein the molar ratio of intermediate IV-1a or IV-1b to 85% hydrazine hydrate is 1:10; the reaction time is 4 to 8 hours at 85°C to obtain intermediate IV-2a or IV-2b.

[0083] (3) Intermediate IV-2a or IV-2b is reacted with different alkyl-substituted isothiocyanates in ethanol at a molar ratio of 1:1.2 at 85°C for 6 to 12 hours to obtain intermediate IV-3a or IV-3b.

[0084] (4) Intermediate IV-3a or IV-3b is reacted in aqueous sodium hydroxide solution at 100°C for 4 to 6 hours to obtain intermediate IV-4a or IV-4b.

[0085] (5) Intermediate IV-4a or IV-4b is reacted with a halide in N,N-dimethylformamide with potassium tert-butoxide as a base to undergo nucleophilic substitution reaction at room temperature for 4 to 6 hours to obtain the target product, i.e., the compound of formula (II) or (I).

[0086] In the preparation method of the present invention, the process described in route 1 is shown in reaction formula (c):

[0087]

[0088] Among them, L, R 2 , R 3 , R 4 The definition of is the same as that of formula (I); X is Cl, Br, I.

[0089] In the preparation method of the present invention, the process described in route 2 is shown in reaction formula (d):

[0090]

[0091] Among them, L, R 1 , R 2 , R 3 The definition is the same as that of formula (I); X is Cl, Br, I; and R is hydroxy or alkoxy.

[0092] The present invention also provides the use of 3,5-disubstituted-1,2,4-triazole compounds represented by formula (I), formula (II), formula (III-1) and formula (III-2) in the preparation of antiviral drugs.

[0093] In the present invention, the 3,5-disubstituted-1,2,4-triazole compounds of formula (I), formula (II), formula (III-1) and formula (III-2) are used to inhibit the growth and replication of viruses, inhibit the recognition, entry, replication and release processes of viruses, and reduce the virus infection rate.

[0094] In the present invention, the virus is severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), new coronavirus (SARS-CoV-2), etc.

[0095] The present invention also provides a medicine / pharmaceutical composition, which contains the 3,5-disubstituted-1,2,4-triazole compound described above.

[0096] Furthermore, the drug / drug composition is used alone and / or in combination with other drugs.

[0097] Furthermore, the drug / drug composition also includes a pharmaceutically acceptable carrier.

[0098] Preferably, the pharmaceutically acceptable carrier refers to a drug that does not produce adverse, allergic or other adverse reactions when the drug is properly administered to animals or humans. Pharmaceutically acceptable carriers include, but are not limited to: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium methylcellulose, ethyl cellulose and methyl cellulose; tragacanth powder; malt; gelatin; talc; solid lubricants, such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils, such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and cocoa butter; polyols, such as propylene glycol, glycerol, sorbitol, mannitol and polyethylene glycol; alginic acid; emulsifiers, such as Tween; wetting agents, such as sodium lauryl sulfate; colorants; flavorings; tablets, stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic saline solution; and phosphate buffer, etc. These materials are used as needed to aid in the stability of the formulation or to help increase the activity or its bioavailability or to produce an acceptable taste or flavor in the case of oral administration.

[0099] Preferably, the drug / drug composition may also contain physiologically compatible excipients, including buffers, diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption enhancers, surfactants, adsorption carriers, lubricants, etc.

[0100] Specifically, the medicine / drug composition includes liquid dosage forms, gas dosage forms, solid dosage forms and semi-solid dosage forms.

[0101] Preferably, the dosage form of the drug / drug composition includes injection, sterile powder for injection, tablet, pill, capsule, lozenge, elixir, powder, granule, syrup, solution, tincture, aerosol, powder spray, or suppository, etc. The above-mentioned drugs / drug compositions in various dosage forms can be prepared according to conventional methods in the pharmaceutical field.

[0102] Specifically, the drug / drug composition is administered orally, by injection, nasally, transdermally or by mucosal administration.

[0103] Preferably, the administration route of the drug / drug composition is preferably parenteral administration, injection or oral administration. The injection preferably includes intravenous injection, intramuscular injection, intraperitoneal injection, intradermal injection or subcutaneous injection. The drug / drug composition is a conventional dosage form in the art, preferably in the form of solid, semi-solid, gas or liquid, that is, it can be an aqueous solution, non-aqueous solution or suspension, more preferably tablets, capsules, granules, injection or infusion, etc. More preferably, it is administered intravascularly, subcutaneously, intraperitoneally or intramuscularly. Preferably, the drug / drug composition can also be administered as an aerosol or a coarse spray, that is, nasally; or, intrathecally, intramedullary or intraventricularly. More preferably, the drug / drug composition can also be administered transdermally, percutaneously, topically, enterally, intravaginally, sublingually or rectally. The drug / drug composition of the present invention can be prepared into various dosage forms as needed, and can be administered by a physician according to factors such as the type of patient, age, weight and general disease condition, and mode of administration to determine the dosage that is beneficial to the patient. The administration method may be, for example, injection or other treatment methods.

[0104] The 3,5-disubstituted-1,2,4-triazole compounds described in the present invention inhibit the early stages of virus recognition, binding and entry into cells, have a novel structure, and are particularly effective against Middle East Respiratory Syndrome Coronavirus (MERS-CoV), and have good prospects in the field of new anti-coronavirus drug discovery. DETAILED DESCRIPTION

[0105] The present invention is further described in detail with reference to the following specific examples. The process, conditions, implementation methods, etc. of the present invention, except for the contents specifically mentioned below, are all common knowledge and common common sense in the art and are not particularly limited by the present invention.

[0106] The following description of at least one exemplary embodiment is actually only illustrative and is by no means intended to limit the present invention and its application or use. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0107] In all examples shown and discussed herein, any specific values ​​should be interpreted as merely exemplary and not as limiting. Therefore, other examples of the exemplary embodiments may have different values.

[0108] The present invention provides a novel 3,5-disubstituted-1,2,4-triazole compound and a preparation method and application thereof. The method involves two synthetic routes. Route 1 is to react substituted o-aminothiophenol with a five-membered or six-membered aromatic heterocycle substituted with an aldehyde or ester group to generate a key ester; route 2 is to Suzuki coupling reaction of a halogen (chlorine, bromine, iodine) substituted aromatic heterocycle with an ester group substituted five-membered or six-membered aromatic heterocycle boric acid or boric acid ester to generate a key ester under alkaline conditions. Both routes generate five-membered or six-membered aromatic heterocycle esters with different aromatic heterocycle substitutions, and then the ester undergoes a series of reactions such as hydrazinolysis, ring closure, and nucleophilic substitution to obtain the target product 3,5-disubstituted-1,2,4-triazole compound. The present invention also discloses the application of the 3,5-disubstituted-1,2,4-triazole compound in the preparation of antiviral drugs, which has certain prospects in the field of discovery of new small molecule drugs for inhibiting coronaviruses.

[0109] Unless otherwise specified, the test materials used in the examples are all conventional biochemical reagents.

[0110] Example 1: Compound Ia ( R 4 =H, R 2 = methyl, Synthesis of n=1).

[0111] o-Aminothiophenol (1.525 g, 12.18 mmol) and methyl p-formylbenzoate (2 g, 12.18 mmol) were dissolved in 20 mL of dimethyl sulfoxide and reacted at 130°C for 6 h. After cooling to room temperature, ice water was added, filtered, washed, and dried to obtain an off-white solid product Ia-1 (3.198 g).

[0112] Ia-1 (3.198 g, 11.87 mmol) and 85% hydrazine hydrate (5.942 g, 118.7 mmol) were refluxed in ethanol at 85°C. After reacting for 8 hours, the solvent was removed, ice water was added, filtered, and dried to obtain an off-white solid product Ia-2 (3.005 g).

[0113] Ia-2 (250 mg, 0.93 mmol) and methyl isothiocyanate (81.9 mg, 1.12 mmol) were refluxed in ethanol, and after reacting for 10 h, the solvent was distilled off under reduced pressure, ice water was added, filtered, washed, and dried to obtain a light yellow solid product Ia-3 (278 mg). Ia-3 was refluxed in a 2N sodium hydroxide solution for 5 h. After the reaction was completed, 1N hydrochloric acid solution was added dropwise to neutralize the system to weak acidity, and a white solid precipitated. After filtering, washing, and drying, an off-white solid Ia-4 (250 mg) was obtained. In the presence of potassium tert-butoxide (345.6 mg, 3.08 mmol), Ia-4 (250 mg, 0.77 mmol) and bromochloromethane (120 mg, 0.924 mmol) underwent nucleophilic substitution reaction in anhydrous N,N-dimethylformamide. After reacting at room temperature for 5 h, water and ethyl acetate were added for extraction, washed with saturated brine, dried over anhydrous sodium sulfate, and distilled under reduced pressure to remove the solvent to obtain a crude product, which was purified by silica gel column chromatography (eluent: petroleum ether and ethyl acetate) to obtain white solid compound Ia (199 mg). 1 H NMR(400MHz,Chloroform-d)δ8.26(d,J=8.1Hz,2H),8.11(d,J=8.2Hz,1H),7.94(d,J=8.0Hz,1H ),7.84(d,J=8.2Hz,2H),7.53(t,J=7.7Hz,1H),7.43(t,J=7.6Hz,1H),5.20(s,2H),3.80(s,3H). 13 C NMR (101MHz, CDCl3) δ = 165.5, 154.9, 153.1, 148.2, 134.3, 134.2, 128.2, 127.9, 127.0, 125.6, 124.7, 122.5, 120.7, 47.9, 31.5.

[0114] Example 2: Compound Ib ( R 4 =H, R 2 = cyclopropyl, Synthesis of n=1).

[0115] The raw material methyl isothiocyanate in Example 1 of the present invention was replaced by cyclopropyl isothiocyanate, and the white solid compound Ib was obtained by referring to the experimental method of Example 1 of the present invention. 1HNMR(400MHz,Chloroform-d)δ8.23(d,J=8.1Hz,2H),8.11(d,J=8.1Hz,1H),8.00–7.91(m,3H),7.53(t,J =7.6Hz,1H),7.43(t,J=7.5Hz,1H),5.39(s,2H),3.36–3.25(m,1H),1.20–1.12(m,2H),0.83–0.76(m,2H). 13 C NMR (101MHz, CDCl3) δ = 165.7, 155.1, 153.1, 150.5, 134.1, 133.9, 128.1, 126.6, 125.5, 124.6, 122.4, 120.7, 44.9, 25.0, 8.5.

[0116] Example 3: Compound Ic ( R 4 =H, R 2 =ethyl, Synthesis of n=1).

[0117] The raw material methyl isothiocyanate in Example 1 of the present invention was replaced by ethyl isothiocyanate, and the white solid compound Ic was obtained by referring to the experimental method of Example 1 of the present invention. 1 H NMR(400MHz,Chloroform-d)δ8.26(d,J=8.0Hz,2H),8.11(d,J=8.1Hz,1H),7.95(d,J=8.0Hz,1H),7.80(d,J=7.9 Hz, 2H), 7.53 (t, J = 7.7Hz, 1H), 7.43 (t, J = 7.7Hz, 1H), 5.27 (s, 2H), 4.18 (q, J = 7.4Hz, 2H), 1.38 (t, J = 7.2Hz, 3H). 13 C NMR (101MHz, CDCl3) δ = 165.5, 154.4, 153.1, 147.6, 134.3, 134.2, 128.2, 128.2, 127.1, 125.6, 124.7, 122.5, 120.7, 47.4, 39.2, 14.8.

[0118] Example 4: Compound Id ( R 4 =H, R 2 = isopropyl, Synthesis of n=1).

[0119] The raw material methyl isothiocyanate in Example 1 of the present invention was replaced by isopropyl isothiocyanate, and the white solid compound Id was obtained by referring to the experimental method of Example 1 of the present invention. 1 HNMR(400MHz,Chloroform-d)δ8.25(d,J=7.8Hz,2H),8.11(d,J=8.1Hz,1H),7.95(d,J=8.0Hz,1H),7.68(d,J=7 .8Hz,2H),7.53(t,J=7.5Hz,1H),7.43(t,J=7.5Hz,1H),5.39(s,2H),4.67–4.57(m,1H),1.55(d,J=6.9Hz,6H). 13 C NMR (101MHz, CDCl3) δ = 165.5, 154.6, 153.1, 146.5, 134.3, 134.2, 129.1, 128.7, 126.8, 125.6, 124.6, 122.5, 120.7, 48.4, 46.5, 20.9.

[0120] Example 5: Compound Ie ( R 4 =H, R 2 = cyclopropyl, Synthesis of n=2).

[0121] The raw material bromochloromethane in Example 1 of the present invention was replaced by 1-bromo-2-chloroethane, and the raw material methyl isothiocyanate was replaced by cyclopropyl isothiocyanate. Referring to the experimental method of Example 1 of the present invention, a white solid compound Ie was obtained. 1 H NMR(400MHz,Chloroform-d)δ8.21(d,J=8.2Hz,2H),8.10(d,J=8.2Hz,1H),7.97–7.90(m,3H),7.52(t,J=7.7Hz,1H),7.42(t, J=7.6Hz,1H),3.98(t,J=6.9Hz,2H),3.70(t,J=6.9Hz,2H),3.24(tt,J=7.1,3.8Hz,1H),1.18–1.09(m,2H),0.82–0.74(m,2H). 13 C NMR (101MHz, CDCl3) δ = 165.8, 154.6, 153.1, 152.6, 134.1, 133.7, 128.2, 128.0, 126.5, 125.5, 124.5, 122.4, 120.7, 41.8, 33.0, 24.7, 8.4.

[0122] Example 6: Compound If ( R 4 =H, R 2 = cyclopropyl, R 3 = methyl) synthesis.

[0123] The raw material bromochloromethane in Example 1 of the present invention was replaced by iodomethane, and the raw material methyl isothiocyanate was replaced by cyclopropyl isothiocyanate. Referring to the experimental method of Example 1 of the present invention, a light yellow solid compound If was obtained. 1 HNMR(400MHz,Chloroform-d)δ8.21(d,J=8.1Hz,2H),8.10(d,J=8.2Hz,1H),7.98–7.90(m,3H),7.53(t,J=7.7 Hz,1H),7.42(t,J=7.6Hz,1H),3.22(tt,J=7.0,3.7Hz,1H),2.80(s,3H),1.16–1.08(m,2H),0.81–0.75(m,2H). 13 C NMR (101MHz, CDCl3) δ = 165.9, 154.6, 154.5, 153.1, 134.1, 133.6, 128.5, 128.0, 126.5, 125.5, 124.5, 122.4, 120.7, 24.6, 13.4, 8.3.

[0124] Example 7: Compound Ig ( R 4 =H, R 2 = cyclopropyl, R 3 =ethyl) synthesis.

[0125] The raw material bromochloromethane in Example 1 of the present invention was replaced by iodoethane, and the raw material methyl isothiocyanate was replaced by cyclopropyl isothiocyanate. Referring to the experimental method of Example 1 of the present invention, a light yellow solid compound Ig was obtained. 1 HNMR(400MHz,Chloroform-d)δ8.21(d,J=8.2Hz,2H),8.10(d,J=8.1Hz,1H),7.97–7.91(m,3H),7.52(t,J=7.6Hz,1H),7.42(t, J=7.6Hz,1H),3.39(q,J=7.4Hz,2H),3.21(tt,J=7.2,3.8Hz,1H),1.51(t,J=7.4Hz,3H),1.15–1.09(m,2H),0.81–0.74(m,2H). 13C NMR (101MHz, CDCl3) δ = 165.9, 154.3, 153.9, 153.1, 134.1, 133.5, 128.6, 128.0, 126.5, 125.5, 124.5, 122.4, 120.7, 25.4, 24.7, 13.8, 8.4.

[0126] Example 8: Compound Ih ( R 4 =H, R 2 = cyclopropyl, ) synthesis.

[0127] The raw material bromochloromethane in Example 1 of the present invention was replaced by bromofluoromethane, and the raw material methyl isothiocyanate was replaced by cyclopropyl isothiocyanate, and the white solid compound Ih was obtained by referring to the experimental method of Example 1 of the present invention. 1 HNMR(400MHz,Chloroform-d)δ8.22(d,J=8.3Hz,2H),8.10(d,J=8.1Hz,1H),7.99–7.91(m,3H),7.52(t,J=7.6Hz,1H ),7.42(t,J=7.6Hz,1H),6.18(d,J=50.7Hz,2H),3.31(tt,J=7.2,3.8Hz,1H),1.20–1.13(m,2H),0.82–0.76(m,2H). 13 C NMR (101MHz, CDCl3) δ = 165.7, 155.2, 153.1, 150.4, 134.1, 133.9, 128.1, 128.1, 126.6, 125.6, 124.6, 122.4, 120.7, 84.7, 82.5, 25.2, 8.6.

[0128] Example 9: Compound Ii ( R 4 =6-F, R 2 = cyclopropyl, Synthesis of n=1).

[0129] 2-Bromo-6-fluorobenzothiazole (300 mg, 1.29 mmol) and 4-methoxycarbonylphenylboronic acid (348 mg, 1.935 mmol) were catalyzed by tetrakistriphenylphosphine palladium (75 mg, 0.0645 mmol) and potassium carbonate (535 mg, 3.87 mmol) was used as a base. Suzuki coupling reaction was carried out at 80°C in a mixed solvent of toluene: ethanol: water = 2:1:1. After the reaction was completed, ethyl acetate and water were added for extraction, and the mixture was purified by silica gel column chromatography (eluent: petroleum ether and ethyl acetate) to obtain a white solid product Ii-1 (119 mg).

[0130] Ii-1 is an ester compound. Then, Ii-1 is reacted with hydrazine hydrate, cyclopropyl isothiocyanate, etc. in accordance with the reaction steps in Example 1 of the present invention. After ring closure in sodium hydroxide aqueous solution, it undergoes nucleophilic substitution reaction with bromochloromethane to obtain the target product, a white solid Ii. 1 H NMR(400MHz,Chloroform-d)δ8.18(d,J=8.0Hz,2H),8.04(dd,J=8.8,4.8Hz,1H),7.95(d,J=8.0Hz,2H),7.62( d,J=7.9Hz,1H),7.26–7.22(m,1H),5.39(s,2H),3.34–3.26(m,1H),1.16(q,J=6.6Hz,2H),0.83–0.77(m,2H). 13 C NMR(101MHz, CDCl3)δ=165.5,160.9,158.4,155.0,150.5,149.7,135.2,135.1,133 .6,128.2,128.1,126.5,123.4,123.3,114.4,114.2,107.1,106.8,44.9,25.0,8.5.

[0131] Example 10: Compound Ij ( R 2 = cyclopropyl, Synthesis of n=1).

[0132] The raw material 2-bromo-6-fluorobenzothiazole in Example 9 of the present invention was replaced by 2-bromobenzothiophene, and a white solid compound Ij was obtained by referring to the experimental method of Example 9 of the present invention. 1H NMR(400MHz,Chloroform-d)δ7.91–7.78(m,6H),7.65(s,1H),7.37(p,J=7.0Hz ,2H),5.39(s,2H),3.31–3.24(m,1H),1.16(q,J=6.7Hz,2H),0.84–0.78(m,2H). 13 C NMR (101MHz, CDCl3) δ = 156.4, 151.2, 142.9, 140.6, 139.7, 135.8, 129.1, 126.5, 126.4, 124.8, 124.7, 123.8, 122.4, 120.5, 46.0, 26.0, 9.5.

[0133] Example 11: Compound Ik ( R 2 = cyclopropyl, Synthesis of n=1).

[0134] The raw material 2-bromo-6-fluorobenzothiazole in Example 9 of the present invention was replaced with 2-bromothiazol[5,4-B]pyridine, and the white solid compound Ik was obtained by referring to the experimental method of Example 9 of the present invention. 1 H NMR(400MHz,Chloroform-d)δ8.61(d,J=4.1Hz,1H),8.33(d,J=8.0Hz,1H),8.24(d,J=8.1Hz,2H),7.98(d,J=8 .1Hz,2H),7.48(dd,J=8.0,4.7Hz,1H),5.40(s,2H),3.35–3.27(m,1H),1.21–1.14(m,2H),0.84–0.77(m,2H). 13 C NMR (101MHz, CDCl3) δ = 167.4, 158.5, 156.0, 151.6, 147.5, 147.3, 134.7, 130.3, 129.7, 129.2, 127.7, 121.7, 45.9, 26.0, 9.6.

[0135] Example 12: Compound I1 ( R 2 = cyclopropyl, Synthesis of n=1).

[0136] The raw material 2-bromo-6-fluorobenzothiazole in Example 9 of the present invention was replaced by 2-bromo-1-benzofuran, and the white solid compound I1 was obtained by referring to the experimental method of Example 9 of the present invention. 1H NMR(400MHz,Chloroform-d)δ7.99(d,J=8.2Hz,2H),7.89(d,J=8.2Hz,2H),7.62(d,J=7.6Hz,1H),7.55(d,J=8.1Hz,1H),7.3 3(t,J=7.6Hz,1H),7.29–7.26(m,1H),7.14(s,1H),5.39(s,2H),3.32–3.25(m,1H),1.15(q,J=6.7Hz,2H),0.83–0.77(m,2H). 13 C NMR (101MHz, CDCl3) δ = 156.5, 155.1, 154.8, 151.2, 131.9, 129.0, 126.7, 124.9, 123.2, 121.2, 111.3, 102.7, 46.0, 26.0, 9.5.

[0137] Example 13: Compound Im( R 4 =H, R 2 = cyclopropyl, Synthesis of n=1).

[0138] The raw material methyl p-formylbenzoate in Example 1 of the present invention is replaced by methyl 5-formylthiophene-2-carboxylate, and the raw material methyl isothiocyanate is replaced by cyclopropyl isothiocyanate, and the yellow solid compound Im is obtained by referring to the experimental method of Example 1 of the present invention. 1 H NMR (400MHz, Chloroform-d) δ8.05(d,J=8.2Hz,1H),7.88(d,J=7.9Hz,1H),7.71(d,J=3.8Hz,1H),7.68(d,J=3.8Hz,1H),7. 50(t,J=7.6Hz,1H),7.40(t,J=7.6Hz,1H),5.37(s,2H),3.25(tt,J=7.0,3.9Hz,1H),1.36–1.31(m,2H),1.09–1.03(m,2H). 13 CNMR (101MHz, CDCl3) δ = 159.3, 152.6, 150.9, 150.2, 138.7, 133.8, 129.8, 128.3, 127.4, 125.7, 124.6, 122.2, 120.5, 44.8, 24.7, 8.9.

[0139] Example 14: Compound In( R 4 =H, R 2= cyclopropyl, Synthesis of n=1).

[0140] The raw material methyl p-formylbenzoate in Example 1 of the present invention is replaced by methyl 5-formyl-1-methyl-1H-pyrrole-2-carboxylate, and the raw material methyl isothiocyanate is replaced by cyclopropyl isothiocyanate, and the white solid compound In is obtained by referring to the experimental method of Example 1 of the present invention. 1 HNMR(400MHz,Chloroform-d)δ8.01(d,J=8.1Hz,1H),7.87(d,J=7.9Hz,1H),7.47(t,J=7.6Hz,1H),7.37(t,J=7.5Hz,1H),6.9 1(d,J=4.0Hz,1H),6.64(d,J=4.0Hz,1H),5.40(s,2H),4.35(s,3H),3.19–3.11(m,1H),1.18–1.11(m,2H),0.87–0.82(m,2H). 13 C NMR (101MHz, CDCl3) δ = 159.8, 154.1, 150.8, 150.2, 134.0, 129.6, 126.2, 125.0, 123.8, 123.0, 121.2, 114.3, 113.6, 45.6, 35.6, 25.8, 9.0.

[0141] Example 15: Compound Io ( R 4 =H, R 2 = cyclopropyl, Synthesis of n=1).

[0142] The raw material methyl p-formylbenzoate in Example 1 of the present invention was replaced by methyl 2-fluoro-4-formylbenzoate, and the raw material methyl isothiocyanate was replaced by cyclopropyl isothiocyanate. Referring to the experimental method of Example 1 of the present invention, a white solid compound Io was obtained. 1 H NMR(400MHz,Chloroform-d)δ8.12(d,J=8.1Hz,1H),8.02(d,J=10.9Hz,1H),7.97(t,J=8.8Hz,2H),7.81(t,J=7.5Hz,1H) ,7.55(t,J=7.6Hz,1H),7.46(t,J=7.6Hz,1H),5.39(s,2H),3.35–3.26(m,1H),1.02(q,J=6.7Hz,2H),0.75–0.68(m,2H). 13CNMR(101MHz, CDCl3)δ=164.2,160.4,157.9,152.9,150.6,136.9,136.8,134.2,131.1,131.1 ,125.8,124.9,122.8,122.8,122.7,120.8,117.0,116.8,113.6,113.4,44.7,25.0,24.9,6.5.

[0143] Example 16: Compound Ip( R 4 =H, R 2 = methyl, Synthesis of n=1).

[0144] The raw material methyl p-formylbenzoate in Example 1 of the present invention was replaced with methyl 6-formylnicotinate, and the light yellow solid compound Ip was obtained by referring to the experimental method of Example 1 of the present invention. 1 H NMR (400MHz, DMSO-d6) δ9.16–9.08(m,1H),8.51(d,J=8.2Hz,1H),8.44(dd,J=8.2,2.0Hz,1H),8.22(d ,J=7.9Hz,1H),8.16(d,J=8.0Hz,1H),7.64–7.57(m,1H),7.57–7.49(m,1H),5.46(s,2H),3.79(s,3H). 13 C NMR (101MHz, DMSO) δ = 168.5, 154.3, 153.7, 151.6, 149.7, 149.4, 137.9, 136.1, 127.3, 126.8, 125.5, 124.0, 123.2, 120.8, 49.3, 32.8.

[0145] Example 17: Compound Iq ( R 4 =H, R 2 = methyl, Synthesis of n=1).

[0146] The raw material methyl p-formylbenzoate in Example 1 of the present invention was replaced with methyl 5-formylthiophene-2-carboxylate, and a light yellow solid compound Iq was obtained by referring to the experimental method of Example 1 of the present invention. 1H NMR (400MHz, DMSO-d6) δ8.16(d,J=7.9Hz,1H),8.06(d,J=8.1Hz,1H),7.99(d,J=4.0Hz,1H),7 .78(d,J=4.0Hz,1H),7.57(t,J=7.6Hz,1H),7.49(t,J=7.6Hz,1H),5.41(s,2H),3.86(s,3H). 13 C NMR (101MHz, DMSO) δ = 160.5, 153.4, 150.9, 149.5, 138.5, 135.0, 131.7, 130.6, 129.4, 127.4, 126.4, 123.2, 122.9, 49.6, 32.9.

[0147] Example 18: Screening of anti-MERS-CoV pseudovirus invasion activity (pseudovirus-cell level model)

[0148] 1.1 Materials: Some compounds described in the present invention

[0149] 1.2 Test Principle: Huh7 cells are used as virus host cells (susceptible cells) to test the activity of the prepared drug in blocking the MERS-CoV S protein from modifying HIV pseudovirus-infected cells, which can reflect the antiviral activity of the sample in intervening in the key targets of MERS-CoV infection. The detection index is the activity level of the reporter gene on the pseudovirus genome.

[0150] 1.3 Test method: Huh7 cells were inoculated into 96-well culture plates one day in advance, and activity plates and cytotoxicity plates were set up respectively, and placed in a 37°C, 5% CO2 incubator for culture. The activity assay plate and the cytotoxicity assay plate were added with drugs (compounds prepared in the embodiments of the present invention) and MERS-CoV pseudovirus suspensions of different dilution concentrations in the same manner. Virus control, cell control and sample control were set up. After continuing to culture for 48-72 hours, the cytotoxicity plate used the CCK-8 method to determine the cell survival rate. After the culture medium was aspirated from the activity plate, 100 μL of cell lysis solution was added to each well. After shaking and lysing for 5 minutes, 100 μL of Lucifrase reaction detection solution was added to each well, and the chemiluminescence value was determined after shaking and incubating for 5 minutes.

[0151] 1.4 Evaluation method: Cytotoxicity (CCK-8 method): By comparing the OD values ​​of the virus control, cell control and sample control, the cell survival rate is calculated, and the cytotoxic effect of the drug is further calculated. Pseudovirus infection rate: Taking the reading of the cell control group as the background, the chemiluminescence values ​​of the virus control and drug control are deducted from the background, and the relative infection rate relative to the virus control well is calculated, and then the protective activity of the drug against virus-infected cells is calculated.

[0152] The present invention provides a compound of general formula (I) for the treatment of MERS-CoV pseudovirus IC 50 Test activity and the effect of compounds on Huh-7 cell CC 50 of the test (see Table 1).

[0153] Table 1 IC of the compounds of the present invention against MERS-CoV pseudovirus 50 Activity and CC 50 Cytotoxicity test

[0154]

[0155]

[0156] Table 1 shows the inhibitory activity and cytotoxicity results of the series of compounds provided by the present invention on MERS-CoV pseudovirus. The small molecule compounds of the present invention have excellent inhibitory activity against MERS-CoV pseudovirus. The inhibitory activity of 10 compounds in the above table against MERS-CoV pseudovirus is less than 0.1 μM, among which compounds Ia, Ib, Im, and In have inhibitory activities against MERS-CoV pseudovirus of about 0.01 μM, compounds Ic, Ii, and Ip have inhibitory activities of about 0.02 μM, and even compound Iq has an inhibitory activity against MERS-CoV pseudovirus of 7 nM. Moreover, while maintaining good virus inhibitory activity, the compounds in Table 1 have low cytotoxicity, most of which are above 50 μM.

[0157] Example 19: Screening for Anti-SARS-CoV-2 Pseudovirus Entry Activity (Pseudovirus-Cell Level Model)

[0158] 2.1 Materials: Some compounds described in the present invention

[0159] 2.2 Test principle: Huh7 cells are used as virus host cells (susceptible cells) to test the activity of the prepared drug in blocking HIV pseudovirus infection cells modified by the spike protein (S protein) of SARS-CoV-2. This inhibitory activity can reflect the antiviral activity of the drug in intervening in the key targets of SARS-CoV-2 infection. The detection index is the activity level of the reporter gene firefly luciferase Lucifrase.

[0160] 2.3 Test method: Huh7 cells were inoculated into 96-well culture plates one day in advance, and activity assay plates and cytotoxicity assay plates were set up respectively, and placed in a 37°C, 5% CO2 incubator for culture. The activity assay plate and the cytotoxicity assay plate were added in the same manner, and different dilution concentrations of drugs (compounds prepared in the embodiments of the present invention) and pseudovirus suspensions modified with SARS-CoV-2S protein were added. Virus control, cell control and sample control were set up. After continuing to culture for 48-72 hours, the cytotoxicity plate used the CCK-8 method to determine the cell viability. After the culture medium was removed from the activity assay plate, 100 μL of cell lysate was added to each well. After shaking and lysing for 5 minutes, 100 μL of Lucifrase reaction detection solution was added to each well, and the chemiluminescence value was determined after shaking and incubating for 5 minutes.

[0161] 2.4 Evaluation method: Cytotoxicity (CCK-8 method): By comparing the OD values ​​of the virus control, cell control and sample control, the cell survival rate is calculated, and the cytotoxic effect of the drug is further calculated. Pseudovirus infection rate: Taking the reading of the cell control group as the background, the chemiluminescence values ​​of the virus control and drug control are deducted from the background, and the relative infection rate relative to the virus control well is calculated, and then the protective activity of the drug against virus-infected cells is calculated.

[0162] The present invention provides some compounds Ia, Ib, Ic for SARS-CoV-2 pseudovirus IC 50 Test activity (see Table 2).

[0163] Table 2 IC of the compounds of the present invention against SARS-CoV-2 pseudovirus 50 Activity and CC 50 Cytotoxicity test

[0164] Compound <![CDATA[IC 50 (μM)]]> <![CDATA[CC 50 (μM)]]> Ia 0.0085 >100 Ic 0.0447 >100 Ib 0.0309 >100

[0165] Table 2 shows the inhibitory activity and cytotoxicity results of the series of compounds provided by the present invention against SARS-CoV-2 pseudovirus. The small molecule compounds of the present invention have excellent inhibitory activity against SARS-CoV-2 pseudovirus, IC 50 All of them are within 0.05μM, among which compound Ia has an inhibitory activity against SARS-CoV-2 pseudovirus of 8.5nM, and its cytotoxicity is very low, all above 100μM. The three compounds Ia, Ib, and Ic all show excellent inhibitory activity against MERS-CoV and SARS-CoV-2 coronaviruses, and can achieve the purpose of inhibiting multiple viruses at the same time.

[0166] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0167] As used in the present invention, the term "comprising" is an open expression, that is, including the contents specified in the present invention but not excluding other contents.

[0168] As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0169] The protection content of the present invention is not limited to the above embodiments. Without departing from the spirit and scope of the inventive concept, changes and advantages that can be thought of by those skilled in the art are included in the present invention and are protected by the attached claims.

Claims

1. A 3,5-disubstituted 1,2,4-triazole compound, characterized in that: The structure of the compound is shown in formula (I): In formula (I), L is any of the following five-membered or six-membered aromatic heterocyclic structures: In formula (I), R 1 Any of the following aromatic heterocyclic structures: Among them, R 4 is hydrogen, halogen, C1-C6 alkyl, C1-C6 alkoxy, hydroxy, amino, alkylamino; In formula (I), R 2 is hydrogen, C1-C6 alkyl, C3-C8 cycloalkyl, benzyl; In formula (I), R 3 is a C1-C6 alkyl group, Among them, n=1, 2, 3.

2. The 3,5-disubstituted-1,2,4-triazole compound according to claim 1, characterized in that: The R 1 Any of the following aromatic heterocyclic structures: Among them, R 4 is hydrogen, halogen, methyl, methoxy, hydroxyl, amino; The R 2 is hydrogen, methyl, ethyl, isopropyl, n-propyl, n-butyl, cyclopropyl, cyclopentyl, cyclohexyl, benzyl; The R 3 Methyl, ethyl, 3. The 3,5-disubstituted 1,2,4-triazole compound according to claim 1, characterized in that: When the R 1 for When the 3,5-disubstituted 1,2,4-triazole compound has a structural formula as shown in formula (II): Among them, L, R 2 , R 3 , R 4 The definition is the same as that of claim 1.

4. The 3,5-disubstituted 1,2,4-triazole compound according to claim 3, characterized in that: When L in formula (II) is a benzene ring and a 2,5-disubstituted thiophene ring, respectively, the structural formulas of the 3,5-disubstituted-1,2,4-triazole compounds are shown in formulas (III-1) and (III-2), respectively: Among them, R 2 , R 3 , R 4 The definition is the same as that of claim 1.

5. A method for preparing a 3,5-disubstituted 1,2,4-triazole compound, characterized in that: The preparation method comprises the following two routes: Route 1: (1) in a first solvent, substituted o-aminothiophenol and a five-membered or six-membered aromatic heterocycle substituted with an aldehyde or ester group react to generate an intermediate IV-1a; (2) In a third solvent, the intermediate IV-1a obtained in step (1) undergoes ester hydrazinolysis reaction with hydrazine hydrate to generate intermediate IV-2a; (3) in a fourth solvent, reacting the intermediate IV-2a obtained in step (2) with isothiocyanates substituted with different alkyl groups to generate an intermediate IV-3a; (4) In a fifth solvent, the intermediate IV-3a obtained in step (3) undergoes a ring-closing reaction in an alkaline aqueous solution to generate an intermediate IV-4a; (5) In a sixth solvent, in the presence of a base, the intermediate IV-4a obtained in step (4) undergoes a nucleophilic substitution reaction with a halide to obtain the target product, i.e., a compound represented by formula (II); The reaction process of route 1 is shown in reaction formula (a): Among them, L, R 2 , R 3 , R 4 The definition is the same as that of claim 1; X is Cl, Br, I; Or, route 2: (1) In a second solvent, in the presence of a base and a palladium catalyst, a halogen-substituted aromatic heterocycle and an ester-substituted five-membered or six-membered aromatic heterocycle boronic acid or boronic ester undergo a palladium-catalyzed Suzuki coupling reaction under alkaline conditions to produce an intermediate IV-1b; (2) In a third solvent, the intermediate IV-1b obtained in step (1) undergoes ester hydrazinolysis reaction with hydrazine hydrate to generate an intermediate IV-2b; (3) in a fourth solvent, reacting the intermediate IV-2b obtained in step (2) with isothiocyanates substituted with different alkyl groups to generate an intermediate IV-3b; (4) In a fifth solvent, the intermediate IV-3b obtained in step (3) undergoes a ring-closing reaction in an alkaline aqueous solution to generate an intermediate IV-4b; (5) In a sixth solvent, in the presence of a base, the intermediate IV-4b obtained in step (4) undergoes a nucleophilic substitution reaction with a halide to obtain the target product, i.e., the compound represented by formula (I); The reaction process of route 2 is shown in reaction formula (b): Among them, L, R 1 , R 2 , R 3 The definition is the same as that of claim 1; X is Cl, Br, I; R is hydroxyl or alkoxy.

6. The preparation method according to claim 5, characterized in that: In route 1, in step (1), the first solvent is one or more of dimethyl sulfoxide, N,N-dimethylformamide, and toluene; and / or the molar ratio of the substituted o-aminothiophenol to the five-membered or six-membered aromatic heterocycle substituted with aldehyde or ester groups is 1:1 to 1.05:1; and / or the reaction temperature for the reaction of the substituted o-aminothiophenol and the five-membered or six-membered aromatic heterocycle substituted with aldehyde or ester groups is 120° C. to 140° C.; and / or the reaction time for the reaction of the substituted o-aminothiophenol and the five-membered or six-membered aromatic heterocycle substituted with aldehyde or ester groups is 4 to 6 hours; and / or, In route 2, in step (1), the second solvent is one or more of methanol, ethanol, 1,4-dioxane, toluene, and water; and / or the molar ratio of the halogen-substituted aromatic heterocycle to the ester-substituted five-membered or six-membered aromatic heterocycle boronic acid or boric ester is 1:1.5 to 1:2; and / or the palladium catalyst is tetrakistriphenylphosphine palladium, bistriphenylphosphine palladium dichloride (II), 1,1'-bis(diphenylphosphino)ferrocene palladium dichloride (II) dichloromethane complex. One or more; and / or, the molar ratio of the halogen-substituted aromatic heterocycle to the palladium catalyst is 1:0.05 to 1:0.10; and / or, the base is one or more of potassium carbonate and sodium carbonate; and / or, the molar ratio of the base to the halogen-substituted aromatic heterocycle is 3:1 to 5:1; and / or, the reaction temperature of the Suzuki coupling reaction is 80°C to 100°C; and / or, the reaction time of the Suzuki coupling reaction is 5 to 12 hours; and / or, In the step (2), the third solvent is one or more of methanol, ethanol, and isopropanol; and / or the molar ratio of the intermediate IV-1a or IV-1b to hydrazine hydrate is 1:5 to 1:10; and / or the reaction temperature is 80°C to 90°C; and / or the reaction time is 4 to 8 hours; and / or, In the step (3), the fourth solvent is one or more of methanol, ethanol, and acetonitrile; and / or the molar ratio of the intermediate IV-2a or IV-2b to the isothiocyanate substituted with different alkyl groups is 1:1.1 to 1:1.5; and / or the reaction temperature is 80°C to 90°C; and / or the reaction time is 6 to 12 hours; and / or, In the step (4), the fifth solvent is one or more of a sodium hydroxide aqueous solution, a sodium bicarbonate aqueous solution, and a potassium carbonate aqueous solution; and / or the reaction temperature is 95° C. to 105° C.; and / or the reaction time is 4 to 6 hours; and / or, In the step (5), the sixth solvent is one or more of N,N-dimethylformamide, acetone, and dichloromethane; and / or the base is one or more of potassium carbonate, potassium tert-butoxide, and sodium tert-butoxide; and / or the molar ratio of the base to the intermediate IV-4a or IV-4b is 2:1 to 5:1; and / or the molar ratio of the intermediate IV-4a or IV-4b to the halide is 1:1.05 to 1:1.2; and / or the reaction temperature is 15°C to 35°C; and / or the reaction time is 4 to 6 hours.

7. Use of the 3,5-disubstituted-1,2,4-triazole compound according to any one of claims 1 to 4 in the preparation of antiviral drugs.

8. The use according to claim 7, characterized in that The 3,5-disubstituted-1,2,4-triazole compound is used to inhibit the recognition, entry, replication and release processes of the virus and reduce the virus infection rate.

9. The use according to claim 7, characterized in that The viruses are severe acute respiratory syndrome coronavirus, Middle East respiratory syndrome coronavirus, and new coronavirus.

10. A drug / drug composition, characterized in that: The drug / drug composition contains the 3,5-disubstituted-1,2,4-triazole compound according to any one of claims 1 to 4.

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