A 3,5-disubstituted-1,2,4-triazole compound and its preparation method and application

Abstract

The present invention discloses a novel structural 3,5-disubstituted-1,2,4-triazole compound and its preparation method and application. The method involves two synthetic routes. Route one is to react a five-membered or six-membered aromatic heterocycle substituted with an aldehyde or ester group to generate a key ester; Route two is to react a halogen-substituted aromatic heterocycle with an ester group to generate a key ester by Suzuki coupling reaction of a five-membered or six-membered aromatic heterocycle boronic acid or boric acid ester under alkaline conditions. Both routes of the present invention generate a five-membered or six-membered aromatic heterocycle ester substituted with different aromatic heterocycles, and the ester then 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 small molecule new drug discovery for inhibiting coronavirus.

Description

Technical Field

[0001] The present 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 novel zoonotic viral pathogen discovered in the Middle East in 2012. The viral infectious disease caused by this coronavirus is known as Middle East Respiratory Syndrome (MERS) (N. Engl. J. Med. 2012, 367, 1814-1820). As of May 31, 2019, at least 27 countries worldwide have reported cases of MERS-CoV infection, with a mortality rate as high as 35%. MERS-CoV is transmitted from animals to humans and then spreads between humans. Infection with the virus can cause severe respiratory illness, 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. It primarily enters cells through its spike protein (S protein) (Viruses 2019, 11, 59; Lancet. 2004, 363, 938-947). The S protein consists of the S1 subunit, which contains the receptor binding domain (RBD), and the S2 subunit, which contains the fusion peptide (FP), the long heptad repeat 1 domain (HR1), and the 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 host cell surface through the RBD in the S protein. Subsequently, S2 changes its conformation and inserts its FP into the plasma membrane or endosomal membrane. HR2 binds to HR1 to form a six-helix bundle (6-HB) fusion core, allowing the virus and cell membrane to tightly bind 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 the MERS-CoV S protein to the host cell receptor is particularly important for the virus to invade the cell. Therefore, blocking the recognition and binding of the viral S protein to the host cell receptor 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 therapy 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. 50 The values ​​were 0.069μM, 0.074μM and 0.77μM respectively (Cell Res. 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. 50 The value is 4.03-21.4 μM (Antimicrob. Agents Chemother. 2014, 58, 4885-4893; J. Virol. 2020, 94, e01218-20).

[0006] Currently, no specific drugs or vaccines have been approved for the prevention or treatment of MERS-CoV infection. Numerous basic and clinical studies are underway on anti-MERS-CoV drugs. Among MERS-CoV inhibitors, antiviral peptides have been the most studied. In 2014, HR2P, discovered to effectively inhibit MERS-CoV replication by interacting with the HR1 domain and preventing S protein-mediated cell-cell fusion, was reported (Nat. Commun. 2014, 5, 3067). In 2016, a short peptide, P9, was reported as a broad-spectrum inhibitor of respiratory viruses. At concentrations above 25 μg / ml, it exhibited inhibition rates exceeding 95% against SARS-CoV and MERS-CoV (Sci. Rep. 2016, 6, 22008). In 2018, a research group designed and synthesized a series of hydrocarbon-stapled peptides that inhibit MERS-CoV pseudovirus infection and S protein-mediated cell-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 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 the S protein, research on small molecule compounds with pan-coronavirus inhibitory activity against the S protein remains limited. In 2018, three natural products were screened and identified: dihydrotanshinone, E-64-C, and E-64-D, which showed strong inhibitory activity against the entry of MERS-CoV S protein pseudoviruses (Sci. Adv. 2019, 5, eaav4580). Additionally, K22, SSAA09E2, luteolin, and quercetin have all been reported as small molecule inhibitors of the S protein, but these inhibitory activities have been limited (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 at 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 a wide range of MERS-CoV inhibitors are currently under basic or clinical research, these drugs still have limited inhibitory effects and toxic side effects against MERS-CoV. There is an urgent need to develop new, highly effective, and low-toxic small-molecule inhibitors to address potential viral outbreaks. Summary of the Invention

[0009] To address the deficiencies in the prior art, the present invention provides a 3,5-disubstituted-1,2,4-triazole compound, a preparation method thereof, and an 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, or 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, or 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 structural formula of the 3,5-disubstituted-1,2,4-triazole compound is as shown in formula (II):

[0025]

[0026] Among them, L, R 2 、R 3 、R 4 The definition of is the same as that of 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 definition of is the same as that of 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 includes 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 form 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 different alkyl-substituted isothiocyanates to generate intermediate IV-3a;

[0035] (4) In a fifth solvent, the intermediate IV-3a obtained in step (3) undergoes a ring-closure 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 the route 1 is shown in reaction formula (a):

[0038]

[0039] Among them, L, R 2 、R 3 、R 4 The same as defined in 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 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 intermediate IV-2b;

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

[0044] (4) In a fifth solvent, the intermediate IV-3b obtained in step (3) undergoes a ring-closure 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; 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 is reacted with a five-membered or six-membered aromatic heterocycle substituted with an aldehyde or ester group in a 1:1 molar ratio in dimethyl sulfoxide at 130°C for 4-6 hours to obtain intermediate IV-1a (Route 1);

[0081] Alternatively, a halogen (chlorine, bromine, iodine)-substituted aromatic heterocycle is reacted with an ester-substituted five-membered or six-membered aromatic heterocycle boronic acid or boronic ester in a molar ratio of 1:1.5. A Suzuki coupling reaction catalyzed by tetrakistriphenylphosphine palladium (5 mol%) is carried out at 85°C in a mixed solvent (toluene:ethanol:water = 2:1:1) under alkaline conditions for 5-12 hours to produce 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 is the same as that of formula (I); X is Cl, Br, or 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; 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 viral 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 further includes a pharmaceutically acceptable carrier.

[0098] Preferably, the pharmaceutically acceptable carrier refers to a drug that does not produce adverse, allergic or other untoward reactions when 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, ethylcellulose and methylcellulose; 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; tableting agents, stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic saline solution; and phosphate buffered saline. 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 further contain physiologically compatible excipients, including buffers, diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption enhancers, surfactants, adsorption carriers, lubricants, etc.

[0100] Specifically, the drug / drug composition includes liquid dosage forms, gaseous dosage forms, solid dosage forms and semisolid dosage forms.

[0101] Preferably, the dosage forms of the drug / pharmaceutical composition include injections, sterile powders for injection, tablets, pills, capsules, lozenges, elixirs, powders, granules, syrups, solutions, tinctures, aerosols, powder sprays, or suppositories. The drugs / pharmaceutical compositions in the above 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 through the mucosa.

[0103] Preferably, the drug / pharmaceutical composition is administered parenterally, by injection, or orally. Injection preferably includes intravenous, intramuscular, intraperitoneal, intradermal, or subcutaneous injection. The drug / pharmaceutical composition is in various dosage forms conventionally used in the art, preferably in the form of a solid, semisolid, gaseous, or liquid solution, i.e., an aqueous solution, non-aqueous solution, or suspension, more preferably a tablet, capsule, granule, injection, or infusion. More preferably, it is administered intravascularly, subcutaneously, intraperitoneally, or intramuscularly. Preferably, the drug / pharmaceutical composition can also be administered as an aerosol or coarse spray, i.e., nasally; or, alternatively, intrathecally, intramedullary, or intraventricularly. More preferably, the drug / pharmaceutical composition can also be administered transdermally, percutaneously, topically, enterally, intravaginally, sublingually, or rectally. The drug / pharmaceutical composition of the present invention can be prepared into various dosage forms as needed, and a physician can determine the dosage that is beneficial to the patient based on factors such as the patient's type, age, weight, general condition, and route of administration. Administration may be by injection or other therapeutic 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. They have a novel structure and are particularly effective against Middle East Respiratory Syndrome Coronavirus (MERS-CoV). They have good prospects in the field of discovering new anti-coronavirus drugs. DETAILED DESCRIPTION

[0105] The present invention will be further described in detail with reference to the following specific examples. Except for the contents specifically mentioned below, the processes, conditions, and methods for implementing the present invention 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 merely illustrative and is in no way intended to limit the present invention, its application, or use. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort are intended to fall 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 limiting. Therefore, other examples of the exemplary embodiments may have different values.

[0108] The present invention provides novel 3,5-disubstituted-1,2,4-triazole compounds, their preparation methods, and applications. The methods involve two synthetic routes: Route 1 involves the reaction of substituted o-aminothiophenol with a five- or six-membered aromatic heterocycle substituted with an aldehyde or ester group to produce a key ester; Route 2 involves a Suzuki coupling reaction of a halogen (chlorine, bromine, or iodine)-substituted aromatic heterocycle with an ester-substituted five- or six-membered aromatic heterocycle boronic acid or ester under alkaline conditions to produce the key ester. Both routes produce five- or six-membered aromatic heterocycle esters with varying aromatic heterocycle substitutions. These esters then undergo a series of reactions, including hydrazinolysis, ring closure, and nucleophilic substitution, to yield the target 3,5-disubstituted-1,2,4-triazole compounds. The present invention also discloses the use of these 3,5-disubstituted-1,2,4-triazole compounds in the preparation of antiviral drugs, which have promising applications in the discovery of new small-molecule drugs for the inhibition of 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] Dissolve o-aminothiophenol (1.525 g, 12.18 mmol) and methyl p-formylbenzoate (2 g, 12.18 mmol) in 20 mL of dimethyl sulfoxide and react at 130°C for 6 h. Cool to room temperature, add ice water, filter, wash, and dry to obtain the product Ia-1 (3.198 g) as an off-white solid.

[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 h, 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 allowed to react for 10 h. Afterwards, the solvent was removed by distillation under reduced pressure, ice water was added, the mixture was filtered, washed, and dried to afford the product Ia-3 (278 mg) as a pale yellow solid. Ia-3 was refluxed in 2N sodium hydroxide solution for 5 h. After completion of the reaction, 1N hydrochloric acid was added dropwise to neutralize the system to a weak acidity. A white solid precipitated, which was filtered, washed, and dried to afford Ia-4 (250 mg) as an off-white solid. 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, and dried over anhydrous sodium sulfate. The solvent was removed by distillation under reduced pressure to obtain a crude product, which was purified by silica gel column chromatography (eluent: petroleum ether and ethyl acetate) to obtain compound Ia (199 mg) as a white solid. 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 experimental method of Example 1 of the present invention was used to obtain a white solid compound Id. 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 1e ( 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. Referring to the experimental method of Example 1 of the present invention, a white solid compound Ih was obtained. 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] A Suzuki coupling reaction was conducted between 2-bromo-6-fluorobenzothiazole (300 mg, 1.29 mmol) and 4-methoxycarbonylphenylboronic acid (348 mg, 1.935 mmol) in the presence of tetrakistriphenylphosphine palladium (75 mg, 0.0645 mmol) and potassium carbonate (535 mg, 3.87 mmol) as a base in a 2:1:1 mixture of toluene, ethanol, and water at 80°C. After completion of the reaction, the product was extracted with ethyl acetate and water, and purified by silica gel column chromatography (eluent: petroleum ether and ethyl acetate) to afford the product Ii-1 (119 mg) as a white solid.

[0130] Ii-1 is an ester compound. Then, Ii-1 is reacted with hydrazine hydrate, cyclopropyl isothiocyanate, etc. according to the reaction steps in Example 1 of the present invention. After ring closure in aqueous sodium hydroxide solution, it undergoes nucleophilic substitution reaction with bromochloromethane to obtain the target product Ii as a white solid. 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 with 2-bromobenzothiophene, and the 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 11 ( R 2 =cyclopropyl, Synthesis of n=1).

[0136] The raw material 2-bromo-6-fluorobenzothiazole in Example 9 of the present invention was replaced with 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 was replaced by methyl 5-formylthiophene-2-carboxylate, and the raw material methyl isothiocyanate was replaced by cyclopropyl isothiocyanate. Referring to the experimental method of Example 1 of the present invention, a yellow solid compound Im was obtained. 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 was replaced with methyl 5-formyl-1-methyl-1H-pyrrole-2-carboxylate, and the raw material methyl isothiocyanate was replaced with cyclopropyl isothiocyanate. Referring to the experimental method of Example 1 of the present invention, a white solid compound In was obtained. 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 experimental method of Example 1 of the present invention was used to obtain a light yellow solid compound Ip. 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 the experimental method of Example 1 of the present invention was referred to to obtain a light yellow solid compound Iq. 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 for 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: Using Huh7 cells as viral host cells (susceptible cells), the prepared drug is tested for its ability to block MERS-CoV S protein-modified HIV pseudovirus infection in cells. This reflects the sample's antiviral activity against key targets of MERS-CoV infection. The assay measures the activity 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 viability. After the culture medium was aspirated from the activity 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. After shaking and incubating for 5 minutes, the chemiluminescence value was measured.

[0151] 1.4 Evaluation Method: Cytotoxicity (CCK-8 Assay): Compare the OD values ​​of the virus control, cell control, and sample control to calculate cell viability and further calculate the drug's cytotoxicity. Pseudovirus Infection Rate: Using the cell control as the background value, the chemiluminescence values ​​of the virus control and drug control are subtracted from the background value to calculate the relative infection rate relative to the virus control wells, thereby calculating the drug's protective activity against virus-infected cells.

[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 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 a series of compounds provided by the present invention against MERS-CoV pseudovirus. The small molecule compounds of the present invention have excellent inhibitory activity against MERS-CoV pseudovirus. Ten compounds in the table above have MERS-CoV pseudovirus inhibitory activities below 0.1 μM, including compounds Ia, Ib, Im, and In with inhibitory activities of around 0.01 μM, compounds Ic, Ii, and Ip with inhibitory activities of around 0.02 μM, and even compound Iq with inhibitory activity of 7 nM against MERS-CoV pseudovirus. Furthermore, while maintaining good viral inhibitory activity, the compounds in Table 1 have low cytotoxicity, with most compounds 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: Using Huh7 cells as viral host cells (susceptible cells), the prepared drug is tested for its ability to inhibit infection of cells by HIV pseudoviruses modified with the SARS-CoV-2 spike protein (S protein). This inhibitory activity reflects the drug's antiviral activity against key targets of SARS-CoV-2 infection. The assay is based on the activity of the reporter gene firefly luciferase.

[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, with different dilution concentrations of drugs (compounds prepared in accordance with the embodiments of the present invention) and pseudovirus suspensions modified with SARS-CoV-2S protein added. Virus control, cell control and sample control were set up. After continuing to culture for 48-72 hours, the cytotoxicity plate was used to determine the cell viability using the CCK-8 method. 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 measured after shaking and incubating for 5 minutes.

[0161] 2.4 Evaluation Method: Cytotoxicity (CCK-8 Assay): Compare the OD values ​​of the virus control, cell control, and sample control to calculate cell viability and further calculate the drug's cytotoxicity. Pseudovirus Infection Rate: Using the cell control as the background value, the chemiluminescence values ​​of the virus control and drug control wells are subtracted from the background value to calculate the relative infection rate relative to the virus control wells, thereby calculating the drug's protective activity against virus-infected cells.

[0162] The present invention provides some compounds Ia, Ib, Ic for SARS-CoV-2 pseudovirus IC 50 Activity was tested (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 were within 0.05 μM, with compound Ia showing an inhibitory activity of 8.5 nM against the SARS-CoV-2 pseudovirus, and exhibiting very low cytotoxicity, all above 100 μM. Compounds Ia, Ib, and Ic exhibited excellent inhibitory activity against both MERS-CoV and SARS-CoV-2 coronaviruses, enabling the simultaneous inhibition of multiple viruses.

[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 aspects.

[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 appended 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 ring 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, hydroxyl, or amino; In formula (I), R 2 is hydrogen, C1-C6 alkyl, C3-C8 cycloalkyl; 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, wherein 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; The R 3 Methyl, ethyl, Where n = 1, 2, 3, Where n = 1, 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 the structural formula shown in formula (II): Among them, L, R 2 、R 3 、R 4 Same as the definition in 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 or a 2,5-disubstituted thiophene ring, 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 Same as the definition in claim 1.

5. A method for preparing a 3,5-disubstituted 1,2,4-triazole compound, characterized in that: The preparation method includes the following two routes: Route 1: (1) In a first solvent, substituted o-aminothiophenol and The reaction occurs to produce 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, the intermediate IV-2a and R obtained in step (2) are reacted 2 NCS reacts to produce intermediate IV-3a; (4) In a fifth solvent, the intermediate IV-3a obtained in step (3) undergoes a ring-closure 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 same as defined in claim 1; X is Cl, Br, I; Or, Route 2: (1) In the second solvent, in the presence of a base and a palladium catalyst, R 1 -X and Palladium-catalyzed Suzuki coupling reaction under alkaline conditions generates 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 intermediate IV-2b; (3) In a fourth solvent, the intermediate IV-2b obtained in step (2) and R 2 NCS reacts to produce intermediate IV-3b; (4) In a fifth solvent, the intermediate IV-3b obtained in step (3) undergoes a ring-closure 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, wherein In route 1, in step (1), the first solvent is one or more of dimethyl sulfoxide, N,N-dimethylformamide, and toluene; and / or the substituted o-aminothiophenol and The molar ratio of the substituted o-aminothiophenol and The reaction temperature is 120°C to 140°C; and / or, the substituted o-aminothiophenol and The reaction time for the reaction to occur 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 R 1 -X and The molar ratio of the amount is 1:1.5 to 1:2; and / or, the palladium catalyst is one or more of tetrakistriphenylphosphine palladium, bistriphenylphosphine palladium dichloride (II), 1,1'-bis(diphenylphosphino)ferrocene palladium dichloride (II) dichloromethane complex; and / or, the R 1 -X and palladium catalyst is used in a molar ratio of 1:0.05 to 1:0.10; and / or, the base is one or more of potassium carbonate and sodium carbonate; and / or, the base and R 1 -X 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 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 intermediate IV-2a or IV-2b and R 2 The molar ratio of NCS 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 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; the virus is severe acute respiratory syndrome coronavirus, Middle East respiratory syndrome coronavirus, or new coronavirus.

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 viruses, thereby reducing the virus infection rate.

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

Images