A benzimidazole compound and its preparation method and application

Abstract

The present invention provides a benzimidazole compound, a preparation method, and an application thereof. The structural formula of the benzimidazole compound is shown below, and the benzimidazole compound is a Z-isomer, wherein R1 is selected from C1-C10 alkyl, C1-C10 alkoxy, C1-C10 haloalkoxy, C1-C10 haloalkyl, C1-C10 hydroxyalkyl, and cyano. The benzimidazole compound has a strong inhibitory effect on arenaviruses, low cytotoxicity, and a high selectivity index, and can be used to prepare anti-arenavirus drugs. The preparation method is stable, simple to operate, reproducible, and has controllable quality.

Description

Technical Field

[0001] The present invention belongs to the technical field of antiviral drugs, and in particular relates to a benzimidazole compound, a preparation method and an application thereof. Background Art

[0002] Arenaviruses are RNA viruses and a type of hemorrhagic fever virus. They are divided into Old World and New World arenaviruses. Old World arenaviruses primarily include Lymphocytic Choriomeningitis Virus (LCMV), Lassa Virus (LASV), and Lujo Virus; New World arenaviruses primarily include Junin Virus (JUNV), Machupo Virus (MACV), Guanarito Virus (GTOV), White Water Arroyo Virus (WWAV), Sabia Virus (SABV), and Tacaribe Virus. Lassa, Junin, Machupo, and Guanarito viruses primarily cause hemorrhagic fever in humans and have been designated as Category A potential bioterrorism agents by the US Centers for Disease Control and Prevention. This type of virus spreads quickly and has a high mortality rate, making it an important potential warfare agent and very likely to become a biological weapon.

[0003] The arenavirus genome consists of two single-stranded RNA segments, each encoding two genes in opposite orientations. The larger segment is the L RNA, measuring 7.2 kb, while the smaller segment is the S RNA, measuring 3.4 kb. The L RNA encodes the L and Z proteins; the L protein is an RNA-dependent RNA polymerase (RdRp), and the Z protein is a small zinc-binding RING finger protein involved in viral budding. The S RNA encodes the nucleoprotein (NP) and the envelope glycoprotein precursor (GPC), which is embedded in the lipid bilayer surrounding the viral nucleocapsid. The arenavirus envelope glycoprotein (GP) functions to attach the virus to specific host cell receptors and mediate fusion of the viral and host membranes, thereby depositing the viral genome within the target cell. Co-translational transport of the envelope glycoprotein throughout the endoplasmic reticulum membrane is facilitated by an N-terminal signal peptide, which is subsequently removed by a signal peptidase. Post-translational proteolysis further processes it into an N-terminal subunit (GP1) and a C-terminal transmembrane subunit (GP2). GP1 contains a receptor binding determinant, and GP2 is capable of undergoing a significant conformational rearrangement associated with membrane fusion. The two subunits remain bound to each other and are assembled into a trimeric complex of the heterodimer. GP1 and GP2 form heterodimers that facilitate entry into host cells by interacting with the α-dystroglycan gene of the Old World viruses LASV and LCMV or the transferrin 1 receptor of the New World viruses.

[0004] Arenaviruses spread rapidly and have a high mortality rate, necessitating the development of safe and effective drugs to protect against potential biological attacks and arenavirus outbreaks. However, no anti-arenavirus drugs are currently available. Previous studies have shown that the arenavirus glycoprotein GP2 is a key protein mediating viral entry into cells. Benzimidazole structures can act on the arenavirus envelope glycoprotein GP2, inhibiting conformational transformation of the viral GP protein and preventing fusion with the cell membrane. This inhibits viral entry and thus achieves antiviral efficacy. However, the anti-arenavirus activity of previously developed compounds remains low. Therefore, further development of drugs with enhanced anti-arenavirus activity, lower cytotoxicity, and higher selectivity is needed. Summary of the Invention

[0005] The technical problem solved by the present invention is to provide a benzimidazole compound and its preparation method and application. The benzimidazole compound has a strong inhibitory effect on arenavirus, and at the same time has low cytotoxicity and a high selectivity index, and can be used to prepare drugs against arenavirus; the preparation method has stable process, simple operation, good reproducibility and controllable quality.

[0006] In order to solve the above problems, the first aspect of the present invention provides a benzimidazole compound or a pharmaceutically acceptable salt thereof, wherein the structural formula of the compound is shown in Formula 1 below, and the benzimidazole compound is a Z-isomer:

[0007]

[0008] Wherein, R1 is selected from and

[0009] Wherein, R2 is selected from C1-C10 alkyl, C1-C10 alkoxy, C1-C10 haloalkoxy, C1-C10 haloalkyl, C1-C10 hydroxyalkyl, and cyano.

[0010] Preferably, R2 is selected from -CH3, -CN.

[0011] Preferably, the structural formula of the compound is shown in Formula 2 below:

[0012]

[0013] The second aspect of the present invention provides a method for preparing the above-mentioned benzimidazole compound, comprising the following steps:

[0014] S1. 4-fluoro-3-nitrobenzonitrile and 4-isopropoxyaniline are reacted in the presence of a catalyst, and the reaction product is separated to obtain an intermediate I. The structural formula of the intermediate I is shown in Formula 3 below;

[0015] S2. The intermediate I is subjected to a reduction reaction, and the reaction product is separated to obtain an intermediate II. The structural formula of the intermediate II is shown in Formula 4 below;

[0016] S3. The intermediate II is mixed with formamidine acetate and the reaction product is separated to obtain an intermediate III. The structural formula of the intermediate III is shown in Formula 5 below;

[0017] S4. The intermediate III is subjected to a reduction reaction, and the reaction product is separated to obtain an intermediate IV. The structural formula of the intermediate IV is shown in Formula 6 below;

[0018] S5. The compound of formula 7 is reacted with triphenylphosphine to obtain intermediate V;

[0019] S6. reacting the intermediate IV with the intermediate V under the action of n-butyl lithium, separating the reaction product to obtain the benzimidazole compound,

[0020]

[0021] Wherein, R1 is selected from

[0022] Wherein, R2 is selected from C1-C10 alkyl, C1-C10 alkoxy, C1-C10 haloalkoxy, C1-C10 haloalkyl, C1-C10 hydroxyalkyl, and cyano.

[0023] Preferably, in step S4, the intermediate III is reduced using Raney nickel.

[0024] Preferably, step S4 is specifically adding the intermediate III to a solvent to obtain a reaction solution, and then adding an aqueous suspension of Raney nickel to the reaction solution to carry out a reduction reaction; the solvent is a mixture of water, acetic acid and pyridine, and the volume ratio of water:acetic acid:pyridine is 1:1:2.

[0025] The third aspect of the present invention provides a pharmaceutical composition comprising the above-mentioned benzimidazole compound or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier or excipient.

[0026] The fourth aspect of the present invention provides a use of the above-mentioned benzimidazole compound or a pharmaceutically acceptable salt thereof or the above-mentioned pharmaceutical composition in the preparation of a drug against arenavirus, wherein the arenavirus is one of Lassa virus, Hunin virus, Machupo virus, Guanarito virus, Sabia virus, Whitewater Arroyo virus, Chapare virus, LCMV, and LCMV-like virus.

[0027] Preferably, the arenavirus is Lassa virus.

[0028] Preferably, the benzimidazole compound or a pharmaceutically acceptable salt thereof or the pharmaceutical composition acts on the arenavirus envelope glycoprotein.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] The benzimidazole compounds or pharmaceutically acceptable salts thereof of the present invention have a strong inhibitory effect on arenaviruses. At the same time, the compounds have low cytotoxicity and a high selectivity index, and can be used to prepare drugs against arenaviruses.

[0031] The preparation method of the benzimidazole compound of the present invention has stable process, simple operation, good reproducibility and controllable quality. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is a high-resolution mass spectrum of compound 7h-Z prepared in an example of the present invention;

[0033] Figure 2 is the hydrogen nuclear magnetic resonance spectrum of compound 7h-Z prepared in the example of the present invention;

[0034] Figure 3is the carbon NMR spectrum of compound 7h-Z prepared in the examples of the present invention;

[0035] Figure 4 This is a graph showing the detection results of the compound 7h-Z prepared in an example of the present invention inhibiting viral infection in a pseudovirus model. DETAILED DESCRIPTION

[0036] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0037] In order to further develop safe and effective drugs that can inhibit arenaviruses, the present invention has improved the compound structure based on previous research and obtained another structure of benzimidazole compounds with anti-arenavirus effects, some of which have higher anti-arenavirus activity, lower cytotoxicity and higher selectivity.

[0038] A first aspect of an embodiment of the present invention provides a benzimidazole compound or a pharmaceutically acceptable salt thereof, wherein the structural formula of the compound is shown in Formula 1 below, and the benzimidazole compound is a Z-isomer:

[0039]

[0040] Wherein, R1 is selected from

[0041] Wherein, R2 is selected from C1-C10 alkyl, C1-C10 alkoxy, C1-C10 haloalkoxy, C1-C10 haloalkyl, C1-C10 hydroxyalkyl, and cyano.

[0042] The Z-isomer refers to the Z-isomer among the E,Z isomers.

[0043] Pharmaceutically acceptable salts include inorganic and organic salts. Inorganic salts include salts of hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like. Organic salts include salts of acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like.

[0044] C1-C10 alkyl represents a branched or straight-chain saturated hydrocarbon group having 1 to 10 carbon atoms, such as methyl, ethyl, propyl, 1-methylethyl (isopropyl), butyl, 1-methylpropyl (sec-butyl), 2-methylpropyl (isobutyl) and tert-butyl, etc. Methyl and tert-butyl are preferred.

[0045] The C1-C10 alkoxy group represents a linear or branched C1-C10 alkyl group linked via an oxygen atom, for example, methoxy, ethoxy, propoxy, 1-methylethoxy (isopropoxy), butoxy, 1-methylpropoxy (sec-butoxy), 2-methylpropoxy (isobutoxy) and 1,1-dimethylethoxy (tert-butoxy), etc. Preferably, it is ethoxy.

[0046] A C1-C10 haloalkoxy group is a group in which one or more hydrogen atoms in a C1-C10 alkoxy group are replaced by halogen atoms. Specifically, one, two, three, or four hydrogen atoms may be replaced by halogen atoms. Halogen atoms include fluorine, chlorine, bromine, and iodine atoms, and are preferably fluoroalkoxy groups. Examples include difluoromethoxy and trifluoromethoxy groups.

[0047] A C1-C10 haloalkyl group is a group in which one or more hydrogen atoms in a C1-C10 alkyl group are replaced by halogen atoms. Specifically, one, two, three, or four hydrogen atoms may be replaced by halogen atoms. Halogen atoms include fluorine atoms, chlorine atoms, bromine atoms, iodine atoms, and are preferably fluoroalkyl groups. Examples include difluoromethyl and trifluoromethyl groups. Trifluoromethyl is preferred.

[0048] A C1-C10 hydroxyalkyl group is a group in which one or more hydrogen atoms in a C1-C10 alkyl group are replaced by a hydroxy group, for example, 2-hydroxy-tert-butyl.

[0049] In some embodiments, R2 is selected from -CH3,

[0050] In some embodiments, the compound has the structural formula shown in Formula 2 below:

[0051]

[0052] Experimental studies have shown that the compound of formula 2 has better activity in inhibiting arenavirus, lower cytotoxicity and higher selectivity.

[0053] A second aspect of the embodiments of the present invention provides a method for preparing the above-mentioned benzimidazole compound, comprising the following steps:

[0054] S1. 4-fluoro-3-nitrobenzonitrile and 4-isopropoxyaniline are reacted in the presence of a catalyst, and the reaction product is separated to obtain an intermediate I. The structural formula of the intermediate I is shown in Formula 3 below;

[0055] S2. The intermediate I is subjected to a reduction reaction, and the reaction product is separated to obtain an intermediate II. The structural formula of the intermediate II is shown in Formula 4 below;

[0056] S3. The intermediate II is mixed with formamidine acetate and the reaction product is separated to obtain an intermediate III. The structural formula of the intermediate III is shown in Formula 5 below;

[0057] S4. The intermediate III is subjected to a reduction reaction, and the reaction product is separated to obtain an intermediate IV. The structural formula of the intermediate IV is shown in Formula 6 below;

[0058] S5. The compound of formula 7 is reacted with triphenylphosphine to obtain intermediate V;

[0059] S6. reacting the intermediate IV with the intermediate V under the action of n-butyl lithium, separating the reaction product to obtain the benzimidazole compound,

[0060]

[0061] Wherein, R1 is selected from

[0062] Wherein, R2 is selected from C1-C10 alkyl, C1-C10 alkoxy, C1-C10 haloalkoxy, C1-C10 haloalkyl, C1-C10 hydroxyalkyl, and cyano.

[0063] In some embodiments, step S1 specifically includes: adding 4-fluoro-3-nitrobenzonitrile to a solvent, stirring until dissolved, adding a catalyst and 4-isopropoxyaniline, then heating to reflux and stirring overnight, cooling and concentrating under reduced pressure, dissolving the residue with ethyl acetate, adding water to wash the liquid, separating the aqueous layer, back-extracting with ethyl acetate, combining the organic layers, adding anhydrous sodium sulfate to dry, concentrating under reduced pressure, adding the crude product to a slurry solvent, slurrying and filtering to obtain intermediate I.

[0064] In some embodiments, triethylamine is used as a catalyst in step S1.

[0065] In some embodiments, the solvent in step S1 is acetonitrile.

[0066] In some embodiments, the slurrying solvent in step S1 is tert-butyl methyl ether.

[0067] In some embodiments, the volume ratio of the crude product to the slurry solvent in step S1 is 1:3-1:5.

[0068] In some embodiments, step S2 specifically includes: adding intermediate I to a solvent, stirring until dissolved, then heating, adding a reducing agent in portions, continuing to heat until no starting material remains, then cooling the reaction solution to room temperature, and adjusting the pH to alkaline, then filtering, washing the precipitate, separating the filtrate, drying the organic layer, and concentrating under reduced pressure to obtain intermediate II.

[0069] In some embodiments, the solvent in step S2 is ethyl acetate.

[0070] In some embodiments, the reducing agent in step S2 is tin chloride dihydrate.

[0071] In some embodiments, diatomaceous earth is used for filtration in step S2.

[0072] In some embodiments, ethyl acetate and water are used to wash the precipitate in step S2.

[0073] In some embodiments, step S2 is to dry the organic layer using anhydrous sodium sulfate.

[0074] In some embodiments, step S3 specifically comprises: adding intermediate II to a solvent, adding formamidine acetate, heating to reflux for 9-10 hours, then cooling to room temperature and stirring overnight, then removing the solvent under reduced pressure, diluting the residue with water, filtering, washing the obtained solid, adding a slurry solvent, then heating and stirring, slurrying, cooling and filtering, and drying the obtained solid to obtain intermediate III.

[0075] In some embodiments, the solvent in step S3 is ethanol.

[0076] In some embodiments, the slurrying solvent in step S3 is methanol.

[0077] In some embodiments, the volume ratio of the residue to the slurry solvent in step S3 is 1:3-1:5.

[0078] In some embodiments, step S4 specifically comprises: adding intermediate III to a solvent, adding sodium hypophosphite, adding a reducing agent, carrying out a reduction reaction, thin layer monitoring indicating that the reaction is complete, then cooling the mixture to room temperature, diluting with water, adding dichloromethane for separation, separating the dichloromethane layer and extracting the aqueous portion with dichloromethane, washing the combined organic layer with water, drying, filtering and evaporating under reduced pressure, adding isopropanol to the residue for slurrying, then heating, stirring at room temperature, placing in a refrigerator, filtering, and obtaining intermediate IV.

[0079] In some embodiments, the reducing agent in step S4 is Raney nickel. Compared with the existing diisobutylaluminum hydride reduction method, which has been found in practice to be incomplete and low in yield, the preparation method of the present invention uses Raney nickel for reduction, which has a complete reaction, a high yield, and greatly reduces the time required for batch production.

[0080] In some embodiments, in step S4, the reduction reaction temperature is 45-50° C., and the reaction time is 3-5 h.

[0081] In some embodiments, the solvent in step S4 is a mixture of water, acetic acid, and pyridine, with a volume ratio of water:acetic acid:pyridine of 1:1:2. During the reduction reaction, the inventors compared the use of acetic acid as the solvent with a 1:1:2 mixed solvent of water:acetic acid:pyridine. The use of the mixed solvent accelerated the reaction and achieved a complete reaction.

[0082] In some embodiments, step S5 specifically comprises: adding the compound of formula 7 to a solvent, adding triphenylphosphine, reacting the reactants under heating reflux, cooling to room temperature after the reaction is completed, filtering the suspension, washing with a solvent and drying to obtain intermediate V.

[0083] In some embodiments, the solvent in step S5 is toluene.

[0084] In some embodiments, step S6 specifically includes: adding intermediate V to a solvent, adding it to the solvent and cooling it to -78°C, adding a hexane solution of n-butyl lithium, and stirring the reactants at -78°C, then adding intermediate IV to react, and allowing the reactants to warm to room temperature. TLC monitors that the reaction is 80% complete, concentrating the reactants to dryness under reduced pressure, purifying the residue on a rapid separation column, and eluting to separate the cis-structured benzimidazole compound, and the product is recrystallized using acetonitrile.

[0085] In some embodiments, the solvent in step S6 is anhydrous tetrahydrofuran.

[0086] In some embodiments, in step S6, elution is performed with a gradient solution of 10% to 80% ethyl acetate in n-hexane (petroleum ether).

[0087] In some embodiments, in step S6, the volume ratio of the recrystallization solvent to the benzimidazole compound is 1:8-1:10.

[0088] A third aspect of the embodiments of the present invention provides a pharmaceutical composition comprising the above-mentioned benzimidazole compound or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier or excipient.

[0089] A fourth aspect of an embodiment of the present invention provides a use of the above-mentioned benzimidazole compound or a pharmaceutically acceptable salt thereof or the above-mentioned pharmaceutical composition in the preparation of a drug against arenavirus, wherein the arenavirus is one of Lassa virus, Hunin virus, Machupo virus, Guanarito virus, Sabia virus, Whitewater Arroyo virus, Chapare virus, LCMV, and LCMV-like virus.

[0090] In some embodiments, the arenavirus is Lassa virus.

[0091] In some embodiments, the benzimidazole compound or a pharmaceutically acceptable salt thereof or the pharmaceutical composition acts on the envelope glycoprotein of arenavirus.

[0092] Example 1

[0093] (1) Preparation of benzimidazole compounds

[0094] The benzimidazole compounds prepared in the examples of the present invention, their R1 groups and corresponding compound numbers are shown in Table 1 (Z represents the Z isomer).

[0095] Table 1 Benzimidazole compounds R1 group and corresponding compound numbers

[0096]

[0097] The synthetic route of benzimidazole compounds is as follows:

[0098]

[0099] The preparation of each compound adopts the same preparation method, which will not be described in detail here. Taking compound 7h-Z as an example, the specific preparation method is:

[0100] S1: Synthesis of 4-((4-isopropoxyphenyl)amino)-3-nitrobenzonitrile (Intermediate I).

[0101] To a reaction flask, add 20 g of 4-fluoro-3-nitrobenzonitrile to 100 ml of acetonitrile and stir until completely dissolved. Then add 19.46 g of triethylamine and 22.76 g of 4-isopropoxyaniline. Heat to reflux and stir overnight. After cooling, concentrate under reduced pressure. Dissolve the residue in 500 ml of ethyl acetate and wash with 200 ml of water. Separate the aqueous layer and back-extract with a small amount of ethyl acetate (3 x 50 ml). Combine the organic layers, dry over anhydrous sodium sulfate, and concentrate under reduced pressure. The crude product is slurried in tert-butyl methyl ether and filtered to obtain 35 g of brownish-red intermediate I in a 97.7% yield.

[0102] S2: Synthesis of 3-amino-4-((4-isopropoxyphenyl)amino)benzonitrile (Intermediate II).

[0103] 24 g of Intermediate I was added to a reaction flask, followed by complete dissolution with 600 ml of ethyl acetate. The mixture was heated to 50°C, and 63.75 g of tin chloride dihydrate was added portionwise over 6 minutes. Heating was continued until no starting material remained. The reaction mixture was cooled to room temperature, and aqueous sodium bicarbonate was added to an alkaline pH. The mixture was stirred for 1 hour, then filtered through celite. The precipitate was washed with 600 ml of ethyl acetate and water. The filtrate was transferred to a separatory funnel and separated. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to yield 20.5 g of brown Intermediate II (95.3% yield).

[0104] S3: Synthesis of intermediate III.

[0105] Add 20.0 g of Intermediate II to 500 ml of ethanol, add 10.13 g of formamidine acetate, heat under reflux for 9-10 hours, cool to room temperature, and stir overnight. Remove the solvent under reduced pressure, dilute the residue with 120 ml of water, stir for 30 minutes, and filter. Rinse the resulting solid with water, add an appropriate amount of methanol, heat and stir to form a slurry, cool, filter, and dry the solid in a vacuum oven at 50°C to obtain 20.75 g of brown-yellow Intermediate III, with a yield of 97.0%.

[0106] S4: Add 8.4 g of intermediate III to 168 ml of a mixed solution of water: acetic acid: pyridine = 1:1:2, and add 12.84 g of hypophosphorous acid. Add an aqueous suspension of 8.49 g of Raney nickel to the reaction solution, maintain the temperature at 45-50°C and react for 3-5 hours. Thin layer monitoring indicates that the reaction is complete. Cool the mixture to room temperature, dilute with water, and add 250 ml of dichloromethane for separation. Separate the organic layer and extract the aqueous layer with a small amount of dichloromethane. Wash the combined organic layers with water, dry them with anhydrous sodium sulfate, filter them and evaporate them under reduced pressure. Add an appropriate amount of isopropanol to the residue to make a slurry, heat it to 50 degrees Celsius, stir it at room temperature for 2 hours, place it in a refrigerator, filter it, and obtain 8.15 g of intermediate IV as an off-white solid with a yield of 95.9%.

[0107] S5: Synthesis of Intermediate V. 5 g of 2-(bromomethyl)naphthalene was added to 100 ml of toluene, followed by 5.93 g of triphenylphosphine. The reaction was heated under reflux for 48 hours and allowed to cool to room temperature. The suspension was filtered, washed with toluene, and dried to afford 9.29 g of Intermediate V as a white solid (85% yield).

[0108] S6: Synthesis of compound of formula I. 1.72 g of intermediate V was added to 60 ml of anhydrous tetrahydrofuran and cooled to -78°C. A 2.5 M hexane solution of n-butyl lithium (0.24 g) was added and the reactants were stirred at -78°C for 1 hour. 1.0 g of intermediate IV was added, reacted for 0.5 h and the reactants were allowed to warm to room temperature. After 12 hours, the reaction was 80% complete as monitored by TLC. The reactants were concentrated to dryness under reduced pressure. The residue was purified on a rapid separation column and eluted with a gradient solution of 10%-80% ethyl acetate in n-hexane (petroleum ether) to separate the target product as the Z isomer. The product was recrystallized from acetonitrile to obtain 0.38 g of an off-white solid with a yield of 26%.

[0109] (II) Structural identification of benzimidazole compounds

[0110] The prepared product was identified by LC-MS, 1H-NMR and 13C-NMR. Figure 1-3 And the following data shows.

[0111] In the embodiment of the present invention, the high-resolution mass spectrometry detection instrument adopts a tandem Fourier transform mass spectrometer produced by BRUKER; and the nuclear magnetic resonance detection instrument adopts an INOVA 600 MHz nuclear magnetic resonance instrument produced by VARIAN.

[0112] The data is as follows:

[0113] Compound LHF-535

[0114] NMR data:

[0115] 1 H NMR(600MHz,Chloroform-d)δ8.00(s,1H),7.76(s,1H),7.37(d,J=7.3Hz,2H),7.33(d,J=7.0Hz,2H),7.30(d,J=8.4Hz,1H),7.27–7.22(m,3H ),7.03(d,J=8.7Hz,2H),6.73(d,J=12.2Hz,1H),6.58(d,J=12.2Hz,1H),4.65-4.55(m,1H),2.01(s,1H),1.56(s,6H),1.39(d,J=6.1Hz,6H). 13 C NMR(151MHz,Chloroform-d)δ157.89,148.09,143.94,142.99,135.90,133.50,132.29,130.49,129 .33,128.89,126.39,125.71,125.10,124.50,120.71,117.03,110.19,72.51,70.61,31.76,22.12.

[0116] Mass spectrometry data:

[0117] HRMS (ESI) m / z: (M+H) + calcd for C 27 H 28 N2O2, 413.2151; found 413.2234.

[0118] Compound 7a-Z

[0119] NMR data:

[0120] 1H NMR (600MHz, Chloroform-d): δ8.06(s,1H),7.84(s,1H),7.42(d,J=8.9Hz,2H),7.35(d,J=8.4Hz,1H),7.30(d,J=8.4Hz,1H),7.28–7.23 (m,4H),7.07(d,J=8.9Hz,2H),6.74(d,J=12.3Hz,1H),6.60(d,J=12.2Hz,1H),4.64(p,J=6.1Hz,1H),1.43(d,J=6.1Hz,6H),1.32(s,9H). 13 C NMR (151MHz, Chloroform-d): δ157.85,150.64,144.46,143.28,134.91,133.96,132.84,128.88 ,128.50,127.62,126.25,125.73,122.38,118.52,116.99,110.63,70.55,34.74,31.44,22.12.

[0121] Mass spectrometry data:

[0122] HRMS(ESI)m / z:(M+H) + calcd for C 28 H 30 N2O 411.2358; found 411.2422.

[0123] Melting point: 172-174℃.

[0124] Compound 7b-Z

[0125] NMR data:

[0126] 1 H NMR (600MHz, Chloroform-d): δ8.03(s,1H),7.71(s,1H),7.47(d,J=8.4Hz,2H),7.37(d,J=8.8Hz,2H),7.34(d,J=8.2Hz,2H),7.31(d,J=8.4Hz,1 H),7.12(dd,J=8.4,1.7Hz,1H),7.04(d,J=8.9Hz,2H),6.91(d,J=12.2Hz,1H),6.58(d,J=12.1Hz,1H),4.65-4.57(m,1H),1.39(d,J=6.1Hz,6H). 13CNMR (151MHz, Chloroform-d): δ157.90,144.12,143.37,142.47,133.91,133.76,132.16,130.97 ,129.69,128.62,127.75,125.69,124.76,120.92,119.11,116.97,110.50,110.40,70.53,22.07.

[0127] Mass spectrometry data:

[0128] HRMS(ESI)m / z:(M+H) + calcd for C 25 H 21 N3O 380.1685; found 380.1747.

[0129] Melting point: 165-166℃.

[0130] Compound 7c-Z

[0131] NMR data:

[0132] 1 H NMR (600MHz, Chloroform-d): δ8.01(s,1H),7.77(s,1H),7.37(d,J=8.5Hz,2H),7.29(d,J=8.5Hz,1H),7.23(d,J=8.5Hz,1H),7.18(d,J =7.9Hz,2H),7.07-6.94(m,4H),6.71(d,J=12.2Hz,1H),6.58(d,J=12.2Hz,1H),4.66-4.57(m,1H),2.30(s,3H),1.39(d,J=6.2Hz,6H). 13 C NMR (151MHz, Chloroform-d): δ157.75,144.06,142.97,136.83,134.54,133.39,132.30,129 .95,129.59,129.06,128.90,125.64,125.04,120.77,116.93,110.06,70.51,22.09,21.34.

[0133] Mass spectrometry data:

[0134] HRMS(ESI)m / z:(M+H) + calcd for C 25 H 24N2O 369.1889; found 369.1955

[0135] Melting point: 168-169℃.

[0136] Compound 7d-Z

[0137] NMR data:

[0138] 1 H NMR (600MHz, Chloroform-d): δ8.05(s,1H),7.82(s,1H),7.41(d,J=8.9Hz,2H),7.3 3(d,J=8.4Hz,1H),7.27(d,J=8.5Hz,1H),7.24(d,J=6.9Hz,2H),7.06(d,J=6.9Hz,2H ),6.77(d,J=6.7Hz,2H),6.69(d,J=12.1Hz,1H),6.58(d,J=12.2Hz,1H),4.64(dtq,J =9.2,5.8,3.2,2.6Hz,1H),4.02(dt,J=7.3,2.4Hz,2H),1.42(dt,J=5.0,2.5Hz,9H). 13 C NMR (151MHz, Chloroform-d): δ158.08,157.72,144.06,142.92,133.30,132.45,130.21,129.75,12 9.24,129.04,128.88,125.60,124.99,120.66,116.91,114.25,110.07,70.48,63.38,22.07,14.94.

[0139] Mass spectrometry data:

[0140] HRMS(ESI)m / z:(M+H) + calcd for C 26 H 26 N2O2,399.1994; found 399.2057.

[0141] Melting point: 161-162℃.

[0142] Compound 7e-Z

[0143] NMR data:

[0144] 1H NMR (600MHz, Chloroform-d): δ8.02 (s, 1H), 7.74 (d, J = 1.6 Hz, 1H), 7.37 (d, J = 8. 9Hz,2H),7.31(d,J=8.4Hz,1H),7.26(d,J=8.7Hz,2H),7.18(dd,J=8.4,1.7Hz,1H ),7.03(d,J=8.9Hz,2H),6.94(d,J=8.6Hz,2H),6.76(d,J=12.1Hz,1H),6.56(d, J=12.1Hz,1H),6.48(t,J=74.1Hz,1H),4.65-4.56(m,1H),1.39(d,J=6.1Hz,6H). 13 C NMR (151MHz, Chloroform-d): δ157.83, 150.20 (t, J = 2.7Hz), 144.09, 143.14, 134.75, 133.57, 131.75, 131.06, 1 30.50,128.79,128.31,125.67,124.90,120.77,119.20,116.96,116.08(t,J=259.0Hz),110.29,70.53,22.08.

[0145] Mass spectrometry data:

[0146] HRMS(ESI)m / z:(M+H) + calcd for C 25 H 22 F2N2O2 421.1649; found 421.1712 Melting point: 173-174℃.

[0147] Compound 7f-Z

[0148] NMR data:

[0149] 1 H NMR (600MHz, Chloroform-d): δ8.03(s,1H),7.75(d,J=1.7Hz,1H),7.37(d,J=8.9Hz,2H),7.31(d,J=8.4Hz,1H),7.28(d,J=8.5Hz,2H),7 .18(dd,J=8.5,1.7Hz,1H),7.07-7.00(m,4H),6.80(d,J=12.2Hz,1H),6.56(d,J=12.1Hz,1H),4.65-4.56(m,1H),1.39(d,J=6.1Hz,6H). 13C NMR (151MHz, Chloroform-d): δ157.87, 148.10 (q, J = 1.5Hz), 144.12, 143.20, 136.18, 133.66, 131.62, 131.57, 1 30.42,128.79,128.08,125.70,124.84,120.85,120.76,120.57(q,J=257.2Hz),116.98,110.35,70.55,22.10.

[0150] Mass spectrometry data:

[0151] HRMS(ESI)m / z:(M+H) + calcd for C 25 H 21 F3N2O2 439.1555; found439.1619.

[0152] Melting point: 161-162℃.

[0153] Compound 7g-Z

[0154] NMR data:

[0155] 1 H NMR (600MHz, Chloroform-d): δ8.03(s,1H),7.74(s,1H),7.45(d,J=8.3Hz,2H),7.39-7.33(m,4H),7.30(dd,J=8.5Hz,1H),7. 16(d,J=8.4Hz,1H),7.06-7.00(m,2H),6.86(d,J=12.2Hz,1H),6.60(d,J=12.2Hz,1H),4.66-4.55(m,1H),1.41-1.35(m,6H). 13 CNMR (151MHz, Chloroform-d): δ157.87,144.10,143.25,141.25,133.76,132.72,131.33,129.27,128.90(q,J=32.0Hz ),128.72,128.09,125.68,125.29(q,J=3.9Hz),124.85,124.29(q,J=272.3Hz),120.89,116.97,110.39,70.53,22.07.

[0156] Mass spectrometry data:

[0157] HRMS(ESI)m / z:(M+H) +calcd for C 25 H 21 F3N2O 423.1606; found 423.1667.

[0158] Melting point: 165-166℃.

[0159] Compound 7h-Z

[0160] NMR data:

[0161] 1 H-NMR(600MHz,Chloroform-d)δ8.02(s,1H),7.80(s,1H),7.77(s,1H),7.74-7.69(m,1H),7.73-7.69(m,1H),7.62(d,J=8.5Hz,1H),7.43-7.40(m,2H) ,7.39–7.35(m,3H),7.28-7.22(m,2H),7.02(d,J=8.9Hz,2H),6.84(d,J=12 .1Hz,1H),6.78(d,J=12.2Hz,1H),4.65-4.56(m,1H),1.38(d,J=6.1Hz,6H). 13 C-NMR(151MHz,Chloroform-d)δ157.79,143.99,143.07,135.23,133.65,133.53,132.67,132.16,131.02,129.56,128 .83,128.11,128.02,127.73,127.65,127.10,126.06,125.91,125.65,125.12,120.99,116.95,110.13,70.52,22.09.

[0162] Mass spectrometry data: HRMS (ESI) m / z: (M+H)+calcd for C28H24N2O, 405.1889; found 405.1955. (M+1) is the molecular ion peak.

[0163] Melting point: 170-171℃.

[0164] Example 2 Determination of cytotoxicity and antiviral activity of benzimidazole compounds

[0165] This study constructs a pseudovirus model based on the envelope glycoprotein of the Lassa virus, using the envelope glycoprotein rather than the virus itself. This allows for safe activity evaluation under normal BSL-2 conditions. The viral entry step is an important target for antiviral drug development, as it is an essential process in every viral life cycle.

[0166] The Lassa fever envelope function was originally evaluated using a Lassa pseudovirus model. This pseudovirus model specimen was generated by co-transfection of the Lassa virus envelope with a replication-defective HIV provirus using a luciferase reporter gene. The provirus is engineered so that the HIV envelope is not expressed, thereby obtaining heterologous viral envelope proteins when budding virions nonspecifically capture cell surface proteins. Pseudotypes prepared in this manner will infect cells via the heterologous envelope and are commonly used to test heterologous envelope function. Infection is measured by the luciferase signal generated from an integrated HIV reporter gene construct. The amount of infectious virus used to infect the cell culture system is proportional to the luciferase-mediated luminescence produced in the infected cells over several orders of magnitude.

[0167] The present invention uses the GP gene of an arenavirus (LASV-Josiah GP) and synthesizes it after codon optimization. The synthesized gene is then cloned into the lentiviral vector pLV[Exp]-CMV, which carries the HIV-1 plasmid enhanced green fluorescent protein and luciferase reporter genes (pLV[Exp]-CMV>EGFP / luc). Plasmid DNA is extracted and transfected into HEK293T cells using the liposome method, and the virus is harvested.

[0168] (I) Culture of HEK 293FT cells and preparation of pseudovirus

[0169] One day before transfection, HEK293T cells were seeded into 10 T175 culture flasks and DMEM medium containing 10% FBS was added. The cells were cultured at 37°C and 5% CO2 for 24 hours. The cell confluency was about 80% to 90% before transfection. One hour before transfection, the medium was changed and Opti-MEM was added. The culture medium was continued to be cultured, and the A and B solutions were mixed separately: the A solution was 45 mL Opti-MEM and 120 μg DNA, which were the packaging plasmids SL3 (gag / pol element), SL4 (pRev element), RP[Exp]-CMV-human betaglobin intron>Lassa virus_gpc (replacing the pVSVG element), and pLV[Exp]-CMV>EGFP / luc, respectively. The plasmids were added to Opti-MEM and then mixed by pipetting with a pipette tip; the B solution was 45 mL Opti-MEM and 1200 μL Lipofectamine 2000, which were mixed by pipetting with a pipette tip and incubated at room temperature for 5 minutes; the A solution was added to the B solution, mixed by pipetting with a pipette tip, and incubated at room temperature for 20 minutes; the incubated transfection complex was slowly added dropwise to 10 T175 culture flasks containing cultured cells, and slightly shaken horizontally to make it evenly distributed, at 37°C, 5% CO2 culture, and replace the culture medium with DMEM medium containing 10% FBS 6 hours after transfection, and continue to culture at 37°C, 5% CO2; 48 hours after transfection, collect the culture supernatant and store it at 4°C, add the same volume of fresh DMEM medium containing 10% FBS and continue to culture, and collect the culture supernatant again after 24 hours; the culture supernatants collected twice are concentrated together, centrifuged at 2000g at 4°C for 30 minutes, and the supernatant obtained by centrifugation is filtered with a 0.45μm filter to remove cell debris; the filtrate is added to a centrifuge tube, centrifuged at 50,000g at 4°C for 2 hours, and the supernatant is discarded. The lentiviral particles are resuspended in 200μL HBSS buffer in each tube of pellet, aliquoted and stored at -80°C. The obtained virus can be used to transduce cells.

[0170] (2) Pseudovirus infection

[0171] To test the antiviral activity, HEK-293 cells were cultured in opaque white 96-well plates. The assay medium consisted of phenol red-free DMEM, 2% FBS, 25 mM HEPES, 4.5 g / L d-glucose, and L-glutamine. The optimal cell density (2 × 10 4 Experiments were performed with different concentrations of the benzimidazole compounds (cells / well) and pseudovirus (MOI = 5). The negative control consisted of medium supplemented with 0.5% DMSO; the positive control consisted of virus without compound. Prior to pseudovirus infection, various concentrations of each benzimidazole compound were prepared and added to the cells. An equal amount of DMSO was used as a solvent control before virus was added. After 48 hours, the supernatant was discarded, the cells were lysed, and the relative luciferase activity in the cell lysate was measured. The infection rate in the drug-treated wells was calculated, with the RLUs value in the DMSO solvent wells set as 100%. Subsequently, HEK-293 cells were infected with Lassa pseudovirus, and luciferase activity was observed 48 hours after infection.

[0172] (III) Luciferase activity analysis

[0173] HEK 293 cells were infected with pseudotyped lentivirus supernatant for 2 days, and the cells were collected and washed with PBS. After centrifugation at 3000 rpm for 5 min, the supernatant was discarded. 90 μL of 1× lysis buffer (CCLR, Promega) was added to the cell pellet to lyse the cells. The cells were thoroughly mixed by pipetting and centrifugation. 10 μL of supernatant was collected and added to a white 96-well plate. 50 μL of luciferase detection substrate was added to each well, and luciferase activity was immediately detected using a detector.

[0174] The experimental data obtained were analyzed and plotted using GraphPad Prism software. A scatter plot of compound concentration versus infection inhibition rate was converted to logarithms and then linearly fitted to obtain correlation, correlation coefficient, and half-maximal inhibitory effective concentration. Based on the linear fit, an inhibition curve for the relevant Lassa virus pseudovirus (LASVpv) was obtained. Each compound had activity to inhibit LASVpv in a dose-dependent manner. Table 2 below shows the inhibitory effect and selectivity index of each benzimidazole compound on the LASVpv pseudovirus, where: a Activity in IC 50 (nM) indicates IC 50 The smaller the value, the higher the activity. b The cytotoxicity of HEK-293 cells was shown to be 50 (μM), CC 50 The larger the value, the less cytotoxic it is; c The selectivity index is expressed as SI. The larger the SI, the better the selectivity. As shown in Table 2, IC 50 The activity is shown to be at the μM level. 50 All reached the nanomolecular level, indicating that each compound possessed excellent activity. Furthermore, each benzimidazole compound exhibited low cytotoxicity and high selectivity. Among them, compound 7h-Z exhibited superior activity against arenavirus infection, lower cytotoxicity, and higher selectivity, taking all factors into consideration.

[0175] Table 2

[0176]

[0177]

[0178] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will readily appreciate that other variations or modifications based on the above descriptions are possible. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A benzimidazole compound or a pharmaceutically acceptable salt thereof, characterized in that: The structural formula of the compound is shown in Formula 2 below, and the benzimidazole compound is a Z-isomer:

2. A method for preparing a benzimidazole compound according to claim 1, characterized in that: The following steps are involved: S1. 4-fluoro-3-nitrobenzonitrile and 4-isopropoxyaniline are reacted in the presence of a catalyst, and the reaction product is separated to obtain an intermediate I. The structural formula of the intermediate I is shown in Formula 3 below; S2. The intermediate I is subjected to a reduction reaction, and the reaction product is separated to obtain an intermediate II. The structural formula of the intermediate II is shown in Formula 4 below; S3. The intermediate II is mixed with formamidine acetate and the reaction product is separated to obtain an intermediate III. The structural formula of the intermediate III is shown in Formula 5 below; S4. The intermediate III is subjected to a reduction reaction, and the reaction product is separated to obtain an intermediate IV. The structural formula of the intermediate IV is shown in Formula 6 below; S5. The compound of formula 7 is reacted with triphenylphosphine to obtain an intermediate V, the structure of the intermediate V is shown in formula 8 below; S6. reacting the intermediate IV with the intermediate V under the action of n-butyl lithium, separating the reaction product to obtain the benzimidazole compound, Among them, R1 is 3. The preparation method according to claim 2, wherein: In step S4, the intermediate III is reduced using Raney nickel.

4. The preparation method according to claim 3, wherein: Step S4 specifically comprises adding the intermediate III to a solvent to obtain a reaction solution, and then adding an aqueous suspension of Raney nickel to the reaction solution to carry out a reduction reaction; the solvent is a mixture of water, acetic acid and pyridine, and the volume ratio of water:acetic acid:pyridine is 1:1:

2.

5. A pharmaceutical composition, characterized in that The invention comprises the benzimidazole compound or a pharmaceutically acceptable salt thereof as claimed in claim 1 and a pharmaceutically acceptable carrier or excipient.

6. Use of the benzimidazole compound or a pharmaceutically acceptable salt thereof according to claim 1 or the pharmaceutical composition according to claim 5 in the preparation of an anti-Lassa virus drug.

7. The use according to claim 6, characterized in that: The benzimidazole compound or a pharmaceutically acceptable salt thereof or the pharmaceutical composition acts on the Lassa virus envelope glycoprotein.

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