CN117551039A - An aryl carboxamide compound, its preparation method and its application against enterovirus EV71 - Google Patents

Abstract

The invention discloses an aryl formamide compound, a preparation method thereof and anti-enterovirus EV71 type applicationIs used, belonging to the technical field of medicines. The structure of the aryl formamide compound is shown as a general formula (I), and the aryl formamide compound is prepared by adopting an aryl formamide raw material containing alcoholic hydroxyl and a carboxylic acid derivative to perform condensation reaction under the action of a condensing agent EDCI and a catalyst DMAP. Cytotoxicity and anti-EV 71 activity results show that the aryl formamide compound has remarkable anti-EV 71 activity, and can be used for preparing anti-enterovirus EV71 medicines or medicines for treating diseases caused by enterovirus EV71 infection.

Description

An aromatic formamide compound and its preparation method and application against enterovirus EV71 type

Technical Field

The invention belongs to the field of medical technology and relates to an arylformamide compound and a preparation method thereof and an application thereof in resisting enterovirus 71 (EV71).

Background Art

Hand, foot and mouth disease (HFMD) is a common infectious disease of infants and young children caused mainly by enterovirus 71 (EV71) and coxsackievirus A16 (CA16). The virus can be transmitted through the fecal-oral route or droplet transmission to cause infection. Its clinical symptoms include fever, sore throat, and vesicular rash on the hands, feet, tongue, and oral mucosa. However, the symptoms of hand, foot and mouth disease caused by EV71 and CA16 are not exactly the same. EV71 infection in infants and young children often leads to a series of serious neurological diseases, such as meningitis, Japanese encephalitis, acute paralysis, and even death in severe cases. Therefore, it poses a greater threat to the life and health of infants and young children.

Structurally, enteroviruses belong to the Picornaviridae family of non-enveloped single-stranded RNA viruses with high mutability. Enteroviruses can be divided into human enteroviruses and non-human enteroviruses, and EV71 belongs to human enteroviruses. EV71 contains approximately 7,400 amino acids, which form four structural proteins VP1-VP4 and seven non-structural proteins (2A-2C and 3A-3D). These proteins are involved in the replication cycle of enteroviruses, and each can be used as a target for antiviral drugs, among which VP1, 2A, 3A and 3C proteins are common drug targets. However, there are currently no specific human enterovirus inhibitors in clinical practice, and a large number of anti-EV71 drug studies that have entered the clinical research stage have all failed due to a series of problems such as poor antiviral activity, obvious side effects and low oral bioavailability. Therefore, it is urgent to develop anti-EV71 drugs with new structural skeletons and targets.

Summary of the invention

The purpose of the present invention is to overcome the deficiencies of the prior art and provide an aryl formamide compound and its application. The aryl formamide compound has anti-enterovirus 71 (EV71) activity and can be developed as a new anti-enterovirus 71 (EV71) drug with broad application prospects. The purpose of the present invention is also to provide a preparation method of the aryl formamide compound.

In order to achieve the above object, the technical solution adopted by the present invention is as follows:

In a first aspect, the present invention provides an arylcarboxamide compound having a structure as shown in the general formula (I) or a pharmacologically or physiologically acceptable salt thereof,

in,

R ¹ is -CH ₃ , -CF ₃ or -CH ₂ CH(CH ₃ ) ₂ ; R ² is -H or -CH ₃ ;

^R3 is

X is CH or N;

Y is

The present invention finds through in vitro anti-EV71 activity test that the above-mentioned aromatic amide compounds have significant inhibitory activity against enterovirus EV71 type and can be used to prepare anti-EV71 drugs.

Preferably, the arylformamide compound is selected from the compounds shown in Table 1 below:

Table 1

More preferably, the aromatic formamide compound is selected from the following compounds: 4-(6,6-dimethyl-4-oxo-3-(trifluoromethyl)-4,5,6,7-tetrahydro-1H-indazol-1-yl)-2-(((1R,4R)-4-hydroxycyclohexyl)amino)benzamide (LJS-2), (1R,4R)-4-((2-carbamoyl-5-(6,6-dimethyl-4-oxo-3-(trifluoromethyl)- 4,5,6,7-tetrahydro-1H-indazol-1-yl)phenyl)amino)glycine cyclohexyl ester (LJS-3), (1R,4R)-4-((2-carbamoyl-5-(6,6-dimethyl-4-oxo-3-(trifluoromethyl)-4,5,6,7-tetrahydro-1H-indazol-1-yl)phenyl)amino)cyclohexyl D-alanine (LJS-5), (1R,4S)-4-((2-carbamoyl-5-( 6,6-dimethyl-4-oxo-3-(trifluoromethyl)-4,5,6,7-tetrahydro-1H-indazol-1-yl)phenyl)amino)cyclohexyl L-alanine (LJS-6), 3-((2-carbamoyl-5-(6,6-dimethyl-4-oxo-3-(trifluoromethyl)-4,5,6,7-tetrahydro-1H-indazol-1-yl)phenyl)amino)glycine propyl ester (LJS-13), 4-((2-carbamoyl-5-(6,6-dimethyl-4-oxo-3-(trifluoromethyl)-4,5,6,7-tetrahydro-1H-indazol-1-yl)phenyl)amino)cyclohexyl L-alanine (LJS-6), 3-((2-carbamoyl-5-(6,6-dimethyl-4-oxo-3-(trifluoromethyl)-4,5,6,7-tetrahydro-1H-indazol-1-yl)phenyl)amino)glycine propyl ester (LJS-13), (1R,4R)-4-((2-carbamoyl-5-(3-isobutyl-6,6-dimethyl-4-oxo-4,5,6,7-tetrahydro-1H-indazol-1-yl)phenyl)amino)glycine cyclohexyl ester (LJS-15).

In a second aspect, the present invention provides the use of any one of the above-mentioned aromatic carboxamide compounds or their pharmacologically or physiologically acceptable salts in the preparation of drugs against enterovirus EV71; or in the preparation of drugs for treating diseases caused by enterovirus EV71 infection, wherein the diseases caused by enterovirus EV71 infection include hand, foot and mouth disease.

In a third aspect, the present invention provides a drug for resisting enterovirus EV71, which comprises one or more of the above-mentioned aromatic carboxamide compounds or pharmacologically or physiologically acceptable salts thereof. The drug may also comprise a pharmaceutically acceptable carrier or excipient, which may be prepared according to existing conventional pharmaceutical techniques.

In a fourth aspect, the present invention provides a method for preparing the arylformamide compound represented by the above general formula (I). The arylformamide compound is prepared by condensing an arylformamide raw material containing an alcoholic hydroxyl group with a carboxylic acid derivative under the action of a condensation agent EDCI and a catalyst DMAP, and specifically comprises the following steps:

(1) A series of indazole derivatives having R ¹ substituents are synthesized by the following reaction (A) and used as the starting materials for the next step.

Under room temperature conditions, commercially available raw materials 2-acetyl-5,5-dimethyl-1,3-cyclohexanedione 1a-1c and N ₂ H ₄ ·H ₂ O solution can be reacted in anhydrous ethanol to obtain compounds 2a-2c.

(2) A series of aromatic carbonitrile derivatives having R ¹ and R ² substituents are synthesized by the following reaction (B) and used as the starting materials for the next step.

Commercially available raw material pyrazole derivatives 2a-2c and 2-bromo-4-fluorobenzonitrile derivatives 3a-3c prepared by formula (A) are reacted in dry DMF at 85°C through C-N coupling reaction to obtain compounds 4a-4e.

(3) A series of aromatic nitrile derivatives having ^R1 and ^R2 substituents and a Z side chain are synthesized by the following reaction (C) and used as the starting materials for the next step.

Compounds 4a-4e prepared by formula (B) and a series of commercially available amino alcohol derivatives can be subjected to coupling reaction catalyzed by Cs ₂ CO ₃ , Pd(OAc) ₂ and DPPF in anhydrous toluene in a sealed tube at 120° C. to obtain compounds 5a-5l.

(4) A series of aromatic carboxamide derivatives having ^R1 and ^R2 substituents and a Z side chain are synthesized by the following reaction (D) and used as the final product or the starting material for the next reaction.

Compounds 5a-51 prepared by formula (C) can be subjected to hydrolysis reaction mediated by NaOH and H ₂ O ₂ in a mixed solution of EtOH/DMSO to obtain compounds 6a-61.

(5) A series of aromatic carboxamide derivatives having R ¹ , R ² and R ³ substituents and a Z side chain are synthesized by the following reaction (E).

wherein R ¹ is -CH ₃ , -CF ₃ or -CH ₂ CH(CH ₃ ) ₂ ; R ² is -H or -CH ₃ ;

^R3 is

^R4 is

X is CH or N;

Y is

The specific steps can be:

At room temperature, compounds 6a-61 prepared by formula (D) were reacted with a series of commercially available carboxylic acid derivatives in dichloromethane via EDCI and DMAP-mediated condensation, followed by TFA removal of the protecting group (-BOC) to obtain compounds LJS-1 and LJS-3-LJS-17.

Advantages and beneficial effects of the present invention: The aromatic carboxamide compounds of the present invention have good anti-enterovirus EV71 activity, and the inhibitory activity of some compounds reaches the nanomolar level. The aromatic carboxamide compounds of the present invention can be developed as new anti-enterovirus 71 drugs and have broad application prospects.

DETAILED DESCRIPTION

The present invention is further described in detail below by way of examples. It should be understood that the examples provided are merely illustrations of the method of the present invention and are not intended to limit the remainder of the present invention in any way.

The preparation of the aromatic formamide compound represented by the general formula (I) of the present invention specifically comprises the following steps:

1. A series of indazole derivatives with R ¹ substituents are synthesized by the following reaction (A) and used as the raw materials for the next step.

Taking the synthetic operation steps of compound 3,6,6-trimethyl-1,5,6,7-tetrahydro-4H-indazol-4-one 2a as an example, the specific operation steps may be:

First, take a 50mL single-mouth round-bottom flask and add anhydrous ethanol (6mL), then dissolve the raw material 2 _- acetyl-5,5-dimethyl-1,3-cyclohexanedione 1a (8.78mmol, 1600mg) in anhydrous ethanol, slowly add _N2H4 · _H2O (3mL) dropwise at room temperature, and after the addition is complete, stir the reaction system vigorously. Monitor the reaction by TLC. After the raw material disappears completely, cool the reaction system to 5°C in an ice bath, add saturated sodium chloride solution dropwise, and a light yellow solid precipitates. Filter, rinse the filter cake three times with water (3×30mL), and dry the filter cake in a vacuum drying oven at 50°C to obtain 1236mg of the corresponding product 2a.

The preparation methods of the remaining compounds 2b-2c are the same as above, and all raw materials 1a-1c can be obtained through commercial channels.

2. A series of aromatic carbonitrile derivatives with R ¹ and R ² substituents are synthesized by the following reaction (B) and used as the raw materials for the next step.

Taking the synthesis of compound 2-bromo-4-(3,6,6-trimethyl-4-oxo-4,5,6,7-tetrahydro-1H-indazol-1-yl)benzonitrile 4a as an example, the specific operation steps may be:

Take a 50mL round-bottom flask, add anhydrous DMSO and raw material 2a (7.01mmol, 1250mg), stir to completely dissolve, then add commercially available raw materials 2-bromo-4-fluorobenzonitrile 3a (7.71mmol, 1543mg) and K ₂ CO ₃ (21.04mmol, 2908mg) to the above reaction solution in sequence, after addition, heat the reaction system to 80°C and react for 2h. Monitor the reaction progress by TLC. After the intermediate 2a completely disappears, cool the reaction system to room temperature and add ice water dropwise to quench the reaction, extract the reaction system with dichloromethane (DCM) (3×30mL), wash the organic phase with saturated sodium chloride, and add anhydrous sodium sulfate to dry. Concentrate the dried organic phase, and purify the concentrated system by column chromatography, with petroleum ether/ethyl acetate (V/V=3/1) as the mobile phase, to obtain 2060mg of a light yellow solid product 4a.

The preparation methods of the remaining compounds 4b-4e are the same as above.

3. A series of aromatic carbonitrile derivatives with ^R1 and ^R2 substituents and Z side chain are synthesized by the following reaction (C) and used as the raw materials for the next step.

Taking the synthesis of compound 2-(((1R, 4R)-4-hydroxycyclohexyl)amino)-4-(3,6,6-trimethyl-4-oxo-4,5,6,7-tetrahydro-1H-indazol-1-yl)benzonitrile 5a as an example, the specific operation steps may be:

Take a 50mL sealed tube, add raw material 4a (2.23mmol, 800mg), trans-4-aminocyclohexanol (6.70mmol, 772mg), _Cs2CO3 ( _6.70mmol , 2183mg), Pd(OAc) ₂ (5mol%) and DPPF (10mol%) into the sealed tube in sequence, introduce argon (repeated three times), add anhydrous toluene (Toluene) to the reaction system under stirring, and after the addition, place the reaction system in a microwave reactor and react at 120℃ for 3 hours. Monitor the reaction progress by TLC. After the raw material disappears completely, cool the reaction system to 50℃, filter it with diatomaceous earth while hot, concentrate the filtrate, and purify the concentrated system by column chromatography with petroleum ether/ethyl acetate (V/V=1/1) as the mobile phase to obtain 622mg of light yellow solid product 5a.

The preparation methods of the remaining compounds 5b-5l are the same as above.

4. A series of aromatic formamide derivatives with ^R1 and ^R2 substituents and Z side chain are synthesized by the following reaction (D), and used as the final product or the raw material for the next reaction.

Taking the synthesis of compound 2-(((1R, 4R)-4-hydroxycyclohexyl)amino)-4-(3,6,6-trimethyl-4-oxo-4,5,6,7-tetrahydro-1H-indazol-1-yl)benzamide 6b (LJS-2) as an example, the specific operation steps may be:

Take a 25mL single-necked flask, add 5mL of mixed solution EtOH/DMSO (V/V＝4:1), dissolve the raw material 5b (1.34mmol, 600mg) in the above solution, then slowly add 1mol/L NaOH solution (1mL), then add 30% H ₂ O ₂ solution (2mL) dropwise, and stir at room temperature for 2 hours. TLC monitors the reaction progress. After the raw material 5b disappears completely, add saturated NH ₄ Cl solution to quench the reaction. After stirring for 5 minutes, extract the reaction system with DCM, wash the organic phase with saturated sodium chloride solution, and finally add anhydrous sodium sulfate for drying. Remove DCM by rotary evaporation under vacuum to obtain a crude product, which is then purified by column chromatography with the mobile phase being DCM/MeOH (V/V＝30:1) to obtain 593mg of white solid product 6b (LJS-2).

The preparation methods of the remaining compounds 6a, 6c-6l are the same as above.

5. A series of aromatic formamide derivatives having R ¹ , R ² and R ³ substituents and a Z side chain are synthesized by the following reaction (E).

Wherein, R ¹ is -CH ₃ , -CF ₃ or -CH ₂ CH(CH ₃ ) ₂ ; R ² is -H or -CH ₃ ;

^R3 is

^R4 is

X is CH or N;

Y is

Taking the synthesis of compound (1R, 4R)-4-((2-carbamoyl-5-(6,6-dimethyl-4-oxo-3-(trifluoromethyl)-4,5,6,7-tetrahydro-1H-indazol-1-yl)phenyl)amino)glycine cyclohexyl ester LJS-3 as an example, the specific operation steps may be:

Take a 50mL single-mouth round-bottom flask, add raw material 6b (0.43mmol, 200mg), BOC-glycine (0.65mmol, 113mg), EDCI (0.86mmol, 165mg) and DMAP (0.65mmol, 79mg) in sequence, then add 6mL of anhydrous DCM, stir at room temperature overnight. TLC monitors the reaction progress. After the raw material 6b disappears completely, add water to quench the reaction, then extract with DCM, add saturated sodium chloride solution and anhydrous sodium sulfate in sequence for washing and drying, then concentrate the organic phase and add 4mL of DCM, add trifluoroacetic acid (TFA) (1mL) dropwise under ice bath conditions, and stir for 2 hours after addition. The reaction was monitored by TLC. After the reaction of the raw materials was completed, the solvent and part of TFA were removed by vacuum rotary evaporation, and then an appropriate amount of DCM was added for dilution, and the pH of the system was adjusted to alkaline with saturated sodium bicarbonate. The mixture was extracted with DCM, dried over anhydrous sodium sulfate, and dried to obtain a crude product. The crude product was purified by column chromatography with the mobile phase being DCM/MeOH (V/V=15:1) to obtain 162 mg of a white solid product LJS-3.

The preparation methods of the remaining compounds LJS-1, LJS-4-LJS-17 are the same as above.

[Example 1] Preparation of (1R, 4R)-4-((2-carbamoyl-5-(3,6,6-trimethyl-4-oxo-4,5,6,7-tetrahydro-1H-indazol-1-yl)phenyl)amino)glycine cyclohexyl ester (LJS-1).

Take a 50mL single-mouth round-bottom flask, add raw material 6a (0.44mmol, 180mg), BOC-glycine (0.66mmol, 115mg), EDCI (0.88mmol, 168mg) and DMAP (0.66mmol, 80mg) in sequence, then add anhydrous DCM, stir at room temperature, and leave overnight. Monitor the reaction progress by TLC. After the raw material 6a disappears completely, add water to quench the reaction, then extract with DCM, add saturated sodium chloride solution and anhydrous sodium sulfate in sequence for washing and drying, then concentrate the organic phase and add DCM, add trifluoroacetic acid (TFA) dropwise under ice bath, and stir for 2 hours after addition. The reaction was monitored by TLC. After the reaction of the raw materials was completed, the solvent and part of TFA (mL) were removed by vacuum rotary evaporation, and then an appropriate amount of DCM was added for dilution, and the pH of the system was adjusted to alkaline with saturated sodium bicarbonate. The mixture was extracted with DCM, dried over anhydrous sodium sulfate, and rotary dried to obtain a crude product, which was purified by column chromatography to obtain a white solid product LJS-1 with a yield of 75%.

¹ H NMR (400MHz, DMSO-d ₆ ) δ8.45(d,J＝7.5Hz,1H),7.95(s,1H),7.75(d,J＝8.5Hz,1H),7.28(s,1H) ,6.81(d,J＝2.2Hz,1H),6.70(dd,J＝8.4,2.0Hz,1H),4.72(dt,J＝10.1,5.7Hz,1H),3.47–3.42(m,1H), 3.24 (s,2H),2.95(s,2H),2.69(d,J＝7.0Hz,2H),2.31(s,2H),2.08–1.98(m,3H),1.96–1.89(m,2H), 1.53(dt,J＝12.4,9.3Hz,2H),1.40–1.30(m,2H),0.99(s,6H),0.89(d,J＝6.6Hz,6H).

[Example 2] Preparation of 4-(6,6-dimethyl-4-oxo-3-(trifluoromethyl)-4,5,6,7-tetrahydro-1H-indazol-1-yl)-2-(((1R,4R)-4-hydroxycyclohexyl)amino)benzamide (LJS-2).

Take a 25mL single-necked flask, add a mixed solution of EtOH/DMSO (V/V=4:1), dissolve the raw material 2-(((1R, 4R)-4-hydroxycyclohexyl)amino)-4-(3,6,6-trimethyl-4-oxo-4,5,6,7-tetrahydro-1H-indazol-1-yl)benzonitrile 5b (1.79mmol, 800mg) in the above solution, then slowly add a 1mol/L NaOH solution (3mL), then add a 30% mass fraction _{H2O2 solution} (6mL) dropwise, and stir at room temperature for 2 hours. TLC monitors the reaction progress. After the raw material disappears completely, add a saturated _NH4Cl solution to quench the reaction. After stirring for 5 minutes, extract the reaction system with DCM, wash the organic phase with a saturated sodium chloride solution, and finally add anhydrous sodium sulfate for drying. The DCM was removed by rotary evaporation under vacuum to obtain a crude product, which was then purified by column chromatography to obtain a white solid product LJS-2 with a yield of 91%.

¹ H NMR (400MHz, Chloroform-d) δ8.11(d,J＝7.6Hz,1H),7.50(d,J＝8.4Hz,1H),6.74(d,J＝2.1Hz,1H),6.61( dd,J＝8.3,2.0Hz,1H),5.74(s,2H),3.77–3.70(m,1H),3.35(s,1H),2.84(s,2H),2.49(s,2H),2.16 –2.04(m,4H),1.42(q,J＝13.0Hz,4H),1.13(s,6H).

[Example 3] Preparation of (1R, 4R)-4-((2-carbamoyl-5-(6,6-dimethyl-4-oxo-3-(trifluoromethyl)-4,5,6,7-tetrahydro-1H-indazol-1-yl)phenyl)amino)glycine cyclohexyl ester (LJS-3).

The preparation method is the same as that described in Example 1, except that the raw material 6a is replaced by the raw material 6b (LJS-2), and a white solid product LJS-3 is obtained with a yield of 69%.

¹ H NMR (400MHz, Chloroform-d) δ8.18 (d, J = 7.4Hz, 1H), 7.56 (d, J = 8.4Hz, 1H), 6.72 (s, 1H), 6.57 (d, J = 8.3 Hz,1H),6.13(s,2H),4.82(s,1H),3.39(s,3H),2.82(s,2H),2.44(s,2H),2.17–2.01(m,6H),1.55 –1.40(m,4H),1.10(s,6H).

[Example 4] Preparation of cyclohexyl (1R, 4R)-4-((2-carbamoyl-5-(6,6-dimethyl-4-oxo-3-(trifluoromethyl)-4,5,6,7-tetrahydro-1H-indazol-1-yl)phenyl)amino)3-aminopropanoate (LJS-4).

The preparation method is the same as that described in Example 1, except that the raw material 6a and BOC-glycine are replaced by raw material 6b and BOC-β-alanine, respectively, to obtain a white solid product LJS-4 with a yield of 73%.

¹ H NMR(400MHz,Chloroform-d)δ8.19(d,J＝7.5Hz,1H),7.54(d,J＝8.4Hz,1H),6.77(d,J＝2.1Hz,1H),6.61(dd,J＝8.4,2.1Hz,1H),6.04(s,2H),4.84(dq,J＝9.6,4.7,4 .2Hz,1H),3.40(dd,J＝13.2,6.2Hz,1H),3.04–2.98(m,1H),2.86(s,2H),2.57(s,2H),2.49(s,4H),2.18–2.03(m,4H),1.61–1.43(m,4H),1.36–1.29 (m,2H),1.14(s,6H).

[Example 5] Preparation of (1R, 4R)-4-((2-carbamoyl-5-(6,6-dimethyl-4-oxo-3-(trifluoromethyl)-4,5,6,7-tetrahydro-1H-indazol-1-yl)phenyl)amino)cyclohexyl D-alanine (LJS-5).

The preparation method is the same as that described in Example 1, except that the raw material 6a and BOC-glycine are replaced by raw material 6b and BOC-L-alanine, respectively, to obtain a white solid product LJS-5 with a yield of 81%.

¹ H NMR (400MHz, Chloroform-d) δ8.21 (d, J = 7.4Hz, 1H), 7.54 (d, J = 8.3Hz, 1H), 6.78 (d, J = 2.1Hz, 1H), 6.61 ( dd,J＝8.4,2.0Hz,1H),5.92(s,2H),4.85(dt,J＝9.5,5.2Hz,1H),3.55(q,J＝7.0Hz,1H),3.50–3.38(m ,1H),2.86(s,2H),2.50(s,2H),2.20–2.03(m,4H),1.63–1.47(m,4H),1.34(d,J＝7.0Hz,3H),1.15( s,6H).

[Example 6] Preparation of (1R, 4S)-4-((2-carbamoyl-5-(6,6-dimethyl-4-oxo-3-(trifluoromethyl)-4,5,6,7-tetrahydro-1H-indazol-1-yl)phenyl)amino)cyclohexyl L-alanine (LJS-6).

The preparation method is the same as that described in Example 1, except that the raw material 6a and BOC-glycine are replaced by raw material 6b and BOC-D-alanine, respectively, to obtain a white solid product LJS-6 with a yield of 65%.

¹ H NMR (400MHz, Chloroform-d) δ8.21(d,J＝7.5Hz,1H),7.54(d,J＝8.4Hz,1H),6.77(d,J＝2.1Hz,1H),6.61( dd,J＝8.4,2.0Hz,1H),5.93(s,2H),4.84(tt,J＝9.4,4.1Hz,1H),3.55(q,J＝7.0Hz,1H),3.48–3.39(m ,1H),2.86(s,2H),2.50(s,2H),2.18–2.03(m,4H),1.64–1.45(m,4H),1.34(d,J＝7.0Hz,3H),1.15( s,6H).

[Example 7] Preparation of (1R, 4S)-4-((2-carbamoyl-5-(6,6-dimethyl-4-oxo-3-(trifluoromethyl)-4,5,6,7-tetrahydro-1H-indazol-1-yl)phenyl)amino)cyclohexyl (S)-2-amino-3,3-dimethylbutanoate (LJS-7).

The preparation method is the same as that described in Example 1, except that the raw material 6a and BOC-glycine are replaced by raw material 6b and BOC-L-tert-leucine, respectively, to obtain a white solid product LJS-7 with a yield of 57%.

¹ H NMR (400MHz, Chloroform-d) δ8.18 (d, J = 7.4Hz, 1H), 7.54 (d, J = 8.4Hz, 1H), 6.74 (s, 1H), 6.58 (d, J = 8.3 Hz,1H),5.99(s,2H),4.92–4.78(m,1H),3.40(d,J＝9.1Hz,1H),3.16–3.04(m,2H),2.83(s,2H),2.46 (s,2H),2.17–2.01(m,4H),1.63–1.45(m,4H),1.35(t,J＝7.4Hz,2H),1.11(s,6H),0.96(s,9H).

[Example 8] Preparation of (1S, 4S)-4-((2-carbamoyl-5-(6,6-dimethyl-4-oxo-3-(trifluoromethyl)-4,5,6,7-tetrahydro-1H-indazol-1-yl)phenyl)amino)glycine cyclohexyl ester (LJS-8).

The preparation method is the same as that described in Example 1, except that the raw material 6a is replaced by the raw material 6c to obtain a white solid product LJS-8 with a yield of 82%.

¹ H NMR (400MHz, DMSO-d ₆ ) δ8.62 (d, J = 7.8 Hz, 1H), 8.04 (s, 1H), 7.81 (d, J = 8.5 Hz, 1H), 7.38 (s, 1H) ,6.89(d,J＝2.1Hz,1H),6.72(dd,J＝8.4,2.0Hz,1H),4.85(t,J＝4.7Hz,1H),3.56(dq,J＝7.9,3.9Hz, 1H),2.97(s,2H),2.43(s,2H),1.85–1.51(m,9H),1.02(s,6H).

[Example 9] Preparation of 2-(((1R, 4R)-4-(2-aminoacetylamino)cyclohexyl)amino)-4-(6,6-dimethyl-4-oxo-3-(trifluoromethyl)-4,5,6,7-tetrahydro-1H-indazol-1-yl)benzamide (LJS-9).

The preparation method is the same as that described in Example 1, except that the raw material 6a is replaced by the raw material 6d to obtain a white solid product LJS-9 with a yield of 66%.

¹ H NMR (400 MHz, DMSO-d ₆ )δ8.39(d,J＝7.5Hz,1H),8.01(s,1H),7.79(d,J＝8.4Hz,1H),7.69(d,J＝7.9Hz,1H),7.37(s,1H),6.89(d,J＝2.1Hz,1H),6.72(dd,J＝8.4,2.0Hz,1H),3.59 (dp,J＝11.2,3.7Hz,1H),3.44–3.27(m,3H),3.05(s,2H),2.98(s,2H),2.44(s,2H),2.07–2.01(m,2H),1.82(dd,J＝13.0,4.0Hz,2H),1.42–1.22(m,4 H),1.03(s,6H).

[Example 10] Preparation of 2-((4-(2-aminoacetylamino)phenyl)amino)-4-(6,6-dimethyl-4-oxo-3-(trifluoromethyl)-4,5,6,7-tetrahydro-1H-indazol-1-yl)benzamide (LJS-10).

The preparation method is the same as that described in Example 1, except that the raw material 6a is replaced by the raw material 6e to obtain a white solid product LJS-10 with a yield of 75%.

¹ H NMR (400MHz, DMSO-d ₆ ) δ10.22 (s, 1H), 7.91 (d, J = 8.5Hz, 1H), 7.67 (d, J = 8.8Hz, 2H), 7.25 (d, J = 8.8Hz,2H),7.20(dd,J＝3.9,2.1Hz,1H),7.00(d,J＝8.5Hz,1H),3.26(s,2H),2.95(s,2H),2.42(s, 2H),1.04(s,6H).

[Example 11] Preparation of (1R, 3R)-3-((2-carbamoyl-5-(6,6-dimethyl-4-oxo-3-(trifluoromethyl)-4,5,6,7-tetrahydro-1H-indazol-1-yl)phenyl)amino)glycine cyclobutyl ester (LJS-11).

The preparation method is the same as that described in Example 1, except that the raw material 6a is replaced by raw material 6f to obtain a white solid product LJS-11 with a yield of 77%.

¹ H NMR (400MHz, Methanol-d ₄ ) δ7.77 (d, J = 8.4Hz, 1H), 6.80 (dd, J = 8.4, 2.1Hz, 1H), 6.64 (d, J = 2.1Hz, 1H) ,4.57–4.41(m,1H),4.05(dt,J＝7.6,3.7Hz,1H),3.37(s,1H),2.98(s,2H),2.49(s,2H),2.39(tt,J ＝7.5,5.3Hz,2H),2.28(ddd,J＝12.6,6.9,3.8Hz,2H),1.11(s,6H).

[Example 12] Preparation of 1-(2-carbamoyl-5-(6,6-dimethyl-4-oxo-3-(trifluoromethyl)-4,5,6,7-tetrahydro-1H-indazol-1-yl)phenyl)piperidin-4-ylglycine ester (LJS-12).

The preparation method is the same as that described in Example 1, except that the raw material 6a is replaced by the raw material 6g to obtain a white solid product LJS-12 with a yield of 80%.

¹ H NMR (400MHz, Methanol-d ₄ ) δ8.00 (dd, J＝12.7, 8.3Hz, 1H), 7.51–7.47 (m, 1H), 7.38 (dd, J＝8.4, 2.0Hz, 1H), 5.08(dp,J＝7.8,3.7Hz,1H),3.82(td,J＝8.4,7.9,3.8Hz,1H),3.59(d,J＝8.1Hz,1H),3.34 –3.27(m,2H),3.07(dq,J＝8.4,5.0,4.1Hz,1H),3.02(s,2H),2.94(ddd,J＝12.2,9.8,2.9Hz,1H),2.51(s ,2H),2.21–1.94(m,3H),1.78(td,J＝9.4,5.7Hz,1H),1.13(s,6H).

[Example 13] Preparation of 3-((2-carbamoyl-5-(6,6-dimethyl-4-oxo-3-(trifluoromethyl)-4,5,6,7-tetrahydro-1H-indazol-1-yl)phenyl)amino)glycine propyl ester (LJS-13).

The preparation method is the same as that described in Example 1 above, except that the raw material 6a is replaced by raw material 6h to obtain a white solid product LJS-13 with a yield of 51%.

¹ H NMR (400MHz, Methanol-d ₄ ) δ7.77 (d, J = 8.4 Hz, 1H), 6.92 ( d, J = 2.1 Hz, 1H), 6.77 ( dd, J = 8.4, 2.1 Hz, 1H) ,3.73(t,J＝6.7Hz,2H),3.42–3.33(m,4H),3.01(s,2H),2.51(s,2H),1.91(p,J＝6.6Hz,2H),1.13( s,6H).

[Example 14] Preparation of 4-((2-carbamoyl-5-(6,6-dimethyl-4-oxo-3-(trifluoromethyl)-4,5,6,7-tetrahydro-1H-indazol-1-yl)phenyl)amino)glycine butyl ester (LJS-14).

The preparation method is the same as that described in Example 1, except that the raw material 6a is replaced by the raw material 6i to obtain a white solid product LJS-14 with a yield of 65%.

¹ H NMR (400MHz, Methanol-d ₄ ) δ7.78 (d, J＝8.4Hz, 1H), 6.90 (s, 1H), 6.77 (d, J＝8.4Hz, 1H), 4.22 (t, J＝ 6.0Hz,2H),3.43(s,2H),3.26(d,J＝5.8Hz,2H),3.00(s,2H),2.50(s,2H),1.80(h,J＝8.2,7.6Hz, 4H),1.13(s,6H).

[Example 15] Preparation of (1R, 4R)-4-((2-carbamoyl-5-(3-isobutyl-6,6-dimethyl-4-oxo-4,5,6,7-tetrahydro-1H-indazol-1-yl)phenyl)amino)glycine cyclohexyl ester (LJS-15).

The preparation method is the same as that described in Example 1, except that the raw material 6a is replaced by raw material 6j to obtain a white solid product LJS-15 with a yield of 74%.

¹ H NMR (400MHz, DMSO-d ₆ ) δ8.45(d,J＝7.6Hz,1H),7.96(s,1H),7.75(d,J＝8.5Hz,1H),7.28(s,1H) ,6.80(d,J＝2.1Hz,1H),6.69(dd,J＝8.4,1.9Hz,1H),4.72(dt,J＝10.2,5.6Hz,1H),3.45(dtd,J＝10.8,7.6 , 7.2,4.1Hz,1H),3.25(s,2H),2.93(s,2H),2.39(s,3H),2.31(s,2H),2.08–2.00(m,2H),1.96–1.88(m ,2H),1.53(dt,J＝12.4,9.3Hz,2H),1.35(td,J＝12.8,6.3Hz,2H),1.00(s,6H).

[Example 16] Preparation of (1R, 4R)-4-((2-carbamoyl-5-(6,6-dimethyl-4-oxo-3-(trifluoromethyl)-4,5,6,7-tetrahydro-1H-indazol-1-yl)-4-methylphenyl)amino)glycine cyclohexyl ester (LJS-16).

The preparation method is the same as that described in Example 1 above, except that the raw material 6a is replaced by raw material 6k to obtain a white solid product LJS-16 with a yield of 69%.

¹ H NMR (400MHz, DMSO-d ₆ ) δ8.09 (d, J = 7.9 Hz, 1H), 8.00 (s, 1H), 7.69 (s, 1H), 7.39 (s, 1H), 6.83 (s, 1H),4.69(dt,J＝10.2,5.8Hz,1H),3.43–3.33(m,2H),3.22(s,2H),2.63(s,2H),2.43(s,2H),2.02–1.94 (m,2H),1.86(s,5H),1.48(dt,J＝12.5,9.3Hz,2H),1.31(td,J＝11.4,10.2,3.0Hz,2H),1.03(s,6H).

[Example 17] Preparation of (1R, 4R)-4-((2-carbamoyl-5-(6,6-dimethyl-4-oxo-3-(trifluoromethyl)-4,5,6,7-tetrahydro-1H-indazol-1-yl)pyridin-3-yl)amino)glycine cyclohexyl ester (LJS-17).

The preparation method is the same as that described in Example 1 above, except that the raw material 6a is replaced by raw material 6l to obtain a white solid product LJS-17 with a yield of 58%.

¹ H NMR (400MHz, Chloroform-d) δ8.67(d,J＝7.7Hz,1H),7.82(d,J＝2.0Hz,2H),7.15(d,J＝2.1Hz,1H),5.82( d,J＝4.4Hz,1H),4.84(dt,J＝9.7,5.0Hz,1H),3.40(s,3H),2.86(s,2H),2.47(s,2H),2.13–2.02(m ,4H),1.55(dd,J＝14.5,6.0Hz,4H),1.12(s,6H).

The chemical structures of the target compounds LJS-1-LJS-17 of the present invention synthesized above are shown in Table 1.

[Example 18] Biological activity test of the above arylbenzamide compounds

(1) Cytotoxicity assay of arylbenzamide compounds:

Yellow Thiazolyl Blue, referred to as MTT, can penetrate the cell membrane and enter the cell. The amber dehydrogenase in the mitochondria of living cells can reduce exogenous MTT to blue-purple needle-shaped Formazan crystals that are insoluble in water and deposited in the cells. The crystals can be dissolved by 20% (mass to volume) SDS. The light absorption value is measured at a wavelength of 595nm using an enzyme-linked immunosorbent assay, which can indirectly reflect the cell number.

During the experiment, RD cells were transferred to a 96-well plate at a density of 2×10 ⁴ per well. After culturing at 37°C for 24 hours, the culture medium was aspirated and cell culture medium containing various concentration gradient compounds was added to each well. After 24 hours, 5 mg/mL of MTT solution was added to each well, and the cell plate was cultured in a CO ₂ incubator at 37°C for 4 hours. Then, the formazan solubilization solution was added to the RD cells, incubated at 37°C for 3 hours, and the OD value at a wavelength of 595 nm was measured by an enzyme marker. The inhibition rate (%) of the compound = [1-(EN)/(PN)]×100, where "E" represents the OD value of the drug group, "P" represents the OD value of the non-drug group, and "N" represents the OD value of the blank group. The half-maximal inhibition concentration (CC ₅₀ ) of the compound is used as an indicator of the cytotoxicity of the compound.

(2) In vitro anti-enterovirus EV-71 activity of arylbenzamide compounds:

The antiviral activity of the compounds was evaluated by the viral plaque reduction assay. EV71 virus was inoculated at 70 PFU/well in a 6-well plate filled with RD cells. After 40 minutes, the virus-containing medium was removed and the medium containing the specific concentration of the drug to be tested was added. The medium contained a final concentration of 2 μg/mL TPCK-trypsin and 0.5% agarose. After 48-72 hours of incubation at 37°C and 5% _CO2 , the cells were fixed with 3% formalin, stained with 0.5% crystal violet, and the number of viral plaques was calculated. _EC50 refers to the concentration required for a specific drug to effectively inhibit the number of viral plaques to 50% of the control wells.

The present invention uses enviroxime as a positive control, evaluates the cytotoxicity and anti-enterovirus EV71 activity of 17 synthesized compounds, and calculates the selectivity index SI of the compounds. The results are shown in Table 2.

Table 2 Results of anti-EV71 activity and cytotoxicity of 17 target compounds (LJS-1-LJS-17) synthesized in the present invention

The above experimental results show that most of the synthesized aromatic carboxamide compounds have good anti-enterovirus EV71 activity, and the inhibitory activity of some compounds reaches the nanomolar level, such as compounds LJS-2 (EC ₅₀ =35nM), LJS-3 (EC ₅₀ =9nM), LJS-5 (EC ₅₀ =18nM), LJS-6 (EC ₅₀ =23nM), LJS-13 (EC ₅₀ =49.6nM), LJS-14 (99nM) and LJS-15 (10nM).

The above embodiments are only preferred embodiments of the present invention, and the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims (10)

1. An aromatic formamide compound, characterized in that the structure is as shown in the general formula (I): in, R ¹ is -CH ₃ , -CF ₃ or -CH ₂ CH(CH ₃ ) ₂ ; R ² is -H or -CH ₃ ; ^R3 is X is CH or N; Y is 2. The arylformamide compound according to claim 1, characterized in that it is selected from the following compounds: (1R,4R)-4-((2-carbamoyl-5-(3,6,6-trimethyl-4-oxo-4,5,6,7-tetrahydro-1H-indazol-1-yl)phenyl)amino)glycine cyclohexyl ester; 4-(6,6-dimethyl-4-oxo-3-(trifluoromethyl)-4,5,6,7-tetrahydro-1H-indazol-1-yl)-2-(((1R,4R)-4-hydroxycyclohexyl)amino)benzamide; (1R,4R)-4-((2-carbamoyl-5-(6,6-dimethyl-4-oxo-3-(trifluoromethyl)-4,5,6,7-tetrahydro-1H-indazol-1-yl)phenyl)amino)glycine cyclohexyl ester; (1R,4R)-cyclohexyl 4-((2-carbamoyl-5-(6,6-dimethyl-4-oxo-3-(trifluoromethyl)-4,5,6,7-tetrahydro-1H-indazol-1-yl)phenyl)amino)3-aminopropanoate; (1R,4R)-4-((2-carbamoyl-5-(6,6-dimethyl-4-oxo-3-(trifluoromethyl)-4,5,6,7-tetrahydro-1H-indazol-1-yl)phenyl)amino)cyclohexyl D-alanine; (1R,4S)-4-((2-carbamoyl-5-(6,6-dimethyl-4-oxo-3-(trifluoromethyl)-4,5,6,7-tetrahydro-1H-indazol-1-yl)phenyl)amino)cyclohexyl L-alanine; (1R,4S)-4-((2-carbamoyl-5-(6,6-dimethyl-4-oxo-3-(trifluoromethyl)-4,5,6,7-tetrahydro-1H-indazol-1-yl)phenyl)amino)cyclohexyl (S)-2-amino-3,3-dimethylbutanoate; (1S,4S)-4-((2-carbamoyl-5-(6,6-dimethyl-4-oxo-3-(trifluoromethyl)-4,5,6,7-tetrahydro-1H-indazol-1-yl)phenyl)amino)glycine cyclohexyl ester; 2-(((1R,4R)-4-(2-aminoacetylamino)cyclohexyl)amino)-4-(6,6-dimethyl-4-oxo-3-(trifluoromethyl)-4,5,6,7-tetrahydro-1H-indazol-1-yl)benzamide; 2-((4-(2-aminoacetylamino)phenyl)amino)-4-(6,6-dimethyl-4-oxo-3-(trifluoromethyl)-4,5,6,7-tetrahydro-1H-indazol-1-yl)benzamide; (1R,3R)-3-((2-carbamoyl-5-(6,6-dimethyl-4-oxo-3-(trifluoromethyl)-4,5,6,7-tetrahydro-1H-indazol-1-yl)phenyl)amino)glycine cyclobutyl ester; 1-(2-Carbamoyl-5-(6,6-dimethyl-4-oxo-3-(trifluoromethyl)-4,5,6,7-tetrahydro-1H-indazol-1-yl)phenyl)piperidin-4-ylglycine ester; 3-((2-carbamoyl-5-(6,6-dimethyl-4-oxo-3-(trifluoromethyl)-4,5,6,7-tetrahydro-1H-indazol-1-yl)phenyl)amino)glycine propyl ester; 4-((2-carbamoyl-5-(6,6-dimethyl-4-oxo-3-(trifluoromethyl)-4,5,6,7-tetrahydro-1H-indazol-1-yl)phenyl)amino)glycine butyl ester; (1R,4R)-4-((2-carbamoyl-5-(3-isobutyl-6,6-dimethyl-4-oxo-4,5,6,7-tetrahydro-1H-indazol-1-yl)phenyl)amino)glycine cyclohexyl ester; (1R,4R)-4-((2-carbamoyl-5-(6,6-dimethyl-4-oxo-3-(trifluoromethyl)-4,5,6,7-tetrahydro-1H-indazol-1-yl)-4-methylphenyl)amino)glycine cyclohexyl ester; (1R,4R)-4-((2-carbamoyl-5-(6,6-dimethyl-4-oxo-3-(trifluoromethyl)-4,5,6,7-tetrahydro-1H-indazol-1-yl)pyridin-3-yl)amino)glycine cyclohexyl ester. 3. The arylformamide compound according to claim 1, characterized in that it is selected from the following compounds: 4-(6,6-dimethyl-4-oxo-3-(trifluoromethyl)-4,5,6,7-tetrahydro-1H-indazol-1-yl)-2-(((1R,4R)-4-hydroxycyclohexyl)amino)benzamide; (1R,4R)-4-((2-carbamoyl-5-(6,6-dimethyl-4-oxo-3-(trifluoromethyl)-4,5,6,7-tetrahydro-1H-indazol-1-yl)phenyl)amino)glycine cyclohexyl ester; (1R,4R)-4-((2-carbamoyl-5-(6,6-dimethyl-4-oxo-3-(trifluoromethyl)-4,5,6,7-tetrahydro-1H-indazol-1-yl)phenyl)amino)cyclohexyl D-alanine; (1R,4S)-4-((2-carbamoyl-5-(6,6-dimethyl-4-oxo-3-(trifluoromethyl)-4,5,6,7-tetrahydro-1H-indazol-1-yl)phenyl)amino)cyclohexyl L-alanine; 3-((2-carbamoyl-5-(6,6-dimethyl-4-oxo-3-(trifluoromethyl)-4,5,6,7-tetrahydro-1H-indazol-1-yl)phenyl)amino)glycine propyl ester; 4-((2-carbamoyl-5-(6,6-dimethyl-4-oxo-3-(trifluoromethyl)-4,5,6,7-tetrahydro-1H-indazol-1-yl)phenyl)amino)glycine butyl ester; (1R,4R)-4-((2-Carbamoyl-5-(3-isobutyl-6,6-dimethyl-4-oxo-4,5,6,7-tetrahydro-1H-indazol-1-yl)phenyl)amino)glycine cyclohexyl ester. 4. A pharmacologically or physiologically acceptable salt of the arylcarboxamide compound according to any one of claims 1 to 3. 5. Use of the aromatic carboxamide compound or a pharmacologically or physiologically acceptable salt thereof according to any one of claims 1 to 3 in the preparation of a drug against enterovirus EV71. 6. Use of the aromatic carboxamide compound or a pharmacologically or physiologically acceptable salt thereof according to any one of claims 1 to 3 in the preparation of a medicament for treating diseases caused by enterovirus EV71 infection. 7. The use according to claim 6, characterized in that the disease caused by the enterovirus EV71 infection includes hand, foot and mouth disease. 8. A drug, characterized in that it comprises one or more of the aromatic carboxamide compounds or pharmacologically or physiologically acceptable salts thereof as described in any one of claims 1 to 3; the drug is an anti-enterovirus EV71 drug or a drug for treating diseases caused by enterovirus EV71 infection. 9 . The anti-enterovirus EV71 drug according to claim 8 , characterized in that it further comprises a pharmaceutically acceptable carrier or excipient. 10. The method for preparing an arylformamide compound according to any one of claims 1 to 3, characterized in that it comprises the following steps: a condensation reaction is carried out between an arylformamide raw material containing an alcoholic hydroxyl group and a carboxylic acid derivative under the action of a condensation agent and a catalyst to prepare the arylformamide compound; The structure of the aryl formamide containing alcoholic hydroxyl group is The structure of the carboxylic acid derivative is Wherein, R ¹ is -CH ₃ , -CF ₃ or -CH ₂ CH(CH ₃ ) ₂ ; R ² is -H or -CH ₃ ; ^R3 is ^R4 is X is CH or N; Y is