阅读说明：本技术 一种4-(苯并噻唑-2-基)-n-取代苯胺化合物及其制备方法和应用 (4- (benzothiazole-2-yl) -N-substituted aniline compound and preparation method and application thereof ) 是由 左建平 汤杰 唐炜 杨帆 胡玟奇 童贤崑 周宇 林颖 何佩岚 张颖 朱峰华 于 于 2020-05-06 设计创作，主要内容包括：本发明提供了4-(苯并噻唑-2-基)-N-取代苯胺化合物及其制备方法,所述方法为：取代4-(苯并噻唑-2-基)苯胺与卤代物发生反应,生成4-(苯并噻唑-2-基)-N-取代苯胺化合物,或者与卤代酰氯反应,再经亲核取代、还原和亲核取代反应生成系列4-(苯并噻唑-2-基)-N-杂环烷基取代苯胺化合物。本发明还提供了所述4-(苯并噻唑-2-基)-N-取代苯胺化合物在抗中东呼吸综合征冠状病毒(MERS-CoV)和新冠病毒(SARS-CoV-2)中的应用,其在抑制冠状病毒新药发现领域具有应用前景。(The invention provides a 4- (benzothiazole-2-yl) -N-substituted aniline compound and a preparation method thereof, wherein the method comprises the following steps: substituted 4- (benzothiazole-2-group) aniline reacts with halide to generate 4- (benzothiazole-2-group) -N-substituted aniline compound, or reacts with halogenated acyl chloride to generate a series of 4- (benzothiazole-2-group) -N-heterocyclic alkyl substituted aniline compounds through nucleophilic substitution, reduction and nucleophilic substitution. The invention also provides application of the 4- (benzothiazole-2-yl) -N-substituted aniline compound in resisting middle east respiratory syndrome coronavirus (MERS-CoV) and new coronavirus (SARS-CoV-2), and the application has application prospects in the field of new drug discovery for inhibiting coronavirus.)

1. A4- (benzothiazol-2-yl) -N-substituted aniline compound is characterized by having a structure shown in a formula (I):

R₁is hydrogen, halogen, alkoxy;

R₂hydrogen, alkyl;

R₃is cycloalkyl, five-membered aromatic heterocycle, substituted benzyl, aromatic heterocyclic methylene,Wherein n is 1-3; x is O, CH₂N; when X is N, G is hydrogen, methyl,

2. The 4- (benzothiazol-2-yl) -N-substituted aniline compound of claim 1 wherein R is R₁Is hydrogen, halogen, C1-C10 alkoxy; r₂Is hydrogen, C1-C10 alkyl; r₃Is cyclopentyl, cyclohexyl, thiophene, pyrrole, furan,

3. The 4- (benzothiazol-2-yl) -N-substituted aniline compound of claim 1 wherein R is R₁Is hydrogen, halogen, methoxy; r₂Hydrogen, methyl, ethyl; r₃Is cyclopentyl, cyclohexyl, thiophene, pyrrole, furan,

4. The 4- (benzothiazol-2-yl) -N-substituted aniline compound of claim 1 wherein R is as defined in formula (I)₂When the compound is hydrogen, the structure of the 4- (benzothiazole-2-yl) -N-substituted aniline compound is shown as the formula (I-1):

wherein R is₁、R₃Is as defined in claim 1.

5. The 4- (benzothiazol-2-yl) -N-substituted aniline compound of claim 1 wherein R in formula (I)₃Is composed ofThe structure of the 4- (benzothiazole-2-yl) -N-substituted aniline compound is shown as the formula (I-2) andrepresented by the formula (I-4):

wherein R is₁、R₂N, X and G are as defined in claim 1.

6. The process for preparing a 4- (benzothiazol-2-yl) -N-substituted aniline compound according to claim 4, wherein the target product I-1 is obtained by nucleophilic substitution of a substituted 4- (benzothiazol-2-yl) aniline and a halide in a solvent under the action of a base, wherein the reaction process is represented by the following reaction formula (a):

wherein the content of the first and second substances,

R₁is hydrogen, halogen, alkoxy;

R₃is cycloalkyl, five-membered aromatic heterocycle, substituted benzyl, aromatic heterocyclic methylene;

y is halogen.

7. The method of claim 6, wherein the solvent is one or both of DMF, acetonitrile; and/or the alkali is one or more of sodium hydride, potassium carbonate, sodium tert-butoxide and potassium tert-butoxide; and/or the molar ratio of the base to the substituted 4- (benzothiazol-2-yl) aniline is 2:1 to 10: 1; and/or the molar ratio of the halogen compound to the substituted 4- (benzothiazol-2-yl) aniline is 1:1 to 2: 1; and/or the reaction temperature is between room temperature and 130 ℃.

8. The method of preparing a 4- (benzothiazol-2-yl) -N-substituted aniline compound according to claim 5, which comprises the steps of:

(1) reacting a substituted 4- (benzothiazol-2-yl) aniline with a haloacyl chloride in a solvent to form an intermediate chloride ii;

(2) in a solvent, under the action of alkali, the intermediate chloride ii and a nitrogen-containing heterocycle undergo nucleophilic substitution reaction to obtain an amide product I-2;

(3) in a solvent, carrying out reduction reaction on the amide product I-2 and lithium aluminum hydride to obtain a reduction product I-3;

(4) in a solvent, under the action of alkali, the reduction product I-3 and alkyl halide undergo nucleophilic substitution reaction to obtain a target product I-4, wherein the reaction process is shown as a reaction formula (b):

R₁is hydrogen, halogen, alkoxy;

R₂hydrogen, alkyl;

n is 1 to 3;

x is O, CH₂、N；

When X is N, G is hydrogen, methyl,

9. The method of claim 8, wherein in step (1), the solvent is acetone; and/or the halogenated acyl chloride is one or two of chloroacetyl chloride or chloropropionyl chloride; and/or the molar ratio of the halogenated acyl chloride to the substituted 4- (benzothiazole-2-yl) aniline is 1:1 to 1.05: 1; and/or the reaction temperature is between room temperature and 70 ℃.

10. The method of claim 8, wherein in step (2), the solvent is one or both of DMF and acetonitrile; and/or the alkali is one or more of potassium carbonate, sodium tert-butoxide and potassium tert-butoxide; and/or the molar ratio of the alkali to the chloride ii is 1.8:1 to 2.2: 1; and/or the molar use ratio of the nitrogen-containing heterocycle to the chloride ii is 1: 1-1.5: 1; and/or the reaction temperature is 60-130 ℃.

11. The method according to claim 8, wherein in the step (3), the solvent is one or more of anhydrous tetrahydrofuran and anhydrous 1, 4-dioxane; and/or the molar usage ratio of the lithium aluminum hydride to the amide product I-2 is 2: 1-4: 1; and/or the temperature of the reaction is 70 ℃.

12. The method of claim 8, wherein in step (4), the solvent is one or both of DMF and acetonitrile; and/or the alkali is one or more of potassium carbonate, sodium tert-butoxide, potassium tert-butoxide and sodium hydride; and/or the molar usage ratio of the alkali to the reduction product I-3 is 1.8: 1-2.2: 1; and/or the molar usage ratio of the alkyl halide to the reduction product I-3 is 1: 1-1.5: 1; and/or the reaction temperature is between room temperature and 130 ℃.

13. Use of a 4- (benzothiazol-2-yl) -N-substituted aniline compound as claimed in any one of claims 1 to 5 in the preparation of an antiviral medicament.

14. The use of claim 13, wherein the 4- (benzothiazol-2-yl) -N-substituted aniline compound is used to inhibit the growth, metastasis, proliferation and promote apoptosis of a virus.

15. The use of claim 13, wherein the virus is MERS-COV, SARS-COV-2, 2013 SARS-COV.

Technical Field

The invention belongs to the technical field of pharmaceutical chemistry, and relates to a 4- (benzothiazole-2-yl) -N-substituted aniline compound, and a preparation method and application thereof.

Background

Middle east respiratory syndrome coronavirus (MERS-CoV) is a novel zoonotic pathogen in the middle east region. Viral infectious diseases caused by such coronavirus infection are called Middle East Respiratory Syndrome (MERS).

In 9 months 2012, the novel coronavirus was found in a 60 year old sauter man with acute pneumonia and acute renal failure. By 31 months 5 and 2019, at least 27 countries around the world report that there are people infected with MERS-CoV and 2442 people infected with the virus, wherein 842 people die and the mortality rate is as high as 35%. MERS viruses are transmitted to humans by animals and subsequently from human to human. After infection with the virus, severe respiratory diseases can be caused, with symptoms including fever, cough and shortness of breath, and with a higher rate of acute renal failure or death.

At present, no specific drug or vaccine for preventing or treating MERS-CoV infection is approved, and many basic and clinical studies on anti-MERS-CoV drugs are in progress.

MERS virus is an enveloped single-stranded RNA virus belonging to the beta coronavirus C lineage. It is mediated into the cell primarily by the spike protein (S protein). The S protein includes an S1 subunit containing the Receptor Binding Domain (RBD), an S2 subunit containing the Fusion Peptide (FP), the long heptad repeat 1 domain (HR1) and the short heptad repeat 2 domain (HR 2). MER-CoV binds the viral particle to the cell receptor dipeptidyl peptidase-4 (DPP4) on the surface of the host cell via the RBD in the spike protein. Subsequently, S2 changes its conformation and inserts its FP into the plasma or endosomal membrane. HR2 binds to HR1 to form the six-helix bundle (6-HB) fusion core, allowing the virus to bind tightly to the cell membrane for fusion and thus entry into the host cell for autonomous replication. From the aspect of pathogenesis, recognition and combination of MERS virus S protein and cell membrane are particularly important for virus propagation, so that the virus S protein is an important target for development of anti-MERS-CoV medicaments.

Disclosure of Invention

The invention provides a 4- (benzothiazole-2-yl) -N-substituted aniline compound and a preparation method and application thereof.

The 4- (benzothiazol-2-yl) -N-substituted aniline compound has the following structural general formula (I):

R₁is hydrogen, halogen, alkoxy;

R₂hydrogen, alkyl;

R₃is cycloalkyl, five-membered aromatic heterocycle, substituted benzyl, aromatic heterocyclic methylene,n is 1 to 3; x is O, CH₂N; when X is N, G is hydrogen, methyl,

Preferably, the first and second electrodes are formed of a metal,

R₁is hydrogen, halogen, C1-C10 alkoxy;

R₂is hydrogen, C1-C10 alkyl;

R₃is cyclopentyl, cyclohexyl, thiophene, pyrrole, furan,

It is further preferred that the first and second liquid crystal compositions,

R₁is hydrogen, halogen, methoxy;

R₂hydrogen, methyl, ethyl;

R₃is cyclopentyl, cyclohexyl, thiophene, pyrrole, furan,

When R in formula (I)₂When hydrogen is used, the structure of the 4- (benzothiazol-2-yl) -N-substituted aniline compound is shown as a formula (I-1):

wherein R is₁、R₃Is as defined in formula (I).

When R in formula (I)₃Is composed ofWhen the compound is used, the structure of the 4- (benzothiazole-2-yl) -N-substituted aniline compound is shown as the following formula (I-2) and (I-4):

wherein R is₁、R₂N, X and G are as defined in formula (I).

The invention also provides a preparation method of the 4- (benzothiazole-2-yl) -N-substituted aniline compound shown in the formula (I-1), in a solvent, under the action of alkali, the substituted 4- (benzothiazole-2-yl) aniline and a halide are subjected to nucleophilic substitution to generate a target product I-1, and the reaction process is shown in the reaction formula (a):

wherein the content of the first and second substances,

R₁is hydrogen, halogen, alkoxy;

R₃is cycloalkyl, five-membered aromatic heterocycle, substituted benzyl, aromatic heterocyclic methylene; wherein, the cycloalkyl is cyclopentyl and cyclohexyl; the five-membered aromatic heterocycle is thiophene, pyrrole and furan; said substituted benzyl isSaid heteroaromatic methylene group is

Y is halogen.

Wherein the solvent is one or more of DMF, acetonitrile and the like; preferably, it is DMF.

Wherein the alkali is one or more of sodium hydride, potassium carbonate or sodium tert-butoxide, potassium tert-butoxide and the like; preferably, it is sodium hydride, potassium carbonate or sodium tert-butoxide.

Wherein the molar ratio of the alkali to the substituted 4- (benzothiazol-2-yl) aniline is 2:1 to 10: 1; preferably, it is 5: 1.

wherein the molar ratio of the halogenated compound to the substituted 4- (benzothiazol-2-yl) aniline is 1:1 to 2: 1; preferably, 1.2: 1.

wherein the reaction temperature is between room temperature and 130 ℃; preferably, it is 80 ℃.

Wherein the reaction time is 5-16 hours; preferably, it is 14 hours.

The invention also provides a preparation method of the 4- (benzothiazole-2-yl) -N-substituted aniline compound shown in the formula (I-4), which specifically comprises the following steps:

(1) in a solvent, a substituted 4- (benzothiazol-2-yl) aniline is reacted with a haloacyl chloride to form an intermediate chloride, ii.

(2) In a solvent, under the action of alkali, the intermediate chloride ii and the nitrogen-containing heterocycle undergo nucleophilic substitution reaction to obtain an amide product I-2.

(3) In a solvent, the amide product I-2 and lithium aluminum hydride are subjected to reduction reaction to obtain a reduction product I-3.

(4) In a solvent, under the action of alkali, the reduction product I-3 and alkyl halide undergo nucleophilic substitution reaction to obtain a target product I-4, wherein the reaction process is shown as a reaction formula (b):

R₁is hydrogen, halogen, alkoxy;

R₂hydrogen, alkyl;

n is 1 to 3;

x is O, CH₂、N；

When X is N, G is hydrogen, methyl,

In step (1), the solvent is preferably acetone.

In the step (1), the halogenated acyl chloride is one or more of chloroacetyl chloride or chloropropionyl chloride; preferably, chloroacetyl chloride or chloropropionyl chloride.

In the step (1), the molar ratio of the acyl chloride to the 4- (benzothiazol-2-yl) aniline is 1: 1-1.05: 1; preferably, 1: 1.

in the step (1), the reaction temperature is room temperature-70 ℃; preferably 70 deg.c.

In the step (1), the reaction time is 0.2-2 hours; preferably, it is 1 hour.

In the step (2), the solvent is one or more of DMF, acetonitrile and the like; preferably, it is DMF.

In the step (2), the alkali is one or more of potassium carbonate, potassium tert-butoxide, sodium tert-butoxide and the like; preferably, it is potassium carbonate.

In the step (2), the molar ratio of the alkali to the chloride ii is 1.8: 1-2.2: 1; preferably, it is 2: 1.

in the step (2), the molar ratio of the nitrogen-containing heterocycle to the chloride ii is 1: 1-1.5: 1; preferably, 1.1: 1.

in the step (2), the reaction temperature is 60-130 ℃; preferably, it is 80 ℃.

In the step (2), the reaction time is 3-6 hours; preferably, it is 4 hours.

In the step (3), the solvent is one or more of anhydrous tetrahydrofuran, anhydrous 1, 4-dioxane and the like; preferably, it is anhydrous tetrahydrofuran.

In the step (3), the molar ratio of the lithium aluminum hydride to the amide product I-2 is 2: 1-4: 1; preferably, is 4: 1.

in the step (3), the temperature of the reaction was 70 ℃.

In the step (3), the reaction time is 10-16 hours; preferably, it is 14 hours.

In the step (4), the solvent is one or more of DMF, acetonitrile and the like; preferably, it is DMF.

In the step (4), the alkali is one or more of potassium carbonate, potassium tert-butoxide, sodium hydride and the like; preferably, it is potassium carbonate.

In the step (4), the molar ratio of the alkali to the reduction product I-3 is 1.8: 1-2.2: 1; preferably, it is 2: 1.

in the step (4), the molar ratio of the alkyl halide to the reduction product I-3 is 1: 1-1.5: 1; preferably, 1.1: 1.

in the step (4), the reaction temperature is between room temperature and 130 ℃; preferably 130 deg.c.

In the step (4), the reaction time is 6-18 hours; preferably, it is 14 hours.

In one embodiment, the process for preparing the 4- (benzothiazol-2-yl) -N-substituted aniline compound comprises the steps of:

(1) substituted 4- (benzothiazole-2-yl) aniline is used as a raw material, sodium hydride, potassium carbonate or sodium tert-butoxide is used as alkali, and nucleophilic substitution is carried out on the substituted 4- (benzothiazole-2-yl) aniline and alkyl halide in DMF to generate a product I-1' (wherein R is₂Is hydrogen; r₃Is cycloalkyl, five-membered aromatic heterocycle, substituted benzyl, aromatic heterocyclic methylene).

(2) Substituted 4- (benzothiazole-2-yl) aniline is used as a raw material and chloroacetyl chloride or chloropropionyl chloride are used for forming a chloro compound intermediate ii in acetone.

(3) Taking potassium carbonate as base in DMF as intermediate chloride ii, and carrying out nucleophilic substitution with nitrogen-containing heterocycle to obtain amide product I-2' (wherein R is₂Is hydrogen; r₃Is composed of )。

(4) The amide product I-2 'is reduced with lithium aluminium hydride in anhydrous tetrahydrofuran to give the reduced product I-3' (wherein R is₂Is hydrogen; r₃Is composed of )。

(5) The reduction product I-3' takes potassium carbonate as alkali in DMF and carries out nucleophilic substitution with alkyl halide to obtain an N-alkylation product I-4' (wherein R is₂Is methyl or ethyl; r₃Is composed of )。

The reaction route of the preparation method of the invention is shown as the following formula (c):

the invention also provides application of the benzothiazole group-containing N-substituted aniline compound shown in the formula (I), the formula (I-1) and the formula (I-4) in preparation of antiviral drugs.

The 4- (benzothiazole-2-yl) -N-substituted aniline compound shown in the formula (I), the formula (I-1) and the formula (I-4) is used for inhibiting the growth, transfer and proliferation of viruses; promoting the apoptosis of the virus.

The virus is MERS-CoV, SARS-CoV-2, SARS-CoV, etc.

In the invention, the 4- (benzothiazole-2-yl) -N-substituted aniline compound takes virus S protein as a target spot to inhibit viruses from entering host cells, thereby achieving the treatment effect.

The 4- (benzothiazole-2-yl) -N-substituted aniline compound has the beneficial effects that the 4- (benzothiazole-2-yl) -N-substituted aniline compound prepared by the invention has a novel structure and an obvious antiviral effect, particularly has an obvious effect on resisting middle east respiratory syndrome coronavirus (MERS-CoV) and new coronavirus (SARS-CoV-2), has an application prospect in the field of new drug discovery for inhibiting coronavirus, and can be used for developing novel anti-coronavirus drugs.

Detailed Description

The present invention will be described in further detail with reference to the following specific examples. The procedures, conditions, implementation methods and the like for carrying out the present invention are general knowledge and well-known attempts in the art, except for those specifically mentioned below, and the present invention is not particularly limited thereto.

Examples1: compound Ia (R)₁＝H，R₂＝H，R₃Cyclohexyl) synthesis

4- (benzothiazol-2-yl) aniline (100mg, 0.44mmol) was dissolved in DMF (3mL), and sodium hydride (105mg, 4.4mmol) and bromocyclohexane (80mg, 0.49mmol) were added and reacted at 60 ℃ for 5 h. Cooling to room temperature, adding water, extracting with ethyl acetate, washing with saturated saline solution, separating, drying with anhydrous sodium sulfate, filtering, and removing solvent. The crude product is chromatographed on a silica gel column (eluent: petroleum ether/ethyl acetate: 30:1) to give 30mg of the product Ia as a white solid.¹HNMR(400MHz,CDCl₃)δ7.98(d,J＝8.0Hz,1H),7.89 (d,J＝8.8Hz,2H),7.84(d,J＝7.9Hz,1H),7.45–7.41(m,1H),7.32–7.28(m,1H), 6.62(d,J＝8.9Hz,2H),3.98(d,J＝6.5Hz,1H),3.37–3.34(m,1H),1.45–1.41(m, 2H),1.81–1.76(m,2H),1.72–1.66(m,1H),1.46–1.35(m,2H),1.29–1.16(m, 3H).¹³CNMR(101MHz,CDCl₃)δ167.75,153.34,148.72,133.46,128.14, 124.92,123.14,121.24,121.00,120.30,111.51,50.35,32.21,24.76,23.87.

Example 2: compound Ib (R)₁＝H，R₂＝H，R_3＝ ) Synthesis of (2)

Compound Ib was obtained as a yellow solid in the same manner as in example 1 except that the starting bromocyclohexane in example 1 was replaced with 2- (bromomethyl) -6-methoxyphenol.¹HNMR(400MHz,DMSO-d₆)δ8.79(s,1H), 8.00(d,J＝7.4Hz,1H),7.89(d,J＝8.1Hz,1H),7.78(d,J＝8.4Hz,2H),7.46– 7.43(m,1H),7.35–7.31(m,1H),6.88–6.84(m,2H),6.82–6.80(m,1H),6.73– 6.68(m,3H),4.30(d,J＝5.9Hz,2H),3.80(s,3H).¹³CNMR(101MHz,DMSO-d₆)δ 168.00,153.86,151.61,147.29,143.82,133.68,128.60,126.17,125.51,124.26, 121.82,121.70,120.04,120.02,118.62,111.93,110.39,55.78,40.78.

Example 3: compound Ic (R)₁＝H，R₂＝H，R₃＝) Synthesis of (2)

The experimental procedure of example 1 was followed except that 3-bromomethylthiophene was used instead of bromocyclohexane as the starting material in example 1 to obtain compound Ic as a white solid.¹HNMR(400MHz,CDCl₃)δ7.99(d,J＝8.1Hz,1H), 7.92(d,J＝8.5Hz,2H),7.84(d,J＝7.9Hz,1H),7.46–7.42(m,1H),7.36–7.28(m, 2H),7.21(s,1H),7.09(d,J＝4.8Hz,1H),6.70(d,J＝8.5Hz,2H),4.41(s,3H).¹³C NMR(101MHz,CDCl₃)δ168.60,154.34,150.26,139.54,134.57,129.14,126.99, 126.49,126.03,124.33,123.06,122.40,122.03,121.39,112.60,43.31.

Example 4: compound Id (R)₁＝H，R₂＝H，R₃＝) Synthesis of (2)

The compound Id was obtained as a green solid by substituting 3-bromothiophene for bromocyclohexane as the starting material in example 1, according to the experimental procedure of example 1.¹HNMR(400MHz,CDCl₃)δ8.01(d,J＝8.1Hz,1H),7.96 (d,J＝8.6Hz,2H),7.86(d,J＝7.9Hz,1H),7.47–7.44(m,1H),7.36–7.30(m,2H), 7.02–6.97(m,3H),6.89(s,1H),6.01(s,1H).¹³CNMR(101MHz,CDCl₃)δ167.14, 153.24,146.32,138.72,133.62,128.05,125.10,124.56,123.79,123.52,122.30, 121.54,120.41,113.59,108.62.

Example 5: compound Ie (R)₁＝H，R₂＝H，R₃＝) Synthesis of (2)

4- (benzothiazol-2-yl) aniline (1g, 4.42mmol) was dissolved in acetone (20mL), and a solution of chloroacetyl chloride (500mg, 4.42mmol) in acetone (10mL) was slowly added dropwise and the reaction was refluxed for 2 h. Cooling to room temperature, and filtering to obtain 1.5g of yellow solid chloride;

the chloride (300mg, 0.99mmol) was dissolved in DMF (4mL) and piperidine (94mg, 1.09mmol) and potassium carbonate (276mg, 1.98mmol) were added and reacted at 60 ℃ for 4 h. Adding water, extracting with dichloromethane, washing with saturated salt waterWashing, separating, drying with anhydrous sodium sulfate, filtering, and removing the solvent. The crude product was subjected to silica gel column chromatography (eluent: dichloromethane) to give 225mg of a white solid compound Ie.¹HNMR(400MHz, CDCl₃)δ9.49(s,1H),8.08–8.04(m,3H),7.89(d,J＝8.0Hz,1H),7.72(d,J＝8.7 Hz,2H),7.50–7.46(m,1H),7.38–7.35(m,1H),3.10(s,2H),2.59–2.53(m,4H), 1.69–1.65(m,4H),1.55–1.43(m,2H).¹³CNMR(101MHz,CDCl₃)δ169.31, 167.56,154.15,140.22,134.92,129.20,128.46,126.31,125.04,122.98,121.59, 119.37,62.83,54.97,26.33,23.60.

Example 6: compound If (R)₁＝H，R₂＝H，R₃＝) Synthesis of (2)

The starting piperidine in example 5 was replaced with piperazine, and a white solid compound If was obtained by the experimental method in example 5.¹HNMR(400MHz,CDCl₃)δ9.35(s,1H),8.08–8.04(m,3H),7.89(d, J＝8.0Hz,1H),7.72(d,J＝8.3Hz,2H),7.50–7.46(m,1H),7.39–7.35(m,1H), 3.15(s,2H),2.99–2.97(m,4H),2.65–2.57(m,4H).¹³CNMR(101MHz,CDCl₃)δ 168.66,167.47,154.15,140.04,134.92,129.36,128.48,126.33,125.06,123.01, 121.59,119.41,62.68,54.79,46.33.

Example 7: compound Ig (R)₁＝H，R₂＝H，R₃＝) Synthesis of (2)

The starting piperidine in example 5 was replaced with N-methylpiperazine, and a white solid compound Ig was obtained according to the experimental procedure of example 5.¹HNMR(400MHz,CDCl₃)δ9.31(s,1H),8.08–8.04(m, 3H),7.89(d,J＝7.9Hz,1H),7.72(d,J＝8.4Hz,2H),7.50–7.46(m,1H),7.39–7.35(m,1H),3.17(s,2H),2.71–2.65(m,4H),2.58–2.50(m,4H),2.34(s,3H).¹³C NMR(101MHz,CDCl₃)δ167.55,166.43,153.13,139.03,133.91,128.36,127.45, 125.29,124.04,121.98,120.55,118.38,60.88,54.21,52.43,44.95.

Example 8: compound Ih (R)₁＝H，R₂＝H，R₃＝) Synthesis of (2)

The raw material chloroacetyl chloride in example 5 was replaced with chloropropionyl chloride, and the raw material piperidine was replaced with 1- (2-hydroxyethyl) piperazine, to obtain a pale blue solid compound Ih according to the experimental procedure of example 5.¹HNMR(400MHz, CDCl₃)δ11.21(s,1H),8.06–8.02(m,3H),7.88(d,J＝7.9Hz,1H),7.67(d,J＝8.7 Hz,2H),7.50–7.44(m,1H),7.39–7.33(m,1H),3.67(t,J＝5.3Hz,2H),2.89– 2.40(m,15H).¹³CNMR(101MHz,CDCl₃)δ169.63,166.56,153.14,140.25,133.86, 127.82,127.43,125.25,123.96,121.93,120.53,118.47,58.23,56.83,52.50,52.10, 51.33,31.53.

Example 9: compound Ii (R)₁＝H，R₂＝H，R₃＝) Synthesis of (2)

Compound Ie (200mg, 0.57mmol) was dissolved in anhydrous tetrahydrofuran (10mL), cooled in an ice-water bath, and lithium aluminum hydride (75mg, 2mmol) was added and refluxed for 16h under nitrogen. Cooling to room temperature, adding saturated sodium bicarbonate solution, extracting with dichloromethane, washing with saturated saline solution, separating, drying with anhydrous sodium sulfate, filtering, and removing solvent. The crude product is chromatographed on a silica gel column (eluent: dichloromethane/methanol 100:1) to give 180mg of compound Ii as a pale yellow solid.¹HNMR(400MHz,CDCl₃)δ7.98(d,J＝8.1Hz, 1H),7.91(d,J＝8.6Hz,2H),7.84(d,J＝7.9Hz,1H),7.45–7.41(m,1H),7.32– 7.28(m,1H),6.67(d,J＝8.6Hz,2H),4.93(m,1H),3.26–3.21(m,2H),2.63(t,J＝ 6.0Hz,2H),2.47–2.41(m,4H),1.64–1.59(m,4H),1.49–1.45(m,2H).¹³CNMR (101MHz,CDCl₃)δ167.79,153.30,149.81,133.45,128.04,124.94,123.17,121.25, 121.23,120.32,111.40,55.91,53.21,38.65,24.78,23.26.

Example 10: compound Ij (R)₁＝H，R₂＝H，R₃＝) Synthesis of (2)

A yellow solid compound Ij was obtained by substituting morpholine for the piperidine starting material in example 5 and the experimental method in example 9.¹HNMR(400MHz,CDCl₃)δ7.98(d,J＝8.1Hz,1H),7.92 (d,J＝8.7Hz,2H),7.84(d,J＝7.7Hz,1H),7.46–7.41(m,1H),7.33–7.29(m,1H), 6.67(d,J＝8.7Hz,2H),4.76(t,J＝4.8Hz,1H),3.74(t,J＝4.6Hz,4H),3.27–3.21 (m,2H),2.66(t,J＝6.2Hz,2H),2.49(t,J＝4.6Hz,4H).¹³CNMR(101MHz,CDCl₃) δ167.68,153.29,149.63,133.47,128.07,124.98,123.24,121.53,121.28,120.33, 111.43,65.93,55.75,52.24,38.29.

Example 11: compound Ik (R)₁＝H，R₂＝H，R₃＝) Synthesis of (2)

A yellow solid compound Ik was obtained by substituting piperazine as a raw material in example 5 and the experimental method in example 5 and example 9.¹HNMR(400MHz,CDCl₃)δ7.98(d,J＝8.3Hz,1H), 7.92(d,J＝8.2Hz,2H),7.84(d,J＝8.0Hz,1H),7.45–7.41(m,1H),7.32–7.28(m, 1H),6.67(d,J＝8.3Hz,2H),4.80–4.76(m,1H),3.25–3.21(m,2H),2.93–2.91 (m,4H),2.66–2.63(m,2H),2.49–2.45(m,4H).¹³CNMR(101MHz,CDCl₃)δ 167.64,153.29,149.50,133.49,128.09,124.99,123.27,121.66,121.31,120.34, 111.46,55.54,51.05,43.94,38.46.

Example 12: compound Il (R)₁＝H，R₂＝H，R₃＝) Synthesis of (2)

The starting piperidine in example 5 was replaced with N-methylpiperazine, and a pale yellow solid compound Il was obtained according to the experimental methods of example 5 and example 9.¹HNMR(400MHz,CDCl₃)δ7.97(d,J＝8.1 Hz,1H),7.91(d,J＝8.4Hz,2H),7.83(d,J＝7.9Hz,1H),7.45–7.41(m,1H),7.33 –7.31(m,1H),6.66(d,J＝8.5Hz,2H),4.78(t,J＝4.8Hz,1H),3.24–3.20(m,2H), 2.80–2.37(m,10H),2.35(s,3H).¹³CNMR(101MHz,CDCl₃)δ168.75,154.32, 150.69,134.49,129.10,126.03,124.28,122.46,122.30,121.39,112.45,56.14,54.87, 52.32,45.75,39.59.

Example 13: compound Im (R)₁＝7-F，R₂＝H，R₃＝) Synthesis of (2)

The starting 4- (benzothiazol-2-yl) aniline in example 5 was replaced with 4- (7-fluorobenzothiazol-2-yl) aniline, and a yellow solid compound Im was obtained by referring to the experimental methods of example 5 and example 9.¹HNMR(400 MHz,CDCl₃)δ7.92(d,J＝8.4Hz,2H),7.77(d,J＝8.1Hz,1H),7.41–7.36(m,1H), 7.05–7.01(m,1H),6.68(d,J＝8.4Hz,2H),4.82–4.80(t,J＝4.8Hz,1H),3.76– 3.73(m,4H),3.25(q,J＝5.5Hz,2H),2.69–2.66(m,2H),2.51–2.49(m,4H).¹³C NMR(101MHz,CDCl₃)δ168.65(d,J＝1.6Hz),156.23(d,J＝2.7Hz),156.06(d,J ＝249.3Hz),149.93,128.24,125.81(d,J＝7.4Hz),120.84,120.40(d,J＝16.6Hz), 117.05(d,J＝3.5Hz),111.42,108.76(d,J＝18.8Hz),65.92,55.69,52.23,38.21.

Example 14: compound In (R)₁＝6-F，R₂＝H，R₃＝) Synthesis of (2)

The starting 4- (benzothiazol-2-yl) aniline In example 5 was replaced with 4- (6-fluorobenzothiazol-2-yl) aniline according to the experimental procedures of example 5 and example 9 to obtain In as a brown solid compound.¹HNMR(400MHz, CDCl₃)δ7.91–7.86(m,3H),7.52(d,J＝8.2Hz,1H),7.18–7.14(m,1H),6.67(d,J ＝8.2Hz,2H),4.78–4.76(m,1H),3.76–3.72(m,4H),3.26–3.22(m,2H),2.67(d, J＝6.2Hz,2H),2.51–2.47(m,4H).¹³CNMR(101MHz,CDCl₃)δ167.42(d,J＝3.0 Hz),158.93(d,J＝244.2Hz),149.91,149.66,134.41(d,J＝11.1Hz),127.95,121.99 (d,J＝9.3Hz),121.24,113.35(d,J＝24.5Hz),111.44,106.64(d,J＝26.8Hz), 65.91,55.74,52.24,38.28.

Example 15: compound Io (R)₁＝5-F，R₂＝H，R₃＝) Synthesis of (2)

The starting 4- (benzothiazol-2-yl) aniline in example 5 was replaced with 4- (5-fluorobenzothiazol-2-yl) aniline according to the experimental procedures of example 5 and example 9 to obtain a white solid compound Io.¹HNMR(400MHz, CDCl₃)δ7.89(d,J＝8.6Hz,2H),7.74(dd,J＝8.7,5.2Hz,1H),7.65(dd,J＝9.7, 2.5Hz,1H),7.09–7.04(m,1H),6.66(d,J＝8.7Hz,2H),4.80(t,J＝4.8Hz,1H), 3.74–3.72(m,4H),3.23(q,J＝5.4Hz,2H),2.67–2.64(m,2H),2.49–2.47(m, 4H).¹³CNMR(101MHz,CDCl₃)δ170.15,160.82(d,J＝242.2Hz),154.23(d,J＝ 12.1Hz),149.84,128.78(d,J＝1.9Hz),128.09,121.16,120.85(d,J＝9.9Hz), 111.61(d,J＝24.9Hz),111.39,107.46(d,J＝23.7Hz),65.90,55.68,52.21,38.20.

Example 16: compound Ip (R)₁＝4-F，R₂＝H，R₃＝) Synthesis of (2)

The starting 4- (benzothiazol-2-yl) aniline in example 5 was replaced with 4- (7-fluorobenzothiazol-2-yl) aniline according to the experimental procedures of example 5 and example 9 to obtain compound Ip as a yellow solid.¹HNMR(400MHz, CDCl₃)δ7.95(d,J＝8.3Hz,2H),7.60(d,J＝8.0Hz,1H),7.25–7.22(m,1H), 7.16–7.11(m,1H),6.66(d,J＝8.3Hz,2H),4.81–4.77(m,1H),3.75–3.73(m, 4H),3.27–3.22(m,2H),2.67(d,J＝6.0Hz,2H),2.51–2.47(m,4H).¹³CNMR (101MHz,CDCl₃)δ168.22,154.40(d,J＝255.1Hz),149.87,142.06(d,J＝13.3Hz), 136.15(d,J＝4.0Hz),128.37,123.78(d,J＝7.0Hz),121.09,116.01(d,J＝4.3Hz), 111.36,110.75(d,J＝18.2Hz),65.93,55.74,52.25,38.26.

Example 17: compound Iq (R)₁＝6-OCH₃，R₂＝H，R₃＝) Synthesis of (2)

The starting material, 4- (benzothiazol-2-yl) aniline, in example 5 was replaced with 4- (6-methoxybenzothiazol-2-yl) aniline, and compound Iq was obtained as a yellow solid according to the experimental procedures of example 5 and example 9.¹HNMR (400MHz,DMSO-d₆)δ7.79(d,J＝8.9Hz,1H),7.74(d,J＝7.8Hz,2H),7.60(s, 1H),7.05(d,J＝8.9Hz,1H),6.69(d,J＝7.8Hz,2H),6.24(t,J＝5.9Hz,1H),3.82 (s,3H),3.61–3.58(m,4H),3.24–3.19(m,2H),2.54–2.52(m,2H),2.44–2.41(m, 4H).¹³CNMR(101MHz,DMSO-d₆)δ165.51,156.67,151.10,148.25,135.07, 128.25,122.23,120.34,115.05,111.83,104.87,66.14,56.92,55.63,53.37,39.65.

Example 18: compound Ir (R)₁＝H，R₂＝H，R₃＝) Synthesis of (2)

The starting material chloroacetyl chloride in example 5 was replaced with chloroacetyl chloride, and a yellow solid compound Ir was obtained according to the experimental methods of example 5 and example 9.¹HNMR(400MHz,CDCl₃)δ7.97(d,J＝8.1 Hz,1H),7.91(d,J＝8.7Hz,2H),7.84(d,J＝7.7Hz,1H),7.45–7.41(m,1H),7.32 –7.28(m,1H),6.63(d,J＝8.7Hz,2H),5.28(t,J＝4.8Hz,1H),3.76(t,J＝4.6Hz, 4H),3.30–3.26(m,2H),2.56–2.41(m,6H),1.86–1.79(m,2H).¹³CNMR(101 MHz,CDCl₃)δ167.75,153.29,149.96,133.44,128.09,124.95,123.18,121.23, 121.18,120.32,111.13,66.07,56.61,52.73,42.04,23.98.

Example 19: compound Is (R)₁＝H，R₂＝H，R₃＝) Synthesis of (2)

The starting material chloroacetyl chloride in example 5 was replaced with chloroacetyl chloride, and the starting material piperidine was replaced with 1- (2-hydroxyethyl) piperazine, to obtain a yellow solid compound Is according to the experimental procedures of examples 5 and 9.¹HNMR (400MHz,CDCl₃)δ7.97(d,J＝8.1Hz,1H),7.90(d,J＝8.7Hz,2H),7.84(d,J＝7.8 Hz,1H),7.45–7.41(m,1H),7.32–7.28(m,1H),6.63(d,J＝8.7Hz,2H),5.41– 5.37(m,1H),3.64(t,J＝5.4Hz,2H),3.28(t,J＝6.3Hz,2H),2.71–2.39(m,12H), 1.87–1.79(m,2H).¹³CNMR(101MHz,CDCl₃)δ167.78,153.33,150.05,133.46, 128.10,124.95,123.18,121.24,121.15,120.31,111.14,58.20,56.68,56.19,52.22, 51.97,42.18,24.23.

Example 20: compound It (R)₁＝H，R₂＝CH₃，R₃＝) Synthesis of (2)

Compound Ij (200mg, 0.59mmol) was dissolved in DMF (4mL), and potassium carbonate (160mg, 1.18mmol) and iodomethane (92mg, 0.65mmol) were added and stirred at room temperature for 6 h. Water was added, extraction was performed with ethyl acetate, the mixture was washed with saturated brine, liquid separation was performed, dried over anhydrous sodium sulfate, filtered, and the solvent was removed, and the obtained crude product was subjected to silica gel column chromatography (eluent: petroleum ether/ethyl acetate ═ 2:1) to obtain 180mg of product It as a white solid.¹HNMR (400MHz,CDCl₃)δ7.99–7.95(m,3H),7.83(d,J＝7.9Hz,1H),7.45–7.41(m, 1H),7.31–7.27(m,1H),6.72(d,J＝8.5Hz,2H),3.71(t,J＝4.6Hz,4H),3.53(t,J ＝7.3Hz,2H),3.02(s,3H),2.54(t,J＝7.3Hz,2H),2.49(t,J＝4.6Hz,4H).¹³CNMR (101MHz,CDCl₃)δ167.59,153.35,149.87,133.46,127.95,124.94,123.15,121.21, 120.31,120.27,110.41,65.92,54.19,53.01,48.81,37.66.

Example 21: screening for anti-MERS-CoV pseudovirus invasion Activity (pseudovirus-cell level model)

Materials: part of the compounds of the invention

1.1 test principle: with Huh7 cells as virus host cells (susceptible cells), the test sample blocks the MERS-CoV membrane protein-modified HIV pseudovirus-infected cells for activity, which reflects the antiviral activity of the sample to interfere with the MERS-CoV infection key target. The detection index is the activity level of a reporter gene on the pseudovirus genome.

1.3 test methods: huh7 cells were inoculated one day in advance in 96-well culture plates, respectively equipped with an active plate and a cytotoxic plate, placed at 37 ℃ with 5% CO₂And (5) culturing. And adding samples with different dilution concentrations and MERS-CoV pseudovirus suspensions into the activity determination plate and the cytotoxicity determination plate according to the same sample adding mode, and setting virus control, cell control and sample control. After further 3 days of culture, the cytotoxic plates were assayed for cell viability using the MTT method. And (3) sucking the culture solution by using an active plate, adding 100 mu L of cell lysate into each hole, performing shake lysis for 5 minutes, adding 100 mu L of Luciferase reaction detection solution into each hole, performing shake incubation for 5 minutes, and determining the chemiluminescence value.

1.4 evaluation method: cytotoxicity (MTT method): by comparing the OD values of the virus control, cell control and sample control, the viability of the cells was calculated and further the cytotoxic effect of the samples was calculated. Pseudoviral infection rate: and (3) taking the reading value of the cell control group as a background, subtracting the background from the chemiluminescence value of the virus control and the sample control, calculating the relative infection rate of the relative virus control hole, and further calculating the protective activity of the sample on virus-infected cells.

The invention provides a compound with a structural general formula (I) to MERS-CoV pseudovirus IC₅₀Test and Huh7 cell CC₅₀Test (see table 1).

Example 22: screening for inhibitory Activity against spike protein of New coronavirus (SARS-CoV-2) Virus (pseudovirus-cell level model)

2.1 materials: part of the compounds of the invention

2.2 testing principle: the Huh7 cell is taken as a virus host cell (susceptible cell), the activity of modifying HIV pseudovirus infected Huh7 cell by blocking spike protein (S protein) of SARS-CoV-2 virus by a test sample, and the inhibitory activity can reflect the activity of interfering SARS-CoV-2 virus infection key target spot by the sample. The detection index is the activity level of reporter gene firefly Luciferase.

2.3 test methods: the Huh7 cells were plated in 96-well culture plates one day in advance, and the activity and cytotoxicity plates were set separately and placed in a 37 ℃ incubator with 5% CO₂And (5) culturing. Adding samples with different dilution concentrations and SARS-CoV-2 virus S protein modified pseudovirus suspension into the activity determination plate and the cytotoxicity determination plate according to the same sample adding mode, and setting virus control, cell control and sample control. After further culturing for 3 days, the cytotoxicity assay plate was used to determine the cell viability by the MTT method. And adding 100 mu L of cell lysate into each hole after the cell culture solution is sucked by the activity determination plate, oscillating and cracking for 5 minutes, adding 100 mu L of Luciferase reaction detection solution into each hole, oscillating and incubating for 5 minutes, and determining the chemiluminescence value.

2.4 evaluation method: cytotoxicity (MTT method): by comparing the OD values of the virus control, cell control and sample control, the viability of the cells was calculated and further the cytotoxic effect of the samples was calculated. Infection rate of pseudovirus: and (3) taking the reading value of the cell control group as a background, deducting the background from the chemiluminescence values of the virus control and the sample control, and calculating the relative infection rate of the relative virus control hole so as to further calculate the protective activity of the sample on virus-infected cells.

The invention provides the 4- (benzothiazol-2-yl) -N-substituted aniline compounds Ii, Ij, Ik, Il and Ir for MERS-CoV pseudovirus IC₅₀Test and Huh7 cell CC₅₀Test (see table 2).

The MERS-CoV inhibitory activity of the series of compounds provided by the invention is shown in Table 1

TABLE 1

The compound of the invention has good inhibitory activity on MERS-CoV, wherein Ik and Il compounds have inhibitory activity IC on MERS-CoV₅₀Reaches 0.01 mu M and shows excellent MERS-CoV activity resistanceAnd (4) sex.

The data of the inhibitory activity of the series of compounds provided by the invention on the novel coronavirus (SARS-CoV-2) are shown in Table 2.

TABLE 2

Compound (I)	IC50:μM	CC50:μM	Compound (I)	IC50:μM	CC50:μM
						Ii	<0.41	11.7	Ij	<0.41	34.4
Ik	<0.41	4.5	Il	<0.41	5.2
						Ir	<0.41	>100

Some of the compounds of the present invention also showed excellent inhibitory activity against SARS-CoV-2.

The protection of the present invention is not limited to the above embodiments. Variations and advantages that may occur to those skilled in the art may be incorporated into the invention without departing from the spirit and scope of the inventive concept, and the scope of the appended claims is intended to be protected.