Preparation method and application of N,N-bis(2-(N'-methylindol-3-yl)-thio-acetyl)-2,6-dichloroaniline

Abstract

The present invention belongs to the field of medical technology, and specifically relates to a method for preparing N,N-bis(2-(N'-methylindol-3-yl)-thio-acetyl)-2,6-dichloroaniline and its application in preparing anti-Zika virus drugs. The N,N-bis(2-(N'-methylindol-3-yl)-thio-acetyl)-2,6-dichloroaniline of the present invention can prevent Zika virus from invading cells and thus block infection, and toxicity studies show that it has good drugability and has good application prospects in preparing anti-Zika virus drugs. N,N-bis(2-(N'-methylindol-3-yl)-thio-acetyl)-2,6-dichloroaniline has excellent anti-Zika virus activity, and the anti-Zika virus activity EC 50 It reaches 60nM, which is significantly better than the control drug ribavirin and is a new type of small molecule inhibitor of Zika virus.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and specifically relates to a preparation method of N,N-bis(2-(N'-methylindol-3-yl)-thio-acetyl)-2,6-dichloroaniline and application thereof in the preparation of anti-Zika virus drugs. Background Art

[0002] Zika virus (ZIKV) is a flavivirus transmitted by Aedes mosquitoes (mainly Aedes aegypti and Aedes albopictus). It was first discovered in the Zika Forest in Uganda in 1947, hence the name. In recent years, Zika virus has attracted widespread attention around the world, especially the large-scale outbreak in Brazil and the Americas from 2015 to 2016.

[0003] Most people infected with Zika virus will experience symptoms such as fever, rash, joint pain, conjunctivitis (red eyes), muscle pain and headache, which usually appear within 2 to 7 days after infection and last for about a week. However, pregnant women infected with Zika virus may cause a variety of abnormalities in the fetus, including microcephaly, brain calcification, placental problems, miscarriage and stillbirth. Microcephaly is a serious congenital defect characterized by abnormal development of the newborn's head and brain, with a head circumference significantly smaller than normal. This condition can cause a series of neurological problems such as intellectual disability, movement disorders and epilepsy. Zika virus infection is also associated with Guillain-Barré Syndrome (GBS), a neurological disease that can cause muscle weakness and even paralysis. Studies have shown that Zika virus infection may cause serious neurological complications such as acute myelitis, encephalitis and myelitis.

[0004] Currently, there is no effective vaccine or small molecule drug for the prevention and treatment of Zika virus infection. For patients infected with Zika virus, symptomatic treatment is mainly used, such as rest, fluid supplementation, analgesics and antipyretics to relieve symptoms. Therefore, the development of new Zika virus inhibitors has important application value. Summary of the invention

[0005] In view of this, the object of the present invention is to provide a method for preparing N,N-bis(2-(N'-methylindol-3-yl)-thio-acetyl)-2,6-dichloroaniline and its application in the preparation of anti-Zika virus drugs. The N,N-bis(2-(N'-methylindol-3-yl)-thio-acetyl)-2,6-dichloroaniline provided by the present invention has a significant inhibitory effect on Zika virus.

[0006] The present invention provides an application of N,N-bis(2-(N'-methylindol-3-yl)-thio-acetyl)-2,6-dichloroaniline in the preparation of an anti-Zika virus drug.

[0007] The present invention provides an anti-Zika virus drug, comprising a pharmaceutically acceptable carrier and an effective ingredient, wherein the effective ingredient is N,N-bis(2-(N'-methylindol-3-yl)-thio-acetyl)-2,6-dichloroaniline.

[0008] The present invention also provides a method for preparing N,N-bis(2-(N'-methylindol-3-yl)-thio-acetyl)-2,6-dichloroaniline, comprising the following steps:

[0009] Compound 1a and compound 1b are subjected to a first substitution reaction to obtain compound 1c;

[0010] Compound 1d is subjected to a second substitution reaction with a halogenating agent to obtain compound 1e;

[0011] Compound 1c and compound 1e are subjected to acylation reaction under alkaline conditions to obtain N,N-bis(2-(N'-methylindol-3-yl)-thio-acetyl)-2,6-dichloroaniline;

[0012] The compounds 1a, 1b, 1c, 1d and 1e respectively have the following structures:

[0013] 1a, 1b, 1c, 1d, 1e,

[0014] In compound 1b, X is Na or K, and in compound 1e, Y is Cl or Br.

[0015] Preferably, the molar ratio of compound 1a to compound 1b is 1:(1.4-1.6).

[0016] Preferably, the first substitution reaction is carried out under the catalysis of elemental iodine, and the molar ratio of the compound 1a to elemental iodine is 1:(0.05~1).

[0017] Preferably, the solvent for the first substitution reaction includes one or more of dimethyl sulfoxide, 1,4-dioxane and tetrahydrofuran.

[0018] Preferably, the temperature of the first substitution reaction is 25-100° C., and the time is 1.8-2.2 h.

[0019] Preferably, the second substitution reaction is a chlorination reaction or a bromination reaction, and the chlorination reagent used in the chlorination reaction includes one or more of thionyl chloride, oxalyl chloride, phosphorus oxychloride, phosphorus trichloride and phosphorus pentachloride; the bromination reagent used in the bromination reaction includes one or more of thionyl bromide, oxalyl bromide, phosphorus tribromide and phosphorus pentabromide.

[0020] Preferably, the molar ratio of the compound 1d to the halogenating agent is 1:(0.33-2).

[0021] Preferably, the alkaline reagent providing the alkaline condition includes one or more of sodium hydride, potassium hydride, calcium hydride, butyl lithium, lithium bistrimethylsilylamide and sodium bistrimethylsilylamide.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The present invention provides an application of N,N-bis(2-(N'-methylindol-3-yl)-thio-acetyl)-2,6-dichloroaniline in the preparation of anti-Zika virus drugs. The N,N-bis(2-(N'-methylindol-3-yl)-thio-acetyl)-2,6-dichloroaniline provided by the present invention can inhibit the invasion of Zika virus into host cells, has a significant inhibitory effect on the life cycle of Zika virus, and toxicity studies show that it has good drugability, indicating that N,N-bis(2-(N'-methylindol-3-yl)acetyl) substituted aniline compounds have good application prospects in the preparation of anti-Zika virus drugs.

[0024] The results of the examples show that the N,N-bis(2-(N'-methylindol-3-yl)-thio-acetyl)-2,6-dichloroaniline provided by the present invention has a strong inhibitory effect on Zika virus, and the anti-Zika virus activity EC 50 Reaching 60nM, it is better than the control drug ribavirin (EC 50 = 23 μM) and had no significant cytotoxicity (CC 50 >100 μM).

[0025] The present invention also provides a method for preparing N,N-bis(2-(N'-methylindol-3-yl)-thio-acetyl)-2,6-dichloroaniline. The method is simple to prepare and separate the target product, and the target product has a high yield and high purity. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0027] Figure 1 This is a graph showing the inhibition of Zika virus and cell viability by compound 1 in Application Example 1;

[0028] Figure 2 This is a graph showing the results of ribavirin's inhibition of Zika virus and cell viability in Application Example 1. DETAILED DESCRIPTION

[0029] The present invention provides application of N,N-bis(2-(N'-methylindol-3-yl)-thio-acetyl)-2,6-dichloroaniline in preparing anti-Zika virus drugs.

[0030] In the present invention, the structural formula of the N,N-bis(2-(N'-methylindol-3-yl)-thio-acetyl)-2,6-dichloroaniline is as shown in Formula I:

[0031] Formula I.

[0032] The N,N-bis(2-(N'-methylindol-3-yl)-thio-acetyl)-2,6-dichloroaniline provided by the present invention has a strong inhibitory effect on Zika virus, and the anti-Zika virus activity EC 50 The concentration of ribavirin was 60 nM, which was superior to the control drug ribavirin (EC50 = 23 μM) and had no obvious cytotoxicity (CC 50 >100 μM).

[0033] The present invention also provides an anti-Zika virus pharmaceutical composition, comprising a pharmaceutically acceptable carrier and an effective ingredient, wherein the effective ingredient is N,N-bis(2-(N'-methylindol-3-yl)-thio-acetyl)-2,6-dichloroaniline.

[0034] In the present invention, the pharmaceutically acceptable carrier includes liquids that can be used in animals, mammals, especially humans as specified in the Chinese Pharmacopoeia, such as water, vegetable oil, animal oil, salt solution, sugar solution or glycerol solution.

[0035] The present invention also provides a method for preparing N,N-bis(2-(N'-methylindol-3-yl)-thio-acetyl)-2,6-dichloroaniline, comprising the following steps:

[0036] Compound 1a and compound 1b are subjected to a first substitution reaction to obtain compound 1c;

[0037] Compound 1d is subjected to a second substitution reaction with a halogenating agent to obtain compound 1e;

[0038] Compound 1c and compound 1e are subjected to acylation reaction under alkaline conditions to obtain N,N-bis(2-(N'-methylindol-3-yl)-thio-acetyl)-2,6-dichloroaniline;

[0039] The compounds 1a, 1b, 1c, 1d and 1e respectively have the following structures:

[0040] 1a, 1b, 1c, 1d, 1e,

[0041] In compound 1b, X is Na or K, and in compound 1e, Y is Cl or Br.

[0042] In the present invention, unless otherwise specified, the materials and equipment used are commercially available products in the art.

[0043] In the present invention, compound 1a and compound 1b are subjected to a first substitution reaction to obtain compound 1c.

[0044] In the present invention, the molar ratio of compound 1a to compound 1b is preferably 1:(1.4-1.6), more preferably 1:1.5. The molar ratio of compound 1a to compound 1b of the present invention provides a high yield of compound 1c and is easy to purify, and excess 1b can be removed by washing with water.

[0045] In the present invention, the solvent for the first substitution reaction preferably includes one or more of dimethyl sulfoxide, 1,4-dioxane and tetrahydrofuran; the amount ratio of the compound 1a to the solvent is preferably 1 g: (27-33) mL, more preferably 1 g: 30 mL.

[0046] In the present invention, the first substitution reaction is preferably carried out under the catalysis of iodine, and the molar ratio of the compound 1a to iodine is preferably 1:(0.05-1), more preferably 1:(0.1-0.2).

[0047] In the present invention, the temperature of the first substitution reaction is preferably room temperature (25°C) to 100°C, more preferably 50 to 60°C, and the time is preferably 1.8 to 2.2 hours, more preferably 2 hours. Taking compound 1b as an example where X is Na, the reaction formula of the first substitution reaction is as follows:

[0048]

[0049] In the present invention, after the first substitution reaction, the following steps are preferably performed: cooling, extracting, washing, drying with a desiccant, concentrating and purifying the obtained reaction solution by column chromatography. The temperature after cooling is preferably room temperature; the extraction reagent is preferably ethyl acetate; the washing preferably includes washing with water, saturated sodium thiosulfate and saturated brine in sequence; the desiccant is preferably anhydrous sodium sulfate; the eluent for column chromatography purification is preferably a mixture of petroleum ether / ethyl acetate, and the volume ratio of petroleum ether to ethyl acetate in the mixture is preferably 10:1 to 3:1.

[0050] In the present invention, compound 1d is subjected to a second substitution reaction with a halogenating agent to obtain compound 1e.

[0051] In the present invention, the second substitution reaction is preferably a chlorination reaction or a bromination reaction, and the chlorination reagent (halogenation reagent) used in the chlorination reaction preferably includes one or more of thionyl chloride, oxalyl chloride, phosphorus oxychloride, phosphorus trichloride and phosphorus pentachloride; the bromination reagent (halogenation reagent) used in the bromination reaction includes one or more of thionyl bromide, oxalyl bromide, phosphorus tribromide and phosphorus pentabromide.

[0052] In the present invention, the molar ratio of the compound 1d to the halogenating agent is preferably 1:(0.33-2), more preferably 1:1.

[0053] In the present invention, the second substitution reaction is preferably carried out in an organic solvent or in the absence of a solvent, and the organic solvent preferably includes one or more of benzene, toluene and carbon tetrachloride. The second substitution reaction is preferably also carried out with a catalyst, and the catalyst is preferably N,N-dimethylformamide. The second substitution reaction is preferably carried out under reflux, and the temperature is preferably 0-120°C, more preferably 20-110°C, and more preferably 40°C. After the second substitution reaction, an acyl chloride compound 1e is obtained. Taking the chlorination reaction as an example, the reaction formula is as follows:

[0054]

[0055] In the present invention, the second substitution reaction preferably further comprises concentration, and the obtained compound 1e is directly used for the next reaction.

[0056] After obtaining compound 1c and compound 1e, the present invention performs acylation reaction on compound 1c and compound 1e under alkaline conditions to obtain N,N-bis(2-(N'-methylindol-3-yl)-thio-acetyl)-2,6-dichloroaniline.

[0057] In the present invention, the alkaline reagent providing the alkaline condition preferably includes one or more of sodium hydride, potassium hydride, calcium hydride, butyl lithium, lithium bis(trimethylsilylamide) and sodium bis(trimethylsilylamide). The alkaline reagent can be used as an acid binding agent to neutralize the generated hydrochloric acid and accelerate the reaction.

[0058] In the present invention, the solvent for the acylation reaction preferably includes one or more of tetrahydrofuran, 1,4-dioxane and N,N-dimethylformamide. The amount ratio of the compound 1c to the solvent is preferably 1mmol:2mL. In the present invention, the compound 1c and the solvent for the acylation reaction are preferably mixed first, cooled, and then an alkaline reagent providing alkaline conditions is added to mix, and then the compound 1e is added for acylation reaction; the temperature after cooling is preferably 0°C.

[0059] In the present invention, the temperature of the acylation reaction is preferably -40 to 40°C, more preferably 0°C to room temperature, and the time is preferably 1.8 to 2.2 hours, more preferably 2 hours. After the acylation reaction, it is preferred to further include: quenching reaction, extraction, drying with a desiccant, concentration and column chromatography purification. The quenching reaction is preferably carried out when the raw material compound 1a disappears in the thin layer detection, and the method of quenching the reaction is preferably to add ice; the extraction reagent is preferably ethyl acetate; the desiccant is preferably anhydrous sodium sulfate; the eluent of the column chromatography purification is preferably a mixture of dichloromethane / methanol, and the volume ratio of dichloromethane to methanol in the mixture is preferably 100:0 to 100:2.

[0060] In order to further illustrate the present invention, the preparation method of N,N-bis(2-(N'-methylindol-3-yl)-thio-acetyl)-2,6-dichloroaniline provided by the present invention and its application in the preparation of anti-Zika virus drugs are described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0061] Example 1

[0062] Preparation of N,N-bis(2-(N'-methylindol-3-yl)-thio-acetyl)-2,6-dichloroaniline (Compound 1), the reaction formula is as follows:

[0063]

[0064] 1) Preparation of compound 1c

[0065] Compound 1a (1 g, 8.33 mmol) and compound 1b (4.21 g, 12.5 mmol, 1.5 eq.) were dissolved in 30 mL of dimethyl sulfoxide, and elemental iodine (0.42 g, 1.67 mmol, 0.2 eq.) was added. The mixture was reacted at 60 °C for 2 h. The thin layer showed that the raw material disappeared. The reaction solution was cooled to room temperature, 400 mL of ethyl acetate was added, and the mixture was washed with water 3 times (300 mL each time), followed by washing with saturated sodium thiosulfate and saturated brine 100 mL each. The organic phase was dried over anhydrous sodium sulfate and concentrated by column chromatography to obtain compound 1c (2.19 g, yield 72%).

[0066] MS: 365.2[M+H] + . 1 H NMR (500 MHz, DMSO- d 6) δ 9.86 (s, 1H), 7.69 (d, J =7.8 Hz, 1H), 7.57 – 7.45 (m, 4H), 7.33 (t, J = 8.1 Hz, 1H), 7.22 (t, J = 7.6 Hz,1H), 7.15 (d, J = 7.5 Hz, 1H), 3.78 (s, 3H), 3.54 (s, 2H).

[0067] 2) Preparation of compound 1e

[0068] Compound 1d (1 g, 4.52 mmol) was dissolved in 10 mL of toluene, and dichlorothionyl (0.54 g, 4.52 mmol) and 1 drop of N,N-dimethylformamide were added as a catalyst. The mixture was refluxed at 110 °C for 3 h and concentrated to dryness to obtain compound 1e, which was used directly in the next step.

[0069] 3) Preparation of compound 1

[0070] Compound 1c (365 mg, 1 mmol) was dissolved in 2 mL of anhydrous N,N-dimethylformamide, cooled to 0°C, and 60% sodium hydride mineral oil (48 mg, 1.2 mmol) was added. After stirring for 5 minutes, compound 1d (240 mg, 1 mmol) was added and stirred at room temperature for 2 h. Crushed ice was added to quench the reaction, extracted with ethyl acetate, dried over anhydrous sodium sulfate, and concentrated. Column chromatography gave compound 1 (494 mg, purity 98.5%, yield 87%).

[0071] MS: 596.2[M+H] + .MS:568.2[M+H] + . 1H NMR (500 MHz, DMSO- d 6) δ 7.64 (d, J =8.1 Hz, 2H), 7.59 (d, J = 7.9 Hz, 2H), 7.56 – 7.47 (m, 5H), 7.24 (t, J = 7.6Hz, 2H), 7.14 (t, J = 7.5 Hz, 2H), 3.78 (s, 6H), 3.71 (s, 4H).

[0072] Comparative Example 1

[0073]

[0074] Compound 1d (1 g, 4.52 mmol) was dissolved in 10 mL of dichloromethane, and DIEA (1.88 mL, 10.85 mmol), HATU (2.06 g, 5.44 mmol) and 2,6-dichloroaniline 1f (0.44 g, 2.71 mmol) were added and reacted at room temperature for 18 h. 50 mL of dichloromethane was added, and the mixture was washed with 0.1 M dilute hydrochloric acid, saturated sodium bicarbonate solution and saturated brine in sequence, and dried over anhydrous sodium sulfate. Concentrated silica gel column chromatography, eluted with dichloromethane / methanol = 100:0~100:1, and the product-containing fractions were collected and concentrated to obtain crude product 1 (430 mg, purity 78.6%). The crude product was chromatographed on a silica gel column again, eluted with petroleum ether: ethyl acetate = 10:1~3:1, and product 1 (282 mg, purity 96.8%, yield 11%) was obtained.

[0075] The mass spectrometry data are consistent with those in Example 1.

[0076] Application Example 1

[0077] 1) This application example uses green monkey kidney cells (Vero) to test the performance of the N,N-bis(2-(N'-methylindol-3-yl)acetyl)substituted aniline compound (Compound 1) in Example 1. Specifically, a concentration of 8×10 4Vero cell suspension of 100 μL / well was inoculated in a 96-well plate. After 24 h of inoculation, when the cells grew to 80%, different concentrations of N,N-bis(2-(N'-methylindol-3-yl)-thio-acetyl)-2,6-dichloroaniline were added. The final concentration of the compound was 20, 4, 0.8, 0.16, 0.032, 0.0064, 0.00128, and 0.000256 μM. ZIKV was infected (MOI=0.01). A negative control DMSO group and a positive control broad-spectrum antiviral nucleoside inhibitor ribavirin group were set. The culture was continued for 6 days, and the culture medium was replaced with serum-free DMEM (containing 10% CCK-8 reagent). The culture was incubated at 37°C for 1.5 h, and the absorbance at OD450 nm was measured by an enzyme reader. The inhibitory activity of compound 1 against ZIKV was calculated using the following formula. The experimental data are given in GraphPadPrism 5.0 was used for graphing and statistical analysis.

[0078]

[0079] 2) CCK-8 (Cell Counting Kit-8) kit was used to detect the cytotoxic effect of the test compound 1. Specifically, the concentration of 8×10 4 Vero cell suspension of 100 μL / mL was inoculated in a 96-well plate. After 24 h of inoculation, when the cells grew to 80%, different concentrations of N,N-bis(2-(N'-methylindol-3-yl)-thio-acetyl)-2,6-dichloroaniline were added. The final concentrations of the compound were 20, 4, 0.8, 0.16, 0.032, 0.0064, 0.00128, and 0.000256 μM. A negative control DMSO group and a positive control broad-spectrum antiviral nucleoside inhibitor ribavirin group were set up. The culture was continued for 6 days, and the culture medium was replaced with serum-free DMEM (containing 10% CCK-8 reagent), incubated at 37°C for 1.5 h, and the absorbance at OD450 nm was measured by an enzyme reader. The cell activity was calculated using the following formula. The experimental data are given in GraphPadPrism 5.0 was used for graphing and statistical analysis.

[0080] Cell viability %=

[0081] Figure 1 This is the result diagram of compound 1's inhibition of Zika virus and cell viability; Figure 2 The results of ribavirin inhibition of Zika virus and cell viability are shown in Figure 2. The experimental results show that compound 1 inhibits Zika virus infection in a dose-dependent manner. The EC value of compound 1 for the inhibition of Zika virus is 50The value was 60.0±10.1nM, which was significantly better than the positive control drug ribavirin (EC 50 The value was 23.08±4.2μM); at the same time, compound 1 showed weaker cytotoxicity than ribavirin. The experimental results showed that N,N-bis(2-(N'-methylindol-3-yl)acetyl)substituted aniline compounds have good application prospects in the preparation of anti-Zika virus drugs.

[0082] Although the above-mentioned embodiments have made a detailed description of the present invention, they are only some embodiments of the present invention, rather than all embodiments. People can also obtain other embodiments based on the embodiments of the present invention without creative work, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. Application of N,N-bis(2-(N'-methylindol-3-yl)-thio-acetyl)-2,6-dichloroaniline in the preparation of anti-Zika virus drugs.

2. An anti-Zika virus drug, characterized in that: The invention comprises a pharmaceutically acceptable carrier and an effective component, wherein the effective component is N,N-bis(2-(N'-methylindol-3-yl)-thio-acetyl)-2,6-dichloroaniline.

3. A method for preparing N,N-bis(2-(N'-methylindol-3-yl)-thio-acetyl)-2,6-dichloroaniline, characterized in that: The following steps are involved: Compound 1a and compound 1b are subjected to a first substitution reaction to obtain compound 1c; Compound 1d is subjected to a second substitution reaction with a halogenating agent to obtain compound 1e; Compound 1c and compound 1e are subjected to acylation reaction under alkaline conditions to obtain N,N-bis(2-(N'-methylindol-3-yl)-thio-acetyl)-2,6-dichloroaniline; The compounds 1a, 1b, 1c, 1d and 1e respectively have the following structures: 1a, 1b, 1c, 1d, 1e, In compound 1b, X is Na or K, and in compound 1e, Y is Cl or Br.

4. The preparation method according to claim 3, characterized in that: The molar ratio of compound 1a to compound 1b is 1:(1.4-1.6).

5. The preparation method according to claim 3, characterized in that: The first substitution reaction is carried out under the catalysis of elemental iodine, and the molar ratio of the compound 1a to elemental iodine is 1:(0.05~1).

6. The preparation method according to claim 3, characterized in that: The solvent for the first substitution reaction includes one or more of dimethyl sulfoxide, 1,4-dioxane and tetrahydrofuran.

7. The preparation method according to any one of claims 3 to 6, characterized in that The temperature of the first substitution reaction is 25-100° C., and the time is 1.8-2.2 h.

8. The preparation method according to claim 3, characterized in that: The second substitution reaction is a chlorination reaction or a bromination reaction. The chlorination reagent used in the chlorination reaction includes one or more of thionyl chloride, oxalyl chloride, phosphorus oxychloride, phosphorus trichloride and phosphorus pentachloride; the bromination reagent used in the bromination reaction includes one or more of thionyl bromide, oxalyl bromide, phosphorus tribromide and phosphorus pentabromide.

9. The preparation method according to claim 3, characterized in that: The molar ratio of the compound 1d to the halogenating agent is 1:(0.33-2).

10. The preparation method according to claim 3, characterized in that: The alkaline reagent providing the alkaline condition includes one or more of sodium hydride, potassium hydride, calcium hydride, butyl lithium, lithium bistrimethylsilylamide and sodium bistrimethylsilylamide.

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