2-arylheterocyclic thiomethyl-indole derivatives, process for their synthesis and use thereof

Abstract

The application provides a 2-aryl heterocyclic thiomethyl-indole derivative and a synthesis method and application thereof, and belongs to the field of medicinal chemistry. In-vitro experiments prove that the compound of the application shows good anti-influenza virus activity, and especially, the inhibition rate of the compound of the application on WSN (H1N1) influenza A virus is up to 72.1%, thereby providing a new selection for anti-influenza A virus.

Description

Technical Field

[0001] This invention relates to the field of medicinal chemistry, specifically to a 2-aromatic heterocyclic thiomethyl-indole derivative, its preparation method, and its application in anti-influenza virus treatment. Background Technology

[0002] The spread of influenza viruses not only poses a significant threat to human health but also places a heavy burden on economic development. Oseltamivir is a preferred treatment option for clinicians. However, with the continuous mutation of influenza viruses, many viruses have acquired resistance to oseltamivir, requiring annual revaccination for effective prevention, and the vaccine must be well-matched to the circulating strain. Therefore, there is an urgent need to continuously search for new, highly effective, and safe antiviral drugs for influenza.

[0003] Arbidol is a broad-spectrum antiviral drug that inhibits HA-mediated membrane fusion by increasing the acid stability of hemagglutinin (HA). It can be used to treat influenza A and H1N1 viruses and influenza B virus infections. Due to its broad-spectrum activity, safety profile, and resistance to drug resistance, it has become a representative drug among hemagglutinin protein inhibitors. However, arbidol requires high doses to achieve effective blood concentrations and exert its therapeutic effect. Some research groups have attempted to improve the therapeutic potential of arbidol by modifying the substituents of the indole core. In 2017, Wright et al. modified the structure of the indole ring of arbidol, disclosing a new arbidol analogue that showed a significantly increased affinity for the H3 (1150-fold) and H1 (98-fold) hemagglutinin isoforms compared to the parent compound (Zoë VF Wright et al., 2017). In 2013, Brancato et al. designed and synthesized a series of indole analogs related to the Arbidol structure, among which compound (15) 5-(hydroxymethyl)-1-methyl-2-(phenylsulfonylmethyl)-1h-indole-3-carboxylic acid ethyl ester was identified as one of the most effective inhibitors against certain influenza A virus subtypes (Virginia Brancato et al., 2013). Compared with Arbidol, compound (15) exhibited greater affinity and preference for HA, and played a greater stabilizing role. However, the greater affinity for HA did not translate into a corresponding increase in antiviral activity, which clearly reflects the complexity of the antiviral activity of Arbidol and its derivatives.

[0004] To date, there are few cases of modifying the 2-arylthiomethyl group. This invention mainly focuses on modifying the 2-arylthiomethyl group in order to obtain an anti-influenza virus lead compound with good therapeutic effect and low dosage. Summary of the Invention

[0005] The present invention aims to provide a 2-aromatic heterocyclic thiomethyl-indole derivative; another objective is to provide its preparation method and its application in anti-influenza virus treatment.

[0006] The technical solution adopted in this invention is as follows:

[0007] 2-Aromatic heterocyclic thiomethyl-indole derivatives or pharmaceutically acceptable salts thereof, having the following structure:

[0008] .

[0009] Preferably, the 2-aromatic heterocyclic thiomethyl-indole derivative or its pharmaceutically acceptable salt is a compound such that:

[0010] .

[0011] The synthetic routes for the above-mentioned 2-aromatic heterocyclic thiomethyl-indole derivatives are as follows:

[0012] ;

[0013] R1 is diethylaminomethyl, dipropylaminomethyl, dibutylaminomethyl, or pyrrolidinemethyl.

[0014] or

[0015] .

[0016] The specific preparation steps are as follows:

[0017] Preparation of compounds I-IV:

[0018] (1) Using methanol as solvent, sodium hydroxide was added, followed by 2-mercaptothiophene. The mixture was stirred for 2 h, and finally compound A was added and stirred for 3 h. After the reaction was completed, the reaction solution was neutralized with acetic acid, and a large amount of brownish-black solid precipitated. The mixture was allowed to stand, filtered, the filter cake was dried, and the filtrate was extracted to obtain the crude product. The crude product was purified by column chromatography (dichloromethane:methanol = 120:1), and then recrystallized with EA to obtain pure compound B1, which was a white solid. The molar ratio of sodium hydroxide, 2-mercaptothiophene and compound A was 2.5:1.5:1.

[0019] (2) Using N,N-dimethylformamide (DMF) as a solvent, compound B1 was dissolved in the solvent, followed by the addition of R1, 37% formaldehyde solution and an appropriate amount of glacial acetic acid. The mixture was stirred at 25°C for 15 minutes, and then at 60–80°C for 10 minutes. oStir for 10 hours at C; adjust pH to 7 with 20% NaOH solution, then extract with EA, dry, and evaporate EA to obtain crude product; purify the crude product by column chromatography (dichloromethane:methanol = 60:1) to obtain pure yellow solids of target compounds I-IV. The molar ratio of compounds B1, R1, and 37% formaldehyde solution is 1:2.5:1.5. Specifically, when R1 is diethylaminomethyl, target compound I is obtained; when R1 is dipropylaminomethyl, target compound II is obtained; when R1 is dibutylaminomethyl, target compound III is obtained; and when R1 is pyrrolidinemethyl, target compound IV is obtained.

[0020] Preparation of compound V:

[0021] Compound B1 was dissolved in a suitable amount of 1,4-dioxane, and 10% NaOH solution was added. Dimethyl sulfate was then added dropwise at 25°C with stirring for 5 h. After the reaction was complete, the solvent was evaporated, water was added, and the mixture was filtered. The filter cake was purified by column chromatography (using dichloromethane as the developing solvent) to obtain pure target compound V as a yellow solid. The molar ratio of compound B1, 10% NaOH, and dimethyl sulfate was 1:14:9.

[0022] Preparation of compound VI:

[0023] The specific steps are the same as those for the preparation of compound II, except that 2-mercaptothiophene in step (1) is replaced with 2-mercaptopyridine.

[0024] Another objective of this invention is to provide the application of the above-mentioned 2-aromatic heterocyclic thiomethyl-indole derivatives in the preparation of anti-influenza drugs, especially in the application of drugs against highly pathogenic H1N1 influenza.

[0025] This invention synthesizes a class of 2-aromatic heterocyclic thiomethyl-indole derivatives, which exhibit good anti-influenza activity, especially against WSN(H1N1) influenza A virus with an inhibition rate of up to 72.1%, providing a new option for anti-influenza A virus. Detailed Implementation

[0026] The present invention will be further described below with reference to the embodiments. The embodiments described below are intended to illustrate the present invention and do not limit the scope of protection of the present invention in any way.

[0027] The processes and methods not described in detail in the following examples are conventional methods known in the art. All reagents used in the examples are analytically pure or chemically pure, and can be commercially available or prepared by methods known to those skilled in the art.

[0028] Example 1: Preparation of Compound I

[0029]

[0030] At 25°C, 0.23 g (2.5 mol) of sodium hydroxide was first added to methanol, followed by 0.44 g (1.7 mol) of 2-mercaptothiophene, and the mixture was stirred for 2 h. Finally, 1 g (1 mol) of compound A was added, and the mixture was stirred for 3 h. After the reaction was complete, the reaction solution was neutralized with acetic acid (pH = 6), precipitating a large amount of brownish-black solid. The solid was allowed to stand, filtered, the filter cake was dried, and the filtrate was extracted to obtain the crude product. The crude product was purified by column chromatography (dichloromethane:methanol = 120:1), and then recrystallized from EA to obtain compound B, which was a white solid with a yield of 97%.

[0031] Dissolve 0.2 g (1 mol) of compound B in DMF, then add 0.3 mL (6.73 mol) of diethylamine, 0.2 mL (3.3 mol) of 37% formaldehyde solution, and 6 mL of glacial acetic acid. Stir at 25°C for 15 min, then allow to cool for 60–80 minutes. o Stir for 10 hours. Add 20% NaOH solution to adjust pH to 7, then extract with EA, dry, and evaporate EA to obtain crude product; purify the crude product by column chromatography (dichloromethane:methanol = 60:1) to obtain compound I.

[0032] Yield: 47.5%. Melting point: 105.0-108.0°C. o C. 1 H NMR (600 MHz, CDCl3) δ7.40 (s, 1H),7.36 – 7.33 (m, 1H), 6.91 (s, 1H), 6.90 (d, J = 1.3 Hz, 1H), 4.39 (s, 2H),4.33 (s, 2H), 4.22 (q, J = 7.1 Hz, 2H), 3.47 (s, 3H), 2.68 (q, J = 7.1 Hz, 4H), 1.36 (t, J = 7.1 Hz, 3H), 1.15 (t, J = 7.2 Hz, 6H); 13 C10 NMR (151 MHz, CDCl3) δ 165.59, 151.93, 141.65, 136.13, 131.83, 131.80, 131.29, 127.91, 124.29, 113.40, 112.49, 108.88, 106.17, 60.42, 54.51, 46.78, 33.84, 30.02, 14.48, 11.34. Molecular formula C10 22 H 27 BrN2O3S2, HRMS-ESI m / zcalcd for [M+H] +511.0720, found 511.0718.

[0033] Example 2 Preparation of Compound II

[0034]

[0035] The procedure was the same as in Example 1, and the reaction was carried out for 10 hours to obtain compound II.

[0036] Yield: 49.0%. Melting point: 100.7-103.2°C. o C. 1 H NMR (600 MHz, CDCl3) δ7.42 (s, 1H),7.36 – 7.34 (m, 1H), 6.90 (t, J = 3.5 Hz, 2H), 4.39 (s, 4H), 4.21 (q, J = 7.1Hz, 2H), 3.48 (s, 3H), 2.61 – 2.56 (m, 4H), 1.67 – 1.59 (m, 4H), 1.36 (t, J =7.1 Hz, 3H), 0.88 (t, J = 7.4 Hz, 6H); 13 C10 NMR (151 MHz, CDCl3) δ 165.60, 151.75, 141.57, 136.14, 131.83, 131.82, 131.29, 127.91, 124.25, 113.66, 112.46, 108.72, 106.23, 60.43, 55.84, 55.75, 33.83, 30.04, 19.63, 14.48, 11.97. Molecular formula C10 24 H 31 BrN2O3S2, HRMS-ESI m / zcalcd for[M+H] + 539.1033, found539.1033.

[0037] Example 3 Preparation of Compound III

[0038]

[0039] The procedure was the same as in Example 1, and the reaction was carried out for 10 hours to obtain compound III.

[0040] Yield: 46.6%. Melting point: 141.8-143.0°C. o C. 1H NMR (600 MHz, CDCl3) δ7.39 (s, 1H),7.36 – 7.33 (m, 1H), 6.93 – 6.88 (m, 2H), 4.39 (s, 2H), 4.27 (s, 2H), 4.22(q, J = 7.1 Hz, 2H), 3.48 (s, 3H), 2.59 – 2.48 (m, 4H), 1.59 – 1.53 (m, 4H), 1.36 (t, J = 7.1 Hz, 3H), 1.32 – 1.26 (m, 4H), 0.88 (t, J = 7.4 Hz, 6H); 13 CNMR (151 MHz, CDCl3) δ 165.64, 151.84, 141.53, 136.14, 131.88, 131.84, 131.29, 127.92, 124.30, 113.66, 112.45, 108.73, 106.30, 60.43, 55.78, 53.60, 33.84, 30.06, 28.60, 20.74, 14.51, 14.03. Molecular formula C 26 H 35 BrN2O3S2, HRMS-ESI m / zcalcd for[M+H] + 567.1346, found 567.1338.

[0041] Example 4 Preparation of Compound IV

[0042]

[0043] The procedure was the same as in Example 1, and the reaction was carried out for 10 hours to obtain compound IV.

[0044] Yield: 42.7%. Melting point: 98.5-100.3°C. o C. 1 H NMR (600 MHz, CDCl3) δ7.49 (s, 1H),7.36 – 7.34 (m, 1H), 6.91 – 6.89 (m, 2H), 4.80 (s, 2H), 4.42 (s, 2H), 4.23(q, J = 7.1 Hz, 2H), 3.50 (s, 3H), 3.02 (s, 4H), 1.99 (s, 4H), 1.38 (t, J =7.1 Hz, 3H); 13C10 NMR (151 MHz, CDCl3) δ 165.60, 151.37, 142.19, 136.18, 132.13, 131.84, 131.36, 127.94, 124.32, 113.78, 113.10, 109.50, 106.12, 60.52, 55.32, 53.24, 33.93, 30.10, 23.83, 14.50. Molecular formula C10 22 H 25 BrN2O3S2, HRMS-ESI m / zcalcd for[M+H] + 509.0563, found 509.0548.

[0045] Example 5 Preparation of Compound V

[0046]

[0047] 0.89 g (1 mol) of compound B was dissolved in 1,4-dioxane, and 0.9 g (10.65 mol) of 10% NaOH solution was added. Then, 0.7 mL (3.5 mol) of dimethyl sulfate was added dropwise at 25 °C, and the mixture was stirred for 5 h. After the reaction was complete, the solvent was evaporated to dryness, filtered with water, and the filter cake was purified by column chromatography (using dichloromethane as the developing solvent) to obtain compound V.

[0048] Yield: 91.2%. Melting point: 141.1-143.3°C. o C. 1 H NMR (600 MHz, CDCl3) δ7.69 (s, 1H),7.48 (s, 1H), 7.34 (dd, J = 5.2, 1.2 Hz, 1H), 6.93 – 6.89 (m, 2H), 4.52 (s,2H), 4.29 (q, J = 7.1 Hz, 2H), 3.96 (s, 3H), 3.49 (s, 3H), 1.39 (t, J = 7.1Hz, 3H); 13 C10 NMR (151 MHz, CDCl3) δ 165.07, 151.92, 143.00, 136.25, 132.24, 131.84, 131.33, 127.84, 126.64, 114.10, 108.20, 105.17, 103.87, 59.82, 56.65, 33.31, 29.98, 14.59. Molecular formula C10 18 H 18BrNO3S2, molecular weight 461.9803.

[0049] Example 6 Preparation of Compound VI

[0050]

[0051] At 25°C, 0.23 g (2.5 mol) of sodium hydroxide was first added to methanol, followed by 0.44 g (1.7 mol) of 2-mercaptopyridine. The mixture was stirred for 2 h, and finally 1 g (1 mol) of compound A was added, followed by stirring for 3 h. After the reaction was complete, the reaction solution was neutralized with acetic acid (pH = 6), precipitating a large amount of brownish-black solid. The solid was allowed to stand, filtered, the filter cake was dried, and the filtrate was extracted to obtain the crude product. The crude product was purified by column chromatography (dichloromethane:methanol = 120:1), and then recrystallized from EA to obtain compound B2, a white solid with a yield of 95%.

[0052] Dissolve 0.2 g (1 mol) of compound B2 in DMF, then add 0.3 mL (6.73 mol) of di-n-propylamine, 0.2 mL (3.3 mol) of 37% formaldehyde solution, and 6 mL of glacial acetic acid. Stir at 25°C for 15 min, then allow to cool for 60–80 minutes. o Stir for 10 hours. Add 20% NaOH solution to adjust pH to 7, then extract with EA, dry, and evaporate EA to obtain crude product; purify the crude product by column chromatography (dichloromethane:methanol = 60:1) to obtain compound VI.

[0053] Yield: 41.2%. Melting point: 104.3-107.8 °C. o C. 1 H NMR (600 MHz, CDCl3) δ8.48 – 8.46 (m,1H), 7.49 (td, J = 7.8, 1.8 Hz, 1H), 7.41 (s, 1H), 7.16 (d, J = 8.0 Hz, 1H), 7.03 – 7.01 (m, 1H), 4.92 (s, 2H), 4.37 (q, J = 7.1 Hz, 2H), 4.33 (s, 2H), 3.71 (s, 3H), 2.57 – 2.51 (m, 4H), 1.64 – 1.57 (m, 4H), 1.38 (t, J = 7.1 Hz,3H), 0.88 (t, J = 7.4 Hz, 6H); 13C10 NMR (151 MHz, CDCl3) δ 165.94, 157.84, 151.89, 149.41, 142.28, 136.33, 131.84, 124.37, 122.39, 120.01, 113.61, 112.51, 108.53, 106.25, 60.53, 55.86, 55.67, 30.51, 24.85, 19.72, 14.50, 11.99. Molecular formula C10 25 H 32 BrN3O3S, HRMS-ESI m / zcalcd for[M+H] + 534.1421, found 534.1409.

[0054] Example 7

[0055] The 2-arylthiomethylindole compounds obtained in Examples 1-6 of this invention exhibit cytotoxicity and anti-influenza virus activity against MDCK.

[0056] 1. Cytotoxicity assay

[0057] Sample: 2-arylthiomethylindole compounds obtained in Examples 1-6 of this invention

[0058] Cell line: Canine kidney (MDCK) cells, provided by American Type Culture Collection.

[0059] Reagents: Minimum essential culture medium (MEM), phosphate-buffered saline (PBS), and fetal bovine serum (FBS) required for cell culture were obtained from Life Technologies (Gibco, NY, USA).

[0060] Experimental procedure:

[0061] (1) Cell culture

[0062] MDCK cells were stored in MEM medium containing 10% FBS and incubated at 37°C. o C, conventional culture under 5% CO2.

[0063] (2) MTT experimental method

[0064] Based on the principle of the MTT assay, the cytotoxicity of the target compounds in Examples 1-6 against MDCK cells was determined. First, a cell suspension was prepared and diluted to the required concentration. An alcohol lamp was lit, and the biosafety cabinet work surface and hands were disinfected with 75% alcohol. MDCK cells were then seeded into 96-well plates, and the cells were mixed after each well to ensure a consistent cell density. The 96-well plates were placed in a CO2 incubator and cultured for 24 h. The MDCK cells in the 96-well plates were washed with PBS, and the target compounds were added at two-fold serial dilutions. The culture was carried out at 37°C. o After incubation at C for 48 h, MTT (final concentration 0.5 mg / mL) was added to the 96-well plate and incubated at 37°C. o After culturing at C for 4 hours, blue formazan crystals formed. The supernatant was then removed, and DMSO was added. The 96-well plate was shaken for 10 minutes to fully dissolve the formazan crystals. The absorbance was measured at 540 nm using a ClarioStar microplate reader. The concentration at which 50% cytotoxicity was achieved was denoted as CC. 50 Values. The test results are shown in the table below.

[0065] Table 1. Cytotoxicity of the compounds of the present invention to MDCK cells

[0066]

[0067] As can be seen from the table, except for compound VI, which has similar cytotoxicity to the positive control Arbidol, most of the target compounds prepared in this invention have lower cytotoxicity than Arbidol.

[0068] 2. Anti-influenza virus experiment

[0069] Sample: 2-arylthiomethylindole compounds obtained in Examples 1-6 of this invention

[0070] Cell line: MDCK cells, provided by American Type Culture Collection.

[0071] Experimental Procedure: The inhibitory effect of the target compound on influenza virus was determined using a cytopathic effect assay (CPE). First, the drug-containing culture medium was prepared. A DMSO stock solution of the target compound was prepared in advance. Before use, different concentrations of the target compound were prepared in the culture medium for testing its anti-influenza virus activity. Second, 1 × 10⁻⁶ cells per well were used. 5 MDCK cells were seeded at a cell density of 37°C and cultured at 37°C. o Cells were cultured at 5% CO2. After 24 hours of culture, different concentrations of drug solution and virus solution were added (10 TCID50 per well). 50(Viral fluid). Finally, three days after infection, cell lysis and shedding were observed under a microscope. The IC50 was calculated based on the degree of improvement of influenza virus-induced cytopathic effects by the target compound. 50 Values. The test results are shown in the table below.

[0072] Table 2. Anti-influenza virus activity of the compounds of the present invention against MDCK cells.

[0073]

[0074] The above results indicate that compounds I-VI have inhibitory activity against WSN(H1N1) influenza virus, with compound I exhibiting the best inhibitory activity. At a concentration of 62.5 μg / mL, compound I achieved the highest inhibition rate of 72.1% against WSN(H1N1) influenza virus.

Claims

1,2-Aromatic heterocyclic thiomethyl-indole derivatives or pharmaceutically acceptable salts thereof, having the following structure: 。 2. The 2-aromatic heterocyclic thiomethyl-indole derivative or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that, The following compounds are examples: 。 3. The method for preparing the 2-aromatic heterocyclic thiomethyl-indole derivative according to claim 1, characterized in that, The synthetic route is as follows: ， R1 is diethylamine, di-n-propylamine, di-n-butylamine, or tetrahydropyrrole; or 。 4. The use of the 2-aromatic heterocyclic thiomethyl-indole derivative of claim 1 or a pharmaceutically acceptable salt thereof in the preparation of an anti-influenza drug.

5. The use of the 2-aromatic heterocyclic thiomethyl-indole derivative of claim 1 or a pharmaceutically acceptable salt thereof in the preparation of an anti-H1N1 influenza drug.

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