Application of Triazole Thione Derivatives in the Preparation of COVID-19 Inhibitors

Abstract

The present invention relates to the use of triazolethione derivatives represented by Structural Formula I or II in the preparation of a novel coronavirus 3CL protease inhibitor. Among them, X is selected from: hydrogen, C1-C2 alkyl, hydroxyl, C1-C2 alkoxy or C2-C5 acyloxy; Y is selected from: hydrogen, C1-C2 alkyl, hydroxyl, C1-C2 alkoxy, C2-C4 acyloxy, 2-hydroxy-3-methoxy, 3-hydroxy-4-methoxy or 4-hydroxy-3-methoxy; R is selected from: hydrogen, methyl, ethyl, C3-C4 straight-chain alkyl or C3-C4 branched-chain alkyl; Z is selected from: CH or N.

Description

Technical Field

[0001] The present invention relates to the application of a class of compounds, specifically the application of triazolethione derivatives in the preparation of novel coronavirus 3CL protease inhibitors. Background Art

[0002] 4-(arylmethyleneamino)-3-alkyl-1H-1,2,4-triazole-5(4H)-thiones (1) and their biological activities are summarized as follows:

[0003]

[0004]

[0005] Siddiqui et al. [Oriental Journal of Chemistry, 2005, 21(2):317-322] described that 3-phenyl-4-benzylmethyleneamino-1,2,4-triazolethione compounds (2) have certain analgesic and anti-inflammatory activities. When R = 4-Cl or 4-NO2, the analgesic and anti-inflammatory activities are better than the control drug diclofenac sodium.

[0006]

[0007] Chinese invention patents [CN109053606A, CN109053607A, published on December 21, 2018] have described the application of 4-(4-hydroxyphenylmethyleneamino)-1H-1,2,4-triazole-5(4H)-thione [3 and 4: J. Heterocyclic Chem., 2019, 56, 2192] in the preparation of influenza virus neuraminidase inhibitors.

[0008]

[0009] Among them, R 1 is selected from: hydrogen, C1-C2 alkyl, C3-C7 straight-chain or C3-C7 branched-chain alkyl; phenyl, 4-fluorophenyl; X is selected from: H, methyl, ethyl, amino or hydroxy; R is selected from: hydrogen, methoxy, ethoxy, C3-C4 straight-chain alkoxy or C3-C4 branched-chain alkoxy; Y is selected from: phenyl, 4-hydroxyphenyl or 4-methoxyphenyl, fluoromethyl, difluoromethyl or trifluoromethyl. Summary of the Invention

[0010] The technical problem to be solved by the present invention is to provide triazolethione derivatives, their preparation methods, pharmaceutical compositions and uses in the preparation of novel coronavirus 3CL protease inhibitors.

[0011] To solve the technical problems of the present invention, the present invention provides the following technical solutions:

[0012] The first aspect of the technical solution of the present invention provides a class of triazolethione derivatives represented by Structural Formula I or II:

[0013]

[0014] In Formula I, X is selected from: hydrogen, 2-methyl, 2-ethyl, 2-hydroxy, 2-methoxy, 2-ethoxy, 2-acetoxy, 3-methyl, 3-ethyl, 3-hydroxy, 3-methoxy, 3-ethoxy, 3-acetoxy, 3-propionoxy, 3-butyryloxy, 3-valeryloxy, 4-methyl, 4-ethyl, 4-hydroxy, 4-methoxy, 4-ethoxy, 4-acetoxy, 4-propionoxy, 4-butyryloxy or 4-valeryloxy; Y is selected from: hydrogen, 2-methyl, 2-ethyl, 2-hydroxy, 2-methoxy, 2-ethoxy, 2-acetoxy, 3-methyl, 3-ethyl, 3-hydroxy, 3-methoxy, 3-ethoxy, 3-acetoxy, 4-methyl, 4-ethyl, 4-hydroxy, 4-methoxy, 4-ethoxy, 4-acetoxy, 2-hydroxy-3-methoxy, 3-hydroxy-4-methoxy or 4-hydroxy-3-methoxy.

[0015] In Formula II, X is selected from: hydrogen, 2-methyl, 2-ethyl, 2-hydroxy, 2-methoxy, 2-ethoxy, 2-acetoxy, 4-methyl, 4-ethyl, 4-hydroxy, 4-methoxy, 4-ethoxy, 4-acetoxy, 4-propionoxy, 4-butyryloxy, 4-valeryloxy, 5-methyl, 5-ethyl, 5-hydroxy, 5-methoxy, 5-ethoxy, 5-acetoxy, 6-methyl, 6-ethyl, 6-hydroxy, 6-methoxy, 6-ethoxy or 6-acetoxy; R is selected from: hydrogen, methyl, ethyl, a C3-C4 straight-chain alkyl or a C3-C4 branched-chain alkyl; Z is selected from: CH or N.

[0016] The first aspect of the technical solution of the present invention also provides a class of triazolethione derivatives selected from the following compounds:

[0017]

[0018] The second aspect of the technical solution of the present invention provides a preparation method of the triazolethione derivative shown in II, which is characterized in that its preparation reaction is as follows:

[0019]

[0020] Among them, X is selected from: hydrogen, 2-methyl, 2-ethyl, 2-hydroxy, 2-methoxy, 2-ethoxy, 2-acetoxy, 4-methyl, 4-ethyl, 4-hydroxy, 4-methoxy, 4-ethoxy, 4-acetoxy, 4-propionoxy, 4-butyryloxy, 4-valeryloxy, 5-methyl, 5-ethyl, 5-hydroxy, 5-methoxy, 5-ethoxy, 5-acetoxy, 6-methyl, 6-ethyl, 6-hydroxy, 6-methoxy, 6-ethoxy or 6-acetoxy; R is selected from: hydrogen, methyl, ethyl, a C3-C4 straight-chain alkyl or a C3-C4 branched-chain alkyl; Z is selected from: CH or N.

[0021] The third aspect of the technical solution of the present invention is to provide a pharmaceutical composition containing the compound described in the first aspect and its pharmaceutically acceptable salt. This pharmaceutical composition contains a therapeutically effective amount of the triazolethione derivative of the present invention and its pharmaceutically acceptable salt, and optionally contains a pharmaceutical carrier. The pharmaceutical carrier mentioned herein refers to a commonly used pharmaceutical carrier in the pharmaceutical field; this pharmaceutical composition can be prepared according to methods well known in the art. By combining the compound of the present invention and its pharmaceutically acceptable salt with one or more pharmaceutically acceptable solid or liquid excipients and / or adjuvants, any dosage form suitable for human or animal use can be prepared. The content of the compound of the present invention and its pharmaceutically acceptable salt in its pharmaceutical composition is usually 0.1% to 95% by weight percentage.

[0022] The compound of the present invention and its pharmaceutically acceptable salt or the pharmaceutical composition containing it can be administered in unit dosage form, and the administration route can be enteral or parenteral, such as oral, intravenous injection, intramuscular injection, subcutaneous injection, nasal cavity, oral mucosa, eye, lung and respiratory tract, skin, vagina, rectum, etc.

[0023] The dosage form for administration can be a liquid dosage form, a solid dosage form or a semi-solid dosage form. The liquid dosage form can be a solution (including a true solution and a colloidal solution), an emulsion (including an O / W type, a W / O type and a multiple emulsion), a suspension, an injection (including an aqueous injection, a powder injection and an infusion), an eye drop, a nasal drop, a lotion and a liniment, etc.; the solid dosage form can be a tablet (including an ordinary tablet, an enteric-coated tablet, a buccal tablet, a dispersible tablet, a chewable tablet, an effervescent tablet, an orally disintegrating tablet), a capsule (including a hard capsule, a soft capsule, an enteric-coated capsule), a granule, a powder, a pellet, a dropping pill, a suppository, a film, a patch, an aerosol (powder aerosol), a spray, etc.; the semi-solid dosage form can be an ointment, a gel, a paste, etc.

[0024] The compound of the present invention and its pharmaceutically acceptable salt can be made into ordinary preparations, and can also be made into sustained-release preparations, controlled-release preparations, targeted preparations and various particulate drug delivery systems.

[0025] In order to prepare the compounds of the present invention and their pharmaceutically acceptable salts into tablets, various excipients well-known in the art can be widely used, including diluents, binders, wetting agents, disintegrants, lubricants, and glidants. The diluents can be starch, dextrin, sucrose, glucose, lactose, mannitol, sorbitol, xylitol, microcrystalline cellulose, calcium sulfate, calcium hydrogen phosphate, calcium carbonate, etc.; the wetting agents can be water, ethanol, isopropanol, etc.; the binders can be starch paste, dextrin, syrup, honey, glucose solution, microcrystalline cellulose, acacia mucilage, gelatin mucilage, sodium carboxymethyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, ethyl cellulose, acrylic resin, carbomer, polyvinylpyrrolidone, polyethylene glycol, etc.; the disintegrants can be dry starch, microcrystalline cellulose, low-substituted hydroxypropyl cellulose, cross-linked polyvinylpyrrolidone, cross-linked sodium carboxymethyl cellulose, sodium carboxymethyl starch, sodium bicarbonate and citric acid, polyoxyethylene sorbitan fatty acid ester, sodium dodecyl sulfate, etc.; the lubricants and glidants can be talc, silica, stearates, tartaric acid, liquid paraffin, polyethylene glycol, etc.

[0026] The tablets can be further prepared into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or bilayer tablets and multilayer tablets.

[0027] In order to prepare the dosage unit into capsules, the active ingredient, the compounds of the present invention and their pharmaceutically acceptable salts, can be mixed with diluents and glidants, and the mixture can be directly placed into hard capsules or soft capsules. Alternatively, the active ingredient, the compounds of the present invention and their pharmaceutically acceptable salts, can be first made into granules or pellets with diluents, binders, and disintegrants, and then placed into hard capsules or soft capsules. The various diluents, binders, wetting agents, disintegrants, and glidants used for preparing the tablets of the compounds of the present invention and their pharmaceutically acceptable salts can also be used for preparing the capsules of the compounds of the present invention and their pharmaceutically acceptable salts.

[0028] In order to prepare the compounds of the present invention and their pharmaceutically acceptable salts into injections, water, ethanol, isopropanol, propylene glycol, or their mixtures can be used as solvents, and appropriate solubilizers, cosolvents, pH adjusters, and osmotic pressure regulators commonly used in the art can be added. The solubilizers or cosolvents can be poloxamer, lecithin, hydroxypropyl-β-cyclodextrin, etc.; the pH adjusters can be phosphates, acetates, hydrochloric acid, sodium hydroxide, etc.; the osmotic pressure regulators can be sodium chloride, mannitol, glucose, phosphates, acetates, etc. When preparing freeze-dried powder injections, mannitol, glucose, etc. can also be added as supporting agents. In addition, if necessary, colorants, preservatives, fragrances, flavoring agents, or other additives can also be added to the pharmaceutical preparations. To achieve the purpose of medication and enhance the therapeutic effect, the drugs or pharmaceutical compositions of the present invention can be administered by any well-known administration methods.

[0029] The fourth aspect of the technical solution of the present invention is to provide the application of the triazolylthione derivatives (I or II) of the present invention and their pharmaceutically acceptable salts, as well as the pharmaceutical composition described in the third aspect, in the preparation of a novel coronavirus 3CL protease inhibitor.

[0030] Beneficial technical effects:

[0031] The triazolylthione derivatives (I or II) of the present invention are a class of new compounds with novel coronavirus 3CL protease inhibitory activity; they can be used to prepare drugs for treating novel coronavirus.

[0032] Detailed implementation manners

[0033] The following examples are intended to illustrate the present invention rather than further limit the present invention.

[0034] Example 1

[0035] Preparation of 4-(4-hydroxyphenylmethyleneamino)-3-(4-hydroxyphenyl)-1H-1,2,4-triazole-5(4H)-thione

[0036]

[0037] 2.0 mmol of 4-amino-3-(4-hydroxyphenyl)-1H-1,2,4-triazole-5(4H)-thione, 2.2 mmol of 4-hydroxybenzaldehyde, and 3 mL of acetic acid were refluxed for 5 h to obtain white solid 4-(4-hydroxyphenylmethyleneamino)-3-(4-hydroxyphenyl)-1H-1,2,4-triazole-5(4H)-thione with a yield of 61.3% and m.p. 239 - 241 °C. 1 H NMR (400 MHz, DMSO-d6) δ: 13.98 (s, 1H, NH), 10.40 (s, 1H, OH), 10.07 (s, 1H, OH), 9.34 (s, 1H, NCH), 7.75 (d, J = 8.0 Hz, 2H, C6H4), 7.71 (d, J = 8.0 Hz, 2H, C6H4), 6.93 (d, J = 8.0 Hz, 2H, C6H4), 6.87 (d, J = 8.0 Hz, 2H, C6H4); 13 C NMR (100 MHz, DMSO-d6) δ: 167.84, 162.43 (d, J = 9.2 Hz), 159.98, 149.00, 131.51, 130.27, 123.35, 116.64 (d, J = 15.9 Hz), 115.95 (s).

[0038] Example 2

[0039] Preparation of 4-(4-Hydroxy-3-methoxybenzylideneamino)-3-(4-hydroxyphenyl)-1H-1,2,4-triazole-5(4H)-thione

[0040]

[0041] Prepared according to the method of Example 1: 4-Amino-3-(4-hydroxyphenyl)-1H-1,2,4-triazole-5(4H)-thione and vanillin were refluxed for 5 h to obtain white solid 4-(4-Hydroxy-3-methoxybenzylideneamino)-3-(4-hydroxyphenyl)-1H-1,2,4-triazole-5(4H)-thione, with a yield of 62.8% and m.p. 233-235 °C. 1 H NMR (400 MHz, DMSO-d6) δ: 13.98 (s, 1H, NH), 10.06 - 10.05 (m, 2H, OH), 9.36 (s, 1H, NCH), 7.73 (d, J = 8.0 Hz, 2H, C6H4 2,6-H), 7.46 (s, 1H, C6H4 2-H), 7.34 (d, J = 8.0 Hz, 1H, C6H3), 6.95 (d, J = 8.0 Hz, 1H, C6H3), 6.88 (d, J = 8.0 Hz, 2H, C6H4 3,5-H), 3.84 (s, 3H, OCH3).

[0042] Example 3

[0043] Preparation of 4-Acetamido-3-(6-methylpyridin-3-yl)-1H-1,2,4-triazole-5(4H)-thione

[0044]

[0045] 0.83 g (4 mmol) of 4-Amino-3-(6-methylpyridin-3-yl)-1H-1,2,4-triazole-5(4H)-thione and 6 ml of glacial acetic acid were added to a reaction flask. Under reflux, 0.82 g (8 mmol) of acetic anhydride was added dropwise. After the reaction was complete, it was cooled to precipitate a white solid, which was filtered by suction. The solid was immersed in 4 mL of ethanol, 2 mL of glacial acetic acid and 0.2 mL of DMF, stirred for 30 min, filtered by suction and dried to obtain white solid 4-Acetamido-3-(6-methylpyridin-3-yl)-1H-1,2,4-triazole-5(4H)-thione, with a yield of 40.2% and m.p. 300-302 °C. 11H NMR (400 MHz, DMSO-d6) δ: 14.23 (s, 1H, CSNH), 11.46 (s, 1H, CONH), 8.74 (s, 1H, C5H3N2-H), 7.94 (d, J = 8.0 Hz, 1H, C5H3N 4-H), 7.45 (d, J = 8.0 Hz, 1H, C5H3N 5-H), 2.54 (s, 3H, C5H3N 6-CH3), 2.03 (s, 3H, CH3); 13 13C NMR (100 MHz, DMSO-d6) δ: 169.28, 168.56, 161.28, 149.10, 147.52, 135.42, 123.83, 118.86, 24.57, 20.99.

[0046] Example 4

[0047] Preparation of 4-Acetamido-3-(4-acetoxyphenyl)-1H-1,2,4-triazole-5(4H)-thione

[0048]

[0049] 0.833 g (4 mmol) of 4-Amino-3-(4-hydroxyphenyl)-1H-1,2,4-triazole-5(4H)-thione and 6 ml of glacial acetic acid were added to a reaction flask. Under reflux, 0.816 g (8 mmol) of acetic anhydride was added dropwise. After the reaction was complete, the mixture was cooled to precipitate a white solid, which was filtered by suction, washed with ethanol, filtered by suction again, and dried to obtain 4-Acetamido-3-(4-acetoxyphenyl)-1H-1,2,4-triazole-5(4H)-thione as a white solid, with a yield of 59.94% and a melting point of 230 - 232 °C. 1 1H NMR (400 MHz, DMSO-d6) δ: 14.16 (s, 1H, CSNH), 11.43 (s, 1H, CONH), 7.74 (d, J = 8.4 Hz, 2H, C6H4 2,6-H), 7.33 (d, J = 8.4 Hz, 2H, C6H4 3,5-H), 2.30 (s, 3H, CH3), 2.04 (s, 3H, CH3); 13 13C NMR (100 MHz, DMSO-d6) δ: 169.60, 168.28, 152.88, 150.28, 129.15, 123.21, 122.49, 21.25, 20.94.

[0050] Example 5

[0051] Preparation of 4-Acetamido-3-(4-hydroxyphenyl)-1H-1,2,4-triazole-5(4H)-thione

[0052]

[0053] 0.1 g (0.34 mmol) of 4-acetamido-3-(4-acetoxyphenyl)-1H-1,2,4-triazole-5(4H)-thione and 3 mL of methanol were taken. At room temperature, an aqueous solution of lithium hydroxide was added dropwise. After the addition was complete, the reaction was carried out at 50 °C for 12 h. After cooling, the pH was adjusted to acidic with dilute hydrochloric acid, and a white solid was precipitated. It was filtered by suction, washed with ethanol, and dried to obtain the compound 4-acetamido-3-(4-hydroxyphenyl)-1H-1,2,4-triazole-5(4H)-thione, yield: 80.0%, m.p. 249 - 251 °C; 1 H NMR (400 MHz, DMSO-d6) δ: 13.95 (s, 1H, CSNH), 11.38 (s, 1H, CONH), 10.18 (s, 1H, OH), 7.53 (d, J = 8.0 Hz, 2H, C6H4 2,6-H), 6.89 (d, J = 8.0 Hz, 2H, C6H4 3,5-H), 2.02 (s, 3H, CH3); 1 H NMR (400 MHz, DMSO-d6 + D2O) δ: 7.54 (d, J = 8.0 Hz, 2H, C6H4 2,6-H)), 6.91 (d, J = 7.9 Hz, 2H, C6H4 3,5-H), 2.03 (s, 3H, CH3); 13 C NMR (100 MHz, DMSO-d6) δ: 169.28, 167.91, 160.07, 151.02, 129.45, 116.17, 115.70, 20.93.

[0054] Example 6

[0055] Antiviral Activity of Triazole Thione Derivatives Against Novel Coronavirus 3CL Protease

[0056] 1 Experimental Principle

[0057] Using the method of fluorescence resonance energy transfer (FRET), the detection principle is as follows: Edans is a fluorescent donor, and Dabcyl is a fluorescent acceptor or a quencher group. The absorption spectra of these two fluorescent groups overlap to a certain extent. When the distance between these two fluorescent groups is appropriate (usually 7-10 nm), the fluorescence energy is transferred from the donor to the acceptor, resulting in the attenuation of the fluorescence intensity of the donor fluorescent molecule itself. Edans and Dabcyl are connected to both ends of the natural substrate of the 2019-nCoV Mpro / 3CLpro protease, namely Dabcyl-KTSAVLQSGFRKME-Edans. When the 2019-nCoV Mpro / 3CLpro protease does not cleave this substrate, the two groups are close enough for fluorescence resonance energy transfer to occur, that is, Dabcyl can quench the fluorescence of Edans and result in undetectable fluorescence; when this substrate is cleaved by the 2019-nCoV Mpro / 3CLpro protease, the head and tail ends of the polypeptide are separated, the two groups are separated, and the fluorescence of Edans is no longer quenched by Dabcyl, so the fluorescence of Edans can be detected. In this way, the enzyme activity of the 2019-nCoV Mpro / 3CLpro protease can be detected very sensitively by fluorescence detection. If an inhibitor of 2019-nCoV Mpro / 3CLpro is added to the reaction system, the generation of fluorescence will be inhibited, and the fluorescence intensity is inversely proportional to the inhibitory effect of the inhibitor. In this way, the inhibitory effect of the 2019-nCoV Mpro / 3CLpro protease inhibitor can be detected. The maximum excitation wavelength of Edans is 340 nm, and the maximum emission wavelength is 490 nm.

[0058] 2 Experimental methods

[0059] 2.1 Preparation of samples and positive drugs

[0060] Take an appropriate amount of the inhibitor sample to be measured and the positive drug Ebselen, and prepare a solution with an appropriate concentration using DMSO.

[0061] 2.2 Preparation of Assay Reagent

[0062] According to the number of samples (including relevant controls), prepare an appropriate amount of Assay Reagent. For every 1 μl of 2019-nCoV Mpro / 3CLpro, 92 μl of Assay Buffer needs to be added to prepare Assay Reagent for detecting one sample.

[0063] 2.3 Sample detection

[0064] The example samples were detected in a 96-well black plate. 93 μl of Assay Reagent and 5 μl of the sample were sequentially added to each sample well, 93 μl of Assay Reagent and 5 μl of the solvent DMSO were added to the model well, and 93 μl of Assay Buffer and 5 μl of the solvent DMSO were added to the blank control. Oscillate for 1 min using an oscillator to mix well. 2 μl of Substrate was quickly added to each well, and then oscillate for 1 min using an oscillator to mix well. After incubating in the dark at 37 °C for 15 - 20 minutes, fluorescence measurement was performed using a multifunctional microplate reader. The excitation wavelength was 340 nm and the emission wavelength was 490 nm.

[0065] 3 Detection of Samples

[0066] The example samples; the positive control drug was ebselen.

[0067] 4 Activity Results

[0068] The inhibitory activity of the 2019 - nCoV Mpro / 3CLpro protease in the reaction system was evaluated at a sample detection concentration of 50 μg / ml. Compounds with an inhibition rate greater than 50% were rescreened, repeated 3 times, and their IC 50 values were calculated according to the rescreening results. The preferred experimental results are as follows.

[0069]

[0070] The triazolethione derivative has anti - novel coronavirus 3CL protease activity and can be used to prepare a novel coronavirus 3CL protease inhibitor. It can be used to prepare a drug for treating novel coronavirus.

Claims

1. A class of triazolethione derivatives represented by structural formula II and their pharmaceutically acceptable salts: Among them, In the triazolethione derivative represented by formula II, X is selected from: 4-acetoxy, 4-propionyloxy, 4-butyryloxy, 4-valeryloxy, 5-acetoxy or 6-acetoxy; R is selected from: methyl, ethyl, C3-C4 straight-chain alkyl or C3-C4 branched-chain alkyl; Z is selected from: CH or N.

2. The triazolylthione derivative according to claim 1, wherein The triazolethione derivatives represented by II are selected from the following compounds:

3. The preparation method of the triazolethione derivative according to claim 1, characterized in that The preparation reaction of the triazolethione derivative represented by II is as follows: Among them, the definitions of R, X and Z are as described in claim 1.

4. Use of the triazolethione derivative according to claim 1 or 2 and its pharmaceutically acceptable salt in the preparation of a novel coronavirus 3CL protease inhibitor.

5. Use of the triazolethione derivative according to claim 1 or 2 and its pharmaceutically acceptable salt in the preparation of an anti-novel coronavirus drug.

6. A pharmaceutical composition comprising at least one compound according to claim 1 and a pharmaceutically acceptable carrier.

Images