Indigo derivatives, pharmaceutically acceptable salts, pharmaceutical compositions, and uses

Abstract

The application relates to an indigo derivative, a pharmaceutically acceptable salt, a pharmaceutical composition and application, which has the following general formula (I): wherein when R1 and R2 are both hydrogen atoms, R3 is selected from one of 8-quinolinylmethyl, 1-carboxylic acid tert-butyl ester-indole-3-methyl, allyl and 2-quinolinylmethyl; and when R1 and R2 are both bromine atoms, R3 is selected from one of 4-phenyl-benzyl, 4-bromo-1-naphthylmethyl and 4-(2-cyano-1-phenyl)-benzyl. The application has a good inhibiting effect on a novel coronavirus 3CL protease.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to an indigo derivative, a pharmaceutically acceptable salt, a pharmaceutical composition, and its applications. Background Technology

[0002] Vaccines are the most effective means of preventing COVID-19, but antiviral drugs are the first-line treatment for novel coronavirus infection. The 3CL protease is highly conserved in the novel coronavirus and is less prone to mutation compared to the spike protein; its active site amino acids are unique to SARS-CoV-2, showing low homology with human proteases, and it has no cytotoxicity or reproductive toxicity to humans, which can greatly improve the safety of such drugs, making its clinical safety profile higher than that of RNA polymerase inhibitors. The crucial role of the 3CL protease in viral replication and its high degree of conservation make it considered the most attractive target for coronavirus drug development.

[0003] Currently reported coronavirus 3CL protease inhibitors can be divided into peptide-mimicking and non-peptide inhibitors based on their compound structure. 3CL protease inhibitors are currently an important target for developing oral antiviral drugs against the novel coronavirus, with more than 10 drugs already developed, many of which have entered Phase II / III clinical trials.

[0004] Research on SARS-CoV-2 3CL protease inhibitors is still in its early stages, with few available products. Their clinical efficacy and safety remain to be observed, while the clinical demand is enormous, making it one of the most important topics of concern for drug researchers worldwide. Summary of the Invention

[0005] This application provides an indigo derivative, a pharmaceutically acceptable salt, a pharmaceutical composition, and its application, which exhibit good inhibitory effects on the 3CL protease of the novel coronavirus.

[0006] In a first aspect, embodiments of this application provide an indigo derivative having the structure of the following general formula (I):

[0007]

[0008] When both R1 and R2 are hydrogen atoms, R3 is selected from one of 8-quinolinemethyl, 1-carboxylic acid tert-butyl ester-indole-3-methyl, allyl, and 2-quinolinemethyl.

[0009] When both R1 and R2 are bromine atoms, R3 is selected from one of 4-phenyl-benzyl, 4-bromo-1-naphthyl, and 4-(2-cyano-1-phenyl)-benzyl.

[0010] Secondly, embodiments of this application provide a pharmaceutically acceptable salt of the indigo derivative as described above.

[0011] Thirdly, embodiments of this application provide a pharmaceutically acceptable salt of an indigo derivative having the structure of the following general formula (I):

[0012]

[0013] In this context, R1 and R2 are both hydrogen atoms, and R3 is selected from one of 1-biphenylmethyl, 1-naphthylmethyl, 2-naphthylmethyl, 1-bromo-2-naphthylmethyl, 4-bromo-1-naphthylmethyl, 3-phenyl-1-benzylmethyl, 4-(2-cyano-1-phenyl)-benzyl, and 4-(2-carboxylic acid methyl ester-1-phenyl)-benzyl.

[0014] In one embodiment, the pharmaceutically acceptable salt is an inorganic acid salt or an organic acid salt.

[0015] In one embodiment, the inorganic acid salt is selected from at least one of hydrochloride, hydrobromide, hydrofluoric acid, sulfate, nitrate and phosphate.

[0016] In one embodiment, the organic acid salt is selected from at least one of formate, acetate, propionate, citrate, methanesulfonate, ethanesulfonate, malonate, succinate, fumarate, maleate, lactate, malate, oxalate, and tartrate.

[0017] Fourthly, embodiments of this application provide a pharmaceutical composition comprising an active substance and a pharmaceutical carrier, wherein the active substance comprises an indigo derivative as described above, and one or more of the pharmaceutically acceptable salts as described above.

[0018] In one embodiment, the active substance accounts for 0.1% to 95% of the weight percentage of the pharmaceutical composition.

[0019] Fifthly, embodiments of this application provide the use of the indigo derivatives as described above, the pharmaceutically acceptable salts as described above, or the pharmaceutical compositions as described above in the preparation of a medicament for inhibiting the main protease activity of the novel coronavirus and its mutant strains.

[0020] In one implementation, the novel coronavirus mutant strain includes one or more of Alpha, Beta, Gamma, Delta, Omicron, and EG.5 novel variants.

[0021] The beneficial effects of the technical solutions provided in this application include at least the following:

[0022] The indigo derivative provided in this application has a good inhibitory effect on the 3CL protease of the novel coronavirus, providing a new strategy for preparing drugs to treat diseases caused by the novel coronavirus and / or novel coronavirus mutant strains.

[0023] The indigo derivative provided in this application has low acute toxicity and high drug safety, and can be used as an anti-novel coronavirus drug in clinical applications.

[0024] The indigo derivatives provided in this application can be used to prepare pharmaceutical compositions for clinical application as anti-novel coronavirus drugs. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of this application, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 A flowchart illustrating the preparation method of indigo derivatives provided in this application embodiment. Detailed Implementation

[0027] To enable those skilled in the art to better understand the present application, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present application, and not all embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present application.

[0028] An indigo derivative provided in this application embodiment has the structure of the following general formula (I):

[0029]

[0030] When both R1 and R2 are hydrogen atoms, R3 is selected from one of 8-quinolinemethyl, 1-carboxylic acid tert-butyl ester-indole-3-methyl, allyl, and 2-quinolinemethyl.

[0031] When both R1 and R2 are bromine atoms, R3 is selected from one of 4-phenyl-benzyl, 4-bromo-1-naphthyl, and 4-(2-cyano-1-phenyl)-benzyl.

[0032] (1) When both R1 and R2 are hydrogen atoms:

[0033] If R3 is selected from 8-quinolinemethyl, then the indigo derivative is designated as compound 1.

[0034] If R3 is selected from tert-butyl 1-carboxylate-indole-3-methyl, then the indigo derivative is designated as compound 2.

[0035] If R3 is selected from allyl, then the indigo derivative is designated as compound 3.

[0036] If R3 is selected from 2-quinoline methyl, then the indigo derivative is designated as compound 4.

[0037] (2) When both R1 and R2 are bromine atoms:

[0038] If R3 is selected from 4-phenyl-benzyl, then the indigo derivative is designated as compound 5.

[0039] If R3 is selected from 4-bromo-1-naphthylmethyl, then the indigo derivative is designated as compound 6.

[0040] If R3 is selected from 4-(2-cyano-1-phenyl)-benzyl, then the indigo derivative is designated as compound 7.

[0041] The structural formulas of compounds 1 to 7 are shown below:

[0042]

[0043] This application also provides a pharmaceutically acceptable salt of the above-mentioned indigo derivative. That is, this application provides a pharmaceutically acceptable salt of compounds 1 to 7.

[0044] This application also provides another class of pharmaceutically acceptable salts of indigo derivatives. These indigo derivatives have the structure of the following general formula (I):

[0045]

[0046] In this context, R1 and R2 are both hydrogen atoms, and R3 is selected from one of 1-biphenylmethyl, 1-naphthylmethyl, 2-naphthylmethyl, 1-bromo-2-naphthylmethyl, 4-bromo-1-naphthylmethyl, 3-phenyl-1-benzylmethyl, 4-(2-cyano-1-phenyl)-benzyl, and 4-(2-carboxylic acid methyl ester-1-phenyl)-benzyl.

[0047] Specifically, if R3 is selected from 1-biphenylmethyl, then the indigo derivative is designated as compound 8.

[0048] If R3 is selected from 1-naphthylmethyl, then the indigo derivative is designated as compound 9.

[0049] If R3 is selected from 2-naphthylmethyl, then the indigo derivative is designated as compound 10.

[0050] If R3 is selected from 1-bromo-2-naphthylmethyl, then the indigo derivative is designated as compound 11.

[0051] If R3 is selected from 4-bromo-1-naphthylmethyl, then the indigo derivative is designated as compound 12.

[0052] If R3 is selected from 3-phenyl-1-benzyl, then the indigo derivative is designated as compound 13.

[0053] If R3 is selected from 4-(2-cyano-1-phenyl)-benzyl, then the indigo derivative is designated as compound 14.

[0054] If R3 is selected from 4-(2-methyl carboxylate-1-phenyl)-benzyl, then the indigo derivative is designated as compound 15. The structural formulas of compounds 8 to 15 are shown below:

[0055]

[0056] Among them, pharmaceutically acceptable salts include, but are not limited to, inorganic acid salts formed by compounds of general formula (I) with inorganic acids such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid and phosphoric acid, as well as organic acid salts formed by various organic acids such as formic acid, acetic acid, propionic acid, citric acid, methanesulfonic acid, ethanesulfonic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, oxalic acid and tartaric acid.

[0057] In a specific embodiment, the pharmaceutically acceptable salt is selected from at least one inorganic acid salt selected from hydrochloride, hydrobromide, hydrofluoric acid, sulfate, nitrate and phosphate, and / or, the pharmaceutically acceptable salt is selected from at least one organic acid salt selected from formate, acetate, propionate, citrate, methanesulfonate, ethanesulfonate, malonate, succinate, fumarate, maleate, lactate, malate, oxalate and tartrate.

[0058] See Figure 1 As shown, this application also provides a method for preparing the above-mentioned indigo derivatives (i.e., compounds 1 to 15), which includes the following steps:

[0059] 101: Dissolve substance A in a solvent at 0-5℃, then add alkali, stir at 0℃ for 30 min, then add the corresponding bromine-substituted product R3Br to react for 30 min, and monitor the reaction using thin-layer chromatography (TLC) after the reaction is complete.

[0060] 102: Extraction was performed using an extraction reagent, followed by washing with saturated brine, and then drying and concentration with a drying agent followed by silica gel column separation to obtain the indigo derivative; wherein, substance A has the structure of the following general formula (II):

[0061]

[0062] The aforementioned alkalis include one or more of sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate, sodium hydride, and potassium hydride.

[0063] The solvents mentioned above include one or more of N,N-dimethylformamide (DMF), methanol, ethanol, tetrahydrofuran, etc.

[0064] The aforementioned desiccants include one or more of anhydrous sodium sulfate, anhydrous magnesium sulfate, etc.

[0065] The extraction reagents mentioned above include one or more of ethyl acetate, chloroform, dichloromethane, etc.

[0066] This application also provides a pharmaceutical composition comprising an active substance and a pharmaceutical carrier, wherein the active substance comprises one or more of the indigo derivatives provided in the above embodiments and pharmaceutically acceptable salts provided in the above embodiments.

[0067] The pharmaceutical carrier refers to a pharmaceutical carrier commonly used in the pharmaceutical field, such as pharmaceutically acceptable solid or liquid excipients and / or adjuvants.

[0068] In other words, the indigo derivatives and / or pharmaceutically acceptable salts provided in the above embodiments can be combined with pharmaceutically acceptable solid or liquid excipients and / or adjuvants to formulate any dosage form suitable for human or animal use.

[0069] Preferably, the active substance accounts for 0.1% to 95% of the weight percentage of the pharmaceutical composition. That is, the indigo derivatives and / or pharmaceutically acceptable salts provided in the above embodiments account for 0.1% to 95% of the weight percentage of the pharmaceutical composition.

[0070] The indigo derivatives and their pharmaceutically acceptable salts or pharmaceutical compositions containing them provided in this application can be administered in unit dose form via enteral or non-enteric routes, such as oral, intravenous, intramuscular, subcutaneous, nasal, oral mucosa, eye, lung and respiratory tract, skin, vagina, rectum, etc.

[0071] Dosage forms can be liquid, solid, or semi-solid. Liquid dosage forms can include solutions (including true solutions and colloidal solutions), emulsions (including O / W, W / O, and double emulsions), suspensions, injections (including aqueous injections, powder injections, and infusions), eye drops, nasal drops, lotions, and liniments, etc.; solid dosage forms can include tablets (including regular tablets, enteric-coated tablets, lozenges, dispersible tablets, chewable tablets, effervescent tablets, and orally disintegrating tablets), capsules (including hard capsules, soft capsules, and enteric-coated capsules), granules, powders, microcapsules, pellets, suppositories, films, patches, aerosols, and sprays, etc.; semi-solid dosage forms can include ointments, gels, and pastes, etc.

[0072] The indigo derivatives and their pharmaceutically acceptable salts provided in this application can be formulated into conventional formulations, as well as sustained-release formulations, controlled-release formulations, targeted formulations, and various microparticle delivery systems.

[0073] In order to formulate the indigo derivatives and their pharmaceutically acceptable salts provided in this application into tablets, a wide variety of excipients known in the art can be used, including diluents, binders, wetting agents, disintegrants, lubricants, and flow aids. Diluents can be starch, dextrin, sucrose, glucose, lactose, mannitol, sorbitol, xylitol, microcrystalline cellulose, calcium sulfate, dicalcium phosphate, calcium carbonate, etc.; wetting agents can be water, ethanol, isopropanol, etc.; binders can be starch paste, dextrin, syrup, honey, glucose solution, microcrystalline cellulose, gum arabic paste, gelatin paste, sodium carboxymethyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, ethyl cellulose, acrylic resin, carbomer, polyvinylpyrrolidone, polyethylene glycol, etc.; disintegrants can be dry starch, microcrystalline cellulose, low-substituted hydroxypropyl cellulose, croscarmellose, croscarmellose sodium carboxymethyl cellulose, sodium carboxymethyl starch, sodium bicarbonate and citric acid, polyoxyethylene sorbitol fatty acid ester, sodium dodecyl sulfonate, etc.; lubricants and flow aids can be talc, silica, stearate, tartaric acid, liquid paraffin, polyethylene glycol, etc.

[0074] Tablets can also be further processed into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or bilayer and multilayer tablets.

[0075] To formulate the drug delivery unit into capsules, the active ingredient, the indigo derivative and its pharmaceutically acceptable salt provided in this application, can be mixed with a diluent and a gliding agent, and the mixture can be placed directly into hard or soft capsules. Alternatively, the active ingredient, the indigo derivative and its pharmaceutically acceptable salt provided in this application, can be first formed into granules or microspheres with a diluent, binder, and disintegrant, and then placed into hard or soft capsules. The diluents, binders, wetting agents, disintegrants, and gliding agents used to prepare tablets of the indigo derivative and its pharmaceutically acceptable salt provided in this application can also be used to prepare capsules of the indigo derivative and its pharmaceutically acceptable salt provided in this application.

[0076] To prepare the indigo derivatives and their pharmaceutically acceptable salts provided in this application into injectable formulations, water, ethanol, isopropanol, propylene glycol, or mixtures thereof can be used as solvents, and appropriate amounts of commonly used solubilizers, co-solvents, pH adjusters, and osmotic pressure adjusters can be added. Solubilizers or co-solvents may include poloxamer, lecithin, hydroxypropyl-β-cyclodextrin, etc.; pH adjusters may include phosphates, acetates, hydrochloric acid, sodium hydroxide, etc.; osmotic pressure adjusters may include sodium chloride, mannitol, glucose, phosphates, acetates, etc. If preparing lyophilized powder injections, mannitol, glucose, etc., may also be added as supporting agents. Furthermore, if necessary, colorants, preservatives, flavorings, tasters, or other additives may be added to the pharmaceutical preparation. To achieve the intended therapeutic purpose and enhance the therapeutic effect, the drugs or pharmaceutical compositions of this application may be administered using any known method of administration.

[0077] This application also provides the use of the above-mentioned indigo derivative, a pharmaceutically acceptable salt of the above-mentioned indigo derivative, or the above-mentioned pharmaceutical composition in the preparation of a drug that inhibits the activity of the main protease of the novel coronavirus and its mutant strains.

[0078] Specifically, the aim is to prepare drugs that inhibit the activity of the novel coronavirus 3CL protease.

[0079] Among them, the novel coronavirus mutant strains include one or more of the following: Alpha, Beta, Gamma, Delta, Omicron, and EG.5, but are not limited to the aforementioned novel coronavirus and its mutant strains.

[0080] The present application is further illustrated below with reference to embodiments and comparative examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the application. Furthermore, it should be understood that after reading the teachings of this application, those skilled in the art can make various alterations or modifications to the application, and these equivalent forms also fall within the scope defined by the appended claims.

[0081] Example 1: Synthesis of Compound 1

[0082]

[0083] Synthesis method: Indigo (70.62 mg, 0.48 mmol) was dissolved in 10 ml DMF at 0 °C. NaOH (19.2 mg, 0.48 mmol) was added to the system and stirred at 0 °C for 30 min. Then 8-quinoline methyl bromide (159.9 mg, 0.72 mmol) dissolved in DMF was added and reacted for 30 min. The mixture was extracted three times with 50 ml of ethyl acetate. The ethyl acetate layer was washed three times with 50 ml of saturated brine. The ethyl acetate layer was dried over anhydrous sodium sulfate and concentrated. The mixture was then separated by silica gel column chromatography (100 mesh, silica gel; eluent: petroleum ether: ethyl acetate = 100:1-10:1) to obtain compound 1, an orange-yellow powder of 104.3 mg, yield 75.4%, melting point mp: 235-237 °C.

[0084] 1 H NMR (600MHz, CDCl3): δ H 9.00(dd,J=4.2,1.8Hz,1H),8.20(dd,J=8.3,1.8Hz,1H),7.78(dd,J=8.2,1.3Hz,1H),7.69(dd,J=7.1,1.3Hz,1H),7.59(dd,J=7.4,1.3Hz,1H), 7.49 (d, J=8.2Hz, 1H), 7.48 (dd, J=8.3, 1.8Hz, 1H), 7.41 (dd, J=7.8, 1.3Hz, 1H), 7.04 (t, J=7.5, 0.8Hz, 1H), 7.02 (t, J=8.0Hz, 1H), 5.69 (s, 2H).

[0085] 13 C NMR (151MHz, CDCl3): δ C 183.7,159.0,151.3,150.0,146.1,138.5,136.6,133.0,128.5,128.4,128.2,126.6,125.3,123.8,121.6,117.8,111.8,39.6.

[0086] HRMS (ESI) + ):m / z calcd C 18 H 12 N₂O₂ for [M + Na] + 311.0796, found 311.0780.

[0087] Example 2: Synthesis of Compound 2

[0088]

[0089] The synthesis method is the same as in Example 1, except that:

[0090] Replacing 8-quinoline methyl bromide with tert-butyl 1-carboxylate-indole-3-methyl bromide yields compound 2, an orange powder, in 70.4% yield, with a melting point mp of 159-161 °C.

[0091] 1 H NMR (600MHz, CDCl3): δ H 8.08(d,J=8.2Hz,1H),7.70–7.62(m,2H),7.58(d,J=7.4Hz,1H),7.51(td,J=7.8,1.2Hz,1H),7.33(td, J=7.5,0.9Hz,1H),7.25(m,1H),7.08(t,J=7.5Hz,1H),6.99(d,J=8.0Hz,1H),5.04(s,2H),1.67(s,9H).

[0092] 13 C NMR (151MHz, CDCl3): δ C 183.3,158.2,150.8,149.7,138.4,135.6,128.8,125.5,125.2,125.2,124.0,123.2,119.5,117.8,115.5,114.2,111.1,84.5,35.8,28.3,28.3,28.3.

[0093] HRMS (ESI) + ):m / z calcd C 22 H 20 N₂O₄ for [M+Na] + 399.1321, found 399.1316.

[0094] Example 3: Synthesis of compound 3

[0095]

[0096] The synthesis method is the same as in Example 1, except that:

[0097] Replacing 8-quinoline methyl bromide with allyl bromide yields compound 3, an orange powder, with a yield of 74.9% and a melting point (mp) of 158-160 °C.

[0098] 1 H NMR (600MHz, CDCl3): δ H7.60(d,J=7.1Hz,1H),7.56(d,J=7.8Hz,1H),7.11(t,J=7.5Hz,1H),6.89(d,J=7.8Hz ,1H),5.84(ddd,J=22.5,10.5,5.3Hz,1H),5.34–5.26(m,2H),4.36(d,J=5.5Hz,2H).

[0099] 13 CNMR (151MHz, CD3OD): δ C 183.4,158.0,150.9,138.4,130.5,125.5,123.9,118.8,117.7,111.0,42.6.

[0100] HRMS (ESI) + ):m / z calcd C 11 H9NO2 for [M+Na] + 210.0531, found210.0519.

[0101] Example 4: Synthesis of compound 4

[0102]

[0103] The synthesis method is the same as in Example 1, except that:

[0104] Replacing 8-quinoline methyl bromide with 2-quinoline methyl bromide yields compound 4, an orange-yellow powder, in 71.0% yield, with a melting point mp of 213-214 °C.

[0105] 1 H NMR (600MHz, CDCl3): δ H 8.14(d,J=8.4Hz,1H),8.07(d,J=8.4Hz,1H),7.80(d,J=8.0Hz,1H),7.73(t,J=7.7Hz,1H),7.60(d,J=7.4Hz,1H),7.55( t,J=7.5Hz,1H),7.46(t,J=7.8Hz,1H),7.42(d,J=8.5Hz,1H),7.07(t,J=7.5Hz,1H),7.02(d,J=8.0Hz,1H),5.22(s,2H).

[0106] 13 C NMR (151MHz, CDCl3): δ C183.3,158.5,154.9,150.9,147.7,138.6,137.8,130.2,129.2,127.8,127.7,127.1,125.4,124.1,119.6,117.8,111.7,46.8.

[0107] HRMS (ESI) + ):m / z calcd C 18 H 12 N₂O₂ for [M+H] + 289.0977, found289.0963.

[0108] Example 5: Synthesis of compound 5

[0109]

[0110] The synthesis method is the same as in Example 1, except that:

[0111] Replacing indigo with 5,7-dibromoindigo and 8-quinoline methyl bromide with 4-phenylbenzyl bromide yields compound 5, a yellow powder with a yield of 77.7% and a melting point (mp) of 145-147 °C.

[0112] 1 H NMR (600MHz, CDCl3): δ H 7.80(d,J=2.0Hz,1H),7.70(d,J=2.0Hz,1H),7.56(d,J=8.0Hz,4H),7.42(m,2H),7.35(m,1H),7.32(d,J=8.2Hz,2H),5.43(s,2H).

[0113] 13 C NMR (151MHz, CDCl3): δ C 181.3,158.4,146.8,145.4,140.7,140.4,134.8,128.9,128.9,127.6,127.6,127.6,127.6,127.3,127.1,127.1,127.1,121.5,117.3,105.3,44.5.

[0114] HRMS (ESI) + ):m / z calcd C 21 H 13 Br2NO2 for [M+Na] + 493.9190, found 493.9137.

[0115] Example 6: Synthesis of Compound 6

[0116]

[0117] The synthesis method is the same as in Example 1, except that:

[0118] Replacing indigo with 5,7-dibromoindigo and 8-quinoline methyl bromide with 4-bromo-1-naphthyl bromide yields compound 6, a yellow powder, with a yield of 71.1% and a melting point (mp) of 209-211 °C.

[0119] 1 H NMR (600MHz, DMSO): δ H 8.23(ddd,J=6.6,3.8,1.9Hz,2H),7.99(d,J=2.0Hz,1H),7.85(d,J=2.0Hz, 1H), 7.79 (d, J = 7.8Hz, 1H), 7.76 (m, 2H), 7.52 (d, J = 7.8Hz, 1H), 5.63 (s, 2H).

[0120] 13 C NMR (151MHz, DMSO)δ C 180.4,159.2,146.1,143.2,132.9,131.1,130.9,129.6,127.9,127.7,127.2,126.3,123.7,123.6,123.0,121.2,115.7,104.3,42.7.

[0121] HRMS (ESI) + ):m / z calcd C 19 H 10 Br3NO2 for [M+Na] + 545.8139, found 545.8105.

[0122] Example 7: Synthesis of Compound 7

[0123]

[0124] The synthesis method is the same as in Example 1, except that:

[0125] Replacing indigo with 5,7-dibromoindigo and 8-quinoline methyl bromide with 4-(2-cyano-1-phenyl)benzyl bromide yields compound 7, a yellow powder, with a yield of 74.8% and a melting point mp of 207-209 °C.

[0126] 1 H NMR (600MHz, CDCl3): δH 7.82(d,J=2.0Hz,1H),7.75(dd,J=7.8,1.3Hz,1H),7.72(d,J=2.0Hz,1H),7.63(td,J=7.8,1.3Hz,1H),7 .53(m,2H),7.48(dd,J=8.0,1.1Hz,1H),7.44(dd,J=7.6,1.2Hz,1H),7.38(d,J=8.0Hz,2H),5.47(s,2H).

[0127] 13 CNMR (151MHz, CDCl3): δ C 181.2,158.4,146.6,145.5,144.7,137.7,136.5,133.9,133.0,130.1,130.1,129.3,127.8,127.7,127.0,127.0,121.5,118.7,117.4,111.2,105.3,44.5.

[0128] HRMS (ESI) + ):m / z calcd C 22 H 12 Br2N2O2 for [M+Na] + 518.90, found 518.9095.

[0129] Example 8: Synthesis of compound 8

[0130]

[0131] The synthesis method is the same as in Example 1, except that:

[0132] Replacing 8-quinoline methyl bromide with 1-biphenylmethyl bromide yields compound 8, an orange-yellow powder, in 74.3% yield, mp: 239-241℃.

[0133] 1 H NMR (600MHz, CDCl3): δ H 7.62(d,J=7.4Hz,1H),7.58–7.55(m,4H),7.50(t,J=7.8Hz,1H),7.44–7.40(m,4H) ,7.35(t,J=7.3Hz,1H),7.10(t,J=7.5Hz,1H),6.83(d,J=8.0Hz,1H),4.97(s,2H).

[0134] 13 CNMR (151MHz, CDCl3): δC 183.4,158.4,150.8,141.3,140.4,138.5,133.6,129.0,129.0,128.0,128.0,127.9,127.9,127.7,127.2,127.2,125.6,124.0,117.8,111.1,43.9.

[0135] HRMS (ESI) + ):m / z calcd C 21 H 15 NO2 for [M+Na] + 336.1000, found 336.0985.

[0136] Example 9: Synthesis of compound 9

[0137]

[0138] The synthesis method is the same as in Example 1, except that:

[0139] Replacing 8-quinoline methyl bromide with 1-naphthylmethyl bromide yielded compound 9, an orange powder, in 72.7% yield, mp: 229.6–233.1 °C.

[0140] 1 H NMR (600MHz, CDCl3): δ H 8.08(d,J=8.3Hz,1H),7.90(d,J=8.0Hz,1H),7.83(d,J=7.3Hz,1H),7.63–7.58(m,2H),7.55( d,J=7.5Hz,1H),7.43–7.38(m,3H),7.07(d,J=7.5Hz,1H),6.74(d,J=8.0Hz,1H),5.41(s,2H).

[0141] 13 C NMR (151MHz, CDCl3): δ C 183.2,158.5,151.2,138.5,134.0,131.0,129.4,129.2,129.0,127.1,126.4,125.5,125.3,125.1,124.0,122.8,117.9,111.6,42.5.

[0142] HRMS (ESI) + ):m / z calcd C 19 H 13 NO2 for [M+Na] +310.0844, found 310.0828.

[0143] Example 10: Synthesis of compound 10

[0144]

[0145] The synthesis method is the same as in Example 1, except that:

[0146] Replacing 8-quinoline methyl bromide with 2-naphthylmethyl bromide yields compound 10, an orange-yellow powder, in 76.3% yield, mp: 233-235℃.

[0147] HRMS (ESI) + ):m / z calcdC 19 H 13 NO2 for [M+Na] + 310.0844, found 310.0832.

[0148] Example 11: Synthesis of compound 11

[0149]

[0150] The synthesis method is the same as in Example 1, except that:

[0151] Replacing 8-quinoline methyl bromide with 1-bromo-2-naphthyl bromide yields compound 11, a yellow powder, in 68.3% yield, mp: 294.5-297.3 °C.

[0152] 1 H NMR (600MHz, CDCl3): δ H 8.36(d,J=8.5Hz,1H),7.81(d,J=8.0Hz,1H),7.75(d,J=8.5Hz,1H),7.67–7.62(m,2H),7.55(d,J=7.5Hz,1 H), 7.44 (d, J = 8.5Hz, 1H), 7.29 (d, J = 8.5Hz, 1H), 7.10 (t, J = 7.8Hz, 1H), 6.77 (d, J = 8.0Hz, 1H), 5.31 (s, 2H).

[0153] 13 C NMR (151MHz, CDCl3): δ C183.1,158.7,150.6,138.7,134.2,132.5,131.7,128.8,128.4,128.2,127.4,127.2,125.6,124.8,124.2,123.4,117.9,111.4,45.1.

[0154] HRMS (ESI) + ):m / zcalcd C 19 H 12 BrNO2 for [M+Na] + 387.9949, found 387.9934.

[0155] Example 12: Synthesis of compound 12

[0156]

[0157] The synthesis method is the same as in Example 1, except that:

[0158] Replacing 8-quinoline methyl bromide with 4-bromo-1-naphthyl bromide yields compound 12, an orange-yellow powder, in 77.3% yield, mp: 247.8-248.6 °C.

[0159] 1 H NMR (600MHz, CDCl3): δ H 8.32(d,J=9.7Hz,1H),8.06(d,J=9.5Hz,1H),7.70(d,J=7.7Hz,1H),7.67–7.64(m,2H),7.62(d,J=7.4Hz,1 H), 7.42 (t, J = 7.8Hz, 1H), 7.23 (d, J = 7.7Hz, 1H), 7.05 (t, J = 7.5Hz, 1H), 6.71 (d, J = 8.0Hz, 1H), 5.35 (s, 2H).

[0160] 13 C NMR (151MHz, CDCl3): δ C 182.9,158.4,150.9,138.5,132.4,132.1,129.5,129.4,128.5,127.9,127.8,125.6,125.4,124.2,123.8,123.3,117.9,111.4,42.3.

[0161] HRMS (ESI) + ):m / zcalcd C 19 H 12 BrNO2 for [M+Na]+ 387.9949, found 387.9938.

[0162] Example 13: Synthesis of compound 13

[0163]

[0164] The synthesis method is the same as in Example 1, except that:

[0165] Replacing 8-quinoline methyl bromide with 3-phenyl-1-benzyl bromide yields compound 13, an orange-yellow powder, in 76.5% yield, mp: 203.8-205.4 °C.

[0166] 1 H NMR (600MHz, CDCl3): δ H 7.61(d,J=7.3Hz,1H),7.57–7.51(m,4H),7.49(d,J=7.8Hz,1H),7.46–7.40(m,3H),7.36(t,J =7.4Hz, 1H), 7.32 (d, J = 7.7Hz, 1H), 7.09 (t, J = 7.5Hz, 1H), 6.82 (d, J = 8.0Hz, 1H), 4.99 (s, 2H).

[0167] 13 C NMR (151MHz, CDCl3): δ C 183.3,158.4,150.8,142.3,140.5,138.5,135.2,129.6,129.0,129.0,127.8,127.3,127.3,127.2,126.4,126.3,125.6,124.0,117.8,111.1,44.3.

[0168] HRMS (ESI) + ):m / zcalcd C 21 H 15 NO2 for [M+H] + 314.1181, found 314.1169.

[0169] Example 14: Synthesis of compound 14

[0170]

[0171] The synthesis method is the same as in Example 1, except that:

[0172] Replacing 8-quinoline methyl bromide with 4-(2-cyano-1-phenyl)-benzyl bromide yields compound 14, an orange-yellow powder, in 78.1% yield, mp: 254.2-256.4 °C.

[0173] 1 H NMR (600MHz, CDCl3): δ H 7.75(d,J=7.7Hz,1H),7.65(d,J=7.0Hz,1H),7.63(d,J=7.6Hz,1H),7.57-7.54(m,2H),7.52( d,J=7.8Hz,1H),7.49-7.43(m,4H),7.12(t,J=7.5Hz,1H),6.84(d,J=8.0Hz,1H),4.99(s,2H).

[0174] 13 C NMR (151MHz, CDCl3): δ C 183.2,158.4,150.7,144.7,138.6,138.2,135.3,133.9,133.1,130.1,129.6,129.6,127.9,127.9,127.9,125.7,124.1,118.7,117.8,111.3,111.1,43.8.

[0175] HRMS (ESI) + ):m / z calcd C 22 H 14 N₂O₂ for [M+H] + 339.1134, found 339.1120.

[0176] Example 15: Synthesis of compound 15

[0177]

[0178] The synthesis method is the same as in Example 1, except that:

[0179] Replacing 8-quinoline methyl bromide with 4-(2-carboxylic acid methyl ester-1-phenyl)-benzyl bromide yields compound 15, an orange-yellow powder, in 78.4% yield, mp: 227.3-228.2 °C.

[0180] 1 H NMR (600MHz, CDCl3): δ H7.83(d,J=7.7Hz,1H),7.63(d,J=7.4Hz,1H),7.51(q,J=7.3Hz,2H),7.41(t,J=7.3Hz,1H),7.37(d,J=8.1Hz,2H),7 .32(d,J=7.5Hz,1H),7.29(d,J=8.1Hz,2H),7.11(t,J=7.5Hz,1H),6.84(d,J=8.0Hz,1H),4.98(s,2H),3.61(s,3H).

[0181] 13 C NMR (151MHz, CDCl3): δ C 183.3,168.9,158.4,150.9,141.9,141.5,138.4,133.5,131.5,130.8,130.7,130.1,129.2,129.2,127.6,127.3,127.3,125.6,124.0,117.9,111.1,52.1,44.0.

[0182] HRMS (ESI) + ):m / z calcd C 23 H 17 N₂O₄ for [M+Na] + 394.1055, found 394.1041.

[0183] Example 16: Indigo derivatives against novel coronavirus 2019-nCoV M pro / 3CL pro Inhibition experiment

[0184] The novel coronavirus 2019-nCoVM produced by Beyotime Biotechnology Co., Ltd. pro / 3CL pro The inhibitor screening kit uses the fluorescence resonance energy transfer (FRET) method.

[0185] 1. Detection Principle: Edans is the fluorescent donor, and Dabcyl is the fluorescent acceptor (or quencher). The absorption spectra of these two fluorescent groups overlap to some extent. When the distance between these two fluorescent groups is appropriate (generally 7-10 nm), fluorescence energy is transferred from the donor to the acceptor, causing the fluorescence intensity of the donor fluorescent molecule to decrease. Edans and Dabcyl are linked to the 2019-nCoV virus. pro / 3CLpro The two ends of the natural substrate of the protease, namely Dabcyl-KTSAVLQSGFRKME-Edans. When 2019-nCoVM pro / 3CL pro When the protease does not cleave the substrate, the two groups are close enough to undergo fluorescence resonance energy transfer, meaning that Dabcyl can quench the fluorescence of Edans, resulting in undetectable fluorescence; when the substrate is reacted with 2019-nCoV M... pro / 3CL pro After protease cleavage, the two ends of the peptide separate, and the two groups are separated. Edans fluorescence is no longer quenched by Dabcyl, allowing for detection of Edans fluorescence. Thus, fluorescence detection provides a highly sensitive way to detect 2019-nCoV M. pro / 3CL pro Enzymatic activity of proteases. If 2019-nCoV M is added to the reaction system... pro / 3CL pro The inhibitor suppresses fluorescence generation, and the fluorescence intensity is inversely proportional to the inhibitor's inhibitory effect. This allows for the detection of 2019-nCoV M. pro / 3CL pro Inhibitory effect of protease inhibitors. Edans has a maximum excitation wavelength of 340 nm and a maximum emission wavelength of 490 nm.

[0186] 2. Experimental Methods

[0187] 1) Sample Preparation. Take an appropriate amount of the sample to be measured and prepare a solution of suitable concentration using a suitable solvent such as assay buffer or dimethyl sulfoxide (DMSO). If necessary, an appropriate concentration gradient can be prepared for later use. The sample is an indigo derivative provided in this application, including compounds 1 to 15.

[0188] 2) Preparation of positive control. The positive control inhibitor Ebselen provided in this kit is at a concentration of 10 mM and is prepared in DMSO. It can be diluted to the required concentration or concentration gradient using the same solvent as the sample as needed.

[0189] 3) Sample determination.

[0190] a. Prepare an appropriate amount of Assay Reagent according to the number of samples (including relevant controls) and refer to Table 1 below.

[0191] Table 1

[0192] 1 sample 5 samples 10 samples 20 samples AssayBuffer 92μl 460μl 920μl 1.84ml <![CDATA[2019-nCoV M pro / 3CL pro ]]> 1μl 5μl 10μl 20μl

[0193] Due to 2019-nCoVM pro / 3CL pro The glycerol content is high, so care should be taken to avoid adsorption loss by the pipette tip and to ensure complete aspiration when taking micro-absorptions. Add 2019-nCoVM pro / 3CL pro Afterwards, it is important to mix thoroughly.

[0194] b. Set up each group using a 96-well blackboard and add the test reagents and samples in the order shown in Table 2 below. After adding the sample to be tested, mix well.

[0195] To obtain more reliable test results, it is recommended that each sample be tested in at least two replicate wells.

[0196] Table 2

[0197] Blank control 100% enzyme activity control Positive inhibitor control sample AssayBuffer 93μl - - - AssayReagent - 93μl 93μl 93μl Ebselen solution - - 5μl - Sample solvent 5μl 5μl - - Sample to be tested - - - 5μl

[0198] Note: The sample solvent refers to the solvent used to prepare and dilute the test compound.

[0199] c. Quickly add 2 μl of substrate to each well and mix well.

[0200] The reaction will start immediately after the substrate is added. If there are many wells, it is recommended to operate at a low temperature or use a multi-pipette to reduce the error caused by the time difference in adding the substrate between wells. The mixing operation can be performed on a culture plate shaker.

[0201] After incubation at 37℃ in the dark for 5 minutes, the signal tends to stabilize, and fluorescence can be measured using a multifunctional enzyme label within 5-20 minutes.

[0202] The excitation wavelength is 340 nm, and the emission wavelength is 490 nm. When the fluorescence reading is low, the incubation time can be extended to 20-30 minutes.

[0203] Alternatively, in step a, the sample can be added and incubated at 37°C for 10 minutes before adding the substrate to the reaction system. However, incubating at 37°C for 10 minutes before adding the substrate may decrease the IC50 of the sample. It is recommended to determine the optimal incubation time for the sample through preliminary experiments.

[0204] 4) Calculation

[0205] a. Calculate the average fluorescence value of each sample well and the blank control well without sample, which can be recorded as RFU. 空白对照 RFU 100％酶活性对照 RFU 阳性对照 and RFU 样品 . RFU stands for Relative Fluorescence Unit.

[0206] b. Calculate the inhibition percentage for each sample. The calculation formula is as follows: Inhibition percentage (%) = (RFU) / (RFU) 100％酶活性对照 -RFU 样品 ) / (RFU 100％酶活性对照 -RFU 空白对照 )×100%.

[0207] c. For inhibitors that are found to be effective, the half-maximal inhibitory concentration (IC50) can be calculated by detecting the dose-response relationship of the inhibitor. 50 .

[0208] 3. Experimental Results

[0209] Table 3. Indigo derivatives against the novel coronavirus 2019-nCoV M pro / 3CL pro Inhibitory activity

[0210]

[0211]

[0212] Table 3 shows that among the 15 compounds of indigo derivatives, compounds 1-4 and 8-15, at a concentration of 50 μM, showed resistance to 2019-nCoV M. pro / 3CL pro The protease inhibition rates ranged from 0.72% to 73.3%, demonstrating varying degrees of inhibitory activity. Compound 5, with bromine double substitutions at positions 5 and 7, showed inhibitory activity against 2019-nCoV at a concentration of 50 μM. pro / 3CL pro The protease inhibition rate was 45.8%. Compared with the positive control drug Ebselen, these compounds showed weak activity. However, it is noteworthy that compounds 6 and 7, with double bromine substitutions at positions 5 and 7, both showed stronger inhibition of 2019-nCoV M. pro / 3CL pro The activity of the protease (novel coronavirus 3CL protease), its half-maximal inhibitory activity (IC50) 50 It is comparable to the positive control drug Ebselen, but with less toxicity, providing a new approach for finding new anti-SARS-CoV-2 drugs.

[0213] Example 17: Acute toxicity test of indigo derivatives

[0214] Compound 7, which showed good activity, was selected for acute toxicity testing. Male KM mice, weighing 36±4g and SPF grade, were randomly divided into a saline group, a low-dose group (200mg / kg), and a high-dose group (800mg / kg), with 6 mice in each group. After 7 consecutive days of administration, there were no significant changes in body weight or hair, and no deaths occurred, indicating that the indigo derivative has low toxicity and can be used for research on drugs against the novel coronavirus.

[0215] In the description of this application, it should be noted that the terms "upper," "lower," etc., indicating the orientation or positional relationship are based on the orientation or positional relationship shown in the accompanying drawings, and are only for the convenience of describing this application and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation, and therefore should not be construed as a limitation of this application. Unless otherwise expressly specified and limited, the terms "installed," "connected," and "linked" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral connection; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; they can refer to the internal communication between two elements. For those skilled in the art, the specific meaning of the above terms in this application can be understood according to the specific circumstances.

[0216] It should be noted that in this application, relational terms such as "first" and "second" are used merely to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0217] The above description is merely a specific embodiment of this application, enabling those skilled in the art to understand or implement this application. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of this application. Therefore, this application is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features claimed herein.

Claims

1. An indigo derivative, characterized in that, It has the following general formula (I) structure: When both R1 and R2 are hydrogen atoms, R3 is selected from one of 1-tert-butyl carboxylate-indole-3-methyl and 2-quinolinemethyl. When both R1 and R2 are bromine atoms, R3 is selected from 4-bromo-1-naphthylmethyl and 4-(2-cyano-1-phenyl)-benzyl.

2. A pharmaceutically acceptable salt of the indigo derivative as described in claim 1.

3. A pharmaceutically acceptable salt of an indigo derivative, characterized in that, This indigo derivative has the following general formula (I): In this context, R1 and R2 are both hydrogen atoms, and R3 is selected from one of 1-biphenylmethyl, 1-naphthylmethyl, 2-naphthylmethyl, 1-bromo-2-naphthylmethyl, 4-bromo-1-naphthylmethyl, 3-phenyl-1-benzylmethyl, 4-(2-cyano-1-phenyl)-benzyl, and 4-(2-carboxylic acid methyl ester-1-phenyl)-benzyl.

4. The pharmaceutically acceptable salt as described in claim 2 or 3, characterized in that: The pharmaceutically acceptable salt is an inorganic acid salt or an organic acid salt.

5. The pharmaceutically acceptable salt as described in claim 4, characterized in that: The inorganic acid salt is selected from at least one of hydrochloride, hydrobromide, hydrofluoric acid, sulfate, nitrate and phosphate.

6. The pharmaceutically acceptable salt as described in claim 4, characterized in that: The organic acid salt is selected from at least one of formate, acetate, propionate, citrate, methanesulfonate, ethanesulfonate, malonate, succinate, fumarate, maleate, lactate, malate, oxalate, and tartrate.

7. A pharmaceutical composition, characterized in that: It includes an active substance and a pharmaceutical carrier, wherein the active substance comprises one or more of the indigo derivatives as described in claim 1 and pharmaceutically acceptable salts as described in any one of claims 2 to 6.

8. The pharmaceutical composition according to claim 7, characterized in that: The active substance accounts for 0.1% to 95% of the weight of the pharmaceutical composition.

9. The use of an indigo derivative as described in claim 1, a pharmaceutically acceptable salt as described in any one of claims 2 to 6, or a pharmaceutical composition as described in any one of claims 7 to 8 in the preparation of a medicament for inhibiting the activity of the main protease of the novel coronavirus and its mutant strains.

10. The application as described in claim 9, characterized in that: The novel coronavirus mutant strains include one or more of the following: Alpha, Beta, Gamma, Delta, Omicron, and EG.5.

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