Indole Compounds, Their Preparation Methods, and Anti-Coronavirus Drugs Containing the Same

Abstract

The present invention relates to an indole compound having a structure shown in Formula I or Formula II: wherein A is a six-membered ring or a seven-membered ring; optionally, the indole compound is compound RU-75 or RU-75A, and the isomer of RU-75A is compound RU-75A'. Compared with the prior art solutions, the present invention has the following advantageous effects: Through a large number of designs and screenings, the inventors of the present application found that the indole compounds of the present application are superior to the lead compound rutaecarpine in the effect of anti-SARS-CoV-2. Moreover, the preparation method route of the present application is reasonably designed, the starting materials are easily available, the reagents are of low price, the post-treatment is simple, and the production cost can be effectively reduced.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine and organic synthesis, and in particular to an indole compound and a preparation method thereof, and an anti-coronavirus drug containing the indole compound. Background Art

[0002] Coronaviruses are a large family of viruses that are known to cause more serious diseases such as colds, Middle East Respiratory Syndrome (MERS) and Severe Acute Respiratory Syndrome (SARS). Among them, the new coronavirus is a new strain of coronavirus that has never been found in humans before. The International Committee on Taxonomy of Viruses named this new pathogen severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Common signs of human infection with the new coronavirus (SARS-CoV-2) include respiratory symptoms, fever, cough, shortness of breath and difficulty breathing. In more severe cases, infection can lead to pneumonia, severe acute respiratory syndrome, renal failure, and even death. Therefore, finding candidate drugs that effectively inhibit coronaviruses is a technical problem that needs to be solved urgently. Summary of the invention

[0003] Based on this, the purpose of the embodiments of the present application includes providing indole compounds, which have good anti-coronavirus effects.

[0004] In the first aspect of the present application, an indole compound or an isomer thereof is provided, wherein the indole compound has a structure as shown in Formula I or Formula II:

[0005]

[0006]

[0007] Wherein, ring A is a 6- to 7-membered monocyclic heterocyclic ring.

[0008] In some embodiments of the present application, ring A is selected from one of the following structures:

[0009]

[0010] In some embodiments of the present application, the indole compound is compound RU-75 or RU-75A.

[0011]

[0012] In some embodiments of the present application, the isomer of RU-75A ​​is compound RU-75A',

[0013]

[0014] In the second aspect of the present application, there is provided the use of the indole compound or its isomer described in the first aspect in the preparation of a drug against coronaviruses.

[0015] In some embodiments of the present application, the coronaviruses include severe acute respiratory syndrome coronavirus 2, severe acute respiratory syndrome coronavirus, Middle East respiratory syndrome coronavirus, or bat SARS-like coronavirus.

[0016] In some embodiments of the present application, the drug includes the indole compound or its isomer described above and a pharmaceutically acceptable excipient.

[0017] In some embodiments of the present application, the dosage form of the drug includes ointment, patch, solution, suspension, emulsion, cream, spray, drop, infusion, granule, capsule, tablet, pill, tea preparation, or tube feeding preparation.

[0018] In the third aspect of the present application, there is provided a drug against coronaviruses, which includes the indole compound or its isomer described in the first aspect and a pharmaceutically acceptable excipient.

[0019] In some embodiments of the present application, the coronaviruses include severe acute respiratory syndrome coronavirus 2, severe acute respiratory syndrome coronavirus, Middle East respiratory syndrome coronavirus, or bat SARS-like coronavirus.

[0020] In some embodiments of the present application, the dosage form of the drug includes ointment, patch, solution, suspension, emulsion, cream, spray, drop, infusion, granule, capsule, tablet, pill, tea preparation, or tube feeding preparation.

[0021] In the fourth aspect of the present application, there is provided a preparation method of the indole compound or its isomer described in the first aspect, and the preparation method includes the step of preparing compound 2 and the step of preparing compound RU-75, compound RU-75A, or compound RU-75A' with the compound 2;

[0022] Wherein,

[0023] The step of preparing compound 2 includes: mixing compound 1, ethyl formate, triethylamine, and methanol for a first-stage reaction to prepare a first intermediate; mixing the first intermediate, phosphorus oxychloride, and dichloromethane for a second-stage reaction to prepare compound 2;

[0024] The step of preparing compound RU-75 with the compound 2 includes:

[0025] React the compound 2, 2 - amino - 4 - nitrobenzoic acid, 1 - ethyl - (3 - dimethylaminopropyl) carbodiimide and dimethylformamide to prepare compound 3;

[0026] React the compound 3, iron powder, ethanol and ammonium chloride to prepare compound 4;

[0027] React the compound 4, bromobenzene, palladium acetate, XPhos, cesium carbonate and 1,4 - di alkane to prepare compound RU - 75;

[0028] The steps of preparing compound RU - 75A or compound RU - 75A' using the compound 2 include:

[0029] React the compound 2, 2 - amino - 4 - nitrobenzoic acid, 1 - ethyl - (3 - dimethylaminopropyl) carbodiimide and dimethylformamide to prepare compound 5;

[0030] React the compound 5, iron powder, ethanol and ammonium chloride to prepare compound 6;

[0031] React the compound 6, bromobenzene, palladium acetate, XPhos, cesium carbonate and 1,4 - di alkane to prepare compound RU - 75A or compound RU - 75A';

[0032]

[0033] In some embodiments of the present application, the preparation method satisfies one or more of the conditions shown in (1) to (8):

[0034] (1) The conditions for the first - stage reaction include: temperature is 95°C - 105°C, time is 15h - 20h;

[0035] (2) The conditions for the second - stage reaction include: temperature is 0°C - 5°C, time is 1.5h - 2.5h;

[0036] (3) In the step of preparing compound 3, the reaction conditions include: temperature is 75°C - 85°C, time is 15h - 20h;

[0037] (4) In the step of preparing compound 4, the reaction conditions include: temperature is 75°C - 85°C, time is 0.5h - 1.5h;

[0038] (5) In the step of preparing compound RU - 75, the reaction conditions include: temperature is 95°C - 105°C, time is 15h - 25h;

[0039] (6) In the step of preparing Compound 5, the reaction conditions include: the temperature is 75°C - 85°C, and the time is 15 h - 25 h;

[0040] (7) In the step of preparing Compound 6, the reaction conditions include: the temperature is 75°C - 85°C, and the time is 0.5 h - 1.5 h; and,

[0041] (8) In the step of preparing Compound RU-75A, the reaction conditions include: the temperature is 95°C - 105°C, and the time is 15 h - 25 h.

[0042] Compared with the traditional technical solution, the present application has the following advantageous effects:

[0043] The inventors of the present application have conducted a large number of designs and screenings and found that the indole compounds of the present application are superior to the lead compound rutaecarpine in the effect of anti-SARS-CoV-2. Moreover, the preparation method route of the present application is reasonably designed, the starting materials are easily available, the reagents are of low price, and the post-treatment is simple, which can effectively reduce the production cost. Description of the Drawings

[0044] In order to more clearly illustrate the technical solutions in the embodiments of the present application and more fully understand the present application and its beneficial effects, the following will briefly introduce the drawings required to be used in the description of the embodiments. Obviously, the drawings in the following description are only some embodiments of the present application, and those skilled in the art can also obtain other drawings based on these drawings without creative efforts.

[0045] Figure 1 It is the preparation route diagram of RU-75 in Example 1 of the present application;

[0046] Figure 2 It is the preparation route diagram of RU-75A in Example 1 of the present application;

[0047] Figure 3 It is the 1H NMR spectrum diagram of Compound RU-75 prepared in Example 1 of the present application;

[0048] Figure 4 It is the 1H NMR spectrum diagram of Compound RU-75A prepared in Example 1 of the present application;

[0049] Figure 5 It is the result of the inhibitory effects of Compound RU-75, RU-75A and the lead compound rutaecarpine (RU) prepared in Example 1 of the present application on SARS-CoV-2 Spike pseudotyped virus;

[0050] Figure 6Quantitative comparison chart of the potential cytotoxicity of the compounds RU-75 and RU-75A prepared in Example 1 of this application and the lead compound rutaecarpine in the MTT assay;

[0051] Figure 7 In Example 4 of this application, molecular docking technology shows that RU-75, RU-75A, and rutaecarpine bind to the RBD site of the spike protein. Detailed implementation manners

[0052] The present invention will be further described in detail below with reference to the drawings, implementation manners, and examples. It should be understood that these implementation manners and examples are only used to illustrate the present invention and not to limit the scope of the present invention. The purpose of providing these implementation manners and examples is to make the understanding of the disclosed content of the present invention more thorough and comprehensive. It should also be understood that the present invention can be implemented in many different forms and is not limited to the implementation manners and examples described herein. Those skilled in the art can make various changes or modifications without departing from the connotation of the present invention, and the equivalent forms obtained also fall within the protection scope of this application. In addition, in the following description, a large number of specific details are given to provide a more thorough understanding of the present invention. It should be understood that the present invention can be implemented without one or more of these details.

[0053] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which this invention belongs. The terms used in the description of the present invention herein are only for the purpose of describing the implementation manners and examples and are not intended to limit the present invention.

[0054] Term

[0055] Unless otherwise stated or there is a contradiction, the terms or phrases used herein have the following meanings:

[0056] As used herein, the alternative ranges of the terms "and / or", "or / and", and "and / or" include any one of two or more related listed items, and also include any and all combinations of the related listed items. The aforesaid any and all combinations include combinations of any two related listed items, any more than two related listed items, or all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in this application, this technical solution undoubtedly includes the technical solution connected by "logical AND", and undoubtedly also includes the technical solution connected by "logical OR". For example, "A and / or B" includes three parallel solutions: A, B, and A + B. Another example is the technical solution of "A, and / or, B, and / or, C, and / or, D", which includes any one of A, B, C, and D (that is, the technical solution connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, combinations of any two or any three of A, B, C, and D, and also includes the four-item combination of A, B, C, and D (that is, the technical solution connected by "logical AND").

[0057] In the present invention, terms such as "multiple", "multiple types", "multiple times", "multiple elements", etc. refer to a quantity greater than or equal to 2 if not otherwise specified. For example, "one or more types" means one type or two or more types.

[0058] In the present invention, "its combination", "any combination thereof", "any combination mode thereof", etc. include all suitable combination modes of any two or more than two items in the listed items.

[0059] In the present invention, the "suitable" in "suitable combination mode", "suitable mode", "any suitable mode", etc. is subject to being able to implement the technical solution of the present invention, solve the technical problems of the present invention, and achieve the expected technical effects of the present invention.

[0060] In the present invention, "preferred", "better", "more preferable", "preferably" are only used to describe the embodiments or examples with better effects, and it should be understood that they do not constitute a limitation to the protection scope of the present invention.

[0061] In the present invention, "further", "even further", "especially", etc. are used for descriptive purposes and represent differences in content, but should not be construed as a limitation to the protection scope of the present invention.

[0062] In the present invention, "optionally", "optional", "optional" mean that it can be either present or absent, that is, it refers to any one of the two parallel solutions of "present" or "absent". If "optional" appears multiple times in a technical solution, and there is no special description, no contradiction or mutual restriction relationship, then each "optional" is independent.

[0063] In the present invention, in "the first aspect", "the second aspect", "the third aspect", "the fourth aspect", etc., the terms "first", "second", "third", "fourth", etc. are only for descriptive purposes and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first", "second", "third", "fourth", etc. only serve the purpose of non-exhaustive enumerative description and should be understood not to constitute a closed limitation on quantity.

[0064] In the present invention, among the technical features described in an open-ended manner, there are included closed technical solutions composed of the listed features, as well as open technical solutions containing the listed features.

[0065] In the present invention, regarding numerical intervals (i.e., numerical ranges), unless otherwise specified, the optional numerical values are considered continuous within the above numerical intervals and include the two numerical endpoints (i.e., the minimum value and the maximum value) of the numerical range, as well as each numerical value between these two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to integers within the numerical interval, it includes the two endpoint integers of the numerical range, as well as each integer between the two endpoints. In this document, it is equivalent to directly listing each integer. For example, when t is an integer selected from 1 to 10, it means that t is any integer selected from the integer group composed of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. In addition, when multiple ranges are provided to describe features or characteristics, these ranges can be combined. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all sub-ranges subsumed therein.

[0066] For the temperature parameter in the present invention, unless otherwise specified, it allows both constant temperature treatment and variation within a certain temperature range. It should be understood that the so-called constant temperature treatment allows the temperature to fluctuate within the accuracy range controlled by the instrument. Fluctuation within a range such as ±5°C, ±4°C, ±3°C, ±2°C, ±1°C is allowed.

[0067] In the present invention, %(w / w) and wt% both represent weight percentage, %(v / v) represents volume percentage, and %(w / v) represents mass-volume percentage.

[0068] All documents mentioned in the present invention are incorporated herein by reference as if each individual document was specifically and individually incorporated by reference. Unless it conflicts with the object and / or technical solution of the present invention, the cited documents related to the present invention are incorporated by reference in their entirety and for all purposes. When referring to a cited document in the present invention, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited document are also incorporated by reference. When referring to a cited document in the present invention, examples and preferred modes of the relevant technical features cited can also be incorporated by reference into the present application, provided that the present invention can be implemented. It should be understood that when the cited content conflicts with the description in the present application, the present application shall prevail or be amended adaptively according to the description in the present application.

[0069] "Monocyclic heterocycle" means that 1, 2 or 3 ring carbon atoms in a saturated or partially unsaturated monocyclic ring are replaced by heteroatoms selected from nitrogen, oxygen or S(O)t (where t is an integer from 0 to 2), but does not include ring moieties of -O-O-, -O-S- or -S-S-, and the remaining ring atoms are carbon. "6- to 7-membered monocyclic heterocycle" has 6 to 7 ring atoms, where 1, 2 or 3 ring atoms are the above-mentioned heteroatoms.

[0070] Evodia fruit is the dried nearly ripe fruit of Evodia rutaecarpa (Juss.) Benth., Evodia rutaecarpa (Juss.) Benth. var. officinalis (Dode) Huang or Evodia rutaecarpa (Juss.) Benth. var. bodinieri (Dode) Huang of the Rutaceae family, also known as spicy fruit, qi spicy fruit, tea spicy fruit, etc. "Chinese Pharmacopoeia" records its efficacy and indications including: warming the middle-jiao, relieving pain, regulating qi, drying dampness, and treating eczema, etc. Modern research shows that the components of Evodia fruit include rutaecarpine, evodiamine, evocarpine, evodolactone, hydroxyevodiamine, etc. At the same time, it is found that the extract of Evodia fruit has varying degrees of inhibitory effects on dermatophytes. In addition, rutaecarpine compounds have shown effects of anti-hypertension, anti-arrhythmia and uterine excitation in research.

[0071] During the research process of traditional Chinese medicine, the inventor screened more than 200 traditional Chinese medicines and their chemical components using three models: a pseudovirus that simulates the infection of the novel coronavirus SARS-CoV-2, cytotoxicity analysis, and molecular docking technology. It was found that rutaecarpine can inhibit the invasion of the novel coronavirus pseudovirus, reduce the binding of the spike protein to angiotensin-converting enzyme 2 (ACE2), and inhibit 3CL protease, thereby playing a role in inhibiting coronaviruses. Based on this discovery, the present invention uses rutaecarpine as a lead compound for chemical structure modification and structure-activity relationship research, and designs and synthesizes two novel indole compounds, RU-75 and RU-75A. It was found that the two indole compounds, RU-75 and RU-75A, synthesized by us have antiviral activity but no cytotoxicity, and bind better to the RBD region of the spike protein. And the antiviral activity is superior to the lead compound rutaecarpine. It provides new ideas and solutions for drugs against coronavirus infection.

[0072] The first aspect of the present application

[0073] The present application provides an indole compound or its isomer, and the indole compound has a structure shown in Formula I or Formula II:

[0074]

[0075] Among them, Ring A is a 6- to 7-membered single heterocyclic ring.

[0076] In one example, Ring A is selected from one of the following structures:

[0077]

[0078] In one example, the indole compound is compound RU-75 or RU-75A,

[0079]

[0080] In one example, the isomer of RU-75A is compound RU-75A',

[0081]

[0082] The second aspect of the present application

[0083] The present application provides the use of the indole compound or its isomer described in the first aspect in the preparation of a drug against coronavirus.

[0084] In one example, the coronavirus includes Severe Acute Respiratory Syndrome Coronavirus 2, Severe Acute Respiratory Syndrome Coronavirus, Middle East Respiratory Syndrome Coronavirus, or Bat SARS-like Coronavirus.

[0085] In one example, the drug includes the indole compound or its isomer and a pharmaceutically acceptable excipient.

[0086] In one example, the dosage form of the drug includes ointment, patch, solution, suspension, emulsion, cream, spray, drop, infusion, granule, capsule, tablet, pill, tea preparation, or tube feeding preparation.

[0087] The diluent of the present application can be selected from, including but not limited to, starches, sugars, celluloses, and inorganic salts, and one or a combination of multiple ones can be selected.

[0088] The wetting agent of the present application can be selected from, including but not limited to, water and ethanol, and one or a combination of multiple ones can be selected.

[0089] The binder of the present application can be selected from, including but not limited to, starch paste, dextrin, sugar, cellulose derivatives, gelatin, polyvinylpyrrolidone, and polyethylene glycol, and one or a combination of multiple ones can be selected.

[0090] The disintegrant of the present application is selected from dry starch, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, cross-linked carboxymethyl cellulose sodium, cross-linked polyvinylpyrrolidone, surfactant, and effervescent disintegrant, and one or a combination of multiple ones can be selected.

[0091] The lubricant of the present application can be selected from, including but not limited to, talc, calcium stearate, magnesium stearate, sodium dodecyl sulfate, colloidal silica, and polyethylene glycol, and one or a combination of multiple ones can be selected.

[0092] The color, flavor, and odor regulator of the present application can be selected from, including but not limited to, pigments, flavors, sweeteners, mucilage, and odor correctors, and one or a combination of multiple ones can be selected.

[0093] The solvent of the present application can be selected from, including but not limited to, water, oil, ethanol, glycerol, propylene glycol, polyethylene glycol, dimethyl sulfoxide, liquid paraffin, fatty oil, and ethyl acetate, and one or a combination of multiple ones can be selected.

[0094] The solubilizer of the present application can be selected from, including but not limited to, Tweens, Myrj, polyoxyethylene fatty alcohol ethers, soaps, sulfates, and sulfonates, and one or a combination of multiple ones can be selected.

[0095] The solubilizer of the present application may be selected from, including but not limited to, organic acids and their salts, amides and amine compounds, inorganic salts, polyethylene glycol, povidone, and glycerol. One kind can be selected, or a combination of multiple kinds can be selected.

[0096] The emulsifier of the present application may be selected from, including but not limited to, span, tween, myrj, benzalkonium, glycerol fatty acid esters, higher fatty acid salts, sulfates, sulfonates, gum arabic, tragacanth, gelatin, pectin, phospholipids, agar, sodium alginate, hydroxides, silicon dioxide, and bentonite. One kind can be selected, or a combination of multiple kinds can be selected.

[0097] The antioxidant of the present application may be selected from, including but not limited to, sulfites, metabisulfites, bisulfites, ascorbic acid, gallic acid and its esters. One kind can be selected, or a combination of multiple kinds can be selected.

[0098] The metal chelating agent of the present application may be selected from, including but not limited to, disodium ethylenediaminetetraacetate and polycarboxylic acid compounds. One kind can be selected, or a combination of multiple kinds can be selected.

[0099] The inert gas of the present application may be selected from, including but not limited to, nitrogen and carbon dioxide. One kind can be selected, or a combination of multiple kinds can be selected.

[0100] The preservative of the present application is selected from parabens, organic acids and their salts, quaternary ammonium compounds, chlorhexidine acetate, alcohols, phenols, and volatile oils. One kind can be selected, or a combination of multiple kinds can be selected.

[0101] The local anesthetic of the present application is selected from benzyl alcohol, chlorobutanol, lidocaine, and procaine. One kind can be selected, or a combination of multiple kinds can be selected.

[0102] The pH regulator of the present application is selected from hydrochloric acid, sulfuric acid, phosphoric acid, citric acid, tartaric acid, acetic acid, sodium hydroxide, sodium bicarbonate, ethylenediamine, meglumine, phosphates, acetates, and citrates. One kind can be selected, or a combination of multiple kinds can be selected.

[0103] The isotonic or isosmotic regulator of the present application is selected from glucose, sodium chloride, sodium citrate, sorbitol, and xylitol. One kind can be selected, or a combination of multiple kinds can be selected.

[0104] In the present invention, "anti" includes aspects such as prevention, treatment, and adjuvant treatment. As used herein, "prevention and treatment" means alleviating, delaying progression, attenuation, prevention, or maintaining an existing disease or disorder. "Prevention and treatment" also includes curing one or more symptoms of a disease or disorder, preventing its development, or alleviating it to a certain extent.

[0105] In the present invention, "drug" includes any agent, compound, composition or mixture that provides physiological and / or pharmacological effects in vivo or in vitro, and often provides beneficial effects. The scope of the physiological and / or pharmacological effects of "drug" in vivo is not particularly limited, and it can be a systemic effect or only a local effect. The activity of the "drug" is not particularly limited, and it can be an active substance that can interact with other substances, or it can be an inert substance that does not interact.

[0106] In the present application, treatment refers to alleviating the symptoms of coronavirus infection in a subject under the condition of administering an effective dose.

[0107] In this application, "therapeutically effective amount" refers to the dosage of the preparation, the minimum amount necessary to improve, cure or treat one or more symptoms of a disease or condition. The "therapeutically effective amount" will vary depending on the specific state, disease, disorder or condition being treated and its severity, as well as the age, weight, physical condition and responsiveness of the individual being treated. Therefore, one or more of these parameters can be used to select and adjust the therapeutically effective amount of Huatuo Zaizao Pills.

[0108] In this application, "subject" refers to a patient taking a drug.

[0109] In the present application, "subject" is an animal, preferably a mammal, more preferably a human, and the subject includes but is not limited to a patient with a disease, condition and / or symptom. The subject in the present invention is preferably a mammal. The term "mammal" mainly refers to warm-blooded vertebrate mammals, including but not limited to: such as cats, dogs, rabbits, bears, foxes, wolves, monkeys, deer, mice (such as rats, mice), pigs, cattle, sheep, horses, humans, etc., preferably primates, more preferably humans.

[0110] In the present application, "patient" refers to an animal, preferably a mammal, such as a human.

[0111] The third aspect of the present application

[0112] The present application provides an anti-coronavirus drug, which includes the indole compound or its isomer described in the first aspect and a pharmaceutically acceptable excipient.

[0113] In one example, the coronavirus includes severe acute respiratory syndrome coronavirus 2, severe acute respiratory syndrome coronavirus, Middle East respiratory syndrome coronavirus, or bat SARS-like coronavirus.

[0114] In the third aspect of the present application, “antibody”, “drug”, “dosage form”, “therapeutically effective amount”, “subject” and “patient” are as defined in the second aspect.

[0115] The fourth aspect of the present application

[0116] The present application provides a method for preparing the indole compound or its isomer described in the first aspect. The preparation method includes the step of preparing Compound 2 and the step of preparing Compound RU-75, Compound RU-75A or Compound RU-75A' using the Compound 2;

[0117] Wherein,

[0118] The step of preparing Compound 2 includes: mixing Compound 1, ethyl formate, triethylamine and methanol for a first-stage reaction to prepare a first intermediate; mixing the first intermediate, phosphorus oxychloride and dichloromethane for a second-stage reaction to prepare Compound 2;

[0119] The step of preparing Compound RU-75 using the Compound 2 includes:

[0120] Mixing the Compound 2, 2-amino-4-nitrobenzoic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide and dimethylformamide for reaction to prepare Compound 3;

[0121] Mixing the Compound 3, iron powder, ethanol and ammonium chloride for reaction to prepare Compound 4;

[0122] Mixing the Compound 4, bromobenzene, palladium acetate, XPhos, cesium carbonate and 1,4-di alkane for reaction to prepare Compound RU-75;

[0123] The step of preparing Compound RU-75A or Compound RU-75A' using the Compound 2 includes:

[0124] Mixing the Compound 2, 2-amino-4-nitrobenzoic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide and dimethylformamide for reaction to prepare Compound 5;

[0125] Mixing the Compound 5, iron powder, ethanol and ammonium chloride for reaction to prepare Compound 6;

[0126] Mixing the Compound 6, bromobenzene, palladium acetate, XPhos, cesium carbonate and 1,4-di alkane for reaction to prepare Compound RU-75A or Compound RU-75A';

[0127]

[0128]

[0129] In one example, the preparation method satisfies one or more of the conditions shown in (1) to (8):

[0130] (1) The conditions for the first-stage reaction include: temperature is 95°C - 105°C (such as 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105°C), and time is 15h - 20h (such as 15, 16, 17, 18, 19, 20h);

[0131] (2) The conditions for the second-stage reaction include: temperature is 0°C - 5°C (such as 0, 1, 2, 3, 4, 5°C), and time is 1.5h - 2.5h (such as 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5h);

[0132] (3) In the step of preparing Compound 3, the reaction conditions include: temperature is 75°C - 85°C (such as 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85°C), and time is 15h - 20h (15, 16, 17, 18, 19, 20h);

[0133] (4) In the step of preparing Compound 4, the reaction conditions include: temperature is 75°C - 85°C (such as 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85°C), and time is 0.5h - 1.5h (such as 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5h);

[0134] (5) In the step of preparing Compound RU-75, the reaction conditions include: temperature is 95°C - 105°C, and time is 15h - 25h (such as 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25h);

[0135] (6) In the step of preparing Compound 5, the reaction conditions include: temperature is 75°C - 85°C (such as 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85°C), and time is 15h - 25h (such as 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25h);

[0136] (7) In the step of preparing Compound 6, the reaction conditions include: temperature is 75°C - 85°C (such as 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85°C), and time is 0.5h - 1.5h (such as 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5h); and,

[0137] In the step of preparing compound RU-75A, the reaction conditions include: the temperature is 95°C - 105°C, and the time is 15h - 25h (such as 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25h).

[0138] The fifth aspect of the present application

[0139] The present application provides a method for treating coronavirus infection, which includes administering to a subject a therapeutically effective amount of a drug containing the indole compound or its isomer in claim 1.

[0140] In one example, the administration methods of the drug include but are not limited to: oral, rectal, parenteral (intravenous, intramuscular or subcutaneous) injection, and topical administration, inhalation.

[0141] In one example, the drug can be administered orally, by enema or parenterally.

[0142] In one example, the dosing period of the drug can be intermittent dosing, periodic dosing, continuous dosing or long-term dosing.

[0143] "Subject" and "therapeutically effective amount" are defined as in the second aspect.

[0144] The embodiments of the present invention will be described in detail below in conjunction with the examples. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. For the experimental methods without specific conditions in the following examples, the guidance given in the present invention is preferably referred to, and it can also be carried out according to the experimental manuals or conventional conditions in the art, or according to the conditions recommended by the manufacturer, or referring to the experimental methods known in the art.

[0145] In the following specific examples, for the measurement parameters of the raw material components, if there is no special description, there may be slight deviations within the weighing accuracy range. For the temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

[0146] Example 1: Preparation of indole compounds RU-75 and RU-75A

[0147] 1. The synthesis route of RU-75 is as Figure 1 . The reaction conditions for each step are as follows:

[0148] Step I: (1) Ethyl formate, triethylamine, methanol, 100°C, 16 hours; (2) Phosphorus oxychloride, dichloromethane, 0 - 5°C, 2 hours;

[0149] Step II: 2-Amino-4-nitrobenzoic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, dimethylformamide, 80°C, 16 hours;

[0150] Step III: Iron powder, ethanol, ammonium chloride, water, 80 °C, 1 hour;

[0151] Step IV: Bromobenzene, palladium acetate, XPhos, cesium carbonate, 1,4 - dibutane, 100 °C, 20 hours. Alkane, 100 °C, 20 hours.

[0152] The 1H NMR data of the obtained compound RU - 75 ( Figure 3 ) are as follows:

[0153] 1H NMR data of RU - 75: 1 1H NMR: (400 MHz, DMSO - d 6 ) δ 3.15 (2H, t, J = 8.0 Hz), 4.40 (2H, t, J = 8.0 Hz), 7.03 - 7.09 (3H, m), 7.19 - 7.26 (1H, m), 7.25 - 7.28 (3H, m), 7.37 - 7.40 (2H, m), 7.45 - 7.47 (1H, m), 7.62 (1H, d, J = 8.0 Hz), 7.96 (1H, d, J = 8.0 Hz), 8.93 (1H, br), 11.78 (1H, br).

[0154] 2. The synthetic route of RU - 75A is as Figure 2 . The reaction conditions for each step are as follows:

[0155] Step I: (1) Ethyl formate, triethylamine, methanol, 100 °C, 16 hours; (2) Phosphorus oxychloride, dichloromethane, 0 - 5 °C, 2 hours;

[0156] Step II: 2 - Amino - 4 - nitrobenzoic acid, 1 - ethyl - (3 - dimethylaminopropyl) carbodiimide, dimethylformamide, 80 °C, 16 hours;

[0157] Step III: Iron powder, ethanol, ammonium chloride, water, 80 °C, 1 hour;

[0158] Step IV: Bromobenzene, palladium acetate, XPhos, cesium carbonate, 1,4 - dibutane, 100 °C, 20 hours. Alkane, 100 °C, 20 hours.

[0159] The 1H NMR data of the obtained compound RU - 75A ( Figure 4 ) are as follows:

[0160] 1H NMR data of RU - 75A: 1 1H NMR: (400 MHz, DMSO - d 6)δ3.13(2H,t,J=8.0Hz),4.38(2H,t,J=8.0Hz),7.05 - 7.11(3H,m),7.21 - 7.23(1H,m),7.25 - 7.28(3H,m),7.37 - 7.40(2H,m),7.45 - 7.47(1H,m),7.62(1H,d,J=8.0Hz),7.96(1H,d,J=8.0Hz),9.02(1H,br),11.79(1H,br).

[0161] Example 2. Inhibitory Effects of Compounds RU - 75 and RU - 75A on SARS - CoV - 2 Spike Pseudotyped Viruses

[0162] (1) Experimental Principle

[0163] Pseudoviruses are a type of chimeric virus particles, which are chimeric virus particles that express the recombinant glycoprotein of another virus on the surface of a replication - defective virus (virus vector). Replacing the envelope glycoprotein in the virus vector with the S protein of the novel coronavirus forms pseudoviruses that mimic the infection of SARS - CoV - 2. If a compound can inhibit the invasion and infection of pseudoviruses, it highly likely means that the compound can also inhibit the invasion of the novel coronavirus into host cells.

[0164] (2) Experimental Materials

[0165] High - glucose Dulbecco's Modified Eagle Medium (DMEM) was purchased from Gibco (UK). Fetal Bovine Serum (FBS) was purchased from Gibco (Brazil). Penicillin and Streptomycin (Pen / Strep) were purchased from Gibco. Trypsin - EDTA (1x) was purchased from Gibco. The luciferase detection system was purchased from Promega. Protein Assay Dye Reagent Concentration was purchased from Bio - Rad. Bovine Serum Albumin (BSA) was purchased from Sigma. The microplate reader was purchased from Thermo Scientific. Microplate Luminometer was purchased from GLOMAX.

[0166] (3) Experimental Procedures

[0167] Cell culture: HEK293T cells were cultured in high - glucose Dullbecco's modified Eagle medium (DMEM) containing 10% (v / v) fetal bovine serum (FBS) and 1% (w / w) Pen / Strep. The HEK293T cell line overexpressing ACE - 2 was transfected with the pcDNA3.1 - hACE2 plasmid.

[0168] Generation and collection of SARS-CoV-2 pseudovirus: HEK293T cells were co-transfected with plasmids NR-52514, NR-52516, NR-52517, NR-52518, and NR-52519 to generate SARS-CoV-2 Spike pseudotyped virus particles. The pseudovirus in the supernatant was collected 48 hours after transfection, filtered through a 0.45-μm pore size filter, and stored at -80 °C.

[0169] Table 1. Types and functions of plasmids co-transfected to establish the SARS-CoV-2 Spike pseudotyped virus model

[0170] Plastid type Embed BEI Resources product number Viral Entry Protein Spike Glycoprotein NR-52514 Lentiviral Backbone Luc2; ZsGreen NR-52516 Helper Plasmid Gag; pol NR-52517 Helper Plasmid Tat1b NR-52518 Helper Plasmid Rev1b NR-52519

[0171] Pseudovirus invasion assay: HEK293T cells overexpressing ACE-2 were seeded in 48-well plates, and 100 μL of pseudovirus (along with test samples) and 400 μL of medium were added to each well and incubated overnight. The medium was replaced with a mixture of DMEM, FBS, and Pen / Strep, and after culturing for 48 hours, it was washed with 1×PBS and then luciferase activity was measured.

[0172] Using neutralizing antibody (A19215) as the positive control, solvent blank and no-pseudovirus groups as negative controls. The test samples were RU-75, RU-75A, and rutaecarpine. The final concentrations of the test samples were controlled to be from 0.1 μM to 10 μM. The luciferase activity was standardized with the luciferase activity detection data of the solvent blank, and the percentage of virus inhibition by the samples was calculated. Each sample was added to three wells, and the average value was taken. The experiment was repeated three times.

[0173] (4) Experimental results

[0174] In the experiment, the neutralizing antibody A19215 solution was used as the positive control. The results are as Figure 5 shown. This antibody was able to exhibit significant inhibitory ability against the pseudovirus channel, indicating the effectiveness and stability of this test method. In addition, the newly designed indole compounds RU-75 and RU-75A also showed excellent inhibitory effects in the concentration range of 0.1 - 10 μM and demonstrated a good dose-effect curve. It is worth noting that in the same concentration range, RU-75 and RU-75A showed a better inhibition rate than the lead compound rutaecarpine, which further indicates that RU-75 and RU-75A can show better effects in inhibiting the invasion of SARS-CoV-2 into host cells at the cellular level.

[0175] Example 3. Cytotoxicity experiment

[0176] 3.1. Method

[0177] The cytotoxic effects of rutaecarpine, RU-75, and RU-75A on human HEK293T cells were observed using the MTT colorimetric assay. HEK293T cells were seeded in 96-well culture plates at a density of 5.0×10 3 cells / mL, 100 μL per well. After the cells grew to 80% confluence, different concentrations of rutaecarpine, RU-75, and RU-75A (0.1, 1, 5, 10, 25 μM) were added to the wells. After 48 hours, 10 μL of MTT solution was added to each well, and the cells were further cultured at 37°C for 4 hours and then terminated. The culture supernatant was discarded, 150 μL of DMSO was added to each well, and the mixture was shaken for 10 min to dissolve completely. The absorbance (OD) value of each well was measured at a wavelength of 490 nm using an enzyme-linked immunosorbent assay (ELISA) reader. The wells without drugs were used as controls, and the cell viability was calculated according to the following formula: Cell viability (%) = (OD sample - OD control) / OD control × 100%.

[0178] 3.2. Results and conclusions

[0179] The results are as Figure 6 shown. In the concentration range of 0.1–25 μM, none of the three compounds showed obvious toxicity in the MTT toxicity assay, indicating that compared with rutaecarpine, RU-75 and RU-75A have enhanced medicinal properties while the compound toxicity has no obvious change.

[0180] Example 4. Analysis of the affinity of RU-75 and RU-75A for spike protein using molecular docking technology

[0181] (1) Experimental principle

[0182] RBD is the region where the spike protein of the virus binds to human cell ACE2. If a chemical component can bind to RBD, it can reduce the chance of the virus binding to human ACE2, thereby preventing human infection with the novel coronavirus. Calculate the binding energy of the chemical component to RBD. The more negative the binding energy, the stronger the binding force to the target protein and the higher the potential efficacy of inhibiting the novel coronavirus.

[0183] (2) Experimental operation and results

[0184] Using the SEESAR software, the RBD (residues 438-506) in the spike protein (PDB code: 7T9L) was selected as the target, and then the affinity of RU-75 and RU-75A for this target was detected.

[0185] The results are as Figure 7As shown, in terms of binding energy, the binding efficiency of rutaecarpine to RBD is -10.2 KJ / mol, while the binding efficiencies of RU-75 and RU-75A to RBD are -16.0 and -12.8 KJ / mol respectively. This proves that RU-75 and RU-75A can not only bind effectively to the spike protein, but also have a higher potential inhibitory efficiency than the lead compound rutaecarpine, thus more efficiently inhibiting the binding of the novel coronavirus to the ACE2 receptor and preventing the virus from attacking host cells. And this result is also consistent with and corroborates Example 2 above.

[0186] Example 5

[0187] This example relates to the application of indole compounds RU-75 and RU-75A in the preparation of anti-SARS-CoV-2 drugs, and the dosage form of the drug is a granule.

[0188] Example 6

[0189] This example relates to the application of indole compounds RU-75 and RU-75A in the preparation of anti-SARS-CoV-2 drugs, and the dosage form of the drug is a powder.

[0190] Example 7

[0191] This example relates to the application of indole compounds RU-75 and RU-75A in the preparation of anti-SARS-CoV-2 drugs, and the dosage form of the drug is a capsule.

[0192] Example 8

[0193] This example relates to the application of indole compounds RU-75 and RU-75A in the preparation of anti-SARS-CoV-2 drugs, and the dosage form of the drug is a tablet.

[0194] Example 9

[0195] This example relates to the application of indole compounds RU-75 and RU-75A in the preparation of anti-SARS-CoV-2 drugs, and the dosage form of the drug is a tea.

[0196] In summary, the inventors found in previous studies that rutaecarpine (RU) can inhibit the invasion of SARS-CoV-2 pseudovirus; reduce the binding of spike protein to angiotensin-converting enzyme 2 (ACE2); and can inhibit 3CL protease, thereby playing an inhibitory role against coronaviruses. Based on this discovery, the present invention uses rutaecarpine as a lead compound, conducts chemical structure modification, and designs and synthesizes novel indole compounds RU-75 and RU-75A. It is found that the two indole compounds RU-75 and RU-75A synthesized by us have antiviral activity but no cytotoxicity, and bind better to the RBD region of the spike protein. And their antiviral activity is superior to that of the lead compound rutaecarpine. It provides new ideas and solutions for drugs against coronavirus infection. The present invention relates to the application of two new indole compounds against SARS-CoV-2, mainly to the application of compounds RU-75 and RU-75A in the preparation of drugs against coronaviruses. Through a large number of screenings, the inventors of the present invention found and synthesized that RU-75 and RU-75A have strong effects on inhibiting coronavirus infection and against SARS-CoV-2, and their effects are significantly superior to those of their lead compound rutaecarpine. Preparing them into drugs against coronaviruses can provide new ideas for preventing and / or treating coronaviruses.

[0197] The technical features of the above-described embodiments and examples can be combined in any suitable manner. For the sake of brevity of description, not all possible combinations of the technical features in the above-described embodiments and examples are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered to be within the scope described in this specification.

[0198] The above-described examples only represent several embodiments of the present invention, which are convenient for understanding the technical solutions of the present invention specifically and in detail, but should not be construed as limiting the protection scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and the equivalent forms obtained also fall within the protection scope of this application. It should also be understood that the technical solutions obtained by those skilled in the art through logical analysis, reasoning or limited experiments based on the technical solutions provided by the present invention are all within the protection scope of the appended claims of the present invention. Therefore, the protection scope of this invention patent should be subject to the content of the appended claims, and the specification and drawings can be used to explain the content of the claims.

Claims

1. Indole compounds, characterized in that, the indole compound is compound RU-75 or RU-75A; 2. Use of the indole compound according to claim 1 in the preparation of a drug against coronaviruses.

3. The use according to claim 2, characterized in that, the coronaviruses include severe acute respiratory syndrome coronavirus 2, severe acute respiratory syndrome coronavirus, Middle East respiratory syndrome coronavirus or bat SARS-like coronavirus.

4. The use according to claim 2 or 3, characterized in that, the drug comprises the indole compound and a pharmaceutically acceptable excipient.

5. The use according to claim 2 or 3, characterized in that, the dosage form of the drug includes ointment, patch, solution, suspension, emulsion, cream, spray, drop, infusion, granule, capsule, tablet, pill, tea or tube feeding preparation.

6. A drug against coronaviruses, characterized in that, the drug comprises the indole compound according to claim 1 and a pharmaceutically acceptable excipient.

7. The drug against coronaviruses according to claim 6, characterized in that, the coronaviruses include severe acute respiratory syndrome coronavirus 2, severe acute respiratory syndrome coronavirus, Middle East respiratory syndrome coronavirus or bat SARS-like coronavirus.

8. The drug against coronaviruses according to claim 6 or 7, characterized in that, the dosage form of the drug includes ointment, patch, solution, suspension, emulsion, cream, spray, drop, infusion, granule, capsule, tablet, pill, tea or tube feeding preparation.

9. Preparation method of the indole compound according to claim 1, characterized in that, the preparation method includes the step of preparing compound 2 and the step of preparing the compound RU-75 or the compound RU-75A with the compound 2; wherein, the step of preparing compound 2 includes: mixing compound 1, ethyl formate, triethylamine and methanol for a first-stage reaction to prepare a first intermediate; mixing the first intermediate, phosphorus oxychloride and dichloromethane for a second-stage reaction to prepare compound 2; the step of preparing the compound RU-75 with the compound 2 includes: mixing the compound 2, 2-amino-4-nitrobenzoic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide and dimethylformamide for reaction to prepare compound 3; mixing the compound 3, iron powder, ethanol and ammonium chloride for reaction to prepare compound 4; Mix the compound 4, bromobenzene, palladium acetate, XPhos, cesium carbonate and 1,4 - di alkane to carry out a reaction to prepare the compound RU - 75; the step of preparing the compound RU-75A with the compound 2 includes: mixing the compound 2, 2-amino-4-nitrobenzoic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide and dimethylformamide for reaction to prepare compound 5; mixing the compound 5, iron powder, ethanol and ammonium chloride for reaction to prepare compound 6; Mix the compound 6, bromobenzene, palladium acetate, XPhos, cesium carbonate and 1,4-dialkane for reaction to prepare the compound RU-75A; ​ 10. Preparation method of the indole compound or its isomer according to claim 9, characterized in that, the preparation method meets one or more of the conditions shown in (1) to (8): (1) The conditions for the first-stage reaction include: temperature is 95°C - 105°C, and time is 15 h - 20 h; (2) The conditions for the second-stage reaction include: temperature is 0°C - 5°C, and time is 1.5 h - 2.5 h; (3) In the step of preparing Compound 3, the reaction conditions include: temperature is 75°C - 85°C, and time is 15 h - 20 h; (4) In the step of preparing Compound 4, the reaction conditions include: temperature is 75°C - 85°C, and time is 0.5 h - 1.5 h; (5) In the step of preparing Compound RU-75, the reaction conditions include: temperature is 95°C - 105°C, and time is 15 h - 25 h; (6) In the step of preparing Compound 5, the reaction conditions include: temperature is 75°C - 85°C, and time is 15 h - 25 h; (7) In the step of preparing Compound 6, the reaction conditions include: temperature is 75°C - 85°C, and time is 0.5 h - 1.5 h; and (8) In the step of preparing Compound RU-75A, the reaction conditions include: temperature is 95°C - 105°C, and time is 15 h - 25 h.

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