TWI680973B - Neutrophil inflammation inhibitor and uses thereof - Google Patents

Abstract

Disclosed herein are compounds of formula (I), and pharmaceutical compositions comprising the same. The compounds of formula (I) are neutrophilic inflammation inhibitors, thus, they are useful for treatment and/or prophylaxis of inflammatory diseases and/or disorders associated with abnormal activation of neutrophils, such as ARDS, ALI, COPD, lung fibrosis, chronic bronchitis, pulmonary emphysema, α-1 anti-trypsin deficiency, cystic fibrosis, idiopathic pulmonary fibrosis, liver injury, steatohepatitis, liver fibrosis, damages caused by ischemia and reperfusion, myocardial infarction, shock, stroke, and organ transplantation, ulcerative cholitis, vasculitis, SLE, sepsis, SIRS, arthritis, psoriasis, atopic dermatitis, and inflammatory skin diseases.

Description

Neutrophilic leukocyte inflammation inhibitor and its use

This disclosure relates generally to neutrophil inflammation inhibitors and the use of this inhibitor to treat or prevent neutrophil-related diseases and/or abnormalities.

Neutrophils are a group of white blood cells that contain a host's defense system against invading bacteria. In response to soluble inflammatory mediators released by cells in the injured area, neutrophils migrate from the blood across the blood vessel wall into the tissue. At the injury site, activated neutrophils will kill foreign cells by phagocytosis and release of cytotoxins (such as oxides, proteases, cytokines, etc.). Although neutrophils are very important to fight infection, neutrophils themselves can also cause damage to tissues. During an abnormal inflammatory response, neutrophils can cause significant tissue damage due to the release of toxic substances to the blood vessel wall or undamaged tissue. Or, neutrophils adhering to the microvessel wall can cause hypoxia and damage the tissue. Such abnormal inflammatory reactions have been considered to be related to the etiology of various clinical diseases, including but not limited to, adult respiratory distress syndrome (Adult respiratory distress syndrome (ARDS), acute lung injury (ALI), chronic obstructive pulmonary disease (COPD), pulmonary fibrosis, liver injury, steatohepatitis, liver fibrosis, ischemia and relapse Perfusion-induced injury, myocardial infarction, shock, stroke, organ transplantation, ulcerative colitis, vasculitis, systemic lupus erythematosus (SLE), sepsis, systemic inflammatory response syndrome (SIRS) ), arthritis, psoriasis, atopic dermatitis, and inflammatory skin diseases.

In view of this, there is an urgent need for a therapeutic agent that can suppress the abnormal activation of neutrophils in order to effectively prevent and/or treat inflammatory diseases and/or diseases related thereto.

This disclosure is based on the unexpected discovery that certain new synthetic compounds are potent inhibitors of neutrophil inflammation, which can antagonize the inflammation caused by the abnormal activation of neutrophils, so these compounds can be used as lead compounds to develop drugs, For the treatment of inflammatory diseases and/or diseases related to abnormal activation of neutrophils, such as ARDS, ALI, COPD, pulmonary fibrosis, chronic bronchitis, emphysema, alpha-1 antitrypsin deficiency, cystic fibers Metastasis, cryptogenic fibrosis alveolitis, liver injury, fatty hepatitis, liver fibrosis, injury caused by ischemia and reperfusion, myocardial infarction, shock, stroke, organ transplantation, ulcerative colitis, vasculitis, SLE, sepsis, SIRS, arthritis, psoriasis, atopic dermatitis, and inflammatory skin diseases.

其中： X是N或O； R ₁是氫、烷基或不存在，其中當X是O時，R ₁不存在，且當X是N時，R ₁是氫或烷基； R ₂、R ₃、R ₄及R ₅係獨立為氫、羥基、氫硫基、鹵素、烷基、鹵烷基、-OR ₆、-SR ₆、-(C=O)R ₆、或-COOH； R ₆是烷基或鹵烷基。 Therefore, the first aspect of the present disclosure is to provide a new compound having the structure of formula (I), a salt or solvate thereof:

Where: X is N or O; R ₁ is hydrogen, alkyl or absent, wherein when X is O, R ₁ does not exist, and when X is N, R ₁ is hydrogen or alkyl; R ₂ , R ₃ , R ₄ and R ₅ are independently hydrogen, hydroxyl, hydrogenthio, halogen, alkyl, haloalkyl, -OR ₆ , -SR ₆ , -(C=O)R ₆ , or -COOH; R ₆ It is alkyl or haloalkyl.

According to a preferred embodiment of the present disclosure, in formula (I), X is N; R ₁ is methyl; R ₂ , R ₃ and R ₅ are independently hydrogen; and R ₄ is hydroxyl.

The disclosure also covers a pharmaceutical composition suitable for treating or preventing diseases caused by abnormal activation of neutrophils. The disclosed pharmaceutical composition comprises the compound of formula (I), its salts or solvates; and a pharmaceutically acceptable excipient.

Also covered by this disclosure is a method for treating and/or preventing an individual with or suspected of having an inflammatory disease and/or a disease associated with abnormal activation of neutrophils. The method of the present invention comprises administering to the individual a therapeutically or prophylactically effective amount of a compound of formula (I), a salt or solvate thereof, to reduce, alleviate and/or prevent the inflammatory disease and/or neutrophils Symptoms of diseases related to abnormal activation of white blood cells.

According to an embodiment of the present disclosure, the inflammatory diseases and/or diseases related to abnormal activation of neutrophils are selected from the group consisting of ARDS, ALI, COPD, pulmonary fibrosis, chronic bronchitis, emphysema , Α-1 antitrypsin deficiency, cystic fibrosis, cryptogenic fibrosis alveolitis, liver injury, steatohepatitis, liver fibrosis, injury caused by ischemia and reperfusion, myocardial infarction, shock, Stroke, organ transplantation, ulcerative colitis, vasculitis, SLE, sepsis, SIRS, arthritis, psoriasis, atopic dermatitis, and inflammatory skin diseases.

According to a preferred embodiment of the present disclosure, the inflammatory disease and/or abnormality is ARDS.

According to another preferred embodiment of the present disclosure, the inflammatory disease and/or abnormality is ALI.

According to yet another preferred embodiment of the present disclosure, the inflammatory disease and/or abnormality is psoriasis.

According to some preferred embodiments of the present disclosure, the amount of the compound of formula (I) administered to the individual is 0.001-100 mg/kg.

According to some preferred embodiments of the present disclosure, the individual is a human.

The details of one or more embodiments of the present disclosure are described in the description of the invention. Through these descriptions and the accompanying claims, other features and advantages of the present disclosure will be more clearly understood.

It should be noted that the foregoing general description and the following detailed description are examples only for the purpose of further explaining the content of the present invention.

In order to make the description of this disclosure more detailed and complete, the following provides an illustrative description of the implementation form and specific embodiments of the present invention; however, this is not the only form for implementing or using specific embodiments of the present invention. The embodiments cover the features of multiple specific embodiments, as well as the method steps and their sequence for constructing and operating these specific embodiments. However, other specific embodiments can also be used to achieve the same or equal functions and sequence of steps.

1. Definition

The term "alkyl" includes substituted and unsubstituted linear, branched, and cyclic alkyl groups. Unless otherwise indicated, each alkyl group may be individually and selectively substituted with one or more substituents to become an unsubstituted alkyl group or an alkyl group with one or more substituents. When a chemical structure or part is described as "substituted", it means that one or more hydrogen atoms of the structure or part are replaced by one or more groups such as halogen, haloalkyl, or hydroxyl The chemical structure produced by the substitution or the derivative of the moiety. In certain embodiments, the alkyl group is an alkyl group without a substituent, especially an alkyl group (or a lower carbon number alkyl group) having 1 to 6 carbon atoms, such as methyl, ethyl, propyl, Butyl and so on. In other specific embodiments, the alkyl group is an alkyl group containing at least one substituent, such as a haloalkyl group.

"Halogen (halo or halogen)" means fluorine, chlorine, bromine or iodine.

"Haloalkyl" refers to an alkyl group as defined above, having one, two, three, four, five, six or more halogen atoms on it. Haloalkyl includes fluoroalkyl, chloroalkyl, bromoalkyl and iodoalkyl. "Fluoroalkyl" refers to an alkyl group having one, two, three, four, five, six, or more fluorine atoms thereon. The haloalkyl group may have more than one halogen atom. For example, the fluoroalkyl group includes an alkyl group having one or more fluorine atoms and one or more chlorine atoms. The haloalkyl group may have one or more substituents other than halogen.

"Alkoxy" refers to a group having the chemical structure -OR', wherein R'may be the aforementioned alkyl group having a selective substituent.

The above substituents are only examples, and the scope of the present invention is not limited to the foregoing.

The term "salts" here refers to pharmaceuticals suitable for contacting human tissues and lower animals in the medical field without causing unnecessary toxicity, irritation, allergies and other adverse reactions, and having a reasonable benefit/risk ratio Acceptable salts. Pharmaceutically acceptable salts are well known in the relevant art. The pharmaceutically acceptable salts of the compound of the present invention are prepared by reacting the compound with a suitable inorganic acid or organic acid or base. Examples of pharmaceutically acceptable acid addition salts of non-toxic acids include amino groups and inorganic acids (eg, hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and hypochlorous acid) or organic acids (For example, acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid) or acid addition salts formed by other methods known in the art (eg, ion exchange) . Other pharmaceutically acceptable salts include, but are not limited to, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate Salt, camphorate, camphorsulfonate, citrate, cyclopentapropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate Salt, gluconate, glycerol phosphate, gluconate, hemisulfate, enanthate, hexanoate, hydroiodic acid, 2-hydroxyethanesulfonate, lactobionate, lactate, laurate, Lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, Oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionic acid Salt, stearate, succinate, sulfate, tartrate, thiocyanate, paratoluenesulfonate, undecanoate, valerate, etc. Salts produced by reaction with appropriate bases include alkali metal salts, alkaline earth metal salts, appropriate ammonium salts and N ⁺ (C _1-4 alkyl) ₄ ^- salts. Representative alkali metal salts and alkaline earth metal salts include sodium salts, lithium salts, potassium salts, calcium salts, magnesium salts, and the like. Other pharmaceutically acceptable salts include appropriately non-toxic ammonium salts, quaternary ammonium salts, and the use of relative ions (such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, lower alkyl sulfate, Salts formed by the reaction of ammonium cations formed with arylsulfonate).

The term "solvate" here refers to the state formed by a compound and its surrounding solvent molecules (such as water, ethanol, etc.) through a solvolysis reaction (solvolysis). This physical state is usually through hydrogen Key formation. Common solvents include water, methanol, ethanol, acetic acid, dimethyl sulfoxide (DMSO), tetrahydrofuran (THF), diethyl ether and the like. The disclosed compound can be made into a crystalline form and can be dissolved by a solvent. Suitable solvents include pharmaceutically acceptable solvents, and further include both stoichiometric and non-stoichiometric solvents. In some examples, the solvate may be isolated, for example, when one or more solvent molecules are incorporated into the crystalline lattice. The term "solvate" here encompasses two major categories of solvent phase solvates and solvates that can be isolated. Representative solvates such as hydrates, ethanolates and methanolates.

The term "hydrate" refers to a compound associated with water molecules. Generally speaking, a certain number of water molecules can be included in the hydrate, so the hydrate of a compound can generally R. xH ₂ O is represented by the general formula, where R is a compound, and x is the number of water molecules, and is greater than zero. A particular compound can form more than one hydrate, such as monohydrate (x=1); low hydrate (x is greater than 0 but less than 1), for example, hemihydrate (R.0.5H ₂ O); multihydrate Substances (x greater than 1), such as dihydrate (R.2H ₂ O) or hexahydrate (R.6H ₂ O).

"Pharmaceutically acceptable excipients" refers to compounds that are compatible with other ingredients in a pharmaceutical composition and do not harm the individual after administration.

Unless otherwise specified, the term "effective amount" refers here to the amount of drug sufficient to produce the desired therapeutic response. For therapeutic purposes, an effective amount also refers to an amount of a compound or composition whose therapeutic benefit exceeds its toxic or deleterious effects. The specific effective amount depends on various factors, such as the specific condition to be treated, the patient's physiological condition (eg, the patient's weight, age, or gender), the type of mammal or animal being treated, the duration of treatment, and current therapy (if available) )) and the specific formulations used and the structure of the compound or its derivatives. The effective amount can be expressed as the total weight of the drug (for example, in grams, milligrams, or micrograms) or as the ratio of the weight of the drug to body weight (the unit is milligrams per kilogram (mg/kg)). Alternatively, the effective amount can be expressed as the concentration of the active ingredient (for example, the compound of formula (I) of the present disclosure), for example, molar concentration, weight concentration, volume concentration, weight molar concentration, molar fraction, weight fraction Rate and mixing ratio. In addition, those skilled in the art can also calculate the human equivalent dose (HED) of drugs (such as the antibodies of the present disclosure) based on the animal model dose. For example, those skilled in the art can refer to the "estimating the maximum safe initial dose of adult healthy volunteers in the initial clinical treatment test" published by the US Food and Drug Administration (FDA). Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers) to estimate the safest dose for human use.

Unless otherwise indicated, the "therapeutically effective amount" of a compound here means sufficient to provide a therapeutic benefit to the treatment or management of a disease, or to delay one or more symptoms associated with the disease or Minimize the amount of this compound. The "therapeutically effective amount" of a compound refers to the amount of the therapeutic agent that can provide therapeutic benefits for the treatment or management of the disease when used alone or in combination with other therapies. The term "therapeutically effective amount" also includes an amount that can improve the overall therapeutic effect, reduce or avoid the symptoms or causes of the disease, or provide another therapeutic agent.

The "prophylatically effective amount" of a compound refers herein to an amount of the compound sufficient to prevent a disease, or prevent one or more symptoms associated with the disease from occurring. The "preventive effective amount" of a compound refers to the amount of the therapeutic agent that is effective for preventing the disease when used alone or in combination with other therapies. The term "preventive effective amount" also includes an amount that can improve the overall preventive effect or enhance the preventive effect of another therapeutic agent.

The term ``treat, treating, treatment'' includes partial or complete prevention, improvement, mitigation, and/or delay of symptoms, secondary disorders, or symptoms of patients suffering from the specific disease ( condition).

The terms "subject" or "patient" are used interchangeably herein to refer to mammals including humans, which can be treated with the compounds of the present invention. "Mammals" means all members of the mammals, including humans, primates, poultry and livestock animals, such as rabbits, pigs, sheep, and cattle, as well as zoo animals, racing animals and pets, and rodents, such as mice And rats. Unless specifically stated otherwise, the term "subject" means both male and female. Therefore, the term "subject" encompasses any mammal that can benefit from the compounds and methods of the present invention. Examples of "subject" or "patient" include, but are not limited to, humans, rats, mice, guinea pigs, monkeys, pigs, sheep, cows, horses, dogs, cats, birds, and birds. In a preferred embodiment, "subject" refers to a human being.

It should be noted that when the stereochemical structure of a structure or a part of a structure is not specifically marked with a thick line or a broken line, the structure or a part of the structure should be interpreted as including all stereoisomers thereof. Similarly, when describing compounds with one or more palm centers, if the stereochemistry of those centers is not specified, they should be interpreted as containing pure stereoisomers and mixtures thereof. In addition, any atom with an unsaturated valence in the diagram should assume that it is combined with enough hydrogen atoms to meet its valence requirements.

Although the numerical ranges and parameters used to define the broader range of the present invention are approximate values, the relevant numerical values in the specific embodiments have been presented as accurately as possible. However, any numerical value inevitably contains standard deviations due to individual test methods. Here, "about" usually means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a specific value or range. Or, the term "about" means that the actual value falls within the acceptable standard error of the average value, depending on the consideration of those with ordinary knowledge in the technical field to which the present invention belongs. Except for experimental examples, or unless clearly stated otherwise, all ranges, quantities, values, and percentages used herein can be understood (for example, to describe the amount of materials, length of time, temperature, operating conditions, quantity ratio, and other similarities All) have been modified by "about". Therefore, unless otherwise stated to the contrary, the numerical parameters disclosed in this specification and the accompanying patent application are approximate values and can be changed as required. At least these numerical parameters should be understood as the indicated significant digits and the values obtained by applying the general rounding method. Here, the numerical range is expressed from one end point to another segment point or between two end points; unless otherwise stated, the numerical range described herein includes end points.

Unless otherwise defined in this specification, singular nouns used in this specification cover the plural form of the noun without conflict with the context; and plural nouns used also cover the singular form of the noun.

2. Neutrophil Leukocyte Inhibitor

The inventors of the present disclosure have unexpectedly discovered that certain synthetic compounds are potent inhibitors of neutrophil inflammation, which can be used to antagonize the inflammatory reaction caused by abnormal activation of neutrophils, so these compounds can be used as lead compounds. Develop drugs suitable for treating diseases associated with abnormal activation of neutrophils.

The disclosed compound is a compound having the structure of formula (I), and its pharmaceutically acceptable salts or solvates:

According to certain embodiments, it may be a compound salt of the structure of formula (I), a solvate, or a hydrate.

In the structure of formula (I), X may be N or O. Alternatively, R ₁ is hydrogen, alkyl or absent, wherein when X is O, R ₁ is absent, and when X is N, R ₁ is hydrogen or alkyl. In addition, R ₁ may or may not have a substituent. In certain instances, R ₁ is methyl.

Alternatively, R ₂ , R ₃ , R ₄ and R ₅ are independently hydrogen, hydroxyl, hydrogenthio, halogen, alkyl, haloalkyl, -OR ₆ , -SR ₆ , -(C=O)R ₆ , Or -COOH; and R ₆ is alkyl or haloalkyl.

According to certain specific embodiments, the compound of formula (I) may be compound 3, wherein X is N, R ₁ is methyl, and R ₂ , R ₃ , R ₄ and R ₅ are independently hydrogen.

According to certain specific embodiments, the compound of formula (I) may be compound 4, wherein X is N, R ₁ is methyl, and R ₂ , R _3, and R ₅ are independently hydrogen, and R ₄ is fluorine.

According to certain specific embodiments, the compound of formula (I) may be compound 7, wherein X is N, R ₁ is methyl, R ₄ is -OR ₆ , wherein R ₆ is methyl, and R ₂ and R ₃ And R ₅ is independently hydrogen.

According to certain specific embodiments, the compound of formula (I) may be compound 8, wherein X is N, R ₁ is methyl, R ₂ is hydrogen, R ₃ , R ₄ and R ₅ are independently -OR ₆ , Where R ₆ is methyl.

According to certain specific embodiments, the compound of formula (I) may be compound 10, wherein X is N, R ₁ is methyl, R ₄ is -(C=O)R ₆ , wherein R ₆ is methyl, and R ₂ , R ₃ and R ₅ are independently hydrogen.

According to certain specific embodiments, the compound of formula (I) may be compound 11, wherein X is N, R ₁ is methyl, R ₂ is hydroxy, and R ₃ , R _4, and R ₅ are independently hydrogen.

According to certain specific embodiments, the compound of formula (I) may be compound 12, wherein X is N, R ₁ is methyl, R ₃ is hydroxy, and R ₂ , R ₄ and R ₅ are independently hydrogen.

According to certain specific embodiments, the compound of formula (I) may be compound 13, wherein X is N, R ₁ is methyl, R ₄ is hydroxy, and R ₂ , R ₃ and R ₅ are independently hydrogen.

According to certain specific embodiments, the compound of formula (I) may be compound 15, wherein X is N, R ₁ is methyl, R ₄ is carboxy, and R ₂ , R ₃ and R ₅ are independently hydrogen.

According to certain specific embodiments, the compound of formula (I) may be compound 16, wherein X is N, R ₁ is methyl, R ₂ is hydrogenthio, and R ₃ , R ₄ and R ₅ are independently hydrogen.

According to certain specific embodiments, the compound of formula (I) may be compound 17, wherein X is N, R ₁ is methyl, R ₄ is chlorine, and R ₂ , R ₃ and R ₅ are independently hydrogen.

According to certain specific embodiments, the compound of formula (I) may be compound 28, wherein X is O, R ₁ is absent, R ₂ is hydroxyl, and R ₃ , R _4, and R _{5 are} independently hydrogen.

According to certain specific embodiments, the compound of formula (I) may be compound 30, wherein X is O, R ₁ is absent, R ₄ is an alkyl group substituted with 3 fluorine atoms (eg, trifluoromethyl), And R ₂ , R ₃ and R ₅ are independently hydrogen.

The compounds of the present invention can selectively inhibit the activity of neutrophils, including the inhibition of one or more of the following neutrophil activities: the release of superoxide anions, the release of reactive oxygen species, the release of bone marrow peroxidase, Elastase release, neutrophil coagulation, adhesion to plastic surface, adhesion to vascular endothelial cells, chemotaxis, and migration across monolayer endothelial cells. In certain embodiments, the compounds of the present invention can inhibit formyl-L-methionyl-L-leucyl-L-phenylalanine, formyl-L-methionyl-L-phenylalanine, fMLF) Superoxide anion (O ₂ ^{• −} ) released from activated neutrophils. In other embodiments, the compounds of the invention can inhibit elastase released from fMLF-activated neutrophils.

The half of the maximum inhibitory concentration (IC ₅₀ ) produced by the compound of formula (I) of the present invention or its salts or solvates directly or indirectly contacting fMLF-activated neutrophils represents the inhibition of the compound of the present invention The degree of superoxide anion formation. According to certain specific embodiments, the compound of the present invention inhibits the superoxide anion generated by neutrophils activated by fMLF with an IC ₅₀ value of up to about 4 nM, up to about 0.5 μM, up to about 5 μM, up to about 8 μM, up to About 10μM. According to other specific embodiments, the compounds of the present invention inhibit fMLF-activated neutrophil-released elastase with an IC ₅₀ value of at least about 30 nM, at least about 2 μM, at least about 5 μM, at least about 10 μM.

3. Method for preparing the compound of the present invention

Any of the compounds disclosed herein can be manufactured by methods commonly used in the related art or methods taught in the examples.

Generally speaking, the pyrazolo[4,3-c]quinoline (ie, compound 2a or 2b of Example 1) and a substituted aniline (for example, 2-methoxyaniline, 4-fluoro Aniline, etc.) are contacted to prepare the compounds disclosed herein (ie, compounds of formula (I)). The contact reaction may be performed in an aprotic solvent, and examples of the aprotic solvent include dimethyl sulfoxide (DMSO), dimethylformamide (DMF), N-methyl-2-pyrrolidone (N-methyl -2-pyrrolidone, NMP), dichloromethane (DCM), tetrahydrofuran (THF), acetone, ethanol, propanol and the like. Thin film chromatography (TLC) or high performance liquid chromatography (HPLC) was used to monitor the progress of the reaction, and the product was identified by mass spectrometry or NMR spectroscopy.

4. Pharmaceutical composition and kit

The disclosure also covers pharmaceutical compositions and kits used to treat and/or prevent inflammatory diseases and/or abnormalities, especially diseases and/or abnormalities caused by abnormal activation of neutrophils. The disclosed pharmaceutical composition comprises a therapeutically effective amount or a prophylactically effective amount of a compound of formula (I) of the present invention (for example, compound 13), a salt or solvate thereof; and a pharmaceutically acceptable excipient .

The weight of the compound of formula (I) of the present invention is about 0.1% to 99% of the total weight of the pharmaceutical composition or drug. In some embodiments, the weight of the compound of formula (I) is at least 1% of the total weight of the pharmaceutical composition or drug. In certain embodiments, the weight of the compound of formula (I) is at least 5% of the total weight of the pharmaceutical composition or drug. In some embodiments, the weight of the compound of formula (I) is at least 10% of the total weight of the pharmaceutical composition or drug. In other embodiments, the weight of the compound of formula (I) accounts for at least 25% of the total weight of the pharmaceutical composition or drug.

The specific pharmaceutical composition is suitable for oral, mucosal (eg, nasal, sublingual, intravaginal, buccal, or rectal), parenteral gastrointestinal (eg subcutaneous, intravenous, single bolus injection, intramuscular injection, or intraarterial A single dosage form administered by injection) or percutaneously. Examples of dosage forms include tablets, and exemplary dosage forms include, but are not limited to, tablets, capsule tablets, capsules (such as soft elastic gelatin capsules), cachets, lozenges, dispersions, suppositories, ointments, Mud, paste, powder, dressing, emulsion, patch, solution, patch, spray (such as nasal spray or inhalation), gel, suitable for oral or mucosal administration to patients Liquid dosage forms (including suspensions (such as water-compatible or insoluble liquid suspensions, oil-in-water emulsions, or water-in-oil emulsions), solutions, and elixirs), suitable for non-oral administration to patients The liquid dosage form and sterile solid (such as crystalline or amorphous solid) can be reconstituted into a liquid dosage form suitable for non-oral administration to the patient.

The dosage form of the present invention can be formulated according to a specific administration route. For example, oral administration requires enteric coating to protect the compound of the present invention from decomposition in the gastrointestinal tract. Similarly, the dosage form may contain additional ingredients by which the active ingredient is delivered to the site of action. For example, the compound can be administered in a liposome dosage form to prevent it from being broken down by enzymes, assist in transport through the individual's circulatory system, and penetrate the cell membrane to the intracellular location.

Similarly, poorly soluble compounds can be formulated with co-solvents, emulsifiers and surfactants into liquid dosage forms (and dosage forms suitable for reconstitution). These co-solvents, emulsifiers and surfactants include, but are not limited to , Cyclodextrin (such as α-cyclodextrin and β-cyclodextrin), and anhydrous solvents, including, but not limited to, alcohol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate Ester, propylene glycol, 1,3-butanediol, dimethylformamide, dimethyl sulfoxide (DMSO), biocompatible oils (e.g. cottonseed oil, peanut oil, corn oil, germ oil, castor oil , Olive oil, sesame oil), glycerin, tetrahydrofuran, polyethylene glycol, sorbitan fatty acid esters and combinations thereof (eg DMSO: corn oil).

The composition, shape and type of the dosage form can be adjusted according to the specific use requirements. For example, compared to the chronic treatment of a disease, when the acute treatment of the same disease is performed, the drug may contain a larger amount of one or more active ingredients. Similarly, non-oral dosage forms may contain a smaller amount of one or more active ingredients when treating the same disease compared to oral dosage forms. Those with ordinary knowledge in the technical field to which the present invention belongs can understand the differences between the specific dosage forms of the present invention. For example, refer to Remington's Pharmaceutical Sciences , 18th ed., Mack Publishing, Easton PA (1990).

4.1 Dosage form suitable for oral administration

The dosage form containing the compound of formula (I) of the present invention can be formulated into discrete dosage forms suitable for oral administration, such as lozenges (eg chewable tablets), capsule-shaped lozenges, capsules and liquids (eg syrup). These dosage forms contain specific amounts of active ingredients and can be prepared according to methods well known to those skilled in the art. For example, refer to Remington's Pharmaceutical Sciences , 18th ed., Mack Publishing, Easton PA (1990).

In general, the traditional technology for manufacturing pharmaceutical compounds can be used to mix the active ingredient with at least one excipient to prepare an oral dosage form. Different types of excipients can be used according to specific administration needs.

Due to the ease of administration, lozenges and capsules are the two most common oral dosage forms. If necessary, tablets can be coated with standard aqueous or non-aqueous techniques. These dosage forms can be prepared by traditional pharmaceutical methods. In general, the active ingredient can be uniformly mixed with a liquid carrier, a finely divided solid carrier, or a combination thereof, and made into a specific shape. A disintegrant can be added to the solid dosage form to assist in rapid solution. Lubricants can also be added to assist in the preparation of dosage forms (e.g. Tablets).

4.2 Suitable for non-oral administration

Non-oral dosage forms can be administered to patients subcutaneously, intravenously (including single full-dose injections), intramuscularly, and intraarterially. Based on this type of administration, the patient's self-defense mechanism against contaminants can be avoided, so non-oral dosage forms are generally sterile, or aseptically processed before administration. Exemplary non-oral dosage forms include, but are not limited to, solutions for direct injection, dry products that can be dissolved or suspended in a pharmaceutically acceptable carrier for injection, suspensions for direct injection, and emulsifiers.

The carrier suitable for preparing the non-oral dosage form of the present invention is a carrier well known to those skilled in the art to which the present invention belongs. Exemplary carriers include, but are not limited to, water, liquid carriers (such as, but not limited to, sodium chloride solution, Ringer's solution, and glucose), water-miscible carriers (such as, but not limited to, ethanol, polyethylene glycol Glycols and polypropylene glycols) and non-aqueous carriers (such as, but not limited to, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate and benzyl benzoate) ).

4.3 Suitable for transdermal, topical and / Or dosage form for mucosal administration

Transdermal, regional, and mucosal dosage forms include, but are not limited to, ophthalmic solutions, sprays, aerosols, emulsions, lotions, ointments, gels, solutions, emulsifiers, suspensions, and others with general knowledge in the field of the invention Well-known form. For example, refer to Remington's Pharmaceutical Sciences , 18th eds., Mack Publishing, Easton PA (1990). Transdermal dosage forms include "reservoir type" or "matrix type" patches, which can be attached to the skin for a specific period of time to promote a specific dose of active ingredients to penetrate the skin.

Suitable excipients (such as carriers and diluents) and other materials that can be used to prepare transdermal, topical, and mucosal dosage forms are substances well known to those of ordinary skill in the pharmaceutical arts, and can be administered to specific tissues and pharmaceutical compositions The proper excipient is used for its dosage form.

Depending on the treatment tissue, additional ingredients may be administered before, simultaneously with, or after administration of the active ingredient of the present invention. For example, penetration enhancers can be used to assist delivery of the active ingredient to the tissue.

The pH of the drug or dosage form can also be adjusted, or the pH of the tissue administered with the drug or dosage form, to improve the delivery of one or more active ingredients. Similarly, the polarity, ionic strength and tension of the solvent carrier can be adjusted to improve the transmission efficiency. Compounds such as stearate can be added to medicines and dosage forms to change the hydrophilicity or lipophilicity of one or more active ingredients, thereby improving delivery efficiency. At this time, the stearate can be used as a lipid carrier of a preparation, an emulsifier or a surfactant, and a delivery enhancer or penetration enhancer. Salts, hydrates or solvates of the active ingredients can be used to further adjust the characteristics of the composition.

4.4 Set

Also included in this disclosure is a manufacturing or "kit" that can be used to treat and/or prevent inflammatory diseases and/or diseases associated with abnormal activation of neutrophils.

In one embodiment, the kit includes a container containing the compound of the present disclosure. The kit is suitable for treating and/or preventing inflammatory diseases, especially diseases related to abnormal activation of neutrophils, such as ARDS, ALI, COPD, pulmonary fibrosis, chronic bronchitis, emphysema, α- 1 Antitrypsin deficiency, cystic fibrosis, cryptogenic fibrosis alveolitis, liver injury, steatohepatitis, liver fibrosis, injury caused by ischemia and reperfusion, myocardial infarction, shock, stroke, organ Transplantation, ulcerative colitis, vasculitis, SLE, sepsis, SIRS, arthritis, psoriasis, atopic dermatitis, and inflammatory skin diseases, etc. Suitable containers include, for example, bottles, vials, syringes, bubble bags, and the like. A variety of different materials, such as glass or plastic, can be used to make the container. The container may contain a pharmaceutically effective amount of the compound of the present invention or a pharmaceutical formulation thereof to treat and/or prevent inflammatory diseases and/or diseases related to abnormal activation of neutrophils; and the container may also have a sterile hole. For example, the container may be an intravenous infusion bag or an intravenous infusion bottle, which is attached with a stopper that can be penetrated by an intravenous needle. The set may further include a label or a copy. The label or copy will indicate what disease and/or condition the composition is used to treat. Alternatively, the kit may further include a second container containing a pharmaceutically acceptable buffer, such as physiological saline, Ringer's solution or glucose solution. The kit may further include other desired materials from a commercial implementation or consumer perspective, such as other buffers, diluents, fillers, needles, and syringes.

The kit may further include instructions for administering the compound of the present invention, or a second formulation for treating and/or preventing inflammatory diseases and/or diseases related to abnormal activation of neutrophils. If the kit includes a first formula containing the compound of the present invention and a second formula, the kit may further include how to administer the first formula simultaneously, continuously or separately to the individual in need And instructions for the second recipe.

In another embodiment, the kit is a solid oral dosage form suitable for administration of the compound of the present invention. For example, the kit contains multiple unit doses. Such kits should include card packaging with unit doses arranged neatly according to the intended use. An example of such a kit is "blister pack". Blister packaging is a unit-dose packaging method well known in the pharmaceutical industry. If necessary, the package will also be provided with numbers, words or other indications indicating when and which unit-dose should be used according to the course of treatment.

According to a particular embodiment, the kit includes at least (a) a first container containing the compound of formula (I) of the present invention; and optionally, (b) a second container containing a second therapeutic agent , Which can be any conventional anti-inflammatory drugs, antibiotics, anti-neoplastic drugs, or cardiovascular drugs; and (c) instructions for use related to the kit to indicate to the user how to use the kit. The instructions can be folded, video, CD, VCD or DVD.

5. treatment method

Since the compounds of the present invention can antagonize the signaling process after the activation of neutrophils, they can be used as lead compounds to develop drugs for the treatment of inflammatory diseases and/or abnormalities (especially diseases related to the abnormal activation of neutrophils) . Therefore, the present disclosure relates to a method for treating or preventing individuals with diseases associated with abnormal activation of neutrophils with compounds of formula (I), including, but not limited to, ARDS, ALI, COPD, pulmonary fibrosis, chronic Bronchitis, emphysema, alpha-1 antitrypsin deficiency, cystic fibrosis, cryptogenic fibrosis alveolitis, liver injury, steatohepatitis, liver fibrosis, injury caused by ischemia and reperfusion , Myocardial infarction, shock, stroke, organ transplantation, ulcerative colitis, vasculitis, SLE, sepsis, SIRS, psoriasis, atopic dermatitis, and inflammatory skin diseases.

The present disclosure therefore includes a method for treating and/or preventing an individual with or suspected of having an inflammatory disease and/or a disease associated with abnormal activation of neutrophils. The method of the present invention comprises administering to the individual a therapeutically effective or prophylactically effective amount of a compound of formula (I), a salt or solvate thereof, to reduce, alleviate and/or prevent the inflammatory disease and/or addiction Symptoms related to abnormal activation of neutral leukocytes.

The disclosed compound of formula (I) can be synthesized or prepared according to the methods provided in the operating examples in this disclosure.

According to the embodiments provided in this disclosure, the diseases and/or abnormalities associated with abnormal activation of neutrophils may be any of the following: ARDS, ALI, COPD, pulmonary fibrosis, chronic bronchitis, emphysema, α- 1 Antitrypsin deficiency, cystic fibrosis, cryptogenic fibrosis alveolitis, liver injury, steatohepatitis, liver fibrosis, injury caused by ischemia and reperfusion, myocardial infarction, shock, stroke, organ Transplantation, ulcerative colitis, vasculitis, SLE, sepsis, SIRS, psoriasis, atopic dermatitis, and inflammatory skin diseases.

The treatment method can also be used together with other treatment methods that can treat the aforementioned diseases today. Therefore, this method also includes before, during or after the administration of the compound of formula (I) to an individual in need of treatment, another administration for the treatment of diseases associated with abnormal activation of neutrophils Pharmacy. This other conventional agent may be an anti-inflammatory agent (eg, cholesterol), an antibiotic (eg, penicillin), an anti-neoplastic agent, or a cardiovascular drug. For example, if the disease to be treated is ARDS, in addition to administering the compound of formula (I), its salts or solvates to the individual, it can also be administered to further alleviate, reduce or alleviate ARDS-related Of bronchial stenosis or bronchospasm.

According to the embodiments provided in this disclosure, the dose administered to individuals in need of treatment is approximately between 0.001 and 100 mg per kilogram of body weight, such as 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007 per kilogram of body weight , 0.008, 0.009, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 , 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37 , 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62 , 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87 , 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100 mg; preferably, about 0.01 to 90 mg per kg of body weight of the individual, such as 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, or 90 mg; more preferably about 1 kg The body weight of the individual is 0.1 to 80 mg, for example 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 5 8, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79 or 80 mg.

The dosage and route of administration of the compound of formula (I), its salts or solvates to be administered can be adjusted according to factors such as the specific disease type to be treated, prevented or managed, the patient's age, weight and gender And/or course of treatment. The role of these factors in dose, route of administration, and or course of treatment is information that people with ordinary skills in the relevant field can obtain on their own without undue experimentation.

A number of experimental examples are presented below to illustrate certain aspects of the present invention, so that those with ordinary knowledge in the technical field to which the present invention pertains can implement the present invention, and these experimental examples should not be regarded as limiting the scope of the present invention. It is believed that the skilled artisans can fully utilize and practice the present invention without over-interpretation after reading the description presented here. All published documents cited herein are deemed to be part of this specification.

Examples

Materials and methods

Preparation of neutrophils

Human neutrophils present in a state free of lipopolysaccharide were obtained by dextran sedimentation and Ficoll centrifugation.

animal

The 7-8 week old BALB/c male rats were purchased from Taiwan Lesco Company (Taipei, Taiwan) and kept in an animal house with 12 hours of light, and they can freely access standard feed and drinking water. All animal testing procedures are in accordance with the guidelines issued by the Changgung University Animal Care and Use Committee.

Measuring O ₂ ^{• −} Yield

The content of O ₂ ^{• −} is determined by the amount of ferricytochrome c that can be inhibited by superoxide dismutase (SOD). Briefly, after adding 0.5 mg/ml of ferrous cytochrome c and 1 mM calcium ions to the cell culture medium, neutrophils (6 x ¹⁰⁵ cells / ml) for about 5 minutes. Then, the neutrophils were treated with cytochalasin B (CB, CB, 1 μg/ml) for about 3 minutes, and then treated with methy-L-methylthiamine-L-leucine-L -Amphetamine (fMLF, 0.1 μM) activation for about 10 minutes. Continuous stirring in a dual-beam, 6-slot positioning spectrometer (Hitachi U-3010, Tokyo, Japan) to continuously monitor the change in absorbance at 550 nm due to the reduction of ferrous cytochrome c with or without the addition of SOD (100 Unit/ml) divided by the extinction coefficient (ε=21.1mM ^-1 /10mm) to calculate O ₂ ^{• −} yield.

Measuring the amount of elastase released

Methoxysuccinyl-alanine-alanine-co-amino acid-valine-p-nitroaniline (Ms-Ala-Ala-Pro-Val-pNA) was used as a matrix to measure the amount of elastase released. Briefly, the culture medium was added to the Ms-Ala-Ala-Pro- Val-pNA (100μM), and let the neutrophils (6x10 ⁵ cells / ml) at 37 [deg.] C for about 2 minutes equilibrium, then with various concentrations of The test compound acts for about 5 minutes. The cells were first treated with CB (0.5 μg/ml) for about 3 minutes, and then activated with fMLF (0.1 μM) for about 10 minutes, and the change in absorbance at a wavelength of 405 nm was measured. The results are expressed as the percentage of elastase released relative to the drug-free control group (DMSO).

Cell survival analysis

The cytotoxicity of a candidate compound on neutrophils was evaluated using a commercial kit (Promaga) by measuring the amount of lactose dehydrogenase (LDH). Since LDH is an enzyme in cells, it is only released when the cell membrane is ruptured (that is, cell death), so it can be used as an indicator of cell survival rate. The amount of LDH can be measured with a commercially available commercial kit. The amount of LDH can be determined by measuring the change in absorbance at 450 nm when LDH reduces NAD to NADH. Cytotoxicity is expressed as the percentage of cell-free medium divided by the total LDH of cells. The total amount of LDH is the total amount of LDH released by treating cells with 0.1% Triton X-100 at 37°C for about 30 minutes to completely lyse the cells.

Animal model of ALI induced by LPS in mice

The test compound (eg, HKI-4971) was dissolved in 50 μl of physiological saline containing 10% DMSO and 20% Kolliphor EL, and then injected into mice by intravenous injection. After 1 hour, the mice were anesthetized with Zoletil 50 (30 mg/kg) and xylazine (6 mg/kg), and 50 μg of LPS ( Escherichia coli 0111:B4; Sigma- Aldrich, USA) (dissolved in 40 microliters of physiological saline). The mice in the control group were given physiological saline. After 5 hours, the mice were sacrificed by cervical dislocation and their lung tissues were removed. The left lobe of the lung was fixed with 10% formalin for tissue section observation, and the right lobe was used to measure MPO activity.

Bone marrow peroxidase (myeloperoxidase, MPO) activity analysis

Using MagNA Lyser instrument (Roche, Germany) in 0.5% (w/v) cetyltrimethylammonium bromide (HETAB, Sigma-Aldrich, USA) in PBS solution (50 mM, pH 6.0) , Homogenize the lung tissue and centrifuge at 12,000xg at 4°C for 20 minutes to remove insoluble materials. The activity of peroxidase is evaluated by the degree to which it can oxidize o-dianisidine dihydrochloride (Sigma-Aldrich, USA). Briefly, ortho-dianisidine dihydrochloride (0.2 mg/ml) containing 0.001% hydrogen peroxide was added to the homogenous substance, and reacted at room temperature for 10 minutes. Measure the absorbance at 405 nm. MPO activity is expressed relative to protein concentration (determined using the Bradford method).

Animal model of psoriasis-like skin inflammation induced by imiquimod (IMQ) in mice

In this experiment, IMQ induced psoriasis-like skin inflammation in BALB/c male mice (7-8 weeks old). The hair on the back of the mice was shaved, and then 62.5 mg of 5% imiquimod (IMQ) ointment (Aldara, MEDA Pharma, Sweeden) was applied daily, and the test compound (eg, HKI) was injected intraperitoneally for 7 consecutive days -4971) or physiological saline.

Observation of histopathology and immunohistochemistry

The tissue was fixed with 10% formalin and embedded in a paraffin block. After cutting out a tissue section with a thickness of about 5 microns, it was stained with hematoxylin-eosin (H&E). In terms of immunohistochemical staining, first recognize with primary antibodies of anti-Ly6g, MPO, ki67, and then proceed with secondary antibody recognition that can recognize the primary antibody. Finally, it is dyed with DAB masterbatch and then contrasted with hematoxylin.

statistics

The results are shown by mean quality + standard deviation (SEM), and compared by student's t -test. Probability less than 0.05 means significant.

Example 1 Preparation and analysis of compound of formula (I)

1.1 Preparation of compound of formula (I)

Generally, the compound of formula (I) is prepared in the manner disclosed in Scheme I.

Process I

Compound of formula (I) Compound number and name X R ₁ R ₂ R ₃ R ₄ R ₅ 3 HKI-4961 N CH ₃ H H H H 4 HKI-4962 N CH ₃ H H F H 5 HKI-4966 N CH ₃ OCH ₃ H H H 6 HKI-4965 N CH ₃ H OCH ₃ H H 7 HKI-4964 N CH ₃ H H OCH ₃ H 8 HKI-4963 N CH ₃ H OCH ₃ OCH ₃ OCH ₃ 9 HKI-4967 N CH ₃ H H SCH ₃ H 10 HKI-4968 N CH ₃ H H -(C=O)CH ₃ H 11 HKI-4973 N CH ₃ OH H H H 12 HKI-4972 N CH ₃ H OH H H 13 HKI-4971 N CH ₃ H H OH H 14 HKI-4974 N CH ₃ H H CF ₃ H 15 HKI-4970 N CH ₃ H H COOH H 16 HKI-4975 N CH ₃ SH H H H 17 TCH-30o N CH ₃ H H Cl H 18 TCH-30p N CH ₃ H H CH ₃ H 19 HKI-4978 O no H H H H 20 HKI-4979 O no H H F H twenty one HKI-4983 O no OCH ₃ H H H twenty two HKI-4982 O no H OCH ₃ H H twenty three HKI-4981 O no H H OCH ₃ H twenty four HKI-4980 O no H OCH ₃ OCH ₃ OCH ₃ 25 HKI-4984 O no H H SCH ₃ H 26 HKI-4986 O no H -(C=O)CH ₃ H H 27 HKI-4987 O no H H -(C=O)CH ₃ H 28 HKI-4990 O no OH H H H 29 HKI-4988 O no H H OH H 30 HKI-4991 O no H H CF ₃ H 31 HKI-4985 O no H H COOH H 32 TCH-30q O no H H Cl H 33 TCH-30r O no H H CH ₃ H

4- chlorine -1- methyl -1 H- Pyrazolo [4,3-c] quinoline -3- amine ( Chemical compound 2a; X = N, R ₁ = CH ₃ )

A mixed solution of 2,4-dichloro-3-cyanoquinoline (5.48 g, 24.6 mmol) dissolved in ethanol (100 ml) and methylhydrazine (2.21 g, 48.0 mmol) was refluxed for 2 hours (Monitored by TLC). The reaction mixture was concentrated under vacuum, water (100 ml) was added, the precipitate was collected after filtration, the precipitate was washed with ethanol and water, and a light yellow solid was obtained after drying. After recrystallization of the crude product with ethanol, compound 2a (4.23 g, 74%) was obtained.

¹ H-NMR (200 MHz, DMSO- d ₆ ): δ 7.65-7.78 (m, 2H, Ar-H), 7.92-7.96 (m, 1H, Ar-H), 7.39-7.44 (m, 1H, Ar -H).

4- chlorine Isoxazole [4,5- c ] quinoline -3- amine ( Chemical compound 2b; X = O, R ₁ = H)

Combine 2,4-dichloro-3-cyanoquinoline (1.12 g, 5 mmol), hydroxylamine hydrochloride (1.03 g, 15 mmol) and K ₂ CO dissolved in ethanol (30 mL) The mixed solution of ₃ (2.07 g, 15 mmol) was refluxed for 1 hour (monitored by TLC). The reaction mixture was concentrated under vacuum, water (100 ml) was added, the precipitate was filtered and collected, the precipitate was washed with water, and a yellow solid was obtained after drying. The crude product was recrystallized with methanol to obtain compound 2b (0.71 g, 65%).

Mp.: 194-196 ^o C.

¹ H-NMR (400 MHz, DMSO- d ₆ ): 7.52-7.60 (m, 1H, Ar-H), 7.73-7.78 (m, 2H, Ar-H), 8.06 (d, 1H, J = 7.6 Hz , Ar-H), 8.72 (br s, 2H, NH ₂ ).

Synthetic 4 Aniline group -1 methyl -1 H- Pyrazolo [4,3- c ] quinoline -3- amine ( Chemical compound 3-18) and First 4 Aniline group Isoxazole [4,5- c ] quinoline -3- amine ( Chemical compound 19-33) General synthesis method

Compound 2a or compound 2b (2 mmol) was stirred with substituted aniline (4 mmol) dissolved in DMF (25 mL) and refluxed for 2-5 hours (monitored by TLC). The mixture was cooled and the reaction mixture was concentrated under vacuum, and water (50 mL) was added to the residue. After collecting the precipitate and washing with water, the remaining precipitate was dried to produce a crude product, which was recrystallized with ethanol.

Each compound will be identified in the following manner: thin layer chromatography (TLC) (with pre-coated (0.2 mm) silicone 60 F ₂₅₄ plates, purchased from EM Laboratories), detected by UV light (254 nm) Measured; melting point (Electrothermal IA9100 digital melting point detector); and ¹ H, ¹³ C NMR spectroscopy (400 and 100 MHz Varian-Unity-400 spectrometer or 200 and 50 MHz Varian-Gemini-200 spectrometer, chemical shift d Expressed in ppm, it uses SiMe ₄ as the internal standard (= 0 ppm), and the coupling constant J is expressed in Hz). Elemental analysis is performed with the Heraeus CHN-O-Rapid elemental analyzer, and the results are within ± 0.4% of the calculated value.

4- aniline -1- methyl -1H- Pyrazolo [4,3- c ] quinoline -3- amine ( Chemical compound 3, HKI-4961)

Yield 40%; melting point: 186-187 ^o C (EtOH).

¹ H-NMR (400 MHz, DMSO- d ₆ ): δ 4.19 (s, 3H, NCH ₃ ), 5.58 (br s, 2H, NH ₂ ), 6.99-7.03 (m, 1H, Ar-H), 7.33 -7.37 (m, 3H, Ar-H), 7.52-7.56 (m, 1H, Ar-H), 7.67-7.70 (m, 1H, Ar-H), 7.95-7.97 (m, 2H, Ar-H) , 8.22-8.24 (m, 1H, Ar-H), 8.25 (br s, 1H, NH).

¹³ C-NMR (100MHz, DMSO- d ₆ ): δ 39.18, 99.13, 114.49, 119.93 (2C), 121.71, 121.96, 122.46, 126.96, 128.45 (2C), 128.51, 140.52, 140.67, 146.07, 147.29, 148.95.

Elemental analysis predicts C ₁₇ H ₁₅ N ₅ : C 70.57, H 5.23, N 24.21; actual: C 70.20, H 5.24, N 24.14.

4-(4- Fluoroaniline )-1- methyl -1H- Pyrazolo [4,3-c] quinoline -3- amine ( Chemical compound 4, HKI-4962)

Yield 62%; melting point: 218-219 ^o C (EtOH).

¹ H-NMR (400 MHz, DMSO- d ₆ ): δ 4.19 (s, 3H, NCH ₃ ), 5.90 (br s, 2H, NH ₂ ), 7.24-7.28 (m, 2H, Ar-H), 7.37 -7.41 (m, 1H, Ar-H), 7.55-7.59 (m, 1H, Ar-H), 7.72-7.74 (m, 1H, Ar-H), 7.84-7.87 (m, 2H, Ar-H) , 8.24-8.26 (m, 1H, Ar-H), 9.26 (br s, 1H, NH).

¹³ C-NMR (100MHz, DMSO- d ₆ ): δ 39.35, 97.75, 113.74, 115.39, 115.61, 112.36 (2C), 123.37, 123.84, 129.14, 135.390, 140.05, 142.33, 148.59, 157.37, 159.74.

Elemental analysis predicts C ₁₇ H ₁₄ FN ₅ ∙ 0.3HCl: C 64.14, H 4.53, N 22.00; Actual: C 63.87, H 4.73, N 21.77. .

4-(2- Methoxyaniline )-1- methyl -1H- Pyrazolo [4,3-c] quinoline -3- amine ( Chemical compound 5, HKI- 4966)

Yield 74%; melting point: 229-230 ^o C (EtOH).

¹ H-NMR (400 MHz, DMSO- d ₆ ): δ 3.90 (s, 3H, OCH ₃ ), 4.21 (s, 3H, NCH ₃ ), 5.21 (br s, 2H, NH ₂ ), 6.98-7.08 ( m, 3H, Ar-H), 7.32-7.36 (m, 1H, Ar-H), 7.53-7.57 (m, 1H, Ar-H), 7.73-7.75 (m, 1H, Ar-H), 8.22- 8.24 (m, 1H, Ar-H), 8.69 (br s, 1H, NH), 8.99-8.02 (m, 1H, Ar-H).

¹³ C-NMR (100MHz, DMSO- d ₆ ): δ 39.11, 56.04, 100.52, 110.55, 114.44, 119.16, 120.50, 121.46, 121.96, 122.43, 127.12, 128.50, 129.78, 140.20, 146.35, 147.28, 148.05, 149.12.

Elemental analysis predicts C ₁₈ H ₁₇ FN ₅ O: C 67.70, H 5.37, N 21.93; actual: C 67.37, H 5.39, N 21.85.

4-(3- Methoxyaniline )-1- methyl -1H- Pyrazolo [4,3-c] quinoline -3- amine ( Chemical compound 6, HKI-4965)

Yield 79%, melting point: 284-286 ^o C (EtOH).

¹ H-NMR (400 MHz, DMSO- d ₆ ): δ 3.80 (s, 3H, OCH ₃ ), 4.19 (s, 3H, NCH ₃ ), 5.58 (br s, 2H, NH ₂ ), 6.58-6.60 ( m, 1H, Ar-H), 7.22-7.26 (m, 1H, Ar-H), 7.32-7.42 (m, 2H, Ar-H), 7.53-7.75 (m, 1H, Ar-H), 7.69- 7.71 (m, 1H, Ar-H), 7.88 (br s, 1H, NH), 8.23-8.26 (m, 2H, Ar-H).

¹³ C-NMR (100MHz, DMSO- d ₆ ): δ 39.17, 54.93, 99.19, 105.43, 107.30, 112.12, 114.45, 121.97, 122.57, 126.97, 128.56, 129.11, 140.48, 141.86, 145.95, 147.24, 148.88, 159.49.

Elemental analysis predicts C ₁₈ H ₁₇ N ₅ O ∙ 0.4HCl: C 64.72, H 5.26, N 20.97; actual: C 64.96, H 5.29, N 21.08.

4-(4- Methoxyaniline )-1- methyl -1H- Pyrazolo [4,3-c] quinoline -3- amine ( Chemical compound 7, HKI-4964)

Yield 62%; melting point: 193-194 ^o C (EtOH).

¹ H-NMR (400 MHz, DMSO- d ₆ ): δ 3.82 (s, 3H, OCH ₃ ), 4.20 (s, 3H, NCH ₃ ), 6.30 (br s, 2H, NH ₂ ), 7.06- 7.08 (m, 2H, Ar-H), 7.43-7.47 (m, 1H, Ar-H), 7.59-7.63 (m, 3H, Ar-H), 7.81-7.83 (m, 1H, Ar-H), 8.26-8.28 (m, 1H, Ar-H), 9.74 (br s, 1H, NH).

¹³ C-NMR (100MHz, DMSO- d ₆ ): δ 39.58, 55.37, 96.37, 112.90, 114.71 (2C), 121.28, 122.79, 124.23, 125.78, 129.59, 129.82, 138.43, 139.55, 148.50, 149.88, 157.43.

Elemental analysis predicts C ₁₈ H ₁₇ N ₅ O ∙ 0.5HCl: C 64.02, H 5.23, N 20.74; Actual: C 64.13, H 5.42, N 20.69.

4-(3,4,5- Trimethoxyaniline )-1- methyl -1H- Pyrazolo [4,3-c] quinoline -3- amine ( Chemical compound 8, HKI-4963)

Yield 63%; melting point: 219-220 ^o C (EtOH).

¹ H-NMR (400 MHz, DMSO- d ₆ ): δ 3.68 (s, 3H, OCH ₃ ), 3.82 (s, 6H, OCH ₃ ), 4.19 (s, 3H, NCH ₃ ), 5.75 (br s, 2H, NH ₂ ), 7.38 (m, 3H, Ar-H), 7.56-7.60 (m, 1H, Ar-H), 7.72-7.76 (m, 1H, Ar-H), 8.24-8.26 (m, 1H , Ar-H).

¹³ C-NMR (100MHz, DMSO- d ₆ ): δ 39.29, 55.79 (2C), 60.12, 98.54, 113.92, 122.24, 122.97, 123.13, 127.10, 129.00, 140.11, 148.59, 152.85.

Elemental analysis predicts C ₂₀ H ₂₁ N ₅ O ₃ ∙ 0.5HCl: C 60.39, H 5.46, N 17.61; actual: C 60.08, H 5.57, N 17.36.

1- methyl -4-(4- Methylthioaniline )- 1H- Pyrazolo [4,3-c] quinoline -3- amine ( Chemical compound 9, HKI-4967)

Yield 84% Melting point: 233-235 ^o C (EtOH).

¹ H-NMR (400 MHz, DMSO- d ₆ ): δ 2.48 (s, 3H, SCH ₃ ), 4.18 (s, 3H, NCH ₃ ), 5.71 (br s, 2H, NH ₂ ), 7.29-7.37 ( m, 3H, Ar-H), 7.53-7.57 (m, 1H, Ar-H), 7.69-7.71 (m, 1H, Ar-H), 7.86-7.88 (m, 2H, Ar-H), 8.22- 8.24 (m, 1H, Ar-H).

¹³ C-NMR (100MHz, DMSO- d ₆ ): δ 15.88, 39.22, 98.58, 114.15, 121.42, 122.10 (2C), 122.77, 125.80, 127.37 (2C), 128.74, 130.67, 137.67, 140.28, 144.66, 147.78, 148.65.

Elemental analysis predicts C ₁₈ H ₁₇ N ₅ S ∙ 0.2HCl: C 63.08, H 5.06, N 20.43, S 9.36; actual: C 63.01, H 5.19, N 20.43, S 9.39.

4-(4- Acetanilide )-1- methyl -1H- Pyrazolo [4,3-c] quinoline -3- amine ( Chemical compound 10, HKI-4968)

60% yield; melting point: 272-274 ^o C (EtOH).

¹ H-NMR (400 MHz, DMSO- d ₆ + TFA- d ): δ 2.65 (s, 3H, COCH ₃ ), 4.28 (s, 3H, OCH ₃ ), 7.62-7.74 (m, 4H, Ar-H ), 7.93-7.95 (m, 1H, Ar-H), 8.14-8.17 (m, 2H, Ar-H), 8.39-8.41 (m, 1H, Ar-H).

¹³ C-NMR (100MHz, DMSO- d ₆ + TFA- d ): δ 26.89, 40.06, 96.94, 113.17, 119.71, 123.61, 123.98 (2C), 126.17, 130.46 (2C), 131.04, 135.22, 135.53, 140.11, 140.97, 147.68, 151.36, 197.17.

Elemental analysis predicts C ₁₉ H ₁₇ N ₅ O: C 68.87, H 5.17, N 21.13; actual: C 68.67, H 5.18, N 20.95

4-(2- Hydroxyaniline )-1- methyl -1H- Pyrazolo [4,3-c] quinoline -3- amine ( Chemical compound 11, HKI-4973)

Yield 64%; melting point: 233-235 ^o C (EtOH).

¹ H-NMR (400 MHz, DMSO- d ₆ ): δ 4.21 (s, 3H, NCH ₃ ), 5.98 (br s, 2H, NH ₂ ), 6.91-6.95 (m, 1H, Ar-H), 7.03 -7.04 (m, 1H, Ar-H), 7.13-7.14 (m, 1H, Ar-H), 7.24-7.45 (m, 1H, Ar-H), 7.58-7.62 (m, 1H, Ar-H) , 7.81-7.83 (m, 2H, Ar-H), 8.26-8.28 (m, 1H, Ar-H).

¹³ C-NMR (100MHz, DMSO- d ₆ ): δ 39.28, 97.06, 113.06, 116.81 (2C), 119.44 (2C), 122.25, 122.76, 123.97, 124.94, 126.56, 129.68, 139.58, 148.58, 149.38, 150.37.

Elemental analysis predicts C ₁₇ H ₁₅ N ₅ O ∙ 1.0HCl: C 59.72, H 4.72, N 20.49; actual: C 59.62, H 5.06, N 20.30.

4-(3- Hydroxyaniline )-1- methyl -1H- Pyrazolo [4,3-c] quinoline -3- amine ( Chemical compound 12, HKI-4972)

70% yield; melting point: 190-191 ^o C (EtOH).

¹ H-NMR (400 MHz, DMSO- d ₆ ): δ 4.21 (s, 3H, NCH ₃ ), 6.23 (br s, 2H, NH ₂ ), 6.66-6.69 (m, 1H, Ar-H), 7.07 -7.09 (m, 1H, Ar-H), 7.22-7.26 (m, 2H, Ar-H), 7.45-7.4 (m, 1H, Ar-H), 7.61-7.65 (m, 1H, Ar-H) , 7.83-7.85 (m, 2H, Ar-H), 8.27-8.29 (m, 1H, Ar-H), 9.75 (br s, 1H, OH).

¹³ C-NMR (100MHz, DMSO- d ₆ ): δ 39.58, 97.14, 110.25, 113.13, 113.59, 122.81, 124.34, 129.83, 130.13, 138.38, 139.77, 148.14, 149.81, 158.31.

Elemental analysis predicts C ₁₇ H ₁₅ N ₅ O ∙ 0.9HCl: C 60.36, H 4.77, N 20.71; actual: C 59.99, H 5.06, N 20.31.

4-(4- Hydroxyaniline )-1- methyl -1H- Pyrazolo [4,3-c] quinoline -3- amine ( Chemical compound 13, HKI-4971)

Yield 79%. Melting point: 320-322 ^o C (EtOH).

¹ H-NMR (400 MHz, DMSO- d ₆ ): δ 4.20 (s, 3H, NCH ₃ ), 6.47 (br s, 2H, NH ₂ ), 6.93-6.97 (m, 2H, Ar-H), 7.34 -7.37 (m, 2H, Ar-H), 7.48-7.52 (m, 1H, Ar-H), 7.62-7.66 (m, 1H, Ar-H), 7.89-7.91 (m, 1H, Ar-H) , 8.27-8.29 (m, 1H, Ar-H), 9.94 (br s, 1H, OH), 11.62 (br s, 1H, NH).

¹³ C-NMR (100MHz, DMSO- d ₆ ): δ 39.29, 95.29, 112.33, 116.47 (2C), 119.27, 123.10, 125.00, 125.97, 127.36, 130.31, 135.59, 139.17, 148.35, 150.77, 157.17.

Elemental analysis predicts C ₁₇ H ₁₅ N ₅ O ∙ 1.0HCl: C 59.72, H 4.72, N 20.49; actual: C 59.64, H 4.85, N 20.41.

4-(4- Trifluoromethylaniline )-1- methyl -1H- Pyrazolo [4,3-c] quinoline -3- amine ( Chemical compound 14, HKI-4974)

Yield 59%. Melting point: 219-220 ^o C (EtOH).

¹ H-NMR (400 MHz, TFA- d ): δ 4.28 (s, 3H, NCH ₃ ), 7.60-7.63 (m, 1H, Ar-H), 7.72-7.81 (m, 3H, Ar-H), 7.90-7.95 (m, 3H, Ar-H), 8.38-8.40 (m, 1H, Ar-H).

Elemental analysis predicts C ₁₈ H ₁₄ F ₃ N ₅ ∙ 1.3HCl: C 53.40, H 3.81, N 17.30; Actual: C 53.00, H 4.12, N 17.20.

4-[(3- Amine -1- methyl -1H- Pyrazolo [4,3-c] quinoline -4- base ) Amine ] benzoic acid ( Chemical compound 15, HKI-4970)

Yield 68%; melting point: 325-326 ^o C (EtOH).

¹ H-NMR (400 MHz, DMSO- d ₆ ): δ 4.24 (s, 3H, NCH ₃ ), 7.59-7.59 (m, 1H, Ar-H), 7.69-7.79 (m, 3H, Ar-H) , 7.87-7.89 (m, 1H, Ar-H), 8.04-8.06 (m, 1H, Ar-H), 8.34-8.37 (m, 1H, Ar-H), 8.27-8.29 (m, 1H, Ar-H) H).

¹³ C-NMR (100MHz, SO- d ₆ ): δ 39.29, 97.33, 112.94, 120.22, 122.67, 123.15, 125.46, 127.67, 130.45, 130.91 (2C), 131.16, 136.34, 139.79, 141.47, 147.39, 150.81, 166.82 .

Elemental analysis predicts C ₁₈ H ₁₅ N ₅ O ₂ ∙ 1.0HCl: C 58.44, H 4.36, N 18.93; actual: C 58.04, H 4.50, N 18.79.

2-[(3- Amine -1- methyl -1H- Pyrazolo [4,3-c] quinoline -4- base ) Amine ] Phenylmercaptan ( Chemical compound 16, HKI-4975)

Yield 42%. Melting point: 235-236 ^o C (EtOH).

¹ H-NMR (400 MHz, DMSO- d ₆ ): δ 3.14 (s, 3H, NCH ₃ ), 5.03 (br s, 1H, SH), 6.05 (br s, 1H, NH ₂ ), 6.93-6.97 ( m, 1H, Ar-H), 6.85-6.87 (m, 1H, Ar-H), 7.02-7.04 (m, 1H, Ar-H), 7.20-7.30 (m, 2H, Ar-H), 7.36- 7.41 (m, 1H, Ar-H), 7.81-7.86 (m, 1H, Ar-H).

¹³ C-NMR (100MHz, DMSO- d ₆ ): δ 35.75, 100.80, 111.46, 115.40, 116.44, 120.82, 121.33, 123.74, 125.82, 131.11, 131.31, 132.51, 140.24, 147.51, 152.11, 153.59, 161.85.

Elemental analysis predicts C ₁₇ H ₁₅ N ₅ S ∙ 0.1HCl: C 62.82, H 4.68, N 21.55, S 9.86; actual: C 63.03, H 4.72, N 21.55, S 9.88.

4-(4- Chloroaniline )-1- methyl -1H- Pyrazolo [4,3-c] quinoline -3- amine ( Chemical compound 17, TCH-30o)

Yield 82%. Melting point: 281-282 ^o C (EtOH).

¹ H-NMR (400 MHz, DMSO- d ₆ ): δ 4.23 (s, 3H, NCH ₃ ), 7.53-7.61 (m, 3H, Ar-H), 7.66-7.71 (m, 3H, Ar-H) , 7.88-7.90 (m, 1H, Ar-H), 8.33-8.34 (m, 1H, Ar-H), 12.19 (br s, 1H, NH).

¹³ C-NMR (100MHz, DMSO- d ₆ ): δ 39.77, 96.36, 112.62, 119.49, 123.18, 125.36, 126.43 (2C), 129.74 (2C), 130.46, 130.99, 135.31, 135.70, 139.49, 147.76, 150.97.

Elemental analysis predicts C ₁₇ H ₁₄ ClN ₅ ∙ 1.5HCl: C 53.93, H 4.13, N 18.50; Actual: C 54.09, H 4.44, N 18.53.

4-(4- Toluidine )-1- methyl -1H- Pyrazolo [4,3-c] quinoline -3- amine ( Chemical compound 18, TCH-30p)

Yield 85%. Melting point: 253-255 ^o C (EtOH).

¹ H-NMR (400 MHz, DMSO- d ₆ ): δ 2.36 (s, 3H, CH ₃ ), 4.20 (s, 3H, NCH ₃ ), 6.22 (br s, 2H, NH ₂ ), 7.27-7.29 ( m, 2H, Ar-H), 7.43-7.47 (m, 1H, Ar-H), 7.559-7.63 (m, 3H, Ar-H), 7.79-7.81 (m, 1H, Ar-H), 8.26- 8.28 (m, 1H, Ar-H), 9.72 (br s, 1H, NH).

¹³ C-NMR (100MHz, DMSO- d ₆ ): δ 20.63, 39.50, 97.08, 113.19, 122.14, 122.67 (2C), 123.14, 124.05, 129.65, 129.78 (2C), 134.30, 135.09, 139.44, 139.76, 148.34, 149.57.

Elemental analysis predicts C ₁₈ H ₁₇ N ₅ ∙ 1.1HCl: C 62.93, H 5.32, N 20.39; actual: C 62.80, H 5.73, N 20.44.

4- Aniline isoxazole [4,5- c ] quinoline -3- amine ( Chemical compound 19, HKI-4978)

Yield 40%. Melting point: 266-267 ^o C (hexane).

¹ H-NMR (400 MHz, DMSO- d ₆ ): δ 7.42-7.45 (m, 2H, Ar-H), 7.54-7.71 (m, 6H, Ar-H), 7.96-7.99 (m, 1H, Ar -H), 10.04 (br s, 2H, NH ₂ ), 11.47 (br s, 1H, 2H, NH, HCl).

¹³ C-NMR (100MHz, DMSO- d ₆ ): δ 82.82, 111.73, 123.07, 125.37, 125.61 (2C), 127.63, 129.80 (2C), 132.22, 135.07, 136.29, 148.52, 155.35, 168.52.

Elemental analysis predicts C ₁₆ H ₁₂ N ₄ O ∙ 1.1HCl: C 60.71, H 4.18, N 17.70; actual: C 60.94, H 4.45, N 17.45.

4-(4- Fluoroaniline ) Isoxazole [4,5- c ] quinoline -3- amine ( Chemical compound 20, HKI- 4979)

Yield: 67%. Melting point: 271-272 ^o C (hexane).

¹ H-NMR (400 MHz, DMSO- d ₆ ): δ 7.39-7.44 (m, 3H, Ar-H), 7.60-7.70 (m, 4H, Ar-H), 7.96-7.98 (m, 1H, Ar -H), 7.96-7.99 (m, 1H, Ar-H), 10.02 (br s, 2H, NH ₂ ), 11.46 (br s, 1H, 2H, NH, HCl).

¹³ C-NMR (100MHz, DMSO- d ₆ ): δ 82.69, 111.71, 116.70 (2C, J = 22.8 Hz), 118.97, 123.11, 125.39, 128.45 (2C, J = 8.3 Hz), 131.16, 132.26, 136.33, 148.87, 155.33, 161.34 ( J = 242.6 Hz), 168.52.

Elemental analysis predicts C ₁₆ H ₁₁ FN ₄ O ∙ 1.0HCl: C 58.08, H 3.66, N 16.94; actual: C 57.97, H 3.95, N 16.85.

4-(2- Methoxyaniline ) Isoxazole [4,5- c ] quinoline -3- amine ( Chemical compound 21, HKI-4983)

Yield 40%. Melting point: 261-262 ^o C (hexane).

¹ H-NMR (400 MHz, DMSO- d ₆ ): δ 3.79 (s, 3H, OCH ₃ ), 7.11-7.15 (m, 1H, Ar-H), 7.26-7.28 (m, 1H, Ar-H) , 7.40-7.52 (m, 3H, Ar-H), 7.61-7.65 (m, 1H, Ar-H), 7.74-7.76 (m, 1H, Ar-H), 7.96-7.98 (m, 1H, Ar-H) H), 9.91 (br s, 2H, NH ₂ ), 10.99 (br s, 1H, 2H, NH, HCl).

¹³ C-NMR (100MHz, DMSO- d ₆ ): δ 55.95, 82.11, 111.55, 113.24, 118.81, 121.19, 122.12, 123.25, 125.54, 128.55, 130.19, 132.50, 136.21, 148.49, 154.43, 155.19, 168.64.

Elemental analysis predicts C ₁₇ H ₁₄ N ₄ O ₂ ∙ 1.1HCl: C 58.92, H 4.40, N 16.17; actual: C 59.01, H 4.58, N 15.92.

4-(3- Methoxyaniline ) Isoxazole [4,5- c ] quinoline -3- amine ( Chemical compound 22, HKI-4982)

Yield 50%. Melting point: 262-263 ^o C (hexane).

¹ H-NMR (400 MHz, DMSO- d ₆ ): δ 3.80 (s, 3H, OCH ₃ ), 6.99-7.02 (m, 1H, Ar-H), 7.26-7.28 (m, 2H, Ar-H) , 7.40-7.48 (m, 2H, Ar-H), 7.62-7.66 (m, 1H, Ar-H), 7.71-7.73 (m, 1H, Ar-H), 7.97-7.98 (m, 1H, Ar-H) H), 10.02 (br s, 2H, NH ₂ ), 11.44 (br s, 2H, NH, HCl).

¹³ C-NMR (100MHz, DMSO- d ₆ ): δ 55.38, 82.50, 111.27, 111.74, 113.43, 117.60, 119.07, 123.05, 125.36, 130.57, 132.23, 136.08, 136.35, 148.48, 155.36, 160.20, 168.51.

Elemental analysis predicts C ₁₇ H ₁₄ N ₄ O ₂ ∙ 1.1HCl: C 58.92, H 4.40, N 16.17; actual: C 58.92, H 4.56, N 15.85.

4-(4- Methoxyaniline ) Isoxazole [4,5- c ] quinoline -3- amine ( Chemical compound 23, HKI-4981)

Yield: 43%. Melting point: 257-259 ^o C (hexane).

¹ H-NMR (400 MHz, DMSO- d ₆ ): δ 3.84 (s, 3H, OCH ₃ ), 7.10-7.13 (m, 2H, Ar-H), 7.38-7.48 (m, 3H, Ar-H) , 7.60-7.63 (m, 1H, Ar-H), 7.70-7.72 (m, 1H, Ar-H), 7.95-7.96 (m, 1H, Ar-H), 9.98 (br s, 2H, NH ₂ ) , 11.17 (br s, 1H, NH), 11.32 (br s, 1H, HCl).

¹³ C-NMR (100MHz, DMSO- d ₆ ): δ 55.45, 82.38, 111.61, 115.06 (2C), 118.97, 123.05, 125.19, 127.13, 127.66 (2C), 132.17, 136.42, 148.83, 155.31, 158.81, 168.42.

Elemental analysis predicts C ₁₇ H ₁₄ N ₄ O ₂ ∙ 1.1HCl: C 58.92, H 4.40, N 16.17; actual: C 58.83, H 4.49, N 16.01.

4-(3,4,5- Trimethoxyaniline ) Isoxazole [4,5- c ] quinoline -3- amine ( Chemical compound 24, HKI-4980)

Yield 63%; melting point: 264-266 ^o C (hexane).

¹ H-NMR (400 MHz, DMSO- d ₆ ): δ 3.74 (s, 3H, OCH ₃ ), 3.79 (m, 6H, OCH ₃ ), 6.95 (s, 2H, Ar-H), 7.42-7.44 ( m, 1H, Ar-H), 7.62-7.67 (m, 1H, Ar-H), 7.76-7.78 (m, 1H, Ar-H), 7.96-7.99 (m, 1H, Ar-H), 10.02 ( br s, 2H, NH ₂ ), 11.34 (br s, 1H, NH), 11.44 (br s, 1H, HCl).

¹³ C-NMR (100MHz, DMSO- d ₆ ): δ 56.12 (2C), 60.01, 82.50, 103.75 (2C), 111.62, 119.01, 123.03, 125.33, 130.14, 132.26, 136.27, 136.87, 148.50, 153.55 (2C) , 155.31, 168.47.

Elemental analysis predicts C ₁₉ H ₁₈ N ₄ O ₄ ∙ 1.2HCl: C 55.64, H 4.72, N 13.66; actual: C 55.65, H 5.01, N 13.51.

4-(4- Methylthioaniline ) Isoxazole [4,5- c ] quinoline -3- amine ( Chemical compound 25, HKI-4984)

Yield 42%. Melting point: 246-247 ^o C (hexane).

¹ H-NMR (400 MHz, DMSO- d ₆ ): δ 2.55 (s, 3H, SCH ₃ ), 7.39-7.52 (m, 5H, Ar-H), 7.61-7.69 (m, 3H, Ar-H) , 7.95-7.97 (m, 1H, Ar-H), 10.02 (br s, 2H, NH ₂ ), 11.39 (br s, 2H, NH, HCl).

¹³ C-NMR (100MHz, DMSO- d ₆ ): δ 14.67, 82.70, 111.69, 119.03, 123.04, 125.25, 126.35 (2C), 126.87 (2C), 131.69, 132.16, 136.44, 137.79, 148.55, 155.33, 168.43.

Elemental analysis predicts C ₁₇ H ₁₄ N ₄ OS ∙ 1.1HCl: C 56.33, H 4.20, N 15.46, S 8.85; actual: C 56.21, H 4.22, N 15.34, S 8.88.

4-(3- Acetylaniline ) Isoxazole [4,5- c ] quinoline -3- amine ( Chemical compound 26, HKI-4986)

Yield 40%. Melting point: 249-250 ^o C (hexane).

¹ H-NMR (400 MHz, DMSO- d ₆ ): δ 2.62 (s, 3H, COCH ₃ ), 7.36 (m, 1H, Ar-H), 7.60-7.65 (m, 3H, Ar-H), 7.89 -7.96 (m, 3H, Ar-H), 8.25 (m, 1H, Ar-H), 9.61 (br s, 2H, NH ₂ ), 10.94 (br s, 1H, NH), 11.27 (br s, 1H , HCl).

¹³ C-NMR (100MHz, DMSO- d ₆ ): δ 26.89, 112.30, 122.96 (2C), 124.10, 124.63, 125.77, 128.83, 129.84, 131.92, 137.90, 148.66, 156,13, 167.86, 197.60.

Elemental analysis predicts C ₁₈ H ₁₄ N ₄ O ₂ ∙ 1.2HCl: C 59.69, H 4.23, N 15.47; actual: C 59.50, H 4.62, N 15.19.

4-(4- Acetanilide ) Isoxazole [4,5- c ] quinoline -3- amine ( Chemical compound 27, HKI-4987)

Yield 50%. Melting point: 255-257 ^o C (hexane).

¹ H-NMR (400 MHz, DMSO- d ₆ ): δ 2.60 (s, 3H, COCH ₃ ), 7.36 (m, 1H, Ar-H), 7.60 (m, 2H, Ar-H), 7.79-7.80 (m, 2H, Ar-H), 7.94-7.96 (m, 1H, Ar-H), 8.04-8.06 (m, 1H, Ar-H), 9.45 (br s, 2H, NH ₂ ), 10.48 (br s, 1H, NH), 11.83 (br s, 1H, HCl).

¹³ C-NMR (100MHz, DMSO- d ₆ ): δ 26.67, 84.16, 112.45, 121.48, 122.95 (2C), 123.33, 124.81, 125.15, 129.63 (2C), 130.57, 131.87, 133.32, 148.15, 156.25, 167.76, 196.85.

Elemental analysis predicts C ₁₈ H ₁₄ N ₄ O ₂ ∙ 1.3HCl: C 59.09, H 4.22, N 15.31; actual: C 59.20, H 4.58, N 14.96.

4-(2- Hydroxyaniline ) Isoxazole [4,5- c ] quinoline -3- amine ( Chemical compound 28, HKI- 4990)

Yield 42%. Melting point: 240-241 ^o C (hexane).

¹ H-NMR (400 MHz, DMSO- d ₆ ): δ 6.87-6.96 (m, 2H, Ar-H), 7.10 (m, 1H, Ar-H), 7.21 (m, 1H, Ar-H), 7.34-7.36 (m, 2H, Ar-H), 7.50 (m, 1H, Ar-H), 7.88 (m, 1H, Ar-H), 8.46 (br s, 1H, NH), 8.78 (br s, 2H, NH ₂ ), 11.42 (br s, 1H, OH).

¹³ C-NMR (100MHz, DMSO- d ₆ ): δ 83.79, 112.90, 119.13, 122.78, 123.03, 124.31, 124.92, 126.09, 128.00, 131.36 (2C), 145.30, 149.84, 150.29, 157.64, 166.34.

Elemental analysis predicts C ₁₆ H ₁₂ N ₄ O ₂ ∙ 0.7HCl: C 60.45, H 4.03, N 17.62; actual: C 60.34, H 4.45, N 17.52.

4-(4- Hydroxyaniline ) Isoxazole [4,5- c ] quinoline -3- amine ( Chemical compound 29, HKI-4988)

Yield 45%; melting point: 236-237 ^o C (hexane).

¹ H-NMR (400 MHz, DMSO- d ₆ ): δ 6.80-6.85 (m, 2H, Ar-H), 7.19-7.23 (m, 1H, Ar-H), 7.44-7.52 (m, 4H, Ar -H), 7.86-7.88 (m, 1H, Ar-H), 8.88 (br s, 2H, NH ₂ ), 9.43 (br s, 1H, NH), 10.84 (br s, 1H, OH).

¹³ C-NMR (100MHz, DMSO- d ₆ ): δ 83.55, 112.63, 113.98, 115.39 (2C), 116.15, 122.67 (2C), 122.98, 125.34, 127.16, 131.27, 149.09, 154.57, 157.10, 166.70.

Elemental analysis predicts C ₁₆ H ₁₂ N ₄ O ₂ ∙ 1.9HCl: C 58.84, H 4.88, N 17.16; actual: C 58.74, H 4.59, N 17.01.

4-(4- Trifluoromethylaniline ) Isoxazole [4,5- c ] quinoline -3- amine ( Chemical compound 30, HKI-4991)

Yield 42%. Melting point: 227-229 ^o C (hexane).

¹ H-NMR (400 MHz, DMSO- d ₆ ): δ 7.34 (m, 1H, Ar-H), 7.57 (m, 2H, Ar-H), 7.81-7.93 (m, 3H, Ar-H), 7.93-7.94 (m, 2H, Ar-H), 9.26 (br s, 2H, NH ₂ ), 10.16 (br s, 1H, NH), 11.65 (br s, 1H, OH).

Elemental analysis predicts C ₁₇ H ₁₁ F ₃ N ₄ O ∙ 0.4HCl: C 56.88, H 3.20, N 15.61; actual: C 56.59, H 3.34, N 15.35.

4-[(3- Aminoisoxazole [4,5- c ] quinoline -4- base ) amine ] benzoic acid ( Chemical compound 31, HKI-4985)

Yield 58%. Melting point: 308-310 ^o C (hexane).

¹ H-NMR (400 MHz, DMSO- d ₆ ): δ 7.44-7.46 (m, 1H, Ar-H), 7.61-7.72 (m, 4H, Ar-H), 7.98-8.09 (m, 3H, Ar -H), 10.05 (br s, 2H, NH ₂ ), 11.62 (br s, 1H, NH), 11.80 (br s, 1H, HCl), 13.01 (br s, 1H, COOH).

¹³ C-NMR (100MHz, DMSO- d ₆ ): δ 83.57, 111.94, 119.23, 123.12, 124.74, 125.61, 129.03, 130.84 (2C), 131.16, 132.29, 136.40, 139.78, 148.40, 155.43, 166.75, 168.60.

Elemental analysis predicts C ₁₇ H ₁₂ N ₄ O ₃ ∙ 1.4HCl: C 54.96, H 3.64, N 15.08; actual: C 54.97, H 3.95, N 15.303.

4-(4- Chloroaniline ) Isoxazole [4,5- c ] quinoline -3- amine ( Chemical compound 32, TCH-30q)

Yield 75%. Melting point: 268-269 ^o C (hexane).

¹ H-NMR (400 MHz, DMSO- d ₆ ): δ 7.41-7.46 (m, 1H, Ar-H), 7.59-7.69 (m, 6H, Ar-H), 7.97-7.99 (m, 1H, Ar -H), 9.94 (br s, 2H, NH ₂ ), 11.31 (br s, 2H, NH, HCl).

¹³ C-NMR (100MHz, DMSO- d ₆ ): δ 83.09, 111.86, 119.17, 123.25, 125.67, 127.79 (2C), 129.99 (2C), 132.22, 132.45, 134.15, 136.45, 148.82, 155.48, 168.68.

Elemental analysis predicts C ₁₆ H ₁₁ ClN ₄ O ∙ 1.0HCl: C 55.33, H 3.49, N 16.140; Actual: C 55.37, H 3.45, N 16.24.

4-(4- Toluidine ) Isoxazole [4,5- c ] quinoline -3- amine ( Chemical compound 33, TCH-30r)

Yield 80%; melting point: 173-175 ^o C (hexane).

¹ H-NMR (400 MHz, DMSO- d ₆ ): δ 2.40 (s, 3H, CH ₃ ), 7.37-7.45 (m, 5H, Ar-H), 7.61-7.65 (m, 1H, Ar-H) , 7.70-7.72 (m, 1H, Ar-H), 7.95-7.98 (m, 1H, Ar-H), 9.92 (br s, 2H, NH ₂ ), 11.20 (br s, 2H, NH, HCl).

¹³ C-NMR (100MHz, DMSO- d ₆ ): δ 20.88, 82.71, 111.75, 119.14, 123.20, 125.50, 125.92 (2C), 130.52 (2C), 132.20, 132.40, 136.42, 137.57, 148.78, 155.44, 168.63.

Elemental analysis predicts C ₁₇ H ₁₄ N ₄ O ∙ 1.1HCl: C 61.78, H 4.61, N 16.96; actual: C 61.63, H 4.62, N 16.97.

1.2 Analysis of the compound of Example 1

1.2.1 Compound of formula (I) inhibits O ₂ ^{• −} production and elastase release in human neutrophils

In this example, the effect of the compound of Example 1 (ie, the compound of formula (I)) on inhibiting the production of O ₂ ^{• −} and the amount of elastase released in human neutrophils was investigated. The results are shown in Table 2.

Table 2 The effect of the compound of Example 1 in inhibiting fMLF/CB-induced O ₂ in human neutrophils ^{• −} Yield and elastase release Chemical compound Superoxide anion Elastase release IC ₅₀ (μM) ^a inhibition% IC ₅₀ (μM) ^a inhibition% 3 HKI-4961 2.39 ± 0.63 93.92 ± 6.32*** 4.09 ± 1.02 80.45 ± 2.57*** 4 HKI-4962 1.44 ± 0.22 91.16 ± 4.43*** 5.50 ± 1.50 68.04 ± 6.80*** 5 HKI-4966 1.37 ± 0.08 90.13 ± 3.38*** 4.65 ± 2.57 66.02 ± 6.51*** 6 HKI-4965 ＞ 10 27.54 ± 5.47** ＞ 10 33.36 ± 6.04** 7 HKI-4964 0.51 ± 0.17 99.26 ± 1.51*** 3.77 ± 0.71 96.67 ± 6.56*** 8 HKI-4963 0.80 ± 0.09 98.19 ± 2.41*** 3.25 ± 0.08 89.77 ± 6.51*** 9 HKI-4967 ＞ 10 47.72 ± 3.32*** ＞ 10 20.84 ± 3.75** 10 HKI-4968 1.58 ± 0.16 85.10 ± 1.66*** 4.63 ± 1.10 57.17 ± 7.34*** 11 HKI-4973 0.73 ± 0.23 93.51 ± 1.75*** 2.24 ± 0.91 101.39 ± 4.50*** 12 HKI-4972 1.89 ± 0.69 93.17 ± 3.66*** 4.62 ± 1.74 94.36 ± 8.34*** 13 HKI-4971 0.00369 ± 0.00174 88.13 ± 1.19*** 0.03210 ± 0.01740 104.56 ± 2.53*** 14 HKI-4974 4.56 ± 0.83 63.09 ± 2.51*** ＞ 10 37.61 ± 6.22** 15 HKI-4970 1.83 ± 0.17 82.93 ± 2.86*** 3.68 ± 0.42 79.42 ± 1.91*** 16 HKI-4975 ＞ 10 4.05 ± 2.95 ＞ 10 -3.34 ± 1.78 17 TCH-30p 3.49 ± 1.25 82.78 ± 5.36*** 5.28 ± 0.66 75.78 ± 6.64*** 18 TCH-30o 5.21 ± 1.90 69.71 ±7.05*** ＞ 10 39.66 ± 3.83*** 19 HKI-4978 ＞ 10 43.73 ± 6.43*** ＞ 10 5.81 ± 5.22 20 HKI-4979 6.16 ± 1.18 63.22 ± 7.76* ＞ 10 10.69 ± 4.00* twenty one HKI-4983 ＞ 10 32.68 ± 4.92** ＞ 10 -8.32 ± 2.23* twenty two HKI-4982 6.53 ± 0.90 69.18 ± 3.14*** ＞ 10 -3.61 ± 0.98* twenty three HKI-4981 8.59 ± 0.29 59.08 ± 2.03*** ＞ 10 -9.67 ± 3.44* twenty four HKI-4980 ＞ 10 17.10 ± 3.77** ＞ 10 -10.55 ± 3.81* 25 HKI-4984 - increase ＞ 10 18.10 ± 3.97** 26 HKI-4986 2.88 ± 0.27 76.17 ± 4.68*** ＞ 10 -4.55 ± 5.83 27 HKI-4987 ＞ 10 37.41 ± 5.43*** ＞ 10 -9.53 ± 5.26 28 HKI-4990 4.54 ± 1.18 91.04 ± 1.69*** 5.77 ± 1.14 77.57 ± 4.52*** 29 HKI-4988 0.65 ± 0.20 91.10 ± 0.38*** ＞ 10 29.85 ± 6.66** 30 HKI-4991 - increase 6.23 ± 0.52 81.50 ± 6.97*** 31 HKI-4985 ＞ 10 7.44 ± 4.23 ＞ 10 -1.40 ± 3.53 32 TCH-30q ＞ 10 38.65 ± 2.30*** ＞ 10 3.69 ± 3.42 33 TCH-30r - Promote 7.24 ± 0.72 67.96 ± 5.23*** % Inhibition refers to the ratio of inhibition at a concentration of 10 μM. The results are expressed as mean + ± standard deviation (n=3-5), * p <0.05, ** p <0.01, *** p <0.001, relative For the control group, ^a refers to the concentration of the compound at 50% inhibition (IC ₅₀ )

As shown in Table 2, most compounds except compounds 6, 9, 16, 19, 21, 24, 25, 27, 30, 31, 32 and 33 can inhibit neutrophils induced by fMLF O ₂ ^{• −} produced (IC ₅₀ between about 4 nM and 8.6 μM) and elastase released (IC ₅₀ between about 32 nM and 7.2 μM). Among these compounds, compound 13 (HKI-4971) has the strongest potency. Its IC _{50 for} inhibiting superoxide anion formation is about 3.7±1.7 nM; the IC _{50 for} inhibiting elastase release is about 32±17 nM. Therefore, in subsequent Examples 2 and 3, compound 13 (HKI-4971) was evaluated.

Example 2 HKI-4971 inhibits the process of imiquimod (IMQ)-induced psoriasis

In this example, the effect of Compound 13 (HKI-4971) on the progress of psoriasis was evaluated in the currently accepted psoriasis animal model. Briefly, HKI-4971 (20 mg/kg) was injected into BALB/c mice by intraperitoneal injection. After 30 minutes, the ointment containing 5% IMQ was applied to the back skin of the mice for 7 days. Next, samples were taken from the skin of these animals, and the collected skin samples were observed with a microscope and stained with immunochemistry; blood samples were taken to analyze liver or kidney function. The results are shown in Figures 1 to 3.

Figure 1(A) is a representative photograph taken from the back skin of a mouse treated with IMQ (left) or IMQ+HKI-4971 (right); as for figures 1(B) and 1(C) The photos are the appearance of the skin of the IMQ control group and the mice treated with IMQ+HKI-4971 under the microscope. After immunostaining with these skin samples, it was confirmed that the amount of the markers representing cell proliferation (ki67) and the number of neutrophils (Ly6g and MPO) were inhibited by HKI-4971 (Figure 2) . Therefore, HKI-4971 can reduce the severity and progression of IMQ-induced psoriasis in experimental animals.

From blood samples taken from mice treated with IMQ- or IMQ+HKI-4971, the amounts of alanine aminotransferase and aspartate aminotransferase, which represent liver function, and blood and urine nitrogen, which represent kidney function, were measured respectively BUN) and creatinine (CRE) content, the results are shown in Figure 3. As shown in Figure 3, using IMQ or HKI-4971 alone or in combination to treat mice does not affect their liver or kidney function.

Overall, the results of this example confirm that HKI-4971 has a protective effect on IMQ-induced psoriasis, so it can be used as psoriasis or due to multiple causes (including, but not limited to bacterial infections, trauma, autoimmunity, and allergies) The treatment of inflammatory diseases of the skin.

Example 3 HKI-4971 regulates LPS-induced ALI

In this example, the effect of HKI-4971 was explored by using the Gram-negative bacterial endotoxin LPS to induce an ALI animal model in mice that can mimic the human ARDS situation. Briefly, BALB/c mice were given intravenous injection of HKI-4971 (20 mg/kg) for about 1 hour, followed by intratracheal injection of LPS (2 mg/kg) for about 5 hours. After sacrificing the mice, their lung tissues were taken and the MPO activity and the amount of neutrophil markers (ie, Ly6g and MPO) were measured. The results are shown in Figure 4.

As shown in Figure 4(A), LPS induces increased MPO activity in the lungs, which represents inflammatory cells, and this increased activity can be inhibited by HKI-4971. Figure 4(B) shows photographs of lung tissue after H&E staining and immunostaining with neutrophil markers (Ly6g and MPO). It can be found that HKI-4971 can obviously reduce LPS-induced neutrophil infiltration and lung injury.

Overall, the results of this example confirm that HKI-4971 has a protective effect on LPS-induced lung injury, so it can be used as a treatment for ALI or ARDS (which is often caused by a variety of factors, including but not limited to, sepsis, trauma , And pneumonia, etc.).

Although the above embodiments disclose specific examples of the present invention, they are not intended to limit the present invention. Those with ordinary knowledge in the technical field to which the present invention belongs, without departing from the principle and spirit of the present invention, should Various changes and modifications can be made to it, so the scope of protection of the present invention shall be defined by the scope of the accompanying patent application.

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In order to make the above and other objects, features, advantages and embodiments of the present invention more obvious and understandable, the description of the attached drawings is as follows: Figure 1 shows the compound HKI-4971 against imiquimod in mice (imiquimod, IMQ) The therapeutic effect of psoriasis induced, (A) Representative mouse phenotypes from the IMQ group (left) and HKI-4971 group (right); (B) Skin from mice in the IMQ group Microscope image; (C) Microscope image of mice skin treated with HKI-4971; scale = 1 mm. Figure 2 shows H&E staining and immunochemical results of skin obtained from psoriasis mice treated with compound HKI-4971 according to an embodiment of the present disclosure, scale = 100 μm. Figure 3 shows the effect of compound HKI-4971 on the liver and kidney function of IMQ-induced psoriasis mice according to an embodiment of the present disclosure. (A) Alanine aminotransferase; (B) Aspartate aminotransferase Enzymes; (C) blood and urine nitrogen content, and (D) creatine; data is expressed as mean + standard deviation (n=3). Figure 4 shows the therapeutic effect of compound HKI-4971 on lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice, (A) activity of bone marrow peroxidase (MPO) in lung tissue, ( B) Upper image: lung photo; lower image: H&E stained photo and immunochemical photo after lung sectioning. The data is expressed as mean + standard deviation (n=3), scale = 100 μm, ^*** P <0.01.

Claims (12)

A compound having the structure of formula (I), its salts or solvates:

Among them: X is N or O; R ₁ is alkyl or does not exist, when X is O, R ₁ does not exist, when X is N, R ₁ is alkyl; R ₂ , R ₃ , R ₄ and R ₅ is independently hydrogen, hydroxyl, hydrogenthio, halogen, alkyl, haloalkyl, -OR ₆ , -SR ₆ , -(C=O)R ₆ , or -COOH; and R ₆ is alkyl or halogen alkyl. The compound according to claim 1, a salt or solvate thereof, wherein X is N, R ₁ is methyl, R ₂ , R ₃ and R ₅ are independently hydrogen, and R ₄ is hydroxyl. A pharmaceutical composition comprising a compound of formula (I), salts or solvates thereof

Among them: X is N or O; R ₁ is alkyl or does not exist, when X is O, R ₁ does not exist, when X is N, R ₁ is alkyl; R ₂ , R ₃ , R ₄ and R ₅ is independently hydrogen, hydroxyl, hydrogenthio, halogen, alkyl, haloalkyl, -OR ₆ , -SR ₆ , -(C=O)R ₆ , or -COOH; and R ₆ is alkyl or halogen Alkyl; and a pharmaceutically acceptable excipient. The pharmaceutical composition according to claim 3, wherein in formula (I), X is N, R ₁ is methyl, R ₂ , R ₃ and R ₅ are independently hydrogen, and R ₄ is hydroxyl. Use of a compound of formula (I), salts or solvates thereof for the preparation of a medicament, wherein the medicament is used to treat an inflammatory disease of a body and/or a disease associated with abnormal activation of neutrophils,

Among them: X is N or O; R ₁ is hydrogen, alkyl or does not exist, when X is O, R ₁ does not exist, when X is N, R ₁ is hydrogen or alkyl; R ₂ , R ₃ , R ₄ and R ₅ are independently hydrogen, hydroxyl, hydrogenthio, halogen, alkyl, haloalkyl, -OR ₆ , -SR ₆ , -(C=O)R ₆ , or -COOH; and R ₆ is Alkyl or haloalkyl. The use according to claim 5, wherein in formula (I), X is N, R ₁ is methyl, R ₂ , R ₃ and R ₅ are independently hydrogen, and R ₄ is hydroxyl. The use according to claim 5, wherein the inflammatory disease and/or the disease associated with abnormal activation of neutrophils are selected from the group consisting of Adult Respiratory Distress Syndrome (ARDS), acute lung injury (acute lung injury, ALI), chronic obstructive pulmonary disease (COPD), pulmonary fibrosis, liver injury, steatohepatitis, liver fibrosis, injury caused by ischemia and reperfusion, myocardial infarction, shock, stroke , Organ transplantation, ulcerative colitis, vasculitis, systemic lupus erythematosus (SLE), sepsis, systemic inflammatory response syndrome (SIRS), arthritis, psoriasis, atopic dermatitis , And inflammatory skin diseases. The use according to claim 7, wherein the inflammatory disease and/or disease associated with abnormal activation of neutrophils is ALI. The use according to claim 7, wherein the inflammatory disease and/or disease associated with abnormal activation of neutrophils is ARDS. The use according to claim 7, wherein the inflammatory disease and/or disease associated with abnormal activation of neutrophils is psoriasis. The use according to claim 7, wherein the compound of formula (I) is administered to the individual at a dose of 0.001-100 mg/kg. The use as claimed in claim 5, wherein the individual is a human.

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