Use of JAK inhibitors in the treatment of ischemic stroke

Abstract

This application provides the use of JAK inhibitors for the treatment of ischemic stroke, specifically relating to the use of compounds of formula (I) or pharmaceutically acceptable salts, solvates, or isomers thereof in the preparation of medicaments for the treatment or prevention of ischemic stroke.

Description

Technical Field

[0001] This application belongs to the field of biomedicine and specifically relates to new uses of JAK inhibitors, particularly their use in the treatment of ischemic stroke. Background Technology

[0002] The core cause of ischemic stroke (IS) is the interruption of cerebral blood flow, which in turn induces cerebral ischemia and hypoxia, disordered energy metabolism, increased oxidative stress, and activation of inflammatory pathways, leading to a series of cascading pathophysiological damages, including inflammatory infiltration, neuronal apoptosis, blood-brain barrier damage, and imbalance in neurorepair. While significant progress has been made in revascularization techniques, the therapeutic window is limited, preventing many patients from benefiting. Furthermore, reperfusion injury can further exacerbate brain tissue damage and accelerate neuronal death; therefore, there is an urgent clinical need for highly effective neuroprotective drugs for stroke.

[0003] Existing technologies disclose various NMDAR antagonists targeting excitotoxicity; however, clinical research data show that these antagonists can cause dose-dependent adverse reactions such as behavioral abnormalities, cognitive impairment, and sedation within the effective dose range, exhibiting a narrow therapeutic window, and none of the phase III clinical trials met the primary efficacy endpoint. Drugs targeting oxidative stress and apoptosis showed no statistically significant differences, failing to provide definitive evidence of clinical benefit. Therefore, there is an urgent clinical need for novel intervention targets and drugs that can effectively intervene in the inflammatory cascade response after acute stroke while also possessing good safety profiles.

[0004] The JAK / STAT (Janus kinase / signal transducer and activator of transcription) signaling pathway is a highly conserved signaling pathway that mediates extracellular cytokine and growth factor signals and intracellular gene expression, participating in biological effects such as cell proliferation, differentiation, apoptosis, inflammation, immunity, and hematopoiesis. Cerebral ischemia and hypoxia activate the JAK / STAT pathway, participating in processes such as apoptosis, angiogenesis, and oxidative stress following cerebral ischemia-reperfusion injury. Inhibiting the JAK / STAT pathway can improve inflammation, oxidative stress, and neuronal apoptosis after cerebral ischemia-reperfusion injury, and this pathway may be a promising area of ​​research for future stroke treatment. Summary of the Invention

[0005] This invention proposes a novel application for non-brain-penetrating JAK inhibitors in the preparation of drugs for the prevention or treatment of ischemic stroke, especially stroke reperfusion injury. Addressing the long-standing technical bias in the field that "stroke treatment relies on central penetration of drugs, and non-brain-penetrating small molecules have no central therapeutic value," and the current lack of clinical research and translational protocols for non-brain-penetrating JAK inhibitors in ischemic stroke, this invention breaks through the existing single-target development approach. It demonstrates that non-brain-penetrating JAK inhibitors can effectively improve blood-brain barrier damage after stroke, block neuroinflammatory cascade responses, and reduce brain reperfusion injury through peripheral inflammatory regulatory pathways, establishing a novel system of action for non-centrally penetrating drugs to intervene in acute ischemic stroke. This invention overcomes the limitations of existing technology in understanding the stroke treatment potential of non-brain-penetrating kinase drugs, fills the gap in clinical translation technology in this field, and avoids the shortcomings of traditional central nervous system protective drugs, such as poor target selectivity, significant adverse reactions, and limited applicable time windows. It provides a new and feasible technical solution for the safe and effective intervention of ischemic stroke, especially reperfusion injury after thrombolysis and thrombectomy.

[0006] This application relates to the use of JAK inhibitors or pharmaceutically acceptable salts, solvates, active metabolites, polymorphs, isotope labels, isomers, or prodrugs thereof for the treatment or prevention of ischemic stroke.

[0007] This application provides the use of the compound of formula (I) or a pharmaceutically acceptable salt, solvate, isomer, or prodrug thereof in the preparation of a medicament for the treatment or prevention of ischemic stroke. I.

[0008] This application provides the use of the compound of formula (I) or a pharmaceutically acceptable salt, solvate or isomer thereof in the preparation of a medicament for the treatment or prevention of reperfusion injury after ischemic stroke. Attached Figure Description

[0009] Figure 1 The neurobehavioral scores of each group of model animals 24 hours after reperfusion are shown. Figure 2 The statistical data of infarct volume after 24 hours of reperfusion are shown for each group of model animals. Detailed Implementation

[0010] The present application will now be described in further detail with reference to the accompanying drawings and embodiments. Through these descriptions, the features and advantages of the present application will become clearer and more apparent.

[0011] The term “exemplary” as used herein means “serving as an example, embodiment, or illustration.” Any embodiment illustrated herein as “exemplary” is not necessarily to be construed as superior to or better than other embodiments. Although various aspects of embodiments are shown in the accompanying drawings, the drawings are not necessarily drawn to scale unless specifically indicated otherwise.

[0012] Furthermore, the technical features involved in the different embodiments of this application described below can be combined with each other as long as they do not conflict with each other.

[0013] definition When a substituent is described using a conventional chemical formula written from left to right, it also includes chemically equivalent substituents obtained when the structural formula is written from right to left. For example, CH2O is equivalent to OCH2.

[0014] The terms “optional” or “optionally” mean that the event or condition described below may or may not occur, including both the occurrence and non-occurrence of the event or condition. For example, the ethyl group “optionally” being halogenated means that the ethyl group can be unsubstituted (-CH2CH3), monosubstituted (e.g., -CH2CH2F), polysubstituted (e.g., -CHFCH2F, -CH2CHF2, etc.), or fully substituted (-CF2CF3). Those skilled in the art will understand that for any group containing one or more substituents, no substitution or substitution pattern that is spatially impossible and / or cannot be synthesized is introduced.

[0015] The term "substitution" refers to the replacement of one or more hydrogen atoms on a specific atom by a substituent, as long as the valence state of the specific atom is normal and the resulting compound is stable. When the substituent is oxo (i.e., =O), it means that two hydrogen atoms are replaced; oxo substitution does not occur on aromatic groups.

[0016] When any variable (e.g., R) appears more than once in the composition or structure of a compound, its definition is independent in each case. Thus, for example, if a group is substituted by 0-2 Rs, the group can optionally be substituted by at most two Rs, and the Rs in each case have independent options. Furthermore, combinations of substituents and / or their variants are only permitted if such combinations produce a stable compound.

[0017] The C used in this article m~n This refers to a portion containing m to n carbon atoms. For example, the "C"... 1~8 "A group refers to a part containing 1-8 carbon atoms, that is, a group containing 1 carbon atom, 2 carbon atoms, 3 carbon atoms... 8 carbon atoms. Therefore, for example, 'C'..." 1~8"Alkyl" refers to an alkyl group containing 1 to 8 carbon atoms, meaning the alkyl group is selected from methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl... octyl, etc. The numerical ranges used in this document, such as "1-8", refer to integers within a given range. For example, "1-8 carbon atoms" means that the group can have 1, 2, 3, 4, 5, 6, 7, or 8 carbon atoms.

[0018] The term "alkyl" refers to a saturated aliphatic hydrocarbon group that is optionally substituted, either straight-chain or branched, and is connected to the rest of the molecule by a single bond. As used herein, "alkyl" can have 1 to about 8 carbon atoms, for example, 1 to 6 carbon atoms, 1 to 4 carbon atoms, or 1 to 3 carbon atoms. The “alkyl” examples in this document include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, 2-methyl-1-propyl, 2-methyl-2-propyl, 2-methyl-1-butyl, 3-methyl-1-butyl, 2-methyl-3-butyl, 2,2-dimethyl-1-propyl, 2-methyl-1-pentyl, 3-methyl-1-pentyl, 4-methyl-1-pentyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 2,2-dimethyl-1-butyl, 3,3-dimethyl-1-butyl, 2-ethyl-1-butyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, tert-pentyl, hexyl, etc., as well as longer alkyl groups such as heptyl and octyl. When the term "alkyl" as defined in this article is used in a numerical range, such as "C1-8 alkyl," it refers to an alkyl group that can be composed of 1, 2, 3, 4, 5, 6, 7, or 8 carbon atoms. Similarly, "C1-4 alkyl" refers to an alkyl group that can be composed of 1, 2, 3, or 4 carbon atoms. The term "alkyl" in this article also includes cases where no numerical range is specified.

[0019] The term "alkenyl" refers to a monovalent hydrocarbon group, optionally substituted straight-chain or optionally substituted branched, having at least one C=C double bond. The alkenyl group has, but is not limited to, 2 to about 8 carbon atoms, such as 2 to about 6 carbon atoms, 2 to about 4 carbon atoms. The double bond in these groups can be in either cis or trans conformation and should be understood to include both isomers. Examples of alkenyl groups include, but are not limited to, vinyl (CH=CH2), 1-propenyl (CH2CH=CH2), isopropenyl (C(CH3)=CH2), butenyl, and 1,3-butadienyl. When an alkenyl group as defined herein uses a numerical range, such as "C2-8 alkenyl," it refers to an alkenyl group that can be composed of 2, 3, 4, 5, 6, 7, or 8 carbon atoms. The use of alkenyl groups herein also includes cases where no numerical range is specified.

[0020] The term "alkynyl" refers to an optionally substituted straight-chain or branched monovalent hydrocarbon group having at least one C≡C triple bond. The alkynyl group has, but is not limited to, 2 to about 8 carbon atoms, for example 2 to about 6 carbon atoms, or 2 to about 4 carbon atoms. Examples of alkynyl groups herein include, but are not limited to, acetylenyl, 2-propynyl, 2-butynyl, and 1,3-butadiynyl. When a numerical range is specified for alkynyl groups as defined herein, such as "C2-8 alkynyl," it refers to an alkynyl group that can consist of 2, 3, 4, 5, 6, 7, or 8 carbon atoms. The use of alkynyl groups herein also includes cases where no numerical range is specified.

[0021] The term "cycloalkyl" refers to a non-aromatic carbon-containing ring, including saturated carbon rings (such as cycloalkyl) or unsaturated carbon rings (such as cycloalkenyl). Carbon rings include monocyclic rings (having one ring), such as monocyclic cycloalkyl; dicyclic rings (having two rings), such as dicyclic cycloalkyl; and polycyclic rings (having more than two rings). The rings can be bridged or spirocyclic. Carbon rings (such as cycloalkyl or cycloalkenyl) can have 3 to 8 carbon atoms, for example, 3 to about 6 cyclic carbon atoms or 3 to about 5 cyclic carbon atoms.

[0022] The term "aryl" refers to an optionally substituted aromatic hydrocarbon group having 6 to about 20, such as 6 to 12 or 6 to 10 cyclic carbon atoms, which can be monocyclic, bicyclic, or more cyclic aryl groups. Bicyclic or more cyclic aryl groups can be a monocyclic aryl group fused with other independent rings, such as alicyclic, heterocyclic, aromatic, or aromatic-heterocyclic rings. Non-limiting examples of monocyclic aryl groups include monocyclic aryl groups with 6 to about 12, 6 to about 10, or 6 to about 8 cyclic carbon atoms, such as phenyl; bicyclic aryl groups, such as naphthyl; and polycyclic aryl groups, such as phenanthryl, anthracene, or azulel.

[0023] The term "heteroaryl" refers to an arbitrarily substituted heteroaryl group comprising about 5 to about 20, such as 5 to 12 or 5 to 10, skeletal cyclic atoms, wherein at least one (e.g., 1-4, 1-3, 1-2) of the cyclic atoms is a heteroatom, which is independently selected from, but not limited to, heteroatoms of oxygen, nitrogen, sulfur, phosphorus, silicon, selenium, and tin. Heteroaryl groups include monocyclic heteroaryl groups (having one ring), bicyclic heteroaryl groups (having two rings), or polycyclic heteroaryl groups (having more than two rings). In embodiments where two or more heteroatoms appear in the ring, the two or more heteroatoms may be identical to each other, or some or all of the two or more heteroatoms may be different from each other. Bicyclic or more cyclic heteroaryl groups can be a monocyclic heteroaryl group fused with other independent rings, such as alicyclic, heterocyclic, aromatic, or aromatic-heterocyclic groups (collectively referred to as fused cyclic heteroaryl groups). Non-limiting examples of heteroaryl groups include, but are not limited to, pyrrole, furanyl, thiophene, imidazolyl, oxazolyl, pyrazolyl, pyridyl, pyrimidinyl, pyrazinyl, quinolinyl, isoquinolinyl, tetrazolyl, triazolyl, triazinyl, benzofuranyl, benzothiophene, indole, isoindole, etc.

[0024] The term "heterocyclic group" refers to a non-aromatic heterocycle, including saturated or unsaturated heterocycles (containing unsaturated bonds), which do not possess a fully conjugated π-electron system. It can be classified into non-aromatic monocyclic, fused polycyclic, bridged, or spirocyclic systems. One or more (e.g., 1-4, 1-3, 1-2) of the cyclic atoms are heteroatoms, such as oxygen, nitrogen, or sulfur atoms. Heterocycles can include monocyclic (having one ring), bicyclic (having two bridging rings), or polycyclic (having more than two bridging rings); spirocyclic groups also include those with 3 to approximately 20 cyclic atoms, such as 3-approximately 10, 3-approximately 8, 4-approximately 8, 4-approximately 7, 5-approximately 8, or 5-approximately 6. Non-limiting examples of heterocyclic groups include ethylene oxide, cyclothioethylene, cycloazoethylene, acridine, oxobutylcycloyl, thiobutylcycloyl, tetrahydrofuranyl, pyrrolyl, oxazolyl, tetrahydropyrazolyl, pyrrolinyl, dihydrofuranyl, dihydrothiophenyl, piperidinyl, tetrahydropyranyl, tetrahydrothiaranyl, morpholinyl, piperazine, dihydropyridinyl, tetrahydropyridinyl, dihydropyranyl, tetrahydropyranyl, dihydrothiaranyl, azirheptanyl, oxaheptanyl, thioheptanyl, oxazadiabicyclo[2.2.1]heptyl and azispir[3.3]heptyl, etc.

[0025] The term "halogenated" or "halogen" refers to the substitution of at least one hydrogen atom in an optionally substituted group (such as alkyl, alkenyl, alkynyl, alkoxy, etc.) with a halogen (such as fluorine, chlorine, bromine, iodine, or a combination thereof). In some embodiments, two or more hydrogen atoms are replaced with the same halogen (e.g., difluoromethyl, trifluoromethyl); in other embodiments, two or more hydrogen atoms are replaced with halogens that are not exactly the same (e.g., 1-chloro-1-fluoro-1-iodoethyl).

[0026] The term "alkoxy" refers to an alkyl ether group (O-alkyl), and non-limiting examples of alkoxy groups include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, and tert-butoxy.

[0027] The term "alkyl acyl" refers to a group formed by an alkyl group bonded to -CO-. Non-limiting examples of this term include formyl, acetyl, propionyl, butyryl, etc. For example, the term "C..." 1-6 "alkyl acyl" refers to C 1-6 An alkyl group formed by attaching an alkyl group to a -CO- group. For example, the term "C 1-4 "alkyl acyl" refers to C 1-4 An alkyl group formed by attaching an alkyl group to a -CO- group.

[0028] The term "alkylsulfonyl" refers to a group consisting of an alkyl group attached to -SO2-. Non-limiting examples of this term include methanesulfonyl, ethanesulfonyl, propanesulfonyl, butanesulfonyl, etc. For example, the term "C..." 1-6 "alkylsulfonyl" refers to C 1-6 An alkyl group formed by attaching an alkyl group to -SO2-. For example, the term "C 1-4 "alkylsulfonyl" refers to C 1-4 An alkyl group formed by attaching an alkyl group to -SO2-.

[0029] The term "amino" refers to the -NH2 group, -NH(C) group, etc. 1~6 alkyl group or -N(C) 1~6 Alkyl)2 group. Specific examples of amino groups include, but are not limited to, -NH2, -NHCH3, -N(CH3)2, -NHC2H5, -N(C2H5)2, -N(C3H7)2, -N(CH3)C2H5, etc.

[0030] Other group terms in this article include: "hydroxyl" refers to the -OH group, "thiol" refers to the -SH group, "cyano" refers to the -CN group, and "carboxyl" refers to the -COOH group.

[0031] The term "membered ring" refers to the number of skeleton atoms that make up the ring. For example, pyridine is a six-membered ring, and pyrrole is a five-membered ring.

[0032] The term "pharmaceutical acceptable" refers to compounds, materials, compositions, and / or dosage forms that, within the bounds of reliable medical judgment, are suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, in proportion to a reasonable benefit / risk ratio.

[0033] The drug refers to a bioactive compound optionally mixed with at least one pharmaceutically acceptable chemical component or reagent, which is a "carrier" that facilitates the introduction of the compound into cells or tissues, including but not limited to stabilizers, diluents, suspending agents, thickeners and / or excipients.

[0034] The term "pharmaceutically acceptable salt" refers to a salt that retains the biological potency of the specified compound's free acid and free base and has no adverse effects in biological or other respects. Unless otherwise specified, the term "salt" in this application may refer to metal salts, ammonium salts, salts formed with organic bases, salts formed with inorganic acids, salts formed with basic or acidic amino acids, etc. Non-limiting examples of metal salts include, but are not limited to, alkali metal salts, such as sodium salts, potassium salts, etc.; alkaline earth metal salts, such as calcium salts, magnesium salts, barium salts, etc.; aluminum salts, etc. Non-limiting examples of salts formed with organic bases include, but are not limited to, salts formed with trimethylamine, triethylamine, pyridine, methylpyridine, 2,6-dimethylpyridine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine, etc. Non-limiting examples of salts formed with inorganic acids include, but are not limited to, salts formed with hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid, etc. Non-limiting examples of salts formed with organic acids include, but are not limited to, salts formed with formic acid, acetic acid, trifluoroacetic acid, fumaric acid, oxalic acid, malic acid, maleic acid, tartaric acid, citric acid, succinic acid, methanesulfonic acid, benzenesulfonic acid, and p-toluenesulfonic acid. Non-limiting examples of salts formed with basic amino acids include, but are not limited to, salts formed with arginine, lysine, and ornithine. Non-limiting examples of salts formed with acidic amino acids include, but are not limited to, salts formed with aspartic acid and glutamic acid.

[0035] Pharmaceutically acceptable salts can be synthesized from parent compounds containing an acid radical or a base using conventional chemical methods. Generally, such salts are prepared by reacting these compounds, in their free acid or base form, with a stoichiometric amount of a suitable base or acid in water, an organic solvent, or a mixture of both. Non-aqueous media such as ethers, ethyl acetate, ethanol, isopropanol, or acetonitrile are generally preferred.

[0036] The term "solvent" refers to a physical aggregate formed by a compound of this application with one or more solvent molecules, including varying degrees of ions and covalent bonds, such as hydrogen bonds. It has been demonstrated that this solvate can be separated, for example, when one or more solvent molecules are mixed in the crystal lattice. "Solvent" comprises both a solvent phase and a separable solvate component. Numerous examples of solvates exist, including ethanol solvates, methanol solvates, etc. "Hydrate" is a solvate that uses water (H₂O) molecules as a solvent. One or more compounds of this application can be freely prepared as solvates. The preparation of solvates is well known. A typical, non-limiting preparation process involves dissolving the compound of the invention in a desired amount of an ideal solvent (organic solvent, water, or a mixture thereof) at a temperature above room temperature, cooling, allowing crystals to crystallize, and then separating and picking out the crystals using standard methods. The presence of the solvent (water) in the crystallization can be confirmed using IR spectroscopy.

[0037] The term "pharmaceuticalally acceptable prodrug" or "prodrug" refers to any pharmaceutically acceptable salt, ester, salt of ester, or other derivative of the compound of this application, which, upon administration to a receptor, can directly or indirectly provide the compound of this application or its pharmaceutically active metabolites or residues. Particularly preferred derivatives or prodrugs are those compounds that, when administered to a patient, can improve the bioavailability of the compound of this application (e.g., make orally administered compounds more readily absorbed into the bloodstream), or those compounds that facilitate the delivery of the parent compound to a biological organ or site of action (e.g., the brain or lymphatic system). Prodrugs can be prepared by modifying functional groups present in the compound in a manner that allows them to decompose into the parent compound, either through conventional procedures or in vivo. Various forms of prodrugs are well known in the art.

[0038] The term "stereoisomer" refers to isomers resulting from different spatial arrangements of atoms in a molecule. The compounds in this application contain asymmetric or chiral centers, double bonds, etc.; therefore, the compounds in this application may include various isomer forms such as optical isomers, geometric isomers, tautomers, and blocked transisomers. These isomers, as well as their single isomers, racemates, etc., are all included within the scope of this application. For example, optical isomers can be prepared by chiral resolution, chiral synthesis, or chiral reagents or other conventional techniques. R )-and( S )-Isomers and D and L Isomers. For example, they can be converted to diastereomers by reacting with suitable optically active substances (such as chiral alcohols or Mosher's chloride), separated, and converted (e.g., hydrolyzed) to the corresponding single isomers. Alternatively, they can be separated by chromatographic column chromatography.

[0039] The drugs described herein can be prepared in a manner well known in the pharmaceutical field and can be administered or applied via a variety of routes, depending on whether local or systemic treatment is required and the area to be treated. They can be administered topically (e.g., transdermal, skin, eye, and mucous membrane delivery, including intranasal, vaginal, and rectal delivery), pulmonaryly (e.g., by inhalation or blowing of powders or aerosols, including via nebulizers; intratracheal, intranasal), or orally or parenterally. Parenterally administration includes intravenous, intra-arterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion; or intracranial, such as intrathecal or intraventricular administration. Parenterally administration can be in the form of a single large dose or via, for example, a continuous infusion pump. The pharmaceutical compositions described herein include, but are not limited to, the following forms: tablets, pills, powders, lozenges, capsules, elixirs, suspensions, emulsions, solutions, syrups, aerosols (solid or soluble in liquid solvents); ointments, soft and hard gelatin capsules, suppositories, sterile injectable solutions, and sterile packaged powders containing, for example, up to 10% by weight of an active compound.

[0040] The drugs described herein can be formulated in unit dosage forms, each containing approximately 0.1–1000 mg, typically approximately 5–1000 mg of active ingredient, and more typically approximately 100–500 mg of active ingredient. The term “unit dosage form” refers to a physically isolated single-dose unit suitable for use in human patients and other mammals, each unit containing a predetermined amount of active substance, calculated to produce the desired therapeutic effect, mixed with a suitable drug carrier.

[0041] The term "treatment" and other similar synonyms include relieving, reducing, or improving symptoms of a disease or condition; preventing other symptoms; improving or preventing the underlying metabolic causes of symptoms; inhibiting a disease or condition, such as preventing its progression; alleviating a disease or condition; improving a disease or condition; relieving symptoms caused by a disease or condition; or stopping the symptoms of a disease or condition. Furthermore, the term may also include a preventative purpose. The term also includes achieving therapeutic and / or preventative effects. A therapeutic effect refers to the cure or improvement of the underlying disease being treated. Additionally, the cure or improvement of one or more physiological symptoms associated with the underlying disease is also a therapeutic effect; for example, an improvement is observed in a patient even though they may still be affected by the underlying disease. In terms of preventative effects, the composition or compound may be administered to a patient at risk of developing a specific disease, or to a patient exhibiting one or more physiological symptoms of a disease, even if no disease diagnosis has been made.

[0042] The terms "dosage to achieve the necessary therapeutic effect" or "therapeutic effective dose" refer to the amount of at least one drug or compound, when administered, sufficient to alleviate one or more symptoms of the disease or condition being treated to some extent. The result may be a reduction and / or relief of signs, symptoms, or causes, or any other desired change in the biological system. Effective doses suitable for any individual case can be determined using techniques such as dose escalation testing. The actual amount of compound, pharmaceutical composition, or drug administered is usually determined by the physician based on relevant circumstances, including the condition being treated, the chosen route of administration, the actual compound administered, the individual patient's age, weight, and response, and the severity of the patient's symptoms.

[0043] The proportion or concentration of the compound in the pharmaceutical composition may not be fixed and depends on various factors, including dosage, chemical properties (e.g., hydrophobicity), route of administration, etc. For example, the compound may be provided in a physiologically buffered aqueous solution containing about 0.1 to 10% w / v of the compound for parenteral administration. Some typical dosage ranges are from about 1 μg / kg to about 1 g / kg body weight / day. In some embodiments, the dosage range is from about 0.01 mg / kg to about 100 mg / kg body weight / day. The dosage is likely to depend on such variables as the type and severity of the disease or condition, the general health status of the specific patient, the relative biological potency of the selected compound, the excipient formulation, and the route of administration.

[0044] The term "administration" refers to a method capable of delivering a compound or composition to the desired site for biological action. These methods include, but are not limited to, oral administration, duodenal administration, parenteral administration (including intravenous, subcutaneous, intraperitoneal, intramuscular, intra-arterial injection or infusion), topical application, and rectal administration. Administration techniques that can be used with the compounds and methods described herein are well known to those skilled in the art.

[0045] This application relates to the use of JAK inhibitors or pharmaceutically acceptable salts, solvates, active metabolites, polymorphs, isotope labels, isomers, or prodrugs thereof in medicaments for the treatment or prevention of ischemic stroke.

[0046] The JAK inhibitors mentioned above can be selected from pyrrolopyrimidine compounds represented by general formula (I): Ⅰ in, L is selected from -C(=O)-, -S(=O)2- or -CONH-; X is selected from CR4 or N; Ar is selected from the following groups, whether substituted or unsubstituted: C 3~8 cycloalkyl, C 3~8 Heterocyclic group, C 6~20 Aryl or C 5~20Heteroaryl groups, with substituents selected from halogens, C 1~8 Alkyl, C 1~8 Halogenated alkyl or C 1~8 Alkoxy; R1 is selected from the following groups, whether substituted or unsubstituted: halogen, amino, C 1~8 Alkyl, C 1~8 Alkoxy, C 2-8 alkenyl, C 2-8 alkynyl group, C 3~8 cycloalkyl, C 3~8 Heterocyclic group, C 6~20 Aryl or C 5~20 heteroaryl; substituents are selected from halogens, C 1~8 Alkyl, C 1~8 Haloalkyl, C 1~8 Alkoxy, C 3~8 cycloalkyl, C 3~8 Heterocyclic group, C 6~20 Aryl, C 5~20 heteroaryl, cyano, amino, hydroxyl, carboxyl, or thiol; R2 is selected from -NHR7, hydroxyl, or mercapto, where R7 is selected from the following substituted or unsubstituted groups: hydrogen, halogen, C 1~6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 1~6 Alkyl acyl, C 1~6 alkylsulfonyl, C 1~6 Halogenated alkyl or C 1~6 Alkoxy group; substituents are selected from cyano, amino, hydroxy, carboxyl, mercapto, C 3~6 cycloalkyl, C 3~8 Heterocyclic group, C 6~20 Aryl or C 5~20 Mixed aromatics; R3 is selected from hydrogen, halogens, and C. 1~6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 1~6 Haloalkyl, C 1~6 Alkoxy, cyano, amino, hydroxy, carboxyl, or thiol groups; R4 is selected from hydrogen, halogen, or C. 1~6 alkyl; R5 is selected from cyano, -CONH2, or carboxyl groups; R6 is selected from hydrogen, halogen, or C. 1~6 alkyl.

[0047] For the compound represented by general formula (Ⅰ), when the substituent Ar is selected from heterocyclic or heteroaryl groups, the substituent L may be attached to the heteroatom of the heterocyclic or heteroaryl group.

[0048] For the compound represented by general formula (Ⅰ), when the substituent L is selected from -CONH-, the substituent Ar is attached to the carbonyl group in the substituent.

[0049] For compounds represented by general formula (I), the substituent Ar can be selected from the following substituted or unsubstituted groups: C 5~7 cycloalkyl, C 6~12 aryl or C containing at least one heteroatom 5~12 Heterocyclic group or C 5~12 The heteroaryl group, wherein the heteroatom is selected from N, O, or S; in a preferred embodiment of the invention, the substituent Ar is selected from substituted or unsubstituted C atoms containing 1 to 3 N atoms. 5~6 Heterocyclic group or C 5~6 Heteroaryl. In a more preferred embodiment of the present invention, Ar may be selected from substituted or unsubstituted piperidinyl, piperazinyl, pyridinyl, pyrazinyl, pyridazinyl, triazinyl, pyrimidinyl, pyrrolyl, pyrrolidinyl, imidazolyl, triazolyl, tetrazolyl, cyclopentyl, cyclohexyl, phenyl, biphenyl, naphthyl, etc.

[0050] For the compound represented by general formula (Ⅰ), the substituent of the substituent group Ar can be a halogen, that is, the substituent group Ar can be replaced by fluorine, chlorine, bromine or iodine atoms, and the number of substituents can be one or more, and the types can be the same or different.

[0051] For compounds represented by general formula (I), the substituent R2 may be selected from -NHR7, wherein R7 may be selected from the following groups, either substituted or unsubstituted: hydrogen, halogen, C 1~4 Alkyl, C 2-4 alkenyl, C 2-4 alkynyl group, C 1~4 Alkyl acyl, C 1~4 alkylsulfonyl, C 1~4 Halogenated alkyl or C 1~4 Alkoxy; substituents can be selected from cyano, amino, hydroxy, carboxyl, mercapto, C 3~5 cycloalkyl, C 3~6 Heterocyclic group, C 6~12 Aryl or C 5~12 Heteroaryl. In a preferred embodiment of the invention, R7 may be selected from hydrogen, methyl, ethyl, propyl, isopropyl, n-butyl, methylacyl, ethylacyl, propylacyl, methylsulfonyl, ethylsulfonyl, propylsulfonyl, methoxy, ethoxy, propoxy, etc., and the optional substituents may be fluorine, chlorine, bromine, iodine, cyano, amino, hydroxyl, carboxyl, mercapto, etc. , It can be phenyl, naphthyl, five- or six-membered heterocyclic or heteroaryl, wherein the heteroatom can be at least one of N, O, and S, and the number of substituents can be one or more, and the types can be the same or different.

[0052] For compounds represented by general formula (Ⅰ), the substituent X may be selected from CH.

[0053] For compounds represented by general formula (I), the substituent R1 may be selected from the following substituted or unsubstituted groups: C 1~6 Alkyl, C 1~6 Alkoxy, C 3~6 cycloalkyl, C 6~12 Aryl, C containing at least one heteroatom 3~6 Heterocyclic group or C 5~12 The heteroaryl group, wherein the heteroatom is selected from N, O, or S. In a preferred embodiment of the invention, the substituent R1 may be selected from the following substituted or unsubstituted groups: C 1~4 Alkyl, C 1~4 Alkoxy, phenyl, biphenyl, naphthyl, and C atoms containing 1 to 3 nitrogen atoms 5~6 Heterocyclic group or C 5~6 Heteroaryl. The optional substituents of the substituent R1 can be halogens, C... 1~4 Alkyl or C 1~4 Halogenated alkyl group. In a more preferred embodiment of the invention, R1 may be selected from methyl, ethyl, propyl, isopropyl, n-butyl, vinyl, propenyl, ethynyl, propynyl, methoxy, ethoxy, propoxy, etc. , Cyclopentyl, cyclohexyl, phenyl, biphenyl, naphthyl, piperidinyl, piperazine, pyridinyl, pyrazine, pyridazine, triazine, pyrimidinyl, pyrrolyl, pyrrolidinyl, imidazolyl, triazolyl, tetrazolyl, etc.; optional substituents can be fluorine, chlorine, bromine, iodine, methyl, ethyl, propyl, isopropyl, n-butyl, trifluoromethyl, tribromomethyl, etc., and the number of substituents can be one or more, and the types of substituents can be the same or different.

[0054] For compounds represented by general formula (Ⅰ), the substituent R3 may be selected from hydrogen, halogen, C 1~4 Alkyl, C 2-4 alkenyl, C 2-4 alkynyl group, C 1~4 Haloalkyl, C 1~4 Alkoxy, cyano, amino, hydroxy, carboxyl, or mercapto. In a preferred embodiment of the invention, the substituent R3 may be selected from hydrogen, methyl, ethyl, propyl, isopropyl, n-butyl, cyano, amino, hydroxy, carboxyl, mercapto, methoxy, ethoxy, propoxy, or trifluoromethyl.

[0055] This application provides the use of the compound of formula (I) or a pharmaceutically acceptable salt, solvate or isomer thereof in the preparation of a medicament for the treatment or prevention of ischemic stroke. Ⅰ in, L is selected from -C(=O)-, -S(=O)2- or -CONH-; X is selected from CH or N; Ar is selected from C6 aryl, 5-6 membered heterocyclic alkyl or 5-6 membered heteroaryl, wherein the heteroatom is selected from N, O or S, and the C6 aryl, 5-6 membered heterocyclic alkyl or 5-6 membered heteroaryl may optionally be substituted with one or more halogens; R1 is selected from C 1~6 Alkyl, C 1~6 alkoxy, C6 aryl, or 5-6 heteroaryl, wherein the heteroatom is selected from N, O, or S, wherein the C 1~6 Alkyl, C 1~6 Alkoxy, C6 aryl, or 5-6 heteroaryl groups may be optionally substituted with one or more of the following substituents: halogen, C 1~4 Alkyl or C 1~4 Halogenated alkyl groups; R2 is selected from -NH2; R3 is selected from hydrogen or -NH2; R5 is selected from cyano groups; R6 is selected from hydrogen.

[0056] The number of heteroatoms can be 1, 2, 3 or 4.

[0057] This application provides the use of the compound of formula (I) or a pharmaceutically acceptable salt, solvate or isomer thereof in the preparation of a medicament for the treatment or prevention of reperfusion injury after ischemic stroke.

[0058] The aforementioned ischemic stroke reperfusion injury refers to the situation where, after an ischemic stroke, when blood flow is restored to the blocked blood vessel (i.e., "reperfusion"), the damage is not immediately repaired. Instead, it may trigger a series of new pathological reactions, such as oxidative stress, inflammatory response, calcium overload, and apoptosis, leading to further damage to brain tissue.

[0059] In some implementations, R1 is selected from C 1~4 Alkyl, C 1~4 alkoxy, C6 aryl, or 6-membered heteroaryl, wherein the heteroatom is selected from N, O, or S, wherein the C 1~4 Alkyl, C 1~4 Alkoxy, C6 aryl, or 6-membered heteroaryl are optionally substituted with one or more of the following substituents: halogen, C 1~4 Alkyl or C 1~4Halogenated alkyl groups.

[0060] In this application, the number of heteroatoms can be 1, 2, 3 or 4.

[0061] In some implementations, R1 is selected from C 1~4 Alkyl, C 1~4 alkoxy, C6 aryl, or 6-membered heteroaryl, wherein the heteroatom is selected from N, O, or S, wherein the C 1~4 The alkyl, C6 aryl, or 6-membered heteroaryl group may be optionally substituted with one or more of the following substituents: -F or -CF3. The number of heteroatoms may be 1, 2, 3, or 4.

[0062] In some implementations, R3 is hydrogen.

[0063] In some embodiments, Ar is selected from C6 aryl, 6-membered heterocyclic alkyl, or 6-membered heteroaryl, wherein the heteroatom is selected from N, O, or S, and the C6 aryl, 6-membered heterocyclic alkyl, or 6-membered heteroaryl may optionally be substituted with one or more halogens.

[0064] In some embodiments, Ar is a group that is optionally substituted with one or more halogens: , in, Indicates the position connected to L. This indicates the position where it is attached to the nitrogen-containing heterocyclic butyl group.

[0065] In some embodiments, the halogen is -F.

[0066] In some embodiments, the compound is selected from one of the following structures:

[0067] To make the objectives, technical solutions, and advantages of the present invention clearer, the technical solutions of exemplary embodiments of the present invention will be further described below.

[0068] The compounds described herein can be prepared using the methods described below. The following methods and examples are for illustrative purposes. These procedures and examples should not be construed as limiting the scope of this application in any way. The compounds described herein can also be synthesized using standard synthetic techniques known to those skilled in the art, or in combination with methods known in the art.

[0069] The chemical reactions in the embodiments of this application are carried out in a suitable solvent, which must be suitable for the chemical changes and the reagents and materials required in this application. In order to obtain the compounds of this application, it is sometimes necessary for those skilled in the art to modify or select the synthesis steps or reaction process based on existing embodiments.

[0070] A crucial consideration in planning any synthetic route in this field is selecting appropriate protecting groups for reactive functional groups, such as the amino groups in this application. For trained practitioners, Greene and Wuts's (Protective Groups in Organic Synthesis, Wiley and Sons, 1991) is the authority on this matter. All references cited in this application are incorporated herein by reference in their entirety.

[0071] The reactions described herein can be monitored using any suitable method known in the art. For example, they can be monitored using broad-spectrum methods such as nuclear magnetic resonance spectroscopy (e.g., 1 H or 13 C) Infrared spectroscopy, spectrophotometry (e.g., UV-Vis light), mass spectrometry, or monitoring of product formation by chromatography such as high performance liquid chromatography (HPLC) or thin layer chromatography.

[0072] This application is illustrated in more detail by way of specific embodiments. The following embodiments are provided for illustrative purposes and are not intended to limit this application in any way. Those skilled in the art will readily recognize that various non-critical parameters can be changed or modified to obtain substantially the same results.

[0073] Example 1 2-(3-(3-amino-4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-1-(1-(3-fluoro-2-(trifluoromethyl)isonicotinyl)piperidin-4-yl)azacyclobut-3-yl)acetonitrile

[0074] Step A: tert-butyl-4-(3-(cyanomethylene)azacyclobut-1-yl)piperidine-1-carbonate

[0075] 40 g (0.3 mol, 1.0 eq) of 2-(azacyclobut-3-yl)acetonitrile hydrochloride (the compound was prepared according to patent US2014 / 256941A1 Paragraph0145) and 61 g (0.3 mol, 1.0 eq) of N-tert-butoxycarbonyl-4-piperidinone were dissolved in 500 mL of dichloromethane and cooled to 0 °C in an ice-water bath under nitrogen protection. oC. Slowly add 130 g (0.6 mol, 2.0 eq) of sodium borohydride acetate in batches, maintaining the reaction in an ice bath and monitoring the reaction for completion by TLC. Slowly pour the reaction solution into water and extract the aqueous phase with dichloromethane. Combine the organic phases, wash them successively with water and saturated sodium bicarbonate, dry the organic phase with anhydrous sodium sulfate, evaporate to dryness under reduced pressure, and precipitate the residue by column chromatography to obtain the product (40 g, yield = 85%).

[0076] Step B: 2-(1-(piperidin-4-yl)azacyclobut-3-ylidene)acetonitrile hydrochloride

[0077] 40 g (0.14 mol, 1.0 eq) of tert-butyl-4-(3-(cyanomethylene)azacyclobutyl-1-yl)piperidine-1-carbonate was dissolved in 250 mL of anhydrous ethanol and cooled to 0 °C in an ice-water bath under nitrogen protection. o C. Slowly add 40g of concentrated hydrochloric acid, and then slowly raise the temperature to room temperature. Monitor the reaction progress by TLC. Filter the suspension, wash the filter cake with anhydrous ethanol, and dry under vacuum to obtain the product (24g, yield = 81%).

[0078] Step C: 2-(1-(1-(3-fluoro-2-(trifluoromethyl)isonicotinyl)piperidin-4-yl)azacyclobut-3-ylidene)acetonitrile

[0079] 650 mg (3.0 mmol, 1.0 eq) of 2-(1-(piperidin-4-yl)azacyclobut-3-ylidene)acetonitrile hydrochloride, 630 mg (3.0 mmol, 1.0 eq) of 2-fluoro-3-trifluoromethylpyridinecarboxylic acid, and 2 g (15 mmol, 5.0 eq) of N,N-diisopropylethylamine were dissolved in 30 mL of dichloromethane at room temperature. 1.4 g (3.6 mmol, 1.2 eq) of HATU was added, and the mixture was stirred overnight at room temperature. The reaction was quenched with 100 mL of water. The aqueous phase was extracted with dichloromethane, and the organic phase was dried over anhydrous sodium sulfate and evaporated under reduced pressure. The residue was subjected to column chromatography (PE:EA = 1:1 to 0:1) to give the product (1.1 g, yield = 98%). 1 H-NMR (400 MHz, DMSO-D6): δ 8.67 (d, J = 4.5 Hz, 1H), 7.91(t, J= 4.5 Hz, 1H), 5.69-5.71(m,1H), 3.90-4.15(m, 5H), 3.25-3.50 (m, 2H), 3.07-3.20 (m, 1H), 2.51-2.64 (m,1H), 1.55-1.85 (m, 2H), 1.10-1.30 (m, 2H).

[0080] Step D: 4-Chloro-7-([2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrole[2,3-d]pyrimidine

[0081] Under ice-bath stirring, 38.4 g (250.4 mmol, 1.0 eq.) of 4-chloro-7H-pyrrolo[2,3-d]pyrimidine was dissolved in 200 mL of dry DMF solution, and 13 g (305 mmol, 1.2 eq.) of 57% NaH was added. The reaction mixture was stirred at room temperature for 1 hour, and then 50.9 g of SEMCl (305 mmol, 1.2 eq.) was added dropwise under ice-bath cooling. After the addition was complete, the reaction mixture was stirred in an ice-bath for 1 hour, quenched with water, extracted with ethyl acetate, and the combined organic phases were washed with brine, dried over sodium sulfate, filtered, and concentrated under vacuum. The target compound (71 g, yield = 100%) was obtained by column chromatography on silica gel column.

[0082] Step E: 2-cyano-2-(7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)ethyl acetate

[0083] Under stirring at room temperature, 33.5 g (118 mmol, 1.0 eq.) of 4-chloro-7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidine was added to a mixture of 40.1 g (354.0 mmol, 3.0 eq.) ethyl cyanoacetate and 33.0 g (238 mmol, 2.0 eq.) potassium carbonate. The reaction mixture was heated to 60 °C for 0.5 h, then heated to 130 °C for 1.0 h. After cooling to room temperature, the reaction mixture was quenched with water, extracted with ethyl acetate, washed with brine, dried over sodium sulfate, filtered, concentrated under vacuum, and separated by silica gel column chromatography to obtain the target compound (30.6 g, yield = 72%).

[0084] Step F: 2-(7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)acetonitrile

[0085] 50 g (855.6 mmol, 10.0 eq.) of sodium chloride was added to a mixture of 30.6 g (84.9 mmol, 1.0 eq.) of ethyl 2-cyano-2-(7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)acetate in DMSO and water under stirring at room temperature. The reaction mixture was reacted at 150 °C for 6 days under nitrogen protection. After cooling to room temperature, the reaction was quenched with water, extracted with ethyl acetate, and the combined organic phases were washed with brine, dried over sodium sulfate, filtered, concentrated under vacuum, and separated by silica gel column chromatography to obtain the target compound (18.1 g, yield = 74%).

[0086] Step G: 4-(7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-3-amino-1H-pyrazole

[0087] Under stirring at room temperature, 37 g of 85% hydrazine hydrate (628 mmol, 10.0 eq.) was added to 18.1 g (62.8 mmol, 1.0 eq.) of 2-(7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)acetonitrile and 22.4 g (188 mmol, 3 eq.) of DMF-DMA in 80 mL of DMF solution. The reaction mixture was stirred and refluxed at 90 °C for 3 hours under nitrogen protection. After cooling to room temperature, 100 mL of water was added and stirred. The mixture was filtered and dried to obtain the target compound (11.0 g, yield = 53%). 1 HNMR (400MHz, DMSO- d 6): δ 12.13 (brs, 1H), 8.65 (s, 1H), 8.18 (brs, 1H), 7.61(d, J =3.2 Hz, 1H), 7.01 (d, J =3.6 Hz, 1H), 6.55 (brs, 2H), 5.60 (s, 2H), 3.52(t, J =8.0 Hz, 2H), 0.83 (t, J =8.0 Hz, 2H), -0.10 (s, 9H). LC-MS: m / z = 331[M+1] + .

[0088] Step H: 2-(3-(3-amino-4-(7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-1-(1-(3-fluoro-2-(trifluoromethyl)isonicotinyl)piperidin-4-yl)azacyclobut-3-yl)acetonitrile

[0089] 800 mg (2.4 mmol, 1.2 eq) 4-(7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-3-amine and 740 mg (2.0 mmol, 1.0 eq) 2-(1-(1-(3-fluoro-2-(trifluoromethyl)isonicotinyl)piperidin-4-yl)azacyclobut-3-ylidene)acetonitrile were dissolved in 15 mL of acetonitrile. 920 mg (6.0 mmol, 3.0 eq) 1,8-diazabicyclo[5.4.0]undec-7-ene was slowly added dropwise to the reaction system at room temperature, and the mixture was stirred overnight at room temperature. The reaction was quenched with 50 mL of saturated sodium bicarbonate aqueous solution, the aqueous phase was extracted with ethyl acetate, the organic phase was dried over anhydrous sodium sulfate, evaporated under reduced pressure, and the residue was subjected to column chromatography (PE:EA = 1:1~0:1) to give the product (878 mg, yield = 63%).

[0090] Step I: 2-(3-(3-amino-4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-1-(1-(3-fluoro-2-(trifluoromethyl)isonicotinyl)piperidin-4-yl)azacyclobut-3-yl)acetonitrile

[0091] Method 1: 260 mg (0.37 mmol, 1.0 eq) of 2-(3-(3-amino-4-(7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-1-(1-(3-fluoro-2-(trifluoromethyl)isonicotinyl)piperidin-4-yl)azacyclobut-3-yl)acetonitrile was dissolved in a mixture of 8 mL dichloromethane and 1 mL trifluoroacetic acid at room temperature. The reaction was carried out overnight at room temperature. The solvent was evaporated under reduced pressure, and the residue was dissolved in 10 mL methanol. 0.5 mL ethylenediamine was added at room temperature, and the mixture was stirred for 30 minutes at room temperature. The reaction solution was poured into 50 mL water, and the aqueous phase was extracted with ethyl acetate. The organic phase was dried over anhydrous sodium sulfate and evaporated under reduced pressure. The residue was subjected to column chromatography (EA:MeOH = 1:0~25:1) to obtain the product (150 mg, (Yield = 70%).

[0092] Method 2: 478 mg (0.7 mmol, 1.0 eq) of 2-(3-(3-amino-4-(7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-1-(1-(3-fluoro-2-(trifluoromethyl)isonicotinyl)piperidin-4-yl)azacyclobut-3-yl)acetonitrile was dissolved in 15 mL of acetonitrile at room temperature. 500 mg (3.5 mmol, 5.0 eq) of boron trifluoride diethyl ether complex was slowly added dropwise under ice bath conditions. After the addition was complete, the reaction was allowed to proceed at room temperature for 4 hours. The reaction system was then cooled to 50°C under ice bath conditions. o Below C, 8 mL of 25% ammonia solution was slowly added dropwise to the system, and the mixture was stirred at room temperature for 60 minutes. The reaction solution was then poured into 80 mL of water, and the aqueous phase was extracted with ethyl acetate. The organic phase was dried over anhydrous sodium sulfate and evaporated under reduced pressure. The residue was subjected to column chromatography (EA:MeOH = 1:0~25:1) to obtain the product (195 mg, yield = 50%).

[0093] 1 H-NMR (400 MHz, CDCl3): δ 10.15 (s, 1H), 8.82 (s, 1H), 8.60 (d, J =4.5 Hz, 1H), 8.06 (s, 1H), 7.56 (t, J = 4.4 Hz, 1H), 7.37 (dd, 1H), 6.69 (dd,1H), 5.74 (s, 2H), 4.17-4.29 (m, 1H), 3.76-3.69 (m, 2H), 3.57-3.64 (m, 2H), 3.42-3.54 (s, 2H), 3.34 (s, 2H), 3.07-3.20 (m, 1H), 2.54-2.64 (m, 1H), 1.60-1.92 (m, 2H), 1.37-1.57 (m, 2H). LC-MS: m / z = 569[M+1] + .

[0094] Example 2 2-(3-(3-amino-4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-1-(1-(2-fluoro-3-(trifluoromethyl)phenyl)piperidin-4-yl)azacyclobut-3-yl)acetonitrile

[0095] Step A: 2-(1-(1-(2-fluoro-3-(trifluoromethyl)benzoyl)piperidin-4-yl)azacyclobutyronin-3-ylidene)acetonitrile

[0096] The compound (550 mg, yield = 98%) was prepared according to step C described in Example 1. 1 H NMR (400MHz, CDCl3): δ 7.71 (t, J = 7.0 Hz, 1H), 7.62 (t, J = 6.2 Hz, 1H), 7.37 (t, J =7.7 Hz, 1H), 5.29-5.36 (m, 1H), 4.20-4.30 (m, 1H), 4.10 (s, 2H), 4.01 (s,2H), 3.48-3.59 (m, 1H), 3.37-3.48 (m, 1H), 3.10-3.25 (m, 1H), 2.52-2.63 (m,1H), 1.65-1.95 (m, 2H), 1.25-1.59 (m, 2H).

[0097] Step B: 2-(3-(3-amino-4-(7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-1-(1-(2-fluoro-3-(trifluoromethyl)benzoyl)piperidin-4-yl)azacyclobut-3-yl)acetonitrile

[0098] The compound (720 mg, yield = 71%) was prepared according to step H described in Example 1.

[0099] Step C: 2-(3-(3-amino-4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-1-(1-(2-fluoro-3-(trifluoromethyl)benzoyl)piperidin-4-yl)azacyclobut-3-yl)acetonitrile

[0100] The compound (210 mg, yield = 36%) was prepared according to method 1 in step E of Example 1. 1 H NMR (400 MHz, DMSO-D6): δ 12.09 (s, 1H), 8.68 (s, 1H), 8.54 (s, 1H), 7.90 (t, J =7.1 Hz, 1H), 7.79 (t, J = 6.6 Hz, 1H), 7.54 (dd,J = 16.7, 8.9 Hz, 2H), 7.09(d, J = 1.7 Hz, 1H), 6.36 (s, 2H), 4.02-4.22 (m, 1H), 3.65-3.78 (m, 2H), 3.46-3.57 (m, 4H), 3.40-3.45 (m, 1H), 3.22-3.33 (m, 1H), 3.03-3.15 (m, 1H), 2.50-2.60 (m, 1H), 1.74-1.84 (m, 1H), 1.61-1.73 (m, 1H), 1.16-1.37 (m, 2H). LC-MS: m / z = 568[M+1] + .

[0101] Example 3 2-(3-(3-amino-4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-1-(1-(1-(pyrimidin-4-formyl)piperidin-4-yl)azacyclobut-3-yl)acetonitrile

[0102] Step A: 2-(1-(1-(pyrimidin-4-formyl)piperidin-4-yl)azacyclobutane-3-ylidene)acetonitrile

[0103] The compound (570 mg, yield = 99%) was prepared according to step C described in Example 1. 1 H NMR (400MHz, CDCl3): δ 9.28 (s, 1H), 8.93 (d, J = 5.0 Hz, 1H), 7.63 (d, J = 4.8 Hz,1H), 5.33 (s, 1H), 4.17-4.26 (m, 1H), 4.11 (s, 2H), 4.01 (s, 2H), 3.70-3.87(m, 1H), 3.40-3.50 (m, 1H), 3.24-3.37 (m, 1H), 2.54-2.65(m, 1H), 1.70-1.92(m, 2H), 1.41-1.59 (m, 2H).

[0104] Step B: 2-(3-(3-amino-4-(7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-1-(1-(pyrimidin-4-formyl)piperidin-4-yl)azacyclobut-3-yl)acetonitrile

[0105] The compound (980 mg, yield = 82%) was prepared according to step H described in Example 1.

[0106] Step C: 2-(3-(3-amino-4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-1-(1-(1-(pyrimidin-4-formyl)piperidin-4-yl)azacyclobut-3-yl)acetonitrile

[0107] The compound (150 mg, yield = 20%) was prepared according to method 1 in step E of Example 1. 1 H NMR (400 MHz, DMSO): δ 12.06 (s, 1H), 9.24 (s, 1H), 8.95 (d, J = 5.1 Hz, 1H), 8.65(s, 1H), 8.50 (s, 1H), 7.65 (d, J = 5.1 Hz, 1H), 7.54 (s, 1H), 7.06 (s, 1H), 6.33 (s, 2H), 4.02-4.13(m, 1H), 3.68 (d, J = 7.7 Hz, 2H), 3.38-3.54 (m, 5H), 3.16-3.26 (m, 1H), 3.03-3.14 (m, 1H), 2.50-2.57 (m, 1H), 1.72-1.82 (m, 1H), 1.59-1.70 (m, 1H), 1.18-1.33 (m, 2H). LC-MS: m / z = 484[M+1] + .

[0108] Example 4 2-(3-(3-amino-4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-1-(1-(2-trifluoromethylpyrimidin-4-formyl)piperidin-4-yl)azacyclobut-3-yl)acetonitrile

[0109] Step A: 2-(1-(1-(2-trifluoromethylpyrimidin-4-formyl)piperidin-4-yl)azacyclobutane-3-ylidene)acetonitrile

[0110] The compound (480 mg, yield = 95%) was prepared according to step C described in Example 1.

[0111] Step B: 2-(3-(3-amino-4-(7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-1-(1-(2-trifluoromethylpyrimidin-4-formyl)piperidin-4-yl)azacyclobut-3-yl)acetonitrile

[0112] The compound (780 mg, yield = 78%) was prepared according to step H described in Example 1.

[0113] Step C: 2-(3-(3-amino-4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-1-(1-(2-trifluoromethylpyrimidin-4-formyl)piperidin-4-yl)azacyclobut-3-yl)acetonitrile

[0114] The compound (185 mg, yield = 32%) was prepared according to method 1 in step E of Example 1. LC-MS: m / z = 552 [M+1] + .

[0115] Example 5 4-(3-(3-amino-4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-(cyanomethyl)azacyclobut-1-yl)-N-(4-fluoro-2-(trifluoromethyl)phenyl)piperidin-1-carboxamide

[0116] Step A: 4-(3-(cyanomethylidene)azacyclobut-1-yl)-N-(4-fluoro-2-(trifluoromethyl)phenyl)piperidine-1-carboxamide

[0117] 325 mg (1.8 mmol, 1.2 eq) of 4-fluoro-2-trifluoromethylaniline and 760 mg (7.5 mmol, 5.0 eq) of triethylamine were dissolved in 20 mL of dichloromethane. 225 mg (0.76 mmol, 0.5 eq) of triphosgene was added under ice bath conditions, and the mixture was stirred for 5 minutes while maintaining the ice bath. Then, 320 mg (1.5 mmol, 1.0 eq) of 2-(1-(piperidin-4-yl)azacyclobutane-3-yl)acetonitrile hydrochloride was added in a single batch. After the addition was complete, the mixture was slowly allowed to rise to room temperature and reacted overnight. The reaction was quenched with 50 mL of water. The aqueous phase was extracted with dichloromethane, and the organic phase was dried over anhydrous sodium sulfate and evaporated to dryness under reduced pressure. The residue was subjected to column chromatography (PE:EA = 1:1~0:1) to give the product (324 mg, yield = 57%). 1 H NMR (400 MHz, CDCl3): δ 7.98 (dd, J = 8.9, 5.0 Hz, 1H),7.25-7.32 (m, 1H), 7.19-7.24 (m, 1H), 6.66 (s, 1H), 5.28 (s, 1H), 4.02-4.09(m, 2H), 3.92-4.01 (m, 2H), 3.77-3.85 (m, 2H), 3.15-3.20 (m, 2H), 2.42-2.53(m, 1H), 1.71-1.82 (m, 2H), 1.44-1.37 (m, 2H).

[0118] Step B: 4-(3-(3-amino-4-(7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-(cyanomethyl)azacyclobut-1-yl)-N-(4-fluoro-2-(trifluoromethyl)phenyl)piperidin-1-carboxamide

[0119] The compound (500 mg, yield = 84%) was prepared according to step H described in Example 1.

[0120] Step C: 4-(3-(3-amino-4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-(cyanomethyl)azacyclobut-1-yl)-N-(4-fluoro-2-(trifluoromethyl)phenyl)piperidin-1-carboxamide

[0121] The compound (90 mg, yield = 22%) was prepared according to method 1 in step E of Example 1.1 H NMR (400 MHz, DMSO): δ 12.09 (s, 1H), 8.68 (s, 1H), 8.53 (s, 1H), 8.25 (s, 1H), 7.62-7.44 (m, 4H), 7.09 (s, 1H), 6.36 (s, 2H), 3.80-3.93 (m, 2H), 3.66-3.75(m, 2H), 3.55-3.46 (m, 4H), 2.94-3.09 (m, 2H), 2.37-2.49 (m, 1H), 1.63-1.75(m, 2H), 1.10-1.24 (m, 2H). LC-MS: m / z = 583[M+1] + .

[0122] Example 6 Methyl 5-(3-(3-amino-4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-(cyanomethyl)azacyclobut-1-yl)pyrazine-2-carboxylic acid

[0123] Step A: Methyl 5-(3-(cyanomethylidene)azacyclobut-1-yl)pyrazine-2-carboxylic acid

[0124] 520 mg (4.0 mmol, 1.0 eq) of methyl 5-chloropyrazine-2-carboxylate, 690 mg (4.0 mmol, 1.0 eq) of 2-(1-(piperidin-4-yl)azacyclobut-3-ylidene)acetonitrile hydrochloride, and 1.6 g (12 mmol, 3.0 eq) of N,N-diisopropylethylamine were dissolved in 20 mL of dioxane. The reaction mixture was heated under reflux with stirring for 2 hours. After the reaction was completed by TLC, the system was cooled to room temperature and poured into 150 mL of water. The suspension was filtered, and the filter cake was washed successively with water and diethyl ether. The filter cake was collected and dried under vacuum to give the product (840 mg, yield = 91%). 1 H NMR (400 MHz, CDCl3): δ 8.85 (s, 1H), 7.89 (s, 1H), 5.53 (s, 1H), 5.01 (s, 2H), 4.93 (s, 2H), 3.97 (s, 3H).

[0125] Step B: Methyl 5-(3-(3-amino-4-(7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-(cyanomethyl)azacyclobut-1-yl)pyrazine-2-carboxylic acid

[0126] The compound (820 mg, yield = 86%) was prepared according to step H described in Example 1.

[0127] Step C: Methyl 5-(3-(3-amino-4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-(cyanomethyl)azacyclobut-1-yl)pyrazine-2-carboxylic acid

[0128] The compound (40 mg, yield = 48%) was prepared according to method 2 in step E of Example 1. 1 H NMR (400 MHz, DMSO-D6): δ 12.09 (s, 1H), 8.71 (d, J = 1.3 Hz, 1H), 8.67 (d, J = 2.6Hz, 2H), 8.07 (d, J = 1.3 Hz, 1H), 7.59-7.54 (m, 1H), 7.11 (dd, J = 3.5, 1.6Hz, 1H), 6.43 (s, 2H), 4.81 (d, J = 10.0 Hz, 2H), 4.48 (d, J = 9.9 Hz, 2H),3.82 (s, 3H), 3.73 (s, 2H). LC-MS: m / z = 431[M+1] + .

[0129] Example 7 (S)-4-(3-(3-amino-4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-(cyanomethyl)azacyclobut-1-yl)-2,5-difluoro-N-(1,1,1-trifluoropropane-2-yl)benzamide

[0130] Step A: (S)-4-chloro-2,5-difluoro-N-(1,1,1-trifluoropropane-2-yl)benzamide

[0131] 1.5 g (10.0 mmol, 1.0 eq) of (S)-1,1,1-trifluoropropane-2-amine hydrochloride and 3.9 g (30.0 mmol, 3.0 eq) of N,N-diisopropylethylamine were dissolved in 30 mL of dichloromethane. 2.11 g (10.0 mmol, 1.0 eq) of 4-chloro-2,5-difluorobenzoyl chloride was slowly added under ice bath conditions. After addition, the mixture was slowly brought to room temperature and reacted for 1 hour. The reaction was monitored by TLC until completion. The reaction was quenched by adding 100 mL of saturated sodium bicarbonate. The aqueous phase was extracted with dichloromethane, and the organic phase was dried over anhydrous sodium sulfate and evaporated under reduced pressure. The residue was purified by column chromatography to give the product (2.5 g, yield = 87%).

[0132] Step B: (S)-4-(3-(cyanomethylene)azacyclobut-1-yl)-2,5-difluoro-N-(1,1,1-trifluoropropane-2-yl)benzamide

[0133] The compound (350 mg, yield = 56%) was prepared according to the method described in patent US20150246046A1 Paragraph0166.

[0134] Step C: (S)-4-(3-(3-amino-4-(7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-(cyanomethyl)azacyclobut-1-yl)-2,5-difluoro-N-(1,1,1-trifluoropropane-2-yl)benzamide

[0135] The compound (260 mg, yield = 82%) was prepared according to step H described in Example 1.

[0136] Step D: (S)-4-(3-(3-amino-4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-(cyanomethyl)azacyclobut-1-yl)-2,5-difluoro-N-(1,1,1-trifluoropropane-2-yl)benzamide

[0137] The compound (46 mg, yield = 53%) was prepared according to method 2 in step E of Example 1. LC-MS: m / z = 546 [M+1] + .

[0138] Example 8 5-(3-(3-amino-4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-(cyanomethyl)azacyclobut-1-yl)-N-isopropylpyrazine-2-carboxamide

[0139] Step A: 5-(3-(3-amino-4-(7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-basic)-3-(cyanomethyl)azacyclobut-1-yl)pyrazine-2-carboxylic acid

[0140] 200 mg (0.37 mmol, 1.0 eq) of methyl 5-(3-(3-amino-4-(7-(((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-(cyanomethyl)azacyclobut-1-yl)pyrazin-2-carboxylic acid was dissolved in a mixture of 10 mL methanol and 3 mL water. 52 mg (1.1 mmol, 3.0 eq) of monohydrate and lithium hydroxide were added, and the mixture was stirred overnight at room temperature. After the reaction was complete as monitored by TLC, the solvent was evaporated to dryness, 15 mL of water was added, and the pH was adjusted to 2-3 with 0.5 N hydrochloric acid solution. The suspension was filtered, the filter cake was washed with water, collected, and dried under vacuum to obtain the product (195 mg, quant).

[0141] Step B: 5-(3-(3-amino-4-(7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-(cyanomethyl)azacyclobut-1-yl)-N-isopropylpyrazine-2-carboxamide

[0142] The compound (100 mg, yield = 49%) was prepared according to step C described in Example 1.

[0143] Step C: 5-(3-(3-amino-4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-(cyanomethyl)azacyclobut-1-yl)-N-isopropylpyrazine-2-carboxamide

[0144] The compound (60 mg, yield = 77%) was prepared according to method 2 in step E of Example 1. 1H NMR (400 MHz, DMSO-D6): δ 12.09 (s, 1H), 8.71-8.62 (m, 3H), 8.13 (d, J = 8.4 Hz, 1H), 7.97 (d, J = 1.4 Hz, 1H), 7.57 (dd, J = 3.5, 2.5 Hz, 1H), 7.11 (dd, J =3.6, 1.8 Hz, 1H), 6.43 (s, 2H), 4.78 (d, J = 9.7 Hz, 2H), 4.45 (d, J = 9.6 Hz, 2H), 4.15-4.06 (m, 1H), 3.73 (s, 2H), 1.17 (d, J = 6.6 Hz, 6H). LC-MS: m / z =458[M+1] + .

[0145] Example 9 2-(3-(3,5-diamino-4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-1-(1-(3-fluoro-2-(trifluoromethyl)isonicotinyl)piperidin-4-yl)azacyclobut-3-yl)acetonitrile

[0146] Step A: 2-(7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)malononitrile

[0147] Under stirring at room temperature, 10.0 g (35.2 mmol, 1.0 eq.) of 4-chloro-7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidine was added to a mixture of 3.5 g (53 mmol, 1.5 eq.) malononitrile and 7.2 g (53 mmol, 1.5 eq.) potassium carbonate. The reaction mixture was heated to 60 °C and reacted for 6 hours. After cooling to room temperature, the reaction was quenched with water, extracted with ethyl acetate, and the combined organic phases were washed with brine, dried over sodium sulfate, filtered, concentrated under vacuum, and separated by silica gel column chromatography to give the target compound (9.3 g, yield = 84%).

[0148] Step B: 4-(7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-3,5-diamino-1H-pyrazole

[0149] 7.5 g of 85% hydrazine hydrate (127 mmol, 10.0 eq.) was added to 100 mL of anhydrous ethanol solution of 4.0 g (12.8 mmol, 1.0 eq.) 2-(7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)malonadionitrile under nitrogen protection with stirring at room temperature. The reaction mixture was stirred and refluxed overnight at 90 °C. After cooling to room temperature, the mixture was concentrated under reduced pressure, filtered, and dried to give the target compound (1.98 g, yield = 45%). LC-MS: m / z = 346 [M+1] + .

[0150] Step C: 2-(3-(3,5-diamino-4-(7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-1-(1-(3-fluoro-2-(trifluoromethyl)isonicotinyl)piperidin-4-yl)azacyclobut-3-yl)acetonitrile

[0151] The compound (110 mg, yield = 60%) was prepared according to step H described in Example 1.

[0152] Step D: 2-(3-(3,5-diamino-4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-1-(1-(3-fluoro-2-(trifluoromethyl)isonicotinyl)piperidin-4-yl)azacyclobut-3-yl)acetonitrile

[0153] The compound (54 mg, yield = 51%) was prepared according to step I described in Example 1. LC-MS: m / z = 584 [M+H] + . Examples 10-16 The following compounds were synthesized using a similar method as described in the examples above.

[0154]

[0155] Example 17 2-(3-(3-amino-4-(9H-purin-6-yl)-1H-pyrazol-1-yl)-1-(1-(3-fluoro-2-(trifluoromethyl)isonicotinyl)piperidin-4-yl)azacyclobut-3-yl)acetonitrile

[0156] Following the steps described in Example 1, 4-chloro-7H-pyrrolo[2,3-d]pyrimidine was replaced with 6-chloropurine to prepare the target compound (67 mg). LC-MS: m / z = 570 [M+H] + . Examples 18-24 The following compounds were synthesized using a similar method as described in the examples above.

[0157]

[0158]

[0159] Example 25 2-(3-(3,5-diamino-4-(9H-purin-6-yl)-1H-pyrazol-1-yl)-1-(1-(3-fluoro-2-(trifluoromethyl)isonicotinyl)piperidin-4-yl)azacyclobut-3-yl)acetonitrile

[0160] Following the steps described in Examples 1 and 9, 4-chloro-7H-pyrrolo[2,3-d]pyrimidine was replaced with 6-chloropurine to prepare the target compound (68 mg). LC-MS: m / z = 585 [M+H] + . Examples 26-32 The following compounds were synthesized using a similar method as described in the examples above.

[0161]

[0162] These compounds can be prepared according to existing methods, such as the method disclosed in Chinese Patent CN201711248947.4.

[0163] Effect evaluation 1. Enzymatic activity of compounds (IC50) 50 ) detection A JAK1 / 2 / 3 kinase activity assay platform was established using the Lance Ultra principle to determine compound activity. In the assay plate, the enzyme, Ulight-labeled peptide substrate, ATP, and the assay compound were mixed and incubated. After the reaction, EDTA was added to terminate the reaction, and Eu-labeled antibody was added simultaneously for detection. The assay plate was analyzed using PE Envision in TR-FRET mode, and the data were expressed as fluorescence signal readings at 665 nm and 615 nm. A high 665 nm / 615 nm ratio indicated high enzyme activity, while a low 665 nm / 615 nm ratio indicated inhibited enzyme activity.

[0164] Reagents: kinases (JAK1 / 2 / 3), substrates (ULight-JAK-1peptide and ATP), and assay reagents (Eu-W1024 Anti-phosphotyrosine and EDTA).

[0165] Instruments: Echo acoustic pipetting system, Envision multi-functional plate reader.

[0166] The test compound was dissolved in 10 mM DMSO solution and stored in a nitrogen cabinet for long-term preservation. 10 μL of the 10 mM test compound solution was diluted to prepare a 1 mM working solution. This solution was then diluted 3-fold using an Echo pump to obtain 11 different concentrations, resulting in a final reaction system with compound concentrations ranging from 10 μM to 0.17 nM. 5 μL of the enzyme and peptide substrate mixture was added to the test plate using a power pipette. The test plate was centrifuged and then placed at room temperature (23°C). o C) Incubate for 15 minutes, add 5 μL of ATP-containing kinase buffer to the test plate using an electric pipette, centrifuge the test plate, seal it with aluminum foil, and place the test plate at room temperature (23°C). o C) Incubate for 90 minutes. Terminate the reaction, add the test reagent to the test plate using an electric pipette, centrifuge the test plate, seal it with aluminum foil, and place the test plate at room temperature (23°C). o C) After incubating for half an hour, the signal value of the reaction plate was detected using an Envision instrument. The results are shown in Table 1.

[0167] Table 1. JAK enzyme inhibitory activity of the compounds

[0168] 2. Pharmacokinetic experiments Female SD rats were divided into groups of three, and each group was given a single oral gavage dose of the suspension of the compound described in this study (5 mg / kg). The animals were fasted overnight before the experiment, from 10 hours before administration to 4 hours after administration. Blood samples were collected from the oral administration group at 0.25, 0.5, 1, 2, 4, 8, 12, and 24 hours after administration. After anesthesia with isoflurane using a small animal anesthesia machine, 0.3 mL of whole blood was collected and placed in heparin anticoagulant tubes. The samples were then collected at 4... o C. Centrifuge at 4000 rpm for 5 min, transfer plasma to centrifuge tubes, and store at -80℃ until analysis. Protein precipitation was used to extract the plasma sample, and the extract was analyzed by LC / MS / MS. Pharmacokinetic results are shown in Table 2.

[0169] Table 2. Pharmacokinetic parameters of different compounds administered by gavage to rats at 5 mg / kg

[0170] As shown in Table 2, the pharmacokinetic properties of the compound in Example 1 of this invention are superior.

[0171] 3. Blood-brain distribution experiment Male SD rats were selected for a blood-brain barrier penetration evaluation experiment. Multiple groups were set up, with three rats in each group. All experimental animals were fasted and allowed free access to water for 10 hours prior to drug administration to ensure stable physiological conditions. Each group of rats received a single intravenous (IV) injection of the test compound at a dose of 2 mg / kg. Rats were sacrificed immediately 0.5 hours after administration, and peripheral whole blood and brain tissue samples were collected. All collected samples were centrifuged at 4 ℃ and 4000 rpm for 5 min to separate plasma samples, which were then aliquoted into centrifuge tubes and frozen at -80 ℃ for later analysis.

[0172] During detection, the sample was pretreated and extracted using a plasma protein precipitation method. The resulting extract was then quantitatively analyzed using an LC / MS / MS mass spectrometry system. The brain-to-blood ratio (BCR) is a widely used parameter for evaluating the brain distribution of compounds. A value below 0.1 (i.e., the drug concentration in brain tissue is less than 10% of the plasma concentration) typically indicates that the compound has difficulty freely crossing the blood-brain barrier. The results of this experiment show that the BCR of the compound in the embodiments of this application is below 0.1, confirming that the compound has difficulty crossing the blood-brain barrier and belongs to a typical brain-impermeable small molecule compound.

[0173] Table 3

[0174] 4. Experiment on the treatment of cerebral ischemia-reperfusion in rats This embodiment was used to evaluate the efficacy of the compound in a middle cerebral artery occlusion (MCAO) model established in SD rats using the suture occlusion method. Neurological behavioral scoring (out of 18 points) and 2,3,5-triphenyltetrazolium chloride staining, TTC staining were used to assess model establishment and compound efficacy.

[0175] Experimental animals and grouping Table 4. SD rats, male, 280-300 g

[0176] Solvent: 5% N-methylpyrrolidone (NMP) + 5% polyethylene glycol-15-hydroxystearate + 90% (20% sulfobutyl ether-β-cyclodextrin).

[0177] Test Arrangement Seven days after the animals were adapted to the environment, they were randomly divided into groups to establish MCAO models. The drug was administered once every 10 minutes after reperfusion (iv), and once every 6 hours thereafter (iv). The mNSS score (modified neurological deficit score) was performed 24 hours after reperfusion, followed by TTC staining.

[0178] MCAO model establishment Embolization time: 60 min Reperfusion time: 24 h The specific process is as follows: (1) Anesthesia, neck preparation, and iodine disinfection; (2) Fix the animal in a supine position and expose its neck; (3) Make a blunt dissection in the middle of the neck with scissors to expose the right common carotid artery, internal carotid artery and external carotid artery. Clamp the common carotid artery with an artery clamp. Tie a dead knot with 5-0 sutures on the side of the external carotid artery near the brain and tie a slip knot on the external carotid artery near the bifurcation of the common carotid artery. (4) Clamp the internal carotid artery with an artery clip, cut the external carotid artery between the two knots with microscissors, insert the suture from the cut into the internal carotid artery, loosen the internal carotid artery clip, insert it into the middle cerebral artery until the suture bends, tighten the slipknot at the bifurcation of the common carotid artery, and the embolization time is 60 min. (5) After the embolization is completed, loosen the slipknot slightly, pull out the suture, immediately tie the external carotid artery slipknot, loosen the carotid artery clamp, suture the neck wound, and disinfect with iodine.

[0179] Neurobehavioral score Table 5. Neurological behavioral scores 24 h after reperfusion

[0180] TTC staining (1) Quick-freeze the sample in a -20℃ freezer for 20 min to facilitate slicing; (2) Cut the frozen sample into sections at 2 mm intervals, for a total of 6 sections; (3) Place the slices in 1% TTC (keep heating at 37 ℃); (4) After 20-30 min, remove the slices and store them in 4% paraformaldehyde overnight; (5) The slices were photographed the next day and the infarct area was calculated.

[0181] (6) Calculate the infarct volume based on the infarct area: Calculate according to the formula [(left side area - right side non-infarct area) × thickness / (2 × left side area × thickness) × 100% - percentage of whole brain infarct volume].

[0182] Data Analysis Behavioral data were analyzed using SPSS data analysis software, and graphs were created using GraphPad software based on the analysis results. IBM SPSS Statistics 26.0 statistical software was used to perform between-group statistical analysis of the data coefficients. Quantitative data are expressed as mean ± standard error (Mean ± SEM). After data aggregation and statistical analysis, one-way ANOVA was performed using SPSS statistical software, and a p-value < 0.05 was considered statistically significant.

[0183] Compound 1 is the compound obtained in Example 1 of this application.

[0184] Experimental results: 1) Neurological score 24 hours after reperfusion: The scoring structure is as follows Figure 1 As shown, n=5, compared with the control group, the mNSS neurobehavioral scores of the compound 1 treatment group were all lower than those of the control group. The specific scores were: Table 6

[0185] Note: In this application, Mean ± SEM refers to the mean ± standard error. The smaller the SEM value, the more reliable the mean of the data and the smaller the fluctuation. n=5 means that the sample size of each group is 5. p<0.05 means that there is a statistically significant difference compared with the control group.

[0186] 2) Infarct volume statistics after 24 hours of reperfusion Statistical results are as follows Figure 2 As shown, with n=5, the infarct volume in the compound 1-treated group was lower than that in the control group. Specifically: Table 7

[0187] Experimental conclusion: Analysis of the mNSS score and infarct volume percentage results from this trial shows that the compound 1 treatment group significantly reduced the mNSS score and infarct volume percentage in the MCAO model compared to the model group, demonstrating a significant therapeutic effect. Compound 1 effectively reduces brain damage caused by reperfusion injury in ischemic stroke and can be considered as a drug for treating ischemic stroke.

[0188] The present application has been described above with reference to preferred embodiments; however, these embodiments are merely exemplary and illustrative. Various substitutions and modifications can be made to the present application based on these embodiments, all of which fall within the protection scope of the present application.

Claims

1. Use of the compound represented by formula (I) or a pharmaceutically acceptable salt, solvate or isomer thereof in the preparation of a medicament for the treatment or prevention of ischemic stroke. Ⅰ in, L is selected from -C(=O)-, -S(=O)2- or -CONH-; X is selected from CH or N; Ar is selected from C6 aryl, 5-6 membered heterocyclic alkyl or 5-6 membered heteroaryl, wherein the heteroatom is selected from N, O or S, and the C6 aryl, 5-6 membered heterocyclic alkyl or 5-6 membered heteroaryl may optionally be substituted with one or more halogens; R1 is selected from C 1~6 Alkyl, C 1~6 alkoxy, C6 aryl, or 5-6 heteroaryl, wherein the heteroatom is selected from N, O, or S, wherein the C 1~6 Alkyl, C 1~6 Alkoxy, C6 aryl, or 5-6 heteroaryl groups may be optionally substituted with one or more of the following substituents: halogen, C 1~4 Alkyl or C 1~4 Halogenated alkyl groups; R2 is selected from -NH2; R3 is selected from hydrogen or -NH2; R5 is selected from cyano groups; R6 is selected from hydrogen.

2. Use of the compound of formula (I) according to claim 1, or a pharmaceutically acceptable salt, solvate or isomer thereof, in the preparation of a medicament for the treatment or prevention of reperfusion injury after ischemic stroke.

3. The use according to claim 1 or 2, wherein, R1 is selected from C 1~4 Alkyl, C 1~4 alkoxy, C6 aryl, or 6-membered heteroaryl, wherein the heteroatom is selected from N, O, or S, wherein the C 1~4 Alkyl, C 1~4 Alkoxy, C6 aryl, or 6-membered heteroaryl are optionally substituted with one or more of the following substituents: halogen, C 1~4 Alkyl or C 1~4 Halogenated alkyl groups.

4. The use according to claim 3, wherein, R1 is selected from C 1~4 Alkyl, C 1~4 alkoxy, C6 aryl, or 6-membered heteroaryl, wherein the heteroatom is selected from N, O, or S, wherein the C 1~4 Alkyl, C6 aryl, or 6-membered heteroaryl groups may be optionally substituted with one or more of the following substituents: -F or -CF3.

5. The use according to claim 1 or 2, wherein, R3 is hydrogen.

6. The use according to claim 1 or 2, wherein, Ar is selected from C6 aryl, 6-membered heterocyclic alkyl or 6-membered heteroaryl, wherein the heteroatom is selected from N, O or S, and the C6 aryl, 6-membered heterocyclic alkyl or 6-membered heteroaryl is optionally substituted with one or more halogens.

7. The use according to claim 6, wherein, Ar is a group that is optionally substituted with one or more halogens: ， in, Indicates the position connected to L. This indicates the position where it is attached to the nitrogen-containing heterocyclic butyl group.

8. The use according to claim 7, wherein, The halogen is -F.

9. The use according to claim 1 or 2, wherein, The compound is selected from one of the following structures:

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