CN113372351A

CN113372351A - JAK kinase inhibitor and preparation and application thereof

Info

Publication number: CN113372351A
Application number: CN202110262564.2A
Authority: CN
Inventors: 李傲; P·K·贾达夫; 姚元山; 陈以乐; 曹国庆
Original assignee: Minghui Pharmaceutical Shanghai Co ltd; Minghui Pharmaceutical Hangzhou Co ltd
Current assignee: Minghui Pharmaceutical Shanghai Co ltd; Minghui Pharmaceutical Hangzhou Co ltd
Priority date: 2020-03-10
Filing date: 2021-03-10
Publication date: 2021-09-10

Abstract

The invention provides a JAK kinase inhibitor and preparation and application thereof, and particularly provides a compound with a structure shown in a formula (I) or a pharmaceutically acceptable salt thereof. The compounds have excellent JAK kinaseEnzyme inhibitory activity and thus may be useful in the treatment of diseases or conditions associated with JAK kinase activity or expression levels.

Description

JAK kinase inhibitor and preparation and application thereof

Technical Field

The invention relates to the field of pharmaceutical compounds, and particularly provides a JAK kinase inhibitor, and preparation and application thereof.

Background

Protein kinases are a family of enzymes that catalyze the phosphorylation of specific residues in proteins, broadly classified as tyrosine kinases and serine/threonine kinases. Abnormal kinase activity caused by mutations, overexpression or inappropriate regulation, dysregulation or imbalance and excess or insufficient production of growth factors or cytokines has been associated with a number of diseases including, but not limited to, cancer, cardiovascular diseases, allergies, asthma and other respiratory diseases, autoimmune diseases, inflammatory diseases, skeletal diseases, metabolic diseases and neurological and neurodegenerative diseases (e.g., alzheimer's disease). Inappropriate kinase activity triggers a variety of biological cellular responses associated with the above and related diseases related to cell growth, cell differentiation, cell function, survival, apoptosis and cell migration.

Thus, protein kinases have become an important class of enzymes as targets for therapeutic intervention. In particular, the JAK family of cellular protein tyrosine kinases play a central role in cytokine signaling. Upon binding to their receptors, cytokines activate JAKs, which then phosphorylate cytokine receptors, establishing docking sites for signaling molecules, particularly members of the Signal Transducer and Activator of Transcription (STAT) families, ultimately leading to gene expression. Therefore, compounds that effectively and highly selectively inhibit specific JAK enzymes may be potential therapeutic agents for a range of diseases or conditions, in particular inhibitors of TYK2 and JAK 1.

TYK2 is a JAK kinase family member, important in the signaling of type I interferons (IFNa, INFb) IL-6, IL-10, IL-12 and IL-23. Thus, TYK2 signals with other members of the JAK kinase family in the following combinations: TYK2/JAK1, TYK2/JAK2, TYK2/JAK1/JAK 2. TYK2 has been shown to be important in the differentiation and function of a variety of cell types that are important in inflammatory and autoimmune diseases, including natural killer, B-cell and T-helper cell types.

JAK1 is expressed at different levels in all tissues. Many cytokine receptors signal through paired JAK kinases in combination: JAK1/JAK2, JAK1/JAK3, JAK1/TYK2, JAK2 TYK2 or JAK2/JAK 2. In this case, JAK1 is the most widely paired JAK kinase and is required for signaling by the gamma-universal (IL-2Ry) cytokine receptor, the IL-6 receptor family, the I, II and III receptor families, and the IL-10 receptor family. Animal studies have shown that JAK1 is essential for immune system development, function and homeostasis. Modulation of immune activity by inhibition of JAK1 kinase activity may prove useful in the treatment of various immune diseases.

In view of the above, there remains a need in the art to develop inhibitors against JAK family protein kinases, particularly TYK2 or JAK1 protein kinases.

Disclosure of Invention

The invention aims to provide an inhibitor aiming at JAK family protein kinase, in particular TYK2 or JAK1 protein kinase.

In a first aspect of the present invention, there is provided a compound having a structure represented by the following formula (I), or a pharmaceutically acceptable salt thereof:

wherein the content of the first and second substances,

ring A is a substituted or unsubstituted 5-6 membered aromatic or heteroaromatic ring;

ring B is a substituted or unsubstituted 3-6 membered carbocyclic ring;

R₁selected from the group consisting of: substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C3-C8 cycloalkyl, substituted or unsubstituted C1-C4 alkoxy, substituted or unsubstituted C1-C4 alkylamino, substituted or unsubstituted 6-to 10-membered aryl, substituted or unsubstituted C1-to 3-membered ring selected from N, S (O)_pAnd a heteroatom of O, a substituted or unsubstituted 4-10 membered heterocyclyl having 1-3 heteroatoms selected from N, S and O;

R₂and R₄Each independently selected from the group consisting of: H. d, halogen, CN, CHF₂、CF₃；

R₃Selected from the group consisting of: H. d, halogen, CN, hydroxyl, amino, substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C1-C4 alkoxy, COOH, CO (C1-C4 alkyl), CONH₂CONH (C1-C4 alkyl), CON (C1-C4 alkyl)₂NH (C1-C4 alkyl), N (C1-C4 alkyl)₂NH (CO) (C1-C4 alkyl), O (CO) (C1-C4 alkyl);

x is selected from a bond, NH, N (C1-C4 alkyl) or (CR)₂)_m；

Y is (CR)₂)_n；

The R is selected from the following group: H. halogen, C1-C6 alkyl, halogenated C1-C6 alkyl, C1-C6 alkoxy, halogenated C1-C6 alkoxy, C3-C8 cycloalkyl, halogenated C3-C8 cycloalkyl, oxo, -CN, hydroxy, amino, carboxy, a group selected from the group consisting of unsubstituted or substituted by one or more substituents selected from the group consisting of: C6-C10 aryl, halogenated C6-C10 aryl, 5-10 membered heteroaryl having 1-3 heteroatoms selected from N, S and O, halogenated 5-10 membered heterocyclyl having 1-3 heteroatoms selected from N, S and O; and the substituents are selected from the group consisting of: halogen, C1-C6 alkoxy;

m and n are respectively and independently selected from 0, 1 or 2;

and when X is selected from (CH)₂)_mWhen m and n are not 0 at the same time;

unless otherwise specified, "substituted" means substituted with one or more (e.g., 2,3, 4, etc.) substituents selected from the group consisting of: halogen, C1-C6 alkyl, halogenated C1-C6 alkyl, C1-C6 alkoxy, halogenated C1-C6 alkoxy, C3-C8 cycloalkyl, halogenated C3-C8 cycloalkyl, oxo, -CN, hydroxy, amino, carboxy, a group selected from the group consisting of unsubstituted or substituted by one or more substituents selected from the group consisting of: C6-C10 aryl, halogenated C6-C10 aryl, 5-10 membered heteroaryl having 1-3 heteroatoms selected from N, S and O, halogenated 5-10 membered heterocyclyl having 1-3 heteroatoms selected from N, S and O; and the substituents are selected from the group consisting of: halogen, C1-C6 alkoxy.

In another preferred embodiment, X is NH or (CH)₂)_m。

In another preferred embodiment, the a ring is a substituted or unsubstituted group selected from the group consisting of:

in another preferred embodiment, R₁Selected from the group consisting of: substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C3-C8 cycloalkyl, substituted or unsubstituted C1-C4 alkoxy, and substituted or unsubstituted C1-C4 alkylamino.

In another preferred embodiment, R is₁Is a halogenated cycloalkyl group having from C3 to C8.

In another preferred embodiment, R is₄Is H.

In another preferred embodiment, said (I) is preferably selected from (IIA) and (IIB).

Wherein ring B is a substituted or unsubstituted 3-4 membered carbocyclic ring.

In another preferred embodiment, R₂Selected from hydrogen, halogen and CN.

In another preferred embodiment, R₂Is hydrogen or F.

In another preferred embodiment, R₃Is a group selected from: H. d, halogen, CN, hydroxyl, amino, substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C1-C4 alkoxy.

In another preferred embodiment, the compound is selected from the group consisting of:

in a second aspect of the present invention, there is provided a pharmaceutical composition comprising (1) a compound according to the first aspect of the present invention, or a stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt, hydrate or solvate thereof; and (2) a pharmaceutically acceptable carrier.

In a third aspect of the present invention, there is provided a use of a compound according to the first aspect of the present invention, or a stereoisomer or a tautomer thereof, or a pharmaceutically acceptable salt, hydrate or solvate thereof, or a pharmaceutical composition according to the second aspect of the present invention, for the preparation of a pharmaceutical composition for the prevention and/or treatment of a disease or condition associated with JAK kinase activity or expression.

In another preferred embodiment, the disease or condition is selected from the group consisting of: inflammation, autoimmune diseases, neuroinflammation, arthritis, rheumatoid arthritis, spondyloosteoarthritis, systemic lupus erythematosus, lupus nephritis, gouty arthritis, pain, fever, pulmonary sarcoidosis, silicosis, cardiovascular disease, atherosclerosis, sarcoidosis, myocarditis and cardiac reperfusion injury, cardiomyopathy, stroke, ischemia, reperfusion injury, cerebral edema, cerebral trauma, neurodegenerative diseases, liver disease, inflammatory bowel disease, Crohn's disease, ulcerative colitis, nephritis, retinitis, retinopathy, macular degeneration, glaucoma, diabetes mellitus (type 1) and type 2), diabetic neuropathy, viral and bacterial infections, myalgia, endotoxic shock, toxic shock syndrome, autoimmune diseases, osteoporosis, multiple sclerosis, endometriosis, menstrual cramps, vaginitis, candidiasis, cancer, fibrosis, obesity, muscular dystrophy, polymyositis, dermatomyositis, autoimmune hepatitis, primary biliary cirrhosis, primary sclerosing cholangitis, vitiligo, alopecia, alzheimer's disease, skin flushing, eczema, psoriasis, atopic dermatitis and sunburn.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Detailed Description

The inventors have conducted extensive and intensive studies for a long time and unexpectedly found a compound represented by formula I. The compounds have unexpected activity in modulating cytokines and/or interferons and are useful in the treatment of diseases mediated by cytokines and/or interferons. Based on the above findings, the inventors have completed the present invention.

Definition of

As used herein, the term "alkyl" includes straight or branched chain alkyl groups. E.g. C₁-C₈Alkyl represents a straight or branched chain alkyl group having 1 to 8 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, and the like.

As used herein, the term "alkenyl" includes straight or branched chain alkenyl groups. E.g. C₂-C₆Alkenyl means a straight or branched alkenyl group having 2 to 6 carbon atoms, such as vinyl, allyl, 1-propenyl, isopropenyl, 1-butenyl, 2-butenyl, or the like.

As used herein, the term "alkynyl" includes straightAlkynyl, chain or branched. E.g. C₂-C₆Alkynyl means straight or branched chain alkynyl having 2 to 6 carbon atoms, such as ethynyl, propynyl, butynyl, or the like.

As used herein, the term "C₃-C₁₀Cycloalkyl "refers to cycloalkyl groups having 3 to 10 carbon atoms. It may be a single ring, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, or the like. It may also be in the form of a double ring, for example a bridged or spiro ring.

As used herein, the term "C₁-C₈Alkylamino "is defined as being substituted by C₁-C₈The amino group substituted by the alkyl can be mono-substituted or di-substituted; for example, methylamino, ethylamino, propylamino, isopropylamino, butylamino, isobutylamino, tert-butylamino, dimethylamino, diethylamino, dipropylamino, diisopropylamino, dibutylamino, diisobutylamino, di-tert-butylamino and the like.

As used herein, the term "C₁-C₈Alkoxy "means a straight or branched chain alkoxy group having 1 to 8 carbon atoms; for example, methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, tert-butoxy and the like.

As used herein, the term "3-10 membered heterocycloalkyl having 1-3 heteroatoms selected from the group consisting of N, S and O" refers to a saturated or partially saturated cyclic group having 3-10 atoms and wherein 1-3 atoms are heteroatoms selected from the group consisting of N, S and O. It may be monocyclic or may be in the form of a double ring, for example a bridged or spiro ring. Specific examples may be oxetane, azetidine, tetrahydro-2H-pyranyl, piperidinyl, tetrahydrofuranyl, morpholinyl, pyrrolidinyl, and the like.

As used herein, the term "C₆-C₁₀Aryl "means an aryl group having 6 to 10 carbon atoms, for example, phenyl or naphthyl and the like.

As used herein, the term "5-12 membered heteroaryl" refers to a heteroaryl group having 5-12 atoms and wherein 1-3 atoms are selected from N, S (O)_pAnd a heteroatom of O (p is 0, 1 or 2). It can be a sheetThe rings may also be fused. Specific examples may be pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, pyrrolyl, pyrazolyl, imidazolyl, (1,2,3) -triazolyl and (1,2,4) -triazolyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, oxazolyl and the like.

Unless specifically stated to be "substituted or unsubstituted", the groups of the present invention may be substituted with a substituent selected from the group consisting of: halogen, nitrile group, nitro group, hydroxyl group, amino group, C₁-C₆Alkyl-amino, C₁-C₆Alkyl radical, C₂-C₆Alkenyl radical, C₂-C₆Alkynyl, C₁-C₆Alkoxy, halo C₁-C₆Alkyl, halo C₂-C₆Alkenyl, halo C₂-C₆Alkynyl, halo C₁-C₆Alkoxy, allyl, benzyl, C₆-C₁₂Aryl radical, C₁-C₆alkoxy-C₁-C₆Alkyl radical, C₁-C₆Alkoxy-carbonyl, phenoxycarbonyl, C₂-C₆Alkynyl-carbonyl, C₂-C₆Alkenyl-carbonyl, C₃-C₆Cycloalkyl-carbonyl, C₁-C₆Alkyl-sulfonyl, and the like.

As used herein, "halogen" or "halogen atom" refers to F, Cl, Br, and I. More preferably, the halogen or halogen atom is selected from F, Cl and Br. "halogenated" means substituted with an atom selected from F, Cl, Br, and I.

Unless otherwise specified, the structural formulae depicted herein are intended to include all isomeric forms (e.g., enantiomers, diastereomers and geometric isomers (or conformational isomers)): for example, R, S configuration containing an asymmetric center, (Z), (E) isomers of double bonds, and the like. Thus, individual stereochemical isomers of the compounds of the present invention or mixtures of enantiomers, diastereomers or geometric isomers (or conformers) thereof are within the scope of the present invention.

As used herein, the term "tautomer" means that structural isomers having different energies may exceed the low energy barrier, thereby converting with each other. For example, proton tautomers (i.e., proton transmutations) include interconversion by proton shift, such as 1H-indazoles and 2H-indazoles. Valence tautomers include interconversion by recombination of some of the bonding electrons.

As used herein, the term "solvate" refers to a complex of a compound of the present invention coordinated to solvent molecules in a specific ratio.

As used herein, the term "hydrate" refers to a complex formed by the coordination of a compound of the present invention with water.

The compounds of the present application may be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combinations of the specific embodiments with other chemical synthetic methods, and equivalents known to those skilled in the art, with preferred embodiments including, but not limited to, the examples of the present application.

The solvent used in the present application can be commercially available. Abbreviations used in this application are as follows: aq represents an aqueous solution; HATU represents O- (7-azabenzotriazol-1-yl) -N, N' -tetramethyluronium hexafluorophosphate; EDC stands for N- (3-dimethylaminopropyl) -N' -ethylcarbodiimide hydrochloride; m-CPBA represents 3-chloroperoxybenzoic acid; eq represents equivalent, equivalent; CDI represents carbonyldiimidazole; DCM represents dichloromethane; PE represents petroleum ether; DIAD represents diisopropyl azodicarboxylate; DMF represents N, N-dimethylformamide; DMSO represents dimethyl sulfoxide; EtOAc for ethyl acetate; EtOH stands for ethanol; MeOH represents methanol; cbz represents benzyloxycarbonyl, an amino protecting group; boc represents tert-butyloxycarbonyl, an amino protecting group; HOAc represents acetic acid; NaCNBH₃Represents sodium cyanoborohydride; r.t. represents room temperature; THF represents tetrahydrofuran; TFA represents trifluoroacetic acid; DIPEA stands for diisopropylethylamine; boc₂O represents di-tert-butyl dicarbonate; LDA stands for lithium diisopropylamide.

The compound is artificially synthesized or

SoftwareThe nomenclature used for commercially available compounds is the supplier catalogue name.

Pharmaceutical compositions and methods of administration

The compound of the present invention and various crystal forms, pharmaceutically acceptable inorganic or organic salts, hydrates or solvates thereof, and pharmaceutical compositions containing the compound of the present invention as a main active ingredient are useful for the prevention and/or treatment (stabilization, alleviation or cure) of various autoimmune and inflammation-related diseases including systemic lupus erythematosus, inflammatory bowel disease, psoriasis, rheumatoid arthritis and the like, because the compound of the present invention has excellent inhibitory activity against cytokines and/or interferons.

The pharmaceutical compositions of the present invention comprise a safe and effective amount of a compound of the present invention in combination with a pharmaceutically acceptable excipient or carrier. Wherein "safe and effective amount" means: the amount of the compound is sufficient to significantly improve the condition without causing serious side effects. Typically, the pharmaceutical composition contains 1-2000mg of a compound of the invention per dose, more preferably, 1-200mg of a compound of the invention per dose. Preferably, said "dose" is a capsule or tablet.

"pharmaceutically acceptable carrier" refers to: one or more compatible solid or liquid fillers or gel substances which are suitable for human use and must be of sufficient purity and sufficiently low toxicity. By "compatible" is meant herein that the components of the composition are capable of intermixing with and with the compounds of the present invention without significantly diminishing the efficacy of the compounds. Examples of pharmaceutically acceptable carrier moieties are cellulose and its derivatives (e.g. sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (e.g. stearic acid, magnesium stearate), calcium sulfate, vegetable oils (e.g. soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (e.g. propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers (e.g. tween, etc.)

) Wetting agents (such as sodium lauryl sulfate), coloring agents, flavoring agents, stabilizers, antioxidants, preservativesPyrogen-free water, etc.

The mode of administration of the compounds or pharmaceutical compositions of the present invention is not particularly limited, and representative modes of administration include (but are not limited to): oral, parenteral (intravenous, intramuscular or subcutaneous).

Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In these solid dosage forms, the active compound is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following ingredients: (a) fillers or extenders, for example, starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) binders, for example, hydroxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and acacia; (c) humectants, for example, glycerol; (d) disintegrating agents, for example, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) slow solvents, such as paraffin; (f) absorption accelerators, e.g., quaternary ammonium compounds; (g) wetting agents, such as cetyl alcohol and glycerol monostearate; (h) adsorbents, for example, kaolin; and (i) lubricants, for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. In capsules, tablets and pills, the dosage forms may also comprise buffering agents.

Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared using coatings and shells such as enteric coatings and other materials well known in the art. They may contain opacifying agents and the release of the active compound or compounds in such compositions may be delayed in release in a certain part of the digestive tract. Examples of embedding components which can be used are polymeric substances and wax-like substances. If desired, the active compound may also be in microencapsulated form with one or more of the above-mentioned excipients.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly employed in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, propylene glycol, 1, 3-butylene glycol, dimethylformamide and oils, in particular, cottonseed, groundnut, corn germ, olive, castor and sesame oils or mixtures of such materials and the like.

In addition to these inert diluents, the compositions can also contain adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

Suspensions, in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances, and the like.

Compositions for parenteral injection may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols and suitable mixtures thereof.

The compounds of the present invention may be administered alone or in combination with other pharmaceutically acceptable therapeutic agents.

When administered in combination, the pharmaceutical composition further comprises one or more (2, 3,4, or more) other pharmaceutically acceptable therapeutic agents. One or more (2, 3,4, or more) of such other pharmaceutically acceptable therapeutic agents may be used simultaneously, separately or sequentially with a compound of the invention for the prevention and/or treatment of cytokine and/or interferon mediated diseases.

When the pharmaceutical composition is used, a safe and effective amount of the compound of the present invention is suitable for mammals (such as human beings) to be treated, wherein the administration dose is a pharmaceutically-considered effective administration dose, and for a human body with a weight of 60kg, the daily administration dose is usually 1 to 2000mg, preferably 1 to 500 mg. Of course, the particular dosage will depend upon such factors as the route of administration, the health of the patient, and the like, and is within the skill of the skilled practitioner.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers. Unless otherwise indicated, percentages and parts are by weight.

Examples

Example 1

First step of

Compound 1a (1.16g, 0.01mol) was dissolved in dichloromethane (10mL), and 3, 4-dihydro-2H-pyran (0.84g, 0.01mol), pyridine p-toluenesulfonate (241mg, 1.00mmol) and reacted at room temperature overnight under nitrogen protection. The reaction system was concentrated under reduced pressure to give a residue, which was purified by column chromatography (ethyl acetate: petroleum ether ═ 0 to 100%) to give compound 1b (1.00g), yield: 50 percent.

Second step of

Dissolving 1b (1.00g, 5.00mmol) in tetrahydrofuran (20mL), adding lithium aluminum hydride (380mg, 10.00mmol) to the reaction system in batches under ice bath and nitrogen protection, reacting at room temperature for 2 hours, slowly dropwise adding water (1mL) to the reaction system to quench the reaction, then slowly dropwise adding 15% sodium hydroxide aqueous solution (1mL) and water (3mL) to the reaction system in sequence, drying over anhydrous magnesium sulfate, filtering, concentrating the filtrate under reduced pressure to obtain crude product 1c (0.86g), and directly using the crude product in the next reaction.

The third step

After crude product 1c (0.86g, 5.00mmol) was dissolved in tetrahydrofuran (10mL), 4-nitropyrazole (565mg, 5.00mmol) and triphenylphosphine (2.60g, 10.00mmol) were added in this order under ice bath and nitrogen protection, diisopropyl azodicarboxylate (2.00g, 10.00mmol) was added dropwise to the reaction system, the reaction was allowed to react overnight at room temperature, the reaction mixture was concentrated under reduced pressure to give a residue, and the residue was purified by a column layer (ethyl acetate: petroleum ether ═ 0 to 100%) to give compound 1d (1.20g) in yield: 89 percent.

MS-ESI calculated value [ M + Na ]]⁺290, found value 290.

The fourth step

Compound 1d (1.76g, 6.60mmol) was dissolved in methanol (50mL), to which was added successively a saturated ammonium chloride solution (20mL) and iron powder (1.85g, 33.00 mmol). The reaction was stirred at 90 ℃ for 1 hour. After the reaction system was cooled to room temperature, it was diluted with water (200mL) and ethyl acetate (100mL) and separated, the organic phase was washed with saturated brine (50mL × 1), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give a residue, which was purified by column chromatography (methanol: dichloromethane ═ 0 to 100%) to give compound 1e (950mg), yield: 60 percent.

MS-ESI calculated value [ M + H%]⁺238, found value 238.

¹H NMR(400MHz,CDCl₃)δ7.28(s,1H),7.15(s,1H),4.68-4.67(m,1H),4.55-4.51(m,1H),3.96-3.93(m,1H),3.85-3.84(m,1H),3.50-3.47(m,1H),2.85(brs,2H),1.63-1.58(m,2H),1.51-1.44(m,4H),1.16-1.11(m,1H),0.90-0.86(m,1H),0.79-0.66(m,2H).

The fifth step

Triethylamine (1.37g, 1.34mmol) was added to a solution of tert-butyl 3, 8-diazabicyclo [3.2.1] octane-8-carboxylate (1.57g, 0.74mmol) and 1f (1.00g, 0.67mmol) in N, N-dimethylformamide (10 mL). Stirred at room temperature for 3 hours. After the reaction was completed, dichloromethane (10mL) was added to the reaction system to dilute, and washed with water (10mL × 3), the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a residue, which was purified by column chromatography (ethyl acetate: petroleum ether ═ 1-100%) to obtain 1g (1.55g) with yield: 71 percent.

MS-ESI calculated value [ M + H%]⁺325, found value 325.

The sixth step

To a solution of 1g (324mg, 1.00mmol), 1e (237mg, 1.00mmol), dibenzylideneacetone dipalladium (45mg, 0.05mmol) and 4, 5-bis-diphenylphosphine-9, 9-dimethylxanthene (59mg, 0.10mmol) in toluene (10mL) was added cesium carbonate (752mg, 2.00mmol) under nitrogen. The reaction was stirred at 120 ℃ overnight. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure to obtain a residue. The residue was purified by column chromatography (ethyl acetate: petroleum ether ═ 0 to 100%) to give 1h (95mg), yield: 18 percent.

MS-ESI calculated value [ M + H%]⁺526, measured value 526.

Seventh step

To a 1h (90mg, 0.17mmol) solution in methanol (2mL) was added a hydrogen chloride/ethyl acetate solution (4M, 2 mL). The reaction was stirred at room temperature for 1 hour. The reaction was concentrated to give crude 1i (90mg) which was used directly in the next reaction.

MS-ESI calculated value [ M + H%]⁺342, found 342.

Eighth step

Crude 1i (90mg, 0.17mmol) and (S) -2, 2-difluorocyclopropanecarboxylic acid (23mg, 0.18mmol) and N, N-diisopropylethylamine (88mg, 0.68mmol) were dissolved in N, N-dimethylformamide (1 mL). 2- (7-Azabenzotriazole) -N, N, N ', N' -tetramethyluronium hexafluorophosphate (84mg, 0.22mmol) was added thereto. The reaction was stirred at room temperature for 1 hour. After completion of the reaction, the reaction mixture was quenched by adding water (40mL), diluted with ethyl acetate (40mL), and separated. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give a residue, which was purified by preparative high performance liquid chromatography to give 1(32mg), yield: two steps 43%.

MS-ESI calculated value [ M + H%]⁺446, found 446.

¹H NMR(400MHz,DMSO-d₆)δ8.91(brs,1H),7.90-7.94(m，2H),7.38-7.44(m，1H),6.12-6.08(m,1H),5.49(s,1H),4.68-4.57(m,2H),4.11-4.09(m,4H),3.22-3.14(m,1.5H),2.94-2.91(m,1.5H),2.03-1.94(m,6H),0.64(s,4H).

Example 2

First step of

To 1g (100mg, 0.31mmol) of the compound in methanol (5mL) was added a 4N ethyl acetate hydrochloride solution (4mL) and the reaction was carried out at room temperature for 1 hour. The reaction mixture was concentrated under reduced pressure to give crude product 2a (68 mg).

MS-ESI calculated value [ M + 1]]⁺225, found value 225.

Second step of

Crude 2a (68mg, 0.31mmol), triethylamine (60mg, 0.60mmol) and 2, 2-difluorocyclopropanecarboxylic acid (37mg, 0.31mmol) were dissolved in N, N-dimethylformamide (2mL), and 2- (7-azabenzotriazole) -N, N, N ', N' -tetramethyluronium hexafluorophosphate (228mg, 0.60mmol) was added to the reaction solution and reacted at room temperature overnight. The reaction was dispersed in water (10mL) and extracted with ethyl acetate (10mL x 2), the organic phase was washed successively with saturated brine (10mL x 1), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give a residue which was purified by preparative HPLC to give compound 2b (49mg) yield: 50 percent.

MS-ESI calculated value [ M + 1]]⁺329 found, value 329.

The third step

Compound 2c (158mg, 1.00mmol) was dissolved in isopropanol (5mL), diisopropylethylamine (258mg, 2.00mmol) and compound 2d (172mg, 1.20mmol) were added thereto in this order, the temperature was raised to 100 ℃ to react overnight, the reaction solution was cooled to room temperature, and concentrated under reduced pressure to give a residue, which was subjected to reverse phase column chromatography (acetonitrile: water ═ 0-100%) to give compound 2e (80mg), yield: 30 percent.

The fourth step

Compound 2e (80mg, 0.35mmol) was dissolved in methanol (10mL), reacted under hydrogen atmosphere (15psi) at room temperature overnight, filtered through celite, and concentrated under reduced pressure to give compound 2f (60mg), yield: 86 percent.

The fifth step

Compound 2f (60mg, 0.30mmol) was dissolved in isopropanol (5mL), diisopropylethylamine (116mg, 0.90mmol) and compound 2b (98mg, 0.30mmol) were added thereto in this order, the temperature was raised to 100 ℃ to react overnight, the reaction was cooled to room temperature, and concentrated under reduced pressure to give a residue, which was purified by preparative HPLC to give compound 2(48mg), yield: 33 percent.

MS-ESI calculated value [ M + 1]]⁺492, measured value 492.

¹H NMR(400MHz,CDCl₃)δ8.25-8.22(m,1H),7.98-7.96(m,1H),7.72-7.70(m，1H),6.60(s,1H),6.39-6.37(m,1H),5.97-5.95(m,1H),4.88-4.81(m,1H),4.56-4.55(m,1H),4.44-4.40(m,1H),4.40-4.11(m,3H),3.13-3.05(m,4H),2.56-2.48(m,3H),2.47-1.71(m,6H).

Example 3

First step of

Reference example 2 synthesis of 2b from 2a synthesis of 3a from 2 a.

MS-ESI calculated value [ M + 1]]⁺329 found, value 329.

Second step of

3b (100mg, 0.58mmol), 2d (126mg, 0.87mmol) and triethylamine (176mg, 1.74mmol) were dissolved in isopropanol (3mL) and the reaction was allowed to warm to 130 ℃ overnight. The reaction solution was cooled to room temperature, and concentrated under reduced pressure to give a residue, which was purified by reverse phase column chromatography (acetonitrile: water 0-100%) to give compound 3c (110mg) in yield: 68 percent.

MS-ESI calculated value [ M + 1]]⁺244, measured value 244.

The third step

3c (110mg, 0.45mmol) was dissolved in methanol (10mL), 10% wet palladium on carbon (20mg) was added, the reaction was carried out overnight at room temperature under hydrogen atmosphere (15psi), the filtrate was filtered through celite, and the filtrate was concentrated under reduced pressure to give compound 3d (90mg), yield: 96 percent.

MS-ESI calculated value [ M + 1]]⁺214, measured value 214.

The fourth step

3a (30mg, 0.09mmol), N, N-diisopropylethylamine (23mg, 0.18mmol) and 3d (19mg, 0.09mmol) were dissolved in isopropanol (4mL) and the reaction was allowed to warm to 130 ℃ overnight. The reaction system was concentrated under reduced pressure to give a residue, which was purified by reverse phase column (acetonitrile: water ═ 0 to 100%) and preparative thin layer chromatography in this order to give compound 3(4mg) in yield: 8 percent.

MS-ESI calculated value [ M + 1]]⁺506, measured value 506.

¹H NMR(400MHz,DMSO-d₆)δ8.64(d,J＝2.4Hz,1H),8.11-8.09(m,1H),7.92-7.90(m,1H),7.59(d,J＝2.8Hz,1H),6.15-6.11(m,1H),5.92(d,J＝6.4Hz,1H),4.78-4.72(m,2H),4.31-3.96(m,3H),3.28-3.08(m,2H),2.95-2.90(m,3H),2.67-2.54(m,2H),2.04(s,3H),2.07-1.60(m,6H).

Example 4

First step of

After adding 60% sodium hydride (142mg, 3.54mmol) to a solution of 4b (200mg, 1.77mmol) in N, N-dimethylformamide (3mL) under nitrogen atmosphere and ice bath for 30 minutes, 4a (460mg, 1.77mmol) was added to the reaction system, and the reaction was allowed to react overnight at room temperature, and the reaction mixture was purified by reverse phase column chromatography (acetonitrile: water ═ 0 to 100%) to obtain compound 4c (240mg) in yield: and 64 percent.

MS-ESI calculated value [ M + H%]⁺212, measured value 212.

¹H NMR(400MHz,CDCl₃)δ8.28(s,1H),8.08(s,1H),3.73(s,3H),1.93-1.90(m,2H),1.73-1.70(m,2H).

Second step of

10% wet palladium on carbon (10mg) was added to a solution of 4c (130mg, 0.62mmol) in methanol (3mL) under nitrogen, the reaction was allowed to react at room temperature for 3 hours, then the system was filtered through celite, and concentrated under reduced pressure to give crude 4d (110 mg).

MS-ESI calculated value [ M + H%]⁺182, measured value 182.

The third step

1g (196mg, 0.61mmol) was added to crude 4d (110mg, 0.61mmol) in n-butanol solution (5mL) and reacted at 130 ℃ for 2 hours, the reaction was cooled to room temperature and concentrated under reduced pressure to give a residue, which was purified by column chromatography (methanol: dichloromethane 0-100%) to give compound 4e (280mg) in yield: 98 percent.

MS-ESI calculated value [ M + H%]⁺470, found 470.

The fourth step

A 1M solution of lithium borohydride in tetrahydrofuran (1.00mL, 1.00mmol) was added to a solution of compound 4e (50mg, 0.11mmol) in methanol (2mL), reacted overnight at room temperature, concentrated under reduced pressure to give a residue, which was purified by reverse phase column chromatography (acetonitrile: water 0-100%) to give the objective compound 4f (23mg) in yield: and 47 percent.

MS-ESI calculated value [ M + H%]⁺442, found 442.

The fifth step

4f (23mg, 0.05mmol) was dissolved in 4M ethyl acetate hydrochloride solution (2mL), reacted at room temperature for 1 hour, and concentrated under reduced pressure to give 4g (17mg) of crude product.

The sixth step

2- (7-Benzotolyltriazole) -N, N, N ', N' -tetramethyluronium hexafluorophosphate (27mg,0.07mmol) was added to a solution of 4g (17mg, 0.05mmol), 2, 2-difluorocyclopropanecarboxylic acid (6mg, 0.05mmol) and triethylamine (10mg,0.10mmol) in N, N-dimethylformamide (2mL) and reacted at room temperature overnight. Concentration under reduced pressure gave a residue, which was purified by reverse phase column chromatography (acetonitrile: water ═ 0 to 100%) to give compound 4(3mg), yield: 13 percent.

MS-ESI calculated value [ M + H%]⁺446, found 446.

¹H NMR(400MHz,CDCl₃)δ7.98-7.93(m,1H),7.78(s,1H),7.58-7.54(s,1H),6.54(s,1H),5.96-5.90(m,1H),4.90-4.76(m,1H),4.43(s,1H),4.26-3.92(m,2H),3.77(s,2H),3.22-3.08(m,2H),2.60-2.48(m,1H),2.30-1.60(m,7H),1.29(t,J＝6.0Hz,2H),1.10(t,J＝6.0Hz,2H).

Example 5

2- (7-Benzotolyltriazole) -N, N, N ', N' -tetramethyluronium hexafluorophosphate (27mg,0.07mmol) was added to a solution of the compounds 4g (17mg, 0.05mmol), (S) -2, 2-difluorocyclopropanecarboxylic acid (6mg, 0.05mmol) and triethylamine (10mg,0.10mmol) in N, N-dimethylformamide (2mL) and reacted at room temperature overnight. Concentration under reduced pressure gave a residue, which was purified by reverse phase column chromatography (acetonitrile: water ═ 0 to 100%) to give compound 5(3mg), yield: 13 percent.

MS-ESI calculated value [ M + H%]⁺446, found 446.

¹H NMR(400MHz,CDCl₃)δ7.98-7.93(m,1H),7.77(s,1H),7.58-7.54(s,1H),6.54(s,1H),5.96-5.90(m,1H),4.90-4.76(m,1H),4.43(s,1H),4.26-3.92(m,2H),3.77(s,2H),3.22-3.08(m,2H),2.50-1.60(m,8H),1.29(t,J＝6.0Hz,2H),1.10(t,J＝6.0Hz,2H).

Example 6

First step of

Triethylamine (465mg, 4.60mmol) was added to a solution of 3b (396mg, 2.30mmol) and 6a (200mg, 2.30mmol) in N, N-dimethylformamide (5mL), the mixture was reacted at room temperature for 3 hours, and the reaction mixture was purified by reverse phase column chromatography (acetonitrile: water ═ 0-100%) to give compound 6b (500mg) in yield: 98 percent.

MS-ESI calculated value [ M + H%]⁺224, found 224.

Second step of

Imidazole (300mg, 3.36mmol) was added to a dichloromethane solution (8mL) of 6b (500mg, 2.46mmol), and tert-butyldimethylsilyl chloride (400mg, 2.46mmol), the reaction system was reacted at room temperature overnight, concentrated under reduced pressure to give a residue, which was purified by column chromatography (ethyl acetate: petroleum ether ═ 0-100%) to give compound 6c (720mg), yield: 96 percent.

MS-ESI calculated value [ M + H%]⁺338, measured value 338.

The third step

10% Wet Palladium on carbon (10mg) was added to a solution of 6c (120mg, 0.36mmol) in methanol (3mL) under a hydrogen atmosphere (15psi) and reacted at room temperature for 3 hours. Filtration through celite and concentration under reduced pressure gave crude 6d (91 mg).

MS-ESI calculated value [ M + H%]⁺308, measured value 308.

The fourth step

1e (94mg, 0.29mmol) was added to 6d (91mg, 0.29mmol) of n-butanol solution (5mL) and the temperature was raised to 130 ℃ for reaction for 2 hours. The reaction solution was cooled to room temperature, and concentrated under reduced pressure to give a residue, which was purified by column chromatography (methanol: dichloromethane ═ 0 to 100%) to give compound 6e (80mg), yield: 46 percent.

MS-ESI calculated value [ M + H%]⁺596, found 596.

The fifth step

A4M ethyl acetate hydrochloride solution (2mL) was added to 6e (80mg, 0.13mmol), reacted at room temperature for 1 hour, and concentrated under reduced pressure to give crude 6f (20 mg).

The sixth step

2- (7-Benzotolyltriazole) -N, N, N ', N' -tetramethyluronium hexafluorophosphate (23mg, 0.06mmol) was added to a solution of crude 6f (20mg, 0.05mmol), (S) -2, 2-difluorocyclopropanecarboxylic acid (6mg, 0.05mmol) and triethylamine (10mg,0.10mmol) in N, N-dimethylformamide (2mL) and reacted at room temperature overnight. The reaction system was concentrated under pressure to give a residue, which was purified by reverse phase column chromatography (acetonitrile: water ═ 0 to 100%) to give compound 6(4mg) in yield: 16 percent.

MS-ESI calculated value [ M + H%]⁺486, measured value 486.

¹H NMR(400MHz,CDCl₃)δ8.12-8.05(m,1H),7.95-7.91(m,1H),7.49-7.42(m,1H),6.76(s,1H),5.94-5.90(m,1H),4.87-4.75(m,1H),4.40-3.95(m,5H),3.20-3.05(m,2H),2.96-2.90(m,2H),2.55-1.65(m,13H).

Example 7

First step of

Compound 4b (500mg, 4.42mmol) was dissolved in tetrahydrofuran (20mL), triphenylphosphine (2.32g, 8.84mmol) and compound 7a (828mg, 4.42mmol) were added thereto, the temperature was reduced to 0 ℃, diisopropyl azodicarboxylate (2.68g, 13.26mmol) was added dropwise, and the mixture was allowed to return to room temperature naturally and reacted overnight. Concentration under reduced pressure gave a residue, which was purified by column chromatography (ethyl acetate: petroleum ether ═ 0 to 100%) and reverse phase column chromatography (acetonitrile: water ═ 0 to 100%) to give compound 7b (1.00g), yield: 80 percent.

Second step of

Compound 7b (300mg, 1.06mmol) was dissolved in methanol (10mL), 10% wet palladium on carbon (30mg) was added, reacted overnight under hydrogen atmosphere (15psi), filtered through celite, and concentrated under reduced pressure to give compound 7c (250mg), yield: 94 percent.

The third step

Compound 7c (38mg, 0.15mmol) was dissolved in isopropanol (5mL), 7d (50mg, 0.15mmol) was added, and the reaction was allowed to warm to 100 ℃ overnight. After completion of the reaction, reverse phase column chromatography (acetonitrile: water ═ 0 to 100%) was performed to purify to obtain compound 7e (70mg), yield: 85 percent.

The fourth step

Compound 7e (70mg, 0.13mmol) was dissolved in methanol (2mL), and 6N ethyl acetate hydrochloride (5mL) was added to the solution to react at room temperature for 2 hours. N, N-diisopropylethylamine was added to the reaction system, and concentration under reduced pressure gave a residue, which was purified by preparative HPLC to give compound 7(30mg), yield: 44 percent.

MS-ESI calculated value [ M + 1]]⁺445, found 445.

¹H NMR(400MHz,CDCl₃)δ7.99(m,1H),7.77(m,1H),7.55(m，1H),6.73(brs,1H),5.97(m,1H),4.87-4.82(m,1H),4.43(m,1H),4.24(s,1H),4.05(m,2H),3.99(s,1H),3.16(m,2H),2.55(m,1H),2.21-1.76(m,6H),0.76-0.70(m,4H).

Example 8

First step of

Compound 8a (100mg,0.602mmol), tert-butyl 3, 8-diazabicyclo [3.2.1] octane-8-carboxylate (140mg, 0.66mmol) and triethylamine (182mg, 1.81mmol) were dissolved in N, N-dimethylformamide (4mL) and reacted at room temperature for 2 hours. Concentration under reduced pressure gave a residue, which was purified by reverse phase column chromatography (acetonitrile: water 0-100%) to give compound 8b (160mg) in yield: 78 percent.

MS-ESI calculated value [ M + 1]]⁺343, found value 343.

Second step of

Compound 8b (160mg, 0.47mmol) and cesium carbonate (180mg, 0.94mmol) were dissolved in N, N-dimethylformamide (6mL), compound 1e (79mg, 0.51 mmol) was added to the system, after displacement of nitrogen gas, xanthphos (28mg, 0.04mmol), bis-dibenzylideneacetone palladium (23mg, 0.04mmol) was added, the temperature was raised to 110 ℃ for reaction for 16 hours, cooled to room temperature, diluted with water (20mL), extracted with ethyl acetate (20mL x 3), the organic phases were combined, washed with saturated brine (20mL x 1), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give a residue, which was purified by column chromatography (ethyl acetate: petroleum ether ═ 0-100%) to give compound 8c (116mg), yield: 54 percent.

MS-ESI calculated value [ M + H%]⁺544, measured value 544.

The third step

8c (116mg, 0.49mmol) was added to a 4N ethyl acetate hydrochloride solution (3mL), reacted at room temperature for 2 hours, and concentrated under reduced pressure to give crude 8d (120 mg).

MS-ESI calculated value [ M + 1]]⁺360, measured value 360.

The fourth step

(S) -2, 2-Difluorocyclopropanecarboxylic acid (45mg, 0.37mmol) and 2- (7-benzotriazol oxide) -N, N, N ', N' -tetramethyluronium hexafluorophosphate (230mg, 0.60mmol) were dissolved in N, N-dimethylformamide (6mL), N, N-diisopropylethylamine (129mg, 1.00mmol) and 8d (120mg, 0.33mmol) were sequentially added thereto, reacted at room temperature for 4 hours, the reaction was quenched with water (5mL), ethyl acetate (5mL x 2) was extracted, the organic phase was combined, washed with saturated saline (5mL x 2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give a residue, which was purified by preparative HPLC to give compound 8(12mg) in yield: 8 percent.

MS-ESI calculated value [ M + 1]]⁺464, found 464.

¹H-NMR(400MHz,CDCl₃)δ7.86-7.84(m,1H),7.76-7.74(t,J＝8.0Hz,1H),7.54-7.51(t,J＝12.0Hz,1H),6.47(s,1H),4.84-4.76(m,1H),4.38-4.20(m,3H),4.16(s,2H),3.27-3.21(m,2H),2.55-2.51(m,1H),2.05-1.67(m,7H),1.25(s,2H),0.93(s,2H).

Example 9

Compound 1(50mg, 0.11mmol) was dissolved in methylene chloride (1mL), and diethylaminosulfur trifluoride (27mg, 0.17mmol) was added to the solution, followed by reaction at room temperature overnight. After quenching with water (10mL), saturated sodium bicarbonate solution (20mL) and dichloromethane (30mL) were added to dilute, the layers were separated, the organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give a residue, which was purified by preparative HPLC to give compound 9(5mg), yield: 9 percent.

MS-ESI calculated value [ M + H%]⁺448, found 448.

¹H NMR(400MHz,CDCl₃)δ7.98(dd,J＝6.0Hz,3.6Hz,1H),7.74(s,1H),7.54(d,J＝4.4Hz,1H),6.58(brs,1H),5.94-5.93(m,1H),5.33(d,J＝2.8Hz,1H),5.21(s,1H),4.83-4.71(m,5H),4.42-4.00(m,1H),4.23-4.22(m,1H),3.95-3.94(m,1H),3.20-3.09(m,2H),2.56-2.52(m,1H),2.26-2.21(m,1H),2.03-1.70(m,5H).

Example 10

First step of

Dissolving compound 10a (1.30g, 0.01mol) in dichloromethane (10mL), adding 3, 4-dihydro-2H-pyran (0.84g, 0.01mol), pyridine p-toluenesulfonate (241mg, 1.00mmol) under nitrogen protection, reacting overnight at room temperature, adding water (10mL), diluting, separating, washing the organic phase with saturated sodium chloride solution (10mL × 2), concentrating under reduced pressure to obtain a residue, and purifying the residue by column chromatography (ethyl acetate: petroleum ether ═ 0-100%) to obtain compound 10b (1.00g), yield: and 47 percent.

Second step of

10b (1.00g, 5.00mmol) was dissolved in tetrahydrofuran (20mL) while cooling on ice, and lithium aluminum hydride (380mg, 10.00mmol) was added in portions and reacted at room temperature for 2 hours. Water (1mL), a 15% aqueous solution of sodium hydroxide (1mL) and water (3mL) were added in this order to quench the reaction while cooling on ice, dried over anhydrous magnesium sulfate, filtered and concentrated under reduced pressure to give crude 10c (0.86g) which was used directly in the next reaction.

The third step

To a solution of crude 10c (0.86g, 5.00mmol) in tetrahydrofuran (10mL) under ice-bath conditions were added compound 4b (565mg, 5.00mmol), followed by triphenylphosphine (2.60g, 10.00 mmol). Diisopropyl azodicarboxylate (2.00g, 10.00mmol) was added dropwise under nitrogen protection, and the reaction was allowed to return to room temperature naturally overnight. The reaction solution was concentrated under reduced pressure to give a residue, which was purified by column chromatography (ethyl acetate: petroleum ether ═ 0 to 100%) to give compound 10d (1.10g), yield: 78 percent.

MS-ESI calculated value [ M + Na ]]⁺304, measured value 304.

The fourth step

To a 10d (140mg, 0.50mmol) solution in methanol (20mL) were added a saturated ammonium chloride solution (20mL) and iron powder (1.85g, 33.00mmol) in this order, and the mixture was heated to 90 ℃ for reaction for 1 hour. The reaction mixture was diluted with water (200mL) and ethyl acetate (100mL), separated, and the organic phase was washed with saturated brine (50mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (methanol: dichloromethane ═ 0 to 100%) to give compound 10e (100mg), yield: 79 percent.

MS-ESI calculated value [ M + H%]⁺252, measured value 252.

The fifth step

To a solution of 1g (194mg, 0.60mmol), 10e (150mg, 0.60mmol), palladium bis-dibenzylideneacetone (35mg, 0.06mmol) and Xantphos (55mg, 0.06mmol) in toluene (10mL) was added cesium carbonate (587mg, 1.80 mmol). After nitrogen gas was replaced, the temperature was raised to 110 ℃ to react overnight. The reaction solution was cooled to room temperature, filtered, and concentrated under reduced pressure to give a residue, which was purified by column chromatography (ethyl acetate: petroleum ether ═ 0 to 100%) to give compound 10f (100mg), yield: 30 percent.

MS-ESI calculated value [ M + H%]⁺540, found 540.

The sixth step

To a 10f (100mg, 0.18mmol) ethyl acetate solution (5mL) was added a 4N ethyl acetate hydrochloride solution (2mL) and the reaction was carried out at room temperature for 1 hour. The reaction mixture was concentrated under reduced pressure to give 10g (50mg) of crude product.

MS-ESI calculated value [ M + H%]⁺356, found 356.

Seventh step

To a solution of crude 10g (50mg, 0.18mmol) and (S) -2, 2-difluorocyclopropanecarboxylic acid (17mg, 0.14mmol) and N, N-diisopropylethylamine (36mg, 0.28mmol) in N, N-dimethylformamide (1mL) was added 2- (7-oxabenzotriazol) -N, N, N ', N' -tetramethyluronium hexafluorophosphate (106mg, 0.28 mmol). The reaction was carried out at room temperature for 1 hour. The reaction was dispersed in water (40mL) and ethyl acetate (40mL), the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give a residue, which was purified by preparative HPLC to give compound 10(27mg) in two-step yield: 42 percent.

MS-ESI calculated value [ M + H%]⁺460, measured value 460.

¹H NMR(400MHz,DMSO-d₆)δ8.85(brs,1H),7.91(d,J＝2.8Hz,1H),7.85(s,1H),7.41(d,J＝6.4Hz,1H),6.09-6.06(m,1H),5.30(s,1H),4.66-4.55(m,2H),4.20-4.08(m,4H),3.10-2.85(m,3H),2.01-1.89(m,9H),1.72-1.40(m,3H).

Example 11

First step of

To a solution of compound 1d (1.80g, 6.67mol) in methanol (10mL) was added a 4N ethyl acetate hydrochloride solution (10mL) and the mixture was reacted at room temperature for 1 hour. Concentration under reduced pressure gave a residue which was diluted with saturated sodium bicarbonate solution (50mL), extracted with ethyl acetate (50mL × 1), the organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to give crude 11a (1.20g) in yield: 100 percent.

Second step of

Dissolving compound 11a (0.90g, 5.00mmol) in N, N-dimethylformamide (10mL) under nitrogen protection, cooling to 0 ℃, adding 60% sodium hydride (400mg, 10.00mmol) to the reaction system, stirring for 30 minutes, adding methyl iodide (1.42g, 10.00mmol), naturally returning to room temperature for overnight reaction, quenching with saturated ammonium chloride solution (10mL), adding water (60mL) and ethyl acetate (60mL), diluting, separating, washing the organic phase with water (50mL x 1), saturated saline (50mL x 1), drying over anhydrous sodium sulfate, filtering, concentrating under reduced pressure to obtain a residue, purifying the residue by column chromatography (ethyl acetate: petroleum ether ═ 0-100%) to obtain compound 11b (450mg) with yield: 46 percent.

The third step

Compound 11b (450mg, 2.30mmol) was dissolved in methanol (10mL), 10% wet palladium on carbon (100mg) was added, and the reaction was carried out under a hydrogen atmosphere (15psi) at room temperature for 1 hour. The reaction was filtered through celite, and the filtrate was concentrated under reduced pressure to give crude 11c (350mg), yield: 91 percent.

MS-ESI calculated value [ M + H%]⁺168, found 168.

The fourth step

To a solution of 1g (611mg, 1.90mmol), 11c (350mg, 2.10mmol), palladium bis dibenzylideneacetone (92mg, 0.10mmol) and Xantphos (116mg, 0.20mmol) in toluene (12mL) was added cesium carbonate (1.40g, 4.20mmol) and the reaction was allowed to warm to 110 ℃ under nitrogen overnight. The reaction solution was cooled to room temperature, filtered, and the filtrate was concentrated to give a residue, which was purified by column chromatography (ethyl acetate: petroleum ether ═ 0 to 100%) to give compound 11d (300mg), yield: 35 percent.

MS-ESI calculated value [ M + H%]⁺456, found 456.

The fifth step

11d (228mg, 0.50mmol) was dissolved in ethyl acetate (10mL), and a 4N ethyl acetate hydrochloride solution (5mL) was added thereto at room temperature, followed by stirring at room temperature for 2 hours. Concentration under reduced pressure gave crude 11e, which was used directly in the next reaction.

MS-ESI calculated value [ M + H%]⁺356, found 356.

The sixth step

(S) -2, 2-Difluoropropanecarboxylic acid (17mg, 0.14mmol) was dissolved in N, N-dimethylformamide (5mL), N, N-diisopropylethylamine (36mg, 0.28mmol) and 2- (7-benzotriazol oxide) -N, N, N ', N' -tetramethylurea hexafluorophosphate (106mg, 0.28mmol) were added under nitrogen protection, and after stirring for 10 minutes, Compound 11e (50mg, 0.14mmol) was added and stirred at room temperature for 2 hours. Diluted with water (20mL) and extracted with ethyl acetate (20mL × 2), washed with saturated sodium chloride (20mL × 1), concentrated under reduced pressure to give compound 11(28mg), yield: 44 percent.

MS-ESI calculated value [ M + H%]⁺460, measured value 460.

¹H NMR(400MHz,DMSO-d₆)δ8.96(brs,1H),7.94-7.89(m,2H),7.42(d,J＝15.6Hz,1H),6.13-6.09(m,1H),4.67-4.56(m,2H),4.24-4.04(m,4H),3.22-3.14(m,4H),3.01-2.91(m,2H),2.02-1.64(m,6H),0.76-0.70(m,4H).

Biological activity assay

Kinase assay

The in vitro inhibition effect of the compound on JAK1, JAK2, JAK3 or TYK2 kinase is detected by using a Caliper mobility shift assay method. Test compounds were dissolved in DMSO to prepare 10mM stock solutions. Gradient dilution of compound stock solution with DMSO to prepare 50 × working solution (total 10 concentrations), and transferring each concentration of working solution to

In the motherboard. By using

A non-contact nano-liter acoustic pipetting system transfers 5. mu.L of compound solution or DMSO at the corresponding concentration from the master plate to a 384-well reaction plate. Then, 10. mu.L of 2.5 Xkinase solution was added to a 384-well reaction plate, and after incubation at room temperature for 10 minutes, 10. mu.L of a 2.5 XFAM-labeled polypeptide and ATP mixed solution (ATP final concentration: 1mM) was added, and after incubation at 28 ℃ for a specific time, 30. mu.L of stop solution was added, and detection was carried out on a Caliper, and the conversion value, i.e., the height of the product peak as compared with the sum of the heights of the substrate peak and the product peak, was calculated. Percent inhibition of kinase by the compound was calculated using the following formula and fitted with XLFit 5.4.0.8 to IC₅₀The value is obtained.

Percent inhibition ═ max-conversion)/(max-min) × 100, where "max" represents the conversion reading for the DMSO control and "min" represents the conversion reading for the low control.

The specific test results are shown in Table-1.

TABLE-1 results of testing of the compounds of the invention for JAK family kinases

N/T: temporarily without data

Note: the activity data were obtained at 1mM ATP concentration.

Cell experiment method

In a murine original B lymphocyte BaF3, a recombinant gene (comprising TEL and human JAK1, TYK2, JAK2 and JAK3 kinase domains) is transferred by using a genetic engineering technology, wherein the TEL can promote phosphorylation and continuous activation of TEL-JAK1 or TEL-TYK2 kinase dimers, so that cells can grow depending on the activity of the recombinant kinase. If the compound has JAK1 or TYK2 kinase inhibitory activity, it can cause cell death by inhibiting kinase activity. The cell titer-Glo method is adopted to detect the inhibition activity of the embodiment on the proliferation of the in vitro cultured genetically engineered cell lines Ba/F3-TEL-JAK1, Ba/F3-TEL-TYK2, Ba/F3-TEL-JAK2 and Ba/F3-TEL-JAK3, Graphpad 7.0 is adopted to fit a curve and calculate IC (integrated circuit) of the cell titer-Glo method₅₀The value is obtained. The specific test results are shown in Table-2.

TABLE-2 inhibitory Activity of the inventive Compounds on the proliferation of murine pro-B lymphocytes BaF3 cells

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Claims

1. A compound having a structure represented by the following formula (I), or a pharmaceutically acceptable salt thereof:

wherein the content of the first and second substances,

ring B is a substituted or unsubstituted 3-6 membered carbocyclic ring;

x is selected from a bond, NH, N (C1-C4 alkyl) or (CR)₂)_m；

Y is (CR)₂)_n；

m and n are respectively and independently selected from 0, 1 or 2;

and when X is selected from (CH)₂)_mWhen m and n are not 0 at the same time;

2. The compound of claim 1, wherein X is NH or (CH)₂)_m。

3. The compound of claim 1, wherein ring a is a substituted or unsubstituted group selected from the group consisting of:

4. the compound of claim 1, wherein R is₁Selected from the group consisting of: substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C3-C8 cycloalkyl, substituted or unsubstituted C1-C4 alkoxy, and substituted or unsubstituted C1-C4 alkylamino.

5. The compound of claim 1, wherein (I) is preferably selected from (IIA) and (IIB):

wherein ring B is a substituted or unsubstituted 3-4 membered carbocyclic ring.

6. The compound of claim 1, wherein R is₂Selected from hydrogen, halogen and CN.

7. The compound of claim 1, wherein said compound is selected from the group consisting of:

8. a pharmaceutical composition comprising (1) a compound of any one of claims 1-7, or a stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt, hydrate, or solvate thereof; and (2) a pharmaceutically acceptable carrier.

9. Use of a compound according to any one of claims 1 to 7, or a stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt, hydrate or solvate thereof, or a pharmaceutical composition according to claim 8, for the preparation of a pharmaceutical composition for the prophylaxis and/or treatment of a disease or disorder associated with JAK kinase activity or expression.

10. The use according to claim 9, wherein the disease or condition is selected from the group consisting of: inflammation, autoimmune diseases, neuroinflammation, arthritis, rheumatoid arthritis, spondyloosteoarthritis, systemic lupus erythematosus, lupus nephritis, gouty arthritis, pain, fever, pulmonary sarcoidosis, silicosis, cardiovascular disease, atherosclerosis, sarcoidosis, myocarditis and cardiac reperfusion injury, cardiomyopathy, stroke, ischemia, reperfusion injury, cerebral edema, cerebral trauma, neurodegenerative diseases, liver disease, inflammatory bowel disease, Crohn's disease, ulcerative colitis, nephritis, retinitis, retinopathy, macular degeneration, glaucoma, diabetes mellitus (type 1) and type 2), diabetic neuropathy, viral and bacterial infections, myalgia, endotoxic shock, toxic shock syndrome, autoimmune diseases, osteoporosis, multiple sclerosis, endometriosis, menstrual cramps, vaginitis, candidiasis, cancer, fibrosis, obesity, muscular dystrophy, polymyositis, dermatomyositis, autoimmune hepatitis, primary biliary cirrhosis, primary sclerosing cholangitis, vitiligo, alopecia, alzheimer's disease, skin flushing, eczema, psoriasis, atopic dermatitis and sunburn.