CN113637019A

CN113637019A - Selective JAK1 inhibitor compound and preparation method and application thereof

Info

Publication number: CN113637019A
Application number: CN202010394301.2A
Authority: CN
Inventors: 李傲; P·K·贾达夫; 姚元山; 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: 2020-05-11
Filing date: 2020-05-11
Publication date: 2021-11-12

Abstract

The invention provides a heterocyclic compound serving as a JAK1 inhibitor and a synthesis and use method thereof, and particularly provides a compound shown as a formula (I), a preparation method thereof and application of the compound serving as a JAK1 inhibitor. The compounds exhibit excellent inhibitory activity against JAK 1.

Description

Selective JAK1 inhibitor compound and preparation method and application thereof

Technical Field

The invention relates to a selective JAK1 inhibitor compound, an isomer, a solvate, a salt of the compound, a medicament taking the compound or the salt as an active ingredient, and application of the compound in medicaments for treating and/or preventing JAK1 related target diseases such as immune system diseases, rheumatoid arthritis, tumors and the like.

Background

The JAK-STAT signaling pathway is a cytokine-stimulated signaling pathway discovered in recent years, JAKs play an important role in cytokine signaling, and downstream substrates of the JAK kinase family include protein-transcribing Signal Transducers and Activators (STATs). JAK proteins are important members of this pathway and abnormal increases in their activity often lead to the development of diseases, many of which are associated with abnormal cellular responses of the JAK-STAT signaling pathway, including autoimmune diseases, inflammatory diseases, bone diseases, metabolic diseases, neurological and neurodegenerative diseases, cancer, cardiovascular diseases, allergies and asthma, alzheimer's disease.

Rheumatoid Arthritis (RA) is a common chronic autoimmune disease in clinic, mainly manifested by joint swelling, pain, stiffness, deformity and severely impaired function, with a population incidence of 0.5% -1.0%. Because the pathogenesis of RA is not clear, the pathological process of RA is difficult to control, the disability rate is high, the physical and mental health of a patient is seriously damaged, and the life quality of the patient is reduced. Currently, non-steroidal anti-inflammatory drugs (NSAIDs), disease-modifying antirheumatic drugs (DMARDs), and antibody-based drugs are mainly used for the treatment of RA. First-line drugs for treating RA have long been DMARDs, and in 1988, the 1 st DMARD drug Methotrexate (MTX) was FDA approved for the treatment of RA, an important milestone in the history of RA treatment. The medicine is widely applied due to the advantages of effectiveness, tolerance, safety and the like, but has adverse reactions including nausea, vomiting, stomach discomfort, hepatotoxicity and the like. In contrast, newly developed antibody drugs have better efficacy and safety indexes for moderate-severe RA, but beneficial people are obviously limited due to targeting of specific cytokines, and the popularization of the drugs is limited due to treatment cost and injection mode administration.

Over the course of the past 20 years, RA treatment has advanced significantly and patient condition has been effectively controlled by existing treatments. Nevertheless, RA patients suffer from disease recurrence, poor therapeutic efficacy, poor long-term tolerance, and some adverse reactions. More importantly, the quality of life of RA patients, including organ function such as joints, has not been truly improved by current therapies, and there is still a great unmet clinical need in this field to address the need to restore normal function in patients.

Research shows that mononuclear/macrophage, lymphocyte and the like infiltrated in RA synovial tissues and cells generate a large amount of cytokines in an autocrine mode, the cytokines interact with each other, JAK/STAT Signal channels (Janus kinase/Signal transducer and activators of transcription signaling pathway) are activated through different ways, and cascade amplification of the cytokines can be blocked by specifically inhibiting the JAK/STAT Signal channels, so that symptoms of damaged joints of RA patients are improved, and the JAK/STAT Signal channels become potential targets for treating RA.

As JAK kinase participates in various important physiological processes in vivo, adverse reactions are possibly generated by the wide inhibition of different subtypes, Tofacitinib is used for moderate and severe RA patients with insufficient MTX reaction or intolerance, and certain adverse reactions accompanied by the Tofacitinib are observed in clinical tests and comprise infection, tuberculosis, tumor, anemia, liver injury, cholesterol increase and the like. Tofacitinib has obvious inhibitory activity on JAK1, JAK2 and JAK3 subtypes, and because JAK2 activity is related to erythrocyte differentiation and lipid metabolism, part of adverse reactions are considered to be related to the non-selective inhibitory characteristics of the medicine. Therefore, the search for selective JAK1 and/or JAK3 inhibitors would be a new direction for the study of RA drugs. At present, JAK inhibitors are proved to be used for treating diseases of a blood system, tumors, rheumatoid arthritis, psoriasis and the like.

In view of the foregoing, there remains a need in the art for the development of more selective JAK inhibitor compounds.

Disclosure of Invention

The invention aims to provide a selective JAK1 inhibitor compound with a novel structure.

In a first aspect of the present invention, there is provided a compound represented by the following formula (I), and isomers, solvates or pharmaceutically acceptable salts thereof:

or a pharmaceutically acceptable salt or hydrate thereof, wherein:

R¹selected from the group consisting of: H. substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C3-C8 cycloalkyl;

R²selected from the group consisting of: h, substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C3-C8 cycloalkyl;

R³selected from the group consisting of: h, substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C3-C8 cycloalkyl;

R⁴selected from the group consisting of: CN, CONH₂；

R⁵、R⁶、R⁷、R⁸Each independently selected from the group consisting of: H. halogen, substituted or unsubstituted C1-C4 alkyl, CN;

R⁹selected from the group consisting of: substituted or unsubstituted C2-C4 alkenyl, substituted or unsubstituted C2-C4 alkynyl, cyano, CONH₂；

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, amide, sulfonamide, sulfone, a group unsubstituted or substituted with 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 alkyl, C1-C6 alkoxy, or ═ O.

In another preferred embodiment, R is¹、R²And R³Each independently selected from the group consisting of: H. substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C3-C8 cycloalkyl; wherein said substituted is substituted with a substituent selected from the group consisting of: CN, OH, OCH₃And a halogen.

In another preferred embodiment, R⁵、R⁶、R⁷、R⁸Each independently selected from the group consisting of: H. f, Cl, Me, and CN.

In another preferred embodiment, R is⁵And R⁶Each independently is halogen.

In another preferred embodiment, theR of (A) to (B)⁷And R⁸Each independently is H.

In another preferred embodiment, R is⁵And R⁶Each independently is F.

In another preferred embodiment, R⁹Selected from the group consisting of: vinyl, ethynyl, CN, CONH₂。

In another preferred embodiment, R¹Is Me, R²And R³Each independently is H.

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

in a second aspect of the invention, there is provided a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a compound, isomer, solvate or pharmaceutically acceptable salt or hydrate thereof according to the first aspect of the invention.

In another preferred embodiment, the pharmaceutical composition is used for treating or preventing diseases related to the activity or expression amount of JAK kinase; preferably, the disease is selected from the group consisting of: cancer, myeloproliferative disease, myelofibrosis, inflammation, immunological disease, organ transplantation, viral disease, cardiovascular disease or metabolic disease, human or animal autoimmune disease, rheumatoid arthritis, skin disorder, multiple sclerosis, rheumatoid arthritis, psoriatic arthritis, inflammatory bowel disease, myasthenia gravis, psoriasis, alopecia.

In a third aspect of the present invention, there is provided a use of the compound according to the second aspect of the present invention, or a pharmaceutically acceptable salt or hydrate thereof, for preparing a pharmaceutical composition for treating or preventing a disease associated with an activity or an expression amount of a JAK kinase.

In another preferred embodiment, the disease is selected from the group consisting of: cancer, myeloproliferative disease, myelofibrosis, inflammation, immunological disease, organ transplantation, viral disease, cardiovascular disease or metabolic disease, human or animal autoimmune disease, rheumatoid arthritis, skin disorder, multiple sclerosis, rheumatoid arthritis, psoriatic arthritis, inflammatory bowel disease, myasthenia gravis, psoriasis, alopecia.

In another preferred embodiment, the pharmaceutical composition is for use in the treatment or prevention of a disease selected from the group consisting of: autoimmune diseases, rheumatoid arthritis, skin disorders, multiple sclerosis, rheumatoid arthritis, psoriatic arthritis, inflammatory bowel disease, myasthenia gravis, psoriasis in humans or animals.

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 present inventors have conducted extensive and intensive studies for a long time and have unexpectedly found a compound represented by the 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-C₄Alkyl represents a straight or branched chain alkyl group having 1 to 4 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, and the like.

As used herein, the term "C₃-C₈Cycloalkyl "refers to cycloalkyl groups having 3 to 8 carbon atoms. It may be a single ring, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, or the like. Also bicyclic, e.g., bridged, fused or spiro forms.

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 "5-10 membered heteroaryl having 1-3 heteroatoms selected from the group consisting of N, S and O" refers to a cyclic aromatic group having 5-10 atoms and wherein 1-3 atoms are heteroatoms selected from the group consisting of N, S and O. It may be a single ring or a condensed ring form. 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 "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

The software names, and the commercial compounds are under the supplier catalog 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 cancer, myeloproliferative diseases, inflammation, immunological diseases, organ transplantation, viral diseases, cardiovascular diseases, metabolic diseases, and the like, because the compound of the present invention has an excellent inhibitory activity against JAK1 kinase.

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 (e.g., sodium lauryl sulfate), coloring agents, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, and the like.

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, subcutaneous or topical).

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. Having described the present application in detail and having disclosed specific embodiments thereof, it will be apparent to one skilled in the art that various changes and modifications can be made to the embodiments of the application without departing from the spirit and scope of the application.

Examples

Example 1

First step of

To 1a (1.00g, 4.73mmol) in 10mL of methanol was added 30% methylamine in methanol (1.00g, 9.66mmol) and the reaction was carried out at room temperature for 3 hours. Sodium borohydride (361mg, 9.54mmol) was added in portions, and the resultant reaction solution was reacted at room temperature overnight. At the end of the reaction, quench with water (20mL), extract with ethyl acetate (30mL x 2), combine the organic phases, wash sequentially with water (20mL x 1) and saturated sodium chloride solution (20mL x 1), dry over anhydrous sodium sulfate, filter, concentrate the filtrate under reduced pressure to give a residue, concentrate the residue under reduced pressure to give crude 1b (1.00g), yield: 93 percent.

¹H NMR(400MHz,CDCl₃)δ3.91(s,2H),3.83(s,2H),3.10-3.06(m,1H),2.46-2.41(m,2H),2.32(s,3H),1.87-1.82(m,2H),1.43(s,9H).

Second step of

A solution of 1b (1.00g, 4.42mmol), 4-chloro-7H-pyrrolo [2,3-d ] pyrimidine (677mg, 4.41mmol) and diisopropylethylamine (1.13g, 8.74mmol) in 10mL of n-butanol was warmed to 100 ℃ and reacted overnight. After the reaction was completed, it was cooled, and the reaction solution was concentrated under reduced pressure to obtain a residue, which was slurried with ethyl acetate (10mL) to obtain 1c (1.00g), yield: 66 percent.

MS-ESI calculated value [ M +1 ]]⁺344, found 344.

The third step

To a solution of 1c (500mg, 1.46mmol) in 5mL of methanol was added dropwise a solution of 5mL of ethyl hydrogen chloride in ethyl acetate (6M). The resulting reaction mixture was reacted at room temperature for 1 hour. After the reaction was completed, the reaction mixture was concentrated under reduced pressure to obtain a white solid 1d which was used in the next reaction.

MS-ESI calculated value [ M +1 ]]⁺244, actually measuringA value of 244.

The fourth step

A solution of 1e (500mg, 3.27mmol), N-bromosuccinimide (872mg, 4.90mmol) and azobisisobutyronitrile (52mg, 0.32mmol) in 5mL of carbon tetrachloride was warmed to 100 ℃ and reacted overnight. After the reaction was completed, it was cooled, and concentrated under reduced pressure to give a residue, which was subjected to column chromatography (ethyl acetate: petroleum ether ═ 0 to 100%) to give 1f (300mg), yield: 48 percent.

¹H NMR(400MHz,CDCl₃)δ7.47-7.42(m,1H),7.37-7.31(m,1H),7.54(s,2H).

The fifth step

To a solution of 1f (220mg, 0.95mmol) in 5mL of anhydrous acetonitrile was added N-methyl-N-morpholine-N-oxide (446mg, 3.81mmol), and the resulting reaction mixture was reacted at room temperature overnight. The reaction solution was concentrated under reduced pressure to give a residue, and the residue was subjected to column chromatography (ethyl acetate: petroleum ether ═ 0 to 100%) to give 1g (35mg) in yield: 22 percent.

¹H NMR(400MHz,CDCl₃)δ10.39(s,1H),7.78-7.73(m,1H),7.56-7.52(m,1H).

The sixth step

After cyanamide (25mg, 0.59mmol) and sodium tert-butoxide (58mg, 0.60mmol) were added in this order to 1g (25mg, 0.15mmol) of a 5mL methanol solution, the reaction was carried out at room temperature for 0.5 hour. N-bromosuccinimide (107mg, 0.60mmol) was added to the reaction mixture, and the mixture was heated to 50 ℃ to react for 12 hours. After the reaction was completed, it was cooled, and 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 1h (5mg) in yield: 19 percent.

¹H NMR(400MHz,CDCl₃)δ7.57-7.52(m,2H),4.15(s,3H).

Seventh step

To a solution of 1h (200mg, 0.90mmol) in 10mL of methanol were added 1d (252mg, 0.90mmol) and triethylamine (272mg, 2.69mmol) in that order, at room temperature overnight. After the reaction was complete, the reaction mixture was directly purified by preparative HPLC to give 1(110 mg). Yield: 28 percent.

¹H NMR(400MHz,CDCl₃)δ11.62(s,1H),8.09(d,1H),8.05-8.00(m,1H),7.70(t,J＝6.8Hz,1H),7.15-7.12(m,1H),6.61-6.56(m,1H),5.19-5.03(m,1H),4.42(s,1H),4.27(s,2H),4.12(s,1H),3.20(d,3H),2.60-2.46(m,4H).

MS-ESI calculated value [ M +1 ]]⁺433, measured value 433.

Example 2

First step of

A solution of 1(50mg, 0.12mmol) in 5mL hydrogen chloride 1, 4-dioxane (4M) was allowed to react overnight at 30 ℃. The reaction was directly purified by preparative HPLC to give 2(12mg) yield: 23 percent.

¹H NMR(400MHz,DMSO-d₆)δ11.62(s,1H),8.17-7.98(m,1H),7.91-7.72(m,1H),7.45-7.25(m,1H),7.13(s,1H),6.57(s,1H),6.47(s,1H),6.11(s,1H),5.25-4.96(m,1H),4.23-3.75(m,4H),3.20(dd,J＝6.9,3.4Hz,3H),2.60-2.51(m,4H).

MS-ESI calculated value [ M +1 ]]⁺451, found value 451.

Example 3

First step of

Methyl iodide (659mg, 4.64mmol) was added to a solution of 3a (1.00g, 4.22mmol) and potassium carbonate (637mg, 4.61mmol) in 10mL of N, N-dimethylformamide, and the resulting reaction mixture was reacted at room temperature overnight. After the reaction was complete, it was diluted with water (20mL), extracted with ethyl acetate (20mL x 1), the organic phases combined and dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give 3b (486mg) which was used directly in the next reaction.

¹H NMR(400MHz,CDCl₃)δ7.69-7.63(m,1H),7.46-7.41(m,1H),3.99(s,3H).

Second step of

A solution of 3b (386mg, 1.54mmol), ethynyltrimethylsilane (226mg, 2.30mmol), bis triphenylphosphine palladium dichloride (21mg, 0.03mmol) and cuprous iodide (3mg, 0.15mmol) in 10mL triethylamine was reacted at 80 ℃ under nitrogen overnight. After the reaction was completed, it was cooled, filtered, and concentrated under reduced pressure to give a residue, which was purified by silica gel column chromatography (ethyl acetate: petroleum ether ═ 0 to 100%) to give 3c (400mg), yield: 97 percent.

¹H NMR(400MHz,CDCl₃)δ7.70-7.65(m,1H),7.29-7.26(m,1H),3.99(s,3H),0.33(s,9H).

The third step

Lithium aluminum hydride (149mg, 3.93mmol) was added portionwise to a solution of 3c (350mg, 1.30mmol) in 5mL tetrahydrofuran under nitrogen at 0 deg.C, and the resulting reaction was allowed to warm to room temperature and allowed to react overnight. After the reaction was complete, it was slowly quenched with water (10mL), extracted with ethyl acetate (10mL × 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give a residue, which was subjected to column chromatography (ethyl acetate: petroleum ether ═ 0-100%) to give 3d (100mg) in yield: 46 percent.

¹H NMR(400MHz,CDCl₃)δ7.27-7.23(m,1H),7.19-7.16(m,1H),4.79(d,J＝5.6Hz,2H),3.35(s,1H).

The fourth step

Manganese dioxide (522mg, 6.00mmol) was added to a solution of 3d (100mg, 0.59mmol) in 5mL of dichloromethane, and the resulting reaction mixture was reacted at room temperature overnight. After the reaction was completed, filtration was performed, and the filtrate was concentrated under reduced pressure to give a residue, which was subjected to column chromatography (ethyl acetate: petroleum ether ═ 0 to 100%) to give 3e (60mg) in yield: 60 percent.

¹H NMR(400MHz,CDCl₃)δ10.33(s,1H),7.62-7.58(m,1H),7.36-7.35(m,1H),3.58(s,1H).

The fifth step

To a solution of 3e (40mg, 0.24mmol) in 5mL of methanol were added cyanamide (40mg, 0.95mmol) and sodium tert-butoxide (92mg, 0.96mmol) in this order, and the resulting reaction mixture was stirred at room temperature for 0.5 hour. N-bromosuccinimide (160mg, 0.90mmol) was added to the above reaction solution, which was then raised to 50 ℃ for reaction for 12 hours. After the reaction was completed, it was cooled, and 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 3f (30mg) in yield: 57 percent.

¹H NMR(400MHz,CDCl₃)δ7.40-7.37(m,2H),4.11(s,3H),3.54(s,1H).

The sixth step

To a solution of 3f (30mg, 0.14mmol) and 1d (33mg, 0.12mmol) in 10mL of methanol was added triethylamine (27mg, 0.27mmol), and the resulting reaction mixture was reacted at room temperature overnight. At the end of the reaction, the reaction was purified by preparative HPLC to give 3(20mg) yield: 34 percent.

¹H NMR(400MHz,CDCl₃)δ9.34(d,J＝13.5Hz,1H),8.27(d,J＝16.2Hz,1H),7.39(dd,J＝8.4,4.5Hz,1H),7.20(t,J＝6.7Hz,1H),7.04(s,1H),6.54(d,J＝3.1Hz,1H),5.14(dd,J＝13.6,8.8Hz,1H),4.37(d,J＝50.1Hz,2H),4.22(s,1H),4.07(s,1H),3.51(d,J＝3.4Hz,1H),3.30(d,J＝14.7Hz,3H),2.79-2.60(m,2H),2.50(ddd,J＝22.2,12.6,9.7Hz,2H).

MS-ESI calculated value [ M +1 ]]⁺432, found 432.

Biological activity assay

The in vitro inhibition effect of the compound on JAK1 or JAK2 kinase was tested by the 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 50X working solution (total 10 concentrations), and transferring 40 μ L of each working solution to

In the motherboard. By using

A non-contact nano-liter acoustic pipetting system transfers 400nL of compound solution or DMSO at a corresponding concentration from a master plate to a 384-well reaction plate. Then 10. mu.L of 2% kinase solution is added into 384-well reaction plates, after incubation for 10 minutes at room temperature, 10. mu.L of 2% FAM-labeled polypeptide and ATP mixed solution is added, after incubation for a specific time at 28 ℃, 30. mu.L of stop solution is added, and detection is carried out on a CaliperThe conversion value, i.e. the height of the product peak compared to the sum of the height 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 is (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 results of the testing of the synthesized compounds of the present invention are shown in table 1.

Inhibitory Activity of the Compounds of Table 1 against JAK1

Compound numbering	JAK1IC₅₀(nM)	JAK2IC₅₀(nM)
			Example 1	27	1408
Example 2	407	NT
			Example 3	4.5	868.4

Note: NT: no test; the ATP concentration tested was 1 mM.

And (4) conclusion: the compound synthesized by the invention has strong inhibition effect on Jak1 and very high selectivity on JAK 2.

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 of the following formula (I):

or a pharmaceutically acceptable salt or hydrate thereof, wherein:

R²selected from the group consisting of: H. substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C3-C8 cycloalkyl;

R³selected from the group consisting of: H. substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C3-C8 cycloalkyl;

R⁴selected from the group consisting of: CN, CONH₂；

2. The compound of claim 1, wherein R is¹、R²And R³Each independently selected from the group consisting of: H. substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C3-C8 cycloalkyl; wherein said substituted is substituted with a substituent selected from the group consisting of: CN, OH, OCH₃And a halogen.

3. The compound of claim 1, wherein R is⁵、R⁶、R⁷、R⁸Each independently selected from the group consisting of: H. f, Cl, Me, and CN.

4. The compound of claim 1, wherein R is⁹Selected from the group consisting of: vinyl, ethynyl, CN, CONH₂。

5. The compound of claim 1, wherein R is¹Is Me, R²And R³Each independently is H.

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

7. a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a compound, isomer, solvate or pharmaceutically acceptable salt or hydrate thereof of claim 1.

8. The pharmaceutical composition of claim 7, wherein the pharmaceutical composition is for treating or preventing a disease associated with the activity or expression level of a JAK kinase; preferably, the disease is selected from the group consisting of: cancer, myeloproliferative disease, myelofibrosis, inflammation, immunological disease, organ transplantation, viral disease, cardiovascular disease or metabolic disease, human or animal autoimmune disease, rheumatoid arthritis, skin disorder, multiple sclerosis, rheumatoid arthritis, psoriatic arthritis, inflammatory bowel disease, myasthenia gravis, psoriasis, alopecia.

9. Use of a compound according to any one of claims 1 to 6, or a pharmaceutically acceptable salt or hydrate thereof, for the preparation of a pharmaceutical composition for the treatment or prevention of a disease associated with the activity or expression level of a JAK kinase.

10. The use according to claim 9, wherein the disease is selected from the group consisting of: cancer, myeloproliferative disease, myelofibrosis, inflammation, immunological disease, organ transplantation, viral disease, cardiovascular disease or metabolic disease, human or animal autoimmune disease, rheumatoid arthritis, skin disorder, multiple sclerosis, rheumatoid arthritis, psoriatic arthritis, inflammatory bowel disease, myasthenia gravis, psoriasis, alopecia.