CN110734427B - Alkenyl-containing pyrimidine formamide compound, composition and application thereof - Google Patents

Abstract

The invention relates to a novel class of compounds, compositions and uses thereof as JAK inhibitors. Specifically, the invention provides a compound (shown as a formula (1)) with strong JAK inhibitory activity, or an isomer, a solvate, a hydrate, a pharmaceutically acceptable salt and a prodrug thereof, and a pharmaceutical composition containing the compound. The invention also discloses the use of a compound or a pharmaceutical composition of the invention in the manufacture of a medicament for the treatment of an autoimmune disease or cancer.

Description

Alkenyl-containing pyrimidine formamide compound, composition and application thereof

Technical Field

The invention belongs to the field of medicinal chemistry, and particularly relates to a pyrimidine carboxamide compound containing alkenyl, an isomer, a hydrate, a solvate, a pharmaceutically acceptable salt or a prodrug thereof, a medicinal composition thereof, and application of the pyrimidine carboxamide compound containing alkenyl in preparation of medicaments for treating autoimmune diseases and cancers related to tyrosine kinases JAK1, JAK2, JAK3 and TYK 2.

Background

JAK kinases (Janus kinases) and their downstream effector, signal transduction and transcriptional activator proteins form an important cytokine signaling pathway, the JAK-STAT pathway. Researches show that the JAK-STAT pathway can be activated by various cytokines, growth factors and receptors and participate in the processes of cell proliferation, differentiation, apoptosis, angiogenesis, immunoregulation and the like. The JAK kinase is a key kinase in the JAK-STAT signal pathway, which was found more than two decades later, the first JAK kinase inhibitor (tofacitinib) was approved for rheumatoid arthritis treatment in 2012 [ Norman p, Selective JAK inhibitors in details for rhematoid arthritis, Expert Opin Investig Drugs,2014,23: 1067-.

JAK1, JAK2, JAK3 and TYK2, relative molecular mass can reach 120000-140000, homology can reach 40% -70%, the JAK kinase family members can be divided into 7 homologous structural domains (JH) from C end to N end, JH1 is a kinase region and is highly conserved in the JAK family; JH2 is a kinase-like region or "pseudo" kinase region, the pseudo kinase domain is a unique property of JAK protein which is different from other tyrosine proteins, the kinase region has no catalytic activity but has a regulating effect on the activity of JH1, and mutations in the domain can often lead to the enhancement or the reduction of the JAK kinase activity and further lead to the occurrence of certain diseases; JH3-JH4 is the SH2 domain (Src homology 2 domain) which contains about 100 amino acid residues that specifically recognize and bind phosphorylated tyrosine residues on ligands; JH5-JH7 are FERM domains that are conserved, primarily regulating JAK binding to receptors. JAK3, a member of the JAK kinase family, structurally contains the kinase domain described above, and changes in kinase activity are caused by mutations in specific amino acids in different domains.

The JAK-STAT signal pathway is an important intracellular signal transduction pathway in the processes of growth, activation, differentiation, apoptosis and function development of various cells. STATs are cytoplasmic proteins that bind to DNA in the regulatory region of target genes and are downstream substrates of JAK. The STAT family includes 7 members including STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B, STAT6, and the like. The interaction between JAKs and STATs plays an important role in Cytokine receptor signaling pathways [ O' Sullivan LA et al, Cytokine receptor signaling through the JAK-Stat-Socs pathway in disease, Mol Immunol,2007,44:2497-2506 ]. Cytokine receptors on cell surfaces, when bound to their respective cytokine ligands, cause dimerization of the receptor molecules, allowing JAK kinases coupled to the receptors to approach each other and become activated by interactive tyrosine phosphorylation. The JAK-STAT signaling pathway is a signaling pathway stimulated by multiple cytokine receptors, JAK kinases mediate signaling of most intracellular cytokines, such as Interleukins (ILs), Interferons (IFNs), Erythropoietins (EPO), Granulocyte and Macrophage Colony Stimulating Factor (GMCSF), somatotropin (GH), Prolactin (PRL), Thrombopoietin (TPO), platelet derived factor (PDGF), and Epidermal Growth Factor (EGF), and the like, and different receptors activate different subtypes of JAK kinases to exhibit differential biological functions [ Pesu m.et al, Therapeutic targeting of Janus kinases, Immunol Rev,2008,223: 132-.

JAK1 and JAK2 are expressed in each tissue cell of the human body, and JAK3 is mainly expressed in each hematopoietic tissue cell, and is mainly present in bone marrow cells, thymocytes, NK cells, activated B lymphocytes, and T lymphocytes. The deletion of JAK1 and JAK2 can cause lethal injury to human body, and the deletion of JAK3 can avoid toxic adverse reaction for damaging other tissue cells [ Yamaoka K., et al., JAK3 novel ligands-cell cytokine production and survival, Blood,2005,106: 3227-. Based on the functional characteristics and special tissue distribution of each subtype in the JAK kinase family, JAK3 has become a popular target for treating autoimmune diseases, and more clinical researches focus on blocking the JAK3 signal transduction path for treating rheumatoid arthritis. In 2012, the selective JAK3 inhibitor Tofacitinib passed clinical trials and was approved for the treatment of rheumatoid arthritis.

Tofacitinib (CP690550) is a pyrrolopyrimidine selective JAK3 kinase inhibitor developed by Pfizer, and has inhibitory activity (IC) on JAK3₅₀1nmol/L) is JAK2 (IC)₅₀20 times of 20nmol/L) and JAK1 (IC)₅₀112nmol/L) of the total. The study on the stereochemistry of Tofacitinib shows that the chiral structure of Tofacitinib determines that Tofacitinib can be specifically bound to JAK3 molecules, so that JAK3 phosphorylation is inhibited, STAT phosphorylation is further inhibited, and downstream inflammatory cytokine synthesis is inhibited. Tofacitinib shows good clinical curative effect in clinical research, in the clinical trial research of rheumatoid arthritis, a 5mg or 10mg Tofacitinib measuring group and an equivalent placebo group show obvious statistical difference in comparison, but Tofacitinib is found to be related to high risk of serious infection in the clinical trial research, and the long-term safety of Tofacitinib is further researched.

The JAK-STAT signal pathway plays an important role in the cell differentiation and proliferation processes, and the change of the JAK activity also causes the change of the signal transmission of the pathway, thereby influencing the cell function. Based on the key role of JAK kinase in JAK-STAT signal transmission and the specific histiocyte distribution of JAK3 kinase, JAK3 becomes a good therapeutic target for diseases such as rheumatoid arthritis.

At present, JAK3 inhibitors are mainly used for treating patients with moderate and severe rheumatoid arthritis, and the drugs show good treatment effect and good safety in treatment, but the long-term safety of the drugs still needs to be further improved. During the clinical research process of Tofacitinib, certain adverse reactions including infection, tuberculosis, tumor and liver injury and the like can be caused after the medicament is used, so that the key problems to be solved urgently in the research field are to improve the drug effect of the JAK3 inhibitor and reduce toxic and side effects.

The JAK kinase has high ATP binding site homology of several subtypes and small structural difference, which is an important reason for low selectivity of JAK inhibitors. There is still room for improvement in therapeutic effect, selectivity and safety of a series of JAK kinase inhibitors disclosed so far, and there is still a need to develop JAK inhibitors having better drug efficacy and better safety. Although a high-selectivity JAK inhibitor is the focus of research in the field at present, in view of the fact that each member of a JAK kinase family is closely related to JAK-STAT signal transmission, the pan-JAK inhibitor can remarkably improve the drug effect and greatly reduce the drug dosage, thereby achieving the purpose of controlling toxic and side effects. In addition, the significant improvement of the drug efficacy will contribute to the development of anti-inflammatory drugs for transdermal administration. The research and development of the medicines provide a new way for treating autoimmune diseases such as psoriasis, leucoderma, dermatitis, alopecia areata, rheumatoid arthritis, colitis, multiple sclerosis, systemic lupus erythematosus, Crohn's disease and the like, and cancers such as leukemia, lymphoma, multiple myeloma and the like. The compounds of the present invention exhibit excellent biological activity as JAK kinase inhibitors.

Disclosure of Invention

In view of the above discussion, the present invention aims to provide an alkenyl-containing pyrimidinecarboxamide compound, its isomer, hydrate, solvate, pharmaceutically acceptable salt or prodrug, and its pharmaceutical composition, and relates to their use in the preparation of medicaments for treating autoimmune diseases and cancers associated with tyrosine kinases JAK1, JAK2, JAK3 and TYK 2.

One aspect of the present invention provides an alkenyl-containing pyrimidinecarboxamide compound having the structural formula (I):

wherein X is N;

R¹is composed of

n₁Is an integer of 0 to 8, n₂Is an integer of 0 to 1, n₃Is an integer of 0 to 8, and n₁、n₂、n₃The sum is 10 or less;

L₁is composed of

R⁴、R⁵Each independently is H, C₁-C₃Alkyl radical, R⁴、R⁵The same or different, or a combination thereof,

R³is unsubstituted or C₁-C₃Alkyl substituted C₂-C₈Alkenyl or unsubstituted or C₁-C₃Alkyl substituted C₄-C₈Cycloalkenyl group of (a);

R²is composed of

n₄Is an integer of 0 to 8, n₅Is an integer of 0 to 1, n₆Is an integer of 0 to 8, and n₄、n₅、n₆The sum is 10 or less;

L₂is composed of

R⁷、R⁸Each independently is H, C₁-C₃Alkyl radical, R⁷、R⁸The same or different, or a combination thereof,

R⁶is-H, hydroxyalkyl, C₁-C₅Alkyl radical, C₃-C₈Cycloalkyl radical, C₁-C₅Alkoxyalkyl group, C₁-C₅Alkylthioalkyl, 5-6 membered heterocyclyl, or-NR 'R ", wherein R', R" are each independently H, C₁-C₃Alkyl group of (1).

Preferably, in the compound of formula (I), R³Is composed of

R⁹、R¹⁰、R¹¹、R¹²、R¹³Each independently is H, C₁-C₃An alkyl group, m is an integer of 0 to 2,

m₁is an integer of 0 to 5, m₂Is an integer of 0 to 5, and m₁,m₂The sum is less than or equal to 5.

Preferably, in the compound of formula (I), n₁Is an integer of 0 to 2, n₂Is an integer of 0 to 1, n₃Is an integer of 0 to 3, R⁴、 R⁵Each independently is H, methyl, R⁴、R⁵The same or different;

R⁹、R¹⁰、R¹¹、R¹²、R¹³each independently is H, methyl or ethyl, and m is 0 or 1. More preferably, n₁、 n₂、n₃The sum of the amounts is 5 or less.

More preferably, in the compound of formula (I), said R³Is composed of

R⁹、R¹¹、R¹²、R¹³Each independently is H, methyl or ethyl.

Preferably, R³Is composed of

R⁹、R¹⁰Each independently is H, C₁-C₃Alkyl radical, R¹¹、R¹²、R¹³Any one of them is C₄-C₆Alkyl, the remainder are each independently H, C₁-C₃Alkyl, m is an integer of 0 to 2.

Preferably, in the compound of formula (I), n₄Is an integer of 0 to 3, n₅Is an integer of 0 to 1, n₆Is an integer of 0 to 5, R⁷、 R⁸Each independently is H, methyl, R⁷、R⁸The same or different;

R⁶is-H, hydroxyethyl, hydroxypropyl, C₁-C₅Alkyl radical, C₃-C₈Cycloalkyl radical, C₁-C₅Alkoxyethyl radical, C₁-C₅Alkoxypropyl group, C₁-C₅An alkylthioethyl group, a 5-6 membered heterocyclyl group, or-NR 'R ", wherein R', R" are each independently H, C₁-C₃Alkyl group of (1). More preferably, n₄、n₅、n₆The sum of the amounts is 5 or less.

More preferably, in the compound of formula (I), R⁶Is H, hydroxyethyl, hydroxypropyl, methyl, ethyl, propyl, isopropyl, t-butyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, methoxyethyl, ethoxyethyl, propoxyethyl, isopropoxyethyl, methoxypropyl, ethoxypropyl, propoxypropyl, isopropoxypropyl, methylthioethyl, ethylthioethyl, propylthioethyl, isopropylthioethyl, a 5-6 membered heterocyclic group, or-NR 'R ", wherein R', R" are each independently H, methyl, ethyl;

the 5-6 membered heterocyclic group is unsubstituted or substituted by 1-2 groups selected from hydroxy, C₁-C₃Alkyl radical, C₁-C₃Acyl substituents substituted heterocyclyl containing 1-2 heteroatoms selected from N, O, S.

More specifically, the heterocyclic group containing 1 to 2 heteroatoms selected from N, O, S is any one selected from the group consisting of:

wherein R is¹⁴H, methyl, ethyl, propyl and isopropyl.

According to some embodiments of the present application, the pharmaceutically acceptable salt of the alkenyl-containing pyrimidinecarboxamide compound is selected from the group consisting of hydrochloride, hydrobromide, hydroiodide, perchlorate, sulfate, nitrate, phosphate, formate, acetate, propionate, glycolate, lactate, succinate, maleate, tartrate, malate, citrate, fumarate, gluconate, benzoate, mandelate of said compound, one or more of mesylate, isethionate, benzenesulfonate, oxalate, palmitate, 2-naphthalenesulfonate, p-toluenesulfonate, cyclamate, salicylate, gluconate, trifluoroacetate, aluminum salt, calcium salt, chloroprocaine salt, choline salt, diethanolamine salt, ethylenediamine salt, lithium salt, magnesium salt, potassium salt, sodium salt, and zinc salt.

In another aspect, the invention relates to the use of the alkenyl-containing pyrimidine carboxamide compounds, isomers, hydrates, solvates, pharmaceutically acceptable salts or prodrugs thereof in the preparation of medicaments for treating autoimmune diseases and cancers associated with tyrosine kinases JAK1, JAK2, JAK3 and TYK2, wherein the autoimmune diseases and cancers associated with tyrosine kinases JAK1, JAK2, JAK3 and TYK2 include fundus diseases, dry eye, psoriasis, vitiligo, dermatitis, alopecia areata, rheumatoid arthritis, colitis, multiple sclerosis, systemic lupus erythematosus, crohn's disease, atheroma, pulmonary fibrosis, hepatic fibrosis, myelofibrosis, non-small cell lung cancer, breast cancer, pancreatic cancer, glioma, glioblastoma, ovarian cancer, cervical cancer, colorectal cancer, melanoma, endometrial cancer, Prostate cancer, bladder cancer, leukemia, gastric cancer, liver cancer, gastrointestinal stromal tumors, thyroid cancer, chronic myelogenous leukemia, acute myelogenous leukemia, non-hodgkin's lymphoma, nasopharyngeal cancer, esophageal cancer, brain tumors, B-cell and T-cell lymphomas, lymphoma, multiple myeloma, biliary sarcoma, cholangiocarcinoma.

In yet another aspect, the present invention provides a pharmaceutical composition comprising an alkenyl-containing pyrimidinecarboxamide compound of the present application, an isomer, hydrate, solvate, pharmaceutically acceptable salt or prodrug thereof, and one or more pharmaceutically acceptable carriers or excipients.

According to some embodiments of the present application, the pharmaceutical composition may further comprise one or more additional therapeutic agents.

Advantageous effects

The alkenyl-containing pyrimidine carboxamide compound shows strong inhibitory activity of tyrosine kinases JAK1, JAK2, JAK3 and TYK2, and the alkenyl-containing pyrimidine carboxamide compound, isomers, hydrates, solvates, pharmaceutically acceptable salts or prodrugs thereof and pharmaceutical compositions thereof are expected to be used for preparing medicines for treating autoimmune diseases and cancers related to tyrosine kinases JAK1, JAK2, JAK3 and TYK2, and are particularly suitable for treating autoimmune diseases (such as psoriasis) and the like through transdermal administration.

Detailed Description

Unless otherwise indicated, the following terms used in the present application (including the specification and claims) have the definitions given below. In this application, the use of "or" and "means" and/or "unless stated otherwise. Furthermore, the use of the terms "including" and other forms, such as "including", "comprising", and "having", are not limiting. The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

"alkyl" refers to aliphatic hydrocarbon groups. The alkyl group is saturated or unsaturated. The alkyl moiety, whether saturated or unsaturated, may be branched or straight chain. The "alkyl group" may have 1 to 5 carbon atoms, preferably 1 to 3 carbon atoms. In one aspect, the alkyl group is selected from the group consisting of methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and tert-butyl. Typical alkyl groups include, but are in no way limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, neopentyl, hexyl, allyl, vinyl, acetylene, but-2-enyl, but-3-enyl and the like.

The term "cycloalkyl" refers to a monocyclic or polycyclic aliphatic nonaromatic group in which each of the atoms making up the ring isA child (i.e., a framework atom) is a carbon atom. Cycloalkyl groups may be saturated or partially unsaturated. The cycloalkyl group may be fused to the aromatic ring and the point of attachment is on a carbon other than a carbon atom of the aromatic ring. Cycloalkyl groups include groups having 3 to 10 ring atoms. In some embodiments, the cycloalkyl group is selected from cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, and cyclooctyl. Cycloalkyl groups may be substituted or unsubstituted. Depending on the structure, cycloalkyl groups can be monovalent or divalent (i.e., cycloalkylene groups such as, but not limited to, cyclopropyl-1, 1-diyl, cyclobutyl-1, 1-diyl, cyclopentyl-1, 1-diyl, cyclohexyl-1, 4-diyl, cycloheptyl-1, 1-diyl, and the like). In one aspect, cycloalkyl is C₃-C₆A cycloalkyl group.

"alkenyl" or "cycloalkenyl" refers to a straight or cyclic hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, which contains at least one double bond.

"alkoxyalkyl" refers to a (alkyl) O (alkyl) -group, and "alkylthio" refers to a (alkyl) S (alkyl) -group, wherein alkyl is as defined herein. Preferably, the alkoxyalkyl group is C₁-C₅Alkoxyalkyl group, more preferably C₁-C₅Alkoxy radical C₁-C₃Alkyl, more preferably C₁-C₅Alkoxyethyl or C₁-C₅An alkoxypropyl group. Preferably, said alkylthioalkyl is C₁-C₅Alkylthioalkyl, more preferably C₁-C₅Alkylthio group C₁-C₃Alkyl, more preferably C₁-C₅An alkylthio ethyl group.

The term "heterocyclyl" in the term "5-6 membered heterocyclyl" refers to both aromatic heterocyclic rings (also known as heteroaryl) and heterocycloalkyl rings (also known as heteroalicyclic groups) containing one or more heteroatoms in the ring, wherein each heteroatom in the ring is selected from the group consisting of O, S and N, and wherein each heterocyclic group contains from 5 to 6 atoms in its ring system. And the 5-6 membered heterocyclic group may be unsubstituted or substituted with 1-2 substituents selected from hydroxy, C₁-C₃Alkyl radical, C₁-C₃Acyl substituents substituted with 1-2 hetero atoms selected from N, O, SA heterocyclic group of a child.

More specifically, the heterocyclic group containing 1 to 2 heteroatoms selected from N, O, S is any one selected from the group consisting of:

wherein R is¹⁴H, methyl, ethyl, propyl and isopropyl.

The term "isomer" in this application is a different compound having the same molecular formula and may include various isomeric forms such as stereoisomers, tautomers and the like. "stereoisomers" are isomers that differ only in the arrangement of the atoms in space. Certain compounds described herein contain one or more asymmetric centers and can therefore give rise to enantiomers, diastereomers, and other stereoisomeric forms which can be defined as (R) -or (S) -in terms of absolute stereochemistry. The chemical entities, pharmaceutical compositions and methods of the present invention are intended to include all such possible isomers, including racemic mixtures, optically pure forms and intermediate mixtures. Optically active (R) -and (S) -isomers can be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques. The optical activity of the compounds can be analyzed by any suitable method, including but not limited to chiral chromatography and polarimetry, and the degree of predominance of one stereoisomer over the other can be determined.

Specifically, for example, for compounds of formula (I) herein, when n is₂Or n₅Is not 0, and R⁴、R⁵Is different from R⁷、R⁸At different times, L₁

Contains a chiral carbon atom in the structure, in which case L₁

The structure of (A) is meant to include all possible isomers, including racemic mixtures, optically active (R) -and (S) -isomers in optically pure form, and the like, i.e., R⁴、R⁵At different times, L₁Is composed of

Comprises that

And racemates; r⁷、R⁸At different times, L₂Is composed of

Comprises that

And racemates.

When a compound described herein contains an olefinic double bond, it is meant that the compound includes various cis-trans isomers, unless otherwise specified.

"tautomers" are structurally different isomers that can be interconverted by tautomerization. "tautomerization" is a form of isomerization and includes proton shift or proton shift tautomerization, which can be considered a subset of acid-base chemistry. "proton shift tautomerization" or "proton shift tautomerization" involves the migration of protons with a shift in the bond order, often an interchange of a single bond with an adjacent double bond. When tautomerization is possible (e.g., in solution), chemical equilibrium of the tautomers can be reached. One example of tautomerization is keto-enol tautomerization.

In the present application, the compounds of structural formula (I), isomers, crystals or prodrugs and pharmaceutically acceptable salts thereof may exist in solvated as well as unsolvated forms. For example, the solvated form may be water soluble. The present invention includes all such solvated and unsolvated forms.

It is a further object of the present invention to provide a method for treating autoimmune diseases and cancer comprising administering to a subject a therapeutically effective amount of a composition comprising a compound of the present invention. Autoimmune diseases, cancers that can be so treated are noted elsewhere herein, including autoimmune diseases, cancers, etc. that are resistant to treatment with Tofacitinib, Pefitinib, Roxolitinib, Decernotinib, or other kinase inhibitors.

One or more other therapies may also be used in combination in the treatment of cancer, including surgery, radiation therapy (e.g., gamma-rays, neutron beam radiation therapy, electron beam radiation therapy, proton therapy, brachytherapy, and systemic radioisotopes, etc.), endocrine therapy, biological response modifiers (e.g., interferons, interleukins, and Tumor Necrosis Factor (TNF)), hyperthermia, cryotherapy, attenuating any adverse effects (e.g., antiemetics), and other therapeutic agents.

The invention also includes the use of a compound of the invention, or a pharmaceutically acceptable derivative thereof, for the manufacture of a medicament for the treatment of diseases such as ocular fundus disease, dry eye, psoriasis, rheumatoid arthritis, skin rash, eczema, alopecia areata, atheroma, pulmonary fibrosis, liver fibrosis, myelofibrosis, inflammatory bowel disease and autoimmune diseases, as well as tumors, including those diseases which develop resistance to one or more other therapeutic agents, as noted elsewhere herein. The compounds of the invention are also useful in medicine for alleviating or preventing disease by inhibiting one or more kinases (e.g., JAK1, JAK2, JAK3, TYK 2).

The invention also provides a method for preparing the corresponding compound, which can be prepared by the following method. The synthetic route for the series (I) of compounds is shown below:

synthetic route to the compounds of formula (I)

Wherein R is¹And R²See the preceding text for a detailed description of (A). The specific reaction conditions of steps 1 and 2 are not particularly limited as long as the reaction can be carried out. One exemplary reaction condition is as follows: 1) DIEA, THF,25 ℃, 2-16 h；2) TFA,s-BuOH,60～100℃,2～16h.

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

For the synthesis of some of the intermediates used in this application, see chinese patent application No. 2017108562180.

Example 14 preparation of- (butyl-3-en-1-ylamino) -2- ((1- (tert-butyl) -1H-pyrazol-4-yl) amino ] pyrimidine-5-carboxamide:

step 1): preparation of 2-chloro-4- (butyl-3-en-1-ylamino) pyrimidine-5-carboxamide:

2, 4-dichloropyrimidine-5-carboxamide (400mg, 2.08mmol) and triethylamine (633mg, 6.25mmol) were dissolved in tetrahydrofuran (10mL), and butyl-3-en-1-amine hydrochloride (225mg, 2.1mmol) was added and reacted at 25 ℃ for 3 hours. Adding saturated saline solution (200mL), stirring for 15 min, filtering, washing the filter cake with petroleum ether to obtain 380mg of white solid, MS:227[ M + H ]]⁺。

Step 2): preparation of 4- (butyl-3-en-1-ylamino) -2- ((1- (tert-butyl) -1H-pyrazol-4-yl) amino ] pyrimidine-5-carboxamide:

2-chloro-4- (butyl-3-en-1-ylamino) pyrimidine-5-carboxamide (70mg, 0.29mmol) was dissolvedTo sec-butanol (3mL) were added 1-tert-butyl-1H-pyrazol-4-amine (49mg, 0.35mmol) and trifluoroacetic acid (0.1mL), and the mixture was subjected to tube-closing reaction at 100 ℃ for 2 hours. The reaction solution was concentrated, filtered, and the solid was washed with acetonitrile to obtain 50mg of a white solid.¹H NMR(400MHz,DMSO-d₆) Delta 10.52(s,1H),10.04(s, 1H),8.53(s,1H), 8.30-8.10 (m,1H),8.02(s,1H), 7.71-7.52 (m,2H), 5.92-5.72 (m,1H), 5.20-4.97 (m,2H), 3.68-3.53 (m,2H), 2.43-2.28 (m,2H),1.52(s,9H)₁₆H₂₃N₇O，MS: 330(M+H)⁺.

Example 22 preparation of- ((1-tert-butyl-1H-pyrazol-4-yl) amino) -4- ((2-methylallyl) amino) pyrimidine-5-carboxamide:

the procedure is as in example 1, replacing butyl-3-en-1-amine hydrochloride in step 1) with 2-methylpropyl-2-en-1-amine to give a white solid,¹H NMR(400MHz,DMSO-d₆) δ 10.36(s,1H),10.12(s,1H),8.55(s,1H), 8.29-8.09 (m,1H), 7.98(s,1H), 7.70-7.49 (m,2H), 4.90-4.80 (m,2H),4.10(d, J ═ 5.7Hz,2H),1.76(s,3H),1.51(s,9H). formula C₁₆H₂₃N₇O，MS: 330(M+H)⁺.

Example preparation of 32- ((1- (tert-butyl) -1H-pyrazol-4-yl) amino) -4- ((3-methylbutyl-2-en-1-yl) amino) pyrimidine-5-carboxamide:

the procedure is as in example 1, 3-methylbutyl-2-en-1-amine is used instead of butyl-3-en-1-amine hydrochloride in step 1) to give a white solid,¹H NMR(400MHz,DMSO-d₆) Delta 10.48 (s,1H),9.94(s,1H),8.51(s,1H), 8.36-8.10 (m,1H),8.06(s,1H), 7.81-7.40 (m,2H), 5.41-5.22 (m,1H), 4.24-3.92 (m,2H),1.71(s, 3H),1.67(s,3H),1.50(s,9H). chemical formula C₁₇H₂₅N₇O，MS:344(M+H)⁺.

Example 42 preparation of- ((1- (tert-butyl) -1H-pyrazol-4-yl) amino) -4- (pentyl-4-en-1-ylamino) pyrimidine-5-carboxamide:

the procedure is as in example 1, pentyl-4-en-1-amine is used instead of butyl-3-en-1-amine hydrochloride in step 1) to give a white solid,¹H NMR(400MHz,DMSO-d₆) Δ 10.39(s,1H), 10.00(s,1H),8.49(s,1H),8.18(s,1H),8.02(s,1H), 7.71-7.50 (m,2H), 5.91-5.73 (m,1H), 5.10-4.91 (m,2H), 3.60-3.45 (m,2H), 2.14-2.02 (m,2H), 1.78-1.63 (m,2H),1.52(s,9H)₁₇H₂₅N₇O，MS:344(M+H)⁺.

Example preparation of- ((2-methylallyl) amino) -2- ((1- (tetrahydro-2H-pyran-4-yl) -1H-pyrazol-4-yl) amino) pyrimidine-5-carboxamide:

the procedure is as in example 1, replacing butyl-3-en-1-amine hydrochloride in step 1) with 2-methylpropyl-2-en-1-amine and 1- (tetrahydro-2H-pyran-4-yl) -1H-pyrazol-4-amine with 1-tert-butyl-1H-pyrazol-4-amine in step 2) to give a white solid,¹H NMR(400MHz,DMSO-d₆) δ 10.55(s,1H),10.16(s,1H), 8.59(s,1H), 8.37-8.16 (m,1H),7.95(s,1H), 7.72-7.53 (m,2H), 4.92-4.76 (m,2H), 4.45-4.31 (m,1H),4.09(d, J ═ 5.8 Hz,2H), 4.01-3.88 (m,2H), 3.48-3.41 (m,2H), 2.02-1.82 (m,4H),1.76(s,3H). formula C₁₇H₂₃N₇O₂，MS:358(M+H)⁺.

Example preparation of 64- ((3-methylbutyl-2-en-1-yl) amino) -2- ((1- (tetrahydro-2H-pyran-4-yl) -1H-pyrazol-4-yl) amino) pyrimidine-5-carboxamide:

the same procedure as in example 1 was followed, except that 3-methylbutyl-2-en-1-amine hydrochloride in step 1) was used in place of butyl-3-en-1-amine hydrochloride and 1- (tetrahydro-2H-pyran-4-yl) -1H-pyrazol-4-amine was used in place of 1-tert-butyl-1H-pyrazol-4-amine in step 2), to give a white solid,¹H NMR(400MHz,DMSO-d₆) Delta 10.47(s,1H),9.93(s,1H),8.50 (s,1H), 8.29-8.07 (m,1H),8.00(s,1H), 7.71-7.50 (m,2H), 5.36-5.24 (m,1H), 4.46-4.30 (m,1H), 4.20-4.06 (m,2H), 4.00-3.87 (m,2H), 3.54-3.38 (m,2H), 2.02-1.80 (m,4H), 1.71(s,3H),1.68(s,3H)₁₈H₂₅N₇O₂，MS:372 (M+H)⁺.

Example preparation of 74- (butyl-3-en-1-ylamino) -2- ((1- (tetrahydro-2H-pyran-4-yl) -1H-pyrazol-4-yl) amino) pyrimidine-5-carboxamide:

the procedure of example 1 was followed, replacing 1-tert-butyl-1H-pyrazol-4-amine in step 2) with 1- (tetrahydro-2H-pyran-4-yl) -1H-pyrazol-4-amine to give a white solid,¹H NMR (400MHz,DMSO-d₆) Delta 10.63(s,1H),10.06(s,1H),8.55(s, 1H), 8.38-8.12 (m,1H),7.99(s,1H), 7.76-7.45 (m,2H), 5.95-5.73 (m,1H), 5.21-5.02 (m,2H), 4.48-4.29 (m,1H), 4.04-3.87 (m,2H), 3.69-3.53 (m,2H), 3.52-3.34 (m,2H), 2.43-2.28 (m,2H), 2.01-1.81 (m,4H) of the formula C₁₇H₂₃N₇O₂，MS:358(M+H)⁺.

Example preparation of 82- ((1- (tetrahydro-2H-pyran-4-yl) -1H-pyrazol-4-yl) amino) -4- (pentyl-4-en-1-ylamino) pyrimidine-5-carboxamide:

the procedure is as in example 1, pentyl-4-en-1-amine is used instead of butyl-3-en-1-amine hydrochloride in step 1) and 1- (tetrahydro-2H-pyran-4-yl) -1H-pyrazol-4-amine is used instead of step 2)Neutralizing 1-tert-butyl-1H-pyrazole-4-amine to obtain white solid,¹H NMR (400MHz,DMSO-d₆) Delta 10.41(s,1H),10.00(s,1H),8.48(s, 1H), 8.28-8.07 (m,1H),7.97(s,1H), 7.70-7.52 (m,2H), 5.91-5.75 (m,1H), 5.10-4.92 (m,2H), 4.46-4.33 (m,1H), 4.01-3.91 (m,2H), 3.57-3.41 (m,4H), 2.15-2.05 (m,2H), 2.02-1.86 (m,4H), 1.77-1.64 (m,2H) formula C₁₈H₂₅N₇O₂，MS:372(M+H)⁺.

Example preparation of 92- ((1- (tert-butyl) -1H-pyrazol-4-yl) amino) -4- ((cyclopentyl-3-en-1-ylmethyl) amino) pyrimidine-5-carboxamide:

step 1): preparation of 2-chloro-4- ((cyclopentyl-3-en-1-ylmethyl) amino) pyrimidine-5-carboxamide:

2, 4-dichloropyrimidine-5-carboxamide (400mg, 2.08mmol) and triethylamine (633mg, 6.25mmol) were dissolved in tetrahydrofuran (10mL), and cyclopentyl-3-en-1-ylmethylamine hydrochloride (281mg, 2.1mmol) was added and reacted at 25 ℃ for 3 hours. Adding saturated saline solution (200mL), stirring for 15 min, filtering, washing the filter cake with petroleum ether to obtain 380mg of white solid, MS:253[ M + H ]]⁺。

Step 2): preparation of 2- ((1- (tert-butyl) -1H-pyrazol-4-yl) amino) -4- ((cyclopentyl-3-en-1-ylmethyl) amino) pyrimidine-5-carboxamide:

2-chloro-4- ((cyclopentyl-3-en-1-ylmethyl) amino) pyrimidine-5-carboxamide (127mg, 0.5mmol) was dissolved in sec-butanol (3mL), 1-tert-butyl-1H-pyrazol-4-amine (84mg, 0.6mmol) and trifluoroacetic acid (0.1mL) were added, and the reaction was blocked at 100 ℃ for 2 hours. The reaction solution was concentrated and purified by column chromatography to obtain 50mg of a white solid.¹H NMR(400MHz,DMSO-d₆)δ10.58(s,1H),10.16(s,1H),8.55(s,1H),8.37–8.14(m,1H),8.02(s,1H),7.71–7.52(m,2H), 5.75–5.63(m,2H),3.57–3.44(m,2H),2.72–2.56(m,1H),2.49–2.39(m,2H),2.15–2.01 (m,2H),1.52(s,9H).MS:356[M+H]⁺。

Example preparation of 104- ((cyclopentyl-3-en-1-ylmethyl) amino) -2- ((1- (tetrahydro-2H-pyran-4-yl) -1H-pyrazol-4-yl) amino) pyrimidine-5-carboxamide:

step 1: same as example 9) step 1;

step 2) preparation of 4- ((cyclopentyl-3-en-1-ylmethyl) amino) -2- ((1- (tetrahydro-2H-pyran-4-yl) -1H-pyrazol-4-yl) amino) pyrimidine-5-carboxamide:

the procedure is as in example 9, step 2) using 1- (tetrahydro-2H-pyran-4-yl) -1H-pyrazol-4-amine instead of 1-tert-butyl-1H-pyrazol-4-amine to give a white solid.¹H NMR(400MHz,DMSO-d₆)δ10.62(s,1H),10.16(s,1H),8.56(s,1H), 8.26(s,1H),7.98(s,1H),7.71–7.53(m,2H),5.75–5.65(m,2H),4.46–4.31(m,1H),4.01– 3.90(m,2H),3.57–3.39(m,4H),2.70–2.55(m,1H),2.48–2.36(m,2H),2.17–2.04(m,2H), 2.03–1.82(m,4H).MS:384[M+H]⁺。

Example 114 preparation of (allylamino) -2- ((1- (tert-butyl) -1H-pyrazol-4-yl) amino) pyrimidine-5-carboxamide:

the procedure is as in example 9, substituting allylamine hydrochloride for cyclopentyl-3-en-1-ylmethylamine hydrochloride to give an off-white solid.¹H NMR(400MHz,DMSO-d₆)δ10.71(s,1H),10.16(s,1H),8.62(s,1H),8.31(s,1H),8.00(s, 1H),7.77–7.54(m,2H),6.07–5.89(m,1H),5.27–5.11(m,2H),4.25–4.12(m,2H),1.51(s, 9H).MS:316[M+H]⁺。

EXAMPLE 12 preparation of (Z) -2- ((1-tert-butyl-1H-pyrazol-4-yl) amino) -4- ((pentan-2-en-1-yl) amino) pyrimidine-5-carboxamide

The procedure is as in example 9, substituting (Z) -pentan-2-en-1-amine for cyclopentyl-3-en-1-ylmethylamine hydrochloride to give an off-white solid.¹H NMR(400MHz,DMSO-d₆)δ9.44(s,1H),9.18(d,J＝28.1Hz,1H),8.46(s,1H),8.02(d,J ＝9.3Hz,1H),7.83–7.56(m,1H),7.48(s,1H),7.23–6.97(m,1H),5.72–5.52(m,2H),4.14 –4.03(m,2H),2.14–1.98(m,2H),1.49(s,9H),0.99–0.91(m,3H).MS:344[M+H]⁺。

Example 13 preparation of (E) -2- ((1-tert-butyl-1H-pyrazol-4-yl) amino) -4- ((4-methylpentan-2-en-1-yl) amino) pyrimidine-5-carboxamide

The procedure is as in example 9, substituting (E) -4-methylpentane-2-en-1-amine for cyclopentyl-3-en-1-ylmethylamine hydrochloride to give an off-white solid.¹H NMR(400MHz,DMSO-d₆)δ9.45(s,1H),9.24(s,1H),8.46(s,1H),8.01(s,1H), 7.87–7.51(m,1H),7.48(s,1H),7.39–6.79(m,1H),5.75–5.39(m,2H),4.25–3.86(m,2H), 2.40–2.10(m,1H),1.49(s,9H),0.94(d,J＝6.7Hz,6H).MS:358[M+H]⁺。

Example 14 preparation of (E) -2- ((1-tert-butyl-1H-pyrazol-4-yl) amino) -4- ((pentan-3-en-1-yl) amino) pyrimidine-5-carboxamide

The procedure is as in example 9, substituting (E) -pentan-3-en-1-amine for cyclopentyl-3-en-1-ylmethylamine hydrochloride to give an off-white solid.¹H NMR(400MHz,DMSO-d₆)δ9.41(s,1H),9.19(s,1H),8.44(s,1H),8.00(s,1H),7.67– 7.53(m,1H),7.50(s,1H),7.21–6.93(m,1H),5.58–5.36(m,2H),3.52–3.47(m,2H),2.37– 2.20(m,2H),1.63(d,J＝5.9Hz,3H),1.49(s,9H).MS:344[M+H]⁺。

Example preparation of 152- ((1-tert-butyl-1H-pyrazol-4-yl) amino) -4- ((3-methylbutyl-3-en-1-yl) amino) pyrimidine-5-carboxamide

The procedure is as in example 9, 3-methylbutyl-3-en-1-ylamine is used instead of cyclopentyl-3-en-1-ylmethylamine hydrochloride to give an off-white solid.¹H NMR(400MHz,DMSO-d₆)δ9.40(s,1H),9.17(s,1H),8.44(s,1H),7.97(s,1H), 7.82–7.59(m,1H),7.52(s,1H),7.18–6.87(m,1H),4.84–4.77(m,1H),4.77–4.72(m,1H), 3.67–3.50(m,2H),2.39–2.22(m,2H),1.73(s,3H),1.49(s,9H).MS:344[M+H]⁺。

Example 162 preparation of- ((1-tert-butyl-1H-pyrazol-4-yl) amino) -4- ((4-methylpentan-3-en-1-yl) amino) pyrimidine-5-carboxamide

The procedure is as in example 9, 4-methylpentane-3-en-1-amine is used instead of cyclopentyl-3-en-1-ylmethylamine hydrochloride to give an off-white solid.¹H NMR(400MHz,DMSO-d₆)δ9.41(s,1H),9.16(s,1H),8.45(s,1H),8.01(s,1H), 7.82–7.55(m,1H),7.50(s,1H),7.24–6.87(m,1H),5.21–5.11(m,1H),3.54–3.43(m,2H), 2.36–2.22(m,2H),1.67(s,3H),1.57(s,3H),1.49(s,9H).MS:358[M+H]⁺。

Example preparation of 172- ((1-tert-butyl-1H-pyrazol-4-yl) amino) -4- ((4-methylpentan-4-en-1-yl) amino) pyrimidine-5-carboxamide

The procedure is as in example 9, 4-methylpentane-4-en-1-amine is used instead of cyclopentyl-3-en-1-ylmethylamine hydrochloride to give an off-white solid.¹H NMR(400MHz,DMSO-d₆)δ9.46(s,1H),9.27(s,1H),8.45(s,1H),8.00(s,1H), 7.86–7.59(m,1H),7.51(s,1H),7.31–6.76(m,1H),4.78–4.63(m,2H),3.52–3.44(m,2H), 2.06(t,J＝7.7Hz,2H),1.80–1.70(m,2H),1.69(s,3H),1.50(s,9H).MS:358[M+H]⁺。

Example 182 preparation of- ((1-tert-butyl-1H-pyrazol-4-yl) amino) -4- ((5-methylhexan-4-en-1-yl) amino) pyrimidine-5-carboxamide

The procedure is as in example 9, 5-methylhexan-4-en-1-amine is used instead of cyclopentyl-3-en-1-ylmethylamine hydrochloride to give an off-white solid.¹H NMR(400MHz,DMSO-d₆)δ9.40(s,1H),9.21(s,1H),8.44(s,1H),7.98(s,1H), 7.82–7.54(m,1H),7.52(s,1H),7.26–6.84(m,1H),5.21–5.01(m,1H),2.07–1.99(m,2H), 1.64(s,6H),1.57–1.53(m,4H),1.49(s,9H).MS:372[M+H]⁺。

Example 19 preparation of (E) -2- ((1-tert-butyl-1H-pyrazol-4-yl) amino) -4- ((5-methylhexan-2-en-1-yl) amino) pyrimidine-5-carboxamide

The procedure is as in example 9, substituting (E) -5-methylhexan-2-en-1-amine for cyclopentyl-3-en-1-ylmethylamine hydrochloride to give an off-white solid.¹H NMR(400MHz,DMSO-d₆)δ9.40(s,1H),9.18(s,1H),8.44(s,1H),8.01(s,1H), 7.77–7.54(m,1H),7.50(s,1H),7.22–6.80(m,1H),5.56–5.44(m,1H),5.44–5.31(m,1H), 3.55–3.42(m,2H),2.35–2.14(m,3H),1.50(s,9H),0.93(d,J＝6.7Hz,6H).MS:372 [M+H]⁺。

Example 202 preparation of- ((1-tert-butyl-1H-pyrazol-4-yl) amino) -4- (((2E,4E) -hexane-2, 4-dien-1-yl) amino) pyrimidine-5-carboxamide

The procedure is as in example 9, replacing cyclopentyl-3-en-1-ylmethylamine hydrochloride with (2E,4E) -hexane-2, 4-dien-1-amine to give an off-white solid.¹H NMR(400MHz,DMSO-d₆)δ9.48(s,1H),9.29(s,1H),8.47(s,1H),7.98(s,1H), 7.86–7.58(m,1H),7.49(s,1H),7.34–6.92(m,1H),6.22–5.99(m,2H),5.82–5.70(m,1H), 5.70–5.55(m,1H),4.19–4.02(m,2H),1.69(d,J＝6.7Hz,3H),1.48(s,9H).MS:356 [M+H]⁺。

Example 212 preparation of- ((1-tert-butyl-1H-pyrazol-4-yl) amino) -4- (((2E,4E) -heptane-2, 4-dien-1-yl) amino) pyrimidine-5-carboxamide

The procedure is as in example 9, replacing cyclopentyl-3-en-1-ylmethylamine hydrochloride with (2E,4E) -heptane-2, 4-dien-1-amine to give an off-white solid.¹H NMR(400MHz,DMSO-d₆)δ9.44(s,1H),9.25(s,1H),8.47(s,1H),7.98(s,1H), 7.88–7.58(m,1H),7.48(s,1H),7.32–6.86(m,1H),6.16(dd,J＝15.1,10.4Hz,1H),6.04(dd, J＝15.1,10.5Hz,1H),5.86–5.60(m,2H),4.21–4.05(m,2H),2.12–1.96(m,2H),1.48(s, 9H),0.94(t,J＝7.5Hz,3H).MS:370[M+H]⁺。

Experimental example 1 test for inhibition of JAK1, JAK2, JAK3, TYK2 kinase Activity by Small molecule Compounds

In the in vitro assembled enzymatic reaction, compounds with different concentrations are added to detect the inhibition of the specific enzymatic reaction by the compounds, and the specific test method is as follows:

instruments, materials and reagents

Second, testing method

Specific experimental conditions for JAK1, JAK2, JAK3 and TYK2, exemplified by JAK3, are described below.

1. Preparing a reagent:

preparation of EDTA (0.5M pH8.0) solution by accurately weighing 14.612g of EDTA powder, adding ultrapure water, and adjusting the volume to 100mL (if insoluble, heating to 37 deg.C, adjusting pH to 8.0 with 1N NaOH solution)

1 × Kinase Assay Buffer: into a reagent bottle were added 25mL of HEPES solution (1M), 190.175mg of EGTA, and 5mL of MgCl₂The solution (1M), 1mL DTT, 50. mu.L Tween-20, and ultrapure water were added to make a volume of 500mL (pH adjusted to 7.5).

1 × Detection Buffer 1mL of 10 × Detection Buffer was added to 9mL of water and mixed.

4 × stop solution: 0.8mL of the above EDTA (0.5M, pH8.0) solution, 1mL of 10 XDetection Buffer, and 8.2mL of ultrapure water were mixed together.

4 XJAK 3 Kinase solution A Kinase stock solution was diluted with 1 XKinase Assay Buffer to a concentration of 0.36nM, mixed well and stored on ice.

4 × substrate solution: substrate ULight was diluted with 1 × Kinase Assay Buffer^TM-Labeled JAK-1(Tyr1023) Peptide stock solution to 200nM and mixed well.

4 × ATP solution: the ATP stock was diluted with 1 XKinase Assay Buffer to a concentration of 40. mu.M and mixed well.

4 × detection solution: the Detection antibody Europium-anti-phosphorus-tyrosine antibody (PT66) was diluted with 1 XDeprotection Buffer to a concentration of 8nM and mixed well.

2 × substrate/ATP mixture: the 4 Xsubstrate solution and 600. mu.l of 4 XATP solution were mixed in equal amounts (prepared before use).

2. Experimental procedure

1) The dilution of the compound is carried out,

in a 96-well plate, compounds were diluted with DMSO solutions at 3-fold ratios to form 11 gradients, and another pure DMSO solution served as a positive control; a new 96-well plate was prepared, and the solution was diluted 25-fold with ultrapure water (DMSO concentration: 4%)

2) Rotating Compounds to 384 well plates

The compound solution diluted with ultrapure water in the above 96-well plate was transferred to the corresponding well of a 384-well plate according to a standard 2-well carousel.

3) Add 4 × kinase solution: mu.l of the 4 Xkinase solution was added to the corresponding reaction well of the 384-well plate using a line gun, and pre-reacted at room temperature for 5 minutes.

4) Add 2 Xsubstrate/ATP mix 5. mu.l of the above 2 Xsubstrate/ATP mix to the corresponding reaction well of a 384 well plate using a discharge gun.

5) Negative control: a negative control well was set in a 384-well plate, and 2.5. mu.l/well of 4 Xsubstrate, 2.5. mu.l of 4 Xenzyme solution, 2.5. mu.l of 1 XKinase Assay Buffer, and 2.5. mu.l of ultrapure water containing 4% DMSO were added to the well.

6) And (4) centrifuging, mixing uniformly, and reacting for 60min at room temperature in the dark.

7) Termination of the enzymatic reaction:

5. mu.l of the 4 Xstop solution was pipetted into the corresponding well of 384-well plate, centrifuged and mixed, and reacted at room temperature for 5 minutes.

8) And (3) color development reaction:

5. mu.l of the 4 × detection solution was pipetted into the mesopores of 384-well plate, centrifuged and mixed, and reacted at room temperature for 60 min.

9) The 384 well plate is placed in a plate reader, and a corresponding program detection signal is called.

10) Inhibition rate and IC₅₀And (3) calculating:

pore reading 10000 × EU 665/EU 615 values

Inhibition rate [1- (experimental well reading-negative control well reading)/(positive control well reading-negative control well reading) ]. 100%

Inputting the drug concentration and the corresponding inhibition rate into GraphPad Prism5 for processing and calculating the corresponding IC₅₀The value is obtained.

Test conditions:

JAK1 kinase activity assay:

JAK1 (final concentration 10 nM); ATP (final concentration 10 μ M); ULight^TM-labeled JAK-1(Tyr1023) Peptide (final concentration 100 nM); the enzymatic reaction time was 2 hours. The maximum final concentration of the compound is 2.5 mu M, and after 3-fold gradient dilution, the total concentration is 11, and the minimum final concentration isThe concentration was 0.042 nM. The final concentration of DMSO is 1%.

JAK2 kinase activity assay:

JAK2 (final concentration 0.25 nM); ATP (final concentration 5 μ M); ULight^TM-labeled JAK-1(Tyr1023) Peptide (final concentration 50 nM); the enzymatic reaction time was 1 hour. The maximum final concentration of the compound was 2.5. mu.M, and after 3-fold gradient dilution, the minimum final concentration was 0.042 nM. The final concentration of DMSO is 1%.

JAK3 kinase activity assay:

JAK3 (final concentration 0.36 nM); ATP (final concentration 10 μ M); ULight^TM-labeled JAK-1(Tyr1023) Peptide (final concentration 50 nM); the enzymatic reaction time was 1 hour. The maximum final concentration of the compound was 2.5. mu.M, and after 3-fold gradient dilution, the minimum final concentration was 0.042 nM. The final concentration of DMSO is 1%.

TYK2 kinase activity assay:

TYK2 (final concentration 8 nM); ATP (final concentration 20 μ M); ULight^TM-labeled JAK-1(Tyr1023) Peptide (final concentration 100 nM); the enzymatic reaction time was 2 hours. The maximum final concentration of the compound was 2.5. mu.M, and after 3-fold gradient dilution, the minimum final concentration was 0.042 nM. The final concentration of DMSO is 1%.

Table 1 shows the results of measurement of inhibitory activity of some of the compounds of the present invention against tyrosine kinases JAK1, JAK2, JAK3, TYK 2. IC in the following Table₅₀The values represent the concentration of compound at which the enzyme inhibits maximally 50%, and NT means that the corresponding enzyme was not tested.

TABLE 1 results of measurement of inhibitory Activity of partial compounds of the present invention against JAK1, JAK2, JAK3 and TYK2 tyrosine kinases

Experimental example 2 test of inhibitory Activity of Compound of the present invention on mouse spleen cell proliferation

The specific experimental steps are as follows:

1) compound dilution: a total of 9 concentrations were obtained starting from the highest concentration of 5000nM after 3-fold gradient dilution (5000 nM for the maximum final concentration of drug and 0.76nM for the minimum final concentration in this experiment).

2) Taking a culture dish with the diameter of 6 cm, placing a cell filter sieve with the aperture of 70 mu m in the culture dish, and then adding 2mL of HBSS solution into the sieve to infiltrate the bottom of the culture dish;

3) euthanasia adult Balb/c mice with carbon dioxide, soaking in 75% alcohol for 1 minute, placing into a safety cabinet, cutting a small opening in the middle of the left ventral side of the mice, exposing the abdominal wall, and finding out the spleen;

4) taking spleen, removing surrounding adipose tissues, putting the spleen on a cell filtering sieve in a culture dish, and properly shearing;

5) gently grinding the spleen with the flat part at the top end of the syringe piston to obtain a cell suspension;

6) collecting cell suspension from a culture dish, and slowly adding the cell suspension into a 15ml centrifuge tube containing 5ml of Ficoll-Paque PLUS;

7) centrifugation was carried out at 400g for 30 minutes at room temperature;

8) after the centrifugation is finished, the upper layer is slowly removed by a suction pipe, and then the middle layer, namely spleen cells, is slowly sucked out;

9) placing the collected spleen cell suspension into another 15ml centrifuge tube, adding 10ml RPMI1640 complete culture medium, centrifuging at 300g and 4 ℃ for 4 minutes;

10) the supernatant was discarded, and the cells were resuspended by adding complete medium, and then subjected to cell counting. The cell suspension is washed repeatedly according to the ninth step;

11) transferring the cells to a culture dish (containing 2.5 mu g/mL of concanavalin A) at a cell density of 200-500 ten thousand/mL for overnight culture;

12) the next day, the cells were transferred to a 15mL centrifuge tube and centrifuged at 300g for 5 min;

13) discarding the supernatant, adding 5mL of RPMI1640 complete culture solution, blowing and beating uniformly, mixing 10 μ L of cell suspension and 10 μ L of trypan blue uniformly, counting by using a cell counter, and recording the number of cells and the survival rate.

14) Inoculating the cell suspension into a 96-well plate, and inoculating 80 mu l of cell suspension into each well, wherein the density of the cell suspension is 100000 cells/well;

15) adding 20 μ L of the 5 × compound solution diluted with the culture solution into each well, and mixing well;

16) after 72 hours of culture, 10. mu.L of CCK-8 reagent is added into each well, and the mixture is incubated for 2 hours (the reaction time can be adjusted according to the color depth);

17) the OD value was read at 450nm on a multifunction plate reader.

18) Data processing:

cell survival (%) - (As-Ab)/(Ac-Ab) ]. 100%

As: OD value of the experimental well (cell-containing medium, CCK-8, compound),

ac: OD value of control well (cell-containing medium, CCK-8),

ab: OD of blank wells (medium without cells and compounds, CCK-8),

the values were then introduced into Graphpad Prism5 software for curve fitting and IC50 was calculated.

Table 2 shows the results of the test of the inhibitory activity of some compounds of the present invention on mouse splenocyte proliferation.

TABLE 2 test results of the inhibitory Activity of some of the Compounds of the invention on mouse splenocyte proliferation

Use, formulation, administration

Medical use and indications

The biological data provided by the invention indicate that the compound of the invention is beneficial for treating or preventing diseases caused by tyrosine kinase (JAK1, JAK2, JAK3 and TYK2) abnormality. The compounds of the present invention have been shown to strongly inhibit JAK tyrosine kinase activity, whereas the JAK kinase family is closely related to the development and metastasis of autoimmune diseases and cancer. Thus, the compounds of the present invention are useful in the treatment of autoimmune diseases, including but not limited to: psoriasis, vitiligo, dermatitis, alopecia areata, rheumatoid arthritis, colitis, multiple sclerosis, systemic lupus erythematosus, and Crohn's disease. The compounds of the invention are also useful in the treatment of cancer, including primary and metastatic cancers, including solid tumors. Such cancers include, but are not limited to, non-small cell lung cancer, breast cancer, pancreatic cancer, glioma, glioblastoma, ovarian cancer, cervical cancer, colorectal cancer, melanoma, endometrial cancer, prostate cancer, bladder cancer, leukemia, gastric cancer, liver cancer, gastrointestinal stromal tumor, thyroid cancer, chronic myelogenous leukemia, acute myelogenous leukemia, non-Hodgkin's lymphoma, nasopharyngeal cancer, esophageal cancer, brain tumors, B-cell and T-cell lymphomas, lymphoma, multiple myeloma, biliary sarcoma, biliary tract cancer. The compounds of the invention also include the treatment of cancer resistant to one or more other therapeutic methods. The compounds of the present invention may also be used in diseases other than autoimmune diseases and cancer, including but not limited to ocular fundus disease, pulmonary fibrosis, liver fibrosis, etc., associated with JAK1 kinase and/or JAK2 and/or JAK3 kinase. The compounds of the present invention may be administered as monotherapy or in combination therapy, in combination with a plurality of the compounds of the present invention or in combination with other drugs other than those of the present invention.

Pharmaceutical process

The pharmaceutical methods of the invention comprise determining a therapeutically effective amount of a compound of the invention to a subject in need thereof. The "therapeutically effective dose" will vary depending on the stage, progression or severity of the disease. The daily dosage of the compounds and compositions of the present invention will depend upon a variety of factors including the condition being treated, the severity of the condition, the pharmaceutical efficacy of the particular compound employed, the particular composition, the age, body weight, general health, sex and diet, the route and schedule of administration, the rate of metabolism and/or excretion of the compound, the duration of treatment, and the like. In addition, the compounds of the invention can be administered to humans and other animals in dosages and in dosages with a pharmaceutically acceptable carrier. Modes of administration include oral, rectal, parenteral, intracisternal, intravaginal, intraperitoneal, topical (e.g., via transdermal patches, powders, ointments, or drops), sublingual, buccal, or nasal spray, and the like. The effective dose of the compounds of the present invention is generally measured in terms of the amount administered per kg of body weight of the patient, preferably 0.1 to 125 mg/kg of body weight, and generally 0.01 to 500 mg/kg of body weight. Administration may be one or more times, daily, weekly, every other day or every other day, or on an intermittent schedule. For example, the compound may be administered daily, weekly (e.g., monday), indefinitely or over a period of weeks (e.g., 4-10 weeks). The effective dosage of the compounds of the present invention will vary depending upon the compound employed, the mode of administration, the severity of the disease, the condition being treated and the various physical factors of the patient involved. In most cases, satisfactory therapeutic results are achieved when the preferred compounds of the invention are administered at a daily dosage of about 0.01 to about 500 mg/kg. The dosage is preferably 0.1-125 mg/kg, and the dosage is more preferably 1-25 mg/kg. Parenteral dosages are generally at an oral dosage level of about 10% to 20%. When the compounds of the present invention are used as part of a combination treatment regimen, the components of each composition will be administered during a desired treatment period. Whether comprising the two components as separate dosage units or as a single dosage form, the components of the composition may be administered simultaneously during the treatment period, may be administered at different times during the treatment period, or may be administered as a pre-treatment of the other.

As to compounds

The compounds of the invention may be used in therapy in free form or, where appropriate, in the form of pharmaceutically acceptable salts or other derivatives. As used herein, the term "pharmaceutically acceptable salts" refers to organic and inorganic salts of the compounds of the present invention which are suitable for use in humans and lower animals without undue toxicity, irritation, allergic response and the like, commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts of amines, carboxylic acids, phosphonates, and other types of compounds are well known in the art. The salts can be formed by reacting the isolated and purified compounds of the present invention with a suitable free base or acid.

Pharmaceutically non-toxic acid salts include, but are not limited to, amino salts with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, perchloric acid or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, malonic acid, or by using methods well known in the art, for example, ion exchange. Other pharmaceutically acceptable salts include adipates, alginates, ascorbates, aspartates, benzenesulfonates, benzoates, bisulfates, borates, butyrates, camphorates, camphorsulfonates, citrates, cyclopentanes, digluconates, dodecylsulfates, ethanesulfonates, formates, fumarates, glucoheptonates, glycerophosphates, gluconates, hemisulfates, heptanes, caproates, hydroiodides, 2-hydroxyethanesulfonates, lactobionates, lactates, laurates, lauryl sulfates, malates, maleates, malonates, methanesulfonates, 2-naphthalenesulfonates, nicotinates, nitrates, oleates, oxalates, palmitates, pamoates, pectinates, persulfates, per3-phenylpropionates, phosphates, picrates, salts of citric acid, salts of lactic acid, salts of 2-naphthalenesulfonates, nicotinates, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate and the like. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Other pharmaceutically acceptable salts include the appropriate non-toxic ammonium, quaternary ammonium, and amine-based cations formed using such salts as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, lower alkyl sulfonates, and aryl sulfonates.

In addition, the term "prodrug" as used herein means a compound which can be converted in vivo to a compound of the formula (I) of the present invention. This conversion is by hydrolysis of the prodrug in the blood or by enzymatic action in the blood or tissue to the parent compound.

Composition comprising a metal oxide and a metal oxide

The compositions described herein consist of any of the compounds (or prodrugs, or pharmaceutically acceptable salts, or other pharmaceutically acceptable derivatives thereof) described herein, and one or more pharmaceutically acceptable carriers or excipients. These compositions may optionally further comprise one or more additional therapeutic agents. The compounds of the invention may be co-administered to a patient in need thereof with one or more other treatment regimens (e.g., administration of Tofacitinib or other kinase inhibitors, interferons, bone marrow transplantation, farnesyl transferase inhibitors, bisphosphonates, thalidomide, cancer vaccines, hormonal therapy, antibodies, radiation, etc.). The pharmaceutical composition of the compound may be another anti-inflammatory or anti-cancer agent or agents.

As described herein, the compositions of the present invention comprise a compound of the present invention in combination with a pharmaceutically acceptable carrier and/or excipient, including any and all solvents, diluents or other carriers, dispersing or suspending aids, surfactants, isotonicity agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as appropriate for the particular dosage form desired. Some examples of pharmaceutically acceptable carrier materials include, but are not limited to, sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; tragacanth powder; malt; gelatin; talc powder; excipients, such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; ethylene glycols, such as propylene glycol; esters such as ethyl oleate and ethyl laurate, agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; ringer's solution; ethanol, and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants, may also be present in the composition.

Formulation of

The invention also encompasses a class of compositions (collectively referred to herein as "carrier" materials) in which the active compounds of the invention are used in combination with one or more pharmaceutically acceptable carriers and/or diluents and/or adjuvants, and also includes other active ingredients, if desired. The active compounds of the present invention may be administered by any suitable route, preferably in the form of pharmaceutical compositions adapted to such route of administration for the effective dosage required for the intended treatment. The compounds and compositions of the present invention may be administered orally, mucosally, topically, rectally, pulmonarily (e.g., by inhalation spray), or parenterally, including intravascular, intravenous, intraperitoneal, subcutaneous, intramuscular, intrasternal, and infusion techniques. The administration is in the form of a dosage unit-based formulation and contains pharmaceutically acceptable carriers, adjuvants, and excipients. For oral administration, the pharmaceutical composition may be in the form of, for example, a tablet, capsule, suspension or liquid. Examples of such dosage units are tablets or capsules. For example, they may contain the active ingredient in an amount of from 1 to 2000 mg, preferably from 1 to 500 mg, more usually from 5 to 200 mg. The appropriate daily dosage for a person or other mammal may vary depending on the patient and other factors, but may be determined again using conventional methods. As previously mentioned, the amount of compound in the administration and dosage regimen of the compounds and/or compositions contemplated by the present invention will depend upon a variety of factors including the age, weight, sex and medical condition of the subject, the type of disease, the severity of the disease, the route and frequency of administration, and the particular compound employed. Thus, dosage regimens can vary widely, but can be determined using standard methods. Typical daily dosage is 0.01-500 mg/kg body weight, preferably 0.1-125 mg/kg body weight, more preferably 1-25 mg/kg body weight.

The active compounds of the present invention are generally combined with one or more adjuvants, excipients or carriers to make up the route of administration. If administered orally, the compounds may be mixed with lactose, sucrose, starch powder, cellulose alkanoates, cellulose alkyl esters, talc, stearic acid, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphoric and sulfuric acids, gelatin, acacia, sodium alginate, polyvinylpyrrolidone and/or polyvinyl alcohol, and then tableted or encapsulated for convenient administration. Such capsules or tablets may contain a controlled release formulation provided by dispersing the active compound in hydroxypropylmethyl cellulose. Formulations suitable for topical administration include liquid or semi-liquid formulations suitable for penetration through the skin (e.g. liniments, lotions, ointments, creams or pastes) and drops suitable for administration to the eye, ear or nose. Suitable topical dosages of the compounds of the invention are 0.1-150 mg, one to four times daily, preferably 1 to 2 times daily. For topical administration, where an ointment is used, the active ingredient may be combined with any paraffin or water-miscible ointment as the base. Alternatively, the active ingredient may be formulated as a water-in-oil emulsion base cream. If desired, the aqueous phase of the cream base may include, for example, at least 30% by weight of a polyhydric alcohol such as propylene glycol, butane-1, 3-diol, mannitol, sorbitol, glycerin, polyethylene glycol, and mixtures thereof. Topical formulations may include compounds that enhance the absorption or penetration of the active ingredient through the skin or other affected areas. Examples of such dermal penetration enhancers include dimethyl sulfoxide and related analogs. The compounds may also be administered by transdermal means. Preferably transdermal administration will be accomplished using a patch containing a reservoir and a porous membrane or solid matrix. The oily phase of the emulsion of the invention may be constituted in a known manner by known ingredients, comprising a mixture of at least one emulsifier with a fat or an oil or with both a fat and an oil. Preferably, the hydrophilic emulsifier is used simultaneously with the lipophilic emulsifier as stabilizer, and it is also preferred that it is used in combination with oils and fats. Emulsifiers and emulsion stabilizers suitable for use in the formulations of the present invention include tween 60, span 80, cetearyl alcohol, myristyl alcohol, glyceryl monostearate, sodium lauryl sulfate, glyceryl distearate alone or with an emulsifying wax, or other materials well known in the art. The cream should preferably be a non-greasy, non-staining and washable product, and have a suitable consistency to avoid leakage from the tube or other container. Straight or branched chain, mono-or dibasic alkyl esters such as diisoadipate, isohexadecyl stearate, propylene glycol diester of coconut fatty acids, isopropyl myristate, decyl oleate, isopropyl palmitate, butyl stearate, 2-ethylhexyl palmitate or mixed branched chain esters may also be used. Alternatively, high melting point lipids such as white soft paraffin and/or liquid paraffin or other mineral oils may be used. Formulations suitable for topical administration to the eye also include eye drops wherein the active ingredient is dissolved or suspended in a suitable carrier, particularly an aqueous solvent for the active ingredient. The active ingredient is preferably present in these formulations in a concentration of 0.5% to 20% by weight, more advantageously 0.5 to 10% by weight, most preferably about 1.5% by weight. The formulations for parenteral administration may be in the form of aqueous or non-aqueous isotonic sterile injection solutions or suspensions. These solutions and suspensions may be prepared from one or more sterile powders or granules using the formulations for oral administration mentioned herein or carriers or diluents using other suitable dispersing or wetting agents and suspending agents. The compounds may be dissolved in water, polyethylene glycol, propylene glycol, ethanol, corn oil, cottonseed oil, peanut oil, sesame oil, benzyl alcohol, sodium chloride, tragacanth gum, and/or various buffers. Other adjuvants and modes of administration are well known in the pharmaceutical art.

The active ingredient may also be administered by injection, in combination with a suitable carrier including saline, dextrose or water, or with cyclodextrin (Captisol), co-solvent solubilization (i.e., propylene glycol), or micelle solubilization (i.e., tween 80). The formulations may also be presented as sterile injectable solutions or suspensions in a non-toxic parenterally-acceptable diluent or solvent, for example as 1, 3-butanediol. Solvents which may be used are water, ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. Any bland fixed oil may be employed for this purpose including synthetic mono-or diglycerides.

For pulmonary administration, the pharmaceutical compositions may be administered in the form of an aerosol or with an inhaler, including a dry powder aerosol. Suppositories for rectal administration of the drug can be prepared by mixing the drug with a suitable non-irritating excipient, such as cocoa butter and polyethylene glycols which are solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum and release the drug. The pharmaceutical compositions may be formulated with conventional pharmaceutical procedures such as sterilization and/or may contain conventional adjuvants such as preservatives, stabilizers, wetting agents, emulsifiers, buffers and the like in tablets and pills or may be formulated with enteric coatings. Such compositions may also contain adjuvants such as wetting agents, sweetening, flavoring and perfuming agents.

The pharmaceutical compositions of the invention comprise a compound of structural formula (I) as described herein or a pharmaceutically acceptable salt thereof, a kinase inhibitor (small molecule, polypeptide, antibody, etc.), an immunosuppressive agent, an anticancer agent, an antiviral agent, an anti-inflammatory agent, an antifungal agent, an antibiotic, or an additional active agent that is an anti-vascular hyperproliferative compound; and any pharmaceutically acceptable carrier, adjuvant or vehicle. Alternative compositions of the invention include a compound having formula (I) as described herein or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier, adjuvant or vehicle. Such compositions may optionally comprise one or more additional therapeutic agents, including, for example, kinase inhibitors (small molecules, polypeptides, antibodies, etc.), immunosuppressive agents, anti-cancer agents, antiviral agents, anti-inflammatory agents, antifungal agents, antibiotics, or anti-vascular hyperproliferative compounds.

The term "pharmaceutically acceptable carrier or adjuvant" refers to a carrier or adjuvant that can be administered to a patient with a compound of the present invention, and which does not destroy pharmaceutical activity and is non-toxic at doses sufficient to deliver a therapeutic amount of administration. Pharmaceutically acceptable carriers, adjuvants and excipients may be used in the pharmaceutical compositions of the present invention, including, but not limited to, ion exchangers, alumina, aluminum stearate, lecithin, Self Emulsifying Drug Delivery Systems (SEDDS), such as d-atopHenol polyethylene glycol 1000 succinate, surfactants used in pharmaceutical dosage forms, such as tweens or other similar polymeric delivery matrices, serum proteins, such as human serum albumin, buffer substances, such as phosphates, glycine, sorbic acid, potassium sorbate, surfactants used in partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene block polymers, polyethylene glycol and lanolin. Cyclodextrins, such as α -, β -, and γ -cyclodextrins, or chemically modified derivatives such as hydroxyalkyl, including 2 and 3-hydroxypropyl-cyclodextrins, or other solubilized derivatives may also be advantageously employed to enhance delivery of the compounds of the formulae described herein. The pharmaceutical compositions may be administered orally in any acceptable dosage form, including but not limited to capsules, tablets, emulsions and aqueous suspensions, dispersions and solutions. In the case of tablets for oral use, carriers which are commonly used include lactose and corn starch. Lubricating agents, such as magnesium stearate, for example, are also commonly added. For oral administration in capsule form, useful diluents include lactose and dried corn starch. When administered orally using aqueous suspensions and/or emulsions, the active ingredient may be suspended or dissolved in an oily phase with emulsifying and/or suspending agents. If desired, certain sweetening, flavoring and/or coloring agents may be added. The pharmaceutical composition may comprise the use of liposomes or microencapsulation techniques, various examples of which are found in the literature. The pharmaceutical composition may be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other solubilizing or dispersing agents, examples of which are also well known in the art.

Combination drug

The compounds of the present invention may be used alone or in combination with one or more other compounds of the present invention or with one or more other agents. When administered in combination, the therapeutic agents may be formulated for simultaneous administration or for sequential administration at different times, or the therapeutic agents may be administered as a single composition. By "combination therapy" is meant the use of a compound of the invention in combination with another agent, either by co-administration of each agent simultaneously or by sequential administration of each agent, in either case, for the purpose of achieving optimal effect of the drug. Co-administration includes simultaneous delivery dosage forms, as well as separate dosage forms for each compound. Thus, administration of the compounds of the invention may be used concurrently with other therapies known in the art, for example, radiation therapy or adjunctive therapies such as cytostatic agents, cytotoxic agents, other anti-cancer agents, etc. in the treatment of cancer to ameliorate the symptoms of the cancer. The present invention is not limited to the order of administration; the compounds of the invention may be administered previously, concurrently, or after other anti-cancer or cytotoxic agents.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.

Claims (11)

1. A compound of structural formula (I), a stereoisomer, or a pharmaceutically acceptable salt thereof:

wherein the content of the first and second substances,

x is N;

R¹is composed of

n₁Is an integer of 0 to 2, n₂Is an integer of 0 to 1, n₃Is an integer of 0 to 3;

L₁is composed of

R⁴、R⁵Each independently is H, methyl, R⁴、R⁵The same or different, or a combination thereof,

R³is composed of

R⁹、R¹⁰、R¹¹、R¹²、R¹³Each independently is H,Methyl or ethyl, m is 0 or 1,

m₁is an integer of 0 to 5, m₂Is an integer of 0 to 5, and m₁,m₂The sum is less than or equal to 5;

R²is composed of

n₄Is an integer of 0 to 3, n₅Is an integer of 0 to 1, n₆Is an integer of 0 to 5;

L₂is composed of

R⁷、R⁸Each independently is H, methyl, R⁷、R⁸The same or different, or a combination thereof,

R⁶is-H, hydroxyethyl, hydroxypropyl, C₁-C₅Alkyl radical, C₃-C₈Cycloalkyl radical, C₁-C₅Alkoxyethyl radical, C₁-C₅Alkoxypropyl group, C₁-C₅An alkylthioethyl group, a 5-6 membered heterocyclyl group, or-NR 'R ", wherein R', R" are each independently H, C₁-C₃Alkyl group of (1).

2. A compound, stereoisomer, or pharmaceutically acceptable salt thereof according to claim 1, wherein R is³Is composed of

R⁹、R¹¹、R¹²、R¹³Each independently is H, methyl or ethyl.

3. A compound, stereoisomer, or pharmaceutically acceptable salt thereof according to claim 1, wherein R⁶Is H, hydroxyethyl, hydroxypropyl, methyl, ethyl, propyl, isopropylPropyl, tert-butyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, methoxyethyl, ethoxyethyl, propoxyethyl, isopropoxyethyl, methoxypropyl, ethoxypropyl, propoxypropyl, isopropoxypropyl, methylthioethyl, ethylthioethyl, propylthioethyl, isopropylthioethyl, a 5-to 6-membered heterocyclic group, or-NR 'R' where R ', R' are each independently H, methyl, ethyl;

the 5-6 membered heterocyclic group is unsubstituted or substituted by 1-2 groups selected from hydroxy, C₁-C₃Alkyl radical, C₁-C₃Acyl substituents substituted heterocyclyl containing 1-2 heteroatoms selected from N, O, S.

4. The compound of claim 3, a stereoisomer, or pharmaceutically acceptable salt thereof, wherein the heterocyclyl containing 1-2 heteroatoms selected from N, O, S is any one selected from:

wherein R is¹⁴H, methyl and ethyl.

5. The compound of claim 1, a stereoisomer, or pharmaceutically acceptable salt thereof, wherein the compound of formula (I) is selected from:

4- (butyl-3-en-1-ylamino) -2- ((1- (tert-butyl) -1H-pyrazol-4-yl) amino ] pyrimidine-5-carboxamide;

2- ((1-tert-butyl-1H-pyrazol-4-yl) amino) -4- ((2-methylallyl) amino) pyrimidine-5-carboxamide;

2- ((1- (tert-butyl) -1H-pyrazol-4-yl) amino) -4- ((3-methylbutyl-2-en-1-yl) amino) pyrimidine-5-carboxamide;

2- ((1- (tert-butyl) -1H-pyrazol-4-yl) amino) -4- (pentyl-4-en-1-ylamino) pyrimidine-5-carboxamide;

4- ((2-methylallyl) amino) -2- ((1- (tetrahydro-2H-pyran-4-yl) -1H-pyrazol-4-yl) amino) pyrimidine-5-carboxamide;

4- ((3-methylbutyl-2-en-1-yl) amino) -2- ((1- (tetrahydro-2H-pyran-4-yl) -1H-pyrazol-4-yl) amino) pyrimidine-5-carboxamide;

4- (butyl-3-en-1-ylamino) -2- ((1- (tetrahydro-2H-pyran-4-yl) -1H-pyrazol-4-yl) amino) pyrimidine-5-carboxamide;

2- ((1- (tetrahydro-2H-pyran-4-yl) -1H-pyrazol-4-yl) amino) -4- (pentyl-4-en-1-ylamino) pyrimidine-5-carboxamide.

6. The compound of claim 1, a stereoisomer, or pharmaceutically acceptable salt thereof, wherein the compound of formula (I) is selected from:

2- ((1- (tert-butyl) -1H-pyrazol-4-yl) amino) -4- ((cyclopentyl-3-en-1-ylmethyl) amino) pyrimidine-5-carboxamide;

4- ((cyclopentyl-3-en-1-ylmethyl) amino) -2- ((1- (tetrahydro-2H-pyran-4-yl) -1H-pyrazol-4-yl) amino) pyrimidine-5-carboxamide;

4- (allylamino) -2- ((1- (tert-butyl) -1H-pyrazol-4-yl) amino) pyrimidine-5-carboxamide;

(Z) -2- ((1-tert-butyl-1H-pyrazol-4-yl) amino) -4- ((pentan-2-en-1-yl) amino) pyrimidine-5-carboxamide;

(E) -2- ((1-tert-butyl-1H-pyrazol-4-yl) amino) -4- ((4-methylpentan-2-en-1-yl) amino) pyrimidine-5-carboxamide;

(E) -2- ((1-tert-butyl-1H-pyrazol-4-yl) amino) -4- ((pentan-3-en-1-yl) amino) pyrimidine-5-carboxamide;

2- ((1-tert-butyl-1H-pyrazol-4-yl) amino) -4- ((3-methylbutyl-3-en-1-yl) amino) pyrimidine-5-carboxamide;

2- ((1-tert-butyl-1H-pyrazol-4-yl) amino) -4- ((4-methylpentan-3-en-1-yl) amino) pyrimidine-5-carboxamide;

2- ((1-tert-butyl-1H-pyrazol-4-yl) amino) -4- ((4-methylpentan-4-en-1-yl) amino) pyrimidine-5-carboxamide;

2- ((1-tert-butyl-1H-pyrazol-4-yl) amino) -4- ((5-methylhexan-4-en-1-yl) amino) pyrimidine-5-carboxamide;

2- ((1-tert-butyl-1H-pyrazol-4-yl) amino) -4- (((2E,4E) -hexane-2, 4-dien-1-yl) amino) pyrimidine-5-carboxamide;

2- ((1-tert-butyl-1H-pyrazol-4-yl) amino) -4- (((2E,4E) -heptane-2, 4-dien-1-yl) amino) pyrimidine-5-carboxamide.

7. The compound of any one of claims 1 to 6, a stereoisomer, or a pharmaceutically acceptable salt thereof, wherein the pharmaceutically acceptable salt of the compound is one or more selected from the group consisting of hydrochloride, hydrobromide, hydroiodide, perchlorate, sulfate, nitrate, phosphate, formate, acetate, propionate, glycolate, lactate, succinate, maleate, tartrate, malate, citrate, fumarate, gluconate, benzoate, mandelate, methanesulfonate, isethionate, benzenesulfonate, oxalate, palmitate, 2-naphthalenesulfonate, p-toluenesulfonate, cyclamate, salicylate, hexonate, and trifluoroacetate of the compound.

8. Use of a compound of any one of claims 1 to 6, a stereoisomer, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for treating an autoimmune disease or cancer associated with tyrosine kinases JAK1, JAK2, JAK3, TYK2, wherein the autoimmune disease or cancer associated with tyrosine kinases JAK1, JAK2, JAK3, TYK2 includes fundus disease, dry eye, psoriasis, vitiligo, dermatitis, alopecia areata, rheumatoid arthritis, colitis, multiple sclerosis, systemic lupus erythematosus, crohn's disease, atheroma, pulmonary fibrosis, liver fibrosis, myelofibrosis, non-small cell lung cancer, breast cancer, pancreatic cancer, glioma, glioblastoma, ovarian cancer, cervical cancer, colorectal cancer, melanoma, endometrial cancer, prostate cancer, bladder cancer, leukemia, gastric cancer, colon cancer, gastric cancer, or a pharmaceutically acceptable salt thereof, Liver cancer, gastrointestinal stromal tumor, thyroid cancer, nasopharyngeal cancer, esophageal cancer, brain tumor, lymphoma, multiple myeloma, biliary tract cancer sarcoma, and cholangiocarcinoma.

9. The use according to claim 8, wherein the leukemia is chronic myeloid leukemia or acute myeloid leukemia and the lymphoma is non-Hodgkin's lymphoma or B-cell or T-cell lymphoma.

10. A pharmaceutical composition comprising a compound of any one of claims 1 to 6, a stereoisomer, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers or excipients.

11. The pharmaceutical composition of claim 10, wherein the pharmaceutical composition further comprises one or more additional therapeutic agents.