WO2022057770A1 - 一种作为jak激酶抑制剂的药物化合物 - Google Patents

一种作为jak激酶抑制剂的药物化合物 Download PDF

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WO2022057770A1
WO2022057770A1 PCT/CN2021/118025 CN2021118025W WO2022057770A1 WO 2022057770 A1 WO2022057770 A1 WO 2022057770A1 CN 2021118025 W CN2021118025 W CN 2021118025W WO 2022057770 A1 WO2022057770 A1 WO 2022057770A1
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pharmaceutical compound
compound
compound according
present
tyk2
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PCT/CN2021/118025
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French (fr)
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邢莉
方文奎
李冠群
蔡雨婷
王晓磊
潘翔
朱文浩
汪杨
王增全
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嘉兴特科罗生物科技有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
    • C07D403/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a chain containing hetero atoms as chain links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/10—Anti-acne agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

Definitions

  • the present invention belongs to the field of pharmaceutical compounds, in particular, relates to a compound that can be used to prevent or treat autoimmune diseases such as rheumatoid arthritis, ankylosing spondylitis, ulcerative colitis, Crohn's disease, systemic lupus erythematosus, Dermatomyositis, multiple sclerosis, type I diabetes, psoriasis, vitiligo, Sjogren's syndrome, etc., or other inflammatory skin diseases such as atopic dermatitis, eczema, lichen planus, lichen glossy, lichen atrophicus, lipid film
  • a pharmaceutical compound for inflammation, acne, hidradenitis suppurativa, etc. in particular, relates to a compound that is a JAK kinase inhibitor.
  • JAK protein kinases catalyze the phosphorylation of amino acids at specific sites in proteins. According to the phosphorylation of amino acids, they can be divided into tyrosine, serine and arginine kinases.
  • JAK is a family of intracellular non-receptor tyrosine protein kinases (Tyrosine Kinase), including four members JAK1, JAK2, JAK3 and Tyk2. JAKs are mainly expressed in hematopoietic cells, leukocytes, and intestinal epithelial cells, and are responsible for mediating signaling of various cytokines involved in inflammatory responses.
  • JAKs When cytokines bind to cell surface receptors, JAKs are activated by phosphorylation, the activated JAKs are then activated by phosphorylating the intracellular portion of the receptors and recruit members of the STAT protein family, which are then activated by JAKs by phosphorylation Dimers are formed, detached from receptors and enter the nucleus to regulate gene transcription, thereby affecting the biological function of cells.
  • the JAK (Janus Kinase)-STAT (Signal Transducer and Activator of Transcription proteins) signaling pathway is the main pathway for intracellular signal transduction triggered by the combination of inflammatory cytokines and receptors.
  • JAK-STAT signaling pathway has an important relationship with many diseases, especially autoimmune diseases such as rheumatoid arthritis, intestinal diseases, allergic diseases and so on. Therefore, these protein kinases have become the most important drug development targets for intervening diseases.
  • many pharmaceutical companies have carried out new drug research and development for members of the JAK family, but most of them focus on inhibiting JAK1, JAK2 and JAK3, and inhibitors against Tyk2 are rarely reported.
  • the JAK-STAT pathway can be activated by more than 50 different cytokines.
  • Pro-inflammatory cytokines IL6, TNF- ⁇ , IL12, IL23
  • anti-inflammatory cytokines IL4, IL10
  • g-CSF hematopoietic growth factors
  • EPO EPO
  • TPO hematopoietic growth factors
  • GH metabolic cytokines
  • Activation of this signaling pathway regulates cell proliferation, differentiation and activation, as well as the transcriptional regulation of multiple genes in human metabolic homeostasis.
  • the Jak/STAT cascade is the confluence of many extracellular regulatory signals within the cell and thus the cell's central communication node.
  • cytokine-mediated signaling system phosphorylates cytokine receptors, recruits STAT through the activation of JAK family members JAK1, JAK2, JAK3, and TYK2 tyrosine kinases, and ultimately leads to intracellular gene expression. Pairing between JAK members is directly related to upstream cytokines. Depending on different cytokine stimuli, differences in upstream cytokine signaling may occur in JAK1/JAK2, JAK1/JAK3, JAK1/TYK2, JAK2/TYK2, JAK2/JAK2 pairings, with JAK1 pairing being the most common.
  • TYK2 is extremely important for the signal transduction of type 1 interferons (IFN- ⁇ , IFN- ⁇ ), IL-6 and IL-23. Immune cell differentiation and function associated with both inflammatory and autoimmune diseases are implicated in TYK2. Usually TYK2 and its family members can appear in the paired form of TYK2/JAK1, TYK2/JAK2, TYK2/JAK1/JAK2 after being activated by the signaling system. Tyk2 plays an extremely important role in the signaling pathway mediating IL12, IL17 and IL23. Biological antibody drugs against IL17 and IL23 have achieved good therapeutic effects on diseases such as psoriasis.
  • TDM-180820 A large number of active compounds, the structure of compound TDM-180820 is as follows:
  • This compound is also active against JAK kinase with IC50 of 0.1-1 ⁇ M or 1-10 ⁇ M. However, the applicant expects to find more targeted and more active compounds on the basis of such prior art.
  • the present invention aims to develop suitable high-efficiency and specific JAK kinase inhibitors, especially Tyk2 inhibitors, and/or JAK1 inhibitors, and/or JAK1/Tyk2 or Tyk2/JAK1 and/or Tyk2/Jak2 dual inhibitors, suitable for Treatment of autoimmune diseases such as rheumatoid arthritis, ulcerative colitis, and inflammatory skin diseases such as eczema, psoriasis as indications.
  • autoimmune diseases such as rheumatoid arthritis, ulcerative colitis
  • inflammatory skin diseases such as eczema, psoriasis as indications.
  • the present invention provides a pharmaceutical compound, which is a compound represented by the following structural formula or a stereoisomer, geometric isomer, tautomer, racemate, hydrate, Solvates, metabolites, and pharmaceutically acceptable salts or prodrugs:
  • the R represents 1 to 3 substituents on the benzene ring, and the 1 to 3 substituents are each independently selected from halogen or cyano.
  • the halogen is F, Cl or Br.
  • the R is ortho-substituted F.
  • the R is meta-substituted F.
  • the R is para-substituted F.
  • the R is meta-substituted cyano.
  • the present invention also provides the use of the above-mentioned pharmaceutical compound in inhibiting JAK kinase.
  • the present invention also provides the use of the above-mentioned pharmaceutical compounds in the preparation of medicines for the prevention or treatment of autoimmune diseases and immune-related inflammatory skin diseases, preferably, the pathogenesis of these diseases is related to Dysregulation of JAK signaling correlates.
  • the autoimmune disease is selected from the group consisting of rheumatoid arthritis, ankylosing spondylitis, ulcerative colitis, Crohn's disease, systemic lupus erythematosus, dermatomyositis, multiple sclerosis, type I At least one of diabetes, Sjögren's syndrome, vasculitis, alopecia areata, psoriasis, and vitiligo.
  • the immune-related inflammatory skin disease is selected from at least the group consisting of atopic dermatitis, eczema, lichen planus, lichen lustrous, lichen atrophicus, panniculitis, acne, and hidradenitis suppurativa A sort of.
  • the present invention carries out the rational design of pharmaceutical compounds with purpose, and the synthesized compound is firstly tested for the kinase biochemical activity of JAK, and SAR (structure-activity relationship) is established according to IC 50 , Potent inhibitors with IC50s below 200 nM were then tested cytologically to determine compound selectivity.
  • SAR structure-activity relationship
  • the compounds involved in the present invention have good inhibitory ability of JAK kinase activity and cell biological activity. More particularly, the pharmaceutical compound of the present invention can exert a better therapeutic effect than when the rightmost benzene ring has no substituent or is substituted with a substituent other than halogen or cyano.
  • the present invention provides pharmaceutical compounds, which are compounds represented by the following structural formulas or stereoisomers, geometric isomers, tautomers, racemates, hydrates, solvates thereof , metabolites, and pharmaceutically acceptable salts or prodrugs:
  • the R represents 1 to 3 substituents on the benzene ring, and the 1 to 3 substituents are each independently selected from halogen or cyano.
  • the pharmaceutical compounds of the present invention contain at least one substituent selected from halogen or cyano on the rightmost benzene ring.
  • the pharmaceutical compound of the present invention contains at least two substituents on the rightmost benzene ring, the substituents may each independently exist at any position of the benzene ring.
  • the species of the halogen can be independently selected from all halogen elements commonly found in the art, such as F, Cl, Br or I.
  • the halogen may be F, Cl or Br.
  • the halogen may be F or Cl.
  • the halogen may be F.
  • the R may be ortho-substituted F.
  • the pharmaceutical compound of the present invention has the following structural formula:
  • the R may be F substituted at the meta position.
  • the pharmaceutical compound of the present invention has the following structural formula:
  • the R may be para-substituted F.
  • the pharmaceutical compound of the present invention has the following structural formula:
  • the R may be meta-substituted cyano.
  • the pharmaceutical compound of the present invention has the following structural formula:
  • the term "pharmaceutically acceptable” refers to a substance that does not affect the biological activity or properties of the compounds of the present invention, and is relatively non-toxic, ie, the substance can be administered to an individual without causing an adverse biological response or in an undesirable manner Interacts with any components contained in the composition.
  • “pharmaceutically acceptable salts” may include inorganic salts and organic salts, wherein the organic salts may include but are not limited to ammonium, lithium, sodium, potassium, cesium, calcium, magnesium, copper, aluminum, Zinc, barium, or quaternary ammonium salts, and the inorganic salts may include, but are not limited to, arginine, tert-butylamine, dimethylamine, diethanolamine, ethanolamine, ethylenediamine, imidazole, lysine, methylamine, pyridine, picolinyl ester, piperazine, triethylamine, triethanolamine, trimethylamine or urea salts.
  • the organic salts may include but are not limited to ammonium, lithium, sodium, potassium, cesium, calcium, magnesium, copper, aluminum, Zinc, barium, or quaternary ammonium salts
  • the inorganic salts may include, but are not limited to, arginine, tert-butylamine, dimethylamine
  • the present invention provides the use of the above-mentioned pharmaceutical compounds for inhibiting JAK kinases, particularly as dual JAK1/Tyk2 inhibitors and Tyk2 specific inhibitors.
  • the present invention also provides the use of the above-mentioned pharmaceutical compounds in the preparation of medicines for the prevention or treatment of autoimmune diseases and immune-related inflammatory skin diseases, preferably, the pathogenesis of these diseases is related to Dysregulation of JAK signaling correlates.
  • treatment refers to any administration of a therapeutic agent according to a therapeutic regimen that achieves the desired effect, ie, partial or complete alleviation, amelioration, remission, inhibition, delay of onset, reduction in severity and/or reducing the incidence of one or more symptoms or characteristics of a particular disease, disorder and/or condition; in some embodiments, administration of a therapeutic agent according to a therapeutic regimen is associated with achievement of a desired effect.
  • Such treatment can be directed to subjects who do not exhibit the relevant disease, disorder and/or condition and/or to subjects exhibiting only early signs of the disease, disorder and/or condition. Alternatively or additionally, such treatment may be directed to subjects exhibiting one or more established signs of the relevant disease, disorder and/or condition.
  • treatment can be directed to a subject who has been diagnosed with the relevant disease, disorder, and/or condition. In some embodiments, treatment may be directed to a subject known to have one or more predisposing factors that are statistically associated with an increased risk of developing the relevant disease, disorder, and/or condition.
  • the medicament prepared in the above-mentioned use may comprise an effective amount of the pharmaceutical compound of the present invention, together with a pharmaceutically acceptable excipient, carrier or diluent.
  • the term "effective amount”, “therapeutically effective amount” or “pharmaceutically effective amount” refers to the amount of a therapeutic agent that confers a therapeutic effect to a treated subject at a reasonable benefit/risk ratio applicable to any drug treatment .
  • Such therapeutic effects may be objective (ie, measurable by some test or marker) or subjective (ie, the subject gives an indication or perceives the effect).
  • a "therapeutically effective amount” refers to an amount effective to treat, ameliorate, or prevent (eg, delay onset), such as by ameliorating symptoms associated with a disease, preventing or delaying the onset of a disease, and/or also reducing the severity or frequency of symptoms of a disease ) associated disease or disorder and/or an amount of a therapeutic agent or composition that exhibits a detectable therapeutic or prophylactic effect.
  • the therapeutically effective amount of the pharmaceutical compound to be administered will vary depending on the nature and severity of the subject and disease, the physical condition of the subject, the treatment regimen (eg whether to use a second therapeutic agent), and the chosen route of administration; appropriate dosages can be readily determined by those skilled in the art. Additionally, the optimal number and interval of individual doses of the drug will be determined by the nature and extent of the condition being treated, the form, route and location of administration, and the age and condition of the particular subject being treated, and the physician will ultimately An appropriate dose to be administered is determined. This dose can be repeated as many times as necessary. If side effects occur, the amount and/or frequency of the dose can be varied or reduced according to normal clinical practice.
  • pharmaceutically acceptable excipient, carrier or diluent includes, but is not limited to, any adjuvant, carrier, excipient, Glidants, sweeteners, diluents, preservatives, dyes/colorants, flavoring agents, surfactants, wetting agents, dispersing agents, suspending agents, stabilizers, isotonic agents, solvents or emulsifiers, etc. .
  • the medicament prepared in the above-mentioned use can also contain other substances that can be used to prevent or treat autoimmune diseases and immune-related inflammatory skin diseases.
  • a medicament for disease as another active ingredient examples include, but are not limited to, vitamin D derivatives, vitamin A derivatives, glucocorticoids, calcineurin inhibitors, or non-steroidal anti-inflammatory drugs, and the like.
  • the medicament contains a plurality of active ingredients, the respective active ingredients may be administered simultaneously, sequentially or separately according to the judgment of the physician.
  • the pharmaceutical compounds of the present invention can be administered to a patient by a variety of routes such as oral, transdermal, subcutaneous, intranasal, intravenous, intramuscular, intrathecal, regional or topical (eg mucosal).
  • routes such as oral, transdermal, subcutaneous, intranasal, intravenous, intramuscular, intrathecal, regional or topical (eg mucosal).
  • the most appropriate route of administration in any given situation will depend on the subject and the nature and severity of the disease, the subject's physical condition, and the like.
  • the pharmaceutical compounds of the present invention may be administered intravenously.
  • the pharmaceutical compounds of the present invention may be administered orally.
  • the medicament of the present invention can be prepared into different dosage forms.
  • the medicament can be prepared as tablets, capsules, pills, granules, aerosols, sprays, or injections.
  • the pharmaceutical compounds of the present invention or the medicines prepared therefrom can exert excellent effects in preventing or treating autoimmune diseases and immune-related inflammatory skin diseases.
  • the autoimmune disease may include, but is not limited to, rheumatoid arthritis, ankylosing spondylitis, ulcerative colitis, Crohn's disease, systemic lupus erythematosus, dermatomyositis, multiple sclerosis, type I Diabetes, Sjögren's syndrome, vasculitis, alopecia areata, psoriasis, or vitiligo; and the immune-related inflammatory skin diseases may include, but are not limited to, atopic dermatitis, eczema, lichen planus, lichen lustrous, lichen atrophicus , panniculitis, acne, or hidradenitis suppurativa. More particularly, the pharmaceutical compound of the present invention can exert a better therapeutic effect than when the rightmost benzene
  • Compound 2 was prepared in a similar manner to Example 1: TDM-180945, i.e. 1-(3-fluorophenyl)-N-(4-(2-(((1-methyl-1H-pyrazol-4-yl)amino)pyrimidin-4-yl)phenyl ) methanesulfonamide (26.1 mg, 19.8% yield).
  • N-chlorosuccinimide (26.71 g, 200 mmol) was dissolved in a mixed solution of 2N hydrochloric acid (13.6 mL) and acetonitrile (80 mL), then compound 4c was added, and the mixture was stirred for 1 hour.
  • the mixture was concentrated under reduced pressure, the residue was added with water and cooled, the solid was collected by filtration, the filter cake was added with water and sonicated, and filtered again to obtain the desired white solid compound 367d (7 g, 67% yield), i.e. (4-fluorophenyl)methanesulfonic acid acid chloride.
  • Kinase reaction buffer prepare the kinase reaction buffer with the following components: 50 mM HEPES, pH 7.5, 1 mM EGTA, 10 mM MgCl 2 , 2 mM DTT, 0.01% Tween 20.
  • 1X detection buffer prepare the detection buffer, deionized water 9: 1 Dilute 10X assay buffer to 1X.
  • 4X Kinase Solution Dilute JAK kinase to 4X final concentration in Kinase Reaction Buffer (JAK1: 80 nM, JAK2/JAK3/Tyk2: 4 nM).
  • 4X Substrate Solution Dilute ULight TM -JAK (Tyr1023) substrate to 200 nM (final concentration: 50 nM) in Kinase Reaction Buffer.
  • 4X ATP solution Dilute ATP to 4X final concentration in Kinase Reaction Buffer (JAK1: 160 ⁇ M, JAK2/JAK3/Tyk2: 40 ⁇ M).
  • 4X test compound solution DMSO dissolves the test compound to 10mM stock solution, 3-fold gradient dilution is configured to the desired concentration, each compound is set to 10 concentration points, and the final concentration range of the test compound is: 10 ⁇ M-0.5nM.
  • 4X Enzyme Reaction Stop Solution 1X Assay Buffer to dissolve EDTA to 40 mM (final EDTA concentration: 10 mM).
  • 4X detection antibody solution Dilute Eu-labeled detection antibody (anti-phosphotyrosine (PT66)) to 8 nM (final antibody concentration: 2 nM) in 1X detection buffer.
  • JAK1 was reacted at room temperature for 2 hours, and JAK2/JAK3/Tyk2 was reacted at room temperature for 1 hour.
  • the final concentrations of each component of the JAK1 reaction were: JAK1: 20 nM, substrate: 50 nM, ATP: 40 uM, and the final concentration of the test compounds ranged from 10 ⁇ M to 0.5 nM.
  • the final concentrations of each component in the JAK2/JAK3/Tyk2 reaction were: JAK2: 1 nM, substrate: 50 nM, ATP: 10 ⁇ M, and the final concentration of the test compounds ranged from 10 ⁇ M to 0.5 nM.
  • the compounds TDM-180958, TDM-180944, TDM-180945 and TDM-180963 are used as the experimental group of this patent, and the applicant's previous application CN110627775A in the TDM-180820 and the prior art CN103298794A in the compound 213 (The data for this compound was taken directly from the source patent) as a positive control.
  • JAK kinase small-molecule inhibitors were evaluated using two cytological assay systems: IL-4-induced pSTAT6 expression in THP-1 cells and IFN- ⁇ -B2-induced U2OS cells.
  • IL-4-induced pSTAT6 expression was used to reflect the cytological activity of JAK1 and Tyk2, respectively.
  • THP-1 or U2OS cells were collected and resuspended in 1*HBSS.
  • Cells were seeded into 384-well plates and incubated in a 37°C & CO 2 incubator.
  • Add 5 ⁇ L of IL4 or IFN- ⁇ -B2 to each well and continue to incubate in a 37°C & CO 2 incubator. Discard the cell culture supernatant, add cell lysate, and incubate at room temperature.
  • Cell lysates were assayed for pSTAT6 with the ⁇ LISA SureFire Ultra p-STAT6HV Kit, or pSTAT1 with the ⁇ LISA SureFire Ultra p-STAT1HV Kit.
  • the reaction plate was incubated at room temperature for 2 hours.
  • the signal for each culture blank was read on an Envision plate reader.
  • Liver microsome stability test The compound is tested in vitro using microsomes isolated and extracted from the liver of experimental animals of a specific species, by detecting the compound and liver microsomes containing hepatocyte phase I metabolic enzymes (mainly P450) After incubation for different lengths of time, the concentration of the prototype compound and its degradation products was used to determine the metabolic stability of the compound in liver microsomes and to preliminarily determine the pharmacokinetic trend of the compound in this species.
  • hepatocyte phase I metabolic enzymes mainly P450
  • the median concentrations of liver microsomes, test compounds, and cofactor NADPH in the tested reaction system were 0.75 mg/mL, 1 ⁇ M, and 2 mM, respectively, and Ketanserin was used as the positive control compound at different incubation time points ( 0, 15, 30 and 60 minutes) take a certain volume of the reaction system solution and terminate the reaction with pre-cooled precipitation solution, after centrifugation, take the supernatant for LC-MS/MS analysis to obtain parameters such as drug solubility.
  • a Conversion factor (microsomal protein per gram of liver) ⁇ (weight of liver per kilogram of body weight)
  • Caco-2 cell permeability test Caco-2 is a human colon cancer cell line.
  • the Caco-2 cell culture model established by Millipore Transwell II 24-well plate is widely used to test the permeability of drugs (drugs). Absorption and efflux), to a certain extent, can reflect the absorption characteristics of drugs in the intestinal tract.
  • a monolayer of Caco-2 cells is seeded at the bottom of an inner chamber (A) lined with a semipermeable membrane, both the inner chamber and the outer chamber (B) containing the inner chamber are filled with culture medium, and the liquid in the inner and outer chambers is added. The surfaces are kept flush, the inner chamber simulates the intestinal lumen, and the outer chamber simulates the intestinal submucosal capillaries.
  • the cells After 21-24 days of culture, the cells will completely cover the bottom surface of the inner chamber, and its integrity can be measured by adding Lucifer yellow (LY) to A or B or detecting transepidermal electrical resistance (TEER), Caco-2 cell culture
  • LY Lucifer yellow
  • TEER transepidermal electrical resistance
  • the drug can be added to the inner chamber medium to detect drug A ⁇ B transport (absorption), or the outer chamber medium to detect B ⁇ A transport (efflux).
  • the solution containing the drug is called the supply solution, while the The drug-free test solution on the other side is called the receiver solution.
  • the present application provides liver microsome stability data and Caco-2 cell model data for the compound TDM-180958 of the present application and the compound TDM-180820 used as a positive control.
  • the specific results are shown in Table 4 below.
  • the compound TDM-180958 of the present application has a significantly higher Papp value (nearly 6 times) in the Caco-2 cell model than the compound TDM-180820 used as a positive control, that is to say , the compound TDM-180958 of the present application has better drug absorption than the compound TDM-180820, which is presumably due to the increase in the number of halogen or cyano groups on the 1-3 positions of the benzene ring on the right side than that of the pure benzene ring. , the reactivity and lipophilicity of the molecule are improved, which is beneficial to enter the cell through the lipid bilayer of the cell membrane.
  • a high Papp value does not necessarily represent a longer plasma half-life or bioavailability, the efficient absorption of drugs by cells is of great significance for topical administration routes such as topical skin, ophthalmic, and ear canal administration.

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Abstract

本发明提供了药物化合物，所述药物化合物为由如下结构式所示的化合物或其立体异构体、几何异构体、互变异构体、消旋体、水合物、溶剂化物、代谢产物以及药学上可接受的盐或前药，其中所述R表示苯环上的1至3个取代基，所述1至3个取代基各自独立地选自卤素或氰基。本发明的药物化合物能够抑制JAK激酶，更特别是作为JAK1/Tyk2双抑制剂和Tyk2特异性抑制剂。更具体地，本发明的药物化合物能够用于预防或治疗自身免疫性疾病如类风湿性关节炎、强直性脊柱炎、溃疡性结肠炎、系统性红斑狼疮、I型糖尿病、干燥综合症、血管炎、斑秃、银屑病和白癜风等，或其它炎症性皮肤病如特应性皮炎、湿疹、痤疮和化脓性汗腺炎等。

Description

一种作为JAK激酶抑制剂的药物化合物
本申请的交叉引用
本发明要求于2020年9月17日提交的申请号为CN202010976485.3、发明名称为“一种作为JAK激酶抑制剂的药物化合物”的专利申请的优先权。
技术领域
本发明属于药物化合物领域，具体地，涉及一种能够用于预防或治疗自身免疫性疾病如类风湿性关节炎、强直性脊柱炎、溃疡性结肠炎、克罗恩病、系统性红斑狼疮、皮肌炎、多发性硬化、I型糖尿病、银屑病、白癜风、干燥综合症等，或其它炎症性皮肤病如特应性皮炎、湿疹、扁平苔藓、光泽苔藓、硬化萎缩性苔藓、脂膜炎、痤疮、化脓性汗腺炎等的药物化合物，具体地，涉及一种作为JAK激酶抑制剂的化合物。
背景技术
蛋白激酶催化蛋白质特殊位点氨基酸的磷酸化，根据氨基酸的磷酸化可分为酪氨酸、丝氨酸和精氨酸激酶。JAK是细胞内非受体性酪氨酸蛋白激酶(Tyrosine Kinase)的一个家族，包括JAK1、JAK2、JAK3和Tyk2四个成员。JAK主要在造血细胞、白细胞和肠道上皮细胞内表达，负责介导参与炎症反应的各种细胞因子的信号传递。当细胞因子和细胞表面受体结合时，JAK通过磷酸化而被激活，激活的JAK随即通过磷酸化受体的细胞内部分使之活化并招募STAT蛋白家族成员，之后STAT被JAK通过磷酸化激活形成二聚体，脱离受体进入细胞核，进行基因转录的调控，从而影响细胞的生物学功能。JAK(Janus Kinase)-STAT(Signal Transducer and Activator of Transcription proteins)信号传导通路是炎症性细胞因子和受体相结合之后激发的信号在细胞内传导的主要通路。许多证据表明，JAK-STAT信号通路与很多疾病，特别是自身免疫性疾病如风湿性关节炎、肠道疾病、过敏性疾病等有重要关联。因此，这类蛋白激酶成 为了干预疾病的最重要药物研发靶点。目前多家制药公司已经针对JAK家族成员进行新药研发，但多集中于抑制JAK1、JAK2和JAK3，而针对Tyk2的抑制剂还极少有报道。
JAK-STAT通路可被50多种不同的细胞因子激活。促炎细胞因子(IL6、TNF-α、IL12、IL23)、抗炎细胞因子(IL4、IL10)、造血细胞生长因子(g-CSF、EPO、TPO)和代谢细胞因子(leptin、GH)等可激活该信号通路，从而调节细胞增殖、分化和活化，以及人体代谢稳态的多种基因转录调控。Jak/STAT级联是细胞内许多细胞外调节信号的汇合点，因此是细胞的中心通讯节点。研究表明，细胞因子介导的信号传导系统通过对JAK家族成员JAK1、JAK2、JAK3和TYK2酪氨酸激酶的激活，磷酸化细胞因子受体，招募STAT，最终导致细胞内的基因表达。JAK成员之间的配对与上游的细胞因子直接有关。取决于不同的细胞因子刺激，上游细胞因子信号的不同可能出现JAK1/JAK2、JAK1/JAK3、JAK1/TYK2、JAK2/TYK2、JAK2/JAK2配对组合，其中JAK1配对最为常见。TYK2对于一型干扰素(IFN-α、IFN-β)、IL-6和IL-23的信号转导极为重要。炎症性疾病和自身免疫性疾病有关的免疫细胞分化和功能都与TYK2有重要关联。通常TYK2与其家族成员在被信号传递系统激活后可以是出现TYK2/JAK1、TYK2/JAK2、TYK2/JAK1/JAK2配对形式。Tyk2在介导IL12、IL17和IL23的信号传导通路上有极为重要的作用。针对IL17和IL23的生物抗体药在银屑病等疾病上取得了很好的治疗效果，可以想象利用高效的小分子对JAK激酶活性，特别是TYK2激酶活性进行抑制可以阻断炎症因子介导的信号通路，控制炎症，有效治疗自身免疫性疾病和/或炎症性皮肤疾病，并减少副作用。
申请人近年来在该领域做了大量的研究，发现了大量具有良好效果JAK激酶抑制剂，例如申请人在先申请CN110627775A(发明名称：一种药物化合物)公开了具体的抑制剂，其中公开了大量的活性化合物，化合物TDM-180820结构如下：
Figure PCTCN2021118025-appb-000001
该化合物的活性针对TYK2、JAK1、JAK2、JAK3的IC 50值小于0.1μM。 此外，其它现有技术CN103298794A(公开日为2013年09月11日)也测试多个化合物的TYK2与JAK2的活性，其中如下化合物213：
Figure PCTCN2021118025-appb-000002
该化合物也针对JAK激酶具有活性，其IC 50为0.1-1μM或1-10μM。然而，申请人期望在这类现有技术的基础上寻找靶向性更强、活性更优的化合物。
发明内容
本发明旨在于开发适合高效和特异的JAK激酶抑制剂，特别是Tyk2抑制剂、和/或JAK1抑制剂、和/或JAK1/Tyk2或Tyk2/JAK1和/或Tyk2/Jak2双重抑制剂，适用于治疗自身免疫性疾病如风湿性关节炎、溃疡性结肠炎，以及炎症性皮肤病如以湿疹，银屑病为适应症。
在一方面，本发明提供了一种药物化合物，所述药物化合物为由如下结构式所示的化合物或其立体异构体、几何异构体、互变异构体、消旋体、水合物、溶剂化物、代谢产物以及药学上可接受的盐或前药：
Figure PCTCN2021118025-appb-000003
其中，所述R表示苯环上的1至3个取代基，所述1至3个取代基各自独立地选自卤素或氰基。
在一个实施方式中，所述卤素为F、Cl或Br。
在另一个实施方式中，所述R为邻位取代的F。
在另一个实施方式中，所述R为间位取代的F。
在另一个实施方式中，所述R为对位取代的F。
在另一个实施方式中，所述R为间位取代的氰基。
在另一方面，本发明还提供了上述药物化合物在抑制JAK激酶中的用途。
在另一方面，本发明还提供了上述药物化合物在制备用于预防或治疗自身免疫性疾病、以及与免疫有关的炎症性皮肤疾病的药物中的用途，优选地，这些疾病的发病机理均与JAK信号传导的失调相关。
在一个实施方式中，所述自身免疫性疾病选自类风湿性关节炎、强直性脊柱炎、溃疡性结肠炎、克罗恩病、系统性红斑狼疮、皮肌炎、多发性硬化、I型糖尿病、干燥综合症、血管炎、斑秃、银屑病和白癜风中的至少一种。
在另一个实施方式中，所述与免疫有关的炎症性皮肤疾病选自特应性皮炎、湿疹、扁平苔藓、光泽苔藓、硬化萎缩性苔藓、脂膜炎、痤疮和化脓性汗腺炎中的至少一种。
本发明的作用和效果：
本发明根据JAK激酶的蛋白结构，特别是Tyk2的蛋白结构，进行了药物化合物有目的的合理设计，合成的化合物首先进行JAK的激酶生化活性检测，根据IC 50建立SAR(structure-activity relationship)，对IC 50在200nM以下的强效抑制剂再进行细胞学的测试，并确定化合物的选择性。参见具体活性实验数据可以发现，本发明涉及的化合物具有良好的JAK激酶活性和细胞生物学活性的抑制能力。更特别地是，本发明的药物化合物在相较于在最右侧苯环上没有取代基或被卤素或氰基以外的取代基所取代时能够发挥出更佳的治疗效果。
具体实施方式
以下对本发明的具体实施方式进行详细说明。应当理解的是，此处所描述的具体实施方式仅用于说明和解释本发明，并不用于限制本发明。
在本文中所披露的范围的端点和任何值都不限于该精确的范围或值，这些范围或值应当理解为包含接近这些范围或值的值。对于数值范围来说，各个范围的端点值之间、各个范围的端点值和单独的点值之间，以及单独的点值之间可以彼此组合而得到一个或多个新的数值范围，这些数值范围应被视为在本文中具体公开。
在详细描述本发明前，应了解，在此使用的术语只在于描述特定的实施方 式，而不希望限制本发明的范围，本发明的范围仅由所附权利要求书限定。为了更完全地了解在此描述的本发明，采用以下术语，它们的定义如下所示。除非另外定义，在此使用的所有技术和科学术语具有与本发明所属领域的普通技术人员所理解的相同的含义。
在一方面，本发明提供了药物化合物，所述药物化合物为由如下结构式所示的化合物或其立体异构体、几何异构体、互变异构体、消旋体、水合物、溶剂化物、代谢产物以及药学上可接受的盐或前药：
Figure PCTCN2021118025-appb-000004
其中，所述R表示苯环上的1至3个取代基，所述1至3个取代基各自独立地选自卤素或氰基。换句话说，本发明的药物化合物在最右侧的苯环上包含至少一个选自卤素或氰基的取代基。进一步地，当本发明的药物化合物在最右侧的苯环上包含至少两个取代基时，所述取代基可以各自独立地存在于该苯环的任何位置上。
根据本发明，当所述至少一个取代基为卤素，所述卤素的种类可以各自独立地选自本领域中常见的所有卤族元素，例如F、Cl、Br或I。在一个优选的实施方式中，所述卤素可以为F、Cl或Br。在一个更优选的实施方式中，所述卤素可以为F或Cl。在一个最优选的实施方式中，所述卤素可以为F。
在一个具体的实施方式中，所述R可以为邻位取代的F。在此情况下，本发明的药物化合物具有以下结构式：
Figure PCTCN2021118025-appb-000005
在一个具体的实施方式中，所述R可以为间位取代的F。在此情况下，本 发明的药物化合物具有以下结构式：
Figure PCTCN2021118025-appb-000006
在一个具体的实施方式中，所述R可以为对位取代的F。在此情况下，本发明的药物化合物具有以下结构式：
Figure PCTCN2021118025-appb-000007
在一个具体的实施方式中，所述R可以为间位取代的氰基。在此情况下，本发明的药物化合物具有以下结构式：
Figure PCTCN2021118025-appb-000008
如本文所用，术语“药学上可接受的”是指不影响本发明化合物的生物活性或性质的物质，并且相对无毒，即该物质可施用于个体而不造成不良的生物反应或以不良方式与组合物中包含的任意组分相互作用。在本发明中，“药学上可接受的盐”可以包括无机盐和有机盐，其中，所述有机盐可以包括但不限于铵、锂、钠、钾、铯、钙、镁、铜、铝、锌、钡或季铵盐，并且所述无机盐可以包括但不限于精氨酸、叔丁胺、二甲胺、二乙醇胺、乙醇胺、乙二胺、咪唑、赖氨酸、甲胺、吡啶、吡啶甲酸酯、哌嗪、三乙胺、三乙醇胺、三甲胺或脲盐。
在另一方面，本发明提供了上述药物化合物在抑制JAK激酶中的用途，特别是作为JAK1/Tyk2双抑制剂和Tyk2特异性抑制剂。
在另一方面，本发明还提供了上述药物化合物在制备用于预防或治疗自身免疫性疾病、以及与免疫有关的炎症性皮肤疾病的药物中的用途，优选地，这些疾病的发病机理均与JAK信号传导的失调相关。
如本文所用，术语“治疗”是指根据治疗性方案的治疗性试剂的任何施用， 所述治疗性方案达到所需效果，即部分或完全减轻、改善、缓解、抑制、延迟发作、降低严重程度和/或降低特定疾病、障碍和/或病症的一种或多种症状或特征的发生率；在一些实施方式中，根据治疗性方案的治疗性试剂的施用与所需效果的实现相关。这种治疗可以针对没有表现出相关疾病、障碍和/或病症的受试者和/或针对仅表现出疾病、障碍和/或病症的早期迹象的受试者。替代地或另外地，这种治疗可以针对表现出相关疾病、障碍和/或病症的一种或多种所确定迹象的受试者。在一些实施方式中，治疗可以针对已被诊断患有相关疾病、障碍和/或病症的受试者。在一些实施方式中，治疗可以针对已知具有一种或多种易感因素的受试者，所述易感因素在统计学上与相关疾病、障碍和/或病症发展的风险增加相关。
根据本发明，上述用途中制得的药物可以包含有效量的本发明的药物化合物，以及药学上可接受的赋形剂、载体或稀释剂。
如本文所用，术语“有效量”、“治疗有效量”或“药学有效量”是指对于治疗的受试者以适用于任何药物治疗的合理受益/风险比赋予治疗效果的治疗性试剂的量。这样的治疗效果可以是客观的(即可以通过某种测试或标记测量)或主观的(即受试者给出指示或感觉到效果)。在一些实施方式中，“治疗有效量”是指诸如通过改善与疾病有关的症状、预防或延迟疾病发作和/或还减轻疾病症状的严重性或频率来有效治疗、改善或预防(例如延迟发作)相关疾病或病症和/或表现出可检测的治疗或预防效果的治疗性试剂或组合物的量。
本领域的技术人员将认识到，待施用的所述药物化合物的治疗有效量将根据以下各项而变化：受试者和疾病的性质和严重程度、受试者的身体状况、治疗方案(例如是否使用第二治疗剂)、以及所选择的施用途径；合适的剂量可以由本领域的技术人员容易地确定。另外，该药物的个体剂量的最佳数量和间隔将通过所治疗的病状的性质和程度、施用的形式、途径和位置、以及所治疗的特定受试者的年龄和病状确定，并且医师将最终确定待施用的合适剂量。此剂量可以视需要重复多次。如果出现副作用，则可以根据正常临床实践改变或减少剂量的量和/或频率。
在本发明中，“药学上可接受的赋形剂、载体或稀释剂”包括但不限于任何被相关的政府管理部门许可为可接受供人类或家畜使用的佐剂、载体、赋形剂、 助流剂、增甜剂、稀释剂、防腐剂、染料/着色剂、矫味剂、表面活性剂、润湿剂、分散剂、助悬剂、稳定剂、等渗剂、溶剂或乳化剂等。
根据本发明，进一步地，上述用途中制得的药物除了可以包含本发明的药物化合物作为有效成分之外，还可以包含其他可用于预防或治疗自身免疫性疾病、以及与免疫有关的炎症性皮肤疾病的药剂作为另一种有效成分。所述药剂的实例包括但不限于维生素D衍生物、维生素A衍生物、糖皮质激素、钙调神经磷酸酶抑制剂或非甾体类抗炎药等。当该药物包含多种有效成分时，各有效成分可以根据医师的判断同时、依次或分开施用。
另外，本发明的药物化合物可以通过多种途径施用于患者，这些途径诸如口服、透皮、皮下、鼻内、静脉内、肌内、鞘内、区域或局部(例如粘膜)。在任何给定情况下最适合的施用途径将取决于受试者和疾病的性质和严重程度、以及受试者的身体状况等。在一个实施方式中，本发明的药物化合物可以经静脉内施用。在另一个实施方式中，本发明的药物化合物可以口服施用。相应地，根据不同的施用方式，本发明的药物可以制备为不同的剂型。例如，在一个实施方式中，所述药物可以制备为片剂、胶囊剂、丸剂、颗粒剂、雾化剂、喷雾剂或注射剂。
经发明人研究发现，本发明的药物化合物或其制得的药物在用于预防或治疗自身免疫性疾病、以及与免疫有关的炎症性皮肤疾病能够发挥优异的效果。具体地，所述自身免疫性疾病可以包括但不限于类风湿性关节炎、强直性脊柱炎、溃疡性结肠炎、克罗恩病、系统性红斑狼疮、皮肌炎、多发性硬化、I型糖尿病、干燥综合症、血管炎、斑秃、银屑病或白癜风；而所述与免疫有关的炎症性皮肤疾病可以包括但不限于特应性皮炎、湿疹、扁平苔藓、光泽苔藓、硬化萎缩性苔藓、脂膜炎、痤疮或化脓性汗腺炎。更特别地是，本发明的药物化合物在相较于在最右侧苯环上没有取代基或被卤素或氰基以外的取代基所取代时能够发挥出更佳的治疗效果。
以下，将通过实施例对本发明的特定药物化合物的效果进行详细描述。
实施例
实施例1 合成化合物1的一般方法(TDM-180944)
Figure PCTCN2021118025-appb-000009
步骤1：化合物1(TDM-180944)的制备
在室温下向化合物1a(60mg，0.225mmol)的吡啶(5mL)溶液中加入化合物1b(65.8mg，0.315mmol)，将混合物加热至70℃并搅拌6h。然后将混合物在减压下浓缩，残余物通过硅胶色谱(二氯甲烷：含10％甲醇的二氯甲烷＝70：30)和甲酸制备纯化，得到黄色固体化合物1，TDM-180944，即1-(2-氟苯基)-N-(4-(2-(((1-甲基-1H-吡唑-4-基)氨基)嘧啶-4-基)苯基)甲磺酰胺(34.5mg，34.97％产率)。
1H NMR(400MHz,DMSO-d 6)δ10.34(s,1H),9.45(s,1H),8.45(d,J＝5.2Hz,1H),8.10(d,J＝8.6Hz,2H),7.93(s,1H),7.55(s,1H),7.45–7.30(m,4H),7.25–7.17(m,3H),4.60(s,2H),3.84(s,3H)。LCMS[M+1] +＝439.2。
实施例2 合成化合物2的一般方法(TDM-180945)
以与实施例1相似的方法制备化合物2：
Figure PCTCN2021118025-appb-000010
TDM-180945，即1-(3-氟苯基)-N-(4-(2-(((1-甲基-1H-吡唑-4-基)氨基)嘧啶-4-基)苯基)甲磺酰胺(26.1mg，19.8％产率)。
1H NMR(400MHz,DMSO-d 6)δ10.25(s,1H),9.45(s,1H),8.45(d,J＝5.2Hz,1H),8.12(d,J＝8.6Hz,2H),7.93(s,1H),7.56(s,1H),7.40(dd,J＝14.3,7.6Hz,1H),7.32(d,J＝8.6Hz,2H),7.26–7.15(m,2H),7.11(d,J＝7.3Hz,2H),4.63(s,2H),3.84(s,3H)。LCMS[M+1] +＝439.2。
实施例3 合成化合物3的一般方法(TDM-180958)
Figure PCTCN2021118025-appb-000011
步骤1：化合物3c(3-氰基苄基氨基硫代氨基甲酸酯)的制备
向化合物3a(1.78g，9.08mmol)的乙醇(13mL)溶液中加入化合物3b(690mg，9.08mmol)，反应液升温至80℃搅拌1小时，LCMS[M+H] +＝192，检测反应完全。后处理：反应液浓缩至干得到白色目标化合物(化合物3c,1.7g,产率97.7％)，LCMS[M+1] +＝192。
步骤2：化合物3d((3-氰基苯基)甲磺酰氯)的制备
向N-氯代丁二酰亚胺(4.85g,36.32mmol)的乙腈(20mL)溶液中加入2N的盐酸溶液(2.5mL)和化合物3c(1.74g，9.08mmol)，反应液室温搅拌30分钟。后处理：反应完成后，将乙腈浓缩除去，加入水(15mL)，有白色固体析出，过滤，固体用油泵拉干得到白色目标化合物(化合物3d，1.776g，产率90.7％)。
步骤3：化合物3(TDM-180958)的制备
向化合物3e(500mg，1.878mmol)的无水吡啶(20mL)溶液中加入化合物3d(1g，4.64mmol)，反应液升温至80℃反应30分钟，LCMS[M+H] +＝446，检测反应完全。后处理：反应液浓缩至干，粗品过柱[洗脱剂:(D:M＝10:1)/DCM＝0～50％]，粗品用DCM/MeOH＝30/1打浆得到黄色固体，再通过制备型HPLC纯化得到黄色目标化合物，即1-(3-氰基苯基)-N-(4-(2-(((1-甲基-1H-吡唑-4-基)氨基)嘧啶-4-基)苯基)甲磺酰胺(化合物3，155.4mg,产率18.6％)。
1H NMR(400MHz,DMSO)δ10.27(s,1H),9.46(s,1H),8.46(d,J＝5.2Hz,1H),8.12(d,J＝8.7Hz,2H),7.93(s,1H),7.84(dt,J＝7.2,1.6Hz,1H),7.71(s,1H),7.61(ddd,J＝17.3,10.8,4.8Hz,3H),7.32(d,J＝8.6Hz,2H),7.24(d,J＝5.2Hz,1H),4.71(s,2H),3.85(s,3H)。LCMS[M+H] +＝446。
实施例4 合成化合物4的一般方法(TDM-180963)
Figure PCTCN2021118025-appb-000012
步骤1：化合物4c的制备
在室温下，向化合物4a(7.2g，50mmol)的乙醇溶液中(50mL)添加化合物4b(3.805g，50mmol)。将混合物加热至80℃，搅拌0.5h，减压浓缩，残余物直接用于下一步反应。LCMS[M+1] +＝185.1。
步骤2：化合物4d的制备
在室温下，将N-氯代丁二酰亚胺(26.71g，200mmol)溶于2N盐酸(13.6mL)和乙腈(80mL)的混合溶液中，然后添加化合物4c，将混合物搅拌1小时。减压浓缩混合物，残余物加水并冷却，过滤收集固体，滤饼加水并超声，再一次过滤获得所需白色固体化合物367d(7g，产率67％)，即(4-氟苯基)甲磺酰氯。
步骤3：化合物4(TDM-180963)的制备
在室温下向化合物4e(600mg，2.25mmol)的吡啶(20mL)溶液中添加化合物4d(940mg，4.5mmol)。将混合物加热至80℃并搅拌1.5小时，然后减压浓缩，向残余物加甲醇，过滤收集固体，用硅胶层析(二氯甲烷：含10％甲醇的二氯甲烷＝30：70)纯化得到黄色固体。将固体用二氯甲烷和甲醇打浆，滤饼加入二甲基亚砜，减压浓缩，然后加入水，重复超声过滤(3次)，最后冻干，得到所需黄色固体化合物4，TDM-180963(化合物363，381.8mg，产率38.7％)，即1-(4-氟苯基)-N-(4-(2-(((1-甲基-1H-吡唑-4-基)氨基)嘧啶-4-基)苯基)甲磺酰胺。
1H NMR(400MHz,DMSO-d 6)δ10.18(s,1H),9.45(s,1H),8.45(d,J＝5.1Hz,1H),8.11(d,J＝8.5Hz,2H),7.93(s,1H),7.56(s,1H),7.37–7.27(m,4H),7.26–7.14(m,3H),4.59(s,2H),3.84(s,3H)。LCMS[M+1] +＝439.1。
测试例1 JAK激酶小分子抑制剂的酶活性抑制检测
实验方案
1、试剂准备
激酶反应缓冲液：配置激酶反应缓冲液，组分如下：50mM HEPES，pH 7.5，1mM EGTA，10mM MgCl 2，2mM DTT，0.01％Tween20。1X检测缓冲液：配置检测缓冲液，去离子水9:1稀释10X检测缓冲液至1X。4X激酶溶液：激酶反应缓冲液稀释JAK激酶至4X终浓度(JAK1：80nM，JAK2/JAK3/Tyk2：4nM)。4X底物溶液：激酶反应缓冲液稀释ULight TM-JAK(Tyr1023)底物至200nM(终浓度：50nM)。4XATP溶液：激酶反应缓冲液稀释ATP至4X终浓度(JAK1：160μM，JAK2/JAK3/Tyk2：40μM)。4X测试化合物溶液：DMSO溶解测试用化合物至10mM储存液，3倍梯度稀释配置成所需浓度，每个化合物设置10个浓度点，测试化合物终浓度范围为：10μM-0.5nM。4X酶反应终止液：1X检测缓冲液溶解EDTA至40mM(EDTA终浓度：10mM)。4X检测抗体溶液：1X检测缓冲液稀释Eu标记检测抗体(抗磷酸酪氨酸(PT66))至8nM(抗体终浓度：2nM)。
2、实验过程
向384微孔板中依次加入2.5μL的4X激酶溶液和2.5μL已经稀释好的不同浓度的4X测试化合物溶液，每个浓度设置2个复孔，同时设置酶溶液空白对照组和阴性对照组(DMSO组)。震荡384多孔板，混匀酶和化合物，1000转，离心1分钟，在室温下孵育60分钟。向384多孔板中加入2.5μL，4X底物溶液，1000转离心1分钟。向384多孔板中加入2.5μL，4XATP溶液，1000转离心1分钟，起始酶反应。JAK1室温反应2小时，JAK2/JAK3/Tyk2室温反应1小时。JAK1反应的各组分终浓度分别为：JAK1：20nM，底物：50nM，ATP：40uM，测试化合物终浓度范围为：10μM-0.5nM。JAK2/JAK3/Tyk2反应的各组分终浓度分别为：JAK2：1nM，底物：50nM，ATP：10μM，测试化合物终浓度范围为：10μM-0.5nM。酶反应结束后，向384多孔板每孔中加入5μL，4X酶反应终止液，1000转，离心1分钟，在室温下孵育5分钟。向384多孔板每孔中加入5μL，4X检测抗体溶液，(检测抗体终浓度为2nM)，1000转，离心1分钟，室温条件下孵育1小时。抗体孵育结束后，在Envision读板仪上测定各孔的信号值
3、数据分析
以酶溶液空白对照组为100％抑制率和阴性对照组(DMSO组)为0％抑制率，计算检测化合物各个浓度对应的百分比抑制率。在GraphPad Prism软件中 对检测化合物的浓度对数值和相对应的百分比抑制率进行非线性回归分析，得到检测化合物的半数抑制浓度(IC 50)，所得实验结果列在下表1中。其中，以化合物TDM-180958、TDM-180944、TDM-180945和TDM-180963作为本专利的实验组，且本申请人的在先申请CN110627775A中的TDM-180820、以及现有技术CN103298794A中的化合物213(该化合物的数据直接来自源专利中)作为阳性对照组。
表1
Figure PCTCN2021118025-appb-000013
从上表1的结果可以看出，本申请的化合物TDM-180958、TDM-180944、TDM-180945和TDM-180963的酶活性数据要明显优于化合物TDM-180820和化合物213的酶活性数据，更特别是化合物TDM-180958，其对于Tyk2和JAK1的半数抑制浓度甚至比作为阳性对照的化合物TDM-180820还要更优一个数量级。因此，通过以上实验已经证明了本申请的化合物是一类针对JAK家族靶向性更强、酶活性更优的化合物。
测试例2 JAK激酶小分子抑制剂的细胞活性抑制检测
我们采用两个细胞学测试系统评价JAK激酶小分子抑制剂对细胞因子在细胞内的信号传导抑制：IL-4诱导的THP-1细胞内pSTAT6表达量和IFN-α-B2诱导的U2OS细胞内pSTAT1表达量，分别用于反映JAK1和Tyk2的细胞学活性。
1、细胞培养、细胞因子刺激和pSTAT检测
收集THP-1或U2OS细胞，重悬于1*HBSS。将细胞接种到384孔板，37℃&CO 2孵箱中孵育。每个孔中加入5μL稀释于DMSO的化合物,DMSO的终浓度为0.1％，继续37℃&CO 2孵箱中孵育。每个孔中加入5μL的IL4或IFN-α-B2，继续在37℃&CO 2孵箱中孵育。弃细胞培育上清，加入细胞裂解液，室温孵育。对细胞裂解混悬液用αLISA SureFire Ultra p-STAT6HV试剂盒检测pSTAT6，或用αLISA SureFire Ultra p-STAT1HV试剂盒检测pSTAT1。反应板室温孵育2小时。在Envision读板仪上读取每个培养空的信号。
2、数据分析
由非线性回归方程：Y＝Bottom+(Top-Bottom)/(1+10^((LogIC 50-X)*HillSlope))拟合化合物IC 50，其中X代表化合物浓度；Y代表抑制％；Top和Bottom与Y单位相同；logIC 50与X相同的log单位；HillSlope代表坡度系数或峰坡度。
根据上述数据分析方法，得到化合物TDM-180958和TDM-180963的细胞活性抑制数据，其半数抑制浓度(IC 50)实验结果列在如下表2中。
表2
编号 JAK1细胞/nM Tyk2细胞/nM
TDM-180958 3.01 4.17
TDM-180963 3.04 4.57
测试例3 JAK激酶小分子抑制剂的肝微粒体稳定性和Caco-2细胞模型试验
肝微粒体稳定性试验：利用从特定种属的实验动物的肝脏分离提取到的微粒体在体外针对化合物进行测试，通过检测化合物与含有肝细胞I相代谢酶(主要是P450)的肝微粒体经过不同时长的温孵后原型化合物及其降解产物的浓度来确定化合物的肝微粒体代谢稳定程度，用于初步判定化合物在该种属动物体内的药代动力学趋势。测试的反应体系中肝微体、待测化合物、辅助因子NADPH的中浓度分别为0.75mg/mL、1μM、2mM，并且阳性对照化合物采用了酮色林(Ketanserin)，在不同的孵育时间点(0、15、30和60分钟)取一定体积的反应体系溶液并用预冷的沉淀溶液终止反应，离心后取上清进行LC-MS/MS分析以获得药物溶度等参数。
实验步骤：1)取10μL配制于DMSO的10mM化合物母液和190μL甲醇(MeOH)混匀制备500μM的化合物工作液；2)取1.5μL的500μM化合物工作液，和18.75μL的肝微粒体(20mg/mL)、479.75μL的PBS混合获得1.5μM的化合物工作液(含肝微粒体0.75mg/mL)；3)在37℃孵箱预热反应板5分钟；4)0分时间点检测样品(每个时间点双孔)的制备：向在冰上预冷的反应板内加入30μL的1.5μM的化合物工作液(含肝微粒体0.75mg/mL)、400μL沉淀溶液、15μL的6mM NADPH(配制于PBS)，混合后震动(600rpm，10分钟)和离心(4℃，3000rpm，10分钟)；5)其它时间点检测样品的制备：取180μL的1.5μM的化合物工作液(含肝微粒体0.75mg/mL)和90μL的6mM NADPH混合并在37℃开始温孵，在不同的时间点取45μL反应液和预冷的400μL沉淀溶液混合、震动和离心；6)取200μL上清液离心10分钟(13000g)，样品随即进行LC-MS/MS定量分析。
数据分析：单一时间点样品内剩下的原型药物百分比(％)＝该时间点的样品原型药物浓度/0分钟时的样品内的原型药物浓度*100；其中多个时间点公式如下：Ke＝-2.303*Slope，T 1/2＝0.693/Ke，内在清除率CL int(mL/min/kg)＝(0.693/T 1/2)x(1/反应体系内肝微粒体蛋白终浓度(0.5mg/mL)x换算系数，CL int(μL·min -1·mg -1蛋白质)＝(0.693*1000/T 1/2)/肝微粒体蛋白终浓度(0.5mg/mL)，结果如下表3所示。
表3
Figure PCTCN2021118025-appb-000014
a换算系数＝(每克肝脏内微粒体蛋白)×(每公斤体重肝脏重量)
Caco-2细胞通透性试验：Caco-2是人结肠癌细胞系，利用Millipore跨孔小室(Transwell II)24孔板建立的Caco-2细胞培养模型广泛用于测试药物的通透 性(药物的吸收和外排)，一定程度上能反映药物在肠道的吸收特点。在这个细胞测试模型中，单层Caco-2细胞接种在铺有半透膜的内室(A)底部，内室和容纳内室的外室(B)都加入培养液、并且内外室的液面保持平齐，内室模拟肠腔，外室模拟肠粘膜下毛细血管。细胞经过21-24天培养后会完整地覆盖内室底部表面，其完整性可通过在A或B加入萤光黄(Lucifer yellow，LY)或检测跨表皮电阻(TEER)，Caco-2细胞培养21天后TEER值应达到250Ω·cm 2以上。测试中，药物可以加入内室培养液以检测药物A→B的转运(吸收)，或加入外室培养液以检测B→A的转运(外排)，含有药物的溶液称为供给液，而另一侧的不含药物的测试溶液称接受液。
实验步骤：1)进行细胞测试前需检测细胞层的完整性，然后用Hanks缓冲液清洗；2)吸收试验(A→B)，在内室加入含有药物的溶液，孵育90分钟，供给液和接收液需留取0点和90分钟样品以备分析药物浓度所需(D0,R0,D90,R90)；3)外排试验(B→A)，在外室加入含有药物的溶液，其余同上；4)对样品进行LC-MS/MS分析以检测药物浓度。
药物通透性计算:Papp＝(VA/(面积×时间))×((接收侧终点药物浓度/供给侧药物起始浓度)×稀释参数)
本申请提供了针对本申请的化合物TDM-180958以及作为阳性对照的化合物TDM-180820的肝微粒体稳定性数据和Caco-2细胞模型数据，具体结果参见下表4。
表4
Figure PCTCN2021118025-appb-000015
从表4的结果可以看出，本申请的化合物TDM-180958相比于作为阳性对照的化合物TDM-180820在Caco-2细胞模型中具有明显更高的Papp值(将近6 倍)，也就是说，本申请的化合物TDM-180958相比于化合物TDM-180820具有更好的药物吸收性，推测是由于右侧苯环上1-3位的卤素或氰基基团比单纯的苯环电子数增加，分子的反应性和亲脂性提高，有利于通过细胞膜脂质双分子层进入细胞内。虽然高Papp值不一定代表更长的血浆半衰期或生物利用度，但是药物能被细胞高效地吸收对于局部给药途径如皮肤外用、眼部用药、耳道给药等具有重要意义。
以上详细描述了本发明的优选实施方式，但是，本发明并不限于上述实施方式中的具体细节，在本发明的技术构思范围内，可以对本发明的技术方案进行多种简单变型，这些简单变型均属于本发明的保护范围。
另外需要说明的是，在上述具体实施方式中所描述的各个具体技术特征，在不矛盾的情况下，可以通过任何合适的方式进行组合，为了避免不必要的重复，本发明对各种可能的组合方式不再另行说明。
此外，本发明的各种不同的实施方式之间也可以进行任意组合，只要其不违背本发明的思想，其同样应当视为本发明所公开的内容。

Claims (10)

  1. 一种药物化合物，其特征在于，所述药物化合物为由如下结构式所示的化合物或其立体异构体、几何异构体、互变异构体、消旋体、水合物、溶剂化物、代谢产物以及药学上可接受的盐或前药：
    Figure PCTCN2021118025-appb-100001
    其中，所述R表示苯环上的1至3个取代基，所述1至3个取代基各自独立地选自卤素或氰基。
  2. 根据权利要求1所述的药物化合物，其特征在于，所述卤素为F、Cl或Br。
  3. 根据权利要求1所述的药物化合物，其特征在于，所述R为邻位取代的F。
  4. 根据权利要求1所述的药物化合物，其特征在于，所述R为间位取代的F。
  5. 根据权利要求1所述的药物化合物，其特征在于，所述R为对位取代的F。
  6. 根据权利要求1所述的药物化合物，其特征在于，所述R为间位取代的氰基。
  7. 根据权利要求1-6中任一项所述的药物化合物在抑制JAK激酶中的用途。
  8. 根据权利要求1-6中任一项所述的药物化合物在制备用于预防或治疗自身免疫性疾病、以及与免疫有关的炎症性皮肤疾病的药物中的用途，这些疾病的发病机理均与JAK信号传导的失调相关。
  9. 根据权利要求8所述的用途，其中，所述自身免疫性疾病选自类风湿性关节炎、强直性脊柱炎、溃疡性结肠炎、克罗恩病、系统性红斑狼疮、皮肌炎、 多发性硬化、I型糖尿病、干燥综合症、血管炎、斑秃、银屑病和白癜风中的至少一种。
  10. 根据权利要求8所述的用途，其中，所述与免疫有关的炎症性皮肤疾病选自特应性皮炎、湿疹、扁平苔藓、光泽苔藓、硬化萎缩性苔藓、脂膜炎、痤疮和化脓性汗腺炎中的至少一种。
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CN117136059A (zh) * 2021-04-22 2023-11-28 深圳微芯生物科技股份有限公司 包含jak3/jak1/tbk1选择性抑制剂的药物组合物及其医药用途
CN113603677B (zh) * 2021-08-06 2022-06-14 嘉兴特科罗生物科技有限公司 一种具有高口服生物利用度的jak抑制剂
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