WO2015188681A1 - 一种新型杂环化合物及其制备方法和作为激酶抑制剂的用途 - Google Patents

一种新型杂环化合物及其制备方法和作为激酶抑制剂的用途 Download PDF

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WO2015188681A1
WO2015188681A1 PCT/CN2015/079384 CN2015079384W WO2015188681A1 WO 2015188681 A1 WO2015188681 A1 WO 2015188681A1 CN 2015079384 W CN2015079384 W CN 2015079384W WO 2015188681 A1 WO2015188681 A1 WO 2015188681A1
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compound
kinase
alkane
preparation
pharmaceutically acceptable
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PCT/CN2015/079384
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French (fr)
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江磊
耿美玉
丁健
刘磊
黄敏
查传涛
艾菁
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上海海和药物研究开发有限公司
中国科学院上海药物研究所
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/04—Ortho-condensed systems

Definitions

  • Tyrosine kinase plays a very important role in the occurrence and development of tumors, and an important one is the non-receptor tyrosine kinase JAK.
  • the JAK family has four members, JAK1, JAK2, JAK3, and TYK2. At present, the correlation between JAK family members and tumors is most detailed and clear. Abnormal activation of JAK2 signaling pathway is most closely related to tumorigenesis and development. Early studies focused on the role of JAK2 gene fusion, amplification and mutation in a variety of abnormal activation forms in hematological tumors.
  • JAK2V617F the 617th proline mutation in phenyl kinase on the pseudokinase domain JH2
  • This mutation causes JAK2 to lose its self-inhibiting function, causing downstream Excessive activation of the signal eventually leads to malignant transformation of the cell.
  • Clinical data indicate that the incidence of this mutation in polycythemia vera, thrombocytopenia, and primary myelofibrosis is 81% to 99%, 41% to 72%, and 39% to 57%, respectively (SzpurkaH et al., Refractory anemia).
  • JAK2/STAT myeloproliferative condition characterized by JAK2V617F mutation
  • RARS-T ringedsideroblasts associated with marked thrombocytosis
  • JAK2V617F mutation another myeloproliferative condition characterized by JAK2V617F mutation [J].
  • RARS-T ringedsideroblasts associated with marked thrombocytosis
  • JAK2/STAT pathway may also play an important role in solid tumors, such as the pro-inflammatory cytokine IL-6, and also as an independent risk factor in liver cancer, mediated by STAT activation.
  • the signaling pathway plays a very important role.
  • IL-6 can only function by forming IL-6/IL-6R/gp130 complex with its receptor, and one of the most important downstream of gp130 is JAK2/STAT.
  • JAK small molecule inhibitors At present, there are more than 10 kinds of targeted JAK small molecule inhibitors at different stages of clinical research, most of which are selective inhibitors of JAK1 and JAK2.
  • the indications are mainly blood system related tumors and a small number of solid tumors.
  • the study found that the drug treatment can improve systemic symptoms such as splenomegaly associated with myeloproliferative neoplasms, but does not significantly reduce or eliminate tumor clones in most patients.
  • the drug has the characteristics of rapid absorption and high efficiency in metabolism, resulting in low bioavailability in patients.
  • R is a C1-C6 linear or branched alkane, a halogenated C1-C6 linear alkane or a branched alkane or a halogen;
  • A is H or CH 3 ;
  • X and Y are each independently selected from: N, CH or C-CH 3 ;
  • Z is N, CH or CX, wherein X is a halogen.
  • R is a C1-C4 linear alkane or a C1-C6 branched alkane, a halogenated C1-C4 linear alkane or a halogenated C1-C6 branched alkane, or a halogen.
  • the stereoisomer of the compound is as shown in Formula 1a,
  • R is a C1-C4 linear alkane or a C1-C6 branched alkane, a halogenated C1-C4 linear alkane or a halogenated C1-C6 branched alkane, a halogen;
  • A is H or CH 3 ;
  • X and Y are each independently selected from: N, CH or C-CH 3 ;
  • Z is N, CH or CX, wherein X is a halogen.
  • R, A, X, Y, and Z are as defined above.
  • the chiral separation step is also included:
  • R, A, X, Y, and Z are as defined above.
  • a pharmaceutical composition comprising (a) an active ingredient: a compound according to the first aspect of the invention, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof; (b) a pharmaceutically acceptable carrier.
  • a compound according to the first aspect of the invention or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to the third aspect of the invention
  • a pharmaceutical composition according to the third aspect of the invention for the preparation of kinase inhibitors or for the preparation of anti-tumor drugs.
  • the kinase is a tyrosine kinase.
  • the kinase is JAK2.
  • C1-C6 linear or branched alkane means a straight or branched alkane having from 1 to 6 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl. , isobutyl, tert-butyl, pentyl and the like.
  • halogenated means fluoro, chloro, bromo, iodo.
  • halogen means fluoro, chloro, bromo, iodo.
  • compound of the invention refers to a compound of formula 1.
  • the term also encompasses various stereoisomers, crystalline forms, pharmaceutically acceptable salts, hydrates or solvates of the compounds of Formula 1.
  • the term "pharmaceutically acceptable salt” refers to a salt of the compound of the invention formed with an acid or base suitable for use as a medicament.
  • Pharmaceutically acceptable salts include inorganic and organic salts.
  • a preferred class of salts are the salts of the compounds of the invention with acids.
  • Suitable acids for forming salts include, but are not limited to, mineral acids such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, phosphoric acid, formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, Organic acids such as maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, methanesulfonic acid, benzoic acid, and benzenesulfonic acid; and acidic amino acids such as aspartic acid and glutamic acid.
  • mineral acids such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, phosphoric acid, formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid,
  • Organic acids such as maleic acid, lactic acid, malic acid, tartaric acid,
  • the preparation method of the structural compound of the formula 1 of the present invention is more specifically described below, but these specific methods do not constitute any limitation to the present invention.
  • the compounds of the present invention may also be conveniently prepared by combining various synthetic methods described in the specification or known in the art, and such combinations are readily made by those skilled in the art to which the present invention pertains.
  • a particularly preferred preparation process is as follows:
  • R, A, X, Y, and Z are as defined above.
  • the method can also include the following chiral separation steps:
  • the compound of the present invention has an excellent inhibitory activity against a tyrosine kinase such as JAK2, the compound of the present invention and a stereoisomer thereof, or a pharmaceutically acceptable inorganic or organic salt, etc., and a compound containing the present invention are main active ingredients.
  • the pharmaceutical composition can be used to treat, prevent, and alleviate diseases mediated by tyrosine kinases.
  • the compounds of the invention are useful in the treatment of diseases such as cancer and the like.
  • compositions of the present invention comprise a safe or effective amount of a compound of the present invention, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient or carrier.
  • safe and effective amount it is meant that the amount of the compound is sufficient to significantly improve the condition without causing serious side effects.
  • the pharmaceutical compositions contain from 1 to 2000 mg of the compound of the invention per agent, more preferably from 10 to 100 mg of the compound of the invention per agent.
  • the "one dose” is a capsule or tablet.
  • “Pharmaceutically acceptable carrier” means: one or more compatible solid or liquid fillers or gel materials which are suitable for human use and which must be of sufficient purity and of sufficiently low toxicity. By “compatibility” it is meant herein that the components of the composition are capable of intermingling with the compounds of the invention and with each other without significantly reducing the efficacy of the compound.
  • pharmaceutically acceptable carriers are cellulose and its derivatives (such as sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid).
  • magnesium stearate magnesium stearate
  • calcium sulfate vegetable oil (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyol (such as propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifier (such as ), a wetting agent (such as sodium lauryl sulfate), a coloring agent, a flavoring agent, a stabilizer, an antioxidant, a preservative, a pyrogen-free water, and the like.
  • the mode of administration of the compound or pharmaceutical composition of the present invention is not particularly limited, and representative modes of administration include, but are not limited to, oral, intratumoral, rectal, parenteral (intravenous, intramuscular or subcutaneous), and topical administration. .
  • Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules.
  • the active compound is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or mixed with: (a) a filler or compatibilizer, for example, Starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) binders, for example, hydroxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and acacia; (c) a humectant such as glycerin; (d) a disintegrant such as agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) a slow solvent, For example, paraffin wax; (f) absorption accelerator, for example, quaternary amine compound; (g) wetting agent such as cetyl alcohol and
  • Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other materials known in the art. They may contain opacifying agents and the release of the active compound or compound in such compositions may be released in a portion of the digestive tract in a delayed manner. Examples of embedding components that can be employed are polymeric and waxy materials. If necessary, the active compound may also be in microencapsulated form with one or more of the above-mentioned excipients.
  • Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or elixirs.
  • the liquid dosage form may contain inert diluents conventionally employed in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1 , 3-butanediol, dimethylformamide and oils, especially cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or a mixture of these substances.
  • inert diluents conventionally employed in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1 , 3-butanediol, dimethyl
  • compositions may contain adjuvants such as wetting agents, emulsifying and suspending agents, sweetening agents, flavoring agents and perfumes.
  • the suspension may contain suspending agents, for example, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar or mixtures of these and the like.
  • suspending agents for example, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar or mixtures of these and the like.
  • compositions for parenteral injection may comprise a physiologically acceptable sterile aqueous or nonaqueous solution, dispersion, suspension or emulsion, and a sterile powder for reconstitution into a sterile injectable solution or dispersion.
  • Suitable aqueous and nonaqueous vehicles, diluents, solvents or vehicles include water, ethanol, polyols, and suitable mixtures thereof.
  • Dosage forms for the compounds of the invention for topical administration include ointments, powders, patches, propellants and inhalants.
  • the active ingredient is admixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or, if necessary, propellants.
  • the compounds of the invention may be administered alone or in combination with other pharmaceutically acceptable compounds.
  • a safe and effective amount of a compound of the invention is administered to a mammal (e.g., a human) in need of treatment wherein the dosage is a pharmaceutically effective effective dosage, for a 60 kg body weight
  • the dose to be administered is usually from 1 to 2000 mg, preferably from 10 to 100 mg.
  • specific doses should also consider factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled physician.
  • the compound of the present invention greatly enhances the metabolic stability of the drug, and the exposure amount (AUC) in the animal is increased by 3-5 times, and the half life is also significantly prolonged. We have reached our research expectations to improve its stability.
  • the enzyme activity reaction experiment is based on the principle of fluorescence resonance energy transfer (FRET) with one donor fluorescence at each end.
  • FRET fluorescence resonance energy transfer
  • the enzymatic reaction substrate of the group and a receptor fluorophore can be phosphorylated by the combination of ATP and kinase, and the substrate is protected from substrate enzyme A, thereby realizing fluorescence resonance energy transfer and adding an inhibitor. After that, the phenomenon is reduced.
  • the reagents used in this experiment are as follows:
  • Enzyme reaction substrate initial concentration of 1 mM, the tyrosine residue on the polypeptide can be phosphorylated by the combination of kinase and ATP; purchased from Invitrogen;
  • 100% phosphorylated substrate initial concentration of 1 mM, the tyrosine residue on the polypeptide has been completely phosphorylated; purchased from Invitrogen;
  • Reaction buffer containing 250 mM HEPES (pH 7.5), 50 mM MgCl 2 , 5 mM EGTA, 0.05% Brij-35; purchased from Invitrogen;
  • Substrate enzyme A the enzyme reaction substrate which is not phosphorylated can be digested; purchased from Invitrogen;
  • Stop solution purchased from Invitrogen.
  • the specific steps of the experiment are summarized as follows: the enzyme reaction substrate, 100% phosphorylated substrate, reaction buffer, ATP, substrate enzyme A, and stop solution were separately equilibrated to room temperature.
  • the JAK kinase domain recombinant proteins JAK1, JAK2JH1JH2, JAK2JH1JH2V617F, JAK3 purchased from Invitrogen
  • the JAK kinase domain recombinant proteins JAK1, JAK2JH1JH2, JAK2JH1JH2V617F, JAK3 purchased from Invitrogen
  • a black 384-well microplate (PerkinElmer) was used, and 2.5 ⁇ L of the enzyme reaction substrate diluted with the reaction buffer was added to each well to a final concentration of 2 ⁇ M.
  • the test compound (compound prepared in the examples of the present invention) was diluted to a suitable concentration with 4% DMSO (the initial concentration was generally set to 3 ⁇ M, diluted to a concentration gradient of 1/3 to 9 concentration gradients), and 2.5 ⁇ L was added per well. Add ATP solution diluted in the reaction buffer to each well.
  • the final concentration corresponds to JAK1, JAK2JH1JH2, JAK2JH1JH2V617F, and JAK3, respectively, 75 ⁇ M, 50 ⁇ M, 50 ⁇ M, 10 ⁇ M, and then add 2.5 ⁇ L of the JAK kinase domain recombinant protein diluted in the reaction buffer to start the reaction.
  • a 0% phosphorylation control group, a 100% phosphorylation control group, and a 0% inhibition control group were required.
  • Each reaction group is defined as follows:
  • 0% phosphorylation control group contains enzyme reaction substrate, ATP, but does not add kinase, or contains enzyme reaction substrate, kinase, but does not add ATP, that is, the control well ensures that the enzyme reaction substrate is not phosphorylated, can be Substrate enzyme A is completely digested;
  • 100% phosphorylation control group 100% phosphorylated substrate was added, that is, without the addition of kinase and ATP, the control group ensured that the substrate was completely phosphorylated and could not be digested by substrate enzyme A;
  • 0% inhibition control group containing enzyme reaction substrate, kinase, ATP, inhibitor solvent, but no inhibitor, the control group is used to indicate that the degree of phosphorylation of the substrate is linear in the kinase reaction system, it is recommended Between 20 and 50%;
  • the shaker plate was mixed for 1 minute, and the reaction was allowed to stand at 27 ° C for 1 hour in the dark. After the reaction is completed, all reactions are completed 5 ⁇ l of substrate enzyme A diluted with the reaction buffer was added to the well, and the dilution ratio was 1:1067. After the addition was completed, the vibrating plate was mixed for 1 minute, and the reaction was allowed to stand at 27 ° C for 1 hour in the dark. Finally, 5 ⁇ l of the stop solution was added to all wells to terminate the reaction. After the shaker was mixed, the fluorescence signal was detected by Synergy 2 Microplate Reader (BioTec) (excitation light wavelength was 400 nm, emission wavelength was 445 nm, 520 nm).
  • the inhibition rate of each well was calculated from the fully active pores and the background signal wells.
  • the data analysis method is as follows:
  • Emission ratio signal intensity of the donor fluorophore (at a wavelength of 445 nm) / signal intensity of the acceptor fluorophore (at a wavelength of 520 nm)
  • Percent phosphorylation 100 ⁇ ⁇ 1 - (emission ratio * signal intensity of the acceptor fluorophore of the 100% phosphorylated control group - signal intensity of the donor fluorophore of the 100% phosphorylated control group) / [0% phosphoric acid Signal intensity of the donor fluorophore in the control group - signal intensity + emission ratio of the donor fluorophore in the 100% phosphorylated control group ⁇ (signal intensity of the acceptor fluorophore in the 100% phosphorylated control group - 0 The signal intensity of the acceptor fluorophore in the % phosphorylation control group)] ⁇
  • Inhibition rate 100 ⁇ (1 - % phosphorylation in test compound group / 0% inhibition of phosphorylation in control group))
  • Test method The inhibitory effect of the compound on BaF3 (mouse pre-B cells) and HEL (human erythroleukemia cells) was examined by a CCK-8 cell counting kit (purchased from Dojindo).
  • the specific steps are as follows: BaF3 and HEL cells in logarithmic growth phase are inoculated into 96-well culture plates at a suitable density (10,000/well) at 90 ⁇ L per well. After 12 hours of culture, different concentrations are added (the initial concentration is generally set). 10 ⁇ L of the test compound (the compound prepared in the Example of the present invention) diluted to a concentration gradient of 50 ⁇ in a ratio of 1/3 was subjected to a reaction for 72 hours, and a solvent control group (negative control) was set. After 72 hours of treatment with the compound, the effect of the compound on cell proliferation was detected by CCK-8 cell counting kit.
  • the inhibition rate of the drug on tumor cell growth was calculated according to the following formula:
  • Inhibition rate (%) (OD control well-OD administration well) / OD control well ⁇ 100%.
  • IC 50 Compound Molecular level activity
  • BaF3, IC 50 Cell level activity 14 ⁇ 100nM ⁇ 500nM 15 ⁇ 100nM ⁇ 500nM 16 ⁇ 100nM 500-10000nM 20 100-500nM N/A twenty four ⁇ 100nM 500-10000nM 25 100-500nM 500-10000nM 26 500-10000nM N/A
  • N/A means not determined.
  • Test method 14 SD rats, males, weighing 200-220 g, were randomly divided into 4 groups, 4/3 each, and the positive compounds Ruxolitinib and Compound 15 were administered by gavage and intravenous administration respectively. Rats were fasted for 12 h before the test, free to drink water, and formulated with 5% DMSO / 5% Tween 80 / 90% 0.5% CMC-Na. The dose of intragastric administration was 50 mg/kg, the dose of intravenous administration was 10 mg/kg, and the rats were fed uniformly 2 hours after administration.
  • the intragastric administration group collected blood at 0.25, 0.5, 1.0, 2.0, 4.0, 6.0, 8.0 and 24 h after administration, and the intravenous administration group was 5 min after administration, 0.25, 0.5, 1.0, 2.0, 4.0, 6.0, 8.0 and Blood was collected for 24 hours, 0.3 ml of venous blood was taken from the posterior venous plexus of the rat, and placed in a heparinized test tube, centrifuged at 11,000 rpm for 5 min, and the plasma was separated and frozen in a refrigerator at -20 °C. The concentration of the drug in the plasma sample was determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS).
  • the plasma sample was separated by protein precipitation and separated by C18 column.
  • TSQ Quantum Ultra triple quadrupole mass spectrometer purchased from Thermo
  • the ion source is a heated electrospray ionization (HESI) source, positive ion detection.
  • HESI heated electrospray ionization
  • the pharmacokinetic parameters after administration were calculated using a non-compartmental model of Phoenix 1.3 software (purchased from Pharsight, USA).
  • the experimental results of the intragastric administration group are shown in Table 2 below, and the experimental results of the intravenous administration group are shown in Table 3.

Abstract

提供一种结构如式1所示的化合物及其制备方法，该化合物是一种有效的激酶抑制剂且具有很高的生物利用度。

Description

一种新型杂环化合物及其制备方法和作为激酶抑制剂的用途 技术领域
本发明属于生物医药领域。具体地，本发明涉及一种新型杂环化合物及其制备方法和作为激酶抑制剂的用途。
背景技术
酪氨酸激酶在肿瘤的发生、发展过程中起着非常重要的作用，其中重要的一类是非受体型酪氨酸激酶JAK。JAK家族有JAK1、JAK2、JAK3、TYK2四个成员。目前，JAK家族各成员与肿瘤的相关性，以JAK2的研究最为详实、明确。JAK2信号通路的异常活化与肿瘤的发生、发展的关系最为密切，早期研究主要关注JAK2基因融合、扩增和突变等多种异常活化形式在血液型肿瘤中的作用。目前，研究较多的是JAK2V617F(假激酶结构域JH2上的第617位缬氨酸突变为苯丙氨酸)在骨髓增生性肿瘤中的作用，该突变发生使JAK2失去自抑制功能，引起下游信号过度激活，最终导致细胞恶变。临床数据表明该突变在真性红细胞增多症、血小板增多症、原发性骨髓纤维化中的发生率分别是81％～99％、41％～72％、39％～57％(SzpurkaH等，Refractory anemia with ringedsideroblasts associated with marked thrombocytosis(RARS-T),another myeloproliferative condition characterized by JAK2V617F mutation[J].Blood,2006,108(7):2173-2181)。近年来，越来越多的研究表明JAK2/STAT通路在实体瘤中可能也发挥了重要作用，如前炎症细胞因子IL-6，同时也作为肝癌中独立风险因子，介导的STAT活化这一信号通路具有非常重要的作用，IL-6只有通过和其受体形成IL-6/IL-6R/gp130复合体才能进行信号转导从而发挥作用，而gp130的最主要下游之一就是JAK2/STAT信号转导通路(Y Liu等，Celecoxib Inhibits IL-6/IL-6R-Induced JAK2/STAT3Phosphorylation in Human Hepatocellular Carcinoma Cells.Cancer Prev.Res.August 20114；1296)。因此，靶向JAK2小分子抑制剂的研究是当前抗肿瘤药物研发的热点之一。
目前，处于临床研究不同阶段的靶向JAK小分子抑制剂有10余种，大多为JAK1、JAK2的选择性抑制剂，适应症主要是血液***相关肿瘤及少部分实体瘤。
目前，靶向JAK2的小分子抑制剂中仅一个药物Ruxolitinib被美国FDA批准上市，主要用于治疗骨髓纤维化(Sara C.Meyer and Ross L.Levine Molecular Pathways:Molecular Basis for Sensitivity and Resistance to JAK Kinase Inhibitors,Clin.Cancer Res.2014；20:2051-2059)。
Figure PCTCN2015079384-appb-000001
Ruxolitinib上市后，研究发现该药物治疗可改善骨髓增生性肿瘤相关的脾肿大等全身症状，但并不显著减少或消除在大多数患者体内的肿瘤克隆。然而该药物在代谢方面具有快速吸收、高效清除的特点，导致其在患者体内的生物利用率低。
因此在实际临床操作中必须要采取每日给药两次的方案以保证Ruxolitinib在病人体内的浓度来达到必要的治疗效果。而在实际操作中，病人往往不能按时服药，因而造成病人体内药物浓度波动过大，影响药物治疗效果。
因此，亟需开发一种代谢稳定的药物，以降低病人服药的频率，并为病人提供更好的治疗效果。
发明内容
本发明的目的是提供一种高效、生物利用度高的小分子激酶抑制剂。
在本发明的第一方面中，提供了结构如式1所示的化合物或其立体异构体，或其药学上可接受的盐，
Figure PCTCN2015079384-appb-000002
其中，R为C1-C6的直链烷烃或支链烷烃、卤代的C1-C6的直链烷烃或支链烷烃或卤素；
A为H或CH3；
X和Y各自独立地选自：N、CH或C-CH3；
Z为N、CH或CX，其中，X为卤素。
在另一优选例中，R为C1-C4的直链烷烃或C1-C6的支链烷烃、卤代的C1-C4的直链烷烃或卤代的C1-C6的支链烷烃、卤素。
在另一优选例中，所述化合物的立体异构体如式1a所示，
Figure PCTCN2015079384-appb-000003
其中，R为C1-C4的直链烷烃或C1-C6的支链烷烃、卤代的C1-C4的直链烷烃或卤代的C1-C6的支链烷烃、卤素；
A为H或CH3；
X和Y各自独立地选自：N、CH或C-CH3；
Z为N、CH或CX，其中，X为卤素。
在本发明第二方面中，提供了本发明第一方面所述的化合物、或其立体异构体的制备方法，包括步骤：
(1)在惰性溶剂中，在碱的存在下，将化合物2和化合物3进行加成反应，得到化合物4；
Figure PCTCN2015079384-appb-000004
(2)在惰性溶剂中，在催化剂和碱的存在下，将化合物4和化合物5进行偶联反应，得到化合物6；
Figure PCTCN2015079384-appb-000005
(3)在惰性溶剂中，在碱的存在下，将化合物6进行脱保护反应，得到化合物1；
Figure PCTCN2015079384-appb-000006
上述各式中，R、A、X、Y、Z定义如前。
在另一优选例中，还包括手性分离步骤：
Figure PCTCN2015079384-appb-000007
式中，R、A、X、Y、Z定义如前。
在本发明第三方面中，提供了一种药物组合物，包含(a)活性成分：如本发明第一方面所述的化合物或其立体异构体，或其药学上可接受的盐；以及(b)药学上可接受的载体。
在本发明第四方面中，提供了本发明第一方面所述的化合物、或其立体异构体，或其药学上可接受的盐或如本发明第三方面所述的药物组合物的用途，用于制备激酶抑制剂或用于制备抗肿瘤药物。
在另一优选例中，所述激酶为酪氨酸激酶。
在另一优选例中，所述激酶为JAK2。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
具体实施方式
发明人经过广泛而深入的研究，意外发现了一类结构新颖的小分子激酶抑制剂。且与现有化合物相比，本发明的化合物具有更高的生物利用度。在此基础上，发明人完成了 本发明。
术语
术语“C1-C6的直链烷烃或支链烷烃”是指具有1-6个碳原子的直链或带有支链的烷烃，例如甲基、乙基、丙基、异丙基、丁基、异丁基、叔丁基、戊基等。
术语“卤代的”是指氟代的、氯代的、溴代的、碘代的。
术语“卤素”是指氟、氯、溴、碘。
活性成分
如本文所用，术语“本发明化合物”指式1所示的化合物。该术语还包括及式1化合物的各种立体异构体、晶型形式、药学上可接受的盐、水合物或溶剂合物。
如本文所用，术语“药学上可接受的盐”指本发明化合物与酸或碱所形成的适合用作药物的盐。药学上可接受的盐包括无机盐和有机盐。一类优选的盐是本发明化合物与酸形成的盐。适合形成盐的酸包括但并不限于：盐酸、氢溴酸、氢氟酸、硫酸、硝酸、磷酸等无机酸，甲酸、乙酸、丙酸、草酸、丙二酸、琥珀酸、富马酸、马来酸、乳酸、苹果酸、酒石酸、柠檬酸、苦味酸、甲磺酸、苯甲磺酸，苯磺酸等有机酸；以及天冬氨酸、谷氨酸等酸性氨基酸。
制备方法
下面更具体地描述本发明式1结构化合物的制备方法，但这些具体方法不对本发明构成任何限制。本发明化合物还可以任选将在本说明书中描述的或本领域已知的各种合成方法组合起来而方便的制得，这样的组合可由本发明所属领域的技术人员容易的进行。
一种特别优选的制备流程如下：
Figure PCTCN2015079384-appb-000008
(1)在一定温度(如70-100℃)下，在惰性溶剂(如乙腈、DMF、DMSO、THF等)中，在碱(如DBU、三乙胺、二异丙基乙胺、吡啶、K2CO3、Na2CO3、NaOH、KOH等)的存在下，将化合物2和化合物3进行Michael加成反应一段时间(如4-20小时)，得到化合物4；
(2)在一定温度(如70-100℃)下，在惰性溶剂(如二氧六环和水、甲苯和水、DMSO、THF、DMF等)中，在催化剂(如四(三苯基膦)钯、三(二亚苄基丙酮)二钯(Pd2(dba)3)、双(二亚苄基丙酮)钯、二氯二(三苯基膦)钯、三苯基膦醋酸钯、双(三邻苯甲基膦)二氯化钯、1，2-二(二苯基膦基)乙烷二氯化钯等)和碱(如碳酸钾、氟化钾、氟化铯、氟化钠、磷酸钾、水合磷酸钾、碳酸钠、碳酸氢钠、1,8-二氮杂二环[5.4.0]十一碳-7-烯、三乙胺、二异丙基乙胺、吡啶或其组合等)的存在下，将化合物4和化合物5进行Suzuki偶联反应一段时间(如1-4小时)，得到化合物6；
(3)在一定温度(如70-100℃)下，在惰性溶剂(如甲醇、乙醇、异丙醇、正丁醇、叔丁醇、异丁醇、苄醇等)中，在碱(如碳酸钾、碳酸钠、氢氧化钠、氢氧化钾等)的存在下，将化合物6进行脱保护反应一段时间(如0.1-4小时)，得到化合物1；
上述各式中，R、A、X、Y、Z定义如前。
该方法还可包括如下手性分离步骤：
Figure PCTCN2015079384-appb-000009
药物组合物和施用方法
由于本发明化合物具有优异的对酪氨酸激酶例如JAK2的抑制活性，因此本发明化合物及其立体异构体，或药学上可接受的无机或有机盐等，以及含有本发明化合物为主要活性成分的药物组合物可用于治疗、预防以及缓解由对酪氨酸激酶介导的疾病。根据现有技术，本发明化合物可用于治疗以下疾病：癌症等。
本发明的药物组合物包含安全有效量范围内的本发明化合物或其药理上可接受的盐及药理上可以接受的赋形剂或载体。其中“安全有效量”指的是：化合物的量足以明显改善病情，而不至于产生严重的副作用。通常，药物组合物含有1-2000mg本发明化合物/剂，更佳地，含有10-100mg本发明化合物/剂。较佳地，所述的“一剂”为一个胶囊或药片。
“药学上可以接受的载体”指的是：一种或多种相容性固体或液体填料或凝胶物质，它们适合于人使用，而且必须有足够的纯度和足够低的毒性。“相容性”在此指的是组合物中各组份能和本发明的化合物以及它们之间相互掺和，而不明显降低化合物的药效。药学上可以接受的载体部分例子有纤维素及其衍生物(如羧甲基纤维素钠、乙基纤维素钠、纤维素乙酸酯等)、明胶、滑石、固体润滑剂(如硬脂酸、硬脂酸镁)、硫酸钙、植物油(如豆油、芝麻油、花生油、橄榄油等)、多元醇(如丙二醇、甘油、甘露醇、山梨醇等)、乳化剂(如
Figure PCTCN2015079384-appb-000010
)、润湿剂(如十二烷基硫酸钠)、着色剂、调味剂、稳定剂、抗氧化剂、防腐剂、无热原水等。
本发明化合物或药物组合物的施用方式没有特别限制，代表性的施用方式包括(但并不限于)：口服、瘤内、直肠、肠胃外(静脉内、肌肉内或皮下)、和局部给药。
用于口服给药的固体剂型包括胶囊剂、片剂、丸剂、散剂和颗粒剂。在这些固体剂型中，活性化合物与至少一种常规惰性赋形剂(或载体)混合，如柠檬酸钠或磷酸二钙，或与下述成分混合：(a)填料或增容剂，例如，淀粉、乳糖、蔗糖、葡萄糖、甘露醇和硅酸；(b)粘合剂，例如，羟甲基纤维素、藻酸盐、明胶、聚乙烯基吡咯烷酮、蔗糖和***胶； (c)保湿剂，例如，甘油；(d)崩解剂，例如，琼脂、碳酸钙、马铃薯淀粉或木薯淀粉、藻酸、某些复合硅酸盐、和碳酸钠；(e)缓溶剂，例如石蜡；(f)吸收加速剂，例如，季胺化合物；(g)润湿剂，例如鲸蜡醇和单硬脂酸甘油酯；(h)吸附剂，例如，高岭土；和(i)润滑剂，例如，滑石、硬脂酸钙、硬脂酸镁、固体聚乙二醇、十二烷基硫酸钠，或其混合物。胶囊剂、片剂和丸剂中，剂型也可包含缓冲剂。
固体剂型如片剂、糖丸、胶囊剂、丸剂和颗粒剂可采用包衣和壳材制备，如肠衣和其它本领域公知的材料。它们可包含不透明剂，并且，这种组合物中活性化合物或化合物的释放可以延迟的方式在消化道内的某一部分中释放。可采用的包埋组分的实例是聚合物质和蜡类物质。必要时，活性化合物也可与上述赋形剂中的一种或多种形成微胶囊形式。
用于口服给药的液体剂型包括药学上可接受的乳液、溶液、悬浮液、糖浆或酊剂。除了活性化合物外，液体剂型可包含本领域中常规采用的惰性稀释剂，如水或其它溶剂，增溶剂和乳化剂，例知，乙醇、异丙醇、碳酸乙酯、乙酸乙酯、丙二醇、1,3-丁二醇、二甲基甲酰胺以及油，特别是棉籽油、花生油、玉米胚油、橄榄油、蓖麻油和芝麻油或这些物质的混合物等。
除了这些惰性稀释剂外，组合物也可包含助剂，如润湿剂、乳化剂和悬浮剂、甜味剂、矫味剂和香料。
除了活性化合物外，悬浮液可包含悬浮剂，例如，乙氧基化异十八烷醇、聚氧乙烯山梨醇和脱水山梨醇酯、微晶纤维素、甲醇铝和琼脂或这些物质的混合物等。
用于肠胃外注射的组合物可包含生理上可接受的无菌含水或无水溶液、分散液、悬浮液或乳液，和用于重新溶解成无菌的可注射溶液或分散液的无菌粉末。适宜的含水和非水载体、稀释剂、溶剂或赋形剂包括水、乙醇、多元醇及其适宜的混合物。
用于局部给药的本发明化合物的剂型包括软膏剂、散剂、贴剂、喷射剂和吸入剂。活性成分在无菌条件下与生理上可接受的载体及任何防腐剂、缓冲剂，或必要时可能需要的推进剂一起混合。
本发明化合物可以单独给药，或者与其他药学上可接受的化合物联合给药。
使用药物组合物时，是将安全有效量的本发明化合物适用于需要治疗的哺乳动物(如人)，其中施用时剂量为药学上认为的有效给药剂量，对于60kg体重的人而言，日给药剂量通常为1～2000mg，优选10～100mg。当然，具体剂量还应考虑给药途径、病人健康状况等因素，这些都是熟练医师技能范围之内的。
本发明主要具有以下优点：
和现有化合物(Ruxolitinib)相比，本发明的化合物大大的提高了药物的代谢稳定性，其在动物体内的暴露量(AUC)提高了3-5倍，半衰期也有明显延长。达到了我们提高其稳定性的研究预期。
下面结合具体实施，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。
实施例1
Figure PCTCN2015079384-appb-000011
将化合物2(250mg，2.1mmol)和化合物10(400mg,2.1mmol)置于50mL单口瓶中，氮气保护下，室温下滴加DBU(1g，4.2mmol)，搅拌均匀。然后加热到80℃，反应8h。反应完毕后，旋干溶剂，柱层析(乙酸乙酯：石油醚＝5：1)得白色固体250mg,产率36％。
1H NMR(CDCl3，400MHz)δppm 7.67(s,1H),4.04(td,1H,J＝10.2Hz,4.0Hz),3.03(dd,1H,J＝8.4Hz,17.2Hz),2.84(dd,J＝4Hz,17.2Hz),2.49(m,1H),2.38(s,3H),1.89(m,1H),1.45-1.70(m,4H),1.20-1.32(m,15H).
Figure PCTCN2015079384-appb-000012
将化合物11(160mg,0.5mmol),化合物12(153mg,0.5mmol)，四三苯基膦钯(29mg，0.025mmol)和碳酸钾(207mg,1.5mmol)，置于50mL单口反应瓶中，氮气保护下，室温下加入1,4-二氧六环(4mL)和H2O(1mL)，搅拌至均匀分散于体系中，然后升温至80℃，在氮气保护下反应2h。反应完毕后，加乙酸乙酯30mL和H2O(10mL)，萃取分液。有机相用硫酸钠干燥，旋干，柱层析(石油醚：乙酸乙酯＝3：1-1：1)得白色固 体132mg,产率55％。
1H NMR(CDCl3，400MHz)δppm 9.00(s,1H),8.10(d,2H,J＝7.2Hz),8.08(s,1H)7.78(d,1H,J＝4.0Hz),7.32(d,2H,7.2Hz),4.14(td,1H,J＝0.2Hz,4.0Hz),3.10(dd,1H,J＝8.4Hz,17.2Hz),2.90(dd,J＝8.4Hz,17.2Hz),2.60(m,1H),2.59(s,3H),2.40(s,3H),1.90(m,1H),1.45-1.76(m,4H),1.20-1.32(m,3H).
Figure PCTCN2015079384-appb-000013
将化合物13(132mg，0.28mmol)和碳酸钾(77mg,0.56mmol)置于50mL的单口瓶中，加入甲醇6mL和水1.5mL，搅拌均匀，加热至60℃反应1小时。反应完毕后，反应完毕后，加乙酸乙酯20mL和H2O(10mL)，萃取分液。有机相用硫酸钠干燥，旋干，柱层析(石油醚：乙酸乙酯＝1：1)得白色固体72mg,产率81％。
1HNMR(CDCl3,400mHz),δppm 0.92(br,1H),8.90(s,1H),8.17(m,1H),7.39(m,1H),6.69(m,1H),4.20(td,1H,J＝10.2Hz,4.0Hz),3.13(dd,1H,J＝8.4Hz,17.2Hz),2.93(dd,J＝8.4Hz,17.2Hz),2.63(m,1H),2.62(s,3H),1.90(m,1H),1.45-1.76(m,4H),1.20-1.32(m,3H).
Figure PCTCN2015079384-appb-000014
将500mg化合物14溶解于流动相，分离条件:仪器：Gi lson 281，柱子:CHIRALPAK IC 30*250mm,5um(Daicel)，流动相:正己烷(0.1％二乙胺):乙醇(0.1％二乙胺)＝70:30，波长:214nm&254nm，流速:1.0ml/min温度:40℃。一个循环时间为：19分钟。分别收集化合物15(174mg)和化合物16(210mg)。
实施例2：
Figure PCTCN2015079384-appb-000015
将化合物17(190mg，0.7mmol)和化合物2(169mg,1.4mmol)置于50mL单口瓶中，氮气保护下，室温下滴加DBU(425mg，2.8mmol)，搅拌均匀。然后加热到80℃，反应8h。反应完毕后，旋干溶剂，柱层析(乙酸乙酯：石油醚＝5：1)得无色油状物60mg,产率17％。
1H NMR(CDCl3，400MHz)δppm 7.56(s,1H),4.21(td,1H,J＝10.2Hz,4.0Hz),3.05(m,1H),2.85(m,1H),2.53(m,1H),1.93(m,1H),1.45-1.80(m,4H),1.20-1.32(m,15H).
Figure PCTCN2015079384-appb-000016
将化合物18(60mg,0.16mmol)，化合物12(48mg，0.16mmol)，四三苯基膦钯(9mg，0.008mmol)和碳酸钾(207mg,1.5mmol)，置于50mL单口反应瓶中，氮气保护下，室温下加入1,4-二氧六环(4mL)和H2O(1mL)，搅拌至均匀分散于体系中，然后升温至80℃，在氮气保护下反应2h。反应完毕后，加乙酸乙酯30mL和H2O(10mL)，萃取分液。有机相用硫酸钠干燥，旋干，柱层析(石油醚：乙酸乙酯＝3：1-1：1)得白色固体24mg,产率30％。
1H NMR(CDCl3，400MHz)δppm 9.04(s,1H),8.12(d,2H,J＝7.2Hz),8.06(s，1H)7.81(d,1H,J＝4.0Hz),7.36(d,2H,j＝7.2Hz),4.30(m,1H,),3.10(m,1H),2.94(m,J1H),2.64(m,1H),2.40(s,3H),1.99(m,1H),1.45-1.77(m,4H),1.20-1.32(m,3H).
Figure PCTCN2015079384-appb-000017
将化合物19(24mg，0.045mmol)和碳酸钾(12mg，0.09mmol)置于50mL的单口瓶中，加入甲醇3mL和水0.75mL，搅拌均匀，加热至80℃反应1小时。反应完毕后，反应完毕后，加乙酸乙酯20mL和H2O(10mL)，萃取分液。有机相用硫酸钠干燥，旋干，柱层析(石油醚：乙酸乙酯＝1：1)得白色固体12mg，产率70％。
1HNMR(CDCl3,400MHz)δppm 10.40(br,1H),8.99(s,1H),8.17(m,1H),7.45(m,1H),6.68(m,1H),4.34(m,1H),3.13(dd,1H,J＝8.0Hz,16.8Hz),2.93(dd,J＝8.0Hz,16.8Hz),2.66(m,1H),2.0(m,1H),1.45-1.76(m,4H),1.20-1.32(m,3H).
以下化合物可按照上述实例用类似的方法合成
Figure PCTCN2015079384-appb-000018
实施例3：化合物在分子水平对JAK酶活性的影响
本酶活反应实验基于荧光共振能量转移(FRET)原理，两端分别带有一个供体荧光 基团和一个受体荧光基团的酶反应底物，在ATP和激酶的共同作用下可以被磷酸化修饰，保护底物不被底物酶A切割，从而实现荧光共振能量转移，加入抑制剂后，则该现象减少。该实验用到的试剂如下：
酶反应底物：初始浓度为1mM，该多肽上的酪氨酸残基在激酶、ATP共同作用下可被磷酸化；购自Invitrogen；
100％磷酸化底物：初始浓度为1mM，该多肽上的酪氨酸残基已被完全磷酸化修饰；购自Invitrogen；
ATP：初始浓度为10mM,购自Invitrogen；
反应缓冲液：含250mM的HEPES(PH为7.5)、50mM的MgCl2，5mM的EGTA,0.05％的Brij-35；购自Invitrogen；
底物酶A：可对未被磷酸化的酶反应底物进行酶切；购自Invitrogen；
终止液：购自Invitrogen。
实验具体步骤概述如下：分别将酶反应底物、100％磷酸化底物、反应缓冲液、ATP、底物酶A、终止液平衡至室温。选择JAK激酶域重组蛋白JAK1、JAK2JH1JH2、JAK2JH1JH2V617F、JAK3(购自Invitrogen)，用反应缓冲液稀释使终浓度分别为0.1ng/L、1ng/L、0.2ng/L、0.08ng/L，置于冰上准备加样。
选用黑色384孔微量加样板(PerkinElmer)，每孔加入用反应缓冲液稀释的酶反应底物2.5μL，终浓度为2μM。待测化合物(本发明实施例制备的化合物)用4％DMSO稀释成合适的浓度(起始浓度一般设为3μM,按1/3的比例稀释成9个浓度梯度)，每孔加入2.5μL。每孔加入反应缓冲液稀释的ATP溶液，终浓度对应JAK1、JAK2JH1JH2、JAK2JH1JH2V617F、JAK3分别为75μM、50μM、50μM、10μM，再加入反应缓冲液稀释的JAK激酶域重组蛋白2.5μL启动反应，每次需设置0％磷酸化对照组、100％磷酸化对照组、0％抑制对照组。各反应组定义如下：
0％磷酸化对照组：含有酶反应底物、ATP，但是不加激酶，或者含有酶反应底物、激酶，但是不加ATP，即该对照孔保证酶反应底物不被磷酸化，可被底物酶A完全酶切；
100％磷酸化对照组：加入100％磷酸化底物，即在不加激酶和ATP的情况下，该对照组保证底物完全被磷酸化，不能被底物酶A酶切；
0％抑制对照组：含有酶反应底物、激酶、ATP、抑制剂溶剂，但是不含抑制剂，该对照组用于指示在该激酶反应体系中，底物的磷酸化程度处于线性范围，建议在20～50％之间；
加样完成后振板混匀1分钟，置27℃避光反应1小时。反应完成后，向所有反应 孔中加入5μl经反应缓冲液稀释的底物酶A，稀释比为1:1067，加样完成后振板混匀1分钟，置27℃避光反应1小时。最后向所有反应孔中加入5μl终止液终止反应，振板混匀后用Synergy 2 Microplate Reader(BioTec)检测荧光信号(激发光波长为400nm，发射光波长为445nm、520nm)。
通过全活性孔和背景信号孔计算出每个孔的抑制率，数据分析方法如下：
发射比率＝供体荧光基团的信号强度(在波长为445nm处)/受体荧光基团的信号强度(在波长为520nm处)
磷酸化百分比＝100×{1-(发射比率*100％磷酸化对照组的受体荧光基团的信号强度-100％磷酸化对照组的供体荧光基团的信号强度)/[0％磷酸化对照组的供体荧光基团的信号强度-100％磷酸化对照组的供体荧光基团的信号强度+发射比率×(100％磷酸化对照组的受体荧光基团的信号强度-0％磷酸化对照组的受体荧光基团的信号强度)]}
抑制率＝100×(1-(测试化合物组的磷酸化百分比/0％抑制对照组的磷酸化百分比))
同时用软件Graph Prism 6对待测化合物进行半数抑制活性(IC50)的拟合，结果见表1。实验重复三次。
实施例4：化合物对细胞增殖的影响
试验方法：化合物对BaF3(小鼠前B细胞)、HEL(人红白血病细胞)的增殖抑制作用以CCK-8细胞计数试剂盒(购自Dojindo)检测。
具体步骤如下：处于对数生长期的BaF3、HEL细胞按合适密度(1万个/孔)接种至96孔培养板中，每孔90μL，培养12小时后，加入不同浓度(起始浓度一般设为50μΜ，按1/3的比例稀释成9个浓度梯度)的待测化合物(本发明实施例制备的化合物)10μL作用72小时，并设定溶剂对照组(阴性对照)。待化合物作用细胞72小时后，化合物对细胞增殖的影响采用CCK-8细胞计数试剂盒检测，每孔加入10μL CCK-8试剂，置于37℃培养箱中放置2-4小时后，用全波长式微孔板酶标仪SpectraMax 190(购自美国Molecular Devices公司)读数，测定波长为450nm。
按以下列公式计算药物对肿瘤细胞生长的抑制率：
抑制率(％)＝(OD对照孔-OD给药孔)/OD对照孔×100％。
同时用软件Graph Prism 6对待测化合物进行半数抑制活性(IC50)的拟合。实验重复三次，结果见表1。可见，本发明的部分化合物已经达到了纳米级。
表1：待测化合物的活性数据
化合物 分子水平活性(IC50) 细胞水平活性(BaF3,IC50)
14 <100nM <500nM
15 <100nM <500nM
16 <100nM 500-10000nM
20 100-500nM N/A
24 <100nM 500-10000nM
25 100-500nM 500-10000nM
26 500-10000nM N/A
注：N/A表示未测定。
实施例5：化合物在大鼠体内的代谢特性研究
试验方法：选择SD大鼠14只，雄性，体重200-220g，随机分成4组，每组4/3只，分别灌胃和静脉给予阳性化合物Ruxolitinib及化合物15。大鼠在试验前禁食12h，自由饮水，用5％DMSO/5％吐温80/90％的0.5％CMC-Na配制药物。灌胃给药剂量为50mg/kg，静脉给药剂量为10mg/kg，大鼠给药后2h统一进食。灌胃给药组在给药后0.25，0.5，1.0，2.0，4.0，6.0，8.0和24h采血，静脉给药组在给药后5min，0.25，0.5，1.0，2.0，4.0，6.0，8.0和24h采血，经大鼠眼球后静脉丛取静脉血0.3ml，置肝素化试管中，11000rpm离心5min，分离血浆，于-20℃冰箱中冷冻。采用液相色谱串联质谱(LC-MS/MS)法测定血浆样品中药物浓度，血浆样品经蛋白沉淀处理后，经C18柱分离，TSQ Quantum Ultra型三重四极杆串联质谱仪(购自Thermo)检测，离子源为加热电喷雾电离(HESI)源，正离子方式检测。
采用Phoenix 1.3软件(购自美国Pharsight公司)的非房室模型计算给药后的药代动力学参数。灌胃给药组的实验结果见下表2，静脉注射给药组的实验结果见表3。
表2：大鼠灌胃给予50mg/kg不同化合物后的药动学参数
Figure PCTCN2015079384-appb-000019
表3：大鼠静脉给予10mg/kg不同化合物后的药动学参数
Figure PCTCN2015079384-appb-000020
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (8)

  1. 结构如式1所示的化合物或其立体异构体，或其药学上可接受的盐，
    Figure PCTCN2015079384-appb-100001
    其中，R为C1-C6的直链烷烃或支链烷烃、卤代的C1-C6的直链烷烃或支链烷烃或卤素；A为H或CH3；
    X和Y各自独立地选自：N、CH或C-CH3；
    Z为N、CH或CX，其中，X为卤素。
  2. 如权利要求1所述的化合物，其特征在于，所述化合物的立体异构体如式1a所示，
    Figure PCTCN2015079384-appb-100002
    其中，R为C1-C4的直链烷烃或C1-C6的支链烷烃、卤代的C1-C4的直链烷烃或卤代的C1-C6的支链烷烃、卤素；
    A为H或CH3；
    X和Y各自独立地选自：N、CH或C-CH3；
    Z为N、CH或CX，其中，X为卤素。
  3. 如权利要求1所述的化合物、或其立体异构体的制备方法，其特征在于，包括步骤：
    (1)在惰性溶剂中，在碱的存在下，将化合物2和化合物3进行加成反应，得到化合物4；
    Figure PCTCN2015079384-appb-100003
    (2)在惰性溶剂中，在催化剂和碱的存在下，将化合物4和化合物5进行偶联反应，得到化合物6；
    Figure PCTCN2015079384-appb-100004
    (3)在惰性溶剂中，在碱的存在下，将化合物6进行脱保护反应，得到化合物1；
    Figure PCTCN2015079384-appb-100005
    上述各式中，R、A、X、Y、Z定义如权利要求1。
  4. 如权利要求3所述的制备方法，其特征在于，还包括手性分离步骤：
    Figure PCTCN2015079384-appb-100006
    式中，R、A、X、Y、Z定义如权利要求1。
  5. 一种药物组合物，其特征在于，包含(a)活性成分：如权利要求1所述的化合物或其立体异构体，或其药学上可接受的盐；以及(b)药学上可接受的载体。
  6. 如权利要求1所述化合物或其立体异构体，或其药学上可接受的盐或如权利要求5所述的药物组合物的用途，其特征在于，用于制备激酶抑制剂或用于制备抗肿瘤药物。
  7. 如权利要求6所述的用途，其特征在于，所述激酶为酪氨酸激酶。
  8. 如权利要求7所述的用途，其特征在于，所述激酶为JAK2。
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