WO2019091440A1 - 作为btk抑制剂的5-氨基吡唑甲酰胺化合物的晶型i - Google Patents

作为btk抑制剂的5-氨基吡唑甲酰胺化合物的晶型i Download PDF

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WO2019091440A1
WO2019091440A1 PCT/CN2018/114666 CN2018114666W WO2019091440A1 WO 2019091440 A1 WO2019091440 A1 WO 2019091440A1 CN 2018114666 W CN2018114666 W CN 2018114666W WO 2019091440 A1 WO2019091440 A1 WO 2019091440A1
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compound
formula
reaction
btk
solvent
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PCT/CN2018/114666
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English (en)
French (fr)
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吴予川
陈曦
黄少强
胡永韩
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苏州信诺维医药科技有限公司
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Priority to EP18876641.4A priority Critical patent/EP3708165B1/en
Priority to CN201880067323.9A priority patent/CN111225669B/zh
Priority to US16/762,830 priority patent/US11180475B2/en
Priority to AU2018364183A priority patent/AU2018364183B2/en
Publication of WO2019091440A1 publication Critical patent/WO2019091440A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/444—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a six-membered ring with nitrogen as a ring heteroatom, e.g. amrinone
    • 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
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B2200/00—Indexing scheme relating to specific properties of organic compounds
    • C07B2200/13—Crystalline forms, e.g. polymorphs

Definitions

  • the invention belongs to the technical field of medicinal chemistry, in particular to a novel high-efficiency, selective and good pharmacokinetic property of a 5-aminopyrazolecarboxamide compound as a BTK inhibitor and a crystal form thereof. And a method of preparing the compound and a crystalline form thereof, and a pharmaceutical composition comprising the compound or a crystalline form thereof.
  • Protein kinases are the largest family of biological enzymes in the human body, including over 500 proteins.
  • the phenolic function on the tyrosine residue can be phosphorylated to exert important biosignaling effects.
  • the tyrosine kinase family has members that control cell growth, migration, and differentiation. Abnormal kinase activity has been elucidated in close association with many human diseases, including cancer, autoimmune diseases, and inflammatory diseases.
  • Bruton's tyrosine kinase is a cytoplasmic non-receptor tyrosine kinase belonging to the TEC kinase family (a total of five members BTK, TEC, ITK, TXK, BMX).
  • the BTK gene is located on Xq21.33-Xq22 of the X-chromosome and shares 19 exons spanning 37.5 kb of genomic DNA.
  • BTK expression plays an essential role in almost all hematopoietic cells, especially in the development, differentiation, signaling and survival of B lymphocytes.
  • B cells are activated by the B cell receptor (BCR), and BTK plays a decisive role in the BCR signaling pathway.
  • BCR B cell receptor
  • Activation of BCR on B cells causes activation of BTK, which in turn leads to an increase in downstream phospholipase C (PLC) concentration and activates the IP3 and DAG signaling pathways. This signaling pathway promotes cell proliferation, adhesion and survival.
  • PLC phospholipase C
  • This signaling pathway promotes cell proliferation, adhesion and survival.
  • Mutations in the BTK gene result in a rare hereditary B cell-specific immunodeficiency disease known as X-Iinked agammaglobulinemia (XLA).
  • XLA X-Iinked agammaglobulinemia
  • BTK In this disease, the function of BTK is inhibited, resulting in the production or maturation of B cells. Men with XLA disease have almost no B cells in their bodies, and there are few circulating antibodies, which are prone to serious or even fatal infections. This strongly proves that BTK plays an extremely important role in the growth and differentiation of B cells.
  • BTK inhibitors bind to BTK, inhibit BTK autophosphorylation, and prevent BTK activation. This can block the signaling of the BCR pathway, inhibit the proliferation of B lymphoma cells, and destroy the adhesion of tumor cells, thereby promoting apoptosis of tumor cells. And induce apoptosis.
  • B-cell lymphomas and leukemias such as non-Hodgkin's lymphoma (NHL), chronic lymphocytic leukemia (CLL), and Recurrent or refractory mantle cell lymphoma (MCL) and the like.
  • BTK inhibitors In addition to fighting against B-cell lymphoma and leukemia, BTK inhibitors also inhibit B cell autoantibodies and cytokine production.
  • B cells present autoantigens, promote the activation and secretion of T cells, cause inflammatory factors, cause tissue damage, and activate B cells to produce a large number of antibodies, triggering autoimmune responses.
  • the interaction of T and B cells forms a feedback regulatory chain, leading to uncontrolled autoimmune response and aggravation of histopathological damage. Therefore, BTK can be used as a drug target for autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus (SLE), and allergic diseases (such as diseases such as esophagitis).
  • BTK inhibitors have been reported to be useful in combination with chemotherapeutic agents or immunological checkpoint inhibitors, and have shown superior therapeutic effects in a variety of solid tumors in clinical trials.
  • Ibrutinib is an irreversible BTK inhibitor developed by Pharmacyclics and Johnson & Johnson, and was approved by the FDA in November 2013 and February 2014 for the treatment of mantle cell lymphocytes.
  • Ibrutinib has been designated by the FDA as a "breakthrough" new drug that works by reacting with the thiol group of cysteine in BTK and forming a covalent bond that inactivates the BTK enzyme.
  • ibrutinib is easily metabolized during metabolism (digested by metabolic enzymes to be dihydroxylated or inactivated by other thiol-containing enzymes, cysteine, glutathione, etc.) Affect the efficacy.
  • the clinically administered dose reached 560 mg per day, which increased the burden on the patient.
  • Ibrutinib also has a certain inhibitory effect on some kinases other than BTK, especially the inhibition of EGFR can lead to more serious rash, diarrhea and other adverse reactions. Therefore, there is still a need in the art to develop a new class of BTK inhibitors that are more efficient, selective, and have good pharmacokinetic properties for the treatment of related diseases.
  • the present inventors have developed a novel 5-aminopyrazolecarboxamide compound which is an effective, safe and selective inhibitor of protein kinase BTK and is a novel covalent bond inhibitor by changing its Cysteine response rate to improve affinity with the target to improve efficacy, selectivity and safety. Its structure is as shown in formula (I):
  • a first object of the present invention is to provide a crystalline form I of the above compound of the formula (I).
  • a second object of the present invention is to provide a process for the preparation of the above Form I of the compound of the formula (I).
  • a third object of the present invention is to provide a pharmaceutical composition comprising the above crystalline form I of the compound of the formula (I).
  • a fourth object of the present invention is to provide the use of the above crystalline form I of the compound of the formula (I).
  • the present invention provides a crystalline form I of a 5-aminopyrazolecarboxamide compound of formula (I), which is At the wavelength, the X-ray powder diffraction pattern measured using Cu-K radiation contains the following characteristic peaks (in terms of 2 ⁇ angle) ( ⁇ 0.2°): 5.8, 9.3, 10.9, 16.4, 18.4, 20.3, 20.9, 21.8, 22.4. 23.0, 24.4, 27.1, 30.0, 31.2, 31.9.
  • the invention provides Form I of a 5-aminopyrazolecarboxamide compound of Formula (I) having an X-ray powder diffraction pattern substantially as shown in Figure 1.
  • the present invention provides a process for the preparation of Form I of a 5-aminopyrazolecarboxamide compound of formula (I), which comprises 5-aminopyrazole as shown in formula (I)
  • the amide compound is dissolved in a good solvent, and then a poor solvent is added to the resulting solution under stirring to form a suspension.
  • the suspension is continuously stirred, and then centrifuged, and the obtained solid is vacuum-dried to obtain 5 as shown in the formula (I).
  • Form I of the aminopyrazolecarboxamide compound is dissolved in a good solvent, and then a poor solvent is added to the resulting solution under stirring to form a suspension.
  • the suspension is continuously stirred, and then centrifuged, and the obtained solid is vacuum-dried to obtain 5 as shown in the formula (I).
  • the good solvent is selected from tetrahydrofuran
  • the poor solvent is selected from the group consisting of water, n-heptane and the like.
  • the ratio of the good solvent to the poor solvent is in the range of 1:2 (v/v) to 1:4 (v/v); more preferably, when the good solvent is tetrahydrofuran and the poor solvent is water, both The ratio between them is 1:2 (v/v). When the good solvent is tetrahydrofuran and the poor solvent is n-heptane, the ratio between them is 1:4 (v/v).
  • the vacuum drying is carried out at room temperature to 100 ° C, more preferably 25 to 80 ° C, more preferably 25 to 50 ° C, most preferably 30 ° C.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising an effective amount of the 5-aminopyrazolecarboxamide compound of the formula (I) of the present invention or a crystalline form I thereof, the pharmaceutical composition further comprising A pharmaceutically acceptable carrier.
  • the pharmaceutical composition of the present invention can be formulated into solid, semi-solid, liquid or gaseous preparations such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres and Aerosol.
  • compositions of the present invention can be prepared by methods well known in the pharmaceutical art.
  • practical methods for preparing pharmaceutical compositions are known to those skilled in the art, for example, see The Science and Practice of Pharmacy, 20th Edition (Philadelphia College of Pharmacy and Science, 2000).
  • dosage forms suitable for oral administration include capsules, tablets, granules, and syrups and the like.
  • the compound of the formula (I) of the present invention contained in these preparations may be a solid powder or granule; a solution or suspension in an aqueous or non-aqueous liquid; a water-in-oil or oil-in-water emulsion or the like.
  • the above dosage forms may be prepared from the active compound and one or more carriers or carriers
  • the above carriers need to be compatible with the active compound or other excipients.
  • non-toxic carriers include, but are not limited to, mannitol, lactose, starch, magnesium stearate, cellulose, glucose, sucrose, and the like.
  • Carriers for liquid preparations include, but are not limited to, water, physiological saline, aqueous dextrose, ethylene glycol, polyethylene glycol, and the like.
  • the active compound can form a solution or suspension with the above carriers. The particular mode of administration and dosage form will depend on the physicochemical properties of the compound itself, as well as the severity of the disease being applied.
  • compositions of the present invention may be presented in unit dosage forms containing a predetermined amount of active ingredient per unit dose.
  • Preferred unit dosage compositions are those containing a daily or sub-dose, or an appropriate fraction thereof, of the active ingredient. Thus, such unit doses can be administered more than once a day.
  • Preferred unit dosage compositions are those containing a daily or sub-dose (more than one administration per day) as hereinbefore described, or an appropriate fraction thereof.
  • compositions of this invention are formulated, quantified, and administered in a manner consistent with medical practice.
  • a "therapeutically effective amount" of a compound of the invention is determined by the particular condition to be treated, the individual being treated, the cause of the condition, the target of the drug, and the mode of administration.
  • the dose for parenteral administration may be 1-200 mg/kg/day
  • the dose for oral administration may be 1-1000 mg/kg/day.
  • the invention provides the use of a 5-aminopyrazolecarboxamide compound of formula (I) and crystalline form I thereof for the manufacture of a medicament for the prevention or treatment of a disease mediated by BTK.
  • the present invention provides a method for inhibiting BTK activity, comprising administering to a biological system a 5-aminopyrazolecarboxamide compound of the formula (I) or a crystalline form I thereof of the present invention or comprising the formula (I) of the present invention A pharmaceutical composition of the 5-aminopyrazolecarboxamide compound or Form I thereof as shown.
  • the biological system is an enzyme, a cell, or a mammal.
  • the invention also provides a method of preventing or treating a disease mediated by BTK comprising administering to a patient in need thereof a therapeutically effective amount of 5 of the invention as shown in formula (I) - an aminopyrazole carboxamide compound or a crystalline form I thereof and one or more drugs selected from the group consisting of immunomodulators, immunological checkpoint inhibitors, glucocorticoids, non-steroidal anti-inflammatory drugs, Cox-2 specificity Inhibitors, TNF- ⁇ binding proteins, interferons, interleukins and chemotherapeutic drugs.
  • one or more drugs selected from the group consisting of immunomodulators, immunological checkpoint inhibitors, glucocorticoids, non-steroidal anti-inflammatory drugs, Cox-2 specificity Inhibitors, TNF- ⁇ binding proteins, interferons, interleukins and chemotherapeutic drugs.
  • the BTK mediated diseases include autoimmune diseases, inflammatory diseases, xenogeneic immune conditions or diseases, thromboembolic diseases, and cancer.
  • the cancer comprises B-cell chronic lymphocytic leukemia, acute lymphocytic leukemia, non-Hodgkin's lymphoma, Hodgkin's lymphoma, acute myeloid leukemia, diffuse large B-cell lymphoma , multiple myeloma, mantle cell lymphoma, small lymphocytic lymphoma, Waldenstrom's macroglobulinemia, solid tumor.
  • the autoimmune disease and inflammatory disease are selected from the group consisting of rheumatoid arthritis, osteoarthritis, juvenile arthritis, chronic obstructive pulmonary disease, multiple sclerosis, systemic lupus erythematosus, psoriasis , psoriatic arthritis, Crohn's disease, ulcerative colitis, and irritable bowel syndrome.
  • the xenogeneic immune condition or disease comprises graft versus host disease, transplantation, blood transfusion, allergic reaction, allergy, type I hypersensitivity, allergic conjunctivitis, allergic rhinitis or atopy dermatitis.
  • Figure 1 shows an X-ray powder diffraction pattern of Form I of a compound of the invention prepared in a different solvent.
  • Figure 2 shows that the compound of the present invention significantly inhibited the growth of the diffuse large B-cell lymphoma cell line TMD-8 in vivo and showed the same antitumor effect as the control compound Ibrutinib.
  • the unit of temperature is Celsius (°C); the definition of room temperature is 18-25 ° C;
  • 200-300 mesh silica gel is used as a carrier for column chromatography separation
  • the progress of the reaction is monitored by thin layer chromatography or LC-MS;
  • the identification of the final product was performed by nuclear magnetic resonance (Bruker AVANCE 300, 300 MHz) and LC-MS (Bruker esquine 6000, Agilent 1200 series).
  • the third step is the preparation of methyl 4-hydroxy-5-nitrovalerate
  • the fourth step is the preparation of 5-hydroxypiperidin-2-one
  • Example 2 The product of Example 2 was obtained by chiral resolution of the product of Example 1.
  • the resolution conditions were: Supercritical fluid chromatography (ChiralPak AD 5 ⁇ , 21 x 250 mm col, 27% methanol, 70 mL/min).
  • the obtained Form I was subjected to X-ray powder diffraction analysis in the same manner as in Example 4 to obtain an X-ray powder diffraction pattern substantially the same as that of the Form I obtained in Example 3, as shown in Fig. 1.
  • the residual enzyme activity in the case of an increase in compound concentration was calculated.
  • the IC50 of each compound was obtained by fitting the data to the four-parameter logistic equation of Graphpad Prism software.
  • the detection platform of EGFR and ITK kinase activity was established by time-resolved fluorescence resonance energy transfer method.
  • the detection platform of LCK, SRC and LYN kinase activity was established by Z'-Lyte method.
  • the detection platform of TEC and JAK3 kinase activity was established by Lance Ultra method.
  • the inhibitory effects of the compounds disclosed herein on different kinase activities were tested separately. Each compound activity data were determined at 11 concentrations of the compound IC 50 value calculated using Graphpad Prism software.
  • the kinase inhibitory activity levels are classified into A, B, C, specifically A (IC 50 ⁇ 100 nM), B (100 nM ⁇ IC 50 ⁇ 1000 nM), C (IC 50 >1000 nM)
  • B cells were purified from healthy donor blood by negative selection using the RosetteSep Human B Cell Enrichment Mix. Cells were plated in growth medium (10% RPMI + 10% fetal bovine serum) and inhibitors of the indicated concentrations were added. After incubating for 1 hour at 37 ° C, the cells were washed three times, and each wash was used for 8-fold dilution in growth medium. The cells were then stimulated with 10 ⁇ g/mL IgM F(ab') 2 for 18 hours at 37 °C. Cells were subsequently stained with anti-CD69-PE antibody and analyzed by flow cytometry using standard conditions.
  • T cells were purified from healthy donor blood by negative selection using the RosetteSep Human T Cell Enrichment Mix.
  • Cells were plated in growth medium (10% RPMI + 10% fetal bovine serum) and inhibitors of the indicated concentrations were added. After incubating for 1 hour at 37 ° C, the cells were washed three times, and each wash was used for 10-fold dilution in growth medium. The cells were then challenged with anti-CD3/CD28 coated beads (bead/cell ratio of 1:1) for 18 hours at 37 °C. Cells were subsequently stained with anti-CD69-PE antibody and analyzed by flow cytometry using standard conditions.
  • Human whole blood was obtained from healthy volunteers and blood was collected by venipuncture into a Vacutainer tube that was anticoagulated with sodium heparin. Test compounds were diluted to 10 times the required initial drug concentration in PBS), followed by three-fold serial dilutions in 10% DMSO in PBS to give a 9 point dose response curve. 5.5 ⁇ L of each compound dilution was added to the aiil 96-well V-bottom plate in duplicate; 5.5 ⁇ L of 10% DMSO in PBS was added to the control and non-stimulated wells. Human whole blood (100 ⁇ L) was added to each well, and after mixing, the plates were incubated for 30 minutes at 37 C, 5% CO 2 , 100% humidity.
  • the sample was then lysed with 1 ml of IX Pharmingen Lyse Buffer (BD Pharmingen) and the plate was centrifuged at 1500 rpm for 5 minutes. The supernatant was removed by aspiration, and the remaining pellet was again lysed with an additional 1 ml of IX Pharmingen Lyse Buffer, and the plate was centrifuged as before. The supernatant was aspirated and the remaining pellet was washed in FACs buffer (PBS + 1% FBQ. After centrifugation and the supernatant was removed, the pellet was resuspended in 150 ⁇ L of FACs buffer. Transfer the sample to a suitable one.
  • IX Pharmingen Lyse Buffer BD Pharmingen
  • 96-well plates run on the HTS 96-well system of the BD LSR II flow cytometer. Data were acquired using excitation and emission wavelengths appropriate for the fluorophore used and percent positive cells were obtained using Cell Quest Software. Results were initially analyzed using FACS analysis (Flow Jo) Analysis. IC50 values were calculated using XLfit v3, Equation 201.
  • phosphate buffer 100 mM, pH 7.4
  • liver microsome protein concentration of 20 mg/ml suspension
  • BD Gentest a species of liver microsomes. Human, dog, rat, and mouse were separately added; 158 ⁇ L of phosphate buffer (100 mM, pH 7.4) was added to the control group.
  • step 3 Prepare the mixed system in step 2, pre-incubated for 3 minutes in a 37 ° C water bath, then add 40 ⁇ L of NADPH production system (containing NADP +: 6.5 mM, glucose 6-phosphate: 16.5 mM, MgCl 2 : 16.5 mM, glucose 6 - Phosphate dehydrogenase: 2 U/ml) The reaction was initiated and incubated for 1 hour in a 37 ° C water bath.
  • NADPH production system containing NADP +: 6.5 mM, glucose 6-phosphate: 16.5 mM, MgCl 2 : 16.5 mM, glucose 6 - Phosphate dehydrogenase: 2 U/ml
  • Preparation method of parallel preparation 0 minute reaction sample The prepared mixed system in step 2 was taken out in a 37 ° C water bath for 3 minutes, and then taken out, 400 ⁇ L of acetonitrile was added, and then 40 ⁇ L of NADPH generation system was added. After vortexing for 3 minutes, centrifugation (13,000 rpm, 4 ° C) for 5 minutes, and the supernatant was taken to detect the drug concentration C0 by HPLC.
  • CYP enzyme metabolism is the main pathway for drug biotransformation, and its quantity and activity directly affect the activation and metabolism of drugs in the body.
  • cytochrome CYP is an important drug phase I metabolizing enzyme that catalyzes the oxidation and reductive metabolism of various exogenous compounds.
  • the CYP enzyme plays a very important role in the elimination of the drug, and is also the main factor in the drug interaction caused by the combination.
  • METHODS This experiment used the cocktail probe drug method to simultaneously determine the inhibitory effect of compounds on five CYP450 enzymes in human liver microsomes.
  • the human microsomes were from BD Gentest.
  • the reaction was carried out in 100 mM phosphate buffer in a total volume of 200 ⁇ L.
  • the concentration of the microsomes in the reaction system was 0.25 mg/mL, and the concentration of the test compound was 20 ⁇ M, 6.67 ⁇ M, 2.22 ⁇ M, 0.74 ⁇ M, 0.25 ⁇ M.
  • the specific probe substrate and concentration were phenacetin (CYP1A2) 40 ⁇ M, respectively.
  • the incubation system was pre-incubated for 5 minutes in a 37-degree constant temperature shaker, and the reaction was started by adding a NADPH-producing system (containing 1.3 mM NADP+, 3.3 mM glucose 6-phosphate, 0.4 U/L glucose 6-phosphate dehydrogenase, 3.3 mM MgCL2). After incubation for 45 minutes, the reaction was stopped by adding an equal volume of acetonitrile, vortexed, centrifuged at 13,000 rpm, and the supernatant was subjected to LC-MS-MS injection to determine the amount of metabolite production.
  • a NADPH-producing system containing 1.3 mM NADP+, 3.3 mM glucose 6-phosphate, 0.4 U/L glucose 6-phosphate dehydrogenase, 3.3 mM MgCL2. After incubation for 45 minutes, the reaction was stopped by adding an equal volume of acetonitrile, vortexed, centrifuged at 13,000 rpm, and
  • the specific metabolites were acetaminophen (CYP1A2), dextrorphan (CYP2D6), 4-hydroxydiclofenac (CYP2C9), 4-hydroxyfenfenin (CYP2C19), and 6 ⁇ -hydroxytestosterone (CYP3A4).
  • the specific inhibitors were furaphylline (CYP1A2), quinidine (CYP2D6), sulfaphenazole (CYP2C9), tranylcypromine (CYP2C19), ketoconazole (CYP3A4).
  • the final result of this experiment is to calculate the IC50 value of the half inhibitory concentration.
  • IC50 ((50% - low inhibition rate %) / (high inhibition rate % - low inhibition rate %)) x (high concentration - low concentration) + low concentration.
  • 2.9 SD rats were randomly divided into 3 groups, 3 in each group, one group was administered by intragastric administration, and the other group was administered by tail vein injection. Rats in the gavage-administered group were fasted overnight before administration.
  • the collected blood samples were centrifuged at 12000 rpm for 5 minutes at 4 ° C, then the upper plasma samples were collected and stored in a refrigerator at -20 ° C for testing.
  • LC-MS/MS liquid phase Waters Acquity UPLC (USA) and mass spectrometry 5500 Q Trap (Applied Biosystem/MDS SCIEX) or HPLC-MS ⁇ MS: liquid phase Agilent 1200 series (USA) and Mass Spectrometry API 4000 (Applied Biosystem/MDS SCIEX) was used to measure the concentration of compounds in plasma.
  • Typical test conditions are as follows:
  • the IC50 value of the compound for hERG inhibition can be determined according to the method described in the patent US20050214870 A1.
  • Compound (R)-5-Amino-3-(4-((5-chloropyridin-2-yl)oxy)phenyl)-1-(4-cyano-4-azaspiro[2.5]octyl- 6-yl)-1H-pyrazole-4-carboxamide (I) has only a weak inhibitory or no inhibitory effect on hERG, and its IC50 value is greater than 1000 nM.
  • the immunodeficiency serious defect NOD.SCID mouse was purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. and was raised in the SPF animal room. After the TMD-8 cells were cultured to a sufficient amount, the cells were collected by centrifugation and washed twice with PBS. Finally, the cells were resuspended in serum-free RPMI 1640 medium and Matrigel (1:1 v/v). Using a 1 ml syringe and a 25G syringe needle, 0.2 ml of the cell suspension was injected into the right flank area of each mouse.

Abstract

本发明公开了一种如式(I)所示的新型5-氨基吡唑甲酰胺化合物的晶型。此外，本发明还公开了上述化合物的晶型的制备方法以及包含上述化合物晶型的药物组合物及其用途。

Description

作为BTK抑制剂的5-氨基吡唑甲酰胺化合物的晶型I 技术领域
本发明属于药物化学技术领域，具体地说，涉及一种新型高效、选择性好的、具有良好的药代动力学性质的、作为BTK抑制剂的5-氨基吡唑甲酰胺化合物及其晶型以及制备该化合物及其晶型的方法和包含该化合物或其晶型的药物组合物。
背景技术
蛋白激酶是人体内生物酶中的最大家族，包括超500种蛋白质。特别地，对于酪氨酸激酶，其酪氨酸残基上的酚官能团能被磷酸化，从而发挥重要的生物信号传导的作用。酪氨酸激酶家族拥有控制细胞生长、迁移和分化的成员。异常的激酶活性己经被阐明了与许多人体疾病密切相关，这些疾病包括癌症、自身免疫疾病和炎性疾病。
布鲁顿酪氨酸激酶(BTK)是一种细胞质非受体酪氨酸激酶，属于TEC激酶家族(共有5个成员BTK，TEC，ITK，TXK，BMX)一员。BTK基因位于X-染色体的Xq21.33-Xq22，共有19外显子，跨越37.5kb基因组DNA。
除了T细胞和浆细胞外，BTK表达在几乎所有造血细胞上，尤其在B淋巴细胞发生，分化，信号和生存中发挥必不可少的作用。B细胞是通过B细胞受体(BCR)被活化的，而BTK在BCR信号通路中起到了决定性的作用。B细胞上的BCR被活化后，会引起BTK的激活，然后导致下游的磷脂酶C(PLC)浓度增加，并激活IP3和DAG信号通路。这一信号通路可以促进细胞的增殖、粘附和存活。BTK基因突变会导致一种罕见的遗传性B细胞特异性免疫缺陷疾病，被称为X-连锁无丙种球蛋白血症(X-Iinked agammaglobulinemia，XLA)。在这种疾病中，BTK的功能被抑制，从而导致了B细胞的产生或成熟受阻。患有XLA疾病的男性，体内基本没有B细胞，循环抗体也很少，容易出现严重甚至致命的感染。这有力证明了BTK在B细胞的生长和分化中起着极其重要的作用。
小分子BTK抑制剂能与BTK结合，抑制BTK自身磷酸化，阻止BTK的激活。这能阻断BCR通路的信号传导，抑制B淋巴瘤细胞的增 殖，破坏瘤细胞的粘附，从而促进瘤细胞的凋亡。并诱导细胞凋亡。这使BTK在B细胞有关的癌症中成为引人注目的药物靶点，尤其是对于B细胞淋巴瘤和白血病，比如非霍奇金淋巴瘤(NHL)、慢性淋巴细胞白血病(CLL)、和抗复发性或难治性套细胞淋巴瘤(MCL)等。
BTK抑制剂除了可以对抗B细胞淋巴瘤和白血病，还可以抑制B细胞自身抗体和细胞因子的产生。在自身免疫性疾病中，B细胞呈递自身抗原，促进T细胞活化分泌致炎症因，既造成组织损伤，同时又激活B细胞产生大量抗体，触发自身免疫反应。T和B细胞相互作用形成反馈调节链，导致自身免疫反应失控，加重组织病理损伤。所以，BTK可以作为自身免疫性疾病，比如类风湿性关节炎、系统性红斑狼疮(SLE)、过敏性疾病(例如食道炎等疾病)的药物靶点。
此外也有报道BTK抑制剂可与化疗药或免疫检查点抑制剂联用，在临床试验中对多种实体瘤表现出较好的治疗效果。
在目前已上市的药物中，依鲁替尼是由Pharmacyclics和强生公司联合开发的一种不可逆BTK抑制剂，己分别于2013年11月和2014年2月获得FDA批准，用于治疗套细胞淋巴瘤(MCL)和慢性淋巴性白血病(CLL)。依鲁替尼被FDA定为“突破性”新药，它通过与BTK中的半胱氨酸的巯基发生反应，并形成共价键，使BTK酶失活而发挥疗效。然而，依鲁替尼在给药过程中，易被代谢(被代谢酶氧化代谢成双羟化产物或者被其他含巯基的酶、半胱氨酸、谷胱甘肽等进攻而失活)而影响药效。其临床给药剂量达到了560mg每天，而使病人负担加重。此外，依鲁替尼对除BTK外的一些激酶也有一定的抑制作用，尤其是对EGFR的抑制可导致较严重的皮疹、腹泻等不良反应。因此，本领域仍需发展新一类更为高效、选择性好，良好的药代动力学性质的BTK抑制剂用于相关疾病的治疗。
发明内容
本发明人研发了一种新型5-氨基吡唑甲酰胺化合物，该化合物是蛋白激酶BTK的有效、安全、选择性高的抑制剂，是一种新的共价键抑制剂，通过改变其和半胱氨酸反应率，来改善与靶标的亲和性，以提高疗效，选择性和安全性。其结构如式(I)所示：
Figure PCTCN2018114666-appb-000001
其化学名称为(R)-5-氨基-3-(4-((5-氯吡啶-2-基)氧基)苯基)-1-(4-氰基-4-氮杂螺[2.5]辛-6-基)-1H-吡唑-4-甲酰胺(简称API)。
本发明的第一个目的是提供上述如式(I)所示化合物的晶型I。
本发明的第二个目的是提供上述如式(I)所示化合物的晶型I的制备方法。
本发明的第三个目的是提供包含上述如式(I)所示化合物的晶型I的药物组合物。
本发明的第四个目的是提供上述如式(I)所示化合物的晶型I的用途。
在一个实施方案中，本发明提供了如式(I)所示的5-氨基吡唑甲酰胺化合物的晶型I，其在
Figure PCTCN2018114666-appb-000002
波长下，使用Cu-K辐射下测定的X-射线粉末衍射图包含以下特征峰(以2θ角度表示)(±0.2°)：5.8、9.3、10.9、16.4、18.4、20.3、20.9、21.8、22.4、23.0、24.4、27.1、30.0、31.2、31.9。
在一个实施方案中，本发明提供了如式(I)所示的5-氨基吡唑甲酰胺化合物的晶型I，其具有基本上如图1所示的X-射线粉末衍射图。
在一个实施方案中，本发明提供了如式(I)所示的5-氨基吡唑甲酰胺化合物的晶型I的制备方法，包括将如式(I)所示的5-氨基吡唑甲酰胺化合物溶解于良溶剂中，然后在搅拌下向生成的溶液中加入不良溶剂形成悬浮液，悬浮液经继续搅拌之后离心分离，将得到的固体真空干燥，得到如式(I)所示的5-氨基吡唑甲酰胺化合物的晶型I。
优选地，良溶剂选自四氢呋喃，不良溶剂选自水、正庚烷等。
优选地，良溶剂与不良溶剂的比例在1∶2(v/v)至1∶4(v/v)的范围内；更优选地，当良溶剂为四氢呋喃且不良溶剂为水时，二者之间比例为1∶2(v/v)，当良溶剂为四氢呋喃且不良溶剂为正庚烷时，二者之间比例为1∶4(v/v)。
优选地，真空干燥在室温至100℃，更优选25至80℃，更优选在25至50℃，最优选在30℃的条件下进行。
在一个实施方案中，本发明提供了一种包含有效剂量的本发明式(I)所示的5-氨基吡唑甲酰胺化合物或其晶型I的药物组合物，所述药物组合物还包括药学上可接受的载体。
本发明的药物组合物可以被配制为固态、半固态、液态或气态制剂，如片剂、胶囊、粉剂、颗粒剂、膏剂、溶液剂、栓剂、注射剂、吸入剂、凝胶剂、微球及气溶胶。
本发明的药物组合物可以通过制药领域中公知的方法制备。例如，制备药物组合物的实际方法为本领域技术人员所已知，例如可参见The Science and Practice of Pharmacy(制药科学与实践)，20th Edition(Philadelphia College of Pharmacy and Science，2000)。
本发明的药物组合物的给药途径包括但不限于口服、局部、经皮、肌肉、静脉、吸入、肠胃外、舌下、直肠、阴道及鼻内。例如，适合口服给药的剂型包括胶囊、片剂、颗粒剂以及糖浆等。这些制剂中包含的本发明的式(I)化合物可以是固体粉末或颗粒；水性或非水性液体中的溶液或是混悬液；油包水或水包油的乳剂等。上述剂型可由活性化合物与一种或多种载体或载体
经由通用的药剂学方法制成。上述的载体需要与活性化合物或其他辅料兼容。对于固体制剂，常用的无毒载体包括但不限于甘露醇、乳糖、淀粉、硬脂酸镁、纤维素、葡萄糖、蔗糖等。用于液体制剂的载体包括但不限于水、生理盐水、葡萄糖水溶液、乙二醇和聚乙二醇等。活性化合物可与上述载体形成溶液或是混悬液。具体的给药方式和剂型取决于化合物本身的理化性质以及所应用疾病的严重程度等。本领域技术人员能够根据上述因素并结合其自身具有的知识来确定具体的给药途径。例如可参见：李俊，《临床药理学》，人民卫生出版社，2008.06；丁玉峰，论临床剂型因素与合理用药，医药导报，26(5)，2007；Howard C.Ansel，Loyd V.Allen，Jr.，Nicholas G.Popovich著，江志强主译，《药物剂型与给药体系》，中国医药科技出版社，2003.05。
本发明的药物组合物可以以每单位剂量含有预定量的活性成分的单位剂型存在。优选的单位剂量组合物为含有日剂量或亚剂量、或其适当分数的活性成分的那些。因此，这种单位剂量可以一天给药多于一次。优选的单位剂量组合物为含有本文如上所述的日剂量或亚剂量(一天给药多于一次)、或其适当分数的活性成分的那些。
本发明的药物组合物以符合医学实践规范的方式配制，定量和给药。本发明化合物的“治疗有效量”由要治疗的具体病症、治疗的个体、 病症的起因、药物的靶点以及给药方式等因素决定。通常，经胃肠道外给药的剂量可以是1-200mg/kg/天，口服给药的剂量可以是1-1000mg/kg/天。
本文中所提供的有效剂量的范围并非意图限制本发明的范围，而是代表优选的剂量范围。但是，最优选的剂量可针对个别个体而进行调整，这是本领域技术人员所了解且可决定的(例如参阅Berkow等人编著，Merck手册，第16版，Merck公司，Rahway，N.J.，1992)。
在一个实施方案中，本发明提供了如式(I)所示的5-氨基吡唑甲酰胺化合物及其晶型I在制备用于预防或治疗由BTK介导疾病的药物中的用途。
本发明提供了一种抑制BTK活性的方法，包含给予生物体系本发明的如式(I)所示的5-氨基吡唑甲酰胺化合物或其晶型I或包含本发明的如式(I)所示的5-氨基吡唑甲酰胺化合物或其晶型I的药物组合物。
在一些实施方案中，所述生物体系是酶、细胞或哺乳动物。
在一个实施方案中，本发明还提供了一种预防或治疗由BTK介导的疾病的方法，其包括对有需要的患者联合给予治疗有效剂量的本发明的如式(I)所示的5-氨基吡唑甲酰胺化合物或其晶型I和一种或多种选自以下的药物：免疫调节剂、免疫检查点抑制剂、糖皮质激素、非甾体抗炎药、Cox-2特异性抑制剂、TNF-α结合蛋白、干扰素、白细胞介素和化疗药物。
在本发明的实施方式中，所述由BTK介导的疾病包括自身免疫性疾病、炎性疾病、异种免疫性情况或疾病、血栓栓塞疾病和癌症。在一些具体实施方式中，所述癌症包括B细胞性慢性淋巴细胞白血病、急性淋巴细胞性白血病、非霍奇金淋巴瘤、霍奇金淋巴瘤、急性髓性白血病、弥漫性大B细胞淋巴瘤、多发性骨髓瘤、套细胞淋巴瘤、小淋巴细胞性淋巴瘤、华氏巨球蛋白血症、实体瘤。在一些具体实施方式中，所述自身免疫性疾病和炎性疾病选自类风湿性关节炎、骨关节炎、青少年关节炎、慢性阻塞性肺疾病、多重硬化、系统性红斑狼疮、银屑病、银屑病关节炎、克罗恩病、溃疡性结肠炎和肠道易激综合症。在一些具体实施方式中，所述异种免疫性情况或疾病包括移植物抗宿主病、移植、输血、过敏反应、变态反应、I型超敏反应、过敏性结膜炎、过敏性鼻炎或特应性皮炎。
实验数据证明，本发明提供的如式(I)所示的5-氨基吡唑甲酰胺化合物及其晶型是蛋白激酶BTK的有效、安全的抑制剂。
附图说明
图1表示的是在不同溶剂中制备的本发明化合物的晶型I的X-射线粉末衍射图。
图2表示的是本发明化合物显著抑制弥漫性大B细胞淋巴瘤细胞株TMD-8体内的生长，并显示出与对照化合物依鲁替尼相同的抗肿瘤效果。
具体实施方式
下文所描述的实验、合成方法以及所涉及的中间体是对本发明的阐明，并不限制本发明的范围。
本发明中实验所使用的起始原料或购买自试剂供应商或经由本领域公知的方法由已知原料制备。除非另有说明，本文的实施例应用下述条件：
温度的单位是摄氏度(℃)；室温的定义是18-25℃；
有机溶剂使用无水硫酸镁或无水硫酸钠干燥；使用旋转蒸发仪在减压升温条件下旋干(例如：15mmHg，30℃)；
柱色谱分离时使用200-300目硅胶作为载体；
通常情况下，反应的进度通过薄层色谱法或LC-MS监测；
最终产品的鉴定由核磁共振(Bruker AVANCE 300，300MHz)和LC-MS(Bruker esquine 6000，Agilent 1200series)完成。
实施例1
5-氨基-3-(4-((5-氯吡啶-2-基)氧基)苯基)-1-(4-氰基-4-氮杂螺[2.5]辛-6-基)-1H-吡唑-4-甲酰胺(II)的制备
Figure PCTCN2018114666-appb-000003
第一步 4-羟基丁酸甲酯的制备
将二氢呋喃-2(3H)-酮(100g，1.163mol)和三乙胺(460g，4.65mol)加入到甲醇(1L)溶液中，反应液在60℃条件下反应24小时，薄层色谱(石油醚∶乙酸乙酯＝2∶1)检测反应完毕，将反应液旋干，得到4-羟基丁酸甲 酯(120g，87.6％，黄色液体)，直接用于下一步反应。
第二步 4-氧代丁酸甲酯的制备
将4-羟基丁酸甲酯(120g，1.02mol)加入到二氯甲烷(1.2L)溶液中，然后将氯铬酸吡啶(330g，1.53mol)加入到上述反应液中，室温条件下反应12小时，薄层色谱(石油醚∶乙酸乙酯＝3∶1)检测反应完毕，将反应液通过硅藻土过滤，旋干，得到4-氧代丁酸甲酯(60g，50％，黄色液体)，直接用于下一步反应。
第三步 4-羟基-5-硝基戊酸甲酯的制备
冰水浴中，将4-氧代丁酸甲酯(60g，0.46mol)，硝基甲烷(42g，0.69mol)，四氢呋喃(300mL)，叔丁醇(300mL)加入到反应瓶中，然后将叔丁醇钾(5g)缓慢加入上述反应体系中，升至室温，反应2小时，薄层色谱(石油醚∶乙酸乙酯＝3∶1)检测反应完毕，加水(30mL)淬灭反应，旋干溶剂，加入水(300mL)和乙酸乙酯(300mL)分液，有机相用饱和食盐水洗涤，无水硫酸钠干燥，旋干，得到粗品4-羟基-5-硝基戊酸甲酯(45g，淡黄色油状液体)直接用于下一步。
第四步 5-羟基哌啶-2-酮的制备
将甲基4-羟基-5-硝基戊酸甲酯(45g，0.23mol)和钯碳(2.1g)加入到甲醇(500mL)中，反应液在60℃，H 2条件下反应24小时，薄层色谱(石油醚∶乙酸乙酯＝1∶1)检测反应完毕，将反应液通过硅藻土过滤，滤液旋干，得到5-羟基哌啶-2-酮(10g，黄色固体，38％)，直接用于下一步反应。
第五步 1-苄基-5-(苄氧基)哌啶-2-酮的制备
室温下，将5-羟基哌啶-2-酮(10g，0.1mol)，加入到二甲基亚砜(100mL)中，然后将氢化钠(10g，0.25mol)缓慢加入到上述反应体系中，加入完毕后，将苄溴(43.5g，0.25mol)滴加到反应液中，搅拌过夜，薄层色谱(石油醚∶乙酸乙酯＝1∶1)检测反应完毕，向反应体系中加入饱和氯化铵(100ml)淬灭反应，乙酸乙酯(100mL*3)萃取三次，饱和食盐水洗涤，无水硫酸钠干燥，旋干，过柱纯化，得到1-苄基-5-(苄氧基)哌啶-2-酮(16g，黄色固体，54％)。
第六步 4-苄基-6-(苄氧基)-4-氮杂螺[2.5]辛烷的制备
-78℃氮气保护下，将1-benzy1-苄基-5-(苄氧基)哌啶-2-酮15g，50mmol)溶于无水四氢呋喃(150mL)中，然后向反应瓶中缓慢滴加乙基溴化镁(150mL)，滴加完毕后，再将钛酸四丙酯(45g，150mmol)加入到上述反应体系中，滴加完毕后，将反应升至室温，搅拌2小时，薄层色谱(石油醚∶乙酸乙酯＝10∶1)检测反应完毕，向反应体系中加入饱和氯化铵水溶液(100ml)淬灭反应，乙酸乙酯(100mL*3)萃取三次，合并有机相，有机相用饱和食盐水洗涤后，加无水硫酸钠干燥，旋干，过柱纯化， 得到4-苄基-6-(苄氧基)-4-氮杂螺[2.5]辛烷(5.1g，黄色固体，31％)。
第七步 4-氮杂螺[2.5]辛-6-醇的制备
将4-苄基-6-(苄氧基)-4-氮杂螺[2.5]辛烷(5.5g，18mmol)和钯碳(2g，1.8mmol)加入到甲醇(200mL)和氯化氢(2mL)溶液中，反应液在60℃，H 2条件下(压力)反应48小时，薄层色谱(石油醚∶乙酸乙酯＝10∶1)检测反应完毕，将反应液通过硅藻土过滤，滤液旋干，得到4-氮杂螺[2.5]辛-6-醇(2.5g，黄色固体)，直接用于下一步反应。
第八步 6-羟基-4-氮杂螺[2.5]辛烷-4-甲酸苄酯的制备
将4-氮杂螺[2.5]辛-6-醇(2.5g，21mmol)和碳酸氢钠(3.8g，45mmol)加入到四氢呋喃(100mL)中，然后将苄氧基甲酰氯(4.25g，25mmol)滴加到上述反应体系中，反应液在室温条件下反应48小时，薄层色谱(石油醚∶乙酸乙酯＝3∶1)检测反应完毕，将反应液过滤，滤液旋干，过柱纯化，得到6-羟基-4-氮杂螺[2.5]辛烷-4-甲酸苄酯(4.2g)。
第九步 6-氧代-4-氮杂螺[2.5]辛烷-4-甲酸苄酯的制备
将6-羟基-4-氮杂螺[2.5]辛烷-4-甲酸苄酯(4.2g，16mmol)和2-碘酰基苯甲酸(6.7g，24mmol)加入到丙酮(100mL)中，然后将反应液升至60℃反应12小时，薄层色谱(石油醚∶乙酸乙酯＝2∶1)检测反应完毕，将反应液萃取，滤液旋干，过柱纯化，得到6-氧代-4-氮杂螺[2.5]辛烷-4-甲酸苄酯(3.6g，85％)。
1H-NMR(400MHz，DMSO-d6)：δppm 7.34-7.35(m，5H)，5.15(s，2H)，4.07(s，2H)，2.55-2.58(m，2H)，1.95-1.98(m，2H)，1.09-1.11(m，2H)，0.85-0.86(m，2H).
第十步 (E)-6-(2-(叔丁氧基羰基)亚肼基)-4-氮杂螺[2.5]辛烷-4-甲酸苄酯的制备
将6-氧代-4-氮杂螺[2.5]辛烷-4-甲酸苄酯(3.6g，13.9mmol)和叔丁氧基羰基肼(1.98g，15mmol)加入到四氢呋喃(50mL)中，然后将反应液升至70℃反应2小时，薄层色谱(石油醚∶乙酸乙酯＝2∶1)检测反应完毕，将反应液萃取，滤液旋干，过柱纯化，得到(E)-6-(2-(叔丁氧基羰基)亚肼基)-4-氮杂螺[2.5]辛烷-4-甲酸苄酯(5.0g，90％)。
第十一步 6-(2-(叔丁氧基羰基)肼基)-4-氮杂螺[2.5]辛烷-4-甲酸苄酯的制备
将(E)-6-(2-(叔丁氧基羰基)亚肼基)-4-氮杂螺[2.5]辛烷-4-甲酸苄酯(5.0g，13.4mmol)和氰基硼氢化钠(1.4g，20mmol)加入到四氢呋喃(100mL)中，然后将对甲苯磺酸(3.8g，20mmol)滴加到上述反应体系中，反应液在室温条件下反应36小时，薄层色谱(石油醚∶乙酸乙酯＝1∶1)检测反应完毕，将反应液萃取，滤液旋干，过柱纯化，得到6-(2-(叔丁 氧基羰基)肼基)-4-氮杂螺[2.5]辛烷-4-甲酸苄酯(4.2g，80.4％)。
第十二步 6-肼基-4-氮杂螺[2.5]辛烷-4-甲酸苄酯的制备
将6-(2-(叔丁氧基羰基)肼基)-4-氮杂螺[2.5]辛烷-4-甲酸苄酯(4.2g，11mmol)加入到氯化氢/乙酸乙酯(50mL)溶液中，室温反应12小时，薄层色谱(石油醚∶乙酸乙酯＝2∶1)检测反应完毕，将反应液萃取，滤液旋干，过柱纯化，得到6-肼基-4-氮杂螺[2.5]辛烷-4-甲酸苄酯(3.5g)。
1H-NMR(400MHz，DMSO-d6)：δppm 7.21-7.35(m，10H)，4.46-4.54(m，2H)，3.89-3.93(m，1H)，3.78-3.81(m，1H)，3.68-3.73(m，1H)，2.86-2.90(m，1H)，2.63-2.68(m，1H)，2.08-2.12(m，1H)，1.57-1.85(m，2H)，1.24-1.29(m，1H)，0.65(s，2H).0.41-0.44(m，2H)
第十三步 4-((5-氯吡啶-2-基)氧基)苯甲酸甲酯的制备
4-羟基苯甲酸甲酯(6.5g，49mmol)、5-氯-2-氟吡啶(5.0g，33mmol)以及碳酸铯(20g，65mmol)溶于N，N-二甲基甲酰胺(50mL)，搅拌。反应液在110℃回流反应12小时，薄层色谱(石油醚∶乙酸乙酯＝5∶1)检测，反应完毕，旋干溶剂。粗品化合物加入乙酸乙酯(250mL)和水(250mL)分液萃取。有机相用无水硫酸钠干燥，过滤，减压蒸干。粗品化合物经柱层析分离纯化，得产品4-((5-氯吡啶-2-基)氧基)苯甲酸甲酯(9.0g，93％)。
MS：m/z 264.2[M+1]
第十四步 4-((5-氯吡啶-2-基)氧基)苯甲酸的制备
4-((5-氯吡啶-2-基)氧基)苯甲酸甲酯(6.5g，49mmol)溶于甲醇(100mL)和水(5mL)中，然后向反应体系中加入氢氧化锂(2.3g)，搅拌。反应液在45℃条件下反应12小时，薄层色谱(石油醚∶乙酸乙酯＝1∶1)检测，反应完毕，旋干溶剂。粗品化合物加入稀盐酸(100mL，1M)调至PH＝7，此时有大量固体生成，将固体过滤，烘干，得到产品4-((5-氯吡啶-2-基)氧基)苯甲酸(6.5g，白色固体，84％)。
MS：m/z 250.1[M+1]
第十五步 4-((5-氯吡啶-2-基)氧基)苯甲酰氯的制备
将4-((5-氯吡啶-2-基)氧基)苯甲酸(6.5g，23mmol)加入到二氯亚砜(20mL)中，反应液在80℃条件下反应3小时。薄层色谱(石油醚∶乙酸乙酯＝3∶1)检测，反应完毕，旋干溶剂，粗品4-((5-氯吡啶-2-基)氧基)苯甲酰氯(7g，黄色固体)直接用于下一步反应。
第十六步 2-((4-((5-氯吡啶-2-基)氧基)苯基)(羟基)亚甲基)丙二腈的制备
0℃条件下，将丙二睛(3.72g，56.4mmol)溶于无水四氢呋喃(50mL)，然后缓慢向其中加入氢化钠(3.6g，90mmol，60％)，加入完毕后， 搅拌，将反应升至室温搅拌1小时，然后再降至0℃，将4-((5-氯吡啶-2-基)氧基)苯甲酰氯(6g，22.2mmol)溶于无水四氢呋喃(50mL)中，缓慢滴加入上述反应液，滴加完毕后，反应液在0℃条件下搅拌1小时。薄层色谱(石油醚∶乙酸乙酯＝3∶1)检测，反应完毕，新点生成。反应液加入饱和氯化铵(100mL)淬灭，乙酸乙酯(100mL*3)萃取3次，有机相分别用饱和食盐水(100mL)洗涤，无水硫酸钠干燥，旋干，粗品化合物用石油醚∶乙酸乙酯＝50∶1打浆，得到2-((4-((5-氯吡啶-2-基)氧基)苯基)(羟基)亚甲基)丙二腈(8.0g，淡黄色固体，82％)。
MS：m/z298[M+1]
第十七步 2-((4-((5-氯吡啶-2-基)氧基)苯基)(乙氧基)亚甲基)丙二腈的制备
将2-((4-((5-氯吡啶-2-基)氧基)苯基)(羟基)亚甲基)丙二腈(5.0g，16.8mmol)加入到原甲酸三乙酯(50mL)中，反应升温至80℃搅拌12小时。薄层色谱(石油醚∶乙酸乙酯＝3∶1)显示有产物生成，将反应液过滤，滤液旋干，得到固体用甲醇打浆，得到2-((4-((5-氯吡啶-2-基)氧基)苯基)(乙氧基)亚甲基)丙二腈(1.5g，白色产物，27.7％)。
MS：m/z 326.0[M+1]
第十八步 6-(5-氨基-3-(4-((5-氯吡啶-2-基)氧基)苯基)-4-氰基-1H-吡唑-1-基)-4-氮杂螺[2.5]辛烷-4-甲酸苄酯的制备
2-((4-((5-氯吡啶-2-基)氧基)苯基)(乙氧基)亚甲基)丙二腈(1.0g，3.09mmol)、6-肼基-4-氮杂螺[2.5]辛烷-4-甲酸苄酯(1.28g，3.71mmol)以及三乙胺(1.56g，15.5mmol)溶于乙醇(20mL)。反应液在25℃反应12小时，薄层色谱(石油醚∶乙酸乙酯＝2∶1)检测，反应完毕，加水淬灭。乙酸乙酯(25mL*3)萃取。有机相用无水硫酸钠干燥，过滤，旋干。粗品化合物用石油醚∶乙酸乙酯＝50∶1打浆2次，得产品6-(5-氨基-3-(4-((5-氯吡啶-2-基)氧基)苯基)-4-氰基-1H-吡唑-1-基)-4-氮杂螺[2.5]辛烷-4-甲酸苄酯(1.5g，88％)。
MS：m/z 555.2[M+1]
第十九步 5-氨基-3-(4-((5-氯吡啶-2-基)氧基)苯基)-1-(4-氮杂螺[2.5]辛-6-基)-1H-吡唑-4-甲酰胺的制备
室温下，将6-(5-氨基-3-(4-((5-氯吡啶-2-基)氧基)苯基)-4-氰基-1H-吡唑-1-基)-4-氮杂螺[2.5]辛烷-4-甲酸苄酯(750mg，1.35mmol)加入到90％的浓硫酸(10分钟)中，搅拌15分钟，然后将反应体系升温至30℃，反应24小时。薄层色谱(二氯甲烷∶甲醇＝10∶1)检测，反应完毕.将反应液缓慢倒入氨水(50ml)中，调至PH＝7，乙酸乙酯(30mL*3)萃取三次，合并的有机相用饱和食盐水(30mL)洗涤，无水硫酸钠干燥，旋干，粗 品化合物过柱纯化，得到5-氨基-3-(4-((5-氯吡啶-2-基)氧基)苯基)-1-(4-氮杂螺[2.5]辛-6-基)-1H-吡唑-4-甲酰胺(480mg，78％，淡黄色固体)。
MS：m/z 439.2[M+1]
第二十步 5-氨基-3-(4-((5-氯吡啶-2-基)氧基)苯基)-1-(4-氰基-4-氮杂螺[2.5]辛-6-基)-1H-吡唑-4-甲酰胺的制备
5-氨基-3-(4-((5-氯吡啶-2-基)氧基)苯基)-1-(4-氮杂螺[2.5]辛-6-基)-1H-吡唑-4-甲酰胺(50mg，0.113mmol)溶于N，N-二甲基甲酰胺(5mL)，加入碳酸铯(110mg，0.342mmol)和溴化氰(12.5mg，0.113mmol)，反应液室温搅拌2小时，点板检测，反应完毕，反应液倒入乙酸乙酯(50mL)，水洗(20mL*3)，无水硫酸钠干燥，过滤，减压蒸干。粗产物制备薄层层析板纯化(二氯甲烷∶甲醇＝50∶1)，得产品5-氨基-3-(4-((5-氯吡啶-2-基)氧基)苯基)-1-(4-氰基-4-氮杂螺[2.5]辛-6-基)-1H-吡唑-4-甲酰胺(15mg，15％)。
1H-NMR(400MHz，DMSO-d6)：δppm 8.12(s，1H)，7.66-7.69(dd，J 1＝2.4Hz，J 2＝8.4Hz 2H)，7.56-7.58(d，J＝8Hz，2H)，7.21-7.23(d，J＝8Hz，2H)，6.92-6.94(d，J＝8Hz，1H)，5.61(s，1H)，5.19-5.30(m，2H)，4.19-4.25(m，1H)，3.52-3.66(m，2H)，2.34-2.45(m，2H)，2.19(s，1H)，1.25-1.30(m，1H)，1.15-1.20(m，1H)，0.78-0.89(m，2H)，0.66-0.71(m，1H).
MS：m/z 446.2[M+1]
实施例2
(R)-5-氨基-3-(4-((5-氯吡啶-2-基)氧基)苯基)-1-(4-氰基-4-氮杂螺[2.5]辛-6-基)-1H-吡唑4-甲酰胺(I)和(S)-5-氨基-3-(4-((5-氯吡啶-2-基)氧基)苯基)-1-(4-氰基-4-氮杂螺[2.5]辛-6-基)-1H-吡唑-4-甲酰胺(III)的制备
Figure PCTCN2018114666-appb-000004
实施例1的产物经手性拆分后可得实施例2化合物。拆分条件为：Supercritical fluid chromatography(ChiralPak AD 5μ，21 x 250mm col， 27％甲醇，70mL/分钟)。
化合物(R)-5-氨基-3-(4-((5-氯吡啶-2-基)氧基)苯基)-1-(4-氰基-4-氮杂螺[2.5]辛-6-基)-1H-吡唑-4-甲酰胺(I)的谱图数据：
1H-NMR(400MHz，CDCl3)：δppm 8.12(s，1H)，7.69-7.67(m，1H)，7.57(d，J＝8.4Hz，2H)，7.22(d，J＝8.4Hz，2H)，6.93(d，J＝8.8Hz，1H)，5.60(s，2H)，5.24(s，2H)，4.25-4.20(m，1H)，3.67-3.48(m，2H)，2.42-2.35(m，2H)，2.19-2.16(m，1H)，1.30-1.26(m，1H)，1.21-1.16(m，1H)，0.90-0.79(m，2H)，0.71-0.67(m，1H).
MS：m/z 464.4[M+H]
化合物(S)-5-氨基-3-(4-((5-氯吡啶-2-基)氧基)苯基)-1-(4-氰基-4-氮杂螺[2.5]辛-6-基)-1H-吡唑-4-甲酰胺(III)的谱图数据：
1H-NMR(400MHz，CDCl3)：δppm 8.12(s，1H)，7.69-7.67(m，1H)，7.57(d，J＝8.4Hz，2H)，7.22(d，J＝8.4Hz，2H)，6.93(d，J＝8.8Hz，1H)，5.60(s，2H)，5.24(s，2H)，4.25-4.20(m，1H)，3.67-3.48(m，2H)，2.42-2.35(m，2H)，2.19-2.16(m，1H)，1.30-1.26(m，1H)，1.21-1.16(m，1H)，0.90-0.79(m，2H)，0.71-0.67(m，1H).
MS：m/z 464.4[M+H]
实施例3
(R)-5-氨基-3-(4-((5-氯吡啶-2-基)氧基)苯基)-1-(4-氰基-4-氮杂螺[2.5]辛-6-基)-1H-吡唑-4-甲酰胺(I)的晶型I的制备
将约20mg的式(I)化合物溶解于1mL四氢呋喃中。向生成的溶液中加入水，其中四氢呋喃与水的体积比为1∶2。将生成的悬浊液在25℃的条件下搅拌24小时。然后通过离心分离悬浊液，将得到的固体在30℃的条件下真空干燥大约3小时，得到黄色的式(I)化合物的晶型I。
对上述晶型I进行X射线粉末衍射分析：
将约10mg样品均匀的分散在单晶硅板上，用于XRPD检测，具体仪器参数如下：
光管：Cu：K-Alpha
Figure PCTCN2018114666-appb-000005
光管电压：40kV
光管电流：40mA
扫描范围：3-40deg.
样品盘转速：15rpm
扫描速率：10deg./min
最终获得上述晶型I的X射线粉末衍射图，如图1所示。
实施例4
(R)-5-氨基-3-(4-((5-氯吡啶-2-基)氧基)苯基)-1-(4-氰基-4-氮杂螺[2.5]辛-6-基)-1H-吡唑-4-甲酰胺(I)的晶型I的制备
将约20mg的式(I)化合物溶解于1mL四氢呋喃中。向生成的溶液中加入正庚烷，其中四氢呋喃与正庚烷的体积比为1∶4。将生成的悬浊液在25℃的条件下搅拌24小时。然后通过离心分离悬浊液，将得到的固体在30℃的条件下真空干燥大约3小时，得到黄色的式(I)化合物的晶型I。
采用与实施例4类似的方法对获得的晶型I进行X射线粉末衍射分析，得到与实施例3获得的晶型I基本相同的X射线粉末衍射图，如图1所示。
实施例5
激酶活性抑制实验(BTK)
在基于时间分辨荧光共振能量转移方法的试验中，测试化合物(R)-5-氨基-3-(4-((5-氯吡啶-2-基)氧基)苯基)-1-(4-氰基-4-氮杂螺[2.5]辛-6-基)-1H-吡唑-4-甲酰胺(I)对BTK激酶活性的抑制作用。重组的Btk与本文公开的化合物在室温下在含有50mM Tris pH7.4、10mM MgCl 2、2mM MnCl 2、0.1mM EDTA、1mM DTT、20nM SEB、0.1％BSA、0.005％tween-20的试验缓冲液中预先温育1小时。通过加入ATP(在ATP Km浓度下)和肽底物(Biotin-AVLESEEELYSSARQ-NH2)来引发反应。在室温下温育1小时后，加入等体积的含有50mM HEPES pH7.0、800mM KF、20mM EDTA、0.1％BSA、连接Eu穴合物的p-Tyr66抗体和链霉亲和素标记的XL665的终止液以终止反应。盘在室温下再温育1小时，然后在BMG PHERAstar FS仪器上读取TR-FRET信号(ex337nm，em 620nm/665nm)。基于615nm处的荧光与665nm处的荧光的比值，计算化合物浓度增加情况下残余酶活性。各化合物的IC50通过Graphpad Prism软件的四参数逻辑方程拟合数据而得到。
按照上述的实验方法，化合物(R)-5-氨基-3-(4-((5-氯吡啶-2-基)氧基)苯基)-1-(4-氰基-4-氮杂螺[2.5]辛-6-基)-1H-吡唑-4-甲酰胺(I)表现出较强的激酶抑制活性(IC50＜100nM)。
实施例6
体外激酶选择性实验
采用基于时间分辨荧光共振能量转移方法建立了EGFR，ITK激酶活性检测平台；采用Z’-Lyte方法建立了LCK，SRC，LYN激酶活性检测平台；采用Lance Ultra方法建立了TEC和JAK3激酶活性检测平台，分别测试本文公开的化合物对不同激酶活性的抑制作用。每个化合物分别在11个浓度下测定酶活性数据，用Graphpad Prism软件计算该化合物的IC 50值。
按照上述的实验方法，化合物(R)-5-氨基-3-(4-((5-氯吡啶-2-基)氧基)苯基)-1-(4-氰基-4-氮杂螺[2.5]辛-6-基)-1H-吡唑-4-甲酰胺(I)表现出很强的激酶选择性，明显优于对照化合物依鲁替尼。结果见下表。
Figure PCTCN2018114666-appb-000006
激酶抑制活性等级分为A、B、C，具体地A(IC 50＜100nM)，B(100nM＜IC 50＜1000nM)，C(IC 50＞1000nM)
实施例7
B细胞抑制实验
在活体外短暂暴露至BTK抑制剂足以抑制正常人类B细胞中的B细胞激活。此方案模拟活体内细胞至抑制剂的预测暴露，并且显示尽管清洗抑制剂但对B细胞的抑制仍得以保持。
B细胞是使用若赛特赛普(RosetteSep)人类B细胞富集混合剂通过阴性选择自健康供体血液纯化得到。将细胞铺板于生长培养基(10％RPMI+10％胎牛血清)中并添加指定浓度的抑制剂。于37℃下培育1小时后，将细胞洗涤三次，每次洗涤均利用于生长培养基中进行8倍稀释。随后将细胞用10μg/mL IgM F(ab′)2在37℃下剌激18小时。随后用抗CD69-PE抗体对细胞进行染色并通过流式细胞术使用标准条件进行分析。
依照以上的方法进行测定，化合物(R)-5-氨基-3-(4-((5-氯吡啶-2-基)氧基)苯基)-1-(4-氰基-4-氮杂螺[2.5]辛-6-基)-1H-吡唑-4-甲酰胺(I)对B细胞具有较强的抑制活性，其IC50值小于10nM。
实施例8
T细胞抑制实验
T细胞是使用若赛特赛普(RosetteSep)人类T细胞富集混合剂通过阴性选择自健康供体血液纯化得到。将细胞铺板于生长培养基(10％RPMI+10％胎牛血清)中并添加指定浓度的抑制剂。于37℃下培育1小时后，将细胞洗涤三次，每次洗涤均利用于生长培养基中进行10倍稀释。随后将细胞用anti-CD3/CD28包被珠(珠/细胞比例为1∶1)在37℃下剌激18小时。随后用抗CD69-PE抗体对细胞进行染色并通过流式细胞术使用标准条件进行分析。依照以上的方法进行测定，化合物(R)-5-氨基-3-(4-((5-氯吡啶-2-基)氧基)苯基)-1-(4-氰基-4-氮杂螺[2.5]辛-6-基)-1H-吡唑-4-甲酰胺(I)对T细胞具有很弱的抑制活性或无抑制，其IC50值大于4000nM。
实施例9
人全血B细胞抑制实验
人全血(hWB)获自健康志愿者，通过静脉穿刺将血液收集到用肝素钠抗凝化的Vacutainer管中。测试化合物在PBS中稀释至10倍所需初始药物浓度)，接着在10％的在PBS中的DMSO中三倍系列稀释，以得到9点的剂量响应曲线。将5.5μL的每种化合物稀释液一式两份添加到aiil 96孔V型底的板上；向对照和无刺激孔中添加5.5μL的10％在PBS中的DMSO。向每孔添加人全血(100μL)，在混合后将板在37C，5％CO 2，100％湿度温育30分钟。在搅拌下向每孔(无刺激孔除外)添加羊F(ab′)2抗人IgM(Southern Biotech)(10μL的500μg/mL溶液，50μg/mL最终浓度)，并且将板温育另外20小时。在20小时温育结束时，将样品与荧光探针标记的20μL APC小鼠抗人CD69(BD Pharmingen)在37C，5％CO 2，100％湿度温育30分钟。包括用于补偿调节和初始电压设置的诱导对照、未染色的和单染色剂。然后将样品用1ml的IX Pharmingen Lyse Buffer(BD Pharmingen)裂解，并且将板在1500rpm离心5分钟。通过抽吸除去上清液，将残留的团粒用另外1ml的IX Pharmingen Lyse Buffer再次裂解，并且将板如前离心。吸出上清液，将残留的团粒在FACs缓冲液(PBS+1％FBQ中洗涤。离心后并除去上清液后，将团粒重悬浮在150μL的FACs缓冲液中。将样品转移至适于在BD LSR II流式细胞器的HTS 96孔体系上运行的96孔板。采用适合所用荧光团的激发和发射波长，获取数据并且采用Cell Quest Software获得百分比阳性细胞值。结果最初用FACS分析软件(Flow Jo)分析。IC50值使用XLfit v3，公式201计算。
依照以上的方法进行测定，化合物(R)-5-氨基-3-(4-((5-氯吡啶-2-基)氧基)苯基)-1-(4-氰基-4-氮杂螺[2.5]辛-6-基)-1H-吡唑-4-甲酰胺(I)对人全血中B细胞具有较强的抑制活性，其IC50值小于200nM。
实施例10
化合物(R)-5-氨基-3-(4-((5-氯吡啶-2-基)氧基)苯基)-1-(4-氰基-4-氮杂螺[2.5]辛-6-基)-1H-吡唑-4-甲酰胺(I)在肝微粒体中的稳定性研究
1.将化合物(R)-5-氨基-3-(4-((5-氯吡啶-2-基)氧基)苯基)-1-(4-氰基-4-氮杂螺[2.5]辛-6-基)-1H-吡唑-4-甲酰胺(I)溶解在乙腈中，制成浓度为0.5mM的储备液。
2.2μL储备液加入1.5ml离心管中，然后加入148μL磷酸缓冲液(100mM，pH 7.4)和10μL肝微粒体(蛋白浓度为20mg/ml)悬液【BD Gentest公司】，肝微粒体的种属分别为人，狗，大鼠，小鼠；对照组加入158μL磷酸缓冲液(100mM，pH 7.4)。
3.步骤2中制备好的混合体系，于37℃水浴中预孵3分钟，然后加入40μL NADPH发生体系(含有NADP+：6.5mM，葡萄糖6-磷酸：16.5mM，MgCl 2：16.5mM，葡萄糖6-磷酸脱氢酶：2U/ml)启动反应，并于37℃水浴中孵育1小时。
4.反应进行1小时后，将离心管从水浴中取出，并加入400μL乙腈终止反应，然后涡旋震荡3分钟，最后离心(13000rpm，4℃)5分钟，取上清液用HPLC检测剩余药物浓度Cr。
5.平行制备0分钟反应样品的制备方法：步骤2中制备好的混合体系，于37℃水浴中预孵3分钟后取出，加入400μL乙腈，然后加入40μL NADPH发生体系。涡旋震荡3分钟后，离心(13000rpm，4℃)5分钟，取上清液用HPLC检测药物浓度C0。
6.经60分钟孵育后，药物在孵育体系中的剩余百分比按照下式计算：
药物剩余(％)＝Cr÷C0×100％
按照上述的实验方法，化合物(R)-5-氨基-3-(4-((5-氯吡啶-2-基)氧基)苯基)-1-(4-氰基-4-氮杂螺[2.5]辛-6-基)-1H-吡唑-4-甲酰胺(I)表现出较好的微粒体稳定性，其在各种属的肝微粒体中的剩余百分比＞30％。
实施例11
评价化合物对CYP酶抑制作用
CYP酶代谢是药物生物转化的主要途径，其数量和活性大小直接影响药物在体内的活化与代谢。作为外源性化合物的主要代谢酶，细胞色 素CYP是重要的药物I相代谢酶，可以催化多种外源性化合物的氧化和还原代谢。CYP酶在药物的消除过程中起着非常重要的作用，同时也是引起联合用药时药物相互作用产生的主要因素。
方法：本实验采用cocktail探针药物法同时测定化合物对人源肝微粒体中五种CYP450酶的抑制作用，人源微粒体来自BD Gentest公司。
实验步骤如下：
反应在100mM磷酸盐缓冲液中进行，总体积200μL。反应体系中微粒体浓度为0.25mg/mL，待测化合物浓度为20μM、6.67μM、2.22μM、0.74μM、0.25μM，特异性探针底物及浓度分别为非那西汀(CYP1A2)40μM、右美沙芬(CYP2D6)5μM、双氯芬酸(CYP2C9)10μM、S-美芬妥英(CYP2C19)40μM、睾酮(CYP3A4)80μM。孵育体系在37度恒温振荡器中预孵育5分钟，加入NADPH发生体系(含1.3mM NADP+、3.3mM葡萄糖6-磷酸、0.4U/L葡萄糖6-磷酸脱氢酶、3.3mM MgCL2)开始反应。孵育45分钟后加入等体积的乙腈终止反应，涡旋，13000rpm离心，取上清LC-MS-MS进样测定代谢产物生成量。特异性代谢产物分别为对乙酰氨基酚(CYP1A2)、右啡烷(CYP2D6)、4-羟基双氯芬酸(CYP2C9)、4-羟基美芬妥英(CYP2C19)、6β-羟基睾酮(CYP3A4)。特异性抑制剂分别为呋拉茶碱(CYP1A2)、奎尼丁(CYP2D6)、磺胺苯吡唑(CYP2C9)、反苯环丙胺(CYP2C19)、酮康唑(CYP3A4)。本实验最终结果为计算半数抑制浓度IC50值。IC50＝((50％-低抑制率％)/(高抑制率％-低抑制率％))×(高浓度-低浓度)+低浓度。
按照上述的实验方法，化合物(R)-5-氨基-3-(4-((5-氯吡啶-2-基)氧基)苯基)-1-(4-氰基-4-氮杂螺[2.5]辛-6-基)-1H-吡唑-4-甲酰胺(I)对各种CYP酶均只有不强的抑制或无抑制，说明其对其它药物的代谢影响较小。
实施例12
化合物(R)-5-氨基-3-(4-((5-氯吡啶-2-基)氧基)苯基)-1-(4-氰基-4-氮杂螺[2.5]辛-6-基)-1H-吡唑-4-甲酰胺(I)在大鼠体内的药物代谢动力学研究方法
1.雄性SD大鼠【华阜康】买入后，在本实验室适应性饲养7天。
2.9只SD大鼠随机分为3组，每组3只，一组用于灌胃给药，另一组用于尾静脉注射给药。灌胃给药组的大鼠，给药前需过夜禁食。
3.大鼠给药后，采用眼眶静脉丛采血的方法在以下时间点采集血样：I.V.：(给药前)，0.08小时，0.25小时，0.5小时，1小时，2小时，4小时，8小时，24小时。P.O.：0.08小时，0.25小时，0.5小时，1小时，2小时，4小时，8小时，24小时。每个采血时间点采血量约为300μl。
4.采集的血样在4℃以12000rpm的转速离心5分钟，然后采集上层血浆样品，并于-20℃冰箱中保存待测。
5.实验操作总结见表4：
表4、化合物(R)-5-氨基-3-(4-((5-氯吡啶-2-基)氧基)苯基)-1-(4-氰基-4-氮杂螺[2.5]辛-6-基)-1H-吡唑-4-甲酰胺(I)在大鼠体内的药物代谢动力学试验设计
Figure PCTCN2018114666-appb-000007
6.使用LC-MS/MS(UPLC-MS/MS：液相Waters Acquity UPLC(USA)和质谱5500 Q Trap(Applied Biosystem/MDS SCIEX)或者HPLC-MS\MS：液相Agilent 1200series(USA)和质谱API 4000(Applied Biosystem/MDS SCIEX))检测血浆中的化合物浓度。典型的检测条件如下：
Figure PCTCN2018114666-appb-000008
Figure PCTCN2018114666-appb-000009
Figure PCTCN2018114666-appb-000010
使用药代动力学专业软件WinNonlin【型号：Phoenix TM
Figure PCTCN2018114666-appb-000011
6.1厂家：Pharsight Corporation】计算药代动力学参数。【Phoenix 1.1 User’s Guide：p251-p300】
按照上述的实验方法，化合物(R)-5-氨基-3-(4-((5-氯吡啶-2-基)氧基)苯基)-1-(4-氰基-4-氮杂螺[2.5]辛-6-基)-1H-吡唑-4-甲酰胺(I)表现出较好的生物利用度(＞40％)。
实施例13
hERG结合实验(Dofetillide法)
依照专利US20050214870 A1上描述的方法，可测定化合物对hERG抑制的IC50值。化合物(R)-5-氨基-3-(4-((5-氯吡啶-2-基)氧基)苯基)-1-(4-氰基-4-氮杂螺[2.5]辛-6-基)-1H-吡唑-4-甲酰胺(I)对hERG只有很弱的抑制作用或无抑制作用，其IC50值大于1000nM。
实施例14
药效学实验
免疫功能严重缺陷NOD.SCID小鼠购自北京维通利华实验动物技术有限公司，饲养于SPF级动物房。TMD-8细胞培养到足够数量后，离心收集细胞，PBS洗2遍。最后细胞用不含血清的RPMI1640培养基和基质胶(1∶1v/v)重悬。采用1ml的注射器和25G注射器针头，将0.2ml的细胞悬液注入每只小鼠右侧翼皮下区域。植入一周左右用游标卡尺测量肿瘤大小，用以下公式计算肿瘤体积：肿瘤体积＝(长×宽 2)/2。当肿瘤体积达到100-200mm 3左右，将小鼠分组灌胃给药，连续给药21天。
化合物(R)-5-氨基-3-(4-((5-氯吡啶-2-基)氧基)苯基)-1-(4-氰基-4-氮杂螺[2.5]辛-6-基)-1H-吡唑-4-甲酰胺(I)能显著抑制弥漫性大B细胞淋巴瘤细胞株TMD-8体内的生长，并显示出与对照化合物依鲁替尼相同 的抗肿瘤效果(实验结果请见图2)。

Claims (8)

  1. 一种如式(I)所示的5-氨基吡唑甲酰胺化合物的晶型I，
    Figure PCTCN2018114666-appb-100001
    其特征在于，所述晶型具有包括以下的X-射线粉末衍射特征峰(±0.2°)：5.8、9.3、10.9、16.4、18.4、20.3、20.9、21.8、22.4、23.0、24.4、27.1、30.0、31.2、31.9。
  2. 根据权利要求1所述的晶型I，其特征在于，所述晶型I具有基本上如图1-5所示的X-射线粉末衍射图。
  3. 根据权利要求1所述的如式(I)所示的5-氨基吡唑甲酰胺化合物的晶型I的制备方法，包括：将如式(I)所示的5-氨基吡唑甲酰胺化合物溶解于良溶剂中，然后在搅拌下向生成的溶液中加入不良溶剂形成悬浮液，悬浮液经继续搅拌之后离心分离，将得到的固体真空干燥，得到如式(I)所示的5-氨基吡唑甲酰胺化合物的晶型I；其中所述良溶剂选自四氢呋喃，所述不良溶剂选自水或正庚烷；所述良溶剂与不良溶剂的比例在1∶2(v/v)至1∶4(v/v)的范围内。
  4. 根据权利要求3所述的制备方法，其中所述良溶剂是四氢呋喃，所述不良溶剂是水或正庚烷；所述良溶剂与不良溶剂的比例为1∶2(v/v)。
  5. 根据权利要求3所述的制备方法，其中所述良溶剂是四氢呋喃，所述不良溶剂是正庚烷；所述良溶剂与不良溶剂的比例为1∶4(v/v)。
  6. 药物组合物，其包含权利要求1所述的式(I)所示的5-氨基吡唑甲酰胺化合物的晶型I和药学上可接受的载体。
  7. 权利要求1所述晶型或权利要求6所述药物组合物在制备BTK抑制剂药物中的用途。
  8. 如权利要求7所述的用途，其中，所述BTK抑制剂是指预防或治疗由BTK介导的疾病，所述疾病选自自身免疫性疾病、炎性疾病、异种免疫性情况或疾病、血栓栓塞疾病和癌症。
PCT/CN2018/114666 2017-11-10 2018-11-08 作为btk抑制剂的5-氨基吡唑甲酰胺化合物的晶型i WO2019091440A1 (zh)

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