WO2019091438A1 - 作为btk抑制剂的5-氨基吡唑甲酰胺化合物的无定型固体分散体 - Google Patents

作为btk抑制剂的5-氨基吡唑甲酰胺化合物的无定型固体分散体 Download PDF

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WO2019091438A1
WO2019091438A1 PCT/CN2018/114658 CN2018114658W WO2019091438A1 WO 2019091438 A1 WO2019091438 A1 WO 2019091438A1 CN 2018114658 W CN2018114658 W CN 2018114658W WO 2019091438 A1 WO2019091438 A1 WO 2019091438A1
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amorphous solid
compound
solid dispersion
formula
nonionic polymer
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PCT/CN2018/114658
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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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    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • 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
    • A61P35/00—Antineoplastic agents
    • 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
    • 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

Definitions

  • the invention belongs to the technical field of medicinal chemistry, in particular to a novel high-efficiency, selective and good pharmacokinetic property of an amorphous solid of a 5-aminopyrazolecarboxamide compound as a BTK inhibitor.
  • Dispersions, methods for their preparation, and pharmaceutical compositions comprising the amorphous solid dispersions of the compounds and uses 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 signal transduction of the BCR pathway, inhibit the proliferation of B lymphoma cells, destroy the adhesion of tumor cells, and promote the 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 an amorphous solid dispersion of the above compound of formula (I) formed on a nonionic polymer.
  • a second object of the present invention is to provide a process for the preparation of an amorphous solid dispersion of the above compound of formula (I) formed on a nonionic polymer.
  • a third object of the present invention is to provide a pharmaceutical composition comprising the above amorphous solid dispersion of the compound of the formula (I) in a nonionic polymer.
  • a fourth object of the present invention is to provide the use of the above amorphous solid dispersion of the compound of formula (I) in a nonionic polymer.
  • the present invention provides an amorphous solid dispersion of a 5-aminopyrazolecarboxamide compound as shown in formula (I) in a nonionic polymer, wherein the nonionic polymer is selected From povidone, cellulose derivatives, polyvinyl amide-polyvinyl ester-polyethylene glycol graft copolymer.
  • the povidone is selected from the group consisting of povidone K30 (PVPK30), povidone VA64 (PVPVA64);
  • the cellulose derivative is selected from the group consisting of hydroxypropyl methylcellulose E5 (HPMCE5) , hydroxypropyl methylcellulose acetate succinate (HPMCAS-MG);
  • the polyvinyl amide-polyvinyl ester-polyethylene glycol graft copolymer is selected from the group consisting of polyvinyl caprolactam-polyvinyl acetate-polyethylene Alcohol graft copolymer.
  • the amorphous solid dispersion of the 5-aminopyrazolecarboxamide compound of formula (I) as shown in formula (I) in a nonionic polymer has an X substantially as shown in FIG. - ray powder diffraction pattern.
  • the present invention provides a process for the preparation of an amorphous solid dispersion of a 5-aminopyrazolecarboxamide compound as shown in formula (I) in a nonionic polymer, comprising: separately After mixing with a nonionic polymer as described above, it was dissolved in a solvent, and after removing the solvent by a rotary evaporation method, the residue solid product was collected and dried under vacuum.
  • the ratio of the compound of formula I to the polymer is from 10:1 to 1:10, more preferably from 5:1 to 1:5, still more preferably from 2:1 to 1:2, most preferably from 1:1 to 1:2. .
  • the solvent is selected from the group consisting of DMF, acetonitrile, methanol, ethanol, propanol, isopropanol, butanol, isobutanol, pentanol, isoamyl alcohol, tert-butanol, acetone, methyl ethyl ketone, cyclopentanone, chloroform, Methylene chloride, ethyl acetate, tetrahydrofuran, diethyl ether, methyl tert-butyl ether or a combination thereof; more preferably, the solvent is selected from the group consisting of a mixture of tetrahydrofuran and water or a mixture of tetrahydrofuran and ethanol; most preferably, the solvent is selected from the group consisting of Tetrahydrofuran: water (7:3) and tetrahydrofuran:ethanol (7:3).
  • the present invention provides a process for the preparation of an amorphous solid dispersion of a 5-aminopyrazolecarboxamide compound of formula (I) in a nonionic polymer, comprising: a compound of formula I Each of the nonionic polymers is mixed and dissolved in a solvent, and spray-dried to obtain an amorphous solid dispersion (SDD for short).
  • the solvent is selected from the group consisting of DMF, acetonitrile, methanol, ethanol, propanol, isopropanol, butanol, isobutanol, pentanol, isoamyl alcohol, tert-butanol, acetone, methyl ethyl ketone, cyclopentanone, chloroform, Methylene chloride, ethyl acetate, tetrahydrofuran, diethyl ether, methyl tert-butyl ether or a combination thereof; more preferably, the solvent is selected from the group consisting of a mixture of tetrahydrofuran and water or a mixture of tetrahydrofuran and ethanol; most preferably, the solvent is selected from the group consisting of Tetrahydrofuran: water (7:3) and tetrahydrofuran:ethanol (7:3).
  • the present invention provides a medicament comprising an effective amount of a 5-aminopyrazolecarboxamide compound of the formula (I) of the present invention or an amorphous solid dispersion thereof in a nonionic polymer.
  • a composition, 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 present invention provides a 5-aminopyrazolecarboxamide compound of formula (I) and an amorphous solid dispersion thereof in a nonionic polymer prepared for prevention or treatment by BTK Use in drugs that mediate disease.
  • the present invention provides a method for inhibiting BTK activity comprising administering to a biological system a 5-aminopyrazolecarboxamide compound of the present invention as shown in formula (I) or an amorphous solid dispersion thereof in a nonionic polymer. Or a pharmaceutical composition comprising the 5-aminopyrazolecarboxamide compound of the present invention as shown in formula (I) or an amorphous solid dispersion thereof in a nonionic polymer.
  • 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)
  • 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 is a perspective view of a polarizing microscope image of an amorphous solid dispersion of a compound of formula I in a nonionic polymer prepared by the rotary evaporation method of the present invention.
  • Figure 2 shows an X-ray powder diffraction pattern of an amorphous solid dispersion of a compound of formula I in a nonionic polymer prepared by the rotary evaporation process of the present invention.
  • Figure 3 shows the results of differential thermal analysis of an amorphous solid dispersion of a compound of formula I in a nonionic polymer prepared by the rotary evaporation process of the present invention.
  • Figure 4 is a polarizing microscope image of the amorphous solid dispersion of the compound of formula I in the nonionic polymer prepared by the rotary evaporation method of the present invention after completion of the dynamic solubility test at 37 ° C in SGF.
  • Figure 5 is a polarizing microscope image of the amorphous solid dispersion of the compound of formula I prepared in the nonionic polymer of the present invention in a non-ionic polymer after completion of the dynamic solubility test at 37 ° C in FaSSIF.
  • Figure 6 is a view showing a polarized microscope image of an amorphous solid dispersion of a compound of the formula I prepared by a rotary evaporation method in a nonionic polymer after storage for 5 days at 40 ° C / 75% RH.
  • Figure 7 is a polarizing microscope image of an amorphous solid dispersion of a compound of formula I prepared in a non-ionic polymer prepared by the rotary evaporation method of the present invention after storage at 50 ° C for 5 days.
  • Figure 8 is a polarized microscope image of an amorphous solid dispersion (SDD) of a compound of formula I prepared in a spray drying process in a nonionic polymer of the present invention.
  • SDD amorphous solid dispersion
  • Figure 9 is a graph showing the X-ray powder diffraction pattern of an amorphous solid dispersion of a compound of formula I in a nonionic polymer prepared by spray drying in accordance with the present invention.
  • Figures 10-11 show the results of dynamic moisture adsorption analysis of an amorphous solid dispersion of a compound of formula I prepared in a spray drying process in a nonionic polymer.
  • Figure 12 is a graph showing the X-ray powder diffraction pattern of an amorphous solid dispersion of a compound of formula I in a nonionic polymer prepared by spray drying after dynamic moisture adsorption experiments.
  • Figure 13 is a graph showing the results of differential calorimetry of an amorphous solid dispersion of a compound of formula I in a nonionic polymer prepared by spray drying in accordance with the present invention.
  • Figure 14 shows the results of thermogravimetric analysis of an amorphous solid dispersion of a compound of formula I prepared in a spray drying process in a nonionic polymer of the present invention.
  • Figure 15 is a polarizing microscope image of an amorphous solid dispersion of a compound of formula I in a nonionic polymer prepared by spray drying after 2 weeks under different conditions.
  • Figure 16 is a polarizing microscope image of an amorphous solid dispersion of a compound of formula I prepared in a spray drying process of the present invention in a nonionic polymer after 4 weeks under different conditions.
  • Figure 17 is a polarizing microscope image of an amorphous solid dispersion of a compound of formula I in a nonionic polymer prepared by spray drying in accordance with the present invention, under different conditions for 3 months.
  • Figure 18 is a graph showing that the compound of the formula I of the present invention significantly inhibits the growth of the diffuse large B-cell lymphoma cell line TMD-8 in vivo and exhibits substantially the same antitumor effect as the control compound ibrutinib.
  • Figure 19 is a graph showing the pharmacokinetics of an amorphous solid dispersion of a compound of formula I in a nonionic polymer prepared by spray drying in accordance with the present invention in a rat.
  • the unit of temperature is Celsius (°C); the definition of room temperature is 18-25 ° C;
  • 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).
  • Step 1 0.210 g of sodium hydroxide, 2.235 g of sodium dihydrogen phosphate and 3.093 g of sodium chloride were added to a 500 mL volumetric flask, about 0.450 L of water was added, and the pH was adjusted to 6.5 with 1 N sodium hydroxide or 1 N hydrochloric acid. Make up the volume (0.5 liters) with pure water.
  • Step 2 1.120 g of SIF powder was placed in a 500 mL volumetric flask with 0.250 L of buffer (Step 1), then water was added to volume and mixed well.
  • -DSC detection method for SDD: preheat the sample to 100 ° C, remove water at 10 ° C / min, hold for 2 minutes, then cool to 30 ° C at 10 ° C / min, hold for 2 minutes, then heat to 250 °C, 10 ° C / min.
  • the temperature was heated from room temperature to 300 ° C at a rate of 10 ° C/min under atmospheric conditions, and the test was completed if the loss weight of the sample was greater than 20%.
  • the hygroscopicity evaluation criteria are shown in the following table:
  • Hygroscopic classification Hygroscopicity* Dissolved Absorb enough water to become liquid Very hygroscopic ⁇ W% ⁇ 15% Moisture absorption 15%> ⁇ W% ⁇ 2% Slightly hygroscopic 2%> ⁇ W% ⁇ 0.2% Not hygroscopic ⁇ W% ⁇ 0.2%
  • the second step is the preparation of methyl 4-oxobutanoate
  • 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).
  • PVPK30, PVPVA64, HPMCE5, HPMCAS-MG and Soluplus were separately selected for the preparation of amorphous solid dispersions of the compounds of formula I using two ratios of compound I to polymer, ie 1:1 and The compound of formula I and the corresponding polymer were separately weighed and dissolved together in a solvent in a round bottom flask at 1:2 (w/w).
  • the solvent for the PVPVA64, HPMCE5, HPMCAS and Soluplus ASD is tetrahydrofuran:water (7:3) and for PVPK30 the solvent is tetrahydrofuran:ethanol (7:3). After removing the solvent by rotary evaporation, the residue solid product was collected and dried in a vacuum oven overnight.
  • PLM microscopy images
  • XRPD X-ray powder diffraction patterns
  • DSC differential thermal analysis
  • DFS dynamic moisture adsorption analysis
  • TGA thermogravimetric analysis
  • Example 2 the compound (R)-5-amino-3-(4-((5-chloropyridin-2-yl)oxy)phenyl)-1-(4-cyano-4) prepared in Example 2 was weighed.
  • -Azaspiro[2.5]oct-6-yl)-1H-pyrazole-4-carboxamide (I) (about 4 mg)
  • ASD amorphous solid dispersion
  • Approximately 4 mg of the compound of formula I was added to a 2 mL HPLC vial and 1 mL of two test solutions, SGF and FaSSIF, were added, respectively.
  • the sample was then placed in a temperature controlled mixer and shaken at 37 ° C, 700 rpm.
  • Table 2 and Table 3 show the kinetic solubility results for 10 amorphous solid dispersions in SGF and FaSSIF, respectively.
  • the original compound (R)-5-amino-3-(4-((5-chloropyridin-2-yl)oxy)phenyl)-1-(4) was prepared as in Example 2.
  • the solubility of the body is improved.
  • the 1:2 ratio of the amorphous solid dispersion showed a higher concentration than the amorphous solid dispersion of the 1:1 ratio, and the highest concentration was 0.5 hours. It can be observed that the concentration of the amorphous solid dispersion containing HPMCE5 can be observed in SGF compared to the original compound (R)-5-amino-3-(4-((5-chloropyridine)-) prepared in Example 2. 2-yl)oxy)phenyl)-1-(4-cyano-4-azaspiro[2.5]oct-6-yl)-1H-pyrazole-4-carboxamide is 11 times higher than FaSSIF is 14 times higher. .
  • Amorphous solid dispersions were prepared by spray drying using HPMCAS and Soluplus, respectively.
  • the drug loading in the prepared amorphous solid dispersion preparation was set at 40%.
  • the solvent used for spray drying was tetrahydrofuran:water (70:30 V:V).
  • the concentration of the amorphous solid dispersion in the solvent was 30 mg/ml.
  • Spray drying parameters are listed in Table 4.
  • the yield of the product was 58.5% for the Soluplus amorphous solid dispersion formulation and 54.5% for the HPMCAS amorphous solid dispersion formulation.
  • the amorphous solid dispersion product was dried in a vacuum oven for 2 days after preparation.
  • Cyclone pressure (mBar) 24.5 Set nozzle airflow (L/min) 9.0 Actual nozzle airflow (L/min) 9.4 Cyclone size medium Pump factor 420 Cooling airflow (m 3 /min) 0.15
  • the above amorphous solid dispersion (SDD) prepared by spray drying was characterized by XRPD, PLM, DVS, DSC and TGA-MS.
  • Figures 8 and 9 show the PLM and XRPD characterization results for the above amorphous solid dispersion, respectively.
  • Figures 10 and 11 show the results of dynamic moisture adsorption analysis (DVS) of the above amorphous solid dispersion, respectively.
  • DVDS dynamic moisture adsorption analysis
  • the original Formula I compound has similar moisture absorption and desorption behavior, while the compound of Formula I - HPMCAS amorphous solid dispersion exhibits relatively less water absorption, which should be attributed to the hygroscopicity of HPMCAS.
  • XRPD showed no crystal formation observed in the above two amorphous solid dispersions after the dynamic moisture adsorption analysis experiment (Fig. 12).
  • DSC analysis showed that the Tg of the above two amorphous solid dispersions were 86.8 ° C (Soluplus amorphous solid dispersion) and 103.5 ° C (HPMCAS amorphous solid dispersion), respectively ( FIG. 13 ).
  • Figure 14 shows the results of TGA-MS analysis of two amorphous solid dispersions. The residual solvent residue of the two amorphous solid dispersions is about 1%.
  • Table 5 and Table 6 list the kinetic solubility results for the two amorphous solid dispersion formulation samples, respectively.
  • the solubility of the compound of formula I in the Soluplus amorphous solid dispersion is improved (about 4 fold increase in SGF buffer and about 10 fold increase in FaSSIF buffer).
  • the solubility in the FaSSIF buffer is increased by more than 10 fold, and the solubility in the SGF buffer is also improved.
  • Table 7 shows the HPLC purity of the amorphous solid dispersion after the above physical stability test. It can be seen that for the compound of formula I - Soluplus amorphous solid dispersion sample, there is no significant change in purity; for the compound of formula I - HPMCAS amorphous solid dispersion sample, the total related substances are at 40 ° C / 75% RH and 50 ° C / There was a slight increase in ambient conditions, about 2% to 3%, while the impurity of the compound of formula I - HPMCAS amorphous solid dispersion did not increase at 25 °C.
  • 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 stimulated 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 5500Q Trap (Applied Biosystem/MDS SCIEX) or HPLC-MS ⁇ MS: liquid phase Agilent 1200 series (USA) and mass spectrometry API 4000 (Applied Biosystem/MDS SCIEX) detects 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 has only a weak inhibitory effect 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.
  • SD rats were randomly divided into groups and administered by intragastric administration.
  • the test group of the API group was administered with 0.5% methylcellulose, 4% Poloxamer 188, 20% PEG400, 4% DMSO as a vehicle, and the test compound of the ASD group was suspended with 0.5% methylcellulose as a solvent. . 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 5500Q Trap (Applied Biosystem/MDS SCIEX) or HPLC-MS ⁇ MS: liquid phase Agilent 1200 series (USA) and mass spectrometry API 4000 (Applied Biosystem/MDS SCIEX) detects the concentration of compounds in plasma.
  • Typical test conditions are as follows:
  • the drug exposure of the amorphous solid dispersion of the compound of the formula I in the nonionic polymer is significantly higher in the rat than in the compound itself, and increases proportionally with the increase in dose, and has a good linear relationship;
  • the exposure of the drug in the same dose range of the amorphous solid dispersion tends to be saturated, showing no linear relationship (see Figure 19 for experimental results).

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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在非离子型聚合物中的无定型固体分散体。此外，本发明还公开了上述化合物在非离子型聚合物中的无定型固体分散体的制备方法以及包含上述化合物在非离子型聚合物中的无定型固体分散体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无定型固体分散体 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的无定型固体分散体及其制备方法以及包含该化合物的无定型固体分散体的药物组合物及其用途。
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 PCTCN2018114658-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)所示化合物在非离子型聚合物上形成的无定型固体分散体。
本发明的第二个目的是提供上述如式(I)所示化合物在非离子型聚合物上形成的无定型固体分散体的制备方法。
本发明的第三个目的是提供包含上述如式(I)所示化合物在非离子型聚合物中的无定型固体分散体的药物组合物。
本发明的第四个目的是提供上述如式(I)所示化合物在非离子型聚合物中的无定型固体分散体的用途。
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在非离子型聚合物中的无定型固体分散体，其中，所述非离子型聚合物选自聚维酮、纤维素衍生物、聚乙烯酰胺-聚乙烯酯-聚乙二醇接枝共聚物。
在一个优选的实施方案中，所述聚维酮选自聚维酮K30(PVPK30)、 聚维酮VA64(PVPVA64)；所述纤维素衍生物选自羟丙基甲基纤维素E5(HPMCE5)、醋酸羟丙基甲基纤维素琥珀酸酯(HPMCAS-MG)；所述聚乙烯酰胺-聚乙烯酯-聚乙二醇接枝共聚物选自聚乙烯己内酰胺-聚乙酸乙烯酯-聚乙二醇接枝共聚物。
在一个实施方案中，本发明提供的如式(I)所示的5-氨基吡唑甲酰胺化合物在非离子型聚合物中的无定型固体分散体具有基本上如图2所示的X-射线粉末衍射图。
在另一个实施方案中，本发明提供的如式(I)所示的5-氨基吡唑甲酰胺化合物在非离子型聚合物中的无定型固体分散体具有基本上如图9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在非离子型聚合物中的无定型固体分散体的制备方法，包括：将式I化合物分别与如上所述非离子型聚合物混合溶解于溶剂中，通过旋转蒸发法除去溶剂之后，收集残余物固体产物并真空干燥。
优选地，式I化合物与聚合物的比例为10∶1至1∶10，更优选5∶1至1∶5，还更优选2∶1至1∶2，最优选1∶1至1∶2。
优选地，溶剂选自DMF、乙腈、甲醇、乙醇、丙醇、异丙醇、丁醇、异丁醇、戊醇、异戊醇、叔丁醇、丙酮、丁酮、环戊酮、氯仿、二氯甲烷、乙酸乙酯、四氢呋喃、乙醚、甲基叔丁基醚或其组合；更优选地，溶剂选自四氢呋喃与水的混合液或四氢呋喃与乙醇的混合液；最优选地，溶剂选自四氢呋喃∶水(7∶3)和四氢呋喃∶乙醇(7∶3)。
在另一个实施方案中，本发明提供了如式(I)所示的5-氨基吡唑甲酰胺化合物在非离子型聚合物中的无定型固体分散体的制备方法，包括：将式I化合物分别与所述非离子型聚合物混合溶解于溶剂中，通过喷雾干燥得到无定型固体分散体(简称SDD)。
优选地，溶剂选自DMF、乙腈、甲醇、乙醇、丙醇、异丙醇、丁醇、异丁醇、戊醇、异戊醇、叔丁醇、丙酮、丁酮、环戊酮、氯仿、二氯甲烷、乙酸乙酯、四氢呋喃、乙醚、甲基叔丁基醚或其组合；更优选地，溶剂选自四氢呋喃与水的混合液或四氢呋喃与乙醇的混合液；最优选 地，溶剂选自四氢呋喃∶水(7∶3)和四氢呋喃∶乙醇(7∶3)。
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在非离子型聚合物中的无定型固体分散体的药物组合物，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在非离子型聚合物中的无定型固体分散体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在非离子型聚合物中的无定型固体分散体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在非离子型聚合物中的无定型固体分散体的药物组合物。
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在非离子型聚合物中的无定型固体分散体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无定型固体分散体是蛋白激酶BTK的有效、安全的抑制剂。
附图说明
图1表示的是本发明采用旋转蒸发法制备的式I化合物在非离子型聚合物中的无定型固体分散体的偏光显微镜图像。
图2表示的是本发明采用旋转蒸发法制备的式I化合物在非离子型聚合物中的无定型固体分散体的X-射线粉末衍射图。
图3表示的是本发明采用旋转蒸发法制备的式I化合物在非离子型聚合物中的无定型固体分散体的差示热量分析结果。
图4表示的是本发明采用旋转蒸发法制备的式I化合物在非离子型聚合物中的无定型固体分散体在SGF中在37℃下的动态溶解度实验结束之后的偏光显微镜图像。
图5表示的是本发明采用旋转蒸发法制备的式I化合物在非离子型聚合物中的无定型固体分散体在FaSSIF中在37℃下的动态溶解度实验结束之后的偏光显微镜图像。
图6表示的是本发明采用旋转蒸发法制备的式I化合物在非离子型聚合物中的无定型固体分散体在40℃/75％RH条件下储存5天后的偏光 显微镜图像。
图7表示的是本发明采用旋转蒸发法制备的式I化合物在非离子型聚合物中的无定型固体分散体在50℃条件下储存5天后的偏光显微镜图像。
图8表示的是本发明采用喷雾干燥法制备的式I化合物在非离子型聚合物中的无定型固体分散体(SDD)的偏光显微镜图像。
图9表示的是本发明采用喷雾干燥法制备的式I化合物在非离子型聚合物中的无定型固体分散体的X-射线粉末衍射图。
图10-11表示的是本发明采用喷雾干燥法制备的式I化合物在非离子型聚合物中的无定型固体分散体的动态水分吸附分析结果。
图12表示的是本发明采用喷雾干燥法制备的式I化合物在非离子型聚合物中的无定型固体分散体在动态水分吸附实验后的X-射线粉末衍射图。
图13表示的是本发明采用喷雾干燥法制备的式I化合物在非离子型聚合物中的无定型固体分散体的差示热量分析结果。
图14表示的是本发明采用喷雾干燥法制备的式I化合物在非离子型聚合物中的无定型固体分散体的热重分析结果。
图15表示的是本发明采用喷雾干燥法制备的式I化合物在非离子型聚合物中的无定型固体分散体的在不同条件下2周后的偏光显微镜图像。
图16表示的是本发明采用喷雾干燥法制备的式I化合物在非离子型聚合物中的无定型固体分散体的在不同条件下4周后的偏光显微镜图像。
图17表示的是本发明采用喷雾干燥法制备的式I化合物在非离子型聚合物中的无定型固体分散体的在不同条件下3个月后的偏光显微镜图像。
图18表示的是本发明的式I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基本相同的抗肿瘤效果。
图19表示的是本发明采用喷雾干燥法制备的式I化合物在非离子型聚合物中的无定型固体分散体与式I化合物在大鼠体内药代动力学的对比
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：
1.合成实验条件：
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℃)；
快速柱色谱分离时使用200-300目硅胶作为载体，TLC表示薄层色谱法；
通常情况下，反应的进度通过TLC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。
2.测试试剂与仪器：
2.1测试试剂
名称 规格 公司
Soluplus N/A BASF
Kollidon VA64 N/A BASF
PVPK30 N/A BASF
HPMC(E5) E5 Colorcon
HPMCAS-MG MG Ashland
SGF(pH 1.2)：
将约950mL的水转移到1L容量瓶中。加入1.4mL浓盐酸(12N)和2g氯化钠，并搅拌以确保均匀性。用水稀释至1L，调节pH至1.2。
FaSSIF(pH 6.5)：
步骤1：在500mL容量瓶中加入0.210g氢氧化钠，2.235g磷酸二氢钠和3.093g氯化钠，加入约0.450L水，并用1N氢氧化钠或1N盐酸调节pH至6.5。用纯水补足体积(0.5升)。
步骤2：将1.120g SIF粉末用0.250L缓冲液(步骤1)放入500mL容量瓶中，然后加水至体积，并充分混合。
2.2仪器型号：
Figure PCTCN2018114658-appb-000002
2.3仪器参数：
2.3.1偏光显微镜(PLM)
测试中使用的偏振光显微镜方法的细节如下：
-尼康LV100POL配备500万像素CCD
-物理镜头：20X/50X
2.3.2 X射线粉末衍射仪(XRPD)
使用以下方法在XRPD上运行样品：
-管：Cu：K-α
Figure PCTCN2018114658-appb-000003
-发电机电压：40kV；电流：40mA。
扫描范围：4～40度；
-样品转速：15rpm。
-扫描速率：10℃/分
2.3.3差示热量分析仪(DSC)
测试中使用的DSC方法的细节如下：
-10℃/min从30℃加热至300℃
-循环DSC条件：加热温度从30℃到235℃，10℃/min，保持2min(加热循环1)；冷却温度由235℃降至30℃，20℃/分钟保持5min；从30℃到250℃，10℃/min(加热循环2)。
-mDSC方法：2℃/min的速度从30-250℃加热，1℃/min的调制速率
-DSC检测方法(用于SDD)：将样品预热至100℃，以10℃/min，除去水分，保持2分钟，然后以10℃/min冷却至30℃，保持2分钟，然后加热至250℃，10℃/min。
2.3.4热重分析仪(TGA)
将样品(2～5mg)放入铝锅中，运行如下：
在大气条件下以10℃/min的速率从室温加热到300℃，如果样品的损失重量大于20％，则试验完成。
-对于TGA-MS方法，使用第二电子倍增器(SEM)检测器进行溶剂检测。
2.3.5动态水分吸附分析(DVS)
将约10mg样品转移到DVS中，记录相对于25℃的大气湿度的重量变化。
使用以下参数：
-平衡：dm/dt：0.01％/min。(最小：10分钟，最大180分钟)。
-干燥：0％RH，持续120分钟
-RH(％)测量步骤：10％
-RH(％)测量步长范围：0-90-0％
吸湿性评估标准如下表所示：
吸湿性评估标准
吸湿性分类 吸湿性标准*
溶解的 吸收足够多的水分变成液体
非常吸湿 ΔW％≥15％
吸湿 15％＞ΔW％≥2％
轻微吸湿 2％＞ΔW％≥0.2％
不吸湿 ΔW％＜0.2％
*在25±1℃以及80±2％RH(欧洲药典6.0)
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 PCTCN2018114658-appb-000004
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)溶液中，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)加入到反应液中，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，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(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)溶液中，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，将反应液萃取，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)溶液中，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)溶液中，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)溶液中，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)。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)。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，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。粗产物用制备薄层层析板纯化(二氯甲烷∶甲醇＝50∶1)，得到产品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 PCTCN2018114658-appb-000005
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，21x 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(API)在非离子型聚合物上的无定型固体分散体的制备
分别选择五种非离子型聚合物PVPK30，PVPVA64，HPMCE5，HPMCAS-MG和Soluplus用于制备式I化合物的无定型固体分散体，使用两种式I化合物与聚合物的比例，即1∶1和1∶2(w/w)，将式I化合物和相应的聚合物分别称重并一起溶解在圆底烧瓶中的溶剂中。对于PVPVA64，HPMCE5，HPMCAS和Soluplus ASD而言溶剂是四氢呋喃∶水(7∶3)，对于PVPK30而言溶剂是四氢呋喃∶乙醇(7∶3)。通过旋转蒸发法除去溶剂之后，收集残余物固体产物并在真空干燥箱中干燥过夜。然后在第二天取出产品，并分别用偏光显微镜图像(PLM)、X-射线粉末衍射图(XRPD)、差示热量分析(DSC)、动态水分吸附分 析(DVS)和热重分析(TGA)来进行表征。
表1
溶质 溶剂
API/PVPK30(1/1)(w/w) 四氢呋喃∶乙醇(7∶3)
API/PVPVA64(1/1)(w/w) 四氢呋喃∶水(7∶3)
API/HPMCE5(1/1)(w/w) 四氢呋喃∶水(7∶3)
API/HPMCAS-MG(1/1)(w/w) 四氢呋喃∶水(7∶3)
API/Soluplus(1/1)(w/w) 四氢呋喃∶水(7∶3)
API/PVPK30(1/2)(w/w) 四氢呋喃∶乙醇(7∶3)
API/PVPVA64(1/2)(w/w) 四氢呋喃∶水(7∶3)
API/HPMCE5(1/2)(w/w) 四氢呋喃∶水(7∶3)
API/HPMCAS-MG(1/2)(w/w) 四氢呋喃∶水(7∶3)
API/Soluplus(1/2)(w/w) 四氢呋喃∶水(7∶3)
实施例4
动态溶解度实验
首先称量实施例2制备得到的化合物(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)(约4mg)和实施例3中制备得到的无定型固体分散体(简称ASD)(相当于约4mg的式I化合物的重量)到2mL HPLC小瓶中，并分别加入1mL的两个测试溶液SGF和FaSSIF。然后将样品放置于控温混合器中，在37℃，700rpm下震荡。在0.5小时，1小时和2小时时取出300μL悬浮液，在14000rpm条件下离心4分钟。然后将上清液以14000rpm条件下重新离心4分钟。之后，用稀释的乙腈∶甲醇(1∶1)稀释上清液三倍以防止沉淀，并通过HPLC分析样品的浓度。测量最终悬浮液的pH值，并在溶解度测试后通过PLM测试固体残留物。
表2和表3分别列出了10种无定型固体分散体在SGF和FaSSIF中的动力学溶解度结果。从数据可以看出，与实施例2制备得到的原始化 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-甲酰胺相比，在0.5h、1h和2h时，实施例3制备的所有无定型固体分散体的溶解度都有所提高。
对于含有HPMCE5的无定型固体分散体，在SGF和FaSSIF中，1∶2比例的无定型固体分散体显示出比1∶1比例的无定型固体分散体更高的浓度，并且最高浓度在0.5小时内可以观测到，在SGF中可以观察到含有HPMCE5的无定型固体分散体的浓度比实施例2制备得到的原始化合物(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-甲酰胺高出11倍，而在FaSSIF中则高出14倍。。
对于含有HPMCAS的无定型固体分散体，可以观察到，相较于实施例2制备得到的原始化合物(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)，含有HPMCAS的无定型固体分散体在FaSSIF缓冲液中的溶解度提高10倍以上，在SGF中的溶解度提高不到4倍。这应该是由于HPMCAS-MG是肠溶性聚合物，它在中性缓冲液(pH 6左右)中可以很好地溶解。所以在FaSSIF中，HPMCAS更有助于提高溶解度。
表2无定型固体分散体在SGF中37℃条件下的动态溶解度结果
Figure PCTCN2018114658-appb-000006
Figure PCTCN2018114658-appb-000007
表3无定型固体分散体在FsSSIF中37℃条件下的动态溶解度结果
Figure PCTCN2018114658-appb-000008
实施例5
物理稳定性实验
在40℃/75％RH和50℃/环境条件下测试实施例3制备的分别含有HPMCAS和Soluplus两种聚合物的无定型固体分散体的物理稳定性，将无定型固体分散体在40℃/75％RH和50℃/环境条件下保持5天后取出进行PLM或XRPD表征，以检查其物理稳定性。图6和图7显示无定型固体分散体在物理稳定性试验后5天后的结果。可以看到，在40℃/75％RH和50℃/环境条件下5天后，对于HPMCAS，所有的无定型固体分散体均保持无定形状态；对于在40℃/75％RH和50℃的环境条件下，含有Soluplus的无定型固体分散体存储产物仍然保持无定型。
实施例6
(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)在非离子型聚合物上的无定型固体分散体的制备
采用喷雾干燥法，分别选择HPMCAS和Soluplus来制备无定型固体分散体。制备的无定型固体分散体制剂中的药物载药量定为40％。用于喷雾干燥的溶剂为四氢呋喃∶水(70∶30V∶V)。溶剂中无定型固体分散体的浓度为30mg/ml。喷雾干燥参数列于表4中。对于Soluplus无定型固体分散体制剂，产物的收率为58.5％，对于HPMCAS无定型固体分散体制剂，产物的收率为54.5％。无定型固体分散体产品在制备完成后在真空烘箱中干燥2天。
表4喷雾干燥参数
参数设定  
溶剂 THF∶H 2O(70∶30)
式I化合物浓度(mg/mL) 30
喷嘴口径(mm) 1.0
设定空速(m 3/min) 0.30
实际空速(m 3/min) 0.28
设定入口温度(℃) 90
实际空气温度(℃) 90
腔室外温度(℃) 54.8
旋风器内温度(℃) 41.2
上气室(mBar) 1.9
参数设定  
旋风器压力(mBar) 24.5
设定喷嘴气流(L/min) 9.0
实际喷嘴气流(L/min) 9.4
旋风器大小 中等
泵因子(Pump factor) 420
冷却气流(m 3/min) 0.15
通过XRPD，PLM，DVS，DSC和TGA-MS来表征上述采用喷雾干燥法制备的无定型固体分散体(简称SDD)。图8和图9分别列出了上述无定型固体分散体的PLM和XRPD表征结果。图10和图11分别显示上述无定型固体分散体的动态水分吸附分析(DVS)结果，从图10和11中可以看出，式I化合物-Soluplus无定型固体分散体显示出与实施例2制备的原始式I化合物相似的吸湿和解吸湿行为，而式I化合物-HPMCAS无定型固体分散体则显示相对来讲较少的吸水性，这应该归因于HPMCAS的耐吸湿性。XRPD表明，在动态水分吸附分析实验后上述两种无定型固体分散体均未观测到结晶形成(图12)。DSC分析结果表明，上述两种无定型固体分散体的Tg分别为86.8℃(Soluplus无定型固体分散体)和103.5℃(HPMCAS无定型固体分散体)(图13)。图14显示了两种无定型固体分散体的TGA-MS分析结果。两种无定型固体分散体的剩余的溶剂残留量约为1％左右。
实施例7
动态溶解度实验
将称重量的化合物(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(约4mg)和根据实施例6制备的两种无定型固体分散体(相当于约4mg的式I化合物的重量)分别称量到2mL HPLC小瓶中，并分别加入1mL的两个缓冲液SGF和FaSSIF。然后将样品在控温混合器中在37℃，700rpm下振荡。在0.5小时，1小时，2小时和4小时时分别取出200μL悬浮液，并以14000rpm离心5分钟。然后将上清液以14000rpm重新离心5分钟。之后，用稀 释的ACN∶MeOH(1∶1)稀释上清液四倍以防止沉淀，并通过HPLC分析浓度。
表5和表6分别列出了两种无定型固体分散体制剂样品的动力学溶解度结果。式I化合物在Soluplus无定型固体分散体中的溶解度得到提高(在SGF缓冲液中提高约4倍，在FaSSIF缓冲液中提高约10倍)。对于含HPMCAS的无定型固体分散体，在FaSSIF缓冲液中溶解度提高10倍以上，在SGF缓冲液中的溶解度也得到提高。
表5两种无定型固体分散体在SGF中37℃条件下的动态溶解度
Figure PCTCN2018114658-appb-000009
表6两种无定型固体分散体在FaSSIF中37℃条件下的动态溶解度
Figure PCTCN2018114658-appb-000010
实施例8
物理稳定性实验
在25℃/密闭、40℃/75％RH和50℃/密闭环境条件下分别测试根据实施例6制备的两种无定型固体分散体的稳定性，分别在2周，1个月和3个月取出样品，用PLM来表征其物理稳定性，并通过HPLC检查样品的杂质。图15、16和17分别显示了几种无定型固体分散体在稳定性测试后2周、4周以及3个月后的PLM表征结果。我们可以看到， 物理稳定性试验3个月后，两种无定型固体分散体都保持为无定形状态。
表7显示了上述物理稳定性测试后无定型固体分散体的HPLC纯度。可以看到，对于式I化合物-Soluplus无定型固体分散体样品，纯度没有太大变化；对于式I化合物-HPMCAS无定型固体分散体样品，总有关物质在40℃/75％RH和50℃/环境条件下有略微增加，约2％至3％，而式I化合物-HPMCAS无定型固体分散体的杂质在25℃下没有增加。
表7两种无定型固体分散体在不同储存条件下HPLC的纯度值
Figure PCTCN2018114658-appb-000011
实施例9
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-甲酰胺表现出较强的激酶抑制活性(IC50＜100nM)。
实施例10
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 PCTCN2018114658-appb-000012
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11
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℃下刺激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-甲酰胺对B细胞具有较强的抑制活性，其IC50值小于10nM。
实施例12
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℃下刺激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13
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14
化合物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15
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16
化合物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、化合物在大鼠体内的药物代谢动力学试验设计
Figure PCTCN2018114658-appb-000013
6.使用LC-MS/MS(UPLC-MS/MS：液相Waters Acquity UPLC(USA)和质谱5500Q 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 PCTCN2018114658-appb-000014
Figure PCTCN2018114658-appb-000015
Figure PCTCN2018114658-appb-000016
使用药代动力学专业软件WinNonlin【型号：Phoenix TM
Figure PCTCN2018114658-appb-000017
6.1厂家：Pharsight Corporation】计算药代动力学参数。【Phoenix 1.1User’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-甲酰胺表现出较好的生物利用度(＞40％)。
实施例17
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18
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18)。
实施例19
式I化合物和其在非离子型聚合物中的无定型固体分散体在大鼠体内的药物代谢动力学比较
实验步骤如下：
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.SD大鼠按要求随机分组，灌胃给药。API组待测化合物用0.5％甲基纤维素，4％Poloxamer 188，20％PEG400，4％DMSO作为溶媒悬浮后给药，ASD组待测化合物用0.5％甲基纤维素作为溶媒悬浮后给药。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：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.使用LC-MS/MS(UPLC-MS/MS：液相Waters Acquity UPLC(USA)和质谱5500Q 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 PCTCN2018114658-appb-000018
Figure PCTCN2018114658-appb-000019
Figure PCTCN2018114658-appb-000020
使用药代动力学专业软件WinNonlin【型号：Phoenix TM
Figure PCTCN2018114658-appb-000021
6.1厂家：Pharsight Corporation】计算药代动力学参数。【Phoenix 1.1User’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-甲酰胺的150和500mpk剂量组的血药浓度明显低于其在非离子型聚合物中的无定型固体分散体50mpk。此外，上述两个剂量组的药物暴露量(AUC0-24h)没有跟给药剂量成比例增加；而两个配方的无定型固体分散体组的药物暴露量跟给药剂量成比例增加。综上，式I化合物在非离子型聚合物中的无定型固体分散体在大鼠体内的药物暴露量要明显高于该化合物本身，同时随剂量的增加成比例增加，具有良好的线性关系；而无定型固体分散体同等剂量范围内药物暴露量趋于饱和，没有表现出应有的线性关系(实验结果见图19)。
应当理解，尽管本文为了示例目的已经描述了本发明的特定实施方案，但是可以在没有背离本发明的精神和范围下进行各种修饰。因此，本发明不限于除了所附权利要求书的部分。

Claims (7)

  1. 一种如式(I)所示的5-氨基吡唑甲酰胺化合物在非离子型聚合物中的无定型固体分散体，
    Figure PCTCN2018114658-appb-100001
    其中，所述非离子型聚合物选自聚维酮、纤维素衍生物、聚乙烯酰胺-聚乙烯酯-聚乙二醇接枝共聚物。
  2. 根据权利要求1所述的无定型固体分散体，其中所述聚维酮选自聚维酮K30(PVPK30)、聚维酮VA64(PVPVA64)；所述纤维素衍生物选自羟丙基甲基纤维素E5(HPMCE5)、醋酸羟丙基甲基纤维素琥珀酸酯(HPMCAS-MG)；所述聚乙烯酰胺-聚乙烯酯-聚乙二醇接枝共聚物选自聚乙烯己内酰胺-聚乙酸乙烯酯-聚乙二醇接枝共聚物。
  3. 根据权利要求1或2所述的如式(I)所示的5-氨基吡唑甲酰胺化合物在非离子型聚合物中的无定型固体分散体的制备方法，包括：将式I化合物分别与所述非离子型聚合物混合溶解于溶剂中，通过旋转蒸发法除去溶剂之后，收集残余物固体产物并真空干燥。
  4. 根据权利要求1或2所述的如式(I)所示的5-氨基吡唑甲酰胺化合物在非离子型聚合物中的无定型固体分散体的制备方法，包括：将式I化合物分别与所述非离子型聚合物混合溶解于溶剂中，通过喷雾干燥得到无定型固体分散体。
  5. 药物组合物，其包含权利要求1或2所述的式(I)所示的5-氨基吡唑甲酰胺化合物在非离子型聚合物中的无定型固体分散体和药学 上可接受的载体。
  6. 权利要求1或2所述的无定型固体分散体或权利要求5所述药物组合物在制备BTK抑制剂药物中的用途。
  7. 如权利要求6所述的用途，其中，所述BTK抑制剂药物是指预防或治疗由BTK介导的疾病的药物，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。
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* Cited by examiner, † Cited by third party
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CN114929203A (zh) * 2019-12-20 2022-08-19 英特维特国际股份有限公司 分散在聚合物基质中的吡唑化合物的药物组合物

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