WO2016155359A1 - 3-羟基吡啶化合物、其制备方法及其制药用途 - Google Patents

3-羟基吡啶化合物、其制备方法及其制药用途 Download PDF

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WO2016155359A1
WO2016155359A1 PCT/CN2015/097246 CN2015097246W WO2016155359A1 WO 2016155359 A1 WO2016155359 A1 WO 2016155359A1 CN 2015097246 W CN2015097246 W CN 2015097246W WO 2016155359 A1 WO2016155359 A1 WO 2016155359A1
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pharmaceutically acceptable
acceptable salt
subject
compound
compound according
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French (fr)
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周云隆
蔡遂雄
王光凤
焦玲玲
闵平
景羽
郭明
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SHENYANG SUNSHINE PHARMACEUTICAL CO Ltd
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Priority to US15/561,435 priority patent/US10149841B2/en
Priority to JP2017551703A priority patent/JP6473519B2/ja
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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
    • A61K31/4412—Non condensed pyridines; Hydrogenated derivatives thereof having oxo groups directly attached to the heterocyclic ring
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D213/78—Carbon atoms having three bonds to hetero atoms, with at the most one bond to halogen, e.g. ester or nitrile radicals
    • C07D213/81—Amides; Imides
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • 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
    • A61P7/06—Antianaemics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D213/62—Oxygen or sulfur atoms
    • C07D213/63—One oxygen atom
    • C07D213/65—One oxygen atom attached in position 3 or 5
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • the present invention relates to the field of medicine, and in particular to a 3-hydroxy compound, a process for the preparation of the compound, and a medicament for the preparation of a medicament for inhibiting the activity of an ischemic inducing factor (HIF) proline hydroxylase application.
  • HIV ischemic inducing factor
  • Hypoxia inducible factor is a transcriptional activator containing alkaline helix-loop-helix (bHLH) and PAS (Per/Arnt/Sim), which mediates a series of genes in biological cells. Regulated in response to cellular hypoxia.
  • bHLH alkaline helix-loop-helix
  • PAS Per/Arnt/Sim
  • HIF erythropoientin
  • HIF-a contains an oxygen-dependent degradation domain (ODDD), which is a key unit in response to cellular oxygen content.
  • ODDD oxygen-dependent degradation domain
  • HIF-a can form a stable dimer with HIF-b. After entering the nucleus, this dimer will activate important enzymes such as glucose metabolism-related enzymes, GLUT-1, erythropoietin, and vascular endothelial growth factor (VEGF). The expression of the line, thereby counteracting the hypoxic state of the cells.
  • HIF-b is a type of aromatic hydrocarbon nuclear translator (ARNT) that forms a heterodimer upon binding to HIF-a to activate downstream gene transcription.
  • HIF-1a was first discovered by wang in 1995 and is widely expressed in humans and mice.
  • HIF-2a was isolated and identified in 1997, and its protein sequence is 48% similar to HIF-1a, so it also has a similar function to HIF-1a, but HIF-2a is only in lung, endothelial cells and carotid arteries. expression.
  • HIF-3a is the newly discovered HIF-a subtype, but so far there has been very little research on it.
  • HIF-a in cells with normal oxygen content, but HIF-a cannot be stably present in cells with normal oxygen content, and the half-life is only 5 minutes. HIF-a can only be used under anoxic conditions. Stable presence allows normal functioning to activate downstream transcription factors.
  • proline at 402 and 564 in the ODDD region of HIF-a is oxidized by proline hydroxylase to form 4-hydroxyproline, resulting in HIF-a not being able to interact with HIF- b dimerization, but soon binds to the pVHL protein and is subsequently degraded, thereby failing to function as an anti-hypoxia.
  • Prolyl hydroxylase (also referred to as PHD or EGLN), which plays a key role in the degradation of HIF-a, is a 2-oxoglutatarate (2-OG)-dependent oxygenation.
  • the enzyme, PHD uses 2-OG and divalent iron ions as a prosthetic group to transfer an oxygen atom to the 4-position of the proline molecule to form hydroxyproline, and converts 2-OG into a molecule of carbon dioxide and succinic acid.
  • 2-OG analogues or divalent nickel, cobalt and manganese ions can antagonize the oxidation of proline by HIF-a by PHD, inhibit the degradation process of HIF-a, and enable HIF-a to dimerize with HIF-b.
  • PHD has three subtypes: PHD1, PHD2, and PHD3. Further research suggests that inhibition of PHD1 can help treat skeletal muscle cell degeneration, protect myofibroblasts in ischemic conditions, treat inflammatory bowel disease and colitis, treat heart failure and ischemia in patients with heart disease and kidney disease. . However, no studies have shown that the other two PHD subtypes differ in function.
  • EPO erythropoietin
  • EPO erythropoietin
  • red blood cell proliferation differentiation and maturation.
  • EPO can stimulate bone marrow hematopoietic function, increase the number of red blood cells in a timely and effective manner, thereby improving the oxygen carrying capacity of blood.
  • EPO can enhance the body's ability to combine oxygen, transport and supply, and improve hypoxia. Under normal physiological conditions, EPO is mainly synthesized and released by kidney tissue, so patients with renal failure suffer from ischemia due to the inability to synthesize EPO in the body.
  • Another object of the invention is to provide a process for the preparation of the above compounds.
  • a further object of the present invention is to provide a pharmaceutical use of the above compound or a pharmaceutically acceptable salt thereof.
  • R 1 and R 2 are independently hydrogen.
  • R 3 is selected from hydrogen, C1-C7 straight chain, branched or cyclic alkyl
  • R 4 , R 5 , R 6 , R 7 and R 8 are each independently selected from a C1-C7 alkyl group, a halogenated C1-C7 alkyl group, a C1-C3 alkoxy group, a halogenated C1-C3 alkoxy group, a halogen. a hydroxy, hydrogen, amino, nitro, cyano substituted or unsubstituted aromatic or aromatic heterocyclic ring. or
  • R 4 , R 5 , R 6 , R 7 and R 8 are linked to each other by an oxygen bridge to form a molecular formula having the structure (II) of the formula: or a pharmaceutically acceptable salt thereof:
  • n is selected from an integer of 1, 2, 3 or 4;
  • a 1 and A 2 are independently selected from oxygen, carbon or nitrogen atoms.
  • the pharmaceutically acceptable salts of the compounds of formula (I) and (II) are preferably formed by reaction with a pharmaceutically acceptable base.
  • the pharmaceutically acceptable bases include, but are not limited to, sodium hydroxide, sodium carbonate, potassium hydroxide, potassium carbonate, magnesium hydroxide, magnesium oxide, calcium hydroxide, calcium oxide, and the like.
  • Preferred compounds according to the invention include those selected from the group consisting of:
  • a method of preparing a compound of the invention comprises the steps of:
  • Step 1 5-Bromo-3-fluoropyridin-2-carbonitrile in the presence of methanol
  • Step 2 The intermediate (III) obtained in the step 1 is mixed with Ar0H and a ligand in a solvent, and the Ullman reaction is carried out to form an ether intermediate (IV) with the participation of a catalyst.
  • R 4 , R 5 , R 6 , R 7 and R 8 are each independently selected from C 1 -C 7 alkyl, halo C 1 -C 7 alkyl, C 1 -C 3 alkoxy, halo C 1 -C 3 alkoxy, Halogen, hydroxy, hydrogen, amino, nitro, cyano substituted or unsubstituted aromatic or aromatic heterocyclic ring;
  • Step 3 The intermediate (IV) obtained in the step 2 is reacted with HBr under reflux to be hydrolyzed to form a 3-hydroxypyridine-2-carboxylic acid derivative (V).
  • Step 4 Amidation reaction of the intermediate (V) obtained in the step 3 with the ⁇ -R 3 substituted amino acid benzyl ester (VI) in the presence of a condensing agent to obtain a 3-hydroxypyridine-2-carboxylic acid benzyl ester intermediate ( VII):
  • Step 5 The intermediate (VII) obtained in the step 4 is subjected to hydrogenolysis under a hydrogenolysis condition in a solvent, in the presence of a catalyst, and at room temperature to remove a benzyl ester protecting group to finally form a compound corresponding to the formula I.
  • the starting material 5-bromo-3-fluoropyridine-2-carbonitrile can be obtained by a commercial route, for example, from sigma or J&K, and the reaction is carried out at room temperature.
  • the catalyst in the above step 2 is preferably copper (I) iodide, and the preferred metal ligands are N,N-dimethylglycine, N-methylproline, N,N-tetramethylethylenediamine, etc.
  • Preferred reaction solvents are 1,4-dioxane, toluene, tetrahydrofuran. The reaction is preferably carried out by heating to 70 ° C to 120 ° C.
  • the step 3 comprises deprotecting the intermediate (IV) in hydrobromic acid/glacial acetic acid and simultaneously hydrolyzing to obtain a 3-hydroxypyridine-2-carboxylic acid intermediate (V), preferably
  • the ratio of hydrobromic acid to glacial acetic acid is from 2:1 to 1:3, the preferred reaction temperature is from 90 to 140 ° C, and the preferred heating reaction time is from 6 to 12 hours.
  • the ⁇ -R 3 amino acid benzyl ester (VI) may be in the form of its hydrochloride, and the amino acid ⁇ -R 3 benzyl ester hydrochloride may be selected from glycine benzyl ester hydrochloride, ( ⁇ ). Or ⁇ ) alanine, ( ⁇ or ⁇ ) valine, ( ⁇ or ⁇ ) leucine, ( ⁇ or ⁇ ) isoleucine, etc., preferred solvents are dichloromethane, chloroform, tetrahydrofuran, 1, 4-dioxane, N,N-dimethylformamide, N-methylpyrrolidone, and the like.
  • Preferred amidation catalysts are 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), 1-hydroxybenzotriazole (HOBT), bicyclohexyl carbon Preferred bases such as diimine (DCC) are triethylamine and N,N-diisopropylethylamine.
  • the reaction of this step is carried out by mixing the intermediate (V) with the amino acid benzyl ester hydrochloride (VI) and a condensing agent at room temperature for 10 hours or more.
  • the preferred catalyst is palladium (0) / C, palladium hydroxide (II), palladium hydroxide (II) / carbon, platinum (IV) dioxide and the like, preferably a solvent for the hydrogenolysis of methanol, ethanol, iso Propanol, tetrahydrofuran, ethyl acetate, and the like.
  • the present invention also relates to the use of a compound of the present invention or a pharmaceutically acceptable salt thereof for the preparation of a medicament for inhibiting HIF proline hydroxylase; the use of the compound of the present invention for the preparation of a medicament for promoting endogenous EPO production; The use of a compound for the preparation of a medicament for stabilizing hypoxia-inducible factor alpha; the use of a compound of the invention in the preparation of a medicament for treating chronic disease-associated anemia in a subject, wherein the chronic disease-associated anemia is selected from the group consisting of rheumatoid joints Inflammation, rheumatic fever and inflammatory bowel disease; use of a compound of the invention in the manufacture of a medicament for increasing the production of inflammatory cytokines in a subject, wherein the inflammatory cytokines include tumor necrosis factor, interleukin and interferon; The use of a compound for the preparation of a medicament for treating an anemia in a subject which is resistant to the external administration of
  • the present invention also relates to a method of inhibiting HIF proline hydroxylase in a subject, comprising administering to a subject a compound of the present invention or a pharmaceutically acceptable salt thereof; and a method for promoting production of endogenous EPO in a subject Including administering a compound of the present invention or a pharmaceutically acceptable salt thereof to a subject; a method for stabilizing hypoxia-inducible factor alpha in a subject, comprising administering to the subject a compound of the present invention or a pharmaceutically acceptable salt thereof; A method for treating chronic disease-associated anemia in a subject, comprising administering to the subject a compound of the present invention or a pharmaceutically acceptable salt thereof, wherein the chronic disease-associated anemia is selected from the group consisting of rheumatoid arthritis, rheumatic fever And inflammatory bowel disease.
  • the invention also relates to a method of increasing the production of a subject's inflammatory cytokine comprising administering to a subject a compound of the invention, or a pharmaceutically acceptable salt thereof, wherein the inflammatory cytokine comprises tumor necrosis factor, interleukin and interferon .
  • a further aspect of the invention relates to a method of treating a treatment in which an anemia of a subject is resistant to external administration of erythropoietin, comprising administering to the subject a compound of the invention or a pharmaceutically acceptable salt thereof, wherein the compound enhances hematopoiesis Presuppose the response of the cells to the erythropoietin.
  • the present invention also relates to a method of increasing the production of a desired factor in iron uptake, iron transport, and iron utilization in a subject, comprising administering to the subject a compound of the present invention or a pharmaceutically acceptable salt thereof, wherein the factor is selected from the group consisting of erythroid cells Aminolevulinate synthase, transferrin, transferrin receptor and ceruloplasmin.
  • Step 4 Preparation of benzyl 2-(3-hydroxy-5-phenoxypyridine-2-carboxamido)acetate
  • Step 5 Preparation of 2-(3-hydroxy-5-phenoxypyridine-2-carboxamido)acetic acid.
  • Benzyl 2-(3-hydroxy-5-phenoxypyridine-2-carboxamido)acetate (106 mg) was dissolved in methanol (10 ml), and 10% palladium(II) hydroxide was added under nitrogen. Carbon (10 mg) was then added to hydrogen and stirred at room temperature for 10 hours. The catalyst was removed by suction filtration, and the filtrate was concentrated to ethyl 2-(3-hydroxy-5-phenoxypyridine-2-carboxamido)acetic acid (46 mg, yield 57%).
  • Step 4 Preparation of ⁇ [5-(2,3-dimethylphenoxy)-3-hydroxy-pyridine-2-carbonyl]-amino ⁇ -benzyl acetate
  • Step 5 Preparation of 2-(5-(2,3-dimethylphenoxy)-3-hydroxypyridine-2-carboxamido)acetic acid. 100 mg (0.25 mmol) of ⁇ [5-(2,3-dimethylphenoxy)-3-hydroxy-pyridine-2-carbonyl]-amino ⁇ -benzyl acetate in 10 ml of methanol under nitrogen Under the environment, 10% palladium(II) hydroxide/carbon 20 mg was added, and then the reaction was stirred at room temperature for 10 hours with hydrogen gas.
  • Step 4 Preparation of ⁇ [5-(3-chlorophenoxy)-3-hydroxy-pyridine-2-carbonyl]-amino ⁇ -benzyl acetate
  • the dichloromethane solution was washed with water (30 ml), the organic phase was collected, the organic solvent was evaporated, and the residue was subjected to column chromatography and washed.
  • the solvent was ethyl acetate/petroleum ether (1/4) to give ⁇ [5-(3-chlorophenoxy)-3-hydroxy-pyridine-2-carbonyl]-amino ⁇ -benzyl acetate as a pale yellow oil. Things.
  • Step 5 Preparation of 2-(5-(3-chlorophenoxy)-3-hydroxypyridine-2-carboxamido)acetic acid.
  • 150 mg of ⁇ [5-(3-chlorophenoxy)-3-hydroxy-pyridine-2-carbonyl]-amino ⁇ -benzyl acetate was dissolved in 10 ml of methanol, and 10% palladium hydroxide (II) was added under nitrogen atmosphere. / Charcoal 15 mg, after which hydrogen was introduced and stirred at room temperature for 10 hours. The catalyst was removed by suction filtration, and the filtrate was concentrated to ethyl 2-(5-(2,3-difluorophenoxy)-3-hydroxypyridine-2-carboxamido)acetic acid as a white solid.
  • Step 4 Preparation of ⁇ [3-hydroxy-5-(naphthalene-2-oxy)-pyridine-2-carbonyl]-amino ⁇ -benzyl acetate
  • Step 4 Preparation of ⁇ [5-(2,4-dimethylphenoxy)-3-hydroxy-pyridine-2-carbonyl]-amino ⁇ -benzyl acetate
  • Step 5 Preparation of 2-(5-(2,4-dimethylphenoxy)-3-hydroxypyridine-2-carboxamido)acetic acid. 50 mg (0.12 mmol) of ⁇ [5-(2,4-dimethylphenoxy)-3-hydroxy-pyridine-2-carbonyl]-amino ⁇ -benzyl acetate in 10 ml of methanol under nitrogen 10% palladium(II) hydroxide/carbon 10 mg was added under the environment, and then the reaction was stirred at room temperature for 10 hours with hydrogen. The catalyst was removed by suction filtration, and the filtrate was concentrated to ethyl 2-(5-(2,4-dimethylphenoxy)-3-hydroxypyridine-2-carboxamido)acetic acid as a white solid, m.
  • reaction solution was concentrated, and then ethyl acetate (50 ml) and water (30 ml) was added and the layers were separated, and the ethyl acetate layer was concentrated and then subjected to column chromatography to give 5-(2,3-difluoro-phenoxy)-3 -Methoxy-pyridine-2-carbonitrile 160 mg, pale yellow solid, 30.5%.
  • Step 4 Preparation of ⁇ [5-(2,3-difluoro-phenoxy)-3-hydroxy-pyridine-2-carbonyl]-amino ⁇ -benzyl acetate
  • the experimental complete liver cancer cell Hep3B (China Type Culture Collection, CTCCC) culture complete medium required for MEM (Cat#GNM 41500, GIBCO, Hangzhou Gino Biomedical Technology Co., Ltd.) plus 10% serum FBS (Cat #10099-141, GIBCO) and 1% double-antibody P/S (Cat#GNM15140, supplied by Hangzhou Gino Biomedical Technology Co., Ltd.).
  • the cells were cultured at 37 ° C in a 5% CO 2 incubator.
  • the experimental ELISA kit was purchased from Quantikine IVD ELISA, Human Erythropoietin (R&D, DEP00).
  • the test control AKB-6548 was obtained by homemade or commercial purchase. The test substance was stored in the dark at -20 ° C.
  • test substance is dissolved in sterile water or DMSO as a solvent in the dark, and the test substance and the positive control are sufficiently dissolved to prepare a stock solution having a concentration of 10-1 mol/L or 10-2 mol/L.
  • the MEM medium containing 0.5% FBS was used as a diluent, and the test substance stock solution was diluted to prepare a test substance dilution solution having a concentration of 100 ⁇ mol/L and 10 ⁇ mol/L.
  • 200 ⁇ l/well (1.5 or 2.0*104 cells/well) of hepatoma cell Hep3B complete medium suspension was added and cultured overnight at 37 ° C in a 5% CO 2 incubator.
  • the old solution in the 96-well culture plate was removed, and the cells were washed once with MEM medium containing 0.5% FBS.
  • 200 ⁇ l/well of the test substance was added in the dark at a dose of 100 ⁇ mol/L and 10 ⁇ mol/L, and 2 wells were prepared for each dose as a test well and a backup.
  • Replace the drug solution with a diluent as a cell control well (excluding the test substance and vehicle).
  • the drug solution was replaced with a diluent containing the corresponding concentration of solvent (DMSO) as a vehicle control well (excluding the test substance). Incubate for 24 hours at 37 ° C in a 5% CO 2 incubator.
  • DMSO solvent
  • the supernatant was aspirated and stored as a sample at -20 ° C for use. Stop solution was added, 100 ⁇ l/well. The OD value was measured by a microplate reader A450nm-A600nm.
  • the EPO expression content (mIU/mL) of the test substance was obtained according to the standard curve, and the ratio of the EPO expression content of the test substance to the expression content of the positive control substance AKB6548EPO was calculated. The test results are shown in the following table:
  • hypoxia-inducible factor HIF-1 ⁇ with VBC complex (von Hippel–Lindau protein-Elongin B–Elongin C, VBC) was detected by fluorescence polarization (FP) method to determine HIF proline hydroxylase PHD2. (Prolyl hydroxylases 2, PHD2) Inhibitor activity of inhibitor compounds.
  • EBC buffer 50 mM Tris.HCl, 120 mM NaCl, 0.5% NP-40
  • a GST-VBC complex at a final concentration of 300 nM was added to the corresponding detection well (with only the EBC buffer well as a blank well).
  • the corresponding PHD2 proline hydroxylation reaction sample was then added as a final concentration of 100 nM substrate in the dark.
  • a full-wavelength multi-plate reader (TECAN infinite M1000) was used to detect lateral and longitudinal fluorescence intensity readings at 407 nM excitation wave and 518 nM emission wave.
  • mP 1000 ⁇ (transverse reading-G factor*longitudinal reading)/(transverse reading+G factor ⁇ longitudinal reading)
  • the lateral reading the lateral fluorescence intensity reading of the test hole - the lateral fluorescence intensity reading of the blank hole
  • the longitudinal reading the longitudinal fluorescence intensity reading of the test hole - the longitudinal fluorescence intensity reading of the blank hole
  • test compound PHD2 inhibition rate (%) was calculated according to the following formula:
  • Inhibition rate (%) 1 - (mP test well - mP negative control well) / (mP positive control well - mP negative control well).

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Abstract

本发明涉及具有下式(I)的化合物或其药学上可接受的盐，其中，R1，R2独立为氢；R3选自氢、C1-C7直链、支链或环状烷基；R4，R5，R6，R7和R8各自独立地选自C1-C7烷基、卤代C1-C7烷基等。本发明还涉及该化合物的制备方法，含有所述化合物或其药学上可接受的盐的药物组合物，以及所述化合物或其药学上可接受的盐在制备用于抑制HIF脯氨酸羟化酶的药物或制备促进生成内源性EPO的药物中的用途。

Description

3-羟基吡啶化合物、其制备方法及其制药用途 技术领域
本发明涉及医药领域，具体来说，本发明涉及3-羟基化合物，所述化合物的制备方法，及其在制备抑制缺血性诱导因子(HIF)脯氨酸羟化酶的活性的药物中的应用。
背景技术
缺氧诱导因子(hypoxia inducible factor,简称HIF)是一段含有碱性helix-loop-helix(bHLH)及PAS(Per/Arnt/Sim)的转录激活因子，在生物细胞内通过介导一系列的基因调节以响应细胞缺氧状态。(Chowdhury,R.,Hardy,A和Schofield,C.J.，人体氧气传感设备和其操控(The human oxygen sensing machinery and its manipulation)，Chem.Soc.Rev.，2008，37,1308-1319页；Kaelin,W.G.,Jr.,和Ratcliffe,P.J.,由后生动物进行的氧气传感：HIF羟化酶途径的中心角色(Oxygen sensing by metazoans:the central role of the HIF hydroxylase pathway)摩尔.Cell,2008,30,393-402页；Schofield,C.J.,和Ratcliffe,P.J.，通过HIF羟化酶进行氧气传感(Oxygen sensing by HIF hydroxylases)Nat.Rev.摩尔.Cell.Biol.，2004,5,343-354页)。
1992年，wang等在研究促红细胞生成素(erythropoientin,简称EPO，一种刺激红细胞生成的激素)时发现了刺激缺氧细胞生成EPO的转录激活因子，并将此因子命名为缺氧诱导因子，简称HIF。HIF对细胞在缺氧状态下的适应和存活至关重要，实验表明在HIF的作用下，即使将细胞的含氧量由正常的20％降低到1％，细胞仍然可以存活。
HIF由两个亚单元—HIF-a和HIF-b组成。HIF-a含有氧气依赖降解区(oxygen-dependent degradation domain,简称ODDD)是对细胞氧气含量响应的关键单元。HIF-a可以与HIF-b形成稳定的二聚体，此二聚体进入细胞核后将激活糖代谢相关酶、GLUT-1、促红细胞生成素以及血管内皮生长因子(VEGF)等重要酶或酶系的表达，从而对抗细胞的缺氧状态。HIF-b是一类芳香碳氢核转录子(aryl hydrocarbon nuclear translator,简称ARNT),在与HIF-a结合后形成杂二聚体从而激活下游的基因转录。
讫今为止，人类一共发现了三种HIF-a亚型:分别是HIF-1a,HIF-2a,HIF-3a。HIF-1a最早由wang于1995年发现，在人类和小鼠体内广泛表达。HIF-2a在1997年被分离识别，其蛋白序列与HIF-1a的有48％的相似性，因此也具有与HIF-1a相似的功能，但HIF-2a只在肺、内皮细胞及颈动脉中表达。HIF-3a是最新发现的HIF-a亚型，但目前为止对它的研究还非常少。
研究表明HIF-a在细胞内的表达不受氧含量的影响，但是HIF-a在含氧量正常的细胞内不能稳定存在，半衰期仅为5分钟，HIF-a只有在缺氧条件下才可以稳定存在从而正常的发挥激活下游转录因子的作用。在含氧量正常的细胞内，HIF-a的ODDD区内402、564两个位置的脯氨酸被脯氨酸羟化酶氧化生成4-羟基脯氨酸，导致HIF-a不能与HIF-b二聚，而是很快与pVHL蛋白结合并随后被降解，从而不能发挥抗缺氧的功能。在HIF-a降解过程起关键作用的脯氨酸羟化酶(Prolyl hydroxylase,也简称PHD或EGLN)是一种2-氧代戊二酸(2-oxoglutatarate，简称2-OG)依赖的加氧酶，PHD以2-OG与二价铁离子为辅基，将一个氧原子传递到脯氨酸分子的4位形成羟基脯氨酸，同时将2-OG转变为一分子二氧化碳及琥珀酸。2-OG类似物或者二价镍、钴、锰离子都可以拮抗PHD对HIF-a中脯氨酸的氧化过程，抑制HIF-a的降解过程，使HIF-a能够顺利与HIF-b二聚，从而激发下游的转录因子，并最终发挥抗缺氧的功能。研究发现PHD共有三种亚型：PHD1,PHD2,PHD3。进一步的研究提示，对PHD1的抑制可以有助于治疗骨骼肌细胞退化，可以在缺血情况下保护成肌纤维细胞，治疗炎症性肠炎及大肠炎，治疗心脏病及肾病患者的心衰及缺血。但是尚无研究表明其它两种PHD亚型在功能上有所区别。
HIF的重要作用之一是激活生物体内促红细胞生成素(EPO)的表达。作为一种糖蛋白激素，EPO能够刺激红细胞增殖、分化及成熟。EPO一方面能够刺激骨髓造血功能，及时有效的增加红细胞的数量，从而提高血液的携氧能力，另一方面EPO能够增强机体对氧的结合、运输及供应的能力，改善缺氧状态。在正常生理状况下，EPO主要由肾脏组织合成并释放，因此肾衰竭的病人因为体内不能正常的合成EPO而饱受缺血之苦。在20世纪八十年代后期，Amgen公司首次成功的将EPO工业化，并逐渐将EPO用于慢性肾衰竭、艾滋病、癌症及化疗导致贫血的病人。但是随着EPO的生产及应用得到了巨大的发展，外源性给以EPO仍然 面临几个问题：1，EPO的使用费用较高，尤其对于需要长期使用的病患是很大的负担。2，作为一种大分子糖蛋白，EPO同样具有生物利用度低，在生物体内半衰期短，易被胃肠道内的酶体水解等特点，因此EPO必须频繁的注射给药，限制了病人自己用药的可能，给病人来了很大的不便。3，工业化合成的EPO仍然不能避免免疫原性问题，产品存在一定的用药风险。
由于外源性大分子EPO在使用中存在的这些问题，开发小分子HIF脯氨酸羟化酶抑制剂，抑制HIF-a的降解，从而刺激人体生成内源性EPO，将很有希望替代外源性EPO，给病人更多的选择。
目前为止，已经有Akebia公司的AKB-6548以及Fibrogen公司的FG-4592两种HIF脯氨酸羟化酶进入临床二期研究中。(参考WO2012170377A1,US2010331374A1,US2010305097A1,P&GUS2007299086A1,US2004254215A1,US2007298104A1,US2009082357A1,US2010113444A1,WO2013134660,WO2010059552A1)。
Figure PCTCN2015097246-appb-000001
发明内容
本发明的目的是提供一种结构新颖的3-羟基吡啶化合物或其药学上可接受的盐。
本发明的另一个目的是提供制备上述化合物的方法。
本发明的再一个目的是提供含有上述化合物的药物组合物。
本发明进一步的目的是提供上述化合物或其药学上可接受盐的制药用途。
本发明的目的是通过下列构思实现的：
一种具有下式(I)结构的化合物或其药学上可接受盐：
Figure PCTCN2015097246-appb-000002
其中，R1，R2独立为氢。
R3选自氢、C1-C7直链、支链或环状烷基；
R4，R5，R6，R7和R8各自独立地选自C1-C7烷基、卤代C1-C7烷基、C1-C3烷氧基、卤代C1-C3烷氧基、卤素、羟基、氢、氨基、硝基、氰基取代或未取代的芳环或芳杂环。或者
R4，R5，R6，R7，R8相互之间以氧桥相联，形成具有下式结构(II)的分子式或其药学上可接受的盐：
Figure PCTCN2015097246-appb-000003
结构式(II)中，n选自1、2、3或4的整数；
A1和A2独立地选自氧、碳或氮原子。
结构式(I)和(II)的化合物的药学上可接受的盐优选地通过与药学上可接受的碱反应生成盐。所述的药学上可接受的碱包括，但不限于，氢氧化钠、碳酸钠、氢氧化钾、碳酸钾、氢氧化镁、氧化镁、氢氧化钙及氧化钙等。
根据本发明的优选化合物包括选自下列化合物：
Figure PCTCN2015097246-appb-000004
Figure PCTCN2015097246-appb-000005
结构式(I)化合物的合成路线如下个反应流程所示：
Figure PCTCN2015097246-appb-000006
根据本发明的一个方面，本发明化合物的制备方法包括下列步骤：
步骤1：在甲醇的存在下，使5-溴-3-氟吡啶基-2-甲腈
Figure PCTCN2015097246-appb-000007
与甲醇钠反应，生成5-溴-3-甲氧基吡啶-2-甲腈(中间体III)
Figure PCTCN2015097246-appb-000008
步骤2：将步骤1得到的中间体(III)与Ar0H和配体在溶剂中混合加热，在催化剂的参与下进行Ullman反应生成醚类中间体(IV)，
Figure PCTCN2015097246-appb-000009
其中Ar代表
Figure PCTCN2015097246-appb-000010
其中R4，R5，R6，R7和R8各自独立地选自C1-C7烷基、卤代C1-C7烷基、C1-C3烷氧基、卤代C1-C3烷氧基、卤素、羟基、氢、氨基、硝基、 氰基取代或未取代的芳环或芳杂环；
步骤3：将步骤2得到的中间体(IV)在回流下与HBr反应，水解生成3-羟基吡啶-2-甲酸衍生物(V)
Figure PCTCN2015097246-appb-000011
步骤4：将步骤3得到的中间体(V)与α-R3取代氨基酸苄酯(VI)在缩合剂存在下进行酰胺化反应，得到3-羟基吡啶-2-羧酸苄酯中间体(VII)：
Figure PCTCN2015097246-appb-000012
步骤5：将步骤4得到的中间体(VII)在氢解条件在溶剂中、催化剂存在和室温下进行氢解反应下脱去苄酯保护基，最终形成对应于I式的化合物。
在上述步骤1中，所用的原料5-溴-3-氟吡啶-2-甲腈可以通过商品化途径获得，例如购自sigma或J&K，所述反应在室温下进行。
上述步骤2中的催化剂优选为碘化亚铜(I)，优选的金属配体是N,N-二甲基甘氨酸、N-甲基脯氨酸、N,N-四甲基乙二胺等，优选的反应溶剂为1,4-二氧六环、甲苯、四氢呋喃。优选地加热到70℃到120℃进行反应。
在一个实施方案中，所述步骤3包括将中间体(IV)在氢溴酸/冰醋酸中进行脱保护及同时水解反应制得3-羟基吡啶-2-甲酸中间体(V)，优选的氢溴酸与冰醋酸的比例为2:1～1:3，优选的反应温度为90～140℃，优选的加热反应时间为6～12小时。
步骤4中，所述的α-R3氨基酸苄酯(VI)可以为其盐酸盐形式，所述的氨基酸α-R3苄酯盐酸盐可以选自甘氨酸苄酯盐酸盐、(α或β)丙氨酸、(α或β)缬氨酸、(α或β)亮氨酸、(α或β)异亮氨酸等，优选的溶剂为二氯甲烷、氯仿、四氢呋喃、1，4-二氧六环、N,N-二甲基甲酰胺、N-甲基吡咯烷酮等。优选的酰胺化催化剂为1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDCI)、1-羟基苯并三氮唑(HOBT)、二环已基碳二亚胺(DCC)等，优选的碱为三乙胺、N,N-二异丙基乙基胺。在一个实施方案中，通过混合中间体(V)与氨基酸苄酯盐酸盐(VI)及缩合剂在室温下搅拌反应10小时以上完成本步骤反应。
步骤5中，优选的催化剂为钯(0)/炭、氢氧化钯(II)、氢氧化钯(II)/炭、二氧化铂(IV)等，氢解的溶剂优选为甲醇、乙醇、异丙醇、四氢呋喃、乙酸乙酯等。
本发明还涉及本发明化合物或其药学上可接受的盐在制备抑制HIF脯氨酸羟化酶的药物中的应用；本发明化合物在制备促进生成内源性EPO的药物中的应用；本发明化合物在制备稳定缺氧诱导因子α的药物中的应用；本发明化合物在制备用于治疗对象的慢性疾病变相关贫血的药物中的应用，其中所述慢性疾病变相关贫血选自类风湿性关节炎，风湿热和炎症性肠道疾病；本发明化合物在制备增加对象炎性细胞因子产生的药物中的应用，其中所述炎性细胞因子包括肿瘤坏死因子，白细胞介素和干扰素；本发明化合物在制备治疗在对象的贫血对外部给予红细胞生成素的治疗具有抗性的药物中的应用，其中所述化合物增强了造血前提细胞对所述红细胞生成素的应答；本发明化合物在制备在对象中增加铁摄取、铁运输和铁利用中所需因子产生的药物中的应用，其中所述因子选自类红细胞氨基乙酰丙酸合酶、运铁蛋白、运铁蛋白受体和血浆铜蓝蛋白。
本发明还涉及一种在对象中抑制HIF脯氨酸羟化酶的方法，包括对对象给予本发明化合物或其药学上可接受的盐；涉及一种在对象中促进生成内源性EPO的方法，包括对对象给予本发明化合物或其药学上可接受的盐；涉及一种在对象中稳定缺氧诱导因子α的的方法，包括对对象给予本发明化合物或其药学上可接受的盐；涉及一种用于治疗对象的慢性疾病变相关贫血的的方法，包括对对象给予本发明化合物或其药学上可接受的盐，其中所述慢性疾病变相关贫血选自类风湿性关节炎，风湿热和炎症性肠道疾病。
本发明还涉及一种增加对象炎性细胞因子产生的方法，包括对对象给予本发明化合物或其药学上可接受的盐，其中所述炎性细胞因子包括肿瘤坏死因子，白细胞介素和干扰素。
本发明再一方面涉及一种治疗在对象的贫血对外部给予红细胞生成素的治疗具有抗性的方法，包括对对象给予本发明化合物或其药学上可接受的盐，其中所述化合物增强了造血前提细胞对所述红细胞生成素的应答。
本发明还涉及一种在对象中增加铁摄取、铁运输和铁利用中所需因子产生的方法，包括对对象给予本发明化合物或其药学上可接受的盐，其中所述因子选自类红细胞氨基乙酰丙酸合酶、运铁蛋白、运铁蛋白受体和血浆铜蓝蛋白。
具体实施例
本发明给出的实施例仅用于解释目的，而不是限制本发明，在不背离本发明的精神和范围的情况下，可以对本发明作出适当的改变。
实施例1
Figure PCTCN2015097246-appb-000013
2-(3-羟基-5-苯氧-2-吡啶甲酰氨基)乙酸(1号化合物)
步骤1：5-溴-3-甲氧基吡啶-2-甲腈的制备
室温下将甲醇钠(9.7克,0.18摩尔)甲醇溶液(50ml)滴入5-溴-3-氟吡啶-2-甲腈(30克,0.15摩尔)的甲醇(150ml)悬浮液中。滴加完毕后，室温反应2小时后反应液变澄清。将反应液中加入少量冰醋酸调节pH至7～8间，加入冰水(300ml)，浓缩反应液至有固体析出后，冷却静置2小时使固体析出更彻底。抽滤析出的固体，以水洗涤滤饼，收集滤饼室温放置风干得白色固体5-溴-3-甲氧基吡啶-2-甲腈(24克，收率75％)。
步骤2：3-甲氧基-5-苯氧基吡啶-2-甲腈的制备
将5-溴-3-甲氧基吡啶-2-甲腈(1.33克,14.1毫摩尔)、苯酚(1克,4.7毫摩尔)、碘化亚铜(266毫克,1.4毫摩尔)、N,N-二甲基甘氨酸(144毫克,1.4毫摩尔)、碳酸铯(2.3克,7.05毫摩尔)及1，4-二氧六环(10ml)混合后，于氮气保护下在120℃搅拌反应过夜。将反应液浓缩后加入乙酸乙酯(50ml)及水(30ml)并分层，取乙酸乙酯层浓缩后柱层析，洗脱得3-甲氧基-5-苯氧基吡啶-2-甲腈固体(769毫克,收率72％)。
步骤3：3-羟基-5-苯氧基吡啶-2-甲酸的制备
将3-甲氧基-5-苯氧基吡啶-2-甲腈(600毫克)溶于冰醋酸(15ml)中，加入氢溴酸液(15ml)，在120℃下反应8小时。冷却反应液至25℃下静置8小时，析出固体。抽滤固体，以水洗涤滤饼，收集滤饼后减压干燥得白色固体3-羟基-5-苯氧基吡啶-2-甲酸(370毫克,收率60％)。
步骤4：2-(3-羟基-5-苯氧基吡啶-2-甲酰氨基)乙酸苄酯的制备
将3-羟基-5-苯氧基吡啶-2-甲酸(89毫克,0.38毫摩尔)、甘氨酸苄酯盐酸盐(116毫克,0.58毫摩尔)、EDCI(110毫克,0.58毫摩尔)、HOBT(52毫克,0.385毫摩尔)加入二氯甲烷(5ml)中形成悬浮液，向此悬浮液中滴入二异丙基乙基胺(75毫克,0.578毫摩尔)。滴完后室温反应8小时，向反应液内加入二氯甲烷(50ml)进行稀释，加水(30ml)洗涤二氯甲烷溶液，收集有机相，蒸去有机溶剂，将残留物进行柱层析，洗脱溶剂为乙酸乙酯/石油醚(1/4)，得2-(3-羟基-5-苯氧基吡啶-2-甲酰氨基)乙酸苄酯(106毫克，收率73％)。
步骤5：2-(3-羟基-5-苯氧基吡啶-2-甲酰氨基)乙酸的制备。将2-(3-羟基-5-苯氧基吡啶-2-甲酰氨基)乙酸苄酯(106毫克)溶于甲醇(10ml)中，在氮气环境下加入10％氢氧化钯(II)/炭(10毫克)，之后通入氢气在室温下搅拌反应10小时。抽滤除去催化剂，将滤液浓缩得到2-(3-羟基-5-苯氧基吡啶-2-甲酰氨基)乙酸固体(46毫克,收率57％)。
1H NMR(400MHz,DMSO-d6)δ12.55(s,1H),9.21(t,J＝6.2Hz,1H),8.00(d,J＝2.4Hz,1H),7.54–7.40(m,2H),7.34–7.17(m,3H),6.83(d,J＝2.4Hz,1H),3.97(d,J＝6.2Hz,1H).
实施例2
Figure PCTCN2015097246-appb-000014
2-(5-(2,3-二甲基苯氧)-3-羟基吡啶-2-甲酰氨基)乙酸(5号化合物)
步骤1：5-溴-3-甲氧基吡啶-2-甲腈的制备
室温下将甲醇钠(9.7克,0.18摩尔)甲醇溶液(50ml)滴入5-溴-3-氟吡啶-2-甲腈(30克,0.15摩尔)的甲醇(150ml)悬浮液中。滴加完毕后，室温反应2小时后反应液变澄清。将反应液中加入少量冰醋酸调节pH至7～8间，加入冰水(300ml)，浓缩反应液至有固体析出后，冷却静置2小时使固体析出更彻底。抽滤析出的固体，以水洗涤滤饼，收集滤饼室温放置风干得白色固体5-溴-3-甲氧基吡啶-2-甲腈(24克，收率75％)。
步骤2：5-(2,3-二甲基苯氧基)-3-甲氧基-吡啶-2-甲腈的制备
将5-溴-3-甲氧基吡啶-2-甲腈426毫克(2.0毫摩尔,1.0当量)、2,3-二甲基苯酚733毫克(6.0毫摩尔,3.0当量)、碘化亚铜114毫克(0.6毫摩尔,0.3当量)、N,N-二甲基甘氨酸61.8毫克(0.6毫摩尔,0.3当量)、碳酸铯1.3克(4.0毫摩尔,2.0当量)及1，4-二氧六环6ml混合后，于氮气保护下在120℃搅拌反应过夜。将反应液浓缩后加入乙酸乙酯(50ml)及水(30ml)并分层，取乙酸乙酯层浓缩后柱层析，洗脱得5-(2,3-二甲基苯氧基)-3-甲氧基-吡啶-2-甲腈410毫克，淡黄色固体，80.7％。
步骤3：5-(2,3-二甲基苯氧基)-3-羟基-吡啶-2-羧酸的制备
将5-(2,3-二甲基苯氧基)-3-甲氧基-吡啶-2-甲腈410毫克(1.61毫摩尔)溶于冰醋酸2ml中，加入氢溴酸液6ml，在120℃下反应8小时。冷却反应液至25℃下静置8小时，析出固体。抽滤固体，以水洗涤滤饼，收集滤饼后减压干燥得5-(2,3-二甲基苯氧基)-3-羟基-吡啶-2-羧酸350毫克红棕色固体，83.9％。
步骤4：{[5-(2,3-二甲基苯氧基)-3-羟基-吡啶-2-羰基]-氨基}-乙酸苄酯的制备
将5-(2,3-二甲基苯氧基)-3-羟基-吡啶-2-羧酸250毫克，(0.96毫摩尔,1.0当量)、甘氨酸苄酯盐酸盐291.0毫克,(1.45毫摩尔,1.5当量)、1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐277.0毫克(1.45毫摩尔,1.5当量)、1-羟基苯并三氮唑195.8毫克 (1.45毫摩尔,1.5当量)加入二氯甲烷10ml中形成悬浮液，向此悬浮液中滴入二异丙基乙基胺187.0毫克(1.45毫摩尔,1.5当量)。滴完后室温反应8小时，向反应液内加入二氯甲烷(50ml)进行稀释，加水(30ml)洗涤二氯甲烷溶液，收集有机相，蒸去有机溶剂，将残留物进行柱层析，洗脱溶剂为乙酸乙酯/石油醚(1/4)，得{[5-(2,3-二甲基苯氧基)-3-羟基-吡啶-2-羰基]-氨基}-乙酸苄酯100毫克油状物，25.6％。
步骤5：2-(5-(2,3-二甲基苯氧)-3-羟基吡啶-2-甲酰氨基)乙酸的制备。将{[5-(2,3-二甲基苯氧基)-3-羟基-吡啶-2-羰基]-氨基}-乙酸苄酯100毫克(0.25毫摩尔)溶于甲醇10ml中，在氮气环境下加入10％氢氧化钯(II)/炭20毫克，之后通入氢气在室温下搅拌反应10小时。抽滤除去催化剂，将滤液浓缩得到2-(5-(2,3-二甲基苯氧)-3-羟基吡啶-2-甲酰氨基)乙酸39毫克，49.4％，白色固体。
1H NMR(400MHz,DMSO-d6)δ12.53(s,1H),9.17(t,J＝6.2Hz,1H),7.97(d,J＝2.4Hz,1H),7.25–7.07(m,2H),7.01–6.94(m,1H),6.55(d,J＝2.4Hz,1H),3.97(d,J＝6.0Hz,1H),2.31(s,3H),2.07(s,3H).
实施例3
Figure PCTCN2015097246-appb-000015
2-(5-(3-氯苯氧)-3-羟基吡啶-2-甲酰氨基)乙酸(22号化合物)
步骤1：5-溴-3-甲氧基吡啶-2-甲腈的制备
室温下将甲醇钠(9.7克,0.18摩尔)甲醇溶液(50ml)滴入5-溴-3-氟吡啶-2-甲腈(30克,0.15摩尔)的甲醇(150ml)悬浮液中。滴加完毕后，室温反应2小时后反应液变澄清。将反应液中加入少量冰醋酸调节pH至7～8间，加入冰水(300ml)，浓缩反应液至有固体析出后，冷却静置2小时使固体析出更彻底。抽滤析出的固体，以水洗涤滤饼，收集滤饼室温放置风干得白色固体5-溴-3-甲氧基吡啶-2-甲腈(24克，收率75％)。
步骤2：5-(3-氯苯氧)-3-甲氧基-吡啶-2-甲腈的制备
将5-溴-3-甲氧基吡啶-2-甲腈400毫克、3-氯苯酚500毫克、碘化亚铜120毫克、N,N-二甲基甘氨酸80毫克、碳酸铯1.5克(4.0毫摩尔,2.0当量)及1，4-二氧六环2ml混合后，于氮气保护下在120℃搅拌反应过夜。将反应液浓缩后加入乙酸乙酯(50ml)及水(30ml)并分层，取乙酸乙酯层浓缩后柱层析，洗脱得5-(3-氯苯 氧)-3-甲氧基-吡啶-2-甲腈390毫克淡黄色固体。
步骤3：5-(3-氯苯氧)-3-羟基-吡啶-2-羧酸的制备
将5-(3-氯苯氧)-3-甲氧基-吡啶-2-甲腈390毫克(0.61毫摩尔)溶于冰醋酸1ml中，加入氢溴酸液3ml，在120℃下反应8小时。冷却反应液至25℃下静置8小时，析出固体。抽滤固体，以水洗涤滤饼，收集滤饼后减压干燥得5-(3-氯苯氧)-3-羟基-吡啶-2-羧酸300毫克红棕色固体。
步骤4：{[5-(3-氯苯氧)-3-羟基-吡啶-2-羰基]-氨基}-乙酸苄酯的制备
将5-(3-氯苯氧)-3-羟基-吡啶-2-羧酸300毫克、甘氨酸苄酯盐酸盐560毫克、1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐460毫克、1-羟基苯并三氮唑330毫克加入二氯甲烷8ml中形成悬浮液，向此悬浮液中滴入二异丙基乙基胺340毫克。滴完后室温反应8小时，向反应液内加入二氯甲烷(50ml)进行稀释，加水(30ml)洗涤二氯甲烷溶液，收集有机相，蒸去有机溶剂，将残留物进行柱层析，洗脱溶剂为乙酸乙酯/石油醚(1/4)，得{[5-(3-氯苯氧)-3-羟基-吡啶-2-羰基]-氨基}-乙酸苄酯150毫克淡黄色油状物。
步骤5：2-(5-(3-氯苯氧)-3-羟基吡啶-2-甲酰氨基)乙酸的制备。将{[5-(3-氯苯氧)-3-羟基-吡啶-2-羰基]-氨基}-乙酸苄酯150毫克溶于甲醇10ml中，在氮气环境下加入10％氢氧化钯(II)/炭15毫克，之后通入氢气在室温下搅拌反应10小时。抽滤除去催化剂，将滤液浓缩得到2-(5-(2,3-二氟苯氧)-3-羟基吡啶-2-甲酰氨基)乙酸70毫克白色固体。
1H NMR(400MHz,DMSO-d6)δ12.57(s,1H),9.24(t,J＝6.2Hz,1H),8.03(d,J＝2.4Hz,1H),7.56–7.30(m,2H),7.30–7.14(m,1H),7.12–6.94(m,2H),3.96(d,J＝6.2Hz,1H).
实施例4
Figure PCTCN2015097246-appb-000016
2-(3-羟基-5-(萘-2-氧基)吡啶-2-甲酰氨基)乙酸(8号化合物)
步骤1：5-溴-3-甲氧基吡啶-2-甲腈的制备
室温下将甲醇钠(9.7克,0.18摩尔)甲醇溶液(50ml)滴入5-溴-3-氟吡啶-2-甲腈(30克,0.15摩尔)的甲醇(150ml)悬浮液中。滴加完毕后，室温反应2小时后反应液变澄清。将反应液中加入少量冰醋酸调节pH至7～8间，加入冰水 (300ml)，浓缩反应液至有固体析出后，冷却静置2小时使固体析出更彻底。抽滤析出的固体，以水洗涤滤饼，收集滤饼室温放置风干得白色固体5-溴-3-甲氧基吡啶-2-甲腈(24克，收率75％)。
步骤2：3-甲氧基-5-(萘-2-氧基)-吡啶-2-甲腈的制备
将5-溴-3-甲氧基吡啶-2-甲腈320毫克(1.5毫摩尔,1.0当量)、2-萘酚649毫克(4.5毫摩尔,3.0当量)、碘化亚铜85.5毫克(0.45毫摩尔,0.3当量)、N,N-二甲基甘氨酸46.4毫克(0.45毫摩尔,0.3当量)、碳酸铯978毫克(3.0毫摩尔,2.0当量)及1，4-二氧六环6ml混合后，于氮气保护下在120℃搅拌反应过夜。将反应液浓缩后加入乙酸乙酯(50ml)及水(30ml)并分层，取乙酸乙酯层浓缩后柱层析，洗脱得3-甲氧基-5-(萘-2-氧基)-吡啶-2-甲腈300毫克，红色固体，72.5％。
步骤3：3-羟基-5-(萘-2-氧基)-吡啶-2-羧酸的制备
将3-甲氧基-5-(萘-2-氧基)-吡啶-2-甲腈300毫克溶于冰醋酸1.5ml中，加入氢溴酸液4.5ml，在120℃下反应8小时。冷却反应液至25℃下静置8小时，析出固体。抽滤固体，以水洗涤滤饼，收集滤饼后减压干燥得3-羟基-5-(萘-2-氧基)-吡啶-2-羧酸200毫克红棕色固体，81.8％。
步骤4：{[3-羟基-5-(萘-2-氧基)-吡啶-2-羰基]-氨基}-乙酸苄酯的制备
将3-羟基-5-(萘-2-氧基)-吡啶-2-羧酸250毫克(0.89毫摩尔,1.0当量)、甘氨酸苄酯盐酸盐268.2毫克(1.33毫摩尔,1.5当量)、1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐254.0毫克(1.33毫摩尔,1.5当量)、1-羟基苯并三氮唑179.6毫克(1.33毫摩尔,1.5当量)加入二氯甲烷10ml中形成悬浮液，向此悬浮液中滴入二异丙基乙基胺171.6毫克(1.33毫摩尔,1.5当量)。滴完后室温反应8小时，向反应液内加入二氯甲烷(50ml)进行稀释，加水(30ml)洗涤二氯甲烷溶液，收集有机相，蒸去有机溶剂，将残留物进行柱层析，洗脱溶剂为乙酸乙酯/石油醚(1/4)，得{[3-羟基-5-(萘-2-氧基)-吡啶-2-羰基]-氨基}-乙酸苄酯100毫克淡黄色油状物，25.4％。
步骤5：2-(3-羟基-5-(萘-2-氧基)吡啶-2-甲酰氨基)乙酸的制备。将{[3-羟基-5-(萘-2-氧基)-吡啶-2-羰基]-氨基}-乙酸苄酯100毫克(0.23毫摩尔)溶于甲醇10ml中，在氮气环境下加入10％氢氧化钯(II)/炭20毫克，之后通入氢气在室温下搅拌反应10小时。抽滤除去催化剂，将滤液浓缩得到2-(3-羟基-5-(萘-2-氧基)吡啶-2-甲酰氨基)乙酸43毫克，56.2％，白色固体。
1H NMR(400MHz,DMSO-d6)δ12.57(s,1H),9.25(t,J＝6.4Hz,1H),8.08(d,J＝2.4Hz,1H),7.98(d,J＝8.0Hz,1H),7.92(d,J＝8.0Hz,1H),7.70-7.68(m,1H),7.60–7.47(m,2H),7.46–7.37(m,1H),7.10(s,1H),6.98(d,J＝2.4Hz,1H),4.00(d,J＝6.4Hz,1H).
实施例5
Figure PCTCN2015097246-appb-000017
2-(5-(2,4-二甲基苯氧)-3-羟基吡啶-2-甲酰氨基)乙酸(19号化合物)
步骤1：5-溴-3-甲氧基吡啶-2-甲腈的制备
室温下将甲醇钠(9.7克,0.18摩尔)甲醇溶液(50ml)滴入5-溴-3-氟吡啶-2-甲腈(30克,0.15摩尔)的甲醇(150ml)悬浮液中。滴加完毕后，室温反应2小时后反应液变澄清。将反应液中加入少量冰醋酸调节pH至7～8间，加入冰水(300ml)，浓缩反应液至有固体析出后，冷却静置2小时使固体析出更彻底。抽滤析出的固体，以水洗涤滤饼，收集滤饼室温放置风干得白色固体5-溴-3-甲氧基吡啶-2-甲腈(24克，收率75％)。
步骤2：5-(2,4-二甲基苯氧基)-3-甲氧基-吡啶-2-甲腈的制备
将5-溴-3-甲氧基吡啶-2-甲腈426毫克(2.0毫摩尔,1.0当量)、2,4-二甲基苯酚733毫克(6.0毫摩尔,3.0当量)、碘化亚铜114毫克(0.6毫摩尔,0.3当量)、N,N-二甲基甘氨酸61.8毫克(0.6毫摩尔,0.3当量)、碳酸铯1.3克(4.0毫摩尔,2.0当量)及1，4-二氧六环6ml混合后，于氮气保护下在120℃搅拌反应过夜。将反应液浓缩后加入乙酸乙酯(50ml)及水(30ml)并分层，取乙酸乙酯层浓缩后柱层析，洗脱得5-(2,4-二甲基苯氧基)-3-甲氧基-吡啶-2-甲腈250毫克，淡黄色固体，49.2％。
步骤3：5-(2,4-二甲基苯氧基)-3-羟基-吡啶-2-羧酸的制备
将5-(2,4-二甲基苯氧基)-3-甲氧基-吡啶-2-甲腈250毫克(0.98毫摩尔)溶于冰醋酸1.5ml中，加入氢溴酸液4.5ml，在120℃下反应8小时。冷却反应液至25℃下静置8小时，析出固体。抽滤固体，以水洗涤滤饼，收集滤饼后减压干燥得5-(2,4-二甲基苯氧基)-3-羟基-吡啶-2-羧酸185毫克红棕色固体，73.1％。
步骤4：{[5-(2,4-二甲基苯氧基)-3-羟基-吡啶-2-羰基]-氨基}-乙酸苄酯的制备
将5-(2,4-二甲基苯氧基)-3-羟基-吡啶-2-羧酸185毫克，(0.71毫摩尔,1.0当量)、甘氨酸苄酯盐酸盐215.4毫克(1.07毫摩尔，1.5当量)、1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐204.4毫克(1.07毫摩尔，1.5当量)、1-羟基苯并三氮唑144.4毫克(1.07毫摩尔，1.5当量)加入二氯甲烷10ml中形成悬浮液，向此悬浮液中滴入二异丙基乙基胺138毫克(1.07毫摩尔，1.5当量)。滴完后室温反应8小时，向反应液内加入二氯甲烷(50ml)进行稀释，加水(30ml)洗涤二氯甲烷溶液，收集有 机相，蒸去有机溶剂，将残留物进行柱层析，洗脱溶剂为乙酸乙酯/石油醚(1/4)，得{[5-(2,4-二甲基苯氧基)-3-羟基-吡啶-2-羰基]-氨基}-乙酸苄酯50毫克淡黄色油状物，17.3％。
步骤5：2-(5-(2,4-二甲基苯氧)-3-羟基吡啶-2-甲酰氨基)乙酸的制备。将{[5-(2,4-二甲基苯氧基)-3-羟基-吡啶-2-羰基]-氨基}-乙酸苄酯50毫克(0.12毫摩尔)溶于甲醇10ml中，在氮气环境下加入10％氢氧化钯(II)/炭10毫克，之后通入氢气在室温下搅拌反应10小时。抽滤除去催化剂，将滤液浓缩得到2-(5-(2,4-二甲基苯氧)-3-羟基吡啶-2-甲酰氨基)乙酸白色固体20毫克，51.4％。
1H NMR(400MHz,DMSO-d6)δ12.52(s,1H),9.17(t,J＝6.4Hz,1H),7.95(d,J＝2.4Hz,1H),7.23-7.18(m,1H),7.15–7.07(m,1H),7.06–6.94(m,1H),6.56(d,J＝2.4Hz,1H),3.98(d,J＝6.4Hz,2H),2.31(s,3H),2.11(s,3H).
实施例6
Figure PCTCN2015097246-appb-000018
2-(5-(2,3-二氟苯氧)-3-羟基吡啶-2-甲酰氨基)乙酸(9号化合物)
步骤1：5-溴-3-甲氧基吡啶-2-甲腈的制备
室温下将甲醇钠(9.7克,0.18摩尔)甲醇溶液(50ml)滴入5-溴-3-氟吡啶-2-甲腈(30克,0.15摩尔)的甲醇(150ml)悬浮液中。滴加完毕后，室温反应2小时后反应液变澄清。将反应液中加入少量冰醋酸调节pH至7～8间，加入冰水(300ml)，浓缩反应液至有固体析出后，冷却静置2小时使固体析出更彻底。抽滤析出的固体，以水洗涤滤饼，收集滤饼室温放置风干得白色固体5-溴-3-甲氧基吡啶-2-甲腈(24克，收率75％)。
步骤2：5-(2,3-二氟-苯氧)-3-甲氧基-吡啶-2-甲腈的制备
将5-溴-3-甲氧基吡啶-2-甲腈426毫克(2.0毫摩尔,1.0当量)、2,3-二氟苯酚786毫克(6.0毫摩尔,3.0当量)、碘化亚铜114毫克(0.6毫摩尔,0.3当量)、N,N-二甲基甘氨酸61.8毫克(0.6毫摩尔,0.3当量)、碳酸铯1.3克(4.0毫摩尔,2.0当量)及1，4-二氧六环6ml混合后，于氮气保护下在120℃搅拌反应过夜。将反应液浓缩后加入乙酸乙酯(50ml)及水(30ml)并分层，取乙酸乙酯层浓缩后柱层析，洗脱得5-(2,3-二氟-苯氧)-3-甲氧基-吡啶-2-甲腈160毫克，淡黄色固体，30.5％。
步骤3：5-(2,3-二氟-苯氧)-3-羟基-吡啶-2-羧酸的制备
将5-(2,3-二氟-苯氧)-3-甲氧基-吡啶-2-甲腈160毫克(0.61毫摩尔)溶于冰醋酸1ml中，加入氢溴酸液3ml，在120℃下反应8小时。冷却反应液至25℃下静置8小时，析出固体。抽滤固体，以水洗涤滤饼，收集滤饼后减压干燥得5-(2,3-二氟-苯氧)-3-羟基-吡啶-2-羧酸150毫克红棕色固体，92.0％。
步骤4：{[5-(2,3-二氟-苯氧)-3-羟基-吡啶-2-羰基]-氨基}-乙酸苄酯的制备
将5-(2,3-二氟-苯氧)-3-羟基-吡啶-2-羧酸150毫克，(0.56毫摩尔,1.0当量)、甘氨酸苄酯盐酸盐169.4毫克(0.84毫摩尔,1.5当量)、1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐160.4毫克(0.84毫摩尔,1.5当量)、1-羟基苯并三氮唑113.4毫克(0.84毫摩尔,1.5当量)加入二氯甲烷8ml中形成悬浮液，向此悬浮液中滴入二异丙基乙基胺108.4毫克(0.84毫摩尔,1.5当量)。滴完后室温反应8小时，向反应液内加入二氯甲烷(50ml)进行稀释，加水(30ml)洗涤二氯甲烷溶液，收集有机相，蒸去有机溶剂，将残留物进行柱层析，洗脱溶剂为乙酸乙酯/石油醚(1/4)，得{[5-(2,3-二氟-苯氧)-3-羟基-吡啶-2-羰基]-氨基}-乙酸苄酯70毫克淡黄色油状物，20.1％。
步骤5：2-(5-(2,3-二氟苯氧)-3-羟基吡啶-2-甲酰氨基)乙酸的制备。将{[5-(2,3-二氟-苯氧)-3-羟基-吡啶-2-羰基]-氨基}-乙酸苄酯70毫克(0.17毫摩尔)溶于甲醇10ml中，在氮气环境下加入10％氢氧化钯(II)/炭15毫克，之后通入氢气在室温下搅拌反应10小时。抽滤除去催化剂，将滤液浓缩得到2-(5-(2,3-二氟苯氧)-3-羟基吡啶-2-甲酰氨基)乙酸30毫克白色固体，54.5％，。
1H NMR(400MHz,DMSO-d6)δ12.82(s,1H),12.59(s,1H),9.25(t,J＝6.4Hz,1H),8.10(d,J＝2.4Hz,1H),7.46–7.35(m,1H),7.34-7.25(m,1H),7.24-7.18(m,1H),7.03(d,J＝2.4Hz,1H),3.99(d,J＝6.4Hz,2H).
生物实施例1：体外检测方法的开发和筛选：HIF-PHD2抑制剂系列化合物体外促肝癌细胞Hep3B促红细胞生成素表达
实验用肝癌细胞Hep3B(中国典型培养物保藏中心，CCTCC)培养所需完全培养基为MEM(Cat#GNM 41500，GIBCO，杭州吉诺生物医药技术有限公司供货)补加10％血清FBS(Cat#10099-141，GIBCO)及1％双抗P/S(Cat#GNM15140，杭州吉诺生物医药技术有限公司供货)。细胞于37℃，5％CO2培养箱中培养。实验试剂包括DMSO(DimethyIsulfoxide,for摩尔ecular biology,>＝99.9％,Catalog#D8418)购自Sigma。实验用ELISA试剂盒购自Quantikine IVD ELISA，Human Erythropoietin(R&D，DEP00)。试验用对照物AKB-6548通过自制或商业购买获得。受试物避光密封于-20℃储存。
避光操作下将受试物以无菌水或DMSO作为溶媒，充分溶解受试物及阳性对照物，配制成浓度为10-1摩尔/L或10-2摩尔/L的储存液。将受试物储存液均置于-20℃存放。以含0.5％FBS的MEM培养基作为稀释液，稀释受试物储存液，配制成浓度为100μ摩尔/L及10μ摩尔/L的受试物稀释液。在96孔培养板中，加入200μl/孔(1.5或2.0*104细胞数/孔)肝癌细胞Hep3B完全培养基悬液，于37℃下5％CO2培养箱中培养过夜。除去96孔培养板中旧液，以含0.5％FBS的MEM培养基清洗细胞一次。避光下加入200μl/孔受试物，剂量为100μ摩尔/L及10μ摩尔/L，每个剂量做2复孔，作为受试孔及备份。以稀释液替代药液，作为细胞对照孔(不含受试物及溶媒)。以含相应浓度溶媒(DMSO)的稀释液替代药液，作为溶媒对照孔(不含受试物)。在37℃，5％CO2培养箱中培养24小时。吸取上清，作为样本-20℃冻存备用。加入终止液，100μl/孔。酶标仪A450nm-A600nm检测OD值。根据标准曲线获得受试物促EPO表达含量(mIU/mL)，再计算出受试物物EPO表达含量与阳性对照品AKB6548EPO表达含量的比值，检测结果如下表所示：
Figure PCTCN2015097246-appb-000019
生物实施例2：检测化合物对PHD2的抑制作用(IC50)
通过荧光偏振(fluorescence polarization，FP)方法检测缺氧诱导因子HIF-1α与VBC复合物(von Hippel–Lindau protein-Elongin B–Elongin C，VBC)的相互作用，测定HIF脯氨酸羟化酶PHD2(prolyl hydroxylases 2，PHD2)抑制剂化合物的酶抑制活性。
在含有抗坏血酸200μM、α–酮戊二酸20μM、FeCl2100μM的NETN(20mM Tris.HCl，100mM NaCl，1mM EDTA，0.5％NP-40，1mM PMSF)缓冲液中避光加入终浓度1μM的FAM-HIF(556-575)。接着加入所需浓度受试化合物或阳性化合物(缓冲液代替化合物作为阴性对照及阳性对照)。最后加入终浓度为0.5μg/μl的PHD2(阴性对照以缓冲液代替PHD2)。混匀，室温避光放置反应30分钟后，95℃水浴1 分钟，终止反应。待温度降至室温，样品备用。在96孔黑色检测板相应孔中加入EBC缓冲液(50mM Tris.HCl、120mM NaCl、0.5％NP-40)。在相应检测孔加入终浓度300nM的GST-VBC复合物(以仅含有EBC缓冲液孔作为空白孔)。随后避光加入相应PHD2脯氨酸羟化反应样本作为终浓度100nM底物。混匀后使用全波长多功能酶标仪(TECAN infinite M1000)，在激发波407nM，发射波518nM条件下检测横向及纵向荧光强度读值。
计算荧光偏振值(mP)：
mP＝1000×(横向读值-G因子*纵向读值)/(横向读值+G因子×纵向读值)
其中，横向读值＝测试孔横向荧光强度读值-空白孔横向荧光强度读值，纵向读值＝测试孔纵向荧光强度读值-空白孔纵向荧光强度读值，
根据以下公式计算受试化合物PHD2抑制率(％)：
抑制率(％)＝1-(mP测试孔–mP阴性对照孔)/(mP阳性对照孔-mP阴性对照孔)。
使用Graphpad Prism 4.0软件(Golden software,Golden,Colorado,USA)的非线性回归数据分析方法计算IC50。
化合物编号 IC50(μM)
1 84
5 237
8 91
9 15
19 63
22 236

Claims (25)

  1. 一种具有下式(I)结构的化合物或其药学上可接受盐：
    Figure PCTCN2015097246-appb-100001
    其中，R1，R2独立为氢。
    R3选自氢、C1-C7直链、支链或环状烷基；
    R4，R5，R6，R7和R8各自独立地选自C1-C7烷基、卤代C1-C7烷基、C1-C3烷氧基、卤代C1-C3烷氧基、卤素、羟基、氢、氨基、硝基、氰基取代或未取代的芳环或芳杂环；
    或者R4，R5，R6，R7，R8相互之间以氧桥相联，形成下式基团：
    Figure PCTCN2015097246-appb-100002
    其中，n选自1、2、3或4的整数；
    A1和A2独立地选自氧、碳或氮原子。
  2. 一种具有下式结构(II)的分子式或其药学上可接受的盐：
    Figure PCTCN2015097246-appb-100003
    其中，n选自1、2、3或4的整数；
    A1和A2独立地选自氧、碳或氮原子。
  3. 如权利要求1所述的化合物或其药学上可接受的盐，其中所述的药学上可接受的盐通过与药学上可接受的碱反应生成盐。
  4. 如权利要求1所述的化合物或其药学上可接受的盐，其中所述的化合物选自下列化合物：
    Figure PCTCN2015097246-appb-100004
    Figure PCTCN2015097246-appb-100005
    Figure PCTCN2015097246-appb-100006
  5. 如权利要求1所述的化合物的制备方法，包括下列步骤：
    步骤1：在甲醇的存在下，使5-溴-3-氟吡啶基-2-甲腈
    Figure PCTCN2015097246-appb-100007
    与甲醇钠反应，生成5-溴-3-甲氧基吡啶-2-甲腈(中间体III)
    Figure PCTCN2015097246-appb-100008
    步骤2：将步骤1得到的中间体(III)与Ar-OH和配体在溶剂中混合加热，在催化剂的参与下进行Ullman反应生成醚类中间体(IV)
    Figure PCTCN2015097246-appb-100009
    步骤3：将步骤2得到的中间体(IV)在回流下在氢溴酸/冰醋酸中水解生成3-羟基吡啶-2-甲酸衍生物(V)
    Figure PCTCN2015097246-appb-100010
    步骤4：将步骤3得到的中间体(V)与α-R3取代氨基酸苄酯(VI)在缩合剂存在下进行酰胺化反应，得到3-羟基吡啶-2-羧酸苄酯中间体(VII)：
    Figure PCTCN2015097246-appb-100011
    步骤5：将步骤4得到的中间体(VII)在氢解条件在溶剂中、催化剂存在和室温下进行氢解反应下脱去苄酯保护基，最终形成对应于式I或式II的化合物；
    其中Ar代表
    Figure PCTCN2015097246-appb-100012
    R1、R3、R4-R8的定义与权利要求1中的相同。
  6. 如权利要求5所述的方法，其中所述步骤2中的催化剂为碘化亚铜(I)，金属配体是N,N-二甲基甘氨酸、N-甲基脯氨酸、N,N-四甲基乙二胺，反应溶剂为1,4-二氧六环、甲苯、四氢呋喃，该步骤包括加热到70℃到120℃进行反应；所述步骤3中氢溴酸与冰醋酸的比例为2:1～1:3，反应温度为90～140℃，加热反应时间为6～12小时；所述步骤4中，所述的α-R3氨基酸苄酯(VI)为选自甘氨酸苄酯盐酸盐、(α或β)丙氨酸、(α或β)缬氨酸、(α或β)亮氨酸、(α或β)异亮氨酸的其盐酸盐形式，所述溶剂为二氯甲烷、氯仿、四氢呋喃、1，4-二氧六环、N,N-二甲基甲酰胺、N-甲基吡咯烷酮；所述的酰胺化催化剂选自1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐、1-羟基苯并三氮唑、二环已基碳二亚胺；所述步骤5中，催化剂选自钯(0)/炭、氢氧化钯(II)、氢氧化钯(II)/炭、二氧化铂(IV)，氢解的溶剂选自甲醇、乙醇、异丙醇、四氢呋喃、乙酸乙酯。
  7. 一种药物组合物，它包含权利要求1-4任一所述的化合物或其药学上可接受的盐以及药学上可接受的载体。
  8. 如权利要求1-4任一所述的化合物或其药学上可接受的盐在制备抑制HIF脯氨酸羟化酶的药物中的应用。
  9. 如权利要求1-4任一所述的化合物或其药学上可接受的盐在制备促进生成内源性EPO的药物中的应用。
  10. 如权利要求1-4任一所述的化合物或其药学上可接受的盐在制备稳定缺氧诱导因子α的药物中的应用。
  11. 如权利要求1-4任一所述的化合物或其药学上可接受的盐在制备用于治疗对象的慢性疾病变相关贫血的药物中的应用。
  12. 如权利要求11所述的应用，其中所述慢性疾病变相关贫血选自类风湿性关节炎，风湿热和炎症性肠道疾病。
  13. 如权利要求1-4任一所述的化合物或其药学上可接受的盐在制备增加对象炎性细胞因子产生的药物中的应用。
  14. 如权利要求13所述的应用，其中所述炎性细胞因子包括肿瘤坏死因子，白细胞介素和干扰素。
  15. 如权利要求1-4任一所述的化合物或其药学上可接受的盐在制备治疗在对象的贫血对外部给予红细胞生成素的治疗具有抗性的药物中的应用，其中所述化合物增强了造血前提细胞对所述红细胞生成素的应答。
  16. 如权利要求1-4任一所述的化合物或其药学上可接受的盐在制备在对象中增加铁摄取、铁运输和铁利用中所需因子产生的药物中的应用，其中所述因子选 自类红细胞氨基乙酰丙酸合酶、运铁蛋白、运铁蛋白受体和血浆铜蓝蛋白。
  17. 一种在对象中抑制HIF脯氨酸羟化酶的方法，包括对对象给予如权利要求1-4任一所述的化合物或其药学上可接受的盐。
  18. 一种在对象中促进生成内源性EPO的方法，包括对对象给予如权利要求1-4任一所述的化合物或其药学上可接受的盐。
  19. 一种在对象中稳定缺氧诱导因子α的的方法，包括对对象给予如权利要求1-4任一所述的化合物或其药学上可接受的盐。
  20. 一种用于治疗对象的慢性疾病变相关贫血的的方法，包括对对象给予如权利要求1-4任一所述的化合物或其药学上可接受的盐。
  21. 如权利要求20所述的方法，其中所述慢性疾病变相关贫血选自类风湿性关节炎，风湿热和炎症性肠道疾病。
  22. 一种增加对象炎性细胞因子产生的方法，包括对对象给予如权利要求1-4任一所述的化合物或其药学上可接受的盐。
  23. 如权利要求22所述的方法，其中所述炎性细胞因子包括肿瘤坏死因子，白细胞介素和干扰素。
  24. 一种治疗在对象的贫血对外部给予红细胞生成素的治疗具有抗性的方法，包括对对象给予如权利要求1-4任一所述的化合物或其药学上可接受的盐，其中所述化合物增强了造血前提细胞对所述红细胞生成素的应答。
  25. 一种在对象中增加铁摄取、铁运输和铁利用中所需因子产生的方法，包括对对象给予如权利要求1-4任一所述的化合物或其药学上可接受的盐，其中所述因子选自类红细胞氨基乙酰丙酸合酶、运铁蛋白、运铁蛋白受体和血浆铜蓝蛋白。
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US10149841B2 (en) 2018-12-11
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