WO2018103600A1 - 一种取代的杂芳基酰胺化合物及包含该化合物的组合物及其用途 - Google Patents

一种取代的杂芳基酰胺化合物及包含该化合物的组合物及其用途 Download PDF

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WO2018103600A1
WO2018103600A1 PCT/CN2017/114399 CN2017114399W WO2018103600A1 WO 2018103600 A1 WO2018103600 A1 WO 2018103600A1 CN 2017114399 W CN2017114399 W CN 2017114399W WO 2018103600 A1 WO2018103600 A1 WO 2018103600A1
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compound
mmol
chlorophenyl
etoac
pharmaceutically acceptable
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PCT/CN2017/114399
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English (en)
French (fr)
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王义汉
任兴业
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深圳市塔吉瑞生物医药有限公司
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Priority to CN201780004842.6A priority Critical patent/CN108401429A/zh
Publication of WO2018103600A1 publication Critical patent/WO2018103600A1/zh

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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
    • 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
    • A61K31/4439—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. omeprazole
    • 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
    • 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

Definitions

  • the invention belongs to the technical field of medicine, in particular to a substituted heteroarylamide compound and a composition comprising the same, and capable of regulating the stability of a hypoxia inducible factor (HIF) subunit and increasing endogenous promotion in vitro and in vivo.
  • HIF hypoxia inducible factor
  • Hypoxia-inducible factor is a basic helix-loop-helix (bHLH) PAS (Per/Arnt/Sim) transcriptional activator that regulates changes in gene expression as a function of cellular oxygen concentration.
  • HIF is a heterodimer containing an oxygen-regulated alpha subunit (HIF ⁇ ) and a constitutively expressed beta subunit (HIF ⁇ ), also known as the aromatic hydrocarbon receptor nuclear transporter (ARNT).
  • ONT aromatic hydrocarbon receptor nuclear transporter
  • the HIF ⁇ subunit is rapidly degraded by a mechanism involving ubiquitination of the retinal hemangiostatin (pVHL) E3 ligase complex.
  • HIF ⁇ does not degrade, and an active HIF ⁇ / ⁇ complex accumulates in the nucleus and activates the expression of several genes, including glycolytic enzymes, glucose transporter (GLUT)-1, and erythropoietin ( EPO) and vascular endothelial growth factor (VEGF).
  • GLUT glucose transporter
  • EPO erythropoietin
  • VEGF vascular endothelial growth factor
  • Erythropoietin is a naturally occurring hormone produced by HIF ⁇ that stimulates the production of red blood cells that carry oxygen throughout the body. EPO is usually secreted by the kidney, and endogenous EPO is increased under conditions of reduced oxygen (anoxia). All types of anemia are characterized by a reduced ability of the blood to carry oxygen and are thus accompanied by similar signs and symptoms, including pale and weak skin, mucous membranes, dizziness, fatigue, and lethargy, leading to a decline in quality of life. Subjects with severe anemia showed difficulty breathing and cardiac malformations. Anemia is usually associated with a lack of blood in red blood cells or hemoglobin.
  • Ischemia and hypoxia are the leading causes of morbidity and mortality.
  • Cardiovascular disease causes at least 15 million deaths each year and is responsible for 30% of deaths worldwide.
  • ischemic heart disease and cerebrovascular disease cause about 17% of deaths.
  • a total of 1.3 million cases of non-fatal acute myocardial infarction are reported each year, constituting an incidence of approximately 300 out of every 100,000 people.
  • treatment of myocardial infarction includes nitroglycerin and analgesics to control pain and reduce cardiac workload.
  • Use other drugs including digoxin, diuretics, amrinone, beta-blockers, lipid lowering agents, and angiotensin-converting enzyme inhibitors to stabilize the condition, but none of these therapies Directly acts on tissue damage caused by ischemia and hypoxia.
  • erythropoietin-related conditions such as anemia, including diabetes, anemia, ulcers, kidney failure, cancer, infection, dialysis.
  • Surgery and chemotherapy-related anemia and conditions involving ischemia and hypoxia such as arterial occlusive disease, angina pectoris, intestinal infarction, pulmonary infarction, cerebral ischemia and myocardial infarction.
  • ischemia and hypoxia such as arterial occlusive disease, angina pectoris, intestinal infarction, pulmonary infarction, cerebral ischemia and myocardial infarction.
  • tissue damage caused by ischemia which occurs due to, for example, atherosclerosis, diabetes, and pulmonary disorders such as pulmonary embolism and the like.
  • the present invention discloses a compound and a composition comprising the same, which can be used to modulate hypoxia-inducible factor (HIF) and/or endogenous erythropoietin (EPO) and/or have better Pharmacodynamics / pharmacokinetic properties.
  • HIF hypoxia-inducible factor
  • EPO endogenous erythropoietin
  • HIF hypoxia-inducible factor
  • EPO endogenous erythropoietin
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 and R 8 are each independently hydrogen, deuterium or halogen;
  • An additional condition is that at least one of R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 and R 8 is deuterated or deuterated.
  • R 1 and R 2 are each independently hydrazine or hydrogen.
  • R 1 and R 2 are deuterium.
  • R 3 and R 4 are each independently hydrazine or hydrogen.
  • R 5 , R 6 , R 7 , and R 8 are each independently hydrazine or hydrogen.
  • the compound may be selected from the group consisting of the compounds or pharmaceutically acceptable salts thereof, but is not limited to the following compounds:
  • the cerium isotope content of the cerium in the deuterated position is at least greater than the natural strontium isotope content (0.015%), preferably greater than 30%, more preferably greater than 50%, and even more preferably greater than 75%.
  • the ground is greater than 95%, more preferably greater than 99%.
  • the osmium isotope content in each of the deuterated positions of R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 and R 8 is at least 5%, preferably more than 10%.
  • the compound does not include a non-deuterated compound.
  • a method of preparing a pharmaceutical composition comprising the steps of: pharmaceutically acceptable carrier and a compound of the first aspect of the invention, or a crystalline form thereof, pharmaceutically acceptable
  • the accepted salt, hydrate or solvate is mixed to form a pharmaceutical composition.
  • a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a compound of the first aspect of the invention, or a crystalline form thereof, a pharmaceutically acceptable salt, hydrated Or a solvate.
  • Pharmaceutically acceptable carriers that can be used in the pharmaceutical compositions of the present invention include, but are not limited to, any glidants, sweeteners, diluents, preservatives, dyes/colorants, flavor enhancers, surfactants, wetting agents A dispersing agent, a disintegrating agent, a suspending agent, a stabilizer, an isotonic agent, a solvent or an emulsifier.
  • the pharmaceutical composition of the present invention can be formulated into solid, semi-solid, liquid or gaseous preparations, such as tablets, pills, capsules, powders, granules, ointments, emulsions, suspensions, solutions, suppositories, injections, inhalants, coagulation Glues, microspheres and aerosols.
  • Typical routes of administration of the pharmaceutical compositions of the invention include, but are not limited to, oral, rectal, transmucosal, enteral, or topical, transdermal, inhalation, parenteral, sublingual, intravaginal, intranasal, intraocular, intraperitoneal , intramuscular, subcutaneous, intravenous administration. Oral administration or injection administration is preferred.
  • the pharmaceutical composition of the present invention can be produced by a method known in the art, such as a conventional mixing method, a dissolution method, a granulation method, a sugar-coating method, a pulverization method, an emulsification method, a freeze-drying method, and the like.
  • halogen means F, Cl, Br, and I unless otherwise specified. More preferably, the halogen atom is selected from the group consisting of F, Cl and Br.
  • deuterated means that one or more hydrogens in the compound or group are replaced by deuterium; deuteration may be monosubstituted, disubstituted, polysubstituted or fully substituted.
  • deuteration may be monosubstituted, disubstituted, polysubstituted or fully substituted.
  • deuterated is used interchangeably with “one or more deuterated”.
  • non-deuterated compound means a compound containing a proportion of germanium atoms not higher than the natural helium isotope content (0.015%).
  • the invention also includes isotopically labeled compounds, equivalent to the original compounds disclosed herein.
  • Examples of the compound isotope which may be listed as the present invention include hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine and chlorine isotopes such as 2 H, 3 H, 13 C, 14 C, 15 N, 17 O, 18 O, respectively. , 31 P, 32 P, 35 S, 18 F and 36 Cl. a compound, or an enantiomer, a diastereomer, an isomer, or a pharmaceutically acceptable salt or solvate of the present invention, wherein an isotope or other isotopic atom containing the above compound is within the scope of the present invention .
  • isotopically-labeled compounds of the present invention such as the radioisotopes of 3 H and 14 C, are also among them, useful in tissue distribution experiments of drugs and substrates. ⁇ , ie 3 H and carbon-14, ie 14 C, are easier to prepare and detect and are preferred in isotopes.
  • Isotopically labeled compounds can be prepared in a conventional manner by substituting a readily available isotopically labeled reagent with a non-isotopic reagent using the protocol of the examples.
  • Pharmaceutically acceptable salts include inorganic and organic salts.
  • a preferred class of salts are the salts of the compounds of the invention with acids.
  • Suitable acids for forming salts include, but are not limited to, mineral acids such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, phosphoric acid; formic acid, acetic acid, trifluoroacetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, Organic acids such as fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, benzoic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, benzenesulfonic acid, naphthalenesulfonic acid; Amino acids such as amino acid, phenylalanine, aspartic acid, and glutamic acid.
  • salts of the compounds of the invention with bases such as alkali metal salts (for example sodium or potassium salts), alkaline earth metal salts (for example magnesium or calcium salts), ammonium salts (for example lower alkanolammonium).
  • bases such as alkali metal salts (for example sodium or potassium salts), alkaline earth metal salts (for example magnesium or calcium salts), ammonium salts (for example lower alkanolammonium).
  • Salts and other pharmaceutically acceptable amine salts such as methylamine, ethylamine, propylamine, dimethylamine, trimethylamine, diethylamine, triethylamine, tert-butyl
  • a base amine salt an ethylenediamine salt, a hydroxyethylamine salt, a dihydroxyethylamine salt, a trihydroxyethylamine salt, and an amine salt formed of morpholine, piperazine, and lysine, respectively.
  • solvate refers to a complex of a compound of the invention that is coordinated to a solvent molecule to form a specific ratio.
  • Hydrophilate means a complex formed by the coordination of a compound of the invention with water.
  • the present invention provides a method of modulating HIF and/or EPO which stabilizes HIF and activates expression of a HIF regulatory gene by inhibiting hydroxylation of HIF ⁇ .
  • the methods are also applicable to the prevention, pretreatment or treatment of HIF and/or EPO related conditions, including anemia, ischemia and anoxic conditions.
  • Ischemia and hypoxia are two conditions associated with HIF and include, but are not limited to, myocardial infarction, hepatic ischemia, renal ischemia and stroke; peripheral vascular disorders, ulcers, burns and chronic wounds; pulmonary embolism; And ischemia-reperfusion injury, including, for example, ischemia-reperfusion injury associated with surgery and organ transplantation.
  • One aspect of the invention provides methods for treating a variety of ischemic and anoxic conditions, particularly using the compounds described herein.
  • the methods of the invention produce a therapeutic benefit when administered after ischemia or hypoxia.
  • the methods of the present invention result in a dramatic reduction in morbidity and mortality and a significant improvement in cardiac structure and performance.
  • the method of the present invention improves liver function when administered after hepatotoxic-ischemic injury.
  • Hypoxia is an important component of liver disease, especially in chronic liver diseases associated with hepatotoxic compounds such as ethanol.
  • HIF ⁇ Induced gene expression is increased in alcoholic liver disease, such as nitric oxide synthase and glucose transporter-1.
  • the present invention provides a method of treating a condition associated with ischemia or hypoxia, the method comprising administering a therapeutically effective amount of a compound or a pharmaceutically acceptable salt thereof, alone or in combination with a pharmaceutically acceptable excipient, Tester.
  • the compound is administered immediately following the development of an ischemic condition, such as myocardial infarction, pulmonary embolism, intestinal infarction, ischemic stroke, and renal ischemia-reperfusion injury.
  • the compound is administered to a patient diagnosed as a condition associated with the development of chronic ischemia, such as cardiogenic cirrhosis, macular degeneration, pulmonary embolism, acute respiratory failure, neonatal respiratory distress synthesis Symptoms and congestive heart failure.
  • Another aspect of the invention provides a method of treating a patient at risk of developing an ischemic or hypoxic condition, such as an atherosclerotic high risk individual, using a compound described herein.
  • Risk factors for atherosclerosis include, for example, hyperlipidemia, smoking, hypertension, diabetes, hyperinsulinemia, and abdominal obesity.
  • the present invention provides a method of preventing ischemic tissue damage, comprising administering a therapeutically effective amount of a compound or a pharmaceutically acceptable salt thereof, alone or in combination with a pharmaceutically acceptable excipient, to a patient in need thereof .
  • the compound can be administered based on a predisposition to a condition such as hypertension, diabetes, arterial occlusive disease, chronic venous insufficiency, Raynaud's disease, chronic skin ulcer, sclerosis, congestive heart failure, and systemic hardening.
  • a condition such as hypertension, diabetes, arterial occlusive disease, chronic venous insufficiency, Raynaud's disease, chronic skin ulcer, sclerosis, congestive heart failure, and systemic hardening.
  • the method is used to increase angiogenesis and/or granulation tissue formation in damaged tissues, wounds, and ulcers.
  • the compounds of the invention have been shown to be effective in stimulating granulation tissue formation during wound healing.
  • Granulation tissue contains newly formed leaky blood vessels and temporary plasma protein matrices such as fibrinogen and plasma fibronectin.
  • the release of growth factors from inflammatory cells, platelets and activated endothelium stimulates the migration and proliferation of fibroblasts and endothelial cells in granulation tissue. If angiogenesis or nerve stimulation is weakened, ulceration can occur.
  • the method of the invention effectively promotes the formation of granulation tissue.
  • the present invention provides methods for treating a patient having a tissue injury due to, for example, an infarction, having a wound induced by, for example, trauma or injury, or having a chronic wound or ulcer resulting from a condition such as diabetes.
  • the method comprises administering to a subject in need thereof a therapeutically effective amount of a compound, or a pharmaceutically acceptable salt thereof, alone or in combination with a pharmaceutically acceptable excipient.
  • Another aspect of the invention provides a method of pre-treating a subject with the compound to reduce or prevent the occurrence of tissue damage associated with ischemia or hypoxia.
  • the methods of the invention produce a therapeutic benefit when administered immediately prior to a condition involving ischemia or hypoxia.
  • the application of the method of the invention prior to induction of myocardial infarction showed a statistically significant improvement in cardiac structure and performance.
  • the methods of the invention when administered immediately before and between ischemia-reperfusion injury, produce a therapeutic benefit that significantly reduces diagnostic parameters associated with renal failure.
  • the present invention provides a method of pretreating a subject to reduce or prevent tissue damage associated with ischemia or hypoxia, the method comprising administering a therapeutically effective amount of a compound or a pharmaceutically acceptable salt thereof, alone or in combination with a medicament Acceptable excipient combination
  • a patient with a history of ischemic conditions such as a myocardial infarction, or a patient with impending ischemic symptoms, such as angina pectoris.
  • the compound can be administered based on physical parameters suggesting possible ischemia, such as for individuals under general anesthesia or temporarily working at high altitudes.
  • the compound can be used in an organ transplant to pre-treat an organ donor or to maintain an organ removed by the body prior to implantation into the recipient.
  • the present invention encompasses a "dual therapy" method for treating or preventing a condition involving ischemia or hypoxia, including Ischemia or hypoxia associated with concurrent reactive fibrosis, such as myocardial infarction and consequent congestive heart failure.
  • the method may use a compound which inhibits more than one 2-ketoglutarate dioxygenase having the same specificity or different specificity, such as HIF prolyl hydroxylase and sol prolyl 4- Hydroxylase.
  • the method may employ a combination of compounds wherein each compound specifically inhibits only one 2-ketoglutarate dioxygenase, for example, one compound specifically inhibits HIF prolyl hydroxylase and the second compound is specific Inhibition of sol-prolyl-hydroxylase.
  • the compounds of the invention inhibit one or more 2-ketoglutarate dioxygenases.
  • the compound inhibits at least two members of the 2-ketoglutarate dioxygenase family having the same specificity or different specificities, such as HIF prolyl hydroxylase and HIF asparagine-hydroxyl Chemical enzyme (FIH-1).
  • the compound is specific for a 2-ketoglutarate dioxygenase, such as HIF prolyl hydroxylase, and exhibits little or no specificity for other family members. Display specificity.
  • the compounds can be administered in combination with a variety of other therapeutic methods.
  • the compound is administered with another 2-ketoglutarate dioxygenase inhibitor, wherein the two compounds have an individual member of the 2-ketoglutarate dioxygenase family.
  • the two compounds can be administered simultaneously in a ratio relative to the other. Determination of the proportions suitable for a given therapeutic procedure or a particular subject is within the skill of the art.
  • the two compounds can be administered continuously over the course of treatment, for example after myocardial infarction.
  • a compound specifically inhibits the activity of HIF prolyl hydroxylase
  • the second compound specifically inhibits the activity of the procollagen prolyl 4-hydroxylase.
  • one compound specifically inhibits the activity of HIF prolyl hydroxylase, and the second compound specifically inhibits the activity of HIF asparaginyl-hydroxylase.
  • the compound is administered with another therapeutic agent having a different mode of action, such as an ACE inhibitor (ACEI), an angiotensin-II receptor blocker (ARB), statin, diuretic Agent, digoxin, carnitine, etc.
  • ACEI ACE inhibitor
  • ARB angiotensin-II receptor blocker
  • statin diuretic Agent
  • digoxin digoxin, carnitine, etc.
  • the present invention provides a method of increasing endogenous erythropoietin (EPO). These methods can be applied in vivo, such as in plasma, or in vitro, such as in a conditioned cell culture medium.
  • the invention further provides a method for increasing the content of endogenous EPO.
  • the method for preventing, pre-treating or treating EPO-related conditions including, for example, conditions associated with anemia and nervous system disorders.
  • Anemia-related conditions include conditions such as acute or chronic kidney disease, diabetes, cancer, ulcers, viral infections (eg, HIV, bacteria or parasites), inflammation, and the like.
  • Anemia conditions may further include conditions associated with a procedure or treatment including, for example, radiation therapy, chemotherapy, dialysis, and surgery.
  • Anemia-related disorders additionally include abnormal hemoglobin and/or red blood cells, such as found in conditions such as small red cell anemia, hypohemoglobinemia, aplastic anemia, and the like.
  • the invention may be used to prophylactically or simultaneously increase endogenous EPO in a subject undergoing a particular treatment or procedure, such as HIV anemia being treated with azidothymidine (Zidovudine) or other reverse transcriptase inhibitors Patients, anemia cancer patients receiving cyclic chemotherapy with cyclohexyl cisplatin or cisplatin, or anemia or non-anemic patients scheduled to undergo surgery.
  • Methods of increasing endogenous EPO can also be used to prevent, pre-treat, or treat EPO-related conditions associated with neurological damage or neuronal degeneration, including, but not limited to, stroke, trauma, epilepsy, spinal cord injury, and neurodegenerative disorders.
  • the method can be used to increase endogenous EPO levels in anemia or non-anemic patients scheduled to undergo surgery to reduce the need for exogenous blood transfusion or to facilitate pre-operative blood storage.
  • a small reduction in blood hematocrit that usually occurs after autologous blood supply before surgery does not stimulate an increase in endogenous EPO or compensatory red blood cell production.
  • pre-surgical stimulation of endogenous EPO will effectively increase red blood cell mass and autologous blood supply volume while maintaining higher hematocrit levels, and the method specificity is encompassed herein.
  • the methods of the present invention can be applied to reduce heterologous blood exposure.
  • the methods of the invention can also be used to enhance athletic performance, improve exercise performance, and promote or enhance aerobic conditioning.
  • an athlete can use the method to facilitate training and the soldier can use the method to improve, for example, stamina and endurance.
  • the methods of the present invention have been shown to increase endogenous erythropoietin levels in the culture medium and in vivo treated animal plasma in vitro for treatment of cultured cells.
  • the kidney is the main source of erythropoietin in the body
  • other organs including the brain, liver and bone marrow, can and do synthesize erythropoietin once properly stimulated.
  • the expression of endogenous erythropoietin in a plurality of body organs, including the brain, kidney and liver, can be increased using the method of the invention.
  • the method of the invention even increases the content of endogenous erythropoietin in animals undergoing double nephrectomy.
  • the method of the present invention demonstrates that the content of erythropoietin can be increased even when renal function is impaired.
  • the present invention is not limited by the mechanism of erythropoietin production, the reduction in erythropoietin secretion normally seen during renal failure can be attributed to hyperoxemia caused by increased flow/perfusion in renal tissue.
  • the methods of the invention increase hematocrit and blood hemoglobin levels in an animal treated in vivo.
  • the dosage regimen can be determined to produce a constant, controllable response level of the compound of the invention.
  • the treatment of the substance can treat anemia, such as anemia caused by toxic compounds such as the chemotherapeutic agent cisplatin, or anemia due to blood loss, such as trauma, injury, parasites or surgery.
  • the present invention encompasses methods of increasing the amount of reticulocytes in the blood of an animal to produce a cell-free reticulocyte lysate by the compounds of the invention (e.g., Pelham and Jackson in Eur. J. Biochem. 67). Use in: 247-256 (described in 1976).
  • the amount of circulating reticulocytes in an animal e.g., rabbit, etc. is increased by treatment with a compound of the invention alone or in combination with another compound, such as acetophenone or the like.
  • the beneficial effects of the present invention are that the compounds of the invention are useful for modulating hypoxia inducible factor (HIF) and/or endogenous erythropoietin (EPO).
  • HIF hypoxia inducible factor
  • EPO endogenous erythropoietin
  • this technique changes the metabolism of the compound in the organism, giving the compound a better pharmacokinetic parameter characteristic.
  • the dosage can be changed and a long-acting preparation can be formed to improve the applicability.
  • Replacing a hydrogen atom in a compound with hydrazine can increase the drug concentration of the compound in an animal to improve the efficacy of the drug due to its strontium isotope effect.
  • Substitution of a hydrogen atom in a compound with hydrazine may increase the safety of the compound due to inhibition of certain metabolites.
  • each reaction is usually carried out in an inert solvent at room temperature to reflux temperature (e.g., 0 ° C to 100 ° C, preferably 0 ° C to 80 ° C).
  • the reaction time is usually from 0.1 to 60 hours, preferably from 0.5 to 24 hours.
  • Step 1 Synthesis of 3-chloro-5-(3-chlorophenyl)cyanopyridine (Compound 3).
  • Step 2 Synthesis of 5-(3-chlorophenyl)-3-methoxycyanopyridine (Compound 4).
  • Step 3 Synthesis of 5-(3-chlorophenyl)-3-hydroxypicolinic acid (Compound 5).
  • Step 1 Synthesis of glycine methyl ester-2,2-d 2 hydrochloride (Compound 7).
  • Step 2 (5- (3-chlorophenyl) -3-hydroxypyridine-formyl) glycine methyl ester synthesis -2,2-d 2 (Compound 8).
  • N,N-diisopropylethylamine DIPEA, 777 mg, 6.02 mmol
  • DIPEA 1-hydroxybenzotriazole
  • HOBT 407 mg, 3.01 mmol
  • 1-ethyl-(3-dimethyl) Aminoaminopropyl)carbodiimide EDCI, 577 mg, 3.01 mmol
  • EDCI 1-ethyl-(3-dimethyl) Aminoaminopropyl)carbodiimide
  • Step 3 Synthesis of (5- (3-chlorophenyl) -3-hydroxypyridine-formyl) glycine -2,2-d 2 (Compound 9).
  • Step 1 Synthesis of 2-carboxy-5-(3-chlorophenyl)-3-hydroxypyridine-1-oxide (Compound 10).
  • Step 2 Synthesis of 2-carboxy-5-(3-chlorophenyl)-3-hydroxypyridine-1-oxide-6-d (Compound 11).
  • Step 3 5-(3-Chlorophenyl)-3-hydroxy-2-((2-methoxy-2-oxoethyl)carbamoyl)pyridine-1-oxide-6-d (Compound 12 )Synthesis.
  • Step 4 Synthesis of (5-(3-chlorophenyl)-3-hydroxypyridinecarbonyl-6-d)glycine methyl ester (Compound 13).
  • Step 5 Synthesis of (5-(3-chlorophenyl)-3-hydroxypyridinecarbonyl-6-d)glycine (Compound 14).
  • Step 1 5-(3-Chlorophenyl)-3-hydroxy-2-((2-methoxy-2-oxoethyl-1,1-d 2 )carbamoyl)pyridine-1-oxide Synthesis of -6-d (Compound 15).
  • Step 2 Synthesis of (5- (3-chlorophenyl) -3-hydroxypyridine-formyl -6-d) glycinate -2,2-d 2 (Compound 16).
  • Step 5 Synthesis of (5-(3-chlorophenyl)-3-hydroxypyridinecarbonyl-6-d)glycine-2,2-d 2 (Compound 17).
  • Step 1 Synthesis of 3-chlorophenyl-2,4,6-d 3 -ammonia (Compound 19).
  • Step 2 Synthesis of 2-(3-chlorophenyl-2,4,6-d 3 )-4,4,5,5-tetramethyl-1,3,2-dioxaborane (Compound 20) .
  • Step 3 Synthesis of 3-chloro-5-(3-chlorophenyl-2,4,6-d 3 )cyanopyridine (Compound 21).
  • Step 4 Synthesis of 5-(3-chlorophenyl-2,4,6-d 3 )-3-methoxycyanopyridine (Compound 22).
  • Step 5 Synthesis of 5-(3-chlorophenyl-2,4,6-d 3 )-3-hydroxypicolinic acid (Compound 23).
  • Step 6 Synthesis of (5-(3-chlorophenyl-2,4,6-d 3 )-3-hydroxypyridinecarbonyl)glycine methyl ester (Compound 24).
  • Step 7 Synthesis of (5-(3-chlorophenyl-2,4,6-d 3 )-3-hydroxypyridinecarbonyl)glycine (Compound 25).
  • Step 1 Synthesis of (5-(3-chlorophenyl-2,4,6-d 3 )-3-hydroxypyridinecarbonyl)glycine methyl ester-2,2-d 2 (Compound 26).
  • Step 2 Synthesis of (5-(3-chlorophenyl-2,4,6-d 3 )-3-hydroxypyridinecarbonyl)glycine-2,2-d 2 (Compound 27).
  • Step 1 Synthesis of 3-chlorophenyl-6-d-ammonia (Compound 29).
  • Step 2 Synthesis of 2-(3-chlorophenyl-6-d)-4,4,5,5-tetramethyl-1,3,2-dioxaborane (Compound 30).
  • Step 3 Synthesis of 3-chloro-5-(3-chlorophenyl-6-d)cyanopyridine (Compound 31).
  • Step 4 Synthesis of 5-(3-chlorophenyl-6-d)-3-methoxycyanopyridine (Compound 32).
  • Step 5 Synthesis of 5-(3-chlorophenyl-6-d)-3-hydroxypicolinic acid (Compound 33).
  • Step 6 Synthesis of (5-(3-chlorophenyl-6-d)-3-hydroxypyridineformyl)glycine methyl ester (Compound 34).
  • Step 7 Synthesis of (5-(3-chlorophenyl-6-d)-3-hydroxypyridinecarbonyl)glycine (Compound 35).
  • Step 1 (5- (3-chlorophenyl -6-d) -3- hydroxypyridine-formyl) glycine methyl ester synthesis -2,2-d 2 (Compound 36).
  • Step 2 (5- (3-chlorophenyl -6-d) -3- hydroxypyridine-formyl) glycine synthesis -2,2-d 2 (Compound 37).
  • the mouse tissues stored at -80 ° C were ground into a powder using a mortar and pestle frozen under liquid nitrogen.
  • Nuclear extracts were prepared using NE-PER kit (Pierce Biotechnology).
  • the nuclear extract was added to the HIF-1 anti-monoclonal antibody at a ratio of tissue to antibody of 200:1.
  • the suspension was incubated for 4 hours at 4 ° C in a conical microcentrifuge tube. Protein A/G coupled agarose beads (40% suspension of 40-lipose beads) was then added to the tube. After rotating at 4 ° C overnight, the beads were washed 3 times with ice-cold phosphate buffer.
  • the beads were then used to prepare the SDS-PAGE with an ice-cold phosphate sample buffer solution after 40 minutes. Proteins separated from SDS-PAGE were transferred to nitrocellulose plates with the XCell-II Blot Module system. The blot was blocked with 5% BSA and then incubated with HIF-1 at a ratio of 1:100 diluted with rabbit antibody. The blots were then washed with Tris buffered saline/Tween-20 buffer and incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody. The blot was visualized with ECL reagent. The blot image was captured with an Epson Expression 1600 scanner.
  • the compounds of the present invention and their non-deuterated compound AKB-6548 were simultaneously tested and compared, and their ability to promote secretion of HIF protein in mouse tissues was evaluated.
  • the experimental results are shown in Table 1 below.
  • the ability of the compounds of the present invention to promote the secretion of HIF protein in mouse tissues was significantly stronger than that of the undeuterated compound AKB-6548.
  • Mouse serum EPO was detected using R&D Systems' mouse Quantikine erythropoietin ELISA kit according to the manufacturer's instructions.
  • the compounds of the present invention and their non-deuterated compound AKB-6548 were simultaneously tested and compared, and their ability to secrete EPO in mice was evaluated.
  • the experimental results are shown in Table 2 below.
  • the compound of the present invention promoted the ability of mice to secrete EPO significantly stronger than the undeuterated compound AKB-6548, indicating that the compound of the present invention can be used for preparing a drug for regulating anemia in humans.
  • Microsomal experiments human liver microsomes: 0.5 mg/mL, Xenotech; rat liver microsomes: 0.5 mg/mL, Xenotech; coenzyme (NADPH/NADH): 1 mM, Sigma Life Science; magnesium chloride: 5 mM, 100 mM phosphate buffer Agent (pH 7.4).
  • phosphate buffer 100 mM, pH 7.4.
  • the pH was adjusted to 7.4, diluted 5 times with ultrapure water before use, and magnesium chloride was added to obtain a phosphate buffer (100 mM) containing 100 mM potassium phosphate, 3.3 mM magnesium chloride, and a pH of 7.4.
  • NADPH regeneration system containing 6.5 mM NADP, 16.5 mM G-6-P, 3 U/mL G-6-P D, 3.3 mM magnesium chloride was prepared and placed on wet ice before use.
  • Formulation stop solution acetonitrile solution containing 50 ng/mL propranolol hydrochloride and 200 ng/mL tolbutamide (internal standard). Take 25057.5 ⁇ L of phosphate buffer (pH 7.4) into a 50 mL centrifuge tube, add 812.5 ⁇ L of human liver microsomes, and mix to obtain a liver microsome dilution with a protein concentration of 0.625 mg/mL. Take 25057.5 ⁇ L of phosphate buffer (pH 7.4) to a 50 mL centrifuge tube In the experiment, 812.5 ⁇ L SD rat liver microsomes were added and mixed to obtain a liver microsome dilution with a protein concentration of 0.625 mg/mL.
  • the corresponding compound had a reaction concentration of 1 ⁇ M and a protein concentration of 0.5 mg/mL.
  • 100 ⁇ L of the reaction solution was taken at 10, 30, and 90 min, respectively, and added to the stopper, and the reaction was terminated by vortexing for 3 min.
  • the stopper plate was centrifuged at 5000 ⁇ g, 40 for 10 min.
  • 100 in supernatant was taken into a 96-well plate pre-charged with 100 ⁇ L of distilled water, mixed, and sample analysis was performed by LC-MS/MS.
  • Rats were fed a standard diet and given water. Fasting began 16 hours before the test.
  • the drug was dissolved with PEG400 and dimethyl sulfoxide. Blood was collected from the eyelids at a time point of 0.083 hours, 0.25 hours, 0.5 hours, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, and 24 hours after administration.
  • Rats were briefly anesthetized after inhalation of ether, and 300 ⁇ L of blood samples were collected from the eyelids in test tubes. There was 30 ⁇ L of 1% heparin salt solution in the test tube. The test tube was placed in 60 liquid before use. Dry overnight. After the blood sample collection was completed at a later time point, the rats were anesthetized with ether and sacrificed.
  • the experimental results show that the compound of the present invention has better pharmacokinetics in animals than the control compound AKB-6548, and thus has better pharmacodynamics and therapeutic effects.

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Abstract

本发明提供了一种取代的杂芳基酰胺化合物及包含该化合物的组合物及其用途，本发明公开了如式(I)所示的杂芳基酰胺化合物，或其晶型、药学上可接受的盐、前药，立体异构体、水合物或溶剂化合物。本发明所述的杂芳基酰胺化合物及包含该化合物的组合物可用于调节缺氧诱导因子(HIF)和/或内源性促红细胞生成素(EPO)，可用于制备调控人体贫血的药物。

Description

一种取代的杂芳基酰胺化合物及包含该化合物的组合物及其用途 技术领域
本发明属于医药技术领域，尤其涉及一种取代的杂芳基酰胺化合物及包含该化合物的组合物，以及能够调节缺氧诱导因子(HIF)亚单位的稳定性和增加体外及体内内源性促红细胞生成素的方法和化合物。
背景技术
缺氧诱导因子(HIF)是一种碱性螺旋-环-螺旋(bHLH)PAS(Per/Arnt/Sim)转录激活剂，其调控随细胞氧浓度改变的基因表达的改变。HIF是一种含有一个氧调节α亚单位(HIFα)和一个组成性表达β亚单位(HIFβ)的杂二聚体，也被称为芳香烃受体核转运蛋白(ARNT)。在氧合(常氧)细胞中，HIFα亚单位通过涉及视网膜血管瘤抑制蛋白(pVHL)E3连接酶复合物泛素化的机制迅速降解。在缺氧条件下，HIFα不降解，且一种活性HIFα/β复合物在细胞核中累积并激活若干基因的表达，包括糖酵解酶、葡萄糖转运蛋白(GLUT)-1、促红细胞生成素(EPO)和血管内皮细胞生长因子(VEGF)。(Maxwell等人，自然，1999，399，271-275)。
促红细胞生成素(EPO)是随HIFα而产生的一种自然存在的激素，其刺激运载氧气贯穿全身的红细胞的产生。EPO通常由肾分泌，且内源性EPO在氧减少(缺氧)的条件下增加。所有类型贫血的特征在于血液运载氧的能力减少，并因而伴有类似体征与症状，包括皮肤及粘膜苍白、虚弱、头晕、易疲劳和嗜睡，导致生活质量的下降。具有严重贫血情况的受试者表现出难以呼吸及心脏畸形。贫血通常与红细胞中或血红蛋白中血液缺乏有关。
局部缺血和缺氧病症是发病和死亡的主要原因。心血管病每年引起至少一千五百万的死亡且是造成全世界30％死亡的原因。在多种心血管病中，缺血性心脏病和脑血管病引起约17％的死亡。每年报道有一百三十万非致命性急性心肌梗塞的病例，构成大约每100,000人中300人的发病率。另一方面，估计每年有五百万美国人患有静脉血栓症，且约600,000这些病例导致肺栓塞。约三分之一的肺栓塞患者最终死亡，使得肺栓塞成为美国人死亡的第三个最普遍原因。
当前，局部缺血和缺氧病症的治疗集中在症状的减轻和致病性病症的治疗上。例如，心肌梗塞的治疗包括用以控制疼痛和减轻心脏工作负荷的硝酸甘油和镇痛药。使用其它药物，包括地高辛(digoxin)、利尿剂、氨利酮(amrinone)、β-阻断剂、降脂剂和血管紧张素转换酶抑制剂来稳定病况，但这些疗法中没有一个可直接作用于由局部缺血和缺氧产生的组织损坏。
由于当前治疗中及生产和使用重组EPO中的不足，所以依然需要有效治疗以下疾病的化合物：促红细胞生成素相关病况，例如贫血，包括与糖尿病、贫血、溃疡、肾衰竭、癌症、感染、透析、手术和化学疗法相关的贫血和涉及局部缺血和缺氧的病况，例如动脉闭塞性疾病、心绞痛、肠梗塞、肺梗塞、脑局部缺血和心肌梗塞。也需要有效预防由局部缺血引起的组织损坏的化合物，所述局部缺血由于例如动脉粥样硬化、糖尿病和例如肺栓塞及其类似病症的肺部病症而发生。总之，在此项技术中需要调节HIF和/或内源性促红细胞生成素，且可用于治疗和预防HIF相关和EPO相关病症的方法和化合物，所述病症包括涉及贫血、局部缺血和缺氧的病况。
发明内容
针对以上技术问题，本发明公开了一种化合物及包含该化合物的组合物，其可用于调节缺氧诱导因子(HIF)和/或内源性促红细胞生成素(EPO)和/或具有更好药效学/药代动力学性能。
对此，本发明采用的技术方案为：
本发明的目的是提供一类新型可用于调节缺氧诱导因子(HIF)和/或内源性促红细胞生成素(EPO)的和/或具有更好药效学/药代动力学性能的化合物。
本发明的第一方面中，提供了一种式(I)所示的化合物，或其晶型、药学上可接受的盐、水合物或溶剂化合物。
Figure PCTCN2017114399-appb-000001
其中，R1、R2、R3、R4、R5、R6、R7、R8各自独立地为氢、氘、卤素；
附加条件是R1、R2、R3、R4、R5、R6、R7和R8中至少一个是氘代的或氘。
在另一优选例中，R1和R2各自独立地为氘或氢。
在另一优选例中，R1、R2是氘。
在另一优选例中，R3和R4各自独立地为氘或氢。
在另一优选例中，R5、R6、R7、R8各自独立地为氘或氢。
在另一优选例中，所述化合物可选自下组化合物或其药学上可接受的盐，但不局限于如下化合物：
Figure PCTCN2017114399-appb-000002
在另一优选例中，氘在氘代位置的氘同位素含量至少是大于天然氘同位素含量(0.015％)，较佳地大于30％，更佳地大于50％，更佳地大于75％，更佳地大于95％，更佳地大于99％。
具体地说，在本发明中R1、R2、R3、R4、R5、R6、R7和R8各氘代位置中氘同位素含量至少是5％，较佳地大于10％，更佳地大于15％，更佳地大于20％，更佳地大于25％，更佳地大于30％，更佳地大于35％，更佳地大于40％，更佳地大于45％，更佳地大于50％，更佳地大于55％，更佳 地大于60％，更佳地大于65％，更佳地大于70％，更佳地大于75％，更佳地大于80％，更佳地大于85％，更佳地大于90％，更佳地大于95％，更佳地大于99％。
在另一优选例中，式(I)中化合物的R1、R2、R3、R4、R5、R6、R7和R8，至少其中一个R含氘，更佳地两个R含氘，更佳地三个R含氘，更佳地四个R含氘，更佳地五个R含氘，更佳地六个R含氘，更佳地七个R含氘，更佳地八个R含氘。
在另一优选例中，所述化合物不包括非氘代化合物。
在本发明的第二方面中，提供了一种制备药物组合物的方法，包括步骤：将药学上可接受的载体与本发明第一方面中所述的化合物，或其晶型、药学上可接受的盐、水合物或溶剂合物进行混合，从而形成药物组合物。
在本发明的第三方面中，提供了一种药物组合物，它含有药学上可接受的载体和本发明第一方面中所述的化合物，或其晶型、药学上可接受的盐、水合物或溶剂合物。
可用于本发明药物组合物中的药学上可接受的载体包括但不限于任何助流剂、增甜剂、稀释剂、防腐剂、染料/着色剂、矫味增强剂、表面活性剂、润湿剂、分散剂、崩解剂、助悬剂、稳定剂、等渗剂、溶剂或乳化剂。
本发明药物组合物可配制成固态、半固态、液态或气态制剂，如片剂、丸剂、胶囊剂、粉剂、颗粒剂、膏剂、乳剂、悬浮剂、溶液剂、栓剂、注射剂、吸入剂、凝胶剂、微球及气溶胶等。
给予本发明药物组合物的典型途径包括但不限于口服、直肠、透黏膜、经肠给药，或者局部、经皮、吸入、肠胃外、舌下、阴道内、鼻内、眼内、腹膜内、肌内、皮下、静脉内给药。优选口服给药或注射给药。
本发明的药物组合物可以采用本领域周知的方法制造，如常规的混合法、溶解法、制粒法、制糖衣药丸法、磨细法、乳化法、冷冻干燥法等。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
本文中，如无特别说明，“卤素”指F、Cl、Br、和I。更佳地，卤原子选自F、Cl和Br。
本文中，如无特别说明，“氘代”指化合物或基团中的一个或多个氢被氘所取代；氘代可以是一取代、二取代、多取代或全取代。术语“一个或多个氘代的”与“一次或多次氘代”可互换使用。
本文中，如无特别说明，“非氘代的化合物”是指含氘原子比例不高于天然氘同位素含量(0.015％)的化合物。
本发明还包括同位素标记的化合物，等同于原始化合物在此公开。可以列为本发明的化合物同 位素的例子包括氢，碳，氮，氧，磷，硫，氟和氯同位素，分别如2H，3H，13C，14C，15N，17O，18O，31P，32P，35S，18F以及36Cl。本发明中的化合物，或对映体，非对映体，异构体，或药学上可接受的盐或溶剂化物，其中含有上述化合物的同位素或其他其他同位素原子都在本发明的范围之内。本发明中某些同位素标记化合物，例如3H和14C的放射性同位素也在其中，在药物和底物的组织分布实验中是有用的。氚，即3H和碳-14，即14C，它们的制备和检测比较容易，是同位素中的首选。同位素标记的化合物可以用一般的方法，通过用易得的同位素标记试剂替换为非同位素的试剂，用示例中的方案可以制备。
药学上可接受的盐包括无机盐和有机盐。一类优选的盐是本发明化合物与酸形成的盐。适合形成盐的酸包括但并不限于：盐酸、氢溴酸、氢氟酸、硫酸、硝酸、磷酸等无机酸；甲酸、乙酸、三氟乙酸、丙酸、草酸、丙二酸、琥珀酸、富马酸、马来酸、乳酸、苹果酸、酒石酸、柠檬酸、苦味酸、苯甲酸、甲磺酸、乙磺酸、对甲苯磺酸、苯磺酸、萘磺酸等有机酸；以及脯氨酸、苯丙氨酸、天冬氨酸、谷氨酸等氨基酸。另一类优选的盐是本发明化合物与碱形成的盐，例如碱金属盐(例如钠盐或钾盐)、碱土金属盐(例如镁盐或钙盐)、铵盐(如低级的烷醇铵盐以及其它药学上可接受的胺盐)，例如甲胺盐、乙胺盐、丙胺盐、二甲基胺盐、三甲基胺盐、二乙基胺盐、三乙基胺盐、叔丁基胺盐、乙二胺盐、羟乙胺盐、二羟乙胺盐、三羟乙胺盐，以及分别由吗啉、哌嗪、赖氨酸形成的胺盐。
术语“溶剂合物”指本发明化合物与溶剂分子配位形成特定比例的配合物。“水合物”是指本发明化合物与水进行配位形成的配合物。
本发明提供调节HIF和/或EPO的方法，其通过抑制HIFα羟基化从而稳定HIF和激活HIF调控基因的表达。所述方法也可应用于预防、预先治疗或治疗HIF和/或EPO相关病况，包括贫血、局部缺血和缺氧病况。
局部缺血和缺氧是两种与HIF有关的病况并包括(但不限于)心肌梗塞、肝局部缺血、肾局部缺血和中风；周围血管病症、溃疡、烧伤和慢性伤口；肺栓塞；和缺血-再灌注损伤，包括例如与手术和器官移植相关的缺血-再灌注损伤。
本发明的一个方面提供用于治疗多种局部缺血和缺氧病况的方法，特别是使用本文中所描述的化合物。在一个实施例中，当在局部缺血或缺氧后投予时，本发明的方法产生治疗益处。例如，在心肌梗塞之后，本发明的方法使得发病率和死亡率惊人的降低，并且显著改善心脏结构和性能。另一方面，当在肝中毒性-局部缺血性损伤之后投予时，本发明的方法改善肝功能。缺氧是肝脏疾病的一个重要组成部分，尤其在与肝毒性化合物，例如乙醇有关的慢性肝病中。另外，已知由HIFα 诱导的基因表达在酒精性肝病中增加，例如一氧化氮合成酶和葡萄糖转运体-1。
因此，本发明提供治疗局部缺血或缺氧相关病况的方法，所述方法包含将治疗有效量的化合物或其医药上可接受的盐单独或与医药上可接受的赋形剂组合投予受试者。在一个实施例中，在产生局部缺血的病况之后立即投予所述化合物，例如心肌梗塞、肺栓塞、肠梗塞、缺血性中风和肾缺血-再灌注损伤。在另一个实施例中，将所述化合物投予诊断为与慢性局部缺血的发生相关的病况的患者，例如心原性肝硬化、黄斑变性、肺栓塞、急性呼吸衰竭、新生儿呼吸窘迫综合症和充血性心力衰竭。
本发明的另一方面提供使用本文中所描述的化合物治疗有发生局部缺血或缺氧病况危险的患者的方法，例如动脉粥样硬化高危个体。动脉粥样硬化的危险因素包括，例如高脂血症、吸烟、高血压、糖尿病、高胰岛素血症和腹部肥胖。因此，本发明提供预防局部缺血性组织损伤的方法，所述方法包含将治疗有效量的化合物或其医药上可接受的盐单独或与医药上可接受的赋形剂组合投予需要的患者。在一个实施例中，可基于素因性病况投予所述化合物，例如高血压、糖尿病、动脉闭塞性疾病、慢性静脉机能不全、雷诺氏病、慢性皮肤溃疡、硬化、充血性心力衰竭和系统性硬化。
在一个特定实施例中，将所述方法用于增加受损组织、伤口和溃疡中的血管形成和/或肉芽组织形成。例如，本发明的化合物已经显示在伤口愈合中可有效刺激肉芽组织形成。肉芽组织含有新形成的渗漏血管和临时血浆蛋白基质，例如纤维蛋白原和血浆纤维结合蛋白。来自炎性细胞、血小板和激活内皮的生长因子的释放刺激成纤维细胞和内皮细胞在肉芽组织中的迁移和增殖。若血管形成或神经刺激削弱，则可发生溃疡。本发明的方法有效促进肉芽组织的形成。因而，本发明提供用于治疗具有由于例如梗塞造成的组织损坏、具有由例如创伤或损伤诱导的伤口或具有由于某种病症(例如糖尿病)而产生的慢性伤口或溃疡的患者的方法。所述方法包含将治疗有效量的化合物或其医药上可接受的盐单独或与医药上可接受的赋形剂组合投予需要的患者。
发明的另一方面提供使用所述化合物预先治疗受试者以减少或预防与局部缺血或缺氧相关的组织损坏发生的方法。当在涉及局部缺血或缺氧的病况之前立即投予时，本发明的方法产生治疗益处。例如，在诱导心肌梗塞之前应用本发明的方法显示心脏结构和性能得到在统计学上有显著意义的改善。另一方面，当在缺血-再灌注损伤之前和之间立即投予时，本发明的方法产生治疗益处，显著减少与肾衰竭相关的诊断参数。
因此，本发明提供预先治疗受试者以减少或预防与局部缺血或缺氧相关的组织损坏的方法，所述方法包含将治疗有效量的化合物或其医药上可接受的盐单独或与医药上可接受的赋形剂组合 投予具有局部缺血病症病史的患者，例如心肌梗塞，或具有迫近局部缺血症状的患者，例如心绞痛。在另一个实施例中，可基于暗示可能局部缺血的物理参数投予所述化合物，例如对于处于全身麻醉下或暂时在高海拔下工作的个体。在又一实施例中，可将所述化合物用于器官移植中，用以预先治疗器官供体或在移植入受体之前，用以维持自身体移除的器官。
先前研究已经显示，在本发明的方法中使用的某些化合物是溶胶原脯氨酰4-羟化酶的有效抑制剂。尽管认识到最初梗塞或伤口的恢复需要结缔组织在坏死区域内沉积，但是本发明证明对于瘢痕形成的治疗无副作用。因而，基于本发明的某些化合物在治疗和预防缺氧组织损坏和纤维化上所提供的益处，本发明涵盖一种治疗或预防涉及局部缺血或缺氧病况的“双重治疗”方法，包括与并发反应性纤维化相关的局部缺血或缺氧，例如心肌梗塞和随之而来的充血性心力衰竭。所述方法可使用一种化合物，其抑制一种以上具有相同特异性或不同特异性的2-酮戊二酸双加氧酶，例如HIF脯氨酰羟化酶和溶胶原脯氨酰4-羟化酶。或者，所述方法可使用化合物的组合，其中每一化合物特异抑制仅一种2-酮戊二酸双加氧酶，例如一种化合物特异抑制HIF脯氨酰羟化酶且第二种化合物特异抑制溶胶原脯氨酰4-羟化酶。
在一个方面，本发明的化合物抑制一种或一种以上2-酮戊二酸双加氧酶。在一个实施例中，所述化合物抑制至少两种具有相同特异性或不同特异性的2-酮戊二酸双加氧酶家族成员，例如HIF脯氨酰羟化酶和HIF天冬酰胺-羟化酶(FIH-1)。在另一实施例中，所述化合物对于一种2-酮戊二酸双加氧酶具有特异性，例如HIF脯氨酰羟化酶，并且对于其它家族成员显示出很少的特异性或不显示特异性。
所述化合物可与多种其它治疗方法组合投予。在一个实施例中，所述化合物和另一种2-酮戊二酸双加氧酶抑制剂一起投予，其中这两种化合物对于个别的2-酮戊二酸双加氧酶家族成员具有不同的特异性。所述两种化合物可同时以一个相对于另一个的比例投予。对于适于给定治疗过程或特定受试者的比例的测定在所述领域的技术水平之内。或者，所述两种化合物可在治疗时程中连续投予，例如在心肌梗塞之后。在一个特定实施例中，一种化合物特异抑制HIF脯氨酰羟化酶的活性，且第二种化合物特异抑制溶胶原脯氨酰4-羟化酶的活性。在另一特定实施例中，一种化合物特异抑制HIF脯氨酰羟化酶的活性，且第二种化合物特异抑制HIF天冬酰胺酰-羟化酶的活性。在另一实施例中，所述化合物与另一具有不同作用模式的治疗剂一起投予，例如ACE抑制剂(ACEI)、血管紧张素-II受体阻断剂(ARB)、抑制素、利尿剂、地高辛、肉毒碱等。
本发明提供增加内源性促红细胞生成素(EPO)的方法。这些方法可在体内应用，例如血浆中，或在体外应用，例如在经调节的细胞培养基中。本发明进一步提供增加内源性EPO含量的方 法，用以预防、预先治疗或治疗EPO相关病况，包括例如与贫血和神经系统紊乱相关的病况。贫血相关病况包括例如急性或慢性肾脏疾病、糖尿病、癌症、溃疡、病毒感染(例如HIV、细菌或寄生虫)、炎症等的病症。贫血病况可进一步包括与程序或治疗相关的病况，所述程序或治疗包括例如放射治疗、化学治疗、透析和手术。贫血相关病症另外包括异常血红蛋白和/或红血球，例如发现于如小红细胞性贫血、低血色素贫血症、再生障碍性贫血等病症中。
本发明可用于预防性或同时增加经历特定治疗或程序的受试者中的内源性EPO，例如正以叠氮胸苷(齐多夫定)或其它逆转录酶抑制剂治疗的感染HIV贫血患者、接受含环顺氯氨铂或不含顺氯氨铂的循环化学疗法的贫血癌症患者、或计划经历手术的贫血或非贫血患者。增加内源性EPO的方法也可用于预防、预先治疗或治疗与神经损坏或神经组织退化相关的EPO相关病况，包括(但不限于)中风、创伤、癫痫、脊髓损伤和神经变性病症。
另外，所述方法可用于增加计划经历手术的贫血或非贫血患者中的内源性EPO含量，用以减少对外源输血的需要或用以便于手术前血液的储存。通常在手术前自体供血后发生的血液血细胞比容的少量减少并不刺激内源性EPO或补偿性红血球生成的增加。然而，内源性EPO的手术前刺激将有效增加红血球质量和自体供血体积，同时维持更高的血细胞比容水平，并且所述方法特异性的涵盖于本文中。在一些手术人群中，特别是手术失血超过2升的个体，可应用本发明的方法来减少异源血液曝露。
本发明的方法也可用于增强运动性能、改善锻炼能力和促进或增强有氧调节。例如，运动员可使用所述方法以促进训练且士兵可使用所述方法以改善例如持久力和忍耐力。
本发明的方法已显示可增加体外治疗培养细胞的培养基中和体内治疗的动物血浆中的内源性促红细胞生成素含量。尽管肾是体内促红细胞生成素的主要来源，但一经适当刺激，其它器官，包括大脑、肝和骨髓可且确能合成促红细胞生成素。使用本发明的方法可增加多个身体器官中内源性促红细胞生成素的表达，包括大脑、肾和肝。实际上，本发明的方法甚至增加在经历双测肾切除术的动物中内源性促红细胞生成素的含量。
本发明的方法证明即使当肾功能损害时，也可增加促红细胞生成素的含量。尽管本发明并不为促红细胞生成素产生的机制所限制，但通常在肾衰竭过程中可见的促红细胞生成素分泌的减少可归因于肾组织中流动/灌注增加所致的高氧症。
另一方面，本发明的方法增加体内治疗的动物中血细胞比容和血液血红蛋白水平。随着化合物在本发明的方法中使用而产生的血浆EPO、血细胞比容和血液血红蛋白的增加具有剂量敏感性，然而可确定剂量方案以产生恒定、可控的本发明化合物的响应水平。另一方面，使用本发明化合 物的治疗可医治贫血，例如由毒性化合物，例如化学治疗剂顺氯氨铂诱导的贫血，或由于失血所致的贫血，例如创伤、损伤、寄生虫或手术。
用本发明的化合物治疗的动物中，在血细胞比容和血液血红蛋白增加之前是血液中循环未成熟红细胞(网织红细胞)百分率的增加。因而，本发明涵盖本发明化合物在增加动物血液中网织红细胞的含量从而产生无细胞网织红细胞溶菌产物的方法(如Pelham和Jackson在《欧洲生物化学杂志》(Eur.J.Biochem.)67:247-256(1976)中所描述)中的用途。通过用本发明的化合物单独治疗或与另一种化合物，例如乙酰苯肼等组合治疗，动物(例如兔子等)中循环网织红细胞的含量增加。
与现有技术相比，本发明的有益效果为：本发明化合物可用于调节缺氧诱导因子(HIF)和/或内源性促红细胞生成素(EPO)。通过氘化这一技术改变化合物在生物体中的代谢，使化合物具有更好的药代动力学参数特性。在这种情况下，可以改变剂量并形成长效制剂，改善适用性。用氘取代化合物中的氢原子，由于其氘同位素效应，能够提高化合物在动物体内的药物浓度，以提高药物疗效。用氘取代化合物中的氢原子，由于某些代谢产物被抑制，可能提高化合物的安全性。
具体实施方式
下面更具体地描述本发明式I结构化合物的制备方法，但这些具体方法不对本发明构成任何限制。本发明化合物还可以任选将在本说明书中描述的或本领域已知的各种合成方法组合起来而方便地制得，这样的组合可由本发明所属领域的技术人员容易地进行。
通常，在制备流程中，各反应通常在惰性溶剂中，在室温至回流温度(如0℃～100℃，优选0℃～80℃)下进行。反应时间通常为0.1小时-60小时，较佳地为0.5-24小时。
制备中间体5-(3-氯苯基)-3-羟基吡啶甲酸(化合物5)：
Figure PCTCN2017114399-appb-000003
步骤1：3-氯-5-(3-氯苯基)氰基吡啶(化合物3)的合成。
氮气保护下将无水N,N-二甲基甲酰胺(DMF，20mL)和水(2mL)加入到(3-氯苯基)硼酸(化合物1，2.00g，12.80mmol)和3,5-二氯氰基吡啶(化合物2，2.40g，14.10mmol)、碳酸钾(2.20 g，15.94mmol)、和PdCl2(dppf)(100mg)的混合物中，反应在氮气保护下，45℃搅拌反应过夜。冷却至室温，加水(50mL)淬灭反应，用乙酸乙酯(50mL x 3)萃取，合并有机层，用饱和食盐水洗涤(50mL x 2)，有机层用无水硫酸钠干燥，减压浓缩滤液，浓缩液进行柱分离，得到2.50g白色固体，收率：78.8％。LC-MS(APCI):m/z＝249.1(M+1)。
步骤2：5-(3-氯苯基)-3-甲氧基氰基吡啶(化合物4)的合成。
室温下，氮气保护下将甲醇钠(3.45g，6.39mmol)加到3-氯-5-(3-氯苯基)氰基吡啶(化合物3，2.50g，10.00mmol)的无水甲醇(50mL)的溶液中，反应回流搅拌过夜。冷却至室温，减压除去反应液，加水(50mL)淬灭反应，用乙酸乙酯(50mL x 3)萃取，合并有机层，用饱和食盐水洗涤(50mL x 2)，有机层用无水硫酸钠干燥，减压浓缩滤液，浓缩液进行柱分离，得到1.05g白色固体，收率：43.0％。LC-MS(APCI):m/z＝245.1(M+1)。
步骤3：5-(3-氯苯基)-3-羟基吡啶甲酸(化合物5)的合成。
室温下，将48％的氢溴酸(10mL)加入到5-(3-氯苯基)-3-甲氧基氰基吡啶(化合物4，1.05g，4.30mmol)中，反应在氮气保护下，100℃搅拌反应过夜。冷却至室温。在冰浴下，用50％的氢氧化钠溶液调pH值约为2，继续搅拌1小时，有大量白色固体析出，过滤，用水洗涤滤饼，真空干燥得到1.05g灰白色固体，收率：99％。LC-MS(APCI):m/z＝250.0(M+1)。
实施例1 制备(5-(3-氯苯基)-3-羟基吡啶甲酰基)甘氨酸-2,2-d2(化合物9)
Figure PCTCN2017114399-appb-000004
步骤1：甘氨酸甲酯-2,2-d2盐酸盐(化合物7)的合成。
冰浴下，将二氯亚砜(0.58mL，7.50mmol)缓慢滴加到甘氨酸-d5(500mg，6.25mmol)的无水甲醇(6.5mL)溶液中。滴加完毕后，反应液升温至65℃下反应4小时。减压浓缩得到750mg白色固体，直接用于下一步。LC-MS(APCI):m/z＝91.0(M+1)；1H NMR(500MHz,DMSO-d6)δ3.71(s,3H)。
步骤2：(5-(3-氯苯基)-3-羟基吡啶甲酰基)甘氨酸甲酯-2,2-d2(化合物8)的合成。
冰浴下，将N，N-二异丙基乙胺(DIPEA，777mg，6.02mmol)，1-羟基苯并三唑(HOBT，407mg，3.01mmol)，1-乙基-(3-二甲基氨基丙基)碳酰二亚胺(EDCI，577mg，3.01mmol)分 别加入到5-(3-氯苯基)-3-羟基吡啶甲酸(化合物5，500mg，2.01mmol)的无水N,N-二甲基甲酰胺(20mL)的溶液中，氮气下搅拌反应15分钟后。加入甘氨酸甲酯-2,2-d2盐酸盐(390mg，3.01mmol)，升温至室温，反应在室温下搅拌反应2天。加水(50mL)淬灭反应，用乙酸乙酯(50mL x 3)萃取，合并有机层，用饱和食盐水洗涤(50mL x 2)，有机层用无水硫酸钠干燥，减压浓缩滤液，浓缩液进行柱分离，得到310mg白色固体，收率：45.8％。LC-MS(APCI):m/z＝323.1(M+1)。
步骤3：(5-(3-氯苯基)-3-羟基吡啶甲酰基)甘氨酸-2,2-d2(化合物9)的合成。
室温下，将氘氧化钠(2.89mL，1M in D2O)加入到(5-(3-氯苯基)-3-羟基吡啶甲酰基)甘氨酸甲酯-2,2-d2(310mg，0.96mmol)的四氢呋喃(5mL)溶液中，反应搅拌反应2小时。减压除去四氢呋喃，用1N的稀盐酸将pH调至2左右，乙酸乙酯(30mL x 3)萃取。合并有机层，用饱和食盐水洗涤(30mL x 2)，有机层用无水硫酸钠干燥，减压浓缩滤液，得到245mg淡黄色固体，收率：82.6％。LC-MS(APCI):m/z＝309.0；1H NMR(500MHz,DMSO-d6)NMR(500MHz,DMSO-9.0(M+1)，得到明化合物还可以任选将在本说明书中描述的或本领域已知的各种合成方法组合起来而方便地制得，这样的组合可由本发明所属领域的技术人员容易地。
实施例2 制备(5-(3-氯苯基)-3-羟基吡啶甲酰基-6-d)甘氨酸(化合物14)
Figure PCTCN2017114399-appb-000005
步骤1：2-羧基-5-(3-氯苯基)-3-羟基吡啶-1-氧化物(化合物10)的合成。
冰浴下，将三氟醋酸酐(2mL，14.38mmol)加入到尿素-过氧化氢的加合物(1.44g，15.31mmol)和5-(3-氯苯基)-3-羟基吡啶甲酸(1.80g，7.23mmol)的无水乙腈(22mL)溶液中。反应液在冰浴下反应6小时。减压除去乙腈，用饱和的硫代硫酸钠淬灭反应，搅拌10分钟，用稀盐酸将体系pH至调至2左右，过滤，水洗涤，真空干燥得到750mg白色固体。LC-MS(APCI):m/z＝220(M-COOH)。
步骤2：2-羧基-5-(3-氯苯基)-3-羟基吡啶-1-氧化物-6-d(化合物11)的合成。
室温下，将氘氧化钠(8mL，40％in D2O)滴加到2-羧基-5-(3-氯苯基)-3-羟基吡啶-1-氧化物(1.80g，6.77mmol)的D2O(30mL)混合物中，反应在100℃下反应过夜。冷却至室温，用2M的稀盐酸调pH值至2左右，有大量白色固体析出，过滤，用水洗涤滤饼，真空干燥得到1.8g白色固体，收率：100％。LC-MS(APCI):m/z＝221.1(M-COOH)。
步骤3：5-(3-氯苯基)-3-羟基-2-((2-甲氧基-2-氧乙基)氨基甲酰基)吡啶-1-氧化物-6-d(化合物12)的合成。
冰浴下，将DIPEA(655mg，5.08mmol)，HOBT(343mg，2.54mmol)，EDCI(487mg，2.54mmol)分别加入到2-羧基-5-(3-氯苯基)-3-羟基吡啶-1-氧化物-6-d(450mg，1.69mmol)的无水N,N-二甲基甲酰胺(10mL)的溶液中，氮气下搅拌反应15min后。加入甘氨酸甲酯盐酸盐(319mg，2.54mmol)，升温至室温，反应在室温下搅拌反应2天。加水(50mL)淬灭反应，用乙酸乙酯(50mL x 3)萃取，合并有机层，用饱和食盐水洗涤(50mL x 2)，有机层用无水硫酸钠干燥，减压浓缩滤液，浓缩液进行柱分离，得到360mg白色固体，收率：80.1％。LC-MS(APCI):m/z＝338.1(M+1)。
步骤4：(5-(3-氯苯基)-3-羟基吡啶甲酰基-6-d)甘氨酸甲酯(化合物13)的合成。
室温下，将三氯化磷(0.56mL，1.12mmol，2M in DCM)加入到5-(3-氯苯基)-3-羟基-2-((2-甲氧基-2-氧乙基)氨基甲酰基)吡啶-1-氧化物-6-d(190mg，0.56mmol)的无水二氯甲烷(10mL)的溶液中，氮气下回流搅拌反应2小时。冷去至室温，加水淬灭反应，用饱和的碳酸氢钠饱和溶液调pH至中性，用二氯甲烷(50mL x 3)萃取，合并有机层，用饱和食盐水洗涤(50mL x 2)，有机层用无水硫酸钠干燥，减压浓缩滤液，浓缩液进行柱分离，得到70mg白色固体，收率：38.9％。LC-MS(APCI):m/z＝322.1(M+1)。
步骤5：(5-(3-氯苯基)-3-羟基吡啶甲酰基-6-d)甘氨酸(化合物14)的合成。
室温下，将氢氧化钠(0.65mL，1M)加入到(5-(3-氯苯基)-3-羟基吡啶甲酰基-6-d)甘氨酸甲酯(70mg，0.22mmol)的四氢呋喃(5mL)溶液中，反应搅拌反应2小时。减压除去四氢呋喃，用1N的稀盐酸将pH调至2左右，乙酸乙酯(30mL x 3)萃取。合并有机层，用饱和食盐水洗涤(30mL x 2)，有机层用无水硫酸钠干燥，减压浓缩滤液，得到65mg白色固体，收率：95.9％，LC-MS(APCI):m/z＝308.0(M+1)；1H NMR(500MHz,DMSO-d6)δ12.81(br,1H),12.36(s,1H),9.35(t,J＝6.0Hz,1H),7.92(s,1H),7.84-7.71(m,2H),7.62-7.45(m,2H),4.00(d,J＝6.1Hz,2H)。
实施例3 制备(5-(3-氯苯基)-3-羟基吡啶甲酰基-6-d)甘氨酸-2,2-d2(化合物17)
Figure PCTCN2017114399-appb-000006
步骤1：5-(3-氯苯基)-3-羟基-2-((2-甲氧基-2-氧乙基-1,1-d2)氨基甲酰基)吡啶-1-氧化物-6-d(化合物15)的合成。
冰浴下，将DIPEA(191mg，1.48mmol)，HOBT(100mg，0.74mmol)，EDCI(142mg，0.74mmol)分别加入到2-羧基-5-(3-氯苯基)-3-羟基吡啶-1-氧化物6-d(450mg，1.69mmol)的无水N,N-二甲基甲酰胺(10mL)的溶液中，氮气下搅拌反应15分钟后。加入甘氨酸甲酯-2,2-d2的盐酸盐(70mg，0.54mmol)，升温至室温，反应在室温下搅拌反应2天。加水(50mL)淬灭反应，用乙酸乙酯(50mL x 3)萃取，合并有机层，用饱和食盐水洗涤(50mL x 2)，有机层用无水硫酸钠干燥，减压浓缩滤液，浓缩液进行柱分离，得到110mg白色固体，收率：66.2％。LC-MS(APCI):m/z＝340.1(M+1)。
步骤2：(5-(3-氯苯基)-3-羟基吡啶甲酰基-6-d)甘氨酸甲酯-2,2-d2(化合物16)的合成。
室温下，将三氯化磷(0.33mL，0.65mmol，2M/DCM)加入到5-(3-氯苯基)-3-羟基-2-((2-甲氧基-2-氧乙基-1,1-d2)氨基甲酰基)吡啶-1-氧化物-6-d(110mg，0.32mmol)的无水二氯甲烷(10mL)的溶液中，氮气下回流搅拌反应2小时。冷去至室温，加水淬灭反应，用饱和的碳酸氢钠饱和溶液调pH至中性，用二氯甲烷(50mL x 3)萃取，合并有机层，用饱和食盐水洗涤(50mL x 2)，有机层用无水硫酸钠干燥，减压浓缩滤液，浓缩液进行柱分离，得到35mg白色固体，收率：33.8％。LC-MS(APCI):m/z＝324.1(M+1)。
步骤5：(5-(3-氯苯基)-3-羟基吡啶甲酰基-6-d)甘氨酸-2,2-d2(化合物17)的合成。
室温下，将氘氧化钠(0.33mL，1M in D2O)加入到(5-(3-氯苯基)-3-羟基吡啶甲酰基-6-d)甘氨酸甲酯-2,2-d2(35mg，0.11mmol)的四氢呋喃(2mL)溶液中，反应搅拌反应2小时。减压除去四氢呋喃，用1N的稀盐酸将pH调至2左右，乙酸乙酯(30mL x 3)萃取。合并有机层，用饱和食盐水洗涤(30mL x 2)，有机层用无水硫酸钠干燥，减压浓缩滤液，得到30mg白色固体，收率：95.9％。LC-MS(APCI):m/z＝310.0(M+1)；1H NMR(500MHz,DMSO-d6)δ12.75(br,1H),12.37(s,1H),9.34(s,1H),7.92(s,1H),7.83-7.72(m,2H),7.61-7.46(m,2H)。
实施例4 制备(5-(3-氯苯基-2,4,6-d3)-3-羟基吡啶甲酰基)甘氨酸(化合物25)
Figure PCTCN2017114399-appb-000007
步骤1：3-氯苯基-2,4,6-d3-氨(化合物19)的合成。
室温下，将DCl(235mg，37％in D2O)加入到3-氯苯胺(800mg，6.27mmol)的重水(17mL)溶液中，微波160℃反应1.5小时。冷却至室温，用饱和的碳酸氢钠溶液调pH至碱性，乙酸乙酯(50mL x 3)，合并有机层，用饱和食盐水洗涤(30mL x 2)，有机层用无水硫酸钠干燥，减压浓缩滤液，得到800mg油状物，收率：100％。LC-MS(APCI):m/z＝131.1(M+1)。
步骤2：2-(3-氯苯基-2,4,6-d3)-4,4,5,5-四甲基-1,3,2-二恶硼烷(化合物20)的合成。
室温下，将稀盐酸(12.5mL，37.5mmol)和水(12mL)依次加入到3-氯苯基-2,4,6-d3-氨(12.30mmol，1.60g)的甲醇(25mL)溶剂中。-10℃下将亚硝酸钠(816mg in 6mL H2O，12.40mmol)溶液滴加到反应液中，反应在-10℃下反应搅拌1小时。然后将联硼酸频那醇酯(9.30g溶于25mL甲醇，37.00mmol)溶液滴加到反应液中，反应在0℃下反应2小时。用乙酸乙酯(60mL x 3)萃取。合并有机层，用饱和碳酸氢钠溶液和食盐水洗涤(30mL x 2)，有机层用无水硫酸钠干燥，减压浓缩滤液，浓缩液进行柱分离，得到1.80g无色油状物。
步骤3：3-氯-5-(3-氯苯基-2,4,6-d3)氰基吡啶(化合物21)的合成。
氮气保护下将无水N,N-二甲基甲酰胺(20mL)和水(2mL)加入到2-(3-氯苯基-2,4,6-d3)-4,4,5,5-四甲基-1,3,2-二恶硼烷(1.80g，7.47mmol)和3,5-二氯氰基吡啶(1.40g，8.22mmol)、碳酸钾(1.30g，9.34mmol)、和PdCl2(dppf)(100mg)的混合物中，反应在氮气保护下，45℃搅拌反应过夜。冷却至室温，加水(50mL)淬灭反应，用乙酸乙酯(50mL x 3)萃取，合并有机层，用饱和食盐水洗涤(50mL x 2)，有机层用无水硫酸钠干燥，减压浓缩滤液，浓缩液进行柱分离，得到1.10g白色固体，收率：35.5％。LC-MS(APCI):m/z＝293.0(M+1)。
步骤4：5-(3-氯苯基-2,4,6-d3)-3-甲氧基氰基吡啶(化合物22)的合成。
室温下，氮气保护下将甲醇钠(1.20g，22.2mmol)加到3-氯-5-(3-氯苯基2,4,6-d3)氰基吡啶(950mg，3.77mmol)的无水甲醇(20mL)的溶液中，反应回流搅拌过夜。冷却至室温，减压除去反 应液，加水(50mL)淬灭反应，用乙酸乙酯(50mL x 3)萃取，合并有机层，用饱和食盐水洗涤(50mL x 2)，有机层用无水硫酸钠干燥，减压浓缩滤液，浓缩液进行柱分离，得到565mg白色固体，收率：60.7％。LC-MS(APCI):m/z＝248.0(M+1)。
步骤5：5-(3-氯苯基-2,4,6-d3)-3-羟基吡啶甲酸(化合物23)的合成。
室温下，将48％的氢溴酸(6mL)加入到5-(3-氯苯基-2,4,6-d3)-3-甲氧基氰基吡啶(565mg，2.28mmol)中，反应在氮气保护下，100℃搅拌反应过夜。冷却至室温。在冰浴下，用50％的氢氧化钠溶液调pH值约为2，继续搅拌1小时，有大量白色固体析出，过滤，用水洗涤滤饼，真空干燥得到525mg灰白色固体，收率：91.4％。LC-MS(APCI):m/z＝253.0(M+1)。
步骤6：(5-(3-氯苯基-2,4,6-d3)-3-羟基吡啶甲酰基)甘氨酸甲酯(化合物24)的合成。
冰浴下，将DIPEA(307mg，2.38mmol)，HOBT(161mg，1.19mmol)，EDCI(228mg，1.19mmol)分别加入到5-(3-氯苯基-2,4,6-d3)-3-羟基吡啶甲酸(200mg，0.79mmol)的无水N,N-二甲基甲酰胺(20mL)的溶液中，氮气下搅拌反应15min后。加入甘氨酸甲酯盐酸盐(150mg，1.19mmol)，升温至室温，反应在室温下搅拌反应2天。加水(50mL)淬灭反应，用乙酸乙酯(50mL x 3)萃取，合并有机层，用饱和食盐水洗涤(50mL x 2)，有机层用无水硫酸钠干燥，减压浓缩滤液，浓缩液进行柱分离，得到120mg白色固体，收率：47.0％。LC-MS(APCI):m/z＝324.0(M+1)。
步骤7：(5-(3-氯苯-2,4,6-d3)-3-羟基吡啶甲酰基)甘氨酸(化合物25)的合成。
室温下，将氢氧化钠(1.11mL，1M)加入到(5-(3-氯苯基-2,4,6-d3)-3-羟基吡啶甲酰基)甘氨酸甲酯(120mg，0.37mmol)的四氢呋喃(6mL)溶液中，反应搅拌反应2小时。减压除去四氢呋喃，用1N的稀盐酸将pH调至2左右，乙酸乙酯(30mL x 3)萃取。合并有机层，用饱和食盐水洗涤(30mL x 2)，有机层用无水硫酸钠干燥，减压浓缩滤液，得到40mg白色固体，收率：35.0％。LC-MS(APCI):m/z＝310.0(M+1)；1H NMR(400MHz,DMSO-d6)δ12.81(br,1H),12.37(s,1H),9.37(t,J＝6.1Hz,1H),8.55(d,J＝1.9Hz,1H),7.79(d,J＝1.9Hz,1H),7.55(s,1H),4.02(d,J＝6.1Hz,2H)。
实施例5 制备(5-(3-氯苯基-2,4,6-d3)-3-羟基吡啶甲酰基)甘氨酸-2,2-d2(化合物27)
Figure PCTCN2017114399-appb-000008
步骤1：(5-(3-氯苯基-2,4,6-d3)-3-羟基吡啶甲酰基)甘氨酸甲酯-2,2-d2(化合物26)的合成。
冰浴下，将DIPEA(500mg，3.87mmol)，HOBT(260mg，1.93mmol)，EDCI(370mg，1.93mmol)分别加入到5-(3-氯苯基-2,4,6-d3)-3-羟基吡啶甲酸(324mg，1.29mmol)的无水N,N-二甲基甲酰胺(20mL)的溶液中，氮气下搅拌反应15分钟后。加入甘氨酸甲酯-2,2-d2盐酸盐(250mg，1.93mmol)，升温至室温，反应在室温下搅拌反应2天。加水(50mL)淬灭反应，用乙酸乙酯(50mL x 3)萃取，合并有机层，用饱和食盐水洗涤(50mL x 2)，有机层用无水硫酸钠干燥，减压浓缩滤液，浓缩液进行柱分离，得到160mg白色固体，收率：38.3％。LC-MS(APCI):m/z＝326.0(M+1)。
步骤2：(5-(3-氯苯-2,4,6-d3)-3-羟基吡啶甲酰基)甘氨酸-2,2-d2(化合物27)的合成。
室温下，将氘氧化钠(1.48mL，1M in D2O)加入到(5-(3-氯苯基-2,4,6-d3)-3-羟基吡啶甲酰基)甘氨酸甲酯-2,2-d2(160mg，0.49mmol)的四氢呋喃(6mL)溶液中，反应搅拌反应2小时。减压除去四氢呋喃，用1N的稀盐酸将pH调至2左右，乙酸乙酯(30mL x 3)萃取。合并有机层，用饱和食盐水洗涤(30mL x 2)，有机层用无水硫酸钠干燥，减压浓缩滤液，得到75mg白色固体，收率：49.2％。LC-MS(APCI):m/z＝312.0(M+1)；1H NMR(400MHz,DMSO-d6)δ12.81(s,1H),12.38(s,1H),9.36(s,1H),8.55(d,J＝1.9Hz,1H),7.78(d,J＝1.9Hz,1H),7.55(s,1H)。
实施例6 制备(5-(3-氯苯基-6-d)-3-羟基吡啶甲酰基)甘氨酸(化合物35)
Figure PCTCN2017114399-appb-000009
步骤1：3-氯苯基-6-d-氨(化合物29)的合成。
室温下，将Pd/C(500mg，55％in D2O)加入到3-氯-6-溴苯胺(3.00g，14.56mmol)的CH3OD(20mL)溶液中，滴加两滴NaOD(40％in D2O)溶液，反应液在D2下室温反应过夜。硅藻土过滤，减压浓缩滤液，得到2.8g黄色固体，收率：92.5％。LC-MS(APCI):m/z＝131.1(M+1)。
步骤2：2-(3-氯苯基-6-d)-4,4,5,5-四甲基-1,3,2-二恶硼烷(化合物30)的合成。
室温下，将稀盐酸(13.6mL，40.9mmol)和水(13mL)依次加入到3-氯苯-6-d-氨(13.40mmol，2.80g)的甲醇(27mL)溶剂中。-10℃下将亚硝酸钠(890mg溶于6.5mL H2O，13.50mmol)溶液滴加到反应液中，反应在-10℃下反应搅拌1小时。然后将联硼酸频那醇酯(10.14g溶于27mL甲醇，40.30mmol)溶液滴加到反应液中，反应在0℃下反应2小时。用乙酸乙酯(60mL x 3)萃取。合并有机层，用饱和碳酸氢钠溶液和食盐水洗涤(30mL x 2)，有机层用无水硫酸钠干燥，减压浓缩滤液，浓缩液进行柱分离，得到3.00g无色油状物。
步骤3：3-氯-5-(3-氯苯基-6-d)氰基吡啶(化合物31)的合成。
氮气保护下将无水N,N-二甲基甲酰胺(20mL)和水(2mL)加入到2-(3-氯苯基-6-d)-4,4,5,5-四甲基-1,3,2-二恶硼烷(3.00g，12.6mmol)和3,5-二氯氰基吡啶(2.40g，13.8mmol)、碳酸钾(2.17g，15.70mmol)、和PdCl2(dppf)(100mg)的混合物中，反应在氮气保护下，45℃搅拌反应过夜。冷却至室温，加水(50mL)淬灭反应，用乙酸乙酯(50mL x 3)萃取，合并有机层，用饱和食盐水洗涤(50mL x 2)，有机层用无水硫酸钠干燥，减压浓缩滤液，浓缩液进行柱分离，得到2.00g白色固体，收率：59.7％。LC-MS(APCI):m/z＝290.0(M+CH3CN)。
步骤4：5-(3-氯苯基-6-d)-3-甲氧基氰基吡啶(化合物32)的合成。
室温下，氮气保护下将甲醇钠(2.60g，48.00mmol)加到3-氯-5-(3-氯苯基6-d)氰基吡啶(2.00g，8.00mmol)的无水甲醇(40mL)的溶液中，反应回流搅拌搅拌过夜。冷却至室温，减压除去反应液，加水(50mL)淬灭反应，用乙酸乙酯(50mL x 3)萃取，合并有机层，用饱和食盐水洗涤(50mL x 2)，有机层用无水硫酸钠干燥，减压浓缩滤液，浓缩液进行柱分离，得到1.30g白色固体，收率：66.0％。LC-MS(APCI):m/z＝245.9(M+1)。
步骤5：5-(3-氯苯基-6-d)-3-羟基吡啶甲酸(化合物33)的合成。
室温下，将48％的氢溴酸(15mL)加入到5-(3-氯苯基-6-d)-3-甲氧基氰基吡啶(1.30g，5.29mmol)中，反应在氮气保护下，100℃搅拌反应过夜。冷却至室温。在冰浴下，用50％的氢氧化钠溶液调pH值约为2，继续搅拌1小时，有大量白色固体析出，过滤，用水洗涤滤饼，真空干燥得到1.30g白色固体，收率：97.9％。LC-MS(APCI):m/z＝251.0(M+1)。
步骤6：(5-(3-氯苯基-6-d)-3-羟基吡啶甲酰基)甘氨酸甲酯(化合物34)的合成。
冰浴下，将DIPEA(307mg，2.40mmol)，HOBT(161mg，1.20mmol)，EDCI(228mg，1.20mmol)分别加入到5-(3-氯苯基-6-d)-3-羟基吡啶甲酸(200mg，0.80mmol)的无水N,N-二甲基甲酰胺(20mL)的溶液中，氮气下搅拌反应15min后。加入甘氨酸甲酯盐酸盐(150mg，1.19mmol)，升温至室温，反应在室温下搅拌反应2天。加水(50mL)淬灭反应，用乙酸乙酯(50mL x 3)萃取，合并有机层，用饱和食盐水洗涤(50mL x 2)，有机层用无水硫酸钠干燥，减压浓缩滤液，浓缩液进行柱分离，得到95mg白色固体，收率：36.9％。LC-MS(APCI):m/z＝321.9(M+1)。
步骤7：(5-(3-氯苯基-6-d)-3-羟基吡啶甲酰基)甘氨酸(化合物35)的合成。
室温下，将氢氧化钠(0.90mL，1M)加入到(5-(3-氯苯基-6-d)-3-羟基吡啶甲酰基)甘氨酸甲酯(95mg，0.30mmol)的四氢呋喃(5mL)溶液中，反应搅拌反应1hrs。减压除去四氢呋喃，用1N的稀盐酸将pH调至2左右，乙酸乙酯(30mL x 3)萃取。合并有机层，用饱和食盐水洗涤(30mL x 2)，有机层用无水硫酸钠干燥，减压浓缩滤液，得到87mg白色固体，收率：100％。LC-MS(APCI):m/z＝307.9(M+1)。
实施例7 制备(5-(3-氯苯基-6-d)-3-羟基吡啶甲酰基)甘氨酸-2,2-d2(化合物37)
Figure PCTCN2017114399-appb-000010
步骤1：(5-(3-氯苯基-6-d)-3-羟基吡啶甲酰基)甘氨酸甲酯-2,2-d2(化合物36)的合成。
冰浴下，将DIPEA(307mg，2.40mmol)，HOBT(161mg，1.20mmol)，EDCI(228mg，1.20mmol)分别加入到5-(3-氯苯基-6-d)-3-羟基吡啶甲酸(200mg，0.80mmol)的无水N,N-二甲基甲酰胺(20mL)的溶液中，氮气下搅拌反应15分钟后。加入甘氨酸甲酯盐酸盐-2,2-d2(150mg，1.19mmol)，升温至室温，反应在室温下搅拌反应2天。加水(50mL)淬灭反应，用乙酸乙酯(50mL x 3)萃取，合并有机层，用饱和食盐水洗涤(50mL x 2)，有机层用无水硫酸钠干燥，减压浓缩滤液，浓缩液进行柱分离，得到86mg白色固体，收率：33.2％。LC-MS(APCI):m/z＝324.0(M+1)。
步骤2：(5-(3-氯苯基-6-d)-3-羟基吡啶甲酰基)甘氨酸-2,2-d2(化合物37)的合成。
室温下，将氘氧化钠(0.80mL，1M in D2O)加入到(5-(3-氯苯基-2,4,6-d3)-3-羟基吡啶甲酰基)甘氨酸甲酯-2,2-d2(86mg，0.27mmol)的四氢呋喃(5mL)溶液中，反应搅拌反应2小时。减压 除去四氢呋喃，用1N的稀盐酸将pH调至2左右，乙酸乙酯(30mL x 3)萃取。合并有机层，用饱和食盐水洗涤(30mL x 2)，有机层用无水硫酸钠干燥，减压浓缩滤液，得到80mg白色固体，收率：49.2％。LC-MS(APCI):m/z＝310.0(M+1)。
生物活性测试。
小鼠组织HIF蛋白质印迹分析。
储存于-80℃的小鼠组织用液氮冷冻下的研钵和杵研成粉末。细胞核提取物使用NE-PER试剂盒(Pierce Biotechnology)制备。为进行免疫沉淀反应，将细胞核提取物以组织比抗体为200：1的比例加入到HIF-1淀反单克隆抗体中。将该悬浮液在4℃下于圆锥形微型离心管中培育4小时。然后将蛋白A/G偶联琼脂糖珠(40脂糖珠蛋％的悬浮液)加入该管中。在4℃下旋转过夜后，用冰冷的磷酸盐缓冲液将该珠洗涤3次。然后将该珠用40后将该珠用冰冷的磷酸盐试样缓冲溶液制备用于SDS-PAGE。自SDS-PAGE上分离的蛋白质转移到带有XCell-II Blot Module系统的硝化纤维板上。将印迹用5％的BSA封闭，然后用HIF-1用维兔抗体以1：100稀释的比例培育。然后用Tris缓冲盐水/Tween-20缓冲液洗涤印迹并用辣根过氧化物酶缀合的羊抗兔二级抗体培育。印迹用ECL试剂显像。印迹图像用爱普生Expression 1600扫描仪捕捉。
对本发明化合物及其未氘代的化合物AKB-6548同时测试比较，评价它们促使小鼠组织分泌HIF蛋白质的能力，实验结果如下表1中。通过实验结果分析，本发明化合物促使小鼠组织分泌HIF蛋白质的能力明显强于未氘代的化合物AKB-6548。
表1：实施例化合物的小鼠组织HIF蛋白质印迹实验结果
化合物编号 HIF-α(pg/mL)
AKB-6548 360.36
化合物9 393.67
化合物14 432.70
化合物17 693.63
化合物25 383.94
化合物27 427.92
化合物35 395.54
化合物37 410.74
小鼠血清EPO试验。
使用R&DSystems的小鼠Quantikine促红细胞生成素ELISA试剂盒根据使用说明书对小鼠血清EPO进行检测。
对本发明化合物及其未氘代的化合物AKB-6548同时测试比较，评价它们促使小鼠分泌EPO的能力，实验结果如下表2中。通过实验结果分析，本发明化合物促使小鼠分泌EPO的能力明显强于未氘代的化合物AKB-6548，说明本发明化合物可用于制备调控人体贫血的药物。
表2：实施例化合物的小鼠血清EPO实验结果
化合物编号 EPO(mlU/mL)
AKB-6548 67.86
化合物9 60.77
化合物14 88.24
化合物17 94.71
化合物25 83.36
化合物27 78.75
化合物35 124.13
化合物37 117.59
代谢稳定性评价。
微粒体实验：人肝微粒体：0.5mg/mL，Xenotech；大鼠肝微粒体：0.5mg/mL，Xenotech；辅酶(NADPH/NADH)：1mM，Sigma Life Science；氯化镁：5mM，100mM磷酸盐缓冲剂(pH为7.4)。
储备液的配制：精密称取一定量的化合物实施例1-7粉末，并用DMSO分别溶解至5mM。
磷酸盐缓冲液(100mM，pH7.4)的配制：取预先配好的0.5M磷酸二氢钾150mL和700mL的0.5M磷酸氢二钾溶液混合，再用0.5M磷酸氢二钾溶液调节混合液pH值至7.4，使用前用超纯水稀释5倍，加入氯化镁，得到磷酸盐缓冲液(100mM)，其中含100mM磷酸钾，3.3mM氯化镁，pH为7.4。
配制NADPH再生系统溶液(含有6.5mM NADP，16.5mM G-6-P，3U/mL G-6-P D，3.3mM氯化镁)，使用前置于湿冰上。
配制终止液：含有50ng/mL盐酸普萘洛尔和200ng/mL甲苯磺丁脲(内标)的乙腈溶液。取25057.5μL磷酸盐缓冲液(pH7.4)至50mL离心管中，分别加入812.5μL人肝微粒体，混匀，得到蛋白浓度为0.625mg/mL的肝微粒体稀释液。取25057.5μL磷酸盐缓冲液(pH7.4)至50mL离心管 中，分别加入812.5μL SD大鼠肝微粒体，混匀，得到蛋白浓度为0.625mg/mL的肝微粒体稀释液。
样品的孵育：用含70％乙腈的水溶液将相应化合物的储备液分别稀释至0.25mM，作为工作液，备用。分别取398μL的人肝微粒体或者大鼠肝微粒体稀释液加入96孔孵育板中(N＝2)，分别加入2μL 0.25mM的的工作液中，混匀。
代谢稳定性的测定：在96孔深孔板的每孔中加入300μL预冷的终止液，并置于冰上，作为终止板。将96孔孵育板和NADPH再生系统置于37系水浴箱中，100转/分钟震荡，预孵5min。从孵育板每孔取出80μL孵育液加入终止板，混匀，补充20μL NADPH再生系统溶液，作为0min样品。再向孵育板每孔加入80再向的NADPH再生系统溶液，启动反应，开始计时。相应化合物的反应浓度为1μM，蛋白浓度为0.5mg/mL。分别于反应10、30、90min时，各取100μL反应液，加入终止板中，涡旋3min终止反应。将终止板于5000×g，40条件下离心10min。取100in上清液至预先加入100μL蒸馏水的96孔板中，混匀，采用LC-MS/MS进行样品分析。
数据分析：通过LC-MS/MS系统检测相应化合物及内标的峰面积，计算化合物与内标峰面积比值。通过化合物剩余量的百分率的自然对数与时间作图测得斜率，并根据以下公式计算t1/2和CLint，其中V/M即等于1/蛋白浓度。
Figure PCTCN2017114399-appb-000011
实验结果如下表2所示，同AKB-6548相比，本发明化合物在人肝微粒体与大鼠肝微粒体实验中都表现出优异的代谢稳定性。
表1 实施例1-10化合物的肝微粒代谢评价
Figure PCTCN2017114399-appb-000012
Figure PCTCN2017114399-appb-000013
大鼠中的药代动力学评价。
6只雄性Sprague-Dawley大鼠，7-8周龄，体重约210g，分成2组，每组3只，经静脉或口服单个剂量的化合物(经静脉3mg/kg，口服10mg/kg)，比较其药代动力学差异。
大鼠采用标准饲料饲养，给予水。试验前16小时开始禁食。药物用PEG400和二甲亚砜溶解。眼眶采血，采血的时间点为给药后0.083小时，0.25小时、0.5小时、1小时、2小时、4小时、6小时、8小时、12小时和24小时。
大鼠吸入乙醚后短暂麻醉，眼眶采集300μL血样于试管。试管内有30μL1％肝素盐溶液。使用前，试管于60液。烘干过夜。在随后一个时间点血样采集完成之后，大鼠乙醚麻醉后处死。
血样采集后，立即温和地颠倒试管至5次，保证混合充分后放置于冰上。血样在4保证混合充分后放置于离心5分钟，将血浆与红细胞分离。用移液器吸出100μL血浆到干净的塑料离心管中，表明化合物的名称和时间点。血浆在进行分析前保存在-80℃。用LC-MS/MS测定血浆中本发明化合物的浓度。药代动力学参数基于每只动物在不同时间点的血药浓度进计算。
实验结果表明，相对于对照化合物AKB-6548，本发明化合物在动物体内具有更好的药物动力学，因而具有更好的药效学和治疗效果。
应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围，实施例中未注明具体条件的实验方法，通常按照常规条件，或按照制造厂商所建议的条件。除非另外说明，否则份数和百分比为重量份和重量百分比。
以上内容是结合具体的优选实施方式对本发明所作的进一步详细说明，不能认定本发明的具体实施只局限于这些说明。对于本发明所属技术领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干简单推演或替换，都应当视为属于本发明的保护范围。

Claims (7)

  1. 一种取代的杂芳基酰胺化合物，其特征在于：如式(I)所示的杂芳基酰胺化合物，或其晶型、药学上可接受的盐、前药、立体异构体、水合物或溶剂化合物，
    Figure PCTCN2017114399-appb-100001
    其中，R1、R2、R3、R4、R5、R6、R7、R8各自独立地为氢、氘、卤素；
    附加条件是R1、R2、R3、R4、R5、R6、R7和R8中至少一个是氘代的或氘。
  2. 根据权利要求1所述的化合物，其特征在于：R1和R2各自独立地为氘或氢。
  3. 根据权利要求1所述的化合物，其特征在于：R3和R4各自独立地为氘或氢。
  4. 根据权利要求1所述的化合物，其特征在于：R5、R6、R7、R8各自独立地为氘或氢。
  5. 根据权利要求1所述的化合物，其特征在于：所述化合物选自下组化合物或其药学上可接受的盐：
    Figure PCTCN2017114399-appb-100002
    Figure PCTCN2017114399-appb-100003
  6. 一种药物组合物，其特征在于：其含有药学上可接受的载体和如权利要求1～5任意一项所述的取代的杂芳基酰胺化合物，或其晶型、药学上可接受的盐、水合物或溶剂合物、立体异构体、前药或同位素变体的药物组合物。
  7. 一种如权利要求1所述的化合物，或其晶型、药学上可接受的盐、水合物或溶剂化合物的用途，其特征在于：制备预防和治疗慢性疾病性贫血、葡萄糖耐受不良和/或肾病相关贫血，以及癌症贫血或血细胞相关病症药物中的用途。
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