CN111518038B

CN111518038B - Substituted pyrimidinetrione compound, composition containing same and application thereof

Info

Publication number: CN111518038B
Application number: CN202010484114.3A
Authority: CN
Inventors: 王义汉; 任兴业
Original assignee: Shenzhen Targetrx Inc
Current assignee: Shenzhen Targetrx Inc
Priority date: 2017-05-26
Filing date: 2018-05-22
Publication date: 2022-04-05
Anticipated expiration: 2038-05-22
Also published as: CN111518038A; WO2018214872A1; CN108623528B; CN108623528A

Abstract

The invention provides a substituted pyrimidinetrione compound, a composition containing the compound and an application of the compound, and discloses a pyrimidinetrione compound shown in a formula (I), or a crystal form, a pharmaceutically acceptable salt, a prodrug, a stereoisomer, a hydrate or a solvate of the pyrimidinetrione compound. The pyrimidinetrione compound and the composition containing the compound can be used for regulating Hypoxia Inducible Factor (HIF) and/or endogenous Erythropoietin (EPO), and can be used for preparing a medicine for regulating and controlling human anemia.

Description

Substituted pyrimidinetrione compound, composition containing same and application thereof

The application is a divisional application of an invention patent application with the application date of 2018, 05 and 22, and the application number of 201810494949.X, and the invention name of 'a substituted pyrimidinetrione compound, a composition containing the compound and application thereof'.

Technical Field

The present invention belongs to the field of pharmaceutical technology, and in particular, relates to a substituted pyrimidinetrione compound, compositions comprising the compound, and methods and compounds capable of modulating the stability of Hypoxia Inducible Factor (HIF) subunits and increasing endogenous erythropoietin in vitro and in vivo.

Background

Hypoxia Inducible Factor (HIF) is a basic helix-loop-helix (bHLH) PAS (Per/Arnt/Sim) transcriptional activator that regulates changes in gene expression as cellular oxygen concentration changes. HIF is a heterodimer comprising an oxygen-regulated alpha subunit (HIF- α) and a constitutively expressed beta subunit (HIF- β), also known as the Aromatic Receptor Nuclear Transporter (ARNT). In oxygenated (normoxic) cells, the HIF- α subunit is rapidly degraded by a mechanism involving ubiquitination of the retinoblastoma inhibitory protein (pVHL) E3 ligase complex. Under hypoxic conditions, HIF- α does not degrade, and an active HIF- α/β complex accumulates in the nucleus and activates the expression of several genes, including glycolytic enzymes, glucose transporter (GLUT) -1, Erythropoietin (EPO), and Vascular Endothelial Growth Factor (VEGF). (Maxwell et al, Nature 1999, 399, 271-.

Erythropoietin (EPO) is a naturally occurring hormone produced by HIF- α that stimulates the production of red blood cells that carry oxygen throughout the body. EPO is normally secreted by the kidney, and endogenous EPO is increased under conditions of reduced oxygen (hypoxia). All types of anemia are characterized by a reduced capacity of the blood to carry oxygen and are therefore accompanied by similar signs and symptoms, including pale skin and mucous membranes, weakness, dizziness, fatigue and lethargy, leading to a reduction in quality of life. Subjects with severe anemia conditions exhibit difficulty breathing and cardiac malformations. Anemia is often associated with a blood deficiency in red blood cells or in hemoglobin.

Ischemic and hypoxic conditions are the leading causes of morbidity and mortality. Cardiovascular disease causes at least fifteen million deaths per year and is responsible for 30% of deaths worldwide. Of the many cardiovascular diseases, ischemic heart disease and cerebrovascular disease cause about 17% of deaths. One hundred and thirty million cases of non-fatal acute myocardial infarction are reported each year, constituting an incidence of approximately 300 per 100,000 people. On the other hand, it is estimated that five million americans suffer from venous thrombosis every year, and about 600,000 of these cases cause pulmonary embolism. About one third of the patients with pulmonary embolism eventually die, making pulmonary embolism the third most common cause of death in americans.

Currently, treatment of ischemic and hypoxic conditions focuses on the alleviation of symptoms and the treatment of pathogenic conditions. For example, treatment of myocardial infarction includes nitroglycerin and analgesics to control pain and reduce cardiac workload. Other drugs, including digoxin (digoxin), diuretics, amrinone (amyinone), beta-blockers, lipid-lowering agents, and angiotensin converting enzyme inhibitors are used to stabilize the condition, but none of these therapies act directly on tissue damage resulting from ischemia and hypoxia.

Daprodustat (chemical name N- [ (1, 3-dicyclohexyl-2, 4, 6-trioxo-5-hexahydropyrimidyl) carbonyl ] glycine, which has the following structural formula) is an oral HIF-alpha prolyl hydroxylase inhibitor developed by Kurarian Schker for the treatment of anemia associated with chronic kidney disease and is currently in phase III clinical trials.

Poor absorption, distribution, metabolism and/or excretion (ADME) properties are known to be the major cause of failure in many drug candidate clinical trials. Many drugs currently on the market also have limited their range of application due to poor ADME properties. The rapid metabolism of drugs can result in the difficulty of obtaining many drugs that are otherwise effective in treating disease due to their rapid metabolic clearance from the body. Although frequent or high dose administration may solve the problem of rapid clearance of the drug, this method may cause problems such as poor patient compliance, side effects caused by high dose administration, and increased treatment costs. In addition, rapidly metabolizing drugs may also expose patients to undesirable toxic or reactive metabolites.

There remains a serious clinical unmet need in the art, and it is a challenging task to find novel compounds with good oral bioavailability and druggability for the treatment of HIF-related and EPO-related diseases.

Disclosure of Invention

In view of the above technical problems, the present invention discloses a compound and a composition comprising the same, which can be used for modulating Hypoxia Inducible Factor (HIF) and/or endogenous Erythropoietin (EPO) and/or have better pharmacodynamic/pharmacokinetic properties.

In contrast, the technical scheme adopted by the invention is as follows:

the object of the present invention is to provide a new class of compounds useful for modulating Hypoxia Inducible Factor (HIF) and/or endogenous Erythropoietin (EPO) and/or having better pharmacodynamic/pharmacokinetic properties.

In a first aspect of the invention, a compound represented by formula (I), or a crystalline form, a pharmaceutically acceptable salt, a hydrate or a solvate thereof is provided.

Wherein R is¹、R²、R³、R⁴、R⁵、R⁶、R⁷、R⁸、R⁹、R¹⁰、R¹¹、R¹²、R¹³、R¹⁴、R¹⁵、R¹⁶、R¹⁷、R¹⁸、R¹⁹、R²⁰、R²¹、R²²、R²³、R²⁴、R²⁵Each independently is hydrogen, deuterium, halogen or trifluoromethyl;

with the proviso that R¹、R²、R³、R⁴、R⁵、R⁶、R⁷、R⁸、R⁹、R¹⁰、R¹¹、R¹²、R¹³、R¹⁴、R¹⁵、R¹⁶、R¹⁷、R¹⁸、R¹⁹、R²⁰、R²¹、R²²、R²³、R²⁴And R²⁵At least one of which is deuterated or deuterium.

In another preferred embodiment, R²⁴And R²⁵Each independently is deuterium or hydrogen.

In another preferred embodiment, R¹、R²、R³、R⁴、R⁵、R⁶、R⁷、R⁸、R⁹、R¹⁰And R¹¹Each independently is deuterium or hydrogen.

In another preferred embodiment, R¹²、R¹³、R¹⁴、R¹⁵、R¹⁶、R¹⁷、R¹⁸、R¹⁹、R²⁰、R²¹And R²²Each independently is deuterium or hydrogen.

In another preferred embodiment, the deuterium isotope content of deuterium at the deuterated position is at least greater than the natural deuterium isotope content (0.015%), preferably greater than 30%, more preferably greater than 50%, more preferably greater than 75%, more preferably greater than 95%, more preferably greater than 99%.

Specifically, in the present invention R¹、R²、R³、R⁴、R⁵、R⁶、R⁷、R⁸、R⁹、R¹⁰、R¹¹、R¹²、R¹³、R¹⁴、R¹⁵、R¹⁶、R¹⁷、R¹⁸、R¹⁹、R²⁰、R²¹、R²²、R²³、R²⁴And R²⁵The deuterium isotope content in each deuterated position is at least 5%, preferably greater than 10%, more preferably greater than 15%, more preferably greater than 20%, more preferably greater than 25%, more preferably greater than 30%, more preferably greater than 35%, more preferably greater than 40%, more preferably greater than 45%, more preferably greater than 50%, more preferably greater than 55%, more preferably greater than 60%, more preferably greater than 65%, more preferably greater than 70%, more preferably greater than 75%, more preferably greater than 80%, more preferably greater than 85%, more preferably greater than 90%, more preferably greater than 95%, more preferably greater than 99%.

In another preferred embodiment, R of the compound of formula (I)¹、R²、R³、R⁴、R⁵、R⁶、R⁷、R⁸、R⁹、R¹⁰、R¹¹、R¹²、R¹³、R¹⁴、R¹⁵、R¹⁶、R¹⁷、R¹⁸、R¹⁹、R²⁰、R²¹、R²²、R²³、R²⁴And R²⁵Preferably, at least one of R comprises deuterium, more preferably two of R comprises deuterium, more preferably three of R comprises deuterium, more preferably four of R comprises deuterium, more preferably five of R comprises deuterium, more preferably six of R comprises deuterium, more preferably seven of R comprises deuterium, more preferably eight of R comprises deuterium, more preferably nine of R comprises deuterium, more preferably ten of R comprises deuterium, more preferably eleven of R comprises deuterium, more preferably twelve of R comprises deuterium, more preferably thirteen of R comprises deuterium, more preferably fourteen of R comprises deuterium, more preferably fifteen of R comprises deuterium, more preferably sixteen of R comprises deuterium, more preferably seventeen of R comprises deuterium, more preferably eighteen of R comprises deuterium, more preferably nineteen of R comprises deuterium, more preferably twenty-one of R comprises deuterium, more preferably twenty-two of R comprises deuterium, more preferably twenty-three of R comprises deuterium, more preferably twenty-four of R comprises deuterium, more preferably twenty-five of R comprises deuterium.

In another preferred embodiment, the compound does not include non-deuterated compounds.

In a second aspect of the present invention, there is provided a method of preparing a pharmaceutical composition comprising the steps of: mixing a pharmaceutically acceptable carrier with a compound described in the first aspect of the invention, or a crystalline form, a pharmaceutically acceptable salt, a hydrate, or a solvate thereof, to form a pharmaceutical composition.

In a third aspect of the invention, there is provided a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a compound of the first aspect of the invention, or a crystalline form, a pharmaceutically acceptable salt, a hydrate or a solvate thereof.

Pharmaceutically acceptable carriers that may be used in the pharmaceutical compositions of the present invention include, but are not limited to, any glidant, sweetener, diluent, preservative, dye/colorant, flavor enhancer, surfactant, wetting agent, dispersant, disintegrant, suspending agent, stabilizer, isotonic agent, solvent, or emulsifier.

The pharmaceutical composition can be prepared into solid, semi-solid, liquid or gaseous preparations, such as tablets, pills, capsules, powder, granules, paste, emulsion, suspension, solution, suppositories, injection, inhalant, gel, microspheres, aerosol and the like.

Typical routes of administration of the pharmaceutical compositions of the present 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 compositions of the present invention may be manufactured by methods well known in the art, such as conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, lyophilizing, and the like.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Herein, "halogen" means F, Cl, Br, and I, unless otherwise specified. More preferably, the halogen atom is selected from F, Cl and Br.

Herein, "deuterated", unless otherwise specified, means that one or more hydrogens of a compound or group are replaced with deuterium; deuterium can be mono-, di-, poly-, or fully substituted. The terms "deuterated one or more" and "deuterated one or more" are used interchangeably.

Herein, unless otherwise specified, "non-deuterated compound" means a compound containing deuterium at an atomic ratio of deuterium not higher than the natural deuterium isotope content (0.015%).

The invention also includes isotopically-labeled compounds, equivalent to those disclosed herein as the original compound. Examples of isotopes that can be listed as compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine and chlorine, respectively²H，³H，¹³C，¹⁴C，¹⁵N，¹⁷O，¹⁸O，³¹P，³²P，³⁵S，¹⁸F and³⁶and (4) Cl. The compounds of the present invention, or enantiomers, diastereomers, isomers, or pharmaceutically acceptable salts or solvates thereof, wherein isotopes or other isotopic atoms containing such compounds are within the scope of the present invention. Certain isotopically-labelled compounds of the invention, e.g.³H and¹⁴among these, the radioactive isotope of C is useful in tissue distribution experiments of drugs and substrates. Tritium, i.e.³H and carbon-14, i.e.¹⁴C, their preparation and detection are relatively easy, and are the first choice among isotopes. Isotopically labeled compounds can be prepared by conventional methods by substituting readily available isotopically labeled reagents for non-isotopically labeled reagents using the protocols set forth in the examples.

Pharmaceutically acceptable salts include inorganic and organic salts. One preferred class of salts is that formed by reacting a compound of the present invention with an acid. Suitable acids for forming the salts include, but are not limited to: inorganic acids such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, phosphoric acid, and the like; organic acids such as formic acid, acetic acid, trifluoroacetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, 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 and the like; and amino acids such as proline, phenylalanine, aspartic acid, glutamic acid, etc. Another preferred class of salts are those of the compounds of the invention with bases, for example alkali metal salts (e.g., sodium or potassium salts), alkaline earth metal salts (e.g., magnesium or calcium salts), ammonium salts (e.g., lower alkanolammonium salts and other pharmaceutically acceptable amine salts), for example methylamine salts, ethylamine salts, propylamine salts, dimethylamine salts, trimethylamine salts, diethylamine salts, triethylamine salts, tert-butylamine salts, ethylenediamine salts, hydroxyethylamine salts, dihydroxyethylamine salts, triethanolamine salts, and amine salts formed from morpholine, piperazine, lysine, respectively.

The term "solvate" refers to a complex of a compound of the present invention coordinated to solvent molecules in a specific ratio. "hydrate" refers to a complex formed by the coordination of a compound of the present invention with water.

The present invention provides methods for modulating HIF and/or EPO, by inhibiting hydroxylation of HIF α, thereby stabilizing HIF and activating expression of HIF regulatory genes. The methods may also be applied to prevent, pre-treat, or treat HIF and/or EPO-associated conditions, including anemia, ischemia, and hypoxia conditions.

Ischemia and hypoxia are two conditions associated with HIF and include, but are not limited to, myocardial infarction, liver ischemia, kidney 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 present invention provides methods for treating a variety of ischemic and hypoxic conditions, in particular using the compounds described herein. In one embodiment, the methods of the invention produce therapeutic benefits when administered after ischemia or hypoxia. For example, following myocardial infarction, the methods of the invention result in dramatic decreases in morbidity and mortality, and significant improvements in cardiac structure and performance. On the other hand, when administered after a toxic-ischemic injury in the liver, the methods of the invention improve liver function. Hypoxia is an important component of liver disease, especially in chronic liver disease associated with hepatotoxic compounds such as ethanol. In addition, expression of genes induced by HIF α is known to be increased in alcoholic liver disease, for example, nitric oxide synthase and glucose transporter-1.

Accordingly, the present invention provides a method of treating an ischemia or hypoxia related condition comprising administering to a subject a therapeutically effective amount of a compound or a pharmaceutically acceptable salt thereof, alone or in combination with a pharmaceutically acceptable excipient. In one embodiment, the compound is administered immediately after the ischemic condition is developed, such as myocardial infarction, pulmonary embolism, intestinal infarction, ischemic stroke, and renal ischemia-reperfusion injury. In another embodiment, the compound is administered to a patient diagnosed with a condition associated with the occurrence of chronic ischemia, such as cardiogenic cirrhosis, macular degeneration, pulmonary embolism, acute respiratory failure, neonatal respiratory distress syndrome, and congestive heart failure.

Another aspect of the invention provides methods of treating a patient at risk of developing an ischemic or hypoxic condition, such as an individual at high risk of atherosclerosis, using the compounds described herein. Risk factors for atherosclerosis include, for example, hyperlipidemia, smoking, hypertension, diabetes, hyperinsulinemia, and abdominal obesity. Accordingly, the present invention provides a method of preventing ischemic tissue injury comprising administering to a patient 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. In one embodiment, the compound may be administered based on a predisposing condition, such as hypertension, diabetes, arterial occlusive disease, chronic venous insufficiency, raynaud's disease, chronic skin ulcers, cirrhosis, congestive heart failure, and systemic sclerosis.

In a particular embodiment, the method is used to increase vascularization and/or granulation tissue formation in damaged tissues, wounds and ulcers. For example, the compounds of the present invention have been shown to be effective in stimulating granulation tissue formation in wound healing. Granulation tissue contains newly formed leaky blood vessels and a temporary plasma protein matrix, 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 impaired, ulceration can occur. The method of the present invention is effective in promoting the formation of granulation tissue. Thus, the present invention provides methods for treating patients with tissue damage due to, for example, infarction, with wounds induced by, for example, trauma or injury, or with chronic wounds or ulcers resulting from certain conditions (e.g., diabetes). The method comprises administering to a patient 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 methods of pre-treating a subject with the compounds to reduce or prevent the occurrence of tissue damage associated with ischemia or hypoxia. The methods of the invention produce therapeutic benefits when administered immediately prior to a condition involving ischemia or hypoxia. For example, application of the methods of the invention prior to induction of myocardial infarction has shown a statistically significant improvement in cardiac structure and performance. On the other hand, when administered immediately before and between ischemia-reperfusion injury, the methods of the invention produce therapeutic benefits that significantly reduce the diagnostic parameters associated with renal failure.

Accordingly, the present invention provides a method of pre-treating a subject to reduce or prevent tissue damage associated with ischemia or hypoxia, the method comprising administering to a patient having a history of ischemic disorders, such as myocardial infarction, or a patient having impending symptoms of ischemia, such as angina pectoris, a therapeutically effective amount of a compound or a pharmaceutically acceptable salt thereof, alone or in combination with a pharmaceutically acceptable excipient. In another embodiment, the compound may be administered based on physical parameters suggestive of possible ischemia, such as for individuals under general anesthesia or temporarily working at high altitude. In yet another embodiment, the compounds may be used in organ transplantation to pre-treat organ donors or to maintain organs removed from the body prior to transplantation into a recipient.

Previous studies have shown that certain compounds used in the methods of the invention are effective inhibitors of prolyl-4-hydroxylase. Although it is recognized that the recovery of the initial infarction or wound requires the deposition of connective tissue in the necrotic area, the present invention demonstrates no adverse effects on the treatment of scarring. Thus, based on the benefits provided by certain compounds of the invention in the treatment and prevention of hypoxic tissue damage and fibrosis, the invention encompasses a "dual therapy" approach to the treatment or prevention of conditions involving ischemia or hypoxia, including ischemia or hypoxia associated with complicated reactive fibrosis, such as myocardial infarction and consequent congestive heart failure. The methods can use a compound that inhibits more than one 2-oxoglutarate dioxygenase enzyme, e.g., a HIF prolyl hydroxylase and a collagenolytic prolyl 4-hydroxylase, of the same specificity or of different specificities. Alternatively, the methods can use a combination of compounds, where each compound specifically inhibits only one 2-oxoglutarate dioxygenase enzyme, e.g., one compound specifically inhibits HIF prolyl hydroxylase and a second compound specifically inhibits collagen prolyl 4-hydroxylase.

In one aspect, the compounds of the invention inhibit one or more 2-oxoglutarate dioxygenase enzymes. In one embodiment, the compound inhibits at least two 2-oxoglutarate dioxygenase family members, e.g., HIF prolyl hydroxylase and HIF asparagine-hydroxylase (FIH-1), having the same specificity or different specificities. In another embodiment, the compound is specific for a 2-oxoglutarate dioxygenase enzyme, such as a HIF prolyl hydroxylase, and exhibits little or no specificity for other family members.

The compounds can be administered in combination with a variety of other methods of treatment. In one embodiment, the compound is administered with another 2-oxoglutarate dioxygenase inhibitor, wherein the two compounds have different specificities for individual 2-oxoglutarate dioxygenase family members. The two compounds may be administered simultaneously in a ratio of one to the other. Determination of the proportion appropriate for a given course of treatment or particular subject is within the level of skill in the art. Alternatively, the two compounds may be administered continuously over the course of treatment, for example following a myocardial infarction. In a particular embodiment, one compound specifically inhibits HIF prolyl hydroxylase activity, and a second compound specifically inhibits collagenolytic prolyl 4-hydroxylase activity. In another particular embodiment, one compound specifically inhibits HIF prolyl hydroxylase activity, and a second compound specifically inhibits HIF asparaginyl-hydroxylase activity. In another embodiment, the compound is administered with another therapeutic agent having a different mode of action, such as an ACE inhibitor (ACEI), angiotensin-II receptor blocker (ARB), statin, diuretic, digoxin, carnitine, and the like.

The present invention provides methods for increasing endogenous Erythropoietin (EPO). These methods can be applied in vivo, e.g., in plasma, or in vitro, e.g., in conditioned cell culture medium. The invention further provides methods of increasing endogenous EPO levels for the prevention, pre-treatment, or treatment of EPO-related conditions, including, for example, conditions associated with anemia and neurological disorders. Anemia-related conditions include disorders such as acute or chronic kidney disease, diabetes, cancer, ulcers, viral infections (e.g., HIV, bacterial or parasitic), inflammation, and the like. Anemic conditions may further include conditions associated with procedures or treatments including, for example, radiation therapy, chemotherapy, dialysis, and surgery. Anemia-related conditions additionally include abnormal hemoglobin and/or red blood cells, such as found in conditions such as microcytic anemia, hypochromic anemia, aplastic anemia, and the like.

The present invention is useful for prophylactically or simultaneously increasing endogenous EPO in a subject undergoing a particular treatment or procedure, such as HIV-infected anemia patients being treated with zidovudine or other reverse transcriptase inhibitors, anemia cancer patients receiving cyclic chemotherapy with or without cisplatin, or anemia or non-anemia 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 nerve damage or degeneration of neural tissue, including (but not limited to) stroke, trauma, epilepsy, spinal cord injury, and neurodegenerative disorders.

In addition, the methods can be used to increase endogenous EPO levels in anemic or non-anemic patients scheduled to undergo surgery, to reduce the need for exogenous blood transfusions, or to facilitate storage of pre-operative blood. The small decrease in blood hematocrit that typically occurs after a preoperative autologous blood supply does not stimulate an increase in endogenous EPO or compensatory erythropoiesis. However, preoperative stimulation of endogenous EPO will effectively increase red blood cell mass and autologous blood supply volume while maintaining higher hematocrit levels, and the method is specifically contemplated herein. In some surgical populations, particularly individuals with surgical blood loss of more than 2 liters, the methods of the invention can be used to reduce allogenic blood exposure.

The methods of the present invention may also be used to enhance athletic performance, improve exercise performance, and promote or enhance aerobic regulation. For example, athletes may use the method to facilitate training and soldiers may use the method to improve endurance and endurance, for example.

The methods of the invention have been shown to increase the endogenous erythropoietin content in the medium of in vitro therapeutic culture cells and in the plasma of in vivo therapeutic animals. Although the kidneys are the primary source of erythropoietin in the body, other organs, including the brain, liver and bone marrow, can and do synthesize erythropoietin upon appropriate stimulation. The methods of the invention can be used to increase the expression of endogenous erythropoietin in a variety of body organs, including the brain, kidney and liver. Indeed, the method of the invention even increases the amount of endogenous erythropoietin in animals undergoing double nephrectomy.

The method of the invention demonstrates that the erythropoietin content can be increased even when kidney function is impaired. Although the present invention is not limited by the mechanism of erythropoietin production, the decrease in erythropoietin secretion that is typically seen during renal failure can be attributed to hyperoxia resulting from increased flow/perfusion in renal tissue.

In another aspect, the methods of the invention increase hematocrit and blood hemoglobin levels in an in vivo treated animal. The increase in plasma EPO, hematocrit, and blood hemoglobin produced as the compounds are used in the methods of the invention are dose sensitive, however the dosage regimen can be determined to produce a constant, controllable level of response of the compounds of the invention. In another aspect, treatment with a compound of the invention can treat anemia, such as that induced by a toxic compound, such as the chemotherapeutic cisplatin, or anemia due to blood loss, such as trauma, injury, parasites, or surgery.

In animals treated with the compounds of the present invention, the increase in hematocrit and blood hemoglobin is preceded by an increase in the percentage of circulating immature red blood cells (reticulocytes) in the blood. Thus, the invention encompasses the use of a compound of the invention in a method of increasing reticulocyte content in the blood of an animal to produce a cell-free reticulocyte lysate, as described by Pelham and Jackson in the J.European biochem (Eur.J.biochem.)67:247-256 (1976). The amount of circulating reticulocytes in an animal (e.g., a rabbit, etc.) is increased by treatment with a compound of the invention, either alone or in combination with another compound, e.g., acetophenylhydrazine, etc.

Compared with the prior art, the invention has the beneficial effects that: the compounds of the invention are useful for modulating Hypoxia Inducible Factor (HIF) and/or endogenous Erythropoietin (EPO). The deuteration technology changes the metabolism of the compound in organisms, so that the compound has better pharmacokinetic parameter characteristics. In this case, the dosage can be varied and a long acting formulation formed, improving the applicability. Deuterium is used for replacing hydrogen atoms in the compound, and due to the deuterium isotope effect, the medicine concentration of the compound in an animal body can be improved, so that the medicine curative effect is improved. Replacement of hydrogen atoms in compounds with deuterium may increase the safety of the compounds as certain metabolites are inhibited.

Detailed Description

Compound (I)

The invention provides a compound shown as a formula (I), or a crystal form, a pharmaceutically acceptable salt, a hydrate or a solvate thereof.

In a particular embodiment, "R¹、R²、R³、R⁴、R⁵、R⁶、R⁷、R⁸、R⁹、R¹⁰、R¹¹、R¹²、R¹³、R¹⁴、R¹⁵、R¹⁶、R¹⁷、R¹⁸、R¹⁹、R²⁰、R²¹、R²²、R²³、R²⁴、R²⁵Each independently hydrogen, deuterium, halogen or trifluoromethyl "includes R¹Selected from hydrogen, deuterium, halogen or trifluoromethyl, R²Selected from hydrogen, deuterium, halogen or trifluoromethyl, R³Selected from hydrogen, deuterium, halogen or trifluoromethyl, and so on, until R²⁵Selected from hydrogen, deuterium, halogen or trifluoromethyl, R³Selected from hydrogen, deuterium, halogen or trifluoromethyl. More specifically, includes R¹Is hydrogen, R¹Is deuterium, R¹Is halogen (F, Cl, Br or I) or R¹Is trifluoromethyl, R²Is hydrogen, R²Is deuterium, R²Is halogen (F, Cl, Br or I) or R²Is trifluoromethyl, R³Is hydrogen, R³Is deuterium, R³Is halogen (F, Cl, Br or I) or R³Trifluoromethyl, and so on, until R²⁵Is hydrogen, R²⁵Is deuterium, R²⁵Is halogen (F, Cl, Br or I) or R²⁵Is trifluoromethyl.

In a preferred embodiment, the present invention relates to a compound of formula (I), or a crystalline form, a pharmaceutically acceptable salt, hydrate or solvate thereof, wherein R is²³Is hydrogen, and R¹-R²²And R²⁴-R²⁵With the proviso that said compound contains at least one deuterium atom, as defined above.

In a preferred embodiment, the present invention relates to a compound of formula (I), or a crystalline form, a pharmaceutically acceptable salt, hydrate or solvate thereof, wherein R is²³Is hydrogen, and R¹-R²²And R²⁴-R²⁵Each independently selected from hydrogen or deuterium, with the proviso that the compound contains at least one deuterium atom.

In a preferred embodiment, the present invention relates to a compound of formula (I), or a crystalline form, a pharmaceutically acceptable salt, hydrate or solvate thereof, wherein R is²³Is hydrogen, R¹-R²²And R²⁴-R²⁵Each independently selected from hydrogen or deuterium, and R¹And R²Are the same, with the proviso that said compound contains at least one deuterium atom.

In a preferred embodiment, the present invention relates to a compound of formula (I), or a crystalline form, a pharmaceutically acceptable salt, hydrate or solvate thereof, wherein R is¹-R⁶、R¹⁴、R¹⁵、R¹⁷、R¹⁸、R²¹-R²³Is hydrogen, R⁷-R¹¹、R¹²、R¹³、R¹⁶、R¹⁹、R²⁰And R²⁴、R²⁵Each independently selected from hydrogen or deuterium, with the proviso that the compound contains at least one deuterium atom.

In a preferred embodiment, R⁷-R¹⁰Are the same.

In a preferred embodiment, R¹²、R¹³、R¹⁹、R²⁰Are the same.

In a preferred embodiment, R²⁴、R²⁵Are the same.

In a preferred embodiment, R¹、R²Are the same.

As a preferred embodiment of the present invention, the compound is of any one of the following structures, or a pharmaceutically acceptable salt thereof, but is not limited to the following structures:

examples

The following describes more specifically the processes for the preparation of the compounds of formula (I) according to the invention, but these particular processes do not constitute any limitation of the invention. The compounds of the present invention may also be conveniently prepared by optionally combining various synthetic methods described in the present specification or known in the art, and such combinations may be readily carried out by those skilled in the art to which the present invention pertains.

In general, in the preparative schemes, each reaction is usually carried out in an inert solvent at a temperature ranging from room temperature to reflux temperature (e.g., from 0 ℃ to 100 ℃, preferably from 0 ℃ to 80 ℃). The reaction time is usually 0.1 to 60 hours, preferably 0.5 to 24 hours.

Example 1N- [ (1, 3-dicyclohexyl-2, 4, 6-trioxo-5-hexahydropyrimidyl) carbonyl]Glycine-2, 2- ₂d, compound T-1, of the formula:

the following synthetic route is adopted:

step 1: synthesis of Compound 2.

Under nitrogen, a solution of malonyl chloride (1.30mL,13.39mmol) in anhydrous chloroform (20mL) was added dropwise to a solution of 1, 3-bis (cyclohexyl) urea (3.00g,13.39mmol) in anhydrous chloroform (80mL), and after completion of the addition, the reaction mixture was heated to 50 ℃ for 4.5 hrs. Cooling to room temperature, quenching the reaction with 1M dilute hydrochloric acid, drying the organic layer over anhydrous sodium sulfate, concentrating under reduced pressure, concentrating the filtrate under reduced pressure, and column separating the concentrated solution (eluent: petroleum ether/ethyl acetate (v/v) ═ 10:1) to obtain 1.50g of a white solid, yield: 38.4 percent，LC-MS(APCI):m/z＝293.2(M+1)⁺。

Step 2: synthesis of Compound 3.

Ethyl isocyanatoacetate (0.70g,5.50mmol) was added to compound 2(1.50g,5.50mmol) and DIPEA (1.40g,11.00mmol) in dry dichloromethane (80mL) at room temperature, the reaction was stirred overnight at room temperature, the washings were quenched with 1M dilute hydrochloric acid (30mL x 2), and the organic layer was concentrated under reduced pressure to give a white solid which was used directly in the next reaction. LC-MS (APCI): M/z 422.3(M +1)⁺。

And step 3: synthesis of Compound 4.

The crude compound 3 was dissolved in ethanol (10mL) at room temperature, 1M sodium hydroxide (5mL) was added, the reaction was stirred at room temperature for 3hrs, and the reaction solution was acidified with 1M dilute hydrochloric acid. Extracting with ethyl acetate (50mL x 2), combining organic layers, washing with 1M diluted hydrochloric acid, concentrating the organic layer under reduced pressure to obtain a white solid, pulping and purifying with a mixed solvent of diethyl ether and n-hexane, filtering the solid, washing a filter cake with a small amount of mixed solvent of diethyl ether and n-hexane, and drying the solid in vacuum to obtain 2.0g of the white solid, wherein the total yield of the two steps is 92.0%. LC-MS (APCI): M/z ═ 394.2(M +1)⁺. Purity 99.21% (HPLC).¹H NMR(400MHz,DMSO-d₆)δ10.18(t,J＝5.7Hz,1H),4.64(t,J＝12.0Hz,2H),4.08(d,J＝5.6Hz,2H),2.28(q,J＝12.3Hz,4H),1.78(d,J＝12.5Hz,4H),1.69-1.49(m,6H),1.27(q,J＝12.9Hz,4H),1.18-1.04(m,2H).

And 4, step 4: synthesis of Compound T-1.

Sodium hydroxide deuterium oxide (40%) was added to a solution of compound 4(300mg, 0.76mmol) in heavy water (15mL) at 40% (0.31mL,3.05mmol), and the reaction mixture was sealed at 140 ℃ overnight. And cooling to room temperature. The pH was adjusted to about 3 with 1M dilute hydrochloric acid, extracted with ethyl acetate (30mL × 3), the organic layer was dried over anhydrous sodium sulfate, concentrated under reduced pressure, the filtrate was concentrated under reduced pressure, and the concentrated solution was subjected to column separation (eluent: dichloromethane/methanol (v/v) ═ 10:1) to give 104mg, yield: 34.6 percent. Purity: 98.15 percent. LC-MS (APCI) M/z 396.2(M +1)⁺。¹H NMR(400MHz,DMSO-d₆)δ12.99(br,1H),10.17(s,1H),4.62(t,J＝12.2Hz,2H),2.26(dd,J＝22.4,12.0Hz,4H),1.77(d,J＝12.7Hz,4H),1.64-1.51(m,6H),1.31-1.22(m,4H),1.17-1.07(m,2H).

₄Example 2N- [ (1, 3-bis (cyclohexyl-2, 2,6,6-d) -2,4, 6-trioxo-5-hexahydropyrimidyl) carbonyl] Glycine, compound T-2, has the following formula:

the following synthetic route is adopted:

step 1: synthesis of Compound 7.

Cyclohexanone (1.0g, 10.00mmol) and 40% NaOD (0.27mL) were added to 10mL of D₂In O, the reaction was refluxed for 2 days at elevated temperature. After cooling to room temperature, hydroxylamine hydrochloride (2.0g) and sodium acetate (4.0g) were added thereto at 0 ℃ and the reaction was stirred for 20min while elevating the temperature to 70 ℃. Then, the mixture was cooled, a solid precipitated, filtered, and the cake was dried and dissolved in 160mL of diethyl ether, and lithium aluminum hydride (2.0g) was slowly added thereto, and the mixed solution was heated under reflux for 45 minutes. Cooling to room temperature, slowly adding saturated sodium bicarbonate solution dropwise to dilute out a large amount of solid, filtering, leaching the filter cake twice with diethyl ether, mixing the organic phases, drying, and concentrating to obtain 400mg of yellow liquid. Yield: 40 percent. LC-MS (APCI) 104.2(M +1) M/z⁺。

Step 2: synthesis of Compound 8.

Compound 8(3.90g, 37.86mmol), ethylene carbonate (1.67g, 18.93mmol) and 1,5, 7-triazabicyclo (4.4.0) dec-5-ene (53mg) were added to a 50mL single-necked flask, and heated to 120 ℃ and stirred for 2hrs to form a solid. Cooled to room temperature, 10mL of water was added, stirred for ten minutes, then filtered, and the filter cake was washed three times with DCM and then oven dried to give 1.2g of a white solid. Yield: 13.6 percent. LC-MS (APCI): M/z 233.2(M +1)⁺。

And step 3: synthesis of Compound 9.

Under nitrogen, a solution of malonyl chloride (365mg,2.59mmol) in anhydrous chloroform (0.3mL) was added dropwise to a solution of compound 8(600mg,2.59mmol) in anhydrous chloroform (16mL), and after completion of the addition, the reaction mixture was heated to 50 ℃ for reaction for 4.5 hrs. Cooling to room temperature, quenching the reaction with 1M dilute hydrochloric acid, drying the organic layer over anhydrous sodium sulfate, concentrating under reduced pressure, concentrating the filtrate under reduced pressure, and column separating the concentrated solution (eluent: petroleum ether/ethyl acetate (v/v) ═ 10:1) to give 190mg of a white solid, yield: 24.5%, LC-MS (APCI): M/z 301.2(M +1)⁺。

And 4, step 4: synthesis of Compound 10.

Ethyl isocyanatoacetate (82mg,0.63mmol) was added to compound 9(190mg,0.63mmol) and DIPEA (164mg,1.26mmol) in dry dichloromethane (10mL) at room temperature, the reaction was stirred at room temperature overnight, the washed reaction was quenched with 1M dilute hydrochloric acid (10mL x 2), and the organic layer was concentrated under reduced pressure to afford a white solid which was used directly in the next reaction. LC-MS (APCI) M/z 430.3(M +1)⁺。

And 5: and (3) synthesizing a compound T-2.

The crude product was dissolved in ethanol (10mL) at room temperature, 1M sodium hydroxide (5mL) was added, the reaction was stirred at room temperature for 3 days, and the reaction solution was acidified with 1M diluted hydrochloric acid. Extracting with ethyl acetate (20x 2), combining organic layers, washing with 1M diluted hydrochloric acid, concentrating the organic layer under reduced pressure to obtain a white solid, pulping and purifying with a mixed solvent of diethyl ether and n-hexane, filtering the solid, washing a filter cake with a small amount of mixed solvent of diethyl ether and n-hexane, and drying the solid in vacuum to obtain 159mg of white solid, wherein the total yield of the two steps is 62.6%. LC-MS (APCI): M/z 402.2(M +1)⁺(ii) a Purity: 99.67% (HPLC).¹H NMR(400MHz,DMSO-d₆):δ10.18(t,J＝5.6Hz,1H),4.60(s,2H),4.11(d,J＝5.7Hz,2H),1.76(d,J＝12.9Hz,4H),1.62(d,J＝12.4Hz,2H),1.25(t,J＝12.7Hz,4H),1.12(t,J＝12.9Hz,2H).

₄Example 3N- [ (1, 3-bis (cyclohexyl-2, 2,6,6-d) -2,4, 6-trioxo-5-hexahydropyrimidyl) carbonyl] ₂Glycine-2, 2-d, compound T-3, of the formula:

the following synthetic route is adopted:

40% sodium hydroxide (0.1mL) was added to a mixed solution of T-2(90mg, 0.22mmol) in heavy water (1mL) and deuterated ethanol (2mL), and the reaction mixture was stirred at room temperature for 2 days, and then heated to 60 ℃ for 4 days. And cooling to room temperature. Acidifying the reaction solution with 1M dilute hydrochloric acid, extracting with ethyl acetate (20mL x 3), drying the organic layer with anhydrous sodium sulfate, concentrating the organic layer under reduced pressure to obtain a white solid, pulping and purifying with a mixed solvent of diethyl ether and n-hexane, filtering the solid, washing the filter cake with a small amount of mixed solvent of diethyl ether and n-hexane, and vacuum drying the solid to obtain 50mg of white solid with a yield of 55.6%. LC-MS (APCI): M/z 404.1(M +1)⁺Purity: 97.12% (HPLC).¹H NMR(400MHz,DMSO-d₆):δ12.92(br,1H),10.17(s,1H),4.60(s,2H),1.83-1.55(m,6H),1.-1.21(m,4H),1.16-1.08(m,2H).

Example 4N- [ (1, 3-bis (cyclohexyl-1-d) -2,4, 6-trioxo-5-hexahydropyrimidyl) carbonyl]The glycine is used for the synthesis of glycine, i.e., compound T-4, of the formula:

the following synthetic route is adopted:

step 1: synthesis of Compound 11.

Activating the fresh material at room temperature

Molecular sieves (5.0g) were added to a solution of cyclohexanone (2.5mL, 23.9mmol) and benzylamine (2.6mL, 23.9mmol) in dry dichloromethane (24mL) and the reaction was stirred at room temperature overnight and concentrated directly for the next reaction. LC-MS (APCI) 188.2(M +1) M/z⁺。

Step 2: synthesis of Compound 12.

Dissolving the crude product in anhydrous methanol (24mL), adding sodium deuteroborohydride (1.0g,23.9mmol) in portions at 0 deg.C, reacting at room temperature for 1.5hrs while stirring, removing methanol under reduced pressure to obtain crude product, dissolving with ether, and filtering. The filtrate was concentrated under reduced pressure and the concentrate was subjected to column separation (eluent: dichloromethane/methanol (v/v) ═ 10:1) to give a pale yellow oil, LC-ms (apci): M/z 191.2(M +1)⁺。

And step 3: synthesis of Compound 13.

In the greenhouse, Pd/C (10%, 250mg) will be mixed with Pd (OH)₂(250mg) was added to a solution of Compound 12 in dry methanol (50mL), the reaction was stirred at room temperature for 72hrs, filtered through celite, and the filtrate was concentrated under reduced pressure to give 1.2g of a colorless liquid, total yield: 50% LC-MS (APCI): M/z 101.2(M +1)⁺.

And 4, step 4: synthesis of Compound 14.

Compound 13(2.0g, 20mmol), ethylene carbonate (0.88g, 10mmol) and 1,5, 7-triazabicyclo (4.4.0) dec-5-ene (28mg) were put into a 50mL single-neck flask, heated to 120 ℃ and stirred for 2hrs, whereby a solid was formed. Cooled to room temperature, 5mL of water was added, stirred for ten minutes, then filtered, and the filter cake was washed three times with DCM and then oven dried to 185mg of a white solid. Yield: 4.07 percent. LC-MS (APCI): M/z 227.1(M +1)⁺。

And 5: synthesis of Compound 15.

Under nitrogen, a solution of malonyl chloride (115mg,0.82mmol) in anhydrous chloroform (0.2mL) was added dropwise to a solution of compound 14(185mg,0.82mmol) in anhydrous chloroform (10mL), and after completion of the addition, the reaction mixture was heated to 50 ℃ for reaction for 4.5 hrs. Cooling to room temperature, quenching the reaction with 1M dilute hydrochloric acid, and removing the organic layerDried over sodium sulfate, concentrated under reduced pressure, the filtrate concentrated under reduced pressure, and the concentrated solution was subjected to column separation (eluent: petroleum ether/ethyl acetate (v/v) ═ 10:1) to obtain 135mg of a white solid in yield: 56.0%, LC-MS (APCI): 295.2(M +1) M/z⁺。

Step 6: synthesis of Compound 16.

Ethyl isocyanatoacetate (61mg,0.48mmol) was added to compound 15(135mg,0.48mmol) and DIPEA (122mg,0.96mmol) in dry dichloromethane (10mL) at room temperature, the reaction was stirred at room temperature overnight, the washed reaction was quenched with 1M dilute hydrochloric acid (10mL x 2), and the organic layer was concentrated under reduced pressure to afford a white solid which was used directly in the next reaction.

LC-MS(APCI):m/z＝424.1(M+1)⁺。

And 7: and (3) synthesizing a compound T-4.

The crude product was dissolved in ethanol (5mL) at room temperature, 1M sodium hydroxide (2mL) was added, the reaction was stirred at room temperature for 3 days, and the reaction solution was acidified with 1M diluted hydrochloric acid. Extracting with ethyl acetate (20mL x 2), combining organic layers, washing with 1M diluted hydrochloric acid, concentrating the organic layer under reduced pressure to obtain a white solid, pulping and purifying with a mixed solvent of diethyl ether and n-hexane, filtering the solid, washing a filter cake with a small amount of mixed solvent of diethyl ether and n-hexane, and drying the solid in vacuum to obtain 120mg of white solid, wherein the total yield of the two steps is 63.1%. LC-MS (APCI) M/z 396.0(M +1)⁺Purity: 94.46% (HPLC).¹H NMR(400MHz,DMSO-d₆):δ10.16(t,J＝5.5Hz,1H),4.04(d,J＝5.6Hz,2H),2.27(t,J＝11.2Hz,4H),1.77(d,J＝12.6Hz,4H),1.65-1.49(m,6H),1.30-1.22(m,4H),1.17-1.05(m,2H).

₄Example 5N- [ (1-cyclohexyl-3- (cyclohexyl-2, 2,6,6-d) -2,4, 6-trioxo-5-hexahydropyrimidyl) Carbonyl radical]Glycine, compound T-5, has the following formula:

the following synthetic route is adopted:

step 1: synthesis of compounds 18 and 19.

Cyclohexylamine (500mg, 5.04mmol), ethylene carbonate (443mg, 5.04mmol) and 1,5, 7-triazabicyclo (4.4.0) dec-5-ene (10mg) were placed in a 50mL single-neck flask and heated at 120 ℃ for 4 hrs. Then, the reaction mixture was cooled to room temperature, and then compound 7(519mg,5.04mmol) was added thereto, followed by warming to 120 ℃ for 4.5 hrs. Cooled to room temperature, 5mL of water was added, a solid precipitated, filtered, the filter cake washed twice with a small amount of DCM and dried in vacuo to give 293mg of a white solid product. LC-MS (APCI) M/z 229.1(M +1)⁺(ii) a Yield: 25.4 percent.

Step 2: synthesis of Compound 20.

Under nitrogen, a solution of malonyl chloride (181mg,1.29mmol) in anhydrous chloroform (0.4mL) was added dropwise to a solution of compound 19(293mg,1.29mmol) in anhydrous chloroform (16mL), and after completion of the addition, the reaction mixture was heated to 50 ℃ for reaction for 4.5 hrs. Cooling to room temperature, quenching the reaction with 1M dilute hydrochloric acid, drying the organic layer over anhydrous sodium sulfate, concentrating under reduced pressure, concentrating the filtrate under reduced pressure, and column-separating the concentrated solution (eluent: petroleum ether/ethyl acetate (v/v) ═ 10:1) to give 238mg of a white solid, yield: 56.0%, LC-MS (APCI): M/z 297.2(M +1)⁺。

And step 3: synthesis of Compound 21.

Ethyl isocyanatoacetate (104mg,0.80mmol) was added to compound 20(238mg,0.80mmol) and DIPEA (207mg,1.60mmol) in dry dichloromethane (10mL) at room temperature, the reaction was stirred at room temperature overnight, the washed reaction was quenched with 1M dilute hydrochloric acid (10mLx 2), and the organic layer was concentrated under reduced pressure to give a white solid which was used directly in the next reaction. LC-MS (APCI): M/z 426.1(M +1)⁺.

And 4, step 4: and (3) synthesizing a compound T-5.

The crude product was dissolved in ethanol (10mL) at room temperature, 1M sodium hydroxide (5mL) was added, the reaction was stirred at room temperature for 3 days, and the reaction solution was acidified with 1M diluted hydrochloric acid. Acetic acid BEster extraction (20mL x 2), combined organic layers are washed by 1M diluted hydrochloric acid, the organic layers are concentrated under reduced pressure to obtain white solid, the white solid is pulped and purified by a mixed solvent of diethyl ether and n-hexane, the solid is filtered, a filter cake is washed by a small amount of mixed solvent of diethyl ether and n-hexane, the solid is dried in vacuum to obtain 218mg of white solid, and the total yield of the two steps is 68.5%. LC-MS (APCI): M/z 398.2(M +1)⁺(ii) a Purity: 98.98% (HPLC),¹H NMR(400MHz,DMSO-d₆)δ10.16(t,J＝5.4Hz,1H),4.70-4.47(m,2H),4.07(d,J＝5.7Hz,2H),2.25(q,J＝11.8Hz,2H),1.75(d,J＝10.1Hz,4H),1.58(t,J＝14.2Hz,4H),1.24(t,J＝12.7Hz,4H),1.11(t,J＝12.7Hz,2H).

₄example 6N- [ (1-cyclohexyl-3- (cyclohexyl-2, 2,6,6-d) -2,4, 6-trioxo-5-hexahydropyrimidyl) ₂Carbonyl radical]Glycine-2, 2-d, compound T-6, of the formula:

the following synthetic route is adopted:

sodium hydroxide deuterium oxide (0.1mL) at 40% concentration was added to compound T-5(100mg) in heavy water (1mL) and deuterated ethanol (2mL), and the reaction mixture was stirred at room temperature for 2 days, then heated to 60 ℃ and reacted for 4 days. And cooling to room temperature. Acidifying the reaction solution with 1M dilute hydrochloric acid, extracting with ethyl acetate (20mL x 3), drying the organic layer with anhydrous sodium sulfate, concentrating the organic layer under reduced pressure to obtain a white solid, pulping and purifying with a mixed solvent of diethyl ether and n-hexane, filtering the solid, washing the filter cake with a small amount of mixed solvent of diethyl ether and n-hexane, and vacuum drying the solid to obtain 80mg of a light yellow solid with a yield of 79.8%. LC-MS (APCI): M/z 400.2(M +1)⁺(ii) a Purity: 99.17% (HPLC),¹H NMR(400MHz,DMSO-d6):δ13.03(br,1H),10.15(s,1H),4.73–4.44(m,2H),2.30–2.18(m,2H),1.82–1.68(m,4H),1.64–1.52(m,4H),1.26–1.18(m,4H),1.15–1.05(m,2H).

example 7N- [ (1, 3-bis (cyclohexyl-1-d) -2,4, 6-trioxo-5-hexahydropyrimidyl) carbonyl]Glycine- ₂2,2-d, Compound T-7, of the formula:

the following synthetic route is adopted:

sodium hydroxide deuterium oxide (0.1mL) at 40% concentration was added to compound T-4(80mg) in heavy water (1mL) and deuterated ethanol (2mL), and the reaction mixture was stirred at room temperature for 2 days, then heated to 60 ℃ and reacted for 4 days. And cooling to room temperature. The reaction solution was acidified with 1M dilute hydrochloric acid, extracted with ethyl acetate (20mL × 3), the organic layer was dried over anhydrous sodium sulfate, the organic layer was concentrated under reduced pressure to obtain a white solid, which was purified by beating with a mixed solvent of diethyl ether and n-hexane, the solid was filtered, the filter cake was washed with a small amount of a mixed solvent of diethyl ether and n-hexane, and the solid was dried under vacuum to obtain 70mg of a yellow solid with a yield of 87.5%. LC-MS (APCI): M/z 398.2(M +1)⁺(ii) a Purity: 94.35% (HPLC),¹H NMR(400MHz,DMSO-d₆)δ10.15(s,1H),2.24(t,J＝11.3Hz,4H),1.76(d,J＝12.6Hz,4H),1.63–1.53(m,6H),1.25–1.19(m,4H),1.13–1.04(m,2H).

and (4) testing the biological activity.

(1) Mouse tissue HIF western blot analysis.

Mouse tissues stored at-80 ℃ were ground to powder using a mortar and pestle frozen in liquid nitrogen. Cell nuclear extracts were prepared using the NE-PER kit (Pierce Biotechnology). For immunoprecipitation, the nuclear extracts were extracted with a tissue ratio antibody of 200: 1 to the HIF-1 alpha monoclonal antibody. The suspension was incubated in a conical microcentrifuge tube for 4 hours at 4 ℃. Protein A/G-coupled agarose beads (40. mu.L of 50% suspension) were then added to the tube. After spinning overnight at 4 ℃, the beads were washed 3 times with ice-cold phosphate buffer. The beads were then prepared for SDS-PAGE using 40. mu.L Laemmli sample buffer. Proteins separated from SDS-PAGE were transferred to nitrocellulose plates with XCell-II Blot Module system. Blots were blocked with 5% BSA, then blotted with HIF-1 α rabbit antibody at 1: incubate at 100 dilution. The blot was 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 images were captured with an epressen Expression 1600 scanner.

(2) Mouse serum EPO assay.

Mouse serum EPO was detected using R & DSystems' mouse Quantikine erythropoietin ELISA kit according to the instructions for use.

The experimental result shows that the compound of the invention has response to the detection of mouse serum EPO, which indicates that the compound of the invention can be used for preparing the medicine for regulating and controlling human anemia.

(3) And (5) evaluating the metabolic stability.

Microsome experiment: rat liver microsomes: 0.5mg/mL, Xenotech; coenzyme (NADPH/NADH): 1mM, Sigma Life Science; magnesium chloride: 5mM, 100mM phosphate buffer (pH 7.4).

Preparing a stock solution: a predetermined amount of the test compound powder was precisely weighed and dissolved in DMSO to 5mM each.

Preparation of phosphate buffer (100mM, pH 7.4): 150mL of 0.5M potassium dihydrogenphosphate and 700mL of a 0.5M dipotassium hydrogenphosphate solution prepared in advance were mixed, the pH of the mixture was adjusted to 7.4 with the 0.5M dipotassium hydrogenphosphate solution, the mixture was diluted 5-fold with ultrapure water before use, and magnesium chloride was added to obtain a phosphate buffer (100mM) containing 100mM potassium phosphate and 3.3mM magnesium chloride at a pH of 7.4.

NADPH regenerating system solution (containing 6.5mM NADP, 16.5mM G-6-P, 3U/mL G-6-P D, 3.3mM magnesium chloride) was prepared and placed on wet ice before use.

Preparing a stop solution: acetonitrile solution containing 50ng/mL propranolol hydrochloride and 200ng/mL tolbutamide (internal standard). 25057.5 mu L of phosphate buffer (pH7.4) is taken to a 50mL centrifuge tube, 812.5 mu L of SD rat liver microsome is respectively added, and the mixture is mixed evenly to obtain liver microsome dilution with the protein concentration of 0.625 mg/mL.

Incubation of the samples: the stock solutions of the corresponding compounds were diluted to 0.25mM each with an aqueous solution containing 70% acetonitrile, and used as working solutions. 398. mu.L of rat liver microsome dilutions were added to 96-well plates (N2), 2. mu.L of 0.25mM working solution, and mixed.

Determination of metabolic stability: 300. mu.L of pre-cooled stop solution was added to each well of a 96-well deep-well plate and placed on ice as a stop plate. The 96-well incubation plate and the NADPH regeneration system are placed in a 37 ℃ water bath box, shaken at 100 rpm and pre-incubated for 5 min. 80. mu.L of the incubation solution was taken out of each well of the incubation plate, added to the stop plate, mixed well, and supplemented with 20. mu.L of NADPH regenerating system solution as a 0min sample. Then 80. mu.L of NADPH regenerating system solution was added to each well of the incubation plate, the reaction was started, and the timer was started. The reaction concentration of the corresponding compound was 1. mu.M, and the protein concentration was 0.5 mg/mL. When the reaction was carried out for 10min, 30 min and 90min, 100. mu.L of each reaction solution was added to the stop plate and vortexed for 3min to terminate the reaction. The stop plates were centrifuged at 5000 Xg for 10min at 4 ℃. And (3) taking 100 mu L of supernatant to a 96-well plate in which 100 mu L of distilled water is added in advance, mixing uniformly, and performing sample analysis by adopting LC-MS/MS.

And (3) data analysis: and detecting peak areas of the corresponding compound and the internal standard through an LC-MS/MS system, and calculating the peak area ratio of the compound to the internal standard. The slope is determined by plotting the natural logarithm of the percentage of compound remaining against time and calculating t according to the following formula_1/2And CL_intWhere V/M is equal to 1/protein concentration.

The results of the metabolic stability experiments are shown in table 2 below:

TABLE 2 metabolic stability in rat liver microsomes

Compound (I)	T_1/2(min)	Rate of elongation
			Darprodustat	267.2	--
T-1	315.1	17.9％
			T-3	479.8	79.5％
T-4	449	68.0％
			T-5	1151.2	330.8％
T-6	483.5	80.9％
			T-7	485.7	81.7％

The experimental result shows that compared with the original medicine Daprodustat, the compound of the invention can obviously prolong the half-life period and has more stable metabolism, and particularly, the half-life period is prolonged by more than 60 percent from the compound T-3 to the compound T-7.

(4) Pharmacokinetic evaluation in rats.

6 male Sprague-Dawley rats, 7-8 weeks old, weighing about 210g, were divided into 2 groups of 3 per group and compared for pharmacokinetic differences with a single intravenous or oral dose of compound (3 mg/kg intravenously, 10mg/kg orally).

Rats were fed with standard feed and given water. Fasting began 16 hours prior to the experiment. The drug was dissolved with PEG400 and dimethyl sulfoxide. Blood was collected from the orbit at 0.083 hr, 0.25 hr, 0.5 hr, 1 hr, 2hr, 4hr, 6 hr, 8 hr, 12 hr and 24 hr post-dose.

The rats were briefly anesthetized after ether inhalation and 300 μ L of blood was collected from the orbit into a test tube. There was 30 μ L of 1% heparin salt solution in the tube. Before use, the tubes were dried overnight at 60 ℃. After completion of blood sample collection at a subsequent time point, rats were sacrificed after ether anesthesia.

Immediately after blood collection, the tubes were gently inverted 5 times to ensure thorough mixing and placed on ice. The blood samples were centrifuged at 5000rpm for 5 minutes at 4 ℃ to separate the plasma from the erythrocytes. Pipette out 100 μ L of plasma into a clean plastic centrifuge tube, indicating the name of the compound and the time point. Plasma was stored at-80 ℃ before analysis. The concentration of the compounds of the invention in plasma was determined by LC-MS/MS. Pharmacokinetic parameters were calculated based on the plasma concentration of each animal at different time points.

The experimental results show that the compound of the invention has better pharmacokinetics in animals and thus better pharmacodynamics and therapeutic effects compared with the control compound AKB-6548.

It is to be understood that these examples are intended to illustrate the invention and are not intended to limit the scope of the invention, and that experimental procedures not specifically identified in the examples will generally be performed under conventional conditions, or under conditions recommended by the manufacturer. Parts and percentages are parts and percentages by weight unless otherwise indicated.

The foregoing is a more detailed description of the invention in connection with specific preferred embodiments and it is not intended that the invention be limited to these specific details. For those skilled in the art to which the invention pertains, several simple deductions or substitutions can be made without departing from the spirit of the invention, and all shall be considered as belonging to the protection scope of the invention.

Claims

1. A compound or a pharmaceutically acceptable salt thereof, wherein the compound is selected from:

2. a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a compound of claim 1 or a pharmaceutically acceptable salt thereof.

3. Use of a compound of claim 1 or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the prevention and treatment of anemia of chronic disease, glucose intolerance and/or anemia associated with kidney disease, as well as anemia of cancer or blood cell related disorders.

4. An intermediate comprising the following compounds: